Study On The Characteristics Of Female Reproductive Tract Microbiota In Vaginal Dysbiosis And The Culture Of Vaginal Bacteria

Background:Due to the development of next-generation sequencing technology and the reduction of sequencing costs,people have a deeper understanding of the human microbiome.Human microbiome includes intestinal microbiome,vaginal microbiome,oral microbiome and airway microbiome.Among them,the intestinal microbiota has the most complex composition and highest diversity,and is closely related to various human diseases.Compared with the intestinal microbiota,the composition of the vaginal microbiota is relatively simple.Healthy women are colonized with plenty of Lactobacillus along with a few other anaerobic bacteria.Vaginal dysbiosis refers to the replacement of the single dominant Lactobacillus in healthy women by Gardnerella vaginalis,Prevotella,and Atopobacterium vaginalis,and the coexistence of multiple facultative and obligate anaerobic bacteria in the vagina.Existing studies have shown that vaginal dysbiosis were associated with preterm birth,HIV,HPV,cervical cancer,and other common sexually transmitted diseases.Vaginal dysbiosis is often clinically diagnosed as bacterial vaginosis.Some patients are accompanied with a series of female reproductive symptoms such as itching,pain,and burning.The specific relationship between these symptoms and vaginal microbiota is still unclear.In this study,we also compared different nucleic acid extraction methods,and tried to define a more efficient and simple experimental method to optimize sample preparation for vaginal microbiome research.Furthermore,obtaining more pure cultures of vaginal bacteria and determining their classification and function are also important aspects of human microbiome research.Objectives:1.To explore the effect of different nucleic acid extraction methods and optimize the experimental process in vaginal microbiome research;2.To explore the correlation between vaginal microbiota and different clinical symptoms in the vaginal dysbiosis;3.To preliminarily explore the vaginal bacterial culture conditions and the basic composition of culturable vaginal bacterial.Methods:At first we compared four methods for vaginal swabs nucleic acid extraction,including magnetic bead fragmentation method,ion column chromatography,magnetic bead ion column chromatography and direct boiling method.Comparative analysis was performed at PCR amplification efficiency and vaginal microbiota composition.At the same time,we recruited a batch of women with obvious vaginal symptoms,excluding other clear vaginal pathogen infection,clinically diagnosed with vaginitis.We made a questionnaire to collect the information on all reproductive tract symptoms,such as vaginal itching,vulvar burning pain and blood and pain during sex.We collected 2 vaginal swabs every women and performed Nugent Score evaluation,16S rRNA sequencing,wet mount microbiology and dysbiosis-associated enzymology indicators examination on the vaginal swabsResults:The results showed that compared with other kits,the boiling method could truly reflect the composition of the vaginal microbiota,but there was still a disadvantage of low nucleic acid yield,and the application to scientific research experiments needed to be improved.Sequencing results showed that symptoms in female reproductive system correlated with specific community state types of vaginal microbiota.We found that urodynia or lumbodynia had a higher positive rate in CST4 women than in CST3 women,and cervical hypertrophy had a higher positive rate in CST4 women than in CST3 and CST2 women,which refers to CST4 correlated with more severe symptoms.The clinicians should pay more attention to CST4 in subsequent treation and research.Later,we have initially explored and established a system of vaginal bacteria isolation and culture conditions,and 112 strains of vaginal bacteria were isolated and preserved,including potential vaginal probiotics Lactobacillus cripatus 9 strains,pathogenic Prevotella spp.2 strains and Gargnerella vaginalis 3 strains,and function undetermined Lactobacillus iners 3 strains.This part of the work provided an important basis for further research on microbial pathogenic mechanisms.Conclusions:Vaginal microbiota research based on sequencing technology provides a deeper understanding of female reproductive tract diseases.In the study,clinical symptoms in vaginal dysbiosis women were related to vaginal microbiota community state types.Women in CST4 dominated with miscellaneous bacteria neither Lactobacillus nor Gardnerella vaginalis were more clinically significant than those in CST3 dominated with Gardnerella vaginalis,suggesting that the clinician put more attention to the diagnosis,treatment and research of such patients.Furthermore,the successful isolation and cultivation of vaginal microorganisms is still an important basis for the next research on the pathogenic mechanism of vaginal microbiota and female reproductive tract diseases,which deserves further exploration.