Study On Circadian Rhythm Disorder Contributed To The Pathogenesis Of Polycystic Ovary Syndrome And Related Mechanism

BackgroundPolycystic ovary syndrome(PCOS)is the most common endocrine and metabolic disorder in reproductive-age women.As one of the important causes resulting in female infertility,the prevalence of PCOS worldwide is 10%~15% while domestic prevalence is 6.51%.The manifestation of PCOS includes oligomenorrhea,hirsute and acne or hyperandrogenism,polyovary morphological changes in ultrasonic examination.PCOS not only damages female fertility,but also increases the risk of metabolic disorders such as insulin resistantce,glucoss and lipid regulation and cardiovascular disease.It has been reported that genetic,environmental factors,lifestyle and intrauterine androgen excess could contribute to the occurrence of PCOS,yet its etiology remains unclear.Recently,the circadian rhythm has drawn a lot of attention in the field of woman reproduction.Circadian rhythm played a vital part in the regulation of menstrual cycle,ovulation,embryo implantation and pregnancy maintainance.Circadian rhythm disturbance could affect female reproductive function,leading to abnormal menstrual cycle,alterations in FSH(Follicle-Stimulating Hormone)and LH(Luteinizing Hormone),premature birth and fertility decline.In the modern lifestyle,people usually travel with transmeridian flight or need to work in a day-night shift pattern.The circadian system is frequently misaligned with the external physical environment,leading to poor health outcomes.Several researches showed that women exposed to night-light shifts had shorten menstrual cycles,frequently associated with dysmenorrhea and consistently associated with metabolic syndrome,insulin resistance and deregulation of glucose metabolism,all of which are recognized risk factors of PCOS.The circadian rhythm disorder is associated with hormone secretion,glucose and lipid metabolism,chronic inflammation and embryo development.However,the are limited studies about the mechanism of of circadian rhythm disturbance on PCOS associated pathologic changes such as hyperandrogenism.Whether melatonin and its receptor take part in this pathogenesis progress is still unknown.On the other hand,a lot of studies have found imbalanced gut microbiota in women with PCOS and its relationship with clinical paramters.It indicated the possible role of intestinal flora in the occurrence and progression of PCOS.This study aimed to clarify the relationship between circadian rhythm disturbance and occurance of PCOS,and investigate the role of melatonin receptor and gut microbiota in the pathologic changes of PCOS.Methods1.The construction of circadian rhythm disorder rat model and relationship with polycystic ovary syndrome:We chose six-week-old female Sprague Dawley rats to construct circadian disrupted animal model in our experiment.All of 90 rats were divided into three groups randomly with 30 rats in each group.The control group was under periodic cycle(12h/12 h light/dark cycle),the light group were exposed to continuous light(12h/12 h light/light cycle)while the dark group were exposed to continuous dark(12h/12 h light/light cycle)for consecutive ten weeks.The body weight gain was measured every week.The rats of each group were anaesthezed and sacrificed at zeitgeber time(ZT)0,ZT4,ZT8,ZT12,ZT16 and ZT20 with 15 rats in each zeitgeber time(n=5/group).The ovarian weigh,estrous cycle,serum sexual hormones and glucose tolerace were detected to identify whether there was relationship between circadian rhythm disorder and occurrence of PCOS.2.The mechanism investigation of continuous dark induced hyperandrogenism:We measured the concentration of melatonin and expression of melatonin receptors in ovary of control and dark group rats.Then we detected the expression of Cyp17a1 and Cyp19a1 and utilized KGN cell line to examine the effect of melatonin receptor 1a(MTNR1A)in androgenism investigation.Small interfering(si)RNA knocking down and transfection of vectors with Lipo3000 were used to change the expression of MTNR1 A and androgen receptor(AR),and further clarify the mechanism of androgenism pathogenesis.3.The effect of continuous light in PCOS-like manifestation development and gut microbiotaWe used six-week-old female Sprague Dawley rats to induce PCOS-like model with continuous light for successive 4 weeks in this part.All of 22 rats were divided into two groups,with 11 rats in each group.The control group was under periodic cycle(12h/12 h light/dark cycle,L/D group)while the light group were exposed to continuous light(12h/12 h light/light cycle,L/L group.The body weight gain was measured every week.The rats of each group were anaesthezed and sacrificed after treatment of 4 weeks.Ovarian weight,estrous cycle,serum sexual hormones and glucose tolerace were detected to identify PCOS model construction success.Before the beginning of experiment,5 rats were randomly chosen from both groups and their fecal samples were colleceted every week during the experimental progress(n=5/group).Gut microbiota was detected with 16 S r RNA sequencing technique to decipher its relationship with the occurrence of PCOS.Results1.Circadian rhythm disorder induced PCOS-like changes in SD rats.The body weight gain and ovarian weight were not different among control,light and dark group of rats.Compared with control group,the estrous cycle of light group was remarkably disordered while the cycle was prolonged in dark group.Rat of light group and dark group presented with polycystic ovary in morphology with thinner follicle wall and enlarged follicles.As for the endocrine hormonal manifestation,there were significant increase in LH concentration and LH/FSH ratio in light group and dark group.The serum testosterone was elevated in dark group compared with control ones.2.MTNR1 A and AR took part in the pathogenesis of hyperandrogenism in the treatment of continuous light deprivation(1)The serum melatonin level was unchanged in control and dark group,while the abundance of Mtnr1 a in m RNA level was significantly decreased at ZT16 in dark group.The protein expression of Mtnr1 a in granular cells in ovaries of dark group rats was lower than that of control group.(2)The expression of Cyp19a1 and Ar in mRNA level was reduced at ZT16 in dark group.(3)After knockdown of MTNR1 A,the protein abundance of AR and CYP19A1 decreased significantly.Treatment with physiological 10 nmol/L testosterone after MTNR1 A knockdown led to down regulation of AR and CYP19A1 in protein level as well.The estradiol concentration of KGN cell cultivated medium was reduced after knockdown of MTNR1 A.(4)After knockdown of AR,the mRNA abundance of CYP19A1 decreased.After treatment with 10 nmol/L testosterone in AR-knockdown KGN cells,the protein abundance of CYP19A1 was reduced as well.To figure out the role of AR in MTNR1A-regulated CYP19A1 expression,we knockdown MTNR1 A and overexpressed AR at the same time.The m RNA abundance of CYP19A1 remained unchanged but the protein abundance was significantly increased compared with ARoverexpression group.3.Altered gut microbiota could partly contribute to the occurrence of PCOS in the treatement of continuous light exposure(1)Light exposure for 4 weeks induced PCOS-like manifestations in reproductive hallmarks and endocrinal interruption.(2)The m RNA abundance of glucose and lipid metabolic gene expression altered between light group and control group.(3)The β-diversity of gut microbiota altered between two groups,and several microbial genus differed in relative abundacen,with Parasutterella enriched while Corynebacterium,Odoribacter and Acinetobacter decreased in light group.diversed gut microbial taxa was in relationship with PCOSrelated pathways such as steroid synthesis,Gn RH signaling and glycan metabolism.(4)As time went by,the α-diversity and β-diversity both differed among different time points in control and light group.The longer exposed to light,the abundance of Firmicutes grew higher while that of Bacteroidetes became lower.ConclusionsOur study found that rhythm disorder,one of the common environmental factors,would cause PCOS-like changes including estrous cycle disorder,polycystic ovary and endocrine hormone level alterations in rats.It confirmed the relationship between disrupted circadian rhythm and occurrence and development of PCOS.Continuous darkness also caused increased testosterone level,impaired fasting glucose tolerance and altered melatonin receptor expression in ovary,suggesting the possible role of MTNR1 A in the pathological progress of hyperandrogenism.In KGN cell line,downregulation of MTNR1 A would decrease the abundance of CYP19A1 and reduce the convertion ability from androgen to estrogen in granulosa cells,thus led to accumulation of androgens.Androgen receptor was involved in this progress.Down-regulation of MTNR1 A caused AR expression reduction,and overexpression of AR blocked the regulatory effect of MTNR1 A on CYP19A1.On the other hand,continuous light induced gut microbiota construction significantly diversed and several microbial taxa abundacen changed,which was possibly in close relationship with PCOS-related metabolic pathways.Our study found that melatonin receptor and gut flora may play vital parts in the pathogenesis of PCOS.It provides a new research perspect for the occurrence mechanism investigation of PCOS and a new thought for the clinical prevention and treatment of PCOS patients with biological rhythm disorder.