Martina Dören, M.D., a Alexander Rübig, M.D., b Herjan J. T. Coelingh Bennink, M.D., c and Wolfgang Holzgreve, M.D. d

FERTILITY AND STERILITY VOL. 75, NO. 3, MARCH 2001 Copyright 2001 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Differential effects on the androgen status of postmenopausal women treated with tibolone and continuous combined estradiol and norethindrone acetate replacement therapy Martina Dören, M.D., a Alexander Rübig, M.D., b Herjan J. T. Coelingh Bennink, M.D., c and Wolfgang Holzgreve, M.D. d Department of Obstetrics and Gynecology, Westfälische Wilhelms-Universität, Münster, Münster, Germany Received June 16, 2000; revised and accepted November 7, 2000. Supported by NV Organon, Oss, The Netherlands. Reprint requests: Martina Dören, M.D, Free University of Berlin, Benjamin Franklin University Hospital, Clinical Research Center for Women s Health, Klingsorstrasse 109a, D- 12203 Berlin, Germany (FAX: 49 30 8441 5819; E-mail: doeren@ukbf.fuberlin.de). a Free University of Berlin, Benjamin Franklin University Hospital, Clinical Research Center for Women s Health, Berlin, Germany. b University of Oviedo, Faculty of Obstetrics and Gynecology, Celestino Villamil, Oviedo, Spain. c Akzo Nobel NV Organon, Oss, The Netherlands. d Department of Obstetrics and Gynecology, Kantonsspital Basel, Basel, Switzerland. 0015-0282/01/$20.00 PII S0015-0282(00)01768-4 Objective: To determine serum parameters reflective of androgen status in postmenopausal women using two types of hormone replacement therapy (HRT). Design: Randomized, double-blind, prospective 1-year trial of two oral HRT regimens. Setting: University hospital, department of obstetrics and gynecology, menopause clinic. Patient(s): 100 postmenopausal women 45 years. Intervention(s): Daily use of the progestogen tibolone (2.5 mg; n 50) or continuous combined 17- estradiol (2 mg) and norethindrone acetate (E NA, 1 mg; n 50). Main Outcome Measure(s): Measurements of total testosterone (total T), dehydroepiandrosterone sulfate (DHEAS), androstenedione (A), FSH, and sex-hormone-binding globulin (SHBG), and calculations of free testosterone (free T). Assessment of changes from baseline within and between groups after 6 and 12 months. Result(s): We found significant differences (% changes) in the tibolone group compared to baseline within the groups after both 6 and 12 months, respectively. Levels of free T doubled, total T decreased slightly, and SHBG decreased by half; DHEAS increased by approximately 20%; and FSH decreased. In the E NA group, levels of free T, total T, androstenedione, and FSH all decreased, and SHBG increased. Pre-trial levels of DHEAS, A, and total T were significantly higher in the E NA group. Between groups throughout the study, the changes from baseline were significant due to the different extent of FSH reduction, and opposite changes of free T, SHBG, and DHEAS. Conclusion(s): Both regimens modify plasma androgens, DHEAS, and SHBG differently. Tibolone decreased the levels of SHBG, and substantially increased free T and to a lesser extent increased DHEAS; this may reflect a modification of adrenal androgen production. Continuous combined estradiol and norethindrone acetate HRT suppressed the peripheral plasma androgens mediated by increased levels of SHBG. (Fertil Steril 2001;75:554 9. 2001 by American Society for Reproductive Medicine.) Key Words: Androgens, DHEAS, androstenedione, testosterone, SHBG, tibolone, estradiol, and norethindrone acetate replacement therapy, hormone replacement therapy, adrenal function The clinical effects of various hormone replacement therapy (HRT) regimens and their impact on estradiol and gonadotropin levels are well characterized; however, much less is known about the changes of plasma androgens. The available data are both largely observational and inconsistent regarding dehydroepiandrosterone sulfate (DHEAS), androstenedione (A), and total testosterone (total T) in women using HRT (1). DHEAS has been discussed in conjunction with a wide variety of diseases and conditions, including breast and prostate cancer, cognition, mood, immune function, and cardiovascular disease (2 5). We report the results of one prespecified secondary objective, which addressed changes of androgen status within a randomized, double-blind, 1-year HRT trial. This study was designed to assess endometrial response. The oral HRT regimen consisted of tibolone (2.5 mg daily), or continuous combined estradiol 554

(2.5 mg) and norethindrone acetate (E NA, 1 mg daily). Tibolone is a 7 -methyl derivative of norethynodrel, a progestogen, which also exerts androgenic and weak estrogenic effects (6). Previous trials of the study regimens have suggested differential effects upon androgens (7, 8). Thus, we measured total T, A, DHEAS, sex-hormone-binding globulin (SHBG), and calculated free testosterone (free T) within a comparative trial. The impact of both regimens on these parameters has not been well characterized and previous data are inconsistent. Both regimens are in clinical use in European countries to treat menopausal symptoms and prevent bone loss without inducing regular withdrawal bleeding. MATERIAL AND METHODS Study Participants and Design The study was approved by the Ethics Committee of the University of Münster. We recruited 100 white women as has been described elsewhere (9). The study s main objective was to assess changes in endometrial thickness. Secondary study objectives were to measure uterine bleeding (9), the impedance to pelvic blood flow (examined via transvaginal color Doppler sonography), and levels of plasma lipids (10). Women who participated had to be 45 years with a natural menopause (last bleeding 1 year prior to enrollment). In cases of previous HRT, a wash-out phase of 2 weeks was mandatory. This short interval allowed for recruitment of symptomatic women who were seeking a new HRT treatment. The exclusion criteria were endometrial thickness greater than 5 mm (double-layer); unexplained uterine bleeding; any cardiovascular or cerebrovascular disease, (para)thyroid or adrenal disease, any chronic disease, thromboembolic disorders, uncorrected hypertension, untreated hypercholesteremia, history of liver or renal disease; any malignancy less than 10 years prior to enrollment, or any history or presence of hormone-dependent malignancy; chronic use of anticonvulsants, diuretics, corticosteroids, antibiotics, heparin, lipidlowering drugs; alcohol and drug abuse; smoking (more than 10 cigarettes a day); and use of investigational drugs less than 30 days prior to enrollment. Women were randomized to receive oral daily treatment with tibolone (2.5 mg; n 50; Livial, NV Organon, Oss, The Netherlands), or 17- -estradiol (2 mg) and norethindrone acetate (1 mg; n 50; Kliogest, Novo Nordisk A/S, Copenhagen, Denmark). They took one active drug together with a visually matching placebo (double-dummy method) in the evenings. Endocrine Parameters The fasting serum samples were assessed at baseline, after 6 months, and after 12 months by one laboratory blinded to the treatment protocol (Bioscientia, Mainz, Germany). Samples were obtained between 0800 and 1400 hours, stored at 20 C, and processed within 72 hours. TABLE 1 Demographic characteristics. Tibolone (2.5 mg/day) (n 49) Estradiol (2 mg) norethisterone acetate (1 mg/day) (n 49) Age (year) 56.4 3.8 56.3 5.1 (46 64) (46 69) Body mass index (kg/m 2 ) 25.9 3.5 25.3 3.2 (16 35) (20 35) Menopausal age (year) 49.5 3.8 49.9 3.7 (37 57) (38 57) Time since menopause (year) 6.9 4.6 6.4 4.9 (1 20) (1 20) Pre-trial use of HRT (%) 57.1% 53.1% (n 28) (n 26) n n Estradiol (valerate) compounds 22 20 CEE 4 4 Transdermal E 2 2 All data are mean values standard deviation, if not stated otherwise. Minimum and maximum values in brackets; OC oral contraceptives. Levels of estradiol (E), total T, DHEAS, and A were measured by radioimmunoassays (DPC-Biermann GmbH, Bad Nauheim, Germany; A: Bioscientia); the levels of FSH were measures by an immunoluminometric (Ciba-Corning, Fernwald, Germany); and the levels of SHBG were measured by an immunoradiometric assay (Orion Diagnostics, Espoo, Finland). To retain the double-blinded nature of the trial, E was only determined at baseline, as an increase (no change) was expected in the E NA (TIB) group, respectively, within the study period. The ranges of inter-assay coefficients of variations (CV) were 7% to 16.9% for E, 5.4% to 14.6% for total T, 6.4% to 9.5% for DHEAS, 6.3% to 9.2% for A, 4.4% to 6.7% for FSH, and 7.2% to 9.8% for SHBG. All intra-assay CVs were less than 10%. The lower limit of detection was 0.02 nmol/l for E, 0.7 nmol/l for total T, 0.3 mol/l for DHEAS, 0.69 nmol/l for A, 0.3 U/L for FSH, and 0.5 nmol/l for SHBG. Free T was calculated as the molar ratio of total T/SHBG (11). Statistics All analyses are based on Statistical Analyzing System version 6.08 or higher (SAS Institute, Cary, NC). Descriptive statistics include means standard deviation (SD), median, range, and absolute number(s) or percentage(s) where appropriate (Table 1). Baseline values, means SD, and medians of baseline endocrine parameters are plotted in Table 2. The P values of the two-sided Wilcoxon signed rank test on percent changes from baseline within and between groups are shown in Table 3. P values.05 (two-tailed) were regarded as statistically significant. FERTILITY & STERILITY 555

TABLE 2 Endocrine parameters baseline value. a Baseline Mean SD Tibolone Median Estradiol norethisterone acetate Baseline Mean SD Median Group comparison P b Estradiol (pmol/l) 37.6 86.4 18.4 49.5 106.8 18.4.077 FSH (U/L) 64.02 25.03 58.50 61.68 23.26 62.50.915 Total testosterone (nmol/l) 0.83 0.24 0.69 0.97 0.37 0.83.049 Free testosterone (total testosterone/shbg) 0.021 0.01 0.019 0.027 0.016 0.023.051 Androstendione (nmol/l) 3.06 0.94 2.85 3.71 1.41 3.53.028 DHEAS ( mol/l) 2.04 0.86 2.00 3.16 1.76 2.71.001 SHBG (nmol/l) 47.79 21.36 42.50 42.81 17.61 40.50.315 a Intent-to-treat-groups; n 48 for each parameter in each group except estradiol in the tibolone group (n 47). b Wilcoxon rank-sum test of baseline values. Results The demographic data of the patients were similar (see Table 1); one patient in each group did not start HRT after randomization. Pre-trial use of HRT was 57.1% in the tibolone group and 53.1% in the E NA group; none of the women used injectables or implants. The duration of HRT use varied between 3 weeks and 8 years in the tibolone group, and 2 months and 9 years in the E NA group. Prior use of oral and transdermal HRT was very similar in both groups. Use of oral estrogen and progestogen regimens was predominant. Thirteen women did not complete the study. In the tibolone group, discontinuations were due to loss to follow-up examinations (n 2), and sleeplessness, facial acne, presence of an uterine fibroid, and apathy (n 1 each). In the TABLE 3 Endocrine parameters percent changes within and between groups. Tibolone Estradiol NETA Parameter Changes from baseline % changes Mean SD P a Mean SD P a Comparison P a FSH (U/L) Month 6 30.0 12.9 (n 44).0001 76.9 21.9 (n 43).0001.000 Month 12 27.6 17 (n 42).0001 69.5 78.9 (n 41).0001.000 Total T (nmol/l) Month 6 6.4 14.4 (n 44).0019 15.1 23.8 (n 43).0001.025 Month 12 6.8 15.1 (n 42).0049 14.1 23.1 (n 41).0001.155 Free T (total T/SHBG) Month 6 109.1 109.6 (n 44).0000 31.2 31.4 (n 43).0000.000 Month 12 121.6 122.9 (n 42).0000 35.9 25.4 (n 41).0000.000 A (nmol/l) Month 6 0.3 26.8 (n 44).465 7.8 33.7 (n 43).0007.022 Month 12 3.8 28.9 (n 42).070 9.6 27.7 (n 41).0012.090 DHEAS ( mol/l) Month 6 18.5 35.8 (n 44).0098 }3.2 33.5 (n 43).0410.002 Month 12 20.4 38.6 (n 42) 0.0119 4.0 26.9 (n 41).0765.003 SHBG (nmol/l) Month 6 49.0 17.5 (n 44).0001 37.8 45.4 (n 43).0001.000 Month 12 52.5 13.5 (n 42).0001 46.5 46.1 (n 41).0001.000 a Two-sided Wilcoxon signed rank tests on % changes from baseline within and between groups, respectively. 556 Dören et al. HRT and androgen status Vol. 75, No. 3, March 2001

FIGURE 1 Changes of DHEAS and SHBG from baseline: percent within 1 year, tibolone group. FIGURE 2 Changes of DHEAS and SHBG from baseline: percent within 1 year, estradiol and norethindrone acetate group. E NA group, terminations were due to uterine bleeding/ spotting (n 3), and fear of thrombosis, flatulence, depression, and weight increase (n 1 each). Compliance according to diaries and drug accountability was, on average, 98.1% in the tibolone and 97.3% in the E NA group. None of the participants presented with clinical signs of hirsutism. The pre-trial levels of DHEAS, A, and total T were significantly different between groups. There was a trend toward lower free T and E levels in the tibolone group (see Table 2). Within both groups, there were significant changes after 6 and 12 months for the levels of FSH, SHBG, and free and total T. Levels of FSH decreased to a greater extent in the E NA group. We measured changes of SHBG levels in opposite directions: a decrease in the tibolone group and an increase in the E NA group. Free T increased significantly it actually doubled (decreased) in the tibolone (E NA) group. Total T levels decreased in both groups, to a relatively larger extent in the E NA group. Within groups after 6 and 12 months, changes of both DHEAS and A were significant for one group only. The DHEAS levels increased in the tibolone group; the decline in the E NA group was significant only after 6 months. The suppression of A was significant in the E NA group; in the tibolone group there was a trend toward lower levels after 12 months (see Table 3). Differences between regimens after 6 and 12 months were significant for FSH, free T, SHBG, and DHEAS. The differences in the extent of suppression of total T and A by both regimens were significant only after 6 months (see Table 3; Figs. 1 and 2 ). DISCUSSION We found several distinctly different changes in the levels of plasma androgens, DHEAS, SHBG, and FSH in both extent and direction, depending on the type of HRT. There is only one 2-year uncontrolled study that has also suggested an increase of DHEAS levels with tibolone treatment in a small group of younger, early postmenopausal women (12). In controlled 2-year-studies, SHBG levels have decreased by 37% to 70% after 1 year (7, 13) and 55% after 2 years (7). Both the decrease of SHBG and FSH levels in our tibolone group are consistent with these trial results. Free T levels doubled in the tibolone group, most likely due to the suppression of SHGB. Previous controlled trials with the same continuous combined HRT did not show changes in the levels of DHEAS, androstenedione, and calculated free T, but did show suppression of SHBG within 2 years (8). One small 1-year study did not suggest any changes for levels of SHBG, total T, and free T (14). Overall, the findings are inconsistent, because SHBG levels increased in our E NA group and both A and total T decreased. Continuous use of NA in conjunction with E may have attenuated an otherwise larger rise of SHBG, compared with its sequential use as reported previously (8). A decrease of SHBG levels with HRT, allowing for higher levels of bioavailable steroids, is relevant for estrogen-dependent tissues. Whether concurrent changes in insulin resistance may be involved is unknown, and was beyond the scope of this trial. There are no long-term data available for the study regimens that address this issue. Tibolone did FERTILITY & STERILITY 557

not negatively influence glucose metabolism (15), as had been suggested by a small uncontrolled 3-month study. On the contrary, 3 months of continuous combined E NA decreased insulin sensitivity (16). Oral E (2 mg daily) may increase insulin sensitivity, but sequential oral E may reverse this effect, as suggested in an uncontrolled 1-year study (17). Thus, it is possible that long-term continuous combined E NA may decrease insulin sensitivity. DHEAS synthesis in women occurs exclusively in the adrenal cortex. In the postmenopause period, estrogen synthesis is completely dependent on peripheral conversion of the adrenal precursor steroids DHEAS, dehydroepiandrosterone (DHEA), and A. DHEA and its sulfate are also essentially involved in the synthesis of androgens in peripheral tissues (18). It is unknown whether HRT interferes with adrenal androgen synthesis and the subsequent peripheral metabolism in postmenopausal women. The hypotheses to explain the differences of HRT-associated changes in DHEAS levels the majority of which were generated from studies with conjugated equine estrogens include the enhancement of both 3 -hydroxysteroid dehydrogenase (3 - HSD) and 17,20-desmolase (19), a dissociation of these two enzyme activities (20), changes in the peripheral hydroxylation (21), and hepatic sulfation of DHEA (22). It is also unknown whether the increase of DHEAS by tibolone, which was also observed after stimulation of ovulation with menotropins (23) or use of danazol (24), could be the result of a specific enhancement of androgen-producing adrenal cells or whether it interferes with the endogenous stimulation by ACTH, which is known to increase DHEAS production. Exogenous E increased the production of DHEAS in one human adrenocortical tumor cell line (25). However, one recent study did not suggest any changes of adrenal enzyme activity, which was assessed by an adrenocorticotropin-hormone-stimulation test before and after use of transdermal E (26). Given that the plasma pool of DHEAS is several hundred times higher compared with testosterone in women, and that DHEAS is mainly bound to albumin not SHBG (5), the increase of DHEAS in the tibolone group by 20% within 1 year appears to be remarkable. Several limitations affect the interpretations of our results. There were significant differences in the baseline levels of DHEAS, A, and total T between the groups. However, statistical analyses controlled for these differences. Baseline DHEAS levels reflect the wide range of interindividual values (22). DHEAS is a specific individual marker, compared to other adrenal hormones such as cortisol (27). It is unknown whether any pre-trial HRT or the short wash-out period could have modified any of the measured parameters. Finally, the fraction of free T was calculated, not measured. In conclusion, both HRT regimens modified the levels of plasma androgens, DHEAS, and SHBG differently. The largest changes were found for the calculated fraction of free testosterone, which doubled in the tibolone group, likely due to a decrease of SHBG. The increase of DHEAS may reflect a modification of adrenal androgen production. Continuous combined E NA treatment suppressed peripheral plasma androgens, probably as a result of an increase in the level of SHBG. There was no apparent impact on DHEAS levels. Further trials should address the potential interference with adrenal steroidogenesis by various types of HRT and could delineate the physiological significance of any interaction. References 1. Tazuke S, Khaw KT, Barrett-Connor E. Exogenous estrogen and endogenous sex hormones. Medicine 1992;71:44 51. 2. Casson PR, Hornsby PJ, Ghusn HF, Buster JE. Dehydroepiandrosterone (DHEA) replacement in postmenopausal women: present status and future promise. Menopause 1997;4:225 31. 3. Barrett-Connor E, Goodman-Gruen D. Dehydroepiandrosterone sulfate does not predict cardiovascular death in postmenopausal women. Circulation 1995;91:1757 60. 4. Johannes CB, Stellato RK, Feldman HA, Longcope C, McKinlay JB. Relation of dehydrepiandrosterone and dehydrepiandrosterone sulfate with cardiovascular disease risk factors in women: longitudinal results from the Massachusetts women s health study. J Clin Epidemiol 1999; 52:95 103. 5. Kroboth PD, Salek FS, Pittenger AL, Fabian TJ, Frye RF. DHEA and DHEA-S: a review. J Clin Pharmacol 1999;39:327 48. 6. Moore RA. Livial: a review of clinical studies. Br J Obstet Gynecol 1999;106(suppl 19):1 21. 7. Milner M., Sinnott M., Gasparo D, Kelly A, Carole BK, Harrison R. Climacteric symptoms, gonadotropins, sex steroids, and binding proteins with conjugated equine estrogen-progestin and tibolone over two years. 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