血清特异性microRNA改善子宫内膜容受性的机制研究

Assisted reproductive technology has helped infertile couples build families for more than 20 years. Implantation of the embryo into the endometrium is an essential step to a healthy ongoing pregnancy. However, pregnancy rates resulting from fresh embryo transfers still hover around 30%, for single embryo transfers, in spite of considerable improvement in egg selection and quality. Preliminary findings showed that intrauterine infusion of G-CSF lead to the improvement of the endometrium, however, a detailed mechanism is unknown. Infertile patients with repeated implantation failure demonstrated endometrial thickness of no more than 7 mm are selected to do the microarray with serum. Dual-luciferase assays and Western Blotting are used to testify the target genes after the bioinformatics assay. Rat thin endometrium model are used to testify the expression level of serum specific microRNA. Overexpress the microRNA mimics, inhibitors or with G-CSF in human and rat endometrial cells to testify the cell proliferation, cell cycle and other biological function. The critical factors such as integrins β3, LIF and VEGF are detected to validate the molecular signaling pathways. Therefore, the specific microRNA has the potential to become novel endometrial receptivity biomarkers and the therapeutic targets.

辅助生殖技术的诞生为不孕不育患者带来了希望，但体外受精-胚胎移植的植入率依然徘徊在30%左右。子宫内膜容受性是决定胚胎能否着床的关键因素。宫腔灌注G-CSF可显著改善子宫内膜容受性，但具体机制不详。本项目拟选取在我院生殖中心就诊的反复种植失败或子宫内膜较薄的患者，对经宫腔灌注G-CSF后子宫内膜厚度≥7的患者的血清小RNA行microRNA芯片，筛选出与子宫内膜容受性显著相关的特异性循环microRNA。生物信息学预测其靶基因并验证其靶向性。宫腔注入无水乙醇贮留5 min 建立大鼠薄型子宫内膜模型，皮下注射G-CSF后检测相关子宫内膜指标。在人和大鼠子宫内膜细胞中分别过表达microRNA、抑制或联合G-CSF,对细胞增殖，周期等生物学功能及关键因子如整合素β3，LIF，VEGF等进行深入研究，为临床上血清microRNA成为子宫内膜容受性的生物标志物及治疗靶标提供理论依据。

辅助生殖技术的诞生为不孕不育患者带来了希望，但体外受精-胚胎移植的植入率依然徘徊在30%左右。移植次数>3次或移植优质胚胎>10个仍未能妊娠者为胚胎反复移植失败,子宫内膜容受性是影响胚胎反复种植失败的关键因素。本项目选取在我院生殖中心就诊的反复种植失败患者和正常对照的血清行microRNA芯片，筛选出与子宫内膜容受性显著相关的特异性循环microRNA 共185 个，其中上调89个，下调96个。通过GO分析和pathway分析显示，差异表达的microRNA主要集中在在内吞，黏附，Wnt、TGF-β及TNF-α信号通路中。通过实时荧光定量PCR分析，我们验证了上调表达的miR-450b，miR-342-3p，miR-582-3p以及下调表达的miR-636，表达情况与芯片结果一致。基于文献检索，选择miR-342-3p进一步研究。在反复种植失败患者内膜组织中，miR-342-3p也表现为上调。外源过表达miR-342-3p后，发现ID4蛋白水平下降。单独过表达miR-342-3p抑制细胞增殖，提示miR-342-3p通过下调ID4水平抑制内膜细胞的增殖，从而改变子宫内膜细胞的容受性，为临床上血清microRNA成为子宫内膜容受性的生物标志物及治疗靶标提供理论依据。