Flow cytometry of human semen: a preliminary study of a non-invasive method for the detection of spermatogenetic defects

Abstract

The pathway of spermatogenesis involves the conversion of diploid stem cells (spermatogonia) to tetraploid primary spermatocytes, followed by meiosis and two cell divisions, first forming diploid secondary spermatocytes and then haploid round spermatids. Differentiation of round spermatids results in spermatozoa containing condensed chromatin. It has long been known that semen from patients with non-obstructive azoospermia or oligospermia contains small numbers of immature germinal cells. In this article, a flow cytometric procedure is described for assessing defects in spermatogenesis by identifying the ploidy of those immature cells. Cells in semen samples from 44 infertile patients and 14 controls were stained with propidium iodide, which displays red fluorescence when intercalated between bases in double-stranded DNA. The resulting cell suspension was examined by quantitative flow cytometry, with excitation by laser light (488 nm) and red fluorescence recorded on a logarithmic scale to allow easy differentiation between intensities of tetraploid, diploid and haploid round spermatids, and spermatozoa containing condensed chromatin. The flow cytometric method differentiated between cases of 'Sertoli cell-only' syndrome (complete absence of tetraploid and haploid cells) and cases where spermatogenesis was blocked in meiosis or in spermiogenesis. Flow cytometric histograms from semen samples from normozoospermic, oligozoospermic and azoospermic patients fell into patterns that correlated well with the results obtained from testis histology findings. The method described may serve as a simple, non-invasive and reliable assay to help clinicians counsel patients with severe male infertility before referring them for testicular surgery to locate spermatozoa for ICSI.