Embryo Quality Research Articles

P-139 Mitochondrial DNA copy number in trophectodermal cells (MITOSCORE) is related to embryo quality, but not to implantation potential

Abstract Study question Is mtDNA copy number in trophectodermal cells (MITOSCORE) related to the quality of euploid embryos in our PGT-A program? Is it related to implantation potential? Summary answer mtDNA copy number in trophectodermal cells (MITOSCORE) is related to embryo quality and morphokinetic, but no to implantation potential in our PGT-A program. What is known already Fertilization and embryonic development require energy, sourced from mitochondria. Research on human embryos indicates associations between aneuploidy and maternal age and increased mtDNA content. Consequently, exploring embryonic mtDNA levels as a quality marker gained momentum. Embryos with high mtDNA levels often correlate with poor implantation potential, leading to its adoption as a biomarker for selection. However, conflicting studies challenge this notion, failing to establish correlations between mtDNA levels and blastocyst grade, implantation, or ongoing pregnancy rates in patients with euploid embryos. Consequently, the predictive value of mtDNA levels for embryo implantation potential and pregnancy outcomes remains uncertain. Study design, size, duration A retrospective study was conducted to establish a relationship between MITOSCORE and the euploid embryo morphokinetics. It involved 59 embryos from 25 PGT-A cycles performed from January 2022 to December 2023. In addition, we retrospectively analysed 143 thawed single embryo transfers from three centres (October 2020 to December 2023) to assess the correlation between MITOSCORE and pregnancy outcomes. Embryos for transfer were selected based on morphology; mtDNA was considered only if two had similar morphology. Participants/materials, setting, methods Morphokinetic was determined using the AI tool CHLOE (Fairtility, Israel), which automatically provides morphokinetic parameters and also offers an additional score of embryo quality, EQ. mtDNA levels were estimated by NSG and the MITOSCORE score was provided by an algorithm (Igenomix, Spain). Pearson’s correlation analysis was employed to investigate the potential relationship between MITOSCORE results and embryonic morphokinetics. Mann-Whitney test was used to analyse the relationship of MITOSCORE levels with pregnancy. Main results and the role of chance A statistically significant correlation (p < 0.05), albeit weak, was observed between MITOSCORE and embryonic quality evaluated through CHLOE EQ (r: -0.3858). It is noted that a higher EQ was associated with a lower value in MITOSCORE. Additionally, upon analysing morphokinetic parameters, it was found that the time to reach the stages of morula (r: 0.6876), early blastocyst (r: 0.6303), cavitated (r: 0.6823), and expanded (r: 0.5776) were significantly correlated (p < 0.05) with MITOSCORE values. The slower the embryo development, the higher the MITOSCORE. Regarding implantation capacity and mtDNA levels, implanted euploid embryos had a MITOSCORE value of 20.35, which was not significantly different from that of non-implanted embryos, which was 21.65. The highest MITOSCORE value at which a full-term pregnancy was achieved was 32.34. However, no pregnancy was obtained with the three embryos with the highest scores (35.02, 45.02, and 48.17). Limitations, reasons for caution Morphokinetics correlation with MITOSCORE was analysed with a limited number of embryos (59). The highest MITOSCORE level was 48.17. The transfers were not fully blinded. A randomized clinical trial would be necessary to determine the true clinical benefits of mtDNA levels in the trophectoderm of euploid embryos in our clinics. Wider implications of the findings Trophectodermal mtDNA level is associated with morphokinetic parameters and blastocyst quality but not embryo implantation potential. In our PGT-A programme, and with our results, trophectodermal mtDNA copy numbers work as a biomarker of blastocyst quality but have not been correlated with clinical outcomes after the transfer of euploid embryos. Trial registration number NOT APPLICABLE

P-255 Nicotinamide mononucleotide (NMN) supplementation during germinal vesicle (GV) rescue could increase the total number of mature oocytes available in women of advanced maternal age (AMA)

Abstract Study question Does NMN supplementation improve nuclear and cytoplasmic competence of AMA oocytes during GV-rescue? Summary answer NMN supplementation enhances GV-rescue efficiency in AMA oocytes by improving nuclear competence and slightly ameliorating cytoplasmic competence. What is known already Oocyte competence drastically declines from the age of 35 onwards. With age, organism NAD+ levels also drop, and NAD+ depletion at the oocyte level is associated with lower oocyte and embryo quality. Over the past years, supplementation with the NAD+ precursor nicotinamide mononucleotide (NMN) has been showed to recover oocyte quality and reproductive potential in aged mice due to NAD+ repletion. Thus, we propose the GV-rescue strategy to assess the impact of NMN in vitro supplementation on rescued-oocyte competence, according to maternal age. Study design, size, duration Experimental, prospective study performed on GVs collected after controlled ovarian stimulation (COS), oocyte pick-up (OPU) and denudation; from November 2023 to January 2024. After signed informed consent, GVs were randomized into the control and study (NMN) groups, in a ratio 1:1. A total of two or four GVs were collected from each cycle, in order to avoid any cycle-related potential bias. Participants/materials, setting, methods Young (≤35y.o) or AMA (>35y.o.) women from an infertility clinic (Spain) donated their GVs. They were cultured in time-lapsed, CSCM-NXC medium with or without 100µM-NMN (37oC,6%CO2,5%O2). Nuclear dynamics (tGVBD, tMI and tMII) were described, using “denudation” as zero-time (t0). Rescued metaphase-II (rMII) were artificially activated (≤2.8h post-rMII; 8uM A23187,5min; 10ug/ml puromycin,5h). Normal oocyte activation (NOA) was assessed: 2ndpolar body (PB) extrusion, one pronucleus. Continuous and categorical variables were compared by paired-samples and χ2 tests, respectively. Main results and the role of chance Two hundred and 82 GVs were collected in the young (n = 73 women) and aged groups (n = 35 women), respectively. In the young group, NMN supplementation did not affect the rescue rate (RR=rMII/GV; 54.0%; p = 0.21) but in the AMA-NMN group RR significantly increased (24.4% vs. 48.8%; p = 0.04). Indeed, rMII from both AMA-NMN and young-control groups showed comparable RR (53.1%; p = 0.37) and nuclear dynamics (tGVBD: 5.4±2.8h [95%CI:4.9-5.9], p = 0.10; tMI: 14.8±2.5h [95%CI:14.3-15.2], p = 0.55; t1PB: 20.1±3.2h [95%CI:19.6-20.7], p = 0.33). In contrast, rMII from AMA-control group extruded the 1PB earlier (18.4±2.6h [95%CI: 16.6-20.2) than young-control rMII (p = 0.02). Concerning artificial activation, a slightly non-significant detrimental effect of NMN was observed on NOA rate (rMII with NOA/incubated rMII) in the young group (69.2% vs. 50.0%; p = 0.17). In contrast, NMN-supplement seems to improve the NOA rate in AMA women (33.3% vs. 42.9%; p = 0.78). Limitations, reasons for caution Recruitment rate is lower in AMA than in younger women. Hence, the number of GVs included was lower in the AMA group, providing a potential bias in data interpretation. Results from this preliminary analysis should be tested in further studies with a larger sample size. Wider implications of the findings GV-rescue strategy might increase the number of MII available. In the particular case of women >35, NMN-GV-rescue protocol improves quantitative and qualitative the rMIIs, which become closer to those from young women. Since GV has not undergone any chromosome segregation, to validate whether NMN-supplementation could assist reliable meiosis are urged. Trial registration number 2303-VLC-047-ME

O-294 Human oocyte survival, early embryo development, metabolic fingerprinting, and pregnancy outcomes following ultra-rapid or standard vitrification and thawing

Abstract Study question Is ultra-rapid vitrification and thawing superior to standard vitrification in terms of preserving oocyte viability, embryo competence and enhancing pregnancy outcomes? Summary answer Ultra-rapid vitrification and thawing preserves oocyte viability and embryo quality as confirmed by their euploidy, non-invasive biomarkers in the culture media, and favorable pregnancy outcomes. What is known already Cryopreservation and vitrification protocols for oocytes and embryos have varying cooling rates, cryoprotectant chemical composition and concentration. While vitrification avoids intracellular ice crystal formation, the elevated concentrations of cryoprotectants may be cytotoxic. Over the past 20 years, parameters for vitrification protocols were optimized to improve efficiency and viability of human oocytes and embryos, however few studies have evaluated the metabolic and secretome profiles in addition to embryology and pregnancy outcomes, particularly following ultra-rapid vitrification and thawing. Mid-infrared spectroscopy is a great option for characterizing environmental components and metabolic profiles using the more spectrally complex fingerprint region from 1450-500 cm-1. Study design, size, duration This prospective study included 1,214 donor oocytes collected between January 2022 and November 2023. Sibling oocytes were assigned to standard or ultra-rapid vitrification and thawing. Corresponding blastocysts underwent the same type of vitrification following trophectoderm biopsies for PGT-A. Spent embryo culture fluid was collected on day 1, 3, and 5 for spectroscopy and PCR analysis, as well as on day 6 for metabolic fingerprinting. Clinical pregnancy was assessed 5 days following single embryo transfer. Participants/materials, setting, methods Vitrified oocyte survival was assessed one hour after thawing. Secretome profiling of day 1, 3, and 5 embryos was conducted by Attenuated Total Reflection Fournier Transform Infrared (ATR-FTIR) spectroscopy. Metabolic profiling of blastocysts (day 6) was conducted by Fournier Transform Infrared (FTIR) spectroscopy. Embryology and pregnancy data together with lipid, protein, and nucleic acid expression profiles were evaluated using the Shapiro-Wilk test followed by the student t-test (parametric variables) or the Mann-Whitney U-Test (non-parametric variables). Main results and the role of chance There was 100% survival in oocytes subject to ultra-rapid vitrification-thawing (n = 531) compared to 94.5% in oocytes that underwent standard vitrification (n = 634). There were no significant differences in the fertilization rates following intracytoplasmic sperm injection (81.3% with ultra-rapid and 83.2% with standard vitrification) or potential to reach day 3 of embryo development[RL1]. Ultra-rapid vitrification-thawing produced significantly more blastocysts than standard vitrification-thawing (67.3% vs. 53.0%, respectively) but both techniques had similar euploidy rates (61.8% vs. 61.4%, respectively; p > 0.05). Spectra in the 4000-450 cm-1 region revealed there were no significant differences in lipid and protein expression profiles of day 1, 3, 5 and 6 embryos. The similar result obtained for the culture media for day 1,3,5 and 6 day embryo development with no significant difference. Day 3 blastomere biopsy samples and Day5, Day 6 trophectoderm biopsy sample showed no significant differences for total protein concentration, lipid and nucleic acid experssion profiles in both group.This finding was corroborated by PCR analysis showing no significant difference in nucleic acid profiles. Finally, clinical pregnancy rates were 65.22% with ultra-rapid and 56.5% [RL2] with standard vitrification. (p < 0.05) Limitations, reasons for caution While both our ultra-rapid and standard vitrification-thawing protocols appear to preserve embryo competence to the blastocyst stage and euploidy, future studies should evaluate whether RNA expression levels are altered with these approaches. Wider implications of the findings This study shows competent and euploid embryos can be generated following ultra-rapid vitrification and provides embryo metabolites that can serve as non-invasive biomarkers for the identification of healthy embryos before embryo transfer. Trial registration number NA

P-429 Measuring serum progesterone levels on the day of a fresh blastocyst transfer is debatable as there is no evidence for correlation with ongoing pregnancy results

Abstract Study question Does serum progesterone (P4) level on the day of a fresh blastocyst transfer relate to ongoing pregnancy rate (OPR)? Summary answer OPR after a fresh single blastocyst transfer seems not to be associated with serum P4 level on the day of transfer. What is known already Several studies have suggested an optimal serum P4 threshold for embryo transfers in frozen embryo transfer cycles. However, for fresh embryo transfers, data are scarce and conflicting. Study design, size, duration A retrospective single-center cohort study was performed on 598 complete data cycles, retained out of 631 consecutive fresh single blastocyst transfer cycles. Data were collected from patients undergoing a fresh IVF/ICSI cycle with autologous oocytes at a tertiary fertility center between June 2022 and August 2023. Participants/materials, setting, methods A total of 482 unique patients underwent one or more single fresh blastocyst transfers using micronized vaginal progesterone (MVP) 800mg BID for luteal phase support starting day after oocyte retrieval. Serum P4 levels were determined on day of transfer. The primary endpoint was OPR, positive hCG-test and early pregnancy loss (EPL) were secondary endpoints. Unadjusted and adjusted generalized estimating equations (GEE) assessed P4 level association with the endpoints, using an exchangeable working correlation matrix. Main results and the role of chance No association was identified between P4 levels on day of transfer and OPR after accounting for maternal age, blastocyst quality, P4 and estradiol level at last ultrasound, BMI and endometrial thickness. Data were grouped according to 10/50/90 and 25/50/75 percentiles, resulting in groups < = or > 27ng/ml and < = or > 40ng/ml, with omission of 90th and 75th percentile groups due to right-skewed distribution. With serum P4 levels higher than 40ng/ml, the probability of a positive hCG-test significantly increased (OR 1.79, CI 1.11-2.91; p = 0.018). No significant difference was observed in patients with serum P4 < = or > 27ng/ml (OR 1.23, CI 0.66-2.30; p = 0.506). Regarding OPR, no significant differences were observed when comparing patients with serum P4 < = or > 27/ng/ml or < = or > 40ng/ml (OR 0.88, CI 0.42-18.85; p = 0.736 and OR 1.41, CI 0.83-2.37; p = 0.203, respectively). Moreover, EPL did not significantly differ between serum P4 level groups (OR 1.93, CI 0.61-6.13; p = 0.265 and OR 1.2, CI 0.51-2.84; p = 0.674). Age (p < 0.001) and embryo quality (p < 0.001) are significantly related with OPR. Additionally, late follicular progesterone levels and OPR are significantly associated (OR 2.39, CI 1.16-4.95 in patients with P4 < = or > 27/ng/ml and OR 2.21, CI 1.07-4.55 in patients with < =40ng/ml and >40ng/ml). Limitations, reasons for caution Limitations include the retrospective design and the lack of live birth outcome data. Progesterone levels exceeding 60 ng/ml were not further analyzed to their precise value but grouped as 60 plus which did not allow to identify potential detrimental effects of very high P4 levels on treatment outcome. Wider implications of the findings Our data suggest that measuring P4 on the day of a single blastocyst transfer with MVP luteal phase support may not be indicative of OPR or the risk of EPL. This raises questions regarding the necessity for routine P4 measurements as well as the development of rescue strategies. Trial registration number ONZ-2023-0628

P-072 Fresh testicular sperm seems to yield more fertilization abnormalities and early pregnancy loss than frozen testicular sperm

Abstract Study question How do ICSI outcomes using fresh testicular sperm, compare to those using frozen samples cryopreserved from the previous fresh testicular sperm extraction (TESE)? Summary answer Although fresh testicular sperm led to a better fertilization and pregnancy rate,it yielded more fertilization abnormalities and miscarriages than frozen sperm for the same couples. What is known already Testicular biopsies can be performed in cases of severe male infertility to search for spermatozoa. In the current era of advanced reproductive techniques, ICSI in association with TESE may be an effective treatment for patients with azoospermia. TESE could be performed the day of oocyte pick-up, or instead, it may be accomplished previously, and in this case TESE spermatozoa are cryopreserved for the future ICSI cycles. However, when reviewing the literature, not a lot of papers tried to compare the ICSI outcomes between fresh and frozen testicular sperm in the same couples. Study design, size, duration This retrospective study included the clinical and embryological data of pairs of fresh and frozen sperm injection cycles from couples after TESE between 2010 and 2023. The first ICSI was performed with fresh spermatozoa retrieved from TESE. The subsequent ICSI cycles used frozen spermatozoa from the first TESE. Participants/materials, setting, methods All infertile couples that underwent ICSI+TESE with testicular sperm cryopreservation in the study period were identified (110 couples). Of whom, 34 couples who underwent 40 subsequent ICSI cycle using frozen spermatozoa from the previous TESE were included. ICSI cycles were divided in: Group 1:ICSI with TESE and fresh testicular sperm injection Group 2:ICSI with frozen testicular sperm injection ICSI outcomes were compared for: fertilization/fertilization abnormalities rate, embryo quality, pregnancy and miscarriages rate, and live birth rate. Main results and the role of chance The average age of men who underwent ICSI was 42 years, while that of women was 33 years. On average, the couples had their ICSI with frozen sperm within 1.7 years after the TESE. There were no significant differences in women’s clinical parameters, the type of ovarian stimulation used nor its primary outcome between the two ICSI attempts. Although the primary outcome of ICSI showed a significantly higher fertilization rate for fresh testicular sperm compared to frozen sperm (64.4% vs 44.8%; p = 0.003), there was a tendency for more fertilization abnormalities when fresh testicular sperm was used (8.3% vs 6.2%, p = 0.4). Despite a better quality of cleavage embryos in ICSI with fresh testicular sperm (43.9% vs 39.2%, p = 0.6) and a higher pregnancy rate for the same group (21.9% vs 15.9%, p = 0.52), the miscarriage rate was twice as high when compared to the miscarriage rate of ICSI with frozen testicular sperm (6.7% vs 3.4%, p = 0.8), indicating that cryopreservation of testicular sperm could play a role in selecting better sperm for micro-injection. As for the live birth rate, ICSI with frozen sperm led to not statistically inferior rates when compared with ICSI with fresh sperm in the same couples (6.7% vs 10%, p = 0.6). Limitations, reasons for caution Due to our inclusion criteria, the study population may seem to be too reduced to reach significant results. To gain more informative insights, we recommend combining results from different centers and referring to the etiology of azoospermia that led to the TESE procedure. Wider implications of the findings Genetic testing and chromosomal aberration detection on testicular spermatozoa before and after cryopreservation could be proposed to compare the proportion of defected spermatozoa in both types of samples. Trial registration number not applicable

P-243 Effect of delayed development of embryos 7 days on embryo quality, ploidy and reproductive cycle outcomes

Abstract Study question How does delay in embryo development before Day7 affect blastocyst quality, ploidy and reproductive cycle outcomes? Summary answer Embryo quality and ploidy are statistically significantly lower in the Day7 group, no difference in implantation rate was found. What is known already In clinics that allow extended culture up to Day7, on average 5% of embryos have a 48-hour delay in development, blastulation occurs on the 7th Day (Hammond et al., 2018). Day7 blastocysts may have good morphology and be euploid, give a pregnancy and birth of a healthy baby. Back in 2013 it has been proven that embryos from patients over 35 years of age take longer to form a blastocyst (Shapiro et al., 2013). Cultivation until Day 7 is justified for couples with a poor prognosis in cycles. Study design, size, duration This was a retrospective cohort study in a private Delta Fertility Clinic of consecutive female infertility patients (n = 422) between Sept 2021 and Sept 2022. Average age of the patients was 39,7 years who underwent frozen embryo transfer (FET) of a single euploid day 5, 6 or 7 blastocyst. Then we compared the outcomes in the 5, 6 and 7 Day groups. Participants/materials, setting, methods The results of culture and subsequent biopsy of trophoectoderm with PGT-A of 2095 blastocysts (1516 were tested with PGT-A) in 482 cycles were analyzed, of these, embryos Day7 suitable for biopsy were 9.8% (149). Blastocysts were classified as good (AA/AB/BA/BB) or poor (AC/BC/CA/CB) (Gardner D.K. et al., 1999). Cultivation performed in single drops, biopsy was performed using the flicking method. Statistical analysis was performed using the Pearson’s Chi-Squared Test. Main results and the role of chance In 3.4% of cases, patients had only Day7 blastocysts. Poor quality blastocysts were predominant in D7 blastocyst group (good quality: 91.0%, 65.2%, 46.3%; poor quality: 9.0%, 34.8%, 53.7%, P < 0.001 for D5, D6 and D7 blastocysts; respectively). The number of euploid embryos was significantly lower in the Day 6 and Day 7 group compared to Day 5 (51.8% and 50.0% vs 64.4%, p < 0.001 and p < 0.001). We found no differences in cycle outcomes, implantation rate was 52,7%, 49,6% and 38,5% for 5, 6 and 7 Day respectively (P = 0,474). We speculate that this may be due to the small number of day 7 embryo transfers (n = 13), but we have 5 live births in our clinic after euploid Day7 embryo transfer. Limitations, reasons for caution Due to the decrease in the percentage of euploid embryos on day 7, such embryos should be transferred only after PGT-A. There is little information about the outcomes of Day 7 embryo transfers because they are the last embryos to be transferred. Wider implications of the findings Cultivation before day 7 should be introduced into routine practice as an option for patients over 35 years of age or with a known poor prognosis. Having previously assessed the laboratory’s capabilities and financial costs. Trial registration number not applicable

O-247 Validation of an AI (Artificial intelligence) model assessing oocyte quality in a single donor program with frozen-thawed oocytes for blastocyst prediction

Abstract Study question Can an AI model predict blastocyst development in a specific subset population of frozen-thawed oocytes in a single donor program? Summary answer An AI oocyte quality score is predictive of blastocyst formation of frozen-thawed oocytes using post-ICSI images. What is known already The ability to score oocytes based on quality is of paramount importance. A methodology to categorize oocyte quality continues to elude IVF professionals. CHLOE OQ™ is an AI oocyte-quality (OQ) score designed to analyse a single oocyte Timelapse image and provide an OQ score. Responsible use of AI needs the validation of models across different clinical scenarios. The increasing use of frozen-thawed oocytes in IVF has necessitated to objectively score the developmental potential of this oocytes. The objective of this study was to determine if the morphological characteristics of frozen-thawed oocyte images are predictive of blastocyst formation. Study design, size, duration Retrospective cohort study using time-lapse videos of 1318 post-ICSI frozen-thawed donated oocytes between January 2021 and August 2023, with known blastulation outcomes from a single private clinic. Participants/materials, setting, methods Oocytes were evaluated using OQ Score and placed into categories according to their OQ score. Group A: >0.6, Group B: 0.3-0.59, Group C: 0.001-0.29 and Group D: 0. The correlation between OQ score, embryo quality (EQ) score and blastocyst development was measured with binary logistic regression (AUC) and a 2-sample t-test. Chi-square was performed to determine if there were significant differences in blastocyst development rates across these distinct oocyte quality groups. Main results and the role of chance Oocytes that underwent blastulation exhibited a significantly higher OQ Score (7.0 ± 2.3) compared to those that did not blastulate (5.3 ± 3.0, p < 0.001). The predictive capacity for blastocyst formation was observed for OQ score (AUC= 0.67) and EQ Score (AUC= 0.93), with a baseline blastulation of 66% (n = 1318, p < 0.001). Of interest, as early as Day 3 a Blastocyst prediction score was highly predictive of blastulation (AUC= 0.92, p < 0.001). When assessing a subset of transferred oocytes, OQ Score had a higher predictive ability (AUC=0.71, n = 163, p < 0.001).Oocyte quality scores within OQ score groups showed a direct association with blastocyst formation rate: [Group A 74% (656/878), Group B 56% (130/231), Group C 43% (70/164), Group D 13% (3/23), p < 0.05]. The highest-scoring group demonstrated a five-fold increase in blastulation rate compared to the lowest-scoring group (p < 0.001). Furthermore, the highest-scoring group resulted in the highest proportion of good-quality embryos compared to the lowest-scoring group [78% (456/582) vs 0.19% (1/582), p < 0.001]. Limitations, reasons for caution Retrospective assessment of a single clinic. Results reflect the use of frozen-thawed donated oocytes. Therefore, the proportion of data within the four oocyte quality groups is not equally distributed and is biased towards high quality oocytes. This may impact the predictive ability within the smaller subgroups. Wider implications of the findings Developing an objective methodology to categorize oocyte quality post-retrieval is crucial. It is important to validate AI models in different clinical scenarios, for example in oocyte donor programs using frozen-thawed oocytes. AI is a tool that can be used to predict blastocyst development rates within a frozen donor oocyte program. Trial registration number not applicable

P-730 A feasibility study of the impact of the level of exposure to ambient air pollutants on outcomes of assisted reproductive technologies

Abstract Study question What is the impact of air pollution exposure on outcomes for couples having IVF treatment? Summary answer Environmental exposure to high levels of air pollutants may negatively influence IVF outcomes with potential implications for fertility treatment. What is known already Air pollutants are harmful substances in the atmosphere that have adverse effects on human health. Increased levels of air pollutants have been associated with respiratory disorders, cardiovascular disease, and increased morbidity in humans across all age groups. Existing evidence suggests that increased exposure to air pollutants may adversely affect fertility. The relationship between air pollutants and IVF outcomes remains poorly understood. Study design, size, duration This was an ethically approved prospective observational feasibility study conducted over a two-year period. Ten couples due to undergo an IVF cycle were recruited from a fertility centre in the UK. Environmental exposure to ambient air pollutant for particulate matter (PM) 2.5 and 10 micron size was measured using portable AirBeam monitors over the course of two weeks from commencement of the ovarian stimulation regime. Participants/materials, setting, methods Eligible participants were those aged 18-35 years old, undergoing fresh IVF, with a good ovarian reserve and ability to provide informed consent. Data of interest included: BMI, age, parity, ethnicity, smoking status. IVF data included: number of oocytes retrieved, embryo quality and cycle outcomes. AirBeam device data was extracted, analysed and correlated with IVF outcomes. Participants feedback was sought for qualitative input for future studies. Main results and the role of chance Total of 28,269 data points of ambient air pollutant data for PM 2.5 and 10 micron size were recorded amongst the ten participants. One participant was exposed to significantly high air pollutant levels during ovarian stimulation on certain days (PM2.5 - 81.06 µg/m³ and PM10 - 154.48 µg/m³) compared to standard annual air pollution level for the UK of 25 µg/m³ for PM2.5 and 40 µg/m³ for PM10. Two women did not have embryo transfer due to embryo arrest. Of those who had transfer, 50% (n = 4) had positive pregnancy tests however three experienced early pregnancy losses. At the time of writing there was one ongoing single viable pregnancy. On correlation of air quality data with clinical outcomes, the one participant who exhibited fluctuant very high exposure levels to the measured air pollutants, had unexpected low fertilization rate (20%) with IVF and no transfer due to embryo arrest. Of the nine participants returning the questionnaire feedback, all but one rated it acceptable to most acceptable to carry the device around. The main barrier was the size of device and battery life. All respondents were willing to recommend others to participate in a future similar study confirming feasibility to recruit. Limitations, reasons for caution As a feasibility study, with a limited sample size, our findings should be interpreted cautiously. The small cohort limits the generalisability of our results. Further research with larger sample sizes is required to validate our findings. Wider implications of the findings Understanding the impact of air pollutants on IVF outcomes is important for developing evidence-based guidance and interventions to optimise fertility treatment efficacy. Our study demonstrates the feasibility of personal air quality monitoring and emphasises the necessity for larger scale research and the practicality of pollutant exposure measurements in fertility treatments. Trial registration number REC reference: 21/NE/0233 IRAS project ID: 294240. Theramex BIRTH Grant

P-435 Superiority of modified natural cycle vs hormonal replacement therapy in non-menopausal egg recipients of advanced maternal age: clinical outcomes from 1273 single blastocyst transfer cycles

Abstract Study question Is the modified natural cycle (mNC) better than hormonal replacement therapy (HRT) for endometrial preparation in non-menopausal egg recipients of advanced maternal age? Summary answer In women above 40 years, mNC shows better reproductive and obstetrical outcomes compared to hormonal replacement therapy, especially in the subgroup 45-50 years old. What is known already There is extensive evidence showing the superiority of mNC over HRT in terms of live birth rates, miscarriages, and even obstetric complications. Apparently, the presence of the corpus luteum plays a major role in endometrial receptivity and embryo implantation, probably allowing for better placentation. Nevertheless, there is a general belief that the ovulatory cycle of older women is not the best choice for endometrial preparation, although paradoxically they are the group of patients who could benefit most from the reduction of obstetric complications. An increase in cancellation rates for mNC is claimed, without consistent data available. Study design, size, duration Retrospective single-centre comparative clinical study, including 1273 endometrial preparation cycles for frozen single-blastocyst transfer between January 2020 and December 2022: 892 (70,2%) on HRT protocol and 381 (29,8%) on mNC protocol. The two groups were homogeneous for donor’s age (25,7 years HRT vs 25,3 years mNC), recipient’s age (42,2 years HRT vs 42,1 years mNC), severe male factor (6% HRT vs 5% mNC), and the need for a sperm donor (28% HRT vs 26% mNC). Participants/materials, setting, methods Among 1273 started non-menopausal recipient’s cycles, we compared cancellation rates between HRT and mNC due to either medical reasons (inadequate endometrium, bleeding, premature ovulation) or other issues (no embryo survival, COVID-19, etc). We studied the outcomes of the 1096 elective frozen single-blastocyst transfers performed, comparing HRT and mNC cycles for: pregnancy, clinical pregnancy, miscarriage, live birth and <35weeks preterm birth rates. We stratified the results by age and used Chi-square test to compare groups (p < 0,05). Main results and the role of chance The overall cancellation rate was 13.9%. No significant differences in cancellation for medical reasons were found between HRT (7%) and mNC (6.8%), p = 0.93. When we analyse results stratified by age, patients aged 39 or less (270 cycles: HRT 75.9%, mNC 24.1%) showed no statistically significant differences in pregnancy (58% vs 64.6%, p = 0.35), clinical pregnancy (50.7% vs 56.9%, p = 0.34), miscarriage (18.3% vs 18.9%, p = 0.93), and live birth rates (41.5% vs 46.2%, p = 0.5). In the 40-44 age group (494 cycles: HRT 64.2%, mNC 35.8%), no statistically significant differences were found between HRT and mNC in pregnancy (58.2% vs 61.5%, p = 0.47), clinical pregnancy (50.8% vs 51.1%, p = 0.96), and live birth rates (40.4% vs 45.8%, p = 0.25). The HRT group had a significantly higher miscarriage rate (21%) compared to mNC (10%), p = 0.03. For patients aged 45-50 years (332 cycles: HRT 72%, mNC 28%), no statistically significant differences were observed in pregnancy (56.5% vs 62.4%, p = 0.33) and miscarriage rates (24.3% vs 15.1%, p = 0.18). Clinical pregnancy (44.8% Vs 57%, p = 0.04) and live birth rates (33.9% vs 48.4%, p = 0.01) were significantly higher in the mNC group compared to HRT. No significant differences were found between HRT and mNC in preterm birth rates <35 weeks (3.6% vs 4.6%, p = 0.75). Limitations, reasons for caution This study is a single-centre retrospective comparative trial. Our homogeneity study included several confounding variables, however other bias such as severe uterine factor, embryo quality or repeated implantation failure cases were not taken into account. Wider implications of the findings Modified natural cycle should be encouraged as the first option for endometrial preparation in non-menopausal egg recipients of advanced maternal age, having shown superiority over hormonal replacement therapy in patients aged 45-50 years. Moreover, modified natural cycles are more patient friendly and cost-effective, with no increase in the cancellation rates. Trial registration number not applicable

P-408 Monozygotic twins from in vitro fertilization: can bring the rate closer to that of natural conception?

Abstract Study question Whether the occurrence of monozygotic twins in human in vitro fertilization is associated with zona manipulations, and can we explore approaches to reduce its rates? Summary answer Monozygotic twinning is partially caused by zona manipulation, and it can be lowered by creating a larger opening in the zona before embryo transfer. What is known already While the rate of monozygotic twins in spontaneous conception is 0.5% of live births, the frequency of monozygotic twins after in vitro fertilization-embryo transfer can be a few times higher. Various factors have been suggested to contribute to this phenomenon, including ovarian stimulation, intracytoplasmic sperm injection, embryo cryopreservation, extended culture, embryo quality, maternal age, genetic factors, and micromanipulation of the zona pellucida, such as assisted hatching and biopsy. Due to the fact that the overall rate is low, it is difficult to pinpoint the main reason(s) contributing to it, and there are no effective approach(es) to lower this rate. Study design, size, duration We studied monozygotic twins in 8,063 clinical pregnancies resulting from single blastocyst transfers from 2016 to 2023. Blastocysts, whether fresh or frozen, were transferred with or without biopsy for preimplantation genetic testing and/or blastocyst collapse. Clinical pregnancy, embryo implantation, including twin implantation, and clinical outcomes such as live birth, ongoing pregnancy, and birth weight were further evaluated. Monozygotic twin rates were compared between partial zona removal (larger hole) and no zona removal (small hole) Participants/materials, setting, methods Monozygotic twins were initially evaluated in blastocysts with or without biopsy in 732 clinical pregnancies after fresh or frozen embryo transfer. Subsequently, monozygotic twins were further evaluated in 7331 clinical pregnancies after an approach to open a larger hole in the zona before blastocyst transfer. Additionally, 56 monozygotic twins were evaluated for live birth and birth weight outcomes. Main results and the role of chance In the context of fresh blastocyst transfer, the monozygotic twin rate following biopsy was significantly higher than that for blastocysts that did not undergo zona manipulation (3.4% vs. 0.29%; p = 0.025). After blastocysts underwent biopsy and cryopreservation/warming, the monozygotic twin rate was 2.79%, a similar rate as observed in fresh blastocyst transfer after biopsy. However, when frozen blastocysts (either biopsied and/or collapsed) underwent partial zona removal before transfer, the rate of monozygotic twins significantly decreased to 0.6% (p = 0.00001). When we further examined whether the biopsy procedure increased the monozygotic twin rate (28/4947) under the conditions that partial zona removal was performed by comparing those with blastocysts without biopsy (16/2384), we did not find a significant difference (0.57% vs. 0.67%, p = 0.587) between the two treatments. In this study, there were 34 twin live births (60.7%) including 5 ongoing twin pregnancies, and 9 (16.1%) singleton live births, contributing to an overall live birth rate of 76.8%. Ten cases (17.8%) experienced pregnancy loss with heartbeat cessation during the first trimester. Furthermore, 3 cases (5.4%) had abnormal pregnancies. When the birth weights were evaluated among the monozygotic twins, it was found that lower birth weight (< 1500g) was only observed in twin live birth. Limitations, reasons for caution This study only included data from one large clinic in the US under the conditions and protocols (embryo culture, embryo biopsy, and cryopreservation) used in this clinic. Different patients and protocols would affect the rate differently. Wider implications of the findings Zona manipulations are associated with an increased monozygotic twin rate in both fresh and frozen embryo transfers. This effect can be mitigated by creating a larger opening in the zona before embryo transfer, and this approach can bring the monozygotic twin rate closer to that of natural conception. Trial registration number NA

O-286 Weight loss interventions prior to infertility treatment does it make sense?

Abstract Obesity in women is negatively associated with reproductive outcomes such as decreased natural conception and pregnancy rate in assisted reproductive technique (ART), as well as increased risks of miscarriage and maternal and foetal complications, including risks of perinatal and neonatal death. The reproductive consequences of obesity stem from a combination of ovarian, uterine and systemic pathophysiological changes, although not all fully elucidated. These alterations contribute to reduced oocyte and embryo quality, reduced uterine receptivity and systemic inflammatory responses. Various guidelines recommend lifestyle interventions based on dietary and/or physical activity targeting at a 5 to 10% reduction in body weight as an initial step prior to fertility treatment for women with infertility and overweight or obesity. However, evidence from well powered randomised controlled trials (RCTs) assessing the effectiveness of lifestyle interventions aiming at weight loss prior to fertility treatments is scarce and not unequivocally positive with respect to reproductive outcomes. First, this presentation will focus on the effectiveness of pre-conception lifestyle intervention targeting weight loss, on reproductive outcomes documented in the latest systematic review (SR) and meta-analysis and preliminary results of an individual participant data meta-analysis (IPDMA). The latest SR in 2021 (Obesity reviews DOI: 10.1111/obr.13325) including 15 RCTs until March 2020 (N = 1852 women) suggests more weight loss in the intervention group. An increase in live birth rates (LBR) in nine RCTs (N = 1203 women) and a higher natural conception rate following lifestyle intervention compared to no intervention were reported. However, no effect of lifestyle intervention preceding ART in six RCTs (N = 1040 women). With respect to potential harm of lifestyle intervention there is no significant increased risk of early pregnancy loss. Complications during pregnancy, such as early pregnancy loss and maternal, fetal and neonatal outcomes are underreported in most included studies. The VENUS IPDMA (BMJ Open 2022;12:e065206) analyzed individual patient data of 11 RCTs of 14 eligible RCTs (N = 1903 participants; 1010 in the intervention group and 893 in the control group). Preliminary data (abstract ESHRE 2023) showed that physical activity and/or dietary interventions prior to fertility treatment resulted in more weight reduction compared to those in the control group in 11 RCTs. The intervention group, however, did not have a significantly higher rate of LBR in nine RCTs (N = 1702 participants). Compared to the control group, the intervention group had smaller waist and hip circumference, lower systolic and diastolic blood pressure, and lower triglycerides, total cholesterol, glucose, and insulin levels at the time of study follow up. Do we have a definitive answer to the question: “weight loss interventions prior to fertility treatment; does it make sense” using data of IPDMA? Shortcomings of the available evidence regarding the effectiveness and safety of dietary and/or physical activity interventions in women with overweight or obesity prior to fertility treatments will be presented. Limitations of the studies and future perspectives and challenges in this field of research will be highlighted. Finally, the question “does it make sense” is broader than the effectivity of the intervention. Do we as clinicians adopt our role as health advocates to advice and guide future parents to adopt a healthy lifestyle, with weight reduction as a result? Could this lead to a healthier lifestyle for the future of parents and children? To conclude: “does it make sense” is not an easy question to answer.

P-666 Comparison of IVF outcomes in women undergoing IVF stimulation using progestin primed ovarian stimulation (PPOS) Vs GnRH antagonist (GnRH-Antag) protocol

Abstract Study question Can PPOS achieve improved patient & provider satisfaction while simultaneously blocking LH-surge, maintaining similar IVF outcomes like oocyte/embryo yield/clinical pregnancy rates(CPR) when compared to GnRH-Antag? Summary answer PPOS can achieve similar IVF outcomes as compared to GnRH-Antag protocol and is a patient friendly, cost-effective choice for ovarian stimulation without affecting oocyte/embryo yield/CPR. What is known already GnRH-Antag protocol is among the most widely used methods, as it blocks LH surge & significantly lowers the risk of OHSS [Lambalk CB,2017]. However, this protocol has disadvantages like frequent monitoring, dose adjustment, high therapeutic cost and multiple injections[Ata B,2015]. PPOS successfully prevents ovulation and provides comparable IVFoutcomes (oocyte yield, blastocyst/euploid embryo rates, CPR) along with patient cost savings and increased convenience across all infertility diagnoses [Annalyn M.Welp, 2023]. It should be noted that most studies are non-randomized and comprise low to moderate-quality evidence. However, the consistency of the findings across different studies from different centres provides assurance in effect estimates. Study design, size, duration A retrospective cohort study conducted at a tertiary care fertility unit. Data for 449 women aged 21 to 40 years undergoing IVF stimulation using PPOS (n = 226) and GnRH-Antag (n = 223) protocol was retrieved from the hospital’s electronic database and analysed for a period between September 2022 to October 2023. Study population consisted of women undergoing freeze-all strategy either for lowering the risk of OHSS (hyper-responders) or for embryo pooling in view of very low ovarian reserve. Participants/materials, setting, methods For both groups either rFSH/rLH/HMG were used & doses were indivisualised based on patient’s age, BMI & ovarian reserve. PPOS group received twice daily dose of medroxyprogesterone acetate tablet(10 mg)/dydrogesterone(10 mg) from Day2 of cycle. GnRH-Antag group received Inj.Cetrorelix acetate (0.25 mg) daily as flexible protocol. Final oocyte maturation was induced with Triptorelin(0.2mg)/Choriogonadotropin alfa(250mcg)/both, once 2-3follicles reached 18-20mm. Transvaginal oocyte pick-up was performed 35-36h later. Women in PPOS group underwent freeze-all strategy followed by Frozen-Thawed Embryo-transfer. Main results and the role of chance Normally distributed continuous variables were compared using independent t-test, and categorical variables were compared by χ2 and Fisher’s exact test, where appropriate. Patient demographics, clinical/ovarian stimulation characteristics, embryological and IVF outcomes were compared for two groups. Patient demographics {Age(p-value:0.08), BMI(p-value:0.06), duration(p-value:0.09)/type(p-value:0.76) of infertlity}, total gonadotropin dose (p-value: 0.39) and duration of ovarian stimulation (p-value: 0.81) were comparable between the two groups. Though the mean LH level on trigger day was slightly higher in PPOS group (5.53 IU/L) than GnRH-Antag group (3.24 IU/L) (p-value: 0.02), statistically significant difference was not seen in the oocyte yield (p-value: 0.18), M-II oocytes (p-value: 0.17) & fertilization rate (p-value: 0.23) in the two groups. The number of Day3 (p-value: 0.65), Day5 (p-value: 0.07) & Day6 (p-value: 0.08) embryos available in the two groups were also comparable. IVF outcomes in terms of positive B-HCG result (p-value: 0.68), clinical pregnancy rate (p-value: 0.61) & miscarriage rate (p-value: 0.27) showed no statistically significant difference in the two groups. Thus, we conclude that PPOS can achieve similar IVF outcomes as compared to the GnRH-Antag protocol, while at the same time proving to be patient friendly by reducing the need of multiple injection pricks, injection cost and the frequency of hospital visits. Limitations, reasons for caution This is a retrospective study and hence randomized comparison was not possible. PPOS can be used only when freeze-all strategy is planned, fresh embryo-transfer cannot be considered if need arises depending upon embryo quality. IVF service providers have to depend on patient compliance for using oral progestins to block LH-surge. Wider implications of the findings Study findings suggest that PPOS is a patient friendly and equally effective and safe IVF stimulation protocol when compared to GnRH-Antagonist. More RCT will be useful for investigating different aspects of PPOS (it’s utility for women with different ovarian reserve functions, conventional versus flexible approach, optimal dose of each progestin). Trial registration number Not applicable

P-122 The impact of severe male factor on morphokinetic embryo development in low-prognosis patients according to the POSEIDON criteria: an analysis of 10366 injected oocytes

Abstract Study question Does severe male factor infertility (SMF) impact embryo morphokinetic behavior in low-prognosis women as stratified by the Patient-Oriented Strategies Encompassing IndividualizeD Oocyte Number (POSEIDON) criteria? Summary answer Embryos from SMF patients showed lower KIDScore ranking than those from non-SMF (nSMF) patients, which may justify the reduced clinical outcomes contrasted to Control group. What is known already It has been reported that 37.1% of poor responder patients (POR) have infertile male partners. While the maternal influence has been well-investigated, the role of seminal quality on reproductive outcomes in POR remains questionable. Embryo monitoring with timelapse imaging (TLI) may be a valuable approach to evaluate whether sperm quality relates to association between POSEIDON classification and the competences of developing embryos. The aim of this study was to investigate whether SMF impacts embryo quality and morphokinetic behavior in the four groups of low-prognosis women as stratified by the POSEIDON criteria. Study design, size, duration This historical cohort-study was performed in a private university–affiliated IVF center, between Mar/2019 and Apr/2022. Kinetic data were analyzed in 10366 embryos cultured in a TLI incubator until day five, derived from 2272 patients undergoing ICSI. Patients were split into eight groups according to the POSEIDON criteria and the presence or absence of SMF (<5 × 106 sperms/mL). Embryo morphokinetics were compared among the groups (post hoc achieved power > 90%). Participants/materials, setting, methods Injected oocytes were cultured in the EmbryoScope+ incubator, which recorded the following kinetic markers: timing to pronuclei appearance and fading (tPNa and tPNf), two (t2), three (t3), four (t4), five (t5), six (t6), seven (t7), and eight cells (t8), morulae (tM), start of blastulation (tSB) and blastulation (tB). Durations of second and third cell cycles (cc2 and cc3) and timing to complete synchronous divisions s1, s2, and s3 were calculated. The KIDScore ranking was recorded. Main results and the role of chance POSEIDON group 1 embryos showed significantly slower tPNa, tPNf, t2, t3, t4, t6, and t7 compared to those from Control group. POSEIDON group 2 embryos showed significantly slower tPNa, t4, t6, t7, t8, and tM compared to those from Control group. Embryos in POSEIDON 2 SMF subgroup took longer than those in POSEIDON 2 nSMF subgroup, and those in both Control subgroups, to achieve tPNf, t2, t3, t5, tSB. POSEIDON group 3 embryos showed significantly slower t8, and cc2 compared to those from Control group. POSEIDON group 4 embryos showed significantly slower tPNf, t2, t3, t4, t5, t6, t7, t8, s2, and s3 compared to those from Control group. For POSEIDON groups 1, 2 and 4, and Control group, the KIDScore ranking was significantly different between SMF and nSFM subgroups. KIDScore ranking was significantly different across all the subgroups in POSEIDON 3. Irrespective of sperm quality, implantation and miscarriage rates were significantly improved in Control subgroups compared to POSEIDON 2 subgroups, there were trends toward improved clinical outcomes in Control subgroups compared to POSEIDON 3 subgroups, and pregnancy, implantation and miscarriage rates were significantly improved in Control subgroups compared to POSEIDON 4 subgroups. Limitations, reasons for caution The use of historical cohort groups is a drawback. Despite the eligibility criteria for inclusion in the analysis, potential differences in the baseline characteristics cannot be ruled out. Wider implications of the findings The results presented contribute to the understating of the relationship between sperm quality and embryo morphokinetic behavior in POSEIDON patients. Embryo quality, as assessed by TLI-imaging, and clinical outcomes were significantly lower in POSEIDON SMF patients, suggesting that both oocyte (quantity and quality) and sperm are determinant of embryo development. Trial registration number N/A

O-249 METAPHOR: METabolic imaging through AI-powered Phasor-based Hyperspectral analysis and Organelle recognition for the classification of human blastocysts

Abstract Study question Can we classify human blastocysts non-invasively using hyperspectral imaging? Summary answer HS imaging can significantly enhance our classification of human embryos and reveal numerous cellular features present in the embryo invisible to brightfield imaging. What is known already Non-invasive measurement of metabolism is a safe and powerful tool to reliably predict oocyte and embryo quality. We have previously shown that our METAPHOR technology, based on HS imaging coupled to artificial intelligence, was able to separate mouse oocyte categories according to maternal age with an average AUC of 96.2% and predict blastocyst formation (AUC 82.2%). Moreover, it was able to classify mouse embryos deprived from different nutrients with an average AUC of 93.7% showing clear superiority to grading provided by experienced embryologists (51%). Study design, size, duration A total of 74 human embryos donated for research at the blastocyst stage have been imaged to date. Embryos were warmed and imaged according to our established protocol and survival was statistically indistinguishable from control embryos. Participants/materials, setting, methods Embryos are imaged using multiphoton illumination near the infra-red. The HS detection covered the simultaneous measurement of the whole visible spectrum. Embryos are then allowed to implant in our proprietary 3D ex vivo platform. Implantation is monitored up until day 9 before immunostaining is performed to quantify presence and prevalence of lineage and implantation markers. Main results and the role of chance After imaging, embryos were placed on the 3D ex vivo implantation platform and let implant. Successful implantation (75.41%) was scored by observation of the characteristic deformation of collagen fibers within the matrix and staining for relevant cell lineages (OCT4, pMLC2). Interestingly, we observed that embryos with a higher trophectoderm (TE) grading tended to implant with a higher efficiency and penetrate deeper into the matrix than embryos with lower grading. HS images have unveiled a profound cellular diversity within embryos, elucidating distinctions between the Inner Cell Mass (ICM) and TE, as well as revealing different metabolic states defined by NADH dominance, FAD dominance, protoporphyrin levels, and apoptosis. HS raw images are processed using our HS-phasor approach which allows quick computing by converting the multidimensional HS data into comprehensive visual representation of bidimensional histograms and phasor plots. In summary, our analysis pipeline encompasses the algorithms to detect and segment the embryos, the computation of histogram and phasor-plot, and the classifier. AI allows us to classify embryos in distinct spectral spaces based on their metabolic features, paving the way to the establishment of a new grading system based on HS imaging. Limitations, reasons for caution The cohort size is still limited, and ongoing experiments will increase the dataset needed for more accurate prediction of embryo implantation. The implantation platform is an in vitro test that will require future clinical validation. Wider implications of the findings The safety and speed of METAPHOR warrants its incorporation at the IVF clinic, adding an additional layer of information to the current embryo selection methods. Moreover, the non-invasive measurement of the metabolic function opens new alleys of research such as the effect of media and culture supplements on embryo culture. Trial registration number Not applicable

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

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

O-127 Blastocyst expansion speed assay: a putative artificial intelligence-powered tool for embryo selection. Retrospective study involving 2184 blastocysts and 786 PGT-A cycles

Abstract Study question Is blastocyst expansion-speed, examined through Artificial-Intelligence (AI) between time of blastulation (tB) and time of expanding blastocyst (tEB), associated with embryo blastulation and reproductive competence? Summary answer Blastocyst expansion-speed-assay (ESA) was significantly associated with euploidy and live-birth rates, being discordant with clinical embryologists’ ranking in 50% of embryonic cohorts. What is known already Blastocyst expansion is one of the earliest morphogenetic events in mammalian development. It is essential for the establishment of the blastocyst layout and requires trophectoderm integrity. Several studies suggested that expansion is a valuable feature for ranking and prioritizing blastocysts for transfer based on their developmental competence. Time-lapse technology (TLT) implementation in IVF allows a deeper understanding of blastocyst expansion dynamics. In this study, we combined TLT and AI to develop a blastocyst-ESA and investigate its association with embryo blastulation and reproductive competence. Study design, size, duration Retrospective study including 2184 blastocysts cultured in EmbryoScope incubators during 786 PGT-A cycles across 2013-2020. Videos were analyzed through an AI-powered tool (CHLOETM, Fairtility). The expansion-speed was calculated as [(Δ embryo proper area at tEB – embryo proper area at tB) / (Δ tEB - tB)]. ESA was tested for its association with embryo quality, euploidy and live-birth rate (LBR) in 548 vitrified-warmed single euploid transfers. A simulation of ESA putative clinical effectiveness was conducted. Participants/materials, setting, methods ICSI, trophectoderm biopsy of fully-expanded blastocysts without day3 zona-drilling, and qPCR/NGS to assess full-chromosome uniform aneuploidies were performed. tB and tEB, expressed as hours-post-insemination (hpi), embryo proper area (in µm2) at both these stages, and blastocyst quality (EQ-Score from 0 to 1) were all automatically recorded through CHLOE. Possible confounders (e.g., maternal age, male factor, and cause of infertility) were considered. Regression analyses were conducted to adjust the data. Main results and the role of chance The average ESA was 705±458 µm2/hour. Higher ESA was associated with higher EQ-Score. Euploid blastocysts showed higher ESA than aneuploid (761±465 µm2/hour versus 667±449 µm2/hour; p < 0.01). ESA was also associated with LBR per euploid blastocyst transfer (LB: 873±438 µm2/hour versus 736±467 µm2/hour; p < 0.01). Of note, maternal age showed no association with ESA. Multivariate regressions outlined a + 5.1%-increase in the chance of euploidy and a + 6.3%-increase in chance of LB, every +100 µm2/hour-increase in ESA. ESA and embryologists were discordant in grading top-quality embryos in each cohort in 50% of the cases. In 59% of the 352 cohorts where both euploid and aneuploid blastocysts were obtained, ESA would have ranked the former embryos as top-quality. In the 216 cycles with ≥2 euploid blastocysts obtained and ≥1 transferred, ESA would have prioritized (i) a competent embryo in 46% of the cases, (ii) an incompetent embryo in 20%, but (iii) in 33% its value could not be assessed (i.e., the top embryo according to ESA has not been transferred yet). When compared to the embryologists, ESA would have been (i) equally-effective in 49% of the cases, (ii) more effective in 12-24%, and (iii) less effective in 5%-26%. Limitations, reasons for caution Retrospective single-center study. Both continuous and sequential media were used, although they were not associated with the outcomes under investigation. To assess the clinical value of ESA, a prospective study among first transfers is warranted. Wider implications of the findings ESA is an automated, unbiased, and easily-applicable measurement that certainly deserves further appraisal. If validated in prospective studies, AI-based selection algorithms could include this assessment to increase their predictive power. Trial registration number None

P-728 Age limits in medically assisted reproduction: Assessing clinical and pregnancy outcomes in women over 50 years of age undergoing donor oocyte in vitro fertilization

Abstract Study question Is pregnancy in women over 50 years of age undergoing donor oocyte IVF associated with adverse clinical and pregnancy outcomes? Summary answer In vitro fertilization treatment in women over 50 years of age using donor oocytes is not associated with adverse clinical and pregnancy outcomes. What is known already In vitro fertilization with donor oocytes (d-IVF) has become a common strategy for managing cases of very advanced maternal age. However, there is ongoing scientific debate entailing legal and societal implications regarding the age limit beyond which d-IVF should no longer be considered safe. Different age limits and guidelines have been proposed, with no globally accepted consensus. The most commonly reported age limit is 50 years, representing the median age of menopause. However, there is a lack of robust data to support that d-IVF beyond the age of 50 is associated with adverse clinical and pregnancy outcomes. Study design, size, duration A retrospective single-centre study was conducted to evaluate medical records of d-IVF cycles between July 2021-January 2023. A total of 575 medical records of women undergoing d-IVF were analyzed. The study population was divided into two groups based on maternal age: women aged 45-49 years (group A, n = 318) and women aged 50-54 years (group B, n = 257). The outcome measures were positive-hCG, clinical pregnancy, viable pregnancy at 12 weeks of gestation, pregnancy loss and live birth. Participants/materials, setting, methods For oocyte recipients advanced maternal age was the sole infertility etiology and endometrial preparation was performed employing conventional protocols. Oocyte donors were up to 35 years of age and oocyte donation cycles were performed according to national guidelines. Single or double embryo transfers (ETs) were performed at the cleavage or blastocyst stage. Embryo quality assessment was performed according to Veeck and Gardner grading systems. Statistical analysis was performed employing R Programming Language for Statistical Purposes. Main results and the role of chance The mean age of group A was 47.02 ± 1.35 while the mean age of group B was 51.84 ± 1.59. No statistically significant difference was observed regarding the age of oocyte donors (25.17 ± 4.18 vs 26.09 ± 4.13; p-value=0.26). Considering embryo transfer, 90 cleavage stage and 228 blastocyst stage transfers were performed in group A, and 75 cleavage stage and 182 blastocyst stage transfers were performed in group B (p-value=0.23). No statistically significant difference was observed between the two groups with regards to the number of transferred embryos (1.89 ± 0.35 vs 1.90 ± 0.36; p-value=0.69) and embryo quality (Group A: Good 157, Fair 143, Poor: 18; Group B: Good 121, Fair 117; Poor 19; p-value=0.66). Considering clinical and pregnancy outcomes no statistically significant difference was observed between the two groups regarding positive hCG rate (147/318 vs 109/257, respectively; RR: 0.91; 95%CI: 0.76-1.10; p-value=0.36), clinical pregnancy rate (129/318 vs 98/257, respectively; RR: 0.94; 95%CI: 0.77-1.15; p-value=0.55), viable pregnancy rate at 12 weeks of gestation (96/318 vs 70/257, respectively; RR: 0.90; 95%CI: 0.70-1.17; p-value=0.44), pregnancy loss rate (39/129 vs 33/98, respectively; RR: 1.13; 95%CI: 0.76-1.63; p-value=0.58) and live-birth rate (90/318 vs 65/257, respectively; RR: 0.91; 95%CI: 0.68-1.17; p-value=0.42). Limitations, reasons for caution The retrospective and the single-centre nature of the study are important reasons for caution. In addition, the relatively small sample size may serve as another limitation. The lack of neonatal outcomes may also be considered an additional limitation when interpreting data on the safety of d-IVF in the over-50s. Wider implications of the findings Women over 50 who undergo d-IVF are not at increased risk of complications. This highlights the need to reconsider age limits and provide robust evidence ascertaining both safety and reproductive autonomy. Studies are needed to assess neonatal outcomes and maternal age impact on long-term infant health from an epigenetic standpoint. Trial registration number Not applicable

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

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

P-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-065 PGT-A reveals triploid embryos among normally fertilized oocytes: Frequency and blastocyst features

Abstract Study question Is there any embryo parameter that could differentiate 2 pronucleus (PN) triploid from 2 PN euploid embryos? Summary answer Accuracy in the fertilization check is still necessary as 2 PN but triploid embryos are due mostly to retention of the second polar body (PB). What is known already Since blastocyst culture has evolved and the development of genetic technologies enables to know the karyotipe of the embryos, the concept of normal fertilization is being revised. Commonly, correct fertilization is denoted by the appearance of 2 PN 16-18h after insemination. Deviation from 2PN is considered evidence of abnormal fertilization and ploidy anomalies. However, not all oocytes exhibiting 2 PN are euploids. After performing NGS in trophectoderm biopsies for preimplantation genetic testing for aneuploidy (PGT-A), a percentage of embryos are categorized as triploids. Study design, size, duration Retrospective study including 1862 cycles from 1605 patients aged 18-45 years old who underwent PGT-A with NGS of trophectoderm biopsies in our IVF center from 2019 to 2023. From 6526 analyzed blastocysts in an independent Genetics laboratory, 59 were categorized as triploids. These 59 blastocysts were obtained from 57 PGT-A cycles and belonged to 54 women. In total, these patients had 266 biopsied blastocysts. From the remaining 207 embryos, 42 were called as euploids. Participants/materials, setting, methods In embryos cultured in time-lapse, fertilization was re-evaluated to check the appearance of 2PN and the second PB extrusion. Genetic triploid embryos were analysed in relation to the day of biopsy (day 5 vs day 6), the trophectoderm quality and the timing of the first cell divisions (time of cleavage from 2 to 6 cells: t2, t3, t4, t5, t6). Categorical variables are shown as rate. T-student tests were performed. P-value<0.05 was considered statistically significant. Main results and the role of chance The re-evaluation of the fertilization of the 59 triploids categorized as 2 PN revealed 50 oocytes 2 PN, 8 embryos 3 PN and 1 embryo 2 PN + 1 micronuclei configuration. Therefore, the prevalence of real triploidy in 2PN embryos was 0,8% (n = 50/6526; CI95%:0,6-1,0%). These data showed that 2PN zygotes may give rise to embryos with altered ploidy. From 27 triploid 2PN embryos was possible to access to the whole development time-lapse images. Just 33,3% of triploid embryos exhibited the extrusion of the second PB, while 100% of euploid sibling embryos showed this extrusion (N = 9/27 vs N = 24/24,;p<0,005). Comparing 2 PN triploid and the rest of the embryos from the same cohorts, there were no differences regarding the day of biopsy (N = 31/176 day 5 biopsy, 17.6% vs N = 19/81 day 6 biopsy, 23.5%; p > 0.05). On the contrary, a worse blastocyst quality showed a significant relation with triploidy (N = 23/161,14,2% of good quality embryos are triploid vs N = 27/96, 38,6% of fair quality embryos are triploids; p:0.006).Embryos cleavage time points up to 6-cell stage did not show any significative difference between triploid and euploid embryos (t2: 27.36h vs 25.8h; t3: 38.5h vs 35.6h; t4 39.4h vs 37.2h; t5 52.8h vs 47.0h; t6 56.2h vs 52.1h; all comparisons p > 0.05). Limitations, reasons for caution The incidence of triplody from 2PN zygotes may vary among IVF laboratory settings. The small sample size may be responsible of no difference in morphokinetics. Wider implications of the findings Fertilization check should persist in IVF procedures. Special consideration should be given to the second PB extrusion as triploidy is the most common form of aneuploidy observed in the first trimester miscarriages. The role of diploid spermatozoa, the size of the pronucleus and location should be explored in further studies. Trial registration number NOT APPLICABLE