Abnormality Of Telomere Length In Polycystic Ovary Syndrome(PCOS)and Their Pathophysiological Role In The Development Of PCOS

Background and ObjectivePolycystic ovary syndrome is the most common reproductive endocrinopathy among women at their reproductive age.The newly reported prevalence of polycystic ovary syndrome among Chinese women is high up to 5.6%.With the progress of modern medical science,people gradually realized that polycystic ovary syndrome does not merely have tremendous impact on reproductive endocrinology of women of reproductive age,more importantly,polycystic ovary syndrome is also implicated in metabolism,cardiovascular system,skin,skeleton and,et al.Polycystic ovary syndrome is a nightmare for any woman who suffers from it.However,the ideology of polycystic ovary syndrome is still poorly established,which greatly hindered the development of diagnosis and treatment of polycystic ovary syndrome.As a complex disease,the genetic etiology of polycystic ovary syndrome plays an essential role in the onset and development of polycystic ovary syndrome.Among all the genetic etiological factors,the dysfunctions of the telomere/telomerase system are potentially importance,yet long been overlooked.Previous studies indicate that the telomere/telomerase system has profound impact on the regulation of reproductive functions and the system is deeply implicated in every epact of reproduction,including folliculogenesis,synthesis of steroid hormones,et al.As a result,researcher propose that the dysfunctions of the telomere/telomerase system may participate in the development of polycystic ovary syndrome.Unfortunately,the results of previous studies exploring this issue failed to provide a convincing and consistent conclusion.Given the unique property of the telomere length,which is tissue and cell-type specific,we collected two types of different cells from women with polycystic ovary syndrome.And using these two kinds of cell types,we systematically explored the difference of telomere length between controls and polycystic ovary syndromes subjects.Based on identifying the difference in telomere length between controls and PCOS patients,we are going further investigate the underlying melocular mechanism.And,at last,the discovered epigenetic mechanisms will validaded in patients with PCOS.In order to initiate a pilot exploration of this novel genetic and epigenetic mechanisms of polycystic ovary syndrome,the following three parts of experiments were designed:1.To explore the abnormalities of telomere length in leucocytes and ovarian granulosa cells in subjects with polycystic ovary syndrome;2.Using mouse in vitro model to explore the underlying mechanisms of the lengthened telomere length in polycystic ovary syndrome;3,to test the existence of abnormalities of epigenetic regulation of telomerase in patients with polycystic ovary syndrome and provide supporting evidence for this hypothesis.Part 1:Comparison of telomere length of leucocytes and ovarian granulosa cells in controls and patients with PCOSMethods:1.To collect leucocytes and luteinized ovarian granulosa cells frorm controls and patients with PCOS2.To compare telomere length of leucocytes and ovarian granulosa cells in controls and patients with PCOS using the method of quantitative PCR3.To explore the correlation between anthropometric measurements and hormone levels.Results:1.No significant difference was found in the leucocyte telomere length between controls and PCOS patients(0.99 ± 0.44 vs.1.00 ± 0.38,p = 0.93).After adjustments for age and body mass index,the p value remained non-significant(p>0.05).2.When comparing telomere length in granulosa cells between controls and PCOS subjects,significantly lengthened telomere length was found in PCOS subjects(1.00 ± 0.37 vs.1.57 ±0.67,p<0.0001).After adjustments for age and body mass index,the p value remained significant(p<0.0001).Statistical analysis also showed the odd ratio is 2.29 for the risk of PCOS.3.We identified that telomere length in granulosa cells is positively correlated with testosterone levels in the serum.Part 2:Exploration of the effects of testosterone on activity of telomerase and methylation level of the promoter region of telomerase in mouse granulosa cellsMethods:1.To establish in-vitro model of mouse granulosa cell culture.2.To explore the effects of testosterone on the transcriptional activity of telomerase in mouse granulosa cells using quantitative PCR.3.To explore the effects of testosterone on methylation level of the promoter region of telomerase in mouse granulosa cells using bisulfite sequencing.Results:1.In-vitro model of mouse granulosa cell culture,we found that testosterone can stimulate transcriptional activity of telomerase in mouse granulosa cells and testosterone can elevate mRNA level of telomerase in mouse granulosa cells.mRNA level of telomerase of mouse granulosa cells treated by high concentration of testosterone(10-7 M testosterone and 10-5 M testosterone)was significantly higher than that of the control group or the group treated with physiological testosterone level.2.In-vitro model of mouse granulosa cell culture,by doing bisulfite sequencing,we found that a certain degree of methylation in the promoter region of the promoter of telomerase in granulosa cells under physiological level of testosterone.Treatment by high concentration of testosterone(10-7 M testosterone and 10-5 M testosterone)can demethylate the promoter region of the promoter of telomerase in granulosa cells,which reached a statistic significant level(P<0.01).Part 3:Comparison of methylation level of the promotor region of telomerase in controls and patients with PCOSMethods:1.To collect leucocytes and luteinized ovarian granulosa cells from controls and patients with PCOS.2.To measure the methylation level of the promoter region of telomerase in ovarian granulosa cells from patients with PCOS using quantitative PCR.Results:1.We sequenced methylation level in 160 clones from the PCOS patients and 160 clones from non-PCOS subjects.After one way ANOVA analysis,we found that in the group of PCOS,the overall methylation level of the promoter region of telomerase was decreased(P=0.004).2.Analysis of independent CpG methylation level revealed that the#1,#2,#7,#9,#17,#18,#19,and#20 CpG sites of the promoter region of telomerase showed signiantly lower level of methylation comparing to controls.Conclusions:1.We have identified a possible pathophysiological feature of PCOS significantly increased telomere length in ovarian granulosa cells.The lengthening of telomere length is positively correlated with serum testosterone level.2.In mouse in-intro models,we evidenced the relationship between hyperandrogenemia and the dysfunction of the telomere/telomerase system.We found that high testosterone level can induce increase of telomerase activity,which is mediated by demethylation of the promoter region of telomerase gene.3.Finally,using bisulfite sequencing,we found that the promoter region of telomerase gene is hypomethalated in patients with PCOS,which reinforced the epigenetic mechanism underlying the lengthened telomere length in PCOS.