Based On The Second-generation High-throughput Sequencing Technology,the Correlation Between Vaginal Microecology And Premature Rupture Of Membranes Of Pregnant Women In The Middle And Late Trimester Of Pregnancy Was Investigated

Objective:Vaginal microecology is in a balanced state under normal circumstances,and generally does not cause disease.During pregnancy,due to the increase of estrogen and the decrease of immunity,vaginal microflora is disturbed,which leads to the occurrence of reproductive tract infection.Reproductive tract infection during pregnancy will cause a variety of inflammation,easy to cause premature rupture of membranes,preterm neonatal infection,and increase the adverse pregnancy outcomes of mother and child.With the rapid development of molecular biology techniques,the second generation of high-throughput sequencing technologies,more and more in clinical application.This topic is proposed to adopt the second generation of high-throughput sequencing technologies to clarify vaginal flora changes during pregnancy and its relationship with premature rupture of membranes,thereby to the abnormal pregnancy vaginal microecological early clinical recognition,reduce the incidence of premature rupture of membranes,premature delivery and other adverse pregnancy outcomes.Methods:A total of 50 pregnant women with PML who delivered at the Second Affiliated Hospital of Kunming Medical University on January 1,2020 and met the inclusion criteria at 16 to 41 weeks of gestation were collected as the experimental group,and another 30 normal pregnant women of the same gestational age were randomly collected as the control group.After signing the informed consent,the vaginal secretions of pregnant women were collected respectively,and the ordinary vaginal secretions were analyzed(bacteria);Culture of vaginal secretions and the second generation high throughput sequencing of vaginal secretions,namely total DNA extraction.Using bacterial 16 s universal primers 16 s rDNA PCR amplification(V3+V4)area,Illumina MiSeq sequencing machine for 2 x 300 bp double-end sequencing,get the original data,after effectively after quality control data table based feature characteristics,and analysis of the Alpha,beta diversity,species annotation and statistical analysis,to find the difference between different groups of species(Biomarker),state analysis of middle-late pregnancy vaginal flora and the correlation of premature rupture of membranes.Results:1.The detection rate of abnormal vaginal flora during pregnancy is low through traditional vaginal secretion analysis(bacteria)and vaginal secretion culture,and most of the PROM group were asymptomatic and normal in traditional examination.2.The Alpha diversity was small and the Beta diversity was significant between the premature rupture group and the normal control group.3.In terms of phylum level,Firmicutes(relative abundance>80%),Actinobacteria,Fusobacteria,Bacteroidetes,Proteobacteria and so on were the main phylum types in the experimental group.Firmicutes(relative abundance>95%)were dominant in control group.4.At the genus level,Lactobacillus was the dominant bacteria in the experimental group,but compared with the control group,the relative abundance of Lactobacillus was decreased,while Gardnerella,Sneathia,Atopobium,Ureaplasma,and Prevotella were significantly increased.In the control group,Lactobacillus was dominant and the relative abundance was greater than 95%.5.At the species level,L.cripatus(CST Ⅰ)was the main Community State Types in the overall vaginal,followed by L.iners(CST Ⅲ),various anaerobes(CST Ⅳ-B),L.jensenii(CST Ⅴ),and L.gasseri(CST Ⅱ).Normal pregnant women in the control group is given priority to with L.cripatus(CST Ⅰ),L.iners(CST Ⅲ)second,there is also a little L.jensenii(CST Ⅴ),Gardnerella,L.jensenii(CST)Ⅱ.Compared with the corresponding normal control group,CST I decreased significantly in the group of premature rupture of membranes,and CST Ⅲ,CST IV-B other anaerobic bacteria increased.6.There were significant differences in the composition of vaginal microflora between the premature rupture of membranes group and the normal control group.The abundance and diversity of vaginal microflora in the premature rupture of membranes were relatively high.The PPROM was relatively rich and had the highest diversity,while the incomplete pregnancy group had the lowest diversity and the lowest richness.7.Through LEfSe(LDA Effect Size)analysis,we found that compared with normal pregnancy,the biomarkers of vaginal flora of pregnant women with PPROM were Gardnerella,Atopobium,Prevotella,Staphylococcus.Gardnerella is the biomarker of the vaginal flora of pregnant women with full-term PROM.Using Barplot variance analysis,we found that PPROM pregnant women vagina microbial there was a significant difference compared with normal full-term pregnancy,and L.jensenii was the biomarker of preterm normal pregnancy,while there was no obvious biomarkers of normal full-term pregnancy.In comparison between the PPROM group and the full-term PROM group,the PPROM group had L.jensenii as the symbolic species,while the PROM group had Pseudomonas as the symbolic species.Conclusion(s):1.Traditional vaginal secretions analysis and culture can not fully understand the vaginal microbial community during pregnancy.2.The diversity of vaginal microflora during pregnancy was small and the microflora structure was relatively stable.The Alpha diversity of vaginal microflora in normal pregnant women and pregnant women with premature rupture of membranes was small,but the Beta diversity was present.Compared with normal pregnancy,the vaginal microflora of pregnant women with premature rupture of membranes was more diverse and abundant.3.The vaginal microbial community during pregnancy can be divided into five types,including L.cripatus(CST I),L.jensenii(CST Ⅱ),L.iners(CST Ⅲ),and various anaerobic bacteria(CST Ⅳ),Lactobacillus Jenneri(CST Ⅴ).In our study,it was found that CST I was dominant in pregnancy,and BV,PROM were prone to occur if CST IV-B(such as gardnella,prevotella,sneathia,atopobium,etc.)increased in the vagina during pregnancy.4.L.cripatus in the vagina plays a key role in the maintenance of normal vaginal microecology during pregnancy.The abnormal increase of lactobacillus inertia gardnella,ureaplasma prevotae,sneathia atopobium and other bacteria in the vagina is associated with the occurrence of premature rupture of membranes.5.The diversity of vaginal flora in normal pregnancy gradually increased with gestational week,and the diversity of vaginal flora in PPROM was also the largest.We speculated that normal full-term vaginal microecological changes during pre-term pregnant period were abnormal,which could easily cause BV and lead to PPROM.6.High-throughput sequencing technology can provide a more comprehensive and systematic understanding of the distribution of vaginal flora during pregnancy.It will be the direction of our next research to study the changes of vaginal microflora in early pregnancy and whether the supplementation of prophylactic vaginal probiotics during pregnancy can improve vaginal microflora.