Blastocyst Stage Research Articles

O-249 METAPHOR: METabolic imaging through AI-powered Phasor-based Hyperspectral analysis and Organelle recognition for the classification of human blastocysts

Abstract Study question Can we classify human blastocysts non-invasively using hyperspectral imaging? Summary answer HS imaging can significantly enhance our classification of human embryos and reveal numerous cellular features present in the embryo invisible to brightfield imaging. What is known already Non-invasive measurement of metabolism is a safe and powerful tool to reliably predict oocyte and embryo quality. We have previously shown that our METAPHOR technology, based on HS imaging coupled to artificial intelligence, was able to separate mouse oocyte categories according to maternal age with an average AUC of 96.2% and predict blastocyst formation (AUC 82.2%). Moreover, it was able to classify mouse embryos deprived from different nutrients with an average AUC of 93.7% showing clear superiority to grading provided by experienced embryologists (51%). Study design, size, duration A total of 74 human embryos donated for research at the blastocyst stage have been imaged to date. Embryos were warmed and imaged according to our established protocol and survival was statistically indistinguishable from control embryos. Participants/materials, setting, methods Embryos are imaged using multiphoton illumination near the infra-red. The HS detection covered the simultaneous measurement of the whole visible spectrum. Embryos are then allowed to implant in our proprietary 3D ex vivo platform. Implantation is monitored up until day 9 before immunostaining is performed to quantify presence and prevalence of lineage and implantation markers. Main results and the role of chance After imaging, embryos were placed on the 3D ex vivo implantation platform and let implant. Successful implantation (75.41%) was scored by observation of the characteristic deformation of collagen fibers within the matrix and staining for relevant cell lineages (OCT4, pMLC2). Interestingly, we observed that embryos with a higher trophectoderm (TE) grading tended to implant with a higher efficiency and penetrate deeper into the matrix than embryos with lower grading. HS images have unveiled a profound cellular diversity within embryos, elucidating distinctions between the Inner Cell Mass (ICM) and TE, as well as revealing different metabolic states defined by NADH dominance, FAD dominance, protoporphyrin levels, and apoptosis. HS raw images are processed using our HS-phasor approach which allows quick computing by converting the multidimensional HS data into comprehensive visual representation of bidimensional histograms and phasor plots. In summary, our analysis pipeline encompasses the algorithms to detect and segment the embryos, the computation of histogram and phasor-plot, and the classifier. AI allows us to classify embryos in distinct spectral spaces based on their metabolic features, paving the way to the establishment of a new grading system based on HS imaging. Limitations, reasons for caution The cohort size is still limited, and ongoing experiments will increase the dataset needed for more accurate prediction of embryo implantation. The implantation platform is an in vitro test that will require future clinical validation. Wider implications of the findings The safety and speed of METAPHOR warrants its incorporation at the IVF clinic, adding an additional layer of information to the current embryo selection methods. Moreover, the non-invasive measurement of the metabolic function opens new alleys of research such as the effect of media and culture supplements on embryo culture. Trial registration number Not applicable

P-218 Should embryos with direct unequal cleavage be given a chance or directly deprioritized? A follow-up on blastocyst formation and ploidy

Abstract Study question Should embryos with direct unequal cleavage (DUC) be given a chance to develop to blastocyst for a quality and ploidy assessment or directly deprioritized? Summary answer Although DUC embryos are clearly associated with poorer outcomes, if DUC embryos develop to good quality blastocysts the euploidy rate is comparable to non-DUC embryos. What is known already DUCs were first defined by time-lapse imaging and their implantation rate was found to be significantly lower than embryos with a normal cleavage pattern (1.2% vs. 20.2%, respectively) leading to the suggestion that rejection of these embryos for transfer could improve implantation rates (Rubio et al., 2012). Another study concluded that blastocyst formation, implantation potential and euploidy rate are significantly reduced in DUC embryos and that they should be deselected for day 3 transfers but should be cultured to blastocyst stage for possible embryo transfer (Zhan et al., 2016). However, in practice, DUCs are deselected and are therefore rarely transferred. Study design, size, duration This retrospective study included 1191 DUC embryos and 5058 non-DUC embryos incubated in the Embryoscope in 2022. Twenty-eight DUC and 185 non-DUC embryos were transferred in a fresh cycle. Preimplantation genetic testing for aneuploidy (PGT-A) was performed on 128 DUC and 1477 non-DUC blastocysts. Five DUC embryos were transferred in a frozen-thawed cycle. Participants/materials, setting, methods A total of 2430 IVF patients treated in a single private clinic were included in this retrospective study. The mean female age of the cohort was 36.5. Some of the blastocysts were subjected to PGT-A mainly for advanced maternal age. CHLOE (Fairtility) software analyzed time-lapse videos and identified pronuclei, DUCs and blastulation. DUC embryo outcomes (ploidy, blastulation and blastocyst quality) were assessed. Direct cleavages from 2 to 5 cells were not included in the study. Main results and the role of chance The outcomes of DUC embryos (n = 1191) were compared to non-DUC embryos (n = 5058). The blastulation rate was 23.8% for DUC embryos and 50.2% for non-DUC embryos. The good-quality blastocyst rate was 15.1% and 42.5%, respectively. The top-quality blastocyst rate was 2.4% and 18.5%, respectively. PGT-A was performed for 128 DUC blastocysts; 52 were euploid (40%) and 14 were mosaic (11%). The euploidy rate for non-DUC blastocysts was 43% and the mosaicism rate was 9.4%. Thirty-three DUC embryos were transferred giving 6 live births (18%). Limitations, reasons for caution The discrimination between fragments and blastomeres within the 5 hours from the first division is challenging for AI algorithms. Therefore, some DUC embryos may have been misclassified. Wider implications of the findings In conclusion, although DUC embryos are not prioritized for transfer, our results show that, once achieving the blastocyst stage with a good quality, their euploidy rate is comparable to non-DUC blastocysts. Morphokinetic analysis may propose additional markers to identify higher implantation potential DUC blastocysts. Trial registration number N/A

O-156 Trophectoderm biopsy for pre-implantation genetic testing (PGT) does not increase rates of placental abnormalities among singleton pregnancies conceived with in-vitro fertilization (IVF)

Abstract Study question Does trophectoderm biopsy for PGT affect placental pathology? Summary answer Trophectoderm biopsy does not increase rates of placental abnormalities among singleton pregnancies conceived with in-vitro fertilization (IVF). What is known already PGT, routinely performed at the blastocyst stage, is used to screen embryos for aneuploidy, structural rearrangements, and single-gene disorders. The association of ART with abnormal placentation and adverse pregnancy outcomes is known. Whether, in a similar fashion, the disruption of the trophectoderm contributes to increased rates of abnormal placentation, remains unknown. Study design, size, duration Data from 987 singleton livebirths with available placental pathology, who conceived following IVF/±PGT at a single academic fertility center between 01/2004 and 12/2019, were retrospectively reviewed. Placental abnormalities were classified as anatomic, inflammatory, infectious, and vascular (any features of fetal or maternal vascular malperfusion), by one expert perinatal pathologist using the Amsterdam Workshop Consensus definitions. Placental pathology was compared between PGT (n = 76) and non-PGT (fresh and frozen, n = 911) cycles. Participants/materials, setting, methods Primary Outcome Measures: incidence of anatomic, vascular (maternal and fetal side), infectious, and inflammatory placental abnormalities. Odds ratios (OR) and 95% confidence intervals (95%CI) were calculated using generalized estimating equations regression to adjust for multiple deliveries per patient and for potential confounders [maternal age, body mass index (BMI:kg/m2), race, parity, infertility diagnosis, gestational age, infant gender, hypertensive disorders of pregnancy, IVF and FET protocol]. Main results and the role of chance Patients undergoing PGT were older than the non-PGT group [36.2(3.5) vs. 34.9(3.8) years, p = 0.006] and did not otherwise differ in AMH, BMI, and race. Overall, adjusted (Adj) OR (95%CI) did not reveal any differences between groups in anatomic [0.92(0.82-1.03)], inflammatory [1.03 (0.94-1.14)]), infectious [1.02 (0.9-1.16)], or vascular abnormalities [1.02(0.88-1.18)]. When limiting analysis to blastocyst biopsies/FET cycles only, adjOR did not show any differences in inflammatory [1.01 (0.89-1.13)]), infectious [1.02 (0.88-1.17)], or vascular abnormalities [1.01 (0.84-1.2)] between the PGT and non-PGT group. Interestingly, adjOR (95%CI) revealed lower odds for anatomic abnormalities in the PGT group (0.86 (0.76-0.98), p = 0.021, non-PGT: ref). A sub-analysis of programmed FET cycles revealed persistently lower odds of anatomic abnormalities among the PGT group [adjOR (95%CI): 0.77 (0.67-0.89), p < 0.001, non-PGT: ref]. Among anatomic abnormalities studied in the programmed FET cycles, marginal cord insertion showed a difference between the groups [adjOR(95%CI): 0.83 (0.73-0.95), p:0.007, non-PGT: ref ]. AdjOR(95%CI) showed no differences in inflammatory [ 1.01 (0.87-1.17)], infectious [1.01 (0.86-1.2)], and vascular [0.9 (0.73-1.1)] abnormalities between groups. Limitations, reasons for caution This study is limited by its retrospective nature. Although a large number of IVF placentas was included, the number of patients in the PGT compared to the non-PGT group was smaller potentially limiting our ability to detect subtle differences between groups. Wider implications of the findings Trophectoderm biopsy for PGT does not appear to increase risk for placental abnormalities, a reassuring finding given the increased incidence of abnormal placentation and adverse perinatal outcomes among IVF pregnancies. Trial registration number not applicable

P-119 Factors affecting male-to-female ratio at birth in IVF/ICSI cycles

Abstract Study question Are there any factors during an IVF cycle that can influence the ratio of male or female preimplantation embryos? Summary answer Semen preparation technique significantly influences the male-female ratio of newborns through IVF-ICSI with a greater proportion of boys with gradients and girls with swim up. What is known already Although some works have related the fertilization technique or the transfer of blastocysts with the male-female ratio in those born through assisted reproduction, there are limited studies that investigate the relationships of the various factors involved during an IVF/ICSI cycle with the ratio male-female at birth. Furthermore, the number of studies that looked at the effect of embryo morphokinetics in sex ratio at birth, produced contradictory results. Study design, size, duration Retrospective, multicenter study at seven reproductive units belonging to the same center in Spain. The study included 202 patients who underwent IVF/ICSI treatment between January 2022 and December 2022, ensuring that 100% implantation was obtained. All cycles were accomplished with time-lapse, and the transfers were performed at the blastocyst stage. The sex was confirmed at birth. Participants/materials, setting, methods Sex ratios of 223 live births were compared regarding to fertilization method (IVF or ICSI), age, culture medium, embryo morphokinetics (t2, t3, t4,t5,tM,tSB,tBc,cc2,s2), twinship, origin of oocytes and sperm, embryo quality and sperm preparation technique (swim-up vs gradients). The Student’s T test was used for metric variables and the χ2 test for dichotomous variables (p < 0.05). To assess if the rest of the metric variables influenced sex, the Kolmogorov-Smirnov test and the Pearson test were used. Main results and the role of chance 223 live births were obtained: 181 single births after the transfer of one blastocyst and 42 double births of the same sex after the transfer of two blastocysts. Among the 223 newborns included in the study, a statistically significant predominance (p < 0.05) of males was found: 58.3% (95%CI:51.8-64.8%) of boys (n:130) compared to 41.7% (95%CI:35.2-48.2%) of girls (n :93). The mean age of the patients was 38.3 years (SD: 4.87), with no significant differences found regarding the sex ratio, as well as with the fertilization technique (p = 0.937), culture medium (p = 0.096), twinning (p = 0.222), the origin of the oocytes (p:0.608), the origin of the semen (p:0.721) and embryo quality (P:0.186). A statistically significant relationship was found between the sex of the newborn and the semen preparation technique (62.1% of male with gradients versus 32.1% with swim up, p:0.003), which was confirmed using a multivariate binary logistic regression model (p:0.0200). Analyzing each of the parameters obtained from the time lapse with respect to the sex of the newborns in a univariate manner with Student’s T for independent samples no significant differences were found in the division times studied or in the times of the cell cycle and synchrony. Limitations, reasons for caution The main limitation is the necessity of a larger sample size and to further investigate the reasons for these results. Further studies with standardized culture conditions and well-justified inclusion criteria are needed to reach robust and reliable conclusion. Wider implications of the findings While the study needs for more extensive sample size for future research, is the first time to suggest that method of sperm preparation may have an impact on the sex ratio at birth. Understanding which factors within ART process can affect sex ratio remain important considerations. Trial registration number not applicable

P-728 Age limits in medically assisted reproduction: Assessing clinical and pregnancy outcomes in women over 50 years of age undergoing donor oocyte in vitro fertilization

Abstract Study question Is pregnancy in women over 50 years of age undergoing donor oocyte IVF associated with adverse clinical and pregnancy outcomes? Summary answer In vitro fertilization treatment in women over 50 years of age using donor oocytes is not associated with adverse clinical and pregnancy outcomes. What is known already In vitro fertilization with donor oocytes (d-IVF) has become a common strategy for managing cases of very advanced maternal age. However, there is ongoing scientific debate entailing legal and societal implications regarding the age limit beyond which d-IVF should no longer be considered safe. Different age limits and guidelines have been proposed, with no globally accepted consensus. The most commonly reported age limit is 50 years, representing the median age of menopause. However, there is a lack of robust data to support that d-IVF beyond the age of 50 is associated with adverse clinical and pregnancy outcomes. Study design, size, duration A retrospective single-centre study was conducted to evaluate medical records of d-IVF cycles between July 2021-January 2023. A total of 575 medical records of women undergoing d-IVF were analyzed. The study population was divided into two groups based on maternal age: women aged 45-49 years (group A, n = 318) and women aged 50-54 years (group B, n = 257). The outcome measures were positive-hCG, clinical pregnancy, viable pregnancy at 12 weeks of gestation, pregnancy loss and live birth. Participants/materials, setting, methods For oocyte recipients advanced maternal age was the sole infertility etiology and endometrial preparation was performed employing conventional protocols. Oocyte donors were up to 35 years of age and oocyte donation cycles were performed according to national guidelines. Single or double embryo transfers (ETs) were performed at the cleavage or blastocyst stage. Embryo quality assessment was performed according to Veeck and Gardner grading systems. Statistical analysis was performed employing R Programming Language for Statistical Purposes. Main results and the role of chance The mean age of group A was 47.02 ± 1.35 while the mean age of group B was 51.84 ± 1.59. No statistically significant difference was observed regarding the age of oocyte donors (25.17 ± 4.18 vs 26.09 ± 4.13; p-value=0.26). Considering embryo transfer, 90 cleavage stage and 228 blastocyst stage transfers were performed in group A, and 75 cleavage stage and 182 blastocyst stage transfers were performed in group B (p-value=0.23). No statistically significant difference was observed between the two groups with regards to the number of transferred embryos (1.89 ± 0.35 vs 1.90 ± 0.36; p-value=0.69) and embryo quality (Group A: Good 157, Fair 143, Poor: 18; Group B: Good 121, Fair 117; Poor 19; p-value=0.66). Considering clinical and pregnancy outcomes no statistically significant difference was observed between the two groups regarding positive hCG rate (147/318 vs 109/257, respectively; RR: 0.91; 95%CI: 0.76-1.10; p-value=0.36), clinical pregnancy rate (129/318 vs 98/257, respectively; RR: 0.94; 95%CI: 0.77-1.15; p-value=0.55), viable pregnancy rate at 12 weeks of gestation (96/318 vs 70/257, respectively; RR: 0.90; 95%CI: 0.70-1.17; p-value=0.44), pregnancy loss rate (39/129 vs 33/98, respectively; RR: 1.13; 95%CI: 0.76-1.63; p-value=0.58) and live-birth rate (90/318 vs 65/257, respectively; RR: 0.91; 95%CI: 0.68-1.17; p-value=0.42). Limitations, reasons for caution The retrospective and the single-centre nature of the study are important reasons for caution. In addition, the relatively small sample size may serve as another limitation. The lack of neonatal outcomes may also be considered an additional limitation when interpreting data on the safety of d-IVF in the over-50s. Wider implications of the findings Women over 50 who undergo d-IVF are not at increased risk of complications. This highlights the need to reconsider age limits and provide robust evidence ascertaining both safety and reproductive autonomy. Studies are needed to assess neonatal outcomes and maternal age impact on long-term infant health from an epigenetic standpoint. Trial registration number Not applicable

O-215 Copper oxide nanoparticles (CuONPs) impairs mitophagy and promotes DNA methylation to suppress mouse embryonic pluripotency

Abstract Study question Does exposure to copper oxide nanoparticle (CuONPs) have adverse effects on early embryonic development? What is the potential mechanism? Summary answer CuONPs exposure can damage mitophagy and lead to α-KG reduction, resulting DNA hypermethylation, thereby reducing the pluripotency of pre-implantation embryos in mice. What is known already Environmental nanomaterials contamination is a great concern for organisms including human. Exposure to nanomaterials have been associated with fecundity and fertility in couples conceiving via assisted reproduction technology. Among these bio-effects, nanomaterials induced epigenetic changes are of particular interest. Copper oxide nanoparticles (CuONPs) are widely used in a huge range of applications which might pose potential risk to organisms. Recent studies reported that CuONPs exposure resulted in transgenerational toxicity in Caenorhabditis elegans associated with possible epigenetic regulation. However, whether CuONPs exposure affect mammalian embryonic development, and if so the underlying mechanisms, are unclear. Study design, size, duration For the human experiments, we measured the copper ion content in women’s urine, and assessed the expression of pluripotent genes in 3PN embryos treated with CuONPs. For the embryo experiments, we collected zygotes from ICR mice and cultured them in KSOM medium with CuONPs (10 μg/mL) until blastocyst stage. For the cell experiments, we treated mouse embryonic stem cells (mESCs) with CuONPs (2 μg/mL) for four days to detect cell proliferation and pluripotency. Participants/materials, setting, methods The ICP-MS method was used to detect copper ion content in women’s urine. Time–lapse incubator was used to document the developmental potential of embryos. To quantify the metabolites of embryo, we employed metabolomics. Single-cell multiomics sequencing that enables profiling of genome-wide chromatin accessibility, DNA methylation and RNA expression were performed to detect genes expression and epigenetic modification. We used real-time PCR, immunofluorescence, and western blot to measure the expression of pluripotency related genes. Main results and the role of chance In human, we discovered a significant negative relationship between the urine’s copper ion content and clinical pregnancy rate. In embryo experiments, CuONPs exposure has been shown to have adverse effects on embryo developmental potential, resulting morula and blastocyst transformation arrest, as well as reduction in blastocyst rate and live birth rate. Furthermore, CuONPs treatment led to a reduction in blastocyst quality, indicating a decrease in blastocyst cell count, particularly in ICM cells. CuONPs also inhibited the pluripotency of blastocysts and mESCs, showing down-regulated expression of pluripotent genes. Mechanistically, CuONPs aggregated in lysosomes and decreased the mitophagic flux of embryos, which caused damaged mitochondria to accumulate and ultimately caused mitochondrial dysfunction. Notably, in embryos treated with CuONPs, there was a decrease in α-ketoglutarate (α-KG) due to the disruption of the tricarboxylic acid (TCA) cycle. α-KG is an essential cofactor for α-KG dependent dioxygenases such as the ten-eleven translocation (TET) family of DNA demethylase. The lack of α-KG in CuONPs-treated embryos led to DNA hypermethylation, which reduced chromatin accessibility, and suppressed the expression of pluripotent genes like sox2, klf2, and klf4. The poor pluripotency ultimately leads to abnormal embryonic development in CuONPs-treated embryos. Limitations, reasons for caution The molecular mechanism by which CuONPs arrest the development of pre-implantation embryos remains to be determined. In addition, future work should be extended to human embryos to determine whether this mechanism is conserved between mice and humans. Wider implications of the findings The findings of this work indicated the relationship between CuONPs and epigenetic modification. The results will support a more thorough understanding into the biosafety of CuONPs, and offer fresh perspectives on the potential uses of nanomaterials in the field of reproductive medicine in the future. Trial registration number Not Applicable

P-516 Minimizing mosaicism: impact of trophectoderm (TE) biopsy specimen size on mosaic blastocysts in preimplantation genetic testing for aneuploidy (PGT-A)

Abstract Study question Is the size of TE biopsy specimens related to the chromosomal status of the blastocysts in PGT-A? Summary answer Smaller biopsy specimens are associated with higher proportion and altered composition of mosaic blastocysts in PGT-A. What is known already Concordance between TE biopsy and inner cell mass or whole embryos is influenced by mosaicism type, degree, biopsy location, number of cells biopsied, and cell loading. Particularly, mosaic embryos and those with structural rearrangements have a higher level of inconsistency. Recent studies have demonstrated the potential reclassification of chaotic embryos as euploid, leading to successful live births. This study aimed to investigate the potential influence of TE biopsy specimen size on the chromosomal status of blastocysts undergoing PGT-A. Study design, size, duration This retrospective study, conducted from October, 2023, to January, 2024, included 246 blastocysts from patients undergoing TE biopsy and PGT-A at the blastocyst stage on days 5, 6, or 7. Next-generation sequencing (NGS) was used to obtain PGT-A results, categorizing blastocysts into euploid, aneuploid, and mosaic based on their genetic composition. Participants/materials, setting, methods The biopsy specimen area was measured using the Image J program. Mosaic blastocysts were categorized into either a chaotic mosaic group or another group, which included blastocysts with fewer than three chromosomal aneuploidies and segmental mosaicism. Further analysis was conducted by classifying blastocysts into high-level mosaic (>30% aneuploidy) and low-level mosaic (≤30% aneuploidy) groups. Group differences were analyzed using t-tests and chi-square, with a significance threshold set at p<0.05. Main results and the role of chance In the analysis of expanded mosaic blastocysts, the chaotic mosaic group had smaller biopsy specimens than those in the comparison group (p<0.05). The high-level mosaic group also had significantly smaller biopsy specimens than the low-level mosaic group (p<0.05). When categorizing the biopsied sample size into two groups—below 1350 µM2 (n = 98) and above 1500 µM2 (n = 97)—the rates of mosaic embryos were 37% (36/98) and 30% (29/97), respectively, showing a numerical difference without statistical significance. These findings suggest a correlation between the size of biopsy specimens and the chromosomal status of mosaic blastocysts, particularly indicating that a smaller sample size is associated with an elevated chance of mosaic embryos showing chaotic or high-level mosaic characteristics. Limitations, reasons for caution The biopsy specimens were photographed, and their areas were measured using the Image J program, lacking a comprehensive representation of the three-dimensional sample morphology. This constraint could potentially affect the accuracy and in-depth understanding of the spatial characteristics of the biopsy specimens. Wider implications of the findings The study emphasizes the crucial role of optimizing biopsy specimen size in PGT-A. The identified correlation with the chromosomal status in mosaic embryos, especially the association with chaotic or high-level mosaic embryo outcomes in smaller samples, highlights the need for careful size considerations. Trial registration number not applicable

O-154 Chromosomal, gestational, and neonatal outcomes of mosaic embryos: analysis of 3074 cases from the international registry of mosaic embryo

Abstract Study question Which are the clinical outcomes and post-natal results of mosaic embryos with low and high-level of mosaicism? Summary answer The level of mosaicism negatively influences implantation and ongoing pregnancy rate. 6/7 cases in which mosaicism persisted in the foetuses were from low-level mosaic. What is known already Chromosomal mosaic embryos are characterized by the presence of chromosomally different cell lines within the same embryo. While the transfer of these embryos is now offered as an option for women who undergo in vitro fertilization (IVF), several concerns remain. For instance, the limited data on pregnancy outcome and the possibility that intra-biopsy mosaicism in the TE is a poor predictor of the ploidy status of the ICM. Therefore, some argue that mosaicism should be not reported until a clear classification of such embryos in relation to their reproductive potential has been defined. Study design, size, duration We collected the clinical outcomes of 3074 mosaic embryos transferred in women who underwent IVF between May 2019-May 2023. All embryos were cultured to blastocyst stage; trophectoderm (TE) biopsy was performed on Day-5 of development or Day6/7 for slow-growing embryos. The clinical outcome obtained after the transfer of mosaic embryos with the different chromosomal constitutions was compared with each other. Prenatal and post-natal outcomes were collected for available cases. Participants/materials, setting, methods Preimplantation genetic testing (PGT) was performed using high-resolution next-generation sequencing (NGS) methodology. TE biopsies were classified as mosaic if they had 20%-80% abnormal cells. For statistical analysis, mosaic embryos were divided into groups based on mosaic levels and chromosomal constitution detected in TE: single mosaic aneuploidy (monosomy/trisomy; SM), double mosaic chromosomes (monosomy/trisomy or combination, DM), complex mosaic aneuploidy (>2 different aneuploidies; CM) and mosaic segmental aneuploidy (single and double deletion/insertion >5Mb, MS). Main results and the role of chance Embryos classified as ‘low-mosaic’ by NGS-based PGT-A have a higher likelihood of achieving implantation compared to ‘high-mosaic’ embryos (48% vs. 39.%; p < 0.05 ), as well as ongoing pregnancy/live birth (40% vs. 29%; p < 0.05). Chromosomal composition of mosaicism abnormalities dictates the success rate of mosaic embryo transfers, with low and high segmental mosaics being preferable over low- and high-mosaics involving whole chromosomes. For 621/670 pregnancies, parental tests and post-natal data were available. The majority (99.75%) of the babies were largely healthy by routine physical inspection by neonatologists (no gross abnormalities in babies from mosaic embryos, n = 495). A combination of NIPT, CVS, and Amniocentesis prenatal testing results were collected for 552 pregnancies of mosaic embryo transfers, with predominantly normal findings. The mosaicism detected at the embryonic stage by PGT-A was reflected in prenatal testing in only 7 out of 552 pregnancies (0.9%), in which the mosaicism identified with PGT-A at the blastocyst stage was reflected in gestation by prenatal chromosomal testing as true fetal mosaicism. Limitations, reasons for caution Additional clinical data must be obtained to evaluate the contribution of each different chromosome before this approach can be evaluated as an additional tool to choose mosaic embryos for transfer. Wider implications of the findings The international registry of mosaic embryo transfers continues to grow in sample size, in turn increasing the power of analysis. The findings of the mosaic embryo transfer registry can help educate the management and selection of embryos in the clinic. Trial registration number no

P-599 Oestradiol/Progesterone ratio Impact on Frozen Embryo Transfer (FET) and pregnancy outcomes

Abstract Study question Are oestradiol (E2) levels and oestradiol/progesterone (P4) ratio related to higher implantation rates and reduced risk of miscarriage? Summary answer We concluded that higher E2 levels and an increased E2/P4 ratio on the day before FET were associated with lower implantation rates. What is known already A progesterone threshold < 9.2 ng/ml on the day of FET has been related to lower rates of clinical pregnancy. Progesterone is responsible for increasing endometrial vascularization and exerting an immunomodulator effect that allows blastocyst implantation. How oestradiol affects implantation is not well described yet. An appropriate luteal phase support is essential to synchronize the endometrium, ensure proper implantation and reduce the risk of abortion. However, no studies exist regarding E2 levels and E2/P4 ratios related to pregnancy outcomes in the first weeks of pregnancy. Study design, size, duration This is a prospective cohort study. From February 2023 to July 2023, 35 patients who underwent IVF in a private ART medical center were enrolled. Only euploid embryos after PGT- A were transferred and only the first single FET per patient was included. All embryos were transferred in blastocyst stage. Mosaic embryos, fresh cycles, patients with no PGT-A, miscarriages beyond 10 weeks and chemical pregnancies (no evidence of gestational sac) were excluded. Participants/materials, setting, methods 35 patients who underwent IVF-ICSI were retrospectively analyzed. Serum oestradiol, serum progesterone and E2/P4 ratios were measured the day before embryo transfer and later in three moments: 15 days after positive BHCG test, when gestational sac was visualized; 10-15 days after visualization of the gestational sac, when positive embryo heart rate was verified, and in any moment of vaginal bleeding until the 10th week of pregnancy. Main results and the role of chance Our study supported the hypothesis that E2/P4 ratio before FET may be an important factor to predict the risk of threatened abortion considering that all patients had P4 levels > 10 ng/ml before FET. Multiple logistic regression revealed that E2 and E2/p4 ratio on the day before FET are variables related to the risk of threat of abortion. The resulting adjusted OR for E2 on the day before FET in patients who suffered a threatened abortion vs patients who did not experience threat of abortion was 1,025 (95% CI: 1,001-1,049), p value 0,041. The OR for E2/p4 ratio before FET in patients with treat of abortion vs those without threat of abortion was 1,235 (95% CI: 1,035-1,472), p value 0,019. ROC curves showed a significant predictive value for E2 levels >220 pg/ml [AUC of 0,88 (95% CI: 0,73-1,0), p value 0,009] and E2/P4 ratio > 23 [AUC 0,91 (95% CI: 0,78-1,0), p < 0,05], the day before FET indicating a higher risk of threat of miscarriage and loss of pregnancy. Multiple logistic regression did not find significant statistical differences, either E2 or E2/P4 ratio, between the two groups of patients in subsequent determinations at 4 and 6 weeks after transfer. Limitations, reasons for caution The main limitation of this study is the sample size with the lack of further serial measurements of oestradiol and p4 in patients that presented threat of abortion/ miscarriage before the 10th week of pregnancy. Wider implications of the findings E2/P4 ratio the day before FET seems to be the most important factor to decrease threat of abortion in patients with P4> 10 ng/ml. Our study may hold predictive value regarding endometrial receptivity and provide clinicians a guide to individualise FET protocols and to prevent early pregnancy losses Trial registration number N/A

P-505 Clinical rescue rate of abnormally fertilized oocytes and the relationship with patient, embryo and laboratory features

Abstract Study question Which are the main patient, embryonic and laboratory variables associated with a diploid result in abnormally fertilized embryos? Summary answer Embryo diploid constitution is associated with maternal age, trophectoderm and inner cell mass quality as well as pronuclear (PN) number predicted by time-lapse incubator. What is known already In conventional in vitro fertilization cycles, morphological PN evaluation can identify proper fertilization and predict embryo ploidy constitution. Atypical PN patterns like 0PN, 1PN or more than 2PN are associated with ploidy abnormalities. However, we have previously shown that PN number is insufficient to predict embryo ploidy composition, and advanced single nucleotide polymorphism (SNP)-based methodologies for preimplantation genetic testing (PGT) detect a significant number of genetically diploid embryos from presumed abnormally fertilized oocytes. In this study, we characterized the diploid rescue rate associated with abnormal PN configurations, uncovering the link between ploidy results and relevant patient, embryonic and clinical/laboratory parameters. Study design, size, duration Retrospective observational study evaluating the association between diploid rescue rate of abnormally fertilized embryos and different patient, embryonic and laboratory features. Embryos were cultured in conventional or EmbryoScope™ time-lapse incubator (Vitrolife) until blastocyst stage and classified according to Gardner embryo grading system. Ploidy assessment was performed on 343 euploid trophectoderm (TE) biopsies collected between January 2021 and November 2023. Results were stratified according to patients age, embryo quality parameters and different laboratory procedures. Participants/materials, setting, methods Multi-center international study involving consenting patients using autologous gametes and undergoing PGT for aneuploidies (PGT-A) combined with ploidy analysis. Ploidy protocol was performed using a validated targeted NGS approach composed of a custom panel of 357 SNPs and a proprietary pipeline of analysis. Reliable ploidy classification was obtained using a combined algorithm strategy based on SNPs allele frequencies. Categorical variables were expressed in percentages. Nominal variables were analysed using the Cochran-Armitage test to detect trends. Main results and the role of chance Among 343 euploid embryos, diploidy rates obtained for 0PN, 1PN, 2.1PN and 3PN groups were 86.8% (n = 118/136;95%CI=79.89-91.96) 43.7% (n = 59/135;95%CI=35.19-52.50), 65.0% (n = 13/20;95%CI=40.78-84.61) and 26.9% (n = 14/52;95%CI=15.57-41.02) respectively. Regarding patient parameters, advanced maternal age, but not paternal age, was significantly associated with lower diploid rescue rates, with a decreasing trend from 72.5% (n = 79/109;95%CI=63.10-80.60) below age 35 to 42.9% (n = 30/70;95%CI=31.09-55.25) above age 40 (p < 0.01). When considering embryo morphological evaluations, Cochran Armitage test revealed a significant link between diploidy rates and TE quality with a decreasing trend going from 72.1% (n = 80/111;95%CI=62.76-80.17) to 52.7% (n = 79/150;95%CI=44.36-60.87) and 45.8% (n = 22/48;95%CI=31.37-60.83) for embryos classified with TE grade as A, B, and C, respectively (p-value<0.01). Similarly, a decreasing trend was reported in relation to inner cell mass (ICM) quality, when moving from grade A (67,1%, n = 94/140;95%CI=58.23-75.33) to grade C (54.8%, n = 17/31;95%CI=36.03-72.68) (p = 0.03). No clear association was reported for embryo expansion degree. Finally, among laboratory variables (oocyte freezing procedure, fertilization method, incubator type), only time-lapse significantly impacted expected diploid results. Overall, if abnormal PN number is confirmed by time-lapse (n = 42 samples), the accuracy in predicting ploidy abnormalities increased from 29.2% (n = 82/281;95%CI=23.93-34.87), in conventional incubators, to 83.3% (n = 35/42;95%CI=68.64-93.03) (p < 0.01). Consequently, expected diploid rescue rate decreased from 65.5% (n = 184/281;95%CI=59.60-71.03) to 16.7% (n = 7/42;95%CI=6.97-31.36) (p < 0.01). Limitations, reasons for caution Parental DNA analysis should be included to rule-out the residual risk of uniparental disomy in diploid embryos. The multi-center nature of the study didn’t support a uniform PN annotation, especially for centers lacking time-lapse observation. Future studies should focus on the clinical follow-up after the transfer of rescued euploid-diploid embryos. Wider implications of the findings We believe we reported the first evidence of maternal age effect on ploidy results in the largest dataset of abnormally fertilized embryos. We revealed a link between ploidy constitution and blastocyst morphological quality, providing important information for patient counselling. Time-lapse incubators offer a more accurate identification of abnormally fertilized oocytes. Trial registration number n/a

P-797 Assessment of embryo development following fertilization with de novo male gametes generated in a three-dimensional culture

Abstract Study question Can de novo male gamete from embryonic stem cell contribute to the full pre-implantation development? Summary answer Gametes generated after 29 days of differentiation in our three-dimensional (3D) culture system can support consistent fertilization and normal embryo development to the blastocyst stage. What is known already In regenerative medicine, several 3D culture systems have been proposed and are capable of producing functional tissue implants. In reproductive biology, recent studies have reported preliminary success in generating functional de novo gametes through soft-agar culture and testicular organoids from mouse embryonic stem cells. However, in order to achieve successful fertilization and satisfactory embryo development, an heterologous transplantation in a host seminiferous tubule is required to allow proper spermiogenesis to obtain functional gametes. Here we attempt to perform neogametogensis in a novel 3D niche to generate de novo gamete ready to be used for ICSI. Study design, size, duration Mouse ESCs were first cultured on a gelatin coated 6-well plate with fibroblasts in monolayer and later spherified using sodium alginate. Spheres were submerged in specifically designed conditioned media to encourage differentiation of the mESCs into germ-like cells. Over the course of differentiation, cells were assessed for germ cell differentiation biomarkers. Considering that a normal spermatogenesis occur in 30 days, utilization of the de novo gametes was planned for day-15, 22, 29 and 36. Participants/materials, setting, methods Mouse ESCs were differentiated by submerging the spheres in EpiLC medium containing Activin A, bFGF and KSR for 3 days followed by PGCLC medium containing BMP4, LIF, SCF and EGF for up to 36 days. Differentiation was assessed for markers DAZL (spermatogonium), VASA (spermatocyte), BOULE (post-meiotic stage) and acrosin (spermatid). Differentiated cells were then injected into oocytes and activated by calcium ionophore. Embryo development was monitored in a time-lapse incubator. Main results and the role of chance The evaluation of germline-specific markers through immunofluorescence revealed consistent levels of Vasa expression, a ubiquitous germline marker, with percentages at 15% on day-3, 18% on day-10, 17% on day-15, 19% on day-22, 20% on day-29, and 13% on day-36. Dazl, a specific marker for spermatogonia, exhibited a peak expression of 45% on day-10 in spherified cells. Boule, expressed by secondary spermatocytes, reached its highest level of 10% on day-15 but progressively decreased to 8% on day-22. Acrosin, a marker for spermatids, initially manifested at day-10 at 2% positivity and increased to 5% on day-15, reaching 7% on day-22, and peaked at 20% on day-29. According to spermatogenic marker expression, the neogametes follows a maturational profile similar to in vivo mouse spermatogenesis. To prove neogametes competence, we inject the neogametes into mature oocytes. The control ICSI cohort achieved 89.2% fertilization and 77.8% blastocyst rates. Neogametes generated on day-15, 22, 29 and 36, achieved fertilization rates of 35.0%, 61.1%, 81.8% and 75.0%, respectively, and yielded blastulation rates of 5.0%, 16.7%, 36.4% and 8.3%, respectively. Resulting embryos had morphokinesis comparable to control up to the 8-cell stage, but a delay was observed from compaction to blastocyst hatching. Limitations, reasons for caution In spite of the ability to fertilize normally and support blastulation, efficiency rate remained suboptimal. More importantly, the ability to generate live offspring still has to be proven. Wider implications of the findings This novel 3D differentiation model is capable of generating competent gametes and obviate the need for allo-/xeno-geneic transplantation. Once reproducibility and ability to obtain healthy offspring are confirmed, this method may represent a novel treatment option for azoospermic men Trial registration number N/A

P-166 An artificial intelligence-powered tool to score fresh donor oocytes and predict blastulation: interim analysis of a prospective investigation conducted at 3 laboratories

Abstract Study question Is the Magenta-score associated with fresh donor oocytes’ blastulation competence, and can it be used to predict blastocyst rates per cohort of inseminated oocytes (BRpC)? Summary answer Developmentally-competent fresh donor oocytes showed significantly higher Magenta-score, although the number of blastocysts obtained was within the predicted range in only 63% of the cycles. What is known already Currently, many couples undergo donor oocyte treatments. However, despite donors being theoretically fertile, not all oocytes are developmentally competent, and many do not reach the blastocyst stage. Even if subjective and ineffective to predict oocyte competence, visual oocyte morphological assessment is still routinely used to evaluate oocytes. Lately, the rise of artificial intelligence (AI) resulted in promising tools to assess oocytes more objectively in a standardized method, possibly providing novel insights into the prediction of their competence. In this context, Magenta-score (Future Fertility), an AI-based image analysis tool, can potentially prove useful to optimize the management of oocyte donation treatments. Study design, size, duration A Blinded cohort study (June-2023/December-2023) was conducted to assess the primary outcome: association between fresh donor-oocytes’ Magenta-Score and their subsequent blastocyst development (assumed to be 50%). A sample size of 779 achieves 80%-power to detect the difference between the null-hypothesis point-biserial-correlation of 0 and the alternative hypothesis of 0.1 using a two-sided test with 5%-significance. Secondary outcomes were differences (i) predicted - true BRpCs, and (ii) predicted - true numbers of blastocysts obtained. Participants/materials, setting, methods Interim-analysis of 514 fresh-oocytes obtained from 63 donors at 3 centers and allocated (7.7±3, range:3-18) to 67 recipients. Pictures of denuded mature oocytes were acquired before ICSI. Magenta-scores were generated blindly (providing only donor age and total number of mature oocytes in cohort to the software). The software also estimated BRpCs and range of blastocysts obtainable from each cohort. Donors’ characteristics, ovarian stimulation/treatment cycle parameters, and sperm analyses were tested as confounders. Main results and the role of chance The only visual oocyte anomaly associated with lower Magenta-score was irregular shape (N = 484,5.4±2.2 vs N = 30,3.3±2.1, Mann-Whitney-U<0.01). Oocytes that developed into blastocysts (N = 194) had significantly higher Magenta-scores versus oocytes that did not reach this milestone (N = 320) (5.8±2.1 vs 4.9±2.3, Mann-Whitney-U<0.01; Odds-Ratio adjusted for sperm motility and incubator [standard/time-lapse]=1.26, 95%CI:1.13-1.34, p < 0.01; power=99.6%). No association was noted with day of blastulation or morphological quality. Data were concordant across centers. The average predicted-BRpC was 39.4±11.4% versus a true-BRpC of 40.6±22.6% (Person’s correlation: -0.24; mean difference: -1.1±27.6%, 95%CI from -7.8% to + 5.6%, p = 0.74). The average predicted number of blastocysts per oocyte cohort was 3.3±1.6 versus a true average of 2.9±1.7 (Person’s correlation: 0.25; mean difference: +0.4±2.0, 95%CI from -0.04 to + 0.9, p = 0.07). In 21% (N = 14) and 31% (N = 21) of cycles, the tool predicted an equal or lower number of blastocysts, respectively. In 5 cycles, no blastocyst was obtained, but the tool did not predict this unexpected outcome. When testing the predicted range of blastocysts obtained, the true number was within the range in 63% (N = 42) of cases and higher than the maximum predicted number in 13% (N = 9) of cases. No confounder was identified on the BRpC in this population of fresh donor cycles. Limitations, reasons for caution Of note, this is an interim analysis of an ongoing study. Not all mature oocytes recovered from a donor were utilized, thus not all oocytes in a cohort were imaged and included in analysis. Other putative confounders on BRpC should be considered in future studies with a larger sample size. Wider implications of the findings Effective management of an oocyte donation program is critical to comply with expected high success rates while minimizing the number of surplus blastocysts produced. The integration of genomic data, recipient couples’ characteristics, and the benefits of objective standardized AI-powered oocyte scoring is the most promising workflow to optimize this task. Trial registration number Not applicable

O-148 Does presence of endometriosis adversely affect oocyte morphology? Evaluation of a large number of oocytes obtained from endometriosis patients, 27204 oocytes

Abstract Study question Does presence of endometriosis adversely affect oocyte morphology? Evaluation of a large number of oocytes obtained from endometriosis patients, 27204 oocytes Summary answer In the presence of endometriosis, oocyte morphology is not impaired. The rate of reaching blastocyst and fertiilization rate, also pregnancy outcomes are not affected negatively. What is known already Although there are hypothesis, theories that altered steroidogenesis and folliculogenesis, higher oxidative stress, reactive oxygen species, altered cell cycle progression, inflammation and angiogenesis in the follicular environment exposes oocytes to a hostile inflammatory environment and alters oocyte quality. There has been much debate and conflicting evidence as to whether the poorer IVF outcomes in women with endometriosis is related to altered oocyte quality. There is some evidence to suggest that impaired oocyte morphology in women with endometriosis may have an adverse impact on fertilization rate, however, most studies have shown that there is no difference in pregnancy outcomes following IVF. Study design, size, duration This retrospective, single center study evaluated 29130 ART cycles from August 2011 to March 2023,based on data obtained from Istanbul Memorial Hospital,ART and Reproductive Genetics Center.Study group included endometriosis patients(n = 4602 cycles, 27204 oocytes) and control group included non-endometriosis patients(n = 24528 cycles, 178774 oocytes).We analyzed demographic and cycle characteristics, oocyte morphology in ART cycles between the two groups.Futhermore, we compared pregnancy outcomes in frozen-thawed embryo transfer(FET) cycles (total number:11116 FET cycles; endometriosis group:2255 cycles,non-endometriosis group:8861 cycles). Participants/materials, setting, methods Patients diagnosed with endometrioma by ultrasound, diagnosed with endometriosis by laparoscopy or patients who underwent endometrioma surgery or adenomyosis detected on ultrasound were included in the study (endometriosis) group. In the control group, the patients without endometriosis were included. Mann Whitney U test and Pearson Chi-square test used. Cliff's Delta effect size (????) for non-parametric tests and Phi effect size (p) for categorical data were reported. Main results and the role of chance The sample was very large, so the statistical results were given in terms of effect size, not only p value calculated. Female age was similar. Female body mass index, number of previous cycles, duration of infertility, AMH, total gonadotropin dosage used, duration of ovarian stimulation, estradiol level on trigger day, number of aspirated oocytes, mature and fertilized oocytes, maturation and fertilization rate, rate of blastulation, rate of usable blastocyst (top and good quality), number of embryos transferred, blastocyst stage embryo transfer cycles were statistically different between the two groups (p < 0.001). However when the effect size examined, all variables were found to have a negligible association by Cliff's Delta or Phi effect size calculations. Oocytes obtained from endometriosis patients had statistically significantly higher severe central granulation, large perivitellin space, thick zona, polar body defect abnormalities compared to non-endometriosis patients (p < 0.001), Phi effect size showed negligible association for all variables. In endometriosis group compared to non endometriosis group; biochemical pregnancy (68.9% vs 72.2%, p:0.002, Phi:0.030), clinical pregnancy (61.5% vs 64.5%, p:0.007, Phi:0.025), total pregnancy loss (22% vs 24.4%, p:0.05, Phi:0.022) were statistically higher, but Phi effect sizes were negligible. Live birth were similar (52.5% vs 53.2 P:0.56) between the two groups. Limitations, reasons for caution The principal limitation of the study is retrospective design of the analysis. But the strength of the study is that included 27204 oocytes from endometriosis patients. Wider implications of the findings To our knowledge, this study includes the largest case group that investigates the endometriosis and oocyte morphology. The results show that endometriosis does not have a negative impact on oocyte morphology. Additionally, it has been shown that the presence of endometriosis does not have a negative effect on pregnancy outcomes. Trial registration number *

P-019 Impact of severely altered sperm parameters on ICSI outcomes: A retrospective analysis of embryo morphokinetics and cumulative clinical outcomes of 10,622 embryos

Abstract Study question How do severely altered parameters in both ejaculated and surgically retrieved testicular sperm affect preimplantation embryo morphokinetics, blastocyst quality and cumulative clinical outcomes? Summary answer Severely altered semen parameters impaired fertilization, embryo morphokinetics, and blastocyst quality, but only the use of testicular sperm reduced cumulative outcomes, including live birth rates. What is known already Semen quality is a significant determinant of reproductive success. Sperm play a crucial role in embryonic genome activation and facilitate embryo progression to the blastocyst stage. Yet, comprehensive analyses of embryo developmental outcomes in cases of male factor infertility remain scarce. Limited by small study cohorts, inconsistent findings and a lack of cumulative outcomes, existing studies have failed to provide a clear understanding of the differential impacts of altered semen parameters on embryo development. Uncertainty about outcomes following the use of poor-quality or testicular sperm complicates clinical management, ultimately leaving patients without comprehensive information to make informed treatment decisions. Study design, size, duration This retrospective study evaluated 10,622 embryos derived from 1,680 patients, across 1,837 ICSI cycles. We included both autologous (n = 429) and donor oocyte (n = 1,407) cycles, performed at a single IVF center between January 2019 and August 2023. To assess differences in critical aspects of reproductive success, we compared various embryo developmental and clinical outcomes across cycles using sperm with severely altered parameters (either ejaculated or obtained through testicular sperm extraction, TESE) to those using normospermic samples. Participants/materials, setting, methods Embryos were distributed into four groups according to sperm quality and origin, as per WHO guidelines: normospermia (n = 8,798), severe oligospermia (<5 million/ml; n = 567), severe oligoastenospermia (<5 million/ml and progressive motility <32%; n = 716) and TESE (n = 541). We assessed differences in embryo morphokinetics using adjusted Cox survival curves. We used multivariate analyses to evaluate associations between sperm groups and clinical outcomes, adjusting for relevant confounders pertaining to patient demographics and treatment characteristics. P-values <0.05 were considered significant. Main results and the role of chance Compared to normospermia, altered semen parameters resulted in lower mean fertilization rates (74.5% normospermia versus 69.7% severe oligospermia, 69.1% severe oligoastenospermia and 60.6% TESE). While oligospermia and oligoastenospermia showed a negative correlation with fertilization rates (R2=-0.05, p = 0.02 and R2=-0.07, p < 0.0001, respectively), this trend was more pronounced for TESE (R2= -0.12, p < 0.0001). Embryo morphokinetics were comparable for normospermia, oligospermia and oligoastenospermia. However, TESE resulted in faster early morphokinetic patterns (tPNf, t2, t3, t4 and t5; p < 0.05), and similar t8, tsB, tB, with comparable cell cycle durations (s2, s3, cc2 and cc3; p > 0.05). Compared to normospermia, severe oligospermia had no effect on inner cell mass (ICM) (OR = 0.93, 95%CI: 0.72-1.21) nor trophectoderm (TE) quality (OR = 0.79, 95%CI: 0.58-1.06), while oligoastenospermia reduced the odds of having a good quality ICM (OR = 0.75, 95%CI: 0.58-0.97) but showed no effect on TE (OR = 0.94, 95%CI: 0.71-1.23). Notably, TESE had the most profound effect on blastocyst quality (ICM, OR = 0.68, 95%CI: 0.49-0.92; TE, OR = 0.68, 95%CI: 0.46-0.98). Following the first embryo transfer, ongoing pregnancy rates were similar across all groups, apart from TESE (R2=-0.217, p = 0.0160). Unlike the other groups (p > 0.05), TESE also led to decreased cumulative implantation, ongoing pregnancy, and live birth rates compared to normospermia (R2=-0.19, p = 0.03; R2=-0.23, p = 0.009; R2=-0.22, p = 0.019, respectively). Limitations, reasons for caution The primary limitation of this study is its retrospective design, which may not account for all confounders. Moreover, lifestyle factors, such as smoking or alcohol consumption were not considered. The reliance on data from a single IVF center may also restrict the generalizability of the results to a broader population. Wider implications of the findings Our findings underscore the imperative need for careful consideration of sperm quality for ICSI, highlighting the distinct challenges presented by TESE sperm for achieving reproductive success. While altered semen parameters compromised fertilization rates and embryo quality, TESE cycles were markedly less efficient, ultimately leading to reduced cumulative live birth rates. Trial registration number not applicable

O-058 Newly identified female mutations causing oocyte maturation defect and embryo developmental arrest

Abstract Female infertility is a complex issue affecting a significant number of women. In the United States, among married women aged 15–49 years with no prior births, approximately one in five (19%) were unable to get pregnant after one year of trying. In addition, approximately one in four (26%) women in this group had difficulty getting pregnant or carrying a pregnancy to term. For women under 35 years of age, infertility is defined as a lack of success within one year, while for women aged 35 and older, it is within six months. Various factors, such as genetic, endocrine, physiological, anatomical, and immunological irregularities within the reproductive system, can affect a woman's chances of achieving pregnancy and delivering a healthy child. Among these factors, oocyte maturation is an important prerequisite for successful fertilization and subsequent embryonic development. In Medically Assisted Reproduction (MAR), the meiotic maturation of oocytes is induced by human chorionic gonadotropin (hCG) injection, which mimics the natural endogenous luteinizing hormone (LH) surge during the menstrual cycle. Although it is common for a few oocytes to remain immature despite ovarian stimulation and hCG administration, generally, immature oocytes have the competency to spontaneously mature in vitro. However, oocyte maturation is a sophisticated process involving multiple genes or protein molecules. Therefore, pathogenic variations in the genomic sequence or functional defects in any molecule that affects meiosis may lead to impaired oocyte maturation. While complete failure of all oocytes to mature in vivo is extremely rare, understanding the molecular aetiology of oocyte maturation is crucial for addressing infertility issues related to this process and improving fertility outcomes. The transition from the cleavage to the blastocyst stage often represents a significant bottleneck, with nearly 10% of fertilized eggs arrested at the cleavage stages. Developmental arrest of the preimplantation embryo is characterized by the cessation of cellular division for at least 24 h. Various factors originating from both embryonic and parental sources contribute to arrest of early stage embryo development. The genetic roots of developmental arrest were examined by considering both the parental causes of infertility and factors within the embryo. Examination revealed shared elements, regardless of the source of origin. Chromosomal abnormalities, abnormal preimplantation development, and monogenic variations have been reported to cause embryonic developmental arrests. Recently, deficiencies in oocyte maturation and embryo development have been categorized as part of the oocyte/zygote/embryo arrest (OZEMA) phenotype in the Online Mendelian Inheritance in Man (OMIM) database. A latest extensive review of monogenic causes associated with female infertility identified 23 genes with a total of 27 gene disease relationships (GDRs) for the OZEMA phenotype. Seventeen GDRs were classified as having at least moderate evidence of involvement in OZEMA, in addition to ten GDRs with less substantial evidence. Several additional genes were subsequently identified. This presentation will provide an overview of gene defects linked to the OZEMA phenotype, discuss the current status and future prospects of pre-MAR screening of females with OZEMA, and assess a novel workflow for modern genetic diagnosis to maximize the benefit of the patient.

O-171 Apicobasal RNA asymmetries regulate cell fate in the early mouse embryo

Abstract Assessing the morphological appearance of an embryo as it progresses from zygote to blastocyst is the primary method used by embryologists to evaluate embryo viability and developmental potential. Standard embryo grading techniques, using time-lapse microscopy, analyse morphological characteristics such as size, shape, number, and appearance of blastomeres. The aim of this study was to determine how individual cells of the embryo function on a subcellular level to build a healthy embryo. RNA localisation plays indispensable roles in establishing asymmetries and coordinating cell fate decisions during early embryogenesis across various non-mammalian species. To direct the spatiotemporal distribution of RNA within the cells of an embryo, the microtubule cytoskeleton provides highly sophisticated trafficking pathways. Yet, it remains unknown whether subcellular heterogeneities exist during mammalian preimplantation development and how they contribute to cell fate determination. Using advanced live imaging, we visualised global RNA transcripts at high spatiotemporal resolution from fertilisation to the blastocyst stage in living preimplantation mouse embryos. We discovered apicobasal RNA asymmetries specific to outer cells of the 16-cell stage mouse embryo, coinciding with cell fate decisions and the emergence of the pluripotent inner cell mass. Highly clustered RNAs accumulated proximal to the basal membrane, while more dispersed RNA foci were identified apically as the embryo reaches the late 16-cell stage. The targeted distribution of membrane-less RNA molecules was facilitated by the microtubule cytoskeleton, associated with lysosomes which serve as RNA transport vehicles. We discovered that apically located RNA foci were more dynamic and accompanied an enrichment of translation components. Furthermore, we uncovered that the established RNA asymmetries enhanced translation capacity in apical regions compared to basal regions. These spatiotemporal RNA heterogeneities were unevenly inherited by outer and inner daughter cells during subsequent cell divisions. Notably, embryos that were unable to establish RNA asymmetries failed to allocate cells to the trophectoderm and inner cell mass. Our study provides insights into a subcellular mechanism driving asymmetric RNA localisation and compartmentalised translational regulation in outer cells of the 16-cell stage embryo, which may contribute to cell fate decisions during mammalian preimplantation embryogenesis. We envision that when paired with classical morphological assessment criteria of the embryo, these findings may assist with selecting the embryo with the highest developmental potential.

P-202 Impact of multinucleation at the two-cell stage on pregnancy outcomes: a day 3 vs. day 5 transfer analysis

Abstract Study question Does multinucleation at the two-cell stage significantly affect pregnancy outcomes, and how does it differ between day 3 and day 5 embryo transfers? Summary answer Multinucleation at the two-cell stage has a negative effect on pregnancy outcomes in day 3 embryo transfers, but not in day 5 transfers. What is known already Time-lapse imaging provides a valuable tool to study multinucleation in human embryos, although the impact on development and pregnancy remains debated. Literature shows conflicting findings, with most studies associating multinucleation in the early stage embryo with altered developmental potential and decreased pregnancy outcomes. However, other studies suggest a positive correlation, indicating that multinucleated embryos can progress successfully through development and lead to positive pregnancy outcomes. These discrepancies underscore the complexity of and highlight the need for understanding the implications of multinucleation in the context of human embryo development and pregnancy. Study design, size, duration A non-interventional retrospective study on multinucleation was performed on all cycles cultured in a time-lapse incubator in a Dutch fertility clinic between November 2021 and July 2023. In total, 915 embryos (out of 406 cycles) were analyzed and 406 resulting transfers (both fresh and frozen-thawed) were included in this study. Participants/materials, setting, methods Fresh single-embryo transfer was performed on day 3 (for cycles with less than 4 2PN-embryos) or day 5 (for cycles with 4 or more 2PN-embryos). Embryos were vitrified on day 5 or 6 (at least B3BB Gardner score). Nucleation status at the two cell stage was annotated for all transferred and vitrified embryos. Ongoing pregnancies were compared for embryos with or without multinucleation (mononucleation) at the two cell stage. Main results and the role of chance Multinucleation at the two cell stage was observed in 40% (n = 366/915) of all embryos. Multinucleated embryos transferred on day 3 showed a decreased ongoing pregnancy result (17%; n = 9/54) compared to the mononucleated day 3 transfers (29%; n = 24/83). For day 5 transfers, no difference in ongoing pregnancy rates was observed between the multinucleated (37%; n = 37/101) and mononucleated (37%; n = 62/168) groups. These results indicate that development to the blastocyst stage negates any possible negative effect multinucleation can have on cleavage stage embryos. Limitations, reasons for caution A mayor limitation to this study is the small sample size of specifically day 3 transfers. Patients with more than one transfer were allowed to be included. Confounding factors were not assessed. Additionally, different types of multinucleation might result in different clinical effects. Wider implications of the findings Although additional research is needed, these results suggest that assessment of nuclear status at the two-cell stage is important for day 3 transfer strategies, but less so for day 5 blastocyst transfers. Trial registration number Not applicable

P-252 Non-invasive artificial intelligence (AI) prediction of blastocyst development from mature oocytes unveils correlation with blastocyst ploidy for enhanced reproductive insight at earliest stage of development

Abstract Study question Can MAGENTA identify mature oocytes more likely to develop into a blastocyst of euploid status? Summary answer Although built to predict blastocyst development, MAGENTA analysis of mature oocytes additionally displays the ability to highlight those with higher potential to become euploid embryos. What is known already MAGENTA (AI image-analysis tool) has been trained to assess and predict blastocyst stage embryo development from mature, denuded oocytes. MAGENTA scores consistently identify oocytes of better quality – more likely to develop into a blastocyst, including blastocysts of higher morphological quality. However, in addition to achieving a blastocyst, the ploidy status of that embryo is crucial in dictating overall chances of implantation and cycle success. Although MAGENTA was not trained to predict blastocyst ploidy from oocytes, most chromosomal errors are of maternal meiotic origin. A correlation between MAGENTA and ploidy status would ignite research possibilities and provide valuable insights. Study design, size, duration A retrospective dataset of 1,324 blastocyst embryos that underwent PGT-A via NGS sequencing, between the years 2021-2023, was assessed. All embryos that were tested for ploidy from a patient cohort were included. Embryos reported as mosaic or ‘no diagnosis’ were excluded from analysis (N = 169). All embryos were cultured in a time-lapse incubator immediately post-ICSI. Of the biopsied blastocysts, 81% were of good morphological quality (expansion grade 1-6, ICM and TE grade A/B). Participants/materials, setting, methods The data includes 292 patients with a mean age of 38.5 (range 19-46) and an average number of tested blastocysts of 4 (range 1-14). Of the 1173 blastocysts, 847 were aneuploid (72.2%) and 326 were euploid (27.8%). 83% of the blastocysts were from patients of advanced maternal age (AMA, ≥38years old). The first time-lapse image, capturing the mature oocyte stage, was extracted, and scored by MAGENTA (scale 0-10). MAGENTA scores were compared by Welch’s t-tests. Main results and the role of chance Impressively, oocytes that developed into euploid blastocysts (n = 326) were identified by MAGENTA with a higher average score of 6.8 compared to those that developed into aneuploid blastocysts (n = 847) with a score of 6.4; a statistically significant differentiation (p < 0.05). 80% of the dataset were high quality blastocysts (expansion 4-6, ICM and TE grade A/B) with an imbalance of euploid (n = 295; mean score 6.8) and aneuploid (n = 643; mean score 6.6) blastocysts—likely a result of AMA. Therefore, subgroup analysis was focused on assessing the ploidy status relationships amongst several patient age groups within these AMA cases. Due to our clinic’s indication for PGT-A, the smallest subset of oocytes was from patients <38 years of age (N = 373, 48% euploid). In this age group there was no significant difference in MAGENTA scores between the oocytes of euploid and aneuploid embryo status (6.9 vs 6.7; p = 0.522). However, in oocytes from patients aged ≥38 (N = 800, 19% euploid), the distinction between those that became euploid and aneuploid blastocysts is evident with a statistically significant difference in MAGENTA scores (6.8 vs 6.3; p < 0.05). This relationship with ploidy status additionally remains with further stratification into the ≥41 age group (N = 467 oocytes, 13% euploid; 6.7 vs 5.9; p < 0.01). Limitations, reasons for caution Majority of the patients in this dataset underwent PGT-A due to AMA, further research is required to include a wider age demographic, particularly in patients <35 years old. Greater representation of varying blastocyst qualities is required to further assess MAGENTA correlation to ploidy outcomes amongst quality groups. Wider implications of the findings This study presents the first non-invasive assessment of mature oocyte quality that indicates oocytes with greater potential for euploid blastocyst development. This application of MAGENTA provides a novel method of gaining ploidy status potential at the mature oocyte stage, which is invaluable to both IVF and cryopreservation cycles. Trial registration number NOT APPLICABLE

O-030 The impact of aneuploidy on human peri-implantation development: findings from the extended in vitro culture of 191 human embryos

Abstract Study question How does aneuploidy affect lineage specification and the developmental potential of human embryos cultured up to 12 days post fertilization (D12)? Summary answer While distinct types of aberrations carry their own risk of embryonic arrest, prevailing aneuploidies appear to compromise lineage specification beyond the blastocyst stage of development. What is known already Chromosomal instability is a hallmark of human preimplantation development, with up to 50% of embryos diagnosed as aneuploid following preimplantation genetic testing (PGT). Aneuploidies are a major cause of implantation failure, pregnancy loss and human congenital defects. Yet despite their clinical relevance, the impact of distinct aberrations on early human development remains poorly understood. Innovative platforms for culturing human embryos up to D12 provide an important opportunity for investigating chromosomal instability. Understanding the effects of specific aneuploidies on human peri-implantation development will be key for advancing human embryo research and improving the interpretation of findings from in vitro embryo models. Study design, size, duration A total of 191 PGT blastocysts obtained from 103 patients were included in the study. Blastocysts were selected based on their original diagnosis, including embryos with single whole-chromosome aneuploidies (single trisomies, n = 37 and single monosomies, n = 71), single segmental aberrations (n = 22), embryos with complex chromosomal constitutions (two or more abnormalities, n = 44), as well as control euploid embryos (n = 17). Participants/materials, setting, methods Vitrified blastocysts were warmed and cultured up to D12 using an optimized protocol to generate embryo outgrowths. We correlated the original PGT diagnoses to developmental outcomes, including outgrowth viability, morphology and outgrowth size. We evaluated blastocyst quality using the Gardner grading system. Following culture, we immunostained 38 viable outgrowths for lineage-specific markers, including OCT4 (epiblast), GATA6 (hypoblast) and CK-7 (trophoblast). Fisher’s exact test was used for statistical analysis (p < 0.05 was considered significant). Main results and the role of chance Following extended culture, 43.5% of embryos remained viable and attached, while 56.5% degenerated and detached. No significant correlation was found between embryo grade and attachment rate. Euploid embryos exhibited the highest attachment rate, with 88.2% (15/17) forming viable outgrowths. Euploid embryos also generated significantly larger outgrowths than trisomic and monosomic embryos (p = 0.01). Embryos diagnosed with trisomies and segmental aberrations exhibited attachment rates comparable to euploid embryos, at 70.3% (p = 0.18) and 68.2% (p = 0.25), respectively. However, embryos with monosomies and complex chromosomal constitutions arrested more readily (28.2% and 15.9% viable, <0.00001, respectively). All euploid embryos (6/6) retained a discernible OCT4+ epiblast-like structure. This was similar to embryos with segmental aberrations and trisomies, with 75% (3/4) and 53.8% (7/13) of outgrowths containing OCT4+ cells, respectively. Nonetheless, distinct trisomies exerted varying effects on peri-implantation development in vitro. For instance, viable trisomies (13, 18, 21) developed similarly to euploid embryos, while outgrowths carrying a trisomy 22 had fewer OCT4+ cells (p = 0.02) and a higher number of GATA6+ cells (p < 0.01), suggesting a potential lineage proliferation defect. Monosomies had the most profound effects on development. Compared to euploid embryos, monosomic embryos formed significantly fewer OCT4+ outgrowths (28.6%, p = 0.02) and contained fewer epiblast (p < 0.0001) and hypoblast-like (p = 0.003) cells. Limitations, reasons for caution The limited number of embryos in certain groups warrants careful interpretation. The central drawback of the models is that embryo outgrowths are predominately flattened, which confounds identification of three-dimensional structures formed during normal embryogenesis. Future studies should encompass additional lineage specification markers and potential culture refinements to enhance our interpretation. Wider implications of the findings Our findings underscore the critical importance of reporting the chromosomal composition of human embryos utilized for research. This practice will enable a more nuanced interpretation of results from in vitro embryo models and ultimately advance our capabilities in human embryo research. Trial registration number not applicable

P-221 The efficiency of a PGT-A program is negatively impacted by the utilization of frozen-thawed oocytes in patients of all ages

Abstract Study question Are there any differences in the use of fresh or frozen-thawed oocytes in a PGT-A program? Summary answer While euploidy rates are comparable, the efficiency of a PGT program is adversely impacted when utilizing frozen-thawed oocytes. What is known already Oocyte vitrification has become one of the key assisted reproduction procedures. Some studies have reported rates of embryonic development, clinical pregnancy, and embryo implantation comparable between fresh and cryopreserved oocytes in egg donor patients. Vitrified oocyte accumulation before the blastocyst biopsy is a commonly used strategy in PGT programs to enhance the chances of obtaining a euploid blastocyst with the patient’s own oocytes. Further research is needed to assess the impact of frozen-thawed oocytes on aneuploidy and the efficiency of PGT programs. Study design, size, duration We conducted a retrospective comparison of 777 PGT-A cycles, using either fresh or frozen-thawed oocytes, regarding embryo development (fertilization rates, blastocyst formation, and rates of good-quality blastocysts), PGT outcomes (euploidy and mosaicism rates), and the overall efficiency of the PGT cycle, measured as the number of euploid blastocyst per available mature oocytes, between April 2018 and November 2023. The study groups were stratified based on the age of the patients (≤35 and >35 years old). Participants/materials, setting, methods PGT-A cycles were categorized into two groups: patients who utilized fresh oocytes (n = 641) and those who utilized frozen-thawed oocytes (n = 136). A total of 6,370 fresh oocytes and surviving oocytes from the initially vitrified ones (972 out of 1,153) were fertilized and cultured to the blastocyst stage, at which point biopsies were performed. The biopsied cells were subjected to NGS screening, and statistical data analysis was conducted using the Chi-square test, with significance set at P < 0.05. Main results and the role of chance In the group of patients aged ≤35 years old, significant differences were observed when comparing PGT cycles using fresh or frozen-thawed oocytes in terms of the fertilization rate (78.4% vs. 72.0%), blastocyst formation rate (47.4% vs. 37.7%), and the efficiency of the PGT cycle (24.0% vs. 16.0%), corresponding to a 33.3% reduction in efficiency. No differences were observed between fresh and frozen-thawed oocytes regarding euploidy rate (64.6% vs. 68.4%), mosaicism rate (4.9% vs. 5.1%), and the percentage of good-quality embryos (60.0% vs. 56.1%). Similarly, in patients aged >35 years old, fertilization (81.5% vs. 73.5%), blastocyst formation (40.5% vs. 33.4%), and the efficiency of the PGT cycle (11.7% vs. 7.2%), which correspond to a 38.5% reduction in efficiency, were improved when using fresh oocytes. No differences were observed regarding euploidy (35.6% vs. 35.3%) or mosaicism rate (3.8% vs. 5.3%), and the percentage of good-quality embryos (55.2% vs. 54.7%). The oocyte survival rate was 84.3%. Limitations, reasons for caution Our study is limited by its sample size and retrospective design, as well as the inclusion of a non-selected and general infertile population, which could amplify the impact of confounding variables. Wider implications of the findings We found that the utilization of frozen-thawed oocytes reduces the PGT program efficiency by decreasing fertilization and blastocyst formation rates. This suggests that the oocyte accumulation strategy should be approached with caution. However, vitrification did not lead to an increase in the rates of aneuploidy, mosaicism, or poor-quality blastocysts. Trial registration number NOT APPLICABLE