Late‐onset hypogonadism: Reductio ad absurdum of the cardiovascular risk‐benefit of testosterone replacement therapy

Low testosterone (T) level is considered a marker of poor cardiovascular health. Ten years ago, the Testosterone in Older Men with Mobility Limitations (TOM) trial was discontinued due to a higher number of adverse events in men receiving T compared with placebo. Since then, several studies have investigated the risks of T replacement therapy (TRT) in late‐onset hypogonadism (LOH).


| INTRODUC TI ON
It is well-known that both total and bioavailable testosterone (T) levels slightly and progressively decrease with aging in men, independently from other confounding factors such as obesity, illness, medications, cigarette smoking, or alcohol intake. 1,2 The rate of decline has been estimated to be 0.8%-2% per year after 40 years of age. 3 Indeed, according to the main observational studies, 12%-38.7% of men aged ≥ 40 years show T deficiency, 1,4-6 with the incidence progressively increasing from 20% in men over 60 years to 50% in those over 80 years. 1 Furthermore, a proportion of these subjects develop symptoms related to low T levels, such as poor morning erection, low libido, erectile dysfunction, inability to perform vigorous activities, depression, and fatigue. 7 In this population, the presence of at least three sexual symptoms associated with low T levels leads to the diagnosis of late-onset hypogonadism (LOH), 7 with a prevalence of 2.1%-6%. 4,6,7 Older men with LOH can be differentiated based on hormone measurements, clinical features, and predisposing risk factors into the following functional categories: primary hypogonadism, which is strongly associated with age, and secondary hypogonadism, which is related to obesity and is potentially reversible. 8 A third category is represented by compensated or subclinical hypogonadism, which should be considered as a specific clinical entity. 8,9 These findings imply that age-related decline in T, mainly caused by testicular failure, 10,11 is not the only factor underlying the pathogenesis of LOH, which also involves hypothalamic-pituitary function impairment. 12 Subsequently, LOH can be defined as mixed or combined hypogonadism. 13,14 In this regard, comorbidities and lifestyle factors play a major role both independently and in combination with aging. 2,15 In particular, metabolic syndrome and type 2 diabetes dramatically contribute to hypothalamic-pituitary-testicular axis suppression and can in turn be promoted by low T levels themselves in a bidirectional relationship.
Moreover, metabolic syndrome and type 2 diabetes are wellknown risk factors of cardiovascular (CV) disease. 17,18 They may increase the risk of premature death in these patients. However, it is now understood that LOH beyond the worst metabolic profile is associated with CV morbidity and mortality per se. [19][20][21] The unfavorable metabolic profile of patients with LOH could be restored by improvements in lifestyle 22 as well as by T replacement therapy (TRT), which both contributes to a decrease in abdominal fat, total cholesterol and triglyceride levels, as well as fasting glycemia and insulin resistance. 23,24 Therefore, the metabolic-related CV risk of these subjects might be reduced. 19,25 Moreover, some studies have demonstrated a protective effect of TRT against CV events and mortality in older men with T deficiency at high CV risk. [26][27][28] Nonetheless, the safety of TRT in LOH is still controversial: Some studies on CV outcomes have shown an increase in CV adverse events. [29][30][31] The Testosterone in Older Men with Mobility Limitations (TOM) trial evaluated the safety and efficacy of TRT in men aged ≥ 65 years with T deficiency and mobility limitation. 29 However, this study was prematurely discontinued due to a higher rate of CV events in the T group than in the placebo group. The results of this trial sparked the debate of the CV safety of TRT as it had several flaws. First, since the primary outcome of the trial was the change in maximal voluntary muscle strength from baseline until after high-dose TRT, the study was not sufficiently powered to evaluate the CV safety of TRT. Second, the study population comprised elderly men with a mean age of 74 and with significantly limited mobility; in addition, the majority of subjects suffered from several chronic illnesses, including preexisting heart disease. Third, ethnicities as well as the baseline characteristics were different between the two groups, with a higher rate of hypertension, dyslipidemia, and statin use in the T group than in the placebo group. Finally, some of the reported adverse CV events should have been considered as minor phenomena, which could have been dependent on the baseline characteristics of the population. The results of two subsequent observational studies 30,31 resulted in the Food and Drug Administration (FDA) restricting the indications for TRT, warning against the possible risk of myocardial infarction and strokes. 32 In line with this view, the current Endocrine Society guidelines suggest individualized TRT only in men aged 65 years or older with symptomatic T deficiency after providing a detailed explanation regarding the potential risks and benefits of TRT. 14 More recently, the European Academy of Andrology (EAA) guidelines for men with functional hypogonadism have underlined the current lack of conclusive long-term data on the CV risk of TRT, 33 following European Medicines Agency position, 34 and suggest TRT for hypogonadal symptomatic men with sexual or erectile dysfunction. 33 Assuming that T is harmful to the CV system, this review aimed to examine the putative detrimental mechanisms that could be involved in the increased CV risk of patients with LOH undergoing TRT. To this end, all putative mechanisms by which TRT may cause adverse CV events ( Figure 1) were analyzed by first presenting the clinical evidence and by then examining the molecular mechanism pathways targeted by androgens.

| ME THODS
A Medline search for articles published in English from inception until April 22, 2020, was performed using the following keywords: "late-onset hypogonadism," "hypogonadism," "testosterone," initial hypothesis. Current evidence indicates that TRT is safe once other comorbidities are addressed.

| Coronary artery vasomotion
The first evidence of the beneficial effects of T for the treatment of angina pectoris dates back to the 1940s. 35 More recent research has shown that T administration has a beneficial effect on exercise-induced myocardial ischemia in men with coronary artery disease. [36][37][38][39][40][41] The anti-ischemic effect is achieved both in acute 36,37 and chronic administration. [38][39][40][41] The response to T is greater in patients with lower serum T levels than in those with higher serum T levels, [37][38][39] implying that coronary arteries are more sensitive to T in men with a more severe degree of T deficiency. However, the beneficial effect of T on exercise-induced myocardial ischemia has also been demonstrated in men with normal plasma T concentrations. 37 The beneficial effect of T on myocardial ischemia persists for at least 12 months and is maintained without tachyphylaxis as long as the treatment is continued. 40 Evidence suggests that the increased angina threshold after T administration in men with coronary artery disease is caused by vasodilation of coronary arteries. The vasodilatory effects of T on coronary arteries have been investigated in both preclinical [42][43][44] and clinical studies. 45 Intracoronary administration of the physiological concentrations of T in eugonadal men with coronary artery disease induces a prompt (within 2-3 minutes) artery dilation up to 4.5% and increases coronary blood flow up to 17.4% following acetylcholine-induced contraction. 45 In summary, clinical studies have shown the favorable effects of short-or long-term T treatment on exercise-induced cardiac ischemia and coronary vasomotion. However, the exact mechanism of action by which T exerts its effects on coronary vasculature remains unknown.

| Peripheral arterial stiffness
Existing evidence has recognized increased arterial stiffness, defined as an increased resistance to deformation or decreased elastic compliance, as a predictor of CV events and mortality. 59 Stiffening of the arterial tree increases cardiac afterload and lowers diastolic pressure, thereby altering the transmural distribution of myocardial blood flow. This results in a decrease in subendocardial perfusion during ventricular contraction, eliciting subendocardial ischemia, regardless of the presence of coronary artery stenosis. 60 Several studies have demonstrated an independent inverse association between T levels and arterial stiffness. [61][62][63][64][65][66] However, this has been confirmed in specific populations, such as older hypogonadal men 63,64 and adults without CV disease. 65,66 The effect of T deficiency on arterial stiffness appears to be more prominent in adult men (<60 years) and in subjects with higher blood pressure (mean pressure ≥ 102 mmHg). 65 T exerts a long-term influence on the arteries, as indicated by the findings of a longitudinal study that demonstrated that low serum T levels are an independent predictor of the arterial stiffness index. 62 Moreover, there is evidence that acute or chronic T treatment has a favorable effect on arterial stiffness in older men with T deficiency and coronary disease. 63,67 TA B L E 1 Summary of the effects of testosterone on vessel and endothelial function: small, medium, large vessels

Peripheral arterial stiffness
Growth arrest-specific gene 6 (GAS6) 70 Activation Reduced apoptosis and calcification of vascular smooth muscle cells (VSMCs) Growth arrest-specific gene 6 (GAS6) 71 Activation with reduced expression and activity of matrix metalloproteinase-2 and collagen synthesis induced by angiotensin II

Reduced endothelial inflammatory response
Reactive oxygen species (ROS) -Vascular smooth muscle cells (VSMCs) 92,93 Induction of ROS production from VSMCs Promotion of ROS induced VSMCs migration and apoptosis Prostate overexpressed protein 1 (PTOV1) 94 Increased expression Induction of VSMCs proliferation Note: Coronary arteries vasomotion: Testosterone exerts a vasodilating action through a rapid, non-genomic, and endothelial-independent action on K and Ca channels. Moreover, it has also an endothelial-dependent vasodilating effect mediated by NO. Peripheral arterial stiffness: Testosterone reduces arterial stiffness through an androgen receptor-dependent modulation of apoptosis and senescence of vascular smooth muscle cells.
Testosterone vasodilating action and anti-inflammatory effect may also have a role. Large vessels atherosclerosis: Testosterone exerts several anti-atherogenic actions, including an anti-inflammatory effect which may hinder the initial development of atheroma. The effect on VSMSs are complex and may be reliant on the stage of plaque development. VSMCs migration and proliferation could be considered protective against plaque destabilization, while VSMCs apoptosis could be responsible of plaque vulnerability.
The mechanisms by which T affects arterial stiffness are poorly understood. Several mechanisms have been proposed, including the vasodilating action of T 47,65 as well as the modulating action of T on the apoptosis and proliferation of vascular smooth muscle cells 47,65 and its anti-inflammatory effects. 47

| Large vessel atherosclerosis
Several studies have demonstrated that low T levels are independently associated with carotid intimal-media thickness (CIMT), 72-75 a surrogate marker of atherosclerosis that predicts CV events. 76,77 Lower levels of T are also associated with accelerated progression of CIMT. 73 The effects of TRT have been evaluated in a randomized placebo-controlled study. This study showed that T treatment (T undecanoate, 1000 mg every 12 weeks) resulted in a significant reduction in CIMT in men with metabolic syndrome and LOH, which was found to be related to the increase in plasma T levels from baseline. 78 More recently, the long-term effects of T administration (T gel 1%; starting dose, 7.5 g daily) on CIMT or the coronary artery calcium score have been studied in older men with low or low-normal T levels. The results of this randomized placebo-controlled trial showed no significant difference in the rate of change of either CIMT or the coronary artery calcium score. A possible confounding factor that might explain these negative results may be the use of statins. Indeed, in exploratory analyses that have only included statin non-users, the annual rate of change of coronary artery calcium score was lower in the T group than in the placebo group; on the other hand, in statin users, no differences were observed between the two groups. 79 In another trial, the effect of T on coronary artery plaque volume was studied in symptomatic hypogonadal men aged > 65 years.
Compared with placebo, T treatment (T gel 1%; starting dose, 5 g daily) was associated with a significant increase in non-calcified plaque volume and total plaque volume without any significant change in coronary calcium score. Moreover, an exploratory analysis showed that T therapy significantly increased fibrous plaque volume compared with placebo. 80 Therefore, T could promote a more stable plaque with a lower risk of rupture. 81 Clearly, metabolic impairment is a major modulator of CV risk, particularly of atherosclerotic risk, in males with hypogonadism. A detailed review of the changes in metabolic status under T replacement has been recently published. 16 The exact mechanism by which T mediates its effects on atherogenesis remains unclear; however, several pathways have been explored. In mouse models, T replacement was shown to inhibit fatty streak formation. 82 The attachment of leukocytes to endothelial surfaces is a key step in the initial development of atheroma, and this process is facilitated by the enhanced expression of vascular cell adhesion protein 1 (VCAM1). [83][84][85] Tumor necrosis factor (TNF)induced expression of VCAM1 can be suppressed by T in human aortic endothelial cells 86 and human umbilical vein endothelial cells. 87 Furthermore, this study has reported that the aromatization of T to estradiol is important in this protective T effect. 87 However, another study has reported divergent results. 88 Another potential mechanism is the anti-inflammatory effect

| Primary hemostasis
It has been reported that T might have prothrombotic effects in androgenic steroids users 95 and could lead to an increased risk of myocardial infarction and stroke. 96 Accordingly, current EAA guidelines suggest that TRT should not be initiated in patients with a recent major acute CV event. 33 Indeed, T enhances thromboxane A2 (TXA2) receptor density in human platelets, thereby increasing ex vivo platelet aggregability. 97 These data have been confirmed in a study conducted in castrated older men showing a lower maximum platelet aggregation response. 98 However, contrasting results have been shown in subsequent animal studies. [99][100][101][102] In castrated rats, the restoration of the physiological doses of androgens inhibited oxidative stressinduced platelet aggregation and reduced TXA2 release from platelets. 100 Moreover, T could inhibit platelet aggregation via increased NO synthesis, which is associated with endothelial cell growth. 101 Similar results were also obtained in a study that included men aged 60-65 years. This study showed that low levels of T and dihydrotestosterone (DHT) were significantly related to platelet activation and reactivity. 103 Moreover, an in vitro model has confirmed that both T and DHT significantly inhibit platelet aggregation triggered by arachidonate or collagen. 103 However, a recent study failed to show any difference in the procoagulant state of treated or untreated men with Klinefelter syndrome (KS) but demonstrated that thrombin generation in men with KS was inversely associated with androgen action and that it was lower in treated subjects than in untreated subjects. 104 Finally, the largest available meta-analysis revealed no risk of venous thromboembolism (VTE) associated with TRT. 105 Because the risk of VTE could be higher in the presence of thrombophilia, 33 EAA guidelines suggest obtaining a detailed personal and family history of VTE and related risk factors before starting TRT. 33 Overall, the currently available data suggest that the effects of T on primary hemostasis are complex and not yet fully understood; however, if any risk exists, it appears to be very low under physiological replacement ( Table 2).

| Cardiac contractility and remodeling
Clinical data suggest that T has an influence on cardiac contractility.

Increased salt and water retention
Note: Primary hemostasis: Testosterone effects on primary hemostasis are complex. TXA2 and its receptor, which stimulate platelets activation and increase platelet aggregation, seem to mediate testosterone action in primary hemostasis. Oxygen delivery: Testosterone promotes erythropoiesis through a stimulation of EPO production, which is the major erythropoiesis-stimulating hormone, and increasing iron incorporation into red blood cells. Salt retention and muscle strength: Testosterone reduces fatigue related to chronic heart failure increasing muscle strength. However, testosterone could exacerbate chronic heart failure increasing salt and water retention.

TA B L E 2
Summary of testosterone molecular mechanisms and effects on primary hemostasis, oxygen delivery, salt retention, and muscle strength reduced and relaxation was delayed compared with controls or T-treated orchiectomized rats. 113 Physiological levels of T increase positive inotropic response and myocardial relaxation to α1-adrenergic and β1-adrenergic receptors stimulation via an AR-dependent action. 114 Other mechanisms by which T can modulate cardiac performance are by regulating the functional expression of the L-type calcium channel and Na/Ca exchanger, 115 increasing calcium release via the ryanodine receptor, and enhancing calcium clearance by increasing sarcoplasmic/endoplasmic reticulum calcium ATPase levels. 114,116 In animal studies, T has been shown to induce the activation of the renin-angiotensin-aldosterone system (RAAS), resulting in increased maladaptive remodeling. 117 In contrast, it has been shown that T treatment can suppress ventricular remodeling and improve cardiac function in rats, diminishing the imbalance between IL-10 and TNF-α. 118 In addition, it has been demonstrated that T, via the AR system, exerts a protective action against angiotensin II-induced cardiac remodeling. 119,120 There is evidence that T has a beneficial effect on myocyte survival. Indeed, the immediate cardioprotection of ischemic preconditioning is lacking in the absence of T. 121 Moreover, T mediates delayed cardioprotection by inducing heat shock protein 70 (HSP-70), 122 while the induction of ATP-sensitive potassium channels in the mitochondrial inner membrane by T could represent another mechanism of myocyte protection (Table 3). 123

| Cardiac electrophysiology
Growing evidence suggests that T has a direct effect on the car-

Activation Facilitated ventricular repolarization
Note: Cardiac contractility and remodeling: Testosterone seems to have an overall favorable effect on ventricular contractility and remodeling, with a protective effect on cardiomyocytes. Cardiac electrophysiology: Testosterone also promotes ventricular repolarization through a direct action on several ion channels involved in the generation of the action potential.

TA B L E 3 Summary of testosterone molecular mechanisms and effects on cardiac tissue
The role of T is corroborated by the evidence that after puberty, the corrected QT (QTc) interval is significantly shorter in boys than in girls. 125 Subsequent studies have found a negative correlation between endogenous serum T levels in adults and the QTc interval, 126 which is prolonged in hypogonadal men. 127,128 Accordingly, men with an early repolarization ECG pattern (rapidly ascending ST-segment) showed higher endogenous serum T levels than those without this pattern. 129 Moreover, the age-related decline in serum T levels could in part explain the age-dependent prolongation of the QTc interval in men. 130 Ventricular arrhythmias, such as torsade de pointes, and sudden cardiac death are associated with prolonged QTc intervals. [131][132][133] Indeed, as shown in a single-center case series of seven patients, male hypogonadism was associated with torsade de pointes, possibly representing a reversible cause of this life-threatening tachyarrhythmia, which could be treated or prevented by TRT. 134 Moreover, T treatment has been shown to shorten the QTc interval length in community-dwelling men 135 as well as in those with chronic heart failure (CHF). 136 In animal models, T administration facilitated cardiomyocyte repolarization and shortened the action potential duration. T can increase the gradual activation of delayed-rectifier potassium currents (IKs), 137 inhibit inward depolarizing L-type calcium currents (ICaLs), 137 increase the rapid activation of delayed-rectifier potassium currents (IKr), 138 and increase ultra-rapid potassium currents (IKur). 139 However, chronic T treatment increased ICaLs via AR activation, 140 while the opposite was observed for acute T administration, 137 suggesting the existence of a complex network of modulation (Table 3).

| Oxygen delivery
The role of T in the regulation of erythropoiesis is well-known, 141,142 and increased hematocrit is the most frequent adverse effect of TRT in men with hypogonadism men. 14 The stimulatory effect of T on erythropoiesis is dose-dependent and is more pronounced in older men. 143 In a recent randomized placebo-controlled trial that included older men with T deficiency, T therapy significantly increased the hemoglobin levels of all anemic patients, including those with unexplained anemia, as well as those who were not anemic. 144 Therefore, given that T treatment can improve unexplained anemia in older men, the evaluation of T levels should be considered in men who have unexplained anemia as well as symptoms suggestive of T deficiency. 144 Furthermore, T therapy could have beneficial effects in men with chronic anemia and CHF. Indeed, an increase in hematocrit may improve the symptoms of CHF. 108 In fact, the oxygen-transporting capacity is improved by an increased number of erythrocytes within a physiologic range. 145 Favorable effects of TRT have also been observed in men with chronic kidney disease (CKD) 146 in whom hypogonadism may be an additional cause of anemia with reduced responsiveness of erythropoiesis-stimulating agents. 147 However, as shown in animal studies, blood viscosity is increased by T-induced erythropoiesis, 148 leading to an increase in blood flow resistance. Moreover, a correlation between hematocrit and platelet aggregation has been demonstrated, suggesting that an increased number of erythrocytes could increase the risk of thrombosis. 149 Therefore, EAA guidelines do not recommend TRT in the presence of elevated hematocrit. 33 T stimulates erythropoiesis, increasing erythropoietin (EPO) levels and iron utilization. 150 EPO secretion could be stimulated by T by inducing hypoxia or hypoxic sensing. 151 Furthermore, it could regulate the expression of hypoxia-inducible factors (HIFs), Von Hippel-Lindau (VHL), prolyl hydroxylase (PHD), or EPO. [150][151][152] Alternatively, T could affect renal physiology by altering EPO secretion from erythropoietin-producing cells. 153 Increased iron utilization is suggested by the reduction of ferritin and hepcidin concentrations induced by T. 150 Indeed, T can regulate hepcidin expression by regulating bone morphogenetic protein (BMP) signaling pathways ( Table 2). 152

| Chronic heart failure
Among men with CHF, approximately one-third show T deficiency. 154 Moreover, in men with CHF, low T levels independently correlate with exercise intolerance 155 and are associated with muscle wasting and cachexia. 156,157 Therefore, it has been postulated that T deficiency is involved in the pathophysiology of CHF, contributing to some extent to its clinical features, such as fatigue and dyspnea. 158,159 It has also been shown that low T levels are related to poor prognosis, increased hospital admissions, and allcause mortality in men with CHF. 160,161 This evidence suggests that T treatment may ameliorate the clinical status of hypogonadal patients with CHF by improving muscle strength and functional pulmonary capacity. 162 However, some of the adverse CV events reported in the TOM trial were related to an exacerbation of CHF. 29 Indeed, T modulates salt and water homeostasis by promoting salt and water retention as well as the expansion of the extracellular water volume. 163 A 2012 meta-analysis of randomized controlled trials strengthened the evidence of a beneficial effect of T treatment on exercise capacity and oxygen consumption in patients with CHF. 164 However, a more recent meta-analysis showed that TRT did not improve the exercise capacity, cardiac function, quality of life, or clinical outcome of patients with CHF. 165 Taken together, current EAA guidelines recommend against the use of TRT in patients with severe CHF given the risk of polycythemia and VTE in a frail population. 33 Impaired skeletal muscle function and muscle atrophy are features of CHF that could be related to the state of chronic inflammation and insulin resistance characterizing these patients. 166,167 Inflammation has been demonstrated to be promoted by TNF-α, IL-1β, and IL-6. 167 Although the anti-inflammatory action of T is well-known, 89 treatment with a T patch for 3 months (5 mg daily) in men with CHF did not show a reduction in TNF-α levels. 168 The insulin resistance of these patients, which is related to worse symptomatic status, 169 could be linked to a decrease in the glucose transporter GLUT4. 166 There is evidence that T treatment improves insulin sensitivity 109,170,171 and reduces fasting glucose levels 170 in men with CHF and metabolic syndrome or type 2 diabetes. Another mechanism that could have a role in the improved exercise capacity and muscle strength promoted by T in these patients is the shift from type II to type I muscle fiber. 172 Salt and water retention promoted by T are mediated by the proximal tubule RAA system by the increased expression of angiotensinogen 173,174 and increased expression and activity of Na/H exchanger 174,175 and aquaporin 1 176 in the proximal tubules and the epithelial sodium channel in the collecting ducts (Table 2). 177

| CON CLUS IONS
Using the reductio ad absurdum logic, we contradicted the initial assumption of an overall harmful effect of T on the CV system.
Although T has been demonstrated to have overall favorable effects on vasomotion, arterial stiffness, atherosclerosis, cardiac electrophysiology, oxygen delivery, cardiac contractility, and remodeling, it possesses a prothrombotic effect due to its action on platelet function and blood viscosity. In summary, based on the current evidence, CV disease does not appear to be increased in patients undergoing TRT. 178,179 Most physicians against TRT will argue that TRT is "neutral" in terms of CV safety. While the latter is possible, the indication for TRT has never been to address CV dysfunction but rather to improve various signs and symptoms in LOH that correlate well with low testosterone levels. Nonetheless, in older patients with a known CV risk factor, a tailored approach is suggested. 14,25 Symptoms, comorbidities, baseline and target levels of T, formulation, and therapy timing 25,180,181 should be considered to improve sexual function, mood, depressive symptoms, and the mobility of patients with low testosterone levels. 13,33,182,183

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.