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