Kentaro Takahashi✱, Atsushi Manabe, Masako Okada, Hiroko Kurioka, Haruhiko Kanasaki, Kohji Miyazaki
Department of Obstetrics and Gynecology, Shimane Medical University, Enya-cho, Izumo 693-8501, Japan
✱ Corresponding author. Fax: + 81-853-20-2264. (K. Takahashi); E-mail: taka27@shimane-med.ac.jp.
Abstract
Objective: to assess the therapeutic efficacy and safety of oral estriol for the treatment of climacteric symptoms in postmenopausal women. Methods: 68 postmenopausal women with climacteric symptoms received oral estriol, 2 mg/day, daily for 12 months. We evaluated the degree of climacteric complaints with estriol therapy; serum levels of gonadotropins, estradiol (E2) and lipids; biochemical markers of bone metabolism; blood pressure; and side effects both at baseline and during treatment. Climacteric symptoms were assessed according to the menopausal index (MI), a version of the Kupperman index that had been modified for Japanese women. Results: oral estriol therapy significantly reduced total MI scores. The greatest relief was noted for hot flushes, night sweats, and insomnia. Estriol treatment significantly lowered serum follicle stimulating hormone (FSH) and luteinizing hormone (LH) concentrations but did not affect any of the other parameters (lipids, bone, liver and blood pressure) during the study period. Slightly vaginal bleeding occurred in 14.3% of those who underwent natural menopausal women. Histologic evaluation of the endometrium and ultrasound assessment of the breasts following 12 months of estriol treatment found normal results in all women. Conclusion: Estriol is a safe and effective alternative for relieving climacteric symptoms in postmenopausal Japanese women. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Estriol; Hormone replacement therapy; Climacteric symptoms.
Introduction
With the increasing longevity of the population age hormone replacement therapy (HRT) has been suggested to be necessary for obtaining a better quality of life and for avoiding metabolic disturbances resulting from hypoestrogenism in postmenopausal women. It has been well estab-lished, however, that unopposed estrogen admin-istration may contribute significantly to the development of uterine endometrial cancer [1,2] and breast cancer [3,4]. Although these potential risks remain controversial, the addition of sequential or combined progesterone thus has been recognized as essential during estrogen replacement therapy [5]. For obtaining a better quality of life relieving climacteric symptoms, and preventing osteoporosis and cardiovascular disease in postmenopausal women it is important to continue HRT for long period of time. Unfortunately, however, major adverse effects, including regular or irregular uterine bleeding, may impede patient compliance with long-term HRT. Consequently, the biologically weak estrogen, estriol, which has a much weaker stimulating effect on endometrial growth than E2 and does not commonly cause vaginal bleeding appears to be best suited for HRT [6]. Although estriol appears to be much safer than estrone or estradiol, its continuous use in high doses may have a stimulatory effect on both breast and endometrial tissue [7]. The efficacy and safety of orally-administered estriol in the treatment of climacteric symptoms, however, has been investigated rarely [8,9].
The present study therefore was designed to assess various indicators of therapeutic efficacy and safety of oral estriol for treating climacteric complains in postmenopausal women.
Materials and methods
The study included 68 postmenopausal women ages 35-62 years (mean ± S.D., 49.9 ± 5.9 years) with climacteric symptoms, including 35 women ages 45-62 years (53.8 ± 4.4 years) who had un-dergone natural menopause (time since menopause, 61.9 ± 28.3 months, range 36-116 months) and 33 women ages 35-54 years (45.8 ± 4.4 years) who had undergone surgically induced menopause (time since hysterectomy with bilateral oophorectomy, 50.7 ± 9.3 months, range 36-68 months). All subjects gave written informed consent. The study was approved by the local ethics committee. None of the patients had received HRT prior to study entry. All patients presented with climacteric complaints, such as hot flushes, sweating, and neurovegetative symptoms, and 600 mg/day calcium L-aspartate (Aspara-Ca, Tanabe, Tokyo, Japan) as a placebo was orally-administered for a few months prior to study entry. However, no change or aggravation of climacteric complaints occurred in all patients. The women were otherwise healthy based on clinical and routine laboratory examination. Exclusion criteria included thromboembolic disease, estrogen-related neoplasms, all breast disorders, urinary tract in-fections, uterine bleeding, viral vaginal infections and a body mass index (BMI) ≥ 25.
The patients received oral estriol (Estriel, Mochida, Tokyo, Japan), 2 mg/day, for 12 months. The intensity of the climacteric symptoms was recorded monthly according to our menopausal index (MI) (Table 1). This index is a modified version of the simplified menopausal index (SMI) which was developed by Koyama and Aso [10] for Japanese women based on various menopausal indices. To obtain the SMI, the authors simplified existing menopausal indices, including the Kupperman index [11] to be more suitable for clinical practice, considering the clinical experience with each clinical symptom compared with the Kupperman index, the questions for the SMI were reduced from 17 to 10, and vasomotor symptoms were emphasized more strongly. The SMI has a maximum score of 100, which is reduced depending on the conditions of each patient. Our MI also has a maximum score of 100, which additionally includes symptoms of sensory disorder, such as feeling of numbness in the arms, and legs.
Table 1. Menopausal index for subjective assessment of menopausal symptoms.
In addition to climacteric symptoms, we evalu-ated patient satisfaction with therapy monthly. Compliance with the regimen was determined by personal interviews and diary cards. Furthermore, serum levels of estradiol (E2), gonadotropins, blood lipids, biochemical markers of bone turn over and liver function, as well as blood pressure were measured. Blood samples were collected from the subjects early in the morning after an overnight fast. The sera were stored at — 30°C. To minimize variation, each subject’s sample was analyzed at the same time. We also assessed the presence of histologic endometrial neoplasma using endometrial biopsy, and the presence of breast tumors by ultrasound before and after treatment. Endometrial biopsies were obtained with a curette without local anesthesia. These specimens were fixed in neutral formalin, embedded in paraffin wax and sectioned at several levels. All biopsies were examined by a pathologist for signs of hormonal effects and atypia.
Serum E2, follicle stimulating hormone (FSH) and luteinizing hormone (LH) were measured with commercially available kits. Serum FSH, LH and E2 concentrations were determined by ra-dioimmunoassay using the Coat-A-Count E2 kit (Nippon DPC, Chiba, Japan), the SPAC-S FSH kit (Daiichi Radioisotope Laboratory, Tokyo,Japan) and SPAC-S LH kit (Daiichi Radioisotope Laboratory, Tokyo, Japan), the intra- and interassay coefficients of variation (CV) were < 7 and 6.2%, 4.8 and 5.3%, and < 8 and 4%, respectively. The reference E2 assay has 0.32% cross-reactivity with estriol.
Serum calcium (Ca) and alkaline phosphatase (ALP), and urinary Ca were measured to bone metabolism. Serum ALP was measured enzymati-cally with an automatic multi-purpose analyzer (TBA200 FR, Toshiba Medical Products, Tokyo, Japan). Serum ALP was measured by the Quick Auto Neo ALP-JSII (Shino test, Tokyo, Japan), intra- and inter-assay CV of 1.00 and 0.82%, respectively. Serum and urinary Ca were also measured by OCPC colorimetric method using OCPC fluid (Eiken chemical, Tokyo, Japan) (intra- and inter-assay CV of 0.71 and 1.37%, respec-tively). The urinary excretion of Ca was corrected for urinary creatinine (Cr). Serum total cholesterol (TC), triglycerides (TG), high density lipoprotein cholesterol (HDL-C), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and γ-glutamyl transpeptidase (γ-GTP) were measured enzymatically by means of an automatic multi-purpose analyzer (TBA200 FR, Toshiba Medical Products, Tokyo, Japan). Total cholesterol was measured enzymatically using Determiner LTC-S (Kyowa Medex, Tokyo, Japan) (intra- and inter-assay CV of 0.96 and 0.94%, respectively). Triglycerides were measured enzymatically using Auto sera STGN (Daiichi chemical, Tokyo Japan) (intra- and inter-assay CV, 0.66 and 1.02%, respectively). The HDL-C level was measured by a direct method [12] with the use of Determiner L HDL-C (Kyowa Medex Tokyo, Japan) (intra- and inter-assay CV of 1.12 and 1.59%, respectively). The level of low density lipoprotein cholesterol (LDL-C) was calculated from the formula; TC-TG/5-HDL-C [13]. The AST, ALT and y-GTP levels were measured enzymatically using L-Type Wako GOT J (Wako Pure Chemical Industry, Tokyo, Japan) (intra- and inter-assay CV, 1.10 and 1.13%, respectively), L- Type Wako GPT J (Wako Pure Chemical Industry, Tokyo, Japan) (intra- and inter-assay CV, 2.69 and 3.15%, respectively) and Lyquitech γ-GT (Roche Diagnostic K.K., Tokyo, Japan) (intra- and inter-assay CV of 0.95 and 1.66%, respectively), respectively.
Statistical analysis
The data are expressed as the mean ± S.D. and were analyzed using a STATVIEW software package (4.51.1) and were tested by either the Kol- mogorov-Smirnov two-sample test or the Scheffe’s multiple comparison test. The one-way analysis of variance for repeated measures was used to evaluate the significance of any changes in the parameters after initiation of estriol administration. P < 0.05 was considered significant.
Results
Of the 68 participants, 88.2% completed the 12-month trial. Eight women dropped out during the trial. Of these women, one had unexpected vaginal spottings during the third month of treat-ment. Three women dropped out for lack of efficacy after 3 -9 months of treatment, two for self-assessed complete relief of climacteric symptoms after 5 -10 months of treatment, and two for unknown reasons after 10-11 months of treatment. Unexpected vaginal bleeding for 2-3 days occurred in five women (14.3%) of those who underwent natural menopausal women. The bleeding stopped of its own accord despite continuation of medication, except in the woman who discontinued treatment. In this woman, endometrial biopsy specimens taken during the bleeding incidences showed an atrophic endometrium without malignancy. Other clinical complications were observed in four women (5.9% of the entire study group): including epigastralgia in two women (2.9%), mastodynia in one woman (1.5%), and palpitation in one woman (1.5%). However, none of the women discontinued treatment due to the above side-effects. Furthermore, histologic evaluation of the endometrium following 12 months of estriol treatment found no atipical endometrium in all women including atrophic endometrium in 28 women (82.4% of natural menopausal women) and weakly proliferative endometrium in six women (17.6%). Ultrasound assessment of the breasts following 12 months of treatment found no tumor in all women.
With respect to clinical characteristics and blood parameters, estriol treatment significantly lowered serum FSH and LH concentrations. However, none of the other parameters evaluated was altered during the 12-month study period (Table 2).
Table 2. Clinical characteristics and the blood parameters of 68 postmenopausal women before and after 12 months of treatment with oral estriol (2 mg/day)a.
The women’s subjective improvement was de-termined using the MI. Total MI scores prior to study entry (mean, 29.3 ± 14.4 points) was signifi-cantly increased at a start of this study. Total MI scores before treatment ranged from six to 75 points (mean, 31.9 ± 15.8 points) (Fig. 1). At the end of the first month, the mean score on the MI already was significantly reduced by 39.1%. Further significant reductions in MI scores occurred over the next 9 months, and the mean MI score at the end of the study period was 12.8 ± 12.8 points. The changes in average MI scores from baseline to the end of treatment for each symptom are summarized in Fig. 2. The changes in MI scores for vasomotor symptoms (Fig. 2A) and psychological symptoms (Fig. 2B) (75.8 and 51.9% reduction, respectively) were similar to those found for total MI. Hot flushes, night sweats and insomnia showed the most significant reduction. Similarly, statistical analysis of mean MI scores for motor symptoms (3.6 ± 3.2 points) after 3 months of treatment demonstrated a significant decrease compared with mean pretreat-ment values (Fig. 2C). This effect persisted until the end of the 12-month treatment period (3.6 + 3.6 points). with respect to perceptive symptoms, statistical analysis of mean MI scores after 1 and 6 months of treatment showed significant decreases compared with mean baseline values. However, no statistically significant changes were observed at the other time points (Fig. 2D).
Figure 1. Mean total scores on the menopausal index (MI) before and after treatment with oral estriol (2 mg/day) for 12 months. Mean values are shown, with error bars indicating S.E. † P < 0.0001 vs. pretreatment values.
Figure 2. Changes in mean scores on the menopausal index (MI) for vasomotor (A), psychological (B), motor (C) and perceptive (D) symptoms during 12 months of treatment with oral estriol (2 mg/day). Mean values are shown, with error bars indicating S.E. * P < 0.05; ** P < 0.01; † P < 0.0001 vs. pretreatment values. § P < 0.05; ¶ P < 0.01 vs. values at the end of the first month of treatment. ‡ P < 0.05 vs. values at the end of the second month of treatment.
The women’s self-assessed satisfaction with es-triol therapy is shown in Fig. 3. At the end of the first month, 72.1% of women were satisfied with the treatment. Treatment continuation appeared to increase their satisfaction. Thus, 84.2 ± 11.4% of women were satisfied with estriol therapy at the end of treatment (P < 0.01). Furthermore, 93.3% of women who completed the 12-month trial wished to continue with the therapy.
Figure 3. Patient satisfaction with estriol therapy during the 12-month treatment period. Mean values are shown, with error bars indicating S.E. ¶P < 0.01 vs. values at the end of the first month of treatment.
Discussion
The present study investigated the efficacy and safety of oral estriol in the treatment of climacteric symptoms in postmenopausal women. Estriol levels in postmenopausal women are likely to be low, even through the metabolic clearance rate of estriol is decreased by 13% [14]. The decrease in estriol levels probably results from the reduction in serum E2 levels, which are significantly lower in postmenopausal women than during the early fol-licular phase of the menstrual cycle. While orally- administered estriol is absorbed rapidly, its elimination is very rapid compared with that of other estrogens [14]. As a result, only 1-2% of orally-administered estriol reaches the circulation [15]. Despite the low estriol levels in the circulation and the fact that estriol cannot be converted to E2, the present study demonstrated that estriol at a dose of 2 mg/day reduced significantly and rapidly climacteric symptoms in postmenopausal women, particularly hot flushes, night sweats, and insomnia. These findings are similar to the results previously by Yang et al. [9].
Of the 68 women admitted to study, 88% com-pleted the 12-month trial period. About 85% of the patients were satisfied with the results at the end of the study period, and 93% of those who completed the trial wanted to continue with the treatment regimen. These observations suggest that oral estriol, 2 mg/day, may be acceptable for treating postmenopausal Japanese women with the climacteric syndrome. Previous studies indicated that the oral administration of higher estriol doses (8 mg/day) also reduced hot flushes [16]. However, these high doses also produced endometrial effects, such as unexpected vaginal bleeding, as well as such side effects as nausea and mastal- gia. In contrast, few side effects were observed in the present study.
It is commonly believed estriol does not con-tribute to endometrial proliferation in the same way as estradiol and estrone. However, Mon- toneri et al. [17] reported that the treatment with oral estriol, 1 mg twice daily for 10-25 days resulted in hyperplasia changes in 70.8% of the women. In the present study, slightly vaginal bleeding occurred in 14.3% of women who underwent natural menopausal women. Weakly proliferative endometrium was observed in 17.6% of women. However, histologic evaluation of the endometrium following 12 months of estriol treatment found no atipical endometrium in all women. These results support the observation that although estriol causes some degree of endometrial stimulation [17,18], it does not contribute to endometrial proliferation to the same extent as do estradiol and estrone. On the other hand, an interesting study compared the effect of varying dosage schedules on endometrial proliferation. At least 4 weeks was necessary to effect a change in the endometrium. Only a slight effect receiving one 8-mg dose daily; but clear proliferative changes were noted when the dose was divided [19]. It may be that estriol’s effect on the endometrium has less to do with the dose and more to do with the frequency of administration, with more frequent dosages being more likely to contribute to endometrial hyperplasia [7]. Fur-thermore, mortality from endometrial cancer was not related to the prescription of estriol [20]. In the present study, estriol did not produce atypical endometrial in all women. Therefore, estriol with a small dose such as 2 mg/day for a period of 1 year is considered to be safe.
Although in vitro studies have demonstrated that estriol, as well as estrone and estradiol, has a stimulatory effect on human breast cancer cells in tissue culture [21], Lemon [22] postulated that estriol is probably a safer form of estrogen re-placement in regard breast cancer for the following reasons: (1) in vitro, when given in conjunction with estradiol, it accelerates the removal of estradiol bound to protein receptors; (2) investigators have been able to initiate very little carcinogenesis in animal studies unless large doses (200-500 mcg/kg per day) were used on a contin-uous basis; (3) in animal studies it has been found to prevent carcinogen-induced mammary tumors; and (4) unlike estrone and estradiol, estriol metabolism does not result in the formation of large numbers of potentially carcinogenic substances. He postulated that lower doses of estriol and an intermittent schedule of dosing, such as every other day, would provide more potential for protection from mammary carcinogenesis and endometrial hyperplasia [22].
In the present study, we did not observed any stimulatory effect on both breast and endometrial tissue. Therefore, estriol appears to be much safer than estrone or estradiol eatment.
In the present study, estriol administration did not alter serum E2 levels, but reduce significantly the levels of LH and FSH. These observations resemble the results of Keller et al. [23]. Furthermore, one study reported that the daily administration of 6 mg of estradiol inhibited of estriol inhibits pituitary LH release to some degree as indicated by a reduction in plasma LH levels [24]. However, other studies have demonstrated that estriol is rather ineffective in suppressing pituitary gonadotropin release [25]. The reasons for these discrepant results are unknown.
Estrogen influences numerous parameters of bone and lipid metabolism. Thus, estrogen admin-istration generally inhibits bone resorption, and can therefore prevent further bone loss in women at high risk for osteoporosis [26]. Recently several investigators [27-29] reported that estriol prevented postmenopausal bone loss, and concluded that based on the placebo results that estriol may be safe and efficacious. Estrogen reduces serum levels of TC and LDL-C while increasing serum levels of HDL-C [30]. As a result, HRT with E2 or conjugated estrogens reduces the risk of cardiovascular disease [31,32]. However the present study did not demonstrate any effect of estriol administration on bone metabolism and lipid levels. The reasons for this lack of effect are unknown, but it is possible that the schedule of estriol administration and duration of treatment may play a role. Further studies involving a larger number of patients are required to evaluate estri- ol’s effects on these metabolic parameters.
It is well-known that there is a marked placebo response in term of improvement of hot flushes and of Kupperman Index [33] and the results of the present study need to be further documented on the bases of a double-blind, placebo-controlled trial. However, all subjects evaluated in the present study treated with 600 mg/day calcium L-aspartate for a few months prior to study entry but had no change or aggravation of climacteric complaints. The present study is an open trial with straight-forward design and results. The prior use of this calcium tablets play the role of placebo for a few months. Therefore, the present study suggests that estriol may be a safe and effective alternative for the relief of climacteric symptoms in postmenopausal women who reject, or who have contraindications, to conventional HRT with E2 or conjugated estrogens.
References
Smith DG, Prentice R, Thompson DJ, Herrmann WL. Association of exogenous estrogen and endometrial carcinoma. N Engl J Med 1975;293:1164-7.
Ziel HK, Finkle WD. Increased risk of endometrial carcinoma among users of conjugated estrogens. N Engl J Med 1975;293:1167-70.
Steinberg KK, Thacker SB, Smith SJ, et al. A meta-analysis of the effect of estrogen replacement therapy on the risk of breast cancer. J Am Med Assoc 1991;265:1985-90.
Colditz GA, Hankinson SE, Hunter DJ, et al. The use of estrogens and progestins and the risk of breast cancer in postmenopausal women. N Engl J Med 1995;332:1589- 93.
Marslew U, Overgaard K, Riis BJ, Christiansen C. The new combinations of estrogen and progestogen for prevention of postmenopausal bone loss: long-term effect on bone, calcium and lipid metabolism, climacteric symptoms, and bleeding. Obstet Gynecol 1992;79:202-10.
Ohta H, Ikeda T, Masuzawa T, Makita K, Suda Y, Nozawa S. Differences in axial bone mineral density, serum levels of sex steroids, and bone metabolism between postmenopausal and age- and body size-matched premenopausal subjects. Bone (NY) 1993;14:1119-26.
Head KA. Estriol: safety and efficacy. Altern Med Rev 1998;3:101-13.
Tzingounis VA, Aksu F, Greenblatt RB. Estriol in the management of the menopause. J Am Med Assoc 1978;239:1638-41.
Yang TS, Tsan SH, Chang SP, Ng HT. Efficacy and safety of estriol replacement therapy for climacteric women. Chin Med J (Taipei) 1995;55:386-91.
Koyama T, Aso T. Kampo medicine for menopausal women: evaluation with simplified menopausal index. Recent Prog Kampo Med Obstet Gynecol 1992;9:30-4.
Kupperman HS, Wetchler BB, Blatt MHG. Contemporary therapy of the menopausal syndrome. J Am Med Assoc 1959;171:1627-37.
Nauck M, Marz W, Haas B, Wieland H. Homogeneous assay for direct determination of high-density lipoprotein cholesterol evaluated. Clin Chem 1996;42:424-9.
Friedewald WT, Levy RI, Fredrickson DS. Estimation of low-density lipoprotein cholesterol in plasma without use of the preparative ultracentrifuge. Clin Chem 1972;18:499-550.
Englund DE, Elamsson KB, Johansson EDB. Bioavailability of oestriol. Acta Endocrinol 1982;99:136-40.
Kuhl H. Pharmacokinetics of oestrogens and proges- terones. Maturitas 1990;12:171-97.
Schneider HPG. Oestriol and the menopause: clinical results from a prospective study. In: Fioretti P, Martini L, Melis GB, Yen SSC, editors.Serono Symposium, The Menopause: Clinical, Endocrinological and Pathophysiological Aspects, vol. 39. London: Academic Press, 1982:523-33.
Montoneri C, Zarbo G, Garofalo A, Giardinella S. Effects of estriol administration on human postmenopausal endometrium. Clin Exp Obstet Gynecol 1987;14:178-81.
Wren BG. Oestriol in the control of postmenopausal symptoms. Med J Aust 1982;1:176-7.
Punnonen R, Soderstrom KO. The effect of oral estriol succinate therapy on the endometrial morphology in postmenopausal women: the significance of fractionation of the dose. Eur J Obstet Gynecol Reprod Biol 1983;14:217- 24.
Scairer C, Adami H-O, Hoover R, Persson I. Cause specific mortality in women receiving hormone replacement therapy. Epidemiology 1997;8:59-65.
Lippman M, Monaco ME, Bolan G. Effects of estrone, estradiol, and estriol on hormone-responsive human breast cancer in long-term tissue culture. Cancer Res 1977;37:1901-7.
Lemon HM. Pathophysiologic consideration in the treatment of menopausal patients with oestrogens; the role of oestriol in the prevention of mammary carcinoma. Acta Endocrinol (Copenh) 1980;223:S17-27.
Keller PJ, Riedmann R, Fischer M, Gerber C. Oestrogens, gonadotropins and prolactin after intra-vaginal administration of oesteriol in post-menopausal women. Maturitas 1981;3:47-53.
Vahapassi J, Adlercreutz H. Effect of continuous oral administration of estriol on corpus luteum function. Con-traception 1975;11:427-39.
Englund DE, Johansson EDB. A pharmacokinetic study on oestriol. Endometrial effect of oestriol treatment in postmenopausal women. Acta Endocrinol 1979;91(Suppl. 225):2109.
Lindsay R, Cosman F. Estrogen in prevention and treatment of osteoporosis. Ann NY Acad Sci 1990;592:326-33.
Nishibe A, Morimoto S, Hirota K, et al. Effect of estriol and bone mineral density of lumbar vertebrae in elderly and postmenopausal women. Jpn J Geriatr 1996;33:353- 9.
Minaguchi H, Uemura T, Shirasu K, et al. Effect of estriol on bone loss in postmenopausal Japanese women: a multicenter prospective open study. J Obstet Gynaecol Res 1996;22:259-65.
Nozaki M, Hashimoto K, Inoue Y, Sano M, Nakano H. Usefulness of estriol for the treatment of bone loss in postmenopausal women. Acta Obstet Gynaecol Jpn 1996;48:83-8.
Knopp RH. The effects of postmenopausal estrogen therapy on the incidence of arteriosclerotic vascular disease. Obstet Gynecol 1988;72(Suppl. 5):23S-30S.
Dubey RK, Gillespie DG, Jackson EK, Keller PJ. 17P- Estradiol, its metabolites, and progesterone inhibit cardiac fibroblast growth. Hypertention 1998;31:522-8.
Modena MG, Rossi R, Muia N Jr, Origliani G, Rombola O, Molinari R. Short-term results of transdermal estrogen replacement therapy in cardiovascular disease-free post-menopausal females with and without hypertension. G Ital Cardiol 1998;28:636-44.
Saletu B, Brandstatter N, Metka M, et al. Double-blind, placebo-controlled, hormonal, syndromal and EEG mapping studies with transdermal oestradiol therapy in menopausal depression. Psychopharmacology 1995;122:321-9.