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 Y chromoso me-bearing spermatozoa using flow cytometry has been reported as a cli nical technique aiding prevention of X-linked diseases. Although this technique has resulted in several hundred normal births in animals and at least one human birth, there is still concern over its genetic saf ety due to the involvement of two potentially mutagenic agents: UV lig ht and the fluorochrome dye, Hoechst 33342 (H33342). Human spermatozoa , particularly those considered abnormal, may be more likely to suffer DNA damage following exposure to mutagenic agents, compared with othe r mammalian species. The stability of normal fresh and decondensed hum an spermatozoa were examined after exposure to a range of levels of UV and H33342 staining, using an assay that detects endogenous nicks in the DNA of spermatozoa. The stability of abnormal and normal, fresh an d frozen-thawed human spermatozoa was examined following UV laser, H33 342 staining and flow cytometry treatments utilizing the same assay. T here wats an increase in the presence of endogenous nicks when spermat ozoa were decondensed compared with fresh spermatozoa. There was no in crease in the incidence of nicks in any group of spermatozoa after UV and fluorochrome exposure compared with controls without exposure.