We prospectively recruited 325 male veterans (80% consent rate) who met our initial screening eligibility criteria. We subsequently excluded 17 patients because of a non-detectable viral load (n=3), positive HIV or HBV surface antigen (n=4), or a history of testicular cancer, medically or surgically managed prostate cancer, or use of a sex hormone-altering pharmacotherapy (n=10).
Most (93%) of our 308 chronically HCV infected male veteran study participants were between ages 45 and 64 years old and were of either African-American or White race/ethnicity (52% and 43%, respectively). () Comorbid risk factors were highly prevalent in this population, with 34% reporting a positive lifetime history of chronic alcohol abuse, 51% a positive history of injection drug use (IDU), and 71% study-classified as overweight or obese (BMI≥25). () A maximum estimated duration of HCV infection of 34 years (or estimated earliest median age at infection of 22 years old) was calculated based upon data provided by the 90% of respondents who reported both a positive history of, as well as correspondent ages for, at least one established risk factor for HCV transmission (e.g., blood transfusion pre-1992 or injection drug use). Less than 5% of study participants had ever received anti-HCV therapy.
Demographic and clinical characteristics of male veterans with chronic Hepatitis C according to FibroSURE-ActiTest FibroSURE)-determined hepatic pathology. (N=308)
FibroSURE-determined METAVIR equivalent hepatic pathology was used to stratify our study sample into 105 cases with advanced fibrosis (nF4=92 and nF3/F4=13) and 203 mild fibrosis controls(nF0=15, nF0/F1=15, nF1=7, nF1/F2=48, nF2=42, nF3=76); and into 88 cases with advanced inflammatory activity (nA3=82 and nA2/A3=6) and 220 mild inflammatory activity controls (nA0=51, nA0/A1=49, nA1=25, nA1/A2=66, and nA2=29). Total serum testosterone generally increased with increased fibrosis and inflammatory activity (F0-F1=4.8, F1-F2=4.5, F2=5.6, F3=5.4, F3/F4-F4= 5.9 ng/ml, and A0-A1=5.2, A1-A1/A2= 5.1, A2=5.6, A2/3-A3=5.7 ng/ml, respectively). These trends were even more apparent when median testosterone was calculated in samples with a restricted age range (e.g., 50–54 yrs) in whom baseline total testosterone levels would be expected to be more similar. (data not shown)
Both advanced fibrosis cases as well as advanced inflammatory activity cases had higher testosterone levels compared to their mild disease controls (6.0 ng/ml vs.5.3 ng/ml and 5.9 ng/ml vs. 5.4 ng/ml, respectively), with this mean difference highly significant for advanced fibrosis (p=0.009). () There was modest significant negative correlation between median age and total serum testosterone and between BMI and testosterone (ρSpearman=−0.12, p=0.04, and ρSpearman=−0.23, p<0.001, respectively), but no significant correlation between testosterone and HCV viral load (ρSpearman=0.03, p=0.56).
Association between total serum testosterone and other potential confounders and risk of FibroSURE-ActiTest-determined advanced hepatic fibrosis (F3/F4 and F4) in male veterans with chronic hepatitis C infection.
Diabetes mellitus was also significantly more prevalent in both advanced hepatic fibrosis cases and advanced hepatic inflammatory activity cases (p<0.001 and p=0.003, respectively). Although mean age was also significantly higher in those with advanced fibrosis compared to mild fibrosis controls (p=0.02), mean age was not similarly higher in advanced inflammation cases. Baseline distribution of all other potential confounders evaluated, including a history of chronic alcohol abuse, viral load, overweight/obesity and ethnicity, were not significantly different among advanced hepatic disease cases and their respective mild disease controls. ()
Higher serum testosterone levels were associated with increased relative risk of advanced FibroSURE-determined hepatic fibrosis (F3/F4 and F4) in both univariate and multivariate analyses. () Further, the adjusted 3.78-fold and 2.18-fold excess fibrosis risk in HCV+ male veterans with medium and upper testosterone levels, respectively, in comparison to those with lower testosterone levels suggests a monotonic positive dose-response relationship. There was no evidence of significant interaction between any main effects. The inclusion of all other known or biologically-plausible confounders like diabetes and adiposity evaluated in the multivariate model further enhanced both the magnitude and apparent dose-response relationship of the observed association between higher total testosterone levels and increased advanced hepatic fibrosis risk. Overall, we observed a significant 27% increase in advanced hepatic fibrosis risk for each 1 ng/ml increase in total serum testosterone (ORadjusted=1.27, 95% CI 1.13–1.44, p<0.001). (data not shown) Median fibrosis progression rates (FPR) also significantly increased across tertiles of total serum testosterone (FPRs=0.067, 0.079, and 0.095 for the lower, middle, and upper tertiles, respectively, p<0.001).
Higher serum testosterone levels were significantly associated with risk of advanced FibroSURE-determined hepatic inflammatory activity (A2/A3 and A3) in both univariate and multivariate analyses (range 1.9–2.7- fold significant excess relative risk). () Also similar were our findings that the magnitude of the association between increased testosterone levels and increased inflammatory disease risk was further modestly strengthened in multivariate in comparison to univariate analysis (e.g., ORunivariate highest testosterone level=1.92, 95% CI vs. ORmultivariate highest testosterone level=2.64, 95% CI, respectively), and that there was no evidence of significant interaction among any main effects. However, in contrast to our findings for advanced fibrosis, there was no apparent dose-response relationship between total serum testosterone level and advanced inflammatory activity risk. () Overall, we found that a 1 ng/ml increase in total serum testosterone was associated with a significant 16% increase in advanced inflammatory activity risk (ORadjusted=1.16, 95% CI 1.02–1.31, p=0.02). (data not shown)
Association between total serum testosterone and other potential confounders and risk of FibroSURE-ActiTest-determined advanced hepatic inflammatory activity (A2/A3 and A3) in male veterans with chronic hepatitis C infection.
The Hosmer-Lemeshow test results were supportive of fit for all reported models. We also performed several sensitivity analyses to evaluate the robustness of our observed associations of excess advanced hepatic disease risk with increased total serum testosterone. In our first set of sensitivity analyses, we restricted our analysis to the 84% of our HCV+ male veteran sample with total testosterone within a normal range for adult males (2.41–8.47 ng/ml). We continued to observe a significant dose-response between higher total serum testosterone levels and advanced hepatic fibrosis (ORadjusted=3.90, 95% CI 1.82–8.38 and ORadjusted =2.25, 95% CI 1.05–4.80 for advanced fibrosis risk for those in the upper and middle testosterone level as compared to those in the lower testosterone level, respectively). () We similarly found that increased testosterone level also continued to be associated with advanced inflammatory activity risk (ORadjusted=2.66, 95% CI 1.19–5.99 and ORadjusted=3.12, 95% CI 1.41–6.92 for advanced inflammatory activity risk for those in the upper and middle testosterone level as compared to those in the lower testosterone level, respectively). In a second set of sensitivity analyses where we changed the advanced disease case definitions to be more and less restrictive respectively, we continued to demonstrate substantial excess risk of both advanced hepatic fibrosis and advanced hepatic inflammatory activity with increased total serum testosterone levels. ()
Multivariate sensitivity analyses evaluating association between total serum testosterone and other potential confounders and risk of FibroSURE-ActiTest -determined advanced hepatic disease in male veterans with chronic hepatitis C infection (N=308).