Skip to main content Accessibility help
Hostname: page-component-77c89778f8-gvh9x Total loading time: 0 Render date: 2024-07-24T10:44:36.915Z Has data issue: false hasContentIssue false

Chapter 7 - Sperm Function Tests

Published online by Cambridge University Press:  16 February 2022

David Mortimer
Oozoa Biomedical Inc., Vancouver
Lars Björndahl
Karolinska Institutet, Stockholm
Christopher L. R. Barratt
University of Dundee
José Antonio Castilla
HU Virgen de las Nieves, Granada
Roelof Menkveld
Stellenbosch University, South Africa
Ulrik Kvist
Karolinska Institutet, Stockholm
Juan G. Alvarez
Centro ANDROGEN, La Coruña
Trine B. Haugen
Oslo Metropolitan University
Get access


Provides methods for a range of common sperm function tests that are structured as standard operating procedures (SOPs) for easy use at the bench. Methods are focussed on objectivity, robustness, standardized reporting, controlling the risk of errors, and minimizing measurement uncertainty. Includes sperm hyperactivation, acrosome reaction testing, and sperm-zone pellucida binding tests (hemi-zona and competititve binding formats). A protocol for using the sperm survival test in also provided. Limitations of the hyaluronan bibding assay, and of sperm fertilizing ability testing using zona pellucida-free hanster oocytes, are summarized.

Publisher: Cambridge University Press
Print publication year: 2022

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)


ESHRE Andrology Special Interest Group.Consensus workshop on advanced diagnostic andrology techniques. Hum Reprod 1996; 11: 1463–79.Google Scholar
Dresdner, RD, Katz, DF. Relationships of mammalian sperm motility and morphology to hydrodynamic aspects of cell function. Biol Reprod 1981; 25: 920–30.CrossRefGoogle ScholarPubMed
Mortimer, ST. A critical review of the physiological importance and analysis of sperm movement in mammals. Hum Reprod Update 1997; 3: 403–39.Google Scholar
Mortimer, ST, Swan, MA. Variable kinematics of capacitating human spermatozoa. Hum Reprod 1995; 10: 3178–82.Google Scholar
ESHRE Andrology Special Interest Group.Guidelines on the application of CASA technology in the analysis of spermatozoa. Hum Reprod 1998; 13: 142–5.Google Scholar
Mortimer, ST. CASA – Practical aspects. J Androl 2000; 21: 515–24.Google Scholar
Mortimer, D, Kossakowski, J, Mortimer, ST, Fussell, S. Prediction of fertilizing ability by sperm kinematics. Abstract OC-05–043. J Assist Reprod Genet 1997; 14 (5) Suppl: 52S.Google Scholar
Mortimer, D, Mortimer, ST. Laboratory investigation of the infertile male. In: Brinsden, PR, ed. A Textbook of In-Vitro Fertilization and Assisted Reproduction, 3rd edn. London, Taylor & Francis Medical Books, 2005, 6191.Google Scholar
Cummins, JM, Pember, SM, Jequier, AM, et al. A test of the human sperm acrosome reaction following ionophore challenge: relationship to fertility and other seminal parameters. J Androl 1991; 12: 98103.Google Scholar
Makkar, G, Ng, EH, Yeung, WS, et al. The significance of the ionophore-challenged acrosome reaction in the prediction of successful outcome of controlled ovarian stimulation and intrauterine insemination. Hum Reprod 2003; 18: 534–9.Google Scholar
Liu, DY, Stewart, T, Baker, HW. Normal range and variation of the zona pellucida-induced acrosome reaction in fertile men. Fertil Steril 2003; 80: 384–9.CrossRefGoogle ScholarPubMed
Liu, DY, Baker, HW. Disordered zona pellucida-induced acrosome reaction and failure of in vitro fertilization in patients with unexplained infertility. Fertil Steril 2003; 79: 7480.Google Scholar
Bastiaan, HS, Menkveld, R, Oehninger, S, et al. Zona pellucida induced acrosome reaction, sperm morphology, and sperm-zona binding assessments among subfertile men. J Assist Reprod Genet 2002; 19: 329–34.Google Scholar
Bastiaan, HS, Windt, ML, Menkveld, R, et al. Relationship between zona pellucida-induced acrosome reaction, sperm morphology, sperm-zona pellucida binding, and in vitro fertilization. Fertil Steril 2003; 79: 4955.Google Scholar
World Health Organization. WHO Laboratory Manual for the Analysis of Human Semen and Sperm-Cervical Mucus Interaction, 4th edn. Cambridge, Cambridge University Press, 1999.Google Scholar
Mortimer, D, Curtis, EF, Camenzind, AR. Combined use of fluorescent peanut agglutinin lectin and Hoechst 33258 to monitor the acrosomal status and vitality of human spermatozoa. Hum Reprod 1990; 5: 99103.Google Scholar
Cross, NL, Morales, P, Overstreet, JW, Hanson, FW. Two simple methods for detecting acrosome-reacted human sperm. Gamete Res 1986; 15: 213–26.CrossRefGoogle Scholar
Talbot, P, Chacon, RS. A triple-stain technique for evaluating normal acrosome reactions of human sperm. J Exp Zool 1981; 215: 201–8.CrossRefGoogle ScholarPubMed
Mortimer, D. Practical Laboratory Andrology. New York, Oxford University Press, 1994.Google Scholar
Burkman, LJ, Coddington, CC, Franken, DR, et al. The hemizona assay (HZA): development of a diagnostic test for binding of human spermatozoa to the human zona pellucida to predict fertilization potential. Fertil Steril 1988; 49: 688–93.Google Scholar
Franken, DR, Oehninger, S, Burkman, LJ, et al. The hemizona assays (HZA): a predictor of human sperm fertilizing potential in in vitro fertilization (IVF) treatment. J in Vitro Fertil Embryo Transf 1989; 6: 44–9.Google Scholar
Liu, DY, Lopata, A, Johnston, WIH, Baker, HWG. A human sperm-zona pellucida binding test using oocytes that failed to fertilize in vitro. Fertil Steril 1988; 50: 782–8.Google Scholar
Franken, DR, Kruger, TF, Menkveld, R, et al. Hemizona assay and teratozoospermia: increasing sperm insemination concentrations to enhance zona pellucida binding. Fertil Steril 1990; 54: 497503.Google Scholar
Menkveld, R, Franken, DR, Kruger, TF, et al. Sperm selection capacity of the human zona pellucida. Mol Reprod Dev 1991; 30: 346–52.CrossRefGoogle ScholarPubMed
Oehninger, S, Coddington, CC, Franken, DA, et al. Hemizona assay: assessment of sperm dysfunction and prediction of in vitro fertilization outcome. Fertil Steril 1989; 51: 665–70.CrossRefGoogle ScholarPubMed
Franken, DR, Oehninger, S. The clinical significance of sperm-zona pellucida binding: 17 years later. Front Biosci 2006; 1: 1227–33.Google Scholar
Janssen, M, Ombelet, W, Cox, A, et al. The hemizona assay: a simplified technique. Arch Androl 1997; 38: 127–31.Google Scholar
Sanchez, R, Finkenzeller, C, Schill, WB, Miska, W. Comparison of two methods to obtain hemizonae pellucidae for sperm function tests. Hum Reprod 1995; 10: 2945–7.Google Scholar
Edenfeld, J, Schöpper, B, Sturm, R, et al. Application of a 1.48‐μm diode laser for bisecting oocytes into two identical hemizonae for the hemizona assay. Int J Androl 2002; 25: 100–5.Google Scholar
Franken, DR, Burkman, LJ, Oehninger, SC, et al. Hemizona assay using salt‐stored human oocytes: evaluation of zona pellucida capacity for binding human spermatozoa. Gamete Res 1989; 22: 1526.CrossRefGoogle ScholarPubMed
Liu, DY, Liu, ML, Garratt, C, Baker, HWG. Comparison of the frequency of defective sperm-zona pellucida (ZP) binding and the ZP-induced acrosome reaction between subfertile men with normal and abnormal semen. Hum Reprod 2007; 22: 1878–84.Google Scholar
Oehninger, S, Franken, DR, Sayed, E, et al. Sperm function assays and their predictive value for fertilization outcome in IVF therapy: a meta-analysis. Hum Reprod Update 2000; 6: 160–8.Google Scholar
De Jonge, CJ, Centola, GM, Reed, ML, et al. Human sperm survival assay as a bioassay for the assisted reproductive technologies laboratory. J Androl 2003; 24: 1618.Google Scholar
Claassens, OE, Wehr, JB, Harrsion, KL. Optimizing sensitivity of the human sperm motility assay for embryo toxicity testing. Hum Reprod 2000; 15: 1586–91.Google Scholar
Coccia, ME, Becattini, C, Criscuoli, L, et al. A sperm survival test and in-vitro fertilization outcome in the presence of male factor infertility. Hum Reprod 1997; 12: 1969–73.Google Scholar
Mortimer, D, Curtis, EF, Camenzind, AR, Tanaka, S. The spontaneous acrosome reaction of human spermatozoa incubated in vitro. Hum Reprod 1989; 4: 5762.CrossRefGoogle ScholarPubMed
Huszar, G, Ozenci, CC, Cayli, S, et al. Hyaluronic acid binding by human sperm indicates cellular maturity, viability, and unreacted acrosomal status. Fertil Steril 2003; 79 Suppl 3: 1616–24.CrossRefGoogle ScholarPubMed
Ye, H, Huang, GN, Gao, Y, Liu, DY. Relationship between human sperm-hyaluronan binding assay and fertilization rate in conventional in vitro fertilization. Hum Reprod 2006; 21: 1545–50.Google Scholar
Tarozzi, N, Nadalini, M, Bizzaro, D, et al. Sperm-hyaluronan-binding assay: clinical value in conventional IVF under Italian law. Reprod Biomed Online 2009; 19 Suppl 3: 3543.Google Scholar
Lee, D, Jee, BC. Evaluation of normal morphology, DNA fragmentation, and hyaluronic acid binding ability of human spermatozoa after using four different commercial media for density gradient centrifugation. Clin Exp Reprod Med 2019; 46: 813.Google Scholar

Save book to Kindle

To save this book to your Kindle, first ensure is added to your Approved Personal Document E-mail List under your Personal Document Settings on the Manage Your Content and Devices page of your Amazon account. Then enter the ‘name’ part of your Kindle email address below. Find out more about saving to your Kindle.

Note you can select to save to either the or variations. ‘’ emails are free but can only be saved to your device when it is connected to wi-fi. ‘’ emails can be delivered even when you are not connected to wi-fi, but note that service fees apply.

Find out more about the Kindle Personal Document Service.

Available formats

Save book to Dropbox

To save content items to your account, please confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your account. Find out more about saving content to Dropbox.

Available formats

Save book to Google Drive

To save content items to your account, please confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your account. Find out more about saving content to Google Drive.

Available formats