Bibliografisk note

Funding Information:
This work was funded by Rigshospitalet, the Novo‐Nordisk Foundation, the Aase & Ejnar Danielsens Foundation, FSS and Candys Foundation.

Funding Information:
This work was funded by Rigshospitalet, the Novo-Nordisk Foundation, the Aase & Ejnar Danielsens Foundation, FSS and Candys Foundation. This investigation is based on predefined secondary endpoints and subgroups from the Copenhagen Bone-Gonadal Study, a single-center, double-blinded, randomized clinical trial conducted at the Department of Growth and Reproduction, Rigshospitalet, Denmark (NCT01304927). The trial was approved by the Danish Medicines Agency (approval no. 2010-024588-42), the Danish National Committee on Health Research Ethics (approval no. H-4-2010-138), and the Danish Data Protection Agency (approval no. 2010124801) and monitored by the Good Clinical Practice (GCP) Unit, Copenhagen University Hospitals. Informed consent was obtained from all the participants. The primary endpoint was difference in semen quality that in concert with differences in serum FSH and inhibin B and metabolic profile have been published.20,21 The study design and sample size calculation have been described previously.20 In brief, a total of 1427 infertile men were referred to our andrological center. Inclusion criteria were male infertility and impaired semen quality defined as sperm concentration <20 million/mL (with a minimum of ≥0.01 million/mL), or <50% progressive motile spermatozoa, or <12% morphological normal spermatozoa using strict criteria and serum 25OHD ≤ 50 nmol/L (vitamin D insufficiency) at screening and no serious comorbidities, such as endocrine diseases, previous cancer, or chromosome abnormalities. A total of 307 idiopathic infertile men were included in the trial. They were all vitamin D insufficient at baseline but only 179 out of these were still vitamin D insufficient on day 1 when the intervention started as most men were included in winter and spring and had increased UVB radiation from the sun. Included men were randomized 1:1 to vitamin D or placebo treatment for 150 days. Men in the treatment group (n = 151) initially received an oral bolus of 300,000 IU cholecalciferol, followed by daily supplementation with 1400 IU cholecalciferol and 500 mg calcium (tablets; Ferrosan/Pfizer). Men in the placebo group (n = 156) received an oral bolus of oil and non-calcium containing placebo tablets for 150 days. There were no cases of serious adverse events, hypercalcemia, or kidney stones. This investigation is based on predefined secondary endpoints and subgroups from the Copenhagen Bone-Gonadal Study, a single-center, double-blinded, randomized clinical trial conducted at the Department of Growth and Reproduction, Rigshospitalet, Denmark (NCT01304927). The trial was approved by the Danish Medicines Agency (approval no. 2010-024588-42), the Danish National Committee on Health Research Ethics (approval no. H-4-2010-138), and the Danish Data Protection Agency (approval no. 2010124801) and monitored by the Good Clinical Practice (GCP) Unit, Copenhagen University Hospitals. Informed consent was obtained from all the participants. The primary endpoint was difference in semen quality that in concert with differences in serum FSH and inhibin B and metabolic profile have been published.20,21 The study design and sample size calculation have been described previously.20 In brief, a total of 1427 infertile men were referred to our andrological center. Inclusion criteria were male infertility and impaired semen quality defined as sperm concentration <20 million/mL (with a minimum of ≥0.01 million/mL), or <50% progressive motile spermatozoa, or <12% morphological normal spermatozoa using strict criteria and serum 25OHD ≤ 50 nmol/L (vitamin D insufficiency) at screening and no serious comorbidities, such as endocrine diseases, previous cancer, or chromosome abnormalities. A total of 307 idiopathic infertile men were included in the trial. They were all vitamin D insufficient at baseline but only 179 out of these were still vitamin D insufficient on day 1 when the intervention started as most men were included in winter and spring and had increased UVB radiation from the sun. Included men were randomized 1:1 to vitamin D or placebo treatment for 150 days. Men in the treatment group (n = 151) initially received an oral bolus of 300,000 IU cholecalciferol, followed by daily supplementation with 1400 IU cholecalciferol and 500 mg calcium (tablets; Ferrosan/Pfizer). Men in the placebo group (n = 156) received an oral bolus of oil and non-calcium containing placebo tablets for 150 days. There were no cases of serious adverse events, hypercalcemia, or kidney stones. Blood sampling was performed between 8.00 and 10.00 a.m. Measurements of 25OHD were conducted by isotope dilution liquid chromatography tandem mass spectrometry, with an inter-assay coefficient of variation (CV) < 10%. Parathyroid hormone (PTH) levels were measured using the Cobas 8000 (Roche) with a CV < 4%. Testosterone and estradiol levels were measured by RIA (Coat-a-Count; Siemens and Pantex, respectively) with a detection limit of 0.23 nmol/L for testosterone and 18 pmol/L for estradiol, and CV < 13%. Serum testosterone, estradiol, and SHBG levels were used to calculate free testosterone and free estradiol using the method of Vermeulen22 and Mazer23 with a fixed albumin concentration of 43.8 g/L. Measurements of serum LH and SHBG levels were obtained using a time-resolved immuno-fluorometric assay (Delfia, Wallac) with CV < 4% and <6%, respectively. Makers of Sertoli cell function (serum FSH, inhibin B, and AMH) were also measured and have previously been published.20,24 Descriptive statistics are presented as mean with SD in Tables 1 and 2 and mean with 95% CI in Tables 3 and 4. Differences on day 1 according to the predefined subgroups were performed by a Kruskal–Wallis test (Table 2). The primary analysis was performed according to randomization, using an unadjusted comparison of means (t-test), followed by a comparison adjusted for age, percentage of total fat mass, smoking, and the corresponding serum concentration at day 1 (Table 3). Further analyses were performed according to predefined subgroups defined as vitamin D deficiency with serum 25OHD < 25 nmol/L, vitamin D insufficiency with serum 25OHD ≤ 50 nmol/L, and vitamin D sufficiency with serum 25OHD > 50 nmol/L, on day 1, respectively. Differences in outcome after intervention according to the predefined subgroups were performed by an adjusted comparison of means (t-test), adjusted for age, percentage of total fat mass, smoking, and the corresponding serum concentration on day 1 (Table 4). No observations were excluded. All statistical analyses were performed by using IBM SPSS Statistics 28. Figures were performed using GraphPad Prism. Data presented as mean (SD) unless otherwise indicated. Season of inclusion: Included at winter or spring. Hypogonadism is defined as serum testosterone <10.4 nmol/L. Serum 25OHD at day 1 <25 nmol/L Serum 25OHD at day 1 25–50 nmol/L Serum 25OHD at day 1 >50 nmol/L Data presented as mean (SD) unless otherwise indicated. Kruskal–Wallis test were used to comparison of differences between groups, unless for differences in Season of inclusion where Chi-squared test were used. Season of inclusion: Included at winter or spring. Hypogonadism is defined as serum testosterone <10.4 nmol/L. Vitamin D Placebo p Data presented as mean with 95% confidence interval. Adjusted for age, percentage of total fat mass, smoking, and the corresponding serum concentration at day 1. Hypogonadism is defined as serum testosterone <10.4 nmol/L. Vit D (n = 15) Mean (CI) Placebo (n = 21) Mean (CI) Vit D (n = 69) Mean (CI) Placebo (n = 70) Mean (CI) Vit D (n = 49) Mean (CI) Placebo (n = 54) Mean (CI) Subgroup analyses of Leydig cell function after intervention, according to vitamin D status at day 1. Data presented as mean ± 95% CI. All analyses were adjusted for age, percentage of total fat mass, smoking, and the corresponding serum concentration on day 1.

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© 2023 The Authors. Andrology published by Wiley Periodicals LLC on behalf of American Society of Andrology and European Academy of Andrology.