Trial Design and Oversight

The MDIG trial was a randomized, double-blind, placebo-controlled, dose-ranging trial of maternal vitamin D supplementation.17 The protocol is available with the full text of this article at NEJM.org, and the statistical analysis plan is included in the Supplementary Appendix, also available at NEJM.org. The trial was overseen by a steering committee and an independent data and safety monitoring board. The protocol was approved by research ethics committees at the Hospital for Sick Children in Toronto and the International Center for Diarrheal Disease Research, Bangladesh (icddr,b). All authors attest to the completeness and accuracy of the data and analyses and for the adherence of the trial to the protocol. The trial funder had no role in the trial design, data collection and analysis, or interpretation of the results. No support from a commercial entity was provided.

Participants

Participants were generally healthy pregnant women between 17 and 24 weeks of gestation. They were enrolled after providing written informed consent between March 2014 and September 2015 at the Maternal and Child Health Training Institute, a public hospital in Dhaka, Bangladesh. The criteria for inclusion and exclusion are shown in Table S1 in the Supplementary Appendix.

Interventions

Participants were randomly assigned at enrollment to one of five groups to receive vitamin D or placebo. One group received placebo throughout the prenatal period and for 26 weeks post partum. Three groups received prenatal supplementation only, in the following doses: 4200 IU per week (prenatal 4200 group), 16,800 IU per week (prenatal 16,800 group), or 28,000 IU per week (prenatal 28,000 group). The fifth group received prenatal supplementation as well as 26 weeks of postpartum supplementation in the amount of 28,000 IU per week (prenatal and postpartum 28,000 group). A computer-generated, simple randomization scheme was created independently by the trial statistician. The master list linking participant identifiers to supplementation groups was held by the supplement manufacturer and not accessed by any trial personnel until final group assignments were revealed. Concealment of trial-group assignments was ensured with the use of prelabeled and sequentially numbered but otherwise identical supplement vials, which were provided to participants in accordance with the assignment sequence. Oral vitamin D 3 and placebo tablets were manufactured by the Toronto Institute for Pharmaceutical Technology (Toronto). The vitamin D content of each batch of tablets was verified in product testing.17 Tablets with different doses were identical in appearance and taste. Tablets were routinely administered under direct observation by trial personnel; however, up to four consecutive doses may have been unobserved when participants were unavailable for scheduled visits. Missed doses were administered up to 7 days after the scheduled date. Calcium (500 mg per day), iron (66 mg per day), and folic acid (350 μg per day) were provided to all participants throughout the intervention phase.17 If a participant reported use of a vitamin D or a calcium supplement for more than 1 week that had not been provided through the trial, trial supplements were suspended until nontrial supplement use was discontinued. Supplementation was discontinued in participants with confirmed hypercalcemia (as defined below), fetal or infant death, or a new condition or medication that could alter vitamin D metabolism.

Assessments and Outcomes

Trial personnel contacted participants weekly from enrollment until 26 weeks post partum, and infants were further assessed at 9 months and 12 months of age. Visits were conducted in the home or at a clinic and included the use of standardized questionnaires, point-of-care tests, anthropometric measurements, and specimen collection (Table S2 in the Supplementary Appendix). Data on socioeconomic and household characteristics were collected at baseline. Weekly prenatal questionnaires included health care encounters and a checklist of clinical symptoms. Postnatal follow-up visits included assessment of both the infant’s health and feeding practices as well as a basic physical examination. Maternal blood pressure was measured at enrollment, at 24 and 30 weeks of gestation, and weekly from 36 weeks of gestation to delivery. Trial personnel tracked pregnancy outcomes, visits with trial physicians, hospitalizations, and deaths, and they attended all facility-based deliveries and home births when feasible. Participants were provided with free medical care and encouraged to seek medical attention from trial physicians and to notify trial personnel of concerns about their health. Pregnancies were completed from June 25, 2014, through February 29, 2016. One-year postnatal visits were conducted from June 24, 2015, through March 1, 2017.

Infant crown-to-heel length (to the last completed millimeter), head circumference, upper-arm length, mid–upper-arm circumference, and rump-to-knee length — all to the last completed millimeter — and weight (to the nearest 5 g up to 10 kg and to the nearest 10 g for >10 kg) were measured by trained personnel according to standardized procedures17 adapted from the protocols of the INTERGROWTH-21st (International Fetal and Newborn Growth Consortium for the 21st Century) Project.18 Each measurement was obtained independently by two trial personnel and repeated if the difference between paired measurements exceeded specified thresholds. Means of the final pair of values were used in analyses. Interrater reliability was high, and few measurements were excluded due to implausibility or temporal inconsistencies (see Methods, section 1, and Table S3 in the Supplementary Appendix).19 Length, weight, weight for length, body-mass index, head circumference, and mid–upper-arm circumference were expressed as sex- and age- (or gestational age–) standardized z scores according to INTERGROWTH-21st standards for newborn size,20 postnatal growth standards for preterm infants to 64 weeks of postmenstrual age (weight, length, and head circumference only),21 or World Health Organization (WHO) child growth standards.22

The primary outcome was length-for-age z score at 1 year (364 to 420 days). Secondary outcomes included other infant anthropometric variables; preterm birth (<37 weeks of gestation); gestational hypertension; delivery characteristics; stillbirth; mother and infant symptoms, clinical encounters, and hospitalizations; deaths; congenital anomalies; infant neurologic disabilities; and infant rickets (for a complete list of secondary outcomes, see Table S4 in the Supplementary Appendix). Biochemical screening for rickets was scheduled at 6 months of age (see Methods, section 2, in the Supplementary Appendix). Radiologic confirmation was based on interpretations of images of the wrist, knee, or both by a pediatric radiologist who was unaware of other data. Post hoc classifications of infants’ neurologic disabilities, congenital anomalies, and physician-assigned diagnostic codes for clinical encounters and hospitalizations were made by trial investigators who were unaware of treatment allocation.

The primary safety measure was maternal total serum calcium concentration at enrollment, 30 weeks of gestation, delivery, 3 months and 6 months post partum, or during hospitalization (if feasible). “Possible hypercalcemia” was defined as any serum calcium concentration of more than 2.60 mmol per liter (>10.4 mg per deciliter), and “confirmed hypercalcemia” (primary safety outcome) as a serum calcium concentration greater than 2.60 mmol per liter on a repeat specimen or a single serum calcium concentration greater than 2.80 mmol per liter (>11.2 mg per deciliter).

Secondary safety indicators included the serum calcium concentration of infants at 3 and 6 months of age and the urinary calcium:creatinine ratio of mothers at delivery. “Possible hypercalciuria” in mothers was defined as a single urinary calcium:creatinine ratio of more than 1, with both calcium and creatinine measured in millimoles (or >0.35, with both measured in milligrams). Participants with a urinary calcium:creatinine ratio of more than 1 in two consecutive specimens (i.e., confirmed hypercalciuria), symptoms of renal colic, or both underwent ultrasonography for urolithiasis or nephrolithiasis. Infant urinary calcium:creatinine ratios were measured at 6 months of age. Serum calcium concentrations and urinary calcium:creatinine ratios were analyzed at icddr,b.

Vitamin D status was based on serum 25-hydroxyvitamin D23 concentration, with deficiency defined as a concentration below 30 nmol per liter (<12 ng per milliliter).24 The C3-epimer fraction was included only in sensitivity analyses. Measurement of 25-hydroxyvitamin D and intact parathyroid hormone (iPTH) was conducted by the Analytical Facility for Bioactive Molecules at the Hospital for Sick Children, Toronto (Methods, section 3, in the Supplementary Appendix).

Statistical Analysis

The primary analysis was a complete-case intention-to-treat analysis. Analysis of variance was performed to compare length-for-age z scores at 1 year of age across all groups. To estimate the effect of weekly administration of prenatal vitamin D, five pairwise comparisons were conducted with the use of t-tests: prenatal 4200 versus placebo, prenatal 16,800 versus placebo, prenatal 16,800 versus prenatal 4200, prenatal 28,000 versus placebo, and prenatal 28,000 versus prenatal 16,800. Statistical significance was tested with a two-sided alpha level of 0.05, with the Holm test applied for multiple comparisons.25

The determination of sample size was based on the conservative assumption that if each between-group comparison had a two-sided alpha of 0.01 and 90% power, 220 participants per group would enable detection of a between-group difference in length-for-age z score of at least 0.40.14 To accommodate an attrition rate of 15%, we aimed to enroll 260 pregnant women in each group.

The effect of postpartum vitamin D on length-for-age z score at 1 year of age was assessed by means of a pairwise comparison of the prenatal and postpartum 28,000 group with the prenatal 28,000group (with a two-sided alpha level of 0.05) with the use of a t-test. Secondary outcomes were compared across groups with the use of analysis of variance for continuous, normally distributed variables and with the Kruskal–Wallis tests for skewed distributions; chi-square and Fischer’s exact tests were used for categorical variables. Zero-inflated negative binomial models were used to compare incidence rates of clinical encounters, hospitalizations, and other adverse events. When the result of a global test was significant (P<0.05), post hoc pairwise comparisons were performed, with the Holm test applied for multiple comparisons.25 We did not control for the multiplicity of comparisons of the secondary outcomes; any significant differences were viewed as exploratory. We conducted sensitivity and stratified analyses of the primary outcome and multiple imputation with chained equations to account for missing anthropometric data at 1 year of age. Trajectories of infant length-for-age z scores and other anthropometric measures were estimated with the use of restricted cubic spline regression models. Per-protocol analyses were restricted to participants who consumed at least 90% of scheduled supplement doses and had no episodes of reported consumption of nontrial vitamin D or calcium (see Methods, sections 4 to 7, in the Supplementary Appendix for additional details). Analyses were performed with the use of Stata, version 13 (StataCorp).