Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Microfluidics facilitating the use of small extracellular vesicles in innovative approaches to male infertility

Abstract

Sperm are transcriptionally and translationally quiescent and, therefore, rely on the seminal plasma microenvironment for function, survival and fertilization of the oocyte in the oviduct. The male reproductive system influences sperm function via the binding and fusion of secreted epididymal (epididymosomes) and prostatic (prostasomes) small extracellular vesicles (S-EVs) that facilitate the transfer of proteins, lipids and nucleic acids to sperm. Seminal plasma S-EVs have important roles in sperm maturation, immune and oxidative stress protection, capacitation, fertilization and endometrial implantation and receptivity. Supplementing asthenozoospermic samples with normospermic-derived S-EVs can improve sperm motility and S-EV microRNAs can be used to predict non-obstructive azoospermia. Thus, S-EV influence on sperm physiology might have both therapeutic and diagnostic potential; however, the isolation of pure populations of S-EVs from bodily fluids with current conventional methods presents a substantial hurdle. Many conventional techniques lack accuracy, effectiveness, and practicality; yet microfluidic technology has the potential to simplify and improve S-EV isolation and detection.

Key points

  • Epididymosomes and prostasomes, the two distinct populations of seminal plasma small extracellular vesicles (S-EVs), have substantial roles in sperm function, survival and fertilization of the oocyte.

  • Male reproductive S-EV protein and microRNA cargo might serve as biomarkers for infertility and reproductive dysfunction.

  • Conventional methods of S-EV isolation require often laborious or costly workflows with suboptimal results but have received substantial interest in cancer therapeutics and diagnostics.

  • Microfluidic technology has the potential for miniaturization and simplification of S-EV isolation and analysis for use in point-of-need diagnostics in male infertility.

  • Infertility treatment can use technology developed for cancer diagnostics and therapeutics to approach idiopathic infertility.

This is a preview of subscription content, access via your institution

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: Biogenesis and release of EVs.
Fig. 2: Male reproductive S-EVs from the epididymis and prostate and their roles in sperm maturation, protection and interaction with the oocyte.
Fig. 3: Potential seminal plasma small extracellular vesicle protein biomarkers and their involvement in key events during sperm development and function.
Fig. 4: Conventional methods of isolating S-EVs and EVs from biofluids.
Fig. 5: Examples of microfluidic-based methods of S-EV isolation and detection.
Fig. 6: Idealized seminal plasma diagnostic systems.

References

  1. Arraud, N. et al. Extracellular vesicles from blood plasma: determination of their morphology, size, phenotype and concentration. J. Thromb. Haemost. 12, 614–627 (2014).

    Article  CAS  Google Scholar 

  2. Pisitkun, T., Shen, R.-F. & Knepper, M. A. Identification and proteomic profiling of exosomes in human urine. Proc. Natl Acad. Sci. USA 101, 13368–13373 (2004).

    Article  CAS  Google Scholar 

  3. Lässer, C. et al. Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages. J. Transl. Med. 9, 1–8 (2011).

    Article  Google Scholar 

  4. Emelyanov, A. et al. Cryo-electron microscopy of extracellular vesicles from cerebrospinal fluid. PLoS ONE 15, e0227949 (2020).

    Article  CAS  Google Scholar 

  5. Vallabhaneni, K. C. et al. Extracellular vesicles from bone marrow mesenchymal stem/stromal cells transport tumor regulatory microRNA, proteins, and metabolites. Oncotarget 6, 4953 (2015).

    Article  Google Scholar 

  6. Yáñez-Mó, M. et al. Biological properties of extracellular vesicles and their physiological functions. J. Extracell. Vesicles 4, 27066 (2015).

    Article  Google Scholar 

  7. Zaborowski, M. P., Balaj, L., Breakefield, X. O. & Lai, C. P. Extracellular vesicles: composition, biological relevance, and methods of study. Bioscience 65, 783–797 (2015).

    Article  Google Scholar 

  8. Deatherage, B. L. & Cookson, B. T. Membrane vesicle release in bacteria, eukaryotes, and archaea: a conserved yet underappreciated aspect of microbial life. Infect. Immun. 80, 1948–1957 (2012).

    Article  CAS  Google Scholar 

  9. Doyle, L. M. & Wang, M. Z. Overview of extracellular vesicles, their origin, composition, purpose, and methods for exosome isolation and analysis. Cells 8, 727 (2019).

    Article  CAS  Google Scholar 

  10. Colombo, M., Raposo, G. & Théry, C. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu. Rev. Cell Dev. Biol. 30, 255–289 (2014).

    Article  CAS  Google Scholar 

  11. Chargaff, E. & West, R. The biological significance of the thromboplastic protein of blood. J. Biol. Chem. 166, 189–197 (1946).

    Article  CAS  Google Scholar 

  12. Bonucci, E. Fine structure and histochemistry of “calcifying globules” in epiphyseal cartilage. Z. Zellforsch. Mikroskop. Anat. 103, 192–217 (1970).

    Article  CAS  Google Scholar 

  13. Pegtel, D. M. & Gould, S. J. Exosomes. Annu. Rev. Biochem. 88, 487–514 (2019).

    Article  CAS  Google Scholar 

  14. Valadi, H. et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat. Cell Biol. 9, 654–659 (2007).

    Article  CAS  Google Scholar 

  15. Men, Y. et al. Exosome reporter mice reveal the involvement of exosomes in mediating neuron to astroglia communication in the CNS. Nat. Commun. 10, 4136 (2019).

    Article  Google Scholar 

  16. Raposo, G. & Stoorvogel, W. Extracellular vesicles: exosomes, microvesicles, and friends. J. Cell Biol. 200, 373–383 (2013).

    Article  CAS  Google Scholar 

  17. Gurung, S., Perocheau, D., Touramanidou, L. & Baruteau, J. The exosome journey: from biogenesis to uptake and intracellular signalling. Cell Commun. Signal. 19, 47 (2021).

    Article  CAS  Google Scholar 

  18. Andaloussi, S. E., Mäger, I., Breakefield, X. O. & Wood, M. J. Extracellular vesicles: biology and emerging therapeutic opportunities. Nat. Rev. Drug Discov. 12, 347–357 (2013).

    Article  Google Scholar 

  19. Ettelaie, C., Collier, M. E., Maraveyas, A. & Ettelaie, R. Characterization of physical properties of tissue factor-containing microvesicles and a comparison of ultracentrifuge-based recovery procedures. J. Extracell. Vesicles 3, 23592 (2014).

    Article  Google Scholar 

  20. Battistelli, M. & Falcieri, E. Apoptotic bodies: particular extracellular vesicles involved in intercellular communication. Biology 9, 21 (2020).

    Article  CAS  Google Scholar 

  21. Ihara, T., Yamamoto, T., Sugamata, M., Okumura, H. & Ueno, Y. The process of ultrastructural changes from nuclei to apoptotic body. Virchows Arch. 433, 443–447 (1998).

    Article  CAS  Google Scholar 

  22. Hristov, M., Erl, W., Linder, S. & Weber, P. C. Apoptotic bodies from endothelial cells enhance the number and initiate the differentiation of human endothelial progenitor cells in vitro. Blood 104, 2761–2766 (2004).

    Article  CAS  Google Scholar 

  23. Elmore, S. Apoptosis: a review of programmed cell death. Toxicol. Pathol. 35, 495–516 (2007).

    Article  CAS  Google Scholar 

  24. Ronquist, G., Brody, I., Gottfries, A. & Stegmayr, B. An Mg2+ and Ca2+‐stimulated adenosine triphosphatase in human prostatic fluid — part II. Andrologia 10, 427–433 (1978).

    Article  CAS  Google Scholar 

  25. Murdica, V. et al. Seminal plasma of men with severe asthenozoospermia contain exosomes that affect spermatozoa motility and capacitation. Fertil. Steril. 111, 897–908. e892 (2019).

    Article  CAS  Google Scholar 

  26. Lin, Y. et al. Proteomic analysis of seminal extracellular vesicle proteins involved in asthenozoospermia by iTRAQ. Mol. Reprod. Dev. 86, 1094–1105 (2019).

    Article  CAS  Google Scholar 

  27. Park, K.-H. et al. Ca2+ signaling tools acquired from prostasomes are required for progesterone-induced sperm motility. Sci. Signal. 4, ra31 (2011).

    Article  Google Scholar 

  28. Aalberts, M. et al. Spermatozoa recruit prostasomes in response to capacitation induction. Biochim. Biophys. Acta 1834, 2326–2335 (2013).

    Article  CAS  Google Scholar 

  29. Whitesides, G. M. The origins and the future of microfluidics. Nature 442, 368–373 (2006).

    Article  CAS  Google Scholar 

  30. Yasui, T. et al. Unveiling massive numbers of cancer-related urinary-microRNA candidates via nanowires. Sci. Adv. 3, e1701133 (2017).

    Article  Google Scholar 

  31. Suwatthanarak, T. et al. Microfluidic-based capture and release of cancer-derived exosomes via peptide–nanowire hybrid interface. Lab Chip 21, 597–607 (2021).

    Article  CAS  Google Scholar 

  32. Xu, H., Liao, C., Zuo, P., Liu, Z. & Ye, B. C. Magnetic-based microfluidic device for on-chip isolation and detection of tumor-derived exosomes. Anal. Chem. 90, 13451–13458 (2018).

    Article  CAS  Google Scholar 

  33. Zhang, P. et al. Ultrasensitive detection of circulating exosomes with a 3D-nanopatterned microfluidic chip. Nat. Biomed. Eng. 3, 438–451 (2019).

    Article  CAS  Google Scholar 

  34. Kanwar, S. S., Dunlay, C. J., Simeone, D. M. & Nagrath, S. Microfluidic device (ExoChip) for on-chip isolation, quantification and characterization of circulating exosomes. Lab Chip 14, 1891–1900 (2014).

    Article  CAS  Google Scholar 

  35. Smith, J. T. et al. Integrated nanoscale deterministic lateral displacement arrays for separation of extracellular vesicles from clinically-relevant volumes of biological samples. Lab Chip 18, 3913–3925 (2018).

    Article  CAS  Google Scholar 

  36. Wang, Y. et al. Microfluidic Raman biochip detection of exosomes: a promising tool for prostate cancer diagnosis. Lab Chip 20, 4632–4637 (2020).

    Article  CAS  Google Scholar 

  37. Machtinger, R., Laurent, L. C. & Baccarelli, A. A. Extracellular vesicles: roles in gamete maturation, fertilization and embryo implantation. Hum. Reprod. Update 22, 182–193 (2016).

    CAS  Google Scholar 

  38. Harding, C. V., Heuser, J. E. & Stahl, P. D. Exosomes: looking back three decades and into the future. J. Cell Biol. 200, 367–371 (2013).

    Article  CAS  Google Scholar 

  39. Zheng, R. et al. Exosome-transmitted long non-coding RNA PTENP1 suppresses bladder cancer progression. Mol. Cancer 17, 1–13 (2018).

    Article  CAS  Google Scholar 

  40. Zamani, P., Fereydouni, N., Butler, A. E., Navashenaq, J. G. & Sahebkar, A. The therapeutic and diagnostic role of exosomes in cardiovascular diseases. Trends Cardiovasc. Med. 29, 313–323 (2019).

    Article  CAS  Google Scholar 

  41. Pan, B.-T. & Johnstone, R. M. Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: selective externalization of the receptor. Cell 33, 967–978 (1983).

    Article  CAS  Google Scholar 

  42. Cocucci, E., Racchetti, G. & Meldolesi, J. Shedding microvesicles: artefacts no more. Trends Cell Biol. 19, 43–51 (2009).

    Article  CAS  Google Scholar 

  43. Denzer, K., Kleijmeer, M. J., Heijnen, H. F. G., Stoorvogel, W. & Geuze, H. J. Exosome: from internal vesicle of the multivesicular body to intercellular signaling device. J. Cell Sci. 113, 3365–3374 (2000).

    Article  CAS  Google Scholar 

  44. Zhang, M., Ouyang, H. & Xia, G. The signal pathway of gonadotrophins-induced mammalian oocyte meiotic resumption. Mol. Hum. Reprod. 15, 399–409 (2009).

    Article  Google Scholar 

  45. Momen-Heravi, F. et al. Impact of biofluid viscosity on size and sedimentation efficiency of the isolated microvesicles. Front. Physiol. 3, 162 (2012).

    Article  CAS  Google Scholar 

  46. Marleau, A. M., Chen, C.-S., Joyce, J. A. & Tullis, R. H. Exosome removal as a therapeutic adjuvant in cancer. J. Transl. Med. 10, 1–12 (2012).

    Article  Google Scholar 

  47. Sandfeld-Paulsen, B. et al. Exosomal proteins as diagnostic biomarkers in lung cancer. J. Thorac. Oncol. 11, 1701–1710 (2016).

    Article  Google Scholar 

  48. Yuyama, K., Sun, H., Mitsutake, S. & Igarashi, Y. Sphingolipid-modulated exosome secretion promotes clearance of amyloid-β by microglia. J. Biol. Chem. 287, 10977–10989 (2012).

    Article  CAS  Google Scholar 

  49. Tomlinson, P. R. et al. Identification of distinct circulating exosomes in Parkinson’s disease. Ann. Clin. Transl. Neurol. 2, 353–361 (2015).

    Article  Google Scholar 

  50. Feng, J., Waqas, A., Zhu, Z. & Chen, L. Exosomes: applications in respiratory infectious diseases and prospects for coronavirus disease 2019 (COVID-19). J. Biomed. Nanotechnol. 16, 399–418 (2020).

    Article  CAS  Google Scholar 

  51. Nagashima, S. et al. Hepatitis E virus egress depends on the exosomal pathway, with secretory exosomes derived from multivesicular bodies. J. Gen. Virol. 95, 2166–2175 (2014).

    Article  Google Scholar 

  52. Martin-DeLeon, P. A. Uterosomes: exosomal cargo during the estrus cycle and interaction with sperm. Front. Biosci. 8, 115–122 (2016).

    Article  Google Scholar 

  53. Panner Selvam, M. K., Agarwal, A., Pushparaj, P. N., Baskaran, S. & Bendou, H. Sperm proteome analysis and identification of fertility-associated biomarkers in unexplained male infertility. Genes 10, 522 (2019).

    Article  Google Scholar 

  54. Rao, M. et al. Humanin levels in human seminal plasma and spermatozoa are related to sperm quality. Andrology 7, 859–866 (2019).

    Article  CAS  Google Scholar 

  55. Poliakov, A., Spilman, M., Dokland, T., Amling, C. L. & Mobley, J. A. Structural heterogeneity and protein composition of exosome‐like vesicles (prostasomes) in human semen. Prostate 69, 159–167 (2009).

    Article  Google Scholar 

  56. Del Giudice, P. T. et al. Determination of testicular function in adolescents with varicocoele — a proteomics approach. Andrology 4, 447–455 (2016).

    Article  Google Scholar 

  57. Ronquist, G. & Hedström, M. Restoration of detergent-inactivated adenosine triphosphatase activity of human prostatic fluid with concanavalin A. Biochim. Biophys. Acta 483, 483–486 (1977).

    Article  CAS  Google Scholar 

  58. Brody, I., Ronquist, G. & Gottfries, A. Ultrastructural localization of the prostasome-an organelle in human seminal plasma. Ups. J. Med. Sci. 88, 63–80 (1983).

    Article  CAS  Google Scholar 

  59. Ronquist, G. & Brody, I. The prostasome: its secretion and function in man. Biochim. Biophys. Acta 822, 203–218 (1985).

    Article  CAS  Google Scholar 

  60. Ronquist, G. & Nilsson, B. O. The Janus-faced nature of prostasomes: their pluripotency favours the normal reproductive process and malignant prostate growth. Prostate Cancer Prostatic Dis. 7, 21–31 (2004).

    Article  CAS  Google Scholar 

  61. Utleg, A. G. et al. Proteomic analysis of human prostasomes. Prostate 56, 150–161 (2003).

    Article  CAS  Google Scholar 

  62. Stegmayr, B. & Ronquist, G. Promotive effect on human sperm progressive motility by prostasomes. Urol. Res. 10, 253–257 (1982).

    Article  CAS  Google Scholar 

  63. Thimon, V., Frenette, G., Saez, F., Thabet, M. & Sullivan, R. Protein composition of human epididymosomes collected during surgical vasectomy reversal: a proteomic and genomic approach. Hum. Reprod. 23, 1698–1707 (2008).

    Article  CAS  Google Scholar 

  64. Sahlén, G. et al. Secretions from seminal vesicles lack characteristic markers for prostasomes. Ups. J. Med. Sci. 115, 107–112 (2010).

    Article  Google Scholar 

  65. Le Tortorec, A. et al. From ancient to emerging infections: the odyssey of viruses in the male genital tract. Physiol. Rev. 100, 1349–1414 (2020).

    Article  Google Scholar 

  66. Agrawal, Y. & Vanha-Perttula, T. Effect of secretory particles in bovine seminal vesicle secretion on sperm motility and acrosome reaction. Reproduction 79, 409–419 (1987).

    Article  CAS  Google Scholar 

  67. Zhang, X., Vos, H. R., Tao, W. & Stoorvogel, W. Proteomic profiling of two distinct populations of extracellular vesicles isolated from human seminal plasma. Int. J. Mol. Sci. 21, 7957 (2020).

    Article  CAS  Google Scholar 

  68. Tauber, P., Zaneveld, L., Propping, D. & Schumacher, G. Components of human split ejaculates. Reproduction 43, 249–267 (1975).

    Article  CAS  Google Scholar 

  69. Taylor, P. & Kelly, R. 19-HydroxyIated E prostaglandins as the major prostaglandins of human semen. Nature 250, 665–667 (1974).

    Article  CAS  Google Scholar 

  70. Robert, M. & Gagnon, C. Semenogelin I: a coagulum forming, multifunctional seminal vesicle protein. Cell. Mol. Life Sci. 55, 944–960 (1999).

    Article  CAS  Google Scholar 

  71. Belleannée, C., Calvo, É., Caballero, J. & Sullivan, R. Epididymosomes convey different repertoires of microRNAs throughout the bovine epididymis. Biol. Reprod. 89, 30 (2013).

    Article  Google Scholar 

  72. Conine, C. C., Sun, F., Song, L., Rivera-Pérez, J. A. & Rando, O. J. Small RNAs gained during epididymal transit of sperm are essential for embryonic development in mice. Dev. Cell 46, 470–480. e473 (2018).

    Article  CAS  Google Scholar 

  73. Grunewald, S., Paasch, U., Glander, H. J. & Anderegg, U. Mature human spermatozoa do not transcribe novel RNA. Andrologia 37, 69–71 (2005).

    Article  CAS  Google Scholar 

  74. Goodrich, R. J., Anton, E. & Krawetz, S. A. in Spermatogenesis (eds Carrell, D. T. & Aston, K.I.) 385–396 (Springer, 2013).

  75. Rodriguez-Caro, H. et al. In vitro decidualisation of human endometrial stromal cells is enhanced by seminal fluid extracellular vesicles. J. Extracell. Vesicles 8, 1565262 (2019).

    Article  CAS  Google Scholar 

  76. Bai, R. et al. Induction of immune-related gene expression by seminal exosomes in the porcine endometrium. Biochem. Biophys. Res. Commun. 495, 1094–1101 (2018).

    Article  CAS  Google Scholar 

  77. Wang, D. et al. Seminal plasma and seminal plasma exosomes of aged male mice affect early embryo implantation via immunomodulation. Front. Immunol. 12, 723409 (2021).

    Article  CAS  Google Scholar 

  78. Yanagimachi, R., Kamiguchi, Y., Mikamo, K., Suzuki, F. & Yanagimachi, H. Maturation of spermatozoa in the epididymis of the Chinese hamster. Am. J. Anat. 172, 317–330 (1985).

    Article  CAS  Google Scholar 

  79. Thimon, V., Koukoui, O., Calvo, E. & Sullivan, R. Region-specific gene expression profiling along the human epididymis. Mol. Hum. Reprod. 13, 691–704 (2007).

    Article  CAS  Google Scholar 

  80. Frenette, G. & Sullivan, R. Prostasome‐like particles are involved in the transfer of P25b from the bovine epididymal fluid to the sperm surface. Mol. Reprod. Dev. 59, 115–121 (2001).

    Article  CAS  Google Scholar 

  81. Frenette, G., Lessard, C. & Sullivan, R. Selected proteins of “prostasome-like particles” from epididymal cauda fluid are transferred to epididymal caput spermatozoa in bull. Biol. Reprod. 67, 308–313 (2002).

    Article  CAS  Google Scholar 

  82. Rejraji, H. et al. Lipid remodeling of murine epididymosomes and spermatozoa during epididymal maturation. Biol. Reprod. 74, 1104–1113 (2006).

    Article  CAS  Google Scholar 

  83. Griffiths, G. S., Galileo, D. S., Reese, K. & Martin‐DeLeon, P. A. Investigating the role of murine epididymosomes and uterosomes in GPI‐linked protein transfer to sperm using SPAM1 as a model. Mol. Reprod. Dev. 75, 1627–1636 (2008).

    Article  CAS  Google Scholar 

  84. Ecroyd, H., Sarradin, P., Dacheux, J.-L. & Gatti, J.-L. Compartmentalization of prion isoforms within the reproductive tract of the ram. Biol. Reprod. 71, 993–1001 (2004).

    Article  CAS  Google Scholar 

  85. Gatti, J.-L. et al. Post-testicular sperm environment and fertility. Anim. Reprod. Sci. 82, 321–339 (2004).

    Article  Google Scholar 

  86. Fornes, M., Barbieri, A. & Cavicchia, J. Morphological and enzymatic study of membrane‐bound vesicles from the lumen of the rat epididymis. Andrologia 27, 1–5 (1995).

    Article  CAS  Google Scholar 

  87. Grimalt, P., Bertini, F. & Fornes, M. High-affinity sites for β-D-galactosidase on membrane-bound vesicles isolated from rat epididymal fluid. Arch. Androl. 44, 85–91 (2000).

    Article  CAS  Google Scholar 

  88. Candenas, L. & Chianese, R. Exosome composition and seminal plasma proteome: a promising source of biomarkers of male infertility. Int. J. Mol. Sci. 21 (2020).

  89. Johnston, D. S. et al. The mouse epididymal transcriptome: transcriptional profiling of segmental gene expression in the epididymis. Biol. Reprod. 73, 404–413 (2005).

    Article  CAS  Google Scholar 

  90. Zhou, W., De Iuliis, G. N., Dun, M. D. & Nixon, B. Characteristics of the epididymal luminal environment responsible for sperm maturation and storage. Front. Endocrinol. 9, 59 (2018).

    Article  Google Scholar 

  91. Nixon, B. et al. The identification of mouse sperm-surface-associated proteins and characterization of their ability to act as decapacitation factors. Biol. Reprod. 74, 275–287 (2006).

    Article  CAS  Google Scholar 

  92. Dacheux, J. & Voglmayr, J. Sequence of sperm cell surface differentiation and its relationship to exogenous fluid proteins in the ram epididymis. Biol. Reprod. 29, 1033–1046 (1983).

    Article  CAS  Google Scholar 

  93. Dacheux, J.-L. et al. Mammalian epididymal proteome. Mol. Cell. Endocrinol. 306, 45–50 (2009).

    Article  CAS  Google Scholar 

  94. Belleannée, C., Thimon, V. & Sullivan, R. Region-specific gene expression in the epididymis. Cell Tissue Res. 349, 717–731 (2012).

    Article  Google Scholar 

  95. Ecroyd, H., Belghazi, M., Dacheux, J.-L. & Gatti, J.-L. The epididymal soluble prion protein forms a high-molecular-mass complex in association with hydrophobic proteins. Biochem. J. 392, 211–219 (2005).

    Article  CAS  Google Scholar 

  96. Girouard, J., Frenette, G. & Sullivan, R. Comparative proteome and lipid profiles of bovine epididymosomes collected in the intraluminal compartment of the caput and cauda epididymidis. Int. J. Androl. 34, e475–e486 (2011).

    Article  CAS  Google Scholar 

  97. Nixon, B. et al. Proteomic profiling of mouse epididymosomes reveals their contributions to post-testicular sperm maturation. Mol. Cell. Proteom. 18, S91–S108 (2019).

    Article  CAS  Google Scholar 

  98. Johnson, A. L. & Howards, S. S. Intratubular hydrostatic pressure in testis and epididymis before and after long-term vasectomy in the guinea pig. Biol. Reprod. 14, 371–376 (1976).

    Article  CAS  Google Scholar 

  99. Turner, T., Gleavy, J. & Harris, J. Fluid movement in the lumen of the rat epididymis: effect of vasectomy and subsequent vasovasostomy. J. Androl. 11, 422–428 (1990).

    CAS  Google Scholar 

  100. Dacheux, J.-L. et al. The contribution of proteomics to understanding epididymal maturation of mammalian spermatozoa. Syst. Biol. Reprod. Med. 58, 197–210 (2012).

    Article  CAS  Google Scholar 

  101. Girouard, J., Frenette, G. & Sullivan, R. Compartmentalization of proteins in epididymosomes coordinates the association of epididymal proteins with the different functional structures of bovine spermatozoa. Biol. Reprod. 80, 965–972 (2009).

    Article  CAS  Google Scholar 

  102. Zhou, W. et al. Mechanisms of tethering and cargo transfer during epididymosome-sperm interactions. BMC Biol. 17, 35 (2019).

    Article  Google Scholar 

  103. Edidin, M. The state of lipid rafts: from model membranes to cells. Annu. Rev. Biophys. Biomol. Struct. 32, 257–283 (2003).

    Article  CAS  Google Scholar 

  104. Sengupta, P., Baird, B. & Holowka, D. Lipid rafts, fluid/fluid phase separation, and their relevance to plasma membrane structure and function. Semin. Cell Dev. Biol. 18, 583–590 (2007).

    Article  CAS  Google Scholar 

  105. Aitken, R. J. & De Iuliis, G. N. Origins and consequences of DNA damage in male germ cells. Reprod. Biomed. Online 14, 727–733 (2007).

    Article  CAS  Google Scholar 

  106. Rooney, I. A., Heuser, J. E. & Atkinson, J. P. GPI-anchored complement regulatory proteins in seminal plasma. An analysis of their physical condition and the mechanisms of their binding to exogenous cells. J. Clin. Invest. 97, 1675–1686 (1996).

    Article  CAS  Google Scholar 

  107. Sloand, E. M. et al. Correction of the PNH defect by GPI-anchored protein transfer. Blood 92, 4439–4445 (1998).

    Article  CAS  Google Scholar 

  108. Samanta, L., Parida, R., Dias, T. R. & Agarwal, A. The enigmatic seminal plasma: a proteomics insight from ejaculation to fertilization. Reprod. Biol. Endocrinol. 16, 41 (2018).

    Article  Google Scholar 

  109. Caballero, J. N., Frenette, G., Belleannée, C. & Sullivan, R. CD9-positive microvesicles mediate the transfer of molecules to bovine spermatozoa during epididymal maturation. PLoS ONE 8, e65364 (2013).

    Article  CAS  Google Scholar 

  110. Sullivan, R., Frenette, G. & Girouard, J. Epididymosomes are involved in the acquisition of new sperm proteins during epididymal transit. Asian J. Androl. 9, 483–491 (2007).

    Article  CAS  Google Scholar 

  111. Sullivan, R. Epididymosomes: a heterogeneous population of microvesicles with multiple functions in sperm maturation and storage. Asian J. Androl. 17, 726 (2015).

    Article  CAS  Google Scholar 

  112. D’Amours, O. et al. Evidences of biological functions of biliverdin reductase A in the bovine epididymis. J. Cell. Physiol. 231, 1077–1089 (2016).

    Article  Google Scholar 

  113. Hu, J. et al. Epididymal cysteine-rich secretory proteins are required for epididymal sperm maturation and optimal sperm function. Mol. Hum. Reprod. 24, 111–122 (2018).

    Article  CAS  Google Scholar 

  114. Martin-DeLeon, P. A. Epididymal SPAM1 and its impact on sperm function. Mol. Cell. Endocrinol. 250, 114–121 (2006).

    Article  CAS  Google Scholar 

  115. Eickhoff, R. et al. Influence of macrophage migration inhibitory factor (MIF) on the zinc content and redox state of protein-bound sulphydryl groups in rat sperm: indications for a new role of MIF in sperm maturation. Mol. Hum. Reprod. 10, 605–611 (2004).

    Article  CAS  Google Scholar 

  116. Saez, F., Frenette, G. & Sullivan, R. Epididymosomes and prostasomes: their roles in posttesticular maturation of the sperm cells. J. Androl. 24, 149–154 (2003).

    Article  Google Scholar 

  117. Patel, R. et al. Plasma membrane Ca2+-ATPase 4 in murine epididymis: secretion of splice variants in the luminal fluid and a role in sperm maturation. Biol. Reprod. 89, 1–11 (2013).

    Article  Google Scholar 

  118. Sullivan, R. Epididymosomes: role of extracellular microvesicles in sperm maturation. Front. Biosci. 8, 106–114 (2016).

    Article  Google Scholar 

  119. Simon, C. et al. Extracellular vesicles in human reproduction in health and disease. Endocr. Rev. 39, 292–332 (2018).

    Article  Google Scholar 

  120. Ishijima, S., Okuno, M. & Mohri, H. Zeta potential of human X‐ and Y‐bearing sperm. Int. J. Androl. 14, 340–347 (1991).

    Article  CAS  Google Scholar 

  121. Cuasnicú, P. S. et al. in The Epididymis: From Molecules to Clinical Practice (eds Robaire, B. & Hinton, B. T.) 389–403 (Springer, 2002).

  122. Kirchhoff, C. & Hale, G. Cell-to-cell transfer of glycosylphosphatidylinositol-anchored membrane proteins during sperm maturation. Mol. Hum. Reprod. 2, 177–184 (1996).

    Article  CAS  Google Scholar 

  123. Cooper, T. G. & Yeung, C.-H. in Sperm Cell: Production, Maturation, Fertilization, Regeneration (eds De Jonge, C. & Barratt, C.) 72–107 (Cambridge Univ. Press, 2006).

  124. Miller, D., Brinkworth, M. & Iles, D. Paternal DNA packaging in spermatozoa: more than the sum of its parts? DNA, histones, protamines and epigenetics. Reproduction 139, 287–301 (2010).

    Article  CAS  Google Scholar 

  125. Cooper, T. in Tissue Renin-Angiotensin Systems (eds Mukhopadhyay, A. K. & Raizada, M. K.) 87–101 (Springer, 1995).

  126. Jones, R. Membrane remodelling during sperm maturation in the epididymis. Oxf. Rev. Reprod. Biol. 11, 285–337 (1989).

    CAS  Google Scholar 

  127. Vickram, A. et al. Seminal exosomes — an important biological marker for various disorders and syndrome in human reproduction. Saudi J. Biol. Sci. 28, 3607–3615 (2021).

    Article  CAS  Google Scholar 

  128. Llorente, A., de Marco, M. C. & Alonso, M. A. Caveolin-1 and MAL are located on prostasomes secreted by the prostate cancer PC-3 cell line. J. Cell Sci. 117, 5343–5351 (2004).

    Article  CAS  Google Scholar 

  129. Llorente, A., van Deurs, B. & Sandvig, K. Cholesterol regulates prostasome release from secretory lysosomes in PC-3 human prostate cancer cells. Eur. J. Cell Biol. 86, 405–415 (2007).

    Article  CAS  Google Scholar 

  130. Aalberts, M., Stout, T. & Stoorvogel, W. Prostasomes: extracellular vesicles from the prostate. Reproduction 147, R1–R14 (2014).

    Article  CAS  Google Scholar 

  131. Sullivan, R. & Saez, F. Epididymosomes, prostasomes, and liposomes: their roles in mammalian male reproductive physiology. Reproduction 146, R21–R35 (2013).

    Article  CAS  Google Scholar 

  132. Brouwers, J. F. et al. Distinct lipid compositions of two types of human prostasomes. Proteomics 13, 1660–1666 (2013).

    Article  CAS  Google Scholar 

  133. Arienti, G., Carlini, E. & Palmerini, C. Fusion of human sperm to prostasomes at acidic pH. J. Membr. Biol. 155, 89–94 (1997).

    Article  CAS  Google Scholar 

  134. Aalberts, M. et al. Identification of distinct populations of prostasomes that differentially express prostate stem cell antigen, annexin A1, and GLIPR2 in humans. Biol. Reprod. 86, 82–82 (2012).

    Article  Google Scholar 

  135. Publicover, S., Harper, C. V. & Barratt, C. [Ca2+]i signalling in sperm — making the most of what you’ve got. Nat. Cell Biol. 9, 235–242 (2007).

    Article  CAS  Google Scholar 

  136. Bailey, J. L. Factors regulating sperm capacitation. Syst. Biol. Reprod. Med. 56, 334–348 (2010).

    Article  CAS  Google Scholar 

  137. Fraser, L. R. The “switching on” of mammalian spermatozoa: molecular events involved in promotion and regulation of capacitation. Mol. Reprod. Dev. 77, 197–208 (2010).

    Article  CAS  Google Scholar 

  138. Harrison, R. & Miller, N. cAMP‐dependent protein kinase control of plasma membrane lipid architecture in boar sperm. Mol. Reprod. Dev. 55, 220–228 (2000).

    Article  CAS  Google Scholar 

  139. Pons-Rejraji, H. et al. Prostasomes: inhibitors of capacitation and modulators of cellular signalling in human sperm. Int. J. Androl. 34, 568–580 (2011).

    Article  CAS  Google Scholar 

  140. García-Rodríguez, A., Gosálvez, J., Agarwal, A., Roy, R. & Johnston, S. DNA damage and repair in human reproductive cells. Int. J. Mol. Sci. 20, 31 (2019).

    Article  Google Scholar 

  141. Clark, G. F. & Schust, D. J. Manifestations of immune tolerance in the human female reproductive tract. Front. Immunol. 4, 26 (2013).

    Article  CAS  Google Scholar 

  142. Vojtech, L. et al. Exosomes in human semen carry a distinctive repertoire of small non-coding RNAs with potential regulatory functions. Nucleic Acids Res. 42, 7290–7304 (2014).

    Article  CAS  Google Scholar 

  143. Kelly, R. et al. Extracellular organelles (prostasomes) are immunosuppressive components of human semen. Clin. Exp. Immunol. 86, 550–556 (1991).

    Article  CAS  Google Scholar 

  144. Johansson, M., Bromfield, J. J., Jasper, M. J. & Robertson, S. A. Semen activates the female immune response during early pregnancy in mice. Immunology 112, 290–300 (2004).

    Article  CAS  Google Scholar 

  145. Robertson, S. A., Guerin, L. R., Moldenhauer, L. M. & Hayball, J. D. Activating T regulatory cells for tolerance in early pregnancy — the contribution of seminal fluid. J. Reprod. Immunol. 83, 109–116 (2009).

    Article  CAS  Google Scholar 

  146. Malla, B., Zaugg, K., Vassella, E., Aebersold, D. M. & Dal Pra, A. Exosomes and exosomal microRNAs in prostate cancer radiation therapy. Int. J. Radiat. Oncol. Biol. Phys. 98, 982–995 (2017).

    Article  CAS  Google Scholar 

  147. Yang, C. et al. Comprehensive proteomics analysis of exosomes derived from human seminal plasma. Andrology 5, 1007–1015 (2017).

    Article  CAS  Google Scholar 

  148. Taylor, D. D. & Gercel-Taylor, C. MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer. Gynecol. Oncol. 110, 13–21 (2008).

    Article  CAS  Google Scholar 

  149. Milardi, D. et al. Proteomic approach in the identification of fertility pattern in seminal plasma of fertile men. Fertil. Steril. 97, 67–73.e61 (2012).

    Article  CAS  Google Scholar 

  150. De Lazari, F. L. et al. Seminal plasma proteins and their relationship with sperm motility and morphology in boars. Andrologia 51, e13222 (2019).

    Article  Google Scholar 

  151. Gilany, K., Minai-Tehrani, A., Savadi-Shiraz, E., Rezadoost, H. & Lakpour, N. Exploring the human seminal plasma proteome: an unexplored gold mine of biomarker for male infertility and male reproduction disorder. J. Reprod. Infertil. 16, 61 (2015).

    Google Scholar 

  152. Vernet, P., Aitken, R. & Drevet, J. Antioxidant strategies in the epididymis. Mol. Cell. Endocrinol. 216, 31–39 (2004).

    Article  CAS  Google Scholar 

  153. Chabory, E. et al. Epididymis seleno-independent glutathione peroxidase 5 maintains sperm DNA integrity in mice. J. Clin. Invest. 119, 2074–2085 (2009).

    CAS  Google Scholar 

  154. Buschow, S. I. et al. MHC II in dendritic cells is targeted to lysosomes or T cell-induced exosomes via distinct multivesicular body pathways. Traffic 10, 1528–1542 (2009).

    Article  CAS  Google Scholar 

  155. Gibbs, G. M. et al. Glioma pathogenesis-related 1-like 1 is testis enriched, dynamically modified, and redistributed during male germ cell maturation and has a potential role in sperm-oocyte binding. Endocrinology 151, 2331–2342 (2010).

    Article  CAS  Google Scholar 

  156. Ronquist, G. The Male Role in Pregnancy Loss and Embryo Implantation Failure (ed. Bronson, R.) 191–209 (Springer, 2015).

  157. Tarazona, R. et al. Human prostasomes express CD48 and interfere with NK cell function. Immunobiology 216, 41–46 (2011).

    Article  CAS  Google Scholar 

  158. Burden, H., Holmes, C., Persad, R. & Whittington, K. Prostasomes — their effects on human male reproduction and fertility. Hum. Reprod. Update 12, 283–292 (2006).

    Article  CAS  Google Scholar 

  159. Garcia-Rodriguez, A., de la Casa, M., Peinado, H., Gosalvez, J. & Roy, R. Human prostasomes from normozoospermic and non-normozoospermic men show a differential protein expression pattern. Andrology 6, 585–596 (2018).

    Article  CAS  Google Scholar 

  160. Ernesto, J. I. et al. CRISP1 as a novel CatSper regulator that modulates sperm motility and orientation during fertilization. J. Cell Biol. 210, 1213–1224 (2015).

    Article  CAS  Google Scholar 

  161. Roberts, K. P. et al. Epididymal secreted protein Crisp-1 and sperm function. Mol. Cell. Endocrinol. 250, 122–127 (2006).

    Article  CAS  Google Scholar 

  162. Roberts, K. P., Wamstad, J. A., Ensrud, K. M. & Hamilton, D. W. Inhibition of capacitation-associated tyrosine phosphorylation signaling in rat sperm by epididymal protein Crisp-1. Biol. Reprod. 69, 572–581 (2003).

    Article  CAS  Google Scholar 

  163. Weigel Muñoz, M. et al. Influence of the genetic background on the reproductive phenotype of mice lacking Cysteine-Rich Secretory Protein 1 (CRISP1). Biol. Reprod. 99, 373–383 (2018).

    Article  Google Scholar 

  164. Maldera, J. A. et al. Human fertilization: epididymal hCRISP1 mediates sperm–zona pellucida binding through its interaction with ZP3. Mol. Hum. Reprod. 20, 341–349 (2014).

    Article  CAS  Google Scholar 

  165. Da Ros, V. G. et al. Impaired sperm fertilizing ability in mice lacking Cysteine-RIch Secretory Protein 1 (CRISP1). Dev. Biol. 320, 12–18 (2008).

    Article  Google Scholar 

  166. Miki, K. Energy metabolism and sperm function. Soc. Reprod. Fertil. Suppl. 65, 309–325 (2007).

    CAS  Google Scholar 

  167. Odet, F. et al. Lactate dehydrogenase C and energy metabolism in mouse sperm. Biol. Reprod. 85, 556–564 (2011).

    Article  CAS  Google Scholar 

  168. Rolland, A. D. et al. Identification of genital tract markers in the human seminal plasma using an integrative genomics approach. Hum. Reprod. 28, 199–209 (2013).

    Article  CAS  Google Scholar 

  169. Li, S. S.-L. et al. Differential activity and synthesis of lactate dehydrogenase isozymes A (muscle), B (heart), and C (testis) in mouse spermatogenic cells. Biol. Reprod. 40, 173–180 (1989).

    Article  CAS  Google Scholar 

  170. O’Flaherty, C., Beorlegui, N. & Beconi, M. Lactate dehydrogenase‐C4 is involved in heparin‐and NADH‐dependent bovine sperm capacitation. Andrologia 34, 91–97 (2002).

    Article  Google Scholar 

  171. Duan, C. & Goldberg, E. Inhibition of lactate dehydrogenase C4 (LDH-C4) blocks capacitation of mouse sperm in vitro. Cytogenet. Genome Res. 103, 352–359 (2003).

    Article  CAS  Google Scholar 

  172. Odet, F. et al. Expression of the gene for mouse lactate dehydrogenase C (Ldhc) is required for male fertility. Biol. Reprod. 79, 26–34 (2008).

    Article  CAS  Google Scholar 

  173. Oddo, M., Calandra, T., Bucala, R. & Meylan, P. R. Macrophage migration inhibitory factor reduces the growth of virulent Mycobacterium tuberculosis in human macrophages. Infect. Immun. 73, 3783–3786 (2005).

    Article  CAS  Google Scholar 

  174. Meinhardt, A. et al. Macrophage migration inhibitory factor production by Leydig cells: evidence for a role in the regulation of testicular function. Endocrinology 137, 5090–5095 (1996).

    Article  CAS  Google Scholar 

  175. Frenette, G., Lessard, C., Madore, E., Fortier, M. A. & Sullivan, R. Aldose reductase and macrophage migration inhibitory factor are associated with epididymosomes and spermatozoa in the bovine epididymis. Biol. Reprod. 69, 1586–1592 (2003).

    Article  CAS  Google Scholar 

  176. Huleihel, M. et al. Production of macrophage inhibitory factor (MIF) by primary Sertoli cells; its possible involvement in migration of spermatogonial cells. J. Cell. Physiol. 232, 2869–2877 (2017).

    Article  CAS  Google Scholar 

  177. Henkel, R., Bittner, J., Weber, R., Hüther, F. & Miska, W. Relevance of zinc in human sperm flagella and its relation to motility. Fertil. Steril. 71, 1138–1143 (1999).

    Article  CAS  Google Scholar 

  178. Frenette, G., Légaré, C., Saez, F. & Sullivan, R. Macrophage migration inhibitory factor in the human epididymis and semen. Mol. Hum. Reprod. 11, 575–582 (2005).

    Article  CAS  Google Scholar 

  179. Aljabari, B. et al. Imbalance in seminal fluid MIF indicates male infertility. Mol. Med. 13, 199–202 (2007).

    Article  CAS  Google Scholar 

  180. Ebert, B., Kisiela, M. & Maser, E. Human DCXR — another ‘moonlighting protein’ involved in sugar metabolism, carbonyl detoxification, cell adhesion and male fertility? Biol. Rev. 90, 254–278 (2015).

    Article  Google Scholar 

  181. Légaré, C., Gaudreault, C., St-Jacques, S. & Sullivan, R. P34H sperm protein is preferentially expressed by the human corpus epididymidis. Endocrinology 140, 3318–3327 (1999).

    Article  Google Scholar 

  182. Sullivan, R., Saez, F., Girouard, J. & Frenette, G. Role of exosomes in sperm maturation during the transit along the male reproductive tract. Blood Cells Mol. Dis. 35, 1–10 (2005).

    Article  CAS  Google Scholar 

  183. Parent, S., Lefievre, L., Brindle, Y. & Sullivan, R. Bull subfertility is associated with low levels of a sperm membrane antigen. Mol. Reprod. Dev. 52, 57–65 (1999).

    Article  CAS  Google Scholar 

  184. Boué, F. & Sullivan, R. Cases of human infertility are associated with the absence of P34H, an epididymal sperm antigen. Biol. Reprod. 54, 1018–1024 (1996).

    Article  Google Scholar 

  185. Frapsauce, C. et al. Proteomic identification of target proteins in normal but nonfertilizing sperm. Fertil. Steril. 102, 372–380 (2014).

    Article  CAS  Google Scholar 

  186. Sullivan, R., Légaré, C., Villeneuve, M., Foliguet, B. & Bissonnette, F. Levels of P34H, a sperm protein of epididymal origin, as a predictor of conventional in vitro fertilization outcome. Fertil. Steril. 85, 1557–1559 (2006).

    Article  Google Scholar 

  187. Moskovtsev, S. I., Jarvi, K., Légaré, C., Sullivan, R. & Mullen, J. B. M. Epididymal P34H protein deficiency in men evaluated for infertility. Fertil. Steril. 88, 1455–1457 (2007).

    Article  Google Scholar 

  188. Oh, J. S., Han, C. & Cho, C. ADAM7 is associated with epididymosomes and integrated into sperm plasma membrane. Mol. Cell 28, 441–446 (2009).

    Article  CAS  Google Scholar 

  189. Han, C. et al. Identification of heat shock protein 5, calnexin and integral membrane protein 2B as Adam7‐interacting membrane proteins in mouse sperm. J. Cell. Physiol. 226, 1186–1195 (2011).

    Article  CAS  Google Scholar 

  190. Légaré, C., Thabet, M., Gatti, J.-L. & Sullivan, R. HE1/NPC2 status in human reproductive tract and ejaculated spermatozoa: consequence of vasectomy. Mol. Hum. Reprod. 12, 461–468 (2006).

    Article  Google Scholar 

  191. Busso, D. et al. Spermatozoa from mice deficient in Niemann-Pick disease type C2 (NPC2) protein have defective cholesterol content and reduced in vitro fertilising ability. Reprod. Fertil. Dev. 26, 609–621 (2014).

    Article  CAS  Google Scholar 

  192. Okamura, N. et al. Molecular cloning and characterization of the epididymis-specific glutathione peroxidase-like protein secreted in the porcine epididymal fluid. Biochim. Biophys. Acta 1336, 99–109 (1997).

    Article  CAS  Google Scholar 

  193. Légaré, C., Thabet, M., Picard, S. & Sullivan, R. Effect of vasectomy on P34H messenger ribonucleic acid expression along the human excurrent duct: a reflection on the function of the human epididymis. Biol. Reprod. 64, 720–727 (2001).

    Article  Google Scholar 

  194. Giacomini, E. et al. Comparative analysis of the seminal plasma proteomes of oligoasthenozoospermic and normozoospermic men. Reprod. Biomed. Online 30, 522–531 (2015).

    Article  CAS  Google Scholar 

  195. Taylor, A. et al. Epididymal specific, selenium-independent GPX5 protects cells from oxidative stress-induced lipid peroxidation and DNA mutation. Hum. Reprod. 28, 2332–2342 (2013).

    Article  CAS  Google Scholar 

  196. Noblanc, A. et al. Glutathione peroxidases at work on epididymal spermatozoa: an example of the dual effect of reactive oxygen species on mammalian male fertilizing ability. J. Androl. 32, 641–650 (2011).

    Article  CAS  Google Scholar 

  197. Rejraji, H., Vernet, P. & Drevet, J. L. R. GPX5 is present in the mouse caput and cauda epididymidis lumen at three different locations. Mol. Reprod. Dev. 63, 96–103 (2002).

    Article  CAS  Google Scholar 

  198. Barranco, I. et al. Glutathione peroxidase 5 is expressed by the entire pig male genital tract and once in the seminal plasma contributes to sperm survival and in vivo fertility. PLoS ONE 11, e0162958 (2016).

    Article  Google Scholar 

  199. Kim, E. et al. Sperm penetration through cumulus mass and zona pellucida. Int. J. Dev. Biol. 52, 677–682 (2004).

    Article  Google Scholar 

  200. Baba, D. et al. Mouse sperm lacking cell surface hyaluronidase PH-20 can pass through the layer of cumulus cells and fertilize the egg. J. Biol. Chem. 277, 30310–30314 (2002).

    Article  CAS  Google Scholar 

  201. Myles, D. G., Hyatt, H. & Primakoff, P. Binding of both acrosome-intact and acrosome-reacted guinea pig sperm to the zona pellucida during in vitro fertilization. Dev. Biol. 121, 559–567 (1987).

    Article  CAS  Google Scholar 

  202. Primakoff, P., Hyatt, H. & Myles, D. G. A role for the migrating sperm surface antigen PH-20 in guinea pig sperm binding to the egg zona pellucida. J. Cell Biol. 101, 2239–2244 (1985).

    Article  CAS  Google Scholar 

  203. Reese, K. L. et al. Acidic hyaluronidase activity is present in mouse sperm and is reduced in the absence of SPAM1: evidence for a role for hyaluronidase 3 in mouse and human sperm. Mol. Reprod. Dev. 77, 759–772 (2010).

    Article  CAS  Google Scholar 

  204. Kimura, M. et al. Functional roles of mouse sperm hyaluronidases, HYAL5 and SPAM1, in fertilization. Biol. Reprod. 81, 939–947 (2009).

    Article  CAS  Google Scholar 

  205. Honbou, K. et al. The crystal structure of DJ-1, a protein related to male fertility and Parkinson’s disease. J. Biol. Chem. 278, 31380–31384 (2003).

    Article  CAS  Google Scholar 

  206. Junn, E., Jang, W. H., Zhao, X., Jeong, B. S. & Mouradian, M. M. Mitochondrial localization of DJ-1 leads to enhanced neuroprotection. J. Neurosci. Res. 87, 123–129 (2009).

    Article  CAS  Google Scholar 

  207. An, C.-N. et al. Down-regulation of DJ-1 protein in the ejaculated spermatozoa from Chinese asthenozoospermia patients. Fertil. Steril. 96, 19–23.e12 (2011).

    Article  CAS  Google Scholar 

  208. Yoshida, K. et al. Immunocytochemical localization of DJ-1 in human male reproductive tissue. Mol. Reprod. Dev. 66, 391–397 (2003).

    Article  CAS  Google Scholar 

  209. Whyard, T. C., Cheung, W., Sheynkin, Y., Waltzer, W. C. & Hod, Y. Identification of RS as a flagellar and head sperm protein. Mol. Reprod. Dev. 55, 189–196 (2000).

    Article  CAS  Google Scholar 

  210. Wang, J. et al. Proteomic analysis of seminal plasma from asthenozoospermia patients reveals proteins that affect oxidative stress responses and semen quality. Asian J. Androl. 11, 484–491 (2009).

    Article  CAS  Google Scholar 

  211. Nishinaga, H. et al. Expression profiles of genes in DJ-1-knockdown and L166P DJ-1 mutant cells. Neurosci. Lett. 390, 54–59 (2005).

    Article  CAS  Google Scholar 

  212. Pegtel, D. M. et al. Functional delivery of viral miRNAs via exosomes. Proc. Natl Acad. Sci. USA 107, 6328–6333 (2010).

    Article  CAS  Google Scholar 

  213. Hergenreider, E. et al. Atheroprotective communication between endothelial cells and smooth muscle cells through miRNAs. Nat. Cell Biol. 14, 249–256 (2012).

    Article  CAS  Google Scholar 

  214. Kalluri, R. & LeBleu, V. S. The biology, function, and biomedical applications of exosomes. Science 367 (2020).

  215. Ma, J. et al. Testosterone-dependent miR-26a-5p and let-7g-5p act as signaling mediators to regulate sperm apoptosis via targeting PTEN and PMAIP1. Int. J. Mol. Sci. 19, 1233 (2018).

    Article  Google Scholar 

  216. Barceló, M., Castells, M., Bassas, L., Vigués, F. & Larriba, S. Semen miRNAs contained in exosomes as non-invasive biomarkers for prostate cancer diagnosis. Sci. Rep. 9, 1–16 (2019).

    Article  Google Scholar 

  217. Twenter, H. et al. Transfer of microRNAs from epididymal epithelium to equine spermatozoa. J. Equine Vet. Sci. 87, 102841 (2020).

    Article  Google Scholar 

  218. Reilly, J. N. et al. Characterisation of mouse epididymosomes reveals a complex profile of microRNAs and a potential mechanism for modification of the sperm epigenome. Sci. Rep. 6, 31794 (2016).

    Article  CAS  Google Scholar 

  219. Alshanbayeva, A., Tanwar, D. K., Roszkowski, M., Manuella, F. & Mansuy, I. M. Early life stress affects the miRNA cargo of epididymal extracellular vesicles in mouse. Biol. Reprod. 105, 593–602 (2021).

    Article  Google Scholar 

  220. Barcelo, M., Mata, A., Bassas, L. & Larriba, S. Exosomal microRNAs in seminal plasma are markers of the origin of azoospermia and can predict the presence of sperm in testicular tissue. Hum. Reprod. 33, 1087–1098 (2018).

    Article  CAS  Google Scholar 

  221. Wang, C. et al. Altered profile of seminal plasma microRNAs in the molecular diagnosis of male infertility. Clin. Chem. 57, 1722–1731 (2011).

    Article  CAS  Google Scholar 

  222. Wu, W. et al. Seminal plasma microRNAs: potential biomarkers for spermatogenesis status. Mol. Hum. Reprod. 18, 489–497 (2012).

    Article  CAS  Google Scholar 

  223. Abu-Halima, M. et al. Altered micro-ribonucleic acid expression profiles of extracellular microvesicles in the seminal plasma of patients with oligoasthenozoospermia. Fertil. Steril. 106, 1061–1069.e3 (2016).

    Article  CAS  Google Scholar 

  224. World Health Organization. Laboratory Manual for the Examination and Processing of Human Semen 6th edn (WHO, 2021).

  225. Santi, D., Spaggiari, G. & Simoni, M. Sperm DNA fragmentation index as a promising predictive tool for male infertility diagnosis and treatment management–meta-analyses. Reprod. Biomed. Online 37, 315–326 (2018).

    Article  CAS  Google Scholar 

  226. Ferlin, A. et al. Male infertility: role of genetic background. Reprod. Biomed. Online 14, 734–745 (2007).

    Article  CAS  Google Scholar 

  227. Vashisht, A. & Gahlay, G. K. Using miRNAs as diagnostic biomarkers for male infertility: opportunities and challenges. Mol. Hum. Reprod. 26, 199–214 (2020).

    Article  CAS  Google Scholar 

  228. Murdica, V. et al. Proteomic analysis reveals the negative modulator of sperm function glycodelin as over-represented in semen exosomes isolated from asthenozoospermic patients. Hum. Reprod. 34, 1416–1427 (2019).

    Article  CAS  Google Scholar 

  229. Madison, M. N., Roller, R. J. & Okeoma, C. M. Human semen contains exosomes with potent anti-HIV-1 activity. Retrovirology 11, 1–16 (2014).

    Article  Google Scholar 

  230. Hiemstra, T. F. et al. Human urinary exosomes as innate immune effectors. J. Am. Soc. Nephrol. 25, 2017–2027 (2014).

    Article  CAS  Google Scholar 

  231. György, B. et al. Detection and isolation of cell-derived microparticles are compromised by protein complexes resulting from shared biophysical parameters. Blood J. Am. Soc. Hematol. 117, e39–e48 (2011).

    Google Scholar 

  232. He, M., Crow, J., Roth, M., Zeng, Y. & Godwin, A. K. Integrated immunoisolation and protein analysis of circulating exosomes using microfluidic technology. Lab Chip 14, 3773–3780 (2014).

    Article  CAS  Google Scholar 

  233. Muller, L., Hong, C.-S., Stolz, D. B., Watkins, S. C. & Whiteside, T. L. Isolation of biologically-active exosomes from human plasma. J. Immunol. Methods 411, 55–65 (2014).

    Article  CAS  Google Scholar 

  234. Das, C. K. et al. Exosome as a novel shuttle for delivery of therapeutics across biological barriers. Mol. Pharm. 16, 24–40 (2018).

    Article  Google Scholar 

  235. Yamashita, T., Takahashi, Y. & Takakura, Y. Possibility of exosome-based therapeutics and challenges in production of exosomes eligible for therapeutic application. Biol. Pharm. Bull. 41, 835–842 (2018).

    Article  CAS  Google Scholar 

  236. Théry, C. et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J. Extracell. Vesicles 7, 1535750 (2018).

    Article  Google Scholar 

  237. Fedder, J. Nonsperm cells in human semen: with special reference to seminal leukocytes and their possible influence on fertility. Arch. Androl. 36, 41–65 (1996).

    Article  CAS  Google Scholar 

  238. Soares Martins, T., Catita, J., Martins Rosa, I., O, A. B. D. C. E. S. & Henriques, A. G. Exosome isolation from distinct biofluids using precipitation and column-based approaches. PLoS ONE 13, e0198820 (2018).

    Article  Google Scholar 

  239. Lane, R. E., Korbie, D., Anderson, W., Vaidyanathan, R. & Trau, M. Analysis of exosome purification methods using a model liposome system and tunable-resistive pulse sensing. Sci. Rep. 5, 1–7 (2015).

    Article  CAS  Google Scholar 

  240. Lobb, R. J. et al. Optimized exosome isolation protocol for cell culture supernatant and human plasma. J. Extracell. Vesicles 4, 27031 (2015).

    Article  Google Scholar 

  241. Coughlan, C. et al. Exosome isolation by ultracentrifugation and precipitation and techniques for downstream analyses. Curr. Protoc. Cell Biol. 88, e110 (2020).

    Article  CAS  Google Scholar 

  242. Cao, F. et al. Proteomics comparison of exosomes from serum and plasma between ultracentrifugation and polymer‐based precipitation kit methods. Electrophoresis 40, 3092–3098 (2019).

    Article  CAS  Google Scholar 

  243. Madison, M. N., Welch, J. L. & Okeoma, C. M. Isolation of exosomes from semen for in vitro uptake and HIV-1 infection assays. Bio Protoc. 7, e2216 (2017).

    Article  Google Scholar 

  244. Kaddour, H. et al. Proteomics profiling of autologous blood and semen exosomes from HIV-infected and uninfected individuals reveals compositional and functional variabilities. Mol. Cell. Proteom. 19, 78–100 (2020).

    Article  CAS  Google Scholar 

  245. Welch, J. L., Kaufman, T. M., Stapleton, J. T. & Okeoma, C. M. Semen exosomes inhibit HIV infection and HIV‐induced proinflammatory cytokine production independent of the activation state of primary lymphocytes. FEBS Lett. 594, 695–709 (2020).

    Article  CAS  Google Scholar 

  246. Welch, J. L., Kaddour, H., Schlievert, P. M., Stapleton, J. T. & Okeoma, C. M. Semen exosomes promote transcriptional silencing of HIV-1 by disrupting NF-κB/Sp1/Tat circuitry. J. Virol. 92, e00731–18 (2018).

    Article  CAS  Google Scholar 

  247. Chang, X. et al. Exosomes from women with preeclampsia induced vascular dysfunction by delivering sFlt (soluble Fms-like tyrosine kinase)-1 and sEng (soluble endoglin) to endothelial cells. Hypertension 72, 1381–1390 (2018).

    Article  CAS  Google Scholar 

  248. Gemoll, T. et al. Protein profiling of serum extracellular vesicles reveals qualitative and quantitative differences after differential ultracentrifugation and ExoQuick™ isolation. J. Clin. Med. 9, 1429 (2020).

    Article  CAS  Google Scholar 

  249. Wang, X. in Extracellular Vesicles (eds Kuo, W. P. & Jia, S.) 351–353 (Springer, 2017).

  250. Yamada, T., Inoshima, Y., Matsuda, T. & Ishiguro, N. Comparison of methods for isolating exosomes from bovine milk. J. Vet. Med. Sci., 12-0032 (2012).

  251. Lin, S. et al. Progress in microfluidics-based exosome separation and detection technologies for diagnostic applications. Small 16, e1903916 (2020).

    Article  Google Scholar 

  252. Théry, C., Amigorena, S., Raposo, G. & Clayton, A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr. Protoc. Cell Biol. 30, 3.22. 21–23.22. 29 (2006).

    Article  Google Scholar 

  253. Contreras-Naranjo, J. C., Wu, H. J. & Ugaz, V. M. Microfluidics for exosome isolation and analysis: enabling liquid biopsy for personalized medicine. Lab Chip 17, 3558–3577 (2017).

    Article  CAS  Google Scholar 

  254. Webber, J. & Clayton, A. How pure are your vesicles? J. Extracell. Vesicles 2, 19861 (2013).

    Article  Google Scholar 

  255. Vlassov, A. V., Magdaleno, S., Setterquist, R. & Conrad, R. Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials. Biochim. Biophys. Acta 1820, 940–948 (2012).

    Article  CAS  Google Scholar 

  256. Liga, A., Vliegenthart, A. D., Oosthuyzen, W., Dear, J. W. & Kersaudy-Kerhoas, M. Exosome isolation: a microfluidic road-map. Lab Chip 15, 2388–2394 (2015).

    Article  CAS  Google Scholar 

  257. Momen-Heravi, F. et al. Current methods for the isolation of extracellular vesicles. Biol. Chem. 394, 1253–1262 (2013).

    Article  CAS  Google Scholar 

  258. Faruqu, F. N., Xu, L. & Al-Jamal, K. T. Preparation of exosomes for siRNA delivery to cancer cells. J. Vis. Exp. 142, e58814 (2018).

    Google Scholar 

  259. Greening, D. W., Xu, R., Ji, H., Tauro, B. J. & Simpson, R. J. in Proteomic Profiling (ed. Posch, A.) 179–209 (Springer, 2015).

  260. Yang, D. et al. Progress, opportunity, and perspective on exosome isolation-efforts for efficient exosome-based theranostics. Theranostics 10, 3684–3707 (2020).

    Article  CAS  Google Scholar 

  261. Baranyai, T. et al. Isolation of exosomes from blood plasma: qualitative and quantitative comparison of ultracentrifugation and size exclusion chromatography methods. PLoS ONE 10, e0145686 (2015).

    Article  Google Scholar 

  262. Guan, S. et al. Characterization of urinary exosomes purified with size exclusion chromatography and ultracentrifugation. J. Proteome Res. 19, 2217–2225 (2020).

    Article  CAS  Google Scholar 

  263. Stranska, R. et al. Comparison of membrane affinity-based method with size-exclusion chromatography for isolation of exosome-like vesicles from human plasma. J. Transl. Med. 16, 1–9 (2018).

    Article  CAS  Google Scholar 

  264. Al Ali, J. et al. TAF1 transcripts and neurofilament light chain as biomarkers for x‐linked dystonia‐parkinsonism. Mov. Disord. 36, 206–215 (2021).

    Article  CAS  Google Scholar 

  265. Vandendriessche, C. et al. Importance of extracellular vesicle secretion at the blood–cerebrospinal fluid interface in the pathogenesis of Alzheimer’s disease. Acta Neuropathol. Commun. 9, 1–25 (2021).

    Article  Google Scholar 

  266. Salarpour, S. et al. Paclitaxel incorporated exosomes derived from glioblastoma cells: comparative study of two loading techniques. Daru 27, 533–539 (2019).

    Article  CAS  Google Scholar 

  267. Macías, M. et al. Comparison of six commercial serum exosome isolation methods suitable for clinical laboratories. Effect in cytokine analysis. Clin. Chem. Lab. Med. 57, 1539–1545 (2019).

    Article  Google Scholar 

  268. Keller, S., Sanderson, M. P., Stoeck, A. & Altevogt, P. Exosomes: from biogenesis and secretion to biological function. Immunol. Lett. 107, 102–108 (2006).

    Article  CAS  Google Scholar 

  269. Kastelowitz, N. & Yin, H. Exosomes and microvesicles: identification and targeting by particle size and lipid chemical probes. Chembiochem Eur. J. Chem. Biol. 15, 923 (2014).

    CAS  Google Scholar 

  270. Song, Z. et al. Development of a CD63 aptamer for efficient cancer immunochemistry and immunoaffinity-based exosome isolation. Molecules 25, 5585 (2020).

    Article  CAS  Google Scholar 

  271. Zarovni, N. et al. Integrated isolation and quantitative analysis of exosome shuttled proteins and nucleic acids using immunocapture approaches. Methods 87, 46–58 (2015).

    Article  CAS  Google Scholar 

  272. Alvarez, M. L., Khosroheidari, M., Ravi, R. K. & DiStefano, J. K. Comparison of protein, microRNA, and mRNA yields using different methods of urinary exosome isolation for the discovery of kidney disease biomarkers. Kidney Int. 82, 1024–1032 (2012).

    Article  CAS  Google Scholar 

  273. Li, P., Kaslan, M., Lee, S. H., Yao, J. & Gao, Z. Progress in exosome isolation techniques. Theranostics 7, 789 (2017).

    Article  CAS  Google Scholar 

  274. Phan, T. H. et al. New multiscale characterization methodology for effective determination of isolation–structure–function relationship of extracellular vesicles. Front. Bioeng. Biotechnol. 9, 358 (2021).

    Article  Google Scholar 

  275. Heinemann, M. L. et al. Benchtop isolation and characterization of functional exosomes by sequential filtration. J. Chromatogr. A 1371, 125–135 (2014).

    Article  CAS  Google Scholar 

  276. Marsh, S. R., Pridham, K. J., Jourdan, J. & Gourdie, R. G. Novel protocols for scalable production of high quality purified small extracellular vesicles from bovine milk. Nanotheranostics 5, 488–498 (2021).

    Article  Google Scholar 

  277. Manz, A., Graber, N. & Widmer, H. Á. Miniaturized total chemical analysis systems: a novel concept for chemical sensing. Sens. Actuators B Chem. 1, 244–248 (1990).

    Article  CAS  Google Scholar 

  278. Chen, Y.-S., Ma, Y.-D., Chen, C., Shiesh, S.-C. & Lee, G.-B. An integrated microfluidic system for on-chip enrichment and quantification of circulating extracellular vesicles from whole blood. Lab Chip 19, 3305–3315 (2019).

    Article  CAS  Google Scholar 

  279. Panesar, S. & Neethirajan, S. Microfluidics: rapid diagnosis for breast cancer. Nanomicro Lett. 8, 204–220 (2016).

    CAS  Google Scholar 

  280. Nosrati, R. et al. Rapid selection of sperm with high DNA integrity. Lab Chip 14, 1142–1150 (2014).

    Article  CAS  Google Scholar 

  281. Quinn, M. M. et al. Microfluidic sorting selects sperm for clinical use with reduced DNA damage compared to density gradient centrifugation with swim-up in split semen samples. Hum. Reprod. 33, 1388–1393 (2018).

    Article  CAS  Google Scholar 

  282. Tung, C.-K. et al. Fluid viscoelasticity promotes collective swimming of sperm. Sci. Rep. 7, 1–9 (2017).

    Article  Google Scholar 

  283. Vasilescu, S. A. et al. A microfluidic approach to rapid sperm recovery from heterogeneous cell suspensions. Sci. Rep. 11, 7917 (2021).

    Article  CAS  Google Scholar 

  284. Son, J. et al. Non-motile sperm cell separation using a spiral channel. Anal. Methods 7, 8041–8047 (2015).

    Article  Google Scholar 

  285. Son, J., Samuel, R., Gale, B. K., Carrell, D. T. & Hotaling, J. M. Separation of sperm cells from samples containing high concentrations of white blood cells using a spiral channel. Biomicrofluidics 11, 054106 (2017).

    Article  Google Scholar 

  286. Roy, T. K. et al. Embryo vitrification using a novel semi-automated closed system yields in vitro outcomes equivalent to the manual Cryotop method. Hum. Reprod. 29, 2431–2438 (2014).

    Article  Google Scholar 

  287. Smith, D., Gaffney, E., Blake, J. & Kirkman-Brown, J. Human sperm accumulation near surfaces: a simulation study. J. Fluid Mech. 621, 289–320 (2009).

    Article  Google Scholar 

  288. Ramadan, S. et al. Carbon-dot-enhanced graphene field-effect transistors for ultrasensitive detection of exosomes. ACS Appl. Mater. Interfaces 13, 7854–7864 (2021).

    Article  CAS  Google Scholar 

  289. Zhand, S. et al. Improving capture efficiency of human cancer cell derived exosomes with nanostructured metal organic framework functionalized beads. Appl. Mater. Today 23, 100994 (2021).

    Article  Google Scholar 

  290. Sayyadi, N., Zhand, S., Razavi Bazaz, S. & Warkiani, M. E. Affibody functionalized beads for the highly sensitive detection of cancer cell-derived exosomes. Int. J. Mol. Sci. 22, 12014 (2021).

    Article  CAS  Google Scholar 

  291. Dorayappan, K. D. P. et al. A microfluidic chip enables isolation of exosomes and establishment of their protein profiles and associated signaling pathways in ovarian cancer. Cancer Res. 79, 3503–3513 (2019).

    Article  CAS  Google Scholar 

  292. Sancho-Albero, M. et al. Isolation of exosomes from whole blood by a new microfluidic device: proof of concept application in the diagnosis and monitoring of pancreatic cancer. J. Nanobiotechnol. 18, 1–15 (2020).

    Article  Google Scholar 

  293. Chen, C. et al. Microfluidic isolation and transcriptome analysis of serum microvesicles. Lab Chip 10, 505–511 (2010).

    Article  CAS  Google Scholar 

  294. Fang, S. et al. Clinical application of a microfluidic chip for immunocapture and quantification of circulating exosomes to assist breast cancer diagnosis and molecular classification. PLoS ONE 12, e0175050 (2017).

    Article  Google Scholar 

  295. Ashcroft, B. A. et al. Determination of the size distribution of blood microparticles directly in plasma using atomic force microscopy and microfluidics. Biomed. Microdevices 14, 641–649 (2012).

    Article  CAS  Google Scholar 

  296. Zhang, P., He, M. & Zeng, Y. Ultrasensitive microfluidic analysis of circulating exosomes using a nanostructured graphene oxide/polydopamine coating. Lab Chip 16, 3033–3042 (2016).

    Article  CAS  Google Scholar 

  297. Shao, H. et al. Chip-based analysis of exosomal mRNA mediating drug resistance in glioblastoma. Nat. Commun. 6, 6999 (2015).

    Article  CAS  Google Scholar 

  298. Im, H. et al. Label-free detection and molecular profiling of exosomes with a nano-plasmonic sensor. Nat. Biotechnol. 32, 490–495 (2014).

    Article  CAS  Google Scholar 

  299. Vaidyanathan, R. et al. Detecting exosomes specifically: a multiplexed device based on alternating current electrohydrodynamic induced nanoshearing. Anal. Chem. 86, 11125–11132 (2014).

    Article  CAS  Google Scholar 

  300. Zhao, Z., Yang, Y., Zeng, Y. & He, M. A microfluidic ExoSearch chip for multiplexed exosome detection towards blood-based ovarian cancer diagnosis. Lab Chip 16, 489–496 (2016).

    Article  CAS  Google Scholar 

  301. Sina, A. A. I. et al. Real time and label free profiling of clinically relevant exosomes. Sci. Rep. 6, 1–9 (2016).

    Article  Google Scholar 

  302. Ko, J. et al. Smartphone-enabled optofluidic exosome diagnostic for concussion recovery. Sci. Rep. 6, 1–12 (2016).

    Article  Google Scholar 

  303. Hisey, C. L., Dorayappan, K. D. P., Cohn, D. E., Selvendiran, K. & Hansford, D. J. Microfluidic affinity separation chip for selective capture and release of label-free ovarian cancer exosomes. Lab Chip 18, 3144–3153 (2018).

    Article  CAS  Google Scholar 

  304. Kang, Y. T. et al. Dual‐isolation and profiling of circulating tumor cells and cancer exosomes from blood samples with melanoma using immunoaffinity‐based microfluidic interfaces. Adv. Sci. 7, 2001581 (2020).

    Article  CAS  Google Scholar 

  305. Tauro, B. J. et al. Two distinct populations of exosomes are released from LIM1863 colon carcinoma cell-derived organoids. Mol. Cell. Proteom. 12, 587–598 (2013).

    Article  CAS  Google Scholar 

  306. Crescitelli, R. et al. Distinct RNA profiles in subpopulations of extracellular vesicles: apoptotic bodies, microvesicles and exosomes. J. Extracell. Vesicles 2, 20677 (2013).

    Article  Google Scholar 

  307. Oliveira-Rodríguez, M. et al. Development of a rapid lateral flow immunoassay test for detection of exosomes previously enriched from cell culture medium and body fluids. J. Extracell. Vesicles 5, 31803 (2016).

    Article  Google Scholar 

  308. Moyano, A. et al. Magnetic lateral flow immunoassay for small extracellular vesicles quantification: application to colorectal cancer biomarker detection. Sensors 21, 3756 (2021).

    Article  CAS  Google Scholar 

  309. Liu, F. et al. The exosome total isolation chip. ACS Nano 11, 10712–10723 (2017).

    Article  CAS  Google Scholar 

  310. Chen, Z., Yang, Y., Yamaguchi, H., Hung, M.-C. & Kameoka, J. Isolation of cancer-derived extracellular vesicle subpopulations by a size-selective microfluidic platform. Biomicrofluidics 14, 034113 (2020).

    Article  CAS  Google Scholar 

  311. Wu, M. et al. Acoustofluidic separation of cells and particles. Microsyst. Nanoeng. 5, 1–18 (2019).

    Article  Google Scholar 

  312. Wu, M. et al. Isolation of exosomes from whole blood by integrating acoustics and microfluidics. Proc. Natl Acad. Sci. USA 114, 10584–10589 (2017).

    Article  CAS  Google Scholar 

  313. Jubery, T. Z., Srivastava, S. K. & Dutta, P. Dielectrophoretic separation of bioparticles in microdevices: a review. Electrophoresis 35, 691–713 (2014).

    Article  CAS  Google Scholar 

  314. Yun, H., Kim, K. & Lee, W. G. Cell manipulation in microfluidics. Biofabrication 5, 022001 (2013).

    Article  Google Scholar 

  315. Zhu, H., Lin, X., Su, Y., Dong, H. & Wu, J. Screen-printed microfluidic dielectrophoresis chip for cell separation. Biosens. Bioelectron. 63, 371–378 (2015).

    Article  CAS  Google Scholar 

  316. Zheng, L., Brody, J. P. & Burke, P. J. Electronic manipulation of DNA, proteins, and nanoparticles for potential circuit assembly. Biosens. Bioelectron. 20, 606–619 (2004).

    Article  CAS  Google Scholar 

  317. Ibsen, S. D. et al. Rapid isolation and detection of exosomes and associated biomarkers from plasma. ACS Nano 11, 6641–6651 (2017).

    Article  CAS  Google Scholar 

  318. Zhao, W. et al. Microsphere mediated exosome isolation and ultra-sensitive detection on a dielectrophoresis integrated microfluidic device. Analyst 146, 5962–5972 (2021).

    Article  CAS  Google Scholar 

  319. Zeming, K. K., Thakor, N. V., Zhang, Y. & Chen, C.-H. Real-time modulated nanoparticle separation with an ultra-large dynamic range. Lab Chip 16, 75–85 (2016).

    Article  CAS  Google Scholar 

  320. Santana, S. M., Antonyak, M. A., Cerione, R. A. & Kirby, B. J. Microfluidic isolation of cancer-cell-derived microvesicles from hetergeneous extracellular shed vesicle populations. Biomed. Microdevices 16, 869–877 (2014).

    Article  CAS  Google Scholar 

  321. Tottori, N., Muramoto, Y., Sakai, H. & Nisisako, T. Nanoparticle separation through deterministic lateral displacement arrays in poly (dimethylsiloxane). J. Chem. Eng. Jpn. 53, 414–421 (2020).

    Article  CAS  Google Scholar 

  322. Calero, V., Garcia-Sanchez, P., Ramos, A. & Morgan, H. Combining DC and AC electric fields with deterministic lateral displacement for micro- and nano-particle separation. Biomicrofluidics 13, 054110 (2019).

    Article  Google Scholar 

  323. Woo, H.-K. et al. Exodisc for rapid, size-selective, and efficient isolation and analysis of nanoscale extracellular vesicles from biological samples. Acs Nano 11, 1360–1370 (2017).

    Article  CAS  Google Scholar 

  324. Liu, C. et al. Field-free isolation of exosomes from extracellular vesicles by microfluidic viscoelastic flows. ACS Nano 11, 6968–6976 (2017).

    Article  CAS  Google Scholar 

  325. Kang, D., Oh, S., Ahn, S.-M., Lee, B.-H. & Moon, M. H. Proteomic analysis of exosomes from human neural stem cells by flow field-flow fractionation and nanoflow liquid chromatography−tandem mass spectrometry. J. Proteome Res. 7, 3475–3480 (2008).

    Article  CAS  Google Scholar 

  326. Wang, Z. et al. Ciliated micropillars for the microfluidic-based isolation of nanoscale lipid vesicles. Lab Chip 13, 2879–2882 (2013).

    Article  CAS  Google Scholar 

  327. Qi, R. et al. Microfluidic device for the analysis of MDR cancerous cell-derived exosomes’ response to nanotherapy. Biomed. Microdevices 21, 1–9 (2019).

    Article  Google Scholar 

  328. Willis, G. R., Kourembanas, S. & Mitsialis, S. A. Toward exosome-based therapeutics: isolation, heterogeneity, and fit-for-purpose potency. Front. Cardiovasc. Med. 4, 63 (2017).

    Article  Google Scholar 

  329. Luo, J. et al. Immunogenicity study of plasmid DNA encoding mouse cysteine‐rich secretory protein‐1 (mCRISP 1) as a contraceptive vaccine. Am. J. Reprod. Immunol. 68, 47–55 (2012).

    Article  CAS  Google Scholar 

  330. Batruch, I. et al. Analysis of seminal plasma from patients with non-obstructive azoospermia and identification of candidate biomarkers of male infertility. J. Proteome Res. 11, 1503–1511 (2012).

    Article  CAS  Google Scholar 

  331. Batruch, I. et al. Proteomic analysis of seminal plasma from normal volunteers and post-vasectomy patients identifies over 2000 proteins and candidate biomarkers of the urogenital system. J. Proteome Res. 10, 941–953 (2011).

    Article  CAS  Google Scholar 

  332. Tang, H., Duan, C., Bleher, R. & Goldberg, E. Human lactate dehydrogenase A (LDHA) rescues mouse Ldhc-null sperm function. Biol. Reprod. 88, 91–96 (2013).

    Article  Google Scholar 

  333. Anahara, R. et al. Deletion of macrophage migration inhibitory factor gene induces down regulation of sex hormones and ultrastructural abnormalities in mouse testes. Reprod. Toxicol. 21, 167–170 (2006).

    Article  CAS  Google Scholar 

  334. Grzmil, P. et al. Human cyritestin genes (CYRN1 and CYRN2) are non-functional. Biochem. J. 357, 551–556 (2001).

    Article  CAS  Google Scholar 

  335. Choi, H. et al. Reduced fertility and altered epididymal and sperm integrity in mice lacking ADAM7. Biol. Reprod. 93, 70 (2015).

    Article  Google Scholar 

  336. Kim, T. et al. Expression and relationship of male reproductive ADAMs in mouse. Biol. Reprod. 74, 744–750 (2006).

    Article  CAS  Google Scholar 

  337. Ronquist, G. K. et al. Biochemical characterization of stallion prostasomes and comparison to their human counterparts. Syst. Biol. Reprod. Med. 59, 297–303 (2013).

    Article  CAS  Google Scholar 

  338. Chotwiwatthanakun, C. et al. Expression of Penaeus monodon ortholog of Niemann–Pick type C‐2 in the spermatic tract, and its role in sperm cholesterol removal. Mol. Reprod. Dev. 83, 259–270 (2016).

    Article  CAS  Google Scholar 

  339. Vilagran, I., Castillo-Martín, M., Prieto-Martínez, N., Bonet, S. & Yeste, M. Triosephosphate isomerase (TPI) and epididymal secretory glutathione peroxidase (GPX5) are markers for boar sperm quality. Anim. Reprod. Sci. 100, 22–30 (2016).

    Article  Google Scholar 

  340. Zhou, C., Kang, W. & Baba, T. Functional characterization of double-knockout mouse sperm lacking SPAM1 and ACR or SPAM1 and PRSS21 in fertilization. J. Reprod. Dev. 58, 330–337 (2012).

    Article  CAS  Google Scholar 

  341. Lin, Y., Mahan, K., Lathrop, W. F., Myles, D. G. & Primakoff, P. A hyaluronidase activity of the sperm plasma membrane protein PH-20 enables sperm to penetrate the cumulus cell layer surrounding the egg. J. Cell Biol. 125, 1157–1163 (1994).

    Article  CAS  Google Scholar 

  342. Primakoff, P. & Myles, D. G. Penetration, adhesion, and fusion in mammalian sperm-egg interaction. Science 296, 2183–2185 (2002).

    Article  CAS  Google Scholar 

  343. Sun, Y. et al. DJ-1 deficiency causes metabolic abnormality in ornidazole-induced asthenozoospermia. Reproduction 160, 931–941 (2020).

    Article  CAS  Google Scholar 

  344. Klinefelter, G., Laskey, J., Ferrell, J., Suarez, J. & Roberts, N. Discriminant analysis indicates a single sperm protein (SP22) is predictive of fertility following exposure to epididymal toxicants. J. Androl. 18, 139–150 (1997).

    CAS  Google Scholar 

  345. Lyu, Y. et al. Human immunodeficiency virus (HIV) infection and use of illicit substances promote secretion of semen exosomes that enhance monocyte adhesion and induce actin reorganization and chemotactic migration. Cells 8, 1027 (2019).

    Article  CAS  Google Scholar 

  346. Fabiani, R., Johansson, L., Lundkvist, Ö. & Ronquist, G. Enhanced recruitment of motile spermatozoa by prostasome inclusion in swim-up medium. Hum. Reprod. 9, 1485–1489 (1994).

    Article  CAS  Google Scholar 

  347. Minelli, A., Moroni, M., Martinez, E., Mezzasoma, I. & Ronquist, G. Occurrence of prostasome-like membrane vesicles in equine seminal plasma. Reproduction 114, 237–243 (1998).

    Article  CAS  Google Scholar 

  348. Carlsson, L. et al. Characteristics of human prostasomes isolated from three different sources. Prostate 54, 322–330 (2003).

    Article  CAS  Google Scholar 

  349. Ronquist, K. G., Ronquist, G., Larsson, A. & Carlsson, L. Proteomic analysis of prostate cancer metastasis-derived prostasomes. Anticancer Res. 30, 285–290 (2010).

    CAS  Google Scholar 

  350. Carlsson, L. et al. Association of cystatin C with prostasomes in human seminal plasma. Int. J. Androl. 34, 363–368 (2011).

    Article  CAS  Google Scholar 

  351. Chevillet, J. R. et al. Quantitative and stoichiometric analysis of the microRNA content of exosomes. Proc. Natl Acad. Sci. USA 111, 14888–14893 (2014).

    Article  CAS  Google Scholar 

  352. Du, J. et al. Boar seminal plasma exosomes maintain sperm function by infiltrating into the sperm membrane. Oncotarget 7, 58832 (2016).

    Article  Google Scholar 

  353. Milutinović, B., Goč, S., Mitić, N., Kosanović, M. & Janković, M. Surface glycans contribute to differences between seminal prostasomes from normozoospermic and oligozoospermic men. Ups. J. Med. Sci. 124, 111–118 (2019).

    Article  Google Scholar 

  354. Lee, K., Shao, H., Weissleder, R. & Lee, H. Acoustic purification of extracellular microvesicles. ACS Nano 9, 2321–2327 (2015).

    Article  CAS  Google Scholar 

  355. Ku, A. et al. Acoustic enrichment of extracellular vesicles from biological fluids. Anal. Chem. 90, 8011–8019 (2018).

    Article  CAS  Google Scholar 

  356. Wu, M. et al. Separating extracellular vesicles and lipoproteins via acoustofluidics. Lab Chip 19, 1174–1182 (2019).

    Article  CAS  Google Scholar 

  357. Shi, L. et al. Rapid and label-free isolation of small extracellular vesicles from biofluids utilizing a novel insulator based dielectrophoretic device. Lab Chip 19, 3726–3734 (2019).

    Article  CAS  Google Scholar 

  358. Wunsch, B. H. et al. Nanoscale lateral displacement arrays for the separation of exosomes and colloids down to 20 nm. Nat. Nanotechnol. 11, 936–940 (2016).

    Article  CAS  Google Scholar 

  359. Bai, Y. et al. Rapid isolation and multiplexed detection of exosome tumor markers via queued beads combined with quantum dots in a microarray. Nanomicro Lett. 11, 59 (2019).

    CAS  Google Scholar 

  360. Davies, R. T. et al. Microfluidic filtration system to isolate extracellular vesicles from blood. Lab Chip 12, 5202–5210 (2012).

    Article  CAS  Google Scholar 

  361. Cho, S. et al. Isolation of extracellular vesicle from blood plasma using electrophoretic migration through porous membrane. Sens. Actuators B Chem. 233, 289–297 (2016).

    Article  CAS  Google Scholar 

  362. Liang, L.-G. et al. An integrated double-filtration microfluidic device for isolation, enrichment and quantification of urinary extracellular vesicles for detection of bladder cancer. Sci. Rep. 7, 1–10 (2017).

    Google Scholar 

  363. Dong, X. et al. Efficient isolation and sensitive quantification of extracellular vesicles based on an integrated ExoID-Chip using photonic crystals. Lab Chip 19, 2897–2904 (2019).

    Article  CAS  Google Scholar 

  364. Casadei, L. et al. Cross‐flow microfiltration for isolation, selective capture and release of liposarcoma extracellular vesicles. J. Extracell. Vesicles 10, e12062 (2021).

    Article  CAS  Google Scholar 

  365. Han, Z. et al. Highly efficient exosome purification from human plasma by tangential flow filtration based microfluidic chip. Sens. Actuators B Chem. 333, 129563 (2021).

    Article  CAS  Google Scholar 

  366. Han, B. H. et al. Isolation of extracellular vesicles from small volumes of plasma using a microfluidic aqueous two-phase system. Lab Chip 20, 3552–3559 (2020).

    Article  CAS  Google Scholar 

  367. Dudani, J. S. et al. Rapid inertial solution exchange for enrichment and flow cytometric detection of microvesicles. Biomicrofluidics 9, 014112 (2015).

    Article  Google Scholar 

  368. Yeo, J. C. et al. Label-free extraction of extracellular vesicles using centrifugal microfluidics. Biomicrofluidics 12, 024103 (2018).

    Article  Google Scholar 

  369. Zhou, Y., Ma, Z., Tayebi, M. & Ai, Y. Submicron particle focusing and exosome sorting by wavy microchannel structures within viscoelastic fluids. Anal. Chem. 91, 4577–4584 (2019).

    Article  CAS  Google Scholar 

  370. Teoh, B. Y. et al. Isolation of exosome from the culture medium of nasopharyngeal cancer (NPC) C666-1 cells using inertial based microfluidic channel. Biomed. Microdevices 24, 12 (2022).

    Article  CAS  Google Scholar 

  371. Linxweiler, J. & Junker, K. Extracellular vesicles in urological malignancies: an update. Nat. Rev. Urol. 17, 11–27 (2020).

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Contributions

D.M.G. researched data for the article. All authors contributed substantially to discussion of the content. D.M.G. wrote the article. D.M.G., S.A.V., D.K.G. and M.E.W. reviewed and/or edited the manuscript before submission.

Corresponding authors

Correspondence to David K. Gardner or Majid E. Warkiani.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Reviews Urology thanks the anonymous reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Goss, D.M., Vasilescu, S.A., Sacks, G. et al. Microfluidics facilitating the use of small extracellular vesicles in innovative approaches to male infertility. Nat Rev Urol 20, 66–95 (2023). https://doi.org/10.1038/s41585-022-00660-8

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41585-022-00660-8

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing