PC1105: Characterization of Salivary Extracellular Vesicles in Preterm Infants

Background: Identifying biomarkers for inflammatory diseases in preterm infants is extremely challenging. Body fluids contain microRNAs (miRNAs) packaged within cell-derived vesicles (EVs) that play critical roles in various physiological and pathological processes. Serum miRNAs have been suggested as biomarkers in preterm infants, including miR146a-5p, a potential biomarker for necrotizing enterocolitis, and miR21-5p, a potential biomarker for neonatal chronic lung disease. However, the delicate clinical status and small blood volume of neonates preclude frequent blood draws for biomarkers examination. Non-invasive sampling such as salivary EVs miRNAs has been studied as a potential biomarker for inflammatory diseases in adults. However, the identification of salivary EVs in preterm infants and their miRNA content have not been previously explored. The primary aim of this study is to identify and characterize the EVs in salivary samples of preterm infants.

Methods: Neonates admitted to the NICU were prospectively enrolled based on gestational age into 3 groups: < 33 weeks gestation (Group 1, n=7), 33 weeks to 36 weeks gestation (Group 2, n= 8), and > 37 weeks gestation (Group 3, n=6). Saliva samples (0.1 mL – 1 mL) were collected weekly for the first 4 weeks of life from the base of the oral cavity using a syringe attached to wall suction. EVs were isolated using differential centrifugation. The concentration and size of EVs were measured using the ZetaView Nanoparticle tracking analyzer (NTA). The miRNA content of EVs was characterized using PCR. The Friedman test compared changes in EVs size and concentration of content over the 4 weeks.

Results: EVs were identified in all samples from all three groups. The average EV size was 185.5 nm (Group 1), 181.1 nm (Group 2), and 155.62 nm (Group 3), p = 0.07. The average concentration of EVs (particles/mL) were 15.5e9 (Group 1), 4.93e9 (Group 2), and 8.98e9 (Group 3), p = 0.4. Most of the EVs were identified as EVs as determined by Alexa405 labeled anti-human CD9 antibodies (Figure 1). There were no significant changes in EV concentration (p = 0.08) or size (p = 0.2) from week 1-4 in Group 1. EVs contained several microRNAs in the preterm infant group, including miR146a-5p and miR21-5p (Figure 2).

Conclusion: To our knowledge, this is the first study to isolate and characterize salivary EVs in preterm infants. EVs size and concentration did not vary significantly by gestational age or week of life, suggesting that changes in miRNA concentrations will reflect total miRNA saliva content, an important feature for reliable biomarkers. The presence of miRNA-146a-5p and miRNA-21-5p suggests their potential use as biomarkers for neonatal inflammatory diseases in future prospective studies. These findings could revolutionize biomarker discovery in preterm infants using non-invasive sampling.