Investigation of the Roles of Cyclooxygenase-2 and Galectin-3 Expression in the Pathogenesis of Premenopausal Endometrial Polyps

The pathogenesis and etiology of endometrial polyps has not been elucidated. In this study, we aimed to examine the pathogenic mechanisms of endometrial polyp development using immunohistochemistry. We evaluated the expression of galectin-3 and cyclooxgenase-2 (COX-2) during the menstrual cycle in premenopausal women with endometrial polyps or normal endometrium. Methods: Thirty-one patients with endometrial polyps and 50 healthy control pa- tients were included in this study. The levels of expression of COX-2 and galectin-3 were studied by immunohistochemistry. Results: The percentage of COX-2–positive cells and the intensity of COX-2 staining in the endometrium did not vary during the menstrual cycle either in the control group or in patients with endometrial polyps. However, expression of galectin-3 was significantly lower in endometrial polyps and during the proliferative phase of the endometrium compared with the secretory phase. Conclusions: Our data suggests that the pathogenesis of endometrial polyps does not involve expression of COX-2 or galectin-3. Key Words: Endometrial polyps; Cyclooxgenase-2; Galectin-3; Immunohistochemistry Received: January 13, 2016 Revised: March 6, 2016 Accepted: March 8, 2016 Corresponding Author Esin Kasap, MD Department of Obstetrics and Gynecology, Sifa University School of Medicine, Fevzipasa Boulvard, No. 172/2, 35240, Basmane/I˙zmir, Türkiye Tel: +90-232-4460880 Fax: +90-232-4460770 E-mail: [email protected] Journal of Pathology and Translational Medicine 2016; 50: 225-230 http://dx.doi.org/10.4132/jptm.2016.03.08 ▒ ORIGINAL ARTICLE ▒ Focal endometrial projections containing endometrial glands and stroma are termed endometrial polyps.1 The reported prev- alence of endometrial polyps in premenopausal women with ab- normal uterine bleeding is 33%, but only 10% in asymptom- atic women.2 While endometrial polyps can occur in women of all ages, they are more common in women between the ages of 40 and 49.3 Because endometrial polyps have not been reported to occur before the onset of menstruation, estrogenic stimulation is thought to be associated with endometrial polyp growth.4 However, the pathogenesis and etiology of endometrial polyps has not been clearly determined. Endometrial polyps are be- lieved to form due to hormonal factors, e.g., estrogen and pro- gesterone, which mediate endometrial proliferation and differen- tiation via steroid receptors;5 however, the mechanisms involved in the development of endometrial polyps are still unclear. Cyclooxygenase (COX) is a key enzyme involved in the con- version of arachidonic acid to prostaglandins and other eico- sanoids. Two isoforms of COX have been identified: COX-1 and COX-2. COX-1 is constitutively expressed in many tissues, whereas COX-2 is induced by a variety of factors including cy- tokines, growth factors, and tumor promoters. COX-2, which is involved in inflammatory responses and production of prosta- glandins mediating uterine contractions, has been shown to in- duce excessive formation of some pro-angiogenic factors when overexpressed in colon cancer cell lines in vitro.6 Furthermore, recent studies have shown the influence of COX-2 in neoplastic development.5 However, the relationship between COX-2 and endometrial polyps has not been well established. Galectin-3 is a β-galactoside–binding animal lectin that con- tains carbohydrate-recognition domains and is involved in a mul- titude of biological tasks,7 including embryogenesis, growth, cell adhesion, proliferation, differentiation, cell-cycle progression, apoptosis, mRNA splicing, and immune system regulation. Ga- lectin-3 is also involved in tumorigenesis, angiogenesis, and tu- mor metastasis, and is expressed in various cells and tissues in- cluding activated macrophages, eosinophils, neutrophils, mast cells, gastrointestinal and respiratory tract epithelial cells, kid- neys cells, and some sensory neurons.7,8 Interestingly, extracel- lular galectin-3 plays a role in inflammation, while intracellular galectin-3 participates in cell growth and anti-apoptotic processes http://jpatholtm.org/ http://dx.doi.org/10.4132/jptm.2016.03.08 226 • Kasap E, et al. and modulates cell adhesion and migration.7 Polyps tend to occur when apoptosis is inhibited and there is unopposed growth.9 However, the mechanisms mediating these endometrial alterations are unknown. Previous studies have sug- gested that endometrial polyps are a result of endometrial inflam- mation, i.e., endometritis, since the vessel axis of polyps has been shown to develop during endometritis.10 This finding suggests that identification of markers of inflammation and tissue growth may help to elucidate the pathogenic mechanisms of endometri- al polyps. Indeed, recent studies have shown that the levels of expression of COX-2 and galectin-3 are increased during the progression from hyperplasia to cancer in the endometrial tis- sue, suggesting that these proteins may play a role in tumor cell function. However, the association of COX-2 and galectin-3 expression with polyps has not yet been established. Therefore, in this study, we analyzed the levels of expression of COX-2 and galectin-3 in endometrial polyps and normal endo- metrium using immunohistochemistry.

Patients We examined a total of 81 cases of endometrial tissues in pa- tients who were treated in the Department of Obstetrics and Gynecology of our hospital. Tissues were sampled between 2012 and 2014. All procedures were approved by the I˙zmir Sifa Uni- versity Human Ethics Committee and followed the principles of the Declaration of Helsinki. All patients were premenopausal (mean age, 37 years; range, 27 to 52 years) and had a recent history of regular menstruation (cycle of 25–35 days). None of the women included in the study used nonsteroidal anti-inflammatory drugs, hormone replace- ment therapy, or any other estrogen-containing pills. Thirty-one of the 81 patients had endometrial polyps, including 10 who had undergone hysterectomy and 21 who had undergone polypecto- my and endometrial curettage. None of the patients had identi- fiable leiomyoma or adenomyosis by ultrasonography and/or magnetic resonance imaging. The control group consisted of samples from a total of 50 additional patients with normal en- dometrium, and included 23 samples collected during the pro- liferative phase and 27 samples collected during the secretory phase. Control patients were recruited from patients with benign ovarian cysts or a uterine prolapse but no other pathology, and the endometrial samples in this group were collected during hys- terectomy procedures. Endometrial samples were grouped ac- cording to menstrual cycle phases: proliferative (days 1–14 of the cycle) and secretory (days 15–28 of the cycle). The day of the menstrual cycle was established from the women’s menstrual his- tory and confirmed by endometrial dating using the criteria de- scribed by Noyes et al.11 Immunohistochemistry All tissue samples were fixed in 10% formalin and sent to pathology for analysis. Routine hematoxylin and eosin staining was carried out in all samples either to confirm the diagnosis of polyps or to date the endometrium. Samples were embedded in paraffin blocks, cut into 4-μm-thick sections, and deparaffinized. The sections were then stained with primary monoclonal anti- bodies against COX-2 (1:100, clone CX-294, Dako, Glostrup, Denmark) and galectin-3 (1:100, NCL-GAL3, clone 9C4, No- vaCastra, Hamburg, Germany) using a Dako Cytomation Au- tostainer (Dako). After staining, each sample was evaluated under a light microscope (200×, Olympus BX53, Olympus, Tokyo, Japan) to determine the percentage of COX-2–positive cells, the intensity of COX-2 staining, and the percentage of galec- tin-3–positive cells. For positive controls, staining of breast car- cinoma tissue for COX-2 and papillary thyroid carcinoma tis- sue for galectin-3 were used. Primary monoclonal antibodies were omitted in negative controls. Assessment of COX-2 and galectin-3 staining Semi-quantitative analysis of immunostaining for COX-2 and galectin-3 was performed as follows based on the percentage of cells with positive cytoplasmic staining: 0%, 0; < 10%, 1; 10%– 50%, 2; 51%–80%, 3; and ≥ 80%, 4. In addition, staining in- tensity was evaluated as either negative (0), weak (1+), moderate (2+), or strong (3+). Semi-quantitative and intensity scores were analyzed separately. Additionally, the positivity of cells was evaluated as positive or negative.12 COX-2 and galectin-3 ex- pression was evaluated in glandular epithelial cells and stromal cells. Assessment of staining results was performed by one ob- server in a blinded fashion. Statistical analysis Statistical analysis was performed using software (Rstudio software ver. 0.98.501 via R language, R Studio Inc., Boston, MA, USA). Data describing continuous variables are presented as the mean ± standard deviation. The Kruskal-Wallis and Pear- son chi-square exact tests were used to compare continuous and categorical variables, respectively. Differences with p-values less than .05 were accepted as significant. http://jpatholtm.org/http://dx.doi.org/10.4132/jptm.2016.03.08 Cox-2 and Galectin-3 in Endometrial Polyps • 227

Patient demographics There was no statistically significant difference between the ages of healthy control individuals and patients with endome- trial polyps. COX-2 expression Immunoreactivity for COX-2 was observed in glandular epi- thelial cells and stromal cells. COX-2–positive cells were pre- dominantly observed in the endometrial glandular epithelium, where expression peaked during the secretory phase. COX- 2-positive cells were also observed in stromal cells, albeit to a lesser extent (Table 1, Fig. 1A). The percentage of COX-2–pos- itive cells and the intensity of COX-2 staining in stromal cells and glandular epithelial cells did not vary during different peri- ods of the menstrual cycle in the control group or in patients with endometrial polyps (Fig. 1B). Mean COX-2 scores in glandular epithelial cells and stromal cells were not significantly different between endometrial polyp specimens and normal en- dometrium specimens (Table 1). Galectin-3 expression Galectin-3 immunoreactivity was present in the endometrial glandular epithelial cells and stromal cells. Immunostaining was typically cytoplasmic. Galectin-3–positive cells were predomi- nantly observed in the endometrial glandular epithelium, where expression levels peaked during the secretory phase. Galectin-3 expression was also observed to a lesser extent in stromal cells (Table 2). The mean percentage score of galectin-3 expression were lower both in endometrial polyps and the proliferative phase in normal endometrium than in the secretory phase in normal endometrium (Table 2, Fig. 1C). In glandular epithelial cells, no statistically significant differences in galectin-3 expres- sion were found between endometrial polyps and normal endo- metrium during the proliferative phase. However, in patients with normal endometrium, galectin-3 expression was higher during the secretory phase (p = .349) (Fig. 1C). Finally, there Table 1. Percentages of COX-2–positive cells and intensity of COX-2 staining Proliferative phase Secretory phase Endometrial polyps p-value Glandular epithelial tissues COX-2 intensity 2.04 ± 1.02 2.07 ± 0.83 1.77 ± 1.11 .489 a 0–1 7 6 12 .398b 2–3 16 21 19 COX-2 percentage score 2.35 ± 1.12 2.67 ± 1.04 2.35 ± 1.38 .614 a 0–1 4 3 8 .351 b 2–4 19 24 23 Endometrial stroma COX-2 intensity 0.65 ± 0.71 0.70 ± 0.67 0.53 ± 0.68 .564 a 0–1 20 24 27 .941 b 2–3 3 3 3 COX-2 percentage score 0.61 ± 0.65 0.81 ± 0.78 0.61 ± 0.76 .525 a 0–1 21 21 26 .430 b 2–4 2 6 5 Values are presented as mean ± standard deviation. COX, cyclooxgenase. ap-value for Kruskal-Wallis tests, bp-value for Pearson chi-square test. A B C Fig. 1. Expression of cyclooxgenase 2 (COX-2) and galectin-3 in endometrium samples. (A) COX-2 expression in both the glandular epitheli- um and stroma during the secretory phase. (B) COX-2 expression in both the glandular epithelium and stroma of an endometrial polyp. (C) Galectin-3 expression in both the glandular epithelium and stroma during the secretory phase. http://jpatholtm.org/ http://dx.doi.org/10.4132/jptm.2016.03.08 228 • Kasap E, et al. were no differences in galectin-3 expression in stromal cells of en- dometrial polyps and those of the endometrium at any phase of the menstrual cycle (Table 2).

In this study, we compared galectin-3 and COX-2 expression and staining patterns using immunohistochemistry in endome- trial polyps and normal endometrium during the secretory and proliferative phases. Our data indicated that both galectin-3 and COX-2 were not associated with the formation of endometrial polyps. Estrogen is known to play a pivotal role in the pathogenesis of endometrial malignant and benign cancers. However, the role of estrogen biosynthesis from stromal cells of the endometrium and its impact on malignancies has not been fully elucidated. Interestingly, estrogens are known to activate COX-2 in endo- metriosis, which increases levels of estradiol and prostaglandin above normal in women.13 Therefore, the pathogenesis of peri- toneal endometriosis may be a consequence of enhanced prosta- glandins in the eutopic endometrium that stimulate COX-2 ex- pression and activity.14,15 This mechanism may also affect other endometrial pathologies, although no specific roles have been described. COX-2 is expressed at higher levels in the glandular epithelial cells of the endometrium during the secretory phase of the men- strual cycle compared with the proliferative phase; however, its expression is similar during the secretory and proliferative phases in endometrial stromal cells.16 In the present study, we observed that COX-2 expression tended to be higher during the secreto- ry phase than in either the proliferative phase or endometrial polyps in glandular epithelial cells. Some studies have suggested that endometrial polyps may originate from endometrial inflammation.17 After demonstrating that COX-2 is expressed in the epithelial lining of postmeno- pausal polyps, Maia et al.18 concluded that COX-2 may be in- volved in the regulation of polyp growth. Nevertheless, COX-2 expression has not been shown to differ significantly between normal endometrial tissue and endometrial polyps.19 In 2006, Maia and colleagues compared COX-2 expression between endometrial polyps and normal endometrium in wom- en with a regular menstrual cycle and found no significant dif- ference in COX-2 expression between polyps and the normal endometrium.19 In addition, Pinheiro et al.20 observed higher COX-2 expression in the glandular epithelium of obese women than in women of normal weight. This finding indicates that ex- pression of COX-2 expression may be influenced by metabolic changes and growth factors associated with obesity.20 Here, we also demonstrated that COX-2 expression in the glandular epi- thelium and stromal cells in endometrial polyps did not differ significantly from that of normal endometrium. Thus, our data suggest that COX-2 expression may not be associated with the formation of endometrial polyps. We previously reported that expression of galectin-3 is aug- mented during the formation of the receptive endometrium and the mid-secretory phase of the menstrual cycle.21 In addition, there is mounting evidence for a strong relationship between embryo implantation and tumor invasion and metastasis. Inter- estingly, galectin-3 has been reported to have anti-apoptotic po- tential in tumor cells, although there is no evidence for the role of galectin-3 in endometrial cell apoptosis. Thus, we hypothe- size that galectin-3 functions as an anti-apoptotic factor in the endometrium and may facilitate the development of endome- trial polyps. Chiu et al.22 found that immunohistochemical analysis of ga- lectin-3 protein expression is a sensitive, specific, and accurate marker for the diagnosis of thyroid cancer and certain other can- cers.23 In endometriosis, Noel et al.24 found that galectin-3 pro- tein expression, as measured using immunohistochemistry, is higher in endometriosis samples compared to eutopic endome- trium, and also higher in the eutopic endometrium of women with endometriosis compared to those without endometriosis. Together, these data suggest that galectin-3 may have a poten- tial role in the development of endometriosis.24 In a study by Brustmann et al.,25 galectin-3 expression was assessed by immu- nohistochemistry in patients with normal endometrium, simple hyperplasia, complex hyperplasia without atypia, atypical hy- perplasia, endometrioid carcinoma, serous papillary carcinoma, and clear cell carcinoma. They showed that in normal endometri- um, simple hyperplasia, and complex hyperplasia without atyp- Table 2. Percentages of galectin-3–positive cells Proliferative phase Secretory phase Endometrial polyp p-value Galectin-3 glandular epithelial tissues (% score) 1.35 ± 1.19 2.19 ± 1.00 1.71 ± 1.16 .039 a Galectin-3 endometrial stroma (% score) 1.43 ± 1.12 1.63 ± 1.15 1.19 ± 0.87 .349 a Values are presented as mean ± standard deviation. ap-value for Kruskal-Wallis tests. http://jpatholtm.org/http://dx.doi.org/10.4132/jptm.2016.03.08 Cox-2 and Galectin-3 in Endometrial Polyps • 229 ia, the galectin-3 expression did not differ significantly among these groups. Immunohistochemical expression of galectin-3 for atypical hyperplasia and endometrioid carcinoma were signifi- cantly higher than those in the aforementioned groups. Serous papillary carcinoma and clear cell carcinoma, which are known to have a poorer prognosis than endometrioid carcinoma,26 also demonstrated significantly elevated galectin-3 expression levels in this study. On the basis of these findings, we assumed that ga- lectin-3 expression may be related to the different biological be- haviors of endometrialcarcinomas.25 Importantly, while there was no difference in galectin-3 expression in glandular epitheli- um and stromal cells of endometrial polyps and normal endo- metrium, our data demonstrated that galectin-3 expression was significantly lower in both endometrial polyps and the prolifer- ative phase of the endometrium than in the secretory phase of the endometrium. This finding suggests that larger-scale stud- ies are needed to measure galectin-3 levels in secretory adeno- carcinomas, a subgroup of endometrioid adenocarcinomas, and in other endometrial cancers. In analyzing our results, we wondered whether galectin-3 ex- pression may be regulated by estrogen and progesterone. Al- though previous reports have shown that estrogen and/or pro- gesterone increases galectin-3 expression in endometrial cells at all concentrations,27 we did not observe any direct effect of these hormones on galectin-3 expression, suggesting that the rela- tionship may be indirect. Thus, further studies are needed to de- termine the exact nature of the relationship of estrogen and pro- gesterone on galectin-3 expression. The small sample size was a limitation of the present study. Therefore, the results of this study must be confirmed in larger longitudinal population studies. In summary, our data suggested that endometrial polyps may form through certain common pathways that do not involve COX-2 and galectin-3. While COX-2 and galectin-3, in addi- tion to angiogenesis, are known to be involved in different steps of carcinogenesis,28 these two target proteins may not be involved in the formation of endometrial polyps. Further large scale stud- ies comparing adjacent endometrial tissue and healthy women at specific phases of the menstrual cycle should be performed. Conflicts of Interest No potential conflict of interest relevant to this article was reported.

1. Neto LC, Soares JM Jr, Giusa-Chiferi MG, Gonçalves WJ, Baracat EC. Expression of p53 protein in the endometrial polyp in post- menopausal women. Eur J Gynaecol Oncol 2013; 34: 509-12. 2. Clevenger-Hoeft M, Syrop CH, Stovall DW, Van Voorhis BJ. Sono- hysterography in premenopausal women with and without abnor- mal bleeding. Obstet Gynecol 1999; 94: 516-20. 3. Droegemueller W. Benign gynecologic lesions. In: Stenchever MA, Droegemueller W, Herbst AL, Mishell DR Jr, eds. Comprehensive gynecology. 5th ed. St. Louis: Mosby Inc., 2001; 440-92. 4. Inceboz US, Nese N, Uyar Y, et al. Hormone receptor expressions and proliferation markers in postmenopausal endometrial polyps. Gynecol Obstet Invest 2006; 61: 24-8. 5. Sant’Ana de Almeida EC, Nogueira AA, Candido dos Reis FJ, Zambelli Ramalho LN, Zucoloto S. Immunohistochemical expres- sion of estrogen and progesterone receptors in endometrial polyps and adjacent endometrium in postmenopausal women. Maturitas 2004; 49: 229-33. 6. Kendall RL, Wang G, Thomas KA. Identification of a natural solu- ble form of the vascular endothelial growth factor receptor, FLT-1, and its heterodimerization with KDR. Biochem Biophys Res Com- mun 1996; 226: 324-8. 7. Lee J, Moon C, Kim J, et al. Immunohistochemical localization of galectin-3 in the granulomatous lesions of paratuberculosis-infect- ed bovine intestine. J Vet Sci 2009; 10: 177-80. 8. Sakaki M, Fukumori T, Fukawa T, et al. Clinical significance of ga- lectin-3 in clear cell renal cell carcinoma. J Med Invest 2010; 57: 152-7. 9. Topcu HO, Erkaya S, Guzel AI, et al. Risk factors for endometrial hyperplasia concomitant endometrial polyps in pre- and post- menopausal women. Asian Pac J Cancer Prev 2014; 15: 5423-5. 10. Costa-Paiva L, Godoy CE Jr, Antunes A Jr, Caseiro JD, Arthuso M, Pinto-Neto AM. Risk of malignancy in endometrial polyps in pre- menopausal and postmenopausal women according to clinicopath- ologic characteristics. Menopause 2011; 18: 1278-82. 11. Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Fertil Steril 1950; 1: 3-25. 12. Harrington DJ, Lessey BA, Rai V , et al. Tenascin is differentially ex- pressed in endometrium and endometriosis. J Pathol 1999; 187: 242-8. 13. Maia H Jr, Maltez A, Studard E, Zausner B, Athayde C, Coutinho E. Effect of the menstrual cycle and oral contraceptives on cyclooxy- genase-2 expression in the endometrium. Gynecol Endocrinol 2005; 21: 57-61. 14. Erdemoglu E, Güney M, Karahan N, Mungan T. Expression of cy- clooxygenase-2, matrix metalloproteinase-2 and matrix metallo- http://jpatholtm.org/ http://dx.doi.org/10.4132/jptm.2016.03.08 230 • Kasap E, et al. proteinase-9 in premenopausal and postmenopausal endometrial polyps. Maturitas 2008; 59: 268-74. 15. Bulun SE, Zeitoun KM, Takayama K, Sasano H. Estrogen biosyn- thesis in endometriosis: molecular basis and clinical relevance. J Mol Endocrinol 2000; 25: 35-42. 16. Wang D, Chen Q, Zhang C, Ren F, Li T. DNA hypomethylation of the COX-2 gene promoter is associated with up-regulation of its mRNA expression in eutopic endometrium of endometriosis. Eur J Med Res 2012; 17: 12. 17. Carvalho FM, Aguiar FN, Tomioka R, de Oliveira RM, Frantz N, Ueno J. Functional endometrial polyps in infertile asymptomatic patients: a possible evolution of vascular changes secondary to en- dometritis. Eur J Obstet Gynecol Reprod Biol 2013; 170: 152-6. 18. Maia H Jr, Correia T, Freitas LA, Athayde C, Coutinho E. Cyclooxy- genase-2 expression in endometrial polyps during menopause. Gynecol Endocrinol 2005; 21: 336-9. 19. Maia H Jr, Pimentel K, Silva TM, et al. Aromatase and cyclooxygen- ase-2 expression in endometrial polyps during the menstrual cycle. Gynecol Endocrinol 2006; 22: 219-24. 20. Pinheiro A, Antunes A Jr, Andrade L, De Brot L, Pinto-Neto AM, Costa-Paiva L. Expression of hormone receptors, Bcl2, Cox2 and Ki67 in benign endometrial polyps and their association with obe- sity. Mol Med Rep 2014; 9: 2335-41. 21. Du GP , Zhang W, Wang L, Liu YK, Zhou JP . Expression of galectin-3 in human endometrium. Fudan Univ J Med Sci 2006; 2: 143-6. 22. Chiu CG, Strugnell SS, Griffith OL, et al. Diagnostic utility of galec- tin-3 in thyroid cancer. Am J Pathol 2010; 176: 2067-81. 23. Cay T. Immunhistochemical expression of galectin-3 in cancer: a review of the literature. Turk Patoloji Derg 2012; 28: 1-10. 24. Nöel JC, Chapron C, Borghese B, Fayt I, Anaf V . Galectin-3 is over- expressed in various forms of endometriosis. Appl Immunohisto- chem Mol Morphol 2011; 19: 253-7. 25. Brustmann H, Riss D, Naudé S. Galectin-3 expression in normal, hyperplastic, and neoplastic endometrial tissues. Pathol Res Pract 2003; 199: 151-8. 26. Silverberg SG, Kurman RJ. Atlas of tumor pathology, 3rd series. Fascicle 3. Tumors of the uterine corpus and gestational trophoblas- tic disease. Washington, DC: Armed Forces Institute of Pathology, 1992. 27. Yang H, Lei C, Cheng C, et al. The antiapoptotic effect of galectin-3 in human endometrial cells under the regulation of estrogen and progesterone. Biol Reprod 2012; 87: 39. 28. Kumar V , Abbas AK, Fausto N. Robbins and Cotran: pathologic ba- sis of disease. 7th ed. Philadelphia: Elsevier Saunders, 2005; 269- 342.