Pınar TURAN, GÖZDE ERKANLI ŞENTÜRK, FERİHA ERCAN

Kriyopreservasyon oligoastenoteratozoospermik erkeklerde DNA fragmantasyonunu ve ultrastrüktürel hasarı tetikler

Amaç: Oligoastenoteratozoospermi (OAT) erkek infertilitisisebeplerinden biridir. Kriyopreservasyon infertilite yönetimiiçin değerlidir. Bu çalışmanın amacı, OAT’lı hastalardasperm motilitesi, morfolojisi, ultrastrüktürel detaylar ve DNAfragmentasyonu üzerine kriyopreservasyonun etkilerini ortayaçıkarmaktır.Gereç ve Yöntem: Bu gözlemsel çalışmada, gönüllü 20normospermik ve 20 OAT’lı hastadan ejakulatlar toplanmıştır.Ejakulatlar, -196oC sıvı nitrojende saklanmış ve çözdürmeden üçve altı ay sonra analiz edilmiştir. Ejakulatlarda, motilite, morfolojive DNA fragmantasyonu dondurma öncesi ve sonrasındadeğerlendirilmiştir.Bulgular: Her iki grupta da sperm motilitesi ve morfolojisikriyopresvasyondan etkilenmiştir. Her iki grupta da çözdürmedensonra morfolojik değişiklikler anlamlı artmıştır. Ultrastrüktürelincelemeler membran bütünlüğünün değiştiğini ve akrozomalhasarın arttığını göstermiştir. Her iki grupta da, dondurma öncesiile karşılaştırıldığında, çözdürme sonrasında DNA kırıklarıolan hücrelerin arttığı gözlenmiştir. Her iki dondurma-çözmeperiyodunda, kontrol grubu ile kıyaslandığında OAT grubundaDNA kırıkları olan spermatozoa sayısının anlamlı olarak dahayüksek olduğu gözlenmiştir.Sonuç: Kriyopreservasyon, hem normospermik hem de OATgruplarında ultrastrüktürel hasar yapmaktadır ve DNA hasarınıindüklemektedir. Bu hasar OAT hastalarında daha ciddidir.

Anahtar Kelimeler:

Kriyopreservasyon, Oligoastenoteratozoospermi, DNA fragmantasyonu, ultrastrüktür

Cryopreservation triggers DNA fragmentation and ultrastructural damage in spermatozoa of oligoasthenoteratozoospermic men

Objective: Oligoasthenoteratozoospermia (OAT) is one of thecauses of male infertility. Cryopreservation is valuable for themanagement of infertility. This study aimed to reveal the effectsof cryopreservation on sperm motility, morphology, ultrastructuraldetails and DNA fragmentation in patients with OAT.Materials and Methods: In this observational study,ejaculates were collected from 40 male volunteers of whom 20were OAT and 20 were normospermic. The ejaculates were storedin liquid nitrogen at -196oC and analysed following thawing after1 or 3 months. Ejaculates were evaluated in terms of motility,morphology and DNA fragmentation before and after thawing.Results: Sperm motility and morphology were both affectedby cryopreservation in samples from both groups. After thawing,spermatozoa with morphological abnormalities were increasedsignificantly in both groups. Ultrastructural investigations showedalteration in integrity of the membranes and increased acrosomaldefects. Post-thaw investigations revealed prominent increasesin the number of DNA fragmented cells in both groups whencompared to the results before freezing. OAT groups revealed asignificantly higher number of DNA fragmented spermatozoacompared to the normospermic group for both time periods.Conclusion: Cryopreservation produces ultrastructuraldamage and induces DNA fragmentation in both normospermicand OAT groups. The damage is more severe in the OAT group.

Keywords:

Cryopreservation, Oligoasthenoteratozoospermia, DNA fragmentation, Ultrastructure,

PDF

___

Oehninger S, Duru NK, Srisombu C, Morshedi M. Assessment of sperm cryodamage and strategies to improve outcome. Mol Cell Endocrinol 2000; 169:3-10. doi: 10.1016/ S0303-7207(00)00343-9
Anger JT, Gilbert BR, Golstein M. Cryopreservation of sperm: indications, methods and results. J Urol 2003; 170(4 Pt1):1079-84. doi: 10.1097/01.ju.0000084820.98430.b8
Glander HJ, Schaller J. Binding of annexin V to plasma membranes of human spermatozoa: a rapid assay for detection of membrane changes after cryostorage. Mol Hum Reprod 1999;5:109-15. doi: 10.1093/molehr/5.2.109
Schiller J, Arnhold J, Glander HJ, Arnold K. Lipid analysis of human spermatozoa and seminal plasma by MALDITOF mass spectrometry and NMR spectroscopy - effects of freezing and thawing. Chem Phys Lipids 2000; 106: 145-56. doi: 10.1016/S0009-3084(00)00148-1
Liu CH, Tsao HM, Cheng TC, et al. DNA fragmentation, mitochondrial dysfunction and chromosomal aneuploidy in the spermatozoa of oligoasthenoteratozoospermic males. J Asist Reprod Genet 2004; 21: 119-26. doi: 10.1023/B:JARG.0000029495.22787.83
Kalthur G, Adiga SK, Upadhya S, Rao S, Kumar P. Effect of cryopreservation on sperm DNA integrity in patients with teratospermia. Fertil Steril 2008; 89: 1723–7. doi: 10.1016/j. fertnstert.2007.06.087
World Health Organisation. WHO laboratory manual for the examination and processing of human semen, 5th ed. Geneva, Switzerland: WHO Press, 2010.
Aydin MS, Senturk GE, Ercan F. Cryopreservation increases DNA fragmentation in spermatozoa of smokers. Acta Histochem 2013; 115: 394-400. doi: 10.1016/j. acthis.2012.10.003
Stanic P, Tandara M, Sonicki Z, Simunic V, Radakovic B, Suchanek E. Comparison of protective media and freezing techniques for cryopreservation of human semen. Eur J Obstet Gyn Reprod Biol 2000; 91: 65-70. doi: 10.1016/ S0301-2115(99)00255-9
Zeron Y, Pearl M, Borochow A, Arav A. Kinetic and temporal factors influence chilling injury to germinal vesicle mature bovine oocytes. Cryobiology 1999; 38: 35-42. doi: 10.1006/ cryo.1998.2139
Giraud MN, Motta C, Boucher D, Grizard G. Membrane fluidity predicts the outcome of cryopreservation of human spermatozoa. Human Reprod 2000; 15: 2160-4. doi: 10.1093/ humrep/15.10.2160
Quinn PJ. A lipid-phase separation model of low-temperature damage to biological membranes. Cryobiology 1985; 22: 128-46. doi: 10.1016/0011-2240(85)90167-1
O’Connell M, McClure N, Lewis SE. The effects of cryopreservation on sperm morphology, motility and mitochondrial function. Human Reprod 2002; 17: 704-9. doi: 10.1093/humrep/17.3.704
Said TM, Gaglani A, Agarwal A. Implication of apoptosis in sperm cryoinjury. Reprod BioMed Online 2010; 21: 456-62. doi: 10.1016/j.rbmo.2010.05.011
Saleh RA, Agarwal A. Oxidative stress and male infertility: from research bench to clinical practice. J Androl 2002; 23: 737-52.
Lasso JL, Noiles EE, Alvarez JG, Storey BT. Mechanism of superoxide dismutase loss from human sperm cells during cryopreservation. J Androl 1994; 15: 255-65.
Gadea J, Molla M, Selles E, Marco MA, Garcia-Vazquez FA, Gardon JC. Reduced glutathione content in human sperm is decreased after cryopreservation: effect of the addition of reduced glutathione to the freezing and thawing extenders. Cryobiology 2011; 62: 40-6. doi: 10.1016/j. cryobiol.2010.12.001
Di Santo M, Tarozzi N, Nadalini M, Borini A. Human sperm cryopreservation: Update on techniques, effect on DNA integrity, and implications for ART. Adv Urol 2012; 2012: 854837. doi: 10.1155/2012/854837
Martínez-Soto JC, Landeras J, Gadea J. Spermatozoa and seminal plasma fatty acids as predictors of cryopreservation success. Andrology 2013; 1: 365-75. doi: 10.1111/j.2047- 2927.2012.00040.x
Check ML, Check JH, Long R. Detrimental effects of cryopreservation on structural and functional integrity of the sperm membrane. Arch Androl 1991; 27: 155-60.
Paasch U, Sharma RK, Grupta AK, et al. Cryopreservation and thawing is associated with varying extent of activation of apoptotic machinery in subsets of ejaculated human spermatozoa. Bio Reprod 2004; 71: 1828-37. doi: 10.1095/ biolreprod.103.025627
Zribi N, Chakroun NF, Euch HE, Gargouri J, Bahloul A, Keskes LA. Effects of cryopreservation on human sperm deoxyribonucleic acid integrity. Fertil Steril 2010; 93: 159- 66. doi: 10.1016/j.fertnstert.2008.09.038
Nogueira D, Bourgain C, Verheyen G, Van Steirteghem AC. Light and electron microscopic analysis of human testicular spermatozoa and spermatids from frozen and thawed testicular biopsies. Hum Reprod 1999; 14: 2041-9. doi: 10.1093/humrep/14.8.2041
Ozkavukcu S, Erdemli E, Işık A, Oztuna D, Karahuseyinoglu S. Effects of cryopreservation on sperm parameters and ultrastructural morphology of human spermatozoa. J Assist Reprod Genet 2008; 25: 403-11. doi: 10.1007/s10815-008- 9232-3
Evenson DP, Jost LK, Marshall D, et al. Utility of the sperm chromatin structure assay as a diagnostic andrognostic tool in the human fertility clinic. Hum Reprod 1999; 14: 1039-49. doi: 10.1093/humrep/14.4.1039
Agarwal A, Saleh RA, Bedaiwy MA. Role of reactive oxygen species in the pathophysiology of human reproduction. Fertil Steril 2003; 79: 829-43. doi: 10.1016/S0015- 0282(02)004948-8
Aitken RJ, De Luliis GN, McLachlan RI. Biological and clinical significance of DNA damage in the male germ line. Int J Androl 2009; 32: 46-56. doi: 10.1111/j.1365- 2605.2008.00943.x
Desai N, Sharma R, Makker K, Sabanegh E, Agarwal A. Physiologic and pathologic levels of reactive oxygen species in neat semen of infertile men. Fertil Steril 2009; 92: 1626- 31. doi: 10.1016/j.fertnstert.2008.08.109
Brugnon F, Ouchchane L, Pons-Rejraji H, Artonne C, Farigoule M, Janny L. Density gradient centrifugation prior to cryopreservation and hypotaurine supplementation improve post-thaw quality of sperm from infertile men with oligoasthenoteratozoospermia. Hum Reprod 2013; 28: 2045- 57. doi: 10.1093/humrep/det253