Endometriotic Mesenchymal Stem Cells Significantly Promote Fibrogenesis In Ovarian Endometrioma Through The Wnt/?-catenin Pathway By Paracrine Production Of TGF-?1 And Wntl

Backround and Objective Endometriosis is histologically characterized by ectopic outgrowth of endometrial stroma and glands surrounded by dense fibrous tissues.Laparoscopy and pathological diagnosis is required for diagnosis of endometriosis.The understanding of the pathophysiology and cause of endometriosis is limited.The most widely accepted theory was proposed by Sampson,who suggested that endometriosis results from retrograde menstruation.It has been reported that up to 90%of women may experience some degree of retrograde menstruation yet not all develop endometriosis,which has led to the hypothesis that immune factors play an important role in the pathogenesis of endometriosis.The stem cell theory is a new hypothesis which may clarify the underlying pathophysiologic mechanisms of endometriosis.Stem cell is active.The secretion of paracrine factors is rich.It is involved in endometrial regeneration and differentiation.The dysregulation of endometriotic stem cells has been proposed as a potential mechanism for seeding ectopic endometriotic lesions.The role of stem cell in pathogenesis of endometriosis is still unknown.Excess fibrosis in endometriotic lesions may lead to pain and scarring,altered tissue functions and affect the quality of life.Medical treatments to inhibit the hormone can not heal the disease.Because of the severe fibrosis,endometriotic cysts of ovarian endometrioma have a detrimental effect on the ovarian reserve.The loss of ovarian parenchyma at the time of surgery is evident.The pathogenesis of endometriosis,especially ovarian endometrioma-associated fibrosis,is still unknown.Lots of cytokines influence the fibrogenic process.Stem cell release plenty of cytokines.The role of Ecto-MSCs in the pathogenesis of fibrosis in ovarian endometrioma need future study.Methods We analyzed endometrial samples from 15 patients of reproductive age with ovarian endometrioma and normal menstrual cycles.Endometrial and endometriotic mesenchymal cells(Euto-and Ecto-MSCs),Endometrial and endometriotic stromal cells(Euto-and Ecto-ESCs)were isolated.Conditioned medium(CM)was collected from Euto-MSCs and Ecto-MSCs.The effect of CM on cell proliferation,migration,invasion and collagen gel contraction of Euto-and Ecto-ESCs in ovarian endometrioma were evaluated by cell counting kit-8,transwell,and collagen gel contraction assays.Effects of CM on fibrotic markers' expression in Euto-and Ecto-ESCs were determined by real-time RT-PCR and western blot(WB).A total of 54 nude mice received a single injection of proliferative endometrial fragments from 14 individuals without endometriosis.Subcutaneous injection of cytokines and CM was employed as the administration route to investigate the effect on fibrogenesis in the animal mode.Results Our results demonstrated that Ecto-MSC CM significantly promoted cell proliferation,migration,invasion and collagen gel contraction of Euto-and Ecto-ESCs from patients with ovarian endometrioma compared to control and Euto-MSC CM.Expression levels of fibrotic markers in Euto-and Ecto-ESCs were dramatically elevated after treatment with Ecto-MSC CM.Ecto-MSCs secreted higher levels of TGF-?1 and Wntl compared with Euto-MSCs.Furthermore,both TGF-?1 and Wntl significantly increased expression of fibrotic markers in Euto-and Ecto-ESCs,which was reversed by an anti-TGF-?1 antibody or Wntl negative regulator,dickkopf-related protein 1(Dkk1).Mechanistic studies demonstrated that Wnt/?-catenin signaling pathways in stromal cells were activated by Ecto-MSC CM.Additionally,fibrogenic effects of Ecto-MSC CM treatment on endometriotic implants were analyzed using a xenograft model of endometriosis in immunodeficient nude mice.Animal experiments showed that TGF-?1 and Wntl as well as Ecto-MSC CM markedly enhanced progression of fibrosis in endometriosis.Conclusion The in vitro results demonstrated that Ecto-MSCs promoted fibrogenesis in ESCs through the Wnt/?-catenin pathway by paracrine production of TGF-?1 and Wntl.The in vivo experiments showed that treatment with Ecto-MSC promoted the progression of fibrosis during the development of endometriosis.In conclusion,Ecto-MSCs may be involved in the pathogenesis of fibrosis in ovarian endometrioma.