Department of Obstetrics and Gynecology, Karolinska Hospital, Stockholm, Sweden

Transcription

1 FERTILITY AND STERILITY VOL. 81, NO. 6, JUNE 2004 Copyright 2004 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A. MENOPAUSE Mammographic breast density, hormones, and growth factors during continuous combined hormone therapy Peter Conner, M.D., a Gunilla Svane, M.D., Ph.D., b Edward Azavedo, M.D., Ph.D., b Gunnar Söderqvist, M.D., Ph.D., a Kjell Carlström, Ph.D., c Thomas Gräser, M.D., d Friedrich Walter, M.D., d and Bo von Schoultz, M.D., Ph.D. a Department of Obstetrics and Gynecology, Karolinska Hospital, Stockholm, Sweden Received October 22, 2003; revised and accepted February 18, Supported by grants from the Swedish Cancer Society, the Swedish Research Council (project 5982), and the Karolinska Institute Research Funds. The clinical trial with E2V/ DNG was supported by Jenapharm GmbH & CO. KG, Jena, Germany. Reprint requests: Peter Conner, M.D., Department of Obstetrics and Gynecology, Karolinska Hospital, SE Stockholm, Sweden (FAX: ; peter.conner@ks.se). a Department of Obstetrics and Gynecology. b Department of Radiology. c Department of Obstetrics and Gynecology, Karolinska Institutet, Huddinge University Hospital, Huddinge, Sweden. d Jenapharm GmbH & Co. KG, Jena, Germany /04/$30.00 doi: /j.fertnstert Objective: To study the effect on mammographic breast density of two different continuous combined regimens for hormone therapy. Design: Randomized clinical study. Setting: University hospital. Patient(s): Postmenopausal women without any previous history of breast disorder. Intervention(s): The women received either estradiol valerate/dienogest or estradiol/norethisterone acetate. Mammograms and venous blood samples were obtained at baseline and after 6 months. Main Outcome Measure(s): Change in mammographic breast density. Correlations with levels of hormones, growth factors, and binding proteins. Result(s): An increase in mammographic density was recorded in approximately 50% of the women, and there were no differences between treatments. Increased density showed a positive correlation with estradiol, estrone, and sex hormone binding globulin and showed a negative association to free T. Among hormonal factors, levels of free T were the most important for predicting increased density. Conclusion(s): Continuous combined hormone therapy with different progestogens has a marked impact on the breast. (Fertil Steril 2004;81: by American Society for Reproductive Medicine.) Key Words: Mammographic breast density, continuous combined hormone therapy, progestogens Mammographic breast parenchymal density is currently discussed as a strong and independent risk factor for breast cancer. Hereditary factors, age, and body composition are important determinants for the amount of epithelium and stroma relative to fat in the breasts of individual women. Breast density is also related to reproductive status, circulating levels of endogenous sex steroids, peptide hormones, growth factors, and their binding proteins (1). There is accumulating evidence that breast density is influenced by hormonal therapies. Postmenopausal hormone therapy (HT) has been associated with an increased risk of breast cancer (2), and an increase in mammographic breast density has been reported to occur in a significant proportion of women during such treatment (3, 4). However, HT is not a uniform concept, and in clinical practice, a variety of different Es and progestogens are used in various doses and combinations. There are indications that different hormones, doses, and regimens may have a diverging influence on the breast. In fact, data from animal (5), clinical (2, 3), and observational (6, 7) studies suggest that E in continuous combination with progestogen has a stronger stimulatory effect on the breast and also may carry a greater risk for cancer than treatment with E alone. Although the evidence may seem compelling, it should be recalled that the effects of progestogen may differ according to type, dosage, duration of exposure, and the estrogenic environment (8). Epidemiological observations that continuous combined HT is associated with an increased risk of breast cancer were recently supported by the Women s Health Initiative (WHI) study (9). The findings of the WHI and 1617

2 the early termination of the combined-treatment arm have raised questions as to whether the observed increase in breast cancer was related only to the specific hormones used in that particular study, that is, conjugated equine Es and medroxyprogesterone acetate, or whether the results may apply also to other continuous combined regimens as well (10). In a recent randomized prospective clinical trial, we found increased breast epithelial proliferation in postmenopausal women during treatment with E 2 in continuous combination with either norethisterone acetate (NETA) or dienogest (DNG) (11). These two progestogens display different pharmacokinetic and pharmacodynamic properties. Here we report the effects of the two treatments on mammographic breast density. The increase in density among individual women was correlated to age, body composition, circulating hormones, and growth factors during 6 months of treatment. MATERIALS AND METHODS Subjects This prospective randomized double-blind study was conducted at the Department of Obstetrics and Gynecology at Karolinska Hospital in Sweden (11). Postmenopausal, apparently healthy women aged years with a BMI of 32 kg/m 2 and without any previous history of breast disorder or suspicious findings at routine mammography were recruited for the study. They all had been postmenopausal for at least 12 months and had FSH levels of 25 IU/L. Thirty-five of the women had never used HT previously, and none of the women had taken any sex steroid hormones during the last 2 months before the study. Exclusion criteria were hypertension, any signs of hepatic dysfunction, or concomitant treatment known to influence the study medication (phenobarbital, carbamazepine, phenytoin, primidone, clomiphene, large doses of vitamin C, or broad-spectrum antibiotic therapy). A total of 50 women were randomized to receive either E 2 valerate (2 mg) and dienogest (2 mg; E 2 V/DNG; Climodien, Schering, Germany) or E 2 (2 mg) and norethisterone acetate (1 mg; E 2 /NETA; Kliogest, Novo Nordisk A/S, Denmark) once daily for 6 months. Compliance was checked after 3 and 6 months from diaries in which the women registered their daily medication intake. Mammography examinations were performed in accordance with the Quality Control Regulations stipulated by the Swedish National Board of Health and Welfare and by the Swedish National Radiation Protection Institute. The study was approved by the Independent Ethics Committee at the Karolinska Institute (IRB Project ), and all women gave their written informed consent before inclusion. Objectives and Outcomes The objective was to study the effects on mammographic breast density in postmenopausal women during 6 months of treatment with E 2 V/DNG and E 2 /NETA. The primary outcome was mammographic status as classified according to Wolfe (12) and on a percentage scale of 5 categories. Secondary objectives were to assess some possible associations between density and demographic and hormonal factors. Interventions Mammographic Breast Density Mammograms were obtained at baseline and at 6 months to determine breast density and to evaluate any abnormalities. Mammography examinations comprised mediolateral oblique and craniocaudal views of both breasts. Only the mediolateral oblique views of each breast were used to assess breast density. All mammograms were assessed at the Karolinska hospital by two independent radiologists (E.A., G.S.) who were blinded to treatments. Any difference of opinion in the classification of a mammogram was resolved with a consensus result. Mammographic density of all coded films was classified according to Wolfe (12) in four categories: N1, essentially normal breast with parenchyma composed primarily of fat and with, at most, a few fibrous connective tissue strands; P1, prominent ductal pattern in up to one fourth of breast volume; P2, prominent ductal pattern in more than one fourth of breast volume; and DY, extremely dense parenchyma, which usually denotes connective tissue hyperplasia. In addition to the Wolfe classification, for each individual woman, all coded mammograms were classified according to a percentage scale with five categories for the amount of dense breast parenchyma in relation to the whole breast volume. The five categories were the following: 0 20%, 21% 40%, 41% 60%, 61% 80%, and 81% 100%. Dual-Energy X-Ray Absorptiometry Measurements Body composition, bone mineral areal density (g/cm 2 ), lean body mass, and fat mass were determined by dual energy X-ray absorptiometry with Lunar Model DPX-L equipment (Lunar Radiation, Madison, WI). Analytical Methods Venous blood samples were drawn at baseline and after 6 months of treatment. Serum concentrations of E 2-17 (E 2 ) and sex hormone-binding globulin (SHBG) were determined by direct chemiluminescence enzyme immunoassay and T by direct RIA with commercial kits (Immulite and Coat-a- Count Testosterone) that were obtained from Diagnostic Products Corporation, Los Angeles, CA. Estrone in serum was determined after extraction with diethyl ether by RIA. Prolactin was determined by time-resolved fluorescence immunoassay with commercial kits obtained from Wallac OY, Turku, Finland (AutoDELFIA). The concentration of PRL was expressed in micrograms per liter of the third PRL International Reference Preparation 84/500. Insulin-like growth factor I (IGF-I) was determined by chemiluminescence enzyme immunoassay using a commer Conner et al. Breast density and continuous combined hormone therapy Vol. 81, No. 6, June 2004

3 cial kit (Advantage) obtained from Nichols Products Corporation, San Juan Capistrano, CA. Insulin-like growth factor binding proteins IGFBP-1 and IGFBP-3 were analyzed by ELISA using commercial kits obtained from Diagnostic Systems Laboratories Inc (Webster). Serum concentrations of norethisterone were analyzed after extraction with diethyl ether by RIA using the WHO antibody against norethisterone. Dienogest in serum was analyzed by an analogous method developed by the manufacturer of the drug. The detection limits and within and between assay coefficients of variation were for E 2 : 73 pmol/l, 8% and 9%; E 1 : 30 pmol/l, 7% and 10%; SHBG: 0.05 nmol/l, 4% and 8%; T: 0.1 nmol/l, 6% and 10%; PRL: 0.04 g/l, 1.9% and 3.2%; IGF-I: 6 g/l, 4.8% and 6.7%; IGFBP-1: 0.25ng/mL, 3% and 7%; IGFBP-3: 0.04 ng/ml, 9% and 10%; norethisterone: 0.12 ng/ml 7% and 9%; and DNG: 1.0 ng/ml, % and % for concentrations between ng/ml, respectively. Apparent concentrations of free T were calculated from values for total T, SHBG and a fixed albumin concentration of 40 g/l by successive approximation using a computer program based upon an equation system derived from the law of mass action (13). Statistical Analysis Differences within and between groups were analysed by the Mann-Whitney U test, the Wilcoxon signed-rank test and the Kruskal-Wallis test. Correlations were assessed by the Spearman s rank correlation test. To validate the results from univariate analyses, multiple regression analyses were performed and the importance of each variable was estimated while controlling for the influence of the others. A P value of.05 was considered significant. RESULTS A total of 66 postmenopausal women were assessed for eligibility and 16 did not fulfill the inclusion criteria. Of the 50 randomized women, 5 were not assessable for various reasons (i.e., discontinued treatment and suspicious lesions on mammography). Thus a total of 45 women, 23 receiving E 2 V/DNG, and 22, E 2 /NETA, completed the study (Fig. 1). The mean values for age, years since menopause, and BMI were as follows: 54.9 vs. 56.2, 4.2 vs. 5.6, and 25.6 vs. 25.6, respectively and did not differ between groups. Also no significant differences in mammographic breast density were found at baseline. Mammographic status at baseline and after 6 months of treatment classified according to Wolfe and the percentage classes is given in Table 1. After 6 months there was an increase of mammographic density in about 50% of women receiving E 2 V/DNG. For E 2 /NETA, an upgrading in 45 and 68% of the women was found according to the Wolfe and percentage classifications, respectively. All these changes were highly significant (P.001), but there were no differences between the two treatment groups. The number of FIGURE 1 Flow diagram of the randomized study. Conner. Breast density and continuous combined hormone therapy. Fertil Steril women with an increase in mammographic density is also shown in Table 1. Figure 2 illustrates the changes in mammographic breast density in 2 individual women during 6 months of treatment. Values for serum parameters at baseline and during treatment did not differ between groups apart from a more pronounced increase in SHBG during treatment with E 2 V/ DNG than during E 2 /NETA (mean values at 6 months: 95 vs. 68 nmol/l; P.01). Therefore, correlation analyses were performed for the total material (Table 2). For mammographic breast density at baseline, we found an inverse correlation with BMI, total and abdominal fat mass (r s 0.5; P.01). A positive association was seen between baseline levels of SHBG and mammographic density (r s 0.3; P.03). For breast density at 6 months, a positive correlation was found with levels of PRL (r s 0.35; P.03), and an inverse association was seen with levels of IGF-I (r s 0.3; P.05). The change in mammographic breast density during hormonal treatment displayed a positive correlation with time after menopause (r s 0.4; P.01). FERTILITY & STERILITY 1619

4 TABLE 1 Mammographic status (number and percentage of women) according to the Wolfe (12) classification and percentage classes for two groups of women before and after 6 months of treatment with either E 2 V/dienogest (n 23) or E 2 /NETA (n 22). The number of women with an increase in mammographic density is indicated. Parameter E 2 V/dienogest, n (%) E 2 /NETA, n (%) Baseline 6 mo Baseline 6 mo Wolfe classification N1 9 (39) 2 (9) 4 (18) 1 (4) P1 7 (30) 10 (43) 10 (45) 5 (23) P2 6 (26) 10 (43) 8 (36) 15 (68) DY 1 (4) 1 (4) 0 (0) 1 (4) Increase 11/23 (48) 10/22 (45) Percentage class (43) 4 (17) 7 (32) 3 (14) (35) 8 (35) 10 (45) 3 (14) (13) 8 (35) 2 (9) 9 (41) (4) 2 (9) 3 (14) 6 (27) (4) 1 (4) 0 (0) 1 (4) Increase 12/23 (52) 15/22 (68) Conner. Breast density and continuous combined hormone therapy. Fertil Steril During treatment, significant increases were seen in levels of E 2,E 1, PRL, IGFBP-1, and SHBG, whereas levels of ft and IGFBP-3 decreased as shown in Table 2. The increase in mammographic density during 6 months of treatment showed a positive correlation with the concomitant increases in E 2,E 1 and SHBG (r s 0.5; P.01). There was also an inverse relationship between increase in density (percentage scale) and levels of free T: (r s 0.3; P.05). Within the two treatment groups, there was for women receiving E 2 /NETA (n 22) a positive correlation between levels of the progestogen (NETA) and change in mammographic density according to the percentage scale (r s 0.45; P.04). No such association was observed in the E 2 /DNG group. The importance of different hormones and binding proteins to predict increase in mammographic density during treatment was assessed by multiple regression analysis. Accordingly, the decline in free T levels was found to be the most important factor followed by the decline in IGFBP-3 and the raised levels of E 2 and PRL (P.01). DISCUSSION In the present study, postmenopausal women treated with E 2 in continuous combination with two different types of progestogens either NETA or DNG were found to react with an increase in mammographic density in approximately 50% TABLE 2 Serum concentrations (mean SEM) of hormones, growth factors, and binding proteins at baseline and after 6 months in 45 postmenopausal women during continuous combined HT. Variable Baseline 6 mo E 2 (pmol/l) E 1 (pmol/l) , T (nmol/l) ft (pmol/l) * SHBG (nmol/l) * PRL (ng/ml) IGF-I ( g/l) IGFBP-1 (ng/ml) * IGFBP-3 (ng/ml) 2, , DNG (ng/ml) Norethisterone (ng/ml) Note. conversion factors are as follows. E 2, 0.272; E 1, 0.270; T, 0.288; and SHBG, * P.01. P.001. Conner. Breast density and continuous combined hormone therapy. Fertil Steril of cases during 6 months of treatment. There were no apparent differences between the two regimens as judged by two different classification systems. It should be noted that the classification systems used here represent a rather crude measurement and that density is likely to be a continuous variable. An increase in density of 20% 25% is required to fulfill the criteria for an upgrading of one class according to both the Wolfe and the percentage scales. Still, we found two different progestogens with different characteristics to have quite the same impact in terms of increase in breast density when used in continuous combination with E 2. Our findings with regard to E 2 /NETA are in agreement with previous data. NETA is a 19-T derived progestogen and has been used in combination with E 2 for many years, particularly in Europe. The effects of E 2 /NETA as an alternative for HRT were recently reviewed (14). In both retrospective and prospective studies treatment with E 2 /NETA induced increased breast density in approximately 50% of women (3). Here the results for 2 mg of E 2 V in continuous combination with 2 mg DNG were no different. Dienogest is a non-ethinylated progestogen with anti-androgenic activity (15). Large clinical trials have shown E 2 V/DNG and E 2 / NETA to be equally effective in treating postmenopausal symptoms (16). We found elsewhere that the combination of conjugated equine estrogens and medroxyprogesterone acetate has a similar effect and causes an upgrading of the Wolfe score in about 40% of women (17). In contrast, only a few women (2% 18%) responded with an increase in density when treated with different types and doses of E alone (3, 17) Conner et al. Breast density and continuous combined hormone therapy Vol. 81, No. 6, June 2004

5 FIGURE 2 Change in mammographic density in two individual women receiving (A) E 2 V/DNG and (B) E 2 /NETA. Mammograms were obtained at baseline and after 6 months of therapy. Conner. Breast density and continuous combined hormone therapy. Fertil Steril The histologic correlates to mammographic breast density are not fully clear, but it has been suggested to at least partly reflect increased proliferation of epithelium and stroma. We previously reported an association between breast cell proliferation and both endogenous and exogenous progestogen levels in premenopausal women (18). In postmenopausal women as well as in macaques, breast epithelial proliferation was much more pronounced when conjugated equine estrogens was given in combination with medroxyprogesterone acetate than when conjugated equine estrogens was given alone (5, 19). In the women of the present study, we recently found a threefold to fourfold increase in proliferation during treatment with E 2 in combination with NETA or dienogest (11). All these data suggest that progestogen is mitogenic in the breast. This concept is further supported by the present finding of a positive correlation between blood levels of NETA and an increase in mammographic density. The breast response to hormonal treatment both in the form of increased cell proliferation and mammographic density appears to be an early event and thereafter seems to remain stable during further treatment (3, 11). However, there is also a marked variation in response between individual women as illustrated in the present study. Among women on the same treatment a considerable amount (40% 50%) displayed no significant increase in density. The factors that regulate this apparent difference in individual sensitivity to hormonal treatment are at present incompletely understood. Body composition seems to be important in this respect, and here we found lean women to have a higher degree of mammographic density at baseline. Epidemiological data also suggest that the increased risk of breast cancer during treatment with HT is primarily related to women with a normal or low body mass index (6, 7). Insulin-like growth factor I (IGF-I) has been shown to stimulate breast cell proliferation and to inhibit apoptosis (20). Observational data have shown that raised levels of circulating IGF-I in combination with low levels of its major FERTILITY & STERILITY 1621

6 FIGURE 2 Continued. binding protein, IGFBP-3, are associated with breast cancer risk in premenopausal women (21). Previously, Boyd and co-workers found IGF-I and IGFBP-3 to be more important factors than E levels in predicting breast density in untreated premenopausal women (1). Here in postmenopausal women during HT, there was an inverse relationship between IGF-I and density after 6 months of treatment. Oral administration of E has previously been shown to decrease levels of IGF-I (22). The same effect was reported for E 2 V/DNG but not for E 2 /NETA in a large clinical study (16). However, in the present study no significant change in levels of IGF-I were recorded during either treatment. In our postmenopausal women, the increased levels of both E 2 and estrone during treatment also displayed a significant association with increase in mammographic density. Androgens are known to influence the development and growth of the mammary gland in women. Both inhibitory and stimulatory effects on normal epithelial and cancer cell proliferation have been found in animal studies and cellculture experiments (23). The mechanisms for these apparent dual functions remain unclear. Irrespective of type of treatment, we found a significant decrease in levels of free T as well as an increase in SHBG concomitant to enhanced mammographic density. We previously reported a significant negative correlation between breast cell proliferation and free T in young women using oral contraceptives (18). Testosterone has also been found to inhibit E-induced breast epithelial proliferation and E receptor expression in rhesus monkeys (24). In clinical practice, T and androgenic compounds like danazol are often used to reduce mastalgia and may possibly reduce proliferation. In the present study, after multiple regression analysis, the decline in free T levels was found among hormonal factors to be the most important predictor of breast density response. We recently observed a similar inverse relationship between free T and breast cell proliferation in women receiving E 2 /NETA or tibolone, a substance with androgenic properties (25). Thus, circulating androgen levels may be an important factor for both breast proliferation and density. Testosterone may have a protective, anti-proliferative effect. In conclusion, our results add to the growing notion that continuous combined E-progestogen treatment with different types of progestogen may have a marked impact on the breast in a significant proportion of women. Accumulating data indicate a proliferative function of progestogens in human breast tissue. Continuous combined treatment may 1622 Conner et al. Breast density and continuous combined hormone therapy Vol. 81, No. 6, June 2004

7 also differ from cyclic and E-only regimens in this respect. The discontinuation of progestogen has been suggested to be a trigger for apoptosis (26). From a clinical perspective, increased mammographic density during HT should be regarded as an unwanted side effect. Efforts should be made to define treatment regimens that minimize breast response but maintain the many advantages of HT. Acknowledgments: Skillful technical assistance was provided by Ms. Catharina Karlsson, Ms. Berit Legerstam, and Ms. Lotta Blomberg. References 1. Boyd NF, Stone J, Martin LJ, Jong R, Fishell E, Yaffe M, et al. The association of breast mitogens with mammographic densities. Br J Cancer 2002;87: Million Women Study Collaborators. Breast cancer and hormone replacement therapy in the Million Women Study. Lancet 2003;362: Lundström E, Wilczek B, von Palffy Z, Söderqvist G, von Schoultz B. Mammographic breast density during hormone replacement therapy: differences according to treatment. 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A physiologic role for testosterone in limiting estrogenic stimulation of the breast. Menopause 2003;10: Conner P, Christow A, Kersemaekers W, Söderqvist G, Skoog L, Carlström K, et al. A comparative study of breast cell proliferation during hormone replacement therapy: effects of tibolone and continuous combined estrogen-progestogen treatment. Climacteric 2004;7: Foidart JM, Colin C, Denoo X, Desreux J, Beilard A, Fournier S, et al. Estradiol and progesterone regulate the proliferation of human breast epithelial cells. Fertil Steril 1998;69: FERTILITY & STERILITY 1623