Table 1 Comparison of assays measuring sperm DNA fragmentation
Assay | Method of detection | Advantages | Disadvantages |
|---|---|---|---|
SCD/Halo [20] | • Sperm loaded in agarose, denatured in acid solution, stained and observed with fluorescence microscopy for chromatin dispersion/halos. • Sperm with nondispersed chromatin (ie small halos) have fragmented DNA. • Interpreted as percentage of sperm with nondispersed chromatin. | • Commercial assay available • Interpretation does not depend on fluorescence or flow cytometry | • Indirect assay only detects ssDNA breaks • Involves acid denaturation |
SCSA [21] | • Acid denaturation, then staining with acridine orange and measurement with flow cytometer. • Green-staining sperm have intact DNA while red-staining sperm have denatured DNA. • Interpreted as percentage of red-staining sperm (denatured sperm). | • Has extensive body of literature and established clinical thresholds • Can be performed in fresh or frozen samples | • Proprietary protocol with no commercial assay available • Indirect assay only detects ssDNA breaks • Involves acid denaturation |
Comet [26] | • With gel electrophoresis, fragmented DNA forms tail while intact DNA stays in head. • Can be performed in alkaline or neutral conditions. • Tail size correlates to amount of DNA fragmentation within individual spermatozoon. • Interpreted as mean amount of DNA damage per individual spermatozoon. | • Requires small number of sperm cells • Direct assay can examine damage at level of individual spermatozoon • Can detect multiple types of DNA fragmentation | • No established standardized protocol • Time and labour intensive • Requires fresh semen sample |
TUNEL [30] | • Labeled nucleotide added to sites of DNA fragmentation with subsequent fluorescence measured by flow cytometry or fluorescence microscopy. • Fluorescent sperm have fragmented DNA. • Interpreted as percentage of fluorescent sperm. | • Direct assay • Commercial assay available • Can be performed in fresh or frozen samples • Detects ssDNA and dsDNA breaks | • No established standardized protocol • Variable clinical thresholds in literature |