Table 1.

Sperm deoxyribonucleic acid fragmentation testing techniques, principle, advantages, and disadvantages

Test	Principle	Method	Interpretation	Advantages	Disadvantages
Direct assays
AO test	AO is nucleic acid selective dye and causes a metachromatic shift in fluorescence upon binding to DNA breaks	Air-dried semen sample smears are first fixed in Carnoy’s fixative for 2 h followed by AO staining for 5 min. Spermatozoa stained with AO are then excited at 488-nm wavelength	Intact DNA gives green fluorescence and damaged DNA red	Rapid, easy, and cost-effective test	Interlaboratory variations
AB staining	AB test detects the degree of sperm chromatin maturation and chromatin defects	Optical microscopic visualization of AB stained chromatin	Histone-rich nuclei of immature sperm stain blue, while mature protamine-rich nuclei remain unstained	Rapid, easy, and cost-effective test	Interlaboratory variations and results are dependent on staining efficiency
TUNEL	TUNEL assay identifies DNA breaks by the addition of template-independent DNA polymerase to the 3′hydroxyl (OH) breaks-ends of ssDNA and dsDNA	Fluorescein isothiocyanate conjugates with dUTPs and the fluorescent signal thus received is measured by flow cytometer or fluorescence microscopy	Sperm having DNA breaks show fluorescence and the results are presented as a percentage of fluorescent sperms	Direct test Can be used to detect dsDNA and ssDNA breaks Can be performed on fresh/frozen samples	Inconsistent with high variability in the reference values Time-consuming
SCGE/comet assay	SCGE assessment of fragmented DNA	Sperm are embedded in agarose and lysed using detergent and high salt to form nucleoids with supercoiled DNA loops followed by electrophoresis	Fragmented DNA appears as a tail, while intact DNA remains in the sperm head	Direct assay High sensitivity and specificity Can detect different types of DNA damage in single sperm	Inter-observer variation Time-consuming Requires fresh semen samples Expensive
Indirect assays
TB staining	TB is an acidophilic metachromatic dye with a high affinity for sperm DNA phosphate residues	Optical microscopic visualization of stained damaged DNA chromatin	Sperm heads with high chromatin DNA integrity are stained blue and damaged ones are stained violet-blue/purple	Rapid, easy, and cost-effective test	Inter-observer variations and results are dependent on staining efficiency
CMA3 staining	CMA3 is an anthraquinone antibiotic glycoside that binds reversibly to DNA and competes with protamine for the same site	Air-dried seminal smear fixed with glacial acetic acid-methanol (1:3) solution for 20 min at 4 °C followed by staining with CMA3	Spermatozoa with low protamination stains light yellow, and those with high DNA damage stains bright yellow	Strong correlation with other SDF assays	Interlaboratory and inter-observer variations
SCSA	SCSA is a flow cytometric test that identifies sperm DNA breaks indirectly by acid-induced DNA denaturation	Acid-induced denaturation of sperm DNA, followed by AO staining and measurement by flow cytometry	Intact DNA fluoresces green and denatured DNA as orange-red	Rapid, simple, precise, and repeatable test Can be performed on fresh/frozen semen samples	Indirect assay Requires expensive set-up and skilled technicians
SCD/halo test	Detects fragmented DNA dispersion after acid denaturation	Sperm embedded in agarose microgel are acid denatured to remove nuclear proteins followed by staining with nuclear stain DAPI	Sperm with fragmented DNA do not produce the halo of dispersed DNA loops as produced by sperm with non-fragmented DNA, following acid denaturation	A simple test with easy availability of commercial kits Can be performed on neat and washed seminal samples	Indirect assay Time-consuming Inter-observer variations

SCD=Sperm chromatin dispersion, TUNEL=Terminal deoxynucleotidyl transferase dUTP nick end labeling, SCSA=Sperm chromatin structure assay, SCGE/COMET: Single-cell gel electrophoresis, AO=Arcidine orange, AB=Anilinie blue, TB=Toluidine blue, DNA=Deoxyribonucleic acid, SDF=Sperm DNA fragmentation, ssDNA=Single-stranded DNA, dsDNA=Double-stranded DNA, CMA3=Chromomycin A3, DAPI=4′,6-diamidino-2-phenylindole, dUTPs= 2’-Deoxyuridine 5’-Triphosphate, OH=Hydroxyl