Abstract
Context
Testosterone treatment of hypogonadal men improves their hemoglobin, but the mechanism is not understood.
Objective
To investigate possible mechanisms by which testosterone stimulates erythropoiesis in hypogonadal older men with unexplained or iron-deficiency anemia.
Design
The Anemia Trial of The Testosterone Trials, a placebo-controlled study in older, hypogonadal men.
Setting
Twelve academic medical centers.
Participants
A total of 95 hypogonadal men (testosterone < 275 ng/mL) ≥65 years with anemia (hemoglobin < 12.7 g/dL). They were classified as having unexplained (n = 58) or iron deficiency anemia (n = 37).
Intervention
Testosterone or placebo gel for 1 year.
Main Outcome Measures
Markers of iron metabolism during the first 3 months of treatment.
Results
Testosterone replacement significantly (P < 0.001) increased hemoglobin in the 58 men who had unexplained anemia (adjusted mean difference 0.58 g/dL; 95% confidence interval, 0.31-0.85). Testosterone replacement tended to increase hemoglobin in the 37 men who had iron deficiency (0.38 g/dL; −0.19, 0.95), but the response was more variable and not statistically significant (P = 0.19). In men with unexplained anemia, testosterone replacement suppressed hepcidin (−8.2 ng/mL; −13.7, −2.7; P = 0.004) and ferritin (−19.6 µg/L; −32.8, −6.3; P = 0.004), but in men with iron deficiency, testosterone replacement did not. The decrease in hepcidin was moderately correlated with the increase in hemoglobin in the men with unexplained anemia (correlation coefficient −0.35, P = 0.01) but not in those with iron deficiency anemia (correlation coefficient −0.07, P = 0.73).
Conclusions
Testosterone replacement of older hypogonadal men with unexplained anemia stimulates erythropoiesis associated with increased iron mobilization. This effect appears to be attenuated by iron deficiency.
Keywords: anemia, hypogonadism, testosterone, hepcidin, iron, erythropoiesis
Anemia is a common finding in older adults and is associated with impaired function and decreased longevity (1-3). The causes of anemia include iron deficiency, chronic inflammatory disease, bone marrow failure syndromes, and chronic kidney disease (4, 5). In approximately one-third of anemic older adults, however, no clear etiology can be identified, and this is known as unexplained anemia of the elderly.
One possible cause of previously unexplained anemia in elderly men is hypogonadism. Testosterone has long been known to stimulate erythropoiesis. Profound hypogonadism induced by medical or surgical castration significantly reduces hemoglobin (6, 7). The less severe decrease of testosterone that occurs with aging (8-10) is associated with a less severe degree of anemia (11, 12).
The mechanisms by which testosterone stimulates erythropoiesis, however, remain poorly understood. In addition to direct simulation of the bone marrow progenitors (13, 14), testosterone has been postulated to dampen inflammation (15) and stimulate erythropoietin production by the kidneys (16). Testosterone has also been postulated to affect iron homeostasis by decreasing hepcidin (17-19). Hepcidin, a protein synthesized in the liver, regulates iron homeostasis by degrading the iron exporter, ferroportin (20), which transports iron from reticuloendothelial cells of the duodenal enterocytes, hepatocytes, and macrophages. Reduced hepcidin thereby increases ferroportin to mobilize iron stores and enhance iron absorption. However, testosterone was found to increase hemoglobin even in mice with genetic disruption of hepcidin (19). The role of hepcidin and iron in mediating testosterone’s stimulation of erythropoiesis, therefore, remain incompletely understood.
We previously reported that testosterone treatment of older hypogonadal men with anemia significantly increased their hemoglobin levels (21). This increase occurred both in men who had unexplained anemia and in those who had anemia of known cause. Here, we sought to clarify the role of iron in mediating testosterone’s effects on hemoglobin by determining the effects of testosterone replacement in iron-replete and iron-deficient older men who participated in the Testosterone Trials (TTrials), by measuring markers of iron metabolism.
Materials and Methods
Study design
The TTrials are 7 coordinated trials of the efficacy of testosterone in older men who have low testosterone concentrations. They were conducted at 12 clinical trial sites in the United States (https://clinicaltrials.gov/ct2/show/NCT00799617) (22). Details have been published (23). Men 65 years and older who had 2 early morning serum testosterone levels <275 ng/mL were eligible. Men who enrolled were assigned to receive testosterone or placebo gel in a double-blind fashion for 1 year. Serum testosterone was measured at 1, 2, 3, 6, and 9 months, and the dose of testosterone gel was adjusted to attempt to keep the testosterone level within the normal range.
Definition and classification of anemia
We defined anemia as a hemoglobin level ≤12.7 g/dL, irrespective of race (24). The TTrials excluded men who had a hemoglobin <10.0 g/dL. Of the 788 participants in the TTrials, 126 were anemic at baseline (21). As previously reported (21), we classified the anemias as iron deficiency (ferritin <40 ng/mL), B12 deficiency (<200 pg/mL), myelodysplastic syndrome (abnormal blood counts or macrocytosis), plasma cell dyscrasia (elevated paraprotein), chronic inflammatory disease (chart review of medical history and medication), or hemolysis (low haptoglobin). We classified men who met none of the above criteria as having unexplained anemia. Hemoglobin was measured every 3 months during the 1 year of testosterone treatment.
Studies of mechanism of erythropoiesis
We measured markers of erythropoiesis in the 116 of the 126 anemic men who had serum samples available from baseline, 1 month, and 3 months. Samples had been frozen at −80 °C. Mayo Clinic Laboratories (Rochester, MN) performed assays for serum ferritin (immunoenzymatic assay, coefficient of variation [CV] 3.57%-6.26%), transferrin (immunoturbimetric assay, CV 1.85%-5.35%), serum soluble transferrin receptor (immunoturbimetric assay, CV 1.97%-2.39%), serum erythropoietin (immunoenzymatic assay, CV 3.9%-5.7%), interleukin-6 (IL-6) (electrochemiluminescence assay, CV 7.6%-9.7%), vascular endothelial growth factor (VEGF) (electrochemiluminescence assay, CV 9%-11%), and C-reactive protein (CRP) (electrochemiluminescence assay, CV 1.7%-3.4%). Intrinsic Life Sciences, La Jolla, CA, assayed serum hepcidin using an enzyme-linked immunosorbent assay (CV 7%-9%; Hepcidin IDX assay) (25).
Statistical analyses
The participants were analyzed in the treatment group to which they were allocated, according to the intention-to-treat principle. The effect of testosterone on change from baseline in each marker was assessed by fitting a linear mixed model with treatment, time in treatment (1 or 3 months), and balancing factors used in the algorithm for assigning treatment by the technique of minimization (age ≤75 years, baseline testosterone ≤200 ng/dL, trial site, use of phosphodiesterase-5 inhibitors, and participation in the 3 main trials) as predictors and stratification by anemia type. Models included a random intercept for participant. Pearson correlation coefficient was used to assess the correlation of change in hemoglobin with change in hepcidin. All hypothesis tests were conducted at a 2-sided significance level of 0.05. No adjustments were made for multiple comparisons; such adjustments would be overly conservative, because markers of iron metabolism would likely be highly correlated with each other, as would inflammatory markers.
Results
Baseline characteristics of the participants
Of the 116 anemic men in the TTrials who were assessed for the mechanism by which testosterone stimulates erythropoiesis, 58 (50%) had unexplained anemia and 37 (32%) had iron deficiency anemia. Baseline characteristics of the 2 groups did not differ except for lower ferritin and hepcidin levels in the men with iron deficiency anemia than the men with unexplained anemia (Table 1).
Table 1.
Characteristics of Participants at Baseline by Type of Anemia
| Unexplained Anemia | Iron Deficiency Anemia | |||
|---|---|---|---|---|
| Placebo | Testosterone | Placebo | Testosterone | |
| N | 34 | 24 | 20 | 17 |
| Demographics | ||||
| Age, y | 76.4 ± 7.6 | 73.8 ± 6.0 | 73.5 ± 5.0 | 73.0 ± 4.7 |
| Racea | ||||
| Caucasian | 28 (82.4%) | 22 (91.7%) | 17 (85.0%) | 15 (88.2%) |
| African American | 5 (14.7%) | 0 | 0 | 1 (5.9%) |
| Other | 1 (2.9%) | 2 (8.3%) | 3 (15.0%) | 1 (5.9%) |
| Erythropoietic Parameters | ||||
| Hemoglobin, g/dL | 12.1 ± 0.6 | 11.9 ± 0.7 | 11.9 ± 0.8 | 12.1 ± 0.4 |
| Erythropoietin, mIU/mL | 16.8 ± 7.8 | 13.6 ± 7.1 | 25.1 ± 19.8 | 20.5 ± 14.8 |
| Iron Parameters | ||||
| Hepcidin, ng/mLb | 30.8 ± 15.6 | 26.3 ± 11.6 | 10.6 ± 8.0 | 10.2 ± 5.8 |
| Ferritin, ng/mLb | 116.6 ± 68.0 | 92.8 ± 55.3 | 21.5 ± 9.7 | 22.1 ± 9.6 |
| sTfR, mg/dL | 3.5 ± 1.2 | 3.0 ± 1.5 | 4.7 ± 2.5 | 4.0 ± 1.9 |
| Transferrin, mg/dL | 222.5 ± 34.5 | 249.4 ± 34.0 | 293.5 ± 37.0 | 297.1 ± 53.6 |
| Inflammatory and Other Parameters | ||||
| IL-6, pg/mL | 11.3 ± 29.2 | 3.7 ± 2.1 | 4.3 ± 5.4 | 3.0 ± 2.0 |
| CRP, mg/dL | 0.6 ± 0.9 | 0.3 ± 0.3 | 0.4 ± 0.4 | 0.2 ± 0.2 |
| VEGF, pg/mL | 65.6 ± 25.5 | 66.5 ± 34.4 | 73.1 ± 48.7 | 77.7 ± 36.9 |
| Concomitant Conditions | ||||
| BMI, kg/m2 | 29.7 ± 4.0 | 31.7 ± 4.0 | 31.6 ± 3.1 | 31.3 ± 3.7 |
| Smoking | ||||
| Currently | 2 (5.9%) | 0 | 1 (5.0%) | 1 (5.9%) |
| Ever | 21 (61.8%) | 16 (66.7%) | 12 (60.0%) | 12 (70.6%) |
| Diabetes | 11 (32.4%) | 16 (66.7%) | 11 (55.0%) | 11 (64.7%) |
| Hypertension | 25 (73.5%) | 21 (87.5%) | 15 (75.0%) | 14 (82.4%) |
| Sleep apnea | 4 (11.8%) | 8 (33.3%) | 3 (15.0%) | 3 (17.6%) |
| Sex Hormones | ||||
| Testosterone, ng/dL | 217.5 ± 77.6 | 202.4 ± 72.5 | 235.0 ± 72.7 | 205.8 ± 60.4 |
| Free testosterone, pg/mL | 51.0 ± 22.4 | 54.5 ± 24.3 | 60.8 ± 19.4 | 51.0 ± 21.5 |
| DHT, ng/dL | 18.7 ± 13.5 | 17.2 ± 9.4 | 18.8 ± 6.7 | 18.0 ± 9.8 |
| Estradiol, pg/mL | 17.8 ± 5.8 | 17.4 ± 6.5 | 18.1 ± 5.0 | 16.4 ± 6.6 |
| SHBG, nM | 38.7 ± 18.4 | 33.6 ± 19.2 | 28.5 ± 8.1 | 35.1 ± 14.8 |
Values displayed are N (%) or mean ± standard deviation
Abbreviations: BMI, body mass index; CRP, C-reactive protein; DHT, dihydrotestosterone; IL-6, interleukin-6; sTfR, soluble transferrin receptor; SHBG, sex hormone–binding globulin; TTrials, Testosterone Trials; VEGF, vascular endothelial growth factor.
a Among men with unexplained anemia, the treatment groups differed with regard to race (P = 0.05).
b T-test (unequal variances) of the mean of all participants with unexplained anemia vs the mean of all men with iron deficiency anemia, P < 0.001
Erythroid responses
Fig. 1 illustrates the absolute hemoglobin concentrations during the 1 year of testosterone or placebo treatment in the 2 groups of anemic men. In the men with unexplained anemia, testosterone significantly increased hemoglobin compared to placebo (P ≤ 0.001); the adjusted mean difference was 0.58 g/dL (95% CI: 0.31, 0.85). In the men with iron deficiency anemia, testosterone tended to increase hemoglobin, but the response was more variable (0.38; −0.19, 1.95) and not significantly different from that of placebo (P = 0.19). Peak hemoglobin levels were reached by month 6 of treatment and persisted until month 12 (Fig. 1).
Figure 1.
The effect of treatment with testosterone or placebo on hemoglobin in men in The Testosterone Trials who had unexplained anemia (top) or iron deficiency anemia (bottom). The values are means and 95% confidence intervals. P values are based on adjusted mean differences.
Mechanisms of erythropoiesis
Iron parameters.
Testosterone replacement significantly changed several parameters of iron mobilization in men with unexplained anemia (Fig. 2). Testosterone decreased serum hepcidin significantly (adjusted mean difference, −8.21 ng/mL; [−13.7, −2.74]; P = 0.004) and ferritin (−19.6 µg/L [−32.8, −6.3]; P = 0.004) levels. Soluble transferrin receptor tended to increase (0.26 mg/L [−0.03, 0.55]; P = 0.07), and transferrin levels did not change significantly (3.0 [−6.0, 12.0]). In contrast, in men with iron deficiency, testosterone replacement did not affect serum hepcidin (−1.7 [−6.0, 2.6]) or transferrin levels (1.2 [−15.8, 18.2]) and possibly led to a modest decline in the serum ferritin level (−2.7 [−8.5, 3.2]). Testosterone replacement increased soluble transferrin receptor (−0.85 [−1.5, −0.2]). The decrease in hepcidin in the first month was moderately correlated with the average increase in hemoglobin over 12 months (correlation coefficient −0.35, P = 0.01) in men with unexplained anemia but not in men with iron deficiency anemia (correlation coefficient −0.07, P = 0.7) (Fig. 3).
Figure 2.
The effect of treatment with testosterone or placebo in men who had unexplained anemia or iron deficiency anemia on hepcidin, ferritin, transferrin, and soluble transferrin receptor. Values are means and 95% confidence intervals. P values are based on adjusted mean differences.
Figure 3.
Correlation of the change in hemoglobin with the change in hepcidin during testosterone treatment of older hypogonadal men with unexplained anemia (top panel) and with iron deficiency anemia (bottom panel).
Erythropoietin.
Testosterone treatment compared to placebo increased erythropoietin in the men with unexplained anemia (Table 2), but the absolute change was small (3.9 mIU/mL [0.58, 7.28]; P = 0.02). Testosterone had a small negative effect of borderline significance in men with iron deficiency (−6.0 mIU/mL [−12.7, 0.73] P = 0.08).
Table 2.
The Effect of Treatment with Testosterone or Placebo in Men in The Testosterone Trials who had Unexplained Anemia or Iron Deficiency Anemia on Erythropoietin, IL-6, CRP, and VEGF
| Parameter | Arm | Unexplained Anemia | Iron Deficiency Anemia | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Month 0 | Month 1 | Month 3 | Effect | P Value | Month 0 | Month 1 | Month 3 | Effect | P Value | ||
| EPO, mIU/mL | Testosterone | 13.6 ± 7.1 | 18.0 ± 8.9 | 19.6 ± 17.7 | 3.93 (0.58, 7.28) | 0.02 | 20.5 ± 14.8 | 24.3 ± 17.0 | 26.6 ± 17.1 | -5.97 (-12.7, 0.73) | 0.08 |
| Placebo | 16.8 ± 7.8 | 17.4 ± 9.5 | 16.0 ± 7.7 | 25.1 ± 19.8 | 25.8 ± 18.3 | 31.8 ± 24.0 | |||||
| IL-6, pg/mL | Testosterone | 3.7 ± 2.1 | 4.6 ± 4.3 | 5.0 ± 5.0 | 0.13 (-1.39, 1.65) | 0.86 | 3.0 ± 2.0 | 3.1 ± 2.2 | 3.7 ± 2.3 | -0.35 (-1.32, 0.61) | 0.46 |
| Placebo | 1.3 ± 29.2 | 11.3 ± 30.7 | 11.2 ± 27.8 | 4.3 ± 5.4 | 3.9 ± 2.5 | 3.5 ± 1.9 | |||||
| CRP, mg/dL | Testosterone | 0.3 ± 0.3 | 0.3 ± 0.3 | 0.4 ± 0.5 | -0.13 (-0.43, 0.17) | 0.39 | 0.2 ± 0.2 | 0.2 ± 0.2 | 0.3 ± 0.2 | -0.07 (-0.23, 0.08) | 0.35 |
| Placebo | 0.6 ± 0.9 | 0.5 ± 1.0 | 0.6 ± 1.2 | 0.4 ± 0.4 | 0.4 ± 0.3 | 0.3 ± 0.2 | |||||
| VEGF, pg/mL | Testosterone | 66.5 ± 34.4 | 90.3 ± 88.0 | 68.0 ± 43.3 | 21.0 (-10.6, 52.6) | 0.19 | 77.7 ± 36.9 | 74.9 ± 60.8 | 95.2 ± 63.3 | 19.8 (-1.0, 40.5) | 0.06 |
| Placebo | 65.6 ± 25.5 | 66.3 ± 33.9 | 62.8 ± 24.2 | 73.1 ± 48.7 | 77.3 ± 44.4 | 72.1 ± 39.2 | |||||
Values are means ± standard deviation at each month. The treatment effect (95% confidence interval) is the adjusted mean difference in average change from baseline for participants in the testosterone versus placebo arms. P values are based on the adjusted mean differences from 0 to 12 months.
Abbreviations: CRP, C-reactive protein; IL-6, interleukin-6; EPO, erythropoietin; VEGF, vascular endothelial growth factor.
Other parameters.
Testosterone replacement did not affect the major markers of inflammation, CRP, and IL-6, in either anemic group (Table 2). Testosterone replacement tended to increase VEGF in both groups, but not significantly in either.
Discussion
These data demonstrate that the increase in hemoglobin in response to testosterone replacement of hypogonadal older men who have unexplained anemia was accompanied by significant changes in markers of iron metabolism, including decreases in hepcidin and ferritin. In contrast, testosterone treatment of hypogonadal older men who also had iron deficiency was associated with a more variable and not significant change in hemoglobin and these markers. These results suggest that testosterone stimulates erythropoiesis in association with increased mobilization of iron and that iron deficiency may restrict this stimulus.
Prior studies have also shown that testosterone replacement suppresses hepcidin in older hypogonadal men with and without anemia, suggesting that hepcidin directly mediates erythropoiesis through enhanced iron availability (17, 18, 26). Other studies reported that testosterone increased serum soluble transferrin receptor, a marker of iron metabolism that is elevated in iron deficiency (17, 27). This is the first study of which we are aware to demonstrate that testosterone specifically decreases hepcidin and ferritin in association with stimulation of erythropoiesis in older men with unexplained anemia or that these effects appear to be attenuated in iron deficiency anemia.
The data presented here do not establish if the beneficial effect of testosterone on iron mobilization is direct or indirect. Testosterone directly suppresses hepcidin transcription (28). Testosterone, by stimulating erythropoiesis, could also indirectly suppress hepcidin (29). In mice, testosterone stimulates erythropoiesis if they are iron-replete, but not if they are iron-deficient (30). However, testosterone still stimulates erythropoiesis in iron-replete mice with a disruption of the hepcidin gene (19). These studies and ours show that iron plays an essential role in mediating testosterone’s stimulation of erythropoiesis in mice and men.
Testosterone replacement in older men with anemia, either unexplained or associated with iron deficiency, did not result in changes in the inflammatory markers CRP or IL-6, suggesting that the erythropoietic stimulus of testosterone was not mediated by dampening inflammation (17, 26). Additionally, it tended to increase erythropoietin levels in men with unexplained anemia. This tendency occurred despite increased hemoglobin, which normally suppresses erythropoietin, suggesting a reset of the hemoglobin-erythropoietin relationship. In other studies, testosterone treatment had mixed effects on erythropoietin. In one study, when testosterone or placebo was administered to 44 men with slightly low testosterone levels but normal hemoglobin, erythropoietin levels did not increase by week 3 but did by week 15 (26). In another study of 166 older men, 20% of whom were anemic, testosterone treatment resulted in an 8 mIU/mL increase in serum erythropoietin at month 1 but a decrease toward baseline by month 6 (17). Together, these data and ours suggest that if testosterone increases erythropoietin, the effect is small and transient.
This study had several strengths, including its randomized, placebo-controlled design, strict criteria for hypogonadism and anemia, excellent participant retention, and prespecified analyses for mechanisms by which testosterone stimulates erythropoiesis. Limitations include applicability of the results only to older hypogonadal men and not to men who are anemic but not hypogonadal. Another limitation is the relatively small sample sizes for anemia subtypes, resulting in low power to detect moderate effects and relatively wide confidence intervals for estimated treatment effects. Unequal sample sizes of men with unexplained anemia and iron deficiency anemia also challenge the interpretation of differential findings between the groups; we cannot rule out that lower power in the iron deficiency group due to a smaller sample size, as well as greater variability, explains the lack of a significant effect of testosterone on hemoglobin in men with iron deficiency anemia. Finally, we did not adjust for multiple comparisons, which raises the possibility of false positives. The proportion of statistically significant results, however, greatly exceeds the type I error rate, lending credibility to our findings.
We conclude that testosterone stimulates erythropoiesis in older hypogonadal men with unexplained anemia in association with increased iron mobilization. The stimulatory effects of testosterone on erythropoiesis appear to be attenuated by iron deficiency.
Acknowledgments
We express our gratitude to Dr Stanley Schrier, the chair of the Steering Committee of the Partnership for Anemia Clinical and Translational Trials in the Elderly consortium. He was instrumental in the design and conduct of this study. He died on August 15, 2019, before this paper was written. The authors also acknowledge the continued guidance and support of Drs Evan Hadley and Sergei Romashkan of the National Institute on Aging, who fostered the collaboration of the PACTTE and TTrials investigators.
Financial Support: The Testosterone Trials were supported by a grant from the National Institute on Aging, National Institutes of Health (U01 AG030644), supplemented by funds from the National Heart, Lung and Blood Institute, National Institute of Neurological Diseases and Stroke, and National Institute of Child Health and Human Development. The Anemia Trial was additionally supported by a grant from the National Institute on Aging, National Institutes of Health (U01 AG034661) to the Partnership for Anemia Clinical and Translational Trials in the Elderly consortium. AbbVie (formerly Solvay and Abbott Laboratories) generously provided funding, AndroGel, and placebo gel. SB and the Boston Center were supported partly by the Boston Claude D. Pepper Older Americans Independence Center grant 5P30AG031679.
Clinical Trial Information: ClinicalTrials.gov identifier: NCT00799617.
Glossary
Abbreviations
- BMI
body mass index
- CRP
C-reactive protein
- CV
coefficient of variation
- IL-6
interleukin-6
- TTrials
Testosterone Trials
- VEGF
vascular endothelial growth factor
Additional Information
Disclosure Summary: Dr Snyder reports grants from National Institute on Aging, grants and nonfinancial support from AbbVie (formerly Solvay and Abbott Laboratories) during the study; Dr Artz has nothing to disclose; Dr Bhasin reports grants from NIA during the study; and research grants from NIA, NINR, and NICHD, research grant and personal fees from Abbvie, research grant from Transition Therapeutics, consulting fees from OPKO, and research grant from Alivegen outside the submitted work; Dr Bhasin has a patent for a free testosterone calculator and has equity interest in FPT, LLC. Dr Ellenberg reports grants from the National Institute on Aging and AbbVie during the course of the study; Dr Cohen has nothing to disclose.
Data Availability
The datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.
