| Objective:Polycystic ovary syndrome(PCOS)is the most common endocrine disorder disease in women of reproductive age,with high incidence,heterogeneity and difficulty in treatment.Excess androgen is an important clinical feature of PCOS and a key pathogenic factor of PCOS.Studies have found that the androgen levels of PCOS women in pregnancy and nonpregnancy both were significantly increased.Animal experiments have confirmed that androgen intervention in pre-youth or pregnancy can establish PCOS animal models of estrous cycle disorder,increased serum androgen level and changes in ovarian sac shape.These results suggest that prenatal hyperandrogen environment and non-prenatal hyperandrogenemia may be involved in the development and development of PCOS.Based on this,this paper focuses on the following three aspects:(1)To study the effect of intrauterine hyperandrogenism environment on the reproduction,metabolism and gut dysbiosis of the offspring;(2)Based on epigenetics,to explore the effects of intrauterine hyperandrogenism environment on the DNA methylation and transcriptome of placenta,and to study the possible mechanism of fetal reprogramming at the level of DNA methylation caused by prenatal hyperandrogen environment;(3)To study the effect of Yangjing Zhongyu Decoction and Shoutai Pill on reproduction and metabolism of PCOS mice.Methods:1.The effect of intrauterine hyperandrogenism environment on the reproduction,metabolism and gut dysbiosis of the offspringEight-week-old SPF female C57BL/6J mice were accommodated for 1 week and randomly divided into prenatal androgen exposure group(PNA group,n=10)and Control group(Control group,n=10).PNA group mice were mating with male mice in proestrus or estrus period,and vaginal plug or vaginal smear was found at 9:00 in the morning of the next day was determined to be 0.5 days of pregnancy(E0.5).50μL sesame oil solution containing 250μg dihydrotestosterone(DHT)was injected subcutaneously into the neck during E16.5E18,5.Mice in Control group were mating with male mice in proestrus or estrus period,and vaginal plug or vaginal smear was found at 9:00 in the morning of the next day was determined to be 0.5 days of pregnancy(E0.5).50μL sesame oil solution was injected subcutaneously into the neck during E16.5-E18.5.Offspring mice were separated from their mothers at 21 days of age.Test indexes:(1)the distance between vagina and anus of 21-dayold mice was measured;The mice were weighed once every 10 days.(2)At the age of 90 days,mice were given 2g/kg glucose by intragastric for glucose tolerance test;Continuous vaginal smears were performed for 10 days to determine whether the estrous cycle was regular.Female mice and corresponding male mice were caged together for 4 days,pregnancy rate was calculated and fertility was evaluated.(3)At the age of 180 days,the rats were anesthetized under isoflurane,and their heart blood was taken for centrifugation.The supernatant was used for the determination of serum testosterone(T),luteinizing hormone(LH)and Insulin.One ovary was fixed with formalin overnight,then the morphology of the ovary was observed by embedding,sectioning and HE staining.The other ovary was used to measure related gene expression.Cecal feces were collected to detect intestinal microbiota.2.The effect of intrauterine hyperandrogenism environment on the DNA methylation and transcriptome of placentaEight-week-old SPF female C57BL/6J mice were accommodated for 1 week and randomly divided into prenatal androgen exposure group(PNA group,n=10)and Control group(Control group,n=10).PNA group mice were mating with male mice in proestrus or estrus period,and vaginal plug or vaginal smear was found at 9:00 in the morning of the next day was determined to be 0.5 days of pregnancy(E0.5).50μL sesame oil solution containing 250μg dihydrotestosterone(DHT)was injected subcutaneously into the neck during E16.5E18.5.Mice in Control group were mating with male mice in proestrus or estrus period,and vaginal plug or vaginal smear was found at 9:00 in the morning of the next day was determined to be 0.5 days of pregnancy(E0.5).50μL sesame oil solution was injectedsubcutaneously into the neck during E16.5-E18.5.(1)The placenta was collected under isoflurane anesthesia,fixed with formalin,observed the placenta morphology by HE staining,and(2)detected the expression-of Ki67 and aSMA by immunohistochemistry.(3)The fetal tail was cut for sex identification,and the placenta corresponding to the female fetus was sequenced by MeDIP sequencing and transcriptomic sequencing(n=3/group)to analyze the diffrentially expressed DNA methylation and genes,and gene enrichment(GO)and KEGG analysis.(4)Real-time fluorescence quantitative PCR and pyrosequencing were used to detect F2 gene expression and DNA methylation levels.(5)Protein interaction network analysis of methylated differential genes.3.To preliminarily investigate the effect of Yangjing Zhongyu Decoction and Shoutai Pill on the reproduction and metabolism of PCOS micePCOS mouse model was established by androgen during pregnancy,and the mice were treated with Yangjing zhongyu Decoction and Shoutai pill for 28 days.The effects of Yangjing zhongyu Decoction and shoutai Pill on the reproduction and metabolism of PCOS mice were evaluated,including body weight,estrous cycle,glucose tolerance test,serum hormone,ovarian morphology and the expression of ovarian aratase 19(CYP19),3βhydroxysteroid dehydrogenase(3βHSD)and inflammatory factors.Results:1.The effect of intrauterine hyperandrogenism environment on the reproduction,metabolism and gut dysbiosis of the offspring.(1)The body weight of mice in PNA group was significantly lower than that in Control group.(2)The distance between vagina and anus in PNA group was significantly increased compared with that in Control group.(3)Estrous cycle of mice in the Control group was regular,and estrous cycle of mice in the PNA group was disorder,mainly in the interestrous period,and the estrous period decreased.(4)The fasting blood glucose of mice in PNA group was significantly higher than that in Control group,and there were no significant differences between PNA group and Control group in 15 min,30 min,60 min and 90 min blood glucose,and no significant differences between PNA group and Control group in BLOOD glucose AUC.(5)The pregnancy rate of mice in Control group was 66.7%,and that of mice in PNA group was 16.7%.The fertility of mice in PNA group was significantly lower than that in Control group.(6)Compared with Control group,serum T level in PNA group was significantly increased,and the difference was statistically significant.There were no significant differences in serum LH and Insulin levels between the two groups.(7)The ovaries of mice in Control group showed primal follicles,primary follicles,secondary follicles,sinus follicles and luteum,while the ovaries of mice in PNA group showed reduced luteal and sinus follicles and increased atresia follicles.(8)Compared with the Control group,the species richness of intestinal microbiota in the PNA group was significantly reduced.Principal Coordinate analysis(PCoA)based on UniFrac unweighted distance showed significant differences in the clustering of fecal samples between the PNA group and the Control group,indicating significant differences in the microbial communities between the PNA group and the Control group.Spearman correlation analysis found that T level was positively correlated with the relative abundance of Desulfovibrio at the genus level.Insulin level was positively correlated with Prevotella.The levels of FBG and HOMA-IR were positively correlated with Oscillospira.2.The effect of intrauterine hyperandrogenism environment on the DNA methylation and transcriptome of placenta.(1)There were no significant differences in the weight of maternal,placental,fetal,uterine and number of fetal between the DHT group and the Control group;(2)Placental HE staining in DHT group showed that the environment of intrauterine hyperandrogenism environment led to the decrease of erythrocytes in placental blood vessels compared with Control group.(3)There was no significant difference in Ki67 and A-SMA expression between DHT group and Control group.(5)The expression pattern of placental DNA methylation in PNA group combined with transcriptome analysis revealed hypomethylation and high expression of thrombin factor(F2)gene.QPCR confirmed that the expression of F2 gene was up-regulated compared with Control group and DHT group.Proteome network interaction analysis(PPI)found that F2 and Kiss gene were correlated.3.To preliminarily investigate the effect of Yangjing Zhongyu Decoction and Shoutai Pill on the reproduction and metabolism of PCOS mice.(1)The body weight of mice in the PCOS+TCM group was significantly lower than that in the Control group.(2)Estrous cycle of mice in Control group was regular,and estrous cycle of mice in PCOS group was disorder,mainly in estrous interphase;The estrous period of mice in PCOS+TCM group was irregular.Compared with PCOS group,the proportion of estrous period of mice in PCOS+TCM group was increased.(3)The blood glucose AUC of mice in PCOS group was not significantly different from that in Control group,and the blood glucose AUC of mice in PCOS+TCM group was significantly lower than that in PCOS group;(4)There were no significant differences in serum T,LH and Insulin levels among all groups.(5)There were no significant diffeerences in the expression of CYP19,CYP17,3βHSD,TNFa,IL-1 and IL-6 genes among all groups.Conclusion:(1)Intrauterine hyperandrogenism environment resulted in weight loss,impaired glucose tolerance,disturbance of estrous cycle,disturbance of fecundity and intestinal flora of offspring mice.(2)Intrauterine hyperandrogenism environment affected placental DNA methylation expression pattern.Transcriptomic analysis revealed that hypomethylated and highly expressed F2 genes may affect GnRh secretion.(3)Yangjing Zhongyu Decoction and Shoutai Pill reduced the body weight and improved the abnormal glucose tolerance in mice model of PCOS. |
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