Cumulus-oocyte Complexes Research Articles

Granulosa cell metabolism at ovulation correlates with oocyte competence and is disrupted by obesity and aging.

Is oocyte developmental competence associated with changes in granulosa cell (GC) metabolism? GC metabolism is regulated by the LH surge, altered by obesity and reproductive aging, and, in women, specific metabolic profiles are associated with failed fertilization versus increased blastocyst development. The cellular environment in which an oocyte matures is critical to its future developmental competence. Metabolism is emerging as a potentially important factor; however, relative energy production profiles between GCs and cumulus cells and their use of differential substrates under normal in vivo ovulatory conditions are not well understood. This study identified metabolic and substrate utilization profiles within ovarian cells in response to the LH surge, using mouse models and GCs of women undergoing gonadotropin-induced oocyte aspiration followed by IVF/ICSI. To comprehensively assess follicular energy metabolism, we used real-time metabolic analysis (Seahorse XFe96) to map energy metabolism dynamics (mitochondrial respiration, glycolysis, and fatty acid oxidation) in mouse GCs and cumulus-oocyte complexes (COCs) across a detailed time course in the lead up to ovulation. In parallel, the metabolic profile of GCs was measured in a cohort of 85 women undergoing IVF/ICSI (n = 21 with normal ovarian function; n = 64 with ovarian infertility) and correlated with clinical parameters and cycle outcomes. Our study reveals dynamic changes in GC energy metabolism in response to ovulatory LH, with mitochondrial respiration and glycolysis differentially affected by obesity versus aging, in both mice and women. High respiration in GCs is associated with failed fertilization (P < 0.05) in a subset of women, while glycolytic reserve and mitochondrial ATP production are correlated with on-time development at Day 3 (P < 0.05) and blastocyst formation (P < 0.01) respectively. These data provide new insights into the cellular mechanisms of infertility, by uncovering significant associations between metabolism within the ovarian follicle and oocyte developmental competence. A larger prospective study is needed before the metabolic markers that were positively and negatively associated with oocyte quality can be used clinically to predict embryo outcomes. This study offers new insights into the importance of GC metabolism for subsequent embryonic development and highlights the potential for therapeutic strategies focused on optimizing mitochondrial metabolism to support embryonic development. National Health and Medical Research Council (Australia). The authors have no competing interests. N/A.

Temporal metabolomics analysis reveals the metabolic patterns in goat cumulus cells during oocyte maturation

Cumulus cells play a crucial role in the oocyte growth and maturation processes through providing necessary nutrients and growth signals by gap junction communication. However, a global overview of metabolic events in goat cumulus cells is still lacking. In the present study, we collected cumulus cells from goat cumulus-oocyte complexes (COCs) at different developmental stages. Metabolomics analysis was performed to investigate the global metabolic patterns in cumulus cells during oocyte in vitro maturation. In particular, we revealed the several significantly altered metabolic pathways and metaboliccharacteristics in goat cumulus cells, including the accumulation of fatty acids, steroid hormones metabolism, active catabolism of arginine during meiotic resumption, and a progressive decline in nucleotide metabolism. In conclusion, the dataset generated by our metabolomic profiling will provide valuable information to understand the key metabolic pathways and metabolites involved in COCs development.

Enhancing the quality of inferior oocytes of buffalo for in vitro embryo production: The impact of melatonin on maturation, SCNT, and epigenetic modifications

Success of animal cloning is limited by oocyte quality, which is closely linked to reprogramming ability. The number of layers of cumulus cells is typically used to assess the quality of oocyte; a minimum of one-third of collected cumulus-oocyte complexes (COCs) are discarded as inferior oocytes because they have less cumulus cells. Melatonin, which has been recognised for its ability to sequester free radicals and perform multiple functions, has emerged as a potentially effective candidate for enhancing inferior oocytes quality and, consequently, embryo development competency. The current study investigates to improve the quality of inferior oocytes by supplementation of melatonin (10−9 M) during in vitro maturation (IVM) and subsequent cloned embryo production and its mechanism. The results indicate that melatonin supplementation significantly (p<0.05) enhances inferior oocytes maturation, reduces oxidative stress by reducing ROS levels, and improves mitochondrial function by boosting GSH levels. The melatonin treatment (10−9 M) enhances the expression of SOD, GPx1, GDF 9, BMP 15, ATPase 6, and ATPase 8 in inferior oocytes. Furthermore, melatonin treatment increases the total cell number in the treated groups, promoting cloned blastocyst formation rates derived from inferior oocytes. Furthermore, compared to the control, 10−9 M melatonin supplementation enhances H3K9ac acetylation and lowers H3K27me3 methylation in cloned blastocysts derived from inferior oocytes. In conclusion, 10−9 M melatonin supplementation during IVM increased inferior oocyte maturation and promoted cloned buffalo embryo development by lowering oxidative stress and promoting epigenetic alterations. These studies show that melatonin may improve the quality of poor oocytes and buffalo cloning.

DNA methylation, but not microRNA expression, is affected by in vitro THC exposure in bovine granulosa cells

BackgroundA global increase in cannabis use has led to questions about its effects on fertility. The rise in consumption amongst women of reproductive age is a growing concern, as this group is vulnerable in terms of reproductive health. Ample evidence suggests that the psychoactive component of cannabis, Δ9-Tetrahydrocannabinol (THC), interacts with the endocannabinoid system (ECS), that helps regulate mammalian reproduction. This study aimed to research the epigenetic effects of THC in bovine granulosa cells (GCs) by (1) investigating global DNA methylation via measuring 5-mC and 5-hmC levels; (2) measuring key methylation regulators, including the methylating enzymes DNMT1, DNMT3a, DNMT3b and the demethylases TDG and TET1/2/3; and (3) assessing fertility-associated miRNAs key in developmental competency, including miR-21, -155, -33b, -324 and -346.MethodsBovine GCs were used as a translational model for reproductive toxicity in humans. To determine THC effects, GCs were isolated from Cumulus-Oocyte-Complexes (COCs) from bovine ovaries, cultured in vitro for 7 days, or until confluent, and cryopreserved at passage 1 (P1). For experimentation, cells were thawed, cultured until passage 2 (P2), serum restricted for 24-h and treated for 24-h in one of five groups: control, vehicle (1:1:18 ethanol: tween: saline) and three clinically relevant THC doses (0.032, 0.32 and 3.2 μM). Global methylation was assessed by measuring 5-mC and 5-hmC levels with flow cytometry. To assess mRNA and protein expression of methylation regulators and miRNA profiles, qPCR and Western Blotting were utilized. Shapiro-Wilk test was used to determine normality within datasets. One-way ANOVA was applied to determine statistical significance using GraphPad Prism 6.0.0.ResultsResults indicate a significant decrease (p = 0.0435) in 5-mC levels following low THC exposure, while no changes were observed in 5-hmC levels. A significant increase in DNMT1 following high THC exposure at the RNA level (p < 0.05) and a significant increase following low THC exposure at the protein level (p = 0.0048) were also observed. No significant differences were observed in DNMT3a/3b, TDG, TET1/2/3 mRNAs or in any of the miRNAs analyzed.ConclusionsThis research suggests that THC mainly affects DNA methylation, but not miRNA profiles, ultimately altering gene expression and likely impairing oocyte competence, maturation, and fertilization potential.

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.

O-003 COC classifier: a new artificial intelligence-based tool for identifying mature oocytes from cumulus- oocyte-complexes

Abstract Study question Can the use of artificial intelligence (AI) enhance the prediction of mature oocytes (MII) from Cumulus-Oocytes-Complexes (COC) significantly surpassing the accuracy of well-trained embryologists? Summary answer The development of a COC classifier, an AI-based tool, improves the prediction of mature oocytes from COCs graded as 3 and 4 before oocyte denudation. What is known already Determining the maturity of Metaphase II oocytes before denudation is a challenge in assisted reproduction due to the presence of a dense cumulus mass surrounding the oocyte and obstructing proper manual scoring, a critical step in intracytoplasmic sperm injection (ICSI) due to risk of granulosa cells removal from immature oocytes. Although there is a certain correlation between COC morphology and nuclear oocyte maturity, it is not 100% accurate. It is reported that approximately 90% of MII oocytes are expected in expanded COCs (grade 1), while in COCs grade 4, more immature oocytes are predicted and only 25% are MII oocytes. Study design, size, duration 931 COC's from 145 patients were submitted to COC grading by both, manual and a new “COC classifier” from October to January 2023. 4 groups were formed according to Ebner criteria, 2008. Grade 1: 192 COC's (suspected mature), fluffy and radiant corona and cumulus; Grade 2: 318 COC's showing fluffy cumulus (suspected mature); Grade 3: 208 COC's with radiant corona (expected mature) and grade 4: 213 COC's (suspected immature) with dense cumulus without visible oocyte. Participants/materials, setting, methods Prospective study included 145 patients (39.3 years ±3.2) from September to December 2023. After COC obtention, excess of granulosa cells, dark and blood cells were removed. 3 hours after egg collection, COCs were photographed and scored manually by 9 well trained embryologists. Before denudation, obtained COCs were classified in 4 morphological stages according to Ebner et al, 2008. Finally, the taken photos from COC's were analysed by the COC classifier, a sypervised deep learning algorithm. Main results and the role of chance After COC's classification as following: Group 1 (suspected mature), fluffy and radiant corona and cumulus with visible oocyte; Group 2, COC's showing dense corona (oocyte clearly visible) but fluffy cumulus (suspected mature); Group 3, COC's with radiant corona (oocyte visible, expected mature); Group 4, COC's (suspected immature) with dense corona and cumulus without visible oocyte. The new software presented an MII oocyte accuracy of 79.16% for group 1, 87.1% for group 2, 83.17% for group 3, and 73.23% for group 4. In comparison, manual COCs grading by nine experienced embryologists yielded accuracy rates of 73.5% MII oocytes in group 1, 79.5% for group 2, 50.3% for group 3, and 46.9% for group 4. Statistical analysis, using the chi-square test, was performed to compare the accuracy rates between the COC classifier and manual grading. The results revealed significant differences in favor of the COC classifier when compared to manual method for the groups 3 and 4 (p &amp;lt; 0.001). These results indicate a superior performance of the COC classifier in predicting MII oocytes for groups with highest rates of inmture oocytes. However, for groups 1 and 2, there were no statistically significant differences between the COC classifier and manual grading. Limitations, reasons for caution Further validation through a large-scale, prospective, randomized study is necessary to confirm the efficacy and reliability of the new COC classifier, particularly in patients with oocyte cohorts enriched with grade 3 and 4 COCs. Ongoing research is currently being conducted in our laboratory. Wider implications of the findings The new COC classifier, an AI-based tool, developed by our group, offers a non-invasive and efficient method in predicting mature oocytes before denudation by identifying more MII oocytes from patients’ cohorts enriched with grade 3 and 4 COC's, postponing denudation and injection timing or destinating them to conventional IVF Trial registration number 5678765

P-230 Use of a novel in vivo biosensor to rapidly determine oocyte developmental competence

Abstract Study question Can a novel in vivo biosensor (IVOS) rapidly determine oocyte developmental competence? Summary answer Data collected with the IVOS biosensor can be used to determine oocyte developmental competence, observed as blastocyst development after in vitro embryo production. What is known already Methods allowing for rapid, non-invasive assessment of oocyte quality are limited. Subjective morphology is used for gamete selection, but cannot discern between cells with subcellular dysfunction. Metabolism is an accurate method to distinguish cellular quality, but current methods are not rapid, require highly artificial environments, or lack appropriate sensitivity. Extracellular indicators associated with metabolic output may correlate to inherent oocyte developmental potential and serve as a biomarker. A published in vivo biosensor (IVOS) which analyses REDOX homeostasis, an indicator of subcellular function, may be optimised to use at the cellular level to assess oocyte quality. Study design, size, duration Experiments were performed in South Australia using abattoir derived cumulus-oocytes complexes (COC) aspirated from ewe ovaries for in vitro embryo production. COCs were group matured then maintained individually after IVOS analysis to permit identification of oocyte measurements to embryo developmental endpoint. Methodology optimisation and experiments were performed in June 2023 and October 2023, respectively. A holding pipette was modified to permit compatibility with the IVOS system using a proprietary method. Participants/materials, setting, methods Matured COCs were denuded to the corona radiata and placed into an air-buffered medium for IVOS analysis. The holding pipette suctioned COCs prior to excitation (488nm) of extracellular molecules at the COC surface. Emission wavelengths (515-520nm) were retrieved for ten consecutive replicates. COCs were subsequently co-incubated with frozen-thawed ram semen. Presumptive zygotes were cleaned of cumulus cells and loose spermatozoa, cultured for seven days, and stained using Hoechst H33342 to obtain total cell number. Main results and the role of chance Embryos were graded into three categories: 1) did not display a blastocoel cavity (not blastocyst); 2) embryo displaying a blastocoel and total cell count below 50 (early blastocyst); and 3) embryo displaying a blastocoel with a total cell count above 50 (blastocyst). Standard deviation calculations for technical replicates of each COC (n = 32) were used to form an equation capable of distinguishing between groups based on developmental endpoint reached. From this equation, IVOS measurements were lower in oocytes reaching the early blastocyst stage compared to those which reached the blastocyst stage (P &amp;lt; 0.001) or did not reach the blastocyst stage (P = 0.042). Data from oocytes that became full blastocysts were higher than those which did not become blastocysts (P = 0.031). Limitations, reasons for caution The technology used in this study has been tested on sheep oocytes using blastocyst development as a measure of developmental competence. Further research is required to determine if it is applicable to other species or reproductive outcomes such as pregnancy and live birth. Wider implications of the findings This study provides a new technology and optimised methodology to improve the selection of high-quality oocytes among gametes graded homogeneously using subjective morphology. It can be utilised with existing assisted reproductive technologies to possibly increase the success rate of these procedures. Trial registration number N/A

P-618 Impact of ovarian stimulation on outcome of oocyte in-vitro maturation (IVM) in women with polycystic ovaries: corifollitropin alfa (CFA) versus follitropin beta (FSH-B)

Abstract Study question Do corifollitropin alfa and follitropin beta have different effects on oocyte yield and live birth rates after IVM in women with polycystic ovaries? Summary answer In patients who underwent IVM, one injection of CFA resulted in lower oocyte retrieval rates, but similar birth rates compared to three injections of FSH-B. What is known already IVM involves the maturation of cumulus oocyte complexes (COCs) from antral follicles and has been offered to women with polycystic ovaries as an alternative for conventional ovarian stimulation (OS). Exogenous FSH is typically administered for two to five days in IVM cycles to enhance meiotic and developmental competence of immature oocytes in vivo. Previous studies have shown that the number of COCs is associated with ongoing pregnancy after IVM. Because one injection of CFA yields more oocytes compared to daily FSH-B injections in conventional OS protocols, CFA has the potential to combine patient-friendliness and maximised COC yield in IVM cycles. Study design, size, duration We conducted a randomised controlled superiority trial from 12/2017 to 12/2023. The primary endpoint was the number of COCs at collection. We randomised 145 patients to either one CFA 100μg injection or three daily injections of FSH-B 150IU. Laboratory and safety parameters, and pregnancy outcomes after frozen embryo transfer (FET) were analysed on an intention-to-treat basis. All cycles were scheduled using oral contraceptive pretreatment. Participants/materials, setting, methods Eligible patients were &amp;lt;37 years, had ≥24 antral follicles, and BMI 18-30 kg/m2. We analysed serum estradiol, progesterone, LH, and FSH on stimulation day 1 and 3, at oocyte retrieval (OR), and at OR + 6d. Oocyte retrieval (OR) was performed five days after the start of OS. No ovulation trigger was given. Monophasic IVM for 30h, ICSI and elective freeze-all were done in a tertiary fertility clinic. Data were analysed using STATA 13.0. Main results and the role of chance After randomisation, 70 patients underwent OR after FSH-B and 72 had OR after CFA. Hormone levels at OR were significantly different between FSH-B-treated (FSH 6.8 ± 2.8IU/L, LH 3.3 ± 2.7IU/L, E2 106.7 ± 147.0ng/L, Prog 0.19 ± 0.16μg/L) and CFA-treated patients (FSH 24.3 ± 7.9IU/L, LH 1.8 ± 1.6IU/L, E2 575.6 ± 516.2ng/L, Prog 0.29 ± 0.21 μg/L, p ≤ 0.001). On average, 43.1 ± 21.7(FSH-B) vs. 50.8 ± 29.2(CFA) follicles, all &amp;lt;10mm, were punctured during OR (p = 0.06). More COCs per follicle were retrieved after FSH-B (68.3 ± 31.5% vs. 51.0 ± 24.7%, p = 0.001), resulting in a tendency towards more COCs after FSH-B (31.4 ± 23.3), compared to CFA (23.4 ± 11.7, p = 0.07). Maturation rates after IVM were similar (49 ± 19% vs. 50 ± 15%, p = 0.79). In spite of a tendency towards more mature oocytes after FSH-B (16.0 ± 14.6 vs. 11.7 ± 7.9, p = 0.09), the number of good-quality cryopreserved embryos was similar (3.9 ± 2.9(FSH-B) vs. 3.5 ± 2.7(CFA), p = 0.36). Live birth rate (LBR) after the first FET (25.7%(FSH-B) vs. 34.7%(CFA), p = 0.24) and cumulative LBR six months after OR (40.0%(FSH-B) vs. 45.8%(CFA), p = 0.48) were comparable. None of the patients developed ovarian hyperstimulation syndrome (OHSS). Limitations, reasons for caution Outcomes are only applicable for patients with high antral follicle count (AFC) who are treated using a monophasic IVM culture system. The sample size was too small to draw significant conclusions for live birth rate. Wider implications of the findings In subfertile patients with high AFC who have IVM treatment, follicle priming with one injection of CFA is safe, convenient, and as efficacious as priming with FSH-B. The observation of lower oocyte retrieval rates after CFA compared to FSH-B in the specific setting of an IVM cycle merits further scrutiny. Trial registration number EudraCT 2017-002571-25

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

P-355 Effect of biphasic CAPA-IVM on ovarian tissue oocytes of transgender men

Abstract Study question Can CAPA-IVM (Biphasic in-vitro maturation) with C-type natriuretic peptide (CNP), followed by in vitro maturation (IVM) improve ovarian tissue oocyte (OTO) maturation in transgender patients? Summary answer CAPA-IVM did not show any significant difference in maturation competence and frequency of chromosomal aberrations when compared with standard and in-house IVM. What is known already OTO-IVM is a method of fertility preservation in patients where prior ovarian stimulation is undesired. It has resulted in live births in cancer and polycystic ovarian syndrome (PCOS) patients. CAPA-IVM has further improved oocyte competency in these patients by synchronizing nuclear and cytoplasmic maturity. Similarly, OTO can be collected from transgender men without ovarian stimulation during gender reassignment surgery. While oocytes do survive and mature, OTO-IVM in transgender men is characterized by decreased fertilization rate and severely compromised developmental competency. We investigated whether CAPA-IVM can improve cytoplasmic immaturity for transgender OTO. Study design, size, duration Patients were recruited from July 2022 to July 2023. Both ovaries were collected from 9 transgender patients (age= 20-24 years, mean age=22 years) who underwent gender reassignment surgery after testosterone treatment (mean length= 34 months). All ovaries were collected in cold medium (4oC) and manipulation was performed within 30 minutes of the collection for the retrieval of cumulus oocyte complexes (COCs). Participants/materials, setting, methods Collected COCs were cultured either in in-house IVM and Standard (Medicult) IVM medium for 48 hours or in biphasic CAPA-IVM for 54 hours (24 hours pre-maturation culture and 30 hours IVM). Following maturation, oocytes were analyzed for calcium (Ca2+)-releasing potential and developmental competency after ICSI. In vitro matured MI (metaphase I) oocytes from stimulated IVF patients served as controls. Genetic analysis was performed on subsequent embryos to detect chromosomal abnormalities in all groups. Main results and the role of chance After culturing 330 COCs, the survival rate following maturation was similar in the in-house IVM (79%) compared to Medicult (72%, p = 0.221) and CAPA-IVM (73%, p = 0.251). A similar maturation rate (i.e. 55-57%) was observed in the different groups. Following ICSI, 15/25 (60%) CAPA-IVM, 13/19 (68.5%) Medicult IVM, and 17/28 (61%) in-house IVM oocytes were normally fertilized, which was lower than controls (24/29, 83%). Blastocyst rate was inferior in all OTO groups and significantly lower (p = 0.031) in Medicult-IVM (0/13) compared to controls (7/24). Ca2+-releasing potential of oocytes was determined as a product of amplitude and frequency, in arbitrary units (AU). The average values for CAPA-IVM (1.74AU), Medicult IVM (1.24AU), and in-house IVM (1.54AU) were significantly lower (p = 0.022, p = 0.008 and p = 0.017 respectively) compared to controls (4.76AU). Shallow whole genome sequencing of developed embryos (Day 2- Day 5) showed that 8/13 (61.5%) in CAPA-IVM, 4/8 (50%) in In-house IVM, 1/6 (17%) in Medicult, and 4/12 (33%) in the Control group were chromosomally normal. Limitations, reasons for caution The limited inclusion of patients warrants caution in the interpretation of the results. Moreover, the lack of a better control group might mask the properties of OTO-IVM. Wider implications of the findings Our preliminary results do not demonstrate an improvement in oocyte maturation and embryo development with the implementation of CAPA-IVM. However, future proteomics and transcriptomic studies would shed more light on the advantages of the current IVM systems and decipher the true potential of OTO-IVM from transgender men. Trial registration number not applicable

P-650 Cumulative live birth rate after use of capacitation in vitro maturation in women with predicted excessive response to ovarian stimulation: an analysis of 1,563 cycles

Abstract Study question What is the cumulative live birth rate in biphasic (capacitation) in vitro maturation (CAPA-IVM) cycles in women with predicted excessive response? Summary answer The cumulative live birth rate achieved after CAPA-IVM in women with predicted excessive response was 37.8%. What is known already In vitro maturation (IVM) is an alternative infertility treatment for women at high risk of complications during conventional in vitro fertilisation with ovarian stimulation. There is debate about the merits of a newer biphasic approach (capacitation [CAPA]-IVM) versus standard IVM. At centres where it is used, CAPA-IVM has increased the maturation rate, and therefore the number of embryos available for transfer. However, there is a lack of data from large numbers of CAPA-IVM cycles, especially cumulative outcomes. Study design, size, duration This analysis included data from 1,563 individuals who underwent CAPA-IVM at a tertiary IVF center in Ho Chi Minh City, Vietnam from January 2016 to December 2023. Participants/materials, setting, methods All CAPA-IVM cycles in women with predicted excessive response (polycystic ovary syndrome or high antral follicle count) were included. Mean age was 29.6±3.5 years. Median duration of infertility was 3.3±2.5 years. CAPA-IVM was the first assisted reproductive technology in 90.5% of participants. Cumulative live birth rate was defined as the number of live births after using all embryos generated from a CAPA-IVM cycle. Main results and the role of chance Cumulative rates of clinical pregnancy, ongoing pregnancy and live birth were 54.9%, 44.4% and 37.8%, respectively. The median proportion of mature oocytes per cumulus-oocyte complex was 61.3% (IQR 50.0–73.3) and the median proportion of fertilized oocytes per cumulus-oocyte complex was 72.2% (IQR 55.6-85.7). The median total number of day-3 embryos per patient was 4.0 [IQR 3.0–7.0] and the total number of good day-3 embryos per patient was 3.0 [IQR 2.0–5.0]. The proportion of patients with no embryo was 5.2%. The number of frozen embryo transfer cycles was 1 (53.6% of patients), 2 (24.9%), 3 (7.0%), 4 (0.8%) or 5 (0.3%); 7.9% of patients had not undergone transfer of day-3 embryos at the time of analysis. Median [IQR] number of embryos and good embryos transferred was 2.0 [2.0–4.0] and 2.0 [1.0–2.0], respectively. No case of ovarian hyperstimulation syndrome recorded. Limitations, reasons for caution The characteristics of the study population (relatively young, lean women from Vietnam with predicted excessive response to ovarian stimulation) limit the external generalisability of the findings. Wider implications of the findings The cumulative live birth rate from all CAPA-IVM cycles performed in women with predicted excessive response at our centre was comparable to that of IVF. CAPA-IVM could be an alternative for this selected group of patients to avoid ovarian hyperstimulation. Trial registration number not applicable

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-647 Association between PCOS phenotype-D and euploid blastocyst rate per cohort of inseminated oocytes: a matched case-control study versus idiopathic infertile women

Abstract Study question Is there any association between PCOS phenotype-D and oocyte competence defined as euploid blastocyst rate (EBR) per cohort of inseminated oocytes? Summary answer Although higher fertilization and blastulation rates were reported in PCOS phenotype-D women versus idiopathic infertile patients, the EBR per cohort of inseminated oocytes was comparable. What is known already Several studies report poorer oocyte quality and cumulative-live-birth-rate (CLBR) in hyperandrogenic PCOS phenotypes, as defined according to the Rotterdam criteria. Increased androgen concentrations in follicular fluids are indeed associated with elevated serum-LH levels, which can block dominant follicle development inducing atresia and negatively impact fertilization rates and embryo development. Limited evidence exists in phenotype-D PCOS patients, namely women characterized by oligomenorrhea, ovarian PCO morphology and absence of hyperandrogenism. Here, we report the embryological and clinical outcomes in these women during ICSI cycles with preimplantation genetic testing for aneuploidies (PGT-A) at the blastocyst stage. Study design, size, duration Retrospective propensity score matched (PSM) case-control study. Among PGT-A cycles conducted by naïve patients with own oocytes (2013-2021), we excluded FNA/TESE. 58 phenotype-D PCOS patients were identified according to the Rotterdam criteria. These patients were matched for maternal age, number of cumulus-oocyte-complexes (COCs), and sperm quality, with 58 patients with idiopathic infertility, regular cycles, normal ovarian morphology, and no sign of clinical hyperandrogenism. The primary outcome was EBR per cohort of inseminated oocytes. Participants/materials, setting, methods GnRH-antagonist ovarian stimulation, ICSI, blastocyst biopsy, qPCR/NGS-analysis to assess uniform aneuploidies and vitrified-warmed single blastocyst transfers were conducted. The PCOS and control groups were similar for age (35.6±3.0 versus 36.0±3.9 years), COCs (23.1±6.7 versus 23.5±7.6), sperm factor, primary/secondary infertility, hormonal values, BMI (23.8±5.3 versus 22.0±2.8), and duration of infertility (3.6±2.2 versus 3.6±2.0 years). All intermediate embryological and clinical outcomes were assessed as secondary endpoints. Linear/logistic regressions adjusted for confounders were conducted to confirm statistically-significant differences. Main results and the role of chance No difference was reported in maturation rates, but women with PCOS showed better fertilization rates per cohort of inseminated oocytes (76.9±15.2% versus 67.3±20.4%, Mann-Whitney-U-test&amp;lt;0.01), and blastulation rates per cohort of 2PN-zygotes (52±20.4% versus 43.7±20.7%, Mann-Whitney-U-test=0.03). Eventually, PCOS patients obtained more blastocysts than controls (5.8±3.1 versus 4.5±2.9, Mann-Whitney-U-test=0.02), despite similar numbers of metaphase-II oocytes in the groups (16.5±6.1 versus 16.5±7.0). All differences remained significant after adjusting for maternal age and sperm factor. Nevertheless, the euploidy rates per cohort of biopsied blastocysts were similar, thus involving comparable EBR per cohort of inseminated oocytes (21.7±14.1% versus 17.2±16%, Mann-Whitney-U-test=0.12). More patients obtained ≥1 euploid blastocyst in the PCOS versus the control group (N = 56/58, 96% versus N = 47/58, 81%, Fisher’s-exact-test=0.02), independently from maternal age, semen quality, and number of inseminated oocytes (Odds-Ratio: 6.5, 95%CI:1.3-33.7, p = 0.03). Lastly, all clinical outcomes per first vitrified-warmed euploid transfer were similar, thereby involving comparable LBRs (N = 28/54, 51.9% versus N = 26/47, 55.3% in the PCOS and control groups, p = 0.8; Odds-Ratio adjusted for blastocyst quality and day: 1.19, 95%CI:0.5-2.7, p = 0.68). Limitations, reasons for caution Although clinical hyperandrogenism was absent in both groups, biochemical hyperandrogenism was not assessed. Nonetheless, this pilot study paves the way for future more detailed investigations. 24% of cycles in the PCOS group were not concluded, therefore a CLBR comparison would be biased and was not reported. Wider implications of the findings The lower developmental competence in the control group suggests oocyte quality issues, other than chromosomal, underlying their “unexplained infertility” condition. In phenotype-D PCOS women, IVF is effective. Since &amp;gt;50% of this subgroup of women had already failed multiple ovulation-induction and/or intrauterine-insemination attempts, an early referral to IVF deserves further investigations. Trial registration number Not applicable

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

P-178 Putrescine supplementation enhances embryonic development in patients with Recurrent Implantation Failure (RIF)

Abstract Study question Does putrescine, a type of polyamine, affect embryonic development in patients with recurrent implantation failure (RIF)? Summary answer Putrescine supplementation on cumulus-oocyte complexes (COCs) was associated with better embryologic development from fertilization to blastulation in patients with RIF. What is known already Putrescine, a type of polyamine, plays a role in cell growth and proliferation. Several animal studies suggest the potential impacts of putrescine on embryo development, but its effects on human IVF have yet to be extensively studied. Study design, size, duration This retrospective study analyzed 442 patients who have undergone IVF cycles three or more times from January to December 2023. The average IVF cycle number of RIF patients was 7.4 times. A total of 1356 oocytes from the putrescine group and 1391 oocytes from the control group underwent aspiration and ICSI. Further analysis was conducted by classifying RIF patients into two groups: patients under 40 years of age and patients over 40 years of age. Participants/materials, setting, methods COCs were cultured for 4 hours in a culture medium supplemented with 0.5mM putrescine. 2 hours after oocyte collection, cumulus cells were denuded, and ICSI was performed. Embryonic development was evaluated based on fertilized oocytes (2PN), good quality embryos (GQE) on day 3, and blastulation rates on day 5/6. Group differences were analyzed using t-tests and chi-square tests (p &amp;lt; 0.05). Main results and the role of chance When maternal age was ≥40, the fertilization rate in the putrescine group was significantly higher than that in the control group (86.6% vs. 80.5%, p=0.002). The putrescine group had a numerically higher proportion of embryos with top-grade morphology on day 3 (47.8%, compared with 53.3% for control; p=0.057), without statistical significance. Additionally, the rate of blastulation on day 5 indicated a significant difference between the two groups (14.5% vs. 6.1%, p=0.0001). Further analysis was conducted in younger patients (&amp;lt;40 years old), and it also showed a significantly higher fertilization rate (89.7% vs. 71.1 p=0.0001) and blastocyst development rates on the day 5 in the putrescine group (22.3% vs. 11.1%, p=0.0001), respectively. This study suggests that putrescine supplementation can be utilized to improve embryo quality and facilitate blastocyst formation in IVF procedures for patients with RIF, irrespective of age. Limitations, reasons for caution Putrescine supplementation in IVF showed improved fertilization and blastulation rates, but caution is needed due to the small sample size used. Therefore, a larger sample size will help improve the robustness of the conclusions drawn with respect to the embryologic development in patients with RIF. Wider implications of the findings The study’s findings suggest that putrescine supplementation in IVF may enhance outcomes for patients with RIF, potentially leading to personalized treatments. 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 &amp;lt;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 &amp;lt; 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 &amp;gt; 0.05) nor oocyte maturation rates (coeff -2.7x10-04, 95% CI (-0,02-0,02), p &amp;gt; 0.05). Our adjusted analysis revealed comparable ongoing pregnancy and live birth rates between the study groups across all 12,952 ICSI cycles (p &amp;gt; 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

O-303 What is the ideal dose of daily FSH after corifollitropin-alfa? A multicentre-randomized control trial

Abstract Study question Does the mean serum trigger-progesterone levels show significant variation based on the administered rFSH dose from day 8 onwards during ovarian stimulation using corifollitropin-alfa? Summary answer Lower rFSH doses from day 8 of ovarian stimulation onwards are linked to reduced serum progesterone (P4) levels without impact on cumulus-oocyte complexes. What is known already Progesterone elevation during ovulation trigger prompts a non-elective freeze-all strategy, given the lower live birth rates in fresh embryo transfers. Ongoing research explores methods to mitigate late follicular phase P4 rise to allow a fresh embryo transfer. Granulosa cells’ progesterone production is an FSH-driven and dose-dependent process, and FSH dose is furthermore pivotal for a good ovarian response. Balancing FSH dose is crucial for both optimal ovarian response and preventing undesirable P4 elevation while maintaining an adequate number of retrieved oocytes and allowing a fresh embryo transfer. Study design, size, duration This study is an interim-analysis of a randomized-controlled multi-center clinical-trial comparing three ovarian-stimulation-protocols: (A) corifollitropin alfa (CFA) followed by rFSH 50 IU/day; (B) CFA followed by rFSH 150 IU/day; (C) CFA followed by rFSH 250 IU/day.The primary outcome is the serum-progesterone-level on the day of hCG among the study groups, secondary outcomes included number of oocytes, clinical pregnancy rates and freeze-all cycles. 167 women (of 261) were randomized between July 2019-November 2023, with a 1:1allocation. Participants/materials, setting, methods Young (&amp;lt;40 years old), normo-ovulatory cycle (25-35 days), with AFC&amp;gt;7 and &amp;lt;20, and a BMI&amp;lt;29 Kg/m2 patients were recruited in the fertility clinic of three different tertiary university hospitals. On day 8 of ovarian stimulation (OS) following CFA administration, patients who needed additional rFSH were randomized into 3 groups (A-B-C). The OS was performed in a GnRH-antagonist protocol and the ovulation triggered with hCG. A fresh embryo-transfer was performed on day 5 following oocyte retrieval. Main results and the role of chance Overall, 167 patients were randomized, 60 in group A, 55 in group B and 52 in group C. The mean age and BMI were comparable between the groups. Mean AMH was respectively 2.2, 2.1 and 2.2 ng/mL, for group A, B and C (p = 0.95). Group A resulted in a significantly lower P4 level at trigger as compared with group B and C (respectively, 0.4ng/mL, 0.9 ng/mL and 1.4 ng/mL; p &amp;lt; 0.001). The mean number of retrieved oocytes was 10.7, 11 and 12.1 for group A, B and C, respectively (p = 0.47), while the mean number of mature oocytes was 7.3, 6.9 and 7.8 for group A, B and C, respectively (p = 0.7). A freeze-all approach was decided for 27%, 29% and 46% for group A, B and C respectively (p = 0.057). No significant differences were found for fresh-embryo-transfer outcomes (chemical pregnancy rates: 33%, 31% and 29%, respectively for A, B and C (p = 0.12).Of interest, comparisons between serum FSH at trigger showed a mean of 12.5, 17 and 20.5 IU/L for group A, B and C, respectively (p &amp;lt; 0.001) while the serum estradiol levels on day of trigger were comparable among the groups (1659, 2092 and 2098 ng/L for group A, B and C, respectively (p = 0.091). Limitations, reasons for caution This is an interim analysis planned to detect the mean difference in COC between groups. Therefore, the mean differences of the primary outcome should be interpreted as exploratory findings which warrant future investigation. Wider implications of the findings Administering a lower rFSH dose from day 8 onward during ovarian stimulation results in comparable numbers of COC but also leads to significantly lower serum P4 levels. Consequently, this finding has the potential to reduce the overall consumption of rFSH, thereby possibly increasing the likelihood of a fresh embryo transfer. Trial registration number NCT03686852

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 &amp;lt;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 &amp;gt; 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 &amp;gt; 0.05). The presence of the device did not affect biochemical pregnancy (69.7% with contraceptive device, 61.1% without contraceptive device, p &amp;gt; 0.05), clinical pregnancy (54.6% with contraceptive device, 52.9% with contraceptive device, p &amp;gt; 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

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 &amp;lt; 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-260 In Vitro Fertilization (IVF) using magnetotactic sperm cells and their prospects for assisted in vivo reproduction

Abstract Study question Are bovine Magnetotactic Sperm Cells (MSC) able to fertilize an oocyte in-vitro and Which would be the main requirements for translating this technology to in-vivo? Summary answer IVF using highly purified MSC result in embryo development until blastocyst stage. We demonstrate different alternatives to control multiple MSC, individually and as a swarm. What is known already Medical microrobots have appeared as the new generation of drug delivery vehicles. They are tiny devices in the order of a few micrometers, able to move in biological environments by different means, physical, chemical, or biohybrid. They are functionalized to deliver targeted therapies for drugs, cells, or other payloads. In assisted reproduction, sperm-hybrid microcarriers have been pioneered by our group, and various in vitro demonstrations have been done to assist sperm cells with motion deficiencies or low count and to deliver anticancer drugs for treating, for example, cervical and ovarian cancer, but so far in-vitro fertilization has never been shown. Study design, size, duration Experimental quantitative study design. A total of 520 bovine cumulus-oocyte complexes were used for in vitro fertilization. The analyzed sample groups were using bovine frozen sperm cells, MSC, or by evaluating parthenogenetic activation. A power of 0.98 with an a-error of 0.05 was used for the IVF experiments. Sperm samples in the control as well as the micromotor groups have been analyzed for different sperm parameters. Participants/materials, setting, methods Bovine MSC are created by thawing frozen sperm cells and adding polystyrene microparticles. Different sperm parameters were evaluated to check the effects of the particles in the cells. After developing a purification method for the MSC, bovine ovaries were collected from a slaughterhouse nearby and, after in vitro maturation, were used for the IVF experiments. Ultrasound, and photoacoustic imaging setups with a set of electromagnets were employed for real-time manipulation and visualization of MSC. Main results and the role of chance Sperm viability was significantly higher in the MSC group compared to the control group 6h after formation (72.3 ± 5.0% vs 35.0 ± 4.1%), and after 24 hours, both viabilities decreased drastically to 16.6 ± 3.9% and 7.7 ± 1.7% respectively, probably a possible local antioxidant effect by the microparticles through the formation of a protein corona. In the case of motility, the MSC group presented significantly higher motility up to 6h (64.0 ± 1.5%), and in all the measured times, the motility of the MSC group was higher than the control group. Purification of the micromotor sample was performed, but an average of 63 free sperm was still on the MSC sample. IVF experiments were performed, and blastocyst development was observed in IVF control (2x106 sperm/ml), medium concentration (same concentration as MSC group), and the MSC groups (275.000 sperm/ml). No blastocyst formation was observed in the parthenogenetic, parthenogenetic + particles, and low-concentration groups (number of free sperm). The MSC group was significantly different in cleavage and blastocyst rates compared to the low concentration groups, with p-values of 0.04552 and 0.005865, respectively, which suggest that fertilization may have occurred by the micromotors instead by the free sperm in the sample. Limitations, reasons for caution More studies of these MSC in the different gynecological tissues and on embryo development are needed to ensure the safety of future human translation. In addition, developing a detection system for micromotors in deep tissue is needed to perform in vivo experiments. Wider implications of the findings MSC show an alternative to efficiently guide efficiently sperm cells in different biological environments and can be a promising alternative method for assisted fertilization, and the treatment of a variety of gynecological diseases. They can also be used as exploratory tools to learn more about sperm migration in-vivo, among others Trial registration number European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program No. 853609