Hoechst staining and exposure to UV laser during flow cytometric sorting does not affect the frequency of detected endogenous DNA nicks in abnormal and normal human spermatozoa

Controlling the sex of offspring by the separation of X and Ychromosome-bearing spermatozoa using flow cytometry has been reported as aclinical technique aiding prevention of X-linked diseases. Although thistechnique has resulted in several hundred normal births in animals and atleast one human birth, there is still concern over its genetic safety dueto the involvement of two potentially mutagenic agents: UV light and thefluorochrome dye, Hoechst 33342 (H33342). Human spermatozoa, particularlythose considered abnormal, may be more likely to suffer DNA damagefollowing exposure to mutagenic agents, compared with other mammalianspecies. The stability of normal fresh and decondensed human spermatozoawere examined after exposure to a range of levels of UV and H33342staining, using an assay that detects endogenous nicks in the DNA ofspermatozoa. The stability of abnormal and normal, fresh and frozen-thawedhuman spermatozoa was examined following UV laser, H33342 staining and flowcytometry treatments utilizing the same assay. There was an increase in thepresence of endogenous nicks when spermatozoa were decondensed comparedwith fresh spermatozoa. There was no increase in the incidence of nicks inany group of spermatozoa after UV and fluorochrome exposure compared withcontrols without exposure.