Fertilization Rate Of Oocytes Research Articles

Factors affecting the outcomes of in vitro fertilization in women with endometriosis: Preliminary data from Vietnam

Objectives: This study aimed to assess the efficacy of in vitro fertilization (IVF) in patients with endometriosis and identify related factors that may influence treatment outcomes. Methods: This retrospective descriptive study was conducted between November 2018 and November 2022. The study group comprised individuals who were diagnosed with endometriosis (endometrioma and/or adenomyosis) indicated for in vitro fertilization. The control group (129 patients with 249 cycles), which included patients without endometriosis and had an equivalent mean age as the study group patients. Results: In the endometriosis group, the quantities of retrieved oocytes and Anti-Müllerian Hormone (AMH) were reduced compared to the control group. The overall dose of gonadotropin and the duration of ovarian stimulation were significantly higher than the control group. The endometriosis group exhibited a Metaphase II (MII) oocyte rate of 82.3% and an oocyte fertilization rate of 82.2%. However, in patients with endometriosis, the rates of implantation, clinical pregnancy, and ongoing pregnancy were significantly lower than in patients without endometriosis. Besides, the clinical pregnancy rate of women with endometriosis was remarkably higher in the groups with maternal age ⩽35 years, a ratio of Follicle-stimulating Hormone to Luteinizing Hormone (FSH/LH) ⩽1.5, and over 10 retrieved oocytes. Conclusion: Endometriosis was associated with a decreased rate of implantation, clinical pregnancy, and ongoing pregnancy after in vitro fertilization treatment compared with patients without endometriosis. In vitro fertilization outcomes in patients with endometriosis were influenced by maternal age ⩽35 years, FSH/LH ratio ⩽1.5, and number of retrieved oocytes over 10.

Environmental exposure to lead and cadmium only minimally affects the redox system of the follicular fluid and the outcome of intracytoplasmic sperm injection.

The purpose of our study was to determine the influence of lead and cadmium in concentrations commonly found in the environment on the redox system of the follicular fluid (FF) and on the results of assisted reproduction. A prospective study of 113 patients with unexplained infertility who qualified for intracytoplasmic sperm injection (ICSI). Patients with moderate or severe endometriosis or poor ovarian reserve were excluded from the study. Biochemical analyses and heavy metal assays of follicular fluid and serum (blood) were followed by statistical analyses of dependencies between lead and cadmium and the components of redox system and results of assisted reproduction. A highly significant linear correlation of lead (Pb) and cadmium (Cd) concentrations in serum and in FF was stated. The number of retrieved oocytes and MII (metaphase II stage) oocytes depended on the malondialdehyde (MDA), catalase (CAT), catalase/g of protein (CAT/g of protein), and glutathione reductase (GR) concentrations. Among biochemical factors, MDA was the only factor that correlated negatively with cadmium concentration in serum and FF and simultaneously influenced the number of retrieved oocytes and MII oocytes. The fertilization rate of MII oocytes was influenced by thiol groups-SH, SH/g of protein, CAT, CAT/g of protein, and glutathione peroxidase/g of protein (GPx/g of protein). The Pb and Cd concentrations in FF did not significantly influence the fertilization rates. Lead as well as cadmium at concentrations commonly found in women of reproductive age despite some adaptive changes in the redox system in follicular fluid do not cause large changes in the ovarian follicular environment as a whole and do not significantly worsen the final results of assisted reproduction.

Oocyte Competence, Embryological Outcomes and miRNA Signature of Different Sized Follicles from Poor Responder Patients

Poor ovarian response (POR) patients often face the risk of not having enough competent oocytes. Then, aspirating small follicles could serve as a strategy to increase their number. Many efforts have been addressed to associate follicular size with oocyte competence, but results are controversial. Therefore, our study aimed to evaluate oocyte maturation and developmental competence, along with a non-invasive oocyte-maturation-related miRNA signature in oocytes retrieved from both large and small follicles. A total of 178 follicles, from 31 POR patients, were aspirated and measured on the day of ovarian puncture. Follicular diameters, oocyte collection, oocyte maturation, fertilization, blastocysts, and good-quality blastocyst rates were recorded. Simultaneously, follicular fluids were collected to quantify their miRNA expression. The efficacy of oocyte retrieval along with oocyte maturation, fertilization, and blastulation rates tended to increase with follicular size, but few significant differences were found. Despite there being significantly more collected oocytes from follicles > 11.5 mm compared to follicles ≤ 11.5 mm (p < 0.05), oocytes from the latter were also mature, with no significant differences in the miRNA signature, but only those > 13.5 mm demonstrated developmental competence. In conclusion, 11.5 mm follicles can produce mature oocytes, but only those larger than 13.5 mm yielded transferable embryos.

Dithiothreitol prevents the spontaneous release of cortical granules in in vitro aged mouse oocytes by protecting regulatory proteins of cortical granules exocytosis and thickening the cortical actin cytoskeleton

In assisted fertility protocols, in vitro culture conditions mimic physiological conditions to preserve gametes in the best conditions. After collection, oocytes are maintained in a culture medium inside the incubator until in vitro fertilization (IVF) is performed. This time outside natural and physiological conditions exposes oocytes to an oxidative stress that renders in vitro aging. It has been described that in vitro aging produces a spontaneous cortical granule (CG) release decreasing the fertilization rate of oocytes. Nevertheless, this undesirable phenomenon has not been investigated, let alone prevented. In this work, we characterized the spontaneous CG secretion in in vitro aged oocytes. Using immunofluorescence indirect, quantification, and functional assays, we showed that the expression of regulatory proteins of CG exocytosis was affected. Our results demonstrated that in vitro oocyte aging by 4 and 8 h altered the expression and localization of alpha-SNAP and reduced the expression of NSF and Complexin. These alterations were prevented by supplementing culture medium with dithiothreitol (DTT), which in addition to having a protective effect on those proteins, also had an unexpected effect on the actin cytoskeleton. Indeed, DTT addition thickened the cortical layer of fibrillar actin. Both DTT effects, together, prevented the spontaneous secretion of CG and recovered the IVF rate in in vitro aged oocytes. We propose the use of DTT in culture media to avoid the spontaneous CG secretion and to improve the success rate of IVF protocols in in vitro aged oocytes.

O-071 Stringent statistical analysis of 108,684 oocyte donation cycles identifies 2.6% of donors with specific oocyte/embryo developmental arrest phenotypes for genome association studies

Abstract Study question Can statistical modelling accurately identify an outlier group of presumed fertile donors with specific reduced embryological outcomes suggestive of an underlying infertility-related genomic condition? Summary answer Statistical modelling identified a highly specific group of donors as embryologically under-performing outliers across multiple treatment cycles for the optimisation of downstream genomic association studies. What is known already Female infertility has a wide range of clinical presentations. While some causes of infertility are well defined, the aetiology of most infertility remains unexplained. Since IVF allows for in vitro monitoring of oocyte and embryo, it is now possible to identify subtle infertility endophenotypes, characterized by specific developmental failure that would otherwise go undetected in natural conception. Currently, the lack of large and well-categorized datasets of infertility endophenotypes has limited research in the area. Therefore, a clear and methodological definition of infertility endophenotypes in IVF is the first crucial step towards a better understanding of their mechanism and biological/genetic aetiology. Study design, size, duration A retrospective analysis was performed on clinical and embryological data from 40497 egg donors, undergoing 108.684 stimulations, with 4.235.036 oocytes retrieved. The data also included information on the 85482 recipients and their 190996 oocyte reception cycles, spanning from 1999 to 2023. Infertility endophenotypes were able to be well categorized as oocyte donors are presumed fertile based on their young age and on normal ranges displayed for the parameters assessed during typical oocyte donation work-up. Participants/materials, setting, methods Donor performance was calculated according to: (I) oocyte maturation rate (MR); (ii) survival rate of cryopreserved oocytes (SR); (iii) oocyte fertilization rate (FR;2PN/Injected MII); and (iii) blastulation rate (BR;blastocyst/2PN). Donors with significantly impaired rates, considering ≥2 stimulations and/or recipients, were identified as outliers using probabilistic modelling (Binomial test, Bonferroni corrected P < 0.05). Outliers were computed for each rate on data subsets after applying this stringent filtering criteria and excluding inconsistent or missing data. Main results and the role of chance 2.6% of donors were identified as underperforming outliers when summing the rate observed for each embryological outcome using the stringent statistical modelling. The average MR was 79.8% (95%CI: 79.6-80). Taking into account the correlation of follicle size during ultrasound tracking and the number of mature oocytes collected, a total of 168/29768 (0.56%) donors with statistically lower MR (mean MR = 43% 95%CI:40.1-45.7) across ≥2 egg collection cycles were identified as outliers. The average SR was 88% (95%CI:87.9-88.2). A total of 176/18544 (0.95%) donors with ≥2 stimulations and ≥2 recipient cycles were deemed outliers, showing a statistically reduced SR (mean SR = 51% 95%CI:48.9-53.1). The average FR was 74% (95%CI: 73.8-74.2). 164/31657 (0.52%) donors were found to have statistically low FR (mean FR = 37% 95%CI:34.6-38.7) and deemed outliers when assessed over ≥2 stimulation cycles with ≥2 recipient cycles. The average BR was 53.4% (95%CI: 53.1-53.7). After considering only donors with ≥2 recipients and ≥2 stimulations, a total of 142/24716 (0.57%) donors with statistically low BR (mean BR = 14% 95%CI:12.4-15.3) were considered outliers. Limitations, reasons for caution The stringent criteria applied to our statistical modelling has likely resulted in underestimating the true level of outlier donors. This is also likely compounded by the selection bias associated with donor recruitment and the retention policies after the first stimulation cycle preventing the evaluation of recurrent patterns in some cases. Wider implications of the findings The analytical tools developed on this unprecedentedly large dataset are fundamental to define a methodological workflow for accurately identifying unexpectedly infertile individuals. This is crucial for investigating the genetic and biological mechanisms underlying infertility endophenotypes, leading to improvements in treatment and developing screening tool to predict infertility and related pathologies. Trial registration number not applicable

P-353 Perfluorooctanoic acid (PFOA) impacts murine ovarian follicle development through apoptotic signaling pathways and exhibits transgenerational effects

Abstract Study question How does Perfluorooctanoic Acid (PFOA) exposure impact ovarian function and the physiological and reproductive health of subsequent offspring? Summary answer PFOA exposure leads to ovarian dysfunction, characterized by follicular depletion and hormonal imbalances, and adversely affects the reproductive health of offspring. What is known already Mass spectrometry reveals higher perfluorinated compound levels in premature ovarian failure patients compared to healthy individuals. PFOA, an environmental contaminant, is known for its endocrine-disrupting effects, but its specific impact on ovarian function and transgenerational health remains less explored. This study addresses the gap by examining PFOA’s direct ovarian effects and its consequential impacts on offspring, using both in vivo and in vitro models. Study design, size, duration This study employs mouse models and ovarian granulosa cells to assess PFOA’s impact. Mice were administered PFOA (50mg/Kg) and evaluated for ovarian function and offspring health. Ovarian tissue underwent histological analysis and hormone assays. RNA transcriptome sequencing identified activated apoptotic pathways post-PFOA exposure. Offspring physiological and reproductive parameters were also examined. Parallel in vitro experiments with ovarian granulosa cells at 50uM PFOA concentration provided insights into cellular apoptosis. Participants/materials, setting, methods Clinically, we collected serum samples from approximately 100 Premature Ovarian Insufficiency (POI) patients and 100 healthy individuals, analyzed using mass spectrometry. In murine experiments, we simulated human environmental exposure to PFOA (particularly in high-exposure professions) using a 50mg/Kg dose administered via gavage. Transcriptome sequencing analyzed potential signaling pathways in both experimental and control mice. Offspring underwent physiological metabolic assessments and fertility evaluations, including fertilization and blastocyst rates. Main results and the role of chance Mass spectrometry results from human samples revealed significantly higher levels of perfluorinated compounds in POI patients compared to healthy controls, suggesting a potential link between PFOA exposure and ovarian dysfunction. In murine models, PFOA administration at 50mg/Kg resulted in notable ovarian changes. Histological analysis showed a reduction in follicle counts and abnormalities in AMH and FSH levels, indicative of premature ovarian failure. Transcriptome sequencing of these mice highlighted the activation of apoptotic pathways, especially the PI3K/Akt/mTOR signaling, post-PFOA exposure. This suggests a mechanistic pathway through which PFOA may induce ovarian dysfunction. Furthermore, offspring of PFOA-exposed mice displayed significant reproductive health challenges. They exhibited irregularities in estrous cycles and a decrease in oocyte fertilization rates, pointing to potential transgenerational effects of PFOA exposure. These findings in murine models align with the elevated perfluorinated compound levels observed in human POI patients, reinforcing the hypothesis of PFOA’s detrimental impact on ovarian health and subsequent generational fertility. The consistency of results across human clinical data and animal models strengthens the argument for a causal relationship between PFOA exposure and reproductive health risks. Limitations, reasons for caution While these findings highlight PFOA’s detrimental effects on ovarian function and offspring health, translating results from animal models to humans necessitates caution. The specific mechanisms and potential variability in human response to environmental PFOA exposure require further investigation. Wider implications of the findings This research underscores the far-reaching impacts of environmental contaminants like PFOA on reproductive health. It emphasizes the need for stringent regulation of such pollutants and further studies to understand their long-term effects on human health, particularly regarding fertility and intergenerational wellbeing. Trial registration number not applicable

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-371 It’s worth the freeze: outcomes from thawing electively cryopreserved oocytes are comparable in patients with reduced vs normal ovarian reserve at the time of cryopreservation

Abstract Study question For patients coming back to use oocytes derived from elective cryopreservation, does their initial ovarian reserve predict thaw survival, embryos’ developmental milestones and pregnancy success? Summary answer Thaw survival, fertilization rate, embryo development and pregnancy success rates do not vary significantly according to AMH level at initial cryopreservation. What is known already Increasing numbers of patients are electing to cryopreserve oocytes for future use. AMH (Anti Mullerian Hormone) is an ovarian reserve marker often assessed prior to initiation of treatment that predicts response to fertility treatment and the resulting oocyte yield. However, it is not known whether oocytes derived from a patient with compromised ovarian reserve (perhaps over multiple treatment cycles) will fare as well as that of a patient with normal ovarian reserve and a higher per cycle oocyte yield. Our study seeks to shed light on whether a lower initial AMH level negatively impacts the prognosis of the thawed oocytes. Study design, size, duration We included in our retrospective study all oocyte thaw cycles resulting from electively cryopreserved oocytes since 1996 at a single academically affiliated fertility center. 325 patients completed a total of 510 thaw cycles. 315 cycles were excluded from analysis for no AMH information. 6376 oocyte cryopreservation cycles were performed over that timeframe. The primary outcomes were laboratory statistics including oocyte survival, fertilization and embryo blastulation rates, while the secondary outcome was cumulative live birth rates. Participants/materials, setting, methods AMH categories of < 1, 1-2 and > 2 were selected to stratify reduced, low-normal, and normal ovarian reserves, respectively. 70.7% inseminated oocytes with partner sperm while the remaining 29.3% used donor sperm. Mean age at time of oocyte cryopreservation was 36.5 (range 27.5 to 46.2) while mean age at time of oocyte thaw was 39.2 (range 27.7 to 47.5). The statistical analysis was performed using ANOVA for mean comparisons and chi-squared for pregnancy data. Main results and the role of chance Average oocyte yield per cryopreservation cycle was significantly different across AMH groups (7.7 oocytes in AMH < 1, 10.5 oocytes in AMH 1-2, and 17.2 oocytes in AMH > 2, p < 0.01). The number of treatment cycles completed was highest in patients with AMH < 1 at 1.26 cycles. Oocyte survival (mean 89.0%), fertilization rate (mean 75.3%), and blastocyst conversion rate (mean 62.4%) did not vary significantly across AMH groups. Time to oocyte use was not significantly impacted by AMH at the time of initial testing. Those with reduced AMH < 1 came back earlier to utilize the oocytes (787 days), vs 1141 days for patients with AMH 1-2 and 1021 days for patients with AMH > 2. However, this difference was not statistically significant, p = 0.4. The rate of PGT-A euploid embryos did not vary between groups (38.8% in AMH < 1, 45.3% in AMH 1-2, 53.0% in AMH > 2, p = 0.07). Clinical pregnancy, miscarriage rate, and live birth rate in the first transfer cycle was not impacted by AMH. Total cumulative live birth rate was not correlated with AMH (31.6% in AMH < 1, 44.4% in AMH 1-2, 47.8% in AMH > 2, p = 0.48). Limitations, reasons for caution This data was collected at a single fertility center and thus has limited generalizability. 8.0% of patients came back to utilize their oocytes, which varies widely across the published literature. 60.6% of patients did not have AMH assessed as part of their pre-treatment testing and were excluded from analysis. Wider implications of the findings Although reduced ovarian reserve impacts oocyte yield, many studies find no impact on resulting oocyte quality, embryo development and pregnancy success rates. Those with compromised AMH have comparable oocyte thaw outcomes to those with normal ovarian reserve, though more oocyte cryopreservation cycles may be needed to achieve a successful outcome. Trial registration number N/A

P-025 Mitochondrial activity in sperm is associated to blastocyst development: a double-donation study

Abstract Study question Do oxidative parameters in sperm affect oocyte fertilization or blastocyst development in double-donation ICSI cycles? Summary answer While intracellular oxidative stress in sperm does not suppose an impact on fertilization or embryo development, mitochondrial activity is related to increased blastocyst development rates What is known already Reactive oxygen species(ROS) are molecules causing a detrimental effect on sperm quality and function, especially when they are imbalanced with respect to the sperm and seminal plasma antioxidant capacity. Previous research have extensively demonstrated that sperm affected by oxidative stress(OS) show reduced fertilizing capacity, and lead to embryos with impaired development after conventional IVF. The role of OS in sperm in the case of ICSI is, however, controversial because many factors may cause a bias. Not only could this explain the inconsistent results reported thus far, but would also support the need of testing healthy donor gametes to reduce variability. Study design, size, duration This research was devised in the context of a collaboration between a sperm bank and a public university, and was set as a blinded, retrospective study that included samples from 45 sperm donors used in a total of 55 oocyte donation cycles. Intracellular levels of total ROS and of superoxides (O2•-), oxidative stress and mitochondrial activity (MA) were evaluated in frozen-thawed sperm donor samples, and related to the embryo outcomes obtained from the same samples. Participants/materials, setting, methods Samples from selected sperm donors were used to fertilize oocytes through ICSI in oocyte donation programs, and fertilization and blastocyst rates were recorded. Sperm viability and intracellular levels of total ROS and superoxides were determined through a combined assay (2’,7’-Dichlorofluorescin diacetate/Hydroethidine/Sytox Red); oxidative stress and sperm viability were assessed through co-staining with CellRox Far Red and propidium iodide (PI); and MA (mitochondrial membrane potential) and sperm viability were evaluated after staining with DiOC6(3) and PI. Main results and the role of chance The average and standard deviation for fertilization and blastocyst rates were 76.8%±17.3% and 67.5%±22.8%, respectively. Taking the viable sperm population into account, percentages of sperm with high ROS and low O2•- were 17.6%±15.9%, those with high ROS and high O2•- were 0.5%±0.4%, percentages of sperm with low ROS and low O2•- were 25.9%±9.2%, and those with low ROS and high O2•- were 54.5%±21.1%. The evaluation of oxidative stress with CellRox revealed that the percentage of viable sperm with high OS was 63.1%±16.5%. Neither intracellular levels of ROS and superoxides, nor oxidative stress in viable sperm were found to be correlated to fertilization or blastocyst rates (P>0.05). In spite of this, the percentage of viable sperm with high MA, which was 20.7%±10.5%, was observed to be positively correlated to blastocyst rates (Rs=0.276; P=0.04). While sperm mitochondria do not participate in further embryo development, a greater mitochondrial activity in post-thawed donor sperm could indicate a healthier sperm function. Mitochondrial activity could, therefore, be used as a predictive biomarker for embryo development. Limitations, reasons for caution The sample size is the main limitation of this study, despite the efforts made to include a high number of sperm donors. Wider implications of the findings Results obtained in the present study show that ICSI evades the detrimental effects of sperm oxidative stress on fertilization and embryo development. Yet, sperm mitochondrial activity could be used as a biomarker to test the ability of a sperm donor to generate healthy embryos that develop up to blastocyst stage. Trial registration number Not applicable

P-663 Comparing clinical outcomes in PGT-A cycles using donated oocytes between progesterone and GnRH-antagonist protocols

Abstract Study question Does Progesterone Primed Ovarian Stimulation (PPOS) demonstrate comparable efficiency to the GnRH-antagonist protocol in terms of laboratory/reproductive outcomes in PGT-A cycles with donated oocytes? Summary answer PPOS demonstrates efficiency in PGT-A cycles with donated oocytes, comparable to the GnRH-antagonist protocol, providing cost-effectiveness and enhanced donor comfort. What is known already The demand for TRA cycles with donated oocytes steadily rises each year. This treatment instills high hopes for success. Ongoing advancements in donor stimulation protocols prioritize safety and comfort. The shift from LH agonist to antagonist suppresses LH peaks, and recently, PPOS has joined, offering a more comfortable, cost-effective, and equally safe alternative with fewer ultrasound checks, mitigating ovarian hyperstimulation syndrome. Study design, size, duration Retrospective assessment of an oocyte donor cohort undergoing ovarian stimulation: 182 cycles with the GnRH-antagonist protocol (Group 1: G1) and 120 with PPOS (Group 2: G2), conducted between July 2017 and June 2023. Ovarian stimulation involved 225 IU/d of FSHr or FSHu, triggering with a GnRH agonist, and subsequent biopsy of evolutionarily robust embryos after extended culture. Both fresh and vitrified oocytes, inseminated with either self or donor sperm, were included. Participants/materials, setting, methods We analyzed 1188 embryos (G1: 676; G2: 512). Trophoectoderm biopsies from D5/D6 blastocysts underwent NGS using the Illumina platform (VeriSeq Illumina®, San Diego, CA, USA). Comparison included total and MII oocyte numbers, fertilization rates, viable embryos, euploid/aneuploid embryos, mosaic embryos, and clinical outcomes: biochemical pregnancy (BPR), biochemical pregnancy loss (BPLR), clinical pregnancy (CPR), early pregnancy loss (EPLR), ongoing pregnancy (OPR), and live birth (LBR) rates. Statistical analyses were carried out using R statistical software (v.4.3.1). Main results and the role of chance The mean ages of donors (25.57±4.05) and recipients (41.82±4.06) were comparable in both groups. The male partner’s age differed (G1: 40.47±7.87; G2: 42.34±7.65) (p = 0.019). Results were adjusted for confounding factors, including recipient and donor ages, oocyte and seminal origin, and male factor variables. Ovarian stimulation duration (9.26 days), total gonadotropin dose (2015.64 IU), and oocyte origin (fresh G1: 69.8 %; G2: 62.5 %; vitrified G1: 30.2 %; G2: 37.5 %) showed equivalence. Statistical significance emerged in total oocytes retrieved (G1: 17.92±7.64; G2: 20.23±7.89) (p = 0.008), mean microinjected MII (G1: 10.25±1.79; G2: 10.07±1.36) (p = 0.040) and fertilization rate (G1: 73.98 %; G2: 79.44 %) (p = 0.001). The usable embryo rate was similar (G1: 54.78 %; G2: 54.47 %). Significant differences arose in mean biopsied embryos/cycle (G1: 3.71; G2: 4.27) (p = 0.016), but not for aneuploid embryos (G1: 29.71 %; G2: 29.49 %) (p = 0,998). Biochemical pregnancy rates were significant (G1: 48.8 %, G2: 60.4 %) (p = 0.022). Other clinical parameters showed no differences, with LBR at 35.5 % in G1 and 37.8 % in G2 (p = 0.752). Limitations, reasons for caution The study is retrospective, where we addressed potential biases by incorporating confounding variables. However, to confirm our initial findings, randomized prospective studies with an appropriate sample size are essential. Wider implications of the findings In egg donation cycles, PPOS shows optimal embryonic performance resulting in a live birth rate comparable to the GnRH antagonist protocol. Additionally, it offers cost-effectiveness and enhanced comfort for donors. Trial registration number Not applicable

O-299 Exposure of antral follicles to progestins during stimulation does not cause molecular perturbations in gonadotropin-responsiveness and steroidogenic function of granulosa cells in progestin-primed IVF cycles

Abstract Study question Does progestin exposure cause any molecular perturbations in the steroidogenic features and gonadotropin responsiveness of the granulosa cells in progestin-primed ovarian stimulation (PPOS) cycles? Summary answer No. PPOS cycles are identical to antagonist cycles for gonadotropin receptor expression, response to gonadotropins, steroidogenic function at molecular level in normal responding IVF patients. What is known already PPOS is commonly used in IVF cycles as an alternative to antagonist cycles. Although accumulating evidence from clinical studies did not indicate any particular differences between these two types of stimulation protocol for IVF cycle characteristics, it is still unknown if exposure of antral follicle cohorts to synthetic progesterone derivatives during ovarian stimulation causes any subtle molecular aberrations in PPOS cycles in terms of steroidogenesis and gonadotropin responsiveness. To address this issue, detailed molecular analyses were conducted in the luteinized mural granulosa cells (GCs) obtained from the normal responding patients undergoing PPOS vs. antagonist IVF cycles. Study design, size, duration A translational research study in IVF patients. Participants/materials, setting, methods 55 normal responding IVF patients underwent ovarian stimulation either with PPOS using medroxy progesterone acetate (5mg twice daily) or, with GnRH antagonist cetrorelix acetate were included in the study. Recombinant forms of FSH and hCG were used for ovarian stimulation and ovulation trigger, respectively. Luteinized mural granulosa cells obtained during oocyte retrieval procedure were used for the experiments. Cell culture, quantitative real-time PCR, immunoblotting, confocal time-lapse live cell imaging and hormone assays were used. Main results and the role of chance Demographic and IVF characteristics of the patients undergoing ovarian stimulation with PPOS vs. GnRH antagonist were similar including ovarian-response, mature oocyte yield and fertilization rates. Molecular analyses revealed that the expression of the enzymes involved in sex-steroid synthesis (StAR, aromatase, 3B-HSD, 17B-HSD) and uptake/storage/utilization of cholesterol (LDL receptor, Hormone-sensitive lipase, hydroxy-methyl glutaryl Co-enzyme-A reductase and Sterol O-acyltransferase1) in the GCs of the PPOS cycles were comparable to the antagonist IVF cycles. Both cycles were also similar for the expression of FSH and LH receptors at transcriptional and translational level. Basal and hCG-induced increases in 3B-HSD expression and P4 production, and basal and FSH-induced increases in aromatase expression and E2 output of the GCs of the PPOS patients did not show any meaningful differences from the antagonist cycles. Similarly, basal and hCG-induced up-regulation in the LDL receptor expression and cholesterol uptake rate did not differ between the groups. Confocal imaging also revealed similar patterns of expression for the steroidogenic enzymes and their co-localization with mitochondria. Lastly, the expression of the other genes regulating cumulus expansion, ovulation and luteal function (Relaxin, ADAMTS-1 and EGF-like growth factor amphiregulin (AREG)) in the GCs of the PPOS cycles were comparable to the antagonist cycles. Limitations, reasons for caution Caution should be exercised when interpreting our data, which was produced in normal responders. It is unclear if the molecular parameters assessed here vary according to infertility etiologies, ovarian response type, mode of trigger and any other underlying ovarian pathology or systemic diseases that are concomitantly present. Wider implications of the findings This study provides reassuring molecular evidence that exposure of antral cohorts to progestins follicular growth phase does not appear to have any detrimental effect on their steroidogenic, ovulatory and luteal functions, suggesting that PPOS IVF cycles are not different from traditional antagonist cycles. Trial registration number not applicable

Follicular Fluid and Blood Monitorization of Infertility Biomarkers in Women with Endometriosis.

Infertility is recognized globally as a social disease and a growing medical condition, posing a significant challenge to modern reproductive health. Endometriosis, the third-most frequent gynecologic disorder, is one of the most common and intricate conditions that can lead to female infertility. Despite extensive research, the etiology, malignant transformation, and biological therapy of endometriosis remain unknown. Blood and follicular fluid are two matrices that have been carefully studied and can provide insights into women's health. These matrices are clinically significant because they contain metabolites closely associated with women's illness stage and reproductive outcomes. Nowadays, the application of metabolomic analysis in biological matrices may be able to predict the outcome of assisted reproductive technologies with greater precision. From a molecular viewpoint on reproductive health, we evaluate and compare the utilization of human follicular fluid and blood as matrices in analysis for diagnostic and assisted reproductive technology (ART) predictors of success for endometriosis patients. In the follicular fluid (FF), plasma, and serum of endometriosis-affected women, researchers identified dysregulations of oxidative stress, upregulation of several immune factors, and aberrations in energy metabolic pathways. The altered signatures negatively correlate with the overall oocyte and embryo quality and fertilization rate.

Role of Menstrual Blood Stem Cell-Derived Secretome, Follicular Fluid, and Melatonin in Oocyte Maturation and Embryo Development in Polycystic Ovary Syndrome.

In vitro maturation has been considered an approach to mature oocytes derived from women with polycystic ovary syndrome (PCOS). It is suggested that the IVM of oocytes may benefit from mesenchymal stem cells derived conditioned medium (CM-MSC). The purpose of this study was to determine the efficacy of a cocktail of menstrual blood stem cell (MenSCs)-derived secretome, along with follicular fluid and melatonin, in oocyte maturation and embryo development in PCOS. Four hundred left germinal vesicle oocytes were collected from 100 PCOS patients and randomly divided into four treatment groups: 1) control, 2) secretome, 3) follicular fluid, and 4) melatonin. Oocyte maturation, fertilization rate, and embryo development were monitored, as well as the expression levels of oocyte-secreted factors (GDF9- BMP15), oocyte maturation (MPK3), and apoptosis (BAX- Bcl2). The rate of oocyte maturation increased in all test groups, but only the results for the SEC group were significant (P= 0.032). There were no significant differences in oocyte fertilization and embryo yield among groups. However, the quality of embryos significantly increased in the melatonin group compared to the control. Cytoplasmic maturation was confirmed by the expression of oocyte maturation-related genes using Real-time PCR. Additionally, the expression level of BCL-2 was significantly higher in the SEC-FF-MEL group than in the control group (p ≤ 0.01). Enrichment of IVM media using MenSCs-secretome, particularly along with melatonin, could be an effective strategy to improve oocyte maturation and embryo development in PCOS.

Association of Anti-Müllerian Hormone on Oocyte Maturation, Fertilization, and Pregnancy Rates in Patients under Assisted Reproductive Technology Cycles: A Cross-Sectional Study.

Ovarian reserve is one of the most important factors that influences the success of assisted reproductive technology (ART). Recently, the role of anti-müllerian hormone (AMH) in ART has been investigated as a marker for the prediction of ovarian response. We aim to examine this relationship within a large Iranian population. In this cross-sectional study, we obtained data from 1000 infertile couples who referred to the Research and Clinical Centre of Yazd Infertility Clinic for in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI). Serum AMH levels, oocyte count, numbers of fertilised oocytes, endometrial thickness, and percentage of mature oocytes were measured. The relationship between AMH serum levels and the number and quality of oocytes and embryos in ART cycles was analysed. In the linear regression model, the log of the variables total dose of gonadotropin, two pronuclei (2PN), log oestradiol, total embryos, duration of stimulation, number of embryos transferred, protocol, and cause of infertility were significant predictors of log AMH. There appears to be a relationship between serum AMH levels in the early follicular phase and ovarian reserve. Higher serum AMH levels were also associated with shorter ART cycles.

Impact of Body Mass Index (BMI) and Age on in Vitro Fertilization (IVF) Outcome in Indian Patients with Polycystic Ovary Syndrome (PCOS)

Aim: The study evaluated the effect of body mass index (BMI) and age on in vitro fertilization (IVF) outcomes in women with polycystic ovary syndrome (PCOS). Methods: A retrospective study of 412 IVF cycles in PCOS patients aged between 20 and 40 years from January 2019 to 2022. Patients were divided into three groups based on their BMI-normal (18.5–24.9[Formula: see text]kg/m2), overweight (25–29.9[Formula: see text]kg/m2), and obese ([Formula: see text][Formula: see text]kg/m2). The patients were divided into two groups based on their age [Formula: see text] and [Formula: see text] years. The IVF cycle outcomes, clinical pregnancy (CPR), and live birth rates (LBR) were studied. The Mantel–Haenszel Chi-square test was used for statistics. Results: The oocyte yield and endometrial thickness, fertilization, CPR, and LBR were significantly higher in the normal BMI group. Similarly, oocyte yield, mature oocytes, and endometrial thickness were significantly higher in the younger age-group. Comparing the CPR in younger and older normal, overweight, and obese groups, it was observed to be highest in the younger, normal BMI group and lowest in the older, obese group. The LBR were significantly higher in the younger, normal-BMI group compared to the older, obese patients. Conclusion: Oocyte yield, CPR, and LBR were higher in the normal-BMI PCOS patients. The oocyte yield, endometrial thickness, and fertilization rates were higher in the younger age group. The CPR and LBR were significantly lower in the older obese patients. Patients with PCOS must be encouraged to normalize BMI before embarking on IVF. Timely treatment must be encouraged for better IVF outcomes.

Pretreatment with oral contraceptive pills in women with PCOS scheduled for IVF: a randomized clinical trial

Abstract STUDY QUESTION What is the effect of pretreatment with oral contraceptive pills (OCPs) on oocyte and embryo quality and pregnancy rates in women with polycystic ovary syndrome (PCOS) scheduled for IVF/ICSI cycles? SUMMARY ANSWER In women with PCOS who underwent a first or second IVF/ICSI cycle with a GnRH antagonist protocol and were randomized to start ovarian stimulation immediately, the quality of cleavage-stage embryos was non-inferior to pretreatment with OCP. WHAT IS KNOWN ALREADY PCOS in Asian populations is characterized by high levels of circulating LH in the early follicular phase. Previous studies indicated that inappropriately high LH levels might affect oocyte maturation and fertilization rates, and impaired embryo quality, consequently resulting in higher rates of impaired pregnancy and miscarriage in women with PCOS. OCPs are frequently used as pretreatment to lower LH levels in PCOS patients. STUDY DESIGN, SIZE, DURATION We performed a randomized controlled trial. After informed consent, women diagnosed with PCOS scheduled for their first or second IVF/ICSI cycle with a GnRH antagonist protocol were randomized to receive OCPs (OCP group) or start ovarian stimulation immediately, regardless of the day of the menstrual cycle (non-OCP group). Using a non-inferiority hypothesis, the sample size was calculated at 242 women. The study lasted from 7 February 2018 to 31 August 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS A total of 242 infertility patients with PCOS undergoing the first or second cycle of IVF or ICSI were enrolled and randomized into two groups. In the OCP group, recombinant FSH was started on Day 7 of the washout period after pretreatment with OCP. In the non-OCP group, recombinant FSH was started immediately regardless of the day of the menstrual cycle. All participants received standardized GnRH antagonist ovarian stimulation. The freeze-all strategy was applied to all participants. The primary outcome was the number of good-quality embryos on Day 3 after insemination. Secondary outcomes included the rates of blastocyst formation, implantation, clinical pregnancy, and live birth from the first frozen/warmed embryo transfer cycles and cumulative live birth rates. MAIN RESULTS AND THE ROLE OF CHANCE We randomized 242 women to receive OCP (n = 121) or start immediately with ovarian stimulation (n = 121). The number of good-quality embryos on Day 3 in the OCP group was non-inferior to the non-OCP group (OCP group versus non-OCP group, 6.58 ± 4.93 versus 7.18 ± 4.39, AD −0.61, 95% CI: −1.86 to 0.65, P = 0.34). The rates of blastocyst formation (55.4% versus 52.9%, relative risk (RR) 1.11, 95% CI: 0.96 to 1.28, P = 0.17), implantation (63.0% versus 65.5%, RR 0.90, 95% CI: 0.53 to 1.53, P = 0.79), clinical pregnancy (67.9% versus 68.8%, RR 0.96, 95% CI: 0.54 to 1.71, P = 1.0), and live birth rate (52.8% versus 55.1%, RR 0.92, 95% CI: 0.53 to 1.56, P = 0.79) of the first frozen/warmed embryo transfer cycles were all comparable between the OCP and non-OCP group, respectively. Cumulative live birth rates were also similar in the OCP and non-OCP groups (78.3% versus 83.5%, respectively RR 0.71, 95% CI: 0.36 to 1.42, P = 0.39). LIMITATIONS, REASONS FOR CAUTION Only patients with PCOS in Southern China were recruited. Therefore, caution is necessary when generalizing our results to all such patients with PCOS. Also, since a freeze-only strategy was used, the results of this study are only applicable when infertile women with PCOS undergo the freeze-only method. The obvious treatment difference between the two groups meant that the study was designed as an open-label study for women and doctors. The study had a randomized controlled design that minimized bias. WIDER IMPLICATIONS OF THE FINDINGS Pretreatment with OCPs to lower LH levels in patients with PCOS before ovarian stimulation in IVF or ICSI cycles may not improve the quality of cleavage-stage embryos. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by the National Key Research and Development Program of China (No. 2023YFC2705503). This study was supported in part by the Investigator-Initiated Studies Program (grant from MSD and Organon). BWM reports consultancy, travel support, and research funding from Merck. He reports consultancy from Organon and Norgine, and also reports holding stock from ObsEva. No conflicts of interest are declared for the other authors. TRIAL REGISTRATION NUMBER Chinese Clinical Trial Registry (No. chiCTR1800014822). URL: https://www.chictr.org.cn/showproj.html?proj=25280 TRIAL REGISTRATION DATE 7 February 2018. DATE OF FIRST PATIENT’S ENROLLMENT 22 February 2018.

Structural and metabolic cumulus cell alteration affects oocyte quality in underweight women.

This study aimed to investigate the structural and metabolic changes in cumulus cells of underweight women and their effects on oocyte maturation and fertilization. The cytoplasmic ultrastructure was analyzed by electron microscopy, mitochondrial membrane potential by immunofluorescence, and mitochondrial DNA copy number by relative quantitative polymerase chain reaction. The expression of various proteins including the oxidative stress-derived product 4-hydroxynonenal (4-HNE) and autophagy and apoptosis markers such as Vps34, Atg-5, Beclin 1, Lc3-I, II, Bax, and Bcl-2 was assessed and compared between groups. Oocyte maturation and fertilization rates were lower in underweight women (P < 0.05), who presented with cumulus cells showing abnormal mitochondrial morphology and increased cell autophagy. Compared with the mitochondrial DNA copies of the control group, those of the underweight group increased but not significantly. The mitochondrial membrane potential was similar between the groups (P = 0.8). Vps34, Atg-5, Lc3-II, Bax, and Bcl-2 expression and 4-HNE levels were higher in the underweight group compared with the control group (P < 0.01); however, the Bax/Bcl-2 ratio was lower in the underweight group compared with the control group (P = 0.031). Additionally, Beclin 1 protein levels were higher in the underweight group compared with the control group but without statistical significance. In conclusion, malnutrition and other conditions in underweight women may adversely affect ovulation, and the development, and fertilization of oocytes resulting from changes to the intracellular structure of cumulus cells and metabolic processes. These changes may lead to reduced fertility or unsatisfactory reproduction outcomes in women.

Automation in vitrification and thawing of mouse oocytes and embryos.

Vitrification is a common technique for cryopreserving oocytes or embryos. However, manual vitrification is tedious and labor-intensive, and can be subject to variations caused by human factors. To address these challenges, we developed an automated vitrification-thawing system (AVTS) based on a cryo-handle. Our study firstly assessed the efficiency of cryoprotectant exchange through comparing the osmolalities of fresh and collected solutions during automated vitrification and thawing, and evaluated the cooling and warming rates of the cryo-handle. We also compared mouse oocyte survival, fertilization and embryo development after thawing and ICSI, and the development of re-frozen cleavage embryos between manual operation and automated system. The results showed that the osmolalities of collected samples were within normal range and comparable to fresh solutions. Furthermore, the automated system could obtain the reliable cooling and warming rates. Particularly, there were no significant differences in oocyte survival rates, fertilization rates, and subsequent embryo development and its quality between two procedures. Our findings suggest that AVTS has no impact on osmolalities of vitrification and thawing solutions, ensuring the proper exchange of cryoprotectants. The cryo-handle also shows the ability to achieve reliable cooling and warming rates, which benefits for the cryopreservation and thawing process. Moreover, the results from mouse oocytes and embryos indicate that automated system has effectively maintained the survival and fertilization of frozen oocytes and supported subsequent embryo development. Therefore, the automated vitrification and thawing system will inevitably represent a superior alternative to manual operation.

138 Evaluation of dimethyl sulfoxide and ethylene glycol on fertilization rate of pig oocytes

138 Evaluation of dimethyl sulfoxide and ethylene glycol on fertilization rate of pig oocytes

#100 : Can the Dual Trigger be Effective in Improving Oocyte Maturation and Embryo Quality Compared to the HCG Trigger or the GnRH-a Trigger?

Background and Aims: HCG was the standard trigger of final oocyte maturation in IVF cycles. An alternative trigger with gonadotropin releasing hormone agonist (GnRH-a) was effective in reducing OHSS. But GnRH-a trigger induced increased luteal insufficiency and cycle cancellation. A dual trigger (HCG with GnRH-a) was introduced to reduce OHSS, luteal insufficiency and cycle cancellation. This study was designed to compare the effect of dual trigger on final oocyte maturation n embryo quality with conventional HCG trigger and GnRH-a trigger. Method: We conducted this retrospective cohort study of patients who underwent IVF antagonist cycles from Jan. 2020 to Dec. 2022. Patients undergoing their oocyte retrieval 36 hours after trigger with only HCG 10,000 units, or only leuprolide acetate 40 units, or a dual trigger of HCG 5000 units with leuprolide acetate 40 units. Male factor infertility and oocyte donor cycle were excluded. The main outcomes were retrieved mature oocyte rate and fertilization rate. Secondary outcomes include implantation rate and pregnancy rate. Results: Total 3266 patients were included. Dual trigger was in 2279, GnRH-a in 282 and HCG in 705 cycles. Patients using GnRH-a were younger (33.7± 4.0 years) compared to those receiving dual trigger (37.8±2.3 years) and HCG only (37.2±3.2 years) (p&lt;0.01). Because GnRH-a groups were younger, despite low total gonadotropin dose, the larger number of oocytes were retrieved. Dual trigger resulted in no difference in mature oocyte ratio, compared to HCG trigger and GnRH-a trigger (63.0%, 58.3%, 62.8% p: 0.074). Analysis did not show any statistical differences in fertilization rate between the types of triggering methods. Cycel cancellation due to no oocyte recovered from multiple follicles aspirated was shown only in GnRH-a cycle (6 cases. 2.1%). Conclusion: Compared to HCG trigger and GnRH-a trigger, the dual trigger showed no difference in mature oocyte rate and fertilization rate. To reduce OHSS and cycle cancellations, dual trigger could be considered the standard care in IVF without decreasing oocyte quality.