Advanced Maternal Age Patients Research Articles

P-397 A retrospective analysis shows a high effect in reproductive outcomes as age increases associated to endometrial factor in IVF advanced maternal age (AMA) patients

Abstract Study question Is there an increasing risk of a negative reproductive outcomes with every passing year associated with endometrial factor in AMA patients? Summary answer The risk of implantation failure and pregnancy loss associated with endometrial factor increase in 2.0% and 1.2% respectively with each passing year in AMA patients. What is known already Nowadays, there is a delay in maternity due to lifestyle, leading to a progressive increase in the age of women at the first childbirth increasing infertility. However, no universal definition of AMA has been established yet. Typically, studies are performed between different groups of age, establishing arbitrary thresholds. Besides, studies about AMA infertility mainly focus on the decline in oocyte quality, but the effects of age on the endometrium are often overlooked. Therefore, there is a lack of studies centered on endometrial factor and considering age year-by-year to be able to strictly establish when fertility decline starts and its magnitude. Study design, size, duration This retrospective study included a total of 33,735 single embryo transfers from 21,506 IVF AMA patients (>35 y.o, BMI 19-25 kg/m2) performed with high quality embryos (embryos from donated oocytes (donor <35 y.o) or euploid embryos from their own oocyte cohort) transferred in day 5 in an endometrial Hormonal Replacement Therapy (HRT) cycle. Evaluated transfers were performed between 2001 and 2022 in 19 clinics in Spain. This study was approved by an IRB (2203-FIVI-030-PD). Participants/materials, setting, methods A logistic regression analysis was performed, considering the patient’s age (42.93±3.45 y.o.) as a continuous and dependent variable and the reproductive outcome as an independent variable. The risk of obtaining a negative pregnancy outcome was estimated as risk ratios at a 95% confidence interval. A p-value<0.05 was considered as statistically significant. Reproductive rates (live birth and pregnancy loss) were calculated and compared each passing year in the studied range of ages (35-51 y.o). Main results and the role of chance The rates of live birth, Implantation failure and pregnancy loss associated to endometrial factor were affected as age increased in IVF AMA patients (>35 y.o). The implantation failure risk was increased in each passing year a 2% (Risk Ratio (RR) = 1.020 (1.003, 1.021) 95% confidence interval (95%CI), p-value=6.0E-09) and pregnancy loss risk was increased in each passing year a 1.2% (RR = 1.012, (1.003, 1.021) 95%CI, p-value=0.0096). Live birth rate decreased progressively from 42% observed in 35 y.o. to 32% in 50 y.o. Nevertheless, the pregnancy loss rate increased progressively from 29% in 35 y.o. to 37% in 50 y.o. Moreover, results shown a more drastic decrease of implantation failure rate after 42 years old and a pregnancy loss rate after 45 years old. Limitations, reasons for caution Only transfers from Spanish population under a HRT cycle were included, decreasing the results variability. However, the results could not be generalized to other hormonal cycles. Moreover, biochemical and clinical pregnancy loss could not be distinguished in this study considering pregnancy loss as a term that includes both of them. Wider implications of the findings To our knowledge, this is the first study in which the endometrial factor in AMA patients was analyzed year by year, helping to define in a more reliable way the ages at which the sharpest changes occur to improve clinical practices on endometrial implantation failure in AMA patients. Trial registration number Not applicable

P-198 Time to maximum blastocyst expansion (tmBE) may represent a novel metric for assessing embryo competence

Abstract Study question Can the time to achieve maximum blastocyst expansion (tmBE) and the frequency of spontaneous collapses serve as useful indicators of embryo competence? Summary answer Compared to the frequency of spontaneous collapses, tmBE was a better indicator of embryo competence, potentially serving as a valuable metric for embryo selection. What is known already Identifying the most viable blastocyst for transfer is essential for success in assisted reproduction. In scenarios with good prognosis, such as oocyte donation, blastocyst selection may require a more nuanced approach, given the high number of available good quality embryos. Exploring indicators of embryo competence beyond traditional morphology-based criteria may be beneficial, however an in-depth understanding is still lacking. Blastocyst expansion and contraction dynamics have been suggested to play a role in developmental and chromosomal fitness. However, their predictive value has not been investigated across a wide range of patient populations. Study design, size, duration This is a single-center retrospective study of 287 blastocysts transferred in autologous (n = 88) and heterologous (n = 199) cycles, performed between September 2019 and September 2022. Only fresh single embryo transfers were considered. We compared blastocysts across two groups: advanced maternal age (AMA) patients (>35 years old, n = 88) and oocyte donors (<35 years old, n = 199). We evaluated well-established indicators of embryo quality, as well as novel metrics, including tmBE and the frequency of spontaneous collapses. Participants/materials, setting, methods Conventional indicators of embryo quality included blastocyst grade and morphokinetic timings (tPNf, t2, t3, t4, t5, t8, tSB and tB). tMBE was defined as the maximum time (hpi) required for the blastocyst to reach its largest diameter, extending beyond tB. Blastocyst contractions were measured as a decrease and subsequent recovery of the blastocoel area over time. ImageJ was employed for size measurements. p<0.05 was considered statistically significant after Two-tailed Student’s t-test or Chi-square test. Main results and the role of chance We observed a significant difference in live birth rates between oocyte donors and AMA patients (40% vs 18%, p=0.0004). Nevertheless, there were no differences across blastocyst expansion scores between oocyte donors and AMA patients (5= 58% vs 65%; 4= 38% vs 30% and 3= 4% vs 5%, p > 0.05), nor in their morphology score (Excellent= 57% vs 61%; Good= 24% vs 18%; Average= 15% vs 16% and Poor= 4% vs 5%). In terms of morphokinetics, when comparing oocyte donor and AMA embryos, the only significant difference was observed at tPNF (22.0±2.4 vs 22.6±2.3; p= 0.04). No differences were found at t2, t3, t4, t5, t8, tSB and tB. Additionally, the frequency of spontaneous collapses showed no significant differences between the oocyte donor and AMA group (1.4±1.2 vs 1.5±1.0, p=0.66). However, blastocysts from oocyte donors reached tmBE significantly faster than AMA embryos (115.0±4.8 vs 117.2±3.4; p = 0.0003). When these variables were further correlated to clinical outcomes, the frequency of spontaneous collapses did not appear to influence live birth in either group. Notably, however, tmBE was significantly shorter for embryos resulting in a live birth, across both the oocyte donor (113.5 vs 115.4, p = 0.04) and AMA group (115.6 vs 117.6, p = 0.04). Limitations, reasons for caution This is a single center retrospective study with a relatively small sample size, especially in the AMA group. Caution should be made when generalizing results to other patient populations, as confounding were not taken into consideration. No data on the chromosomal status of embryos was available. Wider implications of the findings Our results suggest that tMBE may serve as a reliable indicator of embryo potential, allowing a more nuanced embryo selection, particularly in cases with a high number of good quality embryos. This study opens avenues for further research into novel metrics for assessing embryo competence Trial registration number not applicable

P-109 Non-invasive Genetic Testing (niPGTA) and Laser Assisted Hatching (LAH) may improve pregnancy and ongoing pregnancy rates in patients of advanced maternal age

Abstract Study question Does niPGTA and LAH improve pregnancy and ongoing pregnancy rates in patients of advanced maternal age? Summary answer niPGTA increases pregnancy and clinical pregnancy rates in patients aged ≥38 years compared to those without tested embryos, while LAH may further improve pregnancy outcomes. What is known already Embryos release cell-free DNA (cf-DNA) in the IVF culture media, which can be analyzed for chromosomal constitution. The benefit that niPGTA holds over PGT-A is that it is a non-invasive test and could potentially produce comparable results. Therefore, the time to pregnancy and the risk of miscarriage could be reduced in comparison to an IVF treatment with non-tested embryos. Previous studies have shown high concordance with trophectoderm biopsies in PGT-A patients and improved outcomes with LAH in frozen-thawed cycles. However, there is a lack of studies investigating clinical pregnancy rates with niPGTA. Study design, size, duration Retrospective cohort study including 74 patients aged ≥38 years, who underwent Single Frozen Embryo Transfer (FET) from May 2021 to February 2023. Patients who had Endometrial Receptivity Assay (ERA) were excluded. Participants/materials, setting, methods 32 couples had niPGTA; Spent Blastocyst Media (SBM) for each blastocyst were collected on Day 6 and analyzed using the EMBRACE test. Embryos were vitrified. 42 couples had their blastocysts vitrified on Day 5/6 without testing. LAH was introduced to all patients in October 2021. Before that, no LAH was performed. Embryos were transferred to patients 2 hours post-warming after LAH. Pregnancy test was performed 10 days after FET and clinical pregnancy was established. Main results and the role of chance There was a statistical significant increase of pregnancy rate (p = 0.017, Pearson’s Chi-Squared test) and a trend towards higher clinical pregnancy rate for niPGTA patients versus those who had a transfer of not tested embryos. Regarding LAH, there was a trend towards higher pregnancy and clinical pregnancy rates in both niPGTA and not tested group. Results for pregnancies and clinical pregnancies are presented in Table 1. Limitations, reasons for caution More patients are required to confirm clinical significance of pregnancy and clinical pregnancy rates of niPGTA over not tested embryos and if LAH can improve the outcomes. A review of patients who had previous implantation failures and miscarriages in addition to ERA could yield clearer conclusions. Wider implications of the findings The results of this study are encouraging for niPGTA combined with LAH for patients aged ≥38 years. Further studies on niPGTA and modifications of the protocol for increased concordance rates and shorter time in embryo culture should be explored. Trial registration number Not applicable

P-214 Direct unequal cleavage-1 (DUC1) in IVF/ICSI cycles: relationship with patient characteristics and insemination method

Abstract Study question Does the incidence of DUC1 in IVF/ICSI cycles vary with the maternal profile and insemination method? Summary answer The incidence of DUC1 cycles is higher in patients with a more favourable prognosis. DUC1 embryos are more frequent in ICSI than in IVF cycles. What is known already The emergence of Time-Lapse Microscopy (TLM) has enabled a dynamic and thorough evaluation of in vitro embryo development, unveiling the occurrence of morphological phenomena unobserved with static observation, such as direct unequal cleavage (DUC), which is associated with a drastically reduced competence to implant. The incidence of DUC-1 (DUC immediately after syngamy) has been reported to vary from 8.3% to 26%, but the reasons underlying its occurrence and variability are unknown. There is evidence that the maternal profile and insemination method can impact embryo developmental competence, but whether they impact the incidence of DUC is also unknown. Study design, size, duration The study includes 2920 first IVF/ICSI cycles (2018 to 2022) providing 12492 embryos. Cycles presenting male infertility were excluded from the analysis. The percentages of DUC1 embryos in relation to total embryos and of cycles providing at least one DUC1 embryo in relation to total cycles were compared between AMA (advanced maternal age; ≥35) and pre-AMA (<35) cycles. Patient/cycle characteristics were compared between cycles with and without DUC1 occurrence in AMA and pre-AMA cycles, separately. Participants/materials, setting, methods Ovarian stimulation was induced with rFSH or with a combination of FSH and LH using classic antagonist or short agonist protocols. Oocytes were inseminated with either IVF or ICSI standard methodology. Embryo culture was performed in an integrated embryo culture Time-lapse System (EmbryoScope; Vitrolife). Differences between percentages were assessed by the Fisher’s exact test, while differences between continuous variables were assessed with the Wilcoxon sum rank test. Main results and the role of chance The percentage of DUC1 embryos in relation to total embryos did not vary between pre-AMA (5.4%) and AMA (5.2%; p > 0.05) cycles. Alternatively, the incidence of cycles providing at least one DUC1 embryo was higher in pre-AMA (21.7%) as compared with AMA patients (16.0%; p < 0.0001). In both pre-AMA and AMA cycles, DUC1 occurrence was associated with higher oocyte yield (13.5±7.7 vs. 10.6±6.7 and 11.0±6.3 vs. 7.5±5.5, respectively; p < 0.0001), higher AMH serum levels (4.4±4.7 vs. 3.4±3.2 and 3.0±3.6 vs. 2.1±2.1ng/mL, respectively; p < 0.004) and lower basal FSH serum levels (7.0±2.1 vs. 7.6±2.9 and 7.8±3.3 vs. 8.5±3.4mU/mL, respectively; p < 0.0001). Overall, the percentage of DUC1 embryos was higher in ICSI (5.6%) as compared to IVF (4.4%, p < 0.01) cycles. However, when the data was stratified by maternal age, DUC1 incidence was significantly higher in ICSI vs. IVF in AMA (5.6 vs 4.0%; p = 0.002) but not in pre-AMA (5.7 vs 5.0%; p = 0.19). All 21 DUC1 embryos transferred during the study failed to implant. Limitations, reasons for caution Our study is limited by its retrospective nature. The present conclusions must be confirmed in other patient populations with different race/genetics and habits. Wider implications of the findings The occurrence of cycles providing DUC1 embryos is paradoxically associated with a more favourable patient prognosis, and the percentage of DUC1 embryos is higher in ICSI as compared to IVF cycles in AMA patients. Our findings represent new valuable clues for the clarification of DUC1 underlying causes. Trial registration number NA

P-252 Non-invasive artificial intelligence (AI) prediction of blastocyst development from mature oocytes unveils correlation with blastocyst ploidy for enhanced reproductive insight at earliest stage of development

Abstract Study question Can MAGENTA identify mature oocytes more likely to develop into a blastocyst of euploid status? Summary answer Although built to predict blastocyst development, MAGENTA analysis of mature oocytes additionally displays the ability to highlight those with higher potential to become euploid embryos. What is known already MAGENTA (AI image-analysis tool) has been trained to assess and predict blastocyst stage embryo development from mature, denuded oocytes. MAGENTA scores consistently identify oocytes of better quality – more likely to develop into a blastocyst, including blastocysts of higher morphological quality. However, in addition to achieving a blastocyst, the ploidy status of that embryo is crucial in dictating overall chances of implantation and cycle success. Although MAGENTA was not trained to predict blastocyst ploidy from oocytes, most chromosomal errors are of maternal meiotic origin. A correlation between MAGENTA and ploidy status would ignite research possibilities and provide valuable insights. Study design, size, duration A retrospective dataset of 1,324 blastocyst embryos that underwent PGT-A via NGS sequencing, between the years 2021-2023, was assessed. All embryos that were tested for ploidy from a patient cohort were included. Embryos reported as mosaic or ‘no diagnosis’ were excluded from analysis (N = 169). All embryos were cultured in a time-lapse incubator immediately post-ICSI. Of the biopsied blastocysts, 81% were of good morphological quality (expansion grade 1-6, ICM and TE grade A/B). Participants/materials, setting, methods The data includes 292 patients with a mean age of 38.5 (range 19-46) and an average number of tested blastocysts of 4 (range 1-14). Of the 1173 blastocysts, 847 were aneuploid (72.2%) and 326 were euploid (27.8%). 83% of the blastocysts were from patients of advanced maternal age (AMA, ≥38years old). The first time-lapse image, capturing the mature oocyte stage, was extracted, and scored by MAGENTA (scale 0-10). MAGENTA scores were compared by Welch’s t-tests. Main results and the role of chance Impressively, oocytes that developed into euploid blastocysts (n = 326) were identified by MAGENTA with a higher average score of 6.8 compared to those that developed into aneuploid blastocysts (n = 847) with a score of 6.4; a statistically significant differentiation (p < 0.05). 80% of the dataset were high quality blastocysts (expansion 4-6, ICM and TE grade A/B) with an imbalance of euploid (n = 295; mean score 6.8) and aneuploid (n = 643; mean score 6.6) blastocysts—likely a result of AMA. Therefore, subgroup analysis was focused on assessing the ploidy status relationships amongst several patient age groups within these AMA cases. Due to our clinic’s indication for PGT-A, the smallest subset of oocytes was from patients <38 years of age (N = 373, 48% euploid). In this age group there was no significant difference in MAGENTA scores between the oocytes of euploid and aneuploid embryo status (6.9 vs 6.7; p = 0.522). However, in oocytes from patients aged ≥38 (N = 800, 19% euploid), the distinction between those that became euploid and aneuploid blastocysts is evident with a statistically significant difference in MAGENTA scores (6.8 vs 6.3; p < 0.05). This relationship with ploidy status additionally remains with further stratification into the ≥41 age group (N = 467 oocytes, 13% euploid; 6.7 vs 5.9; p < 0.01). Limitations, reasons for caution Majority of the patients in this dataset underwent PGT-A due to AMA, further research is required to include a wider age demographic, particularly in patients <35 years old. Greater representation of varying blastocyst qualities is required to further assess MAGENTA correlation to ploidy outcomes amongst quality groups. Wider implications of the findings This study presents the first non-invasive assessment of mature oocyte quality that indicates oocytes with greater potential for euploid blastocyst development. This application of MAGENTA provides a novel method of gaining ploidy status potential at the mature oocyte stage, which is invaluable to both IVF and cryopreservation cycles. Trial registration number NOT APPLICABLE

O-157 The effect of growth hormone on the euploid rate and morphokinetic signature of blastocysts in patients with advanced maternal age : a randomized controlled trial

Abstract Study question Does growth hormone (GH) supplementation during ovarian stimulation affect the euploid rate and morphokinetic characteristics of euploid embryos in patients with advanced maternal age (AMA)? Summary answer GH supplementation improved the probability of obtaining euploid blastocysts in women with AMA, while did not affect the morphokinetic parameters of the euploid embryos. What is known already Studies have demonstrated that supplementation with GH during ovarian stimulation (OS) has positive impact on the clinical outcomes of in vitro fertilization (IVF). However, few studies have investigated the association of GH supplementation with the ploidy status of embryos. Meanwhile, it is unclear whether GH supplementation affects the morphokinetic signatures and the transfer outcomes of euploid blastocysts. Therefore, the underlying reason for the effect of GH on IVF outcomes remains largely unknown. Study design, size, duration This is a single centre open-labelled randomized controlled trial conducted at single IVF center from August 2022 to August 2023. A total of 441 patients were screened for eligibility and 309 patients were enrolled and allocated. The primary outcome was the proportion of cycles which obtained euploid blastocysts. Secondary outcomes included euploid blastocyst rate per cohort, euploid blastocyst rate per cycle and morphokinetic parameters of euploid embryos obtained with or without GH supplementation. Participants/materials, setting, methods A total of 309 patients aged 38–46 years who underwent their first preimplantation genetic testing for aneuploidy (PGT-A) cycle using antagonist protocol were randomized into the GH group or the control group at 1:1 ratio, with all embryos cultured in the time-lapse monitoring system. Patients in the GH group received 2 IU/day subcutaneous GH from the 2nd day of the previous menstrual period until the day of trigger, for a total of approximately 6 weeks. Main results and the role of chance The baseline and cycle characteristics between the two groups were similar. The proportion of cycles which obtained euploid blastocysts was significantly higher in the GH group (44.74% vs 31.85%, p = 0.02). The GH group had a significantly higher euploid blastocyst rate per cohort (38.46% vs 28.01%, p = 0.01) and mean euploid blastocyst rate per cycle (per biopsy cycle 0.38±0.43 vs. 0.26±0.39, p = 0.02; per OS cycle 0.29±0.41 vs. 0.19±0.36, p = 0.03). Multivariate logistic regression indicated GH supplement was an independent factor for acquisition of euploid embryos (adjusted risk ratio=1.79, 95%CI [1.06-2.99], p = 0.03). We did not observe a statistically significant difference in the morphokinetic parameters and transfer outcomes of the euploid embryos acquired with or without GH supplemented. Limitations, reasons for caution This was a single center study which limited the sample size, further limiting the number of patients with euploid blastocysts analyzed for the morphokinetic parameters and transfer outcomes. Meanwhile, affordability may also introduce selection bias since patients were required to pay for GH. Wider implications of the findings Our research presents new evidence that GH supplementation AMA patients increased the probability of obtaining euploid blastocysts, and explains the underlying reason for the positive effect of GH on IVF outcomes in existing studies. The effect of GH on morphokinetic parameters and transfer outcomes of euploid embryos warrants further investigation. Trial registration number NCT05447208, www.clinicaltrials.gov.

O-152 Pre-implantation genetic testing for aneuploidies (PGT-A) improves reproductive outcomes in advanced maternal age patients undergoing IVF/ICSI: a multicentre retrospective cohort study with propensity score matching

Abstract Study question In patients > 37 years undergoing IVF/ICSI, does PGT-A improve live birth rates (LBR) and cumulative live birth rates (CLBR)? Summary answer In advanced maternal age women with ≥1 embryo to transfer, PGT-A improves the live birth and cumulative live birth rates. What is known already The age-related rate of aneuploidy remains a major cause of IVF failure. Therefore, PGT-A has been proposed as a method to select embryos with the highest implantation potential. Previous studies have shown that women younger than 37 years old do not benefit from PGT-A given the comparable outcomes in terms of implantation, clinical pregnancy, and live-birth rates. Moreover, PGT-A may also be inherently ineffective given the risk of mitotic mosaicism, sampling errors and misinterpretation of results. However, we are currently lacking solid scientific evidence to support whether this approach benefits advanced maternal age women. Study design, size, duration A multicenter retrospective cohort study was conducted including 9328 patients >37y undergoing their first IVF/ICSI cycle with an oocyte retrieval performed between 1/01/2013-31/07/2021 in a multinational private fertility clinic. The study group (n = 4664) included patients who performed PGT-A. The control group included patients without PGT-A (n = 4664) and was selected by propensity score matching adjusted for age at oocyte retrieval, number of oocytes retrieved and year of oocyte retrieval. Participants/materials, setting, methods The primary outcome was CLBR (delivery of at least one live birth per started cycle, including the fresh and subsequent frozen embryo transfer cycles). The secondary outcomes were LBR and time to pregnancy. The analysis was conducted including all patients with an initiated cycle of ovarian stimulation and also per embryo transfer. Comparisons are presented as adjusted odd-ratios (adjOR) and 95% confidence intervals (95%CI) following multivariable logistic regression (LBR) or cox regression (CLBR) analysis. Main results and the role of chance Patients in the PGT-A group had more oocytes retrieved (11.03±6.82 vs 9.24±6.15, p < 0.001), and a higher number of available blastocysts (2.82±2.21 vs 1.22±2.00, p < 0.001). In the PGT-A group, 13.153 embryos were analysed, with an euploidy rate of 29.30% (n = 3854). In total, 49.83% (n = 2324) patients did not undergo embryo transfer due to unavailable euploid embryos. Considering all patients that initiated ovarian stimulation, the PGT-A group presented a higher unadjusted CLBR per started cycle (27.38% vs 21.81%, p < 0.001). Considering only patients who underwent embryo transfer, a total of 5934 embryo transfers were analysed (n = 3166 in the control group and n = 2768 in the PGT-A group). Following multivariable logistic regression analysis, LBR and CLBR were significantly higher in the PGT-A group (respectively, adjOR 1.56 [95% CI 1.38-1.77], adjOR 1.53 [95% CI 1.39-1.68]). Following the transfer of 5 embryos, the CLBR was approximately 100% in the PGT-A group and 74.51% in the control group. Survival analysis was also perfomed to assess the impact of PGT-A on time to pregnancy. Despite the fact that from 6 months onwards the CLBR was higher in the PGT-A group, the median time to pregnancy was higher in the PGT-A group (3.27 (95%CI 3.16-3.40) vs 2.74 months (95%CI 2.57-3.03). Limitations, reasons for caution This study is limited by its retrospective design and low number of women with more than 3 embryos transferred. Also, the fact that only the first IVF/ICSI cycle was analysed provides a proper patient counselling in this specific context but does not provide data on repeated cycle outcomes. Wider implications of the findings Our findings suggest that, in women older than 37 years with at least one available embryo to biopsy, performing PGT-A may improve the reproductive outcomes in terms of LBR and CLBR, providing a useful clinical tool for embryo selection. Trial registration number not applicable

The association between serum uric acid and low birth weight in advance maternal age women with hypertension: An observational study.

In China, the implementation of 2-child policy since 2015 entitles increasing number of advanced maternal age. Recently, Chinese hypertensive disorders of pregnancy (HDP) in advanced-age women have attracted significant clinical and epidemiological research interest. Previous studies have shown an association between serum uric acid (SUA) levels and low birth weight (LBW) in children. Several studies have reported that advanced maternal age is a risk factor for many complications in pregnancy, including LBW. However, it remains unclear whether SUA affects LBW risk in advanced maternal age mothers with hypertensive diseases. The study was observational in nature. A total of 692 advanced maternal age with hypertension were enrolled in our study. A variety of demographic and vital sign data, laboratory test results, and pregnancy outcomes were collected. Children born with LBW served as the clinical endpoint. On admission, blood samples were taken, and women with advanced maternal ages were divided into 2 groups based on their SUA levels. In order to investigate the association between SUA and LBW, a logistic regression model was used. E-value analysis was used to determine the residual unmeasured confounding. The mean SUA level was increased in advanced maternal age patients with HDP. Of 692 newborns, 244 (35.26%) have LBW. With possible confounders adjusted, high SUA levels were independent risk factors for LBW (odds ratio [OR]2.88, 95% confidence intervals [CI]1.22-6.81), multivariate logistic regression analysis using SUA as a continuous variable recapitulated the pattern (OR 1.01, 95% CI 1.00-1.01). In addition, SUA levels in women with advanced maternal age and hypertension were linearly related to LBW incidence. According to this study, SUA levels in patients with advanced maternal age and HDP are associated with LBW incidence.

Intrinsic and Extrinsic Apoptotic Pathways May Dictate In Vitro Fertilization Outcomes in Women of Advanced Maternal Age [ID 2683425

INTRODUCTION: Women of advanced maternal age (AMA) (≥35 years) are at an increased risk for a decline in ovarian reserve and have a decreased rate of live birth after one cycle of in vitro fertilization (IVF). Current practice recommends preimplantation genetic testing for aneuploidy (PGT-A) to women of AMA to increase live birth outcomes. However, recent studies have found that conventional IVF was non-inferior to PGT-A. Identifying additional biomarkers may provide another metric for selecting the most viable embryo. METHODS: Blastocoel fluid-conditioned media was collected from day 5 IVF embryos that underwent PGT-A. RNA was purified from pooled blastocoel fluid samples from four groups based on implantation status and maternal age, and then cDNA was synthesized. RT-qPCR was used to assess expression of CASP3, CASP7, CASP8, and BCL2L12 levels in a total of 64 embryos. RESULTS: CASP3 and CASP7 expression were higher in the media from IVF embryos with positive implantation outcomes from patients aged less than 35 years. There was increased CASP8 expression in media from IVF embryos from AMA patients regardless of implantation status. CONCLUSION: Preliminary analysis suggests increased CASP8 expression is associated with successful implantation outcomes in AMA patients. This suggests that CASP8 could be activated in preimplantation embryos as an aging response as found in AMA media samples. One explanation is that CASP8 is associated with aneuploidy in the AMA samples. Further research will determine whether expression and activation of CASP8 are required for positive implantation outcomes and be used as an additional embryo selection tool for other AMA patients utilizing IVF.

Maternal and Fetal Outcomes in Pregnancy in the Fifth Decade of Life and Beyond

ObjectivesThe objective is to evaluate maternal and fetal outcomes at an extremely advanced maternal age (AMA) (over age 50 years) in Calgary. The secondary objective is to determine if there is a role in protocolizing complex care plans for patients at extreme AMAs. MethodsA retrospective chart review was conducted of all pregnancies ≥20 weeks gestation in patients over the age of 50 years that delivered in Calgary between January 2007 and December 2021. Pregnancy data were collected, including maternal age, pre-existing medical conditions, mode and timing of delivery, neonatal outcomes, neonatal intensive care unit (NICU) and adult intensive care unit (ICU) admissions, postpartum complications, and maternal or neonatal death. Data were extracted for maternity patients as well as neonatal ICU databases. Maternal and neonatal outcomes were assessed until discharge from hospital. ResultsAll 23 pregnancies identified were achieved through assisted reproductive technologies. Comorbidities varied, but the most common comorbidities included hypertension and gestational diabetes. Cesarean delivery was the most common form of delivery. Three cases involved postpartum maternal ICU admission. Neonatal outcomes included gestational ages of 22–39 weeks and birth weights of 486–3593 g, with 8 confirmed NICU admissions. The most common neonatal complications were jaundice and small for gestational age. ConclusionsExtremely AMA patients are more likely to have pre-existing comorbidities and develop comorbidities during pregnancy. The potential for adverse maternal and fetal outcomes is greater for these pregnancies; however, the complications are diverse and developing a universal complex care plan is difficult.

Growth hormone supplementation during ovarian stimulation in women with advanced maternal age undergoing preimplantation genetic testing for Aneuploidy

BackgroundStudies have shown that supplementation with recombinant human GH (rh-GH) during ovarian stimulation (OS) may improve the ovarian response and clinical outcomes of IVF. However, it remains unclear whether GH is associated with the ploidy status of embryos, and therefore, is unable to explain the underlying reason for the effect of GH on IVF outcomes. This study aimed to investigate whether GH supplementation in women with advanced maternal age (AMA) during OS is related to an increased probability of obtaining euploid blastocysts.MethodsThis was a single center retrospective cohort study. The data of all women aged 38–46 years who underwent their first preimplantation genetic testing for aneuploidy (PGT-A) cycle between January 2021 and June 2022 were reviewed. Patients in the GH group received 4 IU/day subcutaneous GH supplementation from the beginning of OS to the trigger day, and patients in the control group did not. A total of 140 patients in the GH group and 272 patients in the control group were included after 1:2 propensity score matching.ResultsThe baseline and cycle characteristics between the two groups were similar. The proportion of cycles which obtained euploid blastocysts was significantly higher in the GH group than that in the control group (41.43% vs. 27.21%, P = 0.00). The GH group had a significantly higher euploid blastocyst rate per cohort (32.47% vs. 21.34%, P = 0.00) and mean euploid blastocyst rate per cycle (per biopsy cycle 0.35 ± 0.40 vs. 0.21 ± 0.33, P = 0.00; per OS cycle 0.27 ± 0.38 vs. 0.16 ± 0.30, P = 0.02). However, the benefit of GH was more significant in patients aged 38–40 years, but not significant in patients aged 41–46 years. Pregnancy outcomes were similar between the two groups after embryo transfer.ConclusionsGH supplementation during OS is associated with a significantly increased probability of obtaining euploid blastocysts in women aged 38–40 years, but this benefit is not significant in women aged 41–46 years. Our results explained the underlying reason for the effect of GH on IVF outcomes in existing studies, and might be helpful for AMA patients undergoing PGT-A cycles to obtain a better outcome meanwhile to avoid over-treatment.Trial registrationNCT05574894, www.clinicaltrials.gov.

Aneuploidy in oocytes from women of advanced maternal age: analysis of the causal meiotic errors and impact on embryo development.

In oocytes of advanced maternal age (AMA) women, what are the mechanisms leading to aneuploidy and what is the association of aneuploidy with embryo development? Known chromosome segregation errors such as precocious separation of sister chromatids explained 90.4% of abnormal chromosome copy numbers in polar bodies (PBs), underlying impaired embryo development. Meiotic chromosomal aneuploidies in oocytes correlate with AMA (>35 years) and can affect over half of oocytes in this age group. This underlies the rationale for PB biopsy as a form of early preimplantation genetic testing for aneuploidy (PGT-A), as performed in the 'ESHRE STudy into the Evaluation of oocyte Euploidy by Microarray analysis' (ESTEEM) randomized controlled trial (RCT). So far, chromosome analysis of oocytes and PBs has shown that precocious separation of sister chromatids (PSSC), Meiosis II (MII) non-disjunction (ND), and reverse segregation (RS) are the main mechanisms leading to aneuploidy in oocytes. Data were sourced from the ESTEEM study, a multicentre RCT from seven European centres to assess the clinical utility of PGT-A on PBs using array comparative genomic hybridization (aCGH) in patients of AMA (36-40 years). This included data on the chromosome complement in PB pairs (PGT-A group), and on embryo morphology in a subset of embryos, up to Day 6 post-insemination, from both the intervention (PB biopsy and PGT-A) and control groups. ESTEEM recruited 396 AMA patients: 205 in the intervention group and 191 in the control group. Complete genetic data from 693 PB pairs were analysed. Additionally, the morphology from 1034 embryos generated from fertilized oocytes (two pronuclei) in the PB biopsy group and 1082 in the control group were used for statistical analysis. Overall, 461/693 PB pairs showed abnormal segregation in 1162/10 810 chromosomes. The main observed abnormal segregations were compatible with PSSC in Meiosis I (MI) (n = 568/1162; 48.9%), ND of chromatids in MII or RS (n = 417/1162; 35.9%), and less frequently ND in MI (n = 65/1162; 5.6%). For 112 chromosomes (112/1162; 9.6%), we observed a chromosome copy number in the first PB (PB1) and second PB (PB2) that is not explained by any of the known mechanisms causing aneuploidy in oocytes. We observed that embryos in the PGT-A arm of the RCT did not have a significantly different morphology between 2 and 6 days post-insemination compared to the control group, indicating that PB biopsy did not affect embryo quality. Following age-adjusted multilevel mixed-effect ordinal logistic regression models performed for each embryo evaluation day, aneuploidy was associated with a decrease in embryo quality on Day 3 (adjusted odds ratio (aOR) 0.62, 95% CI 0.43-0.90), Day 4 (aOR 0.15, 95% CI 0.06-0.39), and Day 5 (aOR 0.28, 95% CI 0.14-0.58). RS cannot be distinguished from normal segregation or MII ND using aCGH. The observed segregations were based on the detected copy number of PB1 and PB2 only and were not confirmed by the analysis of embryos. The embryo morphology assessment was static and single observer. Our finding of frequent unexplained chromosome copy numbers in PBs indicates that our knowledge of the mechanisms causing aneuploidy in oocytes is incomplete. It challenges the dogma that aneuploidy in oocytes is exclusively caused by mis-segregation of chromosomes during MI and MII. Data were mined from a study funded by ESHRE. Illumina provided microarrays and other consumables necessary for aCGH testing of PBs. None of the authors have competing interests. Data were mined from the ESTEEM study (ClinicalTrials.gov Identifier NCT01532284).

P-240 High mitochondrial DNA content in Day6 blastocyst may impair ongoing pregnancy rate

Abstract Study question Is there an association between mitochondrial DNA (mtDNA) content and Ongoing Pregnancy Rate (OPR) in D5 or D6 euploid blastocyst? Summary answer Day6 OPR is decreased in high mtDNA content blastocyst. Day5 and Day6 mtDNA levels are independent of maternal age or standard morphology. What is known already Preimplantation development is an energy-demanding process and mitochondrial ATP production is crucial for cellular activity in fast replicating cells. Mitochondrial content in preimplantation embryo has been proposed as a marker of embryo potential in term of viability and implantation success though the dynamics and distribution of mitochondria in human embryo is still debated. In recent years, it has been suggested that low mtDNA levels were associated with euploid chromosomal complement and higher implantation rate while other studies failed to find a correlation between mtDNA content and reproductive outcome. Study design, size, duration This study is a retrospective cohort analysis from 2020 to 2022 including 343 Day5 and 187 D6 single euploid blastocyst transfer. Primary endpoint was OPR beyond pregnancy week 16. Day5 and Day6 frozen-thawed transfer were divided based on blastocyst mtDNA content in four groups: Day5-high, Day5-low, D6-high, D6-low. Secondary endpoints were relationship between mtDNA content, maternal age and standard morphology in all obtained blastocysts. Statistical differences were compared by Chi-Square test. Participants/materials, setting, methods 771 couples performed IVF cycles with next-generation sequencing (NGS)-preimplantation genetic testing of aneuploidy (PGT-A) (age 19-47yrs; Mean age 37.8yrs). Following culture in sequential media 700 euploid blastocyst were obtained and mitochondrial and chromosomal DNA copy number variation were examined simultaneously with next generation sequencing (NGS) methodology. mtDNA copy numbers was based on the observed ratios of sequence coverages between mtDNA and nuclear DNA. Main results and the role of chance In our clinical setting OPR is similar in Day5 vs. Day6 transfers (55% vs. 49% n.s.). However when mtDNA content is considered D6-low OPR is comparable to D5-high or low (57.3%; 53.3%; 57,3% respectively n.s.) while D6-high transfers yield significantly decreased OPR (31,2% p < 0.05). When stratified by age only Advanced Maternal Age (AMA) patients ( > = 38yrs) showed this decrease at a significant level (Day6 high OPR 20% p < 0,05).This result in transferred blastocyst is obtained despite high:low ratio of all biopsied blastocyst is constant in Day5 (1:1) (47%-53%) and markedly decreased in Day6 (1:4) (25%-75%) either overall or in younger (n.s.) and AMA patients (n.s.). Moreover when standard morphology distribution is evaluated according to Gardner classification criteria top, fair and poor quality blastocysts display the same 1:1 high:low blastocysts mtDNA ratio in Day5 and 1:4 in Day 6 overall and in younger and AMA patients. Hence, the mtDNA blastocyst content is independent of patients’ age and blastocyst quality. The origin of Day6 OPR decrease could therefore depend on the number of Day6-high blastocyst transferred in AMA patients and ultimately in the number of blastocyst available to transfer for each patient. Limitations, reasons for caution Due to the small numbers of Day6-high mtDNA blastocyst, a larger sample size is required to confirm these preliminary findings. Moreover, the retrospective nature of the study may introduce some bias mainly in patients’ characteristics and the strategy of embryo selected for transfer. Wider implications of the findings While Day5-high blastocyst yield higher OPR the persistence of higher mtDNA levels in Day6 blastocyst may hamper their implantation potential. Our findings may help to select the embryo to transfer to maximize transfer success. D6 blastocysts with low mtDNA content should be preferred whenever a choice is possible. Trial registration number Not Applicable

O-307 Aneuploidy in oocytes of women of advanced maternal age: impact on embryo development and evidence for a novel mechanism of meiotic error

Abstract Study question In oocytes of advanced maternal age (AMA) women, what is the association of aneuploidy with embryo development and what are the mechanisms leading to aneuploidy? Summary answer We confirmed precocious separation of sister chromatids as the main cause of aneuploidy. Known chromosome segregation errors explained 90.4% of the observed abnormal chromosome copy numbers in polar bodies. What is known already Meiotic chromosomal aneuploidies in oocytes correlate with AMA (>35 years), and can affect over half of oocytes in this age group. This underlies the rationale for polar body (PB) biopsy as a form of early PGT-A, as performed in the ‘ESHRE STudy into the Evaluation of oocyte Euploidy by Microarray analysis’ (ESTEEM) randomised controlled trial (RCT). So far, chromosome analysis of oocytes and PBs has shown that precocious separation of sister chromatids (PSSC), meiosis II (MII) nondisjunction (ND) and reverse segregation (RS) are the main mechanisms leading to aneuploidy in oocytes. Study design, size, duration Data was sourced from the ESTEEM study, a multicentre RCT from seven European centres to assess the clinical utility of PGT-A on PBs using array comparative genomic hybridisation (aCGH) in patients of AMA (36-40 years). This included data on the chromosome complement in PB pairs, and on embryo morphology in a subset of embryos, up to day 6 post insemination (dpi), from both the intervention (PB biopsy for PGT-A) and from control (no biopsy) arms. Participants/materials, setting, methods ESTEEM recruited 396 AMA patients: 205 in the intervention group and 191 in the control group. Complete genetic data from 693 PB pairs were analysed. Additionally, the morphology from 1034 embryos generated from fertilized oocytes (2 pronuclei) in the PB biopsy group and 1082 in the control group were compared using age-adjusted multilevel mixed-effect ordinal logistic regression for each day of embryo scoring and summarized with adjusted odds ratios (aOR) and 95% confidence intervals (CI). Main results and the role of chance Overall, 461/693 PB pairs showed abnormal segregation in 1162/10810 chromosomes. The main observed abnormal segregations were compatible with PSSC in meiosis I (MI) (n = 568/1162; 48,9%), ND of chromatids in MII or RS (n = 417/1162; 35.9%) and less frequently ND in MI (n = 65/1162; 5.6%). For 112 chromosomes (112/1162; 9,6%), we observed a chromosome copy number in PB1 and PB2 that is not explained by any of the known mechanisms causing aneuploidy in oocytes. We observed that embryos in the PGT-A arm of the RCT did not have a significantly different morphology between 2 and 6 dpi compared to the control group, indicating that polar body biopsy did not affect embryo quality. Following age-adjusted multilevel mixed-effect ordinal logistic regression models performed for each embryo evaluation day (using the before-mentioned embryo scoring subcategories), aneuploidy was associated with a decrease in embryo quality on day 3 (aOR 0.62, 95% CI 0.43-0.90), day 4 (aOR 0.15, 95% CI 0.06-0.39) and day 5 (aOR 0.28, 95% CI 0.14-0.58). Limitations, reason for caution RS cannot be distinguished from normal segregation or MII ND using aCGH. The observed segregations were based on the detected copy number of PB1 and PB2 only and were not confirmed by the analysis of embryos. The embryo morphology assessment was static and single observer. Wider implications of the findings Our finding of frequent unexplained chromosome copy numbers in polar bodies indicates that our knowledge of the mechanisms causing aneuploidy in oocytes is incomplete. It challenges the dogma that aneuploidy in oocytes is exclusively caused by missegregation of chromosomes during meiosis I and II. Study funding/competing interest(s) Data was mined from a study funded by ESHRE. Illumina provided microarrays and other consumables necessary for aCGH testing of polar bodies. None of the authors have competing interests. This work is dedicated to the memory of Professor Joep Geraedts. Trial registration number NCT01532284 Study funding Yes Funding source Funding by national/international organization(s)

P-303 Outcomes of day 5 versus day 6 blastocysts: correlation of euploidy, implantation and embryo quality

Abstract Study question Are outcomes of day 5 (D5) blastocysts better than day 6 (D6) blastocysts in a PGT-A program? Summary answer Outcomes of day 5 embryos show more blastocysts, better quality, more euploidy due to better quality and higher implantation on same quality than day 6. What is known already Human embryos optimally reach the blastocyst stage after five days of culture, but some have a slower development. The superiority of day 5 blastocysts compared to day 6 blastocysts in fresh cycle transfers was previously demonstrated and attributed mainly to endometrial asynchrony. Data from frozen blastocysts transfers showed conflicting results, some studies have announced higher pregnancy rates after day 5 transfer compared with day 6 transfer, while others have shown equivalent outcomes. However, none of these studies have compared outcomes between same quality and euploid day 5 (D5) frozen blastocysts versus those frozen on day 6 (D6). Study design, size, duration Retrospective observational study performed in a private centre between May 2017 and December 2022. The study includes the data analysis of 5599 D5 and D6 blastocysts undergoing PGT-A obtained from 819 patients following 1295 PGS cycles. 948 euploid blastocysts with known implantation outcome were transferred in 789 frozen embryo transfers. Blastocyst morphology was scored in 3 groups: A: excellent (AA, AB, BA), B: good (BB), C: average and poor-quality embryos (BC, CB, CC). (Gardner-Schoolcraft classification) Participants/materials, setting, methods PGT-A with NGS technology was offered to patients of advanced maternal age and/or with repeated IVF failures. Trophectoderm biopsies were performed on D5 and/or D6 embryos, with laser assistance. (Navilase, OCTAX) We compared both populations (D5 and D6) in terms of number of blastocysts achieved and distribution of embryo quality of each population. As primary outcome, same-quality group results of euploid rate and implantation were compared according to the day of blastocyst development (D5 and D6). Main results and the role of chance Vast majority of the blastocysts were biopsied on D5 (71.8%, n = 4017) versus (28.2%, n = 1582) biopsied on D6. The proportion of three quality-categories of blastocysts according to D5/D6 is uneven. Quality A = 18.6% vs 3,5%. Quality B = 72,4% vs 59,6%. Quality C = 9,0% vs 36,9%, respectively, showing significative differences between D5/D6 (Chi^2 p-values of 3 groups< 0.001), concluding that overall D5 quality is better than D6. Overall euploidy rate on D5 population versus D6 was 31,3% vs 23,3% (p value <0.001), showing significant difference in favour of D5. But layering on 3 quality categories on D5/D6, the euploidy rate was 44,7% vs 51,8% for quality A, 29,6% vs 26,6% for quality B and 17,6% vs 14,9% for quality C, respectively, showing no significative differences D5/D6 (Chi^2 p-values: A = 0,331; B = 0,067; C = 0,280). Euploidy is not D5/D6 dependant but quality dependant. Overall implantation on D5 (65,9%) is higher than D6 (40,4%) (p value <0.001). Layering again, on D5 the implantation was 75,0% vs 66,7% for quality A, 63,8% vs 46,4% for quality B and 35,3% vs 26,3% for quality C, respectively. Despite implantation being higher on D5 on all three groups only group B shows D5/D6 significative differences. (Chi^2 p-values: A = 0,651; B < 0.001; C = 0,360). Implantation is embryo quality and D5/D6 dependant. Limitations, reasons for caution The study is limited by its retrospective nature and the low number of grade A D6-euploid blastocysts available to transfer. Additionally, it is common to transfer more than one grade C quality embryo to increase the chances of pregnancy, losing implantation track of these type. Wider implications of the findings It was uncontested that D5 blastocysts had better reproductive potential, but we managed to quantify this potential according to euploidy and implantation rates based on embryo quality, which remains the most important predictive biomarker for selecting the best embryo to transfer and reducing the time to achieve a pregnancy. Trial registration number not applicable

P-639 Are our advanced maternal age population being affected by the shortage of HP-hMG?

Abstract Study question Could we stimulate patients of advanced maternal age in the face of a shortage of HP-hMG without impacting outcomes? Summary answer Given the current low availability of HP-hMG, it is safe to stimulate only with recombinant FSH in women of advanced maternal age without affecting results. What is known already . Studies on the role of luteinizing hormone (LH) supplementation in patients undergoing assisted reproductive treatments use different sources of LH bioactivity. It is well known that the addition of LH supplementation was associated with a tendency towards improved IVF outcome in specific profiles of patients undergoing Assisted Reproductive treatment. However, during the last quarter of 2022, we are facing a temporary fall in the global supply of HP-hMG, which has hCG-driven LH activity, and may impact ovarian stimulation outcomes in women of advanced maternal age undergoing a preimplantation genetic testing treatment (PGT). Study design, size, duration Retrospective and observational study during November-December 2022 in any of the 11 clinics belonging to the IVIRMA group in Spain. Study population included women ≥38 years undergoing PGT treatment who received monotherapy with recombinant FSH (n = 73), a combination of FSH + HMG containing an FSH: LH activity in a ratio 1:1 (n = 103), or a fertility drug with recombinant FSH + recombinant LH in a 2:1 ratio (n = 112). The study was approved by an IRB (2003-MAD-020-AR) Participants/materials, setting, methods All women underwent short GnRH antagonist protocol. Initial doses were adjusted based on weight and body mass index according to the clinician’s experience. Daily doses of 0.25 mg GnRH antagonist were started on day 6 of stimulation. Finally, a single dose of 0.2 mg GnRH agonist was administered to trigger final oocyte maturation. Embryo biopsy was performed on day 5 with a subsequent freeze-all. A frozen single embryo transfer was accomplished in a subsequent cycle. Main results and the role of chance Assuming the limitations of the study design, we observed significant differences in gonadotropin doses but not in retrieved oocytes, metaphase II oocytes or euploidy rate: the results were as follows for recombinant FSH, FHS + hMG and FSH + LH respectively: gonadotropin doses [2254 (692) vs. 2033 (680) vs. 2000 (660), p = 0.0035]; retrieved oocytes [10.1 (5.4) vs. 11.1 (8.0) vs. 9.2 (6.0), p = 0.131], metaphase II oocytes [8.7 (4.7) vs. 9.8 (7.1) vs. 8.3 (5.1), p = 0.147]; and euploidy rate (38.7% vs. 36.3% vs. 31.5%, p = 0.182). Moreover, recombinant FSH in monotherapy in this specific population could guarantee the efficiency of the cycle since there are no significant differences in the usable blastocyst rate. Finally, clinical results were analyzed in those cases in which a frozen single embryo had been performed in a subsequent artificial cycle, in which the effect of LH on the endometrium is obviated; once again, no significant differences were observed between the study groups neither for the implantation rate (50% vs. 56% vs. 45%, p = 0.780) or the clinical pregnancy rate (50% vs. 56% vs. 45%, p = 0.772). Limitations, reasons for caution This is a retrospective and observational study and thus possible confounders cannot be completely excluded. More data are needed to draw firm conclusions and it will be critical to increase the sample size to check if the results observed in this work remains in the general population. Wider implications of the findings In view of tailoring assisted reproductive protocols to meet patient needs and improve the likelihood of positive outcomes, these data suggest that it is optimal to perform an ovarian stimulation protocol only with recombinant FSH in a frozen embryo program in advanced maternal age context without clinical outcomes being affected. Trial registration number Not applicable

P-253 Clinical efficacy of PGT-A according to maternal age and embryo quality in blastocyst stage

Abstract Study question To establish the usefulness of PGT-A as an embryo selection tool based on the quality of the blastocyst and the age of the patient. Summary answer The euploidy rate of good quality blastocysts decreases dramatically with increasing maternal age. What is known already One of the fundamental objectives to achieve an optimal gestation rate is the selection of the embryo with the highest implantation potential. For this purpose, morphokinetics and aneuploidy detection are used. Most aneuploidies arise in maternal meiosis, and increase exponentially in women over the age of 35 years, coinciding with the rapid decline in IVF success and live birth rates in patients of advanced maternal age. Study design, size, duration In the present retrospective study, embryo quality of 321 blastocysts was evaluated from June 2020 to January 2023. They were analyzed according to the ASEBIR (2015) classification and using preimplantation genetic testing for aneuploidy (PGT-A) with next-generation sequencing (NGS). Participants/materials, setting, methods A total of 321 blastocysts were analyzed using PGT-A from which 101 were euploid. This 101 blastocysts were classified according to the morphokinetic (A or B (good quality) and C (medium quality)) and the maternal age (<35, 35-36, 37-38, 39-40, 41-42, 43-44 and 45 -46 years). Out of the 101 euploid blastocysts, 78 embryos were classified as A or B (good quality) and 23 as C (medium quality) according to the ASEBIR classification. Main results and the role of chance The euploidy rate of the embryos decreases as the age of the patient increases, being 73% for good quality embryos in patients under 35, 54% in patients between 35-36, 37% in patients between 37- 38, 31% in patients between 39-40, 23% in patients between 41-42, 5% in patients between 43- 44 and 0 % in patients between 45-46 years. For medium quality embryos, the euploidy rate is 58% in patients under 35, 38% in patients between 35-36, 30% in patients between 37-38, 26% in patients between 39-40, 0% in patients between 41-42, 0% in patients between 43-44 and 0% in patients between 45- 46 years. In patients under 35 years, PGT-A will improve pregnancy rates considering that it is an invasive and costly technique. In patients between 35-36 years, PGT-A is a very useful tool when embryos have C quality as well as, in patients between 37- 40, regardless of embryo quality. Furthermore, patients between 41-42 years with good quality embryos, PGT-A will decrease the abortion rates and increase the pregnancy rates. In contrast, patients with C-quality embryos should not start new cycles, as well as patients with over than 43 years due to the low probability of euploidy. Limitations, reasons for caution Studies of larger sample size and study period are required to validate our current findings. Furthermore, more information about fertilization, pregnancy, birth rate and perinatal outcomes could improve the current results. Wider implications of the findings Making the decision whether or not to use PGT-A on the day of blastocyst formation helps to choose the best strategy by individualizing each case according to the maternal age and the quality of the blastocysts. Decreasing cycle times will always be beneficial to patients and their outcomes. Trial registration number Does not apply

O-146 Oocyte donation does not increase live birth rates in young women suffering from recurrent pregnancy loss

Abstract Study question Does oocyte donation improve reproductive outcomes in recurrent pregnancy loss (RPL) patients? Summary answer Oocyte donation increases live birth and reduces miscarriage rates in RPL women older than 35, but it does not improve reproductive outcomes in younger patients. What is known already Recurrent pregnancy loss (RPL) is defined as 2 or more pregnancy losses prior to 20-24 weeks of gestation and has an incidence of approximately 1 to 2% in couples trying to conceive. Known risk factors for RPL are embryo aneuploidy, advanced maternal age (AMA), previous miscarriages, uterine abnormalities, parental chromosomal abnormalities, antiphospholipid syndrome, endocrine factors, such as thyroid function or obesity, and thrombophilia. However, 50-70% of patients suffering from RPL do not present any known risk factor, making the management of idiopathic RPL patients particularly challenging for ART clinicians. Study design, size, duration This is a cohort retrospective study involving 2 centers. Charts from 18,273 women undergoing IVF treatment between 2011 and 2020 were reviewed. A total of 912 patients (5%) met the definition of RPL and were classified into study groups based on their age and oocyte origin: ≤35 years old receiving oocyte donation (n = 39) or using their own oocytes (n = 35), and AMA women using donor eggs (n = 716) or their own (n = 122). Participants/materials, setting, methods Demographic variables analysed included known risk factors, number of transferred embryos, day of embryo transfer (3 vs 5) and sperm origin (partner/donor). Differences in biochemical, clinical, ongoing pregnancy, miscarriage and live birth rates were assessed by Pearson’s Chi-squared or Fisher’s exact test. P-values <0.05 were considered significant. Main results and the role of chance RPL patients had 2.77±1.27 pregnancy losses overall (2.76±1.37 in women ≤35 and 2.77±1.26 in patients >35). Most RPL patients (91.9%) were of AMA. Other RPL-associated risk factors, including uterine malformations, previous pregnancy losses, chromosomal abnormalities and thrombophilia were identified in 207/912 patients (22.7%). Interestingly, these were more frequent in patients <35 (37.8% vs 21%, p = 0.001). Young RPL women presented higher rates of karyotype abnormalities than older patients (17.6% vs 3.8%, p < 1x10-04), while showing a similar incidence of uterine abnormalities (14.9% vs 12.9%, p > 0.63) and thrombophilia (4.1% vs 4.7%, p > 0.81). RPL patients >35 preferentially underwent oocyte donation (85.4% vs 52.7%, p < 1x10-04), which led to significantly higher biochemical (52.5% vs 17.1%, p < 1x10-04), clinical (42.4% vs. 8.5% p < 1x10-04), ongoing pregnancy (37.9% vs 5.1%, p < 1x10-04), live birth (32% vs 4%, p < 1x10-04) and lower miscarriage rates (10.5% vs 40%, p = 0.0186) than in autologous cycles. In contrast, RPL patients <35 had similar reproductive outcomes: biochemical (41% vs. 52.9), clinical (28.2% vs. 37.1%), ongoing pregnancy (25.6% vs 29.4%), live birth (25.6% vs 26.4%) and miscarriage rates (9% vs 23%), regardless of oocyte origin (donated vs own, p > 0.05 for all cases). Importantly, this was also true for a small subgroup of idiopathic young RPL patients (n = 13). Limitations, reasons for caution The main limitation of this study is its retrospective nature, which does not allow for full elucidation of all potential confounders. The number of young RPL patients undergoing oocyte donation may be too low to draw significant conclusions. Wider implications of the findings RPL can be resolved in AMA patients by using donor oocytes, which points to key roles of oocyte quality and aneuploidy underlying RPL aetiology. However, RPL is not ameliorated by oocyte donation in young women, suggesting an endometrial/systemic origin and highlighting the need to further study mechanisms driving this disorder. Trial registration number Not applicable

P-710 Single-cell proteomic analysis of human oocytes reveals a decreased abundance of meiosis and proteostasis regulators with advanced maternal age

Abstract Study question How does maternal age affect the proteomic content of her oocytes during their final meiotic progression? Summary answer Compared to young oocytes, oocytes obtained from women of advanced maternal age (AMA) show a decreased abundance of meiosis and proteostasis regulators. What is known already It is well established that AMA leads to a decline in oocyte number and quality, key limiting factors of female reproductive success. Poor oocyte quality is tied to aneuploidies and alterations in cytoplasmic fitness, however the precise mechanisms that underlie oocyte aging remain unknown. Currently knowledge has been largely restricted to transcriptomic studies with no information on the proteome of oocytes obtained from women of AMA. Single-cell proteomic studies may shed light on these mechanisms, as proteins and (post)-translational modifications regulate events unfolding at the oocyte-to-embryo transition in transcriptionally silent oocytes. Study design, size, duration A total of 67 oocytes, obtained from 52 women undergoing controlled ovarian stimulation cycles between May 2021 and May 2022, were included in the study. We obtained 18 fresh germinal vesicle oocytes (GVs) and 17 vitrified in vivo matured metaphase II oocytes (MIIs) from young women (oocyte donors), and 18 GVs and 14 MIIs from AMA. Participants/materials, setting, methods The oocytes were obtained from oocyte donors (n = 27) and AMA patients (n = 25) with a mean age of 24 ± 3.9 years and 39 ± 1.8 years respectively. Single denuded oocytes were snap frozen and their proteomic profiles were generated using the mass spectometry based method plexDIA . Mann-Whitney U test (fold change >|1.5|, adjusted p-value <0.05), Spearman correlation (rs ≥|0.4|, p-value <0.05) and ggplot were used in R to compare the different groups. Main results and the role of chance A total of 2105 proteins were quantified across oocytes stages and age groups. Most of proteins were present in oocytes independently of their maturation state and related to vital processes, such as cellular respiration and energy metabolism. Fifty-four proteins varied in abundance between immature and mature oocytes, including RS14, YBOX2 and RL10A (translational regulation), CLUS and AHSA1 (post-translational modifications) and proteins involved in meiosis progression (WEE2, AURKA, BUB1B, SPDLY). Notably, we observed a negative correlation between protein levels and maternal age for meiosis regulators (e.g.1433E, CDK1; rs: -0.7 and -0.5), meiotic spindle proteins (e.g. DYL2, CAPZB, ARP3, GCP3; rs: -0.6, -0.6, -0.5, -0.5 respectively) and for 5 proteasome subunits (PRS8, PRS6A, PRS10, PSA6, PSMF1; rs: -0.4 to -0.7). The proteasome is a degradation machinery with multiple roles, including meiosis regulation and proteostasis maintenance. Additional proteins of the proteostasis network were found to decline with age (e.g., TCPH, TCPA; rs: -0.5, -0.5). Loss of proteostasis is a common feature of aging and diseases. We also observed a positive correlation between mitochondrial and stress response proteins and maternal age (e.g. ATP5L, TMX1; rs: 0.6, 0.5). Limitations, reasons for caution Unlike GVs that were collected fresh, MII oocytes underwent vitrification and warming before being included in the study due to clinical protocols. These procedures may have unknown effects on the proteome. Wider implications of the findings We show that a woman’s age correlates with changes in her oocyte proteome, which may contribute to the deterioration of oocyte quality through dysregulation of meiosis and disturbance of proteostasis. Proteins showing differential expression in oocytes from women of AMA, such as proteasome, may become targets for improving oocyte quality. Trial registration number Not applicable

O-205 Poor mitochondrial metabolism impairs meiosis and contributes to reduced oocyte maturation rates in patients with advanced maternal age

Abstract Study question Is there a relationship between poor mitochondrial metabolism and impaired meiotic progression of germinal vesicle (GVs) oocytes in women of advanced maternal age (AMA)? Summary answer Poor mitochondrial metabolism impairs meiotic progression in human oocytes, contributing to a lower oocyte maturation capacity in women of AMA. What is known already High aneuploidy rates and impaired metabolism are characteristic features of AMA oocytes. Studies in mouse suggest that successful meiotic maturation and metabolic competency may be functionally linked, however this relationship has not been clearly established in human. Metabolic dynamics can be visualized by indirect measurements through mitochondrial staining and quantified more directly using Fluorescence Lifetime Imaging Microscopy (FLIM). This live-imaging approach can generate metabolic timelapse profiles of oocytes throughout meiosis. We performed an extensive characterization of oocyte metabolism in maturing GVs, obtained from both young and AMA patients, to establish the importance of mitochondrial metabolism during meiosis. Study design, size, duration A total of 401 GVs from young (≤30 years, n = 264) and AMA (>37 years, n = 137) women were included in the study. GVs were matured in vitro in G2-plus medium for 30 hours. Maturation was determined by the presence of an extruded polar body. Oocytes were used for FLIM (n = 175) and immunofluorescence (IF) analysis (n = 226). Further, mitochondrial metabolism loss of function studies (n = 64) were performed by treating young GVs with 1μM Trifluoromethoxy-carbonylcyanide-phenylhydrazone (FCCP) for 30 minutes. Participants/materials, setting, methods The proteins Dihydrolipoamide-S-Acetyltransferase (D-LAT) and Translocase-of-outer mitochondrial-membrane (TOMM20) were analysed in young and AMA oocytes by IF (Arbitrary-Unit, AU) to assess mitochondrial activity and localization, respectively. Fluorescence mean intensities were quantified with ImageJ and compared by t-test; maturation rates were compared by Chi-squared test. FLIM comprehensive metabolism (NADH; FAD+) were taken at GV stage. Different FLIM parameters (fluorescence intensity, fraction bound, short/long lifetime) were evaluated individually and combined into the Redox ratio (NADH intensity/FAD+ intensity). Main results and the role of chance Mitochondrial staining showed a common pattern in young and AMA GV oocytes, with a uniform localization of mitochondria in the ooplasm (TOMM20) and a subcortical localization of active organelles (D-LAT). These patterns were confirmed by in-live FLIM analysis. The total mitochondria abundance was comparable between AMA and young GV oocytes (intensity of 61674±24322 AU in young, 32186±33414 AU in AMA, p = 0.195), however, active mitochondria were diminished in AMA compared to young GVs both by IF and FLIM (intensity of 78614±58534 AU in young, 12517±10187 AU in AMA, p = 0.003; Redox ratio in young 2e+00±0.15, in AMA 1e+00 ± 0.16, p = 2.969e-05). Notably, young oocytes matured at a significantly higher rate (86.3%; 63/73) than AMA oocytes (62.3%; 38/61; p = 0.002). Moreover, FLIM imaging revealed that GVs with a higher metabolism were more likely to complete meiosis to MII (Redox ratio 2e+00±0.17 in GVs matured to MII, 1e+00±0.18 in non-matured, p = 1.054e-06). Following treatment with FCCP, young GVs showed a similar phenotype to AMA oocytes, with a significant decrease in mitochondrial activity (intensity of 78614±58534 IU in untreated and 11554±16131 IU in treated GVs, p = 0.019), and an associated drop-in maturation rate. Ultimately, only 39.5% (17/43) of the young treated GVs were able to accomplish meiotic maturation. Limitations, reasons for caution Maturation rates were assessed by the presence of an extruded PB and variations in spindle assembly timings may have been overlooked. The quantification of mitochondrial activity in loss of function studies was assessed only by IF staining. Wider implications of the findings Our findings demonstrate the presence of a functional link between oocyte metabolism and impaired meiosis, which may contribute to the decline of oocyte quality with age. Given that live-metabolic imaging is able to discern GVs that progress through meiosis, this approach may represent a valuable non-invasive tool in oocyte selection. Trial registration number not applicable