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.

Live Birth after Cleavage-Stage versus Blastocyst-Stage Embryo Transfer in Assisted Reproductive Technology: A Randomised Controlled Study.

BACKGROUND: Blastocyst stage transfer appears to improve pregnancy outcomes. The aim of this study is to evaluate the pregnancy results between fresh cycle blastocyst stage embryo transfer and cleavage stage embryo transfer in patients who undergo intracytoplasmic sperm injection (ICSI). MATERIALS AND METHODS: This randomised clinical trial study was conducted at the Infertility Research Centre of Milad Hospital in Mashhad, Iran from 2018 to 2020 on 240 infertile women who presented for their first ICSI procedure. These patients were assigned to receive either cleavage embryo transfer (n=112) or blastocyst stage transfer (n=107). Pregnancy outcomes were measured in both groups. RESULTS: There were no differences regarding age, body mass index (BMI), serum follicle-stimulating hormone (FSH), duration of infertility, and aetiology of infertility between the groups (P>0.05). There were more follicles, total oocytes, and metaphase II (M2) oocytes in the blastocyst stage group. Considerably more cleavage stage embryos were transferred compared to the number of transferred blastocysts (P=0.001). The blastocyst group had more vitrified embryos than the cleavage group (P=0.000). The rates of implantation (P=0.332), chemical pregnancy (P=0.165), clinical pregnancy (P=0.694), and live births (P=0.727) were higher in the blastocyst group, but they were not significantly different. The rate of abortion was also not significantly higher in the blastocyst group (P=0.296). CONCLUSION: Blastocysts transferred in the fresh cycle of an ICSI procedure may be more advantageous compared to cleavage stage embryo transfer (registration number: IRCT20181030041503N1).

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.

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.

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.

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.

Impact of fluazuron on oocyte maturation: May the antiparasitic affect bovine reproduction?

Fluazuron is a novel veterinary pour-on antitick formulation which can be applied simultaneously with bovine reproduction management strategies. Considering the economic importance of the livestock industry in many countries, it is important to know whether antiparasitics such as fluazuron may cause embryonic loss. The aim of this study was to evaluate the toxicological effect of fluazuron on bovine oocytes during in vitro maturation. The best fluazuron concentrations were determined in a preliminary experiment on Chinese hamster ovary (CHO)-K1 cells and further used to compare fluazuron toxicity in both study models. Results of the annexin V and alkaline single cell gel electrophoresis assays demonstrated that fluazuron caused cytotoxicity and genotoxicity in bovine cumulus cells at all the concentrations tested (50, 75 and 100 µg fluazuron/mL). The evaluation of cortical granules and mitochondria distribution showed that cytoplasmic maturation was not affected by fluazuron treatment. However, a decrease in metaphase II + polar body, degenerate oocytes as well as disorganized chromatin in polar body were observed at all concentrations tested. Whereas the fertilization process was not altered by 50 µg/mL fluazuron, the embryo development rate decreased significantly. No significant differences were observed in any of the oxidative stress parameters assessed. This study contributes to a better understanding of fluazuron in bovines, suggesting that the antiparasitic may affect bovine reproduction and might cause embryo loss.

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.

Optimizing trigger timing in minimal ovarian stimulation for In Vitro fertilization using machine learning models with random search hyperparameter tuning.

Various studies have emphasized the importance of identifying the optimal Trigger Timing (TT) for the trigger shot in In Vitro Fertilization (IVF), which is crucial for the successful maturation and release of oocytes, especially in minimal ovarian stimulation treatments. Despite its significance for the ultimate success of IVF, determining the precise TT remains a complex challenge for physicians due to the involvement of multiple variables. This study aims to enhance TT by developing a machine learning multi-output model that predicts the expected number of retrieved oocytes, mature oocytes (MII), fertilized oocytes (2 PN), and useable blastocysts within a 48-h window after the trigger shot in minimal stimulation cycles. By utilizing this model, physicians can identify patients with possible early, late, or on-time trigger shots. The study found that approximately 27 % of treatments administered the trigger shot on a suboptimal day, but optimizing the TT using the developed Artificial Intelligence (AI) model can potentially increase useable blastocyst production by 46 %. These findings highlight the potential of predictive models as a supplementary tool for optimizing trigger shot timing and improving IVF outcomes, particularly in minimal ovarian stimulation. The experimental results underwent statistical validation, demonstrating the accuracy and performance of the model. Overall, this study emphasizes the value of AI prediction models in enhancing TT and making the IVF process safer and more efficient.

Diameter of Human Day Five Blastocysts and Birth Sex.

Background This study aimed to evaluate the offspring sex ratio, born through fresh and cryo-thawed single blastocyst (BL) transfers regarding a single morphological, static parameter, namely, BL diameter. Methodology This retrospective, observational study was conducted at an assisted reproductive technology (ART) center, Kinderwunschzentrum Niederrhein Germany. We conducted a statistical analysis of all births resulting from fresh and thawed in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) cycles after a single embryo transfer (SET). The main outcome measure was the offspring sex ratio after SET of a day five BL in relation to the BL diameter measurement. Results There were more female than male babies born in our study. We observed a tendency for BL to have a higher diameter, resulting in female offspring, which was not statistically relevant. We also compared the BL diameter in the fresh embryo transfer (ET) group with that of the cryo-thawed ET group, showing a tendency toward a larger diameter in the fresh ET group. In the ICSI cycles, there was a higher tendency for a larger BL diameter when compared to IVF cycles. In the fresh ET cycles, BL leading to the male sex at birth had a tendency toward a larger diameter than the female BL. In the cryo-thaw ET cycles, BL leading to the female sex had a tendency toward a larger diameter than the male BL. Conclusions Our results showed a tendency in the sex of offspring toward the female sex and no significant difference in the BL diameter of BL leading to birth after ART and consecutive transfer of day five BL.

The impact of implementing a non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) embryo culture protocol on embryo viability and clinical outcomes.

STUDY QUESTION: Are modifications in the embryo culture protocol needed to perform non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) affecting clinical reproductive outcomes, including blastocyst development and pregnancy outcomes? SUMMARY ANSWER: The implementation of an embryo culture protocol to accommodate niPGT-A has no impact on blastocyst viability or pregnancy outcomes. WHAT IS KNOWN ALREADY: The recent identification of embryo cell-free (cf) DNA in spent blastocyst media has created the possibility of simplifying PGT-A. Concerns, however, have arisen at two levels. First, the representativeness of that cfDNA to the real ploidy status of the embryo. Second, the logistical changes that need to be implemented by the IVF laboratory when performing niPGT-A and their effect on reproductive outcomes. Concordance rates of niPGT-A to invasive PGT-A have gradually improved; however, the impact of culture protocol changes is not as well understood. STUDY DESIGN, SIZE, DURATION: As part of a trial examining concordance rates of niPGT-A versus invasive PGT-A, the IVF clinics implemented a specific niPGT-A embryo culture protocol. Briefly, this involved initial culture of fertilized oocytes following each laboratory standard routine up to Day 4. On Day 4, embryos were washed and cultured individually in 10 µl of fresh media. On Day 6 or 7, blastocysts were then biopsied, vitrified, and media collected for the niPGT-A analysis. Six IVF clinics from the previously mentioned trial were enrolled in this analysis. In the concordance trial, Clinic A cultured all embryos (97 cycles and 355 embryos) up to Day 6 or 7, whereas in the remaining clinics (B-F) (379 cycles), nearly a quarter of all the blastocysts (231/985: 23.5%) were biopsied on Day 5, with the remaining blastocysts following the niPGT-A protocol (754/985: 76.5%). During the same period (April 2018-December 2020), the IVF clinics also performed standard invasive PGT-A, which involved culture of embryos up to Days 5, 6, or 7 when blastocysts were biopsied and vitrified. PARTICIPANTS/MATERIALS, SETTING, METHODS: In total, 428 (476 cycles) patients were in the niPGT-A study group. Embryos from 1392 patients underwent the standard PGT-A culture protocol and formed the control group. Clinical information was obtained and analyzed from all the patients. Statistical comparisons were performed between the study and the control groups according to the day of biopsy. MAIN RESULTS AND THE ROLE OF CHANCE: The mean age, number of oocytes, fertilization rates, and number of blastocysts biopsied were not significantly different for the study and the control group. Regarding the overall pregnancy outcomes, no significant effect was observed on clinical pregnancy rate, miscarriage rate, or ongoing pregnancy rate (≥12 weeks) in the study group compared to the control group when stratified by day of biopsy. LIMITATIONS, REASONS FOR CAUTION: The limitations are intrinsic to the retrospective nature of the study, and to the fact that the study was conducted in invasive PGT-A patients and not specifically using niPGT-A cases. WIDER IMPLICATIONS OF THE FINDINGS: This study shows that modifying current IVF laboratory protocols to adopt niPGT-A has no impact on the number of blastocysts available for transfer and overall clinical outcomes of transferred embryos. Whether removal of the invasive biopsy step leads to further improvements in pregnancy rates awaits further studies. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by Igenomix. C.R., L.N.-S., and D.V. are employees of Igenomix. D.S. was on the Scientific Advisory Board of Igenomix during the study. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov (NCT03520933).

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.

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.

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.

Impact of inclusion of a poor-quality embryo with a good-quality embryo on pregnancy outcomes in vitrified-warmed blastocyst transfers.

RESEARCH QUESTION: Does the co-transfer of a good-quality embryo and a poor-quality embryo influence pregnancy outcomes in comparison to the transfer of a single good-quality embryo in vitrified-warmed blastocyst transfer cycles? DESIGN: This retrospective cohort study involved a total of 11,738 women who underwent IVF/intracytoplasmic sperm injection cycles and vitrified-warmed blastocyst transfer at a tertiary-care academic medical from January 2015 to June 2022. The study population was categorized into two groups: single-blastocyst transfer (SBT; participants who underwent single good-quality embryo transfer, n = 9338) versus double-blastocyst transfer (DBT; participants who underwent transfers with a poor and a good-quality embryo, n = 2400). RESULTS: The live birth rate (LBR) was significantly higher in the DBT group in comparison with the SBT group (65.6% versus 56.3%, P < 0.001). Multivariable logistic regression analysis showed that DBT was an independent predictor for LBR with a strong potential impact (adjusted odds ratio 1.55, 95% confidence interval 1.41-1.71; P < 0.001). However, the multiple birth rate was significantly higher in the good-quality embryo and poor-quality embryo group compared with patients undergoing a single good-quality embryo transfer (41.4% versus 1.8%; P < 0.001). CONCLUSIONS: In vitrified-warmed blastocyst transfer cycles, LBR was higher following DBT with one good-quality and one poor-quality embryo compared with SBT. However, this was at the expense of a marked increase in the likelihood of multiple gestations. Physicians should still balance the benefits and risks of double-embryo transfer.

Trophectoderm biopsy is associated with lower risks of moderate to extreme prematurity and low birthweights - a national registry cohort study of singleton live births from frozen-thawed blastocyst transfers.

BACKGROUND: Trophectoderm biopsy has become the mainstay assisted reproductive technique performed for preimplantation genetic testing, accounting for 43.8% of embryo transfer cycles in the US in 2019 alone. Despite its prevalence, data on the obstetric and perinatal outcomes post trophectoderm biopsy remains sparse and mixed. OBJECTIVE: This study aimed to examine the risks of adverse perinatal outcomes in birth weights and prematurity after transfers of vitrified-thawed blastocyst with trophectoderm biopsy for preimplantation genetic testing. STUDY DESIGN: This was a retrospective observational cohort study of 45,712 singleton live births resulting from autologous vitrified-thawed blastocyst transfer cycles with or without trophectoderm biopsy for preimplantation genetic testing, reported by participating member clinics to the Society for Assisted Reproductive Technology national registry between 2014 and 2017. Adverse perinatal outcomes of preterm births and low birth weights were analyzed. Multivariable regression analyses were performed to control for covariates. Comparing the trophectoderm biopsy (n=21,584) and no trophectoderm biopsy (n=24,128) groups, adjusted odds ratios were calculated for the outcomes of small for gestational age, large for gestational age, low birth weight < 2,500 g, very low birth weight < 1,500 g, extremely low birth weight < 1,000 g, late preterm births < 37 weeks, moderate preterm births < 34 weeks, and extremely preterm births < 28 weeks. RESULTS: Women in the trophectoderm biopsy group were older and more likely to have prior pregnancies, deliveries, and history of spontaneous abortions. Tobacco use, diminished ovarian reserve, and recurrent pregnancy loss were also more prevalent in the trophectoderm biopsy group. Trophectoderm biopsy was not associated with small-for-gestational-age (aOR 0.97, 95% CI 0.85-1.12, p-value 0.72) or large-for-gestational-age newborns (aOR 1.10, 95% CI 0.99-1.22, p-value 0.09). Risks of preterm births < 37 weeks gestation were similar between the biopsy and non-biopsy groups (aOR 0.93, 95% CI 0.85-1.02, p-value 0.11). Trophectoderm biopsy was associated with a significantly lower risk of low birthweight < 2,500 g (aOR 0.80, 95% CI 0.70-0.92, p-value <0.001), very low birthweight < 1,500 g (aOR 0.62, 95% CI 0.46-0.83, p-value <0.001), extremely low birthweight < 1,000 g (aOR 0.48, 95% CI 0.31-0.74, p-value <0.001), moderate preterm birth < 34 weeks (aOR 0.76, 95% CI 0.64-0.91, p-value 0.003), and extreme preterm birth < 28 weeks (aOR 0.63, 95% CI 0.43-0.92, p-value 0.017). CONCLUSION: Trophectoderm biopsy is not associated with increased risks of small for gestational age, large for gestational age, or late preterm birth. Risks of low birthweight, very low birthweight, and extremely low birthweight from moderate and extreme preterm births are lower after trophectoderm biopsy, possibly by selecting against confined placental mosaicism or inducing placental epigenetic changes, the mechanisms of which warrant further investigation.

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.