Fig 2: The androgen receptor is essential for androgen function during decidualization. A Immunohistochemistry staining analysis of AR in human endometrium during the menstrual cycle. B Western blot analysis of AR in human endometrium during the menstrual cycle. C Immunohistochemistry staining analysis of AR in the endometrium of the control individuals and PCOS patients. D Western blot analysis of AR protein levels in the endometrium of the control individuals and PCOS patients. E–H HESCs were treated with 8-Br-cAMP and MPA for three days, followed by treatment with testosterone (100 μM) and ARV-110 (1 nM) for an additional two days. PRL (E), IGFBP1 (F), and PDK4 (G) mRNA levels were further measured using real-time PCR, and PDK4 protein levels (H) were further measured using a western blot analysis. Statistical significance is denoted by *p < 0.05, **p < 0.01. I Multiple fluorescence immunohistochemistry staining analysis of endometrium in the control individuals and PCOS patients

Fig 3: Effects of AMPK on decidualization. A and B HESCs were pretreated with 8-Br-cAMP and MPA for three days, followed by treatment with various concentrations of testosterone (0.001–100 μM) for an additional two days. AMPK (A) and SIRT1 (B) mRNA levels were further measured using real-time PCR. Statistical significance is denoted by *p < 0.05. C HESCs were treated with 8-Br-cAMP and MPA for 1–5 days, and AMPK and SIRT1 mRNA levels were further measured using real-time PCR. D-I HESCs were pretreated with 8-Br-cAMP and MPA for three days, followed by treatment with Dorsomorphin (5 μM) or A-769662 (20 μM) alone or combining Dorsomorphin/A-769662 with testosterone (100 μM) for an additional two days. IGFBP1 (D) and PRL (E) mRNA levels were further measured using real-time PCR. IGFBP1 protein (F) that was released into the medium was measured using ELISA. PDK4 (G) and SIRT1 (H) mRNA levels were further measured using real-time PCR. Statistical significance is denoted by *p < 0.05, **p < 0.01. I Proteins of p-AMPK, SIRT1, and PDK4 were measured using Western blot analysis. Quantitative analysis of p-AMPK (J), SIRT1 (K), and PDK4 (L) protein expression levels were showed in histogram. a represents the treated cells vs. non-decidualized cells. b represents the treated cells vs. decidualized cells without other treatments. Statistical significance is denoted by *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig 4: Impaired decidualization in PCOS endometrial stromal cells. A H&E staining analysis of endometrium in PCOS patients and control individuals. B Representative images displaying the morphology of endometrial stromal cells isolated from the control individuals and the PCOS individuals. The ‘control’ represents the decidualized controls, the ‘dPCOS’ represents the decidualized PCOS samples, and the ‘ndPCOS’ represents the PCOS samples failed to decidualize. The images of decidualized stomal cells were scanned at × 10 magnification. C Endometrial stromal cells from the control individuals and the PCOS individuals were cultured for 48 h, followed by treatment with 8-Br-cAMP and MPA for an additional 1–7 days. IGFBP1 that was released into the medium was measured using ELISA. Statistical significance is denoted by *p < 0.05, **p < 0.01, ***p < 0.001 when compared with endometrial stromal cells from the PCOS individuals (n = 3, one-way analysis of variance). D and E Endometrial stromal cells isolated from controls and PCOS patients were cultured for 48 h, followed by treatment with 8-Br-cAMP and MPA for additional 96 h, PRL (D) and IGFBP1 (E) mRNA levels were further measured by real-time PCR. Statistical significance is denoted by *p < 0.05, **p < 0.01, ***p < 0.001 when compared to endometrial stromal cells treated without 8-Br-cAMP and MPA. F Endometrial stromal cells isolated from controls and PCOS patients were cultured for 48 h, followed by treatment with 8-Br-cAMP and MPA for additional 2–6 days, and immunofluorescence staining analysis were further performed to analyze the morphological transformation, and the fluorescein isothiocyanate-labeled phalloidin was used to label actin filaments

Fig 5: SIRT1 acts as the upstream of PDK4. A Immunohistochemistry staining analysis of SIRT1 in the endometrium during the menstrual cycle. B, C, and D HESCs were pretreated with 8-Br-cAMP and MPA for three days, followed by treatment with EX-527 (1 nM) and SRT1720 (5 nM) for an additional two days. PRL (B), IGFBP1 (C), and PDK4 (D) mRNA levels were further measured using real-time PCR. Statistical significance is denoted by *p < 0.05, **p < 0.01. E PDK4 and SIRT1 protein levels were measured using Western blot analysis in HESCs. F Proteins were extracted from HESCs treated with or without 8-Br-cAMP and MPA for three days. Immunoprecipitation was performed using an anti-PDK4 antibody or an anti-SIRT1 antibody, followed by Western blot analysis to investigate the interaction between PDK4 and SIRT1