Fig 2: Pathology staining analysis and correlation analysis among the cross-sectional area of adrenal glands, citrate synthase, and OGDH. (a) HE staining of the adrenal glands. (b) Immunohistochemical analysis for the expression of citrate synthase protein. The depth and area of brownish-yellow indicate the expression level of citrate synthase protein. (c) Immunohistochemical analysis for the expression of OGDH. The depth and area of brownish-yellow indicate the expression level of OGDH. (d–f) Quantitative analysis showed that the cross-sectional area of adrenal glands in the AC+CS group was larger than in the control group (P < 0.05) and that the expression of citrate synthase (P < 0.05) and OGDH (P < 0.05) proteins was higher in the AS+CS group than in the control group. (g) The heatmap shows the correlation analysis among the cross-sectional area of adrenal glands, citrate synthase, and OGDH. (h) A strong relationship existed between the cross-sectional area of adrenal glands and relative protein expression of citrate synthase. (i) The cross-sectional area of adrenal glands was related to the relative protein expression of OGDH. (j) A strong relationship existed between the cross-sectional area of adrenal glands and the relative protein expression of citrate synthase.

Fig 3: Low villous SDHB expression reduces succinate accumulation and correlates with RSA.a Western blot analysis of glycolysis and citrate cycle enzymes in villi from individuals with RSA or normal controls (n = 6 persons/group). Right panel: quantitative western blot results of six samples per group. b mRNA expression of glycolytic and citrate cycle genes in villi from individuals with RSA and normal controls (n = 6 persons/group). Exact P values in b are listed below: HK1 0.800, GPI 0.326, TPI 0.369, PGK1 0.202, ENO1 0.145, PKM2 0.685, 6PGD 0.715, TKT 0.914, PDHA1 0.691, CS 0.841, IDH1 0.546, IDH2 0.801, IDH3A 0.969, OGDH 0.163, SUCLA2 0.816, SDHA 0.00134, SDHB 0.00243, SDHC 0.000233, FH 0.222, and MDH2 0.938. c Relative SDH activities in SDHB-overexpressing JEG3 and HTR8 cells and control cells (n = 5 independent experiments). The SDHB overexpression efficiency is shown in the right panel. d Succinate levels in SDHB-overexpressing JEG3 and HTR8 cells and control cells (n = 5 independent experiments). e Relative SDH activities in SDHB-knockdown JEG3 and HTR8 cells and control cells (n = 5 independent experiments). The SDHB-knockdown efficiency is shown in the right panel. f Succinate levels in SDHB-knockdown JEG3 and HTR8 cells and control cells (n = 5 independent experiments). g The relative SDH activities in villi and decidua tissues from both RSA patients and normal controls (n = 10 persons/group). a–g Data represent the mean ± standard error; ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ns not significant. Two-tailed unpaired Student’s t tests in a–g. Source data are provided as a Source Data file.

Fig 4: Enzymatic activity of mitochondrial citrate synthase and OGDH, functional verification of citrate synthase and OGDH with overexpression, and knockdown experiments in the corresponding cell lines, target genes-TF regulatory-miRNA regulatory network. (a) Enzymatic activity of mitochondrial citrate synthase. (b) Enzymatic activity of mitochondrial OGDH. (c) Expression of citrate synthase mRNA via RT-PCR after the siRNA. (d) Expression of OGDH mRNA via RT-PCR after the siRNA. (e) Adrenal cell viability decreased after the citrate synthase siRNA. (f) Adrenal cell viability decreased after the OGDH siRNA. (g) Target genes-TF-miRNA regulatory network.

Fig 5: The most significant hub proteins. (a) Overlap between gene lists at the gene level; purple curves link identical genes. (b) Overlap between gene lists at the shared term level; blue curves link genes that belong to the same enriched ontology term. The inner circle represents gene lists, where hits are arranged along the arc. Genes that hit multiple lists are colored in dark orange, and genes unique to a list are shown in light orange. (c) Metascape analysis revealed the PPI network of DEPs between the control and CS groups. (d) Significant module was identified using MCODE. (e) Metascape analysis revealed the PPI network of DEPs between the AS and AS+CS groups. (f) Significant module was identified using MCODE. (g) Eleven common proteins between the two lists. (h) The two most significant hub proteins (OGDH and citrate synthase) were found using Metascape and the animal model.