The health-promoting role of vitamin D during pregnancy is contentiously debated.1–4 Optimal maternal 25-hydroxyvitamin D concentrations are not known. Nearly one out of three pregnant women in the United States has a serum 25-hydroxyvitamin D concentration less than 50 nmol/L,5 and experts agree that this increases the risks of bone-related disease for mothers and fetuses.3,6 Approximately two out of three pregnant women have serum 25-hydroxyvitamin D concentrations less than 75 nmol/L,5 concentrations associated with cancer, diabetes, cardiovascular disease, autoimmune disorders, and other adverse health outcomes in nonpregnant adults.7,8
It remains unclear whether vitamin D deficiency poses risks for nonskeletal outcomes during pregnancy.9,10 The ability of maternal decidual cells to convert 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D, the hormonally active form of the vitamin, as well as the presence of vitamin D receptors on the placenta11 highlight a potentially important role for vitamin D in pregnancy outcomes, including spontaneous preterm birth. 1,25-dihydroxyvitamin D has immunomodulatory and anti-inflammatory properties and regulates key genes for successful implantation,12 yet few research studies have explored the relation between 25-hydroxyvitamin D and preterm birth;13–15 none has examined this association in twin pregnancies. Women with twin gestations have greater nutrient demands16 and dramatically higher rates of preterm birth than singleton gestations.17 Research of the role of maternal vitamin D deficiency, a common and modifiable factor, in poor outcomes in twin gestations may help improve maternal and child health in these vulnerable pregnancies.
Our objective was to assess the association between maternal 25-hydroxyvitamin D concentrations at 24–28 weeks of gestation and preterm birth in a multicenter U.S. cohort of twin pregnancies.
MATERIALS AND METHODS
This is a secondary analysis of data and samples from a randomized, double-blinded, placebo-controlled trial of 17 α-hydroxyprogesterone caproate for the prevention of preterm birth in twin gestations (2004–2006). Details of the study have been published previously.18 Briefly, women with twin pregnancies who were 16 weeks to 20 weeks 3 days of gestation were recruited from 14 study centers in the United States after providing informed written consent. They were randomized to weekly injections of either 17 α-hydroxyprogesterone caproate or placebo until 34 weeks 6 days of gestation or delivery, whichever occurred first. Medical charts were abstracted by trained certified personnel to ascertain data on antepartum and intrapartum events, obstetric interventions, and neonatal outcomes. The study was approved by the institutional review boards at each clinical site and at the data coordinating center.
An ancillary study to the clinical trial to investigate the pharmacokinetics and pharmacodynamics of 17 α-hydroxyprogesterone caproate began with approximately 1 year left in recruitment. The participants of the ancillary study provided a nonfasting blood sample at 24–28 weeks of gestation. These samples were centrifuged and frozen at −80°C for future analysis. Serum samples were analyzed in a single batch for 25-hydroxyvitamin D concentrations (25-hydroxyvitamin D2 plus 25-hydroxyvitamin D3) using liquid chromatography tandem mass spectrometry.19 The assay had a lower detection limit of 1 ng/mL and no upper limit. The intra-assay coefficient of variation was 8.2% and 5.9% for 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, respectively. There is no universally acceptable definition of vitamin D deficiency. Therefore, we explored 25-hydroxyvitamin D as a continuous variable, as a binary variable (less than 75 nmol/L compared with 75 nmol/L or more),6 and categorized based on distribution quartiles.
Gestational age was determined according to an algorithm on the basis of the last menstrual period and the results of ultrasonography of the larger fetus for women who conceived spontaneously.20 For women who conceived by in vitro fertilization, the duration of gestation was calculated on the basis of the date of embryo transfer and the age of the embryos when transferred.18 The primary outcome for the clinical trial was delivery of the first twin or death of either twin at less than 35 weeks of gestation. For the purposes of this analysis, we have termed that outcome “preterm birth” and analyzed by pregnancy, not by individual neonate. Spontaneous preterm birth was defined as a preterm birth at less than 35 weeks of gestation occurring after preterm labor with intact membranes or preterm prelabor rupture of the fetal membranes. Prespecified criteria encoded in the study manual of operations were used to define whether the birth was indicated or spontaneous, and this distinction was made before the measurement of 25-hydroxyvitamin D concentrations.
Because 17 α-hydroxyprogesterone caproate had no significant effect on the occurrence of preterm delivery,18 all women were included in this secondary analysis. Continuous variables were compared using the Wilcoxon rank-sum test. Categorical variables were analyzed using χ2 or Fisher exact test when appropriate. Multivariable logistic regression models were used to quantify the association between maternal 25-hydroxyvitamin D concentration and preterm birth after adjusting for prepregnancy body mass index (BMI, calculated as weight (kg)/[height (m)]2), race and ethnicity, maternal age, parity, smoking, marital status, education, study site, gestational age at blood draw, 17 α-hydroxyprogesterone caproate treatment, and season at blood draw (confounders identified using theory-based causal diagrams21). Nominal two-tailed P values were reported and no adjustments were made for multiple comparisons. P<.05 was considered significant. Statistical analysis was performed using SAS software.
Of the 661 women enrolled in the randomized trial, 211 (31.9%) had a serum sample drawn at 24–28 weeks of gestation for serum 25-hydroxyvitamin D measurement. A total of 20 women in the parent study who were eligible for the ancillary study delivered before 24 weeks of gestation. Participants who had serum available for 25-hydroxyvitamin D measurement were slightly more likely than women without stored serum to be lean and better educated, but there were no significant differences in any other characteristic (Table 1). In the sample with 25-hydroxyvitamin D assayed, no total 25-hydroxyvitamin D concentrations were below the detectable range. There were 75 cases of preterm birth (one of which qualified because of a stillbirth) at less than 35 weeks of gestation (35.6%), including 50 cases of spontaneous preterm birth at less than 35 weeks of gestation (50 out of 186 deliveries that were not indicated preterm birth at less than 35 weeks of gestation [26.9%]) and 25 cases of indicated deliveries before 35 weeks of gestation. Twenty-five women (11.8%) delivered before 32 weeks of gestation, including 10 women who delivered before 30 weeks of gestation. Of the 117 parous patients in this analysis, 16 had a previous delivery at less than 35 weeks of gestation (13.7%).
The mean (standard deviation [SD]) serum 25-hydroxyvitamin D concentration was 82.7 nmol/L (±31.5), and the median was 85.7 nmol/L; 40.3% of women had concentrations less than 75 nmol/L, and 18.0% had concentrations less than 50 nmol/L. Women with serum 25-hydroxyvitamin D concentrations less than 75 nmol/L were more likely than those with concentrations of 75 nmol/L or more to be non-Hispanic black or Hispanic, young, unmarried, obese before pregnancy, to have less than a high school education, and to have delivered earlier (Table 2). The median 25-hydroxyvitamin D concentrations among women identifying their race and ethnicity as non-Hispanic white, non-Hispanic black, and Hispanic or other were 96.6 nmol/L, 50.1 nmol/L, and 73.9 nmol/L, respectively.
Preterm birth at less than 35 weeks of gestation occurred in 49.4% of women with 25-hydroxyvitamin D concentrations less than 75 nmol/L compared with 26.2% among those with concentrations of 75 nmol/L or more (Table 3; P<.001). Maternal serum 25-hydroxyvitamin D concentrations of 75 nmol/L or more were associated with a 60% reduction in the odds of preterm birth compared with those less than 75 nmol/L. This association remained after adjustment for maternal race and ethnicity, study site, parity, prepregnancy BMI, smoking status, marital status, education, 17 α-hydroxyprogesterone caproate treatment, and season at blood sampling. When 25-hydroxyvitamin D concentration was studied as a continuous variable, every 31.5-nmol/L (1 SD) increase in 25-hydroxyvitamin D concentration was associated with a 50% decrease in the odds of preterm birth at less than 35 weeks of gestation. In quartile analysis, women with 25-hydroxyvitamin D concentrations in the highest one fourth of the distribution had a reduced odds of preterm birth at less than 35 weeks of gestation compared with those with concentrations in the bottom one fourth (Table 3).
Results were similar when we limited analysis to spontaneous preterm birth at less than 35 weeks of gestation. Pregnant women with serum 25-hydroxyvitamin D concentrations less than 75 nmol/L had more spontaneous preterm births (29 out of 72 [40.3%]) than did women with concentrations of 75 nmol/L or more (21 out of 114 [18.4%]; P<.01). The adjusted odds ratio for spontaneous preterm birth associated with a 1-SD increase in 25-hydroxyvitamin D concentration was 0.5 (95% confidence interval 0.3–0.9). There were too few cases of indicated preterm births at less than 35 weeks of gestation (n=25) to perform statistical modeling. However, in bivariate analyses, women with serum 25-hydroxyvitamin D concentrations less than 75 nmol/L were more likely to have indicated preterm birth at less than 35 weeks of gestation than were those with concentrations of 75 nmol/L or more (23.2% compared with 11.4%; P<.05).
Maternal 25-hydroxyvitamin D concentration also was negatively associated with preterm birth at less than 32 weeks of gestation (Table 4). Compared with 25-hydroxyvitamin D concentrations less than 75 nmol/L, serum concentrations of 75 nmol/L or more were associated with an 80% reduction in risk of preterm birth at less than 32 weeks of gestation, after adjustment for confounders. A 1-SD increase in 25-hydroxyvitamin D concentration was associated with a 60% decrease in the likelihood of preterm birth at less than 32 weeks of gestation. None of these associations differed by maternal race and ethnicity.
In this multicenter U.S. cohort of women with twin gestations, we observed that serum vitamin D concentrations measured at 24–28 weeks of gestation were inversely associated with the risk of preterm birth at less than 35 weeks of gestation and at less than 32 weeks of gestation and with spontaneous preterm birth at less than 35 weeks of gestation, even after adjusting for covariates such as prepregnancy BMI, race and ethnicity, and season.
Little is known about maternal vitamin D status in relation to risk of preterm birth in twin pregnancies. In a trial of 504 women with singleton pregnancies who were randomized to receive 400, 2,000, or 4,000 international units of vitamin D3 per day from 12–16 weeks of gestation to delivery, there was no difference in rates of preterm birth without preeclampsia by treatment group.22 These authors reported that women with 25-hydroxyvitamin D concentrations less than 80 nmol/L before delivery were more likely to deliver preterm without preeclampsia than were those with higher concentrations, but this finding may be explained by reverse causality. Using 131 women with preterm birth at less than 35 weeks of gestation as cases and 134 women with term births as controls (all of which were singleton pregnancies with a history of preterm birth) from a U.S. multicenter randomized trial of omega-3 fatty acid supplementation, investigators observed no relationship between 25-hydroxyvitamin D concentrations at 12–16 weeks of gestation or 25–28 weeks of gestation and recurrent preterm birth.13 Our findings may differ because all women in this population had a history of preterm birth and received weekly injections of 17 α-hydroxyprogesterone caproate throughout pregnancy. Researchers using a Canadian cohort of 221 singleton pregnancies at high risk for preeclampsia observed no association between 25-hydroxyvitamin D concentration at 19 weeks of gestation and preterm birth at less than 37 weeks of gestation.14 In a sample of 884 human immunodeficiency virus–infected African women with singleton pregnancies, there was no association between 25-hydroxyvitamin D concentration at a mean of 204 weeks of gestation and risk of preterm birth at less than 37 weeks of gestation or less than 34 weeks of gestation after controlling for multivitamin supplementation, age, enrollment CD4 cell count, and human immunodeficiency stage.15 Similarly, gestational age (rather than preterm birth) was studied in three other singleton gestation cohorts, and results were mixed.17,23,24
Our finding that poor vitamin D status is associated with early preterm birth suggests that the anti-inflammatory and immunomodulating roles of vitamin D may be relevant.12 Vitamin D regulates uterine natural killer cells and other immune cells in vitro by suppressing inflammatory cytokine production.25 A similar effect of vitamin D on cultured trophoblastic cells has been observed,26 which implies a general anti-inflammatory role in pregnancy. Vitamin D promotes innate immune responses in monocytes by stimulating antimicrobial activity.27 In addition, vitamin D regulates genes critical for successful implantation, including calbindin-D9K and HOXA through intracrine or paracrine pathways.12
The demands for micronutrients, including vitamin D, in twin pregnancies are believed to be higher than those for singleton pregnancies,16,28 yet a lack of data to inform nutrient needs in twin pregnancy has resulted in the same recommended dietary allowance (600 international units of vitamin D per day) for singleton and multiple pregnancies.3 Therefore, we did not expect to observe a higher median 25-hydroxyvitamin D concentration in this multicenter U.S. cohort of twin pregnancies than what has been reported in other U.S. singleton samples,29–31 including in a nationally representative group of pregnant women (74.4 nmol/L for non-Hispanic whites, 33.2 nmol/L for non-Hispanic blacks, and 54.0 nmol/L for Mexican-Americans).5 The higher median 25-hydroxyvitamin D concentrations may be attributable to differences in intake of vitamin D through supplements or diet, or attributable to our inclusion of women from the southern United States, who may be exposed to greater amounts of solar radiation. Unfortunately, we lacked data regarding vitamin D intake and sunlight exposure to evaluate the contributing factors. However, characteristics such as race and ethnicity and obesity, which typically are associated with vitamin D levels during pregnancy, also were related in our cohort.
Limitations of our study warrant comment. We used a convenience sample of women who provided a blood sample at 24–28 weeks of gestation as part of the ancillary study. Our results may be biased if vitamin D concentrations affected selection into the ancillary study. For instance, if vitamin D deficiency caused preterm birth, excluding the 20 women in the parent study who delivered before 24 weeks of gestation (and therefore could not participate in the ancillary study) would lead to an underestimation of effects. Although we adjusted for many known factors to be associated with both 25-hydroxyvitamin D concentration and preterm birth, unmeasured confounding by intake of other micronutrients, physical activity, and maternal genotype may have biased our findings, and imperfect measurement of prepregnancy obesity using self-reported data and socioeconomic position using education and marital status may have led to residual confounding. Additionally, only one blood sample before the third trimester was obtained in the parent study, so we could not evaluate other windows of vitamin D exposure. Strengths of our study include measurement of 25-hydroxyvitamin D concentration before the clinical onset of preterm delivery using liquid chromatography tandem mass spectrometry (the gold standard method) and estimation of gestational age in pregnancy based on a well-established algorithm.18
Preterm delivery is a major contributor to the excess neonatal morbidity and mortality in twin pregnancies,32 and identifying modifiable risk factors for preterm birth in twins is a major public health priority.33 Observational studies beginning early in pregnancy of large representative cohorts of women with multiple fetuses are the logical next step to assess whether maternal 25-hydroxyvitamin D concentrations are consistently associated with preterm birth. If these results are replicated, randomized trials of vitamin D supplementation may be warranted.
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