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ORIGINAL ARTICLE
Correspondence:
Elisabeth Lerchbaum, Division of Endocrinology
and Metabolism, Department of Internal
Medicine, Medical University of Graz,
Auenbruggerplatz 15, Graz 8036, Austria.
E-mail: elisabeth.lerchbaum@medunigraz.at
Keywords:
androgens, hypogonadism, sex hormones
Received: 11-Mar-2014
Revised: 16-Jun-2014
Accepted: 18-Jun-2014
doi: 10.1111/j.2047-2927.2014.00247.x
Serum vitamin D levels and
hypogonadism in men
1,2
E. Lerchbaum,
1,3
S. Pilz,
1
C. Trummer,
2
T. Rabe,
4
M. Schenk,
5
A. C. Heijboer and
1
B. Obermayer-Pietsch
1
Division of Endocrinology and Metabolism, Department of Internal Medicine, Medical University of
Graz, Graz, Austria,
2
University Women’s Hospital, Heidelberg, Germany,
3
Department of
Epidemiology and Biostatistics and EMGO Institute for Health and Care Research, VU University
Medical Center, Amsterdam, The Netherlands,
4
Das Kinderwunsch Institut, Dobl, Austria, and
5
Department of Clinical Chemistry, VU University Medical Center, Amsterdam, The Netherlands
SUMMARY
There is inconsistent evidence on a possible association of vitamin D and androgen levels in men. We therefore aim to investigate
the association of 25-hydroxyvitamin D (25(OH)D) with androgen levels in a cohort of middle-aged men. This cross-sectional study
included 225 men with a median (interquartile range) age of 35 (30–41) years. We measured 25(OH)D, total testosterone (TT) and
SHBG concentrations. Hypogonadism was defined as TT <10.4 nmol/L. We found no significant correlation of 25(OH)D and andro-
gen levels. Furthermore, androgen levels were not significantly different across 25(OH)D quintiles. The overall prevalence of hypog-
onadism was 21.5% and lowest in men within 25(OH)D quintile 4 (82–102 nmol/L). We found a significantly increased risk of
hypogonadism in men within the highest 25(OH)D quintile (>102 nmol/L) compared to men in quintile 4 (reference) in crude (OR
5.10, 1.51–17.24, p=0.009) as well as in multivariate adjusted analysis (OR 9.21, 2.27–37.35, p=0.002). We found a trend towards
increased risk of hypogonadism in men within the lowest 25(OH)D quintile (≤43.9 nmol/L). In conclusion, our data suggest that men
with very high 25(OH)D levels (>102 nmol/L) might be at an increased risk of hypogonadism. Furthermore, we observed a trend
towards increased risk of hypogonadism in men with very low vitamin D levels indicating a U-shaped association of vitamin D levels
and hypogonadism. With respect to risk of male hypogonadism, our results suggest optimal serum 25(OH)D concentrations of
82–102 nmol/L.
INTRODUCTION
Vitamin D is a steroid hormone and the vitamin D status is
mainly determined by ultraviolet-B-induced vitamin D produc-
tion in the skin, while vitamin D intake by nutrition and supple-
ments plays only a minor role (Holick, 2007). Vitamin D from
either source is hydroxylated in the liver to 25-hydroxyvitamin D
(25(OH)D), which is used to determine a patient’s vitamin D sta-
tus. 25(OH)D is further hydroxylated to its active form, 1,25-di-
hydroxyvitamin D [1,25(OH)2D] in the kidney as well as in other
tissues including human testis (Blomberg Jensen, 2014). Biologi-
cal actions of vitamin D are mediated through the vitamin D
receptor (VDR), which regulates about 3% of the human genome
(Bouillon et al., 2008). The VDR is almost ubiquitously expressed
in human cells, which underlines the clinical significance of the
vitamin D endocrine system (Kinuta et al., 2000; Holick, 2007;
Pludowski et al., 2013). VDR and enzymes that metabolize vita-
min D are concomitantly expressed in Sertoli cells, germ cells,
Leydig cells, spermatozoa and in the epithelial cells lining the
male reproductive tract (Blomberg Jensen et al., 2010; Blomberg
Jensen, 2014).
Interestingly, there is accumulating evidence suggesting a
complex interplay of vitamin D and androgen metabolism. It has
been shown that androgens increase 1-alpha-hydroxylase, a key
enzyme in vitamin D metabolism which converts 25(OH)D to
1,25(OH)2D (Somjen et al., 2007). Furthermore, it has been dem-
onstrated that the regulation of gene expression by vitamin D
metabolites is modified according to androgen levels (Mordan-
McCombs et al., 2010). In 2009, some of us demonstrated for the
first time that androgen levels are associated with 25(OH)D lev-
els in 2299 older men from the LURIC study (Wehr et al., 2010).
Furthermore, previous data indicated that vitamin D therapy
might increase testosterone levels in obese men undergoing
weight reduction (Pilz et al., 2011). Interestingly, men with a
combined vitamin D and androgen deficiency are at high risk for
all-cause and cardiovascular mortality suggesting that a parallel
deficiency is a powerful marker of poor health (Lerchbaum &
©2014 American Society of Andrology and European Academy of Andrology Andrology, 1–7 1
ISSN: 2047-2919 ANDROLOGY
Obermayer-Pietsch, 2012; Lerchbaum et al., 2012). Despite these
previous studies suggesting an association of vitamin D and
androgen levels in men, several recent studies among young and
healthy men failed to find an association of vitamin D and
androgen levels in men (Chen et al., 2008; Ceglia et al., 2011;
Ramlau-Hansen et al., 2011; Hammoud et al., 2012).
Considering these inconsistent previous data, we aim to study
the association of 25(OH)D levels with TT, free testosterone (FT)
and SHBG levels in a cohort of middle-aged men. Moreover, we
aim to examine the association of vitamin D status with
hypogonadism.
METHODS
Subjects
The study cohort consisted of 225 healthy men, aged 20–
58 years, who were routinely referred to the outpatient clinic of
the Department of Gynecology and Obstetrics at the Medical
University of Graz (n=179, trial site 1) and the Kinderwunsch
Institut Dobl (n=46, trial site 2) between 2010 and 2012. These
men were either part of an infertile couple or simply in desire of
having children and therefore came for endocrine evaluation.
None of the subjects had a history of hypogonadism or any
known disease associated with hypogonadism (except obesity).
None of the patients had diabetes and none of the men took
medications known to affect endocrine parameters. Four men
reported intake of vitamin D supplementation. Those men were
included in the analyses as 25(OH)D levels were similar in men
with and without vitamin D supplementation [78 (67.5–87.0)
nmol/L vs. 74 (46.5–94.0) nmol/L, p=0.889]. The study protocol
was approved by the ethics committee of the Medical University
of Graz. Written informed consent was obtained from each
patient.
Data from a part of the cohort (men without azoospermia
from trial site 1) have been published previously (Schwetz et al.,
2013).
Procedures
Standard anthropometric data [height, weight, waist circum-
ference (WC) and hip circumference, blood pressure (BP)] were
obtained from 200 subjects. WC was measured in a standing
position midway between the lower costal margin and the iliac
crest. Hip circumference was measured in a standing position at
the maximum circumference over the buttocks. The BMI was
calculated as the weight in kilograms divided by the square of
height in metres. Overweight was defined as BMI 25.0–29.9 kg/
m²and obesity was defined as BMI ≥30 kg/m².
Basal blood samples for endocrine parameters [25(OH)D, TT,
free testosterone (FT), SHBG, LH, FSH, estradiol] were collected
between 8.00 and 9.00 a.m. after an overnight fast in all 179 men
at trial site 1. At trial site 2, blood samples were collected
between 7.15 and 10.00 a.m. in 23 men, between 10.30 a.m. and
12.00 noon in 10 men and after 12 noon in 13 men. Routine labo-
ratory parameters were immediately measured on a daily
(SHBG, LH, FSH, estradiol) to weekly (FT) basis. Remaining
blood samples were frozen and stored at 80 °C until further
analysis. Serum levels of 25(OH)D and TT were measured by Iso-
tope-Dilution Liquid Chromatography Tandem Mass Spectrom-
etry (ID-LC-MS/MS) in 2013. FT values (FT
Vermeulen
) were
calculated from TT, SHBG and albumin according to Vermeulen
(Vermeulen et al., 1999). The free androgen index (FAI) was cal-
culated as TT (nmol/L)/SHBG (nmol/L) 9100. Male hypogona-
dism was defined as TT levels <10.4 nmol/L measured by ID-
LC-MS/MS according to widely used cut-offs (to convert serum
TT levels to ng per litre, divide by 3.467) (Tajar et al., 2010). Men
with hypogonadism were further classified depending on their
LH concentrations using a cut-off of 7.4 IU/L as suggested by
the manufacturer and validated in our laboratory. A low TT level
and LH ≤7.4 IU was defined as secondary hypogonadism and a
low TT level and LH >7.4 IU/L was classified as secondary hyp-
ogonadism. According to widely used cut-offs for vitamin D sta-
tus classification, subjects were divided into three groups:
vitamin D sufficiency (25(OH)D ≥75.0 nmol/L), vitamin D insuf-
ficiency (25(OH)D 50.0–74.9 nmol/L) and vitamin D deficiency
(<50.0 nmol/L) (to convert serum 25(OH)D levels to lg per litre,
divide by 2.496). As the prevalence of subjects with sufficient
vitamin D levels was high (48%), we further calculated quintiles
of 25(OH)D concentrations. To study the influence of seasonal
variation on hormones, we subdivided the year into 3-month
measurement periods: January–March (season 1); April–June
(season 2); July–September (season 3); October–December (sea-
son 4) to address the seasonal changes in availability of sunlight.
Biochemical analyses
All samples (trial sites 1 and 2) were measured in the same lab-
oratory using the same assays. FT was determined using a radio-
immunoassay (DSL, Webster, TX, USA; intra- and interassay CVs
of <10%). SHBG was measured by luminescence immunoassay
(Cobas; Roche, Basel, Switzerland) with an intra- and interassay
CV of 1.3 and 2.1% respectively. LH and FSH were measured by
enzyme immunoassay (DiaMetra S.r.l., Segrate (MI), Italy; intra-
and interassay CVs of <10%) and estradiol was measured by
chemiluminescent immunoassay (Immulite, SIEMENS Health-
care, UK; intra- and interassay CVs of 15 and 16% respectively).
MS
Total testosterone and 25(OH)D levels were measured by ID-
LC-MS/MS in 225 men. 25(OH)D measurements by ID-XLC-MS/
MS were performed at the Endocrine Laboratory of the VU Uni-
versity Medical Center (Amsterdam, the Netherlands) as
described before (Heijboer et al., 2012), with only minor adjust-
ments. In short, deuterated internal standard (IS) [25(OH)D3-d6]
(Synthetica, Oslo, Norway) was added to the samples and 25
(OH)D was released from its binding proteins with acetonitrile.
Samples were extracted and analysed by XLC-MS/MS [a Symbio-
sis online SPE system (Spark Holland, Emmen, the Netherlands)]
coupled to a Quattro Premier XE tandem mass spectrometer
(Waters Corp., Milford, MA, USA). The limit of quantitation
(LOQ) was 4.0 nmol/L; intraassay CV was <6%, and interassay
CV was <8% for concentrations between 25 and 180 nmol/L. 25
(OH)D2 and 25(OH)D3 were measured separately.
Total testosterone measurements by ID-XLS-MS/MS were per-
formed at the Endocrine Laboratory of the VU University Medi-
cal Center (Amsterdam, the Netherlands) as described before
(Bui et al., 2013). In short, a stable isotopically labelled internal
standard (testosterone-2,2,4,6,6-D5;D5T; CDN Isotopes, Pointe-
Claire, Canada) was added to every sample. Testosterone was
extracted with hexane/ether (4/1, v/v), dried and derivatized
with methoxylamine hydrochloride (T-mox), followed by
another hexane/ether extraction. Separation was achieved on a
2Andrology, 1–7 ©2014 American Society of Andrology and European Academy of Andrology
E. Lerchbaum et al. ANDROLOGY
C8 analytical column (XBridge, 2.1 950 mm, 2.5 lm particle
size: Waters Corp.) by gradient elution, using 0.1% formic acid in
water and 0.1% formic acid in acetonitrile. A Quattro Premier XE
tandem mass spectrometer (Waters Corp.) with electrospray in
positive mode was used for detection. The LOQ was 0.10 nmol/L
(2.88 ng/dL); inter-assay variation at 0.21, 2.1 and 15.8 nmol/L
was 9, 7 and 4% respectively. This method correlates well with a
ID-GC-MS reference method for TT (Thienpont et al., 2008; Bui
et al., 2013).
Semen analyses
Semen analysis was carried out using standard procedures as
recommended by the World Health Organization (World Health
Organization, 2010). Oligozoospermia was defined as a sperm
concentration of <15 mio/mL, azoospermia was defined as a
sperm count of 0/mL and oligo-astheno-teratozoospermia
(OAT) was defined as sperm concentration <15 mio/mL, motility
<40% and strict morphology <4% according to the WHO criteria
2010 (WHO, 2010).
Statistical analyses
Anthropometric and biochemical data were completely avail-
able in 200 men. Thus, all analyses (except correlation analyses
between endocrine parameters) were performed among 200
men. Data are presented as median with interquartile range
unless otherwise stated. The distribution of data was analysed by
descriptive statistics and Kolmogorov–Smirnov test. All parame-
ters except 25(OH)D and TT were found to be non-normally dis-
tributed. All non-normally distributed variables were log-
transformed and checked for normal distribution before being
entered in parametric tests. Pearson correlation analysis was
used for correlation analysis of hormonal and anthropometric
parameters. T-test, ANOVA and chi-squared test were used for
comparisons between groups. We calculated binary logistic
regression analyses using hypogonadism as dependent variables
and 25(OH)D quintiles, age, BMI, trial site and ethnical back-
ground as independent variables. Because TT levels measured in
morning samples are considered more accurate, we repeated all
analyses after excluding 23 men in whom blood samples were
drawn after 10 a.m. Furthermore, we repeated all analyses after
excluding men with primary hypogonadism, azoospermic men
and after adjusting for time of blood sampling. All statistical pro-
cedures were performed with SPSS version 20 (SPSS Inc., Chi-
cago, IL, USA). A p-value <0.05 was considered statistically
significant.
RESULTS
Baseline characteristics of men are presented in Table 1.
Overweight and obesity were prevalent in 44.7% and 14.3% of
men respectively. We found vitamin D deficiency, insufficiency
and sufficiency in 53 (26.5%), 51 (23.5%) and 96 (48%) of 200
men respectively. Furthermore, 126 men (63.0%) had normo-
zoospermia, 47 men (23.5%) had oligozoospermia, 27 men
(13.5%) had azoospermia and 42 men (21.0%) presented with
OAT.
We found no significant correlation of 25(OH)D levels with
other endocrine or metabolic parameters (data not shown). We
observed significant positive correlations between TT and FT
(r=0.379, p<0.001), FT
Vermeulen
(r=0.812, p<0.001), FAI
(r=0.302, p<0.001), SHBG (r=0.574, p<0.001), FSH (r=0.170,
p=0.036) and LH (r=0.342, p<0.001) levels and significant
negative correlations with age (r=0.239, p<0.001), BMI
(r=0.326, p<0.001), WC (r=0.415, p<0.001) and WHR
(r=0.395, p<0.001).
Anthropometric characteristics of subjects according to 25
(OH)D quintiles are shown in Table 2. We found no significant
differences in anthropometric and biochemical parameters
between men in different 25(OH)D quintiles. The prevalence of
hypogonadism and ethnical background was significantly differ-
ent across 25(OH)D quintiles.
Hypogonadism
We found hypogonadism in 43 of 200 men (21.5%); five men
had primary hypogonadism and 38 men had secondary hypog-
onadism. Men with hypogonadism had significantly higher BMI,
WC and WHR and lower FAI, FT, FT
Vermeulen
and SHBG levels,
whereas no significant differences were found in age, 25(OH)D,
estradiol, LH, FSH, ethnic background (Table 1), season or
month of blood sampling and semen parameters (data not
shown).
Table 1 Characteristics of all subjects and according to hypogonadism
All men (n=200) Eugonadal men (n=157) Hypogonadal men (n=43) p-value
Median IQ range Median IQ range Median IQ range
Age (years) 35 31–41 35 31–41 38 33–42 0.056
BMI (kg/m²) 25.7 23.6–28.5 25.3 23.5–27.7 28.6 24.9–31.4 <0.001
WC (cm) 91 85–99 90 84–96 98 90–106 <0.001
WHR 0.89 0.84–0.93 0.88 0.82–0.92 0.92 0.88–0.96 <0.001
Systolic BP 135 124–145 135 124–145 134 122–146 0.909
Diastolic BP 85 79–94 84 78–94 86 80–91 0.552
TT (nmol/L) 13.9 10.6–16.9 15.3 13.2–18.0 8.6 7.2–9.5 <0.001
FT
Vermeulen
(pmol/L) 281 229–350 312 267–364 191 170–220 <0.001
FT (pmol/L) 353 259–469 387 279–486 293 196–350 0.048
FAI 43.48 35.19–54.04 458 357–549 356 300–445 <0.001
SHBG (nmol/L) 32.4 23.81–41.65 34.4 26.3–44.4 23.6 18.0–29.8 <0.001
FSH (IU/L) 4.3 2.8–6.0 4.3 2.9–6.0 3.6 2.3–5.9 0.276
LH (IU/L) 3.2 2.2–4.5 3.5 2.5–4.7 2.3 1.5–3.7 0.527
Estradiol (pmol/L) 100 70–130 100 70–130 100 70–140 0.509
25(OH)D (nmol/L) 67.8 44.9–94.6 65.0 44.4–94.0 70.3 48.4–107.8 0.413
Caucasian white (%) 74.5 72.7 74.5 0.806
Comparisons between men with and without hypogonadism were performed using t-test. BMI, body mass index; WC, waist circumference; WHR, waist-to-hip ratio;
TT, total testosterone; FT, free testosterone; FAI, free androgen index; 25(OH)D, 25-hydroxyvitamin D.
©2014 American Society of Andrology and European Academy of Andrology Andrology, 1–7 3
VITAMIN D AND HYPOGONADISM IN MEN ANDROLOGY
We performed binary logistic regression using hypogonadism
as dependent variable and 25(OH)D quintiles, age, BMI, ethnic
background and study site as explanatory variables. As the prev-
alence of hypogonadism was lowest in men within 25(OH)D
quintile 4 (Table 2), we used quintile 4 as reference. We found a
significantly increased risk of hypogonadism in men within the
highest 25(OH)D quintile compared with men in quintile 4 in
crude as well as in multivariate adjusted analysis (Table 3). Fur-
thermore, we found a trend towards increased risk of hypogona-
dism in men within the lowest 25(OH)D quintile in all models.
As the majority of men had secondary hypogonadism, we
repeated our binary regression analyses after exclusion of men
with primary hypogonadism, but our results remained materially
unchanged (data not shown).
The prevalence of hypogonadism was higher at trial site 2
(16.7% vs. 37%, p=0.003). To explore this difference, we com-
pared all variables displayed in Table 2 between the trial sites.
We found significantly lower TT and FAI levels (p<0.001 for
both) and significantly higher WHR levels (p=0.032) in men
from trial site 2, whereas all other parameters including 25(OH)
D levels were similar (p-value >0.300 for all). After exclusion of
23 men with non-morning blood samples, the prevalence of
hypogonadism still tended to be higher at trial site 2 (p=0.052).
To further address this difference, we excluded men from trial
site 2 and repeated all analyses including only men from trial site
1. This did, however, not significantly change our results (data
not shown).
Furthermore, when we restricted our analyses to Caucasian
white men, or to men with morning blood samples or to men
without azoospermia, and after adjusting our results for time of
blood sampling, our results did not materially change (data not
shown).
Seasonal variation
Vitamin D levels were highest in season 3 [91.0 (73.0–115.0)
nmol/L] and lowest in season 1 [52.5 (40.0–74.0) nmol/L], season
2 [69.0 (46.5–86.5) nmol/L] and season 4 [85.0 (51.0–101.0)
nmol/L] respectively (p<0.001).
We found no significant differences in TT, FT, FT
Vermeulen
, FAI,
SHBG, FSH, LH and estradiol levels or prevalence of hypogona-
dism regarding season or month of blood sampling (p>0.200
for all, data not shown).
DISCUSSION
We present evidence that 25(OH)D levels >102 nmol/L are
associated with increased risk of hypogonadism in otherwise
healthy men with a median age of 35 (31–41) years. 25(OH)D lev-
els between 82 and 102 nmol/L appear most favourable
Table 2 Characteristics of subjects according to 25(OH)D quintiles
25(OH)D quintiles p-value
Quintile 1
(≤43.9 nmol/L)
Quintile 2 (44.0–
67.9 nmol/L)
Quintile 3 (68.0–
82.1 nmol/L)
Quintile 4 (82.2–
101.8 nmol/L)
Quintile 5
(>101.8 nmol/L)
Median IQ range Median IQ range Median IQ range Median IQ range Median IQ range
Age (years) 35 30–38 34 30–39 35 32–44 36 33–41 37 34–41 0.300
BMI (kg/m²) 25.7 23.6–23.4 25.7 23.9–29.0 25.1 23.2–27.9 26.1 23.5–28.3 25.1 24.7–27.4 0.975
WC (cm) 89 86–101 91 85–97 92 85–99 91 84–97 90 84–98 0.926
WHR 0.89 0.84–0.94 0.87 0.84–0.91 0.90 0.83–0.93 0.88 0.83–0.93 0.88 0.82–0.92 0.743
TT (nmol/L) 14.7 10.3–16.8 14.2 10.9–18.2 12.8 10.9–15.9 15.1 11.1–19.0 13.9 9.1–17.3 0.310
FT
Vermeulen
(pmol/L) 279 222–352 312 260–374 277 198–347 291 246–354 258 220–342 0.321
FT (pmol/L) 333 293–422 437 294–500 372 301–437 388 319–426 344 269–477 0.730
FAI 44.98 32.52–53.26 47.27 37.07–58.1 41.13 33.92–53.95 41.37 36.68 41.35 34.57–51.7 0.362
SHBG (nmol/L) 30.4 24.06–38.5 32.6 23.2–38.5 30.5 22.25–37.2 35.9 29.4–47.5 32.5 23.6–46.5 0.327
FSH (IU/L) 4.6 3.6–5.9 4.7 3.6–7.0 3.5 2.6–4.5 4.5 2.3–6.8 3.7 2.8–6.1 0.125
LH (IU/L) 3.6 2.5–4.7 4.0 2.9–5.2 2.6 2.0–3.8 2.9 2.1–4.2 3.0 2.0–4.0 0.400
Estradiol (pmol/L) 94.5 51.6–114.5 117.3 73.4–143.5 101.7 73.4–122.6 103.7 73.4–127.2 110.0 74.1–136.2 0.129
25(OH)D (nmol/L) 32.0 24.0–41.0 57.0 48.0–62.0 74.0 72.0–78.0 91.0 87.0–94.0 118.5 107.0–133.0 <0.001
Hypogonadism (%) 26.2 11.1 21.1 10.5 37.5 0.021
Caucasian white (%) 38.1 68.4 89.5 92.3 75.1 <0.001
Comparisons between 25(OH)D quintiles were performed using ANOVA and chi-squared test. Data are available in 200 men. 25(OH)D, 25-hydroxyvitamin D; BMI, body
mass index; WC, waist circumference; WHR, waist-to-hip ratio; TT, total testosterone; FT, free testosterone; FAI, free androgen index.
Table 3 Odds ratio with 95% CI for hypogonadism according to 25(OH) quintiles
Model 1 Model 2 Model 3
OR p-value OR p-value OR p-value
Quintile 1 (≤43.9 nmol/L) 3.02 (0.87–10.46) 0.082 3.47 (0.95–12.75) 0.060 3.67 (0.84–16.11) 0.085
Quintile 2 (44.0–67.9 nmol/L) 1.06 (0.25–4.61) 0.935 0.97 (0.20–4.67) 0.968 1.05 (0.21–5.37) 0.949
Quintile 3 (68.0–82.1 nmol/L) 2.27 (0.62–8.29) 0.216 2.32 (0.59–9.16) 0.231 2.68 (0.64–11.25) 0.178
Quintile 4 (82.2–101.8 nmol/L) 1.0 (Reference) 1.0 (Reference) 1.0 (Reference)
Quintile 5 (>101.8 nmol/L) 5.10 (1.51–17.24) 0.009 6.39 (1.76–23.22) 0.005 9.21 (2.27–37.35) 0.002
Binary logistic regression analysis was performed using hypogonadism as dependent variable and 25(OH)D quintiles, age, BMI, ethnic background and study site as
explanatory variables. Data available in 200 men. Model 1: crude. Model 2: adjusted for age and BMI. Model 3: adjusted for age, BMI, ethnic background, study site.
25(OH)D, 25-hydroxyvitamin D; OR, odds ratio; BMI, body mass index.
4Andrology, 1–7 ©2014 American Society of Andrology and European Academy of Andrology
E. Lerchbaum et al. ANDROLOGY
regarding male hypogonadism. We further observed a trend
towards increased risk of hypogonadism in men with low 25
(OH)D levels (<44 nmol/L) suggesting a U-shaped association of
vitamin D status and hypogonadism in middle-aged men. Of
note, this is the first study evaluating the association of vitamin
and androgen levels in men using a state-of-the-art ID-LC-MS/
MS for measuring both 25(OH)D and TT levels which is consid-
ered the gold standard. Furthermore, the inclusion of a relatively
large proportion of men with 25(OH)D levels ≥75 nmol/L allows
a balanced evaluation of high 25(OH)D levels with the possibility
to evaluate non-linear associations.
The lack of a significant correlation between 25(OH)D and
androgen levels can be explained by the non-linear relationship
of the parameters. We further observed no significant differences
in androgen levels across 25(OH)D quintiles which is in line with
previous studies showing comparable testosterone levels in
healthy young and middle-aged men (Chen et al., 2008; Ceglia
et al., 2011; Ramlau-Hansen et al., 2011; Hammoud et al., 2012).
Interestingly, one study among 307 healthy young men aged 18–
21 years reported an inverse association of 25(OH)D levels with
FAI in adjusted analyses (Ramlau-Hansen et al., 2011). The sig-
nificantly lower FAI levels were observed in men with 25(OH)D
levels between 94 and 227 nmol/L. Moreover, results from the
Health Professionals Follow-up Study including 1362 healthy
men found a positive association between 25(OH)D and TT and
FT (Nimptsch et al., 2012). This association was linear at lower
25(OH)D levels (<75–85 nmol/L for TT and 75 nmol/L for FT)
reaching a plateau at higher levels. The authors also found a sig-
nificantly decreased risk of hypogonadism in men within the
highest 25(OH)D quintile compared to men within the lowest
quintile. The observed inconsistencies to our results indicating a
U-shaped association might be explained by age (mean age
66 years vs. 35 years in our study), as striking differences
between young and older men have been suggested and attrib-
uted to indirect effects vitamin D in older men (Blomberg Jen-
sen, 2014). This notion is supported by the fact that the positive
association of testosterone with 25(OH)D was mainly observed
in older men (Nimptsch et al., 2012) (Lee et al., 2012) (Wehr
et al., 2010). Indirect vitamin D effects on testosterone levels in
older men might be related to calcium and phosphate homeo-
stasis, SHBG or osteocalcin production (Blomberg Jensen, 2014).
This assumption is corroborated by the fact that Jorde et al.
(2013) found a weak (r=0.08) but significant correlation of vita-
min D and TT levels in 893 healthy men aged 61 years. Consis-
tently, data from the European Male ageing study, including
3369 community-dwelling men aged 40–79 years, suggest a posi-
tive association of 25(OH)D levels with TT and FT, which lost,
however, significance after adjusting for age and lifestyle factors
(Lee et al., 2012). Nevertheless, an independent association was
found between vitamin D deficiency and compensated as well as
secondary hypogonadism. Furthermore, the prevalence of vita-
min D deficiency was higher in diabetic men with hypogona-
dism compared to diabetic men without hypogonadism and
healthy controls (Bellastella et al., 2014). Results from the LURIC
study, including 2299 men aged 67 years at high cardiovascular
risk, demonstrated an independent linear association of high 25
(OH)D levels with higher TT and FAI levels and men with vita-
min D deficiency had an increased risk of hypogonadism com-
pared to men with sufficient vitamin D levels (Wehr et al., 2010).
The studies (Wehr et al., 2010; Lee et al., 2012; Bellastella et al.,
2014) used men with 25(OH)D levels >75 nmol/L as reference
groups and no further evaluation of men with “sufficient” levels
was performed.
As most previous studies investigated the association between
vitamin D and androgens or hypogonadism using men with 25
(OH)D levels >75 nmol/L as reference group, it was not possible
to detect a U-or J-shaped association. Furthermore, most previ-
ous studies include only a small proportion of men with vitamin
D sufficiency and it was therefore not possible to separately ana-
lyse men with higher vitamin D levels (>100 nmol/L). Neverthe-
less, vitamin D concentrations of 75–100 nmol/L have been
previously suggested as optimal and the need for studies using
optimal vitamin D dosage regimes (e.g. with the aim to reach the
25(OH)D target levels of ~75–100 nmol/L) has been underlined
(Pilz et al., 2012). Nevertheless, the increased risk of hypogona-
dism in men with very high vitamin D concentration is some-
what unexpected and we can only speculate on the underlying
mechanisms. In this context, it should be considered that high
vitamin D concentrations may affect vitamin D metabolism
within the target tissue, leading to increased 24-hydroxylation
(Miller et al., 1995). Thus, in the setting of high circulating 25
(OH)D levels, the concentration of the biologically active 1,25
(OH)2D might be reduced in target tissues such as testis and the
pituitary gland. Hence, both high as well as low vitamin D con-
centrations might cause harm. This hypothesis along with our
findings on a U-shaped association between 25(OH)D and hyp-
ogonadism fit well to previous reports on similar association
curves between vitamin D status and adverse outcomes such as
mortality. However, as we found no significant differences in
androgen or LH levels across vitamin D quintiles, our results
remain difficult to interpret.
Our findings are, however, supported by previous inconsistent
results with possible U-shaped or non-linear associations that
have been suggested for vitamin D and cancer (Pilz et al., 2013),
cardiovascular disease (Wang et al., 2008) and mortality (Zitter-
mann et al., 2012; Sempos et al., 2013). Indeed, the literature
reports a non-linear association of 25(OH)D with outcomes such
as breast cancer (Abbas et al., 2009), incident cardiovascular dis-
ease (Wang et al., 2008) and all-cause mortality (Zittermann
et al., 2012; Sempos et al., 2013). Interestingly, recent data from
the NHANES III cohort suggest a reverse J-shaped association
between serum 25(OH)D and all-cause mortality (Sempos et al.,
2013). A meta-analysis including 14 prospective studies involv-
ing 5562 deaths supports this reverse J-shaped association (Zit-
termann et al., 2012). The suggested optimal 25(OH)D
concentrations were 70–90 nmol/L (Sempos et al., 2013) and
75–87.5 nmol/L (Zittermann et al., 2012) respectively. Further-
more, an increased risk at low and high levels has been sug-
gested for prostate cancer (Tuohimaa et al., 2004), overall and
cancer mortality (Micha€
elsson et al., 2010) and incident cardio-
vascular disease (Wang et al., 2008). Of note, previous studies on
vitamin D and female fertility demonstrated promising results
regarding in vitro fertilization outcome as well as other aspects
(Lerchbaum & Obermayer-Pietsch, 2012; Lerchbaum et al., 2012;
Lerchbaum & Rabe, 2014). In light of the results of this study,
future studies should also focus on optimal vitamin D concen-
trations and critically examine the endocrine effects of very high
25(OH)D levels in women of reproductive age. In addition, the
U-shaped association between 25(OH)D and androgens as
reported in our study may also explain inconsistent results from
©2014 American Society of Andrology and European Academy of Andrology Andrology, 1–7 5
VITAMIN D AND HYPOGONADISM IN MEN ANDROLOGY
previous RCTs on the effect of vitamin D supplementation on
testosterone levels (Pilz et al., 2011; Jorde et al., 2013).
Our study has several limitations that should be noted. First,
because of the cross-sectional design of our study no conclu-
sions with respect to causality or directionality of the vitamin D-
hypogonadism association can be drawn. Furthermore, in some
men, blood samples were taken in the afternoon which probably
influences TT levels. To address this limitation, we repeated all
analyses excluding men with non-morning blood samples and
adjusted our analyses for time of blood sampling which did not
materially change our results. Furthermore, as the majority of
men had secondary hypogonadism, we cannot make a statement
on vitamin D and primary hypogonadism. Moreover, diagnosis
of hypogonadism is defined biochemically using TT and LH
without the inclusion of clinical signs or symptoms.
The strengths of our study include the measurement of TT as
well as 25(OH)D levels using a state-of-the-art LC-MSMS which
is considered the gold standard. Of note, this is the first study
evaluating the association of vitamin and androgen levels in
men using ID-LC-MS/MS for measuring both 25(OH)D and TT
levels. The inclusion of a relatively large proportion of men with
25(OH)D levels ≥75 nmol/L allowed a more balanced evaluation
of high 25(OH)D levels compared to previous studies that
included mainly subjects with 25(OH)D levels <75 nmol/L. As
we observed a significantly higher risk of hypogonadism in men
with 25(OH)D levels >102 nmol/L, future studies on vitamin D
and testosterone might also focus on men with 25(OH)D levels
in higher ranges. Overall, our study contributes to literature sug-
gesting that there might be an optimal 25(OH)D concentration
in humans that is between 75 and 100 nmol/L. Future RCTs
might therefore include only subjects with insufficient or defi-
cient vitamin D levels and should titrate vitamin D supplemen-
tation to reach and maintain 25(OH)D concentrations between
75 and 100 nmol/L.
In summary, we found a significantly increased risk of hypog-
onadism in men with 25(OH)D levels >102 nmol/L, whereas
hypogonadism risk was lowest in men with 25(OH)D levels
between 82 and 102 nmol/L. We further observed a trend
towards increased risk of hypogonadism in men with very low
vitamin D levels suggesting a U-shaped association of vitamin D
status and hypogonadism in middle-aged men. Our study there-
fore contributes to the accumulating evidence suggesting that 25
(OH)D concentrations between 75 and 100 nmol/L are optimal
and that not only low but also very high 25(OH)D levels should
be avoided.
ACKNOWLEDGEMENT
This work was supported by the funds of the Oesterreichische
Nationalbank (Oesterreichische Nationalbank, Anniversary
Fund, project number: 14846).
DISCLOSURES
The authors declare no conflict of interest.
AUTHOR CONTRIBUTIONS
E.L. contributed to research design, analysis of data and draft-
ing of the manuscript; S.P. contributed to research design and
revised the manuscript critically; C.T. contributed to acquisition
of data and drafting of the manuscript; T.R. contributed to inter-
pretation of data and revised the manuscript critically; M.S.
contributed to data acquisition and revised the manuscript criti-
cally; A.H. contributed to acquisition and interpretation of data
and drafting of the manuscript; B.O. contributed to research
design and revised the manuscript critically. All authors
approved the final version of the manuscript.
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