IVF Treatment Research Articles

P-062 The impact of isolated teratozoospermia on cumulative live birth rates in IVF and ICSI treatment: a retrospective analysis of 5254 completed cycles

Abstract Study question To assess fecundity using cumulative live birth rate (CLBR) per egg collection to determine if IVF and ICSI outcomes are significantly influenced by sperm morphology. Summary answer The presence of isolated teratozoospermia does not have a significant impact on the CLBR of IVF treatment. What is known already There is ongoing debate regarding the clinical significance. Some studies indicate a pivotal role of sperm morphology in successful pregnancies, others cast doubt on its reliability as a predictive factor. Despite the application of stringent grading criteria, concerns remain about the subjective nature of assessing sperm morphology. Discrepancies in success rates between normozoospermic and isolated teratozoospermic groups shown by previous research findings underscore the uncertainty surrounding this matter. Inconsistent findings in previous research may be explained by limited sample sizes and a focus on immediate successes, neglecting subsequent frozen cycles. This study seeks to address the overlooked aspects of teratozoospermia. Study design, size, duration A retrospective study was conducted at Waterstone Clinic, involving 5254 IVF/ICSI autologous cycles completed between 2009-2022 cycles (completed meaning all embryos were either transferred or disposed of). Cycles were categorised according to sperm morphology and divided into two groups: the isolated teratospermic (ITS) group (1218 cycles) defined as having less than 4% normal morphologic forms with all other semen parameters being normal, and the normal sperm (NS) group (4036 cycles). Participants/materials, setting, methods The primary outcome measures, included fertilisation rate, number of cryopreserved embryos per egg collection, and CLBR. Employing multivariate statistics, the analysis explored the impact of interferons on CLBR in relation to variables such as sperm morphology, female age, egg number, and aetiology. Main results and the role of chance The female base characteristics were similar between the two groups in terms of age (37.1 vs 37.1 y/o), oocyte number (9.4 vs. 9.7) and FSH level (7.6 vs. 7.6 IU/L). Statistically significant differences were observed in fertilisation rates (61.7% vs. 65.8%, p < 0.001) and fertilisation failure (5.9% vs. 3.3%, p < 0.001) from IVF treatments when comparing the ITS and the NS groups. However, there was not any significant difference in the number of cryopreserved embryos between the two groups. Additionally, the CLBR did not show any significant difference between the two groups for IVF (35.8% vs. 37.8%, p > 0.05) or ICSI treatments (38.1% vs. 33.2%, p > 0.05). ICSI did not confer any benefit in improving the CLBR outcome when compared to conventional IVF, when adjusted for female age, number of oocytes collected, and aetiology. Limitations, reasons for caution While this study is retrospective it encompasses a substantial cohort. A potential for bias due to the selection criteria for IVF and ICSI exists. The intrinsic subjectivity in sperm morphology assessment introduces additional potential bias. Wider implications of the findings Despite the lack of support from CLBR to advocate for choosing ICSI in patients with ITS, this study suggests that couples with ITS experience lower fertilisation and higher fertilisation failure rates with IVF. This observation can serve as a valuable indicator for investigating the occurrence of fertilisation failure. Trial registration number not applicable

P-201 Embryo prioritization through an AI-powered evaluation system improves the clinical outcome in IVF cycles

Abstract Study question How can the implantation rate be improved through the prioritization of embryos, particularly in the context of elective single embryo transfer (eSET)? Summary answer Based on the retrospective data of IVF treatment using 965 embryos, the AI-powered embryo-evaluation system, icONE, was developed to provide unbiased selection with promising accuracy. What is known already Over the past decade, numerous predictive models guiding IVF treatments for frozen-thawed embryo transfer (FET) success have emerged. However, most are unidimensional, focusing on factors like patient characteristics (e.g., age, reproductive hormones), embryo quality (e.g., morphokinetics), or biomarkers (e.g., genes, microRNAs). Despite efforts, many models exhibit limited AUROC (area under the receiver operating characteristic curve) values of 0.70∼0.77, suggesting a need for comprehensive consideration of maternal and embryonic factors to enhance predictive accuracy. Study design, size, duration The current study aims to develop an AI-assisted embryo prediction system. Nine hundred and sixty-five embryos from 232 patients (ages ranging from 24 to 50 years old, mean age = 36.9 years) who underwent IVF treatment during 2015–2018 were enrolled for developing an embryo prediction system. The clinical information and detail of IVF procedures of each patient and embryo were recorded and integrated to explore their impact on clinical outcomes. Participants/materials, setting, methods Pregnant outcomes were followed, including hCG examination, sac formation, and live birth. Whole genome sequencing (WGS) analysis using TE biopsy was performe. Genetic features extracted from WGS data and maternal clinicopathological characteristics were integrated and calculated by decision tree algorithm and machine learning algorithms, including Random Forest, XGBoost, LightGBM, Tabnet, etc. Main results and the role of chance To reduce the number of IVF cycles, diminish the risk associated with multiple pregnancy, and maximize the efficacy of IVF treatment, we analyzed genetic data generated from the preimplantation genetic screenings and developed a prototype of an AI-assisted program to evaluate the viability and quality of each embryo. The “intelligent cOmputing Noble Embryo” (icONE) program integrates genetic data and maternal clinical features. icONE showed classification accuracy and prediction specificity of 0.81 and 0.83, respectively, and the overall prediction accuracy of successful implantation reached to 92%. Random Forest outperformed other machine learning tools with overall positive predictive value, negative predictive value, specificity, and sensitivity of 91.8%, 91.0%, 90.3%, and 92.4%, respectively. The overall prediction accuracy of successful implantation reaches was 91.4%. Notably, the preliminary results of a randomized prospective clinical trial showed that the clinical pregnancy rate reached 77.3% (n = 81) based on the icONE’s evaluation, which was significantly higher than that of 50% (n = 85) in the non-icONE group. Patients of both advanced and young age benefited from icONE. Taken together, these results endorse the efficacy of icONE in prioritizing embryos for clinicians in IVF treatment. Limitations, reasons for caution It is noticed that our study is limited by the small sample size and all the data are collected from a single IVF center. The uniform and controlled operation of IVF cycles in a single center may cause selection bias. Wider implications of the findings Given the shift towards eSET in reproductive medicine, prioritizing embryos for implantation potential is vital in infertility treatment. The AI-powered model established in this study enhances clinical practice, optimizing parenthood chances for those facing fertility issues. Trial registration number not applicable

P-246 Use of image recognition to quantify oocyte quality improved the accuracy of prediction of blastulation by 26-48%

Abstract Study question Can an AI oocyte quality model improve blastulation prediction accuracy? Summary answer An AI oocyte quality model presented an overall accuracy of 61-74%. Exhibiting an improvement of 26-48% for prediction of blastulation over random. What is known already Nowadays, the assessment of whether sufficient oocytes have been cryopreserved is purely based on patient age and number of oocytes collected. No morphological abnormalities provide quantitative guidance about the reproductive potential of the oocyte. Additionally, interobserver agreement and prediction of blastulation by experienced embryologists has been reported to be worse than chance (Behr et al., 2018). Therefore, there is a need to implement oocyte quality assessment tools to predict blastulation in oocyte donation, cryopreservation programs and IVF treatments. Study design, size, duration A retrospective study using an AI oocyte quality score (CHLOE-OQ, Fairtility Ltd) was performed in three private clinics evaluating 1749 oocytes that were inseminated with ICSI and cultured between October 2019 and December 2021. Overall and per clinic analysis were conducted (Clinic 1 n = 1131; Clinic 2 n = 576; Clinic 3: n = 42). Participants/materials, setting, methods An AI model provided an oocyte quality (OQ) score to assess prediction of blastulation, measured by binary logistic regression (AUC) per clinic and overall. The correlation between the OQ score and blastulation was measured using t-test. OQ score was classified into groups, Group A: 0.9-1.0, Group B: 0.7-0.9, Group C: 0.4-0.7 and Group D: 0-0.4. Blastulation rate was assessed in each OQ score subgroup using chi-square. Main results and the role of chance OQ score prediction of blastulation presented an overall accuracy of 63% (1107/1749). Accuracy per clinic ranged from 61-74%, Clinic 1: 61% (690/1131), Clinic 2: 67% (386/576) and Clinic 3: 74% (31/42). Exhibiting an improvement of 24-48% for prediction of blastulation over random. Oocyte quality score was predictive of blastulation per clinic and overall Clinic 1: AUC 0.62, n = 1130; Clinic 2: AUC 0.62, n = 575; Clinic 3: AUC 0.78, n = 41; Overall: AUC=0.62, p < 0.001, n = 1749). Overall blastocyst development rate was 53% (929/1749). OQ score was higher in oocytes that blastulated than those that did not blastulate (0.85 ± 0.14 vs 0.74 ± 0.2, p < 0.001). There was a significant direct association between OQ score subgroups and blastulation rate. Group A with the highest score subgroup had the highest blastulation rate [61% (554/903), p < 0.05]. The blastulation rates for Group B [53% (283/539) p < 0.001], Group C [38%, (71/188), p < 0.001] and Group D [18% (21/119), p < 0.001] were decreasingly lower. Limitations, reasons for caution This study was done in 3 centers in the same country. Therefore, further studies should focus on diversifying in terms of geographical location to address generalization of this AI assessment. Further studies should focus on comparing the efficacy of blastulation prediction by humans. Wider implications of the findings Using AI to do an objective oocyte assessment aids clinicians in determining the optimal time to transition to oocyte donor programs. It assists the decision-making for oocyte banking, minimizing unnecessary cycles and guiding timely additional oocyte collections. This approach at the point of cryopreservation, mitigates risks associated with delayed decisions. Trial registration number N/A

P-180 Trophectoderm biopsy technique and its effect on life birth rates: a prospective study

Abstract Study question Do life birth rates of euploid embryos vary based on factors such as laser intensity, number of laser shots, and the biopsy technique employed? Summary answer This study found no correlation between biopsy technique parameters and life birth rates of euploid embryos, underscoring the robustness of current trophectoderm biopsy methods. What is known already Embryo biopsy for Preimplantation Genetic Testing (PGT) must achieve two primary objectives: ensuring that the biopsied material accurately reflects the genetic makeup of the embryo and maintaining the viability of the embryo without compromise. The strategy that most closely aligns with accomplishing these dual goals is trophectoderm (TE) biopsy. However, it’s important to note that there is currently no standardized TE biopsy procedure, leading to the coexistence several techniques. Given the potential impact of TE biopsy on trophectoderm quality and integrity, and consequently on implantation, there is a critical need for studies to assess the effects of different biopsy techniques. Study design, size, duration This prospective observational study spanned from 2019 to 2021 and involved 298 Preimplantation Genetic Testing for Aneuploidy (PGT-A) treatments, incorporating a total of 1187 trophectoderm (TE) biopsies. Embriologyst were trained in both Pulling and Flicking techniques and choose between the two techniques according to their criteria. Pulling involved laser pulses weakening junctions, facilitating TE detachment with moderate tension. Flicking substituted tension with a cutting motion between holding and biopsy pipettes. Participants/materials, setting, methods Biopsy technique (pulling/flicking), number of laser shots (1-10), laser intensity (200µs-600µs), and the embriologyst responsible for the biopsy were annotated for every biopsied embryo. PGT indication, stimulation protocol, patient age (39.32±4.22), embryo quality, and sperm diagnosis were annotated and used as possible confounding factors in a logistic regression model to understand the impact of each independent variable on the log-odds of the outcome. Main results and the role of chance The 1187 trophectoderm biopsies resulted in a total of 410 (34.5%) euploid embryos. Among these, 237 euploid embryos underwent individual transfers, with 153 subjected to Pulling technique and 84 to Flicking technique leading to a total of 120 healthy babies (50.6% life birth rate). Logistic regression, considering potential confounding factors failed to identify any variable significantly influencing the likelihood of life births (p < 0.05). Both Pulling and Flicking techniques demonstrated comparable success rates (52.9% vs 46.4%), with no discernible impact from variations in laser shots (1-3=65.0%; 4-6=45.6%; >6=66.7%) or intensity (200µs=75.0%; 300µs=45.0%; 400µs=50.6%; 500µs=50.94%; 600µs=50.0%). Furthermore, when evaluating the embryologist conducting the biopsy, no significant differences were identified among operators (p < 0.05). This pattern persists for miscarriage rates, indicating no influence from biopsy technique, laser shots, or laser intensity (p < 0.05). Thus, our findings underscore that variations in trophectoderm biopsy variables, when performed by an experienced embryologist, do not adversely impact trophectoderm quality and do not compromise success rates. Limitations, reasons for caution This is a single-center, observational study, conducted from 2019 to 2021, and this may limit generalizability. The sample size variation between biopsy techniques and potential unmeasured confounders pose challenges. The assumption of technique homogeneity, and reliance on binary outcomes underscore caution in interpreting findings. Wider implications of the findings The lack of correlation between biopsy parameters and life birth rates in our study underscores the effectiveness and reliability of current biopsy techniques. This discovery offers reassurance regarding the proficiency of trophectoderm biopsy methods for achieving success in IVF treatments. Trial registration number Not Applicable

P-021 Development of recombinant Phospholipase C Zeta protein as a therapeutic intervention to overcome certain types of male infertility

Abstract Study question Developing recombinant Phospholipase C Zeta (PLCζ) protein using a bacterial expression system to be utilized for potential future replacement of Ca2+ ionophores during IVF treatments. Summary answer Our findings support the use of MBP-PLCζ recombinant protein as a therapeutic tool for treating certain types of male factor infertility associated with fertilization failure What is known already The long-term safety of calcium ionophores, which are exclusively used for treating cases of fertilization failure, is still questionable. PLCζ is an endogenous agent that may represent a better replacement for treating male infertility cases related to fertilization failure Study design, size, duration Maltose binding protein (MBP)-tagged PLCζ was produced by cloning wild-type PLCζ into a pET41MM vector and purified using affinity chromatography techniques. Circular dichroism (CD) was employed to evaluate the secondary structure and the thermal denaturation of PLCζ protein. Moreover, the PIP2 hydrolysis assay was used to test the enzymatic activity of PLCζ protein over time and under different storage conditions. Furthermore, the embryonic toxicity of the protein was tested using a chick embryo model. Participants/materials, setting, methods Several methods were used in this study including Molecular cloning, circular dichroism, and chick embryo model to investigate the embryonic viability. Main results and the role of chance Our findings demonstrated that PLCζ recombinant protein displayed a properly folded secondary structure mainly composed of alpha-helices. Moreover, the protein was effective in maintaining the enzymatic activity and Ca2+ sensitivity for 90 days when stored at -80oC. Moreover, exposing chicken eggs to MBP-PLCζ did not alter the embryonic viability when compared to the control group. Kaplan-Meier survival curve showed that the rate of survival did not differ between the treatment, control and sham groups until the endpoint of the study. Limitations, reasons for caution The toxicology assays used are not sufficient to confirm the safety of the protein. Further in vivo studies are required to investigate the effect of MBP-PLCζ protein on early embryonic development before being used as a replacement for calcium ionophores in cases of oocyte activation failure. Wider implications of the findings These findings can help develop a new therapeutic tool for treating cases of male infertility associated with oocyte activation failure. Trial registration number not applicable

P-764 A decade of transformations in IVF strategies towards enhanced clinical efficacy, efficiency, and safety: the real-life experience of 6610 couples

Abstract Study question To what extent do clinical and laboratory strategies affect the main measures of IVF success? Summary answer Across 10 years, the implementation and increasing adoption of antagonist protocols combined with agonist-trigger, freeze-all, blastocyst culture, PGT, and multicycle counseling approach improved IVF outcomes. What is known already The primary indicator of IVF success is the cumulative-live-birth-rate per intention-to-treat (CLBR per ITT). Since reproductive competence is inherent to every gamete and embryo, IVF treatments cannot guarantee success. However, they can maximize the likelihood of success with the safest approaches available. Furthermore, there are other outcomes to consider for a more thorough assessment of IVF effectiveness and efficiency, including time in treatment (=LB achieved / no LB achieved and no embryo available for transfer; TiT), miscarriage rate, and prevalence of multiple-births. Moreover, a family planning perspective should be strengthened in IVF aiming at ≥ 2 healthy children, whenever feasible. Study design, size, duration Longitudinal observation of 6610 couples undergoing ≤6 ovarian-stimulations (OS) for IVF at a private clinic within 3 years. Couples were clustered in 11 groups according to the year of their first treatment (years: 2010-2020) and compared for (i) CLBR within 3 years, (ii) prevalence of ≥ 1 miscarriage and ≥1 twin-delivery, (iii) TiT (from first-OS to the last IVF procedure before treatment conclusion), (iv) prevalence of ≥ 2 singleton-deliveries within 6 years. Participants/materials, setting, methods The naïve patients included increased from ≈500 in 2010 to ≥ 600 between 2013 and 2020. The maternal age was stable around 38±4 years with ≈20% prevalence of severe male factor. OS was conducted through long, short flare-up, mild, antagonist, progestin-primed, or DuoStim protocols with hCG/agonist-trigger. All patients underwent ICSI on fresh own eggs and cleavage/blastocyst culture, fresh transfer/freeze-all, untested/euploid transfers, single/multiple-embryo transfers. Main results and the role of chance Antagonist-protocols (≈20% in 2010 and ≈80% in 2020) combined with agonist-trigger (0.4% and 76.8%) superseded long/short flare-up (≈60% and <10%) and hCG-trigger (99.6% and 23.2%). DuoStim, introduced in 2015, reached 15%-prevalence by 2020. The MII-oocytes collected at first-retrievals increased from 5±3 (2010) to 7.2±4.9 (2020). The prevalence of first-cycles without embryos raised from 3% to 14% by 2020 primarily due to an evident cleavage-to-blastocyst culture switch (2010:96% versus 4%; 2020:1% versus 99%). Similarly, PGT-A and freeze-all utilization respectively increased from 3% to 80% and from 12% to 93%. Although TiT increased from ≈2 cycles in ≈140 days (2010-2013) to ≈1.6 in ≈160 days (2014-2020), the 3-years CLBR improved from 32% (2010) to 42% (2020), a difference confirmed also adjusting for maternal age and overall number of MII-oocytes collected (multivariate-OR:1.03,95%CI:1.01-1.05;adjusted-p=0.001). This was paralleled by the halving of women suffering ≥1 miscarriage (≈12% to < 6%;p=0.002) and drop in women with ≥1 twin-delivery (7.5% to 0.5%;p<0.001). The latter achievement was due to single-embryo transfer rates increasing from 26% (2010) to 98% (2020). Accordingly, the women starting in 2017 and obtaining ≥2 LBs within 6-years were 9.9%, 98% of which after consecutive singleton-deliveries; these figures were 6.6% and just 22% among women starting in 2010(p = 0.004). Limitations, reasons for caution This study suffers from the inherent limitations of a retrospective design. Temporal changes, other than clinical strategies and laboratory advancements (e.g., personnel), might have influenced the results, although no critical infrastructural changes were implemented. Gestational and neonatal follow-up data are missing in this study. Wider implications of the findings Clinical and laboratory advancements enhanced IVF efficacy and efficiency over time also fulfilling family planning desire. Future strategies and improved workflows should focus on reducing drop-out and time-to-LB. As larger oocyte cohorts led to higher CLBR throughout maternal age, tailored-multicycle approach might represent a valuable option to this end. Trial registration number none

P-474 State-Trait Anxiety Inventory (STAI) punctuations as a non-invasive method for evaluating endometrial prognosis associated with cortisol and estradiol endometrial levels in IVF patients

Abstract Study question Are STAI State punctuations a potential non-invasive marker of endometrial prognosis in IVF patients? Summary answer Being stressed (determined by STAI State punctuations) is positively and negatively correlated with endometrial cortisol and estradiol levels, respectively, and highly associated with pregnancy consecution. What is known already Despite it is well known IVF treatments are a source of stress, this factor is frequently overlooked mainly due to the difficulty of stress evaluation and the lack of accurate biomarkers, not being standardized in the clinical setting. Steroid hormones are essential for reproductive physiology and highly related to well-known stress biomarkers such as cortisol. The lack of correlation between endometrial and serum steroid levels creates the need to look for non-invasive tools reflecting the endometrial microenvironment. In this study, by molecularly and psychologically characterizing patients, we aim to unveil non-invasive potential markers of stress associated to pregnancy consecution. Study design, size, duration This prospective cohort study included a total of 74 IVF patients (<45 years old, no uterine or systemic pathologies and good quality embryos) who underwent endometrial biopsy collection in mid-secretory phase for metabolites measurement between 2019 and 2023. These patients also underwent psychological evaluation by stress related questionnaires. Participants/materials, setting, methods The endometrial concentration of eleven steroid metabolites was measured by ultra-performance liquid chromatography-tandem mass spectrometry. Patients also underwent stress and anxiety evaluation (STAI), and reproductive outcomes in the first embryo transfer after biopsy collection were followed up. Wilcoxon and Barnard’s test were applied to compare the mean and proportion of stressed/unstressed, pregnant/not pregnant patients and metabolites levels. Correlations between metabolites’ levels and the proportion of stressed patients were evaluated by Pearson’s correlation coefficient. Main results and the role of chance Patients with STAI State punctuations > 60 presented higher mean endometrial cortisol levels (8.01 ng/g (n = 23) vs 5.08 ng/g (n = 35), p-value = 0.05) and lower mean estrone levels (54.97 ng/g (n = 23) vs 56.31 ng/g (n = 35), p-value = 0.049) than those patients with STAI State punctuations < 60. Also, in patients with high endometrial estradiol levels (>2 ng/g) a higher significant proportion of unstressed patients (STAI State < 60) was observed (88.9% vs 52% of unstressed patients in > 2 ng/g and < =2 ng/g estradiol concentrations, respectively) (n = 34, p-value = 0.036). As expected, a significant positive correlation between endometrial cortisol levels and stress (measured by STAI State questionnaire) (cor = 0.977, p-value = 0.023) and a negative correlation between endometrial estradiol levels and stress (cor = -0.9945, p-value = 0.0055) was evidenced. Additionally, a higher proportion of pregnant patients was observed when endometrial estradiol concentrations were high (>1 ng/g) (78.9% vs 50% pregnant in > 1 ng/g vs < =1 ng/g estradiol respectively, p-value = 0.035) and a higher proportion of not pregnant patients was observed when endometrial cortisol concentrations were high ( > =13.9 ng/g) (80% vs 34.4% not pregnant in > =13.9 ng/g and <13.9 ng/g cortisol respectively, p-value = 0.029). Limitations, reasons for caution Although we have employed validated psychological questionnaires and stress biomarkers, a causal relationship between stress and reproductive outcomes should be further explored. Wider implications of the findings This study demonstrated a significant correlation between stress and cortisol and estradiol levels. Since both metabolites’ levels were also related to pregnancy chances, STAI State psychological evaluation could be implemented in the clinical practice as a non-invasive method to prevent endometrial failure through psychological counseling. Trial registration number Not applicable

P-601 Monitoring reproductive hormones during ovarian stimulation: performance and usability of a self-operated at-home urine test

Abstract Study question How effective and user-friendly is a non-invasive, patient-operated urine test (Kinder) in tracking reproductive hormone levels during ovarian stimulation (OS) treatment in an IVF cycle? Summary answer Urinary E1-3G correlates well with blood E2 levels and ovarian follicle development. Patients found it easy and stress-free to perform the urine test at home. What is known already Monitoring blood reproductive hormone levels during OS is a widely accepted practice to ensure the optimal ovarian response to gonadotropin treatment, timing of oocyte maturation triggering and mitigating hyperstimulation risk. However, conventional blood testing is invasive, necessitating patients to travel to clinics for blood sampling, adding to their overall burden. Previous studies have revealed a robust correlation between blood E2 levels, and its corresponding urinary metabolites E1-3G. Kinder is a portable device designed for quantifying urinary E1-3G, allowing patients to conduct tests in the privacy of their homes, and for many obviating the need to travel substantial distances for monitoring. Study design, size, duration This is a prospective, single-cohort, study of Caucasian patients (28-34 years), conducted at Fertility Specialists of Western Australia in Perth, Australia, where patients may travel vast distances for treatment. Twenty-five patients were enrolled from Aug 2022-Aug 2023, and performed home urine tests utilizing the Kinder device in parallel with clinic scheduled blood (ELISA) and ultrasound tests. The primary end point of the study was the correlation between urinary and blood reproductive hormone concentrations (E1-3G). Participants/materials, setting, methods Spearman and Pearson correlation coefficients (CC) and 95% confidence intervals were calculated for E1-3G and E2, and E1-3G/E2 and follicle development (total volume of ovarian follicles by ultrasound). To assess patient acceptability patient-reported outcomes (PRO) were assessed using the State Trait Anxiety Inventory (STAI) questionnaire. Further, patient-reported experiences (PRE) were evaluated through the System Usability Scale (SUS) questionnaire, along with assessing their likelihood to recommend Kinder to their friends or family undergoing IVF treatment. Main results and the role of chance Upon analysing 100 concurrent urine and blood samples from 25 patients, a robust correlation was found between E1-3G and E2 levels (Spearman CC: 0.818, 95% CI: 0.680-0.904; Pearson CC: 0.847, 95% CI: 0.712-0.939). Additionally, we observed a moderate correlation between E1-3G levels and ovarian volume (Spearman CC: 0.701, 95% CI: 0.556-0.805; Pearson CC: 0.670, 95% CI: 0.519-0.781, n = 72), resembled the correlation between E2 levels and ovarian volume (Spearman CC: 0.874, 95% CI: 0.804-0.920; Pearson CC: 0.849, 95% CI: 0.770-0.902, n = 75). PRE: 22 out of 23 patients who completed the survey confirmed that they would definitely, or probably, recommend Kinder. The mean SUS score (91.4) signified an excellent user experience. PRO: Anxiety levels were not affected by use of Kinder (STAI score below threshold of 42.38) in 18/20 patients who completed three STAI surveys (first one before and second one after the initial Kinder test; third one after the third/fourth Kinder test). Although 2/20 patients reported a STAI score of 49 in the final survey, suggesting increased anxiety, both affirmed in the PRE survey that they “will definitely recommend Kinder”, suggesting that the elevated STAI score can be attributed to the overall IVF treatment rather than the use of Kinder itself. Limitations, reasons for caution The sample size is relatively small, necessitating further multi-centre studies with a larger sample size to validate the findings. Wider implications of the findings The results suggest an acceptable interchangeability between at-home urine E1-3G and at-clinic blood E2 monitoring hormone levels during OS treatment. A digital health approach, combining tele-counselling, remote at-home hormone monitoring, and follicle tracking, could reduce patient burden and save many patients the inconvenience of travelling large distances for cycle monitoring. Trial registration number ACTRN12622000488707

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

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

O-225 A study investigating whether egg share donors treatment outcome impacted on their overall psychological well-being and treatment experience

Abstract Study question Does unsuccessful egg sharing treatment impact on the egg share donor’s psychological well-being or make her regret her decision to participate? Summary answer Egg share (ES) donors whose treatment was unsuccessful did not regret egg sharing, and were unanimously happy for their recipient if her treatment was successful. What is known already ES programmes are a unique form of egg donation for two main reasons. First, both the donor and her anonymously matched recipient are engaged in the simultaneous pursuit of having a baby. Second, egg sharing schemes offer a benefit for the donor other than altruism. However, a major concern is for the psychological well-being of ES donors whose own treatment is not successful. This concern raised was based on opinion, rather than empirical research. It is therefore vital that the psychological well-being of ES donors through egg sharing is better studied. Study design, size, duration Data for this study were gathered using a questionnaire. Selection criteria for the study were those who had participated in egg donation treatment between 2014-2022 at Lister Fertility Clinic (LFC), Chelsea, London. Only patients who had agreed to participate in research during the consenting process of their treatment were sent an email invitation asking them to participate in the study. 394 ES donors were sent an email invite. Participants/materials, setting, methods After receiving the email link, respondents filled out the questionnaire online using the Qualtrics survey tool. The paper copies received were manually inputted by the research team onto Qualtrics. The questionnaire took on average 15-20 minutes for participants to complete. 197 egg share donors were included in analysis Statistical analysis was conducted using the χ², analysis of variance (ANOVA), Mann Whitney U test, and Fisher’s exact test. Main results and the role of chance This is the largest study to investigate the psychological issue surrounding ES donation. Overall, ES donors rated their treatment ‘very positively’ (85.7%). The vast majority of egg sharers (91.8%) were glad they took part in the programme. Significantly, patients were stratified into whether IVF treatment was successful or not, with successful treatment being defined as a resulting live birth. For egg sharers, there was no difference between positive or negative treatment outcome, and their responses to whether they: would participate in egg sharing again in the future; would recommend egg sharing to a friend or family member who needs fertility treatment; felt well informed of egg sharing; and felt they had sufficient support from the fertility clinic during and after treatment. The only answer that was statistically significant based on treatment outcome was when asked if they were ‘glad they took part’, with 99.4% of successful egg sharers answering yes, compared to 72.7% of unsuccessful egg sharers (Fishers exact, p < 0.05). Of the unsuccessful ES donors who found out their recipient’s treatment was successful, 100% reported they were ‘happy for their recipients’, with the vast majority of this category stating they were glad they took part (85.7%). Limitations, reasons for caution As with all questionnaire generated data, there is a potential for sampling bias. Second, this is a single centre study. LFC is one of the largest ES centres in the UK and has success rates above the national average. Therefore, this data may not represent the ES programme nationally. Wider implications of the findings As with all questionnaire generated data, there is a potential for sampling bias. Second, this is a single centre study. LFC is one of the largest ES centres in the UK and has success rates above the national average. Therefore, this data may not represent the ES programme nationally. Trial registration number n/a

P-517 The effects of various female infertility factors on endometrial receptivity

Abstract Study question Is there a correlation between different female infertility factors and displaced window of implantation? Summary answer Patients with decreased ovarian reserve and endometriosis have higher risk in experiencing displaced window of implantation. What is known already Numerous studies substantiate that infertility stems from various etiology, including ovulatory dysfunction, male factors, tubal disease, endometriosis, uterine factors...etc. MicroRNAs (miRNAs) play a crucial role in initiating these infertility-related disorders and influencing biological processes like gametogenesis, embryogenesis, ovarian, uterine or endometrial condition. Notably, miRNAs regulate endometrial receptivity, suggesting optimal embryo transfer timing during IVF treatment by identifying the window of implantation (WOI). In recognition of this significant function, MIRA, a molecular testing solution that predicts endometrial receptivity for patients undergoing frozen embryo transfer (FET) by targeting miRNA biomarkers, was developed in hopes of bolstering success rate for personalized embryo transfer. Study design, size, duration This study aims to identify if there is a correlation seen between female infertility factors and the WOI. We enrolled a total of 96 patients who demonstrated successful pregnancy outcomes following personalized embryo transfer. The patients’ personalized embryo transfer timing recommendations were assessed based on their miRNA expression profiles using MIRA. Concurrently, the participants’ underlining infertility diseases were classified according to diagnoses guidelines provided by Taiwan’s Ministry of Health and Welfare. Participants/materials, setting, methods Six categories of female infertility diseases were included: decreased ovarian reserve, tubal, endometriosis, uterine abnormalities (excluding myoma and adenomyosis), polycystic ovary syndrome (PCOS), and myoma factors. Infertile factors such as unexplained, aneuploid oocyte (PGT), male factors, and adenomyosis were excluded due to limited sample size. Statistical analysis of endometrial stages and disease categories involved calculating percentage of the window of implantation (WOI) versus displaced WOI (either pre- or post-receptive) within the six types of diseases. Main results and the role of chance Our result showed that overall, 64.58% of 96 patients have a receptive window of implantation. Compared to this baseline, polycystic ovary syndrome (66.67%) does not elevate the risk of displaced endometrial stages. However, patients with decreased ovarian reserve and endometriosis are more likely to display displaced window of implantation, with average rates of 57.14% and 55.56% WOI respectively. Notably, patients with uterine abnormalities (84.62%) and myoma (87.5%) factors exhibit a lower likelihood of having a displaced WOI. The WOI percentage caused by tubal factors is slightly lower at 76.19%. In conclusion, these preliminary findings establishes that categories of female infertility diseases have discernible effects on patients’ WOI. As patients with certain infertility factors, such as decreased ovarian reserve and endometriosis, demonstrated a greater risk in experiencing displaced WOI, patients that fall under these categories could further benefit from utilizing endometrial receptivity testing to determine the optimal timing for their embryo transfer to potentially improve their reproductive outcomes and facilitate shared decision-making (SDM) in patient’s embryo transfer process. Limitations, reasons for caution While this result offers a preliminary overview for the correlation between endometrial stages and female infertility diseases, we cannot confirm whether these conclusions reached statistical significance due to the limitation of data. Wider implications of the findings Further investigation into the specific reasons for the discernable WOI patterns in different diseases is warranted in larger sample sizes to offer more personalized and robust clinical information supporting IVF treatments. Trial registration number ’not applicable’

P-712 A national survey of women’s perspectives and experiences of pursuing donor eggs

Abstract Study question What are the experiences of women who consider a donor egg, and the outcomes of those who receive donor eggs for conception? Summary answer Of 255 respondents, 59 women (23.1%) were formally placed on a donor egg waitlist, despite 80.6% respondents considering donor eggs with an IVF clinic (166/206). What is known already There is a shortage of donor eggs worldwide. Oocyte donation is a treatment option that is increasingly used for infertile women, as seen through the percentage of patients undergoing IVF who are over 40 years. Previous international studies report oocyte donation recipients to be predominantly aged 34-40 years, white, nulliparous, well-educated, married and financially secure. Although there are records in Australia for the number of donor and recipient cycles undertaken, there is no published data on the number of patients considering a donor oocyte, or on oocyte donor waitlists. In Australia egg donation is altruistic, non-commercial and not anonymous. Study design, size, duration A 52-point online survey was distributed via social media to women in Australia who had ever considered or pursued donor eggs, or come to the end of their own IVF treatment. The survey was sent to all clinical members of the Fertility Society of Australia and New Zealand (FSANZ) and available on the Victorian Assisted Reproductive Technology Authority (VARTA) website. The survey was open from 1 February to 30 April 2023. Hospital ethics approved. Participants/materials, setting, methods A cross-sectional, anonymous, online survey of women in Australia who were potential egg donor recipients. Survey questions included demographics, potential donor egg recipient preferences, IVF clinic experiences, barriers, any impact of the COVID-19 pandemic, attitudes around the reimbursement for donor eggs, and fertility costs already incurred. Responses were collected via the Qualtrics XM platform. Statistical analysis for quantitative data was analysed using Microsoft 365 Excel. Qualitative coding performed on the commentary in free text fields. Main results and the role of chance This study identified a large gap between those who considered a donor egg (80.6%), pursue a donor egg on an IVF clinic waitlist (23.1%), and those who actually received a donor egg. Of the 59/255 women who were placed on an oocyte donor waitlist at an IVF clinic in Australia, less than a quarter (23.1%) received a donor egg (13/58), with a pregnancy and live birth rate of 76.9% and 61.5%, respectively. Thus, of those that were on an Australian waitlist, one quarter received donor eggs, with over half of these recipients having a live birth. ‘Women preferred donor eggs from Australia than overseas, preferred an unknown donor from an IVF clinic program, and wished to know source information for themselves and children in the future. Attitudes towards financial reimbursement were generally positive towards compensation. Barriers to pursuing donor eggs included grief of loss of own fertility, cost, availability and time. When on an Australian donor egg list, many respondents still looked at other sources of donor eggs in Australia and overseas. Reasons women came off an Australian donor egg waitlist included receiving donor eggs and donor embryos elsewhere, including from overseas, as well as advancing age and expense. Limitations, reasons for caution Risk of respondent selection bias of those who chose to response to a public survey, with incomplete data for some respondents. Given survey in English, may have underrepresentation of some cultural groups and sampling a limited range of women from diverse social, economic and religious settings. Wider implications of the findings In this first paper of potential egg recipients’ preferences in the Asia-Pacific region, we have identified a gap where demand for donor eggs exceeds local supply. A majority of potential recipients would support financial reimbursement for eggs already cryopreserved by women initially freezing for own elective purposes. Trial registration number not applicable

O-218 Prospective validation of an artificial intelligence platform for automated follicle counting

Abstract Study question How often are fully automated follicle counts and measurements modified in expert review? Summary answer Automatic follicle annotations provided by an artificial intelligence platform in regular operation of a clinic were edited 3.27% of the time. What is known already Ovarian follicle counting is frequently performed, time-consuming, and subject to noticeable inter-observer variability; as such, it is well-suited to automatizing with artificial intelligence. FOLLISCAN (MIM Fertility) is a software platform that automatically annotates follicles on 2D or 3D ultrasound cine videos, with exact outlines and measurements, without any manual pre-processing. It has undergone several retrospective tests of its precision and recall in detecting ovarian follicles, accuracy of measurements, and time-savings compared to manual counting Study design, size, duration The platform was integrated with a clinic’s existing picture archiving and medical record systems. Ultrasound scans of ovaries were performed as part of regular infertility treatment in two IVF centers. Scans were made by 3 experts in a period from November to December 2023, automatically sent to the platform, analyzed, and then immediately visualized for review by the person performing the ultrasound, in place of manual annotation. Participants/materials, setting, methods The study included 294 cine videos from 147 examinations of 101 patients, with 4347 follicles in total (14.7 per ovary on average, from 2mm). The platform allows users to modify proposed measurements and introduce new follicle annotations. After confirming the review, results were automatically sent back to the clinic’s system to be used in medical decisions just as manual annotations would be. Main results and the role of chance Among all videos the average number of editions (follicle additions, modifications, or deletions) was 0.48 (CI: 0.37-0.61). In total, 142 follicles out of 4347 (3.27%, CI: 2.74-3.80) were edited: 66 follicles were added (1.52%, CI: 1.15-1.88), 26 were modified, (0.60%, CI: 0.37-0.83 ), and 50 were deleted (1.15%, CI: 0.83–1.47). Out of the 151 edited follicles, 13 (9.2%) had 2–5 mm, 29 (20.4%) had 5-10 mm, 51 (35.9%) had 10-15 mm, 42 (29.6%) had 15-20 mm, and 7 (4.9%) had 20 mm or more in diameter. Among follicles of 15 mm or more in size, 49 were edited. Out of 294 videos, 87 (29.6%, 95% Confidence Interval: 24.4-34.6) were edited in review in any way. Limitations, reasons for caution The study was limited in scope and did not track patients until success rates in IVF treatments could be observed. A multi-center randomized control trial could compare pregnancy rates for treatments performed with and without the platform. Wider implications of the findings An integrated platform allows for easy review, while significantly reducing the time spent on follicle counting in a real-world setting. Automated annotations result in consistent, reliable, and quick measurements, with the number of expert modifications smaller than the inter-observer reliability reported in previous studies. Trial registration number Project support was provided by the Polish National Center for Research and Development no. POIR.01.01.01-00-1634/20-00 and ERC Consolidator Grant TUgbOAT no. 772346. This study was conducted following the approval of the research protocol by the review board of the Regional Medical Chamber in Gdańsk (approval no. KB – 51/22).

O-012 Three Injections Protocol for IVF; a proof of concept study into elaborating the outmost patient friendly protocol in IVF

Abstract Study question To examine if 3 injections in follicular phase, with long-acting FSH, long-acting antagonist and HCG/Agonist triggering can result in efficient egg retrieval and blastocyst formation. Summary answer In this proof-of-concept study, all patients retrieved mature oocytes and ended having adequate blastocysts by receiving only 3 injections in the follicular phase. What is known already The old-fashioned way with multiple injections (majority minimum 15 injections) is still the golden IVF standard. Twenty years ago, a long-acting FSH gonadotropin brought into market (corifollitropin alpha) which represented a huge step in simplifying the follicular phase Recently, a new long-acting GnRH-antagonist emerged (degarelix) which can downregulate the hypophysis for 30 days. We have published that titrating this long-acting antagonist to 0,1mg single dose we can downregulate hypophysis without LH surge. Study design, size, duration A prospective proof of concept study included in total 15 patients 30-39 years old who followed IVF treatment with homologous oocytes during 2020-2022 in Assisting Nature IVF Clinic, Thessaloniki, Greece. In this study, we test if by delaying one day the long-FSH and also delaying the long-antagonist, still patients can produce mature eggs without needed extra FSH doses. Institutional review board approved the protocol. Participants/materials, setting, methods Patients were enrolled if AMH was 1,5-3,0 ng/ml and antagonist protocol applied. 150IU fixed corifollitropin-a (Elonva) for ovarian stimulation administered either on evening cycle Day-3 or morning day-4. Fixed 0,1 mg of long-acting GnRH-antagonist (Degarelix) administered on cycle Day 8. Fixed 250mcg recombinant-hCG (Ovitrelle) or 0,3 mg of Triptorelin (Arvekap) for ovulation triggering administered as soon as 3 follicles of 18mm were present. Produced blastocysts were all cryopreserved and transferred in subsequent thawed cycles. Main results and the role of chance All 15 patients reached ovum pick-up with mature eggs retrieved (mean 13,5 oocytes). None experienced any LH surge, however, 3/15 (20%) required an extra dose of 300IU FSH (Puregon) to enhance follicular development. The majority 12/15 reached OPU without any FSH adjuvant. All 15 patients had blastocysts formed (mean 3,3) and in their subsequent thawed cycles the cumulative live birth rate was 53,3% (8/15). Patient satisfaction was excellent though closer monitoring was applied due to novelty of the protocol. Limitations, reasons for caution Limited number of patients as it is a complete novel protocol with unknown outcome. Protocol was focused in normo-ovarian-reservers to secure follicular production. Some patients might need an extra dose of gonadotropin. Long-acting antagonist still not registered for IVF use. Wider implications of the findings Current study promising results indicate that a 3-injection follicular stimulation can efficiently produce mature eggs, viable blastocysts and pregnancies. Although, few patients required an extra fourth injection of FSH, if such approach will be tested in larger studies will re-establish our way for stimulation at least for normo-reservers. Trial registration number NCT03877185

P-715 outcomes of in vitro fertilization and embryo transfer among ethnic Tibetan, Yi and Han Chinese women

Abstract Study question Does difference exist in the outcomes of in vitro fertilization and embryo transfer (IVF-ET) among ethnic Tibetan, Yi and Han Chinese women? Summary answer We report that the outcomes of IVF-ET were poorer among ethnic Yi women compared with ethnic Han and Tibetan women. What is known already It has been reported that the outcomes of IVF-ET are correlated with ethnicity/race in Europe and the United States as an independent factor. Sichuan is the only province in China where all 56 ethnic groups reside, with ethnic Han, Yi and Tibetan Chinese having the highest proportions. In Chengdu city (the capital of Sichuan province, basin), the majority ethnic group is Han, whilst in Liangshan and Aba Prefectures (plateau), the major ethnic groups are Yis and Tibetans. The living environment, diet, custom, medical condition and genetic backgrounds are different among different ethnic groups. Study design, size, duration In this multi-center retrospective cohort study, a total of 58374 Chines women aged 20 ∼ 45 had undergoing the first IVF or intracytoplasmic sperm injection (ICSI) ET treatment cycle with non-donor gametes from January 2014 to December 2022 were enrolled. Participants/materials, setting, methods The patients were enrolled from two University Affiliated Hospitals and one Reproductive Hospital in Chengdu. The controlled ovarian stimulation protocols were gonadotropin releasing hormone (GnRH) agonist protocol or GnRH antagonist protocol. The patients were divided into three groups based on their ethnic origins: Tibetans (n = 1069), Yis (n = 2273) and Hans (n = 55032). The outcomes of IVF-ET were compared, with the primary outcome being the live birth rate. Main results and the role of chance The proportion of GnRH agonist protocol and IVF fertilization, the rates of moderate and severe ovarian hyper-stimulation syndrome (OHSS), and cleavage embryo formation in ethnic Han women were significantly higher than in the ethnic Yis and Tibetans (P < 0.05). The rates of implantation, clinical pregnancy and live birth (35.5% vs. 42.4%) were significantly lower, whereas the late abortion rate was significantly higher in Yi women than that in Han women (P < 0.05). The live birth rate was significantly lower in Yi women (35.5%) than in Tibetan (43.3%) and Han women (42.2%) (P < 0.05). Adjustment was made for age, duration, type and etiology of infertility, history of spontaneous abortion, smoking, alcohol, tuberculosis infection, body mass index, antral follicle count, antimüllerian hormone, and so on, by multivariate logistic regression analysis. Yi women had significantly lower rates of moderate and severe OHSS (OR = 0.69, 95% CI = 0.49-0.94, P = 0.024) and clinical pregnancy (OR 0.8, 95% CI 0.70-0.93, P = 0.002) compared with ethnic Hans. Yi women had significantly lower live birth rate compared with ethnic Hans (OR = 0.79, 95% CI = 0.68-0.92, P = 0.002) and Tibetans (OR 0.73, 95% CI 0.58-0.92, P = 0.007). Limitations, reasons for caution The limitations are related with the retrospective study and lack of some demographic characteristics of the patients which may influence the outcomes of IVF-ET, including diet habits, educational level, and economic status. Wider implications of the findings The ethnic group is an independent factor for the outcomes of IVF-ET among ethnic Tibetan, Yi and Han women. Among the three ethnic groups, the Yis had lower live birth rate and moderate and severe OHSS, whilst ethnic Han women had more moderate and severe OHSS. Trial registration number ChiCTR2300070269

P-016 Prevalence of high oxidative stress and poor sperm DNA fragmentation levels in different groups of patients

Abstract Study question What is the prevalence of patients with high oxidation-reduction potential (ORP) and high sperm DNA fragmentation (SDF) in different patient groups with and without varicocele? Summary answer A large percentage of normozoospermic patients and patients who are normally considered of manageable in assisted reproduction show high SDF and/or high ORP. What is known already Standard semen analysis, even according to WHO criteria, is very limited in predicting male fertility potential. Oxidative stress is widely recognized as a major contributor to male infertility. Therefore, sperm DNA fragmentation (SDF) is increasingly recommended as additional test in diagnostics. High levels of oxidative stress and SDF have also been reported in patients with varicocele. Yet, SDF and oxidative stress tests are not routinely tested in ART laboratories. Apart from the cost and/or complexity of these tests, a reason for the lack of usage is the lack of knowledge of the prevalence. Study design, size, duration This is a retrospective analysis of data obtained from 2,946 patients attending the Department of Urology, Hamad Medical Corporation, Doha, Qatar, for fertility diagnosis from January to December 2018. For this analysis only patients (n = 773) who had a full data set for sperm DNA fragmentation (SDF) and seminal oxidative stress as measured by means of the oxidation-reduction potential (ORP) were included. Participants/materials, setting, methods Apart from SDF and ORP, standard semen parameters (ejaculate volume, sperm concentration, total and progressive motility, and normal sperm morphology), and the presence of varicocele were determined. Semen parameters were determined according to WHO 2010 criteria. SDF was tested using the Halosperm G2 test (Halotech DNA SL, Madrid, Spain), while ORP was determined using the MiOXSYS® (Aytu BioScience, Englewood, CO). Patients were categorized as normo-, oligo-, astheno-, and teratozoospermic. Main results and the role of chance The mean sperm concentration of the patients was 33.5 × 106 sperm/mL with a mean total motility of 50.3%. The overall percentage of patients with high (>20% SDF) SDF was 57.4% and those with high (≥1.34 mV/106 sperm/mL) ORP 63.4%. Varicocele was diagnosed in 14.0% of the patients. In normozoospermic patients, 26.8% had high SDF, whereas in the non-normozoospermic group this was 59.8% (P = 0.0108). In non-oligozoospermic (53.7%), non-asthenozoospermic (34.7%) and non-teratozoospermic (43.4%) groups, SDF was significantly higher. High ORP values were recorded in 35.7% of normozoospermic, 53.4% non-oligozoospermic, 47.2% of non-asthenozoospermic, and 49.7% of teratozoospermic patients. The difference between the prevalence of high ORP in the normozoospermic (35.7%) and the non-normozoospermic group is significant (P = 0.0063). When both tests (high SDF or high ORP) were considered, 50.0% of normozoospermic, 76.9% of non-oligozoospermic, 59.7% of non-asthenozoospermic, and 71.2% of non-teratozoospermic patients showed seminal pathology. High SDF was detected in 59.3% of varicocele patients, while 50.0% of varicocele patients had high ORP values. In patients without varicocele, 57.1%, 65.6%, and 82.7% had high SDF, high ORP, and high SDF or high ORP, respectively. These percentages were significantly (P < 0.0003) higher than for the corresponding normal groups. Limitations, reasons for caution Although the overall sample size of 773 is reasonably high, after sub-grouping the sample size is much smaller. Since this study only analyzed the SDF and ORP in patients attending and andrological outpatient clinic, the results could be different if patients in IVF and/or ICSI treatment cycles were analyzed. Wider implications of the findings This study highlights that complementation of semen analysis with SDF and oxidative stress testing will identify a large percentage of patients who would benefit from andrological treatment before assisted reproductive techniques (ART). This approach would result in better chances of these patients for successful ART. Trial registration number not applicable

P-398 Lower ectopic pregnancy prevalence with natural endometrial preparation in single euploid frozen embryo transfers

Abstract Study question Does the endometrial preparation approach for an euploid-frozen embryo transfer have an impact on the incidence of an ectopic pregnancy? Summary answer The natural cycle endometrial preparation approach seems to have a lower incidence of ectopic pregnancies compared to a hormonal replacement therapy cycle. What is known already Ectopic pregnancy (EP) is an uncommon complication of pregnancy and is associated with significant morbidity. IVF pregnancies have been implicated as a risk factor, but this finding is mostly confounded by multiple embryo transfer and complicated medical history of women undergoing IVF treatment (tubal disease, surgery, endometriosis, etc.). Recent studies have suggested that frozen embryo transfer may reduce the risk of EP, but it is not yet known whether embryo ploidy status and different endometrial preparation regimens affect the risk of EP. Study design, size, duration Retrospective cohort of 1517 pregnancies from 2607 single euploid frozen embryo transfers. Comparator estimates were estimated using multiple large cohort studies in the literature including 58,203,239 pregnancies from unselected cohorts and 220,167 pregnancies from IVF-treated women with the transfer of non-tested embryos. Participants/materials, setting, methods The rate of ectopic per pregnancies were compared to ectopic rate derived from multiple cohorts of IVF-treated women and population level reports of unselected pregnancies (natural conception and IVF). Comparator estimates were obtained with proportion meta-analyses using generalized linear mixed models. Factors associated with ectopic pregnancies were investigated with mixed-effects logistic regression analyses. Main results and the role of chance EP rate in our cohort was 10/1000 pregnancies (95% CI: 5 to 16 per 1000) resulting from single euploid embryo transfers (1.1%, 16/1517). The observed prevalence of EP was 12/1000 pregnancies (95% CI: 6.8 to 21 per 1000) in unselected cohorts and 14/1000 pregnancies (95% CI: 13 to 15 per 1000) after IVF-treatment. Ectopic rate in our study was not significantly different than rates reported in the literature from unselected and IVF pregnancies (P = 0.579 and 0.223, respectively). There were no significant differences between women with ectopic pregnancies and those with clinical pregnancies in terms of age (33.6±5.5 vs. 35.1±5.2 years, P = 0.290), BMI (26.7±4.8 vs. 25.8±4.6 kg/m2, P = 0.475) and serum AMH levels (3.3±2.9 vs. 3.4±3.9 ng/mL, P = 0.854), nor between the quality of embryo transferred between top, good, fair and poor (1.8% vs 0.7% vs 1.9% vs 0.9%, p = 0.088, 0.997 and 0.508 respectively). Ectopic rates were also similar between women with history of cesarean section (1.1% vs 1.0%, P = 0.847) or isthmocele (1.0 vs 1.1%, P = 0.857). There were less ectopic pregnancies following transfers with natural endometrial preparation (0.5 vs 1.5%, natural vs programmed) and the effect showed statistical significance in multivariable analyses (OR: 0.28, 95% CI: 0.06 – 0.88, P = 0.049). Limitations, reasons for caution Small sample size as well as infrequent occurrence of the studied event reduces the power of our study to detect small but clinically meaningful effects with statistical significance. Wider implications of the findings We did not observe a lower than reported prevalence of EP among euploid embryo transfer. However, rate of EP was significantly lower in natural endometrial preparation cycles compared to preparation with exogenous hormones, indicating that a natural cycle might have a protective effect against the development of an ectopic pregnancy. Trial registration number Not applicable

P-095 Towards deciphering the molecular architecture of the human sperm connecting piece

Abstract Study question How can we make the human sperm connecting piece accessible for structural analysis by cryo-electron tomography (cryo-ET)? Summary answer Preparation of sperm by chemical thinning or production of lamellae via cryo-focused ion beam (cryo-FIB) milling allows direct imaging of the human sperm connecting piece. What is known already Previous studies from our group and others have demonstrated the versatility of cryo-ET to advance our understanding of sperm biology. Structures of macromolecular complexes have been resolved in vitro and in situ, enabling a functional assessment at molecular or even atomic level. Resolving structures at high resolution further allowed identification of proteins previously not known to have a role in mediating male fertility. Such insights are instrumental to further improve diagnosis and treatment of male infertility. Study design, size, duration In our research study we use sperm samples from patients undergoing IVF treatment or donor material, which we receive through an ongoing collaboration with the Division of Woman and Baby, University Medical Centre Utrecht (UMCU). These samples are processed in our own laboratory and subsequently imaged by cryo-ET at the Electron Microscopy Centre of Utrecht University. Participants/materials, setting, methods Due to its thickness, the connecting piece cannot be directly analyzed by cryo-ET. Thus, we designed two strategies to achieve thinning of this area and allow collection of tomograms for structural analysis. While the first approach involves incubation of sperm with a detergent, a reducing agent and heparin, the second approach utilizes cryo-FIB milling to produce thin lamellae of the connecting piece. Main results and the role of chance Both strategies developed to access the connecting piece with cryo-ET are effective and allowed us a glimpse of the molecular landscape of the link between sperm head and tail. Chemical thinning of the connecting piece enabled imaging of the proximal and distal centriole while still embedded in the segmented columns or outer dense fibers, respectively. Furthermore, electron density within the microtubules of both centrioles indicates that they are decorated by microtubule inner proteins. Data obtained from lamellae produced by cryo-FIB milling complements these findings, displaying regular densities along the microtubules extending from the proximal centriole. Analyzing tomograms collected from cryo-FIB lamellae we further found a regular protein density that appears to be connecting the nuclear envelope to the base plate. Moreover, cryo-ET of lamellae allowed a detailed view of nuclear pore complexes and the postacrosomal sheath in their native environment. Limitations, reasons for caution Altering the native state of the sperm cell by chemical preparation is a limitation of this approach. Cryo-FIB milling retains the native state of the cell but is limited in throughput. To improve our cryo-FIB milling approach, we will implement automated milling procedures. Wider implications of the findings Chemical preparation and cryo-FIB milling of sperm enable the in-depth analysis of the connecting piece by cryo-ET. These strategies can now be applied to provide structural details at the molecular level and thus advance our understanding of male infertility related to defects of the head-tail junction. Trial registration number not applicable

P-803 Intra-ovarian injection of plasma rich in stem cell-secreted and platelet-enclosed factors to promote follicle rescue and reproductive outcomes in women with very low ovarian reserve

Abstract Study question Could intraovarian injection of plasma rich in stem-cell and platelet growth factors optimize ovarian reserve and IVF outcomes in women with very low ovarian reserve? Summary answer Intraovarian injection of G-CSF mobilized plasma rich in grown factors improved follicle count since first follow-up and led to better fecundation, implantation and pregnancy’ rates. What is known already Autologous stem cell ovarian transplantation (ASCOT) of bone marrow derived stem cells optimized ovarian reserve and reproductive outcomes in poor responders (POR) and this effect seems to be due to the growth factors secreted by the stem cells, acting through paracrine pathways. Similarly, platelet-rich Plasma (PRP) has been also offered to promote ovarian reactivation in patients with impaired ovarian reserve by providing positive growth factors contained in platelets. Indeed, a technique that combines both the non-cellular components of ASCOT and PRP (ASCOT-1) has been proposed to optimize follicle rescue and IVF outcomes in women with very low reproductive prognosis. Study design, size, duration Retrospective observational study with 159 women (≤ 45 years) diagnosed with POR and primary ovarian insufficiency (POI) according to ESHRE criteria, who underwent the ASCOT-1 technique after refusing oocyte donation, between June 2021-October 2023 at IVIRMA Alicante, Spain (IRB approval 2310-FIVI-188-SH). The ASCOT-1 consisted in the intraovarian injection of plasma rich in both stem cell-secreted and platelet-enclosed factors obtained after granulocyte colony stimulating factor (G-CSF) mobilization and PRP obtention (Endoret Kit BTI, Biotechnology institute). Participants/materials, setting, methods Medical records of patients who underwent the ASCOT-1 as an ovarian reactivation treatment were examined. Follow up of AFC and AMH was carried out for a maximum of 4 menstrual cycles to evaluate follicular activation. Moreover, when follicle growth was detected, patients underwent a controlled ovarian stimulation (COS). IVF and reproductive outcomes were compared with previous COS cycles started in our clinics, when available. T-test was used where appropriate. Main results and the role of chance Overall 159 POR/POI women (age 38.7±4.2years, AMH0 0.22±0.41ng/ml; AFC0 1.4±1.9) underwent the ASCOT-1 technique. Mean volume of plasma injected was 6.8±0.8ml, distributed equally in both ovaries. First follow-up was done in 131 women showing statistically significant improvements in the AFC during the entire follow-up compared to the basal levels measured before the treatment (AFC1 3.3±3.4, p < 0.001; AFC2 3.3±3.6, p < 0.001; AFC3 2.9±2.6, p < 0.001; AFC4 2.4±2.6, p = 0.07). After ASCOT-1, 98 COS were initiated in 48 women in our clinics, which were compared to their previous 66 cycles. Although age was significantly higher in their posterior cycles (38.0±2.9 vs40.0±3.6; p < 0.001), after ASCOT-1 oocyte retrieval was reached in a greater amount of cases (75.5% vs87.5%, p=NS). ASCOT-1 showed a positive, but non statistically significant effect in the number of MII-oocytes (2.8±4.1 vs3.2±3.9, p=NS) and blastocysts obtained (1.8±2.8 vs2.4±3.1, p=NS), but relevant improvements were observed in quality related parameters such as oocyte maturation (61.5%, 95%IC 45.8-77.3, vs86.1%, 95I%IC 69.9-85.5; p 0.03), fertilization (62.6%, 95%CI 53.0-71.3, vs76.5%, 95%CI 70.0-82.0; p < 0.001), implantation (25.0%, 95%CI 8.3-52.5 vs41.1%, 95%CI 19.4-66.5; p < 0.001) and clinical pregnancy rate (25.0%, 95%CI 8.3-52.5 vs46.6%, 95%CI 22.2-72.5; p < 0.001). Ultimately, 14 clinical pregnancies (7 after ET and 7 by natural conception) were obtained after ASCOT-1. Limitations, reasons for caution This retrospective study involves short periods of follow up and limited amount of IVF attempts. Thus, further prospective studies with increased sample size are required to validate our results, to establish the duration of the effects, and to identify which subgroups of patients could benefit the most from this treatment. Wider implications of the findings Growth factors contained in the G-CSF mobilized PRP could play a role in follicular development, providing a new tool to increase the reproductive potential of women with very low ovarian reserve where oocyte donation is the only practical option, allowing them to access to homologous IVF treatment. Trial registration number 2310-FIVI-188-SH

P-579 Is the use of donated oocytes effective to reestablish live birth rates? Reproductive outcomes of 5313 euploid embryo transfers in autologous versus egg donation cycles

Abstract Study question Is live birth rate of autologous versus donor cycles similar after a euploid embryo transfer? Summary answer The live birth rate was significantly higher in autologous cycles after a euploid embryo transfer. What is known already Advanced maternal age (AMA) is a detrimental factor for patients to achieve a successful outcome in ART. Physiological ovarian aging negatively affects oocyte quality and quantity. Thus, the number of cycles with donated oocytes is increasing for AMA patients worldwide, with the perspective of surpass the oocyte factor and achieve a live birth in similar rates as younger patients. The goal of this study was to compare the live birth rates of autologous versus donor cycles after a euploid embryo transfer. Study design, size, duration Observational cohort study including all patients of IVF cycles with a euploid embryo transfer after PGT-A (NGS technology), according to medical referral between Jan/2016 and Dec/2020 (n = 3623 patients and 4025 cycles) in a private ART center. Participants/materials, setting, methods Reproductive outcomes between autologous and egg donation cycles were compared and data were analyzed in the follow subgroups: cycles with a fresh oocyte and fresh embryo transfer (FRESH), frozen embryo transfer (FET) and fresh embryo transfer from frozen oocytes (FOT). Positive pregnancy test (PPT), clinical pregnancy (CP), clinical miscarriage (CM) and live birth (LB) rates were compared. Kruskal-Wallis and Fisher test were applied for statistical analysis, significance was considered if p < 0,05. Main results and the role of chance In total, 3623 patients – 88.9% autologous and 11.1% with donated oocytes – were included. They performed 4025 ETs (3591/434) with 5313 biopsied blastocysts (4736/577) in a mean of 1,32/1,33 blastocysts per transfer in autologous/donated cycles respectively. The majority of patients using autologous and donated oocytes underwent a FET (97,2% and 63,1%), followed by FOT (1,5% and 34,7%) and FRESH (1,3% and 2,2%, respectively). Mean maternal age in autologous and donor cycles was 37,76±3,9yo and 43,47±4,26yo and oocyte donors were 24,99±3,86yo. In subgroups, autologous patients in FRESH were 38,33±2,69 yo, similar in FET (37,73±3,93 yo) and older in FOT, 39,25±3,93 yo (p = 0,03). Patients in donor cycles showed no difference in age. PPT and CP were similar in autologous and donated cycles (PPT: 59,2% vs 54,8%, p = 0.08; CP: 52,2% vs 48,2%, p = 0.11). In subgroup analysis, PPT and CP were higher in donated cycles vs autologous cycles for FOT (PPT: 50,7% vs 33,3%, p = 0.004; CP: 40,7% vs 20,8%, p = 0.01). Overall, CM were higher in donated cycles (15,5% vs 10,6%, p = 0.03), especially after FET (16,8% vs 10,6%, p = 0.025). LB rates were higher in autologous compared to donated cycles (37,8% vs 29,5%, p = 0.0007). There was no significant difference in LB rates in subgroup analysis. Limitations, reasons for caution This is a large cohort study; however, we cannot exclude potential bias due its retrospective nature. Including only euploid embryos, we excluded bias on ploidy status when comparing autologous versus donated oocytes cycles, yet the potential harmful of embryo biopsy may not be neglect. Wider implications of the findings Live birth rate was significantly higher in autologous cycles after a euploid embryo transfer, regardless of subgroups. AMA patients that choose to undergo an IVF treatment with donated oocytes should be aware that there are other important factors related to a successful reproductive outcome, including immunological adaptation and endometrial aging. Trial registration number Not applicable.