Euploid Blastocysts Research Articles

O-301 Progesterone Primed Ovarian Stimulation (PPOS) for DuoStim combined with PGT-A in poor prognosis patients: a valuable strategy

Abstract Study question Is PPOS rather than conventional GnRH-antagonist a valid strategy in DuoStim protocols? Summary answer PPOS-DuoStim and conventional-DuoStim showed similar mean euploid blastocyst rate (EBR) per inseminated metaphase-II (MII) oocytes. What is known already DuoStim (double stimulation in the same ovarian cycle) is an efficient strategy to increase the number of oocytes retrieved and embryos obtained in the shortest possible timeframe. PPOS is based on the use of exogenous oral progesterone to prevent LH surge during ovarian stimulation (OS). The effectiveness and safety of PPOS have been consistently reported across the last years, supporting it as a clinically-valuable OS protocol. To date, no evidence exists about PPOS application in DuoStim protocols and its association with oocyte competence, defined as EBR per MII-oocytes. Study design, size, duration Retrospective study involving 138 PPOS-DuoStim and 138 matched conventional-DuoStim patients treated at a single clinic between 2021 and 2022 and collecting ≥1 MII-oocyte after both stimulations. Matching was based on maternal age (40.3±2.7 years), number of oocytes collected after the I-stimulation (5.8±3.6 years), and sperm quality (66% normozoospermic). The primary outcome was the EBR per MII-oocytes. All intermediate embryological and clinical outcomes were also compared. Sub-analyses among I- and II-Stimulations were also conducted. Participants/materials, setting, methods DuoStim entailed the same protocol in I- and II-Stimulations with FSH 300IU/day+LH 150IU/day, 5 days spanning the two stimulations and GnRH-agonist to trigger ovulation. The only difference between PPOS-DuoStim and conventional-DuoStim was the administration of medrossiprogesterone acetate10mg/day orally from the first day of stimulation versus flexible GnRH-antagonist from the day the follicles reached a diameter of 13-14 mm. The same laboratory procedures were applied: ICSI, trophectoderm biopsy, comprehensive-chromosome-testing to report uniform aneuploidies and vitrified-warmed single-embryo-transfers. Main results and the role of chance The patients in the two groups were comparable for ovarian reserve markers, BMI, and duration of infertility. All data were comparable in conventional-DuoStim and PPOS-DuoStim (Mann-Whitney U tests were conducted): (i) overall fertilization rate per inseminated MII-oocytes were 73.1±23.2% versus 76.8±17.8% (p = 0.143), (ii) overall blastulation rate per 2PN-zygotes were 46.6±30.4% versus 45.7±25.1%(p = 0.797), (iii) overall euploidy rates per biopsied blastocysts were 24.1±30.9% versus 25.4±31.3%(p = 0.956), (iv) EBR per inseminated MII-oocytes were 9.6±17% versus 9.2±12.7% (p = 0.735). The data among only I- and II-stimulations were also similar. When comparing I- versus II-stimulations, paired t-tests highlighted longer II-stimulations (11±1.9 vs11.6±1.9 days, p = 0.001), resulting in larger cohorts of MII-oocytes (4.3±2.8 vs 5.2±3.3, p < 0.001) with comparable EBR per MII-oocytes (9.7±20.2% vs 11.6±16.2%, p = 0.233), thereby eliciting larger cohorts of euploid blastocysts (0.5±0.07 vs 0.7±0.09, p = 0.005). These data were confirmed also among conventional-DuoStim and PPOS-DuoStim study arms only. From a cycle perspective, among the 138 conventional-DuoStim couples, 31 (22%), 44 (32%) and 59 (43%) obtained ≥1 euploid blastocyst after I-stimulation, II-stimulation and overall, respectively. The same figures among the 138 PPOS-DuoStim were 36 (26%), 44 (32%) and 63 (46%), respectively. No statistical difference was reported (Fisher’s exact test=0.716). Limitations, reasons for caution Retrospective single center design. The current sample size should be doubled in order to reach a 80%-power with a 5%-α to exclude a 5% two-sided difference in the primary outcome. Patients’ compliance and discomfort, as well as clinical, gestational, and perinatal outcomes and cost-effectiveness analyses are needed. Wider implications of the findings PPOS is valuable in unconventional-OS protocols entailing freeze-all, like DuoStim with PGT-A. Future studies should assess if PPOS-DuoStim, by decreasing the number of injections and scans, may improve patient compliance and decrease OS discomfort. Any putative impact on follicular recruitment and oocyte yield should also be determined. Trial registration number not applicable

P-318 Reproductive ageing: the seed, the soil, or both?

Abstract Study question Is it all about the embryo? Can a single euploid blastocyst transfer sustain reproductive outcomes in women with advanced reproductive age? Summary answer Despite euploid embryo transfer, endometrial senescence appears to play a crucial role in determining reproductive success, particularly in older patients undergoing in vitro fertilization (IVF). What is known already Advanced reproductive age significantly impairs oocyte and embryo quality. Effective crosstalk and synchronization between embryo and endometrium are pivotal for successful pregnancies, both in vivo and in vitro. Implantation failure remains a persistent challenge in IVF, exacerbated by delayed family planning trends. The transfer of euploid blastocysts through preimplantation genetic testing for aneuploidy (PGT-A) is a potential strategy to address implantation issues, decrease miscarriages, and shorten time-to-pregnancy. However, a significant proportion of IVF treatments remain unsuccessful, suggesting critical factors beyond the embryo. Study design, size, duration This retrospective study analyzed 457 single euploid blastocyst transfers performed by two doctors at a private IVF center from January to November 2023. Inclusion criteria encompassed cases with PGT-A indications (advanced maternal age, repeated implantation failure, recurrent pregnancy loss, etc.) or cases where embryo biopsy was patient-requested. The primary outcome was ongoing pregnancy rate (OPR), with secondary outcomes including pregnancy rate (PR) and clinical pregnancy rate (CPR). Participants/materials, setting, methods Participants were categorized into four groups: Group 1 (< 35 years old; N = 100), Group 2 (35-37 years old; N = 102), Group 3 (38-40 years old; N = 118), and Group 4 (> 41 years old; N = 87). A control group (oocyte donation; N = 50) with single euploid embryo transfers showed a mean recipient age of 43 years. Donors were always less than 32 years old. Endometrial preparation protocols were consistent across all groups. Chi-square test was used to compare groups. Main results and the role of chance Among the 457 transfers analyzed, with a mean female age of 37 years, the overall PR was 71.3%, CPR was 64.8%, and OPR was 60.2%. Groups showed no differences in demographic variables and cycle parameters. Group 1 demonstrated higher PR (83.3%), CPR (78.4%), and OPR (73.5%) compared to Group 2 (PR 70.9%, p = 0.034; CPR 66%, p = 0.047; OPR 62.1%, p = 0.081), Group 3 (PR 68.2%, p = 0.009; CPR 61.2%, p = 0.005; OPR 56.6%, p = 0.008), and Group 4 (PR 65%, p = 0.002; CPR 56.1%, p < 0.001; OPR 51.2%, p = 0.001). Furthermore, the control group (oocyte recipients) showed significantly lower PR (68%, p = 0.031), CPR (54%, p = 0.002), and OPR (46%, p < 0.001) compared to Group 1, but not with the other groups. These findings demonstrate that even with euploid embryos originating from young donors, older women exhibited lower pregnancy rates than young women transferring single euploid blastocysts from their own oocytes and similar rates compared to those with advanced reproductive age transferring their own euploid blastocysts, implying that endometrial ageing may disproportionately influence IVF treatment outcomes. Limitations, reasons for caution The retrospective design introduces inherent bias, notably selection bias and unmeasured confounders. This preliminary data did not account for male infertility factors and their impact on embryo quality. Wider implications of the findings Reproductive aging appears to be influenced by factors beyond the embryo itself, especially in women above 35 years. Further research is warranted to elucidate the causes and impact of reproductive aging on implantation potential, aiming to refine clinical practices and enhance the success of IVF treatments. Trial registration number not applicable

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

O-025 An artificial intelligence (AI) model non-invasively evaluates endometrial receptivity from ultrasound images, surpassing endometrial thickness (EMT) in predicting implantation

Abstract Study question Can an AI image-based model be trained to predict whether an endometrium, prior to progesterone, is receptive for successful embryo implantation from ultrasound images? Summary answer An endometrial receptivity AI model, utilizing ultrasound images and clinical features, is predictive of successful implantation and outperforms endometrial thickness as a predictor of implantation. What is known already Despite its importance in successful embryo implantation and subsequent live birth, endometrial receptivity is difficult to measure. The standard of care at some clinics is to cancel an embryo transfer if EMT is measuring less than 7 mm on ultrasound assessment – however, achieving an EMT threshold does not guarantee success, even with euploid blastocysts. Currently available receptivity tests (e.g. endometrial receptivity assays) are unreliable and have not shown significant improvements in reproductive outcomes, necessitating the development of new evaluation methods for endometrial receptivity. AI can potentially address this gap in clinical care and understanding, aiming to increase patient success. Study design, size, duration 79,602 ultrasound images (40,910 patients) of the endometrium, on the day of progesterone start according to patient’s frozen embryo transfer cycle, were retrospectively collected from 4 clinic networks including 70 clinic locations. PGT-A outcomes and blastocyst quality were available for 15,466 and 36,070 blastocysts respectively, with 14,538 having labels for both. Different image samples (based on embryo quality) were selected to train and test several AI models, with implantation (positive beta-hCG) as a binary outcome. Participants/materials, setting, methods Three criteria for negative (implantation) class sample selection were tested for receptivity model development: 1) high- or medium-quality and/or euploid blastocysts; 2) high-quality blastocysts; 3) all blastocysts. Models were ensembles consisting of an image-based deep learning (DL)-model and a feature-based machine learning (ML)-model. Model performance was measured by Area-under-the-curve (AUC), sensitivity, and specificity. Feature importance from the ML model was assessed, while receptivity model performance was compared to the power of EMT to predict implantation. Main results and the role of chance A receptivity model trained on scenario 1) (n = 27,424) achieved AUC 0.613, sensitivity 0.716, and specificity 0.439 on a test set of 9197 samples, in predicting implantation. Another receptivity model trained on scenario 2) (n = 25,625) achieved AUC 0.627, sensitivity 0.637, and specificity 0.543 on a test set of 8642. Whereas a third receptivity model trained on scenario 3) (n = 31,238) was selected for further analysis as it attained the best performance with AUC 0.631, sensitivity 0.628, and specificity 0.556 on a test set of 10,422. The ensemble model also utilizes relevant clinical features, which were ranked by importance to model predictions: EMT; progesterone blood test value; age at transfer; previous total embryo transfers; days between ultrasound date and transfer date; age at retrieval; and oocyte origin. EMT was assessed for its ability to predict implantation at thresholds between 5–20 mm. The threshold that best separates positive and negative implantation was determined by evaluating AUC on a validation set of 5682. A threshold of 8.8 mm achieved an AUC 0.576, sensitivity 0.694, and specificity 0.455 in predicting implantation on a test set of 5686. However, the receptivity model AUC (0.631) was significantly higher (p < 0.001; DeLong test for differences in AUC), outperforming EMT predictability. Limitations, reasons for caution The endometrial receptivity model was built and tested on retrospective frozen embryo transfer data. Results should be corroborated in prospective and non-selection studies, as well as in multiple geographical locations (all clinics included reside in the United States). The dataset requires greater diversification, with a greater representation of EMT (<7mm). Wider implications of the findings This study indicates that an AI model can be built to predict implantation from ultrasound images and clinical features, surpassing current standards - EMT alone. Further experimentation, with additional clinical features, may improve performance to facilitate development of a model that can accurately predict the gold-standard of live birth. Trial registration number Not applicable

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

P-419 Relevance of intravenous immunoglobulins in patients with unexplained recurrent pregnancy loss that occurred at the same gestational weeks every time

Abstract Study question Are intravenous immunoglobulins (IVIG) effective for the selected patients who experienced unexplained recurrent pregnancy loss (RPL) that occurred at the same gestational weeks every time? Summary answer IVIG before and after the gestational week-limit (GWL), where miscarriage occurred at the same gestational weeks every time, may improve pregnancy outcomes in RPL patients. What is known already There is no established treatment for patients with unexplained RPL. Some investigators have indicated that IVIG may exhibit therapeutic efficacy in women with unexplained RPL. Since it has been reported that live birth rate significantly increased in women with four or more RPL of unexplained etiology, who received a high dose IVIG at 4-5 weeks of gestation in 2022, ESHRE Guideline Group on RPL (2022) has changed the recommendation to the following: the use of repeated and high doses of IVIG very early in pregnancy may improve live birth rate in women with four or more unexplained RPL. Study design, size, duration We performed a retrospective study between September 2013 to March 2023 in two fertility centers. We included 164 patients in the study who experienced 2 or more miscarriages between 5 and 21 weeks of gestation that occurred at the same gestational weeks every time, who had all negative results for our routine RPL work up and still unsuccessful result after treating these conditions, and whose products of conceptus (POC) revealed at least one normal karyotype. Participants/materials, setting, methods IVIG (Venoglobulin IH 5%, Japan Blood Products Organization, 0.4g/kg) injection was performed twice before and after 1 week of GWL. We divided 164 patients into IVIG group and non-IVIG group by their own choice. The primary outcome was live birth rate, and the secondary outcome was modified live birth rate excluding pregnancies with abnormal fetal karyotype, maternal and neonatal complications. All participants provided written informed consent, and Institutional Review Board approval was obtained. Main results and the role of chance IVIG group (n = 116) and non-IVIG group (n = 48) showed no significant differences in the background of patients on BMI (20.3±3.7 and 21.1±2.8), age (37.4±4.2 and 36.7±3.2) and Childbirth history (20.7% and 23.0%), respectively (average±SD), except for the history of past miscarriages. Average numbers of previous miscarriages on IVIG group and non-IVIG group were 3.2±1.3 and 2.5±0.8 (p < 0.001), respectively. After multivariate analysis for BMI, age, Childbirth history and previous miscarriages, live birth rate of IVIG group (70.7%, 82/116) was significantly higher than that of non-IVIG group (60.4%, 29/48; Odds ratio 3.40, 95% CI: 1.49-7.74, p = 0.003). Similarly, modified live birth rate of IVIG group (73.9%, 82/111) was significantly higher than that of non-IVIG group (63.0%, 29/46; Odds ratio 3.40, 95% CI: 1.49-7.74, p = 0.003). Treatment outcomes when IVIG was used for each number of previous miscarriages, the modified birth rate was 88.3% for two times miscarriages, 77.0% for three times, and 57.1% for four or more miscarriages. Maternal complications were observed in 2 cases in IVIG group (chest tightness, obstetric critical bleeding). There were no significant differences in the birth weight, gestational weeks, preterm birth, or fetal growth restriction of newborns between two groups. Limitations, reasons for caution Since retrospective nature and we did not perform POC tests for all patients whose pregnancies had ended in miscarriage, we could not assert the effectiveness of IVIG. We need to conduct randomized controlled trial using placebo group to demonstrate the IVIG efficacy before and after the GWL using euploid blastocysts. Wider implications of the findings IVIG before and after the GWL adjusted for each patient may improve pregnancy outcomes in RPL patients. Although IVIG is expensive, the current method requires only two IVIG injections, that may be acceptable for much more patients who want to avoid miscarriages and who didn’t use IVIG for economic reasons. Trial registration number NA

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

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

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

P-531 Embryo grading in PGT-A testing: does it really matter?

Abstract Study question Can embryo morphology grading be a good parameter to predict the embryo transfer’s outcome when euploid embryos are used? Summary answer Gardner’s grading is a useful predictor of LBR per euploid embryo transferred. Morphologic grading should be used for the embryo selection among euploid blastocysts. What is known already When preimplantation genetic testing for aneuploidies (PGT-A) is not performed, the selection of the embryo(s) to be transferred is based on embryo morphology. In PGT-A cycle, some studies have minimised the importance of embryo morphology, when euploid embryos are present. However, others still consider it as good predictor. Understanding the role of morphology in biopsied euploid embryos may help in optimizing frozen embryo transfer cycles. Study design, size, duration We retrospectively analysed PGT-A frozen embryo transfers with reported outcome, performed in our clinics between 2018 and 2022. The cycles included were divided into two groups based on Gardner’s grading of euploid embryos transferred: “good” embryos (AA, AB, BA and BB; n = 263) versus “poor” embryos (including AC, CA, BC, CB, and CC; n =112). Participants/materials, setting, methods We included in the study only single euploid embryo transfers, double embryos transfers and mosaic embryo transfers excluded. Body mass index (BMI), oocyte age and outcomes (pregnancy and live birth rates) were compared between the two groups. Data was statistically analysed using MedCalc Software, where P < 0.05 was considered as significant. Main results and the role of chance A total of 375 embryo transfer cycles were included in the study. Maternal ages were not significantly different between groups (good vs poor embryos) (37.5 vs 38.2). Similarly, oocyte age (37.7 vs 38.1, respectively), and BMI (22 vs 22.7, respectively) did not differ between good and poor embryos. On the contrary, clinical pregnancy (56.6% vs 40.1%) and live birth rates (27.7% vs 13.4%) were significantly higher when morphologically good grade embryos were transferred (p < 0.05). In fact, both rates were significantly higher after the transfer of blastocysts that exhibited A-grade or B-grade in inner cell mass and/or trophectoderm, as opposed to the transfers of blastocysts that exhibited a C-grade trophectoderm and/or inner cell mass. Limitations, reasons for caution The findings apply to the participating centres and may not represent worldwide trends. Data was analysed retrospectively, but details of the follow-up on PGT pregnancies and babies born were limited. The degree of blastocyst expansion and the day of biopsy were not taken into consideration in this study. Wider implications of the findings The Gardner embryo quality assessment improves the clinics’ ability to prioritize embryos after PGT. Preferential transfer based on morphologic criteria increases the chances of a positive outcome in terms of live birth rates. Trial registration number not applicable

P-145 Impacts of double biopsy and double vitrification on the clinical outcomes following euploid blastocyst transfer: a systematic review and meta-analysis

Abstract Study question Do “double biopsy + double vitrification” and “single biopsy + double vitrification” compromise subsequent clinical outcomes following euploidy blastocyst transfer? Summary answer Both “double biopsy + double vitrification” and “single biopsy + double vitrification” led to reduced ongoing and clinical pregnancy rates and elevated miscarriage rates What is known already It is not uncommon to have inconclusive results following blastocyst biopsy and preimplantation genetic testing for aneuploidy (PGT-A). One option for these blastocysts is a second biopsy for re-test after warm, followed by a second vitrification. Furthermore, for better workflow, laboratories may choose to routinely vitrify all blastocysts formed followed by a subsequent biopsy post warm at a preferred timing, after which a second vitrification would be required. However, there is currently a lack of systematic evaluation in the literature regarding potential clinical risks associated with the above-mentioned applications in reference to the conventional “single biopsy + single vitrification” approach. Study design, size, duration A systematic review and meta-analysis were performed, with a protocol registered with PROSPERO (CRD42023469143). A search of PUBMED, EMBASE, and the Cochrane Library for relevant studies was carried out on 30 August 2023, using the keywords ‘biopsy’ and ‘vitrification’ and associated variations respectively. Only studies involving frozen transfers of euploid blastocysts tested via PGT-A were included, with those tested via PGT-M or PGT-SR excluded. Participants/materials, setting, methods Study groups included blastocysts having undergone “double biopsy + double vitrification” or “single biopsy + double vitrification”, with “single biopsy + single vitrification” used as control. The primary outcome was live birth rate while secondary outcomes included ongoing pregnancy, clinical pregnancy, miscarriage, and post warm survival rates. Random effects meta-analysis was performed with risk ratios (RR) and 95% confidence interval (CI) used to present outcome comparisons. Main results and the role of chance Following duplicate removal, a total of 607 records were identified after the initial search. After screening of titles and abstracts, 37 records were included for further eligibility assessment. For meta-analysis, 9 studies (5 full articles and 4 abstracts) were eventually included. Compared to “single biopsy + single vitrification”, “double biopsy + double vitrification” was associated with significantly reduced live birth rates (5 studies, n = 10,068; RR = 0.57, 95% CI = 0.35–0.94; I2=82%), ongoing pregnancy rates (2 studies, n = 4,659; RR = 0.69, 95% CI = 0.51–0.94; I2=2%), clinical pregnancy rates (5 studies, n = 17,673; RR = 0.79, 95% CI = 0.71–0.89; I2=0%), and elevated miscarriage rates (7 studies, n = 22,332; RR = 1.64, 95% CI = 1.02–2.64; I2=53%). However, no significant changes were found in post warm survival rates (p > 0.05, respectively). Furthermore, “single biopsy + double vitrification” was also linked with significantly decreased ongoing pregnancy (3 studies, n = 5,980; RR = 0.84, 95% CI = 0.70–1.00; I2=61%), clinical pregnancy rates (5 studies, n = 12,136; RR = 0.84, 95% CI = 0.72–0.99; I2=71%), and increased miscarriage (5 studies, n = 17,781; RR = 1.25, 95% CI = 1.02–1.53; I2=3%) rates. But live birth rate and post warm survival rate were not affected (p > 0.05, respectively). Limitations, reasons for caution All studies included in this meta-analysis were retrospective with varying levels of heterogeneity for different outcomes. Not all studies have accounted for potential confounding factors, therefore only unadjusted data could be used in the main meta-analysis. Only one study reported neonatal outcomes, making it impossible to perform meta-analysis. Wider implications of the findings Our data clearly indicated adverse impacts by “double biopsy + double vitrification” and “single biopsy + double vitrification” on clinical outcomes following euploid blastocyst transfers. If possible, such approaches should be avoided in routine practice, and patients should be carefully consulted about the risks when offered with such approaches. Trial registration number CRD42023469143

P-510 The impact of the severe male factor in the egg donation cycles using preimplantation genetic test for aneuploidy

Abstract Study question Does severe male factor affect the euploidy rate and IVF outcomes in donor egg ICSI cycles using PGT-A? Summary answer Severe male factor affects the euploidy rate in egg donation cycles, however, no statistical differences were reported in live birth and cumulative live birth rate. What is known already Around 50% of couples seeking fertility treatment have male-factor infertility. Intracytoplasmic sperm injection (ICSI) relies on evaluating sperm morphology, but the direct relationship between semen quality and embryonic potential remains unclear. Evidence indicates that poor semen characteristics correlate with an increase in aneuploidy rate of spermatozoa. Therefore, pregenetic testing for aneuploidies (PGT-A) is recommended in cases of male factor infertility. The relationship between aneuploidy rates and abnormal sperm remains a debated topic. The use of the egg donation model offers a valuable chance to isolate the male-related factor and evaluate its influence on the occurrence of embryonic aneuploidies. Study design, size, duration This is a retrospective, observational, cohort study including all European IVI clinics. The study included egg donation cycles using pre-implantation genetic testing for aneuploidies (PGT-A) generated in IVI clinics from January 2017 until December 2023. 690 egg donation cycles were included in the study, N = 202 in the group with sperm < 5mil/ml, N = 102 in the sperm group with 5-15 mil/ml and N = 386 in the group with sperm >15 mil/ml. Participants/materials, setting, methods The study included women between 30-49 years undergoing egg-donor cycles, using fresh/frozen sperm Sperm samples were derived after 2-5 days of abstinence. The cycles of egg donation were divided into three groups according to sperm concentration, severe oligozoospermia (SO, < 5mil/ml), moderate oligozoospermia (MO, between 5-15 mil/ml), and control group(>15 mil/ml). Trophectoderm biopsy was performed on day 5 or 6 and subsequent analysis used the next sequencing generation (NGS). Single euploid blastocyst transfers were included. Main results and the role of chance The fertilization rate was decreased in SO (73.33 ± 16.19) than in the MO group (79.07 ±12.45) and control group (77.80 ±14.72) p = 0.0047. The number of blastocysts was higher in the control (5.80 ± 2.72) and MO (5.97± 2.78) than in the SO group (4.99 ± 2.40), p = <0.001. The number of euploid blastocysts was decreased in SO (3.21 ± 2.07) than in the control (4.09 ± 2.41) and MO group (4.05 ± 2.15), p = <0.001. The euploidy rate of SO decreased (63.32 ±26.39) compared to the control (69.67 ± 25.08) and MO group (68.55 ± 20.37), p = 0.012. The logistic regression model included possible confounders (male age, age donor, male BMI, sperm motility, sperm morphology and sperm concentration) that showed no effect on the euploidy rate. The pregnancy rate was similar across the groups 69.77% vs 66.05% vs 66.23%, p = 0.54. No statistical differences were reported for the clinical pregnancy rate 62.79% vs 61.73% vs 58.40%, p = 0.44, and the live birth rate 62.79% vs 61.73% vs 58.40%, p = 0.44. 48.05 vs 46.38% vs 42.64%, p = 0.37. No differences were reported in the miscarriage rate (p = 0.37) and CLBR (p = 0.26). Limitations, reasons for caution The retrospective nature of the study and the sample size represent the principal limitations of the study. The clinical reason for severe male factors was not considered in the study. Wider implications of the findings SO affects the quality affect the early embryonic potential through an increase in aneuploidy, despite no clinical effects.The use of PGT-A in case of severe male factor remains not justified in the egg donation.Future prospective multi-center studies are needed to highlight the effect of sperm quality on early embryonic potential. Trial registration number not applicable

P-518 Differentially gene expression identified by RNA seq in aneuploid human blastocysts is more influenced by the phenomenon of aneuploidy than the involved chromosome

Abstract Study question What insights do RNA-Seq profiles provide about gene expression in aneuploid blastocysts with small and large chromosomes compared to euploid blastocysts? Summary answer The difference in gene expression is affected by aneuploidy and is more related to the presence of aneuploidy than the size of the aneuploid chromosomes. What is known already Embryonic aneuploidy is one of the main common causes of implantation failure and miscarriage. RNA-Seq investigations of the embryo in the early stages of preimplantation indicate that with the gradual replacement of the embryonic profile and the reduction of the oocyte residues, the gene expression profile in the blastocyst is more dependent on the embryo’s genome. A recent study by comparing the gene expression profile of euploid blastocysts and specific aneuploidies such as trisomy 21, trisomy 18, XXY and XO syndromes using RNA-Seq, showed how the type of aneuploidy might affect gene expression. Study design, size, duration This study was conducted on surplus embryos of the patients with history of recurrent implantation failure or recurrent miscarriage following PGT by Day-5 trophectoderm biopsy. Inclusion criteria involved maternal age ≤37, normal parental karyotypes, and ovulation by long stimulation protocol. As advanced maternal age and ovarian stimulation method can affect the aneuploidy rate and expression profile, embryos belonging to mothers > 37 years and included only patients with the long stimulation protocol were excluded. Participants/materials, setting, methods The normal embryos and those with single aneuploidy were selected for comparison of gene expression profiles using RNA sequencing. The single aneuploid embryos were classified in two groups: with defined aneuploidy(DA) of small chromosomes (including chromosomes 16, 20, 21, 22), and with undefined aneuploidy(UA) as single aneuploids for other chromosomes. To neutralize the effect of genomic content on gene expression profile in DA group, embryos were pooled reciprocally (trisomy and monosomy for the same chromosome). Main results and the role of chance Principal Component Analysis (PCA) validated embryos classification considering their differentially expressed genes (DEG) clustering into three distinct groups. To minimize the role of chance, only genes with significant adjusted P-values in both STAR and Hisat data analyses were included in consequent interpretations. In addition to comparison between and among the DA, UA and normal groups, the entire set of aneuploid groups was compared with the normal group. The DEG in both aneuploid groups compared to the normal group represented eminent upregulation rather than downregulation. Gene Ontology analyses indicated an upregulation of transcription, ubiquitination, autophagy, mitochondrial, chromosome segregation and DNA repair pathways in both DA and UA compared to normal group. Overexpression of the DNA repair pathway and other validated genes of interest in aneuploid embryos was in agreement with qPCR findings. Translation and protein synthesis pathways also showed overexpression as part of the functional response to aneuploidy. Upregulation of apoptotic process pathway was evident in the UA group. While comparison of gene expression between DA and UA showed overexpression of 143 genes, but cellular lipid synthesis was the only pathway with a significant difference. The distribution of DEG on chromosomes in the DA group was not associated with selected aneuploidies. Limitations, reasons for caution The number of biological replicates in each category was limited in order to provide more confident data by minimizing the time interval between providing the samples and RNA sequencing. Larger studies might change some affected pathways due to differences in identified DEG. Wider implications of the findings This study shed light on the black box of human preimplantation aneuploidy. Considering that the transcriptome is affected by the genomic status and some debates on genomic based current PGT approaches, this study is a step for selection of healthy and viable embryos by revealing the molecular mechanisms. Trial registration number not applicable

P-403 Does a thick endometrium really matter? It’s impact on FET cycles results

Abstract Study question Does a thick endometrium negatively affect the results of FET cycles after PGT? Summary answer Endometrial thickness above 10mm did not affect pregnancy rates, and live birth rates were even higher in the group with thick endometrium. What is known already The success of an assisted reproduction technique leads on two main points: getting a high-quality embryo and a functional endometrium. The results of the relationship between a thick endometrium and its influence on the pregnancy rates are contradictory till the date, mainly due to the relatively reduced sample sizes and the presence of confoundings. In the same way, they are scarce the papers that analyze the results of FET under hormone replacement therapy in cycles with euploid blastocysts. These cycles could be considered the gold standard to establish the influence of thick endometria in the implantation rate. Study design, size, duration Retrospective study of FET cycles under hormone replacement therapy with transfer of an euploid blastocyst after PGT. Multicentric analyze of cycles performed between 2015 and 2021 in our Spanish IVIRMA clinics. Participants/materials, setting, methods We analyzed 3158 cycles of FET under hormone replacement therapy with transfer of an euploid blastocyst. Hormone therapy consisted in estrogens (etinil estradiol orally or in patches), plus progesterone (400 mg/ 12 hours, vaginally). Two groups were established: an “optimal” endometrial thickness (7 - 10mm) (80.81%), and those with a higher one (19.19%), the day that the transfer was planned. The objective was to analyze the implantation, miscarriage and live birth rates. Main results and the role of chance Both groups were comparable in terms of age, origin an age of the oocytes, origin of the sperm, and serum estradiol levels the day that the embryo transfer was programmed. There were differences in the BMI of the patients (23.00 Vs 23.51, p = 0.004) and the day of the cycle that the embryo transfer was planned (11.8 Vs 11.78, p < 0.001). There were no differences regarding hormonal treatment protocol or dose, nor regarding cycle monitoring. There were not significant differences in the implantation rate between the group with an endometrial thickness between 7 and 10mm (62.84%; CI 95% 60.90 – 64.78) and the one with a thicker endometrium (66.08%; CI 95% 62.19-69.98; p = 0.143). Neither in the miscarriage rate (15.76%, CI 95% 14.36-17.24; Vs 14.40%, CI 95% 11.70-17.46; p = 0.444). Statistically significant differences were found in the live birth rate, which was of 47.74% in the group of endometrial thickness between 7 and 10mm (CI 95% 45.75-49.73), and 53.20% in the group with a thicker endometrium (CI 95% 49.09-57.27, p = 0.019) Taking these results into account, we could conclude that a thick endometrium does not negatively affect the results of FET cycles. Limitations, reasons for caution The nature of our study was retrospective not randomized. It was performed with euploid embryos under hormone replacement treatment. Thus, results may not be applicable in other settings. The measure of the endometrial thickness is very variable between professionals and there can be more factors involved in the functionality. Wider implications of the findings Embryo transfer should not be canceled in cases of endometrial thickness > 10 mm. More studies are needed to ascertain if it would be beneficial to achieve systematically an endometrial thickness > 10 mm Trial registration number Not applicable

P-194 Developing a classification system for euploid embryos: leveraging morphology grades and biopsy day to accurately rank them based on ongoing pregnancy potential

Abstract Study question Can morphology grades and day of trophectoderm (TE) biopsy be utilized to accurately rank euploid blastocysts according to their potential for ongoing pregnancy (OP)? Summary answer The proposed ranking system categorized euploid blastocysts accurately in terms of their ongoing pregnancy potential. What is known already Implementing a ranking for embryo selection within a pool is a valuable optimization strategy. Many studies have documented the importance of inner cell mass (ICM) and trophectoderm (TE) grading, as well as the day (D) at which blastocysts are biopsied on clinical outcomes. However, literature sparks controversy on clinical findings in relation to blastocyst quality. This is primarily because it reports a diversity in blastocyst grade-ranking systems, and none of them accounting for the day of biopsy. Enhancing consistency and comparability across various research studies could be achieved by establishing a standardized approach for qualitatively classifying and ranking euploid blastocysts. Study design, size, duration This retrospective cohort study included 2209 single euploid frozen embryo transfers (SeFET) between 2017 and 2023. Blastocyst (BL) expansion, TE and ICM were assessed before biopsy according to Gardner’s criteria. Only blastocysts graded ≥BL3CC were biopsied on D5, D6 or D7. Classification models (in-house, logistic regression, gradient boost) were assessed for their calibration, i.e., correct prediction of ongoing pregnancy (OP) probability. Participants/materials, setting, methods In-house ranking system categorized euploid transferred blastocysts (BL3-BL6) as ‘Top-quality’ for blastocysts AA/AB/BA on D5; ‘Good-quality’ for blastocysts BB on D5 and AA/AB/BA/BB on D6; ‘Fair-quality’ for AC/BC/CA/CB on D5, BC on D6 and AA/AB/BA/BB on D7; and ‘Poor-quality’ for blastocysts CC on D5, AC/CA/CB/CC on D6 and CA/CB/AC/BC/CC on D7. The in-house classification ranking system was compared to other developed models to evaluate whether performance could be increased. Main results and the role of chance A total of 406 blastocysts were ranked as Top-quality (100% D5), 1323 as Good-quality (51.3% D5 and 48.7% D6), 190 Fair-quality (57.9% D5, 36.3% D6 and 5.8% D7) and 290 as Poor-quality (12.8% D5, 73.8% D6 and 13.4% D7). The ongoing pregnancy rate was significantly higher for blastocysts ranked as Top compared to Good, Fair and Poor-quality (58.9%, 52.2%, 35.8% and 26.6%, respectively). Miscarriage rates were significantly associated with ranking quality (20.9%, 24.3%, 30.6% and 39.4% for Top, Good, Fair and Poor-quality blastocysts, respectively). We further developed two new classification ranking systems using logistic regression model and XGBoost machine learning model. But the new models provided similar AUC and calibration slope (CS) values as the in-house ranking system (AUC [CS]: 0.604 ± 0.0167 [0.962 ± 0.213] and 0.601 ± 0.0177 [0.949 ± 0.219] vs.0.597 ± 0.015 [0.980 ± 0.230] for logistic regression, XGBoost versus in-house model, respectively). Limitations, reasons for caution The present embryo classification ranking system may not be extrapolated to non-PGT-A cycles. Wider implications of the findings Different grade-ranking systems of blastocysts keep being reported in the different publications. Reaching a consensus to rank euploid blastocysts qualitatively, as the hereby proposed classification, may improve consistency and comparability across different research studies. Crucially, the present ranking-system aids embryo selection for transfer from available pool. Trial registration number not applicable

P-273 Image-based oocyte model predictive of blastocyst development correlates with ploidy status across a broad spectrum of PGT-A indications, accounting for confounding variables

Abstract Study question Does MAGENTA, an AI assessment of oocyte quality, indicate ploidy potential of the developed blastocysts in the presence of confounding variables? Summary answer MAGENTA correlates to oocytes that develop into euploid embryos across diverse demographics, highlighting its robust ability to assess oocyte quality and genetic potential. What is known already MAGENTA is a non-invasive AI-image analysis tool that assesses images of mature oocytes and provides a score from 0-10, where higher scores indicate higher chances of developing into a blastocyst. MAGENTA has also displayed a correlation to blastocyst morphological quality. The ploidy status of blastocysts plays a significant role in cycle success and is often utilized for the selection of embryos to transfer. Despite the selection of embryos based on their genetic normality, many transfers are unsuccessful. With most chromosomal errors arising in the oocyte, assessment at the gamete level is critical to improving outcomes. Study design, size, duration A retrospective study using CRM Weill Cornell Medicine data included 3,342 mature oocytes that developed into blastocysts and underwent PGT-A in 2021. Blastocysts tested as mosaic (n = 753) or inconclusive (n = 13) were excluded. Images of the oocytes were obtained immediately post-ICSI using EmbryoScope time-lapse incubators (Vitrolife, Sweden). Blastocyst morphological grades were categorized by quality: highest (ICM+TE grade A), high (ICM+TE grade A/B), medium (ICM+TE grade B), or low quality (ICM or TE grade C/D). Participants/materials, setting, methods Data was obtained from 629 patients (females 26-45 years old) with an average of 5.3 blastocysts (range 1-20) with PGT-A results per patient. MAGENTA assessed each oocyte image and provided a score (0-10), with higher scores representing a greater potential to develop into a blastocyst embryo. In this dataset, 1827 (55%) blastocysts were tested as euploid and 1515 (45%) were tested as aneuploid. PGT-A was conducted for various specific and non-specific indications in these patients. Main results and the role of chance MAGENTA was positively associated with oocytes that developed into blastocysts of increasing quality, with significance between highest and high-quality (7.4 vs 7.0,p<0.05) and medium and low-quality (6.9 vs 6.1,p<0.01). Of the blastocysts, the MAGENTA score further displayed significant differences between oocytes that developed into euploid (7.0) compared to aneuploid blastocysts (6.6) (p < 0.001;Welch’s t-test). Although MAGENTA does not predict ploidy, a threshold of 8.8 MAGENTA score was determined to best distinguish between ploidy outcomes with 61% euploidy rate above this threshold compared to 51% below, a significantly different proportion (p < 0.001;Two-Sample Proportions z-test). Therefore, oocytes scored ≥8.8 display a 19.6% relative increase in chance of developing into a euploid blastocyst. To account for female age, subgroup analysis was conducted and found MAGENTA scores to be significantly different between oocytes that developed into euploid and aneuploid blastocysts for patients <35 years old (7.2 vs 6.6, p < 0.01;Welch’s t-test) and ≥35 years old (6.8 vs. 6.5, p < 0.05;Welch’s t-test). Blastocyst morphological grade was also investigated as a confounding factor. No significant differences existed between MAGENTA scores of euploid and aneuploid blastocysts among the low, high, or highest-quality. Significantly, differences were only found within medium quality blastocysts (7.0 vs 6.6,p<0.05), which had the greatest sample size (n = 1854). Limitations, reasons for caution MAGENTA was not trained to predict euploid embryo development from images of mature oocytes. Further data is required to discern the correlation of MAGENTA to chromosomal ploidy status, apart from blastocyst quality. Additionally, investigation into sperm DNA fragmentation as a potential contributor to ploidy determination is warranted. Wider implications of the findings MAGENTA can identify oocytes of better quality that not only have higher chances of blastocyst development and higher morphological grades, but also indicates oocytes that are more likely to become euploid embryos across patients of various PGT-A indications and a broad age demographic. Trial registration number not applicable

O-316 Clinical application of PGT-A for recurrent pregnancy loss, what effects live birth after single euploid blastocyst transfer?

Abstract Study question Which factors effect the likelihood of live birth after euploid frozen embryo transfer (FET) in recurrent pregnancy loss (RPL)? Summary answer Female age and number of previous pregnancy loss do not effect the live birth rates after euploid blastocyst FET. What is known already PGT-A is helpful to discard the aneuploid embryos and has been proven to improve pregnancy outcomes and reduce miscarriage rate per embryo transfer. As approximately 50% of clinical miscarriages have chromosomal abnormalities, PGT-A has been suggested as a treatment option for RPL. However it is questionable on the cost effectiveness, effect on prognosis and labor-intensive. In addition, currently PGT-A use is not recommended by any society guidelines in RPL patients. ESHRE guideline on RPL stated ‘All couples with results of an abnormal fetal or parental karyotype may be informed about the possible treatment options available including their advantages and disadvantages.’ Study design, size, duration This retrospective cohort study from January 2018 to January 2023 was conducted at Bahçeci Health Group included patients underwent PGT-A/ICSI cycles. Patients with RPL (2 or more clinical pregnancy loss) had a complete RPL workup recommended by ASRM and reported no etiology for RPL was included in the study. Patients with uterine synechia, hydrosalpinx, endocrinological disorders and thin endometrium (endometrial thickness <6mm) were excluded Participants/materials, setting, methods A total of 366 single euploid blastocyst FETs employing hormone replacement therapy for endometrial preparation were evaluated. Patients were categorized into 2 group regarding female age (female age< 35 years and female age≥35 years. Demographic, embryological and endocrinological parameters were analyzed. Live birth rate (LBR) was the primary outcome. Clinical pregnancy rate (CPR) and miscarriage rate (MR) were the secondary outcome. Main results and the role of chance The mean female age of the study population was 33.9±4.6 years, the number of previous pregnancy loss was 3.2±1.6 and BMI was 23.8 ±7.9 kg/m2. Overall CPR, MR and LBR were 61.7% (226/366), 24.8% (56/226) and 46.5% (170/366) respectively. Comparison of clinical characteristics of the 195 women <35 years and 171 women ≥35 years revealed no significant deference in BMI, number of previous live birth, and number of previous pregnancy loss. CPR, MR and LBR were similar among two groups [ 63.6% (124/195) vs 59.6% (102/171), p = 0.44; 27.4% (34/124) vs 21.6% (22/102), p = 0.31; 46.2% (90/195) vs 46.8% (80/171), p = 0.9; respectively]. Binary logistic regression evaluating the independent factors that effect live birth revealed that female age and number of previous pregnancy loss were not found to be statistically significant factors. The only independent factor for live birth was day of embryo biopsy, transferring the day 6 biopsied embriyo significantly decreased the live birth [p = 0.003, OR: 0.47, 95% CI (0.29-0.77)]. Limitations, reasons for caution The maim limitation of the study is its retrospective nature. Wider implications of the findings PGT-A has been suggested to be a treatment for RPL. However there is insufficient data to call PGT-A as an efficient treatment for RPL. Investigating the factors that effect live birth after PGT-A may be the first step for selecting the most favorable group 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

Comparison of aneuploidy for patients of different ages treated with progestin-primed ovarian stimulation or GnRH antagonist protocols

Does euploidy status differ among patients of different ages treated with progestin-primed ovarian stimulation (PPOS) or gonadotrophin releasing hormone antagonist (GnRH-a) protocols? Patients undergoing PGT-A (n = 418; 440 cycles) were enrolled and grouped according to female age (<35 years and ≥35 years). Protocols were as follows: PPOS: <35 years (n = 131; 137 cycles); ≥35 years (n = 72; 80 cycles); GnRH-a: <35 years (n = 149; 152 cycles); ≥35 years (n = 66; 71 cycles). For cycles treated with PPOS in the older group, rates of euploid blastocyst per metaphase Ⅱ oocyte (15.48% versus 10.47%) and per biopsied blastocyst (54.94% versus 40.88%) were significantly higher than those treated with GnRH-a (P < 0.05). The mosaic rate per biopsied blastocyst was significantly lower for cycles treated with PPOS than cycles treated with GnRH-a (8.64% versus 23.36%) (P < 0.001). In the younger group, no significant difference was found between treatments (P > 0.05). In older and younger groups, the drug to inhibit LH surge was cheaper for cycles treated with PPOS compared with GnRH-a (P < 0.001). Generalized estimation equations based on binomial distribution female age and euploidy rate was significantly negatively correlated for all participants (β -0.109, 95% CI -0.183 to -0.035, P = 0.004), and between GnRH-a protocol (reference: PPOS) and the euploidy rate in the older group (β -0.126, 95% CI -0.248 to -0.004, P = 0.042). Multiple logistic regression indicated that ovarian stimulation protocol was not associated with ongoing pregnancy rate (OR 0.652, 95% CI 0.358 to 1.177; P = 0.14). PPOS is suitable for patients undergoing PGT-A, particularly older patients for the higher euploid blastocyst rate attained by PPOS protocol.

P-586 Mild ovarian stimulation using the aromatase inhibitor letrozole (LTZ) is an effective stimulation protocol to obtain euploid blastocysts for women of an advanced maternal age

Abstract Study question Does MOS with LTZ produce higher numbers of good-quality blastocysts compared with the use of clomiphene citrate (CC) for women of an advanced maternal age? Summary answer MOS using LTZ is a good ovarian stimulation protocol for a higher number of good-quality blastocysts for women of an advanced maternal age (AMA) What is known already The clinical pregnancy rate with a good blastocyst should be 40% or more among younger women, but about 35% for AMA women (≥40 years). Therefore, high-dose gonadotropins are usually used for AMA women. However, the best efforts for both patients and clinicians are sometimes in vain. Many AMA women show a lower ovarian reserve where ovarian stimulation with a lessened dose of gonadotropins is preferred. Taking CC or LTZ with MOS can reduce the amount of gonadotropins used, but it remains uncertain whether it is better to administer LTZ or CC together for MOS. Study design, size, duration This retrospective study was conducted between January 2020 and April 2021. A total of 288 women received MOS. Of course, 153 used CC (CC group) and 133 used LTZ (LTZ group). The ART outcomes were compared between these two groups. Among them, the women who were ≥40 years old were divided into two groups: CC-O40 (n = 61) and LTZ-O40 (n = 54). The ART outcomes were compared between the two groups. Participants/materials, setting, methods All women took either 100 mg of CC or 5 mg of LTZ daily for 7 days between menstrual cycle 3 (MC3) and MC9, and 225 IU of recombinant-FSH were administered on MC3, MC5, MC7 and MC9. On MC 10, when 3 or more well-developed follicles (≥ 20 mm in diameter) were confirmed, a dual-maturation trigger was performed using rec-hCG and GnRH agonist nasal spray, and then oocyte-pick up (OPU) was performed 35-36 hours afterward. Main results and the role of chance The number of retrieved oocytes in the CC group averaged 7.5 ± 5.3 (mean ± S.D.), which was significantly higher than that in the LTZ group (6.3 ± 4.7; p &amp;lt; 0.05). The average number of blastocysts and morphologically good blastocysts was comparable in the CC and LTZ groups (CC group; 2.8 ± 3.0 and 1.8 ± 2.4; LTZ group; 2.4 ± 2.7 and 1.4 ± 2.0, respectively, [P=NS]). For AMA women, the average number of blastocysts in the CC-O40 group was 1.7 ± 1.8, which was similar to that in the LTZ-O40 group (1.7 ± 1.8, P=NS). The blastocyst formation rate in the LTZ-O40 group was 58.5%, which was significantly higher than that in the CC-O40 group (46.6%; P &amp;lt; 0.05), but the rate for good blastocysts in the LTZ-O40 group (30.8%) was comparable to that in the CC-O40 group (23.3%, P=NS). The euploid rates in the LTZ group was 53.4%, which was significantly higher than that in the CC group (38.0%; P &amp;lt; 0.05), and the difference was significant even for AMA women (LTZ-O40; 40.5%, CC-O40; 16.7%, respectively, p &amp;lt; 0.05). Limitations, reasons for caution The study was a controlled trial with a limited number in the study population. Additionally, all cases had not received a trophectoderm (TE) biopsy. Further study is needed to determine the effect that an aromatase inhibitor exerts on obtaining either morphologically good blastocysts or euploid blastocysts. Wider implications of the findings AMA women showed blastocyst formation rates from LTZ with MOS significantly higher than that of the CC group, and euploid rates in the LTZ group were also significantly higher. The characteristics of an LTZ such as aromatase inhibitor enhanced the development of embryos. Trial registration number not applicable

O-293 Mastering consistency: a similar training allows to standardize embryo transfer performance, but experience does not involve further improvements

Abstract Study question Is there any variation in the live birth rate (LBR) per vitrified-warmed single euploid blastocyst transfer (SEBT) among clinicians trained at the same IVF center? Summary answer The LBR per SEBT (overall: 53%) remained consistent among equally trained transfer operators (range: 48%-60%) regardless of the number of prior procedures performed. What is known already The number of ART treatments and practitioners has increased over the years. To ensure high standards, an efficient quality control system should monitor and standardize operators’ performance by leveraging statistically-sound key-performance-indicators (KPIs). The embryo transfer procedure is a clinically relevant step, particularly susceptible to variations among clinicians, irrespective of their experience. However, the outcome of “LBR per transfer” is influenced by various confounders, such as embryo stage, morphology, reproductive history, uterine conditions, and others. Our objective was to assess potential differences among transfer practitioners who underwent the same training at a single IVF center in the least biased scenario possible. Study design, size, duration Retrospective single center study. Out of 8663 transfers performed by 4 operators (all trained by operator-1) between 2013 and 2021, we selected 421 first vitrified-warmed transfers with day5 euploid blastocysts whose morphology was &amp;gt;Gardner’s BB-grade in non-obese women, thereby excluding all main confounders on our primary outcome (= LBR per SEBT across different operators). The study was powered to exclude a 18%-decrease (β = 0.8, α = 0.05) in the LBR versus operator-1 (ratio comparator:control=1:2). Participants/materials, setting, methods We conducted ICSI, blastocyst biopsy without day3 zona-drilling, and aneuploidy testing to exclude full-chromosome uniform aneuploidies. All blastocysts were transferred ∼2 hours after warming. Uterine assessment was conducted to exclude putative conditions associated with lower LBR. Endometrial preparation protocol (modified-natural cycle versus hormone-replacement-therapy), maternal age at transfer, experience of prior implantation(s) (yes/no), the embryologists involved in the transfer procedure, were assessed as putative further confounders. All significant differences were confirmed through regression analyses. Main results and the role of chance The overall LBR per vitrified-warmed SEBT was 53.4% (N = 225/421). Operator-1 conducted 3960 transfer procedures during the study period, with 167 eligible for this study, achieving a 55.7% LBR (N = 93/167). Operator-2,-3, and -4 respectively conducted 2533, 1087, and 1083 procedures during the study period, with 134, 62, and 58 eligible for this study. Their LBRs were 50% (N = 67/134, p-value versus operator-1=0.2), 48.4% (N = 30/62, p-value=0.2), and 60.3% (N = 35/58, p-value=0.3). Multivariate logistic regression analysis, adjusted for endometrial preparation protocol and embryologists involved in the transfer procedure, confirmed the absence of an association (Operator-2 vs. 1 multivariate-OR: 0.83, 95%CI 0.5-1.39, adjusted-p=0.49; Operator-3 vs. 1 multivariate-OR: 0.75, 95%CI 0.40-1.40, adjusted-p=0.37; Operator-4 vs. 1 multivariate-OR: 1.40, 95%CI 0.72-2.70, adjusted-p=0.32). Importantly, the number of prior transfer procedures conducted by each clinician during the study period also showed no association with the primary outcome (OR = 1.0, p-value=0.82). From an IVF center perspective, the LBR per SEBT remained stable across the 9-year study period, showing minimal non-significant fluctuations. These outcomes describe an IVF center where equally-trained clinicians adopt the same standard-operating-procedures to manage vitrified-warmed embryo transfers. In this context, the LBR per SEBT represents an ideal KPI to assess the learning curve of operators in training. Limitations, reasons for caution Over 2500 SEBTs are required to rule out a 5%-decrease in the LBR. Yet, such a substantial number is better suited for comparing centers’ rather than practitioners’ performance. It would be intriguing to investigate potential differences among operators who underwent different training regimens but operate within the same clinic. Wider implications of the findings No correlation exists between the number of prior embryo transfer procedures conducted and further improvements, therefore initial operators’ training is crucial for standardizing their performance. LBR per SEBT is a strong KPI, though poorly-actionable in the short-term. It’s better suited for center comparisons, prompting internal procedure reviews when differences arise. Trial registration number not applicable