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Relation of Testosterone, Dihydrotestosterone, and Estradiol with Changes in Outcomes Measures in the Testosterone Trials (2022)
Alisa J. Stephens-Shields, PhD, Peter J. Snyder, MD, Susan S. Ellenberg, PhD, Lynne Taylor, PhD, Shalender Bhasin, MB, BS
Abstract
Context. Many effects of testosterone are mediated through dihydrotestosterone (DHT) and estradiol. The relative contributions of each hormone to the observed effects of testosterone remain incompletely understood.
Methods. Using data from the Testosterone Trials, we assessed the association of changes in total testosterone, estradiol, and DHT levels over 12-months of testosterone treatment with hemoglobin, HDL cholesterol (HDLC), volumetric bone mineral density (vBMD) of the lumbar spine, sexual desire, and prostate-specific antigen (PSA). We used random forests to model the associations of predicted mean changes in outcomes with change in each hormone at the low, mean, or high change in the other two hormones. Stepwise regression models were run to confirm the findings of random forests.
Results. Predicted increases in hemoglobin and sexual desire were greater with larger increases in estradiol and were larger with high change in DHT compared to the low change in DHT. Greater increases in estradiol were associated with larger decreases in HDLC; this association did not vary according to changes in DHT or testosterone. Change in vBMD was most robustly associated with the change in estradiol and was greater with a high change in testosterone and DHT. There was no consistent relation between change in PSA and change in any hormone.
Conclusions. Change in estradiol level was the best predictor not only of the change in vBMD and sexual desire but also of the changes in hemoglobin and HDLC. Consideration of testosterone, E2, and DHT together offers a superior prediction of treatment response in older hypogonadal men than testosterone alone.
INTRODUCTION
Circulating testosterone is converted in many peripheral tissues to its two active metabolites, 5α dihydrotestosterone (DHT) and 17β estradiol (E2) (1). In many androgen-responsive tissues, a family of steroid 5α reductase enzymes converts testosterone to DHT, and the aromatase enzyme, a product of the CYP19A1 gene, converts it to E2 (1). Many tissue-specific biologic effects of testosterone are mediated through DHT and E2. The data from preclinical gene targeting experiments, observational and Mendelian randomization studies, and randomized clinical trials have provided strong evidence of the important role of testosterone's aromatization to estradiol in mediating its effects on several reproductive and nonreproductive behaviors, fat mass and metabolism, bone mineral density, negative feedback on gonadotropin secretion through KNDy neuronal network, and plasma lipids (2- 5). Mendelian randomization studies have confirmed that genetically determined estradiol levels are more strongly associated with bone mineral density and fracture risk than genetically determined testosterone levels (3). While the effects of testosterone and estradiol on bone may be complementary, some other effects, such as those on erythropoiesis, have been viewed as antagonistic – testosterone is known to stimulate erythropoiesis (6), while E2 has been reported to inhibit erythropoiesis in some experimental models (7). In contrast, circulating testosterone levels are generally believed to be more robustly associated than estradiol levels with their anabolic effects on skeletal muscle mass, maximal voluntary strength, and some types of male behaviors (2, 6, 8-10). DHT is required for masculinization of the urogenital sinus and the genital tubercle in fetal life and possibly for mediating its effects on the prostate and hair follicle (10) but is not obligatory for mediating testosterone's effects on the skeletal muscle, bone, or erythropoiesis (11). DHT is also more potent than testosterone in its nongenomic effects on vascular smooth muscle (12).
The rates of conversion of testosterone to DHT and E2 vary among people due to polymorphisms of genes that encode the steroid 5α reductases and the aromatase enzyme as well as other host-specific factors that affect the activity of these enzymes (3, 13). In hypogonadal men treated with transdermal testosterone gels, the circulating levels of DHT and the ratio of serum DHT to testosterone levels are substantially higher than in hypogonadal men treated with the injectable testosterone esters (14), presumably due to the high activity of steroid 5α reductase enzyme in the skin. Although DHT is recognized as a potent androgen, circulating DHT levels have been ignored in assessing the efficacy of transdermal testosterone, and the circulating DHT levels are not monitored. The mean serum DHT levels in the participants of the TTrials, who were assigned to the testosterone arm of the trial, were 4 to 5 times the mean DHT levels in healthy young men; in some testosterone-treated men, the DHT levels approached the circulating testosterone levels (15). In spite of this recognition, testosterone treatment of men with hypogonadism in clinical practice is guided almost entirely by the monitoring of circulating on-treatment testosterone levels; DHT and E2 levels are rarely considered in evaluating therapeutic response to testosterone treatment or in dose adjustment (16). It is not known how the circulating concentrations of testosterone’s metabolites – DHT and E2 – modulate the effects of testosterone on various outcomes and how their circulating levels rank in their contribution to the observed effects of testosterone treatment on physiologic outcomes.
We performed secondary analyses of data from participants in the treatment arm of The Testosterone Trials (The TTrials) to assess the association of changes in total testosterone, E2, and DHT levels over the 12 months of testosterone treatment with five quantitative continuous endpoints of the trial - the changes over 12 months in hemoglobin, HDL cholesterol levels, volumetric bone mineral density (vBMD) of the trabecular bone of the lumbar spine, sexual desire, and prostate-specific antigen (PSA). We ranked the three hormones for their relative contribution in predicting change in these five biomarker outcomes of the trial.
*The Relation of Changes in Hemoglobin with Changes in Hormone Levels
*The Relation of Changes in HDL Cholesterol with the Changes in Hormone Levels
*The Relation of Changes in Volumetric Bone Mineral Density with the Changes in Each Hormone Levels
*Relation of Changes in Sexual Desire with the Changes in Hormone Levels
*The Relation of Changes in PSA with the Changes in Hormone Levels
DISCUSSION
We show here that the changes in circulating levels of T and its two major active metabolites, DHT and E2, are related in a complex manner to the observed effects of testosterone treatment on hemoglobin, HDL cholesterol, vBMD, and sexual desire. Our analyses that considered the relation between change in each of the three hormones with changes in hemoglobin, HDL cholesterol, vBMD of the trabecular bone of the spine, and sexual desire at low, mean, or high levels of the other two hormones suggest that these relations are far more complex than has been previously appreciated. We found that among the three hormones, the change in E2 level during testosterone treatment was the best predictor not only of the change in vBMD and sexual desire, as was expected but also of the change in hemoglobin and HDL cholesterol levels. Across the range of changes in testosterone, as well as DHT levels, the participants with a high change in E2 levels generally had the greatest change in hemoglobin, HDL-cholesterol, vBMD, and sexual desire. Because E2 and DHT levels during testosterone treatment with a transdermal gel varied substantially among study participants assigned to the testosterone arm of the trial, these findings suggest that simultaneous consideration of changes in on-treatment E2 as well DHT levels might offer a better prediction of response to testosterone treatment than consideration of the testosterone level alone.
These findings should be considered in the context of the study's strengths and limitations. These secondary analyses utilized data from the TTrials, one of the largest controlled clinical trials of testosterone in which subjects were allocated to intervention arms using minimization. The hormone levels were measured using validated LC-MS/MS assays (11, 15), of which testosterone and E2 assays are certified by the CDC's Hormone Standardization Program. We selected five continuous biomarker outcomes that are known to be testosterone-responsive and that could be measured quantitatively with high levels of precision and accuracy. We used random forest analysis because it imposes fewer restrictions on the relationships of hormones with markers. It works well with continuous values. We also used step-wise regression modeling to complement the findings of the random forest analysis. These analyses were not pre-specified and need confirmation in additional studies of other datasets. Our models were modestly predictive, with r-squared values ranging from 0.16 for HDL-cholesterol to 0.27 for vBMD, indicating that there is substantial variation in the testosterone response that is not explained by changes in these three hormones. The observed findings in older men in the TTrials may not be directly generalizable to younger men or men with severe organic hypogonadism.
In summary, secondary analyses of the TTrials data that considered the association of changes in the circulating level of testosterone, DHT, and E2 during testosterone treatment with three testosterone-responsive quantitative biomarker outcomes while concurrently considering the levels of the other two hormones unveiled a more complex relation of the changes in each of the three sex hormones with the outcome variable than had been previously appreciated. The changes in serum E2 levels were the most robustly associated with changes in vBMD and sexual desire during testosterone treatment, as expected, but surprisingly also with the changes in hemoglobin and HDL cholesterol levels. The strength of the relation of each hormone with biomarker outcomes was influenced by the additional consideration of the magnitude of changes in the other two hormones. Because the serum E2 and DHT levels of men treated with the transdermal testosterone gel varied substantially relative to their contemporaneous testosterone level, our findings suggest that consideration of all three sex hormones - testosterone, E2, and DHT - offers a superior prediction of treatment response for at least some of biomarker testosterone-responsive biomarker outcomes than consideration of each hormone alone. These findings have implications for our current practice of guiding the testosterone treatment of men with hypogonadism almost entirely by monitoring the on-treatment testosterone levels. Serum DHT and estradiol levels are rarely considered in evaluating therapeutic response to testosterone treatment or in dose adjustment. Further validation of these findings in other studies could provide the scientific rationale for measuring for DHT and estradiol and using the levels of these hormones as well as that of total testosterone to guide testosterone treatment of hypogonadal men. This may be particularly relevant to transdermal or oral testosterone formulations whose administration is associated with substantially higher DHT levels than with injectable testosterone esters.
Alisa J. Stephens-Shields, PhD, Peter J. Snyder, MD, Susan S. Ellenberg, PhD, Lynne Taylor, PhD, Shalender Bhasin, MB, BS
Abstract
Context. Many effects of testosterone are mediated through dihydrotestosterone (DHT) and estradiol. The relative contributions of each hormone to the observed effects of testosterone remain incompletely understood.
Methods. Using data from the Testosterone Trials, we assessed the association of changes in total testosterone, estradiol, and DHT levels over 12-months of testosterone treatment with hemoglobin, HDL cholesterol (HDLC), volumetric bone mineral density (vBMD) of the lumbar spine, sexual desire, and prostate-specific antigen (PSA). We used random forests to model the associations of predicted mean changes in outcomes with change in each hormone at the low, mean, or high change in the other two hormones. Stepwise regression models were run to confirm the findings of random forests.
Results. Predicted increases in hemoglobin and sexual desire were greater with larger increases in estradiol and were larger with high change in DHT compared to the low change in DHT. Greater increases in estradiol were associated with larger decreases in HDLC; this association did not vary according to changes in DHT or testosterone. Change in vBMD was most robustly associated with the change in estradiol and was greater with a high change in testosterone and DHT. There was no consistent relation between change in PSA and change in any hormone.
Conclusions. Change in estradiol level was the best predictor not only of the change in vBMD and sexual desire but also of the changes in hemoglobin and HDLC. Consideration of testosterone, E2, and DHT together offers a superior prediction of treatment response in older hypogonadal men than testosterone alone.
INTRODUCTION
Circulating testosterone is converted in many peripheral tissues to its two active metabolites, 5α dihydrotestosterone (DHT) and 17β estradiol (E2) (1). In many androgen-responsive tissues, a family of steroid 5α reductase enzymes converts testosterone to DHT, and the aromatase enzyme, a product of the CYP19A1 gene, converts it to E2 (1). Many tissue-specific biologic effects of testosterone are mediated through DHT and E2. The data from preclinical gene targeting experiments, observational and Mendelian randomization studies, and randomized clinical trials have provided strong evidence of the important role of testosterone's aromatization to estradiol in mediating its effects on several reproductive and nonreproductive behaviors, fat mass and metabolism, bone mineral density, negative feedback on gonadotropin secretion through KNDy neuronal network, and plasma lipids (2- 5). Mendelian randomization studies have confirmed that genetically determined estradiol levels are more strongly associated with bone mineral density and fracture risk than genetically determined testosterone levels (3). While the effects of testosterone and estradiol on bone may be complementary, some other effects, such as those on erythropoiesis, have been viewed as antagonistic – testosterone is known to stimulate erythropoiesis (6), while E2 has been reported to inhibit erythropoiesis in some experimental models (7). In contrast, circulating testosterone levels are generally believed to be more robustly associated than estradiol levels with their anabolic effects on skeletal muscle mass, maximal voluntary strength, and some types of male behaviors (2, 6, 8-10). DHT is required for masculinization of the urogenital sinus and the genital tubercle in fetal life and possibly for mediating its effects on the prostate and hair follicle (10) but is not obligatory for mediating testosterone's effects on the skeletal muscle, bone, or erythropoiesis (11). DHT is also more potent than testosterone in its nongenomic effects on vascular smooth muscle (12).
The rates of conversion of testosterone to DHT and E2 vary among people due to polymorphisms of genes that encode the steroid 5α reductases and the aromatase enzyme as well as other host-specific factors that affect the activity of these enzymes (3, 13). In hypogonadal men treated with transdermal testosterone gels, the circulating levels of DHT and the ratio of serum DHT to testosterone levels are substantially higher than in hypogonadal men treated with the injectable testosterone esters (14), presumably due to the high activity of steroid 5α reductase enzyme in the skin. Although DHT is recognized as a potent androgen, circulating DHT levels have been ignored in assessing the efficacy of transdermal testosterone, and the circulating DHT levels are not monitored. The mean serum DHT levels in the participants of the TTrials, who were assigned to the testosterone arm of the trial, were 4 to 5 times the mean DHT levels in healthy young men; in some testosterone-treated men, the DHT levels approached the circulating testosterone levels (15). In spite of this recognition, testosterone treatment of men with hypogonadism in clinical practice is guided almost entirely by the monitoring of circulating on-treatment testosterone levels; DHT and E2 levels are rarely considered in evaluating therapeutic response to testosterone treatment or in dose adjustment (16). It is not known how the circulating concentrations of testosterone’s metabolites – DHT and E2 – modulate the effects of testosterone on various outcomes and how their circulating levels rank in their contribution to the observed effects of testosterone treatment on physiologic outcomes.
We performed secondary analyses of data from participants in the treatment arm of The Testosterone Trials (The TTrials) to assess the association of changes in total testosterone, E2, and DHT levels over the 12 months of testosterone treatment with five quantitative continuous endpoints of the trial - the changes over 12 months in hemoglobin, HDL cholesterol levels, volumetric bone mineral density (vBMD) of the trabecular bone of the lumbar spine, sexual desire, and prostate-specific antigen (PSA). We ranked the three hormones for their relative contribution in predicting change in these five biomarker outcomes of the trial.
*The Relation of Changes in Hemoglobin with Changes in Hormone Levels
*The Relation of Changes in HDL Cholesterol with the Changes in Hormone Levels
*The Relation of Changes in Volumetric Bone Mineral Density with the Changes in Each Hormone Levels
*Relation of Changes in Sexual Desire with the Changes in Hormone Levels
*The Relation of Changes in PSA with the Changes in Hormone Levels
DISCUSSION
We show here that the changes in circulating levels of T and its two major active metabolites, DHT and E2, are related in a complex manner to the observed effects of testosterone treatment on hemoglobin, HDL cholesterol, vBMD, and sexual desire. Our analyses that considered the relation between change in each of the three hormones with changes in hemoglobin, HDL cholesterol, vBMD of the trabecular bone of the spine, and sexual desire at low, mean, or high levels of the other two hormones suggest that these relations are far more complex than has been previously appreciated. We found that among the three hormones, the change in E2 level during testosterone treatment was the best predictor not only of the change in vBMD and sexual desire, as was expected but also of the change in hemoglobin and HDL cholesterol levels. Across the range of changes in testosterone, as well as DHT levels, the participants with a high change in E2 levels generally had the greatest change in hemoglobin, HDL-cholesterol, vBMD, and sexual desire. Because E2 and DHT levels during testosterone treatment with a transdermal gel varied substantially among study participants assigned to the testosterone arm of the trial, these findings suggest that simultaneous consideration of changes in on-treatment E2 as well DHT levels might offer a better prediction of response to testosterone treatment than consideration of the testosterone level alone.
These findings should be considered in the context of the study's strengths and limitations. These secondary analyses utilized data from the TTrials, one of the largest controlled clinical trials of testosterone in which subjects were allocated to intervention arms using minimization. The hormone levels were measured using validated LC-MS/MS assays (11, 15), of which testosterone and E2 assays are certified by the CDC's Hormone Standardization Program. We selected five continuous biomarker outcomes that are known to be testosterone-responsive and that could be measured quantitatively with high levels of precision and accuracy. We used random forest analysis because it imposes fewer restrictions on the relationships of hormones with markers. It works well with continuous values. We also used step-wise regression modeling to complement the findings of the random forest analysis. These analyses were not pre-specified and need confirmation in additional studies of other datasets. Our models were modestly predictive, with r-squared values ranging from 0.16 for HDL-cholesterol to 0.27 for vBMD, indicating that there is substantial variation in the testosterone response that is not explained by changes in these three hormones. The observed findings in older men in the TTrials may not be directly generalizable to younger men or men with severe organic hypogonadism.
In summary, secondary analyses of the TTrials data that considered the association of changes in the circulating level of testosterone, DHT, and E2 during testosterone treatment with three testosterone-responsive quantitative biomarker outcomes while concurrently considering the levels of the other two hormones unveiled a more complex relation of the changes in each of the three sex hormones with the outcome variable than had been previously appreciated. The changes in serum E2 levels were the most robustly associated with changes in vBMD and sexual desire during testosterone treatment, as expected, but surprisingly also with the changes in hemoglobin and HDL cholesterol levels. The strength of the relation of each hormone with biomarker outcomes was influenced by the additional consideration of the magnitude of changes in the other two hormones. Because the serum E2 and DHT levels of men treated with the transdermal testosterone gel varied substantially relative to their contemporaneous testosterone level, our findings suggest that consideration of all three sex hormones - testosterone, E2, and DHT - offers a superior prediction of treatment response for at least some of biomarker testosterone-responsive biomarker outcomes than consideration of each hormone alone. These findings have implications for our current practice of guiding the testosterone treatment of men with hypogonadism almost entirely by monitoring the on-treatment testosterone levels. Serum DHT and estradiol levels are rarely considered in evaluating therapeutic response to testosterone treatment or in dose adjustment. Further validation of these findings in other studies could provide the scientific rationale for measuring for DHT and estradiol and using the levels of these hormones as well as that of total testosterone to guide testosterone treatment of hypogonadal men. This may be particularly relevant to transdermal or oral testosterone formulations whose administration is associated with substantially higher DHT levels than with injectable testosterone esters.
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