Abstract
Testosterone acting via the androgen receptor, and via aromatisation to oestradiol, an activator of the oestrogen receptor, plays key roles in adipose tissue, bone and skeletal muscle biology. This is reflected in epidemiological studies associating obesity and disordered glucose metabolism with lower serum testosterone concentrations and an increased risk of type 2 diabetes (T2D) in men. Testosterone also modulates erythrocytosis and vascular endothelial and smooth muscle cell function, with potential impacts on haematocrit and the cardiovascular system. The Testosterone for the Prevention of Type 2 Diabetes (T4DM) study enrolled men aged 50 years and over with a waist circumference of 95 cm or over, impaired glucose tolerance or newly diagnosed T2D, and a serum testosterone concentration (as measured by chemiluminescence immunoassay) <14.0 nmol/L. The study reported that a 2-year treatment with testosterone undecanoate 1000 mg, administered 3-monthly intramuscularly, on the background of a lifestyle program, reduced the likelihood of T2D diagnosis by 40% compared to placebo. This effect was accompanied by a decrease in fasting serum glucose and associated with favourable changes in body composition, hand grip strength, bone mineral density and skeletal microarchitecture but not in HbA1c, a red blood cell-dependent measure of glycaemic control. There was no signal for cardiovascular adverse events. With the objective of informing translational science and future directions, this article discusses mechanistic studies underpinning the rationale for T4DM and translational implications of the key outcomes relating to glycaemia, and body composition, together with effects on erythrocytosis, cardiovascular risk and slow recovery of the hypothalamo–pituitary–testicular axis.
Introduction
Testosterone, obesity and male ageing
A decrease in serum testosterone concentration is not inevitable in ageing men (Sartorius et al. 2012, Shi et al. 2013, Handelsman et al. 2015, Marriott et al. 2022). The apparent decrease in testosterone seen in unselected middle aged and older men reflects obesity and the accumulation of chronic conditions (Shi et al. 2013), for example, cardiovascular disease (Regadera et al. 1985) and depression (Finkelstein et al. 2013, Shi et al. 2013), medication use (most commonly opioids), health-related behaviours (e.g. excessive alcohol consumption) and psychosocial factors (Wittert & Grossmann 2022) rather than chronological ageing in itself. However, beyond the age of 70 years, declining testosterone concentrations in unselected men with co-morbidities are accompanied by increases in LH, suggesting possibly impaired Leydig cell function (Yeap et al. 2018). The obesity phenotype associated with a decrease in serum testosterone concentration in middle to older aged men is associated with a reduction serum SHBG with mid-normal serum LH and FSH and is characterised by an accumulation of visceral adipose tissue associated with insulin resistance, aberrant glucose metabolism and dyslipidaemia as features of the metabolic syndrome (Umapathysivam et al. 2022). The proportionate fall in serum testosterone and sex hormone binding globulin (SHBG) concentrations under these circumstances is inherently reversible with weight loss (Grossmann 2018) and does not demonstrate androgen deficiency. Although weight loss will both increase serum testosterone and prevent type 2 diabetes (T2D), it is unclear whether this effect is mediated directly by the increase in testosterone or by beneficial effects of testosterone on body composition.
Mechanistic aspects of testosterone action
Testosterone is both a hormone, acting directly at the androgen receptor (AR), and a prohormone, undergoing 5-alpha reduction to DHT, a more potent AR analogue and being aromatised to oestradiol (E2), which acts via oestrogen receptors (ERs). These sex steroid receptors are ligand-dependent transcription factors, and AR and ERs, upon binding of their cognate hormone, are shuttled to the nucleus where they bind on specific DNA sequences (response elements), in gene promotor regions to regulate gene transcription in concert with transcriptional cofactors. In addition, there may be expression of cell membrane-bound sex steroid receptors, which may mediate rapid (non-classical) actions of testosterone and E2 (Handelsman 2000).
Testosterone effects on body composition: basic and translational considerations
There is a positive relationship between circulating testosterone concentration with muscle mass and strength and an inverse relationship with fat mass (Bhasin et al. 2001, Finkelstein et al. 2013). Testosterone treatment decreases fat mass and increases lean body mass (skeletal muscle and bone) and muscle strength in both healthy and hypogonadal men irrespective of age (Bhasin et al. 2001).
In skeletal muscle, testosterone, via the AR, induces hypertrophy of both type I and type II muscle fibres, increases muscle satellite cells, myogenic differentiation of muscle progenitor cells and muscle protein synthesis and inhibits adipogenic differentiation of pluripotent stem cells (Herbst & Bhasin 2004). Testosterone, following aromatisation to E2, increases growth hormone (GH) secretion from the pituitary (Eakman et al. 1996) and expression of insulin-like growth factor I (IGF-I) in skeletal muscle (Ferrando et al. 2002). However, the trophic effects of testosterone on skeletal muscle do not require GH and IGF-I (Serra et al. 2011).
In males, E2 is predominantly synthesised by aromatisation of testosterone in a tissue-specific manner (Russell et al. 2019). In females, but not males, E2 has a protective effect on functional skeletal muscle mass (McMillin et al. 2022, Pellegrino et al. 2022). The effect of testosterone to decrease fat mass is mediated at least in part by aromatisation to E2. Men or male mice with genetic inactivation of ER-alpha or aromatase have increased fat mass (Simpson et al. 2005) and, in healthy men, increasing serum testosterone by administration of aromatase inhibitors does not lead to a reduction in fat mass (Burnett-Bowie et al. 2009). Any beneficial effect of E2 on fat mass appears to require the presence of testosterone because in men who are androgen deficient, supplemental E2 leads to an increase in fat mass (Russell et al. 2022).
In males, E2 promotes insulin sensitivity and glucose metabolism in skeletal muscle via an E2 receptor alpha-mediated mechanism (Inada et al. 2016). E2 may also improve skeletal muscle glucose metabolism indirectly by increasing overnight pulsatile GH secretion from the pituitary while inhibiting the hepatic IGF-1 response to GH (Russell et al. 2019). In middle aged and older males, higher fat mass (particularly visceral far accumulation) increases the risk of T2D, an effect reversed with reduction in fat mass, and there is an inverse relationship between skeletal muscle mass and strength and risk of T2D (Atlantis et al. 2009, Li et al. 2016, Qadir et al. 2021).
Applying both epidemiological and mechanistic perspectives implicating testosterone in adipose and skeletal muscle biology, and diabetes risk, we designed a randomised controlled trial (RCT), ‘The Testosterone for the Prevention of Type 2 Diabetes (T4DM) study’. T4DM sought to determine whether treatment with testosterone, on the background of a lifestyle program, would prevent the progression of prediabetes to T2D or reverse newly diagnosed T2D in men aged 50 and over with visceral obesity as reflected by a waist circumference (WC) of 95 cm or greater and mild lowering of circulating testosterone but excluded those with pathological hypogonadism.
All men in T4DM were enrolled in a lifestyle program as this is the standard of care for T2D prevention and management of T2D. The lifestyle intervention delivered by WW (formerly Weight Watchers) was selected because it is standardised, widely accessible nationally and evidence based for the cost-effective prevention of T2D in men (Fuller et al. 2013).
Design of T4DM
The serum testosterone cut-off for inclusion
To reflect the typical at-risk population, we excluded men with pathological hypogonadism, where the benefit and safety of testosterone is widely accepted. Ultimately, an upper limit of serum testosterone concentration of 14 nmol/L, as measured by a CLIA was adopted as a screening compromise between treatment thresholds of serum testosterone advocated elsewhere with our observations that the association between serum testosterone concentration and T2D risk in men with obesity and the metabolic syndrome, persists up to about 16 nmol/L (Atlantis et al. 2016), implying that higher testosterone concentrations are associated with protection from T2D. After enrolment in the study, serum testosterone was measured at baseline by LCMS resulting in serum concentrations from 4 to 30 nmol/L, a finding confirming the unreliability of the testosterone immunoassays (Sikaris et al. 2005, Rosner et al. 2007). The absence of a relationship between baseline concentration of testosterone regardless of the method of measurement and the primary outcome of the study highlights the pharmacological effect of testosterone treatment in this study.
Waist circumference to assess obesity
The details of the design of the study are published (Wittert et al. 2019). WC rather than body mass index (BMI) was used as the anthropometric criterion for enrolment because an increase, reflecting abdominal obesity, is associated with T2D risk even if BMI is normal (Janiszewski et al. 2007). Abdominal obesity, where fat accumulates around the viscera and in the liver and infiltrates skeletal muscles mechanistically underpins the metabolic syndrome, which is characterised by insulin resistance, dyslipidaemia (typically raised triglycerides and reduced high-density lipoprotein concentrations), abnormal glucose metabolism and hypertension. The metabolic syndrome is associated with a reduction in serum testosterone, but normal LH and FSH concentrations most likely reflecting a reduction in SHBG (Brand et al. 2014). SHBG is synthesised in the liver and tightly coupled to de novo lipogenesis. When de novo lipogenesis increases, SHBG synthesis decreases (Selva et al. 2007). All testosterone assays measure total testosterone which predominantly reflects SHBG-bound testosterone, and therefore, if SHBG decreases, this will be reflected in a decrease in total testosterone concentration (Gleicher et al. 2020, Kim et al. 2020, Zhou et al. 2020). The extent to which, and under what circumstances, there are direct defects of excess adipose tissue on the regulation of the hypothalamic–pituitary–testicular (HPT) axis is not completely resolved, but there is some evidence for mediation by hypothalamic leptin resistance, central effects of inflammatory cytokines and inhibitory effects of increased oxidised low-density lipoprotein, adenosine and dysregulated iron metabolism on Leydig cell function in the testis (Umapathysivam et al. 2022).
Beyond the age of 60 years, skeletal muscle mass decreases and visceral lipid-filled adipose tissue increases. Unless there is a very substantial increase in fat mass, BMI tends to remain stable and may even decrease therefore not reflecting the increase in viscerally located adipose tissue. Furthermore, with advancing age, the risks (including T2D) associated with higher BMI flatten. WC which is simple to perform and reproducible when done correctly (Ross et al. 2020) provides an approximation of visceral fat and on average increases with increasing age; values 95 cm or above predict increased risk for T2D in middle-aged and older Caucasian men (Siren et al. 2012).
Outcome measures
The primary outcome of T4DM was based on an oral glucose tolerance test (OGTT). Although inherently variable, the OGTT has better sensitivity and specificity for diagnosing T2D compared with fasting glucose or with HbA1c, which at its standard cut-point has high specificity but low sensitivity (Jesudason et al. 2003). The OGTT was used to define the primary outcome in the diabetes prevention program (DPP) (Knowler et al. 2002) and remains generally accepted as the gold standard diagnostic test for T2D (Collaboration 2015). Secondary outcome measures relevant to this paper included fasting serum glucose, HbA1c, body weight, WC, body composition (fat mass, skeletal muscle mass and bone density) by dual-xray absorptiometry (DXA) and muscle strength by peak non-dominant handgrip. Haematocrit was measured and data relating to cardiovascular events collected at each clinic visit. Other outcome and safety measures have previously been described in detail (Wittert et al. 2019).
Glycaemic status: main findings and questions arising
Testosterone and prevention/remission of type 2 diabetes
The T4DM study achieved its recruitment target, enrolling 1007 men, across six centres in Australia. All were enrolled in the WW lifestyle programme and randomly allocated to be treated with either 3 monthly intramuscular TU or placebo for 2 years, making it the largest and longest RCT of testosterone treatment at the time it was done. Outcome data available for primary analysis at 2 years comprised of 443 and 413 testosterone and placebo-treated men, respectively. The primary and secondary outcomes are published (Wittert et al. 2021). Key findings and some implications relevant to translational science and future directions are detailed below.
In the testosterone compared to placebo-treated group, the proportion of participants with T2D at 2 years, based on a 2-h glucose ≤11.1mmol/L on the OGTT, was reduced by 40% (55/443, 12.4% vs 87/413, 21%). There was a 0.75 mmol/L greater decrease from baseline in the 2-h OGTT glucose concentration and a 0.17 mmol/L greater decrease in fasting glucose in the testosterone as compared to the placebo group.
Blood glucose normalised (2-h glucose <7.8 mmol/L) in 51.9% of testosterone and in 43.3% of placebo-treated men, respectively. While the difference is significant, the translational importance of over 40% of these high-risk men even if randomised to placebo treatment achieving normal glucose tolerance 2 years after the initiation of a relatively low-intensity lifestyle intervention, of proven cost-effectiveness, is a crucial finding.
Glucose response to oral challenge vs HbA1c: impact of red blood cell dynamics
In the T4DM study, the benefits of injectable testosterone undecanoate (TU) for glycaemia were shown in the 2-h GTT but were not accompanied by any change in HbA1C (Wittert et al. 2021). Previous small studies had previously reported little or no consistent benefit of TU treatment for HbA1C in studies of men without (Svartberg et al. 2008) or with T2D (Gianatti et al. 2014, Hackett et al. 2014, Ramachandran et al. 2020).
HbA1c is a function of the average blood glucose to which haemoglobin in the red blood cell (RBC) is exposed, and the lifespan of that RBC (Cohen et al. 2008). Improved glycaemia would be expected to produce a lower HbA1c so the unchanged HbA1c may reflect modest or minimal chronic glycaemic changes from testosterone treatment. However, the discordance between testosterone effects on acute (fasting glucose and 2-h OGTT) vs chronic (HbA1c) glycaemia may be due to testosterone’s erythropoietic effects influencing RBC lifespan; if testosterone increases RBC lifespan, this could increase HbA1c masking a glycaemic benefit of testosterone treatment. However, RBC lifespan does not differ significantly between males and females (Zhang et al. 2018) making changes in RBC lifespan due to ambient testosterone concentrations unlikely. Other possible explanations for the discrepancy is that testosterone treatment may abrogate the effects of endogenous inhibitors of deglycation such as fructosamine-3-kinase (Szwergold et al. 2001, Delpierre et al. 2002) or alternative deglycation mechanisms (Szwergold & Beisswenger 2003) operative in erythrocytes; however, their testosterone sensitivity has not been established. If testosterone inhibits deglycation enzymes, then HbA1c will increase. This area warrants further investigation.
Pharmacological not physiological effect of testosterone
For purposes of screening, and entry into the study, serum testosterone concentration was measured by platform chemiluminescent immunoassay (CLIA) (Wittert et al. 2019). Blood was subsequently sampled fasting in the morning prior to the first injection of TU or matched placebo and prior to each subsequent TU injection, serum testosterone concentration was measured by LC-MS/MS in batched samples at study end. Based on the CLIA data testosterone concentrations at screening were <8 nmol/L, 8–11 nmol/L and >11–14 nmol/L in approximately 20%, 40% and 40% of participants, respectively. By contrast, baseline analysis by LCMS showed that 67% of participants had serum testosterone concentrations >11 nmol/L and 41% >14 nmol/L. However, there were no significant relationships between serum testosterone concentration measured either by screening (CLIA) or baseline (LC-MS/MS) and glycaemic outcome variables. This suggests that pharmacological studies of testosterone effects on glycaemia in men without pathological hypogonadism should not employ baseline entry thresholds and should avoid the use of unreliable testosterone immunoassays.
Testosterone effects on body composition
Changes in fat and lean mass: translational considerations
In placebo-treated participants, fat mass, skeletal muscle mass and non-dominant hand grip strength decreased. In testosterone-treated participants, there was a greater decrease in fat mass and increase in skeletal muscle mass and non-dominant hand grip strength (Wittert et al. 2021). The magnitude of changes in lean and fat mass and hand grip strength are summarised in the Table 1 below.
Changes in fat mass, skeletal muscle mass and non-dominant hand grip strength after 2 years treatment with testosterone. Data from the T4DM Study (Wittert et al. 2021).
| Placebo | Testosterone | Between group difference | P | |
|---|---|---|---|---|
| Fat mass | −1.9 kg | −4.6 kg | 2.7 | <0.0001 |
| Skeletal muscle mass | −1.3 kg | +0.4 kg | 1.7 kg | <0.0001 |
| Non-dominant hand grip strength | −1.3 kg | +1.7 kg | 2.19 kg | <0.0001 |
Because fat mass is positively, and skeletal muscle mass (Qadir et al. 2021) and grip strength (Li et al. 2016) are inversely, associated with the risk of T2D, we have sought to determine the extent to which changes in total fat mass (kg), abdominal fat mass (%), lean mass (kg) and non-dominant handgrip (kg) mediate the effect of testosterone treatment on glycaemia. Preliminary data (Wittert & Robledo 2022) show that some, but not all, of the testosterone effect (when coupled with lifestyle changes) is mediated via a decrease in fat mass and to some extent also by the increase in E2 concentration in accordance with the known effects of E2 on glucose metabolism in skeletal muscle documented in section 1.3. The observation that there is limited effect mediation by skeletal muscle mass and strength does not exclude non-trophic mechanisms other than E2, for example, increased GH secretion (Eakman et al. 1996), adiponectin production and GLUT-4 expression and translocation (Inada et al. 2016, Antinozzi et al. 2017) that may mediate the beneficial effects of testosterone.
Lifestyle modification programs are effective to reduce weight in obesity when adherence is high, but even under optimal circumstances, engagement is variable and adherence wanes with time (Fabricatore et al. 2009). Because sex steroid receptors are expressed in the brain, a motivational benefit of testosterone to increase adherence to the lifestyle program was proposed at the outset as a hypothetical mechanism (Wittert et al. 2019). However, the T4DM study provided no support for this motivational hypothesis as engagement in the lifestyle program during the study was not sensitive to testosterone treatment, while both groups achieved 70% sufficient physical activity at 2 years. Further we observed no mental or physical quality of life benefits and no improvement in quality of sleep (Wittert et al. 2021), findings that are consistent with data from the smaller and shorter T trials (Snyder et al. 2016)
Bone mineral density and ultrastructure: mechanistic considerations
Ageing is causally associated with decreasing bone mineral density (BMD), a process accelerated by diet-induced weight loss.
In men, testosterone both acting directly via the AR, as well as via its conversion by aromatisation to E2 to act on the ER, is required for the acquisition of peak bone mass attained at the completion of growth, as well as for the maintenance of bone mass during adulthood. Men with untreated congenital hypogonadism (e.g. congenital hypogonadotropic hypogonadism, or those with Klinefelter Syndrome) fail to achieve peak bone mass during puberty, and men with acquired pathological hypogonadism experience accelerated bone loss during adulthood (Vanderschueren et al. 2014). Small and ethically constrained, uncontrolled studies report that testosterone treatment of men with pathological hypogonadism reduces bone turnover markers and increases bone density (Katznelson et al. 1996, Snyder et al. 2000). The role of sex steroids in skeletal health in unselected older men without pathological hypogonadism is less clear. Population-based studies report that circulating sex steroids are inversely associated with accelerated bone loss, both if assessed by conventional DXA measuring areal bone mineral density (aBMD) and by high resolution–-peripheral quantitative computed tomography (HR-pqCT) measuring volumetric BMD (vBMD) and parameters of bone microarchitecture (David et al. 2022). Moreover, circulating sex steroids are inversely associated with fracture risk (Vanderschueren et al. 2014, David et al. 2022). However, such observational studies cannot ascribe causality.
Testosterone effects on bone structure and strength
The effects of testosterone treatment on skeletal outcomes in men without pathological hypogonadism have only come into focus more recently. An early meta-analysis of RCTs enrolling 264 men aged 60–76 years with a baseline serum testosterone of 10–15 nmol/L receiving transdermal or intramuscular testosterone (short acting esters) reported that testosterone treatment, compared to placebo, increased aBMD at the lumbar spine by 3.7%, without significant effect on the femur (Isidori et al. 2005). A more recent large placebo-controlled study of men aged 60 years or older (n = 211) with a baseline testosterone of <9.54 nmol/L showed that daily transdermal testosterone treatment over one year increased vBMD (by quantitative CT (QCT)) predominantly at the trabecular spine (+6.8%) with lesser effects at peripheral spine (+2.9%) (Snyder et al. 2017). Studies using higher resolution HR-pqQCT (82 µm vs ~500 µm with standard QCT) allow assessment cortical and trabecular bone microarchitecture which predicts fracture risk independent of aBMD and the FRAX score, a validated aBMD-independent predictor of fracture risk incorporating clinical risk factors for fracture (Samelson et al. 2019). However, RCTs assessing the effects of testosterone treatment on bone microarchitecture defined by HR-pqQCT in men were lacking. Therefore, we conducted testosterone for bone (T4Bone), a planned sub-study of testosterone for diabetes mellitus (T4DM). In T4Bone over 24 months, testosterone treatment, compared to placebo, increased total vBMD both at the tibia and radius (Fui et al. 2021). vBMD increased predominantly in cortical bone at both sites with potent treatment effect sizes ranging from 2.9% to 3.1%. However, effects on trabecular microarchitecture were minor. Testosterone also significantly increased aBMD at the lumbar spine (+3.3%) and the total hip (+1.9%) (Fui et al. 2021). The predominant effects of testosterone treatment on cortical bone were consistent with observational HR-pqQCT studies in older men with lower sex steroid concentrations displaying deficits predominantly in cortical, rather than trabecular bone (Argoud et al. 2014). Moreover, men commencing androgen deprivation therapy for prostate cancer, which leads to severe sex steroid deficiency experience predominant loss of cortical, rather than trabecular bone (Hamilton et al. 2010).
Testosterone effects on cortical vs trabecular bone
None of the RCTs evaluating the effects of testosterone treatment in older men has been powered for fracture outcomes. Of note, however, the findings of T4DM that testosterone induced increases in cortical bone, which comprises 80% of the human skeleton, suggests that it might. Preclinical studies report that the loss of cortical bone compromises bone strength to a greater extent than the loss of trabecular bone (Vanderschueren et al. 2014), and in observational HR-pqQCT studies in men, deficits in cortical parameters were more consistently associated with fractures than trabecular parameters (Szulc et al. 2011, Fink et al. 2018). Moreover, the effect sizes, ranging from 1.3% to 3.1% for total and cortical vBMD in T4DM compare favourably with those reported for conventional osteoporotic drug treatments. In postmenopausal women with low BMD, antiresorptive drug therapy (using bisphosphonates or denosumab which have proven anti-fracture efficacy) over 12–24 months produced effect sizes on HR-pqQCT indices ranging from 0.3% to 3.8% (Burghardt et al. 2010). Of note men participating T4Bone had a mean baseline serum testosterone of 13.6 nmol/L; there was no evident relationship of baseline serum testosterone or E2 concentrations and bone microarchitecture.
Mechanistic aspects of testosterone action on bone
Testosterone treatment in T4DM was pharmacological and not designed to explore the mechanisms by which testosterone treatment increased vBMD. It remains unclear first how much testosterone effect is direct i.e. mediated by sex steroid receptors in bone as opposed to effects other tissues such as muscle or fat, and secondly, how much of the testosterone effect is mediated via the AR rather than via aromatisation to E2 and the ER.
Regarding the first question, both AR and ER are expressed in bone cells, and targeted deletions of both the AR and the ERs in bone cells (osteoclasts, osteoblasts and osteocytes) have reported skeletal deficits in mice (Vanderschueren et al. 2014) and congruent findings are reported in men with comparable genetic inactivation. Therefore, the actions of sex steroid on skeletal health are likely to be direct, supported by observations in T4Bone that testosterone treatment reduced bone remodelling. However, in addition, testosterone also increases muscle mass (Wittert et al. 2021), and it is possible that increased muscle mass, although modest, promotes skeletal health either by exerting loading on the skeleton and/or by secretion of bone-anabolic myokines. In addition, increased muscle mass may reduce falls risk, a common proximal cause of fragility fractures. Moreover, in T4DM, testosterone reduced fat mass and WC and fat mass (especially visceral fat), all of which are negatively associated with bone mass (Gilsanz et al. 2009). The mechanisms remain unclear but may be via secretion of osteo-catabolic adipo-cytokines and/or associated insulin resistance (Vanderschueren et al. 2014). These metabolically favourable changes in body composition are, at least in part, responsible for the reduced T2D risk observed in T4DM. Insulin resistance and T2D have been associated with increased fracture risk, although precise mechanisms are unclear (Khosla et al. 2021). Therefore, it is possible that testosterone treatment may improve skeletal health, at least in part indirectly via effects on muscle and fat including possibly glucose metabolism. However, future mechanistic and clinical studies are necessary to test these hypotheses.
Androgens promote periosteal apposition to increase bone size in men, explaining why men generally have wider long bones than women. However, regarding the second question, in older men, some observational studies suggest that skeletal outcomes (reduced bone density, impaired microarchitecture and increased fracture risk) are more closely related to reductions in circulating E2, rather than in testosterone (Russell & Grossmann 2019). Experimental studies among men receiving GnRH analogue therapy to suppress gonadal steroids with testosterone addback with or without an aromatase inhibitor (to suppress aromatisation of the administered testosterone to E2) have suggested indirectly that E2 is important for maintaining bone architecture (Finkelstein et al. 2016). However, these studies have inferred E2 effects by its drug-induced absence, rather that assessing its effect directly. To directly assess the effects of E2 on skeletal health in men, a more recent RCT enrolled older men receiving androgen deprivation therapy for prostate cancer, which reduces both circulating testosterone and E2 to castrate levels (Russell et al. 2022). In this RCT, E2 treatment, compared to placebo improved bone microarchitecture and bone density, thus providing direct evidence that E2 can improve skeletal health in men in the absence of testosterone (Russell et al. 2022). From a clinical perspective, this supports the role of testosterone treatment with its full spectrum of androgen actions (rather than non-aromatisable DHT or synthetic androgens) as the prime mode of testosterone replacement in men with organic hypogonadism. Of note, the findings from T4Bone do not endorse testosterone treatment to improve skeletal health in men without pathological hypogonadism. This is because whether testosterone treatment improves patient important health outcomes (such as fractures) remains unknown, and the long-term risks of testosterone treatment are insufficiently explored.
Questions about safety: erythrocytosis and cardiovascular effects
Erythrocytosis: mechanistic considerations
Testosterone has a direct and dose-related effect on bone marrow to increase erythropoiesis (Coviello et al. 2008). Mechanisms of testosterone-induced erythropoiesis include iron mobilisation and incorporation into RBCs, increased haemoglobin synthesis and RBC production (Bachman et al. 2014, Hennigar et al. 2020). Increased iron flux is mediated by changes in erythropoietin (EPO), hepcidin and ferroportin (Roth et al. 2019). The primary stimulus to renal production of the cytokine, EPO, is hypoxia (Bachman et al. 2014). Hepcidin inhibits absorption of iron in the gut by binding to the iron transporter ferroportin and resulting in its degradation thus preventing iron transport across the basolateral membrane of the enterocytes (Atanasiu et al. 2007). Testosterone induces a sustained suppression of hepcidin (Guo et al. 2013).
Murine data suggest that EPO upregulation and hepcidin suppression operate both jointly and independently (Guo et al. 2013, Guo et al. 2020). Although one clinical trial observed no significant increase in serum EPO during testosterone treatment (Maggio et al. 2013), testosterone produces a decrease in the threshold for EPO release (Bachman et al. 2014).
E2 increases haemopoietic stem cell proliferation and survival in vitro (Calado et al. 2009) and high endogenous E2 following IVF hyperstimulation is reported to inhibit hepcidin in women (Lehtihet et al. 2016). However, testosterone alone is sufficient to maintain red cell mass and to increase net erythropoiesis with supra physiological doses (Rochira et al. 2009).
Erythrocytosis: as an adverse event in clinical trials
Erythrocytosis (haematocrit >0.54) is one of the most common adverse effects in clinical trials of testosterone therapy. Advancing age is associated with a higher risk that cannot be explained by differential effects on EPO or soluble transferrin receptor (Coviello 2008). Erythrocytosis was a particularly prominent adverse event in T4DM and occurred in 22% (106) of men treated with testosterone but only 1% (6) of those on placebo. Once the effect of dehydration was eliminated by a second non-fasting test, only 23 men were withdrawn from treatment because of the finding of elevated haematocrit on two occasions. Nevertheless this rate was substantially higher than generally reported (Warren & Grossmann 2022) possibly because we did not exclude men with obstructive sleep apnoea (OSA). Severe OSA leads to a modest increase in haematocrit (Rha et al. 2022) and the best characterised risk factor for androgen-induced erythrocytosis is elevated baseline haemoglobin/haematocrit (Idan et al. 2010) or trough serum testosterone in testosterone-treated men (Ip et al. 2010). The prevalence of moderate or severe OSA in a randomly selected cohort of community-dwelling men aged 40 years and over is approximately 25%, with half having severe OSA (Adams et al. 2016). The prevalence is higher with increasing age and obesity and is over 50% in the presence of T2D (Andayeshgar et al. 2022). At enrolment, 14% of men were using CPAP and this remained relatively constant for the study duration (unpublished data). Therefore, it is possible that the relatively high prevalence of increased erythrocytosis in T4DM is attributable to the combined effects of testosterone and overnight hypoxia.
Another intriguing possibility is an interaction between testosterone and somatic mutations, which occur with increasing frequency over the ageing process, in genes regulating erythrocytosis. For example, somatic mutations of the Janus kinase 2 (JAK2) gene and SH2B adaptor protein 3 (SH2B3) gene cause most cases of primary-acquired erythrocytosis (Kristan et al. 2021). Somatic mutations indicative of clonal haematopoiesis in individuals with primary erythrocytosis have also been found in two homologous genes BCOR and BCORL1, which play key roles in haematopoiesis (Wouters et al. 2020). Moreover, erythrocytosis with clonal haematopoiesis has an independent association with cardiovascular morbidity and mortality (Wouters et al. 2020). This is an area of active investigation we are currently pursuing.
Implications of a high haematocrit
The relationship between high haematocrit (49–70%) and cardiovascular risk remains unresolved, with some suggesting an increase in risk (Gagnon et al. 1994, Kunnas et al. 2009) and others not (Brown et al. 2001, Puddu et al. 2002). The data from these studies cannot necessarily be used to infer the risk of an increase in haematocrit induced by treatment with testosterone. In a recent study, 5842 men who developed polycythaemia (haematocrit ≥52%) after commencing treatment with testosterone had a higher risk of major adverse cardiovascular events/venous thromboembolism (MACE/VTE) (number of outcomes: 301, 5.15%) in 1 year than 5842 matched men who did not develop polycythaemia (number of outcomes 226, 3.87%) (Ory et al. 2022).
In men treated with testosterone, aggressive management of cardiometabolic co-morbidities and risk factors including OSA decreases the risk of erythrocytosis and improves overall symptoms (Farber et al. 2020).
Cardiovascular effects: mechanistic studies
There is ongoing debate over the cardiovascular effects of testosterone. Mechanistic studies have been conducted using in vitro and animal models of atherogenesis. In an experimental study using aortic ring segments cultured in vitro after endothelial denudation, testosterone treatment inhibited neointimal plaque development, and effect associated with increased expression of androgen receptor mRNA in treated segments (Hanke et al. 2001). This concept was supported by an in vivo study in male miniature pigs, where neointima formation in response to induced coronary artery injury was increased in castrated animals compared to intact controls, and effect abrogated by testosterone treatment (Tharp et al. 2009). Testosterone treatment induces apoptosis in vascular smooth muscle cells, another mechanism by which atherosclerotic plaque growth or stability may be affected (Lopes et al. 2014). Of note, testosterone-augmented angiogenesis, a process central to cardiovascular repair/regeneration, in male but not female endothelial cells (Sieveking et al. 2010). Androgen receptor gene knockdown abrogated these effects in male endothelial cells, whereas androgen receptor overexpression in female endothelial cells conferred androgen responsiveness for angiogenesis. Testosterone treatment also increased cholesterol efflux in a monocyte-macrophage cell line (Kilby et al. 2021). Thus, these experimental data suggest an effect of testosterone to modulate mechanisms relevant to atherogenesis.
In castrated male rabbits fed a cholesterol-rich diet, aortic atherosclerosis was highest in placebo-treated rabbits and lowest in testosterone-treated rabbits, as well as in untreated, sham-operated controls (Alexandersen et al. 1999). In cholesterol-fed ovariectomised female and castrated male rabbits, oestrogen in female rabbits and testosterone in male rabbits reduced aortic plaque formation (Bruck et al. 1997, Alexandersen et al. 1999). Similar results were reported in male miniature pigs fed a high-fat and high-cholesterol diet where castration resulted in increased atherosclerotic plaque and intima-media thickness (Deng et al. 2021). In castrated atherosclerosis-prone mice, the anti-atherosclerosis of the effect of testosterone treatment was dependent of aromatisation to E2 (Nathan et al. 2001). Testosterone treatment reduced atherosclerotic lesion area to a greater extent in castrated wild type compared to AR knockout mice (Bourghardt et al. 2010). Testosterone treatment also attenuated fatty streak formation in mice with a non-functioning androgen receptor, suggesting effects independent of the classical androgen receptor (Nettleship et al. 2007). Taken together, these studies are consistent with an effect of testosterone, either directly or via aromatisation to E2, to regulate atherosclerotic plaque formation. However, testosterone treatment increased arterial calcification in apolipoprotein E knockout mice, with a lesser effect of the non-aromatisable androgen dihydrotestosterone (McRobb et al. 2009). Reconciling these experimental findings with the earlier onset and greater severity of atherosclerosis in men than in women remains an ongoing challenge.
Cardiovascular effects: clinical studies
The associations of endogenous testosterone concentrations with the incidence of cardiovascular events in prospective cohort studies and the risk of cardiovascular adverse events in interventional trials of testosterone therapy have been previously reviewed (seeYeap et al. 2022). Several recent studies of note are highlighted as follows. In a large prospective observational cohort study of 210,700 men aged 40–69 years with 8790 cardiovascular events occurring over 9 years follow-up, baseline testosterone concentrations were not associated with the incidence of myocardial infarction, stroke, heart failure or major cardiovascular adverse events (Yeap et al. 2022). An association of lower testosterone concentrations with higher risk of stroke, but not risk of myocardial infarction, has been reported in men aged ≥70 years (Yeap et al. 2014). In a clinical study of overweight men, testosterone treatment did not improve vascular function assessed using flow-mediated dilation nor did testosterone add to the benefits of exercise on vascular health (Chasland et al. 2021). A sub-study from the Testosterone Trials (T-Trials) of 138 men reported an increase in coronary atheroma measured as non-calcified plaque volume using coronary computed tomography angiography in testosterone-treated men over 12 months, insufficient duration to gauge cardiovascular consequences (Budoff et al. 2017). The authors concluded that larger and longer studies were needed to understand the clinical implications of this finding.
An RCT of 6 months transdermal testosterone or placebo gel treatment to increase lower limb strength and physical function in 209 men aged ≥65 years with mobility limitations was stopped prematurely due to excess adverse cardiovascular events in testosterone-treated men (Basaria et al. 2010). However, another trial in a similar population of intermediate-frail or frail men aged ≥65 years randomised 274 men to testosterone gel or placebo treatment for 6 months, finding an improvement in lean mass, knee extension strength and physical function, with no signal for adverse cardiovascular events (Srinivas-Shankar et al. 2010). A registry study of older men treated with testosterone for up to 10 years reported improvements in cardiometabolic risk factors; however, the limitations of such uncontrolled studies need to be considered (Yassin et al. 2016).
Cardiovascular adverse events in T4DM
In T4DM, cardiovascular adverse events were comparably distributed between testosterone and placebo-treated men; 13/503 men in the placebo arm, and 7/504 in the testosterone arm experienced an ischemic heart disease adverse event over the two-year trial duration. When major adverse cardiovascular events (ischemic heart disease, cerebrovascular disease and cardiovascular deaths) were considered, there were 17/503 events in the placebo arm and 12/504 in the testosterone arm (Wittert et al. 2021). This is in keeping with the T-Trials where 788 men were randomly allocated testosterone or placebo for a year, and 7 men in each arm of the study died from cardiovascular causes or had a myocardial infarction or stroke (Snyder et al. 2016). However, none of these trials was long enough to definitively assess adverse cardiovascular events. A recent systematic review and meta-analysis of RCTs including individual patient data, mostly of 12-month duration found no evidence of excess cardiovascular or cerebrovascular adverse events in testosterone-treated men (Hudson et al. 2022). These and the 2-year T4DM data provide some assurance regarding short-term risk; however, much longer-term follow-up over decades is required for a full safety evaluation. Additional information will be forthcoming when the ‘Testosterone Replacement therapy for Assessment of long-term Vascular Events and efficacy ResponSE in hypogonadal men’ (TRAVERSE) study, a Federal Drug Agency-mandated 5-year cardiovascular safety trial of testosterone in middle- to older-aged men with multiple risk factors or pre-existing cardiovascular disease is completed (Bhasin et al. 2022).
Recovery of the hypothalamo–pituitary–testicular axis
Another potentially detrimental outcome arising from prolonged injectable testosterone treatment even at standard testosterone replacement doses is that it creates sustained suppression of the HPT axis. The slow but eventually complete recovery takes up to 12 months after cessation of testosterone treatment (Handelsman et al. 2022) is comparable with the time course of recovery from androgen abuse-induced HPT axis suppression (Shankara-Narayana et al. 2020). The congruence of these findings suggests that the recovery from androgen-induced HPT axis suppression is primarily related to duration since ceasing androgen intake rather than the doses or patterns of androgens used. Prolonged, slow recovery of the HPT axis can cause symptomatic androgen deficiency withdrawal symptoms during recovery and potential androgen dependence. These pose a practical limitation on the wider application of such injectable testosterone treatment, especially in men without pathologic hypogonadism. Whether shorter treatment duration might facilitate faster HPT axis recovery remains an interesting question; however, the minimal duration of injectable testosterone treatment to maintain similar improvements in acute (if not chronic) glycaemia shown in T4DM remains unknown, and careful investigation would be warranted to determine the benefits and risks of shorter duration of testosterone treatment on iatrogenic HPT axis suppression and recovery. The intermediary molecular mechanism of the HPT axis suppression by exogenous testosterone, and whether it can be modified independent of systemic androgen effects, warrants further investigation.
Conclusions
The primary finding was that over and above the effects of a lifestyle intervention, men treated with testosterone had a 40% lower risk of T2D diagnosis (by OGTT) after 2 years relative to placebo-treated men. Further, at 2 years, 40% of men in the placebo group and 50% of men in the testosterone group normalised glycaemia. Yet HbA1c was not reduced so the magnitude of the chronic glycaemic changes requires further long-term evaluation before wider application of pharmacological testosterone treatment to prevent T2DM is justified.
Testosterone treatment appears to have primarily mediated its effect by inducing metabolically favourable changes in body composition. There were no motivational or quality of life benefits from testosterone treatment.
T4DM illustrates how concepts gained from basic and mechanistic studies are translatable via the platform of a placebo-controlled RCT leading to new information and ongoing studies to answer questions generated. These include the importance of central adiposity, and the role of testosterone and E2 in regulation of adipose and skeletal muscle biology. Similarly, the observed improvements in skeletal microarchitecture and BMD are grounded in the role of androgen and OR in bone biology. Whether the benefits on bone are durable and clinically important require further investigation.
While mechanistic studies have explored the potential role of testosterone in mechanisms relevant to atherogenesis, in T4DM there was no signal of either cardiovascular risk or benefit. The risk of an unacceptably high haematocrit may be preventable by screening for and treating sleep apnoea, but other mechanisms remain to be investigated. Suppression of the endogenous HPT axis may last for up to 12 months, and therefore ill-conceived or uncontrolled trials of testosterone treatment may do more harm than good.
From a translational perspective, building on the results of T4DM, data from trials that are adequately powered to examine durability of treatment effect and offset, prevention of diabetes-related complications, fragility fractures, cardiovascular events and mortality would be important. Further studies are also needed to better define mechanisms and magnitude of testosterone effects for glycaemic and skeletal benefit.
The most important, and readily translatable, outcome of T4DM is that, in line with prior observations, T2D is preventable. Lifestyle changes are a logical first step. The health of men is likely to benefit most not from the from the measurement of serum testosterone and prescription of testosterone treatment but from careful risk assessment and an overall holistic approach to care.
Declaration of interest
GW has received research funding from Lawley Pharmaceuticals, Bayer, Weight Watchers and Lilly, speaker’s honoraria from Bayer Pharma and Besins Healthcare, and consulting fees from Bayer Pharma. MG has received research funding from Bayer Pharma, Novartis, Weight Watchers, Otsuka and Lilly and speaker’s honoraria from Bayer Pharma and Besins Healthcare. BBY has received speaker honoraria and conference support from Bayer, Lilly and Besins, held advisory roles for Lilly, Besins, Ferring and Lawley Pharmaceuticals, and received research support from Bayer, Lilly and Lawley Pharmaceuticals. DJH has received institutional funding for investigator-initiated pharmacology research from Lawley Pharmaceuticals and Besins Healthcare and has served as an expert witness in professional standards and antidoping tribunals and testosterone litigation.
Funding
NHMRC Project grant 1030123, Bayer, WW, Eli Lilly.
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