Amniotic fluid (AF) supports the fetus throughout gestation. In addition to many biomolecules, it is a rich source of extracellular vesicles (EVs). Researchers are actively investigating the role of AF-EVs in pregnancy. The aim of our study was to investigate how the characteristics of AF-EVs change with gestational age.
We obtained AF samples from routine amniocentesis during the second trimester and elective Caesarean section at term. All pregnancies were healthy and resulted in clinically healthy infants. We isolated EVs using a combination of differential centrifugation, filtration, and ultracentrifugation and characterised them using nanoparticle tracking analysis, cryo-electron microscopy, and Western blotting. EV proteome was analysed using label-free proteomics.
We found that the biogenesis and the protein cargo of AF-EVs changed according to the gestation stage. Full-term AF-EVs were smaller in diameter compared to those from the second trimester. This supports the enrichment of EVs originating from the endocytic pathway in term AF, as observed in the proteomic profiles. Cryo-electron microscopy imaging revealed various EV morphologies, including those with multiple compartments, multiple membranes, and corona. There was no difference in the abundance of these morphologies between the groups. The concentration of AF-EVs remained consistent throughout gestation. Proteomic analysis revealed the gestation-dependent dynamics of the protein signature. In the second trimester, AF-EV proteins represented fetal organs such as the brain, liver, and pancreas and were involved in biological pathways related to molecule assembly and metabolism, indicating active growth of the fetoplacental unit. The proteome of the term AF-EVs was similar to that of other biological fluids and was implicated in responding to newborn challenges, such as digestion and immunity.
This study illustrates the dynamic nature of fetal EV biogenesis, showing the changing properties of AF-EVs. Additionally, the AF-EV proteome may offer insights into real-time fetal developmental processes related to both physiology and pathology.