Proteomic analysis of plasma total exosomes and placenta-derived exosomes in patients with gestational diabetes mellitus in the first and second trimesters

Abstract

Gestational diabetes mellitus (GDM) is the first spontaneous hyperglycemia during pregnancy. Early diagnosis and intervention are important for the management of the disease. This study compared and analyzed the proteins of total plasma exosomes (T-EXO) and placental-derived exosomes (PLAP-EXO) in pregnant women who subsequently developed GDM (12-16 weeks), GDM patients (24-28 weeks) and their corresponding controls to investigate the pathogenesis and biomarkers of GDM associated with exosomes. The exosomal proteins were extracted and studied by proteomics approach, then bioinformatics analysis was applied to the differentially expressed proteins (DEPs) between the groups. At 12-16 and 24-28 weeks of gestation, 36 and 21 DEPs were identified in T-EXO, while 34 and 20 DEPs were identified in PLAP-EXO between GDM and controls, respectively. These proteins are mainly involved in complement pathways, immunity, inflammation, coagulation and other pathways, most of them have been previously reported as blood or exosomal proteins associated with GDM. The findings suggest that the development of GDM is a progressive process and that early changes promote the development of the disease. Maternal and placental factors play a key role in the pathogenesis of GDM. These proteins especially Hub proteins have the potential to become predictive and diagnostic biomarkers for GDM.

Keywords: Biomarker; Exosome; Gestational diabetes mellitus; Mechanism; Proteomics.

Fig. 1

Flow diagram of study identification

Fig. 2

Exosome validation. (A) Transmission electron microscopy (TEM) analysis. (B) Western blot analysis detection of CD63 and ALIX proteins. (C) Nanoparticle tracking analysis (NTA)

Fig. 3

Characterization of total exosomal protein expression and identification of DPEs at 12–16 and 24–28 weeks of gestation. (A) PCA plot. (B) PLS-DA plot. (C) Volcano plot depicting the distribution of proteins in the GDM-E/CON-E group. (D) Volcano plot depicting the distribution of proteins in the GDM-M/CON-M group. (E) Cluster analysis of DEPs between GDM-E and CON-E. (F) Cluster analysis of DEPs between GDM-M and CON-M. (C and D), green dots indicate down-regulated protein, and red dots indicate up-regulated protein

Fig. 4

Results of GO, KEGG and protein-protein interactions analyses of differentially expressed proteins in total exosomes at 12–16 weeks of gestation. (A) The top 10 biological processes associated with DEPs. (B) The top 10 cell components associated with DEPs. (C) The KEGG and Reactome pathways associated with DEPs. (D) Results of PPI analysis of total exosomal differential expressed proteins in early pregnancy

Fig. 5

Results of GO, protein-protein interactions and hub proteins analyses of differentially expressed proteins in total exosomes at 24–28 weeks of gestation. (A) The top 10 biological processes associated with DEPs. (B) The top 10 cell components associated with DEPs. (C) Results of PPI analysis of total exosomal differential expressed proteins at mid-pregnancy. (D) The top 10 hub proteins related to the DEPs of T-EXO at 12–16 weeks. (E) The top 10 hub proteins related to the DEPs of T-EXO at 24–28 weeks

Fig. 6

Characterization of placenta-derived exosomal protein expression and identification of DPEs at 12–16 weeks and 24–28 weeks of gestation. (A) PCA plot. (B) PLSDA plot. (C) Volcano plot depicting the distribution of proteins in the GDM-E/CON-E group. (D) Volcano plot depicting the distribution of proteins in the GDM-M/CON-M group. (E) Cluster analysis of DEPs between GDM-E and CON-E. (F) Cluster analysis of DEPs between GDM-M and CON-M. (C and D), green dots indicate down-regulated protein, and red dots indicate up-regulated protein

Fig. 7

Results of GO, KEGG and protein-protein interactions analyses of differentially expressed proteins in placenta-derived exosomes at 12–16 weeks of gestation. (A) The top 10 biological processes associated with DEPs. (B) The top 10 cell components associated with DEPs. (C) The KEGG, Reactome and Wiki pathways associated with DEPs. (D) Results of PPI analysis of placenta-derived exosomal differentially expressed proteins in early pregnancy

Fig. 8

Results of GO, protein-protein interactions and hub proteins analyses of differentially expressed proteins in placenta-derived exosomes at 24–28 weeks of gestation. (A) The top 10 biological processes associated with DEPs. (B) The top 10 cell components associated with DEPs. (C) Results of PPI analysis of placenta-derived exosomal differential expressed proteins at mid-pregnancy. (D) The top 10 hub proteins related to the DEPs of PLAP-EXO at 12–16 weeks. (E) The top 10 hub proteins related to the DEPs of PLAP-EXO at 24–28 weeks

Fig. 9

The mechanism of GDM disease associated with DEPs in plasma total exosomes and placental derived exosomes identified in this study. The results suggest that the mechanisms involved in the early and middle stages are not exactly the same, and that there may be an interaction between maternal and placental factors. Complement and coagulation cascades, immunity and inflammation were common mechanisms in the early and middle stages, whereas in the middle stage, changes in metabolism-related protein expression were detected