Embryo Aneuploidy Research Articles

O-290 Clinical utility of putative mosaicism detected using concurrent copy-number and genotyping PGT method: outcomes from multisite, prospective, non-selection study including 9828 single embryo transfer cycles

Abstract Study question What is the clinical utility and associated outcomes of mosaic whole chromosome or segmental aneuploidies detected using concurrent copy-number and genotyping analysis in PGT-A cycles? Summary answer Although high-level whole-chromosome mosaicism is linked to reduced sustained implantation, it has limited clinical significance in PGT-A cycles when co-evaluated with other clinical/ embryological factors What is known already NGS-based PGT-A can detect intermediate chromosomal copy number (CN), commonly interpreted as mosaic chromosomal aneuploidies in embryos. A prospective non-selection approach is the most effective way to assess the clinical utility of reporting putative mosaicism findings in PGT-A, wherein the presence of mosaicism is not disclosed and does not influence embryo selection. Conflicting results have been reported previously, possibly due to technological limitations in mosaicism assessment or retrospective analysis methods. This study reports the results of the largest multisite prospective non-selection clinical study examining the predictive value of whole-chromosome and segmental mosaicism, assessed through combined CN and genotyping data analysis. Study design, size, duration A multisite study involving seven IVF clinics was conducted from Feb 2020 to Oct 2022, including 6951 patients and 9828 single embryo transfers. The study involved a prospective non-selection approach, where embryos suspected of having whole chromosomal or segmental mosaicism were reported as negative for non-mosaic aneuploidies. Embryos were chosen for transfer based solely on standard morphological features. The primary outcome was sustained implantation rate (SIR) defined as pregnancy continuing beyond 8 weeks of gestation. Participants/materials, setting, methods In this study, the trophectoderm biopsies were analyzed using a custom, targeted NGS assay that examined approximately 5000 loci across the genome, providing genotyping information to support aneuploidy classification. Mosaicism was identified by any copy number deviation from the expected two copies (LogR plots) and confirmed by corresponding SNP B-allele frequency (BAF) patterns. Confounding factors, such as clinical and embryological variables, were controlled for in the multivariate analysis. Main results and the role of chance The average female age in this cohort was 34.9 years (SD = 4.1), with aneuploidy rate of 30% in embryos and SIR of 61.2%. Of the embryos transferred, 6.5% (636/9828) were whole chromosomal mosaic (WCM) only; 9.6% (947/9828) were segmental mosaic (SM) only and 1% (83/9829) were a combination of WCM and SM. The rate of putative mosaicism ranged from 15%-89%. The SIR of embryos in the control group (non-mosaic), SM and WCM were 62% (5190/8328; 95%CI), 58% (549/947;95%CI) and 50.3% (320/636; 95%CI P < 0.01) respectively. A logistic model found that the level of WCM was associated to SIR, along with other embryological and clinical factors. In particular, WCM with a CN difference >50% as well as poor embryo morphology were associated with lower SIR (OR = 0.5; 95% CI:0.32-0.76), but low-level (<50%) mosaicism was not significant (NS). Notably, female age (OR = 0.98; 95% CI:0.97-0.99 per year), BMI (0.98; 95% CI:0.98-0.99) and previous ET failures (OR = 0.58; 95% CI:0.5-0.68) were strongly associated with SIR. A predictive model, taking into account all relevant variables, yielded a significant stratification of SIR, from 43% to 68%. Given the low incidence of WCM in our clinical setting, the multivariate SIR prediction (AUC) was 0.580 without WCM and 0.585 with mosaicism included. Limitations, reasons for caution This study did not have prenatal and post-natal data available at the time of the abstract's writing, hence conclusions about these outcomes wasn’t possible. Despite the large sample size, chromosome specific analysis was not feasible. Furthermore, this study’s data is platform-specific and cannot be translated to other PGT-A assays. Wider implications of the findings In this non-selection study, WCM of > 50% variation was associated with lower SIR. However, high-level WCM has a minimal overall impact on SIR when co-evaluating with other clinical/ embryological parameters. Decisions on reporting criteria for these findings must weigh the risk of discarding potentially viable embryos with substantial reproductive potential. Trial registration number Not applicable

P-377 Artificial Intelligence (AI) for Pre-implantation Genetic Test for Aneuploidy (PGT-A): important epistemic and ethical considerations

Abstract Study question What are the epistemic and ethical considerations if it's used AI for PGT-A? Summary answer The AI used to date presents significant epistemic and ethical problems and there are no studies evaluating its clinical efficacy. This topic requires more attention. What is known already The introduction of time-lapse systems offers the possibility of generating large amounts of data to evaluate the state of health of embryos. With the introduction of AI and machine learning this data can be analyzed by algorithms that improve automatically as they are exposed to more data. Almost all AI is based on algorithms developed using neural networks. Many algorithms are difficult to understand or even uninterpretable because they are covered by company secrets. Not having access to the algorithms causes problems of an ethical nature and caution is required in its use for the identification of healthy embryos. Study design, size, duration We performed a study of the articles that mention a possible use of AI as a tool to be used within the PGT-A. The epistemic and ethical implications of current approaches have been considered. We also considered the oral communications presented during the last ESHRE annual meeting and the sensationalist communications associated with them. Participants/materials, setting, methods We used PubMed for article search which includes more than 35 million citations for biomedical literature from MEDLINE, life science journals and online books. To carry out a targeted search we used the following keywords: Artificial Intelligence* OR AI OR Neural Network* OR Machine Learning OR Support Vector Machine OR Automated Classification AND IVF OR IVF OR Embryo OR Pre-implantation Genetic OR PGT*. Main results and the role of chance The results obtained were difficult to interpret. We found articles where they looked at 2 types of results: accuracy for predicting euploidy and agreement with the molecular assay assessment. It has been demonstrated that some algorithms are not always able to totally differentiate a euploid from an aneuploid embryo, especially when speaking of segmental aneuploidies. This data lends many concerns. Almost universally, AI models were opaque in that at least part of the process was not accessible. If not fully accessible, these models are problematic for epistemic and ethical reasons. Epistemic concerns include information asymmetries between algorithm developers and all the operators who routinely perform these analyses. There is another risk of biased predictions caused by known and/or unknown confounders during the algorithm auto-improvement process. Furthermore, we must consider the high difficulty in checking for any errors in real time due to the total non-accessibility. The ethical trap includes: the risk of misrepresenting important values for the health of the embryo and the patient; the concern of incorrect deselection or worse a risk to the health and well-being of future children. There are also possible social implications; and a hole of responsibility, in case of adverse events. Limitations, reasons for caution Our search was limited to a single major medical research database and to what was presented during the last ESHRE annual meeting. Although there are other databases where it might be possible to find different articles. The application of AI in PGT is a very recent topic. Wider implications of the findings It is premature to implement AI for the identification of aneuploidies in embryos. AI for aneuploidy embryo deselection has enormous potential but needs to be done with care and transparency, as epistemic and ethical issues are significant. Currently the only possible use is to identify priority embryos for the transfer. Trial registration number not applicable

O-146 Oocyte donation does not increase live birth rates in young women suffering from recurrent pregnancy loss

Abstract Study question Does oocyte donation improve reproductive outcomes in recurrent pregnancy loss (RPL) patients? Summary answer Oocyte donation increases live birth and reduces miscarriage rates in RPL women older than 35, but it does not improve reproductive outcomes in younger patients. What is known already Recurrent pregnancy loss (RPL) is defined as 2 or more pregnancy losses prior to 20-24 weeks of gestation and has an incidence of approximately 1 to 2% in couples trying to conceive. Known risk factors for RPL are embryo aneuploidy, advanced maternal age (AMA), previous miscarriages, uterine abnormalities, parental chromosomal abnormalities, antiphospholipid syndrome, endocrine factors, such as thyroid function or obesity, and thrombophilia. However, 50-70% of patients suffering from RPL do not present any known risk factor, making the management of idiopathic RPL patients particularly challenging for ART clinicians. Study design, size, duration This is a cohort retrospective study involving 2 centers. Charts from 18,273 women undergoing IVF treatment between 2011 and 2020 were reviewed. A total of 912 patients (5%) met the definition of RPL and were classified into study groups based on their age and oocyte origin: ≤35 years old receiving oocyte donation (n = 39) or using their own oocytes (n = 35), and AMA women using donor eggs (n = 716) or their own (n = 122). Participants/materials, setting, methods Demographic variables analysed included known risk factors, number of transferred embryos, day of embryo transfer (3 vs 5) and sperm origin (partner/donor). Differences in biochemical, clinical, ongoing pregnancy, miscarriage and live birth rates were assessed by Pearson’s Chi-squared or Fisher’s exact test. P-values <0.05 were considered significant. Main results and the role of chance RPL patients had 2.77±1.27 pregnancy losses overall (2.76±1.37 in women ≤35 and 2.77±1.26 in patients >35). Most RPL patients (91.9%) were of AMA. Other RPL-associated risk factors, including uterine malformations, previous pregnancy losses, chromosomal abnormalities and thrombophilia were identified in 207/912 patients (22.7%). Interestingly, these were more frequent in patients <35 (37.8% vs 21%, p = 0.001). Young RPL women presented higher rates of karyotype abnormalities than older patients (17.6% vs 3.8%, p < 1x10-04), while showing a similar incidence of uterine abnormalities (14.9% vs 12.9%, p > 0.63) and thrombophilia (4.1% vs 4.7%, p > 0.81). RPL patients >35 preferentially underwent oocyte donation (85.4% vs 52.7%, p < 1x10-04), which led to significantly higher biochemical (52.5% vs 17.1%, p < 1x10-04), clinical (42.4% vs. 8.5% p < 1x10-04), ongoing pregnancy (37.9% vs 5.1%, p < 1x10-04), live birth (32% vs 4%, p < 1x10-04) and lower miscarriage rates (10.5% vs 40%, p = 0.0186) than in autologous cycles. In contrast, RPL patients <35 had similar reproductive outcomes: biochemical (41% vs. 52.9), clinical (28.2% vs. 37.1%), ongoing pregnancy (25.6% vs 29.4%), live birth (25.6% vs 26.4%) and miscarriage rates (9% vs 23%), regardless of oocyte origin (donated vs own, p > 0.05 for all cases). Importantly, this was also true for a small subgroup of idiopathic young RPL patients (n = 13). Limitations, reasons for caution The main limitation of this study is its retrospective nature, which does not allow for full elucidation of all potential confounders. The number of young RPL patients undergoing oocyte donation may be too low to draw significant conclusions. Wider implications of the findings RPL can be resolved in AMA patients by using donor oocytes, which points to key roles of oocyte quality and aneuploidy underlying RPL aetiology. However, RPL is not ameliorated by oocyte donation in young women, suggesting an endometrial/systemic origin and highlighting the need to further study mechanisms driving this disorder. Trial registration number Not applicable

P-711 Are there any differences among blastocyst euploid and mosaicism rate (high or low) using fresh or frozen ejaculate sperm in donated oocytes?

Abstract Study question To compare euploid and mosaicism rate (high or low) among blastocysts derived from fresh and frozen ejaculate sperm in donated oocytes. Summary answer No statistically significant differences were found on blastocyst euploid and mosaicism rate between blastocysts derived from fresh versus frozen. What is known already Even though 5%-10% autosomal aneuploidies and 5%-100% sex chromosomes aneuploidies are paternally derived, the clinical relevance of paternal contribution to chromosome aneuploidy is frequently overlooked. Besides, male infertility could contribute to the generation of chromosomal abnormalities in the resulting embryos. Sperm cryopreservation is a widely used technique in assisted reproduction for different reasons. However, little is known regarding the potential effect of sperm freezing on embryo aneuploidies. Study design, size, duration Retrospective, observational and multicenter study of patients undergoing Preimplantation Genetic Testing for Aneuploidies (PGT-A) from January 2017 to December 2022 in IVIRMA Madrid, Valencia and Barcelona. To elucidate the effect of advance maternal age, 1,780 biopsied blastocysts coming from the egg donation program were analyzed. Autologous sperm samples with less than 2 million spermatozoa per milliliter and male partners with altered karyotype, abnormal spermatozoa fish or any genetic disease were excluded from the study. Participants/materials, setting, methods Embryos were cultured in regular incubators, with an atmospheric concentration of 5% O2, 6.5% CO2 and a temperature of 37ºC. Blastocyst biopsy was performed either on day 5 or day 6 of embryo development and analyzed through Next Generation Sequencing (NGS). Sperm freezing was done following manufacturer’s instructions (Sperm freeze, Origio). Continuous variables were compared with a student´s t-test and categorical variables with a Chi-square using SPSS statistical program. Main results and the role of chance Mean paternal age (44.3±5.20 years old vs 43.2±5.20 years old), sperm concentration (46.67±33.05 mill/ml vs 35.1±32.92 mill/ml) were not significantly different among fresh versus frozen sperm group (p > 0.05). A total 1780 blastocysts tested for PGT-A were included: 1267 blastocysts coming from fresh ejaculate sperm (n = 194 patients) and 513 blastocysts from frozen sperm (n = 81 patients). No statistically significant differences were found in blastocyst euploid rate between fresh versus frozen ejaculate sperm (64.5% versus 67.2%, p = 0.278 ;) respectively. Blastocyst mosaicism rate: High mosaicism (>50% of aneuploidy trophectoderm cells) and low mosaicism (30-50% of aneuploidy trophectoderm cells) was comparable between fresh versus frozen sperm (2.7% versus 4.5%; p = 0.90) and (3.9% versus 3.7%), respectively. Limitations, reasons for caution This study is based on a retrospective data. Wider implications of the findings According to our findings, there are no statistically significant differences in the blastocyst euploid and mosaicism rate between fresh and frozen ejaculate sperm. Trial registration number Not Applicable

P-793 Human trophoblastic spheroid derived from human expanded potential stem cells (hEPSC) for the identification of adhesion molecules for embryo implantation.

Abstract Study question Are cell surface proteins on trophoblastic spheroid BAP-EB derived from hEPSC involved in the endometrial epithelial cell attachment? Summary answer E-cadherin (CDH1) and Neuropilin-1 (NRP1) are two potential embryonic surface molecules involved in the human implantation process. What is known already Study of the mechanisms of human implantation in vivo is challenging. The availability of high-quality human embryos for in vitro research is limited. We have recently derived morula-like hEPSC lines (hEPSC-em) from donated human embryos. Trophoblastic spheroids (em-BAP-EB) derived from hEPSC-em resemble human trophectoderm during the early implantation process. The em-BAP-EBs attach specifically onto receptive endometrial epithelial cells, suggesting its usefulness as human embryo surrogate for studying embryonic surface molecules involving in early implantation process. Study design, size, duration hEPSC-em was subjected to embryoid body formation and induced to differentiate into trophoblastic spheroids (em-BAP-EB) for 48h and 72h. Endometrial aspirates were collected from infertile women in their natural cycles 2 or 7 days after LH surge (LH + 2/+7). Endometrial epithelial cells (EEC) were isolated and cultured for the attachment assays. Biotin-labelled apical surface proteins on em-BAP-EB were identified by mass spectrometry. Antibody blocking assays were performed to confirm the biological functions. Participants/materials, setting, methods The attachment rates of em-BAP-EB onto EEC were compared between pre-receptive (LH + 2 day) and receptive (LH + 7 day) phases. The differentially expressed apical surface protein on em-BAP-EB-48h and -72h were subjected to plasma membrane proteins (PMP) identification and pathway analysis. The gene and protein expression patterns of the target proteins were analyzed by single cell RNA sequencing (scRNA-seq) and western blotting respectively. The functional roles of the target proteins were studied by antibody blocking during attachment Main results and the role of chance The attachment rates of em-BAP-EB-72h onto receptive stage (LH + 7, n = 17) EEC were significantly higher than those onto pre-receptive stage (LH + 2, n = 5) EEC. Gene Ontology analysis indicated that the PMP with induced expression levels on em-BAP-EB-72h when compared to -48h (72h>48h) were enriched for “protein binding” and “cadherin binding” in molecular functions, and “cell-cell adhesion” in biological processes. Among them, 28 PMP were trophectoderm-specific. CDH1 and NRP1 were selected for further analysis. Analysis based on a published dataset showed that significantly higher expressions of CDH1 and NRP1 were detected in polar trophectoderm as compared to mural trophectoderm of E7 human blastocyst. CDH1 and NRP1 were significantly up-regulated in trophectoderm-like em-BAP-EB by scRNA-seq analysis. Live-cell immunocytochemistry demonstrated more intense apical surface expression of CDH1 and NRP1 on em-BAP-EB-72h as compared to -48h. Antibody blocking experiments indicated that pre-treatment of the em-BAP-EB-72h with specific antibody individually against the ectodomains of CDH1 and NRP1 significantly reduced the attachment rates onto receptive endometrial epithelial cells. The data suggested that CDH1 and NRP1 are two potential apical surface molecules essential for blastocyst attachment. Limitations, reasons for caution The lack of embryonic compartment in the em-BAP-EB and the absence of other key players like decidualized stromal cells and immune cells in the coculture model might not fully represent the early attachment process. The limited sample size in this study may also limit the interpretation of the data. Wider implications of the findings Despite advancement in in vitro fertilization, the success rate remains unsatisfactory. It is not known if embryological factor(s) other than embryo aneuploidy are involved. The current data may suggest the potential roles of CDH1 and NRP1 as non-chromosomal embryological markers of its implantation potential. Trial registration number Not applicable

P-006 In men with Raised Sperm DNA Fragmentation Index, would advance Sperm Preparation techniques alter Embryo Euploidy?

Abstract Study question In men with Raised Sperm DNA Fragmentation Index (DFI), Would advance sperm preparation techniques at Intra-Cytoplasmic Sperm Injection (ICSI) alter embryo euploidy status? Summary answer In men with raised Sperm DFI, use of advance sperm selection techniques are safe and do not appear to alter ploidy status of the embryos What is known already MACS (Magnetic Activated Cell Sorting), Microfluidic Sperm Sorting (MF), surgically retrieved Testicular Sperm (TESA) and Double Density Gradient sperm processing (DDG) have been shown to optimize sperm selection at ICSI for men with raised sperm DFI. Would any of these sperm selection methods alter embryo ploidy status is not proven Study design, size, duration This was a retrospective observational study conducted at a private fertility centre in-between years 2015 & 2022. Couples where women (<37yrs) had idiopathic Recurrent Implantation Failures (RIF) and men had raised sperm DFI (>25% DFI) and underwent Pre-Implantation Genetic Testing (PGT) were evaluated (n = 473). Based on sperm selection method the study population was divided into four groups. TESA (n = 84), MACS (n = 105), MF(n = 135), DDG (n = 148).A total of 1802 embryos were evaluated in this study. Participants/materials, setting, methods All women underwent Controlled Ovarian Stimulation (COS) and oocyte retrieval and male partners underwent sperm processing as per the clinics SOP. Appropriate counselling and written consents were obtained from all couples, where applicable. All oocytes subjected to ICSI, extended blastocyst culture and trophoectoderm (TE) biopsy. Post biopsy embryos were vitrified and TE tissue subjected to Next-Generation Sequencing (NGS) to assess the ploidy status. Ploidy status between various sperm selection methods was compared. Main results and the role of chance Euploidy rates between the study groups were as follows: TESA (n = 311 embryos screened)- 48% MACS (n = 445 embryos screened) – 51% MF (n = 489 embryos screened) – 61% DDG (n = 557 embryos screened)- 54% Though numerically the ploidy status was highest in MF group for euploid embryos, there seems to be no statistical significance between the groups (p-value = 0.973). Different sperm selection techniques do not seem to alter embryo ploidy status. Ejaculated sperm or Testicular sperms had similar euploidy status. Use of Testicular sperm, which is considered immature doesn’t seem to increase embryo aneuploidy. MACS, MF and DDG all seemed to have similar euploid embryos. This further warrants research to validate the role of MACS and MF as a routine intervention to optimize sperm selection and reproductive outcomes. Limitations, reasons for caution Retrospective design, un-equal group size and small sample size, a well-designed study is needed to further validate the findings of this study. Wider implications of the findings Role of advance sperm selection methods to optimize embryo implantation is yet to be proven. Data from this study showed no alarming increase in embryo aneuploidy in younger women and was comparable with published literature. Use of advance sperm selection techniques seems safe and doesn’t alter embryo ploidy status. Trial registration number not applicable

O-103 A method for mapping sex-specific meiotic crossovers from PGT-A data elucidates the role of aberrant recombination in the origins of aneuploidy

Abstract Study question How do the number and location of meiotic crossovers contribute to the formation of aneuploidies observed in preimplantation human embryos? Summary answer Normalized across chromosomes, trisomies possess 35% fewer crossovers on average compared to disomies, while the genomic distribution of crossovers is also substantially altered. What is known already Meiotic recombination is a crucial source of genetic diversity and is also critical for ensuring the accuracy of chromosome segregation. Understanding the landscape of meiotic recombination, its variation across individuals, and the processes by which it goes awry are long-standing goals in human genetics. Current approaches for inferring the landscape of recombination either rely on population genetic patterns of linkage disequilibrium—capturing a time-averaged view—or direct detection of crossovers in gametes or multi-generation pedigrees, limiting the scale and availability of relevant datasets. Moreover, most of these methods are designed for discovering recombination using data from normal, disomic chromosomes. Study design, size, duration We present a method for mapping sex-specific recombination landscapes from low-coverage (<0.1×) data from preimplantation genetic testing for aneuploidy (PGT-A) of embryos with arbitrary ploidy configurations. To overcome the sparsity of these data, our method exploits its inherent relatedness structure, knowledge of haplotypes from external population reference panels, as well as the frequent occurrence of chromosome loss in embryos, whereby the remaining chromosome is phased by default. Participants/materials, setting, methods We benchmarked our method by simulating crossovers between known haplotypes. Encouraged by the performance on simulated data we extended our study to retrospective analysis utilizing de-identified PGT-A data obtained between April 2021 and August 2022 at the CReATe Fertility Centre (Toronto, Canada). The data include 20,160 embryos (2,559 IVF patients) with an average depth of coverage of ∼0.05×, facilitating the mapping of crossovers at an average resolution of ∼150 kbp. Main results and the role of chance Our benchmarking results demonstrate high sensitivity and specificity across all ancestries at a coverage of 0.05x per homolog (AUC = 0.989), with AUC declining by 0.014 and 0.053 for when coverage is reduced to 0.025x and 0.013x, respectively. Extending our analysis to real PGT-A data, we observed that our inferred sex-specific landscapes of meiotic crossovers on disomic chromosomes were strongly correlated with published genetic maps from studies based on high-coverage sequencing of parent-offspring trios (r = 0.86 for female map; r = 0.53 for male map), broadly supporting the accuracy of our method. Notably, the total length of the female genetic map was reduced by 35% for trisomies compared to disomies, consistent with the hypothesized role of reduced crossovers and exchangeless chromosomes in the origins of female meiotic aneuploidy. In addition, the genomic distribution of crossovers is also altered in a chromosome-specific manner. Examples include a reduction in crossovers near the centromere of trisomies versus disomies of chromosome 16, as well as an enrichment of crossovers on the q-arm of trisomies versus disomies of chromosome 22. Together, our results provide a detailed corroboration of the hypothesis that aberrant meiotic recombination contributes to the origins of aneuploidies. Limitations, reasons for caution The accuracy of our method is influenced by genomic heterogeneity in depth of coverage, rates of heterozygosity, and mismatches between the ancestry of the reference panel and the tested sequence. Moreover, technical errors such as spurious alignment and genotyping could hinder analysis in repetitive genomic regions. Wider implications of the findings Together, our study helps clarify the dual function of meiotic recombination in generating genetic diversity while ensuring meiotic fidelity. Our method for patient-specific mapping of meiotic recombination phenotypes may offer clues about how dysregulation of this process contributes to infertility. Trial registration number NIH r R35GM13374

O-078 Mosaic embryos – are they accurately detected by PGT-A?

Abstract Study question Following quantitative NGS for PGT-A, do current thresholds for mosaicism calling give a true reflection of the presumed mitotic origin of mosaic errors? Summary answer A large proportion of embryo mosaicism diagnosed following quantitative NGS-based PGT-A does not correlate with a true and expected mitotic origin, showing instead meiotic signatures. What is known already Mosaicism is a true biological phenomenon that can affect ongoing embryo development. However, the reproductive potential of mosaic embryos in assisted reproduction varies greatly across publications and is not fully understood. While the transfer of mosaic embryos could result in healthy live births, embryos containing meiotic errors almost never result in healthy live births. Since the clinical implications of meiotic and mitotic errors are very different, it is critical to accurately differentiate between the two. However, diagnosing mosaicism using current NGS-based PGT-A is not always accurate due to technical and biological limitations which may misclassify mosaic embryos. Study design, size, duration Follow-up analysis was carried out on 159 embryos previously categorised as mosaic by NGS-based PGT-A. To identify the origin of mosaic calls, SNP genotyping and karyomapping were performed on the same amplified DNA used for the clinical NGS analysis. Trophectoderm biopsy samples were obtained from embryos on days 5,6 or 7 of development from a single IVF centre between 2018-2022. For all PGT-A tested embryos, the reported mosaicism rate at the IVF centre was 12%. Participants/materials, setting, methods Samples were analysed using PGTai from CooperSurgical with a 30-80% CNV mosaicism range. Patients with mosaic embryos consented to karyomapping follow-up using sibling embryo reference DNA. The origin of mosaic events was defined by SNP inheritance patterns for the affected chromosome. Meiotic errors were defined by the presence of both haplotypes from one parent for trisomy, and the absence of a parental haplotype for monosomy. Mitotic errors were inferred following a result of biparental inheritance. Main results and the role of chance A total of 154 diploid embryos identified as mosaic by NGS-based PGT-A were re-analysed by SNP-genotyping. Of these, 30.5% (n = 47) displayed at least one error of meiotic origin following SNP genotyping. Among the 154 embryos, 189 mosaic chromosomal abnormalities were identified. After SNP-genotyping, 30.7% of chromosomal abnormalities (n = 58) showed evidence of meiotic signatures: 52 whole-chromosome (24 gains, 28 losses), and 6 segmental errors (5 gains and 1 loss). Of 73 segmental errors detected in total, only 8.2% of them (n = 6) were identified to be of meiotic origin. Following PGT-A, 7.8% of embryos (n = 12/154) displayed ≥3 chromosomal abnormalities in the mosaic range. Of these embryos, 58.3% (n = 7/12) displayed one or more meiotic aneuploidies. Overall, embryos showing multiple mosaic events were significantly more likely to have a least one error of meiotic origin than embryos containing one abnormality (McNemar’s test p-value= 1.171e-14). Among mosaic calls above 50% CNV, 56.7% (n = 55/97) showed evidence of meiotic origin. In contrast, but importantly, in the low-moderate mosaicism range <50% CNV, 3.3% of errors (3/92) also displayed meiotic signatures, including two whole chromosome errors (chr21 gain at 32% CNV and chr13 loss at 46% CNV), and one segmental error (dup(6)(pter-p12.1) at 33% CNV). Limitations, reasons for caution Since karyomapping relies on recombination events to identify meiotic trisomy, the true origin of intermediate copy-number changes cannot always be identified. Therefore, errors without recombination may be missed, potentially underestimating meiotic gains, especially segmentals. The absence of a parental haplotype cannot entirely rule-out a mitotic error from a biopsy sample. Wider implications of the findings Quantitative NGS methodologies are unable to accurately distinguish the origin of mosaic errors in PGT-A samples. Since the clinical impact of meiotic and mitotic errors differs greatly, this limitation could explain mosaic embryos’ reduced viability. Reliable identification of meiotic aneuploidies in presumed mosaic embryos is likely to improve clinical outcomes. 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

P-477 Bidirectional association between ovarian reserve and spontaneous miscarriage: findings from the clinical IVF data to epidemiological evidence and genetic links

Abstract Study question To determine whether the association between ovarian reserve and spontaneous miscarriage risk is bidirectional and caused by shared genetic risk loci. Summary answer Bidirectional association between ovarian reserve and spontaneous miscarriage was observed. This may be attributed to five shared risk genes (SYCP2L, TP63, GSPT1, RIF1, and PRRC2A). What is known already Previous studies have shown that women with lower serum anti-Müllerian hormone (AMH) had an increased risk of embryo aneuploidy or spontaneous miscarriage risk, compared to women with higher AMH concentration, although not all studies agree. The most recent meta-analysis postulated that the association between diminished ovarian reserve (DOR) and miscarriage may be not causal, but that the two occurrences merely share a common cause, such as other systematic clinical conditions or past exposure. Inspired by this hypothesis, we conducted the present analyses to clarify this bidirectional association between DOR and miscarriage and further elucidate plausible mechanisms by using genetic data. Study design, size, duration There were four data sources. 1) The Clinical IVF data were retrospectively collected from an academically affiliated Reproductive Medicine Center (17,786 cycles included). 2) We further analyzed data from the UK Biobank (UKB) which is a large-scale, population-based, prospective cohort study (35,316 white women included). 3) The GWAS summary statistics for age at natural menopause (ANM) and spontaneous miscarriage were obtained from published GWAS studies. 4) The individual-level genotype data were also extracted from UKB. Participants/materials, setting, methods There were four analysis modules, 1) IVF data to test the association of ovarian reserve and miscarriage risk by using logistic regression models, 2) UKB data to test the association of spontaneous miscarriage history and ANM by using linear regression models, 3) GWAS summary data to test the overall genetic enrichment between miscarriage and ANM by plotting Q-Q plot, and 4) individual-level genotype data to identify specific shared genes by using ‘pleio’ R package. Main results and the role of chance In the analysis of clinical data, the risk of miscarriage was 1.5 (95% CI: 1.22-1.85, p < 0.001) times in the group with AMH<1.1 ng/ml, compared to the group with AMH > =1.1 ng/ml after adjustment. In the analysis of UK Biobank data, one more spontaneous miscarriage history would lead to 0.02 (95% CI: 0.00-0.03, p = 0.032) years earlier in the standardized ANM after adjustment. Participants with > =2 spontaneous miscarriage history would have 0.05 (95% CI:0.01-0.08, p = 0.011) years earlier in standardized ANM, compared with participants with < =1 spontaneous miscarriage. In the analysis of GWAS summary statistics, we found that there were 11,736,532 single nucleotide polymorphisms (SNPs) tested by both GWAS studies of ANM and miscarriage. In the stratified conditional Q-Q plot, successive leftward deflections did not show which suggested that an overall genetic overlap between miscarriage and ANM was not observed. In the analysis of the individual-level genotype data, we identified five shared genetic risk loci that affect both miscarriage and menopause. They were all novel risk genes for spontaneous miscarriage. Of these five genes, SYCP2L, TP63, GSPT1, and RIF1 were associated with congenital malformations and PRRC2A was associated with autoimmune disease, which are factors that possibly play a role in the occurrence of miscarriage. Limitations, reasons for caution The UKB epidemiology data and genetic data were restricted to participants of European ancestry. Further studies are needed in non-white populations. Second, the predictive value of GWAS summary data for miscarriage may be limited which results in a null finding in the conditional Q-Q plot. Wider implications of the findings Our findings could be of interest to IVF clinicians in patient counseling regarding the prognosis of IVF treatment and genetic counseling for miscarriage. Our findings encourage further research focus on the shared genetic architecture and common pathophysiological basis of diminished ovarian reserve and miscarriage which may bring new therapeutic opportunities. Trial registration number not applicable

O-299 Sperm DNA fragmentation by TUNEL influences live birth in egg recipients after euploid embryo transfer

Abstract Study question Does sperm DNA fragmentation (SDF) by TUNEL affect reproductive success measured as live birth rate (LBR) in egg recipients after euploid embryo transfer? Summary answer SDF increases miscarriage rates in egg recipients with PGT-A. Therefore, live birth rates depends on sperm DNA damage in egg recipients after euploid embryo transfer. What is known already Sperm DNA integrity is important for optimal fertilisation, implantation and pregnancy. Although controversial, several studies have shown that sperm DNA fragmentation correlates with adverse pregnancy outcomes in infertile men. Ooplasmic sperm DNA fragmentation repair mechanisms may occur in female gametes, mainly in young women. However, research on the impact of DNA fragmentation on LBR are limited. Also, chromosomally normal embryos analysed by PGT-A offers opportunity to isolate female factor and study male factor. The aim of this study is to assess whether high level of semen DNA damage has an impact on reproductive outcomes in terms of live birth rate. Study design, size, duration This is a retrospective study of 467 PGT-A cycles in egg recipients. The study population consists of infertile couples for euploid embryo transfer (February 2017-November 2022). Semen samples were obtained to measure DNA damage from 447 men. PGT-A was performed in 1831 blastocysts. Trophectoderm biopsies (d5/d6 blastocysts) were analysed by NGS (Veriseq Illumina). Embryos were frozen and 568 blastocysts transferred in the subsequent cycle. Conventional hormone replacement was used for endometrial preparation in recipient patients. Participants/materials, setting, methods Cohort study with 447 infertile couples attending to private fertility clinic undergoing ART with their autologous semen, donated oocytes and PGT-A. SDF was measured by Terminal-deoxyribonucleotidyl-ARTSferase-mediated dUTP Nodule End Labelling (TUNEL) assay using the FITC-labelled-dUTP in situ cell death detection kit (Roche). The cohort was divided into two groups according the DNA fragmentation index (DFI): high SDF group (DFI>20%) and normal SDF group (DFI≤20%). To evaluate reproductive outcomes Chi-square and Linear regression tests were used. Main results and the role of chance Of the 447 couples included (average paternal age 41.29 + 7.32 years), 66.5% of male partners had normal semen parameters. We reported high sperm DNA fragmentation in 8% of patients. Overall, the embryo aneuploidy rate was 31.7%. For the further statistical analysis confounding factors such as, female and male age, embryo quality, day of embryo biopsy were included. PGT-A analysis showed an aneuploidy rate of 32.1% in the high DNA fragmentation group and 30.3% in the normal DNA fragmentation group (p = 0.453). Regarding clinical data, the overall implantation rate was 34.9%. There was no significant difference between elevate DNA fragmentation and normal DNA fragmentation with respect to biochemical miscarriage rate (4.3% vs 7.6%, p = 0.799). Although not significant differences between groups, a lower implantation rate near to reach significance was found in the high SDF group (20.0% vs 34.4%, p = 0.050). Pregnancy and clinical pregnancy rates were significantly lower in the high DNA fragmentation group (26.1% vs 44.9%, p = 0.042; 21.7% vs 31.3%, p = 0.044;), respectively. Also, miscarriage rate was significantly higher in the high SDF group (40% vs 12.5%, p = 0.014). Therefore, the live birth rate was significantly lower in the high SDF group (13.0% vs 32.6%, p = 0.024). Limitations, reasons for caution Larger studies including a higher number of samples are needed to confirm the correlation observed between LBR in eggs recipients with semen DNA fragmentation. Those patients with a DFI higher than control values should be treated with antioxidants. The pregnancy outcomes should be analyzed after treatment of DNA fragmentation. Wider implications of the findings Our findings suggest that autologous semen DNA fragmentation influence in live birth rate in egg recipients. Our data show that live birth rates decreased in patients with a high DFI after PGT-A. This study confirms published studies in the literature showing that ART outcomes are affected by sperm DNA fragmentation. Trial registration number Not Applicable

O-144 Critical assessment of possible clinical benefits of culturing human embryos until day 7

Abstract Study question Is there any benefit of extending culture until day (D) 7 to increase euploid blastocysts yield and clinical outcomes in a PGT-Aneuploidy analysis (PGT-A) program? Summary answer Extended culture until D7 increases euploid blastocysts yield and cumulative live birth. Highest benefit was observed in patients aged ≤41 with ≤2 D5/D6 blastocysts biopsied. What is known already It is well-known that female age is the strongest predictive factor of embryo aneuploidy and has been associated with delayed blastocyst development in vitro. Culturing embryos until D7 might increase biopsied blastocyst yields and euploidy outcomes among women undergoing ART as already been published. However, extended culture until D7 increases IVF workload and implies extra cost. It is unclear whether the practice is justified when a certain number of blastocysts have already been biopsied on D5/D6. Therefore, the additive value of extended culture until D7 needs further evaluation. Studies published in the literature have small sample sizes and are controversial. Study design, size, duration This single center observational study included 30,941 blastocysts from 6,976 consecutive IVF/ICSI PGT-A fresh autologous cycles performed between 2017 and 2022. Patients who underwent PGT-A by Next Generation Sequencing (NGS) were considered for the analysis. Only blastocysts graded ≥BL3CC (Gardner) that underwent TE biopsy on D5, D6 or D7 were included. Data were stratified by age categories (<37, 37-41 and >41) and on number of biopsied blastocysts performed on D5 and/or D6 of embryo culture. Participants/materials, setting, methods The additive value of extended embryo culture was assessed in terms of absolute numbers (percentage of D7 euploid blastocysts obtained) and theoretical benefit to cumulative live birth rate (having at least 1 live birth by using all available euploid blastocysts) as calculated by binomial density function. Results was assessed between female age strata (<37, 37-41, >41) and number of embryos biopsied in D5/D6 (No biopsy, ≤2, 3-4, 5-6 or ≥ 6 biopsied). Main results and the role of chance A total of 18,478 blastocysts were biopsied on D5 (38.5%), D6 (55.2%) or D7(6.3%). Euploidy rate decreased significantly for blastocysts biopsied on D5, D6 and D7 (55.6%, 39.7% and 27.1%, P < 0.001, respectively). When stratifying patients by age, euploidy rates were consistently higher for D5 biopsied blastocysts compared to D6 and D7 (<37: 61.1%, 50.0% and 38.5%, P < 0.001; 37-41: 39.0%, 27.8% and 20.1%, P < 0.001; >41: 18.5%, 9.0% and 3.9%, P < 0.001, for D5, D6 and D7, respectively). The chances of obtaining at least one euploid D7 blastocyst was higher among patients <37 and 37–41 years compared to > 41 years (7.2% and 4.4% vs. 0.6%, P < 0.001). Among women <37, a > 25% increase in cumulative live birth rate (theoretical) was detected in 10.1% with no biopsy on D5/D6, 2.5% with 1-2 blastocysts biopsied on D5/D6 and only 0.3% with ≥3 blastocysts biopsied on D5/D6 (P < 0.001). In older women (37-41), a > 25% increase in cumulative live birth rate (theoretical) was found in 5.6% with no biopsy on D5/D6, 2.5% with 1-2 blastocysts biopsied on D5/D6, and only 0.4% with ≥3 blastocysts biopsied on D5/D6 (P < 0.001). Women >41 years rarely benefited from extended culture util D7 (<1%) regardless of the number of blastocysts biopsied on D5/D6. Limitations, reasons for caution The study was based on a retrospective analysis data and results were calculated on a theoretical cumulative live birth. While this study provides one of the largest number of blastocysts analyzed in PGT-A and “freeze all” strategy, results should not be extrapolated to other populations or different ART routine practices. Wider implications of the findings Usable blastocysts (6.3%) and euploidy rates (27.1%) are significantly lower with D7 blastocysts compared to D5/D6 blastocysts. Patients aged >41 do not benefit from extended embryo culture to D7 (<1%). Culturing blastocysts for 7 days should be mainly decided based on the number of blastocysts biopsied on D5 or D6. Trial registration number not applicable

O-184 Validation of a model capable of estimating the minimum number of mature oocytes needed to obtain at least one euploid blastocyst regarding female age

Abstract Study question Which is the minimum number of mature oocytes needed to obtain at least one euploid blastocyst regarding female age in In Vitro Fertilization (IVF) treatments? Summary answer The validated model estimates with a 74% accuracy the probability of having one euploid blastocyst regarding the number of mature oocytes and female age. What is known already Female age is significantly and directly related to embryo aneuploidy rates, thus lowering the chances of success in IVF treatments. The current delay in motherhood has led to a large proportion of women of advanced maternal age seeking infertility treatment. The aim of the present study is to determine the number of metaphase II (MII) oocytes needed to obtain at least one euploid blastocyst regarding female age. This information will help to decide the best strategy for each patient, taking also into account other variables such as ovarian reserve, semen quality and oocyte quality. Study design, size, duration Retrospective analysis of IVF cycles with pre-implantational genetic testing for aneuploidies (PGT-A) performed over the last 5 years in an infertility clinic in Spain, from January 2017 to March 2022. Participants/materials, setting, methods Patients undergoing an IVF cycle in an infertility clinic with own or donated oocytes, regardless semen origin. Only trophoectoderm biopsies performed on day 5 or 6 of development and analyzed using Next Generation Sequencing (NGS) were included. PGT-A was used for reasons such as advanced maternal age, implantation failure and recurrent miscarriage. PGT-A cycles due to a known abnormal karyotype were excluded. Endpoints were analized using binary logistic regression models. Main results and the role of chance A total of 3840 IVF-PGT-A cycles meeting the inclusion criteria were performed in the study period. Of them, 939 cycles were discarded due to the absence of any biopsied/analyzed embryo (final sample size=2901). A model for the probability of having at least one euploid blastocyst (pEB) regarding female age and the number of MII oocytes retrieved was created with 80% of the sample (n = 2320) and validated in the remaining 20% (n = 581). The validation of this model showed that it was capable of estimate with an accuracy of 73.88%. The pEB was directly related to the number of MII oocytes retrieved (odds ratio (OR) 1.130, confidence interval (IC) 95% (1.110-1.150); p < 0.001), but indirectly related to female age (OR 0.751, IC95% (0.725-0.778); p < 0.001). The ROC curve showed a significant predictive value of the number of MII oocytes (area under the curve (AUC):0.8041 (0.7882-0.82)) for this model. A mathematical formula was created for the calculation of pEB using this model. The number of MII oocytes needed for a pEB of 75% regarding female age (female age:number MII) was 35:6, 36:9, 37:12, 38:15, 39:18, 40:21, 41:24, 42:27, 43:30, 44:32, 45:35, 46:38, 47:41, 48:44, 49:47, 50:50. Limitations, reasons for caution The main limitation of this study is its retrospective design. In addition, it’s important to keep in mind that many other variables should be taken into consideration along with maternal age in the assessment of the number of oocytes needed to obtain at least one euploid blastocyst in IVF treatments. Wider implications of the findings Results from this study may constitute a useful tool for both clinical and patient. The clinician may be able to better decide the best strategy for each patient, while the patient will understand more easily this information, helping her morally with the treatment. Trial registration number 2204-VLC-040-CR

P-737 Progress in non-invasive embryo assessment with cell-free nucleic acids in blastocoel fluid

Abstract Study question Can cell-free nucleic acids (cfDNA) from blastocoel fluid (BF) after a trophectoderm (TE) biopsy be used as a non-invasive sample type to assess embryos? Summary answer Both BF and spent media carry comparable, highly fragmented cfDNA and cell-free RNA (cfRNA) which may reveal more about the quality of the embryo. What is known already Preimplantation genetic testing for aneuploidy (PGT-A) is commonly used by IVF clinics to screen embryos for any chromosomal abnormalities during the IVF process. Noninvasive preimplantation genetic testing (ni-PGT) is an emerging category of tools within the field of assisted reproductive technology. Currently, niPGT products have been developed for embryo aneuploidy detection using cfDNA released by the embryo into the spent media. Embryo spent media (ESM) carries cfDNA, which has the potential to be applied to evaluate the quality of the embryo. Previous studies have demonstrated that cfDNA in ESM is highly fragmented to the nucleosome level. Study design, size, duration The quality and quantity of cell free nucleic acid from forty BF-conditioned media (BFCM) samples were analyzed in this study. Embgenix™ Analysis Software was used to analyze and interpret sequencing data. For each sample, the concentration and fragmentation of cfDNA were examined and compared using generalized linear mixed models. To assess RNA quality at a picogram level, reverse-transcribed and amplified cDNA was used to represent the quality of cfRNA in BFCM. Participants/materials, setting, methods Embryos were washed and placed in individual biopsy media droplets for TE biopsy. After biopsy, blastocysts were collapsed, releasing BF into the droplet. The entire droplet was collected as BFCM. The Embgenix ESM Screen Kit was used to prepare sequencing libraries. The SMART-Seq® Single Cell Kit was used to evaluate the RNA quality. The BFCM cfDNA results were also compared to spent media profile that had already been obtained from a different cohort of embryos. Main results and the role of chance Amounts of cfDNA fragmentation detected in BFCM samples was comparable to ESM. Whole-genome amplification (WGA) yields correlated to the amount of cfDNA input but not the degree of cfDNA fragmentation. Informative sequencing reads and computed copy number variations (CNV) plot noise, as evaluated by distribution of log2 ratio spread (DLRS), also showed correlation and stable performance down to ∼2 pg of cfDNA input. We extracted detectable cfRNA from BFCM and created cDNA for RNA quality analysis. Our model demonstrates a significant correlation between cDNA length and RNA quality metrics, with a low DV200 of cfRNA from BFCM samples. The quantity and quality of cfDNA released into spent culture media by a developing embryo are comparable to the cfDNA released into BFCM upon TE biopsy, indicating that BFCM has potential as a noninvasive method for assessing the ploidy status of embryos. WGA yield, informative reads, and DLRS of BFCM samples were likewise comparable to data previously acquired using spent media samples. However, the severely fragmented cfRNA in these samples illustrate the inherent instability of RNA and the difficulty of utilizing RNA analysis to determine embryo health. Assays compatible with small RNA or microRNA (miRNA) are more appropriate for RNA-seq of BFCM. Limitations, reasons for caution Different clinics use varying methods for embryo culture and biopsy; the data reported here is from a single research center. The characteristic of samples may vary between clinics. Our assay estimates RNA quality using amplified cDNA; nonetheless, discrepancies between fragmented RNA and amplified cDNA may exist. Wider implications of the findings In PGT-A, BFCM samples are routinely discarded. Our findings imply BFCM samples contain considerable levels of cell-free nucleic acids which could also be used to screen embryo quality. BFCM samples, therefore, could be analyzed in conjunction with PGT-A to select and/or rank embryos for implantation. Trial registration number Not applicable

P-220 “Blame it on my youth”: when very young age appears to be associated with poorer embryo development in oocyte donors

Abstract Study question Is a very young donor’s age associated with different blastocyst usable rate and embryo quality? Summary answer Donors <20 years old have a significantly lower blastocyst usable rate but comparable average embryo quality than donors >25. What is known already Embryo development and quality in IVF cycles are affected by increasing female age. However, some authors also described a lower fertilization rate and reduced number of top-quality embryos in very young infertile patients <25 years old as compared to patients aged 25-35. It has even been reported that live birth rates are significantly lower in oocyte recipients when the donors’ age is < 25. Still, the mechanisms underlying these apparently poorer outcomes in very young women remain speculative given that data on embryo development and quality in vitro are scarce. Study design, size, duration Retrospective-observational study of 1274 oocyte donor’s and 1738 oocyte recipient’s cycles with blastocyst transfer carried out in 2016-2022 in the Fertility Unit of a tertiary University Hospital. Cycles with vitrified oocytes, severe male factor or cleavage-stage embryo transfer were excluded. The main parameters analyzed were blastocyst usable rate (% fertilized oocytes that became a blastocyst suitable for transfer/freezing) and blastocyst quality based on 2015 ASEBIR-scoring system (considering day of development and morphology):D5A, D5B, D5C, D6B, D6C. Participants/materials, setting, methods Cycles were categorized according to donor’s age (Group A: <20, Group B: 20-25, Group C: ≥26). Oocyte donors were aged 18-34. Recipients were aged 18-50. Blastocyst usable rate and embryo quality were compared across groups according to donor’s age. For multivariable analysis, a generalized logistic linear mixed model was applied to estimate the odds for every endpoint, taking Group C as a reference group. Donor and recipient were treated as random factors to avoid the repeated-observation effect. Main results and the role of chance A total of 1274 oocyte donor’s cycles and 1738 oocyte recipient’s cycles were analyzed. Mean age was 26.1 ± 4.3 years old for donors, 42.6 ± 3.8 for recipients, and 43.3 ± 5.7 for male partners. Mean donors’ AMH was 3.8 ± 2.1ng/ml, mean number of mature oocytes (MII) retrieved was 15.8 ± 7.7, and mean total dose of gonadotropins was 1413.1 ± 885.4 units. ICSI technique was used in 75.6% of the cycles. The distribution of cycles by donor age was Group A: n = 55 cycles (4.3%), Group B: n = 532 cycles (41.8%) and Group C: n = 687 cycles (53.9%). Blastocyst usable rate was significantly different according to donors’ age: 33.2% (176/530) for Group A, 42.9% (2580/6016) for Group B and 43.5% (3109/7147) for Group C. Regression analysis, adjusting for confounding factors (AMH, MII, gonadotropins total dose, male age, insemination technique), has shown a significantly lower blastocyst usable rate in very young donors Group A (OR: 0.63; CI 95%; 0.48-0.83) as compared to Group C. Although similar differences were observed in the proportion of top-quality blastocysts (D5A+D5B) in the bivariate analysis A: 19.2% (102/530), B: 24.6% (1480/6016) and C: 24.4% (1742/7147), no significant differences were found after adjustment for confounding factors. Limitations, reasons for caution The main limitation of this study is its retrospective design; still multiple adjustments for confounding factors have been performed in order to minimize the risk of confounding bias. Wider implications of the findings Our finding of a lower blastocyst usable rate in very young donors may be associated with a higher aneuploidy embryo rate in this group, previously reported, advocating for further studies with PGT-A and basic research to decipher the underlying molecular mechanisms. Trial registration number not applicable

Association of embryo aneuploidy and sperm DNA damage in unexplained recurrent implantation failure patients under NGS-based PGT-A cycles.

Recurrent implantation failure (RIF) is one of the most common conditions affecting In Vitro Fertilization (IVF)/Intracytoplasmic sperm injection (ICSI) outcomes. Aneuploidy embryos, one of the main types of embryos-related factors, was reported to be a major contributor to RIF. The present study aimed to examine the association between sperm DNA fragmentation index (DFI) and outcomes of next-generation sequencing (NGS)-based preimplantation genetic testing for aneuploidy (PGT-A) in unexplained RIF patients. This study analyzed 119 couples with unexplained RIF who underwent 119 PGT-A cycles between January, 2017 and March, 2022. The 119 males were divided into 3 groups according to their sperm DFI levels: Group1 (low, DFI ≤ 15%, n = 50), Group2 (medium, 15% < DFI < 30%, n = 41) and Group3 (high, DFI ≥ 30%, n = 28). Sperm DFI was measured by sperm chromatin structure analysis (SCSA) technique. Trophectoderm biopsy on day 5 or 6 were performed with NGS technique. The following outcomes of PGT-A were analyzed and compared: fertilization, good-quality embryos, aneuploidy rate, miscarriage, live birth and newborn defects. The component of aneuploidy embryos was significantly higher in high DFI group (42.71%) than that of medium group (28.39%) and low group (27.80%). The miscarriage rate of high DFI group (27.27%) and medium group (14.29%) is significantly higher than that of low group (0.00%). No significant differences were found regarding fertility, good-quality embryo rate, pregnancy rate, live birth rate or newborn defects among three groups. The sperm DNA damage is associated with blastocyst aneuploidy and miscarriage rate in unexplained RIF cases. Embryo selection by PGT-A and efforts to decrease sperm DFI before IVF/ICSI treatments should be considered for those male patients with high DFI.

Impact of male age on paternal aneuploidy: single-nucleotide polymorphism microarray outcomes following blastocyst biopsy

Does advanced paternal age (APA; ≥40 years) contribute to a higher incidence of paternal origin aneuploidy in preimplantation embryos? This was a multicentre retrospective study of single-nucleotide polymorphism (SNP) microarray (Natera and Karyomapping) preimplantation genetic testing (PGT) outcomes of blastocyst-stage embryos. Whole-chromosome aneuploidy analysis was performed on 2409 embryos from 389 male patients undertaking 681 assisted reproductive technology (ART) cycles between 2012-2021. Segmental aneuploidy analysis was performed on 867 embryos from 140 men undertaking 242 ART cycles between 2016-2021. Embryos were grouped based on paternal age at sperm collection: <35, 35-39 and ≥40 years. Paternal and maternal origin aneuploidy rates were compared between groups using chi-squared and/or Fisher's exact tests. There was no significant difference across groups in paternal origin whole-chromosome aneuploidy rate, overall (P=0.7561) or when segregated by type (trisomy and monosomy: P=0.2235 and 0.8156) or complexity (single versus 2, 3 or ≥4 aneuploidies: P=0.9733, 0.7517, 0.669 and 0.1481). Conversely, maternal origin whole-chromosome aneuploidy rate differed across groups (P<0.0001) in alignment with differing mean maternal age (P<0.001). Paternal origin deletions were 2.9-fold higher than maternal origin deletions (P=0.0084), independent of age stratification. No significant difference in paternal origin deletions was observed with APA ≥40 compared with the younger age groups (4.8% versus 2.5% and 2.8%, P=0.5292). Individual chromosome aneuploidy rates were too low to perform statistical comparisons. No significant association was found between APA and the incidence of paternal origin aneuploidy in preimplantation embryos, irrespective of type or complexity. Thus, APA may not be an indication for PGT.

The effect of sperm DNA fragmentation on the incidence and origin of whole and segmental chromosomal aneuploidies in human embryos.

Whether sperm DNA fragmentation (SDF) affects embryo development and clinical outcomes is still controversial, which limits the utility of SDF testing in assisted reproductive technology management. This study demonstrates that high SDF is associated with the incidence of segmental chromosomal aneuploidy and increased paternal whole chromosomal aneuploidies. We aimed to investigate the correlation of sperm DNA fragmentation (SDF) with the incidence and paternal origin of whole and segmental chromosomal aneuploidies of embryos at the blastocyst stage. A retrospective cohort study was conducted with a total of 174 couples (women aged 35 years or younger) who underwent 238 cycles (including 748 blastocysts) of preimplantation genetic testing for monogenic diseases (PGT-M). All subjects were divided into two groups based on the sperm DNA fragmentation index (DFI) level: low DFI (<27%) and high DFI (≥27%). The rates of euploidy, whole chromosomal aneuploidy, segmental chromosomal aneuploidy, mosaicism, parental origin of aneuploidy, fertilization, cleavage, and blastocyst formation were compared between low- and high-DFI groups. We found no significant differences in fertilization, cleavage, or blastocyst formation between the two groups. Compared to that in the low-DFI group, segmental chromosomal aneuploidy rate was significantly higher in the high-DFI group (11.57% vs 5.83%, P = 0.021; OR: 2.32, 95% CI: 1.10-4.89, P = 0.028). The whole chromosomal embryonic aneuploidy of paternal origin was significantly higher in cycles with high DFI than in cycles with low DFI (46.43% vs 23.33%, P = 0.018; OR: 4.32, 95% CI: 1.06-17.66, P = 0.041). However, the segmental chromosomal aneuploidy of paternal origin was not significantly different between the two groups (71.43% vs 78.05%, P = 0.615; OR: 1.01, 95% CI: 0.16-6.40, P = 0.995). In conclusion, our results suggested that high SDF was associated with the incidence of segmental chromosomal aneuploidy and increased paternal whole chromosomal aneuploidies in embryos.

Mitochondria in Human Fertility and Infertility.

In human spermatozoa and oocytes (and their surrounding granulosa cells), mitochondria carry out important functions relating to human fertility and infertility. Sperm mitochondria are not transmitted to the future embryo, but are closely related to the generation of energy needed for sperm movement, capacitation, and acrosome reactions, as well as for sperm-oocyte fusion. On the other hand, oocyte mitochondria produce energy required for oocyte meiotic division and their abnormalities can thus cause oocyte and embryo aneuploidy. In addition, they play a role in oocyte calcium metabolism and in essential epigenetic events during the oocyte-to-embryo transition. They are transmitted to the future embryos and may thus cause hereditary diseases in the offspring. Due to the long life span of the female germ cells, the accumulation of mitochondrial DNA abnormalities often causes ovarian aging. Mitochondrial substitution therapy is the only way of dealing with these issues nowadays. New therapies based on mitochondrial DNA editing are under investigation.

Are systemic progesterone levels in true natural cycle euploid frozen embryo transfers with luteal phase support predictive for ongoing pregnancy rates?

Are serum progesterone (P4) levels on the embryo transfer (ET) day predictive of ongoing pregnancy (OP) following a single euploid blastocyst transfer in a natural cycle (NC) when luteal phase support is routinely given? In single euploid frozen ETs in NC, P4 levels on ET day are not predictive for OP, when luteal phase support (LPS) is routinely added after the ET. In an NC frozen embryo transfer (FET), P4 produced by the corpus luteum initiates secretory transformation of the endometrium and maintains pregnancy after implantation. There are ongoing controversies on the existence of a P4 cutoff level on the ET day, being predictive for the chance of OP as well as of the possible role of additional LPS after ET. Previous studies in NC FET cycles, evaluating and identifying P4 cutoff levels did not exclude embryo aneuploidy as a possible reason for failure. This retrospective study analyzed single, euploid FET in NC, conducted in a tertiary referral IVF centre between September 2019 and June 2022, for which measurement of P4 on the day of ET and the treatment outcomes were available. Patients were only included once into the analysis. Outcome was defined as OP (ongoing clinical pregnancy with heartbeat, >12 weeks) or no-OP (not pregnant, biochemical pregnancy, early miscarriage). Patients with an ovulatory cycle and a single euploid blastocyst in an NC FET cycle were included. Cycles were monitored by ultrasound and repeated measurement of serum LH, estradiol, and P4. LH surge was identified when a rise of 180% above the previous level occurred and P4 levels of ≥1.0 ng/ml were regarded as confirmation of ovulation. The ET was scheduled on the fifth day after P4 rise and vaginal micronized P4 was started on the day of ET after P4 measurement. Of 266 patients included, 159 (59.8%) patients had an OP. There was no significant difference between the OP- and no-OP-groups for age, BMI, and day of embryo biopsy/cryopreservation (Day 5 versus Day 6). Furthermore, P4 levels were not different between the groups of patients with OP (P4: 14.8 ng/ml (IQR: 12.0-18.5 ng/ml)) versus no-OP (P4: 16.0 ng/ml (IQR: 11.6-18.9 ng/ml)) (P = 0.483), and no differences between both groups, when P4 levels were stratified into categories of P4 levels of >5 to ≤10, >10 to ≤15, >15 to ≤20, and >20 ng/ml (P = 0.341). However, both groups were significantly different for the embryo quality (EQ), defined by inner cell mass/trophectoderm, as well as when stratified into three EQ groups (good, fair, and poor) (P = 0.001 and 0.002, respectively). Stratified EQ groups remained the only significant parameter influencing OP in the uni- and multivariate analyses (P = 0.002 and P = 0.004, respectively), including age, BMI, and P4 levels (each in categories) and embryo cryopreservation day. Receiver operator characteristic curve for the prediction of an OP revealed an AUC of 0.648 when age, BMI and EQ groups were included into the model. The inclusion of P4 measurement on ET day into the model did not add any benefit for OP prediction (AUC = 0.665). The retrospective design is a limitation. Monitoring serum P4 levels can be abandoned in NC FET cycles with routine LPS as they do not seem to be predictive of live birth. No external funding was used for this study. The authors state that they do not have any conflicts of interest. N/A.