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Chapter 15 - Oxidative Stress Testing: Indirect Tests

Published online by Cambridge University Press:  05 April 2021

Ashok Agarwal
The Cleveland Clinic Foundation, Cleveland, OH
Ralf Henkel
University of the Western Cape, South Africa
Ahmad Majzoub
Hamad Medical Corporation, Doha
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Oxidative stress (OS) is the consequence of an imbalance between reactive oxygen species (ROS) and the failure of antioxidants to neutralize excessive ROS production. Although many sperm functions require physiological levels of ROS, excessive levels of ROS are detrimental to the sperm [1]. OS is one of the most common etiologies of male infertility affecting 30–80 percent of infertile men [2, 3]. The role of OS in men with unexplained infertility has been clearly established [4]. OS affects sperm quality as a result of alterations in proteins, lipid peroxidation, DNA damage and apoptosis [1]. Damage to sperm DNA can compromise the contribution of paternal genome to the embryo [4]. Hence the advent of numerous tests to diagnose OS in the semen. There are several laboratory tests available to measure OS – both direct and indirect. Direct tests measure OS or free radicals such as ROS and reactive nitrogen species. These include chemiluminescence, nitroblue tetrazolium, cytochrome C reduction test, electron spin resonance, fluorescein isothiocynate (DFITC)-labeled lectins, and measurement of oxidation reduction potential. Indirect tests measure oxidized products resulting from ROS sources such as the oxidized form of nicotinamide adenine dinucleotide (NADPH)-oxidase in the sperm, the reduced form of NAD (NADH)-dependent oxidoreductase in mitochondria, or leukocytospermia. These include myeloperoxidase or Endtz test, antioxidants (both enzymatic and non-enzymatic), lipid peroxidation, and DNA damage. In this chapter we will discuss the indirect tests that are available to assess OS and also elaborate on the interpretation and their clinical significance [4, 5].

Publisher: Cambridge University Press
Print publication year: 2021

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1.Agarwal, A, Durairajanayagam, D, Halabi, J, Peng, J, Vazquez-Levin, M. Proteomics, oxidative stress and male infertility. Reprod Biomed Online 2014; 29(1): 3258.CrossRefGoogle ScholarPubMed
Agarwal, A, Gupta, S, Sikka, S. The role of free radicals and antioxidants in reproduction. Curr Opin Gynecol Obstet 2006; 18(3): 325–32.CrossRefGoogle ScholarPubMed
Aitken, J, Fisher, H. Reactive oxygen species generation and human spermatozoa: the balance of benefit and risk. Bioessays 1994; 16(4): 259–67.CrossRefGoogle ScholarPubMed
Tremellen, K. Oxidative stress and male infertility—a clinical perspective. Hum Reprod Update 2008; 14(3): 243–58.Google Scholar
Sharma, RK, Agarwal, A. Role of reactive oxygen species in male infertility. Urology 1996; 48(6): 835–50.Google Scholar
Shekarriz, M, Sharma, R, Thomas, A, Agarwal, A. Positive myeloperoxidase staining (Endtz test) as an indicator of excessive reactive oxygen species formation in semen. JAssist Reprod Gen 1995; 12(2): 70–4.Google Scholar
Plante, M, de Lamirande, E, Gagnon, C. Reactive oxygen species released by activated neutrophils, but not by deficient spermatozoa, are sufficient to affect normal sperm motility. Fertil Steril 1994; 62(2): 387–93.CrossRefGoogle Scholar
Calogero, AE, Duca, Y, Condorelli, RA, La Vignera, S. Male accessory gland inflammation, infertility, and sexual dysfunctions: a practical approach to diagnosis and therapy. Andrology 2017; 5(6): 1064–72.Google Scholar
Agarwal, A, Saleh, RA, Bedaiwy, MA. Role of reactive oxygen species in the pathophysiology of human reproduction. Fertil Steril 2003; 79(4): 829–43.Google Scholar
World Health Organization (2010). WHO Laboratory Manual for the Examination and Processing of Human Semen. Geneva: The WHO Press.Google Scholar
Kopa, Z, Wenzel, J, Papp, GK, Haidl, G. Role of granulocyte elastase and interleukin-6 in the diagnosis of male genital tract inflammation. Andrologia 2005; 37(5): 188–94.Google Scholar
Jochum, M, Pabst, W, Schill, WB. Granulocyte elastase as a sensitive diagnostic parameter of silent male genital tract inflammation. Andrologia 1986; 18(4): 413–19.Google ScholarPubMed
Politch, JA, Wolff, H, Hill, JA, Anderson, DJ. Comparison of methods to enumerate white blood cells in semen. Fertil Steril 1993; 60(2): 372–5.Google Scholar
Neumann, S, Gunzer, G, Hennrich, N, Lang, H.PMN-elastase assay”: enzyme immunoassay for human polymorphonuclear elastase complexed with α1-proteinase inhibitor. J Clin Chem Clin Biochem 1984; 22(10): 693–8.Google Scholar
Endtz, A. A rapid staining method for differentiating granulocytes from "germinal cells" in Papanicolaou-stained semen. Acta Cytol 1974; 18(1): 2.Google Scholar
Agarwal, A, Gupta, S, Sharma, R. Leukocytospermia quantitation (ENDTZ) test. In Agarwal, A. et al., eds., Andrological Evaluation of Male Infertility. Geneva: Springer International Publishing, pp. 6972.Google Scholar
Alvarez, JG, Sharma, RK, Ollero, M, Saleh, RA, Lopez, MC, Thomas, AJ Jr, Agarwal, A. Increased DNA damage in sperm from leukocytospermic semen samples as determined by the sperm chromatin structure assay. Fertil Steril 2002; 78(2): 319–29.Google Scholar
Aziz, N, Saleh, RA, Sharma, RK, Lewis-Jones, I, Esfandiari, N, Thomas, AJ Jr, Agarwal, A. Novel association between sperm reactive oxygen species production, sperm morphological defects, and the sperm deformity index. Fertil Steril 2004; 81(2): 349–54.CrossRefGoogle ScholarPubMed
Moskovtsev, SI, Willis, J, White, J, Mullen, JBM. Leukocytospermia: relationship to sperm deoxyribonucleic acid integrity in patients evaluated for male factor infertility. Fertil Steril 2007; 88(3): 737–40.Google Scholar
Moubasher, A, Sayed, H, Mosaad, E, Mahmoud, A, Farag, F, Taha, EA. Impact of leukocytospermia on sperm dynamic motility parameters, DNA and chromosomal integrity. Cent European J Urol 2018; 71(4): 470–5.Google ScholarPubMed
Politch, JA, Tucker, L, Bowman, FP, Anderson, DJ. Concentrations and significance of cytokines and other immunologic factors in semen of healthy fertile men. Hum Reprod 2007; 22(11): 2928–35.CrossRefGoogle ScholarPubMed
Agarwal, A, Virk, G, Ong, C, Du Plessis, SS. Effect of oxidative stress on male reproduction. World J Mens Health 2014; 32(1): 117.Google Scholar
Camejo, M, Segnini, A, Proverbio, F. Interleukin-6 (IL-6) in seminal plasma of infertile men, and lipid peroxidation of their sperm. Arch Androl 2001; 47(2): 97101.Google Scholar
Martínez, P, Proverbio, F, Camejo, MI. Sperm lipid peroxidation and pro‐inflammatory cytokines. Asian J Androl 2007; 9(1): 102–7.Google Scholar
Fraczek, M, Sanocka, D, Kamieniczna, M, Kurpisz, M. Proinflammatory cytokines as an intermediate factor enhancing lipid sperm membrane peroxidation in in vitro conditions. J Androl 2008; 29(1): 8592.Google Scholar
Mahfouz, R, Sharma, R, Sharma, D, Sabanegh, E, Agarwal, A. Diagnostic value of the total antioxidant capacity (TAC) in human seminal plasma. Fertil Steril 2009; 91(3): 805–11.CrossRefGoogle ScholarPubMed
Henkel, R, Sandhu, IS, Agarwal, A. The excessive use of antioxidant therapy: a possible cause of male infertility? Andrologia 2019; 51(1): e13162.CrossRefGoogle ScholarPubMed
Roychoudhury, S, Sharma, R, Sikka, S, Agarwal, A. Diagnostic application of total antioxidant capacity in seminal plasma to assess oxidative stress in male factor infertility. J Assist Reprod Gen 2016; 33(5): 627–35.Google Scholar
Benzie, IF, Strain, JJ. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal Biochem 1996; 239(1): 70–6.CrossRefGoogle ScholarPubMed
Cao, G, Alessio, HM, Cutler, RG. Oxygen-radical absorbance capacity assay for antioxidants. Free Radic Biol Med 1993; 14(3): 303–11.Google ScholarPubMed
Pasqualotto, FF, Sharma, RK, Pasqualotto, EB, Agarwal, A. Poor semen quality and ROS-TAC scores in patients with idiopathic infertility. Urol Int 2008; 81(3): 263–70.Google Scholar
Fridovich, I. Superoxide radical and superoxide dismutases. Annu Rev Biochem 1995; 64(1): 97112.Google Scholar
Olson, KR, Gao, Y, DeLeon, ER, Arif, M, Arif, F, Arora, N, Straub, KD. Catalase as a sulfide-sulfur oxido-reductase: an ancient (and modern?) regulator of reactive sulfur species (RSS). Redox Biol 2017; 12: 325–39.Google Scholar
Chance, B, Sies, H, Boveris, A. Hydroperoxide metabolism in mammalian organs. Physiol Rev 1979; 59(3): 527605.Google Scholar
Crisol, L, Matorras, R, Aspichueta, F, Expósito, A, Hernández, ML, Ruiz-Larrea, MB, Ruiz-Larrea, MB, Mendoza, R. Glutathione peroxidase activity in seminal plasma and its relationship to classical sperm parameters and in vitro fertilization-intracytoplasmic sperm injection outcome. Fertil Steril 2012; 97(4): 852–7.Google Scholar
Fraga, CG, Motchnik, PA, Shigenaga, MK, Helbock, HJ, Jacob, RA, Ames, BN. Ascorbic acid protects against endogenous oxidative DNA damage in human sperm. PNAS 1991; 88(24): 11003–6.Google Scholar
Song, GJ, Norkus, EP, Lewis, V. Relationship between seminal ascorbic acid and sperm DNA integrity in infertile men. Int J Androl 2006; 29(6): 569–75.Google Scholar
Micheli, L, Cerretani, D, Collodel, G, Menchiari, A, Moltoni, L, Fiaschi, A, Moretti, E. Evaluation of enzymatic and non‐enzymatic antioxidants in seminal plasma of men with genitourinary infections, varicocele and idiopathic infertility. Andrology 2016; 4(3): 456–64.Google Scholar
Poulos, A, White, I. The phospholipid composition of human spermatozoa and seminal plasma. Reprod 1973; 35(2): 265–72.CrossRefGoogle ScholarPubMed
Mack, S, Everingham, J, Zaneveld, L. Isolation and partial characterization of the plasma membrane from human spermatozoa. J Exp Zool 1986; 240(1): 127–36.Google Scholar
Alvarez, JG, Storey, BT. Differential incorporation of fatty acids into and peroxidative loss of fatty acids from phospholipids of human spermatozoa. Mol Reprod Dev 1995; 42(3): 334–46.CrossRefGoogle ScholarPubMed
Halliwell, B, Chirico, S. Lipid peroxidation: its mechanism, measurement, and significance. Am J Clin Nutr 1993; 57(5): 715S–725S.Google Scholar
Zarkovic, N. 4-Hydroxynonenal as a bioactive marker of pathophysiological processes. Mol Aspects Med 2003; 24(4–5): 281–91.CrossRefGoogle ScholarPubMed
Spickett, CM. The lipid peroxidation product 4-hydroxy-2-nonenal: advances in chemistry and analysis. Redox Biol 2013; 1(1): 145–52.CrossRefGoogle ScholarPubMed
Borovic, S, Rabuzin, F, Waeg, G, Zarkovic, N. Enzyme-linked immunosorbent assay for 4-hydroxynonenal–histidine conjugates. Free Radic Res 2006; 40(8): 809–20.Google Scholar
Baker, MA, Weinberg, A, Hetherington, L, Villaverde, A-I, Velkov, T, Baell, J, Gordon, CP. Defining the mechanisms by which the reactive oxygen species by-product, 4-hydroxynonenal, affects human sperm cell function. Biol Reprod 2015; 92(4): 101–10.Google Scholar
Delanty, N, Reilly, M, Pratico, D, FitzGerald, D, Lawson, J, FitzGerald, G. 8‐Epi PGF2α: specific analysis of an isoeicosanoid as an index of oxidant stress in vivo. Br J Clin Pharmacol 1996; 42(1): 1519.Google Scholar
Signorini, C, Comporti, M, Giorgi, G. Ion trap tandem mass spectrometric determination of F2‐isoprostanes. J Mass Spectrom 2003; 38(10): 1067–74.Google ScholarPubMed
Signorini, C, Perrone, S, Sgherri, C, Ciccoli, L, Buonocore, G, Leoncini, S, Rossi, V, Vecchio, D, Comporti, M. Plasma esterified F 2-isoprostanes and oxidative stress in newborns: role of nonprotein-bound iron. Ped Res 2008; 63(3): 287–91.Google Scholar
Moretti, E, Pascarelli, NA, Federico, MG, Renieri, T, Collodel, G. Abnormal elongation of midpiece, absence of axoneme and outer dense fibers at principal piece level, supernumerary microtubules: a sperm defect of possible genetic origin? Fertil Steril 2008; 90(4): 1201.e3–8.CrossRefGoogle ScholarPubMed
Khosrowbeygi, A, Zarghami, N. Fatty acid composition of human spermatozoa and seminal plasma levels of oxidative stress biomarkers in subfertile males. Prostaglandins Leukot Essent Fatty Acids 2007; 77(2): 117–21.Google Scholar
Tavilani, H, Goodarzi, MT, Doosti, M, Vaisi-Raygani, A, Hassanzadeh, T, Salimi, S, Joshaghani, HR. Relationship between seminal antioxidant enzymes and the phospholipid and fatty acid composition of spermatozoa. Reprod Biomed Online 2008; 16(5): 649–56.Google Scholar
Collodel, G, Moretti, E, Longini, M, Pascarelli, NA, Signorini, C. Increased F2-isoprostane levels in semen and immunolocalization of the 8-iso prostaglandin F2α in spermatozoa from infertile patients with varicocele. Oxid Med Cell Longev 2018; 7508014.Google Scholar
Rao, B, Soufir, J, Martin, M, David, G. Lipid peroxidation in human spermatozoa as related to midpiece abnormalities and motility. Gamete Res 1989; 24(2): 127–34.CrossRefGoogle ScholarPubMed
Grotto, D, Santa Maria, L, Boeira, S, Valentini, J, Charão, M, Moro, A, Nascimento, PC, Pomblum, VJ, Garcia, SC. Rapid quantification of malondialdehyde in plasma by high performance liquid chromatography – visible detection. J Pharm Biomed Anal, 2007; 43(2): 619–24.Google Scholar
Colagar, AH, Karimi, F, Jorsaraei, SGA. Correlation of sperm parameters with semen lipid peroxidation and total antioxidants levels in astheno-and oligoasheno-teratospermic men. Iran Red Crescent Med J 2013; 15(9): 780–5.Google Scholar
Al-Dujaily, SS, Hassan, NA, Bilal, SA, Salman, SL. Malondialdehyde measurements in semen after in vitro sperm activation by pentoxifylline and Glycyrrhiza glabra extract. Int J Biol Sci 2013; 3(4): 828–33.Google Scholar
Henkel, R, Kierspel, E, Stalf, T, Mehnert, C, Menkveld, R, Tinneberg, HR, Schill, WB, Kruger, TF. Effect of reactive oxygen species produced by spermatozoa and leukocytes on sperm functions in non-leukocytospermic patients. Fertil Steril 2005; 83(3): 635–42.CrossRefGoogle ScholarPubMed
Sakkas, D, Mariethoz, E, Manicardi, G, Bizzaro, D, Bianchi, PG, Bianchi, U. Origin of DNA damage in ejaculated human spermatozoa. Rev Reprod 1999; 4(1): 31–7.Google Scholar
Zini, A, Boman, JM, Belzile, E, Ciampi, A. Sperm DNA damage is associated with an increased risk of pregnancy loss after IVF and ICSI: systematic review and meta-analysis. Hum Reprod 2008; 23(12): 2663–8.Google Scholar
Agarwal, A, Varghese, AC, Sharma, RK. (2009). Markers of oxidative stress and sperm chromatin integrity. In Park-Sarge, OK, Curry, T., eds., Molecular Endocrinology. Methods in Molecular Biology (Methods and Protocols), vol. 590. Totowa, NJ: Humana Press, 377402.Google Scholar
Ribas‐Maynou, J, García‐Peiró, A, Fernández‐Encinas, A, Abad, C, Amengual, M, Prada, E, Navarro, J, Benet, J. Comprehensive analysis of sperm DNA fragmentation by five different assays: TUNEL assay, SCSA, SCD test and alkaline and neutral Comet assay. Andrology 2013; 1(5): 715–22.Google Scholar
Garolla, A, Cosci, I, Bertoldo, A, Sartini, B, Boudjema, E, Foresta, C. DNA double strand breaks in human spermatozoa can be predictive for assisted reproductive outcome. Reprod Biomed Online 2015; 31(1): 100–7.CrossRefGoogle ScholarPubMed
Deng, C, Li, T, Xie, Y, Guo, Y, Yang, QY, Liang, X, Deng, CH, Liu, GH. Sperm DNA fragmentation index influences assisted reproductive technology outcome: a systematic review and meta-analysis combined with a retrospective cohort study. Andrologia 2019; 51(6): e13263.CrossRefGoogle ScholarPubMed
Majzoub, A, Agarwal, A, Cho, CL, Esteves, SC. Sperm DNA fragmentation testing: a cross sectional survey on current practices of fertility specialists. Transl Androl Urol 2017; 6(4): S710S719.CrossRefGoogle ScholarPubMed
Evenson, DP, Larson, KL, Jost, LK. Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques. J Androl 2002; 23(1): 2543.Google Scholar
Evenson, DP. Sperm chromatin structure assay (SCSA): 30 years’ experience with the SCSA. In Agarwal, A, Zini, A., eds., Sperm DNA and Male Infertility and ART. New York: Springer Publishers, pp. 125–49.Google Scholar
Evenson, DP. The Sperm Chromatin Structure Assay (SCSA®) and other sperm DNA fragmentation tests for evaluation of sperm nuclear DNA integrity as related to fertility. Anim Reprod Sci 2016; 169:5675.CrossRefGoogle ScholarPubMed
Sharma, RK, Sabanegh, E, Mahfouz, R, Gupta, S, Thiyagarajan, A, Agarwal, A. TUNEL as a test for sperm DNA damage in the evaluation of male infertility. Urology 2010; 76(6): 1380–6.Google Scholar
Sharma, R, Ahmad, G, Esteves, SC, Agarwal, A. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay using bench top flow cytometer for evaluation of sperm DNA fragmentation in fertility laboratories: protocol, reference values, and quality control. J Assist Reprod Genet 2016; 33(2): 291300.Google Scholar
Ribeiro, S, Sharma, R, Gupta, S, Cakar, Z, De Geyter, C, Agarwal, A. Inter-and intra-laboratory standardization of TUNEL assay for assessment of sperm DNA fragmentation. Andrology 2017; 5(3): 477–85.Google Scholar
Sharma, R, Cakar, Z, Agarwal, A. (2018) TUNEL assay by benchtop flow cytometer in clinical laboratories. In Zini, A, Agarwal, A., eds., A Clinician's Guide to Sperm DNA and Chromatin Damage. New York: Springer, pp. 103–18.Google Scholar
Fernández, JL, Muriel, L, Goyanes, V, Segrelles, E, Gosálvez, J, Enciso, M, LaFromboise, M, De Jonge, C. Halosperm® is an easy, available, and cost-effective alternative for determining sperm DNA fragmentation. Fertil Steril 2005; 84(4): 860.Google Scholar
Gosálvez, J, Rodríguez-Predreira, M, Mosquera, A, López-Fernández, C, Esteves, SC, Agarwal, A, Fernández, JL. Characterisation of a subpopulation of sperm with massive nuclear damage, as recognised with the sperm chromatin dispersion test. Andrologia 2014; 46(6): 602–9.Google Scholar
Feijó, CM, Esteves SC. Diagnostic accuracy of sperm chromatin dispersion test to evaluate sperm deoxyribonucleic acid damage in men with unexplained infertility. Fertil Steril 2014; 101(1): 5863.Google Scholar
Cortes-Gutierrez, EI, Davila-Rodriguez, MI, Cerda-Flores, RM, Fernández, JL, López-Fernández, C, Aragón Tovar, AR, Gosálvez, J. Localisation and quantification of alkali-labile sites in human spermatozoa by DNA breakage detection-fluorescence in situ hybridisation. Andrologia 2015; 47(2): 221–7.CrossRefGoogle ScholarPubMed
Cortés-Gutiérrez, EI, Fernández, JL, Dávila-Rodríguez, MI, López-Fernández, C, Gosálvez, J. (2017). Two-tailed comet assay (2T-Comet): simultaneous detection of DNA single and double strand breaks. In Pellicciari, C, Biggiogera, M, eds., Histochemistry of Single Molecules: Methods and Protocols. New York: Springer Science+Business Media, pp. 285–93.Google Scholar
Ashwood-Smith, M, Edwards, R. DNA repair by oocytes. Mol Hum Reprod 1996; 2(1): 4651.CrossRefGoogle ScholarPubMed
Meseguer, M, Santiso, R, Garrido, N, García-Herrero, S, Remohí, J, Fernandez, JL. Effect of sperm DNA fragmentation on pregnancy outcome depends on oocyte quality. Fertil Steril 2011; 95(1): 124–8.Google Scholar
Cozzubbo, T, Neri, QV, Rosenwaks, Z, Palermo, GD. To what extent can oocytes repair sperm DNA fragmentation? Fertil Steril 2014; 102(3): e61.CrossRefGoogle Scholar
Lewis, SEM. Should sperm DNA fragmentation testing be included in the male infertility work-up? Reprod BioMed Online 2015; 31(2): 134–7.Google Scholar
Agarwal, A, Cho, C-L, Esteves, SC, Majzoub, A. Current limitation and future perspective of sperm DNA fragmentation tests. Transl Androl Urol 2017; 6(4): S549S552.Google Scholar
Wang, Y-J, Zhang, R-Q, Lin, Y-J, Zhang, R-G, Zhang, W-L. Relationship between varicocele and sperm DNA damage and the effect of varicocele repair: a meta-analysis. Reprod BioMed Online 2012; 25(3): 307–14.Google Scholar
Esteves, SC, Gosálvez, J, López-Fernández, C, Núñez-Calonge, R, Caballero, P, Agarwal, A, Fernández, JL. Diagnostic accuracy of sperm DNA degradation index (DDSi) as a potential noninvasive biomarker to identify men with varicocele-associated infertility. Int Urol Nephrol 2015; 47(9): 1471–7.Google Scholar
Smit, M, Romijn, JC, Wildhagen, MF, Veldhoven, JL, Weber, RF, Dohle, GR. Decreased sperm DNA fragmentation after surgical varicocelectomy is associated with increased pregnancy rate. J Urol 2013; 189(1): S146S150.Google Scholar
Chen, S-S, Huang, WJ, Chang, LS, Wei, Y-H. Attenuation of oxidative stress after varicocelectomy in subfertile patients with varicocele. J Urol 2008; 179(2): 639–42.CrossRefGoogle ScholarPubMed
Agarwal, A, Hamada, A, Esteves, SC. Insight into oxidative stress in varicocele-associated male infertility: part 1. Nat Rev Urol 2012; 9: 678.Google Scholar
Blumer, CG, Restelli, AE, Giudice, PTD, Soler, TB, Fraietta, R, Nichi, M, Bertolla, RP, Cedenho, AP. Effect of varicocele on sperm function and semen oxidative stress. Br J Urol Int 2012; 109(2): 259–65.CrossRefGoogle ScholarPubMed
Agarwal, A, Majzoub, A, Esteves, SC, Ko, E, Ramasamy, R, Zini, A. Clinical utility of sperm DNA fragmentation testing: practice recommendations based on clinical scenarios. Transl Androl Urol 2016; 5(6): 935–50.CrossRefGoogle ScholarPubMed
Carlini, T, Paoli, D, Pelloni, M, Faja, F, Dal, Lago A, Lombardo, F, Lenzi, A, Gandini, L. Sperm DNA fragmentation in Italian couples with recurrent pregnancy loss. Reprod BioMed Online 2017; 34(1): 5865.Google Scholar
Brohi, RD, Huo, LJ. Posttranslational modifications in spermatozoa and effects on male fertility and sperm viability. OMICS 2017; 21(5): 245–56.Google Scholar
Radi, R. Nitric oxide, oxidants, and protein tyrosine nitration. Pro Nat Acad Sci. 2004; 101(12): 4003–8.Google Scholar
Dalle-Donne, I, Rossi, R, Colombo, R, Giustarini, D, Milzani, A. Biomarkers of oxidative damage in human disease. Clin Chem 2006; 52(4): 601–23.CrossRefGoogle ScholarPubMed
Samanta, L, Swain, N, Ayaz, A, Venugopal, V, Agarwal, A. Post-translational modifications in sperm proteome: the chemistry of proteome diversifications in the pathophysiology of male factor infertility. Biochem Biophys Acta 2016; 1860(7): 1450–65.Google Scholar
Halliwell, B, Gutteridge, J. (2007). Cellular responses to oxidative stress: adaptation, damage, repair, senescence and death. In Halliwell, B, Gutteridge, J, eds., Free Radicals in Biology and Medicine. New York: Oxford University Press, pp. 187267.Google Scholar
Herrero, MB, de Lamirande, E, Gagnon, C. Tyrosine nitration in human spermatozoa: a physiological function of peroxynitrite, the reaction product of nitric oxide and superoxide. Mol Hum Reprod 2001; 7(10): 913–21.Google Scholar
Vignini, A, Nanetti, L, Buldreghini, E, Moroni, C, Ricciardo-Lamonica, G, Mantero, F, Boscaro, M, Mazzanti, L, Balercia, G. The production of peroxynitrite by human spermatozoa may affect sperm motility through the formation of protein nitrotyrosine. Fertil Steril 2006; 85(4): 947–53.Google Scholar
Bollineni, RC, Fedorova, M, Blüher, M, Hoffmann, R. Carbonylated plasma proteins as potential biomarkers of obesity induced type 2 diabetes mellitus. J Proteome Res 2014; 13(11): 5081–93.Google Scholar
Dalle-Donne, I, Giustarini, D, Colombo, R, Rossi, R, Milzani, A. Protein carbonylation in human diseases. Trends Mol Med 2003; 9(4): 169–76.Google Scholar
Engvall, E, Perlmann, P. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 1971; 8(9): 871–4.Google Scholar
El-Taieb, MA, Herwig, R, Nada, EA, Greilberger, J, Marberger, M. Oxidative stress and epididymal sperm transport, motility and morphological defects. Eur J Obstet Gynecol Reprod Biol 2009; 144 (1): S199S203.Google Scholar
Romeo, C, Ientile, R, Impellizzeri, P, Turiaco, N, Teletta, M, Antonuccio, P, Basile, M, Gentile, C. Preliminary report on nitric oxide‐mediated oxidative damage in adolescent varicocele. Hum Reprod 2003; 18(1): 26–9.CrossRefGoogle ScholarPubMed
Stadtman, ER, Berlett, BS. Fenton chemistry. Amino acid oxidation. J Biol Chem 1991; 266(26): 17201–11.Google Scholar
Hamada, A, Sharma, R, du Plessis, SS, Willard, B, Yadav, SP, Sabanegh, E, Agarwal, A. Two-dimensional differential in-gel electrophoresis-based proteomics of male gametes in relation to oxidative stress. Fertil Steril 2013; 99(5): 1216–26.Google Scholar
Sharma, R, Agarwal, A, Mohanty, G, Du Plessis, SS, Gopalan, B, Willard, B, Yadav, SP, Sabanegh, E. Proteomic analysis of seminal fluid from men exhibiting oxidative stress. Reprod Biol Endocrinol 2013; 3 (11): 85.Google Scholar
Sharma, R, Agarwal, A, Mohanty G, Hamada Gopalan B, AJ, Willard, B, Yadav, S, du Plessis, S. Proteomic analysis of human spermatozoa proteins with oxidative stress. Reprod Biol Endocrinol 2013; 11: 48.Google Scholar
Agarwal, A, Ayaz, A, Samanta, L, Sharma, R, Assidi, M, Abuzenadah, A M, Sabanegh, E. Comparative proteomic network signatures in seminal plasma of infertile men as a function of reactive oxygen species. Clin Proteomics 2015; 12(1): 23.Google Scholar
Ayaz, A, Agarwal, A, Sharma, R, Arafa, M, Elbardisi, H, Cui, Z. Impact of precise modulation of reactive oxygen species levels on spermatozoa proteins in infertile men. Clin Proteomics 2015; 12(1): 4.Google Scholar
Agarwal, A, Sharma, R, Samanta, L, Durairajanayagam, D, Sabanegh, E. Proteomic signatures of infertile men with clinical varicocele and their validation studies reveal mitochondrial dysfunction leading to infertility. Asian J Androl 2016; 18(2): 282–91.Google Scholar
Samanta, L, Agarwal, A, Swain, N, Sharma, R, Gopalan, B, Esteves, SC, Durairajanayagam, D, Sabanegh, E. Proteomic signatures of sperm mitochondria in varicocele: clinical use as biomarkers of varicocele associated infertility. J Urol 2018; 200(2): 414–22.Google Scholar
Mathews, ST, Kim, T. Imaging systems for westerns: chemiluminescence vs. infrared detection. Methods Mol Biol 2009; 536: 499513.CrossRefGoogle ScholarPubMed
Nakane, PK, Pierce, GB Jr. Enzyme-labeled antibodies for the light and electron microscopic localization of tissue antigens. J Cell Biol 1967; 33(2): 307–18.Google Scholar
Salvolini, E, Buldreghini, E, Lucarini, G, Vignini, A, Lenzi, A, Di Primio, R, Balercia, G. Involvement of sperm plasma membrane and cytoskeletal proteins in human male infertility. Fertil Steril 2013; 99(3): 697704.Google Scholar

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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