Protective effects of hesperetin on the quality of sperm, apoptosis, lipid peroxidation, and oxidative stress during the process of cryopreservation: An experimental study
Background: Hesperetin is a bioflavonoid compound, largely used in Chinese traditional medicine and found plenty in citrus fruits. Hesperetin has beneficial effects against different diseases. The sperm cryopreservation process is a common method that is used in infertility laboratories. It has been reported that during the cryopreservation process, the quality of sperm is significantly reduced.
Objective: To investigate the effect of hesperetin on the quality of human spermatozoa during the cryopreservation process.
Materials and Methods: In this experimental study, 22 sperm sample of normozoospermia men who referred to the infertility department of the Shariati Hospital (Tehran, Iran) Between October and November 2019 were collect and divided into three groups as: 1) fresh, 2) control (frozen-thawed group without treatment), and 3) treatment group as frozen-thawed samples supplemented with 20 μM hesperetin. Motility, Viability, morphology, Apoptotic-like changes, intracellular H2O2, intracellular O2−, and lipid peroxidation (LPO) was measured.
Results: Hesperetin treatment during the cryopreservation process of human sperm significantly improved the viability, motility, and morphology rates of the spermatozoa after frozen-thawed process in control group (p < 0.01). In addition, it significantly reduced the reactive oxygen species (ROS) level, LPO level and increased the percentage of viable sperm cells with intact plasma membrane (p < 0.01) after frozen-thawed process.
Conclusion: Hesperetin can improve the quality of human sperm and also protect human sperm against reactive oxygen species, LPO, and apoptosis during the cryopreservation-thawing process.
Key words: Cryopreservation, Hesperetin, Spermatozoa, Reactive oxygen species.
 Sherman JK. Synopsis of the use of frozen human semen since 1964: State of the art of human semen banking. Fertil Steril 1973; 24: 397–412.
 Stewart GJ, Cunningham AL, Driscoll GL, Tyler JP, Barr JA, Gold J, et al. Transmission of human tcell lymphotropic virus type III (htlv-III) by artificial insemination by donor. The Lancet 1985; 326: 581–584.
 Sanger WG, Olson JH, Sherman JK. Semen cryobanking for men with cancer–criteria change. Fertil Steril 1992; 58: 1024–1027.
 Donnelly ET, Steele EK, McClure N, Lewis SE. Assessment of DNA integrity and morphology of ejaculated spermatozoa from fertile and infertile men before and after cryopreservation. Hum Reprod 2001; 16: 1191–1199.
 Donnelly ET, McClure N, Lewis SE. Cryopreservation of human semen and prepared sperm: effects on motility parameters and DNA integrity. Fertil Steril 2001; 76: 892–900.
 Bollwein H, Fuchs I, Koess C. Interrelationship between plasma membrane integrity, mitochondrial membrane potential and DNA fragmentation in cryopreserved bovine spermatozoa. Reprod Domest Anim 2008; 43: 189–195.
 O’Connell M, McClure N, Lewis SEM. The effects of cryopreservation on sperm morphology, motility and mitochondrial function. Hum Reprod 2002; 17: 704– 709.
 Zribi N, Feki Chakroun N, El Euch H, Gargouri J, Bahloul A, Ammar Keskes L. Effects of cryopreservation on human sperm deoxyribonucleic acid integrity. Fertil Steril 2010; 93: 159–166.
 Bansal AK, Bilaspuri GS. Impacts of oxidative stress and antioxidants on semen functions. Vet Med Int 2010; 2010: 686137. 1–7.
 Peris SI, Bilodeau JF, Dufour M, BaileyJL. Impact of cryopreservation and reactive oxygen species on DNA integrity, lipid peroxidation, and functional parameters in ram sperm. Mol Reprod Dev 2007; 74: 878–892.
 Wang AW, Zhang H, Ikemoto I, Anderson DJ, Loughlin KR. Reactive oxygen species generation by seminal cells during cryopreservation. Urology 1997; 49: 921–925.
 Agarwal A, Makker K, Sharma R. Clinical relevance of oxidative stress in male factor infertility: An update. Am J Reprod Immunol 2008; 59: 2–11.
 Goncalves FS, Barretto LSS, Arruda RP, Perri SHV, Mingoti GZ. Effect of antioxidants during bovine in vitro fertilization procedures on spermatozoa and embryo development. Reprod Dom Anim 2010; 45: 129–135.
 Numan Bucak M, Sarıözkan S, Barbaros Tuncer P, Sakin F, Atessahin A, Kulaksiz R, et al. The effect of antioxidants on post-thawed Angora goat (Capra hircus ancryrensis) sperm parameters, lipid peroxidation and antioxidant activities. Small Ruminant Research 2010; 89: 24–30.
 Maxwell WMC, Watson PF. Recent progress in the preservation of ram semen. Anim Reprod Sci 1996; 42: 55–65.
 Gil-Izquierdo A, Gil MI, Ferreres F, Tomás-Barberán FA. In vitro availability of flavonoids and other phenolics in orange juice. J Agric Food Chem 2001; 49: 1035–1041.
 Lee NK, Choi SH, Park SH, Park EK, Kim DH. Antiallergic activity of hesperidin is activated by intestinal microflora. Pharmacology 2004; 71: 174–180.
 Kim HK, Jeong TS, Lee MK, Park YB, Choi MS. Lipidlowering efficacy of hesperetin metabolites in highcholesterol fed rats. Clin Chim Acta 2003; 327: 129– 137.
 Fernández-Rojas B, Gutiérrez-Venegas G. Flavonoids exert multiple periodontic benefits including anti-inflammatory, periodontal ligamentsupporting, and alveolar bone-preserving effects. Life Sci 2018; 209: 435–454.
 Shagirtha K, Pari L. Hesperetin, a citrus flavonone, protects potentially cadmium induced oxidative testicular dysfunction in rats. Ecotoxicol Environ Saf 2011; 74: 2105–2111.
 Trivedi PP, Kushwaha S, Tripathi DN, Jena GB. Cardioprotective effects of hesperetin against doxorubicin-induced oxidative stress and DNA damage in rat. Cardiovasc Toxicol 2011; 11: 215–225.
 Trivedi PP, Tripathi DN, Jena GB. Hesperetin protects testicular toxicity of doxorubicin in rat: Role of NF j B , p38 and caspase-3. Food Chem Toxicol 2011; 49: 838–847.
 Samie A, Sedaghat R, Baluchnejadmojarad T, Roghani M. Hesperetin, a citrus flavonoid, attenuates testicular damage in diabetic rats via inhibition of oxidative stress, inflammation, and apoptosis. Life Sci 2018; 210: 132–139.
 Aranganathan S, Nalini N. Efficacy of the potential chemopreventive agent, hesperetin (citrus flavanone), on 1, 2-dimethylhydrazine induced colon carcinogenesis. Food Chem Toxicol 2009; 47: 2594–2600.
 Mahfouz R, Sharma R, Lackner J, Aziz N, Agarwal A. Evaluation of chemiluminescence and flow cytometry as tools in assessing production of hydrogen peroxide and superoxide anion in human spermatozoa. Fertil Steril 2009; 92: 819–827.
 Mahfouz RZ, du Plessis SS, Aziz N, Sharma R, Sabanegh E, Agarwal A. Sperm viability, apoptosis, and intracellular reactive oxygen species levels in human spermatozoa before and after induction of oxidative stress. Fertil Steril 2010; 93: 814–821.
 Mallick Ch, Mandal S, Barik B, Bhattacharya A, Ghosh D. Protection of testicular dysfunctions by MTEC, a formulated herbal drug, in streptozotocin induced diabetic rat. Biol Pharm Bull 2007; 30: 84–90.
 Larsen L, Scheike Th, Jensen TK, Bonde JP, Ernst E, Hjollund NH, et al. Computer-assisted semen analysis parameters as predictors for fertility of men from the general population. Hum Reprod 2000; 15: 1562–1567.
 Rowe PJ, Comhaire FH, Hargreave TB, Mahmoud AM. WHO manual for the standardised investigation, diagnosis and management of the infertile male. Cambridge: Cambridge University Press; 2000. 91.
 Nawroth F, Rahimi G, Isachenko E, Isachenko V, Liebermann M, Tucker MJ, et al. Cryopreservation in assisted reproductive technology: New Trends. Semin Reprod Med 2005; 23: 325–335.
 Partyka A, Łukaszewicz E, Nizanski W. Effect of cryopreservation on sperm parameters, lipid peroxidation and antioxidant enzymes activity in fowl semen. Theriogenology 2012; 77: 1497–1504.
 Martin G, Sabido O, Durand Ph, Levy R. Cryopreservation induces an apoptosis-like mechanism in bull sperm. Biol Reprod 2004; 71: 28–37.
 Said TM, Gaglani A, Agarwal A. Implication of apoptosis in sperm cryoinjury. Reprod Biomed Online 2010; 21: 456–462.
 Shabani Nashtaei M, Amidi F, Sedighi Gilani MA, Aleyasin A, Bakhshalizadeh Sh, Naji M, et al. Protective features of resveratrol on human spermatozoa cryopreservation may be mediated through 5’ AMP-activated protein kinase activation. Andrology 2017; 5: 313–326.
 Zhang J, Song J, Wu D, Wang J, Dong W. Hesperetin induces the apoptosis of hepatocellular carcinoma cells via mitochondrial pathway mediated by the increased intracellular reactive oxygen species, ATP and calcium. Med Oncol 2015; 32: 101–111.
 Celeghini EC, de Arruda RP, de Andrade AF, Nascimento J, Raphael CF, Rodrigues PH. Effects that bovine sperm cryopreservation using two different extenders has on sperm membranes and chromatin. Anim Reprod Sci 2008; 104: 119–131.
 Watson PF. The causes of reduced fertility with cryopreserved semen. Anim Reprod Sci 2000; 60: 481–492.
 Cormier N, Bailey JL. A differential mechanism is involved during heparin- and cryopreservation-induced capacitation of bovine spermatozoa. Biol Reprod 2003; 69: 177–185.
 Baldi E, Luconi M, Bonaccorsi L, Muratori M, Forti G. Intracellular events and signaling pathways involved in sperm acquisition of fertilizing capacity and acrosome reaction. Front Biosci 2000; 3: E110–E123.
 Medeiros CMO, Forell F, Oliveira ATD, Rodrigues JL. Current status of sperm cryopreservation: why isn’t it better? Theriogenology 2002; 57: 327–344.
 Len JS, Koh WSD, Tan SX. The roles of reactive oxygen species and antioxidants in cryopreservation. Biosci Rep 2019; 39: 1497–1504.
 Banihani S, Agarwal A, Sharma R, Bayachou M. Cryoprotective effect of l-carnitine on motility, vitality and DNA oxidation of human spermatozoa. Andrologia 2014; 46: 637–641.
 Bucak MN, Ateşşahin A, Varışlı Ö, Yüce A, Tekin N, Akçay A. The influence of trehalose, taurine, cysteamine and hyaluronan on ram semen: Microscopic and oxidative stress parameters after freeze-thawing process. Theriogenology 2007; 67: 1060–1067.
 Craig LB, Brush RS, Sullivan MT, Zavy MT, Agbaga MP, Anderson RE. Decreased very long-chain polyunsaturated fatty acids in sperm correlates with sperm quantity and quality. J Assist Reprod Genet 2019; 36: 1379–1385.
 Bassiri F, Nasr-Esfahani MH, Forozanfar M, Tavalaee M. Relationship between sperm parameters with sperm function tests in infertile men with at least one failed cycle after intracytoplasmic sperm injection cycle. Int J Fertil Steril 2020; 13: 324–329.
 Aitken RJ. Reactive oxygen species as mediators of sperm capacitation and pathological damage. Mol Reprod Dev 2017; 84: 1039–1052.
 Ozkavukcu S, Erdemli E, Isik A, Oztuna D, Karahuseyinoglu S. Effects of cryopreservation on sperm parameters and ultrastructural morphology of human spermatozoa. J Assist Reprod Genet 2008; 25: 403–411.
 Minaei MB, Barbarestani M, Nekoonam S, Abdolvahabi MA, Takzare N, Asadi MH, et al. Effect of Trolox addition to cryopreservation media on human sperm motility. Iran J Reprod Med 2012; 10: 99–104.