体外受精-胚胎移植中多囊卵巢综合征颗粒细胞凋亡与妊娠结局关系的探讨

目的探讨体外受精-胚胎移植中多囊卵巢综合征（PCOS）患者颗粒细胞凋亡与妊娠结局的关系。方法选择2004年2月至6月在北京大学第三医院体外受精-胚胎移植患者,其中PCOS组30例、对照组30例,收集其颗粒细胞,采用流式细胞技术,检测颗粒细胞亚二倍体的含量及凋亡相关蛋白PDCD5单克隆抗体免疫荧光密度;免疫组化法检测凋亡相关蛋白PDCD5、Bcl-2、Bax在颗粒细胞中的表达情况;同时将两组相应的临床指标进行比较。结果两组年龄、基础卵泡刺激素（FSH）、FSH的用量、大中卵泡数、受精率、优质胚胎率和妊娠率比较差异无显著性意义（P〉0.05）。而PCOS组取卵总数、小卵泡数、HCG日雌二醇水平及颗粒细胞亚二倍体率、PD-CD5荧光密度、免疫组化PDCD5的积分光密度（IOD）值明显增高,免疫组化Bcl-2的IOD值降低,与对照组比较差异有显著性意义（P〈0.05）。PCOS组及对照组的妊娠组颗粒细胞亚二倍体数降低、PDCD5荧光密度降低、免疫组化Bcl-2的IOD值增高,PDCD5的IOD值降低,两组中的妊娠组与非妊娠组比较差异有显著性意义（P〈0.05）。结论PCOS患者颗粒细胞凋亡明显增加,颗粒细胞凋亡增加妊娠率降低。     

凋亡调控蛋白Bcl-2、Bax、P53和PDCD5在多囊卵巢综合征各级卵泡的表达

目的 检测凋亡调控蛋白Bcl-2、Bax、P53和PDCD5在多囊卵巢综合征和对照组各级卵泡中颗粒细胞的表达，探讨颗粒细胞凋亡与PCOS的发病可能的相关关系。方法选择因多囊卵巢综合征行卵巢楔形切除的卵巢病理组织32例，同时选择年龄相匹配并于卵泡期手术的非多囊卵巢综合征卵巢组织15例为对照组，采用免疫组化法检测凋亡调控蛋白Bcl-2、Bax、P53和PDCD5在各级卵泡中颗粒细胞表达情况，由计算机提取，定量分析各级卵泡颗粒细胞免疫组化着色的光密度（OD）值，比较两组问的差异。结果 Bcl-2、Bax、P53和PDCD5蛋白在多囊卵巢综合征始基卵泡、窦前卵泡颗粒细胞的表达与正常对照组比较差异无显著性（P〉0．05）。窦状卵泡颗粒细胞Bcl-2蛋白表达下调，Bax和PDCD5的表达明显上调，与对照组比较其OD值差异有显著性（P〈0．05）。P53蛋白在多囊卵巢综合征各期卵泡颗粒细胞的表达差异无显著性，与正常对照组比较差异无显著性。结论多囊卵巢综合征始基卵泡和窦前卵泡期颗粒细胞的凋亡与对照组比较差异无显著性，窦状卵泡期颗粒细胞的凋亡明显增加。Bcl-2、Bax和PDCD5细胞浆表达凋亡调控蛋白参与了卵巢颗粒细胞的凋亡过程，P53细胞核表达的凋亡调控蛋白在多囊卵巢综合征颗粒细胞的凋亡过程中与对照组比较无明昆差异。     

山奈酚对BaP引起的人绒毛膜外滋养层细胞HTR8/SV neo凋亡的影响

目的：研究山奈酚对苯并芘（BaP）引起的人绒毛膜外滋养层细胞HTR8/SV neo凋亡的影响。方法：通过四甲基偶氮唑盐（MTT）法观察及测定,最终选取10 μmol/L BaP和0.01,0.1,1 μmol/L山奈酚共同处理人HTR8/SV neo细胞48 h,光镜下观察不同浓度BaP作用下细胞生长情况,吉姆萨（Gimesa）染色及Hochest33342荧光染色观察细胞凋亡情况,利用流式细胞术测定细胞凋亡率变化。结果：①流式细胞术测定结果表明BaP单独作用细胞存活率为（72.58±0.29）%,与0.01,0.1,1 μmol/L山奈酚同时作用时细胞存活率分别为（84.96±1.34）%、（89.54±1.64）%和（91.01±1.26）%,两者同时作用组细胞存活率均高于BaP单独作用组,差异均有统计学意义（P＜0.01）。②单独BaP作用可引起细胞染色质浓缩,出现凋亡小体等典型凋亡形态、细胞核致密浓染、碎裂状。结论：山奈酚对BaP引起的HTR8/SV neo细胞凋亡有明显的抑制作用。     

X线辐射对大鼠卵巢形态与功能的影响

目的：探讨X线电离辐射对大鼠卵巢形态与功能的影响。方法：实验分0,0.2,0.4,0.6,0.8,1.0Gy剂量组,每组各10只大鼠。采用HE染色观察各级卵泡数目,免疫组化法观察P53、Bcl-2蛋白表达,TUNEL法测定颗粒细胞中凋亡小体的表达。结果：随大鼠机体接受X线照射剂量增加,始基卵泡、初级卵泡、次级卵泡数目随之减少（P〈0.05或P〈0.01）,以始基卵泡减少为著;不同剂量X线照射后窦状卵泡数目与假照射组差异无统计学意义（P〉0.05）。当吸收剂量为0.2Gy,卵巢颗粒细胞中P53蛋白及凋亡小体表达较假照射组减少,Bcl-2蛋白有所增多（P〈0.05或P〈0.01）,而吸收剂量在0.4～1.0Gy时,P53蛋白及凋亡小体的表达水平随吸收剂量的增加而上升（P〈0.05或P〈0.01）,Bcl-2蛋白表达水平减少（P〈0.01）。结论：X线可使大鼠卵巢组织中始基卵泡的数目有所减少,促进细胞发生凋亡,进一步影响颗粒细胞的内分泌功能。     

细胞凋亡调控蛋白bcl-2和bax在多囊卵巢综合征患者子宫内膜中的表达

目的探讨多囊卵巢综合征（PCOS）患者子宫内膜中细胞凋亡调控蛋白bcl-2和bax的表达及其在PCOS患者子宫内膜病变中的意义。方法应用免疫组织化学SP方法,测定18例PCOS患者子宫内膜和27例月经周期正常、因输卵管不通或男性不育就诊的对照者子宫内膜（10例增殖期、17例分泌期）bcl-2和bax蛋白的表达。结果PCOS患者子宫内膜bcl-2表达较对照者增殖期和分泌期高（P＜0．05和P＜0．01）,但bax表达相似（p＞0．05）。结论PCOS患者子宫内膜bcl-2蛋白表达增高,与bax形成更多的异源二聚体,从而抑制PCOS子宫内膜凋亡,对其子宫内膜的异常增生改变起一定作用。     

多囊卵巢综合征患者种植窗口期子宫内膜容受性的研究

目的研究影响多囊卵巢综合征（PCOS）患者种植窗口期子宫内膜容受性的基因表达。方法9例PCOS患者（PCOS组）及7例非PCOS因男方原因不孕者（对照组）行阴道超声监测结合血清激素测定确定排卵时间,于种植窗口期对两组妇女进行子宫内膜活检,提取cDNA,其中6例PCOS患者应用含有21571个基因序列的寡核苷酸芯片进行扫描,经过分析后得到PCOS患者的子宫内膜与对照组相比的差异表达基因;另外3例PCOS患者进行实时荧光定量PCR实验检测基质金属蛋白酶26（MMP-26）基因的表达。结果PCOS患者种植窗口期子宫内膜的差异表达基因为116个,占全部表达基因的比例为1．23％（116／9421）。PCOS组患者与对照组比较,表达上调的差异表达基因占0．12％（11／9421）;表达下调的差异表达基因占1．11％（105／9421）,其中,MMP-26在基因芯片中表达下调10．6倍。与对照组患者种植窗口期子宫内膜比较,3例PCOS患者子宫内膜MMP-26基因的相对表达率（Ratio值）分别为0．31、0．11和0．05。结论PCOS患者种植窗口期子宫内膜MMP-26基因表达下调,提示PCOS患者存在子宫内膜容受能力下降。     

GATA4基因沉默对多囊卵巢综合征模式大鼠卵巢颗粒细胞凋亡的影响及相关机制

目的:GATA4基因沉默对多囊卵巢综合征(PCOS)模式大鼠卵巢颗粒细胞凋亡的影响。方法:空白对照组(Control)、阴性对照组(NC)和转染GATA4-siRNA沉默组(GATA4-siRNA)转染PCOS卵巢颗粒细胞48h后,Western blot检测各组细胞中GATA4、Cleaved caspase3、β-catenin、Cyclin D1蛋白表达;流式细胞仪检测细胞凋亡。结果:NCsiRNA组的GATA4、Cleaved caspase3、β-catenin、Cyclin D1蛋白表达及细胞凋亡率与对照组差异无统计学意义(P0.05),GATA4-siRNA组的细胞凋亡率及Cleaved caspase3蛋白表达显著高于对照组,GATA4、β-catenin、Cyclin D1蛋白表达显著低于对照组(P0.01)。结论:沉默PCOS模式大鼠卵巢颗粒细胞中GATA4表达,可促进细胞的凋亡,其机制与上调Cleaved caspase3蛋白表达及下调Wnt/β-catenin信号通路有关。     

GPR30/EGFR在多囊卵巢综合征患者卵巢组织中的表达

目的:探讨雌激素膜受体G蛋白偶联受体30(GPR30)及表皮生长因子(EGFR)在多囊卵巢综合征(PCOS)患者卵巢组织中的表达。方法:选取2012~2015年在青岛市妇女儿童医院生殖中心行手术治疗的PCOS患者25例,以及同期因良性卵巢肿瘤行剥除术的25例患者为对照组。采用实时荧光定量PCR技术检测卵巢组织中ERα、ERβ、GPR30及EGFR mRNA的表达水平;Western blot法检测卵巢组织中ERα、ERβ、GPR30及EGFR蛋白表达;免疫组化法检测卵巢组织中ERα、ERβ、GPR30及EGFR蛋白的表达定位。结果:PCOS卵巢组织中GPR30及EGFR mRNA和蛋白表达水平均显著高于对照组,差异均有统计学意义(P0.05)。始基卵泡及初级卵泡中,GPR30蛋白主要定位表达于卵母细胞胞浆与胞膜以及颗粒细胞的细胞核,始基卵泡卵母细胞表达明显高于初级卵泡卵母细胞,较对照组GPR30在PCOS组卵母细胞及颗粒细胞中表达均升高;EGFR蛋白主要表达于颗粒细胞的细胞核,在PCOS组表达升高。结论:GPR30及EGFR在PCOS患者的卵泡发育障碍过程中可能具有重要作用。     

特发性胎儿生长受限与胎盘细胞凋亡及bcl-2基因表达的关系

目的 探讨特发性胎儿生长受限(FGR)与胎盘细胞凋亡及bcl-2基因表达的关系.方法 应用透射电镜、脱氧核苷酸末端转移酶介导的脱氧尿苷三磷酸标记法(TUNEL)、流式细胞技术(FCM)检测特发性FGR孕妇(FGR组)及正常妊娠妇女(对照组)各15例的胎盘细胞凋亡情况,并采用RT-PCR技术观察两组胎盘细胞中bcl-2基因相对表达量.结果 电镜下观察到FGR组胎盘合体滋养细胞核膜皱缩,核仁消失,染色质致密、凝聚;TUNEL 检测 FGR 组胎盘细胞凋亡率为13.68%,高于对照组的4.05%,两组比较,差异有统计学意义(P<0.05);FCM检测胎盘S期细胞比率FGR组为3.4%,对照组为2.2%,两组比较,差异有统计学意义(P<0.05),FCM检测的胎盘细胞凋亡率变化趋势与TUNEL检测结果一致;FGR组胎盘细胞bcl-2基因相对表达量为0.19±0.13,对照组为0.55±0.17,两组比较,差异也有统计学意义(P<0.05).结论 特发性FGR的发生与胎盘细胞凋亡增加有关,胎盘细胞凋亡增加与胎盘细胞中bcl-2基因表达下调有一定关系.     

TRAIL及其受体DR4、DCR1在多囊卵巢综合征大鼠卵泡发育中的作用

目的:探讨肿瘤坏死因子相关凋亡诱导配体(TNF-related apoptosis inducing ligand,TRAIL)及其受体——死亡受体4(death receptor 4,DR4)、诱骗受体1(decoy receptor 1,DcR1)在PCOS大鼠卵泡发育中的作用。方法:采用硫酸普拉睾酮钠诱导大鼠PCOS模型,免疫组织化学染色、RT-PCR分析观察TRAIL及其受体DR4、DcR1在PCOS大鼠卵巢颗粒细胞的表达情况。结果:①免疫组织化学结果显示,TRAIL蛋白在PCOS组大鼠卵巢窦状卵泡颗粒细胞的表达较对照组明显增强(P<0.05),在窦前卵泡颗粒细胞的表达两组无显著性差异(P>0.05)。DR4蛋白在两组大鼠窦前卵泡和窦状卵泡的表达无显著性差异(P>0.05)。DcR1蛋白在PCOS组大鼠卵巢窦状卵泡颗粒细胞的表达较对照组明显减弱(P<0.01),在窦前卵泡颗粒细胞的表达两组无显著性差异(P>0.05)。②RT-PCR分析显示,PCOS组大鼠卵巢颗粒细胞TRAIL mRNA的表达较对照组明显增强(P>0.01),DR4mRNA的表达两组无显著性差异(P>0.05),DcR1 mRNA的表达较对照组明显减弱(P<0.01)。结论:TRAIL及其受体DR4、DcR1在PCOS大鼠卵泡发育颗粒细胞凋亡中发挥了调控作用。TRAIL及其受体DR4、DcR1在PCOS颗粒细胞的表达异常可能是导致PCOS卵泡发育障碍的机制之一。     

卵巢黄素化颗粒细胞瘦素信号转导分子STAT3磷酸化在多囊卵巢综合征发病中的作用

目的:探讨瘦素在多囊卵巢综合征(PCOS)发病中的作用。方法:选择行IVF-ET治疗的30例PCOS患者根据体重指数(BMI)分为肥胖者(BMI≥24)15例和非肥胖者(BMI<24)15例,及同期排卵功能正常或单纯输卵管因素不孕的非PCOS肥胖者和非肥胖者各15例,采用ELISA检测各组血清瘦素水平;采用放射免疫法检测各组空腹血清胰岛素(FIN)水平;采用葡萄糖氧化酶法测定各组空腹血糖(FPG)水平,利用稳态模型(HOMA)计算胰岛素抵抗指数(即HOMA-IR);采用Western blotting检测卵巢黄素化颗粒细胞信号转导分子STAT3磷酸化水平;同法检测不同浓度的瘦素(0ng/ml、10ng/ml、100ng/ml、1000ng/ml)体外对正常人卵巢黄素化颗粒细胞STAT3磷酸化(p-STAT3)的影响。结果:①血清瘦素水平PCOS肥胖者最高,其后依次为对照组肥胖者、PCOS非肥胖者和对照组非肥胖者,各组之间两两比较,差异均具有显著性(P<0.05);②PCOS患者血清瘦素水平与BMI和HOMA-IR均呈显著正相关(r=0.707,P<0.01;r=0.761,P<0.01);③STAT3水平肥胖PCOS组最高,其后依次为肥胖对照组、非肥胖PCOS组和正常对照组,各组间两两比较,差异均具有显著性(P<0.05);④正常人卵巢黄素化颗粒细胞经不同浓度瘦素处理48h后,p-STAT3水平呈不同程度增加,至瘦素浓度达到100ng/ml时,p-STAT3水平达到高峰,随后呈下降趋势。结论:瘦素可能通过激活JAK2/STAT3信号传导通路参与PCOS排卵障碍的发生。     

CHOP/GADD153在多囊卵巢综合征患者卵巢黄体颗粒细胞上的表达

目的:探讨内质网应激相关因子CHOP/GADD153在多囊卵巢综合征(PCOS)患者卵巢黄体颗粒细胞上的表达。方法:收集因PCOS不孕、行IVF-ET取卵时的黄体颗粒细胞(PCOS组,n=45)及因男方因素不孕、行IVF/ICSI-ET取卵时的黄体颗粒细胞(对照组,n=45),应用RT-PCR和Western blotting方法检测患者卵巢黄体颗粒细胞CHOP/GADD153的表达。结果:PCOS组CHOP/GADD153 mRNA水平(0.45±0.20)及蛋白水平(0.62±0.26)均较对照组(分别为0.40±0.18、0.56±0.17)有增高趋势,但差异均无统计学意义(P>0.05)。结论:内质网应激标志蛋白CHOP/GADD153在PCOS患者卵巢黄体颗粒细胞上的表达无明显改变,推测PCOS患者卵巢黄体颗粒细胞内质网应激尚处于保护性阶段。     

卵泡刺激素对多囊卵巢综合征患者卵巢颗粒细胞分泌抗苗勒管激素的影响

目的 研究卵泡刺激素(FSH)对多囊卵巢综合征(PCOS)患者卵巢颗粒细胞分泌抗苗勒管激素(AMH)的影响.方法 选择2008年8月至2009年12月于中山大学附属第六医院生殖中心就诊的33例PCOS患者,从其直径为8～10 mm的窦卵泡中分离颗粒细胞,并分为以下3组进行培养:未经FSH刺激的颗粒细胞(未刺激组,n=12);外源性FSH刺激的颗粒细胞(体外刺激组,n=12),此组颗粒细胞来源同未刺激组;对患者行FSH促排卵治疗,采集FSH体内刺激的颗粒细胞(体内刺激组,n=21).分别采用ELISA法和实时荧光定量PCR技术检测培养液中颗粒细胞分泌的AMH水平,及AHM mRNA表达水平;构建AMH荧光报告载体,检测卵巢颗粒细胞中AMH启动子的活性.结果 培养液中颗粒细胞分泌的AMH水平,未刺激组为(11.4±4.0)μg/L,体外刺激组为(7.9±1.1)μg/L,体内刺激组为(5.6±1.7)μg/L,体内、体外刺激组分别与未刺激组比较,差异均有统计学意义(P均＜0.05).AMH mRNA表达水平未刺激组为2.5±1.2,体外刺激组为1.5±0.5,体内刺激组为1.1±0.7,未刺激组高于体内、外刺激组,分别比较,差异均有统计学意义(P均＜0.05).未刺激组颗粒细胞中AMH启动子的活性为11.5±2.3,体外刺激组为8.7±2.4,体内刺激组为6.8±2.4,未刺激组高于体内、外刺激组,分别比较,差异均有统计学意义(P均＜0.05).结论 FSH可能通过抑制PCOS患者卵巢颗粒细胞中AMH的启动子活性及其mRNA表达,抑制AHM的过度分泌,促进卵泡的生长发育.     

Local effect of bisphenol A on the estradiol synthesis of ovarian granulosa cells from PCOS

Close relationship between polycystic ovary syndrome (PCOS) and bisphenol A (BPA) has drawn much attention in recent years, while the underlying mechanisms are poorly understood. In our study, we aim to detect BPA concentration in the follicular fluid and investigate its effect on estradiol synthesis in human granulosa cells from PCOS and non-PCOS patients. Follicular fluid and granulosa cells were collected from women who underwent controlled ovarian stimulation for in vitro fertilization or intracytoplasmic sperm injection. BPA concentration in the follicular fluid from PCOS patients (440.50?±?63.70?pg/ml) was significantly higher than that from non-PCOS patients (338.00?±?57.88?pg/ml). Expression of aromatase and estradiol synthesis in cultured granulosa cells was examined after treatment with BPA from 0.01 to 1?μM for 24?h. Expression of aromatase and estradiol synthesis was downregulated by BPA in a dose-dependent manner in PCOS, but no effect was observed in granulosa cells from non-PCOS patients. These findings provide evidence that increased BPA concentration in the follicular fluid of PCOS patients may play an important role in its pathogenesis by attenuating the expression of aromatase in granulosa cells.     

The role of androgen in autophagy of granulosa cells from PCOS

Hyperandrogenism is one of the most common causes for anovulation in women and increases the risk for metabolic disorder in PCOS patients. Autophagy plays an important role in dysfunction of endocrine and anovulation. However, the relationship between hyperandrogenism and autophagy in human granulosa cells of PCOS patients remains unclear. By collecting granulosa cells from PCOS patients and non-PCOS patients, we found that the abundance of autophagy-related genes ATG5, ATG7, BECN1 mRNA and the ratio of autophagy marker protein light chain 3B II/I (LC3 II/I) were significantly increased whereas the abundance of the autophagy substrate SQSTM1/p62 was decreased in ovarian granulosa cells from PCOS patients. Furthermore, we demonstrated that BECN1 mRNA abundance in human granulosa cells positively correlated with the basal level of serum total testosterone and androgen up-regulated the abundance of BECN1 mRNA and the ratio of LC3II/LC3I in a dose-dependent manner in cultured granulosa cells. These observations indicated that androgen-induced activation of autophagy may play an important role in the development of PCOS and to explore the autophagy mechanisms involved in PCOS yield new insight into the pathophysiology and therapy of the disorder.     

Concentration of vascular endothelial growth factor released by cultured human luteinized granulosa cells is higher in women with polycystic ovaries than in women with normal ovaries

To test the hypothesis that increased serum levels of vascular endothelial growth factor (VEGF) in women with polycystic ovaries or the polycystic ovary syndrome (PCOS) result from excess release by ovarian granulosa cells.Prospective study.Academic research setting.Twenty women undergoing IVF treatment, of whom 10 had normal ovaries and 10 had polycystic ovaries.Human granulosa lutein cells were isolated from follicular fluid obtained on the day of oocyte retrieval. Release of VEGF was assessed after co-incubation of granulosa lutein cells with gonadotropins and insulin. Serum and follicular fluid concentrations of VEGF were measured.Release of VEGF from granulosa lutein cells and serum levels of VEGF.Incubation with human hCG, and luteinizing hormone increased release of VEGF into the culture medium. Insulin alone did not increase release of VEGF, but addition of insulin increased hCG-stimulated release of VEGF. Serum and follicular fluid VEGF concentrations and the amount VEGF released from granulosa lutein cells obtained from women with polycystic ovaries or PCOS and those who developed the ovarian hyperstimulation syndrome were greater than those from granulosa lutein cells obtained from women with normal ovaries and those who did not develop the ovarian hyperstimulation syndrome.The amount of VEGF released by granulosa lutein cells is gonadotropin dependent and is augmented by insulin. The increased circulating concentrations of VEGF in women with PCOS may not only be due to an increased number of actively secreting granulosa lutein cells but also due to increased secretory capacity of each granulosa cell.     

Increased risk of polycystic ovary syndrome (PCOS) associated with CC genotype of miR-146a gene variation

Polycystic ovary syndrome (PCOS) is an endocrinopathy in reproductive-age women believed to be affected by several genetics and environmental factors or both. Different miRNAs are one of such genetic factors that their associations with PCOS have been implicated. For instance, miR-146a that is well known for strongly regulating the immune response and inflammation was upregulated in serum plasma, follicular fluid and granulosa cells of PCOS patients. Different studies have shown that genetic changes in pre-miRNA can cause change in the expression or biological function of mature miRNA. Therefore, the main aim of this study was to investigate the association of miR-146a gene variation (rs2910164) with the susceptibility to PCOS. This study consists of 180 patients with PCOS and 192 healthy women matched by age and geographical region. Genotyping were determined by using PCR-RFLP in all subjects. The genotype frequency and allele distributions of all subjects were evaluated using Fisher’s exact test directed by SPSS v.20. The genotype and allele frequencies of the miR-146a polymorphism (rs2910164) significantly differ between PCOS and healthy controls. The frequencies of CC genotype (p?=?.054) and ‘C’ allele (p?=?.0001) of the miR-146a variant indicated a significant incidence in cases compared to controls. Such association was obtained in co-dominant (OR?=?3.16) and dominant (OR?=?2.29) models. Result of this study can be proposed that women with miR-146a variation are at a higher risk for developing PCOS, which can be due to up-regulation of miR-146a.     

Analysis of gonadotropin pulsatility in hirsute women with normal menstrual cycles and in women with polycystic ovary syndrome

OBJECTIVE: To study the relation between plasma gonadotropin pulsatility, androgen levels, and estrogen levels in patients with polycystic ovary syndrome (PCOS), in hirsute women with normal menstrual cycles, and in healthy women. DESIGN: Prospective study. SETTING: University medical center-based cellular and molecular endocrinology laboratory. PATIENT(S): Eight healthy women (group 1), 9 hirsute women with normal menstrual cycles (group 2), and 19 women with PCOS (group 3). INTERVENTION(S): Plasma concentrations of LH and FSH were measured by RIA every 15 minutes for 12 hours. Main Outcome Measure(s): Rhythmic parameters of 12-hour LH and FSH secretion. RESULT(S): Rhythmic parameters of 12-hour LH secretion were significantly higher in patients with PCOS (group 3) than in controls (group 1) or in hirsute women with normal menstrual cycles (group 2). The frequency of LH pulses was statistically higher in patients with PCOS (group 3) than in controls (group 1). Statistically significant correlations were found when the frequency of LH pulses was plotted against basal LH concentrations and rhythmic parameters of 12-hour LH secretion. CONCLUSION(S): Luteinizing hormone pulse amplitude was higher in patients with PCOS than in hirsute women with normal menstrual cycles or in healthy women. The LH pulse frequency was increased only in patients with PCOS compared with healthy women and not in hirsute women with normal menstrual cycles.     

Granulosa cell aromatase enzyme activity: effects of follicular fluid from patients with polycystic ovary syndrome, using aromatase conversion and [11C]vorozole-binding assays.

The local regulation of ovarian aromatase enzyme in polycystic ovary syndrome (PCOS) was studied with aromatase conversion and [11C]vorozole-binding assays to analyze aromatase activity, substrate-enzyme affinity and number of aromatase binding sites in non-cultured human granulosa cells (GC) incubated with different sources and preparations of follicular fluid (FF). Incubation with FF from women stimulated in in vitro fertilization cycles with follicle-stimulating hormone yielded higher conversion activity than with FF from healthy women and PCOS patients, paralleled with higher substrate affinity (lower Kd) than with FF from healthy women. In PCOS women, charcoal-pretreated FF yielded higher conversion, whereas the ether-pretreated FF yielded lower conversion activity, than with untreated PCOS FF. Both preparations of FF yielded higher affinity to substrate (lower Kd values) and the ether-pretreated FF a lower number of binding sites (Bmax). It seems that steroids with the presence of proteins in PCOS FF reduced aromatase conversion activity through decreased substrate affinity, whereas FF preparations devoid of proteins reduced the aromatase conversion activity mainly through blocking of aromatase active sites. Identification of specific agents responsible for this rapid regulation of aromatase function might help to understand normal regulation of the menstrual cycle and supposed imbalances of inhibitors/activators in PCOS.     

Hyperandrogen enhances apoptosis of human ovarian granulosa cells via up-regulation and demethylation of PDCD4

Abstract

Apoptosis of granulosa cells (GCs) induced by hyperandrogen plays a key role in the pathogenesis of polycystic ovary syndrome (PCOS). However, the mechanism of androgen-induced apoptosis of GCs has not been clarified to date. Recent studies have reported that PDCD4 expression is higher in PCOS patients and might be a key factor in PCOS progression. In this study, we aimed to investigate the role of PDCD4 in regulating apoptosis of human GCs and whether hyperandrogen regulate PDCD4 expression through DNA methylation. Overexpression of PDCD4 in human ovarian granulosa cell line KGN cells promoted cells apoptosis. Meanwhile, expression of caspase-3 and caspase-9 were significantly elevated. High concentration of testosterone treatment resulted in up-regulation of PDCD4 and a significant increase of apoptosis in KGN cells. In addition, knockdown of PDCD4 in KGN cells treated with high concentration of testosterone abolished the hyperandrogen-induced apoptosis. Furthermore, high concentration of testosterone down-regulated DNMT1, DNMT3A and DNMT3B expression and the methylation level in the promoter region of PDCD4 was decreased. In conclusion, PDCD4 can promote apoptosis of human ovarian GCs. The mechanism of hyperandrogen-induced apoptosis may be mediated by PDCD4. Furthermore, the up-regulation of PDCD4 induced by hyperandrogen may through demethylation of its promoter regions.