IVF Clinic Research Articles

P-304 Does imaging affect embryo health? Comparative analysis of discrete wavelengths of light on the developing embryo

Abstract Study question What is the effect of exposing the embryo to discrete wavelengths of light on preimplantation development and resultant offspring health? Summary answer Exposure of embryos to red or yellow wavelengths negatively impacted embryo health, pregnancy rate and resulted in offspring that were heavier at weaning. What is known already Previous studies have indicated a potential negative impact of shorter wavelengths of light on embryo health. Red and yellow wavelengths are widely considered benign and utilised clinically in time-lapse equipped incubators within IVF clinics. However previous studies had not uniformly and correctly irradiated embryos to enable a fair comparison between different wavelengths. Study design, size, duration A current aim of the field is to use optical imaging to predict embryo developmental potential. Such approaches use varying wavelengths of light. The impact of irradiating the embryo with discrete wavelengths of light is not fully understood. Here, we assess the impact of various wavelengths on the developing embryo and for the first time, ensured that the energy dose applied was consistent between wavelengths, thus mimicking fluorescence and time-lapse imaging (470 – 620 nm). Participants/materials, setting, methods Preimplantation mouse embryos were exposed daily to blue (470 nm), green (520 nm), yellow (590 nm) or red (620 nm) wavelengths and compared to embryos that were not exposed. We assessed embryo development, DNA damage, and postnatal outcomes following transfer to pseudopregnant recipients. Main results and the role of chance We found exposure to the yellow wavelength significantly impaired embryo development to the blastocyst stage (P < 0.05). While exposure to blue, green and red wavelengths resulted in significantly higher levels of DNA damage when compared to unexposed embryos (P < 0.05). The pregnancy rate was significantly lower when embryos were exposed to the red wavelength (P < 0.05). Interestingly, resultant offspring were significantly heavier when derived from red or yellow light exposed embryos compared to those derived from unexposed embryos (P < 0.01). Towards understanding the effect on offspring weight we assessed intracellular lipid abundance in the embryo. We found lipid abundance to be significantly elevated following exposure to yellow wavelength (1.8-fold, P < 0.0001) but not red. We believe that the role of chance is low as results were collected from multiple independent experimental replicates that were tested using appropriate statistical analyses. Limitations, reasons for caution While we demonstrate the distinct impacts of discrete wavelengths of light on the developing mouse embryos including post-natal effects, confirmation of these results in human embryos is required. Wider implications of the findings Red and yellow wavelengths are utilised clinically in time-lapse equipped incubators within IVF clinics. Our results demonstrate the potential need to re-evaluate these assumptions. Mapping the stress tolerance embryos show for each wavelength may be advantageous in identifying how damage can be mitigated in clinical manipulation and imaging techniques. Trial registration number not applicable

O-153 Assisted Reproductive Technology (ART) in Europe 2020 and development of a strategy of vigilance: Preliminary results generated from European registers by the ESHRE EIM Consortium

Abstract Study question What are the reported data on cycles in ART, IUI and fertility preservation interventions in 2020 as compared to previous years and what are the main trends over the years? Summary answer: author The 24rd 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 2020 in 35 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, 1157 clinics offering ART services in 35 countries reported a total of 713 708 treatment cycles, involving 110 097 with IVF, 253 095 with ICSI, 239 759 with frozen embryo replacement (FET), 46 610 with preimplantation genetic testing (PGT), 59 906 with egg donation (ED), 344 with IVM of oocytes and 3897 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 1176 institutions offering IUI in 28 and 18 countries, respectively. A total of 102 702 treatments with IUI-H and 36 476 treatments with IUI-D were included. A total of 18 270 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, 1157 IVF clinics participated (88.5% of registered clinics in the participating countries). Next to these also 1176 IUI units reported their data. In the 35 reporting countries, after IVF the clinical pregnancy rates (PR) per aspiration and per transfer in 2020 were similar to those observed in 2019 (27.9% and 32.9% versus 28.5% and 34.6%, respectively). After ICSI the corresponding rates were also similar to those achieved in 2019 (24.3% en 32.2% versus 26.2% and 33.5%). After FET with own embryos, the PR per thawing is stabilizing, 35.1% in 2019 and 34.6% in 2020. After ED the PR per fresh embryo transfer was 50.4% (50.5% in 2019) and per FOR 45.3% (44.8% in 2019). 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 62.1%, 32.3%, 2.2% and 0.3% of all treatments, respectively (corresponding to 55.4%, 39.9%, 2.6% and 0.2% in 2019). This resulted in a proportion of singleton, twin and triplet DRs of 90.5%, 9.3% and 0.2%, respectively (compared to 87.7%, 12.0% and 0.3%, respectively in 2019). Treatments with FER in 2020 resulted in twin and triplet DR of 7.0% and 0.1%, respectively (versus 9.3% and 0.1% in 2019). After IUI, the DRs remained similar at 8.9% after IUI-H (9.2% in 2019) and at 12.4% after IUI-D (12.1% in 2019). Twin and triplet DRs after IUI-H were 8.3% and 0.4%, respectively (in 2019: 8.7% and 0.3%) and 5.8% and 0.2% after IUI-D (in 2019: 6.2% and 0.2%). The majority of FP interventions included the cryopreservation of oocytes (n = 5 365 from 14 countries) and of ejaculated sperm (n = 11 571 from 14 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 24rd 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. Trial registration number XXXX

P-403 Oocyte usage and disposition outcomes following oocyte cryopreservation

Abstract Study question What is the fate of cryopreserved oocytes? Summary answer Very few people return to use their cryopreserved oocytes in assisted reproduction and while some surplus oocytes are donated for reproduction, the majority are discarded. What is known already The number of oocyte cryopreservation cycles performed around the world dramatically outpaces the number of people who return to use their oocytes in assisted reproduction. This imbalance is leading to the rapid accumulation of oocytes in cryostorage. Previous studies have reported that many people intend to donate their surplus cryopreserved oocytes, and some commentators suggest that this may help relieve donor shortages. However, there have been no reports to date about what actually happens to surplus oocytes following oocyte cryopreservation. Study design, size, duration A retrospective cohort study was performed, analysing the outcomes for cryopreserved oocytes from 2012 - 2022. Participants/materials, setting, methods Data were collected from seven IVF clinics, located in Victoria, Australia. De-identified data were aggregated on the outcomes following oocyte cryopreservation, including duration of cryostorage, assisted reproduction involving thawed oocytes, and disposition outcomes of surplus oocytes. Main results and the role of chance Across the study period, the number of patients with oocytes in storage grew rapidly from 144 in 2012 to 2015 in 2022. Regarding the current status of oocytes in storage, in 2022, 73% of patients had had their oocytes in storage for <5 years; 25% for 5 - 10 years, and 2% for ≥10 years. The majority of oocyte thaw cycles (600/645) involved oocytes frozen for <5 years; of which 47% were frozen for <6 months. Overall, the live birth rate from the total number of oocyte thaw cycles initiated was 12% (78/645), however, this varied depending on the reason oocytes were frozen. In comparison to the number of patients still with oocytes in storage, very few patients relinquished surplus oocytes across the study period (2015 vs 151, respectively). Of the 128 patients whose surplus oocytes were discarded, 32% had had their oocytes stored for <5 years; 32% for 5-10 years, and 36% for >10 years. Among patients who elected to donate their surplus oocytes (n = 23), all but four patients made this decision having had their oocytes in storage for <5 years. Although a legal option in Australia, no oocytes were donated to research over the period analysed. Limitations, reasons for caution Data were collected from one fertility group based in one state of Australia, thus it may not be representative of wider trends seen across other clinics or states/territories. Further, specific state laws govern the management, storage, and use of surplus oocytes, which may influence the reported disposition outcomes. Wider implications of the findings Our findings suggest that cryopreserved oocytes are rarely used or donated, rather they are left in storage, often for long durations. To relieve some of the pressure this may create on cryostorage facilities, more attention is needed to better understand the experience of oocyte disposition and the barriers to donation. Trial registration number not applicable

P-495 What is the optimal cut off value of the number of egg donations from the same egg donor lifetime?

Abstract Study question Do repeated ovarian stimulation cycles in the same donor have a negative impact on clinical outcome? Summary answer There is no negative impact of the consecutive ovarian stimulation cycles on embryological parameters and outcomes from the same donor. What is known already Different country regulations required from single stimulation to maximum 6 stimulations for egg donors in lifetime. Modern assisted reproductive technologies (ART) clinics should consider this issue described above from the point of view of the number of oocytes retrieved during controlled ovarian stimulation cycles - this is a balance between efficacy of the ART in infertility treatment programs and donor safety. Despite some studies have underscored the likely negative impact of repeated cycles on outcomes it remains up for debate. The age groups of 20-30 years old egg donors may give similar clinical outcomes for 1st-6th attempt of stimulation cycles. Study design, size, duration In this single center, 4 years of retrospective study included oocyte donors (n = 96) with repeating cycles of ovarian stimulation (COS). Cycles took place between January 2018 and December 2022 in an IVF clinic with respect to at least 6 months interim between each cycle. We evaluated the concordance between the follicle-to-oocyte index (FOI), number of oocytes retrieved, maturation rate (MR), utilization rate (UR) and the numbers of attempts from 1st to 6th. Participants/materials, setting, methods The mean age of the patients in this study was represented in dynamics (SD) from 1st COS program 23,2 (3,2) to 27,8 (3,7) years during the final 6th cycle, all of them passed through 6 consequence COS programs.. The association between each COS cycle' parameters and the differences in COS output was assessed by T-Student (parametric) or U-Mann-Whitney (non-parametric). Main results and the role of chance We observed that mean number (SD) of mature oocytes retrieved was non significantly different and raised from the 1st attempt –18,46 (8,89), to 5th - 22,03 (10,16), with decreasing during 6th COS – 21,55 (12,06). MR and UR indicators had a fluctuating picture of the dynamics of values. They either increased or decreased from one cycle to another. Mean values of MR was the lowest after 3rd COS (81%) and the highest – 4th COS (84%). UR dynamic was similar with lowest value after 1st cycle (12,0%) and the maximum 17,9% after 6th COS program. FOI parameter was measured within values of 73,82 – 81,02: min value 73,82 (16,40) was observed after the second stimulation cycle and max value – after fifth (mean = 81,02±18,26). These changes were found to be not significantly different (p > 0.05). Regarding egg donation cycle outcome for recipients (age average 34,57) attempt 1st −3rd gave 96,14% fertilization rate, 66,89% used blastocysts rate and 60,77% clinical pregnancy rate. Recipients ( average age 33,19) attempting 4th- 6th gave 94.17% fertilization rate, 64,09% used blastocysts rate and 63,19% clinical pregnancy rate. Limitations, reasons for caution This study is limited regarding retrospective nature and by the small samples size. Expanding the number of participants is the possible next step, in order to challenge or rather confirm our preliminary findings. Wider implications of the findings The controversial dynamic of the changes of the embryological parameters and COS outcomes in the same donor’s pool after repeated stimulation cycles with no statistically observed differences can be grounds for making a positive decision both personally by the donor and the IVF specialist. Trial registration number NA

O-296 Progesterone level is an alternative marker to detect ovulation time in natural cycle frozen-thawed embryo transfers

Abstract Study question Do serum progesterone or LH levels better predict the optimal timing for embryo transfer in true natural cycle frozen-thawed embryo transfers (NC-FET)? Summary answer Performing embryo transfer according to serum progesterone levels (PL) had similar ongoing pregnancy rates compared to embryo transfer according to serum LH levels. What is known already With efficient and safe embryo vitrification techniques, there is an increasing trend in frozen-thawed embryo cycles (FET) over fresh cycles. FET protocols are programmed cycle, NC-FET and modified NC-FET. NC-FET is increasing in popularity, since it is associated with favourable obstetric and perinatal outcomes. In NC-FETs, ovulation time is the critical parameter for synchronization of embryo and endometrium. In the literature, LH surge is the most commonly used test to define ovulation time. Study design, size, duration This prospective cohort study included 183 NC-FET cycles in an IVF clinic between March 2022 and November 2022. Main outcome measure is ongoing pregnancy. Participants/materials, setting, methods 183 subfertile women aged between 18-40, having regular menstruation with a cycle length between 24-38 days are included. Protocol for NC-FET included serial hormone measurements and ultrasound monitoring when the dominant follicle reached the diameter of 15-16 mm. Serum LH level ≥15 IU/l defined as ‘ovulation -1’ in Group 1 and blastocyst embryo transfer was performed after 6 days. Serum PL > 1 ug/ml was defined as the ‘ovulation day’ in Group 2. Main results and the role of chance: There were no significant differences in baseline characteristics including female age, female body mass index, infertility duration, infertility diagnosis and number of failed IVF cycles between two groups. Also, cycle characteristics regarding endometrial thickness, number of embryos transferred, embryo quality and PLs at transfer day were similar. The overall clinical pregnancy was 59.6% and ongoing pregnancy was 50.8%. The implantation rate was 61.7%. The clinical pregnancy and ongoing pregnancy of Group 1 and Group 2 were 49.5%, 50.5% and 47.3%, 52.7%, respectively (p > 0.05). PLs in order to detect ovulation time were calculated according to a mathematical modelling (PL(Ov )=a1 eb1 Ov )(Ov:ovulation day; a1,b1:regression coefficients; e:euler number≌2.718) described in our previous study. Embryo transfer timing was planned after 5,4 or 3 days based on PLs calculated by modelling. Limitations, reasons for caution Since this is the first study planning embryo transfer in NC-FET based on progesterone levels, sample size should be increased and also serial change of progesterone levels were unknown in women >40 years of age. Wider implications of the findings Progesterone has a particular increasing pattern that enables ovulation day prediction accurately and may be used instead of serum LH levels in timing of embryo transfer. Trial registration number NCT05690360

P-242 Genetic laboratories report significantly different aneuploidy rates for identical patient populations, a finding with important implications for clinics using PGT-A

Abstract Study question If a clinic changes the genetic laboratory it works with, should it expect its new PGT-A results to be equivalent to those it received previously? Summary answer Changing the genetic service provider can result in a significant difference in the proportion of embryos classified euploid, with important clinical implications. What is known already Preimplantation genetic testing for aneuploidy (PGT-A) aims to distinguish potentially viable euploid embryos from embryos harbouring lethal chromosome abnormalities. Typically, IVF clinics perform biopsy at the blastocyst stage and send the resulting trophectoderm specimens to specialist genetic laboratories for analysis. However, many commercially available genetic methods have not been subjected to rigorous validation, leading to uncertainty about their accuracy and predictive value. It would be concerning if the classification of embryos, derived from the same patient population, and from the same clinic, varied depending on the company that provides PGT-A services. Such differences would be unlikely to reflect biological reality. Study design, size, duration Our clinic recently began working with a new genetics company. Our previous PGT-A reference laboratory issued results for 1,136 blastocyst biopsy specimens over ∼15 months. Subsequently, the second company tested 784 biopsy samples in 7 months. Patient populations during the two periods were essentially identical (average maternal age 38.0 years). Participants/materials, setting, methods The first company used whole genome amplification followed by next generation sequencing (NGS) to evaluate the relative amount of DNA from each chromosome. The second company’s method was highly validated, involving targeted amplification of thousands of sites in the genome, again followed by NGS and analysis of relative DNA quantity from individual chromosomes. Additionally, they genotyped numerous polymorphisms, which assist in the detection of haploidy/triploidy, and also reveals problems that can compromise accuracy (e.g. contamination). Main results and the role of chance The first PGT-A company classified 44.4% of blastocysts euploid, 4.1% low-level mosaic, 51.5% aneuploid. In contrast, the second company reported significantly fewer embryos aneuploid (44.0%; P = 0.0019). Even if the mosaics reported by the first company are considered for transfer, the second company is still associated with more potentially transferable embryos (relative increase greater than one-sixth). If PGT-A results from the second company are correct, it implies that potentially viable embryos may have been misclassified by the first company, risking their exclusion and loss of the pregnancies they might have produced. Conversely, if the first company is correct, the second company may be failing to detect some aneuploidies, leading to inadvertent transfer of abnormal embryos, lower pregnancy rates and a higher incidence of miscarriage. 299 transfers following PGT-A using the first company produced 171 pregnancies (74.7%), while 64 single embryo transfers have taken place after PGT-A with the second company, resulting in 53 pregnancies (82.8% per transfer). Losses (biochemical or miscarriage) affected 21.6% and 16.9% of pregnancies after PGT-A conducted by the first and second companies, respectively (ongoing pregnancy rates of 58.5% and 68.8% per transfer, respectively) Thus, there is no evidence that aneuploidies are being missed by the second company. Limitations, reasons for caution Although there were no obvious differences between the patients with embryos tested by the two companies, this study was not prospective nor randomized, so a possibility of undetected differences remains. The study was not sufficiently powered to test the significance of apparent differences in implantation, miscarriage and ongoing pregnancy rates. Wider implications of the findings It is important that IVF clinics appreciate that individual PGT-A methods differ significantly, and that some have undergone much more rigorous validation than others. It is recommended that clinics ask PGT providers to share their validation data, especially clinical studies confirming that embryos classified ‘aneuploid’ or ‘abnormal’ are truly non-viable. Trial registration number Not applicable

P-087 Expanding the comparison of an AI automated semen analyses for concentration determinations with manual analyses and a cell counting system

Abstract Study question Can comparable results be obtained for sperm concentrations determinations in differing clinical settings when using an AI driven analyser, a cytometer, and WHO manual counts? Summary answer Outcomes suggest that comparable results can be obtained when utilising the differing counting techniques. What is known already Despite robust methods described by WHO, protocol variations exist, creating a lack of reproducible and consistent semen analysis results in standard laboratories, as described by Bjorndahl etal 2016. Human error can influence results where WHO protocols are not utilised in conjunction with rigorous IQA and EQA. A human performing an analysis is considered the gold standard, but it's apparent, as demonstrated by time-lapse embryo evaluation, that human assessments can be enhanced with machine assistance to increase accuracy. To establish the benefit of machine assistance, it is necessary to demonstrate comparable results with the manual operator and reliable machine cell counters. Study design, size, duration To assess whether comparable results could be obtained with a well-trained reproductive scientist and a cell counting system, two reputable IVF clinics were selected to perform their standard analyses and perform a repeat test with an automated analyser. A number of semen analyses were performed over a six-month time-period for patients attending IVF clinics for a pre-treatment evaluation. Participants/materials, setting, methods 107 samples were collected and analysed between June 2022 to December 2023 in laboratories in Copenhagen and London. Participants were selected on a random basis. The semen analyses were performed in a manner described by the WHO6, with an additional assessment with a NucleoCounter™ chemometec in the Danish laboratory. Simultaneously, a concentration determination was performed on a Mojo AISA ™. (Artificial Intelligence Semen Analysis system). Main results and the role of chance Manual concentration assessments, the cytometer data and the AISA concentration results were plotted and examined. Comparable values were obtained when data for the manual assessment v AI machine assessment and the data obtained for fluorescent marked sperm v AI analysis were assessed. A significant Spearman correlation was obtained by the manual analysis team and the team who used the cytometer counting system, values of 0.93 and 0.99, (error average 17.5 and 24.4) respectively. Limitations, reasons for caution Increasing the sample frame of this current study, including more laboratories for a multi-centre trial would increase the power of the findings. Inter and intra operator and repeat machine evaluations are planned to establish the human variations during analysis and establish if this can be mitigated by machine assistance. Wider implications of the findings The results demonstrate that a machine assisted semen analysis is comparable to a gold standard semen analysis performed by a reputable laboratory staff either when compared to a manual operator and to a cell sorting system where sperm are labelled with a fluorescent probe. Trial registration number N/A

P-603 Viability of home monitoring of estrone-3-glucuronide (E1-3G) urine levels in controlled ovarian stimulation: A pilot study

Abstract Study question Can the regular determination of E1-3G levels in the patient's urine be used for successful monitoring of progestin-primed ovarian stimulation protocol (PPOS)? Summary answer Regular determination of E1-3G levels in urine can be successfully used in ovarian stimulation (OS), significantly reducing patient visits to the clinic. What is known already To reduce direct and indirect costs and to optimize the workload in IVF clinics, innovative approaches are being sought. It has only recently become possible to implement elements of telemedicine in OS monitoring. Patients have begun performing vaginal sonograms on their own. Studies have further shown that there is a close correlation in the growth of E2 in serum and E1-3G in urine during the menstrual cycle and ovarian stimulation. Thus, theoretical models for the use of urinary E1-3G in ovarian stimulation were developed, which are subject to experimental verification and analysis. Study design, size, duration This is a pilot study carried out in a clinic, which aims to establish the growth rate of E1-3G and to determine its upper and lower limits of growth on each consecutive day at which stimulation is adequate and safe. Inclusion criteria were patients aged < 40 years and AMH ≧ 1.1 ng/mL. From September to December 2022, in total 28 first ovarian stimulatiоn cycles from 28 infertility patients were included. Participants/materials, setting, methods A PPOS protocol with fixed doses of gonadotropins was used. From the second day of stimulation, the patients determined the E1-3G levels in their urine every day at home with a portable analyzer. In parallel, a standard ultrasound follow-up protocol was applied, accompanied by determination of E2, LH and P levels, in order to optimally control stimulation. Patients' convenience of this new approach was assessed using a questionnaire developed for the purpose. Main results and the role of chance The average female age was 32,1 years (±4.4), BMI 22,9 kg/m2 (±4,3), AMH 3,9 ng/ml (±2,7), stimulation days 10,1 (±1,2), collected oocytes 12,6 (±8,5), MII oocytes 10,8 (±7,9), fertilization rate 83,4% (±22,7), blastocyst formation 66,9% (±28,6), good quality blastocysts 31,1% (±16,6). The log-linear mixed effect model (LLMM) estimation produced reasonable estimates of 49% average day-to-day growth rates (slope fixed effect), with one standard deviation (SD) range of 25% to 77% across patients (SD of the slope random effect). Moreover, there was a comparatively high correlation of 0.76 between the individual growth rates of E1G estimated over days 3-6 (the slope random effects of the LLMM model) and the E1-3G levels at day 10. In this way, the estimated slope random effects appear to have a prognostic value and may potentially have therapeutic implications, for example, adjustment of the stimulation dose. Moreover, the Spearman correlation between Estradiol and E1-3G was 0.83 After analyzing interviews and questionnaires, patients evaluated the applied method as easy and convenient, with 97% of them preferring OS monitoring to be performed in this manner compared to the standard method, which includes regular ultrasound examinations and determination of serum hormone levels. Limitations, reasons for caution The small statistical sample size is a limitation of this study, which was carried out in a single clinic. The results have to be confirmed in other clinics and with more diverse populations. Wider implications of the findings This is the first study to analyze the dynamics of E1-3G in the PPOS protocol and the limits of its increase during the entire OS. The results confirm our theoretical model for the successful use of urinary E1-3G in OS. The method is easy to apply and well-accepted by patients. Trial registration number N/A

O-290 Clinical utility of putative mosaicism detected using concurrent copy-number and genotyping PGT method: outcomes from multisite, prospective, non-selection study including 9828 single embryo transfer cycles

Abstract Study question What is the clinical utility and associated outcomes of mosaic whole chromosome or segmental aneuploidies detected using concurrent copy-number and genotyping analysis in PGT-A cycles? Summary answer Although high-level whole-chromosome mosaicism is linked to reduced sustained implantation, it has limited clinical significance in PGT-A cycles when co-evaluated with other clinical/ embryological factors What is known already NGS-based PGT-A can detect intermediate chromosomal copy number (CN), commonly interpreted as mosaic chromosomal aneuploidies in embryos. A prospective non-selection approach is the most effective way to assess the clinical utility of reporting putative mosaicism findings in PGT-A, wherein the presence of mosaicism is not disclosed and does not influence embryo selection. Conflicting results have been reported previously, possibly due to technological limitations in mosaicism assessment or retrospective analysis methods. This study reports the results of the largest multisite prospective non-selection clinical study examining the predictive value of whole-chromosome and segmental mosaicism, assessed through combined CN and genotyping data analysis. Study design, size, duration A multisite study involving seven IVF clinics was conducted from Feb 2020 to Oct 2022, including 6951 patients and 9828 single embryo transfers. The study involved a prospective non-selection approach, where embryos suspected of having whole chromosomal or segmental mosaicism were reported as negative for non-mosaic aneuploidies. Embryos were chosen for transfer based solely on standard morphological features. The primary outcome was sustained implantation rate (SIR) defined as pregnancy continuing beyond 8 weeks of gestation. Participants/materials, setting, methods In this study, the trophectoderm biopsies were analyzed using a custom, targeted NGS assay that examined approximately 5000 loci across the genome, providing genotyping information to support aneuploidy classification. Mosaicism was identified by any copy number deviation from the expected two copies (LogR plots) and confirmed by corresponding SNP B-allele frequency (BAF) patterns. Confounding factors, such as clinical and embryological variables, were controlled for in the multivariate analysis. Main results and the role of chance The average female age in this cohort was 34.9 years (SD = 4.1), with aneuploidy rate of 30% in embryos and SIR of 61.2%. Of the embryos transferred, 6.5% (636/9828) were whole chromosomal mosaic (WCM) only; 9.6% (947/9828) were segmental mosaic (SM) only and 1% (83/9829) were a combination of WCM and SM. The rate of putative mosaicism ranged from 15%-89%. The SIR of embryos in the control group (non-mosaic), SM and WCM were 62% (5190/8328; 95%CI), 58% (549/947;95%CI) and 50.3% (320/636; 95%CI P < 0.01) respectively. A logistic model found that the level of WCM was associated to SIR, along with other embryological and clinical factors. In particular, WCM with a CN difference >50% as well as poor embryo morphology were associated with lower SIR (OR = 0.5; 95% CI:0.32-0.76), but low-level (<50%) mosaicism was not significant (NS). Notably, female age (OR = 0.98; 95% CI:0.97-0.99 per year), BMI (0.98; 95% CI:0.98-0.99) and previous ET failures (OR = 0.58; 95% CI:0.5-0.68) were strongly associated with SIR. A predictive model, taking into account all relevant variables, yielded a significant stratification of SIR, from 43% to 68%. Given the low incidence of WCM in our clinical setting, the multivariate SIR prediction (AUC) was 0.580 without WCM and 0.585 with mosaicism included. Limitations, reasons for caution This study did not have prenatal and post-natal data available at the time of the abstract's writing, hence conclusions about these outcomes wasn’t possible. Despite the large sample size, chromosome specific analysis was not feasible. Furthermore, this study’s data is platform-specific and cannot be translated to other PGT-A assays. Wider implications of the findings In this non-selection study, WCM of > 50% variation was associated with lower SIR. However, high-level WCM has a minimal overall impact on SIR when co-evaluating with other clinical/ embryological parameters. Decisions on reporting criteria for these findings must weigh the risk of discarding potentially viable embryos with substantial reproductive potential. Trial registration number Not applicable

O-132 Comparison of pregnancy rates in antagonist cycles after luteal support with GnRH-agonist versus progesterone – prospective randomized study

Abstract Study question Are pregnancy rates in GnRH-antagonist cycles triggered with hCG and luteal phase support (LPS) with intranasal GnRH-agonist or vaginal progesterone comparable? Summary answer Nasal GnRH-agonist for LPS is associated with higher pregnancy rates compared with standard progesterone support in antagonist-based cycles triggered with hCG What is known already Regardless of its mechanism of action, the use of GnRH-a during the luteal phase in addition to progesterone, either in a single or multiple doses, has become a common practice as a part of ART protocols, based on its association with higher pregnancy and live birth rates . Its use as a sole agent for LPS has not gained the same popularity although studies supporting non-inferiority and even higher live birth rates with GnRH-a as a sole LPS agent have been published. Study design, size, duration A single-center, prospective, randomized study. A total of 150 patients underwent 164 GnRH-antagonist-based IVF cycles triggered with hCG and fresh embryo transfer. The study was conducted between June 9th, 2020, and May 5th, 2022. Participants/materials, setting, methods The study was conducted in the IVF clinic at University-affiliated tertiary medical center. Patients meeting the inclusion criteria who underwent antagonist-based IVF cycles and hCG triggering were enrolled. Computer-based randomization (1:1 ratio) was performed on the day of oocyte pickup (OPU). In both groups LPS was initiated on the evening of the OPU day with either GnRH-agonist nasal spray (Nafareline, nasal spray 200 mcg twice daily)or vaginal micronized progesterone (Utrogestan 300 mg, 3 times daily). Main results and the role of chance A total of 150 patients underwent 164 cycles, 127 cycles of which were included in the study cohort. Of them, 64 (50.4%) and 63 (49.6%) cycles were treated with GnRH-agonist or progesterone respectively as sole luteal phase support. A significantly higher pregnancy rate was found in the GnRH-a group compared with the progesterone group (43.8% versus 25.4% respectively; P = 0.030). After adjustment of several potential confounders such as age, body mass index (BMI), past obstetric history, number of IVF cycles, oocytes retrieved and embryos transferred, GnRH-agonist was still associated with a higher pregnancy rate (odds ratio 3.4, 95% confidence interval 1.4-8.3). Ovarian hyperstimulation syndrome (OHSS) rates were similar between the groups. Significantly higher progesterone levels were measured at β-hCG testing day in the GnRH-a group, both for the cleavage stage embryos (129.1±23.7 nmol/L vs. 97.7±48.9 nmol/L, P = 0.024) and for the blastocysts (127.2±0.1 nmol/L vs. 75.3±49.1 nmol/L, P = 0.034). Limitations, reasons for caution This study is not powered for analysis of clinical pregnancy rates, live birth rates and pregnancy outcomes. Larger studies are needed for research of these outcomes. All patients participating were at low risk for OHSS, therefore the study is underpowered to find significant differences in early or late OHSS.Wider implications of the findings Our findings suggest that intranasal GnRH-agonist administration as a sole luteal support in antagonist-based IVF cycles triggered with hCG results in higher pregnancy rates in comparison with traditional irritating vaginal preparations while not mandating additional support in the first weeks of pregnancy. Trial registration number Clinicaltrials.gov - NCT05484193

O-004 Male and female blastocysts display differences in development such that embryos assigned an identical morphological grade may have differing viability dependent on their sex

Abstract Study question Embryo selection methods based upon morphological or morphokinetic evaluation assume that male and female embryos have identical rates of preimplantation development, but is this true? Summary answer The distribution of trophectoderm grades differs for male and female blastocysts. Furthermore, male and female embryos of identical grade may have different probabilities of viability. What is known already Previous studies have suggested that male and female embryos may have subtle differences in their rates of preimplantation development. However, this possibility remains controversial. Apart from being of scientific interest, the question of whether the sex of an embryo can affect its growth trajectory is of clinical importance. Morphological grading, the primary method used by most IVF clinics when deciding which embryo to prioritise for transfer, assumes that developmental rates are independent of sex. Similarly, morphokinetic strategies for embryo evaluation, using data gathered from time-lapse incubators, are likely to be compromised if male and female embryos have differing developmental behaviour. Study design, size, duration 1,241 blastocysts underwent PGT-A and were shown to be euploid. Chromosome analysis also revealed the sex of the embryos, although this was not disclosed to patients. Standard morphological grading was carried out blindly with respect to the sex of the embryo. Information on clinical outcomes following embryo transfer was available for a subset of 336 embryos. Data was evaluated using various statistical methods to reveal any associations between embryo sex, morphology and clinical outcome. Participants/materials, setting, methods Embryos included in this study were derived from patients undergoing routine IVF with PGT-A. The only embryos excluded were those derived from patients carrying a monogenic disease mutation or a chromosome rearrangement. Embryos underwent PGT-A at the blastocyst stage on either day-5 or day-6, using a highly validated method. The euploid embryos were divided into male and female groups and blastocyst morphological grades were considered with respect to rates of implantation, miscarriage and ongoing pregnancy/birth. Main results and the role of chance The proportion of embryos biopsied on day-5 versus day-6 was identical for males and females (68% day-5 for both). No difference was observed in blastocyst expansion or inner cell mass grading on day-5. However, a highly significant difference was noted in the distribution of trophectoderm grades (A, B, C, D according to the system of Gardner and Schoolcraft, 1999) (P < 0.0001). This was characterised by a disproportionate representation of the highest grade amongst male embryos. 21.6% of male blastocysts had trophectoderm graded ‘A’, compared to 14.9% of females. Interestingly, euploid male embryos with a ‘B’ grade trophectoderm were associated with significantly higher implantation rates than females of the same morphological grade (82.1% vs. 58.7%; P = 0.0002), but the males of this grade also experienced a higher incidence of biochemical losses (16.7% vs. 4.2%; P = 0.017). As a group, male embryos do not have greater viability than female embryos, which implies that the greater proportion of high trophectoderm grades amongst these embryos is not an indicator of superior potential. In turn, this suggests that different criteria should be used for grading trophectoderm in males and females. Limitations, reasons for caution It may be desirable to introduce parallel morphological grading systems, one for male embryos and another for female. However, this would only be applicable in cycles involving PGT-A. The observation that embryos of equal grade can have different outcomes depending on their sex should be confirmed in a prospective study. Wider implications of the findings These results suggest the development of sex-specific morphological grading strategies might provide more reliable insights into embryonic potential, increasing the likelihood of selecting a viable embryo for transfer. Consideration of differences in the development of male and female embryos will also be important when developing morphokinetic algorithms for embryo selection. Trial registration number Not applicable

P-562 Elective egg freezers’ disposition decisions: A qualitative study

Abstract Study question What are the factors that influence elective egg freezers’ (EEF) disposition decisions towards their surplus frozen oocytes? Summary answer Achieving motherhood or dealing with grief if motherhood was not achieved, the complexities of donating to others, and a lack of information and professional advice. What is known already Most women who undergo EEF do not use their oocytes. Consequently, there is an abundant, but unquantified, number of women with surplus oocytes in storage globally. Many women are deciding about the disposition of their surplus oocytes due to storage limits in countries such as Australia, Belgium, Finland, and Taiwan. However, no studies have examined the factors that influence EEF oocyte disposition decisions. Research exploring factors relevant to embryo disposition and planned oocyte donation may not be relevant. Consequently, women are making the challenging and stressful decision regarding the fate of their oocytes with limited research available to support them. Study design, size, duration Thirty-one structured interviews took place in Australia between October 2021 and March 2022. Recruitment was via: Facebook (paid advertising, posts on relevant groups and organisation sites), newsletters and emails from universities and professional organizations, emails to eligible patients from an IVF clinic, and snowballing. A reflexive thematic approach was planned; data collection and analysis occurred concurrently. Recruitment occurred until the process of analysis did not identify any new themes and saturation have been reached. Participants/materials, setting, methods Eligible participants (EEF with surplus frozen eggs, 18+, living in Australia) were interviewed and included women who had previously made a disposition decision (n = 7), were currently deciding (n = 6), or who not yet considered the decision (n = 18). Interviews took place on recorded teleconference, were transcribed verbatim and anonymised. Transcripts were iteratively coded via NVivo and analysed, and themes developed inductively. The researcher reflected on their subjectivity with co-authors to ensure accuracy and clarity of data interpretation. Main results and the role of chance Six inter-related themes were identified related to the decision-making process: ‘decisions are dynamic’; ‘triggers for the final decision’; ‘achieving or not achieving motherhood’; ‘conceptualisation of oocytes’; ‘the impacts of egg donation on others’; and ‘external factors affecting the final disposition outcome’. All women reported a type of trigger ‘event’ for making a final decision (e.g. completing their family). Women who achieved motherhood were more open to donating their oocytes to others, wanting to share the joy of motherhood, but were concerned about the implications for their child (e.g. donor-conceived half-siblings) and also felt responsibility for potential donor children. Women who did not achieve motherhood were less likely to donate to others due to the grief of not becoming a mother, often feeling alone, misunderstood, and unsupported. Reclaiming oocytes (e.g. taking them home) and closure ceremonies helped some women process their grief. Donating to research was viewed as an altruistic option as oocytes would not be wasted and did not have the “complication” of a genetically linked child. Decisions were often made based on misinformation and a lack of knowledge of the available disposition options and their consequences, with few women seeking professional advice on their decision. Limitations, reasons for caution Most participants had not considered the decision and their stated intentions may not reflect their final decision. Women who had previously made disposition decisions were difficult to recruit despite comprehensive study advertising. Other limitations were the use of convenience sampling and conduct of interviews via teleconference (due to COVID). Wider implications of the findings Due to a lack of understanding of the disposition options, their impacts, and women not seeking professional advice, decision support (e.g. counselling, decision aids) is suggested. Counselling should occur at least at the beginning and end of the process, address disposition options, impacts, grief, and gaining support from others. Trial registration number Not applicable

P-724 The choice of genetic service provider can have significant implications for the outcome of IVF cycles using preimplantation genetic testing for aneuploidy (PGT-A)

Abstract Study question Does the choice of PGT-A provider have any impact on clinical results, such as rates of aneuploidy, pregnancy and miscarriage? Summary answer The laboratory providing PGT can have a significant impact on various important clinical outcomes. PGT-A providers are not equivalent and should be chosen with care. What is known already A growing number of IVF cycles include PGT-A to assist in the identification of euploid embryos for transfer to the uterus. All modern PGT laboratories utilise a method called next generation sequencing (NGS) to predict the copy number of each chromosome in embryo biopsy samples. Given this technical convergence, it is often supposed that genetic results will be similar regardless which PGT laboratory is used, and consequently a clinic choosing a genetics provider need only consider convenience, quality of support and price. Here we consider whether the choice of PGT-A provider might have more profound affects, potentially impacting clinical results. Study design, size, duration A large network of IVF clinics switched from PGT-A provider ‘A’ to provider ‘B’. The final 6 months of clinical data using provider A was compared to 6 months of data after the switch to B. There were no changes in any aspect of patient population, treatment protocols, or embryological practice occurred between the two time periods evaluated. Genetic results (aneuploidy/euploidy rates) and clinical outcomes (implantation, miscarriage, ongoing pregnancy/birth) were compared to identify any differences. Participants/materials, setting, methods Within the two time periods considered, 9,091 embryos underwent PGT-A with provider A and 10,281 using B. The average female age was 39.0 and 39.3, respectively. Embryos were biopsied at the blastocyst stage and the cells were shipped to the genetics laboratories. Provider A was in the same country as the IVF clinics, while provider B was located overseas. Both PGT-A laboratories used NGS, but employed different methods for DNA amplification, sequencing and data analysis. Main results and the role of chance A higher proportion of embryos were classified euploid by provider B in comparison to A (49.3% versus 39.8%; p < 0.0001). Differences in reported aneuploidy were particularly striking for younger patients (<38 years; 67.5% euploid according to B versus 52.5% reported by A; p < 0.0001). For patients over 37 years, the lower aneuploidy frequency reported by B resulted in fewer cycles with all embryos classified ‘abnormal’ and more cycles with a transfer (52.2% of 2115 cycles in this age group had a transfer, compared to 48.6% of 1915 cycles for A; p = 0.02). Having more embryos classified euploid also benefitted younger patients, providing opportunities to combine morphological selection with genetic evaluation. Improved selection may explain a higher ongoing pregnancy rate observed for patients <38 years (63.2% ongoing after the first embryo transfer for B versus 55.3% for A). Differing miscarriage rates were also observed (18.1% when using A versus 15.1% for B; p = 0.047). We speculate that higher rates of euploidy and pregnancy when using provider B might be a consequence of viable embryos being excluded due to incorrect classification as ‘abnormal’ by A. Additionally, failure to detect some aneuploidies, leading to transfer of abnormal embryos, might explain the higher miscarriage rate associated with A. Limitations, reasons for caution There were no detectable differences in the composition of patient populations receiving PGT-A from the two providers, and no apparent changes to protocols used during the two time periods assessed. Nevertheless, it must be acknowledged that the conclusions are based on a retrospective review of data, not a controlled study. Wider implications of the findings PGT-A and NGS are umbrella terms encompassing multiple methods with widely varying levels of validation and accuracy. Our results suggest that the choice of PGT-A provider has important implications for clinical results, affecting the number of transferrable embryos, pregnancy, and miscarriage rates. The observed differences likely reflect differing PGT-A accuracies. Trial registration number Not applicable

P-078 In vitro effects of polycarbophil vaginal moisturizing gel on sperm motility and vitality

Abstract Study question The aim of this study was to evaluate the possible effects of intravaginal polycarbophil gel in sperm quality in an in vitro model. Summary answer Incubation of human sperm samples with a vaginal moisturizer containing polycarbophil gel 10% resulted in important reduction of sperm motility and vitality. What is known already The possible effect of lubricants on sperm quality has been a matter of debate for over 50 years. Despite the publication of a few studies, the real impact of lubricants on male gametes is remains unknown. Some commercial lubricants have been tested but so far there is a lack of studies evaluating the impact of vaginal moisturizers on seminal quality. Vaginal dryness is a recurring and significant complaint affecting couples trying to conceive who may use lubricants, vaginal moisturizers or even natural oils that could advsersely interfere with seminal quality. Study design, size, duration This paired prospective study involved seminal samples obtained fom 14 men undergoing fertility treatment at a private IVF clinic in Brazil from August/2020 to July/2022. The study was approved by the local review board and each participant signed an informed consent. Each sample was divided into two aliquots: 20% polycarbophil gel (final dilution 10%) or medium only. Statistical analysis was performed using D’Agostino/ Pearson, General Linear Model, Wilcoxon, Friedman’s. P values <0.05 were considered significant. Participants/materials, setting, methods Normozoospermic samples of 14 patients (alpha 0.05, power 0.8)were collected by masturbation, 2 to 5 days after the last ejaculation. After liquefaction ,10 μmL were analyzed using a Makler chamber according to World Helath Organization (WHO) guidelines. Samples were mixed 1:1 v/v with 20% policarbophil gel or with culture medium only, then incubated for 24h at 34 °c. Analysis were performed at 0, 15, 30 min and 24h to determine progressive motility and vitality (eosin-nigrosin test) Main results and the role of chance There was a significant decrease of sperm motility according to the WHO classification in both experimental conditions over time, but the reduction was more evident in the samples treated with polycarbophil gel (p < 0.0001 group x time, General LinearModel for Repeated Measure). The proportion of progressively motile sperm reduced immediately upon contact of the seminal sample with polycarbophil gel (time point 0 min) and decreased throughout the experiment, contrasting to incubation with culture medium (control), which only affected substantially sperm motility after 24h. In the validation cohort, the proportion of progressively motile sperm decreased from a median of 52% in fresh samples to 11% after 24h incubation with culture medium and 0% after 24h incubation with polycarbophil gel (p < 0.0001). Sperm vitality at the end of the experiment also differed significantly between treatments, with medians of 62.5% after exposure to culture medium and 47% after exposure to polycarbophil gel (p = 0.020, Wilcoxon’s test) Limitations, reasons for caution Results represent samples from a selected group of men undergoing fertility treatment at a private center in Brazil. The effect of polycarbophil gel in fertile males may be different. Moreover, the in vitro setting used here does not accurately mimic the vaginal environment which could interfere with the results. Wider implications of the findings Polycarbophil-based gels and similar products may harm sperm vitality and motility, therefore woment trying to conceive should avoid the use of such products until more studies prove otherwise. Trial registration number Not applicable

P-168 Follow-up survey of deliveries derived from Day 7 blastocysts

Abstract Study question Does Day 7 blastocyst-transfer affect prognosis of babies born? Summary answer There was no effect on growth up to 3 years of age in babies from Day 7 blastocyst-derived births. What is known already Culture medium and technology have advanced, and the efficiency to culture up to the blastocyst stage in order to obtain viable embryos has increased dramatically. As a result, blastocyst transfer is associated with higher clinical pregnancy outcomes than cleavage transfer. In 1998, it was first reported that some embryos may develop into blastocysts on Day 7. However, there are few reports on neonatal outcomes from Day 7 blastocyst transfer. Therefore, the utility of Day 7 blastocysts is controversial. Study design, size, duration This was retrospective study which included 23 cases where there was a live birth after single embryo transfer of a Day 7-derived blastocyst. The incidence of birth defects, birth weight was recorded as well as a physical development survey. The height and weight were recorded in 10 children aged 18-months and 4 children aged 3 who responded to follow-up survey. The time period was from January 2013 to December 2021. Participants/materials, setting, methods Patient seeking fertility treatment at an established private IVF clinic. We compared the birth weight, and birth after 18-months and 3 years height and weight of children born to a Day 7 blastocyst with data from babies born from Day 5 and Day 6 derived blastocysts. Statistical significance was determined using Kruskal-Wallis test. Main results and the role of chance The incidence of birth defects was 2.4% (115/4828), 1.7% (16/954) and 0% (0/23) in the Day 5, Day 6 and Day 7 groups, respectively. The average birth weight of the Day 7 group was 3057+/-532 g, which was not significantly different from 3024+/-472 g and 3041+/-480 g in the Day 5 and Day 6 groups. The average height at 18-months, was 80.0+/-5.3 cm, 79.8+/-5.1 cm, 77.8+/-6.9 cm in the Day 5, Day 6 and Day 7 groups, respectively. The average weight at 18-months, was 10.5+/-1.2 kg, 10.4+/-1.1 kg, 10.5+/-1.4 kg. The average height at 3 years old, was 93.2+/-4.8 cm, 92.9+/-4.7 cm, 93.4+/-5.9 cm. The average weight at 3 years old, was 13.9+/-1.6 kg, 13.8+/-1.4 kg, 14.1+/-2.6 kg, respectively. There was no significant difference in average height and weight up-to the 3 years old follow-up survey. Limitations, reasons for caution The incidence of Day 7 blastocyst-transfer is low and the study was limited to cases of single blastocyst embryo transfer. The study was not separated by sex, because sample size was small. Wider implications of the findings There was no difference in growth up to a 3-year-old follow-up survey of the Day 7 blastocyst compared with the Day 5 and Day 6 groups. Transfer of a Day 7-derived blastocyst does not appear to affect infant development and should be considered in cases where blastocyst development is delayed. Trial registration number not applicable

P-142 Alterations in mitochondrial DNA levels in luteal granulosa cells may affect the morphology of mature oocytes and subsequently their fertilization potential: a retrospective multicenter study

Abstract Study question Do alterations in mitochondrial DNA levels in luteal granulosa cells (LGCs) affect the quality of mature oocytes in infertile women? Summary answer Alterations in the mitochondrial DNA levels in LGCs may modify the characteristics of the first polar body in oocytes and subsequently decrease their fertilization potential. What is known already Assisted reproductive treatment outcomes are closely linked to the oocyte quality as shown in several systematic reviews and meta-analyses. Particularly, the oocyte quality is highly affected by the surrounding luteal granulosa cells (LGCs). This is mainly due to the crucial role of the mitochondria in the LGCs in fueling the metabolic processes required for oocyte maturation. Therefore, modifications in the quality of LGCs may have direct effects on the developmental competence of oocytes. However, the exact mechanisms by which this could happen are not fully understood. Study design, size, duration A retrospective multicenter study was conducted on 303 mature oocytes retrieved from 51 women undergoing intracytoplasmic sperm injection (ICSI). It was conducted in Lebanon at Al Hadi IVF center and Azoury IVF clinic, between January 2019 and January 2020. G-Power 3.1 was used to determine the sample size for Generalized Linear Mixed Models through a one-sample t-test power analysis with alpha 0.05, power 0.8, medium effect size (f2 = 0.15), and 5 predictors. Resulting sample size: 43. Participants/materials, setting, methods This study excluded cases of premature ovarian failure, severe oligozoospermia (<2 x 102/ml), or cases using frozen gametes. Mature oocytes, from young women (< 36 years old), were injected and cultured in the Embryoscope where morphometric measurements were performed. LGCs vitality and mitochondrial DNA (mtDNA) levels were analyzed using trypan blue exclusion dye and next-generation sequencing, respectively. Possible associations between these independent variables and the size and integrity of the first polar body were evaluated. Main results and the role of chance The included women presented with primary infertility. Their mean age was 29.98 ± 5.5 years old and their mean body mass index was 23.33 ± 3.32 Kg/m2. 10% of the included women had polycystic ovary syndrome, and 48% were smokers. Regarding the LGCs parameters, a statistically significant negative correlation was found between the mtDNA levels in LGCs and their vitality percentage (r = −0.313, p = 0.029). Interestingly, the average size of the first polar body (PBI) was 346.67 µm. Here we found that the PBI size decreased by 15.05 units when the mtDNA level in LGCs increased by one unit above its average. In parallel, the median percentage of oocytes having a fragmented PBI was 33.33 (0-100). This percentage increased by 4.26% for every one unit increase in the mtDNA level in LGCs (p < 0.0001). In contrast, this percentage decreased by 0.86% for every unit increase in the percentage of LGCs vitality (p < 0.0001). Moreover, the mean percentage of fertilization rate was found to be 70.19 ± 26.7. A higher percentage of fragmented PBI (38.3%) was found among oocytes that did not fertilize compared to those that did successfully fertilize (24%) (p = 0.019). Limitations, reasons for caution This study only analyzed the effects of alterations in mtDNA levels in LGCs on oocytes. It would be worthwhile to also analyze the effects on foetal development and offspring health. Furthermore, it would be interesting to study the relationship between alterations in LGCs and oocyte quality within individual follicles. Wider implications of the findings Several studies have suggested that abnormal size and fragmentation of the PBI may impair embryo development. Therefore, treating factors that affect LGCs, such as obesity and polycystic ovary syndrome, may benefit infertile women. This highlights the need for new clinical trials in this context. Trial registration number Not applicable

P-759 Discard, or not discard, that is the question. An international survey among 117 embryologists about their decision on 50 poor-quality/day7 blastocysts cultured in time-lapse incubators.

Abstract Study question Are decisions on day7 and/or poor-quality blastocysts’ (PQB; <Gardner’s BB) use consistent among embryologists of diverse European countries and what factors influence them? Summary answer “Discard” choice prevalence was ≈40%, however inter-rater agreement was just fair (Fleiss-k=0.26). Country of practice, maternal age and number of sibling embryos influence each decision. What is known already Blastocyst morphokinetics and morphological quality are associated with competence and used to make clinical decisions, even though evaluations are poorly reproducible across embryologists. Moreover, the increasing utilization of PQB and adoption of culture beyond 144 hours-post-insemination is further challenging the decision-making process. Previous surveys reported limited concordance among embryologists in the “discard”/“use” choices made for day7 and/or PQB based on single 2D-pictures without cycle/patient information. However, for instance whole embryo preimplantation development, reproductive history, number of sibling embryos, can influence each decision. Different regulations and mindsets across countries may further influence these choices and require investigation. Study design, size, duration Anonymous survey in Microsoft-Forms run within a large IVF-network. Each laboratory-director shared a link to the survey with his/her colleagues via 3 e-mails sent weekly between 22/12/2023 and 12/1/2023. 117 embryologists participated representing 6 European Countries, 9 IVF-groups, and 28 centers. Raters inspected EmbryoScope videos of 50 PQBs and/or day7 blastocysts’ whole preimplantation development and decided whether to “use” (“transfer-fresh”/“cryopreserve”/“biopsy and cryopreserve”) or “discard” each of them. Inter-rater agreement was measured with Fleiss-k. Participants/materials, setting, methods Couples’ information (age, infertility diagnosis, previous attempts, sperm analysis, number of sibling blastocysts obtained) were provided, and participants were asked their years of experience, center location, average number of cycles and average maternal age, number of colleagues, presence of time-lapse-incubators. The AI-score (CHLOE-EQ, Fairtility and iDAScore v1.0, Vitrolife), chromosomal diagnosis and clinical outcomes were known for all embryos by one embryologist not involved in the survey, but the participants were blinded to them. Main results and the role of chance Participants were Italian (40%,N=47), Spanish (24%,N=28), Portuguese (5%,N=6), Czech (5%,N=6), Swedish (23%,N=27) and Icelandic (3%,N=3). 2263(38.7%) “discard” and 3587(61.3%) “use” decisions were recorded. Czech, Portuguese, and Italian embryologists expressed lower (17±7%,23±14% and 27±18%), Spanish intermediate (37±16%), and Nordic higher (67±11%) “discard” decision rates.The prevalence of “discard” responses per embryo was 37.4±24.2%(2-87). The larger the number of sibling blastocysts the higher the embryologists’ propensity to “discard” PQBs/day7 (unstandardized coefficient-B:+4.4%,95%CI:+0.9 to + 7.9,p=0.015). The most prevalent “use” choice was “biopsy and cryopreserve” (33.1±21.7%,2-78). Maternal age (unstandardized coefficient-B:+2.2%,95%CI:+1.3 to + 3.8,p<0.01) and number of sibling blastocysts (unstandardized coefficient-B:-9.2%,95%CI:-15.2% to -3.3%,p<0.01) influenced this rate.Overall, inter-rater agreement was 0.26 (standard-error:0.003;“fair”), with “moderate” agreement for Czech and Portuguese (0.41,standard-error:0.037), “fair” for Spanish (0.38,standard-error:0.007), Italian and Nordic embryologists (0.28,standard-error:0.006 and 0.009).CHLOE-EQ and iDAScore v1.0 were not associated with embryologists’ decisions. No difference was shown for AI-scores in euploid versus aneuploid blastocysts (CHLOE-EQ: 0.2±0.32,0-0.9 versus 0.2±0.29,0-0.93; iDAScore v1.0: 4.2±1.6,2.2-6.7 versus 4.3±1.5,2.1-9), while the embryologists more often chose “discard” among the latter group (28.3±21%,9-71 versus 41.6±24.8%,2-87). Five blastocysts were transferred and 3 implanted (CHLOE-EQ: 0.001,0.416 and 0.003; iDAScore v1.0: 2.6,5.0 and 6.6; “discard” choice rates: 19%,15% and 10%). Limitations, reasons for caution All embryologists were asked to decide regardless of the regulation enforced in their countries (e.g., PGT-A forbidden, discarding viable embryos not allowed). Nonetheless, the legislative, social, and clinical background might still have affected their choice. The survey included only private IVF clinics located in Europe. Wider implications of the findings European embryologists’ decision-making regarding PQB/day-7 embryos is inconsistent. Mostly, legislative/clinical background and cycles’ characteristics influence each decision. However, this practice has clinical implications, especially in poor-prognosis patients. Artificial-Intelligence may improve the reproducibility by removing intrinsic human subjectivity. However, it is still currently underpowered to assess these outlier embryos Trial registration number Not applicable

P-594 A prevalence study of vitamin D insufficiency among infertile women in Sweden, its determinants and IVF outcome.

Abstract Study question What is the prevalence and the determinants of vitamin D insufficiency and do women with insufficiency have worse IVF outcome compared with them with sufficiency? Summary answer Women from Non-Nordic countries, with avoiding sun behavior and low intake of vitamin supplementation have higher prevalence of vitamin D insufficiency but comparable IVF outcome. What is known already Vitamin D seems to have an important role in human reproduction and infertility. Vitamin D insufficiency is highly prevalent around Europe, yet studies from north latitude countries, where sunlight exposure is undetectable during the winter, describing the prevalence and determinants of this condition in women with infertility lacking. Moreover, there is still discrepancy in the existing evidence whether insufficient vitamin D status is associated with poor IVF/ICSI outcome and no published studies in this field exist from Nordic countries. Study design, size, duration Prospective cohort study that was conducted between September 2020 and August 2021 at Reproductive Medicine, Sahlgrenska University Hospital, Gothenburg in Sweden. Gothenburg is a city located in southwestern Sweden (latitude 57.7o N) with highly variating duration of daylight between seasons. Participants/materials, setting, methods Women with infertility who were scheduled for IVF/ICSI treatment were included. Venous blood samples for measurement of serum 25(OH) vitamin D concentration and questionnaires assessing vitamin D intake and sun exposure habits were collected from 265 women. Main results and the role of chance The overall prevalence of vitamin D insufficiency was 27%, ranging from 7% in summer to 33% in winter. The odds for vitamin D insufficiency were higher for women originating from non-Nordic Europe (OR 3.33, 95% CI 1.26-8.80, adjusted p=0.015), Middle East (OR 14.46, 95% CI 5.20-40.20, adjusted p<0.001) and Asia (OR 6.46, 95% CI 1.79-23.27, adjusted p=0.004) compared with women originating from Nordic countries. It was more likely for women who did not use vitamin D supplements to have insufficiency compared with them using supplements (OR 3.48, 95% CI 1.69-7.20, adjusted p<0.001). The odds for vitamin D insufficiency were also higher for women staying “in the shade all the time” compared with women who were exposing themselves “in the sun all the time” (OR 3.65, 95% CI 1.43-9.27, adjusted p=0.007) and with them staying “both in the sun and shade” (OR 3.57, 95% CI 0.88-14.43, adjusted p=0.074). The reproductive outcome of IVF/ICSI treatment between women with serum 25(OH) vitamin D insufficiency and sufficiency was comparable. Limitations, reasons for caution The use of a questionnaire has intrinsic limitations (recall bias, low accuracy of the provided information). Moreover, the questionnaire did not include questions about sun protection factor usage during sun exposure. Blood sampling was underrepresented during summer due to limited resources during the summer months with closed IVF clinics. Wider implications of the findings Pre-IVF/ICSI treatment vitamin D serum concentration do not seem to influence IVF success. However, information about vitamin D should target women from non-Nordic countries and to advise them for supplementation and sun exposure. The aim is to avoid vitamin D insufficiency and the associated adverse long-term health consequences. Trial registration number not applicable

P-195 Can we predict blastulation, implantation and live birth in early stages of embryo development? Clinical validation of a classification algorithm based on automatic morphokinetic annotations

Abstract Study question Can a classification algorithm based on automatic annotations of early morphokinetic timings improve embryo selection in ICSI cycles? Summary answer The automatic scoring resulted significantly predictive of blastulation, implantation and live birth, especially when combined with morphological evaluation, but not of euploidy status. What is known already The Eeva Test® is an imaging system that automatically annotates morphokinetic parameters of the early embryo development. The latest version features the Xtend® algorithm, which classifies embryos on day 3 of development from highest (1) to lowest (5) potential to reach blastocyst stage, based on four parameters: the automatically recorded parameters P2 (t3-t2) and P3 (t4-t3), plus oocyte age and number of cells. Previous bibliography suggests that the Eeva scoring, in combination with morphologic evaluation, might also be predictive of implantation and live birth potential. However, the efficacy and reproducibility of this algorithm need to be validated with external data. Study design, size, duration Retrospective, observational cohort study, performed over 3736 embryos from oocyte donation cycles and 1291 embryos from autologous cycles with preimplantation genetic testing for aneuploidies (PGT-A) performed in a single IVF clinic over 3 years. All embryos were scored by the Eeva Test on day 3. The performance of the Eeva-Xtend scoring system as predictor of blastocyst development, euploidy, implantation and live birth was assessed. Participants/materials, setting, methods Embryo development was monitored by a time-lapse system. Embryo selection was performed by morphological (ASEBIR) criteria, resulting in the transfer of 959 embryos. The correlation between the Eeva-Xtend scoring, individually and combined with morphological classification, and the study outcomes was quantified by generalized estimating equations (GEE) including main possible confounders, and expressed in terms of odds ratio (OR). The performance of the GEEs was quantified and compared through the Area Under the ROC Curves (AUCs). Main results and the role of chance A positive association (P<0.001) was confirmed between lower Eeva scores and higher odds of reaching blastocyst stage (OR (1 vs 5) = 15.849, 95%CI 12.510-20.078; OR (2 vs 5) = 11.592, 95%CI 9.229-14.560; AUC = 0.768 (95%CI 0.754−0.783)), and consistent in both oocyte donation and PGT-A cycles. Lower morphokinetic embryo scores also resulted significantly associated with higher chances of implantation (OR (1 vs 5) = 3.385, 95%CI 1.507-7.605; P = 0.003) and live birth (OR (1 vs 5) = 5.132, 95%CI 2.089-12.605; P<0.001) in oocyte donation cycles, but not in embryos subjected to PGT-A: OR (1 vs 5) = 2.089, 95%CI 0.321-13.614, P = 0.441 for implantation and OR (1 vs 5) = 0.916, 95%CI 0.161-5.205, P = 0.921 for live birth. In embryos subjected to PGT-A, the automatic scoring system did not result significantly associated with the euploidy status: OR (1 vs 5) = 0.755 (95%CI 0.255−0.981; P = 0.489). In global, the Eeva Test had similar performance than morphological evaluation for implantation (AUC = 0.610 (95%CI 0.572−0.648) vs 0.606 (95%CI 0.568−0.644), respectively) and live birth (AUC = 0.622 (95%CI0.584−0.660) vs 0.609 (95%CI 0.571−0.647)) prediction. However, the highest performance was achieved when combining both scoring systems: AUC for implantation = 0.629 (95%CI 0.592−0.666), AUC for live birth = 0.636 (95%CI 0.598−0.673). Limitations, reasons for caution The retrospective nature of the study introduces some variability in the characteristics of the sample. However, the large sample size partially overcomes said limitation. The automatic annotations of the morphokinetic parameters were not validated in this study. Wider implications of the findings Our results support the applicability and reproducibility of this automatic early embryo evaluation system. Furthermore, our findings support that the use of this automatic embryo evaluation system in combination with morphological evaluation can potentially reduce subjectivity, improve embryo evaluation and increase success rates in IVF treatments. Trial registration number not applicable

P-163 Three-year follow-up of babies born derived from mono-pronuclear zygotes

Abstract Study question Are neonatal outcomes normal up to 3 years of ages from the transfer of blastocysts derived from mono-pronuclear(1PN) zygotes? Summary answer There was no effect on growth up to 3 years of ages in babies born from 1PN-implanted blastocysts. What is known already In human ART, 1PN zygotes are observed at low rates. We have previously reported that 80.7% of 1PN zygotes had a biparental chromosome using a Live-Cell imaging technique, and some of these developed to the blastocyst stage (Tokoro et al. ASRM 2013). Furthermore, we have reported that these blastocysts can result in a viable pregnancy and healthy live birth (Tsuji et al. ASRM 2020), and there was no effect on growth up to 18-months (Tsuji et al. ESHRE 2021). However, there is no information on the long-term prognosis of babies derived from1PN-zygotes. Study design, size, duration This was a retrospective study which included 86 cases where there was a live birth after single embryo transfer of a blastocyst derived from 1PN zygote. The incidence of birth defects, birth weight were recorded as well as a physical development survey of 19 children who responded to a 3-years follow-up survey. The time period was 108 months (January 2013 to December 2021). Participants/materials, setting, methods Patients seeking fertility treatment at an established private IVF clinic. We compared the birth weight, 3-years old height and weight of children born from 1PN zygotes with data from a control, 2PN group. Statistical significance was determined using the t-test (level of P < 0.05). Main results and the role of chance The incidence of birth defects in 1PN embryo-derived infants was 1.2% (1/86). The average birth weight of boys in the 1PN group was 3147.6+/-408.7 g, which was not significantly different from 3070.5+/-479.5 g in the 2PN group. In girls, the average birth weight was 3048.1+/-510.7 g in the 1PN group, which was not significantly different from the 2PN group (2966.2+/-462.7 g). The average height at 18-months, was 81.3+/-2.5 cm vs 80.9+/-3.4 cm for boys; 79.5+/-2.2 cm vs 79.5+/-3.0 cm for girls in the 1PN and 2PN groups, respectively. The average body weights of the 1PN and 2PN groups were 11.0+/-1.2 kg vs 10.8+/-1.1 kg for boys; 9.8+/-1.0 kg vs 10.2+/-1.1 kg for girls, respectively. The average height at 3-years, was 95.2+/-3.1 cm vs 93.8+/-4.5 cm for boys; 91.5+/-2.9 cm vs 92.5+/-4.9 cm for girls in the 1PN and 2PN groups, respectively. The average body weights of the 1PN and 2PN groups were 14.7+/-1.2 kg vs 14.1+/-1.5 kg for boys; 13.2+/-1.9 kg vs 13.6+/-1.5 kg for girls, respectively. There was no significant difference in average height and weight up-to the 3-years follow-up survey. Limitations, reasons for caution The incidence of 1PN- zygotes that develop to blastocysts resulting in births is low and the study was limited to cases of single blastocyst embryo transfer. Wider implications of the findings The incidence of congenital anomalies in Japan is around 1.7 to 2%, and the incidence was similar in the 1PN group. Also, there was no difference in 3-years follow-up survey of the 1PN compared with the 2PN. These data are reassuring for the clinical utility of 1PN derived embryos. Trial registration number not applicable