Sample records for aneuploid blastocysts identified

  1. Metabolomic profiling and genomic analysis of wheat aneuploid lines to identify genes controlling biochemical pathways in mature grain.

    Francki, Michael G; Hayton, Sarah; Gummer, Joel P A; Rawlinson, Catherine; Trengove, Robert D


    Metabolomics is becoming an increasingly important tool in plant genomics to decipher the function of genes controlling biochemical pathways responsible for trait variation. Although theoretical models can integrate genes and metabolites for trait variation, biological networks require validation using appropriate experimental genetic systems. In this study, we applied an untargeted metabolite analysis to mature grain of wheat homoeologous group 3 ditelosomic lines, selected compounds that showed significant variation between wheat lines Chinese Spring and at least one ditelosomic line, tracked the genes encoding enzymes of their biochemical pathway using the wheat genome survey sequence and determined the genetic components underlying metabolite variation. A total of 412 analytes were resolved in the wheat grain metabolome, and principal component analysis indicated significant differences in metabolite profiles between Chinese Spring and each ditelosomic lines. The grain metabolome identified 55 compounds positively matched against a mass spectral library where the majority showed significant differences between Chinese Spring and at least one ditelosomic line. Trehalose and branched-chain amino acids were selected for detailed investigation, and it was expected that if genes encoding enzymes directly related to their biochemical pathways were located on homoeologous group 3 chromosomes, then corresponding ditelosomic lines would have a significant reduction in metabolites compared with Chinese Spring. Although a proportion showed a reduction, some lines showed significant increases in metabolites, indicating that genes directly and indirectly involved in biosynthetic pathways likely regulate the metabolome. Therefore, this study demonstrated that wheat aneuploid lines are suitable experimental genetic system to validate metabolomics-genomics networks. PMID:26032167

  2. Karyomapping identifies second polar body DNA persisting to the blastocyst stage: implications for embryo biopsy.

    Ottolini, Christian S; Rogers, Shaun; Sage, Karen; Summers, Michael C; Capalbo, Antonio; Griffin, Darren K; Sarasa, Jonas; Wells, Dagan; Handyside, Alan H


    Blastocyst biopsy is now widely used for both preimplantation genetic screening (PGS) and preimplantation genetic diagnosis (PGD). Although this approach yields good results, variable embryo quality and rates of development remain a challenge. Here, a case is reported in which a blastocyst was biopsied for PGS by array comparative genomic hybridization on day 6 after insemination, having hatched completely. In addition to a small trophectoderm sample, excluded cell fragments from the subzonal space from this embryo were also sampled. Unexpectedly, the array comparative genomic hybridization results from the fragments and trophectoderm sample were non-concordant: 47,XX,+19 and 46,XY, respectively. DNA fingerprinting by short tandem repeat and amelogenin analysis confirmed the sex chromosome difference but seemed to show that the two samples were related but non-identical. Genome-wide single nucleotide polymorphism genotyping and karyomapping identified that the origin of the DNA amplified from the fragments was that of the second polar body corresponding to the oocyte from which the biopsied embryo developed. The fact that polar body DNA can persist to the blastocyst stage provides evidence that excluded cell fragments should not be used for diagnostic purposes and should be avoided when performing embryo biopsies as there is a risk of diagnostic errors. PMID:26380865

  3. Blastocyst Transfer



    ### Method: Blastocyst transfer is usually performed 24 hours after aggregation when the morulae have become expanded blastocysts and on the same day as injection. A little time is given between injection and transfer to allow blastocysts to re-expand. **The Recipient** Careful selection of the recipient is most important as the pups are the end result of a lot of hard work. Two strains of mice are used:RB Swiss and (CBA*C57BL6/J)f1. RB Swiss are quiet and make excellent mothers ...

  4. Human embryonic stem cells as models for aneuploid chromosomal syndromes.

    Biancotti, Juan-Carlos; Narwani, Kavita; Buehler, Nicole; Mandefro, Berhan; Golan-Lev, Tamar; Yanuka, Ofra; Clark, Amander; Hill, David; Benvenisty, Nissim; Lavon, Neta


    Syndromes caused by chromosomal aneuploidies are widely recognized genetic disorders in humans and often lead to spontaneous miscarriage. Preimplantation genetic screening is used to detect chromosomal aneuploidies in early embryos. Our aim was to derive aneuploid human embryonic stem cell (hESC) lines that may serve as models for human syndromes caused by aneuploidies. We have established 25 hESC lines from blastocysts diagnosed as aneuploid on day 3 of their in vitro development. The hESC lines exhibited morphology and expressed markers typical of hESCs. They demonstrated long-term proliferation capacity and pluripotent differentiation. Karyotype analysis revealed that two-third of the cell lines carry a normal euploid karyotype, while one-third remained aneuploid throughout the derivation, resulting in eight hESC lines carrying either trisomy 13 (Patau syndrome), 16, 17, 21 (Down syndrome), X (Triple X syndrome), or monosomy X (Turner syndrome). On the basis of the level of single nucleotide polymorphism heterozygosity in the aneuploid chromosomes, we determined whether the aneuploidy originated from meiotic or mitotic chromosomal nondisjunction. Gene expression profiles of the trisomic cell lines suggested that all three chromosomes are actively transcribed. Our analysis allowed us to determine which tissues are most affected by the presence of a third copy of either chromosome 13, 16, 17 or 21 and highlighted the effects of trisomies on embryonic development. The results presented here suggest that aneuploid embryos can serve as an alternative source for either normal euploid or aneuploid hESC lines, which represent an invaluable tool to study developmental aspects of chromosomal abnormalities in humans. PMID:20641042

  5. Epigenetic Dysregulation Observed in Monosomy Blastocysts Further Compromises Developmental Potential

    Denomme, Michelle M.; McCallie, Blair R.; Parks, Jason C.; Schoolcraft, William B.; Katz-Jaffe, Mandy G.


    Epigenetic mechanisms such as DNA methylation regulate genomic imprinting and account for the distinct non-equivalence of the parental genomes in the embryo. Chromosomal aneuploidy, a major cause of infertility, distorts this highly regulated disparity by the presence or absence of chromosomes. The implantation potential of monosomy embryos is negligible compared to their trisomy counterparts, yet the cause for this is unknown. This study investigated the impact of chromosomal aneuploidy on strict epigenetically regulated domains, specifically imprinting control regions present on aneuploid chromosomes. Donated cryopreserved human IVF blastocysts of transferable quality, including trisomy 15, trisomy 11, monosomy 15, monosomy 11, and donor oocyte control blastocysts were examined individually for DNA methylation profiles by bisulfite mutagenesis and sequencing analysis of two maternally methylated imprinting control regions (ICRs), SNRPN (15q11.2) and KCNQ1OT1 (11p15.5), and one paternally methylated imprinting control region, H19 (11p15.5). Imprinted genes within the regions were also evaluated for transcript abundance by RT-qPCR. Overall, statistically significant hypermethylated and hypomethylated ICRs were found in both the trisomy and monosomy blastocysts compared to controls, restricted only to the chromosome affected by the aneuploidy. Increased expression was observed for maternally-expressed imprinted genes in trisomy blastocysts, while a decreased expression was observed for both maternally- and paternally-expressed imprinted genes in monosomy blastocysts. This epigenetic dysregulation and altered monoallelic expression observed at imprinting control regions in aneuploid IVF embryos supports euploid embryo transfer during infertility treatments, and may specifically highlight an explanation for the compromised implantation potential in monosomy embryos. PMID:27271036

  6. The mammalian blastocyst.

    Frankenberg, Stephen R; de Barros, Flavia R O; Rossant, Janet; Renfree, Marilyn B


    The blastocyst is a mammalian invention that carries the embryo from cleavage to gastrulation. For such a simple structure, it exhibits remarkable diversity in its mode of formation, morphology, longevity, and intimacy with the uterine endometrium. This review explores this diversity in the light of the evolution of viviparity, comparing the three main groups of mammals: monotremes, marsupials, and eutherians. The principal drivers in blastocyst evolution were loss of yolk coupled with evolution of the placenta. An important outcome of blastocyst development is differentiation of two extraembryonic lineages (trophoblast and hypoblast) that contribute to the placenta. While in many species trophoblast segregation is often coupled with blastocyst formation, in marsupials and at least some Afrotherians, these events do not coincide. Thus, many questions regarding the conservation of molecular mechanisms controlling these events are of great interest but currently unresolved. For further resources related to this article, please visit the WIREs website. PMID:26799266

  7. Blastocyst Morphology Holds Clues Concerning The Chromosomal Status of The Embryo

    Rita de Cassia Savio Figueira


    Full Text Available Background: Embryo morphology has been proposed as an alternative marker of chromosomal status. The objective of this retrospective cohort study was to investigate the association between the chromosomal status on day 3 of embryo development and blastocyst morphology. Materials and Methods: A total of 596 embryos obtained from 106 cycles of intracytoplasmic sperm injection (ICSI followed by preimplantation genetic aneuploidy screening (PGS were included in this retrospective study. We evaluated the relationship between blastocyst morphological features and embryonic chromosomal alteration. Results: Of the 564 embryos with fluorescent in situ hybridization (FISH results, 200 reached the blastocyst stage on day 5 of development. There was a significantly higher proportion of euploid embryos in those that achieved the blastocyst stage (59.0% compared to embryos that did not develop to blastocysts (41.2% on day 5 (P<0.001. Regarding blastocyst morphology, we observed that all embryos that had an abnormal inner cell mass (ICM were aneuploid. Embryos with morphologically normal ICM had a significantly higher euploidy rate (62.1%, P<0.001. As regards to the trophectoderm (TE morphology, an increased rate of euploidy was observed in embryos that had normal TE (65.8% compared to embryos with abnormal TE (37.5%, P<0.001. Finally, we observed a two-fold increase in the euploidy rate in high-quality blastocysts with both high-quality ICM and TE (70.4% compared to that found in low-quality blastocysts (31.0%, P<0.001. Conclusion: Chromosomal abnormalities do not impair embryo development as aneuploidy is frequently observed in embryos that reach the blastocyst stage. A high-quality blastocyst does not represent euploidy of chromosomes 13, 14, 15, 16, 18, 21, 22, X and Y. However, aneuploidy is associated with abnormalities in the ICM morphology. Further studies are necessary to confirm whether or not the transfer of blastocysts with low-quality ICM should be

  8. SNP microarray-based 24 chromosome aneuploidy screening demonstrates that cleavage-stage FISH poorly predicts aneuploidy in embryos that develop to morphologically normal blastocysts.

    Northrop, L E; Treff, N R; Levy, B; Scott, R T


    Although selection of chromosomally normal embryos has the potential to improve outcomes for patients undergoing IVF, the clinical impact of aneuploidy screening by fluorescence in situ hybridization (FISH) has been controversial. There are many putative explanations including sampling error due to mosaicism, negative impact of biopsy, a lack of comprehensive chromosome screening, the possibility of embryo self-correction and poor predictive value of the technology itself. Direct analysis of the negative predictive value of FISH-based aneuploidy screening for an embryo's reproductive potential has not been performed. Although previous studies have found that cleavage-stage FISH is poorly predictive of aneuploidy in morphologically normal blastocysts, putative explanations have not been investigated. The present study used a single nucleotide polymorphism (SNP) microarray-based 24 chromosome aneuploidy screening technology to re-evaluate morphologically normal blastocysts that were diagnosed as aneuploid by FISH at the cleavage stage. Mosaicism and preferential segregation of aneuploidy to the trophectoderm (TE) were evaluated by characterization of multiple sections of the blastocyst. SNP microarray technology also provided the first opportunity to evaluate self-correction mechanisms involving extrusion or duplication of aneuploid chromosomes resulting in uniparental disomy (UPD). Of all blastocysts evaluated (n = 50), 58% were euploid in all sections despite an aneuploid FISH result. Aneuploid blastocysts displayed no evidence of preferential segregation of abnormalities to the TE. In addition, extrusion or duplication of aneuploid chromosomes resulting in UPD did not occur. These findings support the conclusion that cleavage-stage FISH technology is poorly predictive of aneuploidy in morphologically normal blastocysts. PMID:20479065

  9. ET spontaneous hatched blastocyst

    Teplá, O.; Mrázek, M.; Mayer, Z.; Cibulková, E.; Králová, Alena; Strnadová, J.; Hulvert, J.

    Praha : BTO-N, 2009. s. 30. [XV.Symposium českých reprodukčních immunologů s mezinárodní účastí. 29.05.2009-31.05.2009, Žďár nad Sázavou] Institutional research plan: CEZ:AV0Z50520701 Keywords : embryo transfer * blastocyst * hatching Subject RIV: FK - Gynaecology, Childbirth

  10. Identifying MicroRNA and mRNA Expression Profiles in Embryonic Stem Cells Derived from Parthenogenetic, Androgenetic and Fertilized Blastocysts

    Xiang-Shun Cui; Xing-Hui Shen; Shao-Chen Sun; Sun-Wha Cho; Young-Tae Heo; Yong-Kook Kang; Teruhiko Wakayama


    MicroRNAs (miRNAs) are a class of highly conserved small non-coding RNA molecules that play a pivotal role m several cellular functions.In this study,miRNA and messenger RNA (mRNA) profiles were examined by Illumina microarray in mouse embryonic stem cells (ESCs) derived from parthenogenetic,androgenetic,and fertilized blastocysts.The global analysis of miRNA-mRNA target pairs provided insight into the role of miRNAs in gene expression.Results showed that a total of 125 miRNAs and 2394 mRNAs were differentially expressed between androgenetic ESCs (aESCs) and fertilized ESCs (fESCs),a total of 42 miRNAs and 87 mRNAs were differentially expressed between parthenogenetic ESCs (pESCs) and fESCs,and a total of 99 miRNAs and 1788 mRNAs were differentially expressed between aESCs and pESCs.In addition,a total of 575,5 and 376 miRNA-mRNA target pairs were observed in aESCs vs.fESCs,pESCs vs.fESCs,and aESCs vs.pESCs,respectively.Furthermore,15 known imprinted genes and 16 putative uniparentally expressed miRNAs with high expression levels were confirmed by both microarray and real-time RT-PCR.Finally,transfection of miRNA inhibitors was performed to validate the regulatory relationship between putative maternally expressed miRNAs and target mRNAs.Inhibition of miR-880 increased the expression of Peg3,Dyrklb,and Prrg2 mRNA,inhibition of miR-363 increased the expression of Nfat5 and Soatl mRNA,and inhibition of miR-883b-5p increased Nfat5,Tacstd2,and Ppapdcl mRNA.These results warrant a functional study to fully understand the underlying regulation of genomic imprinting in early embryo development.

  11. 陆地棉非整倍体的诱发和鉴定%Induction of Aneuploids and Their Identification in Upland Cotton

    Wang-zhen GUO; Bao-min HU; Cheng-xin YI; Bao-liang ZHOU; Tian-zhen ZHANG; Jia-ju PAN


    @@ Aneuploid lines have been playing an important role in crop genetic studies and breeding utilization. Unfortunately, only several genetic materials of aneuploid can be used in Upland cotton. So far, there are 16 monosomics identified in cotton. In order to fill the gap, we carried on the irradiation research of mature pollen grains with γ-rays at different dosages and further produced F1 generation.

  12. Transfer of spontaneously hatching or hatched blastocyst yields better pregnancy rates than expanded blastocyst transfer

    Natachandra M Chimote; Nishad N Chimote; Nath, Nirmalendu M.; Bindu N Mehta


    Context: Blastocyst stage embryo transfer (ET) has become routine practice in recent years. However, probably due to limitations of assisted hatching techniques, expanded blastocyst transfer (EBT) is still the preferred mode. Inexplicably, not much consideration has been given to spontaneously hatching/hatched blastocyst transfer (SHBT). Aim: This study aimed to investigate developmental potential of spontaneously hatching/hatched blastocyst against EBT in in vitro fertilization (IVF) cycles....

  13. Early cleavages influence the molecular and the metabolic pattern of individually cultured bovine blastocysts.

    Milazzotto, Marcella Pecora; Goissis, Marcelo Demarchi; Chitwood, James Lee; Annes, Kelly; Soares, Carlos Alexandre; Ispada, Jéssica; Assumpção, Mayra Elena Ortiz Ávila; Ross, Pablo Juan


    Embryo morphokinetics suggests that the timing of the first embryonic cell divisions may predict the developmental potential of an embryo; however, correlations between embryonic morphokinetics and physiology are not clear. Here, we used RNA sequencing to determine the gene expression profile of in vitro-produced early- and late-dividing bovine embryos and their respective blastocysts, and compared these profiles to in vivo-produced blastocysts to identify differentially expressed genes (DEGs). Principal component analysis revealed that fast- and slow-dividing embryos possess similar transcript abundance over the first cleavages. By the blastocyst stage, however, more DEGs were observed between the fast- and slow-dividing embryo groups, whereas blastocysts from the slow-dividing group were more similar to in vivo-produced blastocysts. Gene ontology enrichment analysis showed that the slow-dividing and in vivo-produced blastocysts shared biological processes related to groups of up- or down-regulated genes when compared to the fast-dividing blastocysts. Based on these DEG results, we characterized the relationship between developmental kinetics and energy metabolism of in vitro-produced bovine embryos. Embryos from fast- and slow-dividing groups exhibited different pyruvate and lactate metabolism at 22 hr post-in vitro culture (hpc), glucose consumption at 96 hpc, and glutamate metabolism at 168 hpc. Glycogen storage was similar between cleavage-stage and morulae groups, but was higher in the blastocysts of the slow-dividing group. On the other hand, blastocysts of the fast-dividing group had a higher concentration of lipids. Taken together, these data identify transcriptomic and metabolic differences between embryos with different morphokinetics, suggesting that sorting embryos based on cleavage speed may select for different metabolic patterns. Mol. Reprod. Dev. 83: 324-336, 2016. © 2016 Wiley Periodicals, Inc. PMID:26822777

  14. Transfer of spontaneously hatching or hatched blastocyst yields better pregnancy rates than expanded blastocyst transfer

    Natachandra M Chimote


    Full Text Available Context: Blastocyst stage embryo transfer (ET has become routine practice in recent years. However, probably due to limitations of assisted hatching techniques, expanded blastocyst transfer (EBT is still the preferred mode. Inexplicably, not much consideration has been given to spontaneously hatching/hatched blastocyst transfer (SHBT. Aim: This study aimed to investigate developmental potential of spontaneously hatching/hatched blastocyst against EBT in in vitro fertilization (IVF cycles. Settings and Design: Prospective study of 146 women undergoing their first IVF- ET cycle. SUBJECTS AND Methods: On the basis of blastocyst status, women were classified into SHBT and EBT groups. Intracytoplasmic sperm injection cycles were excluded to remove male factor bias. Implantation rate (IR, clinical pregnancy rate, and live birth rate were the main outcome measures. Statistical Analysis: Graph-pad Prism 5 statistical package. Results: SHBT group showed significantly higher blastocyst formation rate (53.3 ± 17.5 vs. 43.1 ± 14.5%, P = 0.0098, top-quality blastocysts (71.8 vs. 53.7%, P = 0.0436, IR (43.6 vs. 27.9%, P = 0.0408, pregnancy rate (59.4 vs. 45.1%, P = 0.0173, and live birth rate (36.8 vs. 22.8%, P = 0.003 compared to EBT group. Multiple pregnancy rates remained comparable between the two groups. Implantation correlated strongly with top-quality blastocysts (Pearson, r = 0.4441 in SHBT group, while the correlation was nonsignificant in EBT group. Conclusion: Extending culture of expanded blastocysts by a few hours to allow transfer of spontaneously hatching/hatched blastocysts gives higher implantation and pregnancy rates with no added risk of multiple gestations. Spontaneously hatching/hatched blastocysts have a better potential to implant and develop into a positive pregnancy.

  15. Flow cytofluorometric evidence for the differential radioresponsiveness of aneuploid and diploid cervix tumours

    348 patients have been included in the study over a 54-month period. Data on these patients have shown that DNA aneuploid tumours are significantly more radioresponsive than diploid cervix tumours. Analysis of the data on 213 patients with a minimum follow-up time of 15 months has, however, failed to show an overall more favourable prognosis conferred by tumour DNA aneuploidy. Analysis of the relationship between clinical stage and disease state and tumour DNA ploidy, however, suggests that (1) aneuploid tumours metastasize to distant sites at an earlier stage of the disease than diploid tumours and (2) local recurrence rates for diploid tumours, in late stage disease, are double those for aneuploid tumours. Improved staining procedures, and instrument modification, has also shown that cervix tumour heterogeneity is of considerably greater frequency than at first appeared to be the case (approximately 75% of DNA aneuploid tumours show heterogeneity). (Auth.)

  16. Making the blastocyst: lessons from the mouse

    Cockburn, Katie; Rossant, Janet


    Mammalian preimplantation development, which is the period extending from fertilization to implantation, results in the formation of a blastocyst with three distinct cell lineages. Only one of these lineages, the epiblast, contributes to the embryo itself, while the other two lineages, the trophectoderm and the primitive endoderm, become extra-embryonic tissues. Significant gains have been made in our understanding of the major events of mouse preimplantation development, and recent discoveri...

  17. A tetraploid intermediate precedes aneuploid formation in yeasts exposed to fluconazole.

    Benjamin D Harrison


    Full Text Available Candida albicans, the most prevalent human fungal pathogen, is generally diploid. However, 50% of isolates that are resistant to fluconazole (FLC, the most widely used antifungal, are aneuploid and some aneuploidies can confer FLC resistance. To ask if FLC exposure causes or only selects for aneuploidy, we analyzed diploid strains during exposure to FLC using flow cytometry and epifluorescence microscopy. FLC exposure caused a consistent deviation from normal cell cycle regulation: nuclear and spindle cycles initiated prior to bud emergence, leading to "trimeras," three connected cells composed of a mother, daughter, and granddaughter bud. Initially binucleate, trimeras underwent coordinated nuclear division yielding four daughter nuclei, two of which underwent mitotic collapse to form a tetraploid cell with extra spindle components. In subsequent cell cycles, the abnormal number of spindles resulted in unequal DNA segregation and viable aneuploid progeny. The process of aneuploid formation in C. albicans is highly reminiscent of early stages in human tumorigenesis in that aneuploidy arises through a tetraploid intermediate and subsequent unequal DNA segregation driven by multiple spindles coupled with a subsequent selective advantage conferred by at least some aneuploidies during growth under stress. Finally, trimera formation was detected in response to other azole antifungals, in related Candida species, and in an in vivo model for Candida infection, suggesting that aneuploids arise due to azole treatment of several pathogenic yeasts and that this can occur during the infection process.

  18. Proteomic Analysis of Human Blastocoel Fluid and Blastocyst Cells

    Linnert Jensen, Pernille; Beck, Hans Christian; Petersen, Jørgen;

    cells of the blastocyst are exposed. The ICM is the starting point for the development of undifferentiated human embryonic stem cells (hESCs), which posses the potential to develop into any cell type present in the adult human body [1,2]. This ability makes hESCs a potential source of cells for...... blastocoel fluid (1-8 nanoliters per blastocyst), has hampered an in-depth study of the human blastocyst proteome. However, recent developments in mass spectrometry-based proteomic techniques allow the identification and characterization of thousands of proteins from low microgram levels of protein extracted......The human blastocyst consists of 100-200 cells that are organized in an outer layer of differentiated trophectoderm (TE) cells lining the blastocyst cavity into which the undifferentiated inner cell mass (ICM) protrudes. The cavity of the blastocyst is filled with blastocoel fluid to which all the...

  19. Association between blastocyst morphology and outcome of single-blastocyst transfer

    Van den Abbeel, Etienne; Balaban, Basak; Ziebe, Søren; Lundin, Kersti; Cuesta, Maria José Gómez; Klein, Bjarke Mirner; Helmgaard, Lisbeth; Arce, Joan-Carles


    prospectively collected in a large multicentre trial. A total of 618 intracytoplasmic sperm injection patients undergoing ovarian stimulation in a gonadotrophin-releasing hormone antagonist cycle with compulsory single-blastocyst transfer on day 5 were included. In the simple logistic regression analysis, all...

  20. Blastocyst development potential of D3 low quality embryos

    Zhen Jing-ran; Sun Zhen-yi; Yu Qi; Deng Cheng-yan; Zhou Yuan-zheng; He Fang-fang


    Objective:To investigate the potential of blastocyst development of D3 low quality embryos for blastocyst culturing and freezing.Methods:Two thousand one hundred and eighty embryos of 398 IVF/ICSI patients' were observed after D3 embryo transfer.The low-quality embryos were cultured by sequential micro-drops and continuously observed blastocyst formation from D3 to D6.Results:(1) A total of 1,546 low-quality embryos were collected,and 426 blastocysts formed in DS-D6.Total blastocyst formation rate (BFR) was 27.56%,of which embryos with grade 6 Ⅲ/Ⅳ were 40.87 % (318/778),5 Ⅰ /Ⅱ 28.85% (30/104),4Ⅰ/Ⅱ 8.88% (16/180),4Ⅲ/IV 19.72% (56/284),2-3 Ⅰ/Ⅱ 3% (6/200).(2) Embryos with more blastomeres had a higher blastocyst formation rate.The lower qualities of embryos resulted in lower blastocyst formation rate.Conclusions:Low-quality embryos still have the potential to develop into blastocysts.Therefore,they should not be discarded on D3.They should be continually cultured to D6 in order to reduce embryo wastage and get good clinical results.

  1. Cytogenetics of semi-fertile triploid and aneuploid intergeneric vine cacti hybrids.

    Tel-Zur, N; Abbo, S; Mizrahi, Y


    Crosses between the diploid Hylocereus polyrhizus, as the female parent, and the tetraploid Selenicereus megalanthus, as the male parent, yielded triploid and aneuploid hybrids. The fruits of these hybrids combined the attractive appearance of Hylocereus fruits with the delicious taste of S. megalanthus fruits. The aim of this work was to assess the fertility and breeding potential of the triploid and aneuploid hybrids with a view to developing an improved vine cactus crop. Pollen mother cells at metaphase I revealed univalents, bivalents, trivalents, and occasionally quadrivalents. Chromosome distribution at anaphase I revealed different classes of chromosome segregation as well as lagging chromosomes. At metaphase II, parallel and tripolar spindles were observed. The occurrence of triads was frequent, whereas dyads were rarely observed. Pollen stainability varied among the clones studied ranging from 9.8% to 18.6%. The diameters of the stained pollen grains varied widely, probably as a result of the number of chromosomes. Despite the allotriploid origin of our hybrids, functional female and male gametes were produced in considerable proportions, most likely as a result of balanced chromosome segregation. The triploid and aneuploid clones studied yielded viable seeds whose number per fruit was strongly dependent on the pollen donor. PMID:15618308

  2. Parthenogenic blastocysts derived from cumulus-free in vitro matured human oocytes.

    Sohyun L McElroy

    Full Text Available BACKGROUND: Approximately 20% of oocytes are classified as immature and discarded following intracytoplasmic sperm injection (ICSI procedures. These oocytes are obtained from gonadotropin-stimulated patients, and are routinely removed from the cumulus cells which normally would mature the oocytes. Given the ready access to these human oocytes, they represent a potential resource for both clinical and basic science application. However culture conditions for the maturation of cumulus-free oocytes have not been optimized. We aimed to improve maturation conditions for cumulus-free oocytes via culture with ovarian paracrine/autocrine factors identified by single cell analysis. METHODOLOGY/PRINCIPAL FINDING: Immature human oocytes were matured in vitro via supplementation with ovarian paracrine/autocrine factors that were selected based on expression of ligands in the cumulus cells and their corresponding receptors in oocytes. Matured oocytes were artificially activated to assess developmental competence. Gene expression profiles of parthenotes were compared to IVF/ICSI embryos at morula and blastocyst stages. Following incubation in medium supplemented with ovarian factors (BDNF, IGF-I, estradiol, GDNF, FGF2 and leptin, a greater percentage of oocytes demonstrated nuclear maturation and subsequently, underwent parthenogenesis relative to control. Similarly, cytoplasmic maturation was also improved as indicated by development to blastocyst stage. Parthenogenic blastocysts exhibited mRNA expression profiles similar to those of blastocysts obtained after IVF/ICSI with the exception for MKLP2 and PEG1. CONCLUSIONS/SIGNIFICANCE: Human cumulus-free oocytes from hormone-stimulated cycles are capable of developing to blastocysts when cultured with ovarian factor supplementation. Our improved IVM culture conditions may be used for obtaining mature oocytes for clinical purposes and/or for derivation of embryonic stem cells following parthenogenesis or nuclear

  3. Effect of genistein on mouse blastocyst development in vitro

    Wen-hsiung CHAN; Hsiang-yu LU; Nion-heng SHIAO


    Aim: To examine the cytotoxic effects of genistein, an isoflavone compound, on early postimplantation embryonic development in vitro. Methods: Mouse blastocysts were incubated in medium with or without genistein (25 or 50 μmol/L) or daidzein (50 μmol/L) for 24 h. Cell proliferation and growth was investigated by dual differential staining, apoptosis was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, and apoptotic or necrotic cells were visualized by Annexin-V and propidium iodide (PI) staining. Implantation and postimplantation development of embryos were measured by in vitro development analysis. Results: TUNEL staining and Annexin-V/PI staining. showed that genistein dose-dependently increased apoptosis in mouse blastocysts, while daidzein, another soy isoflavone, had no such effect. The pretreatment of the blastocysts with genistein caused fewer cells than the control group and this effect was primary in the inner cell mass. The genistein-pretreated blastocysts showed normal levels of implantation on culture dishes in vitro, but significantly fewer genistein-pretreated embryos reached the later stages of embryonic development versus the controls, with many of the former embryos dying at relatively early stages of development. In addition, genistein treatment de-creased the development of morulas into blastocysts, and dietary genistein was found to induce cell apoptosis and decrease cell proliferation in an animal assay model of embryogenesis. Conclusions: Our results collectively indicate that genistein treatment of mouse blastocysts induces apoptosis, decreases cell numbers, retards early postimplantation blastocyst development, and increases early-stage blastocyst death in vitro, while dietary genistein appears to negatively affect mouse embryonic development in vivo by inducing cell apoptosis and inhibiting cell proliferation. These novel findings provide important new insights into the effect of genistein

  4. Proteomic analysis of human blastocoel fluid and blastocyst cells

    Jensen, Pernille; Beck, Hans Christian; Petersen, Jørgen;


    Human embryonic stem cells (hESCs) are derived from the inner cell mass (ICM) of the blastocyst and can differentiate into any cell type in the human body. These cells hold a great potential for regenerative medicine, but to obtain enough cells needed for medical treatment, culture is required on a...... blastocoel fluid, which is in contact with all the cells in the blastocyst, including hESCs. Fifty-three surplus human blastocysts were donated after informed consent, and blastocoel fluid was isolated by micromanipulation. Using highly sensitive nano-high-pressure liquid chromatography-tandem mass...

  5. Choline inhibition of amino acid transport in preimplantation mouse blastocysts

    Addition of 70 mM choline chloride to Brinster's medium (140 mM Na+) inhibited uptake of ∼ 1 μM [3H]glycine, leucine, lysine and alanine in blastocysts by about 50% each during a five-minute incubation period at 370C, whereas 70 mM LiCl, sodium acetate and NaCl or 140 mM mannitol had no effect. They attribute the apparent linear relationship between Gly transport in blastocysts and the square of the [Na+], observed when choline was substituted for Na+ in Brinster's medium, to concomitant, concentration-dependent enhancement and inhibition of transport by Na+ and choline, respectively. As expected, Gly uptake and the [Na+] were linearly related up to 116 mM Na+, when Na+ was replaced with Li+. The rates of Na+-independent Gly and Ala uptake were + or choline replaced Na+. Therefore, neither Li+ nor choline appears to substitute for Na+ in supporting Na+-dependent transport in blastocysts. Na+-independent Leu uptake was 20 times faster than Gly or Ala uptake and appeared to be inhibited by choline in blastocysts since it was about 37% slower when choline instead of Li+ was substituted for Na+. In contrast to blastocysts, choline had no effect on amino acid transport in cleavage-stage mouse embryos. The unexpected sensitivity of transport to choline in blastocysts underscores the importance of testing the effects of this substance when it is used to replace Na+ in new transport studies

  6. Emodin induces embryonic toxicity in mouse blastocysts through apoptosis

    Emodin (1,3,8-trihydroxy-6-methylanthraquinone), a major constituent of rhubarb, has a wide range of therapeutic applications. Previous studies have established that emodin inhibits cell proliferation and induces caspase 3-dependent apoptosis. However, its side-effects, particularly those on embryonic development, have not been well characterized as yet. In the current study, we examined the cytotoxic effects of emodin on mouse embryos at the blastocyst stage, subsequent embryonic attachment and outgrowth in vitro, and in vivo implantation by embryo transfer. Blastocysts treated with 25–75 μM emodin exhibited significantly increased apoptosis and a corresponding decrease in total cell number. Notably, the implantation success rate of blastocysts pretreated with emodin was lower than that of their control counterparts. Moreover, in vitro treatment with 25–75 μM emodin was associated with increased resorption of post-implantation embryos and decreased fetal weight. With the aid of an in vivo mouse model, we showed that consumption of drinking water containing emodin led to apoptosis and decreased cell proliferation, and inhibited early embryonic development to the blastocyst stage. Our findings support a degree of selective inhibition of retinoic acid receptors in blastocysts treated with emodin. In addition, emodin appears to induce injury in mouse blastocysts through intrinsic apoptotic signaling processes to impair sequent embryonic development. These results collectively indicate that emodin has the potential to induce embryonic cytotoxicity.

  7. Prospects for using aneuploids and their derivatives for wheat protein improvement

    The prospects for using the aneuploid technique in studies related to protein improvement in wheat are considered. Chromosomal location of gene(s) governing specific proteins or enzymes, separated by electrophoresis or electrofocusing, can be ascertained using ditelosomics where one arm of a homologous chromosome pair is absent. Reliable quantitative information on chromosomal effects on protein or specific amino-acid content can be obtained only from inter-varietal substitution lines in which a pair of the donor variety chromosomes substitutes for a pair of the recipient variety. Addition or substitution of certain rye chromosomes in wheat is reported to increase lysine of protein conten. Efficiency of mutation experiments, aimed to recover mutants with altered protein characteristics, can be greatly enhanced by treating the monosomic for the dhromosome that is known to carry the gene(s) having such influence. (author)

  8. Chromosome radiosensitivity and kinetics of proliferation of peripheral lymphocytes in individuals with aneuploid karyotype

    Experimentally investigated was the radiosensitivity of chromosomes and the kinetics of the proliferation of peripheral lymphocytes in patients with aneuploid (DS and TS) and normal karyotype irradiated in vitro in the Go stage of the cell cycle. Trisomic lymphocytes were found to proliferate more rapidly in the in vitro culture and to be more sensitive than diploid cell populations. In monosomic lymphocytes in Turner syndrome patients, the proliferation and incidence of chromosomal abberations was similar to the disomic lines in Down's syndrome patients and in Turner syndrome patients, and to that found in persons with a normal karyotype. The results of the experiment show that there is a relationship between the proliferation rate of peripheral lymphocytes cultures in vitro and the radiosensivity of chromosomes. (author). 1 tab., 3 figs., 11 refs

  9. The fate of chrysotile-induced multipolar mitosis and aneuploid population in cultured lung cancer cells.

    Beatriz de Araujo Cortez

    Full Text Available Chrysotile is one of the six types of asbestos, and it is the only one that can still be commercialized in many countries. Exposure to other types of asbestos has been associated with serious diseases, such as lung carcinomas and pleural mesotheliomas. The association of chrysotile exposure with disease is controversial. However, in vitro studies show the mutagenic potential of chrysotile, which can induce DNA and cell damage. The present work aimed to analyze alterations in lung small cell carcinoma cultures after 48 h of chrysotile exposure, followed by 2, 4 and 8 days of recovery in fiber-free culture medium. Some alterations, such as aneuploid cell formation, increased number of cells in G2/M phase and cells in multipolar mitosis were observed even after 8 days of recovery. The presence of chrysotile fibers in the cell cultures was detected and cell morphology was observed by laser scanning confocal microscopy. After 4 and 8 days of recovery, only a few chrysotile fragments were present in some cells, and the cellular morphology was similar to that of control cells. Cells transfected with the GFP-tagged α-tubulin plasmid were treated with chrysotile for 24 or 48 h and cells in multipolar mitosis were observed by time-lapse microscopy. Fates of these cells were established: retention in metaphase, cell death, progression through M phase generating more than two daughter cells or cell fusion during telophase or cytokinesis. Some of them were related to the formation of aneuploid cells and cells with abnormal number of centrosomes.

  10. Modeling abnormal early development with induced pluripotent stem cells from aneuploid syndromes.

    Li, Wen; Wang, Xianming; Fan, Wenxia; Zhao, Ping; Chan, Yau-Chi; Chen, Shen; Zhang, Shiqiang; Guo, Xiangpeng; Zhang, Ya; Li, Yanhua; Cai, Jinglei; Qin, Dajiang; Li, Xingyan; Yang, Jiayin; Peng, Tianran; Zychlinski, Daniela; Hoffmann, Dirk; Zhang, Ruosi; Deng, Kang; Ng, Kwong-Man; Menten, Bjorn; Zhong, Mei; Wu, Jiayan; Li, Zhiyuan; Chen, Yonglong; Schambach, Axel; Tse, Hung-Fat; Pei, Duanqing; Esteban, Miguel A


    Many human diseases share a developmental origin that manifests during childhood or maturity. Aneuploid syndromes are caused by supernumerary or reduced number of chromosomes and represent an extreme example of developmental disease, as they have devastating consequences before and after birth. Investigating how alterations in gene dosage drive these conditions is relevant because it might help treat some clinical aspects. It may also provide explanations as to how quantitative differences in gene expression determine phenotypic diversity and disease susceptibility among natural populations. Here, we aimed to produce induced pluripotent stem cell (iPSC) lines that can be used to improve our understanding of aneuploid syndromes. We have generated iPSCs from monosomy X [Turner syndrome (TS)], trisomy 8 (Warkany syndrome 2), trisomy 13 (Patau syndrome) and partial trisomy 11;22 (Emanuel syndrome), using either skin fibroblasts from affected individuals or amniocytes from antenatal diagnostic tests. These cell lines stably maintain the karyotype of the donors and behave like embryonic stem cells in all tested assays. TS iPSCs were used for further studies including global gene expression analysis and tissue-specific directed differentiation. Multiple clones displayed lower levels of the pseudoautosomal genes ASMTL and PPP2R3B than the controls. Moreover, they could be transformed into neural-like, hepatocyte-like and heart-like cells, but displayed insufficient up-regulation of the pseudoautosomal placental gene CSF2RA during embryoid body formation. These data support that abnormal organogenesis and early lethality in TS are not caused by a tissue-specific differentiation blockade, but rather involves other abnormalities including impaired placentation. PMID:21949351

  11. Animal Experimentation: In Vivo Hatching Phenomenon of Mouse Blastocysts During Implantation

    Lin, Shau-Ping; Lee, Robert Kuo-Kuang; Tsai, Yuan-Jang


    Purpose: To determine whether the blastocyst zona shedding process within the murine uterine cornus in vivo is due to a global lytic process caused by uterine proteolytic enzyme, or is triggered by the blastocyst hatching process as observed in vitro.

  12. Derivation and transcriptional profiling analysis of pluripotent stem cell lines from rat blastocysts

    Chunliang Li; Ying Yang; Junjie Gu; Yu Ma; Ying Jin


    Embryonic stem (ES) cells are derived from blastocyst-stage embryos. Their unique properties of self-renewal and pluripotency make them an attractive tool for basic research and a potential cell resource for therapy. ES cells of mouse and human have been successfully generated and applied in a wide range of research. However, no genuine ES cell lines have been obtained from rat to date. In this study, we identified pluripotent cells in early rat embryos using specific antibodies against markers of pluripotent stem cells. Subsequently, by modifying the culture medium for rat blastocysts, we derived pluripotent rat ES-llke cell lines, which expressed pluripotency markers and formed embryoid bodies (EBs) in vitro. Importantly, these rat ES-like cells were able to produce teratomas. Both EBs and teratomas contained tissues from all three embryonic germ layers, in addition, from the rat ES-like cells, we derived a rat primitive endoderm (PrE) cell line. Furthermore, we conducted transcriptional profiling of the rat ES-like cells and identified the unique molecular signature of the rat pluripotent stem cells. Our analysis demonstrates that multiple signaling pathways, including the BMP, Activin and roTOR pathways, may be involved in keeping the rat ES-like cells in an undifferentiated state. The cell lines and information obtained in this study will accelerate our understanding of the molecular regulation underlying pluripotency and guide us in the appropriate manipulation of ES cells from a particular species.

  13. Dihydrolipoic Acid Induces Cytotoxicity in Mouse Blastocysts through Apoptosis Processes

    Wen-Hsiung Chan


    Full Text Available α-Lipoic acid (LA is a thiol with antioxidant properties that protects against oxidative stress-induced apoptosis. LA is absorbed from the diet, taken up by cells and tissues, and subsequently reduced to dihydrolipoic acid (DHLA. In view of the recent application of DHLA as a hydrophilic nanomaterial preparation, determination of its biosafety profile is essential. In the current study, we examined the cytotoxic effects of DHLA on mouse embryos at the blastocyst stage, subsequent embryonic attachment and outgrowth in vitro, in vivo implantation by embryo transfer, and early embryonic development in an animal model. Blastocysts treated with 50 μM DHLA exhibited significantly increased apoptosis and a corresponding decrease in total cell number. Notably, the implantation success rates of blastocysts pretreated with DHLA were lower than that of their control counterparts. Moreover, in vitro treatment with 50 μM DHLA was associated with increased resorption of post-implantation embryos and decreased fetal weight. Data obtained using an in vivo mouse model further disclosed that consumption of drinking water containing 100 μM DHLA led to decreased early embryo development, specifically, inhibition of development to the blastocyst stage. However, it appears that concentrations of DHLA lower than 50 μM do not exert a hazardous effect on embryonic development. Our results collectively indicate that in vitro and in vivo exposure to concentrations of DHLA higher than 50 μM DHLA induces apoptosis and retards early pre- and post-implantation development, and support the potential of DHLA to induce embryonic cytotoxicity.

  14. Apoptosis of transgenic cloned and recloned bovine blastocysts

    Guojie Sun; Rong Li; Yunping Dai; Haiping Wang; Lili Wang; Ying Liu; Fangrong Ding; Hengxi Wei; Ning Li


    Apoptosis plays an important role in preimplantation embryonic development. Investigating mechanisms of apoptosis can provide useful information for obtaining high-quality embryos and help to improve cloning efficiency. Here, we investigated the incidence of blastomere apoptosis in transgenic blastocysts generated by somatic cell nuclear transfer (SCNT) and recloning using a terminal deoxy-nucleotidyl transferase-mediated d-UTP nick end-labeling (TUNEL) assay. Transgenic recloned embryos were the second generation SCNT embryos derived from the somatic cells of a transgenic SCNT calf. The blastocyst rate of transgenic SCNT embryos was lower than that of nontransgenic SCNT embryos. The incidence of apoptosis in transgenic SCNT embryos was higher than that of nontrans-genie SCNT embryos. The blastocyst rate and the incidence of apoptosis in transgenic recloned embryos were similar to nontransgenic SCNT embryos. The process of donor cell transfection and drug selection may decrease the developmental capacity of transgenic SCNT embryos. Serial cloning did not influence the developmental capacity of transgenic recloned embryos.

  15. High efficiency production and genomic in situ hybridization analysis of Brassica aneuploids and homozygous plants

    LI; Zaiyun; (李再云); M.; Ceccarelli; S.; Minelli; A.; Contento; LIU; Yan; (刘; 焰); P.; G.; Cionini


    Interspecific and intergeneric hybridizations have been widely used in plant genetics and breeding to construct stocks for genetic analysis and to introduce into crops the desirable traits and genes from their relatives. The intergeneric crosses between Brassica juncea (L.) Czern. & Coss., B. carinata A. Braun and Orychophragmus violaceus (L.) O. E. Schulz were made and the plants produced were subjected to genomic in situ hybridization analysis. The mixoploids from the cross with B. juncea were divided into three groups. The partially fertile mixoploids in the first group (2n = 36-42) mainly contained the somatic cells and pollen mother cells (PMCs) with the 36 chromosomes of B. juncea and additional chromosomes of O. violaceus. The mixoploids (2n = 30-36) in the second and third groups were morphologically quite similar to the mother plants B. juncea and showed nearly normal fertility. The plants in the second group produced the majority of PMCs (2n = 36) with their chromosomes paired and segregated normally, but 1-4 pairs of the O. violaceus chromosomes were included in some PMCs. The plants in the third group produced only PMCs with the 36 B. juncea chromosomes, which were paired and segregated normally. The mixoploids (2n = 29-34) from the cross with B. carinata produced the majority of PMCs (2n = 34) with normal chromosome pairing and segregation, but some plants had some PMCs with 1-3 pairs of chromosomes from O. violaceus and other plants had only PMCs with the B. carinata chromosomes. The Brassica homozygous plants and aneuploids with complete or partial chromosome complements of Brassica parents and various numbers of O. violaceus chromosomes were derived from these progeny plants. The results in this study provided the molecular cytogenetic evidence for the separation of parental genomes which was previously proposed to occur in the hybridizations of these two genera.

  16. Cytotoxic Effects of Dillapiole on Embryonic Development of Mouse Blastocysts in Vitro and in Vivo

    Wen-Hsiung Chan


    We examined the cytotoxic effects of dillapiole, a phenylpropanoid with antileishmanial, anti-inflammatory, antifungal, and acaricidal activities, on the blastocyst stage of mouse embryos, subsequent embryonic attachment and outgrowth in vitro, and in vivo implantation via embryo transfer. Blastocysts treated with 2.5–10 μM dillapiole exhibited a significant increase in apoptosis and corresponding decrease in total cell number. Notably, the implantation success rates of blastocysts pretreate...

  17. Utility of FT-IR imaging spectroscopy in estimating differences between the quality of bovine blastocysts

    Wiecheć, A.; Opiela, J.; Lipiec, E.; Kwiatek, W. M.


    This study was conducted to verify whether the FT-IR spectroscopy and Focal Plane Array (FPA) imaging can be successfully applied to estimate the quality of bovine blastocysts (on the basis of the concentration of nucleic acids and amides). The FT-IR spectra of inner cell mass from blastocysts of three different culture systems were examined. The spectral changes between blastocysts were analyzed in DNA (spectral range of 1240-950 cm-1) and protein amides (1800-1400 cm-1). Blastocyst 1 (BL1-HA) was developed from the fertilized oocyte cultured with low concentration of hialuronian (HA), Blastocyst 2 and 3 were developed from the oocytes cultured in standard conditions. Cleavage stage blastocyst 2 (BL2-SOF) has been cultured in SOF medium while blastocyst 3 (BL3-VERO) was cultured in co-culture with VERO cells. The multivariate statistical analysis (Hierarchical Cluster Analysis - HCA and Principal Component Analysis - PCA) of single cells spectra showed high similarity of cells forming the inner cell mass within single blastocyst. The main variance between the three examined blastocysts was related to amides bands. Differences in the intensities of the amides' peaks between the bovine blastocysts derived from different culture systems indicated that specific proteins reflecting the appearance of a new phenotype were produced. However, for the three blastocysts, the α-helix typical peak was twice more intensive than the β-sheet typical peak suggesting that the differentiation processes had been started. Taking into account the quantitative and qualitative composition of the protein into examined blastocysts, it can be assumed, that the quality of the BL1-HA turned out much more similar to BL3-VERO than to BL2-SOF. FT-IR spectroscopy can be successfully applied in reproductive biology research for quality estimation of oocytes and embryos at varied stages of their development. Moreover this technique proved to be particularly useful when the quantity of the

  18. The outcomes of human blastocyst cryopreservation: vitrification using cryoloop versus slow-freezing method

    Sun Zheng-yi; He Fang-fang; Yu Qi; Deng Cheng-yan; Liu Mei-zhi


    Objective: To compare the outcomes of vitrification using cryoloop with slow-freezing method for human blastocyst cryopreservation.Methods: In IVF-ET cycles, supernumerary embryos were cultured to Day 5 or Day 6, blastocysts were cryopreserved by vitrification using cryoloops or slow-freezing method, then blastocyst survival rate and pregnant rate were compared.Results: 115 vitrified blastocysts from 39 cycles were warmed, 104(90.4%) blastocysts survived. After the transfer of 74 blastocysts in 38 cycles, 28(73.7%) women got clinically pregnant, 2(7.1%) of them suffered from miscarriage, 2 healthy babies were born in 2 deliveries, and the other 24 pregnancies are ongoing. As to slow-freezing method, 87 blastocysts from 21 cycles were thawed, 37(42.5%)of them survived, 28 blastocysts were transferred in 15 cycles, 6(40%) women got clinically pregnant, 1 of them miscarried, 3 healthy babies were born in 2 deliveries, and the other 3 pregnancies are ongoing.Conclusion: The survival rate and pregnant rate of vitrification using cryoloop are superior to traditional slow-freezing method, and the transfer cancel rate is lower than that of slow-freezing method. The miscarriage rate is similar in two methods.

  19. No evidence of association between blastocyst aneuploidy and morphokinetic assessment in a selected population of poor-prognosis patients: a longitudinal cohort study.

    Rienzi, L; Capalbo, A; Stoppa, M; Romano, S; Maggiulli, R; Albricci, L; Scarica, C; Farcomeni, A; Vajta, G; Ubaldi, F M


    Recent studies involving a limited number of patients have indicated a correlation between aneuploidy and various morphokinetic parameters during preimplantation development. The results among different groups, however, have been inconsistent in identifying the parameters that are able to predict chromosomal abnormalities. The aim of this study was to investigate whether aneuploidy of human blastocysts was detectable by specific morphokinetic parameters in patients at increased risk of aneuploidy because of advanced maternal age, history of unsuccessful IVF treatments, or both. A longitudinal cohort study was conducted using 455 blastocysts from 138 patients. Morphokinetic features of preimplantation development were detected in a timelapse incubator. Blastocysts were subjected to trophectodermal biopsy and comprehensive chromosomal screening. Analyses were conducted by means of logistic mixed-effects models, with a subject-specific intercept. No statistical correlation between 16 commonly detected morphokinetic characteristics of in-vitro embryo development and aneuploidy was found. Results suggest that morphokinetic characteristics cannot be used to select euploid blastocysts in poor-prognosis patients regarded as candidates for pre-implantation genetic screening. PMID:25458852

  20. Single blastocyst transfer after ICSI from ejaculate spermatozoa, percutaneous epididymal sperm aspiration (PESA) or testicular sperm extraction (TESE)

    Nilsson, Staffan; Waldenström, Urban; Engström, Ann-Britt; Hellberg, Dan


    Purpose: To investigate the outcome of IVF following intracytoplasmic sperm injection (ICSI) from ejaculate, percutaneous epididymal sperm aspiration (PESA) and testicular sperm extraction (TESE), with subsequent blastocyst culture and single blastocyst transfer.

  1. Induction of matrix metalloproteinase-9 and -2 activity in mouse blastocyst by fibronectin-integrin interaction


    Fibronectin, a major extracellular matrix, plays an important role in embryo implantation by mediating embryo adhesion and outgrowth. In this work, mouse blastocysts produced pro-matrix metalloproteinase-9, pro-matrix metalloproteinase-2 and 64 ku matrix metalloproteinase-2 when they were co-cultured with fibronectin. In contrast, mouse blastocysts did not produce these proteinases without fibronectin. Focal adhesion kinase is a fundamental molecule of integrin signaling pathway and its antisense oligodeoxynucleiotide inhibited blastocyst matrix metalloproteinases expression induced by fibronectin. The results indicated that fibronectin triggered matrix metalloproteinase-9 and -2 expression in mouse blastocyst through its integrin receptors and subsequent signaling pathway, which enhanced the synchronization of blastocyst invasiveness and uterine receptivity and ensured the accuracy of events relative to implantation in timing and spatiality.

  2. Lipid-rich bovine serum albumin improves the viability and hatching ability of porcine blastocysts produced in vitro

    Suzuki, Chie; SAKAGUCHI, Yosuke; Hoshi, Hiroyoshi; Yoshioka, Koji


    The effects of lipid-rich bovine serum albumin (LR-BSA) on the development of porcine blastocysts produced in vitro were examined. Addition of 0.5 to 5 mg/ml LR-BSA to porcine blastocyst medium (PBM) from Day 5 (Day 0 = in vitro fertilization) significantly increased the hatching rates of blastocysts on Day 7 and the total cell numbers in Day-7 blastocysts. When Day-5 blastocysts were cultured with PBM alone, PBM containing LR-BSA, recombinant human serum albumin or fatty acid-free BSA, addit...

  3. IGF-Ⅱ and IGFBP-1 reversely regulate blastocyst implantation in mouse


    Insulin-like growth factor (IGF)-Ⅱ and IGF binding protein (IGFBP)-1, members of IGF family, are important in the cyclic development of endometrium and the blastocyst implantation. In the present study, the indirect immunofluorescence showed that IGF-Ⅱ and IGFBP-1 were specifically expressed at the maternal-fetal interface. In a co-culture system, IGF-Ⅱ significantly enhanced the attachment and outgrowth of the blastocyst on monolayer of uterine epithelial cells, while IGFBP-1 did not affect the blastocyst attachment, but markedly inhibited the blastocyst outgrowth. The results of zymography showed that IGF-Ⅱ enhanced the activities of MMP-2 and MMP-9, while IGFBP-1 did not affect the activities of MMP-2 and MMP-9. In conclusion, the equilibrium between the invasion of trophoblast and the inhibition of deciduas may be regulated by the interaction between the IGF-Ⅱ-expressing invading cytotrophoblast and maternal deciduas-derived IGFBP-1.

  4. Developmental competence and ultrastructural changes of heat-stressed mouse early blastocysts produced in vitro

    Mingzhi WANG


    Full Text Available Mouse early blastocysts were exposed to temperatures of 39℃ and 41℃ for 2 h, respectively, to determine their developmental competence and ultrastructural changes. The results showed that heat stress at 41℃ for 2 h, significantly reduced the percentages of expanded and hatched blastocysts, but not at 39℃ for 2 h. The average cell numbers in expanded blastocysts, which developed from early blastocysts heat-stressed at temperatures of 39℃ and 41℃, were significantly reduced. The average cell numbers in hatched blastocysts subjected to heat stress were no different from those in the control group cultured at 37℃. The mitochondria of the early blastocysts heat-stressed at 39℃ for 2 h, were slightly swollen, but they had recovered after culturing at 37℃ for 2 h. However, the mitochondria in the blastocysts heat stressed at 41℃ for 2 h were severely swollen, and their number increased. The ribosomes shed from the rough endoplasmic reticulum, and the number of secondary lysosomes in the plasma increased. The integrity of desmosomes was disrupted. The space between the nuclear envelope and the perivitelline membrane enlarged. The fibre fraction and the particulate fraction of nucleoli were separated. The heterochromatin in nucleoli was also increased in its quantity. There were some lamellar-shape structures and heterogeneous dense materials exhibiting in the cytoplasm. The ultrastructural changes induced by heat shock at 41℃ for 2 h were not reversible. In conclusion, the damage of heat stress to mitochondria, lysosomes, ribosomes and cell nucleus, may be one of the most important factors that inhibit the normal development of mouse early blastocysts [Current Zoology 55(1: 61–66, 2009].

  5. GLUT8 is a glucose transporter responsible for insulin-stimulated glucose uptake in the blastocyst

    Carayannopoulos, Mary O.; Chi, Maggie M.-Y.; Cui, Ying; Pingsterhaus, Joyce M.; McKnight, Robert A.; Mueckler, Mike; Devaskar, Sherin U.; Moley, Kelle H.


    Mammalian preimplantation blastocysts exhibit insulin-stimulated glucose uptake despite the absence of the only known insulin-regulated transporter, GLUT4. We describe a previously unidentified member of the mammalian facilitative GLUT superfamily that exhibits ≈20–25% identity with other murine facilitative GLUTs. Insulin induces a change in the intracellular localization of this protein, which translates into increased glucose uptake into the blastocyst, a process th...

  6. Generation of cardiomyocytes from new human embrionic stem cell lines derived from poor-quanlity blastocysts

    Raya Chamorro, ??ngel; Aran, Bego??a; Consiglio, Antonella; Barri, Pere N.; Veiga, Anna; Izpis??a Belmonte, J. C.; Rodr??guez Piz??, Ignasi


    Human embryonic stem (hES) cells represent a potential source for cell replacement therapy of many degenerative diseases. Most frequently, hES cell lines are derived from surplus embryos from assisted reproduction cycles, independent of their quality or morphology. Here, we show that hES cell lines can be obtained from poor-quality blastocysts with the same efficiency as that obtained from good- or intermediate-quality blastocysts. Furthermore, we show that the self-renewal, pluripotency, and...

  7. Developmental competence and ultrastructural changes of heat-stressed mouse early blastocysts produced in vitro

    Pingping QU; Wenru TIAN; Tao LI; Zhongling JIANG; Shansong GAO; Zhongjie TIAN; Mingzhi WANG


    Mouse early blastocysts were exposed to temporatures of 39℃ and 41℃ for 2 h,respectively,to determine their developmental competence and uhrastructural changes. The results showed that heat stress at 41 ℃ for 2 h,significantly reduced the percentages of expanded and hatched blastocysts,but not at 39℃ for 2 h. The average cell numbers in expanded blastocysts,which developed from early blastocysts heat-stressed at temperatures of 39℃ and 41 ℃,were significantly reduced. The average cell numbers in hatched blastocysts subjected to heat stress were no different from those in the control group cultured at 37℃ . The mitochondria of the early blastocysts heat-stressed at 39T℃ for 2 h,were slightly swollen,but they had recovered after culturing at 37℃ for 2 h. However,the mitochondria in the blastocysts heat stressed at 41 ℃ for 2 h were severely swollen,and their number increased. The ribosomes shed from the rough endoplasmic reticulum,and the number of secondary lysosomes in the plasma increased. The integrity of desmosomes was disrupted. The space between the nuclear envelope and the perivitelline membrane enlarged. The fibre fraction and the particulate fraction of nucleoli were separated. The heterochromatin in nucleoli was also increased in its quantity. There were some lamellar-shape structures and heterogeneous dense materials exhibiting in the cytoplasm. The ultrastructural changes induced by heat shock at 41 ℃ for 2 h were not reversible. In conclusion,the damage of heat stress to mitochondria,lysosomes,ribosomes and cell nucleus,may be one of the most important factors that inhibit the normal development of mouse early blastocysts.

  8. 人胚胎移植后剩余胚胎继续体外培养潜能的研究%Potential development to blastocyst of the surplus embryos from human embryo transfer

    薛侠; 赵皖秋; 张四林; 秦臻; 师娟子


    Objective To explore the developmental potential of the surplus embryos from human embryo transfer during IVF - ET cycles. Methods All embryos with non - pronucleus (0PN), a single pronudeus (1PN), a number of pronucleus (≥3PN) and 2 pronudeus delaying development in cleavage stage (2PN) were cultured into blastula by the sequential method. Results ① 314 Surplus embryos were collected and formed 152 blastulas(48.41% ) after the sequential culture, among which 53 (34.87%) were high-quality blastula. ② The embryo grade on Day 3 was related to blastocyst rate. The higher embryo grade, the higher blastula formation (54.39%, 52.39%, 49.61% and 21.62% ); ③ Blastocyst formation rates of embryos in 1PN embryos,0PN embryos and D3 from blastocyst embryos classified Ⅲ had higher rates of blastula formation than D3 from blastocyst embryos classified beyond Ⅲ ( P < 0.05). Conclusion Embryos of level Ⅲ and above in D3 are still opportunities for blastocyst formation. The 0PN, 1PN embryo cleavage embryos developed from the D3 can continue to develop in high - quality, until after the formation of blastocysts, and then a pre- implantation genetic diagnosis. If the karyotype is aneuploid karyotype, then it should be frozen or transplanted first. The measures above can improve the oocyte retrieval in patients with accumulation of a single pregnancy, which can also provide resources for embryonic stem cell research.%目的 探讨IVF新鲜周期中D3可用胚胎移植和冷冻后剩余胚胎继续培养的价值.方法 通过囊胚序贯培养法将无原核(0PN)、单个原核(1PN)、多个原核(≥3PN)和卵裂期发育延缓的2原核(2PN)废弃胚胎培养至囊胚期.结果 ① 314枚剩余胚胎于D5~D7形成152枚囊胚(48.41%),其中53枚为优质囊胚(34.87%); ② 胚胎级别越高,囊胚形成率越高(54.39%、52.39%、49.61%和21.62%); ③ 0PN和1PN卵裂发育而来的D3优质胚胎、2PN卵裂发育来的D3评分为Ⅲ级

  9. The pig blastocyst: its ultrastructure and the uptake of protein macromolecules.

    Stroband, H W; Taverne, N; vd Bogaard, M


    Between days 8 and 11 of pregnancy spherical blastocysts from 0.3 to 10 mm in diameter were flushed from the uterine horns of Dutch Landrace pigs. A description of their ultrastructure is given, and the uptake of horseradish peroxidase and ferritin is demonstrated. The ultrastructure of the trophoblast was similar at all ages studied. The trophoblast which has many apical microvilli is able to take up and digest the macromolecules which were offered in the in vitro incubation medium. The hypoblast consists of flattened cells. In blastocysts 2 mm and larger, compact cells bearing microvilli are found below the embryoblast. Cell organelles indicating protein synthesis are found within hypoblast cells of such blastocysts. In the embryoblast, local concentrations of cell organelles are visible, indicating that differentiation has started. After the disappearance of Rauber's layer, which takes place when the blastocyst reaches a diameter of about 2 mm, superficial embryoblast cells develop short microvilli. The cells do not absorb ferritin or peroxidase but are dependent on the trophoblast for their food requirements. All cell layers in the blastocyst contain mitochondria that have characteristics of those found in steroid-producing cells. The significance of the uptake and digestion of macromolecules by trophoblast cells, the synthesis of protein by hypoblast cells and the possible synthesis of steroids is discussed with respect to the relationship between the cell layers of the blastocyst and in the context of concepto-maternal relationships. PMID:6705037

  10. Total Cell Number and its Allocation to Trophectoderm and Inner Cell Mass in In Vitro Obtained Cats' Blastocysts.

    Ochota, M; Wojtasik, B; Niżański, W


    The aim of this study was to evaluate the developmental kinetics of cats' blastocysts in connection with their morphology and blastomeres allocation to trophoblast or embryoblast cells. We examined gross blastocyst morphology and the total number of blastomeres together with its allocation to inner cell mass (ICM) or trophectoderm (TE) cells in pre-implantation feline embryos obtained from 6th to 9th day of in vitro culture. From all the investigated embryos, 61.8% developed to early blastocyst, 37.4% to full and 7.6% to hatching blastocyst stage. The total cell number (TCN) varied form 58 cells in early day 6 to 245 in hatching day 8 blastocyst, with the mean 84.9 cells in early, 156.7 in full, and 204.4 in hatching ones. Day 8 blastocyst had the highest number of total cells, together with the highest mean number of ICM regardless of its morphological assessment. Early blastocyst (apart from day 6) had the highest number of arrested cells, while dead cells were the highest in full day 9 blastocyst. More data about the relationship between blastocyst development and morphology would facilitate the selection of optimal blastocysts for further procedures. PMID:26991408

  11. Spatiotemporal Reconstruction of the Human Blastocyst by Single-Cell Gene-Expression Analysis Informs Induction of Naive Pluripotency.

    Durruthy-Durruthy, Jens; Wossidlo, Mark; Pai, Sunil; Takahashi, Yusuke; Kang, Gugene; Omberg, Larsson; Chen, Bertha; Nakauchi, Hiromitsu; Reijo Pera, Renee; Sebastiano, Vittorio


    Human preimplantation embryo development involves complex cellular and molecular events that lead to the establishment of three cell lineages in the blastocyst: trophectoderm, primitive endoderm, and epiblast. Owing to limited resources of biological specimens, our understanding of how the earliest lineage commitments are regulated remains narrow. Here, we examined gene expression in 241 individual cells from early and late human blastocysts to delineate dynamic gene-expression changes. We distinguished all three lineages and further developed a 3D model of the inner cell mass and trophectoderm in which individual cells were mapped into distinct expression domains. We identified in silico precursors of the epiblast and primitive endoderm lineages and revealed a role for MCRS1, TET1, and THAP11 in epiblast formation and their ability to induce naive pluripotency in vitro. Our results highlight the potential of single-cell gene-expression analysis in human preimplantation development to instruct human stem cell biology. PMID:27404362

  12. Occurrence of multipolar mitoses and association with Aurora-A/-B kinases and p53 mutations in aneuploid esophageal carcinoma cells

    Münch Claudia


    Full Text Available Abstract Background Aurora kinases and loss of p53 function are implicated in the carcinogenesis of aneuploid esophageal cancers. Their association with occurrence of multipolar mitoses in the two main histotypes of aneuploid esophageal squamous cell carcinoma (ESCC and Barrett's adenocarcinoma (BAC remains unclear. Here, we investigated the occurrence of multipolar mitoses, Aurora-A/-B gene copy numbers and expression/activation as well as p53 alterations in aneuploid ESCC and BAC cancer cell lines. Results A control esophageal epithelial cell line (EPC-hTERT had normal Aurora-A and -B gene copy numbers and expression, was p53 wild type and displayed bipolar mitoses. In contrast, both ESCC (OE21, Kyse-410 and BAC (OE33, OE19 cell lines were aneuploid and displayed elevated gene copy numbers of Aurora-A (chromosome 20 polysomy: OE21, OE33, OE19; gene amplification: Kyse-410 and Aurora-B (chromosome 17 polysomy: OE21, Kyse-410. Aurora-B gene copy numbers were not elevated in OE19 and OE33 cells despite chromosome 17 polysomy. Aurora-A expression and activity (Aurora-A/phosphoT288 was not directly linked to gene copy numbers and was highest in Kyse-410 and OE33 cells. Aurora-B expression and activity (Aurora-B/phosphoT232 was higher in OE21 and Kyse-410 than in OE33 and OE19 cells. The mitotic index was highest in OE21, followed by OE33 > OE19 > Kyse-410 and EPC-hTERT cells. Multipolar mitoses occurred with high frequency in OE33 (13.8 ± 4.2%, followed by OE21 (7.7 ± 5.0% and Kyse-410 (6.3 ± 2.0% cells. Single multipolar mitoses occurred in OE19 (1.0 ± 1.0% cells. Distinct p53 mutations and p53 protein expression patterns were found in all esophageal cancer cell lines, but complete functional p53 inactivation occurred in OE21 and OE33 only. Conclusions High Aurora-A expression alone is not associated with overt multipolar mitoses in aneuploid ESCC and BAC cancer cells, as specifically shown here for OE21 and OE33 cells, respectively

  13. Effect of clinically-related factors on in vitro blastocyst development after equine ICSI.

    Choi, Young-Ho; Velez, Isabel C; Macías-García, Beatriz; Riera, Fernando L; Ballard, Catherine S; Hinrichs, Katrin


    Equine intracytoplasmic sperm injection (ICSI) is being used clinically for foal production, but little information is available on factors affecting the efficiency of this procedure. We examined factors that may influence blastocyst development when ICSI is performed clinically, i.e., on oocytes recovered from live mares by transvaginal ultrasound-guided follicle aspiration (TVA), using sperm from the stallion of the client's choice. In a clinical setting, there may be a delay from the time of TVA to isolation of oocytes from the aspirated fluid. In a preliminary study, oocytes from fluid held for 1.5 h at ambient temperature (26°C-33°C) yielded 32% blastocysts; however, in experiment 1, fluid held at 32 °C for 2 h after aspiration yielded 16% blastocysts versus 23% for aspirates processed immediately. Performing TVA/ICSI throughout the year would increase production from valuable mares, but efficiency during the nonbreeding season is unknown. In addition, to reduce the possibility of infection after TVA, administration of antibiotics to the mare before TVA is indicated; however, these could affect oocyte quality. In experiment 2, follicle numbers at the time of TVA were significantly higher in December to January than for the same mares during the breeding season. Oocyte recovery rates on TVA were 60% to 66% and the blastocyst rate was 18%. An equivalent blastocyst rate (18%) was achieved after administration of ampicillin and gentamicin to mares before TVA. In experiment 3, we verified that stallion differences exist in rates of cleavage after ICSI with motile sperm. In sperm from a low-performing stallion, density-gradient centrifugation followed by swim-up was associated with significantly higher rates of cleavage (45% vs. 18%) and blastocyst development (14% vs. 0%) than those for density gradient alone. In experiment 4, parthenogenetic activation with ionomycin and 6-dimethylaminopurine yielded 40% blastocysts. Frozen-thawed sperm that were immotile

  14. Derivation of human embryonic stem cell lines from parthenogenetic blastocysts

    Qingyun Mai; Yang Yu; Tao Li; Liu Wang; Mei-jue Chen; Shu-zhen Huang; Canquan Zhou; Qi Zhou


    Parthenogenesis is one of the main, and most useful, methods to derive embryonic stem cells (ESCs), which may be an important source of histocompatible cells and tissues for cell therapy. Here we describe the derivation and characterization of two ESC lines (hPES-1 and hPES-2) from in vitro developed blastocysts following parthenogenetic activation of human oocytes. Typical ESC morphology was seen, and the expression of ESC markers was as expected for alkaline phosphatase, octamer-binding transcription factor 4, stage-specific embryonic antigen 3, stage-specific embryonic antigen 4, TRA-1-60, and TRA-1-81, and there was absence of expression of negative markers such as stage-specific embryonic antigen 1. Expression of genes specific for different embryonic germ layers was detected from the embryoid bodies (EBs) of both hESC lines, suggesting their differentiation potential in vitro. However, in vivo, only hPES-1 formed teratoma consisting of all three embryonic germ layers (hPES-2 did not). Interestingly, after continuous proliferation for more than 100 passages, hPES-1 cells still maintained a normal 46 XX karyotype; hPES-2 displayed abnormalities such as chromosome translocation after long term passages. Short Tandem Repeat (STR) results demonstrated that the hPES lines were genetic matches with the egg donors, and gene imprinting data confirmed the parthenogenetic origin of these ES cells. Genome-wide SNP analysis showed a pattern typical of parthenogenesis. All of these results demonstrated the feasibility to isolate and establish human parthenogenetic ESC lines, which provides an important tool for studying epigenetic effects in ESCs as well as for future therapeutic interventions in a clinical setting.

  15. Superoxide dismutase and taurine supplementation improves in vitro blastocyst yield from poor-quality feline oocytes.

    Ochota, Małgorzata; Pasieka, Anna; Niżański, Wojciech


    Blastocyst production in vitro seems to be crucial part of assisted reproduction techniques in feline species. However, the results of cats' oocyte maturation and embryo development are still lower than those in other species. The aim of this study was to evaluate whether the supplementation with superoxide dismutase (SOD) and taurine during maturation or culture would improve the blastocyst yield obtained from lower grades of oocytes, that are usually discarded, as not suitable for further in vitro purposes. To investigate the effect of antioxidants' addition, the good- and poor-quality oocytes, were cultured with the addition of 10-mmol taurine and 600 UI/mL SOD. The nuclear maturity, embryo development, and blastocyst quality were subsequently assessed. In control group, without antioxidant supplementation, significantly less poor-quality oocytes matured (42% vs. 62%) and more degenerated (35% vs. 20%), comparing to the experimental group supplemented with SOD and taurine. The amount of obtained blastocyst was much higher, when poor quality oocytes were supplemented with SOD and taurine (supplementation to IVM-4%; supplementation to IVC-5.5%; supplementation to IVM and IVC-5.9% of blastocyst), comparing to not supplemented control group (1.3%). The best blastocysts were obtained when poor oocytes had antioxidants added only during embryo culture (185 ± 13.4 blastomeres vs. 100 ± 1.5 in control). In the present study, we reported that the lower grades of oocytes can better mature and form significantly more blastocysts with better quality, when cultured with addition of SOD and taurine. PMID:26643604

  16. Clinical outcome of fresh and vitrified-warmed blastocyst and cleavage-stage embryo transfers in ethnic Chinese ART patients

    Tong Guo; Cao Shan; Wu Xun; Zhang Jun; Cui Ji; Heng Boon; Ling Xiu


    Abstract Objectives This study sought to evaluate the outcome of fresh and vitrified-warmed cleavage-stage and blastocyst-stage embryo transfers in patients undergoing ART treatment within an ethnic Chinese population. Study design We compared the clinical results of embryo transfer on the 3rd (cleavage stage) or 5th (blastocyst stage) day after oocyte retrieval, including clinical pregnancy rates, implantation rates and multiple pregnancy rates. Results Our data showed that blastocyst transf...

  17. Tumor environmental factors glucose deprivation and lactic acidosis induce mitotic chromosomal instability--an implication in aneuploid human tumors.

    Chunyan Dai

    Full Text Available Mitotic chromosomal instability (CIN plays important roles in tumor progression, but what causes CIN is incompletely understood. In general, tumor CIN arises from abnormal mitosis, which is caused by either intrinsic or extrinsic factors. While intrinsic factors such as mitotic checkpoint genes have been intensively studied, the impact of tumor microenvironmental factors on tumor CIN is largely unknown. We investigate if glucose deprivation and lactic acidosis--two tumor microenvironmental factors--could induce cancer cell CIN. We show that glucose deprivation with lactic acidosis significantly increases CIN in 4T1, MCF-7 and HCT116 scored by micronuclei, or aneuploidy, or abnormal mitosis, potentially via damaging DNA, up-regulating mitotic checkpoint genes, and/or amplifying centrosome. Of note, the feature of CIN induced by glucose deprivation with lactic acidosis is similar to that of aneuploid human tumors. We conclude that tumor environmental factors glucose deprivation and lactic acidosis can induce tumor CIN and propose that they are potentially responsible for human tumor aneuploidy.

  18. An Epigenetic Modifier Results in Improved In Vitro Blastocyst production after Somatic Cell Nuclear Transfer

    Zhang, Yunhai; Li, Juan; Villemoes, Klaus; Pedersen, Anette Møjbæk; Purup, Stig; Vajta, Gabor


    significantly improve blastocyst yield compared to the control (46.4 ± 4.6% vs 17.7 ± 4.9% for treated and untreated embryos, respectively; p < 0.05), whereas similar cleavage rate and total cell number per blastocyst were observed. In order to assess if the improvement is cell line specific, three cell lines...... were tested, and for all cell lines an enhancement in blastocyst development compared to their corresponding control was observed. Our data demonstrate that TSA treatment after somatic cell nuclear transfer in the pig can significantly improve the in vitro blastocyst production...

  19. Good quality blastocyst from non-/mono-pronuclear zygote may be used for transfer during IVF.

    Yin, Bao-Li; Hao, Hao-Ying; Zhang, Ya-Nan; Wei, Duo; Zhang, Cui-Lian


    Although healthy infants have developed from non- and mono-pronuclear zygotes, the transfer of embryos from non- and mono-pronuclear zygotes is not recommended because there are no proper selection criteria. In the present study, we discuss how to select non- and mono-pronuclear embryos with the highest developmental potential at 19-20 hours post-insemination. We found that the percentage of blastocysts with normal chromosome constitution in non-pronuclear zygotes was slightly higher than in mono-pronuclear zygotes. Non- and mono-pronuclear embryos that were at the 4-cell stage on D2 and/or at the 6- to 8-cell stage on D3 had higher incidence rates of blastocysts with normal chromosome constitutions. We also found higher incidences of blastocysts with normal chromosome constitution on D6 than on D5. The results suggest that if high quality non- and mono-pronuclear zygotes develop to the 4-cell stage on D2 and the 6-to 8- cell stages on D3, along with high quality D6 blastocysts, the incidence of blastocysts with normal chromosome constitution is higher. PMID:26901373

  20. Effect of fibronectin and leu- kaemia inhibitory factor on matrix metalloproteinases in mouse blastocyst


    This study was designed to detect the effects of fibronectin (FN) and leukaemia inhibitory factor (LIF) on matrix metallopoteinases (MMPs) of mouse blastocysts. The experiments comprised four groups: first, blastocysts grew on the dishes with FN-coated; the second, without FN-coated; the third group, without FN coated, but with 20 ng/mL LIF added to culture medium; and the fourth group, with both FN-coated and 20 ng/mL LIF added. Using MMP-2 and MMP-9 primers respectively, the expressions of MMP-2 and MMP-9 were detected by RT-PCR and cloning identification. The results showed that in the first group, MMP-2 and MMP-9 were produced after 12 and 24 h culturing; in the third group, there were both MMP-2 and MMP-9 bands when blastocysts were cultured for 24 h; and in the fourth group, MMP-2 and MMP-9 bands appeared when blasto-cysts were cultured for 6, 12 and 24 h. But in the second group no MMP-2 or MMP-9 band appeared. These results show that FN may initiate the transcription of MMP-2 and MMP-9 genes to mRNA through signal transduction path-way mediated by integrin receptor and LIF may promote the expression of MMPs mRNA, which guaranteed the accuracy of embryo implantation by associating the invasive blasto-cysts with the receptive endometrium.

  1. Clinical outcome of fresh and vitrified-warmed blastocyst and cleavage-stage embryo transfers in ethnic Chinese ART patients

    Tong Guo


    Full Text Available Abstract Objectives This study sought to evaluate the outcome of fresh and vitrified-warmed cleavage-stage and blastocyst-stage embryo transfers in patients undergoing ART treatment within an ethnic Chinese population. Study design We compared the clinical results of embryo transfer on the 3rd (cleavage stage or 5th (blastocyst stage day after oocyte retrieval, including clinical pregnancy rates, implantation rates and multiple pregnancy rates. Results Our data showed that blastocyst transfer on day 5 did not significantly increase clinical pregnancy rate (41.07% vs 47.08%, p>0.05 and implantation rate (31.8% vs 31.2%, p>0.05 in patients under 35 years of age, in comparison with day 3 cleavage stage embryo transfer. In patients older than 35 years of age, the clinical pregnancy rate after blastocyst transfer was slightly decreased compared with cleavage stage embryo transfer (33.33% vs 42.31%, p>0.05. Unexpectedly, It was found that vitrified-warmed blastocyst transfer resulted in significantly higher clinical pregnancy rate (56.8% and implantation rate (47% compared with fresh blastocyst transfer in controlled stimulation cycles (41.07% and 31.8%, respectively. For patients under 35 years of age, the cumulative clinical pregnancy rate combining fresh and vitrified-warmed blastocyst transfer cycles were significantly higher compared to just cleavage-stage embryo transfer (70.1% versus 51.8%, p Conclusions In an ethnic Chinese patient population, fresh blastocyst transfer does not significantly increase clinical pregnancy rate. However, subsequent vitrified-warmed blastocyst transfer in a non-controlled ovarian hyperstimulation cycle dramatically improves clinical outcomes. Therefore, blastocyst culture in tandem with vitrified-warmed blastocyst transfer is recommended as a favourable and promising protocol in human ART treatment, particularly for ethnic Chinese patients.

  2. Factors Analysis of Spontaneous Abortion after Thawed-vitrified Blastocysts Transfer

    Dong YANG; Zheng-yi SUN; Cheng-yan DENG; Qi YU; Fang-fang HE


    Objective To investigate the factors resulting in spontaneous abortion after transferring frozen-thawing blastocysts. Methods A total of 108 cases transferring vitrified blastocysts were divided into two groups: abortion group (n =20) and ongoing group (n=88). Cytogenetic analysis of apoblemas was performed in 12 cases of the abortion.Results The overall spontaneous abortion rate was 18.50%(20/108) and the early spontaneous rate was 16.67%(18/108). ,4 significant difference in maternal age was observed (abortion group: 33.3±4.0 years, ongoing group: 31.0±3.6 years, P=0.02). No difference in other parameters was found. Cytogenetic analysis of apoblemas was obtained for 12 cases, and 2 specimens were contaminated. Seven of ten patients had abnormal karyotypes. Conclusion The underlying cause of spontaneous abortion after transferring frozen thawing blastocysts appears to be abnormal karyotypes.Advancing maternal age seems to increase the risk of spontaneous abortion.

  3. Generation of parthenogenetic goat blastocysts: effects of different activation methods and culture media.

    Malik, Hruda Nanda; Singhal, Dinesh Kumar; Saugandhika, Shrabani; Dubey, Amit; Mukherjee, Ayan; Singhal, Raxita; Kumar, Sudarshan; Kaushik, Jai Kumar; Mohanty, Ashok Kumar; Das, Bikash Chandra; Bag, Sadhan; Bhanja, Subrata Kumar; Malakar, Dhruba


    The present study was carried out to investigate the effects of different activation methods and culture media on the in vitro development of parthenogenetic goat blastocysts. Calcium (Ca2+) ionophore, ethanol or a combination of the two, used as activating reagents, and embryo development medium (EDM), modified Charles Rosenkrans (mCR2a) medium and research vitro cleave (RVCL) medium were used to evaluate the developmental competence of goat blastocysts. Quantitative expression of apoptosis, stress and developmental competence-related genes were analysed in different stages of embryos. In RVCL medium, the cleavage rate of Ca2+ ionophore-treated oocytes (79.61 ± 0.86) was significantly (P < 0.05) higher than in ethanol (74.90 ± 1.51) or in the combination of both Ca2+ ionophore and ethanol. In mCR2a or EDM, hatched blastocyst production rate of Ca2+ ionophore-treated oocytes (8.33 ± 1.44) was significantly higher than in ethanol (6.46 ± 0.11) or in the combined treatment (6.70 ± 0.24). In ethanol, the cleavage, blastocyst and hatched blastocyst production rates in RVCL medium (74.90 ± 1.51, 18.30 ± 1.52 and 8.24 ± 0.15, respectively) were significantly higher than in EDM (67.81 ± 3.21, 14.59 ± 0.27 and 5.59 ± 0.42) or mCR2a medium (65.09 ± 1.57, 15.36 ± 0.52 and 6.46 ± 0.11). The expression of BAX, Oct-4 and GlUT1 transcripts increased gradually from 2-cell stage to blastocyst-stage embryos, whereas the transcript levels of Bcl-2 and MnSOD were significantly lower in blastocysts. In addition, different activation methods and culture media had little effect on the pattern of variation and relative abundance of the above genes in different stages of parthenogenetic activated goat embryos. In conclusion, Ca2+ ionophore as the activating agent, and RVCL as the culture medium are better than other tested options for development of parthenogenetic activated goat blastocysts. PMID:24405529

  4. Functional challenge affects aquaporin mRNA abundance in mouse blastocysts

    Offenberg, Hanne Kjær; Thomsen, Preben Dybdahl


    The aquaporins (AQPs) are a family of channel proteins that facilitate diffusion of water across cell membranes. Three members of the AQP family have been detected in the mouse blastocyst: AQP 3 and 8 are located in the basolateral domain and AQP 9 predominantly in the apical domain of the tropho...... in vivo developed blastocysts. We found that in vitro culture resulted in lower levels of AQP 8, 9, and 11 compared to in vivo development. These experiments show that mouse embryos are capable of regulating AQP mRNA abundances in response to environmental alterations....

  5. IGF-1/IGFBP-1 increases blastocyst formation and total blastocyst cell number in mouse embryo culture and facilitates the establishment of a stem-cell line

    Hsu Teng-Tsao


    Full Text Available Abstract Background Apoptosis occurs frequently for blastocysts cultured in vitro, where conditions are suboptimal to those found in the natural environment. Insulin-like growth factor-1 (IGF-1 plays an important role in preventing apoptosis in the early development of the embryo, as well as in the progressive regulation of organ development. We hypothesize that IGF-1 and its dephosphorylated binding protein (IGFBP-1 may be able to improve embryo culture with an associated reduced cell death, and that the resultant increase in the total cell number of the embryo could increase the chances of establishing an embryonic stem-cell line. Results In vivo fertilized zygotes were cultured in medium containing supplementary IGF-1, or IGFBP-1/IGF-1. The stages of the resultant embryos were evaluated at noon on day five post-hCG injection. The extent of apoptosis and necrosis was evaluated using Annexin V and propidium iodine staining under fluorescent microscopy. The establishment of embryonic stem-cell lines was performed using the hatching blastocysts that were cultured in the presence of IGF-1 or IGFBP-1/IGF-1. The results show that the rate of blastocyst formation in a tissue-culture system in the presence of IGF-1 was 88.7% and IGFBP-1/IGF-1 it was 94.6%, respectively, and that it was significantly greater than the figure for the control group (81.9%. IGFBP-1/IGF-1 also resulted in a higher hatching rate than was the case for the control group (68.8% vs. 48.6% respectively. IGF-1 also increased the number of Annexin V-free and propidium iodine-free blastocysts in culture (86.8% vs. 75.9% respectively. Total cell number of blastocyst in culture was increased by 18.9% for those examples cultured with dephosphorylated IGFBP-1/IGF-1. For subsequent stem-cell culture, the chances of the successful establishment of a stem-cell line was increased for the IGF-1 and IGFBP-1/IGF-1 groups (IGF-1 vs. IGFBP-1/IGF-1 vs. control: 45.8% vs. 59.6% vs. 27

  6. A prospective randomized comparison of sequential versus monoculture systems for in-vitro human blastocyst development

    N.S. Macklon (Nick); M.H. Pieters; M.A. Hassan; P.H. Jeucken; M.J.C. Eijkemans (René); B.C.J.M. Fauser (Bart)


    textabstractBACKGROUND: Extending the period of in-vitro culture to the blastocyst stage may improve implantation rates in IVF treatment. Recognition of the dynamic nature of early embryo metabolism has led to the development of commercially available sequential culture systems. Ho

  7. Susceptibility of in vitro produced hatched bovine blastocysts to infection with bluetongue virus serotype 8

    Vandaele Leen


    Full Text Available Abstract Bluetongue virus serotype 8 (BTV-8, which caused an epidemic in ruminants in central Western Europe in 2006 and 2007, seems to differ from other bluetongue serotypes in that it can spread transplacentally and has been associated with an increased incidence of abortion and other reproductive problems. For these reasons, and also because BTV-8 is threatening to spread to other parts of the world, there is a need for more information on the consequences of infection during pregnancy. The aim of the present study was to investigate whether hatched (i.e. zona pellucida-free in vitro produced bovine blastocysts at 8-9 days post insemination are susceptible to BTV-8 and whether such infection induces cell death as indicated by apoptosis. Exposure of hatched in vitro produced bovine blastocysts for 1 h to a medium containing 103.8 or 104.9 TCID50 of the virus resulted in active viral replication in between 25 and 100% of the cells at 72 h post exposure. The infected blastocysts also showed growth arrest as evidenced by lower total cell numbers and a significant level of cellular apoptosis. We conclude from this in vitro study that some of the reproductive problems that are reported when cattle herds are infected with BTV-8 may be attributed to direct infection of blastocysts and other early-stage embryos in utero.

  8. 309 proteomic analysis of the blastocoel fluid and remaining cells of bovine blastocysts

    Jensen, P L; Groendahl, M L; Beck, Helle;


    Human embryonic stem cells (hESC) are derived from the human blastocyst and possess the potential to differentiate into any cell type present in the adult human body. Human ESC are considered to have great potential in regenerative medicine for the future treatment of severe diseases and conditions...

  9. Global gene expression of the inner cell mass and trophectoderm of the bovine blastocyst

    Ozawa Manabu


    Full Text Available Abstract Background The first distinct differentiation event in mammals occurs at the blastocyst stage when totipotent blastomeres differentiate into either pluripotent inner cell mass (ICM or multipotent trophectoderm (TE. Here we determined, for the first time, global gene expression patterns in the ICM and TE isolated from bovine blastocysts. The ICM and TE were isolated from blastocysts harvested at day 8 after insemination by magnetic activated cell sorting, and cDNA sequenced using the SOLiD 4.0 system. Results A total of 870 genes were differentially expressed between ICM and TE. Several genes characteristic of ICM (for example, NANOG, SOX2, and STAT3 and TE (ELF5, GATA3, and KRT18 in mouse and human showed similar patterns in bovine. Other genes, however, showed differences in expression between ICM and TE that deviates from the expected based on mouse and human. Conclusion Analysis of gene expression indicated that differentiation of blastomeres of the morula-stage embryo into the ICM and TE of the blastocyst is accompanied by differences between the two cell lineages in expression of genes controlling metabolic processes, endocytosis, hatching from the zona pellucida, paracrine and endocrine signaling with the mother, and genes supporting the changes in cellular architecture, stemness, and hematopoiesis necessary for development of the trophoblast.

  10. Bivariate analysis of basal serum anti-Mullerian hormone measurements and human blastocyst development after IVF

    Sills, E Scott


    Abstract Background To report on relationships among baseline serum anti-Müllerian hormone (AMH) measurements, blastocyst development and other selected embryology parameters observed in non-donor oocyte IVF cycles. Methods Pre-treatment AMH was measured in patients undergoing IVF (n = 79) and retrospectively correlated to in vitro embryo development noted during culture. Results Mean (+\\/- SD) age for study patients in this study group was 36.3 ± 4.0 (range = 28-45) yrs, and mean (+\\/- SD) terminal serum estradiol during IVF was 5929 +\\/- 4056 pmol\\/l. A moderate positive correlation (0.49; 95% CI 0.31 to 0.65) was noted between basal serum AMH and number of MII oocytes retrieved. Similarly, a moderate positive correlation (0.44) was observed between serum AMH and number of early cleavage-stage embryos (95% CI 0.24 to 0.61), suggesting a relationship between serum AMH and embryo development in IVF. Of note, serum AMH levels at baseline were significantly different for patients who did and did not undergo blastocyst transfer (15.6 vs. 10.9 pmol\\/l; p = 0.029). Conclusions While serum AMH has found increasing application as a predictor of ovarian reserve for patients prior to IVF, its roles to estimate in vitro embryo morphology and potential to advance to blastocyst stage have not been extensively investigated. These data suggest that baseline serum AMH determinations can help forecast blastocyst developmental during IVF. Serum AMH measured before treatment may assist patients, clinicians and embryologists as scheduling of embryo transfer is outlined. Additional studies are needed to confirm these correlations and to better define the role of baseline serum AMH level in the prediction of blastocyst formation.

  11. The spatiotemporal hormonal orchestration of human folliculogenesis, early embryogenesis and blastocyst implantation.

    Atwood, Craig S; Vadakkadath Meethal, Sivan


    The early reproductive events starting with folliculogenesis and ending with blastocyst implantation into the uterine endometrium are regulated by a complex interplay among endocrine, paracrine and autocrine factors. This review examines the spatiotemporal integration of these maternal and embryonic signals that are required for successful reproduction. In coordination with hypothalamic-pituitary-gonadal (HPG) hormones, an intraovarian HPG-like axis regulates folliculogenesis, follicular quiescence, ovulation, follicular atresia, and corpus luteal functions. Upon conception and passage of the zygote through the fallopian tube, the contribution of maternal hormones in the form of paracrine secretions from the endosalpinx to embryonic development declines, with autocrine and paracrine signaling becoming increasingly important as instructional signals for the differentiation of the early zygote/morula into a blastocyst. These maternal and embryonic signals include activin and gonadotropin-releasing hormone 1 (GnRH1) that are crucial for the synthesis and secretion of the 'pregnancy' hormone human chorionic gonadotropin (hCG). hCG in turn signals pre-implantation embryonic cell division and sex steroid production required for stem cell differentiation, and subsequent blastulation, gastrulation, cavitation and blastocyst formation. Upon reaching the uterus, blastocyst hatching occurs under the influence of decreased activin signaling, while the attachment and invasion of the trophoblast into the endometrium appears to be driven by a decrease in activin signaling, and by increased GnRH1 and hCG signaling that allows for tissue remodeling and the controlled invasion of the blastocyst into the uterine endometrium. This review demonstrates the importance of integrative endocrine, paracrine, and autocrine signaling for successful human reproduction. PMID:27045358

  12. Comparing thaw survival, implantation and live birth rates from cryopreserved zygotes, embryos and blastocysts

    Mary Ellen Pavone


    Full Text Available Context : Most in vitro fertilization (IVF programs employ embryo cryopreservation to enhance pregnancies from a single ovarian stimulation. More embryos are created, some of which are not transferred to the uterus immediately, generating a need for improved cryopreservation protocols. One protocol may involve growing embryos to a further stage of development, allowing only embryos with proven developmental capabilities to be cryopreserved. Here we examined thaw survival, implantation and live birth rates of embryos cryopreserved at different stages. Aims : We examined thaw survival, implantation and live birth rates of embryos cryopreserved at the zygote, day 3 (D3 embryos or blastocyst stage. Settings and Design : This is a retrospective study from a single academic IVF program. Patients and Methods :0 A retrospective study of all patients who had frozen embryos transferred to their uteri from year 2002 to 2008 at a single academic IVF program was conducted. Statistical Analysis Used : Analysis of variance followed by Fisher′s Exact Test was performed to compare the survival after thaw, implantation and live birth rates between the three groups. Results : One thousand nine hundred and ninety-one zygotes, 2880 D3 embryos and 503 blastocysts were frozen using a slow freeze technique, thawed and transferred. Significantly more D3 embryos and blastocysts survived the thawing process compared to zygotes and significantly higher implantation rate per number of thawed blastocysts was achieved than that for zygotes. Live birth rates were similar between the three groups. Conclusions : Growing embryos to blastocyst stage prior to cryopreservation is associated with fewer frozen embryos but does not appear compromise patients′ chance of achieving pregnancy

  13. Blastocyst rate of in vitro embryo production in sheep is affected by season.

    Mara, L; Sanna, D; Casu, S; Dattena, M; Muñoz, I M Mayorga


    It has been reported that the number and quality of in vitro produced embryos is season related. This study was conducted to assess the effect of season on cleavage, blastocyst and lambing rates of in vitro produced ovine embryos during 3 years of collection data. Ovaries of Sarda sheep were collected from a slaughterhouse. In total, 5035 oocytes were recovered and matured in TCM-199 with 4 mg/ml bovine serum albumin (BSA), 100 μM cysteamine, 0.3 mM Na pyruvate, 0.1 UI/ml recombinant follicle-stimulating hormone (r-FSH), 0.1 UI/ml recombinant luteinising hormone (r-LH), and 1 μg/ml estradiol-17β. Matured oocytes were fertilized with fresh semen in synthetic oviductal fluid (SOF) with 20% heat inactivated estrous sheep serum. The presumptive zygotes were cultured for 6-7 days (blastocyst stage) in SOF medium supplemented with 1% Basel Medium Eagle (BME), 1% Minimum Essential Medium, 1 mM glutamine and 8 mg/ml fatty acid-free BSA. The embryos produced were vitrified and a total of 165 blastocysts (80 from the breeding season and 85 from the anoestrous season) were transferred in pairs into recipient ewes during the reproductive period. There were no significant differences in cleavage rates between seasons in any of the 3 years examined (84% versus 83%, 81% versus 80% and 80% versus 79%, respectively). The blastocyst rate varied significantly between seasons in 2005 and 2007 (P < 0.05), and in 2006 (P < 0.001). There were no differences in pregnancy and lambing rates between embryos during anoestrous versus during the breeding season. In conclusion, only the blastocyst rate appeared to have been affected by season, possibly due to variation in the number of developmentally competent oocytes. PMID:23458093

  14. X-linked gene transcription patterns in female and male in vivo, in vitro and cloned porcine individual blastocysts.

    Chi-Hun Park

    Full Text Available To determine the presence of sexual dimorphic transcription and how in vitro culture environments influence X-linked gene transcription patterns in preimplantation embryos, we analyzed mRNA expression levels in in vivo-derived, in vitro-fertilized (IVF, and cloned porcine blastocysts. Our results clearly show that sex-biased expression occurred between female and male in vivo blastocysts in X-linked genes. The expression levels of XIST, G6PD, HPRT1, PGK1, and BEX1 were significantly higher in female than in male blastocysts, but ZXDA displayed higher levels in male than in female blastocysts. Although we found aberrant expression patterns for several genes in IVF and cloned blastocysts, similar sex-biased expression patterns (on average were observed between the sexes. The transcript levels of BEX1 and XIST were upregulated and PGK1 was downregulated in both IVF and cloned blastocysts compared with in vivo counterparts. Moreover, a remarkable degree of expression heterogeneity was observed among individual cloned embryos (the level of heterogeneity was similar in both sexes but only a small proportion of female IVF embryos exhibited variability, indicating that this phenomenon may be primarily caused by faulty reprogramming by the somatic cell nuclear transfer (SCNT process rather than in vitro conditions. Aberrant expression patterns in cloned embryos of both sexes were not ameliorated by treatment with Scriptaid as a potent HDACi, although the blastocyst rate increased remarkably after this treatment. Taken together, these results indicate that female and male porcine blastocysts produced in vivo and in vitro transcriptional sexual dimorphisms in the selected X-linked genes and compensation of X-linked gene dosage may not occur at the blastocyst stage. Moreover, altered X-linked gene expression frequently occurred in porcine IVF and cloned embryos, indicating that X-linked gene regulation is susceptible to in vitro culture and the SCNT process

  15. Effects of lead on the male mouse as investigated by in vitro fertilization and blastocyst culture

    Johansson, L.; Sjoeblom, P.; Wide, M.


    Long-term exposure of male mice to inorganic lead (lead chloride, 1 g/liter) in the drinking water reduces their fertility. The cause of this reduction, expressed as a decrease in the number of mated females showing inplantations, was investigated, using an in vivo fertilization method. It was found that spermatozoa from lead-exposed males had a significantly lower ability to fertilize mouse eggs than those from unexposed males. Preimplantation embryos, isolated from uterine horns of mice mated with lead-exposed males. Preimplantation embryos, isolated from uterine horns of mice mated with lead-exposed males, were examined. No morphologically abnormal embryos were found. However, when cultured in vitro over the implantation period, blastocysts of the group mated with lead-exposed males showed an increased frequency of delayed hatching from the zona pellucida or an inability to hatch. Among blastocysts from this group a decreased frequency of inner cell mass development was also found.

  16. Monochorionic triamniotic triplets following conventional in vitro fertilization and blastocyst transfer.

    Gurunath, Sumana; Makam, Adinarayana; Vinekar, Sriprada; Biliangady, Reeta H


    Multiple pregnancy in in vitro fertilization (IVF) is on the decline with a reduction in number of embryos transferred. But the risk of monozygotic splitting persists. The risk of monozygotic twinning in women undergoing IVF is reported to be twice that of natural conception, and monochorionic triplets are even rarer at 100 times more than natural conception. We report a case of monochorionic triamniotic (MCTA) triplets following conventional IVF and blastocyst transfer without zona manipulation. This report highlights the possibility of zygotic splitting in IVF in young couples with no family history, in centers with good experience with blastocyst transfer. MCTA triplets carry a high risk of perinatal mortality and morbidity and need multidisciplinary care. Prevention and prediction of zygotic splitting ought to be realized with better reporting and identification of possible risk factors. PMID:25838751

  17. Monochorionic triamniotic triplets following conventional in vitro fertilization and blastocyst transfer

    Sumana Gurunath


    Full Text Available Multiple pregnancy in in vitro fertilization (IVF is on the decline with a reduction in number of embryos transferred. But the risk of monozygotic splitting persists. The risk of monozygotic twinning in women undergoing IVF is reported to be twice that of natural conception, and monochorionic triplets are even rarer at 100 times more than natural conception. We report a case of monochorionic triamniotic (MCTA triplets following conventional IVF and blastocyst transfer without zona manipulation. This report highlights the possibility of zygotic splitting in IVF in young couples with no family history, in centers with good experience with blastocyst transfer. MCTA triplets carry a high risk of perinatal mortality and morbidity and need multidisciplinary care. Prevention and prediction of zygotic splitting ought to be realized with better reporting and identification of possible risk factors.

  18. Blastocyst Injection of Wild Type Embryonic Stem Cells Induces Global Corrections in Mdx Mice

    Stillwell, Elizabeth; Vitale, Joseph; Zhao, Qingshi; Beck, Amanda; Schneider, Joel; Khadim, Farah; Elson, Genie; Altaf, Aneela; Yehia, Ghassan; Dong, Jia-hui; Liu, Jing; Mark, Willie; Bhaumik, Mantu; Grange, Robert; Fraidenraich, Diego


    Duchenne muscular dystrophy (DMD) is an incurable neuromuscular degenerative disease, caused by a mutation in the dystrophin gene. Mdx mice recapitulate DMD features. Here we show that injection of wild-type (WT) embryonic stem cells (ESCs) into mdx blastocysts produces mice with improved pathology and function. A small fraction of WT ESCs incorporates into the mdx mouse nonuniformly to upregulate protein levels of dystrophin in the skeletal muscle. The chimeric muscle shows reduced regenerat...

  19. Human embryonic stem cells derived from abnormal blastocyst donated by Marfan syndrome patient

    Qingqing Yang


    Full Text Available Human embryonic stem cell (hESC line was derived from abnormal blastocyst donated by Marfan syndrome patient after preimpantation genetic diagnosis (PGD treatment. DNA sequencing analysis confirmed that the hESC line carried the heterozygous deletion mutation, c.3536delA, of FBN1 gene. Characteristic tests proved that the hESC line presented typical markers of pluripotency and had the capability to form the three germ layers both in vitro and in vivo.

  20. A prospective randomized comparison of sequential versus monoculture systems for in-vitro human blastocyst development

    Macklon, Nick; Pieters, M.H.; Hassan, M. A.; Jeucken, P.H.; Eijkemans, René; Fauser, Bart


    textabstractBACKGROUND: Extending the period of in-vitro culture to the blastocyst stage may improve implantation rates in IVF treatment. Recognition of the dynamic nature of early embryo metabolism has led to the development of commercially available sequential culture systems. However, their improved efficacy over monoculture systems remains to be demonstrated in prospective studies. METHODS: Embryos obtained from 158 women undergoing IVF treatment were randomized by sealed envelopes to cul...

  1. Uncoupled embryonic and extra-embryonic tissues compromise blastocyst development after somatic cell nuclear transfer

    Degrelle, Severine; Jaffrézic, Florence; Campion, Evelyne; Le Cao, Kim-Anh; Le Bourhis, Daniel; Richard, Christophe; Rodde, Nathalie; Fleurot, Renaud; Everts, Robin E.; Lecardonnel, Jérôme; Heyman, Yvan; Vignon, Xavier; Yang, Xiangzhong; Tian, Xiuchun C.; Lewin, Harris A


    Somatic cell nuclear transfer (SCNT) is the most efficient cell reprogramming technique available, especially when working with bovine species. Although SCNT blastocysts performed equally well or better than controls in the weeks following embryo transfer at Day 7, elongation and gastrulation defects were observed prior to implantation. To understand the developmental implications of embryonic/extra-embryonic interactions, the morphological and molecular features of elongating and gastrulatin...

  2. Comparative Genomic Hybridization Selection of Blastocysts for Repeated Implantation Failure Treatment: A Pilot Study

    Ermanno Greco; Sara Bono; Alessandra Ruberti; Anna Maria Lobascio; Pierfrancesco Greco; Anil Biricik; Letizia Spizzichino; Alessia Greco; Jan Tesarik; Maria Giulia Minasi; Francesco Fiorentino


    The aim of this study is to determine if the use of preimplantation genetic screening (PGS) by array comparative genomic hybridization (array CGH) and transfer of a single euploid blastocyst in patients with repeated implantation failure (RIF) can improve clinical results. Three patient groups are compared: 43 couples with RIF for whom embryos were selected by array CGH (group RIF-PGS), 33 couples with the same history for whom array CGH was not performed (group RIF NO PGS), and 45 good progn...

  3. Vitrification of day 7 hatching blastocyst using hemistraw resulting in a full term delivery: a case study

    Nitin Lad; Neha Lad


    This is probably the first report of a successful delivery following transfer of a vitrified-warmed day 7 slow growing hatching blastocyst using hemistraw-vitriplug as an embryo carrier system. Case Report: A 26 years old Indian woman underwent controlled ovarian stimulation using short antagonist protocol and on day 11 after hCG administration, sixteen mature oocytes obtained were fertilized by ICSI procedure. On day 5, after transfer of two expanded blastocysts, three supernumerary embryos ...

  4. RNA-Seq transcriptome analysis of porcine cloned and in vitro fertilized blastocysts

    XU Wei-hua; LI Zi-cong; OUYANG Zhi-ping; YU Bo; SHI Jun-song; LIU De-wu; WU Zhen-fang


    Somatic nuclear transfer technology has become increasingly promising in biomedicine and agriculture. Whereas the approach remains inefifcient and underlying mechanisms remain ambiguous. Although cloned embryos have similar in vitro developmental capacity as in vitro fertilized (IVF) embryos before implantation, they appeared to have much lower ful-term developmental efifciency in pig and cattle, and thus it would be reasonable to postulate that profound distinction at the molecular level should exist between them. Herein, RNA sequencing technique was used to screen differential y expressed genes in cloned and IVF blastocysts, and in total 628 differential y expressed transcripts were obtained, among which, 280 transcripts are up-regulated and 348 transcripts are down-regulated in cloned blastocysts. Moreover, one statistical y signiifcant pathway associated with endoplasmic reticulum (ER) protein processing was enriched, and some ER-stress markers such as ATF4, ATF6, PDIA3, HSPA1B, HSP40 and HSP90 between cloned and IVF blastocysts were suggested. Additional y, some developmental y important genes such as lipid metabolism related genes (MGLL, DDHD2 and FADS2) and epigenetic modiifcation genes (DNMT1, KDM5C and MBD3L5) were found differential y expressed be-tween cloned and IVF embryos.

  5. Production of transgenic7blastocyst of sheep by somatic cell cloning


    Five samples from primary cultures of five sheep ovarian granulosa cells were transfeeted by pEGFP N1 DNA. Five transgenic positive cell lines, each from one of the five samples above, were used as donor nuclei for somatic nucleus transfer. A total of 352 in vitro matured and enucle ated sheep oocytes were fused electrically with transgenic granulosa cells and 329 reconstructed embryos were ob tained after activation by Ionomycin/6-DMAP, and these embryos were cultured in SOFaaBSA medium for 7 d. The result shows that 312 embryos (94.8%) had gone through cleavage and among them 63 (19.1%) had developed to the blastocyst stage. Expression of GFP gene was detected in various stages of early embryonic development by sampling randomly. Blastocyst rates given by the four cells treated with 0.5% FCS starvation was 19.6% (55/280) and it had not shown difference significantly (P>0.05) with the result ob tained with another cell line that had not gone through se rum starvation (16.3%, 8/49). This experiment indicates that sheep transgenic embryos up to the blastocyst stage can be produced effectively by the combination of gene transfection in somatic cells in culture and somatic cell cloning.

  6. Cytotoxic Effects of Dillapiole on Embryonic Development of Mouse Blastocysts in Vitro and in Vivo

    Wen-Hsiung Chan


    Full Text Available We examined the cytotoxic effects of dillapiole, a phenylpropanoid with antileishmanial, anti-inflammatory, antifungal, and acaricidal activities, on the blastocyst stage of mouse embryos, subsequent embryonic attachment and outgrowth in vitro, and in vivo implantation via embryo transfer. Blastocysts treated with 2.5–10 μM dillapiole exhibited a significant increase in apoptosis and corresponding decrease in total cell number. Notably, the implantation success rates of blastocysts pretreated with dillapiole were lower than those of their control counterparts. Moreover, in vitro treatment with 2.5–10 μM dillapiole was associated with increased resorption of post-implantation embryos and decreased fetal weight. Our results collectively indicate that dillapiole induces apoptosis and retards early post-implantation development, both in vitro and in vivo. However, the extent to which this organic compound exerts teratogenic effects on early human development is not known at present. Further studies are required to establish effective protection strategies against the cytotoxic effects of dillapiole.

  7. Melatonin promotes the in vitro development of pronuclear embryos and increases the efficiency of blastocyst implantation in murine.

    Wang, Feng; Tian, XiuZhi; Zhang, Lu; Tan, DunXian; Reiter, Russel J; Liu, GuoShi


    When a defect occurs in the in vitro development of a pronuclear embryo, the interruption of the subsequent implantation limits the success of assisted conception. This common problem remains to be solved. In this study, we observed that melatonin at its physiological concentration (10(-7)  m) significantly promoted the in vitro development of murine pronuclear embryos. This was indicated by the increased blastocyst rate, hatching blastocyst rate, and blastocyst cell number with melatonin treatment. In addition, when these blastocysts were implanted into female recipient mice, the pregnancy rates (95.0% versus control 67.8%), litter sizes (4.1 pups/litter versus control 2.7 pups/litter), and postnatal survival rates of offspring (96.84% versus control 81.24%) were significantly improved compared with their non-melatonin-treated counterparts. Mechanistic studies revealed that melatonin treatment upregulates gene expression of the antioxidant enzyme, superoxide dismutase (SOD), and the anti-apoptotic factor bcl-2 while downregulating the expression of pro-apoptotic genes p53 and caspase-3. Due to these changes, melatonin treatment reduces ROS production and cellular apoptosis during in vitro embryo development and improves the quality of blastocysts. The implantation of blastocysts with higher quality leads to more healthy offspring and increased pup survival. PMID:23772689

  8. Effect of Retinoic Acid on Apoptosis and Expression of Fas Proteins in Mouse Blastocysts Cultured In Vitro

    Yan'e XIONG; Duanlian ZHANG


    Mouse blastocysts were exposed to doses of 0,1 and 10μmol/L retinoic acid (RA) for 24h and the cytotoxic effect of RA on the mouse blastocysts in vitro was observed. FITC-labeled terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL-FITC) assay was employed to stain apoptotic cells and immunohistochemical S-P staining method was used to detect the expression of Fas protein in mouse blastocysts in vitro. The results showed that RA could induce apoptosis and increase the expression of Fas proteins of trophectoderm (TE) and inner cell mass (ICM) cells in blastocysts. Compared with the findings for the control blastocysts, exposure to RA (10μmol/L) resulted in a more significant apoptosis and higher expression level of Fas proteins (P<0.01). It was concluded that RA could induce apoptosis, which may result in a significant reduction in the average number of total cells and the trophectoderm/inner cell mass in blastocysts and an increased expression of Fas protein, suggesting that RA had a cytotoxic effect on the growth and development of early embryos in mice.

  9. Metabolic induction and early responses of mouse blastocyst developmental programming following maternal low protein diet affecting life-long health.

    Judith J Eckert

    Full Text Available Previously, we have shown that a maternal low protein diet, fed exclusively during the preimplantation period of mouse development (Emb-LPD, is sufficient to induce by the blastocyst stage a compensatory growth phenotype in late gestation and postnatally, correlating with increased risk of adult onset cardiovascular disease and behavioural dysfunction. Here, we examine mechanisms of induction of maternal Emb-LPD programming and early compensatory responses by the embryo. Emb-LPD induced changes in maternal serum metabolites at the time of blastocyst formation (E3.5, notably reduced insulin and increased glucose, together with reduced levels of free amino acids (AAs including branched chain AAs leucine, isoleucine and valine. Emb-LPD also caused reduction in the branched chain AAs within uterine fluid at the blastocyst stage. These maternal changes coincided with an altered content of blastocyst AAs and reduced mTORC1 signalling within blastocysts evident in reduced phosphorylation of effector S6 ribosomal protein and its ratio to total S6 protein but no change in effector 4E-BP1 phosphorylated and total pools. These changes were accompanied by increased proliferation of blastocyst trophectoderm and total cells and subsequent increased spreading of trophoblast cells in blastocyst outgrowths. We propose that induction of metabolic programming following Emb-LPD is achieved through mTORC1signalling which acts as a sensor for preimplantation embryos to detect maternal nutrient levels via branched chain AAs and/or insulin availability. Moreover, this induction step associates with changes in extra-embryonic trophectoderm behaviour occurring as early compensatory responses leading to later nutrient recovery.

  10. Comparison between Cleavage Stage versus Blastocyst Stage Embryo Transfer in an Egyptian Cohort Undergoing in vitro Fertilization: A Possible Role for Laser Assisted Hatching

    Hendawy, Sherif F.; Raafat, TA


    Background Extended in vitro embryo culture and blastocyst transfer have emerged as essential components of the advanced reproductive technology armamentarium, permitting selection of more advanced embryos considered best suited for transfer. Aim of study The aim of this study was to compare between cleavage stage and blastocyst stage embryo transfer in patients undergoing intracytoplasmic sperm injection, and to assess the role of assisted hatching technique in patients undergoing blastocyst...

  11. Morphokinetics of cloned mouse embryos treated with epigenetic drugs and blastocyst prediction.

    Mallol, Anna; Piqué, Laia; Santaló, Josep; Ibáñez, Elena


    Time-lapse monitoring of somatic cell nuclear transfer (SCNT) embryos may help to predict developmental success and increase birth and embryonic stem cells (ESC) derivation rates. Here, the development of ICSI fertilized embryos and of SCNT embryos, non-treated or treated with either psammaplin A (PsA) or vitamin C (VitC), was monitored, and the ESC derivation rates from the resulting blastocysts were determined. Blastocyst rates were similar among PsA-treated and VitC-treated SCNT embryos and ICSI embryos, but lower for non-treated SCNT embryos. ESC derivation rates were higher in treated SCNT embryos than in non-treated or ICSI embryos. Time-lapse microscopy analysis showed that non-treated SCNT embryos had a delayed development from the second division until compaction, lower number of blastomeres at compaction and longer compaction and cavitation durations compared with ICSI ones. Treatment of SCNT embryos with PsA further increased this delay whereas treatment with VitC slightly reduced it, suggesting that both treatments act through different mechanisms, not necessarily related to their epigenetic effects. Despite these differences, the time of completion of the third division, alone or combined with the duration of compaction and/or the presence of fragmentation, had a strong predictive value for blastocyst formation in all groups. In contrast, we failed to predict ESC derivation success from embryo morphokinetics. Time-lapse technology allows the selection of SCNT embryos with higher developmental potential and could help to increase cloning outcomes. Nonetheless, further studies are needed to find reliable markers for full-term development and ESC derivation success. PMID:26621919

  12. Ganglioside GD1a promotes oocyte maturation, furthers preimplantation development, and increases blastocyst quality in pigs

    KIM, Jin-Woo; PARK, Hyo-Jin; CHAE, Sung-Kyu; AHN, Jae-Hyun; DO, Geon-Yeop; CHOO, Young-Kug; PARK, Joung Jun; JUNG, Bae Dong; KIM, Sun-Uk; CHANG, Kyu-Tae; KOO, Deog-Bon


    Gangliosides are key lipid molecules required for the regulation of cellular processes such as proliferation, differentiation, and cell signaling, including signaling of epidermal growth factor receptor (EGFR). Epidermal growth factor (EGF) has long been considered a potential regulator of meiotic and cytoplasmic maturation in mammalian oocytes. However, there is no report on the direct effect of ganglioside GD1a in porcine oocyte maturation. In this study, we first investigated a functional link between GD1a and meiotic maturation during in vitro maturation (IVM) of porcine embryos. Moreover, we confirmed the effect of exogenous GD1a treatment on blastocyst development, quality, and fertilization rate in early embryonic development. First, we observed that the protein level of ST3GAL2, a GD1a synthesizing enzyme, significantly increased (P < 0.01) in cumulus-oocyte-complexes (COCs) during IVM progress. The proportion of arrested germinal vesicles (GV) increased in oocytes treated with EGF+GD1a (41.6 ± 1.5%) at the IVM I stage. Upon completion of meiotic maturation, the proportion of metaphase II (M II) was significantly higher (P < 0.05) in the EGF+GD1a (89.9 ± 3.6%) treated group. After IVF, the percentage of penetrated oocytes was significantly higher (P < 0.05) in the EGF+GD1a (89.1 ± 2.3%) treated group than in the control group. Furthermore, exogenous GD1a treatment improved the developmental competence and quality of blastocysts during preimplantation embryo development stage. These results suggest that ganglioside GD1a may play an important role in IVM mechanisms of porcine maturation capacity. Furthermore, our findings will be helpful for better promoting the embryo development and blastocyst quality in pigs. PMID:26860251

  13. Mouse embryo motion and embryonic development from the 2-cell to blastocyst stage using mechanical vibration systems.

    Asano, Yuka; Matsuura, Koji


    We investigated the effect of mechanical stimuli on mouse embryonic development from the 2-cell to blastocyst stage to evaluate physical factors affecting embryonic development. Shear stress (SS) applied to embryos using two mechanical vibration systems (MVSs) was calculated by observing microscopic images of moving embryos during mechanical vibration (MV). The MVSs did not induce any motion of the medium and the diffusion rate using MVSs was the same as that under static conditions. Three days of culture using MVS did not improve embryonic development. MVS transmitted MV power more efficiently to embryos than other systems and resulted in a significant decrease in development to the morula or blastocyst stage after 2 days. Comparison of the results of embryo culture using dynamic culture systems demonstrated that macroscopic diffusion of secreted materials contributes to improved development of mouse embryos to the blastocyst stage. These results also suggest that the threshold of SS and MV to induce negative effects for mouse embryos at stages earlier than the blastocyst may be lower than that for the blastocyst, and that mouse embryos are more sensitive to physical and chemical stimuli than human or pig embryos because of their thinner zona pellucida. PMID:23697534

  14. Effect of Hyaluronan on Developmental Competence and Quality of Oocytes and Obtained Blastocysts from In Vitro Maturation of Bovine Oocytes

    Jolanta Opiela


    Full Text Available The objective of the present study was to evaluate the effect of hyaluronan (HA during IVM on meiotic maturation, embryonic development, and the quality of oocytes, granulosa cells (GC, and obtained blastocysts. COCs were matured in vitro in control medium and medium with additional 0.035% or 0.07% of exogenous HA. The meiotic maturity did not differ between the analysed groups. The best rate and the highest quality of obtained blastocysts were observed when 0.07% HA was used. A highly significant difference (P<0.001 was noted in the mean number of apoptotic nuclei per blastocyst and in the DCI between the 0.07% HA and the control blastocysts (P<0.01. Our results suggest that addition of 0.035% HA and 0.07% HA to oocyte maturation media does not affect oocyte nuclear maturation and DNA fragmentation. However, the addition of 0.07% HA during IVM decreases the level of blastocysts DNA fragmentation. Finally, our results suggest that it may be risky to increase the HA concentration during IVM above 0.07% as we found significantly higher Bax mRNA expression levels in GC cultured with 0.07% HA. The final concentration of HA being supplemented to oocyte maturation media is critical for the success of the IVP procedure.

  15. Development of In Vitro Derived Sheep Embryos to the Blastocyst Stage

    BİRLER, Sema; PABUÇÇUOĞLU, Serhat; ALKAN, Serhat; ÖZDAŞ, Özen Banu


    The objective of the present study was to mature and fertilize primary sheep oocytes in vitro and to develop them to the blastocyst stage. Ovaries from slaughtered Kıvırcık ewes were used. The ovaries were transported to the laboratory in a vacuum flask in which 30-35 °C PBS (phosphate buffered saline) was included. The cumulus-oocyte complexes were collected by rupturing and washing the follicle wall with oocyte washing medium. They were then selected under a stereo microscope and maturated ...

  16. Numerical chromosome errors in day 7 somatic nuclear transfer bovine blastocysts

    Booth, Paul J; Viuff, Dorthe; Tan, Shijian;


    Day 7 bovine somatic nuclear transfer (NT) embryos reconstructed from granulosa cells were examined for numerical chromosome aberrations as a potential cause of the high embryonic and fetal loss observed in such embryos after transfer. The NT embryos were reconstructed using a zona-free manipulat......Day 7 bovine somatic nuclear transfer (NT) embryos reconstructed from granulosa cells were examined for numerical chromosome aberrations as a potential cause of the high embryonic and fetal loss observed in such embryos after transfer. The NT embryos were reconstructed using a zona...... families, consisting of 112 blastocysts reconstructed from five different primary granulosa cell cultures, were examined. Overall, the mean chromosome complement within embryos was 86.9 +/- 3.7% (mean +/- SEM) diploid, 2.6 +/- 0.5% triploid, 10.0 +/- 3.1% tetraploid, and 0.5 +/- 0.2% pentaploid or greater......; the vast majority (>75%) of the abnormal nuclei were tetraploid. Completely diploid and mixoploid embryos represented 22.1 +/- 4.5% and 73.7 +/- 5.5%, respectively, of all clones. Six totally polyploid blastocysts, containing or=5N chromosome complements, respectively) between two clone families were...

  17. Blastocyst recovery and multifactorial gene expression analysis in the wild guinea pig (Cavia aperea).

    Hribal, Romy; Guenther, Anja; Rübensam, Kathrin; Jewgenow, Katarina


    The expression of specific developmentally important genes in preimplantation embryos is an accepted marker for unraveling the influence of single factors in studies that are mostly related to artificial reproduction techniques. Such studies, however, often reveal high levels of heterogeneity between single embryos, independently of the influence of factors of interest. A possible explanation for this variation could be the large variety of physiological and environmental factors to which early embryos are exposed and their ability to react to them. Here, we investigated several potentially important parameters of development at the same time, in blastocysts of the wild guinea pig (Cavia aperea) generated in vivo after natural mating. The optimal time for flushing fully developed blastocysts was between 123 and 126 hours after mating. The abundance of POU5F1 (P = 0.042), BAX (P sire. In addition, the identity of the father had a significant influence on POU5F1 (P = 0.049) and STAT3 (P < 0.001) mRNA abundances. These data report that the expression of specific genes in early embryos reflects the entire heterogeneity of their surroundings and that it is a plastic reaction toward a multifactorial environment. PMID:27264741

  18. Embryonic stem-like cells derived from in vitro produced bovine blastocysts

    Erika Regina Leal de Freitas


    Full Text Available The aim of this work was to study the derivation of bovine embryonic stem-like (ES-like cells from the inner cell mass (ICM of in vitro produced blastocysts. The ICMs were mechanically isolated and six out of seventeen (35% ICMs could attach to a monolayer of murine embryonic fibroblasts (MEF. Ten days after, primary outgrowths were mechanically dissected into several small clumps and transferred to a new MEF layer. Cells were further propagated and passaged by physical dissociation over a 60 days period. The pluripotency of the bovine ES-like cells was confirmed by RT-PCR of Oct-4 and STAT-3 gene markers. The colonies were weakly stained for alkaline phosphatase and the mesoderm and endoderm differentiation gene markers such as GATA-4 and Flk-1, respectively, were not expressed. Embryoid bodies were spontaneously formed at the seventh passage. Results showed that bovine ES-like cells could be obtained and passaged by mechanical procedures from the fresh in vitro produced blastocysts.

  19. Promotion of human early embryonic development and blastocyst outgrowth in vitro using autocrine/paracrine growth factors.

    Kazuhiro Kawamura

    Full Text Available Studies using animal models demonstrated the importance of autocrine/paracrine factors secreted by preimplantation embryos and reproductive tracts for embryonic development and implantation. Although in vitro fertilization-embryo transfer (IVF-ET is an established procedure, there is no evidence that present culture conditions are optimal for human early embryonic development. In this study, key polypeptide ligands known to be important for early embryonic development in animal models were tested for their ability to improve human early embryo development and blastocyst outgrowth in vitro. We confirmed the expression of key ligand/receptor pairs in cleavage embryos derived from discarded human tri-pronuclear zygotes and in human endometrium. Combined treatment with key embryonic growth factors (brain-derived neurotrophic factor, colony-stimulating factor, epidermal growth factor, granulocyte macrophage colony-stimulating factor, insulin-like growth factor-1, glial cell-line derived neurotrophic factor, and artemin in serum-free media promoted >2.5-fold the development of tri-pronuclear zygotes to blastocysts. For normally fertilized embryos, day 3 surplus embryos cultured individually with the key growth factors showed >3-fold increases in the development of 6-8 cell stage embryos to blastocysts and >7-fold increase in the proportion of high quality blastocysts based on Gardner's criteria. Growth factor treatment also led to a 2-fold promotion of blastocyst outgrowth in vitro when day 7 surplus hatching blastocysts were used. When failed-to-be-fertilized oocytes were used to perform somatic cell nuclear transfer (SCNT using fibroblasts as donor karyoplasts, inclusion of growth factors increased the progression of reconstructed SCNT embryos to >4-cell stage embryos. Growth factor supplementation of serum-free cultures could promote optimal early embryonic development and implantation in IVF-ET and SCNT procedures. This approach is valuable for

  20. Effect of Culture System on Developmental Competence, Cryosurvival and DNA-Fragmentation of In Vitro Bovine Blastocysts

    Mahdi Hajian


    Full Text Available Background: This study investigated the effect of two in vitro embryo culture systems (co-culturesystem versus cell-free sequential-media on developmental competence, cryosurvival and DNAfragmentationof in vitro developed bovine blastocysts.Materials and Methods: Bovine presumptive zygotes were cultured in Ménézo's B2 (B2 plusvero-cells or sequential synthetic oviductal fluid (SOF for eight days. Subsequently, half of theexpanded blastocysts developed in both groups were vitrified, warmed within 30 minutes and postwarmingembryos along with their corresponding non-vitrified embryos were cultured for twoadditional days in the same medium used before vitrification. Embryo development, cryosurvivaland apoptosis were compared between the groups.Results: For non-vitrified embryos, culture in SOF significantly promoted the potency of embryosto develop into blastocysts compared with the co-culture system. The difference in post vitrificationsurvival rate of SOF blastocysts (83.3% was insignificant compared with co-culture (84.3%.However, while total cell number of warmed blastocysts in the co-culture system was significantlyhigher in the co-culture versus the sequential system (215.4 vs. 170.4, the quality of survived embryosin terms of hatching ability and apoptosis was adversely affected by co-culture compared with SOF(65.0% vs. 74.3%, and 13.5% vs. 10.0%, respectively; p<0.05.Conclusion: Although co-culture system may increase the viability of embryos followingcryopreservation, the potency and dynamics of blastocyst formation significantly increased withsequential media compared to the co-culture system which can compensate for the lower efficiency ofsequential media for vitrification/warming purposes.

  1. Linolenic acid improves oocyte developmental competence and decreases apoptosis of in vitro-produced blastocysts in goat.

    Veshkini, Arash; Khadem, Ali Akbar; Mohammadi-Sangcheshmeh, Abdollah; Alamouti, Ali Asadi; Soleimani, Masoud; Gastal, Eduardo L


    The effects of α-linolenic acid (ALA) on developmental competence of oocytes in goats were evaluated in this study. Initially, the level of ALA in small and large antral follicles was determined to be in a range of 0.018-0.028 mg/ml (64.6-100.6 μM, respectively). In vitro maturation was performed in the presence of various concentrations (10, 50, 100, or 200 μM) of ALA. Cumulus expansion, meiotic maturation, levels of intracellular glutathione (GSH), embryonic cleavage, blastocyst formation following parthenogenetic activation (PA) and in vitro fertilization (IVF), number of total and apoptotic cells in blastocyst, and expression of Bax, Bcl-2, and p53 genes in blastocyst cells were determined. Compared with the control, no improvement was observed in cumulus expansion in ALA-treated groups. At 50 μM concentration, ALA increased meiotic maturation rate but had no effect on GSH level. When oocytes treated with 50 μM ALA were subsequently used for PA or IVF, a higher rate of blastocyst formation was observed, and these embryos had a higher total cell number and a lower apoptotic cell number. Expression analyses of genes in blastocysts revealed lesser transcript abundances for Bax gene, and higher transcript abundances for Bcl-2 gene in 50 μM ALA group. Expression of p53 gene was also less observed in ALA-treated blastocysts. Our results show that ALA treatment at 50 μM during in vitro maturation (IVM) had a beneficial effect on maturation of goat oocytes and this, in turn, stimulated embryonic development and regulated apoptotic gene expression. PMID:26584822

  2. Establishment of a pig fibroblast-derived cell line for locus-directed transgene expression in cell cultures and blastocysts

    Jakobsen, Jannik E; Li, Juan; Moldt, Brian;


    We report the establishment of a spontaneously immortalized pig cell line designated Pig Flip-in Visualize (PFV) for locus-directed transgene expression in pig cells and blastocysts. The PFV cell line was isolated from pig ear fibroblasts transfected with a Sleeping Beauty DNA transposon-based do......We report the establishment of a spontaneously immortalized pig cell line designated Pig Flip-in Visualize (PFV) for locus-directed transgene expression in pig cells and blastocysts. The PFV cell line was isolated from pig ear fibroblasts transfected with a Sleeping Beauty DNA transposon...

  3. DNA aneuploidy in colorectal adenomas: Role in the adenoma-carcinoma sequence Aneuploidía del ADN en adenomas colónicos: Papel en la secuencia adenoma-carcinoma

    M. Alcántara Torres


    Full Text Available Introduction: aneuploidy has been observed in 6-27% of lesions known to be precursors of colorectal cancer, such as adenomas or ulcerative colitis. It has been suggested that aneuploidy may predispose to malignancy in these cases. However, its role in the adenoma-carcinoma sequence has not been definitely established. The objective of this study was to assess the incidence of aneuploidy in colon adenomas, as well as to study its possible role in the adenoma-carcinoma sequence. Material and methods: the study was performed on a series of 57 large bowel adenomas measuring 10 mm or more, collected from 54 consecutive patients. All specimens were obtained either by endoscopic or by surgical resection. There were 49 adenomas with low-grade dysplasia, two with high-grade dysplasia, two intramucous carcinomas, and four microinvasive carcinomas. A flow cytometric DNA analysis was performed in fresh specimens following Vindelov´s method. Results: aneuploid DNA was detected in five out of 49 low-grade dysplasia adenomas (10%, in all four high-grade dysplasia adenomas or intramucous carcinomas (100%, and in three out of four microinvasive carcinomas (75%. The association between aneuploidy and high-grade dysplasia adenomas, intramucous, or microinvasive carcinoma was statistically significant (p Introducción: en patología benigna de intestino grueso precursora del cáncer colorrectal, como adenomas o colitis ulcerosa, se ha observado aneuploidía en el 6-27% de los casos y se ha sugerido que su presencia predispone al desarrollo de malignidad. Sin embargo, su papel en la secuencia adenoma-carcinoma no se ha demostrado de forma concluyente. El objetivo de nuestro trabajo fue valorar la incidencia de aneuploidía en adenomas colónicos, con y sin signos de malignidad, y estudiar su posible papel en la secuencia adenoma-carcinoma. Material y métodos: el estudio se realizó en una serie de 57 adenomas de intestino grueso, de 10 o más mil

  4. Effect of Ca Ionophore On Blastocyst Production Following Intracytoplasmic Sperm Injection in Caprine Oocytes.

    Kharche, S D; Pathak, J; Agarwal, S; Kushwah, B; Sikarwar, Aks


    The aim of the present investigation was to study the effect of calcium ionophore activation on blastocyst production following intracytoplasmic sperm injection (ICSI) in in vitro-matured Caprine oocytes. A total of 470 in vitro-matured oocytes were selected and randomly divided in to three groups. Cumulus oocyte complexes (COCs) recovered by slicing the Caprine ovaries were matured in TCM199 supplemented with 10% foetal bovine serum (FBS) + 10% follicular fluid + FSH (5 μg/ml) + LH (10 μg/ml) + estradiol (1 μg/ml) + EGF (10 ng/ml) + BSA (3 mg/ml) for 27 h in humidified atmosphere at 38.5°C with 5% CO2 in CO2 incubator. After 27 h of culture, selected COCs (n = 470) were separated from cumulus cells by treating with 0.1% hyaluronidase enzyme and passing repeatedly through a fine pipette and randomly divided into three groups. In group 1, (n = 168) matured oocytes were injected with injection micropipette without sperm as control. In group 2, (n = 152) capacitated spermatozoa were injected into cytoplasm of in vitro-matured oocytes through injection micropipette. In group 3, (n = 150) capacitated spermatozoa were injected into cytoplasm of in vitro-matured oocytes through injection micropipette and then activated with 5 μm Ca ionophore for 5 min. The oocytes of all groups were then culture in RVCL media for embryo development. The cleavage rate was observed after 48-72 h of injection. The cleavage rate and blastocyst production in group 1, 2 and 3 were 0.00 and 0.00, 18.42 and 3.57 and 61.33% and 16.30%, respectively. The result indicated that mechanical activation failed to induce cleavage in in vitro-matured Caprine oocytes, whereas chemical activation of intracytoplasmic sperm-injected in vitro-matured Caprine oocytes showed significantly higher cleavage rate and blastocyst production as compare to non-activated oocytes. PMID:27170442

  5. Ultrastructure of IVM-IVF bovine blastocysts vitrified after equilibration in glycerol 1,2-propanediol using 2-step and 16-step procedures.

    Kuwayama, M; Fujikawa, S; Nagai, T


    Two experiments were conducted to investigate the relationship between survival rates and ultrastructural appearance, using the freeze-replica technique, of bovine blastocysts after vitrification. In experiment 1, blastocysts obtained from in vitro-matured and in vitro-fertilized (IVM-IVF) bovine oocytes were either equilibrated in a stepwise manner in a vitrification solution (VS; 22.5% glycerol + 22.5% 1,2-propanediol) using 16 steps for 18 min in total (the 16-step method) or equilibrated with 10% glycerol + 20% 1,2-propanediol for 10 min and then exposed to VS (the 2-step method). The blastocysts were then vitrified by plunging them into liquid nitrogen. All samples were subsequently thawed in a water bath at 37 degrees C and cultured in vitro with a monolayer of cumulus cells. The survival rate obtained for blastocysts equilibrated by the 16-step method was 83.3% (25/30). In contrast, no blastocysts survived by the 2-step method (0/30). In experiment 2, freeze-replica observations were carried out on blastocysts vitrified by the 16-step method and the 2-step method. In all the blastocysts, no ice crystals were observed in the cytoplasm, blastocoelic cavity, or extracellular areas, which confirmed the occurrence of complete vitrification. Little ultrastructural change was observed in the plasma membrane of the blastocysts equilibrated by the 16-step method. In contrast, small vesicles and distinct intramembrane particle (IMP) aggregation were frequently observed in the plasma membranes of blastocysts equilibrated by the 2-step method. These results indicate that the successful cryopreservation of blastocysts following the 16-step equilibration requires not only complete vitrification, but also minimization of ultrastructural damage to the plasma membrane. PMID:7988150

  6. Neuroanatomical localization and quantification of amyloid precursor protein mRNA by in situ hybridization in the brains of normal, aneuploid, and lesioned mice

    Amyloid precursor protein mRNA was localized in frozen sections from normal and experimentally lesioned adult mouse brain and from normal and aneuploid fetal mouse brain by in situ hybridization with a 35S-labeled mouse cDNA probe. The highest levels of hybridization in adult brain were associated with neurons, primarily in telencephalic structures. The dense labeling associated with hippocampal pyramidal cells was reduced significantly when the cells were eliminated by injection of the neurotoxin ibotenic acid but was not affected when electrolytic lesions were placed in the medial septum. Since the gene encoding amyloid precursor protein has been localized to mouse chromosome 16, the authors also examined the expression of this gene in the brains of mouse embryos with trisomy 16 and trisomy 19 at 15 days of gestation. RNA gel blot analysis and in situ hybridization showed a marked increase in amyloid precursor protein mRNA in the trisomy 16 mouse head and brain when compared with euploid littermates or with trisomy 19 mice

  7. Derivation of Porcine Embryonic Stem-Like Cells from In Vitro-Produced Blastocyst-Stage Embryos

    Hou, Dao-Rong; Jin, Yong; Nie, Xiao-Wei; Zhang, Man-Ling; Ta, Na; Zhao, Li-Hua; Yang, Ning; Chen, Yuan; Wu, Zhao-Qiang; Jiang, Hai-Bin; Li, Yan-Ru; Sun, Qing-Yuan; Dai, Yi-Fan; Li, Rong-Feng


    Efficient isolation of embryonic stem (ES) cells from pre-implantation porcine embryos has remained a challenge. Here, we describe the derivation of porcine embryonic stem-like cells (pESLCs) by seeding the isolated inner cell mass (ICM) from in vitro-produced porcine blastocyst into α-MEM with basic fibroblast growth factor (bFGF). The pESL cells kept the normal karyotype and displayed flatten clones, similar in phenotype to human embryonic stem cells (hES cells) and rodent epiblast stem cells. These cells exhibited alkaline phosphatase (AP) activity and expressed pluripotency markers such as OCT4, NANOG, SOX2, SSEA-4, TRA-1-60, and TRA-1-81 as determined by both immunofluorescence and RT-PCR. Additionally, these cells formed embryoid body (EB), teratomas and also differentiated into 3 germ layers in vitro and in vivo. Microarray analysis showed the expression of the pluripotency markers, PODXL, REX1, SOX2, KLF5 and NR6A1, was significantly higher compared with porcine embryonic fibroblasts (PEF), but expression of OCT4, TBX3, REX1, LIN28A and DPPA5, was lower compared to the whole blastocysts or ICM of blastocyst. Our results showed that porcine embryonic stem-like cells can be established from in vitro-produced blastocyst-stage embryos, which promote porcine naive ES cells to be established. PMID:27173828

  8. Derivation of Porcine Embryonic Stem-Like Cells from In Vitro-Produced Blastocyst-Stage Embryos.

    Hou, Dao-Rong; Jin, Yong; Nie, Xiao-Wei; Zhang, Man-Ling; Ta, Na; Zhao, Li-Hua; Yang, Ning; Chen, Yuan; Wu, Zhao-Qiang; Jiang, Hai-Bin; Li, Yan-Ru; Sun, Qing-Yuan; Dai, Yi-Fan; Li, Rong-Feng


    Efficient isolation of embryonic stem (ES) cells from pre-implantation porcine embryos has remained a challenge. Here, we describe the derivation of porcine embryonic stem-like cells (pESLCs) by seeding the isolated inner cell mass (ICM) from in vitro-produced porcine blastocyst into α-MEM with basic fibroblast growth factor (bFGF). The pESL cells kept the normal karyotype and displayed flatten clones, similar in phenotype to human embryonic stem cells (hES cells) and rodent epiblast stem cells. These cells exhibited alkaline phosphatase (AP) activity and expressed pluripotency markers such as OCT4, NANOG, SOX2, SSEA-4, TRA-1-60, and TRA-1-81 as determined by both immunofluorescence and RT-PCR. Additionally, these cells formed embryoid body (EB), teratomas and also differentiated into 3 germ layers in vitro and in vivo. Microarray analysis showed the expression of the pluripotency markers, PODXL, REX1, SOX2, KLF5 and NR6A1, was significantly higher compared with porcine embryonic fibroblasts (PEF), but expression of OCT4, TBX3, REX1, LIN28A and DPPA5, was lower compared to the whole blastocysts or ICM of blastocyst. Our results showed that porcine embryonic stem-like cells can be established from in vitro-produced blastocyst-stage embryos, which promote porcine naive ES cells to be established. PMID:27173828

  9. Rat Blastocysts from Nuclear Injection and Time-Lagged Enucleation and Their Commitment to Embryonic Stem Cells.

    Hara, Hiromasa; Goto, Teppei; Takizawa, Akiko; Sanbo, Makoto; Jacob, Howard J; Kobayashi, Toshihiro; Nakauchi, Hiromitsu; Hochi, Shinichi; Hirabayashi, Masumi


    Pronucleus-like vesicle formation following premature chromosome condensation (PCC) of the donor cell nucleus is the key event for successful generation of cloned rodents by nuclear transplantation (NT). However in rat cloning, this change is difficult to induce in enucleated recipient oocytes because of their inability to maintain maturation-promoting factor levels. In this study, intact oocytes retrieved from nuclear-visualized H2B-tdTomato knock-in rats were injected with Venus-labeled cell nuclei. Because the incidence of PCC under MG-132 treatment significantly increased with the culture period (0%, 10.8%, 36.8%, and 87.5% at 0, 0.5, 1, and 2 h postinjection, respectively), the metaphase plate of the oocyte was removed 1-2 h after the nuclear injection. The NT-derived rat zygotes (n = 748) were activated with ionomycin/cycloheximide and transferred into temporal host mothers, resulting in the harvest of three blastocysts (0.4%) with Venus fluorescence. Two blastocysts were examined for their potential to commit to NT-derived embryonic stem cells (ntESCs). One ntESC line was established successfully and found to be competent in terms of karyotype, stem cell marker expression, and pluripotency. In conclusion, time-lagged enucleation of visualized oocyte nuclei allows the PCC incidence of donor nuclei and generation of NT blastocysts, and the blastocysts can commit to germline-competent ntESCs. PMID:26990947

  10. Analysis of Marker Expression in Porcine Cell Lines Derived from Blastocysts Produced In Vitro and In Vivo

    Vacková, I.; Nováková, Z.; Krylov, V.; Okada, K.; Kott, T.; Fulka, H.; Motlík, Jan


    Roč. 57, č. 5 (2011), s. 594-603. ISSN 0916-8818 R&D Projects: GA MZe QI101A166; GA MŠk 1M0538 Institutional research plan: CEZ:AV0Z50450515 Keywords : Blastocyst * In vivo and in vitro * Pig * Stem cells Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.459, year: 2011

  11. Blastocyst injection of wild type embryonic stem cells induces global corrections in mdx mice.

    Elizabeth Stillwell

    Full Text Available Duchenne muscular dystrophy (DMD is an incurable neuromuscular degenerative disease, caused by a mutation in the dystrophin gene. Mdx mice recapitulate DMD features. Here we show that injection of wild-type (WT embryonic stem cells (ESCs into mdx blastocysts produces mice with improved pathology and function. A small fraction of WT ESCs incorporates into the mdx mouse nonuniformly to upregulate protein levels of dystrophin in the skeletal muscle. The chimeric muscle shows reduced regeneration and restores dystrobrevin, a dystrophin-related protein, in areas with high and with low dystrophin content. WT ESC injection increases the amount of fat in the chimeras to reach WT levels. ESC injection without dystrophin does not prevent the appearance of phenotypes in the skeletal muscle or in the fat. Thus, dystrophin supplied by the ESCs reverses disease in mdx mice globally in a dose-dependent manner.

  12. High-Resolution Flow Cytometry: a Suitable Tool for Monitoring Aneuploid Prostate Cancer Cells after TMZ and TMZ-BioShuttle Treatment

    Braun, Klaus; Ehemann, Volker; Wiessler, Manfred; Pipkorn, Ruediger; Didinger, Bernd; Mueller, Gabriele; Waldeck, Waldemar


    If metastatic prostate cancer gets resistant to antiandrogen therapy, there are few treatment options, because prostate cancer is not very sensitive to cytostatic agents. Temozolomide (TMZ) as an orally applicable chemotherapeutic substance has been proven to be effective and well tolerated with occasional moderate toxicity especially for brain tumors and an application to prostate cancer cells seemed to be promising. Unfortunately, TMZ was inefficient in the treatment of symptomatic progressive hormone-refractory prostate cancer (HRPC). The reasons could be a low sensitivity against TMZ the short plasma half-life of TMZ, non-adapted application regimens and additionally, the aneuploid DNA content of prostate cancer cells suggesting different sensitivity against therapeutical interventions e.g. radiation therapy or chemotherapy. Considerations to improve this unsatisfying situation resulted in the realization of higher local TMZ concentrations, sufficient to kill cells regardless of intrinsic cellular sensitivity and cell DNA-index. Therefore, we reformulated the TMZ by ligation to a peptide-based carrier system called TMZ-BioShuttle for intervention. The modular-composed carrier consists of a transmembrane transporter (CPP), connected to a nuclear localization sequence (NLS) cleavably-bound, which in turn was coupled with TMZ. The NLS-sequence allows an active delivery of the TMZ into the cell nucleus after transmembrane passage of the TMZ-BioShuttle and intra-cytoplasm enzymatic cleavage and separation from the CPP. This TMZ-BioShuttle could contribute to improve therapeutic options exemplified by the hormone refractory prostate cancer. The next step was to syllogize a qualified method monitoring cell toxic effects in a high sensitivity under consideration of the ploidy status. The high-resolution flow cytometric analysis showed to be an appropriate system for a better detection and distinction of several cell populations dependent on their different DNA

  13. High-Resolution Flow Cytometry: a Suitable Tool for Monitoring Aneuploid Prostate Cancer Cells after TMZ and TMZ-BioShuttle Treatment

    Klaus Braun, Volker Ehemann, Manfred Wiessler, Ruediger Pipkorn, Bernd Didinger, Gabriele Mueller, Waldemar Waldeck


    Full Text Available If metastatic prostate cancer gets resistant to antiandrogen therapy, there are few treatment options, because prostate cancer is not very sensitive to cytostatic agents. Temozolomide (TMZ as an orally applicable chemotherapeutic substance has been proven to be effective and well tolerated with occasional moderate toxicity especially for brain tumors and an application to prostate cancer cells seemed to be promising. Unfortunately, TMZ was inefficient in the treatment of symptomatic progressive hormone-refractory prostate cancer (HRPC. The reasons could be a low sensitivity against TMZ the short plasma half-life of TMZ, non-adapted application regimens and additionally, the aneuploid DNA content of prostate cancer cells suggesting different sensitivity against therapeutical interventions e.g. radiation therapy or chemotherapy. Considerations to improve this unsatisfying situation resulted in the realization of higher local TMZ concentrations, sufficient to kill cells regardless of intrinsic cellular sensitivity and cell DNA-index. Therefore, we reformulated the TMZ by ligation to a peptide-based carrier system called TMZ-BioShuttle for intervention. The modular-composed carrier consists of a transmembrane transporter (CPP, connected to a nuclear localization sequence (NLS cleavably-bound, which in turn was coupled with TMZ. The NLS-sequence allows an active delivery of the TMZ into the cell nucleus after transmembrane passage of the TMZ-BioShuttle and intra-cytoplasm enzymatic cleavage and separation from the CPP. This TMZ-BioShuttle could contribute to improve therapeutic options exemplified by the hormone refractory prostate cancer. The next step was to syllogize a qualified method monitoring cell toxic effects in a high sensitivity under consideration of the ploidy status. The high-resolution flow cytometric analysis showed to be an appropriate system for a better detection and distinction of several cell populations dependent on their

  14. Conserved roles of fibroblast growth factor receptor 2 signaling in the regulation of inner cell mass development in bovine blastocysts.

    Akizawa, Hiroki; Nagatomo, Hiroaki; Odagiri, Haruka; Kohri, Nanami; Yamauchi, Nobuhiko; Yanagawa, Yojiro; Nagano, Masashi; Takahashi, Masashi; Kawahara, Manabu


    A common process during preimplantation mammalian development is blastocyst formation, which utilizes signaling through fibroblast growth factor receptor 2 (FGFR2), yet the mechanisms through which FGFR2 signaling affect preimplantation development in bovine embryos remain incompletely understood. Here, we used RNA-interference to investigate the in vitro development, the frequency of blastomere apoptosis, and the mRNA expression of developmental marker genes in FGF receptor 2-knockdown (FGFR2-KD) bovine embryos. A reduction in FGFR2 mRNA did not affect preimplantation development or the frequency of apoptotic blastomeres, but did enhanced proliferation of the inner cell mass in blastocysts (P cattle. Mol. Reprod. Dev. 83: 516-525, 2016. © 2016 Wiley Periodicals, Inc. PMID:27060901

  15. Oct4 is required for lineage priming in the developing inner cell mass of the mouse blastocyst

    Le Bin, Gloryn Chia; Muñoz-Descalzo, Silvia; Kurowski, Agata; Leitch, Harry; Lou, Xinghua; Mansfield, William; Etienne-Dumeau, Charles; Grabole, Nils; Mulas, Carla; Niwa, Hitoshi; Hadjantonakis, Anna-Katerina; Nichols, Jennifer


    The transcription factor Oct4 is required in vitro for establishment and maintenance of embryonic stem cells and for reprogramming somatic cells to pluripotency. In vivo, it prevents the ectopic differentiation of early embryos into trophoblast. Here, we further explore the role of Oct4 in blastocyst formation and specification of epiblast versus primitive endoderm lineages using conditional genetic deletion. Experiments involving mouse embryos deficient for both maternal and zygotic Oct4 sug...

  16. The primitive endoderm lineage of the mouse blastocyst: Sequential transcription factor activation and regulation of differentiation by Sox17

    Artus, Jérôme; Piliszek, Anna; Hadjantonakis, Anna-Katerina


    Cells of the primitive endoderm (PrE) and the pluripotent epiblast (EPI), the two lineages specified within the inner cell mass (ICM) of the mouse blastocyst stage embryo, are segregated into adjacent tissue layers by the end of the preimplantation period. The PrE layer which emerges as a polarized epithelium adjacent to the blastocoel, with a basement membrane separating it from the EPI, has two derivatives, the visceral and parietal endoderm. In this study we have investigated the localizat...

  17. Endometrial thickness, Caucasian ethnicity, and age predict clinical pregnancy following fresh blastocyst embryo transfer: a retrospective cohort

    Santoro Nanette


    Full Text Available Abstract Background In-vitro fertilization (IVF with blastocyst as opposed to cleavage stage embryos has been advocated to improve success rates. Limited information exists on which to predict which patients undergoing blastocyst embryo transfer (BET will achieve pregnancy. This study's objective was to evaluate the predictive value of patient and cycle characteristics for clinical pregnancy following fresh BET. Methods This was a retrospective cohort study from 2003–2007 at an academic assisted reproductive program. 114 women with infertility underwent fresh IVF with embryo transfer. We studied patients undergoing transfer of embryos at the blastocyst stage of development. Our main outcome of interest was clinical pregnancy. Clinical pregnancy and its associations with patient characteristics (age, body mass index, FSH, ethnicity and cycle parameters (thickness of endometrial stripe, number eggs, available cleaving embryos, number blastocysts available, transferred, and cryopreserved, and embryo quality were examined using Student's T test and Mann-Whitney-U tests as appropriate. Multivariable logistic regression models were created to determine independent predictors of CP following BET. Receiver Operating Characteristic analyses were used to determine the optimal thickness of endometrial stripe for predicting clinical pregnancy. Results Patients achieving clinical pregnancy demonstrated a thicker endometrial stripe and were younger preceding embryo transfer. On multivariable logistic regression analyses, Caucasian ethnicity (OR 2.641, 95% CI 1.054–6.617, thickness of endometrial stripe, (OR 1.185, 95% CI 1.006–1.396 and age (OR 0.879, 95% CI 0.789–0.980 predicted clinical pregnancy. By receiver operating characteristic analysis, endometrial stripe ≥ 9.4 mm demonstrated a sensitivity of 83% for predicting clinical pregnancy following BET. Conclusion In a cohort of patients undergoing fresh BET, thicker endometrial stripe, Caucasian

  18. Introduction: Examining the many potential reasons why euploid blastocysts do not always result in viable pregnancies: part 1.

    Meldrum, David R


    The environment of the oocyte and embryo includes the metabolic effects of aging, lifestyle issues, psychological stress, nutritional supplements/replacements, cellular energy levels, endocrine and paracrine factors, the cumulus, exogenous gonadotropins and adjunctive medications, culture media, and the in vitro fertilization laboratory's conditions and manipulations. These complex factors, which will be examined in the following series of reviews, explain in part why not all euploid blastocysts result in viable pregnancies. PMID:26730497

  19. Effect of the bovine oviductal fluid on in vitro fertilization, development and gene expression of in vitro-produced bovine blastocysts.

    Cebrian-Serrano, A; Salvador, I; García-Roselló, E; Pericuesta, E; Pérez-Cerezales, S; Gutierrez-Adán, A; Coy, P; Silvestre, M A


    Oviductal microenvironment generally provides better conditions for early embryo development than the conventional in vitro system. In an attempt to simulate the oviduct conditions or the main potentially influencing factors, the effect was studied of a bovine oviductal fluid (bOF) treatment applied prior to IVF on (i) IVF parameters, (ii) cleavage rate, (iii) blastocyst yield and (iv) blastocyst quality. Embryo quality was assessed by morphological embryo quality and relative transcript abundance of several developmental genes in bovine blastocysts. Furthermore, to study the effect of bOF without the male effect and zona-sperm interaction, artificially activated metaphase II oocytes were also treated with bOF. In vitro-matured bovine oocytes from abattoir ovaries were treated or untreated with bOF for 30 min and then washed prior to IVF or activation. Subsequently, in vitro-fertilized and parthenogenetic embryos were in vitro cultured for 7 to 8 days. The bOF treatment had no effect on fertilization parameters, cleavage, blastocyst rates both on parthenogenetic and IVF bovine embryos and neither on morphological quality of IVF blastocysts. G6PD and SOD2 genes from IVF blastocysts showed significant changes in their expression after a bOF treatment. Significant differences were found for the expression of SCL2A1, GPX1, BAX, AKR1B1 and PLAC8 genes between excellent or good blastocysts (Grade 1) and fair blastocysts (Grade 2). To our knowledge, this is the first study that evaluates the effect of bOF oocyte treatment on fertilization parameters, development and quality of bovine embryos. PMID:22908847

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

    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

  1. Both the folate cycle and betaine-homocysteine methyltransferase contribute methyl groups for DNA methylation in mouse blastocysts.

    Zhang, Baohua; Denomme, Michelle M; White, Carlee R; Leung, Kit-Yi; Lee, Martin B; Greene, Nicholas D E; Mann, Mellissa R W; Trasler, Jacquetta M; Baltz, Jay M


    The embryonic pattern of global DNA methylation is first established in the inner cell mass (ICM) of the mouse blastocyst. The methyl donor S-adenosylmethionine (SAM) is produced in most cells through the folate cycle, but only a few cell types generate SAM from betaine (N,N,N-trimethylglycine) via betaine-homocysteine methyltransferase (BHMT), which is expressed in the mouse ICM. Here, mean ICM cell numbers decreased from 18-19 in controls to 11-13 when the folate cycle was inhibited by the antifolate methotrexate and to 12-14 when BHMT expression was knocked down by antisense morpholinos. Inhibiting both pathways, however, much more severely affected ICM development (7-8 cells). Total SAM levels in mouse blastocysts decreased significantly only when both pathways were inhibited (from 3.1 to 1.6 pmol/100 blastocysts). DNA methylation, detected as 5-methylcytosine (5-MeC) immunofluorescence in isolated ICMs, was minimally affected by inhibition of either pathway alone but decreased by at least 45-55% when both BHMT and the folate cycle were inhibited simultaneously. Effects on cell numbers and 5-MeC levels in the ICM were completely rescued by methionine (immediate SAM precursor) or SAM. Both the folate cycle and betaine/BHMT appear to contribute to a methyl pool required for normal ICM development and establishing initial embryonic DNA methylation. PMID:25466894

  2. The impact of food intake and social habits on embryo quality and the likelihood of blastocyst formation.

    Braga, Daniela Paes Almeida Ferreira; Halpern, Gabriela; Setti, Amanda S; Figueira, Rita Cássia S; Iaconelli, Assumpto; Borges, Edson


    The aim of this study was to evaluate the influence of patients' lifestyle factors and eating habits on embryo development. A total of 2659 embryos recovered from 269 patients undergoing intracytoplasmic sperm injection cycles were included. The frequency of intake of food items and social habits were registered and its influences on embryo development evaluated. The consumption of cereals, vegetables and fruits positively influenced the embryo quality at the cleavage stage. The quality of the embryo at the cleavage stage was also negatively correlated with the consumption of alcoholic drinks and smoking habits. The consumption of fruits influenced the likelihood of blastocyst formation, which was also positively affected by the consumption of fish. Being on a weight-loss diet and consumption of red meat had a negative influence on the likelihood of blastocyst formation. The likelihood of blastocyst formation was also negatively influenced by the consumption of alcoholic drinks and by smoking habits. The consumption of red meat and body mass index had a negative effect on the implantation rate and the likelihood of pregnancy. In addition, being on a weight-loss diet had a negative influence on implantation rate. Our evidence suggests a possible relationship between environmental factors and ovary biology. PMID:25982093

  3. Developmental stage on day-5 and fragmentation rate on day-3 can influence the implantation potential of top-quality blastocysts in IVF cycles with single embryo transfer

    Devroey Paul


    Full Text Available Abstract Background In IVF-ICSI cycles with single embryo transfer (SET, embryo selection for transfer is of crucial importance. The present study aimed to define which embryo parameters might be related to the implantation potential of advanced blastocysts. Methods Overall, in 203 cycles with SET, developmental characteristics of 93 implanted (group A and 110 non-implanted (group B advanced blastocysts of good quality were compared. The following developmental parameters were assessed in the two groups: normal fertilization, developmental stage on day 5, number of blastomeres on day 2 and on day 3, fragmentation rate on day 3, compaction on day 4 and cleavage pattern on day 2 and day 3. Results Expanded blastocysts compared to full blastocysts have higher implantation potential (56.5% vs. 29.3%, p 10–50% fragments on day 3 showed a significant lower implantation (29.7% than those with ≤ 10%fragments (49.4%, P = 0.03. All the other parameters analysed were comparable for the two groups. Conclusion Developmental stage on day 5 and fragmentation rate on day 3 were related to the implantation potential of advanced blastocysts and should also be taken into account in the selection of the best advanced blastocyst for transfer.

  4. Influencia de la estimulación ovárica durante un tratamiento de fecundación in vitro en la incidencia de aneuploidías embrionarias

    Labarta Demur, María Elena


    La baja fecundidad humana podría deberse a la alta incidencia de anomalías cromosómicas embrionarias que justifica que hasta un 70% de las concepciones espontáneas no lleguen a término. La elevada tasa de aneuploidías observada en tratamientos de Fecundación in vitro (FIV) podría ser inherente a la especie humana o estar relacionada con los procedimientos derivados de la técnica, como las condiciones de cultivo in vitro o la estimulación ovárica (EO). Se ha especulado que esta última pudiera ...

  5. Daily supplementation with ghrelin improves in vitro bovine blastocysts formation rate and alters gene expression related to embryo quality.

    Dovolou, Eleni; Periquesta, Eva; Messinis, Ioannis E; Tsiligianni, Theodora; Dafopoulos, Konstantinos; Gutierrez-Adan, Alfonso; Amiridis, Georgios S


    Ghrelin is a gastric peptide having regulatory role in the reproductive system functionality, acting mainly at central level. Because the expression of ghrelin system (ghrelin and its receptor) has been detected in the bovine ovary, the objectives of the present study were to investigate whether ghrelin can affect the developmental potential of in vitro-produced embryos, and to test their quality in terms of relative abundance of various genes related to metabolism, apoptosis and oxidation. In the first experiment, in vitro-produced zygotes were cultured in the absence (control [C]) and in the presence of three concentrations of acylated ghrelin (200 pg/mL [Ghr200], 800 pg/mL [Ghr800]; and 2000 pg/mL [Ghr2000]); blastocyst formation rates were examined on Days 7, 8, and 9. In the second experiment, only the 800 pg/mL dose of ghrelin was used. Zygotes were produced as in experiment 1 and 24 hours post insemination they were divided into 4 groups; in two groups (C; without ghrelin; Ghr800 with ghrelin), embryos were cultured without medium replacement; in the remaining two groups (Control N and GhrN), the culture medium was daily renewed. A pool of Day-7 blastocysts were snap frozen for relative mRNA abundance of various genes related to metabolism, oxidation, implantation, and apoptosis. In experiment 3, embryos were produced as in experiment 2, but in the absence of serum (semi-defined culture medium). In experiment 1, no differences were detected between C, Ghr200, and Ghr2000, although fewer blastocysts were produced in group Ghr800 compared with C. In experiment 2, the lowest blastocysts yield was found in Ghr800, whereas daily renewal of ghrelin (Ghr800N) resulted to increased blastocysts formation rate, which on Day 7 was the highest among groups (P quality than controls. Our results imply a specific role of ghrelin in early embryonic development; however, the specific mode of its action needs further investigation. PMID:24332928

  6. Effect of vitrification on number of inner cell mass in mouse blastocysts in conventional straw, closed pulled straw, open pulled straw and cryoloop carriers

    Objective: To compare the effect of using open and closed carriers on count of inner cell mass in vitrified mouse blastocyst after warming. Methods: The experimental study was conducted at Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran, from April to September 2010. Forty female NMRI (Naval Medical Research Institute, USA) mice were injected with pregnant mares serum gonadotropin and human chorionic gonadotropin in order to induce super ovulation. Following the latter injection, two or three females were caged with the same-breed male mice. The presence of vaginal plug was examined the following morning. To collect blastocyst embryos, the pregnant females were sacrificed by cervical dislocation at 88-90 hours after the injection and dissected. Blastocysts were collected in phosphate-buffered saline and allocated to four groups: vitrification in conventional straw, closed pulled straw, open pulled straw and cryoloop. The vitrification solution was ethylene glycol, Ficol and sucrose (EFS) 20% and 40%. After storage for 1 month in liquid nitrogen, the blastocysts were thawed in 0.5 M sucrose then cultured in M16 medium. After 6 hours of culture, the number of expanded blastocysts was recorded and stained by double-dye technique. After staining, the number of total cell and inner cell mass was calculated. Results: The re-expansion rate of blastocysts in the cryoloop group (n=90; 78.26%) was significantly higher (p<0.05) than open pulled straw (n=83; 69.16%), closed pulled straw (n=68; 54.83%) and conventional straws (n=63; 51.21%) groups. Significant differences (p<0.05) in the number of inner cell mass in blastocysts vitrified in open pulled straws, closed straws and cryoloop with blastocysts cryopreserved in conventional straws. Conclusion: The re-expansion rate and total cell number of mouse blastocysts vitrified using open system had a better result compared with the closed system. The value of cryoloop and open pulled straws as carriers in

  7. Elective single blastocyst transfer is more suitable for normal responders than for high responders

    WU Ke-liang; ZHAO Hai-bin; LIU Hui; ZHONG Wan-xia; YU Guan-ling; CHEN Zi-jiang


    Background Embryo quality and receptivity of the endometrium are two factors that determine the results of in vitro fertilization/intra-cytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET).There is no consensus of the optimal transfer strategy for normal responders or high responders.The current study aimed to find the optimal transfer strategy for different subgroups of patients.Methods From April 2010 to December 2010,patients who meet the following criteria were included in this study; primary infertility,female age ≤35 years,FSH level on female cycle day 2-3 ≤12 mlU/ml,at least six good quality embryos available on day three.The clinical outcomes using different transfer strategies between normal responders and high responders were reviewed and compared.Results For the normal responders,the clinical pregnancy rate of day three double-embryo transfer (DET) was comparable to that of day five elective single blastocyst transfer (eSBT),64.04% vs.60.33% (P>0.05).For the high responders,the clinical pregnancy rate of day five eSBT was significantly lower than that of day three DET,43.35% vs.57.21% (P<0.05).For the high responders,the rates of clinical pregnancy and implantation in frozen-thawed embryo transfer (FET) cycles were notably higher than in eSBT cycles (64.56% vs.43.35% and 62.11% vs.43.35% respectively) (P<0.05).Conclusions For normal responders,eSBT might be an applicable strategy to reduce multiple pregnancy rates while maintaining acceptable overall pregnancy rates.And in order to reduce multiple pregnancies and increase the chance of pregnancy of high responders,FET may be a preferable strategy.

  8. Oct4 is required for lineage priming in the developing inner cell mass of the mouse blastocyst.

    Le Bin, Gloryn Chia; Muñoz-Descalzo, Silvia; Kurowski, Agata; Leitch, Harry; Lou, Xinghua; Mansfield, William; Etienne-Dumeau, Charles; Grabole, Nils; Mulas, Carla; Niwa, Hitoshi; Hadjantonakis, Anna-Katerina; Nichols, Jennifer


    The transcription factor Oct4 is required in vitro for establishment and maintenance of embryonic stem cells and for reprogramming somatic cells to pluripotency. In vivo, it prevents the ectopic differentiation of early embryos into trophoblast. Here, we further explore the role of Oct4 in blastocyst formation and specification of epiblast versus primitive endoderm lineages using conditional genetic deletion. Experiments involving mouse embryos deficient for both maternal and zygotic Oct4 suggest that it is dispensable for zygote formation, early cleavage and activation of Nanog expression. Nanog protein is significantly elevated in the presumptive inner cell mass of Oct4 null embryos, suggesting an unexpected role for Oct4 in attenuating the level of Nanog, which might be significant for priming differentiation during epiblast maturation. Induced deletion of Oct4 during the morula to blastocyst transition disrupts the ability of inner cell mass cells to adopt lineage-specific identity and acquire the molecular profile characteristic of either epiblast or primitive endoderm. Sox17, a marker of primitive endoderm, is not detected following prolonged culture of such embryos, but can be rescued by provision of exogenous FGF4. Interestingly, functional primitive endoderm can be rescued in Oct4-deficient embryos in embryonic stem cell complementation assays, but only if the host embryos are at the pre-blastocyst stage. We conclude that cell fate decisions within the inner cell mass are dependent upon Oct4 and that Oct4 is not cell-autonomously required for the differentiation of primitive endoderm derivatives, as long as an appropriate developmental environment is established. PMID:24504341

  9. Embryonic stem cells derived from in vivo or in vitro-generated murine blastocysts display similar transcriptome and differentiation potential.

    Rhodel K Simbulan

    Full Text Available The use of assisted reproductive technologies (ART such as in vitro fertilization (IVF has resulted in the birth of more than 5 million children. While children conceived by these technologies are generally healthy, there is conflicting evidence suggesting an increase in adult-onset complications like glucose intolerance and high blood pressure in IVF children. Animal models indicate similar potential risks. It remains unclear what molecular mechanisms may be operating during in vitro culture to predispose the embryo to these diseases. One of the limitations faced by investigators is the paucity of the material in the preimplantation embryo to test for molecular analysis. To address this problem, we generated mouse embryonic stem cells (mESC from blastocysts conceived after natural mating (mESCFB or after IVF, using optimal (KSOM + 5% O2; mESCKAA and suboptimal (Whitten's Medium, + 20% O2, mESCWM conditions. All three groups of embryos showed similar behavior during both derivation and differentiation into their respective mESC lines. Unsupervised hierarchical clustering of microarray data showed that blastocyst culture does not affect the transcriptome of derived mESCs. Transcriptomic changes previously observed in the inner cell mass (ICM of embryos derived in the same conditions were not present in mESCs, regardless of method of conception or culture medium, suggesting that mESC do not fully maintain a memory of the events occurring prior to their derivation. We conclude that the fertilization method or culture media used to generate blastocysts does not affect differentiation potential, morphology and transcriptome of mESCs.

  10. Effects of oocyte quality, semen donor and embryo co-culture system on the efficiency of blastocyst production in goats.

    Katska-Ksiazkiewicz, L; Opiela, J; Ryńska, B


    The aim of the study was to determine whether the selection of immature oocytes by a combination of cumulus-oocyte-complexes (COCs) morphology and staining with brilliant cresyl blue (BCB) would be helpful in selecting developmentally competent oocytes, and thereby increase the efficiency of blastocyst production from ovarian oocytes of FSH-primed, adult goats. In a second experiment the interaction between oocyte quality and semen donor was assessed. In a third experiment the usefulness of Vero cells for co-culture with goat embryos was investigated. In the pool of morphologically normal COCs recovered from ovaries following slicing (21.9+/-11.0), the mean rate of COCs classified as BCB+ was 85.6%, and the BCB- was approximately 11%. Oocytes classified as grade 1 and BCB+ exhibited the highest developmental competence (Pgrade 1 BCB- and grade 2 BCB+ or BCB-. There were no significant differences in developmental competence in grade 2 oocytes, regardless of BCB coloration. No significant differences in embryo cleavage and blastocyst formation rates among three bucks were observed when morphologically normal, BCB+ oocytes were used. For all tested bucks, differences in embryo production efficiency were related only to the oocyte quality. Similar blastocyst rates were developed from embryos co-cultured with goat oviduct epithelial cells (34.3%) and with Vero cells (33.3%). These results show that the most important criterion for selection of COCs before maturation is the visual assessment of morphological features. Staining with BCB of COCs recovered from adult goats does not enhance efficiency of selection of developmentally competent oocytes for IVF. PMID:17651793

  11. Intactness of zona pellucida does not affect the secretion of a trypsin-like protease from mouse blastocyst.

    Hwang, S.; Lee, E; Chung, Y.; Yoon, B. K.; Lee, J. H.; Choi, D


    Assisted hatching (AH), which is known to improve the hatching potential of mammalian embryos, has been used to increase the pregnancy rate in in vitro fertilization cycles. However, the effect of AH on a trypsin-like protease, which is known to be associated with the hatching process, has not been studied. In this study, we evaluate whether the intactness of zona pellucida affects the secretion of a trypsin-like protease from mouse blastocyst. Four- to 8-cell stage mouse embryos were collect...

  12. Analysis of the effects of blue light on morphofunctional status of in vitro cultured blastocysts from mice carrying gene of enhanced green fluorescent protein (EGFP).

    Sakharova, N Yu; Mezhevikina, L M; Smirnov, A A; Vikhlyantseva, E F


    We studied the effect of blue light (440-490 nm) on the development of late blastocysts of mice carrying the gene of enhanced green fluorescent protein (EGFP). Exposure to blue light for 20 min reduced adhesive properties of blastocysts and their capacity to form primary colonies consisting of the cells of inner cell mass, trophoblast, and extraembryonic endoderm. The negative effects of blue light manifested in morphological changes in the primary colonies and impairment of differentiation and migration of cells of the trophoblast and extraembryonic endoderm. The problems of cell-cell interaction and inductive influences of the inner cell mass on other cell subpopulations are discussed. EGFP blastocysts were proposed as the model for evaluation of the mechanisms underlying the effects of blue light as the major negative factor of visible light used in in vitro experiments on mammalian embryos. PMID:24913583

  13. RNA-Seq analysis uncovers transcriptomic variations between morphologically similar in vivo- and in vitro-derived bovine blastocysts

    Driver Ashley M


    Full Text Available Abstract Background A valuable tool for both research and industry, in vitro fertilization (IVF has applications range from gamete selection and preservation of traits to cloning. Although IVF has achieved worldwide use, with approximately 339,685 bovine embryos transferred in 2010 alone, there are still continuing difficulties with efficiency. It is rare to have more than 40% of fertilized in vitro cattle oocytes reach blastocyst stage by day 8 of culture, and pregnancy rates are reported as less than 45% for in vitro produced embryos. To investigate potential influences in-vitro fertilization (IVF has on embryonic development, this study compares in vivo- and in vitro-derived bovine blastocysts at a similar stage and quality grade (expanded, excellent quality to determine the degree of transcriptomic variation beyond morphology using RNA-Seq. Results A total of 26,906,451 and 38,184,547 fragments were sequenced for in vitro and in vivo embryo pools, respectively. We detected expression for a total of 17,634 genes, with 793 genes showing differential expression between the two embryo populations with false discovery rate (FDR Conclusions Thus, our results support that IVF may influence at the transcriptomic level and that morphology is limited in full characterization of bovine preimplantation embryos.

  14. Doble aneuploidía en un niño Colombiano: síndrome de Down–Klinefelter, con fenotipo de síndrome de Down = Double aneuploidy in a Colombian child: Down-Klinefelter syndrome with Down syndrome phenotype

    Pachajoa Londoño, Harry


    Full Text Available La ocurrencia de una doble aneuploidía en una misma persona es un evento relativamente raro. Se presenta el caso de un niño de siete meses de edad, de padres no consanguíneos con características clínicas de síndrome de Down y cariotipo 48XXY.

  15. The effect of anandamide on uterine nitric oxide synthase activity depends on the presence of the blastocyst.

    Micaela S Sordelli

    Full Text Available Nitric oxide production, catalyzed by nitric oxide synthase (NOS, should be strictly regulated to allow embryo implantation. Thus, our first aim was to study NOS activity during peri-implantation in the rat uterus. Day 6 inter-implantation sites showed lower NOS activity (0.19±0.01 pmoles L-citrulline mg prot(-1 h(-1 compared to days 4 (0.34±0.03 and 5 (0.35±0.02 of pregnancy and to day 6 implantation sites (0.33±0.01. This regulation was not observed in pseudopregnancy. Both dormant and active blastocysts maintained NOS activity at similar levels. Anandamide (AEA, an endocannabinoid, binds to cannabinoid receptors type 1 (CB1 and type 2 (CB2, and high concentrations are toxic for implantation and embryo development. Previously, we observed that AEA synthesis presents an inverted pattern compared to NOS activity described here. We adopted a pharmacological approach using AEA, URB-597 (a selective inhibitor of fatty acid amide hydrolase, the enzyme that degrades AEA and receptor selective antagonists to investigate the effect of AEA on uterine NOS activity in vitro in rat models of implantation. While AEA (0.70±0.02 vs 0.40±0.04 and URB-597 (1.08±0.09 vs 0.83±0.06 inhibited NOS activity in the absence of a blastocyst (pseudopregnancy through CB2 receptors, AEA did not modulate NOS on day 5 pregnant uterus. Once implantation begins, URB-597 decreased NOS activity on day 6 implantation sites via CB1 receptors (0.25±0.04 vs 0.40±0.05. While a CB1 antagonist augmented NOS activity on day 6 inter-implantation sites (0.17±0.02 vs 0.27±0.02, a CB2 antagonist decreased it (0.17±0.02 vs 0.12±0.01. Finally, we described the expression and localization of cannabinoid receptors during implantation. In conclusion, AEA levels close to and at implantation sites seems to modulate NOS activity and thus nitric oxide production, fundamental for implantation, via cannabinoid receptors. This modulation depends on the presence of the blastocyst. These

  16. Establishment of bovine trophoblast stem-like cells from in vitro-produced blastocyst-stage embryos using two inhibitors.

    Huang, Xianghua; Han, Xuejie; Uyunbilig, Borjigin; Zhang, Manling; Duo, Shuguang; Zuo, Yongchun; Zhao, Yuhang; Yun, Ting; Tai, Dapeng; Wang, Chen; Li, Jinhua; Li, Xueling; Li, Rongfeng


    The trophoblast (TR) is the first to differentiate during mammalian embryogenesis and play a pivotal role in the development of the placenta. We used a dual inhibitor system (PD0325901 and CHIR99021) with mixed feeders to successfully obtain bovine trophoblast stem-like (bTS) cells, which were similar in phenotype to mouse trophoblast stem cells (TSCs). The bTS cells that were generated using this system continually proliferated, displayed a normal diploid karyotype, and had no signs of altered morphology or differentiation even after 150 passages. These cells exhibited alkaline phosphatase (AP) activity and expressed pluripotency markers, such as OCT4, NANOG, SOX2, SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81, and TR lineage markers such as CDX2, as determined by both immunofluorescence and reverse transcription-polymerase chain reaction (RT-PCR). Additionally, these cells generated dome-like structures, formed teratomas when injected into NOD-SCID mice, and differentiated into placenta TR cells in vitro. The microarray analysis of bTS cells showed high expression levels of many TR markers, such as TEAD4, EOMES, GATA3, ETS2, TFAP2A, ELF5, SMARCA4 (BRG1), CDH3, MASH2, HSD17B1, CYP11A1, PPARG, ID2, GCM1, HAND1, TDK, PAG, IFN-τ, and THAP11. The expression of many pluripotency markers, such as OCT4, SOX2, NANOG, and GDF3, was lower in bTS cells compared with in vitro-produced blastocysts; however, compared with bovine fetal fibroblasts, the expression of these pluripotency markers was elevated in bTS cells. The DNA methylation status of the promoter regions of OCT4, NANOG, and SOX2 was investigated, which were significantly higher in bTS cells (OCT4 23.90%, NANOG 74.40%, and SOX2 8.50%) compared with blastocysts (OCT4 8.90%, NANOG 34.4%, and SOX2 3.80%). In contrast, two promoter regions of CDX2 were hypomethylated in bTS cells (13.80% and 3.90%) compared with blastocysts (18.80% and 9.10%). The TSC lines that were established in this study may be used either for basic

  17. Developmental potency of mouse primitive ectoderm cells,embryonic ectoderm cells,embryonic ectoderm cells and primordial germ cells after blastocyst injection



    Developmental potency of primitive and embryonic ectoderm cells from 4.50-day to 6.25-day post-coitum (p.c.) mouse embryos and primordial germ cells from 12.50-day p.c.male genital ridges of fetal mice were studied by direct introducing them into 3.50-day p.c.blastocysts.Sixteen (61.5) overt chimaeras out of 26(50%) offsprings were obtained after transfer of 52 blastocysts injected with 4.50-day primitive ectoderm cells;four (16.0%) overt chimaeras were obtained out of 25 (51.0%) offsprings with 4.75-day primitive ectoderm cells from 49 transferred blastocysts.However,no overt chimaera was obtained with either 5.25-day or 6.25-day embryonic ectoderm cells or 12.50-day male primordial germ cells.GPI analysis of mid-gestation conceptuses developed from injected blastocysts showedthat 5.25-day embryonic ectoderm cells could only contributed to yolk sac of conceptus.Results suggested that implantation acts as a trigger for the determination of primitive ectoderm cells,and their developmental potency becomes limited within a short period of time in normal development.

  18. Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF patients: results from a randomized pilot study

    Yang Zhihong


    Full Text Available Abstract Background Single embryo transfer (SET remains underutilized as a strategy to reduce multiple gestation risk in IVF, and its overall lower pregnancy rate underscores the need for improved techniques to select one embryo for fresh transfer. This study explored use of comprehensive chromosomal screening by array CGH (aCGH to provide this advantage and improve pregnancy rate from SET. Methods First-time IVF patients with a good prognosis (age Results For patients in Group A (n = 55, 425 blastocysts were biopsied and analyzed via aCGH (7.7 blastocysts/patient. Aneuploidy was detected in 191/425 (44.9% of blastocysts in this group. For patients in Group B (n = 48, 389 blastocysts were microscopically examined (8.1 blastocysts/patient. Clinical pregnancy rate was significantly higher in the morphology + aCGH group compared to the morphology-only group (70.9 and 45.8%, respectively; p = 0.017; ongoing pregnancy rate for Groups A and B were 69.1 vs. 41.7%, respectively (p = 0.009. There were no twin pregnancies. Conclusion Although aCGH followed by frozen embryo transfer has been used to screen at risk embryos (e.g., known parental chromosomal translocation or history of recurrent pregnancy loss, this is the first description of aCGH fully integrated with a clinical IVF program to select single blastocysts for fresh SET in good prognosis patients. The observed aneuploidy rate (44.9% among biopsied blastocysts highlights the inherent imprecision of SET when conventional morphology is used alone. Embryos randomized to the aCGH group implanted with greater efficiency, resulted in clinical pregnancy more often, and yielded a lower miscarriage rate than those selected without aCGH. Additional studies are needed to verify our pilot data and confirm a role for on-site, rapid aCGH for IVF patients contemplating fresh SET.

  19. The Rate Of Inner Cell Mass Of Blastocysts Which Obtain From Mouse Two Cell Embryos Cultured In Absence And Presence Of Recombinant Human Leukemia Inhibitory Factor

    Akbari M


    Full Text Available Background: This study was performed to investigate the rate of inner cell mass of blastocyst which obtain from culture of mouse two cell embryos in presence and absence of recombinant of human leukemia inhibitory factor. Materials and Methods: ICR female mice that were between 6-8 weeks old received intra peritoneal injection of 7.5 IU of pregnant mare serum gonadotropine for super ovulation, this was followed by intra peritoneal administration of 7.5 of hCG 46-48 hours later. The mice were then mated to mature ICR male mice and were checked for vaginal plug 20 hours later. Female mice were killed by cervical dislocation 48-50 hours after hCG administration and after washing and flushing of the oviduct from the proximal end of the oviduct, two cell embryos were selected and collected by 100 microscopy. All two cell embryos were randomly divided in 4 groups (Groups A, B C and D and culture in special media. Groups A: KSOM+AA, Groups B: KSOM+AA 500 IU/ml LIF. Groups C: KSOM+AA 1000 IU/ml LIF. Groups D: KSOM+AA 1500 IU/ml LIF media until 120 hours in Co2 incubator .After that time all blastocysts collected and the number of ICM was assessed by differential staining technology. Results: The rates of ICM of blastocysts which obtain from groups A, B, C and D were 19 2.6, 28 4.4, 24 2.1, 26 2.2 respectively. This data indicated that the rate of ICM in groups B, C and D was statistically higher than group A (P=0.02 and also there was not statistically different between three groups of B, C and D. Conclusion: Briefly leukemia inhibitory factor can improve the rate of ICM of blastocyst and we suggest that this factor is better added to blastocyst culture medium.

  20. 人废弃胚胎体外培养成囊胚的实验室结局%Laboratory outcomes of discarded embryos cultured into blastocysts in vitro

    王娟; 吕玉珍; 赵芳


    BACKGROUND:Some reports have showed that the discarded embryos can be used to establish the human embryonic stem cell lines, after freezing and thawing, the transplantation of embryos that hysteretic in morphological development still hasmedical value.OBJECTIVE: To study the potential of discarded embryos cultured into blastocyst in vitro.METHODS: Totally 404 discarded embryos were collected from the patients treated with in vitro fertilization and embryo transfer(IVF_ET) or intracytoplasmic sperm injection (ICSI), and then preformed with blastocyst sequential training.RESULTS AND CONCLUSION: ①The 404 discarded embryos formed 65 blastocyst, the total blastocyst formation rate was16.1%. ②There was no relation between the blastocyst formation and the patient's etiology, age, fertilization methods (P > 0.05).③The blastocyst in embryos derived from the ≥ 3 pronuclear (PN) embryos (6-8) cell group had the highest formation rate(P 0.05).③胚胎来源于≥三原核(3PN)的(6~8)细胞组的囊胚形成率最高(P 8细胞囊胚率反而下降、与<3细胞组的结果相近.④Ⅰ,Ⅱ胚胎形成囊胚速度快,形成率显著高于Ⅲ,Ⅳ胚胎(P < 0.05).⑤有囊胚形成患者的临床妊娠率显著高于无囊胚形成患者(P < 0.05).提示囊胚形成与患者病因分类、年龄、受精方式无相关性.优质胚胎与(6~8)细胞的胚胎囊胚形成率高,多原核胚胎形成囊胚的价值应引起关注.废弃胚胎再利用能预测临床结局,为人类胚胎干细胞提供有利资源.

  1. Expression of proposed implantation marker genes CDX2 and HOXB7 in the blastocyst does not distinguish viable from non-viable human embryos

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


    5 after oocyte retrieval. Biopsy was performed only if the blastocyst had a fully expanded blastocoel, a TE consisting of many small cells and an inner cell mass consisting of several tightly packed cells. A non-contact laser was used to create an opening in the zona pellucida permitting TE to...... herniate through the hole. After 4-5 hours of continued culture 5-10 herniating TE cells were aspirated into a biopsy pipette and dissected free of the blastocyst mass using a laser (ZilosTM, Hamilton Thorne Research, wavelength 1480 nm). Biopsied embryos were cultured overnight prior to re-assessment and...... transfer early on day 6 after oocyte retrieval. Immediately following biopsy, cells were lysed and reverse transcriptase PCR (RT-PCR) was carried out directly on the cell lysate using Superscript® III First Strand Synthesis kit (Invitrogen) thus avoiding introducing RNA amplification steps. As part of the...

  2. A combined treatment of ionomycin with ethanol improves blastocyst development of bovine oocytes harvested from stored ovaries and microinjected with spermatozoa

    Abdalla, H; Shimoda, M; M. Hirabayashi; Hochi, S


    Regardless of the presence of sperm-borne oocyte-activating factors, activation of bovine oocytes with exogenous activation stimuli is required for further development after intracytoplasmic sperm injection (ICSI). The current study was designed to develop a new activation regimen for improving the blastocyst yield after ICSI of bovine oocytes harvested from ovaries stored at 10 to 12 degrees C for 24 h. After ICSI, oocytes were treated with 5 mu M ionomycin for 5 min, 7% ethanol for 5 or 10 ...

  3. Effects of bovine serum proteins in culture medium on post-warming survival of bovine blastocysts developed in vitro.

    Ohboshi, S; Etoh, T; Sakamoto, K; Fujihara, N; Yoshida, T; Tomogane, H


    Experiments were conducted to investigate the factors affecting the survival of bovine blastocysts produced in vitro after cryopreservation by vitrification. Zygotes were obtained by in vitro maturation and fertilization of oocytes. Embryos used in this study were developed in vitro at Day 7 and 8 (Day 0 = insemination day) in modified synthetic oviduct fluid medium supplemented with calf serum or BSA. Embryos were cryopreserved in a two-step protocol consisting of exposure to 10% ethylene glycol for 5 min, followed by the original vitrification solution (designated as VS) consisting of 40% (v/v) ethylene glycol, 6% (w/v) polyethylene glycol and 0.5 M sucrose in phosphate-buffered saline for 1 min. After warming, embryos were cultured in modified TCM-199 for an in vitro survival assay. The highest survival rate was obtained from the warmed embryos developed at Day 7 in medium supplemented with BSA (82.6%), and there were significant differences between results with calf scrum and BSA treatment (42.4 and 70.7%, respectively; P < 0.01). However, there were no significant differences in the cell numbers of embryos among the treatments. These results suggest that the survival of embryos developed in medium with BSA is superior to that of embryos developed in medium containing calf serum, although the cell numbers of the embryos developed under both media were similar. PMID:16728072

  4. Laser-assisted blastocyst dissection and subsequent cultivation of embryonic stem cells in a serum/cell free culture system: applications and preliminary results in a murine model

    Sills Eric


    Full Text Available Abstract Background To evaluate embryonic stem cell (ESC harvesting methods with an emphasis on derivation of ESC lines without feeder cells or sera. Using a murine model, laser-assisted blastocyst dissection was performed and compared to conventional immunosurgery to assess a novel laser application for inner cell mass (ICM isolation. Methods Intact blastocysts or isolated ICMs generated in a standard mouse strain were plated in medium with or without serum to compare ESC harvesting efficiency. ESC derivation was also undertaken in a feeder cell-free culture system. Results Although ICM growth and dissociation was comparable irrespective of the media components, an enhanced ESC harvest was observed in our serum-free medium (p Conclusion Achieving successful techniques for human ESC research is fundamentally dependent on preliminary work using experimental animals. In this study, all experimentally developed ESC lines manifested similar features to ESCs obtained from intact blastocysts in standard culture. Cell/sera free murine ESC harvest and propagation are feasible procedures for an embryology laboratory and await refinements for translation to human medical research.

  5. Protective effects of resveratrol on ethanol-induced apoptosis in embryonic stem cells and disruption of embryonic development in mouse blastocysts

    Previous studies have established that ethanol induces apoptosis, but the precise molecular mechanisms are currently unclear. Here, we show that 0.3-1.0% (w/v) ethanol induces apoptosis in mouse blastocysts and that resveratrol, a grape-derived phytoalexin with known antioxidant and anti-inflammatory properties, prevents ethanol-induced apoptosis and inhibition of cell proliferation. Moreover, ethanol-treated blastocysts show normal levels of implantation on culture dishes in vitro but a reduced ability to reach the later stages of embryonic development. Pretreatment with resveratrol prevented ethanol-induced disruption of embryonic development in vitro and in vivo. In an in vitro cell-based assay, we further found that ethanol increases the production of reactive oxygen species in ESC-B5 embryonic stem cells, leading to an increase in the intracellular concentrations of cytoplasmic free Ca2+ and NO, loss of mitochondrial membrane potential, mitochondrial release of cytochrome c, activation of caspase-9 and -3, and apoptosis. These changes were blocked by pretreatment with resveratrol. Based on these results, we propose a model for the protective effect of resveratrol on ethanol-induced cell injury in blastocysts and ESC-B5 cells

  6. The piggyBac-Based Gene Delivery System Can Confer Successful Production of Cloned Porcine Blastocysts with Multigene Constructs.

    Sato, Masahiro; Maeda, Kosuke; Koriyama, Miyu; Inada, Emi; Saitoh, Issei; Miura, Hiromi; Ohtsuka, Masato; Nakamura, Shingo; Sakurai, Takayuki; Watanabe, Satoshi; Miyoshi, Kazuchika


    The introduction of multigene constructs into single cells is important for improving the performance of domestic animals, as well as understanding basic biological processes. In particular, multigene constructs allow the engineering and integration of multiple genes related to xenotransplantation into the porcine genome. The piggyBac (PB) transposon system allows multiple genes to be stably integrated into target genomes through a single transfection event. However, to our knowledge, no attempt to introduce multiple genes into a porcine genome has been made using this system. In this study, we simultaneously introduced seven transposons into a single porcine embryonic fibroblast (PEF). PEFs were transfected with seven transposons containing genes for five drug resistance proteins and two (red and green) fluorescent proteins, together with a PB transposase expression vector, pTrans (experimental group). The above seven transposons (without pTrans) were transfected concomitantly (control group). Selection of these transfected cells in the presence of multiple selection drugs resulted in the survival of several clones derived from the experimental group, but not from the control. PCR analysis demonstrated that approximately 90% (12/13 tested) of the surviving clones possessed all of the introduced transposons. Splinkerette PCR demonstrated that the transposons were inserted through the TTAA target sites of PB. Somatic cell nuclear transfer (SCNT) using a PEF clone with multigene constructs demonstrated successful production of cloned blastocysts expressing both red and green fluorescence. These results indicate the feasibility of this PB-mediated method for simultaneous transfer of multigene constructs into the porcine cell genome, which is useful for production of cloned transgenic pigs expressing multiple transgenes. PMID:27589724

  7. Comparative pluripotency analysis of mouse embryonic stem cells derived from wild-type and infertile hermaphrodite somatic cell nuclear transfer blastocysts

    FAN Yong; YAO RuQiang; YU Yang; LI ZanDong; WANG Liu; ALICE Jouneau; ZHOU Qi; TONG Man; ZHAO ChunLi; DING ChenHui; HAO Jie; LV Zhuo; DAI XiangPeng; HAI Tang; LI XueMei


    Therapeutic cloning, whereby embryonic stem cells (ESCs) are derived from patient-specific cloned blastocysts via somatic cell nuclear transfer (SCNT), holds great promise for treating many human diseases using regenerative medicine. Teratoma formation and germline transmission have been used to confirm the pluripotency of mouse stem cells, but human embryonic stem cells (hESCs) have not been proven to be fully pluripotent owing to the ethical impossibility of testing for germ line transmission, which would be the strongest evidence for full pluripotency. Therefore, formation of differentiated cells from the three somatic germ layers within a teratoma is taken as the best indicator of pluripotency in hESC lines. The possibility that these lines lack full multi- or pluripotency has not yet been evaluated.In this study, we established 16 mouse ESC lines, including 3 genetically defective nuclear transfer-ESC (ntESC) lines derived from SCNT blastocysts of infertile hermaphrodite F1 mice and 13 ntESC lines derived from SCNT blastocysts of normal F1 mice. We found that the defective ntESCs expressed all in vitro markers of pluripotency and could form teratomas that included derivatives from all three germ layers, but could not be transmitted via the germ line, in contrast with normal ntESCs. Our results indicate that teratoma formation assays with hESCs might be an insufficient standard to assess full pluripotency, although they do define multipotency to some degree. More rigorous standards are required to assess the safety of hESCs for therapeutic cloning.

  8. Citrinin induces apoptosis via a mitochondria-dependent pathway and inhibition of survival signals in embryonic stem cells, and causes developmental injury in blastocysts.

    Chan, Wen-Hsiung


    The mycotoxin CTN (citrinin), a natural contaminant in foodstuffs and animal feeds, has cytotoxic and genotoxic effects on various mammalian cells. CTN is known to cause cell injury, including apoptosis, but the precise regulatory mechanisms of CTN action, particularly in stem cells and embryos, are currently unclear. In the present paper, I report that CTN has cytotoxic effects on mouse embryonic stem cells and blastocysts, and is associated with defects in their subsequent development, both in vitro and in vivo. Experiments in embryonic stem cells (ESC-B5) showed that CTN induces apoptosis via ROS (reactive oxygen species) generation, increased Bax/Bcl-2 ratio, loss of MMP (mitochondrial membrane potential), induction of cytochrome c release, and activation of caspase 3. In this model, CTN triggers cell death via inactivation of the HSP90 [a 90 kDa isoform of the HSP (heat-shock protein) family proteins]/multichaperone complex and subsequent degradation of Ras and Raf-1, further inhibiting anti-apoptotic processes, such as the Ras-->ERK (extracellular-signal-regulated kinase) signal transduction pathway. In addition, CTN causes early developmental injury in mouse ESCs and blastocysts in vitro. Lastly, using an in vivo mouse model, I show that consumption of drinking water containing 10 muM CTN results in blastocyst apoptosis and early embryonic developmental injury. Collectively, these findings show for the first time that CTN induces ROS and mitochondria-dependent apoptotic processes, inhibits Ras-->ERK survival signalling via inactivation of the HSP90/multichaperone complex, and causes developmental injury in vivo. PMID:17331071

  9. A comparative study between cleavage stage embryo transfer at day 3 and blastocyst stage transfer at day 5 in in-vitro fertilization/intra-cytoplasmic sperm injection on clinical pregnancy rates

    Prabhleen Kaur


    Full Text Available Objective: To evaluate the efficacy of blastocyst transfer in comparison with cleavage stage transfer. Study Design: A randomized, prospective study was conducted in Infertility clinic, Department of Obstetrics and Gynecology, Mahatma Gandhi Hospital, Jaipur on 300 patients aged 25-40 years undergoing in-vitro fertilization (IVF/intra-cytoplasmic sperm injection (ICSI cycle from May 2010-April 2011. When three or more Grade-I embryos were observed on day 2 of culture, patients were divided randomly into two study groups, cleavage stage transfer and blastocyst transfer group having 150 patients each. Primary outcomes evaluated were, Clinical pregnancy rate and Implantation rate. The results were analyzed using proportions, standard deviation and Chi-square test. Results: Both the groups were similar for age, indication and number of embryos transferred. Clinical pregnancies after blastocyst transfer were significantly higher 66 (44.0% compared to cleavage stage embryo transfer 44 (29.33% (P < 0.01.Implantation rate for blastocyst transfer group was also significantly higher (P < 0.001. Conclusion: Blastocyst transfer having higher implantation rate and clinical pregnancy rate lead to reduction in multiple pregnancies.

  10. Comparison of BALB/c and B6-albino mouse strain blastocysts as hosts for the injection of C57BL6/N-derived C2 embryonic stem cells.

    Alcantar, Tuija M; Wiler, Rhonda; Rairdan, Xin Y


    Embryonic stem (ES) cells from a C57BL/6N (B6N) background injected into B6(Cg)-Tyrc-2J/J (B6-albino) recipient blastocysts are commonly used for generating genetically modified mouse models. To understand the influence of the recipient blastocyst strain on germline transmission, BALB/cAnNTac and B6-albino germline transmission rates were compared using the C57BL6/N-derived C2 ES cell line. A total of 92 ES cell clones from 27 constructs were injected. We compared blastocyst yield, birth rate, chimera formation rate, and high-percentage (>50 %) male chimera formation rate. For germline transmission, we analyzed 24 clones from 19 constructs, which generated high-percentage male chimeras from both donor strains. B6-albino hosts resulted in higher mean blastocyst yields per donor than did BALB/c ones (3.6 vs. 2.5). However, BALB/c hosts resulted in a higher birth rate than B6-albino ones (36 vs. 27 %), a higher chimera formation rate (50 vs. 42 %), a higher high-percentage male chimera rate (10 vs. 8 %), and a higher germline transmission rate (65 vs. 49 %), respectively. Our data suggest that BALB/c is a suitable blastocyst host strain for C2 ES cells and has an advantage over the B6-albino strain for receiving the injection of C2 ES cells. PMID:26852382

  11. Role of cumulus cells during vitrification and fertilization of mature bovine oocytes: Effects on survival, fertilization, and blastocyst development.

    Ortiz-Escribano, N; Smits, K; Piepers, S; Van den Abbeel, E; Woelders, H; Van Soom, A


    This study was designed to determine the role of cumulus cells during vitrification of bovine oocytes. Mature cumulus-oocyte complexes (COCs) with many layers of cumulus cells, corona radiata oocytes (CRs), with a few layers of cumulus cells, and denuded oocytes (DOs) without cumulus cells were vitrified in 15% ethylene glycol, 15% dimethyl sulfoxide, and 0.5-M sucrose. Oocytes that survived the vitrification process were fertilized. Denuded oocytes were fertilized with or without supplementation of intact COCs (DOsCOCs). First, survival and embryo development rates were studied. Higher survival rates were obtained for DOs and DOsCOCs (94% and 95%, respectively) compared with COCs (82.7%, P < 0.05). Corona radiata oocytes showed similar survival rates when compared with DOs. The cleavage and blastocyst rates of vitrified DOs were compromised because cumulus cells were not present during the fertilization (34% and 2.7%, respectively). However, the situation could be reverted when DOs were supplemented with intact COCs (DOsCOCs; 62.7% and 12.7%, respectively, P < 0.05). Vitrified CRs showed similar cleavage and blastocyst rate (49.3% and 7.7%, respectively) compared with COCs (54.8% and 4.9%, respectively). In the second experiment, the penetration rate was analyzed. Removing cumulus cells before fertilization reduced the fertilization of vitrified DOs compared with COCs (24.3% vs. 52.8%, P < 0.05). The supplementation of DOs with intact COCs (DOsCOCs) improved the fertilization rate though (49.6%, P < 0.05). No differences in the fertilization rate were found between CRs and COCs. In the third experiment, parthenogenetic activation was examined. Interestingly, the CRs group showed higher cleavage and blastocyst rates (76.8% and 29.6%, respectively) than the COCs (39.1% and 7.5%, respectively, P < 0.05). Furthermore, oocytes from vitrified CRs had the same odds to become a blastocyst as fresh oocytes (1.1 vs. 1.5, respectively). In conclusion, our data

  12. In vitro production of multigene transgenic blastocysts via sperm-mediated gene transfer allows rapid screening of constructs to be used in xenotransplantation experiments.

    Vargiolu, A; Manzini, S; de Cecco, M; Bacci, M L; Forni, M; Galeati, G; Cerrito, M G; Busnelli, M; Lavitrano, M; Giovannoni, R


    Multigene transgenic pigs would be of benefit for large animal models and in particular for xenotransplantation, where extensive genetic manipulation of donor pigs is required to make them suitable for organ grafting to humans. We have previously produced multitransgenic pigs via sperm-mediated gene transfer (SMGT) using integrative constructs expressing 3 different reporter genes. The aim of the present work was to evaluate the efficacy and safety of using 3 integrative constructs carrying 3 different human genes involved in the modulation of inflammatory responses. We developed an in vitro fertilization system to demonstrate that SMGT can be used to efficiently produce multigene transgenic embryos through a 1-step genetic modification using multiple integrative constructs each carrying a different human gene involved in the modulation of inflammatory processes (hHO1, hCD39, and hCD73). The results suggest that this system allowed an effective preliminary test of transgenesis optimization, greatly reducing the number of animals used in the experiments and fulfilling important ethical issues. We performed 5 in vitro fertilization experiments using sperm cells preincubated with all 3 integrative constructs. A total of 1,498 oocytes were fertilized to obtain 775 embryos, among which 340 further developed into blastocysts. We did not observe any toxicity related to the transgenesis procedure that affected normal embryo development. We observed 68.5% transgenesis efficiency. Blastocysts were 48% single, 31% double, and 21% triple transgenic. PMID:20692428

  13. A randomized assessor-blind trial comparing highly purified hMG and recombinant FSH in a GnRH antagonist cycle with compulsory single-blastocyst transfer

    Devroey, Paul; Pellicer, Antonio; Nyboe Andersen, Anders;


    OBJECTIVE: To compare the efficacy and safety of highly purified menotropin (hphMG) and recombinant FSH (rFSH) for controlled ovarian stimulation in a GnRH antagonist cycle with compulsory single-blastocyst transfer. DESIGN: Randomized, open-label, assessor-blind, parallel groups, multicenter...

  14. Increased blastocyst formation of cloned porcine embryos produced with donor cells pre-treated with digitonin and Xenopus egg extract

    Liu, Ying; Østrup, Olga; Li, Juan;


    Pre-treating donor cells before somatic cell nuclear transfer (SCNT, ‘cloning’) may improve the efficiency of the technology. The aim of this study was to evaluate the early development of cloned embryos produced with porcine fibroblasts pre-treated with a permeabilizing agent and extract from...... Xenopus laevis eggs. In Experiment 1, fetal fibroblasts were permeabilized by digitonin, incubated in egg extract and, after re-sealing of cell membranes, cultured for 3 or 5 days before use as donor cells in handmade cloning (HMC). Controls were produced by HMC with non-treated donor cells. The...... blastocyst rate for reconstructed embryos increased significantly when digitonin-permeabilized, extract-treated cells were used after 5 days of culture after re-sealing. In Experiment 2, fetal and adult fibroblasts were treated with digitonin alone before re-sealing the cell membranes, then cultured for 3 or...

  15. Generation of liver-specific TGF-α/c-Myc-overexpressing porcine induced pluripotent stem-like cells and blastocyst formation using nuclear transfer.

    Park, Kyung-Mee; Lee, Joohyeong; Hussein, Kamal Hany; Hong, Seok-Ho; Yang, Se-Ran; Lee, Eunsong; Woo, Heung-Myong


    Transgenic porcine induced pluripotent stem (iPS) cells are attractive cell sources for the development of genetically engineered pig models, because they can be expanded without senescence and have the potential for multiple gene manipulation. They are also useful cell sources for disease modeling and treatment. However, the generation of transgenic porcine iPS cells is rare, and their embryonic development after nuclear transfer (NT) has not yet been reported. We report here the generation of liver-specific oncogenes (TGF-α/c-Myc)-overexpressing porcine iPS (T/M iPS)-like cells. They expressed stem cell characteristics and were differentiated into hepatocyte-like cells that express oncogenes. We also confirmed that NT embryos derived from T/M iPS-like cells successfully developed blastocysts in vitro. As an initial approach toward porcine transgenic iPS cell generation and their developmental competence after NT, this study provides foundations for the efficient generation of genetically modified porcine iPS cells and animal models. PMID:26725870

  16. Highly Efficient In Vitro Production of Bovine Blastocyst in Cell-Free Sequential Synthetic Oviductal Fluid vs. TCM199 Vero Cell Co-Culture System

    Sayyed Morteza Hosseini


    Full Text Available Background: The aim of this study was to establish a cell-free sequential culture system that cansupport high levels of in vitro embryo development and blastocyst formation from bovine zygotes.To this end, this investigation was carried out to evaluate the effects of glucose, serum and EDTAon bovine zygote in vitro development.Materials and Methods: Bovine presumptive zygotes were derived from oocytes matured, andfertilized in vitro and cultured in synthetic oviductal fluid sequential medium in a two-steps manner;SOF 1 for the first 3 days and SOF 2 for the second 5-6 days of in vitro embryo development. Inorder to evaluate the effect of different modifications of the basic medium on embryo development,glucose was added to the second phase (SOF A, serum was added to the first phase (SOF C andEDTA alone (SOF D or in combination with serum (SOF E was added into the first phase of invitro embryo culture. The results of each composition were compared with each other and with theresults of embryo development in TCM199 vero cell co-culture system.Results: Glucose addition to the second phase of embryo culture, improved the developmentalcompetency; however, the differences were not significant. Serum addition to the first phase ofembryo culture, significantly improved the developmental competency of embryos beyond thecleavage stage, compared to all the treatment and TCM199 co-culture groups. EDTA supplementationof culture medium, either alone or in combination with serum, significantly inhibits the embryodevelopment beyond the morula stage.Conclusion: The results indicated that culture of bovine presumptive zygotes in two steps cell-freeculture system, can support embryo development, and addition of serum throughout the culture andglucose to the second step significantly increased overall developmental competency compared toTCM199 co-culture system.

  17. The Genomic Landscape of Renal Oncocytoma Identifies a Metabolic Barrier to Tumorigenesis

    Shilpy Joshi


    Full Text Available Oncocytomas are predominantly benign neoplasms possessing pathogenic mitochondrial mutations and accumulation of respiration-defective mitochondria, characteristics of unknown significance. Using exome and transcriptome sequencing, we identified two main subtypes of renal oncocytoma. Type 1 is diploid with CCND1 rearrangements, whereas type 2 is aneuploid with recurrent loss of chromosome 1, X or Y, and/or 14 and 21, which may proceed to more aggressive eosinophilic chromophobe renal cell carcinoma (ChRCC. Oncocytomas activate 5′ adenosine monophosphate-activated protein kinase (AMPK and Tp53 (p53 and display disruption of Golgi and autophagy/lysosome trafficking, events attributed to defective mitochondrial function. This suggests that the genetic defects in mitochondria activate a metabolic checkpoint, producing autophagy impairment and mitochondrial accumulation that limit tumor progression, revealing a novel tumor-suppressive mechanism for mitochondrial inhibition with metformin. Alleviation of this metabolic checkpoint in type 2 by p53 mutations may allow progression to eosinophilic ChRCC, indicating that they represent higher risk.

  18. Dosage Compensation of an Aneuploid Genome in Mouse Spermatogenic Cells

    Jansa, Petr; Homolka, David; Blatný, Radek; Mistrik, M.; Bartek, Jiří; Forejt, Jiří


    Roč. 90, č. 6 (2014), 124/1-124/9. ISSN 0006-3363 R&D Projects: GA ČR GA13-08078S Institutional support: RVO:68378050 Keywords : gene dosage * male sterility * segmental trisomy * meiotic silencing of unsynapsed chromatin * DOWN-SYNDROME * MAMMALIAN MEIOSIS Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.318, year: 2014

  19. Identifiability in stochastic models


    The problem of identifiability is basic to all statistical methods and data analysis, occurring in such diverse areas as Reliability Theory, Survival Analysis, and Econometrics, where stochastic modeling is widely used. Mathematics dealing with identifiability per se is closely related to the so-called branch of ""characterization problems"" in Probability Theory. This book brings together relevant material on identifiability as it occurs in these diverse fields.

  20. Identifying Unknown Proteins

    Barker, Winona C.; Dayhoff, Margaret O.


    In this paper we discuss ways to identify a protein, both when its amino acid sequence is known and, particularly, prior to the determination of the complete sequence. If a similar sequence is in the Protein Sequence Database, an unknown may be identified on the basis of partial or ambiguous sequence data, or on the basis of amino acid composition. Identification in the early stages of structural determination can save time and scarce resources by preventing duplicate effort or by suggesting ...

  1. Comparison of the First Trimester Miscarriage Rate between Cleavage Stage Embryo and Blastocyst%囊胚和卵裂期胚胎的早期胚胎流产率比较

    林明媚; 罗璐; 李涛; 张瑞晓; 贾梦希; 王琼


    Objective:To compare the first trimester miscarriage rates between the cleavage stage embryo transfer and blastocyst transfer.Methods:First trimester miscarriage rates of 1 673in vitro fertilization and embryo transfer (IVF-ET) cycles for tubal factor or male factor were retrospectively analyzed.Among them,932 cycles were the fresh embryo transfer and 741 cycles were frozen-thawed embryo transfer.Results:Logistic analysis showed that either fresh embryo transfer group or frozen embryo transfer group,the first trimester miscarriage rate in the blastocyst transfer was lower than that in the cleavage stage embryo transfer,and the miscarriage risk was increased with advanced patient's age.Conclusion:Blastocyst transfer showed a lower first trimester miscarriage rate compared with the cleavage stage embryo transfer,age is still closely related to the first trimester miscarriage.%目的:探讨辅助生殖技术中囊胚培养是否能够筛选出更加具有发育潜能的胚胎,降低胚胎的早期流产率.方法:回顾性分析因单纯输卵管因素或/及男性因素接受囊胚期与第3日卵裂期胚胎移植获得妊娠后患者的流产率.总周期数1 673个,其中新鲜胚胎移植周期(fresh embryo transfer)932个(囊胚移植139个周期,卵裂期胚胎移植793个周期),冻融胚胎移植周期(frozen-thawed embryo transfer)741个(囊胚移植211个周期,卵裂期胚胎移植530个周期).结果:Logistic回归分析表明无论新鲜胚胎移植组还是冻融胚胎移植组,囊胚期胚胎受孕后的早期胚胎流产率均低于第3日的卵裂期胚胎,并且囊胚移植和卵裂期胚胎移植的流产率均与妇女年龄呈正相关.结论:无论新鲜胚胎移植组还是冻融胚胎移植组,囊胚移植均比第3日卵裂期胚胎移植流产风险降低.年龄仍是影响移植后妊娠早期流产的重要因素.

  2. Identifying Knowledge and Communication

    Eduardo Coutinho Lourenço de Lima


    Full Text Available In this paper, I discuss how the principle of identifying knowledge which Strawson advances in ‘Singular Terms and Predication’ (1961, and in ‘Identifying Reference and Truth-Values’ (1964 turns out to constrain communication. The principle states that a speaker’s use of a referring expression should invoke identifying knowledge on the part of the hearer, if the hearer is to understand what the speaker is saying, and also that, in so referring, speakers are attentive to hearers’ epistemic states. In contrasting it with Russell’s Principle (Evans 1982, as well as with the principle of identifying descriptions (Donnellan 1970, I try to show that the principle of identifying knowledge, ultimately a condition for understanding, makes sense only in a situation of conversation. This allows me to conclude that the cooperative feature of communication (Grice 1975 and reference (Clark andWilkes-Gibbs 1986 holds also at the understanding level. Finally, I discuss where Strawson’s views seem to be unsatisfactory, and suggest how they might be improved.

  3. 人类辅助生殖技术中DAY3胚胎质量与囊胚形成相关性分析%Correlation analysis of the quality of the 3th embryo with the blastocyst formation during human ART

    胡煜; 刘吉; 李宝山; 于洪君; 宫立国


    Objective: To analyze the correlation of the quality of the 3th embryo with the blastocyst formation during human ART. Methods: In this study, All 130 patients accorded with our study standard. The 3th surplus embryos after transferred were cultured to the 6th days for the blastocyst formation. We tried to find the relationship among the patient's age, the 3th day embryo quality score and cell number with blastocyst formation. Result; The patient's age was not related with blastocyst formation. The remarkable result was the positive correlation beween blastulation rate with the day 3 embryo cell number or with the day3 embryo quality score. The blastocyst formation rate of the good quality embryo ( I - II ) was significantly higher than that of the poor quality embryo ( ID -IV ). The blastocyst formation rate of the ≥6 cells embryos in the 3th days was higher than that of the ≤5 cells embryos. By comprehensive analysis about the correlation of the blastocyst formation with day3 embryo cell numbe, the day3 embryo quality score and culture time, the result showed that the ≥6 cells I - IIdegree embryos had the highest blastocyst formation rate, the blastocyst formation rate of the≥6 cells III - IV degree embryos was higher. The blastocyst formation rate of the ≤5 cells embryos was lower than the ≥6 cells embryos. In all four groups embryos, the blastocyst formation rates in the 6th day were higher than in the 5th day. Conclusion; The day3 embryo cell number, quality score and culture time were important factors for the blastocyst formation, in which the 3th day embryo cell number was the most important. The culture to the 6th day brought some chance to blastocyst for surplus embryos which quality score was lower or developed slower. This culture progress effectually selected and saved the embryos which had potentiality to form the bastula.%目的 探讨人类辅助生殖技术中,day3胚胎质量与囊胚形成的相关性.方法

  4. Internally readable identifying tag

    A method of identifying non-metallic objects by means of X-ray equipment is described in detail. A small metal pin with a number of grooves cut in a pre-determined equi-spaced pattern is implanted into the non-metallic object and by decoding the groove patterns using X-ray equipment, the object is uniquely identified. A specific example of such an application is in studying the migratory habits of fish. The pin inserted into the snout of the fish is 0.010 inch in diameter, 0.040 inch in length with 8 possible positions for grooves if spaced 0.005 inch apart. With 6 of the groove positions available for data, the capacity is 26 or 64 combinations; clearly longer pins would increase the data capacity. This method of identification is a major advance over previous techniques which necessitated destruction of the fish in order to recover the identification tag. (UK)

  5. Identifying Fiscal Inflation

    De Graeve, Ferre; Queijo von Heideken, Virginia


    Fiscal theorists warn about the risk of future inflation as a consequence of current fiscal imbalances in the US. Because actual inflation remains historically low and data on inflation expectations do not corroborate such risks, warnings for fiscal inflation are often ignored in policy and academic circles. This paper shows that a canonical NK- DSGE model enables identifying an anticipated component of inflation expectations that is closely related to fiscal policy. Estimation results sugges...

  6. On identified predictive control

    Bialasiewicz, Jan T.


    Self-tuning control algorithms are potential successors to manually tuned PID controllers traditionally used in process control applications. A very attractive design method for self-tuning controllers, which has been developed over recent years, is the long-range predictive control (LRPC). The success of LRPC is due to its effectiveness with plants of unknown order and dead-time which may be simultaneously nonminimum phase and unstable or have multiple lightly damped poles (as in the case of flexible structures or flexible robot arms). LRPC is a receding horizon strategy and can be, in general terms, summarized as follows. Using assumed long-range (or multi-step) cost function the optimal control law is found in terms of unknown parameters of the predictor model of the process, current input-output sequence, and future reference signal sequence. The common approach is to assume that the input-output process model is known or separately identified and then to find the parameters of the predictor model. Once these are known, the optimal control law determines control signal at the current time t which is applied at the process input and the whole procedure is repeated at the next time instant. Most of the recent research in this field is apparently centered around the LRPC formulation developed by Clarke et al., known as generalized predictive control (GPC). GPC uses ARIMAX/CARIMA model of the process in its input-output formulation. In this paper, the GPC formulation is used but the process predictor model is derived from the state space formulation of the ARIMAX model and is directly identified over the receding horizon, i.e., using current input-output sequence. The underlying technique in the design of identified predictive control (IPC) algorithm is the identification algorithm of observer/Kalman filter Markov parameters developed by Juang et al. at NASA Langley Research Center and successfully applied to identification of flexible structures.

  7. Identifying Phronotypes in Psychiatry

    F. AndrewKozel


    Full Text Available Refinements in the methods of diagnosis for psychiatric disorders are critically needed. These new methods should be based on objectively measured brain characteristics that provide clinically useful information. Studying the brain with respect to psychiatric disorders, however, faces numerous challenges. Utilizing techniques learned in other areas of medicine to deal with symptoms that lead to complex disorders can provide insight into improving diagnostic models in psychiatry. Specifically, many areas of medicine use objective measures of an organ’s function or characteristic to guide clinical management of particular subjective complaints. In psychiatry, an objectively measured brain characteristic that provides clinically useful information is proposed to be that person’s “phronotype.” Important requirements to developing phronotypes are discussed. Identifying phronotypes in psychiatry will require a specific investigative approach that must be grounded in rigorous scientific methodology. Successfully developing such markers will have a profound impact on clinical care, clinical research, basic science research, and most importantly the lives of those suffering from these illnesses.

  8. Integrative Functional Genomics Analysis of Sustained Polyploidy Phenotypes in Breast Cancer Cells Identifies an Oncogenic Profile for GINS2

    Juha K. Rantala


    Full Text Available Aneuploidy is among the most obvious differences between normal and cancer cells. However, mechanisms contributing to development and maintenance of aneuploid cell growth are diverse and incompletely understood. Functional genomics analyses have shown that aneuploidy in cancer cells is correlated with diffuse gene expression signatures and aneuploidy can arise by a variety of mechanisms, including cytokinesis failures, DNA endoreplication, and possibly through polyploid intermediate states. To identify molecular processes contributing to development of aneuploidy, we used a cell spot microarray technique to identify genes inducing polyploidy and/or allowing maintenance of polyploid cell growth in breast cancer cells. Of 5760 human genes screened, 177 were found to induce severe DNA content alterations on prolonged transient silencing. Association with response to DNA damage stimulus and DNA repair was found to be the most enriched cellular processes among the candidate genes. Functional validation analysis of these genes highlighted GINS2 as the highest ranking candidate inducing polyploidy, accumulation of endogenous DNA damage, and impairing cell proliferation on inhibition. The cell growth inhibition and induction of polyploidy by suppression of GINS2 was verified in a panel of breast cancer cell lines. Bioinformatic analysis of published gene expression and DNA copy number studies of clinical breast tumors suggested GINS2 to be associated with the aggressive characteristics of a subgroup of breast cancers in vivo. In addition, nuclear GINS2 protein levels distinguished actively proliferating cancer cells suggesting potential use of GINS2 staining as a biomarker of cell proliferation as well as a potential therapeutic target.

  9. Knock-in fibroblasts and transgenic blastocysts for expression of human FGF2 in the bovine β-casein gene locus using CRISPR/Cas9 nuclease-mediated homologous recombination.

    Jeong, Young-Hee; Kim, Yeong Ji; Kim, Eun Young; Kim, Se Eun; Kim, Jiwoo; Park, Min Jee; Lee, Hong-Gu; Park, Se Pill; Kang, Man-Jong


    Many transgenic domestic animals have been developed to produce therapeutic proteins in the mammary gland, and this approach is one of the most important methods for agricultural and biomedical applications. However, expression and secretion of a protein varies because transgenes are integrated at random sites in the genome. In addition, distal enhancers are very important for transcriptional gene regulation and tissue-specific gene expression. Development of a vector system regulated accurately in the genome is needed to improve production of therapeutic proteins. The objective of this study was to develop a knock-in system for expression of human fibroblast growth factor 2 (FGF2) in the bovine β-casein gene locus. The F2A sequence was fused to the human FGF2 gene and inserted into exon 3 of the β-casein gene. We detected expression of human FGF2 mRNA in the HC11 mouse mammary epithelial cells by RT-PCR and human FGF2 protein in the culture media using western blot analysis when the knock-in vector was introduced. We transfected the knock-in vector into bovine ear fibroblasts and produced knock-in fibroblasts using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Moreover, the CRISPR/Cas9 system was more efficient than conventional methods. In addition, we produced knock-in blastocysts by somatic cell nuclear transfer using the knock-in fibroblasts. Our knock-in fibroblasts may help to create cloned embryos for development of transgenic dairy cattle expressing human FGF2 protein in the mammary gland via the expression system of the bovine β-casein gene. PMID:26197710

  10. Digital Identifier Systems: Comparative Evaluation

    Hamid Reza Khedmatgozar; Mehdi Alipour-Hafezi; Payam Hanafizadeh


    Identifier is one of the main elements in identifying an object in digital environment. Digital identifier systems were developed followed by a lot of problems such as violation of persistency and uniqueness of physical identifiers and URL in digital environment. These identifiers try to guarantee uniqueness and persistency of hostnames by using indirect names for Domain Name System (DNS). The main objective of this research is to identify qualified digital identifier system among other syste...

  11. Genome-wide uniparental disomy screen in human discarded morphologically abnormal embryos.

    Xu, Jiawei; Zhang, Meixiang; Niu, Wenbin; Yao, Guidong; Sun, Bo; Bao, Xiao; Wang, Linlin; Du, Linqing; Sun, Yingpu


    Uniparental disomy (UPD) has been shown to be rare in human normal blastocysts, but its frequency in discarded morphologically abnormal embryos and its relevance to embryonic self-correction of aneuploid remains unknown. The aim of this study was to detect UPD in discarded morphologically abnormal embryos. Both discarded morphologically abnormal embryos, including zero-pronuclear zygotes (0PN), one-pronuclear zygotes (1PN), three-pronuclear zygotes (3PN) and 2PN embryos scored as low development potential were cultured into blastocysts then underwent trophectoderm biopsy. Genome-wide UPD screening of the trophectoderm of 241 discarded morphologically abnormal embryo sourced blastocysts showed that UPD occurred in nine embryos. Five embryos exhibited UPDs with euploid chromosomes, and four displayed UPDs with chromosomal aneuploid. The percentage of UPDs among the morphologically abnormal sourced blastocysts was 3.73%, which is significant higher than the percentage observed in normal blastocysts. The frequency of UPD in 3PN-sourced blastocysts was 7.69%, which is significantly higher than that in normal blastocysts. This study provides the first systematic genome-wide profile of UPD in discarded morphologically abnormal embryos. Our results indicated that UPD may be a common phenomenon in discarded morphologically abnormal embryos and may be relevant to human embryonic self-correction. PMID:26194013

  12. 小鼠囊胚细胞凋亡和半胱氨酸天冬氨酸蛋白酶3表达与维甲酸的诱导作用%Induction effect of retinoic acid on cell apoptosis and Caspase-3 expression in mouse blastocysts

    熊彦娥; 张端莲


    背景:细胞凋亡是维甲酸导致早期胚胎异常发育的原因之一,而半胱氨酸天冬氨酸蛋白酶3 介导的细胞凋亡是细胞凋亡的主要途径.目的:观察维甲酸对体外培养小鼠囊胚细胞凋亡的影响,以及对囊胚细胞中半胱氨酸天冬氨酸蛋白酶3 表达的改变.方法:获取妊娠3.5 d 小鼠囊胚,分别在含0,1 和10 μmol/L 维甲酸的M199 培养基中培养24 h,用带有荧光标记的原位末端标记法观察维甲酸对小鼠囊胚细胞凋亡的效应;并用免疫组织化学S-P 法检测维甲酸对小鼠囊胚半胱氨酸天冬氨酸蛋白酶3 的表达.结果与结论:所有组均观察到囊胚细胞凋亡和半胱氨酸天冬氨酸蛋白酶3 表达,高剂量维甲酸组中囊胚细胞凋亡荧光值,半胱氨酸天冬氨酸蛋白酶3 表达的平均吸光度值和阳性面积率均显著升高(P < 0.01).结果提示维甲酸可促进体外培养小鼠囊胚细胞凋亡和及半胱氨酸天冬氨酸蛋白酶3 表达,证实了维甲酸对小鼠胚胎的发育的细胞毒性作用.%BACKGROUND:One of the reasons for abnormal development of embryos is the apoptosis induced by retinoic acid, and Caspase-3mediated apoptosis is the main pathway for apoptosis in mouse blastocysts.OBJECTIVE:To observe apoptosis in mouse blastocyst induced by retinoic acid, and to detected expression of Caspase-3 in mouse blastocysts.METHODS:Mouse 3.5 days blastocysts were exposed to doses of 0, 1 and 10 μmol/L retinoic acid for 24 hours. Effects of retinoic acid on mouse blastocysts apoptosis was observed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and the expression of Caspase-3 were detected by immunohistochemical S-P staining.RESULTS AND CONCLUSION:stocysts apoptosis and Caspase-3 expression could be found in all groups, which was significantly higher in the high dosage group than that in the control and low dosage group (P < 0.01). The results suggest that retinoic acid can promote

  13. Digital Identifier Systems: Comparative Evaluation

    Hamid Reza Khedmatgozar


    Full Text Available Identifier is one of the main elements in identifying an object in digital environment. Digital identifier systems were developed followed by a lot of problems such as violation of persistency and uniqueness of physical identifiers and URL in digital environment. These identifiers try to guarantee uniqueness and persistency of hostnames by using indirect names for Domain Name System (DNS. The main objective of this research is to identify qualified digital identifier system among other systems. To achieve the research objective, researchers have considered two major steps: first, identifying main criteria for distinguishing digital identifier based on literature review and focus group interview; and second, performing a comparative evaluation on common identifier systems in the world. Findings of first step demonstrated seven main criteria in three domains for distinguishing digital identifier systems: identifier uniqueness and persistency in the identifier features domain, digital identification, digital uniqueness, digital persistency and digital actionability in the digital coverage domain, and globality in the comprehensiveness of scope domain. In the second step, results of the comparative evaluation on common identifier systems indicated that six identifier systems, included, DOI, Handle, UCI, URN, ARK and PURL, are appropriate choices for using as a digital identifier system. Also, according to these results, three identification systems Including NBN, MARIAM and ISNI were identified as suitable choices for digital identification in certain specialized fields. According to many benefits of using these identifiers in important applied fields, such as, digital content chains and networks integration, digital right management, cross referencing, digital libraries and citation analysis, results of this study can help digital environment experts to diagnose digital identifier and their effective use in applied fields.

  14. Simplified cryopreservation of porcine cloned blastocysts

    Du, Yutao; Zhang, Yunhai; Li, Juan;


    )â€"handmade cloning (HMC)â€"to establish a simplified and efficient cryopreservation system for porcine cloned embryos. In Experiment 1, zonae pellucidae of oocytes were partially digested with pronase, followed by centrifugation to polarize lipid particles. Ninety percent (173/192) oocytes were successfully......). Our results prove that porcine embryos produced from delipated oocytes by PA or HMC can be cryopreserved effectively by ultrarapid vitrification. Further experiments are required to assess the in vivo developmental competence of the cloned-vitrified embryos  ...

  15. New Criteria to Identify Spectrum

    A Jensen; M Krishna


    In this paper we give some new criteria for identifying the components of a probability measure, in its Lebesgue decomposition. This enables us to give new criteria to identify spectral types of self-adjoint operators on Hilbert spaces, especially those of interest.

  16. The Effect of Prolonged Culture of Chromosomally Abnormal Human Embryos on The Rate of Diploid Cells

    Masood Bazrgar


    Full Text Available Background: A decrease in aneuploidy rate following a prolonged co-culture of human blastocysts has been reported. As co-culture is not routinely used in assisted reproductive technology, the present study aimed to evaluate the effect of the prolonged single culture on the rate of diploid cells in human embryos with aneuploidies. Materials and Methods: In this cohort study, we used fluorescence in situ hybridization (FISH to reanalyze surplus blastocysts undergoing preimplantation genetic diagnosis (PGD on day 3 postfertilization. They were randomly studied on days 6 or 7 following fertilization. Results: Of the 30 analyzed blastocysts, mosaicism was observed in 26(86.6%, while 2(6.7% were diploid, and 2(6.7% were triploid. Of those with mosaicism, 23(88.5% were determined to be diploid-aneuploid and 3(11.5% were aneuploid mosaic. The total frequency of embryos with more than 50% diploid cells was 33.3% that was lower on day 7 in comparison with the related value on day 6 (P<0.05; however, there were no differences when the embryos were classified according to maternal age, blastocyst developmental stage, total cell number on day 3, and embryo quality. Conclusion: Although mosaicism is frequently observed in blastocysts, the prolonged single culture of blastocysts does not seem to increase the rate of normal cells.

  17. Author Identifiers in Scholarly Repositories

    Warner, Simeon


    Bibliometric and usage-based analyses and tools highlight the value of information about scholarship contained within the network of authors, articles and usage data. Less progress has been made on populating and using the author side of this network than the article side, in part because of the difficulty of unambiguously identifying authors. I briefly review a sample of author identifier schemes, and consider use in scholarly repositories. I then describe preliminary work at arXiv to implement public author identifiers, services based on them, and plans to make this information useful beyond the boundaries of arXiv.

  18. Identifying Occupationally Specific Affective Behaviors.

    Pucel, David J.


    Data from two groups of cosmetology instructors (n=15) and two groups of machinist instructors (n=17) validated the Occupational Affective Behavior Analysis instrument as capable of identifying affective behaviors viewed as important to success in a given occupation. (SK)

  19. Algorithms to identify failure pattern

    Poudel, Bhuwan Krishna Som


    This project report was written for ?Algorithms to Identify Failure Pattern? at NTNU (Norwegian University of Science and Technology), IME (Faculty of Information Technology, Mathematics and Electrical Engineering) and IDI (Department of Computer Science).In software application, there are three types of failure pattern: point pattern, block pattern and stripe pattern. The purpose of the report is to prepare an algorithm that identifies the pattern in a software application. Only theoretical ...

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

    Adam Burton


    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.

  1. Sparse Linear Identifiable Multivariate Modeling

    Henao, Ricardo; Winther, Ole


    In this paper we consider sparse and identifiable linear latent variable (factor) and linear Bayesian network models for parsimonious analysis of multivariate data. We propose a computationally efficient method for joint parameter and model inference, and model comparison. It consists of a fully...... Bayesian hierarchy for sparse models using slab and spike priors (two-component δ-function and continuous mixtures), non-Gaussian latent factors and a stochastic search over the ordering of the variables. The framework, which we call SLIM (Sparse Linear Identifiable Multivariate modeling), is validated and...... computational complexity. We attribute this mainly to the stochastic search strategy used, and to parsimony (sparsity and identifiability), which is an explicit part of the model. We propose two extensions to the basic i.i.d. linear framework: non-linear dependence on observed variables, called SNIM (Sparse Non-linear...

  2. Identifying tier one key suppliers.

    Wicks, Steve


    In today's global marketplace, businesses are becoming increasingly reliant on suppliers for the provision of key processes, activities, products and services in support of their strategic business goals. The result is that now, more than ever, the failure of a key supplier has potential to damage reputation, productivity, compliance and financial performance seriously. Yet despite this, there is no recognised standard or guidance for identifying a tier one key supplier base and, up to now, there has been little or no research on how to do so effectively. This paper outlines the key findings of a BCI-sponsored research project to investigate good practice in identifying tier one key suppliers, and suggests a scalable framework process model and risk matrix tool to help businesses effectively identify their tier one key supplier base. PMID:23615061

  3. Sparse Linear Identifiable Multivariate Modeling

    Henao, Ricardo; Winther, Ole


    In this paper we consider sparse and identifiable linear latent variable (factor) and linear Bayesian network models for parsimonious analysis of multivariate data. We propose a computationally efficient method for joint parameter and model inference, and model comparison. It consists of a fully...... Bayesian hierarchy for sparse models using slab and spike priors (two-component δ-function and continuous mixtures), non-Gaussian latent factors and a stochastic search over the ordering of the variables. The framework, which we call SLIM (Sparse Linear Identifiable Multivariate modeling), is validated and......-linear Identifiable Multivariate modeling) and allowing for correlations between latent variables, called CSLIM (Correlated SLIM), for the temporal and/or spatial data. The source code and scripts are available from slim/. © 2011 Ricardo Henao and Ole Winther....

  4. Football refereeing: Identifying innovative methods

    Reza MohammadKazemi


    Full Text Available The aim of the present study is to identify the potentials innovation in football industry. Data were collected from 10 national and international referees, assistant referees and referees’ supervisors in Iran. In this study, technological innovations are identified that assist better refereeing performances. The analysis revealed a significant relationship between using new technologies and referees ‘performance. The results indicate that elite referees, assistant referees and supervisors agreed to use new technological innovations during the game. According to their comments, this kind of technology causes the referees’ performance development.

  5. Identifying the Gifted Child Humorist.

    Fern, Tami L.


    This study attempted to identify gifted child humorists among 1,204 children in grades 3-6. Final identification of 13 gifted child humorists was determined through application of such criteria as funniness, originality, and exemplary performance or product. The influence of intelligence, development, social factors, sex differences, family…

  6. Identifying, Assisting the Disturbed Adolescent.

    Carlson, Patricia L.; Schaefer, William


    Educators are in an excellent position to identify seriously troubled young people. Major causes of adolescent problems are discussed, including drugs, parental failure, and biochemical disturbances. Educators can best intervene by becoming aware of support services within their own school and community. (TE)

  7. Identifying Two-Sided Markets

    Filistrucchi, L.; Geradin, D.A.A.G.; van Damme, E.E.C.


    Abstract: We review the burgeoning literature on two-sided markets focusing on the different definitions that have been proposed. In particular, we show that the well-known definition given by Evans is a particular case of the more general definition proposed by Rochet and Tirole. We then identify t

  8. Identifying high-risk medication

    Sædder, Eva; Brock, Birgitte; Nielsen, Lars Peter;


    salicylic acid, and beta-blockers; 30 drugs or drug classes caused 82 % of all serious MEs. The top ten drugs involved in fatal events accounted for 73 % of all drugs identified. CONCLUSION: Increasing focus on seven drugs/drug classes can potentially reduce hospitalizations, extended hospitalizations...

  9. Can Persistent Identifiers Be Cool?

    Barbara Bazzanella


    Full Text Available The fast growth of scientific and non-scientific digital data, as well as the proliferation of new types of digital content, has led – among many other things – to a lot of innovative work on the concept of the identifier. Digital identifiers have become the key to preserving and accessing content, just as physical identifier tags have been the key to accessing paper-based content and other physical entities for millennia. Two main schools of thought have emerged: on the one hand, librarians and public repositories have pushed the concept of the Persistent Identifier (PI as a way to guarantee long term identification and (sometimes access; on the other hand, the extraordinary success of the web has led several researchers and web experts to push the concept of the Cool URI as the universal mechanism for identifying and accessing digital content. Both views have their pros and cons, but so far (with only a few exceptions the two visions have developed in parallel, sometimes with a subtle underlying hostility.In this paper, we present the evolution of the Entity Name System (ENS, an open service-based platform developed as part of the OKKAM EU co-funded project, which can reconcile these two approaches. The new system, called ENS2.0, is currently under development and will enable data creators and curators to combine the technical strengths and opportunities of the (Semantic Web vision with the organizational, economical and social requirements legitimately raised by the PI community and stakeholders.

  10. Giemsa C-banding of Barley Chromosomes. IV. Chromosomal Constitution of Autotetraploid Barley

    Linde-Laursen, Ib


    homologues of each of the chromosomes. The aneuploid C2--seedlings were fairly equally distributed on hypo-and hyperploids, and on the seven chromosome groups. This suggests that a particular chromosome is lost or gained at random in gametes and embryos. The 11 C3--seedlings comprised seven true euploids......The progeny of an autotetraploid barley plant (C1) consisted of 45 tetraploids and 33 aneuploids. Giemsa C-banding was used to identify each of the chromosomes in 20 euploid and 31 aneuploid C2--seedlings, and in 11 C3--offspring of aneuploid C2--plants. The euploid C2--seedlings all had four......, one seedling with 2n=28 having an extra chromosome 6 and missing one chromosome 3, and three seedlings with 2n=29. The chromosomal composition of aneuploid C3--seedlings did not reflect that of their aneuploid C2--parents with respect to missing or extra chromosomes. Two hypohexaploid C2--seedlings...

  11. Identifying the Poorest Older Americans

    Fisher, Johathan; Johnson, David; Marchand, Joseph; Smeeding, Timothy; Boyle Torrey, B.


    Objectives: Public policies generally target a subset of the population defined as poor or needy, but rarely are people poor or needy in the same way. This is particularly true among older adults, as they have fewer options to compensate for financial decisions made earlier in life. This study investigates poverty among this group in order to identify who among them is financially worst off. Methods: We use 20 years of data from the Consumer Expenditure Survey to examine the income and consum...

  12. Identifying Emotion from Natural Walking

    Cui, Liqing; Li, Shun; Zhang, Wan; Zhang, Zhan; Zhu, Tingshao


    Emotion identification from gait aims to automatically determine persons affective state, it has attracted a great deal of interests and offered immense potential value in action tendency, health care, psychological detection and human-computer(robot) interaction.In this paper, we propose a new method of identifying emotion from natural walking, and analyze the relevance between the traits of walking and affective states. After obtaining the pure acceleration data of wrist and ankle, we set a...

  13. Identifying High Performance ERP Projects

    Stensrud, Erik; Myrtveit, Ingunn


    Learning from high performance projects is crucial for software process improvement. Therefore, we need to identify outstanding projects that may serve as role models. It is common to measure productivity as an indicator of performance. It is vital that productivity measurements deal correctly with variable returns to scale and multivariate data. Software projects generally exhibit variable returns to scale, and the output from ERP projects is multivariate. We propose to use Data Envelopment ...

  14. Identifying Network Perturbation in Cancer.

    Maxim Grechkin; Logsdon, Benjamin A.; Gentles, Andrew J.; Su-In Lee


    We present a computational framework, called DISCERN (DIfferential SparsE Regulatory Network), to identify informative topological changes in gene-regulator dependence networks inferred on the basis of mRNA expression datasets within distinct biological states. DISCERN takes two expression datasets as input: an expression dataset of diseased tissues from patients with a disease of interest and another expression dataset from matching normal tissues. DISCERN estimates the extent to which each ...

  15. Identifying Network Perturbation in Cancer

    Grechkin, Maxim; Logsdon, Benjamin A.; Gentles, Andrew J.; Lee, Su-In


    We present a computational framework, called DISCERN (DIfferential SparsE Regulatory Network), to identify informative topological changes in gene-regulator dependence networks inferred on the basis of mRNA expression datasets within distinct biological states. DISCERN takes two expression datasets as input: an expression dataset of diseased tissues from patients with a disease of interest and another expression dataset from matching normal tissues. DISCERN estimates the extent to which each ...

  16. Identifying Network Perturbation in Cancer

    Logsdon, Benjamin A.; Gentles, Andrew J.; Lee, Su-In


    We present a computational framework, called DISCERN (DIfferential SparsE Regulatory Network), to identify informative topological changes in gene-regulator dependence networks inferred on the basis of mRNA expression datasets within distinct biological states. DISCERN takes two expression datasets as input: an expression dataset of diseased tissues from patients with a disease of interest and another expression dataset from matching normal tissues. DISCERN estimates the extent to which each gene is perturbed—having distinct regulator connectivity in the inferred gene-regulator dependencies between the disease and normal conditions. This approach has distinct advantages over existing methods. First, DISCERN infers conditional dependencies between candidate regulators and genes, where conditional dependence relationships discriminate the evidence for direct interactions from indirect interactions more precisely than pairwise correlation. Second, DISCERN uses a new likelihood-based scoring function to alleviate concerns about accuracy of the specific edges inferred in a particular network. DISCERN identifies perturbed genes more accurately in synthetic data than existing methods to identify perturbed genes between distinct states. In expression datasets from patients with acute myeloid leukemia (AML), breast cancer and lung cancer, genes with high DISCERN scores in each cancer are enriched for known tumor drivers, genes associated with the biological processes known to be important in the disease, and genes associated with patient prognosis, in the respective cancer. Finally, we show that DISCERN can uncover potential mechanisms underlying network perturbation by explaining observed epigenomic activity patterns in cancer and normal tissue types more accurately than alternative methods, based on the available epigenomic data from the ENCODE project. PMID:27145341

  17. [Gifted children! How to identify].

    Revol, O; Bléandonu, G


    Recognizing the intellectually gifted child allows one to anticipate the onset of behavioral disorders and to respond to them so as to improve quality of life for these children and their family. Certain signs can draw attention to this phenomenon. The decisive criterion has always been IQ. The notion of intelligence should be broadened and researchers have adventured beyond the purely intellectual competencies, which value most prominently the educational sphere. In addition, the modeling of high potential is currently renewing the approach to this question. This article takes this complexity into account and presents the main criteria used for identifying intellectually gifted children. PMID:22325457

  18. Comprehensive analysis of karyotypic mosaicism between trophectoderm and inner cell mass.

    Johnson, D S; Cinnioglu, C; Ross, R; Filby, A; Gemelos, G; Hill, M; Ryan, A; Smotrich, D; Rabinowitz, M; Murray, M J


    Aneuploidy has been well-documented in blastocyst embryos, but prior studies have been limited in scale and/or lack mechanistic data. We previously reported preclinical validation of microarray 24-chromosome preimplantation genetic screening in a 24-h protocol. The method diagnoses chromosome copy number, structural chromosome aberrations, parental source of aneuploidy and distinguishes certain meiotic from mitotic errors. In this study, our objective was to examine aneuploidy in human blastocysts and determine correspondence of karyotypes between trophectoderm (TE) and inner cell mass (ICM). We disaggregated 51 blastocysts from 17 couples into ICM and one or two TE fractions. The average maternal age was 31. Next, we ran 24-chromosome microarray molecular karyotyping on all of the samples, and then performed a retrospective analysis of the data. The average per-chromosome confidence was 99.95%. Approximately 80% of blastocysts were euploid. The majority of aneuploid embryos were simple aneuploid, i.e. one or two whole-chromosome imbalances. Structural chromosome aberrations, which are common in cleavage stage embryos, occurred in only three blastocysts (5.8%). All TE biopsies derived from the same embryos were concordant. Forty-nine of 51 (96.1%) ICM samples were concordant with TE biopsies derived from the same embryos. Discordance between TE and ICM occurred only in the two embryos with structural chromosome aberration. We conclude that TE karyotype is an excellent predictor of ICM karyotype. Discordance between TE and ICM occurred only in embryos with structural chromosome aberrations. PMID:20643877

  19. Genetic basis for dosage sensitivity in Arabidopsis thaliana.

    Isabelle M Henry


    Full Text Available Aneuploidy, the relative excess or deficiency of specific chromosome types, results in gene dosage imbalance. Plants can produce viable and fertile aneuploid individuals, while most animal aneuploids are inviable or developmentally abnormal. The swarms of aneuploid progeny produced by Arabidopsis triploids constitute an excellent model to investigate the mechanisms governing dosage sensitivity and aneuploid syndromes. Indeed, genotype alters the frequency of aneuploid types within these swarms. Recombinant inbred lines that were derived from a triploid hybrid segregated into diploid and tetraploid individuals. In these recombinant inbred lines, a single locus, which we call SENSITIVE TO DOSAGE IMBALANCE (SDI, exhibited segregation distortion in the tetraploid subpopulation only. Recent progress in quantitative genotyping now allows molecular karyotyping and genetic analysis of aneuploid populations. In this study, we investigated the causes of the ploidy-specific distortion at SDI. Allele frequency was distorted in the aneuploid swarms produced by the triploid hybrid. We developed a simple quantitative measure for aneuploidy lethality and using this measure demonstrated that distortion was greatest in the aneuploids facing the strongest viability selection. When triploids were crossed to euploids, the progeny, which lack severe aneuploids, exhibited no distortion at SDI. Genetic characterization of SDI in the aneuploid swarm identified a mechanism governing aneuploid survival, perhaps by buffering the effects of dosage imbalance. As such, SDI could increase the likelihood of retaining genomic rearrangements such as segmental duplications. Additionally, in species where triploids are fertile, aneuploid survival would facilitate gene flow between diploid and tetraploid populations via a triploid bridge and prevent polyploid speciation. Our results demonstrate that positional cloning of loci affecting traits in populations containing ploidy and

  20. Can tests identify creative people?

    Bell, Peter M.

    It is always a popular pursuit by academic administrators to assess the creativity or innovative qualities of scientists in order to evaluate their research capabilities. Of course, traditionally such evaluations have been fraught with subjectivity (i.e., innovative scientists are commonly thought to be weird, under 40 years old, independent, risk-taking, etc.), and thus such evaluations have not been highly valued. In recent years, through testing, the American Chemical Society (ACS) has attempted to give respectability to the art of predicting the creativity of a scientist. ACS, which draws its members from both industrial and academic laboratories, held a symposium on the subject of evaluating the creativity of scientists. The proceedings were published by ACS as ‘Innovation and U.S. Research: Problems and Recommendations’ (W. N. Smith and C.F. Larson, eds., 1980). In the proceedings, as reported in the July 1982 Chemtec (all quotes here are from the Chemtec article), A. Nisson was able to identify only the following two-part characteristic of an innovative person: (1) a low threshold to ‘a state of discomfort with some aspect of the order of things, the status quo,’ and (2) ‘an extraordinarily high level of mental stamina enabling him or her to persist until the state of discomfort is removed.’

  1. 不同促精子活动剂对水牛卵母细胞体外受精及随后胚胎发育的影响%The effect of sperm-motility enhancing agents on the in vitro fertilization of Bubalus bubalis oocytes and their subsequent development to blastocyst

    Abdul Rahman SESAY; 石德顺


    decrease (12.5% vs 2.6%, P<0.05) in the proportion of cleaved embryos that developed to blastocyst after sperm treatment with trypsin. The treatment of spermatozoa with caffeine and calcium-ionophore A23187 had no significant effect on cleavage and blastocyst yield. There was a minor improvement in blastocyst yield when PHE combined with calcium-ionophore was employed during IVF. However, high concentrations of caffeine seem to be detrimental to cleavage rate and blastocyst development.

  2. Frequency of Aneuploids in Progenies of Autotriploid Barley, Hordeum Vulgare L

    Sandfær, J.


    Chromosome counts of 863 progeny plants originating from 68 autotriploid barley plants revealed a considerable variation in chromosome numbers ranging from the diploid number (2n= 14) to 2n= 39. The most frequent groups were plants with 15 and 16 chromosomes each constituting about 27% of all pro...

  3. The adverse outcome pathway (AOP) for chemical binding to tubulin in oocytes leading to aneuploid offspring.

    Marchetti, Francesco; Massarotti, Alberto; Yauk, Carole L; Pacchierotti, Francesca; Russo, Antonella


    The Organisation for Economic Co-operation and Development (OECD) has launched the Adverse Outcome Pathway (AOP) Programme to advance knowledge of pathways of toxicity and improve the use of mechanistic information in risk assessment. An AOP links a molecular initiating event (MIE) to an adverse outcome (AO) through intermediate key events (KE). Here, we present the scientific evidence in support of an AOP whereby chemicals that bind to tubulin cause microtubule depolymerization resulting in spindle disorganization followed by altered chromosome alignment and segregation and the generation of aneuploidy in female germ cells, ultimately leading to aneuploidy in the offspring. Aneuploidy, an abnormal number of chromosomes that is not an exact multiple of the haploid number, is a well-known cause of human disease and represents a major cause of infertility, pregnancy failure, and serious genetic disorders in the offspring. Among chemicals that induce aneuploidy in female germ cells, a large majority impairs microtubule dynamics and spindle function. Colchicine, a prototypical chemical that binds to tubulin and causes microtubule depolymerization, is used here to illustrate the AOP. This AOP is specific to female germ cells exposed during the periovulation period. Although the majority of the data come from rodent studies, the available evidence suggests that the MIE and KEs are conserved across species and would occur in human oocytes. The development of AOPs related to mutagenicity in germ cells is expected to aid the identification of potential hazards to germ cell genomic integrity and support regulatory efforts to protect population health. Environ. Mol. Mutagen. 57:87-113, 2016. © 2015 Her Majesty the Queen in Right of Canada. PMID:26581746

  4. Adaptive response to ionizing radiation in normal and aneuploid human lymphocytes

    The ability to induce an adaptive response with low doses of gamma rays was studied in normal and trisomic lymphocytes (47, XX or XY, +21). The results indicate the presence of an adaptive response in lymphocytes of 3 normal donors, but in lymphocytes of 5 donors with trisomy 21 no significant adaptive response was found after irradiation with a low dose of gamma rays. In normal and trisomic lymphocytes (47, XX or XY, +21) irradiated with 1.50 Gy, a similar chromosomal radiosensitivity was observed at the 48th hour after stimulation. (author) 2 tabs., 15 ref

  5. Gene expression analysis of induced pluripotent stem cells from aneuploid chromosomal syndromes

    Zhang, Ruosi; Hao, Lili; Wang, Lingping; Chen, Meili; Li, Wen; Li, Rujiao; Yu, Jun; Xiao, Jingfa; Wu, Jiayan


    Background Human aneuploidy is the leading cause of early pregnancy loss, mental retardation, and multiple congenital anomalies. Due to the high mortality associated with aneuploidy, the pathophysiological mechanisms of aneuploidy syndrome remain largely unknown. Previous studies focused mostly on whether dosage compensation occurs, and the next generation transcriptomics sequencing technology RNA-seq is expected to eventually uncover the mechanisms of gene expression regulation and the relat...

  6. Organización espacial y funcional del genoma en células aneuploides

    Paz Gandiaga, Nerea


    198 p. El genoma se encuentra altamente organizado en el espacio nuclear de las células eucariotas. Los cromosomas y los genes ocupan una posición preferente respecto del centro o de la periferia del núcleo, y respecto de otros elementos genéticos. En la actualidad se cuenta con un volumen importante de evidencias que apoyan la noción que la organización espacial del genoma tiene influencia sobre la función del mismo y viceversa. Variaciones en el número y estructura de los cromosomas ...

  7. Cdc20 is critical for meiosis I and fertility of female mice.

    Fang Jin


    Full Text Available Chromosome missegregation in germ cells is an important cause of unexplained infertility, miscarriages, and congenital birth defects in humans. However, the molecular defects that lead to production of aneuploid gametes are largely unknown. Cdc20, the activating subunit of the anaphase-promoting complex/cyclosome (APC/C, initiates sister-chromatid separation by ordering the destruction of two key anaphase inhibitors, cyclin B1 and securin, at the transition from metaphase to anaphase. The physiological significance and full repertoire of functions of mammalian Cdc20 are unclear at present, mainly because of the essential nature of this protein in cell cycle progression. To bypass this problem we generated hypomorphic mice that express low amounts of Cdc20. These mice are healthy and have a normal lifespan, but females produce either no or very few offspring, despite normal folliculogenesis and fertilization rates. When mated with wild-type males, hypomorphic females yield nearly normal numbers of fertilized eggs, but as these embryos develop, they become malformed and rarely reach the blastocyst stage. In exploring the underlying mechanism, we uncover that the vast majority of these embryos have abnormal chromosome numbers, primarily due to chromosome lagging and chromosome misalignment during meiosis I in the oocyte. Furthermore, cyclin B1, cyclin A2, and securin are inefficiently degraded in metaphase I; and anaphase I onset is markedly delayed. These results demonstrate that the physiologically effective threshold level of Cdc20 is high for female meiosis I and identify Cdc20 hypomorphism as a mechanism for chromosome missegregation and formation of aneuploid gametes.

  8. Psychosocial counselling of identifiable sperm donors

    M. Visser; M.H. Mochtar; A.A. de Melker; F. van der Veen; S. Repping; T. Gerrits


    STUDY QUESTION: What do identifiable sperm donors feel about psychosocial counselling? SUMMARY ANSWER: Identifiable sperm donors found it important that psychosocial counselling focused on emotional consequences and on rules and regulations and they expected to have access to psychosocial counsellin

  9. NIH Researchers Identify OCD Risk Gene

    ... News From NIH NIH Researchers Identify OCD Risk Gene Past Issues / Summer 2006 Table of Contents For ... and Alcoholism (NIAAA) have identified a previously unknown gene variant that doubles an individual's risk for obsessive- ...

  10. INVocD: Identifier Name Vocabulary Dataset

    Butler, Simon; Wermelinger, Michel; Yu, Yijun; Sharp, Helen


    NVocD is a dataset of the identifier name declarations and vocabulary found in 60 FLOSS Java projects where the source code structure is recorded and the identifier name vocabulary is made directly available, offering advantages for identifier name research over conventional source code models. The dataset has been used to support a range of research projects from identifier name analysis to concept location, and provides many opportunities to researchers.

  11. Ability of Slovakian Pupils to Identify Birds

    Prokop, Pavol; Rodak, Rastislav


    A pupil's ability to identify common organisms is necessary for acquiring further knowledge of biology. We investigated how pupils were able to identify 25 bird species following their song, growth habits, or both features presented simultaneously. Just about 19% of birds were successfully identified by song, about 39% by growth habit, and 45% of…

  12. IVOA Identifiers Version 2.0

    Demleitner, Markus; Linde, Tony; Williams, Roy; Noddle, Keith


    An IVOA Identifier is a globally unique name for a resource within the Virtual Observatory. This name can be used to retrieve a unique description of the resource from an IVOA-compliant registry or to identify an entity like a dataset or a protocol without dereferencing the identifier. This document describes the syntax for IVOA Identifiers as well as how they are created. The syntax has been defined to encourage global-uniqueness naturally and to maximize the freedom of resource providers to control the character content of an identifier.

  13. De-identifying an EHR Database

    Lauesen, Søren; Pantazos, Kostas; Lippert, Søren


    -identified a Danish EHR database with 437,164 patients. The goal was to generate a version with real medical records, but related to artificial persons. We developed a de-identification algorithm that uses lists of named entities, simple language analysis, and special rules. Our algorithm consists of 3 steps......: collect lists of identifiers from the database and external resources, define a replacement for each identifier, and replace identifiers in structured data and free text. Some patient records could not be safely de-identified, so the de-identified database has 323,122 patient records with an acceptable...... degree of anonymity, readability and correctness (F-measure of 95%). The algorithm has to be adjusted for each culture, language and database....

  14. Identifiable parameters for parallel robots kinematic calibration

    Besnard, Sébastian; Khalil, Wisama


    This paper presents a numerical method for the determination of the identifiable parameters of parallel robots. The special case of Stewart-Gough 6 degrees-offreedom parallel robots is studied for classical and self calibration methods, but this method can be generalized to any kind of parallel robot. The method is based on QR decomposition of the observation matrix of the calibration system. Numerical relations between the identifiable and non identifiable parameters can be obtained.

  15. A uniform resource identifier scheme for SNMP

    Lopes, Rui Pedro; Oliveira, José Luís


    One of the World Wide Web characteristics, besides its omnipresence in computer systems, is the adoption of a universal user interface that is used to access several different services that were previously accessed individually by independent applications. The Internet resources started to be identified by URI schemes, a text string with specific syntax and grammar. Although existing for several services such as http, ftp, gopher and news, these identifiers are not used to identify SNMP resou...

  16. Identifying shortage occupations in the UK

    Anna Downs


    This article describes the top-down methodology used by the Migration Advisory Committee (MAC) to identify shortage occupations. Identifying shortage occupations is difficult and few countries have attempted to do this in a systematic way. The methodology developed by the MAC to identify shortage occupations is the first to use a comprehensive range of indicators. The methodology dovetails national level top-down data with bottom-up micro-level data and qualitative information from stakeholde...

  17. Determination of set-membership identifiability sets

    Ravanbod-Hosseini, Laleh; Verdière, Nathalie; Jauberthie, Carine


    This paper concerns the concept of set-membership identifiability introduced in \\cite{jauberthie}. Given a model, a set-membership identifiable set is a connected set in the parameter domain of the model such that its corresponding trajectories are distinct to trajectories arising from its complementary. For obtaining the so-called set-membership identifiable sets, we propose an algorithm based on interval analysis tools. The proposed algorithm is decomposed into three parts namely {\\it minci...

  18. Moving Beyond Weak Identifiers for Proxemic Interaction

    Sørensen, Henrik; Kjeldskov, Jesper


    significantly closer than what is actually the case. This makes the smartphone a weak identifier for applications that need to track persons. With a focus on feasible interaction design, we present a concept and prototype of a platform, which seek to support proxemic interaction beyond weak identifiers. The...... limitations that need to be addressed....

  19. Identifying Information Focuses in Listening Comprehension

    Zhang, Hong-yan


    The study explains the process of learners' listening comprehension within Halliday's information theory in functional grammar, including the skills of identifying focuses while listening in college English teaching. Identifying information focuses in listening is proved to improve the students' communicative listening ability by the means of a…

  20. EZID: Long term identifiers made easy (Invited)

    Starr, J.


    Scholarly research is producing ever increasing amounts of digital research data, and this data should be managed throughout the research life cycle both as part of good scientific practice, but also to comply with funder mandates, such as the 2013 OSTP Public Access Memo ( By assigning unique and persistent identifiers to data objects, data managers can gain control and flexibility over what can be a daunting task. This is due to the fact that the objects can be moved to new locations without disruption to links, as long as the identifier target is maintained. EZID is a tool that makes assigning and maintaining unique, persistent identifiers easy. It was designed and built by California Digital Library (CDL) and has both a user interface and a RESTful API. EZID currently offers services for two globally unique, persistent identifier schemes: Digital Object Identifiers (DOIs) and Archival Resource Keys (ARKs). DOIs are identifiers originating from the publishing world and are in widespread use for journal articles. CDL is able to offer DOIs because of being a founding member of DataCite (, an international consortium established to provide easier access to scientific research data on the Internet. ARKs are identifiers originating from the library, archive and museum community. Like DOIs, they become persistent when the objects and identifier forwarding information is maintained. DOIs and ARKs have a key role in data management and, therefore, in data management plans. DOIs are the recommended identifier for use in data citation, and ARKs provide the maximum flexibility needed for data documentation and management throughout the early phases of a project. The two identifier schemes are able to be used together, and EZID is made to work with both. EZID clients, coming from education, research, government, and the private sector, are utilizing the

  1. Identifying left-right deterministic linear languages

    CALERA RUBIO, JORGE; Oncina Carratalá, Jose


    Recently an algorithm to identify in the limit with polynomial time and data Left Deterministic Linear Languages (Left DLL) and, consequently Right DLL, was proposed. In this paper we show that the class of the Left-Right DLL formed by the union of both classes is also identifiable. To do that, we introduce the notion of n-negative characteristic sample, that is a sample that forces an inference algorithm to output an hypothesis of size bigger than n when strings from a non identifiable langu...

  2. Identifiable Data Files - Health Outcomes Survey (HOS)

    U.S. Department of Health & Human Services — The Medicare Health Outcomes Survey (HOS) identifiable data files are comprised of the entire national sample for a given 2-year cohort (including both respondents...

  3. Helping You Identify Quality Laboratory Services

    Helping You Identify Quality Laboratory Services Selecting quality health care services for yourself, a relative or friend requires special thought and attention. The Joint Commission has prepared this information ...

  4. Identifying opportunities in online-communities

    Hienerth, C.; Lettl, Christopher

    Previous research has identified innovation as a crucial prerequisite for early entrepreneurial processes. However, the nature of how innovations are developed is changing dramatically, as it is becoming increasingly open, collaborative and democratized. This paper demonstrates in conceptual term...

  5. Researchers Identify Early Sign of Pancreatic Cancer

    ... of pancreatic cancer Researchers identify early sign of pancreatic cancer September 28, 2014 Tags: PancreaticCancer Brian Wolpin, MD ... discovered a sign of the early development of pancreatic cancer – an upsurge in certain amino acids that occurs ...

  6. Identifying motivational factors within a multinational company

    Daniela Bradutanu


    The aim of the study is to identify the main motivational factors within a multinational company. The first objective is to identify work functions, formulated on Abraham Maslow’s pyramid, following the identification of the key characteristics that motivate an employee at the work place and last, but not least, the type of motivation that employees focus, intrinsic or extrinsic. The research method targeted a questionnaire based survey, including various company employees and an interv...

  7. Identifying Domestic and Imported Core Inflation

    Bjørnland, Hilde C.


    This paper estimates core inflation in Norway, identified as that component of inflation that has no long-run effect on GDP. The model distinguishes explicitly between domestic and imported core inflation. The results show that (domestic) core inflation is the main component of CPI inflation. CPI inflation, however, misrepresents core inflation in some periods. The differences are well explained by the other shocks identified in the model, in particular the oil price shocks of the 1970s when ...

  8. ORCID Author Identifiers: A Primer for Librarians.

    Akers, Katherine G; Sarkozy, Alexandra; Wu, Wendy; Slyman, Alison


    The ORCID (Open Researcher and Contributor ID) registry helps disambiguate authors and streamline research workflows by assigning unique 16-digit author identifiers that enable automatic linkages between researchers and their scholarly activities. This article describes how ORCID works, the benefits of using ORCID, and how librarians can promote ORCID at their institutions by raising awareness of ORCID, helping researchers create and populate ORCID profiles, and integrating ORCID identifiers into institutional repositories and other university research information systems. PMID:27054531

  9. Identifying marker typing incompatibilities in linkage analysis.

    Stringham, H M; Boehnke, M.


    A common problem encountered in linkage analyses is that execution of the computer program is halted because of genotypes in the data that are inconsistent with Mendelian inheritance. Such inconsistencies may arise because of pedigree errors or errors in typing. In some cases, the source of the inconsistencies is easily identified by examining the pedigree. In others, the error is not obvious, and substantial time and effort are required to identify the responsible genotypes. We have develope...

  10. Identifying Entity Aspects in Microblog Posts

    Spina, D.; Meij, E.; Rijke, de, M.; Oghina, A.; Bui, M.T.; Breuss, M.


    Online reputation management is about monitoring and handling the public image of entities (such as companies) on the Web. An important task in this area is identifying "aspects" of the entity of interest (such as products, services, competitors, key people, etc.) given a stream of microblog posts referring to the entity. In this paper we compare different IR techniques and opinion target identification methods for automatically identifying aspects and find that (i) simple statistical methods...

  11. Method for detecting and identifying peronospora arborescens

    Muñoz Ledesma, Francisco Javier; Jiménez Díaz, Rafael Manuel; Landa del Castillo, Blanca; Montes Borrego, Miguel


    The method for detecting and identifying Peronospora arborescens is based on amplification of specific regions of said pathogen. To do this, a number of oligonucleotide primers specific for P. arborescens were designed, by means of enzymatic amplification reaction, for identifying and detecting said pathogen. This method is useful in agriculture and provides an early diagnosis of the disease (mildew) during asymptomatic states of plants, which facilitates efficient control thereof, and sanita...

  12. Identifiability analysis in conceptual sewer modelling.

    Kleidorfer, M; Leonhardt, G; Rauch, W


    For a sufficient calibration of an environmental model not only parameter sensitivity but also parameter identifiability is an important issue. In identifiability analysis it is possible to analyse whether changes in one parameter can be compensated by appropriate changes of the other ones within a given uncertainty range. Parameter identifiability is conditional to the information content of the calibration data and consequently conditional to a certain measurement layout (i.e. types of measurements, number and location of measurement sites, temporal resolution of measurements etc.). Hence the influence of number and location of measurement sites on the number of identifiable parameters can be investigated. In the present study identifiability analysis is applied to a conceptual model of a combined sewer system aiming to predict the combined sewer overflow emissions. Different measurement layouts are tested and it can be shown that only 13 of the most sensitive catchment areas (represented by the model parameter 'effective impervious area') can be identified when overflow measurements of the 20 highest overflows and the runoff to the waste water treatment plant are used for calibration. The main advantage of this method is very low computational costs as the number of required model runs equals the total number of model parameters. Hence, this method is a valuable tool when analysing large models with a long runtime and many parameters. PMID:22864432

  13. Exploiting intrinsic fluctuations to identify model parameters.

    Zimmer, Christoph; Sahle, Sven; Pahle, Jürgen


    Parameterisation of kinetic models plays a central role in computational systems biology. Besides the lack of experimental data of high enough quality, some of the biggest challenges here are identification issues. Model parameters can be structurally non-identifiable because of functional relationships. Noise in measured data is usually considered to be a nuisance for parameter estimation. However, it turns out that intrinsic fluctuations in particle numbers can make parameters identifiable that were previously non-identifiable. The authors present a method to identify model parameters that are structurally non-identifiable in a deterministic framework. The method takes time course recordings of biochemical systems in steady state or transient state as input. Often a functional relationship between parameters presents itself by a one-dimensional manifold in parameter space containing parameter sets of optimal goodness. Although the system's behaviour cannot be distinguished on this manifold in a deterministic framework it might be distinguishable in a stochastic modelling framework. Their method exploits this by using an objective function that includes a measure for fluctuations in particle numbers. They show on three example models, immigration-death, gene expression and Epo-EpoReceptor interaction, that this resolves the non-identifiability even in the case of measurement noise with known amplitude. The method is applied to partially observed recordings of biochemical systems with measurement noise. It is simple to implement and it is usually very fast to compute. This optimisation can be realised in a classical or Bayesian fashion. PMID:26672148

  14. Ploidy of Bovine Nuclear Transfer Blastocysts Blastomere Donors

    Booth, P J; VIUFF, D; THOMSEN, P D;


    reconstructed from in vitro produced embryo donors. In vitro matured oocytes were enucleated and then activated using calcium ionophore A23187 followed by 6- dimethylaminopurine (6-DMAP). Subsequently, embryos were reconstructed using blas- tomeres from day 4–5 in vitro produced donors. The embryos were...

  15. Blastocyst Elongation, Trophoblastic Differentiation and Embryonic Pattern Formation

    The molecular basis behind elongation and concomitant gastrulation in ungulates that occurs during pre-implantation is still poorly understood. In-depth transcriptome analysis of the elongating porcine conceptus at specific stages has demonstrated that protein synthesis, protein trafficking, cell g...

  16. Circulating LH/hCG receptor (LHCGR may identify pre-treatment IVF patients at risk of OHSS and poor implantation

    Chambers Anne E


    Full Text Available Abstract Background Successful pregnancy via in vitro fertilization (IVF depends on the recovery of an adequate number of healthy oocytes and on blastocyst implantation following uterine transfer. Two hormones, LH and hCG, utilize a common LH/hCG receptor (LHCGR, variations in which have profound implications in human reproduction. Soluble LHCGR (sLHCGR is released from experimental cell lines and placental explants and it can be detected in the follicular fluid and serum. Methods To evaluate the impact of circulating soluble LHCGR (sLHCGR in fertility treatment, we measured sLHCGR and LH-sLHCGR complex in serum from women seeking IVF using specifically developed quantitative enzyme-linked immunosorbent assays (ELISA. Following an IVF cycle of treatment, patients were grouped according to oocyte yield into low (lower than or equal to 7 oocytes, intermediate (8-14 oocytes and high (greater than or equal to 15 oocytes responders and pregnancy outcome noted. Results Pre-treatment sLHCGR identified many women at risk of ovarian hyperstimulation. Low levels of sLHCGR were associated with pregnancy in both high and low responders but sLHCGR did not significantly affect the treatment outcome of intermediate responders. Low responders who failed to become pregnant had high levels of circulating sLHCGR bound to LH (LH-sLHCGR. Conclusions Pre-treatment measurement of sLHCGR could be used to tailor individual fertility treatment programs and improve outcomes by avoiding ovarian hyperstimulation and poor embryo implantation.

  17. Identifying glass compositions in fly ash

    Aughenbaugh, Katherine; Stutzman, Paul; Juenger, Maria


    In this study, four Class F fly ashes were studied with a scanning electron microscope; the glassy phases were identified and their compositions quantified using point compositional analysis with k-means clustering and multispectral image analysis. The results showed that while the bulk oxide contents of the fly ashes were different, the four fly ashes had somewhat similar glassy phase compositions. Aluminosilicate glasses (AS), calcium aluminosilicate glasses (CAS), a mixed glass, and, in one case, a high iron glass were identified in the fly ashes. Quartz and iron crystalline phases were identified in each fly ash as well. The compositions of the three main glasses identified, AS, CAS, and mixed glass, were relatively similar in each ash. The amounts of each glass were varied by fly ash, with the highest calcium fly ash containing the most of calcium-containing glass. Some of the glasses were identified as intermixed in individual particles, particularly the calcium-containing glasses. Finally, the smallest particles in the fly ashes, with the most surface area available to react in alkaline solution, such as when mixed with portland cement or in alkali-activated fly ash, were not different in composition than the large particles, with each of the glasses represented. The method used in the study may be applied to a fly ash of interest for use as a cementing material in order to understand its potential for reactivity.

  18. Identifying glass compositions in fly ash

    Katherine eAughenbaugh


    Full Text Available In this study, four Class F fly ashes were studied with a scanning electron microscope; the glassy phases were identified and their compositions quantified using point compositional analysis with k-means clustering and multispectral image analysis. The results showed that while the bulk oxide contents of the fly ashes were different, the four fly ashes had somewhat similar glassy phase compositions. Aluminosilicate glasses (AS, calcium aluminosilicate glasses (CAS, a mixed glass, and, in one case, a high iron glass were identified in the fly ashes. Quartz and iron crystalline phases were identified in each fly ash as well. The compositions of the three main glasses identified, AS, CAS, and mixed glass, were relatively similar in each ash. The amounts of each glass were varied by fly ash, with the highest calcium fly ash containing the most of calcium-containing glass. Some of the glasses were identified as intermixed in individual particles, particularly the calcium-containing glasses. Finally, the smallest particles in the fly ashes, with the most surface area available to react in alkaline solution, such as when mixed with portland cement or in alkali-activated fly ash, were not different in composition than the large particles, with each of the glasses represented. The method used in the study may be applied to a fly ash of interest for use as a cementing material in order to understand its potential for reactivity.

  19. Identifying Geographic Clusters: A Network Analytic Approach

    Catini, Roberto; Penner, Orion; Riccaboni, Massimo


    In recent years there has been a growing interest in the role of networks and clusters in the global economy. Despite being a popular research topic in economics, sociology and urban studies, geographical clustering of human activity has often studied been by means of predetermined geographical units such as administrative divisions and metropolitan areas. This approach is intrinsically time invariant and it does not allow one to differentiate between different activities. Our goal in this paper is to present a new methodology for identifying clusters, that can be applied to different empirical settings. We use a graph approach based on k-shell decomposition to analyze world biomedical research clusters based on PubMed scientific publications. We identify research institutions and locate their activities in geographical clusters. Leading areas of scientific production and their top performing research institutions are consistently identified at different geographic scales.

  20. Coherence method of identifying signal noise model

    The noise analysis method is discussed in identifying perturbance models and their parameters by a stochastic analysis of the noise model of variables measured on a reactor. The analysis of correlations is made in the frequency region using coherence analysis methods. In identifying an actual specific perturbance, its model should be determined and recognized in a compound model of the perturbance system using the results of observation. The determination of the optimum estimate of the perturbance system model is based on estimates of related spectral densities which are determined from the spectral density matrix of the measured variables. Partial and multiple coherence, partial transfers, the power spectral densities of the input and output variables of the noise model are determined from the related spectral densities. The possibilities of applying the coherence identification methods were tested on a simple case of a simulated stochastic system. Good agreement was found of the initial analytic frequency filters and the transfers identified. (B.S.)

  1. Identifying Turbulent Structures through Topological Segmentation

    Bremer, Peer-Timo; Gruber, Andrea; Bennett, Janine C.; Gyulassy, Attila; Kolla, Hemanth; Chen, Jacqueline H.; Grout, Ray W.


    A new method of extracting vortical structures from a turbulent flow is proposed whereby topological segmentation of an indicator function scalar field is used to identify the regions of influence of the individual vortices. This addresses a long-standing challenge in vector field topological analysis: indicator functions commonly used produce a scalar field based on the local velocity vector field; reconstructing regions of influence for a particular structure requires selecting a threshold to define vortex extent. In practice, the same threshold is rarely meaningful throughout a given flow. By also considering the topology of the indicator field function, the characteristics of vortex strength and extent can be separated and the ambiguity in the choice of the threshold reduced. The proposed approach is able to identify several types of vortices observed in a jet in cross-flow configuration simultaneously where no single threshold value for a selection of common indicator functions appears able to identify all of these vortex types.

  2. Identifiability of surface sources from Cauchy data

    We explore the reconstruction of surface sources from single Cauchy data on the potential. Identifiability is first proven when the location of these sources is known and their density is to be sought. We also investigate the ill-posedness degree to establish that the problem is severely ill-posed, and exhibit the necessary orthogonality conditions fulfilled by exact Cauchy data, for which the process of recovering the density function of the sources succeeds. We later turn to the more difficult reconstruction of the location, the shape and the density function of the sources. Identifiability cannot be obtained for general geometries. Nevertheless, under some acceptable assumptions on the size and the location of these sources, we are able to state identifiability results

  3. Photoacoustic tomography to identify inflammatory arthritis

    Rajian, Justin Rajesh; Girish, Gandikota; Wang, Xueding


    Identifying neovascularity (angiogenesis) as an early feature of inflammatory arthritis can help in early accurate diagnosis and treatment monitoring of this disease. Photoacoustic tomography (PAT) is a hybrid imaging modality which relies on intrinsic differences in the optical absorption among the tissues being imaged. Since blood has highly absorbing chromophores including both oxygenated and deoxygenated hemoglobin, PAT holds potential in identifying early angiogenesis associated with inflammatory joint diseases. PAT is used to identify changes in the development of inflammatory arthritis in a rat model. Imaging at two different wavelengths, 1064 nm and 532 nm, on rats revealed that there is a significant signal enhancement in the ankle joints of the arthritis affected rats when compared to the normal control group. Histology images obtained from both the normal and the arthritis affected rats correlated well with the PAT findings. Results support the fact that the emerging PAT could become a new tool for clinical management of inflammatory arthritis.

  4. Identifying driver mutations in sequenced cancer genomes

    Raphael, Benjamin J; Dobson, Jason R; Oesper, Layla;


    protein sequence or structure. Finally, we review techniques to identify recurrent combinations of somatic mutations, including approaches that examine mutations in known pathways or protein-interaction networks, as well as de novo approaches that identify combinations of mutations according to......-throughput DNA sequencing data, particularly for tumor samples that comprise heterogeneous populations of cells. Next, we review computational approaches that aim to predict driver mutations according to their frequency of occurrence in a cohort of samples, or according to their predicted functional impact on......, and random mutations. Here, we review computational approaches to identify somatic mutations in cancer genome sequences and to distinguish the driver mutations that are responsible for cancer from random, passenger mutations. First, we describe approaches to detect somatic mutations from high...

  5. Scientometric methods for identifying emerging technologies

    Abercrombie, Robert K; Schlicher, Bob G; Sheldon, Frederick T


    Provided is a method of generating a scientometric model that tracks the emergence of an identified technology from initial discovery (via original scientific and conference literature), through critical discoveries (via original scientific, conference literature and patents), transitioning through Technology Readiness Levels (TRLs) and ultimately on to commercial application. During the period of innovation and technology transfer, the impact of scholarly works, patents and on-line web news sources are identified. As trends develop, currency of citations, collaboration indicators, and on-line news patterns are identified. The combinations of four distinct and separate searchable on-line networked sources (i.e., scholarly publications and citation, worldwide patents, news archives, and on-line mapping networks) are assembled to become one collective network (a dataset for analysis of relations). This established network becomes the basis from which to quickly analyze the temporal flow of activity (searchable events) for the example subject domain.

  6. Identifying motivational factors within a multinational company

    Daniela Bradutanu


    Full Text Available The aim of the study is to identify the main motivational factors within a multinational company. The first objective is to identify work functions, formulated on Abraham Maslow’s pyramid, following the identification of the key characteristics that motivate an employee at the work place and last, but not least, the type of motivation that employees focus, intrinsic or extrinsic. The research method targeted a questionnaire based survey, including various company employees and an interview with the manager. The results confirmed that in Romania, employees put great emphasis on extrinsic motivation, a certain income and job security being primary. These results have implications for managers that in order to effectively motivate staff, first, must know their needs and expectations. To identify the main needs and motivational factors we had as a starting point Maslow's pyramid.

  7. Methods of Identifying and Preventing SQL Attacks

    Bojken Shehu


    Full Text Available The paper begins by identifying the organizations which are vulnerable to the SQL attack referred to as an SQL injection attack. The term SQL injection attack is defined and a diagram is used to illustrate the way that attack occurs. In another section, the paper identifies the methods used to detect an attack to SQL, whereby the techniques are discussed extensively using relevant diagrams for illustration. The other sections cover the preventive methods, where the methods are also discussed with an illustration using diagrams.

  8. Factors identifying pigs predisposed to tail biting

    Beattie, V. E.; Breuer, K.; O Connell, N. E.; Sneddon, I. A.; Mercer, J. T.; Rance, K. A.; Sutcliffe, M. E. M.; Edwards, S A


    Approximately 5% of pigs slaughtered in the UK have been tail-bitten, leading to welfare and production issues. Tail biting is sporadic and not all pigs tail bite. The aim of this study was to identify factors that are common in pigs that perform tail-biting behaviour, and that might be used in a predictive way to identify such animals. The behaviour of 159 pigs was observed in the post-weaning period. Pigs were weaned at 4 weeks of age. In the week prior to weaning and at 6 weeks of age each...

  9. Minimal covariant observables identifying all pure states

    Carmeli, Claudio, E-mail: [D.I.M.E., Università di Genova, Via Cadorna 2, I-17100 Savona (Italy); I.N.F.N., Sezione di Genova, Via Dodecaneso 33, I-16146 Genova (Italy); Heinosaari, Teiko, E-mail: [Turku Centre for Quantum Physics, Department of Physics and Astronomy, University of Turku (Finland); Toigo, Alessandro, E-mail: [Dipartimento di Matematica, Politecnico di Milano, Piazza Leonardo da Vinci 32, I-20133 Milano (Italy); I.N.F.N., Sezione di Milano, Via Celoria 16, I-20133 Milano (Italy)


    It has been recently shown by Heinosaari, Mazzarella and Wolf (2013) [1] that an observable that identifies all pure states of a d-dimensional quantum system has minimally 4d−4 outcomes or slightly less (the exact number depending on d). However, no simple construction of this type of minimal observable is known. We investigate covariant observables that identify all pure states and have minimal number of outcomes. It is shown that the existence of this kind of observables depends on the dimension of the Hilbert space.

  10. Radio Frequency Identifiers: What are the Possibilities?

    Elmorshidy, Ahmed


    This paper defines the components of radio frequency identifiers (RFID). It also explores the different areas and sectors where RFID can be beneficial. The paper discusses the uses and advantages of RFID in deference, consumer packaged goods (CPG), healthcare, logistics, manufacturing, and retail.