Blastocyst Stage Embryos Research Articles

Optimising Electroporation Condition for CRISPR/Cas-Mediated Knockout in Zona-Intact Buffalo Zygotes.

Somatic cell nuclear transfer or cytoplasm microinjection has widely been used to produce genome-edited farm animals; however, these methods have several drawbacks which reduce their efficiency. In the present study, we describe an easy adaptable approach for the introduction of mutations using CRISPR-Cas9 electroporation of zygote (CRISPR-EP) in buffalo. The goal of the study was to determine the optimal conditions for an experimental method in which the CRISPR/Cas9 system is introduced into in vitro-produced buffalo zygotes by electroporation. Electroporation was performed using different combinations of voltage, pulse and time, and we observed that the electroporation in buffalo zygote at 20 V/mm, 5 pulses, 3 msec at 10 h post insemination (hpi) resulted in increased membrane permeability and higher knockout efficiency without altering embryonic developmental potential. Using the above parameters, we targeted buffalo POU5F1 gene as a proof of concept and found no variations in embryonic developmental competence at cleavage or blastocyst formation rate between control, POU5F1-KO, and electroporated control (EC) embryos. To elucidate the effect of POU5F1-KO on other pluripotent genes, we determined the relative expression of SOX2, NANOG, and GATA2 in the control (POU5F1 intact) and POU5F1-KO-confirmed blastocyst. POU5F1-KO significantly (p ≤ 0.05) altered the expression of SOX2, NANOG, and GATA2 in blastocyst stage embryos. In conclusion, we standardized an easy and straightforward protocol CRISPR-EP method that could be served as a useful method for studying the functional genomics of buffalo embryos.

157 Production of β-lactoglobulin gene knockout blastocyst stage embryos of Indian water buffalo using CRISPR and somatic cell nuclear transfer technology

157 Production of β-lactoglobulin gene knockout blastocyst stage embryos of Indian water buffalo using CRISPR and somatic cell nuclear transfer technology

Mouse Trophoblast Cells Have Attenuated Responses to TNF-α and IFN-γ and Can Avoid Synergic Cytotoxicity of the Two Cytokines.

TNF-α and IFN-γ are two inflammatory cytokines that play critical roles in immune responses, but they can also negatively affect cell proliferation and viability. In particular, the combination of the two cytokines (TNF-α/IFN-γ) synergistically causes cytotoxicity in many cell types. We recently reported that mouse embryonic stem cells (ESCs) isolated from the blastocyst stage embryo do not respond to TNF-α and have limited response to IFN-γ, thereby avoiding TNF-α/IFN-γ cytotoxicity. The current study expanded our investigation to mouse trophoblast stem cells (TSCs) and their differentiated trophoblasts (TSC-TBs), the precursors and the differentiated cells of the placenta, respectively. In this study, we report that the combination of TNF-α/IFN-γ does not show the cytotoxicity to TSCs and TSC-TBs that otherwise effectively kills fibroblasts, similar to ESCs. Although ESCs, TSCs, and TSC-TBs are dramatically different in their growth rate, morphology, and physiological functions, they nevertheless share a similarity in being able to avoid TNF-α/IFN-γ cytotoxicity. We propose that this unique immune property may serve as a protective mechanism that limits cytokine cytotoxicity in the blastocyst. With molecular and cellular approaches and genome-wide transcriptomic analysis, we have demonstrated that the attenuated NF-κB and STAT1 transcription activation is a limiting factor that restricts the effect of TNF-α/IFN-γ on TSCs and TSC-TBs.

#92 : An Artificial Intelligence Algorithm Outperforms Highly Variable Embryologist Grading for Predicting the Likelihood of Pregnancy Outcome from Embryo Images

Background and Aims: Embryologist evaluation of embryos is critical for ensuring successful pregnancy outcomes. Standard, manual evaluation is variable, subjective, and time-consuming. The aim of this study was to evaluate whether an artificial intelligence (AI) algorithm can standardize and improve embryo evaluation during IVF. Method: 20 images of blastocyst-stage embryos on day 5 of in vitro development were selected to represent a range of morphological qualities. All embryos had been transferred and the clinical pregnancy outcome was known for each embryo based on detection of fetal heartbeat at first ultrasound scan (∼7-9 weeks gestation). 50% of embryos in the dataset resulted in pregnancy. 158 embryologists made a total of 236 attempts at providing their evaluation of the morphological quality of the 20 embryo images using the Gardner system. The embryologist-assigned grades were then used to generate their prediction of whether that embryo would lead to pregnancy or not (≥ 3BB indicated a pregnancy prediction, and <3BB indicated a non-pregnancy prediction). The same 20 embryo images were also assessed by a previously developed viability AI algorithm for evaluating the likelihood of clinical pregnancy based on embryo images. An AI score of ≥5.0/10 indicated a pregnancy prediction, and <5.0/10 indicated a non-pregnancy prediction. The AI algorithm provided the same score for each embryo image regardless of how many times the analysis was performed. Results: The AI algorithm correctly predicted pregnancy outcome for 14/20 embryo images (70%). Embryologists also correctly predicted 14/20 images in 14/236 attempts (6%), and in 1 attempt correctly predicted 15/20 images. In the remaining 221 attempts (94%) embryologists correctly predicted between 6-13 images, representing a range of accuracies from 30-75%. Conclusion: This study demonstrates the inherent variability and lack of objectivity associated with an embryologist’s evaluation of embryos. It highlights the benefits of accurate AI algorithms for standardizing embryo assessment

Benefits of Blastocyst Transfer With at Least Three Good-Quality Cleavage-stage Embryos in Women of Advanced Maternal Age: A Retrospective Analysis

ObjectivesLimited studies on the benefits of blastocyst transfer in advanced maternal age (AMA) (≥40 years) have been reported. Our objective was to find whether blastocyst-stage embryo transfer improves pregnancy and live birth rates in women ≥40 years who have 3 or more good-quality cleavage-stage embryos. MethodsAll fresh in vitro fertilization-intracytoplasmic sperm injection cycles performed from January 2020 to December 2021 in AMA women that progressed to transfer were considered for analysis. We compared fresh and cumulative ongoing pregnancy rates in AMA women of those who had a cleavage-stage transfer, while meeting the criteria for extended culture (≥3 high-quality embryos, group 1), and those who underwent blastocyst transfer (group 2). Demographic parameters, stimulation, embryology, fresh and cumulative ongoing pregnancy rates, and clinical miscarriage rates were compared. ResultsDuring the study period, 255 cycles were analyzed including group 1 (n = 99) and group 2 (n = 156). Group 1 participants were older and had a greater number of embryos for transfer. Fresh and cumulative ongoing pregnancy rates per transfer were higher in group 2 compared to group 1 (23.4% vs. 13.1%, P = 0.04; 25.5% vs. 14.1%, P = 0.03), while overall miscarriage rates were higher in group 1 than group 2 (51.7% vs. 25%, P = 0.01). ConclusionsBlastocyst culture provides a benefit to AMA women who have at least 3 good-quality embryos on day 3 resulting in significantly higher fresh and cumulative ongoing pregnancy rates and lower miscarriage compared to cleavage-stage transfers.

Meiotic and mitotic aneuploidies drive arrest of in vitro fertilized human preimplantation embryos

BackgroundThe high incidence of aneuploidy in early human development, arising either from errors in meiosis or postzygotic mitosis, is the primary cause of pregnancy loss, miscarriage, and stillbirth following natural conception as well as in vitro fertilization (IVF). Preimplantation genetic testing for aneuploidy (PGT-A) has confirmed the prevalence of meiotic and mitotic aneuploidies among blastocyst-stage IVF embryos that are candidates for transfer. However, only about half of normally fertilized embryos develop to the blastocyst stage in vitro, while the others arrest at cleavage to late morula or early blastocyst stages.MethodsTo achieve a more complete view of the impacts of aneuploidy, we applied low-coverage sequencing-based PGT-A to a large series (n = 909) of arrested embryos and trophectoderm biopsies. We then correlated observed aneuploidies with abnormalities of the first two cleavage divisions using time-lapse imaging (n = 843).ResultsThe combined incidence of meiotic and mitotic aneuploidies was strongly associated with blastocyst morphological grading, with the proportion ranging from 20 to 90% for the highest to lowest grades, respectively. In contrast, the incidence of aneuploidy among arrested embryos was exceptionally high (94%), dominated by mitotic aneuploidies affecting multiple chromosomes. In turn, these mitotic aneuploidies were strongly associated with abnormal cleavage divisions, such that 51% of abnormally dividing embryos possessed mitotic aneuploidies compared to only 23% of normally dividing embryos.ConclusionsWe conclude that the combination of meiotic and mitotic aneuploidies drives arrest of human embryos in vitro, as development increasingly relies on embryonic gene expression at the blastocyst stage.

Intersection clock reveals a rejuvenation event during human embryogenesis.

Recent research revealed a rejuvenation event during early development of mice. Here, by examining epigenetic age dynamics of human embryogenesis, we tested whether a similar event exists in humans. For this purpose, we developed an epigenetic clock method, the intersection clock, that utilizes bisulfite sequencing in a way that maximizes the use of informative CpG sites with no missing clock CpG sites in test samples and applied it to human embryo development data. We observed no changes in the predicted epigenetic age between cleavage stage and blastocyst stage embryos; however, a significant decrease was observed between blastocysts and cells representing the epiblast. Additionally, by applying the intersection clock to datasets spanning pre and postimplantation, we found no significant change in the epigenetic age during preimplantation stages; however, the epigenetic age of postimplantation samples was lower compared to the preimplantation stages. We further investigated the epigenetic age of primed (representing early postimplantation) and naïve (representing preimplantation) pluripotent stem cells and observed that in all cases the epigenetic age of primed cells was significantly lower than that of naïve cells. Together, our data suggest that human embryos are rejuvenated during early embryogenesis. Hence, the rejuvenation event is conserved between the mouse and human, and it occurs around the gastrulation stage in both species. Beyond this advance, the intersection clock opens the way for other epigenetic age studies based on human bisulfite sequencing datasets as opposed to methylation arrays.

Cleavage-stage or blastocyst-stage embryo biopsy has no impact on growth and health in children up to 2 years of age

BackgroundStudies show conflicting results on neonatal outcomes following embryo biopsy for PGT, primarily due to small sample sizes and/or heterogeneity in the timing of embryo biopsy (day 3; EBD3 or day 5/6; EBD5) and type of embryo transfer. Even fewer data exist on the impact on children’s health beyond the neonatal period. This study aimed to explore outcomes in children born after EBD3 or EBD5 followed by fresh (FRESH) or frozen-thawed embryo transfer (FET).MethodsThis single-centre cohort study compared birth data of 630 children after EBD3, of 222 EBD5 and of 1532 after non-biopsied embryo transfers performed between 2014 and 2018. Follow-up data on growth were available for 426, 131 and 662 children, respectively.ResultsEmbryo biopsy, either at EBD3 or EBD5 in FET and FRESH cycles did not negatively affect anthropometry at birth, infancy or childhood compared to outcomes in non-biopsied FET and FRESH cycles.While there was no adverse effect of the timing of embryo biopsy (EBD3 versus EBD5), children born after EBD3 followed by FET had larger sizes at birth, but not thereafter, than children born after EBD3 followed by FRESH.Reassuringly, weight and height gain, proportions of major congenital malformations, developmental problems, hospital admissions and surgical interventions were similar between comparison groups.ConclusionOur study indicated that neither EBD3 nor EBD5 followed by FRESH or FET had a negative impact on anthropometry and on health outcomes up to 2 years of age.

Relationship of quantitative reverse transcription polymerase chain reaction (RT-PCR) to RNA Sequencing (RNAseq) transcriptome identifies mouse preimplantation embryo reference genes†.

Numerous reference genes for use with quantitative reverse transcription polymerase chain reaction (RT-qPCR) have been used for oocytes, eggs, and preimplantation embryos. However, none are actually suitable because of their large variations in expression between developmental stages. To address this, we produced a standardized and merged RNA sequencing (RNAseq) data set by combining multiple publicly available RNAseq data sets that spanned mouse GV oocytes, MII eggs, and 1-cell, 2-cell, 4-cell, 8-cell, morula, and blastocyst stage embryos to identify transcripts with essentially constant expression across all stages. Their expression was then measured using RT-qPCR, with which they did not exhibit constant expression but instead revealed a fixed quantitative relationship between measurements by the two techniques. From this, the relative amounts of total messenger RNA at each stage from the GV oocyte through blastocyst stages were calculated. The quantitative relationship between measurements by RNAseq and RT-qPCR was then used to find genes predicted to have constant expression across stages in RT-qPCR. Candidates were assessed by RT-qPCR to confirm constant expression, identifying Hmgb3 and Rb1cc1 or the geometric mean of those plus either Taf1d or Cd320 as suitable reference genes. This work not only identified transcripts with constant expression from mouse GV oocytes to blastocysts, but also determined a general quantitative relationship between expression measured by RNAseq and RT-qPCR across stages that revealed the relative levels of total mRNA at each stage. The standardized and merged RNA data set should also prove useful in determining transcript expression in mouse oocytes, eggs, and embryos.

Methods to improve frozen-thawed blastocyst transfer outcomes- the IVF laboratory perspective

During the last few years, the number of frozen-thawed embryo transfer cycles (FET) significantly increased due to the universal application of more efficient cryopreservation techniques in the IVF laboratory and the improved survival rates of blastocyst stage embryos and the wide implementation of “freeze all” IVF cycles to prevent OHSS, or for preimplantation genetic testing for aneuploidy (PGT-A). Blastocyst cryopreservation allows single embryo transfers to reduce the rate of multiple pregnancies and improve perinatal outcomes. There is no consensus regarding the optimal laboratory protocol for blastocyst cryopreservation, and research is ongoing for its amelioration. This review summarizes different laboratory methods that may improve frozen-thawed blastocyst embryo transfer outcomes, alone or in combination. Some of the techniques relate to embryo survival; some of them work on endometrial receptivity.

Relationship of morphology and chromatin integrity of sperm in aneuploid blastocyst development: embryos fertilized with sperm diagnosed with teratozoospermia

Objective: Evaluate whether the presence of aneuploid blastocysts is associated with sperm morphology and fragmentation.Methods: Retrospective, observational and cross-sectional study, which included 352 embryos in blastocyst stage, obtained by intracytoplasmic sperm injection (ICSI) from 131 cycles of patients with implantation failure and who decided to perform preimplantation genetic study of aneuploidy (PGT-A) to the embryos that were transferred to the uterus, in order to improve the implantation rate.Results: Of the embryos obtained from donated oocytes, only those fertilized with semen diagnosed with teratozoospermia presented aneuploidy (26.6%). The rate of aneuploid embryos was similar when own oocytes were fertilized with semen diagnosed with normozoospermia or teratozoospermia (38.4 vs 37.07%). Finally, no relationship was observed between chromatin damage and sperm morphology.
 
 

Health assessment of Holstein calves born after in vitro fertilization, biopsy-based genotyping at the blastocyst stage and subsequent embryo transfer

Establishing methods for evaluating genomic estimated breeding values of bovine embryos can potentially increase the efficiency of breeding programs by transferring only embryos with a high genomic estimated breeding value. This may be achieved by analyzing DNA from trophectoderm biopsies. However, manipulation of bovine embryos is associated with a risk of impaired conceptus health. More knowledge on the health implications of embryonic handling procedures is required. In this study, we followed pregnancies after transfer of in vitro-produced (IVP) embryos and assessed the health of the offspring during the first 2 weeks of life. Three groups of calves were studied: i) freshly transferred non-biopsied embryos (39 transfers, 17 calves; Group B-/C-); ii) biopsied and freshly transferred IVP embryos (42 transfers, 21 calves; Group B+/C-); iii) biopsied and cryopreserved IVP embryos (17 transfers, 6 calves; Group B+/C+). Blood biochemical and hematologic values were compared between groups and to a control group of 13 calves produced by conventional artificial insemination. The pregnancy rate on day 50 and the calving rate did not differ among the groups, but the average gestation length of the B+/C+ group was significantly shorter and with wider variation than the two other groups. There was a tendency toward a higher average body weight at birth in group B+/C+ (45.1 kg) and the standard deviation in body weight was larger (11.7 kg) compared to the B-/C- (39.5 kg; 3.2 kg) and B+/C- (41.8 kg; 6 kg) groups. Body weight on day 14 was higher in the B+/C+ calves compared to the other groups. There was no difference in the biochemical and hematological values at birth between the groups and these were within the normal range. However, when compared to a group of calves produced by standard artificial insemination, significantly higher concentrations were found for the hepatic-related enzymes ALAT, ASAT, ALP, and GGT in group B-/C-and B+/C-, while only higher ALP concentrations were found in B+/C+ calves. The biochemical findings indicate higher heterogeneity in IVP calves compared to calves produced by artificial insemination. The more manipulated IVP embryos also showed increased heterogeneity in body weight at birth, with a shift toward heavier calves, which calls for closer attendance at parturition to handle dystocia in a timely manner and minimize fetal losses.

Sex-specific differences in head circumference of term singletons after assisted reproductive technology: a multicentre study in Japan

Research questionDoes fertility treatment, specifically assisted reproductive technology (ART), affect head circumference in term singletons? DesignA total of 32,651 women who delivered at term at 12 maternity hospitals in Japan between 2010 and 2018 were included in the analysis; of these, 1941 (5.9%) and 2984 (9.1%) women conceived through ART and non-ART fertility treatments (timed intercourse, ovulation induction or artificial insemination), respectively. The study evaluated the adjusted odds ratios of head circumference ≥90th percentile stratified by infant sex and type of ART procedure after adjusting for covariates, with natural conception as the reference group. ResultsART significantly increased the risk of head circumference ≥90th percentile (adjusted odds ratio 1.56 [95% confidence interval 1.25–1.96]), whereas non-ART fertility treatment did not increase the risk (1.14 [0.92–1.42]). This increased risk of head circumference ≥90th percentile was observed exclusively in male neonates (1.73 [1.33–2.26]) and not in female neonates (1.18 [0.76–1.85]) in the ART group. Frozen embryo transfer (FET), FET in a hormone replacement cycle (HRC-FET) and blastocyst-stage embryo transfer were significantly associated with head circumference ≥90th percentile (1.60 [1.26–2.02], 1.70 [1.30–2.22] and 1.72 [1.33–2.24], respectively). ConclusionsThe use of ART, particularly FET, HRC-FET or blastocyst-stage embryo transfer, was linked with a heightened risk of head circumference ≥90th percentile compared with non-ART fertility treatment or natural conception. The increased risk was observed only in male neonates.

The time interval between laparoscopic tubal ligation and frozen-thawed embryo transfer does not affect the reproductive outcomes

BackgroundHydrosalpinx may decrease implantation and pregnancy rates after embryo transfer. Laparoscopic tubal ligation after embryo freeze and before frozen-thawed embryo transfer (FET) is effective at improving reproductive outcomes for hydrosalpinx patients. This study is to find out the optimal interval between laparoscopic tubal ligation and FET.MethodsWe retrospectively analyzed 259 infertile women who performed laparoscopic tubal ligation for embryo freeze and FET. Participants were divided into three groups, based on the interval between laparoscopic tubal ligation and FET. Group I: <30 days; Group II: 31– 60 days; Group III: >60 days. Outcomes of cleavage-stage and blastocyst-stage embryo FET were analyzed respectively.ResultsThere was no significant difference in clinical pregnancy rate, live birth rate, implantation rate, biochemical pregnancy rate, ectopic pregnancy rate, miscarriage rate and preterm birth rate among the three groups, in both cleavage-stage and blastocyst-stage embryo FET cycles. In cleavage-stage embryo FET cycle, singleton gestational age was significantly younger in group III (38.11 ± 2.28 weeks) compared with group I (39.29 ± 1.06 weeks, P = 0.001) and group II (38.96 ± 1.05, P = 0.026). Singleton birth weight was significantly heavier in group II (3.65 ± 0.32 Kg) compared with group I (3.38 ± 0.29 Kg, P = 0.001) and group III (3.35 ± 0.60 Kg, P = 0.004). Twin birth weight was significantly heavier in group III (2.72 ± 0.43 Kg) compared to group I (2.23 ± 0.67 Kg, P = 0.002). In blastocyst-stage embryo FET cycles, twin gestational age was significantly younger in group II (34.07 ± 3.18 weeks) compared with group I (35.56 ± 2.27 weeks, P = 0.049) and group III (36.50 ± 1.47 weeks, P = 0.005). Twin birth weight was significantly heavier in group III (2.71 ± 0.39 Kg) compared to group II (2.39 ± 0.67 Kg, P = 0.009).ConclusionsThe duration of the interval between laparoscopic tubal ligation and FET does not affect the reproductive outcomes; however, it may affect the neonate outcomes to some extent.

Relationship of morphology and chromatin integrity of sperm in aneuploid blastocyst development: embryos fertilized with sperm diagnosed with teratozoospermia

Objective: Evaluate whether the presence of aneuploid blastocysts is associated with sperm morphology and fragmentation. Methods: Retrospective, observational and cross-sectional study, which included 352 embryos in blastocyst stage, obtained by intracytoplasmic sperm injection (ICSI) from 131 cycles of patients with implantation failure and who decided to perform preimplantation genetic study of aneuploidy (PGT-A) to the embryos that were transferred to the uterus, in order to improve the implantation rate. Results: Of the embryos obtained from donated oocytes, only those fertilized with semen diagnosed with teratozoospermia presented aneuploidy (26.6%). The rate of aneuploid embryos was similar when own oocytes were fertilized with semen diagnosed with normozoospermia or teratozoospermia (38.4 vs 37.07%). Finally, no relationship was observed between chromatin damage and sperm morphology. Conclusion: In patients who fertilize their oocytes with spermatozoa from samples diagnosed with normozoospermia or teratozoospermia, the rate of aneuploid blastocysts will depend mainly on the female factor, this does not rule out the possibility of aneuploid embryos due to the male factor. Different from what was seen in donated oocytes, where embryos obtained from semen diagnosed with teratozoospermia presented a higher rate of aneuploidy.

Clinical pregnancy rate for frozen embryo transfer with HRT: a randomized controlled pilot study comparing 1 week versus 2 weeks of oestradiol priming

Research questionDoes a frozen-embryo transfer in an artificially-prepared endometrium (FET-HRT) cycle yield similar clinical pregnancy rate with 7 days of oestrogen priming compared to 14 days?DesignThis is a single-centre, randomized, controlled, open-label pilot study. All FET-HRT cycles were performed in a tertiary centre between October 2018 and January 2021. Overall, 160 patients were randomized, with a 1:1 allocation, into two groups of 80 patients each: group A (7 days of E2 prior to P4 supplementation) and group B (14 days of E2 prior to P4 supplementation). Both groups received single blastocyst stage embryos on the 6th day of vaginal P4 administration. The primary outcome was the feasibility of such strategy assessed as clinical pregnancy rate, secondary outcomes were biochemical pregnancy rate, miscarriage rate, live birth rate and serum hormone levels on the day of FET. Chemical pregnancy was assessed by an hCG blood test 12 days after FET and clinical pregnancy was confirmed by transvaginal ultrasound at 7 weeks.ResultsThe analysis included 160 patients who were randomly assigned to either group A or group B on the seventh day of their FET-HRT cycle if the measured endometrial thickness was above 6.5 mm. Following screening failures and of drop-outs, 144 patients were finally included both in group A (75 patients) or group B (69 patients). Demographic characteristics for both groups were comparable. The biochemical pregnancy rate was 42.5% and 48.8% for group A and group B, respectively (p 0.526). Regarding the clinical pregnancy rate at 7 weeks, no statistical difference was observed (36.3% vs 46.3% for group A and group B, respectively, p = 0.261). The secondary outcomes of the study (biochemical pregnancy, miscarriage, and live birth rate) were comparable between the two groups for IIT analysis, as well as the P4 values on the day of FET.ConclusionsIn a frozen embryo transfer cycle, performed with artificial preparation of the endometrium, 7 versus 14 days of oestrogen priming are comparable, in terms of clinical pregnancy rate; the advantages of a seven-day protocol include the shorter time to pregnancy, reduced exposure to oestrogens, and more flexibility of scheduling and programming, and less probability to recruit a follicle and have a spontaneous LH surge. It is important to keep in mind that this study was designed as a pilot trial with a limited study population as such it was underpowered to determine the superiority of an intervention over another; larger-scale RCTs are warranted to confirm our preliminary results.Trial registrationClinical trial number: NCT03930706.

P-239 Comparison of reproductive outcomes for cleavage- and blastocyst-stage frozen embryo transfer: A retrospective study

Abstract Study question Does blastocyst-stage (day 5-6) embryo transfer improve pregnancy and live birth rates per frozen transfer compared to cleavage-stage (day 2-3) embryo transfer? Summary answer Pregnancy rate is significantly higher following frozen blastocyst-stage embryo transfer, while live birth rate does not differ. What is known already The extended culture of cleavage-stage (day 2-3) embryos to blastocyst-stage (day 5-6) embryos has been largely adopted in recent years. In fact, blastocyst culture may allow a better selection of the embryo to be transferred based on its developmental history and morphological criteria, and result in a lower number of exceeded embryos to be frozen for storage. However, the literature comparing pregnancy and live birth rates for cleavage- and blastocyst-stage transfer remains contradictory, and the benefits of frozen blastocyst transfer is still under discussion. Study design, size, duration This retrospective study investigated couples having frozen embryo transfer at the cleavage (days 2 or 3) or the blastocyst-stage (days 5 or 6) at our clinic between 2014–2021. Results from more cycles were analyzed individually. We excluded cycles based on fresh embryo transfer, oocyte or sperm donation. Cycles were further sub-classified based on the type of treatment, as natural or stimulated, and the pregnancy and live birth rates were compared. Participants/materials, setting, methods Data was obtained from patient records at our fertility clinic and sorted by two authors. Variables included female age, treatment plan, the stage of the embryo transferred, and the report of pregnancy and live birth. Statistical analysis was conducted by using MedCalc Software, and Chi-squared test applied to compare the outcomes’ frequency based on the day of frozen embryo transfer. P &amp;lt; 0.05 was considered significant. Main results and the role of chance A total of 2,922 cycles were included for analysis, with age of female patients being 37.2 ± 5.0 years at the time of transfer. Most cycles were stimulated (n = 2,217, 75.9%) compared to natural cycles (n = 705, 24.1%). In all cycles, IVF was used for fertilization. Approximately one third of the cycles (n = 954, 32.6%) underwent frozen embryo transfer at the cleavage-stage (days 2 or 3), while the remaining had transfer at the blastocyst-stage (n = 1,968, 67.4%). Pregnancy rate was significantly (P &amp;lt; 0.0001) higher for blastocyst-stage embryo transfer compared to cleavage-stage embryo transfer (36.2% vs 20.4%, respectively) when all cycles were analyzed. This significance was found when natural (23.3% vs 14.8%, P = 0.0052) or stimulated cycles (38.0% vs 25.8%, P &amp;lt; 0.0001) were sub-analyzed. While we observed 19.0% of successful live birth per embryo transferred in the entire cohort, this did not significantly vary depending on the stage of frozen embryo transferred (cleavage or blastocyst-stage, 13.6% vs 21.5%, respectively) and when patients were sub-classified based on the type of treatment chosen in natural (9.0% vs 14.2% respectively) or stimulated (18.0% vs 22.6%, respectively) cycles. With a large sample size and a very significant P-value for pregnancy rate, the role of chance is limited. Limitations, reasons for caution As a retrospective study, the non-random allocation to treatment groups might hide selection bias and limit the statistical power of the data analysis, especially for the live birth rate. Also, no adjustment for confounders was performed. Wider implications of the findings Frozen embryo transfer at blastocyst-stage may offer an improved chance of pregnancy in both natural and stimulated cycles, possibly resulting in less embryos stored and lower costs for patient treatment. Although no difference was observed, larger prospective studies are needed to clarify the impact on live birth rate. Trial registration number Not applicable

O-005 The ToF-study - comparing the cumulative live birth rate of blastocyst-stage versus cleavage-stage embryo transfers in good prognosis IVF patients: a multicenter randomized controlled trial.

Abstract Study question Does blastocyst-stage embryo transfer in fresh and frozen cycles improve the cumulative live birth rate (cLBR) compared with cleavage-stage embryo transfer in IVF/ICSI treatments? Summary answer In good prognosis IVF patients (≥4 available embryos), a blastocyst-stage transfer policy did not result in a significant higher cLBR compared to cleavage-stage transfer policy. What is known already A recent Cochrane systematic review and meta-analysis concluded that fresh blastocyst-stage transfer in IVF/ICSI treatments is associated with higher rates of pregnancy in comparison to fresh transfer of cleavage-stage embryos. However, it is unknown whether a blastocyst-transfer policy also improves the cumulative live birth rate, i.e. the live birth rate derived from fresh and frozen-thawed embryo transfers following a single oocyte retrieval, in comparison to a cleavage-stage transfer policy in IVF/ICSI. Study design, size, duration In this multicenter randomized controlled trial women were randomly allocated to blastocyst-stage transfers (blastocyst group - fresh embryo transfer on day 5 after oocyte retrieval followed by vitrification of remnant blastocysts on day 5 and 6 following local criteria) or cleavage-stage transfers (cleavage-stage group - fresh embryo transfer on day 3 after oocyte retrieval followed by embryo cryopreservation on day 3 or 4). Randomization was stratified for age (≥36 or &amp;lt; 36 years). Participants/materials, setting, methods Women with a good prognosis after IVF/ICSI (defined as presenting ≥4 available embryos on day 2 of embryo culture), during their first, second, or third treatment cycle, were included. The primary outcome was the cLBR per oocyte retrieval, including associated frozen-thawed embryo transfers within 12 months after randomization (or 17 months during the COVID pandemic). Risk ratios (RR) with 95% CI adjusted for age group were calculated using log-linear binominal regression. Main results and the role of chance A total of 1202 women from 21 Dutch centers were randomly assigned to blastocyst-stage transfers (N = 599) or cleavage stage transfers (N = 603) between 2018 and 2021. At submission of this abstract, data on the primary outcome was available for 1153 (95.9%) women, 577 women in the blastocyst-stage group and 576 women in the cleavage-stage group. The cumulative live birth rate was 58.2% (336/577 women) in the blastocyst-stage group and 57.3% (330/576 women) in the cleavage stage group (RR 1.022, 95% CI 0.844-1.237; p = 0.825). The live birth rate after fresh embryo transfer was 38.0% (219/577 women) versus 29.9% (172/576 women) in the blastocyst-stage group and cleavage-stage group respectively (RR 1.282, 95% CI 1.017-1.615 p = 0.035). Interaction was found between age and day of transfer with a higher cumulative live birth rate and a higher live birth rate after fresh transfer in women of 36 years or older in the blastocyst group. Analyses on other IVF treatment outcomes, obstetrical or neonatal outcomes, patient burden, and cost effectiveness are ongoing. Limitations, reasons for caution Outcomes are only applicable for treatments of women with at least four embryos available on day two of embryo culture. Wider implications of the findings A blastocyst-stage embryo transfer policy did not result in a significant higher cumulative live birth rate in comparison to a cleavage-stage embryo transfer policy in IVF/ICSI treatments. Further research into the interaction of age with outcomes is warranted. Trial registration number NTR7034

P-144 An artificial intelligence algorithm demonstrates optimal performance for evaluating embryo genetic status at 120 hours post-fertilization

Abstract Study question What is the effect of time-point on performance of a non-invasive artificial intelligence (AI) algorithm for evaluating embryo genetic status? Summary answer While predictive ability was maintained across different time-points on day 5, optimal performance for ranking and selecting euploid embryos was observed at 120 hours post-fertilization. What is known already Studies have shown that it is possible to develop computer vision-based AI algorithms capable of predicting embryo ploidy status using single images of blastocyst-stage embryos. The genetic status of embryos is linked to morphokinetic development, with aneuploidy generally resulting in earlier arrest. Given the dynamic nature of embryo development, it might be expected that the time-point selected for analysis could influence AI performance. The key questions remaining to be answered are to what extent is AI analysis affected by expansion grade, and what does this mean for selection of a time-point for evaluation? Study design, size, duration 2,683 images of day 5 blastocyst-stage embryos with matched ploidy outcomes from pre-implantation genetic testing for aneuploidies (PGT-A) were provided by 10 IVF clinics in the USA, Australia, Malaysia, and India. A subset of 182 embryos had images provided at 110, 115, and 120 hour time points (GERI and EmbryoScope time lapse systems). Participants/materials, setting, methods Images were analysed by a previously developed AI algorithm which evaluates the likelihood of an embryo being euploid according to PGT-A (Diakiw et al, 2022. Hum Reprod, Jul 30;37(8):1746-1759). Evaluation was performed on embryos of each expansion grade, and at three time-points on day 5. Correlations were assessed using Chi-squared test for trend, with pair-wise comparisons conducted using Student’s t-test. Performance was evaluated using ROC-AUC, and a simulated cohort ranking analysis method. Main results and the role of chance AI scores positively correlated with expansion grade, and expansion grade likewise correlated with an increasing proportion of euploid embryos. AI scores also increased over time on day 5, consistent with continued embryo expansion. Scores for grade 4 (expanded) embryos increased more than for grade 5 (hatching) embryos (+2.2-fold and +0.8-fold, respectively), indicative of continued expansion becoming limited at later stages. Despite the valid association of AI scores with expansion grade, results showed the AI could predict euploidy even amongst embryos of the same expansion grade (ROC-AUC ranging from 0.61−0.69). While predictive ability was maintained at each time-point on day 5, ROC-AUC values were highest at 120 hours (0.64, 0.64, and 0.68 for 110, 115, and 120 hours, respectively). Simulated cohort ranking analyses also showed that the AI performed best at 120 hours, selecting a euploid embryo as the top-ranked embryo in 77.1% of patient cohorts (71.4%, 75.1%, and 77.1% for 110, 115, and 120 hours, respectively). These results suggest that regardless of expansion grade, all embryos should be assessed at the same time-point on day 5, preferably closer to the 120 hour time-point. Limitations, reasons for caution The time-point analyses were conducted on a relatively small dataset, and therefore findings should be validated on a larger dataset including embryos of other expansion grades. The analysis was limited to day 5 post-fertilization, however it would be interesting to extend the study to embryos on day 6 also. Wider implications of the findings These results suggest that the AI is providing additional information regarding embryo genetic status, over and above that provided by known morphological parameters. The dynamic nature of AI score related to expansion is of interest as it relates to the optimal time-point for conducting analyses for selection of euploid embryos. Trial registration number Not applicable

O-077 Time-lapse imaging analysis of segmental aneuploid embryos: a multicenter study identifies morpho-kinetic patterns associated with chromosomal mosaicism

Abstract Study question Do segmental aneuploid (SA) embryos display unique morpho-kinetic patterns at early developmental stages? Summary answer The first three cell division cycles of embryos carrying segmental aneuploidies are significantly slower than euploid and whole-chromosome aneuploid (WCA) embryos. What is known already Segmental aneuploidies in blastocyst-stage human embryos prevalently originate (i.e., 70% of cases) from mitotic errors. Their clinical management is challenging because it is impossible to establish their meiotic/mitotic origin from PGT-A analysis of a single trophectoderm biopsy. Morpho-kinetic analysis based on Time-lapse microscopy (TLM) has been proposed as a valuable tool for systematic embryo evaluation, although it is unclear whether this approach alone can predict chromosomal abnormalities. This study aims to pinpoint morphokinetic patterns associated with the occurrence of segmental aneuploidies and/or mitotic errors, possibly improving DNA-based diagnostic interpretation and their clinical management. Study design, size, duration This is a retrospective multicenter study including a total of 7,693 embryos from 2,370 IVF cycles and 2,127 couples, cultured between 2016 and 2021 in 4 European IVF centers. Embryos with no more than 4 chromosomal alterations were considered in the analysis, resulting in 3,288 euploids, 3,155 WCA, 715 SA, and 535 complex aneuploids (CA). Overall, the dataset contained 3,742 distinct euploid-SA embryo sibling pairs. Participants/materials, setting, methods Standard morpho-kinetic features were annotated using various TLM systems. Blastocysts were subjected to comprehensive chromosomal screening via PGT-A. Morpho-kinetic timings across different embryo groups were compared using Kolmogorov-Smirnov (KS) test and binomial test, and associations with cleavage features were assessed via two-sided Fisher Exact (FS) test. Multi-center and center-specific logistic regression models were calibrated using a subset of data (70%), and their predictive performance was evaluated on independent test data using Area-Under-ROC curve (AUROC) metrics. Main results and the role of chance SA embryos cleaved at significantly slower rate than their euploid siblings in the first 3 cell cycles, from the appearance of the second blastomere t2 (average delay delta=0.49h, KS p = 0.006, fraction of pairs in which SA was delayed compared to euploid sibling F = 54.2%, binomial p = 0.002) up to t8 (delta=2.16h, KS p = 4e-4, F = 57.9%, binomial p = 1e-8). The developmental delay was partially compensated at full blastocyst stage tB (delta=1.71h, KS = 0.0008, F = 57.5%, binomial p = 5e-8). SA timings also differed from WCA ones: SA embryos were significantly slower than their WCA siblings during early cell cycles, with maximum delay reached at t8 (delta=1.67h, KS p = 0.009, F = 55.4%, binomial p = 0.003), but they fully caught-up at tB, with no significant difference observed. These morpho-kinetic patterns were consistently observed in each center separately, but with different effect sizes. The presence of segmental aneuploidies was significantly associated with multinucleation (OR = 1.99, FS test p = 0.05) and morula cell exclusion (OR = 2.69, p = 0.006). A logistic model based on morpho-kinetic data and cleavage features from a single center dataset and regressed against embryo aneuploidy class (euploid vs. SA embryos) displayed adequate predictive performance on independent data from the same center (AUROC=0.70), although predictivity diminished when tested on data from other centers (AUROC=0.52-0.55). Limitations, reasons for caution We cannot rule out inter-center variability in the annotation procedures of morpho-kinetic parameters. Standardized and automated annotation would increase the performance of predictive models and their transferability. Further work is needed to assess whether the morpho-kinetic signal is driven by mitotic errors and if an association with clinical outcomes exists. Wider implications of the findings These findings might help uncovering biological events contributing to preimplantation mosaicism. Moreover, the developed predictive framework might help improving decision-making in PGT-A cycles, providing interpretation for segmental aneuploidies and possibly helping in the evaluation of embryos showing intermediate copy number values for whole chromosomes that are suggestive of mosaicism. Trial registration number Not applicable