Analysis of human sperm-derived pronuclei by three-colour fluorescent in-situ hybridization

We describe a modification of the human sperm-zona-free hamster egg fusionmethod that permits the study of aneuploidy in sperm-derived pronuclei bymulticolour fluorescent in-situ hybridization (FISH). Zona- free hamstereggs and human spermatozoa were fused and cultured for 15 h in the presenceof colcemid (1 microg/ml of medium) to obtain hamster oocytes arrested atmetaphase II and human spermatozoa at the pronuclear stage. By applying awhole human genomic DNA probe we confirmed that 100% of pronuclei tested(372/372) were of human origin. One-colour fluorescent in-situhybridization using a centromeric 18 probe was applied to 919 pronucleiwith different dithiothreitol (DTT) pretreatments: 50 mM (10 min) or 25 mM(20 and 25 min). The highest hybridization efficiency was obtained withtreatment with 25 mM DTT for 20 min (90.3%). Sex chromosome aneuploidy wasanalysed by three-colour FISH in a total of 2596 pronuclei from a normaldonor. Hybridization efficiency was 98.6%. The disomy rates for X, Y and XYchromosomes (0.11, 0.04 and 0.08% respectively) were similar to datareported for sperm nuclei by three-colour FISH and to those obtained insperm chromosomes. These results suggest that selection of potentiallyfertile spermatozoa (spermatozoa able to fertilize zona-free hamster eggsand produce a pronucleus) does not imply chromosomal selection.