Sonic Hedgehog Signaling Pathway In Endometrium And Its Role In Intrauterine Adhesions

Background:Intrauterine Adheions is reported by Fritsh in 1894 firstly and described by Asherman again in 1948,which ultimately leads to menstrual abnormalities(hypomenorrhea or amenorrhea)?pelvic pain,infertility and habitual abortion,impacting adversely on female physical and mental health and drawing extensive concerns.Transcervical resection of adhesion combined with postoperative therapy is the uppermost treatment to protocols.While there is no unified definition about the clinical application of postoperative therapies,the mainstream is the IUD placement and sodium hyaluronate which can prevent the postoperative re-adhesion.And Periodic treatment of estrogen and progestogen has some actions for repairing endometrium.however,the recurrence rate of severe intrauterine adhesion is 20.0%?62.5%,it faces to dig out some effective treatment methods for repairing endometriumurgently.t has been identified that the tissue injury is the specific risk factor and it is possible that,after an injury to the endometrium,fibrosis may follow with the potential for adhesion formation.Several signal pathways have been identitied to participate in the development of intrauterine adhesion.Hh signaling pathway is one of the most important intracellular pathways,which take part in formation,differentiation and proliferation of embryo development.Previous studies indicated that Shh signaling pathway may play an important role in the development of the disease.The objective of this study was to investigate the Shh signaling pathway's expression in endometrium and its role in intrauterine adhesion,to explore the correlation between the endometrium of IUA patients and non-IUA specimens' expressions and the occurrence of intrauterine adhesion and to to find the theoretical foundation for the effective treatment with a predominance of hysteroscopic operations in the management of IUA.Methods:Endometrial samples from 20 patients who accept TCRA to diagnosis of moderate to severe IUA were selected from Sir Run Run Shaw Hospital from March 2017 to June 2017.The endometrial and scar samples were collected by TCRA to make paraffin section and take immunohistochemistry and Western Blot detection.Meantime,20 patients demanded for TCRA with other reasons found as normal endometrium,10 specimens endometrium at the proliferative stages and other 10 specimens' at secretory stages as the control group.In this study,the determination of location and expression levels of Shh protein and Ptch/Smo/Gli2/Gli3 protein was performed on the endometrium at the proliferative and secretory stages of endometrium.Then to investigate and compare the location and expression levels of Collagen I,E-cadherin,Ki67 protein and Gli2 protein in the endometrium of IUA patients and non-IUA specimens.At last,for interference the expression of Gli2,to construction over-expressed plasmid Gli2 interference vector,use Lipofectamine2000 to transfer human endometrial stromal cells.Physical methods were treated to transfer human endometrial stromal cells to simulating uterine curettage.The Serum-Free DMEM/F12 Culture Medium?Shh pathway inhibitor cyclopamine(Smo-dependent)and recombinant ssh(rShh)were added into the cells as a comparison,to research the role of Shh signaling pathway in proliferation of human endometrial stromal cells and the transformation of fibrosis.Results:In the proliferative stages of endometrium,the expression of Shh protein is moderate,however Smo?Gli2?Gli3 protein shows lower and Ptch protein is no positive results.In the secretory stages of endometrium,the expression of Shh protein is moderate and ptch.Smo protein is lower,however Gli2,Gli3 protein shows strong positive results.The expression of Collagen I,E-cadherin,Ki67 protein in IUAs group was significantly upregulated compared to the control group and the levels of Gli2 in the IUAs group were also increased significantly than those in the control group,while no or low expression in normal endometrium.After physical methods treated to endometrial stromal cells which is constructed over-expressed plasmid Gli2 interference vector,analyzed by western blot,it shows the levels of Collagen I and Gli2 increased.Meanwhile simulating uterine curettage,the proliferation of HESC was significantly increased after rSHH treatment,while it was significantly decreased after cyclopamine treatment.Conclusion:Shh signaling pathway was performed on the endometrium at the proliferative and secretory stages of endometrium,however,the expression is more obvious at secretory stages of endometrium.Endometrial samples from moderate and severe IUA patients exhibited significantly enhanced expression of Collagen I,E-cadherin,Ki67,Gli2 protein compared with normal endometrial samples.The conclusion is that intrauterine adhesions is the results of fibrosis,epitheliumabnomal growth and oral dysplasia.Maybe Shh signaling pathway play important role in the development of intrauterine adhesions and the inhibition of through Shh may be of therapeutic value for delaying the development of intrauterine adhesions.