Placenta-derived Extracellular Vesicles in Maternal Plasma of Hb Bart’s Fetuses

Abstract Introduction: Alpha-thalassemia is the most common cause of hydrops fetalis among Southeast Asians (also called “Bart’s hydrops fetalis). This condition is considered a fatal disorder; therefore, prenatal screening and diagnosis are extremely important. Changes in soluble analytes in the maternal circulation, such as hormones and angiogenic factors are not predictive of Hb Bart’s fetalis condition. This condition is associated with placental hypoxia, which may trigger the release of placenta-derived extracellular vesicles (EVs) in maternal circulation. Therefore, the determination of changes in placenta-derived exosome and its protein content could provide additional insight into the disease pathways of this disorder. Objectives : We aim to characterize: 1) maternal plasma levels of placenta-derived EVs; and 2) proteomics profiles of maternal plasma EVs in women with Bart’s fetalis fetuses. Methods: This prospective cohort study included women with the following groups: 1) normal pregnancy or control group: women with couple at risk for Hb Bart’s fetuses but subsequently proven as non-Hb Bart’s group and delivered at term without maternal or neonatal complications (Group 1) (n=7); 2) Hb Bart’s with hydropic features group (Group 2) (n=4); 3) Hb Bart’s without hydropic features group (Group 3) (n=7); 4) a disease control group which consisted of women who subsequently delivered with placental associated conditions (Group 4) (n=4); and 5) women with hydrops fetalis from non-Bart’s causes (n=5) (Group 5). Maternal plasma EVs were isolated by the combination of stepwise centrifugation, ultrafiltration and qEV size exclusion chromatography. The EVs were characterized by the particle size, morphology and protein markers. Isolated EVs and their plasma were measured placental alkaline phosphatase (PLAP) level using ELISA. Mass spectrometry was used to determine EV proteomics profile. A p-value of <0.05 was used to infer significance, unless multiple testing was involved, with the false discovery rate controlled at the 10% level (q<0.1). Results: 1) EV particle size in Bart fetuses with hydropic features was significantly smaller than that in normal pregnancy and disease control groups; 2) Bart fetuses with hydropic features had higher maternal placenta-derived PLAP-contained EVs (PLAP-EVs) than that in normal pregnancy; although, PLAP-EV level was highest in the placental associated complications group; 3) Bart fetuses without hydropic features tended to have higher maternal PLAP-EV level compared to normal pregnancy group; 4) hydropic fetuses due to non-Bart’s causes had similar PLAP level compared to normal pregnancy group; 5) among the 16/106 differentially expressed EV proteins, hnRNPA2B1 protein was the highest in Bart fetuses with hydropic features group compared to placental associated complication or normal pregnancy groups; 6) sixteen differentially expressed EV proteins were involved in fibrinogen complex, fibrin clot formation and integrin signaling pathway. Conclusions: This is the first study of placenta-derived EVs in women with Hb Bart’s fetalis. EV particle size is significantly smaller but maternal PLAP-EV level is significantly higher in women with Hb Bart’s fetalis fetuses compared to normal pregnancy. In addition, several EV proteins were differential expressed in women with Bart’s fetuses and these proteins involve in aberrant immune response, pro-inflammatory cytokine regulation mediated by TLRs-MyD88-IRAK4-MAPK axis and vascular injuries as part of pathophysiology of placental edema and hypoxia in Bart’s hydrops. The result of this study supports future research to determine whether placenta-derived EVs in maternal circulation can be used as a liquid biopsy or non-invasive prenatal test for the early identification of Bart’s fetuses. This will ultimately reduce the rate of unnecessary invasive prenatal diagnosis testing.