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

Yıl 2020, Cilt: 25 Sayı: 1, 19 - 26, 15.01.2020

Sevilay Erimşah Süheyla Gonca Aysel Kükner Bülent Duran Yusufhan Yazır

Öz

Aim: In this study, we evaluated the DNA integrity of sperms selected by using
the conventional swim-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 intrauterine
insemination treatment of
normozoospermic infertile patients (NIPs).

Materials and Methods: Semen samples obtained from 20 patients were divided into four equal fractions. Control, density gradient centrifugation, swim-up, and microfluidic chip (MC) groups were created, and the untreated (control) and treated (other) sperm samples from the four groups were examined for DNA integrity. Acidic aniline blue staining and the TUNEL method were used respectively for evaluating sperm chromatin condensation defects and DNA fragmentation. Dichlorofluorescein diacetate and flow cytometry were used to determine the reactive oxygen species levels. 

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 the sperms selected with the conventional methods (p<0.0001).

Discussion and Conclusion: We found that the new MC method was more
effective in selecting sperms with high
DNA integrity, compared to the conventional methods. Accordingly, the MC method can be an ideal sperm selection method for
use in the intrauterine insemination treatment of NIPs with high DNA fragmentation,
apoptosis, and reactive oxygen species levels.

Anahtar Kelimeler

DNA integrity, intrauterine insemination, microfluidic chip, sperm selection

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

Öz

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<0,0001).

Tartışma ve Sonuç: Yeni MÇ yönteminin geleneksel yöntemlere göre yüksek DNA bütünlüğüne sahip sperm
seçiminde daha etkili olduğu görüldü. Buna göre MÇ yöntemi yüksek DNA kırığı,
apopitoz ve reaktif oksijen türevi görülen NİH’lerin intrauterin inseminasyon tedavisinde
ideal bir sperm seçim yöntemi olabilir. 

Anahtar Kelimeler

DNA bütünlüğü, intrauterin inseminasyon, mikroakışkan çip, sperm seçimi

Kaynakça

• Agarwal A, Allamaneni SS. Sperm DNA damage assessment: a test whose time has come. Fertil Steril. 2005; 84(4): 850–853.
• Centola G, Ginsburg K. Evaluation and treatment of the infertile male. Cambridge, United Kingdom: Cambridge University Press; 1996.
• 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): 674–677.
• Jones J, Horne G, Fitzgerald C. Who needs ICSI? A nationwide UK survey on ICSI use. Human Fertility. 2012; 15(3): 144-149.
• Benchaib M, Braun V, Lornage J, Hadj S, Salle B, Lejeune H et al. Sperm DNA fragmentation decreases the pregnancy rate in an assiste reproductive technique. Hum Reprod. 2003;18(5): 1023-8.
• 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): 428–432.
• 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):63-68.
• 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):253-262.
• 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): 248-251.
• Ward WS. Function of sperm chromatin structural elements in fertilization and development. Mol Hum Reprod. 2010 ; 16 (1): 30-36.
• Tasoglu S, Safaee H, Zhang X, Kingsley JL, Catalano PN, Gurkan UA, et al. Exhaustion of racing sperm in nature-mimicking microfluidic channels during sorting. Small. 2013; 25: 9(20):3374-84.
• 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):253-262.
• 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.
• Kruger TF, Menkveld R, Stander FS, Lombard CJ, Van der Merwe JP, van Zyl JA, et al. Sperm morphological features as a prognostic factor in in vitro fertilization. Fertil Steril. 1986;46:1118–23.
• Kruger TF, Acosta AA, Simmons KF, Swanson RJ, Matta JF, Oehninger S. Predictive value of abnormal sperm morphology in in vitro fertilization. Fertil Steril. 1988;49:112–7.
• 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): 1671-79.
• 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.
• 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.
• Mortimer ST, Swan MA, Mortimer D. Effect of seminal plasma on capacitation and hyperactivation in human spermatozoa. Hum Reprod. 1998; 13(8): 2139–2146.
• Sharma RK, Agarwal A. Role of reactive oxygen species in male infertility. Urology. 1996; 48(6): 835-50.
• Zini A, Sigman M. Are tests of sperm DNA damage clinically useful? J Androl, 2009; 30(3): 219-29.
• Aitken J, Clarkson JS. Significance of reactive oxygen species and antioxidants in defining the efficacy of sperm preparation techniques. J Androl. 1988; 9(6): 367-376.
• 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): 122-26.
• Hinting A, Lunardhi H. Better sperm selection for intracytoplasmic sperm injection with the side migration technique. Andrologıa, 2001; 33: 343-346.
• Hammadeh ME, Kühnen A, Amer AS, Rosenbaum P, Schmidt W. Comparison of sperm preparation methods: effect on chromatin and morphology recovery rates and their consequences on the clinical outcome after in vitro fertilization embryo transfer. Int J Androl. 2001; 24(6): 360±368.
• 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.
• Wang C. J, Levchenko A. Microfluidics technology for systems biology research. In Systems Biology. New York, NY: Humana Press. 2009. 203-219.
• Alvarez JG1, 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 motil lifetime. Hum Reprod. 1993; 8(7): 1087-92.