Application of the comet assay for the evaluation of DNA damage in mature sperm

Sperm DNA Damage and Its Relevance in Fertility Treatment: A Review of Recent Literature and Current Practice Guidelines

“…This produces a typical ‘comet’ shape, with fragmented DNA in the ‘tail’ and non-fragmented DNA in the ‘head’ [ 20 ]. Mechanistically, sperm are embedded as a single-cell suspension in an agarose matrix and treated with a lysis buffer containing high salt and detergent to remove cell and nuclear membranes [ 26 ]. This processing leaves DNA attached to the nuclear matrix as nucleoids, which are then subjected to electrophoresis [ 26 ].…”

“…Mechanistically, sperm are embedded as a single-cell suspension in an agarose matrix and treated with a lysis buffer containing high salt and detergent to remove cell and nuclear membranes [ 26 ]. This processing leaves DNA attached to the nuclear matrix as nucleoids, which are then subjected to electrophoresis [ 26 ]. Electrophoresis is performed under either alkaline or neutral pH conditions, resulting in the migration of DNA fragments toward the anode.…”

“…This allows for the migration of DNA fragments to form a comet ‘tail’, while intact DNA remains in the ‘head’. After electrophoresis, the samples are rinsed, stained with fluorescent dye, and quantitatively analyzed based on the distribution of the fluorescence pattern to determine the extent of the DNA damage [ 26 ]. The relative fluorescence of the tail versus the head corresponds to the amount of SDF, with increased fluorescence in the tail reflecting high levels of SDF [ 5 ].…”

“…While this assay can be performed with only a small number of sperm, they must be from a fresh sample, and—like the SCD test—interpretation of the results may be time-consuming and prone to inter-observer variability if a manual analysis is performed [ 16 , 17 ]. To reduce such variability, a computerized image analysis system can be used to obtain images, compute the fluorescent intensity profile for each nucleoid, and estimate the relevant comet components [ 26 ]. Regarding cost, this test has been estimated to be approximately USD 400 [ 21 ].…”

DNA damage in prepared semen is associated with fertilisation rate in assisted reproduction technology (ART) treatment

“…Current methods of measuring DNA integrity such as the Comet assay [5], Tunel assay [6], sperm chromatin dispersion assay/ halo assay [7], sperm chromatin structure assay [8] tend to detect a range of different types of DNA damage [3,9]. Other assays that reportedly detect speci cally single or double stranded DNA breaks have also been described [10].…”