The Population of Circulating Extracellular Vesicles Dramatically Alters after Very Premature Delivery- a Previously Unrecognised Postnatal Adaptation Process?
Authors: O'Reilly Daniel , Egan Karl , Burke Oscar , Griffiths Angharad , Neary Elaine , Blanco Alfonso , Szklanna Paulina , Maguire Patricia , McCallion Naomi , Ni Ainle Fionnuala 
Published: October 2019

Abstract
Introduction
Following birth, the transition from intrauterine to extrauterine life is associated with major physiological changes. Many pathological processes linked with mortality and morbidity in preterm infants start at this time. Extracellular vesicles (EVs) are subcellular particles released by all known cell types and readily detectable in large numbers in all biological fluids. EVs are heterogeneous in size and origin, consisting of exosomes (endosomal origin, 30-150 nm), microvesicles (plasma membrane-derived, 50-1000nm), and apoptotic bodies (500-2000 nm). They are linked with a wide variety of processes including coagulation and cell-cell communication, and it has been hypothesized that they may affect preterm morbidities. It is unknown whether circulating EVs can change during this extrauterine transition period.
Aim
Here we investigate if the population of circulating EVs is altered in premature neonates during the extrauterine transition period
Patients and Methods
Preterm neonates were recruited through the Department of Neonatology at the Rotunda Hospital, Dublin, Ireland. Written informed consent was obtained from the parents of all participants. Blood collection was performed during routine phlebotomy. Platelet free plasma was prepared by double centrifugation at 3000g for 10 minutes. 15x Day 1 of life and 14x days 3 of life plasma samples were available from preterm neonates, 8 of which were matched Day 1 and Day 3 samples. EVs were quantified and characterised by both nanoparticle tracking analysis (NTA with a Malvern NanoSight 3000) and flow cytometry (Beckman Coulter CytoFLEX LX).
Results
The extrauterine transition period is characterised by a shift in plasma EVs profile. Using NTA, we observed an increase in the levels of plasma EVs (0-200nm) from Day 1 to Day 3 (Day 1; 4.0 ± 2.5 x 107/µl vs. Day 3; 7.2 ± 4.4 x 107/µl; p = 0.03). This increase in EV levels (0-200nm) was supported by flow cytometry, which also demonstrated an increase in EVs (100-900nm) from day 1 to Day 3 (Day 1; 1.1 ± 0.3 X 106/µl vs. Day 3; 4.2 ± 3.2 x 106/µl, p = 0.0009). There was a highly significant correlation between EV levels measured by NTA and flow cytometry (Spearmann rank correlation coefficient, r = 0.69, p < 0.0001), suggesting simultaneous increases in small and large EVs during the extrauterine transition period. Using flow cytometry, we also observed a change in the composition of plasma EVs during the extrauterine transition period. Flow cytometry data from Day 3 samples were characterised by the presence of a homogenous population of EVs of ~100-300nm in size, which was not observed on Day 1. The presence of this population caused a significant increase in the median side scatter height (SSC-H) value of the plasma EV population (Day 1; 1800 ± 746 vs. Day 3; 3832 ± 1633, p = 0.0013), as well as reduction in the percentage of 100nm EVs (Day 1; 73.9 ± 9.2 % vs. Day 3; 57.4 ± 12.6 %, p = 0.0005) and an increase in the percentage of 100-300nm EVs (Day 1; 19.7 ± 7.7 % vs. Day 3; 38.0 ± 12.9%, p = 0.001). EVs from Day 3 samples were characterised by higher median Red SSC-H values (Day 1; 1688 ± 2902 vs. Day 3; 3641 ± 6247, p = 0.0004) and Violet SSC-H values (Day 1; 42641 ± 21131 vs. Day 3; 97133 ± 38311, p < 0.0001), suggesting a potential change in the membrane or internal composition of EVs in the extrauterine transition period. We also observed a change in protein expression on EVs during the extrauterine transition period. Platelets and platelet activation play a physiological role in the closure of the ductus arteriosus . As such, we assessed the levels of platelet EVs (CD41+/Annexin V+), an established marker of platelet activation in vivo. The percentage of CD41+/Annexin V-EVs significantly decreased from Day 1 to Day 3 (Day 1; 6.5 ± 4.9 % vs. Day 3; 2.4 ± 1.9 %, p = 0.007), suggestive of a platelet activation event early in the extrauterine transition period. Proteomic differences between day 1 and day 3 were analysed using mass spectrometry analysis
Conclusion
In this study, we clearly demonstrate that the extrauterine transition period is characterised by major changes in plasma EVs. These changes include an increase in the levels of EVs, a change in the composition of EVs, and a reduction in the percentage of platelet-derived EVs. The physiological or pathophysiological causes of the changes require further elucidation. In addition, the role of this change of EV profile in the pathogenesis of important preterm morbidities needs to be clarified.

Disclosures
Ni Ainle: Leo Pharma: Research Funding; Actelion: Research Funding; Bayer: Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Boehringer: Membership on an entity's Board of Directors or advisory committees.