Leen Antonio

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.

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.

Intravenous iron supplementation is increasingly used to safely and effectively correct iron deficiency anemia, but some formulations are linked to a renal phosphate wasting syndrome which is mediated by fibroblast growth factor 23. Unawareness among prescribers and the nonspecific clinical symptoms of hypophosphatemia result in underreporting of this complication. Even though it is often an asymptomatic and self-limiting condition, accumulating evidence from case reports and dedicated randomized controlled trials show that IV iron induced hypophosphatemia may be associated with clinical symptoms. If hypophosphatemia is not recognized and treated, a metabolic bone disease phenotype may develop, pathophysiologically reminiscent of hypophosphatemic rickets as seen in X-linked hypophosphatemic rickets or oncogenic osteomalacia. This syndrome is particularly, but not uniquely, associated with formulations containing ferric carboxymaltose, probably due to specific chemical characteristics of its carbohydrate moiety. Risk factors include repeated infusion, severity of iron deficiency, as well as normal kidney function. Coexisting vitamin D deficiency or hyperparathyroidism increase the risk of metabolic bone disease. Complications can be easily prevented by an early diagnosis and switching to another IV iron formulation. In this review, we describe the epidemiology and pathophysiology of this condition, to raise awareness among prescribing clinicians.