Vaginal Microecological Analysis Of High Risk HPV Infection And Cervical Intraepithelial Neoplasia

ObjectiveTo analyze the vaginal microecology of women with high risk human papilloma virus（HR-HPV）and cervical intraepithelial neoplasia（CIN）,and to explore the relationship between HR-HPV,CIN and vaginal microecology.MethodsFrom March 2018 to September 2019,physical examination was collected in the gynecological outpatient department of our hospital,HR-HPV screening positive was transferred to the colposcopy outpatient department of our hospital for examination,146subjects confirmed as CIN by pathological biopsy were taken as the experimental group;139 subjects who were screened at the same time without HPV infection and abnormal cervical fluid based cytology（LCT）were taken as the control group.General clinical data and relevant risk factors of all subjects were collected,and detailed gynecological examination and vaginal microecology examination were carried out.Lactobacilli,trichomonad,gonococcus and vulvovaginal candidiasis VVC were detected by fast gram staining method BV Nugent score was used as the diagnostic standard;precise p H test paper was used to determine the p H of vaginal microenvironment;hydrogen peroxide（H₂O₂）,leukocyte esterase and sialidosidase were tested with vaginal microecological test kit;Mycoplasma was tested with microbiological test;Chlamydia trachomatis test kit was used to detect Chlamydia trachomatis with double antibody sandwich method.Thirty-five samples from the experimental group and the control group were randomly selected for second-generation sequencing,and all bacterial DNA was extracted.Using Illumina misq high-throughput sequencing platform to sequence the amplified products,optimize the data,divide the OTU with 99%similarity threshold,compare the database to analyze the biological information,and determine the species differences between the two groups.ResultTrichomonal vaginitis was detected in the experimental group,There were 3 cases of TV,58 cases of BV,12 cases of VVC,41 cases of Ureaplasma urealyticum,32 cases of Mycoplasma hominis,45 cases of Chlamydia trachomatis CT and 42 cases of lactobacilli negative in the control group.There were 5 cases of TV,13 cases of BV,7cases of VVC,10 cases of UU,9 cases of MH,7 cases of Chlamydia and 5 cases of lactobacilli negative in the control group.48 cases were TV,VVC,BV,UU,MH and CT.The negative detection rate of BV,MH,UU,CT and lactobacilli in the experimental group was significantly higher than that in the control group（P<0.05）,while the infection rate of TV and VVC in the two groups had no significant difference（P>0.05）.There was significant difference between the two groups in the abnormal rate of p H and H₂O₂（P<0.05）.There was no significant correlation between the abnormal rate of vagina cleanliness,sialidosidase and leukocyte esterase and CIN（P>0.05）.Sequencing of V4 hypervariable region of 16S rDNA gene in vaginal microorganism by Illumina sequencing At the level of phylum,the proportion of Firmicutes in the control group was the highest 61.38%,and the other 20 phylum accounted for 38.62%;the proportion of Firmicutes in the experimental group was significantly reduced by46.15%,while the abundance of Actinobacteria,Proteobacteria,fusobacteria and Bacteroidetes increased and Brucella was found.At the genus level,the lactobacilli in the control group accounted for 82.45%;the lactobacilli in the experimental group decreased to 40.84%,while the Gardnerella increased from 4.01%to 18.87%.Multivariate logistic regression analysis showed that BV,CT,UU,lactobacilli and p H abnormality were independent risk factors for CIN（P<0.05）.Conclusion1.BV,UU,MH,CT,lactobacilli were negative and p H value was abnormal.However,there was no significant correlation between the infection of TV and VVC and CIN.2.CIN was positively correlated with p H and H₂O₂,but not with vaginal cleanliness,sialidase and leukocyte esterase.3.The results of 16S rDNA sequencing showed that the diversity of vaginal microflora in HR-HPV infected women increased,and the phylum of sclerenchyma decreased significantly at the phylum level,and Brucella was found;the relative abundance of Lactobacillus decreased significantly at the genus level,while Gardnerella increased positively correlated with HR-HPV infection and CIN.4.BV,lactobacilli negative,CT,UU and p H value abnormality were independent risk factors of CIN,while MH and H₂O₂abnormality were related to CIN but not independent risk factors.