Bacterial vaginosis (BV) is a condition of unknown etiology in which the usual Lactobacillus predominant vaginal bacteria are replaced with overgrowth of Gardnerella vaginalis and mixed anaerobic organisms.1 It is the most common cause of vaginal discharge in reproductive-aged women,2 and women with BV are at increased risk for preterm birth,3 postoperative gynecological infection,4 and acquisition of sexually transmitted diseases,5 including HIV.6 African American women have consistently been demonstrated to have increased BV prevalence compared with white women; the increase is not explained by differences in demographic characteristics, or by differences sexual or hygienic behaviors.7
Recently, reduced serum concentration of 25-hydroxyvitamin D, the standard marker of vitamin D status, has been associated with increased BV prevalence among pregnant women.8–11 However, in only 2 studies of vitamin D and BV have been conducted in nonpregnant women; one failed to find an association8 and the other found vitamin D deficiency to be associated with BV in HIV-positive but not HIV-negative women.12 Because vitamin D is important for immune function and deficiency has been associated with both immune disorders and chronic infection,13,14 an association between vitamin D deficiency and BV is plausible. Synthesis of pre–vitamin D in skin exposed to ultraviolet-B light is the major source of vitamin D in humans15 and due primarily to darker skin color, African American women have lower serum vitamin D concentrations than white women.16 Therefore, insufficient or deficient vitamin D may account for some of the increased BV prevalence seen among African American women. However, serum concentration of free vitamin D, the biologically available fraction, may not differ between African Americans and whites,17 calling into question the role of vitamin D in explaining the racial disparity in BV. Previous studies of the BV–vitamin D association have been cross sectional, in which both BV and vitamin D were assessed at a single point in time. Because serum vitamin D concentration is usually higher in the summer than the winter, if low vitamin D were a cause of BV, then BV ought to be more prevalent in the winter than the summer among women followed up longitudinally. This report describes the seasonality of BV prevalence among nonpregnant women followed up for 1 year.
This report is a secondary analysis of the Longitudinal Study of Vaginal Flora.18 Nonpregnant, 15- to 44-year-old women were recruited from August 1999 to February 2002 upon presentation for a routine health visit to 1 of 12 clinics in the Birmingham, Alabama area. Exclusion criteria were significant medical or gynecological conditions, receiving chronic antibiotics (daily for at least 30 days), planning to leave the area in the next 12 months, and inability to provide informed consent. The study was approved by the institutional review boards of the University of Alabama at Birmingham, the Jefferson County Health Department, and the Eunice Kennedy Shriver National Institute of Child Health and Human Development. All participants provided written informed consent. This secondary analysis of previously collected data was determined not to constitute human subjects research by the Nationwide Children’s Hospital Institutional Review Board.
Women were seen at a research clinic for an initial visit, and then for quarterly visits for up to a year of follow-up. At each visit, the woman was interviewed in a private office by a female interviewer. In addition to demographic factors, the interview included detailed questions on lifestyle, sexual and personal hygienic behaviors during the past 6 months for the initial interval and the past 3 months for subsequent interviews. Also, at each visit, the woman underwent a pelvic examination, at which time a cotton swab was obtained for vaginal Gram stain, which was evaluated according to the Nugent scoring system.19 Bacterial vaginosis was defined as a Nugent score of 7 to 10; 10% of Gram stained slides were reread in a different laboratory, with a high level of agreement for the presence of BV (κ = 0.81). Asymptomatic women who had BV by either Nugent19 or Amsel20 criteria were not routinely treated.
Data were analyzed with the case-crossover method.21 Seasons were defined as winter (December–February), spring (March–May), summer (June–August), and fall (September–November), with winter considered the referent category. The case-crossover design compares each woman’s BV status in spring, summer, and fall with her own status in winter using conditional logistic regression. Because each woman serves as her own control, the method inherently controls for factors that do not vary over time, such as genes, skin type, and most demographic characteristics. Only women who are discordant on the outcome—those who had BV on at least one, but not all visits—contribute to the analysis.21 Characteristics that may vary across the year, such as sexual, hygienic, and health-related behavior, are not inherently controlled by this approach. Therefore, the conditional logistic regression model included these confounding factors, in addition to season. Confounding factors were determined a priori and included vaginal and receptive oral sex frequencies, number of recent sex partners, having a recent new sex partner, current use of hormonal contraception, current douching frequency, type of menstrual protection (with amenorrhea as the referent category), preference for showering versus bathing, use of genital powder, towelettes and sprays, smoking, alcohol drinking, current weight, and receipt of metronidazole since the last study visit (or in the past 2 weeks for the first study visit). We assessed whether these characteristics varied significantly by season using conditional logistic regression (for binary factors) or by fixed-effect linear regression (for continuous factors). We assessed whether the season-BV association varied by race, or by BV status at study entry, by including a product interaction term between season and race (African American vs. all other races) and between season and initial BV status. All calculations were conducted using SAS 9.3 or STATA 12. A 2-tailed P value less than 0.05 was considered statistically significant, and no correction was made for multiple comparisons.22
The Longitudinal Study of Vaginal Flora enrolled 3620 women. This analysis was limited to women who were seen at least once in each season. There were 2337 such women: 714 never had BV at any visit, 288 had BV at all visits, and 1335 had BV at some but not all visits. Thus, the final sample comprised the 1335 women (contributing 6392 visits) whose BV status varied during the study.
The cohort was predominantly African American, low-income, and overweight to obese (Table 1). The frequency of BV at all visits, for all women and separately by BV status at the initial visit, is presented in Table 2. Among women BV negative at entry, there was no difference in BV by season, but among women BV positive at entry, BV was more common in the summer and fall than in winter or spring. The frequency of BV at the enrollment visit did not vary significantly by season of that visit (46.1% in winter, 46.1% in spring, 50.6% in summer, 48.0% in fall; P = 0.62). Dietary intake data23 were available at a single point in time from 909 of the 1335 women, and mean total vitamin D intake, from both diet and supplements, did not differ by season (188 IU in winter, 214 in spring, 192 in summer, 204 in fall; P = 0.47).
Table 3 describes the modest seasonal variation in confounding factors. Both vaginal sex frequency and number of recent sex partners were elevated in the summer. Women weighed more and reported more alcohol consumption at winter visits, but were less likely to have recently received metronidazole at summer visits. They were more likely to use powder on their genitals in the fall, and towlettes in the winter and spring. The prevalence of showering was highest in the summer. None of the other measured behaviors varied significantly by season.
Unadjusted and adjusted odds ratios for BV in each season are presented in Table 4. Among all women, compared with winter, the unadjusted odds ratios for BV were 0.9 (95% confidence interval, 0.8–1.1) in the spring, 1.2 (1.01–1.4) in the summer, and 1.1 (0.97–1.3) in the fall. However, the seasonal effect on BV differed significantly (P < 0.001) by whether the woman was BV negative or positive at study entry. Among women BV negative at entry, the odds ratios for BV, compared with winter, were 1.0 (0.8–1.2) in spring, 1.0 (0.8–1.2) in summer, and 0.9 (0.8–1.1) in fall; the overall association between season and BV was not significant (P = 0.81). However, among women BV positive at entry, the odds ratios were 0.9 (0.7–1.1) in spring, 1.4 (1.2–1.7) in summer, and 1.4 (1.1–1.7) in fall; the overall association between season and BV was highly significant among these women (P < 0.001).
Adjustment for sexual, health, and hygienic behaviors had little impact on the odds ratios. Among all women, BV remained most prevalent in the summer, least prevalent in spring, and intermediate in winter and fall. Among women who were BV negative at entry, season remained unassociated with BV; among women who were BV positive at entry, BV was most common in the summer and fall, least common in the spring, and intermediate in winter. In none of the models did the association between season and BV differ between African Americans and women of other races (all P values for interaction between race and season were >0.4).
This study, which compared the same women across all 4 seasons, did not find that BV was more common in the winter or spring months, when serum vitamin D is expected to be lowest. Rather, season was unassociated with BV among women who were BV negative at study entry, whereas among women who were BV positive at entry; BV was statistically significantly more common in the summer and fall, when serum vitamin D is expected to be highest. Adjustment for numerous sexual, hygienic and other factors that varied with season had minimal impact on the results. Our results suggest that vitamin D insufficiency is unlikely to explain increased BV risk.
Bacterial vaginosis has a high rate of relapse and remission.24,25 It is plausible that women with BV at enrollment are more susceptible to future BV episodes. Residual or unmeasured confounding by sexual, hygienic, or other behavioral factors (which seem to be somewhat more common during the same seasons when BV prevalence was highest), rather than vitamin D insufficiency, may be responsible for the variation in BV by season observed in these women. On the other hand, women who were BV negative at enrollment may have lower susceptibility to BV, such that the variation in BV-associated behaviors by season was not profound enough to lead to differences in BV prevalence by season. Alternatively, seasonal differences in BV by baseline BV status may be explained by seasonal differences in factors associated with BV incidence versus BV remission. Women who were BV negative and then became positive experienced incident BV; in contrast, women who were BV positive at enrollment and became negative during their follow-up experienced a remission. The etiology of BV is not well understood, and it is possible that different factors are associated with the development of BV and with conversion to healthy vaginal flora. However, we believe that in neither cohort (BV positive and BV negative at enrollment) do our results support a strong role for vitamin D insufficiency in the pathogenesis of BV.
Previous studies of the association of vitamin D with BV have had inconsistent results. Reduced serum vitamin D has been associated with increased BV prevalence in pregnant women.8–11 However, there have been few studies conducted in nonpregnant women. One did not find an association between vitamin D and BV,8 and the other found an association between reduced serum vitamin D and increased BV prevalence in HIV-positive but not HIV-negative women.12
Strengths of the study include a large number of women who were followed up over all 4 seasons, systematic assessment of BV, and collection of detailed data on individual behaviors that might confound a seasonal difference in BV. Use of the crossover design has the advantage of inherently controlling for many factors that are difficult to measure and could confound the association between season and BV, yet also brings the limitation that only women who changed BV status over the year were included in the analysis. The crossover design inherently excludes both women who had BV at every visit and women who never had BV at any visit; 43% of women who were seen in every season were not included in the analysis for this reason. Although not biasing per se, this limitation means that the associations in this report apply only to women who were intermittently BV positive.
A second limitation is that season was used as a proxy for vitamin D status, but no actual serum concentrations were measured. In temperate climates, serum vitamin D concentration is generally higher in the summer and fall than in the winter and early spring,26–29 but seasonal fluctuations in serum vitamin D may be less among African Americans than among whites.26,28,29 However, the seasonal changes we observed in BV prevalence were similar among African American and white women. Seasonal variation also may differ by latitude. At 42° latitude or higher, sun exposure in winter did not result in sufficient ultraviolet B radiation to produce previtamin D, but at 34° latitude or less (which includes Birmingham), exposed skin can produce previtamin D in both winter and summer.30 Therefore, the seasonal fluctuation in vitamin D may be less in the southern than in the northern United States.
Behavior may modify seasonal variation, particularly if people in hot climates limit their outdoor activities during summer days and increase them during winter days. Such behavior has been posited to explain a paradoxical seasonal variation in umbilical cord 25-hydroxyvitamin D concentration, a proxy for maternal third trimester concentration, in New Orleans.31 However, a cross-sectional study of umbilical cord serum 25-hydroxyvitamin D concentration in Charleston, SC, which is at comparable latitude to Birmingham, found significantly higher values among both white and African American women from April 1 to October 31 than from November 1 to March 31.29 Dietary changes also can blunt or even reverse the seasonal variation in serum vitamin D,32 although in our population, dietary vitamin D intake did not vary by season. Therefore, we believe that season is a good proxy for serum vitamin D in these predominantly African American women in Birmingham, Alabama.
A final limitation is that although we controlled for numerous factors that could vary by season and also might cause BV, there may be relevant factors that were not measured. Furthermore, adjustment for hygienic behaviors would be inappropriate if these behaviors were undertaken to alleviate symptoms of BV. However, adjustment for hygienic behaviors had little impact on the results. Because visits took place throughout the entire season and the questionnaire asked only about events since the last visit, the timing of behaviors might not correspond to the season of interview, particularly for women interviewed early in the season.
In summary, our findings that BV is more common in the summer than the winter, particularly among women who were BV positive at study entry, do not support an association between vitamin D, measured through the proxy variable of season, and BV. If vitamin D insufficiency is a risk factor for BV, our results suggest that its effect is slight or dwarfed by other, more proximal factors that vary by season. Future studies should assay serum vitamin D concentration in a group of women followed up across multiple seasons, as well as investigate the role of other factors that vary by season, such as physical activity, temperature, and daylight length. Answers to these questions may provide new insights into the enigmatic pathogenesis of BV.
1. Hill GB. The microbiology of bacterial vaginosis. Am J Obstet Gynecol 1993; 169: 450–454.
2. Eschenbach DA, Hillier S, Critchlow C, et al. Diagnosis and clinical manifestations of bacterial vaginosis. Am J Obstet Gynecol 1988; 158: 819–828.
3. Leitich H, Kiss H. Asymptomatic bacterial vaginosis and intermediate flora as risk factors for adverse pregnancy outcome. Best Pract Res Clin Obstet Gynaecol 2007; 21: 375–390.
4. Soper DE, Bump RC, Hurt WG. Bacterial vaginosis and trichomoniasis vaginitis are risk factors for cuff cellulitis after abdominal hysterectomy. Am J Obstet Gynecol 1990; 163: 1016–1021; discussion 1021–1023.
5. Brotman RM, Klebanoff MA, Nansel TR, et al. Bacterial vaginosis assessed by Gram stain and diminished colonization resistance to incident gonococcal, chlamydial, and trichomonal genital infection. J Infect Dis 2010; 202: 1907–1915.
6. Petrova MI, van den Broek M, Balzarini J, et al. Vaginal microbiota and its role in HIV transmission and infection. FEMS Microbiol Rev 2013; 37: 762–792.
7. Koumans EH, Sternberg M, Bruce C, et al. The prevalence of bacterial vaginosis in the United States, 2001–2004: Associations with symptoms, sexual behaviors, and reproductive health. Sex Transm Dis 2007; 34: 864–869.
8. Hensel KJ, Randis TM, Gelber SE, et al. Pregnancy-specific association of vitamin D deficiency and bacterial vaginosis. Am J Obstet Gynecol 2011; 204: e41–e49.
9. Dunlop AL, Taylor RN, Tangpricha V, et al. Maternal vitamin D, folate, and polyunsaturated fatty acid status and bacterial vaginosis during pregnancy. Infect Dis Obstet Gynecol 2011; 2011: 216217.
10. Bodnar LM, Krohn MA, Simhan HN. Maternal vitamin D deficiency is associated with bacterial vaginosis in the first trimester of pregnancy. J Nutr 2009; 139: 1157–1161.
11. Davis LM, Chang SC, Mancini J, et al. Vitamin D insufficiency is prevalent among pregnant African American adolescents. J Pediatr Adolesc Gynecol 2010; 23: 45–52.
12. French AL, Adeyemi OM, Agniel DM, et al. The association of HIV status with bacterial vaginosis and vitamin D in the United States. J Womens Health (Larchmt) 2011; 20: 1497–1503.
13. Liu PT, Stenger S, Tang DH, et al. Cutting edge: Vitamin D–mediated human antimicrobial activity against Mycobacterium tuberculosis
is dependent on the induction of cathelicidin. J Immunol 2007; 179: 2060–2063.
14. Hewison M. Vitamin D and innate immunity. Curr Opin Investig Drugs 2008; 9: 485–490.
15. Holick MF. Vitamin D: A d-lightful solution for health. J Investig Med 2011; 59: 872–880.
16. Ginde AA, Sullivan AF, Mansbach JM, et al. Vitamin D insufficiency in pregnant and nonpregnant women of childbearing age in the United States. Am J Obstet Gynecol 2010; 202: e431–e438.
17. Powe CE, Evans MK, Wenger J, et al. Vitamin D-binding protein and vitamin D status of black Americans and white Americans. N Engl J Med 2013; 369: 1991–2000.
18. Klebanoff MA, Schwebke JR, Zhang J, et al. Vulvovaginal symptoms in women with bacterial vaginosis. Obstet Gynecol 2004; 104: 267–272.
19. Nugent RP, Krohn MA, Hillier SL. Reliability of diagnosing bacterial vaginosis is improved by a standardized method of Gram stain interpretation. J Clin Microbiol 1991; 29: 297–301.
20. Amsel R, Totten PA, Spiegel CA, et al. Nonspecific vaginitis. Diagnostic criteria and microbial and epidemiologic associations. Am J Med 1983; 74: 14–22.
21. Maclure M, Mittleman MA. Should we use a case-crossover design? Annu Rev Public Health 2000; 21: 193–221.
22. Rothman KJ. No adjustments are needed for multiple comparisons. Epidemiology 1990; 1: 43–46.
23. Neggers YH, Nansel TR, Andrews WW, et al. Dietary intake of selected nutrients affects bacterial vaginosis in women. J Nutr 2007; 137: 2128–2133.
24. Bradshaw CS, Morton AN, Hocking J, et al. High recurrence rates of bacterial vaginosis over the course of 12 months after oral metronidazole therapy and factors associated with recurrence. J Infect Dis 2006; 193: 1478–1486.
25. Hay PE, Ugwumadu A, Chowns J. Sex, thrush and bacterial vaginosis. Int J STD AIDS 1997; 8: 603–608.
26. Nesby-O’Dell S, Scanlon KS, Cogswell ME, et al. Hypovitaminosis D prevalence and determinants among African American and white women of reproductive age: Third National Health and Nutrition Examination Survey, 1988–1994. Am J Clin Nutr 2002; 76: 187–192.
27. Klenk J, Rapp K, Denkinger MD, et al. Seasonality of vitamin D status in older people in Southern Germany: Implications for assessment. Age Ageing 2013; 42: 404–408.
28. Harris SS, Dawson-Hughes B. Seasonal changes in plasma 25-hydroxyvitamin D concentrations of young American black and white women. Am J Clin Nutr 1998; 67: 1232–1236.
29. Basile LA, Taylor SN, Wagner CL, et al. Neonatal vitamin D status at birth at latitude 32 degrees 72′: Evidence of deficiency. J Perinatol 2007; 27: 568–571.
30. Webb AR, Kline L, Holick MF. Influence of season and latitude on the cutaneous synthesis of vitamin D3
: Exposure to winter sunlight in Boston and Edmonton will not promote vitamin D3
synthesis in human skin. J Clin Endocrinol Metab 1988; 67: 373–378.
31. Gangat M, Ponnapakkam T, Bradford E, et al. Reversed seasonal variation in maternal vitamin D levels in southern Louisiana. Clin Pediatr (Phila) 2012; 51: 718–722.
32. Brustad M, Edvardsen K, Wilsgaard T, et al. Seasonality of UV-radiation and vitamin D status at 69 degrees north. Photochem Photobiol Sci 2007; 6: 903–908.