Background: Congenital heart disease (CHD) is the leading cause of birth defects globally, impacting 1% of births, resulting in sudden death in children and a significant long-term healthcare burden. Currently, only around 50% of cases are diagnosed in-utero. In 85% of CHD, an abnormal in-utero environment can alter DNA methylation patterns in fetal tissue, resulting in abnormal tissue development and CHD. Fetal extracellular vesicles (EVs) released into maternal circulation carry DNA methylation signatures reflective of fetal development, but their use as biomarkers has not yet been investigated.
Aims: To determine whether EVs isolated from maternal murine plasma carry methylated EV-DNA signatures that can differentiate normal from CHD pregnancies.
Methods: Plasma was purified from C57/B6 pregnant mice in control conditions (n=5), or transiently exposed to 5.5% hypoxia to induce fetal CHD (n=5). Samples underwent EV isolation/purification by size-exclusion chromatography (SEC). EV-rich fractions were identified by nanoparticle tracking analysis (NTA) and BCA assays, and subsequently pooled and concentrated using Amicon columns. Further characterisation was undertaken using western blotting and transmission electron microscopy (TEM).
Results: SEC fractions 1-3 were EV-enriched without protein contamination and pooled for further analysis. TEM imaging confirmed the presence of EVs. NTA demonstrated concentrations of 3.3x109±5.4x108 (mean±SEM) particles/mL across cohorts, with particle sizes 1.2-fold smaller in animals carrying CHD pregnancies compared to normal pregnancies (99.52±3.57nm vs 83.42±3.55nm, p=0.004).
Conclusion: EVs can be successfully isolated from plasma from our murine CHD model. Future work will determine whether EV-DNA carries methylation signatures that can diagnose fetal CHD in-utero.