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.

中文翻译：

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全基因组评估小鼠卵母细胞DNA甲基化揭示了与体外生长，超排卵和性成熟相关的作用。

背景技术如在小鼠系统中应用的体外卵泡培养（IFC）允许大量未成熟的窦前卵泡的生长和成熟成为成熟且能干的卵母细胞。在人类致癌诊所中，人们越来越有兴趣开发这种技术以替代卵巢皮质组织移植并保持青春期前癌症患者的生育能力。但是，IFC和激素刺激对卵母细胞DNA甲基化的影响尚不完全清楚，并且对在辅助生殖技术（ART）期间应用的程序可能诱发的表观遗传异常存在合理关注。结果在这项研究中，我们提出了自然排卵，IFC和超排卵后MII卵母细胞中DNA甲基化的首次全基因组分析。我们还进行了青春期前和成年激素刺激的卵母细胞之间的比较。在全球范围内，保留卵母细胞的独特甲基化景观，包括交替的高甲基化和低甲基化结构域，而与操作无关。甲基化的保守性延伸到印迹基因的种系差异甲基化区域（DMR），这是它们在胚胎中单等位基因表达所必需的。但是，我们确实检测到IFC卵母细胞中以及与性成熟女性相比，从青春期前超排卵后获得的卵母细胞之间，DNA甲基化之间存在特定，一致和一致的差异。几个甲基化差异跨越整个转录单位。其中，我们发现了Tcf4，Sox5，Zfp521和其他与神经系统发育有关的基因发生了改变。结论我们的观察结果表明，IFC与特定位点处甲基化的改变有关。超排卵的青春期前卵母细胞的DNA甲基化与超排卵的成年卵母细胞的DNA甲基化不同，而超排卵的成年雌性卵母细胞的DNA甲基化与天然排卵的卵母细胞的差异很小。重要的是，我们显示，除了印迹的gDMR以外，其他区域还容易发生与小鼠卵母细胞超排卵，IFC和/或性不成熟相关的甲基化变化。我们的结果为卵母细胞的体外生长和成熟，特别是青春期前女性的卵母细胞在辅助生殖治疗或生育能力保存中的应用提供了重要的参考。而超排卵成年雌性的卵母细胞与自然排卵的卵母细胞相差无几。重要的是，我们显示，除了印迹的gDMR以外，其他区域还容易发生与小鼠卵母细胞超排卵，IFC和/或性不成熟相关的甲基化变化。我们的结果为卵母细胞的体外生长和成熟，特别是青春期前女性的卵母细胞在辅助生殖治疗或生育能力保存中的应用提供了重要的参考。而超排卵成年雌性的卵母细胞与自然排卵的卵母细胞相差无几。重要的是，我们显示，除了印迹的gDMR以外，其他区域还容易发生与小鼠卵母细胞超排卵，IFC和/或性不成熟相关的甲基化变化。我们的结果为卵母细胞的体外生长和成熟，特别是青春期前女性的卵母细胞在辅助生殖治疗或生育能力保存中的应用提供了重要的参考。