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Table 1 Comparison of assays measuring sperm DNA fragmentation

From: Sperm DNA damage and its role in IVF and ICSI

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