Table 1. Sperm DNA fragmentation (SDF) testing methods.

	Test	Principle	Advantage	Disadvantage
	AO test	Metachromatic shift in fluorescence of AO when bound to single strand (ss)DNA. Uses fluorescent microscopy	Rapid, simple and inexpensive	Inter-laboratory variations and lack of reproducibility
	AB staining	Increased affinity of AB dye to loose chromatin of sperm nucleus. Uses optical microscopy	Rapid, simple and inexpensive	Inter-laboratory variations and lack of reproducibility
	CMA3 staining	CMA3 competitively binds to DNA indirectly visualizing protamine deficient DNA. Uses fluorescent microscopy	Yields reliable results as it is strongly correlated with other assays	Inter-observer variability
	TB staining	Increased affinity of TB to sperm DNA phosphate residues. Uses optical microscopy	Rapid, simple and inexpensive	Inter-observer variability
	TUNEL	Quantifies the enzymatic incorporation of dUTP into DNA breaks. Can be done using both optical microscopy and fluorescent microscopy. Uses optical microscopy, fluorescent microscopy and flow cytometry	Sensitive, reliable with minimal inter-observer variability. Can be performed on few sperm	Requires standardization between laboratories
	SCSA	Measures the susceptibility of sperm DNA to denaturation. The cytometric version of AO test. Uses flow cytometry	Reliable estimate of the percentage of DNA-damaged sperm	Requires the presence of expensive instrumentation (flow cytometer) and highly skilled technicians
	SCD or Halo test	Assess dispersion of DNA fragments after denaturation. Uses optical or fluorescent microscopy	Simple test	Inter-observer variability
	SCGE or comet assay	Electrophoretic assessment of DNA fragments of lysed DNA. Uses fluorescent microscopy	Can be done in very low sperm count. It is sensitive and reproducible	Requires an experienced observer. Inter-observer variability

[1] Acridine orange (AO) stains normal DNA fluoresces green; whereas denatured DNA fluoresces orange-red. [2] Aniline blue (AB) staining showing sperm with fragmented DNA and normal sperm. [3] Chromomycin A3 (CMA3) staining: protamine deficient spermatozoa appear bright yellow; spermatozoa with normal protamine appear yellowish green. [4] Toulidine blue (TB) staining: normal sperm appear light blue and sperm with DNA fragmentation appear violet. [5] Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay fluorescent activated cell sorting histogram showing percentage of SDF. [6] Sperm chromatin structure assay (SCSA): flow cytometric version of AO staining. [7] Sperm chromatin dispersion (SCD) test: spermatozoa with different patterns of DNA dispersion; large-sized halo; medium-sized halo [2]; very small- sized halo. [8] Comet images showing various levels of DNA damage.