Double probing individual human spermatozoa: aniline blue staining for persistent histones and fluorescence in situ hybridization for aneuploidies

Objective: To study the potential relationship between two sperm nuclear attributes: persistence of histones and occurrence of chromosomal aneuploidies. Design: The two variables were examined by double probing of the same spermatozoa. Setting: Academic Andrology Laboratory. Patient(s): Semen samples subjected for analyses were examined. Intervention(s): We studied >58,000 spermatozoa, in seven men, first with aniline blue histone staining, graded as light (mature sperm), intermediate (moderately immature), and dark (severely arrested maturation). After recording the staining patterns and destaining, the same spermatozoa were subjected to fluorescence in situ hybridization (FISH), using centrometric X, Y, and 17 chromosome probes. Main Outcome Measure(s): Proportions of sperm with light, intermediate, and dark staining were assessed, and ploidy of these sperm was evaluated. Result(s): The aneuploidy frequencies in intermediate versus light (mature) spermatozoa were increased four-to sixfold. In addition, aneuploidy frequencies and proportions of intermediate sperm were related. There was no FISH signal detectable in the darkly stained, severely arrested mature sperm. Conclusion(s): The data suggest that in sperm with arrested maturity and DNA fragmentation, the binding of FISH probes is diminished. DNA damage is further aggravated by the decondensation and denaturation steps of FISH. Thus, there is a strong likelihood that in oligozoospermic men, with a higher proportion of sperm with arrested maturation, the sperm disomy frequencies are historically underestimated. (Fertil Steril (R) 2010;93:2255-61. (C) 2010 by American Society for Reproductive Medicine.)