Effect Of Retinoid Acid Receptor Alpha On Recurrent Implantation Failure And Its Mechanism

The brief period of embryo implantation,also known as window of implantation,coincides with the midluteal phase.During this brief period,the environment and state of the endometrium transfer into the most suitable condition for embryo implantation.It is acknowledged that endometrium get the ability of receiving embryo implantation in a short time span,which is also called endometrial receptivity.Many studies have shown that endometrial receptivity disorder is associated with embryo implantation failure,early pregnancy loss,and some complications during pregnancy.RARA is an important gene involved in physiological processes such as development,differentiation,and proliferation.It is also involved in spermatogenesis,Müller tube differentiation,and other processes.Although the expression level of RARA in the mid-luteal phase lower than in the proliferative period,is there any expression difference in the endometrium of normal people and patients with recurrent implantation failure in the window of implantation.Until now,there are no related studies.Is the expression of RARA in RIF patients different from that in normal women? Which cell types of the endometrial is abnormally expressed? What effect does it have on cell function? What is the mechanism behind it? Those questions around RARA in endometrium are of great significance for finding the etiology of RIF.Chapter 1Expression of RARA gene and its protein RARα in the window of implantation of endometrium in patients with recurrent implantation failurePurpose: To investigate the expression of RARA gene and its protein RARα in the window of implantation of endometrium in RIF patients.Methods: Formulated endometrial sample collection criterion and collected the samples during window of implantation.The mRNA expression level of RARA gene in the endometrium were detected by qPCR.The expression level of RARα protein in the endometrium were detected via western blot.The localization and expression intensity of RARα in the window of implantation of endometrium in RIF patients were detected using immunohistochemistry.Results: The mRNA expression level of RARA in the endometrium of patients with RIF were significantly lower than that in the normal control group.The protein expression level of RARα in the endometrial of patients with RIF was significantly lower than that in the normal control group.In immunohistochemical detection,RARα was mainly expressed in the nucleus of endometrial stroma cells and the average optical density level of RARα in stromal cells of patients with RIF was significantly lower than that in the normal control group.Conclusion: The expression levels of RARA gene mRNA and its protein RARα in the endometrium of patients with RIF are significantly decreased.RARα is mainly expressed in the nucleus of endometrial stroma cells during the implantation window period.The expression level of RARα in endometrial stroma cells of RIF is significantly decreased.Chapter 2 The mechanism of RARA on decidualization in human endometrial stromal cellsPurpose: To investigate the mechanism of RARA on decidualization in human endometrial stromal cellsMethods: Knockdown of RARA using siRNA in the endometrial stroma cells and inducing decidualization in vitro for 4 days.The mRNA expression level of RARA and the decidualization markers PRL,IGFBP-1 and CEBPB were determined by qPCR.The protein expression of PRL and IGFBP-1 was used ELISA.The western blot was used to detect C/EBPβ protein levels.Knockdown of CEBPB using siRNA in the endometrial stroma cells and inducing decidualization in vitro for 4 days.The mRNA expression level of RARA and decidualization markers PRL and IGFBP-1 were detected by qPCR.The protein level of PRL and IGFBP-1 were detected by ELISA.CEBPB-overexpressing adenovirus was used to restore CEBPB expression in the RARA knockdown endometrial stroma undergoing decidualization.CEBPB gene was overexpressed in the endometrial stroma cells,and the mRNA and protein levels of PRL and IGFBP-1 were detected.ChIP was performed to determine whether RARα and RXRα enriched CEBPB promoter region in endometrial stroma cell that had induced decidualization for 4 days in vitro.To further determine the specific sequence of CEBPB promoter region that bind by RARα and RXRα,dual-luciferase experiments were performed.Knockdown of RXRA using siRNA in the endometrial stroma cells and inducing decidualization in vitro for 4 days.The CEBPB transcription level was determined by qPCR.The changes of CEBPB mRNA level in RARA knockdown endometrial stroma cells with cAMP,MPA,cAMP + MPA respectively were detected via qPCR.The expression level of C/EBPβ protein in the endometrium of RIF were detected via western blot.The localization and expression intensity of C/EBPβ in the window of implantation of endometrium in RIF patients were detected using immunohistochemistry.Results: RARA knockdown in endometrial stroma cells significantly decreased mRNA and protein levels of PRL and IGFBP-1 after 4 days in vitro decidualization.In decidualized endometrial stromal cells,RARA knockdown decreased CEBPB and its Protein C/EBPβ expression level.CEBPB knockdown in endometrial stroma cells significantly decreased mRNA and protein levels of PRL and IGFBP-1 after 4 days in vitro decidualization.In ChIP experiment,we found that RARα and RXRα were able to enrich CEBPB chromatin sequence of promoter region(-2009 /-1993).RXRA knockdown in decidualized HESCs did not affect CEBPB transcription level.RARA knockdown did not affect CEBPB transcription level in non-decidualized state,and RARA participated in CEBPB transcription only when stroma cells were induced decidualization by the addition of cAMP and MPA.The protein level of C/EBPβ in endometrium of RIF was lower than normal group.In immunohistochemical detection,C/EBPβ was found mainly expressed in the nucleus of endometrial stroma cells and the average optical density level of C/EBPβ in stromal cells of patients with RIF significantly lower than that in the normal control group.Conclusion: Under the presence of increased progesterone and cAMP,RARα binds CEBPB(-2009 /-1993)to regulate the transcription of CEBPB in the endometrial stroma cells to affect its decidualization.RARA does not participate in the regulation of CEBPB transcription without progesterone and cAMP.Chapter 3 The mechanism of RARA on the migration in human endometrial stromal cellsPurpose: To investigate the mechanism of RARA on the migration in human endometrial stromal cellsMethods: Knockdown of RARA using siRNA in the endometrial stroma cells and inducing decidualization in vitro for 4 days.The wound healing and Transwell experiments were performed to measure the effect on migration.The changes of HOXA10 after RARA knockdown in endometrial stroma cells were determined via qPCR and western blot.Knockdown of HOXA10 using siRNA in the endometrial stroma cells and inducing decidualization in vitro for 4 days,and the wound healing and Transwell experiments were performed to measure the effect on migration.The expression level of HOXA10 protein in the endometrium were detected via western blot.The localization and expression intensity of HOXA10 in the window of implantation of endometrium in RIF patients were detected using immunohistochemistry.Results: knockdown of RARA reduced the ability of migration function of decidualized stroma cells.In decidualized endometrial stroma cells,knockdown of RARA decreased HOXA10 mRNA and protein expression level.knockdown of HOXA10 reduced the ability of migration function of decidualized stroma cells.In immunohistochemical detection,it was found that HOXA10 was mainly expressed in the nucleus of endometrial stroma cells and the average optical density level of HOXA10 in stromal cells of patients with RIF was significantly lower than that in the normal control group.Conclusion: Knockdown of RARA impaired the ability of migration of decidualized stroma cells through decreasing HOXA10 expression.Chapter 4 Effects of RARA,HOXA10,CEBPB on proliferation in endometrial stromal cellsPurpose: To investigate the effects of RARA,HOXA10,CEBPB on proliferation in human endometrial stromal cellsMethods: The effects of RARA knockdown on proliferation of endometrial stroma cells were determined by Ed U and CCK8.The effects of CEBPB,HOXA10 knockdown on proliferation of endometrial stroma cells were determined by Ed U and PCNA;qPCR and western blot were performed to detect effect of knocking down CEBPB and RARA on the expression level of HOXA10 mRNA and protein.Results: After RARA knockdown,the proliferation of endometrial stroma cell decreased as well as HOXA10 expression level.HOXA10 knockdown impaired the proliferation of endometrial stroma cells.CEBPB knockdown decreased the proliferation of endometrial stroma cells as well as HOXA10 expression level.Conclusion: The decreased expression of RARA,HOXA10,and CEBPB in endometrial stroma cells compromised its proliferation,and the decreased expression of RARA and CEBPB decrease HOXA10 expression.