Detection of aromatase cytochrome P-450 in endometrial biopsy specimens as a diagnostic test for endometriosis

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1 FERTILITY AND STERILITY VOL. 72, NO. 6, DECEMBER 1999 Copyright 1999 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Detection of aromatase cytochrome P-450 in endometrial biopsy specimens as a diagnostic test for endometriosis Jo Kitawaki, M.D., Ph.D.,* Izumi Kusuki, M.D.,* Hisato Koshiba, M.D.,* Katsumi Tsukamoto, M.D., Ph.D.,* Shinji Fushiki, M.D., Ph.D., and Hideo Honjo, M.D., Ph.D.* Kyoto Prefectural University of Medicine, Kyoto, Japan Received April 7, 1999; revised and accepted July 9, Reprint requests: Jo Kitawaki, M.D., Ph.D., Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kamigyo-ku, Kyoto , Japan (FAX: ; *Department of Obstetrics and Gynecology. Department of Dynamic Pathology, Research Institute for Neurological Diseases and Geriatrics /99/$20.00 PII S (99) Objective: To evaluate the clinical usefulness of examining endometrial biopsy specimens for aromatase cytochrome P-450 as a diagnostic test for endometriosis. Design: Retrospective, case-controlled study. Setting: Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine, Kyoto, Japan. Patient(s): One hundred five women of reproductive age with normal menstrual cycles underwent endometrial biopsy laparotomy or laparoscopy, and examination of their tissue revealed endometriosis, adenomyosis, and/or leiomyomas. Patients who had cervical carcinoma in situ but no other gynecologic disease were considered to be disease-free. Intervention(s): Endometrial biopsy specimens were collected. Main Outcome Measure(s): The expression of aromatase cytochrome P-450 was examined by reverse transcription-polymerase chain reaction and immunohistochemical analysis. The distribution and intensity of the immunostaining was assessed using a semiquantitative index designed H-score. Result(s): Immunostaining for aromatase cytochrome P-450 was detected in biopsy specimens obtained from patients with endometriosis, adenomyosis, and/or leiomyomas but not in specimens obtained from disease-free patients (H-score 20), with a sensitivity and specificity of 91% and 100%, respectively. Conclusion(s): The expression of aromatase cytochrome P-450 in biopsy specimens of eutopic endometrium distinguishes between disease-free women and women with endometriosis, adenomyosis, and/or leiomyomas. This technique can be used at outpatient infertility clinics as an initial screening procedure to rule out the presence of estrogen-dependent disease. (Fertil Steril 1999;72: by American Society for Reproductive Medicine.) Key Words: Endometriosis, adenomyosis, leiomyomas, infertility, aromatase cytochrome P-450, estrogen, endometrial biopsy, diagnostic test Endometriosis frequently is associated with chronic pelvic pain and infertility. Diagnostic imaging techniques such as transvaginal ultrasonography and magnetic resonance imaging can be used to screen for endometriosis but laparotomy or laparoscopy is necessary to establish a conclusive diagnosis (1). Serologic testing for CA-125 is useful in following the progression of endometriosis but is limited as an initial diagnostic test (2). An accurate and simple diagnostic test for endometriosis is needed. Endometriosis is most prevalent in women of reproductive age and tends to regress after menopause or ovariectomy, suggesting that the disease is estrogen-dependent. Estrogen, progesterone, and androgen receptors have been detected by hormone-ligand binding assays (3) and immunohistochemical analyses (4). The conversion of androgens to estrogens occurs predominantly in the placenta and ovary and is catalyzed by aromatase, the major component of which is aromatase cytochrome P-450. Considerable biochemical evidence indicates that estrogen-dependent diseases of the uterus, such as endometrial carcinoma (5 7), endometriosis (8, 9), adenomyosis (10, 11), and leiomyomas (12, 13), show aromatase activity and the expression of aromatase cytochrome P-450 messenger RNA (mrna). This suggests that, at a local level, these tissues produce estrogens, 1100

2 which may be involved in tissue growth through interaction with the estrogen receptors. A previous study in our laboratory (9) showed that aromatase cytochrome P-450 mrna and protein are expressed in the eutopic endometria of patients with endometriosis, adenomyosis, and/or leiomyomas, whereas neither are expressed in endometrial specimens obtained from normally menstruating women with cervical carcinoma in situ but no other gynecologic disease. This finding indicates that, although the eutopic endometria of patients with endometriosis, adenomyosis, or leiomyomas resemble the endometria of women without gynecologic disease, the estrogen metabolism of these tissues is remarkably different. Endometriosis, adenomyosis, and leiomyomas are separate entities but they share a common pathophysiology in that they develop primarily in women of reproductive age, their growth is estrogen-dependent, and they have a complicated pattern of occurrence. Adenomyosis and leiomyomas are comparatively easy to diagnose with the use of imaging techniques, but mild or asymptomatic endometriosis is difficult to identify. The aim of this study was to determine the clinical usefulness of examining endometrial biopsy specimens for aromatase cytochrome P-450 as a diagnostic test for endometriosis. MATERIALS AND METHODS Tissue Samples Endometrial biopsy specimens were obtained for diagnostic purposes from 105 patients who underwent laparotomy or laparoscopy in the Department of Obstetrics and Gynecology at the Kyoto Prefectural University of Medicine. The study protocol was approved by the Kyoto Prefectural University of Medicine institutional review board, and informed consent was obtained from each patient. All patients were of reproductive age (24 48 years) and had normal menstrual cycles. The patients had not received any endocrine therapy, including GnRH analogues, danazol, and pseudopregnancy therapy. Endometriosis, adenomyosis, and leiomyomas were diagnosed on histologic examination of excised uteruses (91 cases) or biopsy specimens obtained from abdominal lesions at laparoscopy (14 cases). The stage of endometriosis was assigned according to the revised American Fertility Society scoring system (1). Endometrial specimens obtained from patients with cervical carcinoma in situ but no other gynecologic disease were defined as disease-free. Patients with the following conditions were excluded from the study: malignant neoplasms other than cervical carcinoma in situ, ovarian neoplasms, pelvic inflammation, and pregnancy. One hundred five patients met the criteria for enrollment. Fresh tissue samples were divided into three portions; one portion was frozen immediately at 80 C and stored for total RNA extraction and two portions were fixed with 4% paraformaldehyde in 0.05 M of tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer (ph 7.6) at 4 C for 24 hours. One of the latter portions was subjected to histologic examination and the other to immunohistochemical analysis. Blood samples were drawn before the operation and serum CA-125 levels were measured by an immunoradiometric assay kit using the specific monoclonal antibody, M11 (Centocor, Malvern, PA). Isolation of RNA and Reverse Transcription- Polymerase Chain Reaction Southern Blot Analysis The total RNA was extracted as previously described (9) using Trizol reagent (GIBCO BRL, Gaithersburg, MD). The first-strand complementary DNA (cdna) synthesis was catalyzed by Superscript II RT (GIBCO BRL) using oligo(deoxythymidine) and was used for polymerase chain reaction (PCR) amplification as previously described (9). The primers used were 5 -TTGTTGTTAAATATGATGCC-3 (forward) and 5 -ATACCAGGTCCTGGCTACTG-3 (reverse) for human aromatase cytochrome P-450, and the human glyceraldehyde-3-phosphate dehydrogenase (G3PDH) amplimer set (Clontech, Palo Alto, CA) for G3PDH. The PCR mixture comprised 1 L of first-strand cdna, 1 M each of the primers mentioned above, 0.2 mm of deoxyribonucleoside 5 -triphosphates mixture (dntp), and 2.5 U of KOD Dash (Toyobo, Osaka, Japan) in a total volume of 100 L of PCR buffer. The PCR conditions used were 94 C for 3 minutes to denature the RNA/cDNA hybrid, followed by 40 cycles of 94 C for 30 seconds, 45 C (for aromatase cytochrome P-450) or 55 C (for G3PDH) for 2 seconds, and 74 C for 30 seconds. The PCR product was electrophoresed in a 2% agarose gel and transferred to a nylon membrane. An antisense probe 5 -TAATGATTGT- GCTTCATTATGTG-3 for aromatase cytochrome P-450 and a probe for G3PDH (Clontech) were 5 end-labeled with [ - 32 P]ATP. The membrane was hybridized with the labeled probe overnight at 55 C and the hybridized signal was analyzed using a bioimaging analyzer (BAS 2000; Fujix, Tokyo, Japan). Immunohistochemical Analysis Immunostaining was performed as previously described (9) with certain modifications. In brief, tissue samples were fixed with 4% paraformaldehyde solution, embedded in paraffin, and cut into 4- m sections. The sections were deparaffinized and treated for 5 minutes with 3% H 2 O 2 to block the endogenous peroxidase, then washed three times with 0.05 M of Tris-HCl buffer (ph 7.6) containing 0.05% Tween 20 (wash buffer) and incubated for 10 minutes at room temperature with Dako Protein Block Serum-Free (Dako, Carpinteria, CA) to block nonspecific protein binding. The sections were incubated for 24 hours at 4 C with rabbit antihuman placental aromatase cytochrome P-450 antiserum (PAb R-8-2, 1:1,000) diluted in Dako Antibody Diluent FERTILITY & STERILITY 1101

3 (Dako). The characteristics and specificity of the antiserum have been reported previously (14). The sections were washed three times with wash buffer and incubated for 30 minutes at room temperature with Dako Envision peroxidase-labeled polymer conjugated to antirabbit IgG (Dako). The sections again were washed three times with wash buffer, colored with 3,3-diaminobenzidine hydrochloride solution, and counterstained with hematoxylin. Human term placental sections were used for positive controls for aromatase cytochrome P-450. Negative controls for aromatase cytochrome P-450 were incubated with the same dilution of nonimmunized rabbit serum or PAb R-8-2 that had been pretreated with immunopurified human placental aromatase cytochrome P-450 (500 g of aromatase cytochrome P-450 per 1 ml of diluted PAb R-8-2) to block the active site. Positive and negative controls were immunostained simultaneously with each series of endometrial specimens. The intensity of staining was analyzed using a semiquantitative index known as an H-score (15). The H-score was calculated using the following equation: H-score Pi where i is the intensity of staining with a value of 0, 1, 2, or 3 (negative, weak, moderate, or strong, respectively) and P is the percentage of stained cells for each given i (from 0 to 100%). Approximately 500 glandular cells were observed in each slide for determination of the H-score. The H-score was the mean of two values calculated by two independent observers. Statistical Analysis Differences in H-scores among groups were analyzed with the one-factor analysis of variance and multiple comparisons were performed using the Sheffé procedure. A receiver operating characteristic curve was used to evaluate paired sensitivity and for the specificity for increasing H- score cutoff values. Sensitivity and false-positive (1 specificity) rates were calculated at increasing thresholds from Positive and negative predictive values for the cutoff value were calculated. Pearson s correlation coefficient was used to evaluate the relation between serum CA-125 levels and H-scores. RESULTS Of the total 105 patients, the mean ( SD) age and body mass index were years and kg/m 2 for those with endometriosis (n 32), years and kg/m 2 for those with adenomyosis, years and kg/m 2 for those with leiomyomas, and years and kg/m 2 for those free of gynecologic disease (n 21), respectively. The mean age of the patients with leiomyomas was higher than that of the patients with endometriosis (P.001) and the patients free FIGURE 1 Representative Southern blots of aromatase cytochrome P-450 transcripts after reverse transcription-polymerase chain reaction amplification. Total RNA samples obtained from the endometrial biopsy specimens of patients with endometriosis (E), adenomyosis (A), leiomyomas (L), or cervical carcinoma in situ but no other gynecologic disease (DF) were reverse transcribed, amplified with aromatase cytochrome P-450 (P450arom) or G3PDH primers, and hybridized with 32 P-labeled corresponding probes, as described in Materials and Methods. bp base pairs. of gynecologic disease (P.001). There was no difference in the body mass index of the different groups. Reverse transcription-polymerase chain reaction Southern blot analysis for aromatase cytochrome P-450 mrna expression was performed on each of the biopsy specimens obtained throughout the menstrual cycle. Representative experiments are shown in Figure 1, where aromatase cytochrome P-450 transcripts were detected in the specimens obtained from patients with endometriosis, adenomyosis, or leiomyomas. However, aromatase cytochrome P-450 mrna was not detected in any of the specimens obtained from normally menstruating women free of gynecologic disease. In an attempt to increase the sensitivity of detection and to semiquantify the expression of aromatase cytochrome P-450, immunostaining for aromatase cytochrome P-450 was performed in each of the 105 endometrial biopsy specimens. Aromatase cytochrome P-450 is immunolocalized exclusively in the cytoplasm of glandular cells and faintly in the stroma. Immunostaining for aromatase cytochrome P-450 was detected in the specimens obtained from patients with endometriosis in both the proliferative (Fig. 2A) and secretory (Fig. 2B) phases of the menstrual cycle. Similarly, aromatase cytochrome P-450 staining was detected in the specimens obtained from patients with adenomyosis and from those with leiomyomas in both the proliferative and secretory phases. In contrast, however, aromatase cytochrome P-450 was not detected in the endometrial specimens obtained from normally menstruating women free of gyne Kitawaki et al. Aromatase in endometrial biopsy samples Vol. 72, No. 6, December 1999

4 FIGURE 2 Representative immunostaining for aromatase cytochrome P-450 in eutopic endometria of patients with endometriosis (A and B) or cervical carcinoma in situ but no other gynecologic disease (C and D). Endometrial biopsy specimens were obtained during the proliferative (A and C) and secretory (B and D) phases of the menstrual cycle. Formalin-fixed, paraffin-embedded sections were immunostained and developed with the use of peroxidase-labeled polymer using diaminobenzidine as a chromogen (areas of brown staining) and then counterstained with hematoxylin. The H-score was used as a semiquantitative measure of the intensity and distribution of aromatase cytochrome P-450 immunostaining and was calculated as described in Materials and Methods. Original magnification, 100. Bar 30 m. cologic disease in either the proliferative (Fig. 2C) or the secretory (Fig. 2D) phase. The intensity and distribution of aromatase cytochrome P-450 immunostaining were evaluated semiquantitatively using the H-score (Fig. 3). In the specimens obtained from patients with endometriosis, adenomyosis, or leiomyomas, the H-scores varied widely. In contrast, all H-scores of the 21 specimens obtained from patients free of gynecologic disease were 20 and less than the mean H-scores of each of the other three groups (P.0001). There was no difference between the H-scores of specimens obtained in the proliferative and secretory phases of the menstrual cycle (Fig. 3). We grouped specimens with endometriosis, adenomyosis, and leiomyomas into disease categories and attempted to discriminate between diseased and disease-free tissue by setting a cutoff value for the H-score. The receiver operating characteristic curve analysis showed that a cutoff value of 20 provided optimum sensitivity and specificity. The sensitivity was 91%, the specificity was 100%, the positive predictive value was 100%, and the negative predictive value was 72%. The positive ratios for aromatase cytochrome P-450 were 80% in the proliferative and secretory phases for each disease group, regardless of whether the group consisted of one disease or of two or three diseases. Aromatase cytochrome P-450 immunostaining was negligible in the diseasefree group (Table 1). To examine further the relation between the H-score and the severity of endometriosis, the H-scores of specimens with endometriosis were replotted as a function of the stage FERTILITY & STERILITY 1103

5 FIGURE 3 Scattergram of the H-scores for aromatase cytochrome P-450 immunostaining in the endometrial biopsy specimens. The specimens were categorized according to whether they were obtained during the proliferative (open squares) or secretory (solid circles) phase of the menstrual cycle. The H-score, which represents the intensity and distribution of immunostaining, was calculated in each sample as described in Materials and Methods. Bars represent the mean SD of the mixture of specimens in the proliferative and secretory phases. * P.0001 vs. disease-free. of endometriosis (revised American Fertility Society score) or the serum concentration of CA-125. However, no correlation was observed between the H-score and the stage of endometriosis (r 0.87) or between the H-score and the serum CA-125 level (r 0.045, P.82). DISCUSSION Several earlier studies (16 21) reported the presence of aromatase activity and its regulation in normal endometrium of women of reproductive age. However, lack of aromatase expression in normal endometrium was reported by four groups using measurements of catalytic activity (9, 22, 23), immunohistochemical staining (9), and reverse transcription-polymerase chain reaction analysis (9, 24). Careful review of these studies reveals that the normal endometrial specimens were obtained mostly by hysterectomy conducted for various benign diseases, including endometriosis, adenomyosis, and leiomyomas. The specimens therefore were not necessarily obtained from disease-free uteruses. To clarify the controversy, we strictly defined normal endometria as only those obtained from normally menstruating women with cervical carcinoma in situ but no other gynecologic disease, and we showed that neither aromatase cytochrome P-450 activity nor mrna was present in these normal endometria (9). We also showed that aromatase cytochrome P-450 and mrna are expressed in eutopic endometria from patients with endometriosis, adenomyosis, and/or leiomyomas, as well as in ectopic endometriotic implants and adenomyosis tissue (9). The present study confirms that aromatase cytochrome P-450 is expressed in biopsy specimens of eutopic endometrium obtained from patients with endometriosis, adenomyosis, and/or leiomyomas but not in specimens obtained from women free of gynecologic disease. The high sensitivity (91%) and specificity (100%) of this test indicate that the detection of aromatase cytochrome P-450 may distinguish TABLE 1 Positive ratios for aromatase cytochrome P-450 immunostaining in diseased and disease-free endometrial biopsy specimens. No. of cases with positive immunostaining/no. of cases in the indicated phase of each disease group (%) Disease Proliferative Secretory Total Endometriosis 7/7 (100) 4/5 (80) 11/12 (92) Adenomyosis 5/5 (100) 6/7 (86) 11/12 (92) Leiomyomas 10/11 (91) 6/7 (86) 16/18 (89) Endometriosis adenomyosis 2/2 (100) 2/2 (100) 4/4 (100) Endometriosis leiomyomas 5/5 (100) 4/5 (80) 9/10 (90) Adenomyosis leiomyomas 8/10 (80) 12/12 (100) 20/22 (91) Endometriosis adenomyosis leiomyomas 4/5 (80) 1/1 (100) 5/6 (83) Free of disease 0/8 (0) 0/13 (0) 0/21 (0) Note: The cutoff value for the H-score was set at Kitawaki et al. Aromatase in endometrial biopsy samples Vol. 72, No. 6, December 1999

6 between diseased and disease-free tissue. When used in combination with diagnostic imaging techniques, aromatase cytochrome P-450 immunostaining enables us to diagnose endometriosis, adenomyosis, and/or leiomyomas more easily and with greater accuracy. When adenomyosis, leiomyomas, neoplastic diseases, and inflammation have been ruled out, the diagnosis is highly likely to be endometriosis. Although at present laparotomy or laparoscopy is needed to establish the diagnosis of endometriosis (1), endometrial biopsy may be used at outpatient clinics as an initial screening test for infertile women to rule out the presence of estrogen-dependent disease. CA-125 is a useful serologic marker that shows the progression of endometriosis and adenomyosis but its usefulness as a diagnostic test is limited because it is not sensitive enough to identify early stages of endometriosis (2). The present study is the first to use the expression of aromatase cytochrome P-450 in the diagnosis of endometriosis. However, there are several potential problems with our methodology. First, a small number of specimens evaluated in the study were collected retrospectively and the conformation ratio of each as disease or disease-free did not necessarily equal the corresponding prevalence because of a selection bias. A controlled prospective study is needed. Second, in the present study, PCR analysis was less sensitive in detecting aromatase cytochrome P-450 in small biopsy specimens even though it would have been simpler and more objective. Immunohistochemical analysis could be considered less objective and would allow for inherent fluctuation between observers. Nevertheless, it is an advantage that our antibody is sensitive and specific enough to immunostain aromatase cytochrome P-450. There was no difference in the H-scores between specimens obtained in the proliferative and secretory phases of the menstrual cycle. In the specimens with endometriosis, there was no correlation between the H-score and the stage of endometriosis or between the H-score and the serum CA-125 level. Although the stage of endometriosis (1) and the serum CA-125 level (2) in themselves may not correlate well with the progression of endometriosis, our findings indicate that the expression of aromatase cytochrome P-450 may not be associated with disease progression. The mechanism by which aromatase cytochrome P-450 is expressed in the eutopic endometrium of patients with endometriosis, adenomyosis, and/or leiomyomas remains to be determined. Although these diseases are separate entities, they all develop in an estrogen-dependent fashion. Aromatase cytochrome P-450 also is expressed in ectopic implants of endometriosis (8, 9), adenomyotic tissue (9 11), and leiomyomas (12, 13). Prostaglandin E 2 may play a role in stimulating aromatase (25). In conclusion, the expression of aromatase cytochrome P-450 in biopsy specimens of eutopic endometrium facilitates the diagnosis of endometriosis, adenomyosis, and/or leiomyomas with high sensitivity and specificity. This technique may be used at outpatient infertility clinics as an initial screening test to rule out the presence of estrogen-dependent disease. Acknowledgment: The authors thank Miss Yoko Tsutsumi for her skillful technical assistance. References 1. The American Fertility Society. Revised American Fertility Society classification of endometriosis: Fertil Steril 1985;43: Colacurci N, Fortunato N, De Franciscis P, Fratta M, Cioffi M, Zarcone R, et al. Serum and peritoneal CA-125 levels as diagnostic test for endometriosis. Eur J Obstet Gynecol Reprod Biol 1996;66: Tamaya T, Motoyama T, Ohono Y, Ide N, Tsurusaki T, Okada H. Steroid receptor levels and histology of endometriosis and adenomyosis. 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7 of progestogens on aromatase activity in stromal cells of human uterine endometrium. J Endocrinol Invest 1989;12: Huang JR, Bellino FL, Osawa Y, Tseng L. Immunologic identification of the aromatase enzyme system in human endometrium. J Steroid Biochem 1989;33: Baxendale PM, Reed MJ, James VHT. Inability of human endometrium or myometrium to aromatize androstenedione. J Steroid Biochem 1981; 14: Prefontaine M, Shih C, Pan CC, Bhavnani BR. Applicability of the product isolation and the radiometric aromatase assays for the measurement of low levels of aromatase: lack of aromatase activity in the human endometrium. J Endocrinol 1990;127: Bulun SE, Mahendroo MS, Simpson ER. Polymerase chain reaction amplification fails to detect aromatase cytochrome P450 transcripts in normal human endometrium or decidua. J Clin Endocrinol Metab 1993;76: Noble LS, Takayama K, Zeitoun KM, Putman JM, Johns DA, Hinshelwood MM, et al. Prostaglandin E 2 stimulates aromatase expression in endometriosis-derived stromal cells. J Clin Endocrinol Metab 1997;82: Kitawaki et al. Aromatase in endometrial biopsy samples Vol. 72, No. 6, December 1999