Study On The Colonization Of Female Lower Genital Microbiota And Its Relationship With The Pregnancy Outcome Of Pcos Patients

Background & Purpose:In recent years,with the application of 16 Sr RNA sequencing,macro genome sequencing and other technical methods,the role of human microorganisms has been gradually studied and discovered,intestinal microorganisms,oral microorganisms and respiratory microorganisms have been reported to be related to the occurrence and development of a variety of diseases: such as tumors,diabetes,hypertension,obesity,asthma and so on.With the research of microbes implanted in various parts of the human body more and more deeply,reproductive tract microorganisms have also entered people’s field of vision: at present,more research focuses on vaginal microorganisms and their association with gynecological diseases,pregnancy outcomes,etc.As for the composition of reproductive tract microorganisms at different stages of the menstrual cycle in women of childbearing age in China,the study of microorganisms in the cervical tube is still lacking.Polycystic Ovary Syndrome(Polycystic Ovary Syndrome,PCOS)is the most common endocrine disorder in women of childbearing age and is one of the most common factors in female infertility.PCOS is characterized by hyperpositive hormoneemia,abnormal egg development,and the reproductive aspects of patients are often characterized by high abortion rate,premature rupture of membranes and preterm birth,increased incidence of embryo suspension and so on.At present,it is believed that the occurrence of these adverse reproductive phenotypes is caused by the factors such as the immature eggs,poor egg quality,and the sexual hormone disorder caused by the abnormal ovarian function(the antagonistic effect of hyperandrogen and continuous estrogen stimulation lack of progesterone),obesity,abnormal blood glucose level and so on.However,the relationship between these adverse reproductive phenotypes and the reproductive tract microenvironment of PCOS patients has not been explored.The purpose of this topic is to explore the composition of microorganisms in the lower reproductive tract in Chinese women and their relationship with pregnancy outcomes in patients with polycystic ovary syndrome.It is proposed to describe the composition of Chinese female vaginal and cervical tube microorganisms at different stages of the menstrual cycle during normal childbearing age,and to observe whether the hormonal changes in the menstrual cycle in the physiological state will affect the microbial composition of these positions.On this basis,we will further explore the composition of vaginal and cervical tube microorganisms in patients with polycystic ovary syndrome,influencing factors and their correlation with pregnancy outcomes,and from the samples of the same body,we will explore the functional effects of the changes in the reproductive tract microbes on local cells in the cervix of PCOS patients.Methods:1.Vaginal and cervical tube secretion specimens were collected at reproductive clinic from healthy reproductive-aged female(reproductive clinic patients due to male factors)during natural menstrual cycle(excluding drug interference)of the early follicle period(within 5 days of menstrual clean),large follicle period(before ovulation)and implantation period,following by laboratory extraction of microbial DNA,16 Sr RNA sequencing.Then the sequencing data were analyzed to generate OUT table,species diversity,community diversity,abundance difference analysis,etc.,the composition of the reproductive tract microbes(from vaginal and cervical tubes)in healthy reproductive age women are explored in three different stages of the menstrual cycle.2.Vaginal and cervical tube specimens of healthy reproductive-aged women(early follicle period)and PCOS patients(avoiding menstruation)were collected,the diagnosis of PCOS refers to the international Rotterdam standard.After extracting microbial DNA,laboratory procedures of16 Sr RNA sequencing were conducted,data were calculated and analyzed to generate OUT table,species diversity,community diversity,abundance difference analysis,etc.To explore the similarities and differences between vaginal and cervical tube microorganisms and characterize the composition of microbes in the lower reproductive tract of PCOS patients.3.Vaginal and cervical tube swabs were collected from PCOS patients who performed IVF-FET,followed by DNA extraction and 16 S r RNA sequencing.At the same time,the patient’s pregnancy outcome were tracked and recorded,clinical information such as age,height,weight,menstrual cycle,previous reproductive history,the level of testosterone were also recorded.4.Correlation analysis between the lower genital tract microbial diversity,relative abundance of Lactobacillus with clinical indexes such as age,BMI,menstrual cycle,testosterone level were conducted.5.The secretion of vagina and cervical canal of healthy control women and PCOS patients were collected in outpatient department,and the microbial DNA was extracted,the analysis method was the same as the above;at the same time,the cervical exfoliated cells of those patients were also collected.After brief separation in laboratory,Trizol was added for cryopreservation,and RNA was extracted and sequenced.To analyze the effect of lower genital tract microbiome alternation on local cellular function in patients with PCOS.Results:1.As for the natural menstrual cycle of healthy women of reproductive age:early follicular period,pre ovulation period and implantation window period,we found that the composition of microorganisms in the lower genital tract,whether in the vagina or in the cervical canal,were all dominated by Lactobacillus,and there was no significant difference in the three stages of menstrual cycle,the microbial composition was relatively stable.2.The composition of microbes in vagina and cervical canal of reproductive aged women is highly similar,and there is no significant difference in species diversity.3.The composition of bacteria in the lower genital tract of patients with PCOS is different from that of control women,which shows that the beta diversity of bacteria is significantly increased and the relative abundance of lactobacilli is significantly reduced.About 30% of patients with PCOS are no longer dominated by lactobacilli,and some patients even lack the colonization of lactobacilli,instead was abnormal enrichment of some anaerobic bacteria,such as Gardnerella,Prevotella,etc.4.The colonization of microorganisms in the lower genital tract of PCOS patients was significantly related to their embryo implantation rate and clinical pregnancy outcome: the embryo implantation rate and clinical pregnancy rate of PCOS patients with lactobacilli content lower than 95%were lower than those of PCOS patients with lactobacilli content higher than 95%,and the embryo implantation rate between the two groups was statistically different.The microbial diversity of lower genital tract in pregnant group was lower than that in non-pregnant group.5.Alternation of microbial composition in the lower genital tract of PCOS patients were related to their menstrual cycle,BMI and testosterone levels,and the diversity of the lower genital tract microorganisms in obese patients(BMI ≥ 24)was significantly higher than that in non-obese patients.6.Alternation of microorganism in the lower genital tract of PCOS patients affect the expression of related genes in cervical cells.The main influencing cytological functions include intercellular adhesion,biological adhesion,cell adhesion,immune system response,cell conduction,stress and other processes.Conclusion:1.Lactobacilli are the dominant flora in the lower genital tract(vagina and cervical canal)of women of normal childbearing age,which are not affected by the normal menstrual cycle,and the composition is relatively stable;the composition of microorganisms in vagina and cervical canal is similar.2.The microbial composition of the lower genital tract of PCOS patients was different from that of women of normal childbearing age,the species diversity increased significantly,the colonization of lactobacilli decreased significantly,the bacteria in vagina and cervix tube of some patients were no longer dominated by lactobacilli,while the colonization of anaerobic bacteria such as Gardnerella and Prevotella increased.3.There is a tightly correlation between the lower reproductive tract microorganism and the pregnancy outcome in PCOS patients.When the colonization of lactobacilli in the lower reproductive tract microorganism of PCOS patients is reduced and the diversity is increased,the embryo implantation rate and clinical pregnancy rate will be reduced.We think that the lower genital tract microorganism has an important influence on the reproductive outcome of PCOS patients.4.The composition of microorganisms in the lower genital tract of PCOS patients was related to their BMI,menstrual cycle and testosterone level,especially the BMI level.The alpha diversity of microorganisms in the lower genital tract of PCOS patients in obesity group(BMI ≥ 24)was significantly higher than that in normal group(BMI < 24).5.It was found that gene expression in cervical exfoliated cells of PCOS patients with altered lower genital tract microbiome had significant changes,and the main biological functions affected were biological adhesion,immune system response,signal transduction,etc.,suggesting that the changes of lower genital tract microbiome had an impact on the function of female genital tract epithelial cells.