Clinical and genetic determinants of vitamin D receptor expression in cutaneous melanoma patients.

Decrease of vitamin D receptor (VDR) expression is observed in melanocytic naevi and melanoma compared to normal skin. Little is known about factors influencing VDR expression in cutaneous melanoma (CM). We investigated the correlation of VDR expression in CM with 25-hydroxy vitamin D (25OHD) levels, demographic/clinical parameters, genetic variants of VDR and pathology of the primary tumor. Demographic/clinical parameters were recorded in 407 prospectively recruited CM patients of a multi-center controlled study (ViDMe trial). We determined VDR expression both in the nucleus and in the cytoplasm by semi-quantitative assessment in CM tissue using histochemistry in 279 patients, expressed in percentages and histoscore (H-score). Genomic DNA from 332 patients was extracted to genotype thirteen VDR single nucleotide polymorphisms (SNPs) using TaqMan. VDR expression in CM tissue from 279 patients was correlated with clinical/demographic parameters and 25OHD levels (univariable and multivariable analysis), VDR SNPs (univariable analysis) and pathology parameters of primary CM tissue (univariable analysis). Cytoplasmic VDR expression was increased in patients who stated to have a high sun exposure during their life compared to patients with low sun exposure (p H-score,univariable : 0.001, p H-score,multivariable : 0.004). The A allele of the genetic VDR polymorphism Fok1 was associated with a higher expression of the VDR in the cytoplasm (p cytoplasmic, univariable : 0.001 and p H-score, univariable : 0.02). In the primary tumor, presence of mitosis (p nucleus,%, univariable : 0.002) and perineural invasion (p nucleus,%,univariable : 0.03) were significantly associated with low nuclear VDR expression. ClinicalTrials.gov Identifier: NCT01748448.

Does testosterone influence the association between sleep and frailty in men: results from the European Male Aging Study.

BACKGROUND: Previous studies have suggested an association between sleep disturbance and frailty. The mechanism is unknown, although it has been suggested that hormonal factors may play a role. METHODS: The aim was to determine the association between sleep duration, sleep quality and frailty, and to determine whether testosterone influenced this association. Males aged 40-79 years were recruited from eight European centres to the European Male Aging Study (EMAS). Subjects completed an interviewer-assisted questionnaire including questions regarding sleep quality and duration. Sleep quality was scored 0-20 and categorised as 0-4, 5-9, 10-14, and 15-20, with higher scores indicating poorer quality. A 39-component frailty index (FI) was constructed. Total testosterone levels were measured. The association between sleep duration, sleep quality and the FI was assessed using negative binomial regression, with adjustment for putative confounders including testosterone level. RESULTS: Two thousand three hundred ninety-three participants contributed data to the analysis. The mean age was 63.3 years and mean sleep duration was 7.01 h. The mean frailty index was 0.15. Mean testosterone levels declined with decreasing sleep quality. After adjustment, compared to those with a sleep score of 0-4, the FI was 57% (95% CI 38%, 78%) higher among those with a sleep score of 15-20. After adjustment compared to those with normal sleep duration (6-9 h), those with a short (< 6 h) and long (≥ 9 h) sleep duration had a 16% (95% CI 6%, 28%) and 11% (95% CI 0%, 23%) higher FI, respectively. Adjustment for testosterone did not influence the strength of either association. CONCLUSION: Frailty is associated with impaired sleep quality and sleep duration. The association cannot, however, be explained by variation in testosterone levels.

Changes in bone and mineral homeostasis after short-term androgen deprivation therapy with or without androgen receptor signalling inhibitor - substudy of a single-centre, double blind, randomised, placebo-controlled phase 2 trial.

BACKGROUND: Prostate cancer (PCa) patients treated with androgen deprivation therapy (ADT) have an increased fracture risk. Exploring biomarkers for early bone loss detection is of great interest. METHODS: Pre-planned substudy of the ARNEO-trial (NCT03080116): a double blind, randomised, placebo-controlled phase 2 trial performed in high-risk PCa patients without bone metastases between March 2019 and April 2021. Patients were 1:1 randomised to treatment with gonadotropin-releasing hormone antagonist (degarelix) + androgen receptor signalling inhibitor (ARSI; apalutamide) versus degarelix + matching placebo for 12 weeks prior to prostatectomy. Before and following ADT, serum and 24-h urinary samples were collected. Primary endpoints were changes in calcium-phosphate homeostasis and bone biomarkers. FINDINGS: Of the 89 randomised patients, 43 in the degarelix + apalutamide and 44 patients in the degarelix + placebo group were included in this substudy. Serum corrected calcium levels increased similarly in both treatment arms (mean difference +0.04 mmol/L, 95% confidence interval, 0.02; 0.06), and parathyroid hormone and 1,25-dihydroxyvitamin D3 levels decreased. Bone resorption markers increased, and stable calcium isotope ratios reflecting net bone mineral balance decreased in serum and urine similarly in both groups. INTERPRETATION: This exploratory substudy suggests that 12 weeks of ADT in non-metastatic PCa patients results in early bone loss. Additional treatment with ARSI does not seem to more negatively influence bone loss in the early phase. Future studies should address if these early biomarkers are able to predict fracture risk, and can be implemented in clinical practice for follow-up of bone health in PCa patients under ADT. FUNDING: Research Foundation Flanders; KU Leuven; University-Hospitals-Leuven.

Number of CAG repeats and mortality in middle aged and older men.

DESIGN: The androgen receptor (AR) mediates peripheral effects of testosterone. Previous data suggests an association between the number of CAG repeats in exon-1 of the AR gene and AR transcriptional activity. The aim of this analysis was to determine the association between the number of AR CAG repeats and all-cause mortality in men and the influence of testosterone level on the association. PATIENTS AND MEASUREMENTS: Follow-up data to 27 January 2018 were available for men aged 40-79 years recruited across six countries of the European Male Aging Study between 2003 and 2005. Cox proportional hazards modelling was used to determine the association between CAG repeat number/mortality. Results were expressed as hazard ratios (HR)/95% confidence intervals (CI). RESULTS: One thousand nine hundred and seventy-seven men were followed up. Mean baseline age was 60 ± 11.1 years. Mean duration of follow-up was 12.2 years. At follow up 25.1% of men had died. CAG repeat length ranged from 6 to 39, with the highest proportion of CAG repeat number at 21 repeats (16.4%). In a multivariable model, compared to men with 22-23 AR CAG repeats: for men with <22 and >23 AR CAG HR, 95% CI for mortality were, <22 CAG repeats 1.17 (0.93-1.49) and >23 CAG repeats 1.14 (0.88-1.47). In a post-hoc analysis, the association was significant for men in the lowest tertile of baseline testosterone (<14.2 nmol/L) with >23 CAG repeats: in the adjusted model for <22 and >23 CAG repeats, respectively, 1.49 (0.97-2.27) and 1.68 (1.06-2.67) versus 22-23 repeats. CONCLUSIONS: Our European-wide cohort data overall found no association of androgen receptor CAG repeat number and mortality in men. However, post hoc analysis suggested that an association might be present in men with lower baseline testosterone concentrations, which merits further investigation.

Factors Associated With Circulating Sex Hormones in Men : Individual Participant Data Meta-analyses.

BACKGROUND: Various factors modulate circulating testosterone in men, affecting interpretation of testosterone measurements. PURPOSE: To clarify factors associated with variations in sex hormone concentrations. DATA SOURCES: Systematic literature searches (to July 2019). STUDY SELECTION: Prospective cohort studies of community-dwelling men with total testosterone measured using mass spectrometry. DATA EXTRACTION: Individual participant data (IPD) (9 studies; n = 21 074) and aggregate data (2 studies; n = 4075). Sociodemographic, lifestyle, and health factors and concentrations of total testosterone, sex hormone-binding globulin (SHBG), luteinizing hormone (LH), dihydrotestosterone, and estradiol were extracted. DATA SYNTHESIS: Two-stage random-effects IPD meta-analyses found a nonlinear association of testosterone with age, with negligible change among men aged 17 to 70 years (change per SD increase about the midpoint, -0.27 nmol/L [-7.8 ng/dL] [CI, -0.71 to 0.18 nmol/L {-20.5 to 5.2 ng/dL}]) and decreasing testosterone levels with age for men older than 70 years (-1.55 nmol/L [-44.7 ng/dL] [CI, -2.05 to -1.06 nmol/L {-59.1 to -30.6 ng/dL}]). Testosterone was inversely associated with body mass index (BMI) (change per SD increase, -2.42 nmol/L [-69.7 ng/dL] [CI, -2.70 to -2.13 nmol/L {-77.8 to -61.4 ng/dL}]). Testosterone concentrations were lower for men who were married (mean difference, -0.57 nmol/L [-16.4 ng/dL] [CI, -0.89 to -0.26 nmol/L {-25.6 to -7.5 ng/dL}]); undertook at most 75 minutes of vigorous physical activity per week (-0.51 nmol/L [-14.7 ng/dL] [CI, -0.90 to -0.13 nmol/L {-25.9 to -3.7 ng/dL}]); were former smokers (-0.34 nmol/L [-9.8 ng/dL] [CI, -0.55 to -0.12 nmol/L {-15.9 to -3.5 ng/dL}]); or had hypertension (-0.53 nmol/L [-15.3 ng/dL] [CI, -0.82 to -0.24 nmol/L {-23.6 to -6.9 ng/dL}]), cardiovascular disease (-0.35 nmol/L [-10.1 ng/dL] [CI, -0.55 to -0.15 nmol/L {-15.9 to -4.3 ng/dL}]), cancer (-1.39 nmol/L [-40.1 ng/dL] [CI, -1.79 to -0.99 nmol/L {-51.6 to -28.5 ng/dL}]), or diabetes (-1.43 nmol/L [-41.2 ng/dL] [CI, -1.65 to -1.22 nmol/L {-47.6 to -35.2 ng/dL}]). Sex hormone-binding globulin was directly associated with age and inversely associated with BMI. Luteinizing hormone was directly associated with age in men older than 70 years. LIMITATION: Cross-sectional analysis, heterogeneity between studies and in timing of blood sampling, and imputation for missing data. CONCLUSION: Multiple factors are associated with variation in male testosterone, SHBG, and LH concentrations. Reduced testosterone and increased LH concentrations may indicate impaired testicular function after age 70 years. Interpretation of individual testosterone measurements should account particularly for age older than 70 years, obesity, diabetes, and cancer. PRIMARY FUNDING SOURCE: Medical Research Future Fund, Government of Western Australia, and Lawley Pharmaceuticals. (PROSPERO: CRD42019139668).

Methodological aspects of in vivo axial loading in rodents: a systematic review.

Axial loading in rodents provides a controlled setting for mechanical loading, because load and subsequent strain, frequency, number of cycles and rest insertion between cycles, are precisely defined. These methodological aspects as well as factors, such as ovariectomy, aging, and disuse may affect the outcome of the loading test, including bone mass, structure, and bone mineral density. This review aims to overview methodological aspects and modifying factors in axial loading on bone outcomes. A systematic literature search was performed in bibliographic databases until December 2021, which resulted in 2183 articles. A total of 144 articles were selected for this review: 23 rat studies, 74 mouse studies, and 47 knock out (KO) mouse studies. Results indicated that peak load, frequency, and number of loading cycles mainly affected the outcomes of bone mass, structure, and density in both rat and mouse studies. It is crucial to consider methodological parameters and modifying factors such as age, sex-steroid deficiency, and disuse in loading protocols for the prediction of loading-related bone outcomes.

Effects of filgotinib on semen parameters and sex hormones in male patients with inflammatory diseases: results from the phase 2, randomised, double-blind, placebo-controlled MANTA and MANTA-RAy studies.

OBJECTIVES: The phase 2 MANTA and MANTA-RAy studies aimed to determine if the oral Janus kinase 1 preferential inhibitor filgotinib affects semen parameters and sex hormones in men with inflammatory diseases. METHODS: MANTA (NCT03201445) and MANTA-RAy (NCT03926195) included men (21-65 years) with active inflammatory bowel disease (IBD) and rheumatic diseases (rheumatoid arthritis, spondyloarthritis or psoriatic arthritis), respectively. Eligible participants had semen parameters in the normal range per the WHO definition. In each study, participants were randomised 1:1 to receive once-daily, double-blind filgotinib 200 mg or placebo for 13 weeks for pooled analysis of the primary endpoint (proportion of participants with a ≥50% decrease from baseline in sperm concentration at week 13). Participants who met the primary endpoint were monitored over an additional 52 weeks for 'reversibility'. Secondary endpoints included change from baseline to week 13 in: sperm concentration, total motility, normal morphology, total count and ejaculate volume. Sex hormones (luteinising hormone, follicle stimulating hormone, inhibin B and total testosterone) and reversibility were exploratory endpoints. RESULTS: Across both studies, 631 patients were screened, and 248 were randomised to filgotinib 200 mg or placebo. Baseline demographics and characteristics were similar within indications between treatment groups. Numerically similar proportions of filgotinib-treated versus placebo-treated patients met the primary endpoint (8/120 (6.7%) vs 10/120 (8.3%)), Δ-1.7% (95% CI -9.3% to 5.8%)). There were no clinically relevant changes from baseline to week 13 in semen parameters or sex hormones, or patterns of reversibility between treatment groups. Filgotinib was well tolerated, with no new safety events. CONCLUSIONS: Results suggest that once daily filgotinib 200 mg for 13 weeks has no measurable impact on semen parameters or sex hormones in men with active IBD or inflammatory rheumatic diseases.

Testosterone Restores Body Composition, Bone Mass, and Bone Strength Following Early Puberty Suppression in a Mouse Model Mimicking the Clinical Strategy in Trans Boys.

Transgender youth increasingly present at pediatric gender services. Some of them receive long-term puberty suppression with gonadotropin-releasing hormone analogues (GnRHa) before starting gender-affirming hormones (GAH). The impact of GnRHa use started in early puberty on bone composition and bone mass accrual is unexplored. It is furthermore unclear whether subsequent GAH fully restore GnRHa effects and whether the timing of GAH introduction matters. To answer these questions, we developed a mouse model mimicking the clinical strategy applied in trans boys. Prepubertal 4-week-old female mice were treated with GnRHa alone or with GnRHa supplemented with testosterone (T) from 6 weeks (early puberty) or 8 weeks (late puberty) onward. Outcomes were analyzed at 16 weeks and compared with untreated mice of both sexes. GnRHa markedly increased total body fat mass, decreased lean body mass, and had a modest negative impact on grip strength. Both early and late T administration shaped body composition to adult male levels, whereas grip strength was restored to female values. GnRHa-treated animals showed lower trabecular bone volume and reduced cortical bone mass and strength. These changes were reversed by T to female levels (cortical bone mass and strength) irrespective of the time of administration or even fully up to adult male control values (trabecular parameters) in case of earlier T start. The lower bone mass in GnRHa-treated mice was associated with increased bone marrow adiposity, also reversed by T. In conclusion, prolonged GnRHa use started in prepubertal female mice modifies body composition toward more fat and less lean mass and impairs bone mass acquisition and strength. Subsequent T administration counteracts GnRHa impact on these parameters, shaping body composition and trabecular parameters to male values while restoring cortical bone architecture and strength up to female but not male control levels. These findings could help guide clinical strategies in transgender care. © 2023 American Society for Bone and Mineral Research (ASBMR).

Sarcopenia-defining parameters, but not sarcopenia, are associated with cognitive domains in middle-aged and older European men.

BACKGROUND: Previous research suggests that sarcopenia is associated with lower cognitive functioning. Evidence on the longitudinal relationship between cognition and sarcopenia, according to the revised criteria of the European Working Group on Sarcopenia in Older People (EWGSOP2), is scarce. This study aimed to investigate both cross-sectional and longitudinal associations between sarcopenia and its defining parameters (muscle strength, muscle mass and physical performance) and cognitive performance in middle-aged and older men. METHODS: This was a secondary analysis of data from the European Male Ageing Study (EMAS), a multicentre cohort study of men aged 40-79 years, recruited from population registers in eight European centres. Cognitive functioning was assessed by using a battery of three neuropsychological tests, measuring fluid intelligence: Rey-Osterrieth Complex Figure (ROCF-Copy and ROCF-Recall), Camden Topographical Recognition Memory (CTRM) and Digit Symbol Substitution Test (DSST). Sarcopenia-defining parameters appendicular lean mass (aLM), gait speed (GS), chair stand test (CST) and handgrip strength (HGS) were measured. Sarcopenia was diagnosed according to the criteria of the EWGSOP2. All measurements were performed at baseline and after a follow-up of 4.3 years. Cross-sectional associations between cognition, sarcopenia-defining parameters and prevalent sarcopenia (EWGSOP2) were analysed. Longitudinally, the predictive value of baseline cognition on decline in sarcopenia-defining parameters, onset of new sarcopenia and vice versa was examined. Linear and logistic regression were used and adjusted for putative confounders. RESULTS: In the whole cohort (n = 3233), ROCF-Copy (ß = 0.016; P < 0.05), ROCF-Recall (ß = 0.010; P < 0.05), CTRM (ß = 0.015; P < 0.05), DSST score (ß = 0.032; P < 0.05) and fluid cognition (ß = 0.036; P < 0.05) were significantly and independently associated with GS at baseline. In the Leuven + Manchester subcohorts (n = 456), ROCF-Copy (ß = 1.008; P < 0.05), ROCF-Recall (ß = 0.908; P < 0.05) and fluid cognition (ß = 1.482; P < 0.05) were associated with HGS. ROCF-Copy (ß = 0.394; P < 0.05), ROCF-Recall (ß = 0.316; P < 0.05), DSST (ß = 0.393; P < 0.05) and fluid cognition (ß = 0.765; P < 0.05) were associated with aLM. The prevalence of sarcopenia in this population was 17.8%. No associations were detected between cognition and prevalent or incident sarcopenia. Longitudinal analysis showed that low ROCF-Copy score at baseline was associated with an increase in CST in men ≥70 years (ß = -0.599; P < 0.05). In addition, a decrease in ROCF-Recall was associated with a decrease in GS, and a decrease in DSST was associated with an increase in CST (ß = 0.155; P < 0.0001, ß = -0.595; P < 0.001, respectively) in persons with the highest change in both cognition and muscle function. CONCLUSIONS: Sarcopenia was not associated with cognitive performance in this population, whereas several components of sarcopenia were associated with domain-specific cognitive performance. Longitudinally, baseline and change in subdomains of cognition predicted change in muscle function in specific subgroups.

The effect of testosterone treatment on bone mineral density in Klinefelter syndrome: A retrospective cohort study.

BACKGROUND: Although Klinefelter syndrome (KS) is the most frequent sex-hormone disorder, there is ongoing uncertainty about the often associated sex-hormone deficiency, its impact on common comorbidities, and therefore about prevention and treatment. In this study, we focus on bone loss, reported to occur in over 40% of KS patients, and the impact of testosterone replacement therapy (TRT) on this comorbidity. OBJECTIVES: This single-center retrospective cohort study in a tertiary hospital compared the effect of treatment with TRT to no TRT on evolution of bone mineral density (BMD) in KS patients. METHODS: After a medical chart review, a total of 52 KS subjects were included in this study. BMD was measured by dual-energy X-ray absorptiometry (DXA) and expressed as T-scores. RESULTS: The subjects were divided into three groups, according to TRT. In the subgroup that only started TRT after baseline measurements (mean age 31 years), we observed significant gain in BMD T-score at the lumbar spine (0.58 ± 0.60, p = 0.003; mean gain of 0.62% areal BMD per year) and total femur T-score (0.24 ± 0.39, p = 0.041; mean gain of 0.25% areal BMD per year) after a mean follow-up period of 7.5 years. Compared to untreated subjects, a significant difference in evolution was demonstrated at the lumbar level (+0.58 ± 0.60 vs. -0.14 ± 0.42, p = 0.007). In untreated subjects with normal testosterone levels, a loss of BMD (-0.27 ± 0.37, p = 0.029; mean loss of 0.49% areal BMD per year) at the femoral neck was measured. This decline was equal to the predicted loss seen in the general male population. CONCLUSION: TRT results in BMD gain in patients with KS with testosterone deficiency, mainly at the lumbar spine. However, this effect is limited (0.62% per year). Patients who were not treated with TRT because of sufficient endogenous testosterone levels, showed only the predicted age-related bone loss during follow-up. The need for TRT in maintaining bone health in KS should be evaluated on an individual basis according to the degree of sex steroid deficiency.

The influence of proteoforms: assessing the accuracy of total vitamin D-binding protein quantification by proteolysis and LC-MS/MS.

OBJECTIVES: Vitamin D-binding protein (VDBP), a serum transport protein for 25-hydroxyvitamin D [25(OH)D], has three common proteoforms which have co-localized amino acid variations and glycosylation. A monoclonal immunoassay was found to differentially detect VDBP proteoforms and methods using liquid chromatography-tandem mass spectrometry (LC-MS/MS) might be able to overcome this limitation. Previously developed multiple reaction monitoring LC-MS/MS methods for total VDBP quantification represent an opportunity to probe the potential effects of proteoforms on proteolysis, instrument response and quantification accuracy. METHODS: VDBP was purified from homozygous human donors and quantified using proteolysis or acid hydrolysis and LC-MS/MS. An interlaboratory comparison was performed using pooled human plasma [Standard Reference Material® 1950 (SRM 1950) Metabolites in Frozen Human Plasma] and analyses with different LC-MS/MS methods in two laboratories. RESULTS: Several shared peptides from purified proteoforms were found to give reproducible concentrations [≤2.7% coefficient of variation (CV)] and linear instrument responses (R2≥0.9971) when added to human serum. Total VDBP concentrations from proteolysis or amino acid analysis (AAA) of purified proteoforms had ≤1.92% CV. SRM 1950, containing multiple proteoforms, quantified in two laboratories resulted in total VDBP concentrations with 7.05% CV. CONCLUSIONS: VDBP proteoforms were not found to cause bias during quantification by LC-MS/MS, thus demonstrating that a family of proteins can be accurately quantified using shared peptides. A reference value was assigned for total VDBP in SRM 1950, which may be used to standardize methods and improve the accuracy of VDBP quantification in research and clinical samples.

Effects of treatment for diabetes mellitus on testosterone concentrations: A systematic review.

BACKGROUND: Low testosterone levels are frequently present in men with obesity and insulin resistance. Currently available treatment options (testosterone replacement therapy or lifestyle changes) hold possible risks or are insufficient. Since low testosterone levels are closely related to obesity and type 2 diabetes, treatment modalities for these conditions could result into improvement of testosterone levels. OBJECTIVES: To summarize the available evidence on the effects of traditional and recent treatment modalities for diabetes mellitus on testosterone levels and androgen-deficiency-related signs and symptoms. MATERIALS AND METHODS: PubMed was searched from the year 2000 till present using MESH terms: "hypogonadism," "testosterone," "testosterone deficiency," "functional hypogonadism," and the different classes of medications. Studies with observational and experimental designs on humans that evaluated the effect of antidiabetic medications on gonadotropins and testosterone were eligible for inclusion. RESULTS: Current available data show no or only limited improvement on testosterone levels with the classic antidiabetic drugs. Studies with GLP1-receptor analogues show beneficial effects on both body weight and testosterone levels in men with low testosterone levels and obesity with or without type 2 diabetes. However, data are limited to small and heterogeneous study groups and only few studies report data about impact on androgen-deficiency-related signs and symptoms. DISCUSSION AND CONCLUSION: With the recent advances in the knowledge of the pathophysiological pathways in obesity, there is an enormous progress in the development of medications for obesity and type 2 diabetes. Newer incretin-based agents have a great potential for the treatment of functional hypogonadism due to obesity since they show promising weight reducing results. However, before the use of GLP1-receptor analogues can be suggested to treat functional hypogonadism, further studies are needed.

The Leydig cell biomarker INSL3 as a predictor of age-related morbidity: Findings from the EMAS cohort.

Background: Insulin-like peptide 3 (INSL3) is a constitutive hormone secreted in men by the mature Leydig cells of the testes. It is an accurate biomarker for Leydig cell functional capacity, reflecting their total cell number and differentiation status. Objectives: To determine the ability of INSL3 to predict hypogonadism and age-related morbidity using the EMAS cohort of older community-dwelling men. Materials & methods: Circulating INSL3 was assessed in the EMAS cohort and its cross-sectional and longitudinal relationships to hypogonadism, here defined by testosterone (T) <10.5nmol/l, and a range of age-related morbidities determined by correlation and regression analysis. Results & discussion: While INSL3 is an accurate measure of primary hypogonadism, secondary and compensated hypogonadism also indicate reduced levels of INSL3, implying that testicular hypogonadism does not improve even when LH levels are increased, and that ageing-related hypogonadism may combine both primary and secondary features. Unadjusted, serum INSL3, like calculated free testosterone (cFT), LH, or the T/LH ratio reflects hypogonadal status and is associated with reduced sexual function, bone mineral density, and physical activity, as well as increased occurrence of hypertension, cardiovascular disease, cancer, and diabetes. Using multiple regression analysis to adjust for a range of hormonal, anthropometric, and lifestyle factors, this relationship is lost for all morbidities, except for reduced bone mineral density, implying that INSL3 and/or its specific receptor, RXFP2, may be causally involved in promoting healthy bone metabolism. Elevated INSL3 also associates with hypertension and cardiovascular disease. When unadjusted, INSL3 in phase 1 of the EMAS study was assessed for its association with morbidity in phase 2 (mean 4.3 years later); INSL3 significantly predicts 7 out of 9 morbidity categories, behaving as well as cFT in this regard. In contrast, total T was predictive in only 3 of the 9 categories. Conclusion: Together with its low within-individual variance, these findings suggest that assessing INSL3 in men could offer important insight into the later development of disease in the elderly.

N/C Interactions Are Dispensable for Normal In Vivo Functioning of the Androgen Receptor in Male Mice.

The androgen receptor (AR) plays a central role in the development and maintenance of the male phenotype. The binding of androgens to the receptor induces interactions between the carboxyterminal ligand-binding domain and the highly conserved 23FQNLF27 motif in the aminoterminal domain. The role of these so-called N/C interactions in AR functioning is debated. In vitro assays show that mutating the AR in the 23FQNLF27 motif (called ARNoC) attenuates the AR transactivation of reporter genes, has no effect on ligand binding, but does affect protein-protein interactions with several AR coregulators. To test the in vivo relevance of the N/C interaction, we analyzed the consequences of the genomic introduction of the ARNoC mutation in mice. Surprisingly, the ARNoC/Y mice show a normal male development, with unaffected male anogenital distance and normal accessory sex glands, male circulating androgen levels, body composition, and fertility. The responsiveness of androgen target genes in kidney, prostate, and testes was also unaffected. We thus conclude that the N/C interactions in the AR are not essential for the development of a male phenotype under normal physiological conditions.

Bone health in ageing men.

Osteoporosis does not only affect postmenopausal women, but also ageing men. The burden of disease is projected to increase with higher life expectancy both in females and males. Importantly, osteoporotic men remain more often undiagnosed and untreated compared to women. Sex steroid deficiency is associated with bone loss and increased fracture risk, and circulating sex steroid levels have been shown to be associated both with bone mineral density and fracture risk in elderly men. However, in contrast to postmenopausal osteoporosis, the contribution of relatively small decrease of circulating sex steroid concentrations in the ageing male to the development of osteoporosis and related fractures, is probably only minor. In this review we provide several clinical and preclinical arguments in favor of a 'bone threshold' for occurrence of hypogonadal osteoporosis, corresponding to a grade of sex steroid deficiency that in general will not occur in many elderly men. Testosterone replacement therapy has been shown to increase bone mineral density in men, however data in osteoporotic ageing males are scarce, and evidence on fracture risk reduction is lacking. We conclude that testosterone replacement therapy should not be used as a sole bone-specific treatment in osteoporotic elderly men.

Association of age, hormonal, and lifestyle factors with the Leydig cell biomarker INSL3 in aging men from the European Male Aging Study cohort.

BACKGROUND: Aging in men is accompanied by a broad range of symptoms, including sexual dysfunction, cognitive and musculoskeletal decline, obesity, type 2 diabetes, cardiovascular disease and hypertension, organ degeneration/failure, and increasing neoplasia, some of which are associated with declining levels of Leydig cell-produced testosterone. High natural biological variance, together with multiple factors that can modulate circulating testosterone concentration, may influence its interpretation and clinical implications. Insulin-like peptide 3 is a biomarker of Leydig cell function that might provide complementary information on testicular health and its downstream outcomes. OBJECTIVES: To characterize insulin-like peptide 3 as a biomarker to assess gonadal status in aging men. METHODS AND MATERIALS: A large European multicenter (European Male Aging Study) cohort of community-dwelling men was analyzed to determine how insulin-like peptide 3 relates to a range of hormonal, anthropometric, and lifestyle parameters. RESULTS AND DISCUSSION: Insulin-like peptide 3 declines cross-sectionally and longitudinally within individuals at approximately 15% per decade from age 40 years, unlike testosterone (1.9% per decade), which is partly compensated by increasing pituitary luteinizing hormone production. Importantly, lower insulin-like peptide 3 in younger men appears to persist with aging. Multiple regression analysis shows that, unlike testosterone, insulin-like peptide 3 is negatively dependent on luteinizing hormone and sex hormone-binding globulin and positively dependent on follicle-stimulating hormone, suggesting a different mechanism of gonadotropic regulation. Circulating insulin-like peptide 3 is negatively associated with increased body mass index or waist circumference and with smoking, and unlike testosterone, it is not affected by weight loss in obese individuals. Geographic variation in mean insulin-like peptide 3 within Europe appears to be largely explained by differences in these parameters. The results allowed the establishment of a European-wide reference range for insulin-like peptide 3 (95% confidence interval) adjusted for increasing age. CONCLUSION: Insulin-like peptide 3 is a constitutive biomarker of Leydig cell functional capacity and is a robust, reliably measurable peptide not subject to gonadotropin-dependent short-term regulation and within-individual variation in testosterone.

The number of androgen receptor CAG repeats and mortality in men.

INTRODUCTION: The androgen receptor (AR) mediates peripheral effects of testosterone. Evidence suggests that the number of CAG repeats in exon-1 of the AR gene negatively correlates with AR transcriptional activity. The aim of this analysis was to determine the association between CAG repeat number and mortality in men. METHODS: Men aged 40-79 years were recruited from primary care for participation in the UK arm of the European Male Aging Study between 2003 and 2005. Cox proportional hazards modelling was used to determine the association between CAG repeat number/mortality. Results were expressed as hazard ratios(HR)/95% confidence intervals (CI). RESULTS: 312 men were followed up. The mean baseline age was 59.5 years. At follow up, 85/312(27%) men had died. CAG repeat length ranged from 14 to 39, with the highest proportion of CAG repeat number at 21 repeats(16.4%). In a multivariable model, using men with CAG repeat numbers of 22-23 as the reference, men with a lower number of CAG repeats(<22) showed a trend for a higher mortality in the follow-up period (HR 1.46 (0.75, 2.81)) as did men with higher number of repeats (>23) (1.37 (0.65, 2.91)). CONCLUSION: Our data suggest that CAG repeat number may partially influence the risk of mortality in men. Further larger studies are required to quantify the effect.

Inactivation of AR or ERα in Extrahypothalamic Neurons Does not Affect Osteogenic Response to Loading in Male Mice.

Failure of bone mass maintenance in spite of functional loading is an important contributor to osteoporosis and related fractures. While the link between sex steroids and the osteogenic response to loading is well established, the underlying mechanisms are unknown, hampering clinical relevance. Androgens inhibit mechanoresponsiveness in male mice, but the cell type mediating this effect remains unidentified. To evaluate the role of neuronal sex steroid receptor signaling in the male bone's adaptive capacity, we subjected adult male mice with an extrahypothalamic neuron-specific knockout of the androgen receptor (N-ARKO) or the estrogen receptor alpha (N-ERαKO) to in vivo mechanical stimulation of the tibia. Loading increased cortical thickness in the control animals mainly through periosteal expansion, as total cross-sectional tissue area and cortical bone area but not medullary area were higher in the loaded than the unloaded tibia. Trabecular bone volume fraction also increased upon loading in the control group, mostly due to trabecular thickening. N-ARKO and N-ERαKO males displayed a loading response at both the cortical and trabecular bone compartments that was not different from their control littermates. In conclusion, we show that the presence of androgen receptor or estrogen receptor alpha in extrahypothalamic neurons is dispensable for the osteogenic response to mechanical loading in male mice.

MANTA and MANTA-RAy: Rationale and Design of Trials Evaluating Effects of Filgotinib on Semen Parameters in Patients with Inflammatory Diseases.

INTRODUCTION: The phase 2 MANTA and MANTA-RAy studies were developed in consultation with global regulatory authorities to investigate potential impacts of filgotinib, a Janus kinase 1 preferential inhibitor, on semen parameters in men with active inflammatory diseases. Here we describe the methods and rationale for these studies. METHODS AND RATIONALE: The MANTA and MANTA-RAy studies included men (aged 21-65 years) with active inflammatory bowel disease (IBD) and rheumatic diseases, respectively. Participants had no history of reproductive health issues, and the following semen parameter values (≥ 5th percentile of World Health Organization reference values) at baseline: semen volume ≥ 1.5 mL, total sperm/ejaculate ≥ 39 million, sperm concentration ≥ 15 million/mL, sperm total motility ≥ 40% and normal sperm morphology ≥ 30%. Each trial included a 13-week, randomized, double-blind, placebo-controlled period (filgotinib 200 mg vs placebo, up to N = 125 per arm), for pooled analysis of the week-13 primary endpoint (proportion of participants with ≥ 50% decrease from baseline in sperm concentration). All semen assessments were based on two samples (≤ 14 days apart) to minimize effects of physiological variation; stringent standardization processes were applied across assessment sites. From week 13, MANTA and MANTA-RAy study designs deviated owing to disease-specific considerations. All subjects with a ≥ 50% decrease in sperm parameters continued the study in the monitoring phase until reversibility, or up to a maximum of 52 weeks, with standard of care as treatment. Overall conclusions from MANTA and MANTA-RAy will be based on the totality of the data, including secondary/exploratory measures (e.g. sperm motility/morphology, sex hormones, reversibility of any effects on semen parameters). CONCLUSIONS: Despite the complexities, the MANTA and MANTA-RAy studies form a robust trial programme that is the first large-scale, placebo-controlled evaluation of potential impacts of an advanced IBD and rheumatic disease therapy on semen parameters. TRIAL REGISTRATION: EudraCT numbers 2017-000402-38 and 2018-003933-14; ClinicalTrials.gov identifiers NCT03201445 and NCT03926195.

Filgotinib is a treatment for patients with ulcerative colitis and rheumatoid arthritis, and is being studied in other inflammatory diseases. Filgotinib works by blocking Janus kinase 1, an intracellular protein involved in inflammatory signalling processes. We designed the MANTA and MANTA-RAy trials with global health agencies to find out if filgotinib decreases the quality of semen in men with active inflammatory bowel disease (ulcerative colitis or Crohn's disease) (MANTA) or rheumatic disease (rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis or non-radiographic axial spondylitis) (MANTA-RAy). This paper describes the design of the two trials.Patients had normal sperm measurements and could not have had previous reproductive health issues. Nearly 250 patients were included in each trial. In both MANTA and MANTA-RAy, half of the patients were treated with 200 mg of filgotinib once a day for 13 weeks, and the other half with placebo. We determined if any patients had a decrease in number of sperm cells per millilitre (sperm concentration) by at least half after 13 weeks of treatment. We then monitored any patients who had such a decrease in sperm concentration for up to 52 weeks (while they received standard of care treatment) or until the decrease was reversed.The conclusions from the trials will be in a different paper and will be based on all the final data, including changes in sex hormones. This is the first large-scale clinical trial programme to measure the effect of a treatment on sperm in men with inflammatory bowel disease or rheumatic diseases.

Reproductive hormone levels, androgen receptor CAG repeat length and their longitudinal relationships with decline in cognitive subdomains in men: The European Male Ageing Study.

OBJECTIVE: It has been proposed that endogenous sex hormone levels may present a modifiable risk factor for cognitive decline. However, the evidence for effects of sex steroids on cognitive ageing is conflicting. We therefore investigated associations between endogenous hormone levels, androgen receptor CAG repeat length, and cognitive domains including visuoconstructional abilities, visual memory, and processing speed in a large-scale longitudinal study of middle-aged and older men. METHODS: Men aged 40-79 years from the European Male Ageing Study (EMAS) underwent cognitive assessments and measurements of hormone levels at baseline and follow-up (mean = 4.4 years, SD ± 0.3 years). Hormone levels measured included total and calculated free testosterone and estradiol, dihydrotestosterone, luteinizing hormone, follicle-stimulating hormone, dehydroepiandrosterone sulphate and sex hormone-binding globulin. Cognitive function was assessed using the Rey-Osterrieth Complex Figure Copy and Recall, the Camden Topographical Recognition Memory and the Digit Symbol Substitution Test. Multivariate linear regressions were used to examine associations between baseline and change hormone levels, androgen receptor CAG repeat length, and cognitive decline. RESULTS: Statistical analyses included 1,827 and 1,423 participants for models investigating relationships of cognition with hormone levels and CAG repeat length, respectively. In age-adjusted models, we found a significant association of higher baseline free testosterone (ß=-0.001, p=0.005) and dihydrotestosterone levels (ß=-0.065, p=0.003) with greater decline on Rey-Osterrieth Complex Figure Recall over time. However, these effects were no longer significant following adjustment for centre, health, and lifestyle factors. No relationships were observed between any other baseline hormone levels, change in hormone levels, or androgen receptor CAG repeat length with cognitive decline in the measured domains. CONCLUSIONS: In this large-scale prospective study there was no evidence for an association between endogenous sex hormone levels or CAG repeat length and cognitive ageing in men. These data suggest that sex steroid levels do not affect visuospatial function, visual memory, or processing speed in middle-aged and older men.