Characteristics And Risk Prediction Value Of Vaginal Microbiota Of Women With Cervical Lesions Based On Metagenomics

Objective:Cervical cancer is the fourth most malignant tumor in women worldwide,posing a serious threat to women’s health.Although the persistent infection of high-risk human papillomavirus(HR-HPV)has been proved to be the main cause of cervical cancer,it is not a necessary factor.There may be other pathogenic factors involved in the occurrence and development of cervical lesions.The widespread application of metagenomics sequencing technology provides an efficient method to analyze the characteristics of vaginal microbiota at species level and explore the biological functions of microbiota in patients with cervical lesions.It may provide important theoretical basis for early prevention,control,and warning of cervical lesions by exploring the characteristic changes of vaginal microbiota.However,characteristics and functions of vaginal microbiota at species level in the whole ladder of cervical lesion progression,from normal cervix,cervical precancerous lesions to cervical cancer,is still unclear.The purpose of this study is to deeply explore the characteristics and functional changes of vaginal microbiota in different stages of cervical lesions,and comprehensively evaluate the predictive value of vaginal microbiota for the risk of cervical lesions,this study takes normal cervical women(NC),patients with low grade cervical intraepithelial neoplasia(CIN1),patients with high grade cervical intraepithelial neoplasia(CIN2/3)and patients with cervical squamous cell carcinoma(SCC)as the research objectives,and uses the metagenomics sequencing technology to explore the characteristics and functions of vaginal microbiota at species level in patients with different stages of cervical lesions.At the same time,combined with vaginal microbiota information and population characteristics,the random forest algorithm is applied to predict the risk of cervical lesions,aiming to provide scientific evidence for the early prediction and warning of cervical lesions from the perspective of improving vaginal microecology.Methods:A total of 510 women,who meet the inclusion and exclusion criteria and have been confirmed by pathology of different cervical lesions,including 295 with NC,94 with CIN1,105 with CIN2/3,and 16 with SCC,were selected from the Department of Obstetrics and Gynecology in the Second Hospital of Shanxi Medical University from January to June2021.Of the enrolled women,41 NC,39 CIN1,37 CIN2/3,and 15 SCC matched by age and residence(city/county)were selected as the object of study.All these 132 women did not take antibiotics in the past two weeks or received vaginal medications or vaginal douche in the past 48 hours,or had sex in the last three days.A questionnaire was used to collect the demographic characteristics of the subjects and the related factors of cervical lesions.The exfoliated cells of the cervix were collected for HPV typing by diversion hybridization.The vaginal secretions were collected for metagenomics sequencing.The species classification system was used to analyze the composition and dominant bacteria of the vaginal microbiota at the species level.Alpha diversity analysis was used to reflect the diversity of the vaginal microbiota,and LEf Se was applied to analyze the characteristic bacteria.Co-occurrence network was employed to analyze the relationship between vaginal microbiota.KEGG and GO databases were utilized to analyze the function of vaginal microbiota.SPSS 26.0 was performed to complete theχ~2 test,one-way ANOVA analysis and Spearman correlation analysis.The random forest model in R was adopted to predict the risk of cervical lesions,and the model was evaluated using the area under the curve(AUC)of the receiver operating characteristics(ROC).Statistical test significanceα=0.05.Results:1.Characteristics of vaginal microbiota in women with cervical lesions:The diversity of vaginal microbiota in NC,CIN1,CIN2/3 and SCC groups was statistically significant,and the diversity of vaginal microbiota in NC,CIN1 and CIN2/3 groups was significantly lower than that in SCC group(P<0.05).The dominant bacteria in NC,CIN1 and CIN2/3groups were Lactobacillus iners,Lactobacillus crispatus and Gardnerella vaginalis,respectively.With the progression of cervical lesions,the ratio of Lactobacillus crispus gradually decreased,with the ratios of NC,CIN1,CIN2/3,and SCC group being 27.88%,23.64%,16.30%,and 0.32%,respectively;The ratios of Lactobacillus iners in each groups were 32.47%,17.86%,31.66%,and 0.72%,respectively;Gardnerella vaginalis showed a trend of increasing and then decreasing in CIN1,but decreased to the lowest value of4.56%in SCC group;The ratio of Staphylococcus aureus,Phocaeicola vulgatus,Salmonella enterica,Bacteroides fragilis and etc.increased significantly.LEf Se method was used to analyze the characteristic bacteria of vaginal microbiota.The characteristic bacteria of NC group,CIN1 and CIN2/3 were Lactobacillus iners,Bifidobacterium breve and Sneathia amnii,respectively,while Staphylococcus aureus,Salmonella enterica and Porphyromonas asaccharolytica were the characteristic bacteria of SCC group.2.The co-occurrence relationship between vaginal microbiota in cervical lesions:The relationship between vaginal microbiota in different grades of cervical lesions was analyzed by co-occurrence network.In NC and CIN1 group,Lactobacillus crispatus and were significantly negatively correlated with Gardnerella vaginalis(NC:r=-0.512,P<0.05;CIN1:r=-0.711,P<0.001).In CIN1 and CIN2/3 group,Bacteroides fragilis,Bacteroides uniformis,Bacteroides ovatus,Phocaeicola vulgatus and Phocaeicola dorei were positively correlated with each other(P<0.05),in the meanwhile,Sneathia amnii was positively correlated with Shewanella sp.Scap07 in CIN2/3 group(P<0.05).In SCC group,Gardnerella vaginalis was negatively correlated with Staphylococcus aureus and Klebsiella variicola,and a positive correlation was found among various anaerobic bacteria(P<0.05).3.The functional difference of vaginal microbiota in cervical lesions:The KEGG database was used to annotate the metabolic pathway of vaginal microbiota.It was found that the expression of glycosyltransferases of vaginal microbiota in CIN1 patients was down-regulated compared with NC group,while the abundance of pathways such as nicotinate and nicotinamide metabolism and glycerophospholipid metabolism was significantly increased.In particular,the levels of chemical carcinogenesis-reactive oxygen species(ROS)of vaginal microbiota in CIN2/3 and SCC groups were significantly increased.In SCC group,lysine degradation,micro RNAs in cancer,and various types of n-glycan biosynthesis were significantly enriched.According to the difference analysis of the gene and protein functions of the vaginal microbiota in patients with cervical lesions by using the GO annotation results,s-layer protein was significantly reduced in the occurrence and development of cervical lesions,and biological progress such as the cellular amino acid catabolic process,alpha-amino acid catabolic process and etc.were significantly enriched in CIN1 group compared with NC group.Ligase activity in CIN2/3 group was significantly higher than that in NC group.The gene functions such as envelope,cell envelope,metal cluster binding,iron-sulfur cluster binding,carbon-oxygen lyase activity,hydro-lyase activity and etc.were significantly enriched.4.Risk prediction of vaginal microbiota on cervical lesions:The random forest model was used to predict the risk of cervical lesions defining CIN2-(NC and CIN1)and CIN2+(CIN2/3 and SCC)as outcome indicators.When only the relative abundance of vaginal microbiota is included,the order of importance of variables is Porphyromonas asaccharolytica,Anaerococcus prevotii,Curtobacterium sp.Csp2,Klebsiella variicola and Vibrio sp.Scap24.The ROC shows that AUC=0.720(95%CI:0.465-0.975).Further,the importance of the influence of vaginal microbiota on CIN2+under different population characteristics was analyzed.Lactobacillus iners was an important variable under HPV16infection.In particular,Porphyromonas asaccharolytica was an important variable that affects CIN2+under the conditions including unclean vagina after sex,with many sexual partners,menopause and have a history of contraception.In addition,Klebsiella variicola is an important microbiota affecting CIN2+under the condition of HPV+,HPV16+,HPV16-,passive smoking,unclean vagina after sex,have a lot sexual partners,non-menopause and with a history of contraception.After integrating the relative abundance of vaginal microbiota and population characteristics,the ranking results of variable importance showed that HPV16 was an important factor for cervical lesions,especially found that Curtobacterium sp.Csp2,Anaerococcus prevotii,Klebsiella variicola,Bacteroides thetaiotamicron,Porphyromonas asaccharolytica,Bacteroides fragilis,Parabacteroides distasonis and Polynucleobacter necessarius can be used as important biomarkers of CIN2+.Uncleaning vagina after sex is also closely related to CIN2+,with the AUC value increased to 0.837(95%CI:0.685-0.989).Conclusion:1.Vaginal microbiota disorder is closely related to the progression of cervical lesions.With the malignant progress of cervical lesions,the diversity of vaginal microbiota was gradually increasing,and the dominant position of Lactobacillus,represented by Lactobacillus crispatus,in the vaginal microbiota was weakened,while a variety of opportunistic pathogens were significantly enriched.There is a complex positive correlation between various bacteria,suggesting that vaginal microbiota can be used as a biomarker for early screening of cervical lesions,and may provide new ideas for the prevention of cervical precancerous lesions.2.The functional changes associated with vaginal microbiota disorder may play an important role in cervical lesions.The metabolic pathway and gene function of reactive oxygen species(ROS)and amino acids in patients with different stages of cervical lesions were significantly increased.In particular,the s-layer protein of vaginal microbiota was significantly reduced during cervical lesions,suggesting that vaginal microbiota may participate in the occurrence and development of cervical lesions by regulating its own genes and metabolic functions.3.Vaginal microbiota has important predictive value for cervical lesions.In particular,under the conditions of HPV16 infection,Lactobacillus iners was an important variable for cervical lesions.The combination of vaginal microbiota with HPV16 infectious status,whether clean vagina after sex and other disease-related risk factors has a quite good predictive effect on cervical lesions,which suggests that vaginal microbiota plays an important role in the progress of cervical lesions.