Helve et al.

BMC Pediatrics (2017) 17:91

DOI 10.1186/s12887-017-0845-5

STUDY PROTOCOL Open Access

Towards evidence-based vitamin D

supplementation in infants: vitamin D
intervention in infants (VIDI) — study
design and methods of a randomised
controlled double-blinded intervention
study
Otto Helve1* , Heli Viljakainen1,2, Elisa Holmlund-Suila1, Jenni Rosendahl1, Helena Hauta-alus1,
Maria Enlund-Cerullo1, Saara Valkama1, Kati Heinonen3, Katri Räikkönen3, Timo Hytinantti1, Outi Mäkitie1,2,4†
and Sture Andersson1†

Abstract
Background: Vitamin D is important for bone mass accrual during growth. Additionally, it is considered a
requirement for a multitude of processes associated with, for example, the development of immunity. Many
countries apply vitamin D supplementation strategies in infants, but the guidelines are not based on scientific
evidence and aim at prevention of rickets. It remains unclear whether the recommended doses are sufficient for
the wide array of other effects of vitamin D. The VIDI trial performed in Finland is the first large randomised
controlled study for evaluation of the effects of different vitamin D supplemental doses in infancy on:

1. bone strength
2. infections and immunity
3. allergy, atopy and asthma
4. cognitive development
5. genetic regulation of mineral homeostasis
Methods/Design: VIDI, a randomised controlled double-blinded single-centre intervention study is conducted in
infants from the age of 2 weeks to 24 months. Participants, recruited at Helsinki Maternity Hospital, are randomised
to receive daily either 10 μg (400 IU) or 30 μg (1 200 IU) of vitamin D3 supplementation. Both groups are assessed
at 6 months of age for calcium homeostasis, and at 12 and 24 months of age for parameters associated with bone
strength, growth, developmental milestones, infections, immunity, atopy-related diseases, and genetic factors
involved in these functions.
(Continued on next page)

* Correspondence: [email protected]
†
Equal contributors
1
Children’s Hospital, Pediatric Research Center, University of Helsinki and
Helsinki University Hospital, P.O. Box 28100029 HUS Helsinki, Finland
Full list of author information is available at the end of the article

© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Helve et al. BMC Pediatrics (2017) 17:91 Page 2 of 8

(Continued from previous page)

Discussion: The study enables evaluation of short and long term effects of supplemental vitamin D on growth,
immune functions and skeletal and developmental parameters in infants, and the effects of genetic factors therein.
The results enable institution of evidence-based guidelines for vitamin D supplementation in infancy.
Trial registration: ClinicalTrials.gov, NCT01723852, registration date 6.11.2012.
Keywords: Allergy, Asthma, Bone development, Cognitive development, Immunity, Infants, Infections, Vitamin D

Background were vitamin D deficient (<50 nmol/L) during the school

The biologically inactive vitamin D3 (cholecalciferol) is year [9, 10].
produced in the skin after solar UVB exposure. It is
bound to vitamin D binding protein (DBP) and trans- Role of vitamin D in health and disease
ported to the liver for conversion to 25-hydroxy-vitamin Growth and bone mineralisation
D (25-OHD), the most abundant circulating metabolite Serum calcium and phosphate concentrations are tightly
of vitamin D. In the kidney, 25-OHD is further hydrox- regulated by parathyroid hormone (PTH) and 1,25-
ylated to the active form calcitriol, 1,25-OH2D, that con- OH2D by negative feedback. Rickets is caused by insuffi-
tributes to calcium metabolism [1]. Paracrine conversion cient vitamin D supply resulting in the lack of sufficient
of 25-OHD to calcitriol occurs in many tissues, such as calcium and phosphate for bone mineralization at the
dendritic and endothelial cells, brain, placenta and para- growth plates [11]. The worldwide incidence of rickets is
thyroids, underscoring the importance of vitamin D for increasing [11]. Vitamin D deficiency is associated with
these organs [2]. Based on the number of cell types ex- low bone mineral density (BMD) and an unfavourable
pressing vitamin D receptors (VDR), 38 potential target bone development in children aged 6–16 years and in
tissues have been recognised [3]. In target cells, calcitriol newborns [12, 13]. However, data on the relationship be-
regulates the transcription of target genes, including sev- tween vitamin D and BMD in children are somewhat
eral genes involved in cell cycle regulation [4]. Locally contradictory. In healthy children aged 8–17 years with
produced calcitriol in immune cells participates in host S-25-OHD levels above 50 nmol/L, no effect of vitamin
defence reactions and acts as a potent immunomodula- D supplementation on BMD is seen [14], and the rela-
tor [2, 5]. In fact, vitamin D has been estimated to dir- tionship between BMD and S-25-OHD is inconsistent in
ectly and indirectly regulate the expression of 2–3% of infants and young children [15].
the human genome [4]. In infants, severe vitamin D deficiency (<25 nmol/L)
Both cutaneously synthesised and dietary vitamin D associates with poor linear growth and delayed motor
contribute to the circulating 25-OHD concentration. development possibly due to muscle weakness [16, 17].
There is no consensus on the optimal 25-OHD concentra- In vitamin D deficient children, vitamin D supplementa-
tion. In 2011, Institute of Medicine guideline stated that tion improved growth velocity and prevented stunted
25-OHD concentrations above 50 nmol/L are required for growth [18, 19]. The increase in S-25-OHD concentra-
normal body functions including linear growth and bone tion increased circulating IGF-1 level [18].
mass accrual [6]. According to the Endocrine Society [7],
for optimizing long-term health benefits such as the pre- Infections and immunity
vention of diabetes or fractures, concentrations above Rickets commonly presents with comorbidities such as
75 nmol/L may be needed. continuous or recurrent respiratory infections [20, 21].
Vitamin D supplementation has been recommended in Vitamin D has profound effects on the immune system
Finland for all infants since the 1940’s, but in line with by promoting immune responses and inducing innate
the declining prevalence of rickets, the recommended immune defences. Immune cells express vitamin D recep-
doses have gradually decreased. The present Finnish Nu- tor (VDR) and respond to vitamin D [22, 23]. On innate
tritional Council guidelines recommend 10 μg (400 IU) immunity, vitamin D inhibits the expression of pattern-
of vitamin D3 supplementation daily for all infants from recognition receptors, induces autophagy in macrophages
the age of 2 weeks to 24 months, and 7.5 μg (300 IU) to and induces endogenous antimicrobial peptides, such as
children aged 2 to 18 years. Despite these recommenda- cathelicidin. On adaptive immunity, for example, vitamin
tions, 20% of children were shown to be vitamin D defi- D acts by regulating the release of pro-inflammatory
cient (<50 nmol/L) at 14 months of age [8]. In addition, cytokines from mononuclear cells and inhibits T-cell
we have previously shown that more than 70% of 195 proliferation through decreased Th1 cytokine secretion
apparently healthy school children in Helsinki, Finland, [24–26]. Therefore, vitamin D does not only have a role in
Helve et al. BMC Pediatrics (2017) 17:91 Page 3 of 8

modulating host defence to infections, but is likely to con- Genetic regulation of mineral homeostasis
trol factors in autoimmunity, also in allergic disease. S-25-OHD concentration has high heritability (28–80%),
Vitamin D modifies response to both bacterial and which explains differences in responses to vitamin D
viral infections [24, 27]. In adults, vitamin D insuffi- supplementation [53]. Genome-wide association studies
ciency increases the risk of severe infections [28], but on vitamin D insufficiency identified a number of com-
the results from intervention trials are conflicting [29]. mon genetic variants that influence 25-OHD concen-
Vitamin D deficiency may contribute to increased occur- trations and the risk of insufficiency [54]. These
rence of lower respiratory tract infections in children [30]. included single nucleotide polymorphisms in the GC
In 167 Japanese children aged 6–16 years a supplemental gene encoding vitamin D binding protein, locus near
vitamin D3 of 1 200 IU for a period of 4 months resulted the 7-dehydrocholesterol reductase involved in vitamin D
in a significant reduction in influenza A infections [31]. In synthesis in the skin, and CYP2R1 coding cytochrome
addition, vitamin D deficiency is associated with upper P450 responsible for 25-hydroxylation. We have studied
respiratory tract, gastrointestinal and ear infections in GC polymorphisms in relation to vitamin D metabolism
children [32, 33]. Identification, treatment, and prevention and bone strength in children [55] and observed differ-
of vitamin D deficiency in early childhood may therefore ences in S-25-OHD and PTH concentrations between GC
have widespread health effects throughout childhood. genotypes and that variation exists in BMD and bone
strength index as well. The variation in S-25-OHD be-
tween GC genotypes suggests differences in vitamin D
Allergy, atopy and asthma
utilisation. Polymorphisms of the vitamin D receptor gene
In Finland, the prevalence of asthma among children aged
VDR associate with growth pattern in infancy [56, 57] and
7–16 years is 7% [34]. Based on epidemiological studies,
with bone strength [58]. VDR polymorphisms together
low maternal intake of vitamin D at pregnancy and low
with vitamin D supplementation affect bone mineral
cord blood 25-OHD concentration have been associated
accrual suggesting that vitamin D supplementation could
with a higher risk for asthma, allergic rhinitis and atopic ec-
be used to change the developmental trajectory and in-
zema in the offspring, as assessed at 5 years of age [35–38].
duce phenotype with lowered risk for chronic disease or
However, findings have not been consistent [39] and recent
disorder [59, 60]. Other possible candidate genes include
studies on prenatal vitamin D supplementation have not
CYP24A1 that encodes 24-hydroxylase initiating degrad-
demonstrated an effect on wheezing at 3 years of age [40,
ation of both 25-OHD and 1,25-OHD [54].
41]. Vitamin D may, however, have a protective effect
against childhood wheezing and asthma, but the evidence
Overall aims and research questions
is inconclusive [42]. In adults, IgE concentrations are higher
The aim of the study is to investigate long-term health
for those adults with low or very high concentration of
benefits of early vitamin D supplementation with a dose
serum 25-OHD (S-25-OHD) [43].
ensuring effective concentration of 25-OHD.
The primary outcomes of the study are bone strength
Cognitive development and cumulative frequency of infections at 24 months of
The association between concentrations of S-25-OHD age. We hypothesise that a higher vitamin D supplementa-
and cognitive abilities has been widely studied among tion to infants between 2 weeks and 24 months of age will
middle-aged and older individuals [44–46]. Vitamin D increase bone strength and decrease the frequency of
trials are rare and they have not been able to support infections.
beneficial effects of vitamin D supplementation on cogni- Secondary outcomes of the study are occurrence of IgE
tive functions among adults [47, 48]. Little evidence exists mediated allergic symptoms, growth, motor, cognitive and
on the effects of vitamin D on the cognitive development social development, and associations of concentration and
among children and adolescents. Low concentrations of effects of vitamin D with polymorphisms of genes associ-
cord blood 25-OHD increased the risk for delayed mental ated with its synthesis, binding proteins and degradation.
and psychomotor development in 363 16–18-months-old
children with severe vitamin D deficiency [49]. Surpris- Methods/Design
ingly, also the children with the highest S-25-OHD Study design and setting
concentrations had deficits in psychomotor development. This is a randomised on-going controlled double-blinded
However, S-25-OHD concentrations were not associated intervention trial on children of 2 weeks to 24 months of
with cognitive performance in adolescents after control- age (Fig. 1). Vitamin D supplementation was commenced
ling for ethnicity [50–52]. Although preliminary results at 2 weeks of age and continued until 24 months of age.
point to importance of early life exposure, the role of This study is carried out at the Kätilöopisto Maternity
vitamin D in cognitive development during childhood and Hospital, Helsinki, Finland (60°20,40’N). The hospital is
adolescence remains largely unstudied. one of three hospitals providing maternity services for
Helve et al. BMC Pediatrics (2017) 17:91 Page 4 of 8

Fig. 1 Study protocol. Methods are listed in Table 1 and further described in Additional documentation

the greater Helsinki area (a population of 1.5 million) Girls (492) and boys (495) were randomised at recruit-
with 8 000 births annually (15 000 in the whole greater ment in blocks of 50 to receive either 10 or 30 μg of
Helsinki area). vitamin D3 (400 IU or 1 200 IU, respectively) daily dur-
ing the study period. Randomisation was performed by
an external pharmacist and was blinded from the study
Research team
group.
The research team responsible for inception, implemen-
Vitamin D is administered once daily with 5 drops for
tation, and management of the protocol includes paedia-
both concentrations. The families received a study diary
tricians, paediatric specialists in the field of neonatology,
to record treatment compliance prospectively and empty
endocrinology, infectious diseases and immunology, psy-
vitamin D bottles are collected at follow-up visits.
chologists, and nutritionists. The research team and the
Each family keep record of the child’s infections on a
research nurses undertake recruitment, data collection,
study diary throughout the study period. Data on allergic
follow-up visits, and data entry.
symptoms, growth, child’s motor and neurocognitive
functioning, mental and behavioural health, social skills,
Procedure and participants sleep and diet are also collected. Furthermore, mothers
A total of 4 980 infants were assessed for eligibility and report their own well-being at child’s birth (questionnaires
1 572 did not meet the inclusion criteria. Written infor- given at recruitment) and at 12- and 24-month follow-up.
mation of the study was given to 3 408 families at the Follow-up visits for the children are arranged at 6, 12 and
Helsinki Maternity Hospital 1–2 days after delivery from 24 months of age at which blood samples are taken. Bone
which 2 421 declined to participate. Altogether 987 strength is measured with peripheral quantitative com-
healthy term newborns were recruited between January puter tomography (pQCT) at 12 and 24-month follow-up
2013 and June 2014. visits (overview of study follow-up and parameters in
The participating mothers were of Caucasian origin, Table 1). During the visit the families meet a study nurse
without regular medication and with a singleton preg- and have the possibility to meet with a paediatrician.
nancy. The infants were born at term (37 + 0 to 42 + A total of 875 participants have been to their 12-month
0 weeks) with a birth weight appropriate for gestational follow-up visit (a drop-out of 112 children) and the 24-
age (birth weight SD score between −2.0 to +2.0). Exclu- month follow-up visits are ongoing (to date, parents of 36
sion criteria for the infants were nasal continuous posi- children have declined to participate in the 24-month
tive airway pressure treatment for more than 1 day, follow-up visit, Fig. 2).
intravenous glucose infusion, intravenous postnatal anti-
biotic treatment, seizures, duration of phototherapy for Ethical considerations
more than 3 days, and need for nasogastric tube for An informed consent was obtained from the parents at re-
more than 1 day. cruitment. Previous intervention studies have evaluated
Helve et al. BMC Pediatrics (2017) 17:91 Page 5 of 8

Table 1 Methods and timing of sampling. Methods are considered sufficient. Therefore, 10 μg a day was consid-
characterised further in Additional documentation ered adequate and ethically sound to use as the dosage for
Birth 6 months 12 months 24 months the control group. While the group receiving 40 μg/day
of age of age of age did not develop hypercalcaemia, their S-25-OHD reached
Vitamin D x x x levels higher than targeted. Dose of 30 μg/day was there-
Calcium and bone x x x x fore chosen for the present study.
metabolism An external steering group was recruited to monitor
Inflammatory markers x x x the study and possible adverse effects.
DNA x x x The vitamin D3 supplements are prepared by Orion
Bone densitometry x x Pharmaceuticals (Espoo, Finland). The study is researcher
initiated and independent.
Infection records x x x
The Research Ethics Committee of the Hospital
Allergy questionnaire x x x
District of Helsinki and Uusimaa has approved the study
Specific allergic responses x x (ID 107/13/03/03/2012) and it is registered into
(IgA, IgE)
ClinicalTrials.com (NCT01723852).
Iron status x x
a
Dietary survey x x x x
Study measures
Neurological scales x x
An overview of study parameters is shown in Table 1. A
Family sociodemographic x x full description of specific methods used in the study are
and lifestyle
presented in the online Additional documentation. In
Neurocognitive development x x
scales
brief, the parameters used for the assessment of primary
outcomes are as follows: bone parameters assessed with
Mental health and behaviour x x x
pQCT at 12 and 24 months of age, daily infection diary
Clinical assessment x x x filled in by parents from 2 weeks to 24 months of age, and
Health records x x x x S-25-OHD at birth (cord blood) and at 12 and 24 months
Viral and bacterial swab x x of age.
a
retrospectively on maternal diet

Data analyses
the safety of substitution doses of up to 50 μg vitamin Differences between groups are tested with relevant statis-
D in infants [61]. Safety was ensured by performing a tical models, e.g., independent samples t-test, ANOVA
pilot study for the proposed project: 113 children were and analysis of covariance (ANCOVA). Temporal change
randomised into three groups receiving vitamin D3 and difference between intervention groups of continuous
doses of 10, 30 and 40 μg daily for 3 months [62]. No variables (i.e., S-25-OHD) are assessed with repeated mea-
hypercalcemia was observed in this study. Plasma and sures ANCOVA. Furthermore, multivariate and multilevel
urine calcium concentrations did not differ between the regression models adjusted with potential confounders are
three groups and in all groups the mean concentration applied to explore associations between several outcomes
of S-25-OHD reached at least 80 nmol/L, which was when applicable.

Fig. 2 Study recruitment, adherence, and current phase of the study

Helve et al. BMC Pediatrics (2017) 17:91 Page 6 of 8

Sample size D substitution in populations with variable vitamin D

On the basis of sample size calculation for both of the status. In addition, the study is designed to have control
primary outcomes, 400 + 400 study subjects, allowing for over the most common sources of vitamin D.
a 20% drop-out rate, a 0.2 SD difference can be detected If the results from the VIDI-trial confirm the connec-
with p at 0.05 and ß of 0.9. In bone mineral content, a tions between the increased dose of vitamin D and the
0.2 SD increase requires 210 + 210 subjects, and a simi- primary outcomes, the health benefits gained by a
lar increase in bone cross sectional area 297 + 297 sub- population-wide supplementation with higher vitamin D
jects as measured by the same technique as here dose — a simple and low-cost intervention — are obvious.
(pQCT) [63]. The annual rate of infections in children The first results of the VIDI-trial are expected in 2017.
below 2 years of age is 6 (SD ± 1.6) [64, 65]. In the group
receiving 30 μg of vitamin D we expect a decrease from Additional file
12 to 9 infections during the 24-month study period. A
sample size of 220 + 220 is required to achieve statistical Additional file 1: Supplementary documentation. Methods described in
further detail. (DOCX 520 kb)
power.

Abbreviations
Discussion 25-OHD: 25-hydroxy-vitamin D; BMD: Bone mineral density; DBP: Vitamin D
To the best of our knowledge, this is the largest and binding protein; S-25-OHD: serum 25-OHD; VDR: Vitamin D receptor
most comprehensive randomised intervention study to
Acknowledgements
assess the effects of vitamin D supplementation on
We are grateful to all the families participating in the study. Also, we are
skeletal and extra skeletal outcomes in infants. To date, grateful to the research nurses Sirpa Nolvi, Rhea Paajanen, Nea Boman, and
epidemiological studies and randomised intervention Päivi Turunen for their assistance in data collection. Laboratory technician
Sari Linden is acknowledged for her valuable contribution.
trials have demonstrated conflicting results on, for ex-
ample, the effect of vitamin D supplementation on child- Funding
hood infections and on neurocognitive functioning. This This study is being funded by the Governmental Subsidy for Clinical Research,
may be due to difficulties in addressing the multiple Foundation for Pediatric Research, Finska Läkaresällskapet, Folkhälsan Research
Foundation, Sigrid Juselius Foundation, Emil Aaltonen Foundation, Stiftelsen
confounding factors affecting the association between ex- Dorothea Olivia, Karl Walter ooh Jarl Walter Perkléns minne, and Päivikki and
posure and outcome. The present study, based on findings Sakari Sohlberg Foundation.
from a pilot study [62], is designated to specific hypothesis
Availability of data and materials
with adequate statistical power and duration. During the Data from the study are made publicly available in the main papers published
study, we gather data on infant diet at home and day care, from the study, or in their Additional file 1.
psychological profiles and atopy-related symptoms using
Authors’ contributions
questionnaires. Results from this study will likely generate The authors’ contributions are as follows: OH, HV, EH-S, KH, KR, TH, OM and SA
novel evidence-based information for improving vitamin designed the study; OH, HV, EH-S, JR, HH, SV, ME-C and TH conducted the
D status among the paediatric population worldwide. By research; OH, HV, KH, KR, OM and SA wrote the first draft of the manuscript;
OH had primary responsibility for final content; all authors critically reviewed
analysis of the impact of gene polymorphisms on the the manuscript. All of the authors read and approved the final version.
concentration and efficacy of the given vitamin D substi-
tution it may create the basis for individualised dosage Competing interests
The authors declare that they have no competing interests.
guidelines.
Consent for publication
Limitations of the study Not applicable.
Compliance of the study subjects is always an issue in
Ethics approval and consent to participate
intervention trials. We are collecting used vitamin D The Research Ethics Committee of the Hospital District of Helsinki and
bottles to evaluate the amount of vitamin D left after each Uusimaa has approved the study (ID 107/13/03/03/2012) and it is registered
cycle. These data, and parentally kept diaries on the use of into ClinicalTrials.com (NCT01723852). An informed consent was obtained
from the parents at recruitment.
vitamin D are used to evaluate the compliance in vitamin
D intake.
Publisher’s Note
Questionnaires may be improperly filled, resulting in Springer Nature remains neutral with regard to jurisdictional claims in
missing data on, for example, parent-reported infections. published maps and institutional affiliations.
Therefore, national registers can be used for the valid-
Author details
ation of caregiver data. 1
Children’s Hospital, Pediatric Research Center, University of Helsinki and
The results from a study performed in Finland cannot Helsinki University Hospital, P.O. Box 28100029 HUS Helsinki, Finland.
2
be directly generalised to other parts of the world. How- Folkhälsan Research Center, Helsinki, Finland. 3Institute of Behavioral
Sciences, University of Helsinki, Helsinki, Finland. 4Center for Molecular
ever, due to the randomised controlled study design our Medicine, Karolinska Institutet and Clinical Genetics, Karolinska University
study will provide valuable data on the effects of vitamin Hospital, Stockholm, Sweden.
Helve et al. BMC Pediatrics (2017) 17:91 Page 7 of 8

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