Ovarian Cycle Research Articles

Do women with severely diminished ovarian reserve undergoing modified natural-cycle in-vitro fertilization benefit from earlier trigger at smaller follicle size?

To evaluate whether trigger and oocyte collection at a smaller follicle size decreases the risk of premature ovulation while maintaining the reproductive potential of oocytes in women with a severely diminished ovarian reserve undergoing modified natural-cycle in-vitro fertilization. This was a retrospective cohort study including women who had at least one unsuccessful cycle (due to no response) of conventional ovarian stimulation with a high dosage of gonadotropins and subsequently underwent a modified natural cycle with a solitary growing follicle (i.e. only one follicle > 10 mm at the time of trigger). The association between follicle size at trigger and various cycle outcomes was tested using regression analyses. A total of 160 ovarian stimulation cycles from 110 patients were included in the analysis. Oocyte pick-up (OPU) was performed in 153 cycles and 7 cycles were canceled due to premature ovulation. Patients who received their trigger at smaller follicle sizes (≤ 15 mm) had significantly lower rates of premature ovulation and thus higher rates of OPU (98.9% vs 90.8%; odds ratio, 9.56 (95% CI, 1.58-182.9); P = 0.039) compared with those who received their trigger at larger follicle sizes (> 15 mm). On multivariable analysis, smaller follicle sizes at trigger (> 10 to 13 mm, > 13 to 15 mm, > 15 mm to 17 mm) were not associated significantly with a lower rate of cumulus-oocyte complex (COC) retrieval, metaphase-II (MII) oocytes or blastulation when compared to the > 17-mm group. On sensitivity analysis including only the first cycle of each couple, the maturity rate among those with COC retrieval was highest in follicle sizes > 15 to 17 mm (92.3%) and > 13 to 15 mm (91.7%), followed by > 10 to 13 mm (85.7%) and lowest in the > 17-mm group (58.8%). During the study period, five euploid blastocysts developed from 48 fertilized MII oocytes with follicle sizes of 12 mm (n = 3), 14 mm (n = 1) and 16 mm (n = 1) at trigger. Of those, four were transferred and resulted in two live births, both of which developed from follicles with a size at trigger of 12 mm. The ideal follicle size for triggering oocyte maturation may be smaller in women with a severely diminished ovarian reserve managed on a modified natural cycle when compared to conventional cut-offs. The risk of OPU cancellation was significantly higher in women triggered at follicle size > 15 mm and the yield of mature oocytes was not adversely affected in women triggered at follicle size > 13 to 15 mm compared with > 15 to 17 mm. Waiting for follicles to reach sizes > 17mm may be detrimental to achieving optimal outcome. © 2024 International Society of Ultrasound in Obstetrics and Gynecology.

Blood Sampling and Hormone Measurement for Determining the Stage in the Ovarian Cycle in Marmosets.

Common marmosets are small New World monkeys. Since many of their biological mechanisms are similar to those of humans, marmosets are potentially useful for medical and human biology research across a range of fields, such as neuroscience, regenerative medicine, and development. However, there is a lack of literature describing methods for many basic experiments and procedures. Here, detailed methods for determining the levels of sex hormones (progesterone, estradiol, and chorionic gonadotropin) in marmosets are described. The measurement of these hormones enables the prediction of the stage in the ovarian cycle, which is typically 26-30 days in marmosets; accurate determination is essential for the harvesting of oocytes/zygotes at the correct time point and for the preparation of host females for the generation of genetically modified marmosets. Additionally, the measurement of sex hormone levels is useful for endocrinology, ethology, early development, and reproductive biology studies. This protocol provides a detailed description of the methods for blood sampling from the femoral vein, separation of plasma for hormone measurement, measuring chorionic gonadotropin levels using urine and plasma, resetting the ovarian cycle using injections of a prostaglandin F2α analog to shorten and synchronize the cycle, and promoting follicular growth and ovulation by injecting follicle-stimulating hormone and chorionic gonadotropin. Using these protocols, the stages in the ovarian cycle can be determined for the timely collection of oocytes/zygotes.

Novel Methodology for Identifying the Occurrence of Ovulation by Estimating Core Body Temperature During Sleeping: Validity and Effectiveness Study.

Body temperature is the most-used noninvasive biomarker to determine menstrual cycle and ovulation. However, issues related to its low accuracy are still under discussion. This study aimed to improve the accuracy of identifying the presence or absence of ovulation within a menstrual cycle. We investigated whether core body temperature (CBT) estimation can improve the accuracy of temperature biphasic shift discrimination in the menstrual cycle. The study consisted of 2 parts: experiment 1 assessed the validity of the CBT estimation method, while experiment 2 focused on the effectiveness of the method in discriminating biphasic temperature shifts. In experiment 1, healthy women aged between 18 and 40 years had their true CBT measured using an ingestible thermometer and their CBT estimated from skin temperature and ambient temperature measured during sleep in both the follicular and luteal phases of their menstrual cycles. This study analyzed the differences between these 2 measurements, the variations in temperature between the 2 phases, and the repeated measures correlation between the true and estimated CBT. Experiment 2 followed a similar methodology, but focused on evaluating the diagnostic accuracy of these 2 temperature measurement approaches (estimated CBT and traditional oral basal body temperature [BBT]) for identifying ovulatory cycles. This was performed using urine luteinizing hormone (LH) as the reference standard. Menstrual cycles were categorized based on the results of the LH tests, and a temperature shift was identified using a specific criterion called the "three-over-six rule." This rule and the nested design of the study facilitated the assessment of diagnostic measures, such as sensitivity and specificity. The main findings showed that CBT estimated from skin temperature and ambient temperature during sleep was consistently lower than directly measured CBT in both the follicular and luteal phases of the menstrual cycle. Despite this, the pattern of temperature variation between these phases was comparable for both the estimated and true CBT measurements, suggesting that the estimated CBT accurately reflected the cyclical variations in the true CBT. Significantly, the CBT estimation method showed higher sensitivity and specificity for detecting the occurrence of ovulation than traditional oral BBT measurements, highlighting its potential as an effective tool for reproductive health monitoring. The current method for estimating the CBT provides a practical and noninvasive method for monitoring CBT, which is essential for identifying biphasic shifts in the BBT throughout the menstrual cycle. This study demonstrated that the estimated CBT derived from skin temperature and ambient temperature during sleep accurately captures variations in true CBT and is more accurate in determining the presence or absence of ovulation than traditional oral BBT measurements. This method holds promise for improving reproductive health monitoring and understanding of menstrual cycle dynamics.

P-459 Impact of a drop in serum estradiol levels after gonadotropin-releasing hormone (GnRH) antagonist on clinical outcomes of ovarian stimulation cycles in assisted reproductive technology (ART)

Abstract Study question Does a drop in serum estradiol level after the administration of a GnRH antagonist affect outcomes in ovarian stimulation cycles in ART? Summary answer A drop in serum estradiol after administration of a GnRH antagonist is related to changes in luteinizing hormone (LH) and to inferior clinical outcomes. What is known already In recent years, the GnRH antagonist protocol has become widely used to prevent a premature LH surge during ovarian stimulation in ART, with many advantages such as immediate pituitary suppression and a lower risk of ovarian hyperstimulation. However, one phenomenon intriguing professionals monitoring these cycles and reviewing blood analyses, is the decrease in serum estradiol that occasionally occurs after the administration of the antagonist. With the natural menstrual cycle in mind, this could be an undesirable effect possibly affecting clinical outcomes. Study design, size, duration This was a non-interventional, retrospective, observational cohort study. We analyzed 1678 ovarian stimulation cycles in ART in 1143 patients. Patients were treated between January 1st 2015 and November 1st 2022. Patients were 20 to 45 years old, were stimulated with human menopausal gonadotropin (hMG) or recombinant follicle stimulating hormone (follitropin α, β or δ, corifollitropin α and follitropin α + lutropin α) and a GnRH antagonist was used for pituitary suppression in a flexible protocol. Participants/materials, setting, methods Data were extracted from medical records at a tertiary infertility clinic. The difference between serum estradiol level before and after the first antagonist administration was calculated. When this value was negative, the cycle was marked as having an estradiol drop. Influence of treatment and cycle characteristics on estradiol drop, as well as influence of the drop on clinical outcomes were analyzed using generalized linear mixed effects models. Main results and the role of chance The presence of a serum estradiol drop could be calculated in 1552 cycles, of which 153 cycles (9.9%) showed a drop. There was no significant difference in the odds on cycle cancellation between cycles with and without drop (p = 0.87). Type of gonadotrophin used was significantly associated with the presence of an estradiol drop (p < 0.0001), with hMG cycles showing the lowest amount of drops (3.0%). More estradiol drops were observed with the start of antagonist administration on day six of stimulation (10.3% of cycles), compared to starting later than day six (9.1%)(p = 0.001). Serum LH at the start of the cycle was significantly higher in cycles with estradiol drop (mean±SD=4.10±4.13IU/L) than in cycles without (3.88±2.74IU/L) (p = 0.01). Decrease in serum LH levels after antagonist administration was significantly larger in cycles with estradiol drop (-5.30±7.58IU/L versus -1.66±3.13IU/L,p<0.0001). Serum LH on the day of ovulation triggering was also lower in estradiol drop cycles (1.29±1.59IU/L versus 2.24±8.13IU/L,p=0.0007). Estradiol drop was not significantly associated with the number of oocytes retrieved, 2PN fertilized, utilization rate or embryo transfer (p-values>0.05). Ongoing pregnancy and live birth were also significantly lower in cycles with an estradiol drop (13.9%) than in cycles without (25%, p = 0.01). When correcting for age, p-values remained unchanged. Limitations, reasons for caution A limitation of the retrospective study design is that stimulation protocols and any adjustments were chosen by the treating physician according to the patient’s characteristics. The authors did adjust for different cycles per patient by using generalized mixed models. Absolute numbers of estradiol drop are low, limiting optimal multivariable analysis. Wider implications of the findings Because serum estradiol drop after GnRH antagonist administration is related to changes in LH, prospective research could focus on potential optimization of the LH serum levels which could potentially ameliorate clinical outcome. Trial registration number not applicable

P-619 AMH/AFC ratio as a predictive factor for ovarian responsiveness to gonadotropins during ovarian stimulation

Abstract Study question Does AMH/AFC ratio correlate to the number of follicles on the day of trigger and to the number of oocytes retrieved after ovarian stimulation? Summary answer The AMH/AFC ratio correlates significantly to the number of pre-ovulatory follicles and the number of oocytes collected after ovarian stimulation (OS). What is known already Although AFC is a robust marker of oocyte yield after OS, its combination with Anti-Mullerian hormone (AMH) seems to improve the predictability. Besides, Follicular Output Rate [FORT: (number of pre-ovulatory follicles on day of trigger/AFC at baseline) x 100] and Follicle-to-Oocyte Index [FOI: (number of retrieved oocytes/AFC) × 100] evaluate the ovarian responsiveness to gonadotropins during OS, representing its sensitivity to exogenous FSH. Both parameters take antral follicle count (AFC) only into consideration. AMH and AFC as a ratio might optimize the accuracy of FORT and FOI metrics, helping to predict which patients are at higher risk of suboptimal results. Study design, size, duration This retrospective study analyzed 2,163 ovarian stimulation cycles for IVF/ICSI, performed between May 2015 and October 2023 in a tertiary referral IVF centre. For all the cycles, measurement of AMH using Elecsys (Roche®) no longer than 6 months before starting OS, AFC at baseline, number of pre-ovulatory follicles on the day of trigger, and the treatment outcomes were available. Participants/materials, setting, methods Couples with primary/secondary infertility who underwent OS with antagonist protocol, starting on day 2/3 for IVF/ICSI were included, independent of their age or ovarian reserve parameters. Exclusion criteria were patients with body mass index (BMI) >40 kg/m2, non-vaginal ultrasound measurement for AFC and/or follicles during OS, cycles with previous estradiol priming or oral contraceptives. Ethical approval was obtained from the Research Ethics Committee. Main results and the role of chance 2,163 ovarian stimulation cycles were included in the analysis. The mean age, BMI, AMH, AFC, FOI and FORT of the included women were: 35.6±6.1 years, 27.5±4.7 kg/m2, 2.4±2.1 ng/mL, 12±7 follicles, 1.02±0.66 and 1.38±0.79. The correlation of AMH/AFC ratio (coefficient: 0.62, 95% CI: 0.59 to 0.65, p<0.001) to FOI was stronger than the correlation of AMH (coefficient: 0.31, 95% CI: 0.27 to 0.34, p<0.001) or AFC alone (coefficient: -0.09, 95% CI: -0.14 to -0.05, p<0.001). The correlation of AMH/AFC ratio (coefficient: 0.62, 95% CI: 0.59 to 0.64, p<0.001) to FORT was also stronger than the correlation of AMH (coefficient: 0.21, 95% CI: 0.16 to 0.25, p<0.001) or AFC alone (coefficient: -0.23, 95% CI: -0.27 to -0.19, p<0.001). AMH/AFC ratio was better at predicting stimulation efficiency parameters, FOI and FORT, compared to joint use of AMH and AFC (p>0.001 for both). However, regression analysis showed stand-alone use of the AMH/AFC ratio did not provide a better prediction for collected oocyte count compared to joint use of AMH and AFC. In fact, prediction errors were significantly larger for the AMH/AFC ratio (median: -0.4, range: -11 to 11) compared to the model using AMH and AFC separately (median: -0.2, range: -6 to 7). Limitations, reasons for caution Although clinical assessment was performed in the same centre following the same methodology, inter-observer variability for AFC is a limitation. Additionally, AMH levels used in the study were not obtained in the same cycles, which may adversely affect the performance of the ratio. Wider implications of the findings While AMH/AFC ratio was better at predicting stimulation efficiency compared to FOI and FORT parameters, it was not significantly better at predicting absolute oocyte yield. This may imply AMH/AFC ratio can represent the ovarian sensitivity to exogenous FSH. Trial registration number Not applicable

O-292 A three-dimensional ultrasound-based online system for automated ovarian follicle measurement

Abstract Study question Is OSIS Ovary (a three-dimensional ultrasound-based online system for automated ovarian follicle measurement) a reliable tool to track follicle growth during ovarian stimulation cycles? Summary answer We can conclude that OSIS Ovary constitutes a reliable follicle-measurement tool What is known already Ultrasound follicle tracking is a crucial part of cycle monitoring. When the number of follicles increases, the manual measurement process can become tedious, imprecise and can potentially compromise the clinical outcomes The use of automated volumetric software for follicle monitoring makes it possible to overcome these manual limitations, regardless of the moment in the cycle when the metrics are obtained and assessed, providing flexibility for both clinicians and patients At the same time, more objective metrics are obtained with automated volume segmentation than with manual follicle measurement Study design, size, duration A prospective cohort study was performed among 89 patients undergoing ovarian stimulation during in vitro fertilization or oocyte vitrification cycles They were invited to participate in the study between May 2020 and February 2021. All participants signed an informed consent form prior to the start of the study Participants/materials, setting, methods 534 ovarian ultrasound volumes and manual datasets were obtained from ovarian stimulation cycles from the 89 participant patients Three transvaginal (2D and 3D) ultrasounds scans were performed on volunteer patients on the three clinically relevant monitoring days: 4th- 5th day (first follicular assessment), 6th -7th day (introduction of the GnRH antagonist) and 8th-9th day (oocyte pickup scheduling) The mean follicle diameter and the follicle count were estimated manually (2D) and automated (3D with Osis Ovary) Main results and the role of chance Based on the mean follicle diameter and follicle count values obtained, the Pearson/intraclass correlation coefficients were 0.976/0.987 and 0.804/0.889 in ≥ 10mm follicles, 0.989/0.994 and 0.809/0.867 in ≥ 13mm follicles and 0.995/0.997 and 0.791/0.840 in ≥ 16mm follicles. The mean difference (MnD) for the mean diameter and follicle count was,respectively, 0.759/0.161 in ≥ 10mm follicles, 0.486/1.033 in ≥ 13mm follicles and 0.784/0.486 in ≥ 16mm follicles. The upper and lower limits of agreement (ULA and LLA) were 3.641/2.123 and5.392/3.070 in ≥ 10mm follicles, 3.496/2.522 and 4.285/2.218 in ≥ 13mm follicles, and3.723/2.153 and 2.432/1.459 in ≥ 16mm follicles. The limits of agreement range (LoAR) were5.764/8.462 in ≥ 10mm follicles, 6.048/6.503 in ≥ 13mm follicles and 5.876/3.891 in ≥ 16mm follicles Limitations, reasons for caution We can conclude that OSIS Ovary constitutes a reliable follicle-measurement tool. The performance observed can be considered very good and it must be progressively clinical studied to be assessed and optimized in the next future Wider implications of the findings Cloud-based features, such as multi-user functioning in any 3D ultrasound machine, could make OSIS Ovary an attractive resource for reproductive medicine clinics, making it easier to track follicle growth during ovarian stimulation cycles Trial registration number not applicable

P-388 The science of frozen embryo transfer, is modified natural cycle better?

Abstract Study question Do live birth rate, obstetric outcomes and number of clinic visits differ between frozen embryo transfer in modified natural and artificial cycle? Summary answer Modified natural cycle yielded a higher live birth rate for the similar number of visits, less hypertension, but higher rate of gestational diabetes. What is known already A frozen embryo transfer in a modified natural cycle entails procedure timing such that it coincides with the woman’s ovulatory cycle. Conversely, a frozen embryo transfer in an artificial cycle involves controlled hormonal stimulation, with the subsequent scheduling of embryo transfer. Based on the current evidence, there is no difference in pregnancy rates between modified natural cycle and artificial cycle. Frozen embryo transfer during an artificial cycle allows for greater flexibility for the patient and physicians. However, it seems to be associated with higher risk of adverse obstetric and neonatal outcomes. Study design, size, duration 1206 frozen embryo transfer cycles between January 1st, 2022, and December 31st, 2022 were retrospectively studied. Patients older than 40, with recurrent implantation failure, and recurrent pregnancy loss were excluded. Patients were divided according to their age, BMI, AMH, and the type of embryo transfer protocol. Compared outcomes were endometrial thickness, biochemical pregnancy rate, clinical pregnancy rate, miscarriage rate, number of clinic visits prior to transfer, obstetric complications, livebirth rate, and livebirth weight. Participants/materials, setting, methods Patients in the modified natural cycle group were followed by ultrasound and when the endometrium reached a thickness ≥ 7mm, and a dominant follicle ≥ 15 mm, HCG trigger was given, and the embryo (blastocyst) transferred 7 days later. In the artificial cycle group, patients received estrogen supplementation either orally or by patches and when the endometrium reached a thickness ≥ 7 mm, embryo transfer was scheduled following intramuscular progesterone administration for 6 days. Main results and the role of chance Patients with modified natural cycle (n = 250) embryo transfers have better clinical pregnancy rate compared to the embryo transfers (n = 250) in artificial cycles (63,6% vs 55,2% p = 0,05), and significantly better live birth rate (57,2% vs 43,9% p = 0,003) despite similar biochemical pregnancy rate (64% vs 61% p = 0,48). The miscarriage rate was significantly lower in modified natural compared to artificial cycles (10,2 % vs 20,99%, p = 0,008). Similar results were found with the transfer of PGTA tested embryos. There was no difference in the endometrial thickness between the 2 groups. The number of visits was higher in the modified natural cycle group, but the difference was not clinically significant (1,57 days vs 1,2 days). When comparing the obstetric outcomes, patients with a modified natural cycle transfer had a lower risk of hypertension (6,6% vs 13,5 % p = 0,10), but a significantly higher risk of gestational diabetes (19% vs 8,3% p = 0,02). No difference was observed regarding the birth weight, percentage of preterm birth or mode of delivery. Regression analysis was performed to identify any cofounder that might have affected the clinical pregnancy rate, and the results showed that the type of transfer was the only factor affecting the outcomes (OR: 1,23 CI: 1,03- 1,46) Limitations, reasons for caution The main limitation of this study is its retrospective nature. A randomized controlled study should be performed to confirm these findings. Wider implications of the findings Modified natural embryo transfer could be used to improve pregnancy outcomes. With proper scheduling, embryo transfer could be done with a similar number of visits for modified natural cycles and artificial cycles. More studies are needed to assess the increased risk of gestational diabetes with natural cycle embryo transfer. Trial registration number NCT06053827

P-545 Early pregnancy outcomes associated with age and AMH following Next-Generation Sequencing(NGS)-based Preimplantation Genetic Testing for Aneuploidy(PGT-A) in single euploid embryo transfers for advanced age patients

Abstract Study question Does age and AMH impact early pregnancy outcomes in euploid single embryo transfers using NGS-based PGT-A for individuals aged 35 and above? Summary answer In this NGS-based PGT-A study, no significant differences in early pregnancy rates were observed based on age or AMH among individuals aged 35 and above. What is known already A recent meta-analysis suggest a decline in pregnancy rates with advancing age in the context of euploid embryo transfer following PGT-A. However, it’s crucial to acknowledge variations in PGT-A techniques across studies. While individuals with low AMH levels may encounter challenges in obtaining euploid embryos, the precise impact of AMH on pregnancy rates post-embryo acquisition remains unclear. Study design, size, duration A retrospective cohort study was conducted on 428 cycles of single euploid embryo transfers in patients aged 35 and above, performed at the single infertility center from January to December 2022. Participants/materials, setting, methods The study examined frozen-thawed embryo transfer cycles in patients aged 35 and above, analyzing early pregnancy outcomes in single euploid embryo transfers. Participants were grouped by age(35-38, 38-42, 42 and above) and AMH levels(<1, 1-3, >3 ng/ml). NGS-based PGT-A conducted embryo biopsy. The analysis aimed to detect differences in initial pregnancy rates among age and AMH subgroups. The differences between groups were compared using Pearson’s chi-squared test. Main results and the role of chance The average age at transfer was 39.05 ± 2.53 years, with a mean AMH of 2.60 ± 1.86(ng/ml). The mean number of prior frozen embryo transfer (FET) cycles was 1.9 ± 2.04, with 119 patients (27.8%) having undergone three or more transfer cycles. Transfer cycles comprised 363 (84.8%) artificial cycles and 65 (15.2%) ovulatory cycles, with an average endometrial thickness of 8.95 ± 1.73 mm at the time of transfer. The β-hCG positive rate was 57.7% (247/428), and the clinical pregnancy rate was 53.5% (229/428). When analyzing positive b-hCG results by age, rates were 59.9% (82/137) for ages 35-38, 55.5% (121/218) for ages 38-42, and 60.3% (44/73) for ages 42 and above (p = 0.641). In terms of AMH, β-hCG positive rates were 52.1% (37/71) for <1, 59.1% (120/203) for 1-3, and 58.0% (83/143) for ≥3, with no statistically significant differences (p = 0.584). Clinical pregnancy rates were 54.0% for ages 35-38, 51.8% for ages 38-42, and 57.5% for ages 42 and above. For AMH, rates were 50.7% for <1, 53.2% for 1-3, and 54.5% for ≥3, with no statistically significant differences (p = 0.869). Limitations, reasons for caution Being a retrospective study analyzing patients undergoing embryo transfer, our study overlooks variables related to oocyte retrieval. Given the challenges of obtaining euploid embryos in advanced age, further investigation is warranted to explore the probability and duration of embryo acquisition in this population. Wider implications of the findings In this study, utilizing NGS-based PGT-A after generating euploid embryos, no significant differences in early pregnancy outcomes were observed for women aged 35 and older, considering age and AMH levels. The study suggests further research is needed to explore if NGS-based PGT-A can address limitations of traditional chromosomal testing methods. Trial registration number not applicable

P-546 Do patients undergoing PGT-M benefit from the Duostim protocol?

Abstract Study question Do patients undergoing PGT-M benefit from the Duostim protocol? Summary answer PGT-M patients benefit because luteal phase stimulation is as effective as follicular phase stimulation in generating transferable embryos, thereby increasing the chances of treatment success. What is known already Patients undergoing PGT-M often require a larger number of embryos for pregnancy, with 25-50% deemed ineligible for transfer based on the disease indication (recessive, dominant, or sex-linked). DuoStim emerges as a valuable strategy for improving outcomes in poor responders. Progress in blastocyst culture, genetic testing, and vitrification motivates clinicians to optimize ovarian reserve exploitation through tailored stimulation protocols. Luteal Phase Stimulation (LPS) finds success in poor prognosis or oncological cases within DuoStim, a protocol widely used for poor prognosis patients necessitating both Follicular Phase Stimulation (FPS) and LPS. The potential benefits of DuoStim for PGT-M patients remain unexplored. Study design, size, duration Retrospective controlled case-control study conducted with paired FPS- and LPS-derived cohorts of oocytes collected in the same ovarian cycle (DuoStim). The study included 26 patients aged 36.53 ± 4,59 years old, undergoing DuoStim with the indication of PGT-M between July 2022 and October 2023. Participants/materials, setting, methods FPS and LPS were performed using the same daily dose of recombinant gonadotropins in an antagonist protocol. All zygotes underwent blastocyst culture, trophectoderm biopsy, vitrification, and subsequent thawing of one unaffected euploid blastocyst for transfer. The primary outcome was the mean number of transferable blastocysts (not affected euploid ones) obtained per oocyte retrieval from paired FPS- and LPS-derived cohorts. Secondary outcomes included the mean number of oocytes, blastocysts and implantation and pregnancy rates. Main results and the role of chance FPS and LPS were comparable regarding the mean number of mature oocytes collected (7.73 ± 4.27 vs. 7.57 ± 4.56) and the mean number of blastocysts obtained (3.27 ± 2.40 vs. 2.50 ± 1.98). The mean number of transferable blastocysts per retrieval were similar (FPS: 1.21 ± 1.31 vs. LPS: 0.78 ± 0.91). No significant differences in implantation or ongoing pregnancy rates were observed after transfers of euploid single blastocysts derived from FPS or LPS: 70.0% (7/10) vs. 57.14% (4/7). Three patients out of the 26 (11.54%) had transferable embryos only in the luteal phase. Limitations, reasons for caution Our findings warrant additional assessment through studies involving larger sample sizes. While our preliminary data indicate comparable implantation/pregnancy rates for PGT-M blastocysts from both FPS and LPS, further investigation is essential, especially in populations beyond those with a reduced number of usable blastocysts Wider implications of the findings These data underscore that oocytes obtained from LPS are as competent as those derived from FPS. Patients with unaffected euploid embryos solely due to FLS would not have had any transfer without DuoStim. This protocol could be advantageous for women abroad or with time constraints, potentially reducing dropout rates. Trial registration number Not applicable

O-301 Progesterone Primed Ovarian Stimulation (PPOS) for DuoStim combined with PGT-A in poor prognosis patients: a valuable strategy

Abstract Study question Is PPOS rather than conventional GnRH-antagonist a valid strategy in DuoStim protocols? Summary answer PPOS-DuoStim and conventional-DuoStim showed similar mean euploid blastocyst rate (EBR) per inseminated metaphase-II (MII) oocytes. What is known already DuoStim (double stimulation in the same ovarian cycle) is an efficient strategy to increase the number of oocytes retrieved and embryos obtained in the shortest possible timeframe. PPOS is based on the use of exogenous oral progesterone to prevent LH surge during ovarian stimulation (OS). The effectiveness and safety of PPOS have been consistently reported across the last years, supporting it as a clinically-valuable OS protocol. To date, no evidence exists about PPOS application in DuoStim protocols and its association with oocyte competence, defined as EBR per MII-oocytes. Study design, size, duration Retrospective study involving 138 PPOS-DuoStim and 138 matched conventional-DuoStim patients treated at a single clinic between 2021 and 2022 and collecting ≥1 MII-oocyte after both stimulations. Matching was based on maternal age (40.3±2.7 years), number of oocytes collected after the I-stimulation (5.8±3.6 years), and sperm quality (66% normozoospermic). The primary outcome was the EBR per MII-oocytes. All intermediate embryological and clinical outcomes were also compared. Sub-analyses among I- and II-Stimulations were also conducted. Participants/materials, setting, methods DuoStim entailed the same protocol in I- and II-Stimulations with FSH 300IU/day+LH 150IU/day, 5 days spanning the two stimulations and GnRH-agonist to trigger ovulation. The only difference between PPOS-DuoStim and conventional-DuoStim was the administration of medrossiprogesterone acetate10mg/day orally from the first day of stimulation versus flexible GnRH-antagonist from the day the follicles reached a diameter of 13-14 mm. The same laboratory procedures were applied: ICSI, trophectoderm biopsy, comprehensive-chromosome-testing to report uniform aneuploidies and vitrified-warmed single-embryo-transfers. Main results and the role of chance The patients in the two groups were comparable for ovarian reserve markers, BMI, and duration of infertility. All data were comparable in conventional-DuoStim and PPOS-DuoStim (Mann-Whitney U tests were conducted): (i) overall fertilization rate per inseminated MII-oocytes were 73.1±23.2% versus 76.8±17.8% (p = 0.143), (ii) overall blastulation rate per 2PN-zygotes were 46.6±30.4% versus 45.7±25.1%(p = 0.797), (iii) overall euploidy rates per biopsied blastocysts were 24.1±30.9% versus 25.4±31.3%(p = 0.956), (iv) EBR per inseminated MII-oocytes were 9.6±17% versus 9.2±12.7% (p = 0.735). The data among only I- and II-stimulations were also similar. When comparing I- versus II-stimulations, paired t-tests highlighted longer II-stimulations (11±1.9 vs11.6±1.9 days, p = 0.001), resulting in larger cohorts of MII-oocytes (4.3±2.8 vs 5.2±3.3, p < 0.001) with comparable EBR per MII-oocytes (9.7±20.2% vs 11.6±16.2%, p = 0.233), thereby eliciting larger cohorts of euploid blastocysts (0.5±0.07 vs 0.7±0.09, p = 0.005). These data were confirmed also among conventional-DuoStim and PPOS-DuoStim study arms only. From a cycle perspective, among the 138 conventional-DuoStim couples, 31 (22%), 44 (32%) and 59 (43%) obtained ≥1 euploid blastocyst after I-stimulation, II-stimulation and overall, respectively. The same figures among the 138 PPOS-DuoStim were 36 (26%), 44 (32%) and 63 (46%), respectively. No statistical difference was reported (Fisher’s exact test=0.716). Limitations, reasons for caution Retrospective single center design. The current sample size should be doubled in order to reach a 80%-power with a 5%-α to exclude a 5% two-sided difference in the primary outcome. Patients’ compliance and discomfort, as well as clinical, gestational, and perinatal outcomes and cost-effectiveness analyses are needed. Wider implications of the findings PPOS is valuable in unconventional-OS protocols entailing freeze-all, like DuoStim with PGT-A. Future studies should assess if PPOS-DuoStim, by decreasing the number of injections and scans, may improve patient compliance and decrease OS discomfort. Any putative impact on follicular recruitment and oocyte yield should also be determined. Trial registration number not applicable

P-369 DuoStim could be a proper option for women seeking fertility preservation for non-medical reasons

Abstract Study question Is Duostim a suitable option for women who wish to preserve fertility for social reasons? Summary answer Providing the option of DuoStim is an efficient way to expand the cohort of vitrified oocytes in women seeking fertility preservation for non-medical reasons. What is known already A growing number of reproductive-age women are choosing to postpone motherhood, giving rise to the concept of reproductive autonomy. Numerous strategies aim to refine ovarian stimulation protocols. DuoStim, entailing dual ovarian stimulations and retrievals within one menstrual cycle, proves advantageous for those with diminished ovarian reserve or facing cancer. To our knowledge, this is the first study offering DuoStim for social preservation. Study design, size, duration Retrospective controlled case-control study conducted with paired follicular phase stimulation (FPS) and luteal phase stimulation (LPS) derived cohorts of oocytes collected in the same ovarian cycle (DuoStim). The study included 160 patients aged 36.85 ± 3.3 years old, who underwent DuoStim for social egg freezing from October 2018 to September 2023. The primary outcome was the number of MII oocytes retrieved per cycle and secondary outcomes included stimulation duration and gonadotropin dose. Participants/materials, setting, methods FPS and LPS were performed using the same daily dose of recombinant gonadotropins in an antagonist protocol. Oocyte retrieval was performed 36 hours after triggering with gonadotropin-releasing hormone agonist triptorelin acetate (0,2 mg) after the FPS. Luteal phase stimulation protocol began on the 5th day after ovarian puncture using dual trigger, followed by a second oocyte retrieval. Mature oocytes were vitrified 2 hours later following the Kitazato® protocol. Main results and the role of chance Stimulation duration and gonadotropin dose were lower during Follicular Phase Stimulation (FPS) vs. Luteal Phase Stimulation (LPS) (10.8 ± 1.5 days vs. 13.0 ± 2.3, P < 0.05 and 2,487.9 ± 580.8 vs. 3,264.0 ± 942.7, P < 0.05). The number of retrieved oocytes was comparable between the two groups (4.78 ± 3.0 vs. 5.32 ± 3.7; P = 0.15). The mean number of MII oocytes acquired in the LPS group exceeded that in the FPS group (4.16 ± 3.0 vs. 3.58 ± 2.3; P = 0.054), indicating a tendency towards more MII oocytes in the LPS group without achieving statistical significance. A total of 110 patients (69.29%) obtained the same or a greater number of MII during the LPS compared to the FPS. Limitations, reasons for caution Although DuoStim is acknowledged as an option to obtain a greater number of eggs in certain situations, the evidence supporting its utility in social fertility preservation is still limited. It is crucial to recognize the inherent biases in observational retrospective studies. Wider implications of the findings DuoStim could be an advantage for women who live abroad or those who dispose of short periods of time for oocyte vitrification, also potentially reducing the drop-out rate. Trial registration number not applicable

P-749 Maternal and obstetric outcomes following the transfer of embryos warmed with fatty acid supplemented solutions

Abstract Study question Does fatty acid (FA) supplementation into warming solutions influence maternal and obstetric outcomes after single vitrified-warmed cleavage stage embryo transfer (SVCT)? Summary answer FA-supplemented warming solutions improve the live birth rate after SVCTs without exerting any adverse effects on maternal and obstetric outcomes. What is known already Vitrification procedures decrease the intracellular lipid content and impair developmental competence. Adding fatty acids (FAs) to the warming solution has been shown to recover the lipid content of the cytoplasm and improve developmental competence and clinical pregnancy by stimulating the beta-oxidation pathway in warmed embryos. Recently, the FA-supplemented warming solutions have been commercially released. However, influence of the FA-supplemented supplementation on live birth rate after embryo transfers and perinatal outcomes remains unknown. Study design, size, duration The clinical records of 701 treatment cycles in 701 women who underwent SVCTs were retrospectively analysed. Vitrified embryos were warmed using solutions (from April 2022 to June 2022, control group, n = 340) or FA-supplemented solutions (from July 2022 to September 2022, FA group, n = 361). All pregnant women were invited to respond to the questionnaire at 9 weeks of gestation and after delivery. Maternal and obstetric outcomes were obtained from a self-reported questionnaire completed by the patients. Participants/materials, setting, methods The embryos in the FA group were warmed with the FA-supplemented solutions (VT526, Kitazato Corporation) while the embryos in the control group were warmed with the non-supplemented solutions (VT506, Kitazato Corporation). SVCTs were performed on day 2 after ovulation in natural cycles. The live birth rate, incidence of pregnancy complications, and neonatal outcomes were compared between the control and FA groups. Main results and the role of chance The live birth rate was higher in the FA group than in the control group (P = 0.0399). The miscarriage rates were comparable between the groups. Multivariate logistic regression analysis also demonstrated a higher probability of live births in the FA group than in the control group (adjusted odds ratio, 1.46; P = 0.0404). Patients were stratified by median maternal age for further analysis (37 years). This subgroup analysis showed that the live birth rate was significantly higher in the FA group than in the control group in women younger than 37 years of age. However, the live birth rate was comparable between the two groups for women aged 37 years or older. The incidence and types of pregnancy complications were comparable between the control and FA groups. Furthermore, caesarean section rates were similar between the groups. The average gestational age and incidence of preterm delivery were comparable between the groups. Birth length and weight, incidence of low birth weight, incidence of small for gestational age and large for gestational age, and infant sex were comparable between the groups. The incidence of birth defects was similar between the groups. Multivariate logistic regression analysis revealed a similar trend. Limitations, reasons for caution The single-centre retrospective study limits the strength of the study, necessitating further multicentre studies to ascertain the generalizability of these findings to other clinics with different protocols and/or patient demographics. Furthermore, the number of perinatal cycles was low. Wider implications of the findings The FA-supplemented warming solutions improved the live birth rates after SVCTs, particularly in young patients. Furthermore, the incidence of pregnancy complications and infant health was not affected by the FA-supplemented solutions. Therefore, FA-supplemented solutions can be considered safe and effective at improving clinical outcomes and reducing the patient burden. Trial registration number not applicable

O-208 The Programmed Ovulatory Frozen-Thawed Embryo Transfer Cycle (PO-FET): a suggested FET protocol integrating aspects of embryo transfer scheduling, efficacy optimization and maternal/fetal safety

Abstract Study question What is the effect of 30mg oral dydrogesterone (DYD), given to induce endometrial receptivity in natural-proliferative-phase-FET-cycles (NPP-FET), on ovulation, progesterone and DYD/DHD-levels, respectively, and outcomes? Summary answer Oral DYD 30mg allows scheduling endometrial receptivity in natural cycles while not interfering with ovulation in the vast majority of patients. What is known already Globally, approximately 60% of all ETs are performed after cryopreservation. Hormone Replacement Treatment (HRT)-FET cycle-regimens have come under scrutiny, because of i) risk of insufficient progesterone exposure with conventional dosing-schemes and ii) maternal/fetal risks stemming from the iatrogenic lack of a corpus luteum. Oral DYD 10mg (tid) supposedly does not interfere with ovulation/corpus luteum formation, and does not cross-react with progesterone in ELISA. It could thus be used for inducing endometrial receptivity (scheduling, in a natural cycle, the time point of ET) and luteal phase support, while allowing ovulation dissociated from the window-of-implantation and monitoring of endogenous progesterone serum levels. Study design, size, duration Within a multi-centric, prospective, clinical cohort study (NCT03507673), 559 normally cycling women from the routine care population who underwent FET after IVF/ICSI in a natural cycle (2/2021 – 8/2023) had serum/plasma samples prospectively collected/stored on day of FET for measurement of E2, progesterone and DYD/DHD in Roche Elecsys®-Immunoassay and liquid chromatography/tandem mass spectroscopy (LC-MS/MS), respectively. FET was performed on day 2/3 (cleavage-stage) or day 4/5 (morula/blastocyst) in 17.4% and 82.6% of FETs, respectively. Participants/materials, setting, methods From cycle day 10, women underwent endocrine (E2, LH and P) and sonographic monitoring until E2 >180pg/ml, leading follicle >16mm and endometrial thickness was >6mm. Physicians could then initiate oral DYD10mg (tid) or–in absence of LH rise–postpone initiation, under 2-daily monitoring, to meet the organizational preferences of the patient and/or center (for example, avoiding weekends). FET was performed for day 2 (cleavage), 3, 4 or 5 (blastocyst) on day 3-6 of DYD-intake, respectively. Main results and the role of chance Previously reported PK of 10mg DYD (tid) are confirmed/extended. While log-levels of DYD and DHD were highly correlated (0.95), P-levels were not (0.02, 0.02). In 94.4% of patients, ovulation could be confirmed by serum-P levels on the day of FET or in late luteal phase. Medians (IQR) of DYD and P on day 3 and days 5, respectively, were 0.93ng/ml (0.93ng/ml) and 2.96ng/ml (3.19ng/ml), and 1.13ng/ml (1.09ng/ml) and 8.50ng/ml (4.40ng/ml). Serum-P levels increased with the FET day (medians 2.0, 3.0, 6.6, and 8.5 ng/ml on days 2, 3, 4, and 5) and only very weakly correlated with LH, E2 levels, and follicular diameter at last monitoring. 42.5% and 34.8% of patients had positive hCG and ongoing clinical pregnancy, respectively. Ongoing pregnancy was independent from plasma-DYD levels (< 25th percentile [<0.66 ng/ml] 37.9% vs. ≥25thperc. [≥0.66 ng/ml] 34.3%, RD 1.5%, 95%CI: -16.5% to 17.4%, p = 0.334), yet dependent from serum-P levels (< 25thperc [<4.85 ng/ml] 25.9% vs. ≥25thperc [≥4.85 ng/ml] 35.3%, RD 9.4%, 95% CI 0.4% to 17.6%, p = 0.0161).However, neither of the two serum hormone values is predictive of a dichotomous outcome in isolation, nor do they demonstrate predictive interaction when considered together in logistic regression modelling. Limitations, reasons for caution DYD, like hCG or any other progestin given for LPS, may negatively interfere with LH surge, ovulation and luteal phase characteristics. Controlled studies on different progestin types and doses are necessary to better understand how to optimize luteal phase and thereby optimally support embryo nidation, pregnancy maintenance and maternal/fetal health. Wider implications of the findings The PO-FET protocol induces the implantation window, thus offers (some) flexibility in FET timing, shows little interference with ovulation/corpus luteum formation, provides double gestagenic support with no need for monitoring P or DYD levels, however allows corpus luteum activity monitoring in luteal phase and early pregnancy, is injection-free, and low-cost. Trial registration number NCT03507673

P-410 A modified artificial cycle-frozen embryo transfer(mAC-FET) protocol with evidence of corpus luteum(CL) formation as determined by serum relaxin-2 in early pregnancy :a prospective proof-of-concept study

Abstract Study question Can a mAC-FET protocol for endometrial preparation in ovulatory patients lead to formation of a CL as evidenced by serum relaxin-2 in the pregnant patients? Summary answer CL forms in the mAC-FET protocol as serum relaxin-2 in early pregnancy was comparable to that in spontaneous conception.Late luteal rise of relaxin-2 wasn’t demonstrated. What is known already Clinical evidence points to increased incidence of preeclampsia in pregnancies by AC-FET cycles compared to ovulatory FET cycles-natural cycle(NC) and mNC. The absence of CL and it’s secretory products like relaxin-2 is a contributing factor. FET protocols for endometrial preparation in ovulatory patients need to be optimised. NC/mNC-FET protocols need frequent monitoring. Also there is a temporal variability between LH/hCG and progesterone levels. We studied a mAC-FET protocol in ovulatory patients with a delayed start(day3-5 of the cycle),low dose transdermal 17β estradiol gel to see the formation of CL as elucidated by the presence of serum relaxin-2 in early pregnancy. Study design, size, duration A proof-of- concept prospective study was conducted at a tertiary centre (July2023-Dec2023). Serum relaxin-2(R&D/Quantikine/ELISA) was determined in 4 groups of patients- Group-A. mAC-FET(n = 34) 1.On the day of start of progesterone 2.Post-ET day5-9 (along with serum progesterone) 3.In pregnancy(6-9 weeks of gestation) Group-B. AC-FET(n = 8) 1.On the day of start of progesterone 2.In pregnancy(6-9 weeks) Group-C. Spontaneous conception (n = 9) 1.In pregnancy(6-9 weeks) Group-D. Infertility patients ( n = 24) 1.Preovulatory. All patients were ≤40 years with a BMI ≤30kg/m2. Participants/materials, setting, methods In the AC-FET cohort-oral estradiol 6mg/day was started from day1/2 of the cycle. When endometrial thickness was>7mm,progesterone<1ng/ ml then intramuscular progesterone was started- 50mg/day-3 days,100mg on day4-the day of ET(Day-3 embryos) and continued every alternate day.Progesterone gel was added post-ET. In the mAC-FET cohort-17β transdermal estradiol gel(0.06%) 3mg/day was started from day3-5,rest of the protocol was similar. Statistical analysis was performed using appropriate non-parametric tests.Pearson coefficient(r) was used to assess linear-relationship.Two-tailed P<.05 indicated statistical significance. Main results and the role of chance Mean age and BMI of the patients with mAC-FET was 33.3±4.7 years and 24.4±3.7 kg/ m2 which was comparable across all groups (P=.14,P=.52 respectively). Clinical pregnancy rate in the mAC-FET group was 52.94%(18/34). 3 patients had an early abortion. Baseline relaxin-2 levels, median[Interquartile range(IQR)] were comparable- AC-FET cohort-9.1pg/mL(8.6-16.8), mAC-FET cohort-11.5pg/mL(9.6-13.7) and the infertility cohort-10.9pg/mL(8.4-14.5) (P=0.74) The primary outcome was the comparable levels of serum relaxin-2, in early pregnancy in the mAC-FET group and spontaneous conception group - median (IQR) 449.8pg/mL(73.1-492.3) and 487.9pg/mL(419.1-497.8) respectively (P=.12) The relaxin-2 levels, in early pregnancy in the mAC-FET group were significantly higher than the AC-FET group median(IQR) 449.8pg/mL(73.1-492.3) and 7.9pg/mL(7.8-9.5) respectively (P=.0005). As secondary outcomes, we studied the late luteal rise of relaxin-2 in the mAC-FET group and the relationship of late luteal progesterone with relaxin-2 in early pregnancies in the mAC-FET cohort. A late luteal rise of relaxin-2 was not demonstrated in the mAC-FET cohort. The serum relaxin-2, on the day of start of progesterone and on day5-9 post ET- median(IQR) 11.5pg/mL(9.6-13.7) and 11.4pg/mL(9.0-18.5), respectively was comparable (P=.18). The relationship between serum relaxin-2 in early pregnancy and the progesterone levels on day5-9 post ET in the mAC-FET cohort was not significant r = -.059 (P=.07) Limitations, reasons for caution This was a small proof- of-concept single centre study. Due to the limited numbers and short duration of the study, we could not compare efficacy, cancellation rates and pregnancy outcomes of the mAC-FET protocol with other FET protocols. Wider implications of the findings The mAC-FET protocol studied for endometrial preparation was similar to the AC -FET protocol regarding monitoring,flexibility and consistent progesterone exposure with the added advantage of the presence of the CL.This may decrease the risk of maternal/neonatal complications. Larger studies are needed to confirm it’s efficacy and impact on pregnancy outcomes. Trial registration number Not Applicable

P-449 Association of corpus luteum activity in early pregnancy and live birth after assisted reproductive technology (ART) and intrauterine insemination (IUI)

Abstract Study question What is the incidence of low corpus luteum activity in early pregnancy, and how are endogenous progesterone levels and live birth achievement associated? Summary answer Frequently occurring low corpus luteum activity (progesterone-level) in early pregnancy after ART and IUI strongly predicts pregnancy demise, though not independent of trophoblast activity (hCG-level). What is known already Ovarian stimulation and human chorion gonadotropine (hCG) administration may induce a luteal phase defect via negative feedback on the hypothalamic-pituitary-ovarian (HPO) axis. Luteal phase support (LPS) is routinely administered with bio-identical progesterone or oral dydrogesterone (DYD). DYD has high oral availability, minimal interaction with the pituitary gland, and no cross-reactivity in enzyme-linked immunosorbent assay (ELISA) with progesterone. Therefore, utilizing DYD as LPS in IVF/ICSI or ovulatory IUI cycles provides a unique perspective to assess endocrine luteal activity. While various studies explored progesterone and hCG post-IVF or IUI, none focused solely on the corpus luteum’s sole endocrine function and its outcomes. Study design, size, duration This single-center, retrospective study (with institutional review board approval) investigated hCG and progesterone serum levels after IVF/ICSI cycles or IUI cycles supported with DYD as the sole LPS. The study aims to assess their predictive value, individually and in combination, for sustaining pregnancy. Data from 189 IVF/ICSI and 198 IUI early pregnancies, respectively, with positive hCG test between January 2017 and June 2022, was extracted from the patient management system at the University Fertility Center. Participants/materials, setting, methods Included were only first IVF/ICSI and IUI treatment-cycles with serum hCG value ≥ limit of detection (0.6 IU/l) between days 9 and 16 after fresh embryo transfer or IUI, respectively. Additionally to hCG, endogenous progesterone was determined and the relationship between these and pregnancy maintenance was investigated using logistic regression analysis. Inclusion criteria encompassed hCG-triggered cycles using DYD as the sole LPS, administered at 30 and 20 mg daily for IVF/ICSI and IUI therapy, respectively. Main results and the role of chance The 10th, 25th, 50th and 75th quantiles of serum progesterone are at 0.28, 3.75, 33.0 and 98.7 µg/l in IVF/ICSI cycles and 0.48, 2.70, 18.40 and 26.6 µg/l in IUI cycles. Women in the first quartile of progesterone levels had nearly exclusively pregnancy demise: live birth rate 3/47 (7%, 95% confidence interval (CI) 1-19%) and 0/47 (0%, 95% CI 0-7.5%) in IVF/ICSI and IUI cycles, respectively. In both IVF/ICSI and IUI, progesterone and hCG were highly correlated (correlation coefficient 0.83 in both cases). Accordingly, the association of serum hCG levels with live birth achievement was similar in quartile analyses. Using receiver operating characteristic (ROC) curves and Youden’s indices, the optimal cut-off values for predicting live birth were determined as follows: 16.49 µg/l (89 % sensitivity (SN) and 59 % specificity (SP)) for IVF/ICSI and 15.89 µg/l (90 % SN and 70 % SP) for IUI therapy. Logarithmic regression analysis also revealed a significant correlation between progesterone levels and live birth in IVF/ICSI and IUI therapy, with a p-value of < 0.001. Limitations, reasons for caution Low progesterone in early pregnancy may be a consequence or a cause for pregnancy demise. DYD, like any other progesterone for LPS, might adversely impact corpus luteum formation and maintenance through HPO axis interaction, especially in mono-ovulatory cycles or in older women with inherently higher risk of luteal phase defect. Wider implications of the findings Ovarian stimulation and hCG-administration may induce a luteal phase defect with insufficient luteal activity in early pregnancy, even up to luteolysis, with potential consequences for pregnancy maintenance and maternal/fetal health. Future treatment protocols may require exploring an optimal luteotropic state at pregnancy’s outset, necessitating the establishment of relevant markers. Trial registration number not applicable

P-126 The impact of ICSI versus IVF on the reproductive outcomes of couples with non-male factor infertility and frozen-thawed embryo transfer cycles

Abstract Study question Does Intracytoplasmic Sperm Injection (ICSI) improve reproductive outcomes in patients with non-male factor infertility undergoing Frozen-Thawed Embryo Transfer (FET) treatment? Summary answer ICSI showed slightly improved clinical outcomes compared to IVF, but was associated with higher preterm birth rates and low birthweight when twins were born. What is known already The use of ICSI has seen a significant surge globally, particularly in Europe and the USA, as a solution for fertilization failure resulting from male factor infertility. FET presents a viable option for those considering additional children. Numerous studies have investigated the effectiveness of ICSI in couples with non-male factor infertility undergoing fresh embryo transfer in ovarian stimulation cycles. However, there is a dearth of evidence demonstrating whether ICSI, compared to conventional IVF (cIVF), could influence reproductive outcomes in couples with non-male factor infertility undergoing FET treatment. Study design, size, duration This retrospective cohort study included 10,143 cycles from 6,206 couples who underwent FET at the Third Affiliated Hospital of Zhengzhou University between January 2016 and September 2022. Participants/materials, setting, methods Patients were categorized into two groups based on the insemination methods used for the transferred embryos. Patients were excluded if : 1) Embryos were biopsied and tested; 2) Oocytes had been vitrified; 3) Embryos were derived from donor oocytes, AOA treatment, or rescue ICSI; 4) Embryos that were not transferred; and 5) They had undergone fresh embryo transfer. PSM at a ratio of 1:1 was utilized to ensure comparability of reproductive outcomes. Main results and the role of chance Post-PSM, the general characteristics were comparable across both groups. The clinical pregnancy rates (37.6% vs 40.0%, P = 0.311), miscarriage rates (21.4% vs 23.1%, P = 0.597), and live birth rates (28.2% vs 30.4%, P = 0.329) exhibited no significant disparity post-PSM between the two groups. Notably, a marginally elevated live birth rate was observed in the ICSI group when blastocysts were transferred to the women (39.8% vs 48.3%, P = 0.050). However, the GEE models indicated that ICSI did not enhance the likelihood of clinical pregnancy, miscarriage, and live birth relative to IVF in women with non-male factor infertility. Following adjustments for variables, ICSI yielded consistent OR values [1.14(0.92-1.42), P = 0.241; 1.08(0.72-1.61), P = 0.703; and 1.16(0.92-1.46), P = 0.220, respectively]. The perinatal outcomes were similar between the two grousps. However, a significantly higher incidence of preterm birth was observed in the ICSI group compared to the cIVF group (20.0% vs 12.5%, P = 0.026). While pregnancy complications were comparable between the two groups, neonates born as twins in the ICSI group exhibited significantly reduced live birth-weight [2520.8(572.9) vs 2691.9(423.7), P = 0.043] and birth length [47.5(2.8) vs 48.5(2.3), P = 0.031]. Correspondingly, the rate of low birthweight was significantly higher in the ICSI group (38.9% vs 21.0%, P = 0.025). Limitations, reasons for caution The retrospective design may lead to selection bias among the included patients. The use of ICSI in ART has been strictly regulated in our country, limiting the sample size and potentially reducing the statistical power. Wider implications of the findings The value of ICSI should be thoroughly discussed with patients with non-male factor infertility before ovarian stimulation. Further research is needed to better understand the impact of ICSI on reproductive and neonatal outcomes in this population. Trial registration number 2021YFC2700602

P-542 Bidirectional signaling between oocytes and ovarian support cells (OSCs) during in vitro maturation results in improved oocyte gene expression

Abstract Study question Are the gene expression profiles of ovarian support cells (OSCs) and cumulus-free oocytes bidirectionally influenced by co-culture during in vitro maturation (IVM)? Summary answer OSC gene expression is modulated by oocyte presence and OSC-IVM matured oocytes display transcriptomic resemblance to in vivo matured oocytes potentially due to OSC-oocyte crosstalk. What is known already The application of IVM holds a significant interest to improve infertility treatment. We previously showed the development of human OSCs generated from human induced pluripotent stem cells (hiPSCs) with ability to improve the maturation rates of immature human cumulus oocyte complexes (COCs) and embryo formation rates from minimal ovarian stimulation cycles (Piechota et al. 2023). We recently demonstrated that OSC-IVM can also be used to improve the nuclear maturation rate of human denuded oocytes from conventional stimulation cycles. Study design, size, duration We aimed to determine how gene expression changed in oocytes and OSCs by OSC-IVM co-culture. Immature fresh denuded oocytes (n = 141) were collected and randomized for IVM without OSCs (Media-IVM) (MediCult; n = 59) or with OSCs (OSC-IVM) (OSCs+MediCult; n = 82) for 24-28h. Subject enrollment ranged from 12/01/2023-06/30/2023. OSCs were collected for analysis pre-IVM (time 0h; n = 3), and post-IVM with (n = 3) and without (n = 3) immature denuded oocyte co-culture. Participants/materials, setting, methods 47 fertility patients undergoing conventional ovarian stimulation donated immature denuded oocytes (GV or MI oocytes) for research, which were allocated between either the Media-IVM or OSC-IVM conditions. In vivo matured oocytes (IVF-MII) were used as controls. Single oocyte RNA sequencing (RNA-seq) was performed after IVM culture, including both mature oocytes or stalled immature oocytes, alongside IVF-MII oocytes. Bulk RNA-seq was performed on OSCs and primary cumulus cells were used as a reference control (n = 7). Main results and the role of chance RNA-seq analysis demonstrated in the absence of oocytes, OSCs retain their global transcriptomic signature during IVM, maintaining or upregulating expression of CYP19A1, MDK, NR2F2, FOXL2, and CD82 and downregulating FSHR. When co-cultured with oocytes, OSCs shifted their global transcriptomic profile to resemble primary cumulus cells. In OSCs, we identified upregulation of IGF2, EGF, and IGFBP4, and downregulation of AMHR2, EGFR and IGFBP2 in the presence of oocytes, indicating oocyte-directed remodeling of OSC gene expression. Differential gene expression analysis in oocytes comparing MIIs and GVs post-IVM identified 31 genes differentially enriched and 353 genes differentially depleted in MIIs after co-culture with OSCs compared to 37 and 230 genes respectively without OSCs. IVF MII gene expression did not differ from OSC-IVM MII oocytes but did significantly differ from Media-IVM MII gene expression (p = 0.0008). Pathway enrichment analysis demonstrated broad similarity between IVF and OSC-IVM MIIs for Unfolded Protein Response, Myc Targets, and Oxidative Phosphorylation, while Media-IVM uniquely showed enrichment in DNA Damage and Spindle Assembly pathways. Media-IVM produced MIIs enriched uniquely for genes involved in electron transport chain (ETC) and oxidative phosphorylation (OXPHOS), which were not found in OSC-IVM or IVF MIIs. Limitations, reasons for caution The number of oocytes utilized in this study was limited. Additionally, as this study used rescue IVM as a model, it is likely that approaches using cumulus enclosed oocytes and traditional IVM may differ. Wider implications of the findings Bidirectional signaling between OSCs and oocytes may contribute to an improved in vitro environment that supports oocyte nuclear and cytoplasmic maturation. Therefore, oocytes matured in the presence of OSCs are more similar to oocytes matured in vivo and display a pathway enrichment profile associated with a higher oocyte developmental competence. Trial registration number not applicable

O-244 AMH levels in infertile women in APAC region - prospective study in multiple countries, ethnicities and centres

Abstract Secretary General, Asia Pacific Initiative on Reproductive (ASPIRE) President, Taiwan Endometriosis Society Founder & CEO, Taipei Fertility Center AMH is produced by granulosa cells of small growing follicles in the ovary and serum AMH levels strongly correlate with the number of growing follicles, AMH has been used clinically as a marker for ovarian reserve. Despite being of similar ages, with similar AMH levels, durations of ovarian stimulation, and gonadotropin doses, Asian women have been documented as having decreased AFCs and lower yields of total and mature oocytes after ovarian stimulation compared to their Caucasian counter- parts. These earlier data suggested that the effect of ethnicity was as an independent marker of ovarian reserve and potentially subsequent reproductive outcomes. We conducted the first multi-nation, multi-centre, and multi-ethnicity prospective cohort study from January 2020 to May 2021 to investigate serum AMH profiles among infertile Asian women. A total of 4613 women who visited fertility clinics at 12 IVF centres in 11 regions throughout Asia were recruited from Taiwan, China, Hong Kong SAR, India, Indonesia, Japan, Korea, Malaysia (2 centres), Thailand, Vietnam, and Singapore. The inclusion criteria were female partners aged 20–43years and naive to any ovarian stimulation cycle. The AMH measurement was performed using an AMH automated assay on a clinically validated platform. Among the 12 centres that joined the study, most demonstrated racial homogeneity except for Singapore and Malaysia which presented a highly diverse ethnic profile, while others were supposedly monoethnic communities. Unexplained or male infertility is the most common infertility cause in most countries, except for China with a notably high incidence of tubal disease. Age, ethnicity, obesity, and PCOS are significantly associated with ovarian reserve. The highest rates of PCOS were found in Indians (18.6%), Malays (18.9%), and Vietnamese (17.7%), yet remarkably lean with lower obesity rate was found in Vietnamese. Serum AMH levels are not affected by smoking or GnRHa and OCPs pretreatment but are affected by endometrioma surgery. Moreover, the assay can be conveniently conducted on any day of the menstrual cycle and is not influenced by the use of fresh or frozen samples. The rate of decline in ovarian reserve increases with age in Asian women, at 0.13ng/ml/year at 25–33years old and 0.31ng/ ml/year at 33–43 years old. We also found that a cut-off at 4.0 ng/ml could be considered for PCOS diagnosis in the Asian region. Despite limitations, the results can help guide clinicians for personalized infertility treatment, and call for further investigation into the effects of genetic, environmental and lifestyle factors on ovarian reserve.

P-608 Cumulative live birth rates after 3 consecutive embryo transfers in freeze-all cycles following gonadotropin-releasing hormone antagonist protocol versus progesterone-primed ovarian stimulation

Abstract Study question Do cumulative live birth rates in freeze-all cycles differ between gonadotropin-releasing hormone (GnRH) antagonist protocol and progesterone-primed ovarian stimulation (PPOS)? Summary answer GnRH antagonist and PPOS protocols have similar cumulative live birth rates in freeze-all cycles. What is known already GnRH antagonists and progesterone have been used for pituitary suppression in ovarian stimulation cycles. While PPOS protocol has the advantage of oral usage and lower cost, data regarding the laboratory and clinical outcome is limited and confounding. Freeze-all cycles might provide a better model to assess the impact of PPOS protocol and GnRH antagonist protocol on laboratory, clinical outcomes and cumulative live birth rates. Study design, size, duration A retrospective cohort study on IVF cycles performed (n = 568) between January 2020 and May 2022.The entire cohort of embryos were cryopreserved at the blastocyst stage.Inclusion criteria were 18-42 years of age,ICSI for severe male factor infertility.Exclusion criteria were PGD and severe endometriosis.Indications for cryopreservation in the antagonist group were high ovarian response (n:102),endometrial polyps (n:34),high follicular phase P4(n:48) and elective purposes(n:260).PPOS was used due to a transient shortage of GnRH antagonist in the market (n:124). Participants/materials, setting, methods 568 women (GnRH antagonist:444; PPOS:124) were included in the analysis.The primary outcome was the cumulative live birth rate (CLBR) after 3 consecutive embryo transfer cycle, and the secondary outcome parameters were the number of M-II oocytes, fertilization rate and blastocyst utilization rate. Student’s t-test was used for normally distributed continuous data and Fisher’s exact tests for categorical data were used. A generalized estimating equation model and logistic regression analysis were performed to adjust for confounding factors. Main results and the role of chance The mean age of the GnRH antagonist group was 34.1 ± 5.3 and 34.5 ± 5.7 years in PPOS group (p = 0.38). The baseline characteristics including BMI, serum AMH levels, duration of infertility, duration of stimulation and gonadotropin ampules used were similar in both groups. Estradiol and progesterone levels at the time of ovulation trigger were higher in GnRH antagonist cycles (2383.5 ± 1375.4 vs 2034.5 ± 1218.3, p = 0.02 and 0.7 ± 0.6 vs 0.5 ± 0.4, p = 0.01, respectively) The mean number of cumulus-oophorus complexes (12.9 ± 7.4 vs 11.5± 10.0, p = 0.61), M-II oocytes (10.0± 6.1 vs 9.1± 6.1, p = 0.14), frozen blastocysts (4.8± 3.5 vs 4.7± 3.6, p = 0.71) were similar between GnRH antagonist and PPOS groups, respectively. Fertilization rates (84.4% vs 87.2%, p = 0.12) and blastocyst utilization rates (63.4% vs 65.9%, p = 0.34) were also similar between GnRH antagonist and PPOS groups, respectively. The number of transferred embryos and the number of unused blastocysts were similar between groups. The CLBRs after one IVF cycle and 3 consecutive frozen-thawed blastocyst transfers were comparable in GnRH antagonist and PPOS cycles (56.8% and 53.2%, p = 0.37, respectively). Limitations, reasons for caution This was a retrospective study with relatively small sample size performed at a single fertility center, which may limit the generalizability of our findings. Wider implications of the findings If the fresh embryo transfer is not intended, PPOS protocol can be used in all patient types with cost-effective and patient-friendly manner. Trial registration number 2024.013.IRB2.012

P-365 Fertility preservation in patients with borderline ovarian tumors: safety and reproductive outcomes

Abstract Study question Do borderline ovarian tumors (BOT) patients, undergoing controlled ovarian stimulation (COS) face higher recurrence risks, and what are the associated reproductive outcomes? Summary answer Even in advanced-stage or bilateral BOT, COS does not significantly impact recurrence rates. Additionally, COS for fertility preservation(FP)provides a viable option for future reproductive potential. What is known already The primary approach for treating BOTs involves surgical intervention, emphasizing complete tumor removal. Conservative surgery is an option for young women seeking to preserve reproductive potential. However, the recurrence risk following conservative surgery is significant, reaching up to 25%, necessitating repeat surgeries that impact ovarian reserve. Existing evidence on the safety of COS in terms of BOT recurrence remains inconclusive. Concerns about a potential association between COS and tumor progression persist, particularly in advanced-stage and bilateral disease cases. This study address these gaps in knowledge and provide more clarity on the safety of COS in BOT patients. Study design, size, duration A retrospective cohort study was conducted across two tertiary, university-affiliated medical centres: IRCCS San Raffaele Hospital, Milan, Italy, and SHEBA Medical Hospital, Tel-Aviv University, Israel. The study included 165 patients diagnosed with borderline ovarian tumors who underwent conservative surgery between 2012 and 2023. Participants/materials, setting, methods Participants aged 18-42 years with BOT (stages I-III) and undergoing conservative treatment were stratified into two groups: those opting COS for FP(41 patients)and a control group without such intervention (124 patients). COS involved GnRH antagonist random-start protocols utilizing menotropins and/or recombinant FSH. GnRH-agonist or hCG were used for triggering. Oocyte vitrification or fertilization and embryo freezing was performed. Statistical analyses, including Pearson’s chi-square, Fisher’s exact test, T test, and logistic regression, were employed for evaluation. Main results and the role of chance In the course of COS cycles and oocyte retrieval, no complications were observed. Among 165 participants, 33% experienced BOT recurrence over a median of 28.5 months (range: 12.7 to 42.7). Patients with recurrence were younger (29 vs. 32 years, P = 0.04), had advanced-stage (26 vs. 18, p < 0.0001), and bilateral disease (38% vs. 19%, p = 0.01). Of the 41 undergoing COS, 19 experienced recurrence, comparable to the control group (36 patients, p = 0.055). Notably, all COS-related recurrences were BOT histotype, not invasive carcinoma, managed through a second surgery. No significant differences in ovarian stimulation cycles (2.4 vs 1.7, p = 0.7) or retrieved oocytes (11.8 vs 10.6, p = 0.6) were detected between women with and without recurrence. In multivariate analysis, advanced disease significantly increased recurrence odds (aOR=3.7, 95% CI: 1.7-7.9, P < 0.001), while COS did not (aOR=1.7, 95% CI: 0.7-3.9, P = 0.16). This suggests that while age and disease stage play roles, COS alone may not significantly influence recurrence rates. Importantly, among women undergoing COS for FP, 17 achieved at least one pregnancy, totaling 24 pregnancies, with 18 live births. Six were spontaneous, and three used frozen embryos from FP cycles. This underscores the reproductive success of FP through COS, providing hope for fulfilling childbearing desires post-BOT diagnosis. Limitations, reasons for caution Limitations include the retrospective nature, a selection bias in the COS group, and the rarity of the disease leading to a small sample size. Despite these, the recurrence rate in the COS group was comparable to a systematic review, and caution is advised in interpreting these results. Wider implications of the findings Our study suggests that COS does not significantly contribute to recurrence risk in BOT patients, even in advanced stages. FP through COS enhances the likelihood of live births in these patients. While cautious interpretation is warranted, these findings offer hope and call for larger prospective studies to confirm these results. Trial registration number not applicable