In Vitro Maturation System Research Articles

New Insights on In Vitro Maturation of Oocytes for Fertility Preservation

In the last decade, the evolution of oncofertility has sparked a resurgence of interest in in vitro maturation (IVM) due to its suitability in certain oncological scenarios where controlled ovarian hyperstimulation may not be feasible. The retrieval of immature cumulus–oocyte complexes from small antral follicles, regardless of the menstrual cycle phase, presents a swift opportunity to vitrify mature oocytes or embryos post-IVM in urgent situations or when stimulation is not advisable. Harvesting immature cumulus–oocyte complexes and immature oocytes can be achieved transvaginally or directly in the laboratory from extracorporeal ovarian tissue. Although IVM has transitioned from an experimental status due to safety validations, it relies on the intricate process of oocyte maturation. Despite successful live births resulting from IVM in fertility preservation contexts, the comparatively lower developmental competence of in vitro matured oocytes highlights the necessity to enhance IVM culture systems. Recent advancements in IVM systems hold promise in bolstering oocyte competence post-IVM, thereby narrowing the gap between IVM and outcomes from ovarian stimulation. Additionally, for optimizing the chances of conception in cancer survivors, the combination of IVM and ovarian tissue cryopreservation stands as the favored choice when ovarian stimulation is unfeasible.

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

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

Three-dimensional glass scaffolds improve the In Vitro maturation of porcine cumulus-oocyte complexes and subsequent embryonic development after parthenogenetic activation

In vitro maturation (IVM) methods for porcine oocytes are still deficient in achieving full developmental capacity, as the currently available oocyte in vitro culture systems still have limitations. In vitro embryo production must also improve the porcine oocyte IVM system to acquire oocytes with good developmental potential. Herein, we tested a three-dimensional (3D) glass scaffold culture system for porcine oocyte maturation. After 42 h, we matured porcine cumulus-oocyte complexes (COCs) on either two-dimensional glass dishes (2D-B), two-dimensional microdrops (2D-W), or 3D glass scaffolds. The 3D glass scaffolds were tested for porcine oocyte maturation and embryonic development. Among these culture methods, the extended morphology of the 3D group maintained a 3D structure better than the 2D-B and 2D-W groups, which had flat COCs that grew close to the bottom of the culture vessel. The COCs of the 3D group had a higher cumulus expansion index and higher first polar body extrusion rate, cleavage rate, and blastocyst rate of parthenogenetic embryos than the 2D-B group. In the 3D group, the cumulus-expansion-related gene HAS2 and anti-apoptotic gene Bcl-2 were significantly upregulated (p < 0.05), while the pro-apoptotic gene Caspase3 was significantly downregulated (p < 0.05). The blastocysts of the 3D group had a higher relative expression of Bcl-2, Oct4, and Nanog than the other two groups (p < 0.05). The 3D group also had a more uniform distribution of mitochondrial membrane potential and mitochondria (p < 0.05), and its cytoplasmic active oxygen species content was much lower than that in the 2D-B group (p < 0.05). These results show that 3D glass scaffolds dramatically increased porcine oocyte maturation and embryonic development after parthenogenetic activation, providing a suitable culture model for porcine oocytes.

Human-induced pluripotent stem cell-derived ovarian support cell co-culture improves oocyte maturation in vitro after abbreviated gonadotropin stimulation.

Human-induced pluripotent stem cell-derived ovarian support cell co-culture improves oocyte maturation in vitro after abbreviated gonadotropin stimulation.

A Redesigned Method for CNP-Synchronized In Vitro Maturation Inhibits Oxidative Stress and Apoptosis in Cumulus-Oocyte Complexes and Improves the Developmental Potential of Porcine Oocytes.

In vitro embryo production depends on high-quality oocytes. Compared with in vivo matured oocytes, in vitro oocytes undergo precocious meiotic resumption, thus compromising oocyte quality. C-type natriuretic peptide (CNP) is a follicular factor maintaining meiotic arrest. Thus, CNP-pretreatment has been widely used to improve the in vitro maturation (IVM) of oocytes in many species. However, the efficacy of this strategy has remained unsatisfactory in porcine oocytes. Here, by determining the functional concentration and dynamics of CNP in inhibiting spontaneous meiotic resumption, we improved the current IVM system of porcine oocytes. Our results indicate that although the beneficial effect of the CNP pre-IVM strategy is common among species, the detailed method may be largely divergent among them and needs to be redesigned specifically for each one. Focusing on the overlooked role of cumulus cells surrounding the oocytes, we also explore the mechanisms relevant to their beneficial effect. In addition to oocytes per se, the enhanced anti-apoptotic and anti-oxidative gene expression in cumulus cells may contribute considerably to improved oocyte quality. These findings not only emphasize the importance of screening the technical parameters of the CNP pre-IVM strategy for specific species, but also highlight the critical supporting role of cumulus cells in this promising strategy.

Effects of lactate, super-GDF9 and low oxygen tension during biphasic in vitro maturation on the bioenergetic profiles of mouse cumulus-oocyte-complex.

In vitro maturation (IVM) is an alternative assisted reproductive technology (ART) with reduced hormone related side-effects and treatment burden compared to conventional IVF. Capacitation (CAPA)-IVM is a biphasic IVM system with improved clinical outcomes compared to standard monophasic IVM. Yet, CAPA-IVM efficiency compared to conventional IVF is still suboptimal in terms of producing utilizable blastocysts. Previously we have shown that CAPA-IVM leads to a precocious increase in cumulus cell (CC) glycolytic activity during cytoplasmic maturation. In the current study, considering the fundamental importance of CCs for oocyte maturation and cumulus-oocyte complex (COC) microenvironment, we further analyzed the bioenergetic profiles of maturing CAPA-IVM COCs. Through a multi-step approach, we (i) explored mitochondrial function of the in vivo and CAPA-IVM matured COCs through real-time metabolic analysis with Seahorse analyzer; and to improve COC metabolism (ii) supplemented the culture media with lactate and/or super-GDF9 (an engineered form of growth differentiation factor 9) and (iii) reduced culture oxygen tension. Our results indicated that the pre-IVM step is delicate and prone to culture related disruptions. Lactate and/or super-GDF9 supplementations failed to eliminate pre-IVM induced stress on COC glucose metabolism and mitochondrial respiration. However, when performing pre-IVM culture under 5% oxygen tension, CAPA-IVM COCs showed a similar bioenergetic profiles compared to in vivo matured counterparts. This is the first study providing real-time metabolic analysis of the COCs from a biphasic IVM system. The currently used analytical approach provides the quantitative measures and the rational basis to further improve IVM culture requirements.

The effect of antioxidants on increased oocyte competence in IVM: a review

In vitro maturation (IVM) is considered a potential assisted reproductive technology that is a safer and simpler alternative to conventional in vitro fertilization. It is primarily used in patients with impaired oocyte maturation and for the treatment of infertile women who are at risk of fertility loss. In addition, IVM is currently used mainly in polycystic ovarian syndrome patients with a high ovarian response and is still considered an experimental option in fertility preservation. Producing highly competent oocytes during IVM is considered a key step in the success of in vitro production (IVP) of embryos. Some factors, such as culture medium conditions and other supplements, have a significant impact on oocyte IVM performance. One of the known disruptors of oocyte developmental competence in IVP is oxidative stress (OS), which is caused by an imbalance between the production and neutralization of reactive oxygen species (ROS). In vitro conditions induce supraphysiological ROS levels due to exposure to an oxidative environment and the isolation of the oocyte from the follicle protective antioxidant milieu. Given the importance of OS in oocyte competence, the establishment of standardized antioxidant IVM systems is critical for improving the overall success of IVP. This review focuses on the main antioxidants tested to protect oocytes against OS in IVM.

Glutathione ameliorates the meiotic defects of copper exposed ovine oocytes via inhibiting the mitochondrial dysfunctions

Regardless of the essential role of copper (Cu) in the physiological regulation process of mammalian reproduction, excessive exposure to Cu triggers the meiotic defects of porcine oocytes via compromising the mitochondrial functions. However, the connections between the excessive Cu exposure and meiotic defects of ovine oocytes have not been reported. In this study, the effect of copper sulfate (CuSO4) exposure on the meiotic potentials of ovine oocytes was analyzed. Subsequently, the ameliorative effect of glutathione (GSH) supplementation on the meiotic defects of CuSO4 exposed ovine oocytes was investigated. For these purposes, the in vitro maturation (IVM) of ovine cumulus oocyte complexes (COCs) was conducted in the presence of 5, 10, 20 and 40 μg/mL of CuSO4 supplementation. Subsequently, different concentrations of GSH (2, 4 and 8 mM) were added to the IVM medium containing CuSO4 solution. After IVM, the assay, including nuclear maturation, spindle organization, chromosome alignment, cytoskeleton assembly, cortical granule (CGs) dynamics, mitochondrial function, reactive oxygen species (ROS) generation, apoptosis, epigenetic modification and fertilization capacity of ovine oocytes were performed. The results showed that excessive Cu exposure triggered the meiotic defects of ovine oocytes via promoting the mitochondrial dysfunction related oxidative stress damage. Moreover, the GSH supplementation, not only ameliorated the decreased maturation potential and fertilization defect of CuSO4 exposed oocytes, but inhibited the mitochondrial dysfunction related oxidative stress damage, ROS generation, apoptosis and altered H3K27me3 expression in the CuSO4 exposed oocytes. Combined with the gene expression pattern, the finding in the present study provided fundamental bases for the ameliorative effect of GSH supplementation on the meiotic defects of CuSO4 exposed oocytes via inhibiting the mitochondrial dysfunctions, further benefiting these potential applications of GSH supplementation in the mammalian IVM system and livestock breeding suffering from the excessive Cu exposure.

The Science of IVM

Oocyte in vitro maturation (IVM) makes use of oocytes from patients that have received minimal or no gonadotrophin stimulation. Whilst this brings many advantages to patients, this typically means oocytes are collected from small-medium sized (4-12 mm) antral follicles. These oocytes are under-developed, and yet removal of these from the follicle and culture in vitro leads to spontaneous oocyte meiotic maturation, without the benefit of the physiological signals that naturally induce oocyte maturation at ovulation. Using animals models, over the past decade seminal advances have been made in our understanding of the fundamental mechanisms regulating oocyte maturation in vivo. Most IVM systems, as practised clinically today, typically do not recapitulate these key cellular processes, possibly accounting for the lower efficiency of IVM compared to IVF. A key objective of modern approaches to IVM is to restore in vitro, as far as possible, the natural processes that occur during oocyte maturation in vivo. One strategy to achieve this in IVM is to: 1) prevent spontaneous meiotic resumption at oocyte collection using phosphodiesterase inhibitors, then 2) artificially maintain or elevate cumulus-oocyte complex (COC) cAMP levels, and finally to 3) induce oocyte meiotic resumption using EGF-like peptides. This typically requires the use of biphasic or 2-step IVM systems. One such biphasic IVM system is called capacitation-IVM (CAPA-IVM), as the oocyte is “capacitated” for development in vitro. CAPA-IVM uses the follicle’s natural oocyte meiotic inhibitor, c-type natriuretic peptide (CNP), in the pre-IVM phase, and amphiregulin as the oocyte meiotic inducer in the second IVM phase. Such biphasic-IVM systems typically lead to subsequent improvements in embryo yield compared to standard IVM, and indeed this is the case for CAPA-IVM. Several pilot RCTs and a large RCT of CAPA-IVM vs conventional IVF have been completed, and CAPA-IVM is now practiced in a few centres globally and is commencing in Sydney. The development and clinical application of modern IVM systems, built on decades of animal scientific research, is a bench-to-bedside success story of biomedical research. IVM is now a viable minimally invasive procedure for the treatment of infertility and for fertility preservation in female cancer patients.

Supplementation of porcine in vitro maturation medium with FGF2, LIF, and IGF1 enhances cytoplasmic maturation in prepubertal gilts oocytes and improves embryo quality

In porcine in vitro production (IVP) systems, the use of oocytes derived from prepubertal gilts, whilst being commercially attractive, remains challenging due to their poor developmental competence following in vitro maturation (IVM). Follicular fluid contains important growth factors and plays a key role during oocyte maturation; therefore, it is a common supplementation for porcine IVM medium. However, follicular fluid contains many poorly characterized components, is batch variable, and its use raises biosecurity concerns. In an effort to design a defined IVM system, growth factors such as cytokines have been previously tested. These include leukaemia inhibitory factor (LIF), fibroblast growth factor 2 (FGF2), and insulin-like growth factor 1 (IGF1), the combination of which is termed 'FLI'. Here, using abattoir-derived oocytes in a well established porcine IVP system, we compared follicular fluid and FLI supplementation during both IVM and embryo culture to test the hypothesis that FLI can substitute for follicular fluid without compromising oocyte nuclear and cytoplasmic maturation. We demonstrate that in oocytes derived from prepubertal gilts, FLI supplementation enhances oocyte meiotic maturation and has a positive effect on the quality and developmental competence of embryos. Moreover, for the first time, we studied the effects of follicular fluid and FLI combined showing no synergistic effects.

Granulosa Cells Improved Mare Oocyte Cytoplasmic Maturation by Providing Collagens.

Assisted reproductive technology has important clinical applications and commercial values in the horse industry. However, this approach is limited largely by the low efficiency of oocyte in vitro maturation (IVM), especially cytoplasmic maturation. To improve the efficiency of mare oocyte IVM, we evaluated the effects of co-culture with cumulus–oocyte complexes (COCs) and granulosa cells (GCs) from follicles with small (<15 mm) and large diameters (>35 mm). Our results showed that oocyte nucleus maturation was not significantly improved by co-culturing with GCs. Interestingly, the cytoplasmic maturation of oocytes, defined by the distribution of cortical granules and mitochondria, as well as reactive oxygen species (ROS) levels, improved dramatically by co-culture with GCs, especially those derived from small follicles. Moreover, GCs promoted cumulus cell expansion by upregulating the expression of BMP15 in oocytes. To determine the mechanism underlying the effects of GCs, the transcriptomes of GCs from large and small follicles were compared. Expression levels of COL1A2, COL6A1, and COL6A2 were significantly higher in GCs from small follicles than in those from large follicles. These three genes were enriched in the extracellular matrix proteins-receptor interaction pathway and were involved in the regulation of collagens. Taken together, our results suggest that co-culture with GCs is beneficial to oocyte cytoplasmic maturation, and the increased expression of COL1A2, COL6A1, and COL6A2 improve the mare oocyte IVM system via the regulation of collagen.

Effect of cumulin and super-GDF9 in standard and biphasic mouse IVM.

In vitro maturation (IVM) is a technology that generates mature oocytes following culture of immature cumulus-oocyte complexes (COC) in vitro. IVM is characterized by minimal patient stimulation, making it attractive for certain patient groups. Recently, a biphasic IVM system, capacitation (CAPA)-IVM, has shown improved clinical outcomes relative to standard IVM; however, it remains less efficient than IVF. This study assessed whether supplementation of CAPA-IVM culture media with the novel TGFβ superfamily proteins cumulin and super-GDF9 improves subsequent mouse embryo development. Immature mouse COCs were cultured by standard IVM or biphasic IVM ± cumulin or super-GDF9. Both cumulin and super-GDF9 in standard IVM significantly improved day-6 blastocyst rate (53.9% control, 73.6% cumulin, 70.4% super-GDF9; p = 0.006; n = 382-406 oocytes). Cumulin or super-GDF9 in CAPA-IVM did not alter embryo yield or blastocyst cell allocation in an unstimulated model. Moreover, cumulin did not alter these outcomes in a mild PMSG stimulation model. Cumulin in CAPA-IVM significantly increased cumulus cell expression of cumulus expansion genes (Ptgs2, Ptx3, Adamts1, Gfat2) and decreased Lhr expression relative to control. However, cumulin-induced mRNA expression of cumulus cell (Ptgs2, Ptx3) and oocyte genes (Gdf9, Bmp15, Oct4, Stella) in CAPA-IVM remained significantly lower than that of in vivo matured cells. Cumulin did not provide an additional beneficial effect in biphasic IVM in terms of blastocyst yield and cell allocation; however in standard IVM, cumulin and super-GDF9 significantly improve oocyte developmental competence.

Factors Influencing the In Vitro Maturation (IVM) of Human Oocyte.

In vitro maturation (IVM) of oocytes is a promising assisted reproductive technology (ART) deemed as a simple and safe procedure. It is mainly used in patients with impaired oocyte maturation and in fertility preservation for women facing the risk of losing fertility. However, to date, it is still not widely used in clinical practice because of its underperformance. The influencing factors, such as biphasic IVM system, culture medium, and the supplementation, have a marked effect on the outcomes of oocyte IVM. However, the role of different culture media, supplements, and follicular priming regimens in oocyte IVM have yet to be fully clarified and deserve further investigation.

The Role of Mitochondria in Oocyte Maturation

With the nucleus as an exception, mitochondria are the only animal cell organelles containing their own genetic information, called mitochondrial DNA (mtDNA). During oocyte maturation, the mtDNA copy number dramatically increases and the distribution of mitochondria changes significantly. As oocyte maturation requires a large amount of ATP for continuous transcription and translation, the availability of the right number of functional mitochondria is crucial. There is a correlation between the quality of oocytes and both the amount of mtDNA and the amount of ATP. Suboptimal conditions of in vitro maturation (IVM) might lead to changes in the mitochondrial morphology as well as alternations in the expression of genes encoding proteins associated with mitochondrial function. Dysfunctional mitochondria have a lower ability to counteract reactive oxygen species (ROS) production which leads to oxidative stress. The mitochondrial function might be improved with the application of antioxidants and significant expectations are laid on the development of new IVM systems supplemented with mitochondria-targeted reagents. Different types of antioxidants have been tested already on animal models and human rescue IVM oocytes, showing promising results. This review focuses on the recent observations on oocytes’ intracellular mitochondrial distribution and on mitochondrial genomes during their maturation, both in vivo and in vitro. Recent mitochondrial supplementation studies, aiming to improve oocyte developmental potential, are summarized.

P–556 NAT10-mediated N4-acetylcytidine in RNA regulates mouse oocyte maturation in vitro

Abstract Study question Does NAT10-mediated N4-acetylcytidine (ac4C) in RNA, a newly identified mRNA epigenetic modification, participate in modulating in vitro maturation(IVM) of oocytes? Summary answer NAT10-mediated ac4C modification is an important regulatory factor during oocyte maturation in vitro, by regulating genes associated with translation, mitochondrial functions and protein destabilization. What is known already Unlike somatic cells, transcription and translation are uncoupled during oocyte maturation and gene expression is mainly regulated by post-transcriptional modulation, including mRNA degradation, translation and posttranslational modification, which are complex and have not been fully investigated. RNA ac4C is a newly identified mRNA modification and a key determinant of post-transcriptional regulation, which has been shown to promote mRNA stability and translation, and NAT10 is the only known RNA acetyltransferase. Therefore, NAT10-mediated ac4C represents a possible epigenetic regulator in oocyte maturation. Study design, size, duration Oocytes at different stages from mice were collected to detect the changing levels of ac4C and NAT10 during maturation. NAT10 in GV-stage oocytes was knocked down before IVM, to confirm the regulatory role of NAT10-mediated ac4C in meiotic process, followed by further exploration of cellular mechanisms. Each experiment was repeated at least three times, and data were analyzed by chi-square test, one-way ANOVA or unpaired-sample t-test. Participants/materials, setting, methods The expression of ac4C and NAT10 was detected by immunohistochemistry. NAT10 was knocked down in GV-stage oocytes by RNA interference through electroporation. The efficacy of knockdown was confirmed by qPCR and immunohistochemistry targeting ac4C and NAT10, and the percentages of oocytes maturated in vitro were compared among groups. High-throughput sequencing and RNA immunoprecipitation were performed to reveal the modulated genes. Proteins specifically binding to ac4C sites were identified by RNA pulldown and mass spectrometry. Main results and the role of chance We first retrieved publicly available data from GEO and found that transcripts with potential ac4C sites were enriched in genes downregulated during IVM (P &amp;lt; 0.001). The biased distribution of ac4C implicated a possible regulatory role. Then immunohistochemistry revealed significantly decreasing trends of ac4C and NAT10 expression from immature to mature oocytes. With NAT10 knockdown, ac4C modification was reduced and meiotic progression was significantly retarded. Specifically, the rate of first body extrusion was significantly decreased with NAT10 knockdown (34.6%) compared to control oocytes without transfection (74.6%) and oocytes transfected with control siRNA (72.6%) (p &amp;lt; 0.001), while rates of germinal vesicle breakdown were not affected (P = 0.6531). High-throughput sequencing and RNA immunoprecipitation revealed that the modulated genes were enriched in biological processes known to be associated with oocyte maturation, including translation, mitochondrial translational elongation and termination, and protein destabilization. Also, we identified a series of proteins specifically binding to ac4C probes by RNA pulldown and mass spectrometry, through which ac4C modification may exert its function in post-transcriptional modulation. Limitations, reasons for caution This study was performed in vitro. The role of NAT10-mediated ac4C in vivo remains to be elucidated. Also, limited by current techniques, ac4C modification in oocytes cannot be detected. Our exploration of regulated genes and ac4C binding proteins were performed in somatic cell lines. Wider implications of the findings: Post-transcriptional modulation is crucial in oocyte maturation. Our study using in-vitro systems for mouse oocyte identified NAT10-mediated ac4C as an important regulator in IVM. It provided a new insight into the epigenetic mechanisms of IVM, which may lead to improvement of clinical IVM systems. Trial registration number Not applicable

O-231 In vitro maturation of human immature (GV) oocytes after controlled ovarian hormonal stimulation with recombinant AMH in the maturation medium

Abstract Study question Does the addition of recombinant AMH to the in vitro maturation (IVM) medium improve the maturation of GV oocytes after controlled ovarian hormonal stimulation? Summary answer Our results show that the addition of recombinant AMH to the in vitro maturation medium improves the maturation rate of GV oocytes. What is known already Anti-Müllerian hormone (AMH) is an important hormone involved in the process of sex differentiation during embryonic development. At the transition to the 21. century, more and more researchers have studied the role of AMH in ovarian function, especially its impact on folliculogenesis. AMH is becoming one of the main biomarkers of ovarian reserve and ovarian-specific disease, however, little is known about its effect on human oocyte maturation. Therefore, we matured immature GV (germinal vesicle) oocytes in IVM medium with recombinant AMH to assess its effect compared to the conventional IVM procedure with FSH and hCG. Study design, size, duration In this two-year prospective study, we compared the maturation rate of four groups of immature (GV) oocytes matured in maturation medium with added i) AMH (n = 15), ii) AMH+FSH+hCG (n = 44), iii) FSH+hCG (conventional; n = 22), and iv) hormone-free maturation medium (control; n = 15). Each oocyte was matured in vitro for a maximum of 28 hours and monitored by time-lapse microscopy to assess the time of GV breakdown (MI) and extrusion of the polar body (MII). Participants/materials, setting, methods Ninety-six GV oocytes of 46 patients (aged &amp;lt; 38 years, involved in the ICSI programme) after short antagonist protocol of controlled ovarian hormonal stimulation were included after written informed consent. IVM of oocytes was performed in the MediCult IVM System (LAG and IVM medium, Cooper Surgical, Denmark) with added hormones, and in a CO2 incubator equipped with the PrimoVision time-lapse microscope (Vitrolife, Sweden). Main results and the role of chance IVM medium with added recombinant AMH gave the best result with all (100 %) oocytes matured in vitro. In conventional IVM medium with FSH and hCG, the oocyte maturation rate was poorer, with 68 % of oocytes matured in vitro. An even lower oocyte maturation rate (34 %) was observed in IVM medium with AMH, FSH and HCG, which might be explained by the antagonistic action of these hormones. In a group of control oocytes, 25 % of oocytes matured in vitro. The mean time to GV breakdown (MI stage) was 3.7 hours and to polar body release (MII stage) 20,5 hours. The time to MI stage was quite comparable in all groups of oocytes (3.5, 3.8 and 3.7 hours). There was a tendency for the polar body to be released later if AMH was added to the maturation medium (21.5 and 20.2 vs. 19.9 hours) but differences were not statistically significant, as revealed by Student’s t-test. In the control group of oocytes, these times were prolonged (4.2 and 22.2 hours) due to slow spontaneous maturation. These preliminary results demonstrate that AMH could directly affect the oocyte maturation in vitro. Limitations, reasons for caution The limitation is the relatively small number of oocytes included; GV oocytes accounted for less than 10 % of all oocytes in the in vitro fertilisation (ICSI) programme. Moreover, the proportion of GV oocytes spontaneously matured to MI stage before the start of the experiment and were therefore not included. Wider implications of the findings Based on our data, we believe that AMH directly affects human oocyte maturation in vitro. Despite the common knowledge that AMH regulates the recruitment of growing ovarian follicles, it appears that the addition of AMH to the maturation medium can improve the human oocyte maturation in vitro. Trial registration number 0120-546/2018/6

P-556 Pre-selected for an award: NAT10-mediated N4-acetylcytidine in RNA regulates mouse oocyte maturation in vitro

Abstract Study question Does NAT10-mediated N4-acetylcytidine (ac4C) in RNA, a newly identified mRNA epigenetic modification, participate in modulating in vitro maturation(IVM) of oocytes? Summary answer NAT10-mediated ac4C modification is an important regulatory factor during oocyte maturation in vitro, by regulating genes associated with translation, mitochondrial functions and protein destabilization. What is known already Unlike somatic cells, transcription and translation are uncoupled during oocyte maturation and gene expression is mainly regulated by post-transcriptional modulation, including mRNA degradation, translation and posttranslational modification, which are complex and have not been fully investigated. RNA ac4C is a newly identified mRNA modification and a key determinant of post-transcriptional regulation, which has been shown to promote mRNA stability and translation, and NAT10 is the only known RNA acetyltransferase. Therefore, NAT10-mediated ac4C represents a possible epigenetic regulator in oocyte maturation. Study design, size, duration Oocytes at different stages from mice were collected to detect the changing levels of ac4C and NAT10 during maturation. NAT10 in GV-stage oocytes was knocked down before IVM, to confirm the regulatory role of NAT10-mediated ac4C in meiotic process, followed by further exploration of cellular mechanisms. Each experiment was repeated at least three times, and data were analyzed by chi-square test, one-way ANOVA or unpaired-sample t-test. Participants/materials, setting, methods The expression of ac4C and NAT10 was detected by immunohistochemistry. NAT10 was knocked down in GV-stage oocytes by RNA interference through electroporation. The efficacy of knockdown was confirmed by qPCR and immunohistochemistry targeting ac4C and NAT10, and the percentages of oocytes maturated in vitro were compared among groups. High-throughput sequencing and RNA immunoprecipitation were performed to reveal the modulated genes. Proteins specifically binding to ac4C sites were identified by RNA pulldown and mass spectrometry. Main results and the role of chance We first retrieved publicly available data from GEO and found that transcripts with potential ac4C sites were enriched in genes downregulated during IVM (P &amp;lt; 0.001). The biased distribution of ac4C implicated a possible regulatory role. Then immunohistochemistry revealed significantly decreasing trends of ac4C and NAT10 expression from immature to mature oocytes. With NAT10 knockdown, ac4C modification was reduced and meiotic progression was significantly retarded. Specifically, the rate of first body extrusion was significantly decreased with NAT10 knockdown (34.6%) compared to control oocytes without transfection (74.6%) and oocytes transfected with control siRNA (72.6%) (p &amp;lt; 0.001), while rates of germinal vesicle breakdown were not affected (P = 0.6531). High-throughput sequencing and RNA immunoprecipitation revealed that the modulated genes were enriched in biological processes known to be associated with oocyte maturation, including translation, mitochondrial translational elongation and termination, and protein destabilization. Also, we identified a series of proteins specifically binding to ac4C probes by RNA pulldown and mass spectrometry, through which ac4C modification may exert its function in post-transcriptional modulation. Limitations, reasons for caution This study was performed in vitro. The role of NAT10-mediated ac4C in vivo remains to be elucidated. Also, limited by current techniques, ac4C modification in oocytes cannot be detected. Our exploration of regulated genes and ac4C binding proteins were performed in somatic cell lines. Wider implications of the findings Post-transcriptional modulation is crucial in oocyte maturation. Our study using in-vitro systems for mouse oocyte identified NAT10-mediated ac4C as an important regulator in IVM. It provided a new insight into the epigenetic mechanisms of IVM, which may lead to improvement of clinical IVM systems. Trial registration number not applicable

150 Changes in pyruvate metabolism alters the epigenetic and molecular maturation of bovine oocytes

During invitro maturation (IVM), bovine oocytes undergo important metabolic, epigenetic, and transcriptional changes for the acquisition of developmental competence. Particularly, metabolic changes that alter the availability of cytoplasmic acetyl-CoA, the main substrate for histones acetylation, may alter the epigenetic profile of the oocyte, with consequences for correct molecular maturation. To test this hypothesis, cumulus–oocyte complexes (COCs) were IVM in three experimental groups: Control [IVM medium (TCM-199-Bicarbonate, 10% fetal bovine serum, 1µg mL−1 oestradiol, 10µg mL−1 FSH, and 10µg mL−1 human chorionic gonadotrophin)], DCA (IVM medium supplemented with 1.5mM sodium dichloroacetate, a pyruvate to acetyl-CoA conversion stimulator) and IA (IVM medium supplemented with 5µM sodium iodoacetate, a glycolysis inhibitor). Cumulus cells (CC) and oocytes (Oo) were analysed separately at 24h (mitochondrial activity, MA; MitoTracker Red CMXRos, ThermoFisher Scientific] and at 0, 4, 8, 16, and 24h of IVM [lysine 9 histone 3 acetylation (H3K9ac immunofluorescence) and new transcript synthesis (only CC; Click-iT® RNA, ThermoFisher Scientific). The images were acquired using a fluorescence microscope and analysed by Image J software. The results from at least 3 replicates were compared by Student’s t-test (treatment vs. control) or by ANOVA followed by Tukey’s test (comparison within the same group in different time points) considering P&amp;lt;0.05. As expected, DCA treatment led to an increase in MA in CC and oocytes (CC control vs. DCA, P=0.003; Oo control vs. DCA, P=0.003). In CC, during the first 4h, H3K9ac increased significantly in the treated group and decreased in the control group. At 8, 16, and 24h, both groups presented similar tendencies, although H3K9ac levels remained higher in DCA compared with control at all time points (P&amp;lt;0.001). The synthesis of new transcripts in CC was stimulated by DCA compared with control at 8h (P=0.02) and particularly at 16h (P=0.002), when acetylation levels were at the lowest point. Interestingly, in oocytes, the initial trend was reversed. An increase was observed in the H3K9ac levels of the control group (P=0.014), whereas no difference was observed for DCA in the first 4h. Moreover, although acetylation levels followed a downward tendency with time in both groups, oocytes treated with DCA showed lower H3K9ac levels at 4 and 8h and a higher level at 24h (P=0.04) compared with control. Regarding IA, lower MA were verified in CC whereas oocytes had the opposite profile (CC control vs. IA: P=0.0035; Oc control vs. IA: P&amp;lt;0.001). In CC, this decrease in MA was not accompanied by a decrease in H3K9ac. In contrast, H3K9ac increased compared with the control group at 8 and 16h (control 8h vs. IA 8 h: P=0.019 and control 16h vs. IA 16 h: P=0.019). These changes were accompanied by an increase in the synthesis of new transcripts in the IA group over the time of IVM. Based on these data, we can conclude that changes in pyruvate metabolism caused by manipulation of the IVM system lead to epigenetic and molecular changes in both CC and oocytes.

Effects of C-type natriuretic peptide on meiotic arrest and developmental competence of bovine oocyte derived from small and medium follicles

The present study aimed to evaluate the effects of C-type natriuretic peptide (CNP) on meiotic arrest and developmental competence of bovine oocyte derived from follicles of different sizes. Collected immature cumulus-oocyte complexes from small follicles (< 3 mm) and medium follicles (3–8 mm) were cultured for 6 h in basal medium supplementated without or with 200 nM CNP. We observed that CNP effectively sustained meiotic arrest at germinal vesicle stage in in vitro cultured bovine oocytes from follicles of different sizes. Moreover, CNP treatment significantly improved the levels of cGMP in both cumulus cells and oocytes, as well as the levels of cAMP in oocytes regardless of follicle size. Based on the above results, we tested the effect of a novel in vitro maturation (IVM) system based on CNP-pretreatment, including a pre-IVM phase for 6 h using 200 nM CNP, followed by a extended IVM phase for 28 h, on developmental competence of bovine oocyte derived from small follicles (< 3 mm) and medium follicles (3–8 mm) compared to standard IVM system. The results showed that athough the novel IVM system based on CNP-pretreatment enhanced the developmental potencial of oocytes obtained from large follicles, but had no effect on the developmental comptence of oocytes obtained from small follicles.

Steroid-depleted endometriosis serum improves oocyte maturation in IVM systems.

In vitro maturation (IVM) is a novel approach to overcome the adverse effects of human in vitro fertilization (IVF). The aim of the present study is to evaluate the effect of total and steroid-depleted serum obtained from patients with endometriosis on IVM outcome as supplementation for this system. To this purpose, patients with endometriosis were selected according to in/excluding criteria. Germinal vesicles (GVs) and cumulus cells were treated with 10% of each serum. The expression levels of stearoyl CoA desaturase 1 (SCD 1) and cyclooxygenase-2 (COX-2) genes were evaluated by RT-qPCR. Gas-liquid chromatography and flow cytometry were performed to analyze fatty acids composition and apoptosis. The mRNA expression levels of SCD1 (2.47 fold) and COX-2 (6.4 fold), and also the synthesis of oleate, linoleate, and arachidonate were increased (1.19, 1.06, and 2.37 folds, respectively) in cumulus cells treated with steroid-depleted serum (p < .05). The synthesis of palmitate, palmitoleate, and stearate (0.995, 0.67, and 0.7 folds, respectively) and also the rate of apoptosis were significantly decreased in these cells (p < .05). Moreover, GVs cultured in steroid-depleted group showed a significantly higher rate of maturation (p < .001). Overall, our findings imply a new insight into the expansion of IVM system in oocytes development.