Chromosome analysis of spermatozoa extracted from testes of men with non-obstructive azoospermia

Leah Yogev1, Gedalia Paz and Haim Yavetz

Institute for the Study of Fertility, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, Affiliated to the Tel-Aviv University, 6, Weizmann St., Tel Aviv, 64239, Israel

Dear Sir,

We read with great interest the recent article by Martin et al. (Martin et al. 2000Go). This first report on chromosomal aneuploidy in spermatozoa extracted from testes of patients with non-obstructive azoospermia suggested that certain azoospermic men are not at substantially increased risk for chromosomally abnormal spermatozoa. We would like to propose an explanation for the contradiction between this report and that of Huang et al. (Huang et al. 1999Go).

Using the fluorescence in-situ hybridization (FISH) technique, Huang et al. (1999) evaluated germ cells at different stages of spermatogenesis (except for spermatozoa) and found a high frequency of sex chromosome aneuploidy in haploid nuclei, ranging from 26.2 to 34.4% in men with impaired spermatogenesis, and 26% in the controls. This was shown in paraffin-embedded testicular tissue samples, while nuclei were sorted as haploid or diploid on the basis of cell size, and the number of signals for the autosomal chromosome 18 probe.

In Huang's study they misinterpreted the pairing phenomenon that existed during the late zygotene and pachytene stages of the first meiosis, as shown by Scherthan et al. (Scherthan et al. 1996Go). During these stages, all nuclei displayed a paired signal, namely the nuclei have one signal for each autosomal chromosome bivalent, while the X–Y pair forms the sex body structure demonstrating two adjacent signals.

It should be noted that usually most spermatocytes are at the pachytene stage because this is the longest sub-stage during the first meiosis (Metzler-Guillemain et al., 1999Go). Consequently, the striking difference between the two studies of an abnormality frequency of 20–30-fold higher in the study by Huang et al. (1999), may be explained by ignoring the fact that one signal for autosomal chromosome, accompanied by two closed signals for X and Y chromosomes is the normal pattern for spermatocyte nuclei.

In our study, whenever mature spermatids could be found in the tissue, evaluation of spermatocytes by the FISH technique revealed one signal for the two 18 chromosomes in 91 and 95% of the non-obstructive and obstructive azoospermic men respectively, with no statistical difference between the two groups (Yogev et al., 2000Go). This finding supports the results shown by Martin et al. (2000) for normal meiosis, as evaluated by the frequency of aneuploidy of the sperm chromosomes studied by them.

Notes

1 To whom correspondence should be addressed Back

References

Huang, W.J., Lamb, D.J., Kim, E.D. et al. (1999) Germ-cell nondisjunction in testes biopsies of men with idiopathic infertility. Am. J. Hum. Genet., 64, 1638–1645.[ISI][Medline]

Martin, R.H., Greene, C., Rademaker, A. et al. (2000) Chromosome analysis of spermatozoa extracted from testes of men with non-obstructive azoospermia. Hum. Reprod., 15, 1121–1124.[Abstract/Free Full Text]

Metzler-Guillemain, C., Mignon, C., Depetris, D. et al. (1999) Bivalent 15 regularly associates with the sex vesicle in normal male meiosis. Chromosome Res., 7, 369–378.[ISI][Medline]

Scherthan, H., Weich, S., Schwegler, H. et al. (1996) Centromere and telomere movements during early meiotic prophase of mouse and man are associated with the onset of chromosome paring. J. Cell Biol., 134, 1109–1125.[Abstract]

Yogev, L., Gamzu, R., Kleiman, S. et al. (2000) Evaluation of meiotic impairment of azoospermic men by fluorescence in situ hybridization. Fertil. Steril., 74, 228–233[ISI][Medline]


 
Farideh Bischoff1 and Dolores J. Lamb

Department of Obstetrics and Gynecology, Baylor College of Medicine, Departments of Urology and Cell Biology, Baylor College of Medicine 6550 Fannin St, Suite 708, Houston, TX 77030, USA

Dear Sir,

Yogev et al. propose that the difference in the observed frequency of aneuploidy between our study (Huang et al., 1999Go) and that recently published by Martin et al. (Martin et al. 2000Go) is due to the incorrect scoring of the haploid nuclei. Although we would agree that perhaps a small portion of the haploid nuclei scored (among the infertile patients and controls) as aneuploid may be representative of nuclei in the pachytene stage of cell division, the majority of the aneuploid haploid cells scored could not be classified as such. In fact, we refer the authors to Figure 1 (Huang et al., 1999Go), panels B and D where we demonstrate the presence of XY aneuploid haploid cells in which the X and Y signals are close together in one cell and clearly far apart in two other aneuploid cells. In addition to XY aneuploidy, abnormal haploid cells containing either no X/Y signals, two X signals or two Y signals were also scored. Furthermore, because all three of the probes (X, Y and 18) used in the Huang et al. (1999) study were {alpha}-satellite centromere probes, signal size would have been an indicator that the chromosomes were in the pachytene stage. That is, the signal size of the 18 chromosomes together at pachytene would be larger and equivalent to the size of two signals placed together. As shown in Figure 1, panel D, the signal size of the chromosome 18 probe is smaller than that of the X and Y signals that are found close together. In addition, as these are histological sections, the relative position of each cell is apparent and we can distinguish cell types by their position in the seminiferous tubule making it unlikely that pachytene stage cells were identified as haploid cells. The spermatogenic cells in the testis are present in defined cell associations and position within the seminiferous tubule.

In addition to these points, we also wish to restate that the two studies conducted by Martin et al. (2000) and our own were very different in many ways. Our study differed because it compared the incidence of aneuploidy in a much larger group of azoospermic men (n = 15) who are candidates for intracytoplasmic sperm injection (ICSI) to the incidence in men with normal spermatogenesis (n = 5). The Huang et al. (1999) study was strictly focused on fluorescence in-situ hybridization (FISH) detection of aneuploidy during spermatogenesis (in the primary and secondary spermatocytes, not mature spermatozoa) using archived testicular tissue from 15 azoospermic patients with one of three known pathological conditions resulting in severe defects in sperm development as compared with testicular tissue sections derived from five men with confirmed normal spermatogenesis. The direct comparison of the incidence of aneuploidy in testis biopsies of men with normal spermatogenesis with the azoospermic men demonstrated a highly significant difference. This observation again emphasizes that there is a specific defect in a portion of the azoospermic population that puts them at risk for sex chromosome non-disjunction during spermatogenesis. In the report by Martin et al. (2000), FISH was performed on extracted spermatozoa from only three azoospermic patients (no detailed pathology provided) in which >3000 spermatozoa could be recovered from biopsied tissue. Their results were then compared with mature spermatozoa found in semen samples of normal fertile men. The Huang et al. study did not evaluate aneuploidy in mature spermatozoa. The patients studied by Huang et al. have severe spermatogenic defects and certainly do not produce sperm to this level. In fact, in such infertile cases, technicians typically spend many hours to search for spermatozoa and may recover only 6–30 spermatozoa for use in ICSI.

As indicated by Martin et al., the striking difference of 20–30-fold higher aneuploidy in the Huang et al. study is likely to be due to a combination of several factors in addition to patient selection criteria, including sperm selection against aneuploid germ cells. It has long been recognized that normal spermatogenesis is very inefficient and it is estimated that 70% of potential germ cells are lost during spermatogenesis (Huckins, 1978Go). We proposed that a significant number of spermatocytes are likely to undergo germ cell degeneration and are therefore absent in the ejaculate. Although Martin et al. suggest that this is not likely to be a primary mechanism of selection based on their studies of sperm from reciprocal translocation carriers, we propose that the selection mechanism involved is likely to differ when the chromosomal abnormality is constitutional as compared with acquired. Among the Huang et al. infertile cases (n = 15), the increased aneuploidy was clearly acquired in germ cells during mitosis and meiosis when compared to the normal controls (n = 5).

In conclusion, the two studies cannot be directly compared because of differences in the outcome measured (aneuploidy in mature sperm as compared with immature germ cells), the use of testis biopsy specimens as compared with spermiated sperm, the types of FISH probes used for analysis (Y-centromere versus Yq) and most importantly differences in the infertile patient population analysed with different levels of spermatogenic failure. Our results clearly demonstrate that the azoospermic patients are at risk for an increased incidence in sex chromosome non-disjunction and this observation may have implications for the offspring conceived by ICSI.

Notes

2 To whom correspondence should be addressed Back

References

Huckins, C. (1978) The morphology and kinetics of spermatogonial degeneration in normal adult rats: an analysis using a simplified classification of the germinal epithelium. Anat. Rec., 190, 905–926.[ISI][Medline]

Huang, W.J., Lamb, D.J., Kim, E.D. et al. (1999) Germ-cell nondisjunction in testes biopsies of men with idiopathic infertility. Am. J. Hum. Genet., 64, 1638–1645.[ISI][Medline]

Martin, R.H., Greene, C., Rademaker, A. et al. (2000) Chromosome analysis in spermatozoa extracted from testes of men with non-obstructive azoospermia. Hum. Reprod., 15, 1121–1124.[Abstract/Free Full Text]