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Context: Despite clinical guidelines calling for repetitive examination of testicular position during childhood, little is known of normal changes in testicular position during childhood, let alone factors that control it., Objective: To assess changes in and factors associated with testicular position during childhood., Design: Testicular position (the distance from the pubic bone to the upper pole of the testes) at birth, 3 months, 18 months, 36 months, and 7 years and reproductive hormones at 3 months were measured., Setting: Prenatally recruited, prospective longitudinal birth cohort., Participants: A total of 2545 boys were recruited prenatally in a Danish-Finnish birth cohort and had a testicular position examination available. A subset of 680 Danish and 362 Finnish boys had serum reproductive hormone concentrations and insulin-like growth factor I (IGF-I) determined at 3 months., Main Outcome Measures: Testicular distance to pubic bone (TDP), serum reproductive hormone, and IGF-I concentrations., Results: TDP increased from birth to 3 months and decreased thereafter. Length, gestational age, weight for gestational age, and penile length were positively associated with larger TDP and thus lower testicular position in a linear mixed-effect model. Furthermore, IGF-I concentration, inhibin B/follicle-stimulating hormone ratio, and testosterone/luteinizing hormone ratio were all independently and positively associated with longer TDP., Conclusions: We provide longitudinal data on postnatal changes in TDP. TDP is dynamic and associated with Leydig and Sertoli cell function as well as with IGF-I levels during the first months of life at mini-puberty of infancy. TDP may thus be a useful biomarker of postnatal testicular function.

Objective: Male aging is characterized by a decline in testosterone (TS) levels with a substantial variability between subjects. However, it is unclear whether differences in age-related changes in TS are associated with general health. We investigated associations between mortality and intra-individual changes in serum levels of total TS, SHBG, free TS and LH during a ten-year period with up to 18 years of registry follow-up., Design: 1167 men aged 30-60 years participating in the Danish Monitoring Trends and Determinants of Cardiovascular Disease (MONICA1) study and who had a follow-up examination ten years later (MONICA10) were included. From MONICA10, the men were followed up to 18 years (mean: 15.2 years) based on the information from national mortality registries via their unique personal ID numbers., Methods: Cox proportional hazard models were used to investigate the association between intra-individual hormone changes and all-cause, CVD and cancer mortalities., Results: A total of 421 men (36.1%) died during the follow-up period. Men with most pronounced decline in total TS (<10th percentile) had a higher all-cause mortality risk compared to men within the 10th to 90th percentile (hazard ratio (HR): 1.60; 95% confidence interval (CI): 1.08-2.36). No consistent associations were seen in cause-specific mortality analyses., Conclusion: Our study showed that higher mortality rates were seen among the men who had the most pronounced age-related decline in TS, independent of their baseline TS levels., (© 2018 European Society of Endocrinology.)

Based on a large population of 1113 men aged 30-60 at baseline (mean: 44.1 years, standard deviation: 10.5), we investigated whether intra-individual changes in testosterone (T) and related reproductive hormones during a ten year period were dependent of marital status at baseline and follow-up. The studied men were part of a health survey in Denmark, conducted between 1982 and 1984 with a follow-up examination approximately ten years later. Data on reproductive hormones, measured in serum, and lifestyle and marital status were obtained at both time points. As expected, an age-related decline in testosterone was observed. However, independent of age and lifestyle, we observed that men who went from unmarried to married (n=81) during the study period experienced an accelerated age-related decline in testosterone (-6.6nmol/L) whereas men who went from married to unmarried (n=67) experienced an attenuated age-related decline (-2.3nmol/L). Men who were either married or unmarried at both time points (n=167, n=798, respectively) had a testosterone decline in between (-3.7nmol/L and -4.6nmol/L, respectively). Changes in T/LH ratio did not differ according to marital status indicating that the lowered T level is not compensated by increasing LH levels. This could suggest a modification of the gonadostat due to an adaptation to changing life circumstances., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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., (© The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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., (© The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Context: Low serum T levels have been associated with type 2 diabetes (T2D) and cardiovascular disease. However, it is unresolved whether low T is a risk factor or rather a risk marker for these conditions., Objective: The objective of the study was to investigate serum levels of total T, SHBG, free T, estradiol, LH, and FSH and the subsequent risk of T2D and/or cardiovascular disease., Design, Setting, and Participants: A prospective cohort study consisting of 5350 men from the general population aged 30-70 years at baseline, examined between 1982 and 2001 and followed with complete registry follow-up until December 2011, ie, up to 29 years of follow-up., Main Outcome Measures: T2D outcomes defined as the first diagnosis of T2D and cardiovascular outcomes defined as first diagnosis of either ischemic heart disease or stroke (ischemic and hemorrhagic stroke)., Results: In Cox proportional hazards models, a significantly negative association between T quartile and the risk of T2D was seen. Similarly, men with SHBG in the highest quartile had a decreased risk of developing T2D (nonsmokers: hazard ratio 0.30, 95% confidence interval 0.14-0.63; smokers: hazard ratio 0.40, 95% confidence interval 0.20-0.78). Similar trends were seen for free T, however, insignificant in the fully adjusted analysis. No associations were seen for estradiol, LH, and FSH. A less consistent pattern was seen for the hormones in relation to CVD outcomes; nonsmoking men showed a pattern of higher levels of total T, SHBG, and LH being negatively associated with ischemic heart disease and less pronounced for stroke, whereas in smokers higher levels of total T, free T, and LH were positively associated with the two CVD outcomes., Conclusions: The observed negative associations of T and SHBG with T2D, but no association to LH and free T, indicates that low T in men who develop T2D is a marker of the disease rather than primary hypogonadism being a causal risk factor.

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., (© The Author 2015. Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The influence of sex, age, pubertal development and oral contraceptives on dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS), 17α-hydroxyprogesterone (17-OHP), Δ4-androstenedione (Adione), testosterone (T), calculated free testosterone (fT), free androgen index (FAI) and selected ratios in 1798 serum samples from healthy children, adolescents and young adults was evaluated. Samples were analyzed by Turboflow-LC-MS/MS. Sex hormone-binding globulin was analyzed by immunoassay. All steroid metabolite concentrations were positively associated with age and pubertal development in both sexes and generally higher in males than in females except for Adione. The pubertal rise in T in males was more pronounced compared to females, reflecting contribution from the testes. Ratios between steroid metabolites varied and depended on sex and age. All ratios were lower during infancy compared to later in life. Use of oral contraceptives significantly lowered serum concentrations of all steroid metabolites, fT, FAI, the 17-OHP/Adione, the Adione/T and the DHEA/Adione ratios, but not the DHEA/DHEAS ratio. We provide reference ranges for DHEA, DHEAS, 17-OHP, Adione, T, fT, FAI and selected ratios in relation to sex, age and pubertal development. Use of oral contraceptives strongly influences adrenal steroidogenesis and should be considered when diagnosing and monitoring treatment of patients with disorders of sex development., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Insulin-like factor 3 (INSL3) is a promising marker of Leydig cell function with potentially high clinical relevance. Limited data of INSL3 levels in relation to other reproductive hormones in healthy pubertal boys exist. In this study, we aimed to evaluate longitudinal serum changes in INSL3 compared with LH, FSH, testosterone, inhibin B, and anti-Müllerian hormone (AMH) during puberty in healthy boys. Ten boys were included from the longitudinal part of the COPENHAGEN Puberty Study. Pubertal evaluation, including testicular volume, was performed and blood samples were drawn every 6 months for 5 years. Serum concentrations of testosterone were determined by a newly developed LC-MS/MS method, and serum concentrations of INSL3, AMH, inhibin B, FSH, and LH respectively were determined by validated immunoassays. The results showed that serum INSL3 levels increased progressively with increasing age, pubertal onset, and testicular volume. In six of the ten boys, LH increased before the first observed increase in INSL3. In the remaining four boys, the increase in LH and INSL3 was observed at the same examination. The increases in serum concentrations of LH, testosterone, and INSL3 were not parallel or in ordered succession and varied interindividually. We demonstrated that INSL3 concentrations were tightly associated with pubertal onset and increasing testicular volume. However, the pubertal increases in LH, INSL3, and testosterone concentrations were not entirely parallel, suggesting that INSL3 and testosterone may be regulated differently. Thus, we speculate that INSL3 provides additional information on Leydig cell differentiation and function during puberty compared with traditional markers of testicular function.

Background: Accurate and selective assessment of testosterone requires use of a sensitive LC-MS/MS method, especially at low levels as those seen in young children., Methods: The present longitudinal study of 20 healthy children from the Copenhagen Puberty Study followed every 6 months for 5 years evaluates the longitudinal increase in serum testosterone before, during and after pubertal onset quantified by a newly developed LC-MS/MS method in comparison with immunoassay. Testosterone concentrations in serum samples (n = 177) were determined by LC-MS/MS (detection limit 0.1 nmol/l) and by immunoassay (detection limit 0.23 nmol/l)., Results: Serum concentrations of testosterone increased gradually with age by both methods. However, serum testosterone was quantifiable in 9/10 girls prior to pubic hair development measured with LC-MS/MS, and in 2/10 girls measured with immunoassay. In boys, testosterone was quantifiable in 10/10 boys 1 year prior to pubic hair development measured with LC-MS/MS, and only in 1/10 boys measured with immunoassay. Serum testosterone levels were quantifiable 1.5 years (range 0.5-2.5) earlier using LC-MS/MS., Conclusion: Assessment of longitudinal circulating levels of serum testosterone using a selective LC-MS/MS method proved to be more sensitive in predicting early peripubertal changes in healthy children compared to levels determined by immunoassay.

Study Question: Is exposure to perfluorinated compounds (PFCs) associated with testicular function (reproductive hormone levels and semen quality) in healthy men?, Summary Answer: PFOS levels were significantly negatively associated with serum testosterone (total and calculated free), but not with any other reproductive hormones or semen quality., What Is Known Already: In animals, some PFCs have endocrine disrupting potential, but few studies have investigated PFCs in relation to human testicular function. Previously, we and others have observed a negative association between serum PFC levels and sperm morphology. The potential associations with reproductive hormones remain largely unresolved., Study Design, Size, Duration: A cross-sectional study of 247 men was conducted during 2008-2009., Participants/materials, Setting, Methods: Healthy men from the general population, median age of 19 years, gave serum and semen samples. Serum samples were analysed for total testosterone (T), estradiol (E), sex hormone-binding globulin (SHBG), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and inhibin-B and 14 PFCs, including perfluorooctanesulfonate (PFOS). Semen samples were analysed according to the WHO criteria., Main Results and the Role of Chance: PFOS levels were negatively associated with testosterone (T), calculated free testosterone (FT), free androgen index (FAI) and ratios of T/LH, FAI/LH and FT/LH. Other PFCs were found at lower levels than PFOS and did not exhibit the same associations. PFC levels were not significantly associated with semen quality. PFOS levels in these samples collected in 2008-2009 were lower than in our previous study of men participating in 2003., Limitations, Reasons for Caution: Results were robust to adjustment for relevant confounders; however, the possibility of chance associations due to multiple testing or effects of uncontrolled confounding cannot be ruled out., Wider Implications of the Findings: Our previous findings of decreased sperm morphology in the most highly PFC exposed men were not replicated, possibly due to a lack of highly exposed individuals; however, a recent independent study also did corroborate such an inverse association. The negative association between serum PFOS and testosterone indicates that testosterone production may be compromised in individuals with high PFOS exposure., Study Funding/competing Interest(s): The authors received financial support from the European Commission (DEER, FP7-2007-212844), the Danish Agency for Science, Technology and Innovation (grant nos. 27107068 and 09-067180), Rigshospitalet (grant no. 961506336), the University of Copenhagen, the Danish Ministry of Health and the Danish Environmental Protection Agency (MST-621-00013), and Kirsten and Freddy Johansen Foundation (grant no. 95-103-72087). The funding organizations played no role in the design and conduct of the study, in collection, management, analysis and interpretation of the data; or in the presentation, review or approval of the manuscript. The authors declare that they have no competing financial interests.

Purpose: To study the effect of 16 Gy radiotherapy (RT) vs. 20 Gy RT on Leydig cell function in men treated with radiotherapy against carcinoma in situ (CIS) of the testis., Methods and Materials: Fifty-one men who were treated between 1985 and 2005 were included. Fourteen men had been treated with 20 Gy and 37 with 16 Gy RT. Measurements of sex hormone-binding globulin and basic and stimulated testosterone, as well as luteinizing hormone levels were performed., Results: The follow-up periods for the patients treated without additional chemotherapy were for the 20 Gy and 16 Gy group mean/median/min-max: 9.0/10.0/1.0-20.3 years and 4.0/3.1/0.4-14.1 years, respectively. During the follow-up period, men treated with 16 Gy RT had stable testosterone levels (-1.1%/year, p = 0.4), whereas men treated with 20 Gy had an annual decrease of 2.4% (p = 0.008). For the latter group, the testosterone decrease was most pronounced in the first 5 years, leveling off during the following 5 years. Additionally, more men treated with 20 Gy needed androgen substitution treatment. Our study showed an increased luteinizing hormone level for the men treated with 16 Gy, although this was not significant (p = 0.5). We anticipated a similar increase in the patients treated with 20 Gy but instead observed a decrease (-3.1%, p = 0.01)., Conclusion: RT at 16 and 20 Gy seem to affect Leydig cell function differently, with 16 Gy RT better preserving testosterone levels and thus being preferred from an endocrinological point of view.

Context: Adverse secular trends in male reproductive health have been reported to be reflected in increased testicular cancer risk and decreased semen quality in more recently born men. These secular trends may also be reflected by changes in Leydig cell function., Objective: The objective of the study was to examine whether an age-independent time trend in male serum testosterone levels exists., Design and Setting: Testosterone and SHBG were analyzed in 5350 male serum samples from four large Danish population surveys conducted in 1982-1983, 1986-1987, 1991-1992, and 1999-2001. Free testosterone levels were calculated. The effects of age, year of birth, and time period on hormone levels were estimated in a general linear statistical model., Main Outcome Measures: Testosterone, SHBG, and calculated free testosterone levels in Danish men in relation to age, study period, and year of birth were measured., Results: Serum testosterone levels decreased and SHBG levels increased with increasing age. In addition to this expected age effect, significant secular trends in testosterone and SHBG serum levels were observed in age-matched men with lower levels in the more recently born/studied men. No significant age-independent effect was observed for free testosterone. Adjustment for a concurrent secular increase in body mass index reduced the observed cohort/period-related changes in testosterone, which no longer were significant. The observed cohort/period-related changes in SHBG levels remained significant after adjustment for body mass index., Conclusions: The observed age-independent changes in SHBG and testosterone may be explained by an initial change in SHBG levels, which subsequently lead to adjustment of testosterone at a lower level to sustain free testosterone levels.

Background: The diagnosis of androgen deficiency is based on clinical features and confirmatory low serum testosterone levels. In early primary testicular failure, a rise in serum LH levels suggests inadequate androgen action for the individual's physiological requirements despite a serum testosterone level within the normal range. The combined evaluation of serum LH and testosterone levels in the evaluation of testicular failure has not been widely advocated., Patients: Seven hundred and six healthy males and 39 patients with known primary hypogonadism due to Klinefelter's syndrome and SRY-positive 46,XX karyotypes were included in the study., Design: Testosterone, oestradiol and LH serum concentrations were measured in all individuals. Based on the 706 healthy males two-dimensional bivariate LH-testosterone reference charts were constructed., Results: Despite a median serum total and free testosterone and oestradiol levels being reduced (P < 0.001) and LH levels elevated (P < 0.001) in Klinefelter's syndrome and 46,XX-males, many subjects (69%) had total testosterone within the reference range. However, using the bivariate charts all subjects lay outside the 97.5 percentile., Conclusion: Bivariate LH and testosterone charts are useful in the evaluation of men with known primary testicular failure due to sex chromosomal aneuploidy in whom evaluation based on testosterone measurement in isolation underestimates the prevalence of hypogonadism. It is, however, important to emphasize that isolated use of the bivariate evaluation should not form the basis for androgen substitution therapy. Further studies are needed in order to evaluate the use of bivariate LH and testosterone charts in the assessment of any younger man with possible primary testicular failure.

Recent in vitro and animal studies have reported estrogen-like activity of chemicals used in sunscreen preparations. We investigated whether the three sunscreens benzophenone-3 (BP-3), octyl-methoxycinnamate (OMC), and 3-(4-methylbenzylidene) camphor (4-MBC) were absorbed and influenced endogenous reproductive hormone levels in humans after topical application. In this 2-wk single-blinded study 32 healthy volunteers, 15 young males and 17 postmenopausal females, were assigned to daily whole-body topical application of 2 mg per cm(2) of basic cream formulation without (week 1) and with (week 2) the three sunscreens at 10% (wt/wt) of each. Maximum plasma concentrations were 200 ng per mL BP-3, 20 ng per mL 4-MBC, and 10 ng per mL OMC for females and 300 ng per mL BP-3, 20 ng per mL 4-MBC, and 20 ng per mL OMC for men. All three sunscreens were detectable in urine. The reproductive hormones FSH, LH were unchanged but minor differences in testosterone levels were observed between the 2 wk. A minor difference in serum estradiol and inhibin B levels were observed in men only. These differences in hormone levels were not related to sunscreen exposure.

To assess the biological significance of Leydig cell 'hyperplasia' in man, Leydig cell distribution, volume, and function were studied in patients with infertility or testicular cancer and in suddenly deceased controls. A total of 156 biopsies from 95 patients and 18 necropsies from 13 controls were examined using a semi-quantitative stereological method. In patients, serum concentrations of testosterone, sex hormone binding globulin (SHBG), luteinizing hormone (LH), follicle stimulating hormone (FSH), oestradiol and inhibin-B were correlated with the findings on histological examination. Leydig cell clusters of more than 15 cells in a cross-section, for which we proposed the name 'micronodules', were frequently seen in testicles exhibiting Sertoli-cell-only syndrome (SCO), a mixed pattern of impaired spermatogenesis, or complete spermatogenesis in combination with elevated FSH. Median numbers of micronodules per 1.77 mm(2) (four fields of vision) in these three histological patterns were 6, 4, and 3.5, respectively. In contrast, micronodules were only occasionally observed in testicular biopsies from patients with complete spermatogenesis and normal gonadotrophin levels (median 1), and were rare in testes from controls (median = 0, p = 0.02). The proportion of testicular tissue occupied by Leydig cells increased with decreasing spermatogenic capacity. In contrast, the total volume of Leydig cells per testis was roughly comparable irrespective of the histological pattern, with the exception of testes with bilateral micronodules, which had significantly increased Leydig cell volume compared to those without micronodules. The number of micronodules correlated positively to LH (r = 0.577, p < 0.01) and FSH (r = 0.595, p < 0.01) and the presence of micronodules was most pronounced in the hyperstimulated testes, as reflected by an increased LH/testosterone ratio. In conclusion, Leydig cell micronodules were more frequent in biopsies with impaired spermatogenesis and associated with decreased ratios of testicular hormones to gonadotrophins. The presence of micronodules thus seems to be a histological marker of testicular failure in man., (Copyright 2003 John Wiley & Sons, Ltd.)

Background: Testicular function, including compensated Leydig cell function, has been indicated to be an early marker of morbidity. Objective: To study the association of testicular function and markers of metabolic and cardiovascular health in a population of young men. Materials and methods: A cross‐sectional study of 2289 men (median age 19 years, 5–95 percentile 18.4–22.2) from the general population examined between 2012 and 2019. Participants answered a questionnaire, had a blood sample drawn for assessment of reproductive hormone levels and health markers (lipids, glycosylated hemoglobin), delivered a semen sample, underwent physical examination including blood pressure measurements, and dual‐energy X‐ray absorptiometry scan for assessment of body composition. Associations were assessed in both crude and adjusted linear regression analyses. Results: The men were within the normal reference intervals of their age for reproductive and health biomarkers. Compared to the lowest quartile, having luteinizing hormone levels in the highest quartile was associated with higher mean arterial pressure (1.6 [95% confidence interval: 0.8; 2.5] mmHg), cholesterol (0.1 [95% confidence interval: 0.02; 0.18] mmol/L), and total body fat percentage (1.1 [95% confidence interval: 0.4; 1.8] %‐points). Higher serum testosterone levels were associated with more advantageous cardiometabolic health markers and higher total sperm count with a healthier body composition and lower glycosylated hemoglobin. Discussion and conclusion: In this study of young men, unselected regarding reproductive hormones and semen quality, higher luteinizing hormone was associated with cardiovascular risk factors. Higher testosterone and total sperm count were associated with more favorable cardiometabolic indices. Thus, serum reproductive hormones and semen quality may be early appearing biomarkers of cardiovascular health even among young healthy men, which could potentially be useful for preventive initiatives to reduce the excess mortality and morbidity risk among infertile men. However, our study was cross‐sectional and cannot determine causation. Future longitudinal studies of reproductive health in young men are warranted. [ABSTRACT FROM AUTHOR]

Context: Longitudinal data on levels of hypothalamic-pituitary-gonadal axis hormones and insulin-like growth factor I (IGF-I) during puberty in boys with a history of cryptorchidism are largely missing. Objective: We aimed to compare pubertal hormone levels between boys with a history of congenital cryptorchidism who experienced spontaneous testicular descent or underwent orchiopexy and boys without a history of cryptorchidism. Methods: This was a nested case-control study within a population-based birth cohort, with a prospective, longitudinal pubertal follow-up every 6 months (2005 to 2019). Participants were 109 Finnish boys, including boys with a history of unilateral cryptorchidism who underwent orchiopexy (n =15), unilateral cryptorchidism who had spontaneous testicular descent (n =15), bilateral cryptorchidism who underwent orchiopexy (n =9), bilateral cryptorchidism who had spontaneous testicular descent (n=7), and controls (n=63). Serum reproductive hormone levels and testicular volumes were measured. Results: From around onset of puberty, boys with bilateral cryptorchidism who underwent orchiopexy had significantly higher follicle-stimulating hormone (FSH) and lower inhibin B levels than controls. Boys with unilateral cryptorchidism who underwent orchiopexy had significantly higher FSH than controls, whereas inhibin B levels were similar. Testosterone, luteinizing hormone, insulin-like factor 3, and IGF-I were generally similar between groups. Testicular volume of boys with unilateral or bilateral cryptorchidism who underwent orchiopexy was smaller than that of the controls from 1 year after pubertal onset (P<0.05). Conclusion: Cryptorchid boys, particularly those with bilateral cryptorchidism who underwent orchiopexy, had altered levels of serum biomarkers of Sertoli cells and germ cells and smaller testicular volumes compared with controls. [ABSTRACT FROM AUTHOR]

Metformin is the first-line oral treatment for type 2 diabetes mellitus and is prescribed to more than 150 million people worldwide. Metformin's effect as a glucose-lowering drug is well documented but the precise mechanism of action is unknown. A recent finding of an association between paternal metformin treatment and increased numbers of genital birth defects in sons and a tendency towards a skewed secondary sex ratio with less male offspring prompted us to focus on other evidence of reproductive side effects of this drug. Metformin in humans is documented to reduce the circulating level of testosterone in both men and women. In experimental animal models, metformin exposure in utero induced sex-specific reproductive changes in adult rat male offspring with reduced fertility manifested as a 30% decrease in litter size and metformin exposure to fish, induced intersex documented in testicular tissue. Metformin is excreted unchanged into urine and feces and is present in wastewater and even in the effluent of wastewater treatment plants from where it spreads to rivers, lakes, and drinking water. It is documented to be present in numerous freshwater samples throughout the world - and even in drinking water. We here present the hypothesis that metformin needs to be considered a potential reproductive toxicant for humans, and probably also for wildlife. There is an urgent need for studies exploring the association between metformin exposure and reproductive outcomes in humans, experimental animals, and aquatic wildlife. [ABSTRACT FROM AUTHOR]

Study Question: Are use of e-cigarettes and snuff associated with testicular function as previously shown for conventional cigarettes and marijuana?Summary Answer: Use of e-cigarettes is associated with reduced semen quality but not with higher serum testosterone level as observed for conventional cigarette use. Snuff use was not associated with markers of testicular function.What Is Known Already: Cigarette smoking has previously been associated with higher testosterone levels and impaired semen quality, whereas it is unresolved whether use of e-cigarettes or snuff influence the testicular function.Study Design, Size, Duration: This cross-sectional population-based study included 2008 men with information on cigarette and marijuana use (enrolled between 2012 and 2018), among whom 1221 men also had information on e-cigarette and snuff use (enrolled between 2015 and 2018).Participants/materials, Setting, Methods: Men (median age 19.0 years) from the general population provided a semen and blood sample and filled out a questionnaire on lifestyle including information on smoking behaviour. Associations between different types of smoking (e-cigarettes, snuff, marijuana and cigarettes) and reproductive hormones (total and free testosterone, sex hormone-binding globulin, LH, oestradiol and ratios of inhibin B/FSH, testosterone/LH and free testosterone/LH) and semen parameters (total sperm count and sperm concentration) were examined using multiple linear regression analyses adjusted for relevant confounders.Main Results and the Role Of Chance: Approximately half of the men (52%) were cigarette smokers, 13% used e-cigarettes, 25% used snuff and 33% used marijuana. Users of e-cigarettes and marijuana were often also cigarette smokers. Compared to non-users, daily e-cigarette users had significantly lower total sperm count (147 million vs 91 million) as did daily cigarette smokers (139 million vs 103 million), in adjusted analyses. Furthermore, significantly higher total and free testosterone levels were seen in cigarette smoking men (6.2% and 4.1% higher total testosterone and 6.2% and 6.2% higher free testosterone in daily smokers and occasional smokers, respectively, compared to non-smoking men), but not among e-cigarette users. Daily users of marijuana had 8.3% higher total testosterone levels compared to non-users. No associations were observed for snuff in relation to markers of testicular function.Limitations, Reasons For Caution: We cannot exclude that our results can be influenced by residual confounding by behavioural factors not adjusted for. The number of daily e-cigarette users was limited and findings should be replicated in other studies.Wider Implications Of the Findings: This is the first human study to indicate that not only cigarette smoking but also use of e-cigarettes is associated with lower sperm counts. This could be important knowledge for men trying to achieve a pregnancy, as e-cigarettes are often considered to be less harmful than conventional cigarette smoking.Study Funding/competing Interest(s): Funding was received from the Danish Ministry of Health (1-1010-308/59), the Independent Research Fund Denmark (8020-00218B), ReproUnion (20200407) and the Research Fund of the Capital Region of Denmark (A6176). The authors have nothing to disclose.Trial Registration Number: NA. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Reduced androgen action during early fetal development has been suggested as the origin of reproductive disorders comprised within the testicular dysgenesis syndrome (TDS). This hypothesis has been supported by studies in rats demonstrating that normal male development and adult reproductive function depend on sufficient androgen exposure during a sensitive fetal period, called the masculinization programming window (MPW). The main aim of this study was therefore to examine the effects of manipulating androgen production during different timepoints during early human fetal testis development to identify the existence and timing of a possible window of androgen sensitivity resembling the MPW in rats.Methods: The effects of experimentally reduced androgen exposure during different periods of human fetal testis development and function were examined using an established and validated human ex vivo tissue culture model. The androgen production was reduced by treatment with ketoconazole and validated by treatment with flutamide which blocks the androgen receptor. Testicular hormone production ex vivo was measured by liquid chromatography-tandem mass spectrometry or ELISA assays, and selected protein markers were assessed by immunohistochemistry.Results: Ketoconazole reduced androgen production in testes from gestational weeks (GW) 7-21, which were subsequently divided into four age groups: GW 7-10, 10-12, 12-16 and 16-21. Additionally, reduced secretion of testicular hormones INSL3, AMH and Inhibin B was observed, but only in the age groups GW 7-10 and 10-12, while a decrease in the total density of germ cells and OCT4+ gonocytes was found in the GW 7-10 age group. Flutamide treatment in specimens aged GW 7-12 did not alter androgen production, but the secretion of INSL3, AMH and Inhibin B was reduced, and a reduced number of pre-spermatogonia was observed.Conclusions: This study showed that reduced androgen action during early development affects the function and density of several cell types in the human fetal testis, with similar effects observed after ketoconazole and flutamide treatment. The effects were only observed within the GW 7-14 period-thereby indicating the presence of a window of androgen sensitivity in the human fetal testis. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Is there an association between alcohol intake and semen quality and serum reproductive hormones among healthy men from the USA and Europe? SUMMARY ANSWER Moderate alcohol intake is not adversely associated with semen quality in healthy men, whereas it was associated with higher serum testosterone levels. WHAT IS KNOWN ALREADY High alcohol intake has been associated with a wide range of diseases. However, few studies have examined the correlation between alcohol and reproductive function and most have been conducted in selected populations of infertile men or have a small sample size and the results have been contradictory. STUDY DESIGN, SIZE, DURATION A coordinated international cross-sectional study among 8344 healthy men. A total of 1872 fertile men aged 18–45 years (with pregnant partners) from four European cities and four US states, and 6472 young men (most with unknown fertility) aged 18–28 years from the general population in six European countries were recruited. PARTICIPANTS/MATERIALS, SETTING, METHODS The men were recruited using standardized protocols. A semen analysis was performed and men completed a questionnaire on health and lifestyle, including their intake of beer, wine and liquor during the week prior to their visit. Semen quality (semen volume, sperm concentration, percentage motile and morphologically normal sperm) and serum reproductive hormones (FSH, LH, testosterone, sex hormone-binding globulin, and inhibin B and free testosterone) were examined. MAIN RESULTS AND THE ROLE OF CHANCE The participation rate for our populations was 20–30%. We found no consistent association between any semen variable and alcohol consumption, which was low/moderate in this group (median weekly intake 8 units), either for total consumption or consumption by type of alcohol. However, we found a linear association between total alcohol consumption and total or free testosterone in both groups of men. Young and fertile men who consumed >20 units of alcohol per week had, respectively, 24.6 pmol/l (95% confidence interval 16.3–32.9) and 19.7 pmol/l (7.1–32.2) higher free testosterone than men with a weekly intake between 1 and 10 units. Alcohol intake was not significantly associated with serum inhibin B, FSH or LH levels in either group of men. The study is the largest of its kind and has sufficient power to detect changes in semen quality and reproductive hormones. LIMITATIONS, REASONS FOR CAUTION The participation rate was low, but higher than in most previous semen quality studies. In addition, the study was cross-sectional and the men were asked to recall their alcohol intake in the previous week, which was used as a marker of intake up to 3 months before. If consumption in that week differed from the typical weekly intake and the intake 3 months earlier, misclassification of exposure may have occurred. However, the men were unaware of their semen quality when they responded to the questions about alcohol intake. Furthermore, we cannot exclude that our findings are due to unmeasured confounders, including diet, exercise, stress, occupation and risk-taking behavior. WIDER IMPLICATIONS OF THE FINDINGS Our study suggests that moderate alcohol intake is not adversely associated with semen quality in healthy men, whereas it was associated with higher serum testosterone levels which may be due to a changed metabolism of testosterone in the liver. Healthy men may therefore be advised that occasional moderate alcohol intake may not harm their reproductive health; we cannot address the risk of high alcohol consumption of longer duration or binge drinking on semen quality and male reproductive hormones. STUDY FUNDING/COMPETING INTEREST(S) All funding sources were non-profitable and sponsors of this study played no role in ... [ABSTRACT FROM PUBLISHER]

Objective Pubertal gynaecomastia is a very common condition. Although the underlying aetiology is poorly understood, it is generally accepted that excess of oestrogens and deficit of androgens are involved in the pathogenesis. Furthermore, adiposity as well as the GH/ IGF- I axis may play a role. In this study, we elucidate the association of adiposity and levels of follicle-stimulating hormone ( FSH), luteinizing hormone ( LH), sex hormone-binding globulin ( SHBG), testosterone, oestrogen, IGF- I and IGFBP-3 with the presence of pubertal gynaecomastia in a large cohort of healthy boys. Patients A total of 501 healthy Danish school boys (aged 6·1-19·8 year) from the COPENHAGEN Puberty Study. Measurements Anthropometry and pubertal stages ( PH1-6 and G1-5) were evaluated, and the presence of gynaecomastia was assessed. Body fat percentage was calculated by means of four skin folds and impedance. Nonfasting blood samples were analysed for FSH, LH, testosterone, SHBG, oestradiol, IGF- I, IGFBP-3 and prolactin. Results We found that 23% (31/133) of all pubertal boys had gynaecomastia. More specifically, 63% (10/16) of boys in genital stage 4 had gynaecomastia. Boys with gynaecomastia had significantly higher IGF- I levels compared with controls ( IGF- I SD-score 0·72 vs −0·037, P < 0·001). This difference was maintained after adjusting for confounders (age and pubertal stage). Sex steroid levels, oestradiol/testosterone ratio or free testosterone were not associated with the presence of gynaecomastia with or without adjustment for confounders. Conclusions IGF- I levels were elevated in healthy boys with pubertal gynaecomastia compared with boys without gynaecomastia, whereas sex steroid levels did not differ. We speculate that the GH- IGF- I axis may be involved in the pathogenesis of pubertal gynaecomastia. [ABSTRACT FROM AUTHOR]

Several studies have found an association between sleep duration and morbidity and mortality, but no previous studies have examined the association between sleep disturbances and semen quality. We conducted a cross-sectional study among 953 young Danish men from the general population who were recruited in Copenhagen at the time of determination of fitness for military service between January 2008 and June 2011. All of the men delivered a semen sample, had a blood sample drawn, underwent a physical examination, and answered a questionnaire including information about sleep disturbances. Sleep disturbances were assessed on the basis of a modified 4-item version of the Karolinska Sleep Questionnaire, which includes questions on sleep patterns during the past 4 weeks. Sleep disturbances showed an inverse U-shaped association with sperm concentration, total sperm count, percent motile and percent morphologically normal spermatozoa, and testis size. Men with a high level of sleep disturbance (score >50) had a 29% (95% confidence interval: 2, 48) lower adjusted sperm concentration and 1.6 (95% confidence interval: 0.3, 3.0) percentage points’ fewer morphologically normal spermatozoa than men with a sleep score of 11–20. This appears to be the first study to find associations between sleep disturbances and semen quality. In future studies, investigators should attempt to elucidate mechanistic explanations and prospectively assess whether semen quality improves after interventions restoring a normal sleeping pattern. [ABSTRACT FROM PUBLISHER]

Parabens are used as anti-microbial preservatives in a range of consumer products, especially in cosmetics. In vitro and animal studies have shown weak estrogenic and other endocrine disrupting effects of parabens, including reduced testosterone levels in exposed male rats. The knowledge of paraben exposure, distribution and excretion in humans is limited. In this study we determined the concentration of five parabens; methyl-, ethyl-, n-propyl-, n-butyl- and benzylparaben in urine, serum and seminal plasma samples from 60 healthy Danish men. To conduct the study a sensitive and specific method using LC-MS/MS for simultaneous determination of the five parabens was developed for all three different matrices. Highest concentrations of the parabens were found in urine, wherein methyl-, ethyl-, n-propyl- and n-butyl parabens were measurable in 98%, 80%, 98% and 83% of the men, respectively. Benzyl paraben was only measurable in urine from 7% of the men. Methyl- and n-propyl parabens were also measurable in the majority of serum and seminal plasma samples, whereas the other parabens could only be detected in some of the samples. In all the three matrices significant correlations between the parabens were seen. Furthermore, urinary paraben concentrations correlate to the paraben concentrations in both serum and seminal plasma. [ABSTRACT FROM AUTHOR]

We performed a follow-up study of time to pregnancy in a population of first-time pregnancy planners without previous reproductive experience. The objective of this paper is to report and discuss a finding of a strong relationship between glycosylated haemoglobin (HbA1C) and fertility. A total of 165 Danish couples without previous reproductive experience was followed from termination of birth control until pregnancy for a maximum of six menstrual cycles. HbA1C and reproductive hormones were measured at entry. Questionnaire data were collected at entry and once in each cycle during follow-up. The odds ratio (OR) for pregnancy per cycle decreased with increasing concentration of HbA1C (OR per percent HbA1C 0.4, 95% CI 0.2–0.9 for all six cycles and 0.2, 95% CI 0.1–0.5 in the first three cycles). A high concentration of HbA1C was associated with a high concentration of testosterone and a low concentration of inhibin A. No association was found between HbA1C and psychosocial distress. The reduced fertility among women with high HbA1C may be due to an association with subclinical polycystic ovaries as indicated by the hormonal profile. [ABSTRACT FROM PUBLISHER]

• Prenatal exposure to MnBP was related to lower testosterone in 3–4 months old boys. • Maternal phthalate exposure was related to lower T/LH ratio in 3–4 months old boys. • Anti-androgenic phthalates may affect mini-puberty occurring at 3–4 months of age. Phthalates are plastic softeners with anti-androgenic properties. Prenatal exposure has led to lower testosterone (T) levels and smaller testicles in adult rats. To our knowledge, no studies have examined associations between prenatal phthalate exposure and sex hormone concentrations in infants. To study associations between phthalate exposure in Danish pregnant women and T, luteinizing hormone (LH), follicle stimulating hormone (FSH), Δ4-androstenedione (adion), 17α-hydroxyprogesterone (17-OHP) dehydroepiandrosterone sulfate (DHEAS) concentrations in their infants (N = 479) during mini-puberty. Concentrations of 12 phthalate metabolites from six phthalate diesters were measured in urine samples collected from 2010 to 2012 from 479 pregnant women participating in the Odense Child Cohort at gestational week 28 (range 20.4–30.4). Serum T, LH, FSH, adion, 17-OHP, DHEAS, weight and height were measured approximately three months after expected date of birth. Associations between prenatal phthalate exposure and gonadotropin and androgen metabolite concentrations were estimated in boys and girls separately in adjusted linear regression models. T concentration was lower in boys prenatally exposed to phthalates. Maternal urinary concentrations of summed mono- iso -butyl and mono-n-butyl phthalate (∑MBP i+n) and summed metabolites of di- iso -nonyl phthalate (∑DiNP m) were associated with lower T/LH ratio in male offspring and a dose-response association was found. FSH was 14% (95% CI: 1; 25) lower among male offspring from mothers exposed to ∑DiNP m in the highest compared to the lowest tertile. No association was found for girls. Even in these low exposed children, we found a significant decrease in T/LH ratio during mini-puberty in boys prenatally exposed to phthalates, which may suggest impairment of Leydig cells. The children will be followed as they approach adrenarche and pubarche in order to assess if long-term adverse effects persist. [ABSTRACT FROM AUTHOR]