Application of different in-situ hybridization detection methods for human sperm analysis.

The detection of some types of aneuploidy in human spermatozoa can be based on the use of the fluorescence in-situ hybridization technique (FISH). One of the crucial steps for FISH is to achieve a proper decondensation and denaturation of the DNA in the specimen, so as to obtain efficient hybridization results. However, after DNA decondensation the morphology of sperm heads is partly distorted and the majority of the tails is lost. This situation leads to problems in the distinction between disomic and diploid spermatozoa, as well as between abnormal spermatozoa and somatic cells. Double- and triple-target FISH can partly solve this discrimination problem. To improve these procedures we adapted the steps of decondensation and visualization of the single sperm cells. Firstly, DNA decondensation with 25 mM dithiothreitol in 1 M Tris at pH 9.5 resulted in sperm cells with intact morphology of both the head and the tail, and allowed efficient single-, double- and triple-target ISH to be performed. Secondly, we applied a novel detection method, based on enzyme immunocytochemical reactions, with coloured precipitation products. Thirdly, this ISH procedure was combined with Diff-Quik staining and bright-field microscopy. This absorption method has the advantage of a permanent signal, and the adapted cytoplasmic staining of the sperm plasma membrane allows the visualization of the outline of the single spermatozoon. Using this approach, therefore, it is possible to discriminate between disomic, diploid and abnormal spermatozoa, somatic cells and spermatozoa that overlap, because the morphology of the cells is not distorted and the tails of the spermatozoa are intact and properly visualized.