Serum estrogen level after hormone replacement therapy and body mass index in postmenopausal and bilaterally ovariectomized women

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1 Maturitas 50 (2005) Serum estrogen level after hormone replacement therapy and body mass index in postmenopausal and bilaterally ovariectomized women Toshiyuki Yasui a,, Hirokazu Uemura a, Yuka Umino a, Masaya Takikawa a, Seiichiro Saito a, Akira Kuwahara a, Toshiya Matsuzaki a, Masahiko Maegawa a, Hiroyuki Furumoto a, Masakazu Miura b, Minoru Irahara a a Department of Obstetrics and Gynecology, School of Medicine, University of Tokushima, Tokushima , Japan b Department of Pharmaceutical Research, Mitsubishi Kagaku Bio-Clinical Laboratories Inc., Tokyo 174, Japan Received 26 August 2003; received in revised form 19 January 2004; accepted 1 March 2004 Abstract Objective: The objective of this study was to determine the relationships of serum estrogen levels after hormone replacement therapy (HRT) every other day and every day with body mass index (BMI) in postmenopausal and bilaterally ovariectomized women. Methods: Eighty-six postmenopausal and 51 bilaterally ovariectomized women who had been suffering from vasomotor symptoms such as hot flush or atrophy of the vagina were randomly treated with HRT every other day or every day. Seventy-four patients received oral administration of mg conjugated equine estrogen (CEE) and 2.5 mg medroxyprogesterone acetate (MPA) every other day, and 63 patients received oral administration of mg CEE and 2.5 mg MPA every day as conventional HRT. Results: Eighty-four postmenopausal and 50 bilaterally ovariectomized women completed this study. Serum estradiol levels after HRT every day in postmenopausal and bilaterally ovariectomized women were significantly (P <0.05 and <0.01, respectively) correlated with BMI, while those after HRT every other day were not correlated with BMI. The differences between estradiol levels after 12 months of treatment and initial estradiol levels were also significantly (P <0.01) correlated with BMI in both postmenopausal and bilaterally ovariectomized women who received HRT every day but not in women who received HRT every other day. Serum estrone level after HRT every day and the difference between estrone level after 12 months of treatment and initial estrone level were significantly (P <0.05 and <0.01, respectively) correlated with BMI only in bilaterally ovariectomized women. Conclusion: Serum estradiol levels after HRT every day increase more in overweight women than in non-overweight postmenopausal and bilaterally ovariectomized women. The results of the present study regarding the relationship between serum estradiol levels after HRT and BMI should be useful for selecting dosages of drugs to be used in HRT Elsevier Ireland Ltd. All rights reserved. Keywords: Serum estrogen level; Hormone replacement therapy; Ovariectomized women 1. Introduction Corresponding author. Fax: address: yasui@clin.med.tokushima-u.ac.jp (T. Yasui). The effectiveness of hormone replacement therapy (HRT) for treatment of bone loss as well as for relief /$ see front matter 2004 Elsevier Ireland Ltd. All rights reserved. doi: /j.maturitas

2 20 T. Yasui et al. / Maturitas 50 (2005) of vasomotor symptoms and vaginal atrophy in postmenopausal and bilaterally ovariectomized women is well established [1]. The study of Women s Health Initiative (WHI) has stopped because overall health risks exceeded benefits from the use of combined conjugated equine estrogen (CEE) at a dosage of mg per day and medroxyprogesterone acetate (MPA) at a dosage of 2.5 mg per day, and the results indicated that this regimen should not be initiated or continued for the primary prevention of coronary heart disease [2]. The fact that 69% of the postmenopausal women who participated in that trial had a high body mass index (more than 25.0 kg/m 2 ) suggests that body mass index (BMI) may have effects on risks to health and benefits for health of HRT. It is known that endogenous estradiol levels in women with high BMI and those with low BMI are different. Low endogenous estradiol levels in postmenopausal women were related to appearance of hot flush [3] and osteoporosis [4,5]. The relative risk for breast cancer in women with the highest endogenous estradiol level was higher than that in women with the lowest estradiol level [6,7]. Several studies have also shown that BMI is related to various beneficial effects and risks after HRT. It has been reported that HRT reduced the risk of hip fracture more in women with low BMI than in women with high BMI [8]. Genital bleeding after HRT was found to be more frequent in overweight and obese postmenopausal women than in postmenopausal women with normal BMI [9]. However, the relationship between serum estrogen level after CEE treatment and BMI has not been fully investigated. In the present study, we precisely measured serum levels of estradiol and estrone using a highly sensitive and specific assay system in postmenopausal and bilaterally ovariectomized women before and after HRT with CEE and MPA every other day or every day, and we determined the relationships between serum levels of estradiol and estrone after HRT and BMI in these women. 2. Subjects and methods 2.1. Subjects Eighty-six postmenopausal and 51 bilaterally ovariectomized women who had been suffering from vasomotor symptoms such as hot flush or atrophy of the vagina were recruited for this study from the outpatient clinic of the Department of Obstetrics and Gynecology, Tokushima University Hospital, and informed consent for participation in the study was obtained from each woman. Reviews of medical histories and the results of physical examinations and blood chemistry tests showed that all of the women were in good health and that none of the women had previously received any medication before the commencement of hormonal therapy. None of women had a history of estrogen-related cancer, and all women showed negative results on a mammogram and had a transvaginal ultrasonographically normal-appearing endometrium. No abnormalities were found in endometrial smears in any of the women. Serum levels of estradiol and estrone were measured before and at 12 months after the start of HRT, and baseline levels of serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured before treatment. Eligible women were randomly treated with low-dose or conventional HRT. Seventy-four women received oral administration of mg CEE (Premarin, Wyeth) and 2.5 mg MPA (Provera, Upjohn) every other day, and 63 women received oral administration of mg CEE and 2.5 mg MPA every day as conventional HRT. As a preliminary study, serum levels of estradiol and estrone were measured before and at 6, 15 and 24 h after administration of CEE and MPA at 6 and 12 months after the start of HRT every day in eight women and before and at 6, 15, 24, 30 and 48 h after administration of CEE and MPA at 6 and 12 months after the start of HRT every other day in seven women. Endometrial smear and transvaginal ultrasonography were used for endometrial assessment during treatment, and no abnormalities were found in any of the women. Compliance was assessed by pill count, and side-effects were ascertained by questionnaires at 4-week intervals Measurements of estradiol and estrone We have developed a highly sensitive and specific assay for precise measurement of serum estrone and estradiol levels [10]. Estradiol and estrone, obtained by solid-phase extraction using a Sep pak

3 T. Yasui et al. / Maturitas 50 (2005) tc18 cartridge, were purified by high-performance liquid chromatography (HPLC). Estrone and estradiol were sufficiently separated from other steroid hormones. Then levels of estradiol and estrone were measured by radioimmunoassays. Antiserum, 125 I-labeled reagents and a precipitating reagent were obtained from Diagnostic System Laboratories (Texas, USA). The antiserum of estradiol did not react with the corticosterone and aldosterone that were eluted close to a 17 -estradiol by HPLC. The limits of detection of estradiol and estrone, defined as mean blank measurement minus 2S.D. (2 standard deviation of zero bound), were 1.04 and 0.64 pg/ml, respectively. The intra-assay coefficient of variation (CV) of estradiol and that of estrone were 8.5 and 19.5%, respectively (mean serum estradiol concentration: 5.3 pg/ml; mean serum estrone concentration: 22.0 pg/ml). Serum levels of estradiol during the period of treatment with CEE determined by this method were strongly correlated with those determined by gas chromatography mass spectrometry, which was performed at LAB GmbH & Co., Neu-Ulm, Germany. In the normal menstrual cycle, mean levels of estradiol measured by this method in the follicular and preovulatory phases were similar to those reported previously. Based on the finding that estradiol level remained almost constant between 12 and 18 h after single administration of CEE, fasting blood samples in patients treated every other day and every day were drawn h after taking CEE [10] and the assay was run at the same time. The blood samples were centrifuged at 4 C, and the serum samples obtained were frozen until analysis Measurements of serum LH and FSH Blood samples were taken from women at the time of serum estradiol measurement. Serum levels of LH and FSH were measured using radioimmunoassay kits obtained from Daiichi Radioisotope Laboratories (Tokyo, Japan). In these assays, standards of LH and FSH were calibrated by WHO 1st IRP-LH and WHO 2nd IRP-hPG, respectively. The intraassay CV of LH and that of FSH were 6.5 and 4.1%, respectively, and the inter-assay CV of LH and that of FSH were 6.1 and 6.0%, respectively (mean serum LH: 23.5 IU/l; mean serum FSH: 71.8 IU/l) Statistical analysis Data are presented as means ± S.D.s. Statistical analyses among groups were performed by ANOVA. Serum estradiol and estrone levels at each dose in postmenopausal and bilaterally ovariectomized women were tabulated using descriptive statistics and correlated with BMI using linear regression analysis. The differences between serum estradiol and estrone concentrations after HRT and initial concentrations at each dose were also tabulated using descriptive statistics and correlated with BMI using linear regression analysis. P-values <0.05 were considered to be statistically significant. 3. Results Eighty-four of the 86 postmenopausal women and 50 of the 51 bilaterally ovariectomized women completed the 1-year study. There was no difference between compliance of women who received HRT every day and that of women who received HRT every other day. Two of the 86 postmenopausal women treated every day dropped out of the study because of prolonged unscheduled bleeding, and one of the 51 bilaterally ovariectomized women treated every other day dropped out of the study for reasons unrelated to treatment. Baseline characteristics are presented in Table 1. The body mass index was calculated as the weight in kilograms divided by the square of height in meters (kg/m 2 ). Fig. 1 shows the pharmacokinetics of estradiol and estrone after the administration of CEE and MPA every day and every other day as a preliminary study. Mean serum estradiol level in eight women who had undergone HRT every day for 12 months was 31.7 pg/ml at 15 h after administration of CEE and MPA and had decreased to 21.1 pg/ml at 24 h after administration, while that in seven women who had undergone HRT every other day for 12 months was 21.3 pg/ml at 15 h after administration of CEE and MPA and had decreased to 15.8 and 6.7 pg/ml at 24 and 48 h after administration, respectively. On the other hand, mean serum estrone level in eight women who had undergone HRT every day for 12 months was pg/ml at 15 h after administration of CEE and MPA and had decreased to pg/ml at 24 h after administration,

4 22 T. Yasui et al. / Maturitas 50 (2005) Table 1 Characteristics of women at the initial examination Postmenopausal women Bilaterally ovariectomized women HRT every day HRT every other day HRT every day HRT every other day No. of patients Age (years) 53.7 ± ± ± 5.7 a,c 50.1 ± 6.7 b,d Years since menopause (years) 4.9 ± ± ± 3.2 d 5.1 ± 6.9 BMI 23.3 ± ± ± ± 2.6 Estrone (pg/ml) 20.3 ± ± ± ± 13.0 Estradiol (pg/ml) 5.3 ± ± ± ± 1.9 b,d LH (IU/l) 30.8 ± ± ± ± 12.8 FSH (IU/l) 86.1 ± ± ± ± 31.7 Means ± S.D., BMI: body mass index. a P<0.01. b P<0.05 vs. postmenopausal women who received HRT every day. c P<0.01. d P<0.05 vs. postmenopausal women who received HRT every other day. while that in seven women who had undergone HRT every other day for 12 months was pg/ml at 15 h after administration of CEE and MPA and had decreased to 73.8 and 36.6 pg/ml at 24 and 48 h after administration, respectively. Table 2 shows serum estradiol and estrone levels after 12 months in all women treated every other day and every day. The mean levels of estradiol after 12 months in postmenopausal women treated every other day and every day were 14.3 and 31.4 pg/ml, respectively, and those in bilaterally ovariectomized women treated every day and every day were 13.2 and 30.3 pg/ml, respectively. The mean levels of estrone after 12 months in postmenopausal women treated every other day and every day were 72.9 and pg/ml, respectively, and those in bilaterally ovariectomized women treated every day and every day were 75.4 and pg/ml, respectively. There was no significant difference between serum levels of estradiol or between serum levels of estrone at 12 months after the start of treatment in the two treatment groups. Table 3 shows the correlations between endogenous estrone and estradiol levels and BMI. There was no correlation of endogenous estrone or estradiol level with BMI before treatment in either of the groups. There were no significant differences between BMI before and that at 12 months after HRT every day or every other day. The correlations of serum estradiol levels with BMI at 12 months after HRT every day and HRT every other day in postmenopausal and bilaterally ovariectomized women are shown in Fig. 2. The serum estradiol level after HRT every day in postmenopausal women showed a significant (P <0.05) positive correlation with BMI. The Table 2 Serum estrone and estradiol levels in women who received HRT every other day and every day Means ± S.D. Estradiol (pg/ml) Estrone (pg/ml) Postmenopausal women HRT every day 31.4 ± ± 74.7 HRT every other day 14.3 ± ± 37.4 Bilaterally ovariectomized women HRT every day 30.3 ± ± 61.7 HRT every other day 13.2 ± ± 29.3 Serum estrogen levels are measured in 84 postmenopausal women (HRT every day: 38 women; HRT every other day: 46 women) and 50 bilaterally ovariectomized women (HRT every day: 23 women; HRT every other day: 27 women). Fasting blood samples were drawn h after HRT.

5 Fig. 1. Pharmacokinetics of estradiol and estrone in postmenopausal women after 6 months and 12 months of HRT every day and every other day. Closed diamonds are values after HRT every day, and closed square are values after HRT every other day. Data are presented as means ± S.E.s. Serum levels of estradiol and estrone were measured before and at 6, 15 and 24 h after administration of CEE and MPA at 6 and 12 months after the start of HRT every day in 8 postmenopausal women and before and at 6, 15, 24, 30 and 48 h after administration of CEE and MPA at 6 and 12 months after the start of HRT every other day in seven postmenopausal women. T. Yasui et al. / Maturitas 50 (2005)

6 24 T. Yasui et al. / Maturitas 50 (2005) Table 3 Correlations between levels of endogenous estrogens and BMI Postmenopausal women Bilaterally ovariectomized women HRT every day HRT every other day HRT every day HRT every other day Coefficient P-value Coefficient P-value Coefficient P-value Coefficient P-value Estrone (pg/ml) Estradiol (pg/ml) BMI: body mass index; serum estrogen levels are measured in 84 postmenopausal women and 50 bilaterally ovariectomized women. Fig. 2. Correlations between serum estradiol levels at 12 months after HRT every day (upper panel) and every other day (lower panel) and BMI in postmenopausal and bilaterally ovariectomized women. Serum estradiol level in 38 postmenopausal women who received HRT every day showed a significant (P <0.05) positive correlation with BMI (the relationship being expressed by the following equation: estradiol level = 1.21 BMI ; R = 0.402). Estradiol level in 23 bilaterally ovariectomized women who received HRT every day showed a significant (P <0.01) positive correlation with BMI (the relationship being expressed by the following equation: estradiol level = BMI 7.642; R = 0.565).

7 T. Yasui et al. / Maturitas 50 (2005) serum estradiol level after HRT every day in bilaterally ovariectomized women also showed a significant (P < 0.01) positive correlation with BMI. On the other hand, serum estradiol levels after HRT every other day in postmenopausal and bilaterally ovariectomized women were not correlated with BMI. The differences between estradiol levels after 12 months of treatment and initial estradiol levels ( -estradiol) in postmenopausal and bilaterally ovariectomized women who received HRT every day showed a significant (P < 0.01) positive correlation with BMI (data not shown). On the other hand, -estradiol in Fig. 3. Correlations of serum estrone level after HRT every day with BMI in 23 bilaterally ovariectomized women (upper panel) and 38 postmenopausal women (lower panel). Serum estrone level in bilaterally ovariectomized women who received HRT every day showed a significant (P <0.05) positive correlation with BMI (the relationship being expressed by the following equation: estrone level = BMI ; R = 0.504).

8 26 T. Yasui et al. / Maturitas 50 (2005) postmenopausal and bilaterally ovariectomized women treated with every other day were not correlated with BMI. As shown in Fig. 3, the serum estrone level after HRT every day in bilaterally ovariectomized women showed a significant (P <0.05) positive correlation with BMI. The difference between estrone levels after HRT and initial estrone levels ( -estrone) in bilaterally ovariecstomized women treated with HRT every day also showed a significant (P < 0.01) positive correlation with BMI (data not shown). On the other hand, neither the serum estrone level after HRT nor -estrone was correlated with BMI in postmenopausal women treated HRT every day. 4. Discussion Little is known about estradiol levels in women after treatment with CEE because CEE is a complex of at least 10 natural estrogens, including estrone sulfate, equilin sulfate and 17 -estradiol sulfate, and precise measurement of the serum estradiol level during CEE treatment is difficult. Estrone sulfate, which is the major component, is readily absorbed from the gastrointestinal tract, and a fraction of it is rapidly converted into circulating unconjugated estrone and unconjugated 17 -estradiol, which is the most biologically active estrogen [11]. Extraction of sex steroid hormones in serum before the assay is needed for measurement of serum estradiol levels in postmenopausal and bilaterally ovariectomized women receiving CEE for HRT. Lobo et al. reported that serum estradiol levels after 25 days of treatment with CEE at dosages of 0.3 mg and mg per day increased to 18.5 and 39.4 pg/ml, respectively [12], and Jurgens et al. reported that serum estradiol level after administration of CEE at a dosage of mg per day for one month increased to 20.0 pg/ml [13]. These data are consistent with those obtained in the present study. However, serum estradiol levels after long-term treatment with oral CEE have not been reported due to technical difficulties in clinical assays. We have developed a highly sensitive and specific assay using HPLC for purification and a radioimmunoassay for measurement of serum estrone and estradiol levels [10], and we have precisely measured both endogenous estrogen levels and estrogen levels after CEE treatment using this assay. In a pharmacokinetics study, we found that both the levels of estradiol and estrone peaked at about 15 h after CEE administration and that the levels of estradiol and estrone in women treated with CEE every day were about times higher than those in women every other day. After menopause, peripheral aromatization of gonadal steroid hormones in adipose tissue is the primary source of endogenous estrogen. Extraglandular production of estrogen in adipose tissue and circulating estrogen levels increase with increase in body mass in postmenopausal women. Lower levels of endogenous estradiol and estrone have been reported to be found in women with frequent hot flashes, and flushing is more likely in thin women [3]. It has been found that obese postmenopausal women had a significantly higher vertebral BMD than did non-obese women [14] and that a higher endogenous estradiol level is associated with increased BMD, reduced bone loss and reduced fracture incidence in elderly women [4,5]. A high endogenous estradiol level has also been reported to be related to the relative risk for breast cancer in women [6,7]. We did not find a correlation between endogenous estrogen levels and BMI in the subjects in the present study, probably because the subjects in the present study were younger than subjects in previous studies. In the present study, estradiol level after HRT every day showed a significant positive correlation with BMI in postmenopausal and bilaterally ovariectomized women. Moreover, the difference between estradiol level after HRT every day and endogenous estradiol level also showed a significant positive correlation with BMI in these women. Thus, serum estradiol level after HRT every day increases more in obese than in non-obese postmenopausal and bilaterally ovariectomized women. The biological mechanism underlying the effect of HRT on body mass is complex. It might be related to the expression level of estrogen receptors (ERs) in adipose tissue, and the expression levels of ERs in obese women and thin women might be different. Adipose tissue has been reported to increase in female and male ER- knockout mice [15,16]. In obese women, the ER- expression level might be low and there might be a reciprocal relationship between ER- expression level and serum estradiol level. In obese women, a very high estradiol level, exceeding the response threshold, after HRT every day may have not only beneficial effects but also adverse

9 T. Yasui et al. / Maturitas 50 (2005) effects on various parts of the body. Buyuk et al. demonstrated that there is a trend toward a relation between BMI and breakthrough bleeding in postmenopausal women receiving HRT [9]. Women receiving estrogen progestin treatment in the WHI trial had increased relative risks for cardiovascular events and breast cancer. Mean body mass indices at the start of that trial in both the estrogen progestin group and placebo group were 28.5 kg/m 2, and the proportions of women with BMI of more than 25.0 kg/m 2 were 69.5% in the estrogen progestin group and 69.2% in the placebo group [2]. Thus, higher estrogen level due to the additional exogenous estrogen in women with high BMI may be related to these risks. On the other hand, an appropriate estradiol level for response thresholds after HRT every day may have beneficial effects for non-obese women. The association between use of HRT and hip fracture risk was found to be stronger in thin women in a population-based case-control study [8]. Rodriguez et al. found in an observation study that the inverse association between use of estrogen and coronary heart disease mortality was strongest for thin women (BMI < 22 kg/m 2 ) and that estrogen treatment did not reduce the risk of coronary heart disease for obese women whose BMI was more than 30 kg/m 2 [17]. Women with large body mass may have already gained the maximum effect due to increased extraglandular production of estrogen in adipose tissue, and a protective effect of HRT in women with a large body mass has not been observed. HRT resulted in an increase in estrogen to an appropriate level in thin women with frequent hot flashes whose endogenous estrogen level was low, and HRT may improve vasomotor symptoms and quality of life in such women. However, several observational studies have suggested that there are more risks of HRT in thin women than in obese women. An increased risk of breast cancer associated with hormone use has been found for postmenopausal thin women but not for postmenopausal women with high BMI [18]. Schairer et al. demonstrated that relative risk of breast cancer increased in women with a BMI not exceeding 24.4 kg/m 2 but that risk of breast cancer in heavier women (BMI of more than 24.4 kg/m 2 ) was not changed by treatment with estrogen alone or estrogen and progestin [19]. Michaelsson et al. reported that estrogens such as estradiol and conjugated estrogen were most strongly associated with breast cancer as well as protection of hip fracture in relatively lean women [8]. In the present study, both the serum estrone level and the difference between estrone level after HRT every day and endogenous estrone level showed significant positive correlations with BMI in bilaterally ovariectomized women but not in postmenopausal women. It is well known that estrone, which makes up the largest amount of estrogen produced in postmenopausal women, is a result of mainly peripheral conversion of androstenedione in adipose tissue. Thus, the conversion is greater in obese women, and an increased circulating level of estrone is thought to contribute to the association between obesity and endometrial proliferation and cancer. The significant correlation found in bilaterally ovariectomized women may have been because endogenous estrone derived from androstenedione in the adrenal gland and exogenous estrone after CEE administration are only related. On the other hand, the postmenopausal ovary has been shown to be an androgen-producing gland and to produce estrone and estradiol due to aromatization [20]. In postmenopausal women, endogenous estrone in the ovaries together with that produced by the adrenal gland and exogenous estrone after CEE administration are mixed, and a relationship between serum estrone level and BMI might therefore not be clearly observed. Many efforts have been made worldwide to develop low-dose HRT regimens that are effective for achieving long-term benefits of estrogens while minimizing side effects and risks [21 26]. The results of the WHI trial have also indicated that results do not apply to lower dosages of CEE and MPA. The HRT regimen used in the present study, consisting of mg of CEE and 2.5 mg of MPA administered every other day, could be regarded as low-dose HRT. Neither the serum estradiol levels after HRT every other day nor the difference between estradiol levels after HRT every other day and endogenous estradiol levels were correlated with BMI in our study. In obese women, low-dose HRT may be recommended if estrogen levels after low-dose HRT reach response thresholds. It is important to consider the type of hormone regimen as well as the characteristics of each women such as BMI. Our study has several limitations. Since the HRT regimen used in our study was continuous oral administration of CEE and MPA, the results regarding the

10 28 T. Yasui et al. / Maturitas 50 (2005) relationship between serum estrogen levels after CEE and BMI are not applicable to administration of other types of estrogen (e.g., 17 -estradiol administered transdermally, micronized estradiol). Further study using other estrogens is needed. In addition, the range of body mass indices in the subjects in the present study was relatively small. Further study using subjects with a broader range of body mass indices is needed. It is important to titrate the dose of a drug to the needs of the individual. Adjustment of estrogen dosage in women who have a high BMI could be a means for reducing the incidence of breakthrough bleeding in women who are on HRT. Low-dose HRT might be appropriate for obese women. A highly sensitive and specific estrogen assay such as that used in this study is not yet widely available for clinical use. If this estrogen assay is used, estradiol levels in obese women may be an important indicator during HRT every day. The HRT regimen must be based on the needs of each woman and ensure that serum estradiol in the postmenopausal woman receiving HRT will be maintained at an appropriate level. In conclusion, serum estradiol levels after HRT with CEE and MPA every day increase more in overweight than in non-overweight postmenopausal and bilaterally ovariectomized women. The results of the present study regarding the relationship between BMI and estrogen levels during HRT should be useful for choosing appropriate HRT regimens for postmenopausal women. References [1] Belchetz PE. Hormonal treatment of postmenopausal women. N Engl J Med 1994;330: [2] Writing group for the women s health initiative investigators. Risks and benefits of estrogen plus progestin in healthy postmenopausal women. JAMA 2002;288: [3] Erlik Y, Meldrum DR, Judd HL. Estrogen levels in postmenopausal women with hot flashes. Obstet Gynecol 1982;59: [4] Cummings SR, Browner WS, Bauer D, et al. 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11 T. Yasui et al. / Maturitas 50 (2005) [21] Lobo RA, Whitehead MI. Is low-dose hormone replacement therapy for postmenopausal women efficacious and desirable? Climacteric 2001;4: [22] Utian WH, Shoupe D, Bachmann G, Pinkerton JV, Picker JH. Relief of vasomotor symptoms and vaginal atrophy with lower doses of conjugated equine estrogens and medroxyprogesterone acetate. Fertil Steril 2001;75: [23] Lindsay R, Gallagher JC, Kleerekoper M, Pickar JH. Effect of lower doses of conjugated equine estrogens with and without medroxyprogesterone acetate on bone in early postmenopausal women. JAMA 2002;287: [24] Lobo RA, Bush T, Carr BR, Picker JH. Effects of lower doses of conjugated equine estrogens and medroxyprogesterone acetate on plasma lipids and lipoproteins, coagulation factors, and carbohydrate metabolism. Fertil Steril 2001;76: [25] Picker JH, Yeh IT, Wheeler JE, Cunnane MF, Speroff L. Endometrial effects of lower doses of conjugated equine estrogens and medroxyprogesterone acetate. Fertil Steril 2001;76: [26] Archer DF, Dorin M, Lewis V, Schneider DL, Picker JH. Effects of lower doses of conjugated equine estrogens and medroxyprogesterone acetate on endometrial bleeding. Fertil Steril 2001;75: