Diversity Of Vaginal Microbiome On Reproductive-age Women In Guangzhou

Background:Vaginal microbiota form a mutually beneficial relationship with their host and are key components of a multifaceted antimicrobial defense system, which have major impact on health and disease. In recent years our understanding of vaginal bacterial community composition and structure has significantly broadened as a result of investigators using cultivation-independent methods based on the analysis of 16S ribosomal RNA (16S rRNA) gene sequences. In asymptomatic, otherwise healthy women, several kinds of vaginal microbiota exist, the majority often dominated by species of Lactobacillus, while others comprise a diverse array of anaerobic microorganisms. Vaginal pH associated with the vaginal microbiota, low pH (4.5) is considered synonymous with’healthy’. A deeper understanding of the interrelationships of constituent species is needed to understand how they change over time or in response to changes in the host environment. Ecological dynamics of vaginal ecosystems as they respond to disturbances caused by menses and human activities such as intercourse, douching, and other habits and practices, this fundamental knowledge is needed to diagnose and assess risk to disease.A better understanding of ’normal’ and ’healthy’ vaginal ecosystems that is based on its’true’ function and not simply on its composition would help better define health and further improve disease diagnostics as well as the development of more personalized regimens to promote health and treat diseases.The major advantages of using the next generation sequencing (NGS) to determine 16S rRNA tags compared to traditional microbial community profiling methods are cost-effectiveness and high throughput. We could obtain the desired microbial community data with NGS, which provides a sufficient tool to study microbial ecology. BIPES and TSC are two new methods for pre-process data being developed by our lab, QIIME is used to analyze bacterial community. The methods have high efficiency and low cost for processing the high-throughput sequencing tags.In 2008, the NIH (National Institutes of Health, NIH) funded $157 million investment in HMP (Human Microbiome Project, HMP), to establish and improve the microbial gene database, and also to reveal the association between microbiota and health or disease. But, the factor such as race, geography, culture, behavior, etc. can lead to structural differences between the human vaginal microbiota. The vaginal microbial structure of non-pregnant reproductive-age women in Guangzhou, China, commercial sex workers and the vaginal microbial dynamic changes of the menstrual cycle are not clear.Objectives:1、To expound the characteristics of vaginal microbiome on reproductive-age women in southern China and the vaginal microbial community diversity in pH<4.5 and pH> 4.5, by analyzing α, β diversity and microbial composition to show the difference in pH<4.5 and pH> 4.5 groups, using the LEfSe to look for taxa with significant differences in two groups, to further demonstrate the general female vaginal microbial diversity.2、To reveal the characteristics of vaginal microbiome of sex workers, by analyzing α, β diversity and microbiota composition to show the difference in commercial sex workers and the general female vaginal microbial, using the LEfSe to look for taxa with significant differences in two groups.3、To explore whether changes in vaginal flora with the menstrual cycle is relatively stable. Through the menstrual cycle vaginal flora α, β diversity and lactobacilli and vaginal microbiome stability and elasticity analysis,to explore with the change in the menstrual cycle, vaginal flora is still remained relatively stable with its fluctuations.Methods:Part 1Volunteers were engaged between July 2014 and August 2014 during a routine physical examination visit at Nanfang Hospital Healthy Examination Center in China, Guangzhou. Before routine gynecological examination, an informative about the research program and its significance were given to the volunteers to get their consent and cooperation for getting the samples.At last, a total of 99 people,aged between 19-52 years old volunteers were included.The sampling process with a sterile speculum examination was performed by a professional obstetrician. For each individual,3 samples (triplicates) were obtained by sterile plastic swabs from posterior fornix. A total of 297 vaginal swabs were collected from 99 subjects.Swabs were immediately stored at low temperature later frozen after collection within 4 hours and stored at -80℃ until usage.Using bacterial 16S rRNA universal primers, we amplified the bacterial 16S rRNA V46 conservative region. The PCR products were sequenced by platform of Illumina Hiseq 2000 with PE100-bp and then got the high-throughput primitive data. We used BIPES data analysis process to expound the characteristics of vaginal microbiome on reproductive-age women in southern China and the vaginal microbial community diversity in pH≤4.5 and pH> 4.5, by analyzing α, β diversity and microbial composition to show the difference indifferent pH groups. QIIME biological analysis process mainly included the operation classification unit (OTU) and Principal Coordinate Analysis (PCoA), hypothesis testing, the evolutionary tree analysis, species richness and diversity of community structure analysis,. Differentially abundant features were determined using Linear discriminate analysis effect size (LEfSe). LEfSe is an algorithm for high-dimensional biomarker discovery and explanation that identifies genomic features characterizing the differences between two or more biological conditions. LEfSe determines the species most likely to explain differences between classes by coupling standard tests for statistical significance with additional tests encoding biological consistency and effect size. The threshold on the logarithmic LDA score for discriminative features was 3.5. OTUs have been obtained, using Uclust (USEARCH) algorithm to classify; α-diversity using QIIME to get information of the sample. UCLUST similarity algorithm parameter is set to 0.97, indicating that 97% sequence similarity can be attributed to a single OTU. After comparison with the database, using RDP classifier 97% sequence similarity to return the same species. Comprehensive information generated BIOM table format, and then for alpha, beta and a series of analysis.Chaol PD_Whole_tree、Shannon、Observed_speices index used to assess α-diversity. Based on PCoA, β-diversity is analyzed, which is the ecological distance between different individuals. Application SPSS20.0 statistical software is used for statistical analysis, comparison between the two groups with normal distribution using two independent samples t-test, do not meet the normal distribution nonparametric tests take K group, P<0.05 was considered statistically significance.Part 2In May 2013, under the coordination and cooperation with the local health department and Center for Disease Control and Prevention (CDC) Division of STD and AIDS prevention in Guangzhou,we have recruited to provide commercial services among female sex workers to enter the queue. Some STD and AIDS prevention staff were invited to participate in the on-site investigation during sampling. They are mainly responsible for the completion of the study pre-publicity and recruitment work; part of the laboratory personnel responsible for the questionnaire interviews, specimen collection and transport as well as data entry, etc.. Before the study began, all field staff have carried out the principle of confidentiality, as well as project-specific implementation of the program receive training on human subject’s protection of the informed consent process.Sample collection, DNA extraction, PCR amplification, PCR products were sequenced and data processing are the same as "Part 1". We used BIPES data analysis process to character the vaginal microbiome of commercial sex workers(CSW) and the Physical Examination Women (PEW), by analyzing a, (3 diversity and microbial composition to show the difference in CSW and PEW groups.Part 3We have recruited 14 unmarried reproductive-age female students, they reports them in healthy, to join the menstrual cycle changes in vaginal flora longitudinal study. They collected samples from 2014 10 March to 2014 3 May by themselves. Samples were collected for every two days by 10 times gentle rotation of the sterile disposable cotton swab in the vaginal canal. Then it immediately was stored at -80℃, after collection of all samples,transferred to Southern Medical University School of Public Health and Tropical Medicine, Department of Environmental Health Laboratory. Using bacterial 16S rRNA universal primers, we amplified the bacterial 16S rRNA V4 conservative region. The PCR products were sequenced by platform of Illumina Hiseq 2000 with PE100-bp and then got the high-throughput primitive data. We used QIIME data analysis to check whether the vaginal microbiome is stable with the menstrual cycle. Application SPSS20.0 statistical software for statistical analysis, comparing application One-Way ANOVA multiple sets of measurement data, P <0.05 was considered statistically significant.Results:Part 11. We obtained a total number of 1,450,624 high quality 16S rRNA gene sequences from 99 samples, with an average of 14,652 sequences per sample. All samples with more than 2,485 reads per sample are included in the following analysis.2. The community of the vaginal microbiome on southern China were mainly divided into five types:1. L. iners as dominant bacteria; 2 L. crispatus as dominant bacteria; 3 L. gasseri was dominant bacteria; 4 L. crispatus and L. iners as the dominant bacteria; 5 one or two from Atopobium, Sneathia, Prevotella or Gardnerella and other anaerobic bacteria as the main advantage.3. When pH> 4.5, the vaginal microbiota a diversity increases, more types of microorganisms, Prevotella, Sneathia, Anaerococcu, Burkholderia, Bacteroidi, Allisonella, Dialister and Megasphaera have significant differences compared pH≤4.5; also when pH≤4.5 there are significant differences in Lactobacillus, Alliosonella.Part 21. We obtained a total number of 1,177,008 high quality 16S rRNA gene sequences from 71 samples, with an average of 16,577 sequences per sample. All samples with more than 1,212 reads per sample are included in the following analysis.2. At the genus level, Lactobacillus in Sex workers vaginal microbiota is only 44.2%, while Garderella, Prevotella and Sneathia are accounted for approximately 20%,10% and 9% respectively.3. The communities of the sex workers vaginal microbiome are mainly divided into four groups:1. L. iners as dominant bacteria; 2 L. crispatus as dominant bacteria; 3. Gardnerella as the dominant bacteria; 4. One or two from Atopobium, Sneathia or Prevotella and other anaerobic bacteria as the main advantage.4. At the genus level, PEW group mainly enriched by Lactobacillus, Burkholderia, where as CSW group mainly enriched with Prevotella, Sneathia, Gardnerella and Escherichia/Shigella etc.5. There are differences in the microbiota composition of the CSW groups that work in different place.Based on unweighted_unifrac and abund_jaccard distance, the clustering results of the two groups vaginal microbiota can be divided into two distinct parts.6. There are differences in the microbiota composition of the CSW and PEW groups.Based on unweighted_unifrac and binaryjaccard distance, the clustering results of the two groups’ vaginal microbiota can be divided into two distinct parts.Part 31. A total of 3,998,093 high-quality 16S rRNA V4 gene sequences were obtained from the 202 samples, with an average of 19,792 sequences per sample. The sequence depth range from 2,566 to 70,110. All samples with more than 1,283 reads per sample are included in the following analysis.2. Fluctuate with changes in vaginal flora of the menstrual cycle, and during menstruation vaginal flora fluctuate most obvious. Presented alternately dominant flora or change mode to maintain absolute superiority in the menstrual cycle.3. Fluctuations in the size of the vaginal flora vary, some women during menstrual cycle vaginal flora change periodically, and some women vaginal flora is very stable.4. Vaginal flora is affected by environmental hygiene habits significantly, according to the living quarters appear clustering phenomenon.5. Most of the normal vaginal flora dominated by species of Lactobacillus, while others comprise a diverse array of anaerobic microorganismsConclusion:1. The community of the vaginal microbiome on reproductive-age women in Guangzhou are mainly divided into five types:1 L. iners as dominant bacteria; 2 L. crispatus as dominant bacteria; 3 L. gasseri was dominant bacteria; 4 L. crispatus and L. iners as the dominant bacteria; 5 one or two from Atopobium, Sneathia, Prevotella or Gardnerella and other anaerobic bacteria as the main advantage.2. When pH≤4.5 there are significant differences in flora Lactobacillus, When pH> 4.5, the vaginal microbiota were typified by higher proportions of anaerobic bacteria.3. Statistically significant associations are observed between PEW and CSWn within CSW to provide services in hotel and street, vaginal flora affected by environmental hygiene habits significantly.4. Fluctuate with changes in vaginal flora of the menstrual cycle, and during menstruation vaginal flora fluctuate most obvious. Presented alternately dominant flora or change mode to maintain absolute superiority in the menstrual cycle.