Maternal Age Research Articles

O-127 Blastocyst expansion speed assay: a putative artificial intelligence-powered tool for embryo selection. Retrospective study involving 2184 blastocysts and 786 PGT-A cycles

Abstract Study question Is blastocyst expansion-speed, examined through Artificial-Intelligence (AI) between time of blastulation (tB) and time of expanding blastocyst (tEB), associated with embryo blastulation and reproductive competence? Summary answer Blastocyst expansion-speed-assay (ESA) was significantly associated with euploidy and live-birth rates, being discordant with clinical embryologists’ ranking in 50% of embryonic cohorts. What is known already Blastocyst expansion is one of the earliest morphogenetic events in mammalian development. It is essential for the establishment of the blastocyst layout and requires trophectoderm integrity. Several studies suggested that expansion is a valuable feature for ranking and prioritizing blastocysts for transfer based on their developmental competence. Time-lapse technology (TLT) implementation in IVF allows a deeper understanding of blastocyst expansion dynamics. In this study, we combined TLT and AI to develop a blastocyst-ESA and investigate its association with embryo blastulation and reproductive competence. Study design, size, duration Retrospective study including 2184 blastocysts cultured in EmbryoScope incubators during 786 PGT-A cycles across 2013-2020. Videos were analyzed through an AI-powered tool (CHLOETM, Fairtility). The expansion-speed was calculated as [(Δ embryo proper area at tEB – embryo proper area at tB) / (Δ tEB - tB)]. ESA was tested for its association with embryo quality, euploidy and live-birth rate (LBR) in 548 vitrified-warmed single euploid transfers. A simulation of ESA putative clinical effectiveness was conducted. Participants/materials, setting, methods ICSI, trophectoderm biopsy of fully-expanded blastocysts without day3 zona-drilling, and qPCR/NGS to assess full-chromosome uniform aneuploidies were performed. tB and tEB, expressed as hours-post-insemination (hpi), embryo proper area (in µm2) at both these stages, and blastocyst quality (EQ-Score from 0 to 1) were all automatically recorded through CHLOE. Possible confounders (e.g., maternal age, male factor, and cause of infertility) were considered. Regression analyses were conducted to adjust the data. Main results and the role of chance The average ESA was 705±458 µm2/hour. Higher ESA was associated with higher EQ-Score. Euploid blastocysts showed higher ESA than aneuploid (761±465 µm2/hour versus 667±449 µm2/hour; p < 0.01). ESA was also associated with LBR per euploid blastocyst transfer (LB: 873±438 µm2/hour versus 736±467 µm2/hour; p < 0.01). Of note, maternal age showed no association with ESA. Multivariate regressions outlined a + 5.1%-increase in the chance of euploidy and a + 6.3%-increase in chance of LB, every +100 µm2/hour-increase in ESA. ESA and embryologists were discordant in grading top-quality embryos in each cohort in 50% of the cases. In 59% of the 352 cohorts where both euploid and aneuploid blastocysts were obtained, ESA would have ranked the former embryos as top-quality. In the 216 cycles with ≥2 euploid blastocysts obtained and ≥1 transferred, ESA would have prioritized (i) a competent embryo in 46% of the cases, (ii) an incompetent embryo in 20%, but (iii) in 33% its value could not be assessed (i.e., the top embryo according to ESA has not been transferred yet). When compared to the embryologists, ESA would have been (i) equally-effective in 49% of the cases, (ii) more effective in 12-24%, and (iii) less effective in 5%-26%. Limitations, reasons for caution Retrospective single-center study. Both continuous and sequential media were used, although they were not associated with the outcomes under investigation. To assess the clinical value of ESA, a prospective study among first transfers is warranted. Wider implications of the findings ESA is an automated, unbiased, and easily-applicable measurement that certainly deserves further appraisal. If validated in prospective studies, AI-based selection algorithms could include this assessment to increase their predictive power. Trial registration number None

O-321 A foundation model for embryology trained on time-lapse images

Abstract Study question Can a generalizable computer vision foundation model be trained using self-supervised learning on time-lapse embryo images to perform multiple clinically relevant downstream tasks? Summary answer We developed FEMI, a foundation model trained on eight million time-lapse images, to perform multiple clinical tasks, including blastocyst quality scoring, ploidy prediction, and segmentation. What is known already In vitro fertilization success critically depends on choosing viable embryos, a process hampered by current limited diagnostic tools, high costs, and ethical concerns. In recent years, various medical fields have increasingly explored foundation models using vision transformer architectures. These models, trained in a self-supervised manner on vast unlabeled image datasets, perform various clinically relevant tasks. Once trained on a massive unlabeled dataset, the encoder portion of the foundation model can be extracted and subsequently fine-tuned with a labeled dataset to perform various clinically relevant tasks like classification, regression, and segmentation. Study design, size, duration The foundation model was trained on 8 million Embryoscope® (E-SD) and Embryoscope+® (E+) images spanning clinics in the United States, Canada, and Europe. Datasets contained various additional information, such as ploidy status, blastocyst scores (numerical values from 3 to 14 based on Zhan et al. 2020), and segmentation masks (for trophectoderm [TE], inner cell mass [ICM], and zona pellucida [ZP]) that were used for training downstream tasks. Participants/materials, setting, methods A masked autoencoder architecture is trained on time-lapse images to create a foundation model (FEMI). The encoder from the autoencoder is extracted and fine-tuned on three image-based downstream tasks: ploidy prediction, blastocyst scoring, and embryo component segmentation. The performance of the fine-tuned foundation model is compared to VGG16 architectures, specifically trained for each downstream task. We evaluated performances using the area under the receiver-operating-characteristic (AUROC), mean absolute error (MAE), and mean intersection over union (mIoU). Main results and the role of chance The training dataset for the downstream tasks consisted of image data and labels, including PGT-A ploidy results (euploid or aneuploid), blastocyst scores (3-14), and segmentation masks. No clinical data like maternal age was used in order to assess FEMI’s learning capabilities from only time-lapse images against VGG16 model baselines. Both FEMI and baseline models used the same data splits for consistent comparisons. For ploidy prediction, models trained on a Weill Cornell Medicine (WCM) dataset were validated using WCM, Florida, and Spain data. FEMI achieved a 0.610 ± 0.004 AUROC, outperforming the baseline’s 0.590 ± 0.005. In blastocyst score prediction, FEMI attained a superior MAE of 0.099 ± 0.002 compared to the baseline’s 0.118 ± 0.001. For embryo segmentation (TE, ICM, ZP), a publicly available dataset from Simon Fraser University was split for training and validation. FEMI exceeded the baseline in mIoU for TE segmentation (0.738 ± 0.002 vs. 0.726 ± 0.002) and was comparable in ICM (0.832 ± 0.008 vs. 0.821 ± 0.011) and ZP segmentation (0.779 ± 0.002 vs. 0.778 ± 0.010). Limitations, reasons for caution The current version of FEMI is trained on a subset of our available datasets and can be improved through further training. Moreover, the downstream ploidy and blastocyst score models may be biased by errors in PGT results and the subjectivity of embryologists, respectively. Wider implications of the findings A generalizable foundational model for IVF time-lapse imaging aids clinicians in embryo selection by providing various clinical insights for better decision-making. FEMI, once fully trained, will be publicly accessible to the scientific community allowing for researchers to fine-tune FEMI on their own clinic-specific applications with their own image datasets. Trial registration number not applicable

P-176 A novel electrophoretic sperm isolation system achieves equivalent ICSI outcomes to the combined density gradient centrifugation and swim-up method in a significantly shorter processing time

Abstract Study question Does an electrophoretically sperm isolation method, the Felix™ system, have clinical or operational advantages over the combined density gradient centrifugation and swim-up (DGCS) method? Summary answer The Felix™ system produces results that are comparable to the DGCS method, however, Felix™ has the ability to isolate spermatozoa more efficiently. What is known already The isolation of healthy spermatozoa to improve ART outcomes has received enormous attention in the literature recently. Although swim-up and density gradient centrifugation are the most common sperm separation methods, the optimal technique is still unclear. Recently, good sperm selection approaches using various non-centrifugal devices have emerged. Previous reports have shown that a quick and simple sperm separation device, the Felix™ system, can isolate high-quality spermatozoa and improve the results of ART. The Felix™ system combines electrophoresis and separation membranes to collect negatively charged mature normal sperm. Study design, size, duration A prospective oocyte sibling study was conducted between March 2023 and December 2023. The study involved infertile couples (n = 45) with a semen volume of 2.3 mL or more, a motile sperm concentration of 2 million/mL or more, maternal age under 43 years, and at least four mature oocytes at the time of oocyte retrieval. Ejaculated semen was prepared separately using DGCS and Felix™ on the day of oocyte retrieval and subsequent ICSI results were analyzed. Participants/materials, setting, methods In the DGCS group, semen was layered on a 45/90% of sperm grad and centrifuged for 15 minutes, followed by a 30 min swim-up. Subsequently, 0.3 mL of supernatant was collected. In the Felix™ group, 0.3 mL of sperm was collected following six minutes of membrane hydration and electrophoresis. Sperm concentration, total motility, progressive motility and DNA fragmentation (SDFR) were measured. Fertilization, blastocyst development, good-quality blastocyst development, and embryo utilisation rates were also assessed. Main results and the role of chance The average female age at the time of oocyte retrieval was 36.5±4.0 years, and the average male age was 38.1±5.9 years. Of 401 oocytes, 200 and 201 oocytes underwent ICSI using sperm prepared by DGCS and Felix™, respectively. The average processing time of Felix™ was significantly shorter than that of DGCS (54.0±4.3 vs. 7.4±0.2 minutes, p < 0.001, respectively). No significant difference was observed between the DGCS and Felix™ groups for sperm concentration (p = 0.49), motility (p = 0.05), progressive motility (p = 0.34), and SDFR (p = 0.09). Furthermore, there were no significant differences between the two groups with regards to fertilization rate (81.0% vs. 80.6%, p = 0.92), blastocyst development rate (52.9% vs. 58.4%, p = 0.60), good-quality blastocyst development rate (26.1% vs. 35.7%, p = 0.06), and embryo utilisation rate (54.3% vs 58.0%, p = 0.50). Limitations, reasons for caution Although the DGCS method was used as a comparison group, it is necessary to compare the Felix™ system with DGC alone, swim-up alone, or other sperm isolation methods. Larger, more powered studies are required to determine the impact on pregnancy and live birth outcomes. Wider implications of the findings The Felix™ system is a viable option for sperm isolation. While DGCS requires complicated procedures and time-consuming process including centrifugation and incubation; Felix™ requires six minutes only. The Felix™ system reduces processing times significantly and therefore can improve laboratory operations and workflow. Trial registration number Not Applicable

P-784 Visceral and subcutaneous adipose tissue in children born after assisted reproductive technology with frozen and fresh embryo transfer

Abstract Study question Is the distribution of abdominal visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) comparable between children following assisted reproduction and natural conception (NC)? Summary answer Children following frozen embryo transfer (FET) have significantly lower VAT/SAT-ratio than children following NC. However, this is not believed to be clinically important. What is known already It is well-established that an adverse distribution of the abdominal adipose tissue with high VAT/SAT-ratio increases the risk of cardiometabolic diseases, hence the VAT/SAT-ratio is closely related to the metabolic profile. Children conceived after FET and fresh embryo transfer (Fresh-ET) are at risk of being born large-for-gestational-age (LGA) and small-for-gestational-age (SGA), respectively. Both, being born LGA and SGA, increases the risk of obesity in later life. To the best of our knowledge, no studies have reported the VAT/SAT-ratio in children following assisted reproductive technology (ART) (both FET and Fresh-ET). Study design, size, duration Prospective observational cohort study including 150 singletons aged 7- to 10-years old conceived after FET (n = 50), Fresh-ET (n = 50) and NC (n = 50). All children were born in Eastern Denmark. The study is conducted between November 2018 and August 2020. Participants/materials, setting, methods This is a substudy af the “Health in Childhood following Assisted Reproductive Technology” (HiCART) study. The children underwent abdominal magnetic resonance imaging (MRI) for the quantification of abdominal adipose tissue along with measurements of anthropometric measurements, blood pressure, fasting blood samples and Dual-energy X-ray Absorptiometry (DXA) scan. The volumes of VAT and SAT were semi-automatically quantified blinded for the mode of conception. Multivariate linear regression analysis of the VAT/SAT-ratio was performed to adjust for confounders. Main results and the role of chance The VAT/SAT-ratio differed significantly between the three groups (mean SD; FET 0.26 (0.08), Fresh-ET 0.29 (0.07), NC 0.30 (0.08), ANOVA, p = 0.014). Pairwise comparison revealed that children conceived after FET had significantly lower VAT/SAT-ratio as compared to children conceived after NC (p = 0.007). In addition, children following FET tended to have lower VAT/SAT-ratio as compared to the Fresh-ET group (p = 0.059). Multivariate linear regression analysis of the VAT/SAT-ratio confirmed a lower VAT/SAT-ratio in FET after adjustment for potential confounding factors (child age and sex, maternal age at delivery and maternal BMI at pregnancy and child birth weight and BMI). Repeat-analysis of 20 MRI showed good intra-rater reliability of VAT and SAT quantifications. As previously reported in the HiCART studies, there were no differences between the groups in anthropometric measurements including body mass index (BMI), lean body mass, blood pressure or high-sensitivity C-reactive protein (hs-CRP). However, the level of triglycerides was found to be significantly higher in Fresh-ET compared to FET and NC (p = 0.028). Limitations, reasons for caution Selection bias cannot be excluded as only children from Eastern Denmark were invited. The observational nature of a cohort study prevents the establishment of causality. Wider implications of the findings Reassuringly, this study found no clinical important difference in abdominal adipose tissue between children following ART (both FET and Fresh-ET) and NC. However, future studies in larger cohorts are warranted. Trial registration number ClinicalTrials.gov identifier: NCT03719703

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-046 Ejaculatory abstinence: is there any association with embryo developmental competence? A Retrospective Study in time-lapse incubators

Abstract Study question Is ejaculatory abstinence (EA) associated with fertilization and blastocyst development in ICSI cycles with embryo culture in time-lapse incubators? Summary answer There is no association between length of EA and fertilization, blastocyst development or morphology. What is known already Semen analysis is pivotal in medically assisted reproduction (MAR), serving as a significant indicator of male partners’ health. The period of EA is a relevant factor that can affect sperm quality. Numerous studies described EA association with clinical outcomes, while its impact on embryological results remains inconclusive. Recent meta-analyses, despite a conflicting literature, highlighted positive effects of shorter abstinence periods. A recent study suggested that prolonged abstinence might reduce fertilization rate in ICSI cycles without time-lapse imaging, thereby reducing usable zygotes’ rate Study design, size, duration Retrospective study including all oocytes (N = 7164) from 1148 ICSI cycles (N = 902 couples) cultured in time-lapse incubators at a single IVF center between January 2020 and December 2022. Only non-donor fresh gametes were included (Mean Maternal age: 38.5±4 years; Paternal Age: 41±5.6 years). The primary embryological outcomes were fertilization rate and type, and blastocyst development per inseminated oocyte and per 2PN-zygote. Secondary outcomes were euploidy, day of blastocyst development and Gardner’s morphology. Participants/materials, setting, methods Semen analysis was conducted according to WHO. All oocytes were cultured up to day7 in EmbryoScope or GERI incubators with continuous media and refresh in day5 (if needed). If requested, PGT-A was performed to assess full-chromosome uniform aneuploidies. Univariate and multivariate logistic regressions were conducted. Possible confounders were assessed among ovarian stimulation, maternal, paternal, sperm, and IVF cycles characteristics. Associations were confirmed with Generalized estimating equations (GEE) to account for repeated measurements among oocytes’ cohort. Main results and the role of chance Logistic regressions and GEE were conducted to assess associations between possible confounders and blastocyst development per inseminated metaphase-II oocyte. Maternal age, sperm concentration, and motility A+B were associated. The same confounders were reported on blastocyst development among 2PN-zygotes. The days of EA (1-8 days) did not show any association with fertilization (p = 0.198) or blastocyst development per metaphase-II oocyte (p = 0.495). No association was reported with abnormal fertilization, neither combining all abnormalities in a single group, nor clustering them as 1PN, 3PN, 3.1PN, and multi-PN. An increasing trend was reported in blastocyst development among 2PN-zygotes according to the length of EA (1-day: 17/37,45.9%; 2-days: 190/424,44.8%; 3-days: 1405/2574,54.6%; 4-day: 746/1410,52.9%; 5-days: 450/827,54.4%; 6-days: 70/133,52.6%; 7-days: 46/101,45.5%; 8-days: 85/162,52.5%), but this was not significant in a multivariate logistic regression (p = 0.887). Length of EA showed no association with euploidy (p = 0.424), nor with day of development (p = 0.966) and Gardner’s inner cell mass (p = 0.204) or trophectoderm (p = 0.428) morphology. Limitations, reasons for caution This study is inherently limited by its retrospective design. The limited number of cycles included was balanced using time-lapse incubators to improve the accuracy of fertilization check and benefit from an undisturbed embryo culture environment. Artificial Intelligence will be implemented to automate developmental timings’ annotation and objectify embryo quality assessment. Wider implications of the findings The length of EA seems not associated with embryological outcomes, adding to the controversy on this topic. Here, we included only ICSI cycles with blastocyst culture conducted in time-lapse to enhance the quality of the evidence. Larger datasets and diverse settings are now required, especially enriched with poor prognosis patients. Trial registration number not applicable

P-370 The age-associated increase in ovarian stiffness compromises follicle development and induces changes in the follicles’ transcriptome, resulting in poor-quality oocytes

Abstract Study question Does the age-associated increase in ovarian stiffness trigger the decline in oocyte quantity and quality observed in women of advanced maternal age? Summary answer An encapsulated in vitro follicle culture mimicking ovarian biomechanics revealed that age-associated increase in stiffness impacts follicle growth, modulates follicles’ transcriptome and reduces oocyte quality. What is known already Female reproductive aging is characterized by a progressive decline in oocyte quality and quantity, resulting in sub-fertility. We recently discovered that with aging the mouse ovary assumes a pro-fibrotic milieu which induces ovarian stiffness. Moreover, human ovaries also become stiffer with aging; however, it is not known whether this age-associated increase in stiffness impacts ovarian function. The present study aims to examine whether stiffness is a novel mechanism that mediates the age-associated progressive decline in oocyte quality and how stiffness affects the transcriptome of growing follicles, resulting in poor-quality oocytes. Study design, size, duration We used an in vitro alginate-encapsulated follicle culture to mimic young and old environments in which follicles grow. Alginate hydrogel synthesis replicated the soft (0.5% - 1.79±0.08 kPa) and stiff (2% - 4.56±2.03 kPa) environments of young and aged ovaries, respectively. Mouse secondary follicles were cultured in 0.5 or 2% alginate and used for i) long-term culture (day 0-10) to evaluate follicle development and oocyte quality; ii) short-term culture (hour 0-24) to investigate follicles’ transcriptome. Participants/materials, setting, methods During the long-term culture, markers of follicle development were evaluated (n = 31 follicles), including: follicle size, estradiol production (ELISA), cell death (cleaved-caspase3,CC3; immunohistochemistry). At D10, oocyte quality was inspected on isolated oocytes. For the short-term culture experiment, 32 follicles were analyzed by RNAsequencing at 3h (baseline) and 24h. We compared 3h and 24h gene expression (DESeq2,R/Bioconductor) in 0.5% and 2%. Significant differences reported at padj<0.05 and log2FoldChange±2. Two mice strains (CB6F1;CD1) were used in all experiments. Main results and the role of chance Follicles cultured in stiff environment showed a significant reduction in follicle size compared to follicles cultured in soft environment (0.5% 226.9±17.4um, 2% 160.8±9.9um; p < 0.0001). These size differences suggest that granulosa cells are not proliferating in a stiff environment. Estradiol production was reduced in follicles cultured at 2% (0.5% 11.3±15.9ng/ml, 2% 0.3±0.5ng/ml, p = 0.296), while CC3 intensity was increased (CC3 staining intensity/area, 0.5% 20.4±5.8a.u., 2% 27.1±4.3a.u., p < 0.05), suggesting that the stiff environment impacts granulosa cell viability. Oocyte quality significantly declined in follicles cultured in 2%, with 68.9±16.8% of the oocytes degenerated, compared to 23.6±9.2% in 0.5% alginate. The use of two different mice strains yielded comparable results. Using RNAsequencing analysis, we found 1033 differentially expressed genes (DEGs) between 3h and 24h of culture in 2% alginate, mostly involved in apoptosis, inflammation and collagen processes, biological pathways typically associated with aging. The 64% of those DEGs were upregulated at 24h. Conversely, the 1295 DEGs (61% upregulated at 24h) detected comparing 3h and 24h in 0.5% alginate were relate to metabolism, cell cycle and development pathways, biological processes associated to the normal behavior of growing follicles. These differences in gene expression suggest an early-response in the transcriptome of follicles cultured in a stiff environment. Limitations, reasons for caution The stiffness of the alginate gels was based on whole ovary biomechanics; future investigations are needed to measure each follicle-stage-specific stiffness. The changes in follicles’ transcriptome in the stiff environment were evaluated only in short-term culture. This study utilizes in vitro approaches, in vivo modulation of stiffness is currently undergoing. Wider implications of the findings Increased ovarian stiffness is observed in conditions such as aging or ovaries exposed to cancer treatments. Studying how stiffness impacts oocyte quality will allow the development of suitable in vitro systems to support human folliculogenesis and in vivo approaches to modulate biomechanics of the ovary, preserving female fertility. 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-063 How does the exclusion of blastomeres affect the outcome of euploid blastocysts?

Abstract Study question Do euploid blastocysts with excluded blastomere(s) have the same probability of live birth when compared to euploid blastocysts without excluded blastomere(s)? Summary answer Euploid blastocysts with excluded blastomere(s) have significantly worse pregnancy outcomes, with the live birth rate being 10% lower than those without any exclusion pattern. What is known already Blastomeres that have arisen at any time throughout cleavage-stage are sometimes excluded from the formation of the blastocyst and once isolated, those cells do not take further part in preimplantation development. Embryos harboring cell exclusion were found to have altered morphokinetic profiles; S2(second synchrony) and ECC3(embryonic cell cycle 3) were described as useful in identifying increased odds of FHB when a cell exclusion event was present (Kakulavarapua et al., 2023). Besides, pregnancy and live birth rates were reported to progressively decrease in a non-PGT-A (preimplantation genetic testing for aneuploidy) tested cohort with excluded or extruded blastomere(s) (Coticchio et al., 2021). Study design, size, duration This retrospective study included 667 euploid blastocysts with excluded blastomere(s) and 3830 euploid blastocysts without excluded blastomere(s). All were transferred between 2017 and 2023. No discrimination was made between cells failing to compact because of their exclusion from the compaction process (excluded from a partially compacted morula), and cells extruded after an initial inclusion in the compacted embryonic mass (extruded from a partially compacted morula). Participants/materials, setting, methods A total of 4497 IVF patients treated in a single private clinic were included in this retrospective study. Their blastocysts were subjected to PGT-A mainly for advanced maternal age. The analysis was done by next generation sequencing (NGS) (Ion Torrent S5, Thermo Fisher). Vitrified-warmed euploid blastocysts were then transferred in a next cycle. Pregnancy and miscarriage rates were compared between blastocysts with and without excluded blastomere(s) by chi-square test. Main results and the role of chance The outcomes of euploid blastocysts with excluded blastomeres (n = 667) were compared to euploid blastocysts without excluded blastomeres (n = 3830). The biochemical pregnancy rate was 71.6% and 77%, respectively (p = 0.0026). The clinical pregnancy rate was 61.3% and 70.6%, respectively (p = 0.0001). The ongoing pregnancy rate was 53.7% and 63.3%, respectively (p = 0.0001). The biochemical miscarriage rate nearly doubled with the presence of excluded blastomere(s) (14% vs. 8.3%, respectively; p = 0.0001). The clinical miscarriage rate was 12.7% and 10.4%, respectively (ns). Ultimately, the live birth rate was 46.3% for the blastocysts with excluded blastomere(s), compared to 57.6% for the blastocysts without excluded blastomere(s) (p = 0.0001). Limitations, reasons for caution No discrimination was made between cells excluded from a partially compacted morula and cells extruded after an initial inclusion in the compacted embryonic mass. Wider implications of the findings Although blastocysts with excluded blastomere(s) are not the first choice for transfer, our results show that, although at lower rate, successful pregnancies and live births occur. A complete time-lapse culture evaluation not limited to cell division time points may propose indicators to identify higher implantation potential blastocysts with excluded blastomere(s). Trial registration number N/A

P-189 Can artificial intelligence outperform traditional non-invasive assessments in prioritizing euploid blastocysts for transfer? A retrospective intra-cohort analysis of 786 PGT-A cycles

Abstract Study question Are Artificial Intelligence (AI)-powered tools more efficient than traditional non-invasive assessments to prioritize euploid blastocysts for transfer? Summary answer Only one third of cycles yielded ≥3 blastocysts with different diagnoses. In these cycles, AI and traditional grading prioritized euploid embryos with comparable performance. What is known already While embryo morphology and developmental pace to blastocyst are associated with chromosomal and reproductive competence, their evaluation remains subjective and lacks reproducibility. The introduction of Time-lapse technology (TLT) in IVF has provided valuable insights into preimplantation development but has not enhanced embryologists’ reproducibility. Recently, AI models integrated with TLT offered the potential for automating and standardizing assessments. Whole-chromosome testing for uniform aneuploidies still is the strongest predictor of embryo (in)competence. In fact, if transferred, >98% of aneuploid blastocysts fail to result in a live-birth. AI tools strive to non-invasively predict blastocyst (an)euploidy, yet their clinical utility requires intra-cohort testing. Study design, size, duration Retrospective blinded analysis of 786 PGT-A cycles (maternal-age:38.9; years:2013-2020; 2184 blastocysts). For static assessment, 3 embryologists used Gardner’s grading at the time of biopsy (t-biopsy). For morphodynamic assessment, time of blastocyst-expansion (tEB) and embryo-area (embA at tEB) were also annotated. For AI assessment, scores were generated with three commercially-available models. All approaches were compared for their effectiveness in ranking euploid blastocyst(s) as top-quality among cohorts with ≥3 blastocysts and ≥1 euploid and ≥1 aneuploid (N = 279/786,35.5%). Participants/materials, setting, methods Embryologists ranked AA-blastocysts as top-quality (score=1), CC-blastocysts as worst-quality (score=9), prioritizing trophectoderm over ICM, with t-biopsy as third criterion. Morphodynamic assessments followed the same hierarchy but using tEB and embA in place of t-biopsy. For AI, higher scores corresponded to higher priority. For all assessments, coefficients of variation (CV=SD/mean) were calculated to assess data dispersion within cohorts with ≥3 blastocysts (N = 363/786,46.2%). Intra-cohort CVs were tested for their association with the likelihood of prioritizing euploid blastocysts. Main results and the role of chance Intra-cohort CVs were 0.71±0.35, 0.28±0.14, 0.58±0.26, and 0.56±0.34 for Gardner’s score, AI model-1, -2, and -3, respectively. Spearman’s correlations between Gardner’s score and AI model-1, -2, and -3 Intra-cohort CVs were 0.32, 0.34, and 0.14, respectively. AI model-1 and -2 were strongly correlated (0.63), but poorly with model-3 (0.21 and 0.22). Euploid blastocysts were prioritized in 163 cohorts by static assessments (N = 163/279,58.4%), 179 (64.2%) by morphodynamic, 174 (62.4%), 190 (68.1%), and 145 (52%) by AI model-1, -2, and -3. A maternal age-adjusted 0.1-larger Gardner’s grading intra-cohort CV correlated with higher likelihood of prioritizing euploid over aneuploid blastocysts (multivariate-OR:1.1, 95%CI:1.03-1.19, adjusted-p=0.005). No association was reported with AI models intra-cohort CVs. Notably, blastocysts affected from aneuploidies compatible with implantation were prioritized over euploid in 59 cohorts (N = 59/279,21.1%) by static assessment, 54 (19.4%) by morphodynamic, 52 (18.6%), 48 (17.2%), and 54 (19.4%) by AI model-1, -2, and -3. A maternal age-adjusted 0.1-larger Gardner’s grading intra-cohort CV correlated with lower likelihood of prioritizing aneuploid blastocysts compatible with implantation over euploid (multivariate-OR:0.9, 95%CI:0.83-0.98, adjusted-p=0.010). No association was reported with AI models intra-cohort CVs. Limitations, reasons for caution While representing the real-life scenario of a large PGT-A program, this study is a single center retrospective analysis in advanced maternal age women with a limited number of cycles with ≥3 blastocysts showing both euploid and aneuploid. Other AI-powered models exist, some specifically trained to predict euploidy. Wider implications of the findings Trophectoderm biopsy and testing aim to prevent the transfer of chromosomally-abnormal blastocysts. Although traditional/AI-based grading correlate with euploidy, their goal is prioritizing embryos for transfer, irrespective of the IVF setting (with or without PGT-A). Future research should concentrate on emphasizing live-birth prediction over misleadingly considering them as non-invasive PGT-A surrogates. Trial registration number None

P-435 Superiority of modified natural cycle vs hormonal replacement therapy in non-menopausal egg recipients of advanced maternal age: clinical outcomes from 1273 single blastocyst transfer cycles

Abstract Study question Is the modified natural cycle (mNC) better than hormonal replacement therapy (HRT) for endometrial preparation in non-menopausal egg recipients of advanced maternal age? Summary answer In women above 40 years, mNC shows better reproductive and obstetrical outcomes compared to hormonal replacement therapy, especially in the subgroup 45-50 years old. What is known already There is extensive evidence showing the superiority of mNC over HRT in terms of live birth rates, miscarriages, and even obstetric complications. Apparently, the presence of the corpus luteum plays a major role in endometrial receptivity and embryo implantation, probably allowing for better placentation. Nevertheless, there is a general belief that the ovulatory cycle of older women is not the best choice for endometrial preparation, although paradoxically they are the group of patients who could benefit most from the reduction of obstetric complications. An increase in cancellation rates for mNC is claimed, without consistent data available. Study design, size, duration Retrospective single-centre comparative clinical study, including 1273 endometrial preparation cycles for frozen single-blastocyst transfer between January 2020 and December 2022: 892 (70,2%) on HRT protocol and 381 (29,8%) on mNC protocol. The two groups were homogeneous for donor’s age (25,7 years HRT vs 25,3 years mNC), recipient’s age (42,2 years HRT vs 42,1 years mNC), severe male factor (6% HRT vs 5% mNC), and the need for a sperm donor (28% HRT vs 26% mNC). Participants/materials, setting, methods Among 1273 started non-menopausal recipient’s cycles, we compared cancellation rates between HRT and mNC due to either medical reasons (inadequate endometrium, bleeding, premature ovulation) or other issues (no embryo survival, COVID-19, etc). We studied the outcomes of the 1096 elective frozen single-blastocyst transfers performed, comparing HRT and mNC cycles for: pregnancy, clinical pregnancy, miscarriage, live birth and <35weeks preterm birth rates. We stratified the results by age and used Chi-square test to compare groups (p < 0,05). Main results and the role of chance The overall cancellation rate was 13.9%. No significant differences in cancellation for medical reasons were found between HRT (7%) and mNC (6.8%), p = 0.93. When we analyse results stratified by age, patients aged 39 or less (270 cycles: HRT 75.9%, mNC 24.1%) showed no statistically significant differences in pregnancy (58% vs 64.6%, p = 0.35), clinical pregnancy (50.7% vs 56.9%, p = 0.34), miscarriage (18.3% vs 18.9%, p = 0.93), and live birth rates (41.5% vs 46.2%, p = 0.5). In the 40-44 age group (494 cycles: HRT 64.2%, mNC 35.8%), no statistically significant differences were found between HRT and mNC in pregnancy (58.2% vs 61.5%, p = 0.47), clinical pregnancy (50.8% vs 51.1%, p = 0.96), and live birth rates (40.4% vs 45.8%, p = 0.25). The HRT group had a significantly higher miscarriage rate (21%) compared to mNC (10%), p = 0.03. For patients aged 45-50 years (332 cycles: HRT 72%, mNC 28%), no statistically significant differences were observed in pregnancy (56.5% vs 62.4%, p = 0.33) and miscarriage rates (24.3% vs 15.1%, p = 0.18). Clinical pregnancy (44.8% Vs 57%, p = 0.04) and live birth rates (33.9% vs 48.4%, p = 0.01) were significantly higher in the mNC group compared to HRT. No significant differences were found between HRT and mNC in preterm birth rates <35 weeks (3.6% vs 4.6%, p = 0.75). Limitations, reasons for caution This study is a single-centre retrospective comparative trial. Our homogeneity study included several confounding variables, however other bias such as severe uterine factor, embryo quality or repeated implantation failure cases were not taken into account. Wider implications of the findings Modified natural cycle should be encouraged as the first option for endometrial preparation in non-menopausal egg recipients of advanced maternal age, having shown superiority over hormonal replacement therapy in patients aged 45-50 years. Moreover, modified natural cycles are more patient friendly and cost-effective, with no increase in the cancellation rates. Trial registration number not applicable

P-109 Non-invasive Genetic Testing (niPGTA) and Laser Assisted Hatching (LAH) may improve pregnancy and ongoing pregnancy rates in patients of advanced maternal age

Abstract Study question Does niPGTA and LAH improve pregnancy and ongoing pregnancy rates in patients of advanced maternal age? Summary answer niPGTA increases pregnancy and clinical pregnancy rates in patients aged ≥38 years compared to those without tested embryos, while LAH may further improve pregnancy outcomes. What is known already Embryos release cell-free DNA (cf-DNA) in the IVF culture media, which can be analyzed for chromosomal constitution. The benefit that niPGTA holds over PGT-A is that it is a non-invasive test and could potentially produce comparable results. Therefore, the time to pregnancy and the risk of miscarriage could be reduced in comparison to an IVF treatment with non-tested embryos. Previous studies have shown high concordance with trophectoderm biopsies in PGT-A patients and improved outcomes with LAH in frozen-thawed cycles. However, there is a lack of studies investigating clinical pregnancy rates with niPGTA. Study design, size, duration Retrospective cohort study including 74 patients aged ≥38 years, who underwent Single Frozen Embryo Transfer (FET) from May 2021 to February 2023. Patients who had Endometrial Receptivity Assay (ERA) were excluded. Participants/materials, setting, methods 32 couples had niPGTA; Spent Blastocyst Media (SBM) for each blastocyst were collected on Day 6 and analyzed using the EMBRACE test. Embryos were vitrified. 42 couples had their blastocysts vitrified on Day 5/6 without testing. LAH was introduced to all patients in October 2021. Before that, no LAH was performed. Embryos were transferred to patients 2 hours post-warming after LAH. Pregnancy test was performed 10 days after FET and clinical pregnancy was established. Main results and the role of chance There was a statistical significant increase of pregnancy rate (p = 0.017, Pearson’s Chi-Squared test) and a trend towards higher clinical pregnancy rate for niPGTA patients versus those who had a transfer of not tested embryos. Regarding LAH, there was a trend towards higher pregnancy and clinical pregnancy rates in both niPGTA and not tested group. Results for pregnancies and clinical pregnancies are presented in Table 1. Limitations, reasons for caution More patients are required to confirm clinical significance of pregnancy and clinical pregnancy rates of niPGTA over not tested embryos and if LAH can improve the outcomes. A review of patients who had previous implantation failures and miscarriages in addition to ERA could yield clearer conclusions. Wider implications of the findings The results of this study are encouraging for niPGTA combined with LAH for patients aged ≥38 years. Further studies on niPGTA and modifications of the protocol for increased concordance rates and shorter time in embryo culture should be explored. 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

P-254 Optimizing clinical outcomes in failed ICSI Donor cycles: evaluating the efficacy of Assisted Oocyte Activation

Abstract Study question Does Assisted Oocyte Activation (AOA) confer benefits to couples undergoing donor ICSI cycles with a history of poor clinical outcomes and poor sperm quality? Summary answer AOA demonstrates efficacy in enhancing reproductive outcomes in donor ICSI cycles, in cases where oocyte activation deficiency (OAD) is attributed to a sperm factor. What is known already Oocyte activation is a spatial-temporal process caused by a series of intracellular calcium oscillations (Ca2+)triggered by the entry of sperm into the oocyte. Insufficient Ca2+ oscillations, primarily attributed to the absence of sperm-specific phospholipase C zeta (PLCζ) can result in OAD that leads to unsuccessful fertilization and poor embryo growth. Ionomycin and calcimycin, are the most common activating agents used to restart meiosis by raising intercellular Ca2+. As far as our knowledge extends, there aren’t studies reporting clinical outcomes on the use of AOA in patients who undergo cycles both without and with this treatment in donor oocytes. Study design, size, duration This study includes 38 couples undergoing ICSI cycles with donor oocytes from January 2020 to December 2023. After a previous cycle with a fertilization rate of < 50% and poor clinical outcomes, these couples underwent a subsequent attempt with AOA. Maternal age of recipients was 42±4.7 and 42±5 years in cycles without and with AOA (P=NS). Fertilization, blastocyst quality, implantation, clinical pregnancy rates (CPR) and pregnancy loss were compared between the cycles with and without AOA. Participants/materials, setting, methods Oocytes were exposed to a ready-to-use solution of Ca2+-ionophore A23187 (GM508 Cult-Active, Gynemed, Germany) for 15 min after sperm injection. Embryos were cultured in a time-lapse technology (Geri®) andevaluated according to the Gardner blastocyst grading system. Embryo transfer was performed on Day 5 or Day 6. Vitrified and fresh ejaculated sperm samples were used. T-test and Fisher’s Exact test were used for statistical analysis. P-value was considered statistically significant at a threshold of < 0.05. Main results and the role of chance A total of 38 men underwent ICSI cycles alongside their female partners, utilizing donor oocytes. The men’s average semen parameters were as follows: concentration of 15.3±25 x 106/mL, motility of 20.1±15%, and 2.0±1% morphology. In the initial 41 cycles without the AOA procedure, an average and standard deviation of 5.8±3 donor oocytes were injected, resulting in a fertilization rate of 33.9% (81/239) and a blastocyst rate of 30.8% (25/81). Among these cryopreserved embryos, 72% (18/25) were identified as top-quality, characterized by A and/or B quality of both inner cell mass and trophectoderm. Couples (N = 13) who underwent a transfer had an implantation rate of 19% (4/21), with CPR of 28.5% (4/14), which resulted in a pregnancy loss of 75% (3/4). Subsequently, these couples underwent 47 ICSI cycles with AOA, injecting an average of 5±1.7 donor oocytes (P=NS). This led to a significantly higher fertilization rate of 74.2% (176/237, P < 0.0001) and a blastocyst rate of 51.7% (91/176, P < 0.01), with 61% (56/91) identified as top-quality embryos (P=NS). Couples (N = 31) who underwent a transfer had a notable increase in implantation rate to 52% (25/48, P < 0.05), accompanied by a significant rise in CPR, reaching 71.4% (25/35, P < 0.01). Importantly, pregnancy loss decreased to 26% (6/23, P=NS). Limitations, reasons for caution The number of patients included in this study is limited. Although we excluded that OAD could be oocyte-related by using donor gamete, PLCζ or mouse oocyte activation tests were not performed to confirm sperm-related OAD. All patients, diagnosed with teratozoospermia, likely exhibit abnormalities causing PLCζlocalization issues or its absence. Wider implications of the findings In our study, sperm’s inability to generate accurate calcium signaling pathways correlates with poor clinical outcomes, even with donor oocytes. Consequently, for couples facing Oocyte Activation Deficiency attributed to sperm factors, implementing the AOA procedure significantly improves fertilization, blastocyst formation, implantation, and clinical pregnancy rates. Trial registration number na

P-125 Relationship between ooplasmic fluidity and oocyte degeneration examined by image analysis after Piezo-intracytoplasmic sperm injection (ICSI)

Abstract Study question Can image analysis of ooplasmic fluidity in Piezo-ICSI predict unintentional membrane rupture (UMR) and subsequent oocyte degeneration? Summary answer Image analysis of ooplasmic fluidity using “optical flow” can be used to predict UMR and subsequent oocyte degeneration in Piezo-ICSI. What is known already UMR during Piezo-ICSI sometimes leads to oocyte degeneration, but not all oocytes degenerate. Leakage of ooplasm is often observed in degenerating oocytes after Piezo-ICSI. We speculated that the occurrence of ooplasmic leakage could relate to the ooplasmic fluidity. Optical flow is an image processing algorithm that analyses the movement of an object in a motion picture. In this study, the motion vectors for all pixels in the image were calculated using the dense optical flow method. Study design, size, duration We developed an image analysis system that detects the movement of the ooplasm on Piezo-ICSI videos using the OpenCV library. Videos of 96 oocytes recorded during Piezo ICSI (mean maternal age: 39.2±5.4 years) retrieved in our institute between January 2020 and September 2023 were retrospectively analyzed using the system. To minimize the potential influence of technical factors, Piezo-ICSI was performed by two embryologists. Participants/materials, setting, methods The oocytes were characterized by survival or degeneration and labeled with and without UMR. To evaluate the ooplasm fluidity, we analyzed the vertical motion vectors of all pixels between the start to puncture and the rupture of the oolemma (1st period) and between sperm injection and withdrawal of the pipette (2nd period). The velocity between groups was compared at the 95th, 99th, and 99.5th percentiles, followed by statistical analysis by the Mann-Whitney U test. Main results and the role of chance The 95th, 99th, and 99.5th percentiles of the survival group (n = 75) were significantly higher than those of the degeneration group (n = 21) in the 1st period (P < 0.01). Additionally, the 99th, and 99.5th percentiles of the survival group were significantly higher than those of the degenerative group in the 2nd period (P < 0.05). The 95th, 99th, and 99.5th percentiles without UMR (n = 52) were significantly higher than those with UMR (n = 44) in the 1st period (P < 0.001). Similarly, the 95th, 99th, and 99.5th percentiles without UMR were significantly higher than those with UMR in the 2nd period (P < 0.001). ROC curves were generated at the 95th, 99th, and 99.5th percentiles in the 1st period. The resultant areas under the curve (AUC) were 0.73, 0.75, and 0.73, respectively. Using a threshold of 0.75 for the 99th percentile, oocyte degeneration was predicted with a sensitivity of 85% and specificity of 57%. In the 2nd period, ROC curves at the same percentiles resulted in the AUCs of 0.63, 0.69, and 0.67, respectively. Using a threshold of 0.69 for the 99th percentile, oocyte degeneration was predictable with sensitivity as 57% and specificity as 86%. Limitations, reasons for caution This study did not control for variations among the embryologists performing Piezo-ICSI. The culture outcomes and clinical outcomes had not been evaluated. We only showed the relationship between ooplasmic fluidity and oocyte degeneration because it was a retrospective study. A prospective study using image analysis is needed. Wider implications of the findings Before the investigation, we expected that more ooplasmic fluidity (less viscosity) could increase UMR and oocyte degeneration. This study, however, indicated that more ooplasmic fluidity (less viscosity) decreased UMR and oocyte degeneration. Elucidation of the mechanism under this phenomenon may lead to reduced UMR and oocyte degeneration. Trial registration number Not Applicable

P-512 Combined use of genetic diagnosis and algorithms for embryo quality assessment using artificial intelligence improves selection of embryos leading to clinical pregnancy

Abstract Study question Analyze the results of our genetic diagnosis program, evaluating the morphokinetics of the embryo using an automated algorithm, based on its morphokinetic development and quality. Summary answer Transfers with a higher KIDScore D5 v3.2 euploid embryo have a significantly higher chance of achieving pregnancy. What is known already Embryo selection using morphokinetic algorithms is a technique that allows us to select embryos with better quality and perform more transfers of a single embryo improving the clinical outcomes of treatments, this technique combined with the study of aneuploidies and genetic diseases help us to increase the chances of success in embryo implantation and in the results of live newborn at home. Currently, there are techniques available for their diagnoses that, despite the impact of the processes, allow us to select euploid embryos with greater potential for implantation. Study design, size, duration Retrospective study between 1/5/2022 and 31/12/2023 of patients undergoing preimplantation genetic testing for aneuploidy (PGTA) and preimplantation genetic testing for monogenic diseases (PGTM). A single euploid embryo was transferred. Transferred embryos are compared according to KIDScore D5 v3.2. Participants/materials, setting, methods A total of 171 cycles have been performed with PGT, 97 patients have had at least one euploid embryo or transferable mosaic. The single embryos transferred euploids are compared according to the KIDScore D5 v3.2 automated system that ranks each embryo from one to ten, based on their morphokinetic development and quality. Implantation rate, clinical gestation rate and miscarriage are analyzed. Main results and the role of chance A total of 84 single embryo transfers were performed after PGT with an implantation rate of 60.71% and a clinical gestation rate per transfer of 52.38%. According to the morphokinetic KIDScore D5 v3.2 of the transferred euploid embryo, patients with negative pregnancy had a mean KIDScore D5 v3.2 of 6.5±1.3 and for patients with positive gestation it was 7.5±1.4 (S). The mean maternal age of patients with negative and positive pregnancy was 32.8±3.5 and 34.8±3.9, respectively (NS). For the clinical gestation rate, the mean KIDScore D5 v3.2 was 7.48±1.6 (S). The miscarriage rate was 7.84%. Limitations, reasons for caution The main challenges encompass genetic and reproductive counseling and the most efficient approach to blastocyst biopsy. Wider implications of the findings Our data suggest that transfers with a higher KIDScore D5v3.2 euploid embryo have a significantly higher probability of achieving pregnancy. The evolutionary gestation rate is significantly higher when the morphokinetics of the embryo are better. Further studies with a larger number of cases are needed to better assess this trend. Trial registration number not applicable. non-clinical trials

O-129 Live birth of Day 7 blastocyst transfers. Another chance to succeed

Abstract Study question Does the reproductive potential, especially live birth probability, of embryos decrease when blastocyst development takes longer than the traditionally accepted 5 and 6 days?. Summary answer The reproductive potential decreases with blastulation time. Day 7 had the worst live birth (LB), aneuploidy and miscarriage rates compared to Day 5 and 6. What is known already Ending culture on Day 6 is current practice in most clinics, it is important to elucidate whether or not this is a good practice. It has been reported that Day 7 blastocysts represent only 5% of useable blastocysts, although slower growing compared to Day 5 and 6 blastocysts.Day 7 blastocysts can be viable, they can be of top morphological grade, euploid and result in a LB. Furthermore, the effects of prolonged culture to Day 7 in regards to embryo development and their reproductive potential are not well established. Study design, size, duration Retrospective cohort study of patients from US RMA clinics attempting conception through ICSI and utilizing preimplantation genetic testing for aneuploidy screening (PGT-A) from January 2022 to January 2023. A total of 6,132 ICSI PGT-A cycles were included in the analysis; three thousand six hundred and eighty frozen single embryo transfer (SET) from those resulting ICSI cycles occurred during that period of time were included in the study. Participants/materials, setting, methods Patients that performed an ICSI cycle with autologous oocytes were included in the study. Patients with frozen oocytes, donor egg recipients, non PGT-A cases, and conventional insemination were excluded. The main outcome measures include euploidy rates, miscarriage rates and LB rates between Days 5, 6 and 7. Statistical tests were conducted through Chi-Square Test and logistic regression methods adjusted for maternal age at time of transfer and morphological quality of the blastocyst. Main results and the role of chance Euploidy rate was worse on D7 than D5 or D6, with 45.5%95%CI(43,46%-47,50%), 78.1% 95%CI(77,02%-79,13%), and 63.2%95%CI(62,42%-63,96%) respectively, p>0.0001. LB rate per ET (embryo transfer) was also worse on D7, 21.6%95%CI(15.05-28.09), than D5, 62.3%95%CI(59.73-64.95), and D6, 53.3%95%CI(51.23-55.40). We found D5 and D6 statistically significant vs D7, p<0.0001. When adjusted for potential confounders, and establishing D7 as reference, for LB AdjORD5=4.127,95%CI(2.671-6.378), and AdjORD6=3.241, 95%CI(2.145-4.898). Clinical miscarriage was 20.8%, 95%CI(9.84-31.67) on D7, and 11.2%, 95%CI(9.38-13.09) and 14.1%, 95%CI(12.41-15.75) for D5 and D6 respectively, with AdjORD5=0.478, 95%CI(0.230-0.994) and AdjORD6, 95%CI(0.619, 95%CI(0.310-1.239), being statistically different only for AdjORD5 (p=0.048). Finally, when we analyzed the quality of the inner cell mass (ICM) and the trophectoderm among Day 7 ETs, we found that blastocysts with an A quality ICM had a higher chance of LB (40%) when compared with B (25%) and C (7.1%) ICM. The adjusted OR comparing A vs C revealed significant difference (p=0.005), when A ICM is established as reference. Also, ET of blastocysts graded with an A trophectoderm had higher probability of a LB (44%) when compared with B and C (17.3% and17%, respectively). The adjusted OR, with A trophectoderm as reference, B and C trophectoderm revealed significant difference (p=0.012 and p=0.007, respectively). Limitations, reasons for caution The primary limitation is the low number of Day 7 blastocyst transfers. To determine whether Day 7 blastocysts are intrinsically worse from Day 5/6 embryos, prospective randomized studies with patients who have at least one embryo developed on day 5, 6 and 7 suitable for transfer are needed. Wider implications of the findings Expanding blastocyst culture increases the number of useable embryos per cycle and provides further opportunity of LB for patients, especially those with a few or low-quality blastocysts. This study demonstrates that Day 7 blastocysts can achieve live births and challenges the predominant practice of ending culture before Day 7. Trial registration number 2206-SFR-079-MC

P-157 Comparative analysis of iDAScore and KIDScore in predicting live birth following single frozen day 5 blastocyst transfer

Abstract Study question How do iDAScore® and KIDScoreTM perform in predicting live birth outcomes after single frozen Day 5 blastocyst transfers? Summary answer In a euploid major cohort, both KIDScoreTM and iDAScore® demonstrated comparable predictive ability for live birth. However, iDAScore® offers the advantage of being fully automated. What is known already In the field of in vitro fertilization (IVF), the escalating use of artificial intelligence (AI) for predicting clinical outcomes is a burgeoning trend. One key contributor to this trend is time-lapse incubation, which captures images of developing images, generating a wealth of data. This data has facilitated the creation of various predictive models for implantation. KIDScoreTM is one such model that uses morphokinetic data to predict the probability of implantation after a Day 5 embryo transfer. Similarly, iDAScore® is an AI-driven automated scoring system specifically designed to automatically identify embryos with the greatest likelihood of implantation. Study design, size, duration This retrospective study included 779 patients (maternal age range: 19-44, mean age: 33.38) who underwent single frozen day 5 blastocyst transfer cycle from January 2021 to January 2023. Of these, 621 patients transferred euploid blastocysts, 48 transferred mosaic blastocysts, and 110 transferred non-PGT-A tested blastocysts. This study comprised of 12.1% (94/779) fair-to-good and fair grade (below AB and BA), 51.5% (401/779) good grade (AB and BA), and 36.4% (284/779) excellent grade (AA) blastocysts. Participants/materials, setting, methods After PIEZO-ICSI, embryos were cultured in EmbryoScopeTM Plus (Vitrolife, Denmark) until they reached the blastocyst stage. Blastocysts graded at least as fair according to Gardner (1999), were either biopsied for PGT-A and vitrified (Cryotec, Japan), or vitrified without biopsy. Clinical pregnancy and the number of gestational sacs were determined using ultrasound and successful pregnancies were tracked until delivery. KIDScoreTM (KIDScoreTM Day 5 v3.2) and iDAScore® (iDAScore® v2.0) were retrospectively generated for each transferred blastocyst. Main results and the role of chance Box plot showed that both KIDScoreTM and iDAScore® have positive correlation with morphology (Excellent vs. Good vs. Fair-to-Good and Fair; KIDScoreTM median: 8.8 vs. 6.8 vs. 5.7; iDAScore® median: 8.57 vs. 7.68 vs. 6.615), ICM (A vs. B; KIDScoreTM median: 7.5 vs. 6.5; iDAScore® median: 8.14 vs. 7.26) and TE (A vs. B vs. C; KIDScoreTM median: 8.7 vs. 6.5 vs. 4.65; iDAScore® median: 8.41 vs. 7.515 vs. 5.2). In this cohort, the overall implantation rate was 67.91% (529/779), and the live birth rate was 59.31% (462/779). Further statistical analysis revealed interesting correlations. A linear regression analysis indicated a statistically significant positive correlation between KIDScoreTM and iDAScore® (F(1,777)=297.6, p < 0.001, adjusted R2=0.276). Wilcoxon rank sum tests showed that KIDScoreTM has significant higher mean scores in blastocysts resulting in live birth compared to those that did not (7.44 vs. 7.22, w = 66577, p < 0.05). iDAScore® also had higher mean scores in blastocysts resulting in live birth but without statistically significant difference (7.56 vs. 7.48, w = 71328, p > 0.05). ROC curve analysis showed that AUC for KIDScoreTM was 54.4 and AUC for iDAScore® was 51.9, indicating no significant difference in their predictive ability for live birth (p = 0.2455). Limitations, reasons for caution The study’s cohort predominantly consisted of blastocysts with good morphology and were euploid, potentially skewing the results. Therefore, the findings may not be representative of all IVF patients and should be interpreted with caution. Additionally, further study should investigate the capability of iDAScore® 2.0 to predict euploidy. Wider implications of the findings While KIDScoreTM had a higher AUC than iDAScore®, there was no statistically significant difference. The major advantage of iDAScore® lies in its automation, eliminating the need for manual embryo evaluation. This can greatly enhance consistency in embryo grading and selection. Trial registration number NOT APPLICABLE

P-711 Female age at embryo creation and embryo transfer do not impact the ongoing pregnancy rates in single euploid frozen embryo transfers

Abstract Study question Which factors influence the ongoing pregnancy rate in single euploid frozen embryo transfer (seFET) cycles? Summary answer Ongoing pregnancy rates in seFET cycles are significantly influenced by parity, BMI, embryo quality, trophectoderm biopsy day and endometrial preparation, but not by female age. What is known already The probability of a pregnancy is influenced by several factors and female age is the major determinant as the ovarian reserve decreases and embryo aneuploidy increases with age. Preimplantation genetic testing for aneuploidy and embryo selection was considered to mitigate the effect of female age on ART treatment outcomes by selecting euploid embryo(s) for embryo transfer (ET). Previously published data point to a decline in pregnancy rates with increased maternal age even when a euploid embryo is transferred. However, other factors besides embryo ploidy might play a role in the chance of achieving an ongoing pregnancy. Study design, size, duration Retrospective study, including 1923 women who underwent a seFET between 04/2017 and 08/2023. Patients’ (age at oocyte-pick-up and seFET, BMI, parity, endometrial preparation type) and embryo characteristics (Day of biopsy, embryo quality (EQ)) were considered for the analysis. Ongoing pregnancy was defined as viable pregnancy beyond 12 weeks of gestation. Participants/materials, setting, methods Women, undergoing a seFET either in a true natural cycle (NC) t or in a hormonal replacement cycle (HRT). Only cycles with embryos being biopsied on day 5 or day 6 and a known outcome of the cycle were included. Main results and the role of chance There were 990 (51.48%) ongoing pregnancies among 1923 included transfers. The ongoing pregnancy rates were 51.8%, 47.4%, 52.3% and 52.8% for women aged ≤35, >35 to ≤ 37, >37 to ≤ 40, and >40 years-old at seFET, respectively. Cochrane Armitage test did not show a significant trend for decreasing pregnancy rates with increasing female age at OPU (P = 0.679) or at transfer (P = 0.609). Multivariable regression analyses showed primiparous, and multiparous women were more likely to have an ongoing pregnancy compared to nulliparous women (OR: 2.96, 95%CI:2.22-3.94 and 2.73, 95%CI:2.10-3.55, respectively, P < 0.001 for both) and those who had a natural endometrial preparation (OR: 1.53, 95%CI: 1.24-1.88, P < 0.001). Transfers with good quality embryo had higher ongoing pregnancy rates compared to those who had a fair quality embryo (OR: 2.0, 95%CI: 1.37-2.91, P < 0.001). Being overweight or obese at the time of transfer were associated with lower ongoing pregnancy rates compared to normal weight (OR: 0.71, 95%CI: 0.56-0.89, and 0.68, 95%CI: 0.52-0.88, respectively, P = 0.003 for both). Age categories at the time of transfer did not show a significant effect on ongoing pregnancy rates in multivariable analyses (P > 0.05 for all) and there was no significant interaction between age at OPU and embryo quality categories either (P = 0.881 and 0.601). Limitations, reasons for caution The retrospective design of the study is a limitation. Wider implications of the findings This analysis did not confirm the otherwise reported negative impact of female age on the outcome of seFET cycles. Outcomes are significantly influenced by BMI, parity, EQ-related factors as well as the endometrial preparation. As BMI and endometrial preparation can be influenced, the adequate choice might improve seFET outcomes. Trial registration number not applicable

P-391 Association between meteorological season at the time of embryo transfer and pregnancy outcomes in freeze-all cycles: lessons from seasonal reproductive mammals

Abstract Study question Is there any effect of meteorological season at the time of embryo transfer on clinical outcomes of freeze-all cycles? Summary answer Optimal conditions for implantation appear to be associated with spring and summer. Embryo transfers performed in autumn or winter are associated with reduced ongoing pregnancy. What is known already Extensive research has consistently shown that most mammals are seasonal breeders, and as day length increases, gonadotropin secretion increases, to anticipate reproduction in the spring and summer months. There is intriguing evidence that there is a seasonal variance in human reproduction, but the data are inconclusive. Therefore, the goal for the present study was to evaluate the effect the meteorological season at the time of embryo transfer on clinical outcomes of freeze-all cycles. Study design, size, duration This historical cohort study was performed in a private university-affiliated IVF center between February 2018 and December 2022. A total of 4952 frozen-thawed embryos transferred to 3725 cycles in a single clinic were analyzed. Participants/materials, setting, methods Both vitrification and the warming procedures were performed using the Cryotop method. Embryos survival and clinical outcomes were analyzed by the season, at the time of embryo transfer, using generalized linear models adjusted for potential confounders. Main results and the role of chance The mean maternal age was 37.0 ± 4.7 years. Embryos were cryopreserved for a mean of 8.1 ± 17.3 months. Autumn was designated as the reference season for all analyses. No significant differences were observed in embryo survival rates when the seasons were compared, however, lowest implantation and pregnancy rates were observed when embryo transfers occurred in autumn (44.6%), followed by winter (45.8%, OR: 1.049, CI: 0.876 – 1.257, p = 0.603) and summer (49.0%, OR: 1.196, CI: 1.010 – 1.415, p = 0.038), while spring embryo transfers had a 30% increased odds of pregnancy (51.2%, OR: 1.302, CI: 1.100 – 1.541, p = 0.002). An increased miscarriage rate was observed when embryo transfers occurred in autumn (13.9%), followed by winter (11.4%, OR: 0.510, CI: 0.323 – 0.805, p = 0.004) and summer (10.4%, OR: 0.461, CI: 0.292 – 0.727, p = 0.001), while spring embryo transfers had a 63% decreased odds of miscarriage (5.6%, OR: 0.369, CI: 0.230 – 0.592, p < 0.001). Limitations, reasons for caution The use of historical cohort groups is a drawback. Despite the eligibility criteria for inclusion in the analysis, potential differences in the baseline characteristics cannot be ruled out. Wider implications of the findings In seasonal breeding mammals, melatonin increases the gonadotropins’ release during the transition period. Melatonin rise favors embryo implantation and protects offspring by increasing progesterone levels. Our evidence suggests that human reproductive potential may be influenced by climatic variations. Trial registration number N/A