Vitamin D Supplementation and Male Infertility: The CBG-study a Randomized Clinical Trial
Vitamin D Supplementation and Male Infertility: The CBG-study a Randomized Clinical Trial
Today, it is evident that vitamin D (VD) has more widespread effects than the classical
 actions related to bone mineralization and calcium homeostasis1. VD deficiency results in
 impaired reproductive performance in various species of animals, and recently the
 investigators have shown that the VD receptor (VDR), activating (CYP2R1, CYP27A1, CYP27B1)
 and inactivating (CYP24A1) enzymes are expressed in the human testis, epididymis, seminal
 vesicle, prostate and spermatozoa. Our following functional studies showed that VD increases
 intracellular calcium in mature spermatozoa, and hence may be important not only for
 spermatogenesis but also for sperm maturation. A new, and yet unpublished cross sectional
 study of 300 young healthy Danish men showed that men with lower levels of serum VD have
 significantly lower number of normally developed and motile spermatozoa. Hitherto, most cases
 of male infertility have been classified as "idiopathic", and infertile couples have been
 referred to symptomatic treatment at infertility clinics. These fertility treatments are
 often physically demanding for the female partner as well as expensive for the health care
 system. Any treatment that might improve semen quality of involuntary infertile men would be
 beneficial both for the infertile couples and the society in general. Our findings that VD
 may play a role for human semen quality have not yet been tested clinically. However, if VD
 supplementation proves efficient this opens for the first time for a causal, safe and cheap
 treatment of at least some cases of "idiopathic" impaired semen quality. The investigators
 believe our new human data supported by the results from the VD deficient and VDR KO animal
 studies and the high proportion of VD deficient Danish men provide sufficient evidence to
 initiate a randomized clinical trial of VD supplementation to infertile men. Infertile men
 have also have unfavorable altered levels of sex hormones and higher mortality than fertile
 men. Since VD deficiency is associated with increased mortality, regulation of aromatase,
 immune system, bone metabolism, glucose metabolism, cardiovascular system etc. our suggested
 clinical trial may also be able to evaluate several secondary endpoints in addition to the
 potential effect on semen quality.
Background In 2008, approximately 8% of all newborn children were conceived by assisted
 reproduction. Intracytoplasmatic sperm injection (ICSI) accounts for ~50% of all treatments
 and is mainly used in cases of severely reduced semen quality. This is in many cases most
 likely caused by prenatal factors adversely affecting the developing testicles, and it is
 therefore unlikely that any treatment of adult men will be able to completely normalize their
 semen quality. However, less would also be clinically relevant. If reduced semen quality
 could be improved prior to any assisted reproduction a less invasive treatment would be
 needed. Eg. classical in vitro fertilization (IVF) rather than ICSI, simple intrauterine
 insemination (IUI) rather than IVF or natural conception rather than IUI.

 Several endocrine factors have been implicated in sperm production and maturation, but little
 is known about the potential role of VD. VD is a key regulator of calcium homeostasis and
 bone mineralization, although expression of the vitamin D receptor (VDR) in various tissues
 has been related to several diverse actions. VD affects reproduction in several animal
 species, convincingly shown in rodents, where VD deficiency in male rats resulted in reduced
 sperm counts, and female rats inseminated with semen from VD deficient male rats had lower
 fertility rates. The impaired reproductive performance is reversible and can be corrected
 either by supplying VD or by normalizing calcium levels. Supported by VDR knockout mice,
 which showed decreased sperm counts, reduced sperm motility and histological abnormalities of
 the testis, which unlike the VD deficient male rats only partly can be restored by calcium
 supplements.

 The investigators have recently shown expression of VDR and the VD metabolizing enzymes in
 the human testis, ejaculatory tract and in mature spermatozoa. The investigators subsequently
 showed that VD in physiological concentrations increased intracellular calcium in
 spermatozoa. VD acts through a rapid non-genomic response and the VD induced increase in
 calcium may be crucial for the spermatozoa, because VD induced sperm motility and the
 acrosome reaction. Expression analysis of spermatozoa from fertile and infertile men showed
 that men with impaired semen quality have fewer that can metabolize VD than normal men(p <
 0.0005) making presence of one of the proteins a potential marker of semen quality.
 Furthermore, the investigators have just completed a cross sectional study of 300 young men
 from the general population and found that low serum VD was associated with reduced sperm
 motility and morphology.

 SETTING, SCIENTIFIC PLAN AND RECRUITMENT Participants will be included among men referred to
 the Department of Growth and Reproduction (dept. of GR), Rigshospitalet (RH) for evaluation
 of male infertility

 DESIGN This is a prospective, double blinded, two-arm randomized controlled trial

 Group of intervention: Each man will receive 300,000 IU (7500 ug) cholecalciferol (D3) orally
 once after blood- and semen sampling and performed DXA scan. Thereafter they will receive VD
 tablets of 1,400 IU (35 ug) + 500 mg calcium daily for 3 months. Telephone control after 4
 weeks and at 3 months a clinical control and blood sampling will be performed, followed by
 continued daily intake of 1400 IU VD + 500 mg calcium for 2 additional months. At end of
 treatment at five months after inclusion the men deliver two semen samples, have a blood
 sample drawn and a DXA scan performed.

 Group receiving placebo: Will follow the same chain of events, although VD will be replaced
 with placebo.

 PARTICIPANTS Screening of ~700-800 infertile men will be performed. The investigators assume
 that ~340 men will be excluded and ~100 men do not wish to participate. Thus, 300 men will be
 included in the study and half will be randomized to active VD treatment. The investigators
 expect a small drop out (< 20) because of high motivation and no adverse effects.

 ANALYSIS AND INTERVENTION Reproductive hormones and growth factors will be analyzed at dept.
 of GR, Rigshospitalet. An aliquot of the first blood sample will be analyzed before inclusion
 to the study and another kept to be analyzed at the same time as the second blood sample at
 the end of the trial to overcome the interassay variation. Other serum analyses will be
 analyzed at department of clinical biochemistry, Rigshospitalet. Semen analyses and DXA scans
 will be performed at dept. of GR.

 SAMPLE SIZE CALCULATION AND STATISTICS In our association study with 300 participants the
 investigators are able to detect significant dose response relationship between VD status and
 sperm motility and morphology, and found significant differences between men with low and
 high VD levels. Based on these results, the investigators assume to be able to increase serum
 levels of VD with 50 nM with our setup. Including 150 men in each group will enable us to
 detect changes in sperm motility and morphology of 15% and a 25% change in Inhibin B levels.
 Intention to treat principles will be used and co-variate analyzes will be used to analyze
 the potential effect of VD.

 BIOSTATISTICAL ANALYSIS All the analyses will be performed according to Good Clinical
 Practice guidelines and the primary analyses in the intention-to-treat population, which
 included all patients who underwent randomization and received the first dose of medicine on
 day 1. We will analyze the data in 3 ways. The primary analysis will proceed according to the
 randomized vitamin D and calcium dose group assignment. The secondary analysis will be based
 on stratifying the men according to subgroup analyses in relation to the predefined primary
 and secondary endpoints, while the last analysis will try to elucidate any dose-response
 relationship. An author (JHP) who was the statistician for the study and unaware of the
 study-group assignments will perform the primary analysis.

 Data analysis and quality The primary end points for this protocol will be changes in semen
 quality especially sperm motility followed by sperm concentration, morphology and semen
 volume. Multiple secondary endpoints exist but for the initial investigation focus will be on
 changes of the following secondary endpoints: Sperm DFI, pregnancy rate, Inhibin B, vitamin D
 and calcium homeostasis. Subjects who terminate participation after visit at 90 days but
 before visit day 150 will be included for data analysis up to day 90. Men that only deliver
 one semen sample or have missing data at any visit will still be included in the analysis.
 Men with fever up to 3 months prior to semen analysis will be considered as a potential
 confounder. Men that do not meet the criteria in the protocol will be excluded from the
 analysis. Those values will then be carried forward for analyses. A significance level of 5%
 is used. For the primary analyses Bonferronu-Holm p-value correction is calculated
 additionally. For the secondary analysis no multiple test correction are used. Instead
 results are discussed in view of the multiple testing situations.

 1. Analyses between placebo versus active substance Between group analyses placebo versus
 vitamin D + calcium: The first step will be to compare the changes in primary outcomes
 across the two groups placebo versus vitamin D + calcium. This analysis will show if
 there is a significant difference between groups. For outcomes measured repeatedly, this
 will entail comparing the estimated slopes, or rates of change, of each outcome between
 the groups. Mixed models allow for the correlation between the repeated observations
 baseline-day 1-day 90- day 150 from each man to be suitably incorporated into parameter
 estimation. For all endpoints measured at baseline and day 150, paired t-tests will be
 used to assess there is a significant difference between the groups and determine
 whether the mean change within each group differs significantly from zero. In both
 cases, data will be transformed as necessary to meet model assumptions. Afterwards, the
 same analysis will be conducted by using multiple regression with relevant confounders
 such as season, BMI, smoking, duration of abstinence, time from ejaculation to motility
 assessment, fever etc. to see if this changes the results For outcomes measured that
 cannot be compared with t-test or other parametric tests at day 1, day 90 and day 150,
 groups will be compared using non-parametric tests such as Wilcoxon Mann-Whitney test.
 For Binary outcome the data will be compared between the two groups by means of
 conditional logistic-regression analysis with adjustment for relevant confounders
 (defined as being significantly p<0.05 associated).

 .

 2. Analyses after stratification into subgroups Subjects will be grouped according to their
 BMI, calcium, season, semen quality, bonefactors, BMD or vitamin D levels and the effect
 of placebo versus active substance will be evaluated at day 90 and day 150. The subgroup
 analyses will in accordance with normal clinical practice and stratification in
 appropriate groups according to the clinical (25OHD < 25 nmol/l, BMI <25, 25-30, >30
 etc.), tertiles/quartiles/quintiles or highest/lowest versus remaining at baseline.

 3. Data analysis for comparison of changes in vitamin D and calcium We will also compare
 the dose-response relationships between changes in vitamin D and calcium and primary and
 secondary endpoints between placebo and active substance group and in all men. We will
 determine delta values for circulating vitamin D progenitors, calcium ion, total calcium
 and albumin corrected calcium from start to 90 days and 150 days and compare the
 difference with the observed changes in primary and secondary endpoints. We anticipate
 that there is no difference in shapes of the dose-response curve between the different
 endpoints i.e. they will be parallel and linear at least after transformation of the
 data. Afterwards, the same analysis will be conducted by using relevant confounders such
 as season, BMI, smoking, duration of abstinence, time from ejaculation to motility
 assessment etc. to see if this changes the results.

 SCREENING AND TIME COURSE Men, who are investigated at dept. of GR due to infertility will be
 screened for eligibility to the study, and those who meet the criteria for participation will
 be informed, and if they consent allocated to active treatment with VD or placebo. Allocation
 will be done by minimization using minim to avoid unbalanced grouping due to randomization
 failure. Following variables will be balanced: Sperm concentration, BMI, serum inhibin-B, and
 VD level.

 ETHICS AND SIDE EFFECTS All the patients will have fulfilled their investigation, before they
 are invited to the study. They will be informed of potential adverse effects, signs of
 intoxication and they can leave the trial at any point without any consequences. The trial
 will run in accordance with "good clinical practice". VD treatment gives virtually no side
 effects and the risk of intoxication is almost nonexisting in the suggested setting, with
 relatively low doses of VD and close monitoring of VD and calcium status. The participants
 will be exposed to oral VC/placebo, to 2 DXA scans, have 3 extra blood samples drawn and
 deliver 2 extra semen samples. All participants will be informed and counseling according to
 their VD status.

 PUBLICATION OF RESULTS All results, positive or negative will be submitted to peer reviewed
 scientific journals. Data will successively be obtained and transferred to a statistical
 database.

 PRACTICAL ISSUES Dept. of GR investigates annually more than 500 infertile men. Participants
 will be included from that group. The department have the clinical expertise, experience and
 capacity to perform all the investigations, except for the measurements of some of the blood
 samples, which will be analyzed by department of clinical biochemistry. VD and placebo oral
 suspensions are purchased from Glostrup apotek, while VD and placebo tablets are purchased
 from Ferrosan, MD Martin. The investigators are collaborating with associate professor in
 biostatistics J.H. Petersen, University of Copenhagen.
Inclusion Criteria:

 -  Male with an age > 18 years old

 -  Referred for male infertility with sperm concentration >= 0.01 million/ml.
 Additionally, all men must have either sperm concentration < 20 million/ml or < 50%
 progressive motile spermatozoa or < 12% morphological normal spermatozoa using strict
 criteria

 Exclusion Criteria:

 -  Men with chronic diseases (such as diabetes mellitus, Thyroid disease, endocrine
 disturbances in need of treatment, malignant disease, or diseases known to interfere
 with VD intake or very sensitive to VD intake (such as inflammatory disease with
 granuloma: sarcoidoses, tuberculosis, Wegeners, vasculitis, inflammatory bowel disease
 (Crohn's and colitis ulcerosa etc).

 -  Men with present or previous malignant disease

 -  If there is an indication for testis biopsy and it is planned or conducted within the
 next 6 months

 -  Serum 25-hydroxy-D3 > 50 nmol/l at the time of inclusion

 -  Serum Calcium ion > 1,35 mmol/l

 -  Inhibin-B < 30 pg/ml

 -  Intake of vitamin D above 15 ug daily

 -  Allergy towards vitamin D or arachidis oil (peanuts)

 -  Men with total or partly obstructive oligospermia and men who had vasectomy performed

 Criteria for drop out:

 -  Abrogation of the treatment

 -  Newly diagnosed endocrine, calcium metabolic disease, parathyroid, thyroid, diabetes
 or other endocrine disease in need of treatment

 -  New malignant disease

 -  Treatment with chemotherapy, immunomodulating therapy, salazopyrin

 -  Oral or iv treatment with steroid hormones

 -  Treatment with diuretics, antihypertensive treatment, treatment the heart, calcium
 channel blockers

 -  Development of vitamin d intoxication

 -  If testis biopsy is performed or other surgery in the genital region during the trial