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Stem Cells and Development Nov 2018The p66Shc adaptor protein regulates apoptosis and senescence during early mammalian development. However, p66Shc expression during mouse preimplantation development is...
The p66Shc adaptor protein regulates apoptosis and senescence during early mammalian development. However, p66Shc expression during mouse preimplantation development is upregulated at the blastocyst stage. Our objective was to determine the biological function of p66Shc during mouse blastocyst development. In this study, we demonstrate that a reduced p66Shc transcript abundance following its short interfering RNA (siRNA)-mediated knockdown alters the spatiotemporal expression of cell lineage-associated transcription factors in the inner cell mass (ICM) of the mouse blastocyst. P66Shc knockdown blastocysts restrict OCT3/4 earlier to the inner cells of the early blastocyst and have ICMs containing significantly higher OCT3/4 levels, more GATA4-positive cells, and fewer NANOG-positive cells. P66Shc knockdown blastocysts also show a significantly reduced ability to form ICM-derived outgrowths when explanted in vitro. The increase in cells expressing primitive endoderm markers may be due to increased ERK1/2 activity, as it is reversed by ERK1/2 inhibition. These results suggest that p66Shc may regulate the relative abundance and timing of lineage-associated transcription factor expression in the blastocyst ICM.
Topics: Animals; Blastocyst; Blastocyst Inner Cell Mass; Cell Differentiation; Cell Lineage; Cell Proliferation; Embryonic Development; Endoderm; Gene Expression Regulation, Developmental; MAP Kinase Signaling System; Mice; Nanog Homeobox Protein; Octamer Transcription Factor-3; RNA, Small Interfering; Src Homology 2 Domain-Containing, Transforming Protein 1; Transcription Factors
PubMed: 30091687
DOI: 10.1089/scd.2018.0131 -
Fertility and Sterility Jan 2016To assess whether extracellular microRNAs (miRNAs) can be accurately profiled from spent blastocyst culture media (SBM) and used as embryonic biomarkers.
OBJECTIVE
To assess whether extracellular microRNAs (miRNAs) can be accurately profiled from spent blastocyst culture media (SBM) and used as embryonic biomarkers.
DESIGN
Prospective cohort study.
SETTING
Private and academic in vitro fertilization centers.
PATIENT(S)
Inner cell mass-free trophectoderm (TE) samples and their relative SBM from five good-quality human blastocysts.
INTERVENTION(S)
Protocol for miRNA purification and analysis based on quantitative polymerase chain reaction set and validated on human embryonic stem cells (hESCs) and on SBM with and without biological variability.
MAIN OUTCOMES MEASURE(S)
Analysis of miRNAs in culture media in relation with TE cells and comparison of miRNA profiles between implanted and unimplanted euploid blastocysts.
RESULT(S)
Culture media from embryos in the cleavage, morula, and blastocyst stages were collected to investigate the presence of miRNAs. The SBM were prospectively collected from euploid implanted (n = 25) and unimplanted blastocysts (n = 28) for comparison. We hypothesized that human embryos secrete miRNAs in culture media that can be used as biomarkers. The comparative analysis of TE and SBM samples revealed that 96.6% (57 of 59; 95 CI, 88.3-99.6) of the miRNAs detected in the SBM were expressed from TE cells, suggesting a TE origin. The culture media collected from cleavage and morula stage embryos showed a pattern similar to blanks, suggesting that miRNAs profiling from spent culture media applies only for blastocysts. MicroRNAs analysis of SBM from euploid implanted and unimplanted blastocysts highlighted two miRNAs (miR-20a, miR-30c) that showed increased concentrations in the former and were predicted in silico to be involved in 23 implantation-related pathways.
CONCLUSION(S)
MicroRNAs secreted from human blastocysts in culture media can be profiled with high reproducibility, and this approach can be further explored for noninvasive embryo selection.
Topics: Blastocyst; Cell Line; Computational Biology; Computer Simulation; Culture Media, Conditioned; Databases, Genetic; Embryo Culture Techniques; Embryo Implantation; Embryo Transfer; Embryonic Stem Cells; Female; Fertilization in Vitro; Gene Expression Regulation, Developmental; Gene Regulatory Networks; Genetic Markers; Humans; MicroRNAs; Polymerase Chain Reaction; Pregnancy; Prospective Studies
PubMed: 26453979
DOI: 10.1016/j.fertnstert.2015.09.014 -
Fertility and Sterility Apr 2017To determine compositions of commercial single-step culture media and test with a murine model whether differences in composition are biologically relevant. (Comparative Study)
Comparative Study
OBJECTIVE
To determine compositions of commercial single-step culture media and test with a murine model whether differences in composition are biologically relevant.
DESIGN
Experimental laboratory study.
SETTING
University-based laboratory.
ANIMAL(S)
Inbred female mice were superovulated and mated with outbred male mice.
INTERVENTION(S)
Amino acid, organic acid, and ions content were determined for single-step culture media: CSC, Global, G-TL, and 1-Step. To determine whether differences in composition of these media are biologically relevant, mouse one-cell embryos were cultured for 96 hours in each culture media at 5% and 20% oxygen in a time-lapse incubator.
MAIN OUTCOME MEASURE(S)
Compositions of four culture media were analyzed for concentrations of 30 amino acids, organic acids, and ions. Blastocysts at 96 hours of culture and cell cycle timings were calculated, and experiments were repeated in triplicate.
RESULT(S)
Of the more than 30 analytes, concentrations of glucose, lactate, pyruvate, amino acids, phosphate, calcium, and magnesium varied in concentrations. Mouse embryos were differentially affected by oxygen in G-TL and 1-Step.
CONCLUSION(S)
Four single-step culture media have compositions that vary notably in pyruvate, lactate, and amino acids. Blastocyst development was affected by culture media and its interaction with oxygen concentration.
Topics: Amino Acids; Animals; Blastocyst; Cell Hypoxia; Cellular Microenvironment; Culture Media; Embryo Culture Techniques; Embryonic Development; Female; Lactic Acid; Male; Mice; Ovulation Induction; Oxygen; Pyruvic Acid; Superovulation; Time Factors
PubMed: 28238490
DOI: 10.1016/j.fertnstert.2017.01.007 -
Journal of Assisted Reproduction and... Jan 2020To compare a single-step medium with a sequential medium on human blastocyst development rates, aneuploidy rates, and clinical outcomes. (Comparative Study)
Comparative Study
PURPOSE
To compare a single-step medium with a sequential medium on human blastocyst development rates, aneuploidy rates, and clinical outcomes.
METHODS
Retrospective cohort study of IVF cycles that used Sage advantage sequential medium (n = 347) and uninterrupted Sage 1-step medium (n = 519) from July 1, 2016, to December 31, 2017, in an academic fertility center. Main outcome measures are blastocyst formation rates per two-pronuclear (2PN) oocyte and aneuploidy rates per biopsy.
RESULTS
Of all IVF cycles, single-step medium yielded higher blastocyst formation rate (51.7% vs 43.4%) but higher aneuploidy rate (54.0% vs 45.8%) compared with sequential medium. When stratified by maternal age, women under age 38 had no difference in blastocyst formation (52.2% vs 50.2%) but a higher aneuploidy rate (44.5% vs 36.4%) resulting in a lower number of euploid blastocysts per cycle (2.6 vs 3.3) when using single-step medium compared to sequential medium. In cycles used single-step medium, patients ≥ age 38 had higher blastocyst rate (48.0% vs 33.6%), but no difference in aneuploidy rate (68.8% vs 66.0%) or number of euploid embryos (0.8 vs 1.1). For patients reaching euploid embryo transfer, there was no difference in clinical pregnancy rates, miscarriage rates, or live birth rates between two culture media systems.
CONCLUSIONS
Our study demonstrates an increase in aneuploidy in young women whose embryos were cultured in a single-step medium compared to sequential medium. This study highlights the importance of culture conditions on embryo ploidy and the need to stratify by patient age when examining the impact of culture conditions on overall cycle potential.
Topics: Academic Medical Centers; Adult; Aneuploidy; Birth Rate; Blastocyst; Culture Media; Embryo Culture Techniques; Embryo Implantation; Embryo Transfer; Female; Fertilization in Vitro; Humans; Pregnancy; Retrospective Studies
PubMed: 31950455
DOI: 10.1007/s10815-019-01621-8 -
Journal of Assisted Reproduction and... Aug 2021Oocytes and embryos can be vitrified with and without dimethyl sulfoxide (DMSO). Objectives were to compare no vitrification (No-Vitr), vitrification with DMSO (Vitr +...
PURPOSE
Oocytes and embryos can be vitrified with and without dimethyl sulfoxide (DMSO). Objectives were to compare no vitrification (No-Vitr), vitrification with DMSO (Vitr + DMSO), and vitrification without DMSO (Vitr - DMSO) on fresh/warmed oocyte survival, induced parthenogenetic activation, parthenogenetic embryo development, and embryonic maternal imprinted gene expression.
METHODS
In this prospective controlled laboratory study, mature B6C3F1 female mouse metaphase II oocytes were treated as: i) No-Vitr, ii) Vitr + DMSO/warmed, and iii) Vitr - DMSO/warmed with subsequent parthenogenetic activation and culture to the blastocyst stage. Oocyte cryo-survival, parthenogenetic activation and embryo development, parthenogenetic embryo maternal imprinted gene expression were outcome measures.
RESULTS
Oocyte cryo-survival was significantly improved in Vitr + DMSO versus Vitr - DMSO at initial warming and 2 h after warming. Induced parthenogenetic activation was similar between all three intervention groups. While early preimplantation parthenogenetic embryo development was similar between control, Vitr + DMSO, Vitr - DMSO oocytes, the development to blastocysts was significantly inferior in the Vitr - DMSO oocytes group compared to the control and Vitr + DMSO oocyte groups. Finally, maternal imprinted gene expression was similar between intervention groups at both the 2-cell and blastocyst parthenogenetic embryo stage.
CONCLUSION(S)
Inclusion of DMSO in oocyte vitrification solutions improved cryo-survival and developmental potential of parthenogenetic embryos to the blastocyst stage without significantly altering maternal imprinted gene expression.
Topics: Animals; Blastocyst; Cryopreservation; Cryoprotective Agents; Dimethyl Sulfoxide; Embryonic Development; Female; Gene Expression Profiling; Gene Expression Regulation, Developmental; Genomic Imprinting; In Vitro Oocyte Maturation Techniques; Mice; Oocytes; Parthenogenesis; Prospective Studies; Vitrification
PubMed: 34021463
DOI: 10.1007/s10815-021-02221-1 -
Journal of Assisted Reproduction and... Aug 2021To determine whether blastocyst morphology has an impact on sex proportion at pre-implantation and birth in PGT-A and non-PGT-A cycles.
PURPOSE
To determine whether blastocyst morphology has an impact on sex proportion at pre-implantation and birth in PGT-A and non-PGT-A cycles.
METHODS
A total of 1254 biopsied blastocysts from 466 PGT-A cycles were analyzed for sex proportion, day of biopsy, degree of expansion, inner cell mass (ICM), and trophectoderm (TE) morphology. From these, 197 frozen single embryo transfers (SET) were assessed for clinical outcomes and sex proportion of ongoing pregnancies and deliveries. In addition, we evaluated the day of vitrification/embryo transfer, degree of expansion, and TE morphology in a group of 229 births (217 cycles) from frozen or fresh transfers of non-biopsied blastocysts.
RESULTS
Sex proportion was impacted by day of biopsy and TE morphology, but not by ICM morphology, in PGT-A cycles. Therefore, biopsy on day 5 and TE "A" shifted the sex proportion towards males. Interestingly, we noted that our morphology-based embryo selection for SET of euploid blastocysts has favored the choice for XY embryos, generating a 54.3% XY proportion at transfer and 56.1% XY proportion at ongoing pregnancy/delivery. Our models indicate a weaker association between blastocyst morphology parameters and sex proportion of babies in non-PGT-A cycles.
CONCLUSION
Blastocyst features associated with a skewed sex proportion towards XY embryos, such as biopsy on day 5 and top quality TE, are also parameters used for selecting euploid embryos for SET. Therefore, our data suggest that morphology-based embryo selection represents a strong factor responsible for a skewed male sex proportion at birth in PGT-A cycles.
Topics: Adult; Aneuploidy; Biopsy; Blastocyst; Embryo Implantation; Embryo Transfer; Female; Genetic Testing; Humans; Live Birth; Male; Pregnancy; Preimplantation Diagnosis; Single Embryo Transfer; Vitrification
PubMed: 34009630
DOI: 10.1007/s10815-021-02235-9 -
Journal of Assisted Reproduction and... Aug 2013To determine the survival and subsequent in vitro development of human cleavage stage embryos and hatched blastocysts following varying periods of short-term storage at...
PURPOSE
To determine the survival and subsequent in vitro development of human cleavage stage embryos and hatched blastocysts following varying periods of short-term storage at 4 °C, using tripronucleated human embryos (TPN) as a model.
METHODS
TPN cleavage embryos and hatched blastocysts short-term stored at 4 °C for 0 h (control), 24 h and 48 h. The main outcome measures were: survival rates (SR) and in vitro developmental ability (blastocyst rate and blastocyst-re-expansion rate) in each of the groups after storage.
RESULTS
Cleavage-stage TPN survived at comparable rates to controls, regardless of storage time (average: 97.3 %). The in vitro development of cleavage-stage TPN stored for 24 h was comparable to that of controls (average 64.7 %), but was significantly impaired when storage lasted 48-h (20.8 %). After artificial shrinkage, SR was comparable in 24-h-stored and non-stored hatched blastocysts (85.7 %; p > 0.05), but was significantly impaired in the 48-h-stored group (20.0 %). Following 24-h storage, the re-expansion rate of hatched blastocysts was similar to that of controls (average: 57.1 %; p > 0.05), but was higher than that of the 48-h-stored group (15.0 %; p < 0.05).
CONCLUSIONS
TPN human cleavage embryos and blastocysts can be successfully stored short-term for up to 24 h at 4 °C without using cryoprotectants without any significant negative impact on survival or subsequent in vitro development.
Topics: Blastocyst; Embryo Culture Techniques; Embryonic Development; Humans; Reproductive Techniques, Assisted; Time Factors
PubMed: 23820799
DOI: 10.1007/s10815-013-0036-8 -
Fertility and Sterility Aug 2000To determine the relationship between blastocyst quality and the results of embryo transfer at the blastocyst stage. (Clinical Trial)
Clinical Trial
OBJECTIVE
To determine the relationship between blastocyst quality and the results of embryo transfer at the blastocyst stage.
DESIGN
Retrospective case analysis.
SETTING
Tertiary care private hospital IVF center.
PATIENT(S)
A total of 350 blastocyst-stage embryo transfer cycles.
INTERVENTION(S)
In vitro culture to the blastocyst stage was undertaken in 350 ICSI cycles where four or more cleavage-stage embryos were available on day 3.
MAIN OUTCOME MEASURE(S)
Relationship between blastocyst quality and implantation and clinical and multiple pregnancy rates.
RESULT(S)
Transfer of at least one grade 1 or grade 2 blastocyst or one hatching blastocyst was associated with very high implantation and pregnancy rates. However, transfer of grade 3 blastocysts yielded very low implantation and pregnancy rates.
CONCLUSION(S)
There appears to be a strong correlation between blastocyst quality and success of blastocyst transfer.
Topics: Blastocyst; Embryo Implantation; Embryo Transfer; Female; Humans; Pregnancy; Pregnancy Rate; Pregnancy, Multiple; Retrospective Studies
PubMed: 10927045
DOI: 10.1016/s0015-0282(00)00645-2 -
Gynecologic and Obstetric Investigation 2012The importance of oocyte/embryo ploidy to achieve implantation and a subsequent pregnancy.
A linear karyotypic association between PB-I, PB-II and blastomere using sequentially performed comparative genome hybridization with no association established between karyotype, morphologic, biochemical (sHLA-G expression) characteristics, blastocyst formation and subsequent pregnancy outcome.
BACKGROUND
The importance of oocyte/embryo ploidy to achieve implantation and a subsequent pregnancy.
AIM
To correlate first and second polar bodies and day-3 blastomere ploidy, embryo morphology and biochemical (sHLA-G) characteristics with blastocyst development and in vitro pregnancy outcome.
MATERIALS AND METHODS
All oocytes/zygotes and embryos were individually cultured to the blastocyst stage. PB-I, PB-II and blastomeres underwent complete karyotyping and sHLA-G expression was measured on day 2.
RESULTS
57 mature (MII) donor oocytes were obtained, 33/57 (57.9%) were aneuploid, 21/57 (36.8%) were euploid, and 3/57 (5%) were 'inconclusive'. No correlation was found between comparative genomic hybridization (CGH) status of PB-I, PB-II and the graduated embryo score. Furthermore, no correlation was established between PB-I CGH results and blastocyst morphology grade. There was a significant correlation between PB-I CGH and blastomere CGH results. Euploid and aneuploid PB-I developed into 58 and 67% blastocysts, respectively. ĸ statistics (>0.7) revealed a positive correlation between the ploidy of PB-I, PB-II and the blastomeres.
CONCLUSION
Following ICSI and sequential genetic karyotyping of the oocyte/zygote and subsequent blastomeres, the majority of oocytes fertilized and subsequent zygotes developed into blastocysts, despite their ploidy status. We therefore conclude that blastocyst development is not associated with ploidy.
Topics: Adult; Aneuploidy; Blastocyst; Blastomeres; Comparative Genomic Hybridization; Female; HLA-G Antigens; Humans; Karyotype; Oocytes; Polar Bodies; Pregnancy; Pregnancy Outcome; Pregnancy Rate; Sperm Injections, Intracytoplasmic; Statistics, Nonparametric
PubMed: 22890181
DOI: 10.1159/000339632 -
Fertility and Sterility Dec 2011To determine the influence of delayed compaction and fragmentation on the developmental capacity of morulas.
OBJECTIVE
To determine the influence of delayed compaction and fragmentation on the developmental capacity of morulas.
DESIGN
Prospective study.
SETTING
University IVF center.
PATIENT(S)
Intracytoplasmic sperm injection (ICSI) cycles with compact embryos on day 4 or day 5.
INTERVENTION(S)
The embryos were divided into day 4 (n = 329) and day 5 (n = 256) morulas and graded I, II, or III, according to the percentage of fragmentation (<5%, 5%-20%, or >20%). The embryos were measured using Cronus3 software.
MAIN OUTCOME MEASUREMENT(S)
Blastocyst development rate, blastocoel expansion rate, and optimal blastocyst rate. In an optimal blastocyst: surface area, trophectoderm cell number, inner cell mass (ICM) surface area, ICM volume and ICM shape.
RESULT(S)
Day 4 morulas in classes I-III developed into optimal blastocysts in 57.4%, 50%, and 35.6% of the total, respectively, and day 5 morulas in classes I-III in 43.3%, 29.1%, and 13.6% of the total, respectively. A negative association was identified between the amount of morula fragmentation, the blastocyst ICM size, and the number of trophectoderm cells. A delay of 1 day in compaction was associated with a reduced ICM volume.
CONCLUSION(S)
The measurement of compaction timing and cytoplasmic loss in morulas assists in predicting their ability to develop into optimal blastocysts.
Topics: Adult; Blastocyst; Cell Count; Cell Shape; Cells, Cultured; Cleavage Stage, Ovum; Embryonic Development; Female; Fertilization in Vitro; Humans; Infertility; Male; Morula; Pregnancy; Prognosis; Time Factors
PubMed: 21982283
DOI: 10.1016/j.fertnstert.2011.09.015