Fig 2: FBP administration induces COX-2+ dMf differentiation and alleviates pregnancy loss.(A and B) The FBP levels in uterine tissues and blood plasma of normal pregnant mice (Ctrl, n = 8), spontaneous abortion-prone mice (SA; n = 8), or SA mice intraperitoneally injected with FBP (500 mg/kg per day, SA + FBP, n = 8) were detected. (C and D) The IL-27 levels in USCs of pregnant mice from Ctrl (n = 8), SA (n = 8), or SA + FBP (n = 8) group were analyzed by FCM. (E and F) The COX-2 levels in uMf of pregnant mice from Ctrl (n = 8), SA (n = 8), or SA + FBP (n = 8) group were analyzed by FCM. (G) The mRNA levels of decidualization-related genes in uterus of pregnant mice from Ctrl (n = 8), SA (n = 8), or SA + FBP (n = 8) group were detected by RT-PCR. (H) Depth of CK7+ trophoblast infiltration into uterus of pregnant mice from Ctrl (n = 8), SA (n = 8), or SA + FBP (n = 8) group was observed by hematoxylin and eosin staining or immunofluorescence staining. (I and J) Photograph (red arrow: absorption point) of uterus and absorption rates of pregnant mice from Ctrl (n = 8), SA (n = 8), or SA + FBP (n = 8) group. Data were presented as means ± SEM and analyzed by one-way ANOVA test. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 3: FBP increases IL-27 expression in DSCs in a PKM2/ERK1/2/c-FOS–dependent manner.(A and B) Expression of p-c-fos, c-fos, p-ERK, or ERK in FBP (0.5 mM)–treated ESCs (n = 9) was analyzed by Western blotting. (C) The IL-27 levels in supernatants of FBP, FBP, and ERK1/2 inhibitor (U0126, 10 µM) or FBP and c-fos inhibitor (T-5224, 20 mM)–treated ESCs (24 hours, n = 6) were detected by ELISA. (D) Sense proteins of FBP and G6P in ESCs were analyzed by the HuProt 20K protein array. (E) PPI network based on the STRING database between 69 proteins from (D), FOS, MAPK1, and IL27. (F to H) The expression of p-c-fos, c-fos, p-ERK, or ERK and secretion levels of IL-27 in a PKM2 activator (mitapivat, 10 µM) or DMSO (Ctrl, 1‰)–treated ESCs (24 hours, n = 6) were analyzed by Western blotting and ELISA. (I to K) Expression of p-c-fos, c-fos, p-ERK, or ERK in uterus and IL-27 levels in USCs from pregnant mice (n = 8) treated with saline (1%) or 2-DG (50 mg/kg per day) were analyzed by Western blotting and ELISA. Data were presented as means ± SEM and analyzed by t test or one-way ANOVA test. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 4: IL-27 maintains normal pregnancy in a uterine COX-2+ macrophage-dependent manner.(A) Expression of COX-2 in uMfs of WT (mated with male Il27ra-/- mice, n = 8) or Il27ra-/- pregnant mice (mated with male WT mice, n = 8) was detected by FCM. (B) Expression of COX-2 in uMfs of saline (1%, n = 8) or 2-DG (50 mg/kg per day, n = 8)–treated pregnant mice was detected by FCM. (C) Expression of differentiation molecules in uMfs of WT (n = 8) or Ptgs2-/- pregnant mice (n = 8) was detected by FCM. (D to F) Photograph (red arrow: absorption point) of uterus, the CRL of embryos, or absorption rates from WT (n = 8) or Ptgs2-/- pregnant mice (n = 8). (G) Differential proteins of supernatants between IL-27RA+ dMfs and IL-27RA- dMfs were evaluated by the proteomic microarray. (H to J) Transcription levels of decidualization-related genes in uterus, depth of CK7+ trophoblast infiltration into uterus, and embryo absorption rates of Il27ra-/- pregnant mice (mated with male WT mice, n = 8) adopted with WT or Ptgs2-/- macrophages. Data were presented as means ± SEM or median and quartile and analyzed by t test, Mann-Whitney U test, or one-way ANOVA test. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 5: The FBP–IL-27–COX-2 regulatory axis in decidua is insufficient in patients with unexplained RSA.(A) The mRNA levels of FBP1 and PFK1 between DSCs from normal pregnant women (n = 10) or patients with unexplained RSA (n = 10) were detected by RT-PCR. (B and C) Expression of FBP1 and PFK1 between decidua tissues from normal pregnant women (n = 12) or patients with unexplained RSA (n = 12) were detected by immunohistochemistry. (D) The FBP levels in the blood plasma from nonpregnant (NP) or pregnant (P) normal women or patients with unexplained RSA (n = 10 per group) were detected. (E) The mRNA level of IL-27 between DSCs from normal pregnant women (n = 10) or patients with unexplained RSA (n = 10) was detected by RT-PCR. (F and G) Expression of IL-27 between decidua tissues from normal pregnant women (n = 20) or patients with unexplained RSA (n = 20) was detected by immunohistochemistry. (H and I) The levels of COX-2 (H), CD80, CD86, INF-?, CD206, CD209, and IRF4 (I) in dMfs from normal pregnant women (n = 12) or patients with unexplained RSA (n = 12) were detected by FCM. Data were presented as means ± SEM and analyzed by t test or one-way ANOVA test. *P < 0.05, **P < 0.01, and ***P < 0.001.