microRNA对轻度子宫内膜异位症性不孕在位内膜孕激素抵抗的调控作用

Mild/minimal endometriosis associated infertility is one of the common reasons that impacts negatively on women's fecundity and results in female infertility. However, the pathogenesis is unclear. It is considered that the peritoneal microenvironment, ovarian function and endometrial receptivity should be involved. Based on our previous study and published literature, we speculate that some microRNA may regulate steroid nuclear receptor in eutopic endometrium which further result in progestin resistance, then endometrial receptivity in these women decreased. In this study we plan to screen differentiated microRNA in eutopic endometrium of women with mild/minimal endometriosis associated infertility by microRNA chip. At the same time, we will detect the differentiated mRNA. The differentiated microRNAs related with steroid nuclear receptor are further determined and chosen by miRNA-mRNA array analysis and networks analysis. The expression and distribution of target microRNAs and its downstream genes are determined by qRT-PCR, in situ hybridization, western blotting, transfection, and luciferase reporter assays. The endometriosis animal model of rhesus monkey is used to investigate expression of microRNAs and its downstream genes in vivo. The purpose of the study is to explore the pathogenesis of mild/minimal endometriosis associated infertility that provide experimental basis for clinical practice; meantime, the study should service for specific diagnosis and therapeutic method by tracing biological markers and molecule target.

轻度子宫内膜异位症性不孕是育龄妇女不孕不育的常见原因之一，影响妇女的生殖功能，但发病机制不明，可能涉及腹腔内环境、卵巢功能和内膜容受性等方面，我们根据以往研究结果，推测在位内膜的某些miRNA可能通过影响在位内膜雌孕激素核受体的表达异常而导致孕激素抵抗，从而影响内膜容受性致妇女不孕不育。本研究通过miRNA芯片筛查轻度内异症的不孕患者在位内膜中差异性表达的miRNAs，确立miRNA在轻度内异症的不孕患者表达谱，同时通过全基因表达谱芯片检测差异表达的mRNA，进行miRNA-mRNA芯片联合分析及分子网络分析，确定其中与雌孕激素核受体表达和调节相关的miRNA，采用qPCR、原位杂交、转染、荧光素酶实验等确定相应的miRNA及其下游调控基因，并在恒河猴内异症动物模型验证相关miRNA的影响及干预效果。通过研究，探索轻度内异症性不孕患者的发病机制，为临床诊治提供理论依据。

轻度子宫内膜异位症性不孕是育龄妇女不孕不育的常见原因之一，影响妇女的生殖功能，但发病机制不明，可能涉及腹腔内环境、卵巢功能和内膜容受性等方面，我们根据以往研究结果，推测在位内膜的某些miRNA可能通过影响在位内膜雌孕激素核受体的表达异常而导致孕激素抵抗，从而影响内膜容受性致妇女不孕不育。本研究通过miRNA芯片筛查轻度内异症不孕患者在位内膜中差异性表达的miRNAs，确立miRNA在轻度内异症的不孕患者表达谱，同时通过全基因表达谱芯片检测差异表达的mRNA，进行miRNA-mRNA芯片联合分析及分子网络分析，确定与雌孕激素核受体表达和调节相关的miRNA，采用qPCR、原位杂交、转染、荧光素酶实验等确定相应的miRNA及其下游调控基因，并在内异症动物模型验证相关miRNA的影响及干预效果。本研究首次建立了轻度内异症性不孕患者在位内膜的microRNA联合mRNA表达谱。microRNA芯片筛查出显著差异表达大于2倍的microRNA共66个，mRNA表达谱芯片筛查出差异表达大于2倍的mRNA共357个。miR-196a是差异表达最大的microRNA，研究提示miR-196a通过激活MARK信号通路的MEK/ERK，负向调控孕激素受体（PR）-A和PR-B蛋白表达以及PR-A/PR-B蛋白比值升高，且这种调控具有特异性。miR-22-5p在轻度内异症性不孕患者围着床期在位内膜表达降低，直接作用于羟甲基化酶TET2；miR22-5p的异常表达还可促进雌激素受体（ESR）2表达，ESR1/ESR2比值降低，但该调节并不通过ESR2启动子区（-197/+359）甲基化调节。此外，miR-194-3p在轻度内异症在位内膜呈高表达，抑制PR的表达以及内膜间质细胞的蜕膜化，参与孕激素抵抗和不孕；miR-297通过靶向作用于PR的3’UTR区，负向调控PR的异常表达，从而导致在位内膜孕激素抵抗。裸鼠动物模型提示造模后出现内膜孕激素抵抗，本研究从表观遗传学角度探讨内异症性不孕的发病机制，通过研究miRNAs在轻度内异症不孕患者在位内膜孕激素抵抗中的作用，以及对妇女生育力的影响和调控作用，将为这类患者的诊治开拓新的局面。