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Treatment of human sperm with serine protease during density gradient centrifugation

  • Technological Innovations
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Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

Abstract

Purpose

Seminal pathogens can bind specifically or non-specifically to spermatozoa, rendering semen decontamination procedures ineffective, whereby vertical or horizontal transmission of the infection could occur. Serine proteases have been demonstrated to effectively inactivate viruses and to break pathogen-sperm bonds. However, the addition of a protease to density gradient layers during semen processing could negatively impact on sperm parameters. This study investigated the effect of the addition of a recombinant, human-sequence protease (rhProtease) on sperm parameters during density gradient centrifugation.

Methods

(i) Pooled semen samples (n = 9) were split and processed by density gradient centrifugation, with the top density layers supplemented, or non-supplemented with rhProtease at three different concentrations (diluted 2, 10 and 20 times). Sperm parameters were then analysed by flow cytometry and computer-assisted semen analyses. (ii) Semen samples (n = 5) were split and similarly processed using PureSperm® Pro, with rhProtease in the 40 % density gradient layer, or standard PureSperm® not supplemented with rhProtease (Nidacon, International) respectively. The Hemizona assay was then utilized to compare sperm-zona binding post processing.

Results

Evaluation of sperm parameters indicated that rhProtease did not, at any of the tested concentrations, have an impact on (i) mitochondrial membrane potential, vitality, motility, or (ii) zona binding potential.

Conclusion

We report that the addition of rhProtease to density gradients is a non-detrimental approach that could improve the effectiveness of semen processing for the elimination of seminal pathogens, and benefit assisted reproduction outcome.

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References

  1. Bujan L, Pasquier C, Labeyrie E, Lanusse-Crousse P, Morucci M, Daudin M. Insemination with isolated and virologically tested spermatozoa is a safe way for human immunodeficiency type 1 virus-serodiscordant couples with an infected male partner to have a child. Fertil Steril. 2004;82:857–62.

    Article  PubMed  Google Scholar 

  2. Sauer MV, Wang JG, Douglas NC, Nakhuda GS, Vardhana P, Jovanovic V, Guarnaccia MM. Providing fertility care to men seropositive for human immunodeficiency virus: reviewing 10 years of experience and 420 consecutive cycles of in vitro fertilization and intracytoplasmic sperm injection. Fertil Steril. 2009;91:2455–60.

    Article  PubMed  Google Scholar 

  3. Nicopoullos JD, Almeida P, Vourliotis M, Gilling-Smith C. A decade of the sperm-washing programme: correlation between markers of HIV and seminal parameters. HIV Med. 2011;12:195–201.

    Article  PubMed  CAS  Google Scholar 

  4. Tang SB, Levy JA. Inactivation of HIV-1 by trypsin and its use in demonstrating specific virus infection of cells. J Virol Methods. 1991;33:39–46.

    Article  PubMed  CAS  Google Scholar 

  5. Fanibunda SE, Velhal SM, Raghavan VP, Bandivdekar AH. CD4 independent binding of HIV gp120 to mannose receptor on human spermatozoa. J Acquir Immune Defic Syndr. 2008;48:389–97.

    Article  PubMed  CAS  Google Scholar 

  6. Sanchez R, Villagran E, Concha M, Cornejo R. Ultrastructural analysis of the attachment sites of Escherichia coli to the human spermatozoon after in vitro migration through estrogenic cervical mucus. Int J Fertil. 1989;34:363–7.

    PubMed  CAS  Google Scholar 

  7. Diemer T, Huwe P, Michelmann HW, Mayer F, Schiefer HG, Weidner W. Escherichia coli-induced alterations of human spermatozoa. An electron microscopy analysis. Int J Androl. 2000;23:178–86.

    Article  PubMed  CAS  Google Scholar 

  8. Nunez-Calonge R, Caballero P, Redondo C, Baquero F, Martinez-Ferrer M, Meseguer MA. Ureaplasma urealyticum reduces motility and induces membrane alterations in human spermatozoa. Hum Reprod. 1998;13:2756–61.

    Article  PubMed  CAS  Google Scholar 

  9. Diaz-Garcia FJ, Herrera-Mendoza AP, Giono-Cerezo S, Guerra-Infante FM. Mycoplasma hominis attaches to and locates intracellularly in human spermatozoa. Hum Reprod. 2006;21:1591–8.

    Article  PubMed  Google Scholar 

  10. James-Holmquest AN, Swanson J, Buchanan TM, Wende RD, Williams RP. Differential attachment by piliated and nonpiliated Neisseria gonorrhoeae to human sperm. Infect Immun. 1974;9:897–902.

    PubMed  CAS  Google Scholar 

  11. Erbengi T. Ultrastructural observations on the entry of Chlamydia trachomatis into human spermatozoa. Hum Reprod. 1993;8:416–21.

    PubMed  CAS  Google Scholar 

  12. Garrido N, Meseguer M, Bellver J, Remohi J, Simon C, Pellicer A. Report of the results of a 2 year programme of sperm wash and ICSI treatment for human immunodeficiency virus and hepatitis C virus serodiscordant couples. Hum Reprod. 2004;19:2581–6.

    Article  PubMed  Google Scholar 

  13. Paju A, Bjartell A, Zhang WM, Nordling S, Borgstrom A, Hansson J, Stenman UH. Expression and characterization of trypsinogen produced in the human male genital tract. Am J Pathol. 2000;157:2011–21.

    Article  PubMed  CAS  Google Scholar 

  14. Loskutoff NM, Huyser C, Singh R, Walker DL, Thornhill AR, Morris L, Webber L. Use of a novel washing method combining multiple density gradients and trypsin for removing human immunodeficiency virus-1 and hepatitis C virus from semen. Fertil Steril. 2005;84:1001–10.

    Article  PubMed  CAS  Google Scholar 

  15. Bollendorf A, Check JH, Katsoff D, Fedele A. The use of chymotrypsin/galactose to treat spermatozoa bound with anti-sperm antibodies prior to intra-uterine insemination. Hum Reprod. 1994;9:484–8.

    PubMed  CAS  Google Scholar 

  16. Gupta SK, Bansal P, Ganguly A, Bhandari B, Chakrabarti K. Human zona pellucida glycoproteins: functional relevance during fertilization. J Reprod Immunol. 2009;83:50–5.

    Article  PubMed  CAS  Google Scholar 

  17. Silva N, Solana A, Castro JM. Evaluation of the effects of different trypsin treatments on semen quality after BHV-1 inactivation, and a comparison of the results before and after freezing, assessed by a computer image analyzer. Anim Reprod Sci. 1999;54:227–35.

    Article  PubMed  CAS  Google Scholar 

  18. Donnelly ET, Lewis SE, McNally JA, Thompson W. In vitro fertilization and pregnancy rates: the influence of sperm motility and morphology on IVF outcome. Fertil Steril. 1998;70:305–14.

    Article  PubMed  CAS  Google Scholar 

  19. Visagie MH. In vitro cell signalling events of 2-methoxyestradiol-bis-sulphamate in a breast adenocarcinoma- and a non-tumorigenic breast epithelial cell line [Dissertation]. University of Pretoria; 2011, http://upetd.up.ac.za/thesis/available/etd-07112011-123356/

  20. World Health Organization. WHO laboratory manual for the examination and processing of human semen. 5th ed. Cambridge: Cambridge University Press; 2010.

    Google Scholar 

  21. Mahfouz RZ, Sharma RK, Poenicke K, Jha R, Paasch U, Grunewald S, Agarwal A. Evaluation of poly(ADP-ribose) polymerase cleavage (cPARP) in ejaculated human sperm fractions after induction of apoptosis. Fertil Steril. 2009;91:2210–20.

    Article  PubMed  CAS  Google Scholar 

  22. Marchetti C, Obert G, Deffosez A, Formstecher P, Marchetti P. Study of mitochondrial membrane potential, reactive oxygen species, DNA fragmentation and cell viability by flow cytometry in human sperm. Hum Reprod. 2002;17:1257–65.

    Article  PubMed  Google Scholar 

  23. Franken DR. New aspects of sperm-zona pellucida binding. Andrologia. 1998;30:263–8.

    Article  PubMed  CAS  Google Scholar 

  24. StataCorp. Stata statistical software: release 10. 10th ed. College Stattion: StataCorp LP; 2007.

    Google Scholar 

  25. Dunnett CW. New tables for multiple comparisons with a control. Biometrics. 1964;20:482.

    Article  Google Scholar 

  26. Bielanski A, Loewen KG, Hare WCD. Inactivation of bovine herpesvirus-1 (BHV-1) from in vitro infected semen. Theriogenology. 1988;30:649–57.

    Article  PubMed  CAS  Google Scholar 

  27. Esfandiari N, Burjaq H, Gotlieb L, Casper RF. Seminal hyperviscosity is associated with poor outcome of in vitro fertilization and embryo transfer: a prospective study. Fertil Steril. 2008;90:1739–43.

    Article  PubMed  Google Scholar 

  28. Mortimer D, Mortimer ST. Methods of sperm preparation for assisted reproduction. Ann Acad Med Singapore. 1992;21:517–24.

    PubMed  CAS  Google Scholar 

  29. Crittenden JA, Handelsman DJ, Stewart GJ. Semen analysis in human immunodeficiency virus infection. Fertil Steril. 1992;57:1294–9.

    PubMed  CAS  Google Scholar 

  30. Wang Y, Liang CL, Wu JQ, Xu C, Qin SX, Gao ES. Do Ureaplasma urealyticum infections in the genital tract affect semen quality? Asian J Androl. 2006;8:562–8.

    Article  PubMed  Google Scholar 

  31. Zinzendorf N, Kouassi-Agbessi B, Lathro J, Don C, Kouadio L, Loukou Y. Ureaplasma urealyticum or Mycoplasma hominis infections and semen quality of infertile men in Abidjan. J Reprod Contracept. 2008;19:65–72.

    Article  Google Scholar 

  32. Cohen J, Aafjes H. Proteolytic enzymes stimulate human spermatozoal motility and in vitro hamster egg penetration. Life Sci. 1982;30:899–904.

    Article  PubMed  CAS  Google Scholar 

  33. Mendeluk G, Gonzalez Flecha FL, Castello PR, Bregni C. Factors involved in the biochemical etiology of human seminal plasma hyperviscosity. J Androl. 2000;21:262–7.

    PubMed  CAS  Google Scholar 

  34. Fourie JM, Loskutoff N, Huyser C. Elimination of bacteria from human semen during sperm preparation using density gradient centrifugation with a novel tube insert. Andrologia. 2012;44:513–7.

    Article  PubMed  Google Scholar 

  35. Mattson KJ, Devlin BR, Loskutoff NM. Comparison of a recombinant trypsin with the porcine pancreatic extract on sperm used for the in vitro production of bovine embryos. Theriogenology. 2008;69:724–7.

    Article  PubMed  CAS  Google Scholar 

  36. Blevins BA, de la Rey M, Loskutoff NM. Technical note: effect of density gradient centrifugation with trypsin on the in vivo fertilising capability of bovine spermatozoa. Reprod Fertil Dev. 2008;20:784–8.

    Article  PubMed  CAS  Google Scholar 

  37. Figenschau Y, Bertheussen K. Enzymatic treatment of spermatozoa with a trypsin solution, SpermSolute: improved motility and enhanced ATP concentration. Int J Androl. 1999;22:342–4.

    Article  PubMed  CAS  Google Scholar 

  38. Westhoff D, Kamp G. Glyceraldehyde 3-phosphate dehydrogenase is bound to the fibrous sheath of mammalian spermatozoa. J Cell Sci. 1997;110:1821–9.

    PubMed  CAS  Google Scholar 

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Acknowledgments

The authors are grateful to Prof. P. Becker, Biostatistics Unit Medical Research Council (MRC) for the statistical evaluations; to Nidacon International for providing the PureSperm® products–The authors do not have a commercial or other association with the products that might cause a conflict of interest; to Mr. A. Stander, Department Physiology, University of Pretoria for the confirmation of rhProtease activity and the validation of flow cytometry protocols, and to the MRC for funding the research. The views expressed by the authors do not necessarily reflect the views of the MRC.

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Authors and Affiliations

  1. Department of Obstetrics & Gynaecology, Steve Biko Academic Hospital, University of Pretoria, Private Bag x323, Arcadia, Pretoria, Gauteng, 0007, South Africa

    J. Fourie & C. Huyser

  2. The Bill & Berniece Grewcock Center for Conservation & Research, Omaha, NE, USA

    N. Loskutoff

Authors
  1. J. Fourie
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  2. N. Loskutoff
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  3. C. Huyser
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Corresponding author

Correspondence to J. Fourie.

Additional information

Capsule The addition of rhProtease to density gradient layers did not impact negatively on: sperm apoptotic or necrotic status, mitochondrial membrane potential, or motility.

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Fourie, J., Loskutoff, N. & Huyser, C. Treatment of human sperm with serine protease during density gradient centrifugation. J Assist Reprod Genet 29, 1273–1279 (2012). https://doi.org/10.1007/s10815-012-9851-6

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  • DOI: https://doi.org/10.1007/s10815-012-9851-6

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