The influence of body composition on the response to dynamic stimulation of the endocrine pituitary-testis axis.

BACKGROUND: Testosterone treatment is generally not recommended in men with obesity induced low serum testosterone. However, distinguishing this condition from overt testosterone deficiency in men with obesity where treatment should be initiated is a diagnostic challenge and tools to differentiate these conditions are scarce but could be of important clinical relevance. OBJECTIVES: To investigate the association between body composition and dynamic responses of the pituitary-testis axis in men. METHODS: Single-center cross-sectional study including 112 healthy men. Participants went through a full biochemical assessment of the pituitary-testis axis, and dynamic stimulatory tests of luteinizing hormone (LH) secretion (gonadotropin-releasing hormone (GnRH)-test) and testosterone secretion (choriogonadotropin (hCG)-test). A subset (N = 78) further had a DXA-scan performed. RESULTS: A higher body mass index (BMI) was associated with lower basal serum LH (BU = -0.44, 95% CI: -0.88--0.01, p = 0.04). The GnRH-stimulated LH increase was not significantly associated with BMI (BU = -0.10, 95% CI: -0.72-0.51, p = 0.74). Furthermore, a high BMI was associated with low basal testosterone (BU -0.02, 95% CI: -0.03--0.02, p < 0.001), and free testosterone (BU -15.0, 95% CI: -19.9--10.0, p < 0.001) and men with overweight and obesity had significantly lower testosterone (9%, p = 0.003 and 24%, p < 0.001) and free testosterone (25%, p = 0.006 and 50%, p < 0.001) concentrations compared to men with normal weight. The HCG-stimulated testosterone increase was significantly less dependent on BMI compared to the influence of BMI on basal testosterone concentrations (p = 0.04 for the interaction). CONCLUSIONS: Dynamic sex hormone responses following pituitary-testis axis stimulation were less dependent on BMI, compared to the influence of BMI on basal hormone concentrations and could potentially assist clinical decision making in patients with obesity suspected of testosterone deficiency.

Hair cortisol, glucocorticoid gene receptor polymorphisms, stress, and testicular function.

OBJECTIVE: Self-reported psychological stress has been associated with decreased semen quality. Cortisol levels in scalp hair (hair cortisol concentration, HCC) has emerged as a potential objective marker of psychological stress. Thus, we investigated if HCC was associated with markers of testicular function. Furthermore, we examined whether three common single nucleotide polymorphisms in the glucocorticoid-receptor gene (NR3C1, chromosome 5), potentially affecting receptor sensitivity, were associated with HCC and could influence the studied association between HCC and testicular function. DESIGN: Cross-sectional study. METHODS: We analysed HCC, serum-levels of reproductive hormones, semen parameters, and the three NR3C1-polymorphisms; BclI (rs41423247), Tth111I (rs10052957), and 9ß (rs6198), in a population of 696 men from the general population. RESULTS: HCC was not associated with testicular function, and adjustment for the three NR3C1-polymorphisms did not alter the results. However, HCC increased significantly with the number of Tth111I minor-alleles (T) and decreased significantly with the number of 9ß minor-alleles (G). CONCLUSION: Given previously shown associations between stress and semen quality, and that no association between HCC and self-reported stress was observed in the current study, we speculate that negative reproductive effects of stress may not be mediated directly by cortisol. This study demonstrates associations between HCC and glucocorticoid receptor gene variants indicating that these SNPs may influence systemic glucocorticoid levels, but the potential health effects of such alterations are yet unknown.

Long-term testosterone undecanoate treatment in the elderly testosterone deficient male: An observational cohort study.

BACKGROUND: Quarterly intramuscular injections with long-acting testosterone undecanoate (TU) provide stable serum testosterone concentrations over time and are therefore preferred by many testosterone-deficient patients. However, the use of long-acting TU in elderly patients is limited due to lack of safety and feasibility studies. OBJECTIVE: To investigate long-acting TU pharmacokinetics and assess differences in treatment regimens and risk of adverse outcomes in younger versus elderly testosterone-deficient patients. MATERIALS AND METHODS: Single-center longitudinal observational study. Patients who initiated long-acting TU treatment between 2005 and 2010 were included. Elderly patients were born before 1956 and younger patients between 1965 and 1985. TU dose was adjusted yearly through shortening or prolongation of time between injections. Treatment targets were as follows: (1) free testosterone between 0 and -1 SD from the age-adjusted mean, (2) no symptoms of testosterone deficiency, and (3) hematocrit within the normal range. RESULTS: The study population consisted of 63 elderly and 63 younger patients. Median follow-up time during testosterone replacement was 12.1 years. Increasing intervals between TU injections were performed 44% more often in the elderly compared to younger patients and time between TU injections were prolonged 4% more in the elderly patients. The hematocrit, as well as the hematocrit for a given serum testosterone (hematocrit: testosterone ratio), increased with treatment time but did not differ between age groups. During follow-up, 40% of patients-both elderly and younger-experienced polycythemia. Risk of polycythemia did not differ with age. DISCUSSION AND CONCLUSION: An increased number of adjustments of TU dose are necessary in elderly patients in order to reach treatment targets. TU treatment in elderly testosterone-deficient patients is not associated with an increased risk of polycythemia compared to younger patients if age-adjusted treatment targets are reached.

Semen quality and waiting time to pregnancy explored using association mining.

BACKGROUND: Assessment of semen quality is a key pillar in the evaluation of men from infertile couples. Usually, semen parameters are interpreted individually because the interactions between parameters are difficult to account for. OBJECTIVES: To determine how combinations of classical semen parameters and female partner age were associated with waiting time to pregnancy (TTP). MATERIALS AND METHODS: Semen results of 500 fertile men, information of TTP, and partner age were used for regressions and to detect breaking points. For a modified Association Rule Mining algorithm, semen parameters were categorized as High, Medium, and Low. RESULTS: Men ≤32.1 years and women ≤32.9 years had shorter TTP than older. Decreasing TTP was associated with increasing level of individual semen parameters up to threshold values: sperm concentration 46 mill/mL, total sperm count 179 mill, progressive motility 63%, and normal morphology 11.5%. Using association mining, approximately 100 combinations of semen parameters and partner age were associated with TTP. TTP ≤ 1 month often co-occurred with high percentages of progressive motility (≥62%) and morphologically normal spermatozoa (≥10.5%). Furthermore, TTP ≤ 1 did not tend to appear with lower percentages of these two semen parameters or high partner age (≥32 years). However, high percentages of motile or normal spermatozoa could not compensate for sperm concentration ≤42 mill/mL or total sperm count ≤158 mill. The prolonging effect of high partner age could not be compensated for by the man's semen quality. DISCUSSION AND CONCLUSION: Using association mining, we observed that TTP was best predicted when combinations of semen parameters were accounted for. Sperm counts, motility, and morphology were all important, and no single semen parameter was inferior. Additionally, female age above 32 years had a negative impact on TTP that could not be compensated for by high semen parameters of the man.

FSHB and FSHR gene variants exert mild modulatory effect on reproductive hormone levels and testis size but not on semen quality: A study of 2020 men from the general Danish population.

BACKGROUND: Spermatogenesis depends on stimulation by follicle-stimulating hormone (FSH) which binds to FSH receptors (FSHR) on testicular Sertoli cells. Three FSH-related single-nucleotide polymorphisms (SNPs), FSHB -211G>T (rs10835638), FSHR -29G>A (rs1394205) and FSHR 2039A>G (rs6166) affect FSH action, and have been suggested to affect testicular function, but the evidence is uncertain. OBJECTIVE: To describe the associations between the three SNPs and testicular function in a large and well-characterised cohort of men from the general population. MATERIALS AND METHODS: A cross-sectional study of 2020 Danish men unselected regarding testicular function. Outcome variables were semen parameters, reproductive hormones and testis size. Genotyping was done by competitive allele-specific quantitative PCR. Differences in genotype frequencies were tested by chi-square test and associations between genotypes and outcomes were assessed by multivariate linear regressions. RESULTS: The SNPs affected serum FSH; carriers of the variant affecting FSH secretion (FSHB -211G>T) had lower FSH levels while carriers of variants affecting receptor expression (FSHR -29G>A) and receptor sensitivity (FSHR 2039A>G) had higher FSH levels. Carriers of FSHB -211G>T had lower calculated free testosterone/LH ratio. Although both FSHB -211G>T and FSHR 2039A>G were associated with smaller testis size, no clear association was detected in relation to any semen parameters, except a lower total number of morphologically normal spermatozoa in the heterozygous carriers of the FSHB -211G>T DISCUSSION AND CONCLUSION: The studied polymorphisms have only minor modulating influence on testis size and function in healthy men. We detected subtle effects of the three SNPs on FSH levels, but also effects of FSHB -211G>T on calculated free testosterone/LH ratio, compatible with altered Leydig cell function. Thus, the role of these FSH-related polymorphisms is complex and modest in men with normal testicular function, but the possible importance of FSH polymorphisms in men with impaired testicular function should be evaluated in future studies in more detail.

Testicular microlithiasis on scrotal ultrasound in 4850 young men from the general population: associations with semen quality.

BACKGROUND: Testicular microlithiasis (TM) is sometimes found on scrotal ultrasound. The prevalence seems higher in populations of men with testicular dysfunction, and TM may be a risk factor for testicular germ cell neoplasia in situ in men with additional risk factors. The association between TM and testicular function is controversial, especially in incidentally found TM. OBJECTIVES: To determine the frequency of TM in young men from the general population, and associations between TM, semen quality, and reproductive hormones. MATERIALS AND METHODS: A cross-sectional study of 4850 Danish men, median age 19 years. Testicular pattern, including the presence of TM, was assessed by ultrasound examination. Participants provided a questionnaire, one semen sample, and one blood sample. Semen variables and serum reproductive hormones were analyzed as outcomes using multivariable regression analysis to determine associations with TM. RESULTS: TM was detected in 53 men (1%), of which 19 (36%) were unilateral and 34 (64%) were bilateral cases. A history of cryptorchidism was associated with presence of TM. Bilateral TM was associated with slightly lower testicular volume, sperm concentration, and total sperm count. TM was not significantly associated with serum testosterone or other reproductive hormones. DISCUSSION AND CONCLUSION: TM is rare in men from the general population and is associated with lower sperm count if bilateral, although effect sizes were small. Current European guidelines do not recommend any follow-up in cases of TM with no other risk factors for testicular cancer. We suggest that men with incidentally found bilateral TM may be offered a semen analysis, but analysis of reproductive hormones seems unnecessary.

Psychological stress, stressful life events, male factor infertility, and testicular function: a cross-sectional study.

OBJECTIVE: To assess the association between psychological stress and male factor infertility as well as testicular function (semen quality, serum reproductive hormones) and erectile dysfunction. DESIGN: Cross-sectional study. SETTING: University Hospital-based research center. PATIENTS: Men with impaired semen quality were included from infertile couples, and men with naturally conceived pregnant partners were used as a reference population. INTERVENTIONS: Participants completed a questionnaire on health and lifestyle, including a 14-item questionnaire about self-rated psychological stress symptoms and stressful life event (SLEs), had a physical examination performed, delivered a semen sample and had a blood sample drawn. MAIN OUTCOMES: Differences in stress scores (calculated from self-reported stress symptoms) and SLEs between infertile and fertile men were assessed in crude and fully adjusted linear regression models. Secondary outcomes were semen quality, serum reproductive hormones, and erectile dysfunction. RESULTS: Of 423 men, 176 (41.6%) experienced at least one SLE in the 3 months prior to inclusion (50.4%/36.9%: infertile/fertile men, P = .03); ß-coefficient and 95% confidence interval for the difference between the groups on the transformed scale in fully adjusted linear regression models was 0.18 (0.06, 0.30). However, there were no differences in psychological stress symptoms between the two groups (ß-coefficient and 95% confidence interval) on the transformed scale (0.14; -0.02, 0.30). No association between stress (self-reported stress symptoms and SLEs) and testicular function or with erectile dysfunction was found in any of the men. CONCLUSION: Infertile men reported a higher number of SLEs than fertile men but did not report more psychological stress symptoms. Distress and SLEs were not associated with reduced male reproductive function.

Evaluation of Serum Insulin-like Factor 3 Quantification by LC-MS/MS as a Biomarker of Leydig Cell Function.

BACKGROUND: The peptide hormone insulin-like factor 3 (INSL3) is a marker for Leydig cell function and the clinical use of serum INSL3 measurements has been suggested by several groups. AIM: (1) To establish a reference range for liquid chromatography-tandem mass spectrometry (LC-MS/MS) of serum INSL3 in healthy boys and men; and (2) to compare the associations of serum INSL3 and testosterone (T) to pubertal stage, lifestyle factors, diurnal variation, body composition, and human chorionic gonadotropin (hCG) stimulation. RESULTS: In a reference range based on LC-MS/MS analysis of serum from 1073 boys and men, INSL3 increased from levels close to the detection limit (0.03 µg/L) in prepubertal boys to a maximum mean level of 1.3 µg/L (95% CI, 0.9-2.7) in young men (19-40 years of age) and decreased slightly in older men (0.1 µg/L per decade). Serum T, but not INSL3, was associated with body mass index or body fat percentage and with alcohol consumption. Smoking was positively associated with serum T, but negatively associated with INSL3. There were significant diurnal variations in both INSL3 and T in men (P < 0.001), but serum INSL3 varied substantially less, compared with serum T (± 11% vs ± 26%). Mean serum INSL3 increased after hCG stimulation, but less than T (+ 17% vs + 53%). In both healthy men and in patients suspected of testicular failure, baseline serum INSL3 was more closely associated to the hCG-induced increase in serum T than baseline T itself. CONCLUSION: Measurement of serum INSL3 by LC-MS/MS has promise as a marker of testicular disorders.

Development and validation of a mass spectrometry-based assay for quantification of insulin-like factor 3 in human serum.

BACKGROUND: The circulating level of the peptide hormone insulin-like factor 3 (INSL3) is a promising diagnostic marker reflecting Leydig cell function in the male. Few commercial immunoassays of varying quality exist. Therefore, we decided to develop and validate a precise method for quantification of INSL3 by mass spectrometry. METHODS: We developed an assay in which the INSL3 A-chain is released from the INSL3 A-B heterodimer by chemical reduction and alkylation. The alkylated INSL3 A-chain is quantitated by liquid chromatography-tandem mass spectrometry (LC-MS/MS), as substitute for serum INSL3. The method was compared to a validated and sensitive in-house serum INSL3 immunoassay using 97 serum samples from 12 healthy boys during pubertal transition. Adult levels were determined based on sera from 72 adult healthy males aged 18-40 years. RESULTS: An LC-MS/MS assay with limit of detection and limit of quantification (LOQ) of 0.06 and 0.15 ng/mL, respectively, and intra-assay CVs <9% in the relevant ranges was obtained. The LC-MS/MS compared well with the in-house immunoassay (Deming regression slope: 1.28; Pearson correlation: R=0.86). INSL3 concentrations increased with pubertal maturation in healthy boys. INSL3 concentrations were above the LOQ in all samples from the adult men. The mean (±2 SD range)for serum INSL3 concentrations in the adult men was 2.2 (0.5-3.9) ng/mL. CONCLUSIONS: We have developed a robust and sensitive method suitable for quantitation of serum INSL3 in a clinical setting using LC-MS/MS instrumentation available in modern clinical laboratories. The method paves the way for future studies into the clinical role of serum INSL3 measurements.

Viable acrosome-intact human spermatozoa in the ejaculate as a marker of semen quality and fertility status.

STUDY QUESTION: Is it possible, in an unbiased and clinical relevant way, to determine the number of viable acrosome-intact human spermatozoa in ejaculates and to use this as a measure of fertility chances? SUMMARY ANSWER: Image cytometry enables easy and unbiased quantification of viable acrosome-intact spermatozoa and it correlates with semen quality and fertility status. WHAT IS KNOWN ALREADY: The presence of the acrosome and its ability to respond to physiological inducers (e.g. progesterone) in the female reproductive tract at the appropriate time and place is required for fertilization. However, the available assays are labor intensive and therefore not used clinically. STUDY DESIGN, SIZE, DURATION: Washed semen samples and capacitated swim-up fractions from volunteers were used to develop the assay. Subsequently washed ejaculates from patients in fertility treatment (n = 156), proven fertile men (n = 54) and volunteers (n = 10) were assessed to evaluate the number of acrosome-intact spermatozoa in the ejaculate (acrosomal status) and compared to other semen parameters, fertility status, fertility treatments and pregnancy rates. PARTICIPANTS/MATERIALS, SETTING, METHODS: Image cytometry was used to assess the fluorescence intensity of Pisum sativum agglutinin and Propidium iodide. MAIN RESULTS AND THE ROLE OF CHANCE: The assay was validated by inducing the acrosome reaction in swim-up-purified and capacitated spermatozoa with progesterone and ionomycin, and in repeated acrosomal status measurements of washed ejaculates a small coefficient of variation (3.7%) was observed. Men with poor semen quality had fewer viable acrosome-intact spermatozoa in the ejaculate (P = 0.0012; median 32.6% vs. 49.3%). A large proportion (44%) of normozoospermic men from infertile couples had less than the observed median fraction (46%) of viable acrosome-intact spermatozoa in the ejaculate. Furthermore, the total number of viable acrosome-intact spermatozoa was significantly lower among men with male factor infertility compared to fertile men (median 35 vs. 97 mill, P = 1 × 10-7). Men from couples going through one or more ICSI cycles had significant fewer viable acrosome-intact spermatozoa than men from couples who only underwent IUI (P = 0.002; 44.4% vs. 62.0%) and the fraction of viable acrosome-intact spermatozoa appeared better than classical semen parameters in classifying whether or not couples needed ICSI. A positive, although non-significant, tendency toward ongoing pregnancy with an increasing number of viable acrosome-intact spermatozoa was observed (P = 0.2). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Even larger cohorts of infertile couples are needed to substantiate the clinical application of the assay in regard to estimation of fertility potential of an individual. WIDER IMPLICATIONS OF THE FINDINGS: The presented assay makes it possible to measure the number of acrosome competent spermatozoa in an ejaculate in a standardized manner and hence may serve as a new biomarker for male fertility. Few spermatozoa in an ejaculate are acrosome competent and it might be a valuable measure when evaluating male reproductive function. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from the Innovation Fund Denmark. M.G. and S.K. work at ChemoMetec, which produces the image cytometer used in the study, M.G. hold shares in the company. The other authors have no conflict of interest.

Polymorphisms in JMJD1C are associated with pubertal onset in boys and reproductive function in men.

JMJD1C, a member of the Jumonji-domain containing histone demethylases protein family, has been associated with levels of sex-hormone binding globulin (SHBG) and testosterone in men, and knock-out rodent models show age-dependent infertility. The objective of this study was to investigate whether single nucleotide polymorphisms (SNPs) nearby JMJD1C are associated with pubertal onset in boys and with male reproduction. 671 peri-pubertal boys, 1,027 young men, 315 fertile men, and 252 infertile men were genotyped for two JMJD1C SNPs (rs7910927 and rs10822184). rs7910927 and rs10822184 showed high linkage. Boys with the rs7910927 TT genotype entered puberty 3.6 months earlier than their peers (p = 2.5 × 10-2). In young men, the number of T alleles was associated with decreased levels of SHBG, follicle-stimulating hormone (FSH), testosterone, and testosterone x luteinizing hormone, as well as increased levels of Inhibin B, Inhibin B/FSH ratio, and testis size. No significant associations with semen parameters were observed and the genotype distribution was comparable among fertile and infertile men. In conclusion, genetic variation in the vicinity of JMJD1C had a surprisingly large impact on the age at pubertal onset in boys as well as levels of reproductive hormones and testis size in men, emphasizing the relationship between JMJD1C and reproductive functions.

Anti-Müllerian hormone levels and fecundability in women with a natural conception.

OBJECTIVES: To investigate the association between anti-Müllerian hormone (AMH), a well-established marker of the ovarian reserve, and time-to-pregnancy (TTP) in natural conceptions, and to assess changes in serum-AMH in early pregnancy. STUDY DESIGN: A cross sectional study comprising 279 women aged 21-42 years with a natural conception recruited during 2012-2014. AMH was measured in gestational week 10-19. AMH z-scores (z-AMH) adjusted for gestational week at blood sampling were categorised in the 1st, 2nd-4th (reference), and 5th quintile. Data were analysed by discrete-time survival-analysis and results presented as odds ratios (OR), 95% confidence interval (CI); OR <1 indicating a longer TTP and OR >1 indicating a shorter TTP. RESULTS: The median AMH-level was 23.0 (range:<3.0;144.0)pmol/l, and serum-AMH decreased by 7.5% (95% CI:-12.0%;-2.8%) per gestational week. Mean±SD female age was 30.9±3.6years. The median TTP was 2 (range: 1-32) months. After adjustment for possible confounders including total sperm count, TTP was unrelated to female age (aOR:1.0, 95% CI:0.9;1.0) and continuous z-AMH (aOR:0.8, 95% CI:0.7;1.0), but women in the low z-AMH group had a shorter TTP than the reference group (aOR:1.7, 95% CI:1.1;2.7). TTP was prolonged in preconception oral contraceptive (OC) users (aOR:0.7, 95% CI:0.5;1.0, p=0.04). Compared with women having used OC <2 years, TTP was significantly longer in women having used OC for 2-12 years (aOR:0.5, 95% CI:0.2;1.0, p=0.048) and >12 years (aOR:0.4, 95% CI:0.2;0.9, p=0.022) after age-adjustment. CONCLUSIONS: TTP was unrelated with z-AMH when modelled as a continuous covariate. Unexpectedly, TTP was shorter in the low z-AMH group. Natural conception was observed in women with a wide range of AMH-levels including women with undetectable serum-AMH. A continuous decrease in serum-AMH was observed during first and second trimester. Preconception OC-use was identified as an independent predictor of a prolonged TTP, and the duration of OC-use appeared to influence the delay in conception. Although this is presently one of the largest studies investigating the association between AMH and fecundability in fertile women, the study has some limitation including a relatively low participation rate and a risk of selection bias in addition to AMH assessment in pregnancy and a retrospective collection of TTP and OC-use associated with a risk of recall bias. These limitations may explain the unexpected finding of a shorter TTP in the low z-AMH group.

Is Sedentary Lifestyle Associated With Testicular Function? A Cross-Sectional Study of 1,210 Men.

Based on cross-sectional data on 1,210 healthy young Danish men, we investigated whether sedentary lifestyle was associated with testicular function (semen quality and reproductive hormones) independent of physical activity. The men were invited to participate in the study between 2008 and 2012, when they attended a compulsory medical examination to determine their fitness for military service. Information on sedentary behavior (television watching and computer time) and physical activity was obtained by questionnaire. The men had a physical examination, delivered a semen sample, and had a blood sample drawn. Time spent watching television, but not time sitting in front of a computer, was associated with lower sperm counts. Men who watched television more than 5 hours/day had an adjusted sperm concentration of 37 million/mL (95% confidence interval (CI): 30, 44) versus 52 million/mL (95% CI: 43, 62) among men who did not watch television; total sperm counts in those 2 groups were 104 million (95% CI: 84, 126) and 158 million (95% CI: 130, 189), respectively. Furthermore, an increase in follicle-stimulating hormone and decreases in testosterone and the testosterone/luteinizing hormone ratio were detected in men watching many hours of television. Self-rated physical fitness, but not time spent on physical activity, was positively associated with sperm counts.

Vitamin D deficiency and low ionized calcium are linked with semen quality and sex steroid levels in infertile men.

STUDY QUESTION: Are low vitamin D levels linked with semen quality and sex steroids in infertile men? SUMMARY ANSWER: Infertile men with vitamin D deficiency had lower sperm motility, total numbers of motile sperm, Inhibin B, sex-hormone-binding-globulin (SHBG) and testosterone/estradiol ratio, but higher levels of free sex steroids, than infertile men with normal vitamin D levels. WHAT IS KNOWN ALREADY: Low vitamin D levels have been associated with decreased sperm motility in healthy men, but a relationship between vitamin D and calcium with semen quality and especially sex steroids has not been sufficiently described in infertile men. STUDY DESIGN, SIZE, DURATION: This study comprises baseline characteristics of 1427 infertile men screened from 2011 to 2014 for inclusion in a randomized clinical trial, the Copenhagen-Bone-Gonadal Study. PARTICIPANTS/MATERIALS, SETTING, METHODS: In total 1427 infertile men, consecutively referred to our tertiary andrological centre for fertility workup, underwent a physical examination and had semen quality assessed based on two samples and blood analysed for serum testosterone, SHBG, estradiol, inhibin B, luteinizing hormone, follicle-stimulating hormone (FSH), 25-hydroxyvitamin D (25-OHD), ionized calcium (Ca(2+)) and karyotype. There were 179 men excluded due to serious comorbidities or anabolic steroid usage, leaving 1248 patients for analyses. MAIN RESULTS AND THE ROLE OF CHANCE: Men with 25-OHD >75 nmol/l had higher sperm motility and 66 and 111% higher total numbers of motile spermatozoa after 45 and 262 min, respectively, than men with 25-OHD <25 nmol/l (all P < 0.05). SHBG levels and testosterone/estradiol ratios were 15 and 14% lower, respectively, while free testosterone and estradiol ratios were 6 and 13% higher, respectively, in men with 25-OHD <25 nmol/l (all P < 0.05). Men with lower Ca(2+) levels had higher progressive sperm motility and inhibin B/FSH ratio but lower testosterone/estradiol ratio (all P < 0.05). LIMITATIONS, REASONS FOR CAUTION: All outcomes presented are predefined end-points but inferral of causality is compromised by the descriptive study design. It remains to be shown whether the links between vitamin D, calcium, semen quality and sex steroids in infertile men are causal. WIDER IMPLICATIONS OF THE FINDINGS: The associations between vitamin D deficiency and low calcium with semen quality and sex steroids support the existence of a cross-link between regulators of calcium homeostasis and gonadal function in infertile men. STUDY FUNDING/COMPETING INTERESTS: This study was supported by the Danish Agency for Science, Technology and Innovation, Hørslev Fonden, Danish Cancer Society and Novo Nordisk Foundation. There are no conflicts of interest. TRIAL REGISTRATION NUMBER: NCT01304927. DATE OF TRIAL REGISTRATION: 25 February 2011. DATE OF ENROLMENT OF FIRST PATIENT: 8 March 2011.

Self-reported onset of puberty and subsequent semen quality and reproductive hormones in healthy young men.

STUDY QUESTION: Is there an association between pubertal onset and subsequent reproductive health in young men? SUMMARY ANSWER: Self-reported later onset of puberty was associated with reduced semen quality and altered serum levels of reproductive hormones among 1068 healthy, young Danish men. WHAT IS KNOWN ALREADY: The long-term effects of variations in the onset of male puberty on subsequent reproduction remain largely unstudied. STUDY DESIGN, SIZE, DURATION: In a cross-sectional study, young healthy Danish men were approached when they attended a compulsory medical examination to determine their fitness for military service from 2008 to 2012. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: A total of 1068 healthy, young Danish men (mean age 19 years) participated. They were asked to assess whether onset of penile and testicular growth, development of pubic hair and voice break occurred earlier, at the same time as or later than their peers. Their semen quality (semen volume, sperm concentration, total sperm count and percentages of motile and morphologically normal spermatozoa) and serum concentrations of sex hormones (LH, FSH, total testosterone, SHBG, inhibin B) and testicular size were determined. MAIN RESULTS AND THE ROLE OF CHANCE: The response rate was 29%. Of the 1068 men who then participated, 652 answered the questions about penile growth and pubic hair development and were therefore included in the analysis. Self-reported later onset of puberty was associated with a 25% reduction in sperm concentration (95% CI -41%; -4%), a 40% reduction in total sperm count (-55%; -21%), a 1.6% age point reduction in morphological normal spermatozoa (-2.9; -0.3) and a 1.6 ml reduction in testicular size (-2.4 and -0.8 ml), after adjustment for confounders. Self-reported later onset of puberty was also associated with a 9% (3%; 15%) reduction in free testosterone and a 16% (2%; 31%) increase in FSH, after adjustment for confounders. LIMITATIONS, REASON FOR CAUTION: Our study was cross-sectional and reverse causality cannot be ruled out. In addition, we cannot rule out the possibility that the men with late puberty onset had not yet fully matured although most were in Tanner stage 5. WIDER IMPLICATIONS OF THE FINDINGS: Approximately 15% of young Danish men have self-reported later onset of puberty than their peers. We found poorer testicular function in young men with a history of later pubertal development, suggesting that timing of pubertal onset may be a fundamental marker of male reproductive health. However, we cannot exclude the possibility that these men had not fully matured at the time of examination and therefore their semen quality may yet improve, which makes follow-up important. STUDY FUNDING/COMPETING INTERESTS: This work was supported by the Danish Council for Strategic Research, Program Commission on Health, Food and Welfare (project number 2101-08-0058), Rigshospitalet (grants 961506336 and R42-A1326), European Union, DEER (grant agreement no 212844), the Danish Ministry of Health and the Danish Environmental Protection Agency and Kirsten and Freddy Johansens Foundation (grant 95-103-72087). There are no competing interests.

Psychological stress and testicular function: a cross-sectional study of 1,215 Danish men.

OBJECTIVE: To study the associations between self-reported psychological stress, semen quality, and serum reproductive hormones among young Danish men. DESIGN: Cross-sectional study. SETTING: University hospital-based research center. PARTICIPANT(S): Danish men (median age 19 years) from the general population were investigated from 2008 to 2012. INTERVENTION(S): Participants completed a questionnaire on health and lifestyle, including a four-item questionnaire about self-rated stress, had a physical examination performed, delivered a semen sample, and had a blood sample drawn. MAIN OUTCOME MEASURE(S): Semen parameters (semen volume, sperm concentration, and percentages of motile and morphologically normal spermatozoa) and serum levels of reproductive hormones (LH, FSH, T, calculated free T, sex hormone-binding globulin, and inhibin B). RESULT(S): Poorer semen quality was detected among men with self-reported stress scores above an intermediate stress level, in a dose-response manner. For example, men with the highest stress levels had 38% (95% confidence interval [CI] 3%; 61%) lower sperm concentration, 34% (95% CI 59%; 106%) lower total sperm count, and 15% (95% CI 1%; 27%) lower semen volume than men with intermediate stress levels. No significant associations between self-reported stress and levels of reproductive hormones were detected. CONCLUSION(S): A negative association between self-reported stress and semen quality was detected. If causal, stress may be a contributing factor for suboptimal semen quality among otherwise healthy men.

Association Between Use of Marijuana and Male Reproductive Hormones and Semen Quality: A Study Among 1,215 Healthy Young Men.

A total of 1,215 young Danish men aged 18-28 years were recruited between 2008 and 2012 when they attended a compulsory medical examination to determine their fitness for military service. The participants delivered a semen sample, had a blood sample drawn, and underwent a physical examination. They responded to questionnaires including information on marijuana and recreational drug use during the past 3 months (no use, use once per week or less, or use more than once per week). A total of 45% had smoked marijuana within the last 3 months. Regular marijuana smoking more than once per week was associated with a 28% (95% confidence interval (CI): -48, -1) lower sperm concentration and a 29% (95% CI: -46, -1) lower total sperm count after adjustment for confounders. The combined use of marijuana more than once per week and other recreational drugs reduced the sperm concentration by 52% (95% CI: -68, -27) and total sperm count by 55% (95% CI: -71, -31). Marijuana smokers had higher levels of testosterone within the same range as cigarette smokers. Our findings are of public interest as marijuana use is common and may be contributing to recent reports of poor semen quality.

UGT2B17 Genotype and the Pharmacokinetic Serum Profile of Testosterone during Substitution Therapy with Testosterone Undecanoate. A Retrospective Experience from 207 Men with Hypogonadism.

BACKGROUND: Testosterone (T) is mainly excreted in the urine as testosterone glucuronide (TG). This glucuronidation is partly dependent on the UGT2B17 genotype, and TG excretion is therefore lower in men having the UGT2B17 deletion. However, the possible influence of UGT2B17 genotype on serum T during androgen therapy is unknown. We retrospectively investigated the possible association between the UGT2B17 gene polymorphism and serum T levels in hypogonadal men during Testosterone undecanoate (TU) substitution therapy. SUBJECTS AND METHODS: Two hundred and seven patients treated with TU (Nebido(®)) were genotyped by quantitative polymerase chain reaction for the UGT2B17 deletion polymorphism. All were given 1000 mg TU per injection at 0, 6, and 18 weeks. Blood samples were taken 2 and 6 weeks after the first and second injection, prior to the third injection, and after 2-3 years of treatment. We analyzed for the levels of T, luteinizing hormone (LH), sex-hormone-binding globulin, estradiol, prostate specific antigen, hematocrit, hemoglobin, and total cholesterol. RESULTS: The UGT2B17 genotype frequency was: ins/ins: 42%, ins/del: 44%, and del/del: 14%. During the initial 18 weeks of TU treatment, large intra- and inter-individual variations in serum T levels were observed. Large peaks in T levels, ranging from 6.7 to 69.5 nmol/l, were noted 2 weeks after injections, regardless of the genotype. T levels did not differ between the three genotypes prior to the third injection, but the del/del group had significantly lower levels of LH. At follow-up after 2-3 years, the injection interval or daily T dosage was not dependent on the UGT2B17 genotype. CONCLUSION: In conclusion, we found large intra- and inter-individual variations in serum T during standard TU treatment regimen in hypogonadal men. Only subtle differences in serum T and LH were noted according to UGT2B17 genotype, which however suggest that the UGT2B17 genotype exert modest influence on the pharmacokinetic profile of T after TU treatment.