Süheyla Gonca, Yusufhan Yazır, Sevilay Erimşah, Aysel Kükner, Bülent Duran

Normozoospermik İnfertil Hastaların İntrauterin İnseminasyon Tedavisi İçin İdeal Bir Sperm Seçim Yöntemi

Amaç: Bu çalışmada normozoospermik infertil hastaların (NİH) intrauterin inseminasyon tedavisi için ideal sperm seçim yöntemini belirlemek amacıyla geleneksel yüzdürme ve yoğunluk gradyanlı santrifüjleme teknikleri ve yeni nanoteknoloji bazlı mikroakışkan çip yöntemi ile seçilen spermlerin DNA bütünlüğü değerlendirilmiştir. Gereç ve Yöntemler: Yirmi hastadan toplanan semen örnekleri dört eşit parçaya bölündü. Kontrol, yoğunluk gradyanlı santrifüjleme, yüzdürme ve mikroakışkan çip (MÇ) grupları oluşturuldu ve bu dört gruba ait işlem görmemiş (kontrol) ve görmüş (diğer) sperm örnekleri DNA bütünlüğü açısından incelendi. Sperm kromatin yoğunlaşma kusurlarını ve DNA kırıklarını değerlendirmek için asidik anilin mavisi ve TUNEL boyaması kullanıldı. Reaktif oksijen türevi seviyelerini tespit etmek için akış sitometrisi ile diklorofloresan diasetat kullanıldı. Bulgular: MÇ yöntemiyle seçilen spermlerde kromatin kondensasyon defekti, DNA fragmantasyonu ve reaktif oksijen türevi değerleri, geleneksel yöntemlerle seçilen spermlere göre anlamlı şekilde düşük bulundu (p

An Ideal Sperm Selection Method for the Intrauterine Insemination Treatment of Normozoospermic Infertile Patients

Aim: In this study, we evaluated the DNA integrity of sperms selected by using the conventionalswim-up and density gradient centrifugation techniques and the new nanotechnology-based microfluidic chip method in order to determine the ideal sperm selection method for the intrauterineinsemination treatment of normozoospermic infertile patients (NIPs).Materials and Methods: Semen samples obtained from 20 patients were divided into four equalfractions. Control, density gradient centrifugation, swim-up, and microfluidic chip (MC) groupswere created, and the untreated (control) and treated (other) sperm samples from the four groupswere examined for DNA integrity. Acidic aniline blue staining and the TUNEL method were usedrespectively for evaluating sperm chromatin condensation defects and DNA fragmentation. Dichlorofluorescein diacetate and flow cytometry were used to determine the reactive oxygen specieslevels.Results: We measured significantly lower values of chromatin condensation defects, DNA fragmentation and reactive oxygen species for the sperms selected with the MC method, compared to thesperms selected with the conventional methods (p

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1. Agarwal A, Mulgund A, Hamada A, Chyatte MR. A unique view on male infertility around the globe. Reprod Biol Endocrinol. 2015;13:37.
2. Bieniek JM, Drabovich AP, Lo KC. Seminal biomarkers for the evaluation of male infertility. Asian J Androl. 2016;18(3):426–33.
3. Zini A, Bielecki R, Phang D, Zenzes MT. Correlations between two markers of sperm DNA integrity, DNA denaturation and DNA fragmentation, in fertile and infertile men. Fertil Steril. 2001;75(4):674–7.
4. Jones J, Horne G, Fitzgerald C. Who needs ICSI? A nationwide UK survey on ICSI use. Human Fertility. 2012;15(3):144–9.
5. Benchaib M, Braun V, Lornage J, Hadj S, Salle B, Lejeune H, et al. Sperm DNA fragmentation decreases the pregnancy rate in an assisted reproductive technique. Hum Reprod. 2003;18(5):1023–8.
6. Sakkas D, Seli E, Bizzaro D, Tarozzi N, Manicardi GC. Abnormal spermatozoa in the ejaculate: abortive apoptosis and faulty nuclear remodelling during spermatogenesis. Reprod Biomed Online. 2003;7(4):428–32.
7. Shibahara H, Obara H, Ayustawati, Hirano Y, Suzuki T, Ohno A, et al. Prediction of pregnancy by intrauterine insemination using CASA estimates and strict criteria in patients with male factor infertility. Int J Androl. 2004;27(2):63–8.
8. Henkel R, Schill WB. Sperm preparation for ART. Reprod Biol Endocrinol. 2003;1:108.
9. Rappa KL, Rodriguez HF, Hakkarainen GC, Anchan RM, Mutter GL, Asghar W. Sperm processing for advanced reproductive technologies: where are we today? Biotechnol Adv. 2016;34:578–87.
10. Muratori M, Maggi M, Spinelli S, Filimberti E, Forti G, Baldi E. Spontaneous DNA fragmentation in swim‐up selected human spermatozoa during long term incubation. J Androl. 2003;24(2):253–62.
11. Sauer R, Coulam CB, Jeyendran RS. Chromatin intact human sperm recovery is higher following glass wool column filtration as compared with density gradient centrifugation. Andrologia. 2012;44(1):248–51.
12. Ward WS. Function of sperm chromatin structural elements in fertilization and development. Mol Hum Reprod. 2010;16(1):30–6.
13. Tasoglu S, Safaee H, Zhang X, Kingsley JL, Catalano PN, Gurkan UA, et al. Exhaustion of racing sperm in naturemimicking microfluidic channels during sorting. Small. 2013;9(20):3374–84.
14. Cooper TG, Noonan E, von Eckardstein, Auger J, Baker HW, Behre HM, et al. World Health Organization reference values for human semen characteristics. Hum Reprod Update. 2010;16(3):231–45.
15. Önel T, Ayla S, Keskin İ, Parlayan C, Yiğitbaşı T, Kolbaşı, et al. Leptin in sperm analysis can be a new indicator. Acta Histochem. 2019;121(1):43–9.
16. Rouen A, Balet R, Dorna M, Hyon C, Pollet-Villard X, Chantot-Bastaraud S, et al. Discontinuous gradient centrifugation (DGC) decreases the proportion of chromosomally unbalanced spermatozoa in chromosomal rearrangement carriers. Hum Reprod. 2013;28(7):2003–9.
17. Hinting A, Lunardhi H. Better sperm selection for intracytoplasmic sperm injection with the side migration technique. Andrologia, 2001;33:343–6.
18. Asghar W, Velasco V, Kingsley JL, Snoukat MS, Shafiee H, Anchan RM, et al. Selection of functional human sperm with higher DNA integrity and fewer reactive oxygen species. Adv Healthc Mater. 2014;3(10):1671–9.
19. İrez T, Sahmay S, Ocal P, Goymen A, Senol H, Erol N, et al. Investigation of the association between the outcomes of sperm chromatin condensation and decondensation tests, and assisted reproduction techniques. Andrologia. 2015;47(4):438–47.
20. Khalili MA, Nazari S, Dehghani-Firouzabadi R, Talebi A, Baghazadeh-Naeini S, Sadeghian-Nodoshan F, et al. Comparing the roles of sperm chromatin integrity and apoptosis in intrauterine insemination outcomes of couples with mild male and female factor infertility. J Reprod Infertil. 2014;15(1):35–40.
21. Nasr-Esfahani MH, Razavi S, Mardani M. Relation between different human sperm nuclear maturity tests and in vitro fertilization. J Assist Reprod Genet. 2001;18(4):219–25.
22. Björndahl L, Mohammadieh M, Pourian M, Söderlund I, Kvist U. Contamination by seminal plasma factors during sperm selection. J Androl. 2005;26(2):170–3.
23. Mortimer ST, Swan MA, Mortimer D. Effect of seminal plasma on capacitation and hyperactivation in human spermatozoa. Hum Reprod. 1998;13(8):2139–46.
24. Sharma RK, Agarwal A. Role of reactive oxygen species in male infertility. Urology. 1996;48(6):835–50.
25. Zini A, Sigman M. Are tests of sperm DNA damage clinically useful? J Androl. 2009;30(3):219–29.
26. Aitken J, Clarkson JS. Significance of reactive oxygen species and antioxidants in defining the efficacy of sperm preparation techniques. J Androl. 1988;9(6):367–76.
27. Zini A, Bielecki R, Phang D, Zenzes MT. Correlations between two markers of sperm DNA integrity, DNA denaturation and DNA fragmentation, in fertile and infertile men. Fertil Steril. 2001;75(4):674–7.
28. Matsuura K, Takeanmi M, Kuroda Y, Naruse K. Development and performance of plastic microfluidic sperm sorter. Fertil Steril. 2008;90:241.
29. Ward WS. Function of sperm chromatin structural elements in fertilization and development. Mol Hum Reprod. 2010;16(1):30–6.
30. Carrell DT, Kuneck PH, Peterson CM, Hatasaka HH, Jones KP, Campbell BF. A randomized, prospective analysis five sperm preparation techniques before intrauterine insemination of husband sperm. Fertil Steril. 1998;69(1):122–6.
31. Hinting A, Lunardhi H. Better sperm selection for intracytoplasmic sperm injection with the side migration technique. Andrologia. 2001;33:343–6.
32. Tamburrino L, Marchiani S, Montoya M, Elia Marino F, Natali I, Cambi M, et al. Mechanisms and clinical correlates of sperm DNA damage. Asian J Androl. 2012;14(1):24–31.
33. Wang CJ, Levchenko A. Microfluidics technology for systems biology research. Methods Mol Biol. 2009;500:203–19.
34. Alvarez JG, Lasso JL, Blasco L, Nuñez RC, Heyner S, Caballero PP, Storey BT. Centrifugation of human spermatozoa induces sublethal damage; separation of human spermatozoa from seminal plasma by a dextran swim up procedure without centrifugation extends their motile lifetime. Hum Reprod. 1993;8(7):1087–92.
35. Abed F, Zadehmodarres S. A comparative study of swim-up and upstream methods for isolating sperm cell for intra uterine insemination. Int J Women’s Health Reprod Sci. 2015;3:103–6
36. Ricci G, Perticarari S, Boscolo R, Montico M, Guaschino S, Presani G. Semen preparation methods and sperm apoptosis: swim-up versus gradient-density centrifugation technique. Fertil Steril. 2009;91:632–8.
37. Said TM, Land JA. Effects of advanced selection methods on sperm quality and ART outcome: a systematic review. Hum Reprod. 2011;6:719–33.