2.1 Testosterone: reference values and indications for testing

Testosterone circulates predominantly bound to plasma proteins: ~60% to sex hormone-binding globulin (SHBG), 38% to albumin, and only 0.5–2% as free testosterone (FT). The sum of FT and albumin-bound fractions is bioavailable testosterone, which represents the biologically active pool [11, 12]. SHBG, produced in the liver, strongly modulates testosterone availability and is influenced by age, obesity, thyroid status, liver disease, and medications [12, 42, 43]. Measurement of SHBG is, therefore, valuable when total testosterone (TT) and clinical presentation are discordant, enabling the calculation of calculated FT (cFT) and improving diagnostic accuracy [4, 13, 38].

Reference values for TT in healthy, non-obese young men range from 264–916 ng/dL (9.2–31.8 nmol/L) [13, 38]. The AUA defines hypogonadism at TT <300 ng/dL, confirmed by symptoms [11]. During TRT, guidelines recommend targeting mid-normal levels (400–700 ng/dL; 13.9–24.3 nmol/L) to maximize efficacy while minimizing risks [11, 13]. Similar thresholds are endorsed by the EAU and Endocrine Society, emphasizing that biochemical evidence must always be interpreted in conjunction with clinical features [12, 13].

TT is the first-line test, ideally with two early morning samples under fasting, stable conditions. Liquid chromatography-tandem mass spectrometry (LC-MS/MS), remains the gold standard, though automated immunoassays are widely used, despite lower accuracy [12, 19, 37]. FT assessment is indicated when TT is borderline (230–350 ng/dL), SHBG is abnormal, or clinical features and TT are discordant [12, 13, 20]. FT can be measured directly (equilibrium dialysis, ultrafiltration) or calculated (Vermeulen, Zakharov), though assay variability affects accuracy [12, 21, 41]. Salivary testosterone is not recommended due to poor standardization [12].

Screening is not advised in asymptomatic men, but TT measurement should be considered in those with suggestive symptoms or conditions, such as unexplained anemia, bone loss, type 2 diabetes, human immunodeficiency virus (HIV), chronic opioid use, infertility, pituitary dysfunction, or prior testicular damage [11, 12, 43]. Clinical questionnaires, such as the Androgen Deficiency in the Aging Male (ADAM) questionnaire and the Aging Males’ Symptoms (AMS) scale, may support assessment but should not replace laboratory confirmation [12, 20].

2.2 Prolactin, erythrocytosis, and alternative strategies

Secondary hypogonadism may arise from multiple hypothalamic or pituitary disorders. Among these, hyperprolactinemia is clinically relevant because elevated prolactin can suppress gonadotropin release and reduce testosterone production. For this reason, baseline prolactin measurement is recommended in men with suspected central hypogonadism, although it should be interpreted within the broader context of pituitary function [11, 12].

Erythrocytosis is the most frequent dose-limiting adverse effect of TRT. Elevated hematocrit (>54%) increases the risk of thromboembolic events, and major guidelines recommend monitoring hematocrit at baseline, at 3–6 months, and annually thereafter [11, 16]. If erythrocytosis develops, management includes dose adjustment, switching formulation, temporary discontinuation, or therapeutic phlebotomy [11, 13].

2.3 Fertility preservation: alternatives to exogenous TRT

“In selected cases, particularly in younger men wishing to preserve fertility, alternatives to exogenous testosterone can be considered. Agents such as selective estrogen receptor modulators (SERMs), human chorionic gonadotropin (hCG), or aromatase inhibitors may enhance endogenous testosterone production, by selectively stimulating the hypothalamic–pituitary–testicular (HPT) axis. However, their long-term safety and efficacy remain less well established compared with TRT.” [12, 13, 20, 21].

Figs. 1,2 proposes a Diagnostic and therapeutic algorithm for male hypogonadism and testosterone deficiency, outlining the clinical evaluation, biochemical confirmation, and subsequent management strategy.

Fig. 1.Assessment of male hypogonadism: stepwise evaluation including symptoms, risk factors, biochemical confirmation with total testosterone (TT) and free testosterone (cFT), and subsequent endocrine workup (LH and prolactin) to distinguish primary from secondary hypogonadism. Hct: hematocrit; PSA: prostate-specific antigen; DEXA: dual-energy X-ray absorptiometry; CPAP: continuous positive airway pressure.

Fig. 2.Treatment of testosterone deficiency: selection of formulation and dosing, monitoring of efficacy and safety (hematocrit, PSA, lipid profile, and bone density), management of complications, and periodic reevaluation to balance risks, benefits, and patient preferences. SHBG: sex hormone-binding globulin; TT: total testosterone; cFT: calculated free testosterone; LH: luteinizing hormone; MRI: magnetic resonance imaging.

3.1 TD and metabolic syndrome

Well-documented association exists between low testosterone levels and metabolic dysfunction, including obesity, insulin resistance, type 2 diabetes mellitus (T2DM), and metabolic syndrome [12, 20, 43]. Up to 40% of men with T2DM exhibit serum testosterone levels below 300 ng/dL, most often as a form of hypogonadotropic (functional) hypogonadism linked to excess adiposity and systemic inflammation [13, 38].

Visceral obesity, particularly abdominal fat accumulation, is strongly correlated with low testosterone levels. Adipose-derived factors, such as leptin, pro-inflammatory cytokines (e.g., interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α)), and dysregulation of the hypothalamic–pituitary–testicular (HPT) axis contribute to this relationship [13]. Insulin resistance may be both a consequence and a driver of hypogonadism, as low testosterone reduces insulin receptor expression in adipose tissue and impairs glucose transporter type 4 (GLUT4)-mediated glucose uptake in skeletal muscle [20].

Weight loss, improved glycemic control, and reduction of inflammation have been shown to restore endogenous testosterone levels. Lifestyle interventions remain the first-line approach for men with obesity-related or functional hypogonadism. Studies have shown that both caloric restriction and bariatric surgery can significantly raise serum testosterone, even leading to normalization in some men [39].

Testosterone replacement therapy (TRT) aims primarily to alleviate symptoms of hypogonadism, but it also exerts metabolic effects. In hypogonadal men with obesity, metabolic syndrome, or T2DM, TRT has been shown to improve several cardiometabolic risk factors, including body composition, insulin sensitivity, lipid profiles, and glycemic control [19, 20, 21]. Meta-analyses and observational cohorts have reported:

● Improved glycemic control (reduction in fasting glucose and glycated hemoglobin A1c (HbA1c)).

● Increased insulin sensitivity.

● Decreased visceral fat and waist circumference.

● Increased lean body mass.

● Improved lipid profile, with reductions in triglycerides and LDL cholesterol, though accompanied by a consistent reduction in high-density lipoprotein (HDL) cholesterol.

The landmark the Testosterone for the Prevention of Diabetes Mellitus (T4DM) trial further explored whether TRT could prevent the development of T2DM in high-risk men. In this 2-year, double-blind, placebo-controlled study, more than 1000 overweight or obese men aged 50–74 years with abdominal obesity and testosterone ≤14 nmol/L, but without pathological hypogonadism, were randomized to intramuscular testosterone undecanoate or placebo. All participants received an intensive lifestyle programme. At 2 years, TRT significantly reduced the incidence of new-onset diabetes compared with the placebo (12% vs. 21%; relative risk 0.59, 95% confidence interval (CI) 0.43–0.80), independent of baseline testosterone [21]. However, approximately 22% in the TRT arm triggered the hematocrit safety threshold (>54%), versus ~1% with placebo, despite both arms receiving an intensive lifestyle program. These findings suggest that while TRT may reduce diabetes risk beyond lifestyle measures, its long-term safety, durability, and cardiovascular effects remain to be determined.

Despite promising results, TRT is not indicated for diabetes prevention, and major guidelines (e.g., American Diabetes Association (ADA)) do not support its use solely to improve metabolic outcomes. Evidence on cardiovascular benefits remains inconclusive, and risks such as erythrocytosis or exacerbation of sleep apnea must be considered [11, 19, 38]. Professional societies (Endocrine Society, AUA, European Academy of Andrology (EAA)) recommend restricting TRT to men with consistent hypogonadal symptoms and confirmed low testosterone on two separate morning samples. In men with functional hypogonadism—often related to obesity or T2DM—lifestyle modification is first-line, as it may restore endogenous testosterone without the risks of exogenous therapy [19, 38, 40, 44, 45].

3.2 Bone health and fracture risk

Testosterone plays a fundamental role in male bone physiology, promoting bone formation and maintaining bone mineral density (BMD). Its effects are mediated both directly through androgen receptors and indirectly via aromatization to estradiol (E2), which is especially important for trabecular bone preservation [12, 22, 42].

Hypogonadism is a well-established secondary cause of male osteoporosis, and fracture risk related to low testosterone is most evident in older, often obese men with mildly reduced T and non-elevated Luteinizing hormone (LH) (functional, age-related hypogonadism).

Some studies suggest a nearly threefold higher risk of fractures in older men with low testosterone [46, 47]. The European Male Ageing Study (EMAS) and the Osteoporotic Fractures in Men Study (MrOS) Sweden cohorts demonstrated that low serum testosterone is independently associated with decreased BMD and greater fracture risk [46, 47]. A population-based study confirmed that testosterone levels predict fracture risk even in men without overt hypogonadism, reinforcing the hormone’s role in skeletal aging [46].

Routine evaluation of bone health in hypogonadal men includes dual-energy X-ray absorptiometry (DXA) scanning, particularly in those over 50, with previous low-trauma fractures, or additional risk factors, such as corticosteroid use, smoking, or alcohol intake. DXA is also advised before starting TRT in men with severe deficiency or osteopenia, and should be repeated after 12–24 months to monitor response [11, 46].

TRT has consistently been associated with improvements in BMD, especially in the lumbar spine [22, 23]. A meta-analysis reported TRT has consistently been associated with modest but significant increases in BMD at the lumbar spine (≈+3.7% vs. placebo; 95% CI 1.0–6.4%), with no significant change at the femoral neck, and concomitant reductions in bone resorption markers, with the greatest benefits in younger men and those with lower baseline testosterone [22]. Transdermal testosterone has also shown efficacy in increasing both bone and muscle mass in older hypogonadal men [23]. However, these anabolic effects on BMD do not necessarily translate into fracture prevention. In the bone substudy of the TRAVERSE trial—the largest randomized trial of TRT to date—the incidence of clinical fractures was higher in the testosterone group than in placebo (3.50% vs. 2.46%; hazard ratio (HR) 1.43, 95% CI 1.04–1.97) among 5204 hypogonadal men aged ≥45 years [24]. This paradox may reflect improved BMD, which is primarily seen at the lumbar spine, without parallel improvements in bone quality, potentially reflecting an increased fall risk in older, more active men with underlying frailty. The findings underscore that TRT is not a primary osteoporosis treatment, particularly in the LOH population.

Therefore, TRT is not a primary osteoporosis treatment. This is particularly important for older, obese men with functional hypogonadism (mildly low T, non-elevated LH), where TRAVERSE data suggest fracture risk may outweigh BMD benefits.

Fracture prevention evidence in men is limited. Intravenous zoledronic acid is the only drug with proven efficacy for reducing vertebral fractures in randomized controlled trials (RCTs), though data on hip fractures are lacking. Treatment must be individualized: TRT is key for bone mass accrual in young men with pathological hypogonadism (e.g., Klinefelter syndrome), while older men with established osteoporosis may require added bone-specific therapy, considering its finite duration and long-term strategic use.

3.3 Mental health

Testosterone modulates brain regions responsible for mood, cognition, and motivation, including the prefrontal cortex, hippocampus, and amygdala [48, 49, 50]. Low testosterone levels have been consistently associated with increased depressive symptoms, particularly in older men. The European Male Ageing Study (EMAS) reported a higher risk of incident depression among men with hypogonadism (or low testosterone), even after adjustment for comorbidities [6, 47].

Psychological symptoms—irritability, low mood, reduced motivation, subjective cognitive difficulties, poor memory, and decreased concentration—are frequently reported in men with late-onset hypogonadism (LOH). While these features are nonspecific, their presence together with low serum testosterone should prompt clinical evaluation [12, 45].

Several trials show that TRT improves depressive symptoms in men with confirmed hypogonadism. In one RCT, remission occurred in 53% with TRT versus 19% with placebo among men with dysthymia [31]. A meta-analysis confirmed a significant reduction in depressive symptoms, with greater benefits in those with lower baseline testosterone or comorbid mild depression [6].

Evidence on cognition remains inconclusive. Some studies suggest modest improvements (e.g., spatial memory, verbal fluency), whereas others find no significant changes. Marked cognitive impairment is mainly observed in contexts of profound androgen deprivation, such as long-term androgen-deprivation therapy (ADT) for prostate cancer [51].

3.4 Physical strength

Testosterone exerts anabolic effects on skeletal muscle, stimulating protein synthesis, satellite cell activation, and neuromuscular coordination. Hypogonadism leads to reductions in lean body mass, strength, endurance, and physical function, contributing to frailty and risk of falls [23, 52]. TRT has consistently been shown to increase lean body mass and, in some cases, improve muscle strength. The T-Trials, a multicenter series of RCTs, assessed the impact of TRT on physical function and vitality in older men with hypogonadism. The physical function trial reported modest improvements in walking distance and strength, though the magnitude of effect varied [53]. In the TEAAM study, long-term TRT improved leg press and chest press strength and enhanced stair-climbing power [27]. TRT may also improve self-reported energy and reduce fatigue, although some trials report minimal or placebo-driven effects on these domains [54]. While the benefits on muscle and vitality are relevant, it is important to emphasize that supraphysiologic doses of testosterone (e.g., anabolic steroid abuse) carry serious risks, including cardiovascular events, mood instability, and infertility [13, 54].

3.5 TD and sexual function

Testosterone is considered the “fuel” of male sexual function. It plays a key role in regulating libido, erectile function, ejaculatory response, and sexual satisfaction, with androgen receptors distributed throughout the hypothalamus, limbic system, spinal cord, and penile tissue [12, 55, 56].

Sexual symptoms are among the most specific clinical indicators of hypogonadism, especially in men over 50. According to the European Male Ageing Study (EMAS), the presence of at least three sexual symptoms—reduced libido, fewer spontaneous/morning erections, and erectile dysfunction—combined with total testosterone <11 nmol/L (320 ng/dL) and free testosterone <220 pmol/L (64 pg/mL), is the minimum criterion for diagnosing late-onset hypogonadism (LOH) [57].

In the EMAS cohort of over 3400 men, sexual symptoms were the strongest predictor of low testosterone and the most common reason for medical consultation. The first sign to appear is typically the loss of morning erections, followed by decreased libido and, later, erectile dysfunction (ED) [57, 58].

Morning erections are closely related to circulating testosterone levels. Studies show that testosterone below 200 ng/dL is often associated with the complete disappearance of morning erections [39]. Libido is particularly sensitive to testosterone levels: a meta-analysis observed a 31% improvement in sexual desire with testosterone therapy, with the greatest gains in men with the lowest baseline testosterone [59].

Erectile dysfunction is one of the most frequent presenting complaint. About 30% of men with erectile dysfunction (ED) have concurrent testosterone deficiency, and in men with suboptimal response to phosphodiesterase type-5 (PDE5) inhibitors, testosterone should always be assessed [55, 60].

TRT has been shown to improve libido and erectile function in hypogonadal men. Meta-analyses confirm significant improvements in sexual desire, satisfaction, and erectile rigidity, with greater effects in those with lower starting testosterone levels [56].

TRT can restore nocturnal erections, increase ease of initiating and maintaining erections, and improve erection quality sufficient for penetration [59]. In cases of incomplete response, combined therapy with PDE5 inhibitors is often recommended. TRT may enhance PDE5i responsiveness, likely through increased nitric oxide synthase expression and improved smooth muscle relaxation in the corpus cavernosum [60].

Experimental studies further support this: in androgen-deprived rats, testosterone restored cavernosal muscle and endothelial nitric oxide synthase (eNOS) expression, reversing erectile tissue damage [31, 35, 61].

4.1 Cardiovascular risk

The TRAVERSE Trial: Redefining the Evidence:

Published in 2023, the TRAVERSE trial represents a landmark in the evaluation of testosterone therapy, being the largest and most methodologically rigorous RCT to date addressing cardiovascular (CV) safety in hypogonadal men. This multicenter, placebo-controlled study enrolled over 5000 men with confirmed hypogonadism and high CV risk, with a median follow-up of 33 months.

Crucially, TRT did not increase the incidence of major adverse cardiovascular events (MACE) compared with placebo (HR 1.03; 95% CI 0.85–1.25), providing the most robust evidence to date that therapeutic testosterone does not raise CV risk when used appropriately [25]. The trial also confirmed neutral effects on stroke, myocardial infarction, and CV mortality, helping to resolve a decade-long controversy.

Clinical Implications and Risk Management:

The TRAVERSE findings support the CV safety of TRT in symptomatic, well-selected men, but clinical prudence remains essential. Testosterone should be avoided in the first 3–6 months following an acute cardiovascular event, and modifiable risk factors—such as hypertension, dyslipidemia, and smoking—should be optimized prior to initiating therapy [13, 23, 39].

Importantly, these safety data refer only to physiologic TRT, and should not be extrapolated to the misuse of anabolic androgenic steroids (AAS) at supraphysiologic doses, which are clearly associated with severe cardiovascular harm—including sudden death, arrhythmia, thrombosis, and cardiomyopathy [62].

4.2 TD and obstructive sleep apnea

Obstructive sleep apnea (OSA) is an important comorbidity to consider in men being evaluated for TRT. OSA predisposes to nocturnal hypoxia, which may contribute to secondary polycythemia, a well-known adverse effect of testosterone therapy.

Chronic intermittent hypoxia, as observed in untreated OSA, stimulates erythropoietin production and increases the risk of erythrocytosis. In men receiving TRT, this effect may be amplified, particularly when additional risk factors, such as chronic pulmonary disease, are present [41, 51, 63]. One study reported that OSA is independently associated with polycythemia in men with hypogonadism on TRT, suggesting an additive effect [44].

Most clinical guidelines list untreated severe OSA as a relative or absolute contraindication to initiating TRT. Although the mechanisms are not fully established, the concern lies in the synergistic risk of hypoxia and testosterone-induced erythropoiesis, which may raise the likelihood of cardiovascular or thrombotic events [38, 39, 40, 63]. TRT should therefore be avoided in men with severe, untreated OSA until the condition is controlled—typically with continuous positive airway pressure (CPAP) therapy. In men with treated or mild OSA, TRT may be considered with close monitoring of hematocrit and respiratory symptoms.

Evidence suggests that TRT may worsen OSA in susceptible individuals by reducing upper airway muscle tone, altering central respiratory drive, and affecting sleep architecture. In a randomized trial, testosterone administration in older obese men with OSA worsened nocturnal oxygen desaturation and apnea severity [63]. This effect appeared dose-dependent and may be more relevant in men receiving higher doses or with borderline oxygenation at baseline.

Conversely, some studies evaluating the effect of CPAP on testosterone levels in men with OSA have shown modest increases in total testosterone after prolonged use, possibly due to improved sleep quality and reduced nocturnal hypoxia [64].

4.3 TD and lower urinary tract symptoms (LUTS)

Lower urinary tract symptoms (LUTS) are highly prevalent among aging men and often attributed to benign prostatic hyperplasia (BPH). Given the androgen-dependence of the prostate, concerns have historically been raised about the potential for TRT to exacerbate LUTS or accelerate BPH progression [12, 38, 39].

The prostate is an androgen-sensitive organ. Testosterone is converted locally to dihydrotestosterone (DHT) via 5α-reductase, promoting epithelial proliferation. However, the saturation model suggests that androgen receptors in the prostate become fully activated at relatively low testosterone levels, such that further increases do not result in proportional prostatic growth [33, 65].

Multiple clinical trials and meta-analyses have evaluated the impact of TRT on LUTS, with no consistent evidence of symptom worsening. Some studies even suggest improvement, possibly due to enhanced detrusor muscle function, improved insulin sensitivity, or reduced systemic inflammation [31].

A 2016 meta-analysis by Cui et al. [32] reported no significant increase in International Prostate Symptom Score (IPSS) with TRT, and noted improvements in subgroups—particularly men with metabolic syndrome and mild LUTS. Current AUA guidelines state that mild LUTS are not a contraindication to TRT, although caution is advised in men with severe urinary obstruction [17].

Additionally, TRT may confer indirect benefits through reductions in central adiposity and improvement in metabolic parameters, both of which are associated with bladder dysfunction and LUTS. TRT impact on BPH progression appears minimal when patients are appropriately selected and monitored [33].

4.4 Prostate cancer: risk, recurrence, and monitoring

The long-standing belief that testosterone “feeds” prostate cancer originated in 1941, when a seminal study showed that surgical castration reduced acid phosphatase levels in men with metastatic disease, while exogenous testosterone increased them [34].

Contemporary research has introduced a more nuanced understanding. The saturation model proposes that androgen receptors in the prostate become maximally activated at low testosterone concentrations (around <250 ng/dL), and further increases in serum testosterone do not stimulate additional prostate growth [33].

Current evidence does not support an increased incidence of prostate cancer (PCa) in appropriately selected men receiving TRT short-to-intermediate term. A meta-analysis of seven RCTs found no significant difference in prostate cancer diagnosis between TRT and placebo groups [35]. Although some observational studies report more frequent prostatic events—such as prostate-specific antigen (PSA) elevation—these have not translated into increased PCa detection rates [45].

TRT is contraindicated in men with metastatic or high-risk localized PCa. However, cautious use may be considered in select individuals with previously treated, organ-confined disease and undetectable PSA [36].

● Post-Radical Prostatectomy: Retrospective studies suggest that TRT does not increase the risk of biochemical recurrence in low-risk patients with undetectable PSA after surgery [36].

● Post-Radiation Therapy: Similarly, TRT after radiation does not appear to increase recurrence risk, though transient PSA rises may occur [37].

● Active Surveillance: Emerging data from small series suggest TRT may be used without disease progression, but long-term safety remains uncertain [66].

TRT may cause a modest increase in PSA levels, which typically stabilizes within the first year. Professional guidelines vary in their specific monitoring recommendations:

● The American Urological Association (AUA) advises obtaining a baseline PSA level, repeating it at 3–6 months after initiating therapy, and continuing annual monitoring thereafter, particularly in men over 40 or those with additional risk factors [39].

● Other societies, such as the Society for Endocrinology, recommend that men with hypogonadism should follow prostate cancer screening guidelines equivalent to those of the general population, without additional PSA testing solely based testosterone therapy.

Beyond PSA, clinicians should assess individual risk factors for prostate cancer before and during therapy, including age, family history, and genetic background (e.g., Breast Cancer gene 2 (BRCA2) mutations). Current evidence does not support an increased risk of prostate cancer with testosterone therapy in appropriately screened men.

Interestingly, several studies suggest an inverse relationship between serum testosterone levels and prostate cancer aggressiveness. Low testosterone has been associated with higher-grade disease and adverse outcomes after prostatectomy [67].

The historical fear of testosterone promoting prostate cancer is increasingly being challenged by modern evidence. While TRT remains contraindicated in men with active or high-risk prostate cancer, data support its cautious use in selected individuals with treated, localized disease and stable PSA. Proper patient selection, risk stratification, and close PSA monitoring are essential to ensure safety.