Fig 2: Hormones and IFN-t treatment increased CRIM1 and activated autophagy. (A,B) After being treated with or without P4, E2 and IFN-t, total proteins from EECs were subjected to Western blotting. (C,D) Fluorescence microscopy images of TFEB expression in EECs and histograms of fluorescence intensity. Representative images of three independent experiments are shown. Scale bar = 100 µm. (E) The GFP/RFP fluorescence ratio images of EECs with or without P4, E2 and IFN-t treatment are shown. (F) EECs were treated with or without P4, E2 and IFN-t for 12 h. The EECs were fixed and processed for TEM analysis. Scale bar = 2 µm. (G) After being pre-treated with or without CQ, total proteins from EECs were subjected to Western blotting. (H) Scanning electron microscopy (SEM) of EECs, which were treated with or without P4 and E2 followed by IFNT incubation for 12 h. The data are presented as the means ± SEM of three independent experiments. * Significant difference (p < 0.05) compared with other groups; ** Significant difference (p < 0.01) compared with other groups.

Fig 3: Knockdown of CRIM1 jeopardized endometrial receptivity. EECs were infected with a lentivirus specific for CRIM1 and shN for 48 h under P4, E2 and IFN-t treatment. (A) Growth curves of the shN and shCRIM1 groups were determined by the CCK-8 method. (B) The shN and shCRIM1 groups were collected to measure DNA synthesis by EdU proliferation assay. (C) The shN and shCRIM1 groups were collected to measure the cell cycle distributions by flow cytometry. (D) Western blot analysis of pho-CDK4 expression in the shN and shCRIM1 groups. (E) The shN and shCRIM1 groups were treated with P4, E2 and IFN-t for 12 h. The EECs were fixed and processed for SEM analysis. (F) After being treated with P4, E2 and IFN-t, total proteins from the shN and shCRIM1 groups were subjected to Western blotting. (G) The secretion levels of PGE2 and PGF2a were measured in EECs using an ELISA kit. (H) The rate-limiting enzymes of synthesized PGs were measured by real-time quantitative PCR. (I,J) GTCs spheroids were prepared, and the spheroid attachment rate was measured. Representative images of three independent experiments are shown. Scale bar = 1000 µm. (K,L) Fluorescence microscopy images of SPP1 expression in EECs and curves of fluorescence intensity. Representative images of three independent experiments are shown. Scale bar = 100 µm. (M) The cell adhesion molecules were measured by real-time quantitative PCR. The data are presented as the means ± SEM of three independent experiments. * Significant difference (p < 0.05) compared with other groups; ** Significant difference (p < 0.01) compared with other groups.

Fig 4: Proposed network of miR-143-5p–CRIM1 autophagy in regulating endometrial receptivity. Our results suggest that hormones and IFN-t promote CRIM1 expression by inhibiting the level of miR-143-5p, while CRIM1 regulates endometrial receptivity phenotypes through autophagy activation to reduce the production of ROS.

Fig 5: miR-143-5p targeted the CRIM1 3'UTR to regulate endometrial receptivity. (A) The effect of P4, E2 and IFN-t on the expression levels of miR-143-5p. (B) The psiCHECKTM-2 vector map and the insertion site of CRIM1-3'UTR are marked in red. (C) Schematic diagram illustrating the design of luciferase reporters with the WT-CRIM1 3' UTR (WT-CRIM1) or the site-directed mutant CRIM1 3' UTR (Mu-CRIM1). The nucleotides in red represent the “seed sequence” of miR-143-5p. (D) The luciferase activity was measured by the dual-luciferase reporter assay system. (E) The protein level of CRIM1 was measured by Western blot. (F) GTC spheroids were prepared and the spheroid attachment rate was measured. Representative images of three independent experiments are shown. Scale bar = 1000 µm. (G) The NC and mimic groups were treated with P4, E2 and IFN-t for 12 h. The EECs were fixed and processed for SEM analysis. (H,I) Fluorescence microscopy images of SPP1 expression in EECs and curves of fluorescence intensity. Representative images of three independent experiments are shown. Scale bar = 100 µm. (J) The cell adhesion molecules were measured by real-time quantitative PCR. (K) The secretion levels of PGE2 and PGF2a were measured in EECs using an ELISA kit. (L) The rate-limiting enzymes of synthesized PGs were measured by real-time quantitative PCR. The data are presented as the means ± SEM of three independent experiments. * Significant difference (p < 0.05) compared with other groups; ** Significant difference (p < 0.01) compared with other groups.