Abstract
Oxidative stress (OS) is an imbalance between the amount of reactive oxygen species (ROS) produced and the ability of the antioxidants to scavenge these. OS has been established as a major etiological cause of male infertility. High levels of ROS are harmful and cause damage to sperm nuclear DNA. Evaluation of OS-related damage to spermatozoa is therefore highly relevant in assisted reproductive techniques (ART) such as intracytoplasmic sperm injection (ICSI). ICSI is an effective therapy for severe male factor infertility that bypasses the majority of reproductive tract deficiencies. Despite the controversial findings in the existing literature, there is now enough evidence to show that sperm DNA damage is detrimental to reproductive outcomes. In addition to impairment of fertility, such damage might increase the transmission of genetic diseases to the offspring. Standardization of protocols to assess ROS, antioxidant status, and DNA damage is very important for implementation of these tests in clinical practice. Estimation of seminal ROS levels and extent of sperm DNA damage, especially in an infertile male, may help develop new therapeutic strategies and improve the success of ART.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Muratori, M., Piomboni, P., Baldi, E., Filimberti, E., Pecchioli, P., Moretti, E., Gambera, L., et al. (2000) Functional and ultrastructural features of DNA-fragmented human sperm. J Androl. 21, 903–912.
Aitken, R.J., Gordon, E., Harkiss, D., Twigg, J.P., Milne, P.J. Irvine, D.S. (1998) Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa. Biol Reprod. 59 , 1037–1046.
Sun, J.G., Jurisicova A, Casper, R.F. (1997) Detection of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro. Biol Reprod. 56, 602–607.
Lopes, S., Sun, J.G., Jurisicova, A., Meriano, J., Casper, R.F. (1998) Sperm deoxyribonucleic acid fragmentation is increased in poor-quality semen samples and correlates with failed fertilization in intracytoplasmic sperm injection. Fertil Steril. 69 , 528–532.
Host, E., Lindenberg, S., Kahn, J.A., Christensen, F. (1999) DNA strand breaks in human sperm cells: a comparison between men with normal and oligozoospermic sperm samples. Acta Obstet Gynecol Scand. 78,336–339.
Irvine, D.S., Twigg, J.P., Gordon, E.L., Fulton, N., Milne, P.A., Aitken, R.J. (2000) DNA integrity in human spermatozoa: relationships with semen quality. J Androl. 21,33–44.
Sharma, R.K., Agarwal A. (1996) Role of reactive oxygen species in male infertility. Urology 48, 835–850.
Hammadeh, M.E., Radwan, M., Al-Hasani, S., Micu, R., Rosenbaum, P., Lorenz, M., Schmidt W. (2006) Comparison of reactive oxygen species concentration in seminal plasma and semen parameters in partners of pregnant and non-pregnant patients after IVF/ICSI. Reprod Biomed Online 13, 696–706.
Warren, J.S., Johnson, K.J., Ward, P.A. (1987) Oxygen radicals in cell injury and cell death. Pathol Immunopathol Res. 6 , 301–315.
Griveau, J.F., Renard, P., Le Lannou, D. (1995) Superoxide anion production by human spermatozoa as a part of the ionophore-induced acrosome reaction process. Int J Androl. 18,67–74.
Aitken, R.J., Buckingham, D. (1992) Enhanced detection of reactive oxygen species produced by human spermatozoa with 7-dimethyl amino-naphthalin-1, 2-dicarbonic acid hydrazide. Int J Androl. 15, 211–219.
Agarwal, A, Allamaneni, S.S.R., and Said, T.M. (2004) Chemiluminescence technique for measuring reactive oxygen species. Reprod Biomed Online 9, 466–468.
Allamaneni, S.S.R., Agarwal, A., Nallella, K., Sharma, R.K., Thomas, A.J., Jr, Sikka, S.C. (2005) Characterization of oxidative stress status by evaluation of reactive oxygen species levels in whole semen and isolated spermatozoa. Fertil Steril. 83:800–803.
Athayde, K.S., Cocuzza, M., Agarwal, A., Krajcir, N., Lucon, A.M., Srougi, M., Hallak, J. (2007) Development of normal reference values for seminal reactive oxygen species and their correlation with leukocytes and semen parameters in a fertile population. J Androl. 28, 613–620.
Aitken, R.J., Buckingham, D.W., West, K.M. (1992) Reactive oxygen species and human spermatozoa: analysis of the cellular mechanisms involved in luminol- and lucigenin-dependent chemiluminescence. J Cell Physiol. 151, 466–477.
Aitken, R.J., Ryan, A.L., Curry, B.J., and Baker, M.A. (2003) Multiple forms of redox activity in populations of human spermatozoa. Mol Hum Reprod. 9, 645–661.
Said, T., Agarwal, A., Sharma, R.K., Mascha, E., Sikka, S.C., Thomas, A.J.Jr. (2004) Human sperm superoxide anion generation and correlation with semen quality in patients with male infertility. Fertil Steril. 82, 871–877.
Guthrie, H.D., Welch, G.R. (2006) Determination of intracellular reactive oxygen species and high mitochondrial membrane potential in Percoll-treated viable boar sperm using fluorescence-activated flow cytometry. J Anim Sci. 84, 2089–2100.
Mahfouz, R., Sharma, R., Lackner, J., Aziz, N, Agarwal, A. (2008) Evaluation of chemiluminescence and flowcytometry as tools in assessing production of hydrogen peroxide and superoxide anion in human spermatozoa. Fertil Steril. Aug 16. [Epub ahead of print], PMID: 18710706.
Aitken, R.J., Harkiss, D., Buckingham, D. (1993) Relationship between iron-catalysed lipid peroxidation potential and human sperm function. J Reprod Fertil. 98, 257–265.
Sharma, R.K., Pasqualotto, F.F., Nelson, D.R., Thomas, A.J., Jr., and Agarwal, A. (1999) The reactive oxygen species – total antioxidant capacity score is a new measure of oxidative stress to predict male infertility. Hum Reprod. 14, 2801–2807.
Pasqualotto, F.F., Sundaram, A., Sharma, R.K., Borges, E. Jr, Pasqualotto, E.B., Agarwal, A. (2008) Semen quality and oxidative stress scores in fertile and infertile patients with varicocele. Fertil Steril. 89, 602–607.
Hendin, B.N., Kolettis, P.N., Sharma, R.K., Thomas, A.J., Jr., and Agarwal, A. (1999) Varicocele is associated with elevated spermatozoal reactive oxygen species production and diminished seminal plasma antioxidant capacity. J Urol. 161, 1831–1834.
Pasqualotto, F.F., Sharma, R.K., Nelson, D.R., Thomas, A.J., Jr., and Agarwal, A. (2000) Relationship between oxidative stress, semen characteristics, and clinical diagnosis in men undergoing fertility investigation. Fertil Steril. 73, 459–464.
Saleh, R.A., Agarwal A., Kandirali, E., Sharma, R.K., Thomas, A.J. Jr., Nada, E.A., Evenson, D.P., and Alvarez, J.G. (2002) Leukocytospermia is associated with increased reactive oxygen species production by human spermatozoa. Fertil Steril. 78, 1215–1224.
Saleh, R.A., Agarwal, A., Nada, E.A., El-Tonsy, M.H., Sharma, R.K., Meyer, A., et al. (2003) Negative effects of increased sperm DNA damage in relation to seminal oxidative stress in men with idiopathic and male factor infertility. Fertil Steril. 79, 1597–1605.
Pasqualotto, F.F., Sharma, R.K., Pasqualotto, E.B., Agarwal, A. (2008) Poor semen quality and ROS-TAC scores in patients with idiopathic infertility. Urol Int. 3, 263–270.
Mahfouz, R., Sharma, R., Sharma, D., Sabanegh, E., Agarwal, A. (2009) Diagnostic value of the total antioxidant capacity (TAC) in human seminal plasma. Fertil Steril. 91, 805–811.
Said, T.M., Kattal, N., Sharma, R.K., Sikka, S.C., Thomas, A.J., Jr, Mascha, E., Agarwal, A. (2003) Enhanced chemiluminescence assay vs colorimetric assay for measurement of the total antioxidant capacity of human seminal plasma. J Androl. 24, 676–680.
Hoshi, K., Katayose, H., Yanagida, K., Kimura, Y., Sato, A. (1996) The relationship between acridine orange fluorescence of sperm nuclei and the fertilizing ability of human sperm. Fertil Steril. 66, 634–639.
Evenson, D.P., Jost, L.K., Marshall, D., Zinaman, M.J., Clegg, E., Purvis, K., de Angelis, P. et al. (1999) Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic. Hum Reprod. 14, 1039–1049.
Gopalkrishnan, K., Hurkadli, K., Padwal, V., Balaiah, D. (1999) Use of acridine orange to evaluate chromatin integrity of human spermatozoa in different groups of infertile men. Andrologia 31, 277–282.
Larson, K.L., DeJonge, C.J., Barnes, A.M., Jost, L.K., Evenson, D.P. (2000) Sperm chromatin structure assay parameters as predictors of failed pregnancy following assisted reproductive techniques. Hum Reprod. 15, 1717–1722.
Sergerie, M., Laforest, G., Bujan, L., Bissonnette, F., and Bleau, G. (2005) Sperm DNA fragmentation: threshold value in male fertility. Hum Reprod. 20, 3446–3451.
Mahfouz, R.Z., Sharma, R.K., Poenicke, K., Jha, R., Paasch, U., Grunewald, S., Agarwal, A. (2009) Evaluation of poly (ADP-ribose) polymerase cleavage (cPARP) in ejaculated human sperm fractions after induction of apoptosis. Fertil Steril. 91, 2210–20.
Evenson, D.P., Darzynkiewicz, Z., Melamed, M.R. (1980) Relation of mammalian sperm chromatin heterogeneity to fertility. Science 240, 1131–1133.
Boe-Hansen, G.B., Fedder, J., Ersboll, A.K., and Christensen, P. (2006) The sperm chromatin structure assay as a diagnostic tool in the human fertility clinic.Hum Reprod. 21, 1576–1582.
Wyrobek, J., Eskenazi, B., Young, S., Arnheim, N., Tiemann-Boege, I., Jabs, E.W., Glaser, R.L., Pearson, F.S., and Evenson, D. (2006) Advancing age has differential effects on DNA damage, chromatin integrity, gene mutations, and aneuploidies in sperm. PNAS 103, 9601–9606.
Ahmadi, A., Ng, S.C. (1999) Developmental capacity of damaged spermatozoa. Hum Reprod. 14 , 2279–2285.
Spanò M, Bonde JP, Hjøllund HI, Kolstad HA, Cordelli E, Leter G. (2000) Sperm chromatin damage impairs human fertility. The Danish First Pregnancy Planner Study Team. Fertil Steril. 73, 43–50.
Agarwal, A., and Said, T.M. (2003) Role of sperm chromatin abnormalities and DNA damage in male infertility. Hum Reprod Update 9, 331–345.
Larson-Cook, K.L., Brannian, J.D., Hansen, K.A., Kasperson, K.M., Aarnold, E.T. and Evenson, D.P. (2003) Relationship between the outcomes of assisted reproductive techniques and sperm DNA fragmentation as measured by the sperm chromatin structure assay. Fertil Steril. 80, 895–902.
Bungum, M., Humaidan, P., Spano, M., Jepson, K., Bungum, L., and Giwercman, A. (2004) The predictive value of sperm chromatin structure assay (SCSA) parameters for the outcome of intrauterine insemination, IVF and ICSI. Hum Reprod. 19 , 1401–1408.
Erenpreiss, J., Bungum, M., Spano, M., Elzanaty, S., Orbidans, J., and Giwercman, A. (2006) Intra-individual variation in sperm chromatin structure assay parameters in men from infertile couples: clinical implications. Hum Reprod. 21, 2061–2064.
Evenson, E. and Jost, L. (2000) Sperm chromatin structure assay is useful for fertility assessment. Methods Cell Sci 22, 169–189.
Darzynkiewicz, Z., Traganos, F., Sharpless, T., Melamed, M.R. (1975) Thermal denaturation of DNA in situ as studied by acridine orange staining and automated cytofluorometry. Exp Cell Res. 90, 411–428.
Fernandez, J.L., Muriel, L., Rivero, M.T., Goyanes, V., Vazquez, R., Alvarez, J.G. (2003) The sperm chromatin dispersion test: a simple method for the determination of sperm DNA fragmentation. J Androl. 24 , 59–66.
Enciso, M., Lopez-Fernandez, C., Fernandez, J.L., Garcia, P., Gosalbez, A., Gosalvez, J. (2006) A new method to analyze boar sperm DNA fragmentation under bright-field or fluorescence microscopy. Theriogenology 65 , 308–316.
Fernandez, J.L., Muriel, L., Goyanes, V., Segrelles, E., Gosalvez, J., Enciso, M., LaFromboise, M. et al. (2005) Simple determination of human sperm DNA fragmentation with an improved sperm chromatin dispersion test. Fertil Steril. 84 , 833–842.
Tejada, R.I., Mitchell, J.C., Norman, A., Marik, J.J., Friedman, S. (1984) A test for the practical evaluation of male fertility by acridine orange (AO) fluorescence. Fertil Steril. 42, 87–91.
Said, T.M., Paasch, U., Glander, H-J., and Agarwal, A. (204) Role of caspases in male infertility.Hum Reprod Update 10 , 39–51.
Ekert, P.G., Silke, J., Vaux, D.L. (1999) Caspase inhibitors. Cell Death Differ. 6, 1081–1086.
Bedner, E., Smolewski, P., Amstad, P., Darzynkiewicz, Z. (2000) Activation of caspases measured in situ by binding of fluorochrome-labeled inhibitors of caspases (FLICA): correlation with DNA fragmentation. Exp. Cell Res. 259, 308–313.
Paasch, U., Grunewald, S., Agarwal, A., Glander, H.J. (2004a) Activation pattern of caspases in human spermatozoa. Fertil Steril. 81, 802–809.
Paasch, U, Sharma, R.K., Gupta, A.K., Grunewald, S., Mascha, E.J., Thomas, A.J., Jr., Glander, H-J., and Agarwal, A. (2004b) Cryopreservation and thawing is associated with varying extent of activation of apoptotic machinery in subsets of ejaculated human spermatozoa. Biol Reprod. 71, 1828–1837.
Grunewald, S., Paasch, U., Said, T.M., Sharma, R.K., Glander, H.J., Agarwal, A. (2005) Caspase activation in human spermatozoa in response to physiological and pathological stimuli. Fertil Steril. 83, 1106–1112.
Glander, H-J., and Schaller, J. (1999) Binding of annexin V to plasma membranes of human spermatozoa: a rapid assay for detection of membrane changes after cryostorage. Mol Hum Reprod. 5, 109–115.
Barroso, G., Morshedi, M., and Oehninger, S. (2000) Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa. Hum Reprod. 15, 1338–1344.
Said, T., Agarwal, A., Grunewald, S., Rasch, M., Baumann, T., Kriegel, C., Li, L., Glander, H-J., Thomas, A.J., Jr., and Paasch, U. (2006) Selection of nonapoptotic spermatozoa as a new tool for enhancing assisted reproduction outcomes: An in vitro model.Biol Reprod. 74 , 530–537.
Barroso, G., Taylor, S., Morshedi, M., Manzur, F., Gaviño, F. and Oehninger, S. (2006) Mitochondrial membrane potential integrity and plasma membrane translocation of phosphatidylserine as early apoptotic markers: a comparison of two different sperm subpopulations. Fertil Steril. 85, 149–154.
Tesarik, J., Greco, E., Mendoza, C. (2001) Assisted reproduction with in-vitro-cultured testicular spermatozoa in cases of severe germ cell apoptosis: a pilot study. Hum Reprod. 16, 2640–2645.
Henkel, R., Hajimohammad, M., Stalf, T., Hoogendijk, C., Mehnert, C., Menkveld, R., Gips, H., Schill, WB., Kruger, T.F. (2004) Influence of deoxyribonucleic acid damage on fertilization and pregnancy. Fertil Steril. 81,965–972.
Seli, E., Gardner, D.K., Schoolcraft, W.B., Moffatt, O., Sakkas, D. (2004) Extent of nuclear DNA damage in ejaculated spermatozoa impacts on blastocyst development after in vitro fertilization. Fertil Steril. 82, 378–383.
Said, T.M., Grunewald, S., Paasch, U., Glander, H.J., Baumann, T., Kriegel, C., Li, L., Agarwal, A. (2005) Advantage of combining magnetic cell separation with sperm preparation techniques. Reprod Biomed Online 10, 740–746.
Aziz N, Said T, Paasch U, Agarwal A. (2007) The relationship between human sperm apoptosis, morphology and the sperm deformity index. Hum Reprod. 22 , 1413–1419.
Said, T., Agarwal, A., Zborowski, M., Grunewald, S., Glander, H-J., and Paasch U. (2008) Utility of magnetic cell separation as a molecular sperm preparation technique.J Androl. 29 , 134–142.
Said, T., Agarwal, A., Grunewald, S., Rasch, M., Baumann, T., Kriegel, Li, L., Glander, H-J., Thomas, A.J., Jr., and Paasch, U. (2006) Selection of nonapoptotic spermatozoa as a new tool for enhancing assisted reproduction outcomes: An in vitro model. Biol Reprod. 74 , 530–537.
Said, T.M., Grunewald, S., Paasch, U., Rasch, M., Agarwal, A., Glander, H-J. (2005) Effects of magnetic-activated cell sorting on sperm motility and cryosurvival rates. Fertil Steril. 83, 1442–1446.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Agarwal, A., Varghese, A.C., Sharma, R.K. (2009). Markers of Oxidative Stress and Sperm Chromatin Integrity. In: Park-Sarge, OK., Curry, T. (eds) Molecular Endocrinology. Methods in Molecular Biology, vol 590. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-378-7_24
Download citation
DOI: https://doi.org/10.1007/978-1-60327-378-7_24
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60327-377-0
Online ISBN: 978-1-60327-378-7
eBook Packages: Springer Protocols