Evaluation of chromatin integrity in human sperm using acridine orange staining with different fixatives and after cryopreservation

Abstract

Staining of cells with acridine orange (AO) has been widely accepted as a predictor of DNA damage in many cell types. Because of variability of protocols used in previous studies, the AO staining technique has not been widely accepted as a screening test to predict DNA damage in human sperm. In order to further validate the use of AO staining, sperm were evaluated using numerous variations in the staining protocol. This study also elucidated the effects of cryopreservation on sperm DNA. Sperm fixation in Carnoy's solution showed significantly (P < 0.05) more DNA damage (29.9 +/- 4.5%) than 2% glutaraldehyde (14.4 +/- 2.1%), 4% paraformaldehyde (5.5 +/- 1.7%), no fixation (15.8 +/- 4.3%) but did not differ from Diff Quik solution (19.2 +/- 5.8%). No difference was observed for sperm DNA damage assessment using a 0.2 m (15.5 +/- 3.2%) or 0.3 m (14.9 +/- 3.3%) concentration of Na(2)HPO(4).7H(2)O in the AO staining solution. Frozen-thawed semen samples showed increased damage to sperm DNA under both Carnoy's (fresh: 10.9 +/- 1.3%; frozen: 30.8 +/- 2.9%; P < 0.05) and Diff Quik fixation (fresh: 6.2 +/- 0.8; frozen: 17.1 +/- 2.5%P < 0.05). Present data also showed that spermatozoa from some individuals are more prone to DNA damage after freezing and thawing procedures than others. In conclusion, Carnoy's fixative provides a better predictive value for DNA damage to sperm using AO staining. Additionally, cryopreservation increased damage to the sperm DNA.

Addition of zinc to human ejaculate prior to cryopreservation prevents freeze-thaw-induced DNA damage and preserves sperm function

“…Cryopreservation induces chromatin instability, DNA strand breaks and apoptosis in spermatozoa [4,5,24,25] and thereby reduces their functional competence. Poorly organized chromatin of spermatozoa from infertile men [26] increases their susceptibility to freeze-thaw-induced DNA damage compared to fertile men [27], which poses further challenge in semen cryopreservation technology.…”

Vitrification Technique - New Possibilities for Male Gamete Low-Temperature Storage

“…The danger is that such spermatozoa are still able to fertilize the oocyte, however, the mutations and defects did not possible to discover until the embryo has divided and the fetus has developed (Twigg et al, 1998). At present exist the opinion that DNA decondensation or fragmentation may occur in different magnitudes, which will depend on the process or the kind of cryoprotectant used (Schuffner et al, 2001;Chohan et al, 2004;Ngamwuttiwong & Kunathikom, 2007;Yildiz et al, 2007). Unfortunately, until now this question is still open, because it is not entirely clear what the effect of cryopreservation on DNA integrity is, and what would be the ideal conditions of slow freezing to reduce this effect.…”

A single, mild, transient scrotal heat stress causes DNA damage, subfertility and impairs formation of blastocysts in mice

“…Comparison with other DNA strand break assays (TUNEL and COMET) has shown that the DNA denaturation measured in the SCSA is largely due to DNA strand breaks (Gorczyca et al 1993, Chohan et al 2004. The sperm samples in BWW were adjusted to a concentration of 1-2!10 6 cells/ml with TNE (0.15 M NaCl, 0.1 M Tris, 1 mM EDTA pH 7.4); 100 ml of this sample was mixed with 200 ml acid detergent solution (0.1% Triton X-100, 0.15 M NaCl, 0.08 M HCl).…”