Top

Journal of Assisted Reproduction and Genetics

Published in:

01-11-2012 | Technological Innovations

Treatment of human sperm with serine protease during density gradient centrifugation

Authors: J. Fourie, N. Loskutoff, C. Huyser

Published in: Journal of Assisted Reproduction and Genetics | Issue 11/2012

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.

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.PubMedCrossRef

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.PubMedCrossRef

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.PubMedCrossRef

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.PubMedCrossRef

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.PubMedCrossRef

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

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.PubMedCrossRef

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.PubMedCrossRef

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.PubMedCrossRef

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

11.

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

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.PubMedCrossRef

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.PubMedCrossRef

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.PubMedCrossRef

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

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.PubMedCrossRef

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.PubMedCrossRef

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.PubMedCrossRef

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.

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.PubMedCrossRef

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.PubMedCrossRef

23.

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

24.

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

25.

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

26.

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

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.PubMedCrossRef

28.

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

29.

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

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.PubMedCrossRef

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.CrossRef

32.

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

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

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.PubMedCrossRef

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.PubMedCrossRef

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.PubMedCrossRef

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.PubMedCrossRef

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

Title: Treatment of human sperm with serine protease during density gradient centrifugation
Authors: J. Fourie
N. Loskutoff
C. Huyser
Publication date: 01-11-2012
Publisher: Springer US
Published in: Journal of Assisted Reproduction and Genetics / Issue 11/2012
Print ISSN: 1058-0468
Electronic ISSN: 1573-7330
DOI: https://doi.org/10.1007/s10815-012-9851-6

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Treatment of human sperm with serine protease during density gradient centrifugation

Abstract

Purpose

Methods

Results

Conclusion

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Abstract

Purpose

Methods

Results

Conclusion

Please log in to get access to this content

Other articles of this Issue 11/2012

Angiotensin-converting enzyme insertion/deletion (I/D) polymorphisms and recurrent pregnancy loss: a meta-analysis

Sperm head vacuolization affects clinical outcome in ICSI cycle. A proposal of a cut-off value

A review of, and commentary on, the ongoing second clinical introduction of preimplantation genetic screening (PGS) to routine IVF practice

Spontaneous in vitro maturation of oocytes prior to ovarian tissue cryopreservation in natural cycles of oncologic patients

Metaphase II (MII) oocytes obtained at different time points in the same in vitro fertilization cycle

The use of a metal container for vitrification of mouse ovaries, as a clinical grade model for human ovarian tissue cryopreservation, after different times and temperatures of transport