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DNA flow cytometry of human spermatozoa: Consistent stoichiometric staining of sperm DNA using a novel decondensation protocol
Author(s) -
Kovács Tamás,
Békési Gyöngyi,
Fábián Ákos,
Rákosy Zsuzsa,
Horváth Gábor,
Mátyus László,
Balázs Margit,
Jenei Attila
Publication year - 2008
Publication title -
cytometry part a
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.316
H-Index - 90
eISSN - 1552-4930
pISSN - 1552-4922
DOI - 10.1002/cyto.a.20618
Subject(s) - flow cytometry , sperm , ploidy , biology , propidium iodide , staining , cytometry , microbiology and biotechnology , andrology , genetics , apoptosis , gene , medicine , programmed cell death
Abstract Rapid flow cytometric measurement of the frequency of aneuploid human sperms is in increasing demand but development of an exploitable method is hindered by difficulties of stoichiometric staining of sperm DNA. An aggressive decondensation protocol is needed after which cell integrity still remains intact. We used flow cytometry to examine the effect of lithium diiodosalicylate (LIS, chaotropic agent) on fluorescence intensity of propidium iodide‐treated human spermatozoa from 10 normozoospermic men. When flow cytometric identification of diploid spermatozoa was achieved, validation was performed after sorting by three‐color FISH. In contrast with the extremely variable histograms of nondecondensed sperms, consistent identification of haploid and diploid spermatozoa was possible if samples were decondensed with LIS prior to flow cytometry. A 76‐fold enrichment of diploid sperms was observed in the sorted fractions by FISH. A significant correlation was found between the proportion of sorted cells and of diploid sperms by FISH. Application of LIS during the preparation of sperm for flow cytometry appears to ensure the stoichiometric staining of sperm DNA, making quantification of aneuploid sperm percentage possible. To our knowledge this is the first report in terms of separating spermatozoa with confirmedly abnormal chromosomal content. High correlation between the proportion of cells identified as having double DNA content by flow cytometry and diploid sperm by FISH allows rapid calculation of diploidy rate. © 2008 International Society for Advancement of Cytometry