Background: The integrity of DNA in spermatozoa is considered an additional parameter of semen quality and a potential fertility predictor. Significant progress has been made in recent years towards the development of reliable tests for sperm chromatin integrity and DNA damage assessment. However, most of the techniques available are labor intensive, require expensive instrumentation or utilize enzymes whose activity could be compromised by the highly condensed nature of sperm chromatin. In addition, all the methods currently available involve the destruction of the sperm tested; none is able to select intact spermatozoa that could then be used for fertilization. The aim of the present study was to create a peptide ligand-based stain, capable of binding specific DNA structures, thereby revealing the presence of DNA damage, preferably in living cells.
Methods: The peptide was bioinformatically modelled on the critical region of the p53 protein associated with DNA binding and fluorescently labeled with a terminal rhodamine B dye. The ability of this 21 amino acid synthetic peptide (DW1) to detect DNA damage in intact and fixed human spermatozoa was assessed in detail. Human sperm samples (n=20) were treated with reagents that induce single- and/or double-stranded DNA breaks. The effect of these treatments on peptide-labelling was measured and compared with results obtained using established tests for the evaluation of DNA damage, such as comet assay, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and sperm chromatin dispersion test.
Results: The peptide had a high affinity for single-stranded DNA, and DNA lesions such as double- and single-stranded breaks. The proportion of spermatozoa with intense staining was found to be closely associated with the percentage of cells possessing DNA damage. The analysis of 10 sperm samples using DW1 staining and TUNEL technique showed a significant correlation between the extent of DNA fragmentation for the two methods (r=0.892, Pearson's correlation, P<0.05).
Conclusions: We have produced a novel peptide-based stain capable of detecting DNA damage in individual sperm cells. Evaluation of sperm DNA fragmentation using this peptide may be an inexpensive and easier to use alternative to the tests in current use. Additionally, although DW1 currently requires removal of the membrane using a detergent, further research may allow this approach to be applied to the selection of viable spermatozoa with intact DNA for use in ICSI and/or intra-cytoplasmic morphologically selected sperm injection.