BackgroundIn vitro follicle culture (IFC), as applied in the mouse system, allows the growth and maturation of a large number of immature preantral follicles to become mature and competent oocytes. In the human oncofertility clinic, there is increasing interest in developing this technique as an alternative to ovarian cortical tissue transplantation and to preserve the fertility of prepubertal cancer patients. However, the effect of IFC and hormonal stimulation on DNA methylation in the oocyte is not fully known, and there is legitimate concern over epigenetic abnormalities that could be induced by procedures applied during assisted reproductive technology (ART).ResultsIn this study, we present the first genome-wide analysis of DNA methylation in MII oocytes obtained after natural ovulation, after IFC and after superovulation. We also performed a comparison between prepubertal and adult hormonally stimulated oocytes. Globally, the distinctive methylation landscape of oocytes, comprising alternating hyper- and hypomethylated domains, is preserved irrespective of the procedure. The conservation of methylation extends to the germline differential methylated regions (DMRs) of imprinted genes, necessary for their monoallelic expression in the embryo. However, we do detect specific, consistent, and coherent differences in DNA methylation in IFC oocytes, and between oocytes obtained after superovulation from prepubertal compared with sexually mature females. Several methylation differences span entire transcription units. Among these, we found alterations in Tcf4, Sox5, Zfp521, and other genes related to nervous system development.ConclusionsOur observations show that IFC is associated with altered methylation at specific set of loci. DNA methylation of superovulated prepubertal oocytes differs from that of superovulated adult oocytes, whereas oocytes from superovulated adult females differ very little from naturally ovulated oocytes. Importantly, we show that regions other than imprinted gDMRs are susceptible to methylation changes associated with superovulation, IFC, and/or sexual immaturity in mouse oocytes. Our results provide an important reference for the use of in vitro growth and maturation of oocytes, particularly from prepubertal females, in assisted reproductive treatments or fertility preservation.

In vitro maturation (IVM) is an assisted reproduction technique with reduced hormone-related side effects. Several attempts to implement IVM in routine practice have failed, primarily due to its relatively low efficiency compared to conventional in vitro fertilization (IVF). Recently, capacitation (CAPA)-IVM, a novel two-step IVM method, has improved the embryology outcomes through synchronizing the oocyte nuclear and cytoplasmic maturation. However, the efficiency gap between CAPA-IVM and conventional IVF is still noticeable especially in the numerical production of good quality embryos. Considering the importance of glucose for oocyte competence, its metabolization is studied within both in vivo and CAPA-IVM matured mouse cumulus-oocyte-complexes (COCs) through direct measurements in both cellular compartments, from transcriptional and translational perspectives, to reveal metabolic shortcomings within the CAPA-IVM COCs. These results confirmed that within in vivo COC, cumulus cells are highly glycolytic, whereas oocytes, with low glycolytic activity, are deviating their glucose towards pentose phosphate pathway. No significant differences were observed in the CAPA-IVM oocytes compared to their in vivo counterparts. However, their cumulus cells exhibited a precocious increase of glycolytic activity during the pre-maturation culture step and activity was decreased during the IVM step. Here, specific alterations in mouse COC glucose metabolism due to CAPA-IVM culture were characterized using direct measurements for the first time. Present data show that, while CAPA-IVM cumulus cells are able to utilize glucose, their ability to support oocytes during final maturation is impaired. Future CAPA-IVM optimization strategies could focus on adjusting culture media energy substrate concentrations and/or implementing co-culture strategies.

This study received funding by Strategic Research Programs-Groeiers (OZR/2014/97), IWT/TBM/110680 and by UZ Brussel Fonds Willy Gepts (WFWG 2013). There is no conflict of interest.

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Establishing an ideal human follicle culture system for oncofertility patients relies mainly on animal models since donor tissue is scarce and often of suboptimal quality. The in vitro system developed in our laboratory supports the growth of prepubertal mouse secondary follicles up to mature oocytes. Given the importance of glucose in preparing the oocyte for proper maturation, a baseline characterization of follicle metabolism both in the culture system and in vivo was carried out. Markers of glucose-related pathways (glycolysis, tricarboxylic acid (TCA) cycle, pentose phosphate pathway (PPP), polyol pathway, hexosamine biosynthesis pathway (HBP)) as well as for the antioxidant capacity were measured in the different follicle cell types by both enzymatic activities (spectrophotometric detection) and gene expression (qPCR). This study confirmed that in vivo the somatic cells, mainly granulosa, exhibit intense glycolytic activity, while oocytes perform PPP. Throughout the final maturation step, oocytes in vivo and in vitro showed steady levels for all the key enzymes and metabolites. On the other hand, ovulation triggers a boost of pyruvate and lactate uptake in cumulus cells in vivo, consumes reduced nicotinamide adenine dinucleotide phosphate (NADPH) and increases TCA cycle and small molecules antioxidant capacity (SMAC) activities, while in vitro, the metabolic upregulation in all the studied pathways is limited. This altered metabolic pattern might be a consequence of cell exhaustion because of culture conditions, impeding cumulus cells to fulfil their role in providing proper support for acquiring oocyte competence. SUMMARY SENTENCE: In vitro cultured mouse follicles exhibit altered glycolytic activity and redox metabolism in the somatic compartment during meiotic maturation.

Several studies have linked assisted reproductive technologies to aberrant imprinting. We previously showed that 12-day in vitro follicle culture supports normal imprinting establishment in mouse oocytes. The aim of the present study was to assess whether shortened in vitro follicle growth (8 days of culture compared with 12 days, as a model for human in vitro maturation) or preovulatory intrafollicular oocyte "aging" in culture (14 days of culture) leads to imprinting mutations in oocytes. Limiting-dilution bisulphite sequencing showed that shortened in vitro follicle growth (8 days) does not induce oocyte epimutations at the imprinted Snrpn and Mest genes. In contrast, extension of oocyte residence in large unluteinized follicles in vitro was associated with a low level (1 of 54 alleles) of epimutations for Mest but not for Snrpn. The latter condition may occur during controlled ovarian stimulation where the oocyte growth phase may be extended for several days. Furthermore, we studied the dynamics during follicle culture of transcript levels for genes previously shown to be essential for imprinting establishment in oocytes, including Dnmt3a, Dnmt3L, and Zfp57. Oocyte total mRNA levels during in vitro follicle culture showed the timely shutdown in transcription at the antral follicle stage, and total mRNA levels were comparable to those of in vivo grown equine chorionic gonadotropin-stimulated oocytes.

In vitro oocyte growth is widely studied as an alternative fertility preservation approach. Several animal models are used to generate extensive information on this complex process regulated by the constant and dynamic interaction between the oocyte and its somatic compartment throughout follicle growth and maturation. A 2-dimensional (2D) attachment mouse secondary follicle culture system was used to assess the oocyte’s capacity to overcome disconnection from its somatic companions at different developmental stages for final competence acquisition. To test this, complete mechanical denudation of oocytes from preantral and early antral follicles was performed. Established endpoints were the oocyte’s potential to reconnect with somatic cells and the impact of connectivity disruption on mature oocyte quality. This study proves that oocytes from preantral and early antral cultured mouse follicles can overcome complete denudation, restoring likely functional transzonal projections (TZPs) with no significant differences in meiotic and developmental competence compared to those from intact cultured follicles. These novel findings constitute good premises for developing successful strategies to rescue human oocyte competence in the context of in vitro culture approaches such as non-hCG triggered in vitro maturation (IVM).

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