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Cyclooxygenase-2 expression in the endometrium at the end of 2 years’ continuous combined hormone replacement therapy
Maturitas 46 (2003) 295 /299 www.elsevier.com/locate/maturitas
Cyclooxygenase-2 expression in the endometrium at the end of 2 years’ continuous combined hormone replacement therapy Shih-Cheng Hsu a, Cheng-Yu Long a, Cheng-Hui Yang b, Ching-Hu Wu b, Chien-Hsin Chen c, Fang-I. Liu a,d,* a
Department of Obstetrics and Gynecology, Kaohsiung Municipal Hsiao Kang Hospital, 482 Shan-Ming Road, Hsiao-Kang District, 812 Kaohsiung, Taiwan, ROC b Department of Obstetrics and Gynecology, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan, ROC c Department of Pathology, Municipal Hsiao Kang Hospital, Kaohsiung, Taiwan, ROC d Department of Pharmacy, Chia Nan University of Pharmacy and Science, Tainan, Taiwan, ROC Received 27 January 2003; received in revised form 10 April 2003; accepted 23 April 2003
Abstract Objective: To evaluate the change of endometrial histology and the expression of cyclooxygenase-2 (Cox-2) in the endometrium after continuous combined hormone replacement therapy (HRT). Methods: Forty-five postmenopausal women were recruited. All participants received 0.625 mg conjugated equine estrogen (CEE) and 2.5 mg medroxyprogesterone (MPA) daily for 2 years. Endometrial biopsy was performed twice, before medication (baseline) and after 2 years of HRT, respectively. Immunohistochemistry was used to detect the presence of Cox-2 expression. Results: More atrophic and weak secretory features of endometrium were noted after the 2-year HRT. Endometrial hyperplasia and carcinoma were not found and immunohistochemistry results revealed that Cox-2 was not expressed in the endometrium. Conclusion: Cox-2, known to play an important role in the tumorigenesis of cancer, was not stained in endometrium tissue after hormonal induction and more endometrium atrophy was noted after the 2-year HRT. From the results, it is noted that continuous combined HRT may be a relatively safe and appropriate regimen for longterm use in postmenopausal women. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Hormone replacement therapy; Endometrium; Cox-2
1. Introduction Hormone replacement therapy (HRT) is used to treat vasomotor symptoms and conditions such as
* Corresponding author. Tel.: /886-7-803-6783x3450; fax: /886-7-806-5068. E-mail address: [email protected] (F.-I. Liu).
atrophic vaginitis in perimenopausal and postmenopausal women, and its long-term use also has a role in reducing the incidence of osteoporosis and colorectal cancer in postmenopausal women [1,2]. However, the use of HRT has been hindered due to safety concerns and troublesome side effects. It is well documented that prolonged unopposed estrogen use in women increases the risk of
0378-5122/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0378-5122(03)00218-4
296
S.-C. Hsu et al. / Maturitas 46 (2003) 295 /299
endometrial hyperplasia and carcinoma [3]. For women with intact uterus, modern HRT preparation containing both estrogen and progestogen: progestogen is necessary to protect against risk of endometrial hyperplasia and carcinoma [4,5]. There are two commonly used regimens, sequential (cyclic) and continuous combined. Withdrawal bleeding, which usually accompanies cyclical progestogen administration, is a commonly cited reason for HRT noncompliance [6]. In hope of improving compliance, a continuous combined HRT regimen has often been chosen to induce endometrium atrophy and prevent bleeding. Prostaglandins have been recently shown to assert several effects on tumorigenesis and tumor invasion. It has been confirmed that they are strongly expressed in cancer tissues. Cyclooxygenase (Cox) is a key enzyme in synthesis of prostaglanoids. Two isoforms of the enzyme have been identified, Cox-1 and Cox-2, of which Cox-1 is considered a constitutive enzyme being transcripted by the ‘‘housekeeping gene’’ located on chromosome 9. Cox-1 is thought to be responsible for various physiological functions such as vasodilation in the kidney, cytoprotection of the stomach, and production of thromboxane by the platelets. It is not involved in tumorigenesis or tumor invasion [7]. However, some recent studies have shown Cox-1 expression is also upregulated in human cancers [8,9]. In contrast, Cox-2 is regulated by an ‘‘immediate-early gene’’ located on chromosome 1, which is an upregulated mRNA and thus is an inducible enzyme. Its pathophysiological role has been connected to inflammation, ovulation, and carcinogenesis. Under inflammatory and pathologic conditions, Cox-2 could be induced by factors such as growth factors, cytokines, oncogenes, serum, and tumor promoters to synthesize prostaglandins which then suppress tumor apoptosis and resulted in tumorigenesis. High Cox-2 expression was detected in some human cancers [10 /12] and nonsteroidal antiinflammatory drugs (NSAIDs) had also been used for the prevention and therapy of tumor proliferation and metastasis [13 /16]. There have been conflicting results for the Cox-2 expression in normal endometrium in various phases and endometrial hyperplasia [17 /19]. However, heigh-
tened expression of Cox-2 in the endometrial adenocarcinoma was well demonstrated in the studies [18 /23]. The ultimate goal of our study was to ensure long-term safe use of continuous combined HRT. The study was conducted by evaluating the change of the endometrial histology and to find out whether HRT was capable of inducing Cox-2 expression in the endometrium after 2 years of continuous combined HRT.
2. Materials and methods 2.1. Subjects Forty-five postmenopausal women, mean age 55.2 years (range 49 /55), were recruited from the outpatient clinic. All women had had a natural menopause and they were not hysterectomized. The women were healthy and had no sign of pelvic pathology at gynecological examination. Conventional exclusion criteria for estrogen therapy were used. Patients with malignancy of the breast or uterus, chronic liver disease, previous thromboembolic disorders and untreated hypertension were excluded. In addition, patients should be native of any estrogen- or progestogen-containing medication before the study started. The study was approved by the Institutional Review Board of Kaohsiung Medical University. All women gave written, informed consent. The patients received 0.625 mg conjugated equine estrogen (CEE) and 2.5 mg medroxyprogesterone acetate daily. Endometrial biopsies were performed before treatment began (baseline) and after 2 years of treatment. All biopsy specimens were obtained with a Serrated Novak curette and were fixed in formalin, embedded in paraffin and sectioned at several levels. 2.2. Immunohistochemical staining A streptavidin /biotin complex technique was applied to detect the expression of Cox-2 following the method of Hsu and Raine [24] with slight modification. The commercially available polyclonal antibody against Cox-2 was used at a titer of 1:100 (Santa Cruz Biotechnology, Santa Cruz,
S.-C. Hsu et al. / Maturitas 46 (2003) 295 /299
297
CA). The specimens were treated with 3% H2O2 in methanol for 20 min to block the endogenous peroxidase activity, then the sections were incubated with biotinylated secondary antibodies (BioGenex, San Ramon, CA). The streptavidinconjugated peroxidase (BioGenex) was applied and diaminobenzidine (Sigma, St. Louis, MA) was employed as a chromogen. The counterstain was performed with hematoxylin. Appropriate negative control was provided by omitting the primary antibody and replaced with non-immune serum. Tissue of colon cancer from human served as the positive control in the Cox-2 immunostaining.
3. Results Table 1 demonstrates the results of endometrial biopsy performed at baseline and after 2 years of continuous combined HRT. Three of the 45 women withdrew from the study: two because of troublesome bleeding; one lost interest. Compared with histology status at baseline, endometrial histology after 2 years tended to be more atrophic (13.3 vs. 52.4%). Proliferative (14.3%) and secretory (33.3%) histological features were also noted after 2 years of HRT. There were not inadequate specimens obtained for histological examination. Also, endometrial hyperplasia or carcinoma was not detected at baseline and after the 2 year-study period. To investigate the expression of Cox-2, every specimen was examined by Hsu and Chen. As shown in Fig. 1, Cox-2 was not expressed in any cell compartment of endometrium at baseline or after 2 years of HRT. Table 1 Endometrial histology Number of results (%)
Inadequate Atrophic Proliferative Secretory
At baseline (n /45)
After 2 years (n /42)
0 6 (13.3) 13 (28.9) 26 (57.8)
0 22 (52.4) 6 (14.3) 14 (33.3)
Fig. 1. Immunohistochemical staining of Cox-2 in the endometrium at baseline, (A) and after treatment (B). Cox-2 was not stained in any cell compartments of endometrium in both specimens (magnification /400).
4. Discussion It has been well documented that estrogen therapy given without a concomitant progestogen is associated with endometrial hyperplasia and an increased risk of endometrial cancer. It conveys about a 15/20% annual risk of endometrial hyperplasia [25]. Whitehead et al. demonstrated in 1981 that progestogens taken at least 10 days per month reduced the risk of endometrial hyperplasia and cancer to the same level as that for postmenopausal women not undergoing HRT [26]. Recently, there has been some concern that sequential progestogens given for a minimum of 10 days per cycle may not offer adequate endometrial protection. Beresford et al. [27], in a populationbase, case control study, showed an increased
298
S.-C. Hsu et al. / Maturitas 46 (2003) 295 /299
relative risk (1.3) of endometrial cancer in women taking progestogens for 10 /21 days per cycle compared with that in women not undergoing HRT. Weiderpass et al. [28] further reported that continuously added progestins may be needed to minimize the endometrial cancer risk associated with estrogen replacement therapy. Endometrial hyperplasia or cancer was not detected at the baseline and after 2 years of continuous combined HRT in our study. Even though our study may be limited by the sample size, it has further supported these reports. Inadequate endometrium biopsy specimens were frequently obtained from postmenopausal women on HRT using the Pipelle aspiration device. This method often results in very little tissue for histological analysis [29]. In our study endometrial biopsy specimens were collected using the Serrated Novak curette, therefore, the specimens were all adequate for analysis. Studies have shown that continuous combined HRT could induce an atrophic endometrium within 6/12 months [6]. It is known that the continuous combined HRT causes downregulation of estrogen and progestogen receptors, which in turn decreases sensitivity to hormonal stimulation [30]. In addition, progestogen induces 17-a dehydrogenase, which converts estradiol to the less active estrone, thereby reducing the estrogenic stimulus [31]. Accordingly, atrophic and weak secretory endometrium feature (85.7%) was highly expressive after 2 years of HRT in our study. Proliferative activity (14.3%) was also noted in some cases after HRT. It may be due to the relative dose of progestogen that might have been inadequate to suppress totally the estrogenic stimulation of the endometrium. Cox is known to be present in nuclear membrane and microsomal fraction of several tissues [32]. The peroxidase activity of Cox catalyzes the oxidation of a wide range of xenobiotics including several carcinogens. The resultant oxidized amines can react with DNA to produce base adducts that may mutate critical tumor suppressor gene and lead to initiation of the neoplastic process [33]. Therefore, Cox-2 may assume an important role in the pathogenesis of several cancers, and epidemiological studies have suggested the use of NSAIDs
to reduce the risk of cancers [16,34]. Many human studies have reported recently that Cox-2 is highly expressed in the endometrial adenocarcinoma [18 / 23]. In spite of the conflicting results of Cox-2 immunoreactivity in normal endometrium [17 / 19], we could not detect Cox-2 expression in the endometrium after 2 years of continuous combined HRT. The discrepancy may have resulted from use of different antibodies and immunohistochemical methods. Thus, we showed that HRT was not capable of inducing Cox-2 expression in the endometrium. In conclusion, our study has shown that continuous combined HRT is not associated with an increased risk of endometrial hyperplasia and more endometrium atrophy after treatment was noted. It is supported by the report of Weiderpass et al. that either progesterone- or testosteronederived progestins when given in continuous combined way could prevent endometrial carcinoma [28]. If the expression of Cox-2 could serve as a feature of endometrial carcinoma, it also implies that continuous combined HRT may provide a reassuring degree of endometrial safety and is an appropriate regimen for long-term use in postmenopausal women because there was not Cox-2 expression on the endometrium in the study.
References [1] Hammond CB. Menopause and hormone replacement therapy: an overview. Obstet Gynecol 1996;87:2s /15s. [2] Writing Group for the Women’s Health Initiative Investigators. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the Women’s Health Initiative randomized controlled trial. J Am Med Assoc 2002;288:321 /33. [3] Smith DC, Prentice R, Thompson DJ, Herrmann WL. Association of exogenous estrogen and endometrial carcinoma. New Engl J Med 1975;293:1164 /7. [4] Gambrell RD, Jr. Prevention of endometrial cancer with progestogens. Maturitas 1986;8:159 /68. [5] Persson I, Adami HO, Bergkvist L, et al. Risk of endometrial cancer after treatment with oestrogens alone or in conjunction with progestogens. Results of a prospective study. Br Med J 1989;298:147 /51. [6] Ravnikar VA. Barriers for taking long-term hormone replacement therapy: why do women not adhere to therapy. Eur Menopause J 1996;3(Suppl. 2):90 /3.
S.-C. Hsu et al. / Maturitas 46 (2003) 295 /299 [7] Crofford LJ. Cox-1 and Cox-2 tissue expression: implications and predictions. J Rheumatol 1997;24(Suppl. 49):15 / 9. [8] Hwang D, Scollard D, Byrne J, Levine E. Expression of cyclooxygenase-1 and cyclooxygenase-2 in human breast cancer. J Natl Cancer Inst 1998;90:455 /60. [9] Sales KJ, Katz AA, Howard B, Soeters RP, Millar RP, Jabbour HN. Cyclooxygenase-1 is upregulated in cervical carcinomas: autocrine/paracrine regulation of cyclooxygenase-2, prostaglandin E receptors, and angiogenic factors by cyclooxygenase-1. Cancer Res 2002;62:424 /32. [10] Wolff H, Saukkonen K, Anttila S, Karjalainen A, Vainio H, Ristimaki A. Expression of cyclooxygenase-2 in human lung carcinoma. Cancer Res 1998;58:4997 /5001. [11] Sano H, Kawahito Y, Wilder RL, et al. Expression of cyclooxygenase-1 and 2 in human colorectal cancer. Cancer Res 1995;55:3785 /9. [12] Ryu HS, Chang KH, Yang HW, Kim MS, Kwon HC, Oh KS. High cyclooxygenase-2 expression in stage IB cervical cancer with lymph node metastasis or parametrial invasion. Gynecol Oncol 2000;76:320 /5. [13] Subbaramaiah K, Zakim D, Weksler BB, Dannenberg AJ. Inhibition of cyclooxygenase: a novel approach to cancer prevention. Proc Soc Exp Biol Med 1997;216:201 /10. [14] Liu XH, Yao S, Kirschenbaum A, Levine AC. NS 398, a selective cyclooxygenase-2 inhibitor, induces apoptosis and down-regulates bcl-2 expression in LNCaP cells. Cancer Res 1998;58:4245 /9. [15] Schreinemachers DM, Everson RB. Aspirin use and lung, colon, and breast cancer incidence in a prospective study. Epidemiology 1994;5:138 /46. [16] Thun MJ, Namboodiri MM, Calle EE, Flanders WD, Heath CW, Jr. Aspirin use and risk of fatal cancer. Cancer Res 1993;53:1322 /7. [17] Marions L, Danielsson KG. Expression of cyclooxygenase in human endometrium during the implantation period. Mol Hum Reprod 1999;5:961 /5. [18] Uotila PJ, Erkkola RU, Klemi PJ. The expression of cyclooxygenase-1 and -2 in proliferative endometrium and endometrial adenocarcinoma. Ann Med 2002;34:428 /33. [19] Fujiwaki R, Iida K, Kanasaki H, Ozaki T, Hata K, Miyazaki K. Cyclooxygenase-2 expression in endometrial cancer: correlation with microvessel count and expression of vascular endothelial growth factor and thymidine phosphorylase. Hum Pathol 2002;33:213 /9. [20] Cao QJ, Einstein MH, Anderson PS, Runowicz CD, Balan R, Jones JG. Expression of Cox-2, Ki-67, cyclin D1, and P21 in endometrial endometrioid carcinomas. Int J Gynecol Pathol 2002;21(2):147 /54. [21] Tong BJ, Tan J, Tajeda L, et al. Heightened expression of cyclooxygenase-2 and peroxisome proliferator-activated receptor-d in human endometrial adenocarcinoma. Neoplasia 2000;2:483 /90.
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[22] Jabbour HN, Milne SA, Williams ARW, Anderson RA, Boddy SC. Expression of COX-2 and PGE synthase and synthesis of PGE2 in endometrial adenocarcinoma: a possible autocrine/paracrine regulation of neoplastic cell function via EP2/EP4 receptors. Br J Cancer 2001;85(7):1023 /31. [23] Munir I, Fukunaga K, Kanasaki H, et al. Expression of cyclooxygenase 2 by prostaglandin E2 in human endometrial adenocarcinoma cell line HEC-1B. Biol Reprod 2000;63:933 /41. [24] Hsu SM, Raine L. In: Delellis RA, editor. The use of avidin /biotin /peroxidase complex (ABC) in diagnostic and research pathology, in advances in immunohistochemistry. Chicago: Year Book Medical, 1984. [25] The Writing Group for the PEPI Trial. Effects of hormone replacement therapy on endometrial histology in postmenopausal women. The Postmenopausal Estrogen/Progestin Interventions (PEPI) Trial. J Am Med Assoc 1996;275:370 /5. [26] Whitehead ML, Townsend PL, Pryce-Davies J, Ryder TA, King JB. Effects of estrogens and progestins on the biochemistry and morphology of the postmenopausal endometrium. New Engl J Med 1981;305:1599 /605. [27] Beresford SA, Weiss NS, Voigt LF, McKnight B. Risk of endometrial cancer in relation to use of oestrogen combined with cyclic progestagen therapy in postmenopausal women. Lancet 1997;349:458 /61. [28] Weiderpass E, Adami HO, Baron JA, et al. Risk of endometrial cancer following estrogen replacement with and without progestins. J Natl Cancer Inst 1999;91:1131 / 7. [29] Moyer DL, de Lignieres B, Driguez P, Pez JP. Prevention of endometrial hyperplasia by progesterone during longterm estradiol replacement: influence of bleeding pattern and secretory changes. Fertil Steril 1993;59:992 /7. [30] King RJB, Whitehead MI, Campbell S, et al. Biochemical studies on the endometrium from postmenopausal women receiving hormone replacement therapy. Postgrad Med J 1978;54:65 /8. [31] Casper RF. Regulation of estrogen/progestogen receptors in the endometrium. Int J Fertil Menopausal Stud 1996;41:16 /21. [32] Otto JC, Smith WL. The orientation of prostaglandin endoperoxide synthases-1 and /2 in the endoplasmic reticulum. J Biol Chem 1994;269:19868 /75. [33] Dubois RN, Tsujii M, Bishop P, Awad JA, Makita K, Lanahan A. Cloning and characterization of a growth factor-inducible cyclooxygenase gene from rat intestinal epithelial cells. Am J Physiol 1994;266:G822 /7. [34] Giardiello FM, Offerhaus GJ, DuBois RN. The role of nonsteroidal anti-inflammatory drugs in colorectal cancer prevention. Eur J Cancer 1995;31A:1071 /6.
Cyclooxygenase-2 expression in the endometrium at the end of 2 years’ continuous combined hormone replacement therapy Shih-Cheng Hsu a, Cheng-Yu Long a, Cheng-Hui Yang b, Ching-Hu Wu b, Chien-Hsin Chen c, Fang-I. Liu a,d,* a
Department of Obstetrics and Gynecology, Kaohsiung Municipal Hsiao Kang Hospital, 482 Shan-Ming Road, Hsiao-Kang District, 812 Kaohsiung, Taiwan, ROC b Department of Obstetrics and Gynecology, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan, ROC c Department of Pathology, Municipal Hsiao Kang Hospital, Kaohsiung, Taiwan, ROC d Department of Pharmacy, Chia Nan University of Pharmacy and Science, Tainan, Taiwan, ROC Received 27 January 2003; received in revised form 10 April 2003; accepted 23 April 2003
Abstract Objective: To evaluate the change of endometrial histology and the expression of cyclooxygenase-2 (Cox-2) in the endometrium after continuous combined hormone replacement therapy (HRT). Methods: Forty-five postmenopausal women were recruited. All participants received 0.625 mg conjugated equine estrogen (CEE) and 2.5 mg medroxyprogesterone (MPA) daily for 2 years. Endometrial biopsy was performed twice, before medication (baseline) and after 2 years of HRT, respectively. Immunohistochemistry was used to detect the presence of Cox-2 expression. Results: More atrophic and weak secretory features of endometrium were noted after the 2-year HRT. Endometrial hyperplasia and carcinoma were not found and immunohistochemistry results revealed that Cox-2 was not expressed in the endometrium. Conclusion: Cox-2, known to play an important role in the tumorigenesis of cancer, was not stained in endometrium tissue after hormonal induction and more endometrium atrophy was noted after the 2-year HRT. From the results, it is noted that continuous combined HRT may be a relatively safe and appropriate regimen for longterm use in postmenopausal women. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Hormone replacement therapy; Endometrium; Cox-2
1. Introduction Hormone replacement therapy (HRT) is used to treat vasomotor symptoms and conditions such as
* Corresponding author. Tel.: /886-7-803-6783x3450; fax: /886-7-806-5068. E-mail address: [email protected] (F.-I. Liu).
atrophic vaginitis in perimenopausal and postmenopausal women, and its long-term use also has a role in reducing the incidence of osteoporosis and colorectal cancer in postmenopausal women [1,2]. However, the use of HRT has been hindered due to safety concerns and troublesome side effects. It is well documented that prolonged unopposed estrogen use in women increases the risk of
0378-5122/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0378-5122(03)00218-4
296
S.-C. Hsu et al. / Maturitas 46 (2003) 295 /299
endometrial hyperplasia and carcinoma [3]. For women with intact uterus, modern HRT preparation containing both estrogen and progestogen: progestogen is necessary to protect against risk of endometrial hyperplasia and carcinoma [4,5]. There are two commonly used regimens, sequential (cyclic) and continuous combined. Withdrawal bleeding, which usually accompanies cyclical progestogen administration, is a commonly cited reason for HRT noncompliance [6]. In hope of improving compliance, a continuous combined HRT regimen has often been chosen to induce endometrium atrophy and prevent bleeding. Prostaglandins have been recently shown to assert several effects on tumorigenesis and tumor invasion. It has been confirmed that they are strongly expressed in cancer tissues. Cyclooxygenase (Cox) is a key enzyme in synthesis of prostaglanoids. Two isoforms of the enzyme have been identified, Cox-1 and Cox-2, of which Cox-1 is considered a constitutive enzyme being transcripted by the ‘‘housekeeping gene’’ located on chromosome 9. Cox-1 is thought to be responsible for various physiological functions such as vasodilation in the kidney, cytoprotection of the stomach, and production of thromboxane by the platelets. It is not involved in tumorigenesis or tumor invasion [7]. However, some recent studies have shown Cox-1 expression is also upregulated in human cancers [8,9]. In contrast, Cox-2 is regulated by an ‘‘immediate-early gene’’ located on chromosome 1, which is an upregulated mRNA and thus is an inducible enzyme. Its pathophysiological role has been connected to inflammation, ovulation, and carcinogenesis. Under inflammatory and pathologic conditions, Cox-2 could be induced by factors such as growth factors, cytokines, oncogenes, serum, and tumor promoters to synthesize prostaglandins which then suppress tumor apoptosis and resulted in tumorigenesis. High Cox-2 expression was detected in some human cancers [10 /12] and nonsteroidal antiinflammatory drugs (NSAIDs) had also been used for the prevention and therapy of tumor proliferation and metastasis [13 /16]. There have been conflicting results for the Cox-2 expression in normal endometrium in various phases and endometrial hyperplasia [17 /19]. However, heigh-
tened expression of Cox-2 in the endometrial adenocarcinoma was well demonstrated in the studies [18 /23]. The ultimate goal of our study was to ensure long-term safe use of continuous combined HRT. The study was conducted by evaluating the change of the endometrial histology and to find out whether HRT was capable of inducing Cox-2 expression in the endometrium after 2 years of continuous combined HRT.
2. Materials and methods 2.1. Subjects Forty-five postmenopausal women, mean age 55.2 years (range 49 /55), were recruited from the outpatient clinic. All women had had a natural menopause and they were not hysterectomized. The women were healthy and had no sign of pelvic pathology at gynecological examination. Conventional exclusion criteria for estrogen therapy were used. Patients with malignancy of the breast or uterus, chronic liver disease, previous thromboembolic disorders and untreated hypertension were excluded. In addition, patients should be native of any estrogen- or progestogen-containing medication before the study started. The study was approved by the Institutional Review Board of Kaohsiung Medical University. All women gave written, informed consent. The patients received 0.625 mg conjugated equine estrogen (CEE) and 2.5 mg medroxyprogesterone acetate daily. Endometrial biopsies were performed before treatment began (baseline) and after 2 years of treatment. All biopsy specimens were obtained with a Serrated Novak curette and were fixed in formalin, embedded in paraffin and sectioned at several levels. 2.2. Immunohistochemical staining A streptavidin /biotin complex technique was applied to detect the expression of Cox-2 following the method of Hsu and Raine [24] with slight modification. The commercially available polyclonal antibody against Cox-2 was used at a titer of 1:100 (Santa Cruz Biotechnology, Santa Cruz,
S.-C. Hsu et al. / Maturitas 46 (2003) 295 /299
297
CA). The specimens were treated with 3% H2O2 in methanol for 20 min to block the endogenous peroxidase activity, then the sections were incubated with biotinylated secondary antibodies (BioGenex, San Ramon, CA). The streptavidinconjugated peroxidase (BioGenex) was applied and diaminobenzidine (Sigma, St. Louis, MA) was employed as a chromogen. The counterstain was performed with hematoxylin. Appropriate negative control was provided by omitting the primary antibody and replaced with non-immune serum. Tissue of colon cancer from human served as the positive control in the Cox-2 immunostaining.
3. Results Table 1 demonstrates the results of endometrial biopsy performed at baseline and after 2 years of continuous combined HRT. Three of the 45 women withdrew from the study: two because of troublesome bleeding; one lost interest. Compared with histology status at baseline, endometrial histology after 2 years tended to be more atrophic (13.3 vs. 52.4%). Proliferative (14.3%) and secretory (33.3%) histological features were also noted after 2 years of HRT. There were not inadequate specimens obtained for histological examination. Also, endometrial hyperplasia or carcinoma was not detected at baseline and after the 2 year-study period. To investigate the expression of Cox-2, every specimen was examined by Hsu and Chen. As shown in Fig. 1, Cox-2 was not expressed in any cell compartment of endometrium at baseline or after 2 years of HRT. Table 1 Endometrial histology Number of results (%)
Inadequate Atrophic Proliferative Secretory
At baseline (n /45)
After 2 years (n /42)
0 6 (13.3) 13 (28.9) 26 (57.8)
0 22 (52.4) 6 (14.3) 14 (33.3)
Fig. 1. Immunohistochemical staining of Cox-2 in the endometrium at baseline, (A) and after treatment (B). Cox-2 was not stained in any cell compartments of endometrium in both specimens (magnification /400).
4. Discussion It has been well documented that estrogen therapy given without a concomitant progestogen is associated with endometrial hyperplasia and an increased risk of endometrial cancer. It conveys about a 15/20% annual risk of endometrial hyperplasia [25]. Whitehead et al. demonstrated in 1981 that progestogens taken at least 10 days per month reduced the risk of endometrial hyperplasia and cancer to the same level as that for postmenopausal women not undergoing HRT [26]. Recently, there has been some concern that sequential progestogens given for a minimum of 10 days per cycle may not offer adequate endometrial protection. Beresford et al. [27], in a populationbase, case control study, showed an increased
298
S.-C. Hsu et al. / Maturitas 46 (2003) 295 /299
relative risk (1.3) of endometrial cancer in women taking progestogens for 10 /21 days per cycle compared with that in women not undergoing HRT. Weiderpass et al. [28] further reported that continuously added progestins may be needed to minimize the endometrial cancer risk associated with estrogen replacement therapy. Endometrial hyperplasia or cancer was not detected at the baseline and after 2 years of continuous combined HRT in our study. Even though our study may be limited by the sample size, it has further supported these reports. Inadequate endometrium biopsy specimens were frequently obtained from postmenopausal women on HRT using the Pipelle aspiration device. This method often results in very little tissue for histological analysis [29]. In our study endometrial biopsy specimens were collected using the Serrated Novak curette, therefore, the specimens were all adequate for analysis. Studies have shown that continuous combined HRT could induce an atrophic endometrium within 6/12 months [6]. It is known that the continuous combined HRT causes downregulation of estrogen and progestogen receptors, which in turn decreases sensitivity to hormonal stimulation [30]. In addition, progestogen induces 17-a dehydrogenase, which converts estradiol to the less active estrone, thereby reducing the estrogenic stimulus [31]. Accordingly, atrophic and weak secretory endometrium feature (85.7%) was highly expressive after 2 years of HRT in our study. Proliferative activity (14.3%) was also noted in some cases after HRT. It may be due to the relative dose of progestogen that might have been inadequate to suppress totally the estrogenic stimulation of the endometrium. Cox is known to be present in nuclear membrane and microsomal fraction of several tissues [32]. The peroxidase activity of Cox catalyzes the oxidation of a wide range of xenobiotics including several carcinogens. The resultant oxidized amines can react with DNA to produce base adducts that may mutate critical tumor suppressor gene and lead to initiation of the neoplastic process [33]. Therefore, Cox-2 may assume an important role in the pathogenesis of several cancers, and epidemiological studies have suggested the use of NSAIDs
to reduce the risk of cancers [16,34]. Many human studies have reported recently that Cox-2 is highly expressed in the endometrial adenocarcinoma [18 / 23]. In spite of the conflicting results of Cox-2 immunoreactivity in normal endometrium [17 / 19], we could not detect Cox-2 expression in the endometrium after 2 years of continuous combined HRT. The discrepancy may have resulted from use of different antibodies and immunohistochemical methods. Thus, we showed that HRT was not capable of inducing Cox-2 expression in the endometrium. In conclusion, our study has shown that continuous combined HRT is not associated with an increased risk of endometrial hyperplasia and more endometrium atrophy after treatment was noted. It is supported by the report of Weiderpass et al. that either progesterone- or testosteronederived progestins when given in continuous combined way could prevent endometrial carcinoma [28]. If the expression of Cox-2 could serve as a feature of endometrial carcinoma, it also implies that continuous combined HRT may provide a reassuring degree of endometrial safety and is an appropriate regimen for long-term use in postmenopausal women because there was not Cox-2 expression on the endometrium in the study.
References [1] Hammond CB. Menopause and hormone replacement therapy: an overview. Obstet Gynecol 1996;87:2s /15s. [2] Writing Group for the Women’s Health Initiative Investigators. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the Women’s Health Initiative randomized controlled trial. J Am Med Assoc 2002;288:321 /33. [3] Smith DC, Prentice R, Thompson DJ, Herrmann WL. Association of exogenous estrogen and endometrial carcinoma. New Engl J Med 1975;293:1164 /7. [4] Gambrell RD, Jr. Prevention of endometrial cancer with progestogens. Maturitas 1986;8:159 /68. [5] Persson I, Adami HO, Bergkvist L, et al. Risk of endometrial cancer after treatment with oestrogens alone or in conjunction with progestogens. Results of a prospective study. Br Med J 1989;298:147 /51. [6] Ravnikar VA. Barriers for taking long-term hormone replacement therapy: why do women not adhere to therapy. Eur Menopause J 1996;3(Suppl. 2):90 /3.
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