European Journal of Clinical Nutrition (2012), 14

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ORIGINAL ARTICLE
Feto-maternal vitamin D status and infant whole-body
bone mineral content in the rst weeks of life
DK Dror1, JC King2, DJ Durand2, EB Fung2 and LH Allen1

BACKGROUND/OBJECTIVES: Compromised vitamin D status is common in pregnancy and may have adverse impacts on fetal
development. The purpose of this study was to investigate the association of infant whole-body bone mineral content (WBBMC)
at 821 days of age with feto-maternal vitamin D status in a multiethnic population in Oakland, California.
SUBJECTS/METHODS: This was a cross-sectional study of 120 women and their newborn infants. Maternal and cord blood
were collected at delivery. WBBMC was measured by dual-energy X-ray absorptiometry in term-born infants 821days post birth.
RESULTS: No signicant association was observed between unadjusted or size-adjusted WBBMC and feto-maternal vitamin D
status analyzed continuously or categorically. In multivariate modeling, unadjusted WBBMC was predicted by bone area
(Po0.0001), weight-for-age (Po0.0001) and weight-for-length (P 0.0005) Z-scores, but not by feto-maternal vitamin D status.
Anthropometric predictors but not vitamin D remained signicant in the multivariate model after adjustment of WBBMC for weight,
bone area (bone mineral density) or logarithmically derived exponents of the denominators.
CONCLUSIONS: Results of the present study do not support an association between feto-maternal vitamin D status and early infant
WBBMC, raw or adjusted for inter-individual differences in size, in a multiethnic population in Northern California.

European Journal of Clinical Nutrition advance online publication, 11 July 2012; doi:10.1038/ejcn.2012.79
Keywords: vitamin D; infant; bone mineral content

INTRODUCTION
Fetal bone mineralization is determined by placental mineral
transfer and fetal bone turnover, with maternal nutrient status,
disease and conditions affecting placental transfer having possible
impacts on fetal bone.1 Bone mineralization and growth during
fetal development and early infancy may have implications for
childhood growth and development, peak bone mass and later
risk for osteoporosis.2,3
Vitamin D, which in its biologically active form 1,25(OH)2D acts
as a potent genetic regulator, may inuence fetal bone accretion
by upregulating expression of genes encoding rate-limiting
placental calcium transport proteins (plasma membrane calcium
ATPase14).4 Maternal vitamin D status, especially during the third
trimester of gestation, has been associated with neonatal bone
growth or mineralization in some studies57 but not in others.8
In adults, the relationship between serum 25(OH)D and bone
mineral density (BMD) varies among Caucasians, African Americans
and Hispanics.9 A single study with limited sample size (n 50) has
previously investigated feto-maternal vitamin D status and infant
whole-body bone mineral content (WBBMC) in the rst 2 weeks of
life.10 Oakland, California hosts a racially diverse population, with
skin color and genetic variance affecting maternal vitamin D status
and possibly fetal bone mineralization. The purpose of the present
study was to investigate the relation between feto-maternal vitamin
D status and WBBMC at 821 days of age in a multiethnic
population in Oakland, CA (381N).
SUBJECTS AND METHODS
The study was approved by the institutional review boards of the
University of California, Davis, Childrens Hospital & Research Center,
Oakland and Alta Bates Medical Center, Berkeley. Recruitment took place
between December 2006 and January 2008. Study subjects were mothers
obtaining perinatal care at East Bay Perinatal Medical Associates in
Oakland, CA and their infants. Women who were 1845 years of age and
carrying a singleton fetus were eligible for recruitment and infants born at
term (3742 weeks of gestation) with dual-energy X-ray absorptiometry
(DXA) scans between 8 and 21 days were included in analysis. Informed
consent was obtained from all the mothers upon study enrollment during
weeks 3440 of gestation.
An interviewer-administered questionnaire was used to evaluate intake
and lifestyle factors related to vitamin D status as described previously.11
Medical records were reviewed to ascertain pre- or early-pregnancy weight,
length of gestation, pregnancy and delivery events, and infant birth weight.
Pre-gestational maternal body mass index (BMI) was calculated from
reported height and pre- or early-pregnancy weight recorded in the
medical le.
Maternal venous blood was collected upon admission to the Alta Bates
Medical Center Labor and Delivery Unit, and cord blood immediately post
delivery. Blood samples were kept at 4 1C until centrifuged and serum
stored at  80 1C until analysis. Batched samples of serum 25(OH)D were
assayed monthly at ARUP Laboratories (Salt Lake City, UT, USA) using the
DiaSorin radioimmunoassay (DiaSorin Inc., Stillwater, MN, USA). An internal
standard that had been assayed in duplicate in the laboratory of Dr Bruce
Hollis (Medical University of South Carolina, Charleston, SC, USA) was
included with each batch and results were adjusted accordingly. Medical
records were reviewed to ascertain the length of gestation and infant birth
weight.
Motherinfant pairs visited the Childrens Hospital & Research Center
Oaklands Clinical and Translational Science Institute (CTSI) Clinical
Research Center 821 days post birth. The length, weight and head
circumference of the infants were measured by trained research staff using
the World Health Organizations standardized protocol.12 Weight was
measured to the nearest gram using an infant digital scale (Seca 334, Seca
Corp., Hamburg, Germany), length to the nearest 0.1 cm on an infant
length board (Shorr Infant Polylength Measuring Board, Shorr Productions,

1
Allen Lab, USDA, ARS Western Human Nutrition Research Center, Davis, CA, USA and 2Childrens Hospital & Research Center, Oakland, CA, USA. Correspondence: Dr DK Dror,
Allen Lab, USDA, ARS Western Human Nutrition Research Center, 430 West Health Sciences Drive, Davis, CA 95616, USA.
E-mail: daphnadror@gmail.com
Received 22 November 2011; revised 21 May 2012; accepted 7 June 2012
 Vitamin D status and neonatal bone mineral content
DK Dror et al
2
Olney, MD, USA), and head circumference to the nearest 0.1 cm (Shorr
Productions).
Bone mineral content, bone area and mass were determined by DXA in
duplicate using the Hologic Discovery A whole body infant software
package (version 12.6.1, Hologic Inc., Waltham, MA, USA). The instrument
was calibrated daily using a manufacturer-provided spine phantom. Before
imaging, each infant was dressed only in a clean study-provided diaper
and swaddled in a cotton receiving blanket to restrict movement. Infants
were breast- or formula-fed and pacied immediately before the scan, and
the scan with least movement artifact was selected for inclusion in analysis.
A single trained technician evaluated the acceptability of all scans and
compartmentalized the scan into two regions (head and remainder of
body). As the International Society of Clinical Densitometry (ISCD) has no
standard for DXA measurements in children under the age 5 years, WBBMC
was analyzed raw and adjusted for bone area (BMD) or weight in
accordance with the ISCD standards for pediatrics.13
Size for gestational age at birth was calculated based on fetal growth
reference curves with o10th percentile dened as small for gestational
age and 490th percentile dened as large for gestational age.14
Z-scores for infant weight and length were calculated using CDC
reference data sets.15 Seasonal boundaries were based on sine curve
analysis of maternal serum 25(OH)D concentrations and were dened as
21 January20 April, 21 April20 July, 21 July20 October, and 21 October
20 January as described previously.11
Statistical analysis
Statistical analysis was performed using SAS software (version 9.1, SAS
Institute Inc., Cary, NC, USA). Data are presented as meanss.d. 25(OH)D
was analyzed both as a continuous and categorical variable. Normality of
study variables was tested using the ShapiroWilk statistic. Regression
models including the quadratic term of the variable of interest were used
to test for threshold effects. Path analysis was conducted to identify
contributors to the effect of anthropometrics on WBBMC. Differences in
variable means between groups were evaluated by analysis of variance
using Tukeys test to correct for multiple comparison. Analysis of
covariance was used to construct multivariate models utilizing the
backwards elimination method.
Bone mineral content of the infant whole body and body less
the head were highly correlated (r 0.88, Po0.0001), therefore
whole-body variables were used in the remainder of the analyses.
Infant WBBMC was not signicantly correlated with maternal or
cord 25(OH)D (Figure 1), nor did WBBMC differ signicantly by
maternal or cord vitamin D status categorized by cutpoints
(Table 2) or tertiles (data not shown). Furthermore, no threshold
effect was found between maternal or cord 25(OH)D and infant
WBBMC. Excluding morbidly obese women (n 18), women with
gestational diabetes (n 16) or large for gestational age infants
(n 13) did not alter the results. Other infant characteristics,
including length of gestation, size for gestational age, sex, birth
weight and weight or length at the DXA visit, were not associated
with maternal or cord serum 25(OH)D.
To reduce multicollinearities and control for infant age in
multivariate linear models, weight-for-height and height-for-age
Z-scores were substituted for raw anthropometrics. Infant WBBMC
was signicantly predicted by bone area (Po0.0001) and length-
for-age (Po0.0001) and weight-for-length (P 0.0005) Z-scores at
the time of the DXA scan, with an overall model R2 0.84. Factors
not remaining in the nal predictive model were maternal and
cord 25(OH)D; maternal height, pre-pregnancy BMI or gestational
diabetes; and infant age, length of gestation, size for gestational
age and feeding practice. In path analysis, infant age and maternal
pre-pregnancy BMI were found to mediate the inuence of
anthropometrics at the DXA visit on infant WBBMC.
100
90
80
WBBMC (g)

70

RESULTS 60
Of 143 infants born at term who underwent DXA scans at 821 50
days, 11 were excluded due to poor scan quality and 12 due to
missing maternal or cord serum 25(OH)D. The 120 infants included 40
in the analysis (Table 1) did not differ signicantly from those who
underwent DXA scans but were not included (n 23), or who 30
were eligible but did not return for the DXA visit (n 32), in
gender, maternal age, parity, or maternal or cord vitamin D status. 20
0 20 40 60 80 100 120 140 160 180
Maternal 25(OH)D (nmol/L)

Table 1. Characteristics of maternal and infant study subjects (n 120 100

motherinfant pairs)
90
Means.d. Range
80
Infant birth weight (g) 3420542 22904532
WBBMC (g)

Length of gestation (weeks) 39.61.3 3742 70

Maternal 25(OH)Da (nmol/l) 75.532.3 20.8172.8
Cord 25(OH)D (nmol/l) 44.522.1 13.0157.5 60
Infant age at DXA visit (days) 13.33.5 821
Infant weight at DXA (g) 3655544 24135448 50
Weight-for-age Z-scoreb  0.30.9  2.22.3
Infant length at DXA (cm) 50.92.4 43.558.2 40
Length-for-age Z-scoreb  0.40.9  3.51.9
Weight-for-length Z-scoreb 0.10.7  2.32.6 30
Infant WBBMC (full body, g) 62.112.7 35.495.9
20
Infant body BMC (head subtracted, g) 33.57.9 17.358.5 0 20 40 60 80 100
Infant whole-body BMD (g/cm)b 0.200.02 0.150.25
Cord 25(OH)D (nmol/L)
Abbreviations: BMC, bone mineral content; BMD, bone mineral density;
DXA, dual-energy X-ray absorptiometry. aMaternal 25(OH)D drawn at the African American Hispanic Caucasian Asian Mixed
time of delivery. bZ-scores calculated per Centers for Disease Control and
Figure 1. Distribution of WBBMC by maternal (a) and cord (b) serum
Prevention (CDC) reference data set.
25(OH)D.

European Journal of Clinical Nutrition (2012) 1 4 & 2012 Macmillan Publishers Limited
 Vitamin D status and neonatal bone mineral content
DK Dror et al
3
Table 2. Mean WBBMC by category (analysis of variance) predicting size (maternal pre-pregnancy BMI and infant age at the
time of the scan).
N (%) Means.d. P-value Because of the role that vitamin D has in bone mineralization
BMC (g) and the association of mineral accretion and growth early in life
with future bone integrity, several investigators have sought to
Maternal 25(OH)D 0.58
measure the relationship between vitamin D status and in utero or
o27.5 nmol/l 6 (5.0) 67.25.2
27.579.99 nmol/l 67 (55.8) 62.21.6 early postnatal bone mineralization.10,16 Despite the range in
X80 nmol/l 47 (39.2) 61.41.9 serum 25(OH)D demonstrated in the present study, no signicant
association was found between maternal or cord 25(OH)D
Cord 25(OH)D 0.44 concentration and infant WBBMC. This nding contrasts with
o27.5 nmol/l 26 (21.7) 64.92.5 that of Weiler et al.,10 in which both maternal and cord plasma
27.579.99 nmol/l 86 (71.7) 61.31.4 25(OH)D remained signicant in a multivariate model predicting
X80 nmol/l 8 (6.6) 62.74.5 WBBMC in term-born Canadian infants o15 days of age, albeit
with negative and positive coefcients, respectively. However, in
Infant sex 0.39
the same study infants who were vitamin D decient (plasma
Male 58 (48.3) 61.11.7
Female 62 (51.7) 63.11.6 25(OH)Do27.5 nmol/l) or born to vitamin D-decient mothers
(plasma 25(OHD)o37.5 nmol/l) were heavier and longer than
Season of birtha 0.34 those who were not. Therefore, it is possible that the difference in
21 Jan20 April 21 (17.5) 61.42.7 results was mediated by an association between vitamin D status
21 April20 July 30 (25.0) 61.02.8 and size, which was not found in the present study.
21 July20 Oct 28 (23.3) 58.92.4 It is possible that the comparatively better vitamin D status of
21 Oct20 Jan 41 (34.2) 63.22.0 the population included in the present study may have obscured
an association between vitamin D status and infant size and bone
Feeding behavior 0.34
mineral content. A higher prevalence of maternal and cord serum
Breastfed 50 (41.7) 60.21.8
Formula-fed 26 (21.7) 64.12.5 25(OH)D concentrations o50 nmol/l has been measured in
Mixed 44 (36.7) 63.21.9 studies conducted at more northerly latitudes (42% and 35%,
respectively, at 461N and 531N in Canada17 and 6077% and 52%,
Maternal pre-pregnancy 0.04 respectively, at 601N in Finland7).
BMIb (kg/m2) In adults, variance in body size is accounted for by adjusting bone
1924.99 (normal) 18 (16.5) 56.93.0 mineral content for 2-dimensional bone area (areal BMD). In
2529.99 (overweight) 39 (35.8) 62.72.0 children, BMD is a less valid index due to the dynamic process of
3040 (obese) 34 (31.2) 62.72.1 bone turnover and rapid growth leading to asynchronies between
440 (morbidly obese) 18 (16.5) 69.03.0
increase in bone area and bone mineral.18 Nevertheless, to account
Size for GA o0.0001 for individual size differences among infants, investigators have
Small for GA 19 (15.8) 48.02.3 adjusted WBBMC for bone area or infant weight.7,8,10 In the present
(o10th percentile) analyses, such adjustments were made and the power of the
Appropriate for GA 88 (73.4) 62.71.1 denominator further optimized through logarithmic analysis.
Large for GA 13 (10.8) 78.92.8 Despite adjustments, vitamin D status was unassociated with bone
(490th percentile) measurements. That anthropometric indices remained signicant
Abbreviations: BMC, bone mineral content; BMI, body mass index; GA, predictors in the multivariate models of adjusted WBBMC indicates
gestational age; WBBMC, whole-body bone mineral content. aSeasonal that adjustment of the independent variable for weight or bone area
boundaries based on sine curve analysis of maternal serum 25(OH)D alone did not sufciently control for inter-individual variances in size.
concentrations. bMissing values for pre-pregnancy BMI in 11 participants. Limitations of the present study include restricted sample size
and inherent difculty in collecting DXA data in infants. Type II error
and the fact that a large percentage of mothers in the study had
As WBBMC was most strongly inuenced by infant size, adequate vitamin D status may have precluded an ability to detect
adjustments to the independent variable were attempted to differences in infant WBBMC. Sample size was further limited by the
create a size-neutral index and further consider a potential difculty in restricting movement in young infants during the DXA
inuence of vitamin D status. Simple adjustment of WBBMC for scan. To minimize movement artifact, infants were tightly swaddled,
infant weight (kg) or bone area (as determined by DXA; BMD) fed and pacied immediately before the scan, and the better of two
over- or under-adjusted for size differences per analysis of scans was included in data analysis. Nonetheless, conducting DXA
residuals. To best adjust for inter-individual differences in size as scans on a larger sample of infants is warranted to rule out a
determined through logarithmic analysis, WBBMC was divided by threshold effect of vitamin D status on bone mineralization.
weight1.3 or bone area1.4. No signicant correlations were In conclusion, no association was found between early infant
observed between size-adjusted infant WBBMC and maternal or WBBMC, raw or adjusted for inter-individual differences in size,
cord 25(OH)D. In multivariate modeling of size-adjusted infant and maternal or cord vitamin D status in a multiethnic population
WBBMC, anthropometric indicators were signicant predictors in Northern California.
whereas maternal and cord 25(OH)D were not.

DISCUSSION
In this cohort of multiethnic motherinfant pairs in Oakland, CA
(381N), WBBMC in full-term infants at 821 days of age was not
related to maternal or cord vitamin D status, season of birth,
feeding habits or sex but was most strongly inuenced by various
indices of infant size (bone area, height-for-age and weight-
for-age Z-scores) and factors determined to be in the pathway for
CONFLICT OF INTEREST
The authors declare no conict of interest.
ACKNOWLEDGEMENTS
We wish to acknowledge the nancial support for this study from the USDA, ARS
Western Human Nutrition Research Center and the East Bay Neonatology
Foundation. We extend a special thanks to Janet M Peerson at UC Davis for her
assistance with statistical analyses.

& 2012 Macmillan Publishers Limited European Journal of Clinical Nutrition (2012) 1 4
 Vitamin D status and neonatal bone mineral content
DK Dror et al
4
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