Study design and setting

This randomized, placebo-controlled, double-blinded, single-site trial was conducted in a public STD clinic. Trial design and results are reported according to CONSORT guidelines. Participants attended 4 visits: enrollment and after 4, 12, and 24 weeks ( Figure 1 ). The study was approved by the Ohio State University Institutional Review Board on May 25, 2011 (protocol 2011H0089) and was registered at clinicaltrials.gov ( NCT01450462 ).

Flow diagram of screening, enrollment, follow-up, and analysis of study participants

BV , bacterial vaginosis.

Turner. Vitamin D and bacterial vaginosis. Am J Obstet Gynecol 2014 .

Recruitment, eligibility, and informed consent

All female patients received study information at registration. Women who expressed interest were screened for self-reported eligibility criteria. Eligible women provided written consent.

Eligible women were 18-50 years old; premenopausal; had at least 1 ovary; spoke English; and were BV-positive on clinical examination by modified Amsel criteria (vaginal pH >4.5; thin, homogenous discharge; and positive “whiff” test). Excluded women were pregnant (by report or rapid urine test) or planning pregnancy within 6 months; breast-feeding; currently menstruating; or self-reporting kidney disease or kidney stones, hypercalcemia, hypercalciuria, sarcoidosis, histoplasmosis, thyroid disease, lymphoma, or tuberculosis. Concomitant infection with HIV or other STDs was not an exclusion criterion.

Treatment

All participants received standard BV therapy at no cost: 500 mg of metronidazole orally twice daily for 7 days. Women also received no-cost treatment for other infections diagnosed at enrollment according to normal clinic procedures.

Randomization

Using a permuted block design developed using software (SAS, version 9.2; SAS Institute Inc, Cary, NC), participants were randomized into 4 equal-sized treatment arms labeled by color. Two arms received vitamin D and 2 received placebo. The code linking which color corresponded to which product was not known to participants, staff, investigators, or data analysts. Randomization assignments were placed in individual opaque envelopes, each labeled by participant identification number (PIN). Sealed envelopes were stored in PIN order in a locked cabinet. At enrollment, each participant, together with staff, opened the envelope corresponding to the next available PIN to reveal her color group assignment.

Intervention

Women received their assigned study product in prefilled pill boxes. Women in the vitamin D groups received 9 capsules, each containing 50,000 IU of vitamin D ₃ (cholecalciferol) (BioTech Pharmacal, Fayetteville, AR) and women in the control groups received 9 placebo capsules with identical appearance to the vitamin D. Women were instructed to take the capsules 1, 2, 3, 4, 8, 12, 16, 20, and 24 weeks after enrollment. Participants received a pill schedule at enrollment, a duplicate schedule via post 1 week later, and text message reminders each day a capsule was to be taken. The target date for each capsule was also written in permanent marker directly on pill compartments. Women who reported at enrollment that they regularly took vitamin D supplements were requested to stop for the duration of the trial.

Specimen collection, processing, and testing

At the enrollment and 24-week visits, women underwent comprehensive clinical examinations with collection of blood and vaginal and cervical swabs for STD and reproductive tract infection testing. Chlamydia and gonorrhea were diagnosed by nucleic acid amplification testing; syphilis through rapid plasma reagin testing and confirmed by Treponema pallidum particle agglutination assay; HIV by rapid testing on plasma; trichomoniasis by microscopy and culture; and yeast by microscopy. Total serum calcium was assessed within 7 days of enrollment, and women with levels above the normal range–for whom vitamin D supplementation could be unsafe–were unenrolled. At the 4- and 12-week visits, women self-collected vaginal swabs, which were used to create slides of vaginal material. Slides from all 4 visits were stored and underwent Gram staining and Nugent scoring for BV detection after trial conclusion; technicians scoring the slides were blinded to treatment assignment. Stored serum from each visit was used to quantify 25(OH)D at the end of the trial, using the Liaison 25 OH vitamin D total assay (DiaSorin, Saluggia, Italy). Pregnancy was assessed via urine testing at each visit. Women found to be pregnant remained in the study but had their assigned study product discontinued immediately.

Questionnaire

At enrollment and each follow-up visit, women underwent face-to-face interviews to collect data on demographics, sexual behavior, adherence, and other information. Women were asked about 19 individual side effects previously associated with vitamin D supplementation or metronidazole treatment. Endorsement of any side effect was recorded as an adverse event (AE).

Safety

Participants’ serum calcium levels were checked at enrollment, 4 weeks, and 24 weeks. Safety was further monitored by an independent safety committee, which reviewed AEs by blinded study arm after half the anticipated person-time for the trial had been accrued.

Statistical analysis

All analyses were conducted using software (SAS Institute). We first examined changes in serum 25(OH)D levels by randomization group over time. Next, we examined simple BV prevalence at each visit, by randomization group. Nugent score of 7-10 on Gram-stained vaginal smear was interpreted as BV. We estimated BV-free survival time using the Kaplan-Meier method. We tested the homogeneity of survival functions across randomization groups using the log rank test. Our primary intention-to-treat analysis used Cox proportional hazards models to estimate the hazard ratio (HR) and 95% confidence interval (CI) for the effect of randomization assignment on time to BV recurrence. Our secondary analysis used extended Cox proportional hazard models, and modeled the effect of time-varying 25(OH)D level (rather than randomization assignment) on time to BV recurrence; that analysis also controlled for age and race. In all Cox models, women who missed a visit but returned for later visits were assumed to have remained BV-negative during the missing visit.

We performed 3 exploratory sensitivity analyses. First, using a threshold of α = 0.10 to classify imbalance in participant characteristics at enrollment, we ran an adjusted Cox model controlling for those factors which, due to chance, were not evenly balanced by randomization. Second, we limited the analysis to women who had 25(OH)D levels <20 ng/mL (the IOM threshold for vitamin D deficiency) at enrollment, to determine whether the effect of vitamin D supplementation on BV recurrence was different for this subgroup. Third, we assessed the impact of using Amsel criteria to diagnosis BV at enrollment, by restricting the analysis population to women also determined to be BV-positive at enrollment by Nugent scoring.

Power

We anticipated 20% loss to follow-up and BV recurrence in the placebo arm of 50% within 24 weeks. Under these assumptions, with α = 0.05, we estimated that 120 women (60 per group) would yield 80% power to detect an absolute difference in BV recurrence of 24%, comparing the vitamin D and placebo arms.

Screening

From September 2011 through January 2013, 285 women underwent screening and 85 were ineligible for at least 1 reason ( Figure 1 ). Of 200 women eligible by self-reported criteria, 128 were BV-positive by modified Amsel criteria and 126 enrolled. Following randomization, 1 woman began acting erratically and was immediately unenrolled. Seven women (6%) had abnormal serum calcium levels and were unenrolled. The final sample included 59 women randomized to vitamin D and 59 randomized to placebo. In all, 97 participants (82%) returned after enrollment: 51 (86%) in the vitamin D arm and 46 (78%) in the placebo arm returned after 4 weeks, and 38 women (64%) in the vitamin D arm and 40 control women (68%) returned after 24 weeks ( Figure 1 ). Among returning participants, median follow-up was 24.0 weeks in the vitamin D arm and 24.8 weeks in the control arm.

Participant characteristics at enrollment

Most participants (n = 87; 74%) were black, with a median age of 26 years ( Table 1 ). Women’s median lifetime number of male sex partners was 10 (interquartile range [IQR], 5–20 partners). A large minority (n = 48; 41%) reported lifetime experience with anal sex, and 32 (27%) reported sex with women. All women were BV-positive by modified Amsel criteria, and when vaginal smears from enrollment were Nugent scored at the end of the trial, 80% of the sample was found to be BV-positive at enrollment by Nugent score ( Table 1 ).

In all, 91 women (77%) reported a past BV diagnosis before enrollment. Several participants tested positive for STDs at enrollment ( Table 1 ), but only trichomoniasis varied significantly by randomization group ( P = .05).

Participant characteristics were generally balanced between randomization groups, but we also observed some important differences. Women randomized to the control arm were more likely to report concurrent partnerships in the last 3 months ( P = .03). These women were also somewhat less likely than women in the vitamin D arm to be employed ( P = .09) and to have ever douched ( P = .09). Control women had somewhat fewer lifetime male sexual partners than women randomized to vitamin D ( P = .10). Among women with main partners, control women reported significantly younger partners than women in the vitamin D arm ( P = .01) ( Table 1 ).

We also compared the characteristics of women who completed the trial (n = 78) with those who failed to complete the final visit (n = 40). Very few differences were observed ( Table 2 ). Women who completed the trial were somewhat more likely to have ever douched ( P = .07), and they also reported higher median numbers of lifetime male partners ( P = .03) and somewhat higher median numbers of sex acts with men in the last 3 months ( P = .09).

Vitamin D levels

Presupplementation vitamin D levels were similar: women randomized to the vitamin D arm had median 25(OH)D of 16.6 ng/mL (IQR, 12.1–21.2 ng/mL), vs 15.8 ng/mL (IQR, 11.8–23.3 ng/mL) for control women. Vitamin D deficiency at enrollment [25(OH)D <20 ng/mL] was present in 71% of women randomized to vitamin D and 68% of control women. Throughout follow-up, women in the vitamin D arm had significantly higher median vitamin D levels than control women ( Figure 2 ). By the final visit, the median 25(OH)D level in the vitamin D arm was 30.5 ng/mL, vs 17.8 ng/mL in the control arm.

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