Introduction
Introduction
Vitamin D deficiency is currently considered an important public health problem being
associated with musculoskeletal diseases, cardiovascular disease, cancer, and infectious
and autoimmune diseases [1]. The vitamin D receptor (VDR), as well as key enzymes for vitamin D metabolism,
are widely expressed in human tissues and cells [2]. In this context, Blomberg Jensen et al. [3] observed significant expressions of the VDR and vitamin D metabolizing enzymes in
the male reproductive tract including Leydig cells of the testis. These data raised
the question whether vitamin D is able to influence male reproductive hormone production.
The existence of such an effect is supported by previous studies suggesting that vitamin
D deficiency may contribute to reduced fertility and hypogonadism [2]
[3]. These results are of particular interest because both, vitamin D deficiency and
hypogonadism are associated with skeletal diseases (e. g., osteoporosis or muscle
weakness) as well as extra-skeletal disorders (e. g., cardiovascular disease or obesity)
[1]
[4]
[5]. Recently, some of us [6] have shown in 2 299 men referred for coronary angiography that 25-hydroxyvitamin
D [25(OH)D] levels are significantly associated with testosterone levels and that
both hormones reveal similar seasonal variations with a peak at the end of summer.
Whether there exists a causal link between vitamin D and testosterone status is, however,
currently not known. Therefore, a subgroup analysis of a previously published prospective,
randomized vitamin D supplementation trial was performed in overweight subjects [7]. Here, we present results on serum testosterone concentrations in the male participants
of this study.
Subjects and Methods
Subjects and Methods
Study subjects were derived from a weight reduction program over 12 months, which
included a daily supplementation of either 83 μg (3 332 IU) vitamin D or placebo as
part of a double-blind randomized controlled trial [7]. Details on the study design and major outcomes of the trial have been published
previously [7]. Out of 200 nondiabetic individuals (62 men) who were included in the study, 165
(54 men) completed the trial. Only male study subjects were analyzed for the present
work. The number of dropouts (3 men in the placebo and 5 men in the vitamin D group)
did not differ significantly between groups (p=0.745). The original study was registered
at clinical trials.gov as NCT004493012.
Participants were continuously recruited from December 2005 to October 2006 throughout
the year. Fasting blood samples were drawn at study begin and after 1 year. Specimens
were centrifuged at room temperature. Thereafter, serum aliquots were stored at −80°C
until analyses. To avoid inter-assay variations, the samples of each participant were
analyzed within the same assay run. Concentrations of 25(OH)D were determined by means
of a radioimmunoassay (DiaSorin, Stillwater, MN, USA) with an intra-assay CV of <7%.
According to Holick [1], vitamin D deficiency is defined as a 25(OH)D level of less than 50 nmol/l, whereas
a level of 52.5–72.5 nmol/l indicates a relative insufficiency, and a level of 75 nmol/l
or greater indicates sufficient vitamin D. The serum concentrations of 1,25-dihydroxyvitamin
D [1, 25(OH)2D] were measured by a test kit provided by Immundiagnostik (Bensheim, Germany). The
serum levels of parathyroid hormone (PTH), total testosterone (TT), and sex hormone
binding globulin (SHBG) were analyzed by using the Immulite 2000 system (Siemens,
Munich, Germany). The reference range for total testosterone is 9.09–55.28 nmol/l
for males aged 20–49 years and 6.28–26.30 nmol/l for males aged ≥50 years. The SHBG
reference range for males is 13–71 nmol/l. The within-run and total coefficients of
variation for SHBG and testosterone are 2.5% and 5.2%, respectively. Serum albumin
was measured by using the Architect autoanalyzer (Abbott, Wiesbaden, Germany). Bioactive
testosterone (BAT; reference range: 2.14–13.60 nmol/l) and free testosterone (fT;
reference range: 0.090–0.580 nmol/l) were calculated according to Vermeulen et al.
[8].
Baseline characteristics stratified by treatment group (vitamin D vs. placebo) are
presented as means±SD for continuous variables. Intra-group comparisons (paired t-test) rather than inter-group comparisons were used at the end of the study because
no sex-stratified randomization at baseline was performed. Statistical analyses were
performed by SPSS 16.0 (SPSS Inc, Chicago, USA) and a p-value below 0.05 was considered
statistically significant.
Results
Results
Baseline characteristics of the 54 male patients who completed the trial are shown
in [Table 1]
. At study entry, mean 25(OH)D concentrations were in the deficiency range in both
groups. During follow-up, weight loss was 5.9±5.3 kg (p<0.001) in the vitamin D group
and 6.6±5.7 kg in the placebo group (p<0.001), and thus similar in both groups. Circulating
25(OH)D increased by 53.5±65.3 nmol/l to 86.4±68.8 nmol/l in the vitamin D group (p<0.001),
but increased only nonsignificantly in the placebo group (increase by 5.8±21.3 nmol/l
to 35.5±18.0 nmol/l; p=0.215). PTH decreased in the placebo and vitamin D group (decrease
by 0.94±3.09; p=0.035, and 0.60±1.67; p=0.040, respectively), whereas 1,25(OH)2D tended to increase in both groups (increase by 20.4±41.0; p=0.027 and 21.7±106.0;
p=0.100, respectively). At baseline, mean testosterone values were at the lower end
of the reference range in both groups. By comparing baseline testosterone values with
follow-up values in the placebo group no significant change in TT (11.8±4.0 nmol/l
vs. 12.7±5.45 nmol/l, p=0.355), BAT (6.39±2.22 nmol/l vs. 6.59±2.33 nmol/l, p=0.626)
or fT (0.264±0.087 nmol/l vs. 0.278±0.097 nmol/l, p=0.532) was found. In the vitamin
D group, however, a significant increase in all measures of testosterone status was
observed. TT increased from 10.7±3.9 nmol/l to 13.4±4.7 nmol/l (p<0.001), BAT from
5.21±1.87 nmol/l to 6.25±2.01 nmol/l (p=0.001) and fT from 0.222±0.080 nmol/l to 0.267±0.087 nmol/l
(p=0.001). In the placebo group, there were nonsignificant trends for seasonal differences
in 25(OH)D and testosterone values. Compared with men recruited in the summer half-year
(mid April to mid October; n=12), men recruited in the winter half-year (mid October
to mid April; n=11) had lower values of 25(OH)D (21.8±9.8 nmol/l vs. 37.4±30.0 nmol/l;
p=0.113), TT (11.5±4.33 nmol/l vs. 13.29±4.15 nmol/l; p=0.336), BAT (6.04±1.91 nmol/l
vs. 7.37±2.58 nmol/l; p=0.173), and fT (0.255±0.078 nmol/l vs. 0.301±0.104 nmol/l;
p=0.250).
Table 1 Characteristics of the study groups at baseline and at the end of the study
<TD VALIGN="TOP">
Parameter
</TD><TD VALIGN="TOP" COLSPAN="2">
Placebo group
</TD><TD VALIGN="TOP" COLSPAN="2">
Vitamin D group
</TD><TD VALIGN="TOP" COLSPAN="3">
p-Value
</TD>
<TD VALIGN="TOP">
</TD><TD VALIGN="TOP">
Baseline
</TD><TD VALIGN="TOP">
Study end
</TD><TD VALIGN="TOP">
Baseline
</TD><TD VALIGN="TOP">
Study end
</TD><TD VALIGN="TOP">
2 vs. 4
</TD><TD VALIGN="TOP">
2 vs. 3
</TD><TD VALIGN="TOP">
4 vs. 5
</TD>
<TD VALIGN="TOP">
Number
</TD><TD VALIGN="TOP">
23
</TD><TD VALIGN="TOP">
</TD><TD VALIGN="TOP">
31
</TD><TD VALIGN="TOP">
</TD><TD VALIGN="TOP">
–
</TD><TD VALIGN="TOP">
–
</TD><TD VALIGN="TOP">
–
</TD>
<TD VALIGN="TOP">
Age (years)
</TD><TD VALIGN="TOP">
46.8±12.0
</TD><TD VALIGN="TOP">
</TD><TD VALIGN="TOP">
49.4±10.2
</TD><TD VALIGN="TOP">
</TD><TD VALIGN="TOP">
0.387
</TD><TD VALIGN="TOP">
–
</TD><TD VALIGN="TOP">
–
</TD>
<TD VALIGN="TOP">
Smokers (%)
</TD><TD VALIGN="TOP">
56.5
</TD><TD VALIGN="TOP">
</TD><TD VALIGN="TOP">
38.7
</TD><TD VALIGN="TOP">
</TD><TD VALIGN="TOP">
0.271
</TD><TD VALIGN="TOP">
–
</TD><TD VALIGN="TOP">
–
</TD>
<TD VALIGN="TOP">
Alcohol (g/d)
</TD><TD VALIGN="TOP">
20.0±19.5
</TD><TD VALIGN="TOP">
14.1±15.3
</TD><TD VALIGN="TOP">
17.7±15.1
</TD><TD VALIGN="TOP">
15.3±13.8
</TD><TD VALIGN="TOP">
0.646
</TD><TD VALIGN="TOP">
0.138
</TD><TD VALIGN="TOP">
0.703
</TD>
<TD VALIGN="TOP">
25(OH)D (nmol/l)
</TD><TD VALIGN="TOP">
29.7±23.7
</TD><TD VALIGN="TOP">
35.5±8.1
</TD><TD VALIGN="TOP">
32.5±20.0
</TD><TD VALIGN="TOP">
86.4±68.8
</TD><TD VALIGN="TOP">
0.659
</TD><TD VALIGN="TOP">
0.215
</TD><TD VALIGN="TOP">
<0.001
</TD>
<TD VALIGN="TOP">
1,25(OH)2D (pmol/l)
</TD><TD VALIGN="TOP">
77.0±25.9
</TD><TD VALIGN="TOP">
97.4±32.9
</TD><TD VALIGN="TOP">
96.0±39.6
</TD><TD VALIGN="TOP">
127.7±94.3
</TD><TD VALIGN="TOP">
0.053
</TD><TD VALIGN="TOP">
0.027
</TD><TD VALIGN="TOP">
0.100
</TD>
<TD VALIGN="TOP">
PTH (pmol/l)
</TD><TD VALIGN="TOP">
5.07±3.63
</TD><TD VALIGN="TOP">
4.13±1.48
</TD><TD VALIGN="TOP">
4.14±1.98
</TD><TD VALIGN="TOP">
3.54±1.76
</TD><TD VALIGN="TOP">
0.237
</TD><TD VALIGN="TOP">
0.035
</TD><TD VALIGN="TOP">
0.040
</TD>
<TD VALIGN="TOP">
Body weight (kg)
</TD><TD VALIGN="TOP">
105.7±14.3
</TD><TD VALIGN="TOP">
99.0±13.5
</TD><TD VALIGN="TOP">
109.9±16.1
</TD><TD VALIGN="TOP">
104.0±17.2
</TD><TD VALIGN="TOP">
0.323
</TD><TD VALIGN="TOP">
<0.001
</TD><TD VALIGN="TOP">
<0.001
</TD>
<TD VALIGN="TOP">
BMI (kg/m2)
</TD><TD VALIGN="TOP">
32.5±3.8
</TD><TD VALIGN="TOP">
30.5±4.1
</TD><TD VALIGN="TOP">
33.1±3.9
</TD><TD VALIGN="TOP">
31.2±3.9
</TD><TD VALIGN="TOP">
0.609
</TD><TD VALIGN="TOP">
<0.001
</TD><TD VALIGN="TOP">
<0.001
</TD>
<TD VALIGN="TOP">
Albumin (mmol/l)
</TD><TD VALIGN="TOP">
386±182
</TD><TD VALIGN="TOP">
302±125
</TD><TD VALIGN="TOP">
377±194
</TD><TD VALIGN="TOP">
297±186
</TD><TD VALIGN="TOP">
0.087
</TD><TD VALIGN="TOP">
0.210
</TD><TD VALIGN="TOP">
0.896
</TD>
<TD VALIGN="TOP">
SHBG (mmol/l)
</TD><TD VALIGN="TOP">
26.3±13.7
</TD><TD VALIGN="TOP">
29.5±17.3
</TD><TD VALIGN="TOP">
31.0±10.3
</TD><TD VALIGN="TOP">
35.3±13.6
</TD><TD VALIGN="TOP">
0.153
</TD><TD VALIGN="TOP">
0.046
</TD><TD VALIGN="TOP">
0.002
</TD>
<TD VALIGN="TOP">
TT (nmol/l)
</TD><TD VALIGN="TOP">
11.8±4.0
</TD><TD VALIGN="TOP">
12.7±5.5
</TD><TD VALIGN="TOP">
10.7±3.9
</TD><TD VALIGN="TOP">
13.4±4.7
</TD><TD VALIGN="TOP">
0.317
</TD><TD VALIGN="TOP">
0.355
</TD><TD VALIGN="TOP">
<0.001
</TD>
<TD VALIGN="TOP">
BAT (nmol/l)
</TD><TD VALIGN="TOP">
6.39±2.22
</TD><TD VALIGN="TOP">
6.59±2.33
</TD><TD VALIGN="TOP">
5.21±1.87
</TD><TD VALIGN="TOP">
6.25±2.01
</TD><TD VALIGN="TOP">
0.040
</TD><TD VALIGN="TOP">
0.626
</TD><TD VALIGN="TOP">
0.001
</TD>
<TD VALIGN="TOP">
fT (nmol/l)
</TD><TD VALIGN="TOP">
0.264±0.087
</TD><TD VALIGN="TOP">
0.278±0.097
</TD><TD VALIGN="TOP">
0.222±0.080
</TD><TD VALIGN="TOP">
0.267±0.087
</TD><TD VALIGN="TOP">
0.067
</TD><TD VALIGN="TOP">
0.532
</TD><TD VALIGN="TOP">
0.001
</TD>
<TD VALIGN="TOP">
Data are shown as means±SD. Inter-group comparisons were performed by unpaired t-test and intra-group comparisons by paired t-test
</TD>
<TD VALIGN="TOP">
25(OH)D: 25-hydroxyvitamin D; 1,25(OH)2D: 1,25-dihydroxyvitamin D; PTH: parathyroid hormone; BMI: body mass index; SHBG:
sex-hormone binding
</TD>
In the 54 men, body mass index changes were inversely related to SHBG levels (r=–0.485;
p<0.001), but not to other indices of testosterone status.
Discussion
Discussion
In overweight men with deficient vitamin D status a significant increase in testosterone
was observed after intake of 83 μg vitamin D daily for 1 year whereas there was no
significant change in men receiving placebo. This work is, to the best of our knowledge,
the first study, which specifically addresses the effect of vitamin D supplementation
on androgens in men. The results of this study suggest that vitamin D supplementation
might increase testosterone levels in men. Our data support several experimental and
clinical findings: First, VDR knockout mice suffer from hypergonadotropic hypogonadism
[2]. Second, vitamin D status is directly associated with testosterone levels in men
[6]. Third, the male reproductive tract is a target tissue for vitamin D effects [3]. The nonsignificant trend for seasonal differences in both 25(OH)D and testosterone
in the placebo group supports our hypothesis of a vitamin D effect on testosterone.
In our study participants, mean baseline 25(OH)D values were in the deficiency range
and mean testosterone values were at the lower end of the reference range. Traditionally,
low solar ultraviolet B irradiation of the skin is a major cause of vitamin D deficiency
[1]. Both, vitamin D [1] and testosterone [5]
[9] show beneficial effects on the musculoskeletal system. From an evolutionary point
of view it would make sense that an active lifestyle (leading to an adequate skin
synthesis of vitamin D) also has beneficial effects on muscle function, bone health,
and the male reproductive system. We are aware that no final conclusions can be drawn
from our study regarding the effect of vitamin D supplementation on testosterone in
men but we do believe that our work is of great importance because it provides a reasonable
rationale for future studies. Besides the marked increase in 25(OH)D levels in the
vitamin D group, there was also a slight (nonsignificant) increase in 25(OH)D in the
placebo group during follow-up. We assume that the similar decrease in PTH and the
similar trend for an increase in 1,25(OH)2D in both study groups is due to a nonlinear association of these 2 calciotropic hormones
with increasing circulating 25(OH)D levels [10], with a pronounced effect at low and virtually no effect at high 25(OH)D levels.
Nevertheless, the similar changes in these hormones do not exclude group-specific
effects on the reproductive system, since nonclassical target tissues for vitamin
D largely depend on circulating 25(OH)D levels [1], which differed markedly between the vitamin D and placebo group.
Our study has both strengths and limitations. Strengths are the study design, the
use of a daily vitamin D dose that was effective to increase 25(OH)D values from the
deficiency range into the adequate range, and the fact that sample batching was performed
to avoid inter-assay variability. One limitation is the fact that the effect of vitamin
D supplementation on testosterone was not a prespecified study outcome and that we
did not assess testosterone-related functions such as libido, mood, or muscle strengths.
Another limitation is the relatively small number of male study participants. In addition,
future studies have to clarify whether the vitamin D actions are mediated by a pituitary
effect or a testicular one.
In conclusion, our study results suggest that vitamin D supplementation might increase
testosterone levels in men. Further randomized controlled trials are needed to confirm
this hypothesis and to evaluate whether vitamin D driven increases in testosterone
levels contribute to the vitamin D effects on various health outcomes.
Acknowledgements
Acknowledgements
We would like to thank Mrs. Marlen Ewald for excellent technical assistance.
