Epigenetic abnormalities caused by superovulation have recently attracted increasing attention. Superovulation with exogenous hormones may prevent oocytes from establishing an appropriate epigenetic state, and this effect may extend to the methylation programming in preimplantation embryos, as de novo DNA methylation is a function of developmental stage of follicles and oocyte size. Follicle-stimulating hormone (FSH) and human menopausal gonadotropin (hMG) are common gonadotropins used for superovulation, and appropriate concentrations of these gonadotropins might be necessary. However, no systematic study on the effects of DNA methylation alterations in oocytes associated with superovulation with different dosages of FSH/hMG at the single-cell level has yet been reported. In the current study, different dosages of FSH/hMG combined with human chorionic gonadotropin (hCG) were used in female mice to generate experimental groups, while naturally matured oocytes and oocytes superovulated with only hCG were respectively used as controls. Single-cell level DNA methylation sequencing was carried out on all these matured oocytes. In this study, we revealed that the genome-wide methylation pattern and CG methylation level of the maternal imprinting control regions of all mature oocytes were globally conserved and stable. However, methylation alterations associated with superovulation were found at a specific set of loci, and the differentially methylated regions (DMRs) mainly occurred in regions other than promoters. Furthermore, some of the annotated genes in the DMRs were involved in biological processes such as glucose metabolism, nervous system development, cell cycle, cell proliferation, and embryo implantation and were altered in all dosages of FSH/hMG group (for example, Gfod2 and SYF2). Other genes were impaired only after high gonadotropin dosages (for instance, Sox17 and Phactr4). In conclusion, the current study addressed the effects of superovulation on DNA methylation from the perspective of different dosages of gonadotropins at the single-cell level. We found that the genome-wide DNA methylation landscape was globally preserved irrespective of superovulation or of the kind and dosage of gonadotropins used, whereas the methylation alterations associated with superovulation occurred at a specific set of loci. These observed effects reflect that superovulation recruits oocytes that would not normally be ovulated or that have not undergone complete epigenetic maturation. Our results provide an important reference for the safety assessment of superovulation with different dosages of gonadotropins. However, it should be noted that this study has some limitations, as the sample number and library coverage of analyzed oocytes were relatively low. Future studies with larger sample sizes and high-coverage libraries that examine the effects of superovulation on embryo development and offspring health as well as the underlying mechanisms are still needed.

中文翻译：

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单细胞 DNA 甲基化测序揭示了用不同剂量的促性腺激素超排卵的小鼠卵母细胞的表观遗传改变。

由超排卵引起的表观遗传异常最近引起了越来越多的关注。外源激素的超排可能会阻止卵母细胞建立适当的表观遗传状态，这种影响可能会扩展到植入前胚胎的甲基化程序，因为从头 DNA 甲基化是卵泡发育阶段和卵母细胞大小的函数。促卵泡激素 (FSH) 和人类更年期促性腺激素 (hMG) 是用于超数排卵的常见促性腺激素，可能需要适当浓度的这些促性腺激素。然而，尚未报道在单细胞水平上使用不同剂量的 FSH/hMG 对与超数排卵相关的卵母细胞 DNA 甲基化改变的影响进行系统研究。在目前的研究中，不同剂量的FSH/hMG联合人绒毛膜促性腺激素(hCG)用于雌性小鼠产生实验组，而自然成熟的卵母细胞和仅用hCG超排卵的卵母细胞分别作为对照。对所有这些成熟的卵母细胞进行了单细胞水平的 DNA 甲基化测序。在这项研究中，我们揭示了所有成熟卵母细胞的母体印记控制区的全基因组甲基化模式和 CG 甲基化水平是全局保守和稳定的。然而，在一组特定的位点发现了与超数排卵相关的甲基化改变，差异甲基化区域 (DMR) 主要发生在启动子以外的区域。此外，DMRs 中的一些注释基因参与了生物过程，如葡萄糖代谢、神经系统发育、细胞周期、细胞增殖和胚胎植入，并在所有剂量的 FSH/hMG 组（例如 Gfod2 和 SYF2）中发生改变。其他基因仅在高促性腺激素剂量后才会受损（例如，Sox17 和 Phactr4）。总之，目前的研究从单细胞水平的不同促性腺激素剂量的角度探讨了超数排卵对 DNA 甲基化的影响。我们发现，无论超数排卵或所用促性腺激素的种类和剂量如何，全基因组 DNA 甲基化景观在全球范围内均得以保留，而与超排卵相关的甲基化改变发生在一组特定的位点。这些观察到的效果反映了超数排卵招募了通常不会排卵或未经历完全表观遗传成熟的卵母细胞。我们的研究结果为不同剂量促性腺激素超数排卵的安全性评估提供了重要参考。然而，应该指出的是，这项研究有一些局限性，因为分析的卵母细胞的样本数量和文库覆盖率相对较低。未来仍需要更大样本量和高覆盖率文库的研究，以检查超排卵对胚胎发育和后代健康的影响以及潜在机制。因为分析的卵母细胞的样本数量和文库覆盖率相对较低。未来仍需要更大样本量和高覆盖率文库的研究，以检查超排卵对胚胎发育和后代健康的影响以及潜在机制。因为分析的卵母细胞的样本数量和文库覆盖率相对较低。未来仍需要更大样本量和高覆盖率文库的研究，以检查超排卵对胚胎发育和后代健康的影响以及潜在机制。