This is a flow-cytometric method which is based on the susceptibility of the sperm chromatin to undergo partial denaturation in situ under acidic conditions. Spermatozoa with damaged chromatin denature when exposed to acidic conditions, while those with normal chromatin remain stable (Evenson et al. 1980). After exposure of spermatozoa to acidic conditions, spermatozoa are stained with acridine orange (AO), a metachromatic fluorescent dye. When it intercalates with double-stranded DNA, it fluoresces green and fluoresces red when bound to single-stranded DNA. The extent of DNA denaturation following acid treatment is determined by measuring the metachromatic shift from green fluorescence to red fluorescence. Usually five to ten thousand cells are analyzed and assessed using a specific software. Though the method is highly reproducible and quite rapid, it is an expensive method.

This test is the simplified version of SCSA which uses fluorescent microscope to assess the metachromatic shift in the fluorescence by acridine orange (Tejada et al. 1984). It fluoresces green when bound to double-stranded DNA (excitation maximum at 502 nm and an emission maximum at 525 nm) and fluoresces red when bound to single-stranded DNA or RNA (excitation maximum 460 nm and an emission maximum 650 nm). For the assay, spermatozoa are exposed to mild acid treatment and stained with AO. Spermatozoa with intact DNA fluoresce green, and sperm with DNA damage fluoresces red or orange (Fig. 10.2). It is relatively a simple, rapid, and cheap method. The major disadvantages of this method are heterogeneous staining of the slide, rapid quenching, development of series of intermediate colors depending on the extent of chromatin denaturation, and large degree of inter- and intra-observer variations. Therefore, this method is considered to have low sensitivity and specificity to detect sperm DNA damage.

This assay detects the DNA breaks directly without changing the chromatin structure unlike in comet assay or sperm chromatin structure assay. The assay is based on the ability of the terminal deoxynucleotidyl transferase (TdT) to label the 3′-OH end of broken DNA strand with nucleotides (5-bromo-2′-deoxyuridine 5′-triphosphate nucleotide) usually tagged with a fluorochrome (Fig. 10.3) or a chromogen which can be visualized by observing under fluorescent or light microscope, respectively. The advantage of light microscopic method is that the signal obtained is permanent unlike in fluorescent microscopic method. Since it is a kit-based method, the results are reproducible. However, the kits are usually expensive with differences in working protocol, and the threshold level is not defined for clinical application.

This test is based on the principle that mild acidic denaturation of sperm DNA and lysis of protamines will create a halo of chromatin loops around the sperm head when DNA is intact and small or no halo around the sperm head when DNA is fragmented (Fig. 10.4). It is a relatively simple and inexpensive method to detect sperm DNA integrity which was first described by Fernández et al. (2003). However, it does not give information on the extent of DNA damage in spermatozoa, and there are a limited number of studies to support its clinical application.

This is a very simple light microscopic method which is based on the difference in chromatin packing of spermatozoa. It gives an indirect measure of protamine–histone ratio in spermatozoa. Aniline blue is an acidic dye which has a greater permeability/affinity for histones in the sperm nucleus. Increased aniline blue staining of sperm indicates loose chromatin packing. The blue-stained spermatozoa are considered as immature, and pinkish sperm are mature (Fig. 10.5). Even though it is a rapid and cheap method, heterogeneous staining pattern limits its clinical application.

Toluidine blue is a nuclear dye used for metachromatic and orthochromatic staining of chromatin that stains phosphate residues of the sperm DNA with loosely packed chromatin and fragmented ends. When the stain attaches with lysine-rich regions of histone, it produces a violet-bluish intense coloration, whereas a pale-blue color is produced by interactions with protamines in the chromatin. The sample can be analyzed using an ordinary microscope, but intermediate coloration increases inter-user variability.

This method is a simple fluorescence microscopic method which gives information on the degree of protamination and chromatin packing in spermatozoa. CMA3 specifically binds to GC (guanine–cytosine)-rich sequences in DNA. GC-rich region is also the site where protamines bind with DNA. The higher the CMA3 staining, the lower will be the protamine level and the poorer will be the chromatin packing in spermatozoa (Fig. 10.6). The spermatozoa with bright yellow are considered as immature, while with dull yellow are considered as mature. Due to rapid quenching of fluorescence and high inter- and intra-observer variation, this method has low clinical application.