Study The Mechanism For Impaired Embryo Implantation By Overexpressed Endometrial KLF12 In Recurrent Implantation Failure

Object:The aim of this study was to assess the role of KLF12 in the impaired endometrial receptivity and embryo implantation.Methods:The different expression of KLF12、LIF and Nur77 in the proliferative and secretory endometrium were analyzed by qRT-PCR,Western blot and immunohistochemical staining.In addition,the expression of these factors after treatment with estrogen and progesterone were also detected.The expression of KLF12.LIF and Nur77 in endometrium of RIF patients were further detected by qRT-PCR、Western blot and immunohistochemical staining.Western blotting and qRT-PCR were also used to detect the expression of LIF and its downstream genes in adenovirus-mediated KLF12 enhanced Ishikawa cells.At the same time,Nur77 expression was also detected in KLF12-overexpressed hESCs.Luciferase assay,ChIP/PCR and ABCD assay demonstrated that KLF12 could bind to the conserve sequence(CAGTGGG)within the promoter region of LIF and Nur77 and transcriptionally regulate their expression.Moreover,the in vitro embryo adhesion model was employed to explain the LIF-mediated function of increased KLF12 in Ishikawa cells.Detection of dPRL,immunofluorescence and the in vitro embryo implantation model were all used to prove the Nur77-mediated function of enhanced KLF12 on decidualization in hESCs.Furthermore,the uterine K1f12 knockout mice model was used to prove the molecular changes which were detected in vitro.Results:KLF12 expression was higher in the proliferative endometrium,however,LIF and Nur77 expression was increased in the secretory endometrium.In both Ishikawa cells and hESCs,the repression of KLF12 expression was observed by 48 h after treatment with estrogen and progesterone.In contrast,LIF and Nur77 expression was rapidly induced after in vitro stimulation.In addition,KLF12 expression was increased in endometrial tissues from the RIF patients(n=22)compared with those from the fertile controls(n=18)and the opposite results were observed for LIF and Nur77 expression in these tissues.KLF12 repressed embryo adhesion by decreasing LIF expression in Ishikawa cells.At the same time,KLF12 impaired hESC decidualization by repressing Nur77 expression.Mechanistically,KLF12 bound to a conserved site(CAGTGGG)in both LIF and Nur77 promoter region.Last but not least,the expression pattern of KLF12,LIF and Nur77 in uterus of the uterine K1f12 knockout mouse was similar as we detected in vitro.Conclusion:KLF12 expression was significantly reduced in the secretory endometrium,meanwhile,endometrial LIF and Nur77 expression was increased.And KLF12 expression was repressed after treatment with estrogen and progesterone,which indicated that KLF12 was a negative regulator of embryo implantation.The aberrantly increased KLF 12 in the endometrial epithelium of RIF patients impaired embryo adhesion by transcriptionally repressing LIF expression.In addition,aberrant KLF 12 expression in the RIF endometrial stroma inhibited hESC decidualization via transcriptionally reducing Nur77 expression.The uterine expression pattern of KLF 12,LIF and Nur77 in uterus of the uterine K1f12 knockout mouse was similar as we detected in vitro.The study on the mechanism of KLF 12-regulating embryo implantation can provide the theoretical basis for improving the success rate of embryo implantation in IVF-ET.