Association between bacterial vaginosis and 25-Hydroxy vitamin D: a case-control study | BMC Infectious Diseases

Study design

This case-control study was conducted in North-East Iran in 2021. The study report was prepared based on the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) Statement checklist for case-control studies (2010) [19].

Setting and participants

A total of 250 married women (125 in the BV group and 125 in the control groups) who were referred to the Bohlol Hospital, private obstetrics and gynecology clinics, and Integrative Health Centers in Gonabad city participated in the current study. The BV group included married women with complaints of vaginal discharge who were suspected of BV. The control group was chosen from women referring to the centers for routine obstetrics and gynecology evaluation, including preconception counseling, contraception, breast or cervical cancer screening, and midlife care. The BV and control groups were matched based on age (maximum difference of 3 years) and frequency of intercourse (maximum difference of 2 days per week) with a matching ratio of 1:1.

Inclusion criteria for the case group were as follows: [1] Not being pregnant, [2] Not suffering from underlying diseases, including diabetes and immunodeficiency, [3] Not taking immunosuppressive drugs, [4] Willingness to participate in the study, and [5] Being suspected to have BV due to vaginal discharge and subsequent confirmation of BV based on laboratory tests.

Inclusion criteria for the control group were similar to the BV group except for complaining of vaginal discharge and BV suspicion. The exclusion criterion was refusing to participate in the study.

Measurements

The data were collected using a questionnaire including several items about demographic and reproductive characteristics and predisposing factors for BV. Demographic data included participant’s age, spousal age, educational level, spousal educational level, income level, occupation, cigarette/hookah smoking, other questionnaire items included vitamin D supplement consumption, vaginal douching, pregnancy history, delivery history, abortion history, history of premature delivery, history of taking medication for vaginitis, history of cryotherapy for vaginitis, history for cauterization for vaginitis, dairy consumption, menstruation status, family planning method, age at first sexual intercourse, duration of sunlight exposure, frequency of sexual intercourse per week, and serum vitamin D level. Body Mass Index (BMI) was calculated as weight/height2 (kg/m2) and divided into four categories, underweight defined as BMI < 18.5 kg/m2, normal-weight defined as BMI 18.5–24.9 kg/m2, overweight defined as BMI 25.0-29.9 kg/m2, and obese defined as BMI ≥ 30.0 kg/m2, based on the World Health Organization (WHO) criteria [20]. A checklist was also filled for each participant and data regarding the findings of the Whiff test, serum level of 25 hydroxy vitamin D, litmus paper evaluation, and the findings of microscopic evaluation (reporting clue cells) were collected.

Diagnosis of BV

Vaginal samples were obtained from all participants to evaluate BV. Inspection of the physical appearance of the vagina and vaginal discharge was performed using a sterile speculum. The physical appearance characteristics of vaginal discharge, including shape, consistency, and color, were recorded in the study checklist. Then two smears were obtained from the lateral vaginal wall using two sterile cotton swabs. One swab was used to prepare the smear and was sent to the laboratory, while the second swab was used to evaluate vaginal pH by applying the swab on litmus paper. The litmus paper would have a pink discoloration if BV presents. Whiff test was performed by applying a drop of potassium hydroxide to the vaginal smear slides, and the presence of amine odor was evaluated. Vaginal smears were evaluated for BV in the laboratory. Slides were stained and inspected using a light microscope at 100 X magnification to define the percentage of clue cells. Diagnosis of BV was made based on the presence of at least three out of four Amsel criteria, including homogenous vaginal discharge, vaginal pH greater than 4.5, positive Whiff test, and more than 20% clue cells in the microscopic evaluation of vaginal sample [21].

Evaluation of serum 25 hydroxy vitamin D

In order to evaluate serum 25 hydroxy vitamin D level, 0.5 ml venous blood was drawn from all participants. Serum 25 hydroxy vitamin D level was determined by 25(OH) vitamin D kit (Monobind Inc. USA) using ELISA method (Automated ELISA system, Dynex DS2, USA). Briefly, a standard curve is drawn by using the average optical absorption of the standards. Then, the vitamin D concentration of the sample is identified considering the average optical absorption of the sample and the standard curve. Two control serum with specific concentrations were used. According to the kit guideline, the criteria used to define vitamin D deficiency were as follows: serum vitamin D < 10 ng/ml was defined as vitamin D deficiency, serum vitamin D between 10 and 30 ng/ml was defined as vitamin D insufficiency, serum vitamin D between 31 and 100 ng/ml was defined as vitamin D sufficiency, and serum vitamin D > 100 ng/ml was defined as vitamin D toxicity.

Sample size

The study sample size was calculated using the sample size equation for case-control studies [22] based on the reported prevalence of vitamin D deficiency (p = 0.6) from a previous study [23] and considering Type 1 error of 5%, Type 2 error of 20%, and odds ratio (OR) of 2.5. The sample size was increased by 25% based on the confounders. The final sample size was 125 participants in each group.

Statistical analysis

Qualitative variables were described using frequency and percentage. The normality of the quantitative variables was assessed using Kolmogorov–Smirnov test and skewness and kurtosis values. Normal quantitative variables were described using mean and standard deviation (SD) and non-normal quantitative variables were described using median and 1st and 3rd quartiles. The association between serum 25 hydroxy vitamin D level and BV status was evaluated using a conditional logistic regression model, preferred for matched case-control studies analysis, with adjustment for potential confounding variables. Variables with p-value higher than the cut-off value of 0.15 in simple conditional logistic regression were considered as confounders. False discovery rate (FDR) correction was performed by the Benjamin-Hochberg procedure to account for multiple comparisons using Seed-based d Mapping software (SDM, version 6.21, https://www.sdmproject.com). The statistical significance level was set as p-value < 0.05. For multiple comparison correction, the statistical level was set to FDR q-value < 0.10. The results were reported as odds ratios (ORs) and 95% confidence intervals (CIs) for OR. All statistical analyses were performed using the statistical package for social sciences (SPSS) software version 16 (SPSS Inc., Chicago, Ill., USA) and Stata software, version 12 (Stata Corp, College Station, Texas USA).

Ethical considerations

The current study was approved by the Ethical Committee of the Gonabad University of Medical Sciences (IR.GMU.REC.1398.150). All participants were informed about the purpose of the research and signed a written informed consent prior to participating in the study. The identity of the participants was kept confidential. All examinations and laboratory tests were free of charge. As an incentive, a free gynecologist visit was arranged for participants with documented BV. Furthermore, participants with vitamin D deficiency were treated.