【作者】 田玉燕；

【导师】 徐素欣； 郝桂敏；

【作者基本信息】 河北医科大学 ， 妇产科学， 2008， 硕士

【摘要】 目的:多囊卵巢综合征（ polycystic ovarian syndrome,PCOS）是一种常见的生殖功能异常,也是无排卵性不孕的主要病因之一,其主要内分泌特征为高雄激素血症、高胰岛素血症、黄体生成素（LH）水平升高、LH与卵泡刺激素（ FSH）比值升高。临床上常表现闭经或月经稀发,无排卵和不孕。患者须通过药物诱导排卵方能解决生育问题。在药物促排卵过程中,卵巢的反应性存在明显的个体差异,与卵巢功能正常者相比,同样剂量的促排卵药物,PCOS患者更有可能出现OHSS,其原因尚不明了。本研究通过观察在我院接受体外受精与胚胎移植（invitro fertilization-embryo transfer, IVF- ET ）的PCOS患者与卵巢功能正常者在应用促排卵药物后,卵巢颗粒细胞中促卵泡激素受体（FSHR）、黄体生成素受体（LHR）mRNA的表达,并结合基础血清、垂体降调节后月经第2天、HCG日血清中性激素水平及及卵泡液中促卵泡激素（FSH）、黄体生成素（LH）、雌二醇（E₂）、睾酮（T）水平变化,进一步探讨PCOS患者颗粒细胞中促性腺激素受体与对促排卵药物反应的关系。方法:1研究对象:1.1 PCOS组（实验组）:选择2007年5月-2007年10月在河北医科大学第二医院生殖中心接受IVF- ET治疗的多囊卵巢综合征患者24例,平均年龄在28.83±3.16岁。1.2对照组:随机选择同期在本生殖中心接受IVF-ET治疗的不孕患者20例,月经规律,不孕原因为输卵管因素,且基础血（月经第三天） FSH、LH、E₂、T、催乳素（PRL）在正常范围内,平均年龄在30.17±3.21岁。所有患者均为初次进入周期,空腹血糖均正常,无全身性及其他内分泌性疾病,男方精液正常。2研究方法和测定内容:2.1标本采集:PCOS组和对照组均采用口服避孕药（达英-35）控制性超促排卵长方案治疗,根据阴道B超和血清中LH、E₂水平检测卵泡发育。当B超显示下有一个卵泡平均直径≥18mm,两个卵泡平均直径≥17mm,或者三个卵泡平均直径≥16mm时,于当日21:00给予肌肉注射绒促性素（HCG）5000– 10000 iu,36小时后在阴道超声引导下行采卵术。取未被血染的穿刺液经2000 r/min离心10 min,上清液置-20℃冰箱保存。收集所有的卵泡穿刺液, Ficoll分离获得颗粒细胞,加入Trizol（Invitrogen）,置1.5ml EP管中,经液氮快速冷冻后于-70℃冰箱保存。2.2标本测定:RT-PCR测定颗粒细胞FSHR及LHR mRNA相对表达量。2.3内分泌激素测定:化学发光法测定基础血清FSH,LH,T,E₂,空腹胰岛素水平（INS）,垂体降调节后月经第2天血清FSH、LH,HCG日血清LH,E₂以及卵泡液FSH,LH,T,E₂。结果:1 PCOS组和对照组间颗粒细胞FSHR、LHRmRNA相对表达量的比较1.1 PCOS组颗粒细胞FSHRmRNA相对表达量（0.675±0.106）较对照组颗粒细胞FSHRmRNA （0.567±0.132）增高,两组之间比较有统计学意义（P<0.05）。1.2 PCOS组颗粒细胞LHRmRNA相对表达量（0.647±0.123）较对照组颗粒细胞LHRmRNA （0.519±0.156）增高,两组之间比较有统计学意义（P<0.05）。2各指标之间的直线相关关系通过多元线性回归分析,显示获卵数与FSHRmRNA相对表达量呈正相关,与LHRmRNA相对表达量无明显直线相关关系。回归方程分别为PCOS组:Y=-22.18+50.51X, R²=0.66, F=20.36, P=0.0001;对照组:Y=-0.11+26.32X, R²=0.773, F=61.36, P=0.00013 PCOS组和对照组血清性激素浓度的比较基础血清中,LH、T、空腹胰岛素水平在PCOS组高于对照组,FSH,E₂两组无统计学差异;垂体降调节后月经第2天血清FSH、LH在进行rhFSH治疗前无统计学差异。与对照组相比,PCOS组HCG日血清LH增高, E₂两组相近。4 PCOS组和对照组卵泡液中性激素浓度的比较PCOS组患者卵泡液中FSH浓度（4.80±1.44 mIU/ml）,与对照组（6.47±0.95 mIU/ml）相比,明显降低（P =0.003）,而LH浓度、T水平明显升高（ P值分别为0.004,0.005）,有统计学意义。同时,两组患者卵泡液中E₂浓度相近。5 PCOS组和对照组IVF周期特征比较两组患者rhFSH治疗总用量和时间无明显差异,hCG日最大血浆E₂水平相近。然而PCOS组获卵数（21±8）高于对照组（15±4）,且平均卵泡直径PCOS组（18.02±1.80 mm）小于对照组（19.41±2.32mm）,P<0.05,有统计学意义。结论:1与对照组患者相比,PCOS患者基础血清睾酮（T）和胰岛素（INS）增高,超促排卵过程中总卵泡数增加而卵泡直径下降;PCOS卵泡液中FSH、P浓度明显降低,而LH浓度、T水平明显升高,同时,两组患者卵泡液中E₂浓度相近,提示PCOS患者存在多种内分泌异常和卵泡发育异常。2超排卵过程中,颗粒细胞FSHR mRNA表达在PCOS组高于对照组,获卵数与颗粒细胞FSHRmRNA表达均成正相关,提示FSHRmRNA表达量与卵巢对外源性促性腺激素反应有关,FSHRmRNA的表达增高可能引起卵巢反应性增高,PCOS组获卵数及FSHRmRNA的表达高于对照组,推测FSHRmRNA的表达增高可能是PCOS患者易发生卵巢过度刺激的原因之一;而卵巢反应性与颗粒细胞LHR表达量无明显相关性。3超排卵过程中,颗粒细胞LHRmRNA表达在PCOS组高于对照组,推测PCOS卵泡可能存在提早的黄素化。更多还原

【Abstract】 Objective: Polycystic ovarian syndrome（PCOS） is a common reproductive syndrome of women characterized by ovarian hyper-androgenism, hyperinsulinemia , high LH level and LH/FSH. It is also one of the main causes of anovulatory infertility. It is characterized by a myriad of symptoms including oligomenorrhea or amenorrhea, anovulation or infertility, and so on. Drug-induced ovulation was effective in treatment of such infertility. Through controlled-ovarian hyper-stimulation process, ovarian response has obvious individual difference. Compared with normal ovary,patients with PCOS are more likely to OHSS. The mechanism still remains unknown. This study tested the levels of FSHR、LHR mRNA expression on ovarian granulosa cells after ovulatory induction ovarian by RT-PCR .The sexual hormone levels in serum at the day 3、the day 2 after pituitary down-regulation、the day of HCG administered and in follicular fluid were all measured by chemiluminescent method.The aim was to investigate the correlation between the levels of FSHR、LHR mRNA expression on ovarian granulosa cells after ovulatory induction ovarian and ovarian response to exogenous gonadotropins.Methods: 1 The study subject1.1 The PCOS group （The study group）: To select 24 patients with polycystic ovarian syndrome who undergone IVF-ET because of infertility in the Second Hospital of HeBei Medical University from May 2007 to October 2007.Their mean ages were 28.83±3.16 years old.1.2 The control group: To random select 20 patients with eumenorrhea who undergone IVF-ET because of infertility, from May 2007 to October 2007.The patients were tubal infertility and their elementary hormone FSH、LH、E₂、PRL、T（MC:3day） were within normal limits. Their mean ages were 30.17±3.21 years old.The patients of PCOS group and control group should be the first time to receive controlled ovarian hyper- stimulation （COH）. No woman had systemic diseases, galactorrhea, endometriomas and other endocrine diseases. They all have normal blood sugar and husbands’sperm condition in control group and PCOS group.2 Methods and Contents:2.1 Collecte the sample: All women receive OC long down-regulated protocols and controlled ovarian hypers- timulation （COH）, GnRH-a / Gonal-F /HMG/HCG. Serial LH、E₂ levels and two-dimensional TVUS follicle measurements were performed until at least one dominant follicles reached at least 18mm or two dominant follicles reached at least 17mm or three dominant follicles reached at least 16mm in diameter. Then HCG 5000-10000iu was administered, followed by trans- vaginal oocyte retrieval 36h later. Follicular fluid uncontami- nated by blood was selected.They was centrifuged at 2000r for 10min （ordinary temperature）, and the supernatant liquid were storaged at -20℃with refrigerator.At oocyte retrieval, all follicular fluid was collected. Granulosa cells were separated from follicular fluid by Ficoll density gradient centrifugation .They were stored in 1.5 ml Eppendorf tubes in Trizol （Invitrogen）. Samples were snapping frozen in liquid nitrogen and were stored at -70℃for later RNA extraction.2.2 Mensurate the sample: FSHR and LHR mRNA were determined by RT-PCR .2.3 Hormone assays : Baseline serum FSH、LH、T、E₂、insulin as well as serum FSH、LH at pituitary down- regulation, LH、E₂ at the day of HCG administered , and follicular fluid FSH、LH、T、E₂ were all measured by chemiluminescent method.Results:1 The relative expression level of FSHRmRNA、LHRmRNA on ovarian granulosa cells from PCOS patients and control women1.1 The relative expression level of FSHRmRNA on ovarian granulosa cells from PCOS patients （0.675±0.106） was higher than that from control women （0.567±0.132）. There were remarkably statistical significance （P<0.05）.1.2 The relative expression level of LHRmRNA on ovarian granulosa cells from PCOS patients （0.647±0.123） was higher than that from control women （0.519±0.156）. There were remarkably statistical significance （P<0.05）.2 Multivariate linear regression analysis on data showed that there was a positive correlation between the number retrieved oocyte with the expression of FSHRmRNA on ovarian granulosa cells, but not with the expression of LHRmRNA. The regression equations in two groups were as followes: the PCOS group:Y=-22.18+50.51X, R²=0.66, F=20.36, P=0.0001;the control group:Y=-0.11+26.32X, R²=0.773, F=61.36, P=0.00013 To compare outcomes of serum sex hormone levels of the patients of PCOS group and control groupThe mean of baseline serum LH、T、insulin in PCOS group was higher than that in the control group,while FSH and T was similar. At day 2 after pituitary down-regulation, serum FSH、LH levels in two groups were similar. At the day of HCG administered, the mean of serum LH in PCOS group was higher than that in the control group, while E₂ levels in two groups were similar.4 To compare outcomes of follicular fluid sex hormone levels of the patients of PCOS group and control group:The mean of the FSH level in follicular fluid in PCOS group was lower than that in the control group （4.80±1.44 mIU/ml vs. 6.47±0.95 mIU/ml, P =0.003）, while LH and T levels higher （P =0.004,P =0.005）. There was remarkably statistical significance. The E₂ levels in follicular fluid in two groups were similar. 5 IVF cycle characteristics of control women and PCOS patients undergoing GnRH analog/rhFSHThe amount of rhFSH administered and the duration of rhFSH treatment were similar in control women and PCOS patients, causing comparable maximum serum E₂ levels on the day of hCG administration. Total number of oocytes retrieved was greater （21±8 vs. 15±4）, whereas average follicle diameter was smaller, in PCOS patients vs. control women （18.02±1.80mm vs. 19.41±2.32mm）. There were remarkably statistical significance （P<0.05）.Conclusion: 1 The patients with PCOS existe more than one kinds of endocrine abnormality and abnormal growth of follicles.2 There was a positive correlation between the number retrieved oocyte with the expression of FSHRmRNA on ovarian granulosa cells in two groups,and the relative expression level of FSHRmRNA on ovarian granulosa cells from PCOS patients was higher than that from control women. The increase of the level of the expression FSHR may cause enhancement of ovarian response to exogenous gonado- tropins.This may be one of the cause that patient with PCOS are easy to occour OHSS.At the same time there was no correlation between ovarian response with the expression level of LHR.3 The relative expression level of LHRmRNA on ovarian granulosa cells from PCOS patients was higher than that from control women. To hint that PCOS follicles exposed to GnRH analog/FSH stimulation for IVF may exist abnormal luteinization.更多还原