Using High-throughput Sequencing Technology To Analyze The Correlation Between Vaginal Microbial Diversity And Cervical Precancerous Lesions And Cervical Cancer

Aims:This study intends to use high-throughput sequencing technology to compare and analyze the differences in vaginal microbial diversity among patients with cervical intraepithelial neoplasia,cervical cancer,and normal healthy women,and combine clinical test indicators to analyze vaginal microbes and cervical epithelium The relationship between internal neoplasia and cervical cancer is expected to provide new ideas for scientific research and clinical work.Method:1.Enrollment of research subjects and collection of vaginal microorganisms:Under the premise of informed consent and signing of the informed consent form,collect healthy women and patients with cervical precancerous lesions between the ages of 20 and 40 who were in the Second Affiliated Hospital of Nanchang University from July 2018 to August 2019 for the first time.And 30 cases of vaginal posterior fornix secretions of patients with cervical squamous cell carcinoma were named as healthy control group(C Group,hereinafter referred to as C group),cervical precancerous lesion group(CIN Group,hereinafter referred to as CIN group),and cervical squamous cell carcinoma.Cancer group(CER Group,hereinafter referred to as CER group),and store the collected vaginal secretions in a refrigerator at-80 degrees Celsius for later use.2.Collect clinical data of the enrolled patients:Extract the information of study objects including: age,height,weight,body mass index(BMI),age at first sex,number of births,cervical HPV,TCT test results,hemoglobin test value(Hb),Neutrophil ratio,lymphocyte ratio and serum squamous cell associated antigen(SCCA)detection value from the electronic medical record system in our hospital.3.High-throughput DNA sequencing of vaginal microbial samples and prediction of vaginal flora metabolism differences:A total of 90 subjects were included in this study,including 30 in the C group,30 in the CIN group,and 30 in the CER group.After collecting and integrating the clinical data of the subjects,3subjects in the C group,8 cases in the CIN group and 7 cases in the CER group were finally eliminated due to lack of clinical data.Then,2 cases in group C,3 cases in CIN group and 5 cases in CER group were eliminated due to the failure of vaginal microbial DNA extraction.In the end,25 samples of vaginal microbial DNA samples of C group,19 cases of CIN group and 18 cases of CER group were obtained.A total of 62 samples of vaginal microbial DNA samples were sequenced on the computer.The isolated microbial DNA in the vaginal secretion sample was amplified by PCR,and after quality inspection,the V3-V4 region of the microbial DNA was sequenced using the Illumina platform.Then,according to the results of sequencing and species identification,the vaginal flora was classified according to the "world,phyla,and class,Order,family,genus,species" classification method and classification of vaginal flora species diversity analysis.In this study,bacteria that are beneficial to human health are defined as "probiotics",and bacteria that symbiotically live in the human body and only cause harm to the health of the body when proliferation is out of control,and bacteria that are harmful to human health when they exist are called "pathogenic bacteria".Finally,the KEGG database is used to predict the changes in vaginal bacterial metabolism that may be caused by the diversity of vaginal flora.4.Statistical analysis:SPSS19 software was used to analyze the clinical indicators of patients;Graphpad prism 8.0 software was used to analyze the differences in vaginal microbial diversity of the enrolled subjects and analyze their correlation with clinical indicators.Result:(1)Patient clinical data analysis:Analyzing the clinical data of the study subjects,the results showed that: in terms of production times,the number of births was 0.8±0.145 in the C group,0.9±0.146 in the CIN group,and 1.9±0.153 in the CER group,P<0.001,the difference was statistically significant significance.The age at first sex was 24.30±0.384 in group C,23.86±0.548 in CIN group,18.04±0.298 in CER group,P<0.001,the difference was statistically significant.As for the analysis of HPV typing test results,the infection rate of type 16 or 18 in the CER group(82.61%)is higher than that of the CIN group(59.09%),and the CIN group is higher than that of the C group(0%),P<0.001,the difference is Statistical significance.It can be seen that the number of births,age at first sex,and the positive rate of HPV typing 16 or 18 are significantly different among the three groups of subjects.(2)Comparison of differences in vaginal microbial among different groups of study subjects:For the Shannon index,the C group is 6.239,the CIN group is6.458,and the CER group is 6.845,P= 0.2609.Similarly,for the Simpson index,the C Group is 0.2609,the CIN group is 0.8694,and the CER group is 0.8767,P= 0.2245.It can be seen that the Shannon index and Simpson index,which describe the alpha diversity of vaginal microbes,showed an upward trend among the C,CIN and CER groups,that is,the alpha diversity of vaginal microbes was higher than that of the normal healthy control group,and the difference was not statistically significant.In addition,vaginal microbiome comparison at the level of genus showed that the relative abundance of Lactobacillus in the C group(35.59%)was higher than that in the CIN group(25.24%)and the CER group(18.42%),and the difference was statistically significant(P= 0.034).Comparison of the relative abundance of the pathogenic bacteria such as Prevotella spp.(P= 0.029),Sneathiaspp.(P= 0.176),and Pseudomonasspp.(P= 0.0001)reveals that the the C group lower than CIN group and the CER group.(3)Correlation analysis between the differences in vaginal microbial and clinical indicators:In the correlation analysis,the relative abundance of Lactobacillus was negatively correlated with SCCA(r=-0.29,P<0.05)and Neutrophil ratio(r=-0.14,P>0.05).However,Pseudomonas was positively correlated with SCCA(r= 0.32,P<0.05)and Neutrophil ratio(r = 0.18,P> 0.05).Similarly,Prevotella was positively correlated with SCCA(r=0.06,P>0.05)and Neutrophil ratio(r=0.17,P>0.05).(4)Prediction of vaginal microbial metabolism:Based on the structural differences in the DNA group of vaginal microorganisms,the metabolic pathway information of the KEGG database is used to predict the possible metabolic differences of different groups of vaginal microorganisms.The results showed that for Cell Growth and Death and Cell Motility,which reflect the biological processing of bacteria.The relative abundance of the bacteria that promote Cell Growth and Death is in the C group(0.005573),CIN group(0.005416)and CER group(0.00261),P=0.01.The relative abundance of bacteria that promoted Cell Motility showed an upward trend between the C group(0.02459),the CIN group(0.02557)and the CER group(0.02675),P= 0.14.“Folding,Sorting and Degradation” and “Replication and Repair” are indicators that reflect the genetic information processing of the bacterial flora.The relative abundance of bacteria which promote bacterial DNA Folding,Sorting and Degradation in group C(0.02375)is lower than that of CIN group(0.02380)and CER group(0.02318),P= 0.02.The relative abundance of bacteria that promoted bacterial DNA Replication and Repair in group C(0.08063)was higher than that in CIN group(0.07874)and CER group(0.07716),P= 0.23.Conclusion:(1)There are differences in the diversity of vaginal microbes between normal healthy women and patients with cervical squamous intraepithelial neoplasia and cervical cancer.Among the three groups,as for the diversity of vaginal microbes,cervical cancer is the highest,and patients with cervical squamous intraepithelial neoplasia ranked the second,normal healthy women turn out to be the lowest.(2)The decrease in the relative abundance of Lactobacillus in vaginal microorganisms is positively correlated with the occurrence and development of cervical lesions;the increase in the relative abundance of pathogenic bacteria in the vagina is positively correlated with the occurrence and development of cervical lesions.(3)The mechanism of the relationship between vaginal microbial disorders and cervical precancerous lesions and cervical cancer may be related to the local inflammation caused by vaginal microbial disorders,but the specific mechanism needs further research to confirm.