Differential expression of CRH, UCN, CRHR1 and CRHR2 in eutopic and ectopic endometrium of women with endometriosis

Abstract

Endometriosis is considered as a benign aseptic inflammatory disease, characterised by the presence of ectopic endometrium-like tissue. Its symptoms (mostly pain and infertility) are reported as constant stressors. Corticotropin releasing hormone (CRH) and urocortin (UCN) are neuropeptides, strongly related to stress and inflammation. The effects of CRH and UCN are mediated through CRHR1 and CRHR2 receptors which are implicated in several reproductive functions acting as inflammatory components. However, the involvement of these molecules to endometriosis remains unknown. The aim of this study was to examine the expression of CRHR1 and CRHR2 in endometriotic sites and to compare the expression of CRHR1 and CRHR2 in eutopic endometrium of endometriotic women to that of healthy women. We further compared the expression of CRH, UCN, CRHR1 and CRHR2 in ectopic endometrium to that in eutopic endometrium of women with endometriosis. Endometrial biopsy specimens were taken from healthy women (10 patients) and endometrial and endometriotic biopsy specimens were taken from women with endometriosis (16 patients). Τhe expression of CRH, UCN, CRHR1, and CRHR2 was tested via RT-PCR, immunohistochemistry and Western blotting. This study shows for the first time that CRH and UCN receptor subtypes CRHR1β and CRHR2α are expressed in endometriotic sites and that they are more strongly expressed (p<0.01) in eutopic endometrium of women with endometriosis compared to healthy women endometrium at the mRNA and protein level. CRH, UCN, CRHR1 and CRHR2 mRNA were also more highly expressed in ectopic rather than eutopic endometrium (CRH, UCN, CRHR2α: p<0.01, CRHR1β: p<0.05) and protein (CRH and UCN: p<0.05, CRHR1 and CRHR2: p<0.01) in women with endometriosis. These data indicate that CRH and UCN might play an immunoregulatory role in endometriotic sites by affecting reproductive functions such as decidualization and implantation of women with endometriosis.

Figure 1. CRH, UCN, CRHR1 and CRHR2 expression in endometriotic sites.

A: CRH (413 bps), B: UCN(146 bps), C: CRHR1β (554 bps) and D: CRHR2α (322 bps) mRNA expression and CRHR1 (55 kDa) and CRHR2 (55 kDa) protein expression in endometriotic tissue(E) (1E, 1F respectively), having placental tissue (P) and myometrial tissue (M) as positive control, negative sample(-), GAPDH(G) house keeping gene. (representative data).

Figure 2. Higher CRHR1 and CRHR2 expression in eutopic endometrium of endometriotic women at an mRNA and protein level.

A: mRNA expression of CRHR1β(554 bps) and CRHR2α(322 bps) is higher in eutopic endometrium of endometriotic women(ee) compared to eutopic endometrium of healthy women(he). B: protein expression of CRHR1 (55 kDa) and CRHR2 (55 kDa) is higher in ee compared to he. (**p<0.01) (representative data).

Figure 3. CRH, UCN, CRHR1 and CRHR2 excessive expression in endometriotic sites compared to eutopic endometrium of endometriotic women.

3a) A: mRNA expression of CRH (413 bps), UCN (146 bps), CRHR1β (554 bps), CRHR2α (322 bps), GAPDH (483 bps) in endometriotic tissue (e) and the corresponding eutopic endometrium (ee). B: Immunohistochemical expression of CRH(A,D) and UCN(B,E) in eutopic(A,B) and ectopic endometrium(D,E). Both CRH and UCN are mainly expressed in endometriotic lesions.(C: negative eutopic endometrium, F: negative ectopic endometrium). C: Western blot immunodetection of CRHR1 (55 KDa) and CRHR2 (55 KDa), in endometriotic tissue(e) and eutopic endometrium(ee) of endometriotic women.GAPDH (37 kDa) used as a house keeping gene. CRH, UCN, CRHR1 and CRHR2α are significantly more expressed at an mRNA and protein level in endometriotic tissue compared to the corresponding eutopic endometrium. (representative data). 3b) Presentation of the normalized fold-increase of CRH, UCN, CRHR1 and CRHR2 expression in the study population at mRNA level(A) and protein level(B: CRH and UCN protein levels, C: CRHR1 and CRHR2 protein levels) (*, p<0.05, **,p<0.01).