【作者】 郭慧；

【导师】 卢光琇；

【作者基本信息】 中南大学 ， 生殖工程， 2012， 博士

【摘要】 人类胚胎植入是一个十分复杂的过程,在这个过程中胚胎和子宫内膜互相作用,在一段非常短的时期共同作用完成胚胎着床。一个成功的植入过程需要三个重要的条件：优质的胚胎、良好的子宫内膜容受性以及胚胎和子宫内膜的同步发育。子宫内膜仅在一段十分短暂的时间内具有接受胚胎的能力,这一时期叫做“着床窗口期”。子宫内膜的容受性受到多种生物信号的调控,包括：特殊的生长因子、细胞因子、磷脂分子、细胞粘附因子以及转录因子等。这些生物信号受女性甾体激素、前列腺素和肽类激素的调节,它们的变化会造成子宫内膜容受性的改变,导致胚胎植入率和妊娠率的下降。溶血磷脂酸(Lysophosphatidic acid, LPA)是一种具有多生物效应的生长因子。它可以调控细胞的生存、增殖、分化、抑制细胞间的缝隙连接和改变细胞骨架的重新排列。LPA在生殖方面也起到了重要的调控作用,包括刺激卵子的成熟、调节2-4卵裂球的胚胎向囊胚的转化、影响胚胎在输卵管的运输。LPA主要依靠细胞外基质的溶血磷脂酶D水解磷脂生成,也可以由活化的血小板、红细胞、有丝分裂后的神经元细胞、卵巢和宫颈癌细胞、脂肪细胞和肥大细胞分泌产生。LPA通过G偶联蛋白受体LPAR1-4发挥生物学效应。敲除LPAR1和LPAR2的雌性小鼠生育能力正常,但是敲除LPAR3基因的雌性小鼠生育能力发生改变,主要表现为胚胎植入空间的改变、胚胎缠绕以及种植时间延迟。在小鼠交配后的2.5d,小鼠子宫内膜上LPAR3的表达开始上升,在围植入期即交配后的3.5d表达达到顶峰,然后在交配后4.5d下降恢复到基础水平直至分娩后。目前对LPAR3在人类子宫内膜容受性方面的研究很少,仅仅知道反复植入失败的患者和子宫内膜异位症的患者在他们月经周期的分泌期LPAR3在其子宫内膜上的表达较正常妇女低。在控制性超促排卵(Controlled ovarian hyperstimulation, COH)中,由于为了在一个促排卵周期中同时得到多个发育的成熟卵子,因而使用了大量的促排卵药物。长效长方案是目前应用的较多的一种促排卵方案,该方案可以获得优质的卵子,受精率和优胚率都比较高,但是胚胎的植入率比较低。分析原因,多数学者认为是由于促排卵药物的使用造成体内产生超生理浓度的激素,从而干扰了子宫内膜的内环境,降低了子宫内膜的容受性。因此,找到子宫内膜容受性下降的原因并找到解决的方法,可以提高IVF的妊娠率,具有很好的临床意义。至今,还没有文献报道人类长方案促排卵的患者子宫内膜上LPAR3的表达方式。多囊卵巢综合征(Polycystic ovary syndrome, PCOS)患者由于体内内分泌紊乱,造成雌孕激素分泌异常和促黄体生成素(LH)的升高,导致子宫内膜上影响子宫内膜容受性的因素表达异常,从而降低了PCOS患者子宫内膜的容受性。目前对PCOS患者的子宫内膜容受性的研究主要集中在对甾体激素及其受体、整合素、基质金属蛋白酶、同源框基因等因子,对于目前新近发现在生殖过程中发挥重要作用的LPAR3的研究甚少,国内外均未见报道。此外在我们研究的临床部分,我们在改善PCOS患者子宫内膜容受性的原理的基础上,采用了降调节人工周期子宫内膜准备的方法,目的是找到一种有效的提高PCOS患者的子宫内膜的容受性的方法。本研究主要利用RT-PCR, Western blot和免疫组化等技术研究了正常妇女月经周期、IVF促排卵患者围着床期以及PCOS患者月经周期中子宫内膜LPAR3的表达情况和差异。初步说明了LPAR3在子宫内膜容受性建立过程中的重要作用。研究分为四个部分,前三个部分为实验部分,最后一个部分为临床部分,实验方法和结果如下：第一部分LPAR3在正常妇女月经周期子宫内膜上的表达目的：研究溶血磷脂酸受体3(Lysophosphatidic acid receptor3,LPAR3) mRNA及其蛋白在正常妇女月经周期中子宫内膜上的表达规律。方法：采用RT-PCR, Western blot和免疫组化法,检测LPAR3mRNA和蛋白在正常妇女月经周期中子宫内膜上的表达规律。结果：1.在整个人类月经周期中均可在子宫内膜组织中检测到LPAR3mRNA和蛋白的表达。2.在整个增生期LPAR3mRNA和蛋白的表达呈低表达状态,在早分泌期LPAR3mRNA和蛋白表达明显升高,并且达到峰值,但是在中分泌期LPAR3mRNA和蛋白的表达骤然下降,恢复到增生期水平。但在晚分泌期LPAR3mRNA和蛋白表达又上升,逐级恢复到早分泌期水平。3.在早增生期LPAR3蛋白主要分布在子宫内膜的腔上皮、腺上皮的细胞浆中。在中晚增生期虽然间质细胞的细胞浆中LPAR3的阳性着色范围扩大,但仍是主要在子宫内膜的腺上皮和腔上皮细胞的胞浆中呈阳性表达。在早分泌期腺上皮和子宫内膜腔上皮细胞呈强阳性免疫染色,间质细胞也出现强阳性反应。在中分泌期LPAR3在子宫内膜间质细胞和上皮细胞的表达骤然下降,主要表达在子宫内膜腔上皮的细胞浆中,但是表达降低呈弱阳性反应。在晚分泌期LPAR3王要在子宫内膜间质细胞中和血管上表达,呈强阳性反应。在子宫内膜的腔上皮和腺上皮上呈弱阳性表达。4.在早中增生期主要分泌雌激素(estrogen, E), LPAR3处于低表达状态；在晚增生期成熟卵泡的颗粒细胞在黄体生成素(luteinizinghormone, LH)排卵高峰的作用下,开始分泌孕酮(progestin,P),排卵后P逐渐增加,E分泌下降,子宫内膜上LPAR3的表达开始在早分泌期增加。在中分泌期E水平又增加,LPAR3在子宫内膜上的表达下降。在晚分泌期E水平下降,LPAR3在子宫内膜上的表达开始上升结论：1. LPAR3在人类月经周期中子宫内膜上呈周期性的变化,在人类的围着床期LPAR3的表达达到顶峰,这种变化规律揭示LPAR3可能参与了子宫内膜容受性的建立,可作为预测子宫内膜容受性的分子参考指标。2.我们把月经周期中E、LH和P的表达规律和LPAR3在子宫内膜上的表达方式相比较,我们推测LPAR3可能受到LH和P的正向调控,而E可能对这种正向调控有抑制作用(见下图)。第二部分GnRHa长方案促排卵对围着床期人类子宫内膜上LPAR3表达的影响目的：研究GnRHa/rFSH/hCG控制性超促排卵(COH)对围着床期人类子宫内膜溶血磷脂酸受体3(LPAR3)表达的影响。方法：将研究对象分为自然周期对照组(NC组)和COH实验组,用RT-RCR和Western blot方法观察两组排卵后3d～6d子宫内膜上LPAR3mRNA和蛋白的表达变化。结果：1.NC组子宫内膜中LPAR3的表达在排卵后d4达到顶峰,COH组子宫内膜LPAR3的表达高峰较NC组要提前1d,即在排卵后d3达到顶峰。2.COH组子宫内膜LPAR3在各时间点的表达水平均低于对照组,有统计学差异(P<0.05)。3.COH组血清中E2和P浓度水平在相应各时间点均高于NC对照组,E2/P比值也高于NC对照组,有统计学差异(P<0.05)。结论：1. GnRHa控制性超促排卵可能通过改变子宫内膜LPAR3表达的水平和时序变化,一方面降低了子宫内膜的容受性,同时一方面也使子宫内膜与胚胎发育不同步从而降低胚胎的着床率。这提示我们可能通过改善目前移植胚胎的时间,达到子宫内膜和胚胎发育同步,从而提高IVF妊娠率。2.可能由于在GnRHa控制性超促排卵中超生理剂量的E2和P水平,以及不协调的E2/P比造成了LPAR3表达的异常。我们可能通过调节E2/P比值,使LPAR3的表达恢复正常状态,提高IVF妊娠率。第三部分LPAR3在PCOS患者月经周期子宫内膜上的表达目的：研究溶血磷脂酸受体3(Lysophosphatidic acid receptor3,LPAR3) mRNA及其蛋白在PCOS月经稀发患者月经周期中子宫内膜上的表达。方法：采用RT-PCR, Western blot方法,检测LPAR3mRNA和蛋白在PCOS月经稀发患者月经周期中子宫内膜上的表达。结果：1.PCOS组患者月经周期天数为39.75天,明显多于NC对照组月经周期天数30.5天(P<0.01)。在增殖期PCOS组血清中LH水平为8.61miu/ml,高于NC组增殖期血清中LH水平3.10miu/ml,有统计学差异(P<0.01)。在早、中分泌期PCOS组患者血清中P水平1.64ng/ml、6.36ng/ml,明显低于NC组P水平4.46ng/ml、14.2ng/ml,有统计学差异(P<0.05)。2. PCOS组在整个增生期LPAR3mRNA和蛋白在子宫内膜上呈低表达状态,但与NC对照组增生期相比要高于NC对照组,有统计学差异。在早分泌期LPA3mRNA和蛋白表达明显升高,并且达到峰值,在中分泌期LPAR3mRNA和蛋白的表达骤然下降,恢复到增生期水平。PCOS组中LPAR3mRNA和蛋白水平在早分泌期子宫内膜上的表达要明显低于NC对照组,有统计学差异(见下图)。结论：1.PCOS组中增生期LPAR3的异常表达可能和PCOS患者子宫内膜过度增殖相关。2.PCOS组中在早分泌期即围着床期LPAR3的表达较NC对照组低,这可能干扰了其子宫内膜容受性的建立,降低了子宫内膜的容受性3. PCOS组在增生期血清中LH水平的高表达以及在分泌期P的低表达状态可能和其子宫内膜上LPAR3异常表达相关。第四部分两种雌孕激素替代法内膜准备对多囊卵巢综合症冷冻胚胎移植临床效果的比较目的：比较非降调节雌孕激素替代法与降调节雌孕激素替代法作为子宫内膜准备方法对多囊卵巢综合症患者(PCOS)冻融胚胎移植(FET)的妊娠结局。方法：在PCOS患者FET周期中,分别采用非降调节雌孕激素替代法(GnRHa组,n=89)、降调节雌孕激素替代法(HRT组,n=37)准备内膜,比较周期妊娠率、种植率、流产率、宫外孕发生率等指标。结果：1.HRT组和GnRHa组2组患者年龄分别为29.53±3.39岁、29.24+2.85岁(P>0.05);基础体重指数分别为23.07±3.51、22.20+2.96(P>0.05)；不孕年限分别为3.14±1.89年、3.86+136年(P>0.05);2.在黄体支持日HRT组和GnRHa组2组患者E水平分别为189.72±110.88pg/ml、133.08±55.33pg/ml (P<0.05); LH水平分别为11.74±6.49miu/ml、1.3±0.67miu/ml (P<0.01)。3.2组移植周期临床妊娠率为55.06%、72.9%(P<0.05)；种植率分别为38.7%、53.6%(P<0.05)；流产率分别为16.3%、14.8%(P>0.05);宫外孕率分别为4.1%、3.7%(P>0.05)。结论：对于PCOS患者使用降调节雌孕激素替代法可能改善子宫内膜的容受性,是一种合适的子宫内膜准备方案。更多还原

【Abstract】 Implantation in humans is an intricate process that involves complex interactions between the embryo and the uterus during the initial period of gestation. There are three important conditions for successful implantation:good embryo quality, high endometrial receptivity and a synchronized dialogue between maternal and embryonic tissues. The endometrium is not receptive to embryo implantation except for during a strict frame of time called the’implantation window’. The uterine receptivity is regulated by several kinds of molecular signals, such as specific growth factors, cytokines, lipid mediators, adhesion molecules, and transcription factors. They are further regulated by female sex steroids, prostaglandins (PGs) and peptide hormones. The expression level of any factors is abnormal will contribute to change the endometrial receptivity and lead to reduce the pregnancy rate.Lysophosphatidic acid (LPA) is a growth factor that triggers many cellular responses. These responses include cell survive, proliferation, differentiation, inhibition of gapjunction communication and action cytoskeletal rearrangements. LPA also plays an important role on reproduction, including stimulation of oocyte maturation, the preimplantation development of two-or four-cell embryos to the blastocyst stage and embryo transport in the oviduct. It could be produced through the hydrolysis of phospholipids by extracellular lysophospholi-pase D or by activating platelets, erythrocytes, postmitotic neurons, ovarian and cervical cancer cells, adipocytes and mast cells. LPA functions through its G protein-couple receptors, LPA1-4. Mice kicking out LPA1and LPA2reproduce normally, but LPA3-null mice showed reduced litter size and altered embryo spacing, embryo crowding, and delayed implantation. It provided additional evidence for its role in murine implantation. Expression of LPA3in the mouse uterus is strictly regulated during early pregnancy. Within the mouse uterus, LPA3mRNA increased on2.5days post coitus (2.5d.p. c), peaking around3.5d. p.c, peri-implantation period, then returned to the basal levels on4.5d. p. c. through the end of pregnancy. In the generation domain, there is lack of information about the function of LPA3on endometrial receptivity until now. Recent research showed that LPA3expression is low during secretory phase of endometrium in those patients with repeated implantation failure and endometriosis.Controlled ovarian hyperstimulation (COH) can get many germ cell by using large amounts promoting ovulation drug. Long protocol controlled ovarian hyperstimulation is the most commonly used protocol that can get good quality eggs, high fertilization rate and high qualified embryo rate, but the implantation rate is still low. GnRHa long time superovulation program put a negative effect in the balance of internal hormone which decreasing the endometrial receptiblity. So to find the reason of decreasing in endometrial receptiblity and find an efficient means to solve the problem is with good clinical significance. So far, there is no reported about the expression of LPA3with Long protocol controlled ovarian hyperstimulation in human endometrium during periimplantation periode.Polycystic ovary syndrome (PCOS) patients with endocrine disorder caused by abnormal secretion of estrogen and progesterone and rising of luteinizing hormone (LH), which reduces the receptivity of the endometrium of PCOS patients. Endometrial receptivity in patients with PCOS is mainly concentrated in the steroid hormone receptors, integrins, matrix metalloproteinases, and homeobox factor. LPA3play an important role in the reproductive process. In addition, the clinical part of the study, to improve PCOS endometrial receptivity, artificial frozen-thawed embryo transfer cycles with pituitary suppression was used. The purpose is to improve the receptivity of the endometrium of women with PCOS.In this study, by using RT-PCR, Western blot and immunohis-tochemistry study the expression of LPA3with normal menstrual cycle of women, Long protocol controlled ovarian hyperstimulation in human endometrium during periimplantation periode and menstrual cycle of PCOS. This study is to reveal that the important role of LPA3in the process of establishing endometrial receptivity. Divided into four parts, the three parts of the experimental part, the last part of the clinical part, the experimental methods and results are as follows:Part1:Cyclic regulation of LPA3in human endometriumObject:The aims of the present study were to characterize LPA3mRNA and protein in human endometrium during the normal human menstrual cycle.Methods:Forty-three normally cycling volunteers without reproductive disorders were randomized to undergo endometrial sampling on a specific cycle day. Samples were assessed for relative LPA3mRNA expression using real-time PCR and for LPA3protein using immunohistochemistry and Western blot.Results:ⅰ. LPA3mRNA and protein can be detected in endometrial tissues throughout the human menstrual cycle.ⅱ. The expression of LPA3mRNA and protein significantly increased during the early and late secretory phase compared with other menstrual phases. Its expression dropped rapidly in the mid-secretory and remained low in the whole proliferative phase.iii. In proliferative samples, LPA3protein was detected and localized primarily to the cytoplasm of the luminal and glandular epithelial cells. Early secretory immunostaining for LPA3protein was markedly elevated in both the epithelial and stromal cells. Samples from the midsecretory phase (the implantation window) showed a markedly reduced intensity of LPA3staining in glandular epithelial cells and stromal cells. Late secretory immunostaining for LPA3protein expression was elevated in the nuclei of stromal cells. Thus, the intensity of immunohistochemistry staining for LPA3generally parallels the mRNA and Western blot changes.ⅳ. LPA3is in the low expression in the proliferative phase because of secretion of estrogen; In the late proliferative phase, follicular granulose cells begin to secrete progesterone, progensterone increased gradually after ovulation, LPA3increased during early secretory phase. In the mid-secretory phase E levels increased, LPA3decreased expression in uterine endometrial. In the late secretory phase decreased E level, LPA3in uterine endometrium on expression began to rise.Conclusion:ⅰ. LPA3during the human menstrual cycle of endometrium on the cyclic change, LPA3expression peaked in human peri-implantation. LPA3may participate in the establishment of endometrial receptivity, can be used as a predictor of endometrial receptivityⅱ. LPA3is regulated by progesterone and LH, estrogen by positive or negative feedback. Part2The study on the effect of long protocol controlled ovarian hyperstimulation by GnRHa on expression of peri-implantation window LPA3Object:To study on the effect of long protocol controlled ovarian hyperstimulation by GnRHa/rFSH/hCG on the endometrial LPA3expression.Methods:The people were divided into NC group and COH group. LPA3mRNA and protein expression were determined by RT-RCR and Western-blot form pd3to pd6in the two groups.Results:ⅰ. The peak expression was on the pd4in NC group, the surge was on pd3in COH group.ⅱ. The expression of LPA3significantly lower in COH group compared with NC group.ⅲ. COH group serum E2and P levels in the corresponding time points were higher than the NC control group, the E2/P ratio is also higher than the NC control group.Conclusion:ⅰ. Controlled ovarian hyperstimulation by GnRHa/rFSH/hCG may transform the level of LPA3, lead to disorder of implantation window,declin the endometrial receptivity.ⅱ. May be due to the super physiological doses of E2and P levels, as well as uncoordinated E2/P ratio caused the LPA3expression abnormalities duing COH. We may adjust the E2/P ratios, the expression of LPA3return to normal conditions, improve the IVF pregnancy rate. Part3Expression of LPA3in the endometrium of patients with PCOS during menstrual cycleObject:The aims of the present study were to characterize LPA3mRNA and protein in endometrium during the menstrual cycle of patients with PCOS.Methods:Samples were assessed for relative LPA3mRNA expression using real-time PCR and for LPA3protein using Western blot.Results:ⅰ. PCOS group, patients with menstrual cycle days to39.75days, significantly more than the number of days30.5days of the menstrual cycle of the NC control group (P<0.01). In the proliferative phase in the PCOS group, serum LH levels as8.61miu/ml higher than the NC group, proliferation of serum LH levels3.10miu/ml There were significant differences (P<0.01). Early in the secretory phase in patients with PCOS group serum P level of1.64ng/ml,6.36ng/ml, significantly lower than the NC group P level of4.46ng/ml,14.2ng/ml There were significant differences (P<0.05).ⅱ. Low expression of LPA3mRNA and protein in the proliferative phase in PCOS group, but it was higher in PCOS group than in NC control group in the proliferative phase. In the early secretory phase LPA3mRNA and protein expression was significantly increased and reached its peak, the sharp decline in the expression of mRNA and protein in the middle secretory phase. Expression of LPA3in the early secretory phase endometrium in PCOS group was significantly lower than NC control group.Conclusion:ⅰ. Because of LPA3abnormal expression in the proliferative phase,excessive proliferation s with endometrial with PCOS patients.ⅱ. PCOS group in the early secretory phase has low expression of LPA3, which may interfere with the establishment of endometrial receptivity, and reduced endometrial receptivityⅲ. PCOS group in the proliferative phase of serum levels of LH high expression and low P expression in secretory phase endometrium on state may relate to abnormal expression of LPA3. Part4Comparison of clinical outcomes of the two hormone replacement treatment for frozen-thawed embryo transfer cycle in patients with polycystic ovary syndromeObject:This study was designed to compare the clinical outcomes of the two hormone replacement treatment for frozen-thawed embryo transfer cycle in patients with polycystic ovary syndrome.Methods:Retrospective chart review of126patients with PCOS was done,2clinical protocols for preparing endometrium were carried out, including hormone replacement group(HRT, n=89) and artificial frozen-thawed embryo transfer cycles with pituitary suppression(GnRHa, n=37).Results:No significant differences were found in patient’s age(29.53±3.39vs29.24±2.85years), body mass index (23.07±3.51vs22.20±2.96)and duration of infertility(3.14±1.89vs3.86±1.36 years)(P>0.05), also no significant differences were found in early abortion(16.3%vsl4.8%) and ectopic pregnancy risk(4.1%vs3.7%)(P>0.05). However, compared HRT group, GnRHa group achieved significantly higher clinical pregnancy (55.06%vs72.9%, P<0.05) and implantation (38.7%vs53.6%, P<0.05).Conclusion:It is an effective method for patients with PCOS by using hormone replacement with pituitary suppression for artificial frozen-thawed embryo transfer cycle.更多还原