Preimplantation Genetic Testing Research Articles

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-132 Outcomes of blastocyst transfer derived from oocytes with smooth endoplasmic reticulum clusters (sERC)

Abstract Study question Are blastocysts derived from oocytes with smooth endoplasmic reticulum clusters (sERC+) as suitable for transfer as those from oocytes without such clusters (sERC-)? Summary answer Blastocysts from sERC+ oocytes are considered for embryo transfer; however, it is recommended to prioritize them lower than those from sERC- oocytes. What is known already One of the cytoplasmic abnormalities in human oocytes, sERC, typically manifests during the MII stage oocytes and disappears after fertilization when the female and male pronuclei emerge. Despite this, the mechanism behind sERC development remains unclear. While it is suggested that sERC in oocytes may adversely impact embryo development and implantation, a definitive conclusion on its safety or use has not been reached. Although the 2011 Istanbul consensus discouraged injecting or inseminating sERC+ oocytes, a 2017 expert panel reconsidered this recommendation, advocating for a case-by-case approach. Therefore, there are widespread variations in responses to sERC in clinical practices. Study design, size, duration A retrospective review of intracytoplasmic sperm injection (ICSI) cycles between April 2014 and April 2023 was conducted, exclusively on single frozen blastocyst transfers of two pronuclei (2PN)-derived embryos performed before June 2023. A dataset comprising 95 blastocysts from sERC+ oocytes and 8,698 blastocysts from sERC- oocytes was assembled. To reduce bias between groups, maternal age at retrieval and transfer counts were matched in a 1:1 ratio using propensity score matching, yielding 95 comparable cycle pairs. Participants/materials, setting, methods Among all ICSI cycles, 78 exclusively involved embryos from sERC+ oocytes, 847 included embryos from both sERC+ and sERC- oocytes, and 6,599 had no embryos derived from sERC+ oocytes. Among the 8,698 sERC- blastocysts transferred, 1,083 and 7,615 originated from cycles with and without sERC+ oocytes, respectively. Implantation, miscarriage, and live birth rates were then compared in 95 matched pairs of blastocysts derived from sERC+ and sERC- oocytes. Main results and the role of chance Following propensity score matching adjustments, the mean patient age at retrieval (mean ± SD) for blastocyst transfers derived from sERC+ and sERC- oocytes were 38.6 ± 4.7 years and 38.7 ± 4.8 years, respectively (p = 0.927). The transfer counts were 4.2 ± 3.4 times for sERC+ and 4.1 ± 3.1 times for sERC- blastocyst transfers (p = 0.840). The proportion of high-quality blastocysts was 48.4% for sERC+ transfers and 54.7% in sERC- transfers (p = 0.384). The implantation rates were 24.2% for sERC+ and 32.6% for sERC- transfers (p = 0.198), miscarriage rates were 43.5% vs. 35.5% (p = 0.552), and live birth rates were 13.7% vs. 21.1% (p = 0.172), respectively, with no statistically significant differences. Thirteen live births from sERC+ blastocyst transfers were recorded, and no congenital abnormalities were observed. Limitations, reasons for caution This study exclusively centered on oocytes subjected to ICSI, and the inclusion criteria were restricted to embryos derived from sERC+ oocytes in accordance with clinic policy limitations. Preimplantation genetic testing was not performed on the majority of the transferred embryos. Wider implications of the findings No congenital abnormalities were observed in live births resulting from sERC+ derived blastocyst transfers. However, the limited number of embryo transfers involving sERC+ oocytes poses a constraint in drawing a definitive conclusion regarding the safety of embryos from sERC+ oocytes, and further data is required. Trial registration number non-clinical trials

O-122 ICMART Preliminary World Report 2020

Abstract Abstract title: International Committee for Monitoring Assisted Reproductive Technologies (ICMART) Preliminary World Report on ART, 2020 Study question In 2020 what was the global utilization, effectiveness and safety of ART? Summary answer Globally, ART utilization and data collection continue to increase but with wide variations in utilization, effectiveness and safety. What is known already ICMART has continuously monitored utilization, effectiveness and safety since 1991, with increasing number of cycles, higher pregnancy rates and lower multiple birth rates, the latter due to the transfer of fewer embryos. Frozen embryo transfer (FET), donor egg cycles and preimplantation genetic testing (PGT) continue to increase. Wide variations in practice and outcomes exist globally. At least 12 million ART babies have been born. ICMART has helped develop registries internationally. A new electronic data collection platform has improved validation. Nevertheless, data collection and quality remain challenging. Study design, size, duration Countries and regions annually collect ART data, some prospectively and others retrospectively. ICMART retrospectively requested data from all known global sources for 2020, reviewed them for missing or incorrect data, and obtained additional information when possible. The corrected dataset was validated and analyzed in partnership with University of New South Wales (UNSW). ICMART finalized the results tables. Standardized definitions and previously developed methods were used. Preliminary results are presented. Participants/Materials, setting, methods The European IVF Monitoring Consortium (EIM), Latin American Network of Assisted Reproduction (REDLARA), Australian/ New Zealand Registry and African Network and Registry for ART (ANARA) submitted regional data, and other countries contributed national data, through standardized formats, to ICMART. A few individual clinics with no registry access also contributed. Data received were reviewed, corrected, and validated to the extent possible, analyzed and summarized by ICMART using descriptive statistics. Main results and the role of chance Data collection and analysis are ongoing, so the presented results are preliminary. The number of ART cycles continues to increase, but utilization remains highly variable among countries and regions. While historically Europe has reported the largest number of cycles, China now reports well over 1 million cycles and Japan almost half a million, with Asia overall reporting approximately half of global cycles. Regional and country differences persist in the age of women treated, number of embryos transferred, live birth rates, rate of multiple births, use of ICSI and cryopreservation, and other factors. Covid reduced ART utilization in many countries. The role of chance is minimal. Data are limited to reporting countries and clinics, representing 90 to 95% of global cycles. However, this is a very large sample size from which imputation of total global results is performed. Limitations, reasons for caution Most, but not all, countries report to ICMART. Some countries have limited data and many countries have limited data validation. ICMART can perform only minimal verification of submitted data. Widespread adherence to consensus definitions provided in the Glossary takes time and requires translation into multiple languages. Standardization of validation and reporting is an ongoing process because of missing data and continuing changes in clinical practice. Wider implications of the findings ICMART World Reports standardize data, track trends, enable comparisons, stimulate questions and improve ART quality. Better understanding of ART increases societal acceptance and support for equitable access and ART research.

P-180 Trophectoderm biopsy technique and its effect on life birth rates: a prospective study

Abstract Study question Do life birth rates of euploid embryos vary based on factors such as laser intensity, number of laser shots, and the biopsy technique employed? Summary answer This study found no correlation between biopsy technique parameters and life birth rates of euploid embryos, underscoring the robustness of current trophectoderm biopsy methods. What is known already Embryo biopsy for Preimplantation Genetic Testing (PGT) must achieve two primary objectives: ensuring that the biopsied material accurately reflects the genetic makeup of the embryo and maintaining the viability of the embryo without compromise. The strategy that most closely aligns with accomplishing these dual goals is trophectoderm (TE) biopsy. However, it’s important to note that there is currently no standardized TE biopsy procedure, leading to the coexistence several techniques. Given the potential impact of TE biopsy on trophectoderm quality and integrity, and consequently on implantation, there is a critical need for studies to assess the effects of different biopsy techniques. Study design, size, duration This prospective observational study spanned from 2019 to 2021 and involved 298 Preimplantation Genetic Testing for Aneuploidy (PGT-A) treatments, incorporating a total of 1187 trophectoderm (TE) biopsies. Embriologyst were trained in both Pulling and Flicking techniques and choose between the two techniques according to their criteria. Pulling involved laser pulses weakening junctions, facilitating TE detachment with moderate tension. Flicking substituted tension with a cutting motion between holding and biopsy pipettes. Participants/materials, setting, methods Biopsy technique (pulling/flicking), number of laser shots (1-10), laser intensity (200µs-600µs), and the embriologyst responsible for the biopsy were annotated for every biopsied embryo. PGT indication, stimulation protocol, patient age (39.32±4.22), embryo quality, and sperm diagnosis were annotated and used as possible confounding factors in a logistic regression model to understand the impact of each independent variable on the log-odds of the outcome. Main results and the role of chance The 1187 trophectoderm biopsies resulted in a total of 410 (34.5%) euploid embryos. Among these, 237 euploid embryos underwent individual transfers, with 153 subjected to Pulling technique and 84 to Flicking technique leading to a total of 120 healthy babies (50.6% life birth rate). Logistic regression, considering potential confounding factors failed to identify any variable significantly influencing the likelihood of life births (p < 0.05). Both Pulling and Flicking techniques demonstrated comparable success rates (52.9% vs 46.4%), with no discernible impact from variations in laser shots (1-3=65.0%; 4-6=45.6%; >6=66.7%) or intensity (200µs=75.0%; 300µs=45.0%; 400µs=50.6%; 500µs=50.94%; 600µs=50.0%). Furthermore, when evaluating the embryologist conducting the biopsy, no significant differences were identified among operators (p < 0.05). This pattern persists for miscarriage rates, indicating no influence from biopsy technique, laser shots, or laser intensity (p < 0.05). Thus, our findings underscore that variations in trophectoderm biopsy variables, when performed by an experienced embryologist, do not adversely impact trophectoderm quality and do not compromise success rates. Limitations, reasons for caution This is a single-center, observational study, conducted from 2019 to 2021, and this may limit generalizability. The sample size variation between biopsy techniques and potential unmeasured confounders pose challenges. The assumption of technique homogeneity, and reliance on binary outcomes underscore caution in interpreting findings. Wider implications of the findings The lack of correlation between biopsy parameters and life birth rates in our study underscores the effectiveness and reliability of current biopsy techniques. This discovery offers reassurance regarding the proficiency of trophectoderm biopsy methods for achieving success in IVF treatments. Trial registration number Not Applicable

P-212 Balancing success and safety in assisted reproductive technologies cycles: the outcomes of transferring poor-quality blastocyst or day 5 slow-growing embryos with good-quality blastocyst

Abstract Study question Does transferring two mixed-quality embryos improve the live birth rate (LBR) without increasing the multiple gestation rate compared to the single good-quality blastocyst (GQB) transfers? Summary answer The transfers of single GQB and two mixed-quality embryos show similar LBRs, however, the multiple gestation rate was significantly higher in double embryo transfer groups. What is known already Due to higher LBR after blastocyst transfer, the single embryo transfer (SET) strategy has gained popularity worldwide. Despite that double-embryos transfers (DET) demonstrate higher LBR than SET, multiple gestation rates (MGRs) are higher in DET. To reduce MGR but still accomplish high LBR, DETs could be performed using embryos of mixed-quality. It has been suggested that the transfer of GQB with poor-quality blastocyst (PQB) could increase LBR without increasing MGR. Alternatively, when the embryo transfer is scheduled in a fresh cycle, the DET could be performed with GQB and day 5 slow-growing embryo (SGE) – early blastocyst or morula. Study design, size, duration This study included assisted reproductive technologies (ART) cycles with fresh embryo transfer on day 5, between 2015 and 2021, for patients under 34 years. Selected ART cycles were assigned into five groups: SET with GQB (SET-GQB, n = 754); DET with two GQBs (DET-GQB, n = 306); DET with one GQB and one PQB (DET-GP, n = 75); DET with one GQB and one day 5 SGE (DET-GS, n = 386), DET with two day 5 SGE (DET-SGE, n = 209). Participants/materials, setting, methods According to Gardner and Schoolcraft’s grading system, blastocyst morphological assessment was performed on day 5. Blastocysts graded as AA, AB, BA, and BB with a degree of expansion ≥3 were classified as GQBs, whereas blastocysts graded ВС, СВ, СС with a degree of expansion ≥3 were classified as PQBs. Day 5 morulas and blastocysts with degree of expansion 1 were classified as slow-growing embryos. The cycles with donor gametes, preimplantation genetic testing were excluded. Main results and the role of chance The LBR was significantly higher in DET-GQB than in SET-GQB [53,59% (164/306) vs 40,58% (306/754); P < 0,001]. There were no significant differences in implantation rate (IR) and miscarriage rate (MR) between SET-GQB and DET-GQB [47,88% (361/754) vs. 44,12% (270/612) P = 0,165], [12,19% (44/361) vs. 10,00% (27/270) P = 0,389]. However, the MGR was significantly higher in DET-GQB compared to SET-GQB [36,59% (60/164) vs 1,31% (4/306) P < 0,001]. When SET-GQB was compared with DET-GS, DET-GP there were no significant differences in LBR: [40,58% (306/754) vs. 39,64% (153/386) P = 0,757; vs. 41,33% (31/75) P = 0,899]. The IR was significantly higher in SET-GQB than in DET-GS and DET-GP [47,88% (361/754) vs. 30,70% (237/772) P < 0,001; vs. 30,00% (45/150) P < 0,001]. There were no significant differences in MR between SET-GQB and DET-GS, DET-GP [12,19% (44/361) vs. 17,30% (41/237) P = 0,079; vs. 11,11% (5/45) P = 0,834]. However, the MGR was significantly lower in SET-GQB compared to DET-GS, DET-GP [1,31% (4/306) vs. 18,95% (29/153) P < 0,001; vs. 22,58% (7/31) P < 0,001]. The IR and LBR were significantly higher in SET-GQB compared to DET-SGE [47,88% (361/754) vs 16,99% (71/418) P < 0,001], [40,58% (306/754)vs 19,14% (40/209) P < 0,001]. The MR and MGR were significantly lower in SET-GQB than in DET-SGE [12,19% (44/361) vs 32,39% (23/71) P < 0,001], [1,31% (4/306) vs 15,00% (6/40) P < 0,001]. Limitations, reasons for caution The study is limited due to the uneven distribution and a low number of participants in group DET-GP. Blastocyst morphological assessment remains subjected to a human factor. It was impossible to form a group where two PQBs were transferred, as there were only a few cases of such transfers. Wider implications of the findings Transferring an additional poor-quality embryo (poor-quality blastocyst or day 5 slow-growing embryo) does not increase the live birth rates. However, it significantly raises the multiple gestation rate. Despite the low live birth rate when two day 5 slow-growing embryos are transferred, the multiple gestation rate significantly increases, compared to SET. Trial registration number Not applicable

P-208 To assess or not to assess: reconsidering day 3 embryo quality in planned freeze-all blastocyst cycles

Abstract Study question Should day 3 embryo quality assessment be consider in planned freeze-all blastocyst cycles? Summary answer Day 3 embryo assessment may be unnecessary in planned freeze-all blastocyst cycles. What is known already Day 3 embryo quality is a predictor of in vitro fertilizastion (IVF) success rates in cleavage stage embryo transfer. However, the association between day 3 embryo quality and live birth in blastocyst transfer policy is largely unknown. Study design, size, duration This retrospective study included 1074 frozen-thawed single day 5/6 blastocyst transfer between January 2019 to December 2022. Three groups were assessed depending on whether the transferred blastocyst derived from a top-quality, good-quality or poor-quality embryo at day 3. The analysis was conducted independently for each blastocyst quality group (top, good, and poor) using multivariate logistic regression. We applied factor analysis of mixed data to reduce potential collinearity between covariates used in the model. Participants/materials, setting, methods All blastocysts included in this study were obtained from first ICSI freeze-all cycles. Cleavage stage and blastocyst evaluation were performed according to the Istanbul consensus. Cleavage and blastocysts stage were assessed between 67±0.5 (day-3), 115±0.5 (day-5) and 139±0.5 (day-6) hours post-insemination (hpi), respectively. Exclusion criteria were: i) ICSI-cycles performed with surgically retrieved or frozen sperm, (ii) gamete donation cycles, (iii) cryopreserved-thawed oocyte cycles, (iv) preimplantation genetic testing. The primary outcome was live birth rate. Main results and the role of chance After adjusting for the day of transfer (day 5 or day 6) and several confounding variables, no statistical differences in live birth were observed among same quality blastocysts derived from different day 3 embryo quality groups (top=A, good=B and poor=C). The analysis was stratified for different blastocyst quality groups: 1) top quality blastocyst [day 3 quality A =reference group; day 3 quality B= OR 1.04 95% CI (0.62 – 1.74), p value=0.871; day 3 quality C= OR 0.67 95% CI (0.34 – 1.33), p value= 0.254] 2) good quality blastocyst [A =reference group; B= OR 0.80 95% CI (0.49 – 1.30), p value=0.361; C= OR 0.74 95% CI (0.44 – 1.27), p value= 0.276] 3) poor quality blastocyst [A =reference group; B= OR 0.78 95% CI (0.29 – 2.10), p value=0.622; C= OR 0.80 95% CI (0.31 – 2.07), p value= 0.650]. Limitations, reasons for caution The study is limited by its retrospective design. Embryo assessment without time-lapse technology, to assess the blastocysts at the same developmental time-frame, is another limitation; although the static observation time very close and restricted to the Istanbul consensus guidelines may mitigate such a weakness. Wider implications of the findings Our findings showed that day 3 embryo quality assessment may be unnecessary in planned freeze-all blastocyst cycles. In the context of static embryo assessment, this can reduce the stress on embryos by avoiding unnecessary retrievals from incubators. Trial registration number not applicable

P-559 Pregnancy rates in conventional and non-invasive pre-implantation genetic testing – a multicentric retrospective cohort study

Abstract Study question To determine the pregnancy rates with embryo diagnosed as euploid by non-invasive preimplantation genetic testing (niPGT-A) and trophectoderm based pre-implantation genetic testing (cPGT-A) Summary answer Biochemical pregnancy rates are comparable in couples undergoing transfer with embryos diagnosed as euploid as judged by niPGT-A or cPGT-A What is known already NiPGT-A utilizes cell-free DNA in culture media to assess the chromosomal status of embryos. Several studies have shown that the niPGT-A and cPGT-A have a reasonable concordance rate and niPGT-A can be considered as an alternative in predicting the ploidy status of the embryos. However, limited information exist on the clinical outcomes of embryos deemed euploid by niPGT-A. Study design, size, duration Data of 1209 participants between June 2022 and Dec 2022 was collected from 46 centers of Indira IVF Hospital. These were couples who had experienced recurrent implantation failures (>3 failed ET), recurrent miscarriages (>2 miscarriages) or had advanced maternal age (>35 years) and opting either for niPGT-A or cPGT-A at our centres for euploid embryo transfers. Women with anatomical uterine defects and Asherman syndrome were excluded. Participants/materials, setting, methods Trophectoderm biopsy was done in 1383 Day 5 grade A embryos and PGT-A was done using NGS. Culture media of 3083 Grade A embryos were collected on D5 or D6 and frozen. NGS was done at an external facility. Embryos deemed euploid in either of the groups were transferred. Biochemical pregnancy was determined by measurements of beta hCG. Main results and the role of chance There were 906 patients in the niPGT-A group and 303 in the cPGT-A group. The clinical characteristics of the participants were comparable in both groups. Biochemical pregnancy rates were 60% and 69%, respectively. This difference was not statistically significant (p < 0.6). The euploidy rates were comparable in the niPGT-A and cPGT-A groups (40% and 46%) respectively. The aneuploidy rates were lower in the niPGT-A group as compared to the cPGT-A group (15% vs 26%). The mosaicism rates were higher in the niPGT-A group as compared to the cPGT-A group. Limitations, reasons for caution This is a preliminary data analysis of only the biochemical pregnancy rates. Clinical pregnancy, miscarriage, and live birth outcomes are subject to further investigation. Wider implications of the findings The study will aid IVF specialist to consider niPGT-A as a possible alternative to cPGT-A in clinical practice. Trial registration number ’not applicable’

P-150 Predicting fetal heartbeat in vitrified blastocysts through artificial intelligence analysis of post-warming images and videos

Abstract Study question Is it possible to predict the fetal heartbeat of frozen blastocysts from post-warming time-lapse images and videos captured with EmbryoScope? Summary answer A significant positive correlation was found between the score given by the artificial intelligence algorithm and the fetal heartbeat rate of the vitrified blastocysts analyzed. What is known already Vitrification is the safest and most successful technique for cryopreserving human embryos, although some of the best morphologically graded blastocysts before freezing do not recover properly when thawed, collapsing or failing to re-expand. Artificial intelligence (AI) has been applied on videos and images of fresh embryos at different stages, and also on images of vitrified-warmed embryos. However, a single image does not give information about events such as collapses or re-expansion rate. This study evaluated the ability of an AI software to predict the fetal heartbeat of vitrified blastocysts from images and videos taken in the post-warming period. Study design, size, duration This retrospective single-center study included a video of the period between thawing and transfer of 537 vitrified blastocysts, and the last image of that video of 518 blastocysts (Feb 2022-May 2023). All blastocysts were vitrified and warmed using the Cryotop method (Kitazato, Biopharma, Japan), and placed in EmbryoScope (Vitrolife, Denmark) time-lapse incubators for 2-5 hours to let them re-expand until embryo transfer. Warmed embryos were assessed by experienced embryologists according to the ASEBIR morphological criteria. Participants/materials, setting, methods Images and videos were provided to the algorithm (EMBRYOAID v1.0, MIM Fertility), which returned scores from 0 to 10. Multivariate logistic regression analysis included embryo score and confounders: patient age and body mass index, oocyte age, oocyte origin (autologous vs. donated) and handling (fresh vs. vitrified), day of blastulation (5 vs. 6), preimplantation genetic testing (PGT-A), and post-thaw incubation time. Model performance was calculated using the area under the receiver operating characteristic (ROC) curve (AUC). Main results and the role of chance The mean score was different between embryos with negative (5.9±1.2) and positive (6.3±1.2)* fetal heartbeat outcome. The contribution of each increased unit of algorithm score to the fetal heartbeat was significant in both imaging (OR = 1.36 [95%CI: 1.16-1.60]**) and video (OR = 1.29 [95%CI: 1.12-1.50]*). The score predicted fetal heartbeat with AUCs of 0.61 in images and 0.60 in videos, and it performed better than the ASEBIR conventional morphology, which achieved an AUC of 0.58. The algorithm performance was higher in embryos not subjected to PGT-A (AUC=0.62) in images (n = 418) and videos (n = 435). Fetal heartbeat prediction was optimal in images of non-PGT-A embryos taken between 3.3-4h of post-warming incubation (n = 107) (OR = 1.76 [1.22-2.55]*), reaching an AUC of 0.74. In videos, the optimal prediction in non-PGT-A embryos was at 3.3-4.1h (n = 126) (OR = 1.93 [1.36-2.74]*), with an AUC of 0.69. 9.9% of the embryos collapsed and/or did not re-expand after thawing. These damaged embryos had an implantation rate of 15.2%, lower than the rest (46.8%**). Mean score differed between poor prognostic and normal embryos, both in images (4.8±1.1 vs. 6.1±1.1**) and videos (4.9±1.1 vs. 6.2±1.2**). Embryo score was related to the odds of collapse/non-re-expansion in images (OR = 3.07 [2.20-4.29]**) and videos (OR = 2.94 [2.13-4.05]**), with an AUC=0.81 *P<0.01, **P<0.001. Limitations, reasons for caution The algorithm tested in this study was developed externally. It was designed to evaluate videos of fresh embryo development and images at 120 h post-ICSI, but not post-thaw videos and images at days 5 and 6. The developers did not have access to any data or labels from our clinic. Wider implications of the findings Algorithm scoring on videos and images was related to fetal heartbeat rate and to collapse/non-re-expansion of vitrified blastocysts. Fetal heartbeat prediction performance reached 74% in images of non-PGT-A embryos taken at 3.3-4h post-warming. The application of predictive models in vitrified cycles may facilitate the choice of the best frozen embryo. Trial registration number Not applicable

A healthy live birth after mosaic blastocyst transfer in preimplantation genetic testing for GATA1-related cytopenia combined with HLA matching

BackgroundGATA1-related cytopenia (GRC) is characterized by thrombocytopaenia and/or anaemia ranging from mild to severe. Haematopoietic stem cell transplantation (HSCT) is a healing therapeutic choice for GRC patients. We identified a novel pathogenic variant (GATA1: c.1019delG) in a boy with GATA1-related cytopenia. Then we performed preimplantation genetic testing (PGT) in this GRC family. After a mosaic embryo transfered, a healthy and HLA-compatible with the proband baby was delivered.Case presentationThe proband is a 6-year-old boy who was diagnosed to have transfusion-dependent anaemia since 3 year old. Whole-exome sequencing (WES) showed that the proband has a hemizygous variant c.1019delG in GATA1, which is inherited from his mother. His parents decided to undergo PGT to have a health and HLA-compatible offspring. After whole genome amplification (WGA) of biopsied trophectoderm (TE) cells, next generation sequencing (NGS)-based PGT was preformed to analyse embryos on chromosomal aneuploidy, target mutation and HLA typing. There were 3 embryos HLA-matched to the proband. The genotypes of the 3 embryos were heterozygous variant, hemizygous variant, normal respectively. After a heterozygous, mosaic partial trisomy (chr)16, and HLA-matched embryo transfer, a healthy baby was delivered and whose HSCT is compatible with the proband.ConclusionsNGS-based PGT-HLA is a valuable procedure for the treatment of GATA1-related cytopenia caused by GATA1 variants, or other haematological disorders, oncological and immunological diseases. Furthermore, our study reconfirms that mosaic embryos transfer would bring healthy offspring.

O-272 Preimplantation genetic testing of the first polar body is useful for predicting embryo euploidy: data from 1,836 donor oocytes

Abstract Study question Can preimplantation genetic testing of the first polar body (PGT-1BP) accurately identify donor oocytes that will produce euploid embryos? Summary answer Oocyte chromosomal status determined through PGT-1BP had a strong association with embryo euploidy. What is known already Aberrant meiosis during oocyte development leads to aneuploidy, diminished embryo quality, and ultimately, poor reproductive outcomes. Preimplantation genetic testing for aneuploidies (PGT-A) is increasingly employed in donor oocyte cycles, detecting blastocyst euploidy rates ranging from 30% to 80%. However, this variability, together with the clinical and patient resources required to generate blastocysts, support the need for alternative approaches. Oocyte chromosomal status can safely be determined with PGT-1PB. This strategy has shown that 20–30% oocytes obtained from donors with proven fertility are aneuploid, allowing clinicians and patients to make informed reproductive decisions. Study design, size, duration In this prospective study, PGT-1BP was performed on 1,836 fresh donor oocytes collected between September 2017 and January 2022. Vitrified-thawed euploid oocytes were grouped according to donor age (n = 714, n = 554, and n = 568 from 20–24, 25–29, and 30–34-year-old donors, respectively). Oocyte survival and chromosomal status, fertilization through ICSI, blastocyst use and euploidy status were analyzed. Participants/materials, setting, methods Oocytes were derived from donors (n = 115) whose oocytes previously led to at least one live birth. First polar body biopsies were performed 2 hours after in vitro maturation, which corresponds to 38 hours post-hCG trigger. PGT-1BP and PGT-A analyses were performed on the Illumina platform. Oocyte and embryology data as well as the association between oocyte and embryo chromosomal status was evaluated using Mann-Whitney U test. Main results and the role of chance The post-thawing viability rates for the biopsied oocytes were 98.2%, 99.5%, and 96.5% for the three age groups, respectively. Donor oocyte euploidy significantly decreased with advancing age (81.9% vs. 76.9% vs. 70.0%, respectively; p ≤ 0.05). The rate of blastocyst use was comparable across age groups (68.1% vs. 71.7% vs. 68.7%, respectively; p > 0.05). Blastocysts derived from euploid oocytes maintained their euploid status in 88.7%, 90.1%, and 85.6% of cases, respectively. Euploid oocyte-euploid blastocyst matching rates were 89.2%, 90.1%, and 88.1%. Limitations, reasons for caution Although PGT-1BP may not guarantee euploid embryos, it could allow clinicians and couples to make informed decisions about proceeding with fertilization and embryo culture. The associations between euploidy rate of embryos derived from euploid donor oocytes should be confirmed in further studies with larger cohorts. Wider implications of the findings Selecting euploid oocytes with a high probability of becoming euploid embryos may increase the odds of achieving a successful pregnancy at the first attempt. This approach is cost-effective, could prevent unnecessary emotional distress for patients, and is an attractive option for couples with limited sperm available for IVF/ICSI. Trial registration number not applicable

P-710 Comparison of cumulative delivery rates per aspiration cycle in patients fulfilling Bologna and POSEIDON criteria

Abstract Study question Do cumulative delivery rates (CDRs) per aspiration cycle differ in low-prognosis patients according to the Bologna and POSEIDON (Patient-Oriented-Strategies-Encompassing IndividualizeD-Oocyte-Number) classification criteria? Summary answer The CDR was 8.7% in Bologna and 22.7% in POSEIDON. The odds of achieving live-birth were ∼3-fold higher in POSEIDON than in the Bologna group. What is known already Bologna criteria were generated to address the heterogeneity in the definition of the Poor Ovarian Response (POR). However, patients meeting these criteria may not be homogeneous in terms of the number of oocytes retrieved and live birth rate. Subsequently, as a more inclusive classification, the POSEIDON criteria were proposed to stratify low-prognosis patients according to female age and suboptimal response to ovarian stimulation. To our knowledge, there is a paucity of data comparing CDRs between the POSEIDON and Bologna groups. Study design, size, duration Retrospective cohort study including 1250 patients undergoing their first ovarian egg retrieval cycle at Hacettepe University IVF Center in Ankara between 2017 and 2023. Exclusion criteria were female age >45 years old, body mass index >35 kg/m2, azoospermia, pre-implantation genetic testing (PGT) for structural rearrangement or monogenic disorders, fertility preservation cycles, dual-stimulation cycles, premature ovarian insufficiency, and hypogonadotropic hypogonadism. The primary outcome measured was CDR defined by ICMART (International Committee for Monitoring Assisted Reproductive Technologies). Participants/materials, setting, methods The Bologna patients have at least two of the following three features; female-age ≥40 or prior ovarian surgery, AMH<1.1ng/ml or antral-follicle-count (AFC)<5-7, and obtaining ≤3 oocytes in prior conventional stimulation. POSEIDON patients were categorized into four groups; younger(<35) and older(≥35) women with AMH≥1.2/AFC≥5 experiencing an unexpected poor(<4 oocytes-retrieved) or suboptimal(4–9 oocytes-retrieved) response, along with respective younger and older counterparts with AMH<1.1/AFC<5. Non-POSEIDON patients were those with AMH≥1.2/AFC≥5 and >9 oocytes-retrieved. General-Estimated-Equation(GEE) analysis was employed. Main results and the role of chance Out of 1250 patients, 79.6% were classified as POSEIDON, with sub-groups as follows: 5.28% Group 1a, 23.4% Group 1b, 3.6% Group 2a, 10.1% Group 2b, 14.64% Group 3, and 22.48% Group 4. The number of patients meeting Bologna criteria was 17.4% (218). For POSEIDON patients, median[IQR] values were: age 34 [30-38], AMH 1.33 [0.65-2.69], oocytes retrieved 4 [2-6], and embryo transfers 1 [1-2]. For non-POSEIDON patients: age 30 [27-33], AMH 4.63 [2.93-7.07], oocytes retrieved 13 [11-15], and embryo transfers 2 [1-3]. In Bologna patients: age 40 [36-42], AMH 0.46 [0.20-0.76], oocytes retrieved 2 [1-3], and embryo transfers 1 [0-1]. For non-Bologna patients: age 31 [28-36], AMH 2.30 [1.17-4.29], oocytes retrieved 6 [3-9], and embryo transfers 1 [1-2]. CDR for non-POSEIDON: 49.8%; Group 1a, 24.2%; Group 1b, 34.1%; Group 2a, 15.6%; Group 2b, 27.6%; Group 3, 20.8%; Group 4, 10.7%. In clustered-GEE analysis, using non-POSEIDON as reference, odds ratios (ORs) for live birth were: Group 1a OR 0.32 [0.18 - 0.60]; Group 1b OR 0.52 [0.37-0.74]; Group 2a OR 0.19 [0.08-0.43]; Group 2b OR 0.38 [0.24-0.61]; Group 3 OR 0.26 [0.17-0.41]; Group 4 OR 0.12 [0.08-0.19]. CDR for the Bologna group: 8.7% (OR: 0.10, 0.06-0.16); non-Bologna patients: 32.4% [OR: 0.48, 0.39-0.60]. Limitations, reasons for caution The limitations of the study encompass its retrospective cohort design and the variability introduced by different individuals conducting antral follicle counts using 2D ultrasonography. Wider implications of the findings Significant CDR differences exist between Bologna and Poseidon groups. POSEIDON patients show ∼2-fold lower CDR than normal responders, and Bologna patients exhibit ∼4-fold lower CDR than non-Bologna patients. Poseidon criteria cover a broader range of low-prognosis patients, while meeting Bologna criteria indicates a narrower, more specific group with poorer outcomes. Trial registration number N/A

Development of pre-implantation genetic testing protocol for monogenic disorders (PGT-M) of Hb H disease

Hb H disease is the most severe form of α-thalassemia compatible with post-natal life. Compound heterozygous α0-thalassemia− SEA deletion/α+-thalassemia− 3.7kb deletion is the commonest cause of Hb H disease in Thailand. Preimplantation genetics testing for monogenic disorders (PGT-M) is an alternative for couples at risk of the disorder to begin a pregnancy with a healthy baby. This study aims to develop a novel PCR protocol for PGT-M of Hb H disease− SEA/−3.7kb using multiplex fluorescent PCR. A novel set of primers for α+-thalassemia− 3.7kb deletion was developed and tested. The PCR protocol for α0-thalassemia− SEA deletion was combined for Hb H disease− SEA/−3.7kb genotyping. The PCR protocols were applied to genomic DNA extracted from subjects with different thalassemia genotypes and on whole genome amplification (WGA) products from clinical PGT-M cycles of the families at risk of Hb Bart’s. The results were compared and discussed. The results showed three PCR products from α+-thalassemia− 3.7kb primer set, and three from α0thalassemiaSEA primer set. The results were consistent with the known thalassemia genotypes. The novel -α3.7 primers protocol was also tested on 37 WGA products from clinical PGT-M cycles giving accurate genotyping results and a satisfying amplification efficiency with the ADO rates of 2.7%, 0%, and 0% for HBA2, HBA1, and internal control fragments, respectively. This novel PCR protocol can precisely distinguish Hb H disease− SEA/−3.7kb from other genotypes. Additionally, this is the first PCR protocol for Hb H disease− SEA/−3.7kb which is optimal for PGT-M.

P-567 Is mosaic embryo transfer a valid alternative for patients lacking euploid embryos? a multicentric study from Argentina

Abstract Study question How does low-level mosaicism embryo transfer affect reproductive outcomes, including pregnancy rates and live birth success, compared to transfers involving euploid embryos? Summary answer Most mosaic embryo transfers do not affect reproductive outcomes, as live birth rates appear to be similar to those observed with euploid embryos. What is known already Preimplantation genetic testing (PGT) is used to identify embryonic genetic abnormalities and, ideally, select euploid embryos for intrauterine transfer, to improve clinical IVF outcomes. Chromosomal mosaicism is defined as the presence of two or more different chromosomal cell lines within an embryo. Numerous sources describe the proportion of mosaic embryos ranging from 4 to 22%. Several publications report that mosaic embryo transfer can lead to successful pregnancies and healthy deliveries, albeit at a lower frequency and with higher rates of pregnancy loss compared to euploid embryos. However, the impact of mosaicism on IVF outcomes remains a matter of controversy. Study design, size, duration Observational and retrospective multi-center study analyzed 6241 embryos from 1218 patients, across 4 IVF centers, who underwent PGT between 2016 and 2023 in a single genetics laboratory. Embryos were categorized as euploid, aneuploid, or mosaic. Patients in group A (n = 894) were transferred an euploid embryo, while in group B (n = 55), a low-grade mosaic embryo, defined as those having less than 40% (2016-2021) or 50% (2021-2023) aneuploid cells, was transferred. Participants/materials, setting, methods The embryos were biopsied and vitrified for later transfer. Blastocyst biopsy DNA was amplified using the SurePlex kit, processed with the VeriSeq kit (Vitrolife), and sequenced on a MiSeq platform. The results were analyzed using BlueFuse-Multi software. Mosaic embryo transfers were conducted only in the absence of a euploid embryo, following genetic counseling. beta-hCG levels >25 IU were considered positive, and clinical pregnancy was confirmed through ultrasound visualization of the gestational sac with heartbeat. Main results and the role of chance Among the analyzed embryos, the proportion of euploid was 46.2% (n = 2883), while mosaic embryos accounted for 19.3% (n = 1207), with 69.5% classified as low-grade and 30.5% as high-grade. Aneuploid embryos comprised 31.3% (n = 1954), and results could not be obtained for 3.2% (n = 197). Fifty-five low-grade mosaic embryos were transferred, including 19 with complete chromosomal mosaicism, 34 with segmental mosaicism, and 2 cases with 2 chromosomes involved in the mosaicism. The positive beta-hCG rate post-transfer was 50.3% for Group A, compared to 49.1% for Group B. Clinical pregnancy rates were 36.9% and 36.4%, respectively. Regarding live births, in Group A 328 (36.7%) children were born, and in Group B, 16 (29.1%), with 2 ongoing pregnancies. Conversely, pregnancy losses in Group B (9.1%) were higher compared to the euploid group (2.9%). Regarding pregnancy losses, in 3 cases no fetal heartbeats were identified, there was one miscarriage <20 weeks, and one twin gestation was lost after a normal non-invasive prenatal testing. In all cases the newborns were apparently healthy after mosaic embryo transfer, with an average gestational week at birth of 39 (37-41), and an average weight of 3197 (2900-4390) grams. Cesarean section was performed for 15/16 births, and one was born by vaginal delivery. Limitations, reasons for caution The study is limited by its retrospective nature and the small number of samples and mosaics transferred. Prenatal and postnatal tests were not conducted. Furthermore, the health of newborns was evaluated only in the initial months of life. Wider implications of the findings Mosaic embryo transfer is a valid alternative for couples lacking euploid embryos. This multicenter study adds evidence that mosaic embryo transfer does not have a clinically relevant impact on pregnancy and live birth rates, similar to what has been reported by other authors. Trial registration number ‘not applicable’

O-182 To err is human, also for those working in ART

Abstract Medically assisted reproduction (MAR) centers combine clinical and technical expertise with a very high complexity. Over the years, MAR centers have undergone increased intricacy due to incorporating laboratory techniques, such as cryopreservation of oocytes and embryos and micromanipulation, embryo culture, and pre-implantation genetic testing. Not only the technicality of the MAR laboratory profession is challenging, but the complexity of each phase has increased over the years; the patient population undergoing treatment has expanded, where not only the intended parents’ gametes are involved but also the utilization of gametes from egg and sperm donors, as well as including a gestational carrier receiving the embryo. These complexities have led to the implementation of rigorous control measures to mitigate the risk of errors. Nevertheless, a zero-risk fertility clinic is an illusion, given the many necessary interventions and manipulations in each treatment cycle. Errors in fertility practice involving gametes or embryos can be devastating to patients and clinical personnel, often raising psychological, legal, ethical, economic, and practical concerns. The most critical cases of risk of harm in MAR procedures involve misidentifying a patient, gamete, or embryo, called ‘mix-up,’ which could lead to dramatic consequences. Although the frequency is low, mix-up cases have been described worldwide. Other adverse events derive from preventable human actions, e.g., overlooking an embryo when carrying out assessments, dropping a dish, incorrect labeling, improper pipetting technique, unusually poor thawing, and inadvertent discard of the sample. Human error cannot be eradicated. In the old view, human error was perceived as the root cause of problems, with individuals being viewed as variables requiring control. Conversely, contemporary perspectives consider human error a symptom of deeper systemic issues. As suggested by the Institute of Medicine in 2000, which published the report “To Err Is Human,” the core of the problem lies not with the fallibility of healthcare personnel but the flawed systems within which they operate. Preventing errors is possible by incorporating safety into system processes and hospital structures. Latent errors are errors that arise due to system inefficiencies. In the context of MAR, these latent conditions can, amongst others, be work-related pressure, high workload, lack of communication between team members, distraction, a suboptimal setup of the lab, a lack of clarity in the procedures, and insufficient time for thorough communication. In the famous Reason’s Swiss cheese theory of error, these latent conditions are weaknesses (or gaps/ holes) in the system’s defense layers. When all the holes from each layer (i.e., policy, planning, design, communication) align, they will permit an error to cause harm. Understanding the theory of human errors and building safety into various layers of care processes in MAR are effective ways of reducing errors rather than blaming sentinel individuals. Errors within MAR centers can be prevented by designing the healthcare system at all levels to make it safer, making it hard for people to do the wrong thing and easy for people to do the right thing.

P-577 A randomized trial to compare the live birth rate following the progestin-primed ovarian stimulation versus the antagonist protocol in women with anticipated high ovarian responses

Abstract Study question Is there difference in the live birth rate following the progestin-primed ovarian stimulation (PPOS) versus the antagonist protocol in women with anticipated high ovarian responses? Summary answer The live birth rates of the first frozen embryo transfer following PPOS and the antagonist protocol were comparable in women with anticipated high ovarian responses. What is known already Progestin when given orally inhibits the pituitary LH surge during ovarian stimulation when taken orally i.e. progestin-primed ovarian stimulation. Two randomized trials showed similar number of oocytes for PPOS and the antagonist protocol in oocyte donation cycles. However, there is still no randomized trial to compare the live birth rate between PPOS and the antagonist protocol. Study design, size, duration This is a randomized controlled trial of 784 infertile women conducted from June 2020 and October 2021. Participants/materials, setting, methods Infertile women aged <43 years undergoing the first IVF cycle in a tertiary fertility center and having antral follicle count >15 randomly assigned in a 1:1 ratio into two groups: PPOS group and the antagonist group. Medroxyprogesterone 10 mg daily was given from the start of ovarian stimulation until the day of ovulation trigger in the PPOS group. The primary outcome was the live birth rate of the first frozen embryo transfer. Main results and the role of chance 784 women were randomly assigned into two groups: PPOS group (n = 392) and antagonist group (n = 392). Embryo transfer was either cancelled or postponed in 62 (62/392, 15.8%) women in the PPOS group and 65 (65/392, 16.6%) in the antagonist group because of no transferable embryos or no frozen embryo transfer within 6 months following randomization. The two groups were similar in demographic characteristics and number of oocytes obtained/fertilized, the number of cleaving embryos, number of good quality embryos at day 3, number of blastocysts developed and number of embryo/blastocyst frozen. There was no statistically significant difference in the live birth rate of the first frozen embryo transfer cycle between the progestin-primed ovarian stimulation and antagonist groups based on both intention to treat [37.5.0% (147/392) versus 32.7% (128/392), RR 1.148 (95% CI = 0.949–1.390), P = 0.16] and per protocol analysis [44.5% (147/330) versus 39.1.% (128/327), RR 1.138 (95%CI = 0.950–1.364), P = 0.16]. Both groups showed comparable clinical pregnancy, ongoing pregnancy, miscarriage, multiple pregnancy, ectopic pregnancy and cumulative live birth rates. Limitations, reasons for caution Women with anticipated high ovarian responses were recruited and the results may not be extrapolated to the general population seeking IVF. This is not a blind study although the live birth rate as the primary outcome is objection. The duration of the study is 6 months since randomization. Wider implications of the findings In situations in which fresh embryo transfer is not required such as oocyte/embryo freezing for fertility preservation, oocyte donation or preimplantation genetic testing, the PPOS protocol may be preferred over the antagonist protocol. Trial registration number NCT04414761

P-231 How many blastocysts are necessary to reach pregnancy with preimplantation genetic testing?

Abstract Study question What is the influence of the number of blastocysts before biopsy on the prognosis of pregnancy in preimplantation genetic testing for aneuploidy (PGT-A) cycles? Summary answer The number of pre-biopsy blastocysts seems to be a parameter that can be used as a prognostic factor for the pregnancy chance in PGT-A cycles. What is known already Advances in embryo cryopreservation techniques have allowed for a greater use of PGT. Studies showed that PGT-A has improved live birth rates while reducing the number of embryos transferred. There is a lot of information about the role of the oocyte number as a predicting factor for pregnancy rate. However, little information is available about the influence of the number of blastocysts before performing PGT-A on the chance of achieving a pregnancy. Study design, size, duration Retrospective study performed in a reproductive medicine center using the first IVF cycle from 2016 to 2023, including 682 patients with at least one blastocyst before undergoing PGT-A. All patients performed a single embryo transfer in each procedure, in up to 4 transfers. Participants/materials, setting, methods The sample was divided into four groups according to the blastocyst number: G1, 1 to 2 blastocysts (n = 324); G2, 3 to 4 (n = 197); G3, 5 to 9 (n = 137); and G4, ≥ 10 blastocysts (n = 24); each group was divided according to female age (≤ 37 and > 37) Variables: female age, and euploidy, clinical pregnancy per transfer and cumulative clinical pregnancy rates. Data were compared between groups. Statistical analysis: Chi-square test (Bonferroni correction) and Anova (p < 0.05). Main results and the role of chance The results in G1, G2, G3 and G4 were, respectively: female age, yo (40 vs. 38.4 vs. 37.2 vs. 34.7, p < 0.01); euploidy rate (34.6 vs. 67.2 vs. 83.2 vs. 100.0, p < 0.0001), clinical pregnancy rate per transfer, % (45.5 vs. 58.6 vs. 72.8 vs. 95.8 p < 0.0001 ), cumulative clinical pregnancy rate, % (15,7 vs. 39.4 vs. 60.6 vs. 95,8% p < 0.0001). Women with 1 to 2 blastocysts before biopsy had a cumulative pregnancy rate more than 3-fold lower than those with 3 to 4 blastocysts (%, 39.6 vs. 15.3, p < 0.001, OR 3.59, CI 95% 2.32 to 5.57) and more than 8-fold lower than those with 5 to 9 blastocysts (%, 60,6 vs. 15.3, p < 0.001, OR 8.42, CI 95%: 5.20 to 13.64). The comparison in each group according to female age showed that until 9 blastocysts, the younger women had a pregnancy rate higher than the older, respectively (%): G1: 30.8 vs. 12.5, p = 0.001; OR 3.1, CI 95%: 1.44 to 6.48; G2: 57.4 vs. 30.2, p < 0.001; OR 3.10, CI 95%: 1.61 to 5.98; G3: 72.6 vs. 50.7, p = 0.009. OR 2.58; IC 95%: 1.19 to 5.67; and G4: 93.8 vs. 87.5, p = 0.268. Limitations, reasons for caution As a retrospective study, data collection and filling limitations should be considered. Wider implications of the findings The number of pre-biopsy blastocysts appears to be a parameter that can be used as a prognostic factor for the pregnancy chance in PGT-A cycles. Even transferring 1 embryo, the pregnancy rate/transfer was higher with more blastocysts. In patients with less than 10 blastocysts before PGT, age influences pregnancy rates. Trial registration number Not Applicable

P-532 Preimplantation genetic testing versus morphology as selection criteria for single embryo transfer in women aged 35-42: results of a pilot randomized controlled trial

Abstract Study question To assess the feasibility of running a large randomised controlled trial (RCT) evaluating the benefit of preimplantation genetic testing for aneuploidy (PGT-A) for embryo selection. Summary answer Running a PGT-A RCT is feasible. Considerations include participant recruitment, seven-day biopsy-embryologist availability and logistical complexities. What is known already Critics question the effectiveness of PGT-A leading to concerns about false positives and negatives, unnecessary embryo discarding and clinical effectiveness. Past studies have been criticised for only performing biopsy on top quality embryos, their criteria for randomization and the age of the population being assessed. Given these uncertainties there is a need for a rigorous RCT evaluating the role of PGT-A using current methodologies and biopsy techniques in women most affected by aneuploidy. Our pilot study is the first study where all embryos generated will undergo biopsy and ranked in terms in implantation potential prior to transfer. Study design, size, duration In this prospective, concealed allocation two-arm parallel RCT, our goal was to randomise 100 women with ≥3 good quality embryos on day 3 post egg collection into two groups: embryos screened using next-generation sequencing (NGS)-based PGT-A or selection-based on morphology alone. PGT-A patients underwent a trophectoderm biopsy on day 5/6/7 following egg collection. Biopsied embryos were frozen and subsequently transferred. Euploid and embryos described as being low mosaic (<30% abnormal cells) were prioritised for transfer. Participants/materials, setting, methods Women aged between 35–42 years undergoing IVF±ICSI were included, with those undergoing PGT for inherited genetic disorders, gamete donation cycles, untreated hydrosalpinges or untreated uterine abnormalities excluded. Primary objective during the pilot phase was to assess the ability to recruit, randomise, adherence to the study protocol and plan for a full study. Secondary outcomes were clinical pregnancy rate per embryo transferred, clinical pregnancy rate per couple randomised, miscarriage rate and time to pregnancy. Main results and the role of chance Out of 247 eligible women,100 were randomised, and 94 received the assigned treatment. The recruitment rate was 70.18%(173/247, 95%CI 61.65%-78.70%). An average of 6 participants were randomised each month. All randomised participants completed the follow-up. Our main recruitment challenges included 19% of eligible patients choosing to have PGT-A privately and 6% withdrawing their consent choosing to have a fresh transfer. Lack of biopsy-embryologist also meant patients whose potential biopsy would have been on a weekend had to be excluded in the first 4 months of our recruitment process. Baseline characteristics including age, BMI and cause of infertility were comparable in both arms and a total of 56 euploid, 53 aneuploid and 3 low mosaic embryos were identified. There was no statistically difference in CPR (23/50 vs 21/50, RR 1.09 95%CI:0.83-1.15, p-value>0.23) and miscarriage rate (3/50 vs 2/50, RR 1.5 95%CI:0.84-1.75, p-value: 0.15) per single embryo transfer or per woman randomised (23/46 vs 21/48, RR 1.14 95%CI 0.78-1.27, p-value:0.24 and 3/46 vs 2/48, RR 0.72 95%CI 0.58-1.17 p-value: 0.17) in either arm of the study. We cannot yet confirm the clinical benefit of PGT-A owing to the insufficient sample size in this pilot study. No low mosaic embryos were transferred. Limitations, reasons for caution The study was conducted in a single centre. If future definitive trials are conducted in multiple centres, the criteria for recruitment, randomisation, intervention, and follow-up still need to be unified. Many women aged above 40 failed to reach randomization due to challenges in producing three good-quality embryos. Wider implications of the findings The pilot study found that the eligibility rate is adequate however researchers should consider reviewing the conditions for randomisation in main study such as reducing the number of good embryos required to two. In addition, stratified randomization should be used in order to better balance female age within each group. Trial registration number NCT05009745

O-159 Domestic and cross-border reproductive care among same-sex male intended parents: comparing live birth rates (LBRs) in a national retrospective cohort in the United States (U.S.)

Abstract Study question How common is cross-border reproductive care (CBRC) in the U.S. among international same-sex male intended parents? Do CBRC LBRs differ compared to domestic counterparts? Summary answer A substantial number of international same-sex male intended parents seek CBRC in the U.S. LBRs per transfer are reassuring and comparable to domestic counterparts. What is known already Same-sex male intended parents often work with a gestational carrier to have a genetically related child(ren); however, restrictive and/or discriminatory policies in their home countries may prevent access. Intended parents may seek CBRC in countries like the U.S. due to favorable regulations. However, the volume of CBRC activity among same-sex male intended parents has been difficult to estimate accurately due to poor sexual orientation data collection. Capitalizing on national databases that identify same-sex male intended parents is the first step to describing CBRC trends and their associated outcomes in a marginalized population that is often excluded in the literature. Study design, size, duration This national retrospective cohort study included U.S. based gestational carrier embryo transfer cycles from the Society for Assisted Reproductive Technology (SART) database in the years 2017–2020. Among participating clinics (n = 488), 6,168 cycles among same-sex male intended parents were identified. Relative risks (RRs) for LBRs per transfer were compared for cycles including domestic (i.e., U.S. based, n = 3,052) and internationally based (n = 3,080) patients. LBR and country data were missing for 546 and 36 cycles, respectively. Participants/materials, setting, methods The inclusion criteria were transfer cycles among male patients with male partners. Unadjusted and adjusted RRs for LBRs per transfer were calculated to compare domestic (reference) and internationally based groups using multivariable Poisson and logistic regression models with generalized estimating equations (GEEs). Models were adjusted for donor age, transfer type (i.e., fresh, frozen), day of transfer, number of embryos transferred, male infertility, and the use of intracytoplasmic sperm injection, assisted hatching, and preimplantation genetic testing. Main results and the role of chance Between 2017 and 2020, 6,168 cycles with an embryo transfer to a gestational carrier occurred among same-sex male intended parents in the U.S. In half of the cycles, the U.S. was a destination for CBRC (50%, n = 3,080) among which (48%, n = 1,540) were from one of 23 European countries. Regarding countries of residence for CBRC cycles (n = 3,080), those most reported were China (23%), France (19%), Israel (13%), Spain (11%), the United Kingdom (6.1%), Australia (4.1%), Germany (3.7%), Italy (2.6%), Switzerland (1.5%), Canada (1.5%), Belgium (1.4%), Singapore (1.0%), Taiwan (1.2%), Norway (1.2%) and The Netherlands (0.92%). Additionally, there were 76 cycles from 8 countries in South America (2.3%), 9 cycles from 6 countries in Africa (0.27%), and 3 cycles from 2 countries in Central America (0.089%). Most cycles were in the U.S. West (72%, n = 4,441; e.g., California [47%]). Median oocyte age was 26 years (interquartile range 24,28) with a majority of cycles involving single embryo transfer (78%, n = 4,802). LBR per transfer was 69% (n = 3,867). There was no statistical difference between live births for domestic versus CBRC cycles in the unadjusted (RR 1.14, 95% confidence interval [CI] 0.96, 1.37; p: 0.141) or adjusted regression models (aRR 1.06, 95% CI 0.86, 1.32; p: 0.572). Limitations, reasons for caution The SART surveillance system captures most (∼90%) but not all embryo transfer cycles in the US. Current gender identity data were not available. Only the individual identified as the patient (and not the partner) indicated country of residence. The rationale for pursuing CBRC in the U.S could not be explored. Wider implications of the findings This study highlights a substantial amount of CBRC among same-sex male intended parents choosing the US for reproductive care. Implementing improved sexual orientation and gender identity data collection strategies are critical to filling CBRC data deserts not only among same-sex male intended parents but all sexual and gender minority populations. Trial registration number Not applicable

O-273 A transcriptomic approach for embryo selection after preimplantation genetic diagnosis for aneuploidy

Abstract Study question Is it possible to predict an euploid chromosomal constitution from RNA-Seq data and identify a transcriptomic profile compatible with extended embryonic development? Summary answer It has been possible to obtain a karyotype comparable to PGT-A, in addition to acquiring a transcriptomic signature of embryos suggestive of improved implantation capacity What is known already Conventional assessment of embryo competence, based on morphology and morphokinetic, lacks knowledge of molecular aspects and faces controversy in predicting ploidy status. Understanding the embryonic transcriptome is crucial, as gene expression influences development and implantation. Preimplantation genetic testing (PGT) has improved pregnancy rates, but problems persist when high-quality euploid embryos do not reach term. In fact, around 50 to 60% implant, of which 10% miscarry. Comprehensive approaches, including RNA-Seq, offer potential to discover molecular markers of reproductive competence. Extended-embryo culture platforms up to day 14 can be utilized as a proxy to study embryo development at post-implantation stages Study design, size, duration Prospective pilot cohort study conducted from March 2023 to August 2023. A total of 30 vitrified human blastocysts with previous PGT-A diagnosis on day 5 (D5) or day 6 (D6) of development were analysed: n = 15 euploid and n = 15 aneuploid embryos. Finally, 21 embryo samples were included in the study (n = 10 euploid and n = 11 aneuploid), the rest (n = 9) were excluded due to poor quality after sequencing. All embryos were donated for research purposes Participants/materials, setting, methods Following warming and re-expansion, embryos underwent a second trophectoderm biopsy. The remaining embryo was cultured until day 11 to assess its development. After biopsy analysis by RNA-Seq, karyotypes were studied, as well as differential expression gene (DEG) of the comparison: unattached embryos (n = 12) vs embryos attached (n = 9) to the plate (padj<0.05 and FC > 1). In addition, we obtained a transcriptomic signature specific to embryos with a “theoretical” capacity for sustained implantation (raw p-value<0.05 and shrunk LFC>1.1) Main results and the role of chance Digital karyotype concordance was obtained with a sensitivity of 81%, a specificity of 83%, a Kappa index of 65.5% and an area under the ROC curve of 90%. At the gene level, 76 DEGs (56 upregulated and 20 downregulated) were found in the comparison of embryos unattached to the plate in extended culture versus those that were attached. To address the functional implications of these differences, significantly deregulated pathways according to GO and KEGG categories were identified. The mural trophectoderm of the analysed blastocysts showed 63 significantly deregulated terms (30 GO-Biological Process, 13 GO-Cellular Component, 12 GO-Molecular Function and 8 KEGG pathways), displaying upregulation of autophagy, apoptosis, protein kinase and ubiquitin-like protein ligase activity, and downregulation of ribosome, spliceosome, kinetochore, segregation, and chromosome condensation processes. The first described transcriptomic signature specific to embryos with theoretical enhanced implantation capacity consists of 501 genes, including: EMP2, AURKB, FOLR1, NOTCH3, LRP2, FZD5, MDH1, APOD, GPX8, COLEC12, HSPA1A, CMTM7, BEX3, which are related to implantation and embryonic development. These findings indicate that it would be possible to identify euploid embryos with a greater capacity for implantation and development, excluding those embryos that, although they could be implanted and viable, present chromosomal alterations Limitations, reasons for caution This study included a small sample size and a remarkable variability between samples. Structural chromosomal abnormalities were not included. It is not possible to diagnose mosaic embryos. Trophectoderm biopsy does not assure the chromosomal status of the whole embryo. Maximum number of days for in vitro development was 11 Wider implications of the findings Gene expression profiles of embryos with theoretical increased implantation capacity have been obtained. Clinical implementation of this technique, which assesses chromosomal content and transcriptomic profile, could be used to significantly increase clinical outcomes in assisted reproduction, allowing informed decisions to be made about future treatments and the prospects of success Trial registration number Not applicable

O-206 Natural, modified natural or artificial endometrial preparation strategies for frozen embryo transfer in ovulatory women (MONART): a randomized controlled trial

Abstract Study question Are live birth rates after natural (NC), modified natural (mNC) endometrial preparation superior to artificial cycle (AC) strategies for ovulatory women undergoing frozen embryo transfer? Summary answer Live birth rates were comparable between NC, mNC, and AC. However, NC and mNC resulted in a significantly higher cycle cancellation rate. What is known already The number of frozen embryo transfer (FET) cycles has increased dramatically. In ovulatory women undergoing FET, three common strategies can be used for endometrial preparation: NC, mNC (NC + human chorionic gonadotropin trigger), and AC protocols. Previous studies comparing the effectiveness of these strategies had several drawbacks, including small sample size, lack of consistency between studies in the assessment and reporting of outcomes, and the retrospective origin, which introduced potential biases. Therefore, there is a lack of consensus on the optimal endometrial preparation protocol for FET. Study design, size, duration This single-center, open-label, randomized controlled trial was conducted at IVFMD, My Duc Hospital in Ho Chi Minh City, Vietnam. From March 2021 to March 2022, a total number of 1,428 participants were randomized (476 per group). The primary outcome of the study was live birth after one FET. Secondary outcomes were fertility outcomes, early pregnancy complications, obstetrical outcomes, perinatal and neonatal complications, and endometrial preparation cycle cancellation rates. Participants/materials, setting, methods Eligible participants were ovulatory women aged 18–45 with FET treatment, underwent no more than three IVF/intracytoplasmic sperm injection-FET cycles, had no more than two day-3 or one blastocyst transferred. Women with menopause/anovulation, contraindications for hormonal administration, uterine abnormalities who underwent in-vitro maturation, pre-implantation genetic testing, oocyte donation cycles were excluded. The FET strategies included FET after the first endometrial preparation cycles with AC, mNC, or NC, as per randomization, and the second with AC. Main results and the role of chance A total of 4,779 participants were screened, of whom 1,428 eligible individuals (476 per group) were randomized after written informed consent. The live birth rate after one FET was 36.6% (174/476) in the natural cycle strategy group, 33.4% (159/476) in the modified natural cycle strategy group, and 34.0% (162/476) in the artificial cycle strategy group (relative risk [95% confidence interval] for natural versus artificial cycle strategy: 1.07 [0.87–1.33] and for modified natural versus artificial cycle strategy: 0.98 [0.79–1.22]). Maternal and neonatal outcomes were comparable between groups, in particular,hypertensive disorders in pregnancy. For both natural and modified cycle groups, 20.08% (99/476) of the first FET cycles were canceled before subsequent successful ET versus none in the artificial cycle group. Limitations, reasons for caution Due to the nature of the intervention, blinding was not possible. The study was conducted in ovulatory women in a single center in Vietnam, which may limit the generalisability of the findings. Wider implications of the findings Live birth rates and obstetric/neonatal outcomes after the natural cycle, modified natural cycle, or artificial cycle strategies for ovulatory women undergoing FET are comparable, with higher cancellation rates in both the natural cycle and the modified natural cycle groups. Trial registration number NCT04804020