The importance of testosterone (T) for the maintenance

JCEM ONLINE Hot Topics in Translational Endocrinology Endocrine Research The Relationships between Sex Hormones and Sexual Function in Middle-Aged and Older European Men Daryl B. O Connor, David M. Lee, Giovanni Corona, Gianni Forti, Abdelouahid Tajar, Terence W. O Neill, Neil Pendleton, Gyorgy Bartfai, Steven Boonen, Felipe F. Casanueva, Joseph D. Finn, Aleksander Giwercman, Thang S. Han, Ilpo T. Huhtaniemi, Krzysztof Kula, Fernand Labrie, Michael E. J. Lean, Margus Punab, Alan J. Silman, Dirk Vanderschueren, Frederick C. W. Wu, and the European Male Ageing Study Group* Context: Limited data are available exploring the associations between sex hormones, multiple domains of sexual functioning, and sexual function-related distress in nonpatient samples in Europe. Objectives: The aim of the study was to investigate the relationships between serum testosterone (T), estradiol (E2), and dihydrotestosterone (DHT) and sexual function in a multicenter populationbased study of aging in men. Design: Using stratified random sampling, 2838 men aged 40 79 yr completed the European Male Ageing Study-Sexual Function Questionnaire and provided a blood sample for hormone measurements. T, E2, and DHT were measured using gas chromatography-mass spectrometry. Setting: We conducted a community-based population survey in eight European centers. Main Outcome Measures: Self-reported sexual function (overall sexual function, sexual functionrelated distress, erectile dysfunction, masturbation) was measured. Results: Total and free T, but not E2 or DHT, was associated with overall sexual function in middleaged and older men. E2 was the only hormone associated with sexual function-related distress such that higher levels were related to greater distress. Free T levels were associated with masturbation frequency and erectile dysfunction in the fully adjusted models, such that higher T was associated with less dysfunction and greater frequency. Moreover, there was a T threshold for the relationship between total T, sexual function, and erectile dysfunction. At T concentrations of 8 nmol/liter or less, T was associated with worse sexual functioning, whereas at T levels over 8 nmol/liter, the relationship came to a plateau. Conclusions: These findings suggest that different hormonal mechanisms may regulate sexual functioning (T) vs. the psychological aspects (E2) of male sexual behavior. Moreover, there was a T threshold for overall sexual function such that at levels greater than 8 nmol/liter the relationship between T and sexual function did not become stronger. (J Clin Endocrinol Metab 96: E1577 E1587, 2011) The importance of testosterone (T) for the maintenance of normal sexual function is well established in young eugonadal and hypogonadal men (1). However, its role in the sexual function of aging men remains controversial ISSN Print 0021-972X ISSN Online 1945-7197 Printed in U.S.A. Copyright 2011 by The Endocrine Society doi: 10.1210/jc.2010-2216 Received September 20, 2010. Accepted July 11, 2011. First Published Online August 17, 2011 (2 5). Thus, a recent meta-analysis demonstrated a sizable and significant impact of T replacement therapy on libido but a moderate, nonsignificant, and inconsistent effect on erectile function in older patients (mean age, 50 yr) (6). * Author affiliations are shown at the bottom of the next page. Abbreviations: BDI, Beck Depression Inventory; BMI, body mass index; CI, confidence interval; CV, coefficient of variation; DHT, dihydrotestosterone; E2, estradiol; ED, erectile dysfunction; GC-MS, gas chromatography-mass spectrometry; LLOQ, lower limit of quantification; LOWESS, locally weighted scatterplot smoothing; Overall SF, overall sexual functioning; SF Distress, sexual function-related distress; T, testosterone. J Clin Endocrinol Metab, October 2011, 96(10):E1577 E1587 jcem.endojournals.org E1577

E1578 O Connor et al. Sex Hormones and Sexual Function J Clin Endocrinol Metab, October 2011, 96(10):E1577 E1587 The bulk of previous research has been conducted in the United States and focused on patients with erectile dysfunction (ED) using either the International Index of Erectile Dysfunction (IIED) or the single item measure from the Massachusetts Male Aging Study (5, 7 9). Moreover, few of the existing instruments have been specifically developed for administration in large, population-based studies of nonpatient samples, and none of these measures assessed all aspects of sexual functioning [e.g. the IIED (7), Brief Sexual Function Inventory (10), and the Male Sexual Health Questionnaire (11) do not assess frequency of sexual intercourse or masturbation]. In addition, many of the items included in the existing instruments are not appropriate for use in elderly men from the general population, as opposed to patients seeking medical attention for sexual or genitourinary complaints. Recent work has explored the association between a number of sex hormones, broader aspects of sexual function, and psychological symptoms of male aging (2 5, 12 14). This has been prompted by the worldwide increase in the number of men over 65 yr of age, which is predicted to double between 1980 and 2025 (15), and the appreciation that sexual function remains a significant part of life in the elderly population (16, 17). Furthermore, ED has become a more prevalent disorder, possibly related to the rise in obesity and vascular diseases (17 19). Because circulating T declines with aging, androgen deficiency may affect a number of men at the lower extreme end of the physiological distribution. Although T deficiency is found in patients presenting with ED alone, it is commonly not the principal cause. Nevertheless, there is an increasing tendency to consider T treatment, especially in those patients unresponsive to phosphodiesterase type 5 inhibitors (5, 20 22). Moreover, the practice of using T in the management of ED is currently not supported by an adequate evidence base. The causal relationship between low T and ED, unlike loss of sexual interest and spontaneous erection, has not been firmly established, although there is some preliminary animal experimental evidence that T may regulate nitric oxide-mediated vasodilation in ex vivo penile vasculature (5, 17, 23). It is, therefore, important to further investigate the relationships between T (and other sex hormones) and various domains of sexual function in a general population sample of men without the substantial biases inherent in patient samples. Little research has explored the associations between sex hormones other than T and broader domains of sexual function such as sexual function-related distress (SF Distress). Recent work has been mixed, but some evidence has suggested that T metabolites such as estradiol (E2) and dihydrotestosterone (DHT) may be implicated in the agerelated decline of sexual function (13, 17, 24 27). For example, in a randomized, double-blind, placebo-controlled trial, Amory et al. (25) demonstrated that suppression of circulating serum DHT (by 5 -reductase inhibitors) resulted in modest, but reversible, reduction in sexual function in men. In addition, to the best of our knowledge, no previous studies have used mass spectrometry-based methods [e.g. gas chromatography-mass spectrometry (GC-MS)] with improved accuracy and precision in serum T, DHT, and E2 measurements, as recommended by The Endocrine Society (28), in studies of male sexual function. Using baseline data from the European Male Ageing Study (EMAS), an ongoing epidemiological study of aging in middle-aged and older men, Wu et al. (16) recently showed that low T was associated with three sexual symptoms (e.g. decreased morning erection, ED, and decreased frequency of sexual thoughts). However, this work was focused on developing evidence-based criteria for identifying late-onset hypogonadism in the general population. In contrast, the current study aimed to examine the relationships between multiple sex hormones (T, E2, and DHT) measured by GC-MS and all the principal domains of sexual function [i.e. overall sexual functioning (Overall SF), SF Distress, masturbation, and ED (29)] in men with a current sexual partner. A secondary aim was to investigate whether any observed associations could be explained by lifestyle, health, and psychological factors. Subjects and Methods Participants and design Our analyses are based on baseline data from the EMAS, a prospective, noninterventional cohort study of male aging in Institute of Psychological Sciences (D.B.O.), University of Leeds, Leeds LS2 9JT, United Kingdom; Arthritis Research UK Epidemiology Unit (D.M.L., A.T., T.W.O., A.J.S.), The University of Manchester, Manchester M13 9PL, United Kingdom; Andrology Unit (G.C., G.F.), Department of Clinical Physiopathology, University of Florence, 50121 Florence, Italy; School of Community-Based Medicine (N.P.), The University of Manchester, Hope Hospital, Salford M6 8, United Kingdom; Department of Obstetrics, Gynaecology and Andrology (G.B.), Albert Szent-Gyorgy Medical University, H-6720 Szeged, Hungary; Division of Gerontology and Geriatrics and Center for Musculoskeletal Research (S.B.), Department of Experimental Medicine, Katholieke Universiteit Leuven, 3000 Leuven, Belgium; Department of Medicine (F.F.C.), Santiago de Compostela University, Complejo Hospitalario Universitario de Santiago, CIBER de Fisiopatología Obesidad y Nutricion (CB06/03), Instituto Salud Carlos III, 15705 Santiago de Compostela, Spain; Andrology Research Unit (J.D.F., F.C.W.W.), Developmental and Regenerative Biomedicine Research Group, Manchester Royal Infirmary, The University of Manchester, Manchester M13 9PL, United Kingdom; Reproductive Medicine Center (A.G.), Malmö University Hospital, University of Lund, 221 00 Lund, Sweden; Department of Endocrinology (T.S.H.), Royal Free and University College Hospital Medical School, Royal Free Hospital, Hampstead, London NW3 2QG, United Kingdom; Department of Reproductive Biology (I.T.H.), Imperial College London, Hammersmith Campus, London SW7, United Kingdom; Department of Andrology and Reproductive Endocrinology (K.K.), Medical University of Łódź, 90-419 Łódź, Poland; Laboratory of Molecular Endocrinology and Oncology (F.L.), Laval University, Québec, Canada G1V 0A6; Department of Human Nutrition (M.E.J.L.), University of Glasgow, Glasgow G12 8QQ, United Kingdom; Andrology Unit (M.P.), United Laboratories of Tartu University Clinics, 50406 Tartu, Estonia; and Department of Andrology and Endocrinology (D.V.), Katholieke Universiteit Leuven, 3000 Leuven, Belgium

J Clin Endocrinol Metab, October 2011, 96(10):E1577 E1587 jcem.endojournals.org E1579 Europe. Details regarding recruitment, response rates, and assessments have been described elsewhere (30). Briefly, noninstitutionalized men aged 40 79 yr were recruited from municipal or population registers in eight centers: Florence, Italy; Leuven, Belgium; Łódź, Poland; Malmö, Sweden; Manchester, UK; Santiago de Compostela, Spain; Szeged, Hungary; and Tartu, Estonia. For the baseline survey, stratified random sampling was used with the aim of recruiting equal numbers of men into each of four age bands (40 49, 50 59, 60 69, and 70 79 yr). Participants were invited by letter to complete a short postal questionnaire and to attend a screening at a local clinic. Overall, the mean response rate for full participation in the study was 41%. The study was funded by the European Union, and ethical approval was obtained in accordance with local institutional requirements in each center, with written informed consent obtained from all participants. The current analyses focused only on men who reported having a current sexual partner to explore the relationships between hormone levels and all aspects of sexual function. Measures Participants completed a postal questionnaire at baseline that included information about self-reported health, employment, education, smoking, and alcohol consumption as well as the presence of concomitant morbidities (30). The participants also attended a research clinic to complete an interviewer-assisted questionnaire and undergo clinical assessments. The questionnaire included the Beck Depression Inventory (BDI)-II (31) and the EMAS-Sexual Function Questionnaire (EMAS-SFQ) (29). The latter was completed in private and then placed in a sealable envelope by the participants without scrutiny by the researchers. The EMAS-SFQ has been found to exhibit excellent internal and test-retest reliability, as well as convergent, divergent, and discriminant validity in psychometric analyses (29). It consists of 16 items assessing sexual functioning, SF Distress, and change in sexual functioning compared with 1 yr ago. Measurements of height and weight were carried out as described previously (30). Four of the five domains from the EMAS-SFQ were used in the present analysis and are summarized in Supplemental Table 1 (published on The Endocrine Society s Journals Online web site at http://jcem.endojournals.org). These were: Overall SF, SF Distress, frequency of masturbation, and ED. The change in sexual functioning domain results was not reported in the current crosssectional study because it was felt that responses to these items were potentially heavily influenced by retrospective bias and inaccurate recall. The Overall SF and SF Distress scores are all derived from five self-report items. Items in the Overall SF score were: frequency of sexual thoughts, sexual intercourse, petting, morning erection, and orgasm. SF Distress items were worrying about: frequency of desire, sexual intercourse, erection, morning erection, and orgasm. Low scores on the Overall SF and higher scores on the SF Distress scales represent worse sexual functioning. Single item scores were used for frequency of masturbation and ED (see Supplemental Data). Hormone measurements A single fasting morning (before 1000 h) venous blood sample was obtained, and processed serum was stored at 80 C. A validated GC-MS system (32) was used to analyze T [1.7 nmol/ liter; lower limit of quantification (LLOQ), 0.17 nmol/liter; intraassay coefficient of variation (CV), 2.9%; interassay CV, 3.4%], E2 (70 pmol/liter; LLOQ, 7.3 pmol/liter; intraassay CV, 3.5%; interassay CV, 3.7%), and DHT (LLOQ, 0.07 nmol/liter; intraassay CV, 3.1%; interassay CV, 4.1%). All GC-MS analyses were performed in the Laboratory of Molecular Endocrinology, Laval University, Quebec. SHBG was measured by the Modular E170 platform electrochemiluminescence immunoassay (Roche Diagnostics, Mannheim, Germany) as previously described (33), with a detection limit of 0.35 nmol/liter and intra- and interassay CV of 1.7 and 3.2%, respectively. Free T levels were derived from total hormone, SHBG, and albumin concentrations and using mass action equations and association constants from Vermeulen et al. (34). 1 Statistical analysis Statistical analyses were performed using Intercooled Stata version 9.2 (StataCorp, College Station, TX). Subjects without a sexual partner, with prevalent pituitary or testicular disease, or those using medications that could directly impact upon pituitary/testicular function (33) were excluded from this analysis. Hormone levels, age, the BDI, body mass index (BMI), and the Overall SF score were treated as continuous variables. The SF Distress scores, frequency of masturbation, and ED were examined as ordinal variables. Bivariate associations of the four EMAS-SFQ domains with other covariates were examined using Pearson (r p ) or Spearman (r s ) correlation. Linear regression was used to determine the association of hormones (independent variables) with the Overall SF score (dependent variable) because the Overall SF variable approximated a normal distribution. Adjustments were made for age, BMI, center, smoking, alcohol consumption, depression, and number of morbidities. Results are expressed as -coefficients ( ) and 95% confidence intervals (CI). Because the SF Distress scores, frequency of masturbation, and ED variables were not normally distributed, the associations between hormones and these outcomes were explored using ordinal logistic regression (proportional odds models). Ordinal logistic regression is an extension of logistic regression, and the resulting coefficient for a given predictor represents the odds of being in a higher outcome category associated with a unit increase in the value of that variable (35). The SF Distress scores and frequency of masturbation were each reduced into a three-item ordered response outcome, whereas the ED variable was retained as a four-item outcome. Nonviolation of the parallel slope assumption of the ordinal logistic models was assessed using the Brant test in Stata. A full summary of EMAS-SFQ variable recoding for the ordinal logistic regressions is shown in Supplemental Table 2. The ordinal models were also adjusted for the same independent variables used in the linear regression models, and the results were expressed as odds ratios and 95% CI. Where significant, independent associations between hormones and sexual function were observed in the multiple linear regressions or multiple ordinal logistic regressions; an additional model also included the standardized z-scored hormone. This provided a comparative measure of the magnitude of the association between each hormone (per SD change) and the sexual function outcome, independent of the units of concentration. In addition, where significant associations were observed between either T or E2 and the Overall SF or SF Distress domains 1 Note that our results remained unchanged when free T was calculated using the Sartorius formula (47).

E1580 O Connor et al. Sex Hormones and Sexual Function J Clin Endocrinol Metab, October 2011, 96(10):E1577 E1587 TABLE 1. Baseline characteristics: men with a sexual partner (n 2963) Percentiles Mean (SD) (2.5, 97.5) Age (yr) 59.4 (10.9) BDI-II 6.6 (6.3) BMI (kg/m 2 ) 27.6 (4.0) No. of morbidities 0.9 (1.1) Overall SF 19.5 (8.1) (6, 33) SF distress 6.7 (3.4) Total T (nmol/liter) 16.6 (5.9) (7.4, 30.4) Free T (pmol/liter) 293 (87) (145, 485) Total E2 (pmol/liter) 73.9 (25.1) (36.2, 133) DHT (nmol/liter) 1.3 (0.6) (0.4, 2.8) SHBG (nmol/liter) 42.7 (19.8) (16.8, 91.8) n (%) Masturbation ( once/wk) 471 (17) ED a 802 (28) Worried about erectile function b 931 (33) Current smoker 629 (21) Alcohol ( 1 d/wk) 1683 (57) Obese (BMI 30 kg/m 2 ) 700 (24) Depression c 104 (4) Living arrangement Living with wife 2467 (86) Living with partner 198 (7) Partner, living apart 195 (7) a Sometimes able to Never able to attain an erection good enough for sexual intercourse. b A little bit to extremely worried or distressed. c BDI-II score 13 and/or using antidepressant medications. after multivariable adjustment, the analyses were extended to explore the relationships between hormone levels (z-scored) and the individual items making up each domain using ordinal logistic regression. Results Participants A total of 406 participants who had no current sexual partner, had known pituitary or testicular diseases, or used medications that could affect pituitary/testicular functions or sex steroid clearance (33) were excluded from this analysis. The baseline characteristics of the remaining 2963 men are shown in Table 1. Complete data concerning frequency of sexual activities and erectile function were available in 2838 and 2830 subjects, respectively. Among those reporting one or more morbidities (n 1429), the four most common conditions were hypertension (56%), heart condition (31%), prostate disease (20%), and diabetes (15%) (see Supplemental Table 3 for the associations between main morbidities and SF domains). The Overall SF score (r p 0.51; P 0.001) and frequency of masturbation (r s 0.27; P 0.001) were negatively associated with age, whereas ED (r s 0.48; P 0.001) was positively associated. Total T (r p 0.04; P 0.04) and free T (r p 0.37; P 0.001) showed negative, and total E2 (r p 0.12; P 0.001), DHT (r p 0.05; P 0.01), and SHBG (r p 0.36; P 0.001) showed positive cross-sectional relationships with age. Using a multilevel regression model, we found that the majority of the hormone variance was due to interindividual variation, and only around 5% was attributable to contextual effects, i.e. center, such that 94, 95, and 97% of the variance in T, E2, and DHT, respectively, was due to interindividual variation. Having shown that the center effect was relatively minor, we chose to adjust for center in our statistical models, rather than stratify analyses by center. Hormones and Overall SF Relationships between Overall SF and T, free T, E2, and DHT are shown in Table 2. Models are presented unadjusted and adjusted for age, smoking, alcohol consumption, depression, BMI, number of morbidities, and center. Significant univariate associations were observed between the Overall SF score and free T ( 0.02; P 0.001) and total E2 ( 0.02; P 0.01) only. After multivariable adjustment (model a), higher levels of total T, free T, and total E2 were significantly associated with a higher Overall SF score. To facilitate comparisons between regression models, hormones were also z-scored to provide a measure of the relative magnitude of the association between each hormone and the Overall SF score, independent of the units of concentration (model b). For each SD change in hormone, the association with the Overall SF score was greatest for free T ( 0.55; P 0.001), and then for total T( 0.37; P 0.01) and total E2 ( 0.25; P 0.05). When the T models were additionally adjusted for total E2, the results were broadly unchanged (model c). However, when the E2 model was additionally adjusted for total T, the association was no longer significant. There was no significant association between the Overall SF score and DHT. In view of the observed associations between the Overall SF score and higher total and free T, we also examined the relationship between the five individual items making up the Overall SF score and z-scored T. The results of these additional multiple ordinal logistic regressions are summarized in Fig. 1 and show that of the items making up the Overall SF score, only frequency of petting/ kissing was not associated with T. Locally weighted scatterplot smoothing (LOWESS) and spline regression analysis were used to explore whether there were any obvious threshold effects for the association of total T with the Overall SF score. The LOWESS plot suggested that there was an inflection point around 8 nmol/ liter total T (Fig. 2). When modeled separately with two slopes (breakpoint 8 nmol/liter) in an age-adjusted

J Clin Endocrinol Metab, October 2011, 96(10):E1577 E1587 jcem.endojournals.org E1581 TABLE 2. Association between hormones and Overall SF: linear regressions Model Unadjusted health a health b health c Total T (nmol/liter) 0.021 ( 0.026, 0.067) 0.063 (0.019, 0.106)** 0.374 (0.115, 0.633)** 0.325 (0.014, 0.637)* Free T (pmol/liter) 0.019 (0.016, 0.022)*** 0.006 (0.003, 0.009)*** 0.553 (0.285, 0.821)*** 0.557 (0.246, 0.868)*** Total E2 (pmol/liter) 0.019 ( 0.030, 0.008)** 0.010 (0.000, 0.020)* 0.252 (0.012, 0.492)* 0.104 ( 0.185, 0.392) DHT (nmol/liter) 0.340 ( 0.790, 0.110) 0.385 ( 0.036, 0.806) Data are expressed as coefficient (95% CI). Analysis excludes subjects without a sexual partner, but with complete item response for Overall SF domain (n 2838). a Adjusted for age, smoking (nonsmoker vs. current smoker), alcohol consumption ( 1 d/wk vs. 1 d/wk), depression (BDI score), BMI, number of morbidities, and center. b Adjusted for all variables above: hormones z-scored to report Overall SF change per SD change in hormone. c T model additionally adjusted for total E2; E2 model additionally adjusted for total T (all hormones z-scored). *, P 0.05; **, P 0.01; ***, P 0.001. spline regression, there was a significant difference between the models (P 0.001; see Fig. 2 and Supplemental Table 4). Hormones and SF Distress Relationships between SF Distress and sex hormones (ordinal logistic regressions) are summarized in Table 3. A significant univariate association was observed between SF Distress and higher total E2 only, which remained after multivariable adjustment (model a). Using z-scored hormones (model b), the adjusted odds for being in a higher distress group, compared with the reference group (no distress), increased approximately 10% for each SD increase in total E2. This relationship was unchanged after additional adjustment for total T (model c). We additionally examined the associations between the five items making up the SF Distress domain and z-scored total E2. Figure 3 summarizes the results of these multiple ordinal logistic regressions and shows that only distress about the orgasmic experience was not associated with total E2. LOWESS and spline regression analysis were used again to explore whether there were any obvious threshold effects for the association of total E2 with the SF Distress score. The LOWESS plot suggested that there was no inflection point (data not shown). Hormones and masturbation frequency Relationships between frequency of masturbation and sex hormones are shown in Table 4. Significant univariate associations were observed between masturbation frequency and total T and free T, which remained significant after multivariable adjustment (model a). Using z-scored hormones (model b), the adjusted odds for being in an infrequent masturbation group compared with the reference group (frequent), decreased between approximately 9 and 16% for each SD increase in total T or free T. After additional adjustment for total E2, the relationship between total T and masturbation frequency was no longer significant (model c), whereas the relationship with free T remained unchanged. There was no relationship between DHT and frequency of masturbation. Hormones and ED Relationships between ED and sex hormones are shown in Table 5. Significant univariate associations were observed FIG. 1. Association of T with Overall SF items. **, P 0.01; ***, P 0.001. FIG. 2. Relationship between total T and Overall SF score with evidence of a T threshold at 8 nmol/liter. LOWESS plot adjusted for age.

E1582 O Connor et al. Sex Hormones and Sexual Function J Clin Endocrinol Metab, October 2011, 96(10):E1577 E1587 TABLE 3. Association between hormones and overall SF Distress (3 category variable): ordinal logistic regressions Model Unadjusted health a health b health c Total T (nmol/liter) 1.008 (0.996, 1.021) 1.003 (0.988, 1.018) Free T (pmol/liter) 1.000 (0.999, 1.001) 1.000 (0.999, 1.001) Total E2 (pmol/liter) 1.005 (1.002, 1.008)*** 1.004 (1.001, 1.007)* 1.098 (1.013, 1.191)* 1.125 (1.021, 1.239)* DHT (nmol/liter) 1.055 (0.935, 1.189) 1.032 (0.895, 1.189) Data are expressed as odds ratio (95% CI), with odds ratio of being in a higher distress group. Analysis excludes subjects without a sexual partner, but with complete item response for SF Distress domain (n 2717). a Adjusted for age, smoking (nonsmokers vs. current smokers), alcohol consumption ( 1 d/wk vs. 1 d/wk), depression (BDI score), BMI, number of morbidities, and center. b Adjusted for all variables above: hormones z-scored to report odds ratio per SD change in hormone. c E2 model additionally adjusted for total T (all hormones z-scored). *, P 0.05; ***, P 0.001. only between free T (P 0.001) and a decreased likelihood of ED, and between total E2 and an increased likelihood of ED (P 0.001). Only free T and a decreased likelihood of ED remained significant after multivariable adjustment (model a). Using z-scored hormones (model b), the adjusted odds for being in the ED group compared with the reference group (normal erection) decreased approximately 9% for each SD increase in free T. Additional adjustment for total E2 did not change the magnitude of the association between free T and ED (data not shown). In keeping with our earlier work reported by Wu et al. (16), we examined whether there were any obvious threshold effects for the association of total T and likelihood of ED using spline regression analysis. The results showed that there was a significant relationship between T and likelihood of ED at total T concentrations less than or equal to 8 nmol/liter (P 0.01) and that the relationship came to a plateau at total T levels over 8 nmol/liter. FIG. 3. Association of total E2 with SF Distress items. Ordinal logistic regression models adjusted for age, smoking (nonsmoking vs. current), alcohol consumption ( 1 d/wk vs. 1 d/wk), depression (BDI score), BMI, number of morbidities, and center. Hormones z-scored to report odds ratio per SD change in hormone. *, P 0.05; **, P 0.01; ***, P 0.001. Analysis excludes subjects without a sexual partner; hormones z-scored. Discussion Three main findings emerged from the current study. First, T, and not E2 or DHT, was found to be related to Overall SF in middle-aged and older European men. Second, there was a T threshold for the relationship between total T, Overall SF, and ED. Third, E2 was the only hormone associated with SF Distress. Moreover, this is the first study to simultaneously explore, in community-dwelling men, all aspects of sexual function including frequency of sexual behavior activities (pertinent to the wide age range under study) including libido, masturbation, ED, and distress while controlling for lifestyle factors (e.g. smoking, alcohol consumption), the presence of morbidities (e.g. heart disease, diabetes), and the availability of a sexual partner. To the best of our knowledge, this is also the first study using a mass spectrometry-based method to measure T, DHT, and E2 simultaneously in an aging male cohort. We were able to demonstrate that total T levels were positively associated with Overall SF but not directly related to masturbation frequency or ED (in our fully adjusted models) in the entire sample. Importantly, the former finding remained statistically significant after adjusting for total E2 levels, suggesting that T is directly (independent of aromatization or estrogen receptor activation) associated with sexual function in older men (across the entire physiological range). In terms of other previous communitybased surveys, the Olmsted County study showed that age-related decline in sexual function was not associated with reductions in total T (2). Similarly, Ahn et al. (36) in a sample of aging men reported that total T was not associated with ED, whereas consistent with the current study, free T levels were related to ED. A likely explanation for these inconsistencies is that the two earlier studies were underpowered (n 294 and 213, respectively) to detect the relatively small associations observed in the

J Clin Endocrinol Metab, October 2011, 96(10):E1577 E1587 jcem.endojournals.org E1583 TABLE 4. Association between hormones and frequency of masturbation (3 category variable): ordinal logistic regressions Model Unadjusted health a health b health c Total T (nmol/liter) 0.969 (0.958, 0.981)*** 0.983 (0.968, 0.998)* 0.902 (0.825, 0.987)* 0.911 (0.818, 1.015) Free T (pmol/liter) 0.995 (0.994, 0.996)*** 0.998 (0.997, 0.999)*** 0.841 (0.766, 0.922)*** 0.829 (0.744, 0.923)** Total E2 (pmol/liter) 0.999 (0.996, 1.002) 0.997 (0.994, 1.001) DHT (nmol/liter) 0.907 (0.804, 1.022) 0.925 (0.797, 1.073) Data are expressed as odds ratio (95% CI), with odds ratio of being in a lower frequency group. Analysis excludes subjects without a sexual partner, but who answered the frequency of masturbation question (n 2839). a Adjusted for age, smoking (nonsmoker vs. current smoker), alcohol consumption ( 1 d/wk vs. 1 d/wk), depression (BDI score), BMI, number of morbidities and center. b Adjusted for all variables above: hormones z-scored to report odds ratio per SD change in hormone. c T model additionally adjusted for total E2 (all hormones z-scored). *, P 0.05; **, P 0.01; ***, P 0.001. EMAS cohort. Moreover, it is critical to highlight that although the relationship between total T and Overall SF was statistically significant in the current investigation, the magnitude of this association was modest. However, we also found evidence of a threshold for the relationship between total T levels and Overall SF and ED such that at levels less than or equal to 8 nmol/liter, lower levels of T were associated with worse sexual functioning (see Supplemental Table 5 for a summary of recent evidence and Refs. 5 and 6 for related reviews). At levels above 8 nmol/liter, the relationship came to a plateau and importantly did not become stronger at higher levels of T. These results are in keeping with our earlier work on the identification of late-onset hypogonadism (16), whereby low levels of T were found to be associated with measures of Overall SF assessed using the EMAS-SFQ. Moreover, they are in keeping with previous research by Bhasin and colleagues (37, 38). For example, in an experimental study, Gray et al. (37) found evidence of a T dose-response relationship, consistent with the existence of a T threshold effect, in a sample of older men following suppression of endogenous T by a long-acting GnRH agonist. In contrast, an earlier investigation in healthy younger men by the same group, using a similar experimental design, failed to show a dose-response relationship between T and sexual function. Similarly, O Connor et al. (4) also failed to show any effects of exogenous T on sexual function in a sample of healthy young men. Therefore, these findings taken together suggest that: 1) T supplementation will not have additional beneficial effects on sexual function once T levels are restored to the normal male range; 2) a T threshold exists for Overall SF and ED (but not for petting/kissing) in older men; and 3) the relationship between T and sexual function may be different in older compared with younger men. Future research ought to use well-designed, experimental trials to explore the precise nature of the relationship between T and sexual functioning in older and younger men. We also found that total E2 was the only sex hormone associated with SF Distress, even after adjusting for T levels, such that higher E2 levels were related to greater distress. This suggests that the observed effects of E2 are independent of T. Thus, different hormonal mechanisms may regulate the behavioral (T) vs. the psychological/state TABLE 5. Association between hormones and ED (4 category variable): ordinal logistic regressions Model Unadjusted health a health b Total T (nmol/liter) 0.999 (0.988, 1.010) 0.998 (0.984, 1.012) Free T (pmol/liter) 0.995 (0.994, 0.996)*** 0.999 (0.998, 1.000)* 0.910 (0.833, 0.993)* Total E2 (pmol/liter) 1.009 (1.006, 1.012)*** 1.003 (0.999, 1.006) DHT (nmol/liter) 1.111 (0.994, 1.242) 1.053 (0.923, 1.202) Data are expressed as odds ratio (95% CI). Analysis excludes subjects without a sexual partner, but who answered the erectile function question (n 2830). a Adjusted for age, smoking (nonsmoker vs. current smoker), alcohol consumption ( 1 d/wk vs. 1 d/wk), depression (BDI score), BMI, number of morbidities and center. b Adjusted for all variables above: hormones z-scored to report odds ratio per SD change in hormone. *, P 0.05; ***, P 0.001.

E1584 O Connor et al. Sex Hormones and Sexual Function J Clin Endocrinol Metab, October 2011, 96(10):E1577 E1587 (E2) aspects of male sexual functioning, implying that T treatment is unlikely to provide beneficial effects for SF Distress. A possible explanation for our findings is that increasing levels of E2 may be associated with the development of depressed mood related to declining sexual function in aging men. Some evidence in women has shown clear associations between estrogen levels, depression, and dysphoric mood (39, 40), albeit in the opposite direction. However, the importance of E2 in depression in women remains controversial, and it is acknowledged that the findings are not uniform (41). To the best of our knowledge, no studies have provided evidence of either a positive or negative relationship between total E2 and depression in aging men (42). For example, in a sample of community-dwelling older men, Barrett-Connor et al. (42) showed that total E2 was not associated with depressed mood (measured using the BDI), whereas low T was associated with depression. This null finding may reflect that, similar to women, the relationship between E2 and depression is also not uniform in men and/or highlights that other mechanisms might explain the current observed relationship. Age-related increases in E2 may lead to subtle changes in global assessments of sexual function such as satisfaction (not measured in the current study), which may have knock-on effects on the psychological aspects of sexual functioning. Nonetheless, our finding is consistent with that of Basar et al. (13) who showed that low total (and free) T was associated with sexual symptoms of aging (e.g. impaired potency, ED), whereas E2 was positively correlated with the psychological symptoms (e.g. depressed, irritable, anxious) of male aging. It is also noteworthy in the current analyses that the association between E2 and SF Distress persisted after controlling for depression levels (assessed using the BDI) and that E2 levels were not associated with BDI scores (data not shown). Therefore, the current data cannot rule out the possibility that higher E2 levels are associated with the development of subclinical levels of depression (not measured by the BDI) or negative mood related to declining sexual function. Previous studies on E2 in men using immunoassays were potentially hampered by assay imprecision and variability so that data, particularly in aging men, may not always be reliable. Our current results using GC-MS are less likely to suffer from these potential assay problems. Future research should employ mass spectrometry measurements to further explore the relationships between E2 and SF Distress and measures of clinical and subclinical depression [e.g. the profile of mood states has been used successfully in older populations (43)]. In terms of clinical studies, evidence from case studies of men with aromatase deficiency suggests that E2 may play a role in male sexual activity. Carani et al. (44) have observed that normal sexual function was restored in an aromatase-deficient man only after estrogen (and not T) treatment. More recently, the same group reported that sex drive was returned to normal levels in another man with aromatase deficiency and hypogonadism when both T and E levels (and not T alone) were returned to the normal range (45). Therefore, in addition to T, it is possible that optimal levels of E2 are also required to maintain different aspects of sexual function in aging men (similar to components of cognitive function; see Ref. 46). If this is the case, then it is feasible that higher and lower levels of E2 would be associated with negative effects on sexual activities and experience. The findings in relation to free T require further comment. We found that free T levels were associated with ED and masturbation frequency, such that high T was related to less dysfunction and greater frequency in the fully adjusted models. We recognize that the use of calculated hormones such as free T is controversial and that recent studies have demonstrated that empirical formulae (such as the Vermeulen method) overestimate free T relative to laboratory measurement by equilibrium dialysis [e.g. Sartorius et al. (47); Ly et al. (48)]. For example, Ly and colleagues, comparing the accuracy of five different methods, showed that the two empirical equations used by Sartorius et al. (47) and Ly et al. (48) consistently performed better than the equilibrium-binding methods used by Vermeulen et al. (34) and Södergård et al. (49) as well as another empirical formula published by Nanjee and Wheeler (50). These issues notwithstanding, in the current study, we have presented our results using the Vermeulen method to allow direct comparisons with our previous findings and other published studies. Moreover, it is important to note that our results remained unchanged when free T was calculated using the Sartorius formula. It is also possible that the calculation of free T is confounded by age, given that SHBG is itself largely age dependent, and as a result, the free hormone represents age in a covert form. A number of factors indicate that this is not the case. In our preliminary analyses, collinearity diagnostics between the predictor variables (e.g. age, hormones) were found to be low (i.e. all variance inflation factors were around 1.5), indicating an absence of multicollinearity. In addition, the -coefficient between SHBG and age was also small, signifying that although the relationship was statistically significant, only 14% of the variance in SHBG was explained by age. Nevertheless, we cannot completely rule out the possible confounding by age of the observed relationship between free T, masturbation, and ED, and future researchers ought to be mindful of the potential limitation.

J Clin Endocrinol Metab, October 2011, 96(10):E1577 E1587 jcem.endojournals.org E1585 The main strengths of our study are that it was based on a large sample of community-dwelling men and used uniform methods to assess sexual function, hormone levels, and putative confounders. In addition, it employed a stateof-the-art GC-MS methodology for measuring serum T, E2, and DHT levels simultaneously. Practical limitations inherent to the EMAS have been described previously (30), although a number of specific factors need to be considered here. The overall response rate for participation in the study was 41%. Those who participated may have differed, with respect to levels of sexual function/distress and also sex hormone status, from those who did not participate, and some caution is therefore needed in interpreting these data. The main findings, however, were based on internal association among responders, which reduces the risk that selection factors had any important effect on these results. The cross-sectional data cannot offer any explanation for the observed associations or the temporal nature of these relationships. There remains the possibility that some of our findings may be due to unmeasured factors, residual confounding, and/or misclassification bias from self-reported lifestyle factors (e.g. smoking status). In addition, we recognize that the EMAS-SFQ is a self-report instrument, and therefore responses to its questions may be influenced by extraneous variables (e.g. quality of the sexual relationship with the man s partner), and it may not capture all the subtleties of a man s sexual relationship with his partner. We are also mindful that the Overall SF domain may not always be sensitive to detecting sexual dysfunction; however, the EMAS-SFQ does include an established measure of ED. These potential shortcomings notwithstanding, it is important to bear in mind that the instrument has excellent psychometric properties, is sensitive to variations in sex hormones, and was specifically developed to assess all aspects of sexual functioning in community-dwelling middle-aged and older men. In conclusion, using GC-MS, this study showed that T, and not E2 or DHT, was associated with Overall SF in middle-aged and older European men. E2 was the only sex hormone related to SF Distress. Free T, but not total T, was associated with ED or masturbation frequency in the fully adjusted models. Moreover, there was a T threshold for the relationship between total T, sexual function, and ED. At total T concentrations less than or equal to 8 nmol/liter, T was associated with worse sexual functioning, whereas at total T levels over 8 nmol/liter, the relationship came to a plateau. The magnitude of the observed associations was modest, and the relationship between T and Overall SF did not become stronger at higher levels of T. Prospective data may well provide clues to the directionality of these associations, although cause and effect can only be satisfactorily addressed with well-designed and sufficiently powered interventional trials. Appendix The EMAS Study Group: Florence Gianni Forti, Luisa Petrone, and Giovanni Corona; Leuven Dirk Vanderschueren, Steven Boonen, and Herman Borghs; Łódź Krzysztof Kula, Jolanta Slowikowska-Hilczer, and Renata Walczak-Jedrzejowska; London Ilpo Huhtaniemi; Malmö Aleksander Giwercman; Manchester Frederick Wu, Alan Silman, Neil Pendleton, Terence O Neill, Joseph Finn, Philip Steer, Abdelouahid Tajar, David Lee, and Stephen Pye; Santiago Felipe Casanueva, and Mary Lage; Szeged Gyorgy Bartfai, Imre Földesi, and Imre Fejes; Tartu Margus Punab, and Paul Korrovitz; and Turku Min Jiang. The authors thank the men who participated in the eight countries and the research/nursing staff in the eight centers: C. Pott (Manchester), E. Wouters (Leuven), M. Nilsson (Malmö), M. del Mar Fernandez (Santiago de Compostela), M. Jedrzejowska (Łódź), H.-M. Tabo (Tartu), and A. Heredi (Szeged) for their data collection; and C. Moseley (Manchester) for data entry and project coordination. Acknowledgments Address all correspondence and requests for reprints to: Daryl B. O Connor, Institute of Psychological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom. E-mail: d.b.o connor@leeds.ac.uk. The European Male Aging Study is funded by the Commission of the European Communities Fifth Framework Program Quality of Life and Management of Living Resources Grant QLK6-CT-2001-00258. Additional support was also provided by Arthritis Research UK. The sponsor had no role in the study. S.B. is senior clinical investigator of the Fund for Scientific Research, Flanders, Belgium (F.W.O. 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