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In the pre-implantation embryo, aneuploidy resulting from chromosome segregation error is considered responsible for pregnancy loss. However, only a few studies have examined the relationship between chromosome segregation errors during early cleavage and development. Here, we evaluated this relationship by live-cell imaging using the histone H2B-mCherry probe and subsequent single blastocyst transfer using mouse embryos obtained by in vitro fertilization. We showed that some embryos exhibiting early chromosomal segregation error and formation of micronuclei retained their developmental potential; however, the error affected the blastocyst/arrest ratio. Further, single-cell sequencing after live-cell imaging revealed that all embryos exhibiting micronuclei formation during 1 st mitosis showed aneuploidy at the 2-cell stage. These results suggest that early chromosome segregation error causing micronuclei formation affects ploidy and development to blastocyst but does not necessarily cause developmental failure after the blastocyst stage. Our result suggests the importance of the selection of embryos that have reached blastocysts.

This retrospective study aimed to clarify the cumulative live-birth rates (CLBRs) and cost per live-birth (LB) to evaluate the validity of frozen–thawed embryo transfer without preimplantation genetic testing for aneuploidy (PGT-A) in women aged  ≥  40 years. The study included 1,011 patients aged  ≥  40 years who underwent their first oocyte retrieval at our hospital between January 2010 and September 2017. They were followed up for up to two years or until either treatment discontinuation or a pregnancy that resulted in a live birth. The 2-year CLBRs were 55.6%, 39.0%, 31.3%, 19.1%, 10.6%, 4.4%, and 0% for patients aged 40, 41, 42, 43, 44, 45, and > 46 years, respectively. In approximately 80% of LB cases, patients aged 40–42 years and 43–44 years became pregnant by the fourth and second transfers, respectively. Costs per LB were $30,207, $49,034, $66,345, $102,759, and $195,862 for patients aged 40, 41, 42, 43, and 44, respectively. Cost per LB for each number of transfers reached $300,000 and $ 450,000 for the third transfer at 42 and 43 years of age, respectively. For cost-effectiveness, up to two ET cycles are recommended for patients aged 42–43, and none for patients aged  ≥  44 years.

Totipotency emerges in early embryogenesis, but its molecular underpinnings remain poorly characterized. In the present study, we employed DNA fiber analysis to investigate how pluripotent stem cells are reprogrammed into totipotent-like 2-cell-like cells (2CLCs). We show that totipotent cells of the early mouse embryo have slow DNA replication fork speed and that 2CLCs recapitulate this feature, suggesting that fork speed underlies the transition to a totipotent-like state. 2CLCs emerge concomitant with DNA replication and display changes in replication timing (RT), particularly during the early S-phase. RT changes occur prior to 2CLC emergence, suggesting that RT may predispose to gene expression changes and consequent reprogramming of cell fate. Slowing down replication fork speed experimentally induces 2CLCs. In vivo, slowing fork speed improves the reprogramming efficiency of somatic cell nuclear transfer. Our data suggest that fork speed regulates cellular plasticity and that remodeling of replication features leads to changes in cell fate and reprogramming. Totipotent cells in mouse embryos and 2-cell-like cells have slow DNA replication fork speed. Perturbations that slow replication fork speed promote 2-cell-like cell emergence and improve somatic cell nuclear transfer reprogramming and formation of induced pluripotent stem cell colonies.

To improve the performance of assisted reproductive technology, it is necessary to find an indicator that can identify and select embryos that will be born or be aborted. We searched for indicators of embryo selection by comparing born/abort mouse embryos. We found that asynchronous embryos during the 4–8-cell stage were predisposed to be aborted. In asynchronous mouse embryos, the nuclear translocation of YAP1 in some blastomeres and compaction were delayed, and the number of ICMs was reduced. Hence, it is possible that asynchronous embryos have abnormal differentiation. When the synchrony of human embryos was observed, it was confirmed that embryos that did not reach clinical pregnancy had asynchrony as in mice. This could make synchrony a universal indicator common to all animal species.

Purpose In cryptozoospermic subjects, it may often may be difficult to secure motile sperm for assisted reproductive technology (ART). We examined the results of ART with frozen thawed ejaculated sperm in cryptozoospermic subjects and evaluated whether sperm retrieval surgery is necessary for such patients in our clinic. Methods Between 2013 and 2021, we evaluated 197 cryptozoospermic patients. Age, endocrine panel at the time of the initial semen analysis, and anti‐müllerian hormone levels at the time of the spouse's first egg retrieval were examined. Cryopreservation of ejaculated motile sperm collected essentially weekly over a 3‐month period was carried out. ART data recorded was the number of egg retrieval cycles, normal fertilization rate, and clinical pregnancy rate. Results ART using frozen sperm as well as sperm ejaculated on the day of egg retrieval was possible in all cases. The normal fertilization rate was 70.4%, the clinical pregnancy rate per embryo transferred was achieved in 31.5% (870 cycles), and the live birth rate per case was 73.8%. Conclusions Intracytoplasmic sperm injection (ICSI) was possible without sperm retrieval surgery in cryptozoospermia, resulting in 73.8% of live births per patient. Sperm identification, sperm processing, and ICSI technique are especially important in cryptozoospermia. Sperm retrieval surgery can be avoided in cryptozoospermic patients.

Purpose The primary objective of this investigation is to evaluate how morphological quality affects the pregnancy outcomes in euploid embryos determined by preimplantation genetic testing for aneuploidies (PGT‐A). Concurrently, as a secondary objective, we aim to identify which specific aspects of morphological evaluation exert the most significant impact on these outcomes. Methods A retrospective analysis of 451 single euploid embryo transfer cycles at our clinic was conducted. Embryos were evaluated based on the degree of blastocyst expansion, inner cell mass (ICM), trophectoderm (TE) morphology, and the day of blastocyst vitrification. Outcomes between morphologically low‐grade and high‐grade embryos were compared. Additionally, the study analyzed which morphological factors most influenced pregnancy outcomes. Results Pregnancy outcomes were significantly lower in morphologically low‐grade blastocysts compared to high‐grade ones. Among the morphological evaluations, the ICM assessment was significantly associated with the live birth rate. Conclusion Our study indicates that the morphological quality of euploid embryos, particularly the evaluation of the ICM, plays a crucial role in IVF‐ET success. In this study, we investigated the impact of morphological evaluation on the success rates of in vitro fertilization and embryo transfer (IVF‐ET) in euploid embryos. Our results indicated significantly better pregnancy outcomes for embryos with morphologically high‐grade evaluations compared to those with low ones, with the assessment of the inner cell mass (ICM) being notably associated with live birth rates. This suggests the crucial role of morphological quality, especially ICM evaluation, in the success of IVF‐ET with euploid embryos.

Nuclear/chromosomal integrity is an important prerequisite for the assessment of embryo quality in artificial reproductive technology. However, lipid-rich dark cytoplasm in bovine embryos prevents its observation by visible light microscopy. We performed live-cell imaging using confocal laser microscopy that allowed long-term imaging of nuclear/chromosomal dynamics in bovine in vitro fertilised (IVF) embryos. We analysed the relationship between nuclear/chromosomal aberrations and in vitro embryonic development and morphological blastocyst quality. Three-dimensional live-cell imaging of 369 embryos injected with mRNA encoding histone H2B-mCherry and enhanced green fluorescent protein (EGFP)-α-tubulin was performed from single-cell to blastocyst stage for eight days; 17.9% reached the blastocyst stage. Abnormalities in the number of pronuclei (PN), chromosomal segregation, cytokinesis, and blastomere number at first cleavage were observed at frequencies of 48.0%, 30.6%, 8.1%, and 22.2%, respectively, and 13.0%, 6.2%, 3.3%, and 13.4%, respectively, for abnormal embryos developed into blastocysts. A multivariate analysis showed that abnormal chromosome segregation (ACS) and multiple PN correlated with delayed timing and abnormal blastomere number at first cleavage, respectively. In morphologically transferrable blastocysts, 30–40% of embryos underwent ACS and had abnormal PN. Live-cell imaging may be useful for analysing the association between nuclear/chromosomal dynamics and embryonic development in bovine embryos.

Although the current evaluation of human blastocysts is based on the Gardner criteria, there may be other notable parameters. The purpose of our study was to clarify whether the morphology of blastocysts has notable indicators other than the Gardner criteria. To find such indicators, we compared blastocysts that showed elevated human chorionic gonadotropin (hCG) levels after transplantation (hCG-positive group; = 129) and those that did not (hCG-negative group; = 105) using principal component analysis of pixel brightness of the images. The comparison revealed that the hCG-positive group had grainy morphology and the hCG-negative group had non-grainy morphology. Classification of the blastocysts by this indicator did not make a difference in Gardner score. Interestingly, all embryos with ≥20% fragmentation were non-grainy. The visual classification based on this analysis was significantly more accurate than the prediction of implantation using the Gardner score ≥3BB. As graininess can be used in combination with the Gardner score, this indicator will enhance current reproductive technologies.

Purpose To demonstrate the benefits of the freeze‐all strategy for in vitro fertilization treatment based on retrospective analyses. Methods Post‐thaw embryo survival rates of slow‐frozen embryos in 294 cycles and vitrified embryos in 12 195 cycles were assessed. Progesterone (P4) and estradiol (E2) levels per mature oocyte by age category were assessed in 9081 cycles and pregnancy rates with fresh embryo transfer and frozen‐thawed embryo transfer by P4 level were assessed in 1535 cycles. Results The survival rates of frozen‐thawed embryos were 92.5% with slow freezing and 99.1% with vitrification. P4 levels on the day of human chorionic gonadotropin (hCG) injection showed a trend toward an increase with age. The pregnancy rate per mature oocyte with fresh embryo transfer decreased dependently upon P4 level, while that with frozen‐thawed embryo transfer was not affected by P4 level. The pregnancy rates with frozen‐thawed embryo transfer were higher than those with fresh embryo transfer in patients aged 42 years or younger. Conclusions The freeze‐all strategy is a valuable treatment option which allows the separation of an embryo transfer cycle from an oocyte retrieval cycle, especially for patients with high P4 levels at oocyte retrieval and patients of advanced maternal age.

Purpose To assess the appropriateness of human chorionic gonadotropin (hCG) re‐trigger in poor responders to gonadotropin‐releasing hormone agonist (GnRHa) trigger in controlled ovarian stimulation (COS) cycles. Methods The 2251 cycles in 2251 patients triggered with GnRHa for oocyte stimulation, with or without requiring hCG re‐trigger between 2013 and 2018, were retrospectively analyzed to compare gonadotropin levels at the start of COS and the rate of normal fertilization between the re‐trigger and non–re‐trigger group. Furthermore, patients in the re‐trigger group were stratified by the rate of normal fertilization (good: ≥60% or poor: <60%) to compare patient demographics, hormone profiles, and clinical outcome between the subgroups. Results In the re‐trigger group, FSH and LH levels at the start of COS were significantly lower in the good fertilization group than in the poor fertilization group (P < .01). Receiver operating characteristic curves identified cutoff values of the FSH and LH levels of 1.30 and 0.35 mIU/mL, respectively, for predicting ≥60% normal fertilization. Conclusion Gonadotropin levels at the start of COS are predictors of response to GnRHa trigger and hCG re‐trigger necessity, and may serve as indicators to help clinicians appropriately choose hCG re‐trigger rather than abandoning the cycles or continuing the first oocyte aspiration attempt. When FSH and LH levels at the start of controlled ovarian stimulation were higher than the cutoff levels derived from ROC curve, oocytes obtained by re‐triggering with hCG frequently resulted in abnormal fertilization.