Differential Expression of CRH, UCN, CRHR1 and CRHR2 in Eutopic and Ectopic Endometrium of Women with Endometriosis.

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

Galectin-1 overexpression in endometriosis and its regulation by neuropeptides (CRH, UCN) indicating its important role in reproduction and inflammation.

Endometriosis is an inflammatory disease of women of reproductive age featured by the presence of ectopic endometrium and is strongly related to infertility. Galectins, carbonhydrate-binding proteins, have been found to have pro- or anti-inflammatory roles in the reproductive tract and in pathological conditions concerning infertility. Galectin-1, which is expressed at endometrium and decidua, plays a major role in implantation and trophoblast invasion. Also, the neuropeptides, corticotropin releasing hormone (CRH) and urocortin (UCN) and their receptors are expressed in eutopic and ectopic endometrium showing a differential expression pattern in endometriotic women compared to healthy ones. The aim of this study was to examine the galectin-1 expression in endometriotic lesions and compare its expression in eutopic endometrium of endometriotic and healthy women. Furthermore, we examined the effect of CRH and UCN in galectin-1 expression in Ishikawa cell line and macrophages and investigated the implication of CRHR1 in these responses. Eutopic and ectopic endometrium specimens, Ishikawa cell line and mice macrophages were used. Immunohistochemistry and western blot analysis were performed in order to identify galectin-1 expression in ectopic and eutopic endometrium of women with and without endometriosis and the regulatory effect of CRH and UCN on galectin-1 expression. This study presents for the first time that galectin-1 is overexpressed in endometriotic lesions compared to eutopic endometrium of endometriotic women and is more abundantly expressed in eutopic endometrium of disease women compared to healthy ones. Furthermore, it is shown that CRH and UCN upregulate galectin-1 expression in Ishikawa cell line and macrophages and this effect is mediated through CRHR1. These results suggest that galectin-1 might play an important role in endometriosis pathology and infertility profile of women suffering from endometriosis by being at the same time regulated by CRH and UCN interfering in the immune disequilibrium which characterizes this pathological condition

Galectin-1 is upregulated in macrophages upon CRH and UCN stimulation and this is mediated through CRHR1.

<p>Western blot immunodetection of galectin-1 (14 kDa) protein expression in macrophages, regulated by CRH and UCN neuropeptides. GAPDH (37 KDa) was used as a house keeping gene. <b>3a:</b> In mice macrophages, galectin-1 expression is upregulated by CRH mostly at 24 hrs and Antalarmin effect is stronger at 24 hrs. <b>3b:</b> In mice macrophages, galectin-1 expression is mostly upregulated by UCN at 24 hrs.</p

Galectin-1 overexpression in late secretory phase of eutopic endometrium and in endometriotic sites.

<p><b>1a:</b> Immunohistochemical expression of galectin-1 expression through the different phases of the menstrual cycle of the endometrium (A: proliferative, B: early secretory phase, C: late secretory phase) showing a higher expression pattern at the late secretory phase of the endometrium(C), *p<0.05. <b>1b:</b> Western blot immunodetection of galectin-1 (14 kDa) protein expression in eutopic and ectopic endometrium. Galectin-1 is overexpressed in eutopic endometrium of endometriotic women (ee) when compared to eutopic endometrium of healthy women (he), *p<0.05 and galectin-1 shows an abundant expression in ectopic endometrium (e) when compared to eutopic endometrium of the same endometriotic women (ee), **p<0.01. GAPDH (37 KDa) was used as a house keeping gene.</p

IRS Score results.

<p>We have found that 7/16(43,75%) ectopic endometrium samples showed strong CRH expression and 9/16(69,2%) ectopic endometrium samples showed moderate CRH expression compared to 2/16(12,5%) eutopic endometrium samples of strong CRH expression and 14/16(87,5%) eutopic endometrium samples of moderate CRH expression. Concerning the UCN expression in eutopic and ectopic endometium of the same patients, we have found that 5/16(31,25%) ectopic endometrium samples showed strong UCN expression and 11/16(68,75%) ectopic endometrium samples showed moderate UCN expression compared to 1/16(6,25%) eutopic endometrium samples of strong UCN expression and 15/16(93,75%) eutopic endometrium samples of moderate UCN expression.</p

Galectin-1 is upregulated in Ishikawa cell line upon CRH and UCN stimulation and this is mediated through CRHR1.

<p>Western blot immunodetection of galectin-1 (14 kDa) protein expression in Ishikawa cell line, regulated by CRH and UCN neuropeptides. GAPDH (37 KDa) was used as a house keeping gene. <b>2a:</b> In Ishikawa cell line, galectin-1 expression is upregulated by CRH, showing a higher expression at 8 hrs and 24 hrs of stimulation. When antalarmin is added, galectin-1 shows an impaired expression mostly at 24 hrs of stimulation. <b>2b:</b> In Ishikawa cell line, galectin-1 is upregulated by UCN, showing a higher expression at 8 hrs of stimulation.</p

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

<p>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).</p