LPAR3和HB-EGF在RIF患者子宫内膜着床窗期的表达及意义

发布时间：2018-02-24 20:09

本文关键词： 反复移植失败着床窗期溶血磷脂酸受体3 肝素结合表皮生长因子　出处：《郑州大学》2012年硕士论文　论文类型：学位论文

【摘要】：研究背景当胚胎发育到着床状态的时候,子宫内膜也由非接受态发展到接受态,这-极短的允许胚胎植入的时期称为“着床窗期”。多年来,通过不断改进控制性超促排卵方案和实验室胚胎培养技术,胚胎的数量和质量得到了极大优化,特别是1992年卵胞浆内单精子注射(Intracytoplasmic sperm injection, ICSI)技术应用于临床后,解决了很多由于精卵结合障碍带来的胚胎质量问题。但是体外受精-胚胎移植(in vitro fertilization and embryo transfer, IVF-ET)的植入率依然徘徊在30%左右,其中一个很重要的原因就是部分不孕症患者的着床窗期子宫内膜容受性存在缺陷,进而影响了胚胎的成功着床。目前多数学者认为移植次数≥3次或移植优质胚胎≥10个仍然未能妊娠者,可以称为胚胎反复移植失败(repeated implantation failure, RIF)。通常此类患者胚胎质量良好,没有其它明确的不孕因素可寻,子宫内膜容受性缺陷就成为其反复着床失败的最可能原因,如何预测并改善她们的子宫内膜容受性以提高再次助孕时的妊娠率,成为辅助生殖技术领域研究的热点与难点。近三十年,国内外学者们在子宫内膜容受性方面做了大量研究,发现了一系列影响子宫内膜容受性的激素、细胞因子和生长因子,这些因子之间关系错综复杂,构成一张庞大而井然有序的分子网络,双向调控互相影响。本次实验选取溶血磷脂酸受体3(lysophosphatidic acid receptor3,LPAR3)和肝素结合表皮生长因子(heparin-binding epidermal growthfactor,HB-EGF)为切入点进行子宫内膜容受性的相关研究. 研究目的探讨RIF患者子宫内膜容受性下降的可能机制,探寻适合的评价指标,为改善子宫内膜容受性的临床干预措施提供相应的实验依据。材料与方法实验组(RIF组)23例为2010年10月-2011年6月在郑州大学第三附属医院生殖医学中心就诊的胚胎反复移植失败患者。对照组31例为同期初次接受IVF助孕即妊娠的患者。子宫内膜的获取：在超促排卵前一个月经周期使用经阴道彩色多普勒超声监测卵泡,排卵后7天进行预移植,同时采集着床窗期子宫内膜。内膜采集后,分装为两份,第一份用10%中性福尔马林固定,梯度乙醇脱水,二甲苯透明,石蜡包埋,制成切片,以备LPAR3与HB-EGF的免疫组织化学检测；第二份将吸取的新鲜内膜组织直接冻存于液氮中,等待进行LPAR3和HB-EGF mRNA的半定量RT-PCR检测。外周血的获取：获取子宫内膜的当日晨空腹抽取肘静脉血,采用化学发光法检查雌激素(estradiol,E2)和孕激素(progestogen,P)值。详细记录患者年龄,体重指数,移植周期数,不孕原因及类型,基础内分泌情况,着床窗口期E2值、P值和子宫内膜厚度,IVF周期超促排卵情况、获卵数和胚胎质量。数据分析采用SPSS17.0统计学软件。两组定量资料的比较,符合正态分布的采用两组独立样本t检验进行分析；非正态分布采用秩和检验。两组定性资料的比较采用四格表x2检验进行分析。相关性分析采用Pearson相关分析。以双侧α=0.05为检验水准。结果 1.体重指数、不孕原因、不孕类型、基础状态(月经周期第2天-4天)血卵泡刺激素(follicle stimulatin ghormone, FSH)、黄体生成素(luteotropic hormone,LH)和E2水平、着床窗期血E2水平、子宫内膜厚度、垂体降调方案、实验室胚胎质量方面,两组患者的差异均无统计学意义。与对照组相比,RIF组患者的年龄(31.652±3.284vs.29.839±2.423,P=0.023)、促排卵药物(gonadotropin, Gn)使用天数(11.261±1.137vs.10.484±1.262,P=0.024)和剂量(2311.967±565.290vs.1930.694±511.993,P=0.012)均明显增高,而着床窗期血P水平(11.365±3.612vs.15.039±4.127,P=0.001)明显降低。 2. LPAR3和HB-EGF在两组患者的子宫内膜中均有表达,且表达部位和特点上存在相似性,主要分布在子宫内膜腺上皮细胞和部分基质细胞的胞浆,呈顶浆分泌状。两者的平均光密度值比较,RIF组显著低于对照组(LPAR3:0.255±0.105vs.0.359±0.102,P=0.001;HB-EGF:0.298±0.126vs.0.554±0.197,P0.001)。 3.RIF组患者子宫内膜着床窗期LPAR3和HB-EGF mRNA低表达,灰度值比较显著小于对照组(LPAR3：0.481±0.167vs.0.647±0.177,P=0.001；HB-EGF:0.961±0.312vs.1.514±0.346,P0.001)。 4.两组的患者年龄与LPAR3和HB-EGF mRNA表达之间成负相关性P(LPAR3:r=-3.575,P0.001;HB-EGF:r=-6.025,P0.001)。根据目前的数据,尚不能认为Gn使用天数、Gn剂量、获卵数、着床窗期血E2和P值与两组LPAR3和HB-EGF mRNA表达之间存在显著相关性。结论 1. LPAR3在子宫内膜着床窗期的表达部位及特点与HB-EGF相似,可以作为预测人子宫内膜容受性的良好参考指标。 2.RIF患者着床窗期血清P水平降低、子宫内膜组织中LPAR3和HB-EGF低表达,可能是反复种植失败不孕的原因。 3.随着患者年龄的增长,着床窗期子宫内膜组织中LPAR3和HB-EGF的表达降低。
[Abstract]:Research background
When the embryo to implantation endometrial state, also by non state to accept the receiving state, which allows the implantation of an embryo - short period known as the "implantation window". Over the years, through continuous improvement of controlled ovarian hyperstimulation and laboratory embryo culture technique, the number and quality of embryos greatly optimization, especially in 1992 of intracytoplasmic sperm injection (Intracytoplasmic sperm injection ICSI) technology in clinical application, solve a lot of problems due to the quality of embryo sperm egg binding barriers. But in vitro fertilization and embryo transfer (in vitro fertilization and embryo transfer, IVF-ET) the implantation rate is still hovering around 30% and the endometrial receptivity defects during the implantation window in which a very important reason is that some patients with infertility, which affects the success of embryo implantation. At present, many researchers believe that Transplantation of more than 3 times or more than 10 high-quality embryo transplantation still failed to pregnancy, the embryo can be called repeated implantation failure (repeated implantation failure, RIF). These patients usually embryos of good quality, no other clear infertility could be found, endometrial receptivity defects has become the reason of repeated implantation failure is the most likely. How to predict and improve endometrial receptivity to improve their pregnancy when the pregnancy rate again, become a hot and difficult research field. The reproductive assistive technology in recent thirty years, domestic and foreign scholars suffer in the aspects of endometrial do a lot of research, found a series of hormones that influence endometrial receptivity, cell the relationship between factors and growth factors, these factors constitute a large and perplexing, in order of the molecular network, two-way regulation affect each other. This experiment selected by lysophosphatidic acid 3 (lysophosphatidic acid receptor3, LPAR3) and heparin binding epidermal growth factor (heparin-binding epidermal, growthfactor, HB-EGF) as the starting point for the research of endometrial receptivity.
research objective
To explore the possible mechanism of RIF in patients with endometrial receptivity decreased, searching for suitable evaluation index, provide experimental evidence for clinical interventions to improve the endometrial receptivity.
Materials and methods
In the experimental group (group RIF), 23 cases were failed to repeat embryo transfer from October 2010 to June October 2010 in the reproductive medicine center of the Third Affiliated Hospital of Zhengzhou University. 31 cases in the control group received IVF for the first time.
Obtain endometrial hyperstimulation: in a menstrual cycle before ovulation by transvaginal color Doppler ultrasound monitoring of follicular, pre transplant 7 days after ovulation, while collecting the endometrial implantation window. The endometrium specimens separated into two parts, the first with 10% neutral formalin fixed, gradient ethanol dehydration, xylene transparent, paraffin wax embedded and sectioned by immunohistochemical detection by LPAR3 and HB-EGF; second will draw fresh endometrial tissue directly frozen in liquid nitrogen for semi quantitative RT-PCR detection of LPAR3 and HB-EGF mRNA.
Collection of peripheral blood: obtain endometrial morning fasting venous blood by chemiluminescence (estradiol, E2) examination of estrogen and progesterone (progestogen, P). The patients were recorded age, body mass index, transplantation cycles and the causes of infertility and type, endocrine basis, implantation window period E2 value the value of P, and the thickness of the endometrium, IVF hyperstimulation, number of oocytes and embryo quality.
Data were analyzed by SPSS17.0 statistical software. The comparison of two groups of quantitative data, consistent with the normal distribution with two independent samples t test; non normal distribution using the rank sum test. Two groups of qualitative data were compared with four table analysis of x2 test. Correlation analysis using Pearson correlation analysis. The bilateral a =0.05 as the test level.
Result
The body mass index of 1., the causes of infertility, infertility type, basic condition (second -4 days of menstrual cycle) serum follicle stimulating hormone (follicle stimulatin, ghormone, FSH), luteinizing hormone (luteotropic, hormone, LH) and the level of E2, implantation window blood E2 level, endometrial thickness, pituitary down-regulation protocol, laboratory embryo quality and the difference between the two groups were not statistically significant compared with the control group, RIF group of patients (age 31.652 + 3.284vs.29.839 + 2.423, P=0.023), ovulation drugs (gonadotropin, Gn) days (11.261 + 1.137vs.10.484 + 1.262, P=0.024) and dose (2311.967 + 565.290vs.1930.694 + 511.993, P=0.012) were significantly increased however, during the window of implantation blood P level (11.365 + 3.612vs.15.039 + 4.127, P=0.001) was significantly reduced.
2. LPAR3 and HB-EGF in two patients with endometrial tissues, and the expression of similar parts and characteristics, mainly distributed in the cytoplasm of endometrial glandular epithelial cells and stromal cells, with apocrine secretion. The average optical density value comparison, RIF group was significantly lower than the control group (LPAR3:0.255 + 0.105vs.0.359 + 0.102, P=0.001; HB-EGF:0.298 + 0.126vs.0.554 + 0.197, P0.001).
Group 3.RIF had low expression of LPAR3 and HB-EGF mRNA in the implantation window stage of endometrium, and the gray value was significantly smaller than that in the control group (LPAR3:0.481 + 0.167vs.0.647 + 0.177, P=0.001; HB-EGF:0.961 + 0.312vs.1.514 + 0.346, P0.001).
The 4. group of two patients with LPAR3 and HB-EGF age mRNA expression between the negative correlation between P (LPAR3:r=-3.575, P0.001; HB-EGF:r=-6.025, P0.001). According to the present data, still can not believe that the days of using Gn, Gn dose, number of oocytes, there is a significant correlation between the expression of LPAR3 and HB-EGF two group and mRNA window period of blood E2 and P the value of implantation.
conclusion
Similar to 1. LPAR3 expression in endometrial implantation window location and characteristics and HB-EGF, can be used as a good reference index to predict endometrial receptivity.
The decrease of serum P level in 2.RIF patients and low expression of LPAR3 and HB-EGF in endometrium may be the cause of repeated failure of infertility.
3. with the age of the patients, the expression of LPAR3 and HB-EGF in the endometrium of the bed window decreased.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R321

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