Preeclampsia is a multisystem disorder defined as new-onset hypertension and proteinuria. In developed countries, the perinatal mortality rate among preeclamptic pregnancies is five times as great as nonpreeclamptic pregnancies,1 and indicated preterm deliveries for preeclampsia account for 15% of preterm births.2 Mothers who develop preeclampsia are at elevated risk of abruptio placentae, acute renal failure, and neurologic and cardiovascular complications.1 Moreover, preeclampsia contributes to 18% of maternal deaths in the United States and 20–80% of maternal deaths in developing countries.1,3 Delivery is the only known cure for preeclampsia, and interventions have generally been ineffective in preventing the disorder.
There is a growing interest in the role of maternal vitamin D status in the development of preeclampsia. Vitamin D is a prohormone that is either made in the skin through ultraviolet B radiation exposure or ingested orally.4 Vitamin D deficiency is widespread in US pregnant women5–8 due to inadequate sunlight exposure, limited vitamin D–rich food sources, and the use of prenatal vitamins with low doses of vitamin D.4 Vitamin D has diverse and protean functions that may be relevant in the pathophysiology of preeclampsia, including abnormal placental implantation and angiogenesis, excessive inflammation, hypertension, and immune dysfunction.4,9–12 Most research on vitamin D and preeclampsia has been conducted in predominantly white populations with small numbers of preeclampsia cases, and results have been inconsistent.13–21
We sought to determine the association between maternal vitamin D status at ≤26 weeks and the risk of preeclampsia in a large, geographically diverse US multicenter cohort of pregnant women.
We conducted a case–cohort study22 in the Collaborative Perinatal Project (1959–65).23 A total of 55,908 pregnant women were enrolled at their first prenatal visit at 12 US medical centers after providing verbal informed consent for participation (as was common at the time the study was conducted). Detailed data were collected via in-person interviews on maternal sociodemographic factors, medical history, and obstetric history. Mothers provided nonfasting blood samples every 8 weeks. At each visit, medical and obstetric events were recorded, and random urine samples were tested for albumin. Blood pressures were measured at enrollment and each prenatal visit, during labor and delivery, and postpartum. Korotkoff phase 4 (muffling) or phase 5 (disappearance) was used for diastolic blood pressure.24 A validation study in which information on blood pressure and urinary albumin was checked against that in the original medical records showed a high degree of accuracy.24 A labor-and-delivery summary was recorded by the obstetrician responsible for the patient’s care.
The Figure describes the selection of the sample. There were 44,510 singleton deliveries to white, black, or Puerto Rican mothers at 20 to 42 weeks’ gestation. We excluded women with pregestational diabetes, hypertension, or cardiovascular disease, as well as women who entered the study after 26 weeks, leaving a cohort of 28,429 eligible women. We randomly selected 11% of the eligible cohort and augmented this subcohort with all remaining cases of preeclampsia. We used multiple imputation (described below) to address missing data on 25-hydroxyvitamin D (25(OH)D) concentrations, prepregnancy body mass index (BMI), socioeconomic status, or other covariates in 12% of pregnancies. After imputation, the analytic sample included 717 women with preeclampsia (560 mild and 157 severe cases) and 2,986 without preeclampsia. This study used de-identified data and was exempt from ethics review.
We applied a current definition25 of preeclampsia to measurements of blood pressure and urinary protein taken at the time of the study. Preeclampsia was defined as gestational hypertension and proteinuria, with return to normal in the postpartum period.25 Gestational hypertension was defined as two or more measurements of systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg for the first time after 24 weeks of gestation. In the intrapartum period, the first five pressures obtained after hospital admission for delivery were averaged. Proteinuria was defined as two random urine dipsticks of 1+ protein or one dipstick of 2+ protein. Cases of preeclampsia were considered severe if they had at least one of the following: systolic blood pressure ≥160 mmHg, diastolic blood pressure ≥110 mmHg, proteinuria of 5 g/24 hours, proteinuria of 3+ or more, oliguria, pulmonary edema, or convulsions/eclampsia. All other cases were considered mild. The HELLP (hemolysis, elevated liver enzymes, low platelet count) syndrome had not yet been described at the time of the study, and liver function tests and platelet counts were not included in the database.
Maternal serum was stored in glass at −20°C, with no recorded thaws. We randomly selected one banked serum sample drawn at ≤26 weeks for each mother. A 26-week gestational age cutoff was chosen for two reasons. First, many women in the study had registered late for prenatal care, and this cutoff allowed us to capture a large number of preeclampsia cases while also assessing vitamin D status before the clinical onset of disease in most cases. Second, when the analysis was designed, there was no information available to determine the window of gestation that was critical for vitamin D exposure. With limited resources, we chose to randomly select one sample and perform analyses stratified by gestational age at blood sampling.
Sera were shipped to the laboratory of Michael Holick at Boston University, which is designated as a DEQAS (Vitamin D External Quality Assessment Scheme)-proficient laboratory. Samples were assayed for total 25(OH)D [25(OH)D2 + 25(OH)D3] using liquid chromatography–tandem mass spectrometry based on standards of the National Institute of Standards and Technology.26 The assay had a coefficient of variation of 6.0%. 25(OH)D has been proven to be highly stable. No loss of 25(OH)D has been noted after leaving uncentrifuged blood as long as 72 hours at 24°C, after storage of serum for years at 20°C, after exposure to ultraviolet light, or after up to 11 freeze-thaw cycles.27 A pilot study in the Collaborative Perinatal Project samples compared 25(OH)D in these serum with serum frozen for ≤2 years and found that 25(OH)D is unlikely to show significant degradation.28 There is no universally accepted definition of vitamin D deficiency, so we used multiple cut points.29,30
The study defined race as white, black, or Puerto Rican. Prepregnancy BMI (weight (kg)/height (m)2) was based on maternal self-reported pregravid weight and measured height at enrollment. Season of blood sample collection was defined as winter (December–February), spring (March–May), summer (June–August), or fall (September–November). Education, occupation, and family income data were combined into a composite socioeconomic status score.23 Data were also available on parity (primiparous, multiparous), maternal age (<20, 20–29, ≥30 years), smoking status at entry (smoker, nonsmoker), and marital status (unmarried, married). Gestational age was based on the mother’s report of the first day of her last menstrual period.
To address missing data, we used multiple imputation. We created five imputed data sets that assumed a multivariable normal distribution with a Markov Chain Monte Carlo approach31,32 to jointly address missing data in 25(OH)D and key covariates of interest. We imputed prepregnancy weight, height, parity, smoking, socioeconomic status, month of blood sampling, and 25(OH)D (all of which were log-transformed) by including preeclampsia, race, age, marital status, gestational age at prenatal care entry, gestational age at blood sampling, study site, and sample weight in the imputation model. We compared the results based on multiple imputation with those generated using the complete data set (n = 632 women with preeclampsia and 2,609 without preeclampsia).
Absolute risks, adjusted risk differences (RDs), adjusted risk ratios (RRs), and 95% confidence intervals (CIs) were calculated from multivariable log-binomial regression. All subjects were weighted using the inverse of the sampling fraction,22 and a clustered robust variance was used to account for the cases in the subcohort.22 Nonlinearity in 25(OH)D was tested using splines. Effect modification on the risk-difference scale by sample gestational age, parity, race, and pregravid overweight was tested using the synergy index.33 We assessed effect modification rather than interaction because we believe that these are not variables on which one could intervene (as is required for tests of biologic interaction),34 and we studied that additive scale because it is the scale that is argued to be of greatest public health importance.35 Potential confounders (race, prepregnancy BMI, trimester of entry to prenatal care, smoking, parity, age, socioeconomic status, marital status, season of blood draw, gestational age at blood draw, and latitude of study site) were identified using theory-based causal models.36 Only season and gestational age of blood sampling and trimester of entry to prenatal care met our definition of confounding (≥10% change in the magnitude of the association after removal of the variable from the full model).37 However, we also included race, BMI, smoking, latitude of study site, and parity in models to ensure that our results were comparable with previous literature. For some models, there were too few cases to include indicator variables for study site. Therefore, we tested for confounding by study site using multivariable conditional logistic regression conditioned on site.
Finally, we conducted a probabilistic bias analysis for unmeasured confounding by leisure-time physical activity and fish intake, using published methods38 (eAppendix, http://links.lww.com/EDE/A749). These factors have been shown to be associated with higher 25(OH)D concentrations39–41 and preeclampsia42,43 but were unmeasured in our data set. We compared the estimates from the conventional regression model of maternal 25(OH)D ≥50 versus <50 nmol/L and preeclampsia risk with estimates obtained from the sensitivity analysis iterations, which reflected systematic error and random error associated with missing data on each unmeasured covariate.38
The subcohort was 49% white, 44% black, and 7% Puerto Rican. Many women in the subcohort were 20 to 29 years old, normal weight before pregnancy, married, smokers, with less than a high school education, and with at least one previous live-born child (Table 1). In unadjusted analyses, mothers who developed preeclampsia tended to be black, nulliparous, age <20 years or ≥30 years, overweight, unmarried, nonsmokers, less educated, of lower socioeconomic position, and from study centers at ≤40°N latitude. They were more likely to enter care after the first trimester and have blood drawn in the winter months.
In the subcohort, the mean maternal 25(OH)D at ≤26 weeks was 50.7 nmol/L (95% CI = 49.7 to 51.7); 24%, 57%, and 84% of women had serum 25(OH)D <30, <50, and <75 nmol/L, respectively. As expected, 25(OH)D <30 nmol/L was more common among black women (32%) compared with Puerto Rican (23%) and white (13%) mothers and among those blood samples drawn in December through May (33%) compared with June through November (15%).
The cumulative incidence of preeclampsia was 2.6% in the cohort, and most cases were mild (incidence 2.0%). Incidence of overall preeclampsia was lowest among gravidae with 25(OH)D 50 to 74.9 nmol/L at ≤26 weeks and highest with 25(OH)D <30 nmol/L, but there was no absolute or relative difference in risk after adjustment for race, prepregnancy BMI, socioeconomic position, parity, smoking, latitude, and season of blood sampling (Table 2). Results were similar for mild preeclampsia.
There were no differences in the relation between 25(OH)D and preeclampsia across strata of gestational age at blood draw or parity (Table 3). Alternate cut points for gestational age had no effect on conclusions. There was effect modification on the risk-difference scale for prepregnancy overweight (synergy index = 0.46 [95% CI = 0.01 to 0.92]). Serum 25(OH)D ≥75 nmol/L was associated with both absolute and relative increases in the risk of preeclampsia compared with concentrations <30 nmol/L among overweight women (BMI ≥25) but not leaner women. The estimates, however, were highly imprecise.
For severe preeclampsia, each 1-standard-deviation increase in 25(OH)D was associated with absolute and relative reductions in risk before and after confounder adjustment (Table 2). Compared with 25(OH)D <50 nmol/L, mothers who had concentrations ≥50 nmol/L had a reduction of three cases per 1000 pregnancies (adjusted RD = 0.003 [95% CI = −0.005 to 0.0002]) and a 40% reduction in risk (0.65 [0.43 to 0.98]). In formal sensitivity analysis, this conventional RR was attenuated slightly when accounting for unmeasured confounding by exercise (0.71 [95% simulation interval = 0.46 to 1.1]) or fish intake (0.74 [0.48 to 1.1]).
None of our results was meaningfully different when 25(OH)D was specified using flexible spline terms or quantiles, when preeclampsia was defined based only on antepartum blood pressures and protein measurements, or when other covariates including study site were considered (data not shown). Restriction to women who had complete vitamin D and covariate data or to those who delivered at 26 to 42 weeks of pregnancy also did not alter conclusions (data not shown).
Preeclampsia causes maternal morbidity and infant morbidity and mortality; the severe cases and those with early onset are associated with the highest risks of adverse perinatal outcomes, including preterm birth and fetal growth restriction.44,45 We found that the risk of severe preeclampsia was lower for women with vitamin D sufficiency at ≤26 weeks’ gestation compared with those who were deficient. This finding was robust to adjustments for prepregnancy BMI, race, parity, and other measured confounders, as well as exercise and fish intake, which were unmeasured. We found no association with preeclampsia overall or with mild preeclampsia, after controlling for confounders.
The literature on vitamin D status in relation to preeclampsia is mixed.13–19,46,47 Two recent meta-analyses on vitamin D deficiency and preeclampsia arrived at different conclusions about this body of research,21,48 but neither separated studies based on severity of the disease. There is increasing conviction that separating cases of preeclampsia into more homogenous subgroups based on severity, gestational age, recurrence, or pathophysiologic markers may lead to a better understanding of how exposures play a role in the pathogenesis of this complex syndrome.49 We are aware of three studies of maternal vitamin D in relation to severe or early-onset preeclampsia that adjusted for confounders. Although none had >50 cases of severe disease, most of the findings are consistent with ours. In a study of 43 severe preeclampsia cases and 198 controls, investigators reported that 25(OH)D <50 nmol/L was associated with an 80% reduction in risk compared with ≥75 nmol/L, after adjustment for key confounders.15 Canadian researchers studied 32 women who subsequently developed preeclampsia (23 of whom had severe disease) and 665 controls; they found no association between 25(OH)D and preeclampsia at 16 to 18 weeks, but a 30% reduction in risk with each 1-standard-deviation increase in 25(OH)D at 24 to 26 weeks.43 A 70% reduction in severe preeclampsia risk per 25 nmol/L increase in 25(OH)D was reported in a study of 50 severe preeclamptics and 100 controls,50 but this study measured 25(OH)D after clinical onset of the disease and may not reflect exposures relevant in the pathophysiology of severe preeclampsia.
Our finding was based on samples at a median of 21 weeks. It is possible that, as with the two aforementioned studies,18,50 the low 25(OH)D was a consequence of the disease process in some women. Preeclampsia is thought to originate from reduced placental perfusion caused by abnormal placentation.51 The resultant oxidative stress, antiangiogenic factors, and inflammation are hypothesized to lead to maternal systemic disease.51 The placenta expresses 1-α-hydroxylase for the metabolism of 25(OH)D,11 and abnormal placental functioning could alter the influx and efflux of 25(OH)D into maternal circulation. More research is needed to understand the effect of placental vitamin D metabolism on maternal circulating 25(OH)D.
Our results suggesting an increased risk of overall preeclampsia at 25(OH)D ≥75 nmol/L among mothers with a pregravid BMI ≥25 and among nonwhite mothers are puzzling, particularly because there was no dose–response relationship. We have no obvious explanation for this finding, but our ongoing research studying polymorphisms in key vitamin D metabolizing genes may shed light on this result.
Our study could not determine the association between vitamin D and subtypes other than those based on mild and severe symptoms because we lacked adequate numbers of preeclampsia cases delivered at <34 weeks (n = 17) and data on preeclampsia recurrence (though our results did not differ by parity). We also did not measure biological intermediates of preeclampsia or other biomarkers in the vitamin D pathway, including vitamin D–binding protein. Observational studies on vitamin D are susceptible to confounding bias because sufficient 25(OH)D may be a marker of more time spent outdoors or other behaviors that investigators did not measure. We attempted to account for unmeasured confounding by outdoor leisure-time physical activity and fish intake. We did not consider calcium intake, which was also unmeasured, because we have previously shown that it is unlikely to meaningfully confound the vitamin D–preeclampsia relationship.14 However, unmeasured confounding by genetic factors or other lifestyle variables is possible, and those for which data were available may have been measured with error. Selection bias is unlikely to be a major problem in our study because results generated after multiple imputation of vitamin D and covariate data on 13% of our sample agreed with those from the complete case analysis.
Results may not generalize to today’s obstetric population because of differences in population characteristics (eg, higher levels of smoking and lower prevalence of obesity in the 1960s, with potential interrelationships with vitamin D) and differences in preeclampsia management. However, there is no evidence that the pathophysiology of preeclampsia has changed over time. The large sample size and the proven validity of the hypertension and proteinuria data in the study24 gave us the unique opportunity to prospectively study preeclampsia using rigorous, contemporary definitions stratifying by severity. As expectant management of preeclampsia did not typically involve iatrogenic preterm delivery in the 1960s, these data also offered the chance to study the natural progression of the disease.
While the maternal sera was stored for decades, our pilot work suggests that the long-term storage is unlikely to have caused deterioration of 25(OH)D,28 and the high prevalence of maternal vitamin D deficiency that we observed is similar to racially diverse modern cohorts.6–8 Furthermore, if 25(OH)D had deteriorated over time, it would have misclassified both cases and controls and led to bias toward the null. Our ability to replicate factors generally regarded as associated with vitamin D (eg, race, season) and preeclampsia (eg, parity, obesity, smoking) supports the validity of our measurements.
Research in contemporary pregnancy cohorts with a large number of cases and rigorous definitions of preeclampsia separated by clinical subtype is needed to advance the field. Longitudinal measures of vitamin D metabolites and preeclampsia biomarkers, together with 25(OH)D during gestation, will not only help elucidate pathways linking vitamin D to pregnancy outcome but also identify subsets of preeclampsia that may be responsive to vitamin D treatment.
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