Journal of Perinatology https://doi.org/10.1038/s41372-019-0343-8
Kimberly K. Jefferson • Hardik I. Parikh1,2 • Erin M. Garcia • David J. Edwards2,3 • Myrna G. Serrano1 • Martin Hewison • Judith R. Shary5 • Anna M. Powell6 • Bruce W. Hollis 5 • Jennifer M. Fettweis1,7 • Jerome F. Strauss III2,7 • Gregory A. Buck1 • Carol L. Wagner 5
Vitamin D started "within the first 14 weeks after last menstrual period (LMP)"
Treatment = 4,400 IU Vitamin D daily
Control = 400 IU Vitamin D daily
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Vitamin D Response with >40 vs < 30 added by VitaminDWiki
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Table of Contents
Objective Evidence supports an inverse association between vitamin D and bacterial vaginosis (BV) during pregnancy. Furthermore, both the vaginal microbiome and vitamin D status correlate with pregnancy outcome. Women of African ancestry are more likely to experience BV, to be vitamin D deficient, and to have certain pregnancy complications. We investigated the association between vitamin D status and the vaginal microbiome.
Study design Subjects were assigned to a treatment (4400IU) or a control group (400IU vitamin D daily), sampled three times during pregnancy, and vaginal 16S rRNA gene taxonomic profiles and plasma 25-hydroxyvitamin D [25(OH)D] concentrations were examined.
Result Gestational age and ethnicity were significantly associated with the microbiome. Megasphaera correlated negatively (p = 0.0187) with 25(OH)D among women of African ancestry. Among controls, women of European ancestry exhibited a positive correlation between plasma 25(OH)D and L. crispatus abundance.
Conclusion Certain vaginal bacteria are associated with plasma 25(OH)D concentration.
Only a few portions of the text were extracted from the PDF
The composition of the vaginal microflora can significantly impact both reproductive and neonatal health. Vaginal lactobacilli, through the production of lactic acid, create a vaginal environment characterized by a low pH, and this, in combination with bacteriocins and possibly other components, inhibits the colonization and growth of potentially pathogenic microorganisms and reduces the phylogenetic diversity of the vaginal microbiome. In a state of dysbiosis termed bacterial vaginosis (BV), the lactobacilli are depleted and replaced with a polymicrobial, anaerobic microflora that includes Gardnerella vaginalis, Atopobium vaginae, Sneathia, Prevotella spp, Megasphaera, and others. Although BV is frequently asymptomatic, this dysbiotic state is significantly associated with other clinical complications including pelvic inflammatory disease, infertility, and spontaneous abortion. When BV occurs during pregnancy, it is associated with a more than twofold increased risk for preterm birth. This is important because preterm birth accounts for as much as 70% of neonatal mortality, 75% of neonatal morbidity, and nearly 50% of long-term neurologic sequelae. A significant portion of moderate and late preterm births and the majority of very preterm births (<32 weeks) may be attributable to infection and subsequent inflammation and recent microbiome analyses reveal an association between term birth and abundance of healthy lactobacilli and an association between preterm birth and BV-associated bacterial taxa [1, 2].
Numerous studies support the important role for sufficient serum or plasma concentrations of 25-hydroxyvitamin D (25 (OH)D) during pregnancy in preventing negative outcomes [3-10]. Although some studies failed to detect an association, possibly because vitamin D deficiency is low among certain populations, many others and a recent meta-analysis suggest that vitamin D deficiency increases the rate of preterm birth, particularly early preterm birth . It plays roles in maintenance of maternal and fetal calcium homeostasis, which is linked to skeletal integrity, in general fetal growth and development, and in the regulation of immune function [11, 12]. There also appears to be a relationship between vitamin D and BV status during pregnancy. Multiple reports describe higher rates of BV among women with insufficient 25(OH)D concentrations (often defined as <15 ng/mL or <37.5 nmol/L) [13-15]. However, studies in non-pregnant women have yielded conflicting results. One randomized controlled trial that investigated the effectiveness of vitamin D supplementation in conjunction with metronidazole in eliminating BV in non-pregnant women did not detect a positive effect of vitamin D  while another study found that vitamin D supplementation was effective in eliminating BV .
Race/ethnicity has significant population-level impacts on vitamin D status, BV status, and pregnancy outcomes. Vitamin D deficiency is more prevalent among people with limited sun exposure and among people with dark skin. Children of African ancestry are more likely than those of European ancestry to experience hypocalcemic seizures and to develop rickets . Women of African ancestry are also twice as likely to receive a clinical diagnosis of BV, and analyses of the vaginal microbiota reveal that they are more likely to be colonized by certain BV-associated bacteria . Furthermore, African American women (though not African-born women) are nearly twice as likely to give birth preterm (<37 weeks’ gestation) and more than twice as likely to give birth early preterm (<34 weeks) [20, 21] and vitamin D deficiency has been linked to preterm birth and small-for-gestational-age infants born to women of African ancestry [22, 23]. There is an urgent need to understand and address these important health disparities and an important first step in understanding whether or not the three parameters, vitamin D status, vaginal microbiome, and preterm birth, are linked. As a component of a prospective vitamin D supplementation trial in pregnant women funded by the W. F. F. Kellogg Foundation, referred to as the Kellogg Study, we analyzed a cohort of 230 healthy women and investigated the relationship between plasma 25(OH)D concentrations, the vaginal microbiome, and preterm birth.
The hypothesis driving this study was that vitamin D status is associated with the vaginal microbiome. Vitamin D could affect the vaginal microbiome through a number of mechanisms. First, it could promote the barrier integrity of the vaginal epithelium. Vitamin D was shown to upregulate genes encoding epithelial cell junction proteins and stimulated proliferation of the vaginal epithelium [35, 36]. Furthermore, two studies found that postmenopausal women receiving vitamin D treatment had increased numbers of superficial vaginal epithelial cells compared to women not receiving treatment, and that this increase was accompanied by decreases in vaginal pH [37, 38]. Together these studies suggest that vitamin D may promote vaginal epithelial cell growth, differentiation, and function, which may increase vaginal thickness to prevent atrophy and improve barrier function. Second, adequate vitamin D during pregnancy may impact the vaginal environment so that it is more supportive of beneficial flora. It is well established that insulin stimulates glycogen synthesis , and more recently it has been proposed that vitamin D induces insulin synthesis [40, 41]. Vitamin D was also shown to increase the phosphorylation and inactivation of glycogen synthase kinase, an inhibitor of glycogen synthesis, in adipose tissue . Therefore, there is also a possibility that vitamin D sufficiency alters glucose homeostasis in the vagina to promote increased glycogen deposition. Increased vaginal levels of free glycogen positively correlate with Lactobacillus relative abundance , and so if vitamin D is indeed promoting vaginal glycogen production, the accompanying increase in lactobacilli and decrease in pH may result in the decreased abundance of pathogenic and BV-associated organisms. A third possibility is that vitamin D influences abundance of BV-associated bacteria through its effects on immune function. Previous studies have shown that vitamin D treatment induces the expression of the LL-37 antimicrobial peptide in keratinocytes, neutrophils, monocytes, and bladder and gingival epithelial cells as well as p2 defensin in keratinocytes, neutrophils, and monocytes , as well as potentiates the secretion of interleukin-8 and CXCL10 from airway epithelial cells . These effects could promote leukocyte recruitment to the vaginal epithelium and modulate the antimicrobial response of leukocytes upon arrival to the tissue .
In this study, we analyzed a sub-cohort from a randomized, placebo-controlled clinical trial of vitamin D supplementation of pregnant women who were enrolled during the first trimester of pregnancy and followed until delivery. We analyzed 16S rRNA gene survey data to determine whether plasma 25(OH)D concentrations were associated with the vaginal microbiome. Women in both the control (400 IU vitamin D daily) and treatment groups (4400 IU vitamin D daily) displayed higher circulating 25(OH)D concentrations with increasing gestational age. While initial analyses suggested a substantive association between 25 (OH)D and the vaginal microbiome, we found that gestational age was a strong covariate. While multiple studies have shown that the abundances of certain BV- associated bacterial taxa decrease during pregnancy [49, 50], a correlation with gestational age has not been demonstrated and a report of a longitudinal study suggested that the vaginal microbiome is remarkably constant over the course of pregnancy . In our study, however, G. vaginalis decreased significantly in abundance between visit 1 (7.6-15.1 weeks) and visit 7 (31.6-40.4 weeks) (Fig. 5).
A model that took into account the covariation between the vaginal microbiome and gestational age, found only three taxa that were associated with 25(OH)D concentrations, and these associations were dependent upon ethnicity. Specifically, among women of African ancestry, there was a negative correlation between 25(OH)D and abundance of Megasphaera spp. The majority of reads that were categorized with the genus Megasphaera were Megasphaera type 1, which we have found in another study (Glascock, Fettweis, manuscript in preparation) to be the most common vaginal type. Detection of Megasphaera by PCR has been shown to accurately predict of BV  and Megasphaera is associated with bacteria vaginosis and spontaneous preterm delivery [53-55]. Megasphaera has also been linked to genital tract inflammation  and HIV acquisition among African women . Among women of European ancestry in the control group, women with higher 25(OH)D concentrations were more likely to have higher abundance of L. crispatus. L. crispatus is a healthy H2O2-producing lactobacillus species that is associated with decreased risk for developing BV and decreased risk for HIV acquisition [58, 59]. Thus, although only two taxa were significantly associated with 25(OH)D, these results suggest that vitamin D could have a positive impact on the vaginal microbiome.
A limitation of this study is that the cohort contained few women who had profound vitamin D deficiency (defined as 25(OH)D <12 ng/mL). In our prior National Institute of Child Health and Human Development vitamin D pregnancy study involving 350 women randomized to three treatment groups (400, 2000, and 4000 IU vitamin D3/day) , even the control group showed an increase in 25(OH)D over the course of pregnancy, especially in white/Caucasian women. Most women are not taking a prenatal vitamin or a multivitamin at the start of pregnancy and since most women have limited sunlight exposure or use sunscreen limiting their endogenous synthesis of vitamin D, and because the average American woman is provided ~200 IU vitamin D/day from her diet , the addition of 400 IU/day results in a slight but measurable increase in 25(OH)D over time.
Another potential limitation of the study is the loss of subjects during the study period. There were 108 women who exited the study, 60 due to nonadherence to protocol. The women who exited the study tended to be of lower socioeconomic status, were black/African American, less educated, more likely to be of higher gravidity and parity, and with a lower baseline 25(OH)D concentration than those women who completed the study. The loss of these subjects in the study could have created potential bias in the results.
It is possible that within the range of total circulating 25 (OH)D concentration among women in the cohort, there is little impact on the vaginal microbiome, but that severe vitamin D deficiency has a greater effect. In this study, plasma 25(OH)D was the only marker of vitamin D status that was measured; however, maternal 1,25D and other vitamin D metabolites such as 24,25(OH)2D and the 3-epi- 25(OH)D could also be important factors. Because the subjects in this cohort were pregnant, plasma 1,25D concentration would be expected to be higher and could influence immune function and the vaginal microbiota. Another limitation was that the relatively small cohort size may not have provided the study with adequate power to detect significant associations between 25(OH)D concentrations and bacterial taxa, particularly in light of other covariates (gestational age, ethnicity, and random treatment group assignment). While all participants in the study provided a blood sample to measure total circulating 25(OH)D, only a subset of those women provided a vaginal sample at the three time points, which further decreased the sample size. The vaginal microbiome is a complex parameter and consequently, microbiome studies often require large cohorts to detect biologically relevant results. An additional potential confounder is that women who were diagnosed with BV by Nugent score were prescribed treatment, and while treatment occurred after vaginal swab samples were obtained, it could have influenced the composition of the microbiome at subsequent visits.
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