JBMR
CLINICAL TRIALS
Vitamin D Supplementation During Pregnancy:
Double-Blind, Randomized Clinical Trial of Safety
and Effectiveness
Bruce W Hollis , 1 Donna Johnson , 3 Thomas C Hulsey , 2 Myla Ebeling , 2 and Carol L Wagner1
1
Division of Neonatology and Department of Obstetrics and Gynecology, Medical University of South Carolina, Charleston, SC, USA
Division of Epidemiology, Department of Pediatrics, and Medical University of South Carolina, Charleston, SC, USA
3
Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, Medical University of South Carolina, Charleston,
SC, USA
2
ABSTRACT
The need, safety, and effectiveness of vitamin D supplementation during pregnancy remain controversial. In this randomized, controlled
trial, women with a singleton pregnancy at 12 to 16 weeks’ gestation received 400, 2000, or 4000 IU of vitamin D3 per day until delivery.
The primary outcome was maternal/neonatal circulating 25-hydroxyvitamin D [25(OH)D] concentration at delivery, with secondary
outcomes of a 25(OH)D concentration of 80 nmol/L or greater achieved and the 25(OH)D concentration required to achieve maximal
1,25-dihydroxyvitamin D3 [1,25(OH)2D3] production. Of the 494 women enrolled, 350 women continued until delivery: Mean 25(OH)D
concentrations by group at delivery and 1 month before delivery were significantly different ( p < 0.0001), and the percent who achieved
sufficiency was significantly different by group, greatest in 4000-IU group ( p < 0.0001). The relative risk (RR) for achieving a concentration
of 80 nmol/L or greater within 1 month of delivery was significantly different between the 2000- and the 400-IU groups (RR ¼ 1.52, 95% CI
1.24–1.86), the 4000- and the 400-IU groups (RR ¼ 1.60, 95% CI 1.32–1.95) but not between the 4000- and. 2000-IU groups (RR ¼ 1.06, 95%
CI 0.93–1.19). Circulating 25(OH)D had a direct influence on circulating 1,25(OH)2D3 concentrations throughout pregnancy ( p < 0.0001),
with maximal production of 1,25(OH)2D3 in all strata in the 4000-IU group. There were no differences between groups on any
safety measure. Not a single adverse event was attributed to vitamin D supplementation or circulating 25(OH)D levels. It is
concluded that vitamin D supplementation of 4000 IU/d for pregnant women is safe and most effective in achieving sufficiency in
all women and their neonates regardless of race, whereas the current estimated average requirement is comparatively ineffective
at achieving adequate circulating 25(OH)D concentrations, especially in African Americans. ! 2011 American Society for Bone and
Mineral Research.
KEY WORDS: VITAMIN D; CHOLECALCIFEROL; PREGNANCY; NEONATE
Introduction
T
he function of vitamin D during pregnancy for both mother
and fetus remains largely undefined. Vitamin D is known to
be involved in skeletal homeostasis during pregnancy, as
evidenced by a recent publication dealing with craniotabes in
the newborn, and severe vitamin D deficiency may lead to
neonatal seizures in neonates with profound hypocalcemia.(1–5)
The function of vitamin D during this sensitive period, however,
also may have potential effects on other systems, including
immune,(6–10) pancreatic,(11–13) musculoskeletal,(14–17) and cardiovascular function,(18–20) as well as neural development.(21–24)
Recent publications suggest relationships between maternal
vitamin D status and adverse pregnancy outcomes such as
preeclampsia and cesarean section.(25–28)
A Cochrane Review published in 2000 highlighted the relative
dearth of data dealing with vitamin D supplementation during
human pregnancy.(29) This review listed seven studies on the
topic,(30–36) of which four reported clinical outcomes.(30–32,36)
From these limited data, the Cochrane Review concluded that
there was insufficient evidence to evaluate the effects of vitamin
D supplementation during pregnancy.(29) Since that time, few
studies have addressed this issue.(37–39)
In 2004, we initiated a National Institute of Child Health and
Human Development (NICHD)–sponsored 6-year randomized,
double-blind, placebo-controlld trial of vitamin D supplementa-
Received in original form March 29, 2011; revised form June 7, 2011; accepted June 10, 2011. Published online June 27, 2011.
Address correspondence to: Bruce W Hollis, PhD, Medical University of South Carolina, 173 Ashley Avenue, MSC 513, Charleston, SC 29425, USA.
E-mail: hollisb@musc.edu
Presented in part at the 14th Vitamin D Workshop, Brugge, Belgium, October 2009, and the Pediatric Academic Societies Meeting, Vancouver, British Columbia,
Canada, May 2010.
For a Commentary on this article, please see Abrams (J Bone Miner Res. 2011;26:2338-2340. DOI: 10.1002/jbmr.498).
Journal of Bone and Mineral Research, Vol. 26, No. 10, October 2011, pp 2341–2357
DOI: 10.1002/jbmr.463
! 2011 American Society for Bone and Mineral Research
2341
tion during pregnancy to assess safety and pregnancy outcomes
with an approved Investigational Drug Application from the US
Food and Drug Administration (FDA; No. 66,346). We hypothesized that 4000 IU/d of vitamin D3 would be more efficacious and
effective than the standard dosing regimen of 400 IU/d and the
2000 IU/d (the former upper limit for vitamin D) dosing regimen
in achieving a total circulating 25-hydroxyvitamin D [25(OH)D]
level of at least 80 nmoL/L (32 ng/mL) in pregnant women
regardless of race throughout pregnancy and at the time of
delivery without causing any safety concerns. This minimal value
of 80 nmol/L was based on years of research with regard to
circulating 25(OH)D levels suppressing secondary hyperthyroidism and having optimal intestinal calcium absorption and bone
mineral density.(41) These results are presented here.
Methods
Study design
This study was a single-center, randomized, controlled, doubleblind study of vitamin D supplementation stratified by race (FDA
IND No. 66,346; ClinicalTrials.gov No. NCT00292591). Women at
fewer than 16 weeks’ gestation with a singleton pregnancy were
eligible for participation in the study.
Study participants and setting
This study was approved by Medical University of South
Carolina’s (MUSC’s) Institutional Review Board for Human
Research (HR No. 10725) and was conducted from January 4,
2004, through July 31, 2009, at MUSC (Charleston, SC, USA). The
inclusion criteria for the subjects included the following: (1)
maternal age of 16 years or greater at the time of consent, (2)
confirmed singleton pregnancy of fewer than 16 completed
weeks of gestation at the time of consent, (3) planned to receive
ongoing prenatal care in the Charleston, SC, area, and (4) the
ability to provide written informed consent at the first visit. If a
woman received her obstetrical care at a facility separate from
MUSC, then she came to MUSC’s Clinical and Translational
Research Center (CTRC) outpatient research facility for each of
the study visits. Women were consented at their first prenatal
visit, at which time baseline 25(OH)D levels were measured.
Irrespective of gestational age at enrollment, subjects began
vitamin D supplementation between the start of the twelfth and
the start of the sixteenth weeks of gestation (12 0/7th and 16 0/
7th weeks), as defined by their last menstrual period.
Exclusion criteria
Women with a pregnancy at greater than 16 weeks of gestation
as calculated by their last menstrual period were not eligible
to participate. Pregnant women with preexisting calcium or
parathyroid conditions or who required chronic diuretic or
cardiac medication therapy, including calcium channel blockers,
or who suffered chronic hypertension were not eligible for
enrollment in the study. Pregnant women with active thyroid
disease (eg, Graves disease, Hashimoto disease, or thyroiditis)
also were excluded, but mothers on thyroid supplement with
normal serologic parameters could participate in the study if
they were without any other endocrine dysfunction.
2342
Journal of Bone and Mineral Research
Study protocol
Gestational age at enrollment
Subjects could be consented and enrolled into the study before
the initiation of vitamin D supplementation at 12 to 16 weeks of
gestation. Gestational age was based on last menstrual period. If
a woman was unsure of her gestational age, the obstetrical
estimate at the time of the visit was used. If, at the 20-week fetal
ultrasound it was determined by the obstetrician that the
gestational age was incorrect, the revised gestational age was
used and the discrepancy noted.
Initial study visit
Baseline blood and urine samples were obtained following
subject consent at the initial visit, but the earliest time of
randomization following measurement of baseline total circulating 25(OH)D level was 12 weeks’ gestation, with the target
upper limit of gestation of 16 weeks. Irrespective of enrollment
gestational age, vitamin D supplementation did not begin before
the twelfth week of gestation (12 and 0/7th weeks).
Subsequent study visits
Subjects were followed with monthly study visits, which
continued until delivery. The visits coincided with routine
obstetrical visits or were performed in conjunction with those
visits if the obstetrical care was provided outside MUSC. The
subjects also were seen at the GCRC/CTRC for a study visit at
16 weeks of gestation and with their infant at 2 weeks’
postpartum.
Completion of questionnaires
Following their written informed consent, mothers completed
questionnaires regarding sociodemographic information, baseline health status, and medical history at the first visit. At the
second visit, the Block Food Frequency Questionnaire (FFQ) was
completed to ascertain generalized eating pattern, with specific
calculation of calcium and vitamin D intake (Block, Berkeley, CA,
USA).(42–47) Each completed FFQ form was sent to the processing
center (Berkeley, CA, USA), and these data were reviewed later
for accuracy by a registered dietician who was blinded to subject
treatment group assignment. Total caloric, vitamin D, and
calcium intakes were recorded for each subject.
An interim maternal health history questionnaire also was
completed at each visit with the assistance of the study
coordinator to ascertain adverse events, discussing types and
frequencies of acute illnesses such as respiratory, gastrointestinal, and other viral and/or bacterial illnesses. A review of
medications and doctor’s visits was obtained at that time.
After delivery, the newborn record of each infant was
reviewed for mode of delivery and level of neonatal care
required (normal newborn nursery or level 2 or level 3 intensive
care). Birth weight (g) and gestational age also were recorded.
Blood and urine samples
Maternal blood and urine samples were collected at each visit.
Cord blood was obtained at delivery. If the cord blood sample
HOLLIS ET AL.
could not be obtained, a neonatal blood sample was drawn
within 2 weeks of delivery.
Intervention
Multivitamin and vitamin D supplementation
Pregnant women who presented for prenatal care at 16 or fewer
weeks of gestation were randomized into one of three treatment
regimens of vitamin D3 after establishing their baseline serum
25(OH)D level. All patients received a total of two pills daily: a
standard prenatal multivitamin vitamin containing 400 IU of
vitamin D and an additional vitamin D3 supplement of 0 IU
(placebo), 1600 IU, or 3600 IU of vitamin D3 for a total of 400 IU,
2000 IU, and 4000 IU of vitamin D supplementation, respectively.
In order to obtain Institutional Review Board approval for the
study, the following safety measure was put into place: Baseline
total circulating 25(OH)D levels were measured, and women
with levels of 100 nmol/L (40 ng/mL) or less were eligible for
randomization into one of the three arms (400, 2000, or 4000 IU/
d of vitamin D3) with further substratification by race within each
treatment group. Women with baseline 25(OH)D levels greater
than100 to 150 nmol/L (>40 to 60 ng/mL, levels considered to be
in the normal range at the time of study implementation) were
randomized into one of two treatment groups (400 or 2000 IU/d
of vitamin D3), whereas women with a baseline 25(OH)D level
greater than 150 nmol/L (>60 ng/mL) were given 400 IU/d of
vitamin D3. The doses of vitamin D used in our study were
selected based on current recommendations (400 IU/d), the
upper safe intake level established in 1997 (2000 IU/d),(40) and
the amount we calculated to be required to achieve nutritional
vitamin D sufficiency (4000 IU/d).(48).
Adherence to medication regimen
Adherence to the prescribed vitamin D supplementation
regimen of one prenatal vitamin and the vitamin D supplement
was measured by maternal self-report and pill counts at each
follow-up visit.(49) The number of vitamin D pills returned was
divided by the expected number of pills that would have been
taken between study visits to generate a percentage that served
as a measure of adherence of medication regimen between
study visits. The adherence measures were used to generate an
average adherence for each subject.(49) If a woman missed one
prenatal visit, her next month supply of vitamins was either
mailed to her or dropped off at her residence. In such cases,
medication adherence was based on the pill count from the date
of the last visit to the current prenatal visit over the expected
number of pills taken. If a woman had more than two missed
visits or if she failed to take at least 50% of the prescribed vitamin
D pills, she was exited from the study.
Randomization
Our study used stratified blocked randomization to balance by
ethnicity and also to balance by enrollment (as a cautionary
measure against a potential temporal or seasonal bias). A
randomization scheme was developed separately for each of the
three ethnic groups (ie, the strata). Within each stratum, the
treatments were assigned within blocks. Because there were
VITAMIN D SUPPLEMENTATION DURING PREGNANCY
three treatment groups, the block size had to be divisible by 3;
the data team selected a block size of six, which was unknown to
the investigators or the pharmacists. In this way, at the end of
each block (ie, enrollment of six subjects), each ethnic group was
balanced in the number randomly assigned to the 400-, 2000-,
and 4000-IU treatment groups.
Materials
Source of vitamin D
Vitamin D tablets were manufactured by Tishcon Corporation
(Westbury, NY, USA), a Good-Manufacturing-Practice (GMP)
facility. The cholecalciferol contained in the vitamin D tablet was
supplied to Tishcon Corporation by Hoffman-La Roche, Ltd.
(Basel, Switzerland). The tablet vitamin D concentration was
verified by the company every 6 months and by an independent
laboratory chosen by the investigators (Heartland Assays, Ames,
IO, USA) using high-performance liquid chromatography with UV
detector (HPLC-UV) to ensure that the tablets met label claims
throughout the study; these results were reported to the
Investigational Drugs Department at MUSC. Tablets were
maintained in MUSC’s Research Pharmacy until the time that
they were dispensed to each enrolled subject.
Source of prenatal vitamins
Prenatal vitamins prescribed at the time of each subject’s
enrollment were manufactured by Myadec MultivitaminMultimineral Supplement (distributed by Pfizer Consumer
Healthcare, Morris Plains, NJ, USA) with 400 IU of vitamin D3
per tablet. Mothers who were unable to swallow a prenatal
vitamin were given Flintstones Complete chewable vitamin
(Bayer Healthcare, Morristown, NJ, USA), which provided 400 IU
of vitamin D3 per tablet.
Measures
Maternal sociodemographic measures included maternal age at
time of enrollment, her self-defined race, insurance status,
educational status, and occupation and employment outside of
the home.
Pregnancy health status and labor and delivery
characteristics and complications
Characteristics of each mother’s health status and complications
during pregnancy, labor, and delivery were recorded and
reviewed by an obstetrician (DDJ, blinded to treatment). If the
mother required hospitalization, a copy of the hospital record
was obtained after the mother had signed a release of medical
information form. Any acute illnesses, hospitalizations, or
development of pregnancy-related conditions that were not
preexisting also were recorded. The Data Monitoring and Safety
Committee (DSMC) was notified of all such events.
Anthropomorphic measurements
Prepregnancy height and weight of each mother were recorded
at the first outpatient visit to determine BMI (weight [kg]/height2
Journal of Bone and Mineral Research
2343
[m2]). During subsequent visits, only the subject’s weight was
recorded. Birth weight (g) was recorded for each infant.
Laboratory measurements
Maternal and cord blood/neonatal vitamin D and
metabolite assays
Circulating vitamin D2 and D3 were measured in serum using
direct ultraviolet detection preceded by organic extraction and
high-performance liquid chromatography, as described previously.(50) This assay has a coefficient of variation of 10% or less
and a 5 nmol/L vitamin D detection limit. There is no normal
established circulating range of vitamins D2 or D3 in human
subjects.
A rapid, direct RIA developed in the Hollis laboratory and
manufactured by Diasorin Corporation (Stillwater, MN, USA) was
used to measure total circulating 25(OH)D concentration in
serum samples.(51) This RIA is an FDA-cleared device and, in fact,
is the FDA predicate device for the measurement of circulating
25(OH)D in humans.
Based on clinical laboratory classifications,(52,53) a priori,
deficiency was defined as a total circulating 25(OH)D level of
less than 50 nmol/L (20 ng/mL), insufficiency as 50 nmol/L or
greater to less than 80 nmol/L (“20 to
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