GnRH拮抗剂通过S100P介导细胞自噬与凋亡影响子宫内膜容受性的分子机制

It has been widely accepted that the antagonist protocol which using GnRH antagonist is a great progress in the assisted reproduction technology because of it significantly improving the controllability and safety of Controlled Ovarian Hyper-stimulation. However, the lower success rate of fresh embryo transfer caused by impaired endometrial receptivity limits its clinical advantages. In our previous gene chip study, we found that the functions and pathways of the genes which specially expressed in the endometrium of patients who accepting antagonist protocol at the window of receptivity were mainly accumulated to negative regulation of growth. And S100P, a biomarker of endometrial receptivity we found before, was down regulated. It has been reported that both GnRH antagonist and S100P were involved in the regulation of cellular autophagy and apoptosis, and S100P might be the downstream molecule of GnRH antagonist. Our previous studies also supported this point. Therefore, we speculate that GnRH antagonist might affect endometrial receptivity by regulating autophagy and apoptosis of endometrial cells mediated by S100P. From in vitro and in vivo levels, the present study will employ methods such as gene manipulation and signal blocking to verify our hypothesis. This study will help us to further elucidate the molecular mechanism of endometrial receptivity, to find targets favorable to endometrial receptivity of patients who accepting antagonist protocol, thus to improve the clinical pregnancy rate and bring considerable benefits to infertile patients.

使用GnRH-ant的拮抗剂方案明显提高了辅助生殖控制性超促排卵的可控性及安全性，特别适合于卵巢异常反应人群，是辅助生殖技术的重大进步。然而临床研究却发现该方案影响子宫内膜容受性导致新鲜胚胎移植成功率低下。我们前期使用基因芯片研究发现拮抗剂方案组容受窗期差异基因的功能及通路分类主要与生长负调节有关，并且发现拮抗剂方案组容受性标志分子S100P表达下降。我们进一步研究也提示拮抗剂方案和S100P影响子宫内膜细胞的自噬和凋亡，且GnRH-ant可下调子宫内膜细胞中S100P的表达。因此我们推测GnRH-ant通过S100P影响子宫内膜细胞的自噬与凋亡，导致子宫内膜容受性受损。本课题拟通过基因干扰、过表达、信号阻断等方法，进行体内及体外研究，详细阐述GnRH-ant调节S100P影响子宫内膜细胞自噬与凋亡的作用及机制，并探讨使用S100P改善拮抗剂方案子宫内膜容受性的可能，以造福病人。

GnRH拮抗剂方案由于其相对于传统的GnRH激动剂长方案有着明显优势而在临床上广泛应用。但既往的研究发现在胚胎质量无明显差异的情况下，拮抗剂方案新鲜胚胎移植周期妊娠率低，提示主要是由内膜容受性受损所致。S100P是我们前期研究发现的潜在的子宫内膜容受性标志分子之一，它在正常子宫内膜容受窗期特异性高表达。但目前还没有研究报道它是否参与了拮抗剂方案子宫内膜容受性下降的调控。本项目拟从临床及基础研究两方面进一步验证GnRH拮抗剂方案对子宫内膜的潜在不利影响，探讨S100P在该过程中的作用及机制，以期为临床改善拮抗剂方案子宫内膜容受性、提高其新鲜胚胎移植周期助孕结局提供理论依据，具有重要的理论和实践意义。我们的临床研究发现在卵巢储备功能正常的人群中， 灵活地减少GnRH-ant的用量是安全的，有利于提高临床妊娠率。且发现拮抗剂方案患者分泌中期宫腔冲洗液中S100P的浓度也明显下降。这说明拮抗剂方案子宫内膜容受性下降，且与拮抗剂剂量有关，S100P可以很好的指示子宫内膜容受性形成。基础研究发现拮抗剂方案患者容受窗期子宫内膜中S100P表达下降，子宫内膜细胞凋亡增加，抗凋亡蛋白bcl-2表达减少，促凋亡Bax蛋白表达增加，提示拮抗剂方案子宫内膜容受性下降可能与子宫内膜细胞凋亡调节异常有关。体外研究发现：子宫内膜上皮细胞中干扰S100P的表达后，细胞凋亡增加，bcl-2表达减少，Bax表达增加；而在细胞中过表达S100P后细胞凋亡减少；细胞中添加拮抗剂之后出现与S100P干扰后类似的效应，而在过表达S100P的细胞中添加拮抗剂并不能诱导凋亡增加。这说明S100P可以逆转拮抗剂诱导的子宫内膜上皮细胞凋亡，GnRH拮抗剂可能通过降调S100P介导其促凋亡的作用。我们进一步研究发现GnRH 拮抗剂可降调TGFb及其下游分子p-SMAD、SP1的表达。通过生物信息预测及双荧光素酶报告基因系统实验证实GnRH-ant可以通过TGFb-SP1信号通路参与S100P的表达调节。本研究首次证实了拮抗剂可通过TGFb信号通路降调S100P的表达，促进子宫内膜上皮细胞凋亡，导致子宫内膜容受性受损。S100P可以成为一个潜在的监测和质量靶点。