Sperm DNA damage measured by comet assay.

Measurement of sperm DNA damage is a useful tool in the evaluation of male infertility, as the sperm nucleus lacks protection against oxidative stress and is vulnerable to oxidation-mediated DNA damage. The Comet assay or single-cell gel electrophoresis is a relatively simple and sensitive method for measuring strand breaks in DNA in individual sperm. During this procedure, sperm cells are embedded in a thin layer of agarose on a microscope slide and lysed with detergent under high salt conditions. This process removes protamines and histones allowing the nucleus to form a nucleoid-like structure containing supercoiled loops of DNA. Alkaline pH conditions result in unwinding of double-stranded DNA, and subsequent electrophoresis results in the migration of broken strands towards the anode, forming a comet tail, when observed under fluorescence microscope. The amount of DNA in the head and tail is reflected by its fluorescent intensity. The relative fluorescence in the tail compared with its head serves as a measure of the level of DNA damage. In this chapter, we describe the alkaline version of the Comet assay, which is highly sensitive for measuring single- and double-strand DNA breaks.