Low serum vitamin D is associated with axial length and risk of myopia in young children

Abstract

The aim of the study was to investigate the relationship between serum 25(OH)D levels and axial length (AL) and myopia in 6-year-old children. A total of 2666 children aged 6 years participating in the birth-cohort study Generation R underwent a stepwise eye examination. First, presenting visual acuity (VA) and AL were performed. Second, automated cycloplegic refraction was measured if LogMAR VA > 0.1. Serum 25-hydroxyvitamin D [25(OH)D] was determined from blood using liquid chromatography/tandem mass spectrometry. Vitamin D related SNPs were determined with a SNP array; outdoor exposure was assessed by questionnaire. The relationships between 25(OH)D and AL or myopia were investigated using linear and logistic regression analysis. Average 25(OH)D concentration was 68.8 nmol/L (SD ± 27.5; range 4–211); average AL 22.35 mm (SD ± 0.7; range 19.2–25.3); and prevalence of myopia 2.3 % (n = 62). After adjustment for covariates, 25(OH)D concentration (per 25 nmol/L) was inversely associated with AL (β −0.043; P < 0.01), and after additional adjusting for time spent outdoors (β −0.038; P < 0.01). Associations were not different between European and non-European children (β −0.037 and β −0.039 respectively). Risk of myopia (per 25 nmol/L) was OR 0.65 (95 % CI 0.46–0.92). None of the 25(OH)D related SNPs showed an association with AL or myopia. Lower 25(OH)D concentration in serum was associated with longer AL and a higher risk of myopia in these young children. This effect appeared independent of outdoor exposure and may suggest a more direct role for 25(OH)D in myopia pathogenesis.

Electronic supplementary material
The online version of this article (doi:10.1007/s10654-016-0128-8) contains supplementary material, which is available to authorized users.

Full text

OPHTHALMIC EPIDEMIOLOGY
Low serum vitamin D is associated with axial length and risk
of myopia in young children
J. Willem L. Tideman
1,2
•Jan Roelof Polling
1,5
•Trudy Voortman
2
•
Vincent W. V. Jaddoe
2,3
•Andre
´G. Uitterlinden
2,4
•Albert Hofman
2
•
Johannes R. Vingerling
1
•Oscar H. Franco
2
•Caroline C. W. Klaver
1,2
Received: 17 November 2015 / Accepted: 8 February 2016 / Published online: 8 March 2016
ÓThe Author(s) 2016. This article is published with open access at Springerlink.com
Abstract The aim of the study was to investigate the
relationship between serum 25(OH)D levels and axial length
(AL) and myopia in 6-year-old children. A total of 2666
children aged 6 years participating in the birth-cohort study
Generation R underwent a stepwise eye examination. First,
presenting visual acuity (VA) and AL were performed.
Second, automated cycloplegic refraction was measured if
LogMAR VA [0.1. Serum 25-hydroxyvitamin D
[25(OH)D] was determined from blood using liquid chro-
matography/tandem mass spectrometry. Vitamin D related
SNPs were determined with a SNP array; outdoor exposure
was assessed by questionnaire. The relationships between
25(OH)D and AL or myopia were investigated using linear
and logistic regression analysis. Average 25(OH)D con-
centration was 68.8 nmol/L (SD ±27.5; range 4–211);
average AL 22.35 mm (SD ±0.7; range 19.2–25.3); and
prevalence of myopia 2.3 % (n =62). After adjustment for
covariates, 25(OH)D concentration (per 25 nmol/L) was
inversely associated with AL (b-0.043; P\0.01), and
after additional adjusting for time spent outdoors (b-0.038;
P\0.01). Associations were not different between Euro-
pean and non-European children (b-0.037 and b-0.039
respectively). Risk of myopia (per 25 nmol/L) was OR 0.65
(95 % CI 0.46–0.92). None of the 25(OH)D related SNPs
showed an association with AL or myopia. Lower 25(OH)D
concentration in serum was associated with longer AL and a
higher risk of myopia in these young children. This effect
appeared independent of outdoor exposure and may suggest
a more direct role for 25(OH)D in myopia pathogenesis.
Keywords Myopia Vitamin D Axial length Children
Abbreviations
AL Axial length
25(OH)D 25-Hydroxyvitamin D
VA Visual acuity
VDR Vitamin D receptor gene
SE Spherical equivalent
OR Odds ratio
Introduction
In the last decades, the prevalence of myopia has increased
dramatically in Asia as well as in the Western world [1–3].
Prevalence estimates are now around 2 % in 6-year-old
children with European ethnicity, and 12 % in children of
Asian descent [4,5]. These figures rise to 50 % in young
European adults [6] and up to 96 % in students from South
Electronic supplementary material The online version of this
article (doi:10.1007/s10654-016-0128-8) contains supplementary
material, which is available to authorized users.
&Caroline C. W. Klaver
c.c.w.klaver@erasmusmc.nl
J. Willem L. Tideman
j.tideman@erasmusmc.nl
1
Department of Ophthalmology, Erasmus Medical Center,
NA2808, PO Box 5201, 3008 AE Rotterdam, The
Netherlands
2
Department of Epidemiology, Erasmus Medical Center,
Rotterdam, The Netherlands
3
Department of Paediatrics, Erasmus Medical Center,
Rotterdam, The Netherlands
4
Department of Internal Medicine, Erasmus Medical Center,
Rotterdam, The Netherlands
5
Department of Orthoptics and Optometry, Faculty of Health,
University of Applied Sciences, Utrecht, The Netherlands
123
Eur J Epidemiol (2016) 31:491–499
DOI 10.1007/s10654-016-0128-8
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Korea [7]. Although myopic refractive error can be cor-
rected optically by glasses, contact lenses, or refractive
surgery, the longer axial length ([26 mm) increases the
life-time risk of severe visual impairment and blindness
due to retinal complications [8]. The basis of myopia is a
developmental mismatch between the optical components
of the eye [9,10], of which excessive elongation of axial
length (AL) in early youth is the most important [11].
The need to reveal the etiology of myopia and develop
preventive measures is urgent from a public health per-
spective. Associations with genetic risk variants [12,13]
and environmental factors such as time spent outdoors [14–
16] and education [4,12] have been well established [17,
18]. Recent studies reported an association with serum
25-hydroxy vitamin D [25(OH)D] concentration and
myopia in adolescents [19,20]. Whether this reflects the
association between outdoor exposure and myopia, or
whether vitamin D itself plays a role in the pathophysiol-
ogy is unclear. Studies investigating the potential relation
with vitamin D receptor (VDR) polymorphisms found no
consistent relationships [21,22].
Serum 25(OH)D is derived from multiple sources.
Cholecalciferol (vitamin D3) is formed in the skin after
sunlight exposure, and also absorbed by the gut after
dietary intake of e.g., fatty fish. Ergocalciferol (vitamin
D2) results from intake of foods containing yeasts and
fungi [23,24] Both precursors are hydroxylated in the liver
into 25(OH)D. Its active metabolite 1,25(OH)
2
D is formed
after transformation in the kidney [25] and is distributed to
other sites of the body thereafter. In non-supplemented
individuals, sunlight exposure is thought to be the main
determinant of 25(OH)D [24,26–28]. The main function of
1,25(OH)
2
D is regulation of calcium and phosphate meta-
bolism in bone tissue and plasma, but it also has metabolic
functions in insulin metabolism [29,30]. In neuronal dis-
ease such as cognitive decline and Parkinson disease [31,
32], it can be involved in immune responses [33] and in
DNA transcription and methylation [34,35]. Whether
1,25(OH)
2
D has a direct effect on eye growth is currently
unclear.
The aim of this study was to investigate the association
between 25(OH)D levels, AL, and the risk of myopia in
children at age 6 years in a large population-based study.
Additionally, influence of time spent outdoors on these
relationships, and vitamin D related genotypes was studied.
Population and methods
Study population
This study was embedded in the Generation R Study, a
population-based prospective cohort study of pregnant
women and their children in Rotterdam, The Netherlands.
The complete methodology has been described elsewhere
[36,37]. A total of 4154 children underwent an ophthal-
mologic examination by trained nurses at the research
center at age 6 years and underwent blood withdrawal for
serum measurements. The study protocol was approved by
the Medical Ethical Committee of the Erasmus Medical
Center, Rotterdam (MEC 217.595/2002/20), and written
informed consent was obtained from all participants.
Research was conducted according to the declaration of
Helsinki.
Assessment of AL and myopia
The examination included a stepwise ophthalmological
examination. Step 1 consisted of monocular visual acuity
with LogMAR based LEA-charts at 3 meter distance by
means of the ETDRS method, and ocular biometry
including AL (mm) was measured by Zeiss IOL-master
500 (Carl Zeiss MEDITEC IOL-master, Jena, Germany)
per eye; five measurements were averaged to a mean AL
[38]. Step 2 was carried out in children with a LogMAR
visual acuity of [0.1 in at least one eye and in children
wearing prescription glasses, and included performance of
automated cycloplegic refraction [Topcon auto refractor
KR8900 (Topcon, Japan)] and a complete ophthalmologic
work up by an ophthalmologist. Two drops (three in case of
dark irises) of cyclopentolate (1 %) were administered at
least 30 min before refractive error measurement. Pupil
diameter was C6 mm at time of the measurement. Spher-
ical equivalent (SE) was calculated as the sum of the full
spherical value and half of the cylindrical value in accor-
dance with standard practice, and myopia was defined as
SE B-0.5D in at least one eye. Children with LogMAR
visual acuity B0.1, no glasses or ophthalmic history were
classified as non-myopic [39,40].
Assessment of 25(OH)D
At a median age of 6.0 y (95 % range 5.6–7.9), nonfasting
blood samples were drawn by antecubital venipuncture and
stored at -80 °C until analysis. Serum samples were col-
lected in all children on the examination day at the research
center. The measurements of 25(OH)D (nmol/L) in the
samples (110lmL serum per sample) were DEQAS certi-
fied and were conducted at the Endocrine Laboratory of the
VU University Medical Center, Amsterdam, The Nether-
lands between July 2013 and January 2014 [41]. Serum
25(OH)D was measured with the use of isotope dilution
online solid phase extraction liquid chromatography–tan-
dem mass spectrometry, the ‘gold standard’ (LC–MS/MS)
[42] using a deuterated internal standard [IS: 25(OH)D3-
d6] (Synthetica AS, Oslo, Norway). This method is highly
492 J. W. L. Tideman et al.
123
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

sensitive and has been widely used in 25(OH)D studies [43,
44]. The limit of quantitation was 4.0 nmol/L; intra-assay
CV was \6 %, and interassay CV was \8 % for concen-
trations between 25 and 180 nmol/L.
Questionnaire
Each mother completed a questionnaire regarding the daily
life activities of their child. Time spent playing outdoors
and time spent watching television was obtained using
questions such as ‘‘how much time does your child spend
outdoors/watching television in the morning/afternoon/
evening’’. Questions were asked for weekdays and week-
end days separately, and answers were multiple choice
(never, 0–,–1, 1–2, 2–3, 3–4 h). Total time spent in a
week was summed and divided by seven to make an
average h/day.
Genotyping of SNPs in vitamin D pathway
Samples were genotyped using Illumina Infinium II
HumanHap610 Quad Arrays following standard manufac-
turer’s protocols. Intensity files were analyzed using the
Beadstudio Genotyping Module software v.3.2.32, and
genotype calling based on default cluster files. Any sample
displaying call rates below 97.5 %, excess of autosomal
heterozygosity (F \mean -4SD) and mismatch between
called and phenotypic gender were excluded. Genotypes
were imputed for all polymorphic SNPs from phased
haplotypes in autosomal chromosomes using the 1000
Genomes GIANTv3 panel. SNPs located in genes involved
in the Vitamin D metabolic pathway were studied for
association with AL and presence of myopia; i.e., genes
determining serum 25(OH)D levels (GC, DHCR7,
CYP2R1), a gene involved in activation of serum 25(OH)D
(CYP27B1), the vitamin D receptor gene (VDR), and the
gene involved in deactivation of 1,25-(OH)
2
D in mito-
chondria (CYP24A1). A total of 33 SNPs [21,45,46] were
tested, and analyses were adjusted for multiple testing
using Bonferroni adjusted Pvalue 0.05/33, P=0.0015.
Measurement of covariates
Height and weight of children were measured by trained
nurses, and BMI (weight/height
2
) was calculated. Age was
determined at the time of the visit. Income was obtained
using the questionnaire and was clustered in low income
(lowest tertile) and higher income. If income at the time of
the visit was not available, income at birth was used.
Ethnicity was obtained in the questionnaire, according to
standardized criteria employed by ‘Statistics Netherlands’,
the official national statistics agency [47], concerning the
country of birth of parents and child: (1) if both parents
were born in the Netherlands, the ethnicity is Dutch; (2) if
one of the parents was born in another country than the
Netherlands, that country was considered country of birth;
(3) if both parents were born in the same country other than
the Netherlands, that country was represented; (4) if the
parents were born in different countries outside the
Netherlands, then the country of the mother was repre-
sented; and (5) if that child and both parents were born in
different countries outside the Netherlands, the country of
birth of the child was represented. Ethnicity was grouped
into European and non-European. To adjust for seasonality,
four seasons were formed on basis of the month in which
the children participated in the study (Winter: December–
February, Spring: March–May, Summer: June–August,
Autumn: September–November).
Statistical analysis
Separate analyses were performed for AL and myopia.
Differences in covariates between myopia and children
without myopia were tested using logistic regression
analysis adjusting for potentially confounding effects of
age and gender. The relation between 25(OH)D and AL
was investigated using multivariable linear regression
analysis; the relation with myopia (SE B-0.5D) was
analyzed using multivariable logistic regression analysis,
Covariates were only added to the model if they were
significantly related with the outcome as well as with
25(OH)D. Three models were tested: model 1 only adjus-
ted for age and gender; model 2 for age, gender, BMI,
ethnicity, television watching, family income, and season
visiting the research center; model 3 additionally adjusted
for time spent playing outdoors. Effect estimates were
determined per 25 nmol/L 25(OH)D. Beta’s are presented
with SE; Odds Ratios (ORs) with 95 % confidence inter-
vals (95 % CI). Statistical analyses were performed using
SPSS version 21.0 for Windows software (SPSS Inc).
Results
Demographics
A flow diagram presenting the selection of children for the
current analysis is shown in Supplement Figure 1. A total
of 2666 children were available for analysis of serum
Vitamin D and myopia; 2636 children were available for
analysis of serum 25(OH)D and AL. Demographic char-
acteristics are presented in Table 1. Children with myopia
were on average somewhat older. Adjusted for age and
height, girls had smaller AL than boys but not a lower
frequency of myopia. Myopic children had a higher BMI,
watched more television, and spent less time outdoors.
Low serum vitamin D is associated with axial length and risk of myopia in young children 493
123
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Myopia occurred more frequently in children of non-
European ethnicity.
Serum 25(OH)D
The average serum 25(OH)D in the total study population
was lower than the optimal level of 75 nmol/L [23]. Only
37.2 % (1023) of the children reached this optimal level;
these were mostly (41.1 %) children who had been exam-
ined in summer time (Table 2). Figure 1shows an inverse
relation between serum 25(OH)D and AL for the entire
population (P\0.001). Most myopes had high AL and
low serum 25(OH)D levels; only 18 % (11/62) of myopic
children reached serum levels which corresponded to the
optimal level.
Table 3shows associations between serum 25(OH)D
and AL and myopia. Lower serum levels were associated
with higher AL and higher risks of myopia. The estimates
remained statistically significant after adjustment for
covariates. The effect between serum 25(OH)D and AL
remained [beta -0.033 (SE 0.012; P0.02)] after exclusion
of myopic children. The association was similar in children
of European and non-European descent, but the association
with AL in the relatively small non-European group failed
to reach statistical significance.
Search for possible explanations
We hypothesized that our findings could be explained by
outdoor exposure. Figure 2shows the positive relation
between time spent outdoors and serum 25(OH)D (Pear-
son, P=\0.001). Independent of serum 25(OH)D, time
spent outdoors (hr/day) was a risk factor for AL [beta
-0.034 (SE 0.012; P0.003)]. It was not a significant risk
factor for myopia (OR 0.81; 95 % CI 0.61–1.07), possibly
due to the small number of myopes. The association
between serum 25(OH)D and AL and myopia remained
significant after adjustment for time spent outdoors
(model 3). We explored possible interactions as well, but
there was no significant interaction effect between
25(OH)D, ethnicity or income. Additionally, the associa-
tion was tested separately in the small subgroup with
missing data on time spent outdoors. The effect was
similar to the effect in the group with data.
Table 1 Demographic
characteristics of study
participants in Generation R
(N =2666)
All
N=2666
No myopia
N=2604
Myopia
N=62
Pvalue
Characteristics
Age (years) 6.12 (0.44) 6.12 (0.44) 6.28 (0.65) 0.001
Sex, female (%) 49.1 (1308) 49.1 (1278) 48.4 (30) 0.99
BMI (kg/m
2
) 16.09 (1.71) 16.07 (1.69) 16.86 (2.14) 0.005
Low family income (%) 28.0 (747) 27.5 (715) 51.6 (32) \0.001
Axial length (mm) 22.35 (0.7) 22.33 (0.7) 23.14 (0.86) \0.001
Ethnicity (%)
European 75.5 (2013) 76.3 (1986) 43.5 (27) \0.001
Non-European 24.5 (653) 23.7 (618) 56.5 (35)
Activities daily life
Time spent outdoors (h/day) 1.59 (1.14) 1.60 (1.14) 1.16 (0.96) 0.003
Watching television (h/day) 1.34 (0.99) 1.33 (0.97) 1.83 (1.48) 0.001
Values are means (SD), or percentages (absolute numbers)
Pvalues are corrected for age, gender, height in logistic regression
Table 2 Average serum
25(OH)D (nmol/L) per season
in myopic and non-myopic
children
Serum 25(OH)D concentration (nmol/L) N All No myopia Myopia
Child
All seasons 2666 68.8 (27.5) 69.2 (27.4) 50.2 (24.1)
Spring 751 60.8 (21.7) 61.3 (21.6) 42.5 (17.5)
Summer 693 84.2 (28.4) 84.4 (28.4) 69.2(22.6)
Autumn 686 72.9 (26.8) 73.1 (26.8) 63.3 (24.7)
Winter 536 54.7 (23.0) 55.3 (22.9) 36.8 (19.7)
Values are means (SD)
Pvalues are corrected for age, gender, height. Pvalues \0.05 are shown in bold
494 J. W. L. Tideman et al.
123
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

To investigate a possible genetic association between
Vitamin D and eye growth, we studied genes incorporated
in the Vitamin D pathway. We considered single nucleotide
polymorphisms (SNPs) in genes that determine serum
25(OH)D levels, in genes involved in activation of serum
25(OH)D, in the vitamin D receptor gene (VDR), and in
the gene involved in deactivation of 1,25-(OH)
2
D
3
in
mitochondria (CYP24A1) (supplemental Table 1). One
SNP (rs2245153) in the CYP24A1 gene showed a signifi-
cant association with AL (beta 0.039; P0.04) and myopia
(OR 1.55; 95 % CI 1.04–2.31), 2 SNPs in CYP24A1
(rs4809959 beta 0.032; P0.04 and rs3787557 beta 0.046;
P0.04) and one in the VDR (rs11568820 beta -0.042;
P0.03) only showed a significant association with axial
length. Pvalues were all insignificant after adjustment for
multiple testing.
Discussion
In this cohort study of young children, we found a sig-
nificant association between serum 25(OH)D levels, AL
and myopia. In this study children with lower serum
levels of 25(OH)D had longer AL, and those with higher
25(OH)D had a lower risk of myopia (OR 0.65; 95 % CI
0.46–0.92 per 25 nmol/L). The association remained sig-
nificant after adjusting for outdoor exposure, indicating
that these two closely related determinants may have
some overlapping as well as separate effects on the
development of myopia. Genetic variants in the vitamin D
pathway genes appeared not to be related: although SNPs
in the VDR and CYP24A1 genes showed some associa-
tion with AL and myopia, this did not remain after
adjustment for multiple testing.
Fig. 1 Distribution of axial length as a function of serum level of
25(OH)D in the Generation R cohort
Table 3 Multivariate regression analysis of the association between 25(OH)D and axial length and myopia in children at age 6 years
Model 1: Age and sex adjusted
model
Model 2: Multivariate model excluding
outdoor exposure
Model 3: Multivariate model including
outdoor exposure
Association PAssociation PAssociation P
N=2636 N =2636 N =2636
Axial length (mm), beta (SE) of association with 25(OH)D, per 25 nmol/L
All participants -0.054 (0.012) \0.001 -0.043 (0.014) 0.002 -0.038(0.014) 0.007
European ethnicity -0.051 (0.014) \0.001 -0.043 (0.016) 0.006 -0.037 (0.016) 0.02
Non-European ethnicity -0.034 (0.027) 0.20 -0.043 (0.030) 0.16 -0.039 (0.031) 0.20
Model 1: Age and sex adjusted
model
Model 2: Multivariate model excluding
outdoor exposure
Model 3: Multivariate model including
outdoor exposure
Association PAssociation PAssociation P
N=2666 N =2666 N =2666
Myopia, OR (95 % CI) of association with 25(OH)D, per 25 nmol/L
All participants 0.47 (0.35–0.62) \0.001 0.63 (0.45–0.89) 0.008 0.65 (0.46–0.92) 0.01
European ethnicity 0.61 (0.39–0.95) 0.02 0.69 (0.42–1.11) 0.13 0.71 (0.44–1.16) 0.17
Non-European ethnicity 0.56 (0.37–0.85) 0.006 0.59 (0.37–0.95) 0.03 0.61 (0.38–0.98) 0.04
The multivariate model for axial length includes adjustment for model 1 and BMI, season of blood withdrawal, ethnicity, television watching,
family income. The multivariate model for myopia includes adjustment for model 1 and BMI, ethnicity, television watching, education mother.
Outdoor exposure indicates time spent outdoors
Low serum vitamin D is associated with axial length and risk of myopia in young children 495
123
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Our study had strengths and weaknesses. Assets were
the particularly large study sample, the inclusion of the
combination of measurements of AL and myopia, and the
correction for many potential confounders. The young age
of our study population was a benefit as well as a potential
drawback. It allowed for measurements of the determinant
very close to the onset of myopia, leaving less room for
confounding bias. On the other hand, it hampered the study
of large effects as most children did not develop excessive
eye growth yet. There were other drawbacks. We per-
formed cycloplegia only in children with a diminished
visual acuity. Reports show that our cut off value of Log-
MAR VA of [0.1 had a 97.8 % sensitivity to diagnose
myopia [39,40]. We therefore think that our approach did
not substantially affect the number of myopes in our study,
nor biased the observed associations. Finally, as the cor-
relation between serum 25(OH)D level and time playing
outdoors was relatively low in our study, our questionnaire
may not have fully assessed all time spent outdoors. Not all
participants filled in the questionnaire completely and data
on time spent outdoors was partially missing. However,
association in the sample of children without data on time
spent outdoors was similar to the association in those with
complete data.
A novel finding of our study was that the increase in AL
in children with low 25(OH)D was already present in the
physiological range of refractive error, before the onset of
myopia. This implies that Vitamin D has a continuous
effect on AL, and not only determines the development of
myopia. We confirmed that the risk of myopia decreased
with increasing 25(OH)D levels (OR 0.65) with each
25 nmol/L. The association between 25(OH)D and axial
length was also significant in the European children; but
failed to reach significance in the Non-European group due
to low statistical power. Correction for time spent outdoors
demonstrated some attenuation of the association, but did
not explain it entirely. Whether this is due to residual
confounding of time spent outdoors or whether Vitamin D
is truly causally related with AL and myopia remains an
open question. The evidence for a role of time spent out-
doors in myopia is available from cross sectional studies,
intervention and randomized clinical trials as well as from
animal studies [15,16,48,51]. Vitamin D production is
triggered by UV-exposure, not by light exposure per se.
Animal studies have shown that artificial light, free of UV,
can inhibit development of myopia development [48]. This
may suggests that outdoor exposure and Vitamin D are
independent risk factors for axial elongation and myopia.
However, true causality cannot be concluded from a cross
sectional study; longitudinal and functional studies are
needed to provide more profound evidence.
A few previous studies have investigated the role of
serum 25(OH)D in myopia. A South-Korean and an Aus-
tralian study found a positive association in adolescents
and young adults [19,49]. The ALSPAC study found an
association with development of refractive error only for
25(OH)D
2
, not for 25(OH)D
3
in 15 years old children. A
potential drawback of this study was the measurement of
refraction without any cycloplegia [50]. Mutti et al. [21]
found an association between SNPs in the VDR gene and
myopia in a smaller study. We could not validate this
association, as none of the Vitamin D related SNPs were
significant after adjusting for multiple testing.
Various hypotheses underscribe a function of 25(OH)D
in eye growth. One theory focusses on Vitamin D in rela-
tion to dopamine. The current view is that light exposure
initiates the release of dopamine in retinal amacrine cells
[51–53]. The released dopamine appears to influence the
function of gap junctions and the size of receptive fields
[54], an important determinant of eye growth. Vitamin D is
known to influence dopamine metabolism in neurological
disorders, such as Morbus Parkinson and restless legs
syndrome [55]. In particular in Parkinson, Vitamin D
protects against cell death in the substantia nigra of the
dopamine secreting neuron [32,56]. Increased dopamine
metabolism [57] was found in the rat brain under influence
of vitamin D. In the developing rat brain, Vitamin D was
found to upregulate glial derived neurotrophic factor
(GDNF) which increases dopamine neurons [58]. Taken
together, Vitamin D appears to strengthen the function of
dopamine or dopamine secreting cells in neuronal tissues.
Fig. 2 Distribution of serum level of 25(OH)D as a function of time
spent outdoors
496 J. W. L. Tideman et al.
123
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Whether this also accounts for dopamine secreted by
amacrine cells in the retina remains an intriguing question.
Another mechanism may be the regulation of DNA
transcription in genes containing vitamin D response ele-
ments (VDRE, supplemental figure 2). In this case, the
active intracellular 1,25(OH)
2
D binds to VDR binding
protein, enters the nucleus, and forms a complex with
retinoid X receptor in order to bind to VDRE and initiate
transcription. VDREs are located in many genes [59]. It has
been shown that retinal cells can metabolize 1,25(OH)
2
D;
and this active form of vitamin D may interfere with
transcription of genes that promote the myopia signaling
cascade [60].
In conclusion, we found that serum levels of 25(OH)D
were inversely related to AL, and that low levels increased
the risk of myopia. Our data suggest that this relationship
may be independent from time spent outdoors. The
potential role for 25(OH)D in myopia pathogenesis should
be further explored by intervention research and functional
studies.
Acknowledgments The Generation R Study is conducted by the
Erasmus Medical Center in close collaboration with the School of
Law and Faculty of Social Sciences of the Erasmus University Rot-
terdam, the Municipal Health Service Rotterdam area, Rotterdam, the
Rotterdam Homecare Foundation, Rotterdam and the Stichting
Trombosedienst & Artsenlaboratorium Rijnmond (STAR-MDC),
Rotterdam. We gratefully acknowledge the contribution of children
and parents, general practitioners, hospitals, midwives and pharma-
cies in Rotterdam. The generation and management of GWAS
genotype data for the Generation R Study were done at the Genetic
Laboratory of the Department of Internal Medicine, Erasmus MC,
The Netherlands. We thank Mila Jhamai, Manoushka Ganesh, Pascal
Arp, Marijn Verkerk, Lizbeth Herrera and Marjolein Peters for their
help in creating, managing and QC of the GWAS database. Also, we
thank Karol Estrada and Carolina Medina-Gomez for their support in
creation and analysis of imputed data.
Funding The Generation R Study is made possible by financial
support from the Erasmus Medical Center, Rotterdam, the Erasmus
University Rotterdam; the Netherlands Organisation of Scientific
Research (NWO); Netherlands Organization for the Health Research
and Development (ZonMw); the Ministry of Education, Culture and
Science; the Ministry for Health, Welfare and Sports; the European
Commission (DG XII); The author was supported by the following
foundations: MaculaFonds, Novartis Fonds, ODAS, LSBS, Oogfonds
and ANVVB that contributed through UitZicht (Grant 2013-24). The
funding organizations had no role in the design or conduct of this
research. They provided unrestricted grants TV and OHF work in
ErasmusAGE, a center for aging research across the life course fun-
ded by Nestle
´Nutrition (Nestec Ltd.), Metagenics Inc., and AXA.
Nestle
´Nutrition (Nestec Ltd.), Metagenics Inc., and AXA had no role
in design or conduct of the study; collection, management, analysis,
or interpretation of the data; or preparation, review or approval of the
manuscript.
Authors’ contribution Willem Tideman designed and conducted
the research, analyzed the data, wrote the paper and approved the final
manuscript as submitted. He had primary responsibility for final
content. Jan Roelof Polling designed, conducted the research, ana-
lyzed the data and critically revised all versions of the manuscript. He
approved the final manuscript as submitted. Trudy Voortman pro-
vided comments and consultation regarding the analyses and manu-
script and critically revised all versions of the manuscript. She
approved the final manuscript as submitted. Vincent Jaddoe initiated
and designed the original Generation R study, was responsible for the
infrastructure in which the study is conducted, contributed to the
original data collection and critically revised the manuscript. He
approved the final manuscript as submitted. Andre
´Uitterlinden con-
tributed to the analysis, provided comments and consultation
regarding the analyses and manuscript. He approved the final manu-
script as submitted. Albert Hofman initiated and designed the original
Generation R study, was responsible for the infrastructure in which
the study is conducted, contributed to the original data collection and
critically revised the manuscript. He approved the final manuscript as
submitted. Johannes Vingerling provided comments and consultation
regarding the analyses and manuscript and critically revised all ver-
sions of the manuscript. He approved the final manuscript as sub-
mitted. Oscar Franco contributed to the analysis, provided comments
and consultation regarding the analyses and manuscript. He approved
the final manuscript as submitted. Caroline Klaver designed and
conducted the research and wrote the paper and approved the final
manuscript as submitted. She had primary responsibility for final
content.
Compliance with ethical standards
Conflict of interest The authors have indicated they have no
potential conflicts of interest to disclose.
Financial disclosure The authors have no financial relationships
relevant to this article to disclose.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://crea
tivecommons.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.
References
1. Bar Dayan Y, Levin A, Morad Y, et al. The changing prevalence of
myopia in young adults: a 13-year series of population-based
prevalence surveys. Invest Ophthalmol Vis Sci. 2005;46(8):2760–5.
doi:10.1167/iovs.04-0260.
2. Kim EC, Morgan IG, Kakizaki H, Kang S, Jee D. Prevalence and
risk factors for refractive errors: Korean National Health and
Nutrition Examination Survey 2008–2011. PLoS ONE.
2013;8(11):e80361. doi:10.1371/journal.pone.0080361.
3. Vitale S, Sperduto RD, Ferris FL 3rd. Increased prevalence of
myopia in the United States between 1971–1972 and 1999–2004.
Arch Ophthalmol. 2009;127(12):1632–9. doi:10.1001/arch
ophthalmol.2009.303.
4. French AN, Morgan IG, Burlutsky G, Mitchell P, Rose KA.
Prevalence and 5- to 6-year incidence and progression of myopia
and hyperopia in Australian schoolchildren. Ophthalmology.
2013;120(7):1482–91. doi:10.1016/j.ophtha.2012.12.018.
5. Zhan MZ, Saw SM, Hong RZ, et al. Refractive errors in Singa-
pore and Xiamen, China—a comparative study in school children
aged 6 to 7 years. Optom Vis Sci. 2000;77(6):302–8.
Low serum vitamin D is associated with axial length and risk of myopia in young children 497
123
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

6. Williams KM, Verhoeven VJ, Cumberland P, et al. Prevalence of
refractive error in Europe: the European Eye Epidemiology
(E(3)) Consortium. Eur J Epidemiol. 2015;30(4):305–15. doi:10.
1007/s10654-015-0010-0.
7. Jung SK, Lee JH, Kakizaki H, Jee D. Prevalence of myopia and
its association with body stature and educational level in 19-year-
old male conscripts in Seoul, South Korea. Invest Ophthalmol Vis
Sci. 2012;53(9):5579–83. doi:10.1167/iovs.12-10106.
8. Saw SM. How blinding is pathological myopia? Br J Ophthalmol.
2006;90(5):525–6. doi:10.1136/bjo.2005.087999.
9. Hashemi H, Khabazkhoob M, Miraftab M, et al. Axial length to
corneal radius of curvature ratio and refractive errors. J Oph-
thalmic Vis Res. 2013;8(3):220–6.
10. Ojaimi E, Rose KA, Morgan IG, et al. Distribution of ocular
biometric parameters and refraction in a population-based study
of Australian children. Invest Ophthalmol Vis Sci.
2005;46(8):2748–54. doi:10.1167/iovs.04-1324.
11. Jones LA, Mitchell GL, Mutti DO, Hayes JR, Moeschberger ML,
Zadnik K. Comparison of ocular component growth curves
among refractive error groups in children. Invest Ophthalmol Vis
Sci. 2005;46(7):2317–27. doi:10.1167/iovs.04-0945.
12. Verhoeven VJ, Buitendijk GH, Consortium for Refractive E,
et al. Education influences the role of genetics in myopia. Eur J
Epidemiol. 2013;28(12):973–80. doi:10.1007/s10654-013-9856-
1.
13. Stambolian D. Genetic susceptibility and mechanisms for
refractive error. Clin Genet. 2013;84(2):102–8. doi:10.1111/cge.
12180.
14. Sherwin JC, Reacher MH, Keogh RH, Khawaja AP, Mackey DA,
Foster PJ. The association between time spent outdoors and
myopia in children and adolescents: a systematic review and
meta-analysis. Ophthalmology. 2012;119(10):2141–51. doi:10.
1016/j.ophtha.2012.04.020.
15. Wu PC, Tsai CL, Wu HL, Yang YH, Kuo HK. Outdoor activity
during class recess reduces myopia onset and progression in
school children. Ophthalmology. 2013;120(5):1080–5. doi:10.
1016/j.ophtha.2012.11.009.
16. Rose KA, Morgan IG, Ip J, et al. Outdoor activity reduces the
prevalence of myopia in children. Ophthalmology.
2008;115(8):1279–85. doi:10.1016/j.ophtha.2007.12.019.
17. Dharani R, Lee CF, Theng ZX, et al. Comparison of measure-
ments of time outdoors and light levels as risk factors for myopia
in young Singapore children. Eye (Lond). 2012;26(7):911–8.
doi:10.1038/eye.2012.49.
18. Jones-Jordan LA, Sinnott LT, Cotter SA, et al. Time outdoors,
visual activity, and myopia progression in juvenile-onset myopes.
Invest Ophthalmol Vis Sci. 2012;53(11):7169–75. doi:10.1167/
iovs.11-8336.
19. Yazar S, Hewitt AW, Black LJ, et al. Myopia is associated with
lower vitamin D status in young adults. Invest Ophthalmol Vis
Sci. 2014;55(7):4552–9. doi:10.1167/iovs.14-14589.
20. Choi JA, Han K, Park YM, La TY. Low serum 25-hydroxyvi-
tamin D is associated with myopia in Korean adolescents. Invest
Ophthalmol Vis Sci. 2014;. doi:10.1167/iovs.13-12853.
21. Mutti DO, Cooper ME, Dragan E, et al. Vitamin D receptor
(VDR) and group-specific component (GC, vitamin D-binding
protein) polymorphisms in myopia. Invest Ophthalmol Vis Sci.
2011;52(6):3818–24. doi:10.1167/iovs.10-6534.
22. Annamaneni S, Bindu CH, Reddy KP, Vishnupriya S. Associa-
tion of vitamin D receptor gene start codon (Fok1) polymorphism
with high myopia. Oman J Ophthalmol. 2011;4(2):57–62. doi:10.
4103/0974-620X.83654.
23. Holick MF. Vitamin D deficiency. N Engl J Med.
2007;357(3):266–81. doi:10.1056/NEJMra070553.
24. Misra M, Pacaud D, Petryk A, et al. Vitamin D deficiency in
children and its management: review of current knowledge and
recommendations. Pediatrics. 2008;122(2):398–417. doi:10.1542/
peds.2007-1894.
25. Lips P. Vitamin D physiology. Prog Biophys Mol Biol.
2006;92(1):4–8. doi:10.1016/j.pbiomolbio.2006.02.016.
26. Holick MF, Chen TC. Vitamin D deficiency: a worldwide prob-
lem with health consequences. Am J Clin Nutr.
2008;87(4):1080S–1086S.
27. Looker AC, Pfeiffer CM, Lacher DA, Schleicher RL, Picciano
MF, Yetley EA. Serum 25-hydroxyvitamin D status of the US
population: 1988–1994 compared with 2000–2004. Am J Clin
Nutr. 2008;88(6):1519–27. doi:10.3945/ajcn.2008.26182.
28. Moan J, Porojnicu AC, Dahlback A, Setlow RB. Addressing the
health benefits and risks, involving vitamin D or skin cancer, of
increased sun exposure. Proc Natl Acad Sci USA.
2008;105(2):668–73. doi:10.1073/pnas.0710615105.
29. Pittas AG, Harris SS, Stark PC, Dawson-Hughes B. The effects of
calcium and vitamin D supplementation on blood glucose and
markers of inflammation in nondiabetic adults. Diabetes Care.
2007;30(4):980–6. doi:10.2337/dc06-1994.
30. Tsur A, Feldman BS, Feldhammer I, Hoshen MB, Leibowitz G,
Balicer RD. Decreased serum concentrations of 25-hydroxyc-
holecalciferol are associated with increased risk of progression to
impaired fasting glucose and diabetes. Diabetes Care.
2013;36(5):1361–7. doi:10.2337/dc12-1050.
31. Llewellyn DJ, Lang IA, Langa KM, et al. Vitamin D and risk of
cognitive decline in elderly persons. Arch Intern Med.
2010;170(13):1135–41. doi:10.1001/archinternmed.2010.173.
32. Knekt P, Kilkkinen A, Rissanen H, Marniemi J, Saaksjarvi K,
Heliovaara M. Serum vitamin D and the risk of Parkinson dis-
ease. Arch Neurol. 2010;67(7):808–11. doi:10.1001/archneurol.
2010.120.
33. Mora JR, Iwata M, von Andrian UH. Vitamin effects on the
immune system: vitamins A and D take centre stage. Nat Rev
Immunol. 2008;8(9):685–98. doi:10.1038/nri2378.
34. Fetahu IS, Hobaus J, Kallay E. Vitamin D and the epigenome.
Front Physiol. 2014;5:164. doi:10.3389/fphys.2014.00164.
35. MacDonald PN, Baudino TA, Tokumaru H, Dowd DR, Zhang C.
Vitamin D receptor and nuclear receptor coactivators: crucial
interactions in vitamin D-mediated transcription. Steroids.
2001;66(3–5):171–6. doi:10.1016/S0039-128X(00)00200-2
36. Jaddoe VW, van Duijn CM, Franco OH, et al. The Generation R
Study: design and cohort update 2012. Eur J Epidemiol.
2012;27(9):739–56.
37. Kruithof CJ, Kooijman MN, van Duijn CM, et al. The Generation
R Study: Biobank update 2015. Eur J Epidemiol.
2014;29(12):911–27. doi:10.1007/s10654-014-9980-6.
38. Camparini M, Cassinari P, Ferrigno L, Macaluso C. ETDRS-fast:
implementing psychophysical adaptive methods to standardized
visual acuity measurement with ETDRS charts. Invest Ophthal-
mol Vis Sci. 2001;42(6):1226–31.
39. O’Donoghue L, Rudnicka AR, McClelland JF, Logan NS,
Saunders KJ. Visual acuity measures do not reliably detect
childhood refractive error—an epidemiological study. PLoS
ONE. 2012;7(3):e34441. doi:10.1371/journal.pone.0034441.
40. Leone JF, Mitchell P, Morgan IG, Kifley A, Rose KA. Use of
visual acuity to screen for significant refractive errors in ado-
lescents: is it reliable? Arch Ophthalmol. 2010;128(7):894–9.
doi:10.1001/archophthalmol.2010.134.
41. Voortman T, van den Hooven EH, Heijboer AC, Hofman A,
Jaddoe VWV, Franco OH. Vitamin D deficiency in school-age
children is associated with sociodemographic and lifestyle fac-
tors. J Nutr. 2015;145(4):791–8.
42. Vogeser M. Quantification of circulating 25-hydroxyvitamin D
by liquid chromatography–tandem mass spectrometry. J Steroid
Biochem Mol Biol. 2010;121(3–5):565–73. doi:10.1016/j.jsbmb.
2010.02.025.
498 J. W. L. Tideman et al.
123
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

43. Yetley EA, Pfeiffer CM, Schleicher RL, et al. NHANES moni-
toring of serum 25-hydroxyvitamin D: a roundtable summary.
J Nutr. 2010;140(11):2030S–45S. doi:10.3945/jn.110.121483.
44. Tolppanen AM, Sayers A, Fraser WD, Lewis G, Zammit S,
Lawlor DA. The association of serum 25-hydroxyvitamin D3 and
D2 with depressive symptoms in childhood—a prospective
cohort study. J Child Psychol Psychiatry. 2012;53(7):757–66.
doi:10.1111/j.1469-7610.2011.02518.x.
45. Morrison MA, Silveira AC, Huynh N, et al. Systems biology-
based analysis implicates a novel role for vitamin D metabolism
in the pathogenesis of age-related macular degeneration. Hum
Genomics. 2011;5(6):538–68. doi:10.1186/1479-7364-5-6-538
46. Wang TJ, Zhang F, Richards JB, et al. Common genetic deter-
minants of vitamin D insufficiency: a genome-wide association
study. Lancet. 2010;376(9736):180–8. doi:10.1016/S0140-
6736(10)60588-0.
47. Allochtonen in Nederland 2004. Voorburg/Heerlen: Statistics
Netherlands; 2004.
48. Ashby R, Ohlendorf A, Schaeffel F. The effect of ambient illu-
minance on the development of deprivation myopia in chicks.
Invest Ophthalmol Vis Sci. 2009;50(11):5348–54. doi:10.1167/
iovs.09-3419.
49. Choi JA, Han K, Park YM, La TY. Low serum 25-hydroxyvi-
tamin D is associated with myopia in Korean adolescents. Invest
Ophthalmol Vis Sci. 2014;55(4):2041–7. doi:10.1167/IOVS.13-
12853.
50. Guggenheim JA, Williams C, Northstone K, et al. Does vitamin
D mediate the protective effects of time outdoors on myopia?
Findings from a prospective birth cohort. Invest Ophthalmol Vis
Sci. 2014;55(12):8550–8. doi:10.1167/iovs.14-15839.
51. Feldkaemper M, Schaeffel F. An updated view on the role of
dopamine in myopia. Exp Eye Res. 2013;114:106–19. doi:10.
1016/j.exer.2013.02.007.
52. Luft WA, Iuvone PM, Stell WK. Spatial, temporal, and intensive
determinants of dopamine release in the chick retina. Vis Neu-
rosci. 2004;21(4):627–35. doi:10.1017/S0952523804214110.
53. Feldkaemper M, Diether S, Kleine G, Schaeffel F. Interactions of
spatial and luminance information in the retina of chickens during
myopia development. Exp Eye Res. 1999;68(1):105–15. doi:10.
1006/exer.1998.0590.
54. Bloomfield SA, Volgyi B. The diverse functional roles and reg-
ulation of neuronal gap junctions in the retina. Nat Rev Neurosci.
2009;10(7):495–506. doi:10.1038/nrn2636.
55. Oran M, Unsal C, Albayrak Y, et al. Possible association between
vitamin D deficiency and restless legs syndrome. Neuropsychiatr
Dis Treat. 2014;10:953–8. doi:10.2147/NDT.S63599.
56. Cass WA, Peters LE, Fletcher AM, Yurek DM. Calcitriol pro-
motes augmented dopamine release in the lesioned striatum of
6-hydroxydopamine treated rats. Neurochem Res. 2014;39(8):
1467–76. doi:10.1007/s11064-014-1331-1.
57. Jiang P, Zhang LH, Cai HL, et al. Neurochemical effects of
chronic administration of calcitriol in rats. Nutrients.
2014;6(12):6048–59. doi:10.3390/nu6126048.
58. Orme RP, Bhangal MS, Fricker RA. Calcitriol imparts neuro-
protection in vitro to midbrain dopaminergic neurons by upreg-
ulating GDNF expression. PLoS ONE. 2013;8(4):e62040. doi:10.
1371/journal.pone.0062040.
59. de Borst MH, de Boer RA, Stolk RP, Slaets JP, Wolffenbuttel
BH, Navis G. Vitamin D deficiency: universal risk factor for
multifactorial diseases? Curr Drug Targets. 2011;12(1):97–106.
doi:10.2174/138945011793591590
60. Alsalem JA, Patel D, Susarla R, et al. Characterization of vitamin
D production by human ocular barrier cells. Invest Ophthalmol
Vis Sci. 2014;55(4):2140–7. doi:10.1167/iovs.13-13019.
Low serum vitamin D is associated with axial length and risk of myopia in young children 499
123
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

1.
2.
3.
4.
5.
6.
Terms and Conditions
Springer Nature journal content, brought to you courtesy of Springer Nature Customer Service Center GmbH (“Springer Nature”).
Springer Nature supports a reasonable amount of sharing of research papers by authors, subscribers and authorised users (“Users”), for small-
scale personal, non-commercial use provided that all copyright, trade and service marks and other proprietary notices are maintained. By
accessing, sharing, receiving or otherwise using the Springer Nature journal content you agree to these terms of use (“Terms”). For these
purposes, Springer Nature considers academic use (by researchers and students) to be non-commercial.
These Terms are supplementary and will apply in addition to any applicable website terms and conditions, a relevant site licence or a personal
subscription. These Terms will prevail over any conflict or ambiguity with regards to the relevant terms, a site licence or a personal subscription
(to the extent of the conflict or ambiguity only). For Creative Commons-licensed articles, the terms of the Creative Commons license used will
apply.
We collect and use personal data to provide access to the Springer Nature journal content. We may also use these personal data internally within
ResearchGate and Springer Nature and as agreed share it, in an anonymised way, for purposes of tracking, analysis and reporting. We will not
otherwise disclose your personal data outside the ResearchGate or the Springer Nature group of companies unless we have your permission as
detailed in the Privacy Policy.
While Users may use the Springer Nature journal content for small scale, personal non-commercial use, it is important to note that Users may
not:
use such content for the purpose of providing other users with access on a regular or large scale basis or as a means to circumvent access
control;
use such content where to do so would be considered a criminal or statutory offence in any jurisdiction, or gives rise to civil liability, or is
otherwise unlawful;
falsely or misleadingly imply or suggest endorsement, approval , sponsorship, or association unless explicitly agreed to by Springer Nature in
writing;
use bots or other automated methods to access the content or redirect messages
override any security feature or exclusionary protocol; or
share the content in order to create substitute for Springer Nature products or services or a systematic database of Springer Nature journal
content.
In line with the restriction against commercial use, Springer Nature does not permit the creation of a product or service that creates revenue,
royalties, rent or income from our content or its inclusion as part of a paid for service or for other commercial gain. Springer Nature journal
content cannot be used for inter-library loans and librarians may not upload Springer Nature journal content on a large scale into their, or any
other, institutional repository.
These terms of use are reviewed regularly and may be amended at any time. Springer Nature is not obligated to publish any information or
content on this website and may remove it or features or functionality at our sole discretion, at any time with or without notice. Springer Nature
may revoke this licence to you at any time and remove access to any copies of the Springer Nature journal content which have been saved.
To the fullest extent permitted by law, Springer Nature makes no warranties, representations or guarantees to Users, either express or implied
with respect to the Springer nature journal content and all parties disclaim and waive any implied warranties or warranties imposed by law,
including merchantability or fitness for any particular purpose.
Please note that these rights do not automatically extend to content, data or other material published by Springer Nature that may be licensed
from third parties.
If you would like to use or distribute our Springer Nature journal content to a wider audience or on a regular basis or in any other manner not
expressly permitted by these Terms, please contact Springer Nature at
onlineservice@springernature.com