Objective: To understand the clinical significance of immature germ cells commonly found in ejaculates with low sperm counts by a novel and stringent flow cytometric quantitative method. PATIENTS/MEASUREMENTS: A total of 65 azoospermic, 38 cryptozoospermic and 42 severe oligozoospermic patients underwent routine hormone and semen analysis. Cells from each ejaculate were stained for DNA and mitochondria and analysed as spermatozoa (HC), round spermatids (1N), primary spermatocytes (4N) or diploid cells (2N).
Results: About 90% of HC particles were eliminated as contaminants of the spermatozoa population by the analysis of their laser light scatter pattern and mitochondria staining intensity. Ploidy identification accuracy was improved by selection of singlets and elimination of cell aggregates for analysis. Distribution peaks for HC, 1N and 4N cells were displayed in 53%, 56% and 25% ejaculates, respectively, with prevalence in severe oligozoospermia > cryptozoospermia > azoospermia. 1N cell numbers were correlated with 4N and HC cells. For HC and 1N cells, the number/ejaculate and the incidence of distribution peaks were correlated with serum testosterone levels, and inversely with FSH for HC, 1N and 4N cells, suggesting that the abnormal shedding of 1N and 4N germ cells is the consequence rather than the cause of spermatogenic failure in these patients. Ploidy data bear no association with clinical diagnosis except for Klinefelter patients.
Conclusion: Whereas incidence of HC cells in azoospermic ejaculates may suggest minimal spermatogenic activity which evades detection by routine semen analysis, the presence of 1N and 4N cells in semen of patients provides noninvasive information about their spermatogenic status.