Preimplantation Genetic Testing Research Articles

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

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-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

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’

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

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-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

P-033 Microfluific sperm sorting does not impact the primary sex ratio in IVF-ICSI treatments

Abstract Study question Does sperm selection through microfluidics alter the primary sex ratio (PSR) when compared to density gradients? Summary answer The PSR is not modified when employing a microfluidic sperm sorting when comparing to the conventional density gradient technique for sperm selection. What is known already The PSR refers to gender after fertilization, this is often expressed as the male births per female births. Little is known about the sex ratio in natural and assisted reproduction treatments (ART) since it is difficult to establish in ART as it requires preimplantation genetic testing (PGT). Previous studies have been estimating the variation in the PSR based on the fertilization technique used or the day of embryo transfer. Even the influence of sperm selection techniques has not received much attention, using traditional sperm selection methods such as density gradients or swim-up, a PSR of 1.07 has been estimated. Study design, size, duration This is a retrospective study that includes 1764 blastocysts stage embryos biopsied from 522 autologous preimplantation genetic testing for aneuploidies cycles (PGT-A) performed between January of 2020 and December of 2022 and analysed through Next Generation Sequencing (NGS). All the embryos were cultured under the same conditions with one-step media in a time-lapse incubator and the PGT-A was performed in day 5 or day 6 of culture to determine the embryo ploidy status. Participants/materials, setting, methods Two groups were established based on the sperm selection method before ICSI. The study group using a microfluidic system (MS; ZyMōtICSI® or ZyMōtMulti®) included 256 blastocysts from 98 cycles, and a control group using density gradients (DG), with 1058 blastocysts from 424 cycles were compared. PGT-A determined the embryonic sex and for the PSR estimation, number abnormalities in the sexual chromosomes were excluded (i.e. XXY, X0, XYY). Chi-square test was performed to compare the groups. Main results and the role of chance Our data show that there was no statistical significance in the overall ratio of XY:XX embryos biopsied between both groups (p = 0.09). The PSR was 0.89 (47:53) in the study group (MS) and 1.12 (53:47) in the control group (DG). Although not statistically significant, the rate of XX embryos was 6% higher than XY embryos in the MS group. In contrast, in the DG group, the same increase in the rate of XY embryos was observed. If we consider only the euploid embryos which are the potentially born babies, the PSR acquires values of 0.96 for the DG group and 0.92 for the MS group (p = 0.86), showing an increase in euploid female embryos transferred compared to those euploid male embryos. However, it was not statistically significant. Limitations, reasons for caution The performance of PGT-A on blastocysts does not provide an entirely accurate representation of the PSR since it is carried out 5/6 days after the moment of fertilization; nevertheless, it is the technique that comes closest. Also, the low number of blastocysts in the study group represents a limitation. Wider implications of the findings The sperm selection for ICSI with microfluidic technology does not significantly alter the PSR. Trial registration number Not applicable

P-421 Enhancing the success of frozen embryo transfer by human chorionic gonadotropin injection on the day of progesterone supplementation onset - a machine learning study

Abstract Study question Does subcutaneous injection of human chorionic gonadotropin (hCG) at the onset of progesterone supplementation affect clinical pregnancy rate in frozen embryo transfer cycles? Summary answer Clinical pregnancy rates are higher when hCG injection is administered at the onset of 300 mg/day progesterone in patients undergoing frozen embryo transfer. What is known already HCG and progesterone play crucial roles in embryo transfer and implantation during in vitro fertilization. Exisitng studies have demonstrated that pre-administration of hCG at the onset of progesterone supplementation for frozen embryo transfer increases clinical pregnancy rates in patients with endometriosis. However, the synergistic effects of hCG and progesterone co-supplementation in a broader range of patients remain unexplored and require further investigation. A multifactorial analysis of hormone replacement therapy, covering various patient-specific aspects, is necessary to understand the implications of this approach. Study design, size, duration This retrospective cohort study analyzed data from 2,251 single frozen embryo transfers performed between Jan 2019-Aug 2023 at the INVICTA Fertility Centre, Poland. The dataset was selected by the practitioner and included age, AMH, BMI, embryo morphology grade, total number of transfers, medical and embryological procedures, recurrent implantation failure, preimplantation genetic testing, endometrial thickness before transfer (≥ 7 mm), and the menstrual cycle day on which progesterone was started. Participants/materials, setting, methods Patients (21-47 years) receiving 300 or 600 mg/day progesterone for five days were divided into the control and hCG (6,500 IU hCG injection on progesterone onset) groups. A machine learning model was trained using a gradient boosting technique (100 decision trees, four leaves, maximum depth of two nodes, learning rate of 0.05) to identify factors affecting the clinical pregnancy rate. Statistical analysis was performed to compare clinical pregnancy rates between the groups. Main results and the role of chance Our machine-learning model revealed that hCG injection (512 processes vs. 1739 processes without it) at the onset of progesterone administration is an important feature for the clinical pregnancy rate. The performance accuracy of the model was approximately 70%. Of the 1,294 protocols where patients received 300 mg/day progesterone, 311 included hCG injection while 983 did not. The clinical pregnancy rate was significantly higher in the hCG group than that in the control group (54% vs. 46%, P < 0.05), with 167 clinical pregnancies observed in patients receiving hCG injections against 452 pregnancies without it. In contrast, among the 957 protocols where 600 mg/day progesterone was administered, the previously observed effect was not significant (P > 0.05). Comparing 201 processes with hCG injection to 756 processes without it, the incidence of clinical pregnancies was 46.8% and 47.2% (with 94 and 357 confirmed pregnancies), respectively. Other factors such as the morphology grade of the embryo used for transfer, patient’s age, AMH, BMI, total number of transfers performed on the patient, menstrual cycle day on which progesterone was started, occurrence of recurrent implantation failure, and the use of preimplantation genetic testing were not significantly different between the groups. Limitations, reasons for caution This study focused on frozen embryo transfers and validation across other protocols using fresh embryo transfers, and different doses or routes of hCG and progesterone administration are needed. The impact of hCG administration on patients with various causes of infertility should also be explored. Wider implications of the findings Pre-administration of hCG injection along with a daily dose of 300 mg/day progesterone enhanced clinical pregnancy rates for frozen embryo transfer. This holds the potential for the optimization of hormon supplementation protocols to increase overall success rates in assisted reproductive technologies. Trial registration number not applicable

P-557 The contribution of PGT-A to PGT-M and PGT-SR clinical outcomes – A single centre retrospective analysis

Abstract Study question To determine if the introduction of concurrent testing with PGT-A has improved clinical outcomes for PGT-M/SR patients. Summary answer This study demonstrates that the introduction of concurrent testing may have improved clinical outcomes for this patient demographic irrespective of age. What is known already Preimplantation Genetic Testing (PGT) is utilised to investigate the genetic constitution of embryos. PGT-M (monogenic) and PGT-SR (structural rearrangements) are screening techniques considered in treatment for couples at risk of conceiving offspring with known genetic or structural chromosomal abnormalities respectively. Preimplantation Genetic testing for Aneuploidy (PGT-A) was developed in order to screen embryos for numerical chromosomal abnormalities, a leading cause of failed implantation. Nevertheless, routine use of PGT-A is discouraged due to lack of evidence regarding its efficacy. However, PGT-A is routinely used alongside PGT-M and PGT-SR despite the lack of evidence of its effectiveness in improving patient outcomes. Study design, size, duration The retrospective analysis of 496 PGT-M/SR single embryo transfer patient cycles between December 2013 and December 2021. Participants/materials, setting, methods The retrospective analysis of 496 PGT-M/SR single embryo transfer cycles at CRGH Portland was undertaken (single testing n = 77; concurrent testing n = 419). The clinical outcomes (positive urine pregnancy test, clinical pregnancy, live birth and miscarriage rates) were compared between groups using IBM SPSS Statistics (premium 27). Binary logistic regression was performed for statistical analysis, results with p < 0.05 were determined statistically significant. Main results and the role of chance Overall, concurrent testing was associated with statistically significant improved clinical outcomes compared to the reference group. Statistical significance was seen in positive urine pregnancy test (49% vs 68%, p = 0.012), clinical pregnancy (32.5% vs 63%; p = 0.000013), live birth rate (32.5% vs 59%; p = <0.00016) and miscarriage rates (34.2% vs 13%; p = 0.001). The results imply that the introduction of concurrent testing has improved the probability of obtaining a positive clinical outcome and has reduced the chance of miscarriage in this patient demographic. Further analysis showed concurrent testing improved the chances of obtaining a positive clinical outcome irrespective of the patients age or quality of the embryo. Limitations, reasons for caution This study has produced promising results. However, there are several limitations such as the small sample size, embryo biopsy techniques used, and the different molecular diagnostic techniques and providers utilised. Wider implications of the findings The results imply a justification for the use of PGT-A alongside PGT-M/SR screening techniques to improve embryo selection and clinical outcomes for this cohort of patients. Additionally, they demonstrate the efficacy of PGT-A in a previously underrepresented demographic. However, further research is required to confirm the clinical value of PGT-A. Trial registration number not applicable

P-278 The impact of previous pituitary suppression protocols on IVF outcomes in patients with endometriosis undergoing freeze-all approach

Abstract Study question Do embryological parameters and live birth rates differ in patients with endometriosis undergoing freeze-all approach when GnRH-antagonist protocol or progestin primed pituitary suppression(PPOS) was preferred? Summary answer When comparing the GnRH antagonist protocol, the PPOS protocol was associated with more usable cleavage stage embryos but with a similar live birth rate. What is known already Optimal COH protocol for patients with endometriosis is still controversial. Initially, the GnRH agonist protocol was hypothesized to be superior because it was assumed to increase pregnancy rates by improving the inflammatory process of endometriosis. However, available data represented similar pregnancy rates in GnRH antagonist and GnRH agonist protocols. Recently, progestins have been used for suppression of endogenous LH surge instead of GnRH analogs in COH for various indications. Since, recent data indicates that utilization of progestins for more than eight days can reduce the inflammatory process of endometriosis, PPOS protocol might be employed in this patient group. Study design, size, duration This retrospective cohort study conducted at Bahçeci Health Group between January 2021-January 2023. Inclusion criteria were female age between 20-44 years, patients with endometrioma diagnosed by transvaginal ultrasonography and freeze-all cycles. Exclusion criteria were pre-implantation genetic testing (PGT) cycles, patients with uterine myoma distorting the endometrial cavity, congenital uterine anomaly and presence of hydrosalpinx.Each patient was included only once. A total of 607 patients;407 in GnRH antagonist group and 200 in PPOS group were recruited. Participants/materials, setting, methods The study analyzed the embryological parameters and live birth rates undergoing freeze-all cycles. COH was performed with recombinant FSH or HMG at a starting dose 150-300 IU depending on ovarian reserve and body mass index (BMI). LH surge was controlled by a flexible GnRH antagonist or fixed PPOS protocol. Primary outcome for embryological parameters were usable cleavage embryo after OS and live birth after the first frozen embryo transfer (FET) cycles. Main results and the role of chance Female age in PPOS group was older than GnRH antagonist group (33.3±4.9 vs.32.4±4.8,p=0,02).Other demograhic characteristics were similar among two groups.Total gonadotrophin dose and duration of stimulation in PPOS group were lower than antagonist group (2602.3±1083.5 vs.2866.9±1147.9 IU, p = 0,002 and 10.86±1.77 vs. 11.59±1.82 days, p < 0.001). No significant difference regarding endocrinological parameters of OS and number of oocytes retrieved, mature oocyte, maturation and fertilization rates was found in two groups. However, the number of day 3 usable embryos were higher in the PPOS group than GnRH antagonist group (4.66±4.38 vs. 3.84±3.72 p = 0.04). There was no difference in the cancellation rates. A total of 478 FET cycles of 607 were evaluated; 323 in GnRH antagonist and 255 in PPOS group. No significant difference in FET cycle and embryological characteristics was found in two groups. The clinical pregnancy rate, miscarriage rate, live birth rate and multiple pregnancy rates per transfer were comparable between the two groups [59.1% (191/323) vs. 59.4% ( 92/155) p = 0,963;17.3%(33/191) vs 0.9%(10/92) p = 0,16; 48.6%(157/323) vs. 52.9% (82/155) p = 0,379; 7.3%(14/191) vs.10.9% (10/92) p = 0317, respectively].Multivariate binary regression analysis revealed that female age, embryo day embryo quality.number of embryos transferred were significant independent factors for live birth but not the PPOS vs GnRH antagonist. Limitations, reasons for caution Although we used regression models to adjust all possible confounders, it was a retrospective study with its intrinsic limitations. Further prospective RCTs are warranted. Wider implications of the findings Although cleavage stage usable embryo was significantly higher in PPOS group,the pregnancy outcomes were comparable between groups.Those results suggest that theoretical advantages of progestin in preceding OS does not seem to present a significant difference on live birth,but should be confirmed with prospective trials within cohort of women with endometriosis. Trial registration number not applicable ( retrospective study)

O-121 Assisted Reproductive Technology (ART) in Europe 2021 and development of a strategy of vigilance: Preliminary results generated from European registers by the ESHRE EIM Consortium

Study question What are the reported data on cycles in ART, IUI and fertility preservation interventions in 2021 as compared to previous years and what are the main trends over the years? Summary answer The 25th ESHRE report on ART and IUI shows a progressive increase in reported treatment cycle numbers in Europe, a small decrease in the number of transfers (IVF + ICSI) with more than one embryo with a trend to decreasing multiple delivery rates, outcomes for IUI cycles are similar to previous years. What is known already Since 1997, ART aggregated data generated by national registries, clinics or professional societies have been collected, analyzed by the European IVF-monitoring Consortium (EIM) and reported in 23 manuscripts published in Human Reproduction and Human Reproduction Open. Study design, size, duration Yearly collection of European medically assisted reproduction (MAR) data by EIM for ESHRE. The data on treatments performed between January 1 and December 31, 2021, in 34 European countries were provided by either National Registries or registries based on personal initiatives of medical associations and scientific organizations. Participants/materials, setting, methods In all 1155 clinics offering ART services in 34 countries reported a total of 910 470 treatment cycles, involving 115 567 with IVF, 346 183 with ICSI, 311 487 with frozen embryo replacement (FET), 62 694 with preimplantation genetic testing (PGT), 69 883 with egg donation (ED), 246 with IVM of oocytes and 4410 cycles with frozen oocyte replacement (FOR). European data on IUI using husband/partner’s semen (IUI-H) and donor semen (IUI-D) were reported from 1242 institutions offering IUI in 29 and 21 countries, respectively. A total of 139 350 treatments with IUI-H and 45 532 treatments with IUI-D were included. A total of 28 692 fertility preservation (FP) interventions from 15 countries including oocyte, ovarian tissue, semen and testicular tissue banking in pre-and post-pubertal patients were reported. Main results and the role of chance In total, 1155 IVF clinics participated (81.4% of registered clinics in the participating countries). Next to these also 1242 IUI units reported their data. In the 34 reporting countries, after IVF the clinical pregnancy rates (PR) per aspiration and per transfer in 2021 were similar to those observed in 2020 (26.2% and 43.3% versus 25.4% and 40.9%, respectively). After ICSI the corresponding rates were also similar to those achieved in 2020 (23.9% en 45.7% versus 25.2% and 45.3%). Also after FET with own embryos, the PR per thawing is stabilizing, 35.0% in 2020 and 36.2% in 2021. After ED the PR per fresh embryo transfer was slightly increased to 53.1% (51.3% in 2020) and per FOR 45.5% (43.9% in 2020). In IVF and ICSI together, the trend towards the transfer of fewer embryos continues with the transfer of 1, 2, 3 and ≥4 embryos in 60.2%, 34.8%, 1.8% and 0.2% of all treatments, respectively (corresponding to 57.9%, 37.5%, 2.1% and 0.2% in 2020). This resulted in a proportion of singleton, twin and triplet DRs of 90.4%, 9.5% and 0.1%, respectively (compared to 88.9%, 10.9% and 0.2%, respectively in 2020). Treatments with FER in 2021 resulted in twin and triplet DR of 7.0% and 0.1%, respectively (versus 7.9% and 0.1% in 2020). After IUI, the DRs remained similar at 9.2% after IUI-H (8.8% in 2020) and at 14.0% after IUI-D (12.5% in 2020). Twin and triplet DRs after IUI-H were 7.4% and 0.2%, respectively (in 2020: 7.9% and 0.4%) and 5.9% and 0.2% after IUI-D (in 2020: 5.7% and 0.1%). The majority of FP interventions included the cryopreservation of oocytes (n= 11 966 from 15 countries) and of ejaculated sperm (n=14 688 from 15 countries). Limitations, reasons for caution As the methods of data collection and levels of completeness of reported data vary among European countries, the results should be interpreted with caution. For this abstract, some countries were not able to provide adequate data about the number of centers and initiated cycles and deliveries. Wider implications of the findings The 25th ESHRE report on ART and IUI shows a continuous increase of reported treatment numbers and MAR-derived live births in Europe. Being already the largest data collection on MAR in Europe, continuous efforts to stimulate data collection and reporting strive for future quality control and completeness of the data and offer higher transparency and vigilance in the field of reproductive medicine.

P-428 Identifying key factors for achieving biochemical pregnancy in first autologous IVF cycles: an artificial intelligence-assisted approach

Abstract Study question What key factors determine a positive biochemical pregnancy outcome in first autologous IVF cycles, as identified by machine learning (ML) algorithms? Summary answer Maternal age, AMH, BMI, number of day 3 embryos and total embryos transferred emerged as top predictors for biochemical pregnancy across several ML methods. What is known already ML methods are powerful tools that discern patterns from multiple features to predict outcomes. Beyond their clear predictive capabilities, ML algorithms also offer valuable clinical insights by uncovering underlying data trends. These insights come from understanding the algorithms’ decision-making processes, revealing the rationale behind their predictions. While maternal age is known to influence IVF success, other pre-treatment and in-cycle factors also play a significant role. Understanding these factors can enhance patient management and guide clinical advice and interventions. Study design, size, duration This multicenter retrospective study included data from 9,945 cycles performed between January 2010 and July 2021. Only first autologous cycles using fresh oocytes and donor or patient sperm (fresh or frozen) were included. Preimplantation genetic testing (PGT) cycles were excluded. Both pre-treatment and in-cycle variables were considered, with treatment outcome expressed as cumulative biochemical pregnancy. Cases and variables deemed to have poor data quality were excluded. The final dataset comprised 80 variables and 7350 cycles. Participants/materials, setting, methods Five ML classifier algorithms were applied to predict cumulative biochemical pregnancy using pre-treatment and in-cycle variables. The models tested included logistic regression, support vector machine, random forests, gradient boosting and multilayer perceptron. Shapley values analysis was performed to explain the decision-making structures of all the algorithms tested, quantifying the impact of each included feature on outcome prediction. Main results and the role of chance Within our cohort, mean maternal age was 37.3±4.7 years, mean serum AMH was 2.4±2.1 ng/ml, mean BMI was 23.7±4.0 and mean antral follicle count (AFC) was 11.6± 7.6. Notably, 40.1% of cycles used donor sperm, while 33.8% of male patients were normozoospermic. The majority of cycles (90.8%) used antagonist stimulation protocols, yielding an average of 7.8±5.5 oocytes per patient. Fertilization rate was 70.6%, with a mean of 2.7±1.8 usable embryos obtained per cycle. Fresh embryo transfers accounted for 73% of cases, with an average of 1.5±0.9 transfers performed per cycle. The embryo utilization rate was 64.6% and 48.1% of cycles resulted in a biochemical pregnancy. Among the evaluated algorithms, the gradient boosting model demonstrated superior performance, with an accuracy of 70.6±1.4% and AUC of 77.3±1.7%. Shapley values across all five algorithms consistently revealed the negative correlation between maternal age and biochemical pregnancy rates. Among pre-treatment variables, AMH levels had a positive influence on outcomes, followed by BMI, AFC, and parity status. Regarding in-cycle variables, the number of embryos available at day 3 and the total number of embryos transferred, positively affected the chances of pregnancy. The number of mature oocytes was also an important in-cycle predictive feature. Limitations, reasons for caution his retrospective study spans an extended timeframe, suggesting potential variations in clinical and laboratory practices over the years. Therefore, caution is advised in generalizing the results to other patient populations or to contemporary data gathered using newer and enhanced clinical protocols. Wider implications of the findings The consistent identification of maternal age, ovarian reserve markers, and embryo quantity as primary predictors of pregnancy by the five AI models corroborates existing clinical understanding. This consistency emphasizes the strength and dependability of AI to reinforce and complement clinical knowledge with data-driven evidence. Trial registration number not applicable