This is a longitudinal multicenter cohort study aiming to reveal the association between vaginal microbiota and the outcome of HPV infection. This study is also the largest study investigating vaginal microbiota of reproductive women in China.
Volunteers in this study come from patients of gynecology clinic. This group includes both HPV-positive women and HPV-negative controls. At first, the clinician should confirm with the volunteer who participate in our research about completing this study from beginning until six-month follow-up. Women will be considered to have HPV persistence if they are tested positive for any type of 21 HPV genetypes at two consecutive time points. In this research, we use baseline and the follow-up after 6 months to define HPV persistence.
At the stage of volunteer enrollment, we have designed a strict inclusion and exclusion criteria, excluding those who are not appropriated to be included in this research. The clinician from each medical center will comprehensively evaluate whether the volunteer is eligible to participate in this study. After the volunteers sign the informed consent form, the clinical gynecologist will collect three vaginal secretion swabs, one for bacterial DNA extraction, one for wet mount microscopy, enzymatic indicator detection, and Nugent score, and one for cryogenic storage at -80℃; two tubes of cervical exfoliated cells from every volunteer, one for TCT, and one for HPV genotype and other sexually transmitted pathogens detection. All collected samples will be numbered in sequence. The reserved frozen samples and the samples used for sequencing will be transported to the ZhuJiang Hospital through the cold chain from each research center for the unified follow-up processing.
After samples collected, the research assistant will assist each volunteer to complete an enrollment form, which records every volunteer’s demographic information（age, nationality, native place, occupation, address, height, weight, waist, education level and incomes）, clinical data ( history of present illness, genital symptoms and signs, past medical history including diabetes, hypertension, urinary tract infection, gravidity and parity history, times of poor pregnancy, first delivery time, genital tumor and genital surgery) , the history of drug (drugs related to glucose and lipid metabolism, estrogen, oral antibiotics, antifungal drugs, vaginal suppository) use within one month, lifestyle information (smoking, drinking, family disease, menstrual cycle, the number of sexual partners, contraceptive methods). Our research try to control pregnancy[20, 26-28], drugs, lifestyle and other interference factors that have a great impact on the vaginal microbiota in our study population. After the above procedures completed, corresponding medical inspection results such as TCT, 21 HPV genotype and other sexually transmitted pathogens detection will be added to the enrollment form.
Researchers will collect the vaginal and cervical samples again at the follow-up after six months. Medical inspection results including TCT, HPV genetype and so on will be recorded in the follow-up form. For some patients who undergo colposcopy and histopathological examination due to the condition of the disease during this half year, researchers need to supplement the corresponding examination data in the follow-up form. A flowchart displaying the volunteers flow through the study is shown in Figure. 1
Participating center qualification
Researchers from Zhujiang hospital and additional 49 research centers covering most provinces of China will collaborate together in to conduct this study（Figure. 2）. All research centers must be local level A tertiary general hospitals or specialist hospitals, capable of diagnosing and treating various obstetrics and gynecology crises, contain clinical PCR laboratories, capable of carrying out molecular biotechnology and ThinPrep cytologic test (TCT), have colposcopy and clinical pathological diagnosis and other technologies, and commit to complete this research on time.
Recruitment of volunteers
Volunteers in this study will come from gynecology clinic who undergo TCT examination. After this research beginning, volunteers will be enrolled continuously in the clinical center until enough volunteers are included, then they will be selected according to the following selection criteria.
The following three terms must be met.
Age 18-50 years old,
TCT inspection will be performed.
Have a history of sexual experience.
Be in non-menopausal and non-pregnant periods.
Those who meet any of the following conditions should be excluded:
Be pregnant or within 8 weeks of postpartum period.
Have a history of reproductive tract tumor.
Have a history of HPV bivalent/quadrivalent/9-valent vaccination.
Have ahistory of treatment for HPV infection or other sexually transmitted diseases in the past six months.
Have a history of previous hysterectomy, cervical surgery, and pelvic radiotherapy.
Have sexual intercourse, vaginal medication or douching within 72 hours before vaginal sampling.
Apply broad-spectrum antibiotics or topical probiotics or vaginal suppositories to the vagina in the past one month before vaginal sampling.
Any one of the following cases should be eliminated
Samples with incomplete data or data that cannot be traced back to the source.
Unqualified samples, including those containing blood, insufficient sample size, and those that are not stored properly as required.
Volunteers who are asked to withdraw from the study halfway through.
Duplicate samples from the same volunteer.
The samples that researchers deem to be eliminated from the studyconsidering serious violations against clinical research trials program, failure to obtain observation data, etc. Final decision should be made by major researchers, data administrators, and statisticians based on joint discussions.
Sample Size calculation
Previous studies reported that the HPV infection rate ranged from 23.5% to 50.7%[30-32] and the prevalence of 6-month persistent of HPV infection after baseline was 30.3%. Considering above high infection rates, the sample size is calculated by PASS 15.0 with the a 0.5% significance using two-sided statistical test, the confidence level of 0.9, and the drop-out rate of 3%. The calculated number of participants is 9766. In order to ease execution, the study will enroll a total of 10,000 volunteers from 50 hospitals.
Manage of data
The demographic information (name, age, etc.), clinical data (symptoms, signs, etc.), medical history, and lifestyle information will be collected at the baseline. The results of wet mount microscopy, enzymatic indicator detection, Nugent score, TCT, HPV genetype, other sexually transmitted infections and vaginal microbiota will be gathered at the baseline and follow-up. Results of clinical examination items charged by the research centers involved in this project are recorded by the corresponding clinicians or laboratory doctors. Finally, the principal of the research centers in this project will check the data, and then all the data will be sent to the Zhujiang hospital for analysis. Unqualified data will be corrected if possible otherwise will be removed because of inaccurate information. The impact of missing information will be fully considered and subject to special analysis.
An electronic data capture system was constructed for this project. Each center will upload the information collected to this system. Each principal of the research center has an account with this system. Every change in the patient’s information in the database will be recorded by the system.
The results of TCT, HPV genetype, sexually transmitted infections, vaginal microbiota and microecology-related indicators will be integrated. We will also reveal the baseline status of vaginal microbiota of healthy reproductive women and the correlation among HPV infection, cervical lesions and vaginal microbiota. The following factors will be analyzed:
I. The composition and community state types (CSTs) clustering of vaginal microbiota in healthy subjects.
II. The relationship between HPV infection and vaginal microbiota.
a. Comparison of alpha diversity, beta diversity, Lefse analysis and microbial network analysis between the HPV positive group and HPV negative group.
b. Comparison of alpha diversity, beta diversity, Lefse analysis and microbial network analysis between the HPV infection clearance group and HPV persistent infection group.
c. CST cluster analysis of vaginal microbiota of all volunteers, and heatmap will be used to show the correlation among CST cluster and HPV infection, pH, Nugent score, and non-HPV STDs
d. Calculation of the relative risk of persistent infection of women with different CSTs.
e. Spearman, Pearson or logistic regression analysis will be performed between vaginal microbiota and related indicators of vaginal microecology.
III. Characteristics of vaginal microbiota in subjects with different degrees of cervical lesions.
a. Comparison of alpha diversity, beta diversity, Lefse analysis and microbial network analysis among volunteers with different degrees of cervical lesions.
b. Spearman, Pearson correlation will be performed between different degrees of cervical lesions and related indicators of vaginal microecology.
c. Chi square test and linear regression will be used to analyze the relationship between the degree of cervical lesions and HPV infection state.
IV. Characteristics of vaginal microbiota in subjects with non-HPV STIs.
a. Analysis of alpha diversity, beta diversity, Lefse analysis and microbial network analysis among volunteers with non-HPV STIs.
b. CST cluster analysis of vaginal microbiota among volunteers with non-HPV STIs.
c. Chi square test and linear regression will be used to analyze the relationship between non-HPV STIs and HPV infection state.
V. Assessment of influential factors of persistent HPV infection.
Multivariate regression analysis will be used to calculate the OR of high-risk factors for the development of persistent HPV infection. The 95% confidence interval will be calculated. The receiver operating characteristic curve (ROC) will be used to determine the cutoff value with the best sensitivity and specificity for the prediction of persistent infection.
In this study, QIIME and R software will be used in bioinformatics and statistical analysis. T test, analysis of variance, nonparametric test and Chi square test and other methods will be used to evaluate the statistical differences among the groups. P < 0.05 is considered significant.
This study has registered in ClinicalTrials.gov (NCT04694495) as well as China Human Genetic Resources Management Office（2021SLCJ0955）due to large human derived sample sizes. Research has been approved by the Ethics Committee of Zhujiang Hospital (NO. 2020-KY-071-02). It also needs to be approved by the Ethics Committee of every participating center in this study. Every adverse event will be reported to the Ethics Committee in a timely manner. Every revision of the research protocol will be reported to the Ethics Committee. Patient information will be kept confidential throughout the study and the publication process.