在体外受精中uNK细胞对胚胎种植的调控机制

Repeated implantation failure in IVF-ET is a tough problem in the reproductive medicine. Increasing studies have indentified that immune factors may lead to implantation failure, especially the uterine natural killer (uNK) cells, one of the dominant cells in endometrial lymphocytes. Until now, the regulation meachanism of the uNK cells on the embryo implantation is still unknown. Recent years, our research group have established the NK cells deficiency mouse model stablely and made a series of studies on the function of uNK cells.Based on our previous findings, we hypothesize that uNK cells play an important role in embryo implantation. In order to evaluate this hypothesis, we propose to pursue the following specific aims: Examine uNK cell subsets, the maturity, and the function ( including the cytotoxicity, the secreting cytokines and the TLRs signal pathway); Explore the biological function of the protein expressed by uNK cells and lead to clarify the regulation mechanism of the uNK cells on the embryo implantation. To perform these specific aims, we will adopt the clinical endometrial samples ( repeated implantation failure patients and control patients) and in vitro implantation model . This observation will open new area of investigation a therapeutic for implantation failure and lead to future studies to provide the new insight, diagnoses and treatment methods for the repeated implantation failure patients in IVF-ET.

IVF-ET中反复种植失败是辅助生殖技术研究的难点。越来越多的研究表明胚胎种植失败与免疫因素相关，尤其是子宫内膜淋巴细胞优势细胞群uNK细胞相关。但目前uNK细胞对胚胎种植的作用及调控机制不明。本课题组近年建立了NK细胞缺陷小鼠模型，对uNK细胞功能进行了系列研究。因此本项目拟在前期研究工作基础上，采用免疫组化、流式细胞技术等方法，利用临床IVF-ET种植窗期内膜标本（反复种植失败和正常对照组）和体外种植模型，针对uNK细胞亚型构成比、成熟度和功能（杀伤活性、分泌细胞因子、Toll样信息转导调控等）以及对前期筛查的uNK细胞关键分子的生物学功能进行系统研究，以期达到揭示uNK细胞生物学特征，初步阐明uNK细胞对胚胎种植的调控机制，为临床IVF-ET中反复种植失败提供诊断、治疗的理论依据和潜在的治疗靶点。

子宫内膜对胚胎的容受性是胚胎种植的关键，目前关于子宫内膜容受性的生物标记物并不明确。因此本项目通过子宫内膜标本检测和小鼠妊娠模型拟探索影响子宫内膜容受性的关键生物分子。研究结果显示：(1)通过收集人子宫内膜种植窗期和种植窗前期子宫内膜，采用RNA-Seq技术检测种植窗期与种植窗前期基因表达差异，结果显示2372种基因差异表达，其中1099种基因上调，1273种基因下调，并采用Real time PCR技术验证差异表达基因，首次发现金属硫蛋白家族及4个新的转录基因HAP1，ZCCHC12，MRAP2，OVGP1在子宫内膜种植窗发生显著改。并且通过gene co-expression 网络分析发现GLI2，CDC25A,TLR9，MT1G，SLC5A1参与调节子宫内膜容受性。（2）通过建立NOD(NK细胞缺陷）小鼠妊娠模型，发现子宫蜕膜Treg细胞对于妊娠的建立和维持十分重要，体外过继传输Treg细胞可以显著改善妊娠结局。本项目研究成果为预测子宫内膜容受性提供新的可能的生物标记物，进一步可以为ART的治疗策略提供理论依据。