Oocyte Maturation Research Articles

P-609 Impact of low versus air oxygen tension on the oocyte maturation in PCOS women undergoing capacitation IVM: a sibling oocyte pilot study

Abstract Study question Does low or air oxygen tension improve the oocyte maturation rate in biphasic capacitation IVM (CAPA-IVM)? Summary answer Low oxygen tension resulted in a lower maturation rate and two-pronuclei fertilization rate compared with air oxygen tension in the CAPA-IVM culture. What is known already CAPA-IVM, with a pre-maturation step, improves the competence of oocytes matured in-vitro. The current CAPA-IVM culture condition is under air oxygen concentration. However, the impact of oxygen tension on in-vitro oocyte maturation needs to be better understood, and it remains uncertain whether the maturation process aligns with in-vivo conditions. Study design, size, duration A sibling oocyte pilot study of 416 cumulus-oocyte complexes (COCs) recruited from fifteen polycystic ovary syndrome (PCOS) women was performed at IVFMD, My Duc Phu Nhuan Hospital, Ho Chi Minh City, Vietnam from November 2023 to January 2024. The study was approved by the Institutional Ethics Committee (22/23/DD-BVMD, dated October 12th, 2023) and conducted according to Good Clinical Practice and Declaration of Helsinki 2002 principles. All participants provided written informed consent. Participants/materials, setting, methods Women (18–37 years) diagnosed with PCOS undergoing CAPA-IVM were recruited. At collection, COCs from each woman were divided into two groups based on the oxygen tension used in the two steps of CAPA-IVM culture, low oxygen tension (5% O2, 6% CO2 at 37 °C) and air oxygen tension (20% O2, 6% CO2 at 37 °C). Embryos were cultured to blastocyst stage. Primary outcome was the oocyte maturation rate. Secondary outcomes were embryology outcomes. Main results and the role of chance A total of 416 COCs from fifteen eligible women were assigned to the low oxygen (207 COCs) and air oxygen tension (209 COCs) groups. Mean age and body max index were 29.0±2.5 years and 22.6±2.6, respectively. The maturation rate was significantly lower in the low oxygen group compared to the air oxygen group (54.1% vs. 66.5%, p = 0.01, respectively). The two-pronuclei fertilization rate per COC was significantly decreased in the low oxygen group compared to the air oxygen group (35.7% vs. 49.3%, p = 0.005, respectively). Rates of day-3 embryos, blastocyst formation rate, and good quality blastocyst rate were also lower but not significant in the low oxygen tension group as compared to the air oxygen tension group. Limitations, reasons for caution The study’s limitations include a relatively small sample size and a single ethnicity group. There was a lack of data on clinical outcomes after transferring embryos from the study. Wider implications of the findings Oocyte maturation rate and two-pronuclei fertilization rate were significantly lower when oxygen tension was reduced in the process of CAPA-IVM culture. Therefore, air oxygen tension should be considered as a standard for oocyte culture in the pre-maturation step of CAPA-IVM. Trial registration number NCT06087354

O-007 Leveraging warning thresholds of oocyte immaturity rate to identify patients at risk of pathological endophenotypes. Analysis of 16,155 retrievals with implications for rescue-IVM

Abstract Study question How to develop a robust strategy to pinpoint women exhibiting potential oocyte maturity defects, making them candidates for in-depth genetic investigations and rescue-IVM interventions? Summary answer Oocyte immaturity rate warning-limit was 51%. Within three retrievals, 7.8%, 1.5%, and 0.3% of the patients might exceed it once, twice and three times, respectively. What is known already Oocyte maturity involves cytoplasmic and nuclear aspects. While cytoplasmic maturity is challenging to define and monitor in IVF, nuclear maturation is evaluated through germinal vesicle breakdown and first polar body extrusion. Consensus suggests 10-20% immaturity rates post-OS is acceptable, but some patients exceed. From a diagnostic standpoint, women surpassing these rates may be eligible for additional genetic assessments; from a clinical standpoint, instead, exploring rescue-IVM is promising. However, guidelines for both practices are currently lacking. Study design, size, duration Retrospective study including retrievals with ≥1 cumulus-oocyte-complex (COC; years:2008-2022). The weighted-mean immaturity-rate (20%) and the outliers for COCs retrieved (>22) were defined on the whole dataset (N = 16155). Confounders upon immaturity-rates were outlined among first retrievals with ≥5 COCs (N = 7963). Warning immaturity-rate limit was calculated as “weighted-average+2SD” among first retrievals with 5-22 COCs (N = 7523). Conservative and estimated prevalence of facing immaturity-rates higher than the warning limit across multiple retrievals were also calculated. Participants/materials, setting, methods All retrievals were conducted 35.5±0.5 hours after trigger and cumulus-cells were removed to assess oocyte maturity 3.7±1.2 hours later. Rescue-IVM was not conducted at our center, and immature oocytes were discarded. OS, time between pick-up and denudation, and maternal characteristics were tested as putative confounders through linear regressions. Main results and the role of chance Among all retrievals conducted between 2008 and 2022, the weighted-mean oocyte immaturity-rate was 20%, suggesting that normally ≥1 immature oocyte(s) might be obtained with ≥5 COCs. Use of agonist trigger versus hCG (unstandardized coefficient-B: -2.1%,95%CI from -2.8% to -1.4%,adjusted-p<0.001), length of OS (unstandardized coefficient-B:-0.6%,95%CI from -0.8% to -0.4%,adjusted-p<0.001), and ratio COCs:follicles >16 mm at ovulation induction (unstandardized coefficient-B:+4.6%, 95%CI from +3.8% to + 5.3%,adjusted-p<0.001) were associated with the immaturity-rates in a multivariate linear regression. Among first retrievals with ≥5 and ≤22 COCs, the immaturity-rates warning limit was defined as 51%. In 3.6% (N = 286) of the 7962 first retrievals with ≥5 COCs the immaturity-rates were ≥51%. The same prevalence for second and third retrievals were 3.8% (N = 86/2232) and 2.1% (N = 14/667). The conservative prevalence of patients with immaturity-rates ≥51% once, twice and three times among three consecutive retrievals were 4.5% (N = 361/7962), 0.3% (N = 23/7962), and 0.03% (N = 2/7962). The same rates, assuming all patients would conduct three consecutive retrievals, were 7.8% (N = +257), 1.5% ( = +93), and 0.3% (N = +21). Out of the 667 patients that indeed conducted 3 retrievals between 2008-2022, these rates were 7.6% (N = 51), 0.9% (N = 6) and 0.4% (N = 3), supporting the reliability of the predicted estimates. Limitations, reasons for caution Both germinal-vesicles and metaphase-I immature oocytes were not deemed clinically useful here, although this is controversial. Well-designed studies to systematically monitor rescued oocytes’ developmental, chromosomal, and reproductive competence are still needed, especially in patients subject to high immaturity-rates. Wider implications of the findings Oocyte immaturity-rates ≥51% with ≥5 COCs especially when consistently experienced across multiple retrievals, is a statistically-sound criterion to candidate patients to whole-exome-screening. This aims at outlining gene associations with defective oocyte maturation. The clinical effectiveness of rescue-IVM should be primarily studied in these women (prevalence ≤1%). Trial registration number None

O-033 Molecular insights into the decline in oocyte quality with advanced maternal age: findings from comprehensive single cell -omics analysis of 112 human oocytes

Abstract Study question How does maternal age impact the molecular composition of oocytes during their final meiotic progression? Summary answer Maternal age significantly correlates with proteomic changes during oocyte maturation, particularly in proteostasis and meiosis related proteins, with minimal impact on transcriptome and DNA-methylation patterns. What is known already Oocyte quality declines with maternal age, resulting in diminished developmental competence and higher aneuploidy rates. However, the molecular mechanisms behind this decline in oocyte quality remain elusive. Transcriptomic studies have revealed few differentially expressed genes in advanced maternal age (AMA) oocytes, suggesting that age-related changes in oocyte quality result from a complex interplay of molecular factors, rather than a single cause. However, comprehensive methylation and proteomic data are still lacking. We employed advanced -omics to assess the effect of maternal age on the molecular signature of human oocytes, focusing on DNA-methylation, transcriptome and proteome changes during the final meiotic progression. Study design, size, duration This study included a total of 112 oocytes obtained from young (<35 years, n = 35) and AMA women (>37 years, n = 55), who were recruited in the study from October 2021 to October 2023. Both germinal vesicle (GV, n = 68) and metaphase II (MII, n = 44) oocytes were analysed. Additionally, 19 immature oocytes (GV and metaphase I) from 9 young women were used for validation. Participants/materials, setting, methods Parallel single-cell bisulfite and RNA sequencing was applied to 44 oocytes (26 GV-Young, 6 MII-Young, 8 GV-AMA, 4 MII-AMA) and single-cell proteomics to 68 oocytes (18 GV-Young, 18 MII-Young, 18 GV-AMA, 14 MII-AMA). Additionally, 10 GV and 9 MI oocytes were treated with the proteasome inhibitor MG-312 (0 µM, 10 µM) for 6 hours, followed by rescue in vitro maturation (rIVM) for 36 hours. Chromosomal distribution was assessed by immunocytochemistry. Main results and the role of chance Our analysis revealed no significant changes in DNA methylation patterns in either GV nor MII oocytes associated with AMA. Also, very limited changes were detected in the transcriptome, with transcript levels of only 5 genes in GV oocytes and 7 genes in MII oocytes detected as being significantly changed. In contrast, proteomic analysis, particularly in GV oocytes, revealed significant age-related changes, notably in proteins participating in the proteostasis network (signalosome complex, UCHL1) including chaperones (TRiC-complex, HSP7C, STIP1), and in the cell cycle, including signal transduction factors (1433E, integrins) and cytoskeleton regulators (DYL2, CAPZB, ARHGG) (Rs ≤ |0.5|, p ≤ 0.05). The proteasome complex, which plays a crucial role both in meiosis and the proteostasis network, was found to decline with age; changes were evident in several subunits of the complex (e.g., PRS8, PRS6A, and PRS10; Rs ≤ -0.56, p ≤ 0.05). Compared to controls, treatment of immature oocytes with the proteasome inhibitor MG-132 resulted in either maturation failure or chromosome mislocalization in metaphase plate, further validating the essential role of the proteasome during oocyte maturation. In MII oocytes, 7 proteins showed alterations with age, including the oocyte-specific marker DDX4, which significantly declined in abundance (Rs =-0.6, p ≤ 0.05). Limitations, reasons for caution Unlike GVs that were collected fresh, MII oocytes underwent vitrification and warming before being included in the study due to clinical protocols. These procedures may have unknown effects on the transcriptome and proteome. Wider implications of the findings Our findings suggest that age primarily affects oocyte quality at the post-transcriptional level, potentially through meiosis dysregulation and proteostasis disruption. We also demonstrate the proteasome's vital role in oocyte maturation, suggesting that targeting the proteasome complex may improve oocyte quality in AMA women. Trial registration number Not applicable

O-077 Depletion of MFF from oocytes impairs mitochondrial dynamics, leading to inhibited oocyte maturation and early embryonic development in mice

Abstract Study question To investigate the effect of mitochondrial fission factor (MFF) on oocyte competence and female fertility using an oocyte-specific Mff knockout mouse model. Summary answer Oocyte-specific Mff deficiency hindered oocyte maturation and early embryonic development via regulation of mitochondrial dynamics, resulting in female subfertility. What is known already Emerging studies have demonstrated that mitochondrial dynamics play a vital role in oocyte maturation and early embryo development. MFF is one of the crucial proteins regulating mitochondrial dynamics. Our previous studies have indicated that Mff is necessary for oocyte competence and fertility using a global Mff knockout mouse model. However, further investigation is needed to determine whether the deletion of Mff in oocytes could affect oocyte competence and early embryonic development by regulating mitochondrial dynamics. Study design, size, duration The oocyte-specific Mff knockout (Mfffl/fl; Gdf9-Cre) mice were generated using the Cre-LoxP conditional knockout system. The 2-month-old female mice from each group (Mfffl/fl; Gdf9-Cre or Mfffl/fl, n = 6) were mated to assess the fecundity over 10 months. The maturation and mitochondrial dynamics of germinal vesicle (GV) oocytes were determined. The spindle and chromosome morphologies of ovulated oocytes were also analyzed. Additionally, the 2-cell embryos were collected and cultured in vitro to assess early embryonic development. Participants/materials, setting, methods Female mice from each group were mated with adult males to assess the fecundity. GV oocytes, ovulated oocytes, and 2-cell embryos were collected and cultured in vitro after superovulation, as indicated. Mitochondria, spindle, and chromosome were stained and then imaged using confocal microscopy. The microscopic morphology of mitochondria was assessed using electron microscopy. Main results and the role of chance The Mfffl/fl; Gdf9-Cre mice consistently exhibited reduced fertility with a significant decrease in litter size (3.00 vs. 5.42, p < 0.001), number of litters per female (4.33 vs. 7.50, p < 0.001), and number of pups per female (13.00 vs. 40.67, p < 0.001) compared to the Mfffl/fl mice. The numbers of GV oocytes (29.37 vs. 39.10, p < 0.05), ovulated oocytes (9.81 vs. 20.91, p < 0.001) and 2-cell embryos (3.50 vs. 10.17, p < 0.05) were significantly lower in the Mfffl/fl; Gdf9-Cre mice. The blastocyst (32.32% vs. 68.44%, p < 0.05) embryo development rate per 2-cell embryos was also significantly lower. In vitro maturation revealed that of Mfffl/fl; Gdf9-Cre GV oocytes had significantly lower rates of GV break-down (68.89% vs. 91.57%, p < 0.05) and polar body extrusion (42.76% vs. 81.87%, p < 0.05). The ratios of aberrant spindle (71.56% vs. 23.74%, p < 0.001) and misaligned chromosomes (71.82% vs. 23.14%, p < 0.01) in ovulated oocytes from Mfffl/fl; Gdf9-Cre mice were significantly increased. In terms of mitochondrial dynamics, the mitochondrial size (0.59 µm2 vs. 0.15µm2, p < 0.001) and the ratio (62.50% vs. 18.67%, p < 0.01) of abnormal mitochondrial distribution (unevenly aggregated) in Mfffl/fl; Gdf9-Cre oocytes significantly elevated. This study found that the Mff played a crucial role in oocyte maturation and early embryonic development. Limitations, reasons for caution Further research is needed to determine the downstream targets or pathways of Mff in oocytes. Wider implications of the findings These results shed light on the important role played by MFF in ensuring proper mitochondrial dynamics in oocytes and underscore the significance of this protein in female fertility. This study provides insights into potential therapeutic targets for infertility and related diseases in humans. Trial registration number not applicable

P-155 Reproductive Potential of Metaphase-I (M-I) Intra Cytoplasmic Sperm Injection (ICSI)

Abstract Study question To evaluate the reproductive potential of performing ICSI with metaphase-I (M-I) oocytes derived from In Vitro Fertilization (IVF)-ICSI cycles Summary answer M-I ICSI oocytes can fertilize and develop into blastocysts, but with low rates and limited efficiency in producing more embryos, highlighting challenges in reproductive outcomes. What is known already ICSI for initially immature oocytes undergoing in vitro maturation in IVF cycles is a topic of controversy with varying practices. While it has the potential to increase usable embryos, it may be time-intensive and yield uncertain results. Assessing the maturation status of M-I oocytes multiple times on collection day is challenging. If viable embryos can be obtained after ICSI with M-I oocytes, it simplifies workload management in busy embryology labs for patients with a limited number of mature oocytes. Despite a few reported cases of healthy live births resulting from ICSI with M-I oocytes, their reproductive potential remains inadequately explored. Study design, size, duration The study included IVF cycles (n = 929) with both immature and mature oocytes from Jan, 2022-Dec, 2023. Fertilization and blastocyst were assessed based on oocyte maturity. Participants/materials, setting, methods The study included IVF-ICSI cycles obtaining at least one immature (Germinal vesicle (GV), M-I) and one mature (M-II) oocyte at oocyte denudation. Analyzing 9,793 inseminated oocytes from 929 women (mean age: 34.0), we divided them into groups: M-II (Group 1: n = 7678), M-I at ICSI (Group 2; n = 835), M-IàM-II at ICSI (Group 3; n = 643), M-IàM-II on day 1 (Group 4; n = 129), and GVàM-II on day 1 (Group 5: n = 508). Fertilization and blastocyst development were then examined. Main results and the role of chance Overall, embryos originating from initially immature oocytes exhibited lower cleavage and blastulation rates compared to those from initially mature oocytes (P < 0.05). In Group 2, where ICSI was performed with M-I oocytes, fertilization and blastocyst rates were 23.6% and 16.1%, respectively. However, these rates were significantly lower than in other groups (P < 0.05). In Group 3, where ICSI was performed with M-II oocytes matured from M-I within 2 hours after oocyte denudation, the fertilization rate per oocyte (55.2%) was lower than in Group 4 (75.7%) (P < 0.05). However, the blastocyst rate per 2PN embryo was higher (30.7%) in Group 3 than that of Group 4 (18.0%) (P < 0.05). The blastocyst rate from the inseminated oocytes was directly associated with oocyte maturation: 29.3% (2250/7678) for in vivo M-II, 14.9% (96/643) for Group 3, 11.6% (15/129) for Group 4, 8.9% (45/508) for Group 5, and 2.9% (24/835) for M-I (Group 2). There were few pregnancies resulting from fresh transfers with only embryos produced from day 1 M-II oocytes (3/18=16.7%). While there were pregnancies after transferring only embryos generated from M-II matured from M-I on day 0 (4/10=40%), no patients transferred only embryos produced from M-I oocytes. Limitations, reasons for caution Despite the study possessing a substantial sample size, limited availability of embryos for fresh transfers arises from the deprioritization of those originating from immature oocytes. Consequently, it is crucial to conduct further research involving frozen embryo transfers and chromosome analysis to determine the efficacy of M-I ICSI. Wider implications of the findings Immature M-I oocytes exhibit a blastocyst formation rate of less than 3%. Consequently, these immature M-I oocytes demonstrate very limited developmental potential in progressing to the blastocyst stage, and ICSI for the M-I stage should be re-evaluated. Trial registration number Bnoon20240101

P-580 Benzophenone interrupted maturation of oocytes in vitro by promoting mitochondrial activity and inflammatory response in mice

Abstract Study question As an endocrine disrupting chemical, does benzophenone affect the competency of oocytes? Summary answer Benzophenone interfered with the in vitro maturation of oocytes and increased the number of mitochondria in oocytes by inducing of inflammatory response. What is known already Benzophenone (BP) is the organic compound used in ultraviolet (UV) filters due to its ability to absorb UV radiations and the applications of BP have a wide range in commercial/industrial products such as sunscreens, shampoos, lipsticks, food packaging, and plastics. However, BP is suspected to be an endocrine disrupting chemical (EDC) that can cause adverse effects on the endocrine system by mimicking or blocking the response of hormones in the body. Study design, size, duration Intraperitoneal injection of pregnant mare serum gonadotropin was performed at 7-week-old female ICR mice for acquire the number of available cumulus-oocyte-complexes (COCs). After 46 hours post injection, COCs were harvested from antrum follicles in the ovary and cultured in in-vitro maturation medium for 16 hours. After that, oocytes were retrieved. Participants/materials, setting, methods During in vitro maturation, BP was treated at COCs and Metaphase II (MII) oocytes were separated from retrieved oocytes. Intracellular tubulin of MII oocytes was stained for spindle alignment and their mitochondria were quantified by Mito-tracker and quantitative PCR (qPCR). Also, the expression levels of inflammation-related genes were confirmed by reverse transcription-qPCR. Main results and the role of chance In the present study, MII rates (MII/total oocyte) of each group were 77.50% of control, 79.17% of 1 μM BP, 68.10% of 10 μM BP, and 59.35% of 100 μM BP. Compared with the control group, the MII rate were significantly reduced at 100 μM of BP-treated group. We evaluated the morphology of meiotic spindle in MII oocytes with well-organized tubulin fibers and tightly aligned chromosomes. Abnormal morphology of spindle and chromosomal misalignment were found in the oocytes from BP-treated group. Because it has been known that having the optimal number of functional mitochondria is important for the maturation of oocytes, mitochondrial DNA (mtDNA) copy number and the distribution of mitochondria were confirmed in retrieved MII oocytes. There was a significant 4.2-fold increase in the number of mtDNA copies in 100 μM BP treatment group compared to the control group. Also, the number of activated mitochondria was raised when BP was treated, which was assessed using Mitotracker. Additionally, levels of inflammation-related genes (IL-1β, IL-6) were elevated in BP treatment groups. Limitations, reasons for caution Oocytes used in this study were collected from mice, not human. And since in vitro fertilization did not perform by using the MII oocytes, we couldn’t confirm whether BP-treated MII oocytes were capable of fertilization. Wider implications of the findings As industries evolve, novel chemicals, including EDCs, are being developed, used, and exposed to humans. Therefore, we need to understand the effects of these chemicals and assess their reproductive toxicity. Collectively, we suggest that BP causes an inflammatory response which activate mitochondria and has detrimental effects on the oocyte’s maturation. Trial registration number not applicable

P-795 Robust and simple in vitro maturation of ovarian tissue oocytes

Abstract Study question Using in-vitro gametogenesis results in mice, can we perform simple, robust in-vitro maturation (IVM) of oocytes from human ovarian tissue? Summary answer In vitro oogenesis in mice reveals 3 phases of oocyte maturation, IVD, IVG, and IVM. In vivo IVD and IVG simplifes a more robust IVM. What is known already For fertility preservation in cancer patients, IVM of GV’s obtained from ovary tissue with live birth has been achieved, but the results are variable, and great effort has gone into trying to make the “right” culture media, and most are given booster shots of gonadotropin with questionable results. It is easy to obtain many GV oocytes from tiny follicles using open cortical dissection. But it still has remained a puzzle how to do robust IVM in the best way. Study design, size, duration A total of 146 females underwent ovary tissue cryopreservation since 1977. The most recent 38 consecutive such patients between 2 and 38 years of age underwent in-vitro maturation of oocytes (IVM) derived from their ovarian tissue. This is a prospective non-randomized pilot study based upon our results with in vitro oogenesis. Participants/materials, setting, methods After ovarian cortex dissection, the spent media is examined for cumulous complexes (CC’s), which were placed in culture with widely varying concentrations of FSH and HCG, and in a variety of different media. Based on now established mechanisms of in vitro oogenesis, we postulated that the specific media for IVM and the concentrations of HCG would not impact results. There was no prior hormone administration. Main results and the role of chance The harvested germinal vesicle oocyte developed into mature metaphase II oocytes with simple culture media just as well as with “IVM media”, and with HCG concentration from 10 mIU/l to 1000 mIU/l. Of the 38 cases, 18 had had previous chemotherapy or were pre-pubertal. None of these had CC’s. Of the 20 who were post-pubertal and had not yet undergone chemotherapy, the number of CC’s averaged 20 per patient. Of a total of 402 GV oocytes, 137 became mature MII’s (34.1%), varying between 20% and 56%. The only case with low maturation rate (9%) occurred when the cumulus was stripped immediately instead of at 24 hours. Thus mature oocytes can robustly and easily be obtained from ovary tissue with simple media and no need for ovarian stimulation. Furthermore, ovarian stimulation and ovulation are not necessary for oocyte maturation. Oocytes that have just exited IVD that have been exposed to less than 7 days of gonadotropin (IVG) will not mature. Those oocytes exposed to more than 11 days of IVG will degenerate. Thus about 35% of oocytes at any given time will have already been properly prepared in vivo for IVM. Limitations, reasons for caution This is an early proof of concept study in humans with only 30 participants. However, it is completely consistent with what we have predicted from in vitro oogenesis in the mouse. Wider implications of the findings Ovary tissue freezing along with robust IVM will at once provide cancer patients with two possibilities for fertility preservation, without having to delay cancer treatment. Four months of in vivo IVD (non-hormonal) followed by 11 days of IVG (FSH) have already prepared oocytes for IVM. Trial registration number not applicable

P-251 Denudation time is crucial to increase the proportion of top-quality embryos in egg donation cycles

Abstract Study question How do different denudation times affect in the efficiency of the egg donation cycles? Summary answer Undetected in vitro matured oocytes, resulting from delayed denudations, contribute to a decreased top-quality blastocyst ratio. What is known already While the timeframe for sperm microinjection in ICSI cycles is widely agreed upon (2-6 hours post-oocyte retrieval), no definitive denudation time preference has been established. Published studies diverge on the advantages of early or late denudation, assessing the impact of trigger-to-denudation time (T-D time) on oocyte maturation. Advocates of early denudation contend it prevents oocyte aging, reducing in vitro culture and enhancing outcomes. These studies, often using data from cycles with own oocytes, exhibit high variability and inconsistent conclusions. The present work introduces an alternative hypothesis on the benefits of early denudation, employing a more stable study model. Study design, size, duration This retrospective study encompasses oocyte donation cycles, employing a short antagonist protocol and ovum pick-up 35.5 hours post-trigger, conducted from January 2019 to July 2023 including 1,559 stimulations. Additionally, 1,275 ICSI cycles without detected sperm pathologies derived from these stimulations were examined to evaluate the influence of T-D time on the reproductive outcomes. Six comparable groups were established by segmenting the sample according to T-D time, annotated in 30-minute intervals (36.5h to 39h). Participants/materials, setting, methods Rates of MII, MI, and nonviable oocytes were examined among 1,559 stimulations resulting in 29,671 oocytes (36.5h:187-stimulations/3,178-oocytes; 37h:226/3,836; 37.5h:614/11,921; 38h:329/6,884; 38.5h:119/2,322; 39h:84/1,530). Additionally, in 1,275 ICSI cycles involving 11,333 MII-oocytes (36.5h:117-ICSI cycles/1,027-MII; 37h:130/1,133; 37.5h:502/4,426; 38h:316/2,925; 38.5h:120/1,041; 39h:90/781), comparisons were made for fertilization, abnormal fertilization, ICSI degeneration, useful day 5/6 blastocyst (transferred or cryopreserved; >3BB-top and ≤3BB-good quality by Gardner score), pregnancy, and live birth rates. Statistics were ANOVA, Chi-square test and Pearson’s correlation. Main results and the role of chance Oocyte maturation profile assessment reveals that groups with later denudation exhibit a higher proportion of MII-oocytes (36.5h:80.71%; 37h:81.04%; 37.5h:81.26%; 38h:80.83%; 38.5h:84.84%; 39h:85.29%; r:0.8475; p < 0.05), reaching significance from 38.5h onward (p < 0.05). This is complemented by a strong negative correlation between T-D time and MI rate (36.5h:5.51%; 37h:5.32%; 37.5h:4.64%; 38h:4.17%; 38.5%h:3.27%; 39h:2.94%; r:-0.9881; p < 0.05), with significant declines at 38h and 38.5h (p < 0.05). No correlation between useless oocytes proportion and T-D time was observed (r:-0.5983; p = 0.21). This illustrates that certain MI-collected oocytes progressively mature during in vitro culture and subsequently, upon denudation, are classified as MII. This overestimates in vivo-matured oocytes. No differences attributed to T-D time were observed regarding fertilization, abnormal fertilization, or ICSI degeneration rates. While time does not affect the rate of useful embryos achieved (36.5h:49.4%; 37h:46.4%; 37.5h:44.6%; 38h:48.0%; 38.5h:50.6%; 39h:47.2%; r:0.1342; p = 0.80), it has an impact on the blastocysts quality. The proportion of top-quality blastocysts (>BB) progressively decreases with T-D time (36.5h:86.2%; 37h:85.2%; 37.5h:85.4%; 38h:81.2%; 38,5h:80.5%; 39h:80.5%; r:-0.925; p < 0.05). Out of 1,794 single embryo transfers, 1,477 have conclusive outcome. No implication of T-D time was detected in pregnancy rate (36.5h:59.88%; 37h:64.07%; 37.5h:61.54%; 38h:65.02%; 38.5h:59.55%; 39h:62.26%; r:0.0489; p = 0.93) or live birth rate (36.5h:40.31%; 37h:51.85%; 37.5h:39.12%; 38h:45.04%; 38.5h:41.83%; 39h:37.96%; r:-0.375; p = 0.46). Limitations, reasons for caution This study has inherent limitations as a retrospective analysis. Additionally, the potential effects of a denudation earlier than 36.5 hours post-trigger, not yet standardized in the laboratory, were not assessed. Wider implications of the findings Delayed denudation may benefit autologous IVF by fostering in vitro oocyte maturation. In egg donation cycles, early denudation is preferable for stringent selection of in vivo matured oocytes, yielding superior results. Additionally, shortening or eliminating incubation periods would enhance the automated sequence of diverse processes during an IVF cycle. Trial registration number not applicable

P-272 Cannabis negatively impacts female fertility: an in vitro and clinical investigation

Abstract Study question How does tetrahydrocannabinol (THC) impacts human oocyte maturation and female fertility? Summary answer THC alters the oocyte’s RNA profile and affects meiotic spindle formation resulting in an increased rate of aneuploidy in the oocyte and in the blastocyst. What is known already In the past decades, cannabis consumption and legalization has been increasing globally. It is the third most used substance by reproductive aged people and our group demonstrated that tetrahydrocannabinol (THC) reaches the ovarian follicle. THC is known to impact male fertility, but due to the challenges associated with investigating the female gamete in humans, there is no study about the human oocyte. Animal studies have shown that THC impacts oocyte maturation in a concentration dependent manner, but between models, the results are conflicting. Study design, size, duration For the in vitro exposure assessment, GV oocytes naïve to THC were collected from consenting patients undergoing IVF-ICSI from July 2022 to June 2024 (IRB: Veritas #16518). Per patient, oocytes were split into three groups: Control, THC1 (physiological concentration of THC, based on previous human studies) and THC2 (supra-physiological concentration, based on animal studies), and cultured for 24h in our standard clinical in vitro maturation media. Participants/materials, setting, methods Single-cell RNAseq and pathway analysis were conducted to identify key pathways altered by THC treatments (n = 86 from 24 patients). Spindle morphology was assessed using confocal microscopy. Polar body biopsy was performed and ploidy assessed using our clinical low pass whole genome sequencing pipeline. Also, we conducted a multi-year retrospective study (n = 1059 patients) by measuring the concentration of THC in their follicular fluid by mass spectrometry and collecting and comparing IVF outcomes between cases and controls. Main results and the role of chance In vitro, THC treatment marginally increased the maturation rate (46% for Ctrl (n = 103), 51% for THC1 (n = 103) and 58% for THC2 (n = 102)). Using time-lapse imaging, no difference was observed between key developmental stages of oocyte maturation including GVBD and polar body extrusion. Single-cell RNAseq revealed significant alterations to the transcriptome profile of THC treated oocytes. When treated with THC1 (physiological concentration), 89 upregulated and 227 downregulated genes were identified, this was exaggerated when treated with THC2 (supraphysiologic concentration) 402 upregulated and 62 downregulated genes were identified. Pathway analysis showed that differentially expressed genes in THC1 group were associated with immune system and synaptic transmission, while THC2 group showed genes enriched in meiotic spindle formation and apoptosis. Moreover, preliminary results demonstrated abnormal spindle configuration and misaligned chromosomes in THC-treated groups and a concurrent decrease in the number of euploid oocytes. Furthermore, retrospective analysis of clinical IVF outcomes revealed that patients whose FF was positive for THC (62 patients, 6%) also had a slight increased maturation rate (77% vs 73%, p=ns), but a significant reduction of their embryo euploid rate when compared to control patients (59% vs 70%, p < 0.01). Limitations, reasons for caution The in vitro exposure to THC utilized GV oocytes that did not mature after ovarian stimulation, so they might have a reduced capacity of becoming MII. The THC measurement was assessed only on the day of retrieval and we do not know the route, dose, or duration of THC consumption. Wider implications of the findings This is the first study investigating the impact of cannabis on human oocytes. Our data suggests that cannabis consumption may reduce the number of euploid embryos available for transfer. With cannabis legalization increasing worldwide, further investigation into its consequences is critical for clinical consultation and legalization guidelines. Trial registration number Not applicable

P-207 Analysis of oocyte morphology: differences between rescued and non-rescued metaphase II oocytes after conventional ovarian hyperstimulation

Abstract Study question Are there morphological differences between rescued and non-rescued metaphase II oocytes after conventional ovarian hyperstimulation (COH)? Summary answer Rescued metaphase II oocytes have a smaller diameter and a higher zona pellucida/perivitelline space ratio than non-rescued metaphase II oocytes. What is known already The rescue of germinal vesicles and metaphase I in assisted reproduction clinics has increased in recent years, but it is still not widely used because these oocytes have a lower developmental potential compared to oocytes matured in the follicular environment. Recent studies focus on the different strategies for the oocyte maturation process in the laboratory, one of them showed that rescued metaphase II oocytes have a smaller diameter than oocytes matured within the follicular environment. To our knowledge, this is the first study to analyze in detail the morphological differences between in vivo and in vitro matured oocytes. Study design, size, duration This retrospective study includes the analysis of 144 non-rescued metaphase II oocytes images and 181 rescued metaphase II oocytes images obtained after decumulation, or decumulation and in vitro maturation. Images were acquired over four months using Embryoscope TimeLapse systems. Morphological parameters were manually annotated by one single operator, to reduce the discrepancy of measurements, using the drawing tools of the EmbryoViewer software: oocyte area (μm2), polar body area (PBA)(μm2), zona pellucida (μm), perivitelline space (PVS)(μm). Participants/materials, setting, methods First, the different variables measured were analyzed to see if they were related to each other. Then, measurements obtained from images of non-rescued metaphase II oocytes were compared with those of the rescued. In addition, relationships between the different morphological parameters (oocyte area/PBA ratio, oocyte area/zona pellucida ratio, oocyte area/PVS ratio, zona pellucida/PVS ratio, PBA/PVS ratio and PBA/zona pellucida ratio) were calculated and their values were compared between oocytes from different maturation origins. Main results and the role of chance In both rescued and non-rescued metaphase II oocytes, a significantly positive correlation was detected between oocyte area and PBA*, and zona pellucida thickness*. It was also found a significant negative correlation between PVS size and PBA*. For rescued oocytes, a significantly negative correlation was also found between oocyte area and PVS*. Comparison of the means of age, BMI, AMH and all the morphological parameters analyzed between the two groups showed significant differences in age*, oocyte área*, PVS*, oocyte area-zona pellucida ratio*, oocyte area -PVS ratio*, zona pellucida -PVS ratio*, PBA -PVS ratio *. The average of these variables in the rescued oocytes group were: 28.77 ± 6.25, 9734.70 ± 681.28, 8.79 ± 4.43, 628.10 ± 116.29, 1391.86 ± 689.13, 2.24 ± 1.11, 50.84 ± 40.81; while their mean in the non-rescued oocytes group were: 24.41 ± 4.15, 10152.63 ± 711.3, 11.73 ± 5.62, 675,08 ± 111.48, 1063.32 ± 554.35, 1.60 ± 0.82, 37.65 ± 24.55. In addition, multivariable logistic regression showed that the differences in oocyte area size and zona pellucida/PVS ratio remained statistically different between the two groups despite the difference in patient age*. (p<.05*) Limitations, reasons for caution This study is limited by its descriptive and single-center nature. A multicenter study that also evaluates the developmental potential of oocytes would be needed. Wider implications of the findings Our study shows morphological differences between rescued and non-rescued metaphase II oocytes. It could be studied whether these differences are associated with the lower developmental competence of rescued metaphase II oocytes that has been observed in previous studies. Trial registration number Not applicable

P-264 Analysis of embryo development based on time of pronuclei appearance (tPNa) and aspects of overcoming delayed pronuclei appearance through morphokinetic patterns

Abstract Study question How much delay in the appearance of 2PN can be considered normal when predicting embryo transfer potential? Summary answer Embryos with tPNa <13hpi post-insemination develop into good-quality blastocysts; embryos with tPNa ≥13hpi develop blastocysts by shortening the time of tPNf-tPNa. What is known already Oocytes generally exhibit 2PNs at 17–20 hours, with pronuclei fading (time of pronuclei fading; tPNf) at 23–25 hours and developing into two cells (time of 2-cell division; t2) at 25–33 hours. Fertilization confirmation is typically performed 17–18 hours after insemination. However, a few oocytes with no visible PN (0PN) at the time of fertilization confirmation develop morphologically normal blastocysts, ultimately leading to pregnancy. The failure to identify PNs can be attributed to two scenarios: rapid fading or delayed appearance. Notably, there is still a lack of research on the normal range for delayed PN appearance. Study design, size, duration This study was conducted with 2153 embryos obtained from 390 intracytoplasmic sperm injection cycles (August 2021 to June 2023). All embryos were incubated for 5 days using a time-lapse system (EmbryoScopeTM+, Vitrolife). The blastocyst development rate and morphokinetic parameters according to the tPNa of embryos were analyzed using KIDScore D5 v3 and iDAScore v2.0 (VTH server+, Vitrolife). Clinical pregnancy was also analyzed. Participants/materials, setting, methods Morphokinetic parameters were analyzed from time of second polar body (tPB2) ∼ time of expanded blastocyst (tEB). The times taken for PN to appear after the second polar body release (tPNa–tPB2), for PN to fade (tPNf–tPNa) and for 2-cell division (t2–tPNf) were calculated and compared. Blastocysts were graded using the Gardner system, a grade of BB or higher divided into good quality blastocysts (GQ-BL). Main results and the role of chance tPNa was observed as 8.09±2.11hpi [1.93hpi∼32.46hpi; <5hpi (n = 49), 5∼6hpi (n = 474), 7∼8hpi (n = 1220), 9∼10hpi (n = 266), 11∼12hpi (n = 91), 13∼14hpi (n = 29), 15∼20hpi (n = 18) and >20hpi (n = 6)]. The rate of blastocysts was highest at 5∼6hpi (64.98%) and significantly lower at 9∼10hpi (54.14%), 11∼12hpi (42.86%) and 13∼14hpi (31.03%) (p < 0.005). Similarly, the rate of GQ-BL was also highest at 5∼6hpi (29.11%) and significantly lower at 9∼10hpi (19.17%), 11∼12hpi (10.99%) and 13∼14hpi (3.45%) (p < 0.005). No embryos developed into GQ-BL at 15∼20hpi, and no embryos developed into blastocysts at > 20hpi. The iDAScore was significantly different at < 13hpi and ≥13hpi (6.00±1.86 vs. 4.24±2.26, p < 0.005). Similarly, KIDScore D5 showed the same patterns (6.33±1.93 vs. 4.05±1.66, p < 0.005). At ≥ 13hpi, no blastocysts led to pregnancy. Morphokinetic parameters were analyzed to identify the factors influencing the development of blastocysts. The analysis revealed that tPNf-tPNa tended to gradually become shorter with delayed tPNa. Notably, there were significant differences in tPNf-tPNa between <13hpi and ≥13hpi (16.37±4.10 vs. 13.72±6.28, p < 0.005). At ≥ 13hpi, tPNf-tPNa was shorter in blastocysts than in cases of cleavage arrest (10.68±5.74 vs. 15.28±5.97, p < 0.05). It was particularly observed that tPNf-tPNa of GQ-BL in ≥ 13hpi was 7.90hpi, and t2 was not significantly different from blastocysts and GQ-BL in < 13hpi (25.06 vs. 25.66±3.18 vs. 25.19±2.97). Limitations, reasons for caution More data are needed for conclusive pregnancy results due to the small number of samples. Additionally, the maturation of oocytes had yet to be considered; further detailed studies related to oocyte maturity are needed. Wider implications of the findings Embryos with a tPNa of < 13hpi develop into GQ-BL. Embryos with delayed tPNa tend to overcome this by shortening the tPNf-tPNa, resulting in develop into blastocysts. Predicting blastocyst development can be achieved by considering factors such as tPNa∼t2. This can aid in improving the selection of embryos for cleavage-stage ET. Trial registration number N/A

P-598 Novel inactivating homozygous mutation of the luteinizing hormone/choriogonadotropin receptor (LHCGR) and first live birth after in vitro maturation (IVM): a breakthrough

Abstract Study question How can pregnancy and live birth be achieved in patients with inactivating homozygous luteinizing hormone/choriogonadotropin receptor (LHCGR) mutation? Summary answer We report the first live birth after IVM in a patient with a novel homozygous inactivating LHCGR mutation (exon 6,c.470A>G). What is known already LH plays a fundamental role in female reproductive physiology and is responsible for steroidogenesis, oocyte maturation, ovulation and subsequent progesterone production by the corpus luteum. LH binds to the LHCGR located on the membrane of theca cells and mature granulosa cells. Inactivating mutations of the LHCG receptor lead to the impossibility to obtain final oocyte maturation both during natural cycles and after ovarian stimulation for in vitro fertilization purpose. Until now, egg donation represented the only option to treat their infertility. Study design, size, duration A case report. Participants/materials, setting, methods A 35 year-old nulliparous patient was referred to our university hospital for primary infertility. Main results and the role of chance The 35-year-old nulliparous patient presented with primary spaniomenorrhea but timely and spontaneous onset of secondary sexual characteristics. Serum LH levels were high (ranging from 15 to 30 IU/L) and to a lesser extent so were FSH levels. The ovarian reserve was normal for age, as assessed by serum AMH levels and ultrasound. There was no argument for polycystic ovarian syndrome, 21-hydroxylase deficiency, Cushing’s syndrome or hyperprolactinemia. Two previous attempts of controlled ovarian stimulation with gonadotropins and ovulation trigger with hCG and GnRH-agonist trigger had failed, resulting in low estradiol levels despite correct follicular growth on ultrasound and absence of ovulation after trigger. We identified by genetic analysis a novel homozygous inactivating LHCGR mutation (exon 6, c.470A>G) that had never been described previously. IVM was performed. A total of 7 oocytes were obtained after IVM, resulting in 4 Day 3 embryos. All embryos were frozen. Subsequently, 2 Day 3 embryos were replaced after endometrial preparation by hormone replacement therapy. The patient became pregnant and gave birth to a healthy baby. Two Day 3 embryos remain. Limitations, reasons for caution By definition, a case-report requires further studies to confirm our findings. Wider implications of the findings We describe a novel inactivating LHCGR mutation with subsequent live birth after IVM. As a growing number of gonadotropin receptor mutations are identified, this live birth successfully obtained places IVM as the only reliable option for these patients to conceive with their own eggs. Trial registration number not applicable

P-699 High levels of total FSH during COS is detrimental to oocyte development in good responders: results from a three-year retrospective, multicenter study

Abstract Study question Are elevated doses of follicle stimulating hormone (FSH) detrimental to oocyte maturation and the efficacy of ovarian stimulation in good responders? Summary answer Elevated FSH levels reduce the number of oocytes collected from good responders. Patients receiving lower total FSH doses had optimal and higher maturation rates. What is known already There is an inverse relationship between the dose of exogeneous follicle stimulating hormone (FSH) and the number of oocytes retrieved. This relationship is independent of patient age or health and implies that excessive FSH doses administered during artificial reproductive technologies (ART) may be detrimental to oocyte maturation. Study design, size, duration This three-year retrospective, multicenter study included 1,075 oocyte donor cycles performed in three IVF clinics between January 2020 and December 2023 with at least a six-month interval between each cycle. Donors were all considered good responders and divided according to the total dose of FSH received per cycle (n = 289 cycles with <1,700 IU FSH; 400 cycles with 1,700 to 2,900 IU FSH; and 386 cycles with >2,900 IU FSH). Participants/materials, setting, methods The mean oocyte donor age was between 26.13 and 27.45 years old. The mean BMI for all three groups was 21. The association between the total FSH dose and serum anti-Müllerian hormone (AMH) level, follicle-to-oocyte index (FOI), number of oocytes retrieved, and oocyte maturation was assessed using the Student’s t-test (parametric) or Mann-Whitney-U test (non-parametric). Main results and the role of chance There were no significant differences in the mean serum AMH levels (5.31 vs. 5.27 vs. 5.13 ng/mL, respectively) and FOI (81.1 vs. 80.2 vs. 81.5, respectively) of patients who received different total doses of FSH. However, the number of oocytes retrieved from each group was inversely related to the total dose of FSH received (24.4 vs. 22.9 vs. 21.01 oocytes, respectively; p < 0.05) Limitations, reasons for caution Full characterization of the oocytes and their developmental potential was outside the scope of this retrospective study. Wider implications of the findings Gaining a better understanding of the complex interplay of gonadotropins throughout folliculogenesis can help optimize ovarian stimulation protocols to maximize the number of mature and developmentally competent oocytes recovered for clinical use. Trial registration number non applicable

O-190 Hormone-free versus FSH-primed infertility treatment of women with polycystic ovary syndrome using biphasic in vitro maturation: a randomised clinical trial

Abstract Study question Is any hormone stimulation needed to treat infertility in women with polycystic ovary syndrome (PCOS) using in vitro maturation (IVM)? Summary answer Number of matured oocytes and pregnancy outcomes were similar without or with FSH-priming in women with PCOS undergoing IVM. What is known already In vitro maturation (IVM) is a low-intervention alternative to in vitro fertilisation (IVF) for infertility treatment. Current standard clinical practice for human IVM is to prime patients for 2–5 days with follicle-stimulating hormone (FSH) and/or human chorionic gonadotropin (hCG) before harvesting immature or mixed maturity oocytes for infertility treatment. This gonadotropin priming is thought necessary to induce sufficient follicle and oocyte development in vivo to support subsequent oocyte maturation and embryo development in vitro when using a single step (monophasic) oocyte maturation culture system. It is not clear whether hormone-free IVM can generate satisfactory pregnancy and live birth rates. Study design, size, duration This randomised, controlled trial was conducted at an tertiary IVF center, Ho Chi Minh City, Viet Nam. Between January 2023 and June 2023, 120 women (60 per group) were randomised. Participants/materials, setting, methods Eligible women were aged 18–37 years and had PCOS with an indication for biphasic capacitation-IVM (CAPA-IVM). After providing written informed consent, participants were randomised (1:1) to undergo CAPA-IVM with or without FSH-priming. Participants in the FSH-priming group received two days of FSH injections before oocyte pick-up; no FSH was given in the non-FSH group. After CAPA-IVM, day-5 embryos were vitrified for transfer in a subsequent cycle. The primary endpoint was number of matured oocytes. Main results and the role of chance The number [interquartile range] of matured oocytes after CAPA-IVM did not differ significantly between the non-FSH and FSH groups (13 [9; 18] vs. 14 [7; 18]; absolute difference –1 [95% confidence interval –5, 4]). There were also no significant between-group differences in other oocyte and embryology outcomes, including the number of cumulus-oocyte complexes, number of fertilized oocytes, total number of blastocysts and good blastocysts, and total number of frozen embryos. No between-group differences were found in the rates of ongoing pregnancy and live birth, which were both 38.3% in the non-FSH group and both 31.7% in the FSH group; the miscarriage rate at < 12 weeks’ gestation was 5.0% in both groups. Maternal complications were infrequent and occurred at a similar rate in the non-FSH and FSH groups; there were no preterm deliveries before 32 weeks’ gestation. Limitations, reasons for caution The characteristics of the study population (relatively young, lean women with PCOS from Viet Nam) limit the external generalisability of the study findings, and the findings need to be replicated in other populations. Wider implications of the findings Hormone-free assisted reproductive technology (ART) for women with PCOS appears to be feasible when using CAPA-IVM, and does not result in any loss of clinical efficacy. Benefits of hormone-free ART would be a reduction in medical risk, lower cost, fewer monitoring requirements, and greater convenience and flexibility. Trial registration number NCT05600972

P-750 Is maximalization of the ovum pick-up (OPU) procedure beneficial for the outcome of assisted reproductive technology (ART) cycles?

Abstract Study question Is aspiration of more oocytes than the antral follicle count (AFC) beneficial for the current (pregnancy/live birth rates) and future products of an ART cycle? Summary answer Aspirating more oocytes than the AFC does not influence the outcome of the fresh cycle but is beneficial for fertility preservation and cumulative pregnancy rates. What is known already AFC is a reliable and immediate indicator for ovarian reserve assessment and is in correlation with the ovarian response to gonadotropin stimulation. Additionally, correlation between AFC and embryo quality, clinical pregnancy and live birth rates was reported. There is no data concerning the impact of the ratio between the AFC and actual number of oocytes retrieved on cycle outcome. Study design, size, duration Data of the first oocyte aspiration for ICSI performed between 2018-2022 in attempt to conceive (ICSI, n = 399) or for planned oocyte vitrification (fertility preservation FP, n = 283) was retrieved. Each group was divided into two subgroups according to their ‘oocyte/AFC index’ (OAFCI): <1 and ≥1. The stimulation parameters, number of aspirated oocytes, their maturity rate, number of cryopreserved oocytes/embryos, clinical pregnancy (CPR) and live birth rates (LBR) were compared between the subgroups. Participants/materials, setting, methods The AFC was determined by professional sonographers using a 9 MHz transvaginal ultrasound probe. The most adjacent measurement to the stimulation initiation was used. Stimulation protocol and FSH dose were determined according to the AFC and individual clinical consideration. Patients not in their first cycle, natural cycle aspirations, and cases in which no oocytes were retrieved, were excluded. Main results and the role of chance OAFC≥1 was associated with higher pre-trigger estradiol (6159 pmol/L vs. 4304, p < 0.001 for ICSI, 4949 vs. 3488, p < 0.001 for FP), and longer duration of simulation (9.8 days vs. 9, p < 0.001 for both FP and ICSI). More oocytes were aspirated in OAFC≥1 groups in both FP (16.9±11.2 vs. 7.9±5.9, p < 0.001) and ICSI (17.4±9.8 vs. 10.7±6.7, p < 0.001). Oocyte maturity was unaffected by the OAFCI in the ICSI group, but was lower in FP group at OAFC≥1 (0.79 vs.0.84 p = 0.005). OAFCI≥1 was associated with a higher number of cryopreserved embryos (3.9 vs. 2.3, p < 0.001 for ICSI) and a higher number of vitrified oocytes (13.4 vs. 6.4 p < 0.001 for FP). The OAFCI had no impact on CPR (39% vs. 60%, p = 0.27) and LBR (19.9% vs. 24.8%, p = 0.33). On multivariate analysis controlling for age, gonadotropin dosage and duration of stimulation, OAFC≥1 was associated with a higher number of cryopreserved embryos (ICSI group aOR 1.2, 95% CI 1.1-1.3, p < 0.001), and vitrified oocytes (aOR 1.2, 95% CI 1.1-1.2, p < 0.001) and lower maturity index (aOR 0.1, 95% CI 0.03-0.51, p < 0.001) in the FP group. Limitations, reasons for caution Retrospective study analyzing results of treatment cycles without an intention to aspirate differently. Wider implications of the findings Aspiration of a more oocytes than the current AFC was not associated with higher CPR or LBR in the fresh cycle, but harbors the possibility to cryopreserve more oocytes and embryos. This might translate into higher cumulative pregnancy rates later on. Trial registration number Not applicable

P-644 Effect of Letrozole co-stimulation with gonadotropin in women with normal ovarian reserve on oocyte maturation: a Randomized controlled trial

Abstract Study question To define whether letrozole co-stimulation with gonadotropin in normal-ovarian reserve women will improve the number of oocyte maturation. Summary answer Letrozole co-stimulation in normal ovarian reserve resulted in a reduced number of MII. What is known already Letrozole functions as an aromatase inhibitor. By inhibiting this enzyme, letrozole effectively lowers estrogen levels. Thus, letrozole prevents the estrogen-negative feedback to the HPO axis, which can increase the secretion of gonadotropins from the pituitary gland to promote folliculogenesis. The application of letrozole co-stimulation in women with a normal ovarian reserve is still debated. Study design, size, duration This study is an open-labeled, randomized controlled trial. Letrozole co-stimulation with gonadotropin in women with normal ovarian reserve. The RCT was conducted at the Fertility and Reproductive Biology Unit, Department of Obstetrics and Gynecology, Faculty of Medicine, Siriraj Hospital in Thailand, from March 2023 to October 2023. Participants/materials, setting, methods One hundred forty-four infertile women 18-40 years old with normal ovarian reserve (AMH 1.2-7 ng/ml, AFC ≥ 5) were random to gonadotropin co-stimulation with letrozole 5 mg once daily (n = 72) and gonadotropin alone (n = 72). The number of MII oocytes, follicle-to-oocyte index, GDF-9 and BMP-15 in follicular fluid were assessed. Main results and the role of chance Letrozole co-stimulation resulted in a significantly reduced number of MII [8 (4,12) vs 9 (5, 15.8), p = 0.042] and the rate of MII [ 71.7(60, 83.8) vs 80 (66.7, 90), p = 0.013]. However, the number of retrieved oocytes [12 (6,16.8) vs. 12 (6,16), p = 0.335] and FOI [85.7 (57.4,107.5) vs 84.2 [61.8, 112.5], p = 0.396] remained unchanged. The total dose of gonadotropin in the letrozole group was no different from the control group [1,350 (1,200, 1,650) vs 1,500 (1,350, 1,718.8) I.U., p = 0.137]. The duration of ovarian stimulation was shortened in the letrozole group compared to the control group [9 (8, 10) vs 9 (8.3, 10) days, p = 0.027]. The letrozole and control group had a similar level of GDF-9 (0.036 ± 0.003 A.U. vs. 0.028 ± 0.004 A.U., p = 0.119, letrozole and control group, respectively) and BMP-15 (0.034 ± 0.002 A.U. vs. 0.033 ± 0.004 A.U., p = 0.923, letrozole and control group respectively) in follicular fluid. Limitations, reasons for caution This study was open-label, with limited generalizability due to 91% of ovarian stimulation being strict to the research protocol (r-FSH and antagonist protocol) and no pregnancy outcome. Wider implications of the findings The study showed no benefit of letrozole co-stimulation in women with normal ovarian reserve. Trial registration number TCTR20221125002

P-501 Superovulation alters telomere length and telomerase expression in mouse oocytes, possibly by changing estradiol and progesterone levels

Abstract Study question How does the single and repeated superovulation affect the telomerase components expression and telomere length in mouse GV and MII oocytes? Summary answer Superovulation significantly altered the expression of the telomerase components Tert and Terc genes, TERT protein levels, and telomere length in the GV and MII oocytes. What is known already Assisted reproductive technologies (ARTs) are commonly used to treat infertility problems. One of the basic ART steps is controlled ovarian stimulation, also known as superovulation. Superovulation can lead to several adverse effects, such as increased oxidative stress, decreased oocyte and embryo quality, and mitochondrial dysfunction. These effects of superovulation largely overlap with those that causing telomere shortening. Study design, size, duration The effect of superovulation on the telomerase expression and telomere length of GV and MII oocytes is not fully addressed yet. In this study, we investigated the potential effects of single and repeated superovulation on telomerase expression and telomere length in mouse oocytes at germinal vesicle (GV) and metaphase II (MII) stages. In addition, we measured the estradiol and progesterone as well as oxidant and antioxidant capacity in serum samples of the oocytes-collected mice. Participants/materials, setting, methods Four groups of female Balb/C mice were formed: Control (C; n = 12), superovulated once (1S; n = 12), superovulated three times (3S; n = 12), and superovulated five times (5S; n = 12) at one-week intervals. The Tert and Terc gene expression, TERT protein levels, and telomere length in the mouse oocytes were assessed by RT-qPCR, immunofluorescence, and q-PCR, respectively. The hormone levels were analyzed by ELISA, and oxidant-antioxidant capacities were measured. All the ovarian follicles were counted in the H&E stained-sections. Main results and the role of chance We found that superovulation (1S, 3S, and 5S) significantly altered expression of the telomerase components Tert and Terc genes, TERT protein levels, and telomere length in the GV and MII oocytes (P<0.05). While estradiol levels in GV oocyte-collected and progesterone levels of GV and MII oocyte-collected mice were higher in the 1S, 3S, and 5S groups compared with the C group (P<0.01), the estradiol levels in MII oocyte-collected mice decreased progressively from C to 5S groups (P<0.01). However, no significant changes were found in the total oxidant status (TOS), antioxidant status (TAS), and oxidative stress index (OSI) between the groups. Also, primordial and atretic follicle numbers in the ovaries of 3S and 5S groups were significantly decreased compared to the control and 1S groups (P<0.05). Limitations, reasons for caution The limitation of this study is that we could not evaluate telomerase activity and oxidative stress levels in the mouse oocytes. Wider implications of the findings All these results suggest that altered telomerase expression and telomere length in GV and MII oocytes, possibly due to changed estradiol and progesterone levels, may be associated with adverse effects of superovulation on oocyte maturation and even preimplantation embryo development. Trial registration number not applicaple

P-165 Benefits of ultra-low oxygen tension (2%) in patients with low quality embryos or previous arrest: the patient as its own control

Abstract Study question Is reduction in oxygen tension from 5% to 2% after D3 of extended culture beneficial for blastocyst development in patients with previous failed culture? Summary answer Ultra-low oxygen tension in extended culture improves blastocyst development (overall blastulation rate OBR), quality (top blastulation rate TPR) and increases the pregnancy rate (PR). What is known already The practice ESHRE guidelines recommends human embryo culture at low oxygen concentrations (2-8%). Traditionally atmospheric conditions (20% O2) were used but better outcomes were observed at low monophasic 5% O2. Human embryo leaves the oviduct (5-7% O2) to cross the uterus-tubal junction around D3-4 and reaches the uterin ultra-low O2 tension (2%). This decreasing gradient coincides with the shift from pre-compaction oxidative phosphorylation in the oviduct, to post-compaction glycolysis in the uterus. Few studies with ultra-low 2% O2 incubation suggested that the best O2 level may be stage-dependent and its reduction after D3 may be a more physiological approach. Study design, size, duration Prospective observational study in a single private hospital during 2023. 71 patients experienced a failed cycle of extended culture (n = 29 cIVF, n = 37 ICSI and n = 5 Sibling oocytes) with a poor embryo quality and low OBR, under standard monophasic 5% O2 incubation. In the subsequent cycle for these 71 patients (n = 24 cIVF and n = 46 ICSI and n = 1 Sibling oocytes) the O2 level was reduced to 2% from D3 onwards. Participants/materials, setting, methods After a failed extended culture, considered as the control cycle, the patients were enrolled for the study. Blastocysts were rated by Gardner’s classification. In the control cycle embryos were cultured under monophasic 5% O2 until D6. In the subsequent cycle embryos were cultured under 5% O2 until D3 and in ultra-low 2% O2 from D3 to D6. Sage 1-Step™ media was used for both cycles. The statistical analyses were done by chi-square and fisher test. Main results and the role of chance 71 patients were included in the study regardless of age, indication or technique. Each patient was its own control. The mean age was not different between the 2 groups (36.5±4 vs 37.2±4) but the attempt rank was logically statistically higher in the study cycle (2.1±1.4 vs 3.4±1, p<0.001). Oocyte maturity (75% vs 76.4%) and fertilization rate (66.8 vs 70.3%) were similar between groups. The average embryo quality at D3 was poor in both groups. OBR and TBR (40.7% vs 52.4%, p=0.004 and 15.8% vs 37.5%, p<0.001) were statistically higher after culture in ultra-low O2 conditions. Extended culture failure with absence of usable blastocyst (57.7% vs 19.7%, p<0.001) was statistically lower in ultra-low incubation group and the pregnancy rate PR per cycle (3.6% vs 25%, p=0.004) was seven fold higher after ultra-low culture. Limitations, reasons for caution The small size of the sample needs to be expanded in order to confirm the encouraging findings in a larger population. The study will continue during 2024 and will compel the live birth rate and the cumulative live birth rate. Wider implications of the findings Providing embryos with the most physiologic culture system would improve IVF cost-effectiveness and a better infertility management. Embryo energetic adaptation to suboptimal conditions results in impaired viability and lower pregnancy rates. In our study extended culture of low-quality embryos under 2% O2 after D3 yielded better culture and pregnancy outcomes. Trial registration number NA

P-607 Reproductive outcomes of oocyte donors using subdermal etonogestrel implants and levonorgestrel intrauterine devices

Abstract Study question Does the use of progesterone contraceptive devices during controlled ovarian stimulation (COS) affect ovarian response in oocyte donation cycles? Summary answer Progesterone contraceptive devices during COS did not impact ovarian response, measured by oocyte retrieval and maturity rates, in oocyte donation cycles. What is known already Subdermal etonogestrel implants and levonorgestrel intrauterine devices (L-DIU) are widely used contraceptives amongst oocyte donors. While the safe and effective use of L-DIU during COS has been documented, the impact of subdermal etonogestrel implants on ovarian response is less understood. Given their distinct mechanisms of action and varying local and systemic pharmacodynamics, a comprehensive understanding of the impact of these contraceptives on ovarian response during COS will be crucial for guiding informed clinical practice. Study design, size, duration In this retrospective cohort study, we analyzed 3,452 heterologous ICSI cycles from 1,975 oocyte donors (18-35 years), performed between March 2021 and September 2023, across two centers. We identified 40 COS in donors using contraceptive devices, either a subdermal etonogestrel implant (n = 18) or L-DIU, (n = 22). These were matched with 80 COS without a device. We primarily compared ovarian response amongst the two groups. Secondary outcomes included biochemical pregnancy, clinical pregnancy and live birth rates. Participants/materials, setting, methods We applied a propensity matching score model (PsmPy package, Python), trained on 3,388 oocyte donation cycles, for matching donors with and without contraceptive devices. Matching was based on age, BMI, anti-müllerian hormone (AMH) and antral follicle count (AFC), maintaining a 1:2 ratio. Ovarian response outcomes (oocyte retrieval and maturation rates) were analyzed using Student’s t-tests, while and clinical outcomes were evaluated using Fisher’s exact test. P-values <0.05 were considered significant. Main results and the role of chance Mean age was 26.22, BMI 24.7, AMH 2.63 ng/ml and AFC 18.77. We evaluated a total of 120 COS, leading to 176 heterologous ICSI cycles. The oocyte retrieval rate (total number of cumulus-oocyte-complexes (COCs) per total number of follicles > 14mm at final follicular scan), averaged 109.64%. We observed no differences in the number of COCs retrieved from donors with and without contraceptive devices (17.5 ±7.6 and 18.5 ± 9.6, respectively). Similarly, oocyte maturation rates (total number of MII oocytes per total number of COCs), were comparable amongst the two groups (78.6% overall, 77.6% with contraception device and 79.1% without contraception device, p > 0.05). The presence of the device did not affect biochemical pregnancy (69.7% with contraceptive device, 61.1% without contraceptive device, p > 0.05), clinical pregnancy (54.6% with contraceptive device, 52.9% with contraceptive device, p > 0.05) and ongoing pregnancy (36.4% with contraceptive device, 35.3% without contraceptive device). These results were consistent regardless of the type of device used. Limitations, reasons for caution The main limitation is the retrospective nature of the study, potentially influencing causal inferences, and that both devices were analyzed as one same group. The small subset of COS with contraceptive devices may impact statistical power. Perinatal outcomes were not analyzed. Wider implications of the findings This research provides clinicians with reassurance that the use of progesterone contraceptive devices during COS does not adversely affect clinical outcomes. These findings offer valuable insights for patient counseling, affirming the safety of contraceptive methods and encouraging further research for a more comprehensive understanding in diverse populations and clinical settings. Trial registration number not applicable

P-602 Comparative analysis of recombinant human FSH and a biosimilar FSH preparation in controlled ovarian stimulation: a retrospective study of 12,952 oocyte donation and autologous cycles

Abstract Study question Does the type of FSH preparation used for ovarian stimulation, recombinant human FSH (r-hFSH) or biosimilar, influence reproductive success in oocyte donation and autologous cycles? Summary answer Ovarian response and reproductive outcomes, including live birth rates, are comparable in oocyte donation and autologous cycle, irrespective of the type of r-hFSH preparation used. What is known already Exogenous gonadotrophins, particularly FSH, have long been used in assisted reproduction, evolving from urinary to recombinant forms. The first generation of r-hFSH, including Puregon® and Gonal-f®, became available in the 1990s. However, interest in biosimilar FSH products has grown globally following the expiry of original patents. Approved by the European Medicines Agency, two r-hFSH biosimilars, Ovaleap® and Bemfola®, offer cost-effective alternatives for infertility treatments. Regulatory guidelines emphasize non-inferiority to the originator product, with clinical trials showing comparable outcomes between biosimilar and reference FSH preparations. However, debates persist regarding the efficacy and safety of r-hFSH biosimilars, particularly in achieving live births. Study design, size, duration In this retrospective cohort study, we compared reproductive outcomes following 8,429 controlled ovarian stimulations (COS) using r-hFSH Gonal-f® (n = 5,059) or the biosimilar FSH Bemfola® (n = 3,370), performed between January 2015 and December 2022 at a single fertility center. Overall, 242,978 mature (MII) oocytes were retrieved, utilized in 12,952 ICSI cycles (n = 11,421 heterologous and n = 1,531 autologous). We primarily evaluated ovarian response, measured by oocyte retrieval and maturation rates. Secondary outcomes included ongoing pregnancy and live birth rates. Participants/materials, setting, methods Participants included both oocyte donors (6,895 COS, 18-35 years) and patients using autologous oocytes (1,534 COS, 20-46 years). Univariate and multivariate analyses were performed separately across oocyte donor and patient groups. Outcomes in autologous cycles were further stratified by age. Regressions were adjusted for r-hFSH dose, antral follicle count (AFC), days of stimulation, age, BMI, use of menotropins, fertilization rates and number of embryos transferred, as appropriate. All p-values <0.05 were considered statistically significant. Main results and the role of chance Oocyte donors exhibited similar demographics (age, BMI, days of stimulation), however AFC was significantly higher in the biosimilar group compared to donors stimulated with Gonal-f® (11.31 ± 9.72 vs 7.63 ± 7.5). In patients, we observed no difference in demographic variables between study groups. A mean of 20.5 cumulus-oocyte complexes (COCs) and 15.7 MII oocytes were obtained across all COS. In oocyte donation cycles, the number of COCs retrieved was comparable between groups (22.6, Gonal-f®, 22.3, Bemfola®), however stimulation with Gonal-f® led to a slightly higher number of MII oocytes (17.6 vs 16.9, t-test p < 0.0001). Nevertheless, adjusted analyses revealed improved oocyte retrieval rates with Bemfola® (coeff 0.12, 95% CI: (0.05-0.18), p = 0.0006), while oocyte maturity rates were equivalent amongst the two groups (80% Gonal-f®, 77%, Bemfola®). Comparable results were observed in autologous cycles in terms of COCs (13.7, Gonal-f®, 12.7, Bemfola®) and number of MII oocytes (9.6, Gonal-f®, 9.0, Bemfola®). In autologous cycles, r-hFSH choice did not affect oocyte retrieval rates (coeff -3.3x10-04, 95% CI (-0,09-0,09), p > 0.05) nor oocyte maturation rates (coeff -2.7x10-04, 95% CI (-0,02-0,02), p > 0.05). Our adjusted analysis revealed comparable ongoing pregnancy and live birth rates between the study groups across all 12,952 ICSI cycles (p > 0.05 in all cases). Limitations, reasons for caution The main limitation of this study is its non-interventional retrospective cohort design, impacting generalizability to broader populations and diverse clinical settings. Despite its exploratory nature, the study’s scale, encompassing over 8,000 COS and close to 13,000 ICSI cycles, offers valuable insights into routine clinical care. Wider implications of the findings Comparable ovarian response, pregnancy and live birth rates following COS with Gonal-f® and Bemfola®, in both heterologous and autologous cycles, underscore the utility of biosimilars in ART. Our findings support the broader implications of EU’s policy on biosimilar interchangeability, ultimately enhancing patient access to a wider range of treatment options. Trial registration number not applicable