Protocol for Exosome Isolation from Small Volume of Ovarian Follicular Fluid: Evaluation of Ultracentrifugation and Commercial Kits

  • Shlomit KenigsbergEmail author
  • Brandon A. Wyse
  • Clifford L. Librach
  • Juliano C. da Silveira
Part of the Methods in Molecular Biology book series (MIMB, volume 1660)


The ovarian follicular fluid (FF) is a complex fluid that constitutes the microenvironment of developing follicles and contains factors secreted by the surrounding cells and blood plasma compounds that cross the “blood–follicle barrier.” Upon oocyte retrieval (in human, bovine, and equine) the follicular fluid is normally discarded and represents a repertoire of cellular messages exchanged during follicle development, thus providing a suitable sample for performing oocyte quality diagnostics. Several studies report on the presence of extracellular vesicles (EVs) in FF from human, bovine and equine. Here, we describe the process of FF collection from human and bovine and the enrichment and isolation of EVs that we termed folliculosomes (FFEs), using available commercial kits as well as the traditional ultracentrifugation methods.

Key words

Follicular fluid Extracellular vesicles Exosomes Folliculosomes Ultracentrifugation 



The authors would like to thank Arshia Azizeddin and Bahar Beharouzi for technical assistance, and CReATe IVF laboratory staff for help with sample collection.

Conflict of interest: Shlomit Kenigsberg and Brandon A. Wyse were research associates at Create Fertility Centre while conducting this research and writing the manuscript. Clifford L. Librach is the Medical Director and Owner of Create Fertility Centre. The authors have nothing else to declare.


  1. 1.
    Fortune JE, Rivera GM, Yang MY (2004) Follicular development: the role of the follicular microenvironment in selection of the dominant follicle. Anim Reprod Sci 82-83:109–126CrossRefPubMedGoogle Scholar
  2. 2.
    Rodgers RJ, Irving-Rodgers HF (2010) Formation of the ovarian follicular antrum and follicular fluid. Biol Reprod 82(6):1021–1029CrossRefPubMedGoogle Scholar
  3. 3.
    Revelli A et al (2009) Follicular fluid content and oocyte quality: from single biochemical markers to metabolomics. Reprod Biol Endocrinol 7:40CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Baka S, Malamitsi-Puchner A (2006) Novel follicular fluid factors influencing oocyte developmental potential in IVF: a review. Reprod Biomed Online 12(4):500–506CrossRefPubMedGoogle Scholar
  5. 5.
    Ambekar AS et al (2013) Proteomic analysis of human follicular fluid: a new perspective towards understanding folliculogenesis. J Proteome 87:68–77CrossRefGoogle Scholar
  6. 6.
    Dunning KR, Russell DL, Robker RL (2014) Lipids and oocyte developmental competence: the role of fatty acids and beta-oxidation. Reproduction 148(1):R15–R27CrossRefPubMedGoogle Scholar
  7. 7.
    Hsieh M, Zamah AM, Conti M (2009) Epidermal growth factor-like growth factors in the follicular fluid: role in oocyte development and maturation. Semin Reprod Med 27(1):52–61CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    O'Gorman A et al (2013) Metabolic profiling of human follicular fluid identifies potential biomarkers of oocyte developmental competence. Reproduction 146(4):389–395CrossRefPubMedGoogle Scholar
  9. 9.
    Leroy JL et al (2004) Metabolite and ionic composition of follicular fluid from different-sized follicles and their relationship to serum concentrations in dairy cows. Anim Reprod Sci 80(3–4):201–211CrossRefPubMedGoogle Scholar
  10. 10.
    Siu MK, Cheng CY (2012) The blood-follicle barrier (BFB) in disease and in ovarian function. Adv Exp Med Biol 763:186–192PubMedPubMedCentralGoogle Scholar
  11. 11.
    Mantzavinos T et al (1993) Immunoglobulins IgG, IgA, IgM, complement C3, C4 and ferritin and transferrin levels in serum and follicular fluid in IVF patients. Clin Exp Obstet Gynecol 20(1):32–36PubMedGoogle Scholar
  12. 12.
    Vlassov AV et al (2012) Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials. Biochim Biophys Acta 1820(7):940–948CrossRefPubMedGoogle Scholar
  13. 13.
    van der Pol E et al (2016) Recent developments in the nomenclature, presence, isolation, detection and clinical impact of extracellular vesicles. J Thromb Haemost 14(1):48–56CrossRefPubMedGoogle Scholar
  14. 14.
    Witwer KW et al (2013) Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J Extracell Vesicles 2. doi: 10.3402/jev.v2i0.20360
  15. 15.
    Raposo G, Stoorvogel W (2013) Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol 200(4):373–383CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Lasser C et al (2011) Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages. J Transl Med 9:9CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Keller S et al (2011) Body fluid derived exosomes as a novel template for clinical diagnostics. J Transl Med 9:86CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Revenfeld AL et al (2014) Diagnostic and prognostic potential of extracellular vesicles in peripheral blood. Clin Ther 36(6):830–846CrossRefPubMedGoogle Scholar
  19. 19.
    Yanez-Mo M et al (2015) Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicles 4:27066CrossRefPubMedGoogle Scholar
  20. 20.
    da Silveira JC et al (2012) Cell-secreted vesicles in equine ovarian follicular fluid contain miRNAs and proteins: a possible new form of cell communication within the ovarian follicle. Biol Reprod 86(3):71CrossRefPubMedGoogle Scholar
  21. 21.
    Sohel MM et al (2013) Exosomal and non-exosomal transport of extra-cellular microRNAs in follicular fluid: implications for bovine oocyte developmental competence. PLoS One 8(11):e78505CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Santonocito M et al (2014) Molecular characterization of exosomes and their microRNA cargo in human follicular fluid: bioinformatic analysis reveals that exosomal microRNAs control pathways involved in follicular maturation. Fertil Steril 102(6):1751–1761.e1CrossRefPubMedGoogle Scholar
  23. 23.
    da Silveira JC et al (2014) Regulation of ACVR1 and ID2 by cell-secreted exosomes during follicle maturation in the mare. Reprod Biol Endocrinol 12:44CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Machtinger R, Laurent LC, Baccarelli AA (2015) Extracellular vesicles: roles in gamete maturation, fertilization and embryo implantation. Hum Reprod Update 22(2):182–193PubMedPubMedCentralGoogle Scholar
  25. 25.
    Di Pietro C (2016) Exosome-mediated communication in the ovarian follicle. J Assist Reprod Genet 33(3):303–311CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Saez F, Frenette G, Sullivan R (2003) Epididymosomes and prostasomes: their roles in posttesticular maturation of the sperm cells. J Androl 24(2):149–154CrossRefPubMedGoogle Scholar
  27. 27.
    Sullivan R et al (2005) Role of exosomes in sperm maturation during the transit along the male reproductive tract. Blood Cells Mol Dis 35(1):1–10CrossRefPubMedGoogle Scholar
  28. 28.
    Hoog JL, Lotvall J (2015) Diversity of extracellular vesicles in human ejaculates revealed by cryo-electron microscopy. J Extracell Vesicles 4:28680CrossRefPubMedGoogle Scholar
  29. 29.
    Martin-DeLeon PA (2016) Uterosomes: exosomal cargo during the estrus cycle and interaction with sperm. Front Biosci (Schol Ed) 8:115–122CrossRefGoogle Scholar
  30. 30.
    Ng YH et al (2013) Endometrial exosomes/microvesicles in the uterine microenvironment: a new paradigm for embryo-endometrial cross talk at implantation. PLoS One 8(3):e58502CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Al-Dossary AA et al (2015) Oviductosome-sperm membrane interaction in cargo delivery. J Biol Chem 290(29):17710–17723CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Rienzi L et al (2012) The oocyte. Hum Reprod 27(Suppl 1):i2–21CrossRefPubMedGoogle Scholar
  33. 33.
    Lane RE et al (2015) Analysis of exosome purification methods using a model liposome system and tunable-resistive pulse sensing. Sci Rep 5:7639CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Thery C et al (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol. Chapter 3:Unit 3.22Google Scholar
  35. 35.
    Livshts MA et al (2015) Isolation of exosomes by differential centrifugation: theoretical analysis of a commonly used protocol. Sci Rep 5:17319CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Taylor DD, Shah S (2015) Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes. Methods 87:3–10CrossRefPubMedGoogle Scholar
  37. 37.
    Oksvold MP, Neurauter A, Pedersen KW (2015) Magnetic bead-based isolation of exosomes. Methods Mol Biol 1218:465–481CrossRefPubMedGoogle Scholar
  38. 38.
    Tauro BJ et al (2012) Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes. Methods 56(2):293–304CrossRefPubMedGoogle Scholar
  39. 39.
    Yuana Y et al (2015) Handling and storage of human body fluids for analysis of extracellular vesicles. J Extracell Vesicles 4:29260CrossRefPubMedGoogle Scholar
  40. 40.
    Van Deun J et al (2014) The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling. J Extracell Vesicles 3. doi: 10.3402/jev.v3.24858
  41. 41.
    Heinzelman P et al (2016) Nanoscale extracellular vesicle analysis in Alzheimer’s disease diagnosis and therapy. Int J Alzheimers Dis 2016:8053139PubMedPubMedCentralGoogle Scholar
  42. 42.
    Osteikoetxea X et al (2016) Extracellular vesicles in cardiovascular disease: are they Jedi or Sith? J Physiol 594(11):2881–2894CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Chen L et al (2015) Suppression of fibrogenic signaling in hepatic stellate cells by Twist1-dependent microRNA-214 expression: role of exosomes in horizontal transfer of Twist1. Am J Physiol Gastrointest Liver Physiol 309(6):G491–G499CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Ghosh A et al (2014) Rapid isolation of extracellular vesicles from cell culture and biological fluids using a synthetic peptide with specific affinity for heat shock proteins. PLoS One 9(10):e110443CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Hong CS et al (2016) Isolation of biologically active and morphologically intact exosomes from plasma of patients with cancer. J Extracell Vesicles 5:29289CrossRefPubMedGoogle Scholar
  46. 46.
    Vogel R et al (2016) A standardized method to determine the concentration of extracellular vesicles using tunable resistive pulse sensing. J Extracell Vesicles 5:31242CrossRefPubMedGoogle Scholar
  47. 47.
    Pieterse MC et al (1991) Transvaginal ultrasound guided follicular aspiration of bovine oocytes. Theriogenology 35(4):857–862CrossRefPubMedGoogle Scholar
  48. 48.
    Ludwig AK et al (2006) Perioperative and post-operative complications of transvaginal ultrasound-guided oocyte retrieval: prospective study of >1000 oocyte retrievals. Hum Reprod 21(12):3235–3240CrossRefPubMedGoogle Scholar
  49. 49.
    Carnevale EM, Maclellan LJ (2006) Collection, evaluation, and use of oocytes in equine assisted reproduction. Vet Clin North Am Equine Pract 22(3):843–856CrossRefPubMedGoogle Scholar
  50. 50.
    Rider MA, Hurwitz SN, Meckes DG Jr (2016) ExtraPEG: a polyethylene glycol-based method for enrichment of extracellular vesicles. Sci rep 6:23978CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Weng Y et al (2016) Effective isolation of exosomes with polyethylene glycol from cell culture supernatant for in-depth proteome profiling. Analyst 141(15):4640–4646CrossRefPubMedGoogle Scholar
  52. 52.
    Baranyai T et al (2015) Isolation of exosomes from blood plasma: qualitative and quantitative comparison of ultracentrifugation and size exclusion chromatography methods. PLoS One 10(12):e0145686CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Fahiminiya S et al (2011) Proteomic analysis of mare follicular fluid during late follicle development. Proteome Sci 9:54CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Muller L et al (2014) Isolation of biologically-active exosomes from human plasma. J Immunol Methods 411:55–65CrossRefPubMedPubMedCentralGoogle Scholar
  55. 55.
    van der Pol E et al (2014) Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing. J Thromb Haemost 12(7):1182–1192CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Shlomit Kenigsberg
    • 1
    Email author
  • Brandon A. Wyse
    • 1
  • Clifford L. Librach
    • 1
    • 2
    • 3
  • Juliano C. da Silveira
    • 4
  1. 1.CreATe Fertility CentreTorontoCanada
  2. 2.Department of Obstetrics and GynecologyUniversity of TorontoTorontoCanada
  3. 3.Department of GynecologyWomen’s College HospitalTorontoCanada
  4. 4.Department of Veterinary Medicine, Faculty of Animal Sciences and Food EngineeringUniversity of São PauloSão PauloBrazil

Personalised recommendations