Integrated Quantitative Proteomic Analysis of Biomarkers Derived from Fetal Membranes and Plasma Exosomes in Preterm Birth: A Pilot Study

Preterm birth (PTB), a leading cause of neonatal morbidity, remains poorly understood due to its multifactorial etiology. This study integrates quantitative proteomic profiling of paired fetal membrane and plasma exosomes from preterm (<i>n</i> = 5) and term (<i>n</i> = 5) deliveries to identify PTB-associated biomarkers. Using four-dimensional label-free quantitative liquid chromatography-tandem mass spectrometry (4D label-free LC-MS/MS) proteomic analyses, we characterized exosomal proteins and identified 435 and 330 differentially expressed proteins (DEPs) in fetal membranes and plasma, respectively, associated with PTB. Immune-related pathways dominated shared proteins between fetal membranes and plasma. Notably, REEP5 was significantly upregulated in PTB-derived exosomes across both sample types. Immunohistochemistry confirmed elevated levels of REEP5 expression and membrane localization in preterm fetal membranes, aligning with its exosomal enrichment. Additionally, inflammation- (e.g., PLA2G4C and TBXA2R) and oxidative stress-related proteins (e.g., JUN and EDNRA) were uniquely packaged in PTB exosomes. These findings highlight fetal membrane-plasma exosomal crosstalk and propose REEP5 as a potential biomarker for PTB. This study advances the understanding of exosome-mediated mechanisms in PTB and underscores the utility of proteomics in discovering clinically actionable biomarkers. However, due to the small sample size, this study is a small pilot study, and the findings require validation in larger-scale cohorts.