激活的TGF-β1/Smad通路调控内异症在位内膜孕激素受体的作用及其机制探讨

孕激素抵抗是导致内异症不孕患者内膜容受性下降的原因所在，孕激素抵抗的机制研究是目前内异症性不孕的研究热点和难点问题，需要另辟蹊径，探究在位内膜孕激素抵抗的机制。我们对早期内异症在位内膜进行单细胞测序，结果发现在位内膜组织出现异常增多的CD45+免疫细胞，这些免疫细胞分泌大量的TGF-β1，基于以往研究报道TGF-β1/Smad通路参与小鼠孕激素受体调控，我们采用在位内膜免疫细胞制备的条件培养基或TGF-β1作用于体外培养的在位内膜间质细胞，观察其孕激素受体的表达变化，并应用蛋白免疫共沉淀验证Smad3与孕激素受体及相关蛋白的结合，最后通过体外蜕膜化诱导实验和敲入TGF-β1的小鼠模型验证TGF-β1/Smad通路对内膜蜕膜化和内膜容受性的影响，明确TGF-β1/Smad通路对在位内膜孕激素抵抗的作用机制；同时我们建立早期内异症在位内膜单细胞转录图谱，为深入研究内异症不孕发病机制提供新思路。

Endometriosis is a common chronic disease decreased women’s fecundity. Classical studies suggested that 30% to 50% of women with endometriosis are infertile primarily because of impaired endometrial receptivity. Altered immune cell in eutopic endometrium are more related to an inhospitable environment for embryo implantation. The progesterone resistance has been well-established in the endometriotic lesion and eutopic endometrium of women with endometriosis. As endometrium is a complex and dynamic tissue, the single-cell RNA sequencing can offer deep phenotyping of specific cell population, transcriptomics and cell-specific activated marker. Therefore, we collected eutopic endometrium (4 cases of minimal/mild endometriosis and 1 case of normal control) to finish single cell RNA sequencing. The result revealed increased proportion of CD45+ immune cells and secretion of cytokines TGF-β1. We hypothesized that elevated TGF-β1 resourced from CD45+ immune cells activates TGF-β1/Smad signaling in endometrial stromal cells (ESC) to attenuate expression of progesterone receptor (PGR) and further decrease endometrial receptivity to embryo implantation. In this study, we continued our sample collection of eutopic endometrium of minimal/mild endometriosis to establish single cell RNA database; meanwhile, ESC of eutopic endometrium were separated and cultured in vitro, treated by immune-medium made of immune cells in eutopic endometrium or TGF-β1, separately; then PGR mRNA and receptor related protein were analyzed by qRT-PCR and Western blot; ESC in vitro treated by immune-medium or TGF-β1 were induced in decidualization experiments with MPA+cAMP. Co-IP had been used to confirm the combination between Smad3 and PGR-B, SRC1/SRC2, or FKBP52. Finally, targeted-uterus knock-in TGF-β1 mice model (CKI) was used to confirm effect of TGF-β1/Samd signaling on PGR expression and endometrial receptivity in uterus of mice. The study is novel in associating immune cells and related cytokine with progesterone resistance in eutopic endometrium, and may be helpful to explore new therapy for endometriosis-associated infertility such as immunomodulator.