Deficiency of RARa Suppresses Decidualization via Downregulating CEBPB Transcription in Women With Recurrent Implantation Failure

Background: Recurrent implantation failure (RIF) is a disease associated with endometrial receptivity dysfunction. Retinoic acid receptor alpha (RAR alpha) is an important protein in many biological processes, such as differentiation and development. However, the exact underlying mechanism whereby RAR alpha affects RIF remains unknown. This study investigated RAR alpha expression and its contribution in the mid-luteal phase endometria of patients with RIF. Methods: The expression levels of RAR alpha and CCAAT/enhancer-binding protein (C/EBP) beta in the endometria of the RIF and normal group were investigated using western blotting and immunohistochemistry. In in vitro experiments, immortal telomerase-transformed human endometrial stromal cells (T-HESCs) were incubated with medroxyprogesterone-17-acetate (MPA) and cyclic adenosine monophosphate (cAMP) for 4 days to induce decidualization. The expression levels of the decidualization markers prolactin (PRL) and insulin-like growth factor-binding protein-1 (IGFBP-1) were determined using quantitative polymerase chain reaction. RAR alpha was knocked down using a small interfering RNA, and C/EBP beta was overexpressed from an adenoviral vector. The transcriptional regulation of CEBPB by RAR alpha was determined by chromatin immunoprecipitation (ChIP) assay and luciferase assays. Results: We found that the expression levels of RAR alpha decreased in the mid-luteal endometria of RIF patients. After 4 days of decidualization induction in vitro, RAR alpha knockdown impaired the decidualization of T-HESCs and downregulated the expression of C/EBP beta. The restoration of C/EBP beta expression rescued the RAR alpha knockdown-induced suppression of T-HESC decidualization. In ChIP analysis of lysates from decidualized T-HESCs, the CEBPB promoter region was enriched in chromatin fragments pulled down using an anti-RAR alpha antibody. However, the relationship between CEBPB transcription and RAR alpha expression levels was only observed when the decidualization of T-HESCs was induced by the addition of cAMP and MPA. To identify the binding site of RAR alpha/retinoid X receptor alpha, we performed luciferase assays. Mutation of the predicted binding site in CEBPB (-2,009/-1,781) decreased the transcriptional activity of the reporter. To confirm this mechanism, the expression levels of C/EBP beta in the mid-luteal endometria of RIF patients were determined and found to decrease with decreased RAR alpha expression levels. Conclusion: A deficiency of RAR alpha expression in the mid-luteal endometrium inhibits decidualization due to the downregulation of CEBPB transcription. This is a potential mechanism contributing to RIF.