O-GlcNAcylation regulates endometrial decidualization through dual mechanisms: PI3K-AKT axis-mediated phosphorylation and direct modification of FOXO1.

All animal experiments were conducted following approval by the Animal Ethics Committee of Dalian Medical University (project approval no. AEE21086 ). Adult ICR mice (8–10 weeks old) were obtained from the Experimental Animal Center of Dalian Medical University and were maintained at 22 ± 1 °C under a 12-h light/12-h dark cycle with food and water available ad libitum. To establish a pregnant mouse model, female mice were co-housed with fertile male mice at a 2:1 ratio. Gestation was timed from day 0.5 (D0.5), identified by the presence of a vaginal plug the morning after mating. Mice were euthanized from D2.5 to D6.5 for uterine collection. All experiments complied with ARRIVE guidelines. On D3.5, mice underwent intrauterine injections as previously described [ 14 ]. Following anesthesia, the abdominal wall was incised to expose a single uterine horn for injection. Each mouse received the following: experimental horn: OSMI-1 (40 µM, 10 µL, HY-119738, MedChemExpress, Monmouth Junction, NJ, USA), LY294002 (40 µM, 10 µL, HY-10108, MedChemExpress). Contralateral horn (control): 10 µL of 0.9% sterile saline. All mice were euthanized on D6.5. The uteri were then excised to quantify the number of embryo implantation sites. The immortalized human endometrial stromal cell line (T-hESC) was provided by Prof. Haibin Wang (Xiamen University School of Medicine). Before drug treatment or transfection, cells were maintained in phenol red-free DMEM/F12 (PM150316, Procell Life Science & Technology) supplemented with 10% charcoal-stripped FBS (CS-FBS, FBS-CA50, NEWZERUM, Christchurch, New Zealand) and 1% penicillin/streptomycin at 37 °C in 5% CO 2 . To model decidualization in vitro [ 15 ], T-hESC received treatment medium: phenol red-free DMEM/F12 supplemented with 2% CS-FBS, 1 µM medroxyprogesterone acetate (MPA, B1510, APExBIO Technology, Houston, TX, USA), and 0.5 mM dibutyryl cyclic AMP (db-cAMP, B9001, APExBIO Technology). The medium was replaced every 48 h. For one well of a six-well plate, the siRNA transfection complex included 100 µl phenol red-free DMEM/F12, 100 pmol siRNA and 5 µL Lipofectamine 2000 (11668019, Thermo Fisher Scientific, Carlsbad, CA, USA). The plasmid transfection complex included 100 µL phenol red-free DMEM/F12, 3000 ng plasmid DNA and 5 µL Lipofectamine 2000. The transfection complex was mixed thoroughly and placed for 20 min at room temperature before being added to the wells. Cells were harvested 48 h post-transfection for further experiments. All siRNAs were synthesized by GenePharma; target sequences were listed in Table S1 . The FOXO1 overexpression plasmid (pPPL011951-2a) was obtained from the Public Protein/Plasmid Library (PPL, Nanjing, China). Total protein was isolated using lysis buffer (KGB5303-100, KeyGEN BioTECH, Nanjing, China) containing protease/phosphatase inhibitors and 1 mM PMSF. Nuclear and cytoplasmic fractions were separated with a commercial kit (KGB5302, KeyGEN BioTECH). Protein concentration was assessed by BCA Assay (P0011; Beyotime Biotechnology, Shanghai, China) with bovine serum albumin (BSA) standards. Equal protein aliquots (20 µg per lane) underwent separation on 10% or 12% SDS-PAGE gels followed by transfer to nitrocellulose membranes (66485, Pall Corporation). After blocking with 5% skim milk, membranes were incubated overnight at 4 °C with primary antibodies. Table S2 shows specific information of the primary antibodies. The corresponding secondary antibody (1:4000, SA00001-1/2, Proteintech) was incubated for 50 min at 25 °C. Chemiluminescent signals were generated using ECL reagent (180–5001, Tanon) and captured with a Tanon imaging system. Band intensities were quantified using Image-Pro Plus software (Media Cybernetics) and normalized to the respective loading controls. Total RNA was extracted with TRIzol reagent (9109, Takara), and the concentration of the extracted RNA was subsequently measured by a NanoDrop 2000 UV spectrophotometer. cDNA was synthesized by reverse transcription of RNA (1000 ng) using HiScript II Q RT SuperMix (R223-01, Vazyme). qPCR was then conducted on a QuantStudio system (Thermo Fisher Scientific) using TransStart Top Green qPCR SuperMix (AQ131-04, Transgen). qPCR reaction conditions were as follows: initial denaturation at 94 °C for 30 s, denaturation at 94 °C for 5 s, annealing and extension at 60 °C for 30 s, followed by 40 cycles. Gene expression levels were normalized to the GAPDH. Primer sequences are detailed in Table S3 . Paraffin sections were deparaffinized in xylene, rehydrated through graded ethanol, and subjected to antigen retrieval in 10 mM sodium citrate buffer. Endogenous peroxidase was quenched with 3% H 2 O 2 , and non-specific binding was blocked with 5% goat serum for 20 min. Sections were incubated overnight at 4 °C with primary antibodies (anti-OGT, anti- O -GlcNAc, and anti-GFPT1). Sections were then incubated at 37℃ for 30 min with a biotinylated secondary antibody, followed by another 30-minute incubation at 37 ℃ with horseradish peroxidase-conjugated streptavidin (KIT-9720, MXB Biotechnologies). Staining was developed with DAB substrate, imaged under a Nexcope microscope, and quantified using Image-Pro Plus. Following fixation, cells were washed three times with PBS. Cells were incubated with FITC-phalloidin (1:400, RM02836, ABclonal) for 50–60 min at room temperature. Subsequently, nuclei were counterstained with DAPI. Fluorescent images were acquired using a Nexcope fluorescence microscope. Following fixation, cells were permeabilized with 0.1% Triton X-100 and blocked with 5% BSA at room temperature. Primary antibodies against PRL, IGFBP1, FOXO1, and O -GlcNAc (all diluted 1:300) were applied and incubated overnight at 4 °C. After PBS washes, cells were incubated with species-matched secondary antibodies for 1 h at room temperature. Nuclei were counterstained with DAPI. Fluorescent images were obtained on a Nexcope microscope and subsequently quantified using Image-Pro Plus. Cells were lysed in IP buffer (50 mM Tris-HCl, 150 mM NaCl, 0.3% Triton X-100, pH 7.5). After centrifugation, supernatants were collected. The supernatants were incubated with an anti-FOXO1 antibody or a species-matched control IgG overnight at 4 °C with rotation. Protein A/G magnetic beads (P2108, Beyotime) were added, and the mixture was incubated for 12 h at 4 °C with rotation. The immunoprecipitated complexes were boiled in SDS loading buffer for 10 min. Immunoprecipitates were analyzed by Western blot. Data are presented as the mean ± standard deviation (SD). Statistical significance was determined using GraphPad Prism: Student’s t-test (two groups) or One-way ANOVA (≥ 3 groups), with ≥ 3 biological replicates per experiment/analysis. Significance was set at p  < 0.05.

To assess endometrial O -GlcNAcylation levels, uterine tissues were harvested from mouse pregnancy model at peri-implantation stages (D2.5-D6.5). Immunohistochemical analysis showed that both O -GlcNAcylation and OGT exhibited peak levels in decidual tissue on D4.5 (the implantation window) (Fig.  1 A-B). To evaluate O -GlcNAcylation dynamics during decidualization, T-hESCs were exposed to medroxyprogesterone acetate (MPA) and dibutyryl cyclic AMP (db-cAMP) to trigger in vitro decidualization. qPCR and western blot analyses showed upregulated mRNA and protein expression of decidual markers PRL and IGFBP1 (Fig.  1 C-D). IF further confirmed increased PRL and IGFBP1 expression (Fig.  1 E). Morphological assessment via FITC-phalloidin staining for F-actin revealed that spindle-shaped stromal cells transformed into polygonal/round epithelioid cells after 5 days of induction, in contrast to uninduced cells (Fig.  1 E). Moreover, OGT levels and O -GlcNAcylation were upregulated during chemically induced decidualization (Fig.  1 D). Collectively, these results indicated that O -GlcNAcylation was critical for decidualization. To explore the functional role of O -GlcNAcylation, O -GlcNAcylation levels were inhibited during decidualization by OGT siRNA transfection or OSMI-1 (an OGT specific inhibitor) treatment. We first verified the knockdown efficiency of OGT small interfering RNA (Fig.  2 A). Furthermore, results showed that reduced O -GlcNAcylation suppressed the expression of the decidualization marker molecules PRL and IGFBP1 at both protein and mRNA levels (Fig.  2 B-C). IF analysis demonstrated reversion to mesenchymal morphology following O -GlcNAcylation downregulation, confirming impaired decidualization (Fig.  2 D). Collectively, our findings established O -GlcNAcylation as an essential regulator of decidualization. Fig. 1 Increased O -GlcNAcylation during T-hESC decidualization ( A , B ) IHC of O-GlcNAcylation and OGT in mouse uterine tissues during implantation (D2.5-D6.5). NC represents the negative control. LE, luminal epithelium; GE, glandular epithelium; St, stroma; DZ, decidual zone; E, embryo; scale bar = 100 μm/50 µm. ( C ) qPCR analysis of PRL and IGFBP1 mRNA in human endometrial stromal cells (T-hESC) during 0–5 days decidualization induction (1 µM MPA + 0.5 mM db-cAMP). ( D ) Western blot of PRL, IGFBP1, OGT and O -GlcNAcylation levels in decidualizing T-hESCs. ( E ) IF of PRL/IGFBP1 (red) and F-actin (FITC-phalloidin, green) in T-hESC after 5-day decidualization. Nuclei were counterstained with DAPI (blue). Scale bars = 50 μm. * p  < 0.05, ** p  < 0.01, *** p  < 0.001 Increased O -GlcNAcylation during T-hESC decidualization ( A , B ) IHC of O-GlcNAcylation and OGT in mouse uterine tissues during implantation (D2.5-D6.5). NC represents the negative control. LE, luminal epithelium; GE, glandular epithelium; St, stroma; DZ, decidual zone; E, embryo; scale bar = 100 μm/50 µm. ( C ) qPCR analysis of PRL and IGFBP1 mRNA in human endometrial stromal cells (T-hESC) during 0–5 days decidualization induction (1 µM MPA + 0.5 mM db-cAMP). ( D ) Western blot of PRL, IGFBP1, OGT and O -GlcNAcylation levels in decidualizing T-hESCs. ( E ) IF of PRL/IGFBP1 (red) and F-actin (FITC-phalloidin, green) in T-hESC after 5-day decidualization. Nuclei were counterstained with DAPI (blue). Scale bars = 50 μm. * p  < 0.05, ** p  < 0.01, *** p  < 0.001 Fig. 2 Inhibition of O -GlcNAcylation impairs T-hESC decidualization ( A ) qPCR analysis of OGT in T-hESCs transfected with siNC or siOGT for 48 h. NC siRNA: non-targeting control. ( B ) Western blot analysis of O -GlcNAcylation, PRL, and IGFBP1 in decidualizing T-hESCs treated with OGT siRNA or OSMI-1 (40 µM, 24 h). ( C ) qPCR analysis of PRL and IGFBP1 mRNA in decidualizing T-hESCs following O -GlcNAcylation inhibition. ( D ) Cytoskeletal reorganization visualized by F-actin staining (FITC-phalloidin, green), nuclei were counterstained with DAPI (blue), scale bar = 50 μm. * p  < 0.05, ** p  < 0.01, *** p  < 0.001 Inhibition of O -GlcNAcylation impairs T-hESC decidualization ( A ) qPCR analysis of OGT in T-hESCs transfected with siNC or siOGT for 48 h. NC siRNA: non-targeting control. ( B ) Western blot analysis of O -GlcNAcylation, PRL, and IGFBP1 in decidualizing T-hESCs treated with OGT siRNA or OSMI-1 (40 µM, 24 h). ( C ) qPCR analysis of PRL and IGFBP1 mRNA in decidualizing T-hESCs following O -GlcNAcylation inhibition. ( D ) Cytoskeletal reorganization visualized by F-actin staining (FITC-phalloidin, green), nuclei were counterstained with DAPI (blue), scale bar = 50 μm. * p  < 0.05, ** p  < 0.01, *** p  < 0.001 O -GlcNAcylation levels depend on HBP-mediated substrate flux, with glutamine-fructose-6-phosphate transaminase 1 (GFPT1) catalyzing the rate-limiting step [ 16 ]. To investigate whether the decidualization process is associated with HBP, we initially analyzed the expression of GFPT1, a key enzyme in HBP, in endometrial tissue from pregnant mice using IHC. GFPT1 protein expression significantly increased in endometrial stroma starting at implantation D4.5 (Fig. 3 A). Correspondingly, GFPT1 levels increased during in vitro T-hESCs decidualization (Fig. 3 B), indicating HBP activation. To assess HBP’s functional role, we pharmacologically modulated the pathway during the decidualization process to observe changes in O -GlcNAcylation levels and decidualization-related markers. Glucosamine (GlcNH 2 ), a GFPT1 downstream metabolite, was used to activate HBP. The results showed that upon activation of the HBP pathway, there was an increase in O -GlcNAcylation levels, accompanied by elevated expression of decidualization markers PRL and IGFBP1 (Fig. 3 C-D). IF revealed GlcNH 2 -induced epithelioid transformation characterized by cellular enlargement and rounding (Fig. 3 E), suggesting that activating the HBP pathway may promote decidualization by enhancing O -GlcNAcylation levels. Conversely, treatment with the GFPT1 inhibitor 6-diazo-5-oxo-L-norleucine (DON) reduced both O -GlcNAcylation levels and the expression of decidualization markers (Fig. 3 F-G), with corresponding effects on cell morphology (Fig. 3 H). Additionally, GFPT1 siRNA knockdown yielded consistent results (Figure S1 ). Collectively, these data suggest the HBP as a regulator of decidualization through O -GlcNAcylation. Fig. 3 The HBP pathway influences the decidualization of T-hESC ( A ) IHC of GFPT1 in mouse uterus during implantation (D2.5-D6.5). LE, luminal epithelium; GE, glandular epithelium; St, stroma; DZ, decidual zone; E, embryo; scale bar = 100 μm/50 µm. ( B ) Western blot analysis of GFPT1 protein levels during T-hESC decidualization (0–5 days). ( C ) qPCR analysis of PRL and IGFBP1 mRNA levels in T-hESC treated with GlcNH2 (0.1 mM, 0.5 mM) for 24 h during T-hESC decidualization ( D ) Western blot analysis of O -GlcNAcylation, PRL, and IGFBP1 protein levels in T-hESC treated with GlcNH2 for 24 h during T-hESC decidualization. ( E ) F-actin cytoskeleton (FITC-phalloidin, green) with DAPI nuclear stain (blue), scale bar = 50 μm. ( F ) qPCR analysis of PRL and IGFBP1 mRNA levels after DON treatment (50 µM, 24 h) during T-hESC decidualization. ( G ) Western blot analysis of O-GlcNAcylation, PRL, and IGFBP1 protein levels post-DON exposure during T-hESC decidualization. ( H ) F-actin cytoskeleton (FITC-phalloidin, green) with nuclei counterstained by DAPI (blue), scale bar = 50 μm. * p  < 0.05, ** p  < 0.01, *** p  < 0.001 The HBP pathway influences the decidualization of T-hESC ( A ) IHC of GFPT1 in mouse uterus during implantation (D2.5-D6.5). LE, luminal epithelium; GE, glandular epithelium; St, stroma; DZ, decidual zone; E, embryo; scale bar = 100 μm/50 µm. ( B ) Western blot analysis of GFPT1 protein levels during T-hESC decidualization (0–5 days). ( C ) qPCR analysis of PRL and IGFBP1 mRNA levels in T-hESC treated with GlcNH2 (0.1 mM, 0.5 mM) for 24 h during T-hESC decidualization ( D ) Western blot analysis of O -GlcNAcylation, PRL, and IGFBP1 protein levels in T-hESC treated with GlcNH2 for 24 h during T-hESC decidualization. ( E ) F-actin cytoskeleton (FITC-phalloidin, green) with DAPI nuclear stain (blue), scale bar = 50 μm. ( F ) qPCR analysis of PRL and IGFBP1 mRNA levels after DON treatment (50 µM, 24 h) during T-hESC decidualization. ( G ) Western blot analysis of O-GlcNAcylation, PRL, and IGFBP1 protein levels post-DON exposure during T-hESC decidualization. ( H ) F-actin cytoskeleton (FITC-phalloidin, green) with nuclei counterstained by DAPI (blue), scale bar = 50 μm. * p  < 0.05, ** p  < 0.01, *** p  < 0.001 Given that downregulation of O -GlcNAcylation impairs decidualization, we performed transcriptome profiling in decidualizing cells treated with OSMI-1 to further explore its underlying molecular regulatory mechanism. OSMI-1 treatment induced 1845 differentially expressed genes (847 upregulated, 998 downregulated) versus untreated controls (Fig.  4 A-B). Gene Ontology (GO) analysis revealed enrichment in differentiation and proliferation processes (Fig.  4 C). KEGG pathway analysis demonstrated significant enrichment of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT) pathway (Fig.  4 D). Subsequently, we verified whether O -GlcNAcylation regulates decidualization through the PI3K-AKT signaling pathway. The results showed that the ratios of phospho-PI3K (p-PI3K) / total PI3K (t-PI3K) and phospho-AKT (p-AKT) / total AKT (t-AKT) decreased during decidualization (Fig. 4 E), indicating the PI3K-AKT pathway inhibition, consistent with prior reports [ 17 , 18 ]. Conversely, OSMI-1 treatment activated the PI3K-AKT pathway (Fig. 4 E), suggesting O -GlcNAcylation downregulation impairs decidualization via PI3K-AKT activation. To validate these findings, we administered the PI3K inhibitor LY294002 to determine whether it could reverse OSMI-1-induced decidualization impairment. The results demonstrated that LY294002 rescued OSMI-1-mediated suppression of PRL and IGFBP1 expression (Fig. 4 F-G). These findings were further confirmed at the cellular level by IF analysis (Fig. 4 H). Collectively, O -GlcNAcylation facilitates decidualization by suppressing PI3K-AKT signaling. Fig. 4 O -GlcNAcylation regulates decidualization via the PI3K-AKT signaling pathway ( A ) Heatmap of differentially expressed genes between OSMI-1 treated and control decidual cells. ( B ) Volcano plot showing differentially expressed genes affected by OSMI-1 in decidual cells. Genes significantly upregulated (red) or downregulated (blue) are distributed on both sides, while genes with no significant change are in the middle (gray). ( C , D ) GO enrichment analysis and KEGG enrichment analysis of differentially expressed genes. ( E ) Western blot analysis of key molecules in the PI3K-AKT pathway during T-hESC decidualization with or without OSMI-1 treatment. ( F ) qPCR analysis of PRL and IGFBP1 mRNA levels in T-hESCs undergoing decidualization after treatment with OSMI-1 and LY294002 (20 µM, 40 µM). ( G ) Western blot analysis of PRL, IGFBP1, and FOXO1 protein levels in T-hESCs undergoing decidualization after treatment with OSMI-1 and LY294002 (20 µM, 40 µM). ( H ) F-actin cytoskeleton (FITC-phalloidin, green) with nuclei counterstained with DAPI (blue), scale bar = 50 μm. * p  < 0.05, ** p  < 0.01, *** p  < 0.001 O -GlcNAcylation regulates decidualization via the PI3K-AKT signaling pathway ( A ) Heatmap of differentially expressed genes between OSMI-1 treated and control decidual cells. ( B ) Volcano plot showing differentially expressed genes affected by OSMI-1 in decidual cells. Genes significantly upregulated (red) or downregulated (blue) are distributed on both sides, while genes with no significant change are in the middle (gray). ( C , D ) GO enrichment analysis and KEGG enrichment analysis of differentially expressed genes. ( E ) Western blot analysis of key molecules in the PI3K-AKT pathway during T-hESC decidualization with or without OSMI-1 treatment. ( F ) qPCR analysis of PRL and IGFBP1 mRNA levels in T-hESCs undergoing decidualization after treatment with OSMI-1 and LY294002 (20 µM, 40 µM). ( G ) Western blot analysis of PRL, IGFBP1, and FOXO1 protein levels in T-hESCs undergoing decidualization after treatment with OSMI-1 and LY294002 (20 µM, 40 µM). ( H ) F-actin cytoskeleton (FITC-phalloidin, green) with nuclei counterstained with DAPI (blue), scale bar = 50 μm. * p  < 0.05, ** p  < 0.01, *** p  < 0.001 Studies have indicated that FOXO1 is a key downstream target of the AKT pathway [ 19 ] and directly regulates the decidual markers PRL and IGFBP1 as a transcription factor [ 20 , 21 ]. Therefore, we hypothesized that O -GlcNAcylation modulates FOXO1 via the PI3K-AKT pathway. The results showed that OSMI-1 suppressed FOXO1 protein expression, but this suppression was attenuated by LY294002 (Fig. 4 G). Furthermore, FOXO1 knockdown abrogated LY294002-induced upregulation of PRL and IGFBP1 levels (Fig. 5 A-C) and triggered reversion to a fibroblast-like morphology (Fig. 5 D). These data confirmed that the PI3K-AKT pathway regulates decidualization through FOXO1. Prior studies established that AKT-mediated phosphorylation promotes FOXO1 nuclear export, leading to its functional inactivation [ 19 ]. Subsequently, Western blot analysis was performed to assess the expression of FOXO1 and its phosphorylated forms at the AKT-dependent sites Thr24 and Ser256 in the cytoplasmic and nuclear fractions. The results showed that OSMI-1 treatment markedly reduced total FOXO1 protein levels in both the cytoplasm and the nucleus. Further analysis of phosphorylated FOXO1 (p-FOXO1) revealed that OSMI-1 treatment decreased nuclear p-FOXO1 levels, while concomitantly increasing p-FOXO1 levels in the cytoplasmic fraction (Fig. 5 E). These findings suggest that OSMI-1 may enhance PI3K-AKT signaling activity, thereby promoting FOXO1 phosphorylation and nuclear export, accompanied by an overall reduction in total FOXO1 protein levels. Accordingly, co-treatment with the PI3K inhibitor LY294002 partially reversed the OSMI-1 induced changes in FOXO1 subcellular distribution, as evidenced by restoration of nuclear FOXO1 levels and reduction in cytoplasmic p-FOXO1 levels, indicating that this process is dependent on the PI3K-AKT pathway (Fig. 5 E). Immunofluorescence (IF) analysis of FOXO1 subcellular localization yielded results consistent with the Western blot data (Fig. 5 F). These findings suggest that O -GlcNAcylation may regulate FOXO1 phosphorylation through the PI3K-AKT pathway, thereby influencing its transcriptional function within the nucleus. To validate our in vitro findings in an in vivo model that more closely reflects physiological conditions, we administered OSMI-1 and LY294002 to female mice via intrauterine injection on D3.5 of pregnancy. The results showed that, compared with the contralateral control uterine horn, OSMI-1 treatment significantly reduced the number of implantation sites in pregnant mice. Moreover, implantation sites were unevenly distributed and exhibited a smaller diameter, suggesting that OSMI-1 may impair the embryo implantation process and compromise subsequent embryonic development. Notably, co-administration of LY294002 partially alleviated the detrimental effect of OSMI-1 on implantation efficiency (Fig.  5 G). Western blot analysis revealed elevated p-AKT/t-AKT ratios and phospho-FOXO1 (Thr24/Ser256) with concurrent suppression of total FOXO1, PRL and IGFBP1 in OSMI-1-treated endometrium. LY294002 treatment partially reversed PI3K-AKT-FOXO1 pathway activation and decidualization marker suppression (Fig.  5 H). These results suggest that O -GlcNAcylation promotes the dephosphorylation of FOXO1 by inhibiting the activation of the PI3K-AKT pathway, thereby increasing its nuclear accumulation and transcriptional activity, ultimately affecting decidualization. Fig. 5 O -GlcNAcylation mediates the regulation of FOXO1 phosphorylation via the PI3K-AKT pathway ( A ) qPCR analysis of FOXO1 mRNA expression in T-hESCs transfected with siNC or siFOXO1 for 48 h. ( B ) qPCR detection of PRL and IGFBP1 mRNA levels in T-hESCs undergoing decidualization after treatment with 40 µM LY294002 and transfection with siFOXO1. ( C ) Western blot analysis of PRL and IGFBP1 protein levels in T-hESCs undergoing decidualization after treatment with 40 µM LY294002 and transfection with siFOXO1. ( D ) F-actin cytoskeleton (FITC-phalloidin, green) with nuclei counterstained with DAPI (blue), scale bar = 50 μm. ( E ) T-hESCs undergoing decidualization were treated with 40 µM OSMI-1, followed by 40 µM LY294002. Western blot analysis was performed to detect the expression of phospho-FOXO1 (Thr24/Ser256) and total FOXO1 in the cytoplasmic and nuclear fractions. GAPDH and Lamin B1 served as loading controls for cytoplasmic and nuclear fractions, respectively. ( F ) FOXO1 nuclear translocation assessed by IF (red), nuclei were counterstained with DAPI (blue), scale bar = 50 μm. ( G ) Representative images of mouse uteri following intrauterine injection (scale bar = 1 cm). ( H ) Endometrial protein levels of phospho-AKT (Ser473), total AKT, PRL, IGFBP1, phospho-FOXO1 (Thr24/Ser256), and total FOXO1 analyzed by Western blot. * p  < 0.05, ** p  < 0.01, *** p  < 0.001 O -GlcNAcylation mediates the regulation of FOXO1 phosphorylation via the PI3K-AKT pathway ( A ) qPCR analysis of FOXO1 mRNA expression in T-hESCs transfected with siNC or siFOXO1 for 48 h. ( B ) qPCR detection of PRL and IGFBP1 mRNA levels in T-hESCs undergoing decidualization after treatment with 40 µM LY294002 and transfection with siFOXO1. ( C ) Western blot analysis of PRL and IGFBP1 protein levels in T-hESCs undergoing decidualization after treatment with 40 µM LY294002 and transfection with siFOXO1. ( D ) F-actin cytoskeleton (FITC-phalloidin, green) with nuclei counterstained with DAPI (blue), scale bar = 50 μm. ( E ) T-hESCs undergoing decidualization were treated with 40 µM OSMI-1, followed by 40 µM LY294002. Western blot analysis was performed to detect the expression of phospho-FOXO1 (Thr24/Ser256) and total FOXO1 in the cytoplasmic and nuclear fractions. GAPDH and Lamin B1 served as loading controls for cytoplasmic and nuclear fractions, respectively. ( F ) FOXO1 nuclear translocation assessed by IF (red), nuclei were counterstained with DAPI (blue), scale bar = 50 μm. ( G ) Representative images of mouse uteri following intrauterine injection (scale bar = 1 cm). ( H ) Endometrial protein levels of phospho-AKT (Ser473), total AKT, PRL, IGFBP1, phospho-FOXO1 (Thr24/Ser256), and total FOXO1 analyzed by Western blot. * p  < 0.05, ** p  < 0.01, *** p  < 0.001 FOXO1 contains multiple O -GlcNAcylation sites [ 22 ], indicating direct involvement of O -GlcNAcylated FOXO1 in T-hESC decidualization. IP demonstrated elevated FOXO1 O -GlcNAcylation in decidualized T-hESCs compared with non-induced controls after 5-day induction (Fig. 6 A). To further investigate the impact of O -GlcNAcylation on FOXO1 during decidualization, FOXO1-overexpressing T-hESCs were induced to undergo decidualization in the presence of OSMI-1. The results showed that, compared to the control group, FOXO1 overexpression enhanced decidual marker expression, whereas OSMI-1 suppressed this enhancement (Fig. 6 B). IF revealed that FOXO1-overexpressing cells acquired an enlarged, polygonal morphology, whereas OSMI-1 treatment showed a fibroblast-like phenotype of long spindles (Fig. 6 C). These results demonstrated that O -GlcNAcylation regulates T-hESC decidualization through FOXO1. Notably, O -GlcNAcylation can regulate protein stability [ 23 ]. Consistently, our results indicated that FOXO1 protein expression significantly decreased following O -GlcNAcylation inhibition (Fig. 6 B). To investigate whether O -GlcNAcylation affects FOXO1 protein stability, we downregulated global O -GlcNAcylation using siOGT or OSMI-1, then inhibited protein synthesis with cycloheximide (CHX) at indicated time points. Protein degradation assays demonstrated that depleting O -GlcNAcylation significantly reduced the half-life of FOXO1 (Fig. 6 D-E), indicating that O -GlcNAcylation inhibition promotes FOXO1 degradation in T-hESCs. To further determine the pathway responsible for FOXO1 protein degradation, T-hESCs were treated with the proteasome inhibitor MG132 or the lysosomal inhibitor chloroquine (CQ). The results showed that MG132 markedly attenuated OSMI-1 induced FOXO1 degradation, whereas CQ treatment did not significantly block or alleviate FOXO1 protein loss. These findings suggest that FOXO1 degradation triggered by reduced O-GlcNAcylation is mainly mediated through the ubiquitin–proteasome pathway (Fig. 6 F). IP revealed enhanced FOXO1 ubiquitination following O -GlcNAcylation inhibition (Fig. 6 G). As a nuclear transcription factor, FOXO1 exerts its transcriptional activity by binding to target genes within the nucleus [ 24 ]. Consequently, we assessed the impact of O-GlcNAcylation depletion on FOXO1 subcellular localization. IF results showed that following OSMI-1 treatment and a decrease in O -GlcNAcylation, a significant amount of nuclear FOXO1 shifted to the cytoplasm (Fig. 6 H). Collectively, O -GlcNAcylation stabilizes FOXO1 by blocking its ubiquitin-proteasomal degradation and controls its localization, thereby regulating endometrial stromal cell decidualization. Fig. 6 FOXO1 O -GlcNAcylation coordinates decidualization through stability and subcellular localization ( A ) IP assay detecting O -GlcNAcylation of FOXO1 during T-hESCs decidualization. ( B ) T-hESCs were treated with FOXO1 overexpression and OSMI-1 during decidualization. Western blot analysis was performed to assess protein levels of FOXO1, PRL, and IGFBP1. ( C ) F-actin cytoskeleton stained with FITC-phalloidin (green) and nuclei counterstained with DAPI (blue), scale bar = 50 μm. ( D , E ) T-hESCs were treated with DMSO, siOGT ( D ) or OSMI-1 ( E ), followed by incubation with 100 µM CHX. Western blot analysis was performed to determine FOXO1 protein levels. ( F ) Western blot analysis was performed to examine FOXO1 protein expression in T-hESCs treated with MG132 (10 µM) or chloroquine (CQ, 40 µM) in the absence or presence of OSMI-1 (40 µM). ( G ) T-hESCs were treated with or without OSMI-1, followed by incubation with 10 µM MG132 for 6 h. IP was performed to detect ubiquitination of FOXO1. ( H ) IF staining showing the expression and subcellular colocalization of FOXO1 (green), and O -GlcNAcylation (red). Nuclei were counterstained with DAPI (blue), scale bar = 50 μm. * p  < 0.05, ** p  < 0.01, *** p  < 0.001 FOXO1 O -GlcNAcylation coordinates decidualization through stability and subcellular localization ( A ) IP assay detecting O -GlcNAcylation of FOXO1 during T-hESCs decidualization. ( B ) T-hESCs were treated with FOXO1 overexpression and OSMI-1 during decidualization. Western blot analysis was performed to assess protein levels of FOXO1, PRL, and IGFBP1. ( C ) F-actin cytoskeleton stained with FITC-phalloidin (green) and nuclei counterstained with DAPI (blue), scale bar = 50 μm. ( D , E ) T-hESCs were treated with DMSO, siOGT ( D ) or OSMI-1 ( E ), followed by incubation with 100 µM CHX. Western blot analysis was performed to determine FOXO1 protein levels. ( F ) Western blot analysis was performed to examine FOXO1 protein expression in T-hESCs treated with MG132 (10 µM) or chloroquine (CQ, 40 µM) in the absence or presence of OSMI-1 (40 µM). ( G ) T-hESCs were treated with or without OSMI-1, followed by incubation with 10 µM MG132 for 6 h. IP was performed to detect ubiquitination of FOXO1. ( H ) IF staining showing the expression and subcellular colocalization of FOXO1 (green), and O -GlcNAcylation (red). Nuclei were counterstained with DAPI (blue), scale bar = 50 μm. * p  < 0.05, ** p  < 0.01, *** p  < 0.001

Successful embryo implantation occurs exclusively during a brief and restrictive period when the endometrium is in a receptive state, known as the Window of Implantation (WOI) [ 1 , 2 ]. During the WOI, sustained estrogen and progesterone initiate decidualization by driving stromal cell differentiation [ 3 ]. Insulin-like growth factor binding protein-1 (IGFBP1) and prolactin (PRL) are established molecular markers of decidualization, exhibiting significant upregulation during this process [ 4 ]. The transcription factor Forkhead box protein O1 (FOXO1) is one of the important transcription factors in the decidualization process, capable of regulating the transcription of IGFBP1 and PRL genes [ 5 ]. Endometrial decidualization is essential for blastocyst implantation, placental development, and pregnancy maintenance, constituting a fundamental requirement for healthy gestation [ 6 ]. Abnormalities or defects in decidualization contribute to recurrent implantation failure and contribute to pregnancy disorders such as miscarriage, preeclampsia, and fetal growth restriction [ 7 ]. O -GlcNAcylation is a critical post-translational modification (PTM) initiated through the hexosamine biosynthetic pathway (HBP)-mediated conversion of glucose to uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), regulated by enzymes including glutamine-fructose-6-phosphate aminotransferase 1 (GFPT1). UDP-GlcNAc serves as the activated sugar donor for O -GlcNAc transferase (OGT)-mediated O -GlcNAcylation of serine/threonine residues, while O -GlcNAcase (OGA) catalyzes its removal [ 8 ]. Functioning as a nutrient and stress sensor, O -GlcNAcylation modulates diverse cellular processes including signal transduction, energy metabolism, mitochondrial homeostasis, and cell survival [ 9 ]. Dysregulated O -GlcNAcylation is implicated in the pathogenesis of human diseases, including cancer, diabetes mellitus, and gestational disorders [ 10 , 11 ]. We previously demonstrated that O -GlcNAcylation promotes embryo implantation via enhanced proliferation, migration, and invasion of endometrial cells during the WOI, facilitating embryo recognition and adhesion [ 12 , 13 ]. Given that decidualization is essential for embryo implantation, we investigated its potential regulation by O -GlcNAcylation. This study investigated the effect of O -GlcNAcylation on decidualization and its mechanism by using in vitro decidualization models and pregnant mouse systems. We demonstrated that O -GlcNAcylation is elevated during decidualization and promotes decidualization through dual mechanisms: indirectly regulating FOXO1 phosphorylation and directly modifying FOXO1. These findings provide a new mechanistic framework linking O -GlcNAcylation to endometrial receptivity regulation.

O -GlcNAcylation, a widespread PTM, governs essential cellular activities including transcription, translation, signal transduction, and metabolism in multiple cell types [ 25 ]. Functioning as a nutrient sensor, O -GlcNAcylation is essential for embryonic development. Murine OGT resides on the X chromosome, and its systemic knockout causes embryonic lethality [ 26 ]. During murine embryo implantation, the OGA inhibitor Thiamet-G (TMG) elevates O -GlcNAcylation, promoting trophoblast differentiation into invasive lineages and enhancing blastocyst invasion of the endometrial epithelium [ 27 ]. Furthermore, elevated O -GlcNAcylation enhances human endometrial cell proliferation, migration, invasion, and adhesion, thereby modulating receptivity [ 13 ]. Through glucose transporter 1 (GLUT1) and AQP3, endometrial O -GlcNAcylation orchestrates glycolytic reprogramming during implantation, thereby regulating cellular function and embryo implantation [ 12 ]. We demonstrated elevated O -GlcNAcylation in murine endometrium during the implantation window, and the level of O -GlcNAcylation increased during the decidualization of human endometrial stromal cells. Additionally, O -GlcNAcylation downregulation impaired decidualization and compromised embryo implantation efficiency. These findings implicate O -GlcNAcylation as a critical regulator of stromal-to-decidual cell differentiation. To delineate O -GlcNAcylation’s regulatory mechanism in decidualization, we performed transcriptome analysis following O -GlcNAcylation inhibition in decidualizing cells. KEGG analysis identified significant enrichment of differentially expressed genes in the PI3K-AKT pathway. As a critical cellular pathway, PI3K-AKT regulates signal transduction and key processes, including proliferation, apoptosis, metabolism, angiogenesis, underscoring its fundamental role [ 28 , 29 ]. This pathway directly regulates endometrial decidualization and receptivity, as evidenced by accumulating studies. OPG-Syndecan-1 interaction promotes T-hESCs decidualization by suppressing AKT phosphorylation (Ser473/Thr308) [ 30 ]. PI3K-AKT hyperactivation downregulates IGFBP1 expression in both normal and endometriotic stromal cells [ 31 , 32 ]. Consistent with our data, O -GlcNAcylation negatively regulates the PI3K-AKT pathway and promotes the expression of decidual markers PRL and IGFBP1. To elucidate how PI3K-AKT regulates decidualization, we focused on its important downstream molecule, the transcription factor FOXO1, which is also a hallmark molecule of decidualization. FOXO1 enhances IGFBP1 promoter activity by binding HOXA10 in endometrial stromal cells [ 33 ]. SOX4 directly regulates FOXO1 transcription to promote decidualization [ 34 ]. PI3K-AKT activation induces FOXO1 phosphorylation, triggering cytoplasmic translocation and inactivation that abrogates DNA-binding [ 35 ]. In adenomyosis (AMS), SCRIB deficiency causes PI3K-AKT hyperactivation in T-hESCs, promoting FOXO1 phosphorylation/degradation that impairs decidualization and drives pathogenesis [ 36 ]. Our study demonstrates that O -GlcNAcylation-mediated inhibition of the PI3K-AKT signaling pathway significantly reduces FOXO1 phosphorylation, facilitating FOXO1’s entry into the nucleus to exert its transcriptional role and increase the expression of decidual markers PRL and IGFBP1. Notably, PI3K inhibitor LY294002 rescued O -GlcNAcylation downregulation-impaired decidualization via PI3K-AKT inhibition. Collectively, O -GlcNAcylation regulates decidualization via the PI3K-AKT-FOXO1 axis. O -GlcNAcylation modulates transcriptional programs by altering transcription factor binding affinity, subcellular localization, protein stability, and co-regulator interactions [ 37 , 38 ]. FOXO1 undergoes O -GlcNAcylation at Thr317, Ser550, Thr648, Ser654 and Thr646 [ 22 , 39 ]. These findings implicate the direct involvement of O -GlcNAcylation of FOXO1 in the decidualization process of endometrial stromal cells. We demonstrate that O -GlcNAcylation depletion reduces FOXO1 abundance by promoting its ubiquitin-proteasome degradation. During the decidualization of T-hESCs, reduced O -GlcNAcylation of FOXO1 protein leads to decrease nuclear localization. We further assessed the effects of O -GlcNAcylation on FOXO1 and its target genes (PRL and IGFBP1) transcription. FOXO1 O -GlcNAcylation downregulation attenuated transcriptional activation of PRL and IGFBP1. From a clinical perspective, the O -GlcNAcylation–PI3K–AKT–FOXO1 signaling axis identified in this study provides a novel mechanistic explanation for disorders associated with impaired endometrial decidualization. Defective decidualization is considered an important pathological basis for recurrent implantation failure, recurrent pregnancy loss, and endometrium-related diseases such as adenomyosis and endometriosis. Given that O -GlcNAcylation is a pharmacologically tractable post-translational modification, small-molecule modulators targeting O-GlcNAc cycling enzymes (e.g., OGT or OGA) may offer new therapeutic opportunities to improve implantation outcomes. In addition, our observation that PI3K inhibition partially reversed the decidualization defects induced by reduced O -GlcNAcylation suggests that local, short-term, and endometrium-targeted modulation of this pathway could represent a potential strategy to optimize endometrial preparation in assisted reproductive settings; however, its efficacy and safety require further systematic validation. Despite the mechanistic insights provided by this work, several limitations should be acknowledged. First, our conclusions are primarily based on in vitro–cultured human endometrial stromal cells and mouse models; therefore, additional clinical samples are needed to validate the clinical relevance of O -GlcNAcylation and its downstream signaling axis in patients with adverse pregnancy outcomes. Second, although we demonstrate that FOXO1 O -GlcNAcylation is critical for its protein stability and transcriptional activity, the specific modification sites and their functional consequences—such as the independent contributions to protein stability, nuclear trafficking, and transcriptional output—have not been fully elucidated and warrant further investigation using site-specific mutagenesis and functional assays.

O -GlcNAcylation is markedly elevated during murine embryo implantation and in vitro decidualization. Mechanistically, it suppresses PI3K-AKT signaling to reduce FOXO1 phosphorylation. Concomitantly, O -GlcNAcylation stabilizes FOXO1, maintains its nuclear localization, and enhances the transcriptional activation of core decidual marker genes (PRL and IGFBP1) (Fig.  7 ). Thus, O -GlcNAc dynamics during decidualization orchestrate FOXO1 protein abundance and subcellular distribution via integrated phosphorylation regulation and direct modification, critically governing decidualization in both murine and human systems. Fig. 7 Graphical illustration of the mechanism by which O -GlcNAcylation regulates FOXO1 during decidualization. The hexosamine biosynthetic pathway (HBP), catalyzed by GFPT1, generates UDP-GlcNAc, which serves as the donor substrate for OGT-mediated protein O -GlcNAcylation. During decidualization, O -GlcNAcylation suppresses PI3K–AKT signaling, thereby reducing phosphorylation of the key decidualization transcription factor FOXO1 and limiting its degradation; meanwhile, O -GlcNAcylation can directly modify FOXO1, enhancing its protein stability and promoting its nuclear retention. Graphical illustration of the mechanism by which O -GlcNAcylation regulates FOXO1 during decidualization. The hexosamine biosynthetic pathway (HBP), catalyzed by GFPT1, generates UDP-GlcNAc, which serves as the donor substrate for OGT-mediated protein O -GlcNAcylation. During decidualization, O -GlcNAcylation suppresses PI3K–AKT signaling, thereby reducing phosphorylation of the key decidualization transcription factor FOXO1 and limiting its degradation; meanwhile, O -GlcNAcylation can directly modify FOXO1, enhancing its protein stability and promoting its nuclear retention.

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