Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, No. 49 North HuaYuan Road, Hai Dian District, Beijing, 100191, China.
Key Laboratory of Assisted Reproduction, Ministry of Education, No. 49 North HuaYuan Road, Hai Dian District, Beijing, 100191, China.
Clin Epigenetics. 2020 Jun 1;12(1):75. doi: 10.1186/s13148-020-00866-w.
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.
最近，由超排卵引起的表观遗传异常引起了越来越多的关注。 外源性激素的超排卵可能会阻止卵母细胞建立适当的表观遗传状态，并且这种作用可能会扩展到胚胎的甲基化编程中，因为从头 DNA 甲基化是卵泡和卵母细胞大小发育阶段的功能。 卵泡刺激素（FSH）和人绝经促性腺激素（hMG）是用于超排卵的常用促性腺激素，适当的激素浓度可能是必需的。 但是，尚未在单细胞水平上系统地研究与不同剂量的 FSH / hMG 相关的超排卵卵母细胞中 DNA 甲基化改变。 在本研究中，使用不同剂量的 FSH / hMG 联合人绒毛膜促性腺激素（hCG）在雌性小鼠中产生实验组，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。 对所有这些成熟卵母细胞进行单细胞水平的 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迹控制区域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，在特定的一组基因座中发现了与超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，DMR 中的一些注释基因参与葡萄糖代谢，神经系统发育，细胞周期，细胞增殖和胚胎植入等生物学过程，并在所有 FSH / hMG 剂量组中均发生改变（例如，Gfod2 和 SYF2）。 在高激素剂量后，其他基因受到损害（例如，Sox17 和 Phactr4）。
总之，本研究从单细胞水平上的不同促性腺激素剂量的角度探讨了超排卵对 DNA 甲基化的影响。 我们发现，无论是否超排卵或使用何种类型和剂量的促性腺激素，全基因组 DNA 甲基化景观都是全局保存的，而与超排卵相关的甲基化改变则发生在特定的基因座。 这些观察到的作用反映了超排卵招募了通常不会排卵或尚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的促性腺激素超排卵的安全性评估提供了重要参考。 但是，应该注意的是，这项研究存在一些局限性，因为分析卵母细胞的样本数量和文库覆盖率相对较低。 未来需要进行更多样本量更大，文库覆盖率更高的研究，以检查超排卵对胚胎发育和后代健康的影响及其潜在机制。
