IVF周期血清总睾酮与卵巢反应性参数及颗粒细胞FSHRmRNA表达的相关性研究及睾酮对体外培养人黄素化颗粒细胞FSHRmRNA、IGF-ImRNA、IGF-IImRNA表达的影响

【作者】 丁颖；

【导师】 徐苓； 邓成艳；

【作者基本信息】 中国协和医科大学 ， 妇科内分泌， 2010， 博士

【摘要】 研究背景在颗粒细胞内,雄激素在FSH诱导的芳香化酶作用下转化为雌激素。同时,雄激素又能增加颗粒细胞对FSH的敏感性,促进卵泡发育,如：增加颗粒细胞FSH受体的表达,增加FSH对早期卵泡的募集,促进胰岛素样生长因子-Ⅰ的分泌,IGF-Ⅰ与FSH产生协同作用,促进卵泡生长发育。近年来,国外有些临床研究在IVF周期前或周期过程中使用雄激素处理方案(雄激素启动,androgen priming),包括使用睾酮,脱氢表雄酮,黄体生成素,人绒毛膜促性腺激素和芳香化酶抑制剂等,以提高体循环或卵巢局部的雄激素水平,希望增高的雄激素水平能增加卵巢对外源性促性腺激素的敏感性,增加卵巢反应性,改善IVF结局。然而临床研究结果并不完全一致。因此,我们设计了这项实验,观察IVF过程中血清总睾酮的动态变化、测定颗粒细胞FSHRmRNA的表达及不同浓度睾酮作用下颗粒细胞FSHRmRNA、IGF-ⅠmRNA、IGF-ⅡmRNA的表达,初探IVF周期雄激素处理方案改善卵巢反应性的可行性。研究目的1了解IVF周期血清总睾酮与卵巢反应性参数及IVF结局的关系；2分析IVF周期颗粒细胞FSHRmRNA表达与卵巢反应性及血清总睾酮的关系；3睾酮对体外培养人黄素化颗粒细胞FSHRmRNA, IGF-ⅠmRNA, IGF-ⅡImRNA基因表达的影响。研究方法研究过程分为三部分进行：第一部分：IVF常规长方案患者进入周期后,监测卵巢刺激过程中血清总睾酮浓度变化,记录患者每次检查时的卵泡数量及大小、血清性激素、取卵数、成熟卵数、优质胚胎数、囊胚数及妊娠情况及使用rFSH剂量、卵巢刺激天数。观察卵巢刺激后血清总睾酮的浓度变化；分别以是否妊娠和血清基础总睾酮分组,分析基础总睾酮与卵巢反应性及IVF结局的关系；探讨血清总睾酮与卵巢反应性参数的相关性。第二部分：IVF短方案患者进入周期,临床资料的获取方法同第一部分。取卵同时,留取卵泡液,分离颗粒细胞,提取细胞总RNA,反转录cDNA,进行real-time PCR。按照获得的成熟卵数将患者分为卵巢过度反应组、正常反应组和低反应组。比较三组患者的一般情况、卵巢反应性参数及妊娠率、囊胚冷冻率；比较三组血清总睾酮水平及颗粒细胞FSHRmRNA相对表达量,并对血清总睾酮、卵巢反应性参数和FSHRmRNA相对表达量进行相关分析,进一步探讨血清总睾酮、FSHRmRNA表达与卵巢反应性间的关系。第三部分：取卵时获得人黄素化颗粒细胞,以10-8mol／L、10-7mol／L.10-5mol／L浓度睾酮进行刺激。各组分别在睾酮刺激6小时和24小时时终止培养,提取颗粒细胞总RNA,反转录cDNA,进行real-time PCR。以10-8mol／L作为对照组,比较不同浓度睾酮、刺激不同时间对颗粒细胞FSHRmRNA、IGF-ⅠmRNA、IGF-ⅡmRNA表达的影响。结果1第一部分①卵巢刺激过程中,血清总睾酮浓度随着卵泡的发育逐渐上升；②分别以血清基础总睾酮(bTT)≤0.2ng/ml和>0.2ng／ml将患者分组,两组间卵巢反应性参数及IVF结局比较均无显著性差异；同样方法再以bTT≤0.3ng／ml和>0.3ng／ml、bTT≤0.4ng/ml和>0.4ng／ml将患者分组,两组间卵巢反应性参数及IVF结局比较仍然均无显著性差异；③药物刺激卵巢过程中的总睾酮浓度变化与基础总睾酮浓度有平行关系；④血清基础总睾酮与卵巢反应性参数无线性相关关系；药物刺激卵巢过程中,随着卵泡发育,总睾酮水平与基础窦卵泡数、取卵数、成熟卵泡数有正相关关系,与基础FSH值和rFSH剂量有负相关关系；⑤妊娠组OPU14日总睾酮值显著高于未妊娠组。2第二部分①按照卵巢反应性分组研究发现：卵巢刺激过程中,卵巢对促性腺激素的反应性与颗粒细胞FSHRmRNA的表达量有关,卵巢过度反应组颗粒细胞FSHRmRNA表达量显著高于卵巢低反应组颗粒细胞FSHRmRNA表达量；②颗粒细胞FSHRmRNA表达量与rFSH用药剂量呈负相关关系,与E2峰、取卵数呈正相关关系；③血清基础总睾酮与卵巢反应性参数如rFSH剂量、卵巢刺激天数及E2无线性相关关系,与颗粒细胞FSHRmRNA的表达也无线性相关关系；药物刺激卵巢过程中,随着卵泡发育,动态变化的总睾酮水平与rFSH剂量、卵巢刺激天数呈现负相关关系,与E2峰值、取卵数呈现正相关关系,与FSHRmRNA的表达呈现正相关关系。3第三部分①不同浓度睾酮刺激颗粒细胞6小时,显著增加颗粒细胞FSHRmRNA的表达,并呈现浓度依赖关系：睾酮浓度越高,基因表达量越高；刺激时间延长至24小时,各组间颗粒细胞FSHRmRNA的表达无统计学差异；②不同浓度睾酮刺激颗粒细胞6小时,各组间颗粒细胞IGF-ⅠmRNA的表达无统计学差异；刺激时间延长至24小时,颗粒细胞IGF-ⅠmRNA的表达显著降低,并呈现浓度依赖关系：睾酮浓度越高,基因表达量越低；③不同浓度睾酮刺激颗粒细胞6小时,各组间颗粒细胞IGF-ⅡmRNA的表达无统计学差异；刺激时间延长至24小时,10-7mol／L处理组IGF-ⅡmRNA基因表达量显著高于10-8mol／L处理组和10-5 mol／L处理组。结论1基础总睾酮水平在正常范围内的患者,其基础总睾酮水平不能作为预测卵巢反应性及IVF结局的指标；2 IVF药物刺激卵巢过程中的总睾酮水平与卵巢反应性正相关；颗粒细胞FSHRmRNA表达量与卵巢反应性正相关。3高浓度睾酮短时间作用能上调FSHR和IGF-Ⅱ基因表达,但长时间作用对基因表达上调作用消失。高浓度睾酮下调IGF-Ⅰ基因表达,随着作用时间的延长,下调作用更显著。更多还原

【Abstract】 [Background]Androgens play important roles in follicular growth, development, and atresia, as well as in steroidogenesis. First, androgens act as essential substrates for the production of estrogen. Second, results of some animal experiments showed that Androgens appear to be positive regulators of follicular development by increasing the expression of FSHR and promoting the secretion of the IGF-Ⅰin the granulose cells. IGF-Ⅰshowed some synergistic effects with FSH on follicuogeneisis. Therefore androgens may enhance follicular responsiveness and then promote the development of the follicles. At the same time, animal research also suggested that the androgen might have negative effect on the follicle development. In recent years, it has been reported that some clinical trials used the androgen priming protocol before or during the controlled ovarian hyperstimulation (COH) in IVF patients in order to increase the ovary response to the exogenous gonadotrophins. However, the outcomes of these clinical studies did not accord with each other. Our study was designed to explore the feasibility of androgen supplement before or during the IVF cycle to impove the IVF outcome.[Objective]1 To observe the serum total testosterone level during the controlled ovarian stimulation. To assess possible associations between total testosterone level and IVF stimulation parameters or IVF pregnancy outcome.2 To evaluate the relationship between the serum total testosterone level and the expression of the FSHR in the granulosa cells.3 To evaluate the effects of testosterone treatment for different doses and different incubating time on FSHR, IGF-Ⅰand IGF-Ⅱexpression in human luteinized granulusa cells. [Methods]Our study was devided into three parts: Part one:A clinical prospective study was involved in this part. The serum total testosterone levels was measured during the IVF cycle. The quantity and the size of the follicles, the levels of sex hormones, and the amount of oocyte retrieved, mature oocyte, and blastomeres were recorded. Also the duration of controlled ovarian stimulation and the total rFSH doses were noted. The patients were devided into two groups according to the IVF outcome or the basal total testosterone level respectively. We described the changes of total testosterone level during the IVF cycle. Linear regression analysis was used to assess the correlation between linear data.Part two:Hormone levels and the ovarian response variables were recorded as in part one. In addition, preovulatory granulosa cells were collected from follilcular fluid obtained from patients undergoing transvaginal oocyte retrieval with untrasound guidance. According to the quantity of mature oocyte, the patients were grouped as normal response group, low response group and high response group. The mRNA level of FSHR in the granulosa cells from each patient was quantitatively measured using real-time reverse-transcriptase polymerase chain reaction (RT-PCR). The clinical parameters and the expression of FSHRmRNA among the three groups were compared. And finally, linear regression analysis assessed the correlation between linear data.Part three:Preovulatory granulosa cells were collected from follilcular fluid obtained from patients undergoing transvaginal oocyte retrieval with untrasound guidance. After that, granulosa cells were cultured for 2 days in DMEM/F12 medium supplemented with 10%FBS, penicillin/streptomycin, and glutamine. The cells were grouped and incubated in DMEM/F12 medium supplemented with 1%FBS and different concentrations of testosterone (10-8 mol/L,10-7 mol/L,10-5mol/L) for another 6 or 24 hours. The mRNA levels of FSHR, IGF-I and IGF-II were were quantitatively measured using real-time reverse-transcriptase polymerase chain reaction (RT-PCR). We analyzed the effects of testosterone treatment with different doses and different cultured time on FSHR, IGF-I and IGF-II expression in granulusa cells.[Results]Part one:1 During the controlled ovarian stimulation (COH), total testosterone (TT) levels rised in accordance with the follicle development.2 The change of TT level during the COH presented a parallel phenomenon with the basal TT level.3 Patients were grouped by basal TT levels at increments of 0.1ng/ml from 0.2ng/ml to 0.4ng/ml. The basal TT levels did not significantly correlate with any of the stimulation parameters tested and the IVF outcome for each increment.4 TT levels during the COH correlated positively with the antral follicle count, the quantity of oocyte retrieved and mature oocyte. And it correlated negatively with the basal FSH level and the number of gonadrotropin ampoules used.5 TT level measured 14 days after oocyte retrivel in pregnancy group was significantly higher than that in the non-pregnancy group.Part two:1 The expression of FSHR was different among the three different ovarian response groups. FSHRmRNA level s in high response group was significantly higher than that in the low response group.2 The FSHRmRNA correlated negatively with the total doses of the rFSH. And it correlated positively with the peak E2 level on the day of hCG injection and oocyte number retrieved.3 The basal TT levels did not significantly correlate with both any of the stimulation parameters tested and the expression of FSHRmRNA. TT levels during the COH correlated negatively with the duration of COH and the number of gonadrotropin ampoules used. And it correlated positively with the peak E2 on the day of hCG injection, the quantity of oocyte retrieved, and the expression of FSHRmRNA.Part three:1 After the cells were incubated with testosterone of different concentrations for 6 hours, the expression of FSHRmRNA was significantly increased in a dose-depended way. However, when cultured for 24 hours, no statistical significant difference was observed among the three groups.2 After the cells were incubated with testosterone of different concentrations for 6 hours, no statistically significant difference were observed on the expression of IGF-ImRNA among the three groups. However, when cultured for 24 hours, the expression of IGF-ImRNA was significantly decreased in a dose-depended way.3 After the cells were incubated with testosterone of different concentrations for 6 hours, there were no statistically significant differences on the expression of IGF-IImRNA among the three groups. However, when cultured for 24 hours, the expression of IGF-IImRNA in 10-7 mol/L group was higher than that of the 10-8 mol/L group and 10-5mol/L group.[Conclusions]1:Basal serum total testosterone level may not predict the IVF outcome and the ovarian response.2:Total testosterone levels during the controlled ovarian hyperstimulation procedure correlated positively with the ovarian response. The expression of FSHRmRNA in human luteized granulosa cells correlated positively with the ovarian response.3:Testosterone of high concentration for short time could up-regulate the expression of FSHR and IGF-II. But when treated for long time, the up-regulation of the expression disappeared. Testosterone of high concentration could down-regulate the expression of IGF-I, and the down-regulation became stronger as treated for a longer time.[Key words]IVF, testosterone, ovarian response, granulosa cell, FSHRmRNA、IGF-ⅠmRNA、IGF-ⅡmRNA更多还原