DNA flow cytometry of human spermatozoa: Consistent stoichiometric staining of sperm DNA using a novel decondensation protocol

Tamás Kovács, Gyöngyi Békési, Ákos Fábián, Zsuzsa Rákosy, Gábor Horváth, László Mátyus, Margit Balázs, Attila Jenei

Research output: Contribution to journalArticle

13 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)965-970
Number of pages6
JournalCytometry Part A
Volume73
Issue number10
DOIs
Publication statusPublished - Oct 1 2008

Keywords

  • Aneuploid spermatozoa
  • DNA FACS
  • Diploid spermatozoa
  • FISH
  • Lithium diiodosalicylate
  • Propidium iodide

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Histology
  • Cell Biology

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