Fructose-1,6-bisphosphate prevents pregnancy loss by inducing decidual COX-2+ macrophage differentiation

Abstract

Decidualization is an intricate biological process in which extensive remodeling of the endometrium occurs to support the development of an implanting blastocyst. However, the immunometabolic mechanisms underlying this process are still largely unknown. We found that the decidualization process is accompanied by the accumulation of fructose-1,6-bisphosphate (FBP). The combination of FBP with pyruvate kinase M stimulated IL-27 secretion by endometrial stromal cells in an ERK/c-FOS-dependent manner. IL-27 induced decidual COX-2⁺ M2-like macrophage differentiation, which promotes decidualization, trophoblast invasion, and maternal-fetal tolerance. Transfer of Ptgs2⁺/COX-2⁺ macrophages prevented fetal loss in Il27ra-deleted pregnant mice. FBP levels were low in plasma and decidual tissues of patients with unexplained recurrent spontaneous abortion. In therapeutic studies, FBP supplementation significantly improved embryo loss by up-regulation of IL-27-induced COX-2⁺ macrophage differentiation in a mouse model of spontaneous abortion. These findings collectively provide a scientific basis for a potential therapeutic strategy to prevent pregnancy loss.

Fig. 1.. FBP is enriched during decidualization.

(A) Heatmap of differential metabolites in ESCs (n = 12) from healthy control endometrium of secretory phase and DSCs (n = 12) from normal early pregnant women by metabolomics analysis and list of differential metabolites in glycol metabolism. (B and C) Expression of metabolic enzymes of glycol metabolism between ESCs (n = 6) and DSCs (n = 6) was detected by Western blotting. (D) Expression of FBP1 and PFK1 between control endometrium (n = 6) and decidua tissues (n = 6) was detected by immunohistochemistry. (E) The FBP levels in blood plasma from women without (NP; n = 10) or with pregnancy (P; n = 10) and in endometrium (n = 15) or decidua tissues (n = 15) were detected with the FBP kits. (F) Summary of glycolysis characteristics during decidualization. (G and H) FBP1 and PFK1 levels in ESCs (n = 12) were detected by RT-PCR and Western blotting after pretreatment with 1‰ DMSO (Ctrl), adenosine 3′,5′-monophosphate (cAMP; 0.5 μM), and estrogen (E; 10⁻⁸ μM) or plus progesterone (P; 10⁻⁶ μM) or indirectly cocultured with primary trophoblast cells (Tro; ratio 1:1, 48 hours). Data were presented as means ± SEM and analyzed by t test or one-way ANOVA test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 2.. FBP relieves 2-DG–mediated pregnancy loss.

(A) The FBP levels in uterine tissues of pregnant mice treated with saline (1%, n = 8) or 2-DG (50 mg/kg per day, n = 8) were detected with FBP kits. (B to D) Photograph (red arrow: absorption point) of uterus from pregnant mice intraperitoneally injected with 1% saline (n = 8), 2-DG (50 mg/kg per day, n = 8), or 2-DG (50 mg/kg per day) plus FBP (500 mg/kg per day) (n = 8). The absorption rates or crown-rump length (CRL) of embryos from (B) were analyzed in (C) or (D), respectively. 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; NS, no significance.

Fig. 3.. Abnormal FBP-IL-27 regulatory axis in DSCs leads to pregnancy loss.

(A) Heatmap of the differential proteins (differential fold > 3) of supernatants between FBP (0.5 mM)–ESC (n = 3) and Ctrl-ESC (n = 3) by the proteomic microarray. (B) The IL-27 levels in ESCs (n = 9) treated with FBP were detected by FCM. (C) The IL-27 levels in supernatants of control ESCs (FBP1-NC, n = 9) or FBP1-overexpressing ESCs (FBP1^over, n = 9) treated with DMSO (1‰) or progesterone (P; 10⁻⁸ μM) were detected by ELISA. (D) Expression of IL-27 between control endometrium (n = 6) and decidua tissues (n = 6) was detected by immunohistochemistry. (E to G) Photograph (red arrow: absorption point) of uterus, absorption rates, or CRL of embryos from WT pregnant mice (mated with male Il27ra^−/− mice, n = 8) or Il27ra^−/− pregnant mice (mated with male WT mice, n = 8). (H) Transcription levels of decidualization-related genes in uterus from (E) were analyzed by RT-PCR. (I) Depth of CK7⁺ trophoblast infiltration into uterus from (E) was observed by hematoxylin and eosin staining or immunofluorescence assays. DAPI, 4′,6-diamidino-2-phenylindole. Data were presented as means ± SEM and analyzed by t test or one-way ANOVA test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 4.. 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. 5.. IL-27 induces decidual COX-2⁺ macrophage differentiation in vitro.

(A and B) Expression of IL-27RA in DSCs (n = 9), trophoblast cells (Tros; n = 9), DLCs (n = 9), and decidual macrophages (dMφs) (n = 9) was analyzed by FCM. Statistical graph of (A) was shown in (B). (C and D) Expression of p-STAT3 and STAT3 in dMφs (n = 9) after coculture with ESCs pretreated with FBP (0.5 mM for 24 hours) or transfected with IL-27 overexpression plasmids (IL-27^over) for 48 hours was detected by FCM, and the ratios of p-STAT3 to STAT3 were calculated. (E and F) Expression of COX-2 in peripheral blood monocytes (pMos) (n = 12), eMφs (n = 12), and dMφs (n = 12) was analyzed by FCM. Statistical graph of (E) was shown in (F). (G) Expression of COX-2 in dMφs (n = 9) cocultured with Ctrl or FBP (0.5 mM for 24 hours)–treated ESCs was detected by FCM. (H) Expression of COX-2 in dMφs (n = 9) cocultured with FBP (0.5 mM for 24 hours)–pretreated ESCs or IL-27^over ESCs for 48 hours was detected by FCM. Data were presented as means ± SEM or median and quartile and analyzed by t test, one-way ANOVA test, or Kruskal-Wallis test. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 6.. IL-27 maintains normal pregnancy in a uterine COX-2⁺ macrophage-dependent manner.

(A) Expression of COX-2 in uMφs 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 uMφs 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 uMφs 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⁺ dMφs and IL-27RA⁻ dMφs 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. 7.. 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-γ, CD206, CD209, and IRF4 (I) in dMφs 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.

Fig. 8.. FBP administration induces COX-2⁺ dMφ 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 uMφ 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.