Diversity Analysis Of Vaginal Microbiota On Women With High-risk Human Papillomavirus Infection And Different Levels Of Cervical Lesions

Background:Cervical cancer is one of the most serious disease in women worldwide, and the morbidity and mortality of which ranks first in the female genital tract malignant tumors, threatening the health and lives of women. Present research has confirmed that high-risk human papillomavirus (HR-HPV) persistent infection is a necessary condition in cervical intraepithelial neoplasia (CIN) and cervical cancer occurs. Frequently, most women’s vaginal microorganism environment was able to effectively eradicate the HPV virus from vagina after primary infection. Just a small proportion of HPV-infected women will eventually develop into CIN and cervical cancer in 5-10 years. Therefore, there must be other doubtful carcinogenic factors or cofactors to increase the susceptibility of HPV during the development of cervical cancer.Vaginal microbiota play a crucial protective role in the balance of vaginal microecosystem, the association between vaginal flora with persistent HPV infection has been more concerned by people. The female genital tract is a relatively confined space, vaginal flora are mainly anaerobic bacterium, so the cultivation and nutrition conditions were demanding. Isolating, culturing and identificating for a high proportion of bacteria is difficulty. Therefor, it is difficult to fully recognize the vaginal flora composition with traditional culture-depending method. As the rapid development of molecular biology and relative techniques, the train dependency technology invention, such as denaturing gradient gel electrophoresis (DGGE), fluorescence in situ hybridization (FISH), the awareness of vaginal flora has acquired significant breakthroughs. However, these traditional molecular biology techniques also have restrictions and limitations, such as time-consuming, heavy workload, the important information converged is not comprehensive, or even missing.Barcoded Paired-End Illumina Sequencing (BIPES) is a new sequencing method, it based on the Illumina Solexa high-throughput sequencing platform, and combined with 16S rRNA tag sequence. This method has the characteristics of high efficiency, simple operation, and low costs, therefor we can obtained information about the overall structure and composition of vaginal microbial communities rapidly. By using the BIPES sequencing method, this study investigate the microbial population of vagina in HR-HPV-infected and uninfected female, in order to explore the association between HR-HPV infection and vaginal flora. Furthermore, we explore and compare the microbial community structure of women in different levels of cervical lesion, revealing the dynamic changes of microbial species structure during the development and malignant of cervical carcinoma, in order to provide the theory basis for the further study of etiology, prevention, vaginal micro ecological therapy and the development of probiotics of cervical lesions.Objectives:1. To reveal the characteristics of vaginal microbiome in HR-HPV-negative and HR-HPV-positive women. By analyzing the species diversity and microbial composition to show the differences in two group, to explore the relationship between HR-HPV infection and vaginal flora.2. To expound the characteristics of vaginal microbiome in women with different levels of cervical lesion. Through analyzing the differences among the groups, to disclose the dynamic changes of microbial structure during the occurrence and development of cervical carcinoma, and to discover the relationship between them.Methods:Part 11.The experimental object.The participants engaged in this research were those who visited in the department of gynecology and obstetrics of Zhujiang Hospital between September 2014 and September 2015. According to the results of HR-HPV test (hC2), the participants were divided into two groups.① HPV-negative group:33 cases without HR-HPV infection. ② HPV-positive group:98 cases with HR-HPV infection.Inclusion criteria:aged 21 to 65 yeas old, sexual life more than 3 years, non menstrual, pregnancy or puerperium period. Exclusion criteria:age>65 years old, asexual life, combined immunodeficiency or systemic disease. At the same time, The above participants should satisfy the following requirements:no vagina douching within 48 hours, without sexual life and vaginal administration within three days, no systemic application of antifungal agents and antibiotics within one month.2. Specimen Collection:2 swabs of vaginal discharge from the middle of the vagina or vaginal fornix were collected for 16S rRNA genes extraction.3. Total DNA extractionthe:Genomic DNA was extracted by genomic DNA purification kit, and the specific steps reference manual.4. Amplification of 16S rRNA V4 gene region and Illumina sequence:Using extracted genome DNA as template, the 16S rRNA V4 gene fragment were amplified, and then identified by agarose gel electrophoresis. After the qualitative analysis on ultraviolet absorption spectrometry and quantitative calculation on fluorescence spectrometry, the PCR products were mixed with equal final concentration, and then sequenced by platform of Illumina Hiseq 2000 with PE1000-bp (BGI, Shenzhen).5. The original data were pretreated by BIPES data analysis process. Using Two-stage-clustering (TSC) clustering method to extract the representative sequence of each operational taxonomy units (OTU). We carried out similarity analysis by using GAST to classify the OTU, and the subsequent analysis including Alpha and Beta diversity analysis, microbial structure analysis. Based on the weighted_unifrac distance, using QIIME software to carry out the analysis of principal component of vaginal microbiota. Using linear discriminant analysis (LDA) coupled with effect size measurements (LEfse) to analyze the difference of structure and composition of vaginal microbial communities between two groups.6. Statistical processing:SPSS 19.0 software was applicated for statistical analysis. Measurement data were analyzed with the t-test, and the Mann-Whitney U test was performed to compare the diversity index and the relative abundance of each species. P<0.05 was interpreted to be statistically significant.Part 21.The experimental object.The participants engaged in this research were those who visited in the department of gynecology and obstetrics of Zhujiang Hospital between September 2014 and September 2015. According to the results of liquid-based cervical cytology, HR-HPV test (hC2) and the cervical biopsy pathology, the participants were divided into 4 groups. ① Normal group (N):33 cases with negative results of liquid-based cervical cytology and HR-HPV test. ② Low grade group (L):26 cases with positive result of HR-HPV test and/or the result of cervical biopsy pathology shows low-grade squamous intraepithelial lesion (LSIL). ③ High grade group (H):40 cases with positive result of HR-HPV test and cervical biopsy pathology shows high-grade squamous intraepithelial lesion (HSIL). ④ Cervical cancer group (C):32 cases with positive result of HR-HPV test and cervical biopsy pathology suggests cervical cancer.Inclusion and exclusion criteria are the same as the Part 1.Specimen collection, total DNA extraction, PCR amplification, PCR products sequencing and data processing are in common with Part 1. Comparisons between groups were tested by One-Way ANOVA analysis and LSD test.Results:Part 1:1.131 samples were sequenced in this study, and received 971007 sequences totally, with an average of 7412 per sample.2. PD_whole_tree (PD) index and Shannon index were employed to compare the differences of Alpha diversity between HPV-negative and HPV-positive groups. The results suggest that PD index and Shannon index were increased in HPV-positive groups, and the difference of Shannon index was statistically significant (P=0.000).3. At the genus level, Lactobacillus is the predominant species in HPV-negative group, accounting for 81.54%. Microbial community structure was changed remarkablely in HPV-positive group, and Lactobacillus content decreased significantly, only 40.48%, coupled with the rising proportion of Ureaplasma, Mycoplasma and anaerobic bacteria, such as Prevotella, Atopobium and Peptostreptococcus, ect.4. Caning on the Beta diversity analysis through QIIME procedure, the result illustrates that the diversity of vaginal flora in the two groups could be distinctly divided into two clusters based on weighted_unifrac distance.5. Through LEfSe statistics analyzing, we found that HPV-negative group predominately enriched by Lactobacillus, while HPV-positive group mainly enriched by Gardnerella and anaerobic bacteria, such as Prevotella, Atopobium, Anaerococcus, Bacteroides and Acinetobacter, ect. What’s more, Brucella was detected in HPV-positive group.Part 2:1.131 samples were sequenced in this study, and received 971007 sequences totally, with an average of 7412 per sample.2. PD_whole_tree (PD) index and Shannon index were adopted to compare the difference of Alpha diversity among each group. With the occurrence and progression of cervical lesions, the community structure of vaginal microbiota changed significantly. The Alpha diversity was increased gradually, and reached the maximum in cervical cancer group.3. At the genus level, With the occurrence and development of cervical lesions, the gradually decrease in the relative abundance of Lactobacillus was accompanied by an increase in the ratio of anaerobic bacteria and other unknown bacteria. The relative abundance of Gardnerella was first increased, and then decreased.4. Through LEfSe statistics analyzing, we found that Gardnerella, Ureaplasma and Mycoplasma mainly enriched in low grade group, while high grade group and cervical cancer group predominately enriched by anaerobic bacteria.5. Based on the weightedunifrac distance, in spite of the distinction of clustering results between neighboring two groups was not remarkable, the difference increased gradually with the the occurrence and development of cervical lesions.Conclusion:1. The structure of vaginal microbial community in healthy women is relatively simple, while the diversity of vaginal flora increased in HR-HPV infected women. Vaginal dysbacteriosis is associated with HR-HPV infection.2. With the occurrence and development of cervical lesions, the relative abundance of Lactobacillus decreased gradually, and the diversity of vaginal flora gradually increased.3. The decrease of Lactobacillus in vagina flora and/or the appearance of Brucella, may be the cause of persistent infection of HR-HPV and cervical lesions.