p38 (Mapk14/11) occupies a regulatory node governing entry into primitive endoderm differentiation during preimplantation mouse embryo development

Czech Academy of Sciences Publication Activity Database

Thamodaran, V.; Bruce, Alexander

2016-01-01

Roč. 6, č. 9 (2016), č. článku 160190. ISSN 2046-2441 Grant - others:GA ČR(CZ) GA13-03295S Institutional support: RVO:60077344 Keywords : preimplantation mouse embryo * cell-fate * primitive endoderm Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.481, year: 2016 http://rsob.royalsocietypublishing.org/content/6/9/160190

Effect of Intracytoplasmic Sperm Injection (ICSI on Mouse Embryos Preimplantational Development

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Claudia Cârstea

2012-05-01

Full Text Available It is known that the in vitro culture (IVC of preimplantation embryos is associated with changes in gene expression. It is however, not known if the method of fertilization affects the global pattern of gene expression. We compared the development of mouse blastocysts produced by intracytoplasmic sperm injection (ICSI versus blastocysts fertilized in vivo and cultured in vitro from the zygote stage (IVC. At the end of cultivation (96 hrs for blastocyst stage embryos, expanded blastocysts of each group were randomly selected, and ICM and total cells number were differentially stained. The total cell number of blastocysts was estimated by counting the total number of nuclei using DAPI staining. Cell number for inner cell mass (ICM was estimated by counting the OCT4 (POU5FL positive cells. Digitally recombined, composite images were analyzed using the Zeiss Axion Vision software and Zeiss Apotome. All 5–10 optical sections were divided using a standard grid over each layer to count all. Comparing the total cells and the ICM cells number, it appears that each method of fertilization has a unique pattern development. The developmental rate and the total cell number of the blastocyst were significantly lower in ICSI versus in vivo fertilized embryos which affect the embryonic developmental rate and the total cell number of blastocysts.

Radiotoxicity and incorporation of methyl-tritiated-thymidine on preimplantation mouse embryo. In vitro fertilization and culture

International Nuclear Information System (INIS)

Kikuchi, O.K.; Ohyama, H.; Yamada, T.

1993-04-01

In the present work different concentrations of methyl- 3 H-thymidine was added to the culture medium micro drops containing the mouse zygotes at pro nuclear stage and the embryos were cultured in vitro at 37 0 C in a humidified atmosphere with 5% of CO 2 for four days up to the expanded blastocyst stage, the established end point to calculate the L D 50 lethal dose. The blastocyst formation rate decreased with increasing concentration of tritium in medium and a value of 2.4 X 10 3 Bq/ml for L D 50 was obtained. The 3 H-Td R incorporation by the embryo during the preimplantation period was low at the beginning and increased quickly during the morula and the blastocyst development. (author)

Lethality of radioisotopes in early mouse embryos

International Nuclear Information System (INIS)

Macqueen, H.A.

1979-01-01

The development of pre-implantation mouse embryos was found to be prevented by exposure of the embryos to [ 35 S]methionine, but not to [ 3 H]methionine. Such embryos have also been shown to be highly sensitive to [ 3 H]thymidine. These observations are discussed with reference to the path lengths and energies of electrons emitted from the different radioisotopes. (author)

Sexing of Mouse Preimplantation Embryos Using Polymerase Chain Reaction%运用PCR对小鼠植入前胚胎进行性别诊断

Institute of Scientific and Technical Information of China (English)

李汶; 陆长富; 卢光琇

2001-01-01

In order to determine the sex of mouse embryo, 1 or 2 blastomeres were biopsied from Kun-ming-white mouse preimplantation embryo at 4-8 cells stage. The gDNA of the single-blastomere was abstracted. According to the base sequence of 145C5, a repititive sequence of Y chromosome of mouse C57BL6, a pair of primer were asigned and synthesized. The gDNA was amplified using these primers. 108 mouse preimplantation embryos were sexed via this technique. 46 male embryos and 62 female embryos were transfered into five pseudopreganant mothers respectively. 4 male litters and 9 female litters were obtained. The diagnosis positive　rate was 100%(4/4) and 70% (9/13)respectively. The result of PCR indecated that there was no difference between the repititive sequence of Y chromosome in mouse C57BL6 and Kun-ming-white mouse. The technique developed in this study might be further used for preimplantation genetic diagnosis of single-gene defects.%根据C57BL6小鼠Y染色体重复序列145C5的碱基顺序, 设计并合成一对引物, 运用PCR扩增昆明白小鼠植入前胚胎卵裂球DNA, 以确定其性别。共对108枚活检胚胎的相应卵裂球进行了性别诊断, 获雄性胚46枚, 雌性胚62枚, 移植后分别获雄性仔鼠4只, 准确率100%(4/4), 雌性仔鼠9只, 准确率70%(9/13)。本研究结果表明小鼠Y染色体重复序列145C5的碱基顺序在C57BL6小鼠和昆明白小鼠中基本一致；为农牧业动物进行性别选择和运用PCR进行单基因病植入前遗传学诊断提供了方法学基础。

Can a genetically-modified organism-containing diet influence embryo development? A preliminary study on pre-implantation mouse embryos

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B Cisterna

2009-08-01

Full Text Available In eukaryotic cells, pre-mRNAs undergo several transformation steps to generate mature mRNAs. Recent studies have demonstrated that a diet containing a genetically modified (GM soybean can induce modifications of nuclear constituents involved in RNA processing in some tissues of young, adult and old mice. On this basis, we have investigated the ultrastructural and immunocytochemical features of pre-implantation embryos from mice fed either GM or non- GM soybean in order to verify whether the parental diet can affect the morpho-functional development of the embryonic ribonucleoprotein structural constituents involved in premRNA pathways. Morphological observations revealed that the general aspect of embryo nuclear components is similar in the two experimental groups. However, immunocytochemical and in situ hybridization results suggest a temporary decrease of pre-mRNA transcription and splicing in 2-cell embryos and a resumption in 4-8-cell embryos from mice fed GM soybean; moreover, pre-mRNA maturation seems to be less efficient in both 2-cell and 4-8-cell embryos from GM-fed mice than in controls. Although our results are still preliminary and limited to the pre-implantation phases, the results of this study encourage deepening on the effects of food components and/or contaminants on embryo development.

Can a genetically-modified organism-containing diet influence embryo development? A preliminary study on pre-implantation mouse embryos.

Science.gov (United States)

Cisterna, B; Flach, F; Vecchio, L; Barabino, S M L; Battistelli, S; Martin, T E; Malatesta, M; Biggiogera, M

2008-01-01

In eukaryotic cells, pre-mRNAs undergo several transformation steps to generate mature mRNAs. Recent studies have demonstrated that a diet containing a genetically modified (GM) soybean can induce modifications of nuclear constituents involved in RNA processing in some tissues of young, adult and old mice. On this basis, we have investigated the ultrastructural and immunocytochemical features of pre-implantation embryos from mice fed either GM or non- GM soybean in order to verify whether the parental diet can affect the morpho-functional development of the embryonic ribonucleoprotein structural constituents involved in pre-mRNA pathways. Morphological observations revealed that the general aspect of embryo nuclear components is similar in the two experimental groups. However, immunocytochemical and in situ hybridization results suggest a temporary decrease of pre-mRNA transcription and splicing in 2-cell embryos and a resumption in 4-8-cell embryos from mice fed GM soybean; moreover, pre-mRNA maturation seems to be less efficient in both 2-cell and 4-8-cell embryos from GM-fed mice than in controls. Although our results are still preliminary and limited to the pre-implantation phases, the results of this study encourage deepening on the effects of food components and/or contaminants on embryo development.

Chromosomal mosaicism in human preimplantation embryos: a systematic review.

NARCIS (Netherlands)

Echten-Arends, J. van; Mastenbroek, S.; Sikkema-Raddatz, B.; Korevaar, J.C.; Heineman, M.J.; Veen, F. van der; Repping, S.

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

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

Inverted light-sheet microscope for imaging mouse pre-implantation development.

Science.gov (United States)

Strnad, Petr; Gunther, Stefan; Reichmann, Judith; Krzic, Uros; Balazs, Balint; de Medeiros, Gustavo; Norlin, Nils; Hiiragi, Takashi; Hufnagel, Lars; Ellenberg, Jan

2016-02-01

Despite its importance for understanding human infertility and congenital diseases, early mammalian development has remained inaccessible to in toto imaging. We developed an inverted light-sheet microscope that enabled us to image mouse embryos from zygote to blastocyst, computationally track all cells and reconstruct a complete lineage tree of mouse pre-implantation development. We used this unique data set to show that the first cell fate specification occurs at the 16-cell stage.

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.

The fate of paternal mitochondria in marmoset pre-implantation embryos.

Science.gov (United States)

Luetjens, C M; Wesselmann, R

2008-06-01

Sperm-derived mitochondria are integrated into the oocyte at fertilization but seem to vanish during the early cleavage phase. The developmental potential of pre-implantation embryos seems to be closely related to their ability to induce degeneration of these mitochondria, but the mechanisms underlying their loss of function are not yet understood. This study focuses on the fate of paternal mitochondria in pre-implantation embryos. Stimulation, collection and in vitro culture of oocytes from Callithrix jacchus, allows the study of the destiny of paternal mitochondria by utilizing immunostaining of pre-implantation embryos, fluorescence and laserscanning microscopy. Live pre-implantation embryos were stained with a fluorescence indicator reflecting mitochondrial membrane potential. Evidence indicating the loss of mitochondrial function was not found nor that apoptosis pathways were involved in the disappearance of paternally derived mitochondria. These findings may have implications for mitochondrially inherited diseases and could lead to new strategies for improving assisted reproduction.

Risk to preimplantation mouse embryos of combinations of heavy metals and radiation

International Nuclear Information System (INIS)

Mueller, W.-U.; Streffer, C.

1987-01-01

The influence of arsenic, cadmium, lead or mercury on radiation risk to preimplantation mouse embryos in vitro was studied under various conditions. Morphological development, cell proliferation, and formation of micronuclei were used for assessment of risk after combined exposure to these metals and X-rays. No conditions were found under which arsenic altered radiation risk; the effects were merely additive. Cadmium acted similarly, though a few results indicated that morphological development might be impaired more strongly after combined exposure than expected from the addition of the single effects. Lead enhanced radiation risk with regard to micronucleus formation, but had an additive effect only in the case of morphological development and cell proliferation. Of all four metals, mercury had the greatest potential for enhancement of radiation risk, when morphological development and cell proliferation were studied. The observed combination effects exceeded even those effects which were calculated by taking into account the shape of the dose-effect curves (isobologram analysis, envelope of additivity). Mercury neither induced micro-nuclei nor enhanced their formation in combination experiments. (author)

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.

[Traditional and modern approaches to culture of preimplantation mammalian embryos in vitro].

Science.gov (United States)

Brusentsev, E Iu; Igonina, T N; Amstislavskiĭ, S Ia

2014-01-01

This review covers the basic principles and methods of in vitro culture of preimplantation mammalian embryos. The features of in vitro development of embryos of various species of animals with allowance for the composition of nutrient media are described, with special attention paid to those species that have traditionally been consideredas laboratory (i.e., mice, rats, and hamsters). The effects of suboptimal culturing conditions of preimplantation embryos on the formation of the phenotype of individuals developed from these embryos are discussed. New approaches to optimize the conditions of the development of preimplantation mammalian embryos in vitro are analyzed.

Culture medium, gas atmosphere and MAPK inhibition affect regulation of RNA-binding protein targets during mouse preimplantation development.

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Calder, Michele D; Watson, Patricia H; Watson, Andrew J

2011-11-01

During oogenesis, mammalian oocytes accumulate maternal mRNAs that support the embryo until embryonic genome activation. RNA-binding proteins (RBP) may regulate the stability and turnover of maternal and embryonic mRNAs. We hypothesised that varying embryo culture conditions, such as culture medium, oxygen tension and MAPK inhibition, affects regulation of RBPs and their targets during preimplantation development. STAU1, ELAVL1, KHSRP and ZFP36 proteins and mRNAs were detected throughout mouse preimplantation development, whereas Elavl2 mRNA decreased after the two-cell stage. Potential target mRNAs of RBP regulation, Gclc, Slc2a1 and Slc7a1 were detected during mouse preimplantation development. Gclc mRNA was significantly elevated in embryos cultured in Whitten's medium compared with embryos cultured in KSOMaa, and Gclc mRNA was elevated under high-oxygen conditions. Inhibition of the p38 MAPK pathway reduced Slc7a1 mRNA expression while inhibition of ERK increased Slc2a1 mRNA expression. The half-lives of the potential RBP mRNA targets are not regulated in parallel; Slc2a1 mRNA displayed the longest half-life. Our results indicate that mRNAs and proteins encoding five RBPs are present during preimplantation development and more importantly, demonstrate that expression of RBP target mRNAs are regulated by culture medium, gas atmosphere and MAPK pathways.

Laser fusion of mouse embryonic cells and intra-embryonic fusion of blastomeres without affecting the embryo integrity.

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Krivokharchenko, Alexander; Karmenyan, Artashes; Sarkisov, Oleg; Bader, Michael; Chiou, Arthur; Shakhbazyan, Avetik

2012-01-01

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.

Killing of preimplantation mouse embryos by main ingredients of cleansers AS and LAS

International Nuclear Information System (INIS)

Nomura, T.; Hata, S.; Shibata, K.; Kusafuka, T.

1987-01-01

When main ingredients of cleansers, alcohol sulfate (AS) and linear alkylbenzene sulfonate (LAS), were applied to the dorsal skin of pregnant JCL:ICR mice during preimplantation period, significant numbers of embryos collected from the oviducts and uteri on day 3 showed severe deformity or remained at the morula stage. Most of abnormal embryos were fragmented or remained at the 1-8 cell stages, and they were either dead or dying. Similar results were observed with commercially obtained kitchen detergent and hair shampoo. Fertilized eggs may be specifically sensitive to synthetic detergents. Very low doses of X-rays also induced significant yields of abnormal embryos. Major difference between X-rays and detergents was that X-ray-induced abnormality appeared at the morula or blastocyst stage, while detergent-induced one did at the earlier stages. (Auth.)

In vitro culture of pre-implanted mouse embryos. A model system for studying combined effects

International Nuclear Information System (INIS)

Streffer, C.; Beuningen, D. van; Molls, M.; Pon, A.; Schulz, S.; Zamboglou, N.

1978-01-01

Studies on combined effects, e.g. interaction between chemical toxicants and ionizing radiation, are difficult to perform, as they are dependent on many factors (substance concentration, radiation dose, sequence of treatments, etc.). In order to obtain data from such studies it is necessary to establish a comparatively simple experimental model system. We have established such a model system by studying combined effects on pre-implanted mouse embryos cultured in vitro. This system has the following advantages: (1) The embryos can be cultivated for several days in vitro; (2) Their physiological intactness can be tested; and (3) Cell proliferation, cell killing and chromosomal damage can be investigated comparatively easily. The embryos are isolated at the 2-cell stage and incubated in a culture medium in vitro. The development of the embryos is followed under the microscope until the development of blastocysts or the hatching of blastocysts is observed. These blastocysts can be transplanted to fostered mice and the development of normal animals determined. The proliferation kinetics can be studied easily, and the methods are described. A method has also been developed to measure the DNA content of individual cells by microscope fluorometry. After treatment of the embryos with ionizing radiation or drugs the release of micronuclei has been observed from the cell nuclei, which is an expression for chromosomal damage. Substances or radionuclides can be added to the culture medium or external irradiation can be performed during the culture period. Also the combined effects of radiation and heating can be studied. The effects of X-rays and tritiated compounds have also been investigated. The combined effects of radiation with antibiotics such as actinomycin D, and environmental toxicants such as lead, have been determined. The system described has been useful to evaluate cytological, teratogenic and cytogenetic effects

Insulin and branched-chain amino acid depletion during mouse preimplantation embryo culture programmes body weight gain and raised blood pressure during early postnatal life.

Science.gov (United States)

Velazquez, Miguel A; Sheth, Bhavwanti; Smith, Stephanie J; Eckert, Judith J; Osmond, Clive; Fleming, Tom P

2018-02-01

Mouse maternal low protein diet exclusively during preimplantation development (Emb-LPD) is sufficient to programme altered growth and cardiovascular dysfunction in offspring. Here, we use an in vitro model comprising preimplantation culture in medium depleted in insulin and branched-chain amino acids (BCAA), two proposed embryo programming inductive factors from Emb-LPD studies, to examine the consequences for blastocyst organisation and, after embryo transfer (ET), postnatal disease origin. Two-cell embryos were cultured to blastocyst stage in defined KSOM medium supplemented with four combinations of insulin and BCAA concentrations. Control medium contained serum insulin and uterine luminal fluid amino acid concentrations (including BCAA) found in control mothers from the maternal diet model (N-insulin+N-bcaa). Experimental medium (three groups) contained 50% reduction in insulin and/or BCAA (L-insulin+N-bcaa, N-insulin+L-bcaa, and L-insulin+N-bcaa). Lineage-specific cell numbers of resultant blastocysts were not affected by treatment. Following ET, a combined depletion of insulin and BCAA during embryo culture induced a non sex-specific increase in birth weight and weight gain during early postnatal life. Furthermore, male offspring displayed relative hypertension and female offspring reduced heart/body weight, both characteristics of Emb-LPD offspring. Combined depletion of metabolites also resulted in a strong positive correlation between body weight and glucose metabolism that was absent in the control group. Our results support the notion that composition of preimplantation culture medium can programme development and associate with disease origin affecting postnatal growth and cardiovascular phenotypes and implicate two important nutritional mediators in the inductive mechanism. Our data also have implications for human assisted reproductive treatment (ART) practice. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Transcriptome analyses of rhesus monkey preimplantation embryos reveal a reduced capacity for DNA double-strand break repair in primate oocytes and early embryos

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Wang, Xinyi; Liu, Denghui; He, Dajian; Suo, Shengbao; Xia, Xian; He, Xiechao; Han, Jing-Dong J.; Zheng, Ping

2017-01-01

Preimplantation embryogenesis encompasses several critical events including genome reprogramming, zygotic genome activation (ZGA), and cell-fate commitment. The molecular basis of these processes remains obscure in primates in which there is a high rate of embryo wastage. Thus, understanding the factors involved in genome reprogramming and ZGA might help reproductive success during this susceptible period of early development and generate induced pluripotent stem cells with greater efficiency. Moreover, explaining the molecular basis responsible for embryo wastage in primates will greatly expand our knowledge of species evolution. By using RNA-seq in single and pooled oocytes and embryos, we defined the transcriptome throughout preimplantation development in rhesus monkey. In comparison to archival human and mouse data, we found that the transcriptome dynamics of monkey oocytes and embryos were very similar to those of human but very different from those of mouse. We identified several classes of maternal and zygotic genes, whose expression peaks were highly correlated with the time frames of genome reprogramming, ZGA, and cell-fate commitment, respectively. Importantly, comparison of the ZGA-related network modules among the three species revealed less robust surveillance of genomic instability in primate oocytes and embryos than in rodents, particularly in the pathways of DNA damage signaling and homology-directed DNA double-strand break repair. This study highlights the utility of monkey models to better understand the molecular basis for genome reprogramming, ZGA, and genomic stability surveillance in human early embryogenesis and may provide insights for improved homologous recombination-mediated gene editing in monkey. PMID:28223401

Preimplantation development of embryos in women of advanced maternal age

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O. V. Chaplia

2014-04-01

Full Text Available In order to reveal the influence of genetic component on the early embryo development, the retrospective study of morphokinetic characteristics of 717 embryos subjected to preimplantation genetic testing was conducted. Blastomere biopsy for FISH-based preimplantation genetic screening of 7 chromosomes was performed on the third day of culture, while embryo developmental potential and morphological features at the cleavage and blastulation stage were studied regarding maternal age particularly in the group of younger women and patients older than 36. Results of genetic testing revealed that euploid embryos rate gradually decreased with maternal age comprising 39.9% in young women group and 25.3% of specimen belonging to elder patients. At the cleavage stage, morphological characteristics of aneuploid and euploid embryos didn’t differ significantly regardless of the age of patients that could be accounted for the transcriptional silence of embryo genome till the third day of its development. However, in case of prolonged culture chromosomally balanced embryos rarely faced developmental arrest (in 7.9% and formed blastocysts half more frequently compared to aberrant embryos (respectively 75.6 versus 49.8%. Nevertheless, no substantial difference was found between blastocyst formation rate among embryos with similar genetic component regardless of the maternal age. Taking into consideration high rate of chromosomally unbalanced embryos specific to patients of advanced maternal age, the relative proportion of aneuplouid blastocysts was significantly higher in this group of embryos. Thus, without genetic screening there is a possibility of inaccurate selection of embryos for women of advanced reproductive age for transfer procedure even in case of prolonged culture. Consequently, increase of aneuploid embryos frequency associated with permanent preimplantation natural selection effectiveness along with the postimplantation natural selection failure

Effects of a combination of X-rays and caffeine on preimplantation mouse embryos in vitro

International Nuclear Information System (INIS)

Mueller, W.U.; Streffer, C.; Fischer-Lahdo, C.

1983-01-01

The influence of a combination of caffeine (0.1 mM, 1 mM, or 2 mM) and X-rays (0.24 Gy, 0.94 Gy, or 1.88 Gy) on preimplantation mouse embryos in vitro was studied under different conditions. The agents were applied either singly or in combination. The embryos were irradiated in the G 2 -phase of the two-cell stage (28 h p.c. or 32 h p.c.) either 1 h after or immediately before application of caffeine. Caffeine was present during the whole incubation period (until 144 h p.c.). The effects on the microscopic visible development (formation of blastocysts 96 h p.c., hatching of blastocysts 144 h p.c.) and on the cell numbers of embryos at different times (48 h p.c., 56 h p.c., 96 h p.c., 144 h p.c.) were determined. We found conditions under which caffeine markedly enhanced radiation risk, i.e., under which the combination effect exceeded the sum of the single effects. This is true, in particular, for the embryonal development, for which the risk may almost be doubled, whereas the enhancement of risk is not so great for the proliferation of cells. In some cases the combination results lie even outside the envelope of additivity in the range of supra-additivity. The amount of caffeine necessary for such marked effects, however, is so high (at least 1 mM caffeine for rather long times), that it is almost impossible to reach them in vivo by consumption of caffeine-containing beverages. (orig.)

Preimplantation diagnosis of repeated miscarriage due to chromosomal translocations using metaphase chromosomes of a blastomere biopsied from 4- to 6-cell-stage embryos.

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Tanaka, Atsushi; Nagayoshi, Motoi; Awata, Shoichiro; Mawatari, Yoshifumi; Tanaka, Izumi; Kusunoki, Hiroshi

2004-01-01

To evaluate the safety and accuracy of karyotyping the blastomere chromosomes at metaphase in the natural cell cycle for preimplantation diagnosis. A pilot study. A private infertility clinic and a university laboratory. Eleven patients undergoing IVF and preimplantation diagnosis. Intact human embryos at the 4- to 6-cell stage and human-mouse heterokaryons were cultured and checked hourly for disappearance of the nuclear envelope. After it disappeared, the metaphase chromosomes were analyzed by fluorescence in situ hybridization. Percentage of analyzable metaphase plates and safety and accuracy of the method. The success rate of electrofusion to form human-mouse heterokaryons was 87.1% (27/31), and analyzable chromosomes were obtained from 77.4% (24/31) of the heterokaryons. On the other hand, disappearance of the nuclear envelope occurred in 89.5% (17/19) of the human embryos and it began earlier than that in the heterokaryons. Analyzable chromosomes were obtained and their translocation sites were identified in all blastomeres biopsied from the 17 embryos. After the biopsy, 67.0% of the embryos could develop to the blastocyst stage. The natural cell cycle method reported herein requires frequent observation, but it is safe, with no artificial effects on the chromosomes and without loss of or damage to blastomeres, which occurred with the electrofusion method. Using the natural cell cycle method, we could perform preimplantation diagnosis with nearly 100% accuracy.

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

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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.

Automated microinjection of recombinant BCL-X into mouse zygotes enhances embryo development.

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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.

A medium-chain fatty acid as an alternative energy source in mouse preimplantation development.

Science.gov (United States)

Yamada, Mitsutoshi; Takanashi, Kazumi; Hamatani, Toshio; Hirayama, Akiyoshi; Akutsu, Hidenori; Fukunaga, Tomoko; Ogawa, Seiji; Sugawara, Kana; Shinoda, Kosaku; Soga, Tomoyoshi; Umezawa, Akihiro; Kuji, Naoaki; Yoshimura, Yasunori; Tomita, Masaru

2012-01-01

To further optimize the culturing of preimplantation embryos, we undertook metabolomic analysis of relevant culture media using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). We detected 28 metabolites: 23 embryo-excreted metabolites including 16 amino acids and 5 media-derived metabolites (e.g., octanoate, a medium-chain fatty acid (MCFA)). Due to the lack of information on MCFAs in mammalian preimplantation development, this study examined octanoate as a potential alternative energy source for preimplantation embryo cultures. No embryos survived in culture media lacking FAs, pyruvate, and glucose, but supplementation of octanoate rescued the embryonic development. Immunoblotting showed significant expression of acyl-CoA dehydrogenase and hydroxyacyl-CoA dehydrogenase, important enzymes for ß-oxidation of MCFAs, in preimplantation embryo. Furthermore, CE-TOFMS traced [1-(13)C(8)] octanoate added to the culture media into intermediate metabolites of the TCA cycle via ß-oxidation in mitochondria. These results are the first demonstration that octanoate could provide an efficient alternative energy source throughout preimplantation development.

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.

The impact of preimplantation genetic diagnosis on human embryos

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García-Ferreyra J.

2016-12-01

Full Text Available Chromosome abnormalities are extremely common in human oocytes and embryos and are associated with a variety of negative outcomes for both natural cycles and those using assisted reproduction techniques. Aneuploidies embryos may fail to implant in the uterus, miscarry, or lead to children with serious medical problems (e.g., Down syndrome. Preimplantation genetic diagnosis (PGD is a technique that allows the detection of aneuploidy in embryos and seeks to improve the clinical outcomes od assisted reproduction treatments, by ensuring that the embryos chosen for the transfer are chromosomally normal.

Factors affecting the gene expression of in vitro cultured human preimplantation embryos

NARCIS (Netherlands)

Mantikou, E.; Jonker, M. J.; Wong, K. M.; van Montfoort, A. P. A.; de Jong, M.; Breit, T. M.; Repping, S.; Mastenbroek, S.

2016-01-01

What is the relative effect of common environmental and biological factors on transcriptome changes during human preimplantation development? Developmental stage and maternal age had a larger effect on the global gene expression profile of human preimplantation embryos than the culture medium or

Toxicity of beauvericin on porcine oocyte maturation and preimplantation embryo development

NARCIS (Netherlands)

Schoevers, Eric J; Santos, Regiane R; Fink-Gremmels, Johanna; Roelen, Bernard A J

2016-01-01

Beauvericin (BEA) is one of many toxins produced by Fusarium species that contaminate feed materials. The aim of this study was to assess its effects on porcine oocyte maturation and preimplantation embryo development. Cumulus-oocyte-complexes and developing embryos were exposed to BEA and cultured

Histone variant H3.3-mediated chromatin remodeling is essential for paternal genome activation in mouse preimplantation embryos.

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Kong, Qingran; Banaszynski, Laura A; Geng, Fuqiang; Zhang, Xiaolei; Zhang, Jiaming; Zhang, Heng; O'Neill, Claire L; Yan, Peidong; Liu, Zhonghua; Shido, Koji; Palermo, Gianpiero D; Allis, C David; Rafii, Shahin; Rosenwaks, Zev; Wen, Duancheng

2018-03-09

Derepression of chromatin-mediated transcriptional repression of paternal and maternal genomes is considered the first major step that initiates zygotic gene expression after fertilization. The histone variant H3.3 is present in both male and female gametes and is thought to be important for remodeling the paternal and maternal genomes for activation during both fertilization and embryogenesis. However, the underlying mechanisms remain poorly understood. Using our H3.3B-HA-tagged mouse model, engineered to report H3.3 expression in live animals and to distinguish different sources of H3.3 protein in embryos, we show here that sperm-derived H3.3 (sH3.3) protein is removed from the sperm genome shortly after fertilization and extruded from the zygotes via the second polar bodies (PBII) during embryogenesis. We also found that the maternal H3.3 (mH3.3) protein is incorporated into the paternal genome as early as 2 h postfertilization and is detectable in the paternal genome until the morula stage. Knockdown of maternal H3.3 resulted in compromised embryonic development both of fertilized embryos and of androgenetic haploid embryos. Furthermore, we report that mH3.3 depletion in oocytes impairs both activation of the Oct4 pluripotency marker gene and global de novo transcription from the paternal genome important for early embryonic development. Our results suggest that H3.3-mediated paternal chromatin remodeling is essential for the development of preimplantation embryos and the activation of the paternal genome during embryogenesis. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Inherited effects from irradiated mouse immature oocytes detected in aggregation embryo chimeras

International Nuclear Information System (INIS)

Straume, T.; Raabe, O.G.; Walsh, K.J.; Wiley, L.M.

1993-01-01

Data obtained using the mouse-preimplantation-embryo-chimera assay are presented that show a transmitted effect following low-dose irradiation of immature oocytes in vivo. Six-week-old female mice were irradiated using 137 Cs-γ-rays (0.05 Gy, 0.15 Gy, and unexposed controls). At 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 weeks post exposure, the mice were mated and aggregation chimeras made from the 4-cell embryos. Three independent experiments have now been carried out, all showing a significant embryonic cell-proliferation disadvantage of the embryos obtained from the females treated 7 weeks previously, i.e., embryos from oocytes that were immature at the time of radiation exposure. No effect was detected at 1-6 weeks when embryos were obtained from maturing oocytes. Also, the effect was not seen at 8, 9, 10, 11, and 12 weeks post exposure. The implications of these results are discussed in the light of previous studies on mouse oocytes

Quantitative imaging of lipids in live mouse oocytes and early embryos using CARS microscopy

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Bradley, Josephine; Pope, Iestyn; Masia, Francesco; Sanusi, Randa; Langbein, Wolfgang; Borri, Paola

2016-01-01

Mammalian oocytes contain lipid droplets that are a store of fatty acids, whose metabolism plays a substantial role in pre-implantation development. Fluorescent staining has previously been used to image lipid droplets in mammalian oocytes and embryos, but this method is not quantitative and often incompatible with live cell imaging and subsequent development. Here we have applied chemically specific, label-free coherent anti-Stokes Raman scattering (CARS) microscopy to mouse oocytes and pre-implantation embryos. We show that CARS imaging can quantify the size, number and spatial distribution of lipid droplets in living mouse oocytes and embryos up to the blastocyst stage. Notably, it can be used in a way that does not compromise oocyte maturation or embryo development. We have also correlated CARS with two-photon fluorescence microscopy simultaneously acquired using fluorescent lipid probes on fixed samples, and found only a partial degree of correlation, depending on the lipid probe, clearly exemplifying the limitation of lipid labelling. In addition, we show that differences in the chemical composition of lipid droplets in living oocytes matured in media supplemented with different saturated and unsaturated fatty acids can be detected using CARS hyperspectral imaging. These results demonstrate that CARS microscopy provides a novel non-invasive method of quantifying lipid content, type and spatial distribution with sub-micron resolution in living mammalian oocytes and embryos. PMID:27151947

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

African Journals Online (AJOL)

Yomi

2012-03-06

Mar 6, 2012 ... with pregnancy follow-up to October 2008. Hum. Reprod. 25(11):. 2685-2707. Harper JC, Sengupta SB (2012) Preimplantation genetic diagnosis: State of the ART 2011. Hum. Genet. 131(2): 175-186. Hasler JF (2003). The current status and future of commercial embryo transfer in cattle. Anim. Reprod. Sci.

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

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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

Preimplantation Genetic Screening and Preimplantation Genetic Diagnosis.

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Sullivan-Pyke, Chantae; Dokras, Anuja

2018-03-01

Preimplantation genetic testing encompasses preimplantation genetic screening (PGS) and preimplantation genetic diagnosis (PGD). PGS improves success rates of in vitro fertilization by ensuring the transfer of euploid embryos that have a higher chance of implantation and resulting in a live birth. PGD enables the identification of embryos with specific disease-causing mutations and transfer of unaffected embryos. The development of whole genome amplification and genomic tools, including single nucleotide polymorphism microarrays, comparative genomic hybridization microarrays, and next-generation sequencing, has led to faster, more accurate diagnoses that translate to improved pregnancy and live birth rates. Copyright © 2017 Elsevier Inc. All rights reserved.

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

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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.

Assay using embryo aggregation chimeras for the detection of nonlethal changes in X-irradiated mouse preimplantation embryos

International Nuclear Information System (INIS)

Obasaju, M.F.; Wiley, L.M.; Oudiz, D.J.; Miller, L.; Samuels, S.J.; Chang, R.J.; Overstreet, J.W.

1988-01-01

We have developed a short-term in vitro assay for the detection of sublethal effects produced by very low levels of ionizing radiation. The assay utilizes mouse embryo aggregation chimeras consisting of one irradiated embryo paired with an unirradiated embryo whose blastomeres have been labeled with fluorescein isothiocyanate (FITC). X irradiation (from 0.05 to 2 Gy) and chimera construction were performed with four-cell stage embryos, and the chimeras were cultured for 40 h to the morula stage. The morulae were partially dissociated with calcium-free culture medium and viewed under phase contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution of irradiated (unlabeled) and control (FITC labeled) embryos per chimera. In chimeras where neither embryo was irradiated, the ratio of the unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.50 (17.8 +/- 5.6 cells per unlabeled embryo and 17.4 +/- 5.5 cells per FITC-labeled partner embryo). However, in chimeras formed after the unlabeled embryos were irradiated with as little as 0.05 Gy, the ratio of unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.43 (P less than 0.01). The apparent decreases in cell proliferation were not observed in irradiated embryos that were merely cocultured with control embryos, regardless of whether the embryos were zona enclosed or zona free. We conclude that very low levels of radiation induce sublethal changes in cleaving embryos that are expressed as a proliferative disadvantage within two cell cycles when irradiated embryos are in direct cell-to-cell contact with unirradiated embryos

Perturbation of the Developmental Potential of Preimplantation Mouse Embryos by Hydroxyurea

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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

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

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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

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

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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.

Preimplantation maternal stress impairs embryo development by inducing oviductal apoptosis with activation of the Fas system.

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Zheng, Liang-Liang; Tan, Xiu-Wen; Cui, Xiang-Zhong; Yuan, Hong-Jie; Li, Hong; Jiao, Guang-Zhong; Ji, Chang-Li; Tan, Jing-He

2016-11-01

What are the mechanisms by which the preimplantation restraint stress (PIRS) impairs embryo development and pregnancy outcome? PIRS impairs embryo development by triggering apoptosis in mouse oviducts and embryos,and this involves activation of the Fas system. Although it is known that the early stages of pregnancy are more vulnerable than later stages to prenatalstress, studies on the effect of preimplantation stress on embryo developmentare limited. Furthermore, the mechanisms by which psychological stress impairs embryo development are largely unknown. These issues are worth exploring using the mouse PIRS models because restraint of mice is an efficient experimental procedure developed for studies of psychogenic stress. Mice of Kunming strain, the generalized lymphoproliferative disorder (gld) mice with a germline mutation F273L in FasL in a C57BL/6J genomic background and the wild-type C57BL/6J mice were used. Female and male mice were used 8-10 weeks and 10-12 weeks after birth, respectively. Female mice showing vaginal plugs were paired by weight and randomly assigned to restraint treatments or as controls. For restraint treatment, an individual mouse was put in a micro-cage with food and water available. Control mice remained in their cages with food and water during the time treated females were stressed. Female mice were exposed to PIRS for 48 h starting from 16:00 on the day of vaginal plug detection. At the end of PIRS, levels of glucorticoids (GC), corticotropin-releasing hormone (CRH)and redox potential were measured in serum, while levels of GC, GC receptor (GR), CRH, CRH receptor (CRHR), Fas and Fas ligand (FasL) protein, mRNAs for brain derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1), oxidative stress (OS) and apoptosis were examined in oviducts. Preimplantation development and levels of GR, Fas, redox potential and apoptosis were observed in embryos recovered at different times after the initiation of PIRS. The gld mice

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

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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.

Mouse Embryo Compaction.

Science.gov (United States)

White, M D; Bissiere, S; Alvarez, Y D; Plachta, N

2016-01-01

Compaction is a critical first morphological event in the preimplantation development of the mammalian embryo. Characterized by the transformation of the embryo from a loose cluster of spherical cells into a tightly packed mass, compaction is a key step in the establishment of the first tissue-like structures of the embryo. Although early investigation of the mechanisms driving compaction implicated changes in cell-cell adhesion, recent work has identified essential roles for cortical tension and a compaction-specific class of filopodia. During the transition from 8 to 16 cells, as the embryo is compacting, it must also make fundamental decisions regarding cell position, polarity, and fate. Understanding how these and other processes are integrated with compaction requires further investigation. Emerging imaging-based techniques that enable quantitative analysis from the level of cell-cell interactions down to the level of individual regulatory molecules will provide a greater understanding of how compaction shapes the early mammalian embryo. © 2016 Elsevier Inc. All rights reserved.

Effect of Bacterial Endotoxins on Superovulated Mouse Embryos In Vivo: Is CSF-1 Involved in Endotoxin-Induced Pregnancy Loss?

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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.

Pertussis toxin-catalyzed ADP-ribosylation of a G protein in mouse oocytes, eggs, and preimplantation embryos: Developmental changes and possible functional roles

Energy Technology Data Exchange (ETDEWEB)

Jones, J.; Schultz, R.M. (Univ. of Pennsylvania, Philadelphia (USA))

1990-06-01

G proteins, which in many somatic cells serve as mediators of signal transduction, were identified in preimplantation mouse embryos by their capacity to undergo pertussis toxin-catalyzed ADP-ribosylation. Two pertussis toxin (PT) substrates with Mr = 38,000 and 39,000 (alpha 38 and alpha 39) are present in approximately equal amounts. Relative to the amount in freshly isolated germinal vesicle (GV)-intact oocytes, the amount of PT-catalyzed ADP-ribosylation of alpha 38-39 falls during oocyte maturation, rises between the one- and two-cell stages, falls by the eight-cell and morula stages, and increases again by the blastocyst stage. The decrease in PT-catalyzed ADP-ribosylation of alpha 38-39 that occurs during oocyte maturation, however, does not require germinal vesicle breakdown (GVBD), since inhibiting GVBD with 3-isobutyl-1-methyl xanthine (IBMX) does not prevent the decrease in the extent of PT-catalyzed ADP-ribosylation. A biologically active phorbol diester (12-O-tetradecanoyl phorbol 13-acetate), but not an inactive one (4 alpha-phorbol 12,13-didecanoate, 4 alpha-PDD), totally inhibits the increase in PT-catalyzed ADP-ribosylation of alpha 38-39 that occurs between the one- and two-cell stage; TPA inhibits cleavage, but not transcriptional activation, which occurs in the two-cell embryo. In contrast, cytochalasin D, genistein, or aphidicolin, each of which inhibits cleavage of one-cell embryos, or alpha-amanitin or H8, each of which inhibits transcriptional activation but not cleavage of one-cell embryos, have little or inhibitory effects on the increase in PT-catalyzed ADP-ribosylation of alpha 38-39. Results of immunoblotting experiments using an antibody that is highly specific for alpha il-3 reveal the presence of a cross-reactive species of Mr = 38,000 (alpha 38) in the GV-intact oocyte, metaphase II-arrested egg, and one-, two-cell embryos.

Ultrastructural and autoradiographic studies of nucleolar development and rDNA transcription in preimplantation mouse embryos

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Geuskens, M.; Alexandre, H. (Universite Libre de Bruxelles (Belgium). Dep. de Biologie Moleculaire)

1984-06-01

The development of the nucleoli and the sites of rDNA transcription have been studies by high-resolution autoradiography during the cleavage stages of mouse embryos. The appearance of fibrillar centres at the periphery of the fibrillar primary nucleoli has been observed at the 4-cell stage. Several fibrillar centres interconnected by electron-dense fibrillar strands, form a reticulated region around the fibrillar mass at the 6- to 8-cell stage. After a 10 min pulse with (/sup 3/H)uridine, only this peripheral network is labelled. At the late morula and at the blastocyst stage, the fibrillar component (nucleolonema) of the reticulated nucleoli is labelled after 10 min (/sup 3/H)uridine incorporation. When the embryos are reincubated for 2 h in cold medium, the label is localized mainly in the granular component. Fibrillar centres are not labelled. Autoradiograms of in vitro developed embryos pulsed for 2 h with (/sup 3/H)uridine confirm that the central fibrillar core of the nucleoli of 6- to 8-cell embryos is never labelled. Thus, the fibrillar constituent of this core is not homologous to the fibrillar component of the nucleoli of later stage embryos, which is the site of active rDNA transcription. An interpretation of nucleologenesis during early mouse embryogenesis is proposed.

Ultrastructural and autoradiographic studies of nucleolar development and rDNA transcription in preimplantation mouse embryos

International Nuclear Information System (INIS)

Geuskens, M.; Alexandre, H.

1984-01-01

The development of the nucleoli and the sites of rDNA transcription have been studies by high-resolution autoradiography during the cleavage stages of mouse embryos. The appearance of fibrillar centres at the periphery of the fibrillar primary nucleoli has been observed at the 4-cell stage. Several fibrillar centres interconnected by electron-dense fibrillar strands, form a reticulated region around the fibrillar mass at the 6- to 8-cell stage. After a 10 min pulse with ( 3 H)uridine, only this peripheral network is labelled. At the late morula and at the blastocyst stage, the fibrillar component (nucleolonema) of the reticulated nucleoli is labelled after 10 min ( 3 H)uridine incorporation. When the embryos are reincubated for 2 h in cold medium, the label is localized mainly in the granular component. Fibrillar centres are not labelled. Autoradiograms of in vitro developed embryos pulsed for 2 h with ( 3 H)uridine confirm that the central fibrillar core of the nucleoli of 6- to 8-cell embryos is never labelled. Thus, the fibrillar constituent of this core is not homologous to the fibrillar component of the nucleoli of later stage embryos, which is the site of active rDNA transcription. An interpretation of nucleologenesis during early mouse embryogenesis is proposed. (author)

Saviour embryos? Preimplantation genetic diagnosis as a therapeutic technology.

Science.gov (United States)

Sparrow, Robert; Cram, David

2010-05-01

The creation of 'saviour siblings' is one of the most controversial uses of preimplantation genetic diagnosis (PGD). This paper outlines and invites ethical discussion of an extension of this technology, namely, the creation of 'saviour embryos' to serve as a source of stem cells to be used in potentially life-saving therapy for an existing child. A number of analogies between this hypothetical use of PGD and existing uses of IVF are offered and, in addition, between saviour embryos and proposed therapeutic applications of stem cell technology. The ethical significance of a number of disanalogies between these cases are explored and investigated. While the creation of saviour embryos would involve a significant shift in the rationale for IVF and PGD, it is suggested here that the urgent need of an existing individual should be prioritised over any obligations that might exist in relation to the creation or destruction of human embryos. Copyright (c) 2009 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

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.

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

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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.

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.

[Current options of preimplantion genetic screening and preimplantation genetic diagnostics].

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Šimečková, V

The aim of this work is to summarize the current knowledge about preimplantation genetic screening and diagnostics. A review article. Department of Gynecology and Obstetrics, District Hospital Šternberk, IVF Clinic, Olomouc. Preimplantation genetic testing is a complex of genetic and molecular cytogenetic examinations, which can help to detect abnormalities in embryos before transfer into the uterus of the mother. These specialized examinations are based on the latest findings in genetics and assisted reproduction. The preimplantation genetic testing is necessarily associated with a method of in vitro fertilization. It is performed on isolated blastomeres on the third day of embryo cultivation. Nowadays, it is preferred trophectoderm examination of cells from the five-day blastocysts. Generally speaking, after preimplantation genetic testing, we can select only embryos without genetic load to transfer into uterus. Preimplantation genetic testing is an important part of treatment of infertility. Complex diagnostics and treatment of infertile couples are increasingly influenced by the development and use of advanced genomic technologies. Further development and application of these modern methods require close cooperation between the field of assisted reproduction and clinical genetics.

Mouse immature oocytes irradiated in vivo at 14-days of age and evaluated for transmitted effects using the aggregation embryo chimera assay

International Nuclear Information System (INIS)

Straume, T.; Raabe, O.G.; Walsh, K.J.; Wiley, L.M.

1996-01-01

A previous study using the mouse-preimplantation-embryo-chimera assay demonstrated a reproducible transmitted effect (proliferation disadvantage observed in early embryos) from females irradiated as 49-day-old adults using 0.15 Gy of gamma rays and then mated seven weeks later, i.e., embryos were from oocytes that were immature at time of irradiation. Because mouse immature oocytes are known to be much more radiosensitive to cell killing in juveniles than in adults, a follow-on study was performed here using 14-day-old juvenile mice. In contrast to adults, the exposure of juveniles to 0.15 Gy of gamma rays did not result in a detectable transmitted proliferation disadvantage when animals were mated 7 or 12 weeks later. This observation is discussed in light of previous studies on mouse immature oocytes and embryo chimeras

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

Expression of the CTCF gene in bovine oocytes and preimplantation embryos

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Álvaro F.L. Rios

2007-01-01

Full Text Available The CCCTC - binding factor (CTCF is a protein involved in repression, activation, hormone-inducible gene silencing, functional reading of imprinted genes and X-chromosome inactivation. We analyzed CTCF gene expression in bovine peripheral blood, oocytes and in different cellular stages (2-4 cells, 8-16 cells, 16-32 cells, morulae, and blastocysts of in vitro fertilized embryos. This is the first report of CTCF expression in oocytes and preimplantation bovine embryos and has implications for the production of embryonic stem cells and the development of novel medical technologies for humans.

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

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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.

Preimplantation Genetic Diagnosis: Prenatal Testing for Embryos Finally Achieving Its Potential

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Harvey J. Stern

2014-03-01

Full Text Available Preimplantation genetic diagnosis was developed nearly a quarter-century ago as an alternative form of prenatal diagnosis that is carried out on embryos. Initially offered for diagnosis in couples at-risk for single gene genetic disorders, such as cystic fibrosis, spinal muscular atrophy and Huntington disease, preimplantation genetic diagnosis (PGD has most frequently been employed in assisted reproduction for detection of chromosome aneuploidy from advancing maternal age or structural chromosome rearrangements. Major improvements have been seen in PGD analysis with movement away from older, less effective technologies, such as fluorescence in situ hybridization (FISH, to newer molecular tools, such as DNA microarrays and next generation sequencing. Improved results have also started to be seen with decreasing use of Day 3 blastomere biopsy in favor of polar body or Day 5 trophectoderm biopsy. Discussions regarding the scientific, ethical, legal and social issues surrounding the use of sequence data from embryo biopsy have begun and must continue to avoid concern regarding eugenic or inappropriate use of this technology.

Preimplantation Genetic Diagnosis: Prenatal Testing for Embryos Finally Achieving Its Potential

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Stern, Harvey J.

2014-01-01

Preimplantation genetic diagnosis was developed nearly a quarter-century ago as an alternative form of prenatal diagnosis that is carried out on embryos. Initially offered for diagnosis in couples at-risk for single gene genetic disorders, such as cystic fibrosis, spinal muscular atrophy and Huntington disease, preimplantation genetic diagnosis (PGD) has most frequently been employed in assisted reproduction for detection of chromosome aneuploidy from advancing maternal age or structural chromosome rearrangements. Major improvements have been seen in PGD analysis with movement away from older, less effective technologies, such as fluorescence in situ hybridization (FISH), to newer molecular tools, such as DNA microarrays and next generation sequencing. Improved results have also started to be seen with decreasing use of Day 3 blastomere biopsy in favor of polar body or Day 5 trophectoderm biopsy. Discussions regarding the scientific, ethical, legal and social issues surrounding the use of sequence data from embryo biopsy have begun and must continue to avoid concern regarding eugenic or inappropriate use of this technology. PMID:26237262

Preimplantation genetic screening.

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Harper, Joyce C

2018-03-01

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.

Pre implanted mouse embryos as model for uranium toxicology studies

International Nuclear Information System (INIS)

Kundt, Miriam S.

2001-01-01

Full text: The search of 'in vitro' toxicology model that can predict toxicology effects 'in vivo' is a permanent challenge. A toxicology experimental model must to fill to certain requirements: to have a predictive character, an appropriate control to facilitate the interpretation of the data among the experimental groups, and to be able to control the independent variables that can interfere or modify the results that we are analyzing. The preimplantation embryos posses many advantages in this respect: they are a simple model that begins with the development of only one cell. The 'in vitro' model reproduces successfully the 'in vivo' situation. Due to the similarity that exists among the embryos of mammals during this period the model is practically valid for other species. The embryo is itself a stem cell, the toxicology effects are early observed in his clonal development and the physical-chemical parameters are easily controllable. The purpose of the exhibition is to explain the properties of the pre implanted embryo model for toxicology studies of uranium and to show our experimental results. The cultivation 'in vitro' of mouse embryos with uranylo nitrate demonstrated that the uranium causes from the 13 μgU/ml delay of development, decrease the number of cells per embryo and hipoploidy in the embryonic blastomere. (author)

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

DEFF Research Database (Denmark)

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...... microscopy in both treated and untreated blastocysts. Activation of caspase-3 is likely involved in both spontaneous and induced apoptosis in bovine pre-implantation embryos, and immunohistochemical staining of active caspase-3 may be used in combination with other markers to identify apoptosis in pre...... 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...

Transcriptome profiling of human pre-implantation development.

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Pu Zhang

Full Text Available BACKGROUND: Preimplantation development is a crucial step in early human development. However, the molecular basis of human preimplantation development is not well known. METHODOLOGY: By applying microarray on 397 human oocytes and embryos at six developmental stages, we studied the transcription dynamics during human preimplantation development. PRINCIPAL FINDINGS: We found that the preimplantation development consisted of two main transitions: from metaphase-II oocyte to 4-cell embryo where mainly the maternal genes were expressed, and from 8-cell embryo to blastocyst with down-regulation of the maternal genes and up-regulation of embryonic genes. Human preimplantation development proved relatively autonomous. Genes predominantly expressed in oocytes and embryos are well conserved during evolution. SIGNIFICANCE: Our database and findings provide fundamental resources for understanding

Endometrial signals improve embryo outcome: functional role of vascular endothelial growth factor isoforms on embryo development and implantation in mice.

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Binder, N K; Evans, J; Gardner, D K; Salamonsen, L A; Hannan, N J

2014-10-10

Does vascular endothelial growth factor (VEGF) have important roles during early embryo development and implantation? VEGF plays key roles during mouse preimplantation embryo development, with beneficial effects on time to cavitation, blastocyst cell number and outgrowth, as well as implantation rate and fetal limb development. Embryo implantation requires synchronized dialog between maternal cells and those of the conceptus. Following ovulation, secretions from endometrial glands increase and accumulate in the uterine lumen. These secretions contain important mediators that support the conceptus during the peri-implantation phase. Previously, we demonstrated a significant reduction of VEGFA in the uterine cavity of women with unexplained infertility. Functional studies demonstrated that VEGF significantly enhanced endometrial epithelial cell adhesive properties and embryo outgrowth. Human endometrial lavages (n = 6) were obtained from women of proven fertility. Four-week old Swiss mice were superovulated and mated with Swiss males to obtain embryos for treatment with VEGF in vitro. Preimplantation embryo development was assessed prior to embryo transfer (n = 19-30/treatment group/output). Recipient F1 female mice (8-12 weeks of age) were mated with vasectomized males to induce pseudopregnancy and embryos were transferred. On Day 14.5 of pregnancy, uterine horns were collected for analysis of implantation rates as well as placental and fetal development (n = 14-19/treatment). Lavage fluid was assessed by western immunoblot analysis to determine the VEGF isoforms present. Mouse embryos were treated with either recombinant human (rh)VEGF, or VEGF isoforms 121 and 165. Preimplantation embryo development was quantified using time-lapse microscopy. Blastocysts were (i) stained for cell number, (ii) transferred to wells coated with fibronectin to examine trophoblast outgrowth or (iii) transferred to pseudo pregnant recipients to analyze implantation rates, placental and

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

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

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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.

Synthetic profiles of polypeptides of human oocytes and normal and abnormal preimplantation embryos.

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Capmany, G; Bolton, V N

1999-09-01

There is considerable variation in the rate of development in vitro of individual preimplantation human embryos. The relationship between the rate of development and patterns of polypeptide synthesis in individual embryos was examined using SDS-PAGE and autoradiography. After incubation in [35S]methionine, 19 polypeptide bands were identified that change between fertilization and the morula stage. Although changes in two of the bands occurred in embryos that were developing normally and in ageing oocytes, and are thus independent of fertilization, the changes identified in the remaining 17 bands occurred only after fertilization. In embryos that were developing abnormally, as assessed by delayed cleavage, cleavage arrest or extensive fragmentation, the alteration in polypeptide synthetic profiles increased with increasing abnormality.

Automation and Optimization of Multipulse Laser Zona Drilling of Mouse Embryos During Embryo Biopsy.

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Wong, Christopher Yee; Mills, James K

2017-03-01

Laser zona drilling (LZD) is a required step in many embryonic surgical procedures, for example, assisted hatching and preimplantation genetic diagnosis. LZD involves the ablation of the zona pellucida (ZP) using a laser while minimizing potentially harmful thermal effects on critical internal cell structures. Develop a method for the automation and optimization of multipulse LZD, applied to cleavage-stage embryos. A two-stage optimization is used. The first stage uses computer vision algorithms to identify embryonic structures and determines the optimal ablation zone farthest away from critical structures such as blastomeres. The second stage combines a genetic algorithm with a previously reported thermal analysis of LZD to optimize the combination of laser pulse locations and pulse durations. The goal is to minimize the peak temperature experienced by the blastomeres while creating the desired opening in the ZP. A proof of concept of the proposed LZD automation and optimization method is demonstrated through experiments on mouse embryos with positive results, as adequately sized openings are created. Automation of LZD is feasible and is a viable step toward the automation of embryo biopsy procedures. LZD is a common but delicate procedure performed by human operators using subjective methods to gauge proper LZD procedure. Automation of LZD removes human error to increase the success rate of LZD. Although the proposed methods are developed for cleavage-stage embryos, the same methods may be applied to most types LZD procedures, embryos at different developmental stages, or nonembryonic cells.

Insulin-like growth factor-1 protects preimplantation embryos from anti-developmental actions of menadione.

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Moss, James I; Pontes, Eduardo; Hansen, Peter James

2009-11-01

Menadione is a naphthoquinone used as a vitamin K source in animal feed that can generate reactive oxygen species (ROS) and cause apoptosis. Here, we examined whether menadione reduces development of preimplantation bovine embryos in a ROS-dependent process and tested the hypothesis that actions of menadione would be reduced by insulin-like growth factor-1 (IGF-1). Menadione caused a concentration-dependent decrease in the proportion of embryos that became blastocysts. All concentrations tested (1, 2.5, and 5.0 microM) inhibited development. Treatment with 100 ng/ml IGF-1 reduced the magnitude of the anti-developmental effects of the two lowest menadione concentrations. Menadione also caused a concentration-dependent increase in the percent of cells positive for the TUNEL reaction. The response was lower for IGF-1-treated embryos. The effects of menadione were mediated by ROS because (1) the anti-developmental effect of menadione was blocked by the antioxidants dithiothreitol and Trolox and (2) menadione caused an increase in ROS generation. Treatment with IGF-1 did not reduce ROS formation in menadione-treated embryos. In conclusion, concentrations of menadione as low as 1.0 muM can compromise development of bovine preimplantation embryos to the blastocyst stage of development in a ROS-dependent mechanism. Anti-developmental actions of menadione can be blocked by IGF-1 through effects downstream of ROS generation.

A Simple Method for Transportation of Mouse Embryos Using Microtubes and a Warm Box.

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Mikiko Tokoro

Full Text Available Generally, transportation of preimplantation embryos without freezing requires incubators that can maintain an optimal culture environment with a suitable gas phase, temperature, and humidity. Such incubators are expensive to transport. We reported previously that normal offspring were obtained when the gas phase and temperature could be maintained during transportation. However, that system used plastic dishes for embryo culture and is unsuitable for long-distance transport of live embryos. Here, we developed a simple low-cost embryo transportation system. Instead of plastic dishes, several types of microtubes-usually used for molecular analysis-were tested for embryo culture. When they were washed and attached to a gas-permeable film, the rate of embryo development from the 1-cell to blastocyst stage was more than 90%. The quality of these blastocysts and the rate of full-term development after embryo transfer to recipient female mice were similar to those of a dish-cultured control group. Next, we developed a small warm box powered by a battery instead of mains power, which could maintain an optimal temperature for embryo development during transport. When 1-cell embryos derived from BDF1, C57BL/6, C3H/He and ICR mouse strains were transported by a parcel-delivery service over 3 days using microtubes and the box, they developed to blastocysts with rates similar to controls. After the embryos had been transferred into recipient female mice, healthy offspring were obtained without any losses except for the C3H/He strain. Thus, transport of mouse embryos is possible using this very simple method, which might prove useful in the field of reproductive medicine.

Transcriptomic changes in the pre-implantation uterus highlight histotrophic nutrition of the developing marsupial embryo.

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Whittington, Camilla M; O'Meally, Denis; Laird, Melanie K; Belov, Katherine; Thompson, Michael B; McAllan, Bronwyn M

2018-02-05

Early pregnancy is a critical time for successful reproduction; up to half of human pregnancies fail before the development of the definitive chorioallantoic placenta. Unlike the situation in eutherian mammals, marsupial pregnancy is characterised by a long pre-implantation period prior to the development of the short-lived placenta, making them ideal models for study of the uterine environment promoting embryonic survival pre-implantation. Here we present a transcriptomic study of pre-implantation marsupial pregnancy, and identify differentially expressed genes in the Sminthopsis crassicaudata uterus involved in metabolism and biosynthesis, transport, immunity, tissue remodelling, and uterine receptivity. Interestingly, almost one quarter of the top 50 genes that are differentially upregulated in early pregnancy are putatively involved in histotrophy, highlighting the importance of nutrient transport to the conceptus prior to the development of the placenta. This work furthers our understanding of the mechanisms underlying survival of pre-implantation embryos in the earliest live bearing ancestors of mammals.

Microdrop preparation factors influence culture-media osmolality, which can impair mouse embryo preimplantation development.

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Swain, J E; Cabrera, L; Xu, X; Smith, G D

2012-02-01

Because media osmolality can impact embryo development, the effect of conditions during microdrop preparation on osmolality was examined. Various sizes of microdrops were prepared under different laboratory conditions. Drops were pipetted directly onto a dish and covered by oil (standard method) or pipetted on the dish, overlaid with oil before removing the underlying media and replaced with fresh media (wash-drop method). Drops were made at 23°C or on a heated stage (37°C) and with or without airflow. Osmolality was assessed at 5 min and 24h. The biological impact of osmolality change was demonstrated by culturing 1-cell mouse embryos in media with varying osmolality. Reduced drop volume, increased temperature and standard method were associated with a significant increase in osmolality at both 5 min and 24h (P-values media with elevated osmolality (>310mOsm/kg; P<0.05). Procedures in the IVF laboratory can alter osmolality and impact embryo development. Copyright © 2011 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

The environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin disrupts morphogenesis of the rat pre-implantation embryo

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Albertini David F

2008-01-01

Full Text Available Abstract Background Environmental toxicants, whose actions are often mediated through the aryl hydrocarbon receptor (AhR pathway, pose risks to the health and well-being of exposed species, including humans. Of particular concern are exposures during the earliest stages of development that while failing to abrogate embryogenesis, may have long term effects on newborns or adults. The purpose of this study was to evaluate the effect of maternal exposure to the AhR-specific ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD on the development of rat pre-implantation embryos with respect to nuclear and cytoskeletal architecture and cell lineage allocation. Results We performed a systematic 3 dimensional (3D confocal microscopy analysis of rat pre-implantation embryos following maternal exposure to environmentally relevant doses of TCDD. Both chronic (50 ng/kg/wk for 3 months and acute (50 ng/kg and 1 μg/kg at proestrus maternal TCDD exposure disrupted morphogenesis at the compaction stage (8–16 cell, with defects including monopolar spindle formation, f-actin capping and fragmentation due to aberrant cytokinesis. Additionally, the size, shape and position of nuclei were modified in compaction stage pre-implantation embryos collected from treated animals. Notably, maternal TCDD exposure did not compromise survival to blastocyst, which with the exception of nuclear shape, were morphologically similar to control blastocysts. Conclusion We have identified the compaction stage of pre-implantation embryogenesis as critically sensitive to the effects of TCDD, while survival to the blastocyst stage is not compromised. To the best of our knowledge this is the first in vivo study to demonstrate a critical window of pre-implantation mammalian development that is vulnerable to disruption by an AhR ligand at environmentally relevant doses.

Detrimental effects of microgravity on mouse preimplantation development in vitro.

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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.

Use of alpha-amanitin as a transcriptional blocking agent in mouse embryos: a cautionary note

International Nuclear Information System (INIS)

Kidder, G.M.; Green, A.F.; McLachlin, J.R.

1985-01-01

We have tested the effect of alpha-amanitin at 10, 50 and 100 micrograms/ml, on precursor uptake and incorporation into poly(A)+ RNA and poly(A)- RNA of mouse embryos on days 2, 3 and 4 of gestation. Embryos were pretreated with the inhibitor for 2 hr, then labeled for 2 hr in its continued presence. RNA fractions were separated by affinity chromatography on oligo(dT)-cellulose. alpha-Amanitin did not suppress uptake of RNA precursors at any of the concentrations tested in any stage. At 10 micrograms/ml, we could not detect any effect on incorporation into either RNA fraction in any stage. Only the highest concentration tested, 100 micrograms/ml, was effective in all stages in substantially suppressing incorporation into poly(A)+ RNA within 2 hr. Longer treatments increased the level of suppression to a maximum of about 80%. Incorporation into poly(A)- RNA was suppressed to roughly the same extent. Despite previously reported data, it cannot be assumed that alpha-amanitin at concentrations less than 100 micrograms/ml brings about a quick interruption of mRNA synthesis in preimplantation mouse embryos

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

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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

Prediction of in-vitro developmental competence of early cleavage-stage mouse embryos with compact time-lapse equipment.

Science.gov (United States)

Pribenszky, Csaba; Losonczi, Eszter; Molnár, Miklós; Lang, Zsolt; Mátyás, Szabolcs; Rajczy, Klára; Molnár, Katalin; Kovács, Péter; Nagy, Péter; Conceicao, Jason; Vajta, Gábor

2010-03-01

Single blastocyst transfer is regarded as an efficient way to achieve high pregnancy rates and to avoid multiple pregnancies. Risk of cancellation of transfer due to a lack of available embryos may be reduced by early prediction of blastocyst development. Time-lapse investigation of mouse embryos shows that the time of the first and second cleavage (to the 2- and 3-cell stages, respectively) has a strong predictive value for further development in vitro, while cleavage from the 3-cell to the 4-cell stage has no predictive value. In humans, embryo fragmentation during preimplantation development has been associated with lower pregnancy rates and a higher incidence of developmental abnormalities. Analysis of time-lapse records shows that most fragmentation is reversible in the mouse and is resorbed in an average of 9 h. Daily or bi-daily microscopic checks of embryo development, applied routinely in human IVF laboratories, would fail to detect 36 or 72% of these fragmentations, respectively. Fragmentation occurring in a defined time frame has a strong predictive value for in-vitro embryo development. The practical compact system used in the present trial, based on the 'one camera per patient' principle, has eliminated the usual disadvantages of time-lapse investigations and is applicable for the routine follow-up of in-vitro embryo development. Copyright 2009 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

The Construction of cDNA Libraries from Human Single Preimplantation Embryos and Their Use in the Study of Gene Expression During Development

OpenAIRE

Adjaye, James; Daniels, Rob; Monk, Marilyn

1998-01-01

Purpose:The construction and application of polymerase chain reaction (PCR)-based cDNA libraries from unfertilized human oocytes and single preimplantation-stage embryos are described. The purpose of these studies is to provide a readily available resource for the study of gene expression during human preimplantation development.

Chromosomal mosaicism in mouse two-cell embryos after paternal exposure to acrylamide

Energy Technology Data Exchange (ETDEWEB)

Marchetti, Francesco; Bishop, Jack; Lowe, Xiu; Wyrobek, Andrew J

2008-10-14

Chromosomal mosaicism in human preimplantation embryos is a common cause ofspontaneous abortions, however, our knowledge of its etiology is limited. We used multicolor fluorescence in situ hybridization (FISH) painting to investigate whether paternally-transmitted chromosomal aberrations result in mosaicism in mouse 2-cell embryos. Paternal exposure to acrylamide, an important industrial chemical also found in tobacco smoke and generated during the cooking process of starchy foods, produced significant increases in chromosomally defective 2-cell embryos, however, the effects were transient primarily affecting the postmeiotic stages of spermatogenesis. Comparisons with our previous study of zygotes demonstrated similar frequencies of chromosomally abnormal zygotes and 2-cell embryos suggesting that there was no apparent selection against numerical or structural chromosomal aberrations. However, the majority of affected 2-cell embryos were mosaics showing different chromosomal abnormalities in the two blastomeric metaphases. Analyses of chromosomal aberrations in zygotes and 2-cell embryos showed a tendency for loss of acentric fragments during the first mitotic division ofembryogenesis, while both dicentrics and translocations apparently underwent propersegregation. These results suggest that embryonic development can proceed up to the end of the second cell cycle of development in the presence of abnormal paternal chromosomes and that even dicentrics can persist through cell division. The high incidence of chromosomally mosaic 2-cell embryos suggests that the first mitotic division of embryogenesis is prone to missegregation errors and that paternally-transmitted chromosomal abnromalities increase the risk of missegregation leading to embryonic mosaicism.

Preimplantation genetic haplotyping a new application for diagnosis of translocation carrier's embryos- preliminary observations of two robertsonian translocation carrier families.

Science.gov (United States)

Shamash, Jana; Rienstein, Shlomit; Wolf-Reznik, Haike; Pras, Elon; Dekel, Michal; Litmanovitch, Talia; Brengauz, Masha; Goldman, Boleslav; Yonath, Hagith; Dor, Jehoshua; Levron, Jacob; Aviram-Goldring, Ayala

2011-01-01

Preimplantation genetic diagnosis using fluorescence in-situ hybridization (PGD-FISH) is currently the most common reproductive solution for translocation carriers. However, this technique usually does not differentiate between embryos carrying the balanced form of the translocation and those carrying the homologous normal chromosomes. We developed a new application of preimplantation genetic haplotyping (PGH) that can identify and distinguish between all forms of the translocation status in cleavage stage embryos prior to implantation. Polymorphic markers were used to identify and differentiate between the alleles that carry the translocation and those that are the normal homologous chromosomes. Embryos from two families of robertsonian translocation carriers were successfully analyzed using polymorphic markers haplotyping. Our preliminary results indicate that the PGH is capable of distinguishing between normal, balanced and unbalanced translocation carrier embryos. This method will improve PGD and will enable translocation carriers to avoid transmission of the translocation and the associated medical complications to offspring.

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.

Radiosensitive target in the early mouse embryo exposed to very low doses of ionizing radiation

International Nuclear Information System (INIS)

Wiley, Lynn M.; Raabe, Otto G.; Khan, Rakhshi; Straume, Tore

1994-01-01

We exposed mouse preimplantation embryos in vitro to either tritiated water (HTO) or tritiated thymidine (TdR) to determine whether the radiosensitive target was nuclear or extranuclear for embryonic cell proliferation disadvantage in the mouse embryo chimera assay. 8-cell embryos were incubated in either HTO or TdR for 2 h and paired with non-irradiated control embryos to form chimeras. Chimeras were cultured for an average of 20.2 h to allow for 2-3 cell cycles and then partially dissociated to obtain the number of progeny cells contributed by the two partner embryos for each chimera. These values were expressed as a 'proliferation ratio' (number of cells from the irradiated embryo: total number of cells in the chimera). A ratio significantly less than 0.50 indicates that the experimental embryo expressed an embryonic cell proliferation disadvantage, which is the endpoint of this assay. The activity concentrations of HTO and TdR were adjusted so that both would deliver comparable mean absorbed nuclear doses during the combined initial 2-h irradiation incubation and subsequent 20.2 h chimera incubation periods. Although nuclear doses were comparable under these conditions, the extranuclear dose delivered by the uniformly distributed HTO was about 100 times greater than the extranuclear dose delivered by TdR for each given nuclear dose. Consequently, obtaining mean TdR proliferation ratios≤mean HTO proliferation ratios would be evidence for a nuclear target while obtaining mean HTO proliferation ratios< mean TdR proliferation ratios would be evidence for an extranuclear target. TdR consistently produced lower mean proliferation ratios over a range of doses from 0.14 Gy to 0.43 Gy. Therefore, we conclude that the radiosensitive target for this endpoint is nuclear

Clinical applications of preimplantation genetic testing.

Science.gov (United States)

Brezina, Paul R; Kutteh, William H

2015-02-19

Genetic diagnostic technologies are rapidly changing the way medicine is practiced. Preimplantation genetic testing is a well established application of genetic testing within the context of in vitro fertilization cycles. It involves obtaining a cell(s) from a developing embryo in culture, which is then subjected to genetic diagnostic analysis; the resulting information is used to guide which embryos are transferred into the uterus. The potential applications and use of this technology have increased in recent years. Experts agree that preimplantation genetic diagnosis is clinically appropriate for many known genetic disorders. However, some applications of such testing, such as preimplantation genetic screening for aneuploidy, remain controversial. Clinical data suggest that preimplantation genetic screening may be useful, but further studies are needed to quantify the size of the effect and who would benefit most. © BMJ Publishing Group Ltd 2015.

Developmental defects and genomic instability after x-irradiation of wild-type and genetically modified mouse pre-implantation and early post-implantation embryos

International Nuclear Information System (INIS)

Jacquet, P

2012-01-01

Results obtained from the end of the 1950s suggested that ionizing radiation could induce foetal malformations in some mouse strains when administered during early pre-implantation stages. Starting in 1989, data obtained in Germany also showed that radiation exposure during that period could lead to a genomic instability in the surviving foetuses. Furthermore, the same group reported that both malformations and genomic instability could be transmitted to the next generation foetuses after exposure of zygotes to relatively high doses of radiation. As such results were of concern for radiation protection, we investigated this in more detail during recent years, using mice with varying genetic backgrounds including mice heterozygous for mutations involved in important cellular processes like DNA repair, cell cycle regulation or apoptosis. The main parameters which were investigated included morphological development, genomic instability and gene expression in the irradiated embryos or their own progeny. The aim of this review is to critically reassess the results obtained in that field in the different laboratories and to try to draw general conclusions on the risks of developmental defects and genomic instability from an exposure of early embryos to moderate doses of ionizing radiation. Altogether and in the range of doses normally used in diagnostic radiology, the risk of induction of embryonic death and of congenital malformation following the irradiation of a newly fertilised egg is certainly very low when compared to the ‘spontaneous’ risks for such effects. Similarly, the risk of radiation induction of a genomic instability under such circumstances seems to be very small. However, this is not a reason to not apply some precaution principles when possible. One way of doing this is to restrict the use of higher dose examinations on all potentially pregnant women to the first ten days of their menstrual cycle when conception is very unlikely to have occurred

Advances in preimplantation genetic diagnosis/screening.

Science.gov (United States)

Yan, LiYing; Wei, Yuan; Huang, Jin; Zhu, XiaoHui; Shi, XiaoDan; Xia, Xi; Yan, Jie; Lu, CuiLing; Lian, Ying; Li, Rong; Liu, Ping; Qiao, Jie

2014-07-01

Preimplantation genetic diagnosis (PGD) gives couples who have a high risk of transmitting genetic disorders to their baby the chance to have a healthy offspring through embryo genetic analysis and selection. Preimplantation genetic screening (PGS) is an effective method to select euploid embryos that may prevent repeated implantation failure or miscarriage. However, how and to whom PGS should be provided is a controversial topic. The first successful case of PGD of a human being was reported in 1990, and there have been tremendous improvements in this technology since then. Both embryo biopsy and genetic technologies have been improved dramatically, which increase the accuracy and expand the indications of PGD/PGS.

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

Novel technologies emerging for preimplantation genetic diagnosis and preimplantation genetic testing for aneuploidy.

Science.gov (United States)

Sermon, Karen

2017-01-01

Preimplantation genetic diagnosis (PGD) was introduced as an alternative to prenatal diagnosis: embryos cultured in vitro were analysed for a monogenic disease and only disease-free embryos were transferred to the mother, to avoid the termination of pregnancy with an affected foetus. It soon transpired that human embryos show a great deal of acquired chromosomal abnormalities, thought to explain the low success rate of IVF - hence preimplantation genetic testing for aneuploidy (PGT-A) was developed to select euploid embryos for transfer. Areas covered: PGD has followed the tremendous evolution in genetic analysis, with only a slight delay due to adaptations for diagnosis on small samples. Currently, next generation sequencing combining chromosome with single-base pair analysis is on the verge of becoming the golden standard in PGD and PGT-A. Papers highlighting the different steps in the evolution of PGD/PGT-A were selected. Expert commentary: Different methodologies used in PGD/PGT-A with their pros and cons are discussed.

Comparison of toxicity of smoke from traditional and harm-reduction cigarettes using mouse embryonic stem cells as a novel model for preimplantation development.

Science.gov (United States)

Lin, S; Tran, V; Talbot, P

2009-02-01

Embryonic stem cells (ESC), which originate from the inner cell mass of blastocysts, are valuable models for testing the effects of toxicants on preimplantation development. In this study, mouse ESC (mESC) were used to compare the toxicity of mainstream (MS) and sidestream (SS) cigarette smoke on cell attachment, survival and proliferation. In addition, smoke from a traditional commercial cigarette was compared with smoke from three harm-reduction brands. MS and SS smoke solutions were made using an analytical smoking machine and tested at three doses using D3 mESC plated on 0.2% gelatin. At 6 and 24 h, images were taken and the number of attached cells was evaluated. Both MS and SS smoke from traditional and harm-reduction cigarettes inhibited cell attachment, survival and proliferation dose dependently. For all brands, SS smoke was more potent than MS smoke. However, removal of the cigarette filter increased the toxicity of MS smoke to that of SS smoke. Both MS and SS smoke from harm-reduction cigarettes were as inhibitory, or more inhibitory, than their counterparts from the traditional brand. When preimplantation mouse embryos were cultured for 1 h in MS or SS smoke solutions from a harm-reduction brand, blastomeres became apoptotic, in agreement with the data obtained using mESC. mESC provide a valuable model for toxicological studies on the preimplantation stage of development and were used to show that MS and SS smoke from traditional and harm-reduction cigarettes are detrimental to embryonic cells prior to implantation.

Effects of heat stress on bovine preimplantation embryos produced in vitro.

Science.gov (United States)

Sakatani, Miki

2017-08-19

Summer heat stress decreases the pregnancy rate in cattle and has been thought to be associated with the early embryonic death caused by the elevation of maternal body temperature. In vitro cultures have been widely used for the evaluation of effects of heat stress on oocytes, fertilization, preimplantation, and embryonic development. Susceptibility to heat stress is present in developmental stages from oocytes to cleavage-stage (before embryonic gene activation, EGA) embryos, leading to a consequent decrease in developmental competence. On the other hand, advanced-stage embryos such as morula or blastocysts have acquired thermotolerance. The mechanism for the developmental stage-dependent change in thermotolerance is considered to be the accumulation of antioxidants in embryos in response to heat-inducible production of reactive oxygen species. The supplementation of antioxidants to the culture media has been known to neutralize the detrimental effects of heat stress. Besides, EGA could be involved in acquisition of thermotolerance in later stages of embryos. Morulae or blastocysts can repair heat-induced unfolded proteins or prevent DNA damage occurring in processes such as apoptosis. Therefore, embryo transfer (ET) that can bypass the heat-sensitive stage could be a good solution to improve the pregnancy rate under heat stress. However, frozen-thawed ET could not improve the pregnancy rate as expected. Frozen-thawed blastocysts were more sensitive to heat stress and showed less proliferation upon heat exposure, compared to fresh blastocysts. Therefore, further research is required to improve the reduction in pregnancy rates due to summer heat stress.

Effect of the microenvironment and embryo density on developmental characteristics and gene expression profile of bovine preimplantative embryos cultured in vitro.

Science.gov (United States)

Hoelker, Michael; Rings, Franka; Lund, Qamaruddin; Ghanem, Nasser; Phatsara, Chirawath; Griese, Josef; Schellander, Karl; Tesfaye, Dawit

2009-03-01

The Well of the Well (WOW) system has been developed to culture embryos in small groups or to track the development of single embryos. In the present study, we aimed to examine the effects of the microenvironment provided by the WOW system and embryo density on developmental rates, embryo quality and preimplantative gene expression profile of the resulting embryos. Embryos cultured in a group of 16 reached the blastocyst stage at a significantly lower level than zygotes cultured in a group of 50 (22.2 vs 30.3%), whereas zygotes cultured in WOW were able to compensate against low embryo densities, reaching a blastocyst rate as high as embryos cultured in a group of 50 (31.3 vs 30.3%). Moreover, embryos derived from WOW culture did not differ in terms of differential cell counts and apoptotic cell index compared with controls. The gene expression analysis revealed 62 transcripts to be upregulated and 33 transcripts to be downregulated by WOW culture. Comparing the in vivo derived blastocysts with the blastocysts derived from WOW culture, and group culture, expression of ATP5A1, PLAC8 and KRT8 was more similar to the embryos derived from WOW culture, whereas expression of S100A10 and ZP3 genes was more similar to blastocysts cultured in a group. In conclusion, microenvironment as well as embryo density significantly affected developmental rates. While subsequent blastocysts did not differ in terms of differential cell counts and apoptotic cell index, significant differences were observed in terms of the relative abundance of transcripts in the resulting embryos.

Improving embryo quality in assisted reproduction

NARCIS (Netherlands)

Mantikou, E.

2013-01-01

The goal of this thesis was to improve embryo quality in assisted reproductive technologies by gaining more insight into human preimplantation embryo development and by improving in vitro culture conditions. To do so, we investigated an intriguing feature of the human preimplantation embryo, i.e.

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

DNA topoisomerase II enzyme activity appears in mouse sperm ...

African Journals Online (AJOL)

Jane

2011-08-22

Aug 22, 2011 ... 4 mg/ml bovine serum albumin (BSA; Sigma, Chemical Co., U.S.A) and at time of use, it was ..... Differential growth of mouse preimplantation embryos in chemically ... I. In vitro fertilization of eggs by fresh epididymal sperms.

Ouabain-sensitive Rb+ uptake in mouse eggs and preimplantation conceptuses

International Nuclear Information System (INIS)

Van Winkle, L.J.; Campione, A.L.

1991-01-01

The results of histochemical and immunocytochemical studies have been used elsewhere to support the hypothesis that Na+/K(+)-ATPase expression is initiated or increases dramatically in preimplantation mouse conceptuses just before they begin to cavitate. Moreover, localization of the enzyme in the inner membrane of the mural trophoblast is thought to be involved directly in formation and maintenance of the blastocyst cavity. Presumably, Na+/K(+)-ATPase extrudes the cation, Na+, and therefore water into the cavity. The cation transporting activity of the enzyme can be determined by measuring ouabain-sensitive Rb+ uptake by cells. Therefore, we measured Rb+ uptake in mouse eggs and preimplantation conceptuses at various stages of development. 86Rb+ uptake by conceptuses increased linearly with time for at least 60 min in medium containing 0.7 mM total Rb+ plus K+ in the absence or presence of 1.0 mM ouabain, and ouabain inhibited more than 70% of 86Rb+ uptake. The ouabain concentration at 1/2 of maximum inhibition of the ouabain-sensitive component of 86Rb+ uptake was about 10-20 microM in eggs and conceptuses at all stages of preimplantation development. Moreover, ouabain-sensitive Rb+ uptake had a twofold higher Vmax value in blastocysts than in eggs or conceptuses at earlier stages of development (i.e., approximately 173 vs 70-100 fmole.conceptus-1.min-1), although the total cell surface area also was probably about two times greater in blastocysts than in eggs or other conceptuses. Ouabain-sensitive Rb+ transport in eggs and conceptuses may have occurred via a single ouabain-sensitive Rb+ transporter with a Hill coefficient of 1.5-1.8 (Hill plots). When it was assumed that the Hill coefficient had a value of 2.0, however, eggs and conceptuses appeared to contain at least two forms of Na+/K(+)-ATPase activity

A methodological overview on molecular preimplantation genetic diagnosis and screening: a genomic future?

Science.gov (United States)

Vendrell, Xavier; Bautista-Llácer, Rosa

2012-12-01

The genetic diagnosis and screening of preimplantation embryos generated by assisted reproduction technology has been consolidated in the prenatal care framework. The rapid evolution of DNA technologies is tending to molecular approaches. Our intention is to present a detailed methodological view, showing different diagnostic strategies based on molecular techniques that are currently applied in preimplantation genetic diagnosis. The amount of DNA from one single, or a few cells, obtained by embryo biopsy is a limiting factor for the molecular analysis. In this sense, genetic laboratories have developed molecular protocols considering this restrictive condition. Nevertheless, the development of whole-genome amplification methods has allowed preimplantation genetic diagnosis for two or more indications simultaneously, like the selection of histocompatible embryos plus detection of monogenic diseases or aneuploidies. Moreover, molecular techniques have permitted preimplantation genetic screening to progress, by implementing microarray-based comparative genome hybridization. Finally, a future view of the embryo-genetics field based on molecular advances is proposed. The normalization, cost-effectiveness analysis, and new technological tools are the next topics for preimplantation genetic diagnosis and screening. Concomitantly, these additions to assisted reproduction technologies could have a positive effect on the schedules of preimplantation studies.

In vitro development rate of preimplantation rabbit embryos cultured with different levels of melatonin.

Science.gov (United States)

Mehaisen, Gamal Mohamed Kamel; Saeed, Ayman Moustafa

2015-02-01

This study aimed to investigate the effect of melatonin supplementation at different levels in culture medium on embryo development in rabbits. Embryos of 2-4 cells, 8-16 cells and morula stages were recovered from nulliparous Red Baladi rabbit does by laparotomy technique 24, 48 and 72 h post-insemination, respectively. Normal embryos from each stage were cultured to hatched blastocyst stages in either control culture medium (TCM-199 + 20% fetal bovine serum) or control supplemented with melatonin at 10(-3) M, 10(-6) M or 10(-9) M. No effect of melatonin was found on development of embryos recovered at 24 h post-insemination. The high level of melatonin at 10(-3) M adversely affected the in vitro development rates of embryos recovered at 48 h post-insemination (52 versus 86, 87 and 80% blastocyst rate; 28 versus 66, 78 and 59% hatchability rate for 10(-3) M versus 10(-9) M, 10(-6) M and control, respectively, P< 0.05). At the morula stage, melatonin at 10-3 M significantly increased the in vitro development of embryos (92% for 10(-3) M versus 76% for control, P < 0.05), while the hatchability rate of these embryos was not improved by melatonin (16-30% versus 52% for melatonin groups versus control, P < 0.05). Results show that a moderate level of melatonin (10(-6) M) may improve the development and hatchability rates of preimplantation rabbit embryos. The addition of melatonin at a 10-3 M concentration enhances the development of rabbit morulae but may negatively affect the development of earlier embryos. More studies are needed to optimize the use of melatonin in in vitro embryo culture in rabbits.

Preimplantation genetic diagnosis associated to Duchenne muscular dystrophy.

Science.gov (United States)

Bianco, Bianca; Christofolini, Denise Maria; Conceição, Gabriel Seixas; Barbosa, Caio Parente

2017-01-01

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.

Mouse cloning and somatic cell reprogramming using electrofused blastomeres.

Science.gov (United States)

Riaz, Amjad; Zhao, Xiaoyang; Dai, Xiangpeng; Li, Wei; Liu, Lei; Wan, Haifeng; Yu, Yang; Wang, Liu; Zhou, Qi

2011-05-01

Mouse cloning from fertilized eggs can assist development of approaches for the production of "genetically tailored" human embryonic stem (ES) cell lines that are not constrained by the limitations of oocyte availability. However, to date only zygotes have been successfully used as recipients of nuclei from terminally differentiated somatic cell donors leading to ES cell lines. In fertility clinics, embryos of advanced embryonic stages are usually stored for future use, but their ability to support the derivation of ES cell lines via somatic nuclear transfer has not yet been proved. Here, we report that two-cell stage electrofused mouse embryos, arrested in mitosis, can support developmental reprogramming of nuclei from donor cells ranging from blastomeres to somatic cells. Live, full-term cloned pups from embryonic donors, as well as pluripotent ES cell lines from embryonic or somatic donors, were successfully generated from these reconstructed embryos. Advanced stage pre-implantation embryos were unable to develop normally to term after electrofusion and transfer of a somatic cell nucleus, indicating that discarded pre-implantation human embryos could be an important resource for research that minimizes the ethical concerns for human therapeutic cloning. Our approach provides an attractive and practical alternative to therapeutic cloning using donated oocytes for the generation of patient-specific human ES cell lines.

Meiotic and mitotic behaviour of a ring/deleted chromosome 22 in human embryos determined by preimplantation genetic diagnosis for a maternal carrier

Directory of Open Access Journals (Sweden)

Laver Sarah

2009-01-01

Full Text Available Abstract Background Ring chromosomes are normally associated with developmental anomalies and are rarely inherited. An exception to this rule is provided by deletion/ring cases. We were provided with a unique opportunity to investigate the meiotic segregation at oogenesis in a woman who is a carrier of a deleted/ring 22 chromosome. The couple requested preimplantation genetic diagnosis (PGD following the birth of a son with a mosaic karyotype. The couple underwent two cycles of PGD. Studies were performed on lymphocytes, single embryonic cells removed from 3 day-old embryos and un-transferred embryos. Analysis was carried out using fluorescence in situ hybridisation (FISH with specific probe sets in two rounds of hybridization. Results In total, 12 embryos were biopsied, and follow up information was obtained for 10 embryos. No embryos were completely normal or balanced for chromosome 22 by day 5. There was only one embryo diagnosed as balanced of 12 biopsied but that accumulated postzygotic errors by day 5. Three oocytes apparently had a balanced chromosome 22 complement but all had the deleted and the ring 22 and not the intact chromosome 22. After fertilisation all the embryos accumulated postzygotic errors for chromosome 22. Conclusion The study of the preimplantation embryos in this case provided a rare and significant chance to study and understand the phenomena associated with this unusual type of anomaly during meiosis and in the earliest stages of development. It is the first reported PGD attempt for a ring chromosome abnormality.

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

Characterization of bovine embryos cultured under conditions appropriate for sustaining human naïve pluripotency

NARCIS (Netherlands)

Brinkhof, Bas; van Tol, Helena T A; Groot Koerkamp, Marian J A; Wubbolts, Richard W; Haagsman, Henk P; Roelen, Bernard A J

2017-01-01

In mammalian preimplantation development, pluripotent cells are set aside from cells that contribute to extra-embryonic tissues. Although the pluripotent cell population of mouse and human embryos can be cultured as embryonic stem cells, little is known about the pathways involved in formation of a

Expression of HSG is essential for mouse blastocyst formation

International Nuclear Information System (INIS)

Jiang Guangjian; Pan Lei; Huang Xiuying; Han Mei; Wen Jinkun; Sun Fangzhen

2005-01-01

It has been shown recently that hyperplasia suppressor gene (HSG) is a powerful regulator for cell proliferation and has a critical role in mitochondrial fusion in many cells. However, little is known about its expression, localization, and function during oocyte maturation and early embryogenesis. In this study, with indirect immunofluorescent staining and Western blotting, we found that HSG was expressed in mouse oocytes and preimplantation embryos which primarily exhibited a submembrane distribution pattern in the cytoplasm. Moreover, HSG mainly associated with β-tubulin during oocyte maturation and early embryonic development. When mouse zygotes were injected with HSG antisense plasmid and cultured in vitro, their capacity to form blastocysts was severely impaired. Our results indicate that HSG plays an essential role in mouse preimplantation development

Enhancement of NMRI Mouse Embryo Development In vitro

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Abedini, F.

2013-12-01

Full Text Available Most of the systematic studies used in the development of human embryo culture media have been done first on mouse embryos. The general use of NMRI outbred mice is a model for toxicology, teratology and pharmacology. NMRI mouse embryo exhibit the two-cell block in vitro. The objective of this study was to evaluate and compare the effects of four kinds of culture media on the development of zygotes (NMRI after embryo vitrification. One-cell mouse embryos were obtained from NMRI mice after superovulation and mating with adult male NMRI mice. And then randomly divided into 4 groups for culture in four different cultures media including: M16 (A, DMEM/Ham, F-12 (B, DMEM/Ham's F-12 co-culture with Vero cells(C and DMEM/Ham's F-12 co-culture with MEF cells (D. Afterward all of the embryos were vitrified in EFS40 solution and collected. Results of our study revealed, more blastocysts significantly were developed with co-culture with MEF cells in DMEM/Ham's F-12 medium. More research needed to understand the effect of other components of culture medium, and co-culture on NMRI embryo development.

Caspase activity and expression of cell death genes during development of human preimplantation embryos.

Science.gov (United States)

Spanos, S; Rice, S; Karagiannis, P; Taylor, D; Becker, D L; Winston, R M L; Hardy, K

2002-09-01

It has been observed that apoptosis occurs in human blastocysts. In other types of cell, the characteristic morphological changes seen in apoptotic cells are executed by caspases, which are regulated by the BCL-2 family of proteins. This study investigated whether these components of the apoptotic cascade are present throughout human preimplantation development. Developing and arrested two pronucleate embryos at all stages were incubated with a fluorescently tagged caspase inhibitor that binds only to active caspases, fixed, counterstained with 4,6-diamidino-2-phenylindole (DAPI) to assess nuclear morphology and examined using confocal microscopy. Active caspases were detected only after compaction, at the morula and blastocyst stages, and were frequently associated with apoptotic nuclei. Occasional labelling was seen in arrested embryos. Expression of proapoptotic BAX and BAD and anti-apoptotic BCL-2 was examined in single embryos using RT-PCR and immunohistochemistry. BAX and BCL-2 mRNAs were expressed throughout development, whereas BAD mRNA was expressed mainly after compaction. Simultaneous expression of BAX and BCL-2 proteins within individual embryos was confirmed using immunohistochemistry. The onset of caspase activity and BAD expression after compaction correlates with the previously reported appearance of apoptotic nuclei. As in other types of cell, human embryos express common molecular components of the apoptotic cascade, although apoptosis appears to be suppressed before compaction and differentiation.

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.

The influence of serum substituents on serum-free Vero cell conditioned culture media manufactured from Dulbecco's modified Eagle medium in mouse embryo culture.

Science.gov (United States)

Lee, Jong-Seon; Kim, Ju-Hwan; Seo, Young-Seok; Yang, Jung-Bo; Kim, Yong-Il; Kim, Hye-Jin; Lee, Ki-Hwan

2013-09-01

This study was conducted to examine the influences of supplementation of the serum substituents and available period of serum-free Vero cell conditioned media (SF-VCM) manufactured from Dulbecco's modified Eagle medium cultured with Vero cells for in vitro development of mouse preimplantation embryos. A total of 1,099 two-cell embryos collected from imprinting control region mice were cultured in SF-VCM with 10% and 20% human follicular fluid (hFF), serum substitute supplement (SSS), and serum protein substitute (SPS). Development of embryos was observed every 24 hours. Results between different groups were analyzed by chi-square test, and considered statistically significant when P-value was less than 0.05. The rates of embryonic development cultured in SF-VCM supplemented with serum substituents were significantly higher compare with serum-free group (P media up to 4 weeks did not affect on embryonic development.

Parent-of-origin dependent gene-specific knock down in mouse embryos

International Nuclear Information System (INIS)

Iqbal, Khursheed; Kues, Wilfried A.; Niemann, Heiner

2007-01-01

In mice hemizygous for the Oct4-GFP transgene, the F1 embryos show parent-of-origin dependent expression of the marker gene. F1 embryos with a maternally derived OG2 allele (OG2 mat /-) express GFP in the oocyte and during preimplantation development until the blastocyst stage indicating a maternal and embryonic expression pattern. F1-embryos with a paternally inherited OG2 allele (OG2 pat /-) express GFP from the 4- to 8-cell stage onwards showing only embryonic expression. This allows to study allele specific knock down of GFP expression. RNA interference (RNAi) was highly efficient in embryos with the paternally inherited GFP allele, whereas embryos with the maternally inherited GFP allele showed a delayed and less stringent suppression, indicating that the initial levels of the target transcript and the half life of the protein affect RNAi efficacy. RT-PCR analysis revealed only minimum of GFP mRNA. These results have implications for studies of gene silencing in mammalian embryos

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

International Nuclear Information System (INIS)

Jacquet, P.; Buset, J.; Neefs, M.; Vankerkom, J.; Benotmane, M.A.; Derradji, H.; Hildebrandt, G.; Baatout, S.

2010-01-01

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

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

Inhibition of fumonisin B1 cytotoxicity by nanosilicate platelets during mouse embryo development.

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Yu-Jing Liao

Full Text Available Nanosilicate platelets (NSP, the form of natural silicate clay that was exfoliated from montmorillonite (MMT, is widely used as a feed additive for its high non-specific binding capacity with mycotoxins such as fumonisin B1 (FB1, and has been evaluated its safety for biomedical use including cytotoxicity, genotoxicity, and lethal dosage (LD. In the study, we further examined its toxicity on the development of CD1 mouse embryos and its capacity to prevent teratogenesis-induced by FB1. In vitro cultures, NSP did not disturb the development and the quality of intact pre-implantation mouse embryos. Further, newborn mice from females consumed with NSP showed no abnormalities. NSP had an unexpected high adsorption capacity in vitro. In contrast to female mice consumed with FB1 only, a very low residual level of FB1 in the circulation, reduced incidence of neutral tube defects and significantly increased fetal weight were observed in the females consumed with FB1 and NSP, suggesting a high alleviation effect of NSP on FB1 in vivo. Furthermore, FB1 treatment disturbed the gene expression of sphingolipid metabolism enzymes (longevity assurance homolog 5, LASS 5; sphingosine kinase 1, Sphk1; sphingosine kinase 2, Sphk2; sphingosine 1- phosphate lyase, Sgpl1; sphingosine 1-phosphate phosphatase, Sgpp1 in the maternal liver, uterus, fetus, and placenta, but NSP administration reversed the perturbations. Based on these findings, we conclude that NSP is a feasible and effective agent for supplementary use in reducing the toxicity of FB1 to animals.

Gene Coexpression and Evolutionary Conservation Analysis of the Human Preimplantation Embryos

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Tiancheng Liu

2015-01-01

Full Text Available Evolutionary developmental biology (EVO-DEVO tries to decode evolutionary constraints on the stages of embryonic development. Two models—the “funnel-like” model and the “hourglass” model—have been proposed by investigators to illustrate the fluctuation of selective pressure on these stages. However, selective indices of stages corresponding to mammalian preimplantation embryonic development (PED were undetected in previous studies. Based on single cell RNA sequencing of stages during human PED, we used coexpression method to identify gene modules activated in each of these stages. Through measuring the evolutionary indices of gene modules belonging to each stage, we observed change pattern of selective constraints on PED for the first time. The selective pressure decreases from the zygote stage to the 4-cell stage and increases at the 8-cell stage and then decreases again from 8-cell stage to the late blastocyst stages. Previous EVO-DEVO studies concerning the whole embryo development neglected the fluctuation of selective pressure in these earlier stages, and the fluctuation was potentially correlated with events of earlier stages, such as zygote genome activation (ZGA. Such oscillation in an earlier stage would further affect models of the evolutionary constraints on whole embryo development. Therefore, these earlier stages should be measured intensively in future EVO-DEVO studies.

Gestational surrogacy and the role of routine embryo screening: Current challenges and future directions for preimplantation genetic testing.

Science.gov (United States)

Sills, E Scott; Anderson, Robert E; McCaffrey, Mary; Li, Xiang; Arrach, Nabil; Wood, Samuel H

2016-03-01

Preimplantation genetic screening (PGS) is a component of IVF entailing selection of an embryo for transfer on the basis of chromosomal normalcy. If PGS were integrated with single embryo transfer (SET) in a surrogacy setting, this approach could improve pregnancy rates, minimize miscarriage risk, and limit multiple gestations. Even without PGS, pregnancy rates for IVF surrogacy cases are generally satisfactory, especially when treatment utilizes embryos derived from young oocytes and transferred to a healthy surrogate. However, there could be a more general role for PGS in surrogacy, since background aneuploidy in embryos remains a major factor driving implantation failure and miscarriage for all infertility patients. At present, the proportion of IVF cases involving GS is limited, while the number of IVF patients requesting PGS appears to be increasing. In this report, the relevance of PGS for surrogacy in the rapidly changing field of assisted fertility medicine is discussed. © 2015 Wiley Periodicals, Inc.

A systematic analysis of the suitability of preimplantation genetic diagnosis for mitochondrial diseases in a heteroplasmic mitochondrial mouse model.

Science.gov (United States)

Neupane, Jitesh; Vandewoestyne, Mado; Heindryckx, Björn; Ghimire, Sabitri; Lu, Yuechao; Qian, Chen; Lierman, Sylvie; Van Coster, Rudy; Gerris, Jan; Deroo, Tom; Deforce, Dieter; De Sutter, Petra

2014-04-01

What is the reliability of preimplantation genetic diagnosis (PGD) based on polar body (PB), blastomere or trophectoderm (TE) analysis in a heteroplasmic mitochondrial mouse model? The reliability of PGD to determine the level of mitochondrial DNA (mtDNA) heteroplasmy is questionable based on either the first or second PB analysis; however, PGD based on blastomere or TE analysis seems more reliable. PGD has been suggested as a technique to determine the level of mtDNA heteroplasmy in oocytes and embryos to avoid the transmission of heritable mtDNA disorders. A strong correlation between first PBs and oocytes and between second PBs and zygotes was reported in mice but is controversial in humans. So far, the levels of mtDNA heteroplasmy in first PBs, second PBs and their corresponding oocytes, zygotes and blastomeres, TE and blastocysts have not been analysed within the same embryo. We explored the suitability of PGD by comparing the level of mtDNA heteroplasmy between first PBs and metaphase II (MII) oocytes (n = 33), between first PBs, second PBs and zygotes (n = 30), and between first PBs, second PBs and their corresponding blastomeres of 2- (n = 10), 4- (n = 10) and 8-cell embryos (n = 11). Levels of mtDNA heteroplasmy in second PBs (n = 20), single blastomeres from 8-cell embryos (n = 20), TE (n = 20) and blastocysts (n = 20) were also compared. Heteroplasmic mice (BALB/cOlaHsd), containing mtDNA mixtures of BALB/cByJ and NZB/OlaHsd, were used in this study. The first PBs were biopsied from in vivo matured MII oocytes. The ooplasm was then subjected to ICSI. After fertilization, second PBs were biopsied and zygotes were cultured to recover individual blastomeres from 2-, 4- and 8-cell embryos. Similarly, second PBs were biopsied from in vivo fertilized zygotes and single blastomeres were biopsied from 8-cell stage embryos. The remaining embryo was cultured until the blastocyst stage to isolate TE cells. Polymerase chain reaction followed by restriction fragment

New perspectives on preimplantation genetic diagnosis and preimplantation genetic screening

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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

Effect of Cumulus cell co-culture and Protein Supplement on Success of in vitro Fertilization and Development of Pre-implanted Embryos in mice

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Muhammad-Baqir M-R. Fakhrildin

2005-06-01

Full Text Available Successful oocyte fertilization and normal embryonic development of mice were considered the most important diagnostic criteria for the safety of materials and tools used for human in vitro fertilization and embryo transfer (IVF-ET. Therefore, we studied the influence of cumulus cells co-culture and protein supplement within culture medium on percentages of in vitro fertilization (IVF and normal development of early stages of mouse embryo later. Oocytes were collected and treated with hyaluronidase to remove cumulus cells. Oocytes were divided into four groups namely: Group-1: Oocytes incubated within modified Earl’s medium (MEM supplied with 10% inactivated bovine amniotic fluid as a protein source and cumulus cells; Group-2: Oocytes incubated with MEM supplied with cumulus cells only; Group-3: Oocytes incubated with MEM supplied with 10% inactivated bovine amniotic fluid only; and Group-4: Oocytes  incubated with MEM free of both protein source and cumulus cells. For IVF, 5-6 oocytes were incubated with active spermatozoa under paraffin oil for 18-20 hours at 37° oC in 5% CO2. Percentages of IVF and embryonic development were then recorded. Best results for IVF and normal embryonic development were achieved from oocytes of Group-1 when compared to the other groups. As compared to Group-1, the percentage of IVF for Group-2 and Group-3 were decreased insignificantly and significantly (P<0.002, respectively. Significant (P<0.01 reduction in the percentages of IVF and normal embryonic development were reported in Group-4 as compared to Group-1. Therefore, it was concluded that the presence of cumulus cells co-culture and bovine amniotic fluid as a protein source within culture medium may have an important role on the fertilizing capacity of spermatozoa and oocytes and normal development of pre-implanted mouse embryo later.

Toxicity of various combinations of X-rays, caffeine, and mercury in mouse embryos

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Mueller, W-U. (Universitaetklinikum Essen (Germany, F.R.). Inst. fuer Medizinische Strahlenbiologie)

1989-09-01

Preimplantation mouse embryos in vitro were exposed to various doses of X-rays (0.25-2 Gy) and to different concentrations of two chemicals: caffeine (0.5-2 mM) and mercury (0.5-5 mum). X-irradiation was given first, followed immediately by exposure to the chemicals. The effects of the agents, applied either singly or in combination to two or of three, were studied using morphological, proliferative and cytogenetic endpoints (formation of blastocysts, hatching, trophoblast outgrowth, formation of inner cell mass, cell numbers, micro-nucleus frequency). The term 'enhancement in risk' was used whenever the effects observed after combined exposure (two or three agents) significantly exceeded the sum of the effects due to the component individual agents. The enhancement in risk observed after exposure to the three agents could be explained by the interactions already detected at the level of a combined exposure to only two agents. There was no increase in risk specific for the presence of all three agents. (author).

Preferential selection and transfer of euploid noncarrier embryos in preimplantation genetic diagnosis cycles for reciprocal translocations.

Science.gov (United States)

Wang, Li; Shen, Jiandong; Cram, David S; Ma, Minyue; Wang, Hui; Zhang, Wenke; Fan, Junmei; Gao, Zhiying; Zhang, Liwen; Li, Zhifeng; Xu, Mengnan; Leigh, Don A; Trounson, Alan O; Liu, Jiayin; Yao, Yuanqing

2017-10-01

To develop and validate a new strategy to distinguish between balanced/euploid carrier and noncarrier embryos in preimplantation genetic diagnosis (PGD) cycles for reciprocal translocations and to successfully achieve a live birth after selective transfer of a noncarrier embryo. Retrospective and prospective study. In vitro fertilization (IVF) units. Eleven patients undergoing mate pair sequencing for identification of translocation breakpoints, followed by clinical PGD cycles. Embryo biopsy with 24-chromosome testing to determine carrier status of balanced/euploid embryos. Definition of translocation breakpoints and polymerase chain reaction (PCR) diagnostic primers, correct diagnosis of euploid embryos for carrier status, and a live birth with a normal karyotype after transfer of a noncarrier embryo. In 9 of 11 patients (82%), translocation breakpoints were successfully identified. In four patients with a term PGD pregnancy established with a balanced/euploid embryo of unknown carrier status, the correct carrier status was retrospectively determined, matching with the cytogenetic karyotype of the resulting newborns. In a prospective PGD cycle undertaken by a patient with a 46,XY,t(7;14)(q22;q24.3) translocation, the four balanced/euploid embryos identified comprised three carriers and one noncarrier. Transfer of the noncarrier embryo resulted in birth of a healthy girl who was subsequently confirmed with a normal 46,XX karyotype. The combination of mate pair sequencing and PCR breakpoint analysis of balanced reciprocal translocation derivatives is a novel, reliable, and accurate strategy for distinguishing between carrier and noncarrier balanced/euploid embryos. The method has potential application in clinical PGD cycles for patients with reciprocal translocations or other structural rearrangements. Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Preimplantation Genetic Diagnosis for Stargardt Disease

Science.gov (United States)

Sohrab, Mahsa A.; Allikmets, Rando; Guarnaccia, Michael M.; Smith, R. Theodore

2010-01-01

Purpose To report the first use of in vitro fertilization (IVF) and preimplantation genetic diagnosis to achieve an unaffected pregnancy in an autosomal-recessive retinal dystrophy. Design Case report. Methods An affected male with Stargardt disease and his carrier wife underwent IVF. Embryos obtained by intracytoplasmic sperm injection underwent single-cell DNA testing via polymerase chain reaction and restriction enzyme analysis to detect the presence of ABCA4 mutant alleles. Embryos were diagnosed as being either affected by or carriers for Stargardt disease. A single carrier embryo was implanted. Results Chorionic villus sampling performed during the first trimester verified that the fetus possessed only one mutant paternal allele and one normal maternal allele, thus making her an unaffected carrier of the disease. A healthy, live-born female was delivered. Conclusion IVF and preimplantation genetic diagnosis can assist couples with an affected spouse and a carrier spouse with recessive retinal dystrophies to have an unaffected child. PMID:20149343

Action of uranium on pre implanted mouse embryos

International Nuclear Information System (INIS)

Kundt, Miriam S.

2001-01-01

The cultured preimplantation embryos are normally employed to evaluate the effects of environmental pollutants specially metals. Embryos were obtained from hybrid females CBA x C57 Bl following induction of super ovulation. They were incubated from 1 cell stage during 120 hs. in M16 cultured medium. Three different experiments were carried out: A, B and C using uranyl nitrate UO 2 (NO 3 ) 2 6H 2 O as source of uranium. In experiment 'A' the embryos were cultivated in the same culture dish containing final U concentrations of 13, 26, 52, 104 and 208 μgU/ml. In experiment 'B' embryos in a one cell stage were placed in culture medium with uranyl nitrate with final U concentrations of 26, 52, 104 μgU/ml. After 24 hours those embryos which had reached the two-cell stage were transferred to another culture dish to which fresh solutions of uranyl nitrate were added, maintaining the same concentrations of the previous one. In experiment 'C' the embryos were cultivated containing final U concentrations of 26, 52 and 104 μgU/ml and they were transferred to another culture dish every day to which fresh solutions of uranyl nitrate were added. Different embryos parameters were analyzed: 1) Development grade; 2) Number of cell per embryo and metaphases index; and 3) Embryo ploidy. 1) Embryos were observed each 24 hs. to evaluate development grade: 2, 4 and 8 cell stage, morula, early -expanded- hatched blastocysts and atresic embryos. No significant differences were observed in the proportion of embryos arrested either in the one-cell or in the two cell stages in control culture medium regarding different concentrations of U, in a total of 4388 embryos analyzed. From 2 cell stage, moment that the embryo begins to synthesize its own ARNm, the delay in embryonic development increased dose dependent. On the other hand, the toxicological effects in the same concentration are increase from 'A' treatment to 'C' treatment. Embriotoxicology effects are evidenced by an increment in

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...

Effect of quercetin on the number of blastomeres, zona pellucida thickness, and hatching rate of mouse embryos exposed to actinomycin D: An experimental study

Directory of Open Access Journals (Sweden)

Hamid Reza Sameni

2018-02-01

Full Text Available Background: Quercetin is a flavonoid with the ability to improve the growth of embryos in vitro, and actinomycin D is an inducer of apoptosis in embryonic cells. Objective: The aim was to evaluate the effect of quercetin on the number of viable and apoptotic cells, the zona pellucida (ZP thickness and the hatching rate of preimplantation embryos exposed to actinomycin D in mice. Materials and Methods: Two-cell embryos were randomly divided into four groups (Control, Quercetin, actinomycin D, and Quercetin + actinomycin D group. Blastocysts percentage, hatched blastocysts, and ZP thickness of blastocysts was measured. The number of blastomeres was counted by Hoechst and propidium iodide staining and the apoptotic cells number was counted by TUNEL assay. Results: The results showed that the use of quercetin significantly improved the growth of embryos compared to the control group (p=0.037. Moreover, quercetin reduced the destructive effects of actinomycin D on the growth of embryos significantly (p=0.026. Conclusion: quercetin may protect the embryos against actinomycin D so that increases the number of viable cells and decreases the number of apoptotic cells, which can help the expansion of the blastocysts, thinning of the ZP thickness and increasing the hatching rate in mouse embryos.

Cryopreservation of mouse embryos by ethylene glycol-based vitrification.

Science.gov (United States)

Mochida, Keiji; Hasegawa, Ayumi; Taguma, Kyuichi; Yoshiki, Atsushi; Ogura, Atsuo

2011-11-18

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic modifications and the number is consistently increasing year by year. Its technical development started with slow freezing methods in the 1970s(1), then followed by vitrification methods developed in the late 1980s(2). Generally, the latter technique is advantageous in its quickness, simplicity, and high survivability of recovered embryos. However, the cryoprotectants contained are highly toxic and may affect subsequent embryo development. Therefore, the technique was not applicable to certain strains of mice, even when the solutions are cooled to 4°C to mitigate the toxic effect during embryo handling. At the RIKEN BioResource Center, more than 5000 mouse strains with different genetic backgrounds and phenotypes are maintained(3), and therefore we have optimized a vitrification technique with which we can cryopreserve embryos from many different strains of mice, with the benefits of high embryo survival after vitrifying and thawing (or liquefying, more precisely) at the ambient temperature(4). Here, we present a vitrification method for mouse embryos that has been successfully used at our center. The cryopreservation solution contains ethylene glycol instead of DMSO to minimize the toxicity to embryos(5). It also contains Ficoll and sucrose for prevention of devitrification and osmotic adjustment, respectively. Embryos can be handled at room temperature and transferred into liquid nitrogen within 5 min. Because the original method was optimized for plastic straws as containers, we have slightly modified the protocol for cryotubes, which are more easily accessible in laboratories and more resistant to physical damages. We also describe the procedure of thawing vitrified embryos in detail because it is a critical step for efficient recovery of live mice. These methodologies would be helpful to researchers and

Reduction of transgenerational radiation induced genetic damages observed as numerical chromosomal abnormalities in preimplantation embryos by vitamin E

International Nuclear Information System (INIS)

Salimi, M.; Mozdarani, H.

2008-01-01

To study the effects of parental gamma irradiation (4 Gy) of NMRI (Naval Medical Research Institute) mice on the numerical chromosome abnormalities in subsequent preimplantation embryos in the presence of vitamin E (200 IU/kg), super-ovulated irradiated females were mated with irradiated males at weekly intervals in successive 6 weekly periods. About 68 h post coitus, 8-cell embryos were fixed on slides using standard methods in order to screen for abnormalities in chromosome number. In embryos generated by irradiated mice, the frequency of aneuploids dramatically increased compared to control unirradiated groups (p < 0.001), while no significant difference were observed within irradiated groups mated at weekly interval. Administration of vitamin E significantly decreased chromosomal aberrations in all groups (p < 0.05). Data indicate that gamma irradiation affects spermatogenesis and oogenesis and causes DNA alterations that may lead to chromosome abnormalities in subsequent embryos. Vitamin E effectively reduced the frequency of abnormalities. The way vitamin E reduces genotoxic effects of radiation might be via radical scavenging or antioxidative mechanism. (authors)

Obstetric and neonatal outcomes in blastocyst-stage biopsy with frozen embryo transfer and cleavage-stage biopsy with fresh embryo transfer after preimplantation genetic diagnosis/screening.

Science.gov (United States)

Jing, Shuang; Luo, Keli; He, Hui; Lu, Changfu; Zhang, Shuoping; Tan, Yueqiu; Gong, Fei; Lu, Guangxiu; Lin, Ge

2016-07-01

To study whether embryo biopsy for preimplantation genetic diagnosis/preimplantation genetic screening (PGD/PGS) can influence pregnancy complications and neonatal outcomes. Retrospective analysis. University-affiliated center. This study included data from women and their neonates born after PGD/PGS (n = 317). Questionnaires were designed to obtain information relating to pregnancy complications and neonatal outcomes. Two major strategies for PGD/PGS were evaluated. Blastocyst-stage biopsy and frozen embryo transfer (BB-FET) was carried out in 166 patients, and cleavage-stage biopsy and fresh embryo transfer (CB-ET) was carried out in 129 patients. The incidence of gestational hypertension was significantly higher in BB-FET compared with in CB-ET (9.0% vs. 2.3%, adjusted odds ratio [OR] and 95% confidence interval [CI], 4.85 [1.34, 17.56]). In twins, the birthweight (median [range], 2.70 kg [1.55-3.60 kg] vs. 2.50 kg [1.23-3.75 kg]) was higher in BB-FET than in CB-ET and the gestational age was longer in BB-FET than in CB-ET (median [range], 36.71 weeks [31.14-39.29 weeks] vs. 35.57 weeks [30.57-38.43 weeks]). There was no difference in the incidence of singleton births between the two groups except in the incidence of preterm births (28-37 weeks; 5.3% vs. 16.5% in CB-ET and BB-FET). No significant differences were detected in the incidence of perinatal deaths, birth defects, gender of neonates, and large for gestational age in both singletons and twins, although the numbers of some events were small. BB-FET is associated with a higher incidence of gestational hypertension but better neonatal outcomes compared with CB-ET, especially in twins. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

4D atlas of the mouse embryo for precise morphological staging.

Science.gov (United States)

Wong, Michael D; van Eede, Matthijs C; Spring, Shoshana; Jevtic, Stefan; Boughner, Julia C; Lerch, Jason P; Henkelman, R Mark

2015-10-15

After more than a century of research, the mouse remains the gold-standard model system, for it recapitulates human development and disease and is quickly and highly tractable to genetic manipulations. Fundamental to the power and success of using a mouse model is the ability to stage embryonic mouse development accurately. Past staging systems were limited by the technologies of the day, such that only surface features, visible with a light microscope, could be recognized and used to define stages. With the advent of high-throughput 3D imaging tools that capture embryo morphology in microscopic detail, we now present the first 4D atlas staging system for mouse embryonic development using optical projection tomography and image registration methods. By tracking 3D trajectories of every anatomical point in the mouse embryo from E11.5 to E14.0, we established the first 4D atlas compiled from ex vivo 3D mouse embryo reference images. The resulting 4D atlas comprises 51 interpolated 3D images in this gestational range, resulting in a temporal resolution of 72 min. From this 4D atlas, any mouse embryo image can be subsequently compared and staged at the global, voxel and/or structural level. Assigning an embryonic stage to each point in anatomy allows for unprecedented quantitative analysis of developmental asynchrony among different anatomical structures in the same mouse embryo. This comprehensive developmental data set offers developmental biologists a new, powerful staging system that can identify and compare differences in developmental timing in wild-type embryos and shows promise for localizing deviations in mutant development. © 2015. Published by The Company of Biologists Ltd.

New perspectives on preimplantation genetic diagnosis and preimplantation genetic screening.

Science.gov (United States)

Chen, Chun-Kai; Yu, Hsing-Tse; Soong, Yung-Kuei; Lee, Chyi-Long

2014-06-01

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 the haplotype of

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.

Irreversible barrier to the reprogramming of donor cells in cloning with mouse embryos and embryonic stem cells.

Science.gov (United States)

Ono, Yukiko; Kono, Tomohiro

2006-08-01

Somatic cloning does not always result in ontogeny in mammals, and development is often associated with various abnormalities and embryo loss with a high frequency. This is considered to be due to aberrant gene expression resulting from epigenetic reprogramming errors. However, a fundamental question in this context is whether the developmental abnormalities reported to date are specific to somatic cloning. The aim of this study was to determine the stage of nuclear differentiation during development that leads to developmental abnormalities associated with embryo cloning. In order to address this issue, we reconstructed cloned embryos using four- and eight-cell embryos, morula embryos, inner cell mass (ICM) cells, and embryonic stem cells as donor nuclei and determined the occurrence of abnormalities such as developmental arrest and placentomegaly, which are common characteristics of all mouse somatic cell clones. The present analysis revealed that an acute decline in the full-term developmental competence of cloned embryos occurred with the use of four- and eight-cell donor nuclei (22.7% vs. 1.8%) in cases of standard embryo cloning and with morula and ICM donor nuclei (11.4% vs. 6.6%) in serial nuclear transfer. Histological observation showed abnormal differentiation and proliferation of trophoblastic giant cells in the placentae of cloned concepti derived from four-cell to ICM cell donor nuclei. Enlargement of placenta along with excessive proliferation of the spongiotrophoblast layer and glycogen cells was observed in the clones derived from morula embryos and ICM cells. These results revealed that irreversible epigenetic events had already started to occur at the four-cell stage. In addition, the expression of genes involved in placentomegaly is regulated at the blastocyst stage by irreversible epigenetic events, and it could not be reprogrammed by the fusion of nuclei with unfertilized oocytes. Hence, developmental abnormalities such as placentomegaly as

Developmental fate and lineage commitment of singled mouse blastomeres.

Science.gov (United States)

Lorthongpanich, Chanchao; Doris, Tham Puay Yoke; Limviphuvadh, Vachiranee; Knowles, Barbara B; Solter, Davor

2012-10-01

The inside-outside model has been invoked to explain cell-fate specification of the pre-implantation mammalian embryo. Here, we investigate whether cell-cell interaction can influence the fate specification of embryonic blastomeres by sequentially separating the blastomeres in two-cell stage mouse embryos and continuing separation after each cell division throughout pre-implantation development. This procedure eliminates information provided by cell-cell interaction and cell positioning. Gene expression profiles, polarity protein localization and functional tests of these separated blastomeres reveal that cell interactions, through cell position, influence the fate of the blastomere. Blastomeres, in the absence of cell contact and inner-outer positional information, have a unique pattern of gene expression that is characteristic of neither inner cell mass nor trophectoderm, but overall they have a tendency towards a 'trophectoderm-like' gene expression pattern and preferentially contribute to the trophectoderm lineage.

Experience of more than 100 preimplantation genetic diagnosis cycles for monogenetic diseases using whole genome amplification and linkage analysis in a single centre.

Science.gov (United States)

Chow, Judy F C; Yeung, William S B; Lee, Vivian C Y; Lau, Estella Y L; Ho, P C; Ng, Ernest H Y

2015-08-01

To report the outcomes of more than 100 cycles of preimplantation genetic diagnosis for monogenetic diseases. Case series. Tertiary assisted reproductive centre in Hong Kong, where patients needed to pay for the cost of preimplantation genetic diagnosis on top of standard in-vitro fertilisation charges. Patients undergoing preimplantation genetic diagnosis for monogenetic diseases at the Centre of Assisted Reproduction and Embryology, Queen Mary Hospital-The University of Hong Kong between 1 August 2007 and 30 April 2014 were included. In-vitro fertilisation, intracytoplasmic sperm injection, embryo biopsy, and preimplantation genetic diagnosis. Ongoing pregnancy rate and implantation rate. Overall, 124 cycles of preimplantation genetic diagnosis were initiated in 76 patients, 101 cycles proceeded to preimplantation genetic diagnosis, and 92 cycles had embryo transfer. The ongoing pregnancy rate was 28.2% per initiated cycle and 38.0% per embryo transfer, giving an implantation rate of 35.2%. There were 16 frozen-thawed embryo transfer cycles in which, following preimplantation genetic diagnosis, cryopreserved embryos were replaced resulting in an ongoing pregnancy rate of 37.5% and implantation rate of 30.0%. The cumulative ongoing pregnancy rate was 33.1%. The most frequent indication for preimplantation genetic diagnosis was thalassaemia, followed by neurodegenerative disorder and cancer predisposition. There was no misdiagnosis. Preimplantation genetic diagnosis is a reliable method to prevent couples conceiving fetuses severely affected by known genetic disorders, with ongoing pregnancy and implantation rates similar to those for in-vitro fertilisation for routine infertility treatment.

Study of embryonic ploidy: a probable embryo model

Energy Technology Data Exchange (ETDEWEB)

Kundt, Miriam S; Cabrini, Romulo L [Comision Nacional de Energia Atomica, Buenos Aires (Argentina). Dept. de Radiobiologia

2001-07-01

The second polar body (PB) studies in preimplantation mouse embryos were carried out to evaluate the possibility as reference cell to analyze ploidy. For that purpose embryos in a one cell stage [obtained by crossing hybrid females (CBAxC57BL) to NIH males] were cultured in vitro during 72 hs, individually fixed at morula stage and stained with Feulgen. The DNA content of 263 individual nucleus was evaluated cytophotometrically corresponding to 22 compact morulas of normal development. As haploid PB is present in all pre implanted stage, only embryos with one haploid nuclei were considered as normal. In 95.5% (n = 21) of the embryos the PB was present. DNA measurement of 21 PB was 1n {+-} 0.1. By the height sensibility of PB ploidy, the abnormalities were detected by the criterion of >4.1 n and <1.9 n. The results showed that one embryo was completely haploid (1n). The rest of the embryos (n = 20) 222 blastomeres and 20 PB were analyzed. The DNA measurement showed that 92,7% of the blastomeres (n = 206) are between 2 n and 4 n and 7.3% showed ploidy anomalies, regarding the value n of their PB. The period of the cellular cycle was studied in the normal cell ploidy. This study showed that 16.5% of the blastomeres (n = 34) were in the period G1, 70.39% (n =34) in the period S and 13.2% in the period G2 (n = 27). It is concluded that the PB study showed that it has properties as an excellent indicator of internal ploidia: it is present from the moment of the conception, easily recognizable in the perivitelin space in the embryo of one-two cells, remains in interface during the preimplantation development, it is haploid and digitalized pixel by pixel PB study showed the homogeneity of this type of cell, giving a reliable value of ploidy. The properties of the PB and the results showed that the PB could be an excellent indicator for embryonic ploidy studies on genotoxicity, maintaining its original ploidia during the preimplantation development while the blastomeres are

Generating different genetic expression patterns in the early embryo: insights from the mouse model

Czech Academy of Sciences Publication Activity Database

Bruce, Alexander

2013-01-01

Roč. 27, č. 6 (2013), s. 586-592 ISSN 1472-6483 Grant - others:Marie Curie Career Integration Grant(CZ) IDNOVCELFAT2011; Czech Science Foundation(CZ) 13-032955 Institutional support: RVO:60077344 Keywords : cell fate * preimplantation embryo * probabilistic Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.980, year: 2013 http://www.sciencedirect.com/science/article/pii/S1472648313002435

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.

In vivo photoacoustic imaging of mouse embryos

Science.gov (United States)

Laufer, Jan; Norris, Francesca; Cleary, Jon; Zhang, Edward; Treeby, Bradley; Cox, Ben; Johnson, Peter; Scambler, Pete; Lythgoe, Mark; Beard, Paul

2012-06-01

The ability to noninvasively image embryonic vascular anatomy in mouse models is an important requirement for characterizing the development of the normal cardiovascular system and malformations in the heart and vascular supply. Photoacoustic imaging, which can provide high resolution non invasive images of the vasculature based upon optical absorption by endogenous hemoglobin, is well suited to this application. In this study, photoacoustic images of mouse embryos were obtained ex vivo and in vivo. The images show intricate details of the embryonic vascular system to depths of up to 10 mm, which allowed whole embryos to be imaged in situ. To achieve this, an all-optical photoacoustic scanner and a novel time reversal image reconstruction algorithm, which provide deep tissue imaging capability while maintaining high spatial resolution and contrast were employed. This technology may find application as an imaging tool for preclinical embryo studies in developmental biology as well as more generally in preclinical and clinical medicine for studying pathologies characterized by changes in the vasculature.

Comprehensive preimplantation genetic screening and sperm deoxyribonucleic acid fragmentation from three males carrying balanced chromosome rearrangements.

Science.gov (United States)

Ramos, Laia; Daina, Gemma; Del Rey, Javier; Ribas-Maynou, Jordi; Fernández-Encinas, Alba; Martinez-Passarell, Olga; Boada, Montserrat; Benet, Jordi; Navarro, Joaquima

2015-09-01

To assess whether preimplantation genetic screening can successfully identify cytogenetically normal embryos in couples carrying balanced chromosome rearrangements in addition to increased sperm DNA fragmentation. Comprehensive preimplantation genetic screening was performed on three couples carrying chromosome rearrangements. Sperm DNA fragmentation was assessed for each patient. Academic center. One couple with the male partner carrying a chromosome 2 pericentric inversion and two couples with the male partners carrying a Robertsonian translocation (13:14 and 14:21, respectively). A single blastomere from each of the 18 cleavage-stage embryos obtained was analysed by metaphase comparative genomic hybridization. Single- and double-strand sperm DNA fragmentation was determined by the alkaline and neutral Comet assays. Single- and double-strand sperm DNA fragmentation values and incidence of chromosome imbalances in the blastomeres were analyzed. The obtained values of single-strand sperm DNA fragmentation were between 47% and 59%, and the double-strand sperm DNA fragmentation values were between 43% and 54%. No euploid embryos were observed in the couple showing the highest single-strand sperm DNA fragmentation. However, euploid embryos were observed in the other two couples: embryo transfer was performed, and pregnancy was achieved by the couple showing the lowest sperm DNA fragmentation values. Preimplantation genetic screening enables the detection of euploid embryos in couples affected by balanced chromosome rearrangements and increased sperm DNA fragmentation. Even though sperm DNA fragmentation may potentially have clinical consequences on fertility, comprehensive preimplantation genetic screening allows for the identification and transfer of euploid embryos. Copyright © 2015. Published by Elsevier Inc.

Preimplantation genetic diagnosis for cystic fibrosis: a case report

Science.gov (United States)

Biazotti, Maria Cristina Santoro; Pinto, Walter; de Albuquerque, Maria Cecília Romano Maciel; Fujihara, Litsuko Shimabukuro; Suganuma, Cláudia Haru; Reigota, Renata Bednar; Bertuzzo, Carmen Sílvia

2015-01-01

Cystic fibrosis is an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator gene. This disorder produces a variable phenotype including lung disease, pancreatic insufficiency, and meconium ileus plus bilateral agenesis of the vas deferens causing obstructive azoospermia and male infertility. Preimplantation genetic diagnosis is an alternative that allows identification of embryos affected by this or other genetic diseases. We report a case of couple with cystic fibrosis; the woman had the I148 T mutation and the man had the Delta F508 gene mutation. The couple underwent in vitro fertilization, associated with preimplantation genetic diagnosis, and with subsequent selection of healthy embryos for uterine transfer. The result was an uneventful pregnancy and delivery of a healthy male baby. PMID:25993078

Preimplantation genetic diagnosis to improve pregnancy outcomes in subfertility.

Science.gov (United States)

Simpson, Joe Leigh

2012-12-01

Pre-implantation genetic diagnosis provides prenatal genetic diagnosis before implantation, thus allowing detection of chromosomal abnormalities and their exclusion from embryo transfer in assisted reproductive technologies. Polar body, blastomere or trophectoderm can each be used to obtain requisite genetic or embryonic DNA. Pre-implantation genetic diagnosis for excluding unbalanced translocations is well accepted, and pre-implantation genetic diagnosis aneuploidy testing to avoid repeated pregnancy losses in couples having recurrent aneuploidy is efficacious in reducing miscarriages. Controversy remains about whether pre-implantation genetic diagnosis aneuploidy testing improves take home pregnancy rates, for which reason adherence to specific indications is recommended while the issue is being adjudicated. Current recommendations are for obligatory 24 chromosome testing, most readily using array comparative genome hybridisation. Copyright © 2012 Elsevier Ltd. All rights reserved.

Preimplantation Genetic Diagnosis: The Situation in France and in Other European Countries.

Science.gov (United States)

Duguet, Anne-Marie; Boyer-Beviere, Bénédicte

2017-04-01

Preimplantation genetic diagnosis (PGD) relates exclusively to in vitro fertilisation techniques (IVF) that aim to prevent transmission of a serious genetic abnormality to the child. The genetic characteristics of the embryo created through IVF are analysed, and only the embryos free of the genetic abnormality are implanted in the womb. Performed worldwide since 1990, this technique has raised many legal and ethical debates due to the very wide variations of lawgiving between countries. This is shown by the report of the UNESCO IBC (2003), which described the techniques and the issues raised by preimplantation genetic diagnosis. In this article, the authors present the differences between prenatal diagnosis and preimplantation genetic diagnosis, the French legislation, then the range of legislation in Europe and finally the position of the European Court of Human Rights which sanctioned Italy and Latvia for refusing access to PGD.

Factors affecting the gene expression of in vitro cultured human preimplantation embryos

NARCIS (Netherlands)

Mantikou, E.; Jonker, M.J.; Wong, K.M.; van Montfoort, A.P.A.; de Jong, M.; Breit, T.M.; Repping, S.; Mastenbroek, S.

2016-01-01

STUDY QUESTION: What is the relative effect of common environmental and biological factors on transcriptome changes during human preimplantation development? SUMMARY ANSWER: Developmental stage and maternal age had a larger effect on the global gene expression profile of human preimplantation

Inherited effects from mouse immature oocytes following low-dose irradiation

International Nuclear Information System (INIS)

Straume, T.; Khan, R.; Raabe, O.G.; Walsh, K.J.; Wiley, L.M.

1992-07-01

Immature oocytes represent the genetic pool in female mice as well as in women and therefore are principal cells of concern for genetic studies. Previous studies have demonstrated that genetic effects in female mice can be masked by the hypersensitive plasma membrane lethality target of immature oocytes. Studies have also shown that genetic effects can be detected when the plasma mambrane is sufficiently spared. Here, new data obtained using the mouse preimplantation embryo chimera assay are presented and discussed in light of previous findings for irradiated mouse oocytes

Vitrified/warmed single blastocyst transfer in preimplantation genetic diagnosis/preimplantation genetic screening cycles.

Science.gov (United States)

Huang, Jin; Li, Rong; Lian, Ying; Chen, Lixue; Shi, Xiaodan; Qiao, Jie; Liu, Ping

2015-01-01

To investigate the single blastocyst transfer in preimplantation genetic diagnosis (PGD)/preimplantation genetic screening (PGS) cycles. 80 PGD/PGS cycles undergoing blastocyst biopsy were studied. There were 88 warming cycles during the study period. Only one warmed blastocyst was transferred per cycle. The outcomes were followed up to the infants were born. The embryo implantation rate was 54.55% (48/88). The clinical pregnancy rate was 54.55% (48/88) per transfer cycle and 60% (48/80) per initial PGD/PGS cycle. There was no multi-pregnant in this study. The live birth rate was 42.05% (37/88) per transfer cycle and 46.25% (37/80) per initial PGD/PGS cycle. In PGD/PGS cycles, single blastocyst transfer reduces the multiple pregnancy rate without affecting the clinical outcomes.

Imaging and differentiation of mouse embryo tissues by ToF-SIMS

Energy Technology Data Exchange (ETDEWEB)

Wu, L; Lu, X; Kulp, K; Knize, M; Berman, E; Nelson, E; Felton, J; Wu, K J

2006-06-16

Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) equipped with a gold ion gun was used to image mouse embryos and differentiate tissue types (brain, spinal cord, skull, rib, heart and liver). Embryos were paraffin-embedded and then de-paraffinized. The robustness and repeatability of the method was determined by analyzing nine tissue slices from three different embryos over a period of several weeks. Using Principal Component Analysis (PCA) to reduce the spectral data generated by ToF-SIMS, histopathologically identified tissue types of the mouse embryos can be differentiated based on the characteristic differences in their mass spectra. These results demonstrate the ability of ToF-SIMS to determine subtle chemical differences even in fixed histological specimens.

Impact of preimplantation genetic screening on donor oocyte-recipient cycles in the United States.

Science.gov (United States)

Barad, David H; Darmon, Sarah K; Kushnir, Vitaly A; Albertini, David F; Gleicher, Norbert

2017-11-01

Our objective was to estimate the contribution of preimplantation genetic screening to in vitro fertilization pregnancy outcomes in donor oocyte-recipient cycles. This was a retrospective cross-sectional study of US national data from the Society for Assisted Reproductive Technology Clinic Outcome Reporting System between 2005 and 2013. Society for Assisted Reproductive Technology Clinic Outcome Reporting relies on voluntarily annual reports by more than 90% of US in vitro fertilization centers. We evaluated pregnancy and live birth rates in donor oocyte-recipient cycles after the first embryo transfer with day 5/6 embryos. Statistical models, adjusted for patient and donor ages, number of embryos transferred, race, infertility diagnosis, and cycle year were created to compare live birth rates in 392 preimplantation genetic screening and 20,616 control cycles. Overall, pregnancy and live birth rates were significantly lower in preimplantation genetic screening cycles than in control cycles. Adjusted odds of live birth for preimplantation genetic screening cycles were reduced by 35% (odds ratio, 0.65, 95% confidence interval, 0.53-0.80; P cycles over the past 9 years, has not been associated with improved odds of live birth or reduction in miscarriage rates. Copyright © 2017 Elsevier Inc. All rights reserved.

Successful pregnancy with preimplantation genetic diagnosis in a woman with mosaic Turner syndrome.

Science.gov (United States)

Onalan, Gogsen; Yilmaz, Zerrin; Durak, Tulay; Sahin, Feride Iffet; Zeyneloglu, Hulusi Bulent

2011-04-01

To determine the efficacy of the preimplantation cytogenetic analysis of the embryos obtained from patient with mosaic Turner syndrome before an IVF program. Prospective cytogenetic analysis. University-based tertiary medical center. A 29 year-old female, a partner in a couple with male factor infertility, was diagnosed with mosaic Turner syndrome with a 45,X [17]/46,XX [13] karyotype. Preimplantation genetic diagnosis was performed on four blastomeres obtained from four different embryos by fluorescence in situ hybridization probes specific to chromosomes X, Y, 13, 18, 21 in an intracytoplasmic sperm injection cycle. Blastomeres with normal signals. Two blastomeres detected as normal were transferred and pregnancy was achieved. Preimplantation Genetic Diagnose should be considered in the infertility treatment of the patient with mosaic Turner Syndrome. Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

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.

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......), in vitro fertilized (IVF), somatic cell nuclear transfer (SCNT) and parthenogenetically activated (PA) embryos were evaluated for DNA methylation quantification at different developmental stages. Fertilized (IV and IVF) one-cell stages lacked a substantial active demethylation of the paternal genome...

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......), in vitro fertilized (IVF), somatic cell nuclear transfer (SCNT) and parthenogenetically activated (PA) embryos were evaluated for DNA methylation quantification at different developmental stages. Fertilized (IV and IVF) one-cell stages lacked a substantial active demethylation of the paternal genome...

Preimplantation diagnosis of genetic diseases

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Adiga S

2010-01-01

Full Text Available One of the landmarks in clinical genetics is prenatal diagnosis of genetic disorders. The recent advances in the field have made it possible to diagnose the genetic conditions in the embryos before implantation in a setting of in vitro fertilization. Polymerase chain reaction and fluorescence in situ hybridization are the two common techniques employed on a single or two cells obtained via embryo biopsy. The couple who seek in vitro fertilization may screen their embryos for aneuploidy and the couple at risk for a monogenic disorder but averse to abortion of the affected fetuses after prenatal diagnosis, are likely to be the best candidates to undergo this procedure. This article reviews the technique, indications, benefits, and limitations of pre-implantation genetic testing in clinical practice.

Fusion of blastomeres in mouse embryos under the action of femtosecond laser radiation. Efficiency of blastocyst formation and embryo development

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Osychenko, A A; Zalesskii, A D; Krivokharchenko, A S; Zhakhbazyan, A K; Nadtochenko, V A [N N Semenov Institute of Chemical Physics, Russian Academy of Sciences, Moscow (Russian Federation); Ryabova, A V [A M Prokhorov General Physics Institute, Russian Academy of Sciences, Moscow (Russian Federation)

2015-05-31

Using the method of femtosecond laser surgery we study the fusion of two-cell mouse embryos under the action of tightly focused femtosecond laser radiation with the fusion efficiency reaching 60%. The detailed statistical analysis of the efficiency of blastomere fusion and development of the embryo up to the blastocyst stage after exposure of the embryos from different mice to a femtosecond pulse is presented. It is shown that the efficiency of blastocyst formation essentially depends on the biological characteristics of the embryo, namely, the strain and age of the donor mouse. The possibility of obtaining hexaploid embryonal cells using the methods of femtosecond laser surgery is demonstrated. (extreme light fields and their applications)

Strain preservation of experimental animals: vitrification of two-cell stage embryos for multiple mouse strains.

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Eto, Tomoo; Takahashi, Riichi; Kamisako, Tsutomu

2015-04-01

Strain preservation of experimental animals is crucial for experimental reproducibility. Maintaining complete animal strains, however, is costly and there is a risk for genetic mutations as well as complete loss due to disasters or illness. Therefore, the development of effective vitrification techniques for cryopreservation of multiple experimental animal strains is important. We examined whether a vitrification method using cryoprotectant solutions, P10 and PEPeS, is suitable for preservation of multiple inbred and outbred mouse strains. First, we investigated whether our vitrification method using cryoprotectant solutions was suitable for two-cell stage mouse embryos. In vitro development of embryos exposed to the cryoprotectant solutions was similar to that of fresh controls. Further, the survival rate of the vitrified embryos was extremely high (98.1%). Next, we collected and vitrified two-cell stage embryos of 14 mouse strains. The average number of embryos obtained from one female was 7.3-33.3. The survival rate of vitrified embryos ranged from 92.8% to 99.1%, with no significant differences among mouse strains. In vivo development did not differ significantly between fresh controls and vitrified embryos of each strain. For strain preservation using cryopreserved embryos, two offspring for inbred lines and one offspring for outbred lines must be produced from two-cell stage embryos collected from one female. The expected number of surviving fetuses obtained from embryos collected from one female of either the inbred or outbred strains ranged from 2.9 to 19.5. The findings of the present study indicated that this vitrification method is suitable for strain preservation of multiple mouse strains. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Embryonic death, dwarfism and fetal malformations after irradiation of embryos at the zygote stage. Studies on two mouse strains

International Nuclear Information System (INIS)

Jacquet, P.; Saint-Georges, L. de; Baugnet-Mahieu, L.; Vankerkom, J.

1995-01-01

Female mice of the BALB/c and CF1 strains were mated and irradiated with various doses of X-rays 7 h after presumed fertilization. 18 days later, females were killed and their uteri examined for prenatal mortality at the different stages of development. Living fetuses were weighed and examined for the presence of external malformations. A number of them were also examined for skeletal anomalies. Radiation induced mainly a dose-dependent increase of the preimplantation loss in the BALB/c strain and of the early postimplantation loss in the CF1 strain. Embryos of the BALB/c strain were refractory to the induction of teratogenic effects after such preimplantation irradiation. In CF1 mice, the frequency of malformed fetuses increased regularly after irradiation, the difference with controls being significant for the doses of 10, 50 and 100 cGy. Dwarfism occurrence also appeared to be increased by irradiation in this strain, although the importance of this effect varied depending on the criterion chosen for the assessment of dwarfs. With the definition proposed in the present paper, the increase in the frequency of dwarfs paralleled that of malformed fetuses, being significant after doses of 50 and 100 cGy. Irradiation did not increase the frequency of skeletal anomalies. A careful examination of the various data obtained to date led us to conclude that radiation may possibly be teratogenic in several mouse strains, when administered as early as during the one-cell stage and, to a lesser extent, during the following preimplantation stages. However, early prenatal mortality will remain by far the greatest risk associated with an exposure to radiation during this period. Moreover, the relativity of the risk of abnormality due to such irradiation should be considered in the context of the high prevalence of developmental defects spontaneously occurring during human pregnancy

Embryonic death, dwarfism and fetal malformations after irradiation of embryos at the zygote stage. Studies on two mouse strains

Energy Technology Data Exchange (ETDEWEB)

Jacquet, P.; Saint-Georges, L. de; Baugnet-Mahieu, L. [Laboratory of Radiobiology, Department of Radioprotection, CEN/SCK, Mol (Belgium); Vankerkom, J. [Division of Environmental Research, VITO, Mol (Belgium)

1995-11-01

Female mice of the BALB/c and CF1 strains were mated and irradiated with various doses of X-rays 7 h after presumed fertilization. 18 days later, females were killed and their uteri examined for prenatal mortality at the different stages of development. Living fetuses were weighed and examined for the presence of external malformations. A number of them were also examined for skeletal anomalies. Radiation induced mainly a dose-dependent increase of the preimplantation loss in the BALB/c strain and of the early postimplantation loss in the CF1 strain. Embryos of the BALB/c strain were refractory to the induction of teratogenic effects after such preimplantation irradiation. In CF1 mice, the frequency of malformed fetuses increased regularly after irradiation, the difference with controls being significant for the doses of 10, 50 and 100 cGy. Dwarfism occurrence also appeared to be increased by irradiation in this strain, although the importance of this effect varied depending on the criterion chosen for the assessment of dwarfs. With the definition proposed in the present paper, the increase in the frequency of dwarfs paralleled that of malformed fetuses, being significant after doses of 50 and 100 cGy. Irradiation did not increase the frequency of skeletal anomalies. A careful examination of the various data obtained to date led us to conclude that radiation may possibly be teratogenic in several mouse strains, when administered as early as during the one-cell stage and, to a lesser extent, during the following preimplantation stages. However, early prenatal mortality will remain by far the greatest risk associated with an exposure to radiation during this period. Moreover, the relativity of the risk of abnormality due to such irradiation should be considered in the context of the high prevalence of developmental defects spontaneously occurring during human pregnancy.

Generation of single-copy transgenic mouse embryos directly from ES cells by tetraploid embryo complementation

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Zhao Roong

2001-12-01

Full Text Available Abstract Background Transgenic mice have been used extensively to analyze gene function. Unfortunately, traditional transgenic procedures have only limited use in analyzing alleles that cause lethality because lines of founder mice cannot be established. This is frustrating given that such alleles often reveal crucial aspects of gene function. For this reason techniques that facilitate the generation of embryos expressing such alleles would be of enormous benefit. Although the transient generation of transgenic embryos has allowed limited analysis of lethal alleles, it is expensive, time consuming and technically challenging. Moreover a fundamental limitation with this approach is that each embryo generated is unique and transgene expression is highly variable due to the integration of different transgene copy numbers at random genomic sites. Results Here we describe an alternative method that allows the generation of clonal mouse embryos harboring a single-copy transgene at a defined genomic location. This was facilitated through the production of Hprt negative embryonic stem cells that allow the derivation of embryos by tetraploid embryo complementation. We show that targeting transgenes to the hprt locus in these ES cells by homologous recombination can be efficiently selected by growth in HAT medium. Moreover, embryos derived solely from targeted ES cells containing a single copy LacZ transgene under the control of the α-myosin heavy chain promoter exhibited the expected cardiac specific expression pattern. Conclusion Our results demonstrate that tetraploid embryo complementation by F3 hprt negative ES cells facilitates the generation of transgenic mouse embryos containing a single copy gene at a defined genomic locus. This approach is simple, extremely efficient and bypasses any requirement to generate chimeric mice. Moreover embryos generated by this procedure are clonal in that they are all derived from a single ES cell lines. This

The status of preimplantation genetic diagnosis in Japan: a criticism.

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Munné, Santiago; Cohen, Jacques

2004-09-01

Advances in preimplantation genetic diagnosis (PGD) are occurring worldwide. New clinics specializing in this approach to the control of disease genes or imbalanced chromosome numbers in human preimplantation embryos continue to increase. One exception is Japan, where the Japanese Society of Obstetrics and Gynecology disapproves of this practice because it discriminates against people with genetic abnormalities. Yet, some doctors there wish to introduce this method to help their couples to improved forms of IVF. This paper stresses the rights of patients to have a healthy baby, if necessary by the use of PGD. It argues against prohibition, since it complements the current nature of prenatal diagnosis and avoids the need for abortions in case of afflicted embryos. Consideration is also given to other attempts at restriction that have failed.

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

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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.

Male and female meiotic behaviour of an intrachromosomal insertion determined by preimplantation genetic diagnosis

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Doshi Alpesh

2010-02-01

Full Text Available Abstract Background Two related family members, a female and a male balanced carrier of an intrachromosomal insertion on chromosome 7 were referred to our centre for preimplantation genetic diagnosis. This presented a rare opportunity to investigate the behaviour of the insertion chromosome during meiosis in two related carriers. The aim of this study was to carry out a detailed genetic analysis of the preimplantation embryos that were generated from the three treatment cycles for the male and two for the female carrier. Patients underwent in vitro fertilization and on day 3, 22 embryos from the female carrier and 19 embryos from the male carrier were biopsied and cells analysed by fluorescent in situ hybridization. Follow up analysis of 29 untransferred embryos was also performed for confirmation of the diagnosis and to obtain information on meiotic and mitotic outcome. Results In this study, the female carrier produced more than twice as many chromosomally balanced embryos as the male (76.5% vs. 36%, and two pregnancies were achieved for her. Follow up analysis showed that the male carrier had produced more highly abnormal embryos than the female (25% and 15% respectively and no pregnancies occurred for the male carrier and his partner. Conclusion This study compares how an intrachromosomal insertion has behaved in the meiotic and preimplantation stages of development in sibling male and female carriers. It confirms that PGD is an appropriate treatment in such cases. Reasons for the differing outcome for the two carriers are discussed.

New Advances of Preimplantation and Prenatal Genetic Screening and Noninvasive Testing as a Potential Predictor of Health Status of Babies

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Tanya Milachich

2014-01-01

Full Text Available The current morphologically based selection of human embryos for transfer cannot detect chromosome aneuploidies. So far, only biopsy techniques have been able to screen for chromosomal aneuploidies in the in vitro fertilization (IVF embryos. Preimplantation genetic diagnosis (PGD or screening (PGS involves the biopsy of oocyte polar bodies or embryonic cells and has become a routine clinical procedure in many IVF clinics worldwide, including recent development of comprehensive chromosome screening of all 23 pairs of chromosomes by microarrays for aneuploidy screening. The routine preimplantation and prenatal genetic diagnosis (PND require testing in an aggressive manner. These procedures may be invasive to the growing embryo and fetus and potentially could compromise the clinical outcome. Therefore the aim of this review is to summarize not only the new knowledge on preimplantation and prenatal genetic diagnosis in humans, but also on the development of potential noninvasive embryo and fetal testing that might play an important role in the future.

New Advances of Preimplantation and Prenatal Genetic Screening and Noninvasive Testing as a Potential Predictor of Health Status of Babies

Science.gov (United States)

2014-01-01

The current morphologically based selection of human embryos for transfer cannot detect chromosome aneuploidies. So far, only biopsy techniques have been able to screen for chromosomal aneuploidies in the in vitro fertilization (IVF) embryos. Preimplantation genetic diagnosis (PGD) or screening (PGS) involves the biopsy of oocyte polar bodies or embryonic cells and has become a routine clinical procedure in many IVF clinics worldwide, including recent development of comprehensive chromosome screening of all 23 pairs of chromosomes by microarrays for aneuploidy screening. The routine preimplantation and prenatal genetic diagnosis (PND) require testing in an aggressive manner. These procedures may be invasive to the growing embryo and fetus and potentially could compromise the clinical outcome. Therefore the aim of this review is to summarize not only the new knowledge on preimplantation and prenatal genetic diagnosis in humans, but also on the development of potential noninvasive embryo and fetal testing that might play an important role in the future. PMID:24783200

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

A novel embryo culture media supplement that improves pregnancy rates in mice.

Science.gov (United States)

Highet, A R; Bianco-Miotto, T; Pringle, K G; Peura, A; Bent, S; Zhang, J; Nottle, M B; Thompson, J G; Roberts, C T

2017-03-01

The preimplantation embryo in vivo is exposed to numerous growth factors in the female reproductive tract, which are not recapitulated in embryo culture media in vitro The IGF2 and plasminogen activator systems facilitate blastocyst development. We hypothesized that the addition of IGF2 in combination with urokinase plasminogen activator (uPA) and plasminogen could improve rates of blastocyst hatching and implantation in mice. B6BcF1 and CBAB6F2 mouse embryos were divided into one of four supplemented culture media treatment groups: (1) control (media only); (2) 12.5 nM IGF2; (3) 10 µg/mL uPA and 5 µg/mL plasminogen; or (4) a combination of IGF2, uPA and plasminogen treatments. Embryo development to blastocyst stage and hatching were assessed before transfer to pseudopregnant recipient females and implantation, pregnancy rates and postnatal growth were assessed. After 90.5 h of culture, IGF2 + U + P treatment increased the percentage of B6BcF1 embryos that were hatching/hatched and percentage developing to blastocyst stage compared with controls (P culture, IGF2, uPA and plasminogen supplementation of culture media can improve pregnancy success, but the effect of treatment is dependent on the mouse strain. © 2017 Society for Reproduction and Fertility.

[Analysis of clinical outcomes of different embryo stage biopsy in array comparative genomic hybridization based preimplantation genetic diagnosis and screening].

Science.gov (United States)

Shen, J D; Wu, W; Shu, L; Cai, L L; Xie, J Z; Ma, L; Sun, X P; Cui, Y G; Liu, J Y

2017-12-25

Objective: To evaluate the efficiency of the application of array comparative genomic hybridization (array-CGH) in preimplantation genetic diagnosis or screening (PGD/PGS), and compare the clinical outcomes of different stage embryo biopsy. Methods: The outcomes of 381 PGD/PGS cycles referred in the First Affiliated Hospital of Nanjing Medical University from July 2011 to August 2015 were retrospectively analyzed. There were 320 PGD cycles with 156 cleavage-stage-biopsy cycles and 164 trophectoderm-biopsy cycles, 61 PGS cycles with 23 cleavage-stage-biopsy cycles and 38 trophectoderm-biopsy cycles. Chromosomal analysis was performed by array-CGH technology combined with whole genome amplification. Single embryo transfer was performed in all transfer cycles. Live birth rate was calculated as the main clinical outcomes. Results: The embryo diagnosis rate of PGD/PGS by array-CGH were 96.9%-99.1%. In PGD biopsy cycles, the live birth rate per embryo transfer cycle and live birth rate per embryo biopsy cycle were 50.0%(58/116) and 37.2%(58/156) in cleavage-stage-biopsy group, 67.5%(85/126) and 51.8%(85/164) in trophectoderm-biopsy group (both P 0.05). Conclusions: High diagnosis rate and idea live birth rate are achieved in PGD/PGS cycles based on array-CGH technology. The live birth rate of trophectoderm-biopsy group is significantly higher than that of cleavage-stage-biopsy group in PGD cycles; the efficiency of trophectoderm-biopsy is better.

Ethanol impedes embryo transport and impairs oviduct epithelium

International Nuclear Information System (INIS)

Xu, Tonghui; Yang, Qiuhong; Liu, Ruoxi; Wang, Wenfu; Wang, Shuanglian; Liu, Chuanyong; Li, Jingxin

2016-01-01

Most studies have demonstrated that alcohol consumption is associated with decreased fertility. The aim of this study was to investigate the effects of alcohol on pre-implantation embryo transport and/or early embryo development in the oviduct. We reported here that ethanol concentration-dependently suppressed the spontaneous motility of isolated human oviduct strips (EC50 50 ± 6 mM), which was largely attenuated in the present of L-NAME, a classical nitric oxide synthase(NOS) competitive inhibitor. Notably, either acute or chronic alcohol intake delayed egg transport and retarded early development of the embryo in the mouse oviduct, which was largely rescued by co-administration of L-NAME in a acute alcohol intake group but not in chronic alcohol intake group. It is worth mentioning that the oviductal epithelium destruction was verified by scanning electron microscope (SEM) observations in chronic alcohol intake group. In conclusion, alcohol intake delayed egg transport and retarded early development of the embryo in the oviduct by suppressing the spontaneous motility of oviduct and/or impairing oviductal epithelium. These findings suggested that alcohol abuse increases the incident of ectopic pregnancy.

Ethanol impedes embryo transport and impairs oviduct epithelium.

Science.gov (United States)

Xu, Tonghui; Yang, Qiuhong; Liu, Ruoxi; Wang, Wenfu; Wang, Shuanglian; Liu, Chuanyong; Li, Jingxin

2016-05-16

Most studies have demonstrated that alcohol consumption is associated with decreased fertility. The aim of this study was to investigate the effects of alcohol on pre-implantation embryo transport and/or early embryo development in the oviduct. We reported here that ethanol concentration-dependently suppressed the spontaneous motility of isolated human oviduct strips (EC50 50±6mM), which was largely attenuated in the present of L-NAME, a classical nitric oxide synthase(NOS) competitive inhibitor. Notably, either acute or chronic alcohol intake delayed egg transport and retarded early development of the embryo in the mouse oviduct, which was largely rescued by co-administration of L-NAME in a acute alcohol intake group but not in chronic alcohol intake group. It is worth mentioning that the oviductal epithelium destruction was verified by scanning electron microscope (SEM) observations in chronic alcohol intake group. In conclusion, alcohol intake delayed egg transport and retarded early development of the embryo in the oviduct by suppressing the spontaneous motility of oviduct and/or impairing oviductal epithelium. These findings suggested that alcohol abuse increases the incident of ectopic pregnancy. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Preimplantation genetic diagnosis for Down syndrome pregnancy

Institute of Scientific and Technical Information of China (English)

ZHANG Yu; XU Chen-ming; ZHU Yi-min; DONG Min-yue; QIAN Yu-li; JIN Fan; HUANG He-feng

2007-01-01

Objective: To evaluate the effect of preimplantation genetic diagnosis (PGD) conducted for women who had Down syndrome pregnancy previously. Methods: Trisomy 21 was diagnosed by using fluorescence in site hybridization (FISH) before embryo transfer in two women who had Down syndrome pregnancies. Each received one or two PGD cycles respectively. Results:Case 1: one PGD cycle was conducted, two oocytes were fertilized and biopsied. One embryo is of trisomy 21 and the other of monosomy 21. No embryo was transferred. Case 2: two PGD cycles were conducted, in total, sixteen oocytes were fertilized and biopsied. Four embryos were tested to be normal, six of trisomy 21, and one of monosomy 21. Five had no signal. Four normal embryos were transferred but no pregnancy resulted. Conclusion: For couples who had pregnancies with Down syndrome previously, PGD can be considered, and has been shown to be an effective strategy.

Nucleolar re-activation is delayed in mouse embryos cloned from two different cell lines

DEFF Research Database (Denmark)

Svarcova, Olga; Dinnyes, A.; Polgar, Z.

2009-01-01

displayed early NPBs transformation. In conclusion, despite normal onset of EGA in cloned embryos, activation of functional nucleoli was one cell cycle delayed in NT embryos. NT-MEF embryos displayed normal targeting but delayed activation of nucleolar proteins. Contrary, in NT-HM1 embryos, both......Aim of this study was to evaluate and compare embryonic genome activation (EGA) in mouse embryos of different origin using nucleolus as a marker. Early and late 2-cell and late 4-cell stage embryos, prepared by in vitro fertilization (IVF), parthenogenetic activation (PG), and nuclear transfer...... ofmouse embryonic fibroblast (MEF) and mouse HM1 emryonic stem cells (HM1), were processed for autoradiography following 3H-uridine incubation (transcriptional activity), transmission electron microscopy (ultrastructure) and immunofluorescence (nucleolar proteins; upstream binding factor, UBF...

Conversion and non-conversion approach to preimplantation diagnosis for chromosomal rearrangements in 475 cycles.

Science.gov (United States)

Kuliev, Anver; Janzen, Jeanine Cieslak; Zlatopolsky, Zev; Kirillova, Irina; Ilkevitch, Yury; Verlinsky, Yury

2010-07-01

Due to the limitations of preimplantation genetic diagnosis (PGD) for chromosomal rearrangements by interphase fluorescent in-situ hybridization (FISH) analysis, a method for obtaining chromosomes from single blastomeres was introduced by their fusion with enucleated or intact mouse zygotes, followed by FISH analysis of the resulting heterokaryons. Although this allowed a significant improvement in the accuracy of testing of both maternally and paternally derived translocations, it is still labour intensive and requires the availability of fertilized mouse oocytes, also creating ethical issues related to the formation of interspecies heterokaryons. This method was modified with a chemical conversion procedure that has now been clinically applied for the first time on 877 embryos from PGD cycles for chromosomal rearrangements and has become the method of choice for performing PGD for structural rearrangements. This is presented within the context of overall experience of 475 PGD cycles for translocations with pre-selection and transfer of balanced or normal embryos in 342 (72%) of these cycles, which resulted in 131 clinical pregnancies (38%), with healthy deliveries of 113 unaffected children. The spontaneous abortion rate in these cycles was as low as 17%, which confirms an almost five-fold reduction of spontaneous abortion rate following PGD for chromosomal rearrangements. 2010 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

The Past, Present, and Future of Preimplantation Genetic Testing.

Science.gov (United States)

Imudia, Anthony N; Plosker, Shayne

2016-06-01

Preimplantation genetic testing (PGT) of oocytes and embryos is the earliest form of prenatal testing. PGT requires in vitro fertilization for embryo creation. In the past 25 years, the use of PGT has increased dramatically. The indications of PGT include identification of embryos harboring single-gene disorders, chromosomal structural abnormalities, chromosomal numeric abnormalities, and mitochondrial disorders; gender selection; and identifying unaffected, HLA-matched embryos to permit the creation of a savior sibling. PGT is not without risks, limitations, or ethical controversies. This review discusses the techniques and clinical applications of different forms of PGT and the debate surrounding its associated uncertainty and expanded use. Copyright © 2016 Elsevier Inc. All rights reserved.

Gonadotropin Releasing Hormone Agonists or Antagonists for Preimplantation Genetic Diagnosis (PGD)? A Prospective Randomised Trial.

Science.gov (United States)

Verpoest, Willem; De Vos, Anick; De Rycke, Martine; Parikh, Shruti; Staessen, Catherine; Tournaye, Herman; De Vos, Michel; Vloeberghs, Veerle; Blockeel, Christophe

2017-11-10

The use of GnRH analogue medication is essential in reproductive medicine to avoid premature ovulation by pituitary suppression for the duration of ovarian stimulation by gonadotrophins. The type of pituitary suppression by either GnRH agonist analogues versus GnRH antagonist analogues may result in different embryological hence clinical results. Preimplantation genetic diagnosis is a subtype of IVF in which embryos are created for genetic diagnosis of hereditary disorders in order to avoid genetically affected children. Embryological quality hence ovarian stimulation in preimplantation genetic diagnosis is crucial as genetic selection will reduce the number of available embryos to a fraction of the total. The aim of this study was to assess the efficiency of GnRH antagonist versus GnRH agonist treatment for pituitary suppression in ovarian stimulation for PGD, by proxy of number and quality of embryos at cleavage stage available for biopsy. We conducted a prospective randomised controlled trial comparing pituitary suppression by GnRH antagonist versus GnRH agonist in ovarian stimulation for PGD. The primary outcome measure was the number of embryos of sufficient quality for biopsy at cleavage stage. Secondary outcome parameters were the number of blastocysts available of top quality, and clinical pregnancy rate. There was no difference in number of oocytes retrieved, embryos at cleavage stage available for biopsy or embryo quality. The clinical pregnancy rate was higher in the GnRH agonist group; however the sample size was insufficient to allow conclusions. The use of GnRH agonist versus antagonist treatment does not result in differences in a number of oocytes, embryos or embryo quality in ovarian stimulation for preimplantation genetic diagnosis. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Preimplantation genetic diagnosis by fluorescence in situ hybridization of reciprocal and Robertsonian translocations.

Science.gov (United States)

Chen, Chun-Kai; Wu, Dennis; Yu, Hsing-Tse; Lin, Chieh-Yu; Wang, Mei-Li; Yeh, Hsin-Yi; Huang, Hong-Yuan; Wang, Hsin-Shin; Soong, Yung-Kuei; Lee, Chyi-Long

2014-03-01

The presence of reciprocal and Robertsonian chromosomal rearrangement is often related to recurrent miscarriage. Using preimplantation genetic diagnosis, the abortion rate can be decreased. Cases treated at our center were reviewed. A retrospective analysis for either Robertsonian or reciprocal translocations was performed on all completed cycles of preimplantation genetic diagnosis at our center since the first reported case in 2004 until the end of 2010. Day 3 embryo biopsies were carried out, and the biopsied cell was checked by fluorescent in situ hybridization using relevant informative probes. Embryos with a normal or balanced translocation karyotype were transferred on Day 4. Thirty-eight preimplantation genetic diagnosis cycles involving 17 couples were completed. A total of 450 (82.6%) of the total oocytes were MII oocytes, and 158 (60.0%) of the two-pronuclei embryos were biopsied. In 41.4% of the fluorescent in situ hybridization analyses, the results were either normal or balanced. Embryos were transferred back after 21 cycles. Three babies were born from Robertsonian translocation carriers and another two from reciprocal translocation carriers. The miscarriage rate was 0%. Among the reciprocal translocation group, the live delivery rate was 8.3% per ovum pick-up cycle and 18.2% per embryo transfer cycle. Among the Robertsonian translocation group, the live delivery rate was 14.3% per ovum pick-up cycle and 20.0% per embryo transfer cycle. There is a trend whereby the outcome for Robertsonian translocation group carriers is better than that for reciprocal translocation group carriers. Aneuploidy screening may possibly be added in order to improve the outcome, especially for individuals with an advanced maternal age. The emergence of an array-based technology should help improve this type of analysis. Copyright © 2014. Published by Elsevier B.V.

Lack of metformin effect on mouse embryo AMPK activity: implications for metformin treatment during pregnancy.

Science.gov (United States)

Lee, Hyung-Yul; Wei, Dan; Loeken, Mary R

2014-01-01

Adenosine monophosphate-activated protein kinase (AMPK) is stimulated in embryos during diabetic pregnancy by maternal hyperglycaemia-induced embryo oxidative stress. Stimulation of AMPK disrupts embryo gene expression and causes neural tube defects. Metformin, which may be taken during early pregnancy, has been reported to stimulate AMPK activity. Thus, the benefits of improved glycaemic control could be offset by stimulated embryo AMPK activity. Here, we investigated whether metformin can stimulate AMPK activity in mouse embryos and can adversely affect embryo gene expression and neural tube defects. Pregnant nondiabetic mice were administered metformin beginning on the first day of pregnancy. Activation of maternal and embryo AMPK [phospho-AMPK α (Thr172) relative to total AMPK], expression of Pax3, a gene required for neural tube closure, and neural tube defects were studied. Mouse embryonic stem cells were used as a cell culture model of embryonic neuroepithelium to study metformin effects on AMPK and Pax3 expression. Metformin had no effect on AMPK in embryos or maternal skeletal muscle but increased activated AMPK in maternal liver. Metformin did not inhibit Pax3 expression or increase neural tube defects. However, metformin increased activated AMPK and inhibited Pax3 expression by mouse embryonic stem cells. Mate1/Slc47a1 and Oct3/Slc22a, which encode metformin transporters, were expressed at barely detectable levels by embryos. Although metformin can have effects associated with diabetic embryopathy in vitro, the lack of effects on mouse embryos in vivo may be due to lack of metformin transporters and indicates that the benefits of metformin on glycaemic control are not counteracted by stimulation of embryo AMPK activity and consequent embryopathy. Copyright © 2013 John Wiley & Sons, Ltd.

Effect of Short-Term Hypergravity Treatment on Mouse 2-Cell Embryo Development

Science.gov (United States)

Ning, Li-Na; Lei, Xiao-Hua; Cao, Yu-Jing; Zhang, Yun-Fang; Cao, Zhong-Hong; Chen, Qi; Duan, En-Kui

2015-11-01

Though there are numerous biological experiments, which have been performed in a space environment, to study the physiological effect of space travel on living organisms, while the potential effect of weightlessness or short-term hypergravity on the reproductive system in most species, particularly in mammalian is still controversial and unclear. In our previous study, we investigated the effect of space microgravity on the development of mouse 4-cell embryos by using Chinese SJ-8. .Unexpectedly, we did not get any developed embryo during the space-flight. Considering that the process of space experiment is quite different from most experiments done on earth in several aspects such as, the vibration and short-term hypergravity during the rock launching and landing. Thus we want to know whether the short-term hypergravity produced by the launch process affect the early embryo development in mice, and howthe early embryos respond to the hypergravity. In present study, we are mimicking the short-term hypergravity during launch by using a centrifuge to investigate its influence on the development of early embryo (2-cell) in mice. We also examined the actin filament distribution in 2-cell embryos by immunostaining to test their potential capacity of development under short-term hypergravity exposure. Our results showed that most 2-cell embryos in the hypergravity exposure groups developed into blastocysts with normal morphology after 72h cultured in vitro, and there is no obvious difference in the development rate of blastocyst formation compared to the control. Moreover, there were no statistically significant differences in birth rates after oviduct transfer of 2-cell mouse embryos exposed on short-term hypergravity compared with 1 g condition. In addition, the well-organized actin distribution appeared in 2-cell embryos after exposed on hypergravity and also in the subsequent developmental blastocysts. Taken together, our data shows that short-term exposure in

Dynamics and ethics of comprehensive preimplantation genetic testing: a review of the challenges.

Science.gov (United States)

Hens, Kristien; Dondorp, Wybo; Handyside, Alan H; Harper, Joyce; Newson, Ainsley J; Pennings, Guido; Rehmann-Sutter, Christoph; de Wert, Guido

2013-01-01

Genetic testing of preimplantation embryos has been used for preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS). Microarray technology is being introduced in both these contexts, and whole genome sequencing of blastomeres is also expeted to become possible soon. The amount of extra information such tests will yield may prove to be beneficial for embryo selection, will also raise various ethical issues. We present an overview of the developments and an agenda-setting exploration of the ethical issues. The paper is a joint endeavour by the presenters at an explorative 'campus meeting' organized by the European Society of Human Reproduction and Embryology in cooperation with the department of Health, Ethics & Society of the Maastricht University (The Netherlands). The increasing amount and detail of information that new screening techniques such as microarrays and whole genome sequencing offer does not automatically coincide with an increasing understanding of the prospects of an embryo. From a technical point of view, the future of comprehensive embryo testing may go together with developments in preconception carrier screening. From an ethical point of view, the increasing complexity and amount of information yielded by comprehensive testing techniques will lead to challenges to the principle of reproductive autonomy and the right of the child to an open future, and may imply a possible larger responsibility of the clinician towards the welfare of the future child. Combinations of preconception carrier testing and embryo testing may solve some of these ethical questions but could introduce others. As comprehensive testing techniques are entering the IVF clinic, there is a need for a thorough rethinking of traditional ethical paradigms regarding medically assisted reproduction.

Preimplantation genetic diagnosis of X-linked diseases examined by indirect linkage analysis.

Science.gov (United States)

Borgulova, I; Putzova, M; Soldatova, I; Krautova, L; Pecnova, L; Mika, J; Kren, R; Potuznikova, P; Stejskal, D

2015-01-01

Many centers of assisted reproduction in the Czech Republic offer preimplantation genetic diagnosis with fluorescent in situ hybridization (FISH) to couples requiring preimplantation genetic diagnosis (PGD) of X-linked diseases. However, this process results in discarding all male embryos and is not able to distinguish a carrier or healthy female embryo in X-linked recessive disorders. The main aim of this study was to summarize a six-year period of PGD of X-linked monogenic diseases using indirect linkage analysis. We wanted to accentuate the advantage indirect analysis of PGD using multiple displacement amplification (MDA) followed by short tandem repeat (STR) analysis. We present forty-six PGD cycles, including pre-case haplotyping (PGH) panel, for fifteen X-linked diseases. Embryo transfer was made thirty-eight times and gravidity was confirmed in thirteen female probands with a success rate of pregnancy calculated at 42 %. PGD procedure using MDA amplification followed by STR analysis provides help in identifying genetic defects within embryos prior to implantation. The reliability of the method was also supported by high pregnancy rate compared to other publications, which commonly achieved a 30-35 % success rate (Tab. 2, Fig. 1, Ref. 33).

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.

Recent advances in preimplantation genetic diagnosis

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Kahraman S

2015-04-01

Full Text Available Semra Kahraman, Çağri Beyazyürek, Hüseyin Avni Taç, Caroline Pirkevi, Murat Cetinkaya, Neşe Gülüm IVF and Reproductive Genetics Center, Istanbul Memorial Hospital, Istanbul, Turkey Abstract: Preimplantation genetic diagnosis (PGD is an important method for the identification chromosomal abnormalities and genes responsible for genetic defects in embryos that are created through in vitro fertilization before pregnancy. As the list of conditions and indications for PGD testing is continuing to extend enormously, novel in vitro fertilization techniques and newly established genetic analysis techniques have been implemented in clinical settings in the recent years. Blastocyst-stage biopsy, vitrification techniques, time-lapse imaging, whole-genome amplification, array-based diagnostic techniques, and next-generation sequencing techniques are promising techniques for the accurate diagnosis of diverse genetic conditions and also for the selection of the best embryo that has the highest implantation capacity. The timing and technique used for biopsy, the amplification techniques, the genetic diagnosis techniques, and appropriate genetic counseling play important roles in establishing a successful PGD. In this review, those key points of PGD will be reviewed in detail. Keywords: preimplantation genetic diagnosis, array comparative genomic hybridization, single-nucleotide polymorphism arrays, next-generation sequencing, monogenic disorders, aneuploidy testing 

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 ...

Preimplantation genetic diagnosis with HLA matching.

Science.gov (United States)

Rechitsky, Svetlana; Kuliev, Anver; Tur-Kaspa, Illan; Morris, Randy; Verlinsky, Yury

2004-08-01

Preimplantation genetic diagnosis (PGD) has recently been offered in combination with HLA typing, which allowed a successful haematopoietic reconstitution in affected siblings with Fanconi anaemia by transplantation of stem cells obtained from the HLA-matched offspring resulting from PGD. This study presents the results of the first PGD practical experience performed in a group of couples at risk for producing children with genetic disorders. These parents also requested preimplantation HLA typing for treating the affected children in the family, who required HLA-matched stem cell transplantation. Using a standard IVF procedure, oocytes or embryos were tested for causative gene mutations simultaneously with HLA alleles, selecting and transferring only those unaffected embryos, which were HLA matched to the affected siblings. The procedure was performed for patients with children affected by Fanconi anaemia (FANC) A and C, different thalassaemia mutations, Wiscott-Aldrich syndrome, X-linked adrenoleukodystrophy, X-linked hyperimmunoglobulin M syndrome and X-linked hypohidrotic ectodermal displasia with immune deficiency. Overall, 46 PGD cycles were performed for 26 couples, resulting in selection and transfer of 50 unaffected HLA-matched embryos in 33 cycles, yielding six HLA-matched clinical pregnancies and the birth of five unaffected HLA-matched children. Despite the controversy of PGD use for HLA typing, the data demonstrate the usefulness of this approach for at-risk couples, not only to avoid the birth of affected children with an inherited disease, but also for having unaffected children who may also be potential HLA-matched donors of stem cells for treatment of affected siblings.

Radioactive marking of proteins in cultured mouse embryos

International Nuclear Information System (INIS)

Nowak, J.

1984-01-01

The purpose of this work was to build an in vitro test system, with which on the one hand postimplantation embryos of the mouse could be cultured without morphological of physiological damage and on the other hand their protein could be as highly marked as possible. With this radioactively marked proteins were to be won, which are optimally suited for a high separation by two-dimensional electrophoresis. In addition incubation and preparation methods were found for the ages of day 10, 11 and 12 of the embryonic development. With the use of 3 H-marked amino acids in the culture medium it was determined that embryos without embryonic membranes, so-called N-embryos, built in more radioactivity into their proteins than the embryos with embryonic membranes, the so-called DAO-embryos or the DO-embryos. On the contrary, the embryos with intact blood circulation (DO-embryos) showed an even distribution of radioactive marker in their bodies. Since an even distribution of the marker in the embryo is a necessary prerequisite for a representative presentation of the proteins by 2DE, the DO-preparation was considered the best suited method. In order to increase the amount of radioactivity incorporated into the proteins of the DO-embryos, the concentration of the used isotope or the incubation length could be increased. A combination of both proved to be the best method. A 14 C-marked amino acid mixture of 20 μCi/corresponds to 20 μl instead of the usual 150 μCi 3 H-marked amino acids in a culture medium proved to be equally suitable. Notable changes which would have indicated a damaging affect of the used radioactivity or the in vitro culturing were not observed. The achieved methodical conditions were used for the presentation of the embryo proteins by two-dimensional electrophoresis and fluorography. (orig./MG) [de

Toxicity testing of human assisted reproduction devices using the mouse embryo assay.

NARCIS (Netherlands)

Punt-Van der Zalm, J.P.; Hendriks, J.C.M.; Westphal, J.R.; Kremer, J.A.M.; Teerenstra, S.; Wetzels, A.M.M.

2009-01-01

Systems to assess the toxicity of materials used in human assisted reproduction currently lack efficiency and/or sufficient discriminatory power. The development of 1-cell CBA/B6 F1 hybrid mouse embryos to blastocysts, expressed as blastocyst rate (BR), is used to measure toxicity. The embryos were

Preimplantation genetic diagnosis guided by single-cell genomics

Science.gov (United States)

2013-01-01

Preimplantation genetic diagnosis (PGD) aims to help couples with heritable genetic disorders to avoid the birth of diseased offspring or the recurrence of loss of conception. Following in vitro fertilization, one or a few cells are biopsied from each human preimplantation embryo for genetic testing, allowing diagnosis and selection of healthy embryos for uterine transfer. Although classical methods, including single-cell PCR and fluorescent in situ hybridization, enable PGD for many genetic disorders, they have limitations. They often require family-specific designs and can be labor intensive, resulting in long waiting lists. Furthermore, certain types of genetic anomalies are not easy to diagnose using these classical approaches, and healthy offspring carrying the parental mutant allele(s) can result. Recently, state-of-the-art methods for single-cell genomics have flourished, which may overcome the limitations associated with classical PGD, and these underpin the development of generic assays for PGD that enable selection of embryos not only for the familial genetic disorder in question, but also for various other genetic aberrations and traits at once. Here, we discuss the latest single-cell genomics methodologies based on DNA microarrays, single-nucleotide polymorphism arrays or next-generation sequence analysis. We focus on their strengths, their validation status, their weaknesses and the challenges for implementing them in PGD. PMID:23998893

Regulation of cAMP on the first mitotic cell cycle of mouse embryos.

Science.gov (United States)

Yu, Aiming; Zhang, Zhe; Bi, Qiang; Sun, Bingqi; Su, Wenhui; Guan, Yifu; Mu, Runqing; Miao, Changsheng; Zhang, Jie; Yu, Bingzhi

2008-03-01

Mitosis promoting factor (MPF) plays a central role during the first mitosis of mouse embryo. We demonstrated that MPF activity increased when one-cell stage mouse embryo initiated G2/M transition following the decrease of cyclic adenosine 3', 5'-monophosphate (cAMP) and cAMP-dependent protein kinase (PKA) activity. When cAMP and PKA activity increases again, MPF activity decreases and mouse embryo starts metaphase-anaphase transition. In the downstream of cAMP/PKA, there are some effectors such as polo-like kinase 1 (Plk1), Cdc25, Mos (mitogen-activated protein kinase kinase kinase), MEK (mitogen-activated protein kinase kinase), mitogen-activated protein kinase (MAPK), Wee1, anaphase-promoting complex (APC), and phosphoprotein phosphatase that are involved in the regulation of MPF activity. Here, we demonstrated that following activation of MPF, MAPK activity was steady, whereas Plk1 activity fluctuated during the first cell cycle. Plk1 activity was the highest at metaphase and decreased at metaphase-anaphase transition. Further, we established a mathematical model using Gepasi algorithm and the simulation was in agreement with the experimental data. Above all the evidences, we suggested that cAMP and PKA might be the upstream factors which were included in the regulation of the first cell cycle development of mouse embryo. Copyright 2007 Wiley-Liss, Inc.

Mouse model of chromosome mosaicism reveals lineage-specific depletion of aneuploid cells and normal developmental potential.

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Bolton, Helen; Graham, Sarah J L; Van der Aa, Niels; Kumar, Parveen; Theunis, Koen; Fernandez Gallardo, Elia; Voet, Thierry; Zernicka-Goetz, Magdalena

2016-03-29

Most human pre-implantation embryos are mosaics of euploid and aneuploid cells. To determine the fate of aneuploid cells and the developmental potential of mosaic embryos, here we generate a mouse model of chromosome mosaicism. By treating embryos with a spindle assembly checkpoint inhibitor during the four- to eight-cell division, we efficiently generate aneuploid cells, resulting in embryo death during peri-implantation development. Live-embryo imaging and single-cell tracking in chimeric embryos, containing aneuploid and euploid cells, reveal that the fate of aneuploid cells depends on lineage: aneuploid cells in the fetal lineage are eliminated by apoptosis, whereas those in the placental lineage show severe proliferative defects. Overall, the proportion of aneuploid cells is progressively depleted from the blastocyst stage onwards. Finally, we show that mosaic embryos have full developmental potential, provided they contain sufficient euploid cells, a finding of significance for the assessment of embryo vitality in the clinic.

Tripolar chromosome segregation drives the association between maternal genotype at variants spanning PLK4 and aneuploidy in human preimplantation embryos.

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McCoy, Rajiv C; Newnham, Louise J; Ottolini, Christian S; Hoffmann, Eva R; Chatzimeletiou, Katerina; Cornejo, Omar E; Zhan, Qiansheng; Zaninovic, Nikica; Rosenwaks, Zev; Petrov, Dmitri A; Demko, Zachary P; Sigurjonsson, Styrmir; Handyside, Alan H

2018-04-24

Aneuploidy is prevalent in human embryos and is the leading cause of pregnancy loss. Many aneuploidies arise during oogenesis, increasing with maternal age. Superimposed on these meiotic aneuploidies are frequent errors occurring during early mitotic divisions, contributing to widespread chromosomal mosaicism. Here we reanalyzed a published dataset comprising preimplantation genetic testing for aneuploidy in 24,653 blastomere biopsies from day-3 cleavage-stage embryos, as well as 17,051 trophectoderm biopsies from day-5 blastocysts. We focused on complex abnormalities that affected multiple chromosomes simultaneously, seeking insights into their formation. In addition to well-described patterns such as triploidy and haploidy, we identified 4.7% of blastomeres possessing characteristic hypodiploid karyotypes. We inferred this signature to have arisen from tripolar chromosome segregation in normally-fertilized diploid zygotes or their descendant diploid cells. This could occur via segregation on a tripolar mitotic spindle or by rapid sequential bipolar mitoses without an intervening S-phase. Both models are consistent with time-lapse data from an intersecting set of 77 cleavage-stage embryos, which were enriched for the tripolar signature among embryos exhibiting abnormal cleavage. The tripolar signature was strongly associated with common maternal genetic variants spanning the centrosomal regulator PLK4, driving the association we previously reported with overall mitotic errors. Our findings are consistent with the known capacity of PLK4 to induce tripolar mitosis or precocious M-phase upon dysregulation. Together, our data support tripolar chromosome segregation as a key mechanism generating complex aneuploidy in cleavage-stage embryos and implicate maternal genotype at a quantitative trait locus spanning PLK4 as a factor influencing its occurrence.

Radiation effects on cultured mouse embryos in relation to cell division cycle

International Nuclear Information System (INIS)

Domon, M.

1982-01-01

The authors have worked with mouse embryos in vitro asking first, what are the suitable parameters to define the radiation sensitivity of embryos, and second what is a major factor determining it. The LD 50 was adopted as a parameter of the radiation sensitivity of a population in a mouse embryo system in culture. The fertilized ova were collected into Whitten's medium at various times during the pronuclear and 2-cell stages of development. They were irradiated in chambers with X-rays at doses of 0 to 800 rads. After the embryos were cultured, a set of the lethal fractions for various X-ray doses were obtained. Regarding the radiation sensitivity variation of the embryos, the LD 50 varied from 100 to 200 rads during the pronuclear stage and from 100 to 600 rads during the 2-cell stage. The embryos during the pronuclear stage were most radioresistant at early G 2 phase, followed by an increase in the sensitivity. The embryos during the 2-cell stage were also most radioresistant at early G 2 phase and were more sensitive when they got close to either the first or the second cleavage division. Furthermore, it seems that the factor 6 of the large variation was due to the extremely long G 2 period, 14 hrs for the 2-cell embryos. That is, the pooled 2-cell embryos were in a relative sense well synchronized with G 2 phase. In contrast, the synchrony was poor during the pronuclear stage, which led to less variation of the LD 50 for the pronuclear embryos. It is concluded that during the early cleavage stages of mice, radiosensitivity is mainly governed by the content of cells of various cell cycle ages in the embryo. (Namekawa, K.)

Single-Cell Profiling of Epigenetic Modifiers Identifies PRDM14 as an Inducer of Cell Fate in the Mammalian Embryo

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Adam Burton

2013-11-01

Full Text Available Cell plasticity or potency is necessary for the formation of multiple cell types. The mechanisms underlying this plasticity are largely unknown. Preimplantation mouse embryos undergo drastic changes in cellular potency, starting with the totipotent zygote through to the formation of the pluripotent inner cell mass (ICM and differentiated trophectoderm in the blastocyst. Here, we set out to identify and functionally characterize chromatin modifiers that define the transitions of potency and cell fate in the mouse embryo. Using a quantitative microfluidics approach in single cells, we show that developmental transitions are marked by distinctive combinatorial profiles of epigenetic modifiers. Pluripotent cells of the ICM are distinct from their differentiated trophectoderm counterparts. We show that PRDM14 is heterogeneously expressed in 4-cell-stage embryos. Forced expression of PRDM14 at the 2-cell stage leads to increased H3R26me2 and can induce a pluripotent ICM fate. Our results shed light on the epigenetic networks that govern cellular potency and identity in vivo.

Effect of induced peritoneal endometriosis on oocyte and embryo quality in a mouse model.

Science.gov (United States)

Cohen, J; Ziyyat, A; Naoura, I; Chabbert-Buffet, N; Aractingi, S; Darai, E; Lefevre, B

2015-02-01

To assess the impact of peritoneal endometriosis on oocyte and embryo quality in a mouse model. Peritoneal endometriosis was surgically induced in 33 B6CBA/F1 female mice (endometriosis group, N = 17) and sham-operated were used as control (sham group, N = 16). Mice were superovulated 4 weeks after surgery and mated or not, to collect E0.5-embryos or MII-oocytes. Evaluation of oocyte and zygote quality was done by immunofluorescence under spinning disk confocal microscopy. Endometriosis-like lesions were observed in all mice of endometriosis group. In both groups, a similar mean number of MII oocytes per mouse was observed in non-mated mice (30.2 vs 32.6), with a lower proportion of normal oocytes in the endometriosis group (61 vs 83 %, p endometriosis group (21 vs 35.5, p = 0.02) without difference in embryo quality. Our results support that induced peritoneal endometriosis in a mouse model is associated with a decrease in oocyte quality and embryo number. This experimental model allows further studies to understand mechanisms of endometriosis-associated infertility.

Sex and PRNP genotype determination in preimplantation caprine embryos.

Science.gov (United States)

Guignot, F; Perreau, C; Cavarroc, C; Touzé, J-L; Pougnard, J-L; Dupont, F; Beckers, J-F; Rémy, B; Babilliot, J-M; Bed'Hom, B; Lamorinière, J M; Mermillod, P; Baril, G

2011-08-01

The objective of this study was to test the accuracy of genotype diagnosis after whole amplification of DNA extracted from biopsies obtained by trimming goat embryos and to evaluate the viability of biopsied embryos after vitrification/warming and transfer. Whole genome amplification (WGA) was performed using Multiple Displacement Amplification (MDA). Sex and prion protein (PRNP) genotypes were determined. Sex diagnosis was carried out by PCR amplification of ZFX/ZFY and Y chromosome-specific sequences. Prion protein genotype determination was performed on codons 142, 154, 211, 222 and 240. Embryos were collected at day 7 after oestrus and biopsied either immediately after collection (blastocysts and expanded blastocysts) or after 24 h of in vitro culture (compacted morulae). Biopsied embryos were frozen by vitrification. Vitrified whole embryos were kept as control. DNA of biopsies was extracted and amplified using MDA. Sex diagnosis was efficient for 97.4% of biopsies and PRNP genotyping was determined in 78.7% of biopsies. After embryo transfer, no significant difference was observed in kidding rate between biopsied and vitrified control embryos, whereas embryo survival rate was different between biopsied and whole vitrified embryos (p = 0.032). At birth, 100% of diagnosed sex and 98.2% of predetermined codons were correct. Offspring PRNP profiles were in agreement with parental genotype. Whole genome amplification with MDA kit coupled with sex diagnosis and PRNP genotype predetermination are very accurate techniques to genotype goat embryos before transfer. These novel results allow us to plan selection of scrapie-resistant genotypes and kid sex before transfer of cryopreserved embryo. © 2010 Blackwell Verlag GmbH.

Symmetry breaking, germ layer specification and axial organisation in aggregates of mouse embryonic stem cells.

Science.gov (United States)

van den Brink, Susanne C; Baillie-Johnson, Peter; Balayo, Tina; Hadjantonakis, Anna-Katerina; Nowotschin, Sonja; Turner, David A; Martinez Arias, Alfonso

2014-11-01

Mouse embryonic stem cells (mESCs) are clonal populations derived from preimplantation mouse embryos that can be propagated in vitro and, when placed into blastocysts, contribute to all tissues of the embryo and integrate into the normal morphogenetic processes, i.e. they are pluripotent. However, although they can be steered to differentiate in vitro into all cell types of the organism, they cannot organise themselves into structures that resemble embryos. When aggregated into embryoid bodies they develop disorganised masses of different cell types with little spatial coherence. An exception to this rule is the emergence of retinas and anterior cortex-like structures under minimal culture conditions. These structures emerge from the cultures without any axial organisation. Here, we report that small aggregates of mESCs, of about 300 cells, self-organise into polarised structures that exhibit collective behaviours reminiscent of those that cells exhibit in early mouse embryos, including symmetry breaking, axial organisation, germ layer specification and cell behaviour, as well as axis elongation. The responses are signal specific and uncouple processes that in the embryo are tightly associated, such as specification of the anteroposterior axis and anterior neural development, or endoderm specification and axial elongation. We discuss the meaning and implications of these observations and the potential uses of these structures which, because of their behaviour, we suggest to call 'gastruloids'. © 2014. Published by The Company of Biologists Ltd.

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.

Evaluation of cell number and DNA content in mouse embryos cultivated with uranium

International Nuclear Information System (INIS)

Kundt, Mirian S.; Cabrini, Romulo L.

2000-01-01

The evaluation of the degree of development, the number of cells and the DNA content, were used to evaluate the embryotoxicity of uranium. Embryos at a one cell stage were cultured with uranyl nitrate hexahydrate (UN) at a final concentration of uranium (U) of 26, 52 and 104 μgU/ml. At 24 hs of culture, the embryos at the 2 cell stage, were put in new wells with the same concentrations of U as the previous day, until the end of the period of incubation at 72 hs. At 72 hs of culture, 87% of the original one cell embryos were at morula stage, and in those cultivated with uranium, the percentage decreased significantly to 77; 63.24 and 40.79% respectively for the different U concentrations. Those embryos that exhibited a normal morphology, were selected and fixed on slides. The number of cells per embryo was evaluated in Giemsa stained preparations. The DNA content was evaluated cytophotometrically in Feulgen stained nuclei. The number of cells decreased significantly from 20,3 ± 5.6 in the control to 19 ± 6; 14 ± 3 and 13.9 ± 5.6 for the different concentrations. All the embryos evaluated showed one easy recognizable polar body, which was used a haploid indicator (n). The content of DNA was measured in a total of 20 control embryos and 16 embryos cultivated with UN. In control embryos, 92,7% of the nuclei presented a normal ploidy from 2n to 4n, 2,9% nuclei were hypoploid and 4,4% were hyperploid. The percentage of hypoploid nuclei rose in a dose-dependent fashion to 3.45; 44.45 and 50.34% respectively for the embryos cultured at the different U concentrations. The results indicate that U is embryotoxic, that its effects are dose dependent at the concentrations used in this study and that even those embryos that show a normal morphology, can be genetically affected. We show that the model employed is extremely sensitive. It is possible to use the preimplantation embryos, as a model to test the effect of possibly mutagenic agents of the nuclear industry. (author)

MiRNA-mediated regulation of cell signaling and homeostasis in the early mouse embryo.

Science.gov (United States)

Pernaute, Barbara; Spruce, Thomas; Rodriguez, Tristan A; Manzanares, Miguel

2011-02-15

At the time of implantation the mouse embryo is composed of three tissues the epiblast, trophectoderm and primitive endoderm. As development progresses the epiblast goes on to form the foetus whilst the trophectoderm and primitive endoderm give rise to extra-embryonic structures with important roles in embryo patterning and nutrition. Dramatic changes in gene expression occur during early embryo development and these require regulation at different levels. miRNAs are small non coding RNAs that have emerged over the last decade as important post-transcriptional repressors of gene expression. The roles played by miRNAs during early mammalian development are only starting to be elucidated. In order to gain insight into the function of miRNAs in the different lineages of the early mouse embryo we have analysed in depth the phenotype of embryos and extra-embryonic stem cells mutant for the miRNA maturation protein Dicer. This study revealed that miRNAs are involved in regulating cell signaling and homeostasis in the early embryo. Specifically, we identified a role for miRNAs in regulating the Erk signaling pathway in the extra-embryonic endoderm, cell cycle progression in extra-embryonic tissues and apoptosis in the epiblast.

First systematic experience of preimplantation genetic diagnosis for single-gene disorders, and/or preimplantation human leukocyte antigen typing, combined with 24-chromosome aneuploidy testing.

Science.gov (United States)

Rechitsky, Svetlana; Pakhalchuk, Tatiana; San Ramos, Geraldine; Goodman, Adam; Zlatopolsky, Zev; Kuliev, Anver

2015-02-01

To study the feasibility, accuracy, and reproductive outcome of 24-chromosome aneuploidy testing (24-AT), combined with preimplantation genetic diagnosis (PGD) for single-gene disorders (SGDs) or human leukocyte antigen (HLA) typing in the same biopsy sample. Retrospective study. Preimplantation genetic diagnosis center. A total of 238 PGD patients, average age 36.8 years, for whom 317 combined PGD cycles were performed, involving 105 different conditions, with or without HLA typing. Whole-genome amplification product, obtained in 24-AT, was used for PGD and/or HLA typing in the same blastomere or blastocyst biopsy samples. Proportion of the embryos suitable for transfer detected in these blastomere or blastocyst samples, and the resulting pregnancy and spontaneous abortion rates. Embryos suitable for transfer were detected in 42% blastocyst and 25.1% blastomere samples, with a total of 280 unaffected, HLA-matched euploid embryos detected for transfer in 212 cycles (1.3 embryos per transfer), resulting in 145 (68.4%) unaffected pregnancies and birth of 149 healthy, HLA-matched children. This outcome is significantly different from that of our 2,064 PGD cycle series without concomitant 24-AT, including improved pregnancy (68.4% vs. 45.4%) and 3-fold spontaneous abortion reduction (5.5% vs. 15%) rates. The introduced combined approach is a potential universal PGD test, which in addition to achieving extremely high diagnostic accuracy, significantly improves reproductive outcomes of PGD for SGDs and HLA typing in patients of advanced reproductive age. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Preimplantation genetic diagnosis for a patient with multiple endocrine neoplasia type 1: case report.

Science.gov (United States)

Lima, Aline Dt; Alves, Vanessa R; Rocha, Andressa R; Martinhago, Ana C; Martinhago, Ciro; Donadio, Nilka; Dzik, Artur; Cavagna, Mario; Gebrim, Luiz H

2018-03-01

Preimplantation genetic diagnosis was carried out for embryonic analysis in a patient with multiple endocrine neoplasia type 1 (MEN1). This is a rare autosomal-dominant cancer syndrome and the patients with MEN1 are characterized by the occurrence of tumors in multiple endocrine tissues, associated with germline and somatic inactivating mutations in the MEN1 gene. This case report documents a successful preimplantation genetic diagnosis (PGD) involving a couple at-risk for MEN1 syndrome, with a birth of a healthy infant. The couple underwent a cycle of controlled ovarian stimulation and intracytoplasmic sperm injection (ICSI). Embryos were biopsied at the blastocyst stage and cryopreserved; we used PCR-based DNA analysis for PGD testing. Only one of the five embryos analyzed for MEN1 syndrome was unaffected. This embryo was thawed and transferred following endometrial preparation. After positive βHCG test; clinical pregnancy was confirmed by ultrasound, and a healthy infant was born. PGD for single gene disorders has been an emerging therapeutic tool for couples who are at risk of passing a genetic disease on to their offspring.

Enhanced casein kinase II activity during mouse embryogenesis. Identification of a 110-kDa phosphoprotein as the major phosphorylation product in mouse embryos and Krebs II mouse ascites tumor cells

DEFF Research Database (Denmark)

Schneider, H R; Reichert, G H; Issinger, O G

1986-01-01

Mouse embryos at various stages of development were used to study the relationship of protein kinase activities with normal embryogenesis. Casein kinase II (CKII) activity in developing mouse embryos shows a 3-4-fold activity increase at day 12 of gestation. Together with the CKII activity...... mouse tumour cells also show an enhanced CKII activity. Here too, a 110-kDa phosphoprotein was the major phosphoryl acceptor. Partial proteolytic digestion shows that both proteins are identical. Other protein kinases tested (cAMP- and cGMP-dependent protein kinases) only show a basal level of enzyme...

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.

Single-gene testing combined with single nucleotide polymorphism microarray preimplantation genetic diagnosis for aneuploidy: a novel approach in optimizing pregnancy outcome.

Science.gov (United States)

Brezina, Paul R; Benner, Andrew; Rechitsky, Svetlana; Kuliev, Anver; Pomerantseva, Ekaterina; Pauling, Dana; Kearns, William G

2011-04-01

To describe a method of amplifying DNA from blastocyst trophectoderm cells (two or three cells) and simultaneously performing 23-chromosome single nucleotide polymorphism microarrays and single-gene preimplantation genetic diagnosis. Case report. IVF clinic and preimplantation genetic diagnostic centers. A 36-year-old woman, gravida 2, para 1011, and her husband who both were carriers of GM(1) gangliosidosis. The couple wished to proceed with microarray analysis for aneuploidy detection coupled with DNA sequencing for GM(1) gangliosidosis. An IVF cycle was performed. Ten blastocyst-stage embryos underwent trophectoderm biopsy. Twenty-three-chromosome microarray analysis for aneuploidy and specific DNA sequencing for GM(1) gangliosidosis mutations were performed. Viable pregnancy. After testing, elective single embryo transfer was performed followed by an intrauterine pregnancy with documented fetal cardiac activity by ultrasound. Twenty-three-chromosome microarray analysis for aneuploidy detection and single-gene evaluation via specific DNA sequencing and linkage analysis are used for preimplantation diagnosis for single-gene disorders and aneuploidy. Because of the minimal amount of genetic material obtained from the day 3 to 5 embryos (up to 6 pg), these modalities have been used in isolation of each other. The use of preimplantation genetic diagnosis for aneuploidy coupled with testing for single-gene disorders via trophectoderm biopsy is a novel approach to maximize pregnancy outcomes. Although further investigation is warranted, preimplantation genetic diagnosis for aneuploidy and single-gene testing seem destined to be used increasingly to optimize ultimate pregnancy success. Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Toward an ethical eugenics: the case for mandatory preimplantation genetic selection.

Science.gov (United States)

Appel, Jacob M

2012-01-01

Preimplantation genetic diagnosis offers the possibility of screening and terminating embryos with severe and life-threatening disabilities. This article argues that under certain conditions, the use of this technology is not merely desirable as a means to reduce human suffering but also an ethically required duty of a parent to a potential child.

Development of teeth in chick embryos after mouse neural crest transplantations

OpenAIRE

Mitsiadis, Thimios A.; Chéraud, Yvonnick; Sharpe, Paul; Fontaine-Pérus, Josiane

2003-01-01

Teeth were lost in birds 70–80 million years ago. Current thinking holds that it is the avian cranial neural crest-derived mesenchyme that has lost odontogenic capacity, whereas the oral epithelium retains the signaling properties required to induce odontogenesis. To investigate the odontogenic capacity of ectomesenchyme, we have used neural tube transplantations from mice to chick embryos to replace the chick neural crest cell populations with mouse neural crest cells. The mouse/chick ...

Electroporation of Postimplantation Mouse Embryos In Utero.

Science.gov (United States)

Huang, Cheng-Chiu; Carcagno, Abel

2018-02-01

Gene transfer by electroporation is possible in mouse fetuses within the uterus. As described in this protocol, the pregnant female is anesthetized, the abdominal cavity is opened, and the uterus with the fetuses is exteriorized. A solution of plasmid DNA is injected through the uterine wall directly into the fetus, typically into a cavity like the brain ventricle, guided by fiber optic illumination. Electrodes are positioned on the uterus around the region of the fetus that was injected, and electrical pulses are delivered. The uterus is returned to the abdominal cavity, the body wall is sutured closed, and the female is allowed to recover. The manipulated fetuses can then be collected and analyzed at various times after the electroporation. This method allows experimental access to later-stage developing mouse embryos. © 2018 Cold Spring Harbor Laboratory Press.

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.

Controlled hydrostatic pressure stress downregulates the expression of ribosomal genes in preimplantation embryos: a possible protection mechanism?

Science.gov (United States)

Bock, I; Raveh-Amit, H; Losonczi, E; Carstea, A C; Feher, A; Mashayekhi, K; Matyas, S; Dinnyes, A; Pribenszky, C

2016-04-01

The efficiency of various assisted reproductive techniques can be improved by preconditioning the gametes and embryos with sublethal hydrostatic pressure treatment. However, the underlying molecular mechanism responsible for this protective effect remains unknown and requires further investigation. Here, we studied the effect of optimised hydrostatic pressure treatment on the global gene expression of mouse oocytes after embryonic genome activation. Based on a gene expression microarray analysis, a significant effect of treatment was observed in 4-cell embryos derived from treated oocytes, revealing a transcriptional footprint of hydrostatic pressure-affected genes. Functional analysis identified numerous genes involved in protein synthesis that were downregulated in 4-cell embryos in response to hydrostatic pressure treatment, suggesting that regulation of translation has a major role in optimised hydrostatic pressure-induced stress tolerance. We present a comprehensive microarray analysis and further delineate a potential mechanism responsible for the protective effect of hydrostatic pressure treatment.

Response of maternal immune cells of irradiation of mouse embryos

International Nuclear Information System (INIS)

Nicholls, E.M.; Markovic, B.

1988-01-01

This work began as an attempt to explain the paradox of pregnancy - the survival and growth of the semi-allogenic embryo in an immunologically hostile environment. In 1982 and 1983 we reported the tracing of quinacrine labelled maternal leukocytes (WBC) in maternal, placental and embryonic mouse tissues by fluorescence microscopy. We found that cells in the placenta phagocytose labelled WBC, so that after 1-2 hours the labelled nuclear DNA is found as brightly fluorescing particles in the cytoplasm of the phagocytes with no evidence of it in the nuclei. Identical cells were observed in slide preparations of embryos which had been carefully separated from their placentas. We also found a small population of intact labelled lymphocytes, clearly maternal in origin, in the embryos. This seems to be another paradox - placental phagocytes are observed to be phagocytosing maternal WBC in the placenta and embryo, but there are also free maternal cells in the placenta and embryo. A theoretical explanation is that maternal lymphocytes alloreactive against the embryo will attempt to react with placental cells and in the process be phagocytosed, while other maternal cells will be able to enter the embryo where they could have a surveillance function, removing dead or mutant embryonic cells. To test this theory a series of experiments were carried out and are reported

Aberrant behavior of mouse embryo development after blastomere biopsy as observed through time-lapse cinematography.

Science.gov (United States)

Ugajin, Tomohisa; Terada, Yukihiro; Hasegawa, Hisataka; Velayo, Clarissa L; Nabeshima, Hiroshi; Yaegashi, Nobuo

2010-05-15

To analyze whether blastomere biopsy affects early embryonal growth as observed through time-lapse cinematography. Comparative prospective study between embryos in which a blastomere was removed and embryos in which a blastomere was not removed. An experimental laboratory of the university. We calculated the time between blastocele formation and the end of hatching, the time between the start and end of hatching, the number of contractions and expansions between blastocyst formation and the end of hatching, and the maximum diameter of the expanded blastocyst. In blastomere removal embryos, compaction began at the six-cell stage instead of at the eight-cell stage. We also found that hatching was delayed in these embryos as compared with matched controls. Moreover, the frequency of contraction and expansion movements after blastocyst formation was significantly higher in the blastomere removal group as compared with the control group. Finally, the maximum diameter of the expanded blastocyst just before hatching was not significantly different between both groups. These findings suggested that blastomere removal has an adverse effect on embryonic development around the time of hatching. Thus, future developments in preimplantation genetic diagnosis and screening should involve further consideration and caution in light of the influence of blastomere biopsy on embryonal growth. Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Development of teeth in chick embryos after mouse neural crest transplantations.

Science.gov (United States)

Mitsiadis, Thimios A; Chéraud, Yvonnick; Sharpe, Paul; Fontaine-Pérus, Josiane

2003-05-27

Teeth were lost in birds 70-80 million years ago. Current thinking holds that it is the avian cranial neural crest-derived mesenchyme that has lost odontogenic capacity, whereas the oral epithelium retains the signaling properties required to induce odontogenesis. To investigate the odontogenic capacity of ectomesenchyme, we have used neural tube transplantations from mice to chick embryos to replace the chick neural crest cell populations with mouse neural crest cells. The mouse/chick chimeras obtained show evidence of tooth formation showing that avian oral epithelium is able to induce a nonavian developmental program in mouse neural crest-derived mesenchymal cells.

Is the resulting phenotype of an embryo with balanced X-autosome translocation, obtained by means of preimplantation genetic diagnosis, linked to the X inactivation pattern?

Science.gov (United States)

Ferfouri, Fatma; Bernicot, Izabel; Schneider, Anouck; Haquet, Emmanuelle; Hédon, Bernard; Anahory, Tal

2016-04-01

To examine if a balanced female embryo with X-autosome translocation could, during its subsequent development, express an abnormal phenotype. Preimplantation genetic diagnosis (PGD) analysis on two female carriers with maternal inherited X-autosome translocations. Infertility center and genetic laboratory in a public hospital. Two female patients carriers undergoing PGD for a balanced X-autosome translocations: patient 1 with 46,X,t(X;2)(q27;p15) and patient 2 with 46,X,t(X;22)(q28;q12.3). PGD for balanced X-autosome translocations. PGD outcomes, fluorescence in situ hybridization in biopsied embryos and meiotic segregation patterns analysis of embryos providing from X-autosome translocation carriers. Controlled ovarian stimulation facilitated retrieval of a correct number of oocytes. One balanced embryo per patient was transferred and one developed, but the patient miscarried after 6 weeks of amenorrhea. In X-autosome translocation carriers, balanced Y-bearing embryos are most often phenotypically normal and viable. An ambiguous phenotype exists in balanced X-bearing embryos owing to the X inactivation mechanism. In 46,XX embryos issued from an alternate segregation, der(X) may be inactivated and partially spread transcriptional silencing into a translocated autosomal segment. Thus, the structural unbalanced genotype could be turned into a viable functional balanced one. It is relevant that a discontinuous silencing is observed with a partial and unpredictable inactivation of autosomal regions. Consequently, the resulting phenotype remains a mystery and is considered to be at risk of being an abnormal phenotype in the field of PGD. It is necessary to be cautious regarding to PGD management for this type of translocation, particularly in transferred female embryos. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

In vitro culture of mouse embryos amniotic fluid ID human

African Journals Online (AJOL)

1989-07-15

Jul 15, 1989 ... Because human amniotic fluid is a physiological, balanced ultrafiltrate, it has been considered as an inexpensive alternative culture medium in. IVF. A study of the development of mouse embryos in human amniotic fluid was undertaken to assess the suitability of this as an optional culture medium in human ...

Cytogenetic and genetic studies of radiation-induced chromosome damage in mouse oocytes. Part 1. Numerical and structural chromosome anomalies in metaphase II oocytes, pre- and post-implantation embryos

International Nuclear Information System (INIS)

Tease, Charles; Fisher, Graham

1996-01-01

The incidences of X-ray induced numerical and structural chromosome anomalies were screened in a range of developmental stages from metaphase II oocytes through to post-implantation embryos. Following 1 Gy of acute X-rays to immediately preovulatory stage oocytes, the rate of hyperploidy (chromosome gain) was found to be elevated over levels in unirradiated controls, at metaphase II, in 1-cell and 3.5 day pre-implantation embryos but not in 8.5 day post-implantation foetuses. In the latter, however, the frequency of mosaicism was significantly increased. A similar response of an increase in mosaicism but not in hyperploidy in 8.5 day post-implantation embryos was also found after irradiation of dictyate stage oocytes with 4 Gy of acute X-rays. Significantly elevated frequencies of structural chromosome anomalies were present in metaphase II oocytes and pre-implantation embryonic stages, but could not be detected in block-stained chromosome preparations from 8.5 day post-implantation foetuses. However, analysis of chromosome preparations after G-banding showed that almost 14% of 14.5 day foetuses carried a chromosome rearrangement after 1 Gy of X-rays to immediately preovulatory stage oocytes. Overall, our data indicate that the presence of radiation-induced chromosome gains are incompatible with embryonic survival but that a proportion of embryos with structural chromosome damage develop past mid-gestation. These latter embryos are therefore potentially capable of contributing to the genetic burden of the next generation

The future (r)evolution of preimplantation genetic diagnosis/human leukocyte antigen testing: ethical reflections.

Science.gov (United States)

de Wert, Guido; Liebaers, Inge; Van de Velde, Hilde

2007-09-01

There has been increasing support for combining preimplantation genetic diagnosis (PGD) for specific diseases with a test for human leukocyte antigens (HLA) because the generation of HLA-matched umbilical cord blood cells may save the life of a diseased sibling. To date, this procedure has taken place in the context of conceiving another child--PGD/HLA testing type 1. However, it may well become possible to perform PGD/HLA testing outside this context, that is, to select matched embryos from which embryonic stem cells could be derived and used in cell therapy--PGD/HLA testing type 2. A proactive ethical analysis is needed and is presented in this article. Although PGD/HLA testing type 1 can be morally justified, the risks, pitfalls, and practical limitations of this procedure make it necessary to develop alternative strategies. PGD/HLA testing type 2 may provide an alternative strategy. From an ethical point of view, the controversial issue is that this procedure creates embryos purely for instrumental use. However, given the dominant view that the preimplantation embryo has only limited moral value, this alternative may be as morally justified as PGD/HLA testing type 1.

Aberrant epigenetic reprogramming of imprinted microRNA-127 and Rtl1 in cloned mouse embryos

International Nuclear Information System (INIS)

Cui Xiangshun; Zhang Dingxiao; Ko, Yoeung-Gyu; Kim, Nam-Hyung

2009-01-01

The microRNA (miRNA) genes mir-127 and mir-136 are located near two CpG islands in the imprinted mouse retrotransposon-like gene Rtl1, a key gene involved in placenta formation. These miRNAs appear to be involved in regulating the imprinting of Rtl1. To obtain insights into the epigenetic reprogramming of cloned embryos, we compared the expression levels of mir-127 and mir-136 in fertilized mouse embryos, parthenotes, androgenotes and cloned embryos developing in vitro. We also examined the DNA methylation status of the promoter regions of Rtl1 and mir-127 in these embryos. Our data showed that mir-127 and mir-136 were highly expressed in parthenotes, but rarely expressed in androgenotes. Interestingly, the expression levels of mir-127 and mir-136 in parthenotes were almost twice that seen in the fertilized embryos, but were much lower in the cloned embryos. The Rtl1 promoter region was hyper-methylated in blastocyst stage parthenotes (75.0%), moderately methylated (32.4%) in the fertilized embryos and methylated to a much lower extent (∼10%) in the cloned embryos. Conversely, the promoter region of mir-127 was hypo-methylated in parthenogenetically activated embryos (0.4%), moderately methylated (30.0%) in fertilized embryos and heavily methylated in cloned blastocysts (63-70%). These data support a role for mir-127 and mir-136 in the epigenetic reprogramming of the Rtl1 imprinting process. Analysis of the aberrant epigenetic reprogramming of mir-127 and Rtl1 in cloned embryos may help to explain the nuclear reprogramming procedures that occur in donor cells following somatic cell nuclear transfer (SCNT).

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.

Effect of early addition of bone morphogenetic protein 5 (BMP5) to embryo culture medium on in vitro development and expression of developmentally important genes in bovine preimplantation embryos.

Science.gov (United States)

García, Elina V; Miceli, Dora C; Rizo, Gabriela; Valdecantos, Pablo A; Barrera, Antonio D

2015-09-01

blastocyst stage in the BMP5 group. Moreover, reverse transcription quantitative real-time polymerase chain reaction analysis showed a significant increase in the relative abundance of SOX2 in two-cell stage embryos, ID2 and OCT4 in eight-cell stage embryos, and NANOG and OCT4 in blastocysts derived from BMP5-treated embryos. In conclusion, our results report that early addition of BMP5 to the embryo culture medium had a positive effect on the blastocyst rate and affected the relative expression of BMP target and pluripotency genes, suggesting that BMP5 could play an important role in the preimplantation development of bovine embryos. Copyright © 2015 Elsevier Inc. All rights reserved.

Counselling considerations for chromosomal mosaicism detected by preimplantation genetic screening.

Science.gov (United States)

Besser, Andria G; Mounts, Emily L

2017-04-01

The evolution of preimplantation genetic screening (PGS) for aneuploidy to blastocyst biopsy and more sensitive 24-chromosome screening techniques has resulted in a new diagnostic category of PGS results: those classified as mosaic. This diagnosis presents significant challenges for clinicians in developing policies regarding transfer and storage of such embryos, as well as in providing genetic counselling for patients prior to and following PGS. Given the high frequency of mosaic PGS results and the wide range of possible associated outcomes, there is an urgent need to understand how to appropriately counsel patients regarding such embryos. This is the first commentary to thoroughly address pre- and post-test genetic counselling recommendations, as well as considerations regarding prenatal screening and diagnosis. Current data on mosaic PGS results are summarized along with embryo selection considerations and potential outcomes of embryos diagnosed as mosaic. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

The methyltransferase Setdb1 is essential for meiosis and mitosis in mouse oocytes and early embryos.

Science.gov (United States)

Eymery, Angeline; Liu, Zichuan; Ozonov, Evgeniy A; Stadler, Michael B; Peters, Antoine H F M

2016-08-01

Oocytes develop the competence for meiosis and early embryogenesis during their growth. Setdb1 is a histone H3 lysine 9 (H3K9) methyltransferase required for post-implantation development and has been implicated in the transcriptional silencing of genes and endogenous retroviral elements (ERVs). To address its role in oogenesis and pre-implantation development, we conditionally deleted Setdb1 in growing oocytes. Loss of Setdb1 expression greatly impaired meiosis. It delayed meiotic resumption, altered the dynamics of chromatin condensation, and impaired kinetochore-spindle interactions, bipolar spindle organization and chromosome segregation in more mature oocytes. The observed phenotypes related to changes in abundance of specific transcripts in mutant oocytes. Setdb1 maternally deficient embryos arrested during pre-implantation development and showed comparable defects during cell cycle progression and in chromosome segregation. Finally, transcriptional profiling data indicate that Setdb1 downregulates rather than silences expression of ERVK and ERVL-MaLR retrotransposons and associated chimearic transcripts during oogenesis. Our results identify Setdb1 as a newly discovered meiotic and embryonic competence factor safeguarding genome integrity at the onset of life. © 2016. Published by The Company of Biologists Ltd.

The loss of imprinted DNA methylation in mouse blastocysts is inflicted to a similar extent by in vitro follicle culture and ovulation induction.

Science.gov (United States)

Saenz-de-Juano, M D; Billooye, K; Smitz, J; Anckaert, E

2016-06-01

Does in vitro follicle culture (IFC) have an effect on maintenance of imprinted DNA methylation in preimplantation mouse embryos? We report similar alterations in the methylation pattern of H19 imprinted maternally expressed transcript (H19), small nuclear ribonucleoprotein polypeptide N (Snrpn) and mesoderm specific transcript (Mest) imprinted genes in mouse blastocysts obtained after ovulation induction and IFC. Furthermore, we observed no differences in the gene expression of maternal effect proteins related with imprinting maintenance between superovulated in vivo grown or IFC oocytes. Assisted reproductive technology is associated with adverse post-natal outcomes such as increased risk of premature birth, altered birthweight, congenital anomalies and genomic imprinting syndromes in human and in animal models. Previous studies have shown that ovulation induction allowed normal imprinting establishment in mouse oocytes, but interfered with imprinting maintenance during preimplantation . Normal imprinting establishment was also observed in mouse oocytes derived from a standardized IFC from the early pre-antral follicle stage. The methylation profiles of differentially methylated regions (DMRs) of three key imprinted genes (H19, Snrpn and Mest) were compared at hatched blastocyst stage between embryos obtained from IFC or superovulated oocytes, each subjected to IVF and preimplantation in vitro culture (IVC); in non-manipulated in vivo produced late blastocyst (control) and in in vivo produced 2-cell embryos that were in vitro cultured until the hatched blastocyst stage (to assess the effect of IVC). Two different mice strains (Mus musculus C57BL/6J X CBA/Ca and Mus musculus B6 (CAST7)) were used to discriminate between maternal and paternal alleles of imprinted genes. Additionally, a limiting-dilution bisulfite-sequencing technique was carried out on individual embryos in order to avoid amplification bias. To assess whether IFC and ovulation induction

ART culture conditions change the probability of mouse embryo gestation through defined cellular and molecular responses

NARCIS (Netherlands)

Schwarzer, Caroline; Esteves, Telma Cristina; Arau´zo-Bravo, Marcos J.; le Gac, Severine; Nordhoff, Verena; Schlatt, Stefan; Boiani, Michele

2012-01-01

Do different human ART culture protocols prepare embryos differently for post-implantation development? ... Our data promote awareness that human ART culture media affect embryo development. Effects reported here in the mouse may apply also in human, because no ART medium presently available on the

Embryonic Stem Cell Proteins and MicroRNAs in the Etiology of Germ Cell Cancer

NARCIS (Netherlands)

R. Eini (Ronak)

2013-01-01

textabstractIn the early 1980s, a population of unique cells was isolated from the inner cell mass (ICM) of the mouse pre-implantation embryo named embryonic stem (ES) cells. These cells were generated by removing the ICM from pre-implantation blastocysts. The resulting cells were found to be

Number of blastocysts biopsied as a predictive indicator to obtain at least one normal/balanced embryo following preimplantation genetic diagnosis with single nucleotide polymorphism microarray in translocation cases.

Science.gov (United States)

Wang, Yi-Zi; Ding, Chen-Hui; Wang, Jing; Zeng, Yan-Hong; Zhou, Wen; Li, Rong; Zhou, Can-Quan; Deng, Ming-Fen; Xu, Yan-Wen

2017-01-01

The aim of this study is to investigate the minimum number of blastocysts for biopsy to increase the likelihood of obtaining at least one normal/balanced embryo in preimplantation genetic diagnosis (PGD) for translocation carriers. This blinded retrospective study included 55 PGD cycles for Robertsonian translocation (RT) and 181 cycles for reciprocal translocation (rcp) to indicate when only one of the couples carried a translocation. Single-nucleotide polymorphism microarray after trophectoderm biopsy was performed. Reliable results were obtained for 355/379 (93.7 %) biopsied blastocysts in RT group and 986/1053 (93.6 %) in rcp group. Mean numbers of biopsied embryos per patient, normal/balanced embryos per patient, and mean normal/balanced embryo rate per patient were 7.4, 3.1, and 40.7 % in RT group and 8.0, 2.1, and 27.3 %, respectively, in rcp group. In a regression model, three factors significantly affected the number of genetically transferrable embryos: number of biopsied embryos (P = 0.001), basal FSH level (P = 0.040), and maternal age (P = 0.027). ROC analysis with a cutoff of 1.5 was calculated for the number of biopsied embryos required to obtain at least one normal/balanced embryo for RT carriers. For rcp carriers, the cutoff was 3.5. The clinical pregnancy rate per embryo transfer was 44.2 and 42.6 % in RT and rcp groups (P = 0.836). The minimum numbers of blastocysts to obtain at least one normal/balanced embryo for RT and rcp were 2 and 4 under the conditions of female age < 37 years with a basal FSH level < 11.4 IU/L.

β-catenin functions pleiotropically in differentiation and tumorigenesis in mouse embryo-derived stem cells.

Directory of Open Access Journals (Sweden)

Noriko Okumura

Full Text Available The canonical Wnt/β-catenin signaling pathway plays a crucial role in the maintenance of the balance between proliferation and differentiation throughout embryogenesis and tissue homeostasis. β-Catenin, encoded by the Ctnnb1 gene, mediates an intracellular signaling cascade activated by Wnt. It also plays an important role in the maintenance of various types of stem cells including adult stem cells and cancer stem cells. However, it is unclear if β-catenin is required for the derivation of mouse embryo-derived stem cells. Here, we established β-catenin-deficient (β-cat(Δ/Δ mouse embryo-derived stem cells and showed that β-catenin is not essential for acquiring self-renewal potential in the derivation of mouse embryonic stem cells (ESCs. However, teratomas formed from embryo-derived β-cat(Δ/Δ ESCs were immature germ cell tumors without multilineage differentiated cell types. Re-expression of functional β-catenin eliminated their neoplastic, transformed phenotype and restored pluripotency, thereby rescuing the mutant ESCs. Our findings demonstrate that β-catenin has pleiotropic effects in ESCs; it is required for the differentiation of ESCs and prevents them from acquiring tumorigenic character. These results highlight β-catenin as the gatekeeper in differentiation and tumorigenesis in ESCs.

Laboratory techniques for human embryos.

Science.gov (United States)

Geber, Selmo; Sales, Liana; Sampaio, Marcos A C

2002-01-01

This review is concerned with laboratory techniques needed for assisted conception, particularly the handling of gametes and embryos. Such methods are being increasingly refined. Successive stages of fertilization and embryogenesis require especial care, and often involve the use of micromanipulative methods for intracytoplasmic sperm injection (ICSI) or preimplantation genetic diagnosis. Embryologists must take responsibility for gamete collection and preparation, and for deciding on the means of insemination or ICSI. Embryos must be assessed in culture, during the 1-cell, cleaving and morula/blastocyst stages, and classified according to quality. Co-culture methods may be necessary. The best embryos for transfer must be selected and loaded into the transfer catheter. Embryos not transferred must be cryopreserved, which demands the correct application of current methods of media preparation, seeding and the correct speed for cooling and warming. Before too long, methods of detecting abnormal embryos and avoiding their transfer may become widespread.

ART culture conditions change the probability of mouse embryo gestation through defined cellular and molecular responses.

Science.gov (United States)

Schwarzer, Caroline; Esteves, Telma Cristina; Araúzo-Bravo, Marcos J; Le Gac, Séverine; Nordhoff, Verena; Schlatt, Stefan; Boiani, Michele

2012-09-01

Do different human ART culture protocols prepare embryos differently for post-implantation development? The type of ART culture protocol results in distinct cellular and molecular phenotypes in vitro at the blastocyst stage as well as subsequently during in vivo development. It has been reported that ART culture medium affects human development as measured by gestation rates and birthweights. However, due to individual variation across ART patients, it is not possible as yet to pinpoint a cause-effect relationship between choice of culture medium and developmental outcome. In a prospective study, 13 human ART culture protocols were compared two at a time against in vivo and in vitro controls. Superovulated mouse oocytes were fertilized in vivo using outbred and inbred mating schemes. Zygotes were cultured in medium or in the oviduct and scored for developmental parameters 96 h later. Blastocysts were either analyzed or transferred into fosters to measure implantation rates and fetal development. In total, 5735 fertilized mouse oocytes, 1732 blastocysts, 605 fetuses and 178 newborns were examined during the course of the study (December 2010-December 2011). Mice of the B6C3F1, C57Bl/6 and CD1 strains were used as oocyte donors, sperm donors and recipients for embryo transfer, respectively. In vivo fertilized B6C3F1 oocytes were allowed to cleave in 13 human ART culture protocols compared with mouse oviduct and optimized mouse medium (KSOM(aa)). Cell lineage composition of resultant blastocysts was analyzed by immunostaining and confocal microscopy (trophectoderm, Cdx2; primitive ectoderm, Nanog; primitive endoderm, Sox17), global gene expression by microarray analysis, and rates of development to midgestation and to term. Mouse zygotes show profound variation in blastocyst (49.9-91.9%) and fetal (15.7-62.0%) development rates across the 13 ART culture protocols tested (R(2)= 0.337). Two opposite protocols, human tubal fluid/multiblast (high fetal rate) and ISM1/ISM2

Genetic mouse embryo assay: improving performance and quality testing for assisted reproductive technology (ART) with a functional bioassay.

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Gilbert, Rebecca S; Nunez, Brandy; Sakurai, Kumi; Fielder, Thomas; Ni, Hsiao-Tzu

2016-03-24

Growing concerns about safety of ART on human gametes, embryos, clinical outcomes and long-term health of offspring require improved methods of risk assessment to provide functionally relevant assays for quality control testing and pre-clinical studies prior to clinical implementation. The one-cell mouse embryo assay (MEA) is the most widely used for development and quality testing of human ART products; however, concerns exist due to the insensitivity/variability of this bioassay which lacks standardization and involves subjective analysis by morphology alone rather than functional analysis of the developing embryos. We hypothesized that improvements to MEA by the use of functional molecular biomarkers could enhance sensitivity and improve detection of suboptimal materials/conditions. Fresh one-cell transgenic mouse embryos with green fluorescent protein (GFP) expression driven by Pou6f1 or Cdx2 control elements were harvested and cultured to blastocysts in varied test and control conditions to compare assessment by standard morphology alone versus the added dynamic expression of GFP for screening and selection of critical raw materials and detection of suboptimal culture conditions. Transgenic mouse embryos expressing functionally relevant biomarkers of normal early embryo development can be used to monitor the developmental impact of culture conditions. This novel approach provides a superior MEA that is more meaningful and sensitive for detection of embryotoxicity than morphological assessment alone.

The first cell-fate decisions in the mouse embryo: destiny is a matter of both chance and choice.

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Zernicka-Goetz, Magdalena

2006-08-01

Development of the early mouse embryo has always been classified as regulative, meaning that when parts or blastomeres of the embryo are isolated they change their developmental fate and can even reconstruct the whole. However, regulative development does not mean that, in situ, these parts or blastomeres are equivalent; it does not mean that the early mammalian embryo is a ball of identical cells without any bias. Regulative development simply means that whatever bias the regions of the embryo might have they still remain flexible and can respond to experimental interference by changes of fate. This realization -- that regulative development and patterning can co-exist -- has led to a renaissance of interest in the first days of development of the mouse embryo, and several laboratories have provided evidence for some early bias. Now the challenge is to gain some understanding of the molecular basis of this bias.

Ethical issues in new uses of preimplantation genetic diagnosis: should parents be allowed to use preimplantation genetic diagnosis to choose the sexual orientation of their children?

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Dahl, Edgar

2003-07-01

Extending the application of preimplantation genetic diagnosis (PGD) to screen embryos for non-medical traits such as gender, height and intelligence, raises serious moral, legal, and social issues. In this paper I consider the possibility of using PGD to select the sexual orientation of offspring. After considering five potential objections, I conclude that parents should be permitted to use PGD to choose the sexual orientation of their children.

Preimplantation HLA typing for stem cell transplantation treatment of hemoglobinopathies

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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.

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

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

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

The combined effects of radiation and ultrasound on ICR mouse embryos

International Nuclear Information System (INIS)

Hong, A.I.C.; Kusama, T.; Gu, Y.; Aoki, Y.

1993-01-01

We have investigated the combined effects of radiation and ultrasound on the embryos of ICR mice. The pregnant ICR mice on day 8 of gestation were irradiated with 1.0 W ultrasound after exposure to 1.5 Gy radiation immediately or irradiated with time interval of one hour. The incidences of external malformations synergistically increased in the group irradiated with both agents. Especially in the group treated with time interval of one hour, the incidences of external malformations reached to the maximum. The histological examination showed that the frequencies of pyknotic cells in the neutral folds of embryos on day 8 of gestation increased synergistically while the frequencies of mitotic cells decreased steeply in the group treated with both agents. We concluded that the combined effects of radiation and ultrasound on external malformations and the histological changes in mouse embryos were synergistic-sensitization effects. (6 figs.)

Molecular analysis of radiation-induced albino (c)-locus mutations that cause death at preimplantation stages of development

International Nuclear Information System (INIS)

Rinchik, E.M.; Toenjes, R.R.; Paul, D.; Potter, M.D.

1993-01-01

Deletion mutations at the albino (c) locus have been useful for continuing the development of fine-structure physical and functional maps of the Fes-Hbb region of mouse chromosome 7. This report describes the molecular analysis of a number of radiation-induced c deletions that, when homozygous, cause death of the embryo during preimplantation stages. The distal extent of these deletions defines a locus, pid, (preimplantation development) genetically associated with this phenotype. The proximal breakpoints of eight of these deletions were mapped with respect to the Tyr (tyrosinase; albino) gene as well as to anonymous loci within the Fah-Tyr region that are defined by the Pmv-31 viral integration site and by chromosome-microdissection clones. Rearrangements corresponding to the proximal breakpoints of two of these deletions were detected by Southern blot analysis, and a size-altered restriction fragment carrying the breakpoint of one of them was cloned. A probe derived from this deletion fusion fragment defines a locus, D7Rn6, which maps within (or distal to) the pid region, and which discriminates among the distal extents of deletions eliciting the pid phenotype. Extension of physical maps from D7Rn6 should provide access both to the pid region and to loci mapping distal to pid that are defined by N-ethyl-N-nitrosourea-induced lethal mutations. 36 refs., 10 figs

45,X product of conception after preimplantation genetic diagnosis and euploid embryo transfer: evidence of a spontaneous conception confirmed by DNA fingerprinting.

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Bettio, Daniela; Capalbo, Antonio; Albani, Elena; Rienzi, Laura; Achille, Valentina; Venci, Anna; Ubaldi, Filippo Maria; Levi Setti, Paolo Emanuele

2016-09-06

Preimplantation genetic screening (PGS) provides an opportunity to eliminate a potential implantation failure due to aneuploidy in infertile couples. Some studies clearly show that twins following single embryo transfer (SET) can be the result of a concurrent natural conception and an incidence as high as 1 in 5 twins has been reported. In our case PGS was performed on trophectoderm (TE) biopsies by quantitative polymerase chain reaction (qPCR). The product of conception (POC) was cytogenetically investigated after selection of the placental villi by means of the direct method. Molecular cytogenetic characterization of the POC was performed by fluorescence in situ hybridization (FISH) and array-comparative genomic hybridization (a-CGH) analyses. To investigate the possibility of a spontaneous conception, a panel of 40 single nucleotide polymorphisms (SNPs) was used to compare genetic similarity between the DNA of the POC and the DNA leftover of the TE biopsy. We describe a 36-year old infertile woman undergoing PGS who had a spontaneous abortion after a single euploid embryo transfer on a spontaneous cycle. The POC showed a 45,X karyotype confirmed by FISH and a-CGH. DNA fingerprinting demonstrated a genetic similarity of 75 % between the DNA of the POC and TE biopsy, consistent with a sibling status. All supernumerary euploid embryos were also tested showing a non-self relationship with the POC, excluding a mix-up event at the time of fetal embryo transfer. DNA fingerprinting of the transferred blastocyst and POC, confirmed the occurrence of a spontaneous conception. This case challenges the assumption that a pregnancy after assisted reproductive technology (ART) is always a result of ART, and strengthens the importance to avoid intercourses during PGS and natural transfer cycles. Moreover, cytogenetic analysis of the POCs is strongly recommended along with fingerprinting children born after PGS to see what the concordance is between the embryo transferred and

Ouabain stimulates a Na+/K+-ATPase-mediated SFK-activated signalling pathway that regulates tight junction function in the mouse blastocyst.

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Holly Giannatselis

Full Text Available The Na(+/K(+-ATPase plays a pivotal role during preimplantation development; it establishes a trans-epithelial ionic gradient that facilitates the formation of the fluid-filled blastocyst cavity, crucial for implantation and successful pregnancy. The Na(+/K(+-ATPase is also implicated in regulating tight junctions and cardiotonic steroid (CTS-induced signal transduction via SRC. We investigated the expression of SRC family kinase (SFK members, Src and Yes, during preimplantation development and determined whether SFK activity is required for blastocyst formation. Embryos were collected following super-ovulation of CD1 or MF1 female mice. RT-PCR was used to detect SFK mRNAs encoding Src and Yes throughout preimplantation development. SRC and YES protein were localized throughout preimplantation development. Treatment of mouse morulae with the SFK inhibitors PP2 and SU6656 for 18 hours resulted in a reversible blockade of progression to the blastocyst stage. Blastocysts treated with 10(-3 M ouabain for 2 or 10 minutes and immediately immunostained for phosphorylation at SRC tyr418 displayed reduced phosphorylation while in contrast blastocysts treated with 10(-4 M displayed increased tyr418 fluorescence. SFK inhibition increased and SFK activation reduced trophectoderm tight junction permeability in blastocysts. The results demonstrate that SFKs are expressed during preimplantation development and that SFK activity is required for blastocyst formation and is an important mediator of trophectoderm tight junction permeability.

Preimplantation genetic diagnosis for mitochondrial DNA mutations: analysis of one blastomere suffices.

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Sallevelt, Suzanne C E H; Dreesen, Joseph C F M; Coonen, Edith; Paulussen, Aimee D C; Hellebrekers, Debby M E I; de Die-Smulders, Christine E M; Smeets, Hubert J M; Lindsey, Patrick

2017-10-01

Preimplantation genetic diagnosis (PGD) is a reproductive strategy for mitochondrial DNA (mtDNA) mutation carriers, strongly reducing their risk of affected offspring. Embryos either without the mutation or with mutation load below the phenotypic threshold are transferred to the uterus. Because of incidental heteroplasmy deviations in single blastomere and the relatively limited data available, we so far preferred relying on two blastomeres rather than one. Considering the negative effect of a two-blastomere biopsy protocol compared with a single-blastomere biopsy protocol on live birth delivery rate, we re-evaluated the error rate in our current dataset. For the m.3243A>G mutation, sufficient embryos/blastomeres were available for a powerful analysis. The diagnostic error rate, defined as a potential false-negative result, based on a threshold of 15%, was determined in 294 single blastomeres analysed in 73 embryos of 9 female m.3243A>G mutation carriers. Only one out of 294 single blastomeres (0.34%) would have resulted in a false-negative diagnosis. False-positive diagnoses were not detected. Our findings support a single-blastomere biopsy PGD protocol for the m.3243A>G mutation as the diagnostic error rate is very low. As in the early preimplantation embryo no mtDNA replication seems to occur and the mtDNA is divided randomly among the daughter cells, we conclude this result to be independent of the specific mutation and therefore applicable to all mtDNA mutations. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

[Unaffected child born following preimplantation genetic diagnosis with karyomapping].

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Nánássy, László; Téglás, Gyöngyvér; Csenki, Marianna; Vereczkey, Attila

2016-12-01

Preimplantation genetic diagnosis for single gene defects is a well established method in assisted reproductive technologies. Karyomapping is a genome wide parental haplotyping using a high density single nucleotide polymorphism array that allows the diagnosis of any single gene defects. A couple with an affected child with primary congenital glaucoma attended at our clinic. Six oocyte-cumulus-complex was retrieved and all three mature oocytes were inseminated. One zygote showed the signs of normal fertilization and was cultured for five days. Trophectoderm biopsy and karyomapping analysis were carried out. Result showed a heterozygous carrier for primary congenital glaucoma. Embryo was thawed and transferred and a healthy girl was delivered at term. Here we report the first live birth following in vitro fertilization combined with preimplantation genetic diagnosis using karyomapping in Hungary. Karyomapping is able to accurately detect single gene disorders from a limited amount of samples without a significant preclinical workup. Orv. Hetil., 2016, 157(51), 2048-2050.

EXPERIMENTAL TRIES TO ESTABLISH THE PREIMPLANTATIONAL MAMMALIAN EMBRYOS VIABILITY THROUGHOUT STAINING

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IVAN ALEXANDRA

2007-01-01

Full Text Available Presently there are more methods to assess embryo quality but, still the wieldy usedremains the morphological criteria method. In this experiment were tested twostaining methods for embryos and oocytes. The embryos were recovered from mousefemale at 72 hours after mating. The recovered embryos were first evaluated aftermorphological criteria and than by Trypan blue exclusion and Neutral red staining.Using Trypan blue exclusion were evaluated 30 embryos from which 19 (63.3 wereclassified as viable and 11 (36.7 were classified as nonviable. By Neutral redstaining were evaluated 37 embryos from which 24 (64.8 were considered viableand 13 (35.2 were considered nonviable. The oocytes recovered were alsoevaluated using the two methods: using Trypan blue exclusion were stained 10oocytes from which 9 remained uncolored and were considered viable and 1 wasstained in blue and was considered nonviable and using Neutral red 13 oocytes werestained from which 9 were evaluated as viable and 4 as nonviable.

NAD-content and metabolism in the mouse embryo and developing brain

International Nuclear Information System (INIS)

Beuningen, M. van; Streffer, C.; Beuningen, D. van

1986-01-01

Biochemical studies have shown that NAD is not only the coenzyme of dehydrogenase but also the substrate of poly-(ADPR)-synthetase which is involved in processes of cell proliferation and differentiation. The NAD and protein content was determined in the total embryo and in the CNS 9 to 13 days p.c. The embryos were X-irradiated 9 days p.c. The NAD content increased in the total mouse embryo during the early organogenesis. At the later period a decrease of the NAD content per mg protein was observed. This latter effect was apparently due to an increase of the NAD glycohydrolase activity. This enzyme degrades NAD. A similar development was observed in the developing mouse brain. However, the maximal NAD content per mg protein occurred on day 10 p.c. One of the enzyme activities, which are responsible for NAD synthesis, NMN-pyrophosphorylase, also increased in the brain at the same time. After the injection of C 14-nicotinamide, a precursor of NAD, it was observed that the radioactivity mainly appeared in nicotinamide and NAD. With progressing embryological development less nicotinamide was taken up by the embryonic tissue. When the embryos were X-irradiated on day 9 p.c. with 1.8 Gy the increase of NAD was considerably reduced during the next days, so that also the NAD level per mg protein was reduced. Also the NAD biosynthesis apparently decreased. This was shown again by the reduced NMN-pyrophosphorylase activity. The dose dependance of these effects was studied in the dose range 0.48-1.8 Gy. Two days p.r. most of the radiation effects were normalized again and at later periods even an overshoot of the enzyme activity was observed. The possible relevance of these effects for cell proliferation will be discussed. (orig.)

Biopsy of human morula-stage embryos: outcome of 215 IVF/ICSI cycles with PGS.

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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.

SWAP-70 contributes to spontaneous transformation of mouse embryo fibroblasts

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Chang, Yu-Tzu; Shu, Chung-Li; Lai, Jing-Yang; Lin, Ching-Yu; Chuu, Chih-Pin [Institute of Cellular and System Medicine National Health Research Institute, Zhunan Town 35053, Miaoli County, Taiwan, ROC (China); Morishita, Kazuhiro; Ichikawa, Tomonaga [Division of Tumor and Cellular Biochemistry Department of Medical Sciences Faculty of Medicine University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki-shi, Miyazaki 889-1692 Japan (Japan); Jessberger, Rolf [Faculty of Medicine Carl Gustav Carus, Institute of Physiological Chemistry, Dresden University of Technology, Dresden (Germany); Fukui, Yasuhisa, E-mail: 990412@nhri.org.tw [Institute of Cellular and System Medicine National Health Research Institute, Zhunan Town 35053, Miaoli County, Taiwan, ROC (China)

2016-07-15

Mouse embryo fibroblasts (MEFs) grow slowly after cultivation from animals, however, after an extended period of cultivation, their growth accelerates. We found that SWAP-70 deficient MEFs failed to increase growth rates. They maintain normal growth rates and proliferation cycles for at least 5 years. Complementing SWAP-70 deficiency in one of these MEF clones, MEF1F2, by expressing human SWAP-70 resulted in fast growth of the cells after further cultivation for a long period. The resulting cells show a transformation phenotype, since they grow on top of each other and do not show contact inhibition. This phenotype was reverted when sanguinarine, a putative SWAP-70 inhibitor, was added. Two SWAP-70 expressing clones were examined in detail. Even after cell density became very high their cdc2 and NFκB were still activated suggesting that they do not stop growing. One of the clones formed colonies in soft agar and formed tumors in nude mice. Lately, one more clone became transformed being able to make colonies in soft agar. We maintain 4 human SWAP-70 expressing MEF1F2 cell lines. Three out of 4 clones exhibited transforming phenotypes. The mouse SWAP-70 gene also promoted transformation of MEFs. Taken together our data suggest that SWAP-70 is not a typical oncogene, but is required for spontaneous transformation of MEFs. - Highlights: • Mouse embryo fibroblasts (MEFs) lacking SWAP-70 do not cause spontaneous transform. • Adding back of SWAP-70 to SWAP-70-deficient MEFs induces spontaneous transformation. • SWAP-70 is required for spontaneous transformation of MEFs.

Clinical utilisation of a rapid low-pass whole genome sequencing technique for the diagnosis of aneuploidy in human embryos prior to implantation.

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Wells, Dagan; Kaur, Kulvinder; Grifo, Jamie; Glassner, Michael; Taylor, Jenny C; Fragouli, Elpida; Munne, Santiago

2014-08-01

The majority of human embryos created using in vitro fertilisation (IVF) techniques are aneuploid. Comprehensive chromosome screening methods, applicable to single cells biopsied from preimplantation embryos, allow reliable identification and transfer of euploid embryos. Recently, randomised trials using such methods have indicated that aneuploidy screening improves IVF success rates. However, the high cost of testing has restricted the availability of this potentially beneficial strategy. This study aimed to harness next-generation sequencing (NGS) technology, with the intention of lowering the costs of preimplantation aneuploidy screening. Embryo biopsy, whole genome amplification and semiconductor sequencing. A rapid (cost only two-thirds that of the most widely used method for embryo aneuploidy detection. Validation involved blinded analysis of 54 cells from cell lines or biopsies from human embryos. Sensitivity and specificity were 100%. The method was applied clinically, assisting in the selection of euploid embryos in two IVF cycles, producing healthy children in both cases. The NGS approach was also able to reveal specified mutations in the nuclear or mitochondrial genomes in parallel with chromosome assessment. Interestingly, elevated mitochondrial DNA content was associated with aneuploidy (pcost diagnosis of aneuploidy in cells from human preimplantation embryos and is rapid enough to allow testing without embryo cryopreservation. The method described also has the potential to shed light on other aspects of embryo genetics of relevance to health and viability. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Mechanical control of notochord morphogenesis by extra-embryonic tissues in mouse embryos.

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Imuta, Yu; Koyama, Hiroshi; Shi, Dongbo; Eiraku, Mototsugu; Fujimori, Toshihiko; Sasaki, Hiroshi

2014-05-01

Mammalian embryos develop in coordination with extraembryonic tissues, which support embryonic development by implanting embryos into the uterus, supplying nutrition, providing a confined niche, and also providing patterning signals to embryos. Here, we show that in mouse embryos, the expansion of the amniotic cavity (AC), which is formed between embryonic and extraembryonic tissues, provides the mechanical forces required for a type of morphogenetic movement of the notochord known as convergent extension (CE) in which the cells converge to the midline and the tissue elongates along the antero-posterior (AP) axis. The notochord is stretched along the AP axis, and the expansion of the AC is required for CE. Both mathematical modeling and physical simulation showed that a rectangular morphology of the early notochord caused the application of anisotropic force along the AP axis to the notochord through the isotropic expansion of the AC. AC expansion acts upstream of planar cell polarity (PCP) signaling, which regulates CE movement. Our results highlight the importance of extraembryonic tissues as a source of the forces that control the morphogenesis of embryos. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

What Drives Embryo Development? Chromosomal Normality or Mitochondria?

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A. Bayram

2017-01-01

Full Text Available Objective. To report the arrest of euploid embryos with high mtDNA content. Design. A report of 2 cases. Setting. Private fertility clinic. Patients. 2 patients, 45 and 40 years old undergoing IVF treatment. Interventions. Mature oocytes were collected and vitrified from two ovarian stimulations. Postthaw, survived mature oocytes underwent fertilization by intracytoplasmic sperm injection (ICSI. Preimplantation genetic screening (PGS and mitochondrial DNA (mtDNA copy number were done using next generation sequencing (NGS. The only normal embryo among the all-biopsied embryos had the highest “Mitoscore” value and was the only arrested embryo in both cases. Therefore, the embryo transfer was cancelled. Main Outcome Measures. Postthaw survival and fertilization rate, embryo euploidy, mtDNA copy number, and embryo development. Results. In both patients, after PGS only 1 embryo was euploid. Both embryos had the highest mtDNA copy number from all tested embryos and both embryos were arrested on further development. Conclusions. These cases clearly demonstrate the lack of correlation between mtDNA value (Mitoscore and chromosomal status of embryo.

MODELS FOR MOUSE CHIMERA PRODUCTION: AGGREGATION OF ES CELLS WITH CLEAVAGE STAGE EMBRYOS

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STANCA CLAUDIA

2007-01-01

Full Text Available In a mutant ES cells↔ wild-type embryo chimera, ES cells behave more like epiblastcells. They can contribute to the primitive ectoderm layers, which give rise to all theembryonic tissues and some extraembryonic tissues (Beddington and Robertson,1989, but not to trophectoderm or primitive endoderm. Using transgenic ES celllines, aggregated with cleavage stage host embryo, ES cells can integrate randomlyin the embryo proper. If they will be take part in the formation of ICM (inner cellmass, it will be possible to obtain germline chimera animals. To generate ES cells↔ cleavage stage host embryo chimeras, we used (CD-1 mice as donors of hostembryos as well as recipients of manipulated embryos. For chimera production, weused fluorescent-labeled ES cell line (CD1/EGFP, because in this case we canfollow the fate of ES cells during the embryonic development. We produced thechimers using “aggregation chimera technique”. 8 cells stage zona pellucida free,mouse embryos were aggregated in an aggregation plates, with a clump of ES cells(10 – 15 cells. The chimera embryos were cultivated for 24 hours in the incubator(at 37 °C, 5% CO2 in air. The chimera blastocysts resulted after cultivation, weretransferred to the uterus of the 2.5-dpc pseudo pregnant females.

MODELS FOR MOUSE CHIMERA PRODUCTION: AGGREGATION OF ES CELLS WITH CLEAVAGE STAGE EMBRYOS

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CLAUDIA STANCA

2007-05-01

Full Text Available In a mutant ES cells↔ wild-type embryo chimera, ES cells behave more like epiblastcells. They can contribute to the primitive ectoderm layers, which give rise to all theembryonic tissues and some extraembryonic tissues (Beddington and Robertson,1989, but not to trophectoderm or primitive endoderm. Using transgenic ES celllines, aggregated with cleavage stage host embryo, ES cells can integrate randomlyin the embryo proper. If they will be take part in the formation of ICM (inner cellmass, it will be possible to obtain germline chimera animals. To generate ES cells↔ cleavage stage host embryo chimeras, we used (CD-1 mice as donors of hostembryos as well as recipients of manipulated embryos. For chimera production, weused fluorescent-labeled ES cell line (CD1/EGFP, because in this case we canfollow the fate of ES cells during the embryonic development. We produced thechimers using “aggregation chimera technique”. 8 cells stage zona pellucida free,mouse embryos were aggregated in an aggregation plates, with a clump of ES cells(10 – 15 cells. The chimera embryos were cultivated for 24 hours in the incubator(at 37 °C, 5% CO2 in air. The chimera blastocysts resulted after cultivation, weretransferred to the uterus of the 2.5-dpc pseudo pregnant females.

Round Spermatid Injection Rescues Female Lethality of a Paternally Inherited Xist Deletion in Mouse.

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Federica Federici

2016-10-01

Full Text Available In mouse female preimplantation embryos, the paternal X chromosome (Xp is silenced by imprinted X chromosome inactivation (iXCI. This requires production of the noncoding Xist RNA in cis, from the Xp. The Xist locus on the maternally inherited X chromosome (Xm is refractory to activation due to the presence of an imprint. Paternal inheritance of an Xist deletion (XpΔXist is embryonic lethal to female embryos, due to iXCI abolishment. Here, we circumvented the histone-to-protamine and protamine-to-histone transitions of the paternal genome, by fertilization of oocytes via injection of round spermatids (ROSI. This did not affect initiation of XCI in wild type female embryos. Surprisingly, ROSI using ΔXist round spermatids allowed survival of female embryos. This was accompanied by activation of the intact maternal Xist gene, initiated with delayed kinetics, around the morula stage, resulting in Xm silencing. Maternal Xist gene activation was not observed in ROSI-derived males. In addition, no Xist expression was detected in male and female morulas that developed from oocytes fertilized with mature ΔXist sperm. Finally, the expression of the X-encoded XCI-activator RNF12 was enhanced in both male (wild type and female (wild type as well as XpΔXist ROSI derived embryos, compared to in vivo fertilized embryos. Thus, high RNF12 levels may contribute to the specific activation of maternal Xist in XpΔXist female ROSI embryos, but upregulation of additional Xp derived factors and/or the specific epigenetic constitution of the round spermatid-derived Xp are expected to be more critical. These results illustrate the profound impact of a dysregulated paternal epigenome on embryo development, and we propose that mouse ROSI can be used as a model to study the effects of intergenerational inheritance of epigenetic marks.

Preimplantation genetic diagnosis (PGD) for HLA typing: bases for setting up an open international collaboration when PGD is not available.

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Bellavia, Marina; Von Der Weid, Nicolas; Peddes, Christina; Jacquemont, Sebastien; Liebaers, Inge; Hohlfeld, Patrick; Wunder-Galié, Dorothea; de Ziegler, Dominique

2010-08-01

In severe forms of Diamond-Blackfan anemia, preimplantation genetic diagnosis (PGD) of histocompatibility leukocyte antigen-compatible embryos for enabling the next sibling in the family to be a stem-cell transplantation donor constitutes the sole lasting cure capable of terminating the enduring need for iterative transfusions. We report here an open collaboration between two renowned institutions to provide a family desiring this treatment even though they resided where the preimplantation genetic diagnosis procedure is banned. Copyright (c) 2010 American Society for Reproductive Medicine. All rights reserved.

Alternation of apoptotic and implanting genes expression of mouse embryos after re-vitrification

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Majidi Gharenaz, Nasrin; Movahedin, Mansoureh; Mazaheri, Zohreh; Pour beiranvand, Shahram

2016-01-01

Background: Nowadays, oocytes and embryos vitrification has become a routine technique. Based on clinical judgment, re-vitrification maybe required. But little is known about re-vitrification impact on genes expression. Objective: The impact of re-vitrification on apoptotic and implanting genes, Bax, Bcl-2 and ErbB4, at compaction stage embryos were evaluated in this study. Materials and Methods: In this experimental study, 8 cell embryos (n=240) were collected from female mature mice, 60-62 hr post HCG injection. The embryos were divided randomly to 3 groups included: fresh (n=80), vitrified at 8 cell stage (n=80), vitrified at 8 cell stage thawed and re-vitrified at compaction stage (n=80). Embryos were vitrified by using cryolock, (open system) described by Kuwayama. Q-PCR was used to examine the expression of Bax, Bcl2 ErbB4 genes in derived blastocysts. Results: Our result showed that expanded blastocyst rate was similar between vitrified and re-vitrified groups, while re-vitrified embryos showed significant decrease in expanded blastocyst rate comparing with fresh embryos (p=0.03). In addition, significant difference was observed on apoptotic gene expression when comparing re-vitrified and fresh embryos (p=0.004), however expression of Bax and Bcl-2 (apoptotic) genes didn't demonstrate a significant difference between re-vitrified and vitrified groups. The expression rate of ErbB4, an implantation gene was decreased in re-vitrified embryos comparing with fresh embryos (p=0.003), but it was similar between re-vitrified and vitrified embryos. Conclusion: Re-vitrification can alter the expression of Bax, Bcl-2 and ErbB4 genes and developmental rate of mouse embryos in compaction stage. PMID:27679826

Metabolic and mitochondrial dysfunction in early mouse embryos following maternal dietary protein intervention.

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Mitchell, Megan; Schulz, Samantha L; Armstrong, David T; Lane, Michelle

2009-04-01

Dietary supply of nutrients, both periconception and during pregnancy, influence the growth and development of the fetus and offspring and their health into adult life. Despite the importance of research efforts surrounding the developmental origins of health and disease hypothesis, the biological mechanisms involved remain elusive. Mitochondria are of major importance in the oocyte and early embryo, particularly as a source of ATP generation, and perturbations in their function have been related to reduced embryo quality. The present study examined embryo development following periconception exposure of females to a high-protein diet (HPD) or a low-protein diet (LPD) relative to a medium-protein diet (MPD; control), and we hypothesized that perturbed mitochondrial metabolism in the mouse embryo may be responsible for the impaired embryo and fetal development reported by others. Although the rate of development to the blastocyst stage did not differ between diets, both the HPD and LPD reduced the number of inner cell mass cells in the blastocyst-stage embryo. Furthermore, mitochondrial membrane potential was reduced and mitochondrial calcium levels increased in the 2-cell embryo. Embryos from HPD females had elevated levels of reactive oxygen species and ADP concentrations, indicative of metabolic stress and, potentially, the uncoupling of oxidative phosphorylation, whereas embryos from LPD females had reduced mitochondrial clustering around the nucleus, suggestive of an overall quietening of metabolism. Thus, although periconception dietary supply of different levels of protein is permissive of development, mitochondrial metabolism is altered in the early embryo, and the nature of the perturbation differs between HPD and LPD exposure.

Effect of embryo density on in vitro developmental characteristics of bovine preimplantative embryos with respect to micro and macroenvironments.

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Hoelker, M; Rings, F; Lund, Q; Phatsara, C; Schellander, K; Tesfaye, D

2010-10-01

To overcome developmental problems as a consequence of single embryo culture, the Well of the Well (WOW) culture system has been developed. In this study, we aimed to examine the effect of embryo densities with respect to both microenvironment and macroenvironment on developmental rates and embryo quality to get a deeper insight into developmentally important mechanisms. WOW diameter and depth significantly affected developmental rates (p < 0.05). WOWs with diameter of 500 μm reached significantly higher blastocyst rates (32.5 vs 21.1% vs 20.3%) compared to embryos cultured in WOWs of 300 μm diameter or plain cultured controls. Embryos cultured in WOWs with 700 μm depth reached significant higher developmental rates compared with embryos cultured in WOWs of 300 μm depth and control embryos (30.6 vs 22.6% vs 20.3%). Correlation of the embryo per WOW volume with developmental rates was higher (r(2) = 0.92, p = 0.0004) than correlation of WOW diameter or WOW depth with developmental rates. However, the embryo per WOW volume did not affect differential cell counts. An embryo per culture dish volume of 1 : 30 μl was identified to be optimal when the embryo per WOW volume was 1 : 0.27 μl increasing developmental rates up to the level of mass embryo production. Giving the opportunity to track each embryo over the complete culture period while keeping high developmental rates with normal mitotic dynamics, the results of this work will provide benefit for the single culture of embryos in human assisted reproduction, mammalian embryos with high economic interest as well as for scientific purpose. © 2009 Blackwell Verlag GmbH.

Effect of culture medium volume and embryo density on early mouse embryonic development: tracking the development of the individual embryo.

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Dai, Shan-Jun; Xu, Chang-Long; Wang, Jeffrey; Sun, Ying-Pu; Chian, Ri-Cheng

2012-07-01

To determine the optimal volume or density of embryos for the well-of-the-well (WOW) system in order to track the development of individual embryos and to determine whether the WOW system can reverse the negative impact of culturing embryos singly. (1) Mouse embryos (groups of nine at the 2-cell stage) were cultured in 6.25 μl, 12.50 μl, 25.00 μl and 50.00 μl of droplets of culture medium under paraffin oil; (2) Groups of three, six, nine and twelve embryos at the 2-cell stage were cultured in 50 μl of droplet of culture medium under paraffin oil; (3) Groups of nine embryos at the 2-cell stage were cultured in 50 μl of droplet under paraffin oil with or without nine micro-wells made on the bottom of the Petri dish into each of which were placed one of the nine embryos (WOW system). Also single 2-cell stage embryos was cultured individually in 5.5 μl of droplet of culture medium under paraffin oil with or without a single micro-well made on the bottom of the Petri dish (WOW system for single culture). At the end of culture, the percentages of blastocyst development, hatching and hatched blastocysts were compared in each group. The blastocysts were fixed for differential staining. The blastocyst development was significantly higher (P WOW system. The blastocyst development was not improved when single embryo cultured individually in a micro-well was compared to single embryo cultured individually without micro-well. The total cell numbers of blastocysts were significantly higher in group embryo culture than single embryo culture regardless of whether the WOW system was used. In addition, the total cell numbers of blastocysts were significantly higher (P WOW system than without. Group embryo culture is superior to single embryo culture for blastocyst development. The WOW system with 50 μl of droplet of culture medium can be used to track the individual development of embryo cultured in groups while preserving good embryonic development. The reduced

Interspecies chimera between primate embryonic stem cells and mouse embryos: monkey ESCs engraft into mouse embryos, but not post-implantation fetuses.

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Simerly, Calvin; McFarland, Dave; Castro, Carlos; Lin, Chih-Cheng; Redinger, Carrie; Jacoby, Ethan; Mich-Basso, Jocelyn; Orwig, Kyle; Mills, Parker; Ahrens, Eric; Navara, Chris; Schatten, Gerald

2011-07-01

Unequivocal evidence for pluripotency in which embryonic stem cells contribute to chimeric offspring has yet to be demonstrated in human or nonhuman primates (NHPs). Here, rhesus and baboons ESCs were investigated in interspecific mouse chimera generated by aggregation or blastocyst injection. Aggregation chimera produced mouse blastocysts with GFP-nhpESCs at the inner cell mass (ICM), and embryo transfers (ETs) generated dimly-fluorescencing abnormal fetuses. Direct injection of GFP-nhpESCs into blastocysts produced normal non-GFP-fluorescencing fetuses. Injected chimera showed >70% loss of GFP-nhpESCs after 21 h culture. Outgrowths of all chimeric blastocysts established distinct but separate mouse- and NHP-ESC colonies. Extensive endogenous autofluorescence compromised anti-GFP detection and PCR analysis did not detect nhpESCs in fetuses. NhpESCs localize to the ICM in chimera and generate pregnancies. Because primate ESCs do not engraft post-implantation, and also because endogenous autofluorescence results in misleading positive signals, interspecific chimera assays for pluripotency with primate stem cells is unreliable with the currently available ESCs. Testing primate ESCs reprogrammed into even more naïve states in these inter-specific chimera assays will be an important future endeavor. Copyright © 2011 Elsevier B.V. All rights reserved.

The effect of adriamycin exposure on the notochord of mouse embryos.

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Hajduk, Piotr; May, Alison; Puri, Prem; Murphy, Paula

2012-04-01

The notochord has important structural and signaling properties during vertebrate development with key roles in patterning surrounding tissues, including the foregut. The adriamycin mouse model is an established model of foregut anomalies where exposure of embryos in utero to the drug adriamycin leads to malformations including oesophageal atresia and tracheoesophageal fistula. In addition to foregut abnormalities, treatment also causes branching, displacement, and hypertrophy of the notochord. Here, we explore the hypothesis that the notochord may be a primary target of disruption leading to abnormal patterning of the foregut by examining notochord position and structure in early embryos following adriamycin exposure. Treated (n = 46) and control (n = 30) embryos were examined during the crucial period when the notochord normally delaminates away from the foregut endoderm (6-28 somite pairs). Transverse sections were derived from the anterior foregut and analyzed by confocal microscopy following immunodetection of extracellular matrix markers E-cadherin and Laminin. In adriamycin-treated embryos across all stages, the notochord was abnormally displaced ventrally with prolonged attachment to the foregut endoderm. While E-cadherin was normally detected in the foregut endoderm with no expression in the notochord of control embryos, treated embryos up to 24 somites showed ectopic notochordal expression indicating a change in characteristics of the tissue; specifically an increase in intracellular adhesiveness, which may be instrumental in structural changes, affecting mechanical and signaling properties. This is consistent with disruption of the notochord leading to altered signaling to the foregut causing abnormal patterning and congenital foregut malformations. © 2012 Wiley Periodicals, Inc.

Comprehensive embryo testing. Experts' opinions regarding future directions: an expert panel study on comprehensive embryo testing.

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Hens, Kristien; Dondorp, Wybo J; Geraedts, Joep P M; de Wert, Guido M

2013-05-01

What do scientists in the field of preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS) consider to be the future direction of comprehensive embryo testing? Although there are many biological and technical limitations, as well as uncertainties regarding the meaning of genetic variation, comprehensive embryo testing will impact the IVF/PGD practice and a timely ethical reflection is needed. Comprehensive testing using microarrays is currently being introduced in the context of PGD and PGS, and it is to be expected that whole-genome sequencing will also follow. Current ethical and empirical sociological research on embryo testing focuses on PGD as it is practiced now. However, empirical research and systematic reflection regarding the impact of comprehensive techniques for embryo testing is missing. In order to understand the potential of this technology and to be able to adequately foresee its implications, we held an expert panel with seven pioneers in PGD. We conducted an expert panel in October 2011 with seven PGD pioneers from Belgium, The Netherlands, Germany and the UK. Participants expected the use of comprehensive techniques in the context of PGD. However, the introduction of these techniques in embryo testing requires timely ethical reflection as it involves a shift from choosing an embryo without a particular genetic disease (i.e. PGD) or most likely to result in a successful pregnancy (i.e. PGS) to choosing the best embryo based on a much wider set of criteria. Such ethical reflection should take account of current technical and biological limitations and also of current uncertainties with regard to the meaning of genetic variance. However, ethicists should also not be afraid to look into the future. There was a general agreement that embryo testing will be increasingly preceded by comprehensive preconception screening, thus enabling smart combinations of genetic testing. The group was composed of seven participants from

The Influence of Single Nucleotide Polymorphism Microarray-Based Molecular Karyotype on Preimplantation Embryonic Development Potential.

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Gang Li

Full Text Available 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% (P8 cells, embryos with 6-8 blastomeres have higher rate of balanced molecular karyotype and blastocyst formation.

Antigen presenting cells costimulatory signaling during pre-implantation pregnancy 

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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.

Effect of prenatal gamma irradiation on the different gestation days on mouse

International Nuclear Information System (INIS)

Baskar, R.; Uma Devi, P.

1993-01-01

Pregnant Swiss albino mice were given a single local (abdomen) exposure of 50 cGy gamma radiation on days 1.5 to 17.5 post coitus (p.c.). The animals were sacrificed on 18th day of gestation and the fetuses were dissected out and examined for mortality, growth retardation, sex ratio, changes in body weight and length, head length and width, brain weight and incidence of microphthalmia. Preimplantation exposures (days 1.5 and 3.5 p.c.) resulted in a significant increase in embryonic and fetal mortality, while exposure during the postimplantation period (days 5.5 to 12.5 p.c.) produced a significant increase in the number of growth retarded fetuses. A high incidence of growth retarded fetuses was also observed by irradiation at the late fetal period (17.5 days p.c.). Brain weight was significantly affected by exposure on some days during all the three phases of development, i.e. preimplantation (3.5 days p.c.), mager organogenesis (9.5 to 12.5 days p.c.) and fetal (14.5 and 17.5 days p.c.) periods, though the period of sensitiveness was larger during organogenesis period. Eye development was sensitive to radiation effect mainly during the late organogenesis (9.5 to 12.5 days p.c.). It is concluded that the radiosensitivity of mouse embryos to radiation lethality at low dose is confirmed to the preimplantation period, while growth retardation is a prominent effect of irradiation at the early postimplantation embryos. The late organogenesis, especially from 9.5 to 12.5 days p.c. appears to be a particularly sensitive period in the development of brain and eye, though the sensitivity of brain may continue during the fetal life. (author). 9 refs

ROCK and RHO Playlist for Preimplantation Development: Streaming to HIPPO Pathway and Apicobasal Polarity in the First Cell Differentiation.

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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.

Birth of a healthy infant after preimplantation genetic diagnosis by sequential blastomere and trophectoderm biopsy for β-thalassemia and HLA genotyping.

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Milachich, Tanya; Timeva, Tanya; Ekmekci, Cumhur; Beyazyurek, Cagri; Tac, Huseyin Avni; Shterev, Atanas; Kahraman, Semra

2013-07-01

Preimplantation genetic diagnosis (PGD) is a widely used technique for couples at genetic risk and involves the diagnosis and transfer of unaffected embryos generated through in vitro fertilization (IVF) techniques. For those couples who are at risk of transmitting a genetic disease to their offspring, preimplantation embryos can be selected according to their genetic status as well as human leukocyte antigen (HLA) compatibility with the affected child. Stem cells from the resulting baby's umbilical cord blood can be used for transplantation to the affected sibling without graft rejection. Here we report successful hematopoietic stem cell transplantation (HSCT) after the birth of a healthy infant, who was born after successful PGD testing with both cleavage stage and blastocyst stage biopsy for the purpose of diagnosis of β-thalassemia and HLA compatibility. The specific feature of this work is not only to have the first successful HSCT achieved in Bulgaria after using preimplantation HLA typing technique, it also demonstrates how to accomplish this success via cross-border collaboration of different units, which makes the application of these sophisticated methods possible in hospitals not having the necessary equipments and expertise. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Study on preimplantation genetic diagnosis and follow-up for Duchenne muscular dystrophy

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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

Simultaneous preimplantation genetic diagnosis for Tay-Sachs and Gaucher disease.

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Altarescu, Gheona; Brooks, Barry; Margalioth, Ehud; Eldar Geva, Talia; Levy-Lahad, Ephrat; Renbaum, Paul

2007-07-01

Preimplantation genetic diagnosis (PGD) for single gene defects is described for a family in which each parent is a carrier of both Tay-Sachs (TS) and Gaucher disease (GD). A multiplex fluorescent polymerase chain reaction protocol was developed that simultaneously amplified all four familial mutations and 10 informative microsatellite markers. In one PGD cycle, seven blastomeres were analysed, reaching a conclusive diagnosis in six out of seven embryos for TS and in five out of seven embryos for GD. Of the six diagnosed embryos, one was wild type for both TS and GD, and three were wild type for GD and carriers of TS. Two remaining embryos were compound heterozygotes for TS. Two transferable embryos developed into blastocysts (wt/wt and wt GD/carrier TS) and both were transferred on day 5. This single cycle of PGD resulted in a healthy live child. Allele drop-out (ADO) was observed in three of 34 reactions, yielding an 8% ADO rate. The occurrence of ADO in single cell analysis and undetected recombination events are primary causes of misdiagnosis in PGD and emphasize the need to use multiple polymorphic markers. So far as is known, this is the first report of concomitant PGD for two frequent Ashkenazi Jewish recessive disorders.

[Extending preimplantation genetic diagnosis to HLA typing: the French exception].

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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).

Alternation of apoptotic and implanting genes expression of mouse embryos after re-vitrification

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Nasrin Majidi Gharenaz

2016-08-01

Full Text Available Background: Nowadays, oocytes and embryos vitrification has become a routine technique. Based on clinical judgment, re-vitrification maybe required. But little is known about re-vitrification impact on genes expression. Objective: The impact of re-vitrification on apoptotic and implanting genes, Bax, Bcl-2 and ErbB4, at compaction stage embryos were evaluated in this study. Materials and Methods: In this experimental study, 8 cell embryos (n=240 were collected from female mature mice, 60-62 hr post HCG injection. The embryos were divided randomly to 3 groups included: fresh (n=80, vitrified at 8 cell stage (n=80, vitrified at 8 cell stage thawed and re-vitrified at compaction stage (n=80. Embryos were vitrified by using cryolock, (open system described by Kuwayama. Q-PCR was used to examine the expression of Bax, Bcl2 ErbB4 genes in derived blastocysts. Results: Our result showed that expanded blastocyst rate was similar between vitrified and re-vitrified groups, while re-vitrified embryos showed significant decrease in expanded blastocyst rate comparing with fresh embryos (p=0.03. In addition, significant difference was observed on apoptotic gene expression when comparing re-vitrified and fresh embryos (p=0.004, however expression of Bax and Bcl-2 (apoptotic genes didn't demonstrate a significant difference between re-vitrified and vitrified groups. The expression rate of ErbB4, an implantation gene was decreased in re-vitrified embryos comparing with fresh embryos (p=0.003, but it was similar between re-vitrified and vitrified embryos. Conclusion: Re-vitrification can alter the expression of Bax, Bcl-2 and ErbB4 genes and developmental rate of mouse embryos in compaction stage

Hot Topic: Preimplantation aneuploidy screening

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Kayhan Yakın

2009-03-01

Full Text Available Preimplantation Genetic Screening (PGS is a technique that has been introduced into clinical practice to screen and eliminate aneuploid embryos form transfer with the intention to improve implantation rates and decrease pregnancy wastage. Although practiced widely throughout the world the PGS unfortunately has been adopted without being subjected to rigorous scientific validation. Data from recent prospective randomized trials have shed doubt on the efficacy of the procedure when used in women with advanced age, one of the target populations for PGS. Other purported indications for the application of this complicated technique such as recurrent implantation failure and recurrent spontaneous abortion have not been subjected to randomized controlled trials. For the best interest of patients, we feel it is timely for a debate regarding the efficacy and safety of PGS.

Lipofection of siRNA into bovine 8-16-cell stage embryos using zona removal and the well-of-the-well culture system.

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Ikeda, Shuntaro; Sugimoto, Miki; Kume, Shinichi

2018-04-13

Bovine preimplantation embryos exhibit dramatic biological changes between before and after the 8-16-cell stage. Here we report a simple lipofection method to transfect siRNA into bovine 8-16-cell stage embryos using zona removal and the well-of-the-well (WOW) culture system. Bovine one-cell embryos produced in vitro were freed from the zona pellucida and cultured up to the 8-16-cell stage in WOW dishes. The 8-16-cell embryos were lipofected with siRNA and the transfection efficiency was assessed at 48 h of transfection. Lipofection with a red fluorescent non-targeting siRNA revealed the importance of zona removal for transfection of siRNA into embryos. Using this method, we knocked down the methionine adenosyltransferase 2A (MAT2A) gene, achieving a significant reduction in MAT2A expression (P lipofection', may be useful to analyze gene functions in bovine preimplantation embryos without expensive equipment and skill-intensive techniques.

High survival of mouse embryos after rapid freezing and thawing inside plastic straws with 1-2 propanediol as cryoprotectant.

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Renard, J P; Babinet, C

1984-06-01

A method for obtaining a high survival rate of frozen-thawed mouse embryos is presented. Eight-cell mouse embryos were frozen inside small plastic straws in the presence of 1-2 propanediol and stored at -196 C. After thawing, the embryos were diluted for only 5 min in a 1.0 M sucrose solution to remove the 1-2 propanediol from the cells. At high rate of thawing (is equivalent to 2500 C/min) more than 88% of the embryos survived in vitro to the blastocyst stage provided that the dilution of propanediol was performed rapidly during thawing. At a lower rate of thawing (is equivalent to 300 C/min), survival tended to be higher (94.7%) when dilution was done 5 min after thawing. When the frozen-thawed embryos were transferred to the oviducts of day 1 pseudopregnant recipients either directly after the dilution of 1-2 propanediol or after 24 or 48 hr of culture, a high proportion of them (65.9%) develop normally to viable fetuses.

Birth of healthy children after preimplantation diagnosis of β-thalassemia

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焦泽旭; 庄广伦; 周灿权; 舒益民; 李洁; 梁晓燕

2004-01-01

Background Clinical programs for preventing β-thalassemia are presently based on prospective carrier screening and prenatal diagnosis. This paper report an achievement of a pregnancy with unaffected embryos using in vitro fertilization and embryo transfer (IVF-ET), in combination with preimplantation genetic diagnosis (PGD), for a couple at risk of having children with β-thalassemia.Methods A couple carrying different thalassemia mutations, both a codon 41-42 mutation and the IVS Ⅱ 654 mutation, received standard IVF treatment, with intracytoplasmic sperm injection, embryo biopsiy, single cell polymerase chain reaction (PCR) and DNA analysis. Only unaffected or carrier embryos were transferred to the uterine cavity. After confirmation of pregnancy, a prenatal diagnosis was performed.Results Of a total of 13 embryos analyzed for β-globin mutations, PGD indicated that 2 were normal,3 were affected, and 6 were carriers. Diagnosis could not be made in the other 2 embryos. Three embryos were transferred to the uterus on the third day after oocyte retrieval. Ultrasonography revealed a twin pregnancy with one blighted ovum. The prenatal genetic diagnosis revealed that both fetuses were unaffected, and two healthy boys were born, confirming the results of PGD.Conclusions We developed a single-cell based primer extension preamplification (PEP)-PCR assay for the detection of β-thalassemia mutations. The assays were efficient and accurate at all stages of the procedure, and resulted in the birth of PGD-confirmed β-thalassemia free children in China. PEP was used here in PGD for β-thalassemia.

Heart valve cardiomyocytes of mouse embryos express the serotonin transporter SERT

International Nuclear Information System (INIS)

Pavone, Luigi Michele; Spina, Anna; Lo Muto, Roberta; Santoro, Dionea; Mastellone, Vincenzo; Avallone, Luigi

2008-01-01

Multiple evidence demonstrate a role for serotonin and its transporter SERT in heart valve development and disease. By utilizing a Cre/loxP system driven by SERT gene expression, we recently demonstrated a regionally restricted distribution of SERT-expressing cells in developing mouse heart. In order to characterize the cell types exhibiting SERT expression within the mouse heart valves at early developmental stages, in this study we performed immunohistochemistry for Islet1 (Isl1) and connexin-43 (Cx-43) on heart sections from SERT Cre/+ ;ROSA26R embryos previously stained with X-gal. We observed the co-localization of LacZ staining with Isl1 labelling in the outflow tract, the right ventricle and the conal region of E11.5 mouse heart. Cx-43 labelled cells co-localized with LacZ stained cells in the forming atrioventricular valves. These results demonstrate the cardiomyocyte phenotype of SERT-expressing cells in heart valves of the developing mouse heart, thus suggesting an active role of SERT in early heart valve development.

De novo DNA methylation during monkey pre-implantation embryogenesis.

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Gao, Fei; Niu, Yuyu; Sun, Yi Eve; Lu, Hanlin; Chen, Yongchang; Li, Siguang; Kang, Yu; Luo, Yuping; Si, Chenyang; Yu, Juehua; Li, Chang; Sun, Nianqin; Si, Wei; Wang, Hong; Ji, Weizhi; Tan, Tao

2017-04-01

Critical epigenetic regulation of primate embryogenesis entails DNA methylome changes. Here we report genome-wide composition, patterning, and stage-specific dynamics of DNA methylation in pre-implantation rhesus monkey embryos as well as male and female gametes studied using an optimized tagmentation-based whole-genome bisulfite sequencing method. We show that upon fertilization, both paternal and maternal genomes undergo active DNA demethylation, and genome-wide de novo DNA methylation is also initiated in the same period. By the 8-cell stage, remethylation becomes more pronounced than demethylation, resulting in an increase in global DNA methylation. Promoters of genes associated with oxidative phosphorylation are preferentially remethylated at the 8-cell stage, suggesting that this mode of energy metabolism may not be favored. Unlike in rodents, X chromosome inactivation is not observed during monkey pre-implantation development. Our study provides the first comprehensive illustration of the 'wax and wane' phases of DNA methylation dynamics. Most importantly, our DNA methyltransferase loss-of-function analysis indicates that DNA methylation influences early monkey embryogenesis.

Laser confers less embryo exposure than acid tyrode for embryo biopsy in preimplantation genetic diagnosis cycles: a randomized study.

Science.gov (United States)

Geber, Selmo; Bossi, Renata; Lisboa, Cintia B; Valle, Marcelo; Sampaio, Marcos

2011-04-28

We compared two methods of zona pellucida drilling. 213 embryos were biopsied with acid Tyrode. Each biopsy took 3 minutes and the entire procedure ~29 minutes. 5% of blastomeres lysed, 49% of embryos became blastocyst and 36% of patients became pregnant. 229 embryos were biopsied with laser. Each biopsy took 30 seconds and the entire procedure ~7 minutes. 2.5% of blastomeres lysed, 50.6% of embryos became blastocyst and 47% of patients became pregnant. We can conclude that laser can be used for embryo biopsy. Reduction of embryo exposure and of removed blastomeres is associated with increased blastocysts available for transfer and a better clinical outcome.

Laser confers less embryo exposure than acid tyrode for embryo biopsy in preimplantation genetic diagnosis cycles: a randomized study

Directory of Open Access Journals (Sweden)

Valle Marcelo

2011-04-01

Full Text Available Abstract We compared two methods of zona pellucida drilling. 213 embryos were biopsied with acid Tyrode. Each biopsy took 3 minutes and the entire procedure ~29 minutes. 5% of blastomeres lysed, 49% of embryos became blastocyst and 36% of patients became pregnant. 229 embryos were biopsied with laser. Each biopsy took 30 seconds and the entire procedure ~7 minutes. 2.5% of blastomeres lysed, 50.6% of embryos became blastocyst and 47% of patients became pregnant. We can conclude that laser can be used for embryo biopsy. Reduction of embryo exposure and of removed blastomeres is associated with increased blastocysts available for transfer and a better clinical outcome.

Embryo transcriptome response to environmental factors: implication for its survival under suboptimal conditions.

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Salilew-Wondim, Dessie; Tesfaye, Dawit; Hoelker, Michael; Schellander, Karl

2014-09-01

After its formation, the mammalian zygote undergoes a series of morphological, physiological and biochemical alterations prior to undergoing cell differentiation. The zygote is then transformed into a complex multicellular organism in a defined time window which may differ between species. These orderly embryonic developmental events are tightly regulated by temporal and spatial activation and/or deactivation of genes and gene products. This phenomenon may in turn be dependent on the intrinsic characteristics of the embryo itself, the physiological and biochemical composition of the maternal environment or by in vitro culture condition. In fact, when embryos are subjected to suboptimal culture condition, some of the embryos may escape the environmental stress by activating certain transcripts and some others which are unable to activate anti-stress agents may die or exhibit abnormal development. This phenomenon may partly depend on transcripts and proteins stored during oogenesis. Indeed after embryonic genome activation, the embryo destiny is governed by its own transcripts and protein synthesized over time. Therefore, this review begins by highlighting the type and quality of transcripts accumulated or degraded during oogenesis and its impact on the embryo survival. Thereafter, emphasis is given to the transcriptome response of preimplantation embryos to suboptimal culture conditions. In addition, the long term effect of preimplantation culture environment on the transcriptome response embryos/fetus during peri and post implantation has been addressed. Finally, a brief summary of the epigenetic control of culture induced genetic variation of the embryos has been highlighted. Copyright © 2014 Elsevier B.V. All rights reserved.

Preimplantation genetic diagnosis and rational choice under risk or uncertainty.

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Zuradzki, Tomasz

2014-11-01

In this paper I present an argument in favour of a parental duty to use preimplantation genetic diagnosis (PGD). I argue that if embryos created in vitro were able to decide for themselves in a rational manner, they would sometimes choose PGD as a method of selection. Couples, therefore, should respect their hypothetical choices on a principle similar to that of patient autonomy. My thesis shows that no matter which moral doctrine couples subscribe to, they ought to conduct the PGD procedure in the situations when it is impossible to implant all of the created embryos and if there is a significant risk for giving birth to a child with a serious condition. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Single-cell duplex RT-LATE-PCR reveals Oct4 and Xist RNA gradients in 8-cell embryos

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Hartung Odelya

2007-12-01

Full Text Available Abstract Background The formation of two distinctive cell lineages in preimplantation mouse embryos is characterized by differential gene expression. The cells of the inner cell mass are pluripotent and express high levels of Oct4 mRNA, which is down-regulated in the surrounding trophectoderm. In contrast, the trophectoderm of female embryos contains Xist mRNA, which is absent from cells of the inner mass. Prior to blastocyst formation, all blastomeres of female embryos still express both of these RNAs. We, thus, postulated that simultaneous quantification of Oct4 and Xist transcripts in individual blastomeres at the 8-cell stage could be informative as to their subsequent fate. Testing this hypothesis, however, presented numerous technical challenges. We overcame these difficulties by combining PurAmp, a single-tube method for RNA preparation and quantification, with LATE-PCR, an advanced form of asymmetric PCR. Results We constructed a duplex RT-LATE-PCR assay for real-time measurement of Oct4 and Xist templates and confirmed its specificity and quantitative accuracy with different methods. We then undertook analysis of sets of blastomeres isolated from embryos at the 8-cell stage. At this stage, all cells in the embryo are still pluripotent and morphologically equivalent. Our results demonstrate, however, that both Oct4 and Xist RNA levels vary in individual blastomeres comprising the same embryo, with some cells having particularly elevated levels of either transcript. Analysis of multiple embryos also shows that Xist and Oct4 expression levels are not correlated at the 8-cell stage, although transcription of both genes is up-regulated at this time in development. In addition, comparison of data from males and females allowed us to determine that the efficiency of the Oct4/Xist assay is unaffected by sex-related differences in gene expression. Conclusion This paper describes the first example of multiplex RT-LATE-PCR and its utility, when

Utilization of endogenous fatty acid stores for energy production in bovine preimplantation embryos.

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Sutton-McDowall, Melanie L; Feil, Deanne; Robker, Rebecca L; Thompson, Jeremy G; Dunning, Kylie R

2012-05-01

Although current embryo culture media are based on carbohydrate metabolism of embryos, little is known about metabolism of endogenous lipids. L-carnitine is a β-oxidation cofactor absent in most culture media. The objective was to investigate the influence of L-carnitine supplementation on bovine embryo development. Abattoir-derived bovine cumulus oocyte complexes were cultured and fertilized. Post-fertilization, presumptive zygotes were transferred into a basic cleavage medium ± carbohydrates (glucose, lactate and pyruvate) ± 5 mm L-carnitine and cultured for 4 days in vitro. In the absence of carbohydrates during culture, embryos arrested at the 2- and 4-cell stages. Remarkably, +L-carnitine increased development to the morula stage compared to +carbohydrates alone (P levels were higher and ATP: ADP ratio were 1.9-fold lower (main effect, P < 0.05) compared to embryos cultured in -L-carnitine. Therefore, we inferred that +L-carnitine embryos were more metabolically active, with higher rates of ATP-ADP conversion. In conclusion, L-carnitine supplementation supported precompaction embryo development and there was an additive effect of +L-carnitine +carbohydrates on early embryo development, most likely through increased β-oxidation within embryos. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

Comparison of pre- and postimplantation development following the application of three artificial activating stimuli in a mouse model with round-headed sperm cells deficient for oocyte activation

DEFF Research Database (Denmark)

Vanden Meerschaut, Frauke; Nikiforaki, D.; De Roo, C.

2013-01-01

STUDY QUESTION Does the application of three different artificial activating stimuli lead to a difference in pre- and post-implantation embryo development in the wobbler mouse, a mouse model with oocyte activation deficient round-headed sperm cells similar to human globozoospermia? SUMMARY ANSWER...... fertilized by wobbler and wild-type (WT) sperm following ICSI with or without three different artificial activating agents. Preimplantation development was assessed on 70 injected oocytes on average per group. On average, 10 foster mothers were used per activating group to compare post......-implantation development. PARTICIPANTS/MATERIALS, SETTING, METHODS We used the wobbler mouse model that possesses oocyte activation deficient round-headed sperm cells. First, the calcium release following ICSI using wobbler sperm was compared with that of WT sperm. Outcome measures were the percentage of oocytes...

Apoptosis induced by glufosinate ammonium in the neuroepithelium of developing mouse embryos in culture.

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Watanabe, T

1997-01-24

Glufosinate ammonium structurally resembles glutamate and blocks glutamine synthetase. Glufosinate was recently found to be dysmorphogenic in mammals in vitro. The present study examined the cell death induced specifically by glufosinate in the neuroepithelium of mouse embryos. Electron micrograph revealed characteristic chromatin condensation and segregation, extracellular apoptotic bodies, and cell fragments phagocytosed in macrophages in the neuroepithelium of the brain vesicle and neural tube. Moreover neuroepithelial cells undergoing DNA fragmentation were histochemically identified. DNA gel electrophoresis of the neuroepithelial layer revealed a DNA ladder. These observations demonstrate that glufosinate specifically induced apoptosis in the neuroepithelium of embryos.

[Advance in the methods of preimplantation genetic diagnosis for single gene diseases].

Science.gov (United States)

Ren, Yixin; Qiao, Jie; Yan, Liying

2017-06-10

More than 7000 single gene diseases have been identified and most of them lack effective treatment. As an early form of prenatal diagnosis, preimplantation genetic diagnosis (PGD) is a combination of in vitro fertilization and genetic diagnosis. PGD has been applied in clinics for more than 20 years to avoid the transmission of genetic defects through analysis of embryos at early stages of development. In this paper, a review for the recent advances in PGD for single gene diseases is provided.

Effects of Multimodal Analgesia on the Success of Mouse Embryo Transfer Surgery

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Parker, John M.; Austin, Jamie; Wilkerson, James; Carbone, Larry

2011-01-01

Multimodal analgesia is promoted as the best practice pain management for invasive animal research procedures. Universal acceptance and incorporation of multimodal analgesia requires assessing potential effects on study outcome. The focus of this study was to assess effects on embryo survival after multimodal analgesia comprising an opioid and nonsteroidal antiinflammatory drug (NSAID) compared with opioid-only analgesia during embryo transfer procedures in transgenic mouse production. Mice were assigned to receive either carprofen (5 mg/kg) with buprenorphine (0.1 mg/kg; CB) or vehicle with buprenorphine (0.1 mg/kg; VB) in a prospective, double-blinded placebo controlled clinical trial. Data were analyzed in surgical sets of 1 to 3 female mice receiving embryos chimeric for a shared targeted embryonic stem-cell clone and host blastocyst cells. A total of 99 surgical sets were analyzed, comprising 199 Crl:CD1 female mice and their 996 offspring. Neither yield (pups weaned per embryo implanted in the surgical set) nor birth rate (average number of pups weaned per dam in the set) differed significantly between the CB and VB conditions. Multimodal opioid–NSAID analgesia appears to have no significant positive or negative effect on the success of producing novel lines of transgenic mice by blastocyst transfer. PMID:21838973

Blood pressure and anthropometrics of 4-y-old children born after preimplantation genetic screening: follow-up of a unique, moderately sized, randomized controlled trial

NARCIS (Netherlands)

Seggers, Jorien; Haadsma, Maaike L.; Bastide-van Gemert, Sacha la; Heineman, Maas Jan; Kok, Joke H.; Middelburg, Karin J.; Roseboom, Tessa J.; Schendelaar, Pamela; van den Heuvel, Edwin R.; Hadders-Algra, Mijna

2013-01-01

Recent studies suggest that in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are associated with suboptimal cardiometabolic outcome in offspring. It is unknown whether preimplantation genetic screening (PGS), which involves embryo biopsy, affects blood pressure (BP),

Blood pressure and anthropometrics of 4-y-old children born after preimplantation genetic screening : follow-up of a unique, moderately sized, randomized controlled trial

NARCIS (Netherlands)

Seggers, Jorien; Haadsma, Maaike L.; la Bastide-van Gemert, Sacha; Heineman, Maas Jan; Kok, Joke H.; Middelburg, Karin J.; Roseboom, Tessa J.; Schendelaar, Pamela; Van den Heuvel, Edwin R.; Hadders-Algra, Mijna

2013-01-01

BACKGROUND: Recent studies suggest that in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are associated with suboptimal cardiometabolic outcome in offspring. It is unknown whether preimplantation genetic screening (PGS), which involves embryo biopsy, affects blood pressure

Importance of the pluripotency factor LIN28 in the mammalian nucleolus during early embryonic development.

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Vogt, Edgar J; Meglicki, Maciej; Hartung, Kristina Ilka; Borsuk, Ewa; Behr, Rüdiger

2012-12-01

The maternal nucleolus is required for proper activation of the embryonic genome (EGA) and early embryonic development. Nucleologenesis is characterized by the transformation of a nucleolar precursor body (NPB) to a mature nucleolus during preimplantation development. However, the function of NPBs and the involved molecular factors are unknown. We uncover a novel role for the pluripotency factor LIN28, the biological significance of which was previously demonstrated in the reprogramming of human somatic cells to induced pluripotent stem (iPS) cells. Here, we show that LIN28 accumulates at the NPB and the mature nucleolus in mouse preimplantation embryos and embryonic stem cells (ESCs), where it colocalizes with the nucleolar marker B23 (nucleophosmin 1). LIN28 has nucleolar localization in non-human primate (NHP) preimplantation embryos, but is cytoplasmic in NHP ESCs. Lin28 transcripts show a striking decline before mouse EGA, whereas LIN28 protein localizes to NPBs at the time of EGA. Following knockdown with a Lin28 morpholino, the majority of embryos arrest between the 2- and 4-cell stages and never develop to morula or blastocyst. Lin28 morpholino-injected embryos arrested at the 2-cell stage were not enriched with nucleophosmin at presumptive NPB sites, indicating that functional NPBs were not assembled. Based on these results, we propose that LIN28 is an essential factor of nucleologenesis during early embryonic development.

Oligonucleotide arrays vs. metaphase-comparative genomic hybridisation and BAC arrays for single-cell analysis: first applications to preimplantation genetic diagnosis for Robertsonian translocation carriers.

Science.gov (United States)

Ramos, Laia; del Rey, Javier; Daina, Gemma; García-Aragonés, Manel; Armengol, Lluís; Fernandez-Encinas, Alba; Parriego, Mònica; Boada, Montserrat; Martinez-Passarell, Olga; Martorell, Maria Rosa; Casagran, Oriol; Benet, Jordi; Navarro, Joaquima

2014-01-01

Comprehensive chromosome analysis techniques such as metaphase-Comparative Genomic Hybridisation (CGH) and array-CGH are available for single-cell analysis. However, while metaphase-CGH and BAC array-CGH have been widely used for Preimplantation Genetic Diagnosis, oligonucleotide array-CGH has not been used in an extensive way. A comparison between oligonucleotide array-CGH and metaphase-CGH has been performed analysing 15 single fibroblasts from aneuploid cell-lines and 18 single blastomeres from human cleavage-stage embryos. Afterwards, oligonucleotide array-CGH and BAC array-CGH were also compared analysing 16 single blastomeres from human cleavage-stage embryos. All three comprehensive analysis techniques provided broadly similar cytogenetic profiles; however, non-identical profiles appeared when extensive aneuploidies were present in a cell. Both array techniques provided an optimised analysis procedure and a higher resolution than metaphase-CGH. Moreover, oligonucleotide array-CGH was able to define extra segmental imbalances in 14.7% of the blastomeres and it better determined the specific unbalanced chromosome regions due to a higher resolution of the technique (≈ 20 kb). Applicability of oligonucleotide array-CGH for Preimplantation Genetic Diagnosis has been demonstrated in two cases of Robertsonian translocation carriers 45,XY,der(13;14)(q10;q10). Transfer of euploid embryos was performed in both cases and pregnancy was achieved by one of the couples. This is the first time that an oligonucleotide array-CGH approach has been successfully applied to Preimplantation Genetic Diagnosis for balanced chromosome rearrangement carriers.

Oligonucleotide arrays vs. metaphase-comparative genomic hybridisation and BAC arrays for single-cell analysis: first applications to preimplantation genetic diagnosis for Robertsonian translocation carriers.

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Laia Ramos

Full Text Available Comprehensive chromosome analysis techniques such as metaphase-Comparative Genomic Hybridisation (CGH and array-CGH are available for single-cell analysis. However, while metaphase-CGH and BAC array-CGH have been widely used for Preimplantation Genetic Diagnosis, oligonucleotide array-CGH has not been used in an extensive way. A comparison between oligonucleotide array-CGH and metaphase-CGH has been performed analysing 15 single fibroblasts from aneuploid cell-lines and 18 single blastomeres from human cleavage-stage embryos. Afterwards, oligonucleotide array-CGH and BAC array-CGH were also compared analysing 16 single blastomeres from human cleavage-stage embryos. All three comprehensive analysis techniques provided broadly similar cytogenetic profiles; however, non-identical profiles appeared when extensive aneuploidies were present in a cell. Both array techniques provided an optimised analysis procedure and a higher resolution than metaphase-CGH. Moreover, oligonucleotide array-CGH was able to define extra segmental imbalances in 14.7% of the blastomeres and it better determined the specific unbalanced chromosome regions due to a higher resolution of the technique (≈ 20 kb. Applicability of oligonucleotide array-CGH for Preimplantation Genetic Diagnosis has been demonstrated in two cases of Robertsonian translocation carriers 45,XY,der(13;14(q10;q10. Transfer of euploid embryos was performed in both cases and pregnancy was achieved by one of the couples. This is the first time that an oligonucleotide array-CGH approach has been successfully applied to Preimplantation Genetic Diagnosis for balanced chromosome rearrangement carriers.

Oligonucleotide Arrays vs. Metaphase-Comparative Genomic Hybridisation and BAC Arrays for Single-Cell Analysis: First Applications to Preimplantation Genetic Diagnosis for Robertsonian Translocation Carriers

Science.gov (United States)

Ramos, Laia; del Rey, Javier; Daina, Gemma; García-Aragonés, Manel; Armengol, Lluís; Fernandez-Encinas, Alba; Parriego, Mònica; Boada, Montserrat; Martinez-Passarell, Olga; Martorell, Maria Rosa; Casagran, Oriol; Benet, Jordi; Navarro, Joaquima

2014-01-01

Comprehensive chromosome analysis techniques such as metaphase-Comparative Genomic Hybridisation (CGH) and array-CGH are available for single-cell analysis. However, while metaphase-CGH and BAC array-CGH have been widely used for Preimplantation Genetic Diagnosis, oligonucleotide array-CGH has not been used in an extensive way. A comparison between oligonucleotide array-CGH and metaphase-CGH has been performed analysing 15 single fibroblasts from aneuploid cell-lines and 18 single blastomeres from human cleavage-stage embryos. Afterwards, oligonucleotide array-CGH and BAC array-CGH were also compared analysing 16 single blastomeres from human cleavage-stage embryos. All three comprehensive analysis techniques provided broadly similar cytogenetic profiles; however, non-identical profiles appeared when extensive aneuploidies were present in a cell. Both array techniques provided an optimised analysis procedure and a higher resolution than metaphase-CGH. Moreover, oligonucleotide array-CGH was able to define extra segmental imbalances in 14.7% of the blastomeres and it better determined the specific unbalanced chromosome regions due to a higher resolution of the technique (≈20 kb). Applicability of oligonucleotide array-CGH for Preimplantation Genetic Diagnosis has been demonstrated in two cases of Robertsonian translocation carriers 45,XY,der(13;14)(q10;q10). Transfer of euploid embryos was performed in both cases and pregnancy was achieved by one of the couples. This is the first time that an oligonucleotide array-CGH approach has been successfully applied to Preimplantation Genetic Diagnosis for balanced chromosome rearrangement carriers. PMID:25415307

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.

Influence of the radiation (Co60) in pre-implants rabbit embryos: effect on atypic mitotic index and embryo pole development

International Nuclear Information System (INIS)

Approbato, Mario S.; Oliveira Moura, Katia K.V. de; Souza Florencio, Rodopiano de; Garcia, Ricardo; Faria, Renato S.; Benedetti, Leonardo N.; Goulart, Flamarion B.

1995-01-01

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)

Recent advances in preimplantation genetic diagnosis and screening.

Science.gov (United States)

Lu, Lina; Lv, Bo; Huang, Kevin; Xue, Zhigang; Zhu, Xianmin; Fan, Guoping

2016-09-01

Preimplantation genetic diagnosis/screening (PGD/PGS) aims to help couples lower the risks of transmitting genetic defects to their offspring, implantation failure, and/or miscarriage during in vitro fertilization (IVF) cycles. However, it is still being debated with regard to the practicality and diagnostic accuracy of PGD/PGS due to the concern of invasive biopsy and the potential mosaicism of embryos. Recently, several non-invasive and high-throughput assays have been developed to help overcome the challenges encountered in the conventional invasive biopsy and low-throughput analysis in PGD/PGS. In this mini-review, we will summarize the recent progresses of these new methods for PGD/PGS and discuss their potential applications in IVF clinics.

EXPOSURE TO A P13KINASE INHIBITOR PRODUCED DYSMORPHOGENESIS IN NEURULATION-STAGED MOUSE EMBRYOS IN CULTURE

Science.gov (United States)

The haloacetic acids (HAA) are a family of chemicals that are drinking water disinfection byproducts. We previously reported that bromo- and chloro-acetic acids alter embryonic development when mouse conceptuses are directly exposed to these xenobiotics in whole embryo culture. C...

NMR studies of preimplantation embryo metabolism in human assisted reproductive techniques: a new biomarker for assessment of embryo implantation potential.

Science.gov (United States)

Pudakalakatti, Shivanand M; Uppangala, Shubhashree; D'Souza, Fiona; Kalthur, Guruprasad; Kumar, Pratap; Adiga, Satish Kumar; Atreya, Hanudatta S

2013-01-01

There has been growing interest in understanding energy metabolism in human embryos generated using assisted reproductive techniques (ART) for improving the overall success rate of the method. Using NMR spectroscopy as a noninvasive tool, we studied human embryo metabolism to identify specific biomarkers to assess the quality of embryos for their implantation potential. The study was based on estimation of pyruvate, lactate and alanine levels in the growth medium, ISM1, used in the culture of embryos. An NMR study involving 127 embryos from 48 couples revealed that embryos transferred on Day 3 (after 72 h in vitro culture) with successful implantation (pregnancy) exhibited significantly (p < 10(-5) ) lower pyruvate/alanine ratios compared to those that failed to implant. Lactate levels in media were similar for all embryos. This implies that in addition to lactate production, successfully implanted embryos use pyruvate to produce alanine and other cellular functions. While pyruvate and alanine individually have been used as biomarkers, the present study highlights the potential of combining them to provide a single parameter that correlates strongly with implantation potential. Copyright © 2012 John Wiley & Sons, Ltd.

Survival of mouse embryos after vitrification depending on the cooling rate of the cryoprotectant solution.

Science.gov (United States)

Hredzák, R; Ostró, A; Zdilová, Viera; Maracek, I; Kacmárik, J

2006-03-01

The aim of the study was to determine the relationship between the rate of cooling of eight-cell mouse embryos to the temperature of liquid nitrogen (-196 degrees C) and their developmental capacity after thawing on the basis of their ability to leave the zona pellucida ('hatching') during in vitro culturing. Eight-cell embryos were obtained from superovulated female mice and divided into three experimental and one control group. Embryos from the experimental groups were cryopreserved by the vitrification method using ethylene glycol as cryoprotectant. The vitrification protocols used in the study differed in the rate of cooling of the cryoprotectant solution. Embryos from the first group were frozen in conventional 0.25-ml plastic straws, those from the second group in pipetting 'tips', and embryos from the third group, placed in vitrification solution, were introduced dropwise directly into liquid nitrogen. The control group of embryos was cultured in vitro without freezing in a culturing medium in an environment consisting of 95% air and 5% CO2. The developmental capacity of thawed embryos was assessed on the basis of their ability to leave the zona pellucida ('hatching') after three days of in vitro culturing. In the control group 95.1% of embryos 'hatched'. A significantly higher number of embryos that 'hatched' after thawing was observed in the group introduced dropwise directly into liquid nitrogen (60.0%) compared to the group frozen in pipetting 'tips' (37.9%). The group frozen in straws yielded significantly the lowest proportion of 'hatching' embryos (8.1%). These results showed that increasing cooling rates during vitrification of embryos improved their survival.

Effect of in vitro culture of human embryos on birthweight of newborns

NARCIS (Netherlands)

Dumoulin, John C.; Land, Jolande A.; Van Montfoort, Aafke P.; Nelissen, Ewka C.; Coonen, Edith; Derhaag, Josien G.; Schreurs, Inge L.; Dunselman, Gerard A.; Kester, Arnold D.; Geraedts, Joep P.; Evers, Johannes L.

In animal models, in vitro culture of preimplantation embryos has been shown to be a risk factor for abnormal fetal outcome, including high and low birthweight. In the human, mean birthweight of singletons after in vitro fertilization (IVF) is considerably lower than after natural conception, but it

Human oocyte oolemma characteristic is positively related to embryo developmental competence after ICSI procedure

Directory of Open Access Journals (Sweden)

Mohamed A. Danfour

2010-10-01

Conclusion: The current study provides evidence that preselection at a very early stage based on oolemma behavior may be helpful to identify a subgroup of preimplantation embryos with good prognostic to form blastocyst and consequently to implant and to give pregnancy.

Autophagy in human embryonic stem cells

NARCIS (Netherlands)

Tra, Thien; Gong, Lan; Kao, Lin-Pin; Li, Xue-Lei; Grandela, Catarina; Devenish, Rodney J.; Wolvetang, Ernst; Prescott, Mark

2011-01-01

Autophagy (macroautophagy) is a degradative process that involves the sequestration of cytosolic material including organelles into double membrane vesicles termed autophagosomes for delivery to the lysosome. Autophagy is essential for preimplantation development of mouse embryos and cavitation of

Influence of irradiation (Co60) in pre-implant rabbits embryos: effect on blastocyst diameters and embryos smaller than 2 mm

International Nuclear Information System (INIS)

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.

1995-01-01

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)

Post-Transcriptional Control of Gene Expression in Mouse Early Embryo Development: A View from the Tip of the Iceberg

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Claudio Sette

2011-04-01

Full Text Available Fertilization is a very complex biological process that requires the perfect cooperation between two highly specialized cells: the male and female gametes. The oocyte provides the physical space where this process takes place, most of the energetic need, and half of the genetic contribution. The spermatozoon mostly contributes the other half of the chromosomes and it is specialized to reach and to penetrate the oocyte. Notably, the mouse oocyte and early embryo are transcriptionally inactive. Hence, they fully depend on the maternal mRNAs and proteins stored during oocyte maturation to drive the onset of development. The new embryo develops autonomously around the four-cell stage, when maternal supplies are exhausted and the zygotic genome is activated in mice. This oocyte-to-embryo transition needs an efficient and tightly regulated translation of the maternally-inherited mRNAs, which likely contributes to embryonic genome activation. Full understanding of post-transcriptional regulation of gene expression in early embryos is crucial to understand the reprogramming of the embryonic genome, it might help driving reprogramming of stem cells in vitro and will likely improve in vitro culturing of mammalian embryos for assisted reproduction. Nevertheless, the knowledge of the mechanism(s underlying this fundamental step in embryogenesis is still scarce, especially if compared to other model organisms. We will review here the current knowledge on the post-transcriptional control of gene expression in mouse early embryos and discuss some of the unanswered questions concerning this fascinating field of biology.

Embryo splitting

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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.

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.

Neonatal outcome after preimplantation genetic diagnosis.

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Eldar-Geva, Talia; Srebnik, Naama; Altarescu, Gheona; Varshaver, Irit; Brooks, Baruch; Levy-Lahad, Ephrat; Bromiker, Ruben; Schimmel, Michael S

2014-10-01

To examine whether embryo biopsy for preimplantation genetic diagnosis (PGD) influences neonatal outcomes. Prospective follow-up cohort. Tertiary university-affiliated medical center. 242 children born after PGD, 242 children born after intracytoplasmic sperm injection (ICSI) (158 singletons and 42 twins pairs in each group), and 733 children born after a spontaneous conception (SC) (493 singletons, 120 twins pairs), matched for maternal age, parity, and body mass index. None. Gestational age, birth weight, prematurity (<37 and <34 weeks), low birth weight (<2,500 g, very low birth weight, <1,500 g), and intrauterine growth restriction (<10th percentile for gestational age). For singletons, the mean birth weight was higher after SC compared with ICSI but not compared with PGD. Mean gestational ages were lower after PGD and ICSI compared with SC. The low birth weight and intrauterine growth restriction rates were 4.4%, 12.0%, and 5.7% and 5.1%, 9.5%, and 5.5% for PGD, ICSI, and SC, respectively. Similar results were found when controlled for the number of embryos transferred and cryopreservation. The results for twins exhibited similar but less statistically significant trends. Polar body and blastomere biopsies provided similar outcomes. Embryo biopsy itself did not cause intrauterine growth restriction or low birth weight compared with SC, despite lower gestational ages with PGD. The worsened outcomes in ICSI compared with PGD pregnancies may be due to the infertility itself. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Preimplantation Genetic Testing in the 21st Century: Uncharted Territory

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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.

Theory about the Embryo Cryo-Treatment.

Science.gov (United States)

Vladimirov, Iavor K; Tacheva, Desislava; Diez, Antonio

2017-04-01

To create hypothesis, which can give a logical explanation related to the benefits of freezing/thawing embryos. Cryopreservation is not only a technology used for storing embryos, but also a method of embryo treatment that can potentially improve the success rate in infertile couples. From the analysis of multiple results in assisted reproductive technology, which have no satisfactory explanation to date, we found evidence to support a 'therapeutic' effect of the freezing/thawing of embryos on the process of recovery of the embryo and its subsequent implantation. Freezing/thawing is a way to activate the endogenous survival and repair responses in preimplantation embryos. Several molecular mechanisms can explain the higher success rate of ET using thawed embryos compared to fresh ET in women of advanced reproductive age, the higher miscarriage rate in cases of thawed blastocyst ET compared to thawed ET at early cleavage embryo, and the higher perinatal parameters of born children after thawed ET. Embryo thawing induces a stress. Controlled stress is not necessarily detrimental, because it generates a phenomenon that is counteracted by several known biological responses aimed to repair mitochondrial damage of membrane and protein misfolding. The term for favorable biological responses to low exposures to stress is called hormesis. This thesis will summarize the role of cryopreservation in the activation of a hormetic response, preserving the mitochondrial function, improving survival, and having an impact on the process of implantation, miscarriage, and the development of pregnancy.

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.

The evidence base regarding the experiences of and attitudes to preimplantation genetic diagnosis in prospective parents.

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Cunningham, Jenny; Goldsmith, Lesley; Skirton, Heather

2015-02-01

Preimplantation genetic diagnosis was developed as an alternative to prenatal diagnosis for couples with a family history of genetic disease. After in vitro fertilization, the embryos can be analysed to ensure that only healthy embryos are transferred to the uterus. Past studies have suggested that couples who wish to avoid having a child with an inherited genetic condition look favourably on preimplantation genetic diagnosis as it prevents the need for termination of pregnancy following prenatal diagnosis of an affected fetus. However, it is important to understand the experiences of couples who have used or consider using this technique. To ascertain the current evidence base on this topic, we conducted a mixed methods systematic review. Four databases were searched for relevant peer-reviewed papers published between 2000 and 2013. Of 453 papers, nine satisfied the inclusion criteria and were assessed for quality. Results of nine papers were analysed and synthesised using a narrative approach. Three main themes emerged: (1) motivating factors; (2) emotional labour; (3) choices and uncertainty. The review has identified an emotional and difficult journey for couples pursuing preimplantation genetic diagnosis. While use of the technique gives hope to families who wish to prevent transmission of a genetic disease this is not without hard decision-making and periods of uncertainty. Lack of information was perceived as a barrier to access this reproductive option. Recommendations include: training and education in genetics for midwives who are the first point of contact for pregnant women; clinics to use a decision-making tool to emphasise the uncertainty involved in PGD and improved communication and psychological support to couples. Copyright © 2014 Elsevier Ltd. All rights reserved.

Birth of a healthy infant following preimplantation PKHD1 haplotyping for autosomal recessive polycystic kidney disease using multiple displacement amplification

Science.gov (United States)

Janson, Marleen M.; Roesler, Mark R.; Avner, Ellis D.; Strawn, Estil Y.; Bick, David P.

2010-01-01

Purpose To develop a reliable preimplantation genetic diagnosis protocol for couples who both carry a mutant PKHD1 gene wishing to conceive children unaffected with autosomal recessive polycystic kidney disease (ARPKD). Methods Development of a unique protocol for preimplantation genetic testing using whole genome amplification of single blastomeres by multiple displacement amplification (MDA), and haplotype analysis with novel short tandem repeat (STR) markers from the PKHD1 gene and flanking sequences, and a case report of successful utilization of the protocol followed by successful IVF resulting in the birth of an infant unaffected with ARPKD. Results We have developed 20 polymorphic STR markers suitable for linkage analysis of ARPKD. These linked STR markers have enabled unambiguous identification of the PKHD1 haplotypes of embryos produced by at-risk couples. Conclusions We have developed a reliable protocol for preimplantation genetic diagnosis of ARPKD using single-cell MDA products for PKHD1 haplotyping. PMID:20490649

Polo-like kinase 1 is essential for the first mitotic division in the mouse embryo

Czech Academy of Sciences Publication Activity Database

Baran, V.; Šolc, Petr; Kovaríková, V.; Rehák, P.; Šutovský, P.

2013-01-01

Roč. 80, č. 7 (2013), s. 522-534 ISSN 1040-452X R&D Projects: GA ČR(CZ) GC301/09/J036; GA MŠk ED2.1.00/03.0124 Institutional support: RVO:67985904 Keywords : PLK1 * mouse embryo Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.675, year: 2013

Embryo aggregation does not improve the development of interspecies somatic cell nuclear transfer embryos in the horse.

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Gambini, Andrés; De Stéfano, Adrián; Jarazo, Javier; Buemo, Carla; Karlanian, Florencia; Salamone, Daniel Felipe

2016-09-01

from domestic animals tested in our study, the feline ooplasm might be the most appropriate recipient to partially allow preimplantation embryo development of iSCNT equine embryos. Copyright © 2016 Elsevier Inc. All rights reserved.

Igf1r signaling is indispensable for preimplantation development and is activated via a novel function of E-cadherin.

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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.

Functional studies of signaling pathways in peri-implantation development of the mouse embryo by RNAi

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Bell Graham

2005-12-01

Full Text Available Abstract Background Studies of gene function in the mouse have relied mainly on gene targeting via homologous recombination. However, this approach is difficult to apply in specific windows of time, and to simultaneously knock-down multiple genes. Here we report an efficient method for dsRNA-mediated gene silencing in late cleavage-stage mouse embryos that permits examination of phenotypes at post-implantation stages. Results We show that introduction of Bmp4 dsRNA into intact blastocysts by electroporation recapitulates the genetic Bmp4 null phenotype at gastrulation. It also reveals a novel role for Bmp4 in the regulation the anterior visceral endoderm specific gene expression and its positioning. We also show that RNAi can be used to simultaneously target several genes. When applied to the three murine isoforms of Dishevelled, it leads to earlier defects than previously observed in double knock-outs. These include severe delays in post-implantation development and defects in the anterior midline and neural folds at headfold stages. Conclusion Our results indicate that the BMP4 signalling pathway contributes to the development of the anterior visceral endoderm, and reveal an early functional redundancy between the products of the murine Dishevelled genes. The proposed approach constitutes a powerful tool to screen the functions of genes that govern the development of the mouse embryo.

PSK, a biological response modifier, modifies p53 expression, mitosis and apoptosis in X-ray irradiated mouse embryos. Possible cellular mechanism of the anti-teratogenic effect

International Nuclear Information System (INIS)

Kagohashi, Yukiko; Naora, Hiroyuki; Otani, Hiroki

2002-01-01

We previously showed that PSK, a biological response modifier, suppressed X-ray irradiation induced ocular anomalies in mouse embryos. In the present study, in mouse embryos irradiated at E7.5, PSK, when administered immediately after irradiation, suppressed mitosis and increased apoptosis as compared with embryos not treated with PSK at 12 hrs after irradiation. In the irradiated embryos, p53, which is normally expressed at a high level in early embryos, increased at 6 hrs and decreased at 12 hrs after irradiation. In the irradiated and PSK-treated embryos, the p53 level did not change at 6 hrs, increased at 12 hrs and decreased at 24 hrs after irradiation. This timing of PSK-induced delayed increase of p53 coincided with that of the PSK-induced decrease in mitosis and increase in apoptosis. These results suggested that PSK modified the p53 level and affected cell proliferation and apoptosis, which might contribute to the suppression of teratogenesis. (author)

Detecting cardiac contractile activity in the early mouse embryo using multiple modalities

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Chiann-mun eChen

2015-01-01

Full Text Available The heart is one of the first organs to develop during mammalian embryogenesis. In the mouse, it starts to form shortly after gastrulation, and is derived primarily from embryonic mesoderm. The embryonic heart is unique in having to perform a mechanical contractile function while undergoing complex morphogenetic remodelling. Approaches to imaging the morphogenesis and contractile activity of the developing heart are important in understanding not only how this remodelling is controlled but also the origin of congenital heart defects. Here, we describe approaches for visualising contractile activity in the developing mouse embryo, using brightfield time lapse microscopy and confocal microscopy of calcium transients. We describe an algorithm for enhancing this image data and quantifying contractile activity from it. Finally we describe how atomic force microscopy can be used to record contractile activity prior to it being microscopically visible.

Lack of WDR36 leads to preimplantation embryonic lethality in mice and delays the formation of small subunit ribosomal RNA in human cells in vitro.

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Gallenberger, Martin; Meinel, Dominik M; Kroeber, Markus; Wegner, Michael; Milkereit, Philipp; Bösl, Michael R; Tamm, Ernst R

2011-02-01

Mutations in WD repeat domain 36 gene (WDR36) play a causative role in some forms of primary open-angle glaucoma, a leading cause of blindness worldwide. WDR36 is characterized by the presence of multiple WD40 repeats and shows homology to Utp21, an essential protein component of the yeast small subunit (SSU) processome required for maturation of 18S rRNA. To clarify the functional role of WDR36 in the mammalian organism, we generated and investigated mutant mice with a targeted deletion of Wdr36. In parallel experiments, we used RNA interference to deplete WDR36 mRNA in mouse embryos and cultured human trabecular meshwork (HTM-N) cells. Deletion of Wdr36 in the mouse caused preimplantation embryonic lethality, and essentially similar effects were observed when WDR36 mRNA was depleted in mouse embryos by RNA interference. Depletion of WDR36 mRNA in HTM-N cells caused apoptotic cell death and upregulation of mRNA for BAX, TP53 and CDKN1A. By immunocytochemistry, staining for WDR36 was observed in the nucleolus of cells, which co-localized with that of nucleolar proteins such as nucleophosmin and PWP2. In addition, recombinant and epitope-tagged WDR36 localized to the nucleolus of HTM-N cells. By northern blot analysis, a substantial decrease in 21S rRNA, the precursor of 18S rRNA, was observed following knockdown of WDR36. In addition, metabolic-labeling experiments consistently showed a delay of 18S rRNA maturation in WDR36-depleted cells. Our results provide evidence that WDR36 is an essential protein in mammalian cells which is involved in the nucleolar processing of SSU 18S rRNA.

Assessment of the developmental totipotency of neural cells in the cerebral cortex of mouse embryo by nuclear transfer

Science.gov (United States)

Yamazaki, Yukiko; Makino, Hatsune; Hamaguchi-Hamada, Kayoko; Hamada, Shun; Sugino, Hidehiko; Kawase, Eihachiro; Miyata, Takaki; Ogawa, Masaharu; Yanagimachi, Ryuzo; Yagi, Takeshi

2001-01-01

When neural cells were collected from the entire cerebral cortex of developing mouse fetuses (15.5–17.5 days postcoitum) and their nuclei were transferred into enucleated oocytes, 5.5% of the reconstructed oocytes developed into normal offspring. This success rate was the highest among all previous mouse cloning experiments that used somatic cells. Forty-four percent of live embryos at 10.5 days postcoitum were morphologically normal when premature and early-postmitotic neural cells from the ventricular side of the cortex were used. In contrast, the majority (95%) of embryos were morphologically abnormal (including structural abnormalities in the neural tube) when postmitotic-differentiated neurons from the pial side of the cortex were used for cloning. Whereas 4.3% of embryos cloned with ventricular-side cells developed into healthy offspring, only 0.5% of those cloned with differentiated neurons in the pial side did so. These facts seem to suggest that the nuclei of neural cells in advanced stages of differentiation had lost their developmental totipotency. The underlying mechanism for this developmental limitation could be somatic DNA rearrangements in differentiating neural cells. PMID:11698647

De novo formation of nucleoli in developing mouse embryos originating from enucleolated zygotes.

Science.gov (United States)

Kyogoku, Hirohisa; Fulka, Josef; Wakayama, Teruhiko; Miyano, Takashi

2014-06-01

The large, compact oocyte nucleoli, sometimes referred to as nucleolus precursor bodies (NPBs), are essential for embryonic development in mammals; in their absence, the oocytes complete maturation and can be fertilized, but no nucleoli are formed in the zygote or embryo, leading to developmental failure. It has been convincingly documented that zygotes inherit the oocyte nucleolar material and form NPBs again in pronuclei. It is commonly accepted that during early embryonic development, the original compact zygote NPBs gradually transform into reticulated nucleoli of somatic cells. Here, we show that zygote NPBs are not required for embryonic and full-term development in the mouse. When NPBs were removed from late-stage zygotes by micromanipulation, the enucleolated zygotes developed to the blastocyst stage and, after transfer to recipients, live pups were obtained. We also describe de novo formation of nucleoli in developing embryos. After removal of NPBs from zygotes, they formed new nucleoli after several divisions. These results indicate that the zygote NPBs are not used in embryonic development and that the nucleoli in developing embryos originate from de novo synthesized materials. © 2014. Published by The Company of Biologists Ltd.

Comparative preimplantation genetic diagnosis policy in Europe and the USA and its implications for reproductive tourism

OpenAIRE

Bayefsky, Michelle J

2017-01-01

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 p...

[Establishment of a novel HLA genotyping method for preimplantation genetic diagnonis using multiple displacement amplification-polymerase chain reaction-sequencing based technique].

Science.gov (United States)

Zhang, Yinfeng; Luo, Haining; Zhang, Yunshan

2015-12-01

To establish a novel HLA genotyping method for preimplantation genetic diagnonis (PGD) using multiple displacement amplification-polymerase chain reaction-sequencing based technique (MDA-PCR-SBT). Peripheral blood samples and 76 1PN, 2PN, 3PN discarded embryos from 9 couples were collected. The alleles of HLA-A, B, DR loci were detected from the MDA product with the PCR-SBT method. The HLA genotypes of the parental peripheral blood samples were analyzed with the same protocol. The genotypes of specific HLA region were evaluated for distinguishing the segregation of haplotypes among the family members, and primary HLA matching was performed between the embryos. The 76 embryos were subjected to MDA and 74 (97.4%) were successfully amplified. For the 34 embryos from the single blastomere group, the amplification rate was 94.1%, and for the 40 embryos in the two blastomeres group, the rate was 100%. The dropout rates for DQ allele and DR allele were 1.3% and 0, respectively. The positive rate for MDA in the single blastomere group was 100%, with the dropout rates for DQ allele and DR allele being 1.5% and 0, respectively. The positive rate of MDA for the two blastomere group was 100%, with the dropout rates for both DQ and DR alleles being 0. The recombination rate of fetal HLA was 20.2% (30/148). Due to the improper classification and abnormal fertilized embryos, the proportion of matched embryos HLA was 20.3% (15/74),which was lower than the theoretical value of 25%. PGD with HLA matching can facilitate creation of a HLA-identical donor (saviour child) for umbilical cord blood or bone marrow stem cells for its affected sibling with a genetic disease. Therefore, preimplantation HLA matching may provide a tool for couples desiring to conceive a potential donor progeny for transplantation for its sibling with a life-threatening disorder.

Maternal SENP7 programs meiosis architecture and embryo survival in mouse.

Science.gov (United States)

Huang, Chun-Jie; Wu, Di; Jiao, Xiao-Fei; Khan, Faheem Ahmed; Xiong, Cheng-Liang; Liu, Xiao-Ming; Yang, Jing; Yin, Tai-Lang; Huo, Li-Jun

2017-07-01

Understanding the mechanisms underlying abnormal egg production and pregnancy loss is significant for human fertility. SENP7, a SUMO poly-chain editing enzyme, has been regarded as a mitotic regulator of heterochromatin integrity and DNA repair. Herein, we report the roles of SENP7 in mammalian reproductive scenario. Mouse oocytes deficient in SENP7 experienced meiotic arrest at prophase I and metaphase I stages, causing a substantial decrease of mature eggs. Hyperaceylation and hypomethylation of histone H3 and up-regulation of Cdc14B/C accompanied by down-regulation of CyclinB1 and CyclinB2 were further recognized as contributors to defective M-phase entry and spindle assembly in oocytes. The spindle assembly checkpoint activated by defective spindle morphogenesis, which was also caused by mislocalization and ubiquitylation-mediated proteasomal degradation of γ-tubulin, blocked oocytes at meiosis I stage. SENP7-depleted embryos exhibited severely defective maternal-zygotic transition and progressive degeneration, resulting in nearly no blastocyst production. The disrupted epigenetic landscape on histone H3 restricted Rad51C loading onto DNA lesions due to elevated HP1α euchromatic deposition, and reduced DNA 5hmC challenged the permissive status for zygotic DNA repair, which induce embryo death. Our study pinpoints SENP7 as a novel determinant in epigenetic programming and major pathways that govern oocyte and embryo development programs in mammals. Copyright © 2017 Elsevier B.V. All rights reserved.

Preimplantation genetic diagnosis outcomes and meiotic segregation analysis of robertsonian translocation carriers.

Science.gov (United States)

Ko, Duck Sung; Cho, Jae Won; Lee, Hyoung-Song; Kim, Jin Yeong; Kang, Inn Soo; Yang, Kwang Moon; Lim, Chun Kyu

2013-04-01

To investigate the meiotic segregation patterns of cleavage-stage embryos from robertsonian translocation carriers and aneuploidy of chromosome 18 according to meiotic segregation patterns. Retrospective study. Infertility center and laboratory of reproductive biology and infertility. Sixty-two couples with robertsonian translocation carriers. One blastomere was biopsied from embryos and diagnosed with the use of fluorescence in situ hybridization (FISH). Translocation chromosomes were analyzed with the use of locus-specific and subtelomeric FISH probes. Aneuploidy of chromosome 18 was assessed simultaneously with translocation chromosomes. Preimplantation genetic diagnosis (PGD) outcomes, meiotic segregation patterns of robertsonian translocation, and aneuploidy of chromosome 18 depending on meiotic segregation patterns. Two hundred seventy embryos of 332 transferrable embryos were transferred in 113 cycles, and 27 healthy babies were born. The alternate segregation was significantly higher in male carriers than in female carriers (43.9% vs. 29.9%, respectively), and adjacent segregation was higher in female carriers than in male carriers (44.7% vs. 38.7%, respectively). Aneuploidy of chromosome 18 was significantly increased in 3:0-segregated or chaotic embryos. Forty-seven alternate embryos were excluded from embryo replacement owing to aneuploidy of chromosome 18. In carriers of robertsonian translocation, meiotic segregation showed differences between men and women. Frequent meiotic errors caused by premature predivision or nondisjunction and less stringent checkpoint in women might cause such differences between sexes. Aneuploidy of chromosome 18 might be influenced by meiotic segregation of translocation chromosomes. Factors that cause malsegregation, such as 3:0 or chaotic segregation, seem to play a role in aneuploidy of chromosome 18. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

First successful trial of preimplantation genetic diagnosis for pantothenate kinase-associated neurodegeneration.

Science.gov (United States)

Trachoo, Objoon; Satirapod, Chonthicha; Panthan, Bhakbhoom; Sukprasert, Matchuporn; Charoenyingwattana, Angkana; Chantratita, Wasun; Choktanasiri, Wicharn; Hongeng, Suradej

2017-01-01

We aim to present a case of a healthy infant born after intracytoplasmic sperm injection-in vitro fertilization (ICSI-IVF) with a preimplantation genetic diagnosis (PGD) for pantothenate kinase-associated neurodegeneration (PKAN) due to PANK2 mutation. ICSI-IVF was performed on a Thai couple, 34-year-old female and 33-year-old male, with a family history of PKAN in their first child. Following fertilization, each of the embryos were biopsied in the cleavage stage and subsequently processed for whole-genome amplification. Genetic status of the embryos was diagnosed by linkage analysis and direct mutation testing using primer extension-based mini-sequencing. Comprehensive chromosomal aneuploidy screening was performed using a next-generation sequencing-based strategy. Only a single cycle of ICSI-IVF was processed. There were seven embryos from this couple-two were likely affected, three were likely carriers, one was likely unaffected, and one failed in target genome amplification. Aneuploidy screening was performed before making a decision on embryo transfer, and only one unaffected embryo passed the screening. That embryo was transferred in a frozen thawed cycle, and the pregnancy was successful. The diagnosis was confirmed by amniocentesis, which presented with a result consistent with PGD. At 38 weeks of gestational age, a healthy male baby was born. Postnatal genetic confirmation was also consistent with PGD and the prenatal results. At the age of 24 months, the baby presented with normal growth and development lacking any neurological symptoms. We report the first successful trial of PGD for PKAN in a developing country using linkage analysis and mini-sequencing in cleavage stage embryos.

Contribution of Mouse Embryonic Stem Cells and Induced Pluripotent Stem Cells to Chimeras through Injection and Coculture of Embryos

OpenAIRE

Guo, Jitong; Wu, Baojiang; Li, Shuyu; Bao, Siqin; Zhao, Lixia; Hu, Shuxiang; Sun, Wei; Su, Jie; Dai, Yanfeng; Li, Xihe

2014-01-01

Blastocyst injection and morula aggregation are commonly used to evaluate stem cell pluripotency based on chimeric contribution of the stem cells. To assess the protocols for generating chimeras from stem cells, 8-cell mouse embryos were either injected or cocultured with mouse embryonic stem cells and induced pluripotent stem cells, respectively. Although a significantly higher chimera rate resulted from blastocyst injection, the highest germline contribution resulted from injection of 8-cel...

Preimplantation genetic diagnosis: International standards and the law of the republic of Serbia

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Rajić Nataša

2014-01-01

Full Text Available The process of biomedical assisted reproduction, in addition to the treatment of infertility, also can be implemented for the purpose of prevention of transmission of serious hereditary disease to offspring. This is possible thanks to the preimplantation genetic diagnosis, which involves genetic testing of a few cells of the embryo in the early stage of development before implantation in a woman's body, and its elimination in the case of determining the genetic anomaly. The process of the preimplantation genetic diagnosis faces several constitutional values and raises a series of questions. Some of them were answered by European Court of Human Rights in the case Costa and Pavan v. Italiy. The subject of the paper is the analysis of this decision, which is important from a constitutional point of view, because it establishes guidelines for the interpretation of rules of domestic law. The second task of the paper is the analysis of normative solutions of our legal system in this area, in order to test their compliance with the standards set in this Court's decision.

Dissection of culture media for embryos: the most important and less important components and characteristics.

Science.gov (United States)

Gardner, David K

2008-01-01

Improvements in culture media formulations have led to an increase in the ability to maintain the mammalian embryo in culture throughout the preimplantation and pre-attachment period. Amino acids and specific macromolecules have been identified as being key medium components, whereas temporal dynamics have been recognised as important media characteristics. Furthermore, other laboratory factors that directly impact embryo development and viability have been identified. Such factors include the use of a reduced oxygen tension, an appropriate incubation system and an adequate prescreening of all contact supplies. With rigourous quality systems in place, it is possible to obtain in vivo rates of embryo development in vitro using new media formulations while maintaining high levels of embryo viability. The future of embryo culture will likely be based on novel culture chips capable of providing temporal dynamics while facilitating real-time analysis of embryo physiology.

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...

Estimating limits for natural human embryo mortality [version 2; referees: 2 approved

Directory of Open Access Journals (Sweden)

Gavin E. Jarvis

2016-12-01

Full Text Available Natural human embryonic mortality is generally considered to be high. Values of 70% and higher are widely cited. However, it is difficult to determine accurately owing to an absence of direct data quantifying embryo loss between fertilisation and implantation. The best available data for quantifying pregnancy loss come from three published prospective studies (Wilcox, Zinaman and Wang with daily cycle by cycle monitoring of human chorionic gonadotrophin (hCG in women attempting to conceive. Declining conception rates cycle by cycle in these studies indicate that a proportion of the study participants were sub-fertile. Hence, estimates of fecundability and pre-implantation embryo mortality obtained from the whole study cohort will inevitably be biased. This new re-analysis of aggregate data from these studies confirms the impression that discrete fertile and sub-fertile sub-cohorts were present. The proportion of sub-fertile women in the three studies was estimated as 28.1% (Wilcox, 22.8% (Zinaman and 6.0% (Wang. The probability of conceiving an hCG pregnancy (indicating embryo implantation was, respectively, 43.2%, 38.1% and 46.2% among normally fertile women, and 7.6%, 2.5% and 4.7% among sub-fertile women. Pre-implantation loss is impossible to calculate directly from available data although plausible limits can be estimated. Based on this new analysis and a model for evaluating reproductive success and failure it is proposed that a plausible range for normal human embryo and fetal mortality from fertilisation to birth is 40-60%.

A Functional Assay for Putative Mouse and Human Definitive Endoderm using Chick Whole-Embryo Cultures

DEFF Research Database (Denmark)

Johannesson, Martina; Semb, Tor Henrik; Serup, Palle

2012-01-01

. Thus, the purpose of this study is to describe a method whereby the in vivo functionality of DE derived from ESCs can be assessed. Methods: By directed differentiation, putative DE was derived from human and mouse ESCs. This putative DE was subsequently transplanted into the endoderm of chick embryos...... to determine any occurrence of integration. Putative DE was analyzed by gene and protein expression prior to transplantation and 48 h post transplantation. Results: Putative DE, derived from mouse and human ESCs, was successfully integrated within the chick endoderm. Endoderm-specific genes were expressed...... result show that putative DE integrates with the chick endoderm and participate in the development of the chicken gut, indicating the generation of functional DE from ESCs. This functional assay can be used to assess the generation of functional DE derived from both human and mouse ESCs and provides...

Is it time for a paradigm shift in understanding embryo selection?

Science.gov (United States)

Gleicher, Norbert; Kushnir, Vitaly A; Barad, David H

2015-01-11

Embryo selection has been an integral feature of in vitro fertilization (IVF) almost since its inception. Since the advent of extended blastocyst stage embryo culture, and especially with increasing popularity of elective single embryo transfer (eSET), the concept of embryo selection has increasingly become a mainstay of routine IVF. We here, however, argue that embryo selection via blastocyst stage embryo transfer (BSET), as currently practiced, at best improves IVF outcomes only for a small minority of patients undergoing IVF cycles. For a large majority BSET is either ineffective or, indeed, may actually be harmful by decreasing IVF pregnancy chances. Overall, only a small minority of patients, thus, benefit from prolonged embryo culture, while BSET, as a tool to enhance IVF outcomes, is increasingly utilized as routine care in IVF for all patients. Since newer methods of embryo selection, like preimplantation genetic screening (PGS) and closed system embryo incubation with time-lapse photography are practically dependent on BSET, these concepts of embryo selection, currently increasingly adopted in mainstream IVF, require reconsideration. They, automatically, transfer the downsides of BSET, including decreases in IVF pregnancy chances in some patients, to these new procedures, and in addition raise serious questions about cost-effectiveness.

RepSox improves viability and regulates gene expression in rhesus monkey-pig interspecies cloned embryos.

Science.gov (United States)

Zhu, Hai-Ying; Jin, Long; Guo, Qing; Luo, Zhao-Bo; Li, Xiao-Chen; Zhang, Yu-Chen; Xing, Xiao-Xu; Xuan, Mei-Fu; Zhang, Guang-Lei; Luo, Qi-Rong; Wang, Jun-Xia; Cui, Cheng-Du; Li, Wen-Xue; Cui, Zheng-Yun; Yin, Xi-Jun; Kang, Jin-Dan

2017-05-01

To investigate the effect of the small molecule, RepSox, on the expression of developmentally important genes and the pre-implantation development of rhesus monkey-pig interspecies somatic cell nuclear transfer (iSCNT) embryos. Rhesus monkey cells expressing the monomeric red fluorescent protein 1 which have a normal (42) chromosome complement, were used as donor cells to generate iSCNT embryos. RepSox increased the expression levels of the pluripotency-related genes, Oct4 and Nanog (p  0.05), this was not significant. RepSox can improve the developmental potential of rhesus monkey-pig iSCNT embryos by regulating the expression of pluripotency-related genes.

Dose-response relationship of cadmium or radiation-induced embryotoxicity in mouse whole embryo culture

International Nuclear Information System (INIS)

Nakashima, Kiyohito; Kawamata, Akitoshi; Matsuoka, Masato; Wakisaka, Takashi; Fujiki, Yoshishige

1988-01-01

Mouse embryos of B6C3F 1 strain were exposed in vitro to 1.2 to 2.2 μM cadmium chloride (Cd) or to 100 to 320 R x-rays, and the effects of the exposure on development were examined after 39 h of culture. Development of embryos was assessed from lethality, formation of the neural tube defect, diameter and protein of yolk sac, crown-rump and head lengths, embryonic protein, and number of somites. Incidence of the neural tube defect increased from 3.4 to 100% by 1.2 to 2.0 μM Cd, while embryo deaths increased from 13.8 to 93.3% by 2.0 to 2.2 μM Cd. Embryonic protein was significantly reduced at the teratogenic range, but the number of somites was only affected by 1.6 to 2.0 μM Cd. X-irradiation at 100 to 320 R induced the neural tube defect in 2.9 to 72.7% of the embryos. An embryolethal effect was observed only at the 320 R dose. Crown-rump and head lengths and embryonic protein were significantly affected at the teratogenic range, but the diameter and protein of yolk sac and number of somites were hardly affected. Cadmium- or radiation-induced response data of both teratogenicity and endpoints indicating inhibition of embryonic development were acceptably fitted to a linear log-probit regression. These regressions suggest that as an estimation of interference in development of embryos, embryonic protein and head length are sensitive endpoints while the number of somites is an insensitive criterion. (author)

Hematopoietic Stem Cell Transplantation Using Preimplantation Genetic Diagnosis and Human Leukocyte Antigen Typing for Human Leukocyte Antigen-Matched Sibling Donor: A Turkish Multicenter Study.

Science.gov (United States)

Kurekci, Emin; Küpesiz, Alphan; Anak, Sema; Öztürk, Gülyüz; Gürsel, Orhan; Aksoylar, Serap; Ileri, Talia; Kuşkonmaz, Barış; Eker, İbrahim; Cetin, Mualla; Tezcan Karasu, Gülsün; Kaya, Zühre; Fışgın, Tunç; Ertem, Mehmet; Kansoy, Savaş; Yeşilipek, Mehmet Akif

2017-05-01

Preimplantation genetic diagnosis involves the diagnosis of a genetic disorder in embryos obtained through in vitro fertilization, selection of healthy embryos, and transfer of the embryos to the mother's uterus. Preimplantation genetic diagnosis has been used not only to avoid the risk of having an affected child, but it also offers, using HLA matching, preselection of potential HLA-genoidentical healthy donor progeny for an affected sibling who requires bone marrow transplantation. Here, we share the hematopoietic stem cell transplantation results of 52 patients with different benign and malign hematological or metabolic diseases or immunodeficiencies whose donors were siblings born with this technique in Turkey since 2008. The median age of the patients' at the time of the transplantation was 8 years (range, 3 to 16 years) and the median age of the donors was 2 years (range, .5 to 6 years). The most common indication for HSCT was thalassemia major (42 of all patients, 80%). The stem cell source in all of the transplantations was bone marrow. In 37 of the transplantations, umbilical cord blood of the same donor was also used. In 50 of the 52 patients, full engraftment was achieved with a mean of 4.6 × 10 6 CD 34 + cells per kg of recipient weight. Ninety-six percent of the patients have been cured through hematopoietic stem cell transplantation without any complication. Primary engraftment failure was seen in only 2 patients with thalassemia major. All of the donors and the patients are alive with good health status. Preimplantation genetic diagnosis with HLA matching offers a life-saving chance for patients who need transplantation but lack an HLA genoidentical donor. Copyright © 2017 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

DNA repair ability of cultured cells derived from mouse embryos in comparison with human cells

International Nuclear Information System (INIS)

Yaki, T.

1982-01-01

DNA repair in mouse cells derived from embryos of 3 inbred strains were investigated in comparison with that in human cells. The levels of unscheduled DNA synthesis after UV irradiation appeared to change at different passages, but capacities of host-cell reactivation of UV-irradiated herpes simplex virus were always reduced to the same levels as those in xeroderma pigmentosum cells. This implied that mouse cells are reduced in excision-repair capacities and that the apparently high levels of unscheduled DNA synthesis at certain passages are not quantitatively related to high levels of cell survival. Essentially no differences in DNA repair were noted among 3 strains - BALB/c, C3H/He and C57BL/10. (orig.)

DNA damage and repair in mouse embryos following treatment transplacentally with methylnitrosourea and methylmethanesulfonate

International Nuclear Information System (INIS)

Jirakulsomchok, S.; Yielding, K.L.

1984-01-01

Mouse embryos were labeled in vivo at 10 1/2-12 1/2 days of gestation with [ 3 H]-thymidine and subjected to DNA damage using x-ray, methylmethanesulfonate, or methylnitrosourea. DNA damage and its repair were assessed in specific cell preparations from embryos isolated at intervals thereafter using the highly sensitive method of nucleoid sedimentation, which evaluates the supercoiled state of the DNA. Repair of x-ray damage was demonstrated using trypsin-dispersed cells from whole embryos and from homogenized embryonic liver to show the validity of the analytical approach. The effects of the highly teratogenic methylnitrosourea and the much less teratogenic methylmethanesulfonate were compared in the targeted limb buds using equitoxic doses of the two alkylating agents. DNA supercoiling was fully restored after 24 hr in limb bud cells damaged with methylmethanesulfonate, while as much as 48 hr were required for full repair of methylnitrosourea damage. These results demonstrated the feasibility of studying DNA repair in embryonic tissues after damage in vivo and suggest that the potency of methylnitrosourea as a teratogen may be correlated with a prolonged period required for complete repair of DNA

Polypeptide profiles of human oocytes and preimplantation embryos.

Science.gov (United States)

Capmany, G; Bolton, V N

1993-11-01

The polypeptides that direct fertilization and early development until activation of the embryonic genome occurs, at the 4-8 cell stage in the human, are exclusively maternal in origin, and are either synthesized during oogenesis or translated later from maternal mRNA. Using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and silver stain, we have visualized and compared the polypeptides present in different populations of human oocytes and cleavage stage embryos obtained after superovulation and insemination in vitro. Two polypeptide patterns were resolved, differing in the region of mol. wt 69 kDa. The distribution of these patterns showed no correlation with the ability of individual oocytes to achieve fertilization and develop normally to the 8-cell stage.

Long-distance transportation of primate embryos developing in culture: a preliminary study.

Science.gov (United States)

Nichols, Stephanie; Harvey, Alexandra; Gierbolini, Lynette; Gonzalez-Martinez, Janis; Brenner, Carol; Bavister, Barry

2010-03-01

Non-human primate embryos are invaluable for conducting research relevant to human infertility and stem cells, but their availability is restricted. In this preliminary study, rhesus monkey embryos were produced by IVF at the Caribbean Primate Research Centre and shipped in tubes of gassed culture medium within a battery-powered transport incubator by overnight courier to Wayne State University in Michigan. Upon arrival, the embryos were incubated in fresh culture medium to evaluate further development. In 11 shipments comprising 98 cleavage-stage embryos developing from oocytes that were mature (MII) upon collection, 51 (52%) reached advanced preimplantation stages (morula to hatched blastocyst) during prolonged culture following transportation. However, most embryos produced from oocytes that were immature (MI) at collection arrested and only 5/51 (10%) reached advanced stages of development. This study demonstrates that non-cryopreserved primate embryos can be routinely transported between distant sites without loss of developmental ability. In this way, the processes of production and study of non-cryopreserved primate embryos need not be restricted to the same or nearby laboratories. This will expand the use of these embryos for research and facilitate generation of translationally relevant information. Published by Elsevier Ltd.

Beneficial effect of two culture systems with small groups of embryos on the development and quality of in vitro-produced bovine embryos.

Science.gov (United States)

Cebrian-Serrano, A; Salvador, I; Silvestre, M A

2014-02-01

Currently, in vitro-produced embryos derived by ovum pick up (OPU) and in vitro fertilization (IVF) technologies represent approximately one-third of the embryos worldwide in cattle. Nevertheless, the culture of small groups of embryos from an individual egg donor is an issue that OPU-IVF laboratories have to face. In this work, we tested whether the development and quality of the preimplantation embryos in vitro cultured in low numbers (five embryos) could be improved by the addition of epidermal growth factor, insulin, transferrin and selenium (EGF-ITS) or by the WOW system. With this aim, immature oocytes recovered from slaughtered heifers were in vitro matured and in vitro fertilized. Presumptive zygotes were then randomly cultured in four culture conditions: one large group (LG) (50 embryos/500 μl medium) and three smaller groups [five embryos/50 μl medium without (control) or with EGF-ITS (EGF-ITS) and five embryos per microwell in the WOW system (WOW)]. Embryos cultured in LG showed a greater ability to develop to blastocyst stage than embryos cultured in smaller groups, while the blastocyst rate of WOW group was significantly higher than in control. The number of cells/blastocyst in LG was higher than control or WOW, whereas the apoptosis rate per blastocyst was lower. On the other hand, the addition of EGF-ITS significantly improved both parameters compared to the control and resulted in similar embryo quality to LG. In conclusion, the WOW system improved embryo development, while the addition of EGF-ITS improved the embryo quality when smaller groups of embryos were cultured. © 2013 Blackwell Verlag GmbH.

Development and validation of concurrent preimplantation genetic diagnosis for single gene disorders and comprehensive chromosomal aneuploidy screening without whole genome amplification.

Science.gov (United States)

Zimmerman, Rebekah S; Jalas, Chaim; Tao, Xin; Fedick, Anastasia M; Kim, Julia G; Pepe, Russell J; Northrop, Lesley E; Scott, Richard T; Treff, Nathan R

2016-02-01

To develop a novel and robust protocol for multifactorial preimplantation genetic testing of trophectoderm biopsies using quantitative polymerase chain reaction (qPCR). Prospective and blinded. Not applicable. Couples indicated for preimplantation genetic diagnosis (PGD). None. Allele dropout (ADO) and failed amplification rate, genotyping consistency, chromosome screening success rate, and clinical outcomes of qPCR-based screening. The ADO frequency on a single cell from a fibroblast cell line was 1.64% (18/1,096). When two or more cells were tested, the ADO frequency dropped to 0.02% (1/4,426). The rate of amplification failure was 1.38% (55/4,000) overall, with 2.5% (20/800) for single cells and 1.09% (35/3,200) for samples that had two or more cells. Among 152 embryos tested in 17 cases by qPCR-based PGD and CCS, 100% were successfully given a diagnosis, with 0% ADO or amplification failure. Genotyping consistency with reference laboratory results was >99%. Another 304 embryos from 43 cases were included in the clinical application of qPCR-based PGD and CCS, for which 99.7% (303/304) of the embryos were given a definitive diagnosis, with only 0.3% (1/304) having an inconclusive result owing to recombination. In patients receiving a transfer with follow-up, the pregnancy rate was 82% (27/33). This study demonstrates that the use of qPCR for PGD testing delivers consistent and more reliable results than existing methods and that single gene disorder PGD can be run concurrently with CCS without the need for additional embryo biopsy or whole genome amplification. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Tripolar mitosis and partitioning of the genome arrests human preimplantation development in vitro.

Science.gov (United States)

Ottolini, Christian S; Kitchen, John; Xanthopoulou, Leoni; Gordon, Tony; Summers, Michael C; Handyside, Alan H

2017-08-29

Following in vitro fertilisation (IVF), only about half of normally fertilised human embryos develop beyond cleavage and morula stages to form a blastocyst in vitro. Although many human embryos are aneuploid and genomically imbalanced, often as a result of meiotic errors inherited in the oocyte, these aneuploidies persist at the blastocyst stage and the reasons for the high incidence of developmental arrest remain unknown. Here we use genome-wide SNP genotyping and meiomapping of both polar bodies to identify maternal meiotic errors and karyomapping to fingerprint the parental chromosomes in single cells from disaggregated arrested embryos and excluded cells from blastocysts. Combined with time lapse imaging of development in culture, we demonstrate that tripolar mitoses in early cleavage cause chromosome dispersal to clones of cells with identical or closely related sub-diploid chromosome profiles resulting in intercellular partitioning of the genome. We hypothesise that following zygotic genome activation (ZGA), the combination of genomic imbalance and partial genome loss disrupts the normal pattern of embryonic gene expression blocking development at the morula-blastocyst transition. Failure to coordinate the cell cycle in early cleavage and regulate centrosome duplication is therefore a major cause of human preimplantation developmental arrest in vitro.

Triclabendazole sulfoxide causes stage-dependent embryolethality in zebrafish and mouse in vitro.

Directory of Open Access Journals (Sweden)

Nuria Boix

Full Text Available Fascioliasis and paragonimiasis are widespread foodborne trematode diseases, affecting millions of people in more than 75 countries. The treatment of choice for these parasitic diseases is based on triclabendazole, a benzimidazole derivative which has been suggested as a promising drug to treat pregnant women and children. However, at the moment, this drug is not approved for human use in most countries. Its potential adverse effects on embryonic development have been scarcely studied, and it has not been assigned a pregnancy category by the FDA. Thus, to help in the process of risk-benefit decision making upon triclabendazole treatment during pregnancy, a better characterization of its risks during gestation is needed.The zebrafish embryo test, a preimplantation and a postimplantation rodent whole embryo culture were used to investigate the potential embryotoxicity/teratogenicity of triclabendazole and its first metabolite triclabendazole sulfoxide. Albendazole and albendazole sulfoxide were included as positive controls.Triclabendazole was between 10 and 250 times less potent than albendazole in inducing dysmorphogenic effects in zebrafish or postimplantation rodent embryos, respectively. However, during the preimplantation period, both compounds, triclabendazole and triclabendazole sulfoxide, induced a dose-dependent embryolethal effect after only 24 h of exposure in rodent embryos and zebrafish (lowest observed adverse effect concentrations = 10 μM.In humans, after ingestion of the recommended doses of triclabendazole to treat fascioliasis and paragonimiasis (10 mg/kg, the main compound found in plasma is triclabendazole sulfoxide (maximum concentration 38.6 μM, while triclabendazole concentrations are approximately 30 times lower (1.16 μM. From our results it can be concluded that triclabendazole, at concentrations of the same order of magnitude as the clinically relevant ones, does not entail teratogenic potential in vitro during the

Pregnancy outcomes following 24-chromosome preimplantation genetic diagnosis in couples with balanced reciprocal or Robertsonian translocations.

Science.gov (United States)

Idowu, Dennis; Merrion, Katrina; Wemmer, Nina; Mash, Janine Gessner; Pettersen, Barbara; Kijacic, Dusan; Lathi, Ruth B

2015-04-01

To report live birth rates (LBR) and total aneuploidy rates in a series of patients with balanced translocations who pursued in vitro fertilization (IVF)-preimplantation genetic diagnosis (PGD) cycles. Retrospective cohort analysis. Genetic testing reference laboratory. Seventy-four couples who underwent IVF-PGD due to a parental translocation. IVF cycles and embryo biopsies were performed by referring clinics. Biopsy samples were sent to a single reference lab for PGD for the translocation plus 24-chromosome aneuploidy screening with the use of a single-nucleotide polymorphism (SNP) microarray. LBR per biopsy cycle, aneuploidy rate, embryo transfer (ET) rate, miscarriage rate. The LBR per IVF biopsy cycle was 38%. LBR for patients reaching ET was 52%. Clinical miscarriage rate was 10%. Despite a mean age of 33.8 years and mean of 7 embryos biopsied, there was a 30% chance for no chromosomally normal embryos. Maternal age >35 years, day 3 biopsy, and having fewer than five embryos available for biopsy increased the risk of no ET. IVF-PGD for translocation and aneuploidy screening had good clinical outcomes. Patients carrying a balanced translocation who are considering IVF-PGD should be aware of the high risk of no ET, particularly in women ≥35 years old. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

PREIMPLANTATION GENETIC DIAGNOSIS – 4 YEARS’ EXPERIENCE AT THE DEPARTMENT OF GYNECOLOGY, UNIVERSITY MEDICAL CENTRE LJUBLJANA

Directory of Open Access Journals (Sweden)

Karin Writzl

2018-02-01

Full Text Available Background. Preimplantation genetic diagnosis offers early investigation of embryos in couples with a high risk for offspring affected by a genetic disease. We report indications and results associated with the PGD program conducted at Gynecology Clinic Ljubljana from June 2004 to December 2008. Methods. The retrospective analysis includes sixty cycles performed in 34 couples enrolled in the PGD programe. Embryos were biopsied on the third day and the genetic analysis was performed using the FISH and PCR methods. Embryo transfers were carried out on the fifth day. Results. The main indications were chromosomal abnormalities (67 %, followed by recurrent miscarriages (16 %, autosomal dominant and recessive diseases (9 %, and X-linked diseases (6 %. Sixty cycles were performed and 48 embryo transfer procedures. There were 15 clinical pregnancies resulting in clinical pregnancy rate 25 % per cycle and 37.5 % per embryo transfer. A total of eight unaffected children were born, and two pregnancies are still ongoing. Conclusions. PGD is technically a very challenging procedure. Superior knowledge and communication between geneticists and reproductive medicine scientists is mandatory for successful PGD procedures. PGD has gained a place among the choices offered at Gynecology Clinic Ljubljana to couples at risk of transmission of genetic disease.

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

The application of assisted reproductive technologies (ART) has been shown to induce changes in the methylation of the embryonic genome, leading to aberrant gene expression, including that of imprinted genes. Aberrant methylation and gene expression has been linked to the large offspring syndrome...... (LOS) in bovine embryos resulting in increased embryonic morbidity and mortality. In the bovine, limited numbers of imprinted genes have been studied and studies have primarily been restricted to pre-implantation stages. This study reports original data on the expression pattern of 8 putatively...... 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...

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 of...

Embryo developmental capacity of oocytes fertilised by sperm of mouse exposed to forced swimming stress

International Nuclear Information System (INIS)

Ghasem, S.; Majid, J.; Shiva, R.

2013-01-01

Objective: To assess developmental capacity of fertilised oocytes by sperm of mouse exposed to forced swimming stress. Methods: The experimental study was conducted at the Physiology Research Center of Ahvaz Jundishapur University of Medical Sciences, from August 2011 to January 2012. It comprised 20 adult male and 10 female mice. The male mice were randomly divided into two equal groups (n=10): control and experimental. Animals of the experimental group were submitted to forced swimming stress. All male mice were euthanised and the cauda epididymis removed before contents were squeezed out. A pre-incubated capacitated sperm was gently added to the freshly collected ova of the two groups of study. The combined sperm-oocyte suspension was incubated for 4-6 hours under a condition of 5% Carbon dioxide and 37 degree C temperature. The ova were then washed through several changes of medium and finally incubated. Fertilisation was assessed by recording the number of 1-cell embryos 4-6 hours after insemination. The 1-cell embryos were allowed to further develop in vitro for about 120 hours. Development of embryos everyday and during 5 days of culture was observed by using inverted microscope. SPSS 13.0.1 was used for statistical analysis. Results: The percentage of oocytes fertilised was 75:96 (78.12+-4.8%) and 50:10 (49.5+-3.9%) in the control and experimental groups, respectively. The difference was significant (p 0.05) between the two groups in terms of speed and developmental capacity of blastocysts. Conclusions: Fertilisation capacity of male mice affected by forced swimming stress and also the developmental capacity of oocyte fertilised by sperm of mouse exposed to forced swimming stress decreased. (author)

Embryo developmental capacity of oocytes fertilised by sperm of mouse exposed to forced swimming stress.

Science.gov (United States)

Ghasem, Saki; Majid, Jasemi; Shiva, Razi

2013-07-01

To assess developmental capacity of fertilised oocytes by sperm of mouse exposed to forced swimming stress. The experimental study was conducted at the Physiology Research Center of Ahvaz Jundishapur University of Medical Sciences, from August 2011 to January 2012. It comprised 20 adult male and 10 female mice. The male mice were randomly divided into two equal groups (n=10): control and experimental. Animals of the experimental group were submitted to forced swimming stress. All male mice were euthanised and the cauda epididymis removed before contents were squeezed out. A pre-incubated capacitated sperm was gently added to the freshly collected ova of the two groups of study. The combined sperm-oocyte suspension was incubated for 4-6 hours under a condition of 5% Carbon dioxide and 37 degreeC temperature. The ova were then washed through several changes of medium and finally incubated. Fertilisation was assessed by recording the number of 1-cell embryos 4-6 hours after insemination. The 1-cell embryos were allowed to further develop in vitro for about 120 hours. Development of embryos everyday and during 5 days of culture was observed by using inverted microscope. SPSS 13.0.1 was used for statistical analysis. The percentage of oocytes fertilised was 75:96 (78.12+/-4.8%) and 50:10 (49.5+/-3.9%) in the control and experimental groups, respectively. The difference was significant (p 0.05) between the two groups in terms of speed and developmental capacity of blastocysts. Fertilisation capacity of male mice affected by forced swimming stress and also the developmental capacity of oocyte fertilised by sperm of mouse exposed to forced swimming stress decreased.

The effects of MRI on mouse embryos during fetal stage

Energy Technology Data Exchange (ETDEWEB)

Nakamura, Takashi; Sakazaki, Takahiko; Itokawa, Yuka [Suzuka University of Medical Science, Koriyama (Japan)] (and others)

2006-06-15

The effects of Magnetic Resonance Imaging (MRI) on mouse embryos at the early stage of organogenesis were investigated. Pregnant ICR mice were exposed on day 8 of gestation to MRI at 0.5 T for 0.5 hour to 3 hours. The mortality rates of embryos or fetuses, the incidence of external malformations, fetal body weight and sex ratio were observed at day 18 of gestation. A significant increase in embryonic mortality was observed after exposure to either 0.5 T MRI for 0.5 hour or 2 hours. However, the exposure to MRI for 1 hour or 3 hours did not induce any significant increase in embryonic mortality when compared with control. External malformations such as exencephaly, cleft palate and anomalies of tail were observed in all experimental groups exposed to each MRI. A statistically significant increase of external malformations was observed in all groups treated with 0.5 T MRI for 0.5 hour and 3 hours. The incidence of external malformations in the mice group exposed to 0.5 T MRI for 0.5-hour was found to be higher than those of mice group exposed to 0.5 T MRI for 2 hours. The effects of MRI on the external malformations might not to be dose-dependent. There was no statistically significant difference in fetal body weight and sex ratio among each MRI exposure groups.

Embryo density and medium volume effects on early murine embryo development.

Science.gov (United States)

Canseco, R S; Sparks, A E; Pearson, R E; Gwazdauskas, F C

1992-10-01

One-cell mouse embryos were used to determine the effects of drop size and number of embryos per drop for optimum development in vitro. Embryos were collected from immature C57BL6 female mice superovulated with pregnant mare serum gonadotropin and human chorionic gonadotropin and mated by CD1 males. Groups of 1, 5, 10, or 20 embryos were cultured in 5-, 10-, 20-, or 40-microliters drops of CZB under silicon oil at 37.5 degrees C in a humidified atmosphere of 5% CO2 and 95% air. Development score for embryos cultured in 10 microliters was higher than that of embryos cultured in 20 or 40 microliters. Embryos cultured in groups of 5, 10, or 20 had higher development scores than embryos cultured singly. The highest development score was obtained by the combination of 5 embryos per 10-microliters drop. The percentage of live embryos in 20 or 40 microliters was lower than that of embryos cultured in 10 microliters. Additionally, the percentage of live embryos cultured singly was lower than that of embryos cultured in groups. Our results suggest that a stimulatory interaction occurs among embryos possibly exerted through the secretion of growth factors. This effect can be diluted if the embryos are cultured in large drops or singly.

The Nanos3-3'UTR is required for germ cell specific NANOS3 expression in mouse embryos.

Directory of Open Access Journals (Sweden)

Hitomi Suzuki

Full Text Available BACKGROUND: The regulation of gene expression via a 3' untranslated region (UTR plays essential roles in the discrimination of the germ cell lineage from somatic cells during embryogenesis. This is fundamental to the continuation of a species. Mouse NANOS3 is an essential protein required for the germ cell maintenance and is specifically expressed in these cells. However, the regulatory mechanisms that restrict the expression of this gene in the germ cells is largely unknown at present. METHODOLOGY/PRINCIPAL FINDINGS: In our current study, we show that differences in the stability of Nanos3 mRNA between germ cells and somatic cells is brought about in a 3'UTR-dependent manner in mouse embryos. Although Nanos3 is transcribed in both cell lineages, it is efficiently translated only in the germ lineage. We also find that the translational suppression of NANOS3 in somatic cells is caused by a 3'UTR-mediated mRNA destabilizing mechanism. Surprisingly, even when under the control of the CAG promoter which induces strong ubiquitous transcription in both germ cells and somatic cells, the addition of the Nanos3-3'UTR sequence to the coding region of exogenous gene was effective in restricting protein expression in germ cells. CONCLUSIONS/SIGNIFICANCE: Our current study thus suggests that Nanos3-3'UTR has an essential role in translational control in the mouse embryo.

Association of the transcription profile of bovine oocytes and embryos with developmental potential

Czech Academy of Sciences Publication Activity Database

Kaňka, Jiří; Němcová, Lucie; Toralová, Tereza; Vodičková Kepková, Kateřina; Vodička, Petr; Jeseta, M.; Machatková, M.

2012-01-01

Roč. 134, 1-2 (2012), 29-35 ISSN 0378-4320. [Embryo Genomics Meeting /3./. Bonn, 20.08.2012-22.08.2012] R&D Projects: GA ČR GA523/09/1035; GA MZe QI91A018 Institutional support: RVO:67985904 Keywords : oocyte * in vitro maturation * pre-implantation development Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.897, year: 2012

Whole genome amplification in preimplantation genetic diagnosis*

Science.gov (United States)

Zheng, Ying-ming; Wang, Ning; Li, Lei; Jin, Fan

2011-01-01

Preimplantation genetic diagnosis (PGD) refers to a procedure for genetically analyzing embryos prior to implantation, improving the chance of conception for patients at high risk of transmitting specific inherited disorders. This method has been widely used for a large number of genetic disorders since the first successful application in the early 1990s. Polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH) are the two main methods in PGD, but there are some inevitable shortcomings limiting the scope of genetic diagnosis. Fortunately, different whole genome amplification (WGA) techniques have been developed to overcome these problems. Sufficient DNA can be amplified and multiple tasks which need abundant DNA can be performed. Moreover, WGA products can be analyzed as a template for multi-loci and multi-gene during the subsequent DNA analysis. In this review, we will focus on the currently available WGA techniques and their applications, as well as the new technical trends from WGA products. PMID:21194180

Interspecific in vitro assay for the chimera-forming ability of human pluripotent stem cells.

Science.gov (United States)

Masaki, Hideki; Kato-Itoh, Megumi; Umino, Ayumi; Sato, Hideyuki; Hamanaka, Sanae; Kobayashi, Toshihiro; Yamaguchi, Tomoyuki; Nishimura, Ken; Ohtaka, Manami; Nakanishi, Mahito; Nakauchi, Hiromitsu

2015-09-15

Functional assay limitations are an emerging issue in characterizing human pluripotent stem cells (PSCs). With rodent PSCs, chimera formation using pre-implantation embryos is the gold-standard assay of pluripotency (competence of progeny to differentiate into all three germ layers). In human PSCs (hPSCs), however, this can only be monitored via teratoma formation or in vitro differentiation, as ethical concerns preclude generation of human-human or human-animal chimeras. To circumvent this issue, we developed a functional assay utilizing interspecific blastocyst injection and in vitro culture (interspecies in vitro chimera assay) that enables the development and observation of embryos up to headfold stage. The assay uses mouse pre-implantation embryos and rat, monkey and human PSCs to create interspecies chimeras cultured in vitro to the early egg-cylinder stage. Intra- and interspecific chimera assays with rodent PSC lines were performed to confirm the consistency of results in vitro and in vivo. The behavior of chimeras developed in vitro appeared to recapitulate that of chimeras developed in vivo; that is, PSC-derived cells survived and were integrated into the epiblast of egg-cylinder-stage embryos. This indicates that the interspecific in vitro chimera assay is useful in evaluating the chimera-forming ability of rodent PSCs. However, when human induced PSCs (both conventional and naïve-like types) were injected into mouse embryos and cultured, some human cells survived but were segregated; unlike epiblast-stage rodent PSCs, they never integrated into the epiblast of egg-cylinder-stage embryos. These data suggest that the mouse-human interspecies in vitro chimera assay does not accurately reflect the early developmental potential/process of hPSCs. The use of evolutionarily more closely related species as host embryos might be necessary to evaluate the developmental potency of hPSCs. © 2015. Published by The Company of Biologists Ltd.

Preservation of mammalian germ plasm by freezing

Energy Technology Data Exchange (ETDEWEB)

Mazur, P.

1978-01-01

Embryos of several mammalian species can be frozen to -196/sup 0/C (or below) by procedures that result in the thawed embryos being indistinguishable from their unfrozen counterparts. The survival often exceeds 90%, and in liquid nitrogen it should remain at that high level for centuries. Sublethal biochemical changes are also precluded at -196/sup 0/C. No developmental abnormalities have been detected in mouse offspring derived from frozen-thawed embryos, and, since all the manipulations are carried out on the preimplantation stages, none would be expected.

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

[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)'.

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

The expression of nucleolar-related proteins was studied as an indirect marker of the ribosomal RNA (rRNA) gene activation in porcine embryos up to the blastocyst stage produced in vivo and in vitro. A group of the in vivo-developed embryos were cultured with alpha-amanitin to block the de novo...... proteins pRb and p130, which are involved in cell-cycle regulation, was assessed by semiquantitative RT-PCR up to the blastocyst stage. Toward the end of third cell cycle, the nuclei in non-alpha-amanitin-treated, in vivo-produced embryos displayed different stages of transformation of the nuclear...... 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...

The effect of hepatocyte growth factor on mouse oocyte in vitro maturation and subsequent fertilization and embryo development

Directory of Open Access Journals (Sweden)

Mohammad H. Bahadori

2011-05-01

Full Text Available Background: Oocyte invitro maturation is an enormously promising technology for the treatment of infertility, yet its clinical application remains limited owing to poor success rates. Therefore, this study was devised to evaluate the effect of hepatocyte growth factor (HGF on in vitro maturation of immature mouse oocytes and resulting embryos development. Materials and Method: Cumulus – oocyte complex and germinal vesicle were obtained from eighteen 6-8 weeks-old female NMRI mice 46-48 hours after administration of an injection of 5 IU PMSG (Pregnant Mares’ Serum Gonadotrophin. Oocytes were culture in TCM199 (Tissue culture medium-199 supplemented with dosages of 0, 10, 20, 50 and 100 ng/ml of HGF. After 24 hours, metaphase ІІ oocytes were co-incubated with sperms for 4-6 hours in T6 medium. Following isolation of two pronucleus embryos, cleavage of embryos was assessed in the same medium till blastocyst stage. The number of oocytes and embryos was recorded under an invert microscope and the rate of oocyte maturation, fertilization and embryos cleavage until blastocyst stage compared using of student χ2 test. Results: In all compared groups, oocytes growth and embryos development rate in the 20 ng/ml of HGF treatment group was significantly higher (p<0.05 than the control group (p<0.05.Conclusion: 20 ng/ml of HGF improved the nuclear maturation and embryo development up to blastocyst stage during culture condition

Effect of oxygen concentration on human embryo development evaluated by time-lapse monitoring

DEFF Research Database (Denmark)

Ingerslev, Hans Jakob; Hindkjær, Johnny Juhl; Kirkegaard, Kirstine

2012-01-01

recently demonstrated to occur from first cleavage cycle in mice using time-lapse microscopy, with the largest impact on the pre-compaction stages. However, embryonic development in mice differs in many aspects from human embryonic development. The objective of this retrospective, descriptive study...... was to evaluate the influence of oxygen tension on human pre-implantation development using time-lapse monitoring. Materials and methods: Human embryos were cultured to the blastocyst stage in a time-lapse incubator (EmbryoScope™) in 20% O2 (group 1), 20% O2 for 24 hours followed by culture in 5% O2 (group 2......) or in 5% O2 (group 3). Eligible were patients with age 8 oocytes retrieved. Group 1 consisted of 120 IVF/ICSI embryos from 26 patients recruited to a study conducted to evaluate the safety of the time-lapse incubator by randomising 1:1 embryos from a patient to culture...

Where does New Zealand stand on permitting research on human embryos?

Science.gov (United States)

Jones, D Gareth

2014-08-01

In many respects New Zealand has responded to the assisted reproductive technologies (ARTs) as positively as many comparable societies, such as Australia and the UK. Consequently, in vitro fertilisation (IVF) and pre-implantation genetic diagnosis (PGD) are widely available, as is non-commercial surrogacy utilising IVF. These developments have been made possible by the Human Assisted Reproductive Technology (HART) Act 2004, overseen by its two committees, the Advisory Committee on Assisted Reproductive Technology (ACART) and the Ethics Committee (ECART). However, New Zealand stands apart from many of these other societies by the lack of permission for scientists to conduct research using human embryos. There is no doubt this reflects strongly held viewpoints on the part of some that embryos should be protected and not exploited. Legitimate as this stance is, the resulting situation is problematic when IVF is already designated as an established procedure. This is because the development of IVF involved embryo research, and continuing improvements in procedures depend upon ongoing embryo research. While prohibition of research on human embryos gives the impression of protecting embryos, it fails to do this and also fails to enhance the health and wellbeing of children born using IVF. This situation will not be rectified until research is allowed on human embryos.

[The composition of the gastrointestinal bacterial flora of mouse embryos and the placenta tissue bacterial flora].

Science.gov (United States)

Lei, D; Lin, Y; Jiang, X; Lan, L; Zhang, W; Wang, B X

2017-03-02

Objective: To explore the composition of the gastrointestinal bacterial flora of mouse embryos and the placenta tissue bacterial flora. Method: Twenty-four specimens were collected from pregnant Kunming mouse including 8 mice of early embryonic (12-13 days) gastrointestinal tissues, 8 cases of late embryonic (19-20 days)gastrointestinal tissues, 8 of late pregnancy placental tissues.The 24 samples were extracted by DNeasy Blood & Tissue kit for high-throughput DNA sequencing. Result: The level of Proteobacteria, Bacteroidetes, Actino-bacteria and Firmicutes were predominantin all specimens.The relative content of predominant bacterial phyla in each group: Proteobacteria (95.00%, 88.14%, 87.26%), Bacteroidetes(1.71%, 2.15%, 2.63%), Actino-Bacteria(1.16%, 4.10%, 3.38%), Firmicutes(0.75%, 2.62%, 2.01%). At the level of family, there were nine predominant bacterial families in which Enterobacteriaeae , Shewanel laceae and Moraxellaceae were dominant.The relative content of dominant bacterial family in eachgroup: Enterobacteriaeae (46.99%, 44.34%, 41.08%), Shewanellaceae (21.99%, 21.10%, 19.05%), Moraxellaceae (9.18%, 7.09%, 5.64%). From the species of flora, the flora from fetal gastrointestinal in early pregnancy and late pregnancy (65.44% and 62.73%) were the same as that from placenta tissue in the late pregnancy.From the abundance of bacteria, at the level of family, the same content of bacteria in three groups accounted for 78.16%, 72.53% and 65.78% respectively. Conclusion: It was proved that the gastrointestinal bacterial flora of mouse embryos and the placenta tissue bacterial flora were colonized. At the same time the bacteria are classified.

Non-intact zona improves development of murine preimplantation ...

African Journals Online (AJOL)

ajl5

2012-09-25

Sep 25, 2012 ... 2College of Animal Science and Technology, Northwest A & F University, Yangling, ... Key words: Mouse, non-intact zona embryos, adenovirus vector with green fluorescent protein (pAd-GFP), .... Based on microscopic examination, the ZP of some ..... permeable structure of ZP that allowed penetration of.

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

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

Is there an ethical difference between preimplantation genetic diagnosis and abortion?

Science.gov (United States)

Cameron, C; Williamson, R

2003-04-01

When a person at risk of having a child with a genetic illness or disease wishes to have an unaffected child, this can involve difficult choices. If the pregnancy is established by sexual intercourse, the fetus can be tested early in pregnancy, and if affected a decision can be made to abort in the hope that a future pregnancy with an unaffected fetus ensures. Alternatively, preimplantation genetic diagnosis (PGD) can be used after in vitro fertilisation (IVF) to select and implant an unaffected embryo that hopefully will proceed to term and produce a healthy baby. We are aware that many individuals at risk regard the latter as ethically more acceptable than the former, and examine whether there is an ethical difference between these options. We conclude that PGD and implantation of an unaffected embryo is a more acceptable choice ethically than prenatal diagnosis (PND) followed by abortion for the following reasons: Choice after PGD is seen as ethically neutral because a positive result ("a healthy pregnancy") balances a negative result ("the destruction of the affected embryo") simultaneously (assuming the pregnancy proceeds to full term and a healthy baby is born). While there is usually the intention to establish a healthy pregnancy after an abortion, this is not simultaneous; A woman sees abortion as a personal physical violation of her integrity, and as the pregnancy proceeds she increasingly identifies with and gives ethical status to the embryo/fetus as it develops in utero and not in the laboratory; Many people see aborting a fetus as "killing", whereas in the case of PGD the spare embryos are "allowed to die". We argue that this difference of opinion gives further weight to our conclusion, but note that this has been addressed and debated at length by others.

Dysregulated LIF-STAT3 pathway is responsible for impaired embryo implantation in a Streptozotocin-induced diabetic mouse model

Directory of Open Access Journals (Sweden)

Tong-Song Wang

2015-07-01

Full Text Available The prevalence of diabetes is increasing worldwide with the trend of patients being young and creating a significant burden on health systems, including reproductive problems, but the effects of diabetes on embryo implantation are still poorly understood. Our study was to examine effects of diabetes on mouse embryo implantation, providing experimental basis for treating diabetes and its complications. Streptozotocin (STZ was applied to induce type 1 diabetes from day 2 of pregnancy or pseudopregnancy in mice. Embryo transfer was used to analyze effects of uterine environment on embryo implantation. Our results revealed that the implantation rate is significantly reduced in diabetic mice compared to controls, and the change of uterine environment is the main reason leading to the decreased implantation rate. Compared to control, the levels of LIF and p-STAT3 are significantly decreased in diabetic mice on day 4 of pregnancy, and serum estrogen level is significantly higher. Estrogen stimulates LIF expression under physiological level, but the excessive estrogen inhibits LIF expression. LIF, progesterone or insulin supplement can rescue embryo implantation in diabetic mice. Our data indicated that the dysregulated LIF-STAT3 pathway caused by the high level of estrogen results in the impaired implantation in diabetic mice, which can be rescued by LIF, progesterone or insulin supplement.

Impaired imprinted X chromosome inactivation is responsible for the skewed sex ratio following in vitro fertilization

Science.gov (United States)

Tan, Kun; An, Lei; Miao, Kai; Ren, Likun; Hou, Zhuocheng; Tao, Li; Zhang, Zhenni; Wang, Xiaodong; Xia, Wei; Liu, Jinghao; Wang, Zhuqing; Xi, Guangyin; Gao, Shuai; Sui, Linlin; Zhu, De-Sheng; Wang, Shumin; Wu, Zhonghong; Bach, Ingolf; Chen, Dong-bao; Tian, Jianhui

2016-01-01

Dynamic epigenetic reprogramming occurs during normal embryonic development at the preimplantation stage. Erroneous epigenetic modifications due to environmental perturbations such as manipulation and culture of embryos during in vitro fertilization (IVF) are linked to various short- or long-term consequences. Among these, the skewed sex ratio, an indicator of reproductive hazards, was reported in bovine and porcine embryos and even human IVF newborns. However, since the first case of sex skewing reported in 1991, the underlying mechanisms remain unclear. We reported herein that sex ratio is skewed in mouse IVF offspring, and this was a result of female-biased peri-implantation developmental defects that were originated from impaired imprinted X chromosome inactivation (iXCI) through reduced ring finger protein 12 (Rnf12)/X-inactive specific transcript (Xist) expression. Compensation of impaired iXCI by overexpression of Rnf12 to up-regulate Xist significantly rescued female-biased developmental defects and corrected sex ratio in IVF offspring. Moreover, supplementation of an epigenetic modulator retinoic acid in embryo culture medium up-regulated Rnf12/Xist expression, improved iXCI, and successfully redeemed the skewed sex ratio to nearly 50% in mouse IVF offspring. Thus, our data show that iXCI is one of the major epigenetic barriers for the developmental competence of female embryos during preimplantation stage, and targeting erroneous epigenetic modifications may provide a potential approach for preventing IVF-associated complications. PMID:26951653

The use of embryonic stem cell derived bioactive material as a new protein supplement for the in vitro culture of bovine embryos.

Science.gov (United States)

Kim, Eun Young; Lee, Jun Beom; Park, Hyo Young; Jeong, Chang Jin; Riu, Key Zung; Park, Se Pill

2011-06-01

Embryonic stem (ES) cells are expanded versions of the inner cell mass cells that compose the early mammalian blastocyst. Components derived from ES cells may contain various bioactive materials (BM) helpful for early preimplantation embryo growth. In this study, we examined the effect of human ES cell derived BM (hES-BM) on in vitro culture of bovine embryos. When bovine parthenogenetic day 2 embryos were cultured in 10% hES-BM, a significantly higher embryo development rate (44.3%) and increased cell numbers were observed relative to control medium containing 3 mg/ml BSA (19.5%; Pculture environment to support the growth of bovine embryos in vitro (P<0.05). Little difference was observed between 10% hES-BM and 10% FBS treatment in the examined parthenogenetic or in vitro fertilized embryos, although the hES-BM group developed at a slightly better rate. However, the ICM cell numbers were significantly higher in the hES-BM group in irrespective of embryo origin (P<0.05). In addition, the relative levels of pluripotency (Oct4, × 1.8 fold; Nanog. × 3.3 fold), embryogenesis (Stat3, × 2.8 fold; FGF4, × 18.8 fold; E-cad, × 2.0 fold) and growth (Glut5, × 2.6 fold) genes were significantly higher in the 10% hES-BM group than in the 10% FBS group (P<0.05), while the levels of other genes (Bax, Bcl2, MnSOD and Connexin43) were not different. This is the first report examining the positive effects of hES-BM on bovine embryo development in vitro. Based on our results, we conclude that hES-BM can be used as a new protein supplement for bovine preimplantation embryo development.

New models and molecular markers in evaluation of developmental toxicity

International Nuclear Information System (INIS)

Huuskonen, Hannele

2005-01-01

Mammalian and non-mammalian embryos and embryonic stem cells may be used as models in mechanistic studies and in testing embryotoxicity of compounds. In addition to conventional culture methods, genetic modifications and use of molecular markers offer significant advantages in mechanistic studies as well as in developing new test methods for embryotoxicity. Zebrafish model has been used for a long time and at present several applications are available. It is an easy vertebral non-mammalian model, whose genome is largely known and several genetic modifications are easily constructed to study gene expression or knocked down genes. Fluorescent marker proteins can be used also in zebrafish to indicate gene activation in transgenic models. Chemical genetics approach has been developed using zebrafish model. This is a new approach to screen small molecules that regulate signaling pathways. Embryonic stem cells have been used in mechanistic studies and mouse embryonic stem cell test has been validated to study embryotoxicity in vitro. This method has been improved using quantitative measurements of molecular endpoints by real-time RT-PCR or fluorescent activated cell sorting methods (FACS). Methods facilitating differentiation to several different cell types are available. We have studied preimplantation mouse embryos as a possible model for in vitro testing. In this method, superovulated and in vivo fertilized preimplantation embryos were collected at morula stage and cultured up to blastocysts. The mouse preimplantation culture test was improved by quantitative gene expression measurement using two-step real-time RT-PCR methods. New endpoints improve the tests of in vitro embryotoxicity because subjective assessments are replaced by objective measurements. In addition, automation is possible and less time is needed for analysis. Thus, high throughput screening will come possible to test large numbers of compounds

Loss of maternal ATRX results in centromere instability and aneuploidy in the mammalian oocyte and pre-implantation embryo.

Directory of Open Access Journals (Sweden)

Claudia Baumann

2010-09-01

Full Text Available The α-thalassemia/mental retardation X-linked protein (ATRX is a chromatin-remodeling factor known to regulate DNA methylation at repetitive sequences of the human genome. We have previously demonstrated that ATRX binds to pericentric heterochromatin domains in mouse oocytes at the metaphase II stage where it is involved in mediating chromosome alignment at the meiotic spindle. However, the role of ATRX in the functional differentiation of chromatin structure during meiosis is not known. To test ATRX function in the germ line, we developed an oocyte-specific transgenic RNAi knockdown mouse model. Our results demonstrate that ATRX is required for heterochromatin formation and maintenance of chromosome stability during meiosis. During prophase I arrest, ATRX is necessary to recruit the transcriptional regulator DAXX (death domain associated protein to pericentric heterochromatin. At the metaphase II stage, transgenic ATRX-RNAi oocytes exhibit abnormal chromosome morphology associated with reduced phosphorylation of histone 3 at serine 10 as well as chromosome segregation defects leading to aneuploidy and severely reduced fertility. Notably, a large proportion of ATRX-depleted oocytes and 1-cell stage embryos exhibit chromosome fragments and centromeric DNA-containing micronuclei. Our results provide novel evidence indicating that ATRX is required for centromere stability and the epigenetic control of heterochromatin function during meiosis and the transition to the first mitosis.

Prevention of Lysosomal Storage Diseases and Derivation of Mutant Stem Cell Lines by Preimplantation Genetic Diagnosis

Science.gov (United States)

Altarescu, Gheona; Beeri, Rachel; Eiges, Rachel; Epsztejn-Litman, Silvina; Eldar-Geva, Talia; Elstein, Deborah; Zimran, Ari; Margalioth, Ehud J.; Levy-Lahad, Ephrat; Renbaum, Paul

2012-01-01

Preimplantation genetic diagnosis (PGD) allows birth of unaffected children for couples at risk for a genetic disorder. We present the strategy and outcome of PGD for four lysosomal storage disorders (LSD): Tay-Sachs disease (TSD), Gaucher disease (GD), Fabry disease (FD), and Hunter syndrome (HS), and subsequent development of stem cell lines. For each disease, we developed a family-specific fluorescent multiplex single-cell PCR protocol that included the familial mutation and informative markers surrounding the mutation. Embryo biopsy and PGD analysis were performed on either oocytes (polar bodies one and two) or on single blastomeres from a six-cell embryo. We treated twenty families carrying mutations in these lysosomal storage disorders, including 3 couples requiring simultaneous analysis for two disorders (TSD/GD, TSD/balanced Robertsonian translocation 45XYder(21;14), and HS/oculocutaneus albinism). These analyses led to an overall pregnancy rate/embryo transfer of 38% and the birth of 20 unaffected children from 17 families. We have found that PGD for lysosomal disorders is a safe and effective method to prevent birth of affected children. In addition, by using mutant embryos for the derivation of stem cell lines, we have successfully established GD and HS hESC lines for use as valuable models in LSD research. PMID:23320174

Heme synthesis in the lead-intoxicated mouse embryo

Energy Technology Data Exchange (ETDEWEB)

Gerber, G B; Maes, J

1978-02-01

Incorporation of /sup 55/Fe and of (/sup 14/C) glycine was studied in control embryos and mothers and in those which had received lead in the diet from day 7 of pregnancy. Incorporation of Fe into heme of embryonic liver which increases markedly for controls on day 17 of pregnancy was depressed greatly and showed no such increase in lead-intoxicated embryos. These embryos were retarded in growth but had normal heme concentrations in body and liver. Incorporation of glycine into embryonic heme and proteins was not affected. Data on incorporation in the mothers are also presented. It is thought that the impaired synthesis of heme in lead-intoxicated embryos limits their body growth during the late phase of pregnancy.

Lack of centrioles and primary cilia in STIL(-/-) mouse embryos.

Science.gov (United States)

David, Ahuvit; Liu, Fengying; Tibelius, Alexandra; Vulprecht, Julia; Wald, Diana; Rothermel, Ulrike; Ohana, Reut; Seitel, Alexander; Metzger, Jasmin; Ashery-Padan, Ruth; Meinzer, Hans-Peter; Gröne, Hermann-Josef; Izraeli, Shai; Krämer, Alwin

2014-01-01

Although most animal cells contain centrosomes, consisting of a pair of centrioles, their precise contribution to cell division and embryonic development is unclear. Genetic ablation of STIL, an essential component of the centriole replication machinery in mammalian cells, causes embryonic lethality in mice around mid gestation associated with defective Hedgehog signaling. Here, we describe, by focused ion beam scanning electron microscopy, that STIL(-/-) mouse embryos do not contain centrioles or primary cilia, suggesting that these organelles are not essential for mammalian development until mid gestation. We further show that the lack of primary cilia explains the absence of Hedgehog signaling in STIL(-/-) cells. Exogenous re-expression of STIL or STIL microcephaly mutants compatible with human survival, induced non-templated, de novo generation of centrioles in STIL(-/-) cells. Thus, while the abscence of centrioles is compatible with mammalian gastrulation, lack of centrioles and primary cilia impairs Hedgehog signaling and further embryonic development.

Review:Whole genome amplification in preimplantation genetic diagnosis

Institute of Scientific and Technical Information of China (English)

Ying-ming ZHENG; Ning WANG; Lei LI; Fan JIN

2011-01-01

Preimplantation genetic diagnosis(PGD)refers to a procedure for genetically analyzing embryos prior to implantation,improving the chance of conception for patients at high risk of transmitting specific inherited disorders.This method has been widely used for a large number of genetic disorders since the first successful application in the early 1990s.Polymerase chain reaction(PCR)and fluorescent in situ hybridization(FISH)are the two main methods in PGD,but there are some inevitable shortcomings limiting the scope of genetic diagnosis.Fortunately,different whole genome amplification(WGA)techniques have been developed to overcome these problems.Sufficient DNA can be amplified and multiple tasks which need abundant DNA can be performed.Moreover,WGA products can be analyzed as a template for multi-loci and multi-gene during the subsequent DNA analysis.In this review,we will focus on the currently available WGA techniques and their applications,as well as the new technical trends from WGA products.

A role for Aurora C in the chromosomal passenger complex during human preimplantation embryo development

NARCIS (Netherlands)

Santos, Margarida Avo; van de Werken, Christine; de Vries, Marieke; Jahr, Holger; Vromans, Martijn J. M.; Laven, Joop S. E.; Fauser, Bart C.; Kops, Geert J.; Lens, Susanne M.; Baart, Esther B.

BACKGROUND: Human embryos generated by IVF demonstrate a high incidence of chromosomal segregation errors during the cleavage divisions. To analyse underlying molecular mechanisms, we investigated the behaviour of the chromosomal passenger complex (CPC) in human oocytes and embryos. This important

Effect of oxygen concentration on human embryo development evaluated by time-lapse monitoring

DEFF Research Database (Denmark)

Ingerslev, Hans Jakob; Hindkjær, Johnny Juhl; Kirkegaard, Kirstine

2012-01-01

-points for each cell division and blastocyst stages were registered until 120 hours after oocyte retrieval. Only 2PN embryos completing the first cleavage were evaluated. The groups were compared using one-way ANOVA or Kruskall-Wallis test. Estimates are reported as medians with 95% confidence intervals. Time......Introduction: Data from a number of studies indicate -but not unequivocally- that culture of embryos in 5% O2 compared to 20% O2 improves blastocyst formation in humans and various animal species and may yield better pregnancy rates in IVF. The detrimental effects of atmospheric oxygen were...... was to evaluate the influence of oxygen tension on human pre-implantation development using time-lapse monitoring. Materials and methods: Human embryos were cultured to the blastocyst stage in a time-lapse incubator (EmbryoScope™) in 20% O2 (group 1), 20% O2 for 24 hours followed by culture in 5% O2 (group 2...

Noninvasive imaging systems for gametes and embryo selection in IVF programs: a review.

Science.gov (United States)

Omidi, Marjan; Faramarzi, Azita; Agharahimi, Azam; Khalili, Mohammad Ali

2017-09-01

Optimizing the efficiency of the in vitro fertilization procedure by improving pregnancy rates and reducing the risks of multiple pregnancies simultaneously are the primary goals of the current assisted reproductive technology program. With the move to single embryo transfers, the need for more cost-effective and noninvasive methods for embryo selection prior to transfer is paramount. These aims require advancement in a more acquire gametes/embryo testing and selection procedures using high-tech devices. Therefore, the aim of the present review is to evaluate the efficacy of noninvasive imaging systems in the current literatures, focusing on the potential clinical application in infertile patients undergoing assisted reproductive technology treatments. In this regards, three advanced imaging systems of motile sperm organelle morphology examination, polarization microscopy and time-lapse monitoring for the best selection of the gametes and preimplantation embryos are introduced in full. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

Preimplantation genetic diagnosis for chromosomal rearrangements with the use of array comparative genomic hybridization at the blastocyst stage.

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Christodoulou, Christodoulos; Dheedene, Annelies; Heindryckx, Björn; van Nieuwerburgh, Filip; Deforce, Dieter; De Sutter, Petra; Menten, Björn; Van den Abbeel, Etienne

2017-01-01

To establish the value of array comparative genomic hybridization (CGH) for preimplantation genetic diagnosis (PGD) in embryos of translocation carriers in combination with vitrification and frozen embryo transfer in nonstimulated cycles. Retrospective data analysis study. Academic centers for reproductive medicine and genetics. Thirty-four couples undergoing PGD for chromosomal rearrangements from October 2013 to December 2015. Trophectoderm biopsy at day 5 or day 6 of embryo development and subsequently whole genome amplification and array CGH were performed. This approach revealed a high occurrence of aneuploidies and structural rearrangements unrelated to the parental rearrangement. Nevertheless, we observed a benefit in pregnancy rates of these couples. We detected chromosomal abnormalities in 133/207 embryos (64.2% of successfully amplified), and 74 showed a normal microarray profile (35.7%). In 48 of the 133 abnormal embryos (36.1%), an unbalanced rearrangement originating from the parental translocation was identified. Interestingly, 34.6% of the abnormal embryos (46/133) harbored chromosome rearrangements that were not directly linked to the parental translocation in question. We also detected a combination of unbalanced parental-derived rearrangements and aneuploidies in 27 of the 133 abnormal embryos (20.3%). The use of trophectoderm biopsy at the blastocyst stage is less detrimental to the survival of the embryo and leads to a more reliable estimate of the genomic content of the embryo than cleavage-stage biopsy. In this small cohort PGD study, we describe the successful implementation of array CGH analysis of blastocysts in patients with a chromosomal rearrangement to identify euploid embryos for transfer. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Non-destructive monitoring of mouse embryo development and its qualitative evaluation at the molecular level using Raman spectroscopy

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Ishigaki, Mika; Hashimoto, Kosuke; Sato, Hidetoshi; Ozaki, Yukihiro

2017-03-01

Current research focuses on embryonic development and quality not only by considering fundamental biology, but also by aiming to improve assisted reproduction technologies, such as in vitro fertilization. In this study, we explored the development of mouse embryo and its quality based on molecular information, obtained nondestructively using Raman spectroscopy. The detailed analysis of Raman spectra measured in situ during embryonic development revealed a temporary increase in protein content after fertilization. Proteins with a β-sheet structure—present in the early stages of embryonic development—are derived from maternal oocytes, while α-helical proteins are additionally generated by switching on a gene after fertilization. The transition from maternal to embryonic control during development can be non-destructively profiled, thus facilitating the in situ assessment of structural changes and component variation in proteins generated by metabolic activity. Furthermore, it was indicated that embryos with low-grade morphology had high concentrations of lipids and hydroxyapatite. This technique could be used for embryo quality testing in the future.

Clinical applications of MARSALA for preimplantation genetic diagnosis of spinal muscular atrophy.

Science.gov (United States)

Ren, Yixin; Zhi, Xu; Zhu, Xiaohui; Huang, Jin; Lian, Ying; Li, Rong; Jin, Hongyan; Zhang, Yan; Zhang, Wenxin; Nie, Yanli; Wei, Yuan; Liu, Zhaohui; Song, Donghong; Liu, Ping; Qiao, Jie; Yan, Liying

2016-09-20

Conventional PCR methods combined with linkage analysis based on short tandem repeats (STRs) or Karyomapping with single nucleotide polymorphism (SNP) arrays, have been applied to preimplantation genetic diagnosis (PGD) for spinal muscular atrophy (SMA), an autosome recessive disorder. However, it has limitations in SMA diagnosis by Karyomapping, and these methods are unable to distinguish wild-type embryos with carriers effectively. Mutated allele revealed by sequencing with aneuploidy and linkage analyses (MARSALA) is a new method allowing embryo selection by a one-step next-generation sequencing (NGS) procedure, which has been applied in PGD for both autosome dominant and X-linked diseases in our group previously. In this study, we carried out PGD based on MARSALA for two carrier families with SMA affected children. As a result, one of the couples has given birth to a healthy baby free of mutations in SMA-causing gene. It is the first time that MARSALA was applied to PGD for SMA, and we can distinguish the embryos with heterozygous deletion (carriers) from the wild-type (normal) ones accurately through this NGS-based method. In addition, direct mutation detection allows us to identify the affected embryos (homozygous deletion), which can be regarded as probands for linkage analysis, in case that the affected family member is absent. In the future, the NGS-based MARSALA method is expected to be used in PGD for all monogenetic disorders with known pathogenic gene mutation. Copyright © 2016 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

Transition from blastomere to trophectoderm biopsy: comparing two preimplantation genetic testing for aneuploidies strategies.

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Coll, Lluc; Parriego, Mònica; Boada, Montserrat; Devesa, Marta; Arroyo, Gemma; Rodríguez, Ignacio; Coroleu, Bonaventura; Vidal, Francesca; Veiga, Anna

2018-05-25

SummaryShortly after the implementation of comprehensive chromosome screening (CCS) techniques for preimplantation genetic testing for aneuploidies (PGT-A), the discussion about the transition from day 3 to blastocyst stage biopsy was initiated. Trophectoderm biopsy with CCS is meant to overcome the limitations of cleavage-stage biopsy and single-cell analysis. The aim of this study was to assess the results obtained in our PGT-A programme after the implementation of this new strategy. Comparisons between the results obtained in 179 PGT-A cycles with day 3 biopsy (D+3) and fresh embryo transfer, and 204 cycles with trophectoderm biopsy and deferred (frozen-thawed) embryo transfer were established. Fewer embryos were biopsied and a higher euploidy rate was observed in the trophectoderm biopsy group. No differences in implantation (50.3% vs. 61.4%) and clinical pregnancy rate per transfer (56.1% vs. 65.3%) were found. Although the mean number of euploid embryos per cycle did not differ between groups (1.5 ± 1.7 vs. 1.7 ± 1.8), the final number of euploid blastocysts available for transfer per cycle was significantly higher in the trophectoderm biopsy group (1.1 ± 1.3 vs. 1.7 ± 1.8). This factor led to an increased cumulative live birth rate in this last group (34.1% vs. 44.6%). Although both strategies can offer good results, trophectoderm biopsy offers a more robust diagnosis and the intervention is less harmful for the embryos so more euploid blastocysts are finally available for transfer and/or vitrification.

Individual blastomeres of 16- and 32-cell mouse embryos are able to develop into foetuses and mice.

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Tarkowski, Andrzej K; Suwińska, Aneta; Czołowska, Renata; Ożdżeński, Wacław

2010-12-15

Cell and developmental studies have clarified how, by the time of implantation, the mouse embryo forms three primary cell lineages: epiblast (EPI), primitive endoderm (PE), and trophectoderm (TE). However, it still remains unknown when cells allocated to these three lineages become determined in their developmental fate. To address this question, we studied the developmental potential of single blastomeres derived from 16- and 32-cell stage embryos and supported by carrier, tetraploid blastomeres. We were able to generate singletons, identical twins, triplets, and quadruplets from individual inner and outer cells of 16-cell embryos and, sporadically, foetuses from single cells of 32-cell embryos. The use of embryos constitutively expressing GFP as the donors of single diploid blastomeres enabled us to identify their cell progeny in the constructed 2n↔4n blastocysts. We showed that the descendants of donor blastomeres were able to locate themselves in all three first cell lineages, i.e., epiblast, primitive endoderm, and trophectoderm. In addition, the application of Cdx2 and Gata4 markers for trophectoderm and primitive endoderm, respectively, showed that the expression of these two genes in the descendants of donor blastomeres was either down- or up-regulated, depending on the cell lineage they happened to occupy. Thus, our results demonstrate that up to the early blastocysts stage, the destiny of at least some blastomeres, although they have begun to express markers of different lineage, is still labile. Copyright © 2010 Elsevier Inc. All rights reserved.

Successful application of preimplantation genetic diagnosis for beta-thalassaemia and sickle cell anaemia in Italy.

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Chamayou, S; Alecci, C; Ragolia, C; Giambona, A; Siciliano, S; Maggio, A; Fichera, M; Guglielmino, A

2002-05-01

In Italy, the autosomal recessive diseases beta-thalassaemia and sickle cell anaemia are so widespread that in some regions they can be defined as 'social diseases'. In this study, nine clinical applications of preimplantation genetic diagnosis (PGD) were performed for beta-thalassaemia and sickle cell anaemia on seven Sicilian couples and carriers of beta-globin gene mutations. The studied mutations were: Cd39, HbS, IVS1 nt1, IVS1 nt6 and IVS1 nt110. ICSI was performed with partner's sperm on 131 out of 147 retrieved oocytes, and this resulted in 72 zygotes; 32 embryos were successfully biopsied on day 3. The biopsied blastomeres were lysed and the beta-globin alleles amplified by nested PCR. The mutation diagnosis was performed by restriction enzyme digestion and reverse dot-blot. The amplification efficacy was 97.2%. The genotype study of non-transferred and surplus embryos showed that the allele drop-out rate was 8.6%. Seventeen embryos were transferred in utero on day 4. All couples received an embryo transfer; of the four pregnancies obtained, three resulted in live births and one miscarried at 11 weeks. Prenatal diagnosis at the 11th week and miscarriage material analysis confirmed the PGD results. These studies represent the first successful application of PGD for beta-thalassaemia and sickle cell anaemia in Italy.

Super cool X-1000 and Super cool Z-1000, two ice blockers, and their effect on vitrification/warming of mouse embryos.

Science.gov (United States)

Badrzadeh, H; Najmabadi, S; Paymani, R; Macaso, T; Azadbadi, Z; Ahmady, A

2010-07-01

To evaluate the survival and blastocyst formation rates of mouse embryos after vitrification/thaw process with different ice blocker media. We used X-1000 and Z-1000 separately and mixed using V-Kim, a closed vitrification system. Mouse embryos were vitrified using ethylene glycol based medium supplemented with Super cool X-1000 and/or Super cool Z-1000. Survival rates for the control, Super cool X-1000, Super cool Z-1000, and Super cool X-1000/Z-1000 groups were 74%, 72%, 68%, and 85% respectively, with no significant difference among experimental and control groups; however, a significantly higher survival rate was noticed in the Super cool X-1000/Z-1000 group when compared with the Super cool Z-1000 group. Blastocyst formation rates for the control, Super cool X-1000, Super cool Z-1000, and Super cool X-1000/Z-1000 groups were 71%, 66%, 65%, and 72% respectively. There was no significant difference in this rate among control and experimental groups. In a closed vitrification system, addition of ice blocker Super cool X-1000 to the vitrification solution containing Super cool Z-1000 may improve the embryo survival rate. We recommend combined ice blocker usage to optimize the vitrification outcome. Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.

In vitro production of small ruminant embryos: late improvements and further research.

Science.gov (United States)

de Souza-Fabjan, Joanna Maria Gonçalves; Panneau, Barbara; Duffard, Nicolas; Locatelli, Yann; de Figueiredo, José Ricardo; Freitas, Vicente José de Figueirêdo; Mermillod, Pascal

2014-06-01

Beyond the potential use of in vitro production of embryos (IVP) in breeding schemes, embryos are also required for the establishment of new biotechnologies such as cloning and transgenesis. Additionally, the knowledge of oocyte and embryo physiology acquired through IVP techniques may stimulate the further development of other techniques such as marker assisted and genomic selection of preimplantation embryos, and also benefit assisted procreation in human beings. Efficient in vitro embryo production is currently a major objective for livestock industries, including small ruminants. The heterogeneity of oocytes collected from growing follicles by laparoscopic ovum pick up or in ovaries of slaughtered females, remains an enormous challenge for IVM success, and still limits the rate of embryo development. In addition, the lower quality of the IVP embryos, compared with their in vivo-derived counterparts, translates into poor cryosurvival, which restricts the wider use of this promising technology. Therefore, many studies have been reported in an attempt to determine the most suitable conditions for IVM, IVF, and in vitro development to maximize embryo production rate and quality. This review aims to present the current panorama of IVP production in small ruminants, describing important steps for its success, reporting the recent advances and also the main obstacles identified for its improvement and dissemination. Copyright © 2014 Elsevier Inc. All rights reserved.

Tight junction protein ZO-2 expression and relative function of ZO-1 and ZO-2 during mouse blastocyst formation

International Nuclear Information System (INIS)

Sheth, Bhavwanti; Nowak, Rachael L.; Anderson, Rebecca; Kwong, Wing Yee; Papenbrock, Thomas; Fleming, Tom P.

2008-01-01

Apicolateral tight junctions (TJs) between epithelial cells are multiprotein complexes regulating membrane polarity and paracellular transport and also contribute to signalling pathways affecting cell proliferation and gene expression. ZO-2 and other ZO family members form a sub-membranous scaffold for binding TJ constituents. We investigated ZO-2 contribution to TJ biogenesis and function during trophectoderm epithelium differentiation in mouse preimplantation embryos. Our data indicate that ZO-2 is expressed from maternal and embryonic genomes with maternal ZO-2 protein associated with nuclei in zygotes and particularly early cleavage stages. Embryonic ZO-2 assembled at outer blastomere apicolateral junctional sites from the late 16-cell stage. Junctional ZO-2 first co-localised with E-cadherin in a transient complex comprising adherens junction and TJ constituents before segregating to TJs after their separation from the blastocyst stage (32-cell onwards). ZO-2 siRNA microinjection into zygotes or 2-cell embryos resulted in specific knockdown of ZO-2 mRNA and protein within blastocysts. Embryos lacking ZO-2 protein at trophectoderm TJs exhibited delayed blastocoel cavity formation but underwent normal cell proliferation and outgrowth morphogenesis. Quantitative analysis of trophectoderm TJs in ZO-2-deficient embryos revealed increased assembly of ZO-1 but not occludin, indicating ZO protein redundancy as a compensatory mechanism contributing to the mild phenotype observed. In contrast, ZO-1 knockdown, or combined ZO-1 and ZO-2 knockdown, generated a more severe inhibition of blastocoel formation indicating distinct roles for ZO proteins in blastocyst morphogenesis

Evaluation of cell number and DNA content in mouse embryos cultivated with uranium; Evaluacion del numero de celulas y el contenido de DNA en embriones murinos cultivados con uranio

Energy Technology Data Exchange (ETDEWEB)

Kundt, Mirian S; Cabrini, Romulo L [Comision Nacional de Energia Atomica, General San Martin (Argentina). Dept. de Radiobiologia

2000-07-01

The evaluation of the degree of development, the number of cells and the DNA content, were used to evaluate the embryotoxicity of uranium. Embryos at a one cell stage were cultured with uranyl nitrate hexahydrate (UN) at a final concentration of uranium (U) of 26, 52 and 104 {mu}gU/ml. At 24 hs of culture, the embryos at the 2 cell stage, were put in new wells with the same concentrations of U as the previous day, until the end of the period of incubation at 72 hs. At 72 hs of culture, 87% of the original one cell embryos were at morula stage, and in those cultivated with uranium, the percentage decreased significantly to 77; 63.24 and 40.79% respectively for the different U concentrations. Those embryos that exhibited a normal morphology, were selected and fixed on slides. The number of cells per embryo was evaluated in Giemsa stained preparations. The DNA content was evaluated cytophotometrically in Feulgen stained nuclei. The number of cells decreased significantly from 20,3 {+-} 5.6 in the control to 19 {+-} 6; 14 {+-} 3 and 13.9 {+-} 5.6 for the different concentrations. All the embryos evaluated showed one easy recognizable polar body, which was used a haploid indicator (n). The content of DNA was measured in a total of 20 control embryos and 16 embryos cultivated with UN. In control embryos, 92,7% of the nuclei presented a normal ploidy from 2n to 4n, 2,9% nuclei were hypoploid and 4,4% were hyperploid. The percentage of hypoploid nuclei rose in a dose-dependent fashion to 3.45; 44.45 and 50.34% respectively for the embryos cultured at the different U concentrations. The results indicate that U is embryotoxic, that its effects are dose dependent at the concentrations used in this study and that even those embryos that show a normal morphology, can be genetically affected. We show that the model employed is extremely sensitive. It is possible to use the preimplantation embryos, as a model to test the effect of possibly mutagenic agents of the nuclear industry

Surgical manipulation of mammalian embryos in vitro.

Science.gov (United States)

Naruse, I; Keino, H; Taniguchi, M

1997-04-01

Whole-embryo culture systems are useful in the fields of not only embryology but also teratology, toxicology, pharmacology, and physiology. Of the many advantages of whole-embryo culture, we focus here on the surgical manipulation of mammalian embryos. Whole-embryo culture allows us to manipulate mammalian embryos, similarly to fish, amphibian and avian embryos. Many surgical experiments have been performed in mammalian embryos in vitro. Such surgical manipulation alters the destiny of morphogenesis of the embryos and can answer many questions concerning developmental issues. As an example of surgical manipulation using whole-embryo culture systems, one of our experiments is described. Microsurgical electrocauterization of the deep preaxial mesodermal programmed cell death zone (fpp) in the footplate prevented the manifestation of polydactyly in genetic polydactyly mouse embryos (Pdn/Pdn), in which fpp was abolished.

Chromosome fragility at FRAXA in human cleavage stage embryos at risk for fragile X syndrome.

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Verdyck, Pieter; Berckmoes, Veerle; De Vos, Anick; Verpoest, Willem; Liebaers, Inge; Bonduelle, Maryse; De Rycke, Martine

2015-10-01

Fragile X syndrome (FXS), the most common inherited intellectual disability syndrome, is caused by expansion and hypermethylation of the CGG repeat in the 5' UTR of the FMR1 gene. This expanded repeat, also known as the rare fragile site FRAXA, causes X chromosome fragility in cultured cells from patients but only when induced by perturbing pyrimidine synthesis. We performed preimplantation genetic diagnosis (PGD) on 595 blastomeres biopsied from 442 cleavage stage embryos at risk for FXS using short tandem repeat (STR) markers. In six blastomeres, from five embryos an incomplete haplotype was observed with loss of all alleles telomeric to the CGG repeat. In all five embryos, the incomplete haplotype corresponded to the haplotype carrying the CGG repeat expansion. Subsequent analysis of additional blastomeres from three embryos by array comparative genomic hybridization (aCGH) confirmed the presence of a terminal deletion with a breakpoint close to the CGG repeat in two blastomeres from one embryo. A blastomere from another embryo showed the complementary duplication. We conclude that a CGG repeat expansion at FRAXA causes X chromosome fragility in early human IVF embryos at risk for FXS. © 2015 Wiley Periodicals, Inc.

Analysis of structural and numerical chromosomal aberrations at the first and second mitosis after X irradiation of two-cell mouse embryos

International Nuclear Information System (INIS)

Weissenborn, U.; Streffer, C.

1989-01-01

Two-cell mouse embryos were X-irradiated in the late G2 phase in vivo. The first and second postradiation mitoses were analyzed for chromosomal anomalies. The majority of structural aberrations visible at the first mitosis after irradiation were chromatid breaks and chromatid gaps; only a few interchanges and dicentrics were observed. The aberration frequency resulted in a dose-effect relationship which was well described by a linear model. At the second mitosis 29% of the structural aberrations of the first mitosis were counted; the aberration quality changed only slightly. It is discussed whether these aberrations are to be considered new, derived, or unchanged transmitted aberrations. Contrary to the results obtained after irradiation of one-cell embryos, little chromosome loss was induced by radiation in two-cell embryos

Paternal and maternal factors in preimplantation embryogenesis: interaction with the biochemical environment.

Science.gov (United States)

Ménézo, Yves J R

2006-05-01

Paternal effect on embryonic development occurs as early as fertilization. Incorrect formation of the spermatozoon due to centrosome defects and abnormal concentrations of any components involved in the activation process lead to failure immediately or in the subsequent cell cycles. Sperm chromosomal abnormalities result in early embryo developmental arrests. Generally poor spermatozoa lead to poor blastocyst formation. Sperm DNA fragmentation may impair even late post-implantation development. The DNA repair capacity of the oocytes is of major importance. Early preimplantation development, i.e. until maternal to zygotic transition, is maternally driven. Maternal mRNAs and proteins are of major importance, as there is an unavoidable turnover of these reserves. Polyadenylation of these mRNAs is precisely controlled, in order to avoid too early or too late transcription and translation of the housekeeping genes. An important set of maternal regulations, such as DNA stability, transcriptional regulation and protection against oxidative stress, are impaired by age. The embryo biochemical endogenous pool is very important and may depend upon the environment, i.e. the culture medium. Paternal, maternal and environmental factors are unavoidable parameters; they become evident when age impairs oocyte quality.

Research progression on preimplantation genetic diagnosis and screening%胚胎植入前遗传学诊断和筛查的研究进展

Institute of Scientific and Technical Information of China (English)

刘茜桐; 田莉; 师娟子

2016-01-01

胚胎植入前遗传学诊断（ PGD）和筛查（ PGS）是近年来发展的植入前遗传学检测（ PGT）方法。 PGD主要适用于父母携带基因突变或染色体平衡易位，通过体外受精，在胚胎移植前检测特定的突变以及非平衡染色体异常是否传递到卵子或胚胎。 PGS是运用相同的检测方法检测胚胎染色体非整倍性，通过移植正常的胚胎从而提高妊娠率。 PGD／PGS相关检测技术发展日新月异，传统FISH技术逐渐被取代，更多的新技术也在研发中。但是，PGD／PGS仍存在费用昂贵，无法检测所有胚胎异常等不足之处。该文综述PGD／PGS相关进展和PGD／PGS所存在的问题。%Preimplantation genetic diagnosis ( PGD) and preimplantation genetic screening ( PGS) are recently developed preimplantation genetic testing ( PGT) .PGD is applied when one or both genetic parents carry a gene mutation or a balanced chromosomal rearrangement and testing is performed to determine whether that specific mutation or an unbalanced chromosomal complement has been transmitted to the oocyte or embryo .PGS uses the same method for detecting embryo chromosomal aneuploidy in order to improve pregnancy rate .With the development of new technology related with PGD /PGS, FISH is gradually being replaced and new methods are under research .However , PGD/PGS is expensive and can not detect all abnormalities of the embryo .This article reviewed the advancement and shortcomings of PGD/PGS.

Novel Approach of Differential Staining to Detect Necrotic Cells in Preimplantation Embryos

Directory of Open Access Journals (Sweden)

Mohammad Hossein Nasr Esfahani

2007-01-01

Full Text Available Background: This novel approach describes a rapid and simple method for identification of necrotic vs. viable cells within a mammalian blastocyst.Materials and Methods: Hatched bovine blastocysts produced in vitro were first incubated for 30 min in pre-equilibrated culture medium containing propidium iodide (PI; 300μg/ml and bisbenzimide (Hoechst: H33342; 5μg/ml fluorescent dyes. Embryos were then freed from residual dyes by thoroughly washing in warm phosphate buffer saline free of calcium and magnesium (PBS-, fixed in 2.5% glutharaldehyde and washed again in PBS- . Stained embryos afterwards were mounted in a drop of glycerol over a microscopic slide. Prepared samples were examined under an epifluorescent microscope using the same excitation wavelength (330-385nm and barrier filter (400nm to distinguish necrosed vs. viable blastomers as being appeared in red and blue, respectively.Results: Obtained results showed that in cells with altered cell membrane such as late apoptotic or necrotic cells, PI and H33342 readily enter through the cytoplasmic barriers and so the chromatin materials are stained by both, but since PI quenches bisbenzimide fluorescence, necrotic blastomeres are seen in red to pinky red, while live cells are seen just as blue.Conclusion: Obtained results clearly indicated that this novel approach can be used as a simple, feasible and precise method for every embryology lab and with all the mammalian blastocysts produced either in vitro or in vivo. The basic assay can be completed in 60 min, and valuable and reliable information can be obtained about the quality of the embryos.

Establishment of a Simple and Useful Way for Preimplantation Genetic Diagnosis of Chromosomal Diseases

Institute of Scientific and Technical Information of China (English)

LUO Haining; ZHU Guijin; LIU Qun; CHEN Wen; LI Zhou

2007-01-01

In order to establish a simple and useful way for preimplantation genetic diagnosis (PGD)of chromosomal diseases in general IVF laboratory, the methods that are most commonly used in the embryo biopsy, fixation of blastomere and fluorescence in situ hybridization were compared. The three aspects of PGD were analyzed respectively. There was no significant difference in further development capacity of embryos between mechanical (79.7%) and chemical biopsy group (78.6%)(P＞0.05). In this study, more cells were successfully fixed with the Tween/HCL method (93.8%) than with the methanol/acetic acid method (80.5%, P＜0.05). There was no significant difference in cytoplasm remains between methanol/acetic acid method and Tween/HCL method (P＞0.05). The hybridization efficiency of fluorescence in situ hybridization was 89.5% in successive denaturation method and 90.9% in codenaturation method with the difference being not significant (P＞0.05). In conclusion, the mechanical or chemical method, Tween/HCL fixation method and codenaturation fluorescence in situ hybridization method can constitute a simple and useful way for PGD of chromosomal diseases.

[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.

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...

Clinical outcomes for couples containing a reciprocal chromosome translocation carrier without preimplantation genetic diagnosis.

Science.gov (United States)

Yin, Biao; Zhu, Yuanchang; Wu, Tonghua; Shen, Shuqiu; Zeng, Yong; Liang, Desheng

2017-03-01

To evaluate the pregnancy outcomes of couples containing a carrier of a reciprocal chromosome translocation (RCT) after assisted reproductive technology without preimplantation genetic diagnosis. A retrospective study was performed using data for couples with an RCT carrier and control couples with a normal karyotype (1:4 ratio) who underwent assisted reproductive technology cycles at a Chinese fertility center in 2010-2011. The embryos were fertilized via in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Only the first pick-up cycles were used for analysis. Clinical variables were compared. Compared with the control group (n=164), the RCT group (n=41) had a marginally lower clinical pregnancy rate (46.3% [19/41] vs 54.3% [89/164]), implantation rate (21.7% [23/106] vs 26.9% [118/438]), multiple-gestation pregnancy rate (21.1% [4/19] vs 32.6% [29/89]), and delivery rate (36.6% [15/41] vs 47.6% [78/164]), whereas the spontaneous abortion rate was slightly higher (21.1% [4/19] vs 12.4% [11/89]). However, none of these differences were significant. The clinical outcomes for RCT carriers were acceptable after IVF/ICSI without performing preimplantation genetic diagnosis, indicating that this approach might comprise a feasible alternative fertility treatment for RCT carriers. © 2016 International Federation of Gynecology and Obstetrics.

Derivation and characterization of novel nonhuman primate embryonic stem cell lines from in vitro-fertilized baboon preimplantation embryos.

Science.gov (United States)

Chang, Tien-Cheng; Liu, Ya-Guang; Eddy, Carlton A; Jacoby, Ethan S; Binkley, Peter A; Brzyski, Robert G; Schenken, Robert S

2011-06-01

The development of nonhuman primate (NHP) embryonic stem cell (ESC) models holds great promise for cell-mediated treatment of debilitating diseases and to address numerous unanswered questions regarding the therapeutic efficacy of ESCs while supplanting ethical considerations involved with human studies. Here we report successful establishment and characterization of 3 novel baboon (Papio cynocephalus) ESC lines from the inner cell mass of intracytoplasmic sperm injection-derived blastocysts. Embryos were cultured in an improved baboon embryo in vitro culture protocol. The inner cell mass of blastocyst was laser-dissected and plated on mouse embryonic fibroblast feeder cell monolayer in the NHP ESC culture medium. Three cell lines with characteristic ESC morphology have been cultured through an extended period (>14 months), with 2 male cell lines (UT-1 and -2) and 1 female cell line (UT-3) displaying normal baboon karyotypes. Reverse transcription-polymerase chain reaction analysis confirmed that all 3 lines express primate ESC pluripotency markers, including OCT-4, NANOG, SOX-2, TERT, TDGF, LEFTYA, and REX-1. All 3 lines demonstrated positive immunocytochemical staining for OCT-4, stage-specific embryonic antigen-3, stage-specific embryonic antigen-4, TRA-1-60, and TRA-1-81. Baboon ESCs injected into NOD/SCID mice formed teratomas with all 3 germ layers. In addition, embryoid body-like spherical structures were derived and initial outgrowth was observed when embedded into extracellular matrix Matrigel. The ESC lines established in this NHP model have the potential to extend our knowledge in the fields of developmental biology, regenerative medicine, and future applications, including preclinical safety assessment of in vivo stem cell therapy.

[Preimplantation genetic diagnosis and monogenic inherited eye diseases].

Science.gov (United States)

Hlavatá, L; Ďuďáková, Ľ; Trková, M; Soldátová, I; Skalická, P; Kousal, B; Lišková, P

Preimplantation genetic diagnosis (PGD) is an established application of genetic testing in the context of in vitro fertilization. PGD is an alternative method to prenatal diagnosis which aims to prevent the transmission of an inherited disorder to the progeny by implanting only embryos that do not carry genetic predisposition for a particular disease. The aim of this study is to provide an overview of eye disorders for which PGD has been carried out. The European literature search focused on best practices, ethical issues, risks and results of PGD for inherited eye disorders. PGD is performed for a number of ocular disorders; a prerequisite for its application is however, the knowledge of a disease-causing mutation(s). The main advantage of this method is that the couple is not exposed to a decision of whether or not to undergo an abortion. Qualified counselling must be provided prior to the PGD in order to completely understand the risk of disability in any child conceived, consequences of disease manifestation, and advantages as well as limitations of this method. In the group of non-syndromic eye diseases and diseases in which ocular findings dominate, PGD has been performed in European countries for aniridia, choroideremia, congenital fibrosis of extraocular muscles, Leber congenital amaurosis, ocular albinism, retinitis pigmentosa, X-linked retinoschisis, Stargardt disease, blepharophimosis-ptosis-inverse epicanthus syndrome and retinoblastoma. Sexing for X-linked or mitochondrial diseases has been carried out for blue cone monochromatism, choroideremia, familial exudative vitreoretinopathy, Leber hereditary optic neuropathy, macular dystrophy (not further specified), Norrie disease, X-linked congenital stationary night blindness, X-linked retinoschisis and nystagmus (not further specified). In recent years, there has been an increase in potential to use PGD. The spectrum of diseases for this method has widened to include severe inherited eye diseases

Live embryo imaging to follow cell cycle and chromosomes stability after nuclear transfer.

Science.gov (United States)

Balbach, Sebastian T; Boiani, Michele

2015-01-01

Nuclear transfer (NT) into mouse oocytes yields a transcriptionally and functionally heterogeneous population of cloned embryos. Most studies of NT embryos consider only embryos at predefined key stages (e.g., morula or blastocyst), that is, after the bulk of reprogramming has taken place. These retrospective approaches are of limited use to elucidate mechanisms of reprogramming and to predict developmental success. Observing cloned embryo development using live embryo cinematography has the potential to reveal otherwise undetectable embryo features. However, light exposure necessary for live cell cinematography is highly toxic to cloned embryos. Here we describe a protocol for combined bright-field and fluorescence live-cell imaging of histone H2b-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This protocol, which can be adapted to observe other reporters such as Oct4-GFP or Nanog-GFP, allowed us to quantitatively analyze cleavage kinetics of cloned embryos.