Testosterone levels and cause-specific mortality in the older French men without metabolic syndrome

OBJECTIVES Previous studies have reported controversial findings regarding the association of testosterone with mortality in older men. This heterogeneity might be partially explained by comorbidities and the presence of metabolic syndrome, as well as differential associations according to causes of death. METHODS We used data from a random subsample of the Three-City study, in which hormone levels were measured in 338 men ≥65 years without metabolic syndrome who were followed-up for 12 years. Vital status was determined for all participants from different sources. We used inverse-probability-weighted Cox regression to estimate the hazard ratios (HRs) of cause-specific mortality and 95% confidence intervals (CIs). RESULTS Over the follow-up period, 130 men died (30 from cardiovascular disease, 45 from cancer, 55 from other causes). The association of testosterone with mortality showed significant heterogeneity across causes of death (p=0.027 and p=0.022 for total and bioavailable testosterone, respectively). Higher testosterone levels were associated with increased cardiovascular mortality (HR for 1-standard deviation increase, 1.86; 95% CI, 1.28 to 2.71 and 1.50; 95% CI, 1.04 to 2.17 for total and bioavailable testosterone, respectively). By contrast, there were no significant associations of testosterone with mortality from cancer and other causes. CONCLUSIONS Our data suggest that the association of testosterone with mortality in men without metabolic syndrome might be differential according to the cause of death. These findings may partially explain the heterogeneity across studies on the relationship between testosterone levels and mortality.


Introduction
Findings from studies on the relation between testosterone levels and mortality in men are highly heterogeneous, thus making it difficult to draw clear conclusions [1]. Identifying possible sources of heterogeneity may help better understand this relation and identify groups at higher risk. In a previous analysis, we reported that metabolic syndrome (MetS) could explain a part of this heterogeneity, as there was an inverse association between testosterone and all-cause mortality in men with MetS, but not in those without MetS [2]. However, it is possible that testosterone plays a stronger role for specific causes of death, explaining the lack of association between testosterone and all-cause mortality in men without MetS. Indeed, testosterone was positively associated with cardiovascular events [3,4] but negatively with cancer [5], suggesting differential associations of testosterone with health outcomes.
Nevertheless, MetS was not taken into account and no study examined this differential risk in a metabolically "healthy" population. Such specific investigation which allows identifying subgroups of men at high risk would be very important for clinical practice before considering, for example, testosterone therapy or other specific treatment. Therefore, the aim of the present study is to examine the relationship between plasma testosterone and cause-specific mortality in men without MetS.

Subjects and Methods
The protocol of the 3C study, a French prospective cohort study, is available elsewhere [2,6,7]. We used Cox proportional hazards regression models with age as the time scale to estimate hazard ratios (HRs) and 95% confidence intervals (95% CI) for cause-specific mortality [9].
Participants were followed-up from baseline to the date of death from a specific cause or censoring (death from another cause, date of last news alive).
Model 1 was adjusted for center; model 2 was further adjusted for confounders (smoking, alcohol drinking, education level, history of cardiovascular disease, hypertension, hypercholesterolemia, diabetes, BMI). We created extra categories for covariates with missing values to keep the same number of subjects in the analyses.
In sensitivity analyses, we excluded deaths occurring during the first two and four years of follow-up.
We used a stabilized inverse-probability-weighting approach to take into account the fact that testosterone was measured in a random sub-cohort [10]. Weights were calculated for each individual as detailed elsewhere [2].

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Statistical analyses were performed using SAS 9.4 (SAS Institute, Inc., Cary, NC, USA).

Results
From the random sub-cohort of 495 men, we excluded 22 men with treatments that might influence hormone levels or with a history of prostate cancer or prostatitis, 29 men without data on MetS, and 106 men with MetS. The remaining 338 men without MetS constitute the sample for our analysis.
Over 12 years of follow-up, 130 men died (38.5%): 30 from CV disease, 45 from cancer, and 55 from other causes. Compared to survivors, they were older at baseline and more likely to be current smokers and daily alcohol drinkers (for deaths from cancer) than those who were alive at the end of follow-up, independently of the age at inclusion ( Table 1). As expected, men with the highest levels of testosterone were less likely to present diabetes (Table S1).
Associations of baseline endogenous hormone levels with cause-specific mortality are presented in Results were similar after exclusion of deaths occurring during the first two and four years of follow-up (data not shown).

Discussion
In men without MetS, we found no significant association between testosterone and mortality overall, but we observed a differential association according to the cause of death: independently of CV risk factors, higher levels of testosterone increased the risk of CV mortality, but were not associated with mortality from cancer and other causes.
This positive association of testosterone with CV mortality differed from some previous studies that showed an inverse [11][12][13] or no association [14,15]; however, there are important differences between these studies and ours that could account for inconsistent findings. First, subjects were younger than those included in 3C, resulting in lower mortality rates. Second, these studies had shorter follow-ups, and their findings may have been biased by reverse causation, i.e., testosterone level may have been influenced by the diseases that led to death; this is less likely in the 3C study, given the longer follow-up and findings of our analyses based on exclusion of participants who died early during the follow-up. Finally, previous studies did not take MetS status into account, while our analyses are limited to men without MetS who, by definition, have less comorbidities and are healthier compared to men overall. Conversely, our results are consistent with other studies on the risk of death from coronary heart disease [16,17] and CV events [3,4]. This is also in keeping with a meta-analysis of 27 clinical trials (n=2,994 men) showing an increased CV risk in men treated with exogenous testosterone compared to placebo [18].
The mechanisms underlying the positive association between testosterone and CV mortality remain unknown. One study hypothesized that high testosterone levels may promote fluid-sodium retention in aging men and contribute to the development of high blood pressure and heart failure [19]. However, in our study, adjustment for hypertension did not have an important impact.

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Another study suggested that use of anabolic steroids is associated with left ventricular hypertrophy [20], thus increasing the risk of CV mortality. Although our data do not concern exogenous testosterone, we cannot exclude a similar mechanism for endogenous testosterone.
With respect to cancer mortality, we found no significant association with testosterone, in agreement with several previous studies [16,21,22]; there are, however, other studies that showed an inverse association between testosterone and risk of cancer mortality [5,23,24]. We could not examine specific cancers due to low numbers and we cannot exclude that some associations might exist for specific cancers. In addition, persons who accepted to participate to 3C were healthier than the general population [6], and there were few current smokers at baseline. Since smoking is a very strong CV and cancer risks factor, especially for aero-pharyngeal cancers, this low smoking prevalence may have led to reduced statistical power.
The major limitation of our study is that, our analyses are based on a random stratified sub-cohort of men, rather than on the whole cohort. In order to control for sampling-related biases, we used inverse-probability-weight [10]. In addition, testosterone measurement was carried out by RIA rather than the state-of-the-art gas chromatography mass spectrometry method. However, validation studies showed that RIA provided accurate results in terms of relative ranking and was therefore adequate for epidemiological investigations in which population-level inferences are more of interest than subject-specific ones. Finally, the 3C cohort, like most cohort studies, is not representative of the French general population aged 65 years and over. For example, the prevalence of some CV and cancer risks factor such as smoking status and educational level are underestimated when compared with the general population [6]. However, we verified wellknown associations and therefore we expect that it would attenuate, but not bias, the associations.
Nevertheless, extrapolating these results to the general population must therefore be done carefully.

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In conclusion, among men without MetS, high testosterone levels are associated with increased CV but not cancer mortality. These findings may explain part of the heterogeneity of studies on the relation between testosterone and mortality and help clinicians to identify groups at high risk.   Missing data: a n = 1, b n = 6, c n = 2. d p-value from t-test or chi-square tests. e p-value based on Cochran-Mantel-Haenszel statistic or analysis of variance adjusted for age.