Quantification of seminal germ cells in azoospermia: correlations with testicular histology and TESE outcome.

In the treatment of male infertility by intra-cytoplasmic injection of spermatozoa (ICSI) extracted from testicular tissue (TESE), the high incidence of negative TESE outcome calls for non-invasive prognostic methods. Literature suggests that seminal haploid germ cell detection could be one. For this purpose, a multi-parametric stringent flow cytometric method was applied to 50 TESE patients for the quantification of ejaculated germ cells. Cells from 50 ejaculates were identified and quantified as spermatozoa (HC, highly condensed), round spermatids (1N), primary spermatocytes (SPC) (4N) or diploid cells (2N, including somatic and non-testicular cells) by their DNA and mitochondria staining and laser scatter characteristics, and compared with testicular biopsy histology and TESE outcome. Whereas 96% of patients displayed a diploid peak in the distribution histograms, the HC, 1N and 4N peaks were absent from the majority of samples. In 13 ejaculates, either a HC or 1N or 4N peak, or a combination of these, was discernible. Although seminal germ cell numbers bore no overall association with elongated spermatids (ES) in histology or spermatozoa retrieval in TESE outcome, 4N cells per ejaculate were correlated with the percentage of tubule sections showing SPC as the most advanced germ cells. The incidence of HC peaks was higher in patients showing some ES in histology or sperm retrieval than in the sperm-negative groups. In groups with suspected obstruction showing nearly full spermatogenesis and maximal sperm retrieval, there was no incidence of a HC peak. Germ cell peaks were associated with germ cell degeneration noted in testicular histology. In conclusion, seminal germ cells cannot provide good prognosis for TESE, although their presence could indicate the spermatogenic activity in the testis.

The presence of germ cells in the semen of azoospermic, cryptozoospermic and severe oligozoospermic patients: stringent flow cytometric analysis and correlations with hormonal status.

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.