【作者】 吴丽萍；

【导师】 周馥贞；

【作者基本信息】 浙江大学 ， 妇产科学， 2003， 硕士

【摘要】 不孕症是妇科常见病之一，据国外统计发病率约为10—25％。多数病人经过标准的检查程序能明确病因，但仍有许多夫妇虽然经过各项不孕检查仍未能找到确切的不孕原因，这类不孕被称为不明原因不孕症，约占不孕症的10—20％。另外，人群中有22％的妊娠在临床未能诊断前即发生流产；IVF-ET的着床失败率很高，约80—90％的移植胚胎不在子宫种植；ART的妊娠流产率(20—30％)高于自然妊娠流产率(15—19％)。这些现象使得着床缺陷越来越受到人们的关注和重视。随着围着床期各种调节因素的研究的不断深入，发现子宫内膜局部调节失衡导致子宫内膜容受性下降可能是不明原因不孕症的病因之一。 胚泡着床及妊娠成功建立需要胚胎及母体环境之间精细的调节，胚胎和子宫内膜的发育必须同步，其中子宫内膜的容受性非常关键。子宫内膜只有在种植窗期(implantation window)，即在排卵后的6—10天(月经周期第20—24天)，才能接受胚胎植入，过早或过晚则拒受胚胎。胚胎植入是指胚泡附着并侵入子宫内膜的过程，至少需要3个部分构成：(1)胚泡和细胞外基质(ECM)的粘附：(2)着床部位ECM的降解；(3)穿透ECM。其中第一和第三个过程需要基质蛋白的细胞表面受体如整合素的参与，第二阶段则需要能够降解的相应酶类，其中就涉及到近年来越来越为人们所肯定的基质金属蛋白酶(MMPs)及其自身抑制物(TIMPs)。 整合素(integrins)是一个异二聚体粘附受体大家族，由不同的α、β亚单位非共价结合组成，广泛存在于人体各种组织和器官内，是完整的穿膜蛋白，能介导细胞—细胞之间的相互作用及细胞—ECM之间的粘附，参与多种生理过程。已证实，人类子宫内膜有整合素 浙江大学硕士学位论文的丧达，且鳖合素的夫达呈现一个动态的过程，与月经周用有关。整合素Qyp3，在月经周期的20天后突然出现，其表达的开始与“种植窗”的开放一致，并参与滋养层组织一子宫内膜的双向识别过程，捉示这种整合素在胚胎着床过程中有着相当重要的作用，目前己被公认为子宫内膜客受性的标志物。在内异、黄体功能不全、输卵管积水及不明原因不孕妇女，QVp3表达异常或缺乏。 基质金周蛋白醇侧ms）是一类具有许多共同生化特性的蚤白水解酶类，能降解多种细胞外基质蛋白成分，它广泛分布在人体各种组织和器官内。近年来的研究发现，人类子宫内膜也可分泌（ePs），这些酶可能参与子宫内膜的重建、羹胚种植及滋养层侵润。有研究发现，MMP－9及其抑制物＊－1在子宫内膜分泌期表达增加，尤其是在子宫内膜种植窗口期呈现强阳性表达，因此认为MMP－9、TIhe－1的强阳性表达有助于加强表达水解酶活性，完成细胞外基质的转化，促进子宫内膜的分化和蜕膜化，改答子宫内膜的容受性，使子宫内膜更适合胚胎植人，为胚胎着床作准备。 迄今为止，avp3及MMP－9各自在正常于宫内膜的周期性变化、子宫内膜异位症、妊娠等方面的研究较多，但是，有关两者的相互关系的报道较少，尤其是在不明原因不孕症中的相互关系，未见报道。由于着床缺陷可能是不明原因不孕症的病因之一，avp3己是一个公认的子宫内膜容受性标志物，而MMP－9作为子宫内膜容受性标志物尚未得到公认。为此，本实验旨在通过测定子宫内膜着床期avp3、po－9的表达，并比较两者的相互关系，从而为将MMP－9确定为子宫内膜容受性标志提供一个实验依据。 选取18例不明原因不孕患者为实验组，其中原发不孕＊例，继发不孕7例。不孕年限2－10年，平均3．81土2．73年。15例有正常生育功能的妇女为对照组。基础体温上升第6—9天，采血测血清雌二醇（EZ）、孕酮叩）浓度；阴道 B超测定子宫内膜厚度；子宫内膜活检石蜡（冰冻）切片 SP法免疫组化测定子宫内膜 MMP－9、TIgy－1及 Q y p 3，用免疫组化积分法（HSCORE）对免疫组化结果进行半定量分析，并对石蜡切片进行HE染色，根据Nores标准进行组织学分期。co1．子宫内膜MMP－9的表达 MMP－9在着床窗口期子宫内膜表面上皮、腺上皮和间质中呈阳性表达，表达定位于细胞 2 浙江大学硕士学位论文浆，表面上皮、腺上皮的表达强于间质。实验组于g内腹表面上皮、腺上皮回沪｛免疫组化H－Score积分值较对照组低（表面上皮 255．41土34．08 vs 321．84t38．33，腺上皮 266．50土30．41 vs 354．79土24．98），结果有统计学意义（p＜0．05）．但间质NMP－9免疫组化H－Score积分值虽较对照组低（200．67士33．20 ys 203．60士51．84），但结果无统计学意义（p＞0．05）．2．子宜内膜TIMIAnl的表达 TIkP－二在着床窗口期子宫内膜表面上皮、腺上皮和间质中呈阳性表达，表达定位于细胞浆，表面上皮、腺上皮表达强于间质。实验组子宫内膜表面上皮、腺上皮和间质TIny－1免疫组化H－h。＊e积分值均较对照组低（表面上皮20o．18士4更多还原

【Abstract】 As one of the most common diseases, infertility occurred about 10-25% of incidence rate among couples. Even undergone through systemic investigation, there are still 10% of them who could not find the cause of their failure to conceive pregnancy, which is called unexplained infertility. Among various reasons, the failure of implantation becomes an important cause of consideration.The fine regulation between embryo and maternal environment is required for the success of embryo implantation and pregnancy establishment. The development of embryo and endometrium must be synchronized, and uterine receptivity is critical. Only within the implantation window phase, endometrium accepts the embryo implantation, otherwise it rejects embryo. Embryo implantation involves a series of cellular or molecular events, requiring various modulating factors, among which, including integrins and MMPs.Integrins form a great family of heterodimeric transmembrane protein receptors, consists of two subunits, and . They serve as receptors of ECU, mediate the interaction of cell to cell and the adhesion of cell to extracellular matrix, involving various physiological processes. Human endometrium can also express integrins and their distribution has cycle- and cell- specific, v 3 is a special integrin. It appears abruptly after 20 days of the normal menstrual cycle. Theincreased a vfl 3 expression is coincident with a putative "implantation window" . It is a marker of endometrium receptivity. The expression of avP3 is reduced in the endometrium of women with luteal phase deficiency, hydrosalpinx, mild endometriosis and unexplained infertility.The MMPs form a family of structurally related, zinc-dependent endopeptidases that involve in the degradation of extracellular matrix (ECU) and basement membranes (BM). These enzymes and their tissue inhibitors (TIMPs ) play a critical role in normal tissue-remodeling processes, including embryonic growth, development and implantation. Among MMPs, the 92-Kda type IV collagenase (MMP-9, gelatinase B) is a very important enzyme for BM degradation, capable of degrading virtually all components of the ECM, such as collagens, proteoglycans, and glycoproteins. MMP-9 degrades a variety of substrates, particularly collagen type IV and V. Many studies have found that MMP-9 in glandular epithelial cells of endometrium was greatest during the late proliferative phase and just after ovulation; its presence in glandular secretion and the uterine fluid was optimal during the peri-implantation phase. It is possible that this enzyme might be functional in processing specific components of the uterine fluid, thereby contributing to the recognition processing required for blastocyst implantation.TIMPs are the natural inhibitors of all MMPs called tissue inhibitors of metalloproteinases of which four have been described. The TIMPs bind with high affinity and 1 : 1 molar ratio to active MMPs resulting in the loss of proteinase activity. TIMP-1 is the main inhibitor of MMP-9. The secretion of TIMP-1 is increased during peri-implantation phase accompanying with increased secretion of MMP-9. The TIMP-1 which can inhibit the activity of MMP-9 is the important regulator of ECM remodeling, and can limit the extent of trophoblast invasion. It is important for normal embryo implantation.In this study, 18 patients with the diagnosis of unexplained infertility weredesigned to the study group. The mean duration of their infertility was 3.81 2. 73 years ( range 2-10 years ). Of these patients 11 had primary infertility and 7 had secondary infertility. 15 women with normal fertility were selected as fertile controls. Measurement of BBT daily was requested. Ovulation was considered to have occurred when there was a temperature rise of 0.5 , for 12 days immediate preceding a menstruation period. Six to nine days after the onset of BBT ascending, venous blood ’samples were collected and the endometrial biopsies were performed. The blood serum and the tissue were both stored at the -80 . Serum E2更多还原