Journal of Reproductive Immunology 112 (2015) 63–67 Contents lists available at ScienceDirect Journal of Reproductive Immunology jo u r n al homep age: www.elsevier.com/locate/jreprimm COX-2 gene promoter DNA methylation status in eutopic and ectopic endometrium of Egyptian women with endometriosis Haidy E. Zidan a,∗ , Noha A. Rezk a , Amr Abd Almohsen Alnemr b , Amany M. Abd el Ghany b a Medical Biochemistry Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt b Gynecology & Obstetrics Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt a r t i c l e i n f o Article history: Received 9 May 2015 Received in revised form 3 June 2015 Accepted 25 June 2015 Keywords: Endometriosis DNA hypomethylation COX-2 mRNA expression Epigenetics a b s t r a c t The pathophysiology of COX-2 expression in endometriosis is a matter of debate. The aim was to investi- gate the role of DNA methylation of the NF-IL6 site within the promoter of COX-2 gene in the pathogenesis of endometriosis. The endometrial tissues (ectopic and eutopic) were collected from 60 women with endometriosis and 30 women without endometriosis (control group). The methylation status of COX-2 was examined by methylation-specific PCR. Quantitative real-time PCR (RT-PCR) was performed to mea- sure COX-2 mRNA levels in endometrial tissues. We found significantly higher levels of COX-2 in ectopic endometriotic tissue compared with eutopic tissue. Also, we found that the frequencies of methylation status of the NF-IL6 site within the COX-2 promoter in the eutopic and ectopic endometrial tissues of endometriosis groups were significantly decreased in comparison to controls (P = 0.002, P = 0.000 respec- tively). Our study demonstrated that DNA hypomethylation of the NF-IL6 site within the promoter of COX-2 gene could be a key mechanism for its elevated expression in the eutopic and ectopic tissues of endometriosis. © 2015 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Endometriosis, a common gynecological disorder, is defined as the presence of ectopic endometrial tissue outside the uterine cav- ity. It affects about 6–10% of women of reproductive age (Giudice and Kao, 2004; Bulun, 2009). Up to now, the pathophysiology of endometriosis has remained unknown. A combination of various genetic predispositions, impaired immunological response, and an inflammatory component contribute to the etiology of the disease (Bischoff and Simpson, 2004; Podgaec et al., 2007; Fairbanks et al., 2009). Cyclooxygenase (COX) is the rate-limiting enzyme in the metabolic conversion of arachidonic acid to prostaglandins (PGs). Two isoforms of COX have been described: COX-1 is constitutively expressed in many tissues, while COX-2 is inducible by mitogen (Rupesh et al., 2010), growth factors (Kimba et al., 2005), and cytokines (Han et al., 2013). In normal eutopic endometrium, COX-2 is considered to be involved in cell proliferation, regeneration, and Abbreviations: COX-2, cyclooxygenase-2; PGs, prostaglandins; MS-PCR, methyl- ation specific polymerase chain reaction; NF-kB, nuclear factor-Kappa beta; NF-IL6, nuclear factor for interleukin-6 expression; CRE, cyclic AMP response element. ∗ Corresponding author. E-mail address: haidy menna@hotmail.com (H.E. Zidan). the promotion of angiogenesis (Chishima et al., 2002). Although many previous studies have reported the overexpression of COX-2 in endometriosis (Ota et al., 2001; Chishima et al., 2002; Howard, 2009), the pathophysiology of COX-2 expression in endometriosis is still not totally understood. Within the promoter region of the COX-2 gene, the nuclear factor for the interleukin-6 expression site (NF-IL6) is a CpG site that plays a role in the regulation of COX-2 expression (Yamamoto et al., 1995; Murata et al., 2004). A previous study reported that the hypermethylation of CpG islands in the COX-2 promoter region is a powerful mechanism for the suppression of gene activity by transcriptional silencing, and that the treatment with demethylat- ing agents effectively reactivated COX-2 expression (Song et al., 2001). On the basis of these considerations, we constructed this study to investigate whether or not there is a relationship between the hypomethylation of the NF-IL6 site within the promoter region of the COX-2 gene and the COX-2 mRNA level in ectopic and eutopic endometrium of Egyptian women with endometriosis, which could help with future diagnosis and in improving the treatment of this disease. 2. Subjects and methods Sixty women (mean age 32 ± 6.3 years), undergoing operative laparoscopic surgical service for endometriosis at the Department http://dx.doi.org/10.1016/j.jri.2015.06.093 0165-0378/© 2015 Elsevier Ireland Ltd. All rights reserved.