Preimplantation Genetic Testing For Aneuploidy Research Articles

P-541 First live birth report of identical twins from a single low-grade mosaic blastocyst (46,XY/45,XY,-19) transfer: when a small chance becomes a double miracle

Abstract Study question N/A Summary answer First live birth report of identical twins from the transfer of a single low-grade mosaic blastocyst for monosomy on chromosome 19. What is known already The clinical decision-making in cases involving the transfer of low-grade mosaic embryos remains controversial. Recent studies have indicated that low-grade mosaic embryos exhibit pregnancy rates similar to euploid embryos, encouraging their transfer. However, the judgment to proceed in most cases is still challenging. The transfer of a low-grade mosaic embryo for monosomy on chromosome 19 is considered relative safe. The occurrence of human twins resulting from embryo splitting in ART (Assisted Reproductive Technology) cycles is a rare phenomenon. To date, there are no reports of the birth of identical twins from a single low-grade mosaic embryo transfer. Study design, size, duration This is a single-case report detailing an assisted reproduction cycle conducted by a couple. Participants/materials, setting, methods A couple with a nine-year history of infertility, characterized by low ovarian reserve and azoospermia, underwent an ICSI (Intracytoplasmic Sperm Injection) cycle in October 2022. Due to azoospermia, microsurgical epididymal sperm aspiration and subsequent freezing of the retrieved sperm were performed prior to ovum pick-up. PGT-A (Preimplantation Genetic Testing for Aneuploidy) of the resultant blastocysts was conducted, followed by the transfer of a single low-grade mosaic after a modified natural endometrial preparation cycle. Main results and the role of chance Seven mature oocytes were retrieved, and following ICSI with the thawed seminal sample, four blastocysts were developed and biopsied for PGT-A analysis. Out of these four high-quality blastocysts, three were aneuploid, and the remaining one was a low-grade (50%) mosaic for monosomy on chromosome 19. After genetic counselling, the patients decided to proceed with the transfer of a day 5 3AA low-grade mosaic embryo, which led to a successful pregnancy. The transferred embryo split, generating a pregnancy of identical twins. The twins were delivered via cesarean section at 30 weeks and 5 days of gestational age. The premature delivery was necessitated due to the mother developing cardiac insufficiency related to pre-existing valvulopathy and a superimposed venous thromboembolism. Peripheral blood samples from the twins have been collected for a 100-cell karyotype analysis (results will be available by February 9th). This case marks the first reported instance of twins born from the transfer of a single low-grade mosaic blastocyst (46,XY/45,XY,-19). Limitations, reasons for caution This is a retrospective case-report from one couple, describing a rare event. Wider implications of the findings This case-report could elucidate the physician’s decision and slightly encourage the transfer of low-grade mosaic embryos when there are no full euploids available. Trial registration number Not pllicable

P-204 Automated embryo assessment: a simple web application correlates scores with embryo morphology, developmental velocity, ploidy, and clinical outcomes

Abstract Study question How does the novel automated scoring system in our laboratory correlate with treatment success in different subpopulations? Summary answer The web app automates embryo assessment, linking scores to morphology, development speed, ploidy, and clinical outcomes in treatments with both patient and donor oocytes. What is known already In the realm of assisted reproductive technologies, numerous platforms employing artificial intelligence (AI) have emerged for embryo assessment. Substantial research has been dedicated to evaluating the efficacy of AI in predicting pregnancy outcomes, particularly following blastocyst transfer. However, not all available options have undergone external validation, a crucial step deemed necessary before investing in such tools. This project endeavors to validate the utility of EMBRYOAID v1.0, a straightforward web application capable of assessing embryos through photos and/or videos. Study design, size, duration This is a retrospective cohort study including 485 patients who underwent IVF treatments with embryos (n = 5,710) cultured in EmbryoScope® time-lapse systems. Senior embryologists routinely assessed blastocysts using ASEBIR morphological criteria (A-D). Then, time-lapse videos were scored by the EMBRYOAID algorithm (0-10). We investigated the relationship between automated scoring and: (I) conventional morphology, (II) morphokinetic parameters, (III) euploidy rate, and (IV) implantation in cycles with own oocytes, egg donation, and preimplantation genetic testing for aneuploidies (PGT-A). Participants/materials, setting, methods The association with morphology and morphokinetics was investigated in 5,710 embryos. Ploidy correlation involved 258 blastocysts undergoing trophectoderm biopsy and next-generation sequencing analysis. Implantation association was studied in 381 single blastocyst transfers. Finally, a multivariable analysis considered oocyte age, body mass index, type of transfer (fresh-frozen) and day of transfer (5-6) to quantify the relevance of automated scoring in different treatment subpopulations. Model performance was assessed through ROC curves and area under the curve calculations. Main results and the role of chance The results that involved grouping were quartiled based on sample size, calculated by the statistical software. This deep-learning score was related to conventional morphology (7.9±1 for A, n = 295; 6.9±1.3 for B n = 1168; 5.9±1.3 for C, n = 830; and 3.1±1.8 for D, n = 3,417)*. Embryos with faster development in early stages received higher scores, considering division times at 2, 3, 4, and 5 cells*. Euploidy rate increased as the embryo score was higher: 39.1% for ≤5.6 (n = 54), 45.5% for 5.6-6.2 (n = 61), 48.5% for 6.2-6.9 (n = 65) and 58.2% for >6.9 (n = 78). Similarly, the implantation rate was also higher in accordance with the score: 42.7% for ≤6.2 (n = 73), 50.6% for 6.2-7.4 (n = 87), 60.2% for 7.4-8.2 (n = 103) and 69.4% for >8.2 (n = 118). The stepwise multivariate analysis showed that embryo score was related to the odds of implantation in conventional treatments with patient oocytes (OR = 1.4; 95% CI [1.1-1.8]*) and in the oocyte donation program (OR = 1.2; 95% CI [1–1.4]*). The AUCs demonstrated the following performance: 0.643 (0.559-0.727) for patient oocytes and 0.677 (0,624-0,731) for the oocyte donation program. There was no significant association of embryo score with implantation in PGT-A treatments. Limitations, reasons for caution The retrospective nature of the study, despite serving as a thorough external validation, introduces inherent limitations. Notably, in PGT-A treatments, embryos underwent assisted hatching on day 3, potentially influencing the model’s performance. Wider implications of the findings The implications of our findings validate a user-friendly tool for automated embryo scoring in the laboratory. The achieved performance is comparable or superior to other tested models, particularly in egg donation programs, potentially encouraging clinics to adopt this tool routinely. Trial registration number Not applicable

P-524 Ethnicity and aneuploidy rates in a global perspective: a comparative genomic study of 16,543 embryos from the United Kingdom, India and Japan

Abstract Study question Is embryonic aneuploidy, as determined by Next Generation Sequencing (NGS) based Pre-Implantation Genetic Testing for Aneuploidy (PGT-A) related to female ethnicity? Summary answer In this study, when determining aneuploidy through NGS-based PGT-A, female ethnicity did not have an impact on embryonic aneuploidy. What is known already Inequalities in pregnancy outcomes following in-vitro fertilization (IVF) have often been attributed to potential disparities in genetic susceptibility, with past studies showing higher pregnancy rates in older Caucasian patients compared to younger South-Asian patients. With aneuploidy as one of the best-characterized barriers to IVF success, critics argue that potential ethnic variations in aneuploidy rates may contribute to inequalities in IVF success, raising concerns about the appropriateness of standardized protocols. Proponents stress the importance of personalized medicine accounting for individual genetic backgrounds. Acknowledging genetic diversity and ensuring equitable access to successful fertility treatments remains a complex challenge. Study design, size, duration In this retrospective cohort review, data reflecting 4,354 women undergoing an IVF±ICSI cycle with concomitant NGS-based PGT-A from 65 centres in the United Kingdom, India and Japan was requested. Trophectoderm biopsy results from 16,513 embryos tested through a single genetic laboratory over a five-year period (2019-2023) were analysed. Patients were categorised by ethnicity (Caucasian, African, East Asian, or South Asian) and further stratified by age (<35, 35-37, 38-39, 40-42, >42). Participants/materials, setting, methods Women aged between 25–45 years undergoing an IVF±ICSI cycle with concomitant NGS-based PGT-A were included. Patients undergoing PGT for inherited genetic disorders (PGT-M) or gamete donation cycles were excluded, and only autologous oocyte cases were included. Trophectoderm biopsy was performed on day 5, 6 or 7 and NGS was performed from amplified deoxyribonucleic acid (DNA). Primary outcome of interest was aneuploidy rate (>30% abnormality). Statistical analysis included ANOVA, Kruskal Wallis, and linear regression tests analysis. Main results and the role of chance In our analysis, the mean number of embryos tested overall was 3.46 (SD 2.07) and the mode was 4. The mean number was 3.84 for South Asian, 3.47 for Caucasian, 2.4 for African and 2.87 for East Asian. This was not statistically different between groups (P = 0.48). Subgroup analysis did not identify any statistically significant differences in the number of oocytes retrieved, MII oocytes retrieved and fertilization rates. When categorising by female age alone, there was a statistically significant difference in aneuploidy and female age overall (<35: 32.1±29.7%; 35-37: 42.1±31.3%; 37-39: 50.4±29.3%;40-42: 64.7±33.5% and > 42:73.9±38.7%, p-value: <0.001). When subcategorised by ethnicity, linear regression analysis did not identify a significant association with aneuploidy rate and ethnicity. The was no statistically significant difference in mean aneuploidy rate by ethnicity: East Asian 67.2±32.2%, African 58.3±42.2%, European 55.6±35.6%, and South Asian 53±32.4% (p-value 0.57). With Caucasian as the reference population, coefficients for South Asian (3.20, p-value:0.61), East Asian (8.50 p-value: 0.31), and African (7.20, p-value: 0.60) were not statistically significant. No statistically significant differences in aneuploidy rates were observed across female age categories (<35, 35-37, 38-39, 40-42, >42) and ethnicities, with a p-value of 0.17. Limitations, reasons for caution The study type was retrospective in nature. Ethnic groups are not homogenous, and there can be significant genetic, cultural, and environmental diversity within a group. Failure to account for this heterogeneity may result in oversimplification and misinterpretation of findings. There was under representation of patients from African origin (2%). Wider implications of the findings These data do not support differences in embryonic aneuploidy among various ethnicities in patients undergoing IVF±ICSI /NGS-PGT-A. We limited our analysis to a single genetic laboratory to reduce reporting variation and technical confounders. It does not appear that specific recommendations for aneuploidy screening should be made based upon ethnic heritage. Trial registration number Not applicable

P-647 Association between PCOS phenotype-D and euploid blastocyst rate per cohort of inseminated oocytes: a matched case-control study versus idiopathic infertile women

Abstract Study question Is there any association between PCOS phenotype-D and oocyte competence defined as euploid blastocyst rate (EBR) per cohort of inseminated oocytes? Summary answer Although higher fertilization and blastulation rates were reported in PCOS phenotype-D women versus idiopathic infertile patients, the EBR per cohort of inseminated oocytes was comparable. What is known already Several studies report poorer oocyte quality and cumulative-live-birth-rate (CLBR) in hyperandrogenic PCOS phenotypes, as defined according to the Rotterdam criteria. Increased androgen concentrations in follicular fluids are indeed associated with elevated serum-LH levels, which can block dominant follicle development inducing atresia and negatively impact fertilization rates and embryo development. Limited evidence exists in phenotype-D PCOS patients, namely women characterized by oligomenorrhea, ovarian PCO morphology and absence of hyperandrogenism. Here, we report the embryological and clinical outcomes in these women during ICSI cycles with preimplantation genetic testing for aneuploidies (PGT-A) at the blastocyst stage. Study design, size, duration Retrospective propensity score matched (PSM) case-control study. Among PGT-A cycles conducted by naïve patients with own oocytes (2013-2021), we excluded FNA/TESE. 58 phenotype-D PCOS patients were identified according to the Rotterdam criteria. These patients were matched for maternal age, number of cumulus-oocyte-complexes (COCs), and sperm quality, with 58 patients with idiopathic infertility, regular cycles, normal ovarian morphology, and no sign of clinical hyperandrogenism. The primary outcome was EBR per cohort of inseminated oocytes. Participants/materials, setting, methods GnRH-antagonist ovarian stimulation, ICSI, blastocyst biopsy, qPCR/NGS-analysis to assess uniform aneuploidies and vitrified-warmed single blastocyst transfers were conducted. The PCOS and control groups were similar for age (35.6±3.0 versus 36.0±3.9 years), COCs (23.1±6.7 versus 23.5±7.6), sperm factor, primary/secondary infertility, hormonal values, BMI (23.8±5.3 versus 22.0±2.8), and duration of infertility (3.6±2.2 versus 3.6±2.0 years). All intermediate embryological and clinical outcomes were assessed as secondary endpoints. Linear/logistic regressions adjusted for confounders were conducted to confirm statistically-significant differences. Main results and the role of chance No difference was reported in maturation rates, but women with PCOS showed better fertilization rates per cohort of inseminated oocytes (76.9±15.2% versus 67.3±20.4%, Mann-Whitney-U-test<0.01), and blastulation rates per cohort of 2PN-zygotes (52±20.4% versus 43.7±20.7%, Mann-Whitney-U-test=0.03). Eventually, PCOS patients obtained more blastocysts than controls (5.8±3.1 versus 4.5±2.9, Mann-Whitney-U-test=0.02), despite similar numbers of metaphase-II oocytes in the groups (16.5±6.1 versus 16.5±7.0). All differences remained significant after adjusting for maternal age and sperm factor. Nevertheless, the euploidy rates per cohort of biopsied blastocysts were similar, thus involving comparable EBR per cohort of inseminated oocytes (21.7±14.1% versus 17.2±16%, Mann-Whitney-U-test=0.12). More patients obtained ≥1 euploid blastocyst in the PCOS versus the control group (N = 56/58, 96% versus N = 47/58, 81%, Fisher’s-exact-test=0.02), independently from maternal age, semen quality, and number of inseminated oocytes (Odds-Ratio: 6.5, 95%CI:1.3-33.7, p = 0.03). Lastly, all clinical outcomes per first vitrified-warmed euploid transfer were similar, thereby involving comparable LBRs (N = 28/54, 51.9% versus N = 26/47, 55.3% in the PCOS and control groups, p = 0.8; Odds-Ratio adjusted for blastocyst quality and day: 1.19, 95%CI:0.5-2.7, p = 0.68). Limitations, reasons for caution Although clinical hyperandrogenism was absent in both groups, biochemical hyperandrogenism was not assessed. Nonetheless, this pilot study paves the way for future more detailed investigations. 24% of cycles in the PCOS group were not concluded, therefore a CLBR comparison would be biased and was not reported. Wider implications of the findings The lower developmental competence in the control group suggests oocyte quality issues, other than chromosomal, underlying their “unexplained infertility” condition. In phenotype-D PCOS women, IVF is effective. Since >50% of this subgroup of women had already failed multiple ovulation-induction and/or intrauterine-insemination attempts, an early referral to IVF deserves further investigations. Trial registration number Not applicable

P-137 A deep learning model using time-lapse imaging data to predict successful clinical pregnancy and embryo euploidy

Abstract Study question Are the deep learning models helpful in selecting embryos with a high probability of clinical pregnancy and euploidy from time-lapse imaging during embryo growth? Summary answer Our deep learning models using time-lapse imaging data predicted the probabilities of successful clinical pregnancy and euploidy of embryos with high accuracy. What is known already Recent studies have attempted to select the most viable embryo using deep learning of time-lapse imaging during incubation. However, most studies included embryos that were not transferred due to low evaluation by the embryologist as embryos with unsuccessful pregnancies, or they mixed single and multiple embryo transfer cases in the analyses. Some studies’ algorithms rely on humans’ subjective interpretation of morphological and morphokinetic features, leading to embryologist-to-embryologist and within-embryologist variabilities. Some reports, including the study predicting embryo aneuploidy, used a snapshot image for prediction. These approaches have restricted the potential of deep learning in embryo selections. Study design, size, duration Retrospective analyses of time-lapse videos of 2,192 embryos with clinical pregnancy outcomes, defined as confirmed gestational sacs in utero, and 1,467 embryos with karyotype outcomes examined by preimplantation genetic testing for aneuploidy (PGT-A). The data were obtained from two different in-vitro fertilization clinics between November 2017 and December 2021. All transfers were performed as a single embryo transfer. Participants/materials, setting, methods The deep learning model was trained using time-lapse videos with known clinical pregnancy outcomes for predicting the probability of clinical pregnancy with given time-lapse video sequences and patient ages (CP model). Another deep learning model was trained using time-lapse videos with known karyotype outcomes by PGT-A for predicting the probability of euploidy (AP model). The models’ predictive powers were measured using the average area under the curve (AUC) of the receiver operating characteristic curve. Main results and the role of chance The CP model predicted clinical pregnancy with an AUC of 0.693. The AP model predicted embryo aneuploidy with an AUC of 0.702. Limitations, reasons for caution The CP model is based on the embryos that were transferred because the embryologist judged them as having a high probability of pregnancy. Clinical pregnancy may be associated with maternal factors besides embryo factors. Wider implications of the findings Our two deep learning models, the CP and AP models, will be helpful for selecting the embryos to be transferred. The CP model may increase pregnancy rates in patients undergoing assisted reproductive technology. The AP model may reduce the miscarriage caused by embryo aneuploidy. Trial registration number not applicable

P-188 Growth variance score (GVS): a morphokinetic-based model with capability to distinguish euploid from aneuploid embryos

Abstract Study question Does longitudinal growth of euploid embryos differ from aneuploids? Summary answer Euploid embryos had significantly lower growth variance score (GVS), indicating steadier growth when observed with time-lapse monitoring, compared to aneuploid embryos. What is known already A major quest for optimizing embryo selection is the identification of morphokinetic parameters to distinguish euploid from aneuploid embryos. Although some studies have identified altered morphokinetic parameters related to embryo ploidy, outcomes were not reproducible, and could not provide sufficient evidence to apply it. Moreover, longitudinal changes during this temporal process of embryo development, their reference standards, and the impact of such changes on embryonic fate remains unknown. Aiming to develop a growth variance model, using the reference ranges of embryonic morphokinetic growth as a single growth trajectory might provide a better additive value to distinguish euploid from aneuploid embryos. Study design, size, duration This observational, retrospective cohort study was performed in a tertiary referral IVF centre between December 2020 and March 2023. The study included a total of 677 blastocysts from 147 cycles tested for Preimplantation Genetic Testing for aneuploidy (PGT-A) by next generation sequency (NGS). All embryos were monitored by time-lapse (TL) system during culture. Participants/materials, setting, methods Growth variance score (GVS) is a measure of embryonic growth pattern calculated using the TL morphokinetic reference ranges (from tPB2 until tEB), developed for euploid embryos. Higher GVS scores indicate more erratic growth while lower scores indicate steadier growth. Trophectoderm (TE), inner cell mass (ICM), and expansion grade were assessed immediately before biopsy using Gardner’s criteria. Euploid and aneuploid blastocysts were compared in terms of ICM, TE grade, day of biopsy and GVS score. Main results and the role of chance Among tested blastocysts, 383 were euploid (56.6%) and 294 of them were abnormal (43.4%). Euploid embryos had significantly lower GVS compared to aneuploid embryos (mean difference: 0.30 lower, 95% CI: 0.16 to 0.45; P < 0.001). Among day 5 biopsies, euploids had significantly lower GVS compared to aneuploids (mean difference: 0.27 lower, 95% CI: 0.04-0.51; P = 0.019). Among Day 6+ biopsies, euploids also had lower GVS compared to aneuploids, but the difference did not reach statistical significance (mean difference: 0.09 lower, 95% CI: -0.08-0.28, P = 0.30). Regression analysis showed GVS was significantly and independently associated with euploidy after adjusting for day of biopsy and morphological grade (aOR: 0.83, 95% CI: 0.70 – 0.99; P = 0.048). We compared two embryo selection modalities, the first one using morphology and biopsy day, the second one incorporating GVS to morphology and biopsy day. In 90% (95% CI: 87.2 – 92.8%) of the cycles, both modalities made a concordant selection by choosing the same embryo as the best. In the remaining 10% (95% CI: 6.4% - 14.7%) of the cycles, they made a discordant choice and the euploidy rate of embryos chosen by the combination of GVS and morphology were higher by 20.4% (95% CI: -0.06 to 44.0%, P = 0.060). Limitations, reasons for caution To confirm its reproducibility in other clinical and laboratory settings, GVS should be replicated with larger sample sizes and in external cohorts. Wider implications of the findings The GVS model might emerge as a valuable prognostic tool to select euploid blastocysts within patient’s own pool of embryos. GVS could be easily incorporated into laboratory work-flow when a time-lapse monitor is available to prioritize embryos for transfer, given external validations are successful. Trial registration number Not applicable

P-547 Impact of SARS-COV-2 infection on embryo euploidy rate: a retrospective cohort study of 498 PGT-A cycles in China

Abstract Study question Does SARS-CoV-2 infection before oocyte retrieval have any impacts on embryo euploidy rate? Summary answer SARS-CoV-2 infection before oocyte retrieval had no detrimental impact on embryo euploidy rate, indicating that cycle cancellation due to SARS-CoV-2 infection may not be necessary. What is known already SARS-CoV-2 infection has significant effects on the health of people worldwide and poses a serious threat to reproductive health. According to the previous studies, SARS-CoV-2 infection had negative impacts on the blastocyst formation rate, top-quality embryos rate. However, the association between the infection and the embryo euploidy rate is indeterminate. It is still worth exploring the impact of SARS-CoV-2 infection on embryo euploidy rate. Especially this is the key question to be addressed for providing appropriate counseling to couples seeking for preimplantation genetic testing for aneuploidy (PGT-A) treatments during the ongoing pandemic. Study design, size, duration This was a retrospective cohort study of 498 couples who underwent PGT-A cycles with next-generation sequencing technology from November 1st, 2022 to June 30th, 2023 at a tertiary-care medical center in China. Participants/materials, setting, methods Women tested positive for SARS-CoV-2 before oocyte retrieval were included in the COVID-19 group, while patients who were asymptomatic and tested negative during the same period were included in the non-COVID-19 group. The primary outcome was the euploidy rate. Subgroup analysis was conducted according to the time interval between infection and oocyte retrieval. Univariate and multivariate logistic regression models were applied to adjust for potential confounders. Main results and the role of chance A total of 278 and 220 women were included in the COVID-19 and non-COVID-19 group, respectively. In the COVID-19 group, the maturated oocyte rate (79.5% vs. 83.7%, p＜0.001), number of total viable embryos (2 vs. 3, p=0.038), and number of euploid blastocyst (1 vs. 1, p=0.028) were significantly decreased compared with non-COVID-19 group. The euploidy rate (38.5% vs.40.3%, p = 0.451), aneuploidy rate (45.0% vs. 43.8%, p=0.616), and mosaicism rate (16.5% vs. 15.9%, p=0.746) were all comparable between the two groups. Results of multivariate logistic regressions revealed no association between SARS-CoV-2 infection and euploidy rate, aneuploidy rate, or mosaicism rate. Similarly, no association was detected between the time interval of infection and oocyte retrieval (≤30 days, 31-60days, 61-90 days, 91-120 days, or ≥ 120 days prior to oocyte retrieval) and euploidy rate. Limitations, reasons for caution The retrospective nature of the study and limited sample size for subgroup analysis limits the generalizability of the findings. Other potential confounders, such as the level of SARS-CoV-2 antibody, symptoms of infection, are needed to be taken into consideration as well. Large-scale prospective studies are warranted to confirm the conclusion. Wider implications of the findings Our study demonstrated that SARS-CoV-2 infection before oocyte retrieval had no detrimental impact on embryo euploidy rate, indicating that cycle cancellation due to SARS-CoV-2 infection may not be necessary. Trial registration number Not Applicable

P-565 Moving towards non – invasive euploid blastocyst prediction. Retrospective euploid blastocyst evaluation using different morphokinetics and Artificial Intelligence (AI) models

Abstract Study question Could Preimplantation Genetic Testing for Aneuploidies be replaced by an Aritificial Intelligence model which evaluates blastocysts and selects the most suitable ones for embryo transfer? Summary answer The use of AI for euploidy prediction has not been validated in a large scale and thus it should not be used to replace PGT-A. What is known already PGT-A is an invasive method that may affects blastocysts implantation potential. Both the biopsy protocol and the experience of the embryologist who performs the biopsy can influence the outcome. Efforts are made to develop non-invasive methods for PGT. Many AI systems have been developed in assisted reproduction.Time- Lapse systems have a specialized AI software, that assist in the evaluation of the embryos and the selection of the most suitable ones based on their morphokinetic characteristics. Other AI systems, such as ERICA, evaluate embryos and provide a measure of their euploidy, serving as an alternative method for non-invasive preimplantation genetic testing. Study design, size, duration The retrospective study was conducted between January 2018 and December 2020. A total of 585 blastocysts, derived from 144 women who had undergone an IVF – PGT-A cycle, were included. The embryos were cultured in a TL incubator and evaluated using KID Score D5 (Vitrolife), iDa Score (Vitrolife) and ERICA (ERICA). Participants/materials, setting, methods The study was conducted in a private IVF clinic. Embryos from women with an average age of 38 years who had a history of recurrent miscarriages and/or >3 failed IVF cycles were included. Biopsy was performed in all blastocysts that were cultured in TL incubators up to day 5-6. Statistical and logistic regression analysis were conducted to assess the comparison between the score of the three AI programs and the result of the biopsy. Main results and the role of chance ERICA seems uncapable to separate euploid from aneuploid embryos. T-test showed that there is no statistically significant difference between the two categories (mean_difference=0,02, p = 0,3>0,001), while the effect size (clinical value) of the difference was very low (0,085). This means that this program cannot replace PGT-A. According to the iDA Score, although there is a difference in scores between euploid and aneuploid embryos, their difference, according to t-test, is not statistically significant [t(581)=2,56, p = 0,01>0,001], while the effect size is low (0,217). According to KID Score D5, there is a statistically significant difference in scores between euploid and aneuploid embryos [t(514)=4,4, p < 0,001], which shows that this program could replace PGT-A in some cases. However, the effect size of the difference (0,37), as well as the determination coefficients are quite low. Finally, the KID Score D5 succeeds in discovering aneuploid embryos in a higher percentage than euploid ones. Limitations, reasons for caution Women under the age of 35 were not included in the study. Permission from the National Greek Authority of Medical Assisted Reproduction was needed from every patient to undergo PGT-A. The size of the sample might limit the conclusions. A larger study would help verify the results of the study. Wider implications of the findings AI models can be used as good indicators for embryo selection/prioritization and predict the euploid status of the embryos. Non-invasive euploid prediction models could be a very useful tool for embryologists especially in countries where law restrictions are strict against the cohort of patients that are interested in PGT-A cycles. Trial registration number Not Applicable

P-626 Progesterone rescue is effective for clinical pregnancy outcome in women presenting low circulating progesterone levels at frozen embryo transfer day

Abstract Study question Is subcutaneous progesterone (prolutex® 25mg daily) addition effective for clinical pregnancy outcome in women presenting low progesterone(P4) levels at frozen embryo transfer(FET) day? Summary answer Subcutaneous progesterone rescue improved ongoing pregnancy rate and clinical pregnancy rate in women with low P4 results at FET day. What is known already FET has been increased, because preimplantation genetic testing for aneuploidies is performed widely, and FET presents a significantly low risk of ovarian hyperstimulation syndrome.In FET, there is no established optimal protocol for preparing endometrium, but hormonal replacement therapy (HRT) is widely used due to its convenient schedule adjustment. HRT protocol induce anovulation, functioning corpus luteum is lacking and exogenous progesterone administration is very important. Therefore, women showing low P4 levels around FET day have lower pregnancy rate. However, there are currently no data on the effectiveness of progesterone rescue. Study design, size, duration Primary outcomes from a randomized cohort retrospective study investigating subcutaneous progesterone (prolutex® 25mg) daily addition for whom had low P4 levels (<12ng/mL) at FET day. The study was conducted at one infertility center in Korea from January 2022 to December 2023 and pregnancy outcomes of FET transfers registered until Oct. 2023,. 194 patients fulfilled the inclusion criteria. Participants/materials, setting, methods One hundred ninety four women divided into two groups based on progesterone levels, with 12ng/ml as the threshold. Under 12ng/mL group were randomized to subgroup with and without addition of subcutaneous progesterone. In total 278 FET cycles, the effect of progesterone rescue on cumulative clinical pregnancy rate (CPR), Ongoing pregnancy rate (OPR) was reported as primary outcomes. Main results and the role of chance In total 278 cycles, there were 137 cycles with adequate serum P4 levels, while 141 cycles presented with low P4 levels in which randomly rescue subcutaneous progesterone administration added from next day of embryo transfer. When comparing the groups, there was no statistical difference in maternal age, BMI, smokers, duration of infertility, type of infertility, indication of in vitro fertilization(IVF), endometrial preparation method. In low P4 group, with and without subcutaneous progesterone addition subgroups included 51 and 90 cycles, retrospectively. CPR were 52.9% versus 35.6% (P = 0.044; chi-squared test) with a statistically significant higher rate of OPR in progesterone rescue group (45.1% versus 28.9%, p = 0.052). When comparing the group with adequate P4 levels and the group with low P4 levels without progesterone rescue, OPR were 18.2% versus 28.9% (p = 0.06, chi-squared test), CPR was significantly higher in low P4 levels group (23.4% versus 35.6%,p=0.046, chi-squared test). Adequate P4 level group had more IVF attempts (4.6 versus 2.2, p < 0.001, chi-squared test). There is tendency to use more luteal phase support medication with repeated attempt (serum P4 concentration on ET day 39.3 ng/mL versus 8.7 ng/mL, p < 0.001, t-test) Limitations, reasons for caution The main limitation of the study was retrospective, and had small sample size. A control group without progesterone rescue in low P4 levels did not receive any intervention. Wider implications of the findings The progesterone rescue for women presenting low P4 on FET day improved clinical outcome and excessively raising progesterone medication may have adverse effects. Therefore rather than using excessive progesterone administration from luteal support start day, progesterone rescue might be effective when low P4 levels presenting at FET day. 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

O-031 Tacrolimus treatment significantly improved IVF outcomes with euploid embryo transfer in women with repeated implantation failures (RIF) and elevated T helper (Th)1/Th2 cell ratios

Abstract Study question Dose tacrolimus improve implantation rate for RIF women showing elevated T helper (Th)1/Th2 cell ratios, particularly those undergoing IVF/ET with euploid blastocysts? Summary answer Tacrolimus improved the implantation rate in RIF women with elevated Th1/Th2 cell ratios undergoing IVF/ET using euploid blastocysts. What is known already The peripheral blood Th1 and Th2 cells determine immune inflammatory responses at the maternal-fetal interface. Previously, we reported that about 40% of women with RIF (≥4) had elevated Th1/Th2 cell ratios (≥10.3). The pregnancy rate was significantly improved when they were treated with tacrolimus, an immunosuppressive agent [AJRI 2015]. However, genetic embryonic concerns of RIF could not be ruled out since not all transferred embryos were genetically screened. Recently, we reported an adjusted cut-off value of the Th1/Th2 cell ratios (≥11.8) [AJRI 2021] for women with RIF based on the retrospective analysis. However, the prospective study has not been performed. Study design, size, duration A prospective cohort study was performed, including 569 infertile women with more than 4 times of RIF. The study participants received ET with euploid blastocysts from September 2020 to November 2021. All transferred blastocysts were confirmed as euploid or low-frequency mosaic by preimplantation genetic testing for aneuploidy, and the endometrial preparation for ET was made by using either hormone replacement cycle or natural ovulatory cycle. Participants/materials, setting, methods Before ET, all participants were measured for their peripheral blood Th1/Th2 (CD4+IFN-γ+/ CD4+IL-4+) cell ratios in the secretory phase. Women who had elevated Th1/Th2 cell ratios (≥10.3) received tacrolimus (2-4 mg daily). Tacrolimus was started 2 days before ET, and the hCG and gestational sac (GS) rates were calculated in women with or without tacrolimus. Main results and the role of chance Of 569 participants, 174 (30.6%) women showed elevated Th/Th2 cell ratios (≥10.3), whereas the remaining 395 women served as control (Control group). Among 174 women, 148 women received tacrolimus (Tac group), while 26 did not receive tacrolimus (no-Tac group). The hCG and GS rates of the Tac group were 73.0% and 64.2%, respectively, similar to the no-Tac group (69.2% and 57.7%, respectively, P=NS). Meanwhile the hCG and GS rates of the control group were 70.6% and 60.0%, respectively (P=NS). When the Th1/Th2 cell ratio cut-off value was set to ≥ 11.8, 123 women of the 569 participants (21.6%) had elevated Th1/Th2 cell ratios (≥11.8). Among them, 112 received tacrolimus (Tac2 group), and 11 did not tacrolimus (no-Tac2 group). The remaining 410 women were as a control group (Control2 group). The GS rate of the Tac2 group was 67.0%, which was significantly higher than that of the no-Tac group (36.3%, p < 0.05), while the GS rate of the Control2 group was 60.5% (P=NS, vs Control2 group). Tacrolimus dosing data was as follows; 11.8 or more but less than 15.8 of Th1/Th2 cell ratios were needed for 2 mg of tacrolimus daily, and 15.8 pr more was needed for 3 mg of tacrolimus. Limitations, reasons for caution The study was a controlled trial with a limited number of study population. Additionally, the endometrial changes or peripheral immune responses after tacrolimus were not evaluated thoroughly. Further study is needed for the tacrolimus effect on systematic immune responses and local endometrial immune milieu. Wider implications of the findings Before ET, screening for increased Th1/Th2 ratios (Cut-off value ≥11.8) may significantly improve IVF outcomes by selecting proper candidates for tacrolimus treatment and detecting the proper tacrolimus doses. Notably, a significant proportion of women with RIF have additional implantation failures, even with euploid embryos, when immune-inflammatory conditions were not controlled. 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-133 Impaired blastocyst viability induced by vitrification during re-expansion after trophectoderm biopsy can be prevented by artificial shrinkage before vitrification

Abstract Study question Does the duration between trophectoderm biopsy and vitrification or blastocyst re-expansion affect blastocyst adhesion and outgrowth competence? Summary answer Outgrowth was impacted when blastocysts were vitrified during re-expansion, especially at 2 h post-biopsy; however, this can be mitigated using artificial shrinkage pre-vitrification. What is known already Biopsied blastocysts are usually vitrified until the results of preimplantation genetic testing for aneuploidy are available. The viability of biopsied blastocysts after vitrification is affected by various factors; in fact, a rodent study reported that the duration from biopsy to vitrification and re-expansion at vitrification affected subsequent developmental competence. The vitrification of biopsied blastocysts immediately post-biopsy is recommended in the clinical setting. However, in some cases, embryologists cannot perform vitrification immediately after a biopsy because of other procedures. In this case, blastocysts start re-expanding, which requires embryologists to wait for the completion of the blastocyst re-expansion. Study design, size, duration Human blastocysts donated for research by consenting couples were randomly allocated into biopsied (n = 175) and non-biopsied (control; n = 50) groups. The re-expansion degree (%) at vitrification was calculated using the following formula: [1- (distance of perivitelline cavity at vitrification)/(distance of perivitelline cavity post-biopsy)] × 100. The blastocysts were categorized according to expansion degree: I, ≤0%; II, 1–49%; III, 50–99%; and IV, ≥100%. Participants/materials, setting, methods Biopsied and control blastocysts were vitrified 0–3 h post-biopsy. Some blastocysts were artificially shrunken in hypertonic solution before the vitrification. The blastocysts were warmed and plated on fibronectin-coated dishes and cultured for 96 h to assess the competence for blastocyst adhesion and outgrowth. The correlations of outgrowth outcomes with vitrification timing, re-expansion degree, and artificial shrinkage were retrospectively analysed. Main results and the role of chance No significant intergroup difference was observed in blastocyst adhesion rate. The multivariate linear regression analysis showed that the blastocyst outgrowth area was significantly smaller when the blastocysts were vitrified at 2 h post-biopsy (P = 0.0103). Re-expansion degree was positively correlated with duration after biopsy (Spearman’s rank correlation coefficient, 0.6921; P < 0.0001). The proportion of expansion in group III was significantly higher at 2 h post-biopsy (P < 0.0001). On multivariate linear regression analysis, the blastocyst outgrowth area was significantly smaller in group III (P = 0.0134). The outgrowth area of blastocysts vitrified at 2 h post-biopsy was significantly increased by artificial shrinkage before vitrification (P = 0.0025) and comparable with those of blastocysts vitrified at 0, 1, and 3 h post-biopsy. Multivariate linear regression analysis also demonstrated the effectiveness of artificial shrinkage for blastocysts vitrified at 2 h post-biopsy and for blastocysts for which re-expansion was initiated at vitrification. Limitations, reasons for caution Since this study used discarded vitrified blastocysts, double vitrification was performed. This was an experimental study; therefore, clinical studies are required to validate our findings. Wider implications of the findings The blastocysts are recommended to be vitrified before the initiation of re-expansion, which complicates the laboratory workflow. Our data demonstrate that blastocysts that expand post-biopsy can be vitrified after artificial shrinkage without decreasing their implantation competence. Our findings may contribute to improving the day-to-day practice in embryology laboratories. Trial registration number Not Applicable

P-543 Multicenter concordance study of embryo cell-free DNA and trophectoderm biopsies: impact on clinical outcomes

Abstract Study question Are reproductive outcomes of transfers from euploid day-6 biopsied blastocysts different depending on results of the corresponding embryo cell-free DNA (cfDNA) in the culture medium? Summary answer Transfers with euploid trophectoderm biopsies but aneuploid cfDNA showed higher miscarriage rates than those with euploid trophectoderm biopsies and cfDNA, although without reaching statistical significance. What is known already During development, embryos release cfDNA. There is, therefore, genetic material in the droplet of culture medium in which they develop. When analyzing, for the same embryo, the spent blastocyst medium containing cfDNA, trophectoderm (TE) and inner cell mass biopsies or the whole blastocyst, high concordance rates have been observed. This high representativeness shown by cfDNA on the chromosomal content of the blastocysts opens a new era of possibilities for non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) as its analysis avoids the need of an invasive trophectoderm biopsy for aneuploidy detection. Study design, size, duration Prospective, observational, multicenter study conducted in 10 assisted reproductive centers after approval by each local institutional review board. From April 2018 to December 2022, 2539 embryos from 716 patients included in PGT-A cycles were cultured following a niPGT-A culture protocol. Blastocysts underwent TE biopsy and media collection that were subsequently analyzed and compared. 441 frozen single embryo transfers (SET) from euploid embryos, based on the TE biopsy result, were performed and their clinical data registered. Participants/materials, setting, methods Embryos were cultured in routine conditions up to day 4, then washed and transferred to a new 10μl droplet. On day 6, media were collected and frozen at -20 °C; and blastocyst biopsy and vitrification were performed. Samples were analyzed by NGS (Ion ReproSeq PGS kit, ThermoFisher Scientific) and the results processed with customized algorithms for TE/cfDNA. SET were performed based on TE biopsy results, with blinded cfDNA results at the moment of transfer. Main results and the role of chance Clinical outcomes were compared according to whether the TE and cfDNA result was euploid (eu-eu) or with euploid TE and aneuploid cfDNA (eu-aneu). Transfers where cfDNA analysis had provided non-informative results were excluded. The eu-eu group included 288 transfers (mean female age = 35.3 ± 4.9 years), with an ongoing pregnancy rate (OPR) of 44.4% and a miscarriage rate (MR) of 14.7%. Whereas the eu-aneu group included 95 transfers (mean female age = 34.4 ± 5.2 years), with an OPR of 43.2% and a MR of 25.5%. The comparisons of OPR and MR between both groups did not reach statistical significance (Chi-square test), but MR showed a two-fold increase in transfers with aneuploid cfDNA. A sub-analysis was performed, while only considering transfers from patients without endometrial factor history. Eu-eu and eu-aneu groups included 231 and 73 transfers, respectively. The differences in OPR (49.4% vs 43.8%) and MR (14.3% vs 25.6%) were slightly higher between both groups but did not reach statistical significance (Chi-square test). Limitations, reasons for caution To better understand the trends observed, especially on the miscarriage rate, it would be necessary to collect clinical outcomes from more SET. This study could not determine the benefit of embryo selection based only in cfDNA, since SET was performed according to TE biopsy results. Wider implications of the findings The analysis of embryo cfDNA could provide useful information to help select the best embryo for transfer and, therefore, decrease miscarriage rates and increase the chances of having a healthy newborn at home. Trial registration number NCT03520933

P-537 Linking genetic variation to infertility and embryonic genomic instability. Lessons from a database of whole genomes from targeted populations: challenges, and future directions

Abstract Study question Can novel genomic analysis methods be developed to identify genetic variants that predispose to specific types of infertility and genomic instability in embryos? Summary answer Whole genome analysis and the creation of a genomic variation database of highly targeted populations yielded several genomic links to human infertility and genomic instability. What is known already Infertility is a phenotypic manifestation that originates from highly variable causes, like repeated miscarriage (RM), repeated implantation failure (RIF) or various male and female factors. Specific genetic variations can predispose carriers to distinct types of infertility however quantifiable genetic risks are difficult to be established clinically due to the high amount of variables that exist in any relevant infertility studies. In addition, the combination of the maternal and paternal genetics further complicates the interpretation of genomics results especially when genetic instability in embryos are studied. Several studies have indicated multi[le risk variants however few have managed to establish causal relationships. Study design, size, duration The causes of infertility are extremely variable, which presents a challenge in the design and analysis of genome sequencing studies that involve large populations of participants with similar characteristics. In this study a targeted approach highly selected populations allows more detailed examination of the whole genome of the participants and the different subgroups that exist among them. The participants were recruited using specific selection criteria and samples were collected over a period of 5 years. Participants/materials, setting, methods Couples presenting with infertility and went through preimplantation genetic testing for aneuploidy (PGT- A) were invited to participate. Selection criteria were applied: i) referred for PGT-A after >3 assisted reproduction failed cycles, ii) Normal karyotypes, iii) Female age less than 37, iv) Embryonic instability in their subsequent PGT-A cycles in more than 40% of the produced embryos. After whole genome sequencing (WGS) and variant calling, a bespoke genomic SQL database was constructed for genomic analysis. Main results and the role of chance From 118 individuals that consented to participate 28 individuals with repeated implantation failure (RIF) and 8 individuals RIF/RM passed the selection criteria and their DNA was processed with WGS using a short read platform. In addition, WGS Validation was performed using genomes with known mutations. After the WGS quality control and validation. In total 50 genomes were processed and produced variant results. A novel MySQL database was successfully constructed and validated. Variant z scores were calculated for each participant and the significantly flagged variants were pulled and analysed and comparison with other bespoke and publicly available databases was performed. Ethnicity bias in novel variations detected and corrected. Several smaller cohorts were identified with similar genomic characteristics and accumulation of specific sets of polymorphisms, that were associated with certain types of infertility and preimplantation embryo abnormalities. Of particular interest were variants in synaptonemal complex genes associated with gamete failure, meiotic errors and instability in embryos. Structural variations were significant for male factor infertility. The WGS approach also revealed novel intronic and extragenic variants that would not be usually picked up by other methods like exome sequencing but can play a vital role in gene expression. Limitations, reasons for caution As with all genomic studies relating to infertility, association does not mean causation. Further functional analysis would be required to establish definitive causal pathways based on significant risk variants. In addition, although our study is highly targeted more genomic data can enhance to validation of these results. Wider implications of the findings This highly targeted study using a personalised approach to genome analysis combined with WGS can lead to protocols that determine the cumulative clinical risk for infertility, by revelling the accumulation of specific sets of polymorphisms, intronic and exonic, can be associated with certain types of infertility and preimplantation embryo abnormalities. Trial registration number not applicable

P-167 The sperm parameters in doubling oocytes by the 1st polar body transfer technique: does it matter?

Abstract Study question Does sperm parameters affect the effectiveness of the 1st polar body (PB) transfer in oocytes and embryo doubling? Summary answer Certain sperm conditions affect the transfer technique efficiency. What is known already Previous studies have shown the effectiveness of the 1st polar body (PB) transfer. The technique allows for the modification of donor oocytes to increase the number of cells suitable for fertilization and at the same time preserve the genetic relatedness to the patient. We established that the effectiveness depends on the morphology of the 1stPB. We also showed that the yield of high-quality blastocysts does not depend on whether fresh or thawed donor oocytes are used. But it is also important to understand whether sperm parameters have a significant effect on the performance of the technique. Study design, size, duration The study was performed in the Medical Center IGR from December 2017 to December 2023 and involved 93 patients (mean age 39.7±5.4 years) who underwent the 1st PB transfer and had indicated sperm parameters used for fertilization, including: normozoospermia (NS, n = 43), asthenozoospermia (AS, n = 24), asthenoteratozoospermia (ATS, n = 20), oligozoospermia (OS, n = 5), cryptozoospermia (CS, n = 1). We evaluated the pronuclear scoring, number of embryos on the 3rd and the 5th post-fertilization day, euploidy according to sperm conditions. Participants/materials, setting, methods We used donor oocytes, patients’ oocytes and their 1stPBs obtained from 93 women and their partners sperm with defined parameters in accordance with WHO norms for 2021. The procedure of donor oocyte enucleation and the 1stPBs transfers was carried out using Nikon Ti Eclipse(Japan) inverted microscope, Saturn 3 laser console(UK). Preimplantation genetic testing for aneuploidy was performed using trophectoderm biopsy. Samples were diagnosed using Ion S5(USA). Statistical analysis was carried out using Chi-square test. Main results and the role of chance The total number of donor oocytes modified by the 1stPB of patient’ oocytes was 364, which were distributed as follows by sperm status: NS - 159 oocytes, AS - 103 oocytes, ATS - 77 oocytes, OS - 19 oocytes, and CS - 6 oocytes. The mean age of the female patients according to the groups was similar (39.6±4.9, 39.8±6.2, 40.0±3.8, 37.8±6.1,40, respectively). The number of 2PN pattern during pronuclear scoring was distributed among groups (NS:AS:ATS:OS:CS) with the following percentages 69.2:65.1:62.3:73.7:66.7, respectively. Among the developing embryos on the 3rdday the distribution was 46.6:46.5:36.4:57.9, respectively. No viable embryos were formed in the CS group, so the group was not included in further calculations. Statistically significant difference (SSD) was not found between the groups on the above-mentioned stages of development (p0.05). On the 5thday the number of developing embryos (percents) was distributed as follows: 23.3:28.2:11.7:31.6 for NS:AS:ATS:OS, respectively. It is worth noting that SSD was found between NS and ATS, OS and ATS groups (p < 0.05) and AS and ATS groups (p < 0.001). Percentage of euploid embryos relative to the number of examined blastocysts from groups NS:AS:ATS was 30.8:23.1:22.2, respectively, SSD wasn’t found between the groups(p > 0.05). Limitations, reasons for caution To increase performance of the 1st PB transfer technique it is also worth considering such parameters as sperm DNA fragmentation and level of sperm oxidative stress, which reflect the chromatin state of the male gamete. Wider implications of the findings The study showed that among patients with asthenoteratozoospermia, the yield of viable embryos was significantly lower compared to the other sperm conditions studied. This fact must be taken into account when predicting the result of using the 1st PB transfer technique. Trial registration number N/A

P-050 Microfluidic chip-sorted spermatozoa selection yields embryo euploidy rate and reproductive outcomes similar to density-gradient centrifugation

Abstract Study question Does microfluidic chip-sorted spermatozoa selection (MSS) improve good quality blastocyst (GQB) and embryo euploidy rates compared to density gradient centrifugation (DGC)? Summary answer The use of MSS before intracytoplasmatic sperm injection (ICSI) does not improve good quality blastocist and euploidy rates comparing to DGC. What is known already The integrity of sperm DNA plays a crucial role in embryonic development. Conventional sperm selection methods, such as DGC, may potentially damage sperm DNA due to oxidative stress generated during centrifugation. One of the recent advances in sperm selection techniques is the microfluidic chamber, which, requiring less processing time and eliminating the need for centrifugation, allows for the selection of sperm with better mobility, morphology, and reduced DNA damage. Consequently, improved GQB and euploid embryo rates, as well as enhanced reproductive outcomes, are expected. Study design, size, duration Retrospective study of 1739 blastocysts from 404 couples undergoing 544 autologous cycles of ICSI/PGT-A (preimplantation genetic testing for aneuploidies) from January 2020 to December 2022. Those patients with sperm concentration <5 × 106/mL or motility <10%, altered karyotype, women aged > 43 years, uterine anomalies and thrombophilia were excluded. Besides comparing euploidy rates and blastocyst formation, reproductive outcomes were analyzed and compared. Participants/materials, setting, methods According to the sperm selection technique, two groups were established. One group underwent MSS (ZyMōt ICSI® or ZyMōt Multi®), resulting in 295 blastocysts from 96 cycles. It was compared with a control group (DGC) including 1444 blastocysts from 448 ICSI/PGT-A cycles. PGT-A was performed in day 5/6 using Next Generation Sequencing (NGS) technology. For a 95% confidence interval, statistical significance was considered at p < 0.05. Chi-square analysis was used for the statistical evaluation. Main results and the role of chance Both groups were comparable in terms of demographic data, including age, sperm analysis parameters, and cycle characteristics. There were no significant differences between the DGC and MSS groups in the women’s age (mean ± SD) (37.9 ± 3.9 vs. 38 ± 3.9 years, p = 0.99) or men’s age (38.4 ± 7.1 vs. 39.8 ± 5.9 years, p = 0.11). No significant differences were found between the DGC and MSS groups in terms of GQB rate (49.3% vs. 50.7%, p = 0.55), euploidy rate (35.8% vs. 32.2%, p = 0.23), fertilization rate (69.5% vs. 69.5%, p = 0.99), clinical pregnancy rate per transfer (63% vs. 76%, p = 0.13), clinical miscarriage (9% vs. 21%, p = 0.33), and live birth rate per transfer (44% vs. 37%, p = 0.42). The results also showed a higher number of euploid embryos per initiated cycle in the DGC group (1.154) compared to the MSS group (0.989). Despite this difference, the results were not statistically significant (p > 0.05). Limitations, reasons for caution The limitations of this study include the potential impact of selection bias, as it is a retrospective study in which groups were not randomly assigned. Additionally, the low number of blastocysts in the MSS group and the potential bias of DNA fragmentation, which was not evaluated in the study. Wider implications of the findings Both methods demonstrated similar effectiveness. While MSS could potentially be more effective in samples with high DNA fragmentation and they feature a user-friendly application, its major limitation lies in its challenging application with samples having a low concentration of motile sperm. Specific semen samples may need different sperm selection methods. Trial registration number Not Applicable

P-285 Serum progesterone level may not impact ongoing pregnancy rate (OPR) in patients with endometriosis undergoing frozen-warmed-blastocyst transfer employing hormone replacement treatment (HRT) with subcutaneous progesterone

Abstract Study question Do serum progesterone (P4) levels on frozen embryo transfer (FET)-1 day impact OPRs in patients with endometriosis undergoing HRT-FET with subcutaneous progesterone (sc P)? Summary answer In HRT cycles employing sc P, serum P4 levels on FET-1 day do not affect OPRs in patients with or without endometriosis. What is known already In HRT-FET cycles employing vaginal P, reproductive outcomes are improved by serum P4 monitoring and employing a rescue protocol, if needed. However, there is paucity of data for the need of serum P4 monitoring in HRT cycles employing sc P. Endometriosis is associated with P4 resistance. There is only one study evaluating the impact of serum P4 levels on reproductive outcomes in patients with endometriosis; in this study lacking a control group and employing vaginal P gel 90 mg/12 hours supplemented by daily 50 mg intramuscular P, a threshold of 37.1 ng/ml has been reported for improved live birth rates. Study design, size, duration Retrospective cohort study. 451 consecutive patients undergoing their first HRT- vitrified warmed blastocyst transfer during November 2021-June 2023 at Anatolia IVF Centre were included. 44 patients had endometriosis with surgical and/or ultrasound findings and the remaining 407 patients did not harbor endometriosis. The presence of adenomyosis was an exclusion criterion. Following 10-12 days of 6 mg/day estradiol valereate use, sc P 25 mg/12hours (Prolutex, Ibsa) was administered. The primary outcome measure was OPR. Participants/materials, setting, methods Serum P4 levels were measured 4-5 hours after morning administration of sc P on FET-1 day. The impact of serum P4 on OPRs was assessed at various percentiles adjusting for female age, body mass index, prior birth, preimplantation genetic testing for aneuploidy, number/grade of blastocysts transferred. Multivariate regression models were performed in the whole cohort incorporating polynomial P4 terms and endometriosis interactions; optimal model for OPR prediction was selected using the lowest Akaike Information Criteria. Main results and the role of chance Patients with (27/44, 61.4%) and without (214/407, 52.6%) endometriosis had comparable OPRs. In the endometriosis group, the 10th, 50th, and 90th percentiles of serum P4 were 15.6, 25.1, and 35.4 ng/ml, respectively, compared to 14.9, 23.5, and 35.6 ng/ml in the non-endometriosis group. In the endometriosis group, the OPRs were 60.0%, 66.7%, 56.3%, and 60.0% for the <10p, 10-50p, 50-90p, >90p groups, respectively. These figures were 46.3%, 50.3%, 56.8%, and 51.2%, in the non-endometriosis group, respectively. In the endometriosis group, taking the 50-90p group as the reference, the adjusted odds ratios (aORs) for OPR of < 10p, 10-50p, and >90p groups were 1.24 [0.03, 50.45], 1.77 [0.31, 10.24], and 2.08 [0.05, 80.23], respectively. In the non-endometriosis group, the respective aORs were 0.85 [0.40, 1.79], 0.80 [0.49, 1.29], and 0.72 [0.34, 1.50]. In the multivariate regression analysis to determine the optimal model, neither serum P4 levels (aOR for linear term: 1.11 [0.98, 1.26]; for quadratic term: 0.998 [0.996, 1.00]) nor the diagnosis of endometriosis (OR: 1.48 [0.74, 2.98]) significantly predicted ongoing pregnancy after adjusting for confounders. Additionally, the optimal model did not include the interaction term, suggesting that endometriosis does not significantly modify the impact of serum P4 levels on OPRs. Limitations, reasons for caution The study’s limitations include retrospective study design, a limited sample size of the endometriosis cohort, absence of laparoscopy for endometriosis exclusion in the non-endometriosis group, and lack of data on endometriosis phenotypes. Wider implications of the findings In HRT-FET cycles employing sc P 25 mg/12hours neither serum P4 nor the presence of endometriosis have an impact on OPRs. Endometriosis may not modify the impact of serum P4 on OPRs suggesting a lack of P resistance in such cycles or the overcoming of P resistance by this regimen. Trial registration number not applicable

P-588 Serum Luteinizing Hormone levels before progesterone supplementation are significantly associated with live birth rate in single euploid frozen embryo transfers performed in hormonal replacement cycles

Abstract Study question Do luteinizing hormone (LH) levels measured prior to initiating progesterone supplementation influence live birth rate (LBR) in artificial frozen-thawed single euploid blastocyst transfer cycles? Summary answer Serum LH levels, measured prior to progesterone initiation in artificial frozen-thawed single euploid blastocyst transfer cycles, are significantly associated with LBR. What is known already In the absence of a dominant follicle, estrogen supplementation in artificial endometrial preparation for frozen embryo transfer (FRET) can lead to a rise in endogenous LH levels, as observed in a natural cycle. Since the LH rise is a key feature in natural conception and the embryo, the endometrium, and the myometrium display receptors for LH, the rise observed during FRET preparation may have an impact on the clinical outcomes of the treatment. Previous studies found contradictory results hence none of them excluded embryo aneuploidy by implementation of preimplantation genetic testing for aneuploidies and the transfer of euploid embryos. Study design, size, duration Retrospective analysis of 639 frozen-thawed single euploid blastocyst transfer cycles performed in a tertiary referral IVF center from January 2018 to August 2023. Cycles with hormonal pre-treatment or pituitary suppression were excluded. Participants/materials, setting, methods Single euploid FRET cycles were included. Oral estradiol administration commenced on cycle-day 2/3. Estradiol, progesterone, and LH were measured at the start of endometrial preparation and up to 3 days before progesterone initiation, and the slope of LH was calculated. Patients were stratified in groups according to their LH values before progesterone administration, and the impact of LH on LBR and implantation rate was assessed. Logistic regression analysis was performed to adjust for potential confounders. Main results and the role of chance In total 639 cycles were included. Patients’ characteristics (mean+/-SD) were: age 33.2±5.9 years; BMI 27.5±4.9 kg/m2. The median duration of estrogen administration was 15 days (range: 11-27 days). The overall implantation rate and LBR were 65.9% (421/639) and 46.9% (300/639), respectively. Patients were stratified into six groups according to their LH levels prior to progesterone supplementation: <10th percentile (≤6mIU/ml, n = 57), p10th-p25th (>6 to ≤ 8.5mIU/ml, n = 81), p25th-p50th (>8.5 to ≤ 12.4mIU/ml, n = 176), p50th-p75th (>12.4 to ≤ 18.1mIU/ml, n = 180), p75th-p90th (>18.1 to ≤ 25.0mIU/ml, n = 91) and >p90th (>25.0mIU/ml, n = 54). A significant trend for higher LBR with increasing serum LH levels was observed (Cochran-Armitage Test, P = 0.041) but not for implantation rate (P = 0.158). Those with higher basal serum estradiol levels before estrogen supplementation had a steeper LH increase from basal to progesterone-supplementation day (0.40 mIU/mL higher per 10pmol/L increase in basal estradiol, P = 0.027). Regression analysis identified as independent predictors of LBR BMI (OR 0.96, 95%CI: 0.93-0.99) and embryo quality (CC vs. AA p < 0.001, CB vs. AA p = 0.027, BC vs. AA p = 0.013). Patients with steeper LH increases from estrogen to progesterone-supplementation day had higher LBR in both univariable (OR:1.27, 95%CI: 1.00–1.63, P = 0.052) and multivariable analyses (OR:1.26, 95%CI: 0.97–1.64, P = 0.085) but the difference didn’t reach statistical significance. Limitations, reasons for caution The retrospective design of the study is a limitation. Results should be interpreted with caution due to the small number of patients in the lowest and highest LH percentile groups. Wider implications of the findings The results of this study suggest that serum LH levels measured before progesterone supplementation may have an impact on the clinical outcome of artificial frozen-thawed blastocyst transfer cycles. Further studies with a higher sample size are necessary to confirm or refute our findings. Trial registration number not applicable

P-668 Progesterone levels on the thawed embryo transfer day in artificial cycles with vaginal progesterone are not a predictor of clinical pregnancy: a prospective study

Abstract Study question Are serum progesterone (P) levels on the thawed embryo transfer day a good predictor of clinical pregnancy with own oocytes in artificial endometrial preparation cycles? Summary answer In artificial endometrial preparation cycles, serum P levels on the thawed embryo transfer day are not a good predictor of clinical pregnancy with own oocytes. What is known already Some studies have shown that low serum P levels on the thawed embryo transfer (ET) day are associated with a lower pregnancy rate after artificial endometrial preparation (AEP) cycles. Other studies have shown that serum P is not a good predictor of pregnancy rates. Furthermore, studies are inconclusive about which cutoff level for P on ET day correlates with better gestational outcomes. Study design, size, duration Prospective cohort study with 235 patients who underwent ET after AEP cycle with estradiol valerate and vaginal micronized P (200 mg/ 8 h). All patients underwent blastocyst-stage or cleavage-stage ET with embryos from their own oocytes, without preimplantation genetic testing for aneuploidies, after AEP cycle, with a triple layer endometrium > 7 mm. The study was carried out at the Human Reproduction Center of Ribeirão Preto Clinical Hospital from November 2021 to June 2023. Participants/materials, setting, methods Blood was collected between 8–10 am on the thawed ET day and serum P concentration was measured. The primary outcome was clinical pregnancy (CP) beyond pregnancy week 8. A ROC curve detected the best P cutoff level that correlated with higher CP rate. Thereafter, patients were categorized into those with serum P above and below the cutoff level. Multivariate logistic regression was performed to verify which variables were predictors of CP and P levels. Main results and the role of chance Serum P measured on the ET day showed no significant difference between patients who had CP and those who did not (12.76 +4.1 versus 11.9 + 5.6, p 0.3318). The cutoff level for serum P measured on the ET day above which correlates with higher CP rate was 10.6 ng/mL (AUC 0.58), with a sensibility of 72% and specificity of 44%. Patients who had serum P on the day of ET above this value had a significantly higher CP rate than those who had it below this value (25.9% versus 14.6%) (p 0.0372). However, when the multivariate logistic regression model was performed, there was no significant difference in the CP between the groups (OR 2, 95%IC: 0.9-5). Predictors of CP in the study were the presence of at least one top-quality embryo transferred (OR 2.6, 95%IC: 1-6.6) and the transfer of embryo at the blastocyst-stage (OR 5.2, 95%IC: 1.9-14). The presence of endometriosis was negatively associated with CP (OR 0.2, 95%IC: 0.09-0.9). Limitations, reasons for caution The small sample size is a limitation. We included the blastocyst-stage and the cleavage-stage embryo transfer. Only women with vaginal micronized P were included. Extrapolation to other P administration forms needs to be evaluated. Wider implications of the findings Monitoring serum P levels on the day of ET in AEP cycles with vaginal micronized P is not justified as serum P levels are not a good predictor of CP in cycles with own oocytes. Trial registration number Not applicable