Cyclooxygenase-2 expression in deep endometriosis and matched eutopic endometrium

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1 FERTILITY AND STERILITY VOL. 82, NO. 5, NOVEMBER 2004 Copyright 2004 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A. Cyclooxygenase-2 expression in deep endometriosis and matched eutopic endometrium Sachiko Matsuzaki, M.D., a,b Michel Canis, M.D., a Jean-Luc Pouly, M.D., a Arnaud Wattiez, M.D., a Kunihiro Okamura, M.D., b and Gerard Mage, M.D. a Hôtel-Dieu, Polyclinique, CHU, Clermont-Ferrand, France; and Tohoku University Graduated School of Medicine, Sendai, Japan Received October 6, 2003; revised and accepted March 10, Reprint requests: Sachiko Matsuzaki, M.D., Department of Gynecology, Hôtel-Dieu, Polyclinique, CHU, Clermont-Ferrand, b.p. 69, 13 bd, L. Mafreyt, 63000, Clermont-Ferrand, France (FAX: ; sachikoma@aol.com). a Department of Gynecology, Hôtel-Dieu, Polyclinique, CHU, Clermont-Ferrand, Clermont-Ferrand, France. b Department of Obstetrics & Gynecology, Tohoku University Graduated School of Medicine, Sendai, , Japan /04/$30.00 doi: /j.fertnstert Objective: To identify any relationship between cyclooxygenase-2 expression and the intensity of severe, endometriosis-related dysmenorrhea. Design: Prospective study. Setting: University hospital. Patient(s): Patients with deep endometriosis. Intervention(s): During surgery, paired samples of tissue representing deep endometriosis and eutopic endometrium were obtained from 46 patients. Control endometrial tissue samples were obtained from 34 fertile women who underwent laparoscopic tubal ligation or reversal of tubal sterilization. Pain assessment for dysmenorrhea was done with a 10-point linear analogue scale. Main Outcome Measure(s): The percentage of surface immunostained for Cox-2 was determined by an immunohistochemical technique. Relationships between pain score for dysmenorrhea and Cox-2 expression were analyzed. Result(s): Cox-2 expression was significantly higher in eutopic endometrial stromal cells from patients with deep endometriosis than in stroma from controls during the early, mid, and late secretory phases. In endometriosis patients, Cox-2 expression in eutopic endometrial stromal cells was significantly higher in women with more severe dysmenorrhea (pain score 7 vs. 7) during early and mid secretory phases. Conclusion(s): Elevated Cox-2 expression in stromal cells in eutopic endometrium from patients with deep endometriosis may play a role in severe, endometriosis-related dysmenorrhea. (Fertil Steril 2004;82: by American Society for Reproductive Medicine.) Key Words: Cyclooxygenase-2, deep endometriosis, prostaglandins, severe dysmenorrhea Despite the strong clinical correlation, the basic mechanism of endometriosis-related pain remains unclear. A comprehensive understanding of the mechanisms of endometriosis-related pain is essential for clinical management of affected patients. For a long time, researchers have believed that prostaglandins (PGs) may be involved in endometriosis-related severe dysmenorrhea (1 4). Prostaglandins can result in perceptions of pain and are produced during inflammation. Cyclooxygenase (Cox) is the rate-limiting enzyme that catalyzes the initial step in formation of PGs from arachidonic acid (5). Cyclooxygenase is encoded by two separate genes, Cox-1 and Cox-2. Cox-1 is expressed constitutively in a wide range of tissues. In contrast, Cox-2 operates as an inducible enzyme with low or undetectable levels in most tissues; its expression can be markedly increased by a number of inflammatory, mitogenic, and physical stimuli. It is now thought that eicosanoids produced by Cox-1 are important for housekeeping functions, whereas those produced by Cox-2 lead to various pathological changes (6). Thus, PGs in endometriotic tissues may be produced mainly by Cox-2. No study to date has investigated the possible relationships between Cox-2 expression and endometriosisrelated severe dysmenorrhea. As a first step, we decided to investigate Cox-2 expression in cases of endometriosis. Endometriosis is a very heterogeneous disease based on location and clinical outcome. 1309

2 Deep endometriosis is strongly associated with severe dysmenorrhea, pelvic pain, and deep dyspareunia (7, 8). Therefore, with our focus on endometriosis-related severe dysmenorrhea, we selected patients with deep endometriosis for the present study. We investigated two phenomena: Cox-2 expression in eutopic and ectopic endometrium from patients with deep endometriosis, by using an immunohistochemical technique, and relationships between Cox-2 expression and the intensity of endometriosis-related severe dysmenorrhea. MATERIALS AND METHODS Sample Collection During laparoscopy or laparotomy, paired samples of deep endometriotic lesions and eutopic endometrium were obtained from a total of 46 women (median age, 32 years; range, 21 to 39 years). Of these patients, 24 had stage I, 7 had stage II, 6 had stage III, and 9 had stage IV disease according to the latest revision of the classification of the American Society for Reproductive Medicine (9). As control samples, endometrial tissues were obtained from 34 fertile women (median age, 39 years; range, 35 to 44 years) with normal pelvic cavities who underwent laparoscopic tubal ligation or reversal of tubal sterilization. Endometrial tissue biopsies were obtained immediately before the surgical procedure with use of an endometrial suction catheter (Pipelle, Laboratoire CCD, Paris, France). All patients received no hormonal treatments, such as GnRH-a or sex steroids, and used no intrauterine contraception for at least 6 months before surgery. Recruited patients had regular menstrual cycles, confirmed by menstrual history and measurement of serum 17- E 2 and P levels. The endometrial dating criteria as described by Noyes et al. (10) and the menstrual history were used to assess the phase of menstrual cycle. Tissues were fixed immediately after collection in 20% formalin-acetic and were embedded in paraffin for routine histopathological examinations and immunohistochemical analysis. All deep endometriosis samples contained a glandular epithelium surrounded by stromal tissue. The presence of these features was sufficient to meet the criteria for histopathological diagnosis of endometriosis. Sections of 4 m in thickness were routinely prepared, mounted on glass slides, and dried overnight at 37 C before being used for immunohistochemistry. All tissue samples were obtained with the fully informed consent of the patients. The research protocol was approved by the human research board of the ethical committee of Polyclinique de l Hotel- Dieu, Clermont-Ferrand, France. Pain Assessment Questionnaires on dysmenorrhea and other clinical information were administered before surgery during the hospital stay. Pain assessment was done with a 10-point linear analogue scale, with 0 representing no pain and 10 representing the worst possible pain (11, 12). Immunohistochemical Staining Immunohistochemical staining was performed using a mouse monoclonal antibody against human Cox-2 enzyme (Cayman Chemical, Ann Arbor, MI). Fetal membranes were included in each staining run as positive controls (13). Sections were deparaffinized and rehydrated in graded ethanols. After rinsing in distilled water, sections were microwaved for 5 minutes at 600 watts in 0.01 mol/l sodium citrate buffer (ph 6.0); this step was repeated four times. The slides were immersed in 3% H 2 O 2 in distilled water for 5 minutes and then in blocking solution (0.01 mol/l Tris, 0.3% Triton X100, 1% bovine serum albumin, and 3% normal goat serum) for 30 minutes to block endogenous peroxidase activity and unspecific binding sites, respectively. Sections were then rinsed in Trisbuffered saline and incubated at 4 C overnight with the primary antibody, diluted 1:50 in Tris-buffered saline with 3% bovine serum albumin, followed by a rinse in Tris-buffered saline. Negative controls were performed by replacing the primary antibody with normal mouse IgG diluted to the same concentration as the primary antibody. The sections were thereafter treated with biotinylated secondary anti-rabbit antibodies in a dilution of 1:200 (Jackson Immunoresearch Laboratories, Inc., West Grove, PA), and antibody-binding sites were finally visualized by avidin-biotin peroxidase complex solution (Vectastain Elite ABC kit, Vector Laboratories, Inc., Burlingame, CA), using diaminobenzidine as a chromogen. Quantitation of Cox-2 Immunostained Cells The computerized image analysis system consisted of a light microscope (Leica, Lyon, France; 40 objective, 10 ocular) with a color charge coupling device camera (Sony, Paris, France) connected to a SAMBA 2005 computer analysis system (Alcatel-TITN, Grenoble, France). The percentage of immunostained surface (compared with counterstained surface) was obtained (14). In all samples of endometrial tissue, 10 nonoverlapped fields were analyzed. Because of tissue heterogeneity in endometriosis, we analyzed all glandular and stromal cells within each sample. The number of areas analyzed in endometriotic tissues varied from 6 to 15 areas per sample. Statistical Analysis The Statview 4.5 program (Abacus Concepts, Inc., Berkeley, CA) was used for statistical analysis. The Mann- Whitney U test or Kruskal-Wallis test was applied to compare results from different groups. The Wilcoxon signed rank test was performed to compare differences in Cox-2 expression in paired eutopic and ectopic endometrial samples. Statistical significance was defined as a P value of Matsuzaki et al. Cyclooxygenase-2 in deep endometriosis Vol. 82, No. 5, November 2004

3 FIGURE 1 Immunohistochemical staining of Cox-2 protein in ectopic and eutopic endometrium from a single patient and in normal control endometrium. Arrowheads indicate Cox-2 positive stromal cells. (A) Deep endometriosis. (B) Late secretory eutopic endometrium. (C) Late secretory endometrium from the control group. Matsuzaki. Cyclooxygenase-2 in deep endometriosis. Fertil Steril RESULTS Results for Cox-2 expression are shown in Figure 1 and in Tables 1 and 2. Cytoplasmic immunoreactivity to Cox-2 was detected both in glandular epithelial and stromal cells in normal endometrium and in endometriosis samples obtained at different points in the menstrual cycle. Statistically significant cyclical differences were found in Cox-2 expression within stromal cells in control endometrium (P.03, Kruskal-Wallis test). In contrast, although Cox-2 expression in stromal cells from patients with endometriosis was markedly elevated during the late secretory phase, cyclical differences were not statistically significant in eutopic endometrium from these patients (P.06, Kruskal-Wallis test). We detected no significant cyclical differences in expression within glandular epithelial cells from eutopic endometrium of endometriosis patients or those from control endometrium. In addition, there was no significant cyclical difference in Cox-2 expression in glandular epithelial or stromal cells from endometriotic lesions. Cox-2 Expression in Ectopic and Matched Eutopic Endometrium Cox-2 expression in glandular epithelial cells from ectopic endometrium was significantly higher than that of matched eutopic endometrium during the mid (P.02) and late (P.02) proliferative phases and in the early (P.02), mid (P.02), and late (P.02) secretory phases. There were no significant differences in Cox-2 expression throughout FERTILITY & STERILITY 1311

4 TABLE 1 Percentage of immunostained surface for Cox-2 in eutopic and ectopic endometrium from patients with deep endometriosis and control endometrium. Menstrual cycle Endometriosis Eutopic endometrium Controls endometrium Epithelium Stroma Epithelium Stroma Epithelium Stroma a EP (2) (2) (2) (2) (2) (2) MP (8) b (8) (8) (8) (3) (3) LP (10) b (10) (10) (10) (9) (9) ES (9) b (9) (9) (9) c (3) (3) MS (9) b (9) (9) (9) c (10) (10) LS (8) b (8) (8) (8) c (7) (7) Note: All data are expressed as mean percentage SE. Values in parentheses indicate the number of samples examined for Cox-2 expression. EP early proliferative phase; MP mid proliferative phase; LP late proliferative phase; ES early secretory phase; MS mid secretory phase; LS late secretory phase; NA not available. a P.03 among the six phases in controls by Kruskal-Wallis test. b P.02 vs. eutopic endometrium. c P.04 (during ES and MS); P.02 (during LS), both vs. controls. Matsuzaki. Cyclooxygenase-2 in deep endometriosis. Fertil Steril the menstrual cycle in stromal cells of eutopic vs. ectopic endometrium. Eutopic Endometrium From Patients With Endometriosis vs. Control Endometrium There were no significant differences in Cox-2 expression in glandular epithelial cells throughout the menstrual cycle between patients with endometriosis and controls. However, expression levels of Cox-2 in stromal cells from patients with endometriosis were significantly higher than those of controls during the early (P.04), mid (P.04), and late (P.02) secretory phases. Relationships Between Pain Score for Dysmenorrhea and Cox-2 Expression In the present study, all 46 patients with deep endometriosis had dysmenorrhea. Their median linear analogue scale score for pain was 7.0 (ranging from 3.5 to 10.0), compared with 0 (ranging from 0 to 2.0) for the 34 control women. On the basis of median values for pain score, we divided endometriosis patients into two groups (women with score 7 and women with score 7). Cox-2 expression levels in eutopic and ectopic endometrial tissues were compared between the two groups. Cox-2 expression in eutopic stromal cells in Group 1 (pain score for dysmenorrhea 7) was significantly higher than the level in eutopic stromal cells in Group 2 (pain score for dysmenorrhea 7) during early (P.04) and mid (P.04) secretory phases. In contrast, there were no significant differences in Cox-2 expression in eutopic glandular epithelial cells between groups throughout the menstrual cycle. We detected no significant differences in Cox-2 expression in ectopic glandular epithelial and stromal cells between groups. DISCUSSION In the present study, we demonstrated a significantly higher expression of Cox-2 in eutopic endometrial stromal cells from patients with deep endometriosis compared with expression in control endometrium during the early, mid, and late secretory phases of the menstrual cycle. In particularly, we detected markedly higher levels of Cox-2 expression during the late secretory phase. Ota et al. (15) recently reported higher levels of Cox-2 expression in eutopic endometrial stromal cells from patients with endometriosis, compared with controls, during the early and mid secretory phases. However, they detected no significant difference during the late secretory phase. One possible explanation for the difference in findings is that our population of patients with endometriosis may differ from theirs. During the early and mid secretory phases in our endometriosis patients, Cox-2 expression in eutopic endometrial stromal cells was significantly higher in women with a pain score 7 than in women with a pain score 7. However, we detected no significant differences in Cox-2 expression in glandular epithelial cells from eutopic and control endometrium over the course of the menstrual cycle. In addition, we detected no significant differences in expression of Cox-2 in glandular epithelial and stromal cells in endometriotic lesions (ectopic endometrium) between Groups 1 and 2. These findings suggest that elevated levels of Cox-2 expression in eutopic endometrial stromal cells from patients with deep endometriosis may play a role in their symptomatically severe endometriosis-related dysmenorrhea. There is evidence that interleukin-1 may play a central role in the pathogenesis of endometriosis (16 19). Interleu Matsuzaki et al. Cyclooxygenase-2 in deep endometriosis Vol. 82, No. 5, November 2004

5 TABLE 2 Relationships between pain score for dysmenorrhea and Cox-2 expression. Endometrium Endometriosis Menstrual cycle Epithelium Stroma Epithelium Stroma Group 1 Group 2 Group 1 Group 2 Group 1 Group 2 Group 1 Group 2 EP NA (2) NA (2) NA (2) NA (2) MP (4) (4) (4) (4) (4) (4) (4) (4) LP (6) (4) (6) (4) (6) (4) (6) (4) ES (5) (4) (5) a (4) (5) (4) (5) (4) MS (5) (4) (5) a (4) (5) (4) (5) (4) LS (8) NA (8) NA (8) NA (8) NA Note: All data are expressed as mean percentage SE. Group 1, pain score for dysmenorrhea 7; group 2, pain score for dysmenorrhea 7. Values in parentheses indicate the number of samples examined for Cox-2 expression. Abbreviations as in Table 1. a P.04 vs. group 2 (stromal cells of eutopic endometrium). Matsuzaki. Cyclooxygenase-2 in deep endometriosis. Fertil Steril kin-1 expression levels in eutopic endometrium from patients with endometriosis are significantly higher than those of healthy controls (19). Recent studies have demonstrated that interleukin-1 induces an elevated Cox-2 protein level and enzyme activity in endometrial stromal cells in vitro (20, 21). Interleukin-1 may be a potential contributor to induction of Cox-2 expression in eutopic endometrium. Further studies to investigate regulation of mechanisms underlying elevated Cox-2 expression during the premenstrual phase may provide a better understanding of the mechanisms underlying endometriosis-related severe dysmenorrhea. However, in the present study, it is unclear whether elevated Cox-2 expression results in increasing production of PGs, which also may be involved in endometriosis-related severe dysmenorrhea. Because we analyzed Cox-2 protein expression levels by an immunohistochemical technique, our results do not reflect Cox-2 enzymatic activity. In addition, we did not measure amounts of any types of PGs produced in eutopic or ectopic endometrium. It is therefore necessary to investigate whether elevated Cox-2 protein expression in eutopic endometrial stromal cells is related to increased production of PGs, especially during the premenstrual phase. The pathogenesis of endometriosis remains unclear, but it is generally considered that the development of pelvic endometriosis may be a consequence of implantation of viable endometrial tissue in ectopic sites via retrograde menstruation (22). Because retrograde menstruation is a common phenomenon, it remains unknown why endometriosis affects only about 10% of the female population in their reproductive years (23). One possible explanation is that the eutopic endometrium of women who develop endometriosis may be different from that of women who do not develop the disease (24). There is growing evidence to support this hypothesis (25 31), and the findings from the present study add further evidence. Recent studies have demonstrated that angiogenesis requires Cox-2 activity, which promotes the expression of proangiogenic factors such as vascular endothelial growth factor (32). It is now generally accepted that angiogenesis plays an important role in the pathogenesis of endometriosis (27, 28, 33). Studies have demonstrated that angiogenic activity in eutopic endometrium from patients with endometriosis is increased compared with activity in controls, and this difference is most pronounced during the late secretory phase (27, 28, 33). Although it is necessary to clarify the roles of elevated levels of Cox-2 in eutopic endometrium, higher levels of Cox-2 expression in stromal cells from premenstrual endometrium of patients with endometriosis may facilitate attachment and angiogenesis of menstrual fragments in ectopic sites by promoting proangiogenic factors (34). In addition, we detected significantly higher Cox-2 expression in ectopic glandular epithelial cells than in matched eutopic endometrium. Our findings are in agreement with previous studies that reported overexpression of Cox-2 in epithelial cells from endometriotic tissues (15, 35). There are numerous reports that treatment with Cox-2 inhibitors leads to a marked reduction in the growth of a variety of neoplasms, including those of the colon (36) and bladder (37). Recently, Williams et al. (32) clearly demonstrated that tumor growth requires the presence of Cox-2 in the tumor. The investigators suggested that Cox-2 may regulate intratumoral vascular density and that one mechanism for the antineoplastic effects of Cox-2 inhibitors may be to inhibit the production of proangiogenic factors by tumor and stromal cells. Although endometriosis is not a neoplasm, overexpression of Cox-2 in glandular epithelial cells from endometriotic lesions regardless of cycle stage suggests that growth of endometriosis may also require the presence of Cox-2. FERTILITY & STERILITY 1313

6 The development and growth of endometriotic lesions are estrogen dependent, and aberrant aromatase expression may be one of the most important molecular mechanisms in the pathogenesis of endometriosis (38). Aromatase is expressed both in endometriotic lesions (ectopic endometrium) and eutopic endometrium, but expression is much higher in endometriotic lesions (26). Prostaglandin E 2 has been found to be the most potent known inducer of aromatase activity in endometriotic stromal cells (39). Although we did not measure amounts of prostaglandin E 2 or levels of enzyme activity of Cox-2 in the present study, increased Cox-2 expression might plausibly result in increased production of aromatase in endometriotic lesions. Furthermore, the presence of a positive feedback loop in endometriotic tissue for continuous local production of estrogen and PGs has been established (38, 39). Although the number of samples of eutopic endometrium from patients with endometriosis was larger than that of controls in the current study, statistical analysis did not validate significant cyclical changes in Cox-2 expression in patients stromal cells, whereas cyclical changes were significant in stromal cells from control women. The loss of cyclical change of Cox-2 expression in endometriotic stromal cells appears to be more prominent than those in eutopic endometrium. A higher local production of estrogen might cause the prominent loss of cyclical change in Cox-2 expression seen in endometriotic lesions. Further studies to clarify the roles and regulation of mechanisms underlying Cox-2 expression in eutopic (especially during the premenstrual phase) and ectopic endometrium may well provide important insights into endometriosis and give rise to novel therapeutic strategies for this disease. Acknowledgments: The authors thank the surgical theater staff for their cooperation. 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