RELIABILITY OF ESTIMATES OF DIPLOID HUMAN SPERMATOZOA USING MULTICOLOR FLUORESCENCE IN-SITU HYBRIDIZATION

Multicolour fluorescence in-situ hybridization (FISH) analysis permits distinction between disomic and diploid spermatozoa, Thus estimates o f the frequency of diploid spermatozoa can be obtained for human semen samples, The issue of the accuracy and reliability of these diploidy estimates has been addressed by analysing diploidy frequencies in 10 m en using the same sperm sample to estimate diploidy twice - once durin g two-colour FISH analysis of disomy for chromosomes 1 and 12 and a se cond independent analysis of three-colour FISH for disomy estimates fo r chromosomes X and Y (with chromosome 1 used as the autosomal control ), A minimum of 10 000 spermatozoa per hybridization per male was coun ted for a total of over 200 000 spermatozoa analysed. The mean frequen cy of diploid spermatozoa was 0.13% for the autosomal study and 0.14% for the sex chromosomal study, which were not significantly different. One donor had extremely divergent values of diploidy in the two studi es, Analysis of values ire the other nine donors demonstrated no signi ficant difference in the two diploidy estimates, These results indicat e that the FISH technique is an accurate and reliable method for deter mining diploid frequencies in human spermatozoa.