Background
Bacterial vaginosis, a highly prevalent vaginal condition, is correlated with many adverse reproductive outcomes. In some studies, low vitamin D status (measured as serum 25-hydroxyvitamin D, 25[OH]D) has been associated with increased prevalence of bacterial vaginosis.
Objectives
We examined the cross-sectional association between vitamin D status and prevalence of bacterial vaginosis, separately for pregnant and nonpregnant women. Using prospectively collected data, we also characterized the effect of time-varying vitamin D status on incident bacterial vaginosis.
Study Design
We quantified 25(OH)D in stored sera collected quarterly from 571 Zimbabwean women participating in the Hormonal Contraception and Risk of HIV Acquisition Study. The analysis was restricted to women not using hormonal contraception. We characterized associations between vitamin D insufficiency (defined as 25[OH]D ≤ 30 ng/mL vs > 30 ng/mL) and prevalence of bacterial vaginosis among nonpregnant women at the enrollment visit and among pregnant women at the first follow-up visit that pregnancy was detected. Among women who were negative for bacterial vaginosis at enrollment (n = 380), we also assessed the effect of time-varying vitamin D status on incident bacterial vaginosis. We used the Liaison 25(OH)D total assay to measure 25(OH)D. Bacterial vaginosis was diagnosed via Nugent score.
Results
At enrollment, the prevalence of bacterial vaginosis was 31% and overall median 25(OH)D was 29.80 ng/mL (interquartile range, 24.70–34.30 ng/mL): 29.75 ng/mL (interquartile range, 25.15–33.95 ng/mL) among women with bacterial vaginosis, and 29.90 ng/mL (interquartile range, 24.70–34.50 ng/mL) among women without bacterial vaginosis. Among pregnant women, the prevalence of bacterial vaginosis was 27% and overall median 25(OH)D was 29.90 ng/mL (interquartile range, 24.10–34.00 ng/mL): 30.80 ng/mL (interquartile range, 26.10–36.90 ng/mL) among women with bacterial vaginosis, and 29.10 ng/mL (interquartile range, 23.80–33.45 ng/mL) among women without bacterial vaginosis. Vitamin D levels ≤ 30 ng/mL were not associated with a prevalence of bacterial vaginosis in nonpregnant women (adjusted prevalence ratio, 1.04; 95% confidence interval, 0.81–1.34) or pregnant women (adjusted prevalence ratio, 0.88, 95% confidence interval, 0.51–1.54). Vitamin D levels ≤ 30 ng/mL were similarly not associated with incident bacterial vaginosis (adjusted hazard ratio, 0.98, 95% confidence interval, 0.73–1.31). Our findings were robust to alternative specifications of vitamin D status including using a cut point for vitamin D deficiency of < 20 ng/mL vs ≥ 20 ng/mL and modeling 25(OH)D as a continuous variable.
Conclusion
Among reproductive-age Zimbabwean women, insufficient vitamin D was not associated with increased bacterial vaginosis prevalence or incidence. Given established associations between bacterial vaginosis and poor reproductive outcomes, identification of factors leading to high bacterial vaginosis prevalence is urgently needed.
Bacterial vaginosis (BV), a condition characterized by a reduction in the vaginal lactobacilli and overgrowth of largely anaerobic microorganisms, affects nearly 1 in 3 women worldwide. BV is an extremely important determinant of women’s health and is linked to spontaneous abortion, premature delivery, increased human immunodeficiency virus (HIV) transmission, and other reproductive morbidities.
The causes of BV are not clear. This condition occurs more often in women with more sex partners and higher coital frequency, new partners, female partners, unprotected sex, herpes simplex virus type 2 (HSV-2) infection, and some vaginal hygiene practices. In the United States, a persistent association exists between race and BV, even after an adjustment for other BV risk factors : in nationally representative data, 52% of black women vs 23% of white women had prevalent BV.
The strong correlation between race and BV suggests that a factor more common in black women is also a risk factor for BV. We hypothesized that vitamin D insufficiency could explain this racial disparity. Vitamin D stimulates mechanisms associated with pathogen elimination. Like BV, vitamin D insufficiency is significantly more prevalent among black women than white women. Racial differences in vitamin D status are thought to stem from evolutionary adjustments to different levels of sun exposure and reflect corresponding variations in vitamin D metabolism, vitamin D receptor polymorphisms and signaling.
Evaluation of the association between vitamin D levels, measured as serum 25-hydroxyvitamin D, or 25(OH)D, and BV prevalence has been limited. Some cross-sectional studies in pregnant women report significant links between low vitamin D and increased BV prevalence, whereas others, largely in nonpregnant women, find no link. (Low value varies by study, defined as 25[OH]D < 30 ng/mL or < 20 ng/mL. )
Prospective evaluations of the vitamin D-BV relationship are less common. Three randomized trials of vitamin D supplementation reported no difference in BV prevalence or recurrence between women who received, or did not receive, vitamin D supplementation. In contrast, a vitamin D supplementation trial of women with asymptomatic BV reported significantly improved BV cure rates among those receiving vitamin D.
Although a number of studies have reported on the associations between vitamin D and BV among black women in the United States, none to date have focused on African women. Studies from the region report BV a prevalence between 30% and 40%, and some higher than 50%, confirming the high burden of disease among African women. To fill this gap in the existing literature, we present an ancillary study of the Hormonal Contraception and Risk of HIV Acquisition (HC-HIV) study. Among reproductive-age, urban-dwelling women in Zimbabwe, we characterized the association between vitamin D status and BV prevalence (separately for nonpregnant and pregnant women) and time-varying vitamin D status and BV incidence.
Materials and Methods
Study design, setting, and population
The HC-HIV Study was a prospective cohort study conducted from 1999 through 2004 in Zimbabwe, Uganda, and Thailand, which assessed the effect of hormonal contraception on women’s risk of HIV acquisition. Women eligible for HC-HIV were recruited from family-planning and general health care clinics and were aged 18–35 years, HIV negative, nonpregnant, and sexually active.
HC-HIV participants were enrolled in 3 approximately equal-sized contraceptive groups: oral contraceptive pills, injectable depot medroxyprogesterone acetate, and nonhormonal or no contraception. The contraceptive group was not randomized. Women returned quarterly for up to 2 years to assess time-varying contraceptive use and HIV status.
In the present analysis, because hormonal contraception affects BV risk, we included only women using nonhormonal or no contraception. For efficiency, we restricted the sample to Zimbabwean women only. This project quantified vitamin D levels in serum frozen approximately a decade earlier; levels of 25(OH)D have been shown to be stable in frozen sera.
Data collection and assessment of laboratory outcomes
At all study visits, time-varying demographic and sexual behavior data were captured through interviews. Participants received examinations with specimen collection and testing for sexually transmitted and reproductive tract infections, including BV (by both criteria reported by Amsel et al and Nugent scoring ), yeast, HIV, chlamydia, gonorrhea, trichomoniasis, syphilis, and HSV-2. Serum aliquots from each visit were frozen at –80°C. For this analysis, we quantified 25(OH)D in sera using the Liaison 25 OH vitamin D total assay (DiaSorin, Saluggia, Italy). According to current US guidelines, 25(OH)D < 20 ng/mL is considered deficient, 20–30 ng/mL is insufficient, and > 30 ng/mL is adequate.
Ethical approval
Women in the HC-HIV study provided written informed consent; the form specified participants’ approval to store sera for future research. HC-HIV was approved by ethics committees at collaborating institutions in the United States and Zimbabwe. This ancillary study was approved by the Joint Research Ethics Committee at the University of Zimbabwe July 15, 2011 (JREC/192/11), by the Medical Research Council of Zimbabwe on Sept. 30 2011 (MRCZ/A/1635), and by the Ohio State University Institutional Review Board on March 13, 2012 (2011H0265).
Statistical analysis
Statistical analyses were performed using SAS (version 9.3; SAS Institute, Cary, NC). For our primary analyses, we constructed a binary variable dichotomizing vitamin D into deficient/insufficient (25[OH]D ≤ 30 ng/mL) vs adequate (> 30 ng/mL). The primary outcome was BV by Nugent score, dichotomized as BV negative (Nugent score 0–6) vs BV positive (Nugent score 7–10).
Slides of vaginal material were created at the time of physical examination and stored; microscopists performing Nugent scoring were trained and had their readings validated before a large-scale reading at the conclusion of the study. Batches of 25–100 Gram-stained slides were shipped to the University of California, San Francisco, Chlamydia/Virology Research Laboratory regularly throughout the slide-reading period for external quality control. For the small number of visits in which the Nugent score was missing (3%), we used Amsel criteria to classify BV status. To be BV positive by Amsel criteria, 3 of 4 criteria must be met: vaginal pH > 4.5; characteristic vaginal discharge; clue cells on wet mount; and positive whiff test with the addition of KOH to a swab of vaginal material.
To determine whether vitamin D was associated with prevalent BV in nonpregnant women, we examined data from the HC-HIV enrollment visit. We constructed a log-binomial regression model with a generalized estimating approach to produce an unadjusted prevalence ratio (PR) for the association between deficient/insufficient vitamin D (25[OH]D ≤ 30 ng/mL) and BV prevalence in nonpregnant women. We selected log-binomial models because BV prevalence was above 10%, the cutoff value above which logistic regression is not recommended. We obtained the adjusted PR (aPR) using a model that controlled for demographics and previously identified correlates of BV (age, education, parity, partner circumcision status, sexual frequency, vaginal hygiene practices, condom use, and HSV-2 serostatus).
We repeated this analysis to assess whether vitamin D was associated with prevalent BV in pregnant women. Pregnant women were not eligible to enroll in HC-HIV, but for each woman who became pregnant during follow-up, we measured vitamin D and BV at the first visit that pregnancy was detected. As in the previous text, we constructed log-binomial models to characterize the unadjusted and adjusted associations between deficient/insufficient vitamin D status and BV prevalence in pregnant women.
To examine the prospective effect of time-varying vitamin D status on BV incidence, we constructed an analysis data set of women who were BV negative at enrollment and had valid 25(OH)D and BV data during follow-up visits (n = 380). Women were censored after the first visit when BV was detected or the last visit in the study for women who remained BV free throughout follow-up. We specified Cox proportional hazards models using continuous time (measured in days) and robust variance estimation to estimate hazard ratios (HRs) comparing BV incidence among women with deficient/insufficient (25[OH]D ≤ 30 ng/mL) vs adequate (> 30 ng/mL) vitamin D status.
For all analyses, we used the serum vitamin D measure from the same visit when BV status was assessed. Because vitamin D levels change slowly over time in the absence of supplementation, vitamin D status at a given visit is an acceptable representation of women’s vitamin D status at the time that BV (if present) was developing.
Sensitivity analyses
We undertook several sensitivity analyses to evaluate the robustness of our findings. First, we repeated both the prevalence and incidence analyses using a lower cut point for vitamin D: 25(OH)D < 20 ng/mL. Second, we repeated both analyses using 25(OH)D as a continuous variable. (We also examined the association between vitamin D and BV using splines but observed no change in the outcomes or fit of the models, so we present only the results from the continuous coding of vitamin D.) Third, for BV incidence analyses, we examined the effect of baseline Nugent score on the association between vitamin D and BV incidence. Whereas in the primary analysis women with normal vaginal flora at enrollment (Nugent score 0–3) and women with intermediate flora (Nugent 4–6) were both coded BV negative, this sensitivity analysis examined the effect of time-varying vitamin D on BV incidence among these 2 groups separately.
Results
Sample characteristics
Of the Zimbabwean HC-HIV participants (n = 2296), 640 (28%) were using nonhormonal or no contraception at enrollment. When we further restricted the sample to women who had valid BV results from the enrollment visit and retrievable serum for vitamin D testing, 571 women (89% of eligible participants) were included in this ancillary analysis. Included women were similar to excluded women in all assessed characteristics, except that included women were less likely to have chlamydial ( P < .001) or gonococcal infection ( P = .004) at enrollment.
We observed no significant differences between women by vitamin D status at enrollment ( Table 1 ). Median age was 26 years (interquartile range [IQR], 22–30 years). A third of women (31%) had BV. The prevalence of other infections at enrollment was low, except for HSV-2 (57% seropositivity). The median number of lifetime male partners was 1 (IQR, 1–2) and the median number of monthly sex acts was 12 (IQR, 8–21). Half of women (49%) had adequate vitamin D levels (> 30 ng/mL) at enrollment. The median vitamin D level at enrollment was 29.80 ng/mL (IQR, 24.70–34.30 ng/mL): 29.75 ng/mL (IQR, 25.15–33.95 ng/mL) among women with BV and 29.90 ng/mL (IQR, 24.70–34.50 ng/mL) among women without BV.
| Characteristic | All women (n = 571) | Deficient/insufficient vitamin D b (n = 294) | Adequate vitamin D c (n = 277) | P value d |
|---|---|---|---|---|
| n (%) | n (%) | n (%) | ||
| Marital status | .64 | |||
| Single | 18 (3.2) | 11 (3.7) | 7 (2.5) | |
| Married | 499 (87.4) | 257 (87.4) | 242 (87.4) | |
| Divorced/widowed | 54 (9.5) | 26 (8.8) | 28 (10.1) | |
| Employed | 278 (48.7) | 154 (52.4) | 124 (44.8) | .07 |
| Infections detected | ||||
| Bacterial vaginosis e | 176 (30.8) | 93 (31.6) | 83 (30.0) | .67 |
| Normal flora (Nugent 0–3) | 254 (44.5) | 128 (43.5) | 126 (46.5) | |
| Intermediate flora (Nugent 4–6) | 128 (22.4) | 66 (22.5) | 62 (22.4) | |
| BV (Nugent 7–10) | 171 (30.0) | 89 (30.3) | 82 (39.6) | .94 |
| Missing Nugent score | 18 (3.2) | 11 (3.7) | 7 (2.5) | |
| Candidiasis | 87 (15.2) | 41 (14.0) | 46 (16.6) | 0.42 |
| Chlamydia | 2 (0.4) | 1 (0.3) | 1 (0.4) | 1.00 |
| Gonorrhea | 13 (2.3) | 3 (1.0) | 10 (3.7) | 0.05 |
| Trichomoniasis | 34 (6.0) | 13 (4.4) | 21 (7.6) | 0.16 |
| Syphilis | 14 (2.5) | 7 (2.4) | 7 (2.5) | 1.00 |
| HSV-2 seroprevalence | 324 (56.7) | 161 (54.8) | 163 (58.8) | 0.33 |
| Condom use f | 0.77 | |||
| Always | 382 (67.3) | 200 (68.3) | 182 (66.2) | |
| Sometimes | 84 (14.8) | 38 (13.0) | 46 (16.7) | |
| Never | 102 (18.0) | 55 (18.8) | 47 (17.1) | |
| Ever engaged in commercial sex g | 3 (0.5) | 1 (0.3) | 2 (0.7) | 0.61 |
| Any vaginal practice other than using water for cleaning g | 137 (24.0) | 70 (23.8) | 67 (24.2) | .92 |
| Male partner circumcision status | .96 | |||
| Circumcised | 74 (13.0) | 39 (13.4) | 35 (12.6) | |
| Uncircumcised | 437 (76.8) | 223 (76.4) | 214 (77.3) | |
| Do not know | 58 (10.2) | 30 (10.3) | 28 (10.1) | |
| Primary partner risk f , h | 219 (38.4) | 118 (40.1) | 101 (36.5) | .37 |
| Characteristic | Median (IQR) | Median (IQR) | Median (IQR) | P value d |
|---|---|---|---|---|
| Age, y | 26 (22–30) | 26 (22–30) | 26 (22–29) | .62 |
| Education, y | 10 (9–11) | 10 (9–11) | 10 (9–11) | 1.00 |
| Number of male sex partners, lifetime | 1 (1–2) | 1 (1–2) | 1 (1–2) | .47 |
| Number of male sex partners f | 1 (1–1) | 1 (1–1) | 1 (1–1) | .46 |
| Number of live births, lifetime | 2 (1–3) | 2 (1–3) | 2 (1–3) | .19 |
| Number of monthly sex acts f | 12 (8–21) | 12 (8–23) | 12 (8-20) | .91 |
| Vitamin D (25[OH]D) | 29.8 (24.7–34.3) | 24.9 (21.4–27.5) | 34.5 (31.9–38.7) | < .0001 |
a Pregnancy at enrollment was an exclusion criterion, so all women in Table 1 were nonpregnant
b Deficient/insufficient vitamin D was 25(OH)D ≤ 30 ng/mL
c Adequate vitamin D was 25(OH)D > 30 ng/mL
d χ 2 , Fisher exact, or Cochran-Mantel-Haenszel tests were used for categorical variables, and a Wilcoxon-Mann-Whitney test was used for continuous variables
e BV was assessed primarily by Nugent score. For the 3% of visits in which the Nugent score was missing, BV was classified using the criteria of Amsel
f In a typical month in the last 3 months
h Primary partner risk is a composite variable that indicates that the participant’s primary partner is HIV positive or has abnormal discharge from the penis, or has weight loss, or has spent nights away from the home, or that the partner has sex with other women.
We also examined characteristics of the subset of participants who became pregnant during the study, overall and by vitamin D status ( Table 2 ). The general patterns observed in pregnant women were similar to the patterns seen in the overall population. BV prevalence was 27%, and overall median 25(OH)D was 29.90 ng/mL (IQR, 24.10–34.00 ng/mL): 30.80 ng/mL (IQR, 26.10–36.90 ng/mL) among women with BV and 29.10 ng/mL (IQR, 23.80–33.45 ng/mL) among women without BV. Neither gestational age nor pregnancy outcomes were captured in the HC-HIV data.
| Characteristic | All pregnant women (n = 141) | Deficient/Insufficient Vitamin D a (n = 73) | Adequate Vitamin D b (n = 68) | P value c |
|---|---|---|---|---|
| n (%) | n (%) | n (%) | ||
| Marital status | .90 | |||
| Single | 3 (2.1) | 1 (1.4) | 2 (2.9) | |
| Married | 128 (90.8) | 67 (91.8) | 61 (89.7) | |
| Divorced/widowed | 10 (7.1) | 5 (6.9) | 5 (7.4) | |
| Employed | 65 (46.1) | 30 (41.1) | 35 (51.5) | .22 |
| Infections detected | ||||
| Normal flora (Nugent 0–3) | 71 (53.0) | 35 (51.5) | 36 (54.6) | |
| Intermediate flora (Nugent 4–6) | 25 (18.7) | 15 (22.1) | 10 (15.2) | .52 |
| BV (Nugent 7–10) | 38 (27.0) | 18 (24.7) | 20 (29.4) | |
| Candidiasis | 31 (22.0) | 20 (27.4) | 11 (16.2) | .11 |
| Chlamydia | 1 (0.7) | 0 (0.0) | 1 (1.5) | .48 |
| Gonorrhea | 3 (2.2) | 2 (2.8) | 1 (1.5) | 1.00 |
| Trichomoniasis | 7 (5.0) | 4 (5.5) | 3 (4.5) | 1.00 |
| Syphilis | 5 (7.1) | 1 (2.9) | 4 (11.1) | .36 |
| HSV-2 seroprevalence | 92 (65.3) | 47 (64.4) | 45 (66.2) | .82 |
| Use of condoms d | .12 | |||
| Always | 40 (28.8) | 16 (21.9) | 24 (36.4) | |
| Sometimes | 59 (42.5) | 32 (43.8) | 27 (40.9) | |
| Never | 40 (28.8) | 25 (34.3) | 15 (22.7) | |
| Commercial sex e | 0 (0) | 0 (0) | 0 (0) | N/A |
| Any vaginal practice other than using water for cleaning e | 46 (32.6) | 24 (32.88) | 22 (32.4) | .95 |
| Partner circumcision status | .99 | |||
| Circumcised | 18 (13.1) | 9 (12.9) | 9 (13.4) | |
| Uncircumcised | 107 (78.1) | 55 (78.6) | 52 (77.6) | |
| Do not know | 12 (8.8) | 6 (8.6) | 6 (9.0) | |
| Primary partner risk d , f | 56 (39.7) | 27 (37.0) | 29 (42.7) | .49 |
| Inconsistent condom use d | 99 (70.21) | 57 (78.08) | 42 (61.76) | .03 |
| Median (IQR) | Median (IQR) | Median (IQR) | P value c | |
|---|---|---|---|---|
| Age, y | 25 (22–28) | 25 (21–28) | 26 (22–28.5) | .28 |
| Education, y | 10 (9–11) | 10 (8–11) | 10 (9–11) | .91 |
| Number of male sex partners, lifetime | 1 (1–2) | 1 (1–2) | 1 (1–2) | .93 |
| Number of male sex partners d | 1 (1–1) | 1 (1–1) | 1 (1–1) | .31 |
| Number of live birth, lifetime | 2 (1–3) | 2 (1–3) | 2 (1–3) | .54 |
| Number of monthly sex acts d | 14 (8–22) | 15 (9–24) | 12 (8–20) | .26 |
| Vitamin D (25[OH]D | 29.7 (25.3–34.4) | 25.5 (22.6–27.6) | 34.7 (32.1–39.7) | < .0001 |
a Deficient/insufficient vitamin D was 25(OH)D ≤ 30 ng/mL
b Adequate vitamin D was 25(OH)D > 30 ng/mL
c χ 2 , Fisher exact, or Cochran-Mantel-Haenszel tests were used for categorical variables and a Wilcoxon-Mann-Whitney test was used for continuous variables
d In a typical month in the last 3 months
f Primary partner risk is a composite variable that indicates that the participant’s primary partner is HIV positive, or has abnormal discharge from the penis, or has weight loss, or has spent nights away from the home, or that the partner has sex with other women.
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