线粒体基因组变异在多囊卵巢综合征中的作用及机制研究

Polycystic ovary syndrome (PCOS) is a common endocrine disorder and the main cause of anovulatory infertility in reproductive age women. Although it has been studied for many years, the etiology and pathogenesis of PCOS are still unclear. Based on the previous studies, we suppose that the genome variations of mitochondrial DNA (mtDNA) may be involved in the pathogenesis of PCOS. To test this hypothesis, the entire genome variations and also the copy number variations of the mtDNA in granulosa cells of 800 PCOS cases and 800 controls will be analyzed using next-generation sequencing with the coverage of >5000× and real-time fluorescence quantitative PCR, respectively. The effect of mtDNA variations, including point mutations, indels, haplogroups, private mutations, recurrent mutations, and copy number variations on the PCOS will be analyzed systematically combined with a high resolution mitochondrial phylogenetic tree and other bioinformatics methods. Meanwhile, the independent and also the interaction effects of mtDNA various genome variations, including copy number variations, with PCOS, and also PCOS-related endocrine and metabolic phenotypes will be further evaluated to clarify the role of mtDNA in the development of PCOS. Then, in order to verify the research hypothesis further, the mechanism study of the PCOS-related mtDNA variations will be performed using the cytoplasmic hybrid cell lines. The effect of the variations on the mitochondrial function, including oxygen consumption rate, mitochondrial membrane potential, ATP production, ROS production, and mitochondrial protein synthesis, will be studied, thus to explore the mechanism of mitochondrial genome variation in PCOS deeply.

多囊卵巢综合征（PCOS）是育龄妇女常见的生殖内分泌系统疾病，是导致女性无排卵性不孕的主要原因。本项目在前期研究基础上，提出卵巢颗粒细胞线粒体DNA（mtDNA）基因组变异导致的线粒体功能异常可能参与了PCOS的发生发展。我们拟依托新一代测序平台和实时荧光定量PCR技术，对800例PCOS病例及800例对照的卵巢颗粒细胞mtDNA进行全序列测定（拟测序深度>5000×）和拷贝数变异分析，结合mtDNA高分辨率系统发生树，系统分析mtDNA各类型突变（点突变、插入/缺失、单倍群、私有变异、体细胞突变）及拷贝数变异同PCOS发生的关联，并进一步分析mtDNA各类型突变、拷贝数变异同PCOS、及其相关生殖内分泌表型的独立和/或交互作用关系。之后，我们拟依托Cybrid技术，进一步研究相关变异位点对线粒体功能的影响，以更好的验证研究假设，深入探索线粒体基因组变异在PCOS中的作用机制, 为PCOS的病理生理机制研究以及新的治疗手段的开发提供线索。

多囊卵巢综合征（PCOS）是育龄妇女常见的生殖内分泌系统疾病，是导致女性无排卵性不孕的主要原因。本项目依托新一代测序平台和实时荧光定量PCR技术，对PCOS病例及对照的mtDNA进行全序列测定和拷贝数变异分析，系统分析mtDNA各类型突变（点突变/单倍型、私有变异、体细胞突变）及拷贝数变异同PCOS发生的关联，并进一步分析mtDNA各类型突变、拷贝数变异同PCOS、及其相关生殖内分泌表型的独立和/或交互作用关系。.项目受资助以来，我们按照申报书内容逐项进行了研究，发现PCOS患者的mtDNA拷贝数变异(P = 0.003)和mtDNA4977 缺失率(P <0.001)显著高于对照组。在调整BMI、LH/FSH和T后，只有较高的mtDNA4977 DR与PCOS相关(OR: 1.053 (1.024-1.083), P<0.001)。mtDNA4977 DR同PCOS的线性剂量响应趋势也得到四分位数分析的支持。多变量模型表明mtDNA4977 DR水平与PCOS密切相关，是PCOS的独立危险因素。通过二代测序，本研究结果提示mtDNA序列变异、单倍群及异质性变异同PCOS发生的关系不显著，未发现明确相关的此类变异。随后，我们聚焦于PCOS个体一种常见的不良妊娠结局——早期自然流产EPL，对mtDNA在EPL中的作用进行了分析，发现EPL中mtDNA复制与转录、翻译等调控异常,而TEFM转录后调控异常是导致EPL中mtDNA拷贝数升高、但转录下降的主要原因。通过本研究，我们明确了mtDNA拷贝数变异、4977DR同PCOS发生相关，进一步明确了线粒体功能异常在PCOS发生中的作用，为PCOS的病理生理机制研究以及新的治疗手段的开发提供了线索。