title: Proteomic characterization of macro-, micro- and nano-extracellular vesicles derived from the same first trimester placenta: relevance for feto-maternal communication.
abstract: OBJECTIVE
  What proteins are carried by extracellular vesicles (EVs) released from normal first trimester placentae?
  
  
  CONCLUSIONS
  One thousand five hundred and eighty-five, 1656 and 1476 proteins were characterized in macro-, micro- and nano-vesicles, respectively, from first trimester placentae, with all EV fractions being enriched for proteins involved in vesicle transport and inflammation.
  
  
  BACKGROUND
  Placental EVs are being increasingly recognized as important mediators of both healthy and pathological pregnancies. However, current research has focused on detecting changes in specific proteins in particular fractions of vesicles during disease. This is the first study to investigate the full proteome of different-sized fractions of EVs from the same first trimester placenta and highlights the differences/similarities between the vesicle fractions.
  
  
  METHODS
  A well-established ex vivo placental explant culture model was used to generate macro-, micro- and nano-vesicles from 56 first trimester placentae. Vesicle fractions were collected by differential ultracentrifugation, quantified and characterized.
  
  
  METHODS
  Placental macro-, micro- and nano-vesicles were characterized by microscopy, dynamic light scattering and nanoparticle tracking analysis. The proteome of each EV fraction was interrogated using liquid chromatography-coupled tandem mass spectrometry. Results were validated by semi-quantitative western blotting.
  
  
  RESULTS
  A total of 1585, 1656 and 1476 proteins were identified in macro-, micro- and nano-vesicles, respectively. One thousand one hundred and twenty-five proteins were shared between all three fractions while up to 223 proteins were unique to each fraction. Gene Ontology pathway analysis showed an enrichment of proteins involved in vesicle transport and inflammation in all three fractions of EVs. The expression levels of proteins involved in internalization of vesicles (annexin V, calreticulin, CD31, CD47), the complement pathway [C3, decay-accelerating factor (DAF), membrane cofactor protein (MCP), protectin] and minor histocompatibility antigens [ATP-dependent RNA helicase (DDX3), ribosomal protein S4 (RPS4)] were different between different-sized EVs.
  
  
  CONCLUSIONS
  This study is largely hypothesis-generating in nature. It is important to validate these findings using EVs isolated from maternal plasma and the function of the different EV fractions would need further investigation.
  
  
  CONCLUSIONS
  Our results support the concept that various EV factions can interact with different maternal cells and have unique effects to mediate feto-maternal communication during early pregnancy. This study also provides a list of candidate proteins, which may inform the identification of robust markers that can be used to isolate placental vesicles from the maternal blood in the future.
  
  
  BACKGROUND
  M.T. is a recipient of the University of Auckland Health Research Doctoral Scholarship and the Freemasons Postgraduate Scholarship. This project was supported by a School of Medicine Performance-based research fund (PBRF) grant awarded to L.W.C. No authors have any conflicts of interest to disclose.
authors.affiliation: Department of Obstetrics and Gynaecology, Faculty of Medical and Health Sciences, The University of Auckland, 85 Park Road, Auckland 1023, New Zealand mancy.tong@auckland.ac.nz.
authors.forename: Mancy
authors.initials: M
authors.lastname: Tong
authors.affiliation: Centre for Protein Research, Department of Biochemistry, University of Otago, Dunedin 9016, New Zealand.
authors.forename: Torsten
authors.initials: T
authors.lastname: Kleffmann
authors.affiliation: Department of Obstetrics and Gynaecology, Faculty of Medical and Health Sciences, The University of Auckland, 85 Park Road, Auckland 1023, New Zealand.
authors.forename: Shantanu
authors.initials: S
authors.lastname: Pradhan
authors.affiliation: Department of Obstetrics and Gynaecology, Faculty of Medical and Health Sciences, The University of Auckland, 85 Park Road, Auckland 1023, New Zealand Faculty of Medicine and Health Sciences, Linköping University, Linköping SE-581 83, Sweden.
authors.forename: Caroline L
authors.initials: CL
authors.lastname: Johansson
authors.affiliation: Maternal Fetal Medicine, Auckland City Hospital, Auckland 1023, New Zealand.
authors.forename: Joana
authors.initials: J
authors.lastname: DeSousa
authors.affiliation: Department of Obstetrics and Gynaecology, Faculty of Medical and Health Sciences, The University of Auckland, 85 Park Road, Auckland 1023, New Zealand Maternal Fetal Medicine, Auckland City Hospital, Auckland 1023, New Zealand.
authors.forename: Peter R
authors.initials: PR
authors.lastname: Stone
authors.affiliation: Department of Obstetrics and Gynaecology, Faculty of Medical and Health Sciences, The University of Auckland, 85 Park Road, Auckland 1023, New Zealand.
authors.forename: Joanna L
authors.initials: JL
authors.lastname: James
authors.affiliation: Department of Obstetrics and Gynaecology, Faculty of Medical and Health Sciences, The University of Auckland, 85 Park Road, Auckland 1023, New Zealand.
authors.forename: Qi
authors.initials: Q
authors.lastname: Chen
authors.affiliation: Department of Obstetrics and Gynaecology, Faculty of Medical and Health Sciences, The University of Auckland, 85 Park Road, Auckland 1023, New Zealand.
authors.forename: Larry W
authors.initials: LW
authors.lastname: Chamley
mesh_terms: D000029:Abortion, Legal; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D000067493:Dynamic Light Scattering; D000067128:Extracellular Vesicles / Q000737:chemistry / Q000502:physiology* / Q000648:ultrastructure; D005260:Female; D006801:Humans; D008431:Maternal-Fetal Exchange; D046529:Microscopy, Electron, Transmission; D009520:New Zealand; D010316:Particle Size; D010920:Placenta / Q000737:chemistry / Q000502:physiology* / Q000648:ultrastructure; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000737:chemistry / Q000502:physiology*; D011261:Pregnancy Trimester, First; D020543:Proteome / Q000737:chemistry / Q000502:physiology; D040901:Proteomics / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D046509:Tissue Culture Techniques
chemical_list: D011257:Pregnancy Proteins; D020543:Proteome
keywords: exosome; extracellular vesicle; microparticle; syncytial knots; syncytial nuclear aggregates; trophoblast deportation; trophoblastic debris