Immature Oocytes Research Articles

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 < 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-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-228 Competence of injected MI versus MII sibling oocytes in terms of normal fertilization, blastocyst development and euploidy

Abstract Study question Is the injection of immature MI oocytes as effective as injection of their siblings mature MII oocytes regarding normal fertilization, blastocyst development and euploidy? Summary answer MI oocytes show a lower rate of fertilization, a similar blastulation rate, and a slightly lower rate of euploidy compared to their MII sibling oocytes. What is known already Although several studies have been performed on immature MI oocytes evaluating their developmental competence after intracytoplasmic sperm injection (ICSI), all have been performed on oocytes that extruded the first polar body by the time of injection. ICSI is typically performed on MII oocytes which have completed the process of maturation. Some laboratories also inject MI oocytes, nevertheless, they are generally not used due to the lack of required molecular and cellular changes to support normal development. However, there is limited research on the reproductive potential of oocytes in MI. Consequently, our current practices are founded on assumptions. Study design, size, duration This is a retrospective, observational study of 168 oocytes injected in MI and 839 sibling oocytes injected in MII from 82 ICSI cycles with Preimplantation Genetic Testing for Aneuploidies (PGT-A) performed in a single IVF clinic between April 2018 and December 2023. All PGT-A cycles included in this study contained at least one MI oocyte injected. Participants/materials, setting, methods After oocyte retrieval and denudation, the oocytes were classified as MII, MI or GV. MI oocytes with extruded polar body by the moment of ICSI were reclassified as MII. All MII and MI oocytes were inseminated and the embryos that reached blastocyst stage were biopsied on day 5, 6 or 7. Normal fertilization rate, blastocyst formation rate and euploidy rate were compared between MI-derived embryos and their MII-derived siblings. Main results and the role of chance A total of 1,094 oocytes were retrieved from 83 ICSI cycles with PGT-A, comprising 839 (76.7%) MII, 168 (15.4%) MI y 87 (7.9%) VG. The normal fertilization rate was significantly higher in oocytes injected at MII compared to MI (76.9% vs 53.0%, P < 0.001). The rates of abnormal fertilization in MII and MI oocytes were as follows: 1 PN 0.8% vs 0.6%, P = 0.342; 3PN 2.6% vs 1.2%, P = 0.075; 0 PN 19.7% vs 45.2%, P < 0.001. Interestingly, the blastulation rate of zygotes with normal fertilization was very similar between both groups (46.4% vs 43.8%, P = 0.708), as well as the proportion of biopsied blastocysts on day 5 (74.2% vs 71.8%, P = 0.858), on day 6 (23.1% vs 25.6%, P = 0.946), and on day 7 (2.7% vs 2.6%, P = 0.594). The euploidy rate of the 298 biopsied MII-derived blastocyst was 36.2%, compared with 23.1% of the 39 MI-derived blastocysts biopsied (P = 0.077). Despite the limited sample size, it is noteworthy that three out of the four euploid blastocysts derived from MI oocytes, which were transferred in single embryo transfers, successfully led to pregnancy resulting in one healthy live birth, one ongoing pregnancy and one miscarriage at 9 weeks of pregnancy. Limitations, reasons for caution While this study provides valuable insights into the developmental competence of MI oocytes, it is not exempt from certain limitations, such as its retrospective nature, limited sample size and ethical considerations regarding the injection of putative immature oocytes. Wider implications of the findings MI oocytes showed lower fertilization rates compared to their MII siblings. However, once fertilized, MI-derived embryos had similar blastulation rates and a trend towards lower euploidy rates. Our study suggests that MI oocytes may result in successful pregnancies and live birth, but further research is needed to validate these findings. Trial registration number Not applicable

P-558 Characterization of the morphology of the outer-kinetochore expandable module, aka fibrous corona, and its role in ensuring proper chromosome segregation in human and bovine oocytes

Abstract Study question How does the Fibrous Corona (FC) behave in human and bovine oocyte meiosis and what is its function in chromosome segregation during meiosis I? Summary answer In human and bovine oocytes, FC shows extreme plasticity expanding beyond the centromeric region of the chromosomes. Pharmacological inhibition of FC’s expansion induces chromosome mis-segregation. What is known already Embryonic aneuploidy of meiotic origin is one of the major causes of implantation failure and early pregnancy loss. Erroneous microtubule (MT)-kinetochore attachments in human meiosis are the main cause of meiotic chromosome segregation errors. In somatic cells, the FC, a multiprotein complex, assembles on kinetochores and expands around the centromere during prometaphase to facilitate MT interactions and stimulate correct MT-kinetochore attachment. We have shown that in bovine oocytes FC expand beyond the centrosomes and is a potent MTs nucleator (10.1016/j.cub.2023.01.010). However, it is still unknown how FC behaves and what are its functions in MT-kinetochore interactions in human oocytes. Study design, size, duration FC morphology was studied in immature human oocytes discarded during ICSI procedures and donated (informed consent) by the patients for research (Ethical permit TCBio.21-754) during the years 2021-2024. Because of ethical concerns and reduced availability of human oocytes, functional studies on FC were performed on bovine oocytes collected from slaughterhouse ovaries. Participants/materials, setting, methods 180 immature human oocytes were matured in vitro and either fixed at different meiosis I stages or treated 2h with colcemid, a MT depolymerizing drug, to induce maximum FC expansion and fixed. The oocytes were then immuno-stained for chromatin(DAPI), FC(ZW10), MT(alpha tubulin) and kinetochores(CREST) and imaged with super-resolution confocal microscopy. Bovine immature oocytes were matured in vitro with or without lonafarnib (inhibitor of FC expansion). After maturation chromosome spreads were performed to evaluate oocyte ploidy. Main results and the role of chance In human oocytes, after nuclear envelope breakdown, before microtubule appearance when the chromosomes appear compacted, FCs are visible on the top of the kinetochores. During MT nucleation, FCs form thick mesh in the center of the compacted chromosomes. During prometaphase I, FCs expand beyond the centromeric region of the chromosomes forming a cup around the sister kinetochores of the paired homologous chromosomes. When the chromosomes starts to align at the metaphase plate, FCs start to be stripped from the kinetochores and colocalize to the MTs. At metaphase I and II, only kinetochore localization is visible. When oocytes are treated with colcemid, FCs extend to partially cover chromosome arms. We observed a wide variability in FCs expansion between and within patients. Inhibition of FC expansion in bovine oocytes increase the incidence of oocyte aneuploidy (90% in lonafarnib treated vs 37% in control oocytes). Altogether, our data reveal that in human oocytes FCs expand more compared to human somatic cells. Considering that inhibition of FC expansion induces chromosome segregation errors, we propose that the large FC size in human oocytes may play a role in achieving correct chromosome-MT attachments within their extremely large cytoplasm thus ensuring proper chromosome segregation. Limitations, reasons for caution The limited availability of human oocytes requires the use of an animal model for in depth mechanistic studies. Although bovine oocytes have similar aneuploidy rate and spindle assembly mechanisms as human oocytes, they show morphological differences in FC expansion which could underlie intrinsic functional discrepancies between the two organisms. Wider implications of the findings A better understanding of the chromosome segregation mechanisms in human meiosis will help understanding what causes the high incidence of aneuploidies in human oocytes and possibly contribute to the development of interventions or of oocyte selection procedures to reduce the incidence of oocyte aneuploidy during in vitro maturation. Trial registration number Not applicable

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

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

O-058 Newly identified female mutations causing oocyte maturation defect and embryo developmental arrest

Abstract Female infertility is a complex issue affecting a significant number of women. In the United States, among married women aged 15–49 years with no prior births, approximately one in five (19%) were unable to get pregnant after one year of trying. In addition, approximately one in four (26%) women in this group had difficulty getting pregnant or carrying a pregnancy to term. For women under 35 years of age, infertility is defined as a lack of success within one year, while for women aged 35 and older, it is within six months. Various factors, such as genetic, endocrine, physiological, anatomical, and immunological irregularities within the reproductive system, can affect a woman's chances of achieving pregnancy and delivering a healthy child. Among these factors, oocyte maturation is an important prerequisite for successful fertilization and subsequent embryonic development. In Medically Assisted Reproduction (MAR), the meiotic maturation of oocytes is induced by human chorionic gonadotropin (hCG) injection, which mimics the natural endogenous luteinizing hormone (LH) surge during the menstrual cycle. Although it is common for a few oocytes to remain immature despite ovarian stimulation and hCG administration, generally, immature oocytes have the competency to spontaneously mature in vitro. However, oocyte maturation is a sophisticated process involving multiple genes or protein molecules. Therefore, pathogenic variations in the genomic sequence or functional defects in any molecule that affects meiosis may lead to impaired oocyte maturation. While complete failure of all oocytes to mature in vivo is extremely rare, understanding the molecular aetiology of oocyte maturation is crucial for addressing infertility issues related to this process and improving fertility outcomes. The transition from the cleavage to the blastocyst stage often represents a significant bottleneck, with nearly 10% of fertilized eggs arrested at the cleavage stages. Developmental arrest of the preimplantation embryo is characterized by the cessation of cellular division for at least 24 h. Various factors originating from both embryonic and parental sources contribute to arrest of early stage embryo development. The genetic roots of developmental arrest were examined by considering both the parental causes of infertility and factors within the embryo. Examination revealed shared elements, regardless of the source of origin. Chromosomal abnormalities, abnormal preimplantation development, and monogenic variations have been reported to cause embryonic developmental arrests. Recently, deficiencies in oocyte maturation and embryo development have been categorized as part of the oocyte/zygote/embryo arrest (OZEMA) phenotype in the Online Mendelian Inheritance in Man (OMIM) database. A latest extensive review of monogenic causes associated with female infertility identified 23 genes with a total of 27 gene disease relationships (GDRs) for the OZEMA phenotype. Seventeen GDRs were classified as having at least moderate evidence of involvement in OZEMA, in addition to ten GDRs with less substantial evidence. Several additional genes were subsequently identified. This presentation will provide an overview of gene defects linked to the OZEMA phenotype, discuss the current status and future prospects of pre-MAR screening of females with OZEMA, and assess a novel workflow for modern genetic diagnosis to maximize the benefit of the patient.

P-026 Retrospective descriptive analysis of 1300 ICSI-AOA cycles: evaluating clinical outcomes and child health in the context of artificial oocyte activation (AOA)

Abstract Study question How does the outcomes of ICSI-AOA and child health differ from those reported in the literature? Summary answer ICSI-AOA clinical outcomes, including fertilization, embryo quality, pregnancy, live birth rates, and anomaly rates in children, align with those reported for ICSI alone. What is known already The use of AOA to artificially induce calcium rises by employing calcium ionophores has been shown as an effective approach in overcoming fertilization failure following ICSI, especially in couples facing severe male factor infertility. However, a significant gap exists in reporting live birth outcomes. On the other hand, uncertainties surrounding the health implications for children born through this method necessitate further investigation due to the limited sample sizes in previous reports. This descriptive analysis fills that gap by assessing 1300 cases, providing crucial insights into the clinical outcomes and health status of babies resulting from ICSI-AOA treatment. Study design, size, duration This retrospective study analyzes 1300 cases of ICSI-AOA between 2018 and 2020. Clinical outcomes in this study includes: fertilization, embryo quality, pregnancy, and live birth rates. Questionaries regarding the children health were included from birth to two years. Participants/materials, setting, methods The study included 1300 cases that referred to Isfahan Fertility and Infertility Center (Isfahan, Iran) and underwent ICSI-AOA due to: 1) cases with testicular biopsy (N = 458), 2) cases with 99-100% sperm head abnormal morphology and/or 3) previous low fertilization or total fertilization failure after ICSI (N = 842). In all cases, to induce AOA, oocytes were subjected to a 10-minute exposure of 10 micromolar ionomycin following ICSI. Main results and the role of chance From 1300 couples, 11,756 mature, 2,012 immatures, 193 degenerate, and 76 uninjectable oocytes were obtained. Fertilization were 59.69%, and 52.18% for mature and immature oocytes, resulting in cleavage rates of 96.93% and 93.80%, respectively. On day 3, 25.14% grade A, 31.49% grade B, and 43.36% grade C were observed. In 321 couples, 571 fresh embryos were transferred on day 3, resulting in clinical pregnancy and live birth rates of 20.82%, and 19.55%, respectively. For 32 couples with 44 fresh ET on day 5, rates were 9.38%, and 9.38% respectively. Out of 3270 vitrified embryos on day 3, 1743 were thawed (337 couples) and 1147 were transferred in 643 ET cycles. Clinical pregnancy and live birth rates were 21.61%, and 20.37%, respectively. 1527 embryos remain frozen. Out of 2,974 day 5-embryos, 2,571 did not reach blastocyst, leaving 403 vitrified embryos in 699 cases. Among them, 197 embryos remain frozen (80 cases) while 206 were transferred, with clinical pregnancy and live birth rates of 22.11%, and 21.80%, respectively. Cancellation rate, encompassing failed fertilization, cancellations on multiple days, and after freeze-thawing, was 20.16%. Among 266 live births, seven children showed anomalies, including achondroplasia, combined immunodeficiency, left eye deviation, intrauterine growth restriction, hydrocephaly, epilepsy, and umbilical hernia. Limitations, reasons for caution The main limitations of this study were the lack of comprehensive information on previous cycles, the absence of an ICSI control group, and the limited long-term follow-up on the physical and mental health of children born through the ICSI-AOA method beyond age two. Wider implications of the findings ICSI-AOA yields helpful clinical outcomes in high fertilization, pregnancy, and live birth rates in infertile couples, especially those with severe male factor infertility similar to ICSI in literature. Furthermore, presently, there seem to be no real concerns about health of babies born through this method, aligning with ICSI studies. Trial registration number IR.ACECR.ROYAN.REC.1401.038

P-171 Mitochondrial complex I, a key component of the mitochondrial respiratory chain, is necessary for maintaining chromosomal stability during the final stages of oocyte maturation

Abstract Study question What is the role of mitochondrial complex I during the final stages of human oocyte maturation? Summary answer Mitochondrial complex I is necessary for the cytoplasmic maturation of germinal vesicle (GV) oocytes and the subsequent organization of the meiotic spindle. What is known already Mitochondria play a pivotal role in oogenesis. Primordial human oocytes are characterized by an abundance of immature mitochondria, which exhibit low activity and a diminished capacity to produce adenosine triphosphate (ATP). During oocyte recruitment, mitochondrial fusion and fission play an essential role, contributing to oocyte maturation and oocyte developmental competence. Furthermore, an increase in ATP levels during polar body extrusion has been associated with higher fertilization rates, suggesting a reliance on mitochondria as ATP suppliers during oocyte maturation. However, the precise role of mitochondria during the final stages of human oocyte maturation is yet to be understood. Study design, size, duration At total of 99 immature oocytes, including 56 GVs and 40 metaphase I (MI) oocytes, were included in the study. These were obtained from 40 oocyte donation cycles, performed at a single IVF centre between August 2023 and September 2023. Participants/materials, setting, methods Immature denuded oocytes were cultured in G2TM PLUS medium to reach nuclear maturation (rescue in vitro maturation; rIVM). rIVM was performed in the absence (0.1% DMSO, control) or presence of 0.5 µM Rotenone (an inhibitor of mitochondrial complex I) or 10 µM FCCP (an uncoupler of mitochondrial oxidative phosphorylation). To assess mitochondrial and nuclear status, we performed immunofluorescence analyses for TOMM20 and the meiotic spindle (DNA and TUBA). Main results and the role of chance After 40 hours of culture, 64.3% of control GV oocytes progressed to the MII stage, 7.1% reached MI, while 21.4% arrested, and 7.1% degenerated. Control MI oocytes showed a 90% rIVM rate after 24 hours, with none arresting and 10% degenerating. Different mitochondrial inhibitors had varied effects on GV and MI oocytes. FCCP resulted in the degeneration of all treated oocytes. When treated with rotenone, 87.5% of GVs advanced to the MI stage, after which they arrested, but no signs of degeneration were observed. Notably, 100% of MIs and MIIs derived from GVs subjected to rotenone treatment presented with misaligned chromosomes, compared to 33.3% in the control group. When starting from MIs, the rIVM rate decreased to 41.7% following rotenone treatment, with no signs of degeneration. Here, misalignments were observed in 40% of the rIVM-MII oocytes and 43% of those that arrested, compared to 33.3% in controls. These results suggest that both GV and MI oocytes have adequate complex I activity, essential for proper nuclear maturation at the MII stage. We further highlight the importance of complex I activity, especially in GVs, for processes such as polar body extrusion during oocyte maturation. Limitations, reasons for caution Rescue in vitro maturation poses a challenge due to its limited effectiveness in achieving sufficient MII developmental competence. Consequently, mitochondrial inhibitors may have unforeseen effects on mitochondria and spindle formation. The specific role of complex I should be confirmed by employing other complex I inhibitors not related to rotenone. Wider implications of the findings Our results suggest that complex I activity, a key component of the mitochondrial respiratory chain, plays a crucial role in spindle organization during oocyte maturation. This relationship between mitochondrial function and chromosomal stability in oocytes suggests that interventions targeting the mitochondrial respiratory chain may be promising for enhancing oocyte quality. Trial registration number Not applicable

O-005 The in vitro maturation rate of human oocytes is enhanced following uptake of extracellular vesicles derived from mature follicles

Abstract Study question Could follicular fluid derived extracellular vesicles (ffEVs) impact human oocyte in vitro maturation (IVM)? Summary answer Supplementation of IVM culture with ffEVs isolated from mature follicles enhanced oocyte maturation rates by > 20%, inducing changes in oocyte protein profile and organelle distribution. What is known already IVM involves the culture of immature germinal vesicle (GV) oocytes under set laboratory conditions to allow for their transition to mature metaphase II (MII) stage, which is confirmed by the extrusion of the first polar body. Efficient IVM could circumvent aggressive controlled ovarian stimulation (COS), reduce the cost and broaden the repertoire of infertility treatments. Animal studies suggest that extracellular vesicles (EVs), membranous nanosized vesicles containing different molecular content (e.g. nucleic acids, proteins) and present in the ovarian follicular fluid could facilitate oocyte maturation. The uptake of ffEVs by bovine, equine and feline oocytes, but not human, has been demonstrated. Study design, size, duration Women undergoing transvaginal oocyte retrieval after COS (n = 83) at University Hospital Zurich donated follicular fluid (n = 54 single follicles) and/or immature GV oocytes (n = 95). We aimed to: a) define differences in the protein cargo of ffEVs derived from follicles containing mature oocytes (MII-ffEVs, n = 10) versus (vs.) immature oocytes (GV-ffEVS, n = 5; metaphase I MI-ffEVs, n = 5), b) demonstrate the capacity of GV oocytes to uptake mature MII-ffEVs and c) determine the effect of MII-ffEVs supplementation on oocyte maturation. Participants/materials, setting, methods ffEVs were isolated by ultracentrifugation. The protein content of ffEVs was analysed by mass spectrometry. The uptake of fluorescently-labelled MII-ffEVs by GV oocytes (n = 15) was assessed by confocal microscopy. GVs were cultured for rescue-IVM in a timelapse incubator with MII-ffEVs (n = 45 GVs) or without (n = 40 GVs) and extrusion of polar body denoted maturation. The impact of MII-ffEVs supplementation on IVM-matured oocytes was assessed through single-cell proteomics and intracellular organelles appearance with transmission electron microscopy (TEM). Main results and the role of chance Successful isolation of ffEVs was confirmed by TEM and Western blotting analysis for characteristic protein markers of EVs (TSG101 and CD63). Mass spectrometry identified a total of 1340 proteins across ffEVs samples. Statistical analysis revealed differentially abundant proteins (DAPs) in ffEVs: 61 DAPs in the comparison MII-ffEVs vs. GV-ffEVs, 15 DAPs in MII-ffEVs vs. MI-ffEVs and 14 DAPs in MI-ffEVs vs GV-ffEVs (FDR<0.2). Among the 61 DAPs, 7 proteins in higher abundance in MII-ffEVs (>log2 fold change) were related to oocyte maturation, lipid accumulation, calcium binding and the coagulation cascade (e.g. FABP5, S100A9, F12, SERPINA10). Confocal microscopy confirmed the uptake of MII-ffEVs by GV oocytes after 24 hours of co-incubation. Supplementation of IVM culture with MII-ffEVs for 48 hours significantly increased the oocyte maturation rate compared to control by 22.8±9.4% (77.8% vs 55% mature oocytes respectively; P-value=0.0372). Single oocyte proteomics of IVM-matured oocytes revealed 59 DAPs (FDR 0.1) between ffEVs-supplemented and control groups. Among them, 37 DAPs were in higher abundance in ffEVs-supplemented mature oocytes (>log2 FC) including Hyaluronan Synthase 1 (HAS1) that is associated with oocyte maturation (6.55 fold increase). TEM revealed differences in oocyte organelle distribution and appearance, particularly of endoplasmic reticulum (RE) and RE-mitochondria complexes. Limitations, reasons for caution This study utilised immature oocytes from stimulated cycles, therefore these results should be interpreted within the context of rescue-IVM potential. Wider implications of the findings Our findings pave the way for further mechanistic studies on the role of ffEVs in supporting oocyte maturation and inspire the development of clinical IVM supplements as well as novel complementary regimes for COS based on ffEVs or their associated specific cargo. Trial registration number N/A

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

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

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

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

P-117 Extracellular vesicles from fallopian tubal fluid benefit human embryo development in vitro

Abstract Study question Do extracellular vesicles (EVs) from Fallopian tubes benefit human embryo development, and how do they affect preimplantation embryo development? Summary answer The Fallopian tubal EVs can be internalized by human embryos, and they contain miR-21-5p that downregulated P53 in blastocysts. What is known already EVs in the Fallopian tubes are identified as key mediators in embryo-oviduct interactions that contribute to a successful pregnancy in vivo. We previously showed Fallopian tubal EVs can benefit mouse embryo development in vitro. However, if a similar phenomenon can be observed in human embryos and what is the key component in Fallopian tubal EVs remains elusive. Study design, size, duration The EVs were isolated from the luminal fluid of human Fallopian tubes (n = 23). Immature human oocytes were collected and in vitro matured and fertilized, and 2-pronuclei embryos were included in this study and randomly divided (n = 87). We cocultured these zygotes with Fallopian tubal EVs until the blastocyst stage. Further, two-cell mouse embryos were used to explore the expression levels of miRNAs in Fallopian tubal EVs. The effects of miR-21-5p on mouse embryos were analyzed. Participants/materials, setting, methods EVs were isolated using ultracentrifugation. The uptake of labeled EVs by human embryos was analyzed using confocal microscopy. The fragmentation of the day-3 embryos and the blastocyst rate were compared between the control and the EV-treatment group. Then the stage-specific expression of miRNAs was compared in mouse embryos that were either developed in vivo or cultured in vitro. miR-21-5p were supplemented to the culture medium and the p53-related apoptosis in embryos was evaluated. Main results and the role of chance EVs were measured less than 200 nm in diameter with a typical bi-layer membrane structure and expressed protein markers Heat shock protein 70 (HSP70) and CD9. Most of the fluorescence-labeled EVs were attached to the zona pellucida within 2 h of co-culture. However, human embryos internalized EV within 4 h, with fluorescence signals observed in embryonic cells. The fragmentation rate was decreased and the blastocyst rate was increased by EVs. miR-21-5p, which was highly enriched in EVs, was found to be expressed at a significantly lower level in in-vitro-cultured mouse embryos compared to the in-vivo-developed embryos, through the two-cell stage to the eight-cell stage. miR-21-5p agomir supplementation to the culture medium was found to increase the mouse blastocyst formation rate, decrease apoptosis, and downregulate the P53 expression level in mouse blastocysts. Limitations, reasons for caution Though we observed that Fallopian tubal EVs could be internalized by human embryos and improve embryo development morphologically, the subsequent mechanism exploration was conducted using mouse embryos. Whether the stage-specific expression level of miR-21-5p and the effects of miR-21-5p are the same in human embryos remains to be determined. Wider implications of the findings This study showed miRNAs in Fallopian tubal EVs can act as embryonic survival signals, which opens the insight into further explorations to demonstrate the molecular mechanisms during early embryo development, as well as holds the potential to optimize the culture medium used in ART in the future. Trial registration number 2022YFC2702503

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 <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 <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

O-275 Prenatal genetic screening to assess mtDNA disease occurrence in children

Abstract Study question Can prenatal genetic testing accurately predict mtDNA mutation load anddisease transmission to children for families affected by Leigh Syndrome? Summary answer Whole genome mtDNA sequencing for 13513G>A mutation in chorionic villi and amniotic fluid was similar to those measured in babies suggesting accuracy of disease prediction. What is known already Severity of mtDNA disease in children depends on heteroplasmy levels(proportion of mutant to wild-type mtDNA). There is a minimum threshold for the mutant mtDNA heteroplasmy (typically 60%) above which oxidative metabolism is impaired to cause disease. Predicting the disease based on the mutation load measured during embryonic or fetal development is critically needed. However, preimplantation genetics screening for many mtDNA mutations has limitations due to the potential increase in heteroplasmy levels after implantation. We hypothesized that prenatal screening of chorionic villi and/or amniotic fluid during the first trimester of pregnancy could provide more reliable diagnosis of disease occurrence in children. Study design, size, duration We evaluated mtDNA heteroplasmy in biopsies collected from chorionic villi and amniotic fluid at 10- and 16-weeks of gestation, respectively, for two consecutive, naturally conceived pregnancies for a carrier of 13513G>A mutation. Sequencing results were compared to mutations in the blood, skin, and urine of children after birth. Both children were also extensively monitored for a few years for disease occurrence. Participants/materials, setting, methods A family with maternally transmitted Leigh Syndrome including both parents and two existing children were enrolled during the initial phase. Whole mtDNA sequencing was used to confirm the maternal transmission of the mutation by analysis of blood, skin, and urine from asymptomatic parents and the older son and the severely affected younger son. Furthermore, prenatal diagnostics were performed for two consecutive pregnancies and children after birth. Main results and the role of chance The mother carried 13513G>A mutation in the skin (91% heteroplasmy), urine(39%), and blood (6.8%), while no mutation was detected in the father’s samples. The asymptomatic eldest child revealed a low mutation in the skin (14%), urine(23%), and blood (10%). While the younger son diagnosed with Leigh syndrome carried 56% mutation in blood, 86% in skin, and 97% in urine. Heteroplasmy ranging from 0.6% to 40% was also measured in 4 individual immature oocytes retrieved upon hormonal stimulation of the carrier. The family later naturally conceived 2 consecutive pregnancies and requested help with prenatal diagnostics. In the first pregnancy, mutation loads were 27.5% in chorionic villi and 29.5% in amniotic fluid. The family decided to keep the pregnancy which resulted in the birth of a healthy child with low mutations in blood (0.07%) and skin (14.5%). In the second pregnancy, prenatal diagnostics in CVS showed low mutation at 0.11%, resulting in the birth of an unaffected child with mutation loads of 1.4% in blood and 0% in skin. Limitations, reasons for caution Genetic counseling for mtDNA disease transmission is difficult due tocomplex inheritance of heteroplasmic variants for various pathogenicmutations. Inheritance and distribution of mtDNA mutations also influencedby individual family genetics. Therefore, prenatal diagnostic screeningshould be cautiously considered based on the specific circumstances for eachfamily. Wider implications of the findings Prenatal genetic diagnostics for families with mtDNA disease based on whole mtDNA sequencing by the qualified laboratory may accurately predict heteroplasmy levels and disease occurrence in children. However, careful analysis of family history and the likelihood of having low mutation load oocytes was critical for the success of our case. Trial registration number N/A

P-197 Current status and hotspots of in vitro oocyte maturation: a bibliometric study of the past two decades

Abstract Study question What is the current research status and hotspot of in vitro maturation? And how could we improve the efficiency of IVM technique? Summary answer Five hotspots in IVM field were found, including culturing system, supplementation, cooperation in the ovarian follicle, gene expression, and cryopreservation of oocytes. What is known already In vitro maturation (IVM) of oocytes is a promising technique among assisted reproductive technologies. Because ovarian stimulation is minimal or absent in an IVM cycle, IVM technique reduces the risk of ovarian hyperstimulation syndrome (OHSS) and captures the reproductive potential of immature oocytes. However, the developmental competence of IVM oocytes is generally lower than the oocytes matured in vivo. A recent study showed the cumulative live birth rate in IVM is still inferior to standard IVF. The protocol for IVM still needs to be improved in some details. Study design, size, duration The articles and reviews related to IVM in the Web of Science Core Collection (WoSCC) were retrieved on January 16th, 2023. Three bibliometric tools including VOSviewer 1.6.18, CiteSpace 6.1.R6, and Bibliometrix R package 4.1.0 were used to generate network maps and explore frontier knowledge and trends. Participants/materials, setting, methods The data was retrieved and downloaded from WoSCC using the search formula: TS = (“in vitro maturation” OR “IVM”) AND TS = (follic* OR oocyte*) NOT TS=“ivermectin”. The timespan was set from 2003 to 2022. The language was limited to English, and publication types to article or review. VOSviewer detected indicators, including country, institution, journal, and author. CiteSpace analyzed reference co-citation and identified emerging keywords. Bibliometrix explored trending topics in IVM field. Main results and the role of chance A total of 5133 publications about IVM were retrieved from WoSCC, including 4706 articles and 427 reviews. The number of publications in IVM has increased steadily. China and University of Adelaide were the most prolific country and institution in IVM. Theriogenology was the most published and cited journal. Thompson, Jeremy G was the most productive author in the field of IVM, and Eppig, JJ’s papers were cited most. Furthermore, five hotspots were summarized and introduced, including culturing system, supplementation, cooperation in the ovarian follicle, gene expression, and cryopreservation of oocytes. This study comprehensively and visually analyzed the hotspots and frontiers of IVM in the past two decades. In our conclusion, further study could concentrate on: (1) the mechanism of oocytes matured in vivo and in vitro, especially in energy metabolism and intercellular communications; (2) establishment of IVM culture system, including standardized biphasic IVM culturing system and standardized supplementation; (3) the genetic differences between oocytes matured in vivo and in vitro; (4) the mechanism of cryopreservation-inflicted damage and the solution. Limitations, reasons for caution Due to the limitations of the software, the data we used for bibliometric analysis was only retrieved from WoSCC. Also, while bibliometric analysis can help us understand a field visually, it cannot completely replace system retrieval. Wider implications of the findings In this review, we explored and analyzed the field of IVM from 2003 to 2022 comprehensively and visually. Possible trends and hotspots in future were predicted, which may pave the way for future study directions and developments. Trial registration number not applicable

P-141 Elevating human oocyte vitrification and warming to a new level: fast, efficient, and effective

Abstract Study question Due to the rising trend for oocyte cryopreservation and increasing demand, there is an urgency to make oocyte vitrification and warming protocols more efficient. Summary answer Oocyte vitrification and warming can be done successfully in 4 minutes, reducing the time of commonly used current protocols by up to 25 minutes. What is known already Vitrification and warming of oocytes is widely used in many IVF clinics worldwide and routinely utilized as a dominant technique for fertility preservation and freezing of donor as well as autologous oocytes in many clinical settings. Outcome data using cryopreserved oocytes is comparable to fresh oocytes. However, vitrification and warming of human oocytes can be time consuming. Usually, vitrification of oocytes is done in about 11-16 minutes, and warming requires another 11 minutes. Also, it is a common practice to load not more than 2 oocytes per carrier at time of vitrification. Study design, size, duration From November 2022 to November 2023, 504 germinal vesicles (GV) and 207 metaphase I (MI) oocytes, which did not convert into mature oocytes at day of oocyte retrieval, were vitrified in Vit Kit Freeze (FujiFilm Irvine Scientific, USA), and RapidVit Oocyte (Vitrolife, Sweden). Their survival rates were compared, and as an additional viability indicator, post-warming developmental capabilities were evaluated after 24 and 48hrs of culture in Global High Protein (LifeGlobal, USA). Participants/materials, setting, methods A group of 4 GV or MI oocytes was vitrified at room temperature for 1 minute in Equilibration solution (ES) and 1 minute in Vitrification solution (VS), loaded on a S-Cryolock (Biotech, USA) and then plunged into liquid nitrogen. Warming was done at 37ºC in 0.5M sucrose for 1 minute and Wash solution (WS) for another minute. Post warming, GV and MI oocytes were cultured for developmental evaluation. Statistical significance was defined as P < 0.05. Main results and the role of chance There was no statistically significant difference in the survival rate of GV oocytes between Irvine (209/219; 95.4%), and Vitrolife media (270/285; 94.7%; P = 0.72). The survival of MI oocytes was also comparable and not significantly different between both media; Irvine: 103/106; 97.2%, vs Vitrolife: 96/101; 95.0%; P = 0.43). Because of the similar survival rates of oocytes in both groups, for the developmental potential and conversion rates, oocytes of both groups were combined. Of the 504 GV, 479 survived (95.0%) and after 24hrs of culture, 441 converted to MI (92.1%). After a culture of 48hrs, 56.5% (249/441) converted to metaphase II (MII) oocytes. Combining the vitrified and warmed MI oocytes from both groups, 96.1% (199/207) survived the warming, and after 24hrs of culture, 168 (84.4%) converted to MII oocytes. Vitrification and warming of oocytes at different developmental stages can be done with a short exposure time to hypertonic (ES/VS) and hypotonic (0.5M sucrose) solutions in a total of 4 minutes. Limitations, reasons for caution Immature oocytes were used as study material to evaluate a new protocol with significantly reduced vitrification and warming times. At this moment we cannot provide data on fertilization, embryo development, and blastocyst formation. Wider implications of the findings Oocytes are exposed to less suboptimal temperatures during dehydration and rehydration and help to maintain their developmental potential. Loading 4 oocytes per carrier combined with the decrease in time resulted in increased efficiency. Warming in 0.5M instead of 1M sucrose contributes positively to survival and allows earlier resumption of functionality. Trial registration number not applicable

Novel PATL2 variants cause female infertility with oocyte maturation defect.

Oocyte maturation defect (OOMD) is a rare cause of in vitro fertilization failure characterized by the production of immature oocytes. Compound heterozygous or homozygous PATL2 mutations have been associated with oocyte arrest at the germinal vesicle (GV), metaphase I (MI), and metaphase II (MII) stages, as well as morphological changes. In this study, we recruited three OOMD cases and conducted a comprehensive multiplatform laboratory investigation. Whole exome sequence (WES) revealed four diagnostic variants in PATL2, nonsense mutation c.709C > T (p.R237*) and frameshift mutation c.1486_1487delinsT (p.A496Sfs*4) were novel mutations that have not been reported previously. Furthermore, the pathogenicity of these variants was predicted using in silico analysis, which indicated detrimental effects. Molecular dynamic analysis suggested that the A496S variant disrupted the hydrophobic segment, leading to structural changes that affected the overall protein folding and stability. Additionally, biochemical and molecular experiments were conducted on cells transfected with wild-type (WT) or mutant PATL2 (p.R237* and p.A496Sfs*4) plasmid vectors. The results demonstrated that PATL2A496Sfs*4 and PATL2R237* had impacts on protein size and expression level. Interestingly, expression levels of specific genes involved in oocyte maturation and early embryonic development were found to be simultaneously deregulated. The findings in our study expand the variation spectrum of the PATL2 gene, provide solid evidence for counseling on future pregnancies in affected families, strongly support the application of in the diagnosis of OOMD, and contribute to the understanding of PATL2 function.

Characterization of bovine long non-coding RNAs, OOSNCR1, OOSNCR2 and OOSNCR3, and their roles in oocyte maturation and early embryonic development

In mammals, early embryogenesis relies heavily on the regulation of maternal transcripts including protein-coding and non-coding RNAs stored in oocytes. In this study, the expression of three bovine oocyte expressed long non-coding RNAs (lncRNAs), OOSNCR1, OOSNCR2, and OOSNCR3, was characterized in somatic tissues, the ovarian follicle, and throughout early embryonic development. Moreover, the functional requirement of each transcript during oocyte maturation and early embryonic development was investigated using a siRNA-mediated knockdown approach. Tissue distribution analysis revealed that OOSNCR1, OOSNCR2 and OOSNCR3 are predominantly expressed in fetal ovaries. Follicular cell expression analysis revealed that these lncRNAs are highly expressed in the oocytes, with minor expression detected in the cumulus cells (CCs) and mural granulosa cells (mGCs). The expression for all three genes was highest during oocyte maturation, decreased at fertilization, and ceased altogether by the 16-cell stage. Knockdown of OOSNCR1, OOSNCR2 and OOSNCR3 in immature oocytes was achieved by microinjection of the cumulus-enclosed germinal vesicle (GV) oocytes with siRNAs targeting these lncRNAs. Knockdown of OOSNCR1, OOSNCR2 and OOSNCR3 did not affect cumulus expansion, but oocyte survival at 12 h post-insemination was significantly reduced. In addition, knockdown of OOSNCR1, OOSNCR2 and OOSNCR3 in immature oocytes resulted in a decreased rate of blastocyst development, and reduced expression of genes associated with oocyte competency such as nucleoplasmin 2 (NPM2), growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), and JY-1 in MII oocytes. The data herein suggest a functional requirement of OOSNCR1, OOSNCR2, and OOSNCR3 during bovine oocyte maturation and early embryogenesis.

How germ granules promote germ cell fate.

Germ cells are the only cells in the body capable of giving rise to a new organism, and this totipotency hinges on their ability to assemble membraneless germ granules. These specialized RNA and protein complexes are hallmarks of germ cells throughout their life cycle: as embryonic germ granules in late oocytes and zygotes, Balbiani bodies in immature oocytes, and nuage in maturing gametes. Decades of developmental, genetic and biochemical studies have identified protein and RNA constituents unique to germ granules and have implicated these in germ cell identity, genome integrity and gamete differentiation. Now, emerging research is defining germ granules as biomolecular condensates that achieve high molecular concentrations by phase separation, and itis assigning distinct roles to germ granules during different stages of germline development. This organization of the germ cell cytoplasm into cellular subcompartments seems to be critical not only for the flawless continuity through the germline life cycle within the developing organism but also for the success of the next generation.

Exploring the Potential of In vitro Maturation (IVM) of Oocytes: Indications, Applications, and Treatment Protocols.

This review addresses the current understanding of In Vitro Maturation (IVM) treatment, including indications and effective treatment protocols influencing oocyte developmental competence. A comprehensive literature search was performed to gather relevant studies, clinical trials, and reviews related to IVM. Databases such as PubMed, MEDLINE, and pertinent medical journals were searched. The selected literature was analyzed and synthesized to offer a comprehensive overview. IVM has emerged as a promising technique for inducing maturation in immature oocytes across various developmental stages. Its applications extend to areas utilizing In Vitro Fertilization (IVF), gaining traction as a treatment option for Polycystic Ovary Syndrome (PCOS) and fertility preservation in cancer patients. Recent advancements have led to improved global pregnancy rates, resulting in successful births. IVM also holds potential in reducing risks associated with conventional IVF, including ovarian hyperstimulation syndrome and multiple pregnancies. Despite these advantages, IVM adoption in clinical practice remains limited. Ongoing research aims to refine therapeutic protocols and expand clinical indications. IVM holds promise in assisted reproductive technology, spanning applications from cancer patient fertility preservation to addressing PCOS. Enhanced pregnancy rates highlight efficacy, while risk reduction compared to IVF underscores its importance. Further research is needed for optimal use across patient groups, emphasizing protocol refinement and expanded applications.