Combination of swim-up and density gradient separation methods effectively eliminate DNA damaged sperm

Abstract

Objective: The aim of this experimental prospective study was to investigate the efficacy of single and combination sperm wash methods for their ability to isolate DNA intact spermatozoa. Material and Methods:Sperm DNA damage was introduced by local testicular irradiation in male mice and the extent of damage was quantified by comet assay. The spermatozoa were subjected to single (swim up or density gradient method) and also a combination of sperm wash techniques. The DNA integrity in various sub-fractions of wash techniques was evaluated. Results:The amount of DNA damaged sperm did not differ between individual fractions when single wash technique was applied. However, a combination of density gradient and swim-up techniques significantly reduced (p<0.01) the number of DNA damaged sperm in the final population. Conclusion:The combination of density gradient separation and swim-up method is effective in eliminating DNA damaged spermatozoa. (J Turkish-German Gynecol Assoc 2011; 12: 148-52) Key words: Sperm wash, DNA damage, swim-up, density gradient separation, comet assay Received: 13 June, 2011 Accepted: 19 July, 2011 Amaç: Bu deneysel prospektif çalışmanın amacı tek ve kombinasyon sperm yıkama metotlarının, DNA'sı sağlam spermatozoa izolasyon etkinliklerini araştırmaktı. Gereç ve Yöntemler:Sperm DNA hasarı erkek farelerde lokal testiküler ışınlama ile oluşturuldu ve hasarın büyüklüğü comet assay ile öl-çüldü. Spermatozoa'ya tek (yüzdürme veya dansite gradient metodu) ve ayrıca kombinasyon sperm yıkama teknikleri uygulandı. Yıkama tekniklerinin çeşitli alt fraksiyonlarında DNA'nın bütünlüğü değerlen-dirildi. Bulgular:Tek yıkama tekniği uygulandığında DNA hasarlı sperm miktarı fraksiyonlar arasında farklılık göstermedi. Bununla beraber, dansite gradienti ve yüzdürme tekniklerinin kombinasyonu son popü-lasyondaki DNA hasarlı sperm sayısını anlamlı olarak (p<0.01) azalttı.Sonuç: Dansite gradient ayırma ve yüzdürme mettotlarının kombinasyonu DNA hasarlı spermatozoa'nın uzaklaştırılmasında etkilidir.

A Simple, Centrifugation-Free, Sperm-Sorting Device Eliminates the Risks of Centrifugation in the Swim-Up Method While Maintaining Functional Competence and DNA Integrity of Selected Spermatozoa

“…Though few studies have suggested that centrifugation of semen samples is associated with the generation of increased reactive oxygen species and a high DNA damage [ 8 ], the others failed to show such association [ 18 , 29 ]. These conflicting observations may be related to the extent of DNA fragmentation in sperm cells, duration of the ejaculatory abstinence, quality of the ejaculate, and/or the technique used for sperm selection [ 30 – 32 ]. The swim-up method significantly reduces sperm DNA fragmentation rates and may have prognostic value on IUI in patients with decreased sperm DNA integrity [ 8 , 33 ], which is in agreement with our observation.…”

“…Ultrastructural sperm morphology was analyzed using scanning electron microscopy (SEM) as described earlier [24] with minor modifications. Briefly, sperm cells were centrifuged and fixed on a clean slide using 2.5% glutaraldehyde in 0.1 M sodium acetate buffer (pH 7.3) for 1 h. The fixed cells were washed using 0.1 M sodium acetate and subsequently dehydrated using increasing concentration of ethanol (30,50,70,80,90, and 100% for 10 min each). Following dehydration, the cells were air-dried and subjected to critical point drying using carbon dioxide.…”

Sperm Selection for ICSI: Do We Have a Winner?

“…Unfortunately, by relying only on motility, these techniques cannot be associated with an optimal quality of spermatozoa [ 71 ]. Further studies [ 21 ] have shown that by using both methods in combination, better results can be obtained in separating sperm cells with damaged DNA, removing them from the rest of the sample [ 22 ]. It has also been observed that spermatozoa with less nuclear fragmentation are correlated with an improvement of the embryo quality and a reduced probability of miscarriage [ 72 ].…”

Improving ICSI: A review from the spermatozoon perspective

“…In DGC, sperm cells are separated according to their density, following centrifugation through two discrete layers of discontinuous density gradients [Pousette et al 1986]. Moreover, the combination of DGC and S-U can also yield favorable results especially with respect to eliminating DNA damaged sperm [Ghumman et al 2011;Jayaraman et al 2012].…”