ABSTRACT: BACKGROUND:In 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). RESULTS:In 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. CONCLUSIONS:Our 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.
