子宫内膜异位症卵巢颗粒细胞SF-1的表达及其启动子甲基化的研究

发布时间：2018-05-26 21:58

本文选题：子宫内膜异位症 + 体外受精-胚胎移植　；参考：《上海交通大学》2015年博士论文

【摘要】：第一部子宫内膜异位症对体外受精-胚胎移植治疗临床结果的影响目的:回顾性比较分析子宫内膜异位症(EMs)合并不孕和对照组(单纯输卵管因素性不孕)行体外受精-胚胎移植(IVF-ET)治疗的临床数据,探讨EMs对于IVF-ET治疗结局的影响。方法:选取107例腹腔镜诊断EMs的不孕女性(其中58例为I、II期EMs,49例为III、IV期EMs)和121例对照组女性纳入研究,比较两组一般情况及IVF-ET治疗结局相关指标。结果:(1)EMs和对照组两组患者的年龄、不孕年数、体重指数、基础FSH值、HCG日E2水平和移植日子宫内膜厚度和移植胚胎数进行比较,差异均无统计学意义;(2)EMs组FSH使用总量显著高于对照组(P0.05);(3)两组患者的获卵数和受精率相比无差异,但EMs组的优质胚胎率(P0.01)和可冷冻胚胎数(P0.05)均显著低于对照组,临床妊娠率和种植率在EMs组有下降趋势但差异无统计学意义。结论:EMs对于IVF-ET治疗的临床结果具有不良影响。EMs致卵巢对促排卵药物的反应性下降,降低优质胚胎率和可用胚胎数,使得种植率和临床妊娠率呈现下降趋势。第二部分SF-1和LRH-1在子宫内膜异位症卵巢颗粒细胞中的表达目的:通过测定P450芳香化酶编码基因CYP19、CYP19 PII、孤核受体家族成员类固醇生成因子-1(steroidogenic-1,SF-1)和肝受体类似物-1(liver receptor homolog-1,LRH-1)在EMs卵巢颗粒细胞中的表达及其相关性,探讨SF-1和LRH-1在EMs卵巢颗粒细胞P450芳香化酶表达下降机制中的可能作用。方法:选取16例腹腔镜诊断EMs的不孕女性(其中7例为I、II期EMs,9例为III、IV期EMs)和28例对照组女性纳入研究。收集IVF-ET治疗中的卵泡液分离纯化颗粒细胞,应用QT RT-PCR技术检测颗粒细胞中CYP19,CYP19 PII以及SF-1、LRH-1 m RNA水平并作比较和相关分析。结果:(1)EMs卵巢颗粒细胞中CYP19(P0.05),CYP19 PII(P0.001)以及SF-1(P0.05)、LRH-1(P0.001)m RNA的表达水平均较正常对照组降低;(2)Spearman相关分析结果显示仅EMs组颗粒细胞中SF-1和LRH-1 m RNA的表达水平与CYP19 PII m RNA呈正相关关系(P0.05)。结论:EMs卵巢颗粒细胞中SF-1和LRH-1在转录水平的表达下降可部分解释EMs性不孕卵巢反应下降和卵子质量受损的机制,而SF-1、LRH-1和CYP19 PII在EMs颗粒细胞中的表达下降相关性是探讨EMs颗粒细胞P450芳香化酶活性下降机制的初步研究。第三部分子宫内膜异位症卵巢颗粒细胞SF-1基因启动子甲基化状态的研究目的:通过检测并比较EMs和正常女性卵巢颗粒细胞中SF-1基因启动子区5’Cp G位点甲基化水平探讨EMs卵巢颗粒细胞中SF-1表达下降的机制。方法:应用亚硫酸盐测序即BSP克隆测序法(Bisulfite Sequencing PCR,BSP)检测EMs和正常女性卵巢颗粒细胞SF-1基因启动子区5’Cp G位点甲基化水平,将测得的序列与原始序列比对,统计和比较甲基化位点、数量并分析两组甲基化程度。结果:亚硫酸盐测序片段涵盖SF-1基因转录起始位点附近的13个Cp G位点(从Cp G-84到Cp G+168),正常对照组颗粒细胞SF-1基因启动子区域的Cp G位点表现为差异甲基化状态,而EMs组SF-1基因启动子区域的整体甲基化水平显著高于对照组(P0.05),逐个位点的分析显示EMs组Cp G+7,+18,+21,+42,+54,+60,+132以及+146位点相较于正常对照组呈现高甲基化水平(P0.05);而EMs组Cp G+77,+121和+141位点的高甲基化水平尤为显著(P0.001)。结论:EMs卵巢颗粒细胞SF-1基因启动子区5’Cp G位点甲基化水平显著高于对照组,尤其是+77,+121和+141Cp G位点,可能是EMs卵巢颗粒细胞SF-1 m RNA表达降低的机制。
[Abstract]:The effect of the first endometriosis on the clinical outcome of in vitro fertilization and embryo transfer: a retrospective comparison and analysis of the clinical data of EMs combined with infertility and the control group (simple fallopian factor infertility) in vitro fertilization and embryo transfer (IVF-ET) treatment, and to explore the effect of EMs on the outcome of IVF-ET treatment. Methods: 107 infertile women diagnosed by laparoscopy (58 with I, II EMs, 49 for III, IV EMs) and 121 in the control group were included in the study to compare the general conditions of the two groups and the related indexes of the IVF-ET treatment outcome. Results: (1) the years of age, the number of infertility, the body mass index, the base FSH value, HCG E2 level and shift of two groups of patients in the two groups were compared. The difference between the thickness of endometrium and the number of transplanted embryos was not statistically significant. (2) the total use of FSH in group EMs was significantly higher than that of the control group (P0.05); (3) the number of acquired eggs and fertilization rate in the two groups was not different, but the high quality embryo rate (P0.01) and the number of frozen embryos (P0.05) in the group of EMs were significantly lower than those in the control group, and the clinical pregnancy rate and species were significantly lower than those of the control group. There was a downward trend in the rate of implantation in the EMs group, but there was no statistical difference. Conclusion: EMs has adverse effects on the clinical outcome of IVF-ET treatment with.EMs induced ovarian hyperstimulation, reducing the quality of embryo and the number of embryos available, making the implantation rate and the clinical pregnancy rate descending. Second parts of SF-1 and LRH-1 are in the uterus. The expression and correlation of P450 aromatase encoding gene CYP19, CYP19 PII, -1 (steroidogenic-1, SF-1) and -1 (liver receptor homolog-1, LRH-1) in ovarian granulosa cells and their correlation were investigated. The possible role of the decrease mechanism of P450 aromatase expression in EMs ovarian granulosa cells. Methods: 16 cases of infertility women diagnosed by EMs were selected by laparoscopy (7 cases were I, II phase EMs, 9 cases III, IV stage EMs) and 28 control women were included in the study. The follicle fluid in IVF-ET treatment was collected and purified, and the QT RT-PCR technique was used to detect the granule cells. The levels of CYP19, CYP19 PII, SF-1, LRH-1 m RNA in granulocytes were compared and analyzed. Results: (1) the expression level of CYP19 (P0.05), CYP19 PII (CYP19 PII) in EMs ovarian granulocytes were all lower than that of the normal control group; (2) the results of correlation analysis showed that only granular cells in the group of ovarian granulocytes were only in the granular cells. There was a positive correlation between the expression level of RNA and CYP19 PII m RNA (P0.05). Conclusion: the decline of SF-1 and LRH-1 at the transcription level in the EMs ovarian granulosa cells could partly explain the mechanism of the decline of the EMs sex and the loss of the ovaries and the loss of the egg mass. Preliminary study on the mechanism of decreasing the activity of P450 aromatase in granulocyte. Third Research on the methylation status of SF-1 gene promoter in ovarian granulosa cells of endometriosis: the study of EMs ovarian granular cells by detecting and comparing the level of the 5 'Cp G site methylation in the promoter region of SF-1 gene in EMs and normal female ovarian granulosa cells Methods: the mechanism of decreasing expression of SF-1. Methods: using BSP cloning sequencing (Bisulfite Sequencing PCR, BSP) to detect the methylation level of the 5 'Cp G loci of the SF-1 gene promoter region of the ovarian granulosa cells of normal women, and to compare the measured sequences with the original sequences, to count and compare the methylation sites, and to analyze the number and analyze the two groups. The degree of methylation. Results: the sulfite sequencing fragment covers 13 Cp G loci (from Cp G-84 to Cp G+168) near the starting site of the SF-1 gene transcription site. The Cp G loci of the SF-1 gene promoter region of the normal control group are differential methylation, while the overall methylation level of the SF-1 gene promoter region of the EMs group is significantly higher than that in the control group. P0.05, the analysis of one by one showed that the EMs group Cp G+7, +18, +21, +42, +54, +60, +132, and +146 loci showed higher methylation level compared to the normal control group, and the high methylation level was particularly significant. The level was significantly higher than that in the control group, especially the +77, +121 and +141Cp G loci, which may be the mechanism of the decrease of SF-1 m RNA expression in EMs granulosa cells.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R714.8

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