Nick Narinx

Objective: Lower free testosterone (T) appears more strongly associated with male sexual dysfunction than total T, but most evidence is based on calculated free T (cFT) rather than measured free T (mFT). This study examined associations between mFT and sexual function in community-dwelling men. Design: Cross-sectional analysis of 526 European Male Ageing Study participants. Methods: Sexual function was assessed by validated questionnaire. mFT was measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) following equilibrium dialysis. Vermeulen-derived cFT was calculated from LC-MS/MS total T and sex-hormone binding globulin. Associations of sexual outcomes with total T, cFT and mFT were analysed using proportional ordinal logistic regression and restricted cubic splines adjusted for age, body mass index (BMI) and centre. Results: Participants had a mean age 70.5 ± 8.5 years, BMI 27.3 ± 3.3 kg/m2, cFT 269.2 ± 86.6 pmol/L and mFT 198 ± 82 pmol/L. Lower mFT was significantly associated with erectile dysfunction (odds ratio (OR) per 10 pmol/L decrease 1.04[1.01-1.06]), fewer sexual intercourse (OR 1.03[1.00-1.06]), lower orgasm frequency (OR 1.03[1.01-1.06]) and reduced sexual desire (OR 1.03[1.01-1.05]). Total T was not associated with any outcome and cFT only with erectile dysfunction (OR 1.03[1.01–1.05]) and reduced sexual desire (OR 1.03[1.00–1.05]). Non-linear associations were observed for mFT with erectile function and orgasm frequency, with markedly steeper increase in predicted probability below 163 and 138 pmol/L, respectively. Conclusion: In community-dwelling men, lower mFT was associated with worse erectile function, fewer sexual intercourse attempts, reduced orgasm frequency, and lower sexual desire, with apparent non-linear thresholds for erectile dysfunction and orgasm frequency at 163 and 138 pmol/L, respectively.

Equilibrium dialysis followed by liquid chromatography–tandem mass spectrometry is considered the gold standard for the isolation and quantification of free hormones. However, this technique has not been described for the analysis of free 25-hydroxyvitamin D3. This study investigated analytical challenges, focusing on stability and adsorption of 25-hydroxyvitamin D3 in aqueous buffer environments, to be considered prior to method development of equilibrium dialysis for free vitamin D. Experiments revealed that 25-hydroxyvitamin D3 is highly prone to adsorption, and to a minor extent degradation, resulting in severe analyte loss already upon introduction of 25-hydroxyvitamin D3 into the aqueous environment, far exceeding observations in serum where protein binding provides significant protection. While chemical degradation only slightly contributes, strong surface adsorption accounts for the majority of 25-hydroxyvitamin D3 loss observed. In contrast, the addition of methanol to the buffer environment completely mitigated analyte loss, confirming the severe adsorption of 25-hydroxyvitamin D3 to recipient surfaces. However, incorporating methanol as a buffer component fundamentally altered experimental conditions, thereby potentially disrupting the physiological equilibrium between 25-hydroxyvitamin D3, vitamin D-binding protein, and albumin, and by extension the free fraction of vitamin D. Consequently, the large extent of, primarily, adsorption of 25-hydroxyvitamin D3 in aqueous environments represents an important consideration in setting up equilibrium dialysis experiments for free vitamin D. These findings additionally highlight a critical need for novel analytical strategies that prevent adsorption while maintaining physiological equilibrium in the assessment of free vitamin D.

Transgender individuals are increasingly seeking gender-affirming therapy. For children and adolescents, this typically involves puberty suppression followed by hormone treatment. However, potential long-term adverse effects on the growing skeleton remain poorly characterized, largely due to the lack of appropriate preclinical animal models. Existing models often rely on surgical puberty suppression and, in the case of estradiol (E2) administration, cause a high bone mass-phenotype that is not observed in the clinical setting. To address this, we developed a novel preclinical mouse model that mimics the medical approach used in transgirls and analyzed the effects of gender-affirming therapy on bone. Four-week-old male mice were treated with the gonadotropin-releasing hormone analogue degarelix (DGX) for pharmacological puberty suppression. After 4 wk, E2 was administered at different doses, and bone properties were analyzed after an additional 8 wk. DGX treatment effectively suppressed sex steroid signaling, leading to reduced bone mass and strength, which were dose-dependently restored by E2. While high E2 doses resulted in an excessive increase in bone mass, thereby precluding further mechanistic study, lower doses resulted in a bone phenotype resembling that of native females. At the cellular level, DGX-mediated puberty suppression resulted in increased bone resorption that exceeded bone formation, and also caused an accumulation of marrow adipocytes. Subsequent low-dose E2 administration reduced bone resorption, stimulated bone formation, and prevented the increase in bone marrow adiposity. In summary, we established and validated a mouse model that accurately mimics gender-affirming therapy initiated during early puberty and enables the study of its effects on bone.

Context: Direct measurement of serum free testosterone (FT) may help evaluate hypogonadism in men. However, to date, availability of reference ranges for measured FT (mFT) is limited. Objective: This work aimed to establish age-stratified reference ranges for mFT in healthy and community-dwelling adult men. Methods: Serum samples of 1202 White men were collected from large population cohort studies. FT was measured using equilibrium dialysis followed by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Sex hormone–binding globulin was measured using immunoassay, and total testosterone was measured using LC-MS/MS. Reference ranges per age decade and for men aged 18 to 39 years with a body mass index (BMI) less than 30 were established following Clinical & Laboratory Standards Institute guidelines. Reference ranges were established as the 2.5th and 97.5th percentiles. Results: The reference range for mFT in healthy, nonobese men aged 18 to 39 years was 184 to 749 pmol/L. mFT levels showed a decrease with aging of −4.5 pmol/L per year. Having a BMI of 30 and 35 resulted in a decrease in mFT of 14.4% and 22.2%, respectively, compared to a reference BMI of 22. Conclusion: This study provides age-stratified reference ranges for mFT in adult men, established in the largest cohort to date, using published methodology. Our results also show that mFT levels are influenced by age and BMI. Future work should focus on whether age-stratified or reference ranges for young men should be employed, and how mFT can best be implemented in clinical practice including the improved application of calculated FT.

Background: Serum free testosterone is commonly used as a parameter to evaluate testosterone exposure and is mostly calculated using mathematical approximations. As the principal testosterone-binding protein, SHBG concentration is always included in such calculations. However, variability in SHBG measurements may affect reported SHBG levels and consequently free testosterone calculations. In this study, we re-evaluate the effects of SHBG assay choice and interlaboratory variability on calculated free testosterone (cFT). Methods: Serum samples from 113 men and 106 women were collected. SHBG levels were measured using three different SHBG immunoassays (Roche, Abbott and Siemens). Testosterone levels were measured using LC-MS/MS. Afterwards, cFT was calculated using the Vermeulen formula and measured directly. SHBG concentrations, and derived cFT concentrations, from different assays were compared. To simulate interlaboratory SHBG variability, measured levels were modified by 15% after which cFT was recalculated using the Vermeulen, Ly, Sartorius and Södergard formulae. The proportions of diagnoses of hypogonadism or hyperandrogenism were compared. Results: Assessed SHBG assays showed very good conformity. The largest difference was 7%, between the Abbott and Siemens assay. The difference in cFT levels was at most 3% between the Abbott and Siemens assay. Interlaboratory variability affected the proportion of diagnoses depending on the used formula. Conclusions: Our results do not show large differences between SHBG assays and only minor effects on cFT levels. Therefore, SHBG assay choice is not expected to greatly influence clinical decision making. In contrast, interlaboratory variation in SHBG measurements and choice of formula might considerably affect cFT results and their interpretation.

Objectives: To compare clinical laboratory workflows for the assessment of androgens in men, focusing on total testosterone (T), sex hormone-binding globulin (SHBG) and free T, in clinical laboratories throughout Europe. Methods: An internet-based survey that included questions related to pre-analytical, analytical and post-analytical phases of androgen measurements was distributed between December 2022 and December 2023 by clinical laboratory/chemistry and endocrine societies. A total of 124 unique records from clinical laboratories in 27 European countries were analyzed. Results: Pre-analytical requirements for total T are subject to improvement as less than half of clinical laboratories recommended adequate morning sampling time and/or sampling in a fasting state. Total T was predominantly quantified using enzyme-linked immunoassay (IA) on automated platforms, with only one in four centers using mass spectrometry (MS), while SHBG was exclusively measured by IA. Additionally, free T was used by a majority of clinical laboratories, mainly reported as approximation by calculation of free T (cFT) using the Vermeulen formula. Generally, age-stratification was the preferred means of reporting reference ranges for total T, SHBG and cFT. However, considerate variability was observed in reported lower and upper limits, leading to the necessity of interpreting test results against assay-specific reference ranges, thereby hindering comparability of results between clinical laboratories. Conclusions: Our survey highlights significant inter-laboratory variability for the assessment of androgen status in men, implying non-commutability of clinical test results between different centers. In addition, we observed poor adherence to pre-analytical recommendations. These findings advocate for continued harmonization efforts of measurement procedures for SHBG and total/free T.

Sociodemographic, lifestyle, and medical variables influence total testosterone (T) and sex hormone-binding globulin (SHBG) concentrations. The relationship between these factors and “free” T remains unclear. We examined 21 sociodemographic, lifestyle, and medical predictors influencing calculated free T (cFT) in community-dwelling men across ages. This is a cross-sectional analysis in 20 631 participants in the Androgens in Men Study. Individual participant data (IPD) were provided by 9 cohorts. Total T was determined using mass spectrometry, SHBG using immunoassays, and cFT using the Vermeulen formula. Associations were analyzed using 2-stage random effects IPD meta-analyses. Cohort median ages ranged from 40 to 76 years and median cFT concentrations from 174.3 to 422.8 pmol/L. In men aged 17-99 years, there was a linear inverse association of cFT with age (−57.2 pmol/L [95% confidence interval, −69.4, −44.9] per 1 SD increase in age). Calculated free T increased with increasing baseline body mass index (BMI) among men with BMI < 23.6 kg/m², but decreased among men with BMI > 23.6 kg/m² (−24.7 pmol/L [−29.1, −20.3] per 1 SD increase in the 25.4-29.6 kg/m² BMI range). Calculated free T was lower in younger men, who were married or in a de facto relationship (−18.4 pmol/L [−27.6, −9.3]) and in men who formerly smoked (−5.7 pmol/L [−8.9, −2.6]), were in poor general health (−14.0 pmol/L [−20.1, −7.8]), and had diabetes (−19.6 pmol/L [−23.0, −16.3]), cardiovascular disease (−5.8 pmol/L [−8.3, −3.2]), or cancer (−19.2 pmol/L [−24.4, −14.1]). Calculated free T was most prominently associated with age and BMI. The linear, inverse association with age, nonlinear association with BMI, and presence of diabetes, cancer, and sociodemographic factors should be considered when interpreting cFT values.

Biological sex affects the activity of the hypothalamus-pituitary-adrenal (HPA) axis. However, how androgen deprivation affects this axis remains largely unknown. In this study, we investigated the effect of androgen status on different components of the HPA axis in male mice. Two weeks of androgen deprivation did not affect total plasma corticosterone levels but led to increased pituitary ACTH levels. Stress-induced total plasma corticosterone levels were increased, whereas the suppression of corticosterone after dexamethasone treatment under basal conditions was attenuated. Androgen-deprived mice displayed a 2-fold increase in plasma levels of corticosteroid binding globulin (CBG). A similar increase in CBG was observed in global androgen receptor knock-out animals, compared to wild-type littermates. Androgen deprivation was associated with a 6-fold increase in CBG mRNA in the liver and enhanced transcriptional activity at CBG regulatory regions, as evidenced by increased H3K27 acetylation. We propose that the induction of CBG as a consequence of androgen deprivation, together with the unaltered total corticosterone levels, results in lower free corticosterone levels in plasma. This is further supported by mRNA levels of androgen-independent GR target genes in the liver. The reduction in negative feedback on the HPA axis under basal condition would suffice to explain the enhanced stress reactivity after androgen deprivation. Overall, our data demonstrate that, in mice, tonic androgen receptor activation affects CBG levels in conjunction with effects on gene expression and HPA-axis reactivity.