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Sample records for bovine preimplantation embryos

  1. Preimplantation death of xenomitochondrial mouse embryo harbouring bovine mitochondria

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    Kawahara, Manabu; Koyama, Shiori; Iimura, Satomi; Yamazaki, Wataru; Tanaka, Aiko; Kohri, Nanami; Sasaki, Keisuke; Takahashi, Masashi

    2015-01-01

    Mitochondria, cellular organelles playing essential roles in eukaryotic cell metabolism, are thought to have evolved from bacteria. The organization of mtDNA is remarkably uniform across species, reflecting its vital and conserved role in oxidative phosphorylation (OXPHOS). Our objectives were to evaluate the compatibility of xenogeneic mitochondria in the development of preimplantation embryos in mammals. Mouse embryos harbouring bovine mitochondria (mtB-M embryos) were prepared by the cell-fusion technique employing the haemagglutinating virus of Japan (HVJ). The mtB-M embryos showed developmental delay at embryonic days (E) 3.5 after insemination. Furthermore, none of the mtB-M embryos could implant into the maternal uterus after embryo transfer, whereas control mouse embryos into which mitochondria from another mouse had been transferred developed as well as did non-manipulated embryos. When we performed quantitative PCR (qPCR) of mouse and bovine ND5, we found that the mtB-M embryos contained 8.3% of bovine mitochondria at the blastocyst stage. Thus, contamination with mitochondria from another species induces embryonic lethality prior to implantation into the maternal uterus. The heteroplasmic state of these xenogeneic mitochondria could have detrimental effects on preimplantation development, leading to preservation of species-specific mitochondrial integrity in mammals. PMID:26416548

  2. Sexing bovine pre-implantation embryos using the polymerase ...

    African Journals Online (AJOL)

    The paper aims to present a bovine model for human embryo sexing. Cows were super-ovulated, artificially inseminated and embryos were recovered 7 days later. Embryo biopsy was performed; DNA was extracted from blastomeres and amplified using bovine-specific and bovine-Y-chromosomespecific primers, followed ...

  3. Sexing bovine pre-implantation embryos using the polymerase ...

    African Journals Online (AJOL)

    Yomi

    2012-03-06

    Mar 6, 2012 ... can also be applied to human embryos, using different primers, designed for human DNA. Key words: sexing, embryo, PCR, bovine. INTRODUCTION. In vitro fertilization represents nowadays a modern assisted reproductive technology that can be applied to couples with fertility problems that make natural ...

  4. Expression of microRNAs in bovine and human pre-implantation embryo culture media

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    Kropp, Jenna; Salih, Sana M.; Khatib, Hasan

    2014-01-01

    MicroRNAs (miRNA) are short non-coding RNAs which act to regulate expression of genes driving numerous cellular processes. These RNAs are secreted within exosomes from cells into the extracellular environment where they may act as signaling molecules. In addition, they are relatively stable and are specifically expressed in association to certain cancers making them strong candidates as biological markers. Moreover, miRNAs have been detected in body fluids including urine, milk, saliva, semen, and blood plasma. However, it is unknown whether they are secreted by embryonic cells into the culture media. Given that miRNAs are expressed throughout embryonic cellular divisions and embryonic genome activation, we hypothesized that they are secreted from the embryo into the extracellular environment and may play a role in the developmental competence of bovine embryos. To test this hypothesis, bovine embryos were cultured individually from day 5 to day 8 of development in an in vitro fertilization system and gene expression of 5 miRNAs was analyzed in both embryos and culture media. Differential miRNA gene expression was observed between embryos that developed to the blastocyst stage and those that failed to develop from the morula to blastocyst stage, deemed degenerate embryos. MiR-25, miR-302c, miR-196a2, and miR-181a expression was found to be higher in degenerate embryos compared to blastocyst embryos. Interestingly, these miRNAs were also found to be expressed in the culture media of both bovine and human pre-implantation embryos. Overall, our results show for the first time that miRNAs are secreted from pre-implantation embryos into culture media and that miRNA expression may correlate with developmental competence of the embryo. Expression of miRNAs in in vitro culture media could allow for the development of biological markers for selection of better quality embryos and for subsequent successful pregnancy. PMID:24795753

  5. Expression of microRNAs in bovine and human pre-implantation embryo culture media

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    Jenna eKropp

    2014-04-01

    Full Text Available MicroRNAs (miRNA are short non-coding RNAs which act to regulate expression of genes driving numerous cellular processes. These RNAs are secreted within exosomes from cells into the extracellular environment where they may act as signaling molecules. In addition, they are relatively stable and are specifically expressed in association to certain cancers making them strong candidates as biological markers. Moreover, miRNAs have been detected in body fluids including urine, milk, saliva, semen, and blood plasma. However, it is unknown whether they are secreted by embryonic cells into the culture media. Given that miRNAs are expressed throughout embryonic cellular divisions and embryonic genome activation, we hypothesized that they are secreted from the embryo into the extracellular environment and may play a role in the developmental competence of bovine embryos. To test this hypothesis, bovine embryos were cultured individually from day 5 to day 8 of development in an in vitro fertilization system and gene expression of 5 miRNAs was analyzed in both embryos and culture media. Differential miRNA gene expression was observed between embryos that developed to the blastocyst stage and those that failed to develop from the morula to blastocyst stage, deemed degenerate embryos. MiR-25, mir-302c, miR-196a2, and miR-181a expression was found to be higher in degenerate embryos compared to blastocyst embryos. Interestingly, these miRNAs were also found to be expressed in the culture media of both bovine and human pre-implantation embryos. Overall, our results show for the first time that miRNAs are secreted from pre-implantation embryos into culture media and that miRNA expression may correlate with developmental competence of the embryo. Expression of miRNAs in in vitro culture media could allow for the development of biological markers for selection of better quality embryos and for subsequent successful pregnancy.

  6. Melatonin inhibits paraquat-induced cell death in bovine preimplantation embryos.

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    Pang, Yun-Wei; Sun, Ye-Qing; Sun, Wei-Jun; Du, Wei-Hua; Hao, Hai-Sheng; Zhao, Shan-Jiang; Zhu, Hua-Bin

    2016-03-01

    Preimplantation embryos are sensitive to oxidative stress-induced damage that can be caused by reactive oxygen species (ROS) originating from normal embryonic metabolism and/or the external surroundings. Paraquat (PQ), a commonly used pesticide and potent ROS generator, can induce embryotoxicity. The present study aimed to investigate the effects of melatonin on PQ-induced damage during embryonic development in bovine preimplantation embryos. PQ treatment significantly reduced the ability of bovine embryos to develop to the blastocyst stage, and the addition of melatonin markedly reversed the developmental failure caused by PQ (20.9% versus 14.3%). Apoptotic assay showed that melatonin pretreatment did not change the total cell number in blastocysts, but the incidence of apoptotic nuclei and the release of cytochrome c were significantly decreased. Using real-time quantitative polymerase chain reaction analysis, we found that melatonin pre-incubation significantly altered the expression levels of genes associated with redox signaling, particularly by attenuating the transcript level of Txnip and reinforcing the expression of Trx. Furthermore, melatonin pretreatment significantly reduced the expression of the pro-apoptotic caspase-3 and Bax, while the expression of the anti-apoptotic Bcl-2 and XIAP was unaffected. Western blot analysis showed that melatonin protected bovine embryos from PQ-induced damage in a p38-dependent manner, but extracellular signal-regulated kinase (ERK) and c-JUN N-terminal kinase (JNK) did not appear to be involved. Together, these results identify an underlying mechanism by which melatonin enhances the developmental potential of bovine preimplantation embryos under oxidative stress conditions. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. Preimplantation bovine embryos: Pathobiology of Haemophilus somnus exposure and resistance mechanisms to vesicular stomatitis virus

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    Thomson, M.S.

    1988-01-01

    Preimplantation bovine embryos were exposed in vitro to H. somnus to determine if the bacteria would adhere to zona pellucida-intact (ZP-I) embryos or adhere to or infect ZP-free embryos. The effect of H. somnus on embryonic development in vitro was also investigated. Electrophoretic comparisons of outer membrane proteins of H. somnus revealed 2 major protein bands common to 10 H. somnus isolates. A monoclonal antibody produced against the outer membrane proteins reacted to one of the major protein bands. The sensitivity of a nucleic acid probe for detection of vesicular stomatitis virus (VSV) was validated in cells in culture and used to determine if the synthetic double-stranded complex of polyriboinosinic and polyribocytidylic acids (poly I:C) would induce viral resistance in cultured bovine embryos. Two {sup 32}P-nick translated probes of high specific activity prepared from plasmids containing nucleic acid sequences of VSV virus were employed for viral mRNA detection in the tissue culture cells using a DNA-hybridization dot-blot technique. Using one of the probes, the technique was applied to detect differences in viral replication between four groups of bovine embryos (nonexposed, exposed to VSV virus, poly I:C-treated, and poly I:C-treated and exposed to VSV). The nucleic acid probe was sufficiently sensitive to detect differences in quantities of VSV mRNA among embryo treatment groups, resulting in the demonstration that resistance to viral infection was induced in day 9 bovine embryos.

  8. Sex determination and milk protein genotyping of preimplantation stage bovine embryos using multiplex PCR.

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    Agrawala, P L; Wagner, V A; Geldermann, H

    1992-11-01

    A method for determining the sex and milk protein genotypes (RFLPs) of preimplantation stage bovine embryos using multiplex polymerase chain reaction (PCR) is described. Day 6 to 7 embryos were micromanipulated to isolate 5 to 6 cells. These cells were then dried in reaction tubes for transport to the laboratory. Subsequently, two sets of PCRs were performed using Y chromosome, k-casein and beta-lactoglobulin gene specific primers, followed by electrophoretic analysis of the PCR products. The presence or absence of the Y chromosome was ascertained in 90 of 92 embryos. Moreover, the k-casein specific fragment was amplified and detected in all these embryos. The PCR products were digested in order to genotype the k-casein gene. In 70% of the embryos, the beta-lactoglobulin specific fragment was amplified, although together with some unspecific fragments.

  9. PreImplantation Factor (PIF correlates with early mammalian embryo development-bovine and murine models

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    Coulam Carolyn B

    2011-05-01

    Full Text Available Abstract Background PreImplantation Factor (PIF, a novel peptide secreted by viable embryos is essential for pregnancy: PIF modulates local immunity, promotes decidual pro-adhesion molecules and enhances trophoblast invasion. To determine the role of PIF in post-fertilization embryo development, we measured the peptide's concentration in the culture medium and tested endogenous PIF's potential trophic effects and direct interaction with the embryo. Methods Determine PIF levels in culture medium of multiple mouse and single bovine embryos cultured up to the blastocyst stage using PIF-ELISA. Examine the inhibitory effects of anti-PIF-monoclonal antibody (mAb added to medium on cultured mouse embryos development. Test FITC-PIF uptake by cultured bovine blastocysts using fluorescent microscopy. Results PIF levels in mouse embryo culture medium significantly increased from the morula to the blastocyst stage (ANOVA, P = 0.01. In contrast, atretic embryos medium was similar to the medium only control. Detectable - though low - PIF levels were secreted already by 2-cell stage mouse embryos. In single bovine IVF-derived embryos, PIF levels in medium at day 3 of culture were higher than non-cleaving embryos (control (P = 0.01 and at day 7 were higher than day 3 (P = 0.03. In non-cleaving embryos culture medium was similar to medium alone (control. Anti-PIF-mAb added to mouse embryo cultures lowered blastocyst formation rate 3-fold in a dose-dependent manner (2-way contingency table, multiple groups, X2; P = 0.01 as compared with non-specific mouse mAb, and medium alone, control. FITC-PIF was taken-up by cultured bovine blastocysts, but not by scrambled FITC-PIF (control. Conclusions PIF is an early embryo viability marker that has a direct supportive role on embryo development in culture. PIF-ELISA use to assess IVF embryo quality prior to transfer is warranted. Overall, our data supports PIF's endogenous self sustaining role in embryo development and the

  10. Supplementation of fetal bovine serum alters histone modification H3R26me2 during preimplantation development of in vitro produced bovine embryos

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    Daniel R. Arnold

    2015-07-01

    Full Text Available Abstract In vitro production (IVP of bovine embryos is not only of great economic importance to the cattle industry, but is also an important model for studying embryo development. The aim of this study was to evaluate the histone modification, H3R26me2 during pre-implantation development of IVP bovine embryos cultured with or without serum supplementation and how these in vitro treatments compared to in vivo embryos at the morula stage. After in vitro maturation and fertilization, bovine embryos were cultured with either 0 or 2.5% fetal bovine serum (FBS. Development was evaluated and embryos were collected and fixed at different stages during development (2-, 4-, 8-, 16-cell, morula and blastocyst. Fixed embryos were then used for immunofluorescence utilizing an antibody for H3R26me2. Images of stained embryos were analyzed as a percentage of total DNA. Embryos cultured with 2.5% FBS developed to blastocysts at a greater rate than 0%FBS groups (34.85±5.43% vs. 23.38±2.93%; P<0.05. Levels of H3R26me2 changed for both groups over development. In the 0%FBS group, the greatest amount of H3R26me2 staining was at the 4-cell (P<0.05, 16-cell (P<0.05 and morula (P<0.05 stages. In the 2.5%FBS group, only 4-cell stage embryos were significantly higher than all other stages (P<0.01. Morula stage in vivo embryos had similar levels as the 0%FBS group, and both were significantly higher than the 2.5%FBS group. These results suggest that the histone modification H3R26me2 is regulated during development of pre-implantation bovine embryos, and that culture conditions greatly alter this regulation.

  11. Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos

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    Van Zeveren Alex

    2005-12-01

    Full Text Available Abstract Background Real-time quantitative PCR is a sensitive and very efficient technique to examine gene transcription patterns in preimplantation embryos, in order to gain information about embryo development and to optimize assisted reproductive technologies. Critical to the succesful application of real-time PCR is careful assay design, reaction optimization and validation to maximize sensitivity and accuracy. In most of the studies published GAPD, ACTB or 18S rRNA have been used as a single reference gene without prior verification of their expression stability. Normalization of the data using unstable controls can result in erroneous conclusions, especially when only one reference gene is used. Results In this study the transcription levels of 8 commonly used reference genes (ACTB, GAPD, Histone H2A, TBP, HPRT1, SDHA, YWHAZ and 18S rRNA were determined at different preimplantation stages (2-cell, 8-cell, blastocyst and hatched blastocyst in order to select the most stable genes to normalize quantitative data within different preimplantation embryo stages. Conclusion Using the geNorm application YWHAZ, GAPD and SDHA were found to be the most stable genes across the examined embryonic stages, while the commonly used ACTB was shown to be highly regulated. We recommend the use of the geometric mean of those 3 reference genes as an accurate normalization factor, which allows small expression differences to be reliably measured.

  12. Active caspase-3 and ultrastructural evidence of apoptosis in spontaneous and induced cell death in bovine in vitro produced pre-implantation embryos

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    Gjørret, Jakob O.; Fabian, Dusan; Avery, Birthe

    2007-01-01

    In this study we investigated chronological onset and involvement of active caspase-3, apoptotic nuclear morphology, and TUNEL-labeling, as well as ultrastructural evidence of apoptosis, in both spontaneous and induced cell death during pre-implantation development of bovine in vitro produced...... embryos. Pre-implantation embryos (2-cell to Day 8 blastocysts) were cultured with either no supplementation (untreated) or with 10 µM staurosporine for 24 hr (treated). Embryos were subjected to immunohistochemical staining of active caspase-3, TUNEL-reaction for detection of DNA degradation and DAPI...

  13. Genetic variation in resistance of the preimplantation bovine embryo to heat shock.

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    Hansen, Peter J

    2014-12-01

    Reproduction is among the physiological functions in mammals most susceptible to disruption by hyperthermia. Many of the effects of heat stress on function of the oocyte and embryo involve direct effects of elevated temperature (i.e. heat shock) on cellular function. Mammals limit the effects of heat shock by tightly regulating body temperature. This ability is genetically controlled: lines of domestic animals have been developed with superior ability to regulate body temperature during heat stress. Through experimentation in cattle, it is also evident that there is genetic variation in the resistance of cells to the deleterious effects of elevated temperature. Several breeds that were developed in hot climates, including Bos indicus (Brahman, Gir, Nelore and Sahiwal) and Bos taurus (Romosinuano and Senepol) are more resistant to the effects of elevated temperature on cellular function than breeds that evolved in cooler climates (Angus, Holstein and Jersey). Genetic differences are expressed in the preimplantation embryo by Day 4-5 of development (after embryonic genome activation). It is not clear whether genetic differences are expressed in cells in which transcription is repressed (oocytes >100 µm in diameter or embryos at stages before embryonic genome activation). The molecular basis for cellular thermotolerance has also not been established, although there is some suggestion for involvement of heat shock protein 90 and the insulin-like growth factor 1 system. Given the availability of genomic tools for genetic selection, identification of genes controlling cellular resistance to elevated temperature could be followed by progress in selection for those genes within the populations in which they exist. It could also be possible to introduce genes from thermotolerant breeds into thermally sensitive breeds. The ability to edit the genome makes it possible to design new genes that confer protection of cells from stresses like heat shock.

  14. Validating reference microRNAs for normalizing qRT-PCR data in bovine oocytes and preimplantation embryos.

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    Mahdipour, Mahdi; van Tol, Helena T A; Stout, Tom A E; Roelen, Bernard A J

    2015-06-12

    MicroRNAs (miRNAs) are small noncoding RNAs that act as post-transcriptional regulators of gene targets. Accurate quantification of miRNA expression using validated internal controls should aid in the understanding of their role in epigenetic modification of genome function. To date, most studies that have examined miRNA expression levels have used the global mean expression of all expressed genes or the expression of reference mRNAs or nuclear RNAs for normalization. We analyzed the suitability of a number of miRNAs as potential expression normalizers in bovine oocytes and early embryos, and porcine oocytes. The stages examined were bovine oocytes at the germinal vesicle (GV) and metaphase II stages, bovine zygotes, 2, 4 and 8 cell embryos, morulae and blastocysts, as well as porcine cumulus oocyte complexes, GV, metaphase I and II oocytes. qRT-PCR was performed to quantify expression of miR-93, miR-103, miR-26a, miR-191, miR-23b, Let-7a and U6 for bovine samples and miR-21, miR-26a, miR-93, miR-103, miR-148a, miR-182 and miR-191 for porcine oocytes. The average starting material for each sample was determined using specific standard curves for each primer set. Subsequently, geNorm and BestKeeper software were used to identify a set of stably expressed miRNAs. Stepwise removal to determine the optimum number of reference miRNAs identified miR-93 and miR-103 as the most stably expressed in bovine samples and miR-26a, miR-191 and miR-93 in porcine samples. The combination of miR-93 and miR-103 is optimal for normalizing miRNA expression for qPCR experiments on bovine oocytes and preimplantation embryos; the preferred combination for porcine oocytes is miR-26a, miR-191 and miR-93.

  15. Male fertility status is associated with DNA methylation signatures in sperm and transcriptomic profiles of bovine preimplantation embryos.

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    Kropp, Jenna; Carrillo, José A; Namous, Hadjer; Daniels, Alyssa; Salih, Sana M; Song, Jiuzhou; Khatib, Hasan

    2017-04-05

    stage, preimplantation embryos derived from high and low fertility bulls displayed significant transcriptomic differences. The relationship between the paternal contribution and the embryonic transcriptome is unclear, although differences in methylated regions were identified which could influence the reprogramming of the early embryo. Further characterization of paternal factors delivered to the oocyte could lead to the identification of biomarkers for better selection of sires to improve reproductive efficiency.

  16. The role of RNA polymerase I transcription and embryonic genome activation in nucleolar development in bovine preimplantation embryos

    DEFF Research Database (Denmark)

    Østrup, Olga; Strejcek, F.; Petrovicova, I.

    2008-01-01

    was lacking and clustering of nucleolar proteins was hampered. In conclusion, rDNA transcription is not required for targeting of rRNA processing proteins, rRNA is maternally inherited and target to rDNA independent of transcription, and de novo transcription is required for proper nucleologenesis in cattle.......The aim of the present study was to investigate the role of RNA polymerase I (RPI) transcription in nucleolar development during major transcriptional activation (MTA) in cattle. Late eight-cell embryos were cultured in the absence (control group) or presence of actinomycin D (AD) (RPI inhibition......, Ad 0.2 µg/ml; total transcriptional inhibition, AD 2.0 µg/ml). Late four-cell embryos were cultured to late eight-cell stage in 0.2 µg/ml AD (MTA prevention, ADLT (long-term total transcriptional inhibition group). Embryos were processed for autoradiography, transmission electron microscopy...

  17. Chromosomal mosaicism in human preimplantation embryos : a systematic review

    NARCIS (Netherlands)

    van Echten-Arends, Jannie; Mastenbroek, Sebastiaan; Sikkema-Raddatz, Birgit; Korevaar, Johanna C.; Heineman, Maas Jan; van der Veen, Fulco; Repping, Sjoerd

    2011-01-01

    BACKGROUND: Although chromosomal mosaicism in human preimplantation embryos has been described for almost two decades, its exact prevalence is still unknown. The prevalence of mosaicism is important in the context of preimplantation genetic screening in which the chromosomal status of an embryo is

  18. Interaction of bluetongue virus with preimplantation embryos from mice and cattle.

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    Bowen, R A; Howard, T H; Pickett, B W

    1982-11-01

    Preimplantation embryos from mice and cattle were exposed to bluetongue virus in vitro to determine whether the virus would replicate in these early embryos and, if so, what pathologic consequences would ensue. A high proportion of zona pellucida-free, 2-cell embryos and morulae from mice, and morulae from cattle became infected. The infection was rapidly cytopathic in embryos from both species. Indirect immunofluorescence was used to demonstrate accumulation of virus antigen in the blastomeres of these embryos. The zona pellucida of both murine and bovine embryos provided effective protection from virus present in culture fluid.

  19. Epigenetics and chromosome segregation in human pre-implantation embryos

    NARCIS (Netherlands)

    C. van de Werken (Christine)

    2015-01-01

    markdownabstractAbstract Chapter 1 Currently, the average pregnancy rate per embryo transfer after in vitro fertilization (IVF) is around 32%. In order to achieve better results in the future, we need to gain knowledge on all aspects of the treatment, including pre-implantation embryo

  20. Dilemmas encountered with preimplantation diagnosis of aneuploidy in human embryos.

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    Allan, John; Edirisinghe, Rohini; Anderson, Jasen; Jemmott, Rodney; Nandini, A V; Gattas, Michael

    2004-04-01

    An increased embryo aneuploidy rate is associated with advancing maternal age. Preimplantation genetic diagnosis (PGD) using fluorescence in situ hybridisation (FISH) coupled with in vitro fertilisation (IVF)/embryo biopsy provides a powerful tool to improve the take home baby rates for this poor prognostic group. To report the preliminary findings of a PGD study for aneuploidy screening and to discuss the dilemmas encountered. Preimplantation genetic diagnosis was offered in egg pick up-PGD and frozen embryo transfer-PGD cycles. Embryo biopsy was carried out on day 3 and FISH was used to detect chromosomal abnormalities. The outcome of 75 patients, 100 treatment cycles; 62 egg pick up-PGD and 38 frozen embryo transfer-PGD are presented. The embryo biopsy rate, blastomere survival, presence of nuclei and successful FISH rates for egg pick-up and frozen embryo transfer cycles were similar giving a chromosomal abnormality rate of 57.5 and 51.2% for the respective treatment group. The positive pregnancy, clinical pregnancy and implantation rates were, for egg pick up-PGD 22.7, 13.6 and 21.1% and for frozen embryo transfer-PGD 13.8, 10.3 and 10.0%, respectively. Preimplantation genetic diagnosis coupled with IVF treatment seems to give satisfactory pregnancy rates. The high embryo aneuploidy rates, chromosomal mosaicism and other issues have presented significant ethical and management dilemmas for our physicians and patients alike. These issues highlight the importance of skillful pretreatment counselling for patients considering PGD.

  1. Actions of activin A, connective tissue growth factor, hepatocyte growth factor and teratocarcinoma-derived growth factor 1 on the development of the bovine preimplantation embryo.

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    Kannampuzha-Francis, Jasmine; Tribulo, Paula; Hansen, Peter J

    2017-07-01

    The reproductive tract secretes bioactive molecules collectively known as embryokines that can regulate embryonic growth and development. In the present study we tested four growth factors expressed in the endometrium for their ability to modify the development of the bovine embryo to the blastocyst stage and alter the expression of genes found to be upregulated (bone morphogenetic protein 15 (BMP15) and keratin 8, type II (KRT8)) or downregulated (NADH dehydrogenase 1 (ND1) and S100 calcium binding protein A10 (S100A10)) in embryos competent to develop to term. Zygotes were treated at Day 5 with 0.01, 0.1 or 1.0nM growth factor. The highest concentration of activin A increased the percentage of putative zygotes that developed to the blastocyst stage. Connective tissue growth factor (CTGF) increased the number of cells in the inner cell mass (ICM), decreased the trophectoderm:ICM ratio and increased blastocyst expression of KRT8 and ND1. The lowest concentration of hepatocyte growth factor (HGF) reduced the percentage of putative zygotes becoming blastocysts. Teratocarcinoma-derived growth factor 1 increased total cell number at 0.01nM and expression of S100A10 at 1.0nM, but otherwise had no effects. Results confirm the prodevelopmental actions of activin A and indicate that CTGF may also function as an embryokine by regulating the number of ICM cells in the blastocyst and altering gene expression. Low concentrations of HGF were inhibitory to development.

  2. Cryopreservation of preimplantation embryos of cattle, sheep, and goats.

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    Youngs, Curtis R

    2011-08-05

    Preimplantation embryos from cattle, sheep, and goats may be cryopreserved for short- or long-term storage. Preimplantation embryos consist predominantly of water, and the avoidance of intracellular ice crystal formation during the cryopreservation process is of paramount importance to maintain embryo viability. Embryos are placed into a hypertonic solution (1.4 - 1.5 M) of a cryoprotective agent (CPA) such as ethylene glycol (EG) or glycerol (GLYC) to create an osmotic gradient that facilitates cellular dehydration. After embryos reach osmotic equilibrium in the CPA solution, they are individually loaded in the hypertonic CPA solution into 0.25 ml plastic straws for freezing. Embryos are placed into a controlled rate freezer at a temperature of -6°C. Ice crystal formation is induced in the CPA solution surrounding the embryo, and crystallization causes an increase in the concentration of CPA outside of the embryo, causing further cellular dehydration. Embryos are cooled at a rate of 0.5°C/min, enabling further dehydration, to a temperature of -34°C before being plunged into liquid nitrogen (-196°C). Cryopreserved embryos must be thawed prior to transfer to a recipient (surrogate) female. Straws containing the embryos are removed from the liquid nitrogen dewar, held in room temperature air for 3 to 5 sec, and placed into a 37°C water bath for 25 to 30 sec. Embryos cryopreserved in GLYC are placed into a 1 M solution of sucrose for 10 min for removal of the CPA before transfer to a recipient (surrogate) female. Embryos cryopreserved in EG, however, may be directly transferred to the uterus of a recipient.

  3. Causes and consequences of chromosome segregation error in preimplantation embryos.

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    Vázquez-Diez, Cayetana; FitzHarris, Greg

    2018-01-01

    Errors in chromosome segregation are common during the mitotic divisions of preimplantation development in mammalian embryos, giving rise to so-called 'mosaic' embryos possessing a mixture of euploid and aneuploid cells. Mosaicism is widely considered to be detrimental to embryo quality and is frequently used as criteria to select embryos for transfer in human fertility clinics. However, despite the clear clinical importance, the underlying defects in cell division that result in mosaic aneuploidy remain elusive. In this review, we summarise recent findings from clinical and animal model studies that provide new insights into the fundamental mechanisms of chromosome segregation in the highly unusual cellular environment of early preimplantation development and consider recent clues as to why errors should commonly occur in this setting. We furthermore discuss recent evidence suggesting that mosaicism is not an irrevocable barrier to a healthy pregnancy. Understanding the causes and biological impacts of mosaic aneuploidy will be pivotal in the development and fine-tuning of clinical embryo selection methods. © 2018 Society for Reproduction and Fertility.

  4. Effects of steroidal glycoalkaloids from potatoes (Solanum tuberosum) on in vitro bovine embryo development.

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    Wang, S; Panter, K E; Gaffield, W; Evans, R C; Bunch, T D

    2005-02-01

    alpha-Solanine and alpha-chaconine are two naturally occurring steroidal glycoalkaloids in potatoes (Solanum tuberosum), and solanidine-N-oxide is a corresponding steroidal aglycone. The objective of this research was to screen potential cyto-toxicity of these potato glycoalkaloids using bovine oocyte maturation, in vitro fertilization techniques and subsequent embryonic development as the in vitro model. A randomized complete block design with four in vitro oocyte maturation (IVM) treatments (Experiment 1) and four in vitro embryo culture (IVC) treatments (Experiment 2) was used. In Experiment 1, bovine oocytes (n=2506) were matured in vitro in medium supplemented with 6 microM of alpha-solanine, alpha-chaconine, solanidine-N-oxide or IVM medium only. The in vitro matured oocytes were then subject to routine IVF and IVC procedures. Results indicated that exposure of bovine oocytes to the steroidal glycoalkaloids during in vitro maturation inhibited subsequent pre-implantation embryo development. Potency of the embryo-toxicity varied between these steroidal glycoalkaloids. In Experiment 2, IVM/IVF derived bovine embryos (n=2370) were cultured in vitro in medium supplemented with 6 microM of alpha-solanine, alpha-chaconine, solanidine-N-oxide or IVC medium only. The results showed that the pre-implantation embryo development is inhibited by exposure to these glycoalkaloids. This effect is significant during the later pre-implantation embryo development period as indicated by fewer numbers of expanded and hatched blastocysts produced in the media containing these alkaloids. Therefore, we conclude that in vitro exposure of oocytes and fertilized ova to the steroidal glycoalkaloids from potatoes inhibits pre-implantation embryo development. Furthermore, we suggest that ingestion of Solanum species containing toxic amounts of glycoalkaloids may have negative effects on pre-implantation embryonic survival.

  5. Sperm as a noninvasive gene delivery system for preimplantation embryos.

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    Chan, P J; Kalugdan, T; Su, B C; Whitney, E A; Perrott, W; Tredway, D R; King, A

    1995-05-01

    To determine if sperm could be manipulated to be a noninvasive transport carrier for the delivery of gene fragments to the blastocyst. Sperm cells carrying foreign DNA fragments from human papillomavirus (HPV) types 16, 18, 31, and 33 were allowed to migrate from one end of an artificial reproductive tube and to come in contact with hatching mouse blastocysts at the other end of the tube. The blastocysts were then washed and analyzed for the presence of the foreign DNA fragments. Clinical and academic research environment. Detection of amplified products from transferred foreign DNA using the polymerase chain reaction and primers targeted at the E6-E7 region for different HPV types. Polymerase chain reaction analyses showed transference of DNA HPV type 18 to the blastocysts. Not all types of DNA fragments were transferred equally. The results suggested the possibility of using sperm as a noninvasive gene delivery system for passing on gene fragments to preimplantation embryos. It was demonstrated that certain DNA fragments were easier to deliver than others, indicating the necessity for exploring all the factors involved in the mechanism of the transference process. The study also serves to highlight the possibility of unintentional transmission of viral or bacterial DNA to the developing embryo via the sperm.

  6. Frequency of chromosomal aneuploidy in high quality embryos from young couples using preimplantation genetic screening

    Directory of Open Access Journals (Sweden)

    Farzaneh Fesahat

    2017-09-01

    Full Text Available Background: Selection of the best embryo for transfer is very important in assisted reproductive technology (ART. Using morphological assessment for this selection demonstrated that the correlation between embryo morphology and implantation potential is relatively weak. On the other hand, aneuploidy is a key genetic factor that can influence human reproductive success in ART. Objective: The aim of this lab trial study was to evaluate the incidence of aneuploidies in five chromosomes in the morphologically high-quality embryos from young patients undergoing ART for sex selection. Materials and Methods: A total of 97 high quality embryos from 23 women at the age of 37or younger years that had previously undergone preimplantation genetic screening for sex selection were included in this study. After washing, the slides of blastomeres from embryos of patients were reanalyzed by fluorescence in-situ hybridization for chromosomes 13, 18 and 21. Results: There was a significant rate of aneuploidy determination in the embryos using preimplantation genetic screening for both sex and three evaluated autosomal chromosomes compared to preimplantation genetic screening for only sex chromosomes (62.9% vs. 24.7%, p=0.000. The most frequent detected chromosomal aneuploidy was trisomy or monosomy of chromosome 13. Conclusion: There is considerable numbers of chromosomal abnormalities in embryos generated in vitro which cause in vitro fertilization failure and it seems that morphological characterization of embryos is not a suitable method for choosing the embryos without these abnormalities

  7. Frequency of chromosomal aneuploidy in high quality embryos from young couples using preimplantation genetic screening.

    Science.gov (United States)

    Fesahat, Farzaneh; Montazeri, Fatemeh; Sheikhha, Mohammad Hasan; Saeedi, Hojjatollah; Dehghani Firouzabadi, Razieh; Kalantar, Seyed Mehdi

    2017-05-01

    Selection of the best embryo for transfer is very important in assisted reproductive technology (ART). Using morphological assessment for this selection demonstrated that the correlation between embryo morphology and implantation potential is relatively weak. On the other hand, aneuploidy is a key genetic factor that can influence human reproductive success in ART. The aim of this lab trial study was to evaluate the incidence of aneuploidies in five chromosomes in the morphologically high-quality embryos from young patients undergoing ART for sex selection. A total of 97 high quality embryos from 23 women at the age of 37or younger years that had previously undergone preimplantation genetic screening for sex selection were included in this study. After washing, the slides of blastomeres from embryos of patients were reanalyzed by fluorescence in-situ hybridization for chromosomes 13, 18 and 21. There was a significant rate of aneuploidy determination in the embryos using preimplantation genetic screening for both sex and three evaluated autosomal chromosomes compared to preimplantation genetic screening for only sex chromosomes (62.9% vs. 24.7%, p=0.000). The most frequent detected chromosomal aneuploidy was trisomy or monosomy of chromosome 13. There is considerable numbers of chromosomal abnormalities in embryos generated in vitro which cause in vitro fertilization failure and it seems that morphological characterization of embryos is not a suitable method for choosing the embryos without these abnormalities.

  8. Proteomic analysis of the early bovine yolk sac fluid and cells from the day 13 ovoid and elongated preimplatation embryos

    DEFF Research Database (Denmark)

    Jensen, Pernille L.; Beck, Hans Christian; Petersen, Tonny S.

    2014-01-01

    The bovine blastocyst hatches 8 to 9 days after fertilization, and this is followed by several days of preimplantation development during which the embryo transforms from a spherical over an ovoid to an elongated shape. As the spherical embryo enlarges, the cells of the inner cell mass differenti......The bovine blastocyst hatches 8 to 9 days after fertilization, and this is followed by several days of preimplantation development during which the embryo transforms from a spherical over an ovoid to an elongated shape. As the spherical embryo enlarges, the cells of the inner cell mass...... fluid and cellular components were isolated from 12 ovoid and three elongated embryos and using nano-high-performance liquid chromatography, tandem mass spectrometry, and isobaric tag for relative and absolute quantitation proteomic analysis, a total of 9652 unique proteins were identified. We performed...

  9. Differences in gene expression profiles between human preimplantation embryos cultured in two different IVF culture media.

    Science.gov (United States)

    Kleijkers, Sander H M; Eijssen, Lars M T; Coonen, Edith; Derhaag, Josien G; Mantikou, Eleni; Jonker, Martijs J; Mastenbroek, Sebastiaan; Repping, Sjoerd; Evers, Johannes L H; Dumoulin, John C M; van Montfoort, Aafke P A

    2015-10-01

    Is gene expression in human preimplantation embryos affected by the medium used for embryo culture in vitro during an IVF treatment? Six days of in vitro culture of human preimplantation embryos resulted in medium-dependent differences in expression level of genes involved in apoptosis, protein degradation, metabolism and cell-cycle regulation. Several human studies have shown an effect of culture medium on embryo development, pregnancy outcome and birthweight. However, the underlying mechanisms in human embryos are still unknown. In animal models of human development, it has been demonstrated that culture of preimplantation embryos in vitro affects gene expression. In humans, it has been found that culture medium affects gene expression of cryopreserved embryos that, after thawing, were cultured in two different media for 2 more days. In a multicenter trial, women were randomly assigned to two culture medium groups [G5 and human tubal fluid (HTF)]. Data on embryonic development were collected for all embryos. In one center, embryos originating from two pronuclei (2PN) zygotes that were not selected for transfer or cryopreservation on Day 2 or 3 because of lower morphological quality, were cultured until Day 6 and used in this study, if couples consented. Ten blastocysts each from the G5 and HTF study groups, matched for fertilization method, maternal age and blastocyst quality, were selected and their mRNA was isolated and amplified. Embryos were examined individually for genome-wide gene expression using Agilent microarrays and PathVisio was used to identify the pathways that showed a culture medium-dependent activity. Expression of 951 genes differed significantly (P differences observed between the study groups are caused by factors that we did not investigate. Extrapolation of these results to embryos used for transfer demands caution as in the present study embryos that were not selected for either embryo transfer or cryopreservation have been used for the

  10. Localization and expression of histone H2A variants during mouse oogenesis and preimplantation embryo development.

    Science.gov (United States)

    Wu, B J; Dong, F L; Ma, X S; Wang, X G; Lin, F; Liu, H L

    2014-08-07

    Epigenetic modifications of the genome, such as histone H2A variants, ensure appropriate gene activation or silencing during oogenesis and preimplantation embryo development. We examined global localization and expression of the histone H2A variants, including H2A.Bbd, H2A.Z and H2A.X, during mouse oogenesis and preimplantation embryo development. Immunocytochemistry with specific antibodies against various histone H2A variants showed their localization and changes during oogenesis and preimplantation development. H2A.Bbd and H2A.Z were almost absent from nuclei of growing oocytes (except 5-day oocyte), whereas H2A.X was deposited in nuclei throughout oogenesis and in preimplantation embryos. In germinal vesicle (GV) oocyte chromatin, H2A.Bbd was detected as a weak signal, whereas no fluorescent signal was detected in GV breakdown (GVBD) or metaphase II (MII) oocytes; H2A.Z showed intense signals in chromatin of GV, GVBD and MII oocytes. H2A. Bbd showed very weak signals in both pronucleus and 2-cell embryo nuclei, but intense signals were detected in nuclei from 4-cell embryo to blastula. The H2A.Z signal was absent from pronucleus to morula chromatin, whereas a fluorescent signal was detected in blastula nuclei. Our results suggest that histone H2A variants are probably involved in reprogramming of genomes during oocyte meiosis or after fertilization.

  11. Maternal Diabetes Leads to Unphysiological High Lipid Accumulation in Rabbit Preimplantation Embryos

    NARCIS (Netherlands)

    Schindler, Maria; Pendzialek, Mareike; Santos, Alexander Navarrete; Ploesch, Torsten; Seyring, Stefanie; Guerke, Jacqueline; Haucke, Elisa; Knelangen, Julia Miriam; Fischer, Bernd; Santos, Anne Navarrete

    According to the "developmental origin of health and disease" hypothesis, the metabolic set points of glucose and lipid metabolism are determined prenatally. In the case of a diabetic pregnancy, the embryo is exposed to higher glucose and lipid concentrations as early as during preimplantation

  12. Dehydroepiandrosterone (DHEA) reduces embryo aneuploidy: direct evidence from preimplantation genetic screening (PGS)

    OpenAIRE

    Gleicher, Norbert; Weghofer, Andrea; Barad, David H

    2010-01-01

    Abstract Background Dehydroepiandrosterone (DHEA) has been reported to improve pregnancy chances in women with diminished ovarian reserve (DOR), and to reduce miscarriage rates by 50-80%. Such an effect is mathematically inconceivable without beneficial effects on embryo ploidy. This study, therefore, assesses effects of DHEA on embryo aneuploidy. Methods In a 1:2, matched case control study 22 consecutive women with DOR, supplemented with DHEA, underwent preimplantation genetic screening (PG...

  13. The Impact of Biopsy on Human Embryo Developmental Potential during Preimplantation Genetic Diagnosis

    Directory of Open Access Journals (Sweden)

    Danilo Cimadomo

    2016-01-01

    Full Text Available Preimplantation Genetic Diagnosis and Screening (PGD/PGS for monogenic diseases and/or numerical/structural chromosomal abnormalities is a tool for embryo testing aimed at identifying nonaffected and/or euploid embryos in a cohort produced during an IVF cycle. A critical aspect of this technology is the potential detrimental effect that the biopsy itself can have upon the embryo. Different embryo biopsy strategies have been proposed. Cleavage stage blastomere biopsy still represents the most commonly used method in Europe nowadays, although this approach has been shown to have a negative impact on embryo viability and implantation potential. Polar body biopsy has been proposed as an alternative to embryo biopsy especially for aneuploidy testing. However, to date no sufficiently powered study has clarified the impact of this procedure on embryo reproductive competence. Blastocyst stage biopsy represents nowadays the safest approach not to impact embryo implantation potential. For this reason, as well as for the evidences of a higher consistency of the molecular analysis when performed on trophectoderm cells, blastocyst biopsy implementation is gradually increasing worldwide. The aim of this review is to present the evidences published to date on the impact of the biopsy at different stages of preimplantation development upon human embryos reproductive potential.

  14. The Impact of Biopsy on Human Embryo Developmental Potential during Preimplantation Genetic Diagnosis

    Science.gov (United States)

    Cimadomo, Danilo; Capalbo, Antonio; Ubaldi, Filippo Maria; Scarica, Catello; Palagiano, Antonio; Canipari, Rita; Rienzi, Laura

    2016-01-01

    Preimplantation Genetic Diagnosis and Screening (PGD/PGS) for monogenic diseases and/or numerical/structural chromosomal abnormalities is a tool for embryo testing aimed at identifying nonaffected and/or euploid embryos in a cohort produced during an IVF cycle. A critical aspect of this technology is the potential detrimental effect that the biopsy itself can have upon the embryo. Different embryo biopsy strategies have been proposed. Cleavage stage blastomere biopsy still represents the most commonly used method in Europe nowadays, although this approach has been shown to have a negative impact on embryo viability and implantation potential. Polar body biopsy has been proposed as an alternative to embryo biopsy especially for aneuploidy testing. However, to date no sufficiently powered study has clarified the impact of this procedure on embryo reproductive competence. Blastocyst stage biopsy represents nowadays the safest approach not to impact embryo implantation potential. For this reason, as well as for the evidences of a higher consistency of the molecular analysis when performed on trophectoderm cells, blastocyst biopsy implementation is gradually increasing worldwide. The aim of this review is to present the evidences published to date on the impact of the biopsy at different stages of preimplantation development upon human embryos reproductive potential. PMID:26942198

  15. Expression of Aquaporins in Human Embryos and Potential Role of AQP3 and AQP7 in Preimplantation Mouse Embryo Development

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    Yun Xiong

    2013-05-01

    Full Text Available Background/Aims: Water channels, also named aquaporins (AQPs, play crucial roles in cellular water homeostasis. Methods: RT-PCR indicated the mRNA expression of AQPs 1-5, 7, 9, and 11-12, but not AQPs 0, 6, 8, and 10 in the 2∼8-cell stage human embryos. AQP3 and AQP7 were further analyzed for their mRNA expression and protein expression in the oocyte, zygote, 2-cell embryo, 4-cell embryo, 8-cell embryo, morula, and blastocyst from both human and mouse using RT-PCR and immunofluorescence, respectively. Results: AQP3 and AQP7 were detected in all these stages. Knockdown of either AQP3 or AQP7 by targeted siRNA injection into 2-cell mouse embryos significantly inhibited preimplantation embryo development. However, knockdown of AQP3 in JAr spheroid did not affect its attachment to Ishikawa cells. Conclusion: These data demonstrate that multiple aquaporins are expressed in the early stage human embryos and that AQP3 and AQP7 may play a role in preimplantation mouse embryo development.

  16. Advances in embryo culture platforms: novel approaches to improve preimplantation embryo development through modifications of the microenvironment.

    Science.gov (United States)

    Swain, J E; Smith, G D

    2011-01-01

    The majority of research aimed at improving embryo development in vitro has focused on manipulation of the chemical environment, examining details such as energy substrate composition and impact of various growth factors or other supplements. In comparison, relatively little work has been done examining the physical requirements of preimplantation embryos and the role culture platforms or devices can play in influencing embryo development. Electronic searches were performed using keywords centered on embryo culture techniques using PUBMED through June 2010 and references were searched for additional research articles. Various approaches to in vitro embryo culture that involve manipulations of the physical culture environment are emerging. Novel culture platforms being developed examine issues such as media volume and embryo spacing. Furthermore, methods to permit dynamic embryo culture with fluid flow and embryo movement are now available, and novel culture surfaces are being tested. Although several factors remain to be studied to optimize efficiency, manipulations of the embryo culture microenvironment through novel culture devices may offer a means to improve embryo development in vitro. Reduced volume systems that reduce embryo spacing, such as the well-of-the-well approach, appear beneficial, although more work is needed to verify the source of their true benefit in human embryos. Emerging microfluidic technology appears to be a promising approach. However, along with the work on specialized culture surfaces, more information is required to determine the impact on human embryo development.

  17. Principles guiding embryo selection following genome-wide haplotyping of preimplantation embryos.

    Science.gov (United States)

    Dimitriadou, Eftychia; Melotte, Cindy; Debrock, Sophie; Esteki, Masoud Zamani; Dierickx, Kris; Voet, Thierry; Devriendt, Koen; de Ravel, Thomy; Legius, Eric; Peeraer, Karen; Meuleman, Christel; Vermeesch, Joris Robert

    2017-03-01

    genome-wide methods for PGD in many different centers world-wide as well as the results of ongoing scientific research. Our embryo selection principles have a profound impact on the organization of PGD operations and on the information that is transferred among the genetic unit, the fertility clinic and the patients. These principles are also important for the organization of pre- and post-counseling and influence the interpretation and reporting of preimplantation genotyping results. As novel genome-wide approaches for embryo selection are revolutionizing the field of reproductive genetics, national and international discussions to set general guidelines are warranted. The European Union's Research and Innovation funding programs FP7-PEOPLE-2012-IAPP SARM: 324509 and Horizon 2020 WIDENLIFE: 692065 to J.R.V., T.V., E.D. and M.Z.E. J.R.V., T.V. and M.Z.E. have patents ZL910050-PCT/EP2011/060211-WO/2011/157846 ('Methods for haplotyping single cells') with royalties paid and ZL913096-PCT/EP2014/068315-WO/2015/028576 ('Haplotyping and copy-number typing using polymorphic variant allelic frequencies') with royalties paid, licensed to Cartagenia (Agilent technologies). J.R.V. also has a patent ZL91 2076-PCT/EP20 one 3/070858 ('High throughout genotyping by sequencing') with royalties paid. N/A.

  18. A Legal Jurisprudential Deliberation on Lineage and Inheritance of the Pre-Implantation Embryo

    Directory of Open Access Journals (Sweden)

    Mohammad Taqi Rafiei

    2012-01-01

    Full Text Available Today, in vitro fertilization (IVF is not just a medical issue, but certainly also a complex legal issue, in which lawyers can play an important role by establishing the suitable legal conditions to regulate legal relations by introducing necessary theories. One of the important and controversial issues, which can be approached legally, is the study of the pre-implantation embryo with regard to property law, especially the inheritance of the pre-implantation embryo. Article 3 of the Conditions of Embryo Donation to Infertile Couples Act of the year 2003 only stipulates the responsibilities of the intended couple and born child in terms of support, upbringing, maintenance and respect; it does not specify any law regarding the other financial outcomes of lineage like "inheritance", which makes this law imperfect. Also, in view of the fact that lineage is one of the causes of inheritance; studying inheritance without analyzing lineage in the pre-implantation embryo is not possible. Therefore, it is recommended to study lineage and inheritance simultaneously. Some questions arise in this regard, including whether it is possible to prove lineage between the genetic father and mother with a laboratory child, and between the owner of the womb (that is intended wife and the child. Supposing the lineage is proved, what is the state of inheritance between them? Lawyers and Islamic jurists have different opinions regarding the lineage of the pre-implantation embryo and inheritance. The author believes that the owner of the sperm is regarded as the genetic father of the child and in terms of lineage between laboratory child and mother two genetic and carrying factors must be considered. Thereby, considering inheritance between the genetic father and the child is possible according to inheritance law. Regarding the inheritance state of a laboratory child from two mothers the problem can be solved by using the equality rule within the framework of inheritance

  19. Pro-apoptotic Effect of Pifithrin-α on Preimplantation Porcine Fertilized Embryo Development

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    Brendan Mulligan

    2012-12-01

    Full Text Available The aim of this study was to investigate the impact of a reported p53 inhibitor, pifithrin-α (PFT-α, on preimplantation porcine in vitro fertilized (IVF embryo development in culture. Treatment of PFT-α was administered at both early (0 to 48 hpi, and later stages (48 to 168 hpi of preimplantation development, and its impact upon the expression of five genes related to apoptosis (p53, bak, bcl-xL, p66Shc and caspase3, was assessed in resulting d 7 blastocysts, using real-time quantitative PCR. Total cell numbers, along with the number of apoptotic nuclei, as detected by the in situ cell death detection assay, were also calculated on d 7 in treated and non-treated control embryos. The results indicate that PFT-α, when administered at both early and later stages of porcine IVF embryo development, increases the incidence of apoptosis in resulting blastocysts. When administered at early cleavage stages, PFT-α treatment was shown to reduce the developmental competence of porcine IVF embryos, as well as reducing the quality of resulting blastocysts in terms of overall cell numbers. In contrast, at later stages, PFT-α administration resulted in marginally increased blastocyst development rates amongst treated embryos, but did not affect cell numbers. However, PFT-α treatment induced apoptosis and apoptotic related gene expression, in all treated embryos, irrespective of the timing of treatment. Our results indicate that PFT-α may severely compromise the developmental potential of porcine IVF embryos, and is a potent apoptotic agent when placed into porcine embryo culture media. Thus, caution should be exercised when using PFT-α as a specific inhibitor of p53 mediated apoptosis, in the context of porcine IVF embryo culture systems.

  20. The Responses of Mouse Preimplantation Embryos to Leptin In Vitro in a Transgenerational Model for Obesity

    Directory of Open Access Journals (Sweden)

    Martina Kšiňanová

    2017-09-01

    Full Text Available The aim of the present study was to test the hypothesis that leptin can directly mediate the negative effect of maternal obesity on preimplantation embryos. As previously shown, maternal obesity retards early embryonic development in vivo and increases the incidence of apoptosis in blastocysts. When two-cell embryos isolated from control and obese mice were transferred to identical (leptin free conditions in vitro, no differences in any growth or quality parameters were recorded, including apoptosis incidence in blastocysts. Embryos isolated from control mice responded to transfer to environments with a high concentration of leptin (10 ng/mL with a significant increase in arrest at the first or subsequent cell cycle. However, the majority of non-arrested embryos developed into blastocysts, showing morphology comparable to those cultured in the leptin-free group. On the other hand, the exposure of embryos isolated from obese mice to high leptin concentration in vitro did not retard their development. Furthermore, these embryos developed into blastocysts, showing a lower incidence of apoptosis. In vivo-developed blastocysts recovered from obese mice showed elevated expression levels of the proapoptotic gene BAX and the insulin-responsive glucose transporter gene SLC2A4. In conclusion, elevated leptin levels have both positive and negative effects on preimplantation embryo development in vitro, a response that likely depends on the body condition of the embryo donor. Moreover, these results suggest that leptin acts as a survival factor rather than an apoptotic inductor in embryonic cells. Since no elevations in the expression of the leptin receptor gene (LEPR or fat metabolism-associated genes (PLIN2, SLC27A4 were recorded in blastocysts recovered from obese mice, the role of leptin in mediating the effects of obesity on embryos at the peripheral level is likely lower than expected.

  1. Specific gene-regulation networks during the pre-implantation development of the pig embryo as revealed by deep sequencing.

    Science.gov (United States)

    Cao, Suying; Han, Jianyong; Wu, Jun; Li, Qiuyan; Liu, Shichao; Zhang, Wei; Pei, Yangli; Ruan, Xiaoan; Liu, Zhonghua; Wang, Xumin; Lim, Bing; Li, Ning

    2014-01-03

    Because few studies exist to describe the unique molecular network regulation behind pig pre-implantation embryonic development (PED), genetic engineering in the pig embryo is limited. Also, this lack of research has hindered derivation and application of porcine embryonic stem cells and porcine induced pluripotent stem cells (iPSCs). We identified and analyzed the genome wide transcriptomes of pig in vivo-derived and somatic cell nuclear transferred (SCNT) as well as mouse in vivo-derived pre-implantation embryos at different stages using mRNA deep sequencing. Comparison of the pig embryonic transcriptomes with those of mouse and human pre-implantation embryos revealed unique gene expression patterns during pig PED. Pig zygotic genome activation was confirmed to occur at the 4-cell stage via genome-wide gene expression analysis. This activation was delayed to the 8-cell stage in SCNT embryos. Specific gene expression analysis of the putative inner cell mass (ICM) and the trophectoderm (TE) revealed that pig and mouse pre-implantation embryos share regulatory networks during the first lineage segregation and primitive endoderm differentiation, but not during ectoderm commitment. Also, fatty acid metabolism appears to be a unique characteristic of pig pre-implantation embryonic development. In addition, the global gene expression patterns in the pig SCNT embryos were different from those in in vivo-derived pig embryos. Our results provide a resource for pluripotent stem cell engineering and for understanding pig development.

  2. Expression of plasminogen activators in preimplantation rat embryos developed in vivo and in vitro

    Directory of Open Access Journals (Sweden)

    Har-Vardi Iris

    2005-02-01

    Full Text Available Abstract Background Embryo implantation plays a major role in embryogenesis and the outcome of pregnancy. Plasminogen activators (PAs have been implicated in mammalian fertilization, early stages of development and embryo implantation. The invasion of trophoblast cells into the endometrium during the implantation process can be blocked by inhibitors of serine proteases, illustrating the role of these enzymes in the invasion process. As in vitro developing embryos resulted in lower implantation rate than those developed in vivo we assume that a reduced PAs activity may lead to it. There is hardly any information regarding qualitative or quantitative differences in expression of PAs in preimplantation embryos, or comparisons between in vivo and in vitro developed embryos. The purpose of this study was to assess the expression of urokinase type (uPA and tissue type (tPA plasminogen activators in in vivo and in vitro preimplantation development in rat embryos using immunofluorescence confocal microscopy and computerized image analysis. Methods Zygotes, 2-cell, 4-cell, 8-cell, morula and blastocyst stages of development were flushed from the reproductive tract (control groups of Wistar rats. Zygotes were flushed and grown in vitro to the above mentioned developmental stages and comprised the experimental groups. Immunofluorescence microscopy and computerized image analysis were used to evaluate both qualitative (localization and quantitative expression of plasminogen activators. Results uPA and tPA were found to be expressed in rat embryos throughout their preimplantation development, both in vivo and in vitro. While uPA was localized mainly in the cell cytoplasm, the tPA was detected mainly on cell surface and in the perivitelline space. In blastocysts, both in vivo and in vitro, uPA and tPA were localized in the trophectoderm cells. Total uPA content per embryo was higher in the in vivo as compared with the in vitro developed embryos at all stages

  3. Protein Expression Landscape of Mouse Embryos during Pre-implantation Development

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    Yawei Gao

    2017-12-01

    Full Text Available Pre-implantation embryo development is an intricate and precisely regulated process orchestrated by maternally inherited proteins and newly synthesized proteins following zygotic genome activation. Although genomic and transcriptomic studies have enriched our understanding of the genetic programs underlying this process, the protein expression landscape remains unexplored. Using quantitative mass spectrometry, we identified nearly 5,000 proteins from 8,000 mouse embryos of each stage (zygote, 2-cell, 4-cell, 8-cell, morula, and blastocyst. We found that protein expression in zygotes, morulas, and blastocysts is distinct from 2- to 8-cell embryos. Analysis of protein phosphorylation identified critical kinases and signal transduction pathways. We highlight key factors and their important roles in embryo development. Combined analysis of transcriptomic and proteomic data reveals coordinated control of RNA degradation, transcription, and translation and identifies previously undefined exon-junction-derived peptides. Our study provides an invaluable resource for further mechanistic studies and suggests core factors regulating pre-implantation embryo development.

  4. Is preimplantation genetic diagnosis the ideal embryo selection method in aneuploidy screening?

    Science.gov (United States)

    Sahin, Levent; Bozkurt, Murat; Sahin, Hilal; Gürel, Aykut; Yumru, Ayse Ender

    2014-10-01

    To select cytogenetically normal embryos, preimplantation genetic diagnosis (PGD) aneuploidy screening (AS) is used in numerous centers around the world. Chromosomal abnormalities lead to developmental problems, implantation failure, and early abortion of embryos. The usefulness of PGD in identifying single-gene diseases, human leukocyte antigen typing, X-linked diseases, and specific genetic diseases is well-known. In this review, preimplantation embryo genetics, PGD research studies, and the European Society of Human Reproduction and Embryology PGD Consortium studies and reports are examined. In addition, criteria for embryo selection, technical aspects of PGD-AS, and potential noninvasive embryo selection methods are described. Indications for PGD and possible causes of discordant PGD results between the centers are discussed. The limitations of fluorescence in situ hybridization, and the advantages of the array comparative genomic hybridization are included in this review. Although PGD-AS for patients of advanced maternal age has been shown to improve in vitro fertilization outcomes in some studies, to our knowledge, there is not sufficient evidence to use advanced maternal age as the sole indication for PGD-AS. PGD-AS might be harmful and may not increase the success rates of in vitro fertilization. At the same time PGD, is not recommended for recurrent implantation failure and unexplained recurrent pregnancy loss. Copyright © 2014. Published by Elsevier Taiwan.

  5. Is preimplantation genetic diagnosis the ideal embryo selection method in aneuploidy screening?

    Directory of Open Access Journals (Sweden)

    Levent Sahin

    2014-10-01

    Full Text Available To select cytogenetically normal embryos, preimplantation genetic diagnosis (PGD aneuploidy screening (AS is used in numerous centers around the world. Chromosomal abnormalities lead to developmental problems, implantation failure, and early abortion of embryos. The usefulness of PGD in identifying single-gene diseases, human leukocyte antigen typing, X-linked diseases, and specific genetic diseases is well-known. In this review, preimplantation embryo genetics, PGD research studies, and the European Society of Human Reproduction and Embryology PGD Consortium studies and reports are examined. In addition, criteria for embryo selection, technical aspects of PGD-AS, and potential noninvasive embryo selection methods are described. Indications for PGD and possible causes of discordant PGD results between the centers are discussed. The limitations of fluorescence in situ hybridization, and the advantages of the array comparative genomic hybridization are included in this review. Although PGD-AS for patients of advanced maternal age has been shown to improve in vitro fertilization outcomes in some studies, to our knowledge, there is not sufficient evidence to use advanced maternal age as the sole indication for PGD-AS. PGD-AS might be harmful and may not increase the success rates of in vitro fertilization. At the same time PGD, is not recommended for recurrent implantation failure and unexplained recurrent pregnancy loss.

  6. Assessment of the long-term and transgenerational consequences of perturbing preimplantation embryo development in mice.

    Science.gov (United States)

    Mahsoudi, B; Li, A; O'Neill, C

    2007-11-01

    Perturbations of the development of preimplantation embryos may have long-term consequences for the health of progeny. There are no standardized methods for assessing such risks. The OECD/OCDE 416 Guideline for Testing of Chemicals (Two-Generation Reproduction Toxicity Study) is a standardized assay for detecting potential toxic effects of chemicals. The present study assessed the utility of this guideline for identifying long-term consequences of perturbing preimplantation development. Extended culturing of mammalian zygotes commonly results in retarded preimplantation development. Mouse zygotes were cultured in vitro for 96 h until the blastocyst stage (cultured blastocysts) or blastocysts were collected from the Day-3.5 uterus (in vivo blastocysts). The resulting blastocysts were transferred to the uteri of pseudopregnant recipients (P generation). Progeny from both treatments were mated for a further two generations (F1 and F2 generations). There was no effect of treatment group on gross fertility across the generations tested. Progeny of the cultured blastocysts had lower body weights to the time of weaning compared to in vivo blastocysts in the P and F1 generations, but not in the F2 generation. At maturity, there was no effect of treatment group on body weight, although thyroid weight was higher in the in vivo blastocyst group in the P generation, while the brain, pituitary, and kidneys were larger in the progeny of the cultured blastocysts of the F1 generation. The OECD/OCDE 416 assessment may have a role as a standardized test for the assessment of the biological consequences of perturbing the growth environment of the preimplantation embryo. Embryo culture influenced the somatometric parameters of the resulting progeny, some of which were maintained across a generation.

  7. Mouse preimplantation embryo responses to culture medium osmolarity include increased expression of CCM2 and p38 MAPK activation

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    Watson Andrew J

    2007-01-01

    Full Text Available Abstract Background Mechanisms that confer an ability to respond positively to environmental osmolarity are fundamental to ensuring embryo survival during the preimplantation period. Activation of p38 mitogen-activated protein kinase (MAPK occurs following exposure to hyperosmotic treatment. Recently, a novel scaffolding protein called Osmosensing Scaffold for MEKK3 (OSM was linked to p38 MAPK activation in response to sorbitol-induced hypertonicity. The human ortholog of OSM is cerebral cavernous malformation 2 (CCM2. The present study was conducted to investigate whether CCM2 is expressed during mouse preimplantation development and to determine whether this scaffolding protein is associated with p38 MAPK activation following exposure of preimplantation embryos to hyperosmotic environments. Results Our results indicate that Ccm2 along with upstream p38 MAPK pathway constituents (Map3k3, Map2k3, Map2k6, and Map2k4 are expressed throughout mouse preimplantation development. CCM2, MAP3K3 and the phosphorylated forms of MAP2K3/MAP2K6 and MAP2K4 were also detected throughout preimplantation development. Embryo culture in hyperosmotic media increased p38 MAPK activity in conjunction with elevated CCM2 levels. Conclusion These results define the expression of upstream activators of p38 MAPK during preimplantation development and indicate that embryo responses to hyperosmotic environments include elevation of CCM2 and activation of p38 MAPK.

  8. Identification of chromosomal errors in human preimplantation embryos with oligonucleotide DNA microarray.

    Directory of Open Access Journals (Sweden)

    Lifeng Liang

    Full Text Available A previous study comparing the performance of different platforms for DNA microarray found that the oligonucleotide (oligo microarray platform containing 385K isothermal probes had the best performance when evaluating dosage sensitivity, precision, specificity, sensitivity and copy number variations border definition. Although oligo microarray platform has been used in some research fields and clinics, it has not been used for aneuploidy screening in human embryos. The present study was designed to use this new microarray platform for preimplantation genetic screening in the human. A total of 383 blastocysts from 72 infertility patients with either advanced maternal age or with previous miscarriage were analyzed after biopsy and microarray. Euploid blastocysts were transferred to patients and clinical pregnancy and implantation rates were measured. Chromosomes in some aneuploid blastocysts were further analyzed by fluorescence in-situ hybridization (FISH to evaluate accuracy of the results. We found that most (58.1% of the blastocysts had chromosomal abnormalities that included single or multiple gains and/or losses of chromosome(s, partial chromosome deletions and/or duplications in both euploid and aneuploid embryos. Transfer of normal euploid blastocysts in 34 cycles resulted in 58.8% clinical pregnancy and 54.4% implantation rates. Examination of abnormal blastocysts by FISH showed that all embryos had matching results comparing microarray and FISH analysis. The present study indicates that oligo microarray conducted with a higher resolution and a greater number of probes is able to detect not only aneuploidy, but also minor chromosomal abnormalities, such as partial chromosome deletion and/or duplication in human embryos. Preimplantation genetic screening of the aneuploidy by DNA microarray is an advanced technology used to select embryos for transfer and improved embryo implantation can be obtained after transfer of the screened normal

  9. Preimplantation genetic diagnosis in Welsh pony embryos after biopsy and cryopreservation.

    Science.gov (United States)

    Guignot, F; Reigner, F; Perreau, C; Tartarin, P; Babilliot, J M; Bed'hom, B; Vidament, M; Mermillod, P; Duchamp, G

    2015-11-01

    Preimplantation genetic diagnosis and embryo cryopreservation are important tools to improve genetic management in equine species with marked consequences on the economic value, health, biodiversity, and preservation of the animals. This study aimed to develop a biopsy method at the blastocyst stage that provides viable genotyped cryopreserved Welsh pony embryos. Embryos were collected at d 6.75 to 7 after ovulation. Biopsies were performed with either a microblade or a micropipette. After biopsy, embryos were cryopreserved. The survival rate of biopsied embryos was evaluated on fresh and cryopreserved embryos either 24 h after in vitro culture or after transfer to recipients. Fresh and nonbiopsied embryos were used as controls. Sex, coat color genes, myotony (neuromuscular disorder) diagnosis, and markers of parentage were investigated using PCR on biopsied cells after whole-genome amplification and on remaining embryos. The embryo survival rate after transfer was not affected by the micropipette biopsy (50%, = 8; 43%, = 7; and 50%, = 12, at d 30 for fresh biopsied embryos, vitrified biopsied embryos, and control embryos, respectively) but was significantly reduced by the use of microblade biopsy: 9 ( = 11) vs. 67% ( = 12) for control embryos. Successful sex determination was achieved for 82% ( = 28) of the micropipette biopsies and 100% ( = 50) of the microblade biopsies. Sex determined on biopsied cells was found to correspond completely (100%) with that determined on the remaining embryo ( = 37). More than 90% of the parentage checking markers, coat color, and myotony diagnosis were successfully determined on biopsies obtained with either a micropipette or a microblade. Mendelian incompatibility (7.5 and 5.5%) and embryo genotyping errors (6.6 and 8.6%) were low and not significantly different between the 2 methods. In conclusion, for the first time, pregnancy at Day 30 was obtained after transfer of Welsh pony biopsied and vitrified embryos >300 μm in

  10. A mRNA landscape of bovine embryos after standard and MAPK-inhibited culture conditions : a comparative analysis

    OpenAIRE

    Brinkhof, Bas; Helena T. A. van Tol; Groot Koerkamp, Marian J A; Riemers, Frank M; IJzer, Sascha G; Mashayekhi, Kaveh; Haagsman, Henk P; Roelen, Bernard A. J.; Holstege, FCP

    2015-01-01

    BACKGROUND: Genes and signalling pathways involved in pluripotency have been studied extensively in mouse and human pre-implantation embryos and embryonic stem (ES) cells. The unsuccessful attempts to generate ES cell lines from other species including cattle suggests that other genes and pathways are involved in maintaining pluripotency in these species. To investigate which genes are involved in bovine pluripotency, expression profiles were generated from morula, blastocyst, trophectoderm a...

  11. A mRNA landscape of bovine embryos after standard and MAPK-inhibited culture conditions: a comparative analysis.

    OpenAIRE

    Brinkhof, B.; van Tol, H.T.; Groot Koerkamp, M J; Riemers, F M; IJzer, S.G.; Mashayekhi, K.; Haagsman, H P; Roelen, B.A.

    2015-01-01

    Background Genes and signalling pathways involved in pluripotency have been studied extensively in mouse and human pre-implantation embryos and embryonic stem (ES) cells. The unsuccessful attempts to generate ES cell lines from other species including cattle suggests that other genes and pathways are involved in maintaining pluripotency in these species. To investigate which genes are involved in bovine pluripotency, expression profiles were generated from morula, blastocyst, trophectoderm an...

  12. Expression and localization of heterogeneous nuclear ribonucleoprotein K in mouse ovaries and preimplantation embryos

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Ping [The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai (China); Wang, Ningling [Department of Assisted Reproduction, Shanghai Ninth People' s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai (China); Lin, Xianhua; Jin, Li [The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai (China); Xu, Hong, E-mail: xuhong1168@126.com [The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai (China); Li, Rong [The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai (China); Huang, Hefeng, E-mail: huanghefg@hotmail.com [The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai (China)

    2016-02-26

    Heterogeneous nuclear ribonucleoprotein K (hnRNP K), an evolutionarily conserved protein, is involved in several important cellular processes that are relevant to cell proliferation, differentiation, apoptosis, and cancer development. However, details of hnRNP K expression during mammalian oogenesis and preimplantation embryo development are lacking. The present study investigates the expression and cellular localization of K protein in the mouse ovaries and preimplantation embryos using immunostaining. We demonstrate, for the first time, that hnRNP K is abundantly expressed in the nuclei of mouse oocytes in primordial, primary and secondary follicles. In germ vesicle (GV)-stage oocytes, hnRNP K accumulates in the germinal vesicle in a spot distribution manner. After germinal vesicle breakdown, speckled hnRNP K is diffusely distributed in the cytoplasm. However, after fertilization, the K protein relocates into the female and male pronucleus and persists in the blastomere nuclei. Localization of K protein in the human ovary and ovarian granulosa cell tumor (GCT) was also investigated. Overall, this study provides important morphological evidence to better understand the possible roles of hnRNP K in mammalian oogenesis and early embryo development. - Highlights: • HnRNP K localizes in the nucleus of GV-stage oocyte in a punctate distribution. • HnRNP K strongly accumulates in zygotic pronuclei as condensed spots. • The localization of hnRNP K during oogenesis and embryogenesis is characteristic. • HnRNP K might have an important role in oogenesis and embryonic development.

  13. Kinetics data from bovine sex-specific embryo development from three different bulls

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    C.S. Oliveira

    2016-06-01

    Full Text Available Here we present kinetics data from bovine sex-specific embryo development. Embryos were originated using sex-sorted semen from three different Nelore bulls, and semen from the same batch was used for X-and Y-chromosome spermatozoa sorting. Data was obtained for six time points (24, 48, 96, 120, and 144 h.p.i.. Analyses for each bull׳s embryos (1, 2 and 3 is presented for female and male groups separately. Also, grouped data analysis, considering bull and sex interaction, is shown. For further interpretation and discussion, see "Cell death is involved in sexual dimorphism during preimplantation development" (Oliveira et al., 2015 [1].

  14. Growth factors effects on preimplantation development of mouse embryos exposed to tumor necrosis factor alpha.

    Science.gov (United States)

    Głabowski, Wojciech; Kurzawa, Rafał; Wiszniewska, Barbara; Baczkowski, Tomasz; Marchlewicz, Mariola; Brelik, Paweł

    2005-03-01

    The success rates of assisted reproduction techniques are still unsatisfactory. Relatively few in vitro cultured embryos reach the blastocyst stage. The purpose of the study was to evaluate the protective potential of epidermal growth factor (EGF), insulin-like growth factors 1 and 2 (IGF-I, IGF-II) and stem cell factor (SCF) on in vitro development of pre-implantation mouse embryos exposed to tumor necrosis factor alpha (TNFalpha). C3B6F1 female mice were superovulated with 5 IU of pregnant mare serum gonadotropin (PMSG) and 48 h later with 5IU of equine chorionic gonadotropin (eCG). Following the second injection females were mated with DBA males. Two cell embryos were flushed out from the fallopian tubes 40 h after eCG administration. After retrieval, the embryos were divided into control and experimental media and incubated in groups of ten for 96 h (37 degrees C, 5%CO(2), in droplets of 50 microl under mineral oil). In the first part of experiment, the embryo development was tested in media containing EGF, IGF-I, IGF-II, SCF, TNF-alpha (1 to 1000 ng/ml). In the second part of the study, the development of embryos was examined in medium containing 100 ng/ml TNFalpha and one of following factors: IGF-I, IGF-II; EGF or SCF (100 ng/ml). During the culture embryos were examined at 24 hours intervals to assess the embryo development. Blastocyst rate was determined following 96 hours of culture. Evaluation of total blastocyst cell number (TB) and inner cell mass (ICM) was also performed. TNFalpha significantly reduced (p<0.05) the blastocyst rates as well as TB and ICM. The examined growth factors improved the development of embryos exposed to TNFalpha. Thus, in this study, the protective action of IGF-I and II, EGF and SCF against the detrimental influence of TNFalpha was demonstrated.

  15. Dehydroepiandrosterone (DHEA) reduces embryo aneuploidy: direct evidence from preimplantation genetic screening (PGS).

    Science.gov (United States)

    Gleicher, Norbert; Weghofer, Andrea; Barad, David H

    2010-11-10

    Dehydroepiandrosterone (DHEA) has been reported to improve pregnancy chances in women with diminished ovarian reserve (DOR), and to reduce miscarriage rates by 50-80%. Such an effect is mathematically inconceivable without beneficial effects on embryo ploidy. This study, therefore, assesses effects of DHEA on embryo aneuploidy. In a 1:2, matched case control study 22 consecutive women with DOR, supplemented with DHEA, underwent preimplantation genetic screening (PGS) of embryos during in vitro fertilization (IVF) cycles. Each was matched by patient age and time period of IVF with two control IVF cycles without DHEA supplementation (n = 44). PGS was performed for chromosomes X, Y, 13, 16, 18, 21 and 22, and involved determination of numbers and percentages of aneuploid embryos. DHEA supplementation to a significant degree reduced number (P = 0.029) and percentages (P DHEA effects on DOR patients, at least partially, are the likely consequence of lower embryo aneuploidy. DHEA supplementation also deserves investigation in older fertile women, attempting to conceive, where a similar effect, potentially, could positively affect public health.

  16. Dehydroepiandrosterone (DHEA reduces embryo aneuploidy: direct evidence from preimplantation genetic screening (PGS

    Directory of Open Access Journals (Sweden)

    Weghofer Andrea

    2010-11-01

    Full Text Available Abstract Background Dehydroepiandrosterone (DHEA has been reported to improve pregnancy chances in women with diminished ovarian reserve (DOR, and to reduce miscarriage rates by 50-80%. Such an effect is mathematically inconceivable without beneficial effects on embryo ploidy. This study, therefore, assesses effects of DHEA on embryo aneuploidy. Methods In a 1:2, matched case control study 22 consecutive women with DOR, supplemented with DHEA, underwent preimplantation genetic screening (PGS of embryos during in vitro fertilization (IVF cycles. Each was matched by patient age and time period of IVF with two control IVF cycles without DHEA supplementation (n = 44. PGS was performed for chromosomes X, Y, 13, 16, 18, 21 and 22, and involved determination of numbers and percentages of aneuploid embryos. Results DHEA supplementation to a significant degree reduced number (P = 0.029 and percentages (P Discussion Beneficial DHEA effects on DOR patients, at least partially, are the likely consequence of lower embryo aneuploidy. DHEA supplementation also deserves investigation in older fertile women, attempting to conceive, where a similar effect, potentially, could positively affect public health.

  17. Nucleolar changes in bovine nucleotransferred embryos.

    Science.gov (United States)

    Baran, V; Vignon, X; LeBourhis, D; Renard, J P; Fléchon, J E

    2002-02-01

    This study focused on nucleolar changes in bovine embryos reconstructed from enucleated mature oocytes fused with blastomeres of morulae or with cultured, serum unstarved bovine fetal skin fibroblasts (embryonic vs. somatic cloning). The nucleotransferred (NT) embryos were collected and fixed at time intervals of 1-2 h (early 1-cell stage), 10-15 h (late 1-cell stage), 22-24 h (2-cell stage), 37-38 h (4-cell stage), 40-41 h (early 8-cell stage), 47-48 h (late 8-cell stage), and 55 h (16-cell stage) after fusion. Immunocytochemistry by light and electron microscopy was used for structure-function characterization of nucleolar components. Antibodies against RNA, protein B23, protein C23, and fibrillarin were applied. In addition, DNA was localized by the terminal deoxynucleotidyl transferase (TdT) technique, and the functional organization of chromatin was determined with the nick-translation immunogold approach. The results show that fully reticulated (active) nucleoli observed in donor cells immediately before fusion as well as in the early 1-cell stage after fusion were progressively transformed into nucleolar bodies displaying decreasing numbers of vacuoles from the 2- to 4-cell stage in both types of reconstructed embryos. At the late 8-cell stage, morphological signs of resuming nucleolar activity were detected. Numerous new small vacuoles appeared, and chromatin blocks reassociated with the nucleolar body. During this period, nick-translation technique revealed numerous active DNA sites in the periphery of chromatin blocks associated with the nucleolar body. Fully reticulated nucleoli were again observed as early as the 16-cell stage of embryonic cloned embryos. In comparison, the embryos obtained by fetal cloning displayed a lower tendency to develop, mainly during the first cell cycle and during the period of presumed reactivation. Correlatively, the changes in nucleolar morphology (desegregation and rebuilding) were at least delayed in many somatic NT

  18. EGF increases expression and activity of PAs in preimplantation rat embryos and their implantation rate

    Directory of Open Access Journals (Sweden)

    Har-Vardi Iris

    2007-01-01

    Full Text Available Abstract Background Embryo implantation plays a major role in embryogenesis and the outcome of pregnancy. Plasminogen activators (PAs have been implicated in mammalian fertilization, early stages of development and embryo implantation. As in-vitro developing embryos resulted in lower implantation rate than those developed in-vivo we assume that a reduced PAs activity may be involved. In the present work we studied the effect of EGF on PAs activity, quantity and embryo implantation. Methods Zygotes were flushed from rat oviducts on day one of pregnancy and grown in-vitro in R1ECM supplemented with EGF (10 ng/ml and were grown up to the blastocyst stage. The control groups were grown in the same medium without EGF. The distribution and quantity of the PAs were examined using fluorescence immunohistochemistry followed by measurement of PAs activity using the chromogenic assay. Implantation rate was studied using the embryo donation model. Results PAs distribution in the embryos was the same in EGF treated and untreated embryos. Both PAs were localized in the blastocysts' trophectoderm, supporting the assumption that PAs play a role in the implantation process in rats. EGF increased the quantity of uPA at all stages studied but the 8-cell stage as compared with controls. The tissue type PA (tPA content was unaffected except the 8-cell stage, which was increased. The activity of uPA increased gradually towards the blastocyst stage and more so due to the presence of EGF. The activity of tPA did not vary with the advancing developmental stages although it was also increased by EGF. The presence of EGF during the preimplantation development doubled the rate of implantation of the treated group as compared with controls.

  19. Modulation of the maternal immune system by the pre-implantation embryo

    Directory of Open Access Journals (Sweden)

    Walker Caroline G

    2010-08-01

    Full Text Available Abstract Background A large proportion of pregnancy losses occur during the pre-implantation period, when the developing embryo is elongating rapidly and signalling its presence to the maternal system. The molecular mechanisms that prevent luteolysis and support embryo survival within the maternal environment are not well understood. To gain a more complete picture of these molecular events, genome-wide transcriptional profiles of reproductive day 17 endometrial tissue were determined in pregnant and cyclic Holstein-Friesian dairy cattle. Results Microarray analyses revealed 1,839 and 1,189 differentially expressed transcripts between pregnant and cyclic animals (with ≥ 1.5 fold change in expression; P-value Conclusion The maternal immune system actively surveys the uterine environment during early pregnancy. The embryo modulates this response inducing the expression of endometrial molecules that suppress the immune response and promote maternal tolerance to the embryo. During this period of local immune suppression, genes of the innate immune response (in particular, antimicrobial genes may function to protect the uterus against infection.

  20. Bovine in-vitro embryo production and its contribution towards ...

    African Journals Online (AJOL)

    Mimi

    ... of the Kenyan livestock farmers and boost food security. It discusses the technical aspects of the procedures involved in the in-vitro production of bovine embryos and embryo transfer, with special reference to the application of the techniques in Kenya. MATERIALS AND METHODS. Production of embryos in the laboratory ...

  1. Vitrification of early-stage bovine and equine embryos.

    Science.gov (United States)

    Campos-Chillòn, L F; Suh, T K; Barcelo-Fimbres, M; Seidel, G E; Carnevale, E M

    2009-01-15

    The objectives of this study were to: (1) determine an optimal method and stage of development for vitrification of bovine zygotes or early embryos; and (2) use the optimal procedure for bovine embryos to establish equine pregnancies after vitrification and warming of early embryos. Initially, bovine embryos produced by in-vitro fertilization (IVF) were frozen and vitrified in 0.25mL straws with minimal success. A subsequent experiment was done using two vitrification methods and super open pulled straws (OPS) with 1- or 8-cell bovine embryos. In Method 1 (EG-O), embryos were exposed to 1.5M ethylene glycol (EG) for 5min, 7M ethylene glycol and 0.6M galactose for 30s, loaded in an OPS, and plunged into liquid nitrogen. In Method 2 (EG-DMSO), embryos were exposed to 1.1M ethylene glycol and 1.1M dimethyl sulfoxide (DMSO) for 3min, 2.5M ethylene glycol, 2.5M DMSO and 0.5M galactose for 30s, and loaded and plunged as for EG-O. Cryoprotectants were removed after warming in three steps. One- and eight-cell bovine embryos were cultured for 7 and 4.5 d, respectively, after warming, and control embryos were cultured without vitrification. Cleavage rates of 1-cell embryos were similar (P>0.05) for vitrified and control embryos, although the blastocyst rates for EG-O and control embryos were similar and higher (Pvitrification and warming. In summary, a successful method was established for vitrification of early-stage bovine embryos, and this method was used to establish equine pregnancies after vitrification and warming of 2- to 8-cell embryos produced by ICSI.

  2. Japanese Society for Animal Reproduction: award for outstanding research 2002. Cryopreservation of follicular oocytes and preimplantation embryos in cattle and horses.

    Science.gov (United States)

    Hochi, Shinichi

    2003-02-01

    Factors affecting sensitivity of preimplantation embryos and follicular oocytes to cryopreservation were analyzed in the equine and bovine species. (1) Survival of equine blastocysts after two-step freezing in the presence of glycerol as the cryoprotective agent (CPA) was influenced by development of the embryonic capsule. The use of ethylene glycol (EG) with sucrose as CPAs improved the post-thaw survival of blastocysts and made it possible to transfer the embryos into recipient mares without removing the CPAs. In addition, early blastocysts cryopreserved by vitrification could develop both in vitro and in vivo when the embryos were exposed to vitrification solution in a stepwise manner. The vitrification procedure was also applied to the relatively large expanded blastocysts. (2) Bovine embryos produced in vitro have been considered to be highly sensitive to the process of cryopreservation. To solve this problem, Day-7 blastocysts produced in a serum-free system were cooled at 0.3 C/min rather than 0.6 C/min before being plunged into liquid nitrogen, resulting in no loss of the post-thaw viability. The supplementation of LAA in IVM/IVF media or IVC medium was effective in producing pronuclear-stage zygotes or morula-stage embryos relatively tolerable to freezing, respectively. (3) Transmission electron microscopic observation of immature equine oocytes showed that cellular injury occurred near the sites of gap-junctions between cumulus cells and the oocyte. In cattle, higher fertilization rates of oocytes were obtained when the oocytes were subjected to cryopreservation at an intermediate stage during IVM (GVBD for freezing, Met-I for vitrification). Vitrification of bovine Met-II oocytes in open-pulled glass capillaries, characterized by an ultra-rapid cooling rate (3,000-5,000 C/min), was found to avoid any harmful influence of vitrification and warming.

  3. Culture media for human pre-implantation embryos in assisted reproductive technology cycles.

    Science.gov (United States)

    Youssef, Mohamed M A; Mantikou, Eleni; van Wely, Madelon; Van der Veen, Fulco; Al-Inany, Hesham G; Repping, Sjoerd; Mastenbroek, Sebastiaan

    2015-11-20

    Many media are commercially available for culturing pre-implantation human embryos in assisted reproductive technology (ART) cycles. It is unknown which culture medium leads to the best success rates after ART. To evaluate the safety and effectiveness of different human pre-implantation embryo culture media in used for in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) cycles. We searched the Cochrane Menstrual Disorders and Subfertility Group's Trials Register, Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE, the National Research Register, the Medical Research Council's Clinical Trials Register and the NHS Center for Reviews and Dissemination databases from January 1985 to March 2015. We also examined the reference lists of all known primary studies, review articles, citation lists of relevant publications and abstracts of major scientific meetings. We included all randomised controlled trials which randomised women, oocytes or embryos and compared any two commercially available culture media for human pre-implantation embryos in an IVF or ICSI programme. Two review authors independently selected the studies, assessed their risk of bias and extracted data. We sought additional information from the authors if necessary. We assessed the quality of the evidence using Grades of Recommendation, Assessment, Development and Evaluation (GRADE) methods. The primary review outcome was live birth or ongoing pregnancy. We included 32 studies in this review. Seventeen studies randomised women (total 3666), three randomised cycles (total 1018) and twelve randomised oocytes (over 15,230). It was not possible to pool any of the data because each study compared different culture media.Only seven studies reported live birth or ongoing pregnancy. Four of these studies found no evidence of a difference between the media compared, for either day three or day five embryo transfer. The data from the fifth study did not appear reliable

  4. PRMT5 Protects Genomic Integrity during Global DNA Demethylation in Primordial Germ Cells and Preimplantation Embryos

    Science.gov (United States)

    Kim, Shinseog; Günesdogan, Ufuk; Zylicz, Jan J.; Hackett, Jamie A.; Cougot, Delphine; Bao, Siqin; Lee, Caroline; Dietmann, Sabine; Allen, George E.; Sengupta, Roopsha; Surani, M. Azim

    2014-01-01

    Summary Primordial germ cells (PGCs) and preimplantation embryos undergo epigenetic reprogramming, which includes comprehensive DNA demethylation. We found that PRMT5, an arginine methyltransferase, translocates from the cytoplasm to the nucleus during this process. Here we show that conditional loss of PRMT5 in early PGCs causes complete male and female sterility, preceded by the upregulation of LINE1 and IAP transposons as well as activation of a DNA damage response. Similarly, loss of maternal-zygotic PRMT5 also leads to IAP upregulation. PRMT5 is necessary for the repressive H2A/H4R3me2s chromatin modification on LINE1 and IAP transposons in PGCs, directly implicating this modification in transposon silencing during DNA hypomethylation. PRMT5 translocates back to the cytoplasm subsequently, to participate in the previously described PIWI-interacting RNA (piRNA) pathway that promotes transposon silencing via de novo DNA remethylation. Thus, PRMT5 is directly involved in genome defense during preimplantation development and in PGCs at the time of global DNA demethylation. PMID:25457166

  5. Changes in histone H3 lysine 36 methylation in porcine oocytes and preimplantation embryos.

    Directory of Open Access Journals (Sweden)

    Yun Fei Diao

    Full Text Available Histone H3 lysine 36 (H3K36 methylation is known to be associated with transcriptionally active genes, and is considered a genomic marker of active loci. To investigate the changes in H3K36 methylation in pig, we determined the mono-, di-, and tri-methylations of H3K36 (H3K36me1, H3K36me2 and H3K36me3, respectively in porcine fetal fibroblasts, oocytes and preimplantation embryos by immunocytochemistry using specific antibodies and confocal microscopy. These analyses revealed that only H3K36me3 in porcine fetal fibroblasts consistently colocalized with transcription sites identified as actively synthesizing RNA based on fluorouridine (FU incorporation. Treatment of cells with flavopiridol, which blocks transcription elongation, completely abrogated both H3K36me3 signals and RNA synthesis. All three types of H3K36 methylation were present and did not significantly differ during oocyte maturation. In parthenogenetic embryos, H3K36me1 and -me2 were detected in 1-cell through blastocyst-stage embryos. In contrast, H3K36me3 was not detected in most 1-cell stage embryos. H3K36me3 signals became detectable in 2-cell stage embryos, peaked at the 4-cell stage, decreased at the 8-cell stage, and then became undetectable at blastocyst stages in both parthenogenetic and in vitro-fertilized (IVF embryos. Unlike the case in IVF embryos, H3K36me3 could not be demethylated completely during the 1-cell stage in somatic cell nuclear transfer (SCNT embryos. These results collectively indicate that H3K36me3, but not H3K36me1 or -me2, is associated with transcription elongation in porcine fetal fibroblasts. H3K36me3 is developmentally regulated and may be a histone mark of embryonic gene activation in pig. Aberrant H3K36 tri-methylation occurred during the nuclear reprogramming of SCNT embryos.

  6. The effect of biopsy during precompacted morula stage on post vitrification development of blastocyst derived bovine embryos.

    Science.gov (United States)

    Shirazi, Abolfazl; Borjian, Sara; Ahmadi, Ebrahim; Nazari, Hassan; Heidari, Banafsheh

    2010-04-01

    Improvements on embryo micromanipulation techniques led to the use of embryo biopsy in commercial embryo transfer programs for genetic analysis of preimplantation bovine embryos. The aim of this study was to evaluate the quality of bovine blastocyst derived from embryos biopsied at different pre-compacted morulae stages by assessment of cryosurvivability of the resulting blastocysts. The in vitro produced bovine embryos were subjected to biopsy at days 2, 3, and 4 post-insemination with different cell numbers (4 to 16-cells). Embryo cell biopsy was carried out in a 100 µl drop of H-SOF following pronase drilling by aspiration of one blastomere. The biopsied embryos were then cultured in SOFaaBSA co-cultured with oviduct cells-monolayer until blastocyst formation. The blastocysts were cryopreserved at room temperature after exposure of equilibration (glycerol 1.4 M for 5 min and then glycerol 1.4 M and ethylene glycol 3.6 M for 5 min) and vitrification solutions (3.4 M glycerol and 4.6 M ethylene glycol). The blastocysts were loaded into the center of 0.25 ml straws separated by air bubbles from 2 columns of sucrose 0.5 M and plunged immediately into liquid nitrogen. There was no significant difference in cryosurvivability of vitrified-warmed blastocysts derived form biopsied embryos at different pre-compacted morula stages. The quality of biopsy derived blastocysts was identical to that of non-biopsy derived ones in terms of post vitrifcation survival and hatching rates. In conclusion there was no preference between different times of embryo biopsy at precompacted morula stages in term of cryosurvivability of biopsy derived bovine blastocysts.

  7. Mycotoxin alpha-zearalenol impairs the quality of preimplantation porcine embryos.

    Science.gov (United States)

    Wang, Hongfeng; Camargo Rodriguez, Omar; Memili, Erdogan

    2012-01-01

    Alpha-Zearalenol (α-ZEA) is one of derivatives from Zearalenone (ZEA) which impacts mammalian reproduction and development. Previous studies have shown that pigs are sensitive to the estradiol-like effects of α-ZEA. However, the effect of α-ZEA for the early embryonic development has not been fully studied. The objective of this study was to identify the direct toxicity of α-ZEA on porcine preimplantation embryonic development, embryo quality and expression of developmentally important genes. Presumptive zygotes were cultured in porcine zygote medium 3 (PZM-3) in the presence of α-ZEA (n=2,957) or 17β-estradiol (E2) (n=1,333) dissolved in 0.1% Dimethyl Sulfoxide (DMSO) from 24 to 84 h post insemination followed by determination of apoptotic cell numbers and transcript levels of BAX, BCL2L1 and POU5F1 in blastocysts. Cleavage rates on day 2 were significantly decreased in 10, 30 and 60 µM α-ZEA groups; whereas blastocyst rates on day 6 were significantly decreased in the 30 and 60 µM of α-ZEA groups. Only the 100 µM E2 group significantly decreased cleavage and blastocyst rates. Total cell numbers (TCN) in blastocysts were significantly lower in the 10 µM α-ZEA group, but no differences in apoptotic cell rates were found. The expression levels of POU5F1 and BCL2L1 transcripts were similar; however, levels of BAX transcripts and the BAX/BCL2L1 ratio were increased in both α-ZEA groups. Since α-ZEA and E2 did not elicit similar effects, results suggest that α-ZEA might impact porcine preimplantation embryonic development through pathways other than estrogen receptor binding.

  8. Comparison of transcriptomic landscapes of bovine embryos using RNA-Seq

    Directory of Open Access Journals (Sweden)

    Khatib Hasan

    2010-12-01

    Full Text Available Abstract Background Advances in sequencing technologies have opened a new era of high throughput investigations. Although RNA-seq has been demonstrated in many organisms, no study has provided a comprehensive investigation of the bovine transcriptome using RNA-seq. Results In this study, we provide a deep survey of the bovine embryonic transcriptomes, the first application of RNA-seq in cattle. Embryos cultured in vitro were used as models to study early embryonic development in cattle. RNA amplified from limited amounts of starting total RNA were sequenced and mapped to the reference genome to obtain digital gene expression at single base resolution. In particular, gene expression estimates from more than 1.6 million unannotated bases in 1785 novel transcribed units were obtained. We compared the transcriptomes of embryos showing distinct developmental statuses and found genes that showed differential overall expression as well as alternative splicing. Conclusion Our study demonstrates the power of RNA-seq and provides further understanding of bovine preimplantation embryonic development at a fine scale.

  9. A simple and efficient method to transfect small interference RNA into bovine SCNT embryos.

    Science.gov (United States)

    Zhang, Hui; Wang, LiJun; Li, WenZhe; Mao, QingFu; Wang, YongSheng; Li, Qian; Hua, Song; Zhang, Yong

    2015-10-01

    RNA interference is an important tool to study the gene function. Microinjection and electroporation are usually used to transfer DNA, small interference RNA (siRNA), morpholinos, and protein into oocytes or embryos. This study used a simple and effective method to transfect siRNA into bovine somatic cell nuclear transfer (SCNT) embryos. In this method, siRNA transfection and electrofusion of SCNT were combined. A pair of platinum microelectrodes was used during SCNT to complete electrofusion. A CY3-labeled siRNA-targeted DNA methyltransferase-1 (DNMT1) was chosen to verify the siRNA transfection efficiency of this approach. First, a suitable concentration of siRNA was mixed with Zimmermann's fusion medium. Reconstructed embryos were then added into the microdrops of the mixed fusion medium to simultaneously transfect the siRNA and electrofuse the SCNT embryos. Our results showed that transfecting DNMT1 siRNA via the proposed method caused obvious CY3 fluorescence and significant downregulation of DNMT1 messenger RNA, DNMT1 protein, and global DNA methylation levels in the SCNT embryos. Meanwhile, the survival rate after electrofusion (90.4% vs. 89.4% vs. 89.1%, P > 0.05) and developmental rates of the SCNT embryos (72.8% vs. 74.9% vs. 72.4%, P > 0.05; 29.7% vs. 31.7% vs. 29.7%, P > 0.05) were not significantly affected. In summary, siRNAs were effectively transfected into the SCNT embryos via the proposed method and exert their functions, and the normal development of preimplantation SCNT embryos was not affected by the method used. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Specific gene-regulation networks during the pre-implantation development of the pig embryo as revealed by deep sequencing

    OpenAIRE

    Cao, Suying; Han, Jianyong; Wu, Jun; Li, Qiuyan; Liu, Shichao; Zhang, Wei; Pei, Yangli; Ruan, Xiaoan; Liu, Zhonghua; Wang, Xumin; Lim, Bing; Li, Ning

    2014-01-01

    Background Because few studies exist to describe the unique molecular network regulation behind pig pre-implantation embryonic development (PED), genetic engineering in the pig embryo is limited. Also, this lack of research has hindered derivation and application of porcine embryonic stem cells and porcine induced pluripotent stem cells (iPSCs). Results We identified and analyzed the genome wide transcriptomes of pig in vivo-derived and somatic cell nuclear transferred (SCNT) as well as mouse...

  11. Remodeling of the Nuclear Envelope and Lamina during Bovine Preimplantation Development and Its Functional Implications.

    Directory of Open Access Journals (Sweden)

    Jens Popken

    Full Text Available The present study demonstrates a major remodeling of the nuclear envelope and its underlying lamina during bovine preimplantation development. Up to the onset of major embryonic genome activation (MGA at the 8-cell stage nuclei showed a non-uniform distribution of nuclear pore complexes (NPCs. NPCs were exclusively present at sites where DNA contacted the nuclear lamina. Extended regions of the lamina, which were not contacted by DNA, lacked NPCs. In post-MGA nuclei the whole lamina was contacted rather uniformly by DNA. Accordingly, NPCs became uniformly distributed throughout the entire nuclear envelope. These findings shed new light on the conditions which control the integration of NPCs into the nuclear envelope. The switch from maternal to embryonic production of mRNAs was accompanied by multiple invaginations covered with NPCs, which may serve the increased demands of mRNA export and protein import. Other invaginations, as well as interior nuclear segments and vesicles without contact to the nuclear envelope, were exclusively positive for lamin B. Since the abundance of these invaginations and vesicles increased in concert with a massive nuclear volume reduction, we suggest that they reflect a mechanism for fitting the nuclear envelope and its lamina to a shrinking nuclear size during bovine preimplantation development. In addition, a deposit of extranuclear clusters of NUP153 (a marker for NPCs without associated lamin B was frequently observed from the zygote stage up to MGA. Corresponding RNA-Seq data revealed deposits of spliced, maternally provided NUP153 mRNA and little unspliced, newly synthesized RNA prior to MGA, which increased strongly at the initiation of embryonic expression of NUP153 at MGA.

  12. Acrylamide causes preimplantation abnormalities in embryos and induces chromatin-adducts in male germ cells of mice.

    Science.gov (United States)

    Holland, N; Ahlborn, T; Turteltaub, K; Markee, C; Moore, D; Wyrobek, A J; Smith, M T

    1999-01-01

    Acrylamide, a known male postmeiotic germ cell mutagen, caused a dose-dependent increase in the frequency of morphologic abnormalities in preimplantation embryos. Single-cell eggs, growth retardation, and blastomere lysis were detected after paternal treatment with acrylamide (10 to 50 mg/kg, 5 d). The major effects were seen at weeks 1 to 3 after male treatment, with the highest level of abnormalities at the first week (> 90% vs. 5% in control). The frequency of abnormal four-day embryos was similar to preimplantation loss assessed at 15 to 16 d p.c. A > 100-fold elevation of chromatin adducts in sperm was observed during 1st and 2nd week after treatment, after which adduct levels decreased to baseline level. However, morphologic defects in embryos are not fully explained by the spermatid adduct curve. These findings demonstrate the effects of paternal exposure to acrylamide on preimplantation development and indicate a potential risk to the offspring of men exposed to acrylamide.

  13. Perturbation of the Developmental Potential of Preimplantation Mouse Embryos by Hydroxyurea

    Directory of Open Access Journals (Sweden)

    Edward R. Hills

    2010-04-01

    Full Text Available Women are advised not to attempt pregnancy while on hydroxyurea (HU due to the teratogenic effects of this agent, based on results obtained from animal studies. Several case reports suggest that HU may have minimal or no teratogenic effects on the developing human fetus. Fourteen cases of HU therapy in pregnant patients diagnosed with acute or chronic myelogenous leukemia, primary thrombocythemia, or sickle cell disease (SCD have been reported. Three pregnancies were terminated by elective abortion; 1 woman developed eclampsia and delivered a phenotypically normal stillborn infant. All other patients delivered live, healthy infants without congenital anomalies. We contend that case studies such as these have too few patients and cannot effectively address the adverse effect of HU on preimplantation embryo or fetuses. The objective of this study was to assess the risks associated with a clinically relevant dose of HU used for the treatment of SCD, on ovulation rate and embryo development, using adult C57BL/6J female mice as a model. In Experiment 1, adult female mice were randomly assigned to a treatment or a control group (N = 20/group. Treatment consisted of oral HU (30 mg/kg for 28 days; while control mice received saline (HU vehicle. Five days to the cessation of HU dosing, all mice were subjected to folliculogenesis induction with pregnant mare serum gonadotropin (PMSG. Five mice/group were anesthetized at 48 hours post PMSG to facilitate blood collection via cardiac puncture for estradiol-17β (E2 measurement by RIA. Ovulation was induced in the remaining mice at 48 hours post PMSG with human chorionic gonadotropin (hCG and immediately caged with adult males for mating. Five plugged female mice/group were sacrificed for the determination of ovulation rate. The remaining mated mice were sacrificed about 26 hours post hCG, ovaries excised and weighed and embryos harvested and cultured in Whitten’s medium (WM supplemented with CZBt. In

  14. Ambient ionisation mass spectrometry for lipid profiling and structural analysis of mammalian oocytes, preimplantation embryos and stem cells.

    Science.gov (United States)

    Ferreira, Christina R; Jarmusch, Alan K; Pirro, Valentina; Alfaro, Clint M; González-Serrano, Andres F; Niemann, Heiner; Wheeler, Matthew B; Rabel, Rathnaweera A C; Hallett, Judy E; Houser, Rebecca; Kaufman, Annemarie; Cooks, R Graham

    2015-05-01

    Lipids play fundamental roles in mammalian embryo preimplantation development and cell fate. Triacylglycerol accumulates in oocytes and blastomeres as lipid droplets, phospholipids influence membrane functional properties, and essential fatty acid metabolism is important for maintaining the stemness of cells cultured in vitro. The growing impact that lipids have in the field of developmental biology makes analytical approaches to analyse structural information of great interest. This paper describes the concept and presents the results of lipid profiling by mass spectrometry (MS) of oocytes and preimplantation embryos, with special focus on ambient ionisation. Based on our previous experience with oocytes and embryos, we aim to convey that ambient MS is also valuable for stem cell differentiation analysis. Ambient ionisation MS allows the detection of a wide range of lipid classes (e.g. free fatty acids, cholesterol esters, phospholipids) in single oocytes, embryos and cell pellets, which are informative of in vitro culture impact, developmental and differentiation stages. Background on MS principles, the importance of underused MS scan modes for structural analysis of lipids, and statistical approaches used for data analysis are covered. We envisage that MS alone or in combination with other techniques will have a profound impact on the understanding of lipid metabolism, particularly in early embryo development and cell differentiation research.

  15. The Necessity of OCT-4 and CDX2 for Early Development and Gene Expression Involved in Differentiation of Inner Cell Mass and Trophectoderm Lineages in Bovine Embryos.

    Science.gov (United States)

    Sakurai, Nobuyuki; Takahashi, Kazuki; Emura, Natsuko; Fujii, Takashi; Hirayama, Hiroki; Kageyama, Soichi; Hashizume, Tsutomu; Sawai, Ken

    2016-10-01

    The functions of POU class 5 transcription factor 1 (Oct-4) and caudal-type homeobox 2 (Cdx2) in the differentiation of the murine inner cell mass (ICM) and trophectoderm (TE) have been described in detail. However, little is known about the roles of OCT-4 and CDX2 in preimplantation bovine embryos. To elucidate their functions during early development in bovine embryos, we performed OCT-4 and CDX2 downregulation using RNA interference. We injected OCT-4- or CDX2-specific short interfering RNAs (siRNAs) into bovine zygotes. The rate of blastocyst development of OCT-4-downregulated embryos was lower compared with uninjected or control siRNA-injected embryos. Gene expression analysis revealed decreased CDX2 and fibroblast growth factor 4 expression in OCT-4-downregulated embryos. CDX2-downregulated embryos developed to the blastocyst stage; however, in most cases, blastocoel formation was delayed. Gene expression analysis revealed decreased GATA3 expression and elevated NANOG expression in CDX2-downregulated embryos. In conclusion, OCT-4 and CDX2 are essential for early development and gene expression involved in differentiation of ICM and TE lineages in bovine embryos.

  16. Essential role of maternal UCHL1 and UCHL3 in fertilization and preimplantation embryo development

    Science.gov (United States)

    Mtango, Namdori R.; Sutovsky, Miriam; Susor, Andrej; Zhong, Zhisheng; Latham, Keith E.; Sutovsky, Peter

    2015-01-01

    Posttranslational protein modification by ubiquitination, a signal for lysosomal or proteasomal proteolysis, can be regulated and reversed by deubiquitinating enzymes (DUBs). This study examined the roles of UCHL1 and UCHL3, two members of ubiquitin C-terminal hydrolase (UCH) family of DUBs, in murine fertilization and preimplantation development. Before fertilization, these proteins were associated with the oocyte cortex (UCHL1) and meiotic spindle (UCHL3). Intracytoplasmic injection of the general UCH-family inhibitor ubiquitin-aldehyde (UBAL) or antibodies against UCHL3 into mature metaphase II oocytes blocked fertilization by reducing sperm penetration of the zona pellucida and incorporation into the ooplasm, suggesting a role for cortical UCHL1 in sperm incorporation. Both UBAL and antibodies against UCHL1 injected at the onset of oocyte maturation (germinal vesicle stage) reduced the fertilizing ability of oocytes. The subfertile Uchl1gad−/− mutant mice showed an intriguing pattern of switched UCH localization, with UCHL3 replacing UCHL1 in the oocyte cortex. While fertilization defects were not observed, the embryos from homozygous Uchl1gad−/− mutant females failed to undergo morula compaction and did not form blastocysts in vivo, indicating a maternal effect related to UCHL1 deficiency. We conclude that the activity of oocyte UCHs contributes to fertilization and embryogenesis by regulating the physiology of the oocyte and blastomere cortex. PMID:21678411

  17. Dynamic CpG island methylation landscape in oocytes and preimplantation embryos.

    Science.gov (United States)

    Smallwood, Sébastien A; Tomizawa, Shin-Ichi; Krueger, Felix; Ruf, Nico; Carli, Natasha; Segonds-Pichon, Anne; Sato, Shun; Hata, Kenichiro; Andrews, Simon R; Kelsey, Gavin

    2011-06-26

    Elucidating how and to what extent CpG islands (CGIs) are methylated in germ cells is essential to understand genomic imprinting and epigenetic reprogramming. Here we present, to our knowledge, the first integrated epigenomic analysis of mammalian oocytes, identifying over a thousand CGIs methylated in mature oocytes. We show that these CGIs depend on DNMT3A and DNMT3L but are not distinct at the sequence level, including in CpG periodicity. They are preferentially located within active transcription units and are relatively depleted in H3K4me3, supporting a general transcription-dependent mechanism of methylation. Very few methylated CGIs are fully protected from post-fertilization reprogramming but, notably, the majority show incomplete demethylation in embryonic day (E) 3.5 blastocysts. Our study shows that CGI methylation in gametes is not entirely related to genomic imprinting but is a strong factor in determining methylation status in preimplantation embryos, suggesting a need to reassess mechanisms of post-fertilization demethylation.

  18. Application of next-generation sequencing for 24-chromosome aneuploidy screening of human preimplantation embryos.

    Science.gov (United States)

    Zheng, Haiyan; Jin, Hua; Liu, Lian; Liu, Jianqiao; Wang, Wei-Hua

    2015-01-01

    Aneuploidy is a leading cause of repeat implantation failure and recurrent miscarriages. Preimplantation genetic screening (PGS) enables the assessment of the numeral and structural chromosomal errors of embryos before transfer in patients undergoing in vitro fertilization. Array comparative genomic hybridization (aCGH) has been demonstrated to be an accurate PGS method and in present thought to be the gold standard, but new technologies, such as next-generation sequencing (NGS), continue to emerge. Validation of the new comprehensive NGS-based 24-chromosome aneuploidy screening technology is still needed to determine the preclinical accuracy before it might be considered as an alternative method for human PGS. In the present study, 43 human trophectoderm (TE) biopsy samples and 5 cytogenetically characterized cell lines (Coriell Cell Repositories) were tested. The same whole genome amplified product of each sample was blindly assessed with Veriseq NGS and Agilent aCGH to identify the aneuploidy status. The result showed that the NGS identified all abnormalities identified in aCGH including the numeral chromosomal abnormalities (again or loss) in the embryo samples and the structural (partial deletion and duplication) in the Coriell cell lines. Both technologies can identify a segmental imbalance as small as 1.8 Mb in size. Among the 41 TE samples with abnormal karyotypes in this study, eight (19.5 %) samples presented as multiple chromosome abnormalities. The abnormalities occurred to almost all chromosomes, except chromosome 6, 7, 17 and Y chromosome. Given its reliability and high level of consistency with an established aCGH methodology, NGS has demonstrated a robust high-throughput methodology ready for extensive clinical application in reproductive medicine, with potential advantages of reduced costs and enhanced precision. Then, a randomized controlled clinical trial confirming its clinical effectiveness is advisable to obtain a larger sequencing dataset

  19. ESHRE PGD Consortium/Embryology Special Interest Group--best practice guidelines for polar body and embryo biopsy for preimplantation genetic diagnosis/screening (PGD/PGS)

    DEFF Research Database (Denmark)

    Harton, G L; Magli, M C; Lundin, K

    2011-01-01

    In 2005, the European Society for Human Reproduction and Embryology (ESHRE) Preimplantation Genetic Diagnosis (PGD) Consortium published a set of Guidelines for Best Practice to give information, support and guidance to potential, existing and fledgling PGD programmes (Thornhill AR, De Die...... and embryo biopsy for preimplantation genetic diagnosis/screening (PGD/PGS). Here we have updated the sections that pertain to embryology (including cryopreservation) and biopsy of embryos prior to PGD or PGS. Topics covered in this guideline include uses of embryo biopsy, laboratory issues relating...

  20. Blocking connexin channels improves embryo development of vitrified bovine blastocysts

    OpenAIRE

    Ortiz Escribano, Nerea; Szymanska, Katarzyna; BOL, MELISSA; Vandenberghe, Lynn; Decrock, Elke; Van Poucke, Mario; Peelman, Luc; Van den Abbeel, Etienne; Soom, Ann Van; Leybaert, Luc

    2017-01-01

    Connexins (Cxs) are required for normal embryo development and implantation. They form gap junctions (GJs) connecting the cytoplasm of adjacent cells and hemichannels (HCs), which are normally closed but open in response to stress conditions. Excessive HC opening is detrimental for cell function and may lead to cell death. We found that hatching of in vitro-produced bovine embryos, matured in serum-containing conditions, was significantly improved when vitrification/warming was done in the pr...

  1. Proof of concept: preimplantation genetic screening without embryo biopsy through analysis of cell-free DNA in spent embryo culture media.

    Science.gov (United States)

    Shamonki, Mousa I; Jin, Helen; Haimowitz, Zachary; Liu, Lian

    2016-11-01

    To assess whether preimplantation genetic screening (PGS) is possible by testing for free embryonic DNA in spent IVF media from embryos undergoing trophectoderm biopsy. Prospective cohort analysis. Academic fertility center. Seven patients undergoing IVF and 57 embryos undergoing trophectoderm biopsy for PGS. On day 3 of development, each embryo was placed in a separate media droplet. All biopsied embryos received a PGS result by array comparative genomic hybridization. Preimplantation genetic screening was performed on amplified DNA extracted from media and results were compared with PGS results for the corresponding biopsy. [1] Presence of DNA in spent IVF culture media. [2] Correlation between genetic screening result from spent media and corresponding biopsy. Fifty-five samples had detectable DNA ranging from 2-642 ng/μL after a 2-hour amplification. Six samples with the highest DNA levels underwent PGS, rendering one result with a derivative log ratio SD (DLRSD) of media and a result that is consistent with trophectoderm biopsy. Improvements in DNA collection, amplification, and testing may allow for PGS without biopsy in the future. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  2. Nucleolar ultrastructure in bovine nuclear transfer embryos

    DEFF Research Database (Denmark)

    Kaňka, Jiří; Smith, Steven Dale; Soloy, Eva

    1999-01-01

    (nonactivated) or S phase (activated) cytoplasts. Control embryos were fixed at the two-, four-, early eight- and late eight-cell stages; nuclear transfer embryos were fixed at 1 and 3 hr post fusion and at the two-, four-, and eight-cell stages. Control embryos possessed a nucleolar precursor body throughout...... at 1 hr after fusion and, by 3 hr after fusion, it was restored again. At this time, the reticulated fibrillo-granular nucleolus had an almost round shape. The nucleolar precursor body seen in the two-cell stage nuclear transfer embryos consisted of intermingled filamentous components and secondary...... time intervals after fusion. In the two-cell stage nuclear transfer embryo, the originally reticulated nucleolus of the donor blastomere had changed into a typical nucleolar precursor body consisting of a homogeneous fibrillar structure. A primary vacuole appeared in the four-cell stage nuclear...

  3. mRNA fragments in in vitro culture media are associated with bovine preimplantation embryonic development.

    Science.gov (United States)

    Kropp, Jenna; Khatib, Hasan

    2015-01-01

    In vitro production (IVP) systems have been used to bypass problems of fertilization and early embryonic development. However, embryos produced by IVP are commonly selected for implantation based on morphological assessment, which is not a strong indicator of establishment and maintenance of pregnancy. Thus, there is a need to identify additional indicators of embryonic developmental potential. Previous studies have identified microRNA expression in in vitro culture media to be indicative of embryo quality in both bovine and human embryos. Like microRNAs, mRNAs have been shown to be secreted from cells into the extracellular environment, but it is unknown whether or not these RNAs are secreted by embryos. Thus, the objective of the present study was to determine whether mRNAs are secreted into in vitro culture media and if their expression in the media is indicative of embryo quality. In vitro culture medium was generated and collected from both blastocyst and degenerate (those which fail to develop from the morula to blastocyst stage) embryos. Small-RNA sequencing revealed that many mRNA fragments were present in the culture media. A total of 17 mRNA fragments were differentially expressed between blastocyst and degenerate conditioned media. Differential expression was confirmed by quantitative real-time PCR for fragments of mRNA POSTN and VSNL-1, in four additional biological replicates of media. To better understand the mechanisms of mRNA secretion into the media, the expression of a predicted RNA binding protein of POSTN, PUM2, was knocked down using an antisense oligonucleotide gapmer. Supplementation of a PUM2 gapmer significantly reduced blastocyst development and decreased secretion of POSTN mRNA into the media. Overall, differential mRNA expression in the media was repeatable and sets the framework for future study of mRNA biomarkers in in vitro culture media to improve predictability of reproductive performance.

  4. Embryo genome profiling by single-cell sequencing for preimplantation genetic diagnosis in a β-thalassemia family

    DEFF Research Database (Denmark)

    Xu, Yanwen; Chen, Shengpei; Yin, Xuyang

    2015-01-01

    for a β-thalassemia-carrier couple to have a healthy second baby. We carried out sequencing for single blastomere cells and the family trio and further developed the analysis pipeline, including recovery of the missing alleles, removal of the majority of errors, and phasing of the embryonic genome...... leukocyte antigen matching tests. CONCLUSIONS: This retrospective study in a β-thalassemia family demonstrates a method for embryo genome recovery through single-cell sequencing, which permits detection of genetic variations in preimplantation genetic diagnosis. It shows the potential of single...

  5. Genome stability of bovine in vivo-conceived cleavage-stage embryos is higher compared to in vitro-produced embryos.

    Science.gov (United States)

    Tšuiko, Olga; Catteeuw, Maaike; Zamani Esteki, Masoud; Destouni, Aspasia; Bogado Pascottini, Osvaldo; Besenfelder, Urban; Havlicek, Vitezslav; Smits, Katrien; Kurg, Ants; Salumets, Andres; D'Hooghe, Thomas; Voet, Thierry; Van Soom, Ann; Robert Vermeesch, Joris

    2017-11-01

    Is the rate and nature of chromosome instability (CIN) similar between bovine in vivo-derived and in vitro-cultured cleavage-stage embryos? There is a major difference regarding chromosome stability of in vivo-derived and in vitro-cultured embryos, as CIN is significantly lower in in vivo-derived cleavage-stage embryos compared to in vitro-cultured embryos. CIN is common during in vitro embryogenesis and is associated with early embryonic loss in humans, but the stability of in vivo-conceived cleavage-stage embryos remains largely unknown. Because human in vivo preimplantation embryos are not accessible, bovine (Bos taurus) embryos were used to study CIN in vivo. Five young, healthy, cycling Holstein Friesian heifers were used to analyze single blastomeres of in vivo embryos, in vitro embryos produced by ovum pick up with ovarian stimulation (OPU-IVF), and in vitro embryos produced from in vitro matured oocytes retrieved without ovarian stimulation (IVM-IVF). Single blastomeres were isolated from embryos, whole-genome amplified and hybridized on Illumina BovineHD BeadChip arrays together with the bulk DNA from the donor cows (mothers) and the bull (father). DNA was also obtained from the parents of the bull and from the parents of the cows (paternal and maternal grandparents, respectively). Subsequently, genome-wide haplotyping and copy-number profiling was applied to investigate the genomic architecture of 171 single bovine blastomeres of 16 in vivo, 13 OPU-IVF and 13 IVM-IVF embryos. The genomic stability of single blastomeres in both of the in vitro-cultured embryo cohorts was severely compromised (P vitro than in embryos derived in vivo. Only 18.8% of in vivo-derived embryos contained at least one blastomere with chromosomal anomalies, compared to 69.2% of OPU-IVF embryos (P vitro procedures exacerbate chromosomal abnormalities during early embryo development. Hence, the present study highlights that IVF treatment compromises embryo viability and should be

  6. Injection of ligand-free gold and silver nanoparticles into murine embryos does not impact pre-implantation development

    Directory of Open Access Journals (Sweden)

    Ulrike Taylor

    2014-05-01

    Full Text Available Intended exposure to gold and silver nanoparticles has increased exponentially over the last decade and will continue to rise due to their use in biomedical applications. In particular, reprotoxicological aspects of these particles still need to be addressed so that the potential impacts of this development on human health can be reliably estimated. Therefore, in this study the toxicity of gold and silver nanoparticles on mammalian preimplantation development was assessed by injecting nanoparticles into one blastomere of murine 2 cell-embryos, while the sister blastomere served as an internal control. After treatment, embryos were cultured and embryo development up to the blastocyst stage was assessed. Development rates did not differ between microinjected and control groups (gold nanoparticles: 67.3%, silver nanoparticles: 61.5%, sham: 66.2%, handling control: 79.4%. Real-time PCR analysis of six developmentally important genes (BAX, BCL2L2, TP53, OCT4, NANOG, DNMT3A did not reveal an influence on gene expression in blastocysts. Contrary to silver nanoparticles, exposure to comparable Ag+-ion concentrations resulted in an immediate arrest of embryo development. In conclusion, the results do not indicate any detrimental effect of colloidal gold or silver nanoparticles on the development of murine embryos.

  7. Confirmation rates of array-CGH in day-3 embryo and blastocyst biopsies for preimplantation genetic screening.

    Science.gov (United States)

    Mir, P; Mateu, E; Mercader, A; Herrer, R; Rodrigo, L; Vera, M; Peinado, V; Milán-Sánchez, M; Campos-Galindo, I; García-Herrero, S; Simón, C; Rubio, C

    2016-01-01

    The purpose of this study was to compare the confirmation rate of day-3 embryo biopsy (blastomere) and trophectoderm biopsy using array-comparative genomic hybridization (array-CGH) technology. A blinded study was conducted to re-analyse 109 embryos previously diagnosed as chromosomally abnormal by array-CGH. Preimplantation genetic screening (PGS) was performed using array-CGH on day 3 (n = 50) or day 5 (n = 59). Partial chromosome gains or losses were excluded (n=6), and only whole chromosome aneuploidies were considered. Re-analysis of whole blastocysts was carried out following the same array-CGH protocol used for PGS. The PGS result was confirmed in the whole blastocyst in (a) 49/50 (98 %) abnormal embryos after day-3 biopsy and (b) 57/59 (96.6 %) abnormal embryos after trophectoderm biopsy. One embryo (1/50; 2 %) was diagnosed as abnormal, with monosomy 18, on day 3, and software analysis of the whole blastocyst gave a euploid result; however, a mosaic pattern was observed for monosomy 18 in the whole blastocyst. Two trophectoderm biopsy cases (3.4 %) did not have the abnormalities (trisomy 7, and trisomy 1 and 4, respectively) verified in the whole embryo. Concordance rates for both biopsy strategies and for individual chromosomes were evaluated by Fisher's exact test and showed no significant differences. Both types of biopsies showed similar high concordance rates with whole blastocyst results. Therefore, regarding the confirmation rates shown in this work, day-3 embryo biopsies can be representative of the whole embryo and both types of biopsy can be used for clinical analysis in PGS following the described array-CGH protocol.

  8. Preimplantation diagnosis of a human beta-globin transgene in biopsied trophectoderm cells and blastomeres of the mouse embryo.

    Science.gov (United States)

    Sheardown, S A; Findlay, I; Turner, A; Greaves, D; Bolton, V N; Mitchell, M; Layton, D M; Muggleton-Harris, A L

    1992-10-01

    The preimplantation diagnosis of a HbSA-globin transgene in biopsied trophectoderm cells and blastomeres in embryos using a transgenic mouse model for the trait of human sickle-cell anaemia has been undertaken. A sensitive procedure was developed for the amplification of the human beta-globin gene sequence flanking the sickle mutation. Polymerase chain reaction (PCR) assays were undertaken on one to five biopsied trophectoderm cells and isolated blastomeres of the preimplantation mouse embryo. After biopsy the blastocysts were cultured whilst the cells were analysed for the presence of the transgene, and a high proportion (82-91%) were viable as assessed by the presence of a blastocoele cavity within a 5-h period. The majority of the biopsied cultured blastocysts were frozen and used to confirm the diagnosis; 90 biopsied cultured blastocysts were transferred to pseudopregnant recipients and 34% established pregnancy. Material from day 13.5 post-coitum fetuses was also used to confirm the original diagnosis. The time (4-5 h) required to carry out the analysis obviates a need for extended culture or cryopreservation of the biopsied embryo. In individual experiments under optimal conditions, the presence of the transgene in biopsied cells was detected with 100% accuracy, and the PCR analysis was sensitive at the 1-cell level. The overall success rate of diagnosis and confirmation of the presence or absence of the human beta-globin sequence in the biopsied embryo was 70%. Over the entire experimental period (14 months) DNA contamination from a variety of sources did occasionally occur; the methods used to overcome this problem are discussed.

  9. In vitro production of bovine embryos

    DEFF Research Database (Denmark)

    Stroebech, L.; Mazzoni, Gianluca; Pedersen, Hanne Skovsgaard

    2015-01-01

    be improved, and aspects related to the oocyte donor, oocyte maturation and the recipients are addressed in the following. Also, some of the future aspects of genomic selection and systems biology are addressed with particular focus on the Brazilian-Danish collaboration in the so-called GIFT-project.......In vitro production (IVP) of bovine embryos has become a widespread technology implemented in cattle breeding and production. The implementation of genomic selection and systems biology adds great dimensions to the impact of bovine IVP. The physical procedures included in the IVP process can still...

  10. Increasing of blastocyst rate and gene expression in co-culture of bovine embryos with adult adipose tissue-derived mesenchymal stem cells.

    Science.gov (United States)

    Miranda, Moysés S; Nascimento, Hamilton S; Costa, Mayra P R; Costa, Nathália N; Brito, Karynne N L; Lopes, Cinthia T A; Santos, Simone S D; Cordeiro, Marcela S; Ohashi, Otávio M

    2016-10-01

    Despite advances in the composition of defined embryo culture media, co-culture with somatic cells is still used for bovine in vitro embryo production (IVEP) in many laboratories worldwide. Granulosa cells are most often used for this purpose, although recent work suggests that co-culture with stem cells of adult or embryonic origin or their derived biomaterials may improve mouse, cattle, and pig embryo development. In experiment 1, in vitro produced bovine embryos were co-cultured in the presence of two concentrations of bovine adipose tissue-derived mesenchymal cells (b-ATMSCs; 103 and 104 cells/mL), in b-ATMSC preconditioned medium (SOF-Cond), or SOF alone (control). In experiment 2, co-culture with 104 b-ATMSCs/mL was compared to the traditional granulosa cell co-culture system (Gran). In experiment 1, co-culture with 104 b-ATMSCs/mL improved blastocyst rates in comparison to conditioned and control media (p culture with 104 b-ATMSCs/mL improved not only blastocyst rates but also quality as assessed by increased total cell numbers and mRNA expression levels for POU5F1 and G6PDH (p culture of bovine embryos with b-ATMSCs was more beneficial than the traditional co-culture system with granulosa cells. We speculate that the microenvironmental modulatory potential of MSCs, by means of soluble substances and exosome secretions, could be responsible for the positive effects observed. Further experiments must be done to evaluate if this beneficial effect in vitro also translates to an increase in offspring following embryo transfer. Moreover, this study provides an interesting platform to study the basic requirements during preimplantation embryo development, which, in turn, may aid the improvement of embryo culture protocols in bovine and other species.

  11. The Role of Peroxisome Proliferator-Activated Receptors in the Development and Physiology of Gametes and Preimplantation Embryos

    Directory of Open Access Journals (Sweden)

    Jaou-Chen Huang

    2008-01-01

    Full Text Available In several species, a family of nuclear receptors, the peroxisome proliferator-activated receptors (PPARs composed of three isotypes, is expressed in somatic cells and germ cells of the ovary as well as the testis. Invalidation of these receptors in mice or stimulation of these receptors in vivo or in vitro showed that each receptor has physiological roles in the gamete maturation or the embryo development. In addition, synthetic PPARγ ligands are recently used to induce ovulation in women with polycystic ovary disease. These results reveal the positive actions of PPAR in reproduction. On the other hand, xenobiotics molecules (in herbicides, plasticizers, or components of personal care products, capable of activating PPAR, may disrupt normal PPAR functions in the ovary or the testis and have consequences on the quality of the gametes and the embryos. Despite the recent data obtained on the biological actions of PPARs in reproduction, relatively little is known about PPARs in gametes and embryos. This review summarizes the current knowledge on the expression and the function of PPARs as well as their partners, retinoid X receptors (RXRs, in germ cells and preimplantation embryos. The effects of natural and synthetic PPAR ligands will also be discussed from the perspectives of reproductive toxicology and assisted reproductive technology.

  12. Single nucleotide polymorphism microarray-based concurrent screening of 24-chromosome aneuploidy and unbalanced translocations in preimplantation human embryos.

    Science.gov (United States)

    Treff, Nathan R; Northrop, Lesley E; Kasabwala, Khushabu; Su, Jing; Levy, Brynn; Scott, Richard T

    2011-04-01

    To develop, validate, and apply a single nucleotide polymorphism (SNP) microarray-based method for simultaneous preimplantation genetic diagnosis (PGD) of unbalanced inheritance of rearranged chromosomes and 24-chromosome aneuploidy screening. Prospective clinical research study. Academic reproductive medicine center. Eighteen couples carrying a balanced reciprocal or Robertsonian chromosomal rearrangement. PGD on blastocyst trophectoderm biopsy specimens. Aneuploidy, implantation, pregnancy, and delivery rates after SNP microarray-based aneuploidy and translocation screening. Single nucleotide polymorphism microarray was capable of detecting translocation-associated imbalances as small as 9.0 megabases. In the 12 transfers performed, sustained implantation occurred for 9 (45%) of 20 balanced-normal and euploid embryos replaced. The clinical pregnancy rate in patients receiving a transfer was 75% with six singleton deliveries and three ongoing singleton pregnancies thus far. Significantly fewer embryos were eligible for transfer with the incorporation of simultaneous 24-chromosome aneuploidy screening. Arrested embryos were also significantly more likely to possess unbalanced chromosomes when compared with developmentally competent blastocysts. This SNP microarray-based method provides the first opportunity to improve outcomes through comprehensive identification of euploid embryos from translocation carrier couples. Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  13. Discovery of pluripotency-associated microRNAs in rabbit preimplantation embryos and embryonic stem-like cells.

    Science.gov (United States)

    Maraghechi, Pouneh; Hiripi, László; Tóth, Gábor; Bontovics, Babett; Bősze, Zsuzsanna; Gócza, Elen

    2013-04-01

    MicroRNAs (miRNAs) are small non-coding RNAs that regulate multiple biological processes. Increasing experimental evidence implies an important regulatory role of miRNAs during embryonic development and in embryonic stem (ES) cell biology. In the current study, we have described and analyzed the expression profile of pluripotency-associated miRNAs in rabbit embryos and ES-like cells. The rabbit specific ocu-miR-302 and ocu-miR-290 clusters, and three homologs of the human C19MC cluster (ocu-miR-512, ocu-miR-520e, and ocu-miR-498) were identified in rabbit preimplantation embryos and ES-like cells. The ocu-miR-302 cluster was highly similar to its human homolog, while ocu-miR-290 revealed a low level of evolutionary conservation with its mouse homologous cluster. The expression of the ocu-miR-302 cluster began at the 3.5 days post-coitum early blastocyst stage and they stayed highly expressed in rabbit ES-like cells. In contrast, a high expression level of the ocu-miR-290 cluster was detected during preimplantation embryonic development, but a low level of expression was found in rabbit ES-like cells. Differential expression of the ocu-miR-302 cluster and ocu-miR-512 miRNA was detected in rabbit trophoblast and embryoblast. We also found that Lefty has two potential target sites in its 3'UTR for ocu-miR-302a and its expression level increased upon ocu-miR-302a inhibition. We suggest that the expression of the ocu-miR-302 cluster is characteristic of the rabbit ES-like cell, while the ocu-miR-290 cluster may play a crucial role during early embryonic development. This study presents the first identification, to our knowledge, of pluripotency-associated miRNAs in rabbit preimplantation embryos and ES-like cells, which can open up new avenues to investigate the regulatory function of ocu-miRNAs in embryonic development and stem cell biology.

  14. In vitro culture of individual mouse preimplantation embryos: the role of embryo density, microwells, oxygen, timing and conditioned media.

    Science.gov (United States)

    Kelley, Rebecca L; Gardner, David K

    2017-05-01

    Single embryo culture is suboptimal compared with group culture, but necessary for embryo monitoring, and culture systems should be improved for single embryos. Pronucleate mouse embryos were used to assess the effect of culture conditions on single embryo development. Single culture either before or after compaction reduced cell numbers (112.2 ± 3.1; 110.2 ± 3.5) compared with group culture throughout (127.0 ± 3.4; P media volume from 20 µl to 2 µl increased blastocyst cell numbers in single embryos cultured in 5% oxygen (84.4 ± 3.2 versus 97.8 ± 2.8; P Culture in microwell plates for the EmbryoScope and Primo Vision time-lapse systems changed cleavage timings and increased inner cell mass cell number (24.1 ± 1.0; 23.4 ± 1.2) compared with a 2 µl microdrop (18.4 ± 1.0; P media to single embryos increased hatching rate and blastocyst cell number (91.5 ± 4.7 versus 113.1 ± 4.4; P culture before or after compaction is therefore detrimental; oxygen, media volume and microwells influence single embryo development; and embryo-conditioned media may substitute for group culture. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  15. Effects of embryo-derived exosomes on the development of bovine cloned embryos.

    Science.gov (United States)

    Qu, Pengxiang; Qing, Suzhu; Liu, Ruiqi; Qin, Hongyu; Wang, Weiwei; Qiao, Fang; Ge, Hui; Liu, Jun; Zhang, Yong; Cui, Wei; Wang, Yongsheng

    2017-01-01

    The developmental competence of in vitro cultured (IVC) embryos is markedly lower than that of their in vivo counterparts, suggesting the need for optimization of IVC protocols. Embryo culture medium is routinely replaced three days after initial culture in bovine, however, whether this protocol is superior to continuous nonrenewal culture method under current conditions remains unclear. Using bovine somatic cell nuclear transfer (SCNT) embryos as the model, our results showed that compared with routine renewal treatment, nonrenewal culture system significantly improved blastocyst formation, blastocyst quality (increased total cell number, decreased stress and apoptosis, enhanced Oct-4 expression and ratio of ICM/TE), as well as following development to term. Existence and function of SCNT embryo-derived exosomes were then investigated to reveal the cause of impaired development induced by culture medium replacement. Exosomes were successfully isolated through differential centrifugation and identified by both electron microscopy and immunostaining against exosomal membrane marker CD9. Supplementation of extracted exosomes into freshly renewed medium significantly rescued not only blastocyst formation and quality (in vitro development), but also following growth to term (in vivo development). Notably, ratio of ICM/TE and calving rate were enhanced to a similar level as that in nonrenewal group. In conclusion, our results for the first time indicate that 1: bovine SCNT embryos can secrete exosomes into chemically defined culture medium during IVC; 2: secreted exosomes are essential for SCNT blastocyst formation, blastocyst quality, and following development to term; 3: removal of exosomes induced by culture medium replacement impairs SCNT embryo development, which can be avoided by nonrenewal culture procedure or markedly recovered by exosome supplementation.

  16. Lower total cell numbers in mouse preimplantation embryos cultured in human assisted reproductive technique (ART) media are not induced by apoptosis.

    Science.gov (United States)

    Schulte, Katharina; Ehmcke, Jens; Schlatt, Stefan; Boiani, Michele; Nordhoff, Verena

    2015-12-01

    A common feature of assisted reproductive techniques such as IVF or intracytoplasmic sperm injection is the IVC of oocytes or preimplantation embryos in artificial culture media. The IVC conditions are selected to mimic the environment of the female genital tract. We have shown that murine preimplantation embryos respond to different culture media with changes in developmental rates, cellular lineage composition, and gene expression patterns. In this study, we explored whether apoptosis is responsible for cell loss in mouse preimplantation embryos after exposure to different human culture media. We examined total embryonic cell count as well as the pattern of protein expression for caspase-9 (intrinsic pathway), caspase-8 (extrinsic pathway), and the executioner caspase-3 via immunohistochemical staining. Total cell counts decline in embryos cultured either in innovative sequential medium 1 and Blast Assist (Origio) or human tubal fluid and MultiBlast (Irvine Scientific) when compared to KSOM(aa). Few cells were caspase-9 and -3 positive in all experimental groups. Staining for caspase-8 was almost undetectable. We conclude that embryonic cell loss is not associated with higher rates of intrinsic apoptotic cell loss. Our results suggest that the culture medium-dependent decline in total cell count and the developmental restriction in embryos cultured in innovative sequential medium 1/Blast Assist and human tubal fluid/MultiBlast are related to processes affecting cell proliferation. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. The Lc3-synthase gene B3gnt5 is essential to pre-implantation development of the murine embryo

    Directory of Open Access Journals (Sweden)

    Cinelli Paolo

    2008-11-01

    Full Text Available Abstract Background Glycosphingolipids (GSL are integral components of mammalian cell membranes that are involved in cell adhesion and cell signaling processes. GSL are subdivided into structural series, like ganglio-, lacto/neolacto-, globo- and isoglo-series, which are defined by distinct trisaccharide cores. The β1,3 N-acetylglucosaminyltransferase-V (B3gnt5 enzyme catalyzes the formation of the Lc3 structure, which is the core of lactoseries derived GSL. Results The biological significance of the glycoconjugates produced by the B3gnt5 enzyme was investigated by inactivating the B3gnt5 gene in the mouse germline. The disruption of the B3gnt5 protein-coding region in mouse embryonic stem cells resulted in reduced Lc3-synthase activity, supporting its specific contribution to lactoseries derived GSL synthesis. Breeding of heterozygous mutant mice failed to produce any viable progeny homozygous for the B3gnt5-null allele. The genotypic examination of embryos from heterozygous crosses showed that the disruption of the B3gnt5 gene leads to pre-implantation lethality. This finding was compatible with the expression pattern of the B3gnt5 gene in the pre-implantation embryo as shown by in situ hybridization. The analysis of GSL profiles in embryonic stem cells heterozygous for the B3gnt5-null allele confirmed the reduced levels of lactoseries derived GSL levels and of other GSL species. Conclusion The disruption of the B3gnt5 gene in mice affected the expression of lactoseries derived GLS and possibly of protein-bound β3GlcNAc-linked glycans, thereby demonstrating an essential contribution of these glycoconjugates in early embryonic development, and supporting the importance of these glycoconjugates in cell differentiation and adhesion processes.

  18. Pregnancy after preimplantation diagnosis for a deletion in the dystrophin gene by polymerase chain reaction in embryos obtained after intracytoplasmic sperm injection

    Energy Technology Data Exchange (ETDEWEB)

    Lissens, W.; Liu, J.; Van Broeckhoven, C. [University Hospital, Brussels (Belgium)] [and others

    1994-09-01

    Duchenne muscular dystrophy (DMD) is one of the most common X-linked recessive diseases. In order to be able to perform a DMD-specific preimplantation diagnosis (PID) in a female carrier of a deletion of exons 3 to 18 in the dystrophin gene, we have developed a PCR assay to detect the deletion based on sequences of exon 17. The efficiency of this PCR was evaluated on 50 single blastomeres from 12 normal control embryos and on 41 blastomeres for 9 male and 3 female embryos from the female DMD carrier, obtained after a first preimplantation diagnosis by sexing. The exon 17 region was amplified with 100% efficiency, except in all 21 blastomeres from 6 male embryos from the carrier where no PCR signals were observed. The negative results in these blastomeres were interpreted as being found only in male embryos carrying the deletion. Intracytoplasmic sperm injection was carried out on the carrier`s metaphase II oocytes retrieved after ovarian stimulation. Embryos were analyzed for the presence of exon 17 and 2 male embryos were found to be deleted, while 4 embryos showed normal amplification signals. Three of the latter embryos were replaced, resulting in a singleton pregnancy. Amniotic cell analysis showed a normal female karyotype and DNA analysis indicated a non-carrier.

  19. Raman spectroscopy analysis of differences in composition of spent culture media of in vitro cultured preimplantation embryos isolated from normal and fat mice dams.

    Science.gov (United States)

    Fabian, Dušan; Kačmarová, Martina; Kubandová, Janka; Čikoš, Štefan; Koppel, Juraj

    2016-06-01

    The aim of the present study was to compare overall patterns of metabolic activity of in vitro cultured preimplantation embryos isolated from normal and fat mice dams by means of non-invasive profiling of spent culture media using Raman spectroscopy. To produce females with two different types of body condition (normal and fat), a previously established two-generation model was used, based on overfeeding of experimental mice during prenatal and early postnatal development. Embryos were isolated from spontaneously ovulating and naturally fertilized dams at the 2-cell stage of development and cultured to the blastocyst stage in synthetic oviductal medium KSOMaa. Embryos from fat mice (displaying significantly elevated body weight and fat) showed similar developmental capabilities in vitro as embryos isolated from normal control dams (displaying physiological body weight and fat). The results show that alterations in the composition of culture medium caused by the presence of developing mouse preimplantation embryos can be detected using Raman spectroscopy. Metabolic activity of embryos was reflected in evident changes in numerous band intensities in the 1620-1690cm(-1) (amide I) region and in the 1020-1140cm(-1) region of the Raman spectrum for KSOMaa. Moreover, multivariate analysis of spectral data proved that the composition of proteins and other organic compounds in spent samples obtained after the culture of embryos isolated from fat dams was different from that in spent samples obtained after the culture of embryos from control dams. This study demonstrates that metabolic activity of cultured preimplantation embryos might depend on the body condition of their donors. Copyright © 2016 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  20. Embryo cryopreservation and in vitro culture of preimplantation embryos in Campbell's hamster (Phodopus campbelli).

    Science.gov (United States)

    Amstislavsky, Sergei; Brusentsev, Eugeny; Kizilova, Elena; Igonina, Tatyana; Abramova, Tatyana; Rozhkova, Irina

    2015-04-01

    The aims of this study were to compare different protocols of Campbell's hamster (Phodopus campbelli) embryos freezing-thawing and to explore the possibilities of their in vitro culture. First, the embryos were flushed from the reproductive ducts 2 days post coitum at the two-cell stage and cultured in rat one-cell embryo culture medium (R1ECM) for 48 hours. Most (86.7%) of the two-cell embryos developed to blastocysts in R1ECM. Second, the embryos at the two- to eight-cell stages were flushed on the third day post coitum. The eight-cell embryos were frozen in 0.25 mL straws according to standard procedures of slow cooling. Ethylene glycol (EG) was used either as a single cryoprotectant or in a mixture with sucrose. The survival of frozen-thawed embryos was assessed by double staining with fluorescein diacetate and propidium iodide. The use of EG as a single cryoprotectant resulted in fewer alive embryos when compared with control (fresh embryos), but combined use of EG and sucrose improved the survival rate after thawing. Furthermore, granulocyte-macrophage colony-stimulating factor rat (2 ng/mL) improved the rate of the hamster frozen-thawed embryo development in vitro by increasing the final cell number and alleviating nuclear fragmentation. Our data show the first attempt in freezing and thawing Campbell's hamster embryos and report the possibility of successful in vitro culture for this species in R1ECM supplemented with granulocyte-macrophage colony-stimulating factor. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Effects of oocyte quality, incubation time and maturation environment on the number of chromosomal abnormalities in IVF-derived early bovine embryos.

    Science.gov (United States)

    Demyda-Peyrás, Sebastian; Dorado, Jesus; Hidalgo, Manuel; Anter, Jaouad; De Luca, Leonardo; Genero, Enrique; Moreno-Millán, Miguel

    2013-01-01

    Chromosomal aberrations are one of the major causes of embryo developmental failures in mammals. The occurrence of these types of abnormalities is higher in in vitro-produced (IVP) embryos. The aim of the present study was to investigate the effect of oocyte morphology and maturation conditions on the rate of chromosomal abnormalities in bovine preimplantational embryos. To this end, 790 early cattle embryos derived from oocytes with different morphologies and matured under different conditions, including maturation period (24 v. 36h) and maturation media (five different serum supplements in TCM-199), were evaluated cytogenetically in three sequential experiments. The rates of normal diploidy and abnormal haploidy, polyploidy and aneuploidy were determined in each embryo. Throughout all the experiments, the rate of chromosomal abnormalities was significantly (P<0.05) affected by oocyte morphology and maturation conditions (maturation time and culture medium). Lower morphological quality was associated with a high rate of chromosome abnormalities (P<0.05). Moreover, polyploidy was associated with increased maturation time (P<0.01), whereas the maturation medium significantly (P<0.05) affected the rates of haploidy and polyploidy. In general, supplementing the maturation medium with oestrous cow serum or fetal calf serum resulted in higher rates of chromosomal aberrations (P<0.05) compared with the other serum supplements tested (bovine steer serum, anoestroues cow serum, bovine amniotic fluid and bovine serum albumin). On the basis of the results of the present study, we conclude that the morphological quality of oocytes and the maturation conditions affect the rate of chromosomal abnormalities in IVP bovine embryos.

  2. Cryopreservation and In Vitro culture of Preimplantation Embryos in Djungarian Hamster (Phodopus sungorus).

    Science.gov (United States)

    Brusentsev, E Yu; Abramova, T O; Rozhkova, I N; Igonina, T N; Naprimerov, V A; Feoktistova, N Yu; Amstislavsky, S Ya

    2015-08-01

    Although embryo cryobanking was applied to Syrian golden and to Campbell's hamsters, no attempt has been made at freezing embryos in Djungarian hamsters. Four-cell stage embryos were flushed from the reproductive ducts of pregnant females before noon of the third-day post coitum and frozen in 0.25-ml straws according to standard procedures of slow cooling. A mixture of permeating (ethylene glycol) and non-permeating (sucrose) cryoprotectants was used. The thawing was performed by incubating at RT for 40 s followed by 40 s in a water bath at 30.0°C. Most (66.7%) of the non-frozen four-cell embryos developed up to the morula stage in rat one-cell embryo culture medium (R1ECM). The use of hamster embryo culture medium (HECM) yielded fewer morulas (18.2%) during the same 24-h period of culture. The rate of embryo's surviving the freezing-thawing procedures, as estimated by light microscopy, was 60.7-68.8%. After 24-h culturing in R1ECM, 64.7% of frozen-thawed four-cell embryos developed and all of them reached the morula stage. Supplementation of R1ECM with GM-CSF (2 ng/ml) improved the rate of Djungarian hamster frozen-thawed embryo development: 100% of the four-cell stage embryos developed, 50% of them achieved the morula stage, and 50% developed even further and reached the blastocyst stage within 24 h of culturing. This study reports the world's first successful transfer of frozen-thawed Djungarian hamster embryos yielding term pups. Taken together, the results of this study demonstrate the possibility of applying some key reproductive technologies, that is, embryo freezing/cryopreservation and in vitro culture, to Djungarian hamsters. © 2015 Blackwell Verlag GmbH.

  3. Timing of human preimplantation embryonic development is confounded by embryo origin

    DEFF Research Database (Denmark)

    Kirkegaard, Kirstine Kjær; Sundvall Germeys, Linda Karin M; Erlandsen, M.

    2016-01-01

    STUDY QUESTION To what extent do patient- and treatment-related factors explain the variation in morphokinetic parameters proposed as embryo viability markers? SUMMARY ANSWER Up to 31% of the observed variation in timing of embryo development can be explained by embryo origin, but no single facto...... by a grant from the Danish Council for Independent Research Medical Sciences. The authors declare no competing interest....... embryos from one patient as independent observations, and only very few studies that evaluate the influence from patient- and treatment-related factors on timing of development or time-lapse parameters as predictors of viability have controlled for confounding, which implies a high risk of overestimating...

  4. What next for preimplantation genetic screening? A polar body approach!

    NARCIS (Netherlands)

    Geraedts, Joep; Collins, John; Gianaroli, Luca; Goossens, Veerle; Handyside, Alan; Harper, Joyce; Montag, Markus; Repping, Sjoerd; Schmutzler, Andreas

    2010-01-01

    Screening of human preimplantation embryos for numerical chromosome abnormalities has been conducted mostly at the preimplantation stage using fluorescence in situ hybridization. However, it is clear that preimplantation genetic screening (PGS) as it is currently practiced does not improve live

  5. Near-infrared laser irradiation improves the development of mouse pre-implantation embryos.

    Science.gov (United States)

    Yokoo, Masaki; Mori, Miho

    2017-05-27

    The aim of the present study was to assess the effects of near-infrared laser irradiation on the in vitro development of mouse embryos. Female ICR mice were superovulated with pregnant mare serum gonadotropin and human chorionic gonadotropin (hCG), and mated with male mice. Two-cell stage embryos were collected 40 h after administering hCG and cultured in M16 medium. Two-cell embryos (0 h after culture), 8-cell embryos (approx. 30 h after culture), morula (approx. 48 h after culture), and blastocysts (approx. 73 h after culture) were irradiated at 904 nm for 60 s. These embryos were cultured in a time-lapse monitoring system and the timing of blastocyst hatching was evaluated. Some of the irradiated blastocysts were transferred to the uterine horns of pseudopregnant recipients immediately after irradiation. Pregnancy rates, and offspring growth and fertility, were evaluated. Near-infrared laser irradiation increased the speed of in vitro mouse embryo development. In irradiated blastocysts, hatching was faster than in control (non-irradiated) blastocysts (18.4 vs. 28.2 h, P infrared laser irradiation improves the quality of mouse embryo development in vitro, and increases the live birth rate without affecting the normality of the offspring. Thus, the near-infrared laser method may enhance the quality of embryos and contribute to improvements in reproductive technologies in mammals. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. OCT-4 expression is essential for the segregation of trophectoderm lineages in porcine preimplantation embryos.

    Science.gov (United States)

    Emura, Natsuko; Sakurai, Nobuyuki; Takahashi, Kazuki; Hashizume, Tsutomu; Sawai, Ken

    2016-08-25

    Oct-4, a member of the POU family of transcription factors, is a key factor that regulates the segregation of the inner cell mass (ICM) and the trophectoderm (TE) during the transition from morula to blastocyst in mice. However, little is known about its role in porcine early embryogenesis. To determine the function of OCT-4 in the ICM and TE segregation of porcine embryos, we studied the developmental morphology of porcine embryos using RNA interference technology. Our experiments demonstrated that when 1-cell stage embryos were co-injected with the small interfering RNA (siRNA)for targeted knockdown of OCT-4 (OCT-4-siRNA) and tetramethylrhodamine isothiocyanate (TRITC)-dextran conjugate (Dx), they failed to form blastocysts. Therefore, in this study, we constructed chimeric embryos comprising blastomeres that either expressed OCT-4 normally or showed downregulated OCT-4 expression by co-injection of OCT-4-siRNA and Dx into one blastomere in 2- to 4-cell stage embryos. In control embryos, which were co-injected with control siRNA and Dx, Dx-positive cells contributed to the TE lineage in almost all the blastocysts examined. In contrast, Dx-positive cells derived from a blastomere co-injected with OCT-4-siRNA and Dx were degenerated in almost half the blastocysts. This was probably due to the inability of these cells to differentiate into the TE lineage. Real-time RT-PCR analysis revealed no difference in the levels of SOX2, TEAD4, FGF4 and FGFR1-IIIc, all of which are known to be regulated by OCT-4, between the OCT-4-siRNA-injected morulae and the control ones. However, the level of CDX2, a molecule specifically expressed in the TE lineage, was significantly higher in the former than in the latter. Our results indicate that continuous expression of OCT-4 in blastomeres is essential for TE formation of porcine embryos.

  7. Perkembangan Praimplantasi Embrio Mencit dengan Materi Genetik yang Berasal dari Parental, Maternal, dan Inti Sel Somatik (PRE-IMPLANTATION DEVELOPMENT OF MOUSE EMBRYO WITH GENETIC MATERIAL DERIVED FROM PARENTAL, MATERNAL AND SOMATIC CELL NUCLEUS

    Directory of Open Access Journals (Sweden)

    Harry Murti

    2014-05-01

    Full Text Available Cloned embryo and parthenogenetic embryo are a potential source of stem cells for regenerativemedicine. Stem cells derived from those embryos are expected to overcome the ethical issues to the use offertilization embryos for therapeutic purposes. The pre-implantation development is a critical step fordeveloping embryos reach the blastocyst stage. The objectives in vivo of this research are to produce mousecloned embryo, parthenogenetic embryo, and fertilized embryo and to study stages of  in vitro pre-implantation development culture. In vivo fertilized embryos, mouse oocytes, and cumulus cells were usedin this study. Treatment was performed on female mice superovulated with PMSG and hCG injections.Two-cell stage of in vivo fertilized embryos were collected on the second day post hCG injection. Clonedembryos were produced through Somatic Cell Nuclear Transfer (SCNT, which included enucleation, nucleartransfer and artificial activation. Parthenogenetic embryos were produced with artificial activationtechnique. The result of the research indicated that SCNT application was able to produce cloned embryos which could develop to blastocyst stage (3,2%. In addition, artificial activation of oocytes could produceparthenogenetic embryos which were able to develop up to the blastocyst stage (8,6%. In conclusion,efficiency level of parthenogenetic embryos that is able to reach the blastocyst stage was higher than in thecloned embryos. Fertilized embryos shows a better development and more efficient compared to in vitrocloned embryos and parthenogenetic embryos cultures.

  8. mRNA Fragments in In-Vitro Culture Media are Associated with Bovine Preimplantation Embryonic Development

    Directory of Open Access Journals (Sweden)

    Jenna eKropp

    2015-08-01

    Full Text Available In vitro production (IVP systems have been used to bypass problems of fertilization and early embryonic development. However, embryos produced by IVP are commonly selected for implantation based on morphological assessment, which is not a strong indicator of establishment and maintenance of pregnancy. Thus, there is a need to identify additional indicators of embryonic developmental potential. Previous studies have identified microRNA expression in in vitro culture media to be indicative of embryo quality in both bovine and human embryos. Like microRNAs, mRNAs have been shown to be secreted from cells into the extracellular environment, but it is unknown whether or not these RNAs are secreted by embryos. Thus, the objective of the present study was to determine whether mRNAs are secreted into in vitro culture media and if their expression in the media is indicative of embryo quality. In vitro culture medium was generated and collected from both blastocyst and degenerate (those which fail to develop from the morula to blastocyst stage embryos. Small-RNA sequencing revealed that many mRNA fragments were present in the culture media. A total of 17 mRNA fragments were differentially expressed between blastocyst and degenerated conditioned media. Differential expression was confirmed by quantitative real-time PCR for

  9. Differential regulation of H3S10 phosphorylation, mitosis progression and cell fate by Aurora Kinase B and C in mouse preimplantation embryos

    Directory of Open Access Journals (Sweden)

    Wenzhi Li

    2017-04-01

    Full Text Available ABSTRACT Coordination of cell division and cell fate is crucial for the successful development of mammalian early embryos. Aurora kinases are evolutionarily conserved serine/threonine kinases and key regulators of mitosis. Aurora kinase B (AurkB is ubiquitously expressed while Aurora kinase C (AurkC is specifically expressed in gametes and preimplantation embryos. We found that increasing AurkC level in one blastomere of the 2-cell embryo accelerated cell division and decreasing AurkC level slowed down mitosis. Changing AurkB level had the opposite effect. The kinase domains of AurkB and AurkC were responsible for their different ability to phosphorylate Histone H3 Serine 10 (H3S10P and regulate metaphase timing. Using an Oct4-photoactivatable GFP fusion protein (Oct4-paGFP and fluorescence decay after photoactivation assay, we found that AurkB overexpression reduced Oct4 retention in the nucleus. Finally, we show that blastomeres with higher AurkC level elevated pluripotency gene expression, which were inclined to enter the inner cell mass lineage and subsequently contributed to the embryo proper. Collectively, our results are the first demonstration that the activity of mitotic kinases can influence cell fate decisions in mammalian preimplantation embryos and have important implications to assisted reproduction.

  10. Expression analysis of the endogenous Zscan4 locus and its coding proteins in mouse ES cells and preimplantation embryos.

    Science.gov (United States)

    Ishiguro, Kei-Ichiro; Nakatake, Yuhki; Chikazawa-Nohtomi, Nana; Kimura, Hiromi; Akiyama, Tomohiko; Oda, Mayumi; Ko, Shigeru B H; Ko, Minoru S H

    2017-02-01

    Mouse Zinc finger and SCAN domain containing 4 (Zscan4) is encoded in multiple copies of Zscan4 genes, which are expressed in late two-cell stage preimplantation embryos and in 1-5% of the embryonic stem (ES) cell population at a given time. Due to the highly identical nucleotide sequences of multiple copies of Zscan4 paralogs and pseudogenes in the mouse Zscan4 genomic cluster, previous analyses have been done using exogenous transgenes under the regulation of Zscan4c promoter. In this manuscript, we generated knock-in mouse ES cell lines and mouse lines, in which the expression of endogenous Zscan4c, one of the Zscan4 genes, can be specifically monitored with a green fluorescent protein variant, Emerald. Interestingly, we found that only ∼30% of Zscan4-immunopositive ES cells were Emerald positive, suggesting that even when the Zscan4 locus is active, not all Zscan4 genes are expressed synchronously. We also carried out mass spectrometry of protein complexes associated with endogenous Zscan4 proteins. Taken together, our genetic engineering at an endogenous Zscan4c gene provides the first clue for the expression and function of each gene copy of Zscan4 locus in a physiological context.

  11. Expression of nucleolar-related proteins in porcine preimplantation embryos produced in vivo and in vitro

    DEFF Research Database (Denmark)

    Bjerregaard, Bolette; Wrenzycki, Christine; Strejcek, Frantisek

    2004-01-01

    embryonic mRNA transcription. Localization of proteins involved in the rRNA transcription (upstream binding factor [UBF], topoisomerase I, RNA polymerase I [RNA Pol I], and the RNA Pol I-associated factor PAF53) and processing (fibrillarin, nucleophosmin, and nucleolin) was assessed by immunocytochemistry....... Moreover, immunolocalization of RNA Pol I, but not of UBF, and the mRNA expression of PAF53 and UBF were significantly reduced or absent after culture with alpha-amanitin, indicating that RNA Pol I, PAF53, and presumably, UBF are derived from de novo embryonic transcription. Embryonic genomic activation...... was delayed in porcine embryos produced in vitro compared to the in vivo-derived counterparts with respect to mRNAs encoding PAF53 and UBF. Moreover, differences existed in the mRNA expression patterns of pRb between in vivo- and in vitro-developed embryos. These findings show, to our knowledge for the first...

  12. CRISPR/Cas9 as tool for functional study of genes involved in preimplantation embryo development.

    Directory of Open Access Journals (Sweden)

    Jeongwoo Kwon

    Full Text Available The CRISPR/Cas9 system has proven to be an efficient gene-editing tool for genome modification of cells and organisms. However, the applicability and efficiency of this system in pig embryos have not been studied in depth. Here, we aimed to remove porcine OCT4 function as a model case using the CRISPR/Cas9 system. Injection of Cas9 and single-guide RNA (sgRNA against OCT4 decreased the percentages of OCT4-positive embryos to 37-50% of total embryos, while ~100% of control embryos exhibited clear OCT4 immunostaining. We assessed the mutation status near the guide sequence using polymerase chain reaction (PCR and DNA sequencing, and a portion of blastocysts (20% in exon 2 and 50% in exon 5 had insertions/deletions near protospacer-adjacent motifs (PAMs. Different target sites had frequent deletions, but different concentrations of sgRNA made no impact. OCT4 mRNA levels dramatically decreased at the 8-cell stage, and they were barely detectable in blastocysts, while mRNA levels of other genes, including NANOG, and CDX2 were not affected. In addition, the combination of two sgRNAs led to large-scale deletion (about 1.8 kb in the same chromosome. Next, we injected an enhanced green fluorescent protein (eGFP vector targeting the OCT4 exon with Cas9 and sgRNA to create a knockin. We confirmed eGFP fluorescence in blastocysts in the inner cell mass, and also checked the mutation status using PCR and DNA sequencing. A significant portion of blastocysts had eGFP sequence insertions near PAM sites. The CRISPR/CAS9 system provides a good tool for gene functional studies by deleting target genes in the pig.

  13. Noninvasive metabolic profiling using microfluidics for analysis of single preimplantation embryos.

    Science.gov (United States)

    Urbanski, John Paul; Johnson, Mark T; Craig, David D; Potter, David L; Gardner, David K; Thorsen, Todd

    2008-09-01

    Noninvasive analysis of metabolism at the single cell level will have many applications in evaluating cellular physiology. One clinically relevant application would be to determine the metabolic activities of embryos produced through assisted reproduction. There is increasing evidence that embryos with greater developmental capacity have distinct metabolic profiles. One of the standard techniques for evaluating embryonic metabolism has been to evaluate consumption and production of several key energetic substrates (glucose, pyruvate, and lactate) using microfluorometric enzymatic assays. These assays are performed manually using constriction pipets, which greatly limits the utility of this system. Through multilayer soft-lithography, we have designed a microfluidic device that can perform these assays in an automated fashion. Following manual loading of samples and enzyme cocktail reagents, this system performs sample and enzyme cocktail aliquotting, mixing of reagents, data acquisition, and data analysis without operator intervention. Optimization of design and operating regimens has resulted in the ability to perform serial measurements of glucose, pyruvate, and lactate in triplicate with submicroliter sample volumes within 5 min. The current architecture allows for automated analysis of 10 samples and intermittent calibration over a 3 h period. Standard curves generated for each metabolite have correlation coefficients that routinely exceed 0.99. With the use of a standard epifluorescent microscope and CCD camera, linearity is obtained with metabolite concentrations in the low micromolar range (low femtomoles of total analyte). This system is inherently flexible, being easily adapted for any NAD(P)H-based assay and scaled up in terms of sample ports. Open source JAVA-based software allows for simple alterations in routine algorithms. Furthermore, this device can be used as a standalone device in which media samples are loaded or be integrated into microfluidic

  14. Evaluation of the Cryotech Vitrification Kit for bovine embryos.

    Science.gov (United States)

    Gutnisky, C; Alvarez, G M; Cetica, P D; Dalvit, G C

    2013-12-01

    The purpose of this work was to assess commercially available Cryotech Vitrification Kit, in terms of survival, in vitro development and pregnancy rate for bovine embryos. Cumulus-oocyte complexes (COCs) were recovered from ovaries obtained from slaughtered cows and then matured in vitro for 22 h. COCs were fertilized by sex-sorted sperm in IVF-mSOF and cultured in IVC-mSOF for 7 days to the blastocyst stage. Blastocysts were vitrified with the Cryotech Vitrification Kit(®) and then either warmed to check viability or transferred to synchronized heifers. We observed 100% survival of the in vitro produced blastocysts and obtained the same pregnancy rate (46.8%) as that obtained using fresh in vitro produced blastocysts. We thus conclude that the Cryotech vitrification method is a valid alternative to other vitrification or slow-cooling methods in the bovine species and that it is ready for livestock production. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Involvement of mouse and porcine PLCζ-induced calcium oscillations in preimplantation development of mouse embryos

    Energy Technology Data Exchange (ETDEWEB)

    Yoneda, Akihiro, E-mail: ayoneda@sci.hokudai.ac.jp [Laboratory of Animal Breeding and Reproduction, Graduate School of Agriculture, Hokkaido University (Japan); Division of Molecular Therapeutics, Center for Food & Medical Innovation, Hokkaido University (Japan); Watanabe, Tomomasa [Laboratory of Animal Breeding and Reproduction, Graduate School of Agriculture, Hokkaido University (Japan)

    2015-05-01

    In mammals, phospholipase Cζ (PLCζ) has the ability to trigger calcium (Ca{sup 2+}) oscillations in oocytes, leading to oocyte activation. Although there is a species-specific difference in the PLCζ-induced Ca{sup 2+} oscillatory pattern, whether PLCζ-induced Ca{sup 2+} oscillations affect preimplantation embryonic development remains unclear. Here, we show that Ca{sup 2+} oscillations in mouse PLCζ cRNA-injected oocytes stopped just before pronuclear formation, while that in porcine PLCζ cRNA-injected oocytes continued for several hours after pronuclei had been formed. This difference of Ca{sup 2+} oscillations in oocytes after pronuclear formation was dependent on the difference in the nuclear localization signal (NLS) sequence of PLCζ between the mouse and pig. However, mouse and porcine PLCζ cRNA-injected oocytes parthenogenetically developed to blastocysts regardless of the absence or presence of Ca{sup 2+} oscillations after pronuclear formation. Furthermore, the developmental rate of mouse or porcine PLCζ-activated oocytes injected with round spermatids to the blastocyst stage was not significantly different from that of strontium-activated oocytes injected with round spermatids. These results suggest that the PLCζ-induced Ca{sup 2+} oscillatory pattern in mouse oocytes is dependent on the NLS sequence of PLCζ and injection of PLCζ may be a useful method for activation of round spermatid-injected and somatic nuclear transferred oocytes. - Highlights: • Porcine PLCζ-induced Ca{sup 2+} oscillations continued after pronuclear formation. • The Ca{sup 2+} oscillatory pattern was dependent on the difference in the NLS sequence of PLCζ. • PLCζ-activated oocytes parthenogenetically developed to blastocysts. • PLCζ-activated oocytes injected with round spermatids developed to blastocysts.

  16. Proteome analysis of early lineage specification in bovine embryos.

    Science.gov (United States)

    Demant, Myriam; Deutsch, Daniela R; Fröhlich, Thomas; Wolf, Eckhard; Arnold, Georg J

    2015-02-01

    During mammalian embryo development, the zygote undergoes embryonic cleavage in the oviduct and reaches the uterus at the morula stage, when compaction and early lineage specification take place. To increase knowledge about the associated changes of the embryonic protein repertoire, we performed a comprehensive proteomic analysis of in vitro produced bovine morulae and blastocysts (six biological replicates), using an iTRAQ-based approach. A total of 560 proteins were identified of which 502 were quantified. The abundance of 140 proteins was significantly different between morulae and blastocysts, among them nucleophosmin (NPM1), eukaryotic translation initiation factor 5A-1 (EIF5A), receptor of activated protein kinase C 1 (GNB2L1/RACK1), and annexin A6 (ANXA6) with increased, and glutathione S-transferase mu 3 (GSTM3), peroxiredoxin 2 (PRDX2), and aldo-keto reductase family 1 member B1 (AKR1B1) with decreased abundance in blastocysts. Seventy-three percent of abundance altered proteins increased, reflecting an increase of translation activity in this period. This is further supported by an increase in the abundance of proteins involved in the translation machinery and the synthesis of ATP. Additionally, a complementary 2D saturation DIGE analysis led to the detection of protein isoforms, e.g. of GSTM3 and PRDX2, relevant for this period of mammalian development, and exemplarily verified the results of the iTRAQ approach. In summary, our systematic differential proteome analysis of bovine morulae and blastocysts revealed new molecular correlates of early lineage specification and differentiation events during bovine embryogenesis. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Technique of the `in vitro` fertilization and the culture of mouse embryos at preimplantation; Tecnica de fertilizacao `in vitro` e cultura de embrioes de camundongo durante a pre-implantacao

    Energy Technology Data Exchange (ETDEWEB)

    Kikuchi, Olivia Kimiko [Instituto de Pesquisas Energeticas e Nucleares (IPEN), Sao Paulo, SP (Brazil); Yamada, Takeshi [National Inst. of Radiological Sciences, Chiba (Japan)

    1993-03-01

    The mammal embryo is an intensive cellular proliferating system, very radiosensitive and therefore adequate to the study of the biological effects of ionizing radiation. The technique of the in vitro fertilization and the culture of mouse embryos at preimplantation period, modified by Yamada et al (1982) to improve the efficiency of more than 95% of blastocyst formation is described. (author) 2 refs., 7 figs.

  18. Impact of protamine transcripts and their proteins on the quality and fertilization ability of sperm and the development of preimplantation embryos.

    Science.gov (United States)

    Depa-Martynow, Magdalena; Kempisty, Bartosz; Jagodziński, Paweł Piotr; Pawelczyk, Leszek; Jedrzejczak, Piotr

    2012-03-01

    Protamine 1 (PRM1) and 2 (PRM2) are major nuclear proteins in spermatozoa known to bind to chromatin during early spermatogenesis. The aim of this study was to evaluate the impact of protamine transcripts and proteins on human spermatozoa and their fertilization ability as well as the development of preimplantation embryos. Oocytes selected from 92 patients were fertilized in vitro (IVF) using their partners' sperm after evaluating its concentration, motility and morphology. Reverse transcription and real-time quantitative PCR (RQ-PCR) were applied to determine protamine mRNA level, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were used to quantify the protamine protein concentration. Next, protamine mRNA and protein levels were correlated with sperm concentration, motility and morphology as well as with the fertilization ability of mature spermatozoa and embryos of different quality obtained from the IVF program. A significant correlation has been found between protamine transcripts/proteins and: sperm concentration, progressive sperm motility, sperm fertilization ability as well as embryo quality. Protamine transcripts and proteins contribute to the quality of spermatozoa, successful fertilization and may have a significant influence on the development of preimplantation embryos.

  19. Aspects of energetic substrate metabolism of in vitro and in vivo bovine embryos

    Directory of Open Access Journals (Sweden)

    D.K. de Souza

    2015-03-01

    Full Text Available Although the metabolism of early bovine embryos has not been fully elucidated, several publications have addressed this important issue to improve culture conditions for cattle reproductive biotechnologies, with the ultimate goal of producing in vitro embryos similar in quality to those developing in vivo. Here, we review general aspects of bovine embryo metabolism in vitro and in vivo, and discuss the use of metabolic analysis of embryos produced in vitro to assess viability and predict a viable pregnancy after transference to the female tract.

  20. Aspects of energetic substrate metabolism of in vitro and in vivo bovine embryos

    Energy Technology Data Exchange (ETDEWEB)

    Souza, D.K. de [Laboratório de Biotecnologia da Saúde, Faculdade de Medicina, Universidade de Brasília, Brasília, DF (Brazil); Faculdade da Ceilândia, Universidade de Brasília, Brasília, DF (Brazil); Salles, L.P. [Laboratório de Biotecnologia da Saúde, Faculdade de Medicina, Universidade de Brasília, Brasília, DF (Brazil); Departamento de Biologia Molecular, Instituto de Biologia, Universidade de Brasília, Brasília, DF (Brazil); Rosa e Silva, A.A.M. [Laboratório de Biotecnologia da Saúde, Faculdade de Medicina, Universidade de Brasília, Brasília, DF (Brazil)

    2015-01-23

    Although the metabolism of early bovine embryos has not been fully elucidated, several publications have addressed this important issue to improve culture conditions for cattle reproductive biotechnologies, with the ultimate goal of producing in vitro embryos similar in quality to those developing in vivo. Here, we review general aspects of bovine embryo metabolism in vitro and in vivo, and discuss the use of metabolic analysis of embryos produced in vitro to assess viability and predict a viable pregnancy after transference to the female tract.

  1. Plasma membrane block to polyspermy in human oocytes and preimplantation embryos.

    Science.gov (United States)

    Sengoku, K; Tamate, K; Horikawa, M; Takaoka, Y; Ishikawa, M; Dukelow, W R

    1995-09-01

    The existence and time course of the human plasma membrane block to polyspermy were investigated by an in vitro fertilization assay using zona pellucida-free unfertilized oocytes, pronuclear oocytes and embryos. In the time course study using a high sperm concentration (10(5) spermatozoa ml-1), the number of penetrating spermatozoa at 30 and 60 min after insemination were 1.3 +/- 0.3 and 2.9 +/- 0.4, respectively. By 2 h after insemination, spermatozoa penetration reached a maximum. A lower maximum number of penetrating spermatozoa was observed at a low sperm concentration (10(4) spermatozoa ml-1), but the number of penetrating spermatozoa still reached a maximum by 2 h after insemination. A reinsemination experiment demonstrated that the number of penetrating spermatozoa was not significantly different between control and reinseminated oocytes, while sperm penetration was not observed in the oocyte beyond the two-cell stage. Furthermore, the number of binding spermatozoa decreased after fertilization and most of the four-cell stage embryos displayed no sperm binding. These results suggest that the plasma membrane block plays an important role in the prevention of polyspermy in the human oocyte, and that the plasma membrane block may involve permanent changes in the binding or fusion ability of spermatozoa in the oolemma after fertilization.

  2. Lipid content and cryotolerance of in vitro-produced bovine embryos treated with forskolin before vitrification

    National Research Council Canada - National Science Library

    Melissa Meneghel; Priscila Chediek Dall’Acqua; Marcela Ambrogi; Beatriz C.S. Leão; Nathália A.S. Rocha-Frigoni; Gisele Z. Mingoti

    The aim of the present study was to evaluate the intracytoplasmic lipid content, development and cryotolerance of in vitro-produced bovine embryos treated with different concentrations of forskolin before vitrification...

  3. Effects of bovine oviduct epithelial cells, fetal calf serum and bovine serum albumin on gene expression in single bovine embryos produced in the synthetic oviduct fluid culture system.

    Science.gov (United States)

    Pedersen, Mona E; Øzdas, Øzen Banu; Farstad, Wenche; Tverdal, Aage; Olsaker, Ingrid

    2005-01-01

    In this study the synthetic oviduct fluid (SOF) system with bovine oviduct epithelial cell (BOEC) co-culture is compared with an SOF system with common protein supplements. One thousand six hundred bovine embryos were cultured in SOF media supplemented with BOEC, fetal calf serum (FCS) and bovine serum albumin (BSA). Eight different culture groups were assigned according to the different supplementation factors. Developmental competence and the expression levels of five genes, namely glucose transporter-1 (Glut-1), heat shock protein 70 (HSP), connexin43 (Cx43), (2)-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), analysed as mRNA by using reverse transcription-polymerase chain reaction, were measured on bovine embryos cultured for 9 days. Gene expression of these in vitro-produced embryos was compared with the gene expression of in vivo-produced embryos. There was no significant difference found in embryo developmental competence between the Day 9 embryos in BOEC co-culture, FCS and BSA supplements in SOF media. However, differences in gene expression were observed. With respect to gene expression in in vivo and in vitro embryos, BOEC co-culture affected the same genes as did supplementation with FCS and BSA. HSP was the only gene that differed significantly between in vitro and in vivo embryos. When the different in vitro groups were compared, a significant difference between the BOEC co-culture and the FCS supplementation groups due to Glut-1 expression was observed.

  4. Closed pulled straw vitrification of in vitro-produced and in vivo-produced bovine embryos.

    Science.gov (United States)

    Yu, X L; Deng, W; Liu, F J; Li, Y H; Li, X X; Zhang, Y L; Zan, L S

    2010-03-01

    The objective of this study was to evaluate the efficiency of the closed pulled straw (CPS) method for cryopreserving in vitro-produced and in vivo-produced bovine (Bos taurus) embryos. Based on the open pulled straw (OPS) protocol, the top end of a CPS was closed by tweezers (heated in a flame) to prevent the cryoprotectant medium containing embryos from contacting the liquid nitrogen. Bovine in vitro or in vivo morulae and early blastocyst embryos were frozen by slow cryopreservation, OPS vitrification, or CPS vitrification. Morphology of postthawed embryos was evaluated, and normal embryos were used for successive culture for 72h. There were no significant differences between OPS and CPS freezing groups in postthawed in vitro-produced embryos with respect to rates of morphologically normal embryos (mean+/-SD, 87.9+/-5.2% vs. 85.4+/-4.9%), survival at 24h (58.0+/-6.8% vs. 56.3+/-4.4%), and survival at 72h (35.2+/-6.0% vs. 34.9+/-6.7%). However, both OPS and CPS vitrification resulted in higher postthaw rates of morphologically normal embryo and survival at 24 and 72h than those of the slow-freezing method (Pvitrification was a feasible method to cryopreserve both in vitro-derived and in vivo-derived bovine embryos. This method not only eliminated the risk of embryo contamination by preventing contact with liquid nitrogen but also retained the advantages of the OPS vitrification method. Copyright 2010. Published by Elsevier Inc.

  5. DNA methylation in porcine preimplantation embryos developed in-vivo or produced by in-vitro fertilization, parthenogenetic activation and somatic cell nuclear transfer

    DEFF Research Database (Denmark)

    Deshmukh, Rahul Shahaji; Østrup, Olga; Østrup, Esben

    2011-01-01

    DNA demethylation and remethylation are crucial for reprogramming of the differentiated parental/somatic genome in the recipient ooplasm upon somatic cell nuclear transfer. Here, we analyzed the DNA methylation dynamics during porcine preimplantation development. Porcine in vivo developed (IV....... Embryos produced under in vitro conditions had higher levels of DNA methylation than IV. A lineage-specific DNA methylation (hypermethylation of the inner cell mass and hypomethylation of the trophectoderm) was observed in porcine IV late blastocysts, but was absent in PA- and SCNT-derived blastocysts...

  6. DNA methylation in porcine preimplantation embryos developed in vivo and produced by in vitro fertilization, parthenogenetic activation and somatic cell nuclear transfer

    DEFF Research Database (Denmark)

    Deshmukh, Rahul Shahaji; Østrup, Olga; Østrup, Esben

    2011-01-01

    DNA demethylation and remethylation are crucial for reprogramming of the differentiated parental/somatic genome in the recipient ooplasm upon somatic cell nuclear transfer. Here, we analyzed the DNA methylation dynamics during porcine preimplantation development. Porcine in vivo developed (IV....... Embryos produced under in vitro conditions had higher levels of DNA methylation than IV. A lineage-specific DNA methylation (hypermethylation of the inner cell mass and hypomethylation of the trophectoderm) was observed in porcine IV late blastocysts, but was absent in PA- and SCNT-derived blastocysts...

  7. Preimplantation genetic screening.

    Science.gov (United States)

    Harper, Joyce C

    2017-01-01

    Preimplantation genetic diagnosis was first successfully performed in 1989 as an alternative to prenatal diagnosis for couples at risk of transmitting a genetic or chromosomal abnormality, such as cystic fibrosis, to their child. From embryos generated in vitro, biopsied cells are genetically tested. From the mid-1990s, this technology has been employed as an embryo selection tool for patients undergoing in vitro fertilisation, screening as many chromosomes as possible, in the hope that selecting chromosomally normal embryos will lead to higher implantation and decreased miscarriage rates. This procedure, preimplantation genetic screening, was initially performed using fluorescent in situ hybridisation, but 11 randomised controlled trials of screening using this technique showed no improvement in in vitro fertilisation delivery rates. Progress in genetic testing has led to the introduction of array comparative genomic hybridisation, quantitative polymerase chain reaction, and next generation sequencing for preimplantation genetic screening, and three small randomised controlled trials of preimplantation genetic screening using these new techniques indicate a modest benefit. Other trials are still in progress but, regardless of their results, preimplantation genetic screening is now being offered globally. In the near future, it is likely that sequencing will be used to screen the full genetic code of the embryo.

  8. In vitro production of bovine embryos derived from individual donors in the Corral® dish.

    Science.gov (United States)

    Catteeuw, Maaike; Wydooghe, Eline; Mullaart, Erik; Knijn, Hiemke M; Van Soom, Ann

    2017-06-15

    Since the identity of the embryo is of outmost importance during commercial in vitro embryo production, bovine oocytes and embryos have to be cultured strictly per donor. Due to the rather low yield of oocytes collected after ovum pick-up (OPU) per individual cow, oocyte maturation and embryo culture take place in small groups, which is often associated with inferior embryo development. The objective of this study was to improve embryonic development in small donor groups by using the Corral® dish. This commercial dish is designed for human embryo production. It contains two central wells that are divided into quadrants by a semi-permeable wall. In human embryo culture, one embryo is placed per quadrant, allowing individual follow-up while embryos are exposed to a common medium. In our study, small groups of oocytes and subsequently embryos of different bovine donors were placed in the Corral® dish, each donor group in a separate quadrant. In two experiments, the Corral® dish was evaluated during in vitro maturation (IVM) and/or in vitro culture (IVC) by grouping oocytes and embryos of individual bovine donors per quadrant. At day 7, a significantly higher blastocyst rate was noted in the Corral® dish used during IVM and IVC than when only used during IVM (12.9% ± 2.10 versus 22.8% ± 2.67) (P vitro embryo production (IVP) in cattle; allowing to allocate oocytes and/or embryos per donor. As fresh embryo transfers on day 7 have higher pregnancy outcomes, the Corral® dish offers an added value for commercial OPU/IVP, since a higher blastocyst development at day 7 is obtained when the Corral® dish is used during IVM and IVC.

  9. Analysis of the expression of putatively imprinted genes in bovine peri-implantation embryos

    DEFF Research Database (Denmark)

    Tveden-Nyborg, Pernille Yde; Alexopoulos, N.I.; Cooney, M.A.

    2008-01-01

    imprinted genes (Ata3, Dlk1, Gnas, Grb10, Magel2, Mest-1, Ndn and Sgce) in bovine peri-implantation embryos. Two embryonic developmental stages were examined, Day 14 and Day 21. The gene expression pattern of single embryos was recorded for in vivo, in vitro produced (IVP) and parthenogenetic embryos....... The IVP embryos allow us to estimate the effect of in vitro procedures and the analysis of parthenogenetic embryos provides provisional information on maternal genomic imprinting. Among the 8 genes investigated, only Mest-1 showed differential expression in Day 21 parthenogenetic embryos compared...... to in vivo and IVP counterparts, indicating maternal imprinting of this gene. In addition, our expression analysis of single embryos revealed a more heterogeneous gene expression in IVP than in in vivo developed embryos, adding further to the hypothesis of transcriptional dysregulation induced by in vitro...

  10. In-straw cryoprotectant dilution for bovine embryos vitrified using Cryotop.

    Science.gov (United States)

    Inaba, Yasushi; Aikawa, Yoshio; Hirai, Tomokazu; Hashiyada, Yutaka; Yamanouchi, Tadayuki; Misumi, Koji; Ohtake, Masaki; Somfai, Tamas; Kobayashi, Shuji; Saito, Norio; Matoba, Satoko; Konishi, Kazuyuki; Imai, Kei

    2011-09-01

    The aim of this study was to develop an in-straw dilution method suitable for 1-step bovine embryo transfer of vitrified embryos using the Cryotop vitrification-straw dilution (CVSD) method. The development of embryos vitrified using the CVSD method was compared with those of embryos cryopreserved using in-straw vitrification-dilution (ISVD) and conventional slow freezing, outside dilution of straw (SFODS) methods. In Experiment 1, in vitro-produced (IVP) embryos cryopreserved using the CVSD method were diluted, warmed and exposed to the dilution solution at various times. When vitrified IVP embryos were exposed to the dilution solution for 30 min after warming, the rates of embryos developing to the hatched blastocyst stage after 72 h of culture (62.0-72.5%) were significantly lower (Pembryos exposed to the solution for 5 and 10 min (82.4-94.3%), irrespective of supplementation with 0.3 M sucrose in the dilution solution. In Experiment 2, the rate of embryos developing to the hatching blastocyst stage after 48 h of culture in IVP embryos cryopreserved using the SFODS method (75.0%) was significantly (Pembryos cryopreserved using the CVSD and ISVD methods (93.2 and 97.3%, respectively). In Experiment 3, when in vivo-produced embryos that had been cryopreserved using the CVSD, ISVD and SFODS methods and fresh embryos were transferred to recipient animals, no significant differences were observed in the conception and delivery rates among groups. In Experiment 4, when IVP embryos derived from oocytes collected by ovum pick-up that had been cryopreserved using the CVSD and ISVD methods and fresh embryos were transferred to recipient animals, no significant differences were observed in the conception rates among groups. Our results indicate that this simplified regimen of warming and diluting Cryotop-vitrified embryos may enable 1-step bovine embryo transfer without the requirement of a microscope or other laboratory equipment.

  11. Initial maternal serum human chorionic gonadotropin levels in pregnancies achieved after assisted reproductive technology are higher after preimplantation genetic screening and after frozen embryo transfer: a retrospective cohort.

    Science.gov (United States)

    Hobeika, Elie; Singh, Sonali; Malik, Shaveta; Knochenhauer, Eric S; Traub, Michael L

    2017-10-01

    Few published articles have compared initial hCG values across all different types of ART cycles, including cycles with fresh or frozen embryo transfer. No articles have compared initial hCG values in cycles utilizing preimplantation genetic screening (PGS). The purpose of this study is to compare initial hCG values after fresh embryo transfer, frozen embryo transfer, and after PGS. This was a single-center retrospective cohort study at an academically affiliated private IVF center. All fresh and frozen embryo transfers between January 2013 and December 31, 2015 were included. We compared mean initial serum hCG values 14 days after oocyte retrieval for fresh cycles and 9 days after frozen embryo transfer. We examined cycles of single embryo transfer (SET) and double embryo transfer (DET). Two hundred elven IVF (fresh embryo transfer), 128 FET (frozen embryo transfer cycles, no PGS), and 111 PGS cycles (ovarian stimulation with embryo cryopreservation, PGS, and frozen transfer in a subsequent estrogen-primed cycle) with initial positive hCG values were analyzed. In patients achieving a positive hCG after SET, initial hCG values were higher after PGS compared to FET (182.4 versus 124.0 mIU/mL, p = 0.02) and IVF (182.4 versus 87.1 mIU/mL, p < 0.001) as well as FET compared to IVF (124.0 versus 87.1 mIU/mL, p < 0.01). After DET, initial hCG values were higher after PGS (222.8 mIU/mL) compared to FET (182.1 mIU/mL, p = 0.02) and IVF (131.1 mIU/mL, p = 0.001). Our study suggests that initial serum hCG values are higher after using PGS and higher after the transfer of a frozen embryo compared to a fresh embryo. This suggests that initial hCG values relate to the chromosomal status of embryos. Initial hCG values may help determine intervention and monitoring later in pregnancy.

  12. Transcription of ribosomal RNA genes is initiated in the third cell cycle of bovine embryos

    DEFF Research Database (Denmark)

    Jakobsen, Anne Sørig; Avery, Birthe; Dieleman, Steph J.

    2006-01-01

    Transcription from the embryos own ribosomal genes is initiated in most species at the same time as the maternal-embryonic transition. Recently data have indicated that a minor activation may take place during the third embryonic cell cycle in the bovine, one cell cycle before the major activation...... bovine embryos were investigated to allow comparison of transcription initiation. Signs of active transcription of rRNA were observed in the third cell cycle in 29% of the in vitro produced embryos (n=35) and in 58% of the in vivo developed embryos (n=11). Signs of active transcription of rRNA were...... not apparent in the early phase of the fourth cell cycle but restarted later on. All embryos in the fifth or later cell cycles were all transcribing rRNA. The signs of rRNA synthesis during the third and fourth embryonic cell cycles could be blocked by actinomycin D, which is a strong inhibitor of RNA...

  13. Characteristics of pregnancies and offspring following transfer of bovine in vivo embryos assessed by nanorespirometry

    DEFF Research Database (Denmark)

    Lopes, Ana Sofia; Madsen, S E; Greve, Torben

    2008-01-01

    It has been speculated whether the metabolism of the pre-implantation embryo may be reflected on the pregnancy and characteristics of the newborn animal. The present study investigated whether respiration rates of individual embryos were correlated with gestation length, type of parturition, birth......, III), stage of development, and diameter and were subsequently transferred individually (n = 43) to synchronized recipients. Gestation length of the recipients (n = 22) was calculated and the type of parturition (no assistance, light traction, heavy traction, or caesarean section) recorded. Sex...... as the interaction of weight at birth and type of parturition (P type of parturition, which was not affected by sex. When embryos were divided into two even-sized categories according to their respiration rates...

  14. Vitrification of in vitro produced bovine embryos: in vitro and in vivo evaluations.

    Science.gov (United States)

    Martínez, A G; Valcárcel, A; de las Heras, M A; de Matos, D G; Furnus, C; Brogliatti, G

    2002-09-16

    The efficacy of different vitrification solutions to cryopreserve in vitro produced bovine blastocysts was evaluated based upon in vitro development of embryos in culture and on in vivo development of embryos transferred into recipients. In the first experiment, ethylene glycol + glycerol (Eg + Gly) + different sucrose concentrations were evaluated. There were no significant differences in development rates among solutions. As for hatching, the Eg + Gly + 0.1 M sucrose group had a greater rate as compared with Eg + Gly + 0 M sucrose and Eg + Gly + 0.5 M sucrose groups in the evaluations of Day 6, Day 7 and Day 6 + Day 7 embryos; and, Eg + Gly + 0.3 M sucrose group had a greater rate as compared with the Eg + Gly + 0 M sucrose and Eg + Gly + 0.5 M sucrose groups in evaluations of Day 6 and Day 6 + Day 7 embryos. There were no significant differences in development and hatching rates between Day 6 and 7 in in vitro produced bovine embryos within each treatment group. There were significant differences in nuclei number after vitrification between Eg + Gly + 0.1 M and Eg + Gly + 0 M sucrose groups and the Eg + Gly + 0.5 M sucrose group. Pregnancy after 60 days of transfer and calving rates showed a difference between in vivo produced embryos freshly transferred and in vitro produced embryos vitrified with Eg + Gly + 0.3 M. There were no significant differences in gestation length and sex ratio between treatments. As for birth weight, there were significant differences between fresh in vivo produced embryos and all treatments of in vitro produced embryos. There were significant differences in dystocial parturition between in vivo produced embryos and all treatments with in vitro produced embryos. These results demonstrate that vitrification can be used successfully in the cryopreservation of in vitro produced bovine embryos, and that it might be considered for use in commercial programs. Copyright 2002 Elsevier Science B.V.

  15. Effect of copper during bovine oocytes maturation on preimplantation embryo development

    OpenAIRE

    Rosa, Diana E.

    2015-01-01

    El presente trabajo de tesis consistió en evaluar el efecto de la adición de Cu durante la maduración in vitro (MIV) de ovocitos bovinos y sus consecuencias en el desarrollo embrionario preimplantacional. Para tal fin, se establecieron los siguientes objetivos: 1) evaluar la asociación entre las concentraciones de Cu en licor folicular y en plasma; 2) estudiar el efecto del Cu sobre la MIV; 3) evaluar el efecto del Cu durante la MIV sobre la capacidad de desarrollo embrionario y 4) determinar...

  16. Sequential comprehensive chromosome analysis on polar bodies, blastomeres and trophoblast: insights into female meiotic errors and chromosomal segregation in the preimplantation window of embryo development.

    Science.gov (United States)

    Capalbo, Antonio; Bono, Sara; Spizzichino, Letizia; Biricik, Anil; Baldi, Marina; Colamaria, Silvia; Ubaldi, Filippo Maria; Rienzi, Laura; Fiorentino, Francesco

    2013-02-01

    What is the optimal stage from oocyte through preimplantation embryo development for biopsy and preimplantation genetic screening (PGS) to detect abnormal chromosome segregation patterns in eggs or embryos from advanced maternal age (AMA) patients? Testing at the polar body (PB) stage was the least accurate mainly due to the high incidence of post-zygotic events. This suggests that postponing the time of biopsy to the blastocyst stage of preimplantation embryo development may provide the most reliable results for PGS. In the PGS field there is an ongoing debate about the optimal biopsy stage for PGS. This is a result of the lack of understanding of how aneuploidy arises in the human embryo. To date, most of the cytogenetic data obtained during PGS investigations have been derived through the analysis of cells at isolated points in the preimplantation window, thus potentially missing critical information on chromosomal segregation. Understanding the chromosome segregation patterns during preimplantation development holds the potential to significantly increase the success rates of IVF. In this study, a sequential comprehensive chromosome analysis of both the PBs and the corresponding embryos at both the cleavage and the blastocyst stages is presented. This is a prospective longitudinal cohort study performed between October 2009 and August 2011 involving 9 infertile couples and 21 sets of complete comprehensive chromosomal screening data, including PB1, PB2, corresponding blastomeres and trophectoderm (TE) samples. Infertile couples undergoing IVF cycles with PGS where the female partner was older than 40 years and with a good response to controlled ovarian stimulation (>10 MII oocytes retrieved) were enrolled into the study. The exclusion criteria were (i) patients presenting with abnormal karyotype; (ii) specific ovarian pathologies including polycystic ovary syndrome, endometriosis grade III or higher and premature ovarian failure and (iii) severe male factor

  17. Evaluation of developmental changes in bovine in vitro produced embryos following exposure to bovine Herpesvirus type 5

    Directory of Open Access Journals (Sweden)

    Brenner Mariana PC

    2012-07-01

    Full Text Available Abstract Background Bovine Herpesvirus type-5 (BoHV-5 is a neurovirulent α-Herpesvirus which is potentially pathogenic for cows and suspected to be associated with reproductive disorders. Interestingly, natural transmission of BoHV-5 by contaminated semen was recently described in Australia. Additionally, BoHV-5 was also isolated from the semen of a healthy bull in the same country and incriminated in a natural outbreak of reproductive disease after artificial insemination. In contrast with BoHV-1, experimental exposure of in vitro produced bovine embryos to BoHV-5 does not affect embryo viability and seems to inhibit some pathways of apoptosis. However, the mechanisms responsible for these phenomena are poorly understood. In this study, we examined mitochondrial activity, antioxidant protection, stress response and developmental rates of in vitro produced bovine embryos that were exposed and unexposed to BoHV-5. Methods For this purpose, bovine embryos produced in vitro were assayed for cell markers after experimental infection of oocytes (n = 30; five repetitions, in vitro fertilization and development. The indirect immunofluorescence was employed to measure the expression of superoxide dismutase 1 (SOD1, anti-oxidant like protein 1 (AOP-1, heat shock protein 70.1 (Hsp 70.1 and also viral antigens in embryos derived from BoHV-5 exposed and unexposed oocytes. The determination of gene transcripts of mitochondrial activity (SOD1, antioxidant protection (AOP-1 and stress response (Hsp70.1 were evaluated using the reverse transcriptase polymerase chain reaction (RT-PCR. MitoTracker Green FM, JC-1 and Hoechst 33342-staining were used to evaluate mitochondrial distribution, segregation patterns and embryos morphology. The intensity of labeling was graded semi-quantitatively and embryos considered intensively marked were used for statistical analysis. Results The quality of the produced embryos was not affected by exposure to BoHV-5. Of the 357

  18. Vitrification of in vitro produced bovine embryos: effect of embryonic block and developmental kinetics.

    Science.gov (United States)

    Asgari, V; Hosseini, S M; Forouzanfar, M; Hajian, M; Nasr-Esfahani, M H

    2012-12-01

    In order to investigate whether the kinetics and stage of embryo development affect cryosurvival of in vitro produced bovine embryos, cleaved embryos were categorized in six groups based on their developmental kinetics regarding the stage of embryonic block in bovine (8-16 cell stage): I and II--early (day 2) and late (day 3) 5-8 cell, III and IV--early (day 3) and late (day 4) 8-16 cell, and V and VI--early (day 4) and late (day 5) morula. The cryosurvival and developmental competence of these embryos were compared with each other and also with the corresponding control groups. The potential of 5-8 cell stage embryos to survive vitrification and further develop towards blastocyst stage was significantly lower than vitrified and un-vitrified 8-16 cell and morula stage embryos. These results suggest that, the survival rate and potential of embryos to develop towards blastocyst stage might be affected by the kinetic of the embryo development. Moreover, the results of this study indicated that the optimal stages of early embryo vitrification are post-embryonic block. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Preimplant preparation of the extraction alveolus with the deproteinized bovine bone and calcium-sulphate

    Directory of Open Access Journals (Sweden)

    Brković Božidar

    2006-01-01

    Full Text Available Background. Different materials are used to prevent the resorption of alveolar bone. The aim of this report was to show the radiographical and histological results prior to implant insertion, when a deproteinized bovine bone mineral (BioOss and calcium-sulphate were placed into the extraction socket immediately after the tooth removal. Case report. A 22-year-old woman was scheduled for the removal of the second lower molar when the extraction socket was filled with BioOss covered with calcium-sulphate as a resorbable membrane. Primary closure of the surgical site was performed. Radiography was done 4 and 12 months later. One year after the surgery, when the implant was inserted, a biopsy of the new regenerated bone was obtained for the histological evaluation. The lamellar bone was evident using both materials. The resorption of BioOss was slow and the connective tissue was observed. Conclusion. Both materials had biocompatible and oseoconductive properties. One year after the grafting procedure, we observed the lamellar bone and partial resorption of BioOss, while calciumsulphate showed no significant effect as a resorbable membrane.

  20. Developmental kinetics of in vitro-produced bovine embryos: An aid for making decisions.

    Science.gov (United States)

    Carrocera, S; Caamaño, J N; Trigal, B; Martín, D; Díez, C

    2016-03-15

    Embryo developmental kinetics and embryo survival after cryopreservation have been correlated with embryo quality and viability. The main objectives of this work were to analyze developmental ability and quality of in vitro-produced bovine embryos in relation to their kinetics and to establish a criterion of quality to predict further viability. Embryos were classified and grouped by their specific stage of development (2, 3-4, or ≥ 5 cells) at 44 hours post insemination (hpi) and cultured separately up to Day 8. On Days 7 and 8, good quality expanded blastocysts were vitrified or frozen. Cryopreserved surviving hatched embryos were stained for cell counts. Embryos at a more advanced stage (3-4 cells, and ≥5 cells) developed to morulae (P embryos that had cleaved once by 44 hpi. Vitrification improved the hatching rates of blastocysts at 48 hours (P embryos (3-4 cells and ≥5 cells). After vitrification/warming, blastocysts coming from 3- to 4-cell embryos had higher hatching rates at 48 hours than those that came from ≥5-cell embryos. With regard to differential cell counts, no effect of the initial developmental stage was observed after warming/thawing. However, trophectoderm and total cells were higher in vitrified/warmed than in the frozen/thawed embryos (P embryos at 44 hpi, after the evaluation of their in vitro embryo development, could be used as noninvasive markers of embryo developmental competence and may help to select IVF embryos that would be more suitable for cryopreservation. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Phosphorylated H2AX in parthenogenetically activated, in vitro fertilized and cloned bovine embryos.

    Science.gov (United States)

    Pereira, A F; Melo, L M; Freitas, V J F; Salamone, D F

    2015-08-01

    In vitro embryo production methods induce DNA damage in the embryos. In response to these injuries, histone H2AX is phosphorylated (γH2AX) and forms foci at the sites of DNA breaks to recruit repair proteins. In this work, we quantified the DNA damage in bovine embryos undergoing parthenogenetic activation (PA), in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) by measuring γH2AX accumulation at different developmental stages: 1-cell, 2-cell and blastocyst. At the 1-cell stage, IVF embryos exhibited a greater number of γH2AX foci (606.1 ± 103.2) and greater area of γH2AX staining (12923.6 ± 3214.1) than did PA and SCNT embryos. No differences at the 2-cell stage were observed among embryo types. Although PA, IVF and SCNT were associated with different blastocyst formation rates (31.1%, 19.7% and 8.3%, P DNA damage was comparable among those embryos developing to the blastocyst stage among different methods for in vitro embryo production. While IVF resulted in increased damage at the 1-cell embryo stage, no difference was observed between PA and SCNT embryos at any developmental stage. The decrease in the number of double-stranded breaks at the blastocyst stage seems to indicate that DNA repair mechanisms are functional during embryo development.

  2. Influence of recipient cytoplasm cell stage on transcription in bovine nucleus transfer embryos

    DEFF Research Database (Denmark)

    Smith, Steven D.; Soloy, Eva; Kanka, Jiri

    1996-01-01

    Nucleus transfer for the production of multiple embryos derived from a donor embryo relies upon the reprogramming of the donor nucleus so that it behaves similar to a zygotic nucleus. One indication of nucleus reprogramming is the RNA synthetic activity. In normal bovine embryogenesis, the embryo...... relies upon maternally derived RNA transcripts up to the 8-cell stage, at which time it begins to transcribe its own RNA. In this experiment, RNA synthesis was detected in nucleus transfer embryos (NTE) and control embryos by pulsing with 3H-uridine, fixation, and autoradiography on semithin sections....... NTE were produced using either a MII phase (nonactivated) cytoplasts at 32 hr of maturation or S-phase (activated) cytoplasts activated with calcium ionophore A23187 and cycloheximide treatment approximately 8 hr prior to fusion with a blastomere from an in-vitro-produced morula stage embryo at 32 hr...

  3. Cryopreservation affects the quality of in vitro produced bovine embryos at the molecular level.

    Science.gov (United States)

    Stinshoff, H; Wilkening, S; Hanstedt, A; Brüning, K; Wrenzycki, C

    2011-11-01

    There are still immense differences in the quality of in vitro produced embryos compared to their in vivo generated counterparts. These differences include a higher sensitivity of in vitro produced embryos towards cryopreservation. The quality of such embryos has been evaluated by morphological examination as well as the assessment of total cell numbers, pregnancy rates and in few cases through the analysis of their gene expression. The aim of the present study was to determine whether different cryopreservation methods have an influence on the quality of in vitro produced embryos after thawing. Bovine blastocysts were produced in a standard culture system (SOFaa). Having reached the stage of an expanding blastocyst on day 7, embryos were randomly either vitrified (n = 106) or cryopreserved conventionally (n = 131). Reexpansion (24h post thawing; 86/106 [81.1 ± 20.3%] of the vitrified embryos, 104/131 [79.4 ± 16.5%] of the conventionally cryopreserved embryos respectively) and hatching rates (48 h post thawing; 67/106 [63.2 ± 24.5%] of the vitrified embryos, 80/131 [61.1 ± 29.3%] of the conventionally cryopreserved embryos respectively) were similar. No significant differences in total cell numbers as well as live-dead cell ratio could be seen in hatched blastocysts of either cryopreservation group as well as in a control group (embryos cultured up to the stage of a hatched blastocyst and not cryopreserved by either method). Additionally, RT-qPCR was used to assess the relative abundance of eight developmentally important genes (HSPA1A, SLC2A1, SLC2A3, DSC2, CDH1, TJP1, DNMT3A, IFNT2) in hatched embryos of all groups. The results revealed significant differences in 4 of 8 (HSPA1A, SLC2A1, TJP1, DSC2) gene transcripts when comparing vitrified embryos to embryos of the control group and in 6 of 8 gene transcripts (HSPA1A, SLC2A1, TJP1, SLC2A3, DNMT3A, IFNT2) when comparing slow frozen embryos to embryos of the control group. Furthermore, the comparison of

  4. Routine use of next-generation sequencing for preimplantation genetic diagnosis of blastomeres obtained from embryos on day 3 in fresh in vitro fertilization cycles.

    Science.gov (United States)

    Łukaszuk, Krzysztof; Pukszta, Sebastian; Wells, Dagan; Cybulska, Celina; Liss, Joanna; Płóciennik, Łukasz; Kuczyński, Waldemar; Zabielska, Judyta

    2015-04-01

    To determine the usefulness of semiconductor-based next-generation sequencing (NGS) for cleavage-stage preimplantation genetic diagnosis (PGD) of aneuploidy. Prospective case-control study. A private center for reproductive medicine. A total of 45 patients underwent day-3 embryo biopsy with PGD and fresh cycle transfer. Additionally, 53 patients, matched according to age, anti-Müllerian hormone levels, antral follicles count, and infertility duration were selected as controls. Choice of embryos for transfer was based on the PGD NGS results. Clinical pregnancy rate (PR) per embryo transfer (ET) was the primary outcome. Secondary outcomes were implantation and miscarriage rates. The PR per transfer was higher in the NGS group (84.4% vs. 41.5%). The implantation rate (61.5% vs. 34.8%) was higher in the NGS group. The miscarriage rate was similar in the 2 groups (2.8% vs. 4.6%). We demonstrate the technical feasibility of NGS-based PGD involving cleavage-stage biopsy and fresh ETs. Encouraging data were obtained from a prospective trial using this approach, arguing that cleavage-stage NGS may represent a valuable addition to current aneuploidy screening methods. These findings require further validation in a well-designed randomized controlled trial. ACTRN12614001035617. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  5. Inclusion of bovine lipoproteins and the vitamin E analogue, Trolox, during in vitro culture of bovine embryos changes both embryo and fetal development.

    Science.gov (United States)

    Rooke, J A; Watt, R G; Ashworth, C J; McEvoy, T G

    2012-01-01

    This experiment investigated effects of lipoproteins and Trolox (vitamin E analogue) on bovine embryo and fetal development. The treatments were: in vitro culture (IVC) in synthetic oviducal fluid alone (SOF); with bovine lipoproteins (2% v/v; SOFLP); with Trolox (100μM; SOFT); and with lipoproteins and Trolox (SOFLPT). In vitro culture with lipoproteins increased fatty acid content of blastocysts (PTrolox had no effect (P>0.05). Whereas lipoproteins reduced zygote development to blastocysts (P=0.03), Trolox facilitated increased development (PTrolox (P>0.05) had no effect on blastocyst morphological grade. Pregnancy rates resulting from synchronous transfer of IVP embryos were not affected by IVC treatment. At Day 70 of pregnancy, compared with SOF, fetal weight was lower in SOFLP but not SOFLPT (interaction, PTrolox. Placentome numbers were greater in SOF and SOFLPT compared with SOFLP and SOFT (interaction, P=0.002); superior embryo grades were also associated with increased numbers of placentomes (P=0.024). In conclusion, the interactive effects of lipoprotein and Trolox inclusion on in vitro embryo development were also evident in fetal development at Day 70.

  6. The CDK9/Cyclin T1 subunits of P-TEFb in mouse oocytes and preimplantation embryos: A possible role in embryonic genome activation

    Directory of Open Access Journals (Sweden)

    Park Chang S

    2011-06-01

    Full Text Available Abstract Background Two stages of genome activation have been identified in the mouse embryo. Specifically, minor transcriptional activation is evident at the one-cell stage and a second major episode of activation occurs at the two-cell stage. Nuclear translocation of RNA polymerase II and phosphorylation of the C-terminal domain (CTD of the largest enzyme subunit are major determinants of embryonic genome activation. P-TEFb, the Pol II CTD kinase, regulates transcriptional elongation via phosphorylation of the serine 2 residues of the CTD. Results Here, we show that the CDK9 and cyclin T1 subunits of P-TEFb are present in mouse oocytes and preimplantation embryos. Both proteins translocate to pronuclei at the late one-cell stage and are predominantly localized in nuclei at the two-cell stage. We additionally examine the effects of the CDK9-specific inhibitor, flavopiridol, on mouse preimplantation development. Our data show that treatment with the drug results in mislocalization of CDK9, cyclin T1, and phosphorylated Pol II, as well as developmental arrest at the two-cell stage. Conclusions A change in CDK9 localization from the cytoplasm to the pronucleus occurs at the time of minor embryonic genome activation, and CDK9 accumulation at the two-cell stage is evident, concomitant with major transcriptional activation of the embryonic genome. Moreover, CDK9 inhibition triggers a developmental block at the two-cell stage. Our findings clearly indicate that CDK9 is essential for embryonic genome activation in the mouse.

  7. Isolation of bovine herpesvirus-1 (BHV-1) and bovine viral diarrhea virus (BVDV) in association with the in vitro production of bovine embryos.

    Science.gov (United States)

    Bielanski, A; Loewen, K S; Del Campo, M R; Sirard, M A; Willadsen, S

    1993-09-01

    The purpose of this study was to determine whether oocytes obtained from bovine ovaries collected at commercial abattoirs for use in in vitro fertilization programs would be contaminated with bovine herpesvirus-1 (BHV-1) and/or bovine viral diarrhea virus (BVDV). In total, of 85 samples tested containing 759 embryos produced by in vitro fertilization, 2 (2.4%) were positive for BHV-1 while none were positive for BVDV. The follicular fluid collected during oocyte aspiration tested positive in 11.8% for BVH-1 and in 4.7% for BVDV. Oviductal cells used to co-culture zygotes/embryos tested positive for BHV-1 and BVDV in 6.2% and 1.2% samples respectively.

  8. Phytohemagglutinin facilitates the aggregation of blastomere pairs from Day 5 donor embryos with Day 4 host embryos for chimeric bovine embryo multiplication.

    Science.gov (United States)

    Simmet, Kilian; Reichenbach, Myriam; Reichenbach, Horst-Dieter; Wolf, Eckhard

    2015-12-01

    Multiplication of bovine embryos by the production of aggregation chimeras is based on the concept that few blastomeres of a donor embryo form the inner cell mass (ICM) and thus the embryo proper, whereas cells of a host embryo preferentially contribute to the trophectoderm (TE), the progenitor cells of the embryonic part of the placenta. We aggregated two fluorescent blastomeres from enhanced green fluorescent protein (eGFP) transgenic Day 5 morulae with two Day 4 embryos that did not complete their first cleavage until 27 hours after IVF and tested the effect of phytohemagglutinin-L (PHA) on chimeric embryo formation. The resulting blastocysts were characterized by differential staining of cell lineages using the TE-specific factor CDX2 and confocal laser scanning microscopy to facilitate the precise localization of eGFP-positive cells. The proportions of blastocyst development of sandwich aggregates with (n = 99) and without PHA (n = 46) were 85.9% and 54.3% (P multiplication of genetically valuable donor embryos. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Comparison of the efficacy of conventional slow freezing and rapid cryopreservation methods for bovine embryos

    NARCIS (Netherlands)

    Wagtendonk-de Leeuw, van A.M.; Daas, den J.H.; Kruip, T.A.; Rail, W.F.

    1995-01-01

    Day 7 bovine morulae and early blastocysts were randomly assigned to one of four cryopreservation methods: (i) a modified conventional controlled slow freezing and stepwise dilution after thawing; and three methods which enable direct transfer of the embryo into the recipient upon thawing: (ii)

  10. Influence of antral follicle size on oocyte characteristics and embryo development in the bovine

    DEFF Research Database (Denmark)

    Lequarre, Anne Sophie; Vigneron, Céline; Ribaucour, Fabrice

    2005-01-01

    The developmental competence of bovine oocytes isolated from antral follicles of different sizes was assessed in three European laboratories (Belgium, UCL; Denmark, DIAS; France, INRA). Using the same protocol for in vitro production of embryos, the oocytes isolated from follicles with a diameter...... ≥6 mm always gave a higher blastocyst rate than oocytes from follicles...

  11. Different co-culture systems have the same impact on bovine embryo transcriptome.

    Science.gov (United States)

    Carvalho, A Vitorino; Canon, E; Jouneau, L; Archilla, C; Laffont, L; Moroldo, M; Ruffini, S; Corbin, E; Mermillod, P; Duranthon, V

    2017-11-01

    During the last few years, several co-culture systems using either BOEC or VERO feeder cells have been developed to improve bovine embryo development and these systems give better results at high oxygen concentration (20%). In parallel, the SOF medium, used at 5% O2, has been developed to mimic the oviduct fluid. Since 2010s, the SOF medium has become popular in improving bovine embryo development and authors have started to associate this medium to co-culture systems. Nevertheless, little is known about the putative benefit of this association on early development. To address this question, we have compared embryo transcriptomes in four different culture conditions: SOF with BOEC or VERO at 20% O2, and SOF without feeders at 5% or 20% O2 Embryos have been analyzed at 16-cell and blastocyst stages. Co-culture systems did not improve the developmental rate when compared to 5% O2 Direct comparison of the two co-culture systems failed to highlight major differences in embryo transcriptome at both developmental stages. Both feeder cell types appear to regulate the same cytokines and growth factors pathways, and thus to influence embryo physiology in the same way. In blastocysts, when compared to culture in SOF at 5% O2, BOEC or VERO seems to reduce cell survival and differentiation by, at least, negatively regulating STAT3 and STAT5 pathways. Collectively, in SOF medium both blastocysts rate and embryo transcriptome suggest no influence of feeder origin on bovine early development and no beneficial impact of co-culture systems when compared to 5% O2. © 2017 Society for Reproduction and Fertility.

  12. Cryotop vitrification for in vitro produced bovine and buffalo (Bubalus bubalis embryos at different stages of development

    Directory of Open Access Journals (Sweden)

    B. Gasparrini

    2010-02-01

    Full Text Available The aim of this study was to evaluate the possibility to vitrify in vitro produced (IVP buffalo and bovine embryos at different stages of development by an advanced version of the “minimal volume approaches”: the Cryotop method. In both experiments, the embryos were vitrified at the tight morula (TM, early blastocyst (eBl, blastocyst (Bl, expanded blastocyst (xBl and, only for buffalo, at the hatched blastocyst (hBl stage. After warming, the embryos were cultured in vitro for 24 hours. Stage of development affected the freezability of IVP embryos of both species with the highest embryo survival rates at advanced stages (xBl=76% and hBl=75% for buffalos and xBl=75% for bovine. These results suggest that Cryotop vitrification is an efficient method for buffalo and bovine IVP embryo cryopreservation.

  13. Factors that affect the reproductive efficiency of the recipient within a bovine embryo transfer program

    Directory of Open Access Journals (Sweden)

    Arturo Duica A.

    2007-12-01

    Full Text Available The embryo transfer is a biotechnological technique that allows increasing the descendant of animals with high genetic value. The positive results, represented in pregnancy after the application of this technique, are affected by some factors that are inherent to the donor, the embryo, the technique, and the recipients which receive a strange embryo in the uterus allowing pregnancy. This review describes some factors affecting the reproductive efficiency of the recipients of bovine embryos within a program of embryo transfer. Its important to evaluate the parameters in this kind of recipients, as race, age, physiological status, health status, weight, reproductive tract integrity and management, and also too monitoring the ovarian structures while the estrus synchronization, and within previous and posterior stages in embryo transfer procedure. Therefore an optimum follicular development will be determinant to corpus luteum formation which generates enough serum progesterone concentrations to offer a right uterine environment allowing the optimum embryo development. Controlling the factors that affect the efficiency of the embryo transfer, it will obtain an increasing of positive results represented in pregnancies and births of individuals come from animals with high genetic value.

  14. RESEARCHES REGARDING THE INFLUENCE OF RECOVERY MEDIA ON THE IN VITRO DEVELOPMENT CAPACITY OF THE PREIMPLANTATIONAL MOUSE EMBRYO

    Directory of Open Access Journals (Sweden)

    ADA CEAN

    2009-05-01

    Full Text Available Phosphate Bufered Saline with 0.4% BSA and M2 medium are one of the most common media used in embryorecovery. The aim of our paper was to investigate if the recovery media used for the recovery of the mouseembryo is influencing in vitro developmental capacity. As biological material we used 10 used were mousefemales, age 2 months superovulated with 5UI PMSG (Pregnant Mare Serum Gonadotropine and 5 UI hCG(human Corionic Gonadotropine. The embryos used were recovered, by oviduct flushing, at 24 hours from theidentification of the vaginal plug. The majority of the embryos (78.3% were in two cells stage. A total of 123, 2cells embryos were cultivated in M16 medium. The evolution of the embryos was examined at 24, 48 and 72hours interval. The proportion of hatched blastocyst was higher at the embryos recovered with M2 (53.7%compared with the embryos recovered with PBS 0.4% BSA. The difference is statistically very significant(p<0.001. Embryos recovered in M2 media have a higher in vitro developmental capacity compared with theembryos recovered in PBS media supplemented with 0,4% BSA, possibly because of the sodium bicarbonate andlactate used in M2 media for pH regulation.

  15. Vitrification of bovine embryos followed by in vitro hatching and expansion.

    Science.gov (United States)

    Souza, J F; Oliveira, C M; Lienou, L L; Cavalcante, T V; Alexandrino, E; Santos, R R; Rodrigues, A P R; Campello, C C; Figueiredo, J R; Dias, F E F

    2017-12-18

    The objective of this study was to assess the effects of bovine embryo vitrification by applying three different vitrification solutions containing ethylene glycol (EG) and dimethylsulphoxide (DMSO) at different concentrations (10, 20 or 25% each) combined with 1.0 M glucose or 1.0 M sucrose, on the in vitro hatching and expansion rates. Healthy oocytes were selected for in vitro maturation and fertilization from 200 bovine ovaries, and subsequently cultured up to the blastocyst stage (n = 800). Control (n = 200) and vitrified cells (n = 100 per treatment; 600 in total) were cultured for an extra 24 or 48 h to evaluate hatching and expansion, respectively. Vitrification significantly decreased embryonic re-expansion and hatching rates independently of the tested solution when compared with control embryos, but solutions with 25% EG + 25% DMSO resulted in the highest re-expansion (75%) and hatching (70%) rates, independently of the added sugar. The addition of sucrose resulted in higher rates of re-expanded and hatched embryos when compared with glucose addition. We concluded that the combination of 25% EG + 25% DMSO and 1.0 M sucrose allowed hatching and expansion of vitrified-warmed bovine embryos produced in vitro.

  16. Vitrification of immature feline oocytes with a commercial kit for bovine embryo vitrification.

    Science.gov (United States)

    Apparicio, M; Ruggeri, E; Luvoni, G C

    2013-04-01

    The aim of this study was to evaluate the suitability of a commercial kit for bovine embryo vitrification for cryopreserving cat oocytes and to evaluate comparatively the effects of its use with slow freezing procedure on cryotolerance in terms of morphology and oocyte resumption of meiosis. Germinal vesicle stage oocytes isolated from cat ovaries were either vitrified (n = 72) using a vitrification kit for bovine embryo or slow frozen (n = 69) by exposing oocyte to ethylene glycol solution before being transferred to a programmable embryo freezer. After thawing and warming, oocytes were cultured for 48 h and then were examined for meiosis resumption using bisbenzimide fluorescent staining (Hoechst 33342). Fresh immature oocytes (n = 92) were used as the control group. The proportion of oocytes recovered in a morphologically normal state after thawing/warming was significantly higher in frozen oocytes (94.5%) than in the vitrified ones (75%, p vitrification compared to 60.9% of those submitted to slow freezing procedure (p bovine embryos retain their capacity to resume meiosis after warming and culture, albeit at lower rates than slow frozen oocytes. Vitrification and slow freezing methods show similar proportions of oocytes with normal morphology after culture, which demonstrate that thawed and warmed oocytes that resist to cryodamage have the same chances to maintain their integrity after 48 h of culture. © 2012 Blackwell Verlag GmbH.

  17. The effects of ovalbumin as a protein source during the in vitro production of bovine embryos

    Directory of Open Access Journals (Sweden)

    Tatiane Almeida Drummond Tetzner

    2011-10-01

    Full Text Available Embryo quality is influenced by the culture conditions that affect in vitro maturation (IVM, fertilization (IVF and culture (IVC rates. The present study investigated the feasibility of producing bovine embryos after the replacement of fetal calf serum (FCS and bovine serum albumin (BSA by ovalbumin (OVA. The IVM and IVC medium were supplemented with 10% FCS, 4 mg/mL BSA, or 4 mg/mL OVA. The IVF medium was supplemented with 6 mg/mL BSA or OVA. For IVM, supplementation with FCS, BSA, and OVA did not affect nuclear maturation or cortical granule migration. Higher rates of formation of two pronuclei were obtained when FCS was employed for IVM (79.97%, regardless of the supplement used for IVF, and when BSA was used for IVF (59.4%, regardless of the supplement used for IVM. Supplementation with OVA for IVM+IVC (20.40% and for IVF (22.15% was inferior to supplementation with FCS for IVM+IVC (30.47% and with BSA for IVF (28.91% for blastocyst development. Hatching rates were lower using OVA for IVM+IVC (23.02% and for IVF (28.93% compared with FCS and BSA under the same conditions (40.78 and 34.82%, respectively and BSA for IVF (36.82%. Supplementation with OVA for IVM+IVC and IVF resulted in reduced inner cell mass, trophectoderm cells and total blastocyst cell numbers (17.29, 37.88, and 55.17, respectively. In conclusion, OVA is a protein source for bovine in vitro embryo production, although the quantity and quality of bovine blastocysts using only ovalbumin in the entire in vitro production process are lower than those obtained in the presence of FCS and BSA, when used as supplements in any step of bovine in vitro embryo production.

  18. Serum free embryo culture medium improves in vitro survival of bovine blastocysts to vitrification.

    Science.gov (United States)

    Gómez, E; Rodríguez, A; Muñoz, M; Caamaño, J N; Hidalgo, C O; Morán, E; Facal, N; Díez, C

    2008-05-01

    The aim of this study was to examine the effects of co-culture with Vero cells during the in vitro maturation (IVM) and three culture media, B2+5% fetal calf serum (FCS) on Vero cells, synthetic oviduct fluid (SOF)+5% FCS, and SOF+20 gL(-1) bovine serum albumin (BSA), on the developmental competence of the embryos and their ability to survive vitrification/warming. We also tested the effect of morphological quality and the age of the embryo on its sensitivity to vitrification. The IVM system neither affects the embryo development up to Day 7 nor survival rates after vitrification. The culture of embryos in SOF+FCS and in Vero cells+B2 allowed obtaining more Day 6 and Day 7 blastocysts, and a higher % of Day 7 blastocysts vitrified than culture in SOF+BSA. Contrarily, on Day 8, more blastocysts were vitrified in SOF+BSA than in SOF+FCS. Blastocysts quality affected development after vitrification/warming, and Day 7 embryos showed higher survival rates than their Day 8 counterparts. Day 7 blastocysts produced in Vero cells or in SOF+BSA survived at higher rates than those produced in SOF+FCS at 24 and 48 h after warming. Embryo culture with BSA allows obtaining hatching rates after vitrification/warming higher than those obtained after co-culture with Vero cells in B2 and FCS. Moreover, this system provides hatching rates from Day 8 blastocysts comparable to those obtained on Day 7 in Vero cells. Further studies, including embryo transfer to recipients, are needed to clarify factors affecting the freezability of in vitro produced bovine embryos.

  19. Effect of vitrification using the Cryotop method on the gene expression profile of in vitro-produced bovine embryos.

    Science.gov (United States)

    de Oliveira Leme, Ligiane; Dufort, Isabelle; Spricigo, José Felipe Warmling; Braga, Thiago Felipe; Sirard, Marc-André; Franco, Maurício Machaim; Dode, Margot Alves Nunes

    2016-03-01

    The present study analyzed the changes in gene expression induced by the Cryotop vitrification technique in bovine blastocyst-stage embryos, using Agilent EmbryoGENE microarray slides. Bovine in vitro-produced embryos were vitrified and compared with nonvitrified (control) embryos. After vitrification, embryos were warmed and cultured for an additional 4 hours. Survived embryos were used for microarray analysis and quantitative polymerase chain reaction (qPCR) quantification. Survival rates were higher (P vitrification seems to be the activation of the apoptosis pathway in some cells. Indeed, FOSL1 is part of the activating protein 1 transcription factor complex and is implicated in a variety of cellular processes, including proliferation, differentiation, and apoptosis. Therefore, our results suggest that a limited increase in the rate of apoptosis was the only detectable response of the embryos to vitrification stress. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Physiology and Endocrinology Symposium: influence of cattle genotype (Bos indicus vs. Bos taurus) on oocyte and preimplantation embryo resistance to increased temperature.

    Science.gov (United States)

    Paula-Lopes, F F; Lima, R S; Satrapa, R A; Barros, C M

    2013-03-01

    High environmental temperatures during the hot months of the year reduce reproductive performance in cattle. Summer heat stress depression in fertility is a multifactorial problem; however, there is evidence that the bovine germinal vesicle and maturing oocyte, as well as the early embryo, are major targets of the deleterious effects of heat stress. Such adverse effects are less pronounced in heat-tolerant breeds (Bos indicus) than heat-sensitive breeds (Bos taurus). This genetic variation results from the greater thermoregulatory ability and cellular thermoresistance of heat-tolerant breeds. Heat-induced oocyte cellular damage occurs in both cytoplasmic and nuclear compartments. Heat shock has been shown to reduce oocyte nuclear maturation, induce apoptosis, compromise oocyte cytoskeleton, and impair oocyte mitochondrial function and developmental competence. However, the oocyte cytoplasm is more susceptible to heat shock than the nucleus. This effect is greater for Bos taurus than Bos indicus oocytes. The detrimental effects of heat shock are also critical during the first cleavage divisions when most of the embryonic genome is inactive; however, the bovine embryo becomes more resistant to increased temperature as it proceeds through development. Several studies demonstrated that Bos indicus embryos are more thermotolerant than Bos taurus embryos. Adaptive changes involved in acquisition of thermotolerance are likely derived from changes in gene expression and (or) activity of biochemical molecules that control cellular functions against stress. Recently, molecules such as IGF-I and caspase inhibitor z-DEVD-fmk have been shown to exert a thermoprotective role, rescuing heat-induced oocyte and embryo cellular damage and developmental competence. Therefore, cattle genotype and thermoprotective molecules can be considered as an alternative to modulate the effects of increased temperature in reproductive function.

  1. Improved cryopreservation of in vitro-produced bovine embryos using a chemically defined freezing medium.

    Science.gov (United States)

    Bruyère, P; Baudot, A; Guyader-Joly, C; Guérin, P; Louis, G; Buff, S

    2012-10-01

    This study evaluates a new synthetic substitute (CRYO3, Ref. 5617, Stem Alpha, France) for animal-based products in bovine embryo cryopreservation solutions. During the experiment, fetal calf serum (FCS) and bovine serum albumin (BSA) were used as references. A combination of a thermodynamic approach using differential scanning calorimetry and a biological approach using in vitro-produced bovine embryo slow-freezing was used to characterize cryopreservation solutions containing CRYO3, FCS and BSA. The CRYO3 and fetal calf serum (FCS) slow-freezing solutions were made from Dulbecco's phosphate-buffered saline containing 1.5 m ethylene glycol, 0.1 m sucrose and 20% (v.v(-1)) of CRYO3 or FCS. The bovine serum albumin (BSA) solution was made by adding 0.1 m sucrose to a commercial solution containing 1.5 m ethylene glycol and 4 g L(-1) BSA. These solutions were evaluated using three characteristics: the end of melting temperature, the enthalpy of crystallization (thermodynamic approach) and the embryo survival and hatching rates after in vitro culture (biological approach). The CRYO3 and FCS solutions had similar thermodynamic properties. In contrast, the thermodynamic characteristics of the BSA solution were different from those of the FCS and CRYO3 solutions. Nevertheless, the embryo survival and hatching rates obtained with the BSA and FCS solutions were not different. Similar biological properties can thus be obtained with slow freezing solutions that have different physical properties within a defined range. The embryo survival rate after 48 h of in vitro culture obtained with the CRYO3 solution (81.5%) was higher than that obtained with the BSA (42.2%, P = 0.000 12) and FCS solutions (58%, P = 0.016). Similarly, the embryo hatching rate after 72 h of in vitro culture was higher with the CRYO3 solution (61.1%) than with the BSA (31.1%, P = 0.0055) and FCS solutions (36%, P = 0.018). We conclude that CRYO3 can be used as a chemically defined substitute for animal

  2. Developmental, qualitative, and ultrastructural differences between ovine and bovine embryos produced in vivo or in vitro.

    Science.gov (United States)

    Rizos, Dimitrios; Fair, Trudee; Papadopoulos, Serafeim; Boland, Maurice P; Lonergan, Patrick

    2002-07-01

    The objective of this study was to compare bovine and ovine oocytes in terms of (1) developmental rates following maturation, fertilization, and culture in vitro, (2) the quality of blastocysts produced in vitro, assessed in terms of their ability to undergo cryopreservation, and (3) the ultrastructural morphology of these blastocysts. In vitro blastocysts were produced following oocyte maturation/fertilization and culture of presumptive zygotes in synthetic oviduct fluid. In vivo blastocysts were used as a control from both species. In Experiment 1, the cleavage rate of bovine oocytes was significantly higher than that of ovine oocytes (78.3% vs. 58.0%, respectively, P bovine and ovine, respectively, P vitrification, there was no difference in survival between in vivo produced bovine and ovine blastocysts (72 hr: 85.7% vs. 75.0%). However, IVP ovine blastocysts survived at significantly higher rates than IVP bovine blastocysts at all time points (72 hr: 47.4% vs. 18.1%, P bovine IVP blastocysts, which also displayed electron-lucent mitochondria and large intercellular cavities. These observations may in part explain the species differences observed in terms of cryotolerance. In conclusion, the quality of ovine blastocysts was significantly higher than their bovine counterparts produced under identical in vitro conditions suggesting inherent species differences between these two groups affecting embryo quality. Copyright 2002 Wiley-Liss, Inc.

  3. High survival and hatching rates following vitrification of embryos at blastocyst stage: a bovine model study.

    Science.gov (United States)

    Huang, Jack Y J; Chung, Jin-Tae; Tan, Seang Lin; Chian, Ri-Cheng

    2007-04-01

    Cryopreservation of embryos at the blastocyst stage may provide an effective method to increase the cumulative pregnancy rate for each treatment cycle of ovarian-stimulated IVF. The objective of this study was to evaluate the survival rate and hatching rate of bovine blastocysts following vitrification using a method designed for oocytes, with a view to introducing this methodology into human assisted reproduction technology and reproductive medicine. Bovine blastocysts were produced from abattoir materials subjected to in-vitro maturation and in-vitro fertilization. Survival rate of the bovine blastocysts was 100% (94/94) following vitrification using a method designed for oocyte cryopreservation. There was no difference in the hatching rate of the bovine blastocysts between control (62.5%: 60/96) and vitrified (61.7%: 58/94) groups. The number of dead cells in the blastocysts was not significantly different between control (5.0 +/- 2.9) and vitrified (9.5 +/- 4.0) groups. In conclusion, the results of this study indicate that bovine blastocysts can be vitrified successfully using a procedure designed for oocyte cryopreservation. It is possible that this method may also be successful for the cryopreservation of human embryos. A further study into this is currently being organized.

  4. Serum free embryo culture medium improves in vitro survival of bovine blastocysts to vitrification

    OpenAIRE

    Gómez, E.; Rodríguez, A; Muñoz, M.; Caamaño, J.N. (José); Hidalgo, C.O. (Carlos); Morán, E.; Facal, Nieves; Díez, C.

    2013-01-01

    The aim of this study was to examine the effects of co-culture with Vero cells during the in vitro maturation (IVM) and three culture media, B2+5% fetal calf serum (FCS) on Vero cells, synthetic oviduct fluid (SOF)+5% FCS, and SOF+20 gL(-1) bovine serum albumin (BSA), on the developmental competence of the embryos and their ability to survive vitrification/warming. We also tested the effect of morphological quality and the age of the embryo on its sensitivity to vitrification. The IVM system ...

  5. Freezing of in vitro produced bovine embryos in animal protein-free medium containing vegetal peptones.

    Science.gov (United States)

    George, F; Vrancken, M; Verhaeghe, B; Verhoeye, F; Schneider, Y-J; Massip, A; Donnay, I

    2006-09-15

    Successful cryopreservation is essential for a large-scale dispersal of bovine in vitro produced (IVP) embryos that have been shown to be more sensitive to cryopreservation than their in vivo counterparts. On the other hand, the use of animal proteins in freezing media increases sanitary risks. We first replaced animal proteins, such as bovine serum albumin (BSA) in the freezing medium by plant-derived peptides (vegetal peptones). A batch of wheat peptones was selected after a preliminary experiment showing the absence of toxicity of concentrationsanimal protein-free freezing medium containing 1.8 mg/mL peptones. No beneficial effect of adding 1 mg/mL sodium hyaluronate or 100 microM beta-mercaptoethanol was observed on embryo survival or quality. In conclusion, we have demonstrated that vegetal peptones can replace BSA in freezing media without affecting blastocyst survival and quality.

  6. Nucleolar development and allocation of key nucleolar proteins require de novo transcription in bovine embryos

    DEFF Research Database (Denmark)

    Svarcova, Olga; Laurincik, Jozef; Avery, Birthe

    2007-01-01

    The goal of the present study was to investigate whether key nucleolar proteins involved in ribosomal RNA (rRNA) transcription and processing are transcribed de novo or from maternally inherited messenger RNAs (mRNA) in bovine embryos, and to which extent de novo transcription of these proteins mRNA...... embryos cultured from the 2-cell stage with or without (control groups) a-amanitin, which blocks the RNA plymerases II and III transcription and, thus the synthesis of mRNA. In the control groups, weak autoradiographic labelling was initially observed in the periphery of few nuclei at the 4-cell...... is required for the development of functional nucleoli during the major activation of the embryonic genome. Immunofluorescence for localization of key nucleolar proteins, autoradiography for detection of transcriptional activity, and transmission electron microscopy were applied to in vitro produc ed bovine...

  7. No cytotoxic effects from application of pentoxifylline to spermatozoa on subsequent pre-implantation embryo development in mice

    Directory of Open Access Journals (Sweden)

    Mohammad Ali Khalili

    2017-06-01

    Full Text Available The aim was to assess the effect of spermatozoa exposed to PTX on the rates of fertilization and embryo development and apoptotic cells within blastocysts in an animal model. Mice Oocytes were inseminated with spermatozoa exposed to 3.6 mmol PTX for 30 min, or with neat spermatozoa. Then fertilization and embryo development rate, blastocyst formation and quality, as well as total cell number of blastocyst, and DNA fragmentation index (DFI in blastocysts were surveyed in both groups. Fertilization and embryo development rate were similar between the groups. The rates of blastocyst formation did not differ significantly between control and PTX groups (52.4% vs. 51.8%. The average of total cell count in blastocysts and DFI in control and PTX groups were also insignificant (31.08 ± 1.5 vs. 34.14 ± 1.5 and 9.76 ± 5.0 vs. 11.77 ± 5.4. Application of PTX for enhancing sperm motility does not cause a cytotoxic effect on subsequent embryo development and embryo genome integrity.

  8. Cryopreservation and sexing of in vivo- and in vitro-produced bovine embryos for their practical use.

    Science.gov (United States)

    Tominaga, Keiichiro

    2004-02-01

    My research awarded includes contributions to cryopreservation and sexing of bovine embryos produced in vitro and in vivo, as follows; (1) In vivo-derived morulae and blastocysts were cryopreserved in the presence of 10% glycerol, and the embryos were transferred into recipients after two-step dilution of glycerol in straw, with a practically acceptable pregnancy rate. (2) The survival rate of 16-cell stage embryos frozen in the medium with ethylene glycol was higher than that with DMSO or 1,2-propanediol. Addition of linoleic acid-albumin to culture medium enhanced the survival rate of post-thaw bovine 16-cell stage in vitro-produced (IVP) embryos. (3) Polarization of cytoplasmic lipid droplets by centrifugation of 2-cell stage embryos was found effective to increase freezing tolerance in 16-cell stage embryos developed from the centrifuged embryos, because blastomeres of 16-cell stage embryos were mostly lipid-free. (4) The usefulness of gel-loading tip (GL-Tip) as a container for ultra-rapid vitrification was demonstrated in IVP embryos from 2-cell to blastocyst stages, with a higher in vitro survival than the conventional two-step freezing. (5) PCR analysis for sexing of in vivo-derived Day-7 embryos indicated that male embryos developed faster and graded higher than female embryos. But such correlation between genetic sex and embryonic development was not found in IVP embryos obtained from individual cows. (6) Addition of 0.1-1.0% deproteinized hemodialysate product from calf blood to culture medium increased the producing efficiency of demi-embryos with good quality. Female embryos rather than male embryos required a longer time to repair after bisection. (7) In vivo-derived bovine embryos after biopsy for sexing by PCR analysis and subsequent vitrification using GL-Tips are available to practical use in the field. (8) Introduction of primer extension preamplification-PCR and purification of DNA product before standard sexing PCR of biopsy samples from Day 3

  9. Differential glycolytic and glycogenogenic transduction pathways in male and female bovine embryos produced in vitro.

    Science.gov (United States)

    Garcia-Herreros, M; Aparicio, I M; Rath, D; Fair, T; Lonergan, P

    2012-01-01

    Previous studies have shown that developmental kinetic rates following IVF are lower in female than in male blastocysts and that this may be related to differences in glucose metabolism. In addition, an inhibition of phosphatidylinositol 3-kinase (PI3-K) inhibits glucose uptake in murine blastocysts. Therefore, the aim of this study was to identify and compare the expression of proteins involved in glucose metabolism (hexokinase-I, HK-I; phosphofructokinase-1, PFK-1; pyruvate kinase 1/2, PK1/2; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; glucose transporter-1, GLUT-1; and glycogen synthase kinase-3, GSK-3) in male and female bovine blastocysts to determine whether PI3-K has a role in the regulation of the expression of these proteins. Hexokinase-I, PFK-1, PK1/2, GAPDH and GLUT-1 were present in bovine embryos. Protein expression of these proteins and GSK-3 was significantly higher in male compared with female blastocysts. Inhibition of PI3-K with LY294002 significantly decreased the expression of HK-I, PFK-1, GAPDH, GSK-3A/B and GLUT-1. Results showed that the expression of glycolytic proteins HK-I, PFK-1, GAPDH and PK1/2, and the transporters GLUT-1 and GSK-3 is regulated by PI3-K in bovine blastocysts. Moreover, the differential protein expression observed between male and female blastocysts might explain the faster developmental kinetics seen in males, as the expression of main proteins involved in glycolysis and glycogenogenesis was significantly higher in male than female bovine embryos and also could explain the sensitivity of male embryos to a high concentration of glucose, as a positive correlation between GLUT-1 expression and glucose uptake in embryos has been demonstrated.

  10. Leucemia inhibitory factor; investigating the time-dependent effect on viability of vitrified bovine embryos.

    Science.gov (United States)

    Kocyigit, A; Cevik, M

    2017-12-01

    Leucemia inhibitory factor (LIF) is involved in various reproductive processes, including sperm development, regulation of ovulation, as well as blastocyst formation, hatching and implantation in embryos. Moreover, LIF has also been shown significantly to enhance the blastocyst formation rates of bovine embryos, a finding that remains controversial. Our purpose was to investigate time-dependent effect of LIF on bovine embryo culture, especially in terms of addition timing. Presumptive zygotes were cultured in five different groups. In this study, 100 ng/ml LIF was added to the culture medium were as follows; control: SOF alone, group A: at day 0 (fertilization day), group B: at day 4 post-insemination (p.i.), group C: at day 4 to 7 (p.i. before vitrification) and group D: at day 8 (p.i. after thawing). Addition of LIF to the culture medium at day 4 significantly increased the percentage of blastocyst rate when compared day 0, day 4 at 6/7 and control group (41.8% versus 24.3%, 19.7%, 34.6%). In conclusion, the addition of LIF only on day 4 (p.i.) to the culture medium was found to be beneficial for bovine embryonic development based on several measures, including blastocysts rate, re-expansion rate and cellular cryotolerance after vitrification. © 2017 Blackwell Verlag GmbH.

  11. Evaluation of different culture systems on the in vitro production of bovine embryos.

    Science.gov (United States)

    Pereira, Daniela Costa; Dode, Margot Alves Nunes; Rumpf, Rodolfo

    2005-03-01

    The objective of this study was to determine the development potential and quality of in vitro produced bovine embryos cultured individually or in groups. After IVM and IVF, presumptive zygotes were cultured in groups or individually, either in drops or in the modified "well of the well" (mWOW) system. In Experiment 1, four culture systems were utilized: T1: drop in group (control); T2: mWOW in groups; T3: mWOW individually; and T4: drop individually. Cleavage and blastocyst rates at Days 6, 7 and 8 and total cell number of Day 6 blastocysts were similar (P > 0.05) for all treatments. However, in Day 7 blastocysts, total cell number was lower (P embryos cultured individually in a small drop than those cultured in the mWOW. In Experiment 2, blastocysts of T1, T2 and T3 were allocated into two groups, control and vitrified. After warming, the vitrified embryos were cultured for 72 h. At 48 h, the development of the Days 6 and 7 embryos was similar (P > 0.05) for all treatments in the control group. For the vitrified embryos, lower hatching rates (P embryos cultured in groups in the mWOW system had the same blastocyst rates but better quality (measured by their survival after vitrification) than those cultured individually in the mWOW system.

  12. Crucial surviving aspects for vitrified in vitro-produced bovine embryos.

    Science.gov (United States)

    Sudano, Mateus José; Paschoal, Daniela Martins; da Silva Rascado, Tatiana; Crocomo, Letícia Ferrari; Magalhães, Luis Carlos Oña; Junior, Alício Martins; Machado, Rui; da Cruz Landim-Alvarenga, Fernanda

    2014-05-01

    The objective of the present study was to correlate some parameters (cleavage, blastocyst production, quality degree score, total cell number, fresh apoptosis and lipid content) with embryo survival after cryopreservation. A total of 1727 in vitro-produced bovine blastocysts were used to establish the parameters (mean ± standard error of the mean (SEM)) for cleavage (85.6 ± 0.8), blastocyst production (39.9 ± 1.4), quality degree score (1.6 ± 0.1), total cell number (140.1 ± 2.9), fresh apoptosis (20.8 ± 1.1) and lipid content (21.3 ± 0.8 droplets). On the same way 1316 blastocysts were vitrified for the determination of post-cryopreservation embryo survival (49.4 ± 1.9). Fresh apoptosis rate and total lipid droplets value were correlated (P embryo survival after cryopreservation (r = 0.91 and r = 0.59; respectively). However, cleavage, blastocyst production, quality degree score and total cell number were not correlated (P > 0.05) with embryo cryotolerance (r = 0.23, r = 0.38, r = 0.22 and r = 0.28; respectively). Therefore, the increased lipid content was moderately correlated with apoptosis in vitrified blastocysts. On the other hand, increased apoptosis in fresh blastocysts was strongly correlated with apoptosis in vitrified blastocysts, which indicated that the apoptosis rate in fresh embryos was a better parameter than the lipid content to predict post-vitrification embryo survival.

  13. In vitro development of preimplantation porcine embryos using alginate hydrogels as a three-dimensional extracellular matrix

    Science.gov (United States)

    Between day 10 and 12 of gestation, porcine embryos undergo a dramatic morphological change, known as elongation, with a corresponding increase in estrogen production for maternal recognition of pregnancy. Elongation deficiencies contribute to ~20% of embryonic loss, but exact mechanisms of elongati...

  14. In vitro and in vivo quality of bovine embryos in vitro produced with sex-sorted sperm

    OpenAIRE

    Trigal, B.; Gómez, E.; Caamaño, J.N. (José); Muñoz, M.; Moreno, Javier; Carrocera, S.; Martín, D.; Díez, C.

    2013-01-01

    In this work we analyzed the effects of three culture systems on developmental ability of bovine embryos in vitro produced with sexed sperm, the survival to vitrification (cryologic vitrification method) of such blastocysts, and their pregnancy rates after embryo transfer to recipients, both as fresh and after vitrification/warming. Finally, we measured the accuracy of the sorting protocol by a polymerase chain reaction-based method to validate the embryo sex at blastocyst stages. We confirme...

  15. Cryopreservation of bovine in vitro produced embryos using ethylene glycol in controlled freezing or vitrification.

    Science.gov (United States)

    Sommerfeld, V; Niemann, H

    1999-03-01

    In this study, the cryoprotectant ethylene glycol (EG) was tested for its ability to improve and facilitate the cryopreservation of in vitro produced (IVP) bovine embryos. Embryos were cryopreserved in EG solutions supplemented with either newborn calf serum (NBCS) or polyvinyl alcohol (PVA). To assess EG toxicity, the embryos were equilibrated in EG concentrations from 1.8 to 8.9 M at room temperature for 10 min and then cultured for 72 h on a cumulus cell monolayer. The hatching rate was highest for day 7 blastocysts frozen in 3.6 M EG (98%) and was not different from the control group (85%). The controlled freezing (0.3 degrees C/min to -35 degrees C) of expanded day 7 blastocysts resulted in a hatching rate of 81%, which was similar to that of the nonfrozen controls (76%). Differential staining revealed only very few degenerate blastomeres attributed to freezing and thawing. Upon direct nonsurgical transfer of day 7 expanded blastocysts frozen in 3.6 M EG, a pregnancy rate of 43% was achieved, while the pregnancy rate after transfer of other developmental stages was significantly lower (22% with expanded day 8 blastocysts). When bovine IVP embryos were incubated at room temperature in 7.2 M EG preceded by preequilibration in 3.6 M EG, the hatching rate of day 7 expanded blastocysts reached 93%. Upon vitrification of IVP day 7 and day 8 blastocysts and expanded blastocysts in 7.2 M EG, the latter showed a higher hatching rate (42%) than blastocysts (12%). Overall, PVA as supplement to the basic freezing solution instead of NBCS had deleterious effects on survival after controlled freezing or vitrification. The simple cryopreservation protocol employed in this study and the low toxicity of ethylene glycol highlight the usefulness of this approach for controlled freezing of IVP embryos. However, further experiments are needed to improve the pregnancy rate following embryo transfer and to enhance survival after vitrification. Copyright 1999 Academic Press.

  16. Embryo genome profiling by single-cell sequencing for successful preimplantation genetic diagnosis in a family harboring COL4A1 c.1537G>A; p.G513S mutation

    Science.gov (United States)

    Patel, Nayana H.; Bhadarka, Harsha K.; Patel, Kruti B.; Vaniawala, Salil N.; Acharya, Arpan; Mukhopadhyaya, Pratap N.; Sodagar, Nilofar R.

    2016-01-01

    CONTEXT: Genetic profiling of embryos (also known as preimplantation genetic diagnosis) before implantation has dramatically enhanced the success quotient of in vitro fertilization (IVF) in recent times. The technology helps in avoiding selective pregnancy termination since the baby is likely to be free of the disease under consideration. AIM: Screening of embryos free from c.1537G>A; p.G513S mutation within the COL4A1 gene for which the father was known in before be in heterozygous condition. SUBJECTS AND METHODS: Processing of trophectoderm biopsies was done from twelve embryos for c.1537G>A; p.G513S mutation within the COL4A1 gene. DNA extracted from isolated cells were subjected to whole genome amplification using an isothermal amplification and strand displacement technology. Oligonucleotide primers bracketing the mutation were synthesized and used to amplify 162 base pairs (bp) polymerase chain reaction amplicons originating from each embryo which were subsequently sequenced to detect the presence or absence of the single base polymorphism. RESULTS: Three out of 12 embryos interrogated in this study were found to be normal while 9 were found to harbor the mutation in heterozygous condition. Implantation of one of the normal embryos following by chorionic villus sampling at 11th week of pregnancy indicated that the baby was free from c.1537G>A; p.G513S mutation within the COL4A1 gene. CONCLUSIONS: Single-cell sequencing is a helpful tool for preimplantation embryo profiling. This is the first report from India describing the birth of a normal child through IVF procedure where a potential pathogenic COL4A1 allele was avoided using this technology. PMID:27803589

  17. Embryo genome profiling by single-cell sequencing for successful preimplantation genetic diagnosis in a family harboring COL4A1 c.1537G>A; p.G513S mutation

    Directory of Open Access Journals (Sweden)

    Nayana H Patel

    2016-01-01

    Full Text Available CONTEXT: Genetic profiling of embryos (also known as preimplantation genetic diagnosis before implantation has dramatically enhanced the success quotient of in vitro fertilization (IVF in recent times. The technology helps in avoiding selective pregnancy termination since the baby is likely to be free of the disease under consideration. AIM: Screening of embryos free from c.1537G>A; p.G513S mutation within the COL4A1 gene for which the father was known in before be in heterozygous condition. SUBJECTS AND METHODS: Processing of trophectoderm biopsies was done from twelve embryos for c.1537G>A; p.G513S mutation within the COL4A1 gene. DNA extracted from isolated cells were subjected to whole genome amplification using an isothermal amplification and strand displacement technology. Oligonucleotide primers bracketing the mutation were synthesized and used to amplify 162 base pairs (bp polymerase chain reaction amplicons originating from each embryo which were subsequently sequenced to detect the presence or absence of the single base polymorphism. RESULTS: Three out of 12 embryos interrogated in this study were found to be normal while 9 were found to harbor the mutation in heterozygous condition. Implantation of one of the normal embryos following by chorionic villus sampling at 11 th week of pregnancy indicated that the baby was free from c.1537G>A; p.G513S mutation within the COL4A1 gene. CONCLUSIONS: Single-cell sequencing is a helpful tool for preimplantation embryo profiling. This is the first report from India describing the birth of a normal child through IVF procedure where a potential pathogenic COL4A1 allele was avoided using this technology.

  18. In vitro evaluation and pregnancy rates after vitrification of in vitro produced bovine embryos.

    Science.gov (United States)

    Martínez, A G; de Matos, D G; Furnus, C C; Brogliatti, G M

    1998-10-01

    The efficacy of different vitrification solutions to cryopreserve in vitro-produced bovine blastocysts was evaluated based on in vitro development of embryos in culture and on in vivo development of embryos transferred into recipients. In the first experiment, 2 vitrification solutions were compared: propylene glycol + glycerol (Pg + Gly) and ethylene glycol + Ficoll + sucrose (EFS). Differences in the overall development and hatching rates in favor of EFS were found (56.4 vs 33.3% and 35.4 vs 13.3%; P vitrification solutions were compared: EFS, modified EFS (EFSm) and ethylene glycol + glycerol (Eg + Gly). The vitrification solutions EFSm and Eg + Gly yield higher hatching rates than did EFS (57.7 vs 59.6 vs 35.7%; P vitrification solutions: EFSm and Eg + Gly. There were no differences between them based on the results obtained after transfer (35.2 vs 43.7%). The vitrification solutions EFSm and Eg + Gly have resulted in good pregnancy rates. These results demonstrated that vitrification can be used successfully in the cryopreservation of in-vitro produced bovine embryos, and it might be considered for use in commercial programs.

  19. Preimplantation development and expression of Hsp-70 and Bax genes in bovine blastocysts derived from oocytes matured in alpha-MEM supplemented with growth factors and synthetic macromolecules.

    Science.gov (United States)

    Vireque, A A; Camargo, L S A; Serapião, R V; Rosa E Silva, A A M; Watanabe, Y F; Ferreira, E M; Navarro, P A A S; Martins, W P; Ferriani, R A

    2009-03-01

    In vitro culture conditions affect both the maternal and embryonic expression of genes and is likely to alter both oocyte and embryo developmental competence. The search for better and less variable culture conditions simulating those in vivo has led to the development of defined culture media, with lower impact on the molecular reprogramming of oocytes and embryos. We evaluated embryo development and relative abundance (RA) of Hsp-70 and Bax transcripts in bovine blastocysts produced from oocytes matured in a chemically defined IVM system with synthetic polymers. Immature cumulus oocyte complexes (COCs) were matured for 22-24h in alpha-MEM supplemented with IGF-1, insulin, 0.1% polyvinyl alcohol (PVA), or 0.1% polyvinylpyrrolidone (PVP), but without FSH or LH. The control group consisted of COCs matured in TCM plus FSH and 10% estrous cow serum. After fertilization, presumptive zygotes were co-cultured with cumulus cells until 224 h post-insemination. Total RNA was isolated from embryo pools, reverse transcribed into cDNA, and subjected to transcript analysis by real-time PCR. Cleavage rate was higher (P<0.05) for the control group (68.3%) than for the PVA (54.4%) and PVP-40 (58.3%) groups. Nevertheless, there was no difference among the PVA, PVP-40 and control groups in blastocyst or hatching rates. Similarly, no difference in relative abundance of Hsp-70 and Bax transcripts was detected in comparison to the control group. We inferred that bovine oocytes can be matured in serum- and gonadotrophin-free medium supplemented with PVA or PVP, enriched with IGF-I and insulin, without altering post-cleavage development and relative abundance of some genes associated with stress and apoptosis.

  20. Relation between physical properties of the zona pellucida and viability of bovine embryos after slow-freezing and vitrification.

    Science.gov (United States)

    Moreira da Silva, F; Metelo, R

    2005-06-01

    In vitro-produced bovine morulae/blastocyst embryos (n = 119) were slow-frozen and vitrified and the physical alterations of the zona pellucida (ZP) was observed by scanning electron microscopy (SEM) to find an explanation for the loss of developmental capacity of the embryos after freezing/thawing. A control group was provided, in which embryos (n = 38) were neither frozen nor vitrified. Embryos were in vitro-cultured in a standard CO2 Heraeus incubator and their viability was assessed 24 and 48 h after the start of culture, evaluating their morphological aspect. After 24 h of culture, embryo survival rate for slow-freezing/thawed (n = 23), vitrified/thawed (n = 20) and control embryos (n = 20) was 39, 27 and 90%, and 35, 14 and 65% after 48 h of culture, respectively. For evaluation of physical changes occurring in ZP, 20 embryos were slow-frozen, 18 were vitrified and 18 were used as control. All embryos were fixed, dried and examined under an SEM. Embryo's diameter, as well as the number of pores and their diameter was measured in squares of 6.4 microm width. We observed that, on average, the diameter of the embryos (92.26 +/- 10.15 microm) did not differ significantly among all embryos. As far as the diameter of the pores in the outer surface of the ZP is concerned, the results revealed a significant difference (P embryos. For the number of pores, statistical differences (p embryos (45.4 +/- 7.3 vs 38.2 +/- 8.2). It is possible that ZP functions as a barrier which is positive when dealing with pathogens, but is harmful when nutrients were supplied from the outside, especially at 48 h of culture. Results indicate that the steps of cryopreservation cause alterations in ZP, with irreversible damage on the further developmental competence of bovine embryos.

  1. Expression and localization of transcription factors SNAIL and SLUG in mouse ovaries and pre-implantation embryos.

    Science.gov (United States)

    Guo, Cui; Meng, Xiaoqian; Bai, Jing; Chen, Cheng; Liu, Tao; Liu, Shuzhen; Zhang, Cong; Li, Wei-Ping

    2014-11-01

    SNAIL and SLUG are zinc-finger transcription factors that participate in the regulation of cell division, cell survival, mesoderm formation and epithelial-to-mesenchymal transition. We investigate the expression of SNAIL and SLUG during follicular maturation, ovulation and luteinization in the ovaries of both neonatal mice and gonadotropin-induced immature mice. Furthermore, we examine the expression and localization of these transcription factors during early embryonic cleavage. Our data demonstrate that both SNAIL and SLUG are present in the epithelial cells of the ovarian surface in immature mice. SNAIL is first evident in the interstitial cells and theca cells by postnatal day (PD) 6 and then appears in the oocytes by PD 8, remaining at a constant expression level for all stages studied thereafter. SLUG is expressed in oocytes as early as PD 1. Its expression also increases with the development of the follicles in theca and interstitial cells but not in granulosa cells. In gonadotropin-induced immature mice, both SNAIL and SLUG are expressed in the corpora lutea. During early embryo cleavage, SNAIL occurs in the nucleus and cytoplasm of the majority of the embryo, excluding the nucleolus from the germinal vesicle breakdown (GVBD) to the 8-cell stage and is then localized in the cytoplasm during the morula stage and in the nucleus during the blastocyst stage. SLUG has an identical expression pattern as SNAIL from GVBD until the morula stage, except that it is localized in the cytoplasm during the blastocyst stage. Taken together, these different localization patterns suggest that SNAIL and SLUG probably play important roles during follicular development, luteinization and early embryonic development.

  2. Nuclear and nuclear reprogramming during the first cell cycle in bovine nuclear transfer embryos

    DEFF Research Database (Denmark)

    Østrup, Olga; Petrovicova, Ida; Strejcek, Frantisek

    2009-01-01

    Abstract The immediate events of genomic reprogramming at somatic cell nuclear transfer (SCNT) are to high degree unknown. This study was designed to evaluate the nuclear and nucleolar changes during the first cell cycle. Bovine SCNT embryos were produced from starved bovine fibroblasts and fixed......, somatic cell nuclei introduced into enucleated oocytes displayed chromatin condensation, partial nuclear envelope breakdown, nucleolar desegregation and transcriptional quiescence already at 0.5 hpa. Somatic cell cytoplasm remained temporally attached to introduced nucleus and nucleolus was partially...... restored indicating somatic influence in the early SCNT phases. At 1-3 hpa, chromatin gradually decondensed toward the nucleus periphery and nuclear envelope reformed. From 4 hpa, the somatic cell nucleus gained a PN-like appearance and displayed NPBs suggesting ooplasmic control of development....

  3. Vitrification of in vitro-produced bovine embryos matured inmodified TCM-199 medium

    OpenAIRE

    SANDAL, ASİYE İZEM; ÖZDAŞ, Özen Banu

    2015-01-01

    In vitro-produced bovine embryos matured in modified Tissue Culture Medium 199 (TCM-199) were vitrified at the 7th and 8th days of culture, and development at 48 h after warming was recorded. Ovaries were obtained from a local abattoir, and the oocytes were recovered by slicing method and divided into two main groups. The first group included cysteamine in the TCM-199 (Group A) and the second did not (Group B). They were incubated for 24 h at 38.8 °C in an atmosphere of 5% CO2 in humidified a...

  4. Efficient introgression of allelic variants by embryo-mediated editing of the bovine genome.

    Science.gov (United States)

    Wei, Jingwei; Wagner, Stefan; Lu, Dan; Maclean, Paul; Carlson, Daniel F; Fahrenkrug, Scott C; Laible, Götz

    2015-07-09

    The recent development of designer nucleases allows for the efficient and precise introduction of genetic change into livestock genomes. Most studies so far have focused on the introduction of random mutations in cultured cells and the use of nuclear transfer to generate animals with edited genotypes. To circumvent the intrinsic uncertainties of random mutations and the inefficiencies of nuclear transfer we directed our efforts to the introduction of specific genetic changes by homology-driven repair directly in in vitro produced embryos. Initially, we injected zinc finger nuclease (ZFN)-encoding mRNA or DNA into bovine zygotes to verify cleavage activity at their target site within the gene for beta-lactoglobulin (LGB) and detected ZFN-induced random mutations in 30% to 80% of embryos. Next, to precisely change the LGB sequence, we co-injected ZFNs or transcription activator-like effector nucleases (TALENs) with DNA oligonucleotides (ODNs). Analysis of co-injected embryos showed targeted changes in up to 33% (ZFNs) and 46% (TALENs) of blastocysts. Deep sequence analysis of selected embryos revealed contributions of the targeted LGB allele can reach 100% which implies that genome editing by zygote injections can facilitate the one-step generation of non-mosaic livestock animals with pre-designed biallelic modifications.

  5. Effect of bovine oviductal extracellular vesicles on embryo development and quality in vitro.

    Science.gov (United States)

    Lopera-Vasquez, Ricaurte; Hamdi, Meriem; Maillo, Veronica; Gutierrez-Adan, Alfonso; Bermejo-Alvarez, Pablo; Ramírez, Miguel Ángel; Yáñez-Mó, María; Rizos, Dimitrios

    2017-04-01

    The aim of this study was to evaluate the effect of extracellular vesicles (EV) from oviductal fluid (OF), either from the ampulla or isthmus, on the development and quality of in vitro-cultured bovine embryos. Zygotes were cultured in synthetic oviduct fluid (SOF + 3 mg/mL BSA) without calf serum (C- group), in the presence of 3 × 105 EV/mL from ampullary or isthmic OF at either 1 × 104 g (10 K) or 1 × 105 g (100 K), and compared with SOF + 5% FCS (C+ group). OF-EV size and concentration were assessed by electron microscopy and nanotracking analysis system. Embryo development was recorded on Days 7-9, and blastocyst quality was assessed through cryotolerance and gene expression analysis. Lower blastocyst yield was observed on Day 7 in the C- and OF-EV groups (12.0-14.3%) compared with C+ (20.6%); however, these differences were compensated at Days 8 and 9 (Day 9: 28.5-30.8%). Importantly, the survival rate of blastocysts produced with isthmic 100 K OF-EV was higher than that of C+ and C- group at 72 h after vitrification and warming (80.1 vs 34.5 and 50.5% respectively, P produced in the presence of 100 K isthmic OF-EV upregulated the water channel AQP3 and DNMT3A and SNRPN transcripts compared with the C+, with the expression in C- being intermediate. The lipid receptor LDLR was downregulated in C+ compared with all other groups. In conclusion, the addition of oviductal fluid extracellular vesicles from isthmus, to in vitro culture of bovine embryos in the absence of serum improves the development and quality of the embryos produced. © 2017 Society for Reproduction and Fertility.

  6. Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos

    Directory of Open Access Journals (Sweden)

    Page Grier P

    2009-04-01

    Full Text Available Abstract Background Successful reprogramming of a somatic genome to produce a healthy clone by somatic cells nuclear transfer (SCNT is a rare event and the mechanisms involved in this process are poorly defined. When serial or successive rounds of cloning are performed, blastocyst and full term development rates decline even further with the increasing rounds of cloning. Identifying the "cumulative errors" could reveal the epigenetic reprogramming blocks in animal cloning. Results Bovine clones from up to four generations of successive cloning were produced by chromatin transfer (CT. Using Affymetrix bovine microarrays we determined that the transcriptomes of blastocysts derived from the first and the fourth rounds of cloning (CT1 and CT4 respectively have undergone an extensive reprogramming and were more similar to blastocysts derived from in vitro fertilization (IVF than to the donor cells used for the first and the fourth rounds of chromatin transfer (DC1 and DC4 respectively. However a set of transcripts in the cloned embryos showed a misregulated pattern when compared to IVF embryos. Among the genes consistently upregulated in both CT groups compared to the IVF embryos were genes involved in regulation of cytoskeleton and cell shape. Among the genes consistently upregulated in IVF embryos compared to both CT groups were genes involved in chromatin remodelling and stress coping. Conclusion The present study provides a data set that could contribute in our understanding of epigenetic errors in somatic cell chromatin transfer. Identifying "cumulative errors" after serial cloning could reveal some of the epigenetic reprogramming blocks shedding light on the reprogramming process, important for both basic and applied research.

  7. Expression and distribution of cell adhesion-related proteins in bovine parthenogenetic embryos: The effects of oocyte vitrification.

    Science.gov (United States)

    Zeng, Yan; Fu, Xiangwei; Zhou, Guangbin; Yue, Mingxing; Zhou, Yanhua; Zhu, Shien

    2013-07-01

    The objective was to investigate expression of cell adhesion-related proteins (E-cadherin, β-catenin, and the cytoskeletal protein F-actin) in bovine parthenogenetic embryos derived from vitrified-warmed oocytes. Bovine oocytes at metaphase II were randomly allocated into three groups: (1) untreated (control); (2) exposed to vitrification solution without freezing (toxicity); and (3) vitrified and warmed by the open-pulled straw method (vitrification). After parthenogenetic activation, in the vitrification group compared with the control, the timing of compaction was delayed in (108-120 vs. 96-108 hours, respectively), and the percentage of blastocysts that developed from eight-cell embryos was lower (32.08% vs. 61.03%; P vitrification delayed embryo compaction by affecting adhesion junction formation and function, immunostaining and quantitative reverse transcription polymerase chain reaction were done to characterize distribution patterns (E-cadherin, β-catenin, and the cytoskeletal protein F-actin) and expression levels of cell adhesion-related proteins (β-catenin). Distribution of β-catenin in eight-cell embryos from the vitrification group changed dramatically compared with the control and toxicity groups. Relative expression of β-catenin at the mRNA and protein levels was lower (P bovine parthenogenetic eight-cell embryos derived from vitrified-warmed oocytes were associated with embryo compaction and reduced competence for subsequent embryo development. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. Homologous recombination and non-homologous end-joining repair pathways in bovine embryos with different developmental competence

    Energy Technology Data Exchange (ETDEWEB)

    Henrique Barreta, Marcos [Universidade Federal de Santa Catarina, Campus Universitario de Curitibanos, Curitibanos, SC (Brazil); Laboratorio de Biotecnologia e Reproducao Animal-BioRep, Universidade Federal de Santa Maria, Santa Maria, RS (Brazil); Garziera Gasperin, Bernardo; Braga Rissi, Vitor; Cesaro, Matheus Pedrotti de [Laboratorio de Biotecnologia e Reproducao Animal-BioRep, Universidade Federal de Santa Maria, Santa Maria, RS (Brazil); Ferreira, Rogerio [Centro de Educacao Superior do Oeste-Universidade do Estado de Santa Catarina, Chapeco, SC (Brazil); Oliveira, Joao Francisco de; Goncalves, Paulo Bayard Dias [Laboratorio de Biotecnologia e Reproducao Animal-BioRep, Universidade Federal de Santa Maria, Santa Maria, RS (Brazil); Bordignon, Vilceu, E-mail: vilceu.bordignon@mcgill.ca [Department of Animal Science, McGill University, Ste-Anne-De-Bellevue, QC (Canada)

    2012-10-01

    This study investigated the expression of genes controlling homologous recombination (HR), and non-homologous end-joining (NHEJ) DNA-repair pathways in bovine embryos of different developmental potential. It also evaluated whether bovine embryos can respond to DNA double-strand breaks (DSBs) induced with ultraviolet irradiation by regulating expression of genes involved in HR and NHEJ repair pathways. Embryos with high, intermediate or low developmental competence were selected based on the cleavage time after in vitro insemination and were removed from in vitro culture before (36 h), during (72 h) and after (96 h) the expected period of embryonic genome activation. All studied genes were expressed before, during and after the genome activation period regardless the developmental competence of the embryos. Higher mRNA expression of 53BP1 and RAD52 was found before genome activation in embryos with low developmental competence. Expression of 53BP1, RAD51 and KU70 was downregulated at 72 h and upregulated at 168 h post-insemination in response to DSBs induced by ultraviolet irradiation. In conclusion, important genes controlling HR and NHEJ DNA-repair pathways are expressed in bovine embryos, however genes participating in these pathways are only regulated after the period of embryo genome activation in response to ultraviolet-induced DSBs.

  9. Transgenerational developmental effects and genomic instability after X-irradiation of preimplantation embryos: Studies on two mouse strains

    Energy Technology Data Exchange (ETDEWEB)

    Jacquet, P., E-mail: pjacquet@sckcen.be [Molecular and Cellular Biology, Institute for Environment, Health and Safety, SCK.CEN, Boeretang 200, B-2400 Mol (Belgium); Buset, J.; Neefs, M. [Molecular and Cellular Biology, Institute for Environment, Health and Safety, SCK.CEN, Boeretang 200, B-2400 Mol (Belgium); Vankerkom, J. [Division of Environmental Research, VITO, Boeretang 200, B-2400 Mol (Belgium); Benotmane, M.A.; Derradji, H. [Molecular and Cellular Biology, Institute for Environment, Health and Safety, SCK.CEN, Boeretang 200, B-2400 Mol (Belgium); Hildebrandt, G. [Department of Radiotherapy and Radiation Oncology, University of Leipzig, Stephanstrasse 9a, D-04103 Leipzig (Germany); Department of Radiotherapy, University of Rostock, Suedring 75, D-18059 Rostock (Germany); Baatout, S. [Molecular and Cellular Biology, Institute for Environment, Health and Safety, SCK.CEN, Boeretang 200, B-2400 Mol (Belgium)

    2010-05-01

    Recent results have shown that irradiation of a single cell, the zygote or 1-cell embryo of various mouse strains, could lead to congenital anomalies in the fetuses. In the Heiligenberger strain, a link between the radiation-induced congenital anomalies and the development of a genomic instability was also suggested. Moreover, further studies showed that in that strain, both congenital anomalies and genomic instability could be transmitted to the next generation. The aim of the experiments described in this paper was to investigate whether such non-targeted transgenerational effects could also be observed in two other radiosensitive mouse strains (CF1 and ICR), using lower radiation doses. Irradiation of the CF1 and ICR female zygotes with 0.2 or 0.4 Gy did not result in a decrease of their fertility after birth, when they had reached sexual maturity. Moreover, females of both strains that had been X-irradiated with 0.2 Gy exhibited higher rates of pregnancy, less resorptions and more living fetuses. Additionally, the mean weight of living fetuses in these groups had significantly increased. Exencephaly and dwarfism were observed in CF1 fetuses issued from control and X-irradiated females. In the control group of that strain, polydactyly and limb deformity were also found. The yields of abnormal fetuses did not differ significantly between the control and X-irradiated groups. Polydactyly, exencephaly and dwarfism were observed in fetuses issued from ICR control females. In addition to these anomalies, gastroschisis, curly tail and open eye were observed at low frequencies in ICR fetuses issued from X-irradiated females. Again, the frequencies of abnormal fetuses found in the different groups did not differ significantly. In both CF1 and ICR mouse strains, irradiation of female zygotes did not result in the development of a genomic instability in the next generation embryos. Overall, our results suggest that, at the moderate doses used, developmental defects

  10. Successful pregnancy outcome after in vitro fertilisation following Pre-implantation Genetic Diagnosis/Polymerase Chain Reaction screening for single gene disorder (sickle cell anaemia) before embryo transfer: The clinical experience of an in vitro fertilisation clinic in Nigeria.

    Science.gov (United States)

    Okeke, Chizara; Ailoje-Ibru, Kemi; Olukoya, Kemi; Ogbeche, Rose; Adewusi, Abiola; Iloabachie, Ebele; Ashiru, Oladapo

    2014-01-01

    A couple, both carriers of the sickle cell anaemia trait (Genotype HbAS) with an offspring already affected with the genetic disease underwent a Pre-implantation Genetic Diagnosis/Polymerase Chain Reaction screening of biopsied blastomeres. DNA analysis of single blastomeres was carried out to find out indicated a viable intra-uterine pregnancy with embryos which carried the sickle cell mutation, which resulted in a livebirth (HbAS). PGD/PCR in combination with IVF appears to be the most suitable treatment plan for patients who are at a higher risk of reproducing offspring affected with inheritable genetic diseases.

  11. Successful pregnancy outcome after in vitro fertilisation following Pre-implantation Genetic Diagnosis/Polymerase Chain Reaction screening for single gene disorder (sickle cell anaemia) before embryo transfer: The clinical experience of an in vitro fertilisation clinic in Nigeria

    OpenAIRE

    Chizara Okeke; Kemi Ailoje-Ibru; Kemi Olukoya; Rose Ogbeche; Abiola Adewusi; Ebele Iloabachie; Oladapo Ashiru

    2014-01-01

    A couple, both carriers of the sickle cell anaemia trait (Genotype HbAS) with an offspring already affected with the genetic disease underwent a Pre-implantation Genetic Diagnosis/Polymerase Chain Reaction screening of biopsied blastomeres. DNA analysis of single blastomeres was carried out to find out indicated a viable intra-uterine pregnancy with embryos which carried the sickle cell mutation, which resulted in a livebirth (HbAS). PGD/PCR in combination with IVF appears to be the most suit...

  12. Whole Genome Amplification of Day 3 or Day 5 Human Embryos Biopsies Provides a Suitable DNA Template for PCR-Based Techniques for Genotyping, a Complement of Preimplantation Genetic Testing

    Directory of Open Access Journals (Sweden)

    Elizabeth Schaeffer

    2017-01-01

    Full Text Available Our objective was to determine if whole genome amplification (WGA provides suitable DNA for qPCR-based genotyping for human embryos. Single blastomeres (Day 3 or trophoblastic cells (Day 5 were isolated from 342 embryos for WGA. Comparative Genomic Hybridization determined embryo sex as well as Trisomy 18 or Trisomy 21. To determine the embryo’s sex, qPCR melting curve analysis for SRY and DYS14 was used. Logistic regression indicated a 4.4%, 57.1%, or 98.8% probability of a male embryo when neither gene, SRY only, or both genes were detected, respectively (accuracy = 94.1%, kappa = 0.882, and p<0.001. Fluorescent Capillary Electrophoresis for the amelogenin genes (AMEL was also used to determine sex. AMELY peak’s height was higher and this peak’s presence was highly predictive of male embryos (AUC = 0.93, accuracy = 81.7%, kappa = 0.974, and p<0.001. Trisomy 18 and Trisomy 21 were determined using the threshold cycle difference for RPL17 and TTC3, respectively, which were significantly lower in the corresponding embryos. The Ct difference for TTC3 specifically determined Trisomy 21 (AUC = 0.89 and RPL17 for Trisomy 18 (AUC = 0.94. Here, WGA provides adequate DNA for PCR-based techniques for preimplantation genotyping.

  13. Effect of addition of hyaluronan to embryo culture medium on survival of bovine embryos in vitro following vitrification and establishment of pregnancy after transfer to recipients.

    Science.gov (United States)

    Block, J; Bonilla, L; Hansen, P J

    2009-04-15

    Two experiments were conducted to determine whether addition of hyaluronan to culture medium could improve survival of bovine embryos after vitrification or following embryo transfer. In Experiment 1, embryos were produced in vitro and cultured for 7 days in modified synthetic oviductal fluid (SOF) containing one of four concentrations of hyaluronan (0, 0.1, 0.5, or 1mg/mL), with or without 4 mg/mL of bovine serum albumin (BSA). On Day 7 after insemination, blastocysts and expanded blastocysts were vitrified using open-pulled straws. At a concentration of 1mg/mL, hyaluronan increased (Pembryo hatching rate at 24 and 72 h. Treatment with BSA caused a slight reduction in cleavage rate (Pembryos were produced in vitro and cultured in modified SOF containing 4 mg/mL BSA, with or without 1mg/mL hyaluronan. At 159-162 h after insemination, grade 1 morula, blastocysts and expanded blastocysts were harvested for embryo transfer. Harvested embryos were transferred individually to lactating Holstein recipients with a palpable corpus luteum on Day 7 after presumptive ovulation. There was an interaction (Pembryo stage on pregnancy rate. Recipients that received morula and blastocyst stage embryos treated with hyaluronan had a higher pregnancy rate than recipients that received control embryos of the same stage. There was no effect of hyaluronan on pregnancy rates of recipients that received expanded blastocysts. In conclusion, addition of hyaluronan to embryo culture enhanced blastocyst yield, improved survival following vitrification, and enhanced the post-transfer survival of fresh morula and blastocyst stage embryos.

  14. Nucleologenesis and embryonic genome activation are defective in interspecies cloned embryos between bovine ooplasm and rhesus monkey somatic cells

    Directory of Open Access Journals (Sweden)

    Han Yong-Mahn

    2009-07-01

    Full Text Available Abstract Background Interspecies somatic cell nuclear transfer (iSCNT has been proposed as a tool to address basic developmental questions and to improve the feasibility of cell therapy. However, the low efficiency of iSCNT embryonic development is a crucial problem when compared to in vitro fertilization (IVF and intraspecies SCNT. Thus, we examined the effect of donor cell species on the early development of SCNT embryos after reconstruction with bovine ooplasm. Results No apparent difference in cleavage rate was found among IVF, monkey-bovine (MB-iSCNT, and bovine-bovine (BB-SCNT embryos. However, MB-iSCNT embryos failed to develop beyond the 8- or 16-cell stages and lacked expression of the genes involved in embryonic genome activation (EGA at the 8-cell stage. From ultrastructural observations made during the peri-EGA period using transmission electron microscopy (TEM, we found that the nucleoli of MB-iSCNT embryos were morphologically abnormal or arrested at the primary stage of nucleologenesis. Consistent with the TEM analysis, nucleolar component proteins, such as upstream binding transcription factor, fibrillarin, nucleolin, and nucleophosmin, showed decreased expression and were structurally disorganized in MB-iSCNT embryos compared to IVF and BB-SCNT embryos, as revealed by real-time PCR and immunofluorescence confocal laser scanning microscopy, respectively. Conclusion The down-regulation of housekeeping and imprinting genes, abnormal nucleolar morphology, and aberrant patterns of nucleolar proteins during EGA resulted in developmental failure in MB-iSCNT embryos. These results provide insight into the unresolved problems of early embryonic development in iSCNT embryos.

  15. In vitro assessment of a direct transfer vitrification procedure for bovine embryos.

    Science.gov (United States)

    Campos-Chillòn, L F; Walker, D J; de la Torre-Sanchez, J F; Seidel, G E

    2006-04-01

    We developed a simple vitrification technique for bovine embryos that could permit direct transfer. Embryos were produced in-vitro by standard procedures. The base medium for cryopreservation was a chemically defined medium similar to SOF + 25 mM Hepes and 0.25% fatty acid free bovine serum albumin (FAF-BSA) (HCDM2). In experiment 1, embryos were first exposed to 3.5M ethylene glycol (V1) for 1, 2 or 3 min at room temperature (20-24 degrees C), and then moved to 7 M ethylene glycol (V2) at 4 or 20-24 degrees C and loaded in 0.25-mL straws. After 45 s in 7 M ethylene glycol, straws were placed in liquid nitrogen. Embryos that were loaded at 20-24 degrees C had higher survival rates than those loaded at 4 degrees C (Pembryos survived well after 15 min in straws if warmed at 37 degrees C. In experiment 3, ethylene glycol concentration (3, 4 or 5 M) and exposure time (0.5 or 1 min) during two-step addition of cryoprotectant were studied for bovine morulae. In experiment 4, morulae were exposed to V2 for 30 or 45 s in HCDM2 or Vigro holding medium and then held in 22-24 degrees C air or 37 degrees C water post-warming. Experiment 5 was like experiment 4 except blastocysts were used. Overall survival rates of blastocysts in experiment 5 averaged 80% of non-vitrified controls after 48 h culture. The survival rates with in vitro-produced morulae in experiments 1, 3 and 4 were unacceptable. Vitrification solutions based on Vigro tended to result in higher survival than HCDM2 for blastocysts, but not morulae. In experiment 6, the survival rate in vitro of in vivo-produced morulae and blastocysts after two-step vitrification was nearly 100%. Our vitrification technique was very effective for in vitro produced blastocysts, but not for in vitro-produced morulae.

  16. In vitro maturation and embryo development of bovine oocytes after meiosis blockage with MPF inhibitors

    Directory of Open Access Journals (Sweden)

    Mariana Groke Marques

    2011-12-01

    Full Text Available This study evaluated the bovine oocyte maturation and embryo development after in vitro fertilization. The maturation of the oocytes was blocked using Butyrolactone I and Roscovitine using pre-maturation medium supplemented with fetal calf serum (FCS. The ocytes were divided in four groups: Control 0 hour, Control (24 hours of maturation, Roscovitine (maturation blockage with 50mM Roscovitine during 24 hours followed by 24 hours of maturation, and Butyrolactone I (maturation blockage with 150mM Butyrolactone I during 24 hours followed by 24 hours of maturation. The oocytes were fixed and stained with aceto orcein to evaluate the nuclear maturation. After the maturation period, the remaining oocytes of the Control group, Roscovitine, and Butyrolactone I were fertilized in vitro. Embryo development was assessed by the cleavage rate (D3 and blastocysts formation (D7. The Butyrolactone I group had similar rates of germinal vesical stage oocytes during blockage, and Metaphase 2 after maturation, comparing to Control group at 0 hour and Control group, respectively. On the other hand, the Roscovitine group had lower rates of vesical stage oocytes during blockage, and Metaphase 2 after maturation comparing to Control groups. After in vitro fertilization, higher rates of cleavage were observed in Control and Butyrolactone I groups. For the blastocyst formation rate, the Control group showed better results than Roscovitine group. In summary, Butyrolactone I group had similar results to the Control group, and for this reason, is suitable for pre-maturation of bovine oocytes using FCS. In contrast, Roscovitine group had lower oocyte maturation and embryo development.

  17. Oil-free culture system for in vitro bovine embryo production

    Directory of Open Access Journals (Sweden)

    Paulo B.D. Gonçalves

    2010-04-01

    Full Text Available The use of oil to avoid water evaporation from cell culture has several disadvantages, amongst which there is the migration of compounds from media to oil and from oil to media. The aim of this study was to evaluate the osmolality of a culture system using four-well plates with water in the central hole as an alternative to in vitro bovine embryo production (IVP. In addition, the osmolality changes of the oocyte washing medium were assessed in 35mm dishes with or without 2 mL of silicon oil overlay. Osmolality of oocyte washing medium changed a great deal over time after 60 minutes on a 39°C heated plate (291 mOsm kg-1, which was not detected when the medium was overlaid with silicon oil (280 mOsm kg-1; P0.05. Blastocyst rates were higher when embryos were cultured in presence of water or oil (29.7 and 29.9% for water and 33% in oil conventional microdrop system, except in the group that oocytes were washed in hyperosmotic washing medium (15.1%; P<0.05. Groups cultured in absence of water in the central hole had lower blastocyst rates (P<0.05 independently of exposure (15.5% or not (16.2 and 16.8% to hyperosmotic washing medium. In conclusion, four-well plates with water in the central hole can be an alternative to replace oil overlay for bovine IVP, maintaining stable osmolality and embryo development rates.

  18. In vitro and in vivo quality of bovine embryos in vitro produced with sex-sorted sperm.

    Science.gov (United States)

    Trigal, B; Gómez, E; Caamaño, J N; Muñoz, M; Moreno, J; Carrocera, S; Martín, D; Diez, C

    2012-10-15

    In this work we analyzed the effects of three culture systems on developmental ability of bovine embryos in vitro produced with sexed sperm, the survival to vitrification (cryologic vitrification method) of such blastocysts, and their pregnancy rates after embryo transfer to recipients, both as fresh and after vitrification/warming. Finally, we measured the accuracy of the sorting protocol by a polymerase chain reaction-based method to validate the embryo sex at blastocyst stages. We confirmed an individual effect of the bull as well as development rates of embryos produced with sorted sperm lower than embryos with unsorted sperm, independent of the culture system used. The cryoresistance to vitrification of embryos produced with sexed sperm did not differ from that of conventionally produced embryos (re-expansion rates at 24 and 48 h: 74.6% vs. 75.5%, and 64.5% vs. 68.1% for embryos produced with conventional and sorted sperm, respectively; hatching rates at 48 h: 63.55% vs. 55.5% for embryos produced with conventional and sorted sperm, respectively). Finally, no significant differences were found in pregnancy rates after the embryo transfer of fresh and vitrified/warmed blastocysts (52.8% vs. 42.0%, respectively; P > 0.05). Male and female embryos produced with sorted sperm showed the same quality in terms of developmental ability, cryoresistance, and pregnancy rates after transfer. Our culture system, coupled with the vitrification in fiber plugs, provides good quality sex-known embryos which survive vitrification at similar rates than embryos produced with conventional unsorted sperm; also it produces good pregnancy rates after transfer of sexed embryos both fresh and after vitrification and warming. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Lipid content and cryotolerance of in vitro-produced bovine embryos treated with forskolin before vitrification

    Directory of Open Access Journals (Sweden)

    Melissa Meneghel

    Full Text Available ABSTRACT: The aim of the present study was to evaluate the intracytoplasmic lipid content, development and cryotolerance of in vitro-produced bovine embryos treated with different concentrations of forskolin before vitrification. Embryos were produced from abattoir-derived ovaries and allocated into four groups. In the treatment groups, forskolin was added to the in vitro culture medium on Day 6 and incubated for 24 hours in one of the following concentrations: 2.5μM (Forsk 2.5 group, 5.0μM (Forsk 5.0 group or 10.0μM (Forsk 10.0 group. Embryos from the control group were cultured without forskolin. On Day 7 of culture, the expanded blastocysts were stained with the lipophilic dye Sudan Black B for determination of the intracytoplasmic lipid content or were cryopreserved via the Vitri-Ingá® procedure. Although there were no significant differences (P>0.05 in the blastocyst rates between the Control group (44.9% and the other treatments, the embryo production was lower (P0.05 to that found in Forsk 2.5 (0.92±0.03 and Forsk 10.0 groups (1.06±0.03 groups; however the lipid accumulation in blastocysts from Forsk 5.0 group (0.82±0.04 was lower than in the Control group (P<0.05. Based on these results, Forsk 5.0 treatment was tested for cryotolerance and it was observed that the blastocoel re-expansion rate evaluated 24 hours after warming was greater (P<0.05 in Forsk 5.0 group (72.2% compared to the Control group (46.2%. In conclusion, pre-treatment with forskolin at a concentration of 5.0 μM for 24 hours before vitrification is effective in reducing the intracytoplasmic lipid content and, consequently, improves cryotolerance of IVP bovine embryos.

  20. Osmotic challenge and expression of aquaporin 3 and Na/K ATPase genes in bovine embryos produced in vitro.

    Science.gov (United States)

    Camargo, Luiz Sergio Almeida; Boite, Mariana Cortes; Wohlres-Viana, Sabine; Mota, Gustavo Bruno; Serapiao, Raquel Varela; Sa, Wanderlei Ferreira; Viana, Joao Henrique Moreira; Nogueira, Luiz Altamiro Garcia

    2011-12-01

    The aim of this study was to evaluate the effect of culture media and stage of development in the osmotic ability of in vitro-fertilized bovine embryos and the expression of aquaporin 3 (Aqp3) and Na/K ATPase isoform 1 (ATPAse1) genes in embryos (i) with different ability to undergo rehydration and (ii) following vitrification. In experiment 1, in vitro fertilized presumptive zygotes were co-cultured in SOFaac or modified CR2aa medium and embryos at blastocyst and expanded blastocyst stages at day 7 post-insemination were exposed to NaCl hypertonic medium (900 mOsm) for 5 min following 120 min of culture in isotonic medium in order to evaluate dehydration and rehydration, respectively. No difference (P>0.05) on blastocyst rate was found between CR2aa and SOFaac medium but embryos co-cultured in SOFaac medium underwent greater (PEmbryos at expanded blastocyst stage underwent greater dehydration but slower rehydration than embryos at blastocysts stage (P0.05) on relative amount of transcripts was found in either genes. In the experiment 3, expanded blastocysts produced in a co-culture system were vitrified, warmed and then cultured for 72 h for analysis of embryo survival and amount of Aqp3 and ATPase1 transcripts. Lower (Pembryo survival rate was found for vitrified-warmed embryos (57.9%) than for their fresh counterparts (84.6%). There was no difference on expression of ATPase1 gene but lower (Pembryos. In conclusion, embryo ability to undergo shrinkage and swelling is influenced by medium used in a co-culture system and by embryo stage. Rehydrating ability of embryos after exposure to NaCl hypertonic medium is not associated with variations on expression of Aqp3 and ATPase1 genes, but the vitrification can alter gene expression of in vitro-fertilized bovine embryos produced in a co-culture system. Copyright © 2011 Elsevier Inc. All rights reserved.

  1. Effects of oocyte vitrification on epigenetic status in early bovine embryos.

    Science.gov (United States)

    Chen, Huanhuan; Zhang, Lei; Deng, Tengfei; Zou, Pengda; Wang, Yongsheng; Quan, Fusheng; Zhang, Yong

    2016-08-01

    Oocyte cryopreservation has a great impact on subsequent embryonic development. Currently, several studies have primarily focused on the consequences of vitrification and the development potential of cellular structures. This study determined whether oocyte vitrification caused epigenetic instabilities of bovine embryos. The effects of oocyte vitrification on DNA methylation, histone modifications, and putative imprinted genes' expression in early embryos derived by intracytoplasmic sperm injection were examined. Results showed that oocyte vitrification did not affect zygote cleavage rates (67.0% vs. 73.8% control, P > 0.05) but reduced the blastocyst rate (9.6% vs. 23.0%, P vitrification group during the early cleavage phases. No differences were observed for DNA methylation, H3K9me3, and acH3K9 in the inner cell mass of blastocysts, whereas decreased levels of DNA methylation and acH3K9 (P vitrification. The expression of putative-imprinted genes PEG10, XIST, and KCNQ1O1T was upregulated in blastocysts. These epigenetic abnormalities may be partially explained by altered expression of genes associated with epigenetic regulations. DNA methylation and H3K9 modification suggest that oocyte vitrification may excessively relax the chromosomes of oocytes and early cleavage embryos. In conclusion, these epigenetic indexes could be used as damage markers of oocyte vitrification during early embryonic development, which offers a new insight to assess oocyte vitrification. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. New device for the vitrification and in-straw warming of in vitro produced bovine embryos.

    Science.gov (United States)

    Morató, Roser; Mogas, Teresa

    2014-04-01

    Two experiments were designed to test the use of a new device designed to vitrify and in-straw warm in vitro produced (IVP) embryos, which can potentially be used for their direct transfer to recipient females in field conditions. In experiment 1, IVP embryos from both prepubertal and adult animals were vitrified on cryotops and warmed in steps (1, 0.5 and 0M sucrose; protocol W3) or directly in 0.5M (protocol W1/0.5) or 0M sucrose (protocol W1/0). Similar survival rates were recorded 24h after warming for calf embryos irrespective of the warming procedure (W3: 79.2%, W1/0.5: 62.5%, W1/0: 66.7%). For cow embryos, survival rates at 24h post-warming were significantly higher when embryos were warmed using the W3 (85.7%) or W1/0.5 (89.1%) protocols compared to the W1/0 protocol (70.5%). In experiment 2, IVP embryos were vitrified on the new designed device followed by their in-straw cryoprotectant (0.5M sucrose) dilution/warming and different warming temperatures (45, 50, 60 and 70°C) were tested. When warming solution passed through the new vitrification/warming device at 45°C, 61.5% of blastocysts were fully re-expanded or hatched at 24h post-warming, being not significantly different to the control (65%). Other warming temperatures triggered significantly lower survival rates at 24h post-warming. No significant differences were detected in total cell numbers and blastocyst apoptosis indices in response to vitrification followed by warming at 45°C respect to the control. Our findings indicate that the new device allows vitrification and in-straw warming of IVP bovine embryos, being a useful option for their direct transfer in field conditions. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Cryotop vitrification for in vitro produced bovine and buffalo (Bubalus bubalis) embryos at different stages of development

    OpenAIRE

    B. Gasparrini; G. Campanile; D. Vecchio; L. Boccia; L. Attanasio; De Rosa, A.

    2010-01-01

    The aim of this study was to evaluate the possibility to vitrify in vitro produced (IVP) buffalo and bovine embryos at different stages of development by an advanced version of the “minimal volume approaches”: the Cryotop method. In both experiments, the embryos were vitrified at the tight morula (TM), early blastocyst (eBl), blastocyst (Bl), expanded blastocyst (xBl) and, only for buffalo, at the hatched blastocyst (hBl) stage. After warming, the embryos were cultured in vitro fo...

  4. Reprogenetics: preimplantational genetics diagnosis

    Directory of Open Access Journals (Sweden)

    Roberto Coco

    2014-01-01

    Full Text Available Preimplantational Genetics Diagnosis (PGD is requested by geneticists and reproductive specialists. Usually geneticists ask for PGD because one or both members of the couple have an increased genetic risk for having an affected offspring. On the other hand, reproductive specialists ask for embryo aneuploidy screening (PGS to assures an euploid embryo transfer, with the purpose to achieve an ongoing pregnancy, although the couple have normal karyotypes. As embryonic aneuploidies are responsible for pre and post implantation abortions, it is logical to considerer that the screening of the embryonic aneuploidies prior to embryo transfer could improve the efficiency of the in vitro fertilization procedures. Nevertheless, it is still premature to affirm this until well-designed clinical trials were done, especially in women of advanced age where the rate of embryos with aneuploidies is much greater. Although the indications of PGD are similar to conventional prenatal diagnosis (PND, PGD has less ethical objections than the PND. As with the PGD/PGS results only unaffected embryos are transferred, both methods can avoid the decision to interrupt the pregnancy due to a genetic problem; this makes an important difference when compared to conventional prenatal diagnosis.

  5. Survival and developmental competence of bovine embryos at different developmental stages and separated blastomeres after vitrification in different solutions.

    Science.gov (United States)

    Juanpanich, Theesit; Suttirojpattana, Tayita; Takayama, Mari; Liang, Yuanyuan; Dochi, Osamu; Parnpai, Rangsun; Imai, Kei

    2018-01-01

    Generating techniques to enhance the success of blastomere separation is important for bovine economy, because it increases the number of transferable embryos. This study aimed to identify the optimum cryoprotectants for the vitrification of bovine embryos and the separation of blastomeres at different stages. In experiment 1, expanded blastocysts were vitrified in two different vitrification solutions, either (1) ethylene glycol (EG) + propylene glycol (PG) or (2) EG. The survival rate of blastocysts in the EG + PG was higher than that of the EG. In experiment 2, intact two-cell and eight-cell stage embryos were vitrified in the same solutions used in experiment 1. The EG + PG produced more dead embryos than the EG (P < 0.05). In the EG, the rate of blastocyst formation was similar for the vitrified two- and eight-cell embryos and the non-vitrified ywo-cell embryos. In experiment 3, separated blastomeres of two- and eight-cell embryos were vitrified in EG. There was no difference in the rate of blastocyst formation and total number of cells between the two vitrified groups. In summary, at the blastocyst stage, EG + PG was superior, based on both survival rates and cell numbers; however, at the 2-8 cell stage, the use of EG alone was better than the other groups. © 2017 Japanese Society of Animal Science.

  6. Vitrification of in vitro produced bovine embryos at different ages using one- and three-step addition of cryoprotective additives.

    Science.gov (United States)

    Saha, S; Suzuki, T

    1997-01-01

    The effect of embryo age on development and ratio of live: dead cells after vitrification and warming was examined. One-step and three-step addition of cryoprotectants in vitrification solution (40% ethylene glycol, 0.3 M trehalose and 12% polyvinylpyrrolidone) were compared using in vitro produced (IVP) bovine blastocysts and expanded blastocysts. Rates of development and hatching were 74.2% and 41.9% for Day 7, 57.8% and 23.8% for Day 8, 33.7% and 6.1% for Day 9 embryos with one-step addition. In three-step addition, those rates were 86.2% and 77.3% for Day 7, 72.3% and 39.0% for Day 8, 47.3% and 10.5% for Day 9 embryos. Day 7 embryos showed highest (P embryos with three-step addition was higher (P embryos (P bovine embryos against vitrification and the potential for three-step addition of cryoprotectants to yield a higher survival rate after warming than with one-step addition.

  7. Effect of breed and corpus luteum on pregnancy rate of bovine embryo recipients

    Directory of Open Access Journals (Sweden)

    Ériklis Nogueira

    2012-09-01

    Full Text Available The objective of this study was to evaluate pregnancy rates of recipients of different breed groups (Nellore and crossbreed, as well as the effects of size and type of the corpus luteum (CL on plasmatic concentrations of progesterone and pregnancy rates of embryo recipients. A total of 152 heifers were synchronized with progesterone implants and on the day of embryo transfer, previously obtained by superovulation and frozen in ethylene glycol, the diameter and type of the corpus luteum (cavitary and compact was measured and blood was collected for progesterone measurement. The pregnancy rate was 44.1%, with a diameter of corpus luteum higher in recipients that became pregnant (2.03±0.41 compared with non-pregnant ones (1.86±0.34 cm. Plasmatic concentrations of progesterone did not differ between pregnant (1.50±1.05 and non-pregnant (1.31±0.91 ng/mL animals. The type of corpus luteum did not influence the pregnancy rates. Only Angus and crossbred Marchigiana differ among themselves in pregnancy rates (33.3 and 59.2%, respectively. The pregnancy probability was affected only by CL diameter, but not by P4 plasmatic concentration. Selection of the corpus luteum size at the time of embryo transfer is an important factor to increase pregnancy rates in recipients, and compact and cavitary corpora lutea do not influence the pregnancy rates of bovine embryo recipients. Nellore recipients have pregnancy rates that are satisfactory and comparable to crossbred (Bos taurus × Bos indicus recipients.

  8. Factors affecting the outcome of in vitro bovine embryo production using ovum pick-up-derived cumulus oocyte complexes

    NARCIS (Netherlands)

    Merton, J.S.

    2013-01-01

    Optimization of bovine ovum pick up (OPU) followed by in vitro embryo production (IVP) has been driven by the desire of both beef and dairy cattle breeders to enhance genetic improvement. The work presented in this thesis focuses on optimizing the efficiency and efficacy of the OPU-IVP program.

  9. Effect of estrous cow serum during bovine embryo culture on blastocyst development and cryotolerance after slow freezing or vitrification.

    Science.gov (United States)

    Mucci, N; Aller, J; Kaiser, G G; Hozbor, F; Cabodevila, J; Alberio, R H

    2006-05-01

    The present study investigated the effect of estrous cow serum (ECS) during culture of bovine embryos on blastocyst development and survival after cryopreservation by slow freezing or vitrification. Embryos were derived from in vitro maturation (IVM) and in vitro fertilization (IVF) of abbatoir-derived oocytes. At Day 3, embryos were cultured in three different media: Charles Ronsenkrans medium + amino acids (CR1aa; without bovine serum albumin (BSA)) + 5% estrous cow serum (CR1-ECS), CR1aa + 3 mg/mL BSA (CR1-BSA) or CR1aa + 5% ECS + 3 mg/mL BSA (CR1-ECS-BSA). At 7.5 d post-insemination (PI), blastocyst yield and quality were evaluated; blastocysts and expanded blastocysts from each media were cryopreserved by Open Pulled Straw (OPS) vitrification method or slow freezing (1.5 M ethylene glycol, EM). Total blastocyst yield did not differ among CR1-ECS, CR1-BSA and CR1-ECS-BSA (30.9, 33.1 and 32.9%, respectively, P Embryo survival (hatching rate) was higher in vitrified versus slow-frozen embryos (43% versus 12%, respectively, P embryos cultured in CR1-BSA (40.3%) compared with those cultured in serum-containing media (CR1-ECS, 21.5% and CR1-ECS-BSA, 19.8%; P produce in vitro bovine embryos in serum-free culture medium without affecting blastocyst yield and quality; (b) serum-free medium produced the best quality embryos (in terms of post-cryopreservation survival); and (c) vitrification yielded the highest post-cryopreservation survival rates, regardless of the presence of serum in the culture medium.

  10. A SIMPLE AND EFFICIENT VITRIFICATION METHOD FOR IN-STRAW DILUTION AND DIRECT TRANSFER OF BOVINE EMBRYOS.

    Science.gov (United States)

    Zhang, Youwen; Fu, Xiangwei; Chen, Long; Feng, Chuntao; Bi, Jianghua; Mo, Xianhong; Cheng, Keren; Zhang, Rina; Li, Shujing; Zhu, Shien

    2015-01-01

    An easy and user friendly protocol that produces consistent results will facilitate the commercial application of embryo vitrification technology in the field. This study was designed to develop a simple and efficient vitrification, in-straw dilution and direct transfer method for bovine embryos. After being vitrified and in-straw thawed, in vivo-derived and in vitro-produced bovine embryos were subjected to in vitro culture or embryo transplantation. There were no significant differences (P > 0.05) in survival rates (100.0% vs. 93.9%) and expansion rates (93.8% vs. 87.5%) between in vivo-derived and in vitro-produced blastocysts after vitrification and in-straw dilution. And there was also no significant difference (P > 0.05) in conception rates (56.5% vs. 58.8%) after ET between cryopreserved and fresh in vivo-derived blastocysts. Vitrification using EG-based vitrification solution and in-straw dilution with PBS-based diluent is a simple and efficient method for cryopreservation and direct transfer of bovine embryos.

  11. Validating reference microRNAs for normalizing qRT-PCR data in bovine oocytes and preimplantation embryos

    NARCIS (Netherlands)

    Mahdipour, Mahdi; van Tol, Helena T A; Stout, Tom A E; Roelen, Bernard A J

    2015-01-01

    BACKGROUND: MicroRNAs (miRNAs) are small noncoding RNAs that act as post-transcriptional regulators of gene targets. Accurate quantification of miRNA expression using validated internal controls should aid in the understanding of their role in epigenetic modification of genome function. To date,

  12. Oocyte pre-IVM with caffeine improves bovine embryo survival after vitrification.

    Science.gov (United States)

    Bernal-Ulloa, Sandra Milena; Lucas-Hahn, Andrea; Herrmann, Doris; Hadeler, Klaus-Gerd; Aldag, Patrick; Baulain, Ulrich; Niemann, Heiner

    2016-09-15

    Cryopreservation of in vitro produced bovine embryos is associated with significantly reduced survival rates, mainly due to insufficient quality of the embryos. Caffeine supplementation during IVM has been used to delay meiotic resumption and concomitantly also increased embryo quality. Here, we investigated the influence of pre-IVM with caffeine on oocyte maturation, intraoocyte cAMP concentration, developmental competence after IVF, and blastocyst cryotolerance. Oocytes were obtained by slicing of ovaries and were submitted to either 2 hours culture before IVM with or without caffeine (0, 1, 5, 10, 20, 30 mM), or standard IVM (no pre-IVM). Oocytes were in vitro matured and fertilized and zygotes were cultured under standard in vitro conditions until Day 8. Expanded blastocysts derived from either standard control or the 10-mM caffeine treatments were submitted to vitrification. Caffeine delayed meiotic resumption after 9-hour IVM in a concentration-dependent manner. The cAMP levels were similar before and after IVM. Matured oocytes, cleavage, and blastocyst rates were reduced in the 30-mM caffeine concentration and were similar among the other treatment groups. Number and proportion of inner cell mass and trophectoderm cells in blastocysts did not differ among treatments. Forty-eight hours after thawing, hatching rates were higher in the 10-mM caffeine group (73.8%) compared with the standard control (59.7%). Reexpansion rates and total number of cells after 48 hours were similar in both treatments. The ratio of live/total cells was higher in the caffeine treatment. These results suggest that caffeine supplementation before IVM delayed meiotic resumption and improved blastocyst quality shown in higher cryotolerance. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. No beneficial effect of preimplantation genetic screening in women of advanced maternal age with a high risk for embryonic aneuploidy

    NARCIS (Netherlands)

    Twisk, Moniek; Mastenbroek, Sebastiaan; Hoek, Annemieke; Heineman, Maas-Jan; van der Veen, Fulco; Bossuyt, Patrick M.; Repping, Sjoerd; Korevaar, Johanna C.

    2008-01-01

    Human preimplantation embryos generated through in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) treatments show a variable rate of numerical chromosome abnormalities or aneuploidies. Preimplantation genetic screening (PGS) has been designed to screen for aneuploidies in high

  14. Vitrification of in vitro-produced bovine embryos by addition of ethylene glycol in one-step.

    Science.gov (United States)

    Walker, D J; Campos-Chillon, L F; Seidel, G E

    2006-10-01

    The objective of this study was to simplify two-step addition of cryoprotectant for vitrification of bovine embryos by developing a one-step procedure. Survival was calculated as a percentage of non-vitrified controls developed from the same batch of oocytes. In experiment 1, bovine blastocysts were vitrified following one- or two-step addition of cryoprotectant. Exposure of embryos to cryoprotectant in one-step resulted in survival rates not significantly lower (p > 0.1) than those obtained by two-step addition (85% vs 98%, respectively). Based on these results, experiments 2-4 were designed to test one-step addition of cryoprotectant more rigorously. Experiment 2 exposed day 7 blastocysts to 6, 7 or 8 M ethylene glycol for 2.5 or 3.5 min. At 24 h post-vitrification, survival of embryos was similar, irrespective of ethylene glycol concentration or exposure time (6 M 38%, 7 M 51%, 8 M 59%; 2.5 min 54%, 3.5 min 45%). In experiment 3, blastocysts were exposed to 7 M ethylene glycol for shorter times (30 or 60 s); 30 s exposure resulted in decreased survival (8% vs 31%, p bovine morulae, exposed to 7 M ethylene glycol for 1 or 1.5 min. There was no difference in survival between exposure times of 1 or 1.5 min (28% vs 45%, respectively; p > 0.1). It is unclear why many embryos survive vitrification with one-step addition of cryoprotectant, but others do not. Although, one-step addition of cryoprotectant simplifies the vitrification procedure, survival rates were inadequate for routine cryopreservation of in vitro-produced bovine embryos.

  15. Characterization of microRNA in bovine in vitro culture media associated with embryo quality and development.

    Science.gov (United States)

    Kropp, Jenna; Khatib, Hasan

    2015-09-01

    Dairy cattle fertility has declined over time due to factors including reduced fertilization and early embryonic loss. To counter fertility problems and better study preimplantation embryonic development, in vitro production systems have been developed. These systems largely assess embryos based on their morphology, which is not a strong indicator of developmental potential. Currently, no biomarkers can be used to noninvasively survey an embryo's potential in terms of its development and ability to establish a pregnancy. Thus, the objective of this study was to characterize and identify microRNA (miRNA) in culture media of embryos of differing developmental competence for future development as noninvasive biomarkers of embryo quality. The MiRNA sequencing of media conditioned by blastocyst and degenerate (those that failed to develop from the morula to blastocyst stage) embryos, revealed 11 differentially expressed miRNA; all were higher in concentration in degenerate conditioned media. Differential expression of mature microRNA (miR)-24, miR-191, and miR-148a was further validated using quantitative real-time PCR. Functional analysis of miR-24 revealed that addition of a mimic miRNA to culture media of morulae embryos resulted in a 27.3% decrease in development to the blastocyst stage. Furthermore, expression of miR-24 was 44.29-fold higher in blastocysts cultured with a miR-24 mimic compared with control blastocysts. Interestingly, the expression of CDKN1b, a target gene of miR-24 was repressed in embryos grown in the presence of the miRNA mimic. Mimic supplementation experiments suggest that miRNA are taken up by the embryo and that extracellular miRNA affect embryonic development. Overall, identification of a rich extracellular milieu in conditioned media sets the framework for future studies to determine the long-term predictive ability of embryo-based miRNA biomarkers on pregnancy outcome. Copyright © 2015 American Dairy Science Association. Published by

  16. L-ergothioneine supplementation during culture improves quality of bovine in vitro-produced embryos.

    Science.gov (United States)

    Zullo, G; Albero, G; Neglia, G; De Canditiis, C; Bifulco, G; Campanile, G; Gasparrini, B

    2016-03-01

    The aim of this study was to evaluate whether supplementation of bovine culture medium with the natural antioxidant L-ergothioneine (LE), improves in vitro blastocyst development and quality, assessed as resistance to cryopreservation, total cells number, cellular differentiation, and apoptosis index. Abattoir-derived oocytes were matured and fertilized in vitro according to standard procedure. Twenty hours after IVF, presumptive zygotes were cultured in synthetic oviduct fluid with 0, 0.05 mM, 0.1 mM, 0.5 mM, and 1 mM of LE (experiment 1) at 39 °C under humidified air with 5% CO2, 7% O2, and 88% N2. On the basis of the results of this dose-response trial, the range of concentrations to test was reduced in experiment 2, in which presumptive zygotes were cultured with 0, 0.05 mM, and 0.1 mM of LE. On Day 7, embryo yields were assessed, and the blastocysts (BL) were vitrified by Cryotop method in 16.5% ethylene glycol, 16.5% DMSO and 0.5 M sucrose. Finally, BL produced on Day 8 in the absence (control) and presence of 0.1 mM LE were used for transferase-mediated dUTP nick end labeling and differential staining to evaluate, respectively the apoptotic rate and the allocation of cells into inner cell mass (ICM) and trophectoderm lineages (experiment 3). Despite similar blastocyst yields, supplementation of culture medium with 0.1 mM LE improved the cryotolerance of in vitro-produced (IVP) embryos compared to the control group, as indicated by higher (P culture (48.5%, 50.0%, and 63.8%, respectively with 0, 0.05, and 0.1 mM LE). Interestingly, when embryos were cultured in the presence of 0.1 mM LE, the percentage of BL with the most physiological ICM:total cells ratio (20%-40%) increased (85.1 vs. 66.0%, P culture medium with 0.1 mM LE improves embryo quality, as indicated by the improved cryotolerance, the lower apoptotic rate, and the higher percentage of BL with the most physiological ICM:total cells ratio. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Low serum concentration in bovine embryo culture enhances early blastocyst rates on Day-6 with quality traits in the expanded blastocyst stage similar to BSA-cultured embryos.

    Science.gov (United States)

    Murillo, A; Muñoz, M; Martín-González, D; Carrocera, S; Martínez-Nistal, A; Gómez, E

    2017-06-01

    In bovine, single in vitro embryo culture in protein-free medium from Day-6 to Day-7 leads to expanded blastocyst (XB) with improved pregnancy and birth rates after cryopreservation. Under these conditions, early blastocysts (EB) progress to the XB stage at higher rates than morulae (M). However, embryo production with BSA in culture prior to Day-6 leads to low EB rates. We investigated whether a very low FCS concentration (0.1%) in culture from Day-1 to Day-6 would improve EB rates and, subsequently, increase XB rates on Day-7 after single culture in protein-free medium. The quality of embryos produced was evaluated in terms of survival to cryopreservation, apoptosis percentage, lipid accumulation and transfer to recipients. On Day-6, EB rates from embryos cultured with FCS were higher than with BSA (P=0.022). On Day-7, XB rates were higher in embryos from Day-6 EB than from Day-6M, both with and without FCS (Pvitrification/warming of Day-7 XB, 100% embryos survived at 24h in all treatments, and total cell number and apoptosis percentage were not affected by the presence of FCS or embryonic stage on Day-6. Cryopreserved and fresh embryos produced with FCS until Day-6, and then deprived of protein and cultured individually, led to pregnancies after ET. In conclusion, minute FCS concentration improves EB rates on Day-6 leading, after one-day single culture without protein, to more XBs. The quality of XB produced with FCS compares well with XB produced with BSA in terms of apoptosis, lipid accumulation and pregnancy. Copyright © 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  18. Effects of different cryopreservation methods on post-thaw culture conditions of in vitro produced bovine embryos.

    Science.gov (United States)

    Nicacio, Alessandra Corallo; Simões, Renata; de Paula-Lopes, Fabiola Freitas; de Barros, Flavia Regina Oliveira; Peres, Maria Angelica; Assumpção, Mayra Elena Ortiz D'Avila; Visintin, Jose Antonio

    2012-05-01

    The aim of this work was to evaluate the effect of cryopreservation protocols on subsequent development of in vitro produced bovine embryos under different culture conditions. Expanded in vitro produced blastocysts (n = 600) harvested on days 7-9 were submitted to controlled freezing [slow freezing group: 10% ethylene glycol (EG) for 10 min and 1.2°C/min cryopreservation]; quick-freezing [rapid freezing group: 10% EG for 10 min, 20% EG + 20% glycerol (Gly) for 30 s]; or vitrification [vitrification group: 10% EG for 10 min, 25% EG + 25% Gly for 30 s] protocols. Control group embryos were not exposed to cryoprotectant or cryopreservation protocols and the hatching rate was evaluated on day 12 post-insemination. In order to evaluate development, frozen-thawed embryos were subjected to granulosa cell co-culture in TCM199 or SOFaa for 4 days. Data were analyzed by PROC MIXED model using SAS Systems for Windows®. Values were significant at p embryos cultured in TCM199, slow freezing and vitrification group hatching rates were 44.65 ± 5.94% and 9.43 ± 6.77%, respectively. In embryos cultured in SOFaa, slow freezing and vitrification groups showed hatching rates of 11.65 ± 3.37 and 8.67 ± 4.47%, respectively. In contrast, the rapid freezing group embryos did not hatch, regardless of culture medium. The slow freezing group showed higher hatching rates than other cryopreservation groups. Under such conditions, controlled freezing (1.2°C/min) can be an alternative to cryopreservation of in vitro produced bovine embryos.

  19. Effect of cysteamine supplementation of in vitro matured bovine oocytes on chilling sensitivity and development of embryos.

    Science.gov (United States)

    Balasubramanian, S; Rho, Gyu-Jin

    2007-04-01

    In vitro techniques for production of bovine embryos including in vitro oocyte maturation (IVM), fertilization (IVF) and culture (IVC) are becoming increasingly employed for a variety of research purposes. However, decreased viability following cryopreservation by conventional methods has limited commercial applications of these technologies. A practical alternative to facilitate transport would be to arrest development by chilling without freezing. The present research was undertaken to evaluate chilling sensitivity of IVM-IVF embryos at different stages of development, and to determine possible beneficial effects of cysteamine treatment during IVM, previously shown to enhance embryo development in culture, on survival following chilling at different stages. Embryos produced by standard IVM-IVF-IVC methods were chilled to 0 degrees C for 30 min at 2-cell (30-34 h post-insemination, hpi), 8-cell (48-52 hpi) or blastocyst (166-170 hpi) stages. Viability after chilling was assessed by IVC with development to expanded blastocyst stage determined on days 7 and 8 post-insemination (pi) and hatching blastocyst stage determined on days 9 and 10 pi. Control embryos at the same stages were handled similarly, but without chilling, and development during culture similarly assessed. The effect of cysteamine supplementation (100 microM) of the IVM medium was determined for both chilled and non-chilled (control) embryos. Cysteamine supplementation during IVM had no significant effect on oocyte maturation or fertilization, but increased the proportions of oocytes developing to blastocyst stage by day 7 (13.7+/-0.9% versus 7.2+/-0.9%; Pchilling of blastocysts produced by IVM-IVF of oocytes matured in media supplemented with cysteamine offers promise for applications requiring short-term storage to facilitate transport of in vitro produced bovine embryos.

  20. Production of female bovine embryos with sex-sorted sperm using intracytoplasmic sperm injection: efficiency and in vitro developmental competence.

    Science.gov (United States)

    Jo, Hyun-Tae; Bang, Jae-Il; Kim, Seong-Su; Choi, Byung-Hyun; Jin, Jong-In; Kim, Heyng-Lyool; Jung, In-Suk; Suh, Tae-Kwang; Ghanem, Nasser; Wang, Zhongde; Kong, Il-Keun

    2014-03-15

    The production of embryos with a preselected sex sperm is important in the livestock industry. In this study, we examined the efficiency of producing female embryos by intracytoplasmic sperm injection (ICSI) with flow cytometry sorted (ssICSI) and unsorted (usICSI) bovine sperm, and their developmental competence in vitro. For comparison, bovine embryos were also produced by in vitro fertilization (IVF) with sorted (ssIVF) and unsorted (usIVF) bovine sperm. The semen used in this study was from a bull selected for its high fertility and blastocyst developmental competence among four bulls. We first examined and compared pronuclear (PN) formation and cleavage rates of the produced embryos among the treatment groups. Our results demonstrated that PN formation rates (judged by two pronucleus [2PN]) and cleavage rates in ssIVF group (23.1% and 43.6%) were lower than those in the usIVF (71.1% and 71.6%), usICSI (73.1% and 92.8%) and ssICSI (75% and 79.1%) groups, respectively (P sex-sorting. Of note, we achieved a blastocyst formation rate in the ssICSI group to be comparable with the usIVF group. We then examined embryo quality by counting the number of normal and apoptotic cells in blastocysts. It was found that, despite the fact that blastocyst formation rate in the ssIVF group was significantly lower than those in the usIVF, usICSI and ssICSI groups, there was no difference in total and apoptotic cell numbers among these groups (P > 0.05). Finally, karyotyping analysis demonstrated that the proportion of female embryos in the ssICSI and ssIVF groups was 100%, whereas it was 58.8% and 57.8% in the usIVF and usICSI groups, respectively. In conclusion, ICSI with flow cytometry sorted bovine sperm provides an alternative approach to produce embryos with predetermined sex. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Association between fertilin beta, protamines 1 and 2 and spermatid-specific linker histone H1-like protein mRNA levels, fertilization ability of human spermatozoa, and quality of preimplantation embryos.

    Directory of Open Access Journals (Sweden)

    Bartosz Kempisty

    2008-04-01

    protamines contribute not only to successful fertilization, but may have an important impact in development of preimplantation embryos.

  2. In vitro maturation supplements affect developmental competence of bovine cumulus-oocyte complexes and embryo quality after vitrification.

    Science.gov (United States)

    Räty, Mervi; Ketoja, Elise; Pitkänen, Timo; Ahola, Virpi; Kananen, Kirsi; Peippo, Jaana

    2011-12-01

    Oocyte quality affects subsequent embryo development and quality. We examined the impact of bovine oocyte in vitro maturation (IVM) conditions on subsequent embryo yield, quality and cryosurvival. Cumulus-oocyte complexes (COCs) were sampled for cytological and gene expression analysis after IVM in TCM199 supplemented with 10% fetal bovine serum (FBS), 4 mg/ml of fatty-acid-free bovine serum albumin (FAFBSA), 4 mg/ml of polyvinylpyrrolidone (PVP), FAFBSA with epidermal growth factor (EGF, 100 ng/ml) and insulin-like growth factor 1 (IGF-I, 100 ng/ml) (FAFBSAGF), PVP with EGF and IGF-I (PVPGF) or PVP with single strength BME and MEM amino acids (PVPAA). The remaining COCs were fertilized. On day 7 (IVF=day 0) quality 1 blastocysts were vitrified or analyzed for glucose transporter 1 (Glut-1) expression levels. The remaining blastocysts (days 7-9) were evaluated for morphology and total cell counts. After warming, survival and hatching rates were evaluated followed by total cell counts and Glut-1 expression levels. Only PVPGF IVM resulted in embryo production rates comparable to those recorded with FBS IVM. Growth factors with FAFBSA and amino acids with PVP reduced embryo production rates whereas the effect of the growth factors with PVP was negligible. Insulin-like growth factor 2 binding protein 3 (IGF2BP3) and beta cell translocation gene 4 (BTG4) were revealed as potential candidates for oocyte developmental competence, and secreted protein, acidic and rich in cysteine (SPARC) for cumulus cell expansion. There were no differences among treatments in hatching rates of vitrified embryos after warming. However, total cell numbers and Glut-1 expression levels at 72 h were affected. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Effects of Differences in Dietary Protein and Varying the Interval from Collection of Bovine Embryos to Freezing on Embryo Quality and Viability

    OpenAIRE

    Jousan, Frank Dean

    2002-01-01

    High levels of dietary protein may be detrimental to reproductive performance in cattle. The objective of Exp. 1 was to determine the effects of differences in dietary protein on the production and quality of bovine embryos collected from superovulated donors. Angus cows were randomly assigned to receive one of three experimental diets: a daily ration of 5.7 kg poultry litter, 2.0 kg hay, 3.1 kg corn, and 0.5 kg peanut hulls (LITTER; n = 15); a daily ration of 6.2 kg peanut hulls, 2.2 k...

  4. In vitro fertilization with preimplantation genetic screening

    NARCIS (Netherlands)

    Mastenbroek, Sebastiaan; Twisk, Moniek; van Echten-Arends, Jannie; Sikkema-Raddatz, Birgit; Korevaar, Johanna C.; Verhoeve, Harold R.; Vogel, Niels E. A.; Arts, Eus G. J. M.; de Vries, Jan W. A.; Bossuyt, Patrick M.; Buys, Charles H. C. M.; Heineman, Maas Jan; Repping, Sjoerd; van der Veen, Fulco

    2007-01-01

    BACKGROUND: Pregnancy rates in women of advanced maternal age undergoing in vitro fertilization (IVF) are disappointingly low. It has been suggested that the use of preimplantation genetic screening of cleavage-stage embryos for aneuploidies may improve the effectiveness of IVF in these women.

  5. Melatonin improves the quality of in vitro produced (IVP) bovine embryos: implications for blastocyst development, cryotolerance, and modifications of relevant gene expression.

    Science.gov (United States)

    Wang, Feng; Tian, XiuZhi; Zhou, YanHua; Tan, DunXian; Zhu, ShiEn; Dai, YunPing; Liu, GuoShi

    2014-01-01

    To evaluate the potential effects of melatonin on the kinetics of embryo development and quality of blastocyst during the process of in vitro bovine embryo culture. Bovine cumulus-oocyte complexes (COCs) were fertilized after in vitro maturation. The presumed zygotes were cultured in in vitro culture medium supplemented with or without 10(-7) M melatonin. The cleavage rate, 8-cell rate and blastocyst rate were examined to identify the kinetics of embryo development. The hatched blastocyst rate, mortality rate after thawing and the relevant transcript abundance were measured to evaluate the quality of blastocyst. The results showed that melatonin significantly promoted the cleavage rate and 8-cell embryo yield of in vitro produced bovine embryo. In addition, significantly more blastocysts were observed by Day 7 of embryo culture at the presence of melatonin. These results indicated that melatonin accelerated the development of in vitro produced bovine embryos. Following vitrification at Day 7 of embryo culture, melatonin (10(-7) M) significantly increased the hatched blastocyst rate from 24 h to 72 h and decreased the mortality rate from 48 h to 72 h after thawing. The presence of melatonin during the embryo culture resulted in a significant increase in the gene expressions of DNMT3A, OCC, CDH1 and decrease in that of AQP3 after thawing. In conclusion, melatonin not only promoted blastocyst yield and accelerated in vitro bovine embryo development, but also improved the quality of blastocysts which was indexed by an elevated cryotolerance and the up-regulated expressions of developmentally important genes.

  6. Melatonin improves the quality of in vitro produced (IVP bovine embryos: implications for blastocyst development, cryotolerance, and modifications of relevant gene expression.

    Directory of Open Access Journals (Sweden)

    Feng Wang

    Full Text Available To evaluate the potential effects of melatonin on the kinetics of embryo development and quality of blastocyst during the process of in vitro bovine embryo culture. Bovine cumulus-oocyte complexes (COCs were fertilized after in vitro maturation. The presumed zygotes were cultured in in vitro culture medium supplemented with or without 10(-7 M melatonin. The cleavage rate, 8-cell rate and blastocyst rate were examined to identify the kinetics of embryo development. The hatched blastocyst rate, mortality rate after thawing and the relevant transcript abundance were measured to evaluate the quality of blastocyst. The results showed that melatonin significantly promoted the cleavage rate and 8-cell embryo yield of in vitro produced bovine embryo. In addition, significantly more blastocysts were observed by Day 7 of embryo culture at the presence of melatonin. These results indicated that melatonin accelerated the development of in vitro produced bovine embryos. Following vitrification at Day 7 of embryo culture, melatonin (10(-7 M significantly increased the hatched blastocyst rate from 24 h to 72 h and decreased the mortality rate from 48 h to 72 h after thawing. The presence of melatonin during the embryo culture resulted in a significant increase in the gene expressions of DNMT3A, OCC, CDH1 and decrease in that of AQP3 after thawing. In conclusion, melatonin not only promoted blastocyst yield and accelerated in vitro bovine embryo development, but also improved the quality of blastocysts which was indexed by an elevated cryotolerance and the up-regulated expressions of developmentally important genes.

  7. Derivation of HVR1, HVR2 and HVR3 human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis (PGD for monogenic disorder

    Directory of Open Access Journals (Sweden)

    Abdelkrim Hmadcha

    2016-05-01

    Full Text Available From 106 human blastocyts donate for research after in vitro fertilization (IVF and preimplantation genetic diagnosis (PGD for monogenetic disorder, 3 human embryonic stem cells (hESCs HVR1, HVR2 and HVR3 were successfully derived. HVR1 was assumed to be genetically normal, HVR2 carrying Becker muscular dystrophy and HVR3 Hemophilia B. Despite the translocation t(9;15(q34.3;q14 detected in HVR2, all the 3 cell lines were characterised in vitro and in vivo as normal hESCs lines and were registered in the Spanish Stem Cell Bank.

  8. Supplementation with small-extracellular vesicles from ovarian follicular fluid during in vitro production modulates bovine embryo development

    Science.gov (United States)

    Andrade, Gabriella M.; del Collado, Maite; Sampaio, Rafael V.; Sangalli, Juliano R.; Silva, Luciano A.; Pinaffi, Fábio V. L.; Jardim, Izabelle B.; Cesar, Marcelo C.; Nogueira, Marcelo F. G.; Cesar, Aline S. M.; Coutinho, Luiz L.; Pereira, Rinaldo W.; Perecin, Felipe; Meirelles, Flávio V.

    2017-01-01

    Pregnancy success results from the interaction of multiple factors, among them are folliculogenesis and early embryonic development. Failure during these different processes can lead to difficulties in conception. Alternatives to overcome these problems are based on assisted reproductive techniques. Extracellular vesicles are cell-secreted vesicles present in different body fluids and contain bioactive materials, such as messenger RNA, microRNAs (miRNAs), and proteins. Thus, our hypothesis is that extracellular vesicles from follicular fluid from 3–6 mm ovarian follicles can modulate bovine embryo development in vitro. To test our hypothesis follicular fluid from bovine ovaries was aspirated and small-extracellular vesicles (extracellular vesicles (EVs) were utilized for functional experiments investigating their role in modulating messenger RNA, microRNA as well as global DNA methylation and hydroxymethylation levels of bovine blastocysts. EVs from 3–6 mm follicles were used for RNA-seq and miRNA analysis. Functional annotation analysis of the EVs transcripts revealed messages related to chromatin remodeling and transcriptional regulation. EVs treatment during oocyte maturation and embryo development causes changes in blastocyst rates, as well as changes in the transcription levels of genes related to embryonic metabolism and development. Supplementation with EVs from 3–6 mm follicles during oocyte maturation and early embryo development (until the 4-cell stage) increased the levels of bta-miR-631 (enriched in EVs from 3–6 mm follicles) in embryos. Interestingly, the addition of EVs from 3–6 mm follicles induced changes in global DNA methylation and hydroxymethylation levels compared to embryos produced by the standard in vitro production system. Our results indicate that the supplementation of culture media with EVs isolated from the follicular fluid of 3–6 mm follicles during oocyte maturation and early embryo development can partially modify

  9. Cloned embryos from semen. Part 2: Intergeneric nuclear transfer of semen-derived eland (Taurotragus oryx) epithelial cells into bovine oocytes

    Science.gov (United States)

    Nel-Themaat, L.; Gomez, M.C.; Pope, C.E.; Lopez, M.; Wirtu, G.; Jenkins, J.A.; Cole, A.; Dresser, B.L.; Bondioli, K.R.; Godke, R.A.

    2008-01-01

    The production of cloned offspring by nuclear transfer (NT) of semen-derived somatic cells holds considerable potential for the incorporation of novel genes into endangered species populations. Because oocytes from endangered species are scarce, domestic species oocytes are often used as cytoplasts for interspecies NT. In the present study, epithelial cells isolated from eland semen were used for intergeneric transfer (IgNT) into enucleated bovine oocytes and compared with bovine NT embryos. Cleavage rates of bovine NT and eland IgNT embryos were similar (80 vs. 83%, respectively; p > 0.05); however, development to the morula and blastocyst stage was higher for bovine NT embryos (38 and 21%, respectively; p progress through the early cleavage stage arrest can (a) synthesize DNA, (b) progress through subsequent cell cycles, and (c) may have the potential to develop further. ?? 2008 Mary Ann Liebert, Inc.

  10. Inhibition of apoptosis by caspase inhibitor Z-VAD-FMK improves cryotolerance of in vitro derived bovine embryos.

    Science.gov (United States)

    Pero, Maria Elena; Zullo, Gianluigi; Esposito, Luigi; Iannuzzi, Alessandra; Lombardi, Pietro; De Canditiis, Carolina; Neglia, Gianluca; Gasparrini, Bianca

    2017-12-02

    The aim of this work was to evaluate whether the treatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) during cryopreservation and post-warming in vitro culture improves cryotolerance of bovine in vitro produced (IVP) embryos. Abattoir derived bovine oocytes were in vitro matured, fertilized and cultured according to standard procedure. On Day 7, embryo yields were assessed and blastocysts randomly divided in 2 groups: vitrification and post-warming culture in the absence (n = 184) or presence (n = 156) of 20 μM Z-VAD-FMK. Resistance to cryopreservation was evaluated post-warming culture by assessing the survival rate and hatching rate. Differential staining combined with in situ terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL) technique was performed to evaluate total cells number, cell allocation into inner cell mass (ICM) and trophectoderm (TE) lineages, as well as the DNA fragmentation rate of vitrified blastocysts, while immunohystochemical staining was used to assess the level of cleaved-caspase 3. It was demonstrated that inhibition of caspase activity by Z-VAD-FMK increases embryo cryotolerance, as indicated by higher survival (76.1 vs 51.1%; P vitrification/warming and post-warming culture partially inhibits cryopreservation-induced apoptosis by reducing the level of active caspase 3, suggesting a potential use as an additive to ameliorate the efficiency of embryo cryopreservation in cattle, critical for a further diffusion of IVEP technology in the field. Further studies are though needed to evaluate the effect of Z-VAD-FMK on post-transfer embryo development before considering a commercial application. Copyright © 2017. Published by Elsevier Inc.

  11. Ultra-Structural Alterations in In Vitro Produced Four-Cell Bovine Embryos Following Controlled Slow Freezing or Vitrification.

    Science.gov (United States)

    Cavusoglu, T; Popken, J; Guengoer, T; Yilmaz, O; Uyanikgil, Y; Ates, U; Baka, M; Oztas, E; Zakhartchenko, V

    2016-08-01

    Cryopreservation is the process of freezing and preserving cells and tissues at low temperatures. Controlled slow freezing and vitrification have successfully been used for cryopreservation of mammalian embryos. We investigated the effect of these two cryopreservation methods on in vitro produced four-cell stage bovine embryos which were classified according to their quality and separated into three groups. The first group was maintained as untreated controls (n = 350). Embryos of the second (n = 385) and the third (n = 385) groups were cryopreserved either by controlled slow freezing or by vitrification. Embryos in groups 2 and 3 were thawed after 1 day. Hundred embryos were randomly selected from the control group, and 100 morphologically intact embryos from the second and third group were thawed after 1 day and cultured to observe the development up to the blastocyst stage. The blastocyst development rate was 22% in the control group, 1% in the slow-freezing group and 3% in the vitrification group. Remaining embryos of all three groups were examined by light microscopy, transmission electron microscopy and immunofluorescence confocal microscopy with subsequent histological staining procedures. Cryopreservation caused degenerative changes at the ultra-structural level. Compared with vitrification, slow freezing caused an increased mitochondrial degeneration, cytoplasmic vacuolization, disruption of the nuclear and plasma membrane integrity, organelle disintegration, cytoskeletal damage, a reduced thickness of the zona pellucida and a formation of fractures in the zona pellucida. Further studies are required to understand and decrease the harmful effects of cryopreservation. © 2015 Blackwell Verlag GmbH.

  12. Comparison on in vitro fertilized bovine embryos cultured in KSOM or SOF and cryopreserved by slow freezing or vitrification.

    Science.gov (United States)

    Nedambale, T L; Dinnyés, A; Groen, W; Dobrinsky, J R; Tian, X C; Yang, X

    2004-08-01

    The objectives of this study were to identify an improved in vitro cell-free embryo culture system and to compare post-warming development of in vitro produced (IVP) bovine embryos following vitrification versus slow freezing. In Experiment 1, non-selected presumptive zygotes were randomly allocated to four medium treatments without co-culture: (1) SOF + 5% FCS for 9 days; (2) KSOM + 0.1% BSA for 4 days and then KSOM + 1% BSA to Day 9; (3) SOF + 5% FCS for 4 days and then KSOM + 1% BSA to Day 9; and (4) KSOM + 0.1% BSA for 4 days and then SOF + 5% FCS to Day 9. Treatment 4 (sequential KSOM-SOF culture system) improved (P > 0.05) morulae (47%), early blastocysts (26%), Day-7 blastocysts (36%), cell numbers, as well as total hatching rate (79%) compared to KSOM alone (Treatment 2). Embryos cultured in KSOM + BSA alone developed slowly and most of them hatched late on Day 9, compared to other treatments. In Experiment 2, the sequential KSOM-SOF culture system was used and Day-7 blastocysts were subjected to following cryopreservation comparison: (1) vitrification (VS3a, 6.5 M glycerol); or (2) slow freezing (1.36 M glycerol). Warmed embryos were cultured in SOF with 7.5% FCS. Higher embryo development and hatching rates (P vitrification at 6h (71%), 24h (64%), and 48h (60%) post-warming compared to slow freezing (48, 40, and 31%, respectively). Following transfer of vitrified embryos to synchronized recipients, a 30% pregnancy rate was obtained. In conclusion, replacing KSOM with SOF after 4 days of culture produced better quality blastocysts. Vitrification using VS3a may be used more effectively to cryopreserve in vitro produced embryos than the conventional slow freezing method.

  13. Oocyte maturation, embryo development and gene expression following two different methods of bovine cumulus-oocyte complexes vitrification.

    Science.gov (United States)

    Azari, Mehdi; Kafi, Mojtaba; Ebrahimi, Bita; Fatehi, Roya; Jamalzadeh, Mahboobeh

    2017-03-01

    To examine the maturational competence, embryo development and expression of genes involved in oocyte maturation and cumulus expansion (GDF9, BMP15, HAS2, TNFAIP6, FGF17 and FSHr) following two standard methods of bovine COCs vitrification. Bovine cumulus-oocyte complexes (COCs) were aspirated from slaughtered ovaries and then distributed into three groups: non-vitrified COCs (control), vitrification 1 group (V1); vitrification was performed by 15% ethylene glycol (EG) and 15% DMSO in holding media (TCM-199 with 20% FCS); and vitrification 2 group (V2); vitrification was performed by 40% EG in holding media. After vitrification, COCs were warmed in two steps and cultured and then evaluated for nuclear maturation, embryo development and gene expressions. The mean (±SD) percentages of nuclear maturation and blastocyst/cleaved were higher in control group (79.5 ± 8.0 and 31.0 ± 5.1%) than the V1 (34.8 ± 9.1 and 4.4 ± 5.1%) and V2 (47.8 ± 11.7 and 7.1 ± 5.8%) groups (P vitrification groups (P vitrification procedure and conditions. Using EG alone for vitrification of bovine immature COCs, resulted in higher expression of GDF9, BMP15 and production of more in vitro matured and cleaved oocytes.

  14. Solid-surface vitrification and in-straw dilution after warming of in vitro-produced bovine embryos.

    Science.gov (United States)

    Rodriguez-Villamil, P; Ongaratto, F L; Fernandez Taranco, M; Bó, G A

    2014-02-01

    Three experiments were designed to test a solid-surface vitrification system for bovine in vitro-produced embryos and to develop a simple method of in-straw dilution after warming, which can be potentially used for direct transfer in the field. Experiment 1 evaluated embryo survival rates (i.e. re-expansion and hatching) after vitrification and warming in three different solutions: VS1 (20% ethylene glycol (EG) + 20% propanediol (PROH) + 0.25 m trehalose (Tr)), VS2 (20% EG + 1M Tr) or VS3 (30% EG + 0.75 m Tr). Re-expansion and hatching rates were higher (p vitrification: glass micropipettes or solid surface, using the VS1 or VS3 solutions. No significant differences were detected between the two methods; but re-expansion and hatching rates were higher (p vitrification using simplified EG-based solutions and in-straw dilution with holding media may be a practical alternative for cryopreservation and direct transfer of in vitro-produced bovine embryos. © 2013 Blackwell Verlag GmbH.

  15. Successful application of the strategy of blastocyst biopsy, vitrification, whole genome amplification, and thawed embryo transfer for preimplantation genetic diagnosis of neurofibromatosis type 1

    Directory of Open Access Journals (Sweden)

    Yi-Lin Chen

    2011-03-01

    Conclusion: We first demonstrate successful application of blastocyst biopsy, vitrification, WGA, and thawed embryo transfer for PGD of a monogenic disease. Vitrification of blastocysts after biopsy permits sufficient time for shipment of samples and operation of molecular diagnosis.

  16. Prediction of a rare chromosomal aberration simultaneously with next generation sequencing-based comprehensive chromosome screening in human preimplantation embryos for recurrent pregnancy loss.

    Science.gov (United States)

    Lee, Yi-Xuan; Chen, Chien-Wen; Lin, Yi-Hui; Tzeng, Chii-Ruey; Chen, Chi-Huang

    2017-09-30

    Preimplantation genetic testing has been used widely in recent years as a part of assisted reproductive technology (ART) owing to the breakthrough development of deoxyribonucleic acid (DNA) sequencing. With the advancement of technology and increased resolution of next generation sequencing (NGS), extensive comprehensive chromosome screening along with small clinically significant deletions and duplications can possibly be performed simultaneously. Here, we present a case of rare chromosomal aberrations: 46,XY,dup(15)(q11.2q13),t(16;18)(q23;p11.2), which resulted in a normally developed adult but abnormal gametes leading to recurrent pregnancy loss (RPL). To our best knowledge, this is the first report of t(16;18) translocation with such a small exchanged segment detected by NGS platform of MiSeq system in simultaneous 24-chromosome aneuploidy screening.

  17. Insight into PreImplantation Factor (PIF* mechanism for embryo protection and development: target oxidative stress and protein misfolding (PDI and HSP through essential RIKP [corrected] binding site.

    Directory of Open Access Journals (Sweden)

    Eytan R Barnea

    Full Text Available Endogenous PIF, upon which embryo development is dependent, is secreted only by viable mammalian embryos, and absent in non-viable ones. Synthetic PIF (sPIF administration promotes singly cultured embryos development and protects against their demise caused by embryo-toxic serum. To identify and characterize critical sPIF-embryo protein interactions novel biochemical and bio-analytical methods were specifically devised.FITC-PIF uptake/binding by cultured murine and equine embryos was examined and compared with scrambled FITC-PIF (control. Murine embryo (d10 lysates were fractionated by reversed-phase HPLC, fractions printed onto microarray slides and probed with Biotin-PIF, IDE and Kv1.3 antibodies, using fluorescence detection. sPIF-based affinity column was developed to extract and identify PIF-protein interactions from lysates using peptide mass spectrometry (LC/MS/MS. In silico evaluation examined binding of PIF to critical targets, using mutation analysis.PIF directly targets viable cultured embryos as compared with control peptide, which failed to bind. Multistep Biotin-PIF targets were confirmed by single-step PIF-affinity column based isolation. PIF binds protein disulfide isomerases a prolyl-4-hydroxylase β-subunit, (PDI, PDIA4, PDIA6-like containing the antioxidant thioredoxin domain. PIF also binds protective heat shock proteins (70&90, co-chaperone, BAG-3. Remarkably, PIF targets a common RIKP [corrected] site in PDI and HSP proteins. Further, single PIF amino acid mutation significantly reduced peptide-protein target bonding. PIF binds promiscuous tubulins, neuron backbones and ACTA-1,2 visceral proteins. Significant anti-IDE, while limited anti-Kv1.3b antibody-binding to Biotin-PIF positive lysates HPLC fractions were documented.Collectively, data identifies PIF shared targets on PDI and HSP in the embryo. Such are known to play a critical role in protecting against oxidative stress and protein misfolding. PIF-affinity-column is a

  18. The influence of in vitro fertilization and embryo culture on the embryo epigenetic constituents and the possible consequences in the bovine model.

    Science.gov (United States)

    Sirard, M-A

    2017-08-01

    Medically assisted reproductive technologies, such as in vitro embryo production, are increasingly being used to palliate infertility. Eggs are produced following a hormonal regimen that stimulates the ovaries to produce a large number of oocytes. Collected oocytes are then fertilized in vitro and allowed to develop in vitro until they are either frozen or transferred to mothers. There are controversial reports on the adverse impacts of these technologies on early embryos and their potential long-term effects. Using newly developed technological platforms that enable global gene expression and global DNA methylation profiling, we evaluated gene perturbations caused by such artificial procedures. We know that cells in the early embryo produce all cells in the body and are able to respond to their in vitro environment. However, it is not known whether gene perturbations are part of a normal response to the environment or are due to distress and will have long-term impacts. While the mouse is an established genetic model used for quality control of culture media in clinics, the bovine is a large mono-ovulating mammal with similar embryonic kinetics as humans during the studied developmental window. These model systems are critical to understand the effects of assisted reproduction without the confounding impact of infertility and without the limitations imposed by the scarcity of donated human samples and ethical issues. The data presented in this review come mostly from our own experimentation, publications, and collaborations. Together they demonstrate that the in vitro environment has a significant impact on embryos at the transcriptomic level and at the DNA methylation level.

  19. Technical Update: Preimplantation Genetic Diagnosis and Screening.

    Science.gov (United States)

    Dahdouh, Elias M; Balayla, Jacques; Audibert, François; Wilson, R Douglas; Audibert, François; Brock, Jo-Ann; Campagnolo, Carla; Carroll, June; Chong, Karen; Gagnon, Alain; Johnson, Jo-Ann; MacDonald, William; Okun, Nanette; Pastuck, Melanie; Vallée-Pouliot, Karine

    2015-05-01

    To update and review the techniques and indications of preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS). Discussion about the genetic and technical aspects of preimplantation reproductive techniques, particularly those using new cytogenetic technologies and embryo-stage biopsy. Clinical outcomes of reproductive techniques following the use of PGD and PGS are included. This update does not discuss in detail the adverse outcomes that have been recorded in association with assisted reproductive technologies. Published literature was retrieved through searches of The Cochrane Library and Medline in April 2014 using appropriate controlled vocabulary (aneuploidy, blastocyst/physiology, genetic diseases, preimplantation diagnosis/methods, fertilization in vitro) and key words (e.g., preimplantation genetic diagnosis, preimplantation genetic screening, comprehensive chromosome screening, aCGH, SNP microarray, qPCR, and embryo selection). Results were restricted to systematic reviews, randomized controlled trials/controlled clinical trials, and observational studies published from 1990 to April 2014. There were no language restrictions. Searches were updated on a regular basis and incorporated in the update to January 2015. Additional publications were identified from the bibliographies of retrieved articles. Grey (unpublished) literature was identified through searching the websites of health technology assessment and health technology-related agencies, clinical practice guideline collections, clinical trial registries, and national and international medical specialty societies. The quality of evidence in this document was rated using the criteria described in the Report of the Canadian Task Force on Preventive Health Care. (Table 1) BENEFITS, HARMS, AND COSTS: This update will educate readers about new preimplantation genetic concepts, directions, and technologies. The major harms and costs identified are those of assisted reproductive

  20. Effect of maternal age on the ratio of cleavage and mitochondrial DNA copy number in early developmental stage bovine embryos.

    Science.gov (United States)

    Takeo, Shun; Goto, Hiroya; Kuwayama, Takehito; Monji, Yasunori; Iwata, Hisataka

    2013-01-01

    Age-associated deterioration in both the quality and quantity of mitochondria occurs in older women. The main aim of this study was to examine the effect of age on mitochondrial DNA copy number (mtDNA number) in early developmental stage bovine embryos as well as the dynamics of mtDNA number during early embryo development. Real-time PCR was used to determine mtDNA number. In vitro-produced embryos 48 h after insemination derived from Japanese black cows, ranging in age from 25 to 209 months were categorized based on their cleavage status. There was an overall negative relationship between the age of the cow and cleavage status, to the extent that the ratio of embryos cleaved over the 4-cell stage was greater in younger cows. The mtDNA number did not differ among the cleaved status of embryos. In the next experiment, oocytes collected from each donor cow were divided into 2 groups containing 10 oocytes each, in order to compare the mtDNA number of mature oocytes and early developmental stage embryos within individuals. Upon comparing the mtDNA number between oocytes at the M2 stage and early developmental stage 48 h post insemination, mtDNA number was found to decrease in most cows, but was found to increase in some cows. In conclusion, age affects the cleaving ability of oocytes, and very old cows (> 180 months) tend to have lower mtDNA numbers in their oocytes. The change in mtDNA number during early development varied among individual cows, although overall, it showed a tendency to decrease.

  1. Application of hollow fiber vitrification for cryopreservation of bovine early cleavage stage embryos and porcine morula-blastomeres.

    Science.gov (United States)

    Uchikura, Ayuko; Matsunari, Hitomi; Nakano, Kazuaki; Hatae, Shota; Nagashima, Hiroshi

    2016-04-22

    A novel hollow fiber vitrification (HFV) method was applied to materials that have previously been difficult to cryopreserve, thereby expanding the potential application of this method. The results showed that zona-free porcine morulae and their isolated blastomeres remained viable even after vitrification. The rate of development to blastocysts after vitrification was similar for zona-free and zona-intact morulae (21/23, 91.3% for both). Vitrified blastomeres had a developmental potential equal to that of non-vitrified blastomeres (blastocyst formation rate after reaggregation: 16/17, 94.1% for both). The HFV method was also effective for the cryopreservation of in vitro matured/fertilized bovine embryos at the 2- to 4-cell, 8- to 16-cell and morula stages. The blastocyst formation rates of vitrified embryos (66.1-82.5%) were similar to those of non-vitrified embryos (74.5-82.5%). These results indicate that this novel HFV method is an effective tool for embryo cryopreservation that can enhance current practices in reproductive biology.

  2. Lipid content and apoptosis of in vitro-produced bovine embryos as determinants of susceptibility to vitrification.

    Science.gov (United States)

    Sudano, Mateus José; Paschoal, Daniela Martins; Rascado, Tatiana da Silva; Magalhães, Luis Carlos Oña; Crocomo, Letícia Ferrari; de Lima-Neto, João Ferreira; Landim-Alvarenga, Fernanda da Cruz

    2011-04-15

    The objective was to evaluate supplementation of fetal calf serum (FCS) and phenazine ethosulfate (PES), a metabolic regulator that inhibits fatty acid synthesis, in culture media during in vitro production (IVP) of bovine embryos. Taking oocyte fertilization (n = 4,320) as Day 0, four concentrations of FCS (0, 2.5, 5, and 10%) and three periods of exposure to PES (without addition-CONTROL; after 60 h-PES Day 2.5 of embryo culture; and after 96 h-PES Day 4) were evaluated. Increasing FCS concentration in the culture media enhanced lipid accumulation (P vitrification (2.5%: 81.6 ± 2.5 vs 10%: 67.3 ± 3.5, P vitrification ( 72.0 ± 3.0 vs PES Day 2.5: 79.9 ± 2.8 or PES Day 4: 86.2 ± 2.4, P vitrification. Moreover, embryo quality, represented by the fresh apoptosis rate, was better than lipid content for predicting embryo survival after vitrification. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Effect of resveratrol supplementation during culture on the quality and cryotolerance of bovine in vitro produced embryos.

    Science.gov (United States)

    Salzano, A; Albero, G; Zullo, G; Neglia, G; Abdel-Wahab, A; Bifulco, G; Zicarelli, L; Gasparrini, B

    2014-12-30

    The aim of the study was to evaluate whether resveratrol supplementation of bovine culture medium improves in vitro blastocyst development, embryo cryotolerance and cell numbers. Abattoir-derived oocytes were matured and fertilized in vitro according to standard procedure. Twenty hours after IVF, zygotes were cultured in SOF medium, supplemented with 0 (control, n=439), 0.25μM (n=422), 0.5μM (n=447) and 1μM resveratrol (n=416). On Day 7 (IVF=Day 0) blastocysts were vitrified by cryotop in 16.5% ethylene glycol, 16.5% dimethyl sulfoxide and 0.5M sucrose. Development rate, i.e. the percentage of embryos resuming development to reach a more advanced stage, and hatching rate were evaluated after 24 and 48h culture. Blastocysts cultured with (0.5μM) and without resveratrol underwent differential staining to count inner cell mass (ICM) and trophectoderm (TE) cells. Resveratrol during culture did not increase blastocyst yields (57.1, 57.7, 59.2 and 46.6%, respectively in 0, 0.25, 0.5 and 1μM resveratrol). However, 0.5μM resveratrol improved embryo cryotolerance compared to the control, as indicated by higher development rates (67.3% vs 50.3%, respectively; Pculture. Blastocysts produced in the control and in 0.5μM resveratrol groups had similar numbers of ICM (34.1 and 36.4, respectively), TE (88.1 and 85.3, respectively) and total (122.2 and 121.7, respectively) cells. In conclusion, low levels of resveratrol during in vitro culture improve the quality of IVP bovine embryos, as indicated by their increased resistance to cryopreservation. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. New perspectives on preimplantation genetic diagnosis and preimplantation genetic screening

    Directory of Open Access Journals (Sweden)

    Chun-Kai Chen

    2014-06-01

    Full Text Available Preimplantation genetic diagnosis is a procedure that involves the removal of one or more nuclei from oocytes (a polar body or embryos (blastomeres or trophectoderm cells in order to test for problems in genome sequence or chromosomes of the embryo prior to implantation. It provides new hope of having unaffected children, as well as avoiding the necessity of terminating an affected pregnancy for genetic parents who carry an affected gene or have balanced chromosomal status. Polymerase chain reaction-based molecular techniques are the methods used to detect gene defects with a known sequence and X-linked diseases. The indication for using this approach has expanded for couples who are prevented from having babies because they carry a serious genetic disorder to couples with conditions that are not immediately life threatening, such as cancer predisposition genes and Huntington disease. In addition, fluorescent in situ hybridization (FISH has been widely applied for the detection of chromosome abnormalities. FISH allows the evaluation of many chromosomes at the same time, up to 15 chromosome pairs in a single cell. Preimplantation genetic screening, defined as a test that screens for aneuploidy, has been most commonly used in situations of advanced maternal age, a history of recurrent miscarriage, a history of repeated implantation failure, or a severe male factor. Unfortunately, randomized controlled trials have as yet shown no benefit with respect to preimplantation genetic screening using cleavage stage biopsy, which is probably attributable to the high levels of mosaicism at early cleavage stages and the limitations of FISH. Recently, two main types of array-based technology combined with whole genome amplification have been developed for use in preimplantation genetic diagnosis; these are comparative genomic hybridization and single nucleotide polymorphism-based arrays. Both allow the analysis of all chromosomes, and the latter also allows

  5. Developmental kinetics of the first cell cycles of bovine in vitro PRODUCED EMBRYOS IN RELATION TO THEIR IN VITRO VIABILITY AND SEX

    DEFF Research Database (Denmark)

    Holm, P; Shukri, N.N; Vajta, Gabor

    1998-01-01

    The development of bovine IVP-embryos was observed in a time-lapse culture system to determine cell cycle lengths of 1) embryos that developed into compact morulae (CM) or blastocysts (BL) within 174 h after insemination (viable), 2) embryos that arrested during earlier stages (nonviable) and 3......) male and female embryos. In 4 replicates, inseminated oocytes were cultured on a microscope stage in 3 to 4 groups on a granulosa cell monolayer in supplemented TCM 199. Images were sequentially recorded and stored at 30-min intervals. All embryos that could be identified throughout the culture period...... were included (n=392), and the times of cleavage events noted. After culture, 100 CM or BL were randomly selected for sexing by PCR. BL developed equally well in the time-lapse and control culture systems (36 vs 38. The respective lengths of the first 4 cell cycles of viable embryos were 32.0 + 3.9, g...

  6. Development of pre-implantation porcine embryos cultured within alginate hydrogel systems either supplemented with secreted phosphoprotein 1 or conjugated with arg-gly-asp peptide

    Science.gov (United States)

    Although deficiencies in porcine embryo elongation play a significant role on early embryonic mortality and establishment of within–litter developmental variation, the exact mechanisms of elongation are poorly understood. Secreted phosphoprotein 1 (SPP1) is increased within the uterine milieu during...

  7. Finding biomarkers in non-model species: literature mining of transcription factors involved in bovine embryo development.

    Science.gov (United States)

    Turenne, Nicolas; Tiys, Evgeniy; Ivanisenko, Vladimir; Yudin, Nikolay; Ignatieva, Elena; Valour, Damien; Degrelle, Séverine A; Hue, Isabelle

    2012-08-29

    Since processes in well-known model organisms have specific features different from those in Bos taurus, the organism under study, a good way to describe gene regulation in ruminant embryos would be a species-specific consideration of closely related species to cattle, sheep and pig. However, as highlighted by a recent report, gene dictionaries in pig are smaller than in cattle, bringing a risk to reduce the gene resources to be mined (and so for sheep dictionaries). Bioinformatics approaches that allow an integration of available information on gene function in model organisms, taking into account their specificity, are thus needed. Besides these closely related and biologically relevant species, there is indeed much more knowledge of (i) trophoblast proliferation and differentiation or (ii) embryogenesis in human and mouse species, which provides opportunities for reconstructing proliferation and/or differentiation processes in other mammalian embryos, including ruminants. The necessary knowledge can be obtained partly from (i) stem cell or cancer research to supply useful information on molecular agents or molecular interactions at work in cell proliferation and (ii) mouse embryogenesis to supply useful information on embryo differentiation. However, the total number of publications for all these topics and species is great and their manual processing would be tedious and time consuming. This is why we used text mining for automated text analysis and automated knowledge extraction. To evaluate the quality of this "mining", we took advantage of studies that reported gene expression profiles during the elongation of bovine embryos and defined a list of transcription factors (or TF, n = 64) that we used as biological "gold standard". When successful, the "mining" approach would identify them all, as well as novel ones. To gain knowledge on molecular-genetic regulations in a non model organism, we offer an approach based on literature-mining and score

  8. Finding biomarkers in non-model species: literature mining of transcription factors involved in bovine embryo development

    Directory of Open Access Journals (Sweden)

    Turenne Nicolas

    2012-08-01

    Full Text Available Abstract Background Since processes in well-known model organisms have specific features different from those in Bos taurus, the organism under study, a good way to describe gene regulation in ruminant embryos would be a species-specific consideration of closely related species to cattle, sheep and pig. However, as highlighted by a recent report, gene dictionaries in pig are smaller than in cattle, bringing a risk to reduce the gene resources to be mined (and so for sheep dictionaries. Bioinformatics approaches that allow an integration of available information on gene function in model organisms, taking into account their specificity, are thus needed. Besides these closely related and biologically relevant species, there is indeed much more knowledge of (i trophoblast proliferation and differentiation or (ii embryogenesis in human and mouse species, which provides opportunities for reconstructing proliferation and/or differentiation processes in other mammalian embryos, including ruminants. The necessary knowledge can be obtained partly from (i stem cell or cancer research to supply useful information on molecular agents or molecular interactions at work in cell proliferation and (ii mouse embryogenesis to supply useful information on embryo differentiation. However, the total number of publications for all these topics and species is great and their manual processing would be tedious and time consuming. This is why we used text mining for automated text analysis and automated knowledge extraction. To evaluate the quality of this “mining”, we took advantage of studies that reported gene expression profiles during the elongation of bovine embryos and defined a list of transcription factors (or TF, n = 64 that we used as biological “gold standard”. When successful, the “mining” approach would identify them all, as well as novel ones. Methods To gain knowledge on molecular-genetic regulations in a non model organism, we offer an

  9. Cell apoptosis and lipid content of in vitro-produced, vitrified bovine embryos treated with forskolin.

    Science.gov (United States)

    Paschoal, Daniela Martins; Sudano, Mateus José; Schwarz, Kátia Regina Lancellotti; Maziero, Rosiára Rosário Dias; Guastali, Midyan Daroz; Crocomo, Letícia Ferrari; Magalhães, Luis Carlos Oña; Martins, Alício; Leal, Claudia Lima Verde; Landim-Alvarenga, Fernanda da Cruz

    2017-01-01

    The presence of fetal calf serum in culture medium influences embryo quality, causing a reduction in postcryopreservation survival. Forskolin has been used to induce lipolysis and increase cryotolerance, functioning as an activator of adenylate cyclase and elevating cAMP levels. In the present experiment, bovine zygotes were cultured in synthetic oviduct fluid with amino acid plus 2.5% fetal calf serum for 6 days, when forskolin was added in three concentrations: 2.5, 5, and 10 μM. Treatment with forskolin lasted for 24 hours. Blastocyst formation rate, quantification of lipid granules, total cell numbers, and apoptosis rate were evaluated. In a second assessment, embryos were vitrified, and warming, re-expansion rate, total cell numbers, and apoptosis rate were also evaluated. There was no difference due to forskolin in blastocyst formation or re-expansion rates after vitrification. However, lipid measurements were lower (control: 136.8 and F 2.5 μM: 128.5; P < 0.05), and number of cells per embryo higher (control: 140.1 and F 2.5 μM: 173.5; P < 0.05) than controls for 2.5 μM forskolin but not for higher forskolin concentrations. The number of intact cells per embryo was higher, and the rate of apoptosis was lower in fresh than in vitrified embryos (number of cells of warmed embryos, control: 104.1, F 2.5 μM: 101.3, F 5 μM: 115.4, F 10 μM: 95.1; apoptotic of fresh cells, control: 12.1%, F 2.5 μM: 16.7%, F 5 μM: 11.1%, F 10 μM: 14.2%; and apoptotic warmed embryos, control: 22.3%, F 2.5 μM: 37.3%, F 5 μM: 33.2%, F 10 μM: 30.3%; P < 0.05). It was concluded that forskolin is an effective lipolytic agent even at low concentrations, leading to formation of blastocysts with a comparatively larger number of cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Screening of biotechnical parameters for production of bovine inter-subspecies embryonic chimeras by the aggregation of tetraploid Bos indicus and diploid crossbred Bos taurus embryos.

    Science.gov (United States)

    Razza, Eduardo M; Satrapa, Rafael A; Emanuelli, Isabele P; Barros, Ciro M; Nogueira, Marcelo F G

    2016-03-01

    The aggregation of a tetraploid zebu embryo (Bos indicus, a thermotolerant breed) with a diploid taurine embryo (Bos taurus, a thermosensitive breed) should create a complete taurine fetus, whose extra-embryonic components, e.g., the chorion, is derived mainly from the zebu embryo. These zebu-derived extra-embryonic components may interact positively with the taurine embryo/fetus during pregnancy in a tropical environment. We tested different parameters for the production of tetraploid Nelore (Bos indicus) embryos to be combined via aggregation with crossbred Bos taurus (diploid) embryos in order to produce viable chimeric blastocysts. Bovine (Bos indicus or crossbred Bos taurus) embryos were produced in vitro according to standard procedures. Two-cell Bos indicus embryos were submitted to electrofusion with varying numbers of pulses (1 or 2), voltages (0.4, 0.5, 0.75, 1.0, 1.4 and 5.0 kV/cm) and time (20, 25, 50 and 60 μs) to produce tetraploid embryos. Electrofused embryos were cultured with crossbred non-fused embryos to form chimeras that developed until the blastocyst stage. The best fusion parameter was 0.75 kV/cm for 60 μs. Four chimeric blastocysts (tetraploid Nelore with diploid crossbred Holstein) were formed after 31 attempts in 4 replicates (13%). We established an optimal procedure for the production of tetraploid Bos indicus (4n) embryos and embryonic chimeras by aggregation of crossbred Bos taurus (2n) with Bos indicus (4n) embryos. This technique would be valid in applied research, by producing exclusively taurine calves, but with placental elements from the Bos indicus breed, following transfer of these chimeras into recipient cows. Copyright © 2015 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  11. Improved bovine embryo production in an oviduct-on-a-chip system : prevention of poly-spermic fertilization and parthenogenic activation

    NARCIS (Netherlands)

    Ferraz, Marcia A.M.M.; Henning, Heiko H.W.; Costa, Pedro F.; Malda, Jos; Melchels, Ferry P.W.; Wubbolts, Richard; Stout, Tom A.E.; Vos, Peter L.A.M.; Gadella, Bart M.

    2017-01-01

    The oviduct provides the natural micro-environment for gamete interaction, fertilization and early embryo development in mammals, such as the cow. In conventional culture systems, bovine oviduct epithelial cells (BOEC) undergo a rapid loss of essential differentiated cell properties; we aimed to

  12. In vitro production of bovine embryos: cumulus/granulosa cell gene expression patterns point to early atresia as beneficial for oocyte competence

    DEFF Research Database (Denmark)

    Mazzoni, Gianluca; Razza, Eduardo; Pedersen, Hanne S.

    2017-01-01

    In vitro production (IW) of bovine embryos has become widespread technology implemented in cattle breeding and production. Here, we review novel data on cumulus/granulosa cell gene expression, as determined by RNAseq on cellular material from pooled follicular fluids at the single animal level, a...

  13. Effect of cryopreservation and in vitro culture of bovine fibroblasts on histone acetylation levels and in vitro development of hand-made cloned embryos

    Science.gov (United States)

    Chacon, L.; Gomez, M.C.; Jenkins, J.A.; Leibo, S.P.; Wirtu, G.; Dresser, B.L.; Pope, C.E.

    2011-01-01

    In this study, the relative acetylation levels of histone 3 in lysine 9 (H3K9ac) in cultured and cryopreserved bovine fibroblasts was measured and we determined the influence of the epigenetic status of three cultured (C1, C2 and C3) donor cell lines on the in vitro development of reconstructed bovine embryos. Results showed that cryopreservation did not alter the overall acetylation levels of H3K9 in bovine fibroblasts analysed immediately after thawing (frozen/thawed) compared with fibroblasts cultured for a period of time after thawing. However, reduced cleavage rates were noted in embryos reconstructed with fibroblasts used immediately after thawing. Cell passage affects the levels of H3K9ac in bovine fibroblasts, decreasing after P1 and donor cells with lower H3K9ac produced a greater frequency of embryo development to the blastocyst stage. Cryopreservation did not influence the total cell and ICM numbers, or the ICM/TPD ratios of reconstructed embryos. However, the genetic source of donor cells did influence the total number of cells and the trophectoderm cell numbers, and the cell passage influenced the total ICM cell numbers. ?? Copyright Cambridge University Press 2010.

  14. Cellular alteration after dilution of cryoprotective solutions used for the vitrification of in vitro-produced bovine embryos.

    Science.gov (United States)

    Kaidi, S; Van Langendonckt, A; Massip, A; Dessy, F; Donnay, I

    1999-08-01

    Embryo quality of in vitro-produced bovine blastocysts was assessed at several steps of a vitrification procedure in which glycerol and ethylene glycol were used as cryoprotectants (3-step equilibration with cryoprotectants followed by vitrification, dilution of the cryoprotectants in 0.85 M galactose then in embryo transfer freezing medium [ETF], and finally co-culture for periods). To visualize cell membrane alterations, double staining was performed using a cell permeant fluorochrome (bisbenzimide--BIS) and a nonpermeant one (propidium iodide--PI). In Experiment 1, the effect of the vitrification procedure on the hatching rate and total cell number was assessed 72 h after treatment. Hatching rate and the number of stained nuclei were decreased in comparison with untreated embryos when blastocysts were exposed to the whole procedure with or without vitrification (respectively 42 and 53% vs 76% for hatching and 128 +/- 17 and 141 +/- 17 vs 226 +/- 13 for stained nuclei). In Experiment 2, the effect of cryoprotectants and their dilution was evaluated on membrane permeability and total cell numbers at various steps of the vitrification procedure. Blastocysts exposed only to cryoprotectant solutions and stained immediately after dilution in galactose showed no modification. After dilution in ETF, the total number of stained nuclei decreased, and the number of blastomeres showing membrane permeabilization (PI-stained) increased (respectively, 74 +/- 5 vs 110 +/- 5 and 32 +/- 2% vs 0.1 +/- 1.8%). In Experiment 3, we demonstrated that the total number of stained nuclei after ethanol fixation (membrane permeabilization) was higher when embryos treated up to dilution in ETF were stained with PI than when the same embryos were stained with BIS. This suggests that, for unknown reasons, some nuclei of the treated embryos were not stained with BIS. Membrane permeabilization and inability of BIS to stain some nuclei were the most obvious alterations probably induced by osmotic

  15. Effect of insulin-like growth factor-1 during in vitro oocyte maturation and in vitro culture of bovine embryos

    Directory of Open Access Journals (Sweden)

    Quetglas M.D.

    2001-01-01

    Full Text Available The effects of insulin-like growth factor-I (IGF-I on in vitro maturation (IVM (experiment I and on in vitro embryo development (experiment II of bovine oocytes fertilized in vitro, were evaluated in terms of cleavage (CR, blastocyst (BR and hatching (HR rates. For IVM, immature cumulus-oocyte complexes were cultured in TCM-199 medium supplemented with Hepes, sodium bicarbonate, sodium pyruvate, additives, fetal calf serum (B-199 medium and gonadotropins (14 U/ml PMSG and 7 U/ml hCG. For embryo development, the oocytes/zygotes were cultured in B-199 medium with bovine oviduct epithelial cells in suspension under silicon oil. Treatments for in vitro culture conditions for both experiments were: 1- B-199 + 200 ng/ml IGF-I; 2- B-199 + 100 ng/ml IGF-I; 3- B-199 + 50 ng/ml IGF-I; 4- B-199 + 10 ng/ml IGF-I; 5- B-199 + 0 ng/ml IGF-I. All cultures were performed at 38.5ºC in 5% CO2 in air and the data were analyzed by chi-square test. In experiment I, there were no differences (P>0.05 among treatments for CR, BR or HR. In experiment II, the addition of IGF-I to the embryo culture medium (ECM resulted in a significant increase in CR while for BR and HR this effect was not observed. The addition of 200 ng/ml IGF-I to ECM increased CR (71.1% when compared to 100 ng/ml IGF-I (57.6% or control (56.7% groups, however, there were no differences when compared to 50 (69.4% or 10 ng/ml (73.1% groups. There was no beneficial effect of the addition of IGF-I in the IVM or ECM media on the in vitro development of embryos produced from oocytes matured and fertilized in vitro.

  16. Supplementation of l-carnitine during in vitro maturation improves embryo development from less competent bovine oocytes.

    Science.gov (United States)

    Knitlova, Drahomira; Hulinska, Pavlina; Jeseta, Michal; Hanzalova, Katerina; Kempisty, Bartosz; Machatkova, Marie

    2017-10-15

    The present study was designed to define the impact of l-carnitine, supplemented during maturation, on bovine oocytes with different meiotic competence in terms of their IVF outcomes. Meiotically more competent (MMC) and less competent (MLC) oocytes were obtained separately from differently sized follicles at selected phases of folliculogenesis. The oocytes were matured with or without l-carnitine, fertilized and cultured to the blastocyst stage. The oocytes were examined for nuclear maturation, mitochondrial cluster formation, lipid consumption, fertilization and embryo development. The proportion of oocytes at metaphase II was significantly higher in the l-carnitine-treated MMC than that in the l-carnitine-treated MLC oocytes. However in comparison with the untreated controls, the proportion of MII oocytes with mitochondrial clusters was significantly higher only in the l-carnitine-treated MLC oocytes, which also showed a significantly lower mean lipid content. The l-carnitine-treated MLC oocytes showed significantly higher fertilization and syngamy rates than the untreated MLC oocytes. On the other hand, in the l-carnitine-treated MMC oocytes, the fertilization rate was similar to that of the untreated controls and the syngamy rate was significantly delayed. Although no significant differences in cleavage on Day 2 were found among all oocyte categories, l-carnitine treatment resulted in a significantly higher blastocyst yield in the MLC oocytes on Day 7 and Day 8 and a significantly higher proportion of expanded blastocysts in relation to the total number of blastocysts in MMC oocytes on Day 8. It can be concluded that l-carnitine supplementation during maturation improves the development of bovine embryos from meiotically less competent oocytes and accelerates blastocyst formation from more competent oocytes. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Carbon-activated gas filtration during in vitro culture increased pregnancy rate following transfer of in vitro-produced bovine embryos.

    Science.gov (United States)

    Merton, J S; Vermeulen, Z L; Otter, T; Mullaart, E; de Ruigh, L; Hasler, J F

    2007-04-15

    Many environmental conditions for in vitro embryo production (IVP) systems for cattle have been relatively standardised, e.g. media composition, temperature, pH, water quality, and atmospheric composition. However, little attention has been paid to the quality of ambient laboratory air and the gas environment in incubators. Although a few studies have examined the effects of chemical air contamination on IVP of human embryos, there are no published accounts for domestic animal embryos. Therefore, this study investigated the effects of an intra-incubator carbon-activated air filtration system (CODA) during in vitro culture (IVC) on embryonic development and subsequent pregnancy rate of bovine embryos. Immature cumulus-oocyte-complexes (COCs) were obtained twice-weekly by ultrasonic-guided transvaginal oocyte aspiration. The COCs were matured in TCM199/FCS/LH/FSH, fertilized with frozen-thawed Percoll-separated semen, and subsequently cultured for 7 day in SOFaaBSA. Day 7 embryos were transferred either fresh or frozen/thawed. The experimental design was a 2 x 2 factorial; presumptive zygotes were placed either in a conventional CO(2)-O(2)-N(2) incubator (Control group) or in an identical CO(2)-O(2)-N(2) incubator with a CODA intra-incubator air purification unit (CODA group) for IVC. The embryo production rate at Day 7 was not affected by the CODA air purification unit (23.4 and 24.7% morulae and blastocysts per oocyte for control and CODA, respectively) nor was there any significant effect on embryo stage or quality. However, the pregnancy rate was improved (P=0.043) for both fresh (46.3% versus 41.0%) and frozen/thawed embryos (40.8% versus 35.6%). In conclusion, atmospheric purification by the CODA intra-incubator air purification unit significantly increased pregnancy rate following transfer of in vitro-produced bovine embryos.

  18. Effects of fetal calf serum, phenazine ethosulfate and either glucose or fructose during in vitro culture of bovine embryos on embryonic development after cryopreservation.

    Science.gov (United States)

    Barceló-Fimbres, M; Seidel, G E

    2007-11-01

    This study investigated effects of hexoses, fetal calf serum (FCS), and phenazine ethosulfate (PES) during the culture of bovine embryos on blastocyst development and survival after cryopreservation by slow freezing or vitrification. The basal, control medium was chemically defined (CDM) plus 0.5% fatty acid-free BSA. In vitro-produced bovine zygotes were cultured in CDM-1 with 0.5 mM glucose; after 60 hr, 8-cell embryos were cultured 4.5 days in CDM-2. The 8-cell embryos were randomly allocated to a 2 x 3 x 2 x 3 factorial experimental design with two energy substrates (2 mM glucose or fructose); three additives (0.3 microM PES, 10% FCS, and control); two cryopreservation methods using no animal products (conventional slow freezing or vitrification); and semen from three bulls with two replicates for each bull. A total of 1,107 blastocysts were produced. Fructose resulted in 13% more blastocysts per oocyte than glucose (37.2% vs. 32.9%), and per 8-cell embryo (51.3% vs. 45.3%; P 0.1) control, FCS, or PES for blastocysts per oocyte or per 8-cell embryo. There was a significant interaction (P embryos with PES, which reduces cytoplasmic lipid content, improved cryotolerance of bovine embryos; post-cryopreservation survival of blastocysts averaged over vitrification and slow freezing (between which there was no difference) was 91.9%, 84.9%, and 60.2% of unfrozen controls (P < 0.01) for PES, control, and FCS groups, respectively. (c) 2007 Wiley-Liss, Inc.

  19. FISH preimplantation diagnosis of chromosome aneuploidy in recurrent pregnancy wastage.

    Science.gov (United States)

    Vidal, F; Giménez, C; Rubio, C; Simón, C; Pellicer, A; Santaló, J; Egozcue, J

    1998-05-01

    Our purpose was to detect aneuploidy for chromosomes 13, 16, 18, 21, 22, X, and Y in preimplantation embryos from patients with a history of unexplained recurrent miscarriage. Three patients with a history of unexplained recurrent spontaneous abortion were included in this study. Embryos were biopsied at the eight-cell stage, individually fixed on slides, and processed for fluorescent in situ hybridization (FISH). A multiple FISH protocol for seven chromosomes pairs (13, 16, 18, 21, 22, X, and Y) has been developed. A total of 39 embryos was studied with the multiple FISH protocol developed. Successful analysis of the biopsied embryos was achieved within the time limits usually allowed in a preimplantation diagnosis program. Analysis of the blastomeres showed that 17 embryos were chromosomally normal for the probes used, 16 embryos were aneuploid, and in 6 embryos no informative results were obtained. In the patients studied, a large proportion of embryos (41%) exhibited chromosomal abnormalities for the probes used. Preimplantation diagnosis to screen for chromosome abnormalities could be a feasible approach to improve the possibility of successful pregnancy in these couples.

  20. Effects of flunixin meglumine and prostaglandin F2 α treatments on the development and quality of bovine embryos in vitro.

    Science.gov (United States)

    Kim, S-S; Bang, J-I; Fakruzzaman, M; Lee, K-L; Ko, D-H; Ghanem, N; Wang, Z; Kong, I-K

    2014-12-01

    Assisted reproduction procedures, such as embryo transfer (ET) and artificial insemination (AI), in cattle could induce the secretion of prostaglandin F2 -alpha (PGF2 α) from uterine horns which may in turn interrupt embryo development and implantation. This study investigated the effect of flunixin meglumine (FM), prostaglandin F2 alpha (PGF2α) and FM combined with PGF2α supplementation in culture medium (IVC-II) on the development and quality of in vitro produced bovine embryos. The development rate of embryos was significantly higher in the FM group (33.3%) than in control (24.3%), PGF2 α (23.9%) and FM + PGF2 α groups (24.5%). The percentage of hatched blastocysts was also higher (p < 0.05) in the FM group (41.2%) than in the control (27.8%) and PGF2 α groups (19.8%). While, there was no significant difference in total cell number in all experimental groups, the number of apoptotic cells was significantly higher in the PGF2 α group (8.2 ± 6.6) than in the control (4.7 ± 3.2), FM (4.7 ± 2.5) and FM + PGF2 α (4.9 ± 3.4) groups. Detected by real-time PCR, secreted vesicle seminal protein 1 (SSLP1) and prostaglandin G/H synthase 2 (PTGS2) gene expression decreased (p < 0.05) in the PGF2 α group. However, SSLP1 and PTGS2 gene expression in the FM + PGF2 α group returned to their baseline levels, similar to the control and FM groups. Caspase 3 (CAPS3) gene expression increased in the PGF2 α group compared with other groups (p < 0.05). In conclusion, addition of FM in vitro culture significantly improved embryo development as well as alleviated the negative impact of PGF2 α. © 2014 Blackwell Verlag GmbH.

  1. Reduction in cytoplasmic lipid content in bovine embryos cultured in vitro with linoleic acid in semi-defined medium is correlated with increases in cryotolerance.

    Science.gov (United States)

    Accorsi, Mônica F; Leão, Beatriz Caetano da Silva; Rocha-Frigoni, Nathália Alves de Souza; Perri, Silvia Helena Venturoli; Mingoti, Gisele Zoccal

    2016-08-01

    We examined whether culturing embryos with linoleic acid (LA) in semi-defined medium reduces lipid accumulation and improves cryosurvival after vitrification. Embryos were cultured with LA (100 μM) and a semi-defined medium was used during in vitro culture (IVC), in which the fetal calf serum was substituted by bovine serum albumin (BSA). There was a reduction (P embryos stained with the fluorescent dye Nile Red), consequently increasing (P = 0.0490) the embryo survival after 24h of culture post-warming ( 50.0% versus LA: 71.7%). The results question the criteria used to evaluate the efficiency of an in vitro production system specifically with relation to the maximum number of blastocysts produced and suggest that might be more appropriate to improve the desired characteristics of embryos generated in accordance with the specific purpose of in vitro embryo production, commercial or scientific. In conclusion, supplying LA to serum-free culture medium was found to adversely affect the rates of embryo development to the blastocyst stage, but significantly reduced embryo lipid accumulation and improved cryopreservation survival.

  2. Effect of Temporary Meiotic Attenuation of Oocytes with Butyrolactone I and Roscovitine in Resistance to Bovine Embryos on Vitrification.

    Science.gov (United States)

    Maziero, R R D; Guaitolini, C R F; Paschoal, D M; Kievitsbosch, T; Guastali, M D; Moraes, C N; Landim-Alvarenga, F C

    2016-04-01

    This study aimed to produce in vitro bovine embryos by the addition of two drugs, which is responsible for oocyte meiosis inhibition: roscovitine (ROS) and butyrolactone I (BL-I). Oocytes were recovered from slaughtered cows and matured in a commercial medium and maintained in a 5% CO2 atmosphere. Oocytes were maintained for 6 h in an in vitro maturation (IVM) medium containing ROS (12.5 μm), BL-I (50 μm) and association of drugs (ROS 6.25 μm and BL-I 25 μm). Oocytes were cultured for 18 h in an agent-free medium for the resumption of meiosis. After 24 h of maturation, oocytes were inseminated in the commercial in vitro fertilization (IVF) medium. Presumptive zygotes were cultured in SOFaa medium in a 5% CO2 atmosphere. On day 3, rate of cleavage was evaluated and on days 6 and 7, rate of blastocyst formation. BL-I and its association with the ROS increased the rates of cleavage and blastocyst formation (p vitrification process, presenting a higher rate of embryonic re-expansion (p < 0.05). In conclusion, block of meiosis using BL-I or its association with ROS increased the rate of blastocyst formation, and the association of ROS+BL-I resulted in a better resistance to the embryo cryopreservation process. © 2016 Blackwell Verlag GmbH.

  3. Production of bovine cloned embryos with donor cells frozen at a slow cooling rate in a conventional freezer (20 C)

    Science.gov (United States)

    Chacon, L.; Gomez, M.C.; Jenkins, J.A.; Leibo, S.P.; Wirtu, G.; Dresser, B.L.; Pope, C.E.

    2009-01-01

    Summary Usually, fibroblasts are frozen in dimethyl sulphoxide (DMSO, 10% v/v) at a cooling rate of 1 C/min in a low-temperature (80 C) freezer (LTF) before storage in liquid nitrogen (LN2); however, a LTF is not always available. The purpose of the present study was to evaluate apoptosis and viability of bovine fibroblasts frozen in a LTF or conventional freezer (CF; 20 C) and their subsequent ability for development to blastocyst stage after fusion with enucleated bovine oocytes. Percentages of live cells frozen in LTF (49.5%) and CF (50.6%) were similar, but significantly less than non-frozen control (88%). In both CF and LTF, percentages of live apoptotic cells exposed to LN2 after freezing were lower (4% and 5%, respectively) as compared with unexposed cells (10% and 18%, respectively). Cells frozen in a CF had fewer cell doublings/24 h (0.45) and required more days (9.1) to reach 100% confluence at the first passage (P) after thawing and plating as compared with cells frozen in a LTF (0.96 and 4.0 days, respectively). Hypoploidy at P12 was higher than at P4 in cells frozen in either a CF (37.5% vs. 19.2%) or in a LTF (30.0% vs. 15.4%). A second-generation cryo-solution reduced the incidence of necrosis (29.4%) at 0 h after thawing as compared with that of a first generation cryo-solution (DMEM + DMSO, 60.2%). The percentage of apoptosis in live cells was affected by cooling rate (CF = 1.9% vs. LFT = 0.7%). Development of bovine cloned embryos to the blastocyst stage was not affected by cooling rate or freezer type. ?? 2009 Cambridge University Press.

  4. Preimplantation genetic diagnosis and screening by array comparative genomic hybridisation: experience of more than 100 cases in a single centre.

    Science.gov (United States)

    Chow, J Fc; Yeung, W Sb; Lee, V Cy; Lau, E Yl; Ho, P C; Ng, E Hy

    2017-04-01

    Preimplantation genetic screening has been proposed to improve the in-vitro fertilisation outcome by screening for aneuploid embryos or blastocysts. This study aimed to report the outcome of 133 cycles of preimplantation genetic diagnosis and screening by array comparative genomic hybridisation. This study of case series was conducted in a tertiary assisted reproductive centre in Hong Kong. Patients who underwent preimplantation genetic diagnosis for chromosomal abnormalities or preimplantation genetic screening between 1 April 2012 and 30 June 2015 were included. They underwent in-vitro fertilisation and intracytoplasmic sperm injection. An embryo biopsy was performed on day-3 embryos and the blastomere was subject to array comparative genomic hybridisation. Embryos with normal copy numbers were replaced. The ongoing pregnancy rate, implantation rate, and miscarriage rate were studied. During the study period, 133 cycles of preimplantation genetic diagnosis for chromosomal abnormalities or preimplantation genetic screening were initiated in 94 patients. Overall, 112 cycles proceeded to embryo biopsy and 65 cycles had embryo transfer. The ongoing pregnancy rate per transfer cycle after preimplantation genetic screening was 50.0% and that after preimplantation genetic diagnosis was 34.9%. The implantation rates after preimplantation genetic screening and diagnosis were 45.7% and 41.1%, respectively and the miscarriage rates were 8.3% and 28.6%, respectively. There were 26 frozen-thawed embryo transfer cycles, in which vitrified and biopsied genetically transferrable embryos were replaced, resulting in an ongoing pregnancy rate of 36.4% in the screening group and 60.0% in the diagnosis group. The clinical outcomes of preimplantation genetic diagnosis and screening using comparative genomic hybridisation in our unit were comparable to those reported internationally. Genetically transferrable embryos replaced in a natural cycle may improve the ongoing pregnancy rate

  5. Correlation between the cryosurvival, cell number and diameter in bovine in vitro produced embryos.

    Science.gov (United States)

    Kocyigit, Alper; Cevik, Mesut

    2016-10-01

    The selection of quality embryos is a prerequisite of cryopreservation process. Present study was conducted to examine the correlation between the diameter and cryotolerance, on the cell number of the cryopreserved embryos. The blastocyst stage embryos were collected at culture days 7-9, evaluated morphologically under a microscope and divided according to the diameter into three groups: Group 1; (larger than 150 μm), Group 2; (diameter of 100-150 μm), Group 3; (smaller than 100 μm). Blastocysts were vitrified-thawed using the classical vitrification method and then cultured in SOF medium drops at 24 h. Blastocysts were considered viable if they re-expanded or hatched from the zona pellucidae. Finally re-expanded blastocysts from the Group 1 and Group 2 to determine the differential count of cells in the ICM and TE. The re-expansion ability of blastocysts 100-150 μm in diameter (69.56%) was significantly higher than other groups (52.17 and 47.36%). The value of the correlation coefficient between the re-expansion rate and cell number of blastocysts in the group 2 (r = 0.784) tended to be higher than that in the group 1 (r = 0.512) and group 3 (r = 0.491) (p < 0.05). For ICM/total cell ratio yield group 2 embryos showed higher rate (0.28), compared to the other groups (0.19 and 0.16). In conclusion, the present study demonstrates that the correlation between diameter of embryos and their cryosurvival based on re-expansion ability and cell allocation. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Comparison of different fertilisation media for an in vitro maturation?fertilisation?culture system using flow-cytometrically sorted X chromosome-bearing spermatozoa for bovine embryo production.

    Science.gov (United States)

    Ferré, Luis B; Bogliotti, Yanina; Chitwood, James L; Fresno, Cristóbal; Ortega, Hugo H; Kjelland, Michael E; Ross, Pablo J

    2015-05-13

    High demand exists among commercial cattle producers for in vitro-derived bovine embryos fertilised with female sex-sorted spermatozoa from high-value breeding stock. The aim of this study was to evaluate three fertilisation media, namely M199, synthetic oviductal fluid (SOF) and Tyrode's albumin-lactate-pyruvate (TALP), on IVF performance using female sex-sorted spermatozoa. In all, 1143, 1220 and 1041 cumulus-oocyte complexes were fertilised in M199, SOF and TALP, respectively. There were significant differences among fertilisation media (P fertilisation medium. Embryos derived from SOF and TALP fertilisation media had higher numbers of ICM, TE and total cells than those fertilised in M199. In conclusion, fertilisation media affected cleavage rate, as well as subsequent embryo development, quality and hatching ability. SOF and TALP fertilisation media produced significantly more embryos of higher quality than M199.

  7. Preimplantation genetic diagnosis

    DEFF Research Database (Denmark)

    Bay, Bjorn; Ingerslev, Hans Jakob; Lemmen, Josephine Gabriela

    2016-01-01

    OBJECTIVE: To study whether women conceiving after preimplantation genetic diagnosis (PGD) and their children have greater risks of adverse pregnancy and birth outcomes compared with children conceived spontaneously or after IVF with or without intracytoplasmic sperm injection (ICSI). DESIGN...

  8. Expression of intracellular interferon-alpha confers antiviral properties in transfected bovine fetal fibroblasts and does not affect the full development of SCNT embryos.

    Directory of Open Access Journals (Sweden)

    Dawei Yu

    Full Text Available Foot-and-mouth disease, one of the most significant diseases of dairy herds, has substantial effects on farm economics, and currently, disease control measures are limited. In this study, we constructed a vector with a human interferon-α (hIFN-α (without secretory signal sequence gene cassette containing the immediate early promoter of human cytomegalovirus. Stably transfected bovine fetal fibroblasts were obtained by G418 selection, and hIFN-α transgenic embryos were produced by somatic cell nuclear transfer (SCNT. Forty-six transgenic embryos were transplanted into surrogate cows, and five cows (10.9% became pregnant. Two male cloned calves were born. Expression of hIFN-α was detected in transfected bovine fetal fibroblasts, transgenic SCNT embryos, and different tissues from a transgenic SCNT calf at two days old. In transfected bovine fetal fibroblasts, expression of intracellular IFN-α induced resistance to vesicular stomatitis virus infection, increased apoptosis, and induced the expression of double-stranded RNA-activated protein kinase gene (PKR and the 2'-5'-oligoadenylate synthetase gene (2'-5' OAS, which are IFN-inducible genes with antiviral activity. Analysis by qRT-PCR showed that the mRNA expression levels of PKR, 2'-5' OAS, and P53 were significantly increased in wild-type bovine fetal fibroblasts stimulated with extracellular recombinant human IFN-α-2b, showing that intracellular IFN-α induces biological functions similar to extracellular IFN-α. In conclusion, expression of intracellular hIFN-α conferred antiviral properties in transfected bovine fetal fibroblasts and did not significantly affect the full development of SCNT embryos. Thus, IFN-α transgenic technology may provide a revolutionary way to achieve elite breeding of livestock.

  9. Survival of vitrified in vitro-produced bovine embryos after a one-step warming in-straw cryoprotectant dilution procedure.

    Science.gov (United States)

    Caamaño, J N; Gómez, E; Trigal, B; Muñoz, M; Carrocera, S; Martín, D; Díez, C

    2015-03-15

    Vitrification is an alternative to slow-rate freezing for cryopreserving bovine embryos. However, this technology requires simplification if it is to be used under field conditions. The main objective of this work was to develop a new system for the direct transfer of vitrified embryos to be used under farm conditions. For this, three objectives were set: (1) to compare the effect of vitrification, using the cryologic vitrification method (CVM), and slow-rate freezing on bovine embryo development and quality; (2) to develop a one-step warming procedure for bovine in vitro-produced (IVP) vitrified (by CVM) embryos; and (3) to assess the effects on embryo survival of a new method for the direct transfer of vitrified IVP bovine blastocysts. In vitro-produced blastocysts were initially either vitrified by CVM or subjected to slow freezing to compare embryo survival and quality (experiment 1). No differences were detected between these cryopreservation techniques in terms of the survival and quality variables at 24 hours or in terms of the proteins expressed. However, at 48 hours the vitrified embryos showed higher hatching rates, greater total cell numbers, and lower apoptotic indices (P < 0.05). In experiment 2, CVM-vitrified IVP blastocysts were warmed by the conventional two-step or one-step warming procedure by incubating them at 41 °C in 0.25 M sucrose for 10 minutes, 0.15 M sucrose for 10 minutes, or 0.25 M sucrose for 5 minutes. In addition, embryo transfer (ET) was performed using vitrified embryos warmed by the one-step procedure in 0.25 M sucrose solution for 5 minutes. As a control group, IVP fresh embryos were transferred to recipient females. No differences were observed in embryo survival or total cell number between any of the warming procedures. Moreover, no significant differences for pregnancy at 60 days were found between the ET groups. In experiment 3, expanded IVP blastocysts were then either vitrified using a conventional or a

  10. Mitochondria-targeted DsRed2 protein expression during the early stage of bovine somatic cell nuclear transfer embryo development.

    Science.gov (United States)

    Park, Hyo-Jin; Min, Sung-Hun; Choi, Hoonsung; Park, Junghyung; Kim, Sun-Uk; Lee, Seunghoon; Lee, Sang-Rae; Kong, Il-Keun; Chang, Kyu-Tae; Koo, Deog-Bon; Lee, Dong-Seok

    2016-09-01

    Somatic cell nuclear transfer (SCNT) has been widely used as an efficient tool in biomedical research for the generation of transgenic animals from somatic cells with genetic modifications. Although remarkable advances in SCNT techniques have been reported in a variety of mammals, the cloning efficiency in domestic animals is still low due to the developmental defects of SCNT embryos. In particular, recent evidence has revealed that mitochondrial dysfunction is detected during the early development of SCNT embryos. However, there have been relatively few or no studies regarding the development of a system for evaluating mitochondrial behavior or dynamics. For the first time, in mitochondria of bovine SCNT embryos, we developed a method for the visualization of mitochondria and expression of fluorescence proteins. To express red fluorescence in mitochondria of cloned embryos, bovine ear skin fibroblasts, nuclear donor, were stably transfected with a vector carrying mitochondria-targeting DsRed2 gene tagged with V5 epitope (mito-DsRed2-V5 tag) using lentivirus-mediated gene transfer because of its ability to integrate in the cell genome and the potential for long-term transgene expression in the transduced cells and their dividing cells. From western blotting analysis of V5 tag protein using mitochondrial fraction and confocal microscopy of red fluorescence using SCNT embryos, we found that the mitochondrial expression of the mito-DsRed2 protein was detected until the blastocyst stage. In addition, according to image analysis, it may be suggested possible use of the system for visualization of mitochondrial localization and evaluation of mitochondrial behaviors or dynamics in early development of bovine SCNT embryos.

  11. Effects of vitrification medium composition on the survival of bovine in vitro produced embryos, following in straw-dilution, in vitro and in vivo following transfer.

    Science.gov (United States)

    Pugh, P A; Tervit, H R; Niemann, H

    2000-02-28

    This study examined the effects of adding a macromolecule, polyvinylpyrrolidone (10% PVP) and a sugar (0.3 M trehalose) to vitrification solutions (VS) containing either one (40% ethylene glycol [EG], two (25% EG+25% DMSO) or three (20% EG+20% DMSO+10% 1, 3-butanediol [BD]) permeable cryoprotectants on the survival and hatching of IVP bovine embryos, following vitrification, warming and in-straw cryoprotectant dilution. Grade 1 and 2 compact morulae and blastocysts were selected on Day 7 (Day 0=IVF) of culture in SOFaaBSA and equilibrated for 10 min at room temperature in 10% EG. Following exposure, for up to 1 min at 4 degrees C, to one of the above VS (with or without PVP+trehalose), the embryos were loaded into straws and immersed in liquid nitrogen. Following warming and in-straw cryoprotectant dilution, the embryos were cultured for 48 h to assess hatching. There was no effect of VS on the survival of embryos after 24 h, however fewer compact morulae than blastocysts survived after 24 h (24% vs. 75%; Pvitrification (fresh vs. vitrified; 1/5 [20%] vs. 3/18 [17]). These data demonstrate that a VS comprising three cryoprotectants, rather than one, enables more embryos to hatch during post-thaw culture and that the survival, following direct transfer of these vitrified embryos, is not different to non-vitrified embryos.

  12. Application of preimplantation genetic diagnosis in equine blastocysts

    Directory of Open Access Journals (Sweden)

    Grady ST

    2016-08-01

    Full Text Available Pre-implantation genetic diagnosis (PGD is a procedure used to screen in vitroproduced embryos or embryos recovered after uterine flush to determine genetic traits by DNA testing prior to transfer into the uterus. Biopsy methods to obtain a sample of cells for genetic analysis before implantation have been successful in small embryos (morulae and blastocysts 300 µm diameter. The successful biopsy of expanded equine blastocysts via micromanipulation, with subsequent normal pregnancy rates, was first reported in 2010. Direct PCR may be performed when evaluating only one gene, such as for embryo sexing, while whole genome amplification is effective for subsequent multiplex PCR of multiple genes.

  13. Membrane lipid profile monitored by mass spectrometry detected differences between fresh and vitrified in vitro-produced bovine embryos.

    Science.gov (United States)

    Leão, Beatriz C S; Rocha-Frigoni, Nathália A S; Cabral, Elaine C; Franco, Marcos F; Ferreira, Christina R; Eberlin, Marcos N; Filgueiras, Paulo R; Mingoti, Gisele Z

    2015-10-01

    This study aimed to evaluate the impact of vitrification on membrane lipid profile obtained by mass spectrometry (MS) of in vitro-produced bovine embryos. Matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) has been used to obtain individual embryo membrane lipid profiles. Due to conditions of analysis, mainly membrane lipids, most favorably phosphatidylcholines (PCs) and sphingomyelins (SMs) have been detected. The following ions described by their mass-to-charge ratio (m/z) and respective attribution presented increased relative abundance (1.2-20×) in the vitrified group: 703.5 [SM (16:0) + H]+; 722.5 [PC (40:3) + Na]+; 758.5 [PC (34:2) + H]+; 762.5 [PC (34:0) + H]+; 790.5 [PC (36:0) + H]+ and 810.5 [PC (38:4) + H]+ and/or [PC (36:1) + Na]+. The ion with a m/z 744.5 [PCp (34:1) and/or PCe (34:2)] was 3.4-fold more abundant in the fresh group. Interestingly, ions with m/z 722.5 or 744.5 indicate the presence of lipid species, which are more resistant to enzymatic degradation as they contain fatty acyl residues linked through ether type bonds (alkyl ether or plasmalogens, indicated by the lowercase 'e' and 'p', respectively) to the glycerol structure. The results indicate that cryopreservation impacts the membrane lipid profile, and that these alterations can be properly monitored by MALDI-MS. Membrane lipids can therefore be evaluated by MALDI-MS to monitor the effect of cryopreservation on membrane lipids, and to investigate changes in lipid profile that may reflect the metabolic response to the cryopreservation stress or changes in the environmental conditions.

  14. Array comparative genomic hybridization: its role in preimplantation genetic diagnosis.

    Science.gov (United States)

    Fiorentino, Francesco

    2012-08-01

    Embryo assessment is a crucial component to the success of IVF. A high rate of embryos produced in vitro present chromosomal abnormalities and have reduced potential for achieving a viable pregnancy. The use of preimplantation genetic diagnosis by array comparative genomic hybridization, for comprehensive aneuploidy screening of embryos, to improve IVF outcomes, is reviewed. Data from comprehensive aneuploidy screening of embryos showed that aneuploidies may occur in any of the 24 chromosomes, indicating that aneuploidy screening of all chromosomes is necessary to determine whether an embryo is chromosomally normal. Initial studies on clinical application of this technology have documented improved pregnancy outcomes following transfer of screened embryos. The optimal stage of preimplantation development at which preimplantation genetic screening (PGS) should be performed still remains to be determined. Although clinical results have been promising, further evidence is required to establish whether PGS results in enhanced live birth rate, and if this is the case, to identify which patients may benefit from the procedure. The results from several ongoing randomized controlled trials, performed at different cell biopsy stage and categories of patients, will provide the data needed to accept or reject the clinical efficacy of PGS.

  15. Forskolin effect on the cryosurvival of in vitro-produced bovine embryos in the presence or absence of fetal calf serum.

    Science.gov (United States)

    Paschoal, Daniela Martins; Sudano, Mateus José; Guastali, Midyan Daroz; Dias Maziero, Rosiára Rosária; Crocomo, Letícia Ferrari; Oña Magalhães, Luis Carlos; da Silva Rascado, Tatiana; Martins, Alicio; da Cruz Landim-Alvarenga, Fernanda

    2014-05-01

    The objective of this study was to assess the viability and cryotolerance of zebu embryos produced in vitro with or without the addition of fetal calf serum (FCS) and forskolin (F). Embryos produced in vivo were used as a control. Presumptive zygotes were cultured in modified synthetic oviductal fluid supplemented with amino acids (SOFaa), bovine serum albumin (BSA) and with (2.5%) or without (0%) FCS. On day 6 of growth, the embryos from each group were divided into treatments with or without 10 μM F to induce embryonic lipolysis, comprising a total of four experimental groups: 2.5% FCS, 0% FCS, 2.5% + F and 0% + F. For vitrification, embryos were exposed to vitrification solution 1 (5 M EG (ethylene glycol)) for 3 min and then transferred to vitrification solution 2 (7 M EG, 0.5 M galactose solution and 18% (w/v) Ficoll 70) before being introduced to liquid nitrogen. The presence of FCS in the culture medium resulted in the production of embryos with a similar rate of damaged cells compared with in vivo-produced embryos. After vitrification, the 2.5% FCS group had a significantly higher rate of damaged cells when compared with the other groups (P vitrification.

  16. Effects of fatty acid-free bovine serum albumin and fetal calf serum supplementing repair cultures on pre- and post-warm viability of biopsied bovine embryos produced in vitro.

    Science.gov (United States)

    Yotsushima, Kenji; Sakaguchi, Minoru; Shimizu, Manabu; Okimura, Tomoko; Izaike, Yoshiaki

    2004-08-01

    The objective of this study was to investigate the influence of fatty acid-free bovine serum albumin (BSA) or fetal calf serum (FCS) on the re-expansion of biopsied blastocysts and post-warm viability of subsequently vitrified embryos. Firstly, blastocysts produced in vitro were biopsied at Day 7 and cultured to allow repair in TCM199 with 0.3% BSA or 5% FCS for 24 h. The re-expansion rates and mean total numbers of cells of the re-expanded embryos after the repair culture with BSA were almost the same as that with FCS. Secondly, after biopsied embryos were similarly cultured for repair with BSA or FCS, re-expanded embryos were selected for vitrification. After warming and exposure to 0.5 M sucrose with 20% FCS in mPBS, the embryos were cultured in TCM199 with 5% FCS for 24 h. The re-expansion rate and mean total number of cells in re-expanded blastocysts in the BSA treatment group (97.4 +/- 2.9% and 106 +/- 42) was significantly higher than that in the FCS treatment group (51.6 +/- 9.1% and 61 +/- 38), respectively (Pbovine biopsied blastocysts; but, compared with BSA supplementation, FCS supplementation during repair culture reduces the post-warm viability of biopsied and subsequently vitrified embryos.

  17. MODELAGEM BIOECONÔMICA DA TRANSFERÊNCIA DE EMBRIÕES EM BOVINOS BIOECONOMIC MODEL IN BOVINE EMBRYO TRANSFER

    Directory of Open Access Journals (Sweden)

    Renato Travassos Beltrame

    2010-04-01

    Full Text Available

    O objetivo deste trabalho foi desenvolver um modelo matemático orientado a eventos de simulação, para auxiliar tomadas de decisão relativas à transferência de embriões em bovinos, considerando-se as dinâmicas de dois componentes da transferência de embriões: receptoras e embriões. Na simulação, não se avaliaram respostas individuais de doadoras a coletas consecutivas e eventos correspondentes na transferência de embriões. Simulou-se o mesmo protocolo para superovulação a todas as doadoras. Receptoras foram sincronizadas simulando-se o uso de prostaglandina. O número de embriões viáveis produzido por doadora e sua variabilidade tiveram como base um processo aleatório de simulação de Monte Carlo, que pressupôs uma distribuição exponencial negativa de densidade de probabilidade. Custos e receitas foram inseridos no modelo por meio de um cenário-base para calcular indicadores econômicos de rentabilidade. A análise sugeriu a impraticabilidade da atividade, se realizada diante do cenário proposto (VPL – R$: 57.596,69. A partir do cenário proposto, o custo médio estimado foi de R$ 1.178,19, e de R$ 980,03, para se obter uma prenhez a partir de uma situação otimizada, sugerida pelo modelo (5/100; 5/190.

    PALAVRAS-CHAVES: Otimização, receptoras, simulação, transferência de embriões, viabilidade econômica.

    A simulation model related to embryo transfer programs in bovine was carried out through a mathematical model directed to events, considering the dynamic of two resources: recipients and embryos. Individual answers of donors to consecutive collections and corresponding events in embryo transfer were not evaluated. The same protocol for superovulation was simulated for all the donor collections, using similar doses of hormones and drugs for all the animals. Recipients were synchronized using prostaglandin. Meantime, the number of viable embryos produced by donor and its variability were based at

  18. A microfluidic system supports single mouse embryo culture leading to full-term development

    NARCIS (Netherlands)

    Esteves, Telma Cristina; van Rossem, F.; Nordhoff, Verena; Schlatt, Stefan; Boiani, Michele; le Gac, Severine

    2013-01-01

    The present study demonstrates the feasibility of application of a microfluidic system for in vitro culture of pre-implantation mouse embryos, with subsequent development to full-term upon embryo transfer. Specifically, embryos cultured in groups in nL volume chambers achieve pre-implantation

  19. Paternal breed effects on expression of IGF-II, BAK1 and BCL2-L1 in bovine preimplantation embryos

    DEFF Research Database (Denmark)

    Valleh, Mehdi Vafaye; Tahmoorespur, Mojtaba; Joupari, Morteza Daliri

    2015-01-01

    reaction using the comparative Cp method. Our data revealed that the paternal breed did not influence cleavage rate, blastocyst rate and relative abundance of IGF-II, BAK1 and BCL2-L1 in day 8 blastocysts (P > 0.05). Nevertheless, IGF-II expression levels were highly correlated with embryonic developmental...

  20. MicroRNA-130b is involved in bovine granulosa and cumulus cells function, oocyte maturation and blastocyst formation.

    Science.gov (United States)

    Sinha, Pritam Bala; Tesfaye, Dawit; Rings, Franca; Hossien, Munir; Hoelker, Michael; Held, Eva; Neuhoff, Christaine; Tholen, Ernst; Schellander, Karl; Salilew-Wondim, Dessie

    2017-06-19

    Oocyte maturation and preimplantation embryo development are controlled by array of genes that are post-transcriptionally regulated by microRNAs. With respect to this, previously, we identified altered expression of microRNA-130b (miR-130b) during oocyte maturation. Here, we aimed to investigate the role of miR-130b in bovine granulosa and cumulus cell function, oocyte maturation and preimplantation embryo development using gain- and loss-of- function approach. For this study, the granulosa cells, cumulus cells and the oocytes were collected from ovaries obtained from slaughterhouse. The genes targeted by miR-130b were identified using dual-luciferase reporter assay. The role of miR-130b in granulosa and cumulus cell function was investigated by increasing and inhibiting its expression in in vitro cultured cells using miR-130b precursor and inhibitor, respectively while the role of miR-130b on oocyte development, immature oocytes were microinjected with miR-130b precursor and inhibitor and the polar body extrusion, the proportion of oocytes reaching to metaphase II stage and the mitochondrial were determined in each oocyte group 22 h after microinjection. Moreover, to investigate the role of miR-130b during preimplantation embryo development, zygote stage embryos were microinjected with miR-130b precursor or inhibitor and the cleavage rate, morula and blastocyst formation was analyzed in embryos derived from each zygote group after in vitro culture. The luciferase assay showed that SMAD5 and MSK1 genes were identified as the direct targets of miR-130b. Overexpression of miR-130b increased the granulosa and cumulus cell proliferation, while inhibition showed the opposite phenotype. Apart from these, modulation of miR-130b altered the lactate production and cholesterol biosynthesis in cumulus cells. Furthermore, inhibition of miR-130b expression during oocyte in vitro maturation reduced the first polar body extrusion, the proportion of oocytes reaching to metaphase

  1. bovine

    African Journals Online (AJOL)

    of various breeds under local conditions of management. (Hale, 1974b). AdditionaIly, this procedure has been used to assess the production of LH by the bovine anterior pituitary in vitro and to study the relationships between this production and the activity of the pineal- hypothalamic axis (Hayes, Knight & Symington, 1974;.

  2. Effect of culture system on survival rate of vitrified bovine embryos produced in vitro.

    Science.gov (United States)

    Shirazi, A; Nazari, H; Ahmadi, E; Heidari, B; Shams-Esfandabadi, N

    2009-12-01

    This study was designed to evaluate the effect of in vitro culture system on bovine blastocyst yield and quality after vitrification. In Experiment 1, IVM/IVF zygotes were allocated to three culture conditions: (I) Oviductal cells-SOF (OCM-SOF); (II) Oviductal cells-TCM (OCM-TCM); and (III) SOF for 8 days. There was no significant difference between blastocyst rates among groups. In Experiment 2, the IVP-blastocysts in three above culture conditions were vitrified within groups segregated according to age (Day 7 and 8) and blastocoelic cavity size (early and expanded blastocysts). A trend of higher survival rate was obtained in vitrified/warmed early blastocysts compared with expanded ones, so that the difference in OCM-TCM group was significant (P<0.001). Higher survival and hatching rates (P<0.001) were obtained in OCM-SOF and OCM-TCM groups (co-culture) compared with SOF group and the age of blastocyst had no effect on post-thaw survival and hatching rates. In Experiment 3, after staining of blastocysts, in fresh blastocysts the highest number of trophectoderm cells was observed in OCM-TCM group and the number of inner cell mass (ICM) was higher in co-culture groups than SOF group (P<0.001). In vitrified/warmed blastocysts the number of ICM and trophectoderm cells in co-culture groups was higher than SOF group (P<0.001) except for the ICM of expanded blastocysts. In conclusion, in our culture conditions, the blastocyst yield is not influenced by culture system, while the cryotolerance of IVP-blastocysts is positively influenced by the presence of somatic cells. Moreover, the expanded blastocysts are more susceptible to cryoinjury than early blastocysts.

  3. The effects of macromolecular and serum supplements and oxygen tension during bovine in vitro procedures on kinetics of oocyte maturation and embryo development.

    Science.gov (United States)

    Mingoti, Gisele Zoccal; Castro, Viviane Sggobi Dias Caiado; Méo, Simone Cristina; Sá Barretto, Letícia Siqueira; Garcia, Joaquim Mansano

    2011-06-01

    Aiming to standardize in vitro production of bovine embryos and to obtain supplements to replace serum in culture media, this study evaluated the nuclear maturation kinetics and embryonic development in bovine after in vitro maturation (IVM) and culture (IVC) with several macromolecules (animal origin: bovine serum albumin (BSA), fetal calf serum (FCS); synthetic: polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), Ficoll, and Knockout) at two oxygen tensions (20% and 5% O(2)). Regarding nuclear kinetics, neither the presence of the expected stage (metaphase I, transition anaphase to telophase, and metaphase II) at each evaluation moment (6, 18, and 24 h after IVM, respectively) nor the accelerated polar body emission (at 18 h after IVM) related developmental competence to blastocyst stage when different supplements were compared. Independently of supplement, cleavage rates at 20% O(2) (61.6-79.2%) were higher than at 5% O(2) (38.9-58.7%). At 20% O(2), higher blastocyst and hatching rates, respectively, were obtained in treatments BSA, FCS, Knockout, and control group (IVM with FCS and IVC with BSA + FCS, 14.0-23.5% and 6.8-15.4%) in comparison to PVA, PVP, and Ficoll (0%). The same was observed at 5% O(2) for blastocyst rates with BSA, FCS, Knockout, and control (5.4-16.8%) and for hatching rates with BSA, FCS, and control (2.0-11.1%). We can conclude that producing bovine embryos at 20% O(2) during the entire IVP process resulted in higher developmental rates than at 5% O(2). In addition, while defined macromolecules PVA, PVP, and Ficoll were not suitable for embryonic development, the synthetic serum Knockout was able to replace serum and albumin for IVP in bovine at 20% O(2).

  4. Effect of the time interval between fusion and activation on epigenetic reprogramming and development of bovine somatic cell nuclear transfer embryos.

    Science.gov (United States)

    Liu, Jun; Wang, Yongsheng; Su, Jianmin; Wang, Lijun; Li, Ruizhe; Li, Qian; Wu, Yongyan; Hua, Song; Quan, Fusheng; Guo, Zekun; Zhang, Yong

    2013-04-01

    Previous studies have shown that the time interval between fusion and activation (FA interval) play an important role in nuclear remodeling and in vitro development of somatic cell nuclear transfer (SCNT) embryos. However, the effects of FA interval on the epigenetic reprogramming and in vivo developmental competence of SCNT embryos remain unknown. In the present study, the effects of different FA intervals (0 h, 2 h, and 4 h) on the epigenetic reprogramming and developmental competence of bovine SCNT embryos were assessed. The results demonstrated that H3 lysine 9 (H3K9ac) levels decreased rapidly after fusion in all three groups. H3K9ac was practically undetectable 2 h after fusion in the 2-h and 4-h FA interval groups. However, H3K9ac was still evidently detectable in the 0-h FA interval group. The H3K9ac levels increased 10 h after fusion in all three groups, but were higher in the 2-h and 4-h FA interval groups than that in the 0-h FA interval group. The methylation levels of the satellite I region in day-7 blastocysts derived from the 2-h or 4-h FA interval groups was similar to that of in vitro fertilization blastocysts and is significantly lower than that of the 0-h FA interval group. SCNT embryos derived from 2-h FA interval group showed higher developmental competence than those from the 0-h and 4-h FA interval groups in terms of cleavage rate, blastocyst formation rate, apoptosis index, and pregnancy and calving rates. Hence, the FA interval is an important factor influencing the epigenetic reprogramming and developmental competence of bovine SCNT embryos.

  5. Ratio of proliferating cell nuclear antigen-positive nuclei to total cell number is higher in day 7 than in day 8 vitrified in vitro-produced bovine embryos.

    Science.gov (United States)

    Markkula, M; Räty, M; Jauhiainen, L; Paranko, J; Raula, J; Makarevich, A

    2001-07-01

    The aim of the present study was to find a reliable functional criterion for the evaluation of the proliferation potential of bovine in vitro-produced embryos. We used immunocytochemical detection of proliferating cell nuclear antigen (PCNA) combined with propidium iodide (PI) staining and subsequent confocal laser scanning microscopy together with routine morphological evaluation under a stereomicroscope to study fresh Day 7, 8, and 9, and cryopreserved Day 7 and 8 embryos. The ratio of PCNA/PI-positive nuclei was equal in fresh Day 7 and Day 8 embryos and significantly lower in Day 9 embryos. In general, Day 7 embryos tolerated the cryopreservation treatments better than Day 8 embryos. Vitrification in normal straws was especially detrimental to Day 8 embryos. In fresh Day 7 and 8 embryos, the PCNA results were in agreement with stereomicroscopic evaluation. However, in Day 9 fresh and in Day 7 and 8 treated embryos, the missing PCNA revealed disorders that were not observed under morphological evaluation. PCNA immunocytochemistry is an effective method to obtain information about the functional state of nuclei. The ratio of PCNA-positive nuclei can provide more information and numerical data about the developmental potential of bovine embryos after cryopreservation.

  6. Genome-Wide DNA Methylation Patterns of Bovine Blastocysts Developed In Vivo from Embryos Completed Different Stages of Development In Vitro.

    Directory of Open Access Journals (Sweden)

    Dessie Salilew-Wondim

    imprinting and chromosome segregation in IVP blastocyst groups. Furthermore, 1.6, 3.4, 3.9 and 9.4% of the differentially methylated regions that were overlapped to the transcriptome profile data were negatively correlated with the gene expression patterns in ZY, 4C, 16C and IVP blastocyst groups, respectively. Therefore, this finding indicated that suboptimal culture condition during preimplantation embryo development induced changes in the DNA methylation landscape of the resulting blastocysts in a stage dependent manner and the altered DNA methylation pattern was only partly explained the observed aberrant gene expression patterns of the blastocysts.

  7. The effects of conjugated linoleic acid isomers cis-9,trans-11 and trans-10,cis-12 on in vitro bovine embryo production and cryopreservation.

    Science.gov (United States)

    Absalón-Medina, V A; Bedford-Guaus, S J; Gilbert, R O; Siqueira, L C; Esposito, G; Schneider, A; Cheong, S H; Butler, W R

    2014-10-01

    Conjugated linoleic acid (CLA) isomers can affect the lipid profile and signaling of cells and thereby alter their function. A total of 5,700 bovine oocytes were used in a structured series of experiments to test the effects of CLA cis-9,trans-11 and CLA trans-10,cis-12 in vitro. In experiment 1, high doses of each CLA isomer during in vitro maturation (IVM) were compared with high or low doses during the entire in vitro culture (IVC) of parthenogenetic embryos. High doses of the CLA isomers ranged from 50 to 200 μM and low doses were 15 and 25 μM. In experiment 2, the low doses of each CLA isomer were tested during IVM/IVC on embryos produced by in vitro fertilization (IVF). Experiment 3 compared the effects of 15 μM doses of each CLA isomer during IVM or IVC of IVF embryos. In experiment 4, post-rewarming survival rates and blastomere counts were assessed for embryos supplemented with each CLA isomer during IVM or for 36 h before vitrification. In experiment 1, when either CLA isomer was provided only during IVM, we observed no effects on overall rates of maturation, cleavage, or blastocysts (92.2 ± 1.6%, 78.3 ± 4.1%, and 28.9 ± 5.1%, respectively). However, high doses of each CLA isomer, but not low doses, during the entire embryo culture period decreased blastocyst rates (5-20%) in a dose-dependent manner. Cleavage rates improved with 15 or 50 μM CLA trans-10,cis-12. Progesterone concentrations in maturation media were significantly increased by high doses of each CLA isomer compared with control, but low doses of CLA isomers had no effect. In experiment 2 with IVF embryos, low doses of each CLA isomer did not alter cleavage rates (average 84.9 ± 1.9%) and only 25 μM CLA trans-10,cis-12 during IVC reduced blastocyst rates below those of controls (25.5 ± 2.1 vs. 38.2 ± 2.3%). The lipid content of embryos was increased and relative expression of the BIRC5 (baculoviral IAP repeat containing 5) gene was depressed by CLA trans-10,cis-12. In experiment 3

  8. Myo-Inositol Safety in Pregnancy: From Preimplantation Development to Newborn Animals

    Directory of Open Access Journals (Sweden)

    Nilay Kuşcu

    2016-01-01

    Full Text Available Myo-inositol (myo-Ins has a physiological role in mammalian gametogenesis and embryonic development and a positive clinical impact on human medically assisted reproduction. We have previously shown that mouse embryo exposure to myo-Ins through preimplantation development in vitro increases proliferation activity and blastocyst production, representing an improvement in culture conditions. We have herein investigated biochemical mechanisms elicited by myo-Ins in preimplantation embryos and evaluated myo-Ins effects on postimplantation/postnatal development. To this end naturally fertilized embryos were cultured in vitro to blastocyst in the presence or absence of myo-Ins and analyzed for activation of the PKB/Akt pathway, known to modulate proliferation/survival cellular processes. In parallel, blastocyst-stage embryos were transferred into pseudopregnant females and allowed to develop to term and until weaning. Results obtained provide evidence that myo-Ins induces cellular pathways involving Akt and show that (a exposure of preimplantation embryos to myo-Ins increases the number of blastocysts available for uterine transfer and of delivered animals and (b the developmental patterns of mice obtained from embryos cultured in the presence or absence of myo-Ins, up to three weeks of age, overlap. These data further identify myo-Ins as a possibly important supplement for human preimplantation embryo culture in assisted reproduction technology.

  9. Effect of mating between the donor cow and bull (Holstein versus Gyr on the in vitro production of bovine embryos

    Directory of Open Access Journals (Sweden)

    Ana Paula Toledo Barbosa da Silva

    2015-03-01

    Full Text Available The objective of this study was to evaluate the effect of the breed of the oocyte donor cow and bull (Holstein versus Gyr on in vitro production (IVP parameters of bovine embryos comparing the mean number of recovered oocytes and oocytes suitable for culture, the rate of suitable oocytes, and cleavage and blastocyst rates. Data from 1,000 follicular aspiration sessions (OPU, including 500 in donor cows of the Holstein breed and 500 of the Gyr breed, were collected. The results were analyzed by the unpaired Student t-test and chi-square test, adopting a level of significance of 5%. The mean number and standard deviation of recovered oocytes and oocytes suitable for culture were 15.1±13.0 and 8.7±7.6 for the Holstein breed and 15.5±11.9 and 9.1±7.9 for the Gyr breed. The rates of suitable oocytes were 57.7% and 58.5% for Holstein and Gyr breeds, respectively. A significant difference between breeds was observed for the number of oocytes suitable for culture (P<0.05, but not for the number of recovered oocytes or rates of suitable oocytes (P>0.05. Similarly, the breed of the oocyte donor cow and bull influenced cleavage and blastocyst rates (P<0.05. The cleavage rates were 65.7, 60.3, 59.6 and 56.5% for the combinations (donor breed x bull breed Holstein x Holstein (G1, Holstein x Gyr (G2, Gyr x Holstein (G3 and Gyr x Gyr (G4, respectively, with G1>G2, G1>G3, G1>G4, G2=G3, G2>G4, and G3>G4. The blastocyst rates were 28.1, 33.3, 26.8 and 31.0%, respectively, with G1>G2, G1=G3, G1

  10. Effects of Two Types of Melatonin-Loaded Nanocapsules with Distinct Supramolecular Structures: Polymeric (NC) and Lipid-Core Nanocapsules (LNC) on Bovine Embryo Culture Model.

    Science.gov (United States)

    Komninou, Eliza Rossi; Remião, Mariana Härter; Lucas, Caroline Gomes; Domingues, William Borges; Basso, Andrea Cristina; Jornada, Denise Soledade; Deschamps, João Carlos; Beck, Ruy Carlos Ruver; Pohlmann, Adriana Raffin; Bordignon, Vilceu; Seixas, Fabiana Kömmling; Campos, Vinicius Farias; Guterres, Silvia Stanisçuaski; Collares, Tiago

    2016-01-01

    Melatonin has been used as a supplement in culture medium to improve the efficiency of in vitro produced mammalian embryos. Through its ability to scavenge toxic oxygen derivatives and regulate cellular mRNA levels for antioxidant enzymes, this molecule has been shown to play a protective role against damage by free radicals, to which in vitro cultured embryos are exposed during early development. In vivo and in vitro studies have been performed showing that the use of nanocapsules as active substances carriers increases stability, bioavailability and biodistribution of drugs, such as melatonin, to the cells and tissues, improving their antioxidant properties. These properties can be modulated through the manipulation of formula composition, especially in relation to the supramolecular structures of the nanocapsule core and the surface area that greatly influences drug release mechanisms in biological environments. This study aimed to evaluate the effects of two types of melatonin-loaded nanocapsules with distinct supramolecular structures, polymeric (NC) and lipid-core (LNC) nanocapsules, on in vitro cultured bovine embryos. Embryonic development, apoptosis, reactive oxygen species (ROS) production, and mRNA levels of genes involved in cell apoptosis, ROS and cell pluripotency were evaluated after supplementation of culture medium with non-encapsulated melatonin (Mel), melatonin-loaded polymeric nanocapsules (Mel-NC) and melatonin-loaded lipid-core nanocapsules (Mel-LNC) at 10-6, 10-9, and 10-12 M drug concentrations. The highest hatching rate was observed in embryos treated with 10-9 M Mel-LNC. When compared to Mel and Mel-NC treatments at the same concentration (10-9 M), Mel-LNC increased embryo cell number, decreased cell apoptosis and ROS levels, down-regulated mRNA levels of BAX, CASP3, and SHC1 genes, and up-regulated mRNA levels of CAT and SOD2 genes. These findings indicate that nanoencapsulation with LNC increases the protective effects of melatonin

  11. Expression profile of genes as indicators of developmental competence and quality of in vitro fertilization and somatic cell nuclear transfer bovine embryos.

    Directory of Open Access Journals (Sweden)

    Maria Jesús Cánepa

    Full Text Available Reproductive biotechnologies such as in vitro fertilization (IVF and somatic cell nuclear transfer (SCNT enable improved reproductive efficiency of animals. However, the birth rate of in vitro-derived embryos still lags behind that of their in vivo counterparts. Thus, it is critical to develop an accurate evaluation and prediction system of embryo competence, both for commercial purposes and for scientific research. Previous works have demonstrated that in vitro culture systems induce alterations in the relative abundance (RA of diverse transcripts and thus compromise embryo quality. The aim of this work was to analyze the RA of a set of genes involved in cellular stress (heat shock protein 70-kDa, HSP70, endoplasmic reticulum (ER stress (immunoglobulin heavy chain binding protein, Bip; proteasome subunit β5, PSMB5 and apoptosis (BCL-2 associated X protein, Bax; cysteine aspartate protease-3, Caspase-3 in bovine blastocysts produced by IVF or SCNT and compare it with that of their in vivo counterparts. Poly (A + mRNA was isolated from three pools of 10 blastocysts per treatment and analyzed by real-time RT-PCR. The RA of three of the stress indicators analyzed (Bax, PSMB5 and Bip was significantly increased in SCNT embryos as compared with that of in vivo-derived blastocysts. No significant differences were found in the RA of HSP70 and Caspase-3 gene transcripts. This study could potentially complement morphological analyses in the development of an effective and accurate technique for the diagnosis of embryo quality, ultimately aiding to improve the efficiency of assisted reproductive techniques (ART.

  12. Obesity does not aggravate vitrification injury in mouse embryos: a prospective study

    Directory of Open Access Journals (Sweden)

    Ma Wenhong

    2012-08-01

    Full Text Available Abstract Background Obesity is associated with poor reproductive outcomes, but few reports have examined thawed embryo transfer in obese women. Many studies have shown that increased lipid accumulation aggravates vitrification injury in porcine and bovine embryos, but oocytes of these species have high lipid contents (63 ng and 161 ng, respectively. Almost nothing is known about lipids in human oocytes except that these cells are anecdotally known to be relatively lipid poor. In this regard, human oocytes are considered to be similar to those of the mouse, which contain approximately 4 ng total lipids/oocyte. To date, no available data show the impact of obesity on vitrification in mouse embryos. The aim of this study was to establish a murine model of maternal diet-induced obesity and to characterize the effect of obesity on vitrification by investigating the survival rate and embryo developmental competence after thawing. Methods Prospective comparisons were performed between six–eight-cell embryos from obese and normal-weight mice and between fresh and vitrified embryos. Female C57BL/6 mice were fed standard rodent chow (normal-weight group or a high-fat diet (obese group for 6 weeks. The mice were mated, zygotes were collected from oviducts and cultured for 3 days, and six–eight-cell embryos were then selected to assess lipid content in fresh embryos and to evaluate differences in apoptosis, survival, and development rates in response to vitrification. Results In fresh embryos from obese mice, the lipid content (0.044 vs 0.030, Pvs.9.3%, Pvs. 93.1%, P Conclusions This study demonstrated that differences in survival and developmental rates between embryos from obese and normal-weight mice were eliminated after vitrification. Thus, maternal obesity does not aggravate vitrification injury, but obesity alone greatly impairs pre-implantation embryo survival and development.

  13. Effect of two activation treatments and age of blastomere karyoplasts on in vitro development of bovine nuclear transfer embryos

    DEFF Research Database (Denmark)

    Booth, P J; Holm, P; Vajta, G

    2001-01-01

    .7 +/- 5.1 vs. 31.4 +/- 4.5 [mean +/- SEM]). In contrast, nuclear transfer blastocyst rates per fused embryo were lower (P rates using day 3, 4, and 5 donors and using CHX and DMAP activation treatments were 31.9 +/- 5.0, 31.7 +/- 6.2, 20.......4 +/- 7.3 and 27.8 +/- 4.7, 20.1 +/- 7.5, 12.7 +/- 8.3, respectively. Blastocyst rate per fused embryo was negatively correlated (P = 0.0091) with the total number of blastomeres per donor embryo. Despite this inverse relationship, the calculated potential blastocyst yield per donor embryo was positively...

  14. Effect of container, vitrification volume and warming solution on cryosurvival of in vitro-produced bovine embryos.

    Science.gov (United States)

    Rios, G L; Mucci, N C; Kaiser, G G; Alberio, R H

    2010-03-01

    The aim of the present research was to develop a low cost and easy to perform vitrification method for in vitro-produced cattle embryos. Effect of container material was evaluated (plastic straw compared to glass capillary, experiment 1), two volume sample (1 compared to 0.5 microL, experiment 2) and warming solution composition medium (Tissue Culture Medium 199 (TCM-199) compared to phosphate buffered saline (PBS), experiment 3) as modifications of the open pulled straw (OPS) system in order to reduce embryo damage caused by exposure to cold. In all experiments, day 7 and expanded blastocysts of cattle were exposed to the vitrification solution 1 for 3 min and 30s in solution 2. After this, embryos were placed in a droplet and loaded in a narrow end container, and immediately submerged into liquid nitrogen. For warming, vitrified embryos were plunged into warming solution 1 for 3 min, and transferred into warming solution 2 for 1 min. Fresh embryos kept in culture were used as control group. Hatching rates were recorded in all cases at day 13. In experiment 1 there was no significant effect of container material on hatching rates. Postwarming survival rate of vitrified embryos was lower than control (27.5% plastic straws, 18.9% glass capillary and 80.5% control, Pstraw (OPS) procedure, and that PBS can replace TCM-199 in warming solutions, but lesser hatching rates should be expected.

  15. Hyperspectral microscopy can detect metabolic heterogeneity within bovine post-compaction embryos incubated under two oxygen concentrations (7% versus 20%).

    Science.gov (United States)

    Sutton-McDowall, Melanie L; Gosnell, Martin; Anwer, Ayad G; White, Melissa; Purdey, Malcolm; Abell, Andrew D; Goldys, Ewa M; Thompson, Jeremy G

    2017-10-01

    Can we separate embryos cultured under either 7% or 20% oxygen atmospheres by measuring their metabolic heterogeneity? Metabolic heterogeneity and changes in metabolic profiles in morula exposed to two different oxygen concentrations were not detectable using traditional fluorophore and two-channel autofluorescence but were detectable using hyperspectral microscopy. Increased genetic and morphological blastomere heterogeneity is associated with compromised developmental competence of embryos and currently forms the basis for embryo scoring within the clinic. However, there remains uncertainty over the accuracy of current techniques, such as PGS and time-lapse microscopy, to predict subsequent pregnancy establishment. The impact of two oxygen concentrations (7% = optimal and 20% = stressed) during post-fertilisation embryo culture was assessed. Cattle embryos were exposed to the different oxygen concentrations for 8 days (D8; embryo developmental competence) or 5 days (D5; metabolism measurements). Between 3 and 4 experimental replicates were performed, with 40-50 embryos per replicate used for the developmental competency experiment, 10-20 embryos per replicate for the fluorophore and two-channel autofluorescence experiments and a total of 21-22 embryos used for the hyperspectral microscopy study. In-vitro produced (IVP) cattle embryos were utilised for this study. Post-fertilisation, embryos were exposed to 7% or 20% oxygen. To determine impact of oxygen concentrations on embryo viability, blastocyst development was assessed on D8. On D5, metabolic heterogeneity was assessed in morula (on-time) embryos using fluorophores probes (active mitochondria, hydrogen peroxide and reduced glutathione), two-channel autofluorescence (FAD and NAD(P)H) and 18-channel hyperspectral microscopy. Exposure to 20% oxygen following fertilisation significantly reduced total blastocyst, expanded and hatched blastocyst rates by 1.4-, 1.9- and 2.8-fold, respectively, compared to 7% oxygen

  16. Factors affecting survival rates of in vitro produced bovine embryos after vitrification and direct in-straw rehydration.

    Science.gov (United States)

    Vajta, G; Holm, P; Greve, T; Callesen, H

    1996-12-16

    The aim of this work was to investigate the possibilities of simplification, and to outline the limits of application, of a vitrification method for cow embryos. Morulae and blastocysts were produced by in vitro fertilization of slaughterhouse-derived, in vitro matured oocytes with frozen-thawed bull semen, and subsequent culture on a granulosa cell monolayer. Vitrification was performed by equilibration of embryos with 12.5% ethylene glycol and 12.5% dimethylsulphoxide at 20-22 degrees C for 60 s, then with 25% ethylene glycol and 25% dimethylsulphoxide at 4 degrees C for another 60 s. Embryos were then loaded in straws, placed in liquid nitrogen vapour for 2 min, and then plunged. Straws were thawed in a 22 degrees C water-bath, the embryos were directly rehydrated and further incubated in straw, and were then expelled and cultured in vitro for 72 h. In the first experiment, embryos of different age and developmental stage (Day 5 compacted morulae, Day 6 early blastocysts, Days 6 and 7 blastocysts, Day 7 expanded blastocysts and Day 8 hatched blastocysts) as well as Days 7 and 5 blastocysts previously subjected to partial zone dissection were vitrified. After thawing, the re-expansion rates of blastocysts and zona-dissected embryos did not differ (67 and 87%, respectively), and hatching was more frequent for blastocysts frozen in advanced developmental stages (34, 47 and 63% for early blastocysts, blastocysts and expanded blastocysts, respectively). The re-expansion rate of morulae was lower (10%) and no hatching of these embryos was observed. In the second experiment, Day 7 expanded blastocysts were vitrified using PBS, PBS+albumin, TCM199 and TCM199+calf serum as holding media. No differences in re-expansion and hatching rates were seen. However, when incubation with the concentrated cryoprotectant solution was performed at 20-22 degrees C, the embryo survival rate decreased (PBS+albumin) or no embryo survived (TCM199+calf serum) the vitrification procedure. In

  17. Genetic divergence in cellular resistance to heat shock in cattle: differences between breeds developed in temperate versus hot climates in responses of preimplantation embryos, reproductive tract tissues and lymphocytes to increased culture temperatures.

    Science.gov (United States)

    Paula-Lopes, F F; Chase, C C; Al-Katanani, Y M; Krininger, C E; Rivera, R M; Tekin, S; Majewski, A C; Ocon, O M; Olson, T A; Hansen, P J

    2003-02-01

    The detrimental effects of heat stress on fertility in cattle are less pronounced in heat-tolerant breeds. Although these genetic differences reflect differences in thermoregulation, cells from heat-tolerant breeds are less adversely compromised by increased temperature (that is, heat shock) than cells from heat-sensitive breeds. Experiments were performed to test the hypothesis that cells and tissues from two thermotolerant breeds (Brahman and Senepol) are better able to survive and function after exposure to increased temperature than cells and tissues from two thermosensitive breeds (Holstein and Angus). Exposure of embryos at>eight-cell stage at day 5 after insemination to heat shock of 41.0 degrees C for 6 h decreased development to the blastocyst stage and the number of cells per embryo. However, the deleterious effect of heat shock on blastocyst formation and the number of cells per embryo was less pronounced for Brahman than for Holstein and Angus breeds. Embryos from Senepol cows had very low development and it was not possible to determine heat shock effects in this breed. In contrast to the sensitivity of embryos to heat shock, there was no effect of a 41.0 degrees C heat shock on [(3)H]leucine incorporation into proteins secreted by oviductal or endometrial explants. Lymphocytes from Brahman and Senepol cows were more resistant to heat-induced apoptosis than lymphocytes from other breeds. Heat shock reduced lymphocyte glutathione content but the magnitude of the decrease was not affected by breed. In conclusion, embryos from Brahman cows are more resistant to heat shock than embryos from Holstein or Angus cows. Genetic differences are also present in thermotolerance for apoptosis response in lymphocytes, with Brahman and Senepol cattle being more resistant to heat shock than Angus and Holstein breeds. It is likely that the evolutionary forces that led to the Brahman and Senepol breeds being adapted to hot climates resulted in the selection of genes

  18. Efficient edition of the bovine PRNP prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system.

    Science.gov (United States)

    Bevacqua, R J; Fernandez-Martín, R; Savy, V; Canel, N G; Gismondi, M I; Kues, W A; Carlson, D F; Fahrenkrug, S C; Niemann, H; Taboga, O A; Ferraris, S; Salamone, D F

    2016-11-01

    The recently developed engineered nucleases, such as zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease (Cas) 9, provide new opportunities for gene editing in a straightforward manner. However, few reports are available regarding CRISPR application and efficiency in cattle. Here, the CRISPR/Cas9 system was used with the aim of inducing knockout and knock-in alleles of the bovine PRNP gene, responsible for mad cow disease, both in bovine fetal fibroblasts and in IVF embryos. Five single-guide RNAs were designed to target 875 bp of PRNP exon 3, and all five were codelivered with Cas9. The feasibility of inducing homologous recombination (HR) was evaluated with a reporter vector carrying EGFP flanked by 1 kbp PRNP regions (pHRegfp). For somatic cells, plasmids coding for Cas9 and for each of the five single-guide RNAs (pCMVCas9 and pSPgRNAs) were transfected under two different conditions (1X and 2X). For IVF zygotes, cytoplasmic injection was conducted with either plasmids or mRNA. For plasmid injection groups, 1 pg pCMVCas9 + 0.1 pg of each pSPgRNA (DNA2X) was used per zygote. In the case of RNA, two amounts (RNA1X and RNA2X) were compared. To assess the occurrence of HR, a group additionally cotransfected or coinjected with pHRegfp plasmid was included. Somatic cell lysates were analyzed by polymerase chain reaction and surveyor assay. In the case of embryos, the in vitro development and the genotype of blastocysts were evaluated by polymerase chain reaction and sequencing. In somatic cells, 2X transfection resulted in indels and large deletions of the targeted PRNP region. Regarding embryo injection, higher blastocyst rates were obtained for RNA injected groups (46/103 [44.6%] and 55/116 [47.4%] for RNA1X and RNA2X) than for the DNA2X group (26/140 [18.6%], P < 0.05). In 46% (26/56) of the total sequenced blastocysts, specific gene editing was

  19. Effect of the cryopreservation method used, the embryonic stage and the use of conjugated linoleic acid isomers on the cryotolerance of in vitro-produced bovine embryos

    Directory of Open Access Journals (Sweden)

    Luciana Simões Rafagnin Marinho

    2015-12-01

    Full Text Available Conjugated linoleic acid (CLA might be able to improve the cryotolerance of in vitro-produced (IVP embryos. The effect of two CLA isomers on the cryotolerance of bovine IVP embryos, as well as that of the stage of embryonic development and the method used for cryopreservation was evaluated by three experiments. In Experiment 1, oocytes (n = 3,917 were fertilized in vitro and cultured with 0, 50, 100, or 200 ?M trans-10, cis-12 (t10, c12 CLA. In Experiment 2, fertilized oocytes (n = 2,131 were cultured with 100 ?M t10, c12 or cis-9, trans-11 (c9, t11 CLA, or a combination of both isomers. The embryos were vitrified at the blastocyst (BL or the expanded blastocyst (EB stage. In Experiment 3, oocytes (n = 1,720 were fertilized and cultured with or without 100 ?M t10, c12 CLA, and the blastocysts were vitrified or frozen. Blastocyst development rate as well as the rates of re-expansion and hatching after thawing was recorded. Moreover, the mean cell number and mRNA expression of acetyl-CoA carboxylase (ACC1 and stearoyl-CoA desaturase (SCD1 as well as fatty acid synthase (FASN multienzyme complex were determined. In Experiment 1, the highest concentration of t10, c12 CLA that did not reduce blastocyst development rate was 100 ?M. In Experiment 2, the rates of re-expansion and hatching among the EBs obtained through IVP after supplementation with t10, c12 CLA (73.1% and 57.7%, with c9, t11 CLA (80.0% and 68.6%, with the combination (78.3% and 52.2%, and with the control group (85.4% and 58.3% were similar. At the BL stage, the rates of re-expansion and hatching were lower than those at the EB stage, and CLA combination allowed a hatching rate (8.0% lower than that observed in the control group (40.0%. In Experiment 3, the hatching rates for vitrified EBs (vitrified control; 67.4% and vitrified CLA EBs (65.8% were higher than those obtained for frozen EBs, exposed (13.3% or not exposed (28.6% to CLA. In addition, in Experiment 3, the hatching rate was

  20. Vitrification of immature bovine cumulus-oocyte complexes: effects of cryoprotectants, the vitrification procedure and warming time on cleavage and embryo development.

    Science.gov (United States)

    Prentice-Biensch, Jennifer R; Singh, Jaswant; Mapletoft, Reuben J; Anzar, Muhammad

    2012-09-06

    The present studies evaluated the effects of cryoprotectants, the vitrification procedure and time in the warming solution containing sucrose on cleavage and embryo development of immature (GV stage) bovine cumulus-oocyte complexes (COCs). Two experiments were conducted. In Experiment 1, COCs (n = 420) were randomly assigned to four groups: 1) CONTROL GROUP: no treatment; 2) VS1 group: COCs were exposed to vitrification solution 1 (VS1) containing 7.5% ethylene glycol [EG] + 7.5% dimethyl sulfoxide [DMSO] + 20% calf serum [CS] in TCM-199 at 37 C for 5 min; 3) VS1 + VS2 group: COCs were exposed to VS1 for 5 min followed by VS2 (15% EG + 15% DMSO + 17.1% sucrose + 20% CS) at 37 C for 45-60 sec; and 4) Vitrified group: COCs were exposed to VS1 and VS2, loaded on cryotops, vitrified in liquid nitrogen and then warmed in TCM-199 + 17.1% sucrose + 20% CS at 37 C for 1 min. In Experiment 2, COCs (n = 581) were assigned to the same groups, but those in VS1, VS1 + VS2 and Vitrified groups were sub-divided and exposed to the warming solution for either 1 or 5 min. After treatment and/or warming, all COCs in both experiments underwent in vitro maturation, in vitro fertilization and in vitro culture. Cleavage and blastocyst rates did not differ among Control, VS1 and VS1 + VS2 groups in either experiment. In Experiment 2, there was no effect of time in the warming solution.However, both cleavage and blastocyst rates were lower (P bovine COCs. However, cleavage rate and early embryo development were reduced following the vitrification and warming.

  1. Maintenance of meiotic arrest in bovine oocytes using the S-enantiomer of roscovitine: effects on maturation, fertilization and subsequent embryo development in vitro.

    Science.gov (United States)

    Coy, Pilar; Romar, Raquel; Payton, Rebecca R; McCann, Lisa; Saxton, Arnold M; Edwards, J Lannett

    2005-01-01

    The overall objective was to evaluate the effectiveness of the S-enantiomer of roscovitine (inhibitor of p34cdc2/cyclin B kinase) to maintain bovine cumulus-oocyte complexes at the germinal vesicle (GV) stage for extended times after removal from antral follicles without compromising subsequent maturation, fertilization and embryo development. Oocytes were cultured in 0, 12.5, 25 or 50 micromol/l S-roscovitine for 24 h. Hoechst staining showed that 50 micromol/l S-roscovitine maintained >90% of oocytes at the GV stage and inhibited gonadotropin-induced cumulus expansion. Fewer oocytes underwent nuclear maturation after in vitro maturation (Hoechst staining) when cultured in 50 micromol/l S-roscovitine for 66 versus 21 or 42 h. Zona pellucida (ZP) hardening (pronase resistance), cortical granule types (lens culinaris agglutinin-fluorescein isothiocyanate), nuclear maturation and fertilization with frozen-thawed spermatozoa (Hoechst staining) were assessed after culture of oocytes in 50 micromol/l S-roscovitine for 0, 24 or 48 h. Neither ZP hardening, nor nuclear maturation nor fertilization were altered by roscovitine culture for 48 h. A higher proportion of oocytes had a type III cortical granule pattern (premature translocation to the oolemma) after roscovitine culture for 48 h. However, embryo development was not compromised as cleavage, development to 8-16 cell and blastocyst stages were at least comparable in control and roscovitine-treated oocytes. In conclusion, the studies have shown that S-roscovitine reversibly maintained bovine oocytes at the GV stage for 48 h. However, maintenance of oocytes in static culture for 48 h was not sufficient to improve development above non-treated controls.

  2. Efficacy of mechanical micro-vibration in the development of bovine embryos during in vitro maturation and culture.

    Science.gov (United States)

    Takahashi, Masahiro; Honda, Tatsutoshi; Hatoya, Shingo; Inaba, Toshio; Kawate, Noritoshi; Tamada, Hiromichi

    2018-02-06

    It is currently unclear how mechanical micro-vibration affects the in vitro culture of embryos in Japanese Black cow. In the experimental groups, immature oocytes and fertilized embryos were cultured using the micro-vibration culture system with the vibration set for 5 sec at intervals of 60 min and frequency of 20, 40, or 80 Hz, respectively, during in vitro maturation and in vitro development. Compared with the control group, the rate of blastocyst development significantly increased in the 40 Hz group. In addition, the number of blastocyst cells reduced significantly in the 80 Hz group. In conclusion, the development of blastocysts in cows is facilitated by providing moderate mechanical micro-vibration to immature oocytes and embryos during the in vitro maturation and in vitro development.

  3. Improvement of bovine in vitro embryo production by ovarian follicular wave synchronization prior to ovum pick-up.

    Science.gov (United States)

    Cavalieri, F L B; Morotti, F; Seneda, M M; Colombo, A H B; Andreazzi, M A; Emanuelli, I P; Rigolon, L P

    2017-11-26

    This study evaluated the effects of the synchronization of ovarian follicular wave emergence on the efficiency of in vitro embryo production. Bos indicus cows (n = 20) were divided into two groups (control vs. synchronization) and subjected to repeated ovum pick-up (OPU) sessions (8 replicates each, with an interval of 21 days in a 2 × 2 crossover design) and subsequent in vitro embryo production. Cows in the control group (n = 10) were submitted to OPU procedures without any stimulation every 21 days. Animals in the synchronization group received a protocol-based progesterone implant, estradiol benzoate and prostaglandin on a random day of the estrus cycle (Day 0) and the OPU was performed on Day 5. After in vitro production, embryos were transferred to recipients synchronized at a fixed time and the diagnosis was performed 60 days later. An evaluation of the parameters for each OPU session revealed that donors that received the synchronization protocol pre-OPU showed a greater number of embryos (5.9 ± 0.5 vs. 4.5 ± 0.4; P = 0.037), higher rate of embryo production (45.8% vs. 38.5%; P = 0.001) and higher mean number of conceptions per group (2.2 ± 0.2 vs. 1.6 ± 0.2; P = 0.07) in relation to the group that did not receive hormonal treatment. We concluded that synchronization of the follicular wave prior to OPU showed positive effects on in vitro embryo production as well as on pregnancy rates. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Hepes na produção de embriões bovinos in vitro Hepes on in vitro production of bovine embryos

    Directory of Open Access Journals (Sweden)

    Marcelo Marcos Montagner

    2000-06-01

    of pH changes in maturation and embryo development media, buffered with different HEPES concentrations. Initially, the effect of different concentrations of HEPES (0, 12.5 and 25.0mM on the variation of pH in the maturation (modified TCM-199 and embryonic development (modified KSOM media was evaluated at room temperature (25ºC and in an atmosphere of 5% CO2 in air at 39ºC. In another experiment, the effect of HEPES on in vitro oocyte maturation was determined. Oocytes were maturated in TCM-199 modified either with 25.0mM of HEPES (HEPES group; n = 137 or without HEPES (control group; n = 142, performing 7 replicates and evaluating the rate of blastocyst. In this study, the medium used for fertilization was Fert-TALP while for embryo development was KSOM with 10% of fetal bovine serum with monolayer of oviduct epithelial cells. A third experiment was designed to determine the effect of HEPES on embryo development. The zygotes were divided in two groups and co-incubated with oviduct epithelial cells in modified KSOM with 10% of fetal bovine serum without HEPES (n = 95 or with 25.0mM of HEPES (n = 92. For this experiment, it was used embryos with two or more cells and the embryo development was considered from cleavage to expanded blastocyst (Bx, 7 and 9 days after insemination. The oocytes and embryos were incubated at temperature of 39ºC, an atmosphere containing 5% CO2 in air and saturated humidity. The media with 25.0mM of HEPES were more efficient in minimizing the range of pH than those with 12.5mM or without HEPES. To determine the effect of HEPES during in vitro oocyte maturation, the percentage of Bl considered either the total number of oocytes or the total number of cleavages was higher in the HEPES group (21.9% or 42.9%, respectively than those obtained in the control group (10.56% or 16.67%, respectively. When HEPES was added to embryo culture medium, the percentage of Bx (45.65% was higher than that obtained in medium without HEPES (11.58%; p<0.01. The

  5. Effects of sphingosine-1-phosphate on gene expression of two cell mouse embryos induced by C2-Ceramide

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    Xujing Geng

    2014-06-01

    Conclusions: This study provides a map of genes in the pre-implantation two cell mouse embryo. Further investigation based on these data will provide a better understanding of the effects of S1P on the pre-implantation embryos in other mammalian species, especially human.

  6. Effect of urea during in vitro maturation on nuclear maturation and embryo development of bovine cumulus-oocyte-complexes

    NARCIS (Netherlands)

    Wit, de A.A.C.; Cesar, M.L.F.; Kruip, T.A.M.

    2001-01-01

    High concentrations of urea in reproductive tract fluids are detrimental to bovine reproduction. Therefore, in experiment 1, the effect of 6 mM urea on nuclear maturation of cumulus-oocyte-complexes (COC) collected from abattoir ovaries was studied. After 4, 8, 12, 16, 20, and 24 h of in vitro

  7. Production of bovine cloned embryos with donor cells frozen at a slow cooling rate in a conventional freezer (-20 degrees C).

    Science.gov (United States)

    Chacón, Liliana; Gómez, Martha C; Jenkins, Jill A; Leibo, Stanley P; Wirtu, Gemechu; Dresser, Betsy L; Pope, C Earle

    2009-11-01

    SummaryUsually, fibroblasts are frozen in dimethyl sulphoxide (DMSO, 10% v/v) at a cooling rate of 1 degrees C/min in a low-temperature (-80 degrees C) freezer (LTF) before storage in liquid nitrogen (LN2); however, a LTF is not always available. The purpose of the present study was to evaluate apoptosis and viability of bovine fibroblasts frozen in a LTF or conventional freezer (CF; -20 degrees C) and their subsequent ability for development to blastocyst stage after fusion with enucleated bovine oocytes. Percentages of live cells frozen in LTF (49.5%) and CF (50.6%) were similar, but significantly less than non-frozen control (88%). In both CF and LTF, percentages of live apoptotic cells exposed to LN2 after freezing were lower (4% and 5%, respectively) as compared with unexposed cells (10% and 18%, respectively). Cells frozen in a CF had fewer cell doublings/24 h (0.45) and required more days (9.1) to reach 100% confluence at the first passage (P) after thawing and plating as compared with cells frozen in a LTF (0.96 and 4.0 days, respectively). Hypoploidy at P12 was higher than at P4 in cells frozen in either a CF (37.5% vs. 19.2%) or in a LTF (30.0% vs. 15.4%). A second-generation cryo-solution reduced the incidence of necrosis (29.4%) at 0 h after thawing as compared with that of a first generation cryo-solution (DMEM + DMSO, 60.2%). The percentage of apoptosis in live cells was affected by cooling rate (CF = 1.9% vs. LFT = 0.7%). Development of bovine cloned embryos to the blastocyst stage was not affected by cooling rate or freezer type.

  8. Developmental disparity between in vitro-produced and somatic cell nuclear transfer bovine days 14 and 21 embryos

    DEFF Research Database (Denmark)

    Alexopoulos, Natalie I.; Maddox-Hyttel, Poul; Tveden-Nyborg, Pernille Yde

    2008-01-01

    , immunohistochemistry, and transmission electron microscopy to establish in vivo developmental milestones. Following morphological examination, samples were characterized for the presence of epiblast (POU5F1), mesoderm (VIM), and neuroectoderm (TUBB3). On D14, only 25, 15, and 7% of IVP, SUZI, and HMC embryos were...

  9. Gene expression profiles of vitrified in vitro- and in vivo-derived bovine blastocysts.

    Science.gov (United States)

    Aksu, Digdem Aktoprakligil; Agca, Cansu; Aksu, Soner; Bagis, Haydar; Akkoc, Tolga; Caputcu, Arzu Tas; Arat, Sezen; Taskin, Ali Cihan; Kizil, Sedat H; Karasahin, Tahir; Akyol, Numan; Satilmis, Muharrem; Sagirkaya, Hakan; Ustuner, Burcu; Nur, Zekeriya; Agca, Yuksel

    2012-09-01

    Vitrification is becoming a preferred method for pre-implantation embryo cryopreservation. The objective of this study was to determine the differentially expressed genes of in vivo- and in vitro-produced bovine embryos after vitrification. In vitro- (IVF) and in vivo-derived (IVV) bovine blastocysts were identified as follows: in vitro-produced fresh (IVF-F), in vitro-produced vitrified (IVF-V), in vivo-derived fresh (IVV-F), in vivo-derived vitrified (IVV-V). The microarray results showed that 53 genes were differentially regulated between IVF and IVV, and 121 genes were differentially regulated between fresh and vitrified blastocysts (P vitro bovine blastocyst production protocols used in this study caused no major gene expression differences compared to those of in vivo-produced blastocysts. After vitrification, however, in vitro-produced blastocysts showed major gene expression differences compared to in vivo blastocysts. This study suggests that in vitro-produced embryos are of comparable quality to their in vivo counterparts. Vitrification of in vitro blastocysts, on the other hand, causes significant up-regulation of genes that are involved in stress responses. Copyright © 2012 Wiley Periodicals, Inc.

  10. OPTIMISATION OF TOTAL RNA EXTRACTION FROM BOVINE OOCYTES AND EMBRYOS FOR GENE EXPRESSION STUDIES AND EFFECTS OF CRYOPROTECTANTS ON TOTAL RNA EXTRACTION.

    Science.gov (United States)

    Pavani, K C; Baron, E E; Faheem, M; Chaveiro, A; Da Silva, F Moreira

    2015-01-01

    Gene expression is required for understanding bovine oocytes meiotic maturation as well as the potential of embryonic development. In the present study a standardized reagent protocol for total RNA extraction was designed for bovine oocytes and embryos, which is considered specific and less expensive. For such purpose oocytes (n = 795) recovered from about 80 ovaries were divided in three groups: Group 1 modified Trizol (MTP, n = 355); Group 2 Guanidinium thiocyanate protocol (GNTC, n = 140) and Group 3 Commercial Kit protocol (CKP, n = 60). Oocytes belonging to group 1 (n = 100) and 3 (n = 20) were subjected to vitrification using two cryoprotectants 1,2 propandiol (PROH) or Dimethylsulfoxide (DMSO). The 240 remaining oocytes were divided into 3 groups in which 100 were used, in fresh, for in vitro fertilization, and 140 oocytes were vitrified using PROH (n = 70) and DMSO (n = 70) as cryoprotectants, being then fertilized in vitro after thawing. Embryos were used nine days after fertilization. Gene amplification (SDHA, (GAPDH and DNMT1) was performed in oocytes, and gene quantification (DNMT1) in in vitro produced embryos at the stage of blastocyst (n = 10). Efficiency of the extraction was further compared. The purity of all samples to different protocols ranged from 1.10 to 1.25 for GNTC protocol; from 2.05 to 2.63 for the CKP and from 1.50 to 2.11 for the developed MTP, being the last one nearest to the expected purity levels for RNA samples (1.7 to 2.0). On average, for 30 fresh oocytes, from spectrophotometer readings, total RNA concentration was 127.8 ± 9.3 ng μl(-1) for MTP, against 46.4 ± 9.5 ng μl(-1) from CKP and 476 ± 12.9 ng μl(-1) for GNTC protocol. Using the MTP to evaluate RNA in 30 vitrified/thawed oocytes, resulted in a total RNA concentration of 61.3 ± 3.3 ng μl(-1) and 40.0 μ 12.4 ng μ(-1), respectively for DMSO and PROH. Regarding total RNA concentration and purity, in blastocyst stage, more purity was observed in DMSO as compared to

  11. Preimplantation HLA typing for stem cell transplantation treatment of hemoglobinopathies

    Directory of Open Access Journals (Sweden)

    Anver Kuliev

    2014-09-01

    Full Text Available Preimplantation genetic diagnosis (PGD for HLA typing is steadily becoming an option for at risk couples with thalassemic children, requiring HLA matched bone marrow transplantation treatment. The paper presents the world’s largest PGD experience of 475 cases for over 2 dozens thalassemia mutations, resulting in birth of 132 unaffected children. A total of 146 cases were performed together with preimplantation HLA typing, resulting in detection and transfer of HLA matched unaffected embryos in 83 of them, yielding the birth of 16 HLA matched children, potential donors for their affected siblings. The presented experience of HLA matched stem cell transplantation for thalassemia, following PGD demonstrated a successful hematopoietic reconstitution both for younger and older patients. The data show that PGD is an efficient approach for HLA matched stem cell transplantation treatment for thalassemia.

  12. Optimization of embryo culture conditions for increasing efficiency of cloning in buffalo (Bubalus bubalis) and generation of transgenic embryos via cloning.

    Science.gov (United States)

    Wadhwa, Neerja; Kunj, Neetu; Tiwari, Shuchita; Saraiya, Megha; Majumdar, Subeer S

    2009-09-01

    Cloning in bovine species is marred by low efficiency of blastocyst formation. Any increase in the efficiency of blastocyst formation upon nuclear transfer will greatly enhance the efficiency of cloning. In the present study, the effect of various media, protein sources, and growth factors on the development of cloned buffalo embryos was evaluated. Among various combinations tested, culture of cloned embryos in TCM-199 media on the feeder layer of Buffalo Oviductal Epithelial Cells (BOEC) in the presence of bovine serum albumin-free fatty acid (BSA-FFA) and leukemia inhibitory factor (LIF) provided most suitable environment for efficient development of cloned blastocysts. Under these conditions, we achieved a blastocyst formation rate of 43%, which is better than those reported previously. Because preimplantation embryonic development, in vivo, occurs in an environment of oviductal cells, the blastocysts generated by this method may presumably be more suitable for implantation and further development. Additionally, we generated green blastocysts from enucleated oocytes by transfer of nuclei from cells transfected with EGFP transgene, showing possibility of transgenesis via cloning in this species. To our knowledge, this is the first report regarding the production of transgenic cloned buffalo embryos and their developmental competence with respect to various media, cocultures, and supplements.

  13. Preimplantation genetic diagnosis for X;autosome translocations : lessons from a case of misdiagnosis

    NARCIS (Netherlands)

    Van Echten-Arends, J.; Coonen, E.; Reuters, B.; Suijkerbuijk, R. F.; Dul, E. C.; Land, J. A.; van Ravenswaaij-Arts, C. M. A.

    2013-01-01

    Preimplantation genetic diagnosis (PGD) is offered to couples carrying a reciprocal translocation in an attempt to increase their chance of phenotypically normal offspring. For the selection of embryos that are balanced for the translocation chromosomes, it is critical to use a combination of DNA

  14. Opportunity, obligation, risk – The representation of preimplantational genetic tests in the Hungarian online news media

    OpenAIRE

    Kádár, Eszter

    2017-01-01

    Preimplantational genetic technologies (PG technologies) used in the processes of reproduction under laboratory conditions are considered to be one of the most controversial fields of gene technology. These tests are performed on embryos and result in the selection of the most capable embryo, while destroying the rest. Several moral questions arise regarding the ethical application of these procedures. This study investigated the representation of this topic in the Hungarian online media, app...

  15. Factors affecting survival rates of in vitro produced bovine embryos after vitrification and direct in-straw rehydration

    DEFF Research Database (Denmark)

    Vajta, Gábor; Holm, Peter; Greve, Torben

    1996-01-01

    and developmental stage (Day 5 compacted morulae, Day 6 early blastocysts, Days 6 and 7 blastocysts, Day 7 expanded blastocysts and Day 8 hatched blastocysts) as well as Days 7 and 5 blastocysts previously subjected to partial zona dissection were vitrified. After thawing, the re-expansion rates of blastocysts...... and zona-dissected embryos did not differ (67 and 87%, respectively), and hatching was more frequent for blastocysts frozen in advanced developmental stages (34, 47 and 63% for early blastocysts, blastocysts and expanded blastocysts, respectively). The re-expansion rate of morulae was lower (10......%) and no hatching of these embryos was observed. In the second experiment, Day 7 expanded blastocysts were vitrified using PBS, PBS + albumin, TCM199 and TCM 199 + calf serum as holding media. No differences in re-expansion and hatching rates were seen. However, when incubation with the concentrated cryoprotectant...

  16. Influence of the radiation (Co{sub 60}) in pre-implants rabbit embryos: effect on atypic mitotic index and embryo pole development; Influencia da radiacao ionizante (bomba de cobalto) em embrioes de coelha na fase de pre-implantacao: influencia no numero de mitoses atipicas e no grau de desenvolvimento do polo

    Energy Technology Data Exchange (ETDEWEB)

    Approbato, Mario S.; Oliveira Moura, Katia K.V. de; Souza Florencio, Rodopiano de; Garcia, Ricardo; Faria, Renato S.; Benedetti, Leonardo N.; Goulart, Flamarion B. [Goias Univ., Goiania, GO (Brazil). Faculdade de Medicina. Dept. de Ginecologia e Obstetricia

    1995-08-01

    We studied the effect of ionizing irradiation on 12 New Zealand rabbits (65 embryos), at three different times: at match time (zero hour), two days after and four days after, with two different irradiation doses: five c Gy and ten c Gy. Six rabbits (36 blastocysts) were used as controls. the matching instant was the zero hour. Exactly six days after ({+-} 60 minutes) the embryos of each rabbit was picked up by flushing the uterus with culture media. the embryos were fixed in methanol for 48 hours, and colored with acid Mayer hematoxylin. The following embryo parameters were studied: embryo pole development; percentage of abnormal mitotic figures. irradiation time was associated with lower scores of embryo pole development, but not with irradiation dose. There were no gross abnormalities of embryo pole. The abnormal mitotic cells was affected both by the time and dose of irradiation. (author). 12 refs., 4 figs.

  17. Selection of the In Vitro Culture Media Influences mRNA Expression of Hedgehog Genes, Il-6, and Important Genes regarding Reactive Oxygen Species in Single Murine Preimplantation Embryos

    Directory of Open Access Journals (Sweden)

    N. Pfeifer

    2012-01-01

    Full Text Available Background. The aim of this paper was to determine the influence of different in vitro culture media on mRNA expression of Hedgehog genes, il-6, and important genes regarding reactive oxygen species in single mouse embryos. Methods. Reverse transcription of single embryos either cultured in vitro from day 0.5 until 3.5 (COOK’s Cleavage medium or Vitrolife’s G-1 PLUS medium or in vivo until day 3.5 post coitum. PCR was carried out for β-actin followed by nested-PCR for shh, ihh, il-6, nox, gpx4, gpx1, and prdx2. Results. The number of murine blastocysts cultured in COOK medium which expressed il-6, gpx4, gpx1, and prdx2 mRNA differed significantly compared to the in vivo group. Except for nox, the mRNA profile of the Vitrolife media group embryos varied significantly from the in vivo ones regarding the number of blastocysts expressing the mRNA of shh, ihh, il-6, gpx4, gpx1 and prdx2. Conclusions. The present study shows that different in vitro culture media lead to different mRNA expression profiles during early development. Even the newly developed in vitro culture media are not able to mimic the female reproductive tract. The question of long-term consequences for children due to assisted reproduction techniques needs to be addressed in larger studies.

  18. Cryosurvival of in vitro produced bovine embryos supplemented with l-Carnitine and concurrent reduction of fatty acids.

    Science.gov (United States)

    Held-Hoelker, E; Klein, S L; Rings, F; Salilew-Wondim, D; Saeed-Zidane, M; Neuhoff, C; Tesfaye, D; Schellander, K; Hoelker, M

    2017-07-01

    Lipid accumulation is associated with reduced embryonic quality, causing limited survival after cryopreservation. Therefore, in the present study we aimed to reveal the effects of supplementation of a lipid reducing agent, l-carnitine and the removal of fatty acids during in vitro culture on the morphological as well as on the molecular level. To accomplish that, presumptive zygotes were cultured in 4 contrasting groups: namely SOFaa medium supplemented with BSA, (BSA), SOFaa medium supplemented with fatty acid free BSA (FAF), SOFaa medium supplemented with BSA as well as l-Carnitine (BSA + LC) and SOFaa medium concurrently supplemented with fatty acid free BSA and l-Carnitine (FAF + LC). Considering the developmental rates, no impact of different treatments was observed. Conversely, treatment groups clearly affected lipid content, with the lowest amounts detected in embryos derived from FAF and BSA + LC groups, implicating that both removal of fatty acids and supplementation of LC reduces lipid content effectively. Importantly, survival rates after cryopreservation show that LC significantly affects the kinetics of re-expansion, with the highest hatching rates detected for embryos cultured in FAF + LC (p < 0.05). Noteworthy, the highest cryotolerance did not go along with lowest lipid contents. Finally, metabolic alterations between the groups were reflected in different abundances of selected candidate genes related to lipid metabolism and oxidative stress response, like AMPKA1, ACC and PGC1 α or KEAP1 and SOD1. All in all, highly beneficial effects on survival rates after cryopreservation have been detected when embryos were cultured in absence of fatty acids and concurrent presence of l-Carnitine. Highest cryotolerance, however, did not correlate with lowest lipid contents. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Preimplantation genetic diagnosis and screening: Current status and future challenges

    Directory of Open Access Journals (Sweden)

    Hsin-Fu Chen

    2018-02-01

    Full Text Available Preimplantation genetic diagnosis (PGD is a clinically feasible technology to prevent the transmission of monogenic inherited disorders in families afflicted the diseases to the future offsprings. The major technical hurdle is it does not have a general formula for all mutations, thus different gene locus needs individualized, customized design to make the diagnosis accurate enough to be applied on PGD, in which the quantity of DNA is scarce, whereas timely result is sometimes requested if fresh embryo transfer is desired. On the other hand, preimplantation genetic screening (PGS screens embryo with aneuploidy and was also known as PGD-A (A denotes aneuploidy in order to enhance the implantation rates as well as livebirth rates. In contrasts to PGD, PGS is still under ferocious debate, especially recent reports found that euploid babies were born after transferring the aneuploid embryos diagnosed by PGS back to the womb and only very few randomized trials of PGS are available in the literature. We have been doing PGD and/or PGS for more than 10 years as one of the core PGD/PGS laboratories in Taiwan. Here we provide a concise review of PGD/PGS regarding its current status, both domestically and globally, as well as its future challenges.

  20. Vitrification of immature bovine cumulus-oocyte complexes: effects of cryoprotectants, the vitrification procedure and warming time on cleavage and embryo development

    Directory of Open Access Journals (Sweden)

    Prentice-Biensch Jennifer R

    2012-09-01

    Full Text Available Abstract Background The present studies evaluated the effects of cryoprotectants, the vitrification procedure and time in the warming solution containing sucrose on cleavage and embryo development of immature (GV stage bovine cumulus-oocyte complexes (COCs. Methods Two experiments were conducted. In Experiment 1, COCs (n = 420 were randomly assigned to four groups: 1 Control group: no treatment; 2 VS1 group: COCs were exposed to vitrification solution 1 (VS1 containing 7.5% ethylene glycol [EG] + 7.5% dimethyl sulfoxide [DMSO] + 20% calf serum [CS] in TCM-199 at 37 C for 5 min; 3 VS1 + VS2 group: COCs were exposed to VS1 for 5 min followed by VS2 (15% EG + 15% DMSO + 17.1% sucrose + 20% CS at 37 C for 45–60 sec; and 4 Vitrified group: COCs were exposed to VS1 and VS2, loaded on cryotops, vitrified in liquid nitrogen and then warmed in TCM-199 + 17.1% sucrose + 20% CS at 37 C for 1 min. In Experiment 2, COCs (n = 581 were assigned to the same groups, but those in VS1, VS1 + VS2 and Vitrified groups were sub-divided and exposed to the warming solution for either 1 or 5 min. After treatment and/or warming, all COCs in both experiments underwent in vitro maturation, in vitro fertilization and in vitro culture. Results Cleavage and blastocyst rates did not differ among Control, VS1 and VS1 + VS2 groups in either experiment. In Experiment 2, there was no effect of time in the warming solution. However, both cleavage and blastocyst rates were lower (P  Conclusions The permeating cryoprotectants (EG and DMSO present in VS1 and VS2 solutions and the time in the warming solution containing sucrose had no adverse effects on cleavage and blastocyst rates of immature bovine COCs. However, cleavage rate and early embryo development were reduced following the vitrification and warming.

  1. Influence of irradiation (Co{sub 6}0) in pre-implant rabbits embryos: effect on blastocyst diameters and embryos smaller than 2 mm; Influencia da radiacao ionizante (bomba de cobalto) em embrioes de coelha na fase de pre-implantacao: efeito no diametro das blastulas e embrioes com menos de 2mm

    Energy Technology Data Exchange (ETDEWEB)

    Approbato, Mario S.; Oliveira Moura, Katia K.V. de; Souza Florencio, Rodopiano de; Cunha Junior, Carlos; Garcia, Ricardo; Faria, Renato S.; Benedetti, Leonardo N.; Goulart, Flamarion B.

    1995-06-01

    We studied the effect of ionizing irradiation on 12 New Zealand rabbits (65 embryos), in three different times: at match time (zero hour), two days after and four days after, with two different irradiation doses, 5 c Gy and 10 c Gy. Six rabbits (36 blastocysts) were used as controls. The matching instant was the zero hour. Exactly six days after ({+-} 60 minutes) the embryos of each rabbit was picked up by flushing the uterus with culture media. The embryos were fixed in methanol for 48 hours, and colored with acid Mayer hematoxylin. The following embryos parameters were studied: diameter growth; percentage of embryos smaller than 2 mm. We observed that only the irradiation time influenced the blastocysts diameter (no irradiation dose). There was no relation between percentage of embryos smaller than 2 mm and the irradiation. (author). 11 refs., 2 figs.

  2. Embryo transfer and related technologies in sheep reproduction

    OpenAIRE

    Loi, Pasqualino; Ptak, Grazyna; Dattena, Maria; Ledda, Sergio; Naitana, Salvatore; Cappai, Pietro

    1998-01-01

    This paper reviews the status of embryo transfer and the major technologies applied to preimplantation of embryos in sheep. Embryo production from superovulated ewes is hindered by an unpredictable response to hormonal treatment. Progress in this area should be expected by an appropriated control of follicular development with gonadotropin-releasing hormone (GnRH) agonist or antagonist prior to gonadotrophin administration. Simple protocols for the cryopreservation of sheep embryos by vitrifi...

  3. Microorganisms in cryopreserved semen and culture media used in the in vitro production (IVP) of bovine embryos identified by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS).

    Science.gov (United States)

    Zampieri, Dávila; Santos, Vanessa G; Braga, Patrícia A C; Ferreira, Christina R; Ballottin, Daniela; Tasic, Ljubica; Basso, Andréa C; Sanches, Bruno V; Pontes, José H F; da Silva, Bárbara Pereira; Garboggini, Fabiana Fantinatti; Eberlin, Marcos N; Tata, Alessandra

    2013-09-01

    Commercial cattle breeders produce their own herd offspring for the dairy and beef market using artificial insemination. The procedure involves sanitary risks associated with the collection and commercialization of the germplasm, and the in vitro production and transfer of the bovine embryos must be monitored by strict health surveillance. To avoid the spreading of infectious diseases, one must rely on using controlled and monitored germplasm, media, and reagents that are guaranteed free of pathogens. In this article, we investigated the use of a new mass spectrometric approach for fast and accurate identification of bacteria and fungi in bovine semen and in culture media employed in the embryo in vitro production process. The microorganisms isolated from samples obtained in a commercial bovine embryo IVP setting were identified in a few minutes by their conserved peptide/protein profile, obtained applying matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), matched against a commercial database. The successful microorganisms MS identification has been confirmed by DNA amplification and sequencing. Therefore, the MS technique seems to offer a powerful tool for rapid and accurate microorganism identification in semen and culture media samples. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. Application of reproductive biotechnology for the recovery of endangered breeds: birth of the first calf of Murciana-Levantina bovine breed derived by OPU, in vitro production and embryo vitrification.

    Science.gov (United States)

    Ruiz, S; Romero-Aguirregomezcorta, J; Astiz, S; Peinado, B; Almela, L; Poto, A

    2013-12-01

    In a conservation project, reproductive biotechnology was implemented for the recovery and conservation of an endangered bovine breed in Spain. The breed Murciana-Levantina, declared to be worthy of special protection status (http://www.fao.org/newsroom/en/news/2006/1000464/index.html), is of great interest because of its hardness, longevity, docility and disease resistance. This contribution describes the birth of the first calf of this breed obtained by reproductive biotechnology, using ultrasound-guided punction and aspiration of ovarian follicles, in vitro embryo production, vitrification of embryos by a cryotop device and, finally, the transfer of cryopreserved embryos to recipient heifers of a commercial dairy herd. © 2013 Blackwell Verlag GmbH.

  5. Reduced levels of intracellular reactive oxygen species and apoptotic status are not correlated with increases in cryotolerance of bovine embryos produced in vitro in the presence of antioxidants.

    Science.gov (United States)

    Rocha-Frigoni, Nathália A S; Leão, Beatriz C S; Nogueira, Ériklis; Accorsi, Mônica F; Mingoti, Gisele Z

    2014-01-01

    The effects of intracellular (cysteine and β-mercaptoethanol) and extracellular (catalase) antioxidant supplementation at different times during in vitro production (IVM and/or in vitro culture (IVC)) on bovine embryo development, intracellular reactive oxygen species (ROS) levels, apoptosis and re-expansion rates after a vitrification-thawing process were examined. Blastocyst frequencies were not affected by either antioxidant supplementation (40.5%-56.4%) or the timing of supplementation (41.7%-55.4%) compared with control (48.7%; P>0.05). Similarly, antioxidants and the moment of supplementation did not affect (P>0.05) the total number of blastomeres (86.2-90.5 and 84.4-90.5, respectively) compared with control (85.7). However, the percentage of apoptotic cells was reduced (P0.05) from that in the control group (1.00). Re-expansion rates were not affected (P>0.05) by the treatments (50.0%-93.0%). In conclusion, antioxidant supplementation during IVM and/or IVC reduces intracellular ROS and the rate of apoptosis; however, supplementation does not increase embryonic development and survival after vitrification.

  6. Effect of B-mercaptoethanol on the viability of IVM/IVF/IVC bovine embryos during long-distance transportation in plastic straws.

    Science.gov (United States)

    Takahashi, H; Kuwayama, M; Hamano, S; Takahashi, M; Okano, A; Kadokawa, H; Kariya, T; Nagai, T

    1996-10-15

    Experiments were conducted to assess the effect of beta-mercaptoethanol (beta-ME) on the quality and viability of bovine blastocysts derived from in-vitro culture (IVC) of in-vitro matured and fertilized (TVM-IVF) oocytes during their transport between 2 distant places. Follicular oocytes were collected from ovaries obtained at a slaughterhouse and were cultured for 20 to 21 h in modified TCM-199. The IVM oocytes were fertilized in vitro with frozen-thawed spermatozoa. Fertilized oocytes were cultured for 7 d, and embryos that developed to the blastocyst stage were used for the experiments. The blastocysts, packed in straws with transportation medium that consisted of modified TCM-199 with HEPES equilibrated in air and supplemented with 20 % calf serum and 0, 10, 50, 100 or 150 microM beta-ME, were transported at 37 degrees C from Tokyo to Sapporo by air (18.3 h). The quality of blastocysts was assessed and ranked as excellent (A), good (B), fair (C) or poor (D) after transportation. The percentages of blastocysts ranked as A or B were significantly higher (P plastic straws for several hours without control of CO2 and that the concentration of beta-ME used in this experiment is not detrimental to the blastocysts.

  7. Improved bovine embryo production in an oviduct-on-a-chip system: prevention of poly-spermic fertilization and parthenogenic activation.

    Science.gov (United States)

    Ferraz, Marcia A M M; Henning, Heiko H W; Costa, Pedro F; Malda, Jos; Melchels, Ferry P; Wubbolts, R; Stout, Tom A E; Vos, Peter L A M; Gadella, Bart M

    2017-02-28

    The oviduct provides the natural micro-environment for gamete interaction, fertilization and early embryo development in mammals, such as the cow. In conventional culture systems, bovine oviduct epithelial cells (BOEC) undergo a rapid loss of essential differentiated cell properties; we aimed to develop a more physiological in vitro oviduct culture system capable of supporting fertilization. U-shaped chambers were produced using stereo-lithography and mounted with polycarbonate membranes, which were used as culture inserts for primary BOECs. Cells were grown to confluence and cultured at an air-liquid interface for 4 to 6 weeks and subsequently either fixed for immune staining, incubated with sperm cells for live-cell imaging, or used in an oocyte penetration study. Confluent BOEC cultures maintained polarization and differentiation status for at least 6 weeks. When sperm and oocytes were introduced into the system, the BOECs supported oocyte penetration in the absence of artificial sperm capacitation factors while also preventing polyspermy and parthenogenic activation, both of which occur in classical in vitro fertilization systems. Moreover, this "oviduct-on-a-chip" allowed live imaging of sperm-oviduct epithelium binding and release. Taken together, we describe for the first time the use of 3D-printing as a step further on bio-mimicking the oviduct, with polarized and differentiated BOECs in a tubular shape that can be perfused or manipulated, which is suitable for live imaging and supports in vitro fertilization.

  8. The Influence of Single Nucleotide Polymorphism Microarray-Based Molecular Karyotype on Preimplantation Embryonic Development Potential.

    Science.gov (United States)

    Li, Gang; He, Nannan; Jin, Haixia; Liu, Yan; Guo, Yihong; Su, Yingchun; Sun, Yingpu

    2015-01-01

    In order to investigate the influence of the molecular karyotype based on single nucleotide polymorphism (SNP) microarray on embryonic development potential in preimplantation genetic diagnosis (PGD), we retrospectively analyzed the clinical data generated by PGD using embryos retrieved from parents with chromosome rearrangements in our center. In total, 929 embryos from 119 couples had exact diagnosis and development status. The blastocyst formation rate of balanced molecular karyotype embryos was 56.6% (276/488), which was significantly higher than that of genetic imbalanced embryos 24.5% (108/441) (P35 respectively. Blastocyst formation rates of male and female embryos were 44.5% (183/411) and 38.8% (201/518) respectively, with no significant difference between them (P>0.05). The rates of balanced molecular karyotype embryos vary from groups of embryos with different cell numbers at 68 hours after insemination. The blastocyst formation rate of embryos with 6-8 cells (48.1%) was significantly higher than that of embryos with 8 cells (42.9%) (Pabnormal molecular karyotypes in the subgroup of the arrest, morula and blastocyst. Thus, we conclude that embryos with balanced molecular karyotype have significant higher development potential than those with imbalanced molecular karyotype whilst maternal age, embryo gender and types of abnormal molecular karyotype have no significant influence on blastocyst formation. Compared with embryos with 8 cells, embryos with 6-8 blastomeres have higher rate of balanced molecular karyotype and blastocyst formation.

  9. Holding immature bovine oocytes in a commercial embryo holding medium: High developmental competence for up to 10 h at room temperature.

    Science.gov (United States)

    Pascottini, Osvaldo Bogado; Catteeuw, Maaike; Van Soom, Ann; Opsomer, Geert

    2017-11-04

    Bovine in vitro embryo production (IVP) following Ovum Pick Up (OPU) is all too often hampered by a large time gap between the harvest of oocytes of the first and last OPU session of the day. Immediately after retrieval, oocyte maturation is initiated, resulting in oocytes maturing at different time points which necessitates laborious scheduling of the IVP process. In this study, the potential of a commercial embryo holding medium (EHM; Syngro, Bioniche Inc.) to hold immature bovine oocytes was validated. We assessed the effect of holding time and temperature on (1) oocytes' maturation; (2) blastocyst development and quality at day 8 post insemination; and (3) blastocyst yield in small groups of oocytes/zygotes simulating OPU settings. Oocytes, harvested from slaughterhouse ovaries, were held for 6 h (either at 4 °C, room temperature [RT; 22-25 °C], or 38.5 °C), for 10 h (at 4 °C or RT), and for 14 h (only at RT) in 1 mL sterile glass osmometer tubes filled with EHM prior to standard maturation (22 h at 38.5 °C) and subsequent IVP. Results were compared with controls in which no prior holding was applied. Differences between the treated and control groups were assessed by generalized mixed-effects models and considered significant at P h in EHM at different temperatures remained at the germinal vesicle stage. Holding immature oocytes in EHM for 6 h at 38.5 °C and for 10 h at 4 °C significantly decreased maturation (57.1 ± 4.1% VS 80.9 ± 3.2% and 68.6 ± 3.5% VS 80.7 ± 2.9%; respectively), and development (11.0 ± 1.8% VS 36.2 ± 2.8% and 20.1 ± 3.3% VS 40.6 ± 4.6%) (P h at RT, did not affect the maturation rates (83.2 ± 2.9% and 78.9 ± 3.2%) nor day 8 blastocyst rates (35.2 ± 2.7% and 40.2 ± 4.5%). Prolonging holding time to 14 h in RT decreased maturation and day 8 blastocyst yield (71.9 ± 3.5% VS 84.5 ± 2.7% and 25.7 ± 2.5% VS 39.5 ± 2.8%, respectively) (P h at RT. When subsequently

  10. Lipid characterization of embryo zones by silica plate laser desorption ionization mass spectrometry imaging (SP-LDI-MSI).

    Science.gov (United States)

    Ferreira, Mônica S; de Oliveira, Diogo N; Gonçalves, Roseli F; Catharino, Rodrigo R

    2014-01-07

    Lipid pathways play important biological roles in mammalian embryology, directing early developmental pathways to differentiation. Phospholipids and triglycerides, among others, are the main composing lipids of zona pellucida in several embryo species. Lipid analysis in embryos by mass spectrometry usually requires sample preparation and/or matrix application. This novel approach using silica plate laser desorption/ionization mass spectrometry imaging (SP-LDI-MSI) allows direct single-cell imaging and embryo region discrimination with no matrix coating. Its application is herein described for two- and eight-cell embryos. Lipid biomarkers for blastomere and intact zona pellucida are reported and corroborated by both fragmentation reactions (MS/MS) and images. Results obtained in this work are understood to be of great use for further developments on in vitro bovine fertilization. Since much of the processes can be monitored by characteristic biomarkers, it is now possible to precisely identify cell division errors during early embryo stages, as well as evaluate pre-implantation conditions. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Identification and characteristics of extracellular vesicles from bovine blastocysts produced in vitro.

    Science.gov (United States)

    Mellisho, Edwin A; Velásquez, Alejandra E; Nuñez, María J; Cabezas, Joel G; Cueto, Juan A; Fader, Claudio; Castro, Fidel O; Rodríguez-Álvarez, Lleretny

    2017-01-01

    Extracellular vesicles (EVs) have been identified within different body fluids and cell culture media. However, there is very little information on the secretion of these vesicles during early embryonic development. The aims of this work were first to demonstrate the secretion of extracellular vesicles by pre-implantation bovine embryos and second to identify and characterize the population of EVs secreted by bovine blastocysts during the period from day seven to nine of embryo culture and its correlation with further embryo development up to day 11. Bovine embryos were produced by in vitro fertilization (IVF) or parthenogenetic activation (PA) and cultured until blastocyst stage. Blastocyst selection was performed at day 7 post IVF/PA considering two variables: stage of development and quality of embryos. Selected blastocysts were cultured in vitro for 48 hours in groups (exp. 1) or individually (exp. 2) in SOF media depleted of exosomes. At day 9 post IVF/PA the media was collected and EVs isolated by ultracentrifugation. Transmission electron microscopy revealed the presence of heterogeneous vesicles of different sizes and population: microvesicles (MVs) and exosomes (EXs) of rounded shape, enclosed by a lipid bi-layer and ranging from 30 to 385 nm of diameter. Flow cytometry analysis allowed identifying CD63 and CD9 proteins as exosome markers. Nanoparticle tracking analysis generated a large number of variables, which required the use of multivariate statistics. The results indicated that the concentration of vesicles is higher in those blastocysts with arrested development from day 9 up to day 11 of in vitro development (6.7 x 108 particles/ml) derived from IVF (p <0.05), compared to PA blastocysts (4.7 x 108 particles/ml). Likewise, the profile (concentration and diameter) of particles secreted by embryos derived from IVF were different from those secreted by PA embryos. In conclusion, we demonstrated that bovine blastocysts secrete MVs/EXs to the culture

  12. Obesity does not aggravate vitrification injury in mouse embryos: a prospective study.

    Science.gov (United States)

    Ma, Wenhong; Yang, Xing; Liang, Xiaoyan

    2012-08-31

    Obesity is associated with poor reproductive outcomes, but few reports have examined thawed embryo transfer in obese women. Many studies have shown that increased lipid accumulation aggravates vitrification injury in porcine and bovine embryos, but oocytes of these species have high lipid contents (63 ng and 161 ng, respectively). Almost nothing is known about lipids in human oocytes except that these cells are anecdotally known to be relatively lipid poor. In this regard, human oocytes are considered to be similar to those of the mouse, which contain approximately 4 ng total lipids/oocyte. To date, no available data show the impact of obesity on vitrification in mouse embryos. The aim of this study was to establish a murine model of maternal diet-induced obesity and to characterize the effect of obesity on vitrification by investigating the survival rate and embryo developmental competence after thawing. Prospective comparisons were performed between six-eight-cell embryos from obese and normal-weight mice and between fresh and vitrified embryos. Female C57BL/6 mice were fed standard rodent chow (normal-weight group) or a high-fat diet (obese group) for 6 weeks. The mice were mated, zygotes were collected from oviducts and cultured for 3 days, and six-eight-cell embryos were then selected to assess lipid content in fresh embryos and to evaluate differences in apoptosis, survival, and development rates in response to vitrification. In fresh embryos from obese mice, the lipid content (0.044 vs 0.030, Pvitrification, no significant difference was found between thawed embryos from obese and normal-weight mice in apoptosis, survival, and development rates on days 4 and 5. In both groups, pre- and post-vitrification embryo apoptosis, survival, and development rates were similar. This study demonstrated that differences in survival and developmental rates between embryos from obese and normal-weight mice were eliminated after vitrification. Thus, maternal obesity does

  13. Preimplantation Genetic Testing in the 21st Century: Uncharted Territory

    Directory of Open Access Journals (Sweden)

    Paul R. Brezina MD, MBA

    2013-01-01

    Full Text Available The past hundred years have given birth to arguably the most profound changes in society, medicine, and technology the world has ever witnessed. Genetics is one such field that has enjoyed a meteoric rise during this time. Progressing from Mendelian genetics to the discovery of DNA to the ability to sequence the human genome, perhaps no other discipline holds more promise to affect future change than genetics. Technology currently exists to evaluate some of the genetic information held by developing embryos in the context of an in vitro fertilization (IVF cycle. This information is then used to determine which embryos are selected for uterine transfer. Many societies have enacted legislation to protect against possible abuses utilizing this technology. However, it is incumbent upon society to continue ensuring that preimplantation genetic diagnosis (PGD–-and genetic testing in general–-is applied in a way that utilizes its potential in a responsible manner to improve health care.

  14. Preimplantation genetic testing in the 21st century: uncharted territory.

    Science.gov (United States)

    Brezina, Paul R

    2013-02-10

    The past hundred years have given birth to arguably the most profound changes in society, medicine, and technology the world has ever witnessed. Genetics is one such field that has enjoyed a meteoric rise during this time. Progressing from Mendelian genetics to the discovery of DNA to the ability to sequence the human genome, perhaps no other discipline holds more promise to affect future change than genetics. Technology currently exists to evaluate some of the genetic information held by developing embryos in the context of an in vitro fertilization (IVF) cycle. This information is then used to determine which embryos are selected for uterine transfer. Many societies have enacted legislation to protect against possible abuses utilizing this technology. However, it is incumbent upon society to continue ensuring that preimplantation genetic diagnosis (PGD)-and genetic testing in general-is applied in a way that utilizes its potential in a responsible manner to improve health care.

  15. [Extending preimplantation genetic diagnosis to HLA typing: the French exception].

    Science.gov (United States)

    Steffann, Julie; Frydman, Nelly; Burlet, Philippe; Gigarel, Nadine; Hesters, Laetitia; Kerbrat, Violaine; Lamazou, Frédéric; Munnich, Arnold; Frydman, René

    2011-01-01

    Umut-Talha, a "sibling savior", was born on 26 January 2011 at Beclère Hospital after embryo selection at the Paris preimplantation genetic diagnosis (PGD) center. His birth revived the controversy over "double PGD". This procedure, authorized in France since 2006, allows couples who already have a child with a serious, incurable genetic disease, to opt for PGD in order to select a healthy embryo that is HLA-matched to the affected sibling and who may thus serve as an ombilical cord blood donor. The procedure is particularly complex and the baby take-home rate is still very low. Double PGD is strictly regulated in France, and candidate couples must first receive individual authorization from the Biomedicine Agency. In our experience, these couples have a strong desire to have children, as reflected by the large number of prior spontaneous pregnancies (25% of couples). Likewise, most of these couples request embryo transfer even when there is no HLA-matched embryo, which accounts for more than half of embryo transfers. The controversy surrounding this practice has flared up again in recent weeks, over the concepts of "designer babies" and "double savior siblings" (the baby is selected to be free of the hereditary disease, and may also serve as a stem cell donor for the affected sibling).

  16. Practices and ethical concerns regarding preimplantation diagnosis. Who regulates preimplantation genetic diagnosis in Brazil?

    Directory of Open Access Journals (Sweden)

    B.B. Damian

    2015-01-01

    Full Text Available Preimplantation genetic diagnosis (PGD was originally developed to diagnose embryo-related genetic abnormalities for couples who present a high risk of a specific inherited disorder. Because this technology involves embryo selection, the medical, bioethical, and legal implications of the technique have been debated, particularly when it is used to select features that are not related to serious diseases. Although several initiatives have attempted to achieve regulatory harmonization, the diversity of healthcare services available and the presence of cultural differences have hampered attempts to achieve this goal. Thus, in different countries, the provision of PGD and regulatory frameworks reflect the perceptions of scientific groups, legislators, and society regarding this technology. In Brazil, several texts have been analyzed by the National Congress to regulate the use of assisted reproduction technologies. Legislative debates, however, are not conclusive, and limited information has been published on how PGD is specifically regulated. The country requires the development of new regulatory standards to ensure adequate access to this technology and to guarantee its safe practice. This study examined official documents published on PGD regulation in Brazil and demonstrated how little direct oversight of PGD currently exists. It provides relevant information to encourage reflection on a particular regulation model in a Brazilian context, and should serve as part of the basis to enable further reform of the clinical practice of PGD in the country.

  17. Purification of binder of sperm protein 1 (BSP1) and its effects on bovine in vitro embryo development after fertilization with ejaculated and epididymal sperm.

    Science.gov (United States)

    Rodríguez-Villamil, P; Hoyos-Marulanda, V; Martins, J A M; Oliveira, A N; Aguiar, L H; Moreno, F B; Velho, A L M C S; Monteiro-Moreira, A C; Moreira, R A; Vasconcelos, I M; Bertolini, M; Moura, A A

    2016-02-01

    The present study evaluated functional aspects of binder of sperm 1 (BSP1) in the bovine species. In a first experiment, cumulus-oocyte complexes (n = 1274) were incubated with frozen-thawed ejaculated sperm (18 hours) in Fert-TALP medium containing: heparin, 10, 20, or 40 μg/mL BSP1. Heparin followed by gelatin affinity chromatography was used for purification of BSP1 from bovine seminal vesicle fluid. With ejaculated sperm, cleavage rates were similar when Fert-TALP medium was incubated with heparin (74.1 ± 2.7%), 10 μg/mL BSP1 (77.8 ± 3.1%), or 20 μg/mL BSP1 (74 ± 2.0%). Day-7 blastocyst rates were equivalent after incubations with heparin (40.8 ± 5.0%) and 10 μg/mL BSP1 (34.1 ± 4.4%), but reduced after 20 μg/mL BSP1 (22.4 ± 2.9%) and 40 μg/mL BSP1 (19.3 ± 4.1%; P epididymal sperm (18 hours) in Fert-TALP medium containing: no heparin, heparin, 10, 20, or 40 μg/mL. Cleavage and blastocyst rates were similar after treatments with heparin (68.5 ± 1.3% and 24.7 ± 3.2%, respectively) or without heparin (65.5 ± 1.8% and 27.3 ± 1.6%, respectively). Cleavage was higher after treatment with any BSP1 concentrations (74.2 ± 2.7%-79.0 ± 1.1%) than without heparin (P epididymal sperm, as observed with ejaculated sperm. On the basis of immunocytochemistry, there was BSP1 binding to frozen-thawed ejaculated but not to epididymal sperm. Also, anti-BSP1 reaction remained on ejaculated sperm (as expected) and appeared on epididymal sperm after incubation with purified BSP1. Acrosome reaction of ejaculated and epididymal sperm was induced after incubation with purified BSP1 as well, indicating an effect of BSP1 on capacitation. In conclusion, purified BSP1 from bull seminal vesicles was able to bind to and induce capacitation of ejaculated and epididymal sperm. Also, BSP1 added to fertilization media and allowed proper cleavage and embryo development, with the effects being modulated by previous exposure or not of spermatozoa to seminal plasma. Copyright

  18. Detrimental effects of microgravity on mouse preimplantation development in vitro.

    Directory of Open Access Journals (Sweden)

    Sayaka Wakayama

    Full Text Available Sustaining life beyond Earth either on space stations or on other planets will require a clear understanding of how the space environment affects key phases of mammalian reproduction. However, because of the difficulty of doing such experiments in mammals, most studies of reproduction in space have been carried out with other taxa, such as sea urchins, fish, amphibians or birds. Here, we studied the possibility of mammalian fertilization and preimplantation development under microgravity (microG conditions using a three-dimensional (3D clinostat, which faithfully simulates 10(-3 G using 3D rotation. Fertilization occurred normally in vitro under microG. However, although we obtained 75 healthy offspring from microG-fertilized and -cultured embryos after transfer to recipient females, the birth rate was lower than among the 1G controls. Immunostaining demonstrated that in vitro culture under microG caused slower development and fewer trophectoderm cells than in 1G controls but did not affect polarization of the blastocyst. These results suggest for the first time that fertilization can occur normally under microG environment in a mammal, but normal preimplantation embryo development might require 1G.

  19. Impaired mitotic progression and preimplantation lethality in mice lacking OMCG1, a new evolutionarily conserved nuclear protein

    DEFF Research Database (Denmark)

    Artus, Jérôme; Vandormael-Pournin, Sandrine; Frödin, Morten

    2005-01-01

    -type-specific function, indicating that many aspects of cell cycle regulation during mammalian embryo development remain to be elucidated. Here, we report on the characterization of a new gene, Omcg1, which codes for a nuclear zinc finger protein. Embryos lacking Omcg1 die by the end of preimplantation development....... In vitro cultured Omcg1-null blastocysts exhibit a dramatic reduction in the total cell number, a high mitotic index, and the presence of abnormal mitotic figures. Importantly, we found that Omcg1 disruption results in the lengthening of M phase rather than in a mitotic block. We show that the mitotic...... delay in Omcg1-/- embryos is associated with neither a dysfunction of the spindle checkpoint nor abnormal global histone modifications. Taken together, these results suggest that Omcg1 is an important regulator of the cell cycle in the preimplantation embryo....

  20. SSH adequacy to preimplantation mammalian development: Scarce specific transcripts cloning despite irregular normalisation

    Directory of Open Access Journals (Sweden)

    Renard JP

    2005-11-01

    Full Text Available Abstract Background SSH has emerged as a widely used technology to identify genes that are differentially regulated between two biological situations. Because it includes a normalisation step, it is used for preference to clone low abundance differentially expressed transcripts. It does not require previous sequence knowledge and may start from PCR amplified cDNAs. It is thus particularly well suited to biological situations where specific genes are expressed and tiny amounts of RNA are available. This is the case during early mammalian embryo development. In this field, few differentially expressed genes have been characterized from SSH libraries, but an overall assessment of the quality of SSH libraries is still required. Because we are interested in the more systematic establishment of SSH libraries from early embryos, we have developed a simple and reliable strategy based on reporter transcript follow-up to check SSH library quality and repeatability when starting with small amounts of RNA. Results Four independent subtracted libraries were constructed. They aimed to analyze key events in the preimplantation development of rabbit and bovine embryos. The performance of the SSH procedure was assessed through the large-scale screening of thousands of clones from each library for exogenous reporter transcripts mimicking either tester specific or tester/driver common transcripts. Our results show that abundant transcripts escape normalisation which is only efficient for rare and moderately abundant transcripts. Sequencing 1600 clones from one of the libraries confirmed and extended our results to endogenous transcripts and demonstrated that some very abundant transcripts common to tester and driver escaped subtraction. Nonetheless, the four libraries were greatly enriched in clones encoding for very rare (0.0005% of mRNAs tester-specific transcripts. Conclusion The close agreement between our hybridization and sequencing results shows that the

  1. SSH adequacy to preimplantation mammalian development: scarce specific transcripts cloning despite irregular normalisation.

    Science.gov (United States)

    Bui, L C; Léandri, R D; Renard, J P; Duranthon, V

    2005-11-08

    SSH has emerged as a widely used technology to identify genes that are differentially regulated between two biological situations. Because it includes a normalisation step, it is used for preference to clone low abundance differentially expressed transcripts. It does not require previous sequence knowledge and may start from PCR amplified cDNAs. It is thus particularly well suited to biological situations where specific genes are expressed and tiny amounts of RNA are available. This is the case during early mammalian embryo development. In this field, few differentially expressed genes have been characterized from SSH libraries, but an overall assessment of the quality of SSH libraries is still required. Because we are interested in the more systematic establishment of SSH libraries from early embryos, we have developed a simple and reliable strategy based on reporter transcript follow-up to check SSH library quality and repeatability when starting with small amounts of RNA. Four independent subtracted libraries were constructed. They aimed to analyze key events in the preimplantation development of rabbit and bovine embryos. The performance of the SSH procedure was assessed through the large-scale screening of thousands of clones from each library for exogenous reporter transcripts mimicking either tester specific or tester/driver common transcripts. Our results show that abundant transcripts escape normalisation which is only efficient for rare and moderately abundant transcripts. Sequencing 1600 clones from one of the libraries confirmed and extended our results to endogenous transcripts and demonstrated that some very abundant transcripts common to tester and driver escaped subtraction. Nonetheless, the four libraries were greatly enriched in clones encoding for very rare (0.0005% of mRNAs) tester-specific transcripts. The close agreement between our hybridization and sequencing results shows that the addition and follow-up of exogenous reporter transcripts

  2. Diagnostic accuracy: theoretical models for preimplantation genetic testing of a single nucleus using the fluorescence in situ hybridization technique

    NARCIS (Netherlands)

    Scriven, P. N.; Bossuyt, P. M. M.

    2010-01-01

    The aim of this study was to develop and use theoretical models to investigate the accuracy of the fluorescence in situ hybridization (FISH) technique in testing a single nucleus from a preimplantation embryo without the complicating effect of mosaicism. Mathematical models were constructed for

  3. Case report: birth of healthy twins after preimplantation genetic diagnosis of propionic acidemia.

    Science.gov (United States)

    Alberola, Trinitat M; Bautista-Llácer, Rosa; Vendrell, Xavier; García-Mengual, Elena; Pardo, Merche; Vila, Maria; Calatayud, Carmen

    2011-03-01

    Development of an ad hoc protocol for the preimplantion genetic diagnosis of propionic acidemia in a couple carrying the mutations c.737G>T (G246V) and c.1218del14ins12 (ins/del) in the PCCB gene. Propionic acidemia is an autosomal recessive metabolic disorder where the body is unable to process certain parts of proteins and lipids. Symptoms manifest few days after birth and sometimes progress to more serious medical problems, including heart abnormalities, coma and death. Four short tandem repeat markers closely linked to the PCCB gene were tested, in order to support the direct mutation detection diagnosis. Multiplex fluorescent heminested polymerase chain reaction followed by fragment analysis and minisequencing was used. Fourteen single blastomeres from nine embryos were tested and two carrier embryos were transferred, resulting in the birth of two healthy boys. Preimplantation genetic diagnosis represents a valid reproductive option for couples affected of propionic acidemia, in order to avoid transmission to offspring.

  4. Preimplantation genetic diagnosis for gender selection in the United States

    Energy Technology Data Exchange (ETDEWEB)

    Colls, P.; Silver, L.; Olivera, G.; Weier, J.; Escudero, T.; Goodall, N.; Tomkin, G.; Munne, S.

    2009-08-20

    Preimplantation genetic diagnosis (PGD) of gender selection for non medical reasons has been considered an unethical procedure by several authors and agencies in the Western society on the basis of disrupting the sex ratio, being discriminatory againsts women and disposal of normal embryos of the non desired gender. In this study, the analysis of a large series of PGD procedures for gender selection from a wide geographical area in the United States, shows that in general there is no deviation in preference towards any specific gender except for a preference of males in some ethnic populations of Chinese, Indian and Middle Eastern origin that represent a small percentage of the US population. In cases where only normal embryos of the non-desired gender are available, 45.5% of the couples elect to cancel the transfer, while 54.5% of them are open to have transferred embryos of the non-desired gender, this fact being strongly linked to cultural and ethnical background of the parents. In addition this study adds some evidence to the proposition that in couples with previous children of a given gender there is no biological predisposition towards producing embryos of that same gender. Based on these facts, it seems that objections to gender selection formulated by ethics committees and scientific societies are not well-founded.

  5. Timing embryo biopsy for PGD - before or after cryopreservation?

    Science.gov (United States)

    Shinar, S; Kornecki, N; Schwartz, T; Mey-Raz, N; Amir, H; Almog, B; Shavit, T; Hasson, J

    2016-09-01

    Pre-implantation genetic diagnosis (PGD) is required in order to screen and diagnose embryos of patients at risk of having a genetically affected offspring. A biopsy to diagnose the genetic profile of the embryo may be performed either before or after cryopreservation. The aim of this study was to determine which biopsy timing yields higher embryo survival rates. Retrospective cohort study of all PGD patients in a public IVF unit between 2010 and 2013. Inclusion criteria were patients with good-quality embryos available for cryopreservation by the slow freezing method. Embryos were divided into two groups: biopsy before and biopsy after cryopreservation. The primary outcome was embryo survival rates post thawing. Sixty-five patients met inclusion criteria. 145 embryos were biopsied before cryopreservation and 228 embryos were cryopreserved and biopsied after thawing. Embryo survival was significantly greater in the latter group (77% vs. 68%, p Cryopreservation preceding biopsy results in better embryo survival compared to biopsy before cryopreservation.

  6. Preimplantation Genetic Screening: An Effective Testing for Infertile and Repeated Miscarriage Patients?

    OpenAIRE

    Wang, Ning; Zheng, Ying-Ming; Li, Lei; Jin, Fan

    2010-01-01

    Aneuploidy in pregnancy is known to increase with advanced maternal age (AMA) and associate with repeated implantation failure (RIF), and repeated miscarriage (RM). Preimplantation genetic screening (PGS) has been introduced into clinical practice, screening, and eliminating aneuploidy embryos, which can improve the chance of conceptions for infertility cases with poor prognosis. These patients are a good target group to assess the possible benefit of aneuploidy screening. Although practiced ...

  7. Preimplantation alcohol exposure and developmental programming of FASD: An epigenetic perspective.

    Science.gov (United States)

    Legault, Lisa-Marie; Bertrand-Lehouillier, Virginie; McGraw, Serge

    2017-10-31

    Alcohol exposure during in utero development can permanently change the developmental programming of physiological responses, thereby increasing the risk of childhood neurological illnesses and later adverse health outcomes associated with fetal alcohol spectrum disorders (FASD). There is an increasing body of evidence indicating that alcohol exposure during gestation triggers lasting epigenetic alterations in offspring long after the initial insult; together, these studies support the role of epigenetics in FASD etiology. However, we still have little information about how ethanol interferes with the fundamental epigenetic reprogramming wave (e.g., erasure and re-establishment of DNA methylation marks) that characterizes preimplantation embryo development. This article will review key epigenetic processes occurring during preimplantation development and especially focus on the current knowledge regarding how a prenatal alcohol exposure during this period could affect the developmental programming of the early stage preimplantation embryo. We will also outline current limitations of studies examining the in vivo and in vitro effects of alcohol exposure on embryos as well as underline the next critical steps to be taken if we want to better understand the implicated mechanisms in order to strengthen the translational potential for non-invasive epigenetic diagnosis markers and the treatment of newborns that have higher risks of developing FASD.

  8. The experience of two European preimplantation genetic diagnosis centres on human leukocyte antigen typing.

    Science.gov (United States)

    Van de Velde, Hilde; De Rycke, Martine; De Man, Caroline; De Hauwere, Kim; Fiorentino, Francesco; Kahraman, Semra; Pennings, Guido; Verpoest, Willem; Devroey, Paul; Liebaers, Inge

    2009-03-01

    Two European centres report on human leukocyte antigen (HLA) typing of preimplantation embryos for haematopoietic stem cell (HSC) transplantation: 'UZ Brussel' in Brussels and 'Genoma' in Rome. Both centres have 6 years' experience with technical and clinical aspects of this type of genetic analysis on single blastomeres. Both centres apply a similar technique for preimplantation HLA typing using short tandem repeats linked to the HLA locus in multiplex PCR for haplotyping. At present, a conclusive HLA diagnosis could be assured in 92.8% and 90.3% of the embryos at UZ Brussel and at Genoma, respectively. The implantation rates were 32.4% and 28.2%, respectively, and the birth rates per cycle were 9.4% and 18.6%, respectively. The HLA programme at UZ Brussel and at Genoma resulted in the birth of 9 babies and 3 successful HSC transplantations, and 42 babies and 7 successful HSC transplantations, respectively, so far. Drastic embryo selection for preimplantation HLA typing (in theory 1/4 for HLA, 1/8 for HLA in combination with sexing for X-linked recessive diseases, 3/16 for HLA in combination with autosomal recessive disorders) resulted overall in the birth of 51 babies (15.9% live birth rate per started cycle) in two European centres.

  9. A repetitive probe for FISH analysis of bovine interphase nuclei

    Directory of Open Access Journals (Sweden)

    Cribiu Edmond

    2000-03-01

    Full Text Available Abstract The purpose of this study was to generate repetitive DNA sequence probes for the analysis of interphase nuclei by fluorescent in situ hybridisation (FISH. Such probes are useful for the diagnosis of chromosomal abnormalities in bovine preimplanted embryos. Of the seven probes (E1A, E4A, Ba, H1A, W18, W22, W5 that were generated and partially sequenced, five corresponded to previously described Bos taurus repetitive DNA (E1A, E4A, Ba, W18, W5, one probe (W22 shared no homology with other DNA sequences and one (H1A displayed a significant homology with Rattus norvegicus mRNA for secretin receptor transmembrane domain 3. Fluorescent in situ hybridisation was performed on metaphase bovine fibroblast cells and showed that five of the seven probes hybridised most centromeres (E1A, E4A, Ba, W18, W22, one labelled the arms of all chromosomes (W5 and the H1A probe was specific to three chromosomes (ch14, ch20, and ch25. Moreover, FISH with H1A resulted in interpretable signals on interphase nuclei in 88% of the cases, while the other probes yielded only dispersed overlapping signals.

  10. Influence of embryo culture medium (G5 and HTF) on pregnancy and perinatal outcome after IVF: a multicenter RCT

    NARCIS (Netherlands)

    Kleijkers, Sander H. M.; Mantikou, Eleni; Slappendel, Els; Consten, Dimitri; van Echten-Arends, Jannie; Wetzels, Alex M.; van Wely, Madelon; Smits, Luc J. M.; van Montfoort, Aafke P. A.; Repping, Sjoerd; Dumoulin, John C. M.; Mastenbroek, Sebastiaan

    2016-01-01

    Does embryo culture medium influence pregnancy and perinatal outcome in IVF? Embryo culture media used in IVF affect treatment efficacy and the birthweight of newborns. A wide variety of culture media for human preimplantation embryos in IVF/ICSI treatments currently exists. It is unknown which

  11. Influence of embryo culture medium (G5 and HTF) on pregnancy and perinatal outcome after IVF: a multicenter RCT

    NARCIS (Netherlands)

    Kleijkers, S.H.; Mantikou, E.; Slappendel, E.; Consten, D.; Echten-Arends, J. van; Wetzels, A.M.M.; Wely, M. van; Smits, L.J.; Montfoort, A.P. van; Repping, S.; Dumoulin, J.C.; Mastenbroek, S.

    2016-01-01

    STUDY QUESTION: Does embryo culture medium influence pregnancy and perinatal outcome in IVF? SUMMARY ANSWER: Embryo culture media used in IVF affect treatment efficacy and the birthweight of newborns. WHAT IS KNOWN ALREADY: A wide variety of culture media for human preimplantation embryos in

  12. Influence of embryo culture medium (G5 and HTF) on pregnancy and perinatal outcome after IVF : a multicenter RCT

    NARCIS (Netherlands)

    Kleijkers, Sander H. M.; Mantikou, Eleni; Slappendel, Els; Consten, Dimitri; van Echten - Arends, Jannie; Wetzels, Alex M.; van Wely, Madelon; Smits, Luc J. M.; van Montfoort, Aafke P. A.; Repping, Sjoerd; Dumoulin, John C. M.; Mastenbroek, Sebastiaan

    2016-01-01

    Does embryo culture medium influence pregnancy and perinatal outcome in IVF? Embryo culture media used in IVF affect treatment efficacy and the birthweight of newborns. A wide variety of culture media for human preimplantation embryos in IVF/ICSI treatments currently exists. It is unknown which

  13. Differences in cumulus cell gene expression indicate the benefit of a pre-maturation step to improve in-vitro bovine embryo production.

    Science.gov (United States)

    Dieci, Cecilia; Lodde, Valentina; Labreque, Rémi; Dufort, Isabelle; Tessaro, Irene; Sirard, Marc-André; Luciano, Alberto M

    2016-12-01

    Does the gene expression profile of cumulus cells (CC) accompanying oocytes with different degrees of chromatin compaction within the germinal vesicle (GV) reflect the oocyte's quality and response in culture during in-vitro embryo production (IVP). The transcriptomic profile of the CC is related to oocyte competence, setting the stage for the development of customized pre-maturation strategies to improve IVP. Oocytes complete the acquisition of their competence during antral follicle development. During this period, the chromatin configuration within the GV changes dynamically and is indicative of oocyte's developmental potential. The interactions between somatic and germ cells modulate chromatin morphology and function and are critical for acquisition of oocyte competence. Bovine cumulus-oocyte complexes (COC) were isolated from 0.5 to 6 mm antral follicles. Surrounding CC were separated from the oocyte and classified as GV0, GV1, GV2 and GV3 according to the degree of the oocyte's chromatin compaction. RNA extracted from CC of each group was amplified and hybridized on a bovine embryo-specific 44 K Agilent slide. The CC_GV1, CC_GV2 and CC_GV3 classes were each hybridized against the CC_GV0 class, representing an early oocyte differentiation stage with poor development competence. The data were normalized and fold changes of the differentially expressed genes were determined. Microarray data were validated using quantitative RT-PCR on selected targets. Microarray data were further analyzed through: (i) between-group analysis (BGA), which classifies the samples according to their transcriptomic profiles; (ii) cluster analysis according to the expression profile of each gene; and (iii) Ingenuity Pathway Analysis (IPA) to study gene regulation patterns and predicted functions. Furthermore, CC of each GV group were cultured and apoptotic cells were assessed after 3 h by caspase analysis. Finally, based on the analysis of CC transcriptomic profiles and the

  14. Preimplantation genetic diagnosis associated to Duchenne muscular dystrophy.

    Science.gov (United States)

    Bianco, Bianca; Christofolini, Denise Maria; Conceição, Gabriel Seixas; Barbosa, Caio Parente

    2017-09-21

    Duchenne muscular dystrophy is the most common muscle disease found in male children. Currently, there is no effective therapy available for Duchenne muscular dystrophy patients. Therefore, it is essential to make a prenatal diagnosis and provide genetic counseling to reduce the birth of such boys. We report a case of preimplantation genetic diagnosis associated with Duchenne muscular dystrophy. The couple E.P.R., 38-year-old, symptomatic patient heterozygous for a 2 to 47 exon deletion mutation in DMD gene and G.T.S., 39-year-old, sought genetic counseling about preimplantation genetic diagnosis process. They have had a 6-year-old son who died due to Duchenne muscular dystrophy complications. The couple underwent four cycles of intracytoplasmic sperm injection (ICSI) and eight embryos biopsies were analyzed by polymerase chain reaction (PCR) for specific mutation analysis, followed by microarray-based comparative genomic hybridisation (array CGH) for aneuploidy analysis. Preimplantation genetic diagnosis revealed that two embryos had inherited the maternal DMD gene mutation, one embryo had a chromosomal alteration and five embryos were normal. One blastocyst was transferred and resulted in successful pregnancy. The other embryos remain vitrified. We concluded that embryo analysis using associated techniques of PCR and array CGH seems to be safe for embryo selection in cases of X-linked disorders, such as Duchenne muscular dystrophy. RESUMO A distrofia muscular de Duchenne é a doença muscular mais comum observadas em crianças do sexo masculino. Atualmente, não há terapia eficaz disponível para distrofia muscular de Duchenne, portanto, é essencial o diagnóstico pré-natal e o aconselhamento genético para reduzir o nascimento desses meninos. Relatamos um caso de diagnóstico genético pré-implantação associado à distrofia muscular de Duchenne. O casal E.P.R., 38 anos, heterozigota, sintomática para uma mutação de deleção dos éxons 2 a 47 no gene

  15. [The Cagliari (Italy) Court authorizes the preimplantation genetic diagnosis].

    Science.gov (United States)

    Jorqui Azofra, María

    2007-01-01

    Today, preimplantation genetic diagnosis (PGD) has been greatly accepted within the framework of positive law of many European countries. Nevertheless, in other countries, such as Italy, it is forbidden by law. The ruling of the Civil Court of Cagliari which has authorized its use to a Sardinian couple, has opened, in this way, a small crack to be able to asses possible modifications to the Italian regulation on this matter. This article analyses the ruling of the Civil Court of Cagliari (Italy) from an ethical and legal perspective. The criteria which is used to analyse the legitimacy or illegitimacy of the practice of PGD is analysed. That is, on reasons which could justify or not the transfer of embryos in vitro to the woman. With this objective in mind, the Italian and Spanish normative models which regulates this controversial subject are looked at. As a conclusion, a critical evaluation of the arguments presented is made.

  16. LIF supports primitive endoderm expansion during pre-implantation development

    DEFF Research Database (Denmark)

    Morgani, Sophie M; Brickman, Joshua M

    2015-01-01

    Embryonic stem cells (ESCs) are pluripotent cell lines that can be maintained indefinitely in an early developmental state. ESC culture conditions almost always require the cytokine LIF to maintain self-renewal. As ESCs are not homogeneous but contain multiple populations reminiscent of the blast......Embryonic stem cells (ESCs) are pluripotent cell lines that can be maintained indefinitely in an early developmental state. ESC culture conditions almost always require the cytokine LIF to maintain self-renewal. As ESCs are not homogeneous but contain multiple populations reminiscent...... report that LIF has two distinct roles: it blocks early epiblast (Epi) differentiation, and it supports the expansion of primitive endoderm (PrE)-primed ESCs and PrE in vivo. We find that activation of JAK/STAT signalling downstream of LIF occurs initially throughout the pre-implantation embryo...

  17. [Extending preimplantation genetic diagnosis to HLA typing: the Paris experience].

    Science.gov (United States)

    Steffann, J; Frydman, N; Burlet, P; Gigarel, N; Feyereisen, E; Kerbrat, V; Tachdjian, G; Munnich, A; Frydman, R

    2005-10-01

    Preimplantation genetic diagnosis (PGD) consists in the genetic analysis of one or two cells. These cells (blastomeres) are sampled from embryos, obtained by in vitro fertilization, at the third day of development. Since 1998, the bioethical laws (1994) and their decrees restricted PGD practices in France, strictly to the avoidance of the birth of a child affected with a genetic defect. In parallel, works on blood cord transplantation, taken at the birth of a compatible HLA sibling, showed very encouraging results, particularly for the treatment of Fanconi anemia. In 2001, Verlinsky et al., have reported the first PGD for Fanconi anaemia combined with HLA typing, allowing the birth of a healthy child, HLA-identical with his affected sister. The "designer baby" concept was born. The French law, which allowed PGD under specific conditions, i.e. when the genetic defect has been characterized in one parent at least, recently extended PGD to HLA typing when embryos are at risk of a genetic disorder. Article L.2131-4-1 (August 2004) allows the practice of HLA typing for PGD embryos when an elder sibling is affected with a genetic disorder and need stem cell transplantation. The HLA-matched offspring resulting from PGD can give cord blood at birth to supply the necessary therapy. This double selection give rise to serious ethical problems, but technical difficulties and legal restrictions will probably limit the development of such a procedure.

  18. In vitro development of embryos from experimentally Kerack-addicted Mice

    Directory of Open Access Journals (Sweden)

    Elham Mohammadzadeh

    2017-08-01

    Full Text Available Background: Prenatal drug exposure, as a common public health concern, is associated with an increased risk of adverse effects on early embryo development. Objective: To investigate the in vitro development of - embryo from experimentally Kerack-addicted mice. Materials and Methods: Twenty-five female mice were studied in five groups: control, vehicle, and three experimental groups of Kerack-dependent mice (I, II, and III which received different doses of Kerack for 14 days. After the establishment of addiction model (7 days, experimental groups I, II, and III were given Kerack intraperitoneally at the doses of 5, 35, and 70 mg/kg, twice a day for a period of 7 days, respectively. The vehicle group received normal saline and lemon juice whilst the control group just received water and food. Morulae were obtained through oviduct flashing. The survived embryos were cultured in T6+ 5mg/ml bovine serum albumin. The developmental rates up to hatched stage daily and embryo quality (differential staining and Tunnel staining were also assessed Results: The developmental potential of embryos obtained from the addicted mother was significantly decreased in comparison with control group. There was a significant reduction in the rate of blastocyst formation in the high dose Kerack dependent group. However, in addicted mice there was reduction in the total cell number (40.92% vs. 65.08% in control and, inner cell mass percentage (17.17% vs. 26.15% in control while apoptotic cells numbers were increased (7.17 vs. 1.46 in control (p<0.05. Conclusion: The Kerack addiction during pregnancy retards preimplantation development and induces apoptosis.

  19. Carbon monoxide and the embryo.

    Science.gov (United States)

    Robkin, M A

    1997-04-01

    Mammals are homeotherms and expend considerable energy maintaining their body temperatures. The temperature of a mammalian embryo on the other hand is maintained by the mother and the embryo can devote its metabolic energy to growth and development. The mammalian embryo is acting as a poikilotherm and its energy needs are thus considerably less than if it were a comparably sized homeotherm. The energy requirements of the preimplantation rat embryo are generated by anaerobic metabolism. As it grows, aerobic metabolism develops. In culture, the addition of carbon monoxide to the perfusing gas for early rat embryos has a much smaller effect than decreasing the oxygen concentration. Carbon monoxide appears to be a relatively mild toxicant until the embryo is much larger, is depending much more on transport of oxygen by red blood cells, and the fraction of required metabolic energy produced by anaerobic metabolism has become quite small. The effect from smoking during gestation may be either by the concomitant reduction in food intake or a more direct toxic effect from some components in the smoke. Carbon monoxide does not seem to be the culprit. The possible mitigating effect of a compensatory increase in fetal hematocrit in response to any hypoxia must also be considered. Humans have no yolk sac placenta as rodents do, but if the switch from anaerobic to aerobic metabolism is correlated with the stage of development, then carbon monoxide exposure should not represent any significant risk to the human embryo until later in gestation.

  20. Oviduct: roles in fertilization and early embryo development.

    Science.gov (United States)

    Li, Shuai; Winuthayanon, Wipawee

    2017-01-01

    Animal oviducts and human Fallopian tubes are a part of the female reproductive tract that hosts fertilization and pre-implantation development of the embryo. With an increasing understanding of roles of the oviduct at the cellular and molecular levels, current research signifies the importance of the oviduct on naturally conceived fertilization and pre-implantation embryo development. This review highlights the physiological conditions within the oviduct during fertilization, environmental regulation, oviductal fluid composition and its role in protecting embryos and supplying nutrients. Finally, the review compares different aspects of naturally occurring fertilization and assisted reproductive technology (ART)-achieved fertilization and embryo development, giving insight into potential areas for improvement in this technology. © 2017 Society for Endocrinology.

  1. Seminal Fluid Regulates Accumulation of FOXP3(+) Regulatory T Cells in the Preimplantation Mouse Uterus Through Expanding the FOXP3(+) Cell Pool and CCL19-Mediated Recruitment

    NARCIS (Netherlands)

    Guerin, Leigh R.; Moldenhauer, Lachlan M.; Prins, Jelmer R.; Bromfield, John J.; Hayball, John D.; Robertson, Sarah A.

    Regulatory T (Treg) cells facilitate maternal immune tolerance of the semiallogeneic conceptus in early pregnancy, but the origin and regulation of these cells at embryo implantation is unclear. During the preimplantation period, factors in the seminal fluid delivered at coitus cause expansion of a

  2. Post-hatching development of the porcine and bovine embryo-defining criteria for expected development in vivo and in vitro

    DEFF Research Database (Denmark)

    Vejlsted, Morten; Du, Yutao; Vajta, Gábor

    2006-01-01

    Particular attention has been paid to the pre-hatching period of embryonic development although blastocyst development is a poor indicator of embryo viability. Post-hatching embryonic dev elopment in vitro would allow for establishment of more accurate tools for evaluating developmental potential...... without the need for transfer to recipient animals. Such a system would require (1) definition of milestones of expected post-hatching embryonic development in vivo; and (2) development of adequate culture systems. We propose a stereomicroscopical staging system for post-hatching embryos defining...

  3. The role of preimplantation sonographic exposure in postimplantation development and pregnancy outcome.

    Science.gov (United States)

    Iannaccone, P M; Van Gorder, M; Madsen, E L; Martin, A O; Garton, R

    1991-12-01

    To determine what, if any, effects exposure to ultrasonic beams has on preimplantation embryos we isolated mouse blastocysts and assessed resorption rate, pregnancy rate, decidual swelling volume, and live birth rate after in vitro exposure with subsequent transfer to surrogate mothers. The blastocyst stage embryos were isolated by flushing the uteri of pregnant animals with a phosphate buffered medium. Blastocysts at a discrete stage of development were pooled and transferred to a PB1-methylcellulose medium. This medium is specifically designed to maintain the embryos in a high viscosity solution during the sonographic exposure to prevent microcavitation. After the exposure, the embryos were washed free of methylcellulose and transferred to the uteri of pseudopregnant surrogate mothers. A total of 660 blastocyst stage embryos were distributed among seven treatment groups. After exposure, the embryos were transferred to 54 surrogate mothers. A total of 199 embryos were implanted successfully for postimplantation evaluation. An additional 427 blastocysts were distributed among four treatment groups and transferred to 46 surrogate mothers to assess the effect of sonographic exposure on birth rate. The results indicate possible deleterious effects (decreased implantation rate, increased resorption rate, decreased decidual swelling volume, and increased stillbirth rate) of short ultrasonic exposures (1 min and 5 min) on mouse blastocyst function.

  4. Efeito do ibuprofeno administrado uma hora antes da inovulação de embriões bovinos Effect of ibuprofen administered one hour before the bovine embryo transfer

    Directory of Open Access Journals (Sweden)

    H.J. Narváez

    2010-06-01

    Full Text Available Avaliou-se o efeito do ibuprofeno administrado uma hora antes da inovulação de embriões bovinos, com o objetivo de melhorar a taxa de prenhez. Após a avaliação da resposta ao protocolo de sincronização do estro, 76 fêmeas selecionadas como receptoras de embriões foram distribuídas em três grupos (G experimentais: G1 (n=25 receptoras usadas como controle, G2 (n=30 receptoras que receberam ibuprofeno 5mg/kg, I.M, uma hora antes da inovulação dos embriões, e G3 (n=21 receptoras que receberam uma matriz polimérica de liberação controlada de ibuprofeno administrado por via subcutânea. As taxas de prenhez foram de 16% (4/25, 43,3% (13/30 e 14,2% (3/21, para G1, G2 e G3, respectivamente. Observou-se diferença (PThe effect of the administered ibuprofen was evaluated one hour before the embryo transfer of bovine embryos in order to improve pregnancy rates. After evaluating the response to protocol synchronization of estrus, 76 Females selected as the recipients of embryos were distributed into three experimental groups: G1 (n = 25 surrogate cows used as control, G2 (n = 30 surrogate cows that received 5mg/kg ibuprofen, IM, one hour before the embryo transfer, and G3 (n = 20 surrogate cows that received an array polymeric release of controlled ibuprofen subcutaneously administered. The pregnancy rates were 16% (4/25, 43.3% (13/30, and 14.2% (3/21 for G1, G2, and G3, respectively. There was statistical difference (P<0.024 on pregnancy rate of G2, in comparison with those of G1 and G3. The administration of ibuprofen intramuscularly one hour before the embryo transfer resulted in better pregnancy rate in Nellore surrogate cows.

  5. Human endometrial cell coculture reduces the endocrine disruptor toxicity on mouse embryo development

    Directory of Open Access Journals (Sweden)

    Lee Myeong-Seop

    2012-04-01

    Full Text Available Abstract Backgrounds Previous studies suggested that endocrine disruptors (ED are toxic on preimplantation embryos and inhibit development of embryos in vitro culture. However, information about the toxicity of endocrine disruptors on preimplantation development of embryo in human reproductive environment is lacking. Methods Bisphenol A (BPA and Aroclor 1254 (polychlorinated biphenyls were used as endocrine disruptors in this study. Mouse 2-cell embryos were cultured in medium alone or vehicle or co-cultured with human endometrial epithelial layers in increasing ED concentrations. Results At 72 hours the percentage of normal blastocyst were decreased by ED in a dose-dependent manner while the co-culture system significantly enhanced the rate and reduced the toxicity of endocrine disruptors on the embryonic development in vitro. Conclusions In conclusion, although EDs have the toxic effect on embryo development, the co-culture with human endometrial cell reduced the preimplantation embryo from it thereby making human reproductive environment protective to preimplantation embryo from the toxicity of endocrine disruptors.

  6. Uso da uréia como suplemento protéico na dieta de doadoras e receptoras de embriões bovinos Urea as a protein supplementation in the diet of bovine embryo donors and recipients

    Directory of Open Access Journals (Sweden)

    Amílcar Gasperin Barreto

    2003-02-01

    degenerated embryos (0.5, 1.0 and 1.83, as well as in vitro eclosion rate (81.48, 78.57 and 84.62%, for the groups S, S+U and U, respectively. The 66 recipients were kept on Braquiaria decumbens pasture with 1.25kg of concentrate supplements for the S, S+U and U groups. Frozen embryos were thawed and transferred after 37 days. There was no significant statistical difference in pregnancy rates at 30 days (25, 28 and 28.57%, and 60 days of pregnancy (16.67, 28 and 25%. It may be concluded that urea can replace the soybean meal in concentrated rations for supplementation of bovine embryo donors and recipients since there were no negative effects in embryo quality, eclosion rate and recipient fertility.

  7. Pre-implantation diagnosis of aneuploidy by polar body and blastomere FISH analysis

    Energy Technology Data Exchange (ETDEWEB)

    Munne, S.; Cohen, J.; Grifo, J. [Cornell Univ., New York, NY (United States)] [and others

    1994-09-01

    For preimplantation genetic diagnosis (PGD) of aneuploidy in human in-vitro fertilization (IVF), two blastomeres per embryo should be analyzed to minimize errors caused by FISH and mosaicism. But the biopsy of two cells from an 8-cell embryo can be detrimental. This can be substituted by initial FISH analysis of the first polar body (PB) and subsequent single blastomere analysis. Simultaneous FISH analysis of chromosomes X, Y, 18, 13/21 was used for first polar body aneuploidy analysis. Normal divalents appeared as single-dotted signals corresponding to their two chromatids. We found that pre-division of chromatids increased dramatically with time in culture. All but three pre-division events involved separation of chromatids within the PB or the egg, with a total of two chromatids in each. We concluded that PB aneuploidy analysis is safe when performed within 6 hours after egg retrieval. For our first clinical case we chose a 39 year-old female carrier of an X-linked disease already selected for FISH pre-implantation diagnosis. Eight polar bodies from 12 eggs were analyzed: six showed a normal X181321 complement of divalents; one had an extra chromatid for 13/21 (egg {number_sign}8); and one had a missing chromatid for 13/21 (egg {number_sign}10). After insemination, six fertilized eggs developed into embryos, including egg {number_sign}10 but not egg {number_sign}8. At day 3 of development, a single blastomere per embryo was analyzed by FISH. According to the blastomere analysis, one embryo was haploid, one tetraploid. The two normal female embryos were replaced and pregnancy and CFS results are pending. These results suggest that this technique can be successfully applied for PGD of major aneuploidies in IVF patients over 35. In addition, it indicates that studies on pre-division should be performed on eggs within six hours of retrieval.

  8. A feasible strategy of preimplantation genetic diagnosis for carriers with chromosomal translocation: Using blastocyst biopsy and array comparative genomic hybridization

    OpenAIRE

    Huang, Chu-Chun; Chang, Li-Jung; Tsai, Yi-Yi; Hung, Chia-Cheng; Fang, Mei-Ya; Su, Yi-Ning; Chen, Hsin-Fu; Chen, Shee-Uan

    2013-01-01

    Patients with chromosomal translocation are highly vulnerable to produce unbalanced gametes that result in recurrent miscarriages, affected offspring, or infertility. Preimplantation genetic diagnosis (PGD) with blastomere biopsy and fluorescent in-situ hybridization (FISH) has been used to select normal/balanced embryos for transfer. However, FISH is inherent with some technical difficulties such as cell fixation and signal reading. Here we introduce a strategy of PGD using blastocyst biopsy...

  9. Embryo splitting

    Directory of Open Access Journals (Sweden)

    Karl Illmensee

    2010-04-01

    Full Text Available Mammalian embryo splitting has successfully been established in farm animals. Embryo splitting is safely and efficiently used for assisted reproduction in several livestock species. In the mouse, efficient embryo splitting as well as single blastomere cloning have been developed in this animal system. In nonhuman primates embryo splitting has resulted in several pregnancies. Human embryo splitting has been reported recently. Microsurgical embryo splitting under Institutional Review Board approval has been carried out to determine its efficiency for blastocyst development. Embryo splitting at the 6–8 cell stage provided a much higher developmental efficiency compared to splitting at the 2–5 cell stage. Embryo splitting may be advantageous for providing additional embryos to be cryopreserved and for patients with low response to hormonal stimulation in assisted reproduction programs. Social and ethical issues concerning embryo splitting are included regarding ethics committee guidelines. Prognostic perspectives are presented for human embryo splitting in reproductive medicine.

  10. Influence of Sex on Basal and Dickkopf-1 Regulated Gene Expression in the Bovine Morula.

    Science.gov (United States)

    Denicol, Anna C; Leão, Beatriz C S; Dobbs, Kyle B; Mingoti, Gisele Z; Hansen, Peter J

    2015-01-01

    Sex affects function of the developing mammalian embryo as early as the preimplantation period. There were two goals of the current objective. The first was to determine the degree and nature of differences in gene expression between female and male embryos in the cow at the morula stage of development. The second objective was to determine whether DKK1, a molecule known to alter differentiation of the blastocyst, would affect gene expression differently for female and male morulae. In Experiment 1, female and male embryos were treated with DKK1 at Day 5 after insemination. Morulae were harvested 24 h after treatment, pooled in groups of 20 for microarray analysis and RNA subjected to analysis of gene expression by microarray hybridization. There were 662 differentially expressed genes between females and males and 128 of these genes had a fold change ≥ 1.5 between the two sexes. Of the genes upregulated in females, 49.5% were located in the X chromosome. Functional analysis predicted that cell survival was greater in female embryos. Experiment 2 involved a similar design except that transcripts for 12 genes previously reported to be affected by sex, DKK1 or the interaction were quantified by quantitative polymerase chain reaction. Expression of all genes tested that were affected by sex in experiment 1 was affected in a similar manner in Experiment 2. In contrast, effects of DKK1 on gene expression were largely not repeatable in Experiment 2. The exception was for the Hippo signaling gene AMOT, which was inhibited by DKK1. In Experiment 3, embryos produced by fertilization with unsorted sperm were treated with DKK1 at Day 5 and abundance of transcripts for CDX2, GATA6, and NANOG determined at Days 5, 6 and 7 after insemination. There was no effect of DKK1 on expression of any of the three genes. In conclusion, female and male bovine embryos have a different pattern of gene expression as early as the morula stage, and this is due to a large extent to expression

  11. m-Calpain is required for preimplantation embryonic development in mice

    Directory of Open Access Journals (Sweden)

    Williams Karen

    2006-01-01

    Full Text Available Abstract Background μ-calpain and m-calpain are ubiquitously expressed proteases implicated in cellular migration, cell cycle progression, degenerative processes and cell death. These heterodimeric enzymes are composed of distinct catalytic subunits, encoded by Capn1 (μ-calpain or Capn2 (m-calpain, and a common regulatory subunit encoded by Capn4. Disruption of the mouse Capn4 gene abolished both μ-calpain and m-calpain activity, and resulted in embryonic lethality, thereby suggesting essential roles for one or both of these enzymes during mammalian embryogenesis. Disruption of the Capn1 gene produced viable, fertile mice implying that either m-calpain could compensate for the loss of μ-calpain, or that the loss of m-calpain was responsible for death of Capn4-/- mice. Results To distinguish between the alternatives described above, we deleted an essential coding region in the mouse Capn2 gene in embryonic stems cells and transmitted this mutant allele through the mouse germline. Breeding of heterozygous animals failed to produce homozygous mutant live offspring or implanted embryos. A nested PCR genotyping protocol was established, and homozygous preimplantation mutant embryos were detected at the morula but not at the blastocyts stage. Conclusion We conclude that homozygous disruption of the Capn2 gene results in pre-implantation embryonic lethality between the morula and blastocyst stage. This establishes that μ-calpain and m-calpain have distinct functions, and that m-calpain is vital for development of the preimplantation murine embryo.

  12. Effect of spermatozoa motility hyperactivation factors and gamete coincubation duration on in vitro bovine embryo development using flow cytometrically sorted spermatozoa.

    Science.gov (United States)

    Ferré, Luis B; Bogliotti, Yanina; Chitwood, James L; Fresno, Cristóbal; Ortega, Hugo H; Kjelland, Michael E; Ross, Pablo J

    2016-02-24

    The aim of the present study was to evaluate the effects of sperm motility enhancers and different IVF times on cleavage, polyspermy, blastocyst formation, embryo quality and hatching ability. In Experiment 1, sex-sorted X chromosome-bearing Bos taurus spermatozoa were incubated for 30 min before 18 h fertilisation with hyperactivating factors, namely 10 mM caffeine (CA), 5 mM theophylline (TH), 10 mM caffeine and 5 mM theophylline (CA + TH); and untreated spermatozoa (control). In Experiment 2, matured B. taurus oocytes were fertilised using a short (8 h) or standard (18 h) fertilisation length, comparing two different fertilisation media, namely synthetic oviducal fluid (SOF) fertilisation medium (SOF-FERT) and M199 fertilisation medium (M199-FERT). Cleavage and blastocyst formation rates were significantly higher in the CA + TH group (77% and 27%, respectively) compared with the control group (71% and 21%, respectively). Cleavage rates and blastocyst formation were significantly lower for the shortest fertilisation time (8 h) in M199-FERT medium (42% and 12%, respectively). The SOF-FERT medium with an 8 h fertilisation time resulted in the highest cleavage rates and blastocyst formation (74% and 29%, respectively). The SOF-FERT medium produced the highest embryo quality (50% Grade 1) and hatching rate (66%). Motility enhancers did not affect polyspermy rates, whereas polyspermy was affected when fertilisation length was extended from 8 h (3%) to 18 h (9%) and in M199-FERT (14%) compared with SOF-FERT (6%). We conclude that adding the motility enhancers CA and TH to sex sorted spermatozoa and Tyrode's albumin lactate pyruvate (TALP)-Sperm can improve cleavage and embryo development rates without increasing polyspermy. In addition, shortening the oocyte-sperm coincubation time (8 h) resulted in similar overall embryo performance rates compared with the prolonged (18 h) interval.

  13. [BETWEEN USAGE AND POLEMIC, AN ARGUMENT IN FAVOUR OF CLARIFYING THE TERMINOLOGY FOR PREIMPLANTATION GENETIC DIAGNOSIS].

    Science.gov (United States)

    Côté, Stéphanie; Ravitsky, Vardit; Hamet, Pavel; Bouffard, Chantal

    2015-12-01

    Over 30 years ago, preimplantation genetic diagnosis (PGD) was developed to help couples at risk of transmitting a serious genetic disease to their offspring. Today, the range of medical and non-medical uses of PGD has expanded considerably and some raise much controversy. This is the case, for example, with In-Vitro Fertilization to select embryos as 'saviour siblings' or to screen for susceptibility and predisposition to late onset diseases or conditions of variable penetrance. The situation is even more problematic in the case of sex selection or selection of traits that are culturally valued or discredited (such as deafness, behavioral traits, or height). The debate surrounding PGD has been employing terms to describe these particular uses that have contributed to a focus on the negative effects, thus preventing a distinction between the abuses and the benefits of this reproductive technology. In this context, this paper proposes a terminological clarification that would allow distinguishing medical and non-medical use and, therefore, the issues relevant to each. A more accurate and less generic nomenclature could prevent a conflation of different levels of ethical, clinical and social issues under the single term 'PGD'. For the vast majority of medical uses, we propose to keep: 'preimplantation genetic diagnosis (PGD)', which emphasizes that it is a genetic diagnosis. For non-medical uses, we suggest: 'preimplantation genetic trait selection (PGTS)'.

  14. Influence of radiation (Co{sub 60}) in pre-implant rabbit embryos: effect on mitotic index and embryonic pole malformations; Influencia da radiacao ionizante (bomba de cobalto) em embrioes de coelha na fase de pre-implantacao: influencia no indice mitotico e malformacoes no polo embrionario

    Energy Technology Data Exchange (ETDEWEB)

    Approbato, M.S.; Moura, K.K.V.O.; Florencio, R.S.; Cunha Junior, C.; Garcia, R.; Faria, R.S.; Benedetti, L.N.; Goulart, F.B. [Goias Univ., Goiania, GO (Brazil). Faculdade de Medicina. Dept. de Ginecologia e Obstetricia

    1995-03-01

    We studied the effect of ionizing irradiation on 12 New Zealand rabbits (65 embryos), at three different times: at match time (zero hour), two days after and four days after, with two different irradiation doses: five c Gy and ten c Gy. Six rabbits (36 blastocysts) were used as controls. the matching instant was the zero hour. Exactly six days after ({+-} 60 minutes) the embryos of each rabbit was picked up by flushing the uterus with culture media. the embryos were fixed in methanol for 48 hours, and colored with acid Mayer hematoxylin. The following embryo parameters were studied: mitotic index; embryonic pole malformations. There were no gross abnormalities of embryo pole. The mitotic index were altered both by the time and doses. (author). 12 refs., 3 figs.

  15. Preimplantation Genetic Diagnosis (PGD) for Monogenic Disorders: the Value of Concurrent Aneuploidy Screening.

    Science.gov (United States)

    Goldman, Kara N; Nazem, Taraneh; Berkeley, Alan; Palter, Steven; Grifo, Jamie A

    2016-12-01

    Pre-implantation genetic diagnosis (PGD) has changed the landscape of clinical genetics by helping families reduce the transmission of monogenic disorders. However, given the high prevalence of embryonic aneuploidy, particularly in patients of advanced reproductive age, unaffected embryos remain at high risk of implantation failure or pregnancy loss due to aneuploidy. 24-chromosome aneuploidy screening has become widely utilized in routine in vitro fertilization (IVF) to pre-select embryos with greater pregnancy potential, but concurrent 24-chromosome aneuploidy screening has not become standard practice in embryos biopsied for PGD. We performed a retrospective cohort study of patients who underwent PGD with or without 24-chromosome aneuploidy screening to explore the value of concurrent screening. Among the PGD + aneuploidy-screened group (n = 355 blastocysts), only 25.6 % of embryos were both Single Gene Disorder (SGD)-negative (or carriers) and euploid; thus the majority of embryos were ineligible for transfer due to the high prevalence of aneuploidy. Despite a young mean age (32.4 ± 5.9y), 49.9 % of Blastocysts were aneuploid. The majority of patients (53.2 %) had ≥1 blastocyst that was Single Gene Disorder (SGD)-unaffected but aneuploid; without screening, these unaffected but aneuploid embryos would likely have been transferred resulting in implantation failure, pregnancy loss, or a pregnancy affected by chromosomal aneuploidy. Despite the transfer of nearly half the number of embryos in the aneuploidy-screened group (1.1 ± 0.3 vs. 1.9 ± 0.6, p aneuploidy screening when performed concurrently with PGD provides valuable information for embryo selection, and notably improves single embryo transfer rates.

  16. Supravital fluorometric apoptosis detection in a single mouse embryo using lab-on-a-chip.

    Science.gov (United States)

    Walczak, Rafał; Śniadek, Patrycja; Dziuban, Jan A; Kluger, Joanna; Soyta, Anna Chełmońska

    2011-10-07

    Detection of apoptosis is one of the main criteria of preimplantation embryo growth potential assessment. Recent developments in lab-on-a-chip techniques has led to apoptosis detection and monitoring on a single cell or embryo level. However, single embryo apoptosis detection without a change in embryo developmental competence and post-examination "recovery" still remains a challenge. In this paper we present a lab-on-a-chip, co-working with miniaturized optical instrumentation, which allows supravital examination of single embryos for the presence of apoptotic blastomers with full after lab-on-a-chip study "recovery" and maintenance of their further developmental capacity.

  17. Efeito de diferentes meios de cultivo no desenvolvimento e proporção do sexo de embriões bovinos produzidos in vitro Effect of different culture media on development and sex ratio of bovine embryos fertilized in vitro

    Directory of Open Access Journals (Sweden)

    S.G.T. Gilardi

    2004-10-01

    Full Text Available Avaliou-se o efeito da suplementação de meios de cultivo sobre o desenvolvimento e proporção do sexo de embriões bovinos fertilizados in vitro. Complexos cumulus-oócitos obtidos de ovários de matadouro foram maturados e fertilizados in vitro. Os zigotos (n= 484 foram distribuídos aleatoriamente em meio CR2aa, contendo soro fetal bovino (SFB (T1, albumina sérica bovina (BSA (T2 ou BSA mais insulina:transferrina:selênio e vitaminas (BSA+ (T3, no cultivo embrionário in vitro, a uma atmosfera de 5% CO2 a 38,8ºC em ar. A taxa de clivagem foi observada 72-76 horas pós-fertilização (PF e a taxa de blastocistos com sete e oito dias PF. Os blastocistos (n= 63 foram sexados pela técnica de reação em cadeia de polimerase. A taxa de clivagem em T2 foi maior (P0,05 entre T2 e T3, porém menor (P0,05 entre os tratamentos. O T1 influenciou o desenvolvimento de blastocistos, mas não teve efeito sobre a proporção do sexo.The effect of culture media on the development and on the sex ratio of bovine embryos fertilized in vitro was studied. Cumulus oocyte-complexes from slaughterhouse ovaries were matured and fertilized in vitro. Zygotes (n= 484 were randomly allotted to different culture media and cultured with their cumulus cells in CR2aa medium and an atmosphere of 5% CO2 in air at 38.8ºC. The fetal calf serum (FCS, bovine seric albumin (BSA or BSA plus insulin:transferrin:selenium and vitamins (BSA+ supplementation effect on embryo culture was evaluated. Cleavage rate was assessed at 72-76h post-fertilization (PF and blastocyst rate on days 7 and 8 PF. The blastocysts (n= 63 were also sexed using polymerase chain reaction. Cleavage rate for BSA medium supplemented was higher (P0.05, but lower (P<0.01 than FCS. Culture medium FCS supplemented affected blastocyst development but not the sex ratio.

  18. Cryotops versus open-pulled straws (OPS) as carriers for the cryopreservation of bovine oocytes: effects on spindle and chromosome configuration and embryo development.

    Science.gov (United States)

    Morató, Roser; Izquierdo, Dolors; Paramio, Maria Teresa; Mogas, Teresa

    2008-10-01

    Two experiments were designed to assess the effectiveness of cryopreserving bovine MII oocytes using cryotops as the carrier system for vitrification. In the first experiment, we examined the developmental competence of oocytes after: (i) vitrification in open-pulled straws (OPS method); or (ii) vitrification in plastic handle (Cryotop method). In the second experiment, warmed oocytes that had been vitrified in OPS or cryotops were fixed to analyze spindle and chromosome configuration. In all experiments both cow and calf oocytes were used. Significantly different fertilization rates were observed between the vitrification groups: 31.5% and 20.2% for the cow and calf oocytes vitrified in OPS, respectively, versus 46.1% and 46.4% for the oocytes vitrified using cryotops. After in vitro fertilization, 3.8% of the calf oocytes and 5.3% of the cow oocytes developed to the blastocyst stage. All blastocysts from vitrified oocytes resulted from the Cryotop method. A significantly lower percentage of the OPS-vitrified calf oocytes showed a normal spindle configuration (37.8%) compared to control fresh oocytes (69.9%), while normal spindle and chromosome configurations were observed in a significantly higher proportion of the cryotop-vitrified calf oocytes (60.2%). For the cow oocytes, 60.6% in the OPS group and 60.3% in the Cryotop group exhibited a normal morphology after warming. These findings suggest the cryotop system is a more efficient carrier for vitrification than OPS for the cryopreservation of bovine oocytes.

  19. Karyomapping allows preimplantation genetic diagnosis of a de-novo deletion undetectable using conventional PGD technology.

    Science.gov (United States)

    Giménez, Carles; Sarasa, Jonás; Arjona, César; Vilamajó, Ester; Martínez-Pasarell, Olga; Wheeler, Kenny; Valls, Gemma; Garcia-Guixé, Elena; Wells, Dagan

    2015-12-01

    Preimplantation genetic diagnosis (PGD) was carried out for a couple carrying a de-novo deletion in the TSC2 gene, responsible for tuberous sclerosis. Karyomapping, a method employing genome-wide analysis of single nucleotide polymorphisms (SNP), was used as PGD protocol. Analysis of DNA from the affected parent using karyomapping confirmed the region covered by the deletion and revealed more than 30 SNP located within the affected region. These SNP were subsequently used for embryo diagnosis (deletion revealed by hemizygosity and/or reduced probe intensity). Seven blastocyst embryos underwent trophectoderm biopsy followed by vitrification. Biopsied cells were subjected to comprehensive aneuploidy screening using microarray comparative genomic hybridization (aCGH), with karyomapping for the detection of embryos carrying the mutant TSC2 gene carried out in tandem. Two embryo transfers were performed, the second of which resulted in the birth of a child. This study highlights that karyomapping may be applicable to a subset of de-novo mutations undetectable using standard PGD strategies. Additionally, karyomapping results were in complete concordance with aCGH, both methods revealing the same aneuploidies in the embryos tested. It was concluded that karyomapping may represent a valuable advance in cases of PGD for monogenic diseases. Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  20. Parenting in the Age of Preimplantation Gene Editing.

    Science.gov (United States)

    Klipstein, Sigal

    2017-12-01

    Medical science at its core aims to preserve health and eliminate disease, but a common theme in scientific discovery is the application of findings in ways that were not the primary intent. The development of diagnostic modalities to predict the health of resulting children has been a fundamental aim underpinning research into prenatal and preimplantation diagnostic modalities; however, the knowledge gained has in some cases been utilized for nonmedical purposes. As an example, amniocentesis developed to determine whether the pregnancy is chromosomally normal also provides information about the sex of the fetus, which normally does not affect health. The emerging gene-editing technologies that could be used to repair mutated disease-causing genes in an embryo will presumably also be able to be used to alter traits unrelated to disease. And yet, I will argue, the desire to preserve the mystery of reproduction remains a central value in humans' quest to reproduce. This yearning to maintain the mysteries will likely temper the development of strategies to alter our genome and affect the genetic identities of our offspring. In my experience as an obstetrician and reproductive endocrinology and infertility subspecialist, people want to have, not the best possible baby, but rather their own baby. © 2017 The Hastings Center.

  1. Efeito do transporte no desenvolvimento de embriões bovinos cultivados in vitro a fresco ou reaquecidos após vitrificação Effect of transportation on development of fresh or vitrified-warmed bovine embryos

    Directory of Open Access Journals (Sweden)

    Alessandra de Almeida Ramos

    2006-12-01

    bovine embryos, fresh or warmed, after submitted to different periods of transportation (6h-12h. Oocytes obtained from ovaries collected from slaughterhouse were matured, fertilized and cultured in vitro. After seven days, grades I and II blastocysts (according to IETS manual were selected and vitrified after exposition to PBS solution with 5% fetal calf serum (HM, added with 10% ethylene glycol (EG and 10% of dymetil sulfoxide (DMSO, for one minute, followed by HM solution with 20% EG and 20% DMSO, for 20 seconds. Embryos were loaded into open pulled straws (OPS and plunged into liquid nitrogen. Warming was performed at 39ºC by embryo exposure to decreasing concentration of sucrose (0.25 and 0.15M, for five minutes in each step. The warmed embryos were distributed in three groups: V0: in vitro cultured after warmed; V6: embryos loaded into straws and kept for 6 hours at 35ºC, before in vitro culture; and V12: embryos loaded into straws and kept for 12 hours at 35ºC, before in vitro culture. Each group was evaluated by control groups of fresh embryos (C0, C6 and C12, respectively. The embryos were co-cultured with cumulus cells in TCM-199 micro droplets added with SFB. Re-expansion and hatching rates after 48 hours in culture were evaluated and results were compared by the Chi-square test. Re-expanded rates among groups V0, V6 and V12 as well as hatching rates among vitrified groups and among control groups did not differ. However, hatching rates were different between vitrified groups and their respective controls. The satisfactory rates of hatching suggest that it is possible to transport warmed and fresh in vitro produced embryos for periods up to 12 hours.

  2. Use of preimplantation genetic diagnosis and preimplantation genetic screening in the United States: a Society for Assisted Reproductive Technology Writing Group paper.

    Science.gov (United States)

    Ginsburg, Elizabeth S; Baker, Valerie L; Racowsky, Catherine; Wantman, Ethan; Goldfarb, James; Stern, Judy E

    2011-10-01

    To comprehensively report Society for Assisted Reproductive Technology (SART) member program usage of preimplantation genetic testing (PGT), preimplantation genetic diagnosis (PGD) for diagnosis of specific conditions, and preimplantation genetic screening for aneuploidy (PGS). Retrospective study. United States SART cohort data. Women undergoing a PGT cycle in which at least one embryo underwent biopsy. PGT. PGT use, indications, and delivery rates. Of 190,260 fresh, nondonor assisted reproductive technology (ART) cycles reported to SART CORS in 2007-2008, 8,337 included PGT. Of 6,971 cycles with a defined indication, 1,382 cycles were for genetic diagnosis, 3,645 for aneuploidy screening (PGS), 527 for translocation, and 1,417 for elective sex election. Although the total number of fresh, autologous cycles increased by 3.6% from 2007 to 2008, the percentage of cycles with PGT decreased by 5.8% (4,293 in 2007 and 4,044 in 2008). As a percentage of fresh, nondonor ART cycles, use dropped from 4.6% (4,293/93,433) in 2007 to 4.2% (4,044/96,827) in 2008. The primary indication for PGT was PGS: cycles performed for this indication decreased (-8.0%). PGD use for single-gene defects (+3.2%), elective sex selection (+5.3%), and translocation analysis (+0.5%) increased. PGT usage varied significantly by geographical region. PGT usage in the United States decreased between 2007 and 2008 owing to a decrease in PGS. Use of elective sex selection increased. High transfer cancellation rates correlated with reduced live-birth rates for some PGT indications. Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  3. Falha na sexagem por inibição do desenvolvimento de embri��es bovinos produzidos in vitro com anticorpos anti H-Y Failure of sexing by developmental arrest of bovine embryos in vitro produced with H-Y antisera

    Directory of Open Access Journals (Sweden)

    M.V. Resende

    2008-06-01

    Full Text Available Embriões bovinos produzidos in vitro, em estádio de mórula, foram cultivados em meio contendo anticorpos anti H-Y de alto título proveniente de ratos por 24h e, após este tempo, classificados em dois grupos: 1 embriões inibidos em estádio de mórula (classificados como machos e 2 embriões que se desenvolveram e formaram a blastocele (classificados como fêmeas. O sexo de 311 embriões, distribuídos em três grupos de concentração dos anticorpos, 3%, 5% ou 7%, foi identificado pela reação em cadeia da polimerase. Não houve desvio da proporção entre machos e fêmeas (P>0,05 nos grupos em que se utilizaram os anticorpos anti H-Y, quando comparadas ao grupo-controle, sem adição de anticorpos anti H-Y. Diferentemente dos resultados obtidos utilizando-se embriões bovinos produzidos in vivo, a sexagem com anticorpos anti H-Y de alto título em embriões produzidos in vitro não propiciou sucesso.In vitro produced bovine embryos at morula stage were cultured in medium containing high titer of rat H-Y antisera for 24h. The embryos were classified in two groups: 1 embryos arrested at morula stage (classified as males; and 2 embryos that developed and formed a blastocoele (classified as female. The sex of 311 embryos, divided in three groups of concentration of H-Y antisera, 3%, 5% or 7%, was identified by polimerase chain reaction. The results showed no difference (P>0.05 on sexual deviation in groups in which the H-Y antisera was added, in relation to control group, in which no H-Y antisera was added. In contrast with results obtained with in vivo produced bovine embryos, the sexing of in vitro produced bovine embryos with high H-Y antisera titer did not succed.

  4. Potential of human twin embryos generated by embryo splitting in assisted reproduction and research.

    Science.gov (United States)

    Noli, Laila; Ogilvie, Caroline; Khalaf, Yacoub; Ilic, Dusko

    2017-03-01

    Embryo splitting or twinning has been widely used in veterinary medicine over 20 years to generate monozygotic twins with desirable genetic characteristics. The first human embryo splitting, reported in 1993, triggered fierce ethical debate on human embryo cloning. Since Dolly the sheep was born in 1997, the international community has acknowledged the complexity of the moral arguments related to this research and has expressed concerns about the potential for reproductive cloning in humans. A number of countries have formulated bans either through laws, decrees or official statements. However, in general, these laws specifically define cloning as an embryo that is generated via nuclear transfer (NT) and do not mention embryo splitting. Only the UK includes under cloning both embryo splitting and NT in the same legislation. On the contrary, the Ethics Committee of the American Society for Reproductive Medicine does not have a major ethical objection to transferring two or more artificially created embryos with the same genome with the aim of producing a single pregnancy, stating that 'since embryo splitting has the potential to improve the efficacy of IVF treatments for infertility, research to investigate the technique is ethically acceptable'. Embryo splitting has been introduced successfully to the veterinary medicine several decades ago and today is a part of standard practice. We present here an overview of embryo splitting experiments in humans and non-human primates and discuss the potential of this technology in assisted reproduction and research. A comprehensive literature search was carried out using PUBMED and Google Scholar databases to identify studies on embryo splitting in humans and non-human primates. 'Embryo splitting' and 'embryo twinning' were used as the keywords, alone or in combination with other search phrases relevant to the topics of biology of preimplantation embryos. A very limited number of studies have been conducted in humans and non

  5. The oviduct: from sperm selection to the epigenetic landscape of the embryo.

    Science.gov (United States)

    Pérez-Cerezales, Serafín; Ramos-Ibeas, Priscila; Acuña, Omar Salvador; Avilés, Manuel; Coy, Pilar; Rizos, Dimitrios; Gutiérrez-Adán, Alfonso

    2017-12-08

    The mammalian oviduct is the place where life begins as it is the site of fertilization and preimplantation embryo development. Recent research has highlighted the important role played by the oviduct both in sperm selection for natural fertilization and in the genetic and epigenetic reprogramming of pre-implantation embryo development. This review examines oviduct fluid composition with a special emphasis on exosomes and the role played by the oviduct in sperm selection, early embryo development, and in reshaping the epigenetic landscape of the embryo. In addition, the implications of data obtained for improving assisted reproductive technologies are discussed. © The Author(s) 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  6. PreImplantation Factor (PIF*) Regulates Stress-Induced Adrenal Steroidogenesis and Anti-Inflammatory Cytokines: Potential Application for Bioartificial Adrenal Transplant.

    Science.gov (United States)

    Balyura, Mariya; Gelfgat, Evgeny; Ullmann, Enrico; Ludwig, Barbara; Barnea, Eytan R; Bornstein, Stefan R

    2017-10-24

    The main treatment algorithm for adrenal insufficiency is hormonal replacement, however, inadequate hormone substitution often leads to severe side effects. Adrenal cell transplantation could be a more effective alternative but would require life-long immune suppressive therapy. PreImplantation Factor (PIF) is an endogenous peptide secreted by viable human embryos that leads to maternal tolerance without immunosuppression. PIF could be effective for xenogeneic cell transplantation such as of bovine adrenocortical cells (BAC), which are used for bioartificial adrenal gland development that may more effectively restore complex adrenal functions. We report here that PIF exerts a dual regulatory effect on BAC by targeting mostly hyper-activated cells to specifically reduce adrenocorticotropic hormone (ACTH)-stimulated cortisol secretion. Reverse transcription real time PCR analysis revealed that PIF modulates the expression of two genes in the cortisol synthesis pathway, Steroidogenic Factor 1 (SF1), an activator of steroidogenesis, and the downstream steroidogenic enzyme Cytochrome P450 17A1 (CYP17A1). PIF increased basal expression of SF1 and CYP17A1 regardless of the activation level of the adrenocortical cells. In contrast, following ACTH stimulation, PIF reduced SF1 expression and induced expression of the immune suppressing anti-inflammatory cytokine IL10 only in the hyper-activated cells, suggesting both a protective and immune tolerant function. In conclusion, PIF regulates stress-induced adrenal steroidogenesis and immune tolerance in BAC, supporting a potential clinical application to reduce rejection by the host's immune response following xenotransplantation. © Georg Thieme Verlag KG Stuttgart · New York.

  7. Human embryos from induced pluripotent stem cell-derived gametes: ethical and quality considerations.

    Science.gov (United States)

    Ilic, Dusko; Ogilvie, Caroline; Noli, Laila; Kolundzic, Nikola; Khalaf, Yacoub

    2017-09-01

    Protocols for successful differentiation of male and female gametes from induced pluripotent stem cells have been published. Although culture of precursor cells in a natural microenvironment remains necessary to achieve terminal differentiation, the creation of human preimplantation embryos from induced pluripotent stem cell-derived gametes is technically feasible. Such embryos could provide a solution to the scarcity of human cleavage-stage embryos donated for research. Here, we discuss current technology, major research-related ethical concerns and propose the norms that would assure the quality and reliability of such embryos.

  8. Healthy Baby Born to a Robertsonian Translocation Carrier following Next-Generation Sequencing-Based Preimplantation Genetic Diagnosis: A Case Report

    OpenAIRE

    Lukaszuk, Krzysztof; Pukszta, Sebastian; Ochman, Karolina; Cybulska, Celina; Liss, Joanna; Pastuszek, Ewa; Zabielska, Judyta; Woclawek-Potocka, Izabela

    2015-01-01

    Preimplantation genetic diagnosis (PGD) is well established method for treatment of genetic problems associated with infertility. Moreover, PGD with next-generation sequencing (NGS) provide new possibilities for diagnosis and new parameters for evaluation in, for example, aneuploidy screening. The aim of the study was to report the successful pregnancy outcome following PGD with NGS as the method for 24 chromosome aneuploidy screening in the case of Robertsonian translocation. Day 3 embryos s...

  9. Study on preimplantation genetic diagnosis and follow-up for Duchenne muscular dystrophy

    Directory of Open Access Journals (Sweden)

    Juan YANG

    2015-07-01

    Full Text Available Objective  To carry out preimplantation genetic diagnosis (PGD for Duchenne muscular dystrophy (DMD carrier, so as to prevent the birth of affected infants with DMD.  Methods  One DMD gene carrier with a deletion of exon 10-30 received fertilization with intracytoplasmic sperm injection (ICSI. DMD gene and haplotype were tested after amplification of genome DNA in multiple displacement amplification (MDA, then healthy embryos were transferred to uterus according to the genetic results. Genetic testing was made in second trimester and after delivery, and also periodic follow-up was made for over 3 years.  Results  The second cycle of PGD was successful, and a total of 14 single blastomeres obtained from 7 embryos were used for genetic analysis. The success rate of MDA was 13/14, and the allele dropout rate was 18.75% (18/96. Three unaffected embryos were transferred, resulting in twin pregnancy. One healthy boy and one healthy girl were born in cesarean section at the pregnant week of 35. Genetic results on DNA from both amniotic fluid at 16 weeks of gestation and peripheral blood after birth were normal. During the 3-year follow-up, both 2 infants were normal in growth and development, motor function and dynamic monitor of serum creatine kinase (CK.  Conclusions  Preimplantation genetic diagnosis can help DMD gene carrier give birth to healthy infants, and these infants have normal development. DOI: 10.3969/j.issn.1672-6731.2015.06.008

  10. Embryo culture media and IVF/ICSI success rates: a systematic review

    NARCIS (Netherlands)

    Mantikou, E.; Youssef, M. A. F. M.; van Wely, M.; van der Veen, F.; Al-Inany, H. G.; Repping, S.; Mastenbroek, S.

    2013-01-01

    BACKGROUND The media that are used to culture human preimplantation embryos are considered to be an important factor for the success rates of IVF/ICSI. Here, we present a systematic review of randomized controlled trials (RCTs) on the effect of culture media on IVF/ICSI success rates. METHODS RCTs

  11. ADAM10 Is Involved in Cell Junction Assembly in Early Porcine Embryo Development.

    Directory of Open Access Journals (Sweden)

    Jeongwoo Kwon

    Full Text Available ADAM10 (A Disintegrin and Metalloprotease domain-containing protein 10 is a cell surface protein with a unique structure possessing both potential adhesion and protease domains. However, the role of ADAM10 in preimplantation stage embryos is not clear. In this study, we examined the expression patterns and functional roles of ADAM10 in porcine parthenotes during preimplantation development. The transcription level of ADAM10 dramatically increased from the morula stage onward. Immunostaining revealed that ADAM10 was present in both the nucleus and cytoplasm in early cleavage stage embryos, and localized to the apical region of the outer cells in morula and blastocyst embryos. Knockdown (KD of ADAM10 using double strand RNA did not alter preimplantation embryo development until morula stage, but resulted in significantly reduced development to blastocyst stage. Moreover, the KD blastocyst showed a decrease in gene expression of adherens and tight junction (AJ/TJ, and an increase in trophectoderm TJ permeability by disrupting TJ assembly. Treatment with an ADAM10 specific chemical inhibitor, GI254023X, at the morula stage also inhibited blastocyst development and led to disruption of TJ assembly. An in situ proximity ligation assay demonstrated direct interaction of ADAM10 with coxsackie virus and adenovirus receptor (CXADR, supporting the involvement of ADAM10 in TJ assembly. In conclusion, our findings strongly suggest that ADADM10 is important for blastocyst formation rather than compaction, particularly for TJ assembly and stabilization in preimplantation porcine parthenogenetic development.

  12. In Vitro Elongation of Porcine Embryos Using Alginate Hydrogels as a Three-Dimensional Extracellular Matrix

    Science.gov (United States)

    In the pig, the pre-implantation period of pregnancy is highly influential on sow productivity and therefore the profitability of swine production. Between Day 11 and 12 of gestation, the embryo undergoes a significant morphological change, during which it transforms from an ovoid structure of about...

  13. Laser fusion of mouse embryonic cells and intra-embryonic fusion of blastomeres without affecting the embryo integrity.

    Directory of Open Access Journals (Sweden)

    Alexander Krivokharchenko

    Full Text Available Manipulation with early mammalian embryos is the one of the most important approach to study preimplantation development. Artificial cell fusion is a research tool for various biotechnological experiments. However, the existing methods have various disadvantages, first of them impossibility to fuse selected cells within multicellular structures like mammalian preimplantation embryos. In our experiments we have successfully used high repetition rate picosecond near infrared laser beam for fusion of pairs of oocytes and oocytes with blastomeres. Fused cells looked morphologically normal and keep their ability for further divisions in vitro. We also fused two or three blastomeres inside four-cell mouse embryos. The presence of one, two or three nuclei in different blastomeres of the same early preimplantation mouse embryo was confirmed under UV-light after staining of DNA with the vital dye Hoechst-33342. The most of established embryos demonstrated high viability and developed in vitro to the blastocyst stage. We demonstrated for the first time the use of laser beam for the fusion of various embryonic cells of different size and of two or three blastomeres inside of four-cell mouse embryos without affecting the embryo's integrity and viability. These embryos with blastomeres of various ploidy maybe unique model for numerous purposes. Thus, we propose laser optical manipulation as a new tool for investigation of fundamental mechanisms of mammalian development.

  14. Embryo development and embryo transfer in the European mink (Mustela lutreola), an endangered mustelid species.

    Science.gov (United States)

    Amstislavsky, S; Kizilova, E; Ternovskaya, Y; Zudova, G; Lindeberg, H; Aalto, J; Valtonen, M

    2006-01-01

    The European mink is an endangered Mustelidae species and thus requires effective conservation measures, although little is known about reproduction in this species. In particular, preimplantation development has not been studied and, therefore, embryonic development and the growth of embryos was documented in the present study for European mink using light and fluorescent microscopy. Embryos develop in the oviducts and then migrate into the uterus on Day 6 post coitum (p.c.) at the morula stage. Embryos expanded as blastocysts from Day 7 until implantation on Day 12 p.c. Based on these findings, the use of embryo transfer for a conservation programme for the European mink was evaluated. Embryos were flushed from European mink resource females and transferred into the uterine horns of recipient hybrid females (honoriks and nohoriks). These hybrids were obtained by mating European polecat males with European mink females and vice versa. A total of 40 embryos was transferred and 20 live kits were born. The rates of pre- and postnatal survival were 50% and 70%, respectively. Both male and female offspring were lighter at birth in the embryo transfer group compared with naturally born controls, but there was no difference at 3 months of age.

  15. Is the hypothesis of preimplantation genetic screening (PGS) still supportable? A review.

    Science.gov (United States)

    Gleicher, Norbert; Orvieto, Raoul

    2017-03-27

    The hypothesis of preimplantation genetic diagnosis (PGS) was first proposed 20 years ago, suggesting that elimination of aneuploid embryos prior to transfer will improve implantation rates of remaining embryos during in vitro fertilization (IVF), increase pregnancy and live birth rates and reduce miscarriages. The aforementioned improved outcome was based on 5 essential assumptions: (i) Most IVF cycles fail because of aneuploid embryos. (ii) Their elimination prior to embryo transfer will improve IVF outcomes. (iii) A single trophectoderm biopsy (TEB) at blastocyst stage is representative of the whole TE. (iv) TE ploidy reliably represents the inner cell mass (ICM). (v) Ploidy does not change (i.e., self-correct) downstream from blastocyst stage. We aim to offer a review of the aforementioned assumptions and challenge the general hypothesis of PGS. We reviewed 455 publications, which as of January 20, 2017 were listed in PubMed under the search phrase . The literature review was performed by both authors who agreed on the final 55 references. Various reports over the last 18 months have raised significant questions not only about the basic clinical utility of PGS but the biological underpinnings of the hypothesis, the technical ability of a single trophectoderm (TE) biopsy to accurately assess an embryo's ploidy, and suggested that PGS actually negatively affects IVF outcomes while not affecting miscarriage rates. Moreover, due to high rates of false positive diagnoses as a consequence of high mosaicism rates in TE, PGS leads to the discarding of large numbers of normal embryos with potential for normal euploid pregnancies if transferred rather than disposed of. We found all 5 basic assumptions underlying the hypothesis of PGS to be unsupported: (i) The association of embryo aneuploidy with IVF failure has to be reevaluated in view how much more common TE mosaicism is than has until recently been appreciated. (ii) Reliable elimination of presumed aneuploid

  16. Igf1r signaling is indispensable for preimplantation development and is activated via a novel function of E-cadherin.

    Directory of Open Access Journals (Sweden)

    Ivan Bedzhov

    Full Text Available Insulin-like growth factor I receptor (Igf1r signaling controls proliferation, differentiation, growth, and cell survival in many tissues; and its deregulated activity is involved in tumorigenesis. Although important during fetal growth and postnatal life, a function for the Igf pathway during preimplantation development has not been described. We show that abrogating Igf1r signaling with specific inhibitors blocks trophectoderm formation and compromises embryo survival during murine blastocyst formation. In normal embryos total Igf1r is present throughout the membrane, whereas the activated form is found exclusively at cell contact sites, colocalizing with E-cadherin. Using genetic domain switching, we show a requirement for E-cadherin to maintain proper activation of Igf1r. Embryos expressing exclusively a cadherin chimera with N-cadherin extracellular and E-cadherin intracellular domains (NcEc fail to form a trophectoderm and cells die by apoptosis. In contrast, homozygous mutant embryos expressing a reverse-structured chimera (EcNc show trophectoderm survival and blastocoel cavitation, indicating a crucial and non-substitutable role of the E-cadherin ectodomain for these processes. Strikingly, blastocyst formation can be rescued in homozygous NcEc embryos by restoring Igf1r signaling, which enhances cell survival. Hence, perturbation of E-cadherin extracellular integrity, independent of its cell-adhesion function, blocked Igf1r signaling and induced cell death in the trophectoderm. Our results reveal an important and yet undiscovered function of Igf1r during preimplantation development mediated by a unique physical interaction between Igf1r and E-cadherin indispensable for proper receptor activation and anti-apoptotic signaling. We provide novel insights into how ligand-dependent Igf1r activity is additionally gated to sense developmental potential in utero and into a bifunctional role of adhesion molecules in contact formation and signaling.

  17. Increasing live birth rate by preimplantation genetic screening of pooled polar bodies using array comparative genomic hybridization.

    Directory of Open Access Journals (Sweden)

    Michael Feichtinger

    Full Text Available Meiotic errors during oocyte maturation are considered the major contributors to embryonic aneuploidy and failures in human IVF treatment. Various technologies have been developed to screen polar bodies, blastomeres and trophectoderm cells for chromosomal aberrations. Array-CGH analysis using bacterial artificial chromosome (BAC arrays is widely applied for preimplantation genetic diagnosis (PGD using single cells. Recently, an increase in the pregnancy rate has been demonstrated using array-CGH to evaluate trophectoderm cells. However, in some countries, the analysis of embryonic cells is restricted by law. Therefore, we used BAC array-CGH to assess the impact of polar body analysis on the live birth rate. A disadvantage of polar body aneuploidy screening is the necessity of the analysis of both the first and second polar bodies, resulting in increases in costs for the patient and complex data interpretation. Aneuploidy screening results may sometimes be ambiguous if the first and second polar bodies show reciprocal chromosomal aberrations. To overcome this disadvantage, we tested a strategy involving the pooling of DNA from both polar bodies before DNA amplification. We retrospectively studied 351 patients, of whom 111 underwent polar body array-CGH before embryo transfer. In the group receiving pooled polar body array-CGH (aCGH analysis, 110 embryos were transferred, and 29 babies were born, corresponding to live birth rates of 26.4% per embryo and 35.7% per patient. In contrast, in the control group, the IVF treatment was performed without preimplantation genetic screening (PGS. For this group, 403 embryos were transferred, and 60 babies were born, resulting in live birth rates of 14.9% per embryo and 22.7% per patient. In conclusion, our data show that in the aCGH group, the use of aneuploidy screening resulted in a significantly higher live birth rate compared with the control group, supporting the benefit of PGS for IVF couples in

  18. Antigen presenting cells costimulatory signaling during pre-implantation pregnancy 

    Directory of Open Access Journals (Sweden)

    Anna Sławek

    2012-09-01

    Full Text Available  Success of pregnancy depends on many factors. Three phenomena inducing immune tolerance against semi-allogeneic conceptus may play a crucial role in the pre-implantation period of pregnancy: influence of sex hormones in sex cycle, presence of oocyte or embryo and the presence of semen in the female reproductive tract. On the other hand dendritic cells are the most effective antigen-presenting cells in regulation of immune phenomena and also are considered as potent participants in inducing immune tolerance in the pregnancy. They communicate with T cells in cell contact-dependent manner or via cytokines. During cell-cell contacts, costimulatory molecules play a key role and their expression is often dependent on cytokines milieu. Both costimulatory molecules and cytokines influence generation of T regulatory cells. Interactions of these molecules are closely related. In this paper we would like to pay attention to the importance of antigen presenting cells costimulatory potency in immune regulation during a pre-implantation period of pregnancy.

  19. Lower implantation rates in high responders: evidence for an altered endocrine milieu during the preimplantation period.

    Science.gov (United States)

    Pellicer, A; Valbuena, D; Cano, F; Remohí, J; Simón, C

    1996-06-01

    To determine serum E2 and P levels around the time of implantation in normal and high IVF responders. In Vitro Fertilization program at the Instituto Valenciano de Infertilidad. Twenty-nine women undergoing IVF, who accepted to be studied daily, were classified according to the number of oocytes retrieved in normal (n = 16) and high responders (n = 13). Prospective study in which blood was drawn daily from the day of hCG administration (day 0) up to 7 days later (day 6). In vitro fertilization parameters (number of ampules, FSH-hMG, number of oocytes, fertilization rates, number of transferred embryos, implantation rates, and pregnancy rates); serum E2 and P levels during the 7 days of the study. Implantation rate was significantly higher in normal (18.5%) as compared with high (0%) responders. Estradiol and P levels were elevated significantly in high responders. The E2:P ratio was significantly different between normal and high responders during the preimplantation period. Pregnancy and implantation rates decreased as serum E2 levels increased on days 4 to 6 of the study. A different endocrine milieu between normal and high responders is detected by daily steroid measurements up to the preimplantation period, suggesting that this difference could be responsible for an impaired implantation in high responder patients undergoing IVF. An increase in serum E2 levels seems to be the cause of this difference.

  20. A shift from kinesin 5-dependent metaphase spindle function during preimplantation development in mouse.

    Science.gov (United States)

    Fitzharris, Greg

    2009-06-01

    Microtubules within meiotic and mitotic spindles continually move towards spindle poles in a process termed poleward flux, which is essential for spindle integrity and faithful chromosome segregation. Kinesin 5 is a longstanding candidate for a molecular motor that might drive poleward flux, and has been shown to drive flux and to be necessary for spindle bipolarity in Xenopus egg extracts. However, kinesin 5 is not necessary for poleward flux or for maintaining metaphase spindle bipolarity in intact mammalian cells, and the reason for the different results in these systems is unknown. The experiments presented here test the hypothesis that these results might reflect developmental differences in spindle function by examining the role of kinesin 5 in mouse eggs and preimplantation embryos. In contrast to cultured somatic cells, poleward flux in mouse eggs is critically dependent upon kinesin 5. Inhibition of poleward flux leads to spindle shortening as a result of continued microtubule depolymerisation at the pole, and eventual loss of spindle bipolarity. Spindle bipolarity is also dependent upon kinesin 5 during the first three embryonic cleavages, but becomes kinesin 5-independent in the majority of spindles by the blastocyst stage. This switch occurs asynchronously in different blastomeres but is independent of clonal cell heritage and of whether the blastomere is within the inner cell mass or the trophoectoderm. These experiments reveal a novel developmental switch in the requirements for spindle function and chromosome segregation during preimplantation development.

  1. Ploidy of Bovine Nuclear Transfer Blastocysts Blastomere Donors

    DEFF Research Database (Denmark)

    Booth, P J; VIUFF, D; THOMSEN, P D

    2000-01-01

    The higher rate of embryonic loss in nuclear transfer compared to in vitro produced embryos may be due to chromosome abnormalities that occur during preimplantation in vitro devel- opment. Because little is known about ploidy errors in nuclear transfer embryos, this was ex- amined using embryos...... cultured until day 7 at which time blastocyst nuclei were extracted and chromosome abnormalities were evaluated by fluorescent in situ hybridization using two probes that bind to the subcentromeric regions on chromosomes 6 and 7. In 16 nuclear transfer blastocysts generated from 5 donor embryos, 53.8 6 20...... fertil- ization but the distribution of chromosome abnormalities was different (p 5 0.0002). In conclusion, nuclear transfer embryos reconstructed using blastomere cells can produce over 50% blastocysts with a diploid chromosome complement. However, the contribution of chro- mosome abnormalities...

  2. Designer babies on tap? Medical students' attitudes to pre-implantation genetic screening.

    Science.gov (United States)

    Meisenberg, Gerhard

    2009-03-01

    This paper describes two studies about the determinants of attitudes to pre-implantation genetic screening in a multicultural sample of medical students from the United States. Sample sizes were 292 in study 1 and 1464 in study 2. Attitudes were of an undifferentiated nature, but respondents did make a major distinction between use for disease prevention and use for enhancement. No strong distinctions were made between embryo selection and germ line gene manipulations, and between somatic gene therapy and germ line gene manipulations. Religiosity was negatively associated with acceptance of "designer baby" technology for Christians and Muslims but not Hindus. However, the strongest and most consistent influence was an apparently moralistic stance against active and aggressive interference with natural processes in general. Trust in individuals and institutions was unrelated to acceptance of the technology, indicating that fear of abuse by irresponsible individuals and corporations is not an important determinant of opposition.

  3. Preimplantation Genetic Screening: An Effective Testing for Infertile and Repeated Miscarriage Patients?

    Directory of Open Access Journals (Sweden)

    Ning Wang

    2010-01-01

    Full Text Available Aneuploidy in pregnancy is known to increase with advanced maternal age (AMA and associate with repeated implantation failure (RIF, and repeated miscarriage (RM. Preimplantation genetic screening (PGS has been introduced into clinical practice, screening, and eliminating aneuploidy embryos, which can improve the chance of conceptions for infertility cases with poor prognosis. These patients are a good target group to assess the possible benefit of aneuploidy screening. Although practiced widely throughout the world, there still exist some doubts about the efficacy of this technique. Recent randomized trials were not as desirable as we expected, suggesting that PGS needs to be reconsidered. The aim of this review is to discuss the efficacy of PGS.

  4. Preimplantational genetic diagnosis and mutation detection in a family with duplication mutation of DMD gene.

    Science.gov (United States)

    Ye, Yinghui; Yu, Ping; Yong, Jing; Zhang, Ting; Wei, Xiaoming; Qi, Ming; Jin, Fan

    2014-01-01

    Duchenne muscular dystrophy (DMD) is an X-linked recessive neuromuscular disease caused by mutation in the DMD gene. A 38-year-old woman was referred to our hospital with her son who was diagnosed with DMD. Multiplex PCR failed to detect DMD mutations in the affected child. The female carrier underwent preimplantation genetic diagnosis by linkage analysis and gender determination. Eight embryos were biopsied after in vitro fertilization. Two healthy embryos determined both as females (E1 and E3) were transferred. Although the paternal allele was absent in E3, it was considered to be a result of allele dropout for the STR-49 marker. Surprisingly, a female and a male baby were delivered at 38 gestational weeks, suggesting that E3 was a male embryo with the allele dropout occurring at the SRY gene. Exon 2 duplication was detected in the DMD patient and the carrier mother using next-generation sequencing and multiple ligation-dependent probe amplification. Next, we verified the duplication of exon 2 by real-time PCR, using a special primer at 3' of intron 1, very close to exon 2. Finally, we confirmed that both newborns inherited the normal allele, using quantitative real-time PCR and linkage analysis. © 2014 S. Karger AG, Basel.

  5. Ultrastructure of preimplantation genetic diagnosis-derived human blastocysts grown in a coculture system after vitrification.

    Science.gov (United States)

    Escribá, María-José; Escobedo-Lucea, Carmen; Mercader, Amparo; de los Santos, María-José; Pellicer, Antonio; Remohí, José

    2006-09-01

    To evaluate ultrastructural features of preimplantation genetic diagnosis (PGD) blastocysts before and after vitrification. Descriptive study of both vitrified and fresh hatching blastocysts. PGD program at the Instituto Universitario, Instituto Valenciano de Infertilidad. Patients undergoing PGD donated their abnormal embryos for research (n = 26). Biopsied embryos were cultured in the presence of human endometrial cells until day 6. Sixteen blastocysts were vitrified. A total of 11 high-scored hatching blastocysts, 6 warmed and 5 fresh, were fixed for ultrastructure. The cytoskeleton structure, type of intercellular junctions, and basic intracellular organelles in trophoectoderm cells and the inner cell mass were analyzed. Ten of 16 blastocysts (62%) survived the warming process. Six of these showed no signs of cell degeneration and light microscopy revealed similar ultrastructural characteristics to those of controls. However, in trophoectoderm cells from both fresh and cryopreserved blastocysts, a reduced number of tight junctions and the presence of degradation bodies were detected. The particular ultrastructural features observed in PGD-derived blastocysts could be related to embryo manipulation and culture conditions. Vitrification does not seem to alter blastocysts, as those that survive hatching do not display detectable cellular alterations when observed through electron microscopy.

  6. Evidence of Selection against Complex Mitotic-Origin Aneuploidy during Preimplantation Development

    Science.gov (United States)

    McCoy, Rajiv C.; Demko, Zachary P.; Ryan, Allison; Banjevic, Milena; Hill, Matthew; Sigurjonsson, Styrmir; Rabinowitz, Matthew; Petrov, Dmitri A.

    2015-01-01

    Whole-chromosome imbalances affect over half of early human embryos and are the leading cause of pregnancy loss. While these errors frequently arise in oocyte meiosis, many such whole-chromosome abnormalities affecting cleavage-stage embryos are the result of chromosome missegregation occurring during the initial mitotic cell divisions. The first wave of zygotic genome activation at the 4–8 cell stage results in the arrest of a large proportion of embryos, the vast majority of which contain whole-chromosome abnormalities. Thus, the full spectrum of meiotic and mitotic errors can only be detected by sampling after the initial cell divisions, but prior to this selective filter. Here, we apply 24-chromosome preimplantation genetic screening (PGS) to 28,052 single-cell day-3 blastomere biopsies and 18,387 multi-cell day-5 trophectoderm biopsies from 6,366 in vitro fertilization (IVF) cycles. We precisely characterize the rates and patterns of whole-chromosome abnormalities at each developmental stage and distinguish errors of meiotic and mitotic origin without embryo disaggregation, based on informative chromosomal signatures. We show that mitotic errors frequently involve multiple chromosome losses that are not biased toward maternal or paternal homologs. This outcome is characteristic of spindle abnormalities and chaotic cell division detected in previous studies. In contrast to meiotic errors, our data also show that mitotic errors are not significantly associated with maternal age. PGS patients referred due to previous IVF failure had elevated rates of mitotic error, while patients referred due to recurrent pregnancy loss had elevated rates of meiotic error, controlling for maternal age. These results support the conclusion that mitotic error is the predominant mechanism contributing to pregnancy losses occurring prior to blastocyst formation. This high-resolution view of the full spectrum of whole-chromosome abnormalities affecting early embryos provides insight

  7. Evidence of Selection against Complex Mitotic-Origin Aneuploidy during Preimplantation Development.

    Directory of Open Access Journals (Sweden)

    Rajiv C McCoy

    2015-10-01

    Full Text Available Whole-chromosome imbalances affect over half of early human embryos and are the leading cause of pregnancy loss. While these errors frequently arise in oocyte meiosis, many such whole-chromosome abnormalities affecting cleavage-stage embryos are the result of chromosome missegregation occurring during the initial mitotic cell divisions. The first wave of zygotic genome activation at the 4-8 cell stage results in the arrest of a large proportion of embryos, the vast majority of which contain whole-chromosome abnormalities. Thus, the full spectrum of meiotic and mitotic errors can only be detected by sampling after the initial cell divisions, but prior to this selective filter. Here, we apply 24-chromosome preimplantation genetic screening (PGS to 28,052 single-cell day-3 blastomere biopsies and 18,387 multi-cell day-5 trophectoderm biopsies from 6,366 in vitro fertilization (IVF cycles. We precisely characterize the rates and patterns of whole-chromosome abnormalities at each developmental stage and distinguish errors of meiotic and mitotic origin without embryo disaggregation, based on informative chromosomal signatures. We show that mitotic errors frequently involve multiple chromosome losses that are not biased toward maternal or paternal homologs. This outcome is characteristic of spindle abnormalities and chaotic cell division detected in previous studies. In contrast to meiotic errors, our data also show that mitotic errors are not significantly associated with maternal age. PGS patients referred due to previous IVF failure had elevated rates of mitotic error, while patients referred due to recurrent pregnancy loss had elevated rates of meiotic error, controlling for maternal age. These results support the conclusion that mitotic error is the predominant mechanism contributing to pregnancy losses occurring prior to blastocyst formation. This high-resolution view of the full spectrum of whole-chromosome abnormalities affecting early embryos

  8. Superovulation and embryo recovery in Boer goats treated with ...

    African Journals Online (AJOL)

    Lehloenya

    semen were performed 36 h and 48 h following CIDR removal and the embryos surgically flushed six days after the second AI. The oestrous response, onset- and ..... Increased embryo development and metabolism following short term storage of bovine IVP blastocysts at 25 ºC in EmcareTM compared to ovum culture.

  9. Automated microinjection of recombinant BCL-X into mouse zygotes enhances embryo development.

    Science.gov (United States)

    Liu, Xinyu; Fernandes, Roxanne; Gertsenstein, Marina; Perumalsamy, Alagammal; Lai, Ingrid; Chi, Maggie; Moley, Kelle H; Greenblatt, Ellen; Jurisica, Igor; Casper, Robert F; Sun, Yu; Jurisicova, Andrea

    2011-01-01

    Progression of fertilized mammalian oocytes through cleavage, blastocyst formation and implantation depends on successful implementation of the developmental program, which becomes established during oogenesis. The identification of ooplasmic factors, which are responsible for successful embryo development, is thus crucial in designing possible molecular therapies for infertility intervention. However, systematic evaluation of molecular targets has been hampered by the lack of techniques for efficient delivery of molecules into embryos. We have developed an automated robotic microinjection system for delivering cell impermeable compounds into preimplantation embryos with a high post-injection survival rate. In this paper, we report the performance of the system on microinjection of mouse embryos. Furthermore, using this system we provide the first evidence that recombinant BCL-XL (recBCL-XL) protein is effective in preventing early embryo arrest imposed by suboptimal culture environment. We demonstrate that microinjection of recBCL-XL protein into early-stage embryos repairs mitochondrial bioenergetics, prevents reactive oxygen species (ROS) accumulation, and enhances preimplantation embryo development. This approach may lead to a possible treatment option for patients with repeated in vitro fertilization (IVF) failure due to poor embryo quality.

  10. Automated microinjection of recombinant BCL-X into mouse zygotes enhances embryo development.

    Directory of Open Access Journals (Sweden)

    Xinyu Liu

    Full Text Available Progression of fertilized mammalian oocytes through cleavage, blastocyst formation and implantation depends on successful implementation of the developmental program, which becomes established during oogenesis. The identification of ooplasmic factors, which are responsible for successful embryo development, is thus crucial in designing possible molecular therapies for infertility intervention. However, systematic evaluation of molecular targets has been hampered by the lack of techniques for efficient delivery of molecules into embryos. We have developed an automated robotic microinjection system for delivering cell impermeable compounds into preimplantation embryos with a high post-injection survival rate. In this paper, we report the performance of the system on microinjection of mouse embryos. Furthermore, using this system we provide the first evidence that recombinant BCL-XL (recBCL-XL protein is effective in preventing early embryo arrest imposed by suboptimal culture environment. We demonstrate that microinjection of recBCL-XL protein into early-stage embryos repairs mitochondrial bioenergetics, prevents reactive oxygen species (ROS accumulation, and enhances preimplantation embryo development. This approach may lead to a possible treatment option for patients with repeated in vitro fertilization (IVF failure due to poor embryo quality.

  11. Effect of Bacterial Endotoxins on Superovulated Mouse Embryos In Vivo: Is CSF-1 Involved in Endotoxin-Induced Pregnancy Loss?

    Directory of Open Access Journals (Sweden)

    Yogesh Kumar Jaiswal

    2006-01-01

    Full Text Available Mammalian embryonic development is regulated by several cytokines and growth factors from embryonic or maternal origins. Since CSF-1 plays important role in embryonic development and implantation, we investigated its role in gram-negative bacterial LPS-induced implantation failure. The effect of LPS on normal (nonsuperovulated and superovulated in vivo-produced embryos was assessed by signs of morphological degeneration. A significantly similar number of morphologically degenerated embryos recovered from both nonsuperovulated and superovulated LPS treated animals on day 2.5 of pregnancy onwards were morphologically and developmentally abnormal as compared to their respective controls (P < .001. Normal CSF-1 expression level and pattern were also altered through the preimplantation period in the mouse embryos and uterine horns after LPS treatment. This deviation from the normal pattern and level of CSF-1 expression in the preimplantation embryos and uterine tissues suggest a role for CSF-1 in LPS-induced implantation failure.

  12. Nucleolar remodeling in nuclear transfer embryos

    DEFF Research Database (Denmark)

    Laurincik, Jozef; Maddox-Hyttel, Poul

    2007-01-01

    Transcription of the ribosomal RNA (rRNA) genes occurs in the nucleolus and results in ribosome biogenesis. The rRNA gene activation and the associated nucleolus formation may be used as a marker for the activation of the embryonic genome in mammalian embryos and, thus serve to evaluate the devel...... reprogramming and may help to explain the abnormalities observed in a proportion of fetuses and offspring derived from nuclear transfer embryos....... the developmental potential of embryos originating from varied nuclear transfer protocols. In bovine in vivo developed embryos, functional ribosome-synthesizing nucleoli become structurally distinct toward the end of the 4th post-fertilization cell cycle. In embryonic cell nuclear transfer embryos, fully developed...... nucleoli are not apparent until the 5th cell cycle, whereas in somatic cell nuclear transfer embryos the functional nucleoli emerge already during the 3rd cell cycle. Intergeneric reconstructed embryos produced by the fusion of bovine differentiated somatic cell to a nonactivated ovine cytoplast fail...

  13. Early preimplantation cells expressing Cdx2 exhibit plasticity of specification to TE and ICM lineages through positional changes.

    Science.gov (United States)

    Toyooka, Yayoi; Oka, Sanae; Fujimori, Toshihiko

    2016-03-01

    The establishment of the trophectoderm (TE) and the inner cell mass (ICM) is the first cell lineage segregation to occur in mouse preimplantation development. These two cell lineages arise in a position-dependent manner at the blastocyst stage: the outer cells form TE, which will generate the future placenta, while the inner cells give rise to the ICM, from which the epiblast (EPI) and primitive endoderm (PrE) arise. Previous studies have shown that a portion of cells relocate from the outside position to the inside during this preimplantation stage, but few studies have investigated the correlation between cell relocation and the expression of key transcription factors critical for cell differentiation. To monitor cell movement and the status of the TE-specification pathway in living embryos, we established Cdx2-GFP reporter mice allowing us to visualize the expression of Caudal-type transcriptional factor (Cdx2), a key regulator of the initiation of TE differentiation. Observation of Cdx2-GFP preimplantation embryos by live cell imaging revealed that all cells localized in an initial outer position initiated the expression of Cdx2. Subsequently, cells that changed their position from an outer to an inner position downregulated Cdx2 expression and contributed to the ICM. Finally we showed that internalized cells likely contribute to both the EPI and PrE. Our datas indicate that cells expressing even high levels of Cdx2 can internalize, deactivate an activated TE-specification molecular pathway and integrate into the pluripotent cell population. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Increasing uterine receptivity by decreasing estradiol levels during the preimplantation period in high responders with the use of a follicle-stimulating hormone step-down regimen.

    Science.gov (United States)

    Simón, C; Garcia Velasco, J J; Valbuena, D; Peinado, J A; Moreno, C; Remohí, J; Pellicer, A

    1998-08-01

    To analyze the effect on uterine receptivity of a decrease in E2 levels during the preimplantation period with the use of a step-down regimen in high responders undergoing IVF. Prospective controlled clinical study. The Instituto Valenciano de Infertilidad. High responders in whom at least one previous IVF attempt failed in which 3-4 good-quality embryos were transferred and E2 levels were >3,000 pg/mL on the day of hCG administration. Gonadotropins were administered according to two different protocols. Blood samples were collected and IVF was performed. Serum E2 levels and reproductive outcome of IVF. Estradiol levels on the day of hCG administration and throughout the preimplantation period and the number of oocytes collected were significantly lower with the use of the step-down regimen than during the previous failed cycle in which the standard protocol was used. The fertilization rate was similar and the number of good-quality embryos transferred was comparable. However, the implantation and pregnancy rates were significantly improved in patients who underwent the step-down regimen compared with those who received the standard protocol. With the use of a step-down regimen with FSH in high responders, our clinical results demonstrate that uterine receptivity can be improved when E2 levels are decreased during the preimplantation period.

  15. Lack of carbon air filtration impacts early embryo development.

    Science.gov (United States)

    Munch, Erika M; Sparks, Amy E; Duran, Hakan E; Van Voorhis, Bradley J

    2015-07-01

    To assess human fertilization and preimplantation embryo development in the presence and in the absence of carbon filtration This is a retrospective cohort analysis of fresh, controlled ovarian hyperstimulation cycles as well as previously cryopreserved pronuclear stage embryo transfer cycles in a single IVF center. Embryo development and cycle-based outcomes were compared among three groups: 1) when carbon filtration was present, 2) when carbon filtration was absent, and 3) when carbon filtration had been restored. A total of 524 fresh cycles and 156 cryopreserved embryo cycles were analyzed. Fertilization, cleavage, and blastocyst conversion rates for fresh cycles all declined during the period of absent carbon filtration and recovered after the restoration of carbon filtration. Cryopreserved embryos that were thawed and cultured during the period of absent filtration did not have changes in cleavage or blastocyst conversion rates compared to periods where carbon filtration was present. Clinical pregnancy and live birth rates were unchanged among the three time periods. The absence of carbon filtration in an IVF laboratory air handler is associated with poor fertilization and early embryo development for fresh cycles. Because development of previously frozen pronuclear stage embryos was unaffected, the lack of carbon filtration may preferentially affect embryos in the peri-fertilization period. Carbon filtration is an integral part to a successful human in-vitro fertilization laboratory.

  16. 3D-FISH analysis of embryonic nuclei in mouse highlights several abrupt changes of nuclear organization during preimplantation development

    Directory of Open Access Journals (Sweden)

    Aguirre-Lavin Tiphaine

    2012-10-01

    Full Text Available Abstract Background Embryonic development proceeds through finely tuned reprogramming of the parental genomes to form a totipotent embryo. Cells within this embryo will then differentiate and give rise to all the tissues of a new individual. Early embryonic development thus offers a particularly interesting system in which to analyze functional nuclear organization. When the organization of higher-order chromatin structures, such as pericentromeric heterochromatin, was first analyzed in mouse embryos, specific nuclear rearrangements were observed that correlated with embryonic genome activation at the 2-cell stage. However, most existing analyses have been conducted by visual observation of fluorescent images, in two dimensions or on z-stack sections/projections, but only rarely in three dimensions (3D. Results In the present study, we used DNA fluorescent in situ hybridization (FISH to localize centromeric (minor satellites, pericentromeric (major satellites, and telomeric genomic sequences throughout the preimplantation period in naturally fertilized mouse embryos (from the 1-cell to blastocyst stage. Their distribution was then analyzed in 3D on confocal image stacks, focusing on the nucleolar precursor bodies and nucleoli known to evolve rapidly throughout the first developmental stages. We used computational imaging to quantify various nuclear parameters in the 3D-FISH images, to analyze the organization of compartments of interest, and to measure physical distances between these compartments. Conclusions The results highlight differences in nuclear organization between the two parental inherited genomes at the 1-cell stage, i.e. just after fertilization. We also found that the reprogramming of the embryonic genome, which starts at the 2-cell stage, undergoes other remarkable changes during preimplantation development, particularly at the 4-cell stage.

  17. Efeito do citrato e taurina em meio CR2aa no desenvolvimento de embriões bovinos fecundados in vitro Effect of citrate and taurine added to CR2aa medium on the development of in vitro-fertilized bovine embryos

    Directory of Open Access Journals (Sweden)

    L.S.A. Camargo

    2009-02-01

    Full Text Available Avaliou-se o efeito do citrato em meio CR2aa suplementado com soro fetal bovino (SFB ou livre de proteínas séricas e sua associação com taurina no desenvolvimento de embriões bovinos fecundados in vitro. Embriões foram cultivados em CR2aa contendo 0, 0,5, 1,0 e 3,0mM citrato, suplementado com 10% SFB (experimento 1 ou com álcool polivinil (PVA; experimento 2. No terceiro experimento, embriões foram cultivados em meio com 0,5mM citrato, ou 7mM taurina, ou com a associação de ambos, suplementado com SFB. Os cultivos foram realizados com células do cumulus em ambiente a 38,8ºC com 5% de CO2 em ar atmosférico. Melhora no desenvolvimento embrionário foi observado no cultivo de embriões em CR2aa com 0,5 e 1,0mM citrato na ausência de SFB (P0,05 a produção de embriões ou o número de células. Citrato em meio CR2aa pode ser uma alternativa para cultivo embrionário em condições atmosféricas com 5% de CO2 em ar na ausência de proteína sérica.The effect of citrate added to CR2aa medium supplemented with fetal calf serum (FCS or serum-proteinfree and its association with taurine on the development of in vitro-fertilized bovine embryos was evaluated. Embryos were cultured with 0, 0.5, 1.0, and 3.0mM citrate, in CR2aa supplemented with 10% FCS (experiment 1, or polyvinyl alcohol (PVA; experiment 2. In experiment 3, embryos were cultured with 0.5mM citrate, 7.0mM taurine or with association of both, in medium supplemented with FCS. Embryo culture was performed with cumulus cells at 38.8ºC in 5% CO2 under air for all experiments. Positive effect on embryo development was only observed with 0.5 and 1.0mM citrate in FCS-free CR2aa (P0.05 embryo rate nor total cell number. Citrate in CR2aa medium can be an alternative for serumfree embryo culture under 5% CO2 in air, absence of serum protein.

  18. Biopsy of human morula-stage embryos: outcome of 215 IVF/ICSI cycles with PGS.

    Directory of Open Access Journals (Sweden)

    Elena E Zakharova

    Full Text Available Preimplantation genetic diagnosis (PGD is commonly performed on biopsies from 6-8-cell-stage embryos or blastocyst trophectoderm obtained on day 3 or 5, respectively. Day 4 human embryos at the morula stage were successfully biopsied. Biopsy was performed on 709 morulae from 215 ICSI cycles with preimplantation genetic screening (PGS, and 3-7 cells were obtained from each embryo. The most common vital aneuploidies (chromosomes X/Y, 21 were screened by fluorescence in situ hybridization (FISH. No aneuploidy was observed in 72.7% of embryos, 91% of those developed to blastocysts. Embryos were transferred on days 5-6. Clinical pregnancy was obtained in 32.8% of cases, and 60 babies were born. Patients who underwent ICSI/PGS treatment were compared with those who underwent standard ICSI treatment by examining the percentage of blastocysts, pregnancy rate, gestational length, birth height and weight. No significant differences in these parameters were observed between the groups. Day 4 biopsy procedure does not adversely affect embryo development in vitro or in vivo. The increased number of cells obtained by biopsy of morulae might facilitate diagnostic screening. There is enough time after biopsy to obtain PGD results for embryo transfer on day 5-6 in the current IVF cycle.

  19. Influence of the breed of bull (Bos taurus indicus vs. Bos taurus taurus) and the breed of cow (Bos taurus indicus, Bos taurus taurus and crossbred) on the resistance of bovine embryos to heat.

    Science.gov (United States)

    Eberhardt, Bruno G; Satrapa, Rafael A; Capinzaiki, Cláudia R L; Trinca, Luzia A; Barros, Ciro M

    2009-08-01

    In vitro studies have shown that Bos taurus indicus (B. t. indicus) embryos submitted to heat shock at early stages of development are better able to survive as compared to Bos taurus taurus embryos. Embryo genotype influences resistance to heat shock thus leading to the question as to whether embryos sired by thermo-tolerant breeds exhibit the same resistance to heat shock. In the present study the influence of both oocyte and semen, on the resistance to heat shock (HS) at early stages of in vitro development, was assessed in B. t. indicus [Nelore (N) breed], B. t. taurus [Holstein (H) and Angus (A) breeds] and crossbreds. In Experiment 1, Nelore and crossbred oocytes were collected from slaughterhouse ovaries and fertilized with spermatozoa from Nelore and Angus bulls. Presumptive embryos were collected and randomly assigned to control (39 degrees C) or HS at 12, 48 or 96 h post insemination (hpi; 41 degrees C for 12h) treatments. The cleavage rates and proportion of embryos developing to the blastocyst and hatched blastocyst stages were recorded on Days 2, 8 and 10, respectively. Heat shock treatment decreased development of both Nelore and crossbred embryos. There was a significant interaction between time (12, 48 or 96 hpi) and temperature for blastocyst rates, i.e., the embryos became more thermotolerant as development proceeded. In Experiment 2, oocytes from Nelore and Holstein cows were fertilized with semen from bulls of either Nelore or Angus breeds, and subjected to 12 h HS at 96 hpi. Heat shock at 96 hpi, decreased embryo development. Additionally, cowxtreatment and bullxtreatment interactions were significant for blastocyst rates, i.e., both breed of cow and breed of bull affected the decline in blastocyst rate caused by heat shock treatment. In conclusion, the present results indicate that Nelore embryos (indicus) are more resistant to heat shock than Holstein (taurus) at early stages of in vitro development, and that embryos become more thermo

  20. Healthy Baby Born to a Robertsonian Translocation Carrier following Next-Generation Sequencing-Based Preimplantation Genetic Diagnosis: A Case Report.

    Science.gov (United States)

    Lukaszuk, Krzysztof; Pukszta, Sebastian; Ochman, Karolina; Cybulska, Celina; Liss, Joanna; Pastuszek, Ewa; Zabielska, Judyta; Woclawek-Potocka, Izabela

    2015-10-01

    Preimplantation genetic diagnosis (PGD) is well established method for treatment of genetic problems associated with infertility. Moreover, PGD with next-generation sequencing (NGS) provide new possibilities for diagnosis and new parameters for evaluation in, for example, aneuploidy screening. The aim of the study was to report the successful pregnancy outcome following PGD with NGS as the method for 24 chromosome aneuploidy screening in the case of Robertsonian translocation. Day 3 embryos screening for chromosomal aneuploidy was performed in two consecutive in vitro fertilization (IVF) cycles, first with fluorescent in situ hybridization (FISH), and then with NGS-based protocol. In each IVF attempt, three embryos were biopsied. Short duration of procedures enabled fresh embryo transfer without the need for vitrification. First IVF cycle with the embryo selected using PGD analysis with the FISH method ended with pregnancy loss in week 8. The second attempt with NGS-based aneuploidy screening led to exclusion of the following two embryos: one embryo with 22 monosomy and one with multiple aneuploidies. The transfer of the only euploid blastocyst resulted in the successful pregnancy outcome. The identification of the euploid embryo based on the NGS application was the first successful clinical application of NGS-based PGD in the case of the Robertsonian translocation carrier couple.

  1. Chromothripsis: potential origin in gametogenesis and preimplantation cell divisions. A review.

    Science.gov (United States)

    Pellestor, Franck; Gatinois, Vincent; Puechberty, Jacques; Geneviève, David; Lefort, Geneviève

    2014-12-01

    To review the discovery of chromothripsis and analyze its impact on human reproduction. Database and literature analysis. University hospital. Carriers of massive and complex chromosomal rearrangements. Cytogenetic analysis and molecular testing (fluorescence in situ hybridization, microarray, whole-genome sequencing). Chromothripsis occurrence in human gametes and preimplantation embryos, with regard to the potential causative mechanisms described in literature. Databases were searched for the literature published up to March 2014. Chromothripsis is characterized by the shattering of one (or a few) chromosome segments followed by a haphazard reassembly of the fragments generated, arising through a single initial catastrophic event. Several mechanisms involving abortive apoptosis, telomere erosion, mitotic errors, micronuclei formation, and p53 inactivation might cause chromothripsis. The remarkable point is that all these plausible mechanisms have been identified in the field of human reproduction as causal factors for reproductive failures and the genesis of chromosomal abnormalities. Specific features of gametogenesis and early embryonic development such as the weakness of cell cycle and mitosis checkpoints and the rapid kinetics of division in germ cells and early cleavage embryos may contribute to the emergence of chromothripsis. The discovery of this new class of massive chromosomal rearrangement has deeply modified our understanding on the genesis of complex genomic rearrangements. Data presented in this review support the assumption that chromothripsis could operate in human germlines and during early embryonic development. Chromothripsis might arise more frequently than previously thought in both gametogenesis and early human embryogenesis. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  2. Preimplantation genetic diagnosis for sickle-cell anemia and for beta-thalassemia.

    Science.gov (United States)

    De Rycke, M; Van de Velde, H; Sermon, K; Lissens, W; De Vos, A; Vandervorst, M; Vanderfaeillie, A; Van Steirteghem, A; Liebaers, I

    2001-03-01

    We developed single-cell polymerase chain reaction (PCR) assays for preimplantation genetic diagnosis (PGD) in couples carrying mutations in the beta-globin gene. With PGD the genetic status of an embryo obtained after intracytoplasmic sperm injection (ICSI) is determined by PCR analysis in single blastomeres, allowing only healthy embryos to be transferred to the uterus. We carried out nine PGD cycles using fluorescent PCR for two couples in whom the partners carried sickle-cell trait. Both couples achieved pregnancies, one of which was spontaneously aborted. We have developed two beta-thalassemia PGD protocols: one for the analysis of the 25-26delAA and the IVS2+1G>A mutation, and the other for the simultaneous detection of the IVS1+6T>C and the IVS1+110G>A mutations. For the second protocol, both non-labelled PCR and later fluorescent PCR were used. Both protocols were applied in clinical cycles (two non-labelled PCR cycles and one fluorescent PCR cycle) for two couples. The patient with the fluorescent PCR-PGD cycle became pregnant. Overall, the three fluorescent PCR assays were accurate and reliable with amplification efficiencies of minimum 93% and allele dropout (ADO) rates between 0 and 12%. Copyright 2001 John Wiley & Sons, Ltd.

  3. Preimplantation genetic diagnosis in the prevention of the haemoglobin disorders

    Directory of Open Access Journals (Sweden)

    S. Kahraman

    2011-12-01

    Full Text Available Preimplantation Genetic Diagnosis (PGD is currently an alternative for couples with high risk of pregnancies with genetic anomalies; it offers the possibility of avoiding the need to terminate affected pregnancies, since it allows the selection of unaffected embryos for transfer. PGD for inherited disorders has become extremely accurate (99.5%, and may currently be performed for any single gene disorders in which mutation is identified. PGD has been performed for more than 100 different conditions resulting in the birth of at least 1000 healthy children free of genetic disorder. PGD is presently also used together with preimplantation HLA typing for treatment of affected sibling with genetic and acquired disorders requiring HLA matched stem cell transplantation. This is not only to allow couples to have an unaffected child but also to select a potential donor progeny for stem cell transplantation. In Turkey, thalassemia is the most commonly seen genetic disorder the rate of thalassemia carriers is about 3 - 4% in Turkey. The majority of our PGD cases are thalassemia carriers. They do not only require thalassemia mutation analysis but also HLA typing for their affected child. In this study PGD results of 236 Turkish couples with or without HLA typing will be presented and discussed. A full diagnosis was achieved in 91.0% of the biopsied samples. In Group I, 17.8% of the analyzed embryos were found to be HLA compatible. HLA compatible and disease free embryos were 12.9% of all diagnosed embryos. In group II, 17.2% of embryos were found to be HLA matched and 71.4% HLA non-matched. The majority of our HLA typing combined with PGD cases were β-Thalassemia carriers (87.9%. The mutations analyzed have high heterogeneity, the most frequent mutation was IVS-I-110 G-A and comprised 46.2% of all mutations. To date, 70 healthy and HLA compatible children have been born. Twenty-five sick children have already been cured with cord blood cell and/or bone

  4. In vivo and in vitro development of Tibetan antelope (Pantholops hodgsonii interspecific cloned embryos

    Directory of Open Access Journals (Sweden)

    Guanghua SU,Lei CHENG,Yu GAO,Kun LIU,Zhuying WEI,Chunling BAI,Fengxia YIN,Li GAO,Guangpeng LI,Shorgan BOU

    2014-02-01

    Full Text Available The Tibetan antelope is endemic to the Tibetan Plateau, China, and is now considered an endangered species. As a possible rescue strategy, the development of embryos constructed by interspecies somatic cell nuclear transfer (iSCNT was examined. Tibetan antelope fibroblast cells were transferred into enucleated bovine, ovine and caprine oocytes. These cloned embryos were then cultured in vitro or in the oviducts of intermediate animals. Less than 0.5% of the reconstructed antelope-bovine embryos cultured in vitro developed to the blastocyst stage. However, when the cloned antelope-bovine embryos were transferred to caprine oviducts, about 1.6% of the embryos developed to the blastocyst stage. In contrast, only 0.7% of the antelope-ovine embryos developed to the morula stage and none developed to blastocysts in ovine oviducts. The treatment of donor cells and bovine oocytes with trichostatin A did not improve the embryo development even when cultured in the oviducts of ovine and caprine. When the antelope-bovine embryos, constructed from oocytes treated with roscovitine or trichostatin A, were cultured in rabbit oviducts 2.3% and 14.3% developed to blastocysts, respectively. It is concluded that although some success was achieved with the protocols used, interspecies cloning of Tibetan antelope presents difficulties still to be overcome. The mechanisms resulting in the low embryo development need investigation and progress might require a deeper understanding of cellular reprogramming.

  5. Transcriptional profiling of mouse uterus at pre-implantation stage under VEGF repression.

    Directory of Open Access Journals (Sweden)

    Yan Ji

    Full Text Available Uterus development during pre-implantation stage affects implantation process and embryo growth. Aberrant uterus development is associated with many human reproductive diseases. Among the factors regulating uterus development, vascular remodeling promoters are critical for uterus function and fertility. Vascular endothelial growth factor (VEGF, as one of the major members, has been found to be important in endothelial cell growth and blood vessel development, as well as in non-endothelial cells. VEGF mediation in reproduction has been broadly studied, but VEGF-induced transcriptional machinery during implantation window has not been systematically studied. In this study, a genetically repressed VEGF mouse model was used to analyze uterus transcriptome at gestation 2.5 (G2.5 by Solexa/Illumina's digital gene expression (DGE system. A number of 831 uterus-specific and 2398 VEGF-regulated genes were identified. Gene ontology (GO analysis indicated that genes actively involved in uterus development were members of collagen biosynthesis, cell proliferation and cell apoptosis. Uterus-specific genes were enriched in activities of phosphatidyl inositol phosphate kinase, histone H3-K36 demethylation and protein acetylation. Among VEGF-regulated genes, up-regulated were associated with RNA polymerase III activity while down-regulated were strongly related with muscle development. Comparable numbers of antisense transcripts were identified. Expression levels of the antisense transcripts were found tightly correlated with their sense expression levels, an indication of possibly non-specific transcripts generated around the active promoters and enhancers. The antisense transcripts with exceptionally high or low expression levels and the antisense transcripts under VEGF regulation were also identified. These transcripts may be important candidates in regulation of uterus development. This study provides a global survey on genes and antisense transcripts

  6. Biopsy of embryos produced by in vitro fertilization affects development in C57BL/6 mouse strain

    OpenAIRE

    Sugawara, Atsushi; Ward, Monika A.

    2012-01-01

    Preimplantation genetic diagnosis (PGD) is considered highly successful in respect to its accuracy in detecting genetic anomalies but the effects of embryo biopsy on embryonic/fetal growth and development are less known, particularly in conjunction with in vitro fertilization (IVF). Here, we compared biopsied (B) and non-biopsied (NB) mouse embryos for their developmental competence. Embryos C57BL/6 (B6) and B6D2F2 (F2) generated by IVF were subjected to single blastomere biopsy at the 4-cell...

  7. In vitro and in vivo Development of Cloned Ovine Embryos using in vitro and in vivo Matured Oocytes

    DEFF Research Database (Denmark)

    Holm, P; Nagashima, H; Sun, F-J

    1995-01-01

    Cloning of sheep embryos by nucleus transplantation can be achieved by using in vivo matured (oviductal) oocytes and in vivo culture. However, these steps involve cumbersome procedures. Therefore, the effects of in vivo vs. the equivalent in vitro procedures on the pre-implantation development...... matured oocytes were enucleated and fused with inserted blastomeres from donor embryos. In vitro matured oocytes were enucleated and allowed to age prior to blastomere insertion and electrofusion. Fused embryos were cultured for approximately 132 h either in vivo in ligated sheep oviducts or in vitro...

  8. Embryo transfer and related technologies in sheep reproduction.

    Science.gov (United States)

    Loi, P; Ptak, G; Dattena, M; Ledda, S; Naitana, S; Cappai, P

    1998-01-01

    This paper reviews the status of embryo transfer and the major technologies applied to preimplantation of embryos in sheep. Embryo production from superovulated ewes is hindered by an unpredictable response to hormonal treatment. Progress in this area should be expected by an appropriated control of follicular development with gonadotropin-releasing hormone (GnRH) agonist or antagonist prior to gonadotrophin administration. Simple protocols for the cryopreservation of sheep embryos by vitrification are already available and the development of frozen-thawed blastocysts to term is close to the fresh ones. Further research is required to identify factors able to promote the maturation in vitro of oocytes, namely those obtained from prepubertal animals. Semen and embryo sexing procedures are available in cattle although much less attention was paid to their application to sheep. Among all the reproductive technologies, cloning with embryonic and foetal cells has progressed dramatically in sheep and nuclear transfer has been used to produce transgenic animals as an alternative to pronuclear injection. The production of the first lamb cloned from a somatic cell opened new opportunities in animal breeding as well as exciting lines of basic research. The overall conclusions are that, apart from superovulation, the application of in vitro technologies is likely to evolve rapidly and once applied, a great impact on traditional and new animal productions should be expected. However, a better understanding of the changes in gene expression, induced in embryos by different in vitro manipulation procedures, is necessary to prevent abnormal foetal development.

  9. Attitude towards pre-implantation genetic diagnosis for hereditary cancer

    NARCIS (Netherlands)

    Lammens, Chantal; Bleiker, Eveline; Aaronson, Neil; Vriends, Annette; Ausems, Margreet; Jansweijer, Maaike; Wagner, Anja; Sijmons, Rolf; van den Ouweland, Ans; van der Luijt, Rob; Spruijt, Liesbeth; Garcia, Encarna Gomez; Ruijs, Marielle; Verhoef, Senno

    2009-01-01

    The use of pre-implantation genetic diagnosis (PGD) for hereditary cancer is subject to on-going debate, particularly among professionals. This study evaluates the attitude towards PGD and attitude-associated characteristics of those concerned: family members with a hereditary cancer predisposition.

  10. Embryo selection in IVF

    National Research Council Canada - National Science Library

    Mastenbroek, Sebastiaan; van der Veen, Fulco; Aflatoonian, Abbas; Shapiro, Bruce; Bossuyt, Patrick; Repping, Sjoerd

    2011-01-01

    To optimize success rates of IVF, selection of the most viable embryo(s) for transfer has always been essential, as embryos that are cryopreserved are thought to have a reduced chance of implanting after thawing...

  11. ROCK and RHO Playlist for Preimplantation Development: Streaming to HIPPO Pathway and Apicobasal Polarity in the First Cell Differentiation.

    Science.gov (United States)

    Alarcon, Vernadeth B; Marikawa, Yusuke

    2018-01-01

    In placental mammalian development, the first cell differentiation produces two distinct lineages that emerge according to their position within the embryo: the trophectoderm (TE, placenta precursor) differentiates in the surface, while the inner cell mass (ICM, fetal body precursor) forms inside. Here, we discuss how such position-dependent lineage specifications are regulated by the RHOA subfamily of small GTPases and RHO-associated coiled-coil kinases (ROCK). Recent studies in mouse show that activities of RHO/ROCK are required to promote TE differentiation and to concomitantly suppress ICM formation. RHO/ROCK operate through the HIPPO signaling pathway, whose cell position-specific modulation is central to establishing unique gene expression profiles that confer cell fate. In particular, activities of RHO/ROCK are essential in outside cells to promote nuclear localization of transcriptional co-activators YAP/TAZ, the downstream effectors of HIPPO signaling. Nuclear localization of YAP/TAZ depends on the formation of apicobasal polarity in outside cells, which requires activities of RHO/ROCK. We propose models of how RHO/ROCK regulate lineage specification and lay out challenges for future investigations to deepen our understanding of the roles of RHO/ROCK in preimplantation development. Finally, as RHO/ROCK may be inhibited by certain pharmacological agents, we discuss their potential impact on human preimplantation development in relation to fertility preservation in women.

  12. Pre-implantation genetic screening using fluorescence in situ hybridization in couples of Indian ethnicity: Is there a scope?

    Directory of Open Access Journals (Sweden)

    Shailaja Gada Saxena

    2014-01-01

    Full Text Available Context: There is a high incidence of numerical chromosomal aberration in couples with repeated in vitro fertilization (IVF failure, advanced maternal age, repeated unexplained abortions, severe male factor infertility and unexplained infertility. Pre-implantation genetic screening (PGS, a variant of pre-implantation genetic diagnosis, screens numerical chromosomal aberrations in couples with normal karyotype, experiencing poor reproductive outcome. The present study includes the results of the initial pilot study on 9 couples who underwent 10 PGS cycles. Aim: The aim of the present study was to evaluate the beneficial effects of PGS in couples with poor reproductive outcome. Settings and Design: Data of initial 9 couples who underwent 10 PGS for various indications was evaluated. Subjects and Methods: Blastomere biopsy was performed on cleavage stage embryos and subjected to two round fluorescence in situ hybridization (FISH testing for chromosomes 13, 18, 21, X and Y as a two-step procedure. Results: Six of the 9 couples (10 PGS cycles conceived, including a twin pregnancy in a couple with male factor infertility, singleton pregnancies in a couple with secondary infertility, in three couples with adverse obstetric outcome in earlier pregnancies and in one couple with repeated IVF failure. Conclusion: In the absence of availability of array-comparative genomic hybridization in diagnostic clinical scenario for PGS and promising results with FISH based PGS as evident from the current pilot study, it is imperative to offer the best available services in the present scenario for better pregnancy outcome for patients.

  13. Methods for assessing the quality of mammalian embryos: How far we are from the gold standard?

    Science.gov (United States)

    Rocha, José C; Passalia, Felipe; Matos, Felipe D; Maserati, Marc P; Alves, Mayra F; Almeida, Tamie G de; Cardoso, Bruna L; Basso, Andrea C; Nogueira, Marcelo F G

    2016-08-01

    Morphological embryo classification is of great importance for many laboratory techniques, from basic research to the ones applied to assisted reproductive technology. However, the standard classification method for both human and cattle embryos, is based on quality parameters that reflect the overall morphological quality of the embryo in cattle, or the quality of the individual embryonic structures, more relevant in human embryo classification. This assessment method is biased by the subjectivity of the evaluator and even though several guidelines exist to standardize the classification, it is not a method capable of giving reliable and trustworthy results. Latest approaches for the improvement of quality assessment include the use of data from cellular metabolism, a new morphological grading system, development kinetics and cleavage symmetry, embryo cell biopsy followed by pre-implantation genetic diagnosis, zona pellucida birefringence, ion release by the embryo cells and so forth. Nowadays there exists a great need for evaluation methods that are practical and non-invasive while being accurate and objective. A method along these lines would be of great importance to embryo evaluation by embryologists, clinicians and other professionals who work with assisted reproductive technology. Several techniques shows promising results in this sense, one being the use of digital images of the embryo as basis for features extraction and classification by means of artificial intelligence techniques (as genetic algorithms and artificial neural networks). This process has the potential to become an accurate and objective standard for embryo quality assessment.

  14. Development of a new clinically applicable device for embryo evaluation which measures embryo oxygen consumption.

    Science.gov (United States)

    Kurosawa, Hiroki; Utsunomiya, Hiroki; Shiga, Naomi; Takahashi, Aiko; Ihara, Motomasa; Ishibashi, Masumi; Nishimoto, Mitsuo; Watanabe, Zen; Abe, Hiroyuki; Kumagai, Jin; Terada, Yukihiro; Igarashi, Hideki; Takahashi, Toshifumi; Fukui, Atsushi; Suganuma, Ryota; Tachibana, Masahito; Yaegashi, Nobuo

    2016-10-01

    Does a new system-the chip-sensing embryo respiration monitoring system (CERMs)-enable evaluation of embryo viability for potential application in a clinical IVF setting? The system enabled the oxygen consumption rate of spheroids, bovine embryos and frozen-thawed human embryos to be measured, and this rate corresponded to the developmental potential of embryos. To date, no reliable and clinically suitable objective evaluation methods for embryos are available, which circumvent the differences in inter-observer subjective view. Existing systems such as the scanning electrochemical microscopy (SECM) technique, which enables the measurement of oxygen consumption rate in embryos, need improvement in usability before they can be applied to a clinical setting. This is a prospective original research study. The feasibility of measuring the oxygen consumption rate was assessed using CERMs for 9 spheroids, 9 bovine embryos and 30 redundant frozen-thawed human embryos. The endpoints for the study were whether CERMs could detect a dissolved oxygen gradient with high sensitivity, had comparable accuracy to the SECM measuring system with improved usability, and could predict the development of an embryo to a blastocyst by measuring the oxygen consumption rate. The relationship between the oxygen consumption rate and standard morphological evaluation was also examined. We developed a new CERMs, which enables the oxygen consumption rate to be measured automatically using an electrochemical method. The device was initially used for measuring a dissolved oxygen concentration gradient in order to calculate oxygen consumption rate using nine spheroids. Next, we evaluated data correlation between the CERMs and the SECM measuring systems using nine bovine embryos. Finally, the oxygen consumption rates of 30 human embryos, which were frozen-thawed on 2nd day after fertilization, were measured by CERMs at 6, 24, 48, 72 and 96 h after thawing with standard morphological evaluation

  15. The Effects of Progesterone on Oocyte Maturation and Embryo Development

    Directory of Open Access Journals (Sweden)

    Saeed Zavareh

    2013-01-01

    Full Text Available Oocyte maturation and embryo development are controlled by intra-ovarian factors suchas steroid hormones. Progesterone (P4 exists in the follicular fluid that contributes tonormal mammalian ovarian function and has several critical functions during embryodevelopment and implantation, including endometrial receptivity, embryonic survivalduring gestation and transformation of the endometrial stromal cells to decidual cells.It is well known that the physiological effects of P4 during the pre-implantation stages ofsome mammal’s embryos are mediated by P4 receptors and their gene expression is determined.The effects of P4 on oocytes and embryo development have been assessed bysome investigations, with contradictory results. P4, a dominant steroid in follicular fluidat approximately 18 hours after the luteinizing hormone (LH surge may have a criticalrole in maturation of oocytes at the germinal stage. However, it has been shown that differentconcentrations of P4 could not improve in vitro maturation rates of germinal vesicles(GV in cumulus oocyte complexes (COCs and cumulus denuded oocytes (CDOs.Culture media supplemented with P4 significantly improved mouse embryo development.In addition, an in vivo experimental design has shown high blastocyst survival andimplantation rates in P4-treated mice.In this review we explain some of the findings that pertain to the effects of P4 onoocyte maturation and embryo development both in vitro and in vivo.

  16. Autophagic activity as an indicator for selecting good quality embryos.

    Science.gov (United States)

    Tsukamoto, Satoshi

    2015-04-01

    Is it possible to predict the quality of embryos that appear to be morphologically identical when viewed under a microscope? Thirty-five years have passed since the world's first human birth from in vitro fertilization. While the dissemination of assisted reproduction technologies during this time has been remarkable, the evaluation of embryo quality in both humans and mice currently relies entirely on morphological observation. More efficient infertility treatments will likely be possible if high-quality embryos can be selected by screening. To develop a novel quality evaluation method that does not rely on morphology, we focused on autophagy, one of the molecular mechanisms essential for the early embryonic development. Autophagy is a massive cytoplasmic degradation pathway mediated by the lysosome. Our previous studies have demonstrated that fertilization-induced autophagy is essential for preimplantation embryonic development. This autophagy is thought to supply the nutrients and amino acids necessary for maintaining subsequent embryo development, through the bulk degradation of maternal cytoplasmic factors that are accumulated during oogenesis. Here, we briefly summarize autophagy and its physiological function, and describe a recently developed method for using autophagic activity as an indicator to predict embryo quality.

  17. Comparative preimplantation genetic diagnosis policy in Europe and the USA and its implications for reproductive tourism

    Directory of Open Access Journals (Sweden)

    Michelle J Bayefsky

    2016-12-01

    Full Text Available Unlike many European nations, the USA has no regulations concerning the use of preimplantation genetic diagnosis (PGD, a technique employed during some fertility treatments to select embryos based on their genes. As such, PGD can and is used for a variety of controversial purposes, including sex selection, selection for children with disabilities such as deafness, and selection for ‘saviour siblings’ who can serve as tissue donors for sick relatives. The lack of regulation, which is due to particular features of the US political and economic landscape, has ethical and practical implications for patients seeking PGD around the world. This paper contrasts the absence of PGD oversight in the USA with existing PGD policies in Switzerland, Italy, France and the UK. The primary reasons why PGD is not regulated in the USA are addressed, with consideration of factors such as funding for assisted reproductive technology treatmemt and the proximity of PGD to the contentious abortion debate. The obstacles that would need to be overcome in the USA for PGD to be regulated in the future are outlined. Then, the significance of the current divergence in PGD policy for patients around the world are discussed. Regulatory differences create opportunities for reproductive tourism, which result in legal, health and moral challenges. The paper concludes with comments on the need for policymakers around the world to balance respect for the characters and constitutions of their individual countries with appreciation of the needs of infertile patients across the globe.

  18. Comparative preimplantation genetic diagnosis policy in Europe and the USA and its implications for reproductive tourism.

    Science.gov (United States)

    Bayefsky, Michelle J

    2016-12-01

    Unlike many European nations, the USA has no regulations concerning the use of preimplantation genetic diagnosis (PGD), a technique employed during some fertility treatments to select embryos based on their genes. As such, PGD can and is used for a variety of controversial purposes, including sex selection, selection for children with disabilities such as deafness, and selection for 'saviour siblings' who can serve as tissue donors for sick relatives. The lack of regulation, which is due to particular features of the US political and economic landscape, has ethical and practical implications for patients seeking PGD around the world. This paper contrasts the absence of PGD oversight in the USA with existing PGD policies in Switzerland, Italy, France and the UK. The primary reasons why PGD is not regulated in the USA are addressed, with consideration of factors such as funding for assisted reproductive technology treatmemt and the proximity of PGD to the contentious abortion debate. The obstacles that would need to be overcome in the USA for PGD to be regulated in the future are outlined. Then, the significance of the current divergence in PGD policy for patients around the world are discussed. Regulatory differences create opportunities for reproductive tourism, which result in legal, health and moral challenges. The paper concludes with comments on the need for policymakers around the world to balance respect for the characters and constitutions of their individual countries with appreciation of the needs of infertile patients across the globe.

  19. Rethinking In Vitro Embryo Culture: New Developments in Culture Platforms and Potential to Improve Assisted Reproductive Technologies1

    Science.gov (United States)

    Smith, Gary D.; Takayama, Shuichi; Swain, Jason E.

    2011-01-01

    ABSTRACT The preponderance of research toward improving embryo development in vitro has focused on manipulation of the chemical soluble environment, including altering basic salt composition, energy substrate concentration, amino acid makeup, and the effect of various growth factors or addition or subtraction of other supplements. In contrast, relatively little work has been done examining the physical requirements of preimplantation embryos and the role culture platforms or devices can play in influencing embryo development within the laboratory. The goal of this review is not to reevaluate the soluble composition of past and current embryo culture media, but rather to consider how other controlled and precise factors such as time, space, mechanical interactions, gradient diffusions, cell movement, and surface interactions might influence embryo development. Novel culture platforms are being developed as a result of interdisciplinary collaborations between biologists and biomedical, material, chemical, and mechanical engineers. These approaches are looking beyond the soluble media composition and examining issues such as media volume and embryo spacing. Furthermore, methods that permit precise and regulated dynamic embryo culture with fluid flow and embryo movement are now available, and novel culture surfaces are being developed and tested. While several factors remain to be investigated to optimize the efficiency of embryo production, manipulation of the embryo culture microenvironment through novel devices and platforms may offer a pathway toward improving embryo development within the laboratory of the future. PMID:21998170

  20. Rethinking in vitro embryo culture: new developments in culture platforms and potential to improve assisted reproductive technologies.

    Science.gov (United States)

    Smith, Gary D; Takayama, Shuichi; Swain, Jason E

    2012-03-01

    The preponderance of research toward improving embryo development in vitro has focused on manipulation of the chemical soluble environment, including altering basic salt composition, energy substrate concentration, amino acid makeup, and the effect of various growth factors or addition or subtraction of other supplements. In contrast, relatively little work has been done examining the physical requirements of preimplantation embryos and the role culture platforms or devices can play in influencing embryo development within the laboratory. The goal of this review is not to reevaluate the soluble composition of past and current embryo culture media, but rather to consider how other controlled and precise factors such as time, space, mechanical interactions, gradient diffusions, cell movement, and surface interactions might influence embryo development. Novel culture platforms are being developed as a result of interdisciplinary collaborations between biologists and biomedical, material, chemical, and mechanical engineers. These approaches are looking beyond the soluble media composition and examining issues such as media volume and embryo spacing. Furthermore, methods that permit precise and regulated dynamic embryo culture with fluid flow and embryo movement are now available, and novel culture surfaces are being developed and tested. While several factors remain to be investigated to optimize the efficiency of embryo production, manipulation of the embryo culture microenvironment through novel devices and platforms may offer a pathway toward improving embryo development within the laboratory of the future.

  1. Prenatal diagnosis and preimplantation genetic diagnosis: novel technologies and state of the art of PGD in different regions of the world.

    Science.gov (United States)

    Peyvandi, F; Garagiola, I; Mortarino, M

    2011-07-01

    Prenatal diagnosis (PND) aims to provide accurate, rapid results as early in pregnancy as possible. Conventional PND involves sampling cells of foetal origin by chorionic villus sampling at 11-14th weeks of pregnancy or amniocentesis after 15th week. These are invasive procedures and have a small but significant rate of 0.5% to 1% for loss of pregnancy. An alternative to existing methods for conventional PND for couples at risk of transmitting a genetic disease to their child is preimplantation genetic diagnosis (PGD). PGD is a newly emerging form of a very early prenatal diagnosis. The technique combines assisted reproductive technology with molecular genetics and cytogenetics to allow the identification of abnormality in embryos prior to implantation. The diagnosis of genetic disease in human preimplantation embryos was pioneered in the late 1980s for testing of aneuploidy, single gene and X-linked disease, such as cystic fibrosis, haemophilia and chromosomal abnormalities. The PGD-related legal and ethical issues have been debated at many levels both nationally and internationally. The attitude towards PGD varies substantially not only in different parts of the world but also within the Europe, owing to scientific, cultural and religious differences. PGD has become widely practised throughout the world for various indications and can substantially decrease the eventual risks of passing a genetic undesired condition of the offspring. Nevertheless, its extension to some new and non-medical indications has raised ethical concerns, in particular its potential eugenic dimension. © 2011 Blackwell Publishing Ltd.

  2. Melatonin in maturation media fails to improve oocyte maturation, embryo development rates and DNA damage of bovine embryos Melatonina no meio de maturação não melhorou as taxas de maturação dos ovócitos, de desenvolvimento embrionário e a fragmentação do DNA dos embriões bovinos

    Directory of Open Access Journals (Sweden)

    Luciana Takada

    2010-08-01

    Full Text Available Melatonin (MEL acts as a powerful scavenger of free radicals and direct gonadal responses to melatonin have been reported in the literature. Few studies, however, have evaluated the effect of MEL during in vitro maturation (IVM on bovine embryos. This study tested the addition of MEL to maturation medium (MM with no gonadotropins on nuclear maturation and embryo development rates and the incidence of DNA damage in resulting embryos. Cumulus-oocyte complexes were aspirated from abattoir ovaries and cultured in MM (TCM-199 medium supplemented with 10% fetal calf serum - FCS at 39ºC and 5% CO2 in air. After 24 hours of culture in MM with 0.5 µg mL-1 FSH and 5.0 µg mL-1 LH; 10-9 M MEL or 10-9 M MEL, 0.5 µg mL-1 FSH and 5.0 µg mL-1 LH, the oocytes were stained with Hoechst 33342 to evaluate nuclear maturation rate. After in vitro fertilization and embryo culture, development rates were evaluated and the blastocysts were assessed for DNA damage by Comet assay. There was no effect of melatonin added to the MM, alone or in combination with gonadotropins, on nuclear maturation, cleavage and blastocyst rates. These rates ranged between 88% to 90%, 85% to 88% and 42% to 46%, respectively. The extent of DNA damage in embryos was also not affected by MEL supplementation during IVM. The addition of 10-9 M MEL to the MM failed to improve nuclear maturation and embryo development rates and the incidence of DNA damage in resulting embryos, but was able to properly substitute for gonadotropins during IVM.Melatonin (MEL atua como um potente redutor de radicais livres. Efeito direto da MEL na função gonadal também foi observado. Existem poucos estudos relacionados ao efeito da MEL durante a maturação no desenvolvimento embrionário in vitro. Avaliou-se a adição de MEL no meio de maturação (sem gonadotrofinas nas taxas de maturação nuclear e de desenvolvimento embrionário e na incidência de fragmentação do DNA nos embriões produzidos in vitro

  3. Development and validation of concurrent preimplantation genetic diagnosis for single gene disorders and comprehensive chromosomal aneuploidy screening without whole genome amplification.