ABSTRACT

 

Testosterone is the principal male sex hormone or androgen, which regulates sexual characteristics and body composition. Testosterone is converted to bioactive metabolites dihydrotestosterone and estradiol. Circulating testosterone peaks in early adulthood and declines gradually across middle and older age: older men exhibit lower testosterone and dihydrotestosterone concentrations compared to younger men. In older men, lower testosterone concentrations are associated with higher incidence of cardiovascular events. Lower testosterone and dihydrotestosterone concentrations have also been associated with higher cardiovascular mortality in older men. However, causation is unproven as a randomized placebo-controlled trial of testosterone treatment sufficiently powered to examine outcomes of cardiovascular events or mortality has yet to be reported. Potential mechanisms by which testosterone could exert beneficial actions in the vasculature include reduction in cholesterol accumulation and modulation of inflammation. Smaller randomized trials of testosterone therapy have shown improvements in surrogate endpoints related to cardiovascular risk. However, other trials of testosterone have not shown improvements in carotid atherosclerosis, as assessed by carotid intima-media thickness. One study reported an increase in coronary atheroma assessed using coronary computed tomography angiography in older men receiving testosterone therapy over 12 months. Although one randomized trial of testosterone therapy in older men with mobility limitations reported an excess of adverse events in the treatment arm, larger recent trials in middle-aged to older men did not find any excess of cardiovascular adverse events with testosterone treatment. Meta-analyses of testosterone trials generally have not shown an increase in cardiovascular adverse events. Retrospective case-control studies of health insurance databases have major methodological limitations. The results from such studies are inconsistent, associating testosterone prescriptions with either increased or decreased risk of cardiovascular events, and with lower mortality. While androgen deprivation in men with prostate cancer results in adverse metabolic effects, abuse of high dosages of androgenic steroids is associated with harm. Thus, while some epidemiological studies associate higher circulating concentrations (but within the normal range) of endogenous androgens with lower risk of cardiovascular events and mortality, the effects of exogenous androgens in the form of testosterone therapy seeking to maintain physiological circulating androgen concentrations on the cardiovascular system remain uncertain. This evidence gap has to be accommodated in the current clinical management of hypogonadal men and should be addressed by further randomized interventional studies to clarify whether testosterone treatment has beneficial, neutral or adverse effects on the cardiovascular system.

 

INTRODUCTION

 

Androgen Physiology

 

THE HYPOTHALAMIC-PITUITARY AXIS

 

Testosterone (T) is the principal male sex hormone or androgen that regulates sexual maturation and secondary sexual characteristics and body composition in adult men (1). T undergoes conversion into two major bioactive metabolites, dihydrotestosterone (DHT) a more potent ligand for the androgen receptor, and estradiol (E2), a ligand for estrogen receptors (2). T is produced primarily by the testis, under stimulation of luteinizing hormone (LH) from the pituitary gland, itself under regulation of gonadotrophin releasing hormone (GnRH) from the hypothalamus. The hypothalamic-pituitary-testicular (HPT) axis is under negative feedback regulation via T and E2, acting on the central components of the HPT axis (1,3-5).

 

CONSEQUENCES OF ANDROGEN DEFICIENCY    

 

Androgens play diverse roles in the body, and androgen deficiency in men results in multiple symptoms and signs extending from loss of libido, lethargy, fatigue, poor concentration to gynecomastia, accumulation of fat, loss of muscle mass, and osteopenia or osteoporosis (6,7). Actions of T are amplified by local conversion to DHT in tissues such as the prostate and skin by the enzyme 5α-reductase (8). Of note, some of actions of T, such as in bone and adipose tissue, are mediated via conversion to E2 by the enzyme aromatase (9-11).

 

MEASUREMENT OF CIRCULATING ANDROGEN CONCENTRATIONS

 

Immunoassays have been the standard method for measurement of circulating sex hormones: however, these can exhibit non-specificity and method-dependent bias, particularly at lower hormone concentrations (12,13). Mass spectrometry is regarded as the gold standard for assay of T concentrations (14). Although mass spectrometry is preferred, it is not widely available and validated immunoassays can be informative (6,7). In the circulation, T (and DHT and E2) are bound with high affinity to sex hormone-binding globulin (SHBG), T is also bound with lower affinity to albumin with a small fraction unbound or free (15). However, whether unbound or “free” T represents a more biologically active form of the hormone in vivo is controversial (16). A major difficulty is that measurement of free T using equilibrium dialysis is technically demanding and thus rarely performed. Instead, free T is typically calculated, and this result may vary according to the equations used (15-18). Furthermore, established reference ranges for free T are lacking (7,19). Thus, analysis of actions of T in the male body and cardiovascular system involves consideration of not only its bioactive metabolites, but also the accuracy of assays used and the limitations of calculated free T as a biomarker.

 

Androgens and Male Ageing

 

DECLINE IN CIRCULATING ANDROGENS WITH MALE AGING

 

Circulating T peaks in early adulthood and declines gradually across middle and older age, thus older men exhibit lower T and DHT concentrations compared to younger men (20-22). Observational studies have shown longitudinal declines in T and DHT in ageing men, with parallel increases in LH and SHBG (23-25). This phenomenon suggests that in some men, there is progressive impairment of testicular endocrine function with ageing (25). However, whether older men with symptoms consistent with androgen deficiency and lower circulating concentrations of T compared to younger men, have an androgen deficiency state remains unclear (26,27). The bioactive metabolites of T, DHT and E2 measured with mass spectrometry have been associated with longer leucocyte telomere length, a measure of slower biological ageing, in middle-aged and older men (28,29). However, a study in mostly middle-aged men showed no association between T measured with immunoassay and leucocyte telomere length (30). Thus, there is considerable interest in the question of whether lower T concentrations might contribute to various manifestations of ill health in ageing men.

 

CONTROVERSIES OVER FUNCTIONAL HYPOGONADISM

 

Men with disorders of the hypothalamus, pituitary, or testes resulting in hypogonadism have symptoms and signs of androgen deficiency, and low circulating T concentrations (6). These men are classified as having pathological (or classic or organic) hypogonadism, and T treatment is routinely offered to improve symptoms and restore body composition (6,7). However, low circulating T concentrations are found in many conditions where the HPT axis is intact, including ageing, obesity, and systemic illnesses (7,31). In this context, low T concentrations result from reduction or suppression of HPT axis function, rather than an intrinsic disorder of the HPT axis, with the label of “functional hypogonadism” applied (7,31). Obesity is closely associated with reduced circulating T and SHBG, and loss of excess weight restores endogenous production of T (32-35). Thus, low T might be a biomarker for the presence of systemic illnesses, rather than a contributing or causal factor. Whether or not, and if so which, men with functional hypogonadism should receive T treatment, remains the subject of debate (7,19,20).

 

Androgens and Risks of Cardiovascular Disease

 

AGE AND OBESITY AS COMMON RISK FACTORS

 

Advancing age is an established risk factor for chronic diseases including cardiovascular disease (CVD) and mortality (36,37). Age is a component of all major cardiovascular risk calculators (38). Similarly, obesity, with its close associations with insulin resistance and diabetes risk, is a robust cardiovascular risk factor, and - unlike age - is a potentially modifiable one, whether via bariatric surgery or incretin-based medical therapy (41-44). This is illustrated by observational studies which show a reduction in cardiovascular events and mortality following bariatric surgery in patients who are obese (mean age 48 years) (41,42). Thus, advancing age and obesity are both associated with low circulating T concentrations, and are also risk factors for CVD. 

 

CONFLUENCE OF AGE, OBESITY, LOW TESTOTERONE AND CARDIOVASCULAR DISEASE

 

Given that age and obesity are associated with both low circulating T concentrations and increased risk of CVD, understanding the relationship between these factors becomes vitally important (45). Demographic change will result in increasing numbers of older men in communities worldwide, who will have lower circulating T concentrations and who are at risk of ill health including from CVD (36). If low T contributes to CVD risk, either directly or via its association with obesity, then this represents a potential pathway for intervention to preserve health in ageing men. Conversely, if T is a biomarker for CVD, it may still have a role in risk stratification and identification of men at risk who may benefit from non-hormonal interventions directed at conventional risk factors for CVD.

 

EPIDEMIOLOGICAL STUDIES

 

Associations of Androgens with Cardiovascular Events

 

PROSPECTIVE COHORT STUDIES: IMMUNOASSAY RESULTS

 

Prospective cohort studies report the association of endogenous sex hormones with incidence of cardiovascular events (Table 1). Analyses invariably adjust for age, and typically also include adjustment for body mass index (or waist circumference) and other conventional cardiovascular risk factors. From studies reporting sex hormone results based on immunoassays, some longitudinal analyses have shown no association of total T concentrations with incidence of myocardial infarction (MI) or ischemic heart disease (IHD) events (46,49,53,63,64). In an analysis of 3,443 men aged ≥70 years from the Western Australian Health In Men Study (HIMS), low total T concentrations were associated with an increased incidence of stroke (50). This finding was confirmed by later studies (57,59). In another analysis from HIMS, higher LH was associated with incident IHD (52). One smaller study reported a U-shaped association of total T with incidence of cardiovascular events (54). There were conflicting associations of total E2 with stroke (47,48), and there was no association of total E2 with the incidence of MI (49). In one study, men with lower total T had a higher risk of heart failure, but this was not confirmed in another study (65,68).

 

Table 1. Cohort Studies Examining Associations Between Sex Hormones with Cardiovascular Events in Middle-Aged and Older Men
Study author and year	Size (n of men)	Follow-up (yr)	Age (yr)	Summary of results
Smith GD, 2005 (46)	2,512	16.5	45-59	No association of total T with IHD events or deaths.
Arnlov J, 2006 (47)	2,084	10	56	Higher total E2 at baseline associated with lower incidence of CVD events, total T was not associated.
Abbott RD, 2007 (48)	2,197	≤7	71-93	Baseline total E2 in top quintile (≥125 pmol/L) associated with higher risk of stroke, total T was not associated.
Vikan T, 2009 (49)	1,568	≤13	59.6	No association of total T or E2 with incident MI, or with CVD or IHD mortality.
Yeap BB, 2009 (50)	3,443	3.5	≥70	Total and free T in the lowest quartiles (<11.7 nmol/liter and <222 pmol/liter) predicted increased incidence of stroke or TIA.
* Ohlsson C, 2011 (51)	2,416	5	69-81	Men with total Ta in highest quartile (≥19 mol/L) had lower risk of CVD events. E2 was not associated.
Hyde Z, 2011 (52)	3,637	5.1	70-88	Higher LH was associated with incident IHD.
Haring R, 2013 (53)	254	5, 10	75.5	No associations of baseline total T or total E2 with incident CVD events.
Soisson V, 2013 (54)	495; 146	4	>65	Total T in lowest and highest quintiles associated with CHD or stroke.
* Shores MM, 2014 (55)	1,032	9	76	DHTb <1.7 or >2.6 nmol/L associated with cardiovascular events. Total T was not associated.
* Shores MM, 2014 (56)	1,032	10	76	Non-linear association of DHTb with stroke with lowest risk in men with DHT 1.7-2.6 nmol/L. Total Tb was not associated with stroke. DHT <0.86 nmol/L associated with CVD mortality.
* Yeap BB, 2014 (57)	3,690	6.6	70-89	Higher total Tc (>12.6 nmol/L) or DHT (>1.34 nmol/L) associated with lower incidence of stroke. Tc, DHT and E2 were not associated with MI.
* Srinath R, 2015 (58)	1,558	12.8	63.1	Td was not associated with incidence of CHD events, or cardiac-related mortality.
Holmegard HN, 2016 (59)	4,602	20	57	Total T in lowest decile (0-10thpercentile) associated with stroke.
* Chan YX, 2016 (60)	1,804	14.9	50.3	Total Tc, DHT and E2 were not associated with CVD events.
* Srinath R, 2016 (61)	1,558	14.1	63.1	Td was not associated with stroke.
Wang A, 2019 (62)	5,553	6	63.5	Neither total T nor free T were associated with CVD events.
* Gyawali P, 2019 (63)	1,492	4.9	54.2	Higher total Tc associated with lower risk of incident CVD events, but not with CVD mortality. E2 not associated.
Hatami H, 2020 (64)	816	12	46.1	Total T was not associated with risk of CVD events.
Zhao D, 2020 (65)	4,107	19.2	63.2	Lower total T associated with increased risk of incident heart failure.
* Collett T-H, 2020 (66)	552	7.4	72.4	Total Td not associated with CVD events.
* Boden WE, 2020 (67)	2,118	3	≥40	643 men with total Tb <10.4 nmol/L had higher risk of combined endpoint of CHD death, MI or stroke, compared with 1,475 men with total T ≥10.4 nmol/L.
Shafer S, 2021 (68)	3,865	13.8	48.2	Lower total T not associated with incident heart failure.
Yeap BB, 2022 (69)	210,700	9	58	Total T not associated with incident MI, stroke, heart failure or MACE. Calculated free T not associated with incident MI, stroke or heart failure, but associated with incidence of MACE.

IHD=ischemic heart disease, CVD=cardiovascular disease, MI=myocardial infarction, TIA=transient ischemic attack, CHD=coronary heart disease, MACE=major cardiovascular adverse event. * denotes studies where total T, DHT and/or E2 were measured using mass spectrometry. ^aT and E2 assayed using gas chromatography-mass spectrometry (GC-MS), ^bT and DHT assayed using liquid chromatography-tandem mass spectrometry (LC-MS), ^cT, DHT and E2 assayed by LC-MS, ^dT assayed using LC-MS

 

Recently the association of testosterone with CVD events was examined in the largest prospective cohort study to date, the United Kingdom (UK) Biobank (69). In this study of 210,700 men aged 40-69 years at baseline, with 9 years follow-up, 8,790 had an incident CVD event. Total T was not associated with risk of incident MI, ischemic stroke, hemorrhagic stroke, heart failure, nor major cardiovascular adverse events (MACE) defined as the composite endpoint of non-fatal MI, non-fatal ischemic stroke, and CVD death. The large size of the UK Biobank, and accumulation of outcome events over the period of follow-up, provided power to examine these associations in a robust fashion. UK Biobank used an immunoassay for measurement of serum T which may underestimate results compared to mass spectrometry (70), and UK Biobank men were generally healthier than the UK male population as a whole (71). Therefore, while these results are convincing, their generalizability to different populations in other regions needs to be established.

 

PROSPECTIVE COHORT STUDIES: MASS SPECTROMETRY RESULTS

 

Prospective cohort studies where sex hormones were measured using mass spectrometry are shown (Table 1, marked with *). In the Osteoporotic Fractures in Men Study in Sweden (MrOS), a large prospective cohort study of 2,416 men aged 69-81 years, men with higher total T had a lower incidence of CVD events (51). In HIMS, a later analysis of 3,690 men aged 70-89 years with sex hormones measured using mass spectrometry confirmed the association of low total T with higher incidence of stroke (57). In these analyses, total E2 was not associated with these outcomes (51,57). Analyses from the Cardiovascular Health Study (CHS) of 1,032 men aged 76 years suggested a U-shaped association of DHT with CVD events and stroke risk (55,56). Of note, an analysis from the Busselton Health Study (BHS) of 1,804 predominantly middle-aged men found no association of sex hormones with incidence of CVD events (60). Analyses from the Atherosclerosis Risk in Communities Study of 1,558 men aged 63 years found no association of sex hormones with CVD events (58,61), similar to the findings from a small subset of the MrOS USA study (66). By contrast, in the Men Androgen Inflammation Lifestyle Environment and Stress (MAILES) cohort of 1,492 men followed for 4.9 years, higher total T was associated with a lower risk of CVD events (63). Furthermore, in a post-hoc analysis of 2,118 men with metabolic syndrome participating in a trial of niacin or placebo plus simvastatin, men who had a baseline total T <10.4 nmol/L had higher risk of the combined endpoint of coronary heart disease death, MI or stroke, compared with men with higher total T concentrations (65).

 

SUMMARY: ENDOGENOUS SEX HORMONES VS. CARDIOVASCULAR DISEASE

 

Taken together, these epidemiological studies suggest that there may be an association of lower endogenous T concentrations with increased risk of CVD events in middle-aged and older men. However, the studies provide a mix of positive, equivocal and negative results. Major cohort studies using mass spectrometry for assay of sex hormones associated higher total T concentrations with lower risk of CVD events in older (51,57) and middle-aged to older men (63,67). There may be a predilection for lower testosterone, measured by mass spectrometry, to be associated with stroke risk (57). In one study lower DHT was associated with CVD events including stroke, with a non-linear association (55,56). Inconsistent results in other studies may have been due to smaller cohort sizes and fewer outcome events reducing the power available to detect underlying associations. However, in the largest ever prospective cohort study, the UK Biobank, there was no association of testosterone with a range of CVD events (69). Overall, epidemiological studies would suggest a possible protective effect of endogenous androgens against CVD events in the older population of men, rather than in relatively healthy middle-aged men.

 

Associations of Androgens with Cardiovascular Mortality

 

PROSPECTIVE COHORT STUDIES: IMMUNOASSAY RESULTS

 

Several of the studies in Table 1 reported CVD-related mortality in addition to events. Other studies where the outcome was based on CVD-related mortality are summarized in Table 2. Studies almost invariably adjusted for age, and typically adjusted for BMI and other cardiovascular risk factors. In two studies using immunoassay for assay of sex hormones, T was not associated with CVD or ischemic heart disease (IHD) mortality (46,49). However, several other studies using immunoassay for sex hormones did find associations of lower endogenous total T concentrations with increased risk of CVD-related death (72,74,77,81). Lower calculated free T was also associated with increased CVD-related mortality in some studies (76,78,81), but was associated with lower IHD mortality in one study (73). Lower total E2 was associated with CVD mortality in one study (76). In an analysis from the UK Biobank of 149,436 men followed for 11.3 years, there was no association of either total or calculated free T with risk of CVD mortality (84).

 

Table 2. Cohort Studies Examining Associations Between Sex Hormones and CVD-Related Mortality in Middle-Aged and Older Men
Study author and year	Size (n of men)	Follow-up (yr)	Age (yr)	Summary of results
Khaw K-T, 2007 (72)	825 and 1489	≤10	40-79	Total T inversely related to mortality from all causes, CVD and cancer.
Araujo AB, 2007 (73)	1,686	15.3	40-70	Lower free T associated with lower IHD mortality. Equivocal association of lower DHT with IHD mortality.
Laughlin GA, 2008 (74)	794	11.8	50-91	Total T in the lowest quartile (<8.4 nmol/L) predicted increased mortality from all causes and from CVD and respiratory causes.
* Tivesten A, 2009 (75)	3,014	4.5	75	Total Ta and E2 levels in the lowest quartiles predicted all-cause and non-CVD mortality. T and E2 were not associated with CVD mortality.
Menke A, 2010 (76)	1,114	18	≥20	Lower free T associated with overall and CVD mortality in first 9 years of follow-up. Lower total E2 associated with CVD mortality. (Difference between 90th and 10thpercentiles for free T and total E2)
Haring R, 2010 (77)	1,954	7.2	20-79	Total T <8.7 nmol/L associated with increased all-cause, CVD and cancer mortality.
Hyde Z, 2012 (78)	3,637	5.1	70-88	Lower free T (100 vs 280 pmol/L) predicted all-cause and CVD mortality.
* Yeap BB, 2014 (79)	3,690	7.1	70-89	Optimal total Tb (9.8-15.8 nmol/L) predicted lower all-cause mortality. Higher DHT (>1.3 nmol/L) predicted lower IHD mortality. E2 was not associated with mortality.
* Pye SR, 2014 (80)	2,599	4.3	40-79	Presence of sexual symptoms and total Tc <8 nmol/L associated with all-cause and CVD mortality, total or free T not associated.
Holmboe SA, 2015 (81)	5,323	18.5	30-70	Higher T or free T (highest vs lowest quartile) associated with lower CVD mortality.
* Hsu B, 2016 (82)	1,705, 1,367 and 958	0, 2 and 5	≥70	Decrease in total Td over time associated with all-cause but not CVD mortality. Decrease in total E2d was associated with all-cause and CVD mortality.
* Chasland L, 2017 (83)	1,649	20	49.8	Higher physical activity and total Tb, DHT and E2 were not associated with CVD events. Men with higher physical activity and DHT had the lowest risk of CVD death. Men with lower physical activity and higher E2 had greater risk of CVD death.
Yeap BB, 2021 (84)	149,436	11.3	58.0	Men with lower total T had higher all-cause and cancer-related mortality, no association with CVD deaths.

IHD=ischemic heart disease, CVD=cardiovascular disease, CHD=coronary heart disease. * denotes studies where total T, DHT and/or E2 were measured by mass spectrometry; free T was calculated. ^aT and E2 measured using gas chromatography-mass spectrometry (GC-MS). ^bT, DHT and E2 measured using liquid chromatography-tandem mass spectrometry (LC-MS), ^cT measured using GC-MS, ^dT and E2 measured using LC-MS.

 

PROSPECTIVE COHORT STUDIES: MASS SPECTROMETRY RESULTS

 

Prospective cohort studies using mass spectrometry for assay of sex hormones are of interest. In an analysis from MrOS in Sweden, lower endogenous total T and E2 concentrations were associated with all-cause and non-CVD mortality, but not with CVD mortality (75). Interestingly, in an analysis from HIMS of 3,690 men aged 70-89 years at baseline, optimal endogenous total T concentrations were associated with survival, and higher DHT predicted lower IHD mortality (79). The CHS study reported consistent findings with lower DHT concentrations being associated with CVD mortality (56). An analysis from the European Male Ageing Study found that the combination of sexual symptoms and lower total T was associated with all-cause and CVD mortality, rather than total T or free T on their own (80). In an analysis from the Concord Health and Ageing in Men Project (CHAMP), longitudinal decreases in total T, DHT or E2 were associated with all-cause mortality, but only the longitudinal decrease in total E2 was predictive of CVD mortality (82). Finally, in an analysis from BHS in which physical activity and sex hormones concentrations were analyzed, men with higher levels of physical activity and higher DHT concentrations had the lowest risk of CVD death (83).

 

SUMMARY: ENDOGENOUS SEX HORMONES VS. CARDIOVASCULAR MORTALITY

 

Several cohort studies have reported an association between lower endogenous T concentrations and increased mortality related to CVD, after adjusting for age and other cardiovascular risk factors. Of large studies using mass spectrometry for assay of sex steroids, MrOS in Sweden found an association of lower total T and E2 with all-cause rather than CVD mortality, while HIMS found an optimal total T to be associated with survival (75,79). HIMS found higher DHT was associated with lower IHD mortality (79), consistent with results from CHS (56), and in BHS the combination of higher DHT and higher levels of physical activity was associated with lower risk of death from CVD (83). Declining E2 may also have a role, being associated with CVD mortality in CHAMP (82). However, in relatively healthy middle-aged men (UK Biobank), there was no evidence of an association between total T and CVD mortality risk (84).

 

Therefore, allowing for some heterogeneity in cohort characteristics and results, lower endogenous T concentrations, measured using mass spectrometry, may be predictors of CVD related deaths in older men, as might lower or declining concentrations of its bioactive metabolites DHT and E2. However, this may not be the case in generally healthy middle-aged men. Whether lower concentrations of endogenous sex hormones are biomarkers or possibly contributing factors to these outcomes remains unclear from these observational studies, as proof of causality ultimately requires interventional studies and randomized controlled trials (RCTs).

 

MECHANISTIC STUDIES

 

Potential Mechanisms

 

Knowledge of potential mechanisms by which androgens might exert protective effects against atherosclerosis and reduce the risk of cardiovascular events would bridge the findings from epidemiological studies and clinical investigation. There are a substantial number of such studies with diverse models and results, a comprehensive discussion of each being beyond the scope of this chapter (for reviews, see (85,86)). Selected studies are discussed briefly in this context.

 

Cholesterol Accumulation in Animal Models

 

Experimental studies in castrated male rabbits fed a high cholesterol diet reported effects of testosterone treatment to reduce accumulation of cholesterol in the aortic wall and to reduce atheromatous plaque area and aortic intimal thickness (87-90). Similar results have also been reported in miniature pigs (91). Castration of low-density lipoprotein receptor (LDLR)-deficient male mice results in increased fatty steak lesion formation in the aorta compared to non-castrated controls, which is attenuated with testosterone supplementation (92). At least part of this effect may be mediated via conversion of T to E2. Of note, T and DHT increased calcification of plaque in apolipoprotein E (ApoE)-null mice, even as T had a neutral effect on plaque volume and DHT decreased plaque volume (93). In testicular feminized mice with a non-functional androgen receptor (AR) and low circulating T concentrations, T supplementation to physiological levels reduced fatty streak formation (94). Similarly, AR knockout mice (ARKO) showed increased aortic atherosclerosis, and atherosclerotic lesion area that was reduced with T treatment (95). In wild-type mice, T treatment reduced the presence of necrotic cores within plaque compared with placebo. Therefore, these animal studies suggest an effect of T treatment in reducing cholesterol accumulation and the development of atheromatous plaque, while increasing calcification. However, the actions of sex hormones are complex, being mediated partly via aromatization of T to E2, and occurring at least to an extent via AR-independent mechanisms.

 

Neointimal Formation and Vascular Smooth Muscle Proliferation

 

NEOINTIMAL RESPONSES TO INJURY

 

In a male rabbit aorta model of neointimal plaque formation induced by endothelial denudation, T treatment in vitroinhibited plaque development (96). In a male porcine model of coronary neointimal plaque formation following moderate angioplasty-induced arterial injury, castrated males exhibited greater intimal area compared to intact males and castrated males treated with T (97). T inhibited proliferation and increased expression of the cell-cycle regulator p27^kip1 during neointimal formation. However, despite castration of wild-type mice resulting in increased neointimal formation following wire injury, selective deletion of AR from endothelial cells or smooth muscle cells did not affect lesion size (98). Therefore, effects of T on neointimal formation may be indirect, or mediated by AR-independent mechanisms.

 

VASCULAR SMOOTH MUSCLE 

 

Vascular smooth muscle cells contribute to progression of atherosclerotic lesions and formation of the fibrous cap (99). T was shown to regulate expression of proliferation-associated genes in skeletal myocytes and in myofibers in different muscles (100,101). Its role in smooth muscle cells in the vasculature is not well defined. In one study, T exerted a pro-proliferative effect on vascular smooth muscle cells in vitro, with increased DNA synthesis assessed using a thymidine incorporation assay (102). In that study the effect of T was blocked by the AR antagonist flutamide. In another study, T induced apoptosis in cultured vascular smooth muscle cells, in an AR-dependent manner (103). In one study, deletion of the AR in vascular smooth muscle cells did not change atherosclerotic plaque size in LDLR knockout mice (104). However, another study demonstrated that T, acting via the AR in vascular smooth muscle cells, might be involved in promoting vascular calcification (105). Therefore, T seems to exert indirect effects on neointimal proliferation in response to injury and may play a secondary role in the development and calcification of atheromatous plaque via complex actions in vascular smooth muscle.

 

Inflammation

 

INFLAMMATION AND ATHEROTHROMBOSIS

 

A mechanistic link likely exists between inflammation and atherothrombosis (for reviews, see (106,107)). Statin therapy lowered both LDL cholesterol and C-reactive protein (CRP) concentrations, reducing the risk of cardiovascular events in a primary prevention setting in adults with LDL <3.4 mmol/L and high-sensitivity CRP ≥2.0 mg/L (108). Recently, anti-inflammatory intervention utilizing canakinumab, a monoclonal antibody targeting interleukin-1β, in a secondary prevention setting in adults with high-sensitivity CRP ≥2.0 mg/L demonstrated a modest reduction in major cardiovascular events (109). However, a major trial using low-dose methotrexate showed no benefit in adults with previous MI or multivessel coronary artery disease and either type 2 diabetes or metabolic syndrome (110). By contrast, colchicine has shown promise in major RCTs as an anti-inflammatory agent to reduce cardiovascular risk in both acute and chronic secondary prevention settings (111,112). These results underscore the relationship between inflammation and atherosclerosis.

 

TESTOSTERONE EFFECTS ON IMMUNE CELLS

 

Of note, in vitro studies have shown effects of T to reduce production of inflammatory cytokines from monocytes, macrophages and endothelial cells (113-115). Deletion of monocyte-macrophage AR in LDLR knockout mice resulted in reduced atherosclerosis compared to LDLR knockout mice, suggesting a role for AR-mediated actions in inflammatory cells (104). In an elegant study, pre-pubertal castration of male ApoE knockout mice increased atherosclerotic lesion area, which was abolished by an anti-CD3 antibody targeting T cells, linking hormonal and immunologic regulation of atherosclerosis (116). In that study, both castration and depletion of AR in epithelial cells resulted in increased thymus weight, and mice with depletion of AR in epithelial cells showed increased atherosclerosis and increased infiltration of T cells in the vascular adventitia (116). These findings support a mechanism by which deficiency of androgen action modulates immune/inflammatory responses to promote atherosclerosis.

 

TESTOSTERONE EFFECTS ON CYTOKINES

 

Older men treated with gonadotrophin-releasing hormone agonists to suppress HPT axis function showed increased concentrations of circulating tumor necrosis factor-α and interleukin-6 (117). In a randomized cross-over trial of 27 men ranging in age from 36-78 years, T treatment given via intramuscular injections over one month reduced circulating concentrations of tumor necrosis factor-α and interleukin-1β, and increased concentrations of the anti-inflammatory cytokine interleukin-10 (118). In another study of 20 men with type 2 diabetes, there was an inverse correlation of baseline T and interleukin-6, but T treatment over three months, while reducing waist circumference, did not alter tumor necrosis factor-α, interleukin-6, or C-reactive protein (CRP) concentrations (119). In the Testosterone Trials (T-Trials), T treatment via transdermal gel over 12 months in men aged ≥65 years did not change concentrations of high-sensitivity CRP or interleukin-6 (120). In a study of men treated with DHT or with recombinant human chorionic gonadotrophin over three months, neither intervention affected markers of endothelial cell activation or inflammation (121). By contrast, in a trial of men with metabolic syndrome, men in the T treatment arm showed a reduction in high sensitivity CRP after 12 months of treatment (122). In a trial of 76 men with newly diagnosed type 2 diabetes, T treatment over 9 months reduced markers of endothelial cell activation and inflammation, namely circulating concentrations of intracellular adhesion molecule type 1, p-selectin, and CRP (123). Therefore, the results of clinical studies are not wholly consistent. In summary, although the concept that T might exert anti-inflammatory actions protective against atherosclerosis is plausible, more evidence is needed using a direct measure of atherosclerosis.

 

CLINICAL TRIALS WITH SURROGATE ENDPOINTS

 

Testosterone Effects on Angina and Vascular Function

 

EFFECTS OF EXOGENOUS TESTOSTERONE ON ANGINA

 

Mechanistic studies in cell and animal models provide a plausible rationale for the epidemiological findings associating lower endogenous T concentrations with higher risk of CVD. However, clinical studies are necessary to clarify whether administration of T modulates clinical manifestations of CVD in vivo. Case series from the 1940s reported a beneficial effect of T therapy using intramuscular testosterone propionate to decrease the frequency and severity of angina attacks in an era where nitrate therapy was the mainstay of therapy (124-126). These early reports in men (and a small number of women) describe gradual improvements in symptoms over periods ranging from weeks to months. Conversely, a study in the 1960s found that administration of oral conjugated estrogen (that would suppress the HPT axis and serum androgen concentrations) to men resulted in adverse cardiovascular effects (127). In any case, as T is the native hormone which is metabolized in vivo to DHT and E2 (2), it is the preferred treatment for hypogonadal men (128), and represents the logical candidate for interventional studies.

 

More recent RCTs have revisited the issue of T treatment in men with CAD (Table 3A). In studies lasting from eight weeks to 12 months, T supplementation in men with CAD increased post-exercise ST segment depression (129), time to ischemia on exercise testing (130,132) and in a study in older men with diabetes, reduced the frequency of angina and silent myocardial ischemia during ECG Holter monitoring (133). These findings suggest either a protective effect of T on the myocardium, or an improvement in exercise capacity. A cross-over study found increased perfusion of myocardium supplied by unobstructed arteries, consistent with a vasodilatory action (131). Therefore, while existing data are limited, contemporary RCTs support historical observations suggesting a potentially beneficial effect of T supplementation in men with CAD.

 

Table 3. Selected Randomized Controlled Trials (RCTs) of Testosterone Supplementation in Middle-Aged and Older Men Reporting Outcomes Related to Angina (A), Artery Health (B), and Cardiovascular Adverse Events (C)
Study author and year	Population (men)	Formulation of T	N active	N placebo	Duration	Result
A
Jaffe MD, 1977 (129)	Men with ST segment depression on exercise (mean age 58 years)	T cypionate 200 mg weekly	25	25	8 weeks	Decreased postexercise ST segment depression in T-treated but not placebo group
English KM, 2000 (130)	Men with coronary artery disease (mean age 62 years)	Transdermal patch 5 mg	22	24	12 weeks	Increased time to 1-mm ST- segment depression during treadmill exercise
Webb CM, 2008 (131)	Men with angiographically proven coronary artery disease, 40-75 years	Oral T undecanoate 80 mg bd	22		8 weeks, cross-over	No difference in angina or endothelial function, decreased arterial stiffness, increased perfusion of myocardium
Mathur A, 2009 (132)	Men with chronic stable angina (men age 65 years)	Depot intramuscular T undecanoate	7	6	12 months	Increased time to ischemia, non-significant trend for decreased CIMT
Cornoldi A, 2009 (133)	Men with proven coronary artery disease and type 2 diabetes (mean age 74 years)	Oral T undecanoate 40 mg tds	45	44	12 weeks	Reduced number of angina attacks and silent ischemic episodes on ECG Holter monitoring
B
Aversa A, 2010 (122)	Men with metabolic syndrome, T ≤11 nmol/L or free T ≤250 pmol/L (mean age 57 years)	Depot intramuscular T undecanoate 1,000 mg every 12 weeks	40	10	12 months*	Decreased high sensitivity CRP, improvement in CIMT
Basaria S, 2015 (164)	≥60 years, T 3.5-13.9 nmol/L or free T <173 pmol/L	Transdermal T gel 75 mg daily	156	152	3 years	No difference in rates of change in CIMT or coronary artery calcium
Budoff MJ, 2017 (168)	Men aged ≥65 years with T <9.5 nmol/L	Transdermal T gel 50 mg daily	73	65	12 months	Greater increase in non-calcified coronary plaque volume
Hildreth KL, 2018 (141)	Mean age 66 years, T 6.9-12.1 nmol/L	Transdermal gel, titrated to 13.9-19.1 or 20.8-34.7 nmol/L	41, 43	40	12 months	No effect of T on endothelial function or on CIMT
C
Basaria S, 2010 (172)	≥65 years, T 3.5-12.1 nmol/L or free T <173 pmol/L, mobility limitation	Transdermal gel 100 mg daily	106	103	6 months	Trial stopped prematurely due to excess cardiovascular events in T arm
Srinivas-Shankar U, 2010 (173)	≥65 years, T ≤12 nmol/L or free T ≤250 pmol/L, frail or intermediate frail	Transdermal gel 50 mg daily	138	136	6 months	T improved muscle strength and physical function, no signal for cardiovascular adverse events
Snyder P, 2016 (174)	≥65 years, T <9.5 nmol/L, sexual dysfunction (A), diminished vitality (B) and/or mobility limitations (C)	Transdermal gel 50 mg daily	395 (A 230, B 236, C 193)	395 (A 229, B 238, C 197)	12 months	Modest benefit of T on sexual function, no signal for cardiovascular adverse events
Wittert GA, 2021 (175)	50-74 years, waist ≥95 cm, T ≤14 nmol/L, and impaired glucose tolerance or newly diagnosed type 2 diabetes	Depot intramuscular testosterone undecanoate 1000 mg every 3 months	504	503	24 months	All men received background lifestyle intervention (Weight Watchers). T reduced risk of type 2 diabetes at 2 years by 40%.

T=testosterone, CIMT=carotid intima media thickness, CRP=C-reactive protein. * men in placebo group switched to T after 12 months, extension study to 24 months no longer randomized.

 

EFFECTS OF EXOGENOUS TESTOSTERONE ON VASCULAR FUNCTION

 

Brachial artery endothelial function is an established measure of cardiovascular health examining both endothelial and vascular smooth muscle function, which mirrors responses in the coronary arteries (134). Assessment of arterial stiffness provides complementary insights into vascular health (135,136). In uncontrolled open-label studies in men with low baseline T concentrations, T supplementation improved both endothelial function and arterial stiffness (137,138). A study in hypogonadal older men found an improvement in arterial stiffness with transdermal T therapy (139). In a RCT of 55 obese men with type 2 diabetes, one year’s treatment with T undecanoate given as a depot intramuscular injection every 10 weeks improved endothelial function compared to placebo (140). However, other studies in middle-aged and older men did not show any effect of transdermal T treatment on endothelial function (141,142). There is a pathway by which T treatment is expected to improve endothelial function as in vitro studies demonstrate stimulation of nitric oxide synthesis in human aortic endothelial cells exposed to T (143). T might exert beneficial effects in the vasculature via actions to improve endothelial function and arterial stiffness, but additional studies are needed before a definitive conclusion can be made.

 

Testosterone and Atherosclerosis

 

CIRCULATING CHOLESTEROL CONCENTRATION

 

Clinical studies of T have shown consistent results for T treatment to reduce circulating concentrations of total cholesterol to a modest degree (144-146). A trend for T to lower LDL cholesterol has been noted (144). T treatment appears to lower high density lipoprotein (HDL) cholesterol again to a small degree (147). HDL cholesterol is involved in reverse cholesterol transport thus exerting anti-atherogenic activity, such that HDL function is an independent predictor of cardiovascular events (148,149). However, T may modulate HDL concentrations without a corresponding effect on HDL function (150). Of note, in observational studies, endogenous T concentrations correlated with circulating HDL and were inversely associated with total cholesterol (151,152). Thus, the prognostic significance of T-induced changes in lipid profiles, and the effect of T treatment on HDL-mediated anti-atherogenic action in men at risk for CVD remains unclear.

 

CAROTID ATHEROSCLEROSIS

 

Carotid intima-media thickness (CIMT) and the presence of carotid plaque are measures of preclinical carotid atherosclerosis, which can be assessed non-invasively using ultrasound (153). While low endogenous T concentrations are associated with increased CIMT in observational studies (154,155), it is less clear whether low endogenous T (or E2) predicts progression of CIMT (156-158). One study implicated low-grade inflammation in this process, finding an association of low non-SHBG-bound T with CIMT in older men with CRP ≥2 mg/L, but not in those with CRP <2 mg/L (159). Two cohort studies observed cross-sectional associations of low T concentrations with greater carotid plaque area (158,160). However, no association was found between baseline sex hormone concentrations and change in plaque area during follow-up, possibly due to increased use of anti-hypertensive and lipid-lowering therapy (158). One study reported an association of higher T concentrations with reduced CIMT and lower prevalence of carotid plaque in a cohort of community-dwelling men, but not in a cohort of men with angiographically proven CAD (161). In men with proven CAD, higher DHT was associated with less carotid plaque. Of note, E2 was associated with increased CIMT in community-dwelling men, but with less carotid plaque in men with CAD (161). One study found that higher E2 was associated with the presence of lipid core in carotid plaques in men, with no association of T concentrations (162). In a study of men who underwent carotid endarterectomy, the ratio of circulating T/E2 was inversely associated with plaque calcifications, macrophage staining and plaque neutrophil content, as well as plaque IL-6 protein (163). These findings associate sex hormone concentrations with CIMT and carotid plaque in men.

 

Several interventional studies reporting CIMT as an outcome are summarized (Table 3B). Of note, in a RCT of intramuscular T undecanoate 1,000 mg given every 12 weeks to men with metabolic syndrome, there was improvement in CIMT after 12 months of treatment (122). However, a larger RCT, Testosterone Effects on Atherosclerosis Progression in Aging Men (TEAAM), conducted over three years showed no effect of transdermal T treatment on rates of progression of CIMT (164). A smaller study, which tested both exercise training and transdermal T treatment over a period of 12 months, found no effect of either intervention on CIMT (141). Thus, while the RCT data are limited, the effects of T treatment on preclinical carotid atherosclerosis may be beneficial or neutral, but are unlikely to be adverse. See table 3, part B.

 

CORONARY ATHEROSCLEROSIS

 

Coronary computed tomography angiography (CCTA) has emerged as a non-invasive method for imaging coronary atherosclerosis (165-167). Normal findings on CCTA are associated with a low risk of cardiovascular events, while the presence and extent of CAD demonstrated on CCTA are risk predictors for future cardiovascular events in large epidemiological studies (165-167). Therefore, there is considerable interest in the Cardiovascular sub-study of the T-Trials which reported CCTA outcomes for 73 men treated with transdermal T and 65 men receiving placebo over a 12-month period (168). In this study, men in the T-treated group experienced a greater increase in non-calcified and total coronary artery plaque volume, compared to men in the placebo group (168). However, the groups were unbalanced with men in the T-treated group having considerably lower non-calcified and total plaque volumes at baseline and at the end of the study compared with placebo-treated men. The fact that the two groups of men differed substantively in a key baseline characteristic makes the result of the study challenging to interpret (169). There was no difference in the rate of change of coronary calcium score between groups, albeit men in the T-treated group had lower coronary calcium scores at baseline and at the end of the study compared with men in the placebo group (168). The results for coronary calcium scores are concordant with the TEAAM trial that also showed no difference in coronary calcium scores with T treatment (164). Men in the T-Trials Cardiovascular sub-study overall had a high burden of plaque at baseline with 32% of T-treated men and 38% of placebo recipients having baseline Agatston scores ≥300 (168).

 

Of note, in the T-Trials Cardiovascular sub-study, data on plaque volume were not analyzed relative to vessel lumen to address the issue of whether vascular remodeling was occurring (170). Since then, CCTA technology has progressed to allow more detailed and sophisticated analysis of plaque characteristics associated with higher risk of coronary events, which were not applied in that study (171). While these findings are important and noteworthy, a larger RCT with balanced groups using current CCTA methodology would be needed to clarify the effect of T on coronary plaque characteristics. The findings are a timely reminder that until definitive RCTs are available, the effects of T on the cardiovascular system remain uncertain and may be beneficial, neutral or adverse.

 

TRIALS REPORTING ADVERSE EVENTS

 

Testosterone RCTs and Cardiovascular Adverse Events

 

Selected T RCTs, which have influenced this field, are summarized (Table 3, part C). These studies were underpowered for cardiovascular events and the possibility of type 1 and type 2 errors should be considered. A key RCT randomized 209 men aged 65 years and older, with mobility limitations and low or low-normal baseline T or free T, to transdermal T gel vs placebo for six months (172). Of note the starting dose of transdermal T (100 mg daily) was greater than the usual recommended starting dose (50 mg daily). The study was discontinued after an excess of adverse events was noted in the T arm (172). A contemporaneous RCT in a comparable population of 274 older men who were frail or intermediate frail, using a 50 mg daily dose of transdermal T over 6 months, was successfully completed showing improved muscle strength and physical function, with no signal for cardiovascular adverse events (173). The Testosterone Trials (T-Trials) has reported results from the main study and component sub-studies (174,176). These have been extensively reviewed (169,176). In T-Trials, 790 men aged 65 years and older, with symptoms of sexual dysfunction, diminished vitality or mobility limitations and baseline T <9.5 nmol/L (<275 ng/dL), were randomized to transdermal T gel at a starting dose of 50 mg daily vs placebo for 12 months (174). In T-Trials, T treatment improved sexual function to a moderate degree, while the primary outcomes for physical function and vitality were not met (174). T treatment improved anemia and volumetric bone density, with a neutral effect on cognition (176). The effects on coronary artery plaque volume have been discussed (see Section 4.2.3). T-Trials had a low rate of major cardiovascular adverse events (7 in each of the T and placebo arms).

 

Recently, a larger Australian RCT, Testosterone for the Prevention of Type 2 Diabetes Mellitus (T4DM) has been reported (175). T4DM was a randomized, double-blind, placebo-controlled, 2-year, phase 3b trial done at six Australian tertiary care centers, which randomized 1,007 men to depot intramuscular testosterone undecanoate injections given every three months for two years, vs placebo, on a background of a lifestyle intervention (Weight Watchers) given to all participants. Inclusion criteria were age 50-74 years, waist circumference ≥95 cm and baseline T ≤14.0 nmol/L (≤403.8 ng/dL), and the presence of either impaired glucose tolerance or newly diagnosed type 2 diabetes based on oral glucose tolerance testing (175). In T4DM, testosterone treatment reduced the risk of type 2 diabetes at two years by 40% beyond the effect of the lifestyle intervention (relative risk 0·59, 95% confidence interval 0·43 to 0·80; p=0·0007). T4DM is the largest T RCT reported to date, and the incidence of cardiovascular adverse effects were similar in both T and placebo arms. In T4DM, 17 men in the placebo group and 12 in the T group had a major cardiovascular adverse event (13 men in the placebo arm and 7 in the testosterone arm had an ischemic heart disease event, 3 and 4 had cerebrovascular disease events respectively, and one in each group died from a cardiovascular-related cause) (175). Therefore, in keeping with T-Trials, the rates of major cardiovascular adverse events in T4DM were low, and comparable in testosterone and placebo-treated men.

 

At this point, it is worth commenting on the outcome of cardiorespiratory fitness. Low cardiorespiratory fitness, assessed as maximal oxygen consumption during exercise testing (VO₂peak), is a strong independent predictor of all-cause and CVD mortality in apparently healthy men and in men with established CVD (177-179). Cardiorespiratory fitness has been recommended as a vital sign for use in clinical assessment (180). An earlier study over 12 months did not show an effect of T treatment on fitness (141), nor did a more recent study with a 12-week intervention (181). That study used a 2x2 factorial design, while exercise training resulted in improved VO₂ peak within the 12-week period of intervention, T treatment did not, and there was no evidence within this relatively short timeframe of additive benefit (181). In the TEAAM study conducted over 3 years, placebo-treated men showed a decline in VO₂peak over time, but the decline was attenuated in T-treated men (182). In TEAAM, there was no signal for cardiovascular adverse events with T (164). Part of the beneficial effect of T treatment on VO₂peak might be mediated via its effect to raise hemoglobin concentrations (147,169,176), or its action on skeletal muscle (183,184). The net effect might be to preserve (or at least attenuate the loss of) cardiorespiratory fitness in ageing men, that could translate into a reduction in cardiovascular risk. However, in keeping with the results of T4DM, an extended duration of T intervention may be required to realize these benefits.

 

Meta-analyses of Cardiovascular Adverse Events in Testosterone RCTs

 

To date, no T RCT large and long enough to be powered for the outcome of cardiovascular events has been reported. However, meta-analyses of reported T RCTs have been performed to determine whether T is associated with a difference in the rate of cardiovascular adverse events (Table 4). Earlier meta-analyses done in 2007 and 2010 had found no significant difference in risk for cardiovascular adverse events (147,185). One analysis done in 2013 claimed an association of T treatment with increased risk of cardiovascular-related adverse events (186). However, subsequent meta-analyses done from 2013 to 2020 have not supported this finding (187-193). Instead, these have found no association of T treatment with risk of cardiovascular adverse events (Table 4).

 

Table 4. Meta-Analyses of Cardiovascular Adverse Events in Randomized Controlled Trials (RCTs) of T Supplementation in Men
Study characteristics				Results
Study author and year	N of RCTs	N active	N placebo	Adverse signal	No adverse signal
Haddad RM, 2007 (185)	30	808	834		No significant difference in odds ratio for any cardiovascular adverse event or MI.
Fernandez-Balsells MM, 2010 (147)	51	2,716			No significant difference for all-cause mortality, coronary bypass surgery or MI.
Xu L, 2013 (186)	27	2,994		T associated with increased risk cardiovascular-related event (OR 1.54, 95% CI=1.09-2.18)*.
Ruige JB, 2013 (187)	10 (>100 participants)	1,289	848		No significant difference in cardiovascular adverse events.
Corona G, 2014 (188)	75	3,016	2,448		No association of T supplementation with cardiovascular risk. For MACE OR=1.01 (95% CI 0.57-1.77).
Borst SE, 2015 (189)	35	3,703			No significant risk for cardiovascular-related adverse events.
Alexander GC, 2017 (190)	39, cut-off for T 10.4-16.7 nmol/L	3,230	2,221		No significant increase in risk of MI OR=0.87 (95% CI 0.39-1.93)*, stroke or mortality.
Elliott J, 2017 (191)	87, cut-off for T 12 nmol/L or cFT 225 pmol/L	1,462-2088	1,372-1,851		Improved QoL, libido, depression and erectile function. No increase in risk of adverse events.
Corona G, 2018 (192)	93	4,653	3,826		No clear effect of T on incidence of CVD events. For MACE OR=0.97 (95% CI 0.64-1.46).
Diem SJ, 2020 (193)	38	N/A	N/A		Small improvement in sexual function and quality of life. Pooled risk for adverse cardiovascular outcomes did not differ between groups (OR=1.22, 95% CI 0.66-2.23).*
Hudson J, 2022 (194)	35 RCTs: 17 included in IPD meta-analysis	1,750 (IPD)	1,681 (IPD)		No significant difference between groups. For cardiovascular or cerebrovascular events OR=1·07 (95% CI 0·81–1·42).

MI=myocardial infarction, MACE=major adverse cardiovascular events, OR=odds ratio, 95% CI=confidence interval. QoL=quality of life, IPD=individual participant data. Unless otherwise specified, meta-analyses were conducted using random effects models. *fixed effects model

 

It is worth commenting on more recent meta-analyses that have included the results of the T-Trials. In the meta-analysis by Alexander et al. (2017), 39 RCTs were included. The meta-analysis found no significant increase in risk of MI (data from 30 RCTs utilized), stroke (9 RCTs) or mortality (20 RCTs) (190). However, caveats were noted with respect to the quality of the available evidence. In a network meta-analysis, Elliott et al. (2017) included RCTs that enrolled men with baseline T ≤12 nmol/L (≤346 ng/dl) or free T ≤225 pmol/L, including 87 RCTs overall (191). T treatment was associated with improved quality of life and libido, improvement in depression and in erectile function. There was no increase in risk of adverse events such as cardiovascular death, MI or stroke (191). Corona et al. (2018) studied 93 RCTs and found no clear effect of T on incidence of CVD events, with an odds ratio of 0.97 (95% confidence interval 0.64-1.46) for major cardiovascular adverse events (192).

 

Diem et al. (2020) examined 38 RCTs of at least six months duration, noting that few exceeded a 1-year duration, there was a lack of power to assess important harms, and limited data for men aged 18-50 years (193). They concluded that in older men with lower testosterone concentrations, in the absence of organic hypogonadism, T treatment resulted in small improvements in in sexual functioning and quality of life, with long-term safety and efficacy still uncertain. Recently Hudson et al. (2022) analyzed RCTs involving men with T ≤12 nmol/L and minimum duration of three months, identifying 35 studies and obtaining individual participant data from 17 of these with 3,431 participants (including T-Trials but not T4DM) (194). There was no significant difference in cardiovascular adverse events in testosterone vs placebo-treated men. The authors concluded that their results provide some reassurance about the short- to medium-term safety of T treatment for male hypogonadism (194). Clearly, the results of the TRAVERSE study, a testosterone cardiovascular safety trial, will be of considerable interest (195). For now, bearing in mind the limitations of meta-analyses of RCTs using reported adverse events as the endpoint, the weight of the currently available evidence from these sources indicates that T treatment is not associated with risk of cardiovascular adverse events.

 

RETROSPECTIVE CASE-CONTROL STUDIES

 

Retrospective Studies of Testosterone Prescriptions 

 

Pending an adequately powered T RCT to clarify its effect on the risk of cardiovascular events, retrospective case-control studies have sought to fill this gap (Table 5). These studies are typically based on health insurance databases recording prescriptions for T and subsequent outcomes in men prescribed or not prescribed T. Limitations of these studies include lack of clinical data such as indications for prescribing, the absence of randomization and the possibility of recall and misclassification bias (196). Initial studies in male veterans and in men with type 2 diabetes associated T prescriptions with lower mortality (197,198). By contrast, two studies associated T prescriptions with increased risk of major cardiovascular events (199,200). Both have been subject of criticisms: the first over confusing statistical methodology and data inaccuracies (resulting in publication of an erratum), the second over the lack of an appropriate comparison group (209,210). Subsequent studies have associated T prescriptions with no increase in risk of MI (201), and with lower risk of death, MI and stroke (202,203). An interesting distinction was made in the studies by Sharma et al. (2015) and Andersen et al. (2016) in that men who were prescribed T who then had “normal” T concentrations, did better than men who had persistently low T concentrations or who did not receive T (202,203). However, it is important to note that these studies did not systematically assess testosterone concentrations at multiple times or multiple days. A single measurement of testosterone on a particular day, may not be an accurate reflection of testosterone concentrations achieved over sustained periods of time with different testosterone formulations (211).

 

Table 5. Retrospective Case-Control Studies of Men Prescribed T that Examined Associations of T Prescriptions with Cardiovascular Events and Mortality in Middle-Aged and Older Men
Study characteristics				Results
Study author and year	Size (n of men)	Follow-up (yr)	Age (yr)	Favors no T	Favors T
Shores MM, 2012 (197)	1,031	3.4	62.1		Male veterans with total T ≤8.7 nmol/L, T prescribed in 398. T supplementation associated with lower mortality.
Muraleedharan V, 2013 (198)	581	5.8	59.5		Men with Type 2 diabetes, 238 with total T ≤10.4 nmol/L. T supplementation associated with lower mortality.
Vigen R, 2013 (199)	8,709	2.3	63.4	Male veterans who had coronary angiography and total T ≤10.4 nmol/L. T prescription associated with increased risk of death, MI or stroke.
Finkle WD, 2014 (200)	55,593	90 days	54.4	Men prescribed T. Higher rate of non-fatal MI in 90 days following prescription compared to preceding 1 year.
Baillargeon J, 2014 (201)	6,355; 19,065	4.1; 3.3	≥66		6,355 men prescribed T vs 19,065 matched non-users. T prescription not associated with increased risk of MI. For men with worse prognostic scores, T associated with reduced risk of MI.
Sharma R, 2015 (202)	83,010	6.2; 4.6; 4.7	66		Male veterans with low T. TRT resulting in normalization of circulating T (n=43,931) was associated with lower risk of death, MI and stroke, compared to TRT without normalization of T (n=25,701) or no TRT (n=13,378).
Anderson J, 2016 (203)	4,736	≥3	61.2		Men with T <7.4 nmol/L. T therapy achieving normal T (n=2,241) was associated with reduced risk of MACE compared to persistent low T (n=801). T therapy achieving either normal T or high T (n=1,694) associated with lower all-cause mortality compared to persistent low T.
Wallis CJD, 2016 (204)	10,311: 28,029	5.3	≥66		Men treated with T. T treatment associated with lower mortality HR=0.88 (95% CI=0.84-0.93) and prostate cancer risk HR=0.86 (95% CI=0.75-0.99). Shorter exposure (2 months) associated with increased risk of cardiovascular events and mortality, longer exposure (35 months) with reduced risk.
Cheetham TC, 2017 (205)	8,808: 35,527	3.2	58.4		Men ≥40 years, diagnosis or T <10.4 nmol/L. T associated with reduced risk of outcome of MACE, unstable angina, coronary revascularization, TIA. HR=0.67 (95% CI=0.62-0.73).
Loo SY, 2019 (206)	15,401	4.7	≥45	Men with low T and no evidence of HPT axis disease. T associated with increased risk of composite outcome of stroke/TIA/MI (HR=1.21, 95% CI 1.00-1.46), with risk highest in first 6 months to 2 years of T use (HR 1.35, 95% CI, 1.01-1.79). Risk of all-cause mortality lower with current T use (HR=0.64, 95% CI 0.52-0.78) and higher with past T use (HR=1.72, 95% CI 1.21-2.45), compared with non-use.
Oni OA, 2019 (207)	1,470	3.2-4.0	≥50		Male veterans with low total T and history of MI. All-cause mortality lower in men treated with T who normalized total T (N=755), vs men treated with T who did not normalize total T (N=542, HR=0.76, 95% CI 0.64-0.90), or men not treated with T (N=173, HR=0.76, 95% CI 0.60-0.98). No significant difference in the risk of recurrent MI between groups.
Shores MM, 2021 (208)	204,857	4.3	60.9		Male veterans with low T. Current transdermal T use not associated with risk for incident MI/ischemic stroke/venous thromboembolism (HR=0.89, 95% CI 0.76-1.05) in men without prevalent CVD, and in those with prevalent CVD was associated with lower risk (HR=0.80; 95% CI, 0.70-0.91). Current intramuscular T use not associated with risk for composite endpoint in men without or with prevalent CVD (HR=0.91, 95% CI 0.80-1.04; HR=0.98, 95% CI 0.89-1.09, respectively).

MI=myocardial infarction, TRT=testosterone replacement therapy, MACE=major cardiovascular adverse event comprising death, non-fatal MI and non-fatal stroke, TIA=transient ischemic attack.

 

A study by Wallis et al. (2016) found that T treatment was associated with lower mortality overall, but men who had T for a relatively shorter duration of exposure had increased risk, while men with longer duration of exposure had reduced risk (204). In a study by Cheetham et al. (2017) of men aged ≥40 years diagnosed with low T or with T <10.4 nmol/L, T treatment was associated with a reduced risk of major cardiovascular adverse events (205). Loo et al. (2019) reported contrary findings: analyzing a cohort of men with no evidence of HPT axis disease via the UK Clinical Practice Research Datalink, they associated use of testosterone with higher risk of a composite outcome of stroke, transient ischemic attack or MI, with the risk highest in the first six to 24 months of T use (206). In that study all-cause mortality risk was lower with current T use, and higher with past T use. Those findings are not supported by two more recent analyses, involving men with prior heart disease or with multiple comorbidities (207,208). In a study of male veterans with low T concentrations and a history of MI, men receiving T treatment who had a subsequent normal testosterone concentration had a lower risk of death from any cause compared to men receiving T treatment who had a subsequent low T concentration (207). Those men also had a lower risk of death compared with men who did not receive T treatment. Finally, in a cohort of 204,857 male veterans with a mean age of 60.9 years and 4.7 chronic medical conditions who were followed for 4.3 years, current transdermal T use was not associated with risk for the composite outcome of incident MI, ischemic stroke or venous thromboembolism in men without prevalent CVD (208). On the other hand, it was associated with lower risk in men with prevalent CVD. In that study, current intramuscular T use not associated with risk for the composite endpoint in men without or with prevalent CVD (208).

 

In an earlier retrospective cohort study that compared the use of T gel with T injections, T injections were associated with greater risk of cardiovascular adverse events (212). Bearing in mind the limitations of non-randomized studies and the possibility of bias, and the absence of a control group not receiving T, an additional factor is that more than 90% of the T injections were T cypionate, enanthate or propionate (212), which are short acting formulations typically requiring fortnightly administration with marked fluctuations in blood concentrations. The analysis would not apply to long-acting depot injections of T undecanoate typically administered every 12 weeks, which provide more stable pharmacokinetics (128). It is worth noting in this context that a population-based case-control study (involving 19,215 patients with confirmed venous thromboembolism and 909,530 age-matched controls) found an increased risk of venous thromboembolism within the first six months of T treatment but not thereafter (213). By contrast a systematic review and meta-analysis including six RCTs (2,236 participants) and five observational studies (1,249,640 participants) found no evidence of an association between T treatment and venous thromboembolism (214). However, the authors of that study noted that the available RCT data might have had inadequate power to detect an increased risk.

 

In summary, earlier findings associating T prescriptions with adverse cardiovascular outcomes were echoed in a more recent study. However, most studies do not show such adverse signals, and associated T use with lower risk of adverse cardiovascular events or mortality, including several recent large studies. There is a suggestion that T treatment which achieves normal T concentrations may relate to lower risk of cardiovascular events and mortality. Bearing in mind the limitations of these retrospective, observational and non-randomized studies, which cannot prove causality, the available data provide some reassurance but are far from definitive.

 

Abuse of Androgenic Steroids

 

Androgenic steroids can serve as appearance and performance-enhancing drugs and are abused by some competitive athletes, recreational sportspersons and body builders (1,215,216). The use/abuse of androgenic steroids occurs in contravention of medical advice and applicable sporting regulations, typically without medical supervision using unapproved formulations often in excessive doses (216). The use/abuse can be interspersed with periods of non-use. Adverse effects include suppression of the endogenous HPT axis, reduced spermatogenesis and impaired fertility, decreased testicular volume, hair loss and gynecomastia and are well-recognized (1,215). There is also an appreciation that long-term abuse results in cardiovascular toxicity in the form of myocardial dysfunction and accelerated coronary atherosclerosis (217). However, this study could be confounded as men who abuse androgenic steroids may also consume many other substances and the androgen preparations might have harmful contaminants. Abuse using pharmacological dosing of various products is very distinct from medically supervised T therapy aiming to achieve physiological circulating concentrations of T (128). Nevertheless, this is a reminder that excessive exposure to androgens carries the risk of harm (1).

 

DISCUSSION

 

Lessons from the Available Evidence

 

Epidemiological data are consistent with a protective role for endogenous androgens against CVD. In some studies of middle-aged and older men, lower circulating concentrations of endogenous T are associated with higher incidence of cardiovascular events, particularly stroke. Lower circulating T and DHT concentrations have also been associated with higher cardiovascular mortality (discussed in sections 2.1-2.2). Potential mechanisms by which T could exert beneficial actions in the vasculature have been explored in experimental models. These include reduced cholesterol accumulation and modulation of inflammation (sections 3.1-3.4). Clinical studies have reported favorable effects of T treatment on angina symptoms and exercise tolerance, but its effect on subclinical atherosclerosis remains uncertain (sections 4.1-4.2). The T-Trials, T4DM and meta-analyses of existing T RCTs in general do not show any signal for cardiovascular adverse events (sections 5.1-5.2). Retrospective case-control studies have reported contrasting results but in general, men receiving T prescriptions appear to have lower risk of major cardiovascular events and lower mortality compared to men who did not receive T, particularly if T treatment was associated with subsequent normal concentrations of circulating T (section 6.1). It is important to bear in mind that there are contrasting findings, and beneficial associations of T with cardiovascular outcomes may be less evident in healthier middle-aged men. Therefore, epidemiological evidence and mechanistic data could be used to argue for an anti-atherogenic or a protective effect of T on the cardiovascular system, as could the majority of retrospective case-control studies. However, this remains to be proven in the context of prospective RCTs of T intervention.

 

Gaps in the Current Evidence Base

 

RCT data are lacking as to whether treatment of middle-aged and older men with T would reduce the risk of cardiovascular events. The T-Trials which used transdermal T gel over a 12-month intervention offer important evidence as to benefits of T treatment for sexual function, anemia and bone density in older men without apparent diseases of the HPT axis, who had lower circulating T concentrations compared with younger men and symptoms suggestive of (but not diagnostic for) androgen deficiency (174,176). T4DM demonstrated the benefit of T treatment to reduce the risk of type 2 diabetes in men at high risk, beyond the effects of a lifestyle intervention (175). T4DM also showed a beneficial effect of T treatment on sexual function, and on bone microarchitecture and density (175,218). The T-Trials and T4DM are also noteworthy for the absence of any adverse cardiovascular safety signal for T treatment in these populations of men (174,175). However, the findings of the T-Trials Cardiovascular sub-study regarding an increase in total coronary atheroma plaque volume, in men with substantial baseline atheromatous disease, require clarification (168).

 

Major evidence gaps pertain to the effects of T on the cardiovascular system, as to whether T acts to slow development or progression of coronary or carotid atheromatous plaque in middle-aged and older men, in the differing contexts of either primary or secondary prevention for CVD. If the action of T is to reduce cholesterol accumulation, and to reduce inflammation and neointimal response to injury (sections 3.1-3.4) then these actions may have more impact to prevent or reduce progression of early atherosclerosis, rather than to reverse established disease. The related questions are whether T intervention in a primary prevention setting will reduce growth of coronary or carotid atheromatous plaque, or whether in a secondary prevention setting T intervention would influence the incidence of cardiovascular events. Another important question relates whether transdermal vs depot intramuscular (T undecanoate) formulations of T have similar or differing effects on the cardiovascular system.

 

Neither T-Trials nor T4DM had any cardiovascular endpoints and will not answer the question as to whether T exerts beneficial, neutral or adverse effects on the cardiovascular system. The US multicenter RCT “A study to evaluate the effect of testosterone replacement therapy (TRT) on the incidence of major adverse cardiovascular events (MACE) and efficacy measures in hypogonadal men (TRAVERSE)” commenced recruitment in 2018 of men aged 45-80 years with T <10.4 nmol/L (<300 ng/dl) with evidence of CVD or at increased risk for CVD (195). TRAVERSE was designed as a cardiovascular safety study with the endpoint of myocardial infarction, stroke or death due to cardiovascular causes, aimed to enroll 6,000 men randomized to transdermal T gel or placebo, and is planned to complete in 2022. TRAVERSE will also examine outcomes of prostate cancer, sexual function, bone fractures, depression, anemia and diabetes (195). TRAVERSE will address the issue of the cardiovascular safety of T treatment in what would largely be a secondary prevention setting. This leaves unanswered the question of whether T intervention in a primary prevention setting would reduce development or progression of coronary or carotid atheroma.

 

Application to Clinical Practice

 

Current clinical practice recommendations prioritize the identification of men with classical or pathological hypogonadism who are androgen deficient due to diseases of the hypothalamus, pituitary or testes (6,7). In such men, T treatment consistently resolves symptoms and signs of androgen deficiency (6,7,19). In men with classical or pathological hypogonadism the benefits of T treatment likely outweigh possible cardiovascular risks. In any case, individualized assessment and management of cardiovascular risk factors and disease should be part of routine clinical care. Of note, the US regulatory agency required labelling to warn of a possible increased risk of cardiovascular events with T, but the European regulatory agency concluded there was no consistent evidence of increased risk of coronary heart disease with T therapy (19). Until more evidence is available, it may be prudent to adopt a degree of caution in older men who are frail or who have known CVD, and to optimize management of cardiovascular risk factors and disease before starting T treatment. Treatment should aim for physiological replacement of T using approved formulations and avoid excessive doses (128).

 

It is beyond the scope of this chapter to discuss controversies regarding the management of men with low endogenous T concentrations due to obesity or presence of systemic illnesses where the HPT axis is intact, but its activity may be suppressed (19,31). However, it is worth noting that in men where a clear indication for T treatment is lacking, the risks and benefits of an intervention need to be considered with special care. Further research is needed to determine whether and how T treatment might impact on the risk of CVD in men.

 

Conclusions

 

Some epidemiological studies have associated higher circulating concentrations (but within the normal range) of endogenous androgens with lower risk of cardiovascular events and mortality. In men with pathological hypogonadism, the benefits of T treatment likely outweigh putative risks of cardiovascular adverse events. However, the effects of exogenous androgens in the form of T therapy seeking to maintain physiological circulating androgen concentrations on the cardiovascular system remain uncertain. Additional information will be forthcoming once the results of the TRAVERSE trial are known. Current clinical care of hypogonadal men should recognize this evidence gap and allow for individualized assessment and management of pre-existing cardiovascular risk factors and disease in men requiring T therapy. Well-designed and adequately powered RCTs are needed to clarify whether T treatment has beneficial, neutral or adverse effects on the cardiovascular system in the general population of middle-aged and older men.

 

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ABSTRACT

 

Men continue to have a strong interest and commitment to effective family planning. Traditional methods of male contraception have long included periodic abstinence, non-vaginal ejaculation, condoms, and vasectomy, the latter two representing physical methods to prevent sperm from reaching the site of fertilization. However, for male contraception the reversible methods are not reliable, and the only reliable method, vasectomy, is not intended as reversible. During the 20^th century, a wide array of reversible and highly reliable female hormonal contraceptive methods was marketed; however, no new methods for male fertility regulation have been introduced for centuries. For men to share more equally the burdens as well as the benefits of family planning, more effective reversible male contraceptive methods need to be available. Most studies into male contraception have been conducted with hormonal methods, analogous to well-known female hormonal contraceptives, making them the closest to introducing a reliable, reversible male contraceptive method. The most promising hormonal approach is the combination of an androgen (usually testosterone) with a progestin and multiple studies have shown such combinations of depot steroids displays high contraceptive efficacy, based on reliable and reversible suppression of sperm output, with few side effects. While research into novel methods for male fertility regulation has continued in the public sector, private sector research into male contraception, essential for effective commercial product development, has stalled in recent decades.

 

BACKGROUND

 

A male contraceptive must reduce the number of fertile sperm ejaculated into the vagina to levels that reliably prevent fertilization (1). Conception can be prevented by diverting or suppressing sperm output and/or inhibiting sperm fertilizing capacity. So far, all male methods depend on mechanical means to reduce female exposure to sperm using either traditional drug and device-free methods (abstinence, withdrawal, non-vaginal intercourse) or contemporary male methods comprising condoms and vasectomy.  Unfortunately, among current male contraceptive options, the reversible methods are not reliable, and the reliable method is not intended as reversible. No new male contraceptive methods were introduced during the 20^th century contrasting with highly reliable, reversible hormonal contraceptives developed for women over the last half century. Yet male involvement in family planning remains extensive. Globally, one third of couples using contraception rely on methods requiring active male participation (2) and current male methods remain (together with medicated intrauterine devices) the most cost-effective contraceptive methods in the USA (3). While reflecting traditional and ongoing reliance of family planning on male involvement, greater participation by men in sharing the burdens as well as the benefits of effective family planning require developing more effective, reversible male methods. Such greater participation could ameliorate the still large unmet need for contraception globally as 40% of pregnancies are still unintended with half of unintended pregnancies ending in abortion and those rates are higher among developed countries (4). Family planning must always remain a matter of personal choice by couples. This is highlighted by the human rights abuses of coercive national family planning programs enforcing family limitation (“one-child family”), including through forced sterilization and abortion, in India and China (5, 6). Such coercive national programs originated in the 1970’s prompted by uncritical belief in the “population explosion”, notably from influential publications such as the Club of Rome’s “Limits to Growth” and Ehrlich’s “Population Bomb”, now discredited by their failed, alarmist predictions. India’s forced sterilization program was overturned by popular democratic action within a few years of its imposition whereas China’s one-child family policy operated for nearly 4 decades before its ending in 2016. These issues echo in medical mistrust as a barrier to acceptance of male contraception among the Black American minority (7).  

 

Extensive public sector clinical research over the last 4 decades (8), despite minimal pharmaceutical industry involvement, has proven in principle that hormonal male contraception is feasible. Proof-of-principle is established for androgen-based hormonal suppression of spermatogenesis (9) achieving full reversibility (10), sufficient short-term safety (11) and acceptability to men and women (12, 13) culminating in the crucial proof of reliable contraceptive efficacy using androgen alone (14-17) or depot (18) or daily use (19) androgen-progestin combinations. Demographic modelling shows that introducing new male contraceptive methods, even if adopted by only a minority of men, would significantly reduce unintended pregnancies in Nigeria, South Africa, and the USA (20). Surveys of participants in clinical male contraceptive studies suggest that men may prefer a male contraceptive to be provided by their family doctor rather than specialists (21); however, the practical knowledge of non-specialists remains unsatisfactory and would require specific training (22). In addition to boutique contraception for adventurous early adopters, practical niches for male contraception include contraception after birth, during breast-feeding, or after abortion (23). Nevertheless, despite the proof-of-principle, strong community interest, and medical priority on the need for new, reversible male contraceptives, pharmaceutical industry development has ceased. This market failure reflects pharmaceutical industry perceptions of low profitability coupled with high risk in the context where a better return on investment is expected for developing drugs in more lucrative, less risky areas of chronic disease compared with the higher product liability risk of developing novel contraceptives for healthy men to compete with existing low-cost female methods. The possibility that Western market failure might be overcome if the growing perception of needs for voluntary family planning as a national priority in rapidly industrializing countries such as China, India and Brazil (24), has proved a failed hope. Novel approaches to finance further research such as philanthropic private sector funding (25) and public-private partnerships  (26) have been developed. The 2022 overturning by the US Supreme Court of what was long accepted as a constitutional right to abortion has refreshed interest in developing wider coverage of effective contraception, including for men, to reduce the demand for abortion.

 

NON-HORMONAL METHODS

 

Traditional Methods

 

PERIODIC ABSTINENCE

 

Although theoretically effective, neither celibacy nor castration is an acceptable or practical contraceptive method. Periodic abstinence, the limiting of sexual intercourse to "safe" days (27), has high contraceptive efficacy if the rules are followed perfectly but the failure rates rise steeply with rule breaking (28). This cost- and device-free method is used by >40 million couples world-wide for family planning (2). The typical use 1^st year failure rate is ~25% with ~47% of couples continuing use at 1 year in USA (29) but 8% and 52%, respectively, in France (30, 31). While inherently safe, it has limited acceptability due to low reliability, inflexibility, and interference in the spontaneity of sex.

 

EX-VAGINAL EJACULATION

 

Withdrawal is a traditional male method of contraception whereby intercourse culminates in extra-vaginal ejaculation (32). Often overlooked as a contraceptive method, withdrawal, together with abortion, was the major pre-industrial method of family planning largely responsible for the demographic transition from high to relatively low birth rates in industrial nation states and continues to be used by 40 million couples (2). This cost- and device-free method has limited reliability in its demanding requirement for skill and self-control. The typical use 1^st year failure rate is 18-22% with 46% continuing use at 1 year in the USA (29, 33) and 10% and 55%, respectively, in France (30, 31). While safe and reasonably effective for experienced users, interfering with the pleasure of coitus leads to a correspondingly high failure rate in practice. Other sexual practices that avoid intravaginal ejaculation have also been used traditionally to avoid conception. These include masturbation, oral and anal intercourse, deliberate anejaculation and retrograde ejaculation (34).

 

CONDOM

 

After centuries of use in preventing sexually transmitted infections and pregnancy, now over 50 million couples rely on condoms for contraception (2). Condoms provide safe, cheap, widely available, user-controlled, and reversible contraception with few side-effects. In case of latex allergy, non-rubber (polyurethane, natural membrane) condoms can be substituted. Latex condoms are moderately effective at preventing pregnancy with a typical 1^st year failure rate of 18% with 43% continuing use at 1 year in the USA (29, 33) and 3.3% and 47%, respectively, in France (30, 31). Recent US data suggest a reduction in failure rate to 13% (35). The discrepancy from the estimated 2% perfect-use failure rate (29) is mainly to human error, notably rushed use (36), misuse, or non-use rather than mechanical failure (breakage or slippage) (37). Differences in contraceptive use behaviors may explain lower reported 1^st year failure rates for condoms in France (31). The major limitations of condoms for contraception are relatively high failure rates and interference with sexuality. The requirement for regular and correct application during foreplay disturbs the spontaneity of sex and dulls erotic sensation. These aesthetic drawbacks limit the popularity of condoms especially among stable couples (38). Latex condoms are perishable through tears or snagging on fingernails, clothing, or jewelry as well as deterioration from exposure to light, heat, humidity, or organic oils. 

 

Polyurethane condoms with improved tactile sensitivity were developed in the 1990’s to enhance acceptability (39), but they have shown reduced efficacy and mechanical performance compared with latex condoms in prospective randomized controlled clinical trials (40). Although the theoretical requirements for condom use to protect against sexually transmitted infections differ from those to prevent pregnancy, in practice the protections are similar (41). Laboratory testing of condoms standardizes integrity and durability for strength and leakage. Although viral penetration is not routinely tested, synthetic (latex or polyurethane) but not natural membrane condoms are not perfect at preventing passage of prototype human pathogenic viruses (42). Using a sensitive, objective biochemical marker (PSA) for seminal plasma exposure in vaginal swabs, vaginal exposure to semen was reduced by 50-80% even after mechanical condom failure (breakage, slippage) (43). Novel spermicides with virucidal properties that are being developed to provide dual antimicrobial and contraceptive protection (44)  might not enhance condom efficacy for either function because non-compliance is the major cause of failure for both (37).

 

VASECTOMY

 

Vasectomy, used by over 40 million couples for family planning (2), varies widely in prevalence between countries depending upon cultural factors, public education, and availability of male-oriented facilities (45). Recent data suggests a steady decline in vasectomy in the US over the first two decades of the 21^st century (46). Having overcome a sordid history of applications to eugenics and other historical misadventures into the 20^th century (47), vasectomy is now more widely used than female sterilization in some economically developed countries although female sterilization remain ~4 times more frequent worldwide (48). For men having completed their family and fit for minor surgery, vasectomy is a very safe and highly effective office procedure (49, 50). Relative contraindications include risks from office-type surgery (bleeding disorders, allergy to local anesthetic) or scrotal pathology (post-inguinal surgery scarring, keloid-proneness, active genitourinary or groin infections). Vasectomy is performed under local anesthesia via scrotal incisions and usually involves excising a segment of both vasa deferentia. Interposing a fascial barrier between the occluded cut ends significantly reduces the risk of failure due to recanalization (51, 52). The Chinese-developed "no-scalpel" technique (53)minimizes skin incision and reduces immediate side-effects (bleeding, infection) 10-fold to 0.3% compared with conventional vasectomy (54, 55). Chronic post-vasectomy pain (56) occurs with an estimated prevalence of ~15% at 7 months after vasectomy (57). It may be reported as groin or testicular pain precipitated or aggravated by intercourse, ejaculation, or exertion and accompanied by tender, distended epididymides (56, 57). Fascial interposition significantly reduces failure rate (52, 58) so that, in the hands of an experienced practitioner, no-scalpel vasectomy with fascial interposition is now the method of choice (55, 59). Additional studies suggest that cautery may further enhances reliability (60, 61) and that leaving an open testicular end reduces retrograde pressure-related complications (pain, sperm granuloma, epididymal and testicular damage) thereby better preserving reversibility (62-64).

 

Vasectomy is highly effective once sperm are cleared from the distal vasa deferentia. However, flushing with saline or water (65-69) or spermicides (nitrofurazone (70), euflavine (71, 72) or chlorhexidine (73)) during surgery does not accelerate sperm clearance, but the evidence remains weak (74). Non-irritant spermicides that inhibit sperm function without chemical sclerosis of the vasa that would impair potential reversibility, might have promise (75). Immediately following vasectomy, additional contraception must continue until azoospermia or near azoospermia (<0.1 million non-motile sperm per mL) is demonstrated usually at 3 months post-vasectomy when at least 95% of men reach this clearance criterion (76-79). Although azoospermia might occur sooner (80), reliable evidence is lacking to support the reliability of earlier time points as recanalization, where motile sperm persist in the ejaculate, may occur within the first few weeks after vasectomy (81) and persistence of motile sperm in the ejaculate indicates technical failure. Although detailed information on the rate of sperm clearance from the ejaculate after vasectomy remains sparse, time since vasectomy rather than number of ejaculations is more predictive of sperm clearance  (80). Contraceptive failures are rare; early failures are most often due to not awaiting sperm clearance (82) or occasionally misidentification or duplication of the vasa deferentia whereas late failures are due to spontaneous vas(a) recanalization (~0.1%) (83). Complications of vasectomy include post-surgical bleeding, wound or genito-urinary infections and fistulae, and chronic scrotal pain (50, 84) with risk of death estimated at ~1 per million vasectomies in developed countries although higher in developing countries (85). Vasectomy causes no consistent changes in circulating hormones (86), sexual function, or risk of cardiovascular or other diseases (49, 87, 88) including testicular cancer (89-91). A small increased risk of prostate cancer after vasectomy has been observed in case-control studies (92-94), but the increased risk is unaffected by vasectomy reversal (95, 96). This risk seems to be attributable to surveillance and detection bias, rather than a biological effect as indicated by consistent evidence from numerous cohort studies (96-102). Sperm antibodies develop in most vasectomized men but have no known deleterious health effects apart from a possible role in reducing fertility after vasectomy reversal (103) when sperm antibody titers are very high (>512) (104).

 

Vasectomy is a quick, simple, highly effective, and convenient method of permanent sterilization; its major drawback as a male contraceptive is its limited reversibility. Elective sperm cryostorage is occasionally useful but may reflect ambivalence about the irreversible intent of vasectomy. Cumulative rate of requests for reversal, mostly prompted by remarriage, are 2.4% at 10 year post-vasectomy but exceed 10% for young men (aged <25 years at vasectomy) (105).  Failed vasectomy reversal is now a significant cause of male infertility. Following microsurgical vasovasostomy, 80-100% have any sperm in the ejaculate ("patency") but normal sperm concentration is less common, and cumulative conception rate at 12 months is only ~50% compared to 80-85% at 12 months in healthy young couples not using contraception (105). This discrepancy is most probably attributable to technical limitations of microsurgery as even the lowest reported rates of azoospermia (bilateral non-patency) after microsurgical vasovasostomy indicate that nearly half such men have at least one non-patent vas deferens (106) so that re-operation should be a prominent consideration if pregnancy does not ensue. After technical failure, the wife’s age and time since vasectomy appear to be the dominant predictors of successful reversal (107, 108) Whether robotic microsurgery can improve the technical success of vasovasostomy to become a cost-effective and widely available alternative to human microsurgery remains to be established (109-111). Reversibility is better with microsurgery (106), presence of sperm in the vasal fluids from testicular end of stump (112), in younger men with shorter duration since vasectomy (105) and possibly with longer testicular vasal stump (113, 114); unfavorable predictors include non-microsurgical techniques, older age of wife (especially after 40 yr) (106, 115), high titers of sperm antibodies (104), and long duration (>10 years) since vasectomy (116-118) due to long-term epididymal (119), vasal (120) and testicular damage (116, 117, 121, 122).  Experimentally in a variety of mammalian species, open-ended vasectomy is preferable to complete vas occlusion in preventing the back pressure-induced damage to spermatogenesis and seminiferous tubular integrity which are important contributors to failure of vasectomy reversal (123, 124). An alternative to surgical vasectomy reversal either instead of, or after failed vaso-vasostomy, is sperm harvesting from epididymis or testis in conjunction with intracytoplasmic sperm injection (ICSI)/in-vitro fertilization. Currently cost-benefit analyses suggest that microsurgical vaso-vasostomy is more cost-effective and safer in both North America (125) and Europe (126), with the wife’s age being a key determinant (107). However, optimal management depends on local clinical expertise as well as access to microsurgery and reproductive technologies. The feasibility of successful vasectomy reversal has led to the proposal that, although vasectomy is intended as permanent, some individuals may consider it a reversible contraceptive method (127). USA national economic indicators strikingly influence rates of vasectomy and of vasectomy reversal, which are increased and decreased, respectively, according to the unemployment rate and personal income (128); whether this applies in countries with national health schemes that reduce financial limitations on access to elective health care remains unknown.

 

Modern Methods

 

VAS OCCLUSION

 

The efficacy, safety, simplicity, and acceptability of vasectomy suggest that a reversible mechanical method of vasa occlusion would be an attractive male contraceptive option. Since vasectomy reversal is neither cheap nor widely available, more reversible vasa occlusion methods are needed (129). A nonsurgical, potentially reversible technique involving percutaneous injections of polymers that harden in-situ to form occluding plugs which might be later removed to restore fertility was reported (130) but, despite preliminary positive findings (131), formal evaluation showed vasa occlusion had lower efficacy (inducing azoospermia) than vasectomy (132). In a phase II randomized clinical trial, urethane-coated nylon thread intra-vasa devices were more acceptable with fewer complications but were less effective in producing azoospermia, compared with no-scalpel vasectomy (133). A hydrophilic gel, composed of co-polymer of styrene and maleic anhydride delivered in dimethyl sulfoxide, forms a charged spermicidal biopolymer when injected into the vas deferens that is stable but potentially removable. Experiments in rats favor sodium bicarbonate over dimethyl sulphoxide for intravasal injection for effective reversal of occlusion (134). Preliminary non-comparative clinical evaluation showed azoospermia in 12 men with no pregnancies in their wives for 12 months following intravasal injection suggesting effective vasal occlusion (135); however, some morphologically damaged and non-functional sperm persist in the ejaculate (136) but the mechanisms of the deleterious effects on sperm structure and function remain unexplained(137). In an extended multi-center phase III study of 139 fertile married men who had a single, bilateral intravasal injection of a styrene maleic anhydride copolymer in 120 µl of dimethyl sulphoxide solution (Reversible Inhibition of Sperm under Guidance, RISUG) were followed for six months after injection. In six men, the injections failed to be delivered, but in the remainder, 110 achieved azoospermia within 1 month and 23 achieved azoospermia 3 to 6 months after the procedure. After ceasing condom use in the first 2 months after injection, there were no pregnancies in the 133 men with successful occlusion over the next 4 months whereas pregnancies were reported in two of six men who had failed injections; however, reversibility was not reported (138). Another phase III randomized controlled clinical trial has shown that a prototype implantable non-occlusive intra-vasal device, comprising a nylon thread encased in barium-impregnated polyurethane, is as effective for sterilization as standard no-scalpel sterilization with no more adverse effects (139); however the reversibility of this device remains to be demonstrated.   A promising development for a reversible vas occlusion is a solution for transcutaneous intravasal injection which forms a hydrogel in the vas to reversibly occlude it. The injection solution, comprising sodium alginate conjugated with thioketal and including titanium dioxide and calcium chloride, transforms after injection into the vas deferens into an occluding hydrogel. Subsequently, the occluding hydrogel can be cleared in vivo by non-invasive application of ultrasound that generates reactive oxygen species in the hydrogel causing cleavage of the thioketal and recanalizing the vas deferens. Experimental studies in rats demonstrated fully effective contraception and reversibility (140). Other technical developments including percutaneous injection of sclerosants and transcutaneous delivery of physical agents (ultrasound, lasers) continue to be developed slowly (141).

 

HEATING

 

It has long been known (142) that even brief elevations of testicular temperature can profoundly suppress spermatogenesis (143) while sustained elevation may contribute to testicular pathology in cryptorchidism, varicocele, and occupational male infertility (144). A highly novel experimental approach has been reported using intravenously injected iron oxide nanoparticles that accumulate in the testis where they can be activated by an external alternating magnet field to generate testicular heating and damage to spermatogenesis (145); however, safe clinical application, and notably reversibility, remain to be demonstrated. Clinical studies evaluating the potential for tight scrotal supports as a practical male contraceptive method (146, 147) showed a reversible decrease in sperm output but of inadequate magnitude for reliable contraception. Experience of small numbers of men utilizing thermal male contraception has been favorable, apart from discomfort (148) but wider uptake would require substantial population education (149). Given the dubious acceptability and safety (150) of heat-induced suppression of sperm output, the feasibility of a male contraceptive method based on testicular heating remains to be established.

 

IMMUNOCONTRACEPTION  

 

Sperm vaccines to interrupt fertility have long been of interest (151) with a 1937 patent issued for vaccination with semen (152). Sperm express unique epitopes within the immunologically protected adluminal compartment of the seminiferous tubules at puberty, long after the definition of immune self-tolerance hence explaining their potential autoimmunogenicity. Sperm autoimmunity may contribute to subfertility after vasectomy reversal and in ~7% of infertile otherwise healthy men (other than orchitis). Experimental models for an effective multivalent chimeric protein sperm vaccine targeting surface-expressed antigens involved in fertilization have been reported (153) but remain untested in men. Practical application of this method requires resolving problems of the millions of sperm ejaculated, representing a large antigenic load requiring virtually complete functional blockade, variability of individual immune responses, restricted access of antibodies into the seminiferous tubules and epididymis and the risks of autoimmune orchitis or immune-complex disease. Passive immunization may overcome the present limited predictability of active immunization with sperm antigens to reach quickly and maintain, as well as allowing for volitionally controllable offset, of effective immunocontraceptive titers (154). The smaller antigenic burden in the female reproductive tract requiring complete neutralization suggests that a sperm vaccine, using modern genetic engineering of sperm epitopes (155), may be better targeted for administration to women. However, the most suitable targets for contraceptive vaccines might be feral and wild animals (156, 157).

 

CHEMICAL (NON-HORMONAL) METHODS

 

The lack of commercial product development for hormonal male contraceptives has made attractive the alternative option of innovative non-hormonal mechanisms to inhibit sperm production and/or function. These approaches have focused on developing feasible, druggable targets lacking off-target adverse effects for novel product development using either opportunistic or planned approaches to identify novel leads.

 

Opportunistic approaches include the identification through fortuitous pharmacological observation of male reproductive effects of drugs or natural products. Among older drugs, an orally active spermicide concentrated in semen (158), drugs inhibiting spermatogenesis  (159), ejaculation (160) or epididymal sperm function (161) have been identified. Further, among numerous plant products and natural medicines reputed to inhibit male fertility, the most widely tested was gossypol, a polyphenolic yellow pigment identified in China as causing epidemic infertility among workers ingesting raw cottonseed oil. In over 10,000 men purified gossypol reduced sperm output to <4 million/ml in >98% within 75 days with suppression maintained by a lower weekly maintenance dose (162). Although an effective male contraceptive, the systemic toxicity of gossypol, due to mitochondrial apoptosis (163), and irreversibility of sperm suppression precluded further clinical development (164) although it has potential anti-cancer applications (163). Subsequently, extracts of Tripterygium wilfordii, a traditional Chinese herbal medicine for rheumatoid arthritis and skin disorders, decrease sperm output and function and inhibit fertility in rodents and men. Studies aiming to characterize triptolide, an active alkaloid as a potential lead for an orally effective sperm function inhibitor, reveal prominent induction of germ cell apoptosis (165) at testicular (166) in addition to a post-testicular site of action (167). Additional opportunistic approaches include recognizing that the rapidly proliferating germinal epithelium is highly susceptible to cytotoxins such as drugs, heat, or ionizing irradiation which damage germ cell replication, resulting in inhibition of spermatogenesis. However, complete elimination of sperm by non-specific toxicity compromises full reversibility and the accompanying mutagenic risk from direct interference with DNA replication precludes safe use for reversible male contraception.

 

Alternatively, potential non-hormonal approaches to developing chemical male contraception focus on sperm development maturation and function (168) ensuring that targets are specific for sperm (169). Such approaches may exploit the numerous biological processes required for developing functionally mature, fertile sperm that create abundant targets for reversible inhibition of male fertility (170) . Prominent targets include either reducing sperm output via non-hormonal mechanisms regulating spermatogenesis or post-testicular inhibition of sperm functions. One approach to reduce sperm output is by exploiting the essential requirement for retinoic acid in spermatogenesis (171). Experimental genetic or pharmacological inhibition of vitamin A action inhibits the generation of mature sperm and male fertility (172, 173); however, the ubiquitous roles of vitamin A in cellular replication and differentiation requires thorough safety evaluation for off-target effects in clinical trials. More specific focus on the retinoic acid alpha receptor by developing alpha-specific inhibitors continue in pre-clinical development (174). Another series of orally active indazole carboxylic acid analogs, adjudin, gamendazole and indenopyridine derivatives (175-178), have been developed from the compound lonidamine but aiming to eliminating its non-specific muscle and liver toxicity while retaining its mechanism of action in causing reversible male infertility. The indazole carboxylic acid analogs cause reversible subfertility by disrupting highly specialized intercellular junctions between elongating spermatids and Sertoli cells leading to precocious detachment of immature, elongating spermatids that are then shed prematurely from the germinal epithelium. Clinical evaluation of such drugs remains at an early pre-clinical stage. Other discoveries of molecules with specific expression in sperm and contributions to sperm development and/or fertilizing ability may provide clues to novel leads for chemical non-hormonal male contraceptive drug development. These include protein phosphatase complex (179), cyclin dependent kinase 2 (180), testis-specific bromodomain (181), homeodomain-interacting protein kinase 4 (182) and histone demethylase KDM5B (183).

 

The seclusion of functionally immature post-meiotic, haploid sperm during their transit through seminiferous tubules and epididymis offers targets for chemical methods to regulate male fertility as sperm are stored and mature functionally. Post-testicular targets offer the advantages of fast onset and offset of action compared with hormonal methods; however, specific target identification, selective drug targeting to the epididymis or testis and human dose optimization remain challenging problems. A model, rapid-onset oral spermicide was first provided by the chlorosugars that showed rapid, irreversible effect on rodent epididymal sperm (184) but proved too toxic for clinical development. A recent promising drug lead was the recognition that an alkylated iminosugar drugs that inhibit glucosyltransferase, used therapeutically to reduce lysosomal glycosphingolipid accumulation in storage disorder type 1 (Gaucher's disease), miglustat, was a potent and reversible oral inhibitor of male mouse fertility but free from apparent systemic toxicity (161, 185). Miglustat treatment produced structural malformation of sperm acrosome, head and mid-piece with consequential impaired motility although sperm retain the ability to fertilize oocytes in-vitro and produce normal offspring. However, miglustat effects were species- and mouse strain-dependent and were not effective in rabbits (186) or men (187). Numerous proteins identified as specifically or uniquely expressed in the epididymis provide additional opportunities for development of novel non-hormonal male contraceptive targets (188-192). Signaling enzymes involved in the epididymal acquisition of sperm motility and capacity for hyperactivation and fertilization such as sperm-specific protein phosphatase PPIγ2, glycogen synthase kinase 3 (GSK3), and calcium regulated phosphatase calcineurin (PP2B) and protein kinase A (PKA) have key role in sperm maturation, activation and fertilization that might provide clues to novel chemical, non-hormonal male contraceptive drugs (193). Post-testicular inhibition of purinergic and adrenergic receptors in the vas deferens to inhibit sperm movement through the ejaculatory ducts are feasible (194, 195), and a series of these receptor antagonists have been developed (196).  However, interference with ejaculation is unlikely to be an acceptable form of male contraception. An opportunistic approach based on a FDA-approved excipient, N,N dimethylacetamide that produced a reversible reduction in fertility of rats without hormonal effects has been reported (197).

 

The most rapidly growing area of opportunity arises from serendipitous discoveries of genes found to be necessary for normal fertility, often from gene knock-out mouse models displaying unexpected male sterility or subfertility (170). This rapidly expanding list includes distinctive steps in spermatogenesis involving either biological processes unique to spermatogenesis, notably meiosis (two-stage reductive division of diploid germinal cells into haploid gametes) and spermiogenesis (metamorphosis of haploid round spermatids into spermatozoa) or inhibiting functions essential to sperm fertilizing ability such as flagellar motility and sperm motion (including hyperactivation (198)), excurrent ductular transport, acrosome reaction (199), chemoattraction to and fertilization of oocytes (194, 200), acrosomal function (201)and testis-specific serine/threonine kinases involved in development of normal sperm morphology and function (202, 203). The most frequent mechanisms involves inhibition of ion channels in sperm (200, 204-211) or vas deferens smooth muscle (194, 195) leading to inhibition of sperm motility, hyperactivation and/or transport required for fertilization. One leading candidate is CatSper, the principal calcium channel activated by progesterone (212-216) and essential for the sperm hyperactivation required for fertilization (217). Experimental vaccines against CatSper epitopes inhibit murine fertility (218, 219).

 

In addition, drug screening programs using extensive high-throughput chemical libraries can be employed to identify suitable lead compounds using selective sperm function targets for further development including target optimization (220). For example, the inhibitor analogs of CatSper using patch clamping to identify channel blockade have identified probes suitable for drug discovery development (221). Similarly, screening of a chemical library identified phosducin-like 2, a testis specific phosphoprotein, which when knocked-out in mice, induced sterility due to globozoospermia with impaired sperm head formation (222) as well as screening of natural plant (223-225) and microbial (226) products continue as a source for lead compounds continues. Other products of drug screening that may interfere with DNA replication by targeting meiosis, incur additional theoretical safety risks of mutagenesis if intended for male contraception (227).

 

HORMONAL METHODS

 

Hormonal methods are the closest to meeting the requirement for a reliable, reversible, safe, and acceptable male contraceptive. Although reliability is judged by the efficacy in preventing pregnancy in fertile female partners, as a hormonal male contraceptive aims to prevent pregnancy by reversible inhibition of sperm output, the suppression of spermatogenesis constitutes a useful surrogate marker for development and evaluation of prototype male contraceptive regimens. This makes defining the degree of suppression of sperm output required a key strategic issue in developing a hormonal male contraceptive (228).

 

Two landmark WHO studies, the first ever male contraceptive efficacy studies, involving 671 men from 16 centers in 10 countries established the proof of principle that hormonally-induced azoospermia provides highly reliable, reversible contraception (14, 15). Among the minority (~25%) of men who remained severe oligozoospermic (0.1-3 million sperm/ml) using weekly testosterone enanthate injections, contraceptive failure rate (~8% per annum) was directly proportional to their sperm output. Hence to achieve highly effective contraception, azoospermia is analogous to anovulation as a sufficient, but not necessary, requirement. Nevertheless, reliable contraception by modern standards (29) requires uniform azoospermia as the desirable target for male contraceptive regimens (229). No regimen yet achieves this consistently in all men, although in some Asian countries (e.g. China (15), Indonesia (230, 231)) an approximation to uniform azoospermia can be achieved by a variety of regimens. A study involving 308 Chinese men in 6 centers has shown that monthly injections of testosterone undecanoate provides highly effective and reversible contraception (17). No pregnancies were recorded among men who were azoospermic or severely oligozoospermic (≤3 million sperm per mL) providing a 95% upper confidence limit of pregnancy (contraceptive failure) rate of 2.5% per annum. The overall failure rate based on suppression of spermatogenesis was <4%. The prototype regimen was well tolerated apart from injection site discomfort due to large oil injection volume (4 mL) and reversible androgenic effects (acne, weight gain, hemoglobin, lipids). Nevertheless, despite these promising findings, non-Chinese men require combination hormonal regimens involving a 2^nd gonadotropin suppressing agent, notably progestins, together with testosterone to ensure uniformly adequate spermatogenic suppression. Proof-of-principle for this combination approach was provided by a depot androgen/progestin regimen that observed no pregnancies among 55 couples during 35.5 person-years of exposure (95% upper limit of failure rate ~8%) in a study with satisfactory tolerability and reversibility for a prototype regimen (18). Hence, contraceptive efficacy studies show that highly effective contraception can be achieved with suppression of sperm output to near azoospermia (≤1 million per mL) (229). Ultimately, the efficacy of male contraception must be established by enumerating pregnancies prevented, and not counting sperm. The present paucity of male contraceptive efficacy studies, for which placebo controls are ethically impossible (232), makes systematic evaluations comparing practical clinical regimens a task for the future (233).

 

The reversibility of hormonal male contraceptive regimens is clearly established by an integrated re-analysis that pooled primary data from over 90% of all hormonal male contraceptive studies reported to show that all regimens show full reversibility within a predictable time course (234). This comprehensive review of the recovery of 1549 healthy eugonadal men, aged 18-51 years, who underwent 1283.5 man-years of treatment and 705 man-years of post-treatment recovery, showed the median times for recover to sperm densities of 10 and 20 million per mL were 2.5 months (2.4-2.7) and 3.0 months (2.9-3.1), respectively. Covariables such as age, ethnicity and hormonal or sperm output kinetics had significant but minor influence on the rate, but not the extent, of recovery.

 

Acceptability of a hormonal male contraceptive is high across a wide range of countries and cultures in potential male as well as female users (12). Willingness to use a hypothetical hormonal male contraceptive averaged 55% (range 29-71%) in an extensive, population representative survey of 9342 men aged 18-50 yr from 9 countries (4 Europe, 3 South America, Indonesia and USA) with consistency across a wide range of socio-economic and cultural settings (235, 236). Similar findings are reported in a 4-country study (UK, South Africa, Hong Kong, Shanghai) with 44-83% in each center (237) as well as 75% in Australia willing to try a hormonal male contraceptive (238). A majority of men in a US study were satisfied with and would recommend a transdermal gel-based hormonal contraceptive (239) and a majority of Chinese men were satisfied with monthly injections (240). Female partners from a variety of cultures also indicate high acceptability in a survey of 1894 women in 4 countries, among whom 40-78% would support and trust their male partners in stable relationships to use a hormonal male contraceptive (241). Corroborating the acceptability of hormonal male contraception are findings from experimental studies of prototype regimens for up to 12 months usage in which most participants confirm high levels of satisfaction and willingness to try a commercial product (239, 240, 242, 243).

 

Modern safety evaluation for hormonal contraceptive methods requires long-term, large scale studies of marketed products (244). Consequently, in the absence of any marketed male contraceptive, no long-term safety profile can be discerned. Nevertheless, 4 decades of clinical trials have consistently identified mainly minor adverse effects in short- or medium-term studies of prototype male hormonal contraception regimens (1, 8, 228). This was verified in an unique, placebo-controlled study of a combined androgen-progestin regimen which again proved highly effective at reversible suppression of sperm output although the inclusion of a placebo arm rendered it ethically impossible to evaluate contraceptive efficacy but provided insight into drug-related side effects (11). The study confirmed the benign medium-term safety profile for this prototype regimen in observing few serious adverse effects, none attributable to the steroid regimen, and, among the non-serious adverse effects reported, expected androgenic effects (acne, weight gain, sweating, changes in mood or libido) were more frequent than placebo but mild and rarely led to discontinuation (11). Nevertheless, the long-term safety evaluation of novel contraceptives requires large scale observational studies of extensively used drugs to define low frequency risks but can only occur after the marketing of suitable products.

 

Hence, prototype hormonal methods have proven reliability and reversibility and reasonable prospects for being well accepted and safe. Although they are the most likely opportunity in the foreseeable future to develop a practical contraceptive method for men, progress depends on pharmaceutical industry development. However, leadership in male contraceptive development has come almost exclusively from university-based investigators working with public sector organizations (8), notably WHO (245), CONRAD (246), and the Population Council (247). By contrast, commitment from pharmaceutical companies, including those that flourished in the post-war decades through developing female hormonal contraception, continued to languish over recent decades (24, 248) and is effectively ceased (249).

 

Steroidal Methods

 

ANDROGEN ALONE

 

Testosterone provides both gonadotropin suppression and androgen replacement making it an obvious first choice as a single agent for a reversible hormonal male contraceptive. Although androgen-induced, reversible suppression of human spermatogenesis has long been known (250-253), systematic studies of androgens for male contraception began in the 1970's (254, 255). Feasibility and dose-finding studies (256), mostly using testosterone enanthate (TE) in an oil vehicle as a prototype, showed that weekly im injections of 100-200 mg TE induce azoospermia in most Caucasian men (257)but less frequent or lower doses fail to sustain suppression (258-261). The largest experience with an androgen alone regimen arises from the two WHO studies in which over 670 men from 16 centers in 10 countries received weekly injections of 200 mg TE. In these studies ~60% of non-Chinese and >90% of Chinese men became azoospermic and the remainder were severely oligozoospermic (14, 15) (Figure 1). The high efficacy among Chinese men has also been replicated using monthly TU injections (16, 17). Effective gonadotropin suppression is a prerequisite for effective testosterone-induced spermatogenic suppression in human (18, 262-266) and non-human primates (267, 268). However, the reasons for within and between population differences in susceptibility to hormonally-induced azoospermia remain largely unexplained (269). Possible factors include population differences in reproductive physiology of environmental (270, 271), genetic (272-274) or uncertain (275, 276) origin that may lead to differences in rate and extent of suppression of circulating gonadotropins and/or depletion of intratesticular androgens. Limited invasive studies measuring intratesticular testosterone (and DHT) suggest that the degree of depletion may not predict reliably complete suppression of sperm output  (277-279) but other more widely applicable, non-invasive markers of endogenous Leydig cell function such as circulating epitestosterone (262) or 17-hydroxyprogesterone (280) or non-steroidal testicular products such as INSL3 (281) may be more analytically informative as to the relative roles of gonadotropin suppression and intratesticular androgen depletion. Exogenous testosterone causes suppression of sperm output with an average of 13 weeks to reach severe oligozoospermia (<1 million per mL) or azoospermia with suppression being maintained consistently during ongoing treatment (282) (Figure 2). Following cessation of treatment, sperm reappear within 3 months to reach sperm densities of 10 and 20 million per mL at an average of 11.5 and 13.6 weeks, respectively (282)with ultimately full recovery (10) (Figure 3). Apart from intolerance of weekly injections, there were few discontinuations due to acne, weight gain, polycythemia, or behavioral effects and these were reversible as were changes in hemoglobin, testis size, and plasma urea. There was no evidence of liver, prostate, or cardiovascular disorders (14, 15, 283).

 

The pharmacokinetics of testosterone products are crucial for suppressing sperm output. Oral androgens have major first-pass hepatic effects producing prominent route-dependent effects on hepatic protein secretion (e.g., SHBG, HDL cholesterol) and inconsistent bioavailability. Short-acting testosterone products requiring daily or more frequent administration (oral, transdermal patches or gels) which may be acceptable for androgen replacement therapy present serious feasibility challenges in compliance for effective long-term hormonal contraception. Weekly TE injections required for maximal suppression of spermatogenesis (256) are far from ideal (284) and cause supraphysiological blood testosterone levels risking both excessive androgenic side effects and preventing maximal depletion of intratesticular testosterone for optimal efficacy (285, 286). Other currently available oil-based testosterone esters (cypionate, cyclohexane-carboxylate, propionate) do not differ from the enanthate ester (287), but longer-acting depot preparations such as injectable testosterone undecanoate are a substantial improvement. Subdermal testosterone pellets sustain physiological testosterone levels for 4-6 months (288) and the newer injectable preparations testosterone undecanoate (17), testosterone-loaded biodegradable microspheres (289) and testosterone buciclate (290) provide 2-3 months duration of action. Depot androgens suppress spermatogenesis faster, at lower doses and with fewer metabolic side effects than TE injections but azoospermia is still not achieved uniformly (291) although when combined with a depot progestin, this goal is achievable (18).

 

Oral synthetic 17-a alkylated androgens such as methyltestosterone (292), fluoxymesterone (293), methandienone (294)and danazol (295, 296) suppress spermatogenesis but azoospermia is rarely achieved and the inherent hepatotoxicity of the 17-a alkyl substitutent (297, 298) renders them unsuitable for long-term use. Athletes self-administering supratherapeutic doses of androgens also exhibit suppression of spermatogenesis (294, 299). Synthetic androgens lacking the 17-a alkyl substituent have been little studied although injectable nandrolone esters produce azoospermia in 88% of European men (300, 301) whereas oral mesterolone is ineffective (302). On the other hand, nandrolone hexyloxyphenylpropionate alone was unable to maintain spermatogenic suppression induced by a GnRH antagonist (303) in a prototype hybrid regime (where induction and maintenance treatment differ) whereas testosterone appears more promising (304). A 7-methyl derivative of nandrolone (MeNT), which is partly aromatisable but resistant to 5α-reductive amplification of androgenic potency, has been studied as a non-oral androgen for hormonal male contraceptive regimens (305). While its non-amplification by 5α reduction may be  theoretically prostate-sparing (306), dose titration to achieve essential androgen replacement at each relevant tissue might be more difficult to achieve than for testosterone (307) or impossible. Non-aromatisable androgens which lack estrogen- receptor-mediated effects such as on bone density maintenance (308) and sexual function (309, 310) may not be ideal for long-term hormonal contraception. More potent, synthetic androgens lacking 17-a alkyl groups (311, 312) remain to be evaluated.

 

Adverse effects due to testosterone administration in prototype hormonal contraceptive regimens include (asymptomatic) polycythemia, weight (muscle) gain, and acne as well as changes in mood or sexual behavior. These are usually minor in severity, reversible upon cessation of treatment and of minimal clinical significance (11).

 

The safety of androgen administration concerns mainly potential effects on cardiovascular and prostatic disease As the explanation for the higher male susceptibility to cardiovascular disease is not well understood, the risks of exogenous androgens are not clear (313, 314). In clinical trials, lipid changes are minimal with depot (non-oral) hormonal regimens (18, 291, 315, 316). Changes in blood cholesterol fractions observed during high hepatic exposure to testosterone and/or progestins, due to either oral first pass effects or high parenteral doses, have unknown clinical significance but, in any case, maintenance of physiological blood testosterone concentrations is the prudent and preferred objective. The real cardiovascular risks or benefits of hormonal male contraception will require long-term surveillance of cardiovascular outcomes (317).

 

The long-term effects of exogenous androgens on the prostate also require monitoring since prostatic diseases are both age and androgen dependent. Exposure to adult testosterone levels is required for prostate development and disease (318-320). The precise relationship of androgens to prostatic disease and in particular any influence of exogenous androgens remains poorly understood. Ambient blood testosterone or DHT levels do not predict development of prostatic cancer over future decades in prospective studies of adults (321). A genetic polymorphism, the CAG (polyglutamine) triplet repeat in exon 1 of the androgen receptor, is an important determinant of prostate sensitivity to circulating testosterone with short repeat lengths leading to increased androgen sensitivity (322), however the relationship of the CAG triplet repeat length polymorphism to late-life prostate diseases remains unclear (323). Among androgen deficient men, prostate size and PSA concentrations are reduced and returned towards normal by testosterone replacement without exceeding age-matched eugonadal controls (322, 324-326). In healthy middle-aged men without known prostate disease, very high doses of the potent natural androgen DHT for 2 years did not increase prostate size or age-related growth rate compared with placebo indicating that effects of even high dose exogenous androgen treatment has much less effect than age on the human prostate (308).  Similarly, self-administration of massive androgen over-dosage does not increase total prostate volume or PSA in anabolic steroid abusers although central prostate zone volumes increases (327). In-situ prostate cancer is common in all populations of older men whereas rates of invasive prostate cancer differ many-fold between populations despite similar blood testosterone concentrations. This suggests that early and prolonged exposure to androgens may initiate in-situ prostate cancer, but later androgen-independent environmental factors promote the outbreak of invasive prostate cancer. Therefore, it is prudent to maintain physiological androgen levels with exogenous testosterone, which then might be no more hazardous than exposure to endogenous testosterone. Prolonged surveillance comparable with that for cardiovascular and breast disease in users of female hormonal contraception would be equally essential to monitor both cardiovascular and prostatic disease risk in men receiving exogenous androgens for hormonal contraception.

 

Extensive experience with testosterone in doses equivalent to replacement therapy in normal men indicates minimal effects on mood or behavior (14, 15, 256, 328-330). A careful placebo-controlled, cross-over study showed that a 1000 mg TU injection in healthy young men produces minor mood changes without any detectable increase in self or partner-reported aggressive, non-aggressive or sexual behaviors (331). By contrast, extreme androgen doses used experimentally in healthy men can produce idiosyncratic hypomanic reactions in a minority (332). Aberrant behavior in observational studies of androgen-abusing athletes or prisoners are difficult to interpret particularly to distinguished genuine androgen effects from the influence of self-selection for underlying psychological morbidity (333).

 

ANTI-ANDROGENS

 

Antiandrogens have been used to selectively inhibit epididymal and testicular effects of testosterone without impeding systemic androgenic effects (334). Cyproterone acetate, a steroidal antiandrogen with progestational activity, suppresses gonadotropin secretion without achieving azoospermia but leads to androgen deficiency when used alone (335). In contrast, pure non-steroidal antiandrogens lacking androgenic or gestagenic effects such as flutamide, nilutamide and casodex fail to suppress spermatogenesis when used alone (336, 337). Two studies evaluating the hypothesis that incomplete suppression of spermatogenesis is due to persistence of testicular DHT have reported no additional suppression from administration of finasteride, a type II 5a reductase inhibitor (338, 339); however as testes express predominantly the type I isoforms (340), further studies are required to conclusively test this hypothesis using an inhibitor of type I 5-a reductase (341).

 

ANDROGEN COMBINATION REGIMENS

 

Combination steroid regimens use non-androgenic steroids (estrogens, progestins) to suppress gonadotropins, in conjunction with testosterone for androgen replacement, have shown the most promising efficacy with enhanced rate and extent of spermatogenic suppression compared with androgen alone regimens (315, 342, 343). Synergistic combinations reduce the effective dose of each steroid and minimizing testosterone dosage could enhance spermatogenic suppression if high blood testosterone levels counteract the necessary maximal depletion of intratesticular testosterone (344, 345) as well as reducing androgenic side-effects (Figure 4).

 

 

Progesterone is a key precursor and steroidogenic intermediate for all bioactive natural steroids and the progesterone receptors A and B are structurally and evolutionarily the closest members of the nuclear receptor superfamily to the androgen receptor. Yet, although progesterone has crucial gestational and lactational roles in female reproductive physiology, it has no well-established role in male reproductive physiology apart from a possible role in sperm function (200, 346), possibly via non-genomic rather than a classically genomic mechanism (347). Nevertheless functional nuclear progesterone receptors are expressed in male brain, smooth muscle and reproductive, but not most non-reproductive, tissues (348). Synthetic progestins, steroidal structural agonistic analogs of progesterone, are potent inhibitors of pituitary gonadotropin secretion used widely for female contraception and hormonal treatment of disorders such as endometriosis, uterine myoma and mastalgia. Used alone, progestins suppress spermatogenesis but cause androgen deficiency including impotence (349, 350) so androgen replacement is necessary. Non-human primate studies indicate that this is mediated via a central hypothalamic-pituitary site of action rather than direct effects on the testis (351). Extensive feasibility studies concluded that progestin-androgen combination regimens had promise as hormonal male contraceptives if more potent and durable agents were developed (256, 352). The largest and most detailed multi-center study of the contraceptive efficacy of a androgen-progestin combination involving 320 healthy men aged 18-45 years together with their 18-38 year old female partners, both without known fertility problems, studied for up to 56 weeks while having intramuscular injections of 200 mg norethisterone acetate and testosterone undecanoate every 8 weeks that produced near-complete suppression (96% declined to <1 million/ml by 24 weeks) and reversible recovery (95% recovered by 52 weeks) of sperm output (353). Four pregnancies occurred in partners of 266 men with a pregnancy failure rate of 1.6% (95% confidence interval 0.6-4.1%). Prior information on androgen/progestin regimens derives from studies with medroxyprogesterone acetate (MPA) combined with testosterone. Monthly injections of both agents or daily oral progestin with dermal androgen gels produce azoospermia in ~60% of fertile men of European background with the remainder having severe oligozoospermia and impaired sperm function (256, 352, 354). Nearly uniform azoospermia is produced in men treated with depot MPA and either of two injectable androgens in Indonesian men (230, 231) or testosterone depot implants in Caucasian men (315). Smaller studies with other oral progestins such as levonorgestrel (342, 355, 356) and norethisterone (357, 358) combined with testosterone demonstrate similar efficacy to oral MPA whereas cyproterone acetate with its additional anti-androgenic activity has higher efficacy in conjunction with TE (343, 359) but not oral testosterone undecanoate (360). Highly effective suppression of spermatogenesis is reported with depot progestins in the form of non-biodegradable implants of norgestrel (361-363) or etonorgestrel (364, 365) or depot injectable medroxyprogesterone acetate (18, 315, 366, 367) or norethisterone enanthate (368, 369)coupled with testosterone (370). A comparative study showed that all four progestins (cyproterone acetate, levonorgestrel, norethisterone acetate and nesterone) displayed significant short-term gonadotrophin suppression when combined with testosterone (371). The pharmacokinetics of the testosterone preparation is critical to efficacy of spermatogenic suppression with long-acting depots being most effective while transdermal delivery is less effective than injectable testosterone (361). Progestin side-effects are few if sexual function and well-being are maintained by adequate doses of testosterone replacement. The metabolic effects depend on specific regimen with oral administration and higher testosterone doses exhibiting more prominent hepatic effects such as lowering SHBG and HDL cholesterol. After treatment ceases with depletion or withdrawal of hormonal depots, spermatogenesis recovers completely but gradually consistent with the time-course of the spermatogenic cycle (234, 282). Steroids with dual androgen and progestin properties may be potentially well suited to male contraception if the balance between these two bioactivities is properly balanced (372). A study comparing a gel combining nesterone (8.3 mg) with testosterone (62.5 mg) with the same dose of testosterone alone for daily transdermal application for 28 days demonstrated suppression of sperm output and serum gonadotropin to levels consistent with contraceptive efficacy and superior to testosterone alone indicating  the suitability of the combination gel for further product development (373). Two orally active nandrolone derivatives, dimethandrolone (374-376) and 11-methyl nandrolone dodecylcarbonate (377), are undergoing promising, early stage clinical development showing high acceptability in a short-term, placebo-controlled clinical trials (378, 379). Whether such daily use oral or transdermal gel (239, 380)-based male contraceptives will have sufficient user compliance to attain high contraceptive efficacy remains to be evaluated.

 

Estradiol augments testosterone-induced suppression of primate spermatogenesis (381) and fertility (382) but estrogenic side-effects (gynecomastia) and modest efficacy at tolerable doses make estradiol-based combinations impractical for male contraception (383). The efficacy and tolerability of newer estrogen analogs in combination with testosterone remain to be evaluated.

 

GONADOTROPIN-RELEASING HORMONE BLOCKADE  

 

The pivotal role of GnRH in the hormonal control of testicular function makes it an attractive target for biochemical regulation of male fertility. Blockade of GnRH action by GnRH receptor blockade with synthetic analogs or GnRH immunoneutralization would eliminate LH and testosterone secretion requiring testosterone replacement. Many superactive GnRH agonists are used to induce reversible medical castration for androgen-dependent prostate cancer by causing a sustained, paradoxical inhibition of gonadotropin and testosterone secretion and spermatogenesis due to pituitary GnRH receptor downregulation. When combined with testosterone, GnRH agonists suppress spermatogenesis but rarely achieve azoospermia (344, 345, 384) being less effective than androgen/progestin regimens. By contrast, pure GnRH antagonists create and sustain immediate competitive blockade of GnRH receptors (385, 386) and, in combination with testosterone, are highly effective at suppressing spermatogenesis. Early hydrophobic GnRH antagonists were difficult to formulate and irritating, causing injection site mast cell histamine release. Newer more potent but less irritating GnRH antagonists produce rapid, reversible and complete inhibition of spermatogenesis in monkeys (387-389) and men (390, 391) when combined with testosterone. The striking superiority of GnRH antagonists over agonists may be due to more effective and immediate inhibition of gonadotropin secretion and thereby more effective depletion of intratesticular testosterone. Due to their highly specific site of action, GnRH analogs have few unexpected side-effects. Depot GnRH antagonist plus testosterone formulations suitable for administration at up to 3-month intervals could be promising as a hormonal male contraceptive regimen. Whether GnRH antagonists are more cost-effective than progestins as the second, non-androgenic component of combination male hormonal contraceptive regimens remains to be established (278, 303, 367, 392). The drawback of higher cost might be overcome by hybrid regimens using GnRH antagonists to initiate suppression followed by a switch to more economical steroids for maintenance of spermatogenic suppression in humans (304). A GnRH vaccine could intercept GnRH in the pituitary-portal bloodstream preventing its reaching pituitary GnRH receptors. A limitation of a GnRH vaccine is its uncertain and/or unpredictable reversibility. If it is not irreversible, the offset of contraceptive efficacy may vary between individuals and create practical difficulties in ensuring reliable contraception. Gonadotropin-selective immunocastration would require androgen replacement in men (393) and pilot feasibility studies in advanced prostate cancer are underway (394)but the prospects for acceptably safe application for male contraception remain doubtful (395). By contrast there are growing applications for anti-hormonal contraceptive vaccines in control of companion (pet), agricultural, zoo, feral and wild animal populations (156, 396).

 

FOLLICLE STIMULATING HORMONE BLOCKADE  

 

Selective FSH blockade theoretically offers the opportunity to reduce spermatogenesis without inhibiting endogenous testosterone secretion. FSH action could be abolished by selective inhibition of pituitary FSH secretion with inhibin (397)or novel steroids (398), by FSH vaccine (399, 400) or by FSH receptor blockade with peptide antagonists (401). Although FSH was considered essential to human spermatogenesis, spermatogenesis and fertility persist in rodents (402-404) and humans (405) lacking FSH bioactivity. Even complete FSH blockade alone might produce insufficient reduction in sperm output and function required for adequate contraceptive efficacy (406). In addition to the usual safety concerns of contraceptive vaccines including autoimmune hypophysitis, orchitis or immune-complex disease, an FSH vaccine might be overcome by reflex increases in pituitary FSH secretion. Hence, FSH suppression is a necessary but not sufficient for a hormonal male contraceptive regimen.

 

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