Session 60: Endometrium, endometriosis

Session 60: Endometrium, endometriosisIntroduction: Endometriosis, the presence of endometrium outside the uterine cavity, affects 6–10% of women during their reproductive life [1]. It is one of the most common benign gynaecological diseases however, little is known about its pathogenesis. A postulated theory on the aetiology of endometriosis is that viable endometrial cells reach the peritoneal cavity through retrograde menstruation, adhere and form endometriotic lesions. Endometriosis is an estrogen dependent disorder, though the relation between steroids and its pathogenesis remains unclear. Recently stem/progenitor cells have been identified in the endometrium [2, 3]. They exhibit clonogenic activity and have high selfrenewal capability. Therefore, we hypothesize that a reservoir of endometrial epithelial and stromal stem/progenitor cells may contribute to the development of endometriotic lesions. The aims of this study were to 1) determine the clonogenic capacity of human endometrial epithelial and stromal cells from ovarian endometriotic lesions, 2) assess the self renewal capacity of endometriotic clonogenic cells and, 3) examine the cellular composition of the endometriotic clones using phenotypic markers. Materials and Methods: Ovarian endometriotic cysts were obtained from 19 women undergoing laparoscopy. The lesions were dissociated with collagenase to achieve single cell suspensions. Epithelial and stromal cells were separated using Ber-EP4 Dynabeads and cultured at low seeding density (500 cells/cm2) for 21 days. Colonies were stained with hematoxylin to determine the cloning efficiencies (CE) and some individual clones were serially subcloned every 3 –6 weeks to determine self renewal capacity. Immunohistochemistry was performed with: anti-human fibroblast (CD90), anti-human epithelial antigen (Ber-EP4, CD49f), anti-human endometriotic stromal marker (CD10) and estrogen receptor-a (ER-a). Results: Two types of colonies were observed: small loosely packed colonies containing large cells and large colonies with small, densely packed cells. The total CE for epithelial endometriotic cells was 2.92+2.49% (n ¼ 19); 1.70+1.47% for large and 1.22+1.02% for small colonies. Whereas, the total CE for stromal endometriotic cells was 4.67+2.71% (n ¼ 19); 1.54+0.92% for large and 3.13+1.80% for small colonies. There was no significant difference between the two cell types. Single cells derived from large epithelial and stromal endometriotic clones could be subcloned four times more when compared to those from small clones (n ¼ 5). Small epithelial colonies were positive for epithelial marker and negative for the fibroblast marker. However, large colonies were negative for BerEP4 and CD49f suggesting these epithelial markers are lost during in vitro proliferation. Both large and small stromal colonies were positive for CD90 and CD10 and were negative for epithelial markers. Interestingly, both large epithelial and stromal endometriotic clones possessed ER-a immunoreactivity. Conclusions: These results suggest that a small population of cells with stemlike cell properties of clonogenic and self renewal activity may be responsible for the development and progression in endometriosis. References Wheeler, J.M., Epidemiology of endometriosis-associated infertility. J Reprod Med, 1989. 34(1): p. 41–6. Chan, R.W., K.E. Schwab, and C.E. Gargett, Clonogenicity of human endometrial epithelial and stromal cells. Biol Reprod, 2004. 70(6): p. 1738–50. Chan, R.W. and C.E. Gargett, Identification of label-retaining cells in mouse endometrium. Stem Cells, 2006. 24(6): p. 1529–38.link_to_OA_fulltextabstrac