Kisspeptin、GPR54和LIF在反复移植失败患者植入窗期子宫内膜表达的研究

发布时间：2018-03-02 14:03

本文选题：Kisspeptin　切入点：GPR54　出处：《郑州大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景胚胎着床(Embryo Implantation)也称胚胎植入,是具有植入能力的胚泡和具备容受性的子宫内膜互相接受的过程。胚胎植入成功与否,不仅取决于胚胎能否发育成具有着床能力的囊胚,还取决于子宫内膜协同性地进入对胚胎具有容受性的状态。随着辅助生殖技术(assisted reproductive technology,ART)的不断发展和优化,使胚胎质量有了很大改善和提高,但临床上胚胎移植的着床率不到50%,仍徘徊在较低的水平,甚至出现多次移植失败。目前多数学者认为反复移植失败(recurrent implantation failure,RIF)是指40岁以下的不孕女性既往接受新鲜或冻融周期移植次数≥3次且移植优质胚胎数≥4个后仍不能正常妊娠。导致RIF的因素包括配子/胚胎因素、子宫因素、免疫因素、血栓形成等,但RIF发生的确切机制目前仍不明确,已知2/3的RIF是由子宫内膜容受性失调引起的,子宫内膜组织在性激素、细胞因子、粘附分子和糖蛋白等多种因素调节下发生一系列变化从而具备容受胚胎着床的能力称为子宫内膜容受性,是胚胎正常着床的关键。Kisspeptin是由KiSS-1基因编码的多肽类激素,与其特异性受体GPR54结合后可抑制肿瘤的转移,并能够在下丘脑垂体水平调控促性腺激素的释放,进而调节生殖功能和参与青春期的启动,调控卵泡发育和促进排卵等多种生物学进程。进一步研究发现Kisspeptin/GPR54系统在子宫内膜表达,能够调节胚胎滋养层在子宫内膜的迁移和侵袭,参与子宫内膜蜕膜化、胚胎着床、胎盘形成等生物学进程,在妊娠的建立和维持中发挥着重要作用。分泌型糖蛋白LIF属于白细胞介素-6(interleukin-6,IL-6)家族的一员,在子宫内膜不同月经周期差异性表达,其表达量于黄体中、晚期达到峰值,是目前国内外公认的能够反映子宫内膜容受性的标记物分子之一,在子宫内膜容受性的建立和子宫内膜蜕膜化进程中起着极其重要的作用。近年研究发现,Kisspeptin/GPR54系统于小鼠妊娠早期时空性表达于母胎界面,其表达水平受E2、P4影响;KiSS-1-/-基因敲除的小鼠,其子宫内膜LIF的表达极低,在补充外源性LIF后,能够部分补救突变型雌鼠(KiSS-1-/-)的胚胎着床情况。据此推测Kisspeptin/GPR54信号通路可能通过调控子宫内膜上LIF的表达参与小鼠胚胎着床过程,以上研究数据均来源于动物实验,那么Kisspeptin、GPR54、和LIF在人类RIF患者植入窗期子宫内膜中的表达较正常对照组是否发生变化,这种变化是否与RIF患者子宫内膜容受性受损有关,本课题拟通过相关实验对以上问题进行初步探讨。目的:检测正常妊娠组女性和RIF患者子宫内膜上Kisspeptin、GPR54和LIF的表达和定位,探索导致RIF患者子宫内膜容受性受损的因素,为改善RIF患者子宫内膜容受性和提高ART中胚胎着床率和妊娠率提供理论依据。方法:选取2016年10月至2017年3月期间拟于郑州大学第一附属医院生殖医学中心行新鲜或冻融试管婴儿助孕并符合入选标准的助孕患者,于黄体中后期行宫腔镜检查或子宫内膜刺激时收集其子宫内膜组织,并采集当日空腹静脉血,通过常规病理学检查和血清中E2、P的水平确定所有标本均为分泌期子宫内膜;应用实时荧光定量PCR(quantitative real-time PCR,qPCR)和免疫组织化学技术,检测Kisspeptin、GPR54和LIF在正常妊娠女性和RIF患者植入窗期子宫内膜上的表达和定位,并进行统计学分析。结果:1.实时荧光定量PCR结果显示:相对于正常妊娠组,KiSS-1、GPR54和LIF的mRNA在RIF组女性子宫内膜中的表达均显著降低,差异有统计学意义;2.免疫组化结果显示:子宫内膜植入窗期,LIF主要表达于子宫内膜腺上皮和腔上皮细胞的细胞浆和细胞膜,在间质细胞中可见少量表达,Kisspeptin、GPR54主要表达于子宫内膜腺上皮细胞的细胞浆和细胞膜,Kisspeptin在间质细胞中几乎未见表达,GPR54在间质细胞有少量表达;经积分光密度分析,相对于正常妊娠组,三者在RIF组女性子宫内膜中的表达显著降低。结论:1.Kisspeptin、GPR54和LIF在反复移植失败患者植入窗期子宫内膜中表达均下降;2.Kisspeptin、GPR54在正常女性和RIF患者植入窗期子宫内膜上的表达和变化特点与LIF一致,Kisspeptin、GPR54可能也参与RIF患者子宫内膜容受性的改变。
[Abstract]:The background of embryo implantation (Embryo Implantation) is also known as the embryo implantation, implantation process has ability of blastocyst and endometrial receptivity have to accept each other. Embryo implantation success depends not only on whether embryo develops into a blastocyst implantation, but also depends on the coordination of endometrial receptivity for the embryo has entered the state of with assisted reproductive technology (assisted reproductive technology, ART) of the continuous development and optimization, the embryo quality has been greatly improved and enhanced, but the embryo implantation rate in clinic is less than 50%, still hovering at a relatively low level, even repeated graft failure. At present, most scholars think that the repeated implantation failure (recurrent implantation failure, RIF) refers to the previous infertile women under the age of 40 to accept fresh or frozen transfer cycles with more than 3 times and the number of high-quality embryos transferred more than 4 still can not Normal pregnancy. Factors leading to RIF including the gamete / embryo factors, uterine factors, immune factors, such as thrombosis, but the exact pathogenesis of RIF remains unclear, known 2/3 RIF is caused by uterine receptivity disorders, endometrial tissue factor in sex hormones, cell adhesion molecules, ability to regulate a variety of factors under the glycoprotein and a series of changes which have the receptivity of embryo implantation is called endometrial receptivity and embryo implantation is the key to.Kisspeptin normal is a polypeptide hormone KiSS-1 gene encoding, metastasis and its specific receptor GPR54 binding could inhibit tumor, and gonadotropin-releasing hormone in the regulation of the hypothalamic pituitary level release. To regulate reproduction and participate in adolescent initiation, regulation of follicular development and ovulation promoting a variety of biological processes. Further study found that Kisspeptin/ in GPR54 system Endometrial expression can regulate trophoblast migration and invasion in endometrial, involved in endometrial decidualization, embryo implantation, placental formation and other biological processes, plays an important role in the establishment and maintenance of pregnancy. The LIF secreted glycoprotein belonging to the interleukin -6 (interleukin-6, IL-6) is a member of the family, the expression of in the endometrium of different menstrual cycle differences, the expression level in corpus luteum, late peak, is recognized at home and abroad can reflect one of the molecular markers of endometrial receptivity, capacity plays a very important role of the establishment and decidualization process in the endometrium. Recent studies have found that the system of Kisspeptin/GPR54 in mice during early pregnancy time expressed in maternal fetal interface, P4 influence the expression level of E2, KiSS-1-/-; gene knockout mice, the expression of LIF in endometrium in very low. The supplement of exogenous LIF, can remedy the mutant female mice (KiSS-1-/-) embryo implantation. It is inferred that Kisspeptin/GPR54 signaling pathway may be involved in the process of embryo implantation in mice by regulating the expression of LIF in endometrium, the above data were obtained from animal experiments, then Kisspeptin, GPR54, and LIF expression in human RIF patients implantation window endometrium in compared with normal control group is changed, if the change in patients with RIF impaired endometrial receptivity, this project intends to carry on to the above problem related experiment preliminarily. Objective: to detect the normal pregnancy group and female RIF patients with endometrial Kisspeptin expression and localization of GPR54 and LIF, explore cause RIF factors in patients with impaired uterine receptivity, in order to improve RIF in patients with endometrial receptivity and improve ART in embryo implantation rate and pregnancy rate theory Basis. Methods: from October 2016 to March 2017 in the First Affiliated Hospital of Zhengzhou University for fresh or frozen thawed IVF pregnancy and eligible patients with progesterone in luteal stage, hysteroscopy or endometrial stimulation when collecting the endometrial tissue, and collect the venous blood examination and E2 in serum by routine pathology, P to determine the level of all specimens for secretory endometrium; real-time fluorescence quantitative PCR (quantitative real-time PCR qPCR application, Kisspeptin) detection and immunohistochemical techniques, GPR54 and LIF in the expression and localization of women and RIF in patients with endometrial implantation window of normal pregnancy, and statistical analysis. Results: 1. real time fluorescence quantitative PCR results showed that: compared with the normal pregnancy group, KiSS-1, GPR54 and LIF mRNA expression in RIF group of women of the uterus Decreased significantly, the difference was statistically significant; 2. immunohistochemistry results showed that: the endometrial implantation window, LIF is mainly expressed in endometrial glandular and luminal epithelial cell cytoplasm and membrane of the cells in the stromal cells of the visible expression of a small amount of Kisspeptin, GPR54 is mainly expressed in endometrial epithelial cell cytoplasm and the cell membrane, almost no expression of Kisspeptin in stromal cells, GPR54 was expressed in mesenchymal cells; analysis of the integral optical density, compared with the normal pregnancy group, the expression of RIF in three groups of women in the endometrium were significantly decreased. Conclusion: 1.Kisspeptin, GPR54 and LIF in repeated implantation failure decreased expression in patients with implantation the window phase of endometrium; 2.Kisspeptin, GPR54 and RIF in normal female patients with endometrial implantation window on the expression and characteristics of changes consistent with LIF, Kisspeptin, GPR54 may also be involved in the endometrium of patients with RIF Receptivity to change.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R714.8

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