Abstract Study question What are reproductive outcomes in Fragile X premutation carriers (FXPM) undergoing either preimplantation genetic testing for monogenic disorders (PGT-M) or treatment using oocyte donation (OD)? Summary answer In FXPM with normal ovarian reserve, autologous PGT-M treatment yields a favorable live birth rate (LBR), comparable to outcomes from OD treatment. What is known already Fragile X syndrome, caused by an expanded CGG repeat (>200) in the FMR1 gene, is characterized by intellectual disability. Individuals carrying a premutation (55–200 repeats) risk further expansion in subsequent generations. PGT-M can be offered to prevent the transfer of embryos with such an expansion. However, the premutation is also associated with diminished ovarian reserve, potentially impairing ovarian stimulation response—a key factor for successful PGT-M treatment. Consequently, some patients may require OD as a final treatment option. Additionally, the impact of the premutation on oocyte quality remains to be investigated. Study design, size, duration A retrospective analysis at a university affiliated hospital was performed including all assisted reproductive technology (ART) cycles in FXPM undergoing either PGT-M treatment or OD from January 2010 until December 2022. A total of 93 patients (61 PGT-M and 32 OD) undergoing 422 ART cycles (291 PGT-M and 131 OD) were included in the study. Participants/materials, setting, methods All FXPM performing ART in the study period were included. Patients opting for autologous treatment without PGT were excluded. Patients carrying an intermediate allele (46-54 repeats) or a full mutation (>200 repeats) were not included. The primary outcome focused on LBR and cumulative LBR in both treatment groups. Secondary outcomes included the median number of embryos transferred before achieving live birth and median number of PGT-M cycles required to achieve a transferable embryo. Main results and the role of chance Median number of CGG repeats (PGT-M group 82.5 repeats, OD group 75 repeats), age and BMI were similar in both groups. Median anti-Müllerian hormone (AMH) was higher in PGT-M patients (0.98 µg/L) compared to OD patients (0.11 µg/L). In PGT-M treatment exclusively blastocyst transfers were performed, whereas cleavage stage transfers made up 72.90% of embryo transfers in OD treatment. LBR per cycle was 14.78% for PGT-M treatment compared to 19.85% for OD treatment. In PGT-M treatment 14.09% of cycles were cancelled, compared to 10.79% in OD treatment. Cumulative LBR for PGT-M treatment was 48.65% overall, increasing to 61.91% when limited to patients with AMH levels exceeding 0.5 µg/L, compared to 42.11% in patients with AMH levels below 0.5 µg/L. Cumulative LBR for OD treatment was 71.88%. The small sample size and heterogeneity within the patient population and treatment protocols (e.g. PGT-M or OD, stage of transfer) precluded direct statistical comparison. In both groups a median of two embryos were transferred before achieving live birth. In PGT-M, 93 out of 193 stimulation cycles did not yield a transferable embryo, with 24.59% of PGT-M patients never achieving one. Those who did, achieved a transferable embryo after a median of one stimulation cycle. Limitations, reasons for caution Although the study was conducted in a high-volume tertiary hospital for PGT and provides clinically relevant insights, the results require careful interpretation due to retrospective bias and a limited sample size, reflecting the low incidence of Fragile X premutation. Wider implications of the findings In FXPM with normal ovarian reserve, the LBR following PGT-M is favorable and approaching those observed in patients undergoing OD treatment. However, in cases of diminished ovarian reserve, success rates are reduced in autologous cycles. Trial registration number Yes

Abstract Study question What are the number of collapses and the percentage of embryo surface reduction during blastocyst collapse from which clinical outcomes are impaired? Summary answer Results showed that a single embryo collapse can reduce clinical outcomes, but the threshold of embryo surface reduction varies with the patient’s age. What is known already Embryo collapse before hatching is frequently observed in blastocysts obtained from Assisted Reproductive Technologies (ART). During the collapse, the blastocyst surface reduces transiently and further expands once or more times. Weak contractions seem to be beneficial for blastocyst hatching, but intense or vigorous collapses impair both hatching and embryo development. The incidence of embryo collapse varies between studies, from 15 to 55%, mainly because the embryos with weak collapses may be considered as non-collapsed. Yet, a clear shrinkage threshold for substantial collapses has not been consistently established, the cut-off values ranging from 20 to 50% of blastocyst surface reduction. Study design, size, duration This retrospective study included 1,548 blastocysts obtained by ICSI fertilization of the patient’s own oocytes between January 2016 and December 2022. Patients were admitted to four sanitary centres, but oocyte fertilization and embryo culture were conducted at the same IVF lab. Patients (n = 461) were split into three groups: ≤34 years old (y/o), 35-39 y/o, and ≥40 y/o. All patients consented to participate. Participants/materials, setting, methods Fertilized oocytes were incubated in single-step medium until day+6 in an Embryoscope™ TL incubator. Blastocyst collapse was assessed by counting the number and intensity of collapses (% reduction of embryo surface in each collapse). Surface measures were conducted with Elypse-EmbryoviewerTM using the trophectoderm area in contact with the inner surface of the zona pellucida as a reference. Blastocysts were distributed into three groups according to the maternal age. Results are expressed as mean ± SD. Main results and the role of chance The number and intensity of blastocyst collapses impaired clinical outputs, but differences existed based on the maternal age. In the group of patients ≤ 34 y/o, the pregnancy and live birth rates did not differ between non-collapsed and single-collapse blastocysts (P > 0.05), but clinical outcomes decreased significantly if a second collapse occurs (P < 0.05). When patient’s age was equal to or higher than 35 y/o, a single collapse significantly affected clinical outcomes (P < 0.05). Regarding their intensity, embryo collapses associated with a surface reduction of 10-15% did not affect implantation rates in any of the maternal groups, but significantly impaired pregnancy and live birth rates in patients ≥ 40 y/o (P < 0.05). When embryo collapses resulted in a surface reduction ≥ 20%, all clinical outcomes were significantly lower in patients ≥ 35 y/o than in those ≤ 34 y/o. It should be noted that, in the youngest group, the threshold of surface reduction during the collapse significantly affecting clinical outcomes was higher (40%). Limitations, reasons for caution This preliminary study examined embryos from patients admitted to four different IVF clinics that were fertilized and incubated in a single IVF lab. To strengthen the robustness of our results, the study should be extended to other centres and increase the sample size. Wider implications of the findings Blastocyst collapse should be used as a criterion for embryo selection in ART when using patient own’s oocytes. A single collapse impairs clinical outcomes when its intensity is above the threshold 10% for patients ≥ 40 y/o, 20% for patients of 35-39 y/o, and 40% for patients ≤ 34 y/o. Trial registration number No

Abstract Study question Are single-step warming protocols efficient with the practice of universal warming? Summary answer Universal warming remains efficient with single-step warming protocols, in term of embryology and clinical outcomes. What is known already Clinics receive reproductive cells that have been cryopreserved at other facilities using various vitrification protocols/media/cryo-device. Oocytes and embryos are routinely cryopreserved using different commercial brands of ready-to-use vitrification solutions which differ only slightly in their composition. It has previously been demonstrated that the combined use of different vitrification/warming kits is feasible. However, this concept has not been tested in conjunction with single-step warming protocols using only the first elution step with 1M non-permeating solutes (NPS). Study design, size, duration This retrospective observational study was performed on 329 blastocysts vitrified and warmed between 01/01/2024 and 31/12/2024. Blastocysts were vitrified in Kitazato, SAGE, or Irvine media and warmed with warming media from one of the three manufacturers using a single-step protocol. Blastocyst survival (SR), clinical pregnancy (CPR), implantation (IR) and clinical loss (CLR) rates were calculated for all nine combinations of vitrification and warming media. Statistical analysis: Chi Square/Yates and Fisher exact tests. Participants/materials, setting, methods Vitrification and warming were performed using the 9 possible combinations of kits by Irvine-Fujifilm [I], Kitazato [K], and SAGE CooperSurgical [S]. Blastocysts were vitrified using a “universal” protocol performed at room temperature: 12-15min in ES, 50sec in VS, then transferring the embryo to a second drop of VS, loading and plunging within 70sec. Warming was performed using 4 ml of 1M non permeating solute (NPS) at 37 °C. Transfer was performed after 1-2hrs incubation. Main results and the role of chance Group first initials indicate the vitrification media and the second the warming. Survival rate was 100% in all groups. Overall, 329 blastocysts were warmed and transferred in 303 cycles, resulting in a CPR of 45.2%, IR of 42.3% and CLR of 12.4%; to date, 10 babies are already born. There were no significant difference in CPR, IR or CLR between groups KK (CPR=50.0%, IR = 50.0%, CLR=33.3%), KI (CP = 48.R5%, IR = 45.4%, CLR=10.2%) and KS (CPR=51.3%, IR = 50.0%, CLR=20.0%) or between groups II (CPR=40.0%, IR = 34.8%, CLR=31.3%) and IS (CPR=31.6%, IR = 36.8%, CLR=16.7%). Group IK was excluded due to very low numbers (3 blastocysts). Where embryos were vitrified with SAGE, there was no significant difference in CPR, IR, or CLR rate between group SK (CPR=50.0%, IR = 50.0%, CLR=0.0%) and group SS (CPR=62.5%, IR = 60.0%, CLR=6.7%) or between SK and group SI (CP = 38.8%, IR = 34.6%, CLR=0.0%). However, there was a significantly lower implantation rate in group SI compared to group SS (p = 0.044) although the difference in CP was not quite significant (p = 0.0779), and no significant difference was observed in CLR. Limitations, reasons for caution While maternal age was comparable across all groups, the study was not controlled in terms of patient history or embryo quality. CLR was calculated on the basis of combining livebirth and ongoing pregnancy rates. Wider implications of the findings The introduction of a universal single-step warming (USSW) will reduce costs, facilitate embryo exchange between centres and to streamline the lab workflow Trial registration number No

Abstract Dr. Karina Banasik on behalf of the Copenhagen Pregnancy Loss Study (COPL) consortium Our large-scale study of 3,000 trios (mother, father, fetus) in the Copenhagen Pregnancy Loss Study (COPL) reveals critical insights into the genetic architecture of pregnancy loss, which affects approximately 25% of all pregnancies. Our study is the first to apply large-scale recruitment of consecutive patients with clinically diagnosed pregnancy loss, detailed pheno- and genotyping, and comprehensive sampling of material from both the fetus and parents. By employing whole-genome sequencing and sequencing of cell free fetal DNA from maternal blood samples, we identify chromosomal abnormalities, copy number variants and sequence variants, their functional consequences, and inheritance patterns associated with pregnancy loss. Additionally, we evaluate parental contributions through polygenic risk scores and sperm DNA fragmentation analysis (paternal), providing a comprehensive assessment of how parental genetic factors may influence pregnancy outcomes. We observe that for 48% of the cohort, pregnancy loss is a recurrent event and the women either report having previous losses or return with one or more losses during the study period. By being able to distinguish between aneuploid and euploid losses we hope to advance our understanding of reproductive biology and improve outcomes in couples experiencing recurrent pregnancy loss. By focusing on euploid losses, which account for approximately half of all pregnancy losses and have been particularly challenging to explain, our work opens new avenues for research into the complex genetics of pregnancy loss, where polygenic risk of factors known to impact risk of pregnancy loss (e.g., metabolic, endocrine factors) can help to elucidate etiology.

Abstract Study question Does the administration of micronutrients and probiotics supplement during an oocyte donation program improve fertilization rate, blastocyst development, and pregnancy outcomes? Summary answer Micronutrients and probiotics supplementation in oocyte donors does not influence fertilization or blastocyst rates, but significantly improved pregnancy outcomes of recipients that used vitrified oocytes. What is known already Micronutrient supplementation could have a positive impact on IVF clinical results. Evidence about its role in improving oocytes’ quality and reproductive outcomes in normoresponders patients are still lacking. The OvoVid double-blind, randomized, controlled trial Phase 1 suggested that a micronutrients-probiotics supplementation enhances oocyte yield and quality in normoresponder oocyte donors. A significantly higher number of total oocytes retrieved and mature MII were achieved in the supplement group, which also showed a better follicular response to controlled ovarian hyperstimualtion (COH) than placebo in term of more follicles at the end of COH, despite a similar initial antral follicle count. Study design, size, duration Multicentric, double-blind, randomized controlled trial involving 130 oocyte donors and 107 recipients during a two-year period involving three Fertility Clinics in Spain. Oocyte donors were randomized in supplement or placebo group, taking one tablet a day at least for 30 days before and during COH. The micronutrients-probiotc supplement was a combination of Myoinositol+d-chiro-inositol (ratio 40:1), folic acid, melatonin, coenzyme Q10, vitamin D3, B6, B12, E, iron, zinc, Lactobacillus Gasseri and Crispatus. Participants/materials, setting, methods The Ovovid Phase 2 study analyzed the reproductive outcomes of 107 oocyte donation treatments in recipients, classified in supplement (n = 62) or placebo (n = 45) group depending on the oocytes’ origin, realized with donors’ oocytes of Ovovid Phase 1. Primary outcomes included fertilization and blastocyst development rates, while secondary outcomes encompassed pregnancy rates and oocyte survival after thawing. Statistical analysis was performed using Fisher’s exact test and Mann-Whitney U test, with significance set at p<0.05. Main results and the role of chance No significant differences were observed in fertilization rate (76,7% vs. 78%, p > 0,05) comparing supplement and placebo group, as well as in blastulation development (43,4% vs. 45,4%, p = 0,61), despite similar number of inseminated oocytes (6,7 ± 1,3 vs. 6,3 ± 1,2, p = 0,13). When vitrified oocytes were used, similar survival rate after thawing were described in the two groups (94,3% vs. 93,8%, p = 0,3). Pregnancy rate was similar regardless of multivitamin-probiotics supplement use (44,4% vs. 68,2%, p = 0,07). However, in the sub-analysis for type of oocyte, pregnancy rates with vitrified oocytes (Supplement n = 24; Placebo n = 18) were significantly higher in the supplement group compared to placebo (50% vs. 0%, p = 0,02). No significant differences were found for fresh oocytes (n = 65; Supplement n = 38; Placebo n = 27) outcomes. Limitations, reasons for caution The study was limited by the relatively small sample size for subgroup analyses and the lack of long-term follow-up for pregnancy outcomes. Further studies are warranted to confirm these observations and optimize supplementation strategies in oocyte donation programs and assisted reproductive technologies. Wider implications of the findings Micronutrients and probiotics supplementation during oocyte donors’ COH did not seem to improve overall fertilization or blastulation rates. However, it may be a valuable adjunct in improving recipients’ pregnancy outcomes with vitrified oocytes, suggesting a role in enhancing oocyte competence during vitrification, potentially optimizing outcomes for specific protocols. Trial registration number No

Abstract Study question What is the outcome for non-expanded (NE) blastocysts at the time of frozen embryo transfer? Is this affected by cycle preparation (hormone-programmed vs natural cycles)? Summary answer NE blastocysts transferred 4-hours after thawing have lower pregnancy and clinical pregnancy rates than expanded blastocysts. These pregnancy outcomes can be rescued in natural cycles. What is known already Research has shown that the quality of the blastocyst is a crucial factor influencing pregnancy outcomes. The expansion of the blastocyst at the time of frozen embryo transfer (FET) can vary, impacted by its quality and the duration since thawing, a detail that is often omitted from reports. Additionally, there are differences in outcomes between natural cycle FETs and hormone-programmed FETs, though the underlying reasons for these differences remain unclear. Consequently, a thorough evaluation of these factors is essential for refining IVF practices. Study design, size, duration A retrospective cohort analysis was conducted of 458 single blastocyst FET cycles between January 2023 and August 2024 at a single public university affiliated IVF center. At transfer all embryos included in this study were of Gardner’s grade, AA, AB, or BA. Embryos were classified as non-expanded when they had not yet re-expanded to fill the zona pellucida post thaw, and did not have a large cavity. Participants/materials, setting, methods 152 women had NE embryos and 306 controls with full embryo expansion matched for age from the same study period. Patients undergoing PGT-A (preimplantation genetic test) or oocyte donation cycles were excluded. All embryos were vitrified and warmed using Kitazato media and transferred using Wallace catheters with trans-abdominal ultrasound guidance. Data was compared using chi-square or t-tests where appropriate and multivariate logistic regression to control for confounding effects was planned (baseline characteristic with P < 0.10). Main results and the role of chance The analysis demonstrated that the patient characteristics were comparable between the two groups, including antral follicle count (AFC), serum anti-Müllerian hormone (AMH) level, stimulations protocols, gonadotropins dose, ICSI use, oocytes collected, fertilization rate, blastocyst quality and number of frozen blastocysts. The pregnancy rate for NE (46/152, 30.3%) was significantly lower than that of expanded blastocyst(154/306, 50.3%) (p < 0.001). Clinical pregnancy rates differed (NE: 43/152, 28.3% vs. expanded: 126/306, 41.2%, p = 0.006). Miscarriage rates comparing both embryo groups were similar 25.5% vs. 29.4%, (p = 0.74). Comparing protocols with NE, natural cycle FETs exhibited a higher pregnancy rate (20/27, 74.1%) compared to the hormone programed FETs (26/125, 20.8%), (p < 0.001) and subsequently higher clinical pregnancy rates (20/27, 74.1% vs 23/125, 18.4% respectively p < 0.001). When analyzing the expanded blastocyst (all FET types) and NE blastocysts in natural cycles, no statistically significant improvements were found in pregnancy or clinical pregnancy rates (p > 0.05 in both cases) with expanded embryos. Limitations, reasons for caution The retrospective study design may introduce undetected bias. The relatively small number of patients comprising the natural cycle FET group reduces the overall statistical power of this study group Wider implications of the findings Transferring expanded blastocysts four hours after thawing resulted in better clinical outcomes. Natural cycle FETs seem to rescue NE blastocyst potential to some degree, but the underlying mechanism requires further study. Large studies with more natural cycle FETs are needed to verify the reported observations. Trial registration number No

Abstract Study question To determine the influence of the shape of the uterus and the volume of the favorable implantation zone (Z) on the effectiveness of embryo transfer. Summary answer The reason for implantation failure may be a decrease of favorable zone of implantation which needs more objective and measurable criteria What is known already The uterine cavity abnormalities could be the cause of reproductive disorders, including failure of embryo implantation in ART programs. At the same time, there is no consensus regarding the negative impact of minor changes of the uterine cavity on the embryo transfer success. If the uterus has a small notch in the bottom and slight thickening of the side walls, it can fall into the normal category. But the space in the centre of the uterine cavity with the thickest endometrium, which we call favorable implantation zone will be reduced in these cases. Study design, size, duration We retrospectively analyzed the results of transfers of genetically tested embryos between March 2018 and March 2024 in patients with preserved coronary 3D images of the uterine cavity. Participants/materials, setting, methods The 3D images of uterine cavity were obtained on the day of embryo transfer in 2184 cycles. We divided all cases depending on the volume of Z. The volume of Z was calculated automatically, taking into account the height and the width of the zone and the thickness of the endometrium. Main results and the role of chance In the first group (Z volume > 15 mm3) there were 1134 transfers, pregnancy rate (PR) was 64%, live birth rate (LBR) - 52%, in the second group (Z volume 10.1-14.9 mm3) there were 960 transfers with PR - 34% and LBR - 29%, in the third group (Z volume ˂ 10 mm3) there were only 90 transfers, PR - 13.3%, LBR - 2.2%. The difference between the groups was statistically significant. The last group had the highest percentage of early pregnancy losses (10 out of 12 pregnancies). The part of the patients with an endometrium less than 8 mm in the third group was also the largest. Limitations, reasons for caution Our data were recieved in retrospective study. The results need confirmation by prospective one. Wider implications of the findings Potential possibility to influence implantation rate in the group of patients with decreased favorable implantation zone. Trial registration number No

Abstract Study question Is there a distinction in the correlation between AFC and retrieved oocytes when AFC is measured during the same-retrieval-cycle or a different-cycle’s early follicular phase? Summary answer AFC assessment at the beginning of the cycle demonstrated a stronger and statistically significant correlation with retrieved oocyte numbers compared to the different-cycle assessment. What is known already AFC is a widely used biomarker for ovarian reserve and plays a role in predicting ovarian response in IVF cycles. The inter-cycle variability of AFC is generally considered to be of minimal clinical relevance in the response prediction, therefore, it is commonly accepted that repeated AFC assessment is unlikely to confer additional value. However, the impact of measurement timing on predictive accuracy for retrieved oocytes on the same cycle remains unclear. Study design, size, duration A retrospective cohort study including patients undergoing their first antagonist cycle performed in the United States between 2022–2023. The study group included 1,414 cycles with an AFC assessed at the beginning of the retrieval cycle (same-cycle). The control group included 1,414 cycles with an AFC assessment performed at the early follicular phase within a year of the retrieval cycle but not at the beginning of the specific cycle (different-cycle). Participants/materials, setting, methods A propensity score matching analysis was performed on 5,538 cycles, comprising 4,124 same-cycle and 1,414 from different-cycle AFC. For each cycle in the different-cycle group, the cycle with the most similar propensity score from the same-cycle group was matched. The propensity scores were calculated using patient age, AFC, anti-Müllerian hormone (AMH), initial stimulation dosage, and the number of retrieved oocytes. Main results and the role of chance Baseline characteristics, mean absolute error (MAE), R², and Pearson correlation between AFC and retrieved oocytes were computed. The difference between the groups’ Pearson correlations was calculated using the Fisher-Z test. Baseline cycle characteristics were similar in the study and control group including patients’ age (36.7±4.1 vs. 36.7±4.2; p = 0.84), AMH (2.3±1.9 vs. 2.2±2.1; p = 0.72), AFC (14.1±8.2 vs. 13.9±8.6; p = 0.63, initial stimulation dosage (402±123 vs. 401±123; p = 0.87) and retrieved oocytes (13.1±7.6 vs. 12.9±8.0; p = 0.56) respectively. Results showed an MAE of 5.6 vs. 6.18, R² of 0.19 vs. 0.08, and Pearson Correlation of 0.57 vs. 0.52 between the AFC and retrieved oocytes, for the study and control group, respectively. The MAE indicates that same-cycle AFC assessment is more accurate for understanding the relationship between AFC and retrieved oocytes compared to different-cycle AFC. The R² values demonstrate limited explanatory power, with AFC accounting for 19% of the variance in retrieved oocytes in the same-cycle study group and only 8% in the different control group. Pearson correlations were moderately positive and stronger in the study group (0.57 vs. 0.52). Fisher-Z test for the group’s Pearson correlations (p = 0.03) suggested a significantly higher correlation between the AFC and the retrieved oocytes in the study group. Limitations, reasons for caution The main limitation of this study is its retrospective design, which may introduce biases due to unaccounted factors. The analysis is limited to IVF antagonist cycles within the United States, further research should be performed to ensure it is generalizable across different protocols and regions. Wider implications of the findings These findings suggest that AFC within the treatment cycle provides a more accurate prediction tool for the number of oocytes retrieved than a different-cycle AFC. Statistical methods can leverage the distinction between same-cycle and different-cycle AFC measurements to enhance prediction accuracy, particularly when combined with other biomarkers such as AMH. Trial registration number No

Abstract Study question What are the metabolome differences between the serum of patients with endometriosis and control subjects? What metabolic traits are characteristic of peritoneal and/or ovarian endometriosis? Summary answer Endometriosis patients have high abundance of riboflavin, tryptophan and purine metabolites in serum. Peritoneal endometriosis has stronger metabolic signature than ovarian endometriosis compared to controls. What is known already Endometriosis patients exhibit different metabolic traits compared to healthy subjects. Several metabolomics studies were conducted on different types of samples (serum, plasma, peritoneal fluid, follicular fluid, eutopic/ectopic endometrial tissue, urine, cervical swabs). However, the sample sizes are limited, and inclusion/exclusion criteria vary. Alterations in amino acid levels, (phospho)lipid metabolites, and purine metabolites are described, though results are controversial depending on sample type and study design. Currently, there is no biomarker for endometriosis and its pathomechanism is still unclear. Study design, size, duration Serum samples were obtained before surgery from 338 dysmenorrheic and/or infertile patients undergoing laparoscopic surgery at a university-affiliated fertility center from February 2019 to October 2024. Patients with hormonal treatment, psychiatric medication, immunosuppressive treatment, hyperthyroidism, autoimmune diseases, hyperprolactinaemia, malignancy, systemic diseases, liver-, kidney diseases, and infection were excluded. Patients were divided into groups based on their laparoscopic diagnosis: peritoneal endometriosis (PE, n = 61), ovarian endometriosis (OE, n = 33), or control group (n = 87). Participants/materials, setting, methods Peritoneal group included ASRM I-II (#Enzian P1-2, O0, T0-2), ovarian group included ASRMI-IV (#Enzian P0-3, O1-2, T0-1) endometriosis stages. Controls had a negative laparoscopic diagnosis for endometriosis. Metabolite profiles were investigated by liquid chromatography (UHPLC-MS) coupled with high-resolution mass spectrometry. Semitargeted and untargeted metabolomics analyses were performed. Semitargeted analysis monitored around 50 metabolites, including vitamins, amino acids, sugar, tryptophan-derivates, and nucleotides. Statistical analyses were conducted to identify metabolic differences between endometriosis patients and controls. Main results and the role of chance Endometriosis groups (PE, OE) have different metabolic profiles compared to symptomatic controls. Interestingly, PE group is well-separated metabolically from both the control and OE groups. OE group shows an intermediate metabolic profile between PE and control groups. Peritoneal endometriosis has a stronger metabolic signature than ovarian endometriosis. Semitargeted analysis revealed significant metabolic alterations in the tryptophan and riboflavin pathways, including kynurenines, indols, and riboflavin intermediates. Limitations, reasons for caution A dietary questionnaire was not conducted among patients. No information is available on the diet of patients, which could alter their metabolic profiles. The #Enzian score was established in 2021; we evaluated it retrospectively for older samples based on the operation reports. Wider implications of the findings A well-described metabolic signature of endometriosis might allow the identification of early biomarkers for endometriosis. It could also give deeper insight into the pathomechanism of the disease. Furthermore, it might provide hypotheses for designing nutritional interventions to prevent progression or reduce postoperative recurrence of endometriosis. Trial registration number Yes

Abstract Study question What are the decision-making pathways for oocyte disposition (e.g., discard, donate) among elective egg freezers? Summary answer Distinct decision-making pathways were identified for each disposition outcome, with positive attitude towards an outcome as the most consistent and influential factor across all decisions. What is known already Despite the increasing prevalence of elective egg freezing and the growing number of oocytes in storage, little is known about how women navigate the decision to donate or discard their stored surplus oocytes. Existing research on embryo disposition highlights complex emotional, ethical, and practical considerations, but no prior studies have explored the psychosocial and behavioural pathways specific to egg freezing disposition decisions. Study design, size, duration This cross-sectional, observational, survey study recruited 175 individuals who had electively frozen their eggs. The study was guided by the Prototype Willingness Model (PWM) as the model has successfully predicted future-oriented fertility decisions. The four PWM predictor are attitude, perception of others’ approval, and similarity and favourability towards the typical person who would choose this outcome. The PWM was supplemented with seven additional theoretical constructs identified through our prior qualitative research. Participants/materials, setting, methods Elective egg freezers, 18+ years, residing in Australia with at least one oocyte in storage. Recruitment was via social media, newsletters, emails to women’s groups, universities, IVF clinics, and snowball sampling. The study examined decision-making pathways for (donation to; (1) friends or family, (2) couples advertising for an egg, (3) egg bank/fertility clinic,) (4) research, (5) discarding eggs or (6) reclaiming eggs. Structural equation modelling indicated the distal and proximal predictors for each outcome. Main results and the role of chance There was no single decision-making pathway for all disposition outcomes. All variables were significant for at least one outcome. Positive attitude was the strongest predictor across all disposition choices. Factors shaping attitudes varied by outcome and included: Concerns about others raising their donor-conceived child (barrier to donating to known and unknown others), desire for recipient contact (enabler for donating to known others, e.g. friends/family and couples seeking donors), empathy (facilitator for donating to friends and family members), and concerns about the well-being of donor-conceived children (barrier to donating to egg banks). Positive perceptions of how others viewed their decision increased willingness to donate to research and higher empathy was a barrier to donate to research. Favourable perception of the typical women who would discard their eggs was associated with increased willingness to discard oocytes. Women who had achieved motherhood increased willingness to reclaim their eggs. The revised model explained the following variance in willingness decisions: 75% (donation to an egg bank/fertility clinic), 68% (donation to a couple advertising online), 51% (discarding their eggs), 50% (reclaiming their eggs), 48% (donation to friends or family members), and 45% (donation to research). Limitations, reasons for caution Generalisability to populations in other countries is limited to whether they have similar disposition options available. Wider implications of the findings This study highlights distinct decision-making pathways for oocyte disposition among elective egg freezers. Given the individualized and emotionally complex nature of these decisions, there is a critical need for tailored counselling and evidence-based decision support tools. Decision tools could help patients, clinicians, and fertility counsellors navigate oocyte disposition decisions effectively. Trial registration number No