Comparison of pregnancy outcomes between 4th day morula and 5th day blastocyst after embryo transfer: a retrospective cohort study.

BACKGROUND: This study was designed to evaluate pregnancy outcomes between morulae transferred on day 4 (D4) and blastocysts transferred on day 5 (D5). METHODS: From September 2017 to September 2020, 1963 fresh transfer cycles underwent early follicular phase extra-long protocol for assisted conception in our fertility center were divided into D4 (324 cases) and D5 (1639 cases) groups, and the general situation and other differences of patients in both groups were compared. To compare the differences in pregnancy outcomes, the D4 and D5 groups were further divided into groups A and B based on single and double embryo transfers. Furthermore, the cohort was divided into two groups: those with live births (1116 cases) and those without (847 cases), enabling a deeper evaluation of the effects of D4 or D5 transplantation on assisted reproductive outcomes. RESULTS: In single embryo transfer, there was no significant difference between groups D4A and D5A (P > 0.05). In double embryo transfer, group D4B had a lower newborn birthweight and a larger proportion of low birthweight infants (P < 0.05). The preterm delivery rate, twin delivery rate, cesarean delivery rate, and percentage of low birthweight infants were lower in the D5A group than in the D5B group (P < 0.05). Analysis of factors influencing live birth outcomes further confirmed the absence of a significant difference between D4 and D5 transplantation in achieving live birth (P > 0.05). CONCLUSION: When factors such as working life and hospital holidays are being considered, D4 morula transfer may be a good alternative to D5 blastocyst transfer. Given the in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) success rate and risk of twin pregnancy, D4 morula transfer requires an adapted decision between single and double embryo transfer, although a single blastocyst transfer is recommended for the D5 transfer in order to decrease the twin pregnancy rate. In addition, age, endometrial thickness and other factors need to be taken into account to personalize the IVF program and optimize pregnancy outcomes.

A healthy live birth after mosaic blastocyst transfer in preimplantation genetic testing for GATA1-related cytopenia combined with HLA matching.

BACKGROUND: GATA1-related cytopenia (GRC) is characterized by thrombocytopaenia and/or anaemia ranging from mild to severe. Haematopoietic stem cell transplantation (HSCT) is a healing therapeutic choice for GRC patients. We identified a novel pathogenic variant (GATA1: c.1019delG) in a boy with GATA1-related cytopenia. Then we performed preimplantation genetic testing (PGT) in this GRC family. After a mosaic embryo transfered, a healthy and HLA-compatible with the proband baby was delivered. CASE PRESENTATION: The proband is a 6-year-old boy who was diagnosed to have transfusion-dependent anaemia since 3 year old. Whole-exome sequencing (WES) showed that the proband has a hemizygous variant c.1019delG in GATA1, which is inherited from his mother. His parents decided to undergo PGT to have a health and HLA-compatible offspring. After whole genome amplification (WGA) of biopsied trophectoderm (TE) cells, next generation sequencing (NGS)-based PGT was preformed to analyse embryos on chromosomal aneuploidy, target mutation and HLA typing. There were 3 embryos HLA-matched to the proband. The genotypes of the 3 embryos were heterozygous variant, hemizygous variant, normal respectively. After a heterozygous, mosaic partial trisomy (chr)16, and HLA-matched embryo transfer, a healthy baby was delivered and whose HSCT is compatible with the proband. CONCLUSIONS: NGS-based PGT-HLA is a valuable procedure for the treatment of GATA1-related cytopenia caused by GATA1 variants, or other haematological disorders, oncological and immunological diseases. Furthermore, our study reconfirms that mosaic embryos transfer would bring healthy offspring.

Effects of blastocyst elongation and implantation chamber formation on the alignment of the embryonic axis and uterine axis in mice.

Embryo implantation involves a series of events that bring the embryo and maternal tissues into contact to support post-implantation development in mammals. During implantation, alignment of the embryonic-abembryonic (E-Ab) axis of the blastocyst with the mesometrial-antimesometrial (M-AM) axis of the uterus precedes post-implantation embryonic development and placentation. In the present study, we observed the morphological changes in blastocysts and the endometrial luminal epithelium (LE) that occur during the alignment of the embryonic and the uterine axes. We found that at the time that the blastocysts attached to the LE at the mural trophectoderm, the embryonic axis was not aligned with the uterine axis. Alignment of the embryonic E-Ab axis with the uterine M-AM axis occurred after E4.0, and the embryo was significantly elongated during the process. The depth of the implantation chamber (IC) correlated with the degree of alignment, suggesting that elongated embryos are oriented along the M-AM axis during IC formation. Transplantation of the Concanavalin A (Con A)-coated beads induced IC formation, and the alignment of two Con A-coated beads present in the same IC in the M-AM direction suggested that elongated materials can align along the M-AM axis. These data suggest that an elongated shape of the embryo and IC formation coordinate the alignment of the embryonic and uterine axes.

Total gonadotropin dose did not affect euploid blastocyst rates: an analysis of more than 19,000 oocytes.

BACKGROUND: To evaluate whether increasing total gonadotropin (Gn) dose is associated with changes in euploid blastocyst rate in preimplantation genetic testing (PGT) oocytes. METHODS: This retrospective cohort study was conducted between 2017 and 2022, and 19,246 oocytes were grouped and analyzed based on tri-sectional quantiles of total Gn doses. SETTING: Single reproductive medical center. SUBJECTS: All the patients who underwent PGT cycles, including PGT for aneuploidy, monogenic disorders, and structural rearrangements, were included. EXPOSURE: Next-generation sequencing platforms for chromosomal analysis. MAIN OUTCOME MEASURES: Blastocyst formation and euploid blastocyst rates. RESULTS: In total, 19,246 oocytes and 5375 PGT blastocysts were analyzed. There were significant differences in blastocyst formation and euploid blastocyst rates among the groups classified according to tri-sectional quantiles of total Gn doses. Significant differences in age, body mass index (BMI), proportion of primary infertility, anti-Müllerian hormone (AMH) levels, number of oocytes retrieved, controlled ovarian stimulation (COS) regimen, type of Gn, and PGT category were observed among the three groups. After stratifying the analysis by age, BMI, infertility diagnosis, AMH levels, number of oocytes retrieved, PGT category, type of Gn, and COS regimen, significant differences were only seen in a small number of specific subgroups. Furthermore, the results of the multiple logistic regression analysis showed that the blastocyst formation and euploid blastocyst rates did not significantly increase or decrease with the total Gn dose, whether treated as a continuous variable or divided into three Gn groups as categorical variables. Notably, advancing age was a risk factor for blastocyst formation and euploid blastocyst rates. PGT for structural rearrangements was a risk factor for blastocyst formation and euploid blastocyst rates as compared with PGT for aneuploidy. CONCLUSION: In the total PGT cycles, advancing age, and preimplantation genetic testing for structural rearrangements negatively affected blastocyst formation and euploid blastocyst rates; however, the total Gn dose did not affect blastocyst formation and euploid blastocyst rates.

The fertility outcome of assisted oocyte activation combined with spindle view-assisted intracytoplasmic sperm injection in patients with low fertilization rate.

OBJECTIVE: To examine the possible synergic effect of spindle view-assisted intracytoplasmic sperm injection (SV-ICSI) with assisted oocyte activation (AOA) for low fertilization rate. MATERIALS AND METHODS: A single-center retrospective study from 2019/09-2023/06, a total of 47 patients, autologous IVF cycle, and low fertilization rate history, including control group (SV-ICSI, 33 patients) and intervention group (AOA-SV-ICSI, 14 patients), comparing fertilization rate, blastocyst formation rate, and clinical pregnancy rate. RESULTS: The blastocyst formation rate was significantly higher (p = 0.020) in the AOA-SV-ICSI group than in the SV-ICSI group. The fertilization rate (P = 0.468) and clinical pregnancy rate (p = 0.057) were non-significant between groups. CONCLUSION: The AOA-SV-ICSI group's blastocyst formation rate significantly improved in patients with previous low fertilization rates, which might help them obtain more useable embryos for further embryo implantation.

The Impact of Embryo Quality on Pregnancy Outcomes in Single Day 5 versus Day 6 Euploid Blastocyst Transfer: A Retrospective Cohort Study.

BACKGROUND: Selecting embryos with the highest implantation potential is crucial for in vitro fertilization (IVF) success. Both the timing of blastulation, day 5 (D5) or D6, and the embryo quality have been suggested as influential factors in determining the clinical outcome of single euploid blastocyst transfers. However, evidence supporting the superiority of D5 over D6 blastocysts remains inconclusive. The aim of this study was to compare clinical outcomes following the transfer of euploid blastocysts with different quality and timing of blastulation. MATERIALS AND METHODS: A retrospective cohort study was conducted at our Assisted Reproductive Center, analyzing the outcome of 774 transfers with D5 euploids and 155 transfers with D6 euploids performed between January 2019 and February 2022. RESULTS: The live birth rate was significantly lower in the euploid D6 group compared to the euploid D5 group (38.71vs. 55.04%, P=0.001). The outcome was significantly influenced by the quality of the embryos. Live birth rates were 62.14 and 53.61% following transfers of D5 and D6 excellent embryos respectively, 45.18 and 32.21% following transfer of D5 and D6 good embryos but only 28.64 and 19.32% following transfer of D5 and D6 fair embryos. The outcome difference was statistically significant across embryo quality categories (P=0.001). The adjusted risk ratios (RR) of clinical outcomes indicated that excellent euploid D5 embryos consistently outperformed other types of embryo quality. CONCLUSION: The timing of blastulation and embryo quality are crucial factors in determining the success of single euploid blastocyst transfers. Excellent euploid D5 transfers yielded superior clinical outcomes, providing valuable insights for IVF teams and patients when selecting embryos to be transferred.

What sperm parameters effect blastocyst formation and quality during ICSI with severe male infertility.

Sperm-derived genetic material contributes half of the genome to the embryo, hence it's crucial to investigate which sperm parameter influences blastocyst formation in the intracytoplasmic sperm injection (ICSI) cycles with severe male infertility. The retrospective study analyzed 296 ICSI cycles with severe oligoasthenoteratozoospermia (OAT) and 99 ICSI cycles with preimplantation genetic testing for aneuploidy (PGT-A). Following the correlation analysis, data stratifications were performed in the OAT ICSI subgroup. The results showed that the matching blastocyst in the OAT ICSI cycles had inferior sperm parameters. DFI and sperm morphology had an influence on the blastocyst formation rate and the high-quality blastocysts formation rate on Day6, but no significant effect on the blastocyst development on Day 5. The high-quality blastocysts formation rate and ratio of high-quality blastocyst on Day 6 were demonstrably better in the subgroup of the teratozoospermic morphology when DFI was within the normal range. In the case of the normal sperm morphology, no statistically significant difference was found in blastocyst development, although there were numerical differences within different DFI subgroups. It was concluded that the blastocyst quality and development declined with the decreased sperm qualities.

The density of the inner cell mass is a new indicator of the quality of a human blastocyst: a valid supplement to the Gardner scoring system.

STUDY QUESTION: Can the density of the inner cell mass (ICM) be a new indicator of the quality of the human blastocyst? SUMMARY ANSWER: The densification index (DI) developed in this study can quantify ICM density and provide positive guidance for ploidy, pregnancy, and live birth. WHAT IS KNOWN ALREADY: In evaluating the quality of ICM, reproductive care clinics still use size indicators without further evaluation. The main disadvantage of this current method is that the evaluation of blastocyst ICM is relatively rough and cannot meet the needs of clinical embryologists, especially when multiple blastocysts have the same ICM score, which makes them difficult to evaluate further. STUDY DESIGN, SIZE, DURATION: This observational study included data from 2272 blastocysts in 1991 frozen-thawed embryo transfer (FET) cycles between January 2018 to November 2021 and 1105 blastocysts in 430 preimplantation genetic testing cycles between January 2019 and February 2023. PARTICIPANTS/MATERIALS, SETTING, METHODS: FET, ICSI, blastocyst culture, trophectoderm biopsy, time-lapse (TL) monitoring, and next-generation sequencing were performed. After preliminary sample size selection, the 11 focal plane images captured by the TL system were normalized and the spatial frequency was used to construct the DI of the ICM. MAIN RESULTS AND THE ROLE OF CHANCE: This study successfully constructed a quantitative indicator DI that can reflect the degree of ICM density in terms of fusion and texture features. The higher the DI value, the better the density of the blastocyst ICM, and the higher the chances that the blastocyst was euploid (P < 0.001) and that pregnancy (P < 0.001) and live birth (P = 0.005) were reached. In blastocysts with ICM graded B and blastocysts graded 4BB, DI was also positively associated with ploidy, pregnancy, and live birth (P < 0.05). ROC analysis showed that combining the Gardner scoring system with DI can more effectively predict pregnancy and live births, when compared to using the Gardner scoring system alone. LIMITATIONS, REASONS FOR CAUTION: Accurate calculation of the DI value places high demands on image quality, requiring manual selection of the clearest focal plane and exposure control. Images with the ICM not completely within the field of view cannot be used. The association between the density of ICM and chromosomal mosaicism was not evaluated. The associations between the density of ICM and different assisted reproductive technologies and different culture conditions in embryo laboratories were also not evaluated. Prospective studies are needed to further investigate the impact of ICM density on clinical outcomes. WIDER IMPLICATIONS OF THE FINDINGS: ICM density assessment is a new direction in blastocyst assessment. This study explores new ways of assessing blastocyst ICM density and develops quantitative indicators and a corresponding qualitative evaluation scheme for ICM density. The DI of the blastocyst ICM developed in this study is easy to calculate and requires only TL equipment and image processing, providing positive guidance for clinical outcomes. The qualitative evaluation scheme of ICM density can assist embryologists without TL equipment to manually evaluate ICM density. ICM density is a simple indicator that can be used in practice and is a good complement to the blastocyst scoring systems currently used in most centers. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Key Research & Development Program of China (2021YFC2700603). The authors report no financial or commercial conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Multiple collapses of blastocysts after full blastocyst formation is an independent risk factor for aneuploidy - a study based on AI and manual validation.

BACKGROUND: The occurrence of blastocyst collapse may become an indicator of preimplantation embryo quality assessment. It has been reported that collapsing blastocysts can lead to higher rates of aneuploidy and poorer clinical outcomes, but more large-scale studies are needed to explore this relationship. This study explored the characteristics of blastocyst collapse identified and quantified by artificial intelligence and explored the associations between blastocyst collapse and embryo ploidy, morphological quality, and clinical outcomes. METHODS: This observational study included data from 3288 biopsied blastocysts in 1071 time-lapse preimplantation genetic testing cycles performed between January 2019 and February 2023 at a single academic fertility center. All transferred blastocysts are euploid blastocysts. The artificial intelligence recognized blastocyst collapse in time-lapse microscopy videos and then registered the collapsing times, and the start time, the recovery duration, the shrinkage percentage of each collapse. The effects of blastocyst collapse and embryo ploidy, pregnancy, live birth, miscarriage, and embryo quality were studied using available data from 1196 euploid embryos and 1300 aneuploid embryos. RESULTS: 5.6% of blastocysts collapsed at least once only before the full blastocyst formation (tB), 19.4% collapsed at least once only after tB, and 3.1% collapsed both before and after tB. Multiple collapses of blastocysts after tB (times ≥ 2) are associated with higher aneuploid rates (54.6%, P > 0.05; 70.5%, P < 0.001; 72.5%, P = 0.004; and 71.4%, P = 0.049 in blastocysts collapsed 1, 2, 3 or ≥ 4 times), which remained significant after adjustment for confounders (OR = 2.597, 95% CI 1.464-4.607, P = 0.001). Analysis of the aneuploid embryos showed a higher ratio of collapses and multiple collapses after tB in monosomies and embryos with subchromosomal deletion of segmental nature (P < 0.001). Blastocyst collapse was associated with delayed embryonic development and declined blastocyst quality. There is no significant difference in pregnancy and live birth rates between collapsing and non-collapsing blastocysts. CONCLUSIONS: Blastocyst collapse is common during blastocyst development. This study underlined that multiple blastocyst collapses after tB may be an independent risk factor for aneuploidy which should be taken into account by clinicians and embryologists when selecting blastocysts for transfer.

Cellular mechanisms of monozygotic twinning: clues from assisted reproduction.

BACKGROUND: Monozygotic (MZ) twins are believed to arise from the fission of a single fertilized embryo at different stages. Monochorionic MZ twins, who share one chorion, originate from the splitting of the inner cell mass (ICM) within a single blastocyst. In the classic model for dichorionic MZ twins, the embryo splits before compaction, developing into two blastocysts. However, there are a growing number of ART cases where a single blastocyst transfer results in dichorionic MZ twins, indicating that embryo splitting may occur even after blastocyst formation. OBJECTIVE AND RATIONALE: For monochorionic MZ twins, we conducted a comprehensive analysis of the cellular mechanisms involved in ICM splitting, drawing from both ART cases and animal experiments. In addition, we critically re-examine the classic early splitting model for dichorionic MZ twins. We explore cellular mechanisms leading to two separated blastocysts in ART, potentially causing dichorionic MZ twins. SEARCH METHODS: Relevant studies including research articles, reviews, and conference papers were searched in the PubMed database. Cases of MZ twins from IVF clinics were found by using combinations of terms including 'monozygotic twins' with 'IVF case report', 'ART', 'single embryo transfer', or 'dichorionic'. The papers retrieved were categorized based on the implicated mechanisms or as those with unexplained mechanisms. Animal experiments relating to MZ twins were found using 'mouse embryo monozygotic twins', 'mouse 8-shaped hatching', 'zebrafish janus mutant', and 'nine-banded armadillo embryo', along with literature collected through day-to-day reading. The search was limited to articles in English, with no restrictions on publication date or species. OUTCOMES: For monochorionic MZ twins, ART cases and mouse experiments demonstrate evidence that a looser ICM in blastocysts has an increased chance of ICM separation. Physical forces facilitated by blastocoel formation or 8-shaped hatching are exerted on the ICM, resulting in monochorionic MZ twins. For dichorionic MZ twins, the classic model resembles artificial cloning of mouse embryos in vitro, requiring strictly controlled splitting forces, re-joining prevention, and proper aggregation, which allows the formation of two separate human blastocysts under physiological circumstances. In contrast, ART procedures involving the transfer of a single blastocysts after atypical hatching or vitrified-warmed cycles might lead to blastocyst separation. Differences in morphology, molecular mechanisms, and timing across various animal model systems for MZ twinning can impede this research field. As discussed in future directions, recent developments of innovative in vitro models of human embryos may offer promising avenues for providing fundamental novel insights into the cellular mechanisms of MZ twinning during human embryogenesis. WIDER IMPLICATIONS: Twin pregnancies pose high risks to both the fetuses and the mother. While single embryo transfer is commonly employed to prevent dizygotic twin pregnancies in ART, it cannot prevent the occurrence of MZ twins. Drawing from our understanding of the cellular mechanisms underlying monochorionic and dichorionic MZ twinning, along with insights into the genetic mechanisms, could enable improved prediction, prevention, and even intervention strategies during ART procedures. REGISTRAITON NUMBER: N/A.

Human post-implantation blastocyst-like characteristics of Muse cells isolated from human umbilical cord.

Muse cells, identified as cells positive for the pluripotent surface marker SSEA-3, are pluripotent-like endogenous stem cells located in the bone marrow (BM), peripheral blood, and organ connective tissues. The detailed characteristics of SSEA-3(+) cells in extraembryonic tissue, however, are unknown. Here, we demonstrated that similar to human-adult tissue-Muse cells collected from the BM, adipose tissue, and dermis as SSEA-3(+), human-umbilical cord (UC)-SSEA-3(+) cells express pluripotency markers, differentiate into triploblastic-lineage cells at a single cell level, migrate to damaged tissue, and exhibit low telomerase activity and non-tumorigenicity. Notably, ~ 20% of human-UC-SSEA-3(+) cells were negative for X-inactive specific transcript (XIST), a naïve pluripotent stem cell characteristic, whereas all human adult tissue-Muse cells are XIST-positive. Single-cell RNA sequencing revealed that the gene expression profile of human-UC-SSEA-3(+) cells was more similar to that of human post-implantation blastocysts than human-adult tissue-Muse cells. The DNA methylation level showed the same trend, and notably, the methylation levels in genes particularly related to differentiation were lower in human-UC-SSEA-3(+) cells than in human-adult tissue-Muse cells. Furthermore, human-UC-SSEA-3(+) cells newly express markers specific to extraembryonic-, germline-, and hematopoietic-lineages after differentiation induction in vitro whereas human-adult tissue-Muse cells respond only partially to the induction. Among various stem/progenitor cells in living bodies, those that exhibit properties similar to post-implantation blastocysts in a naïve state have not yet been found in humans. Easily accessible human-UC-SSEA-3(+) cells may be a valuable tool for studying early-stage human development and human reproductive medicine.

Double vitrification and warming of blastocysts does not affect pregnancy, miscarriage or live birth rates.

RESEARCH QUESTION: Does double blastocyst vitrification and warming affect pregnancy, miscarriage or live birth rates, or birth outcomes, from embryos that have undergone preimplantation genetic testing for aneuploidies (PGT-A) testing? DESIGN: This retrospective observational analysis of embryo transfers was performed at a single centre between January 2017 and August 2022. The double-vitrification group included frozen blastocysts that were vitrified after 5-7 days of culture, warmed, biopsied (either once or twice) and re-vitrified. The single vitrification (SV) group included fresh blastocysts that were biopsied at 5-7 days and then vitrified. RESULTS: A comparison of the 84 double-vitrification blastocysts and 729 control single-vitrification blastocysts indicated that the double-vitrification embryos were frozen later in development and had expanded more than the single-vitrification embryos. Of the 813 embryo transfer procedures reported, 452 resulted in the successful delivery of healthy infants (56%). There were no significant differences between double-vitrification and single-vitrification embryos in the pregnancy, miscarriage or live birth rates achieved after single-embryo transfer (55% versus 56%). Logistic regression indicated that while reduced live birth rates were associated with increasing maternal age at oocyte collection, longer culture prior to freezing and lower embryo quality, double vitrification was not a significant predictor of live birth rate. CONCLUSIONS: Blastocyst double vitrification was not shown to impact pregnancy, miscarriage or live birth rates. Although caution is necessary due to the study size, no effects of double vitrification on miscarriage rates, birthweight or gestation period were noted. These data offer reassurance given the absence of the influence of double vitrification on all outcomes after PGT-A.

[Clinical outcome after transfer of vitrified blastocysts in relation to freezing indication]. / Devenir clinique après transfert de blastocystes vitrifiés selon l'indication de congélation.

OBJECTIVES: In France, embryo thawing concern 45.8% of attempts at assisted reproductive technologies excluding artificial inseminations. This proportion is constantly increasing for various reasons. The main objective of this study is to compare the live birth rate following frozen blastocyst transfer (FBT) according to the initial indication for freezing. METHODS: This is a retrospective study including patients who underwent FBT between 01/01/2020 and 06/30/2022 at the Regional University Hospital Center of Tours. The results were compared (univariate and multivariate analyses) between the three main indications for freezing: freezing of the complete cohort of blastocysts for risk of ovarian hyperstimulation (=OHS), freezing of supernumerary blastocysts after fresh blastocyst transfer (BT) with pregnancy (=second request) or without pregnancy (=BT failure). Results have also been described for other indications. RESULTS: Among the 963 FBT cycles selected, 28% of live births by thawing were obtained, all indications of freezing combined. A significantly lower rate was identified in the FBT failure group compared to the OHS group. However, after adjustment, the results remained significant for the age of the patient on the freezing cycle but not for the indication for freezing. CONCLUSIONS: The outcome of a FBT does not seem significantly impacted by the indication of freezing considering the confounding factors. The prospective analysis of more data from a multicenter study would be necessary to confirm these results.

In vitro fertilization and perinatal outcomes of patients with advanced maternal age after single frozen euploid embryo transfer: a propensity score-matched analysis of autologous and donor cycles.

OBJECTIVE: To evaluate in vitro fertilization (IVF) and perinatal outcomes of donor egg and autologous cycles in patients with advanced reproductive age after undergoing single frozen euploid embryo transfer. DESIGN: A multicenter, retrospective, cohort study. SETTING: University-affiliated and private IVF centers. PATIENT(S): Patients aged 39-46 years who underwent IVF with intracytoplasmic sperm injection and preimplantation genetic testing for aneuploidy using whole-chromosome sequencing with donor (n = 278) or autologous (n = 278) oocytes between October 2017 and October 2021. INTERVENTION(S): Single frozen euploid embryo transfer with donor or autologous euploid embryo. MAIN OUTCOME MEASURE(S): The main outcome measure was the live birth rate (LBR) after the first embryo transfer, calculated per embryo transfer. The secondary outcomes included the implantation rate, ectopic pregnancy rate, miscarriage rate, and gestational age and birth weight at the time of delivery. RESULT(S): Patients using donor or autologous oocytes had a similar likelihood of implantation (57.91% [51.87-63.78] vs. 57.19% [51.15-63.09]) and LBR (41.01% [95% confidence interval {CI}, 35.17-47.04] vs. 42.45% [95% CI, 36.56-48.49]). Furthermore, there were no significant differences in the ectopic pregnancy rate (0.72% [0.09-2.57] vs. 0.36% [0.01-1.99]), miscarriage rate (16.19% [12.06-21.05] vs. 14.39% [95% CI, 10.48-19.08]), gestational age (38.50 [38.08-38.92] vs. 39.16 [38.25-40.07] weeks), or birth weight of infants (2,982.25 [2,606.69-3,357.81] vs. 3,128.24 [2,962.30-3,294.17] kg). The univariate analysis showed no association between advanced maternal age and the LBR (relative risk, 1.03 [95% CI, 0.84-1.25]). Multivariate analysis using putative confounders for embryo competency found no associations with LBR (adjusted relative risk, 1.22 [95% CI, 0.75-1.98]). CONCLUSION(S): Patients with euploid blastocysts derived from donor or autologous oocytes did not reveal statistically significant differences in the LBR, implantation rate, ectopic pregnancy rate, miscarriage rate, duration of gestation, or infant birth weight. These findings suggest that age-related reproductive decline and/or poor IVF outcomes associated with women with advanced reproductive age undergoing IVF are heavily driven by embryonic aneuploidy.

Capturing totipotency in human cells through spliceosomal repression.

The cleavage of zygotes generates totipotent blastomeres. In human 8-cell blastomeres, zygotic genome activation (ZGA) occurs to initiate the ontogenesis program. However, capturing and maintaining totipotency in human cells pose significant challenges. Here, we realize culturing human totipotent blastomere-like cells (hTBLCs). We find that splicing inhibition can transiently reprogram human pluripotent stem cells into ZGA-like cells (ZLCs), which subsequently transition into stable hTBLCs after long-term passaging. Distinct from reported 8-cell-like cells (8CLCs), both ZLCs and hTBLCs widely silence pluripotent genes. Interestingly, ZLCs activate a particular group of ZGA-specific genes, and hTBLCs are enriched with pre-ZGA-specific genes. During spontaneous differentiation, hTBLCs re-enter the intermediate ZLC stage and further generate epiblast (EPI)-, primitive endoderm (PrE)-, and trophectoderm (TE)-like lineages, effectively recapitulating human pre-implantation development. Possessing both embryonic and extraembryonic developmental potency, hTBLCs can autonomously generate blastocyst-like structures in vitro without external cell signaling. In summary, our study provides key criteria and insights into human cell totipotency.

Transwell Culture with Adipose Tissue-Derived Stem Cells and Fertilized Eggs Mimics the In Vivo Development of Fertilized Eggs to Blastocysts in the Fallopian Tube: An Animal Study.

Many countries, including Japan, are experiencing declining birth rates. Assisted reproductive technologies have consistently demonstrated good results in resolving infertility. Although the development of fertilized eggs into blastocysts has been recognized as a crucial step in assisted reproductive technologies, the involved mechanisms are currently unclear. Here, we established a new culture system for the in vitro development of fertilized eggs into blastocysts. In the Transwell culture system, the rate of blastocysts hatching from fertilized eggs cultured with adipose-derived stem cells (ASCs) was significantly higher than that of blastocysts cultured only with fertilized eggs. Gene ontology analysis revealed that the developed blastocysts displayed essential gene expression patterns in mature blastocysts. Additionally, when cultured with 3rd-passage ASCs, the developed blastocysts expressed the core genes for blastocyst maturation and antioxidant properties compared to those cultured only with fertilized eggs or cultured with 20th-passage ASCs. These results suggest that the Transwell culture system may imitate the in vivo tubal culture state for fertilized eggs. Exosomes derived from stem cells with stemness potential play a powerful role in the development of blastocysts from fertilized eggs. Additionally, the exosomes expressed specific microRNAs; therefore, the Transwell culture system resulted in a higher rate of pregnancy. In future, the extraction of their own extracellular vesicles from the culture medium might contribute to the development of novel assisted reproductive technologies.

To Assess or Not to Assess: Reconsidering Day 3 Embryo Quality in Planned Freeze-All Blastocyst Cycles.

Day 3 embryo quality is a predictor of in vitro fertilization (IVF) success rates in cleavage-stage embryo transfer. However, the association between day 3 embryo quality and clinical outcomes in blastocyst transfer policy is largely unknown. This retrospective study included 1074 frozen-thawed single day 5/6 blastocyst transfers between January 2019 and December 2022. Three groups were assessed depending on whether the transferred blastocyst derived from a top-quality, good-quality, or poor-quality embryo at day 3. The analysis was conducted independently for each blastocyst quality group (top, good, and poor) using multivariable logistic regression. We applied a Factorial Analysis of Mixed Data (FAMD) to reduce the potential collinearity between the covariates used in the model. All the blastocysts included in this study were obtained from the first ICSI freeze-all cycles. The cleavage and blastocysts stages were assessed between 67 ± 0.5 (day 3), 115 ± 0.5 (day 5), and 139 ± 0.5 (day 6) hours post-insemination (hpi), respectively. After adjusting for the day of transfer (day 5 or day 6) and FAMD dimensions, no statistical differences in a ß-HCG, clinical pregnancy, and live birth were observed among the same-quality blastocysts derived from different day 3 embryo quality groups (top = A, good = B, and poor = C). Our findings showed that a day 3 embryo quality assessment may be unnecessary in planned freeze-all blastocyst cycles.

Vitrification of pig embryos dysregulates the microRNA transcriptome profile.

This study examined how the vitrification of pig blastocysts using either the superfine open pulled straw (SOPS) or Cryotop method affects the expression profile of embryonic microRNA (miRNA) transcriptomes, as well as its relation to changes in the expression of target genes (TGs). Surgically collected pig blastocysts were vitrified using either the SOPS method (n = 60; 4-6 embryos/device) or the Cryotop system (n = 60; 20 embryos/device). Embryos were cultured in vitro for 24 h after warming. Fresh blastocysts (n = 60) cultured for 24 h served as controls. After in vitro culture, five pools of eight viable blastocysts from each group were prepared for miRNA expression analysis based on a microarray approach. Then, biological interpretation of miRNAs profiles and integrative analysis of miRNA and mRNA transcriptome data were performed. Survival after 24 h of in vitro culture was similar (>96 %) for both the vitrification systems and the control group (100 %). Compared with the controls, the SOPS-vitrified blastocysts had 94 (one upregulated and 93 downregulated) differentially expressed (DE) miRNAs, and the Cryotop-vitrified blastocysts had 174 DE miRNAs (one upregulated and 173 downregulated). One DE miRNA (miR-503) in the SOPS group and three DE miRNAs (miR-7139-3p, miR-214 and miR-885-3p) in the Cryotop group were annotated for Sus scrofa. The integrative analysis showed that 27 and 61 DE TGs were regulated by the DE miRNAs in blastocysts vitrified with the SOPS and Cryotop systems, respectively. The TGs enriched one pathway (the TGF-ß signaling pathway) for the SOPS system and four pathways (HIF-1, Notch, ascorbate and aldarate metabolism and glycosphingolipid biosynthesis-ganglio series) for the Cryotop system. In summary, vitrification via the SOPS and Cryotop systems dysregulates miRNAs, with slight differences between methods. The altered miRNAs identified in this study were related mainly to cell proliferation, apoptosis, and the response to cell stress. Further studies are needed to clarify the consequences of dysregulation of miRNAs involved in the TGF-ß (SOPS-vitrified blastocyst) and Notch (Cryotop-vitrified blastocyst) signaling pathways, particularly if they can affect embryonic development.

Does cleavage- versus blastocyst-stage embryo transfer improve fertility rates in women over 38 years of age undergoing assisted reproductive technology?

OBJECTIVE: To evaluate whether extending embryo culture to day 5 (D5) affects pregnancy rates in women older than 38 years undergoing in vitro fertilization (IVF). METHODS: This retrospective, observational cohort study included data from fresh IVF cycles of women over 38 years, during 2011-2021. The cohort was divided according to day 3 (D3) versus D5 embryo transfer (ET). RESULTS: A total of 346 patients (ages 38-45 years) who underwent 496 IVF cycles were included, each yielding one to six embryos. A total of 374 (75%) fresh D3 ETs were compared with 122 (25%) D5 ETs. Demographically, there were more nulliparas in the D3 group (189 [50.9%] vs 47 [38.8%], P = 0.021). Higher gonadotropin dosage was used (3512 ± 1346 vs 3233 ± 1212 IU, P = 0.045) and lower maximum estradiol levels were reached in the D3 group (1129 ± 685 vs 1432 ± 708 pg/mL, P = 0.002). Thirty-three (27%) of the D5 cycles resulted in transfer cancelation due to failure of blastocyst formation (P = 0.001). However, clinical pregnancy rates (P = 0.958), live birth rates (P = 0.988), and miscarriage rates (P = 0.710) did not differ between D3 and D5 ETs. Multivariable logistic regression for clinical pregnancy rate showed that day of transfer did not have a significant effect on the odds (P = 0.376), but maternal age (P = 0.001) and number of retrieved oocytes (P = 0.009) were significant variables. CONCLUSIONS: In older women, culturing embryos to blastocyst stage can decrease invalid ETs without reducing pregnancy rates. Cancelation rates are higher but it may avoid interventions and conserve valuable time.