Sex Chromosome Alignment at Meiosis of Azoospermic Men With Azoospermia Factor Microdeletion
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《男科医学杂志》
The Institute for the Study of Fertility and Obstetrics and Gynecology, Lis Maternity Hospital, and Institute of Pathology, Tel Aviv Sourasky Medical Center, affiliated to the Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Israel, and Obstetrics & Gynecology Department, Barzilai Medical Centre, Ashkelon, Israel.
Abstract
Deletions in the q arm of the Y chromosome result in spermatogenesis impairment. The aim of the present study was to observe the X and Y chromosome alignment in the spermatocytes of men with Y chromosome microdeletion of the azoospermia factor (AZF) region. This was performed by multicolor fluorescence in situ hybridization probes for the centromere and telomere regions. Testicular biopsies were performed in a testicular sperm extraction-intracytoplasmic sperm injection set-up in 11 azoospermic men: 8 (nonobstructive) with AZF deletions and 3 (obstructive) controls. Histological sections, cytology preparations of the testicular biopsies, and evaluation of the meiosis according to the percentage of XY and 18 bivalents formation were assessed. Spermatozoa were identified in at least one location in controls and specimens with AZFc-deleted Y chromosomes. Complete spermatocyte arrest was found in those with a deletion that included the entire AZFb region. Bivalent formation rate of chromosome 18 was high in all samples (81%-99%). In contrast, the rate of bivalent X-Y as determined by centromeric probes was lower but in the range favorable with spermatozoa findings in controls and patients with the AZFc deletion (56%-90%), but not in those with AZFb-c deletions (28%-29%). A dramatic impairment in the normal alignment of X and Y telomeres in the specimen with AZFb-c deletion was shown (29%), compared to the specimens with AZFc deletion (70%-94%). It is suggested that the absence of sperm cells in specimens with the entire AZFb and with AZFb-c deletions is accompanied by meiosis impairment, perhaps as a result of the extent of the deletion or because of the absence of genes that are involved in the X and Y chromosome alignment.
Key words: Azoospermia, chromosome pairing, FISH, Y chromosome microdeletion
Deletions of the long arm of the human Y chromosome resulted in cessation of spermatogenesis, and subsequently, infertility (Chandley and Edmond, 1971; Tiepolo and Zuffardi, 1976). Further study led to the proposal of the existence of 3 azoospermia factor (AZF) subregions termed AZFa,b,c (Vogt et al, 1996).
Deletion of the entire AZFa region (0.8 Mb; Sun et al, 2000) is associated with lack of germ cells (Sertoli-cell-only syndrome), and complete deletion of the AZFb region (6.23 Mb; Repping et al, 2002) is generally associated with arrest of spermatogenesis (Krausz et al, 2000; Foresta et al, 2001; Kleiman et al, 2001; Luetjens et al, 2002). Deletion of AZFc is the most common, and is associated either with severe oligozoospermia, or azoospermia, accompanied by a wide spectrum of histological defects (Reijo et al, 1995, 1996). It is remarkably uniform, spanning a 3.5-Mb segment (Kuroda-Kawaguchi et al, 2001).
Both AZFc and AZFa deletions, as well as most AZFb deletions, appear to ensue from homologous recombination between direct repeats on the Y chromosome (Kamp et al, 2000; Sun et al, 2000; Kuroda-Kawaguchi et al, 2001; Repping et al, 2002). Yq microdeletions may be associated with Y chromosomal instability, leading to the formation of the 45,X0 cell in lymphocytes and sperm cell nullisomic for the Y chromosome (Siffroi et al, 2000). The use of sperm retrieval from testes of infertile men to achieve fertilization through intracytoplasmic sperm injection (ICSI) raises the possibility of transmission of Y-related infertility to the male offspring (Kent-First et al, 1996; Vogt et al, 1996; Kleiman et al, 1999b).
During late zygotene/early pachytene stages of the first meiotic division, X and Y chromosomes form a special structure, known as the sex vesicle (Pathak and Elder, 1980) or the XY body (Solari, 1999), which is characterized by a particular chromatin condensation and transcriptional inactivation. The special chromatin condensation of the XY body during meiotic prophase is thought to serve to prevent accidental recombination events between nonhomologous regions of the X and Y chromosomes (Handel and Hunt, 1992; Mckee and Handel, 1993). In the XY body structure chromosomes X and Y are organized into 2 completely separate chromosomal domains (Metzler-Guillemain et al, 2000). Despite the separation, the ends of the X and Y chromosome domains are joined by their pseudoautosomal regions, as was demonstrated by short synaptonemal complex formation (Pathak and Elder, 1980) and by the clustering of their 4 telomeres (Metzler-Guillemain et al, 2000).
The length of the synaptic region varies from cell to cell (Pathak and Elder, 1980). It is not limited to the pseudoautosomal region, and can include a significant portion of the Y chromosome, leading to apparent nonhomologous pairing (Chandley et al, 1984). However, normal sequence exchange occurs exclusively within two stretches of homology, and is most prevalent within the 2.5 Mb of DNA, adjacent to the short arm telomeres of both the X and Y chromosomes.
Crossing within the pseudoautosomal region is functionally significant in that it generates the chiasma that is required to hold the sex chromosomes together during the first metaphase of meiosis. Frequency of recombination within the long arm pseudoautosomal region (320 kb) is high; however, it is neither necessary nor sufficient for successful meiosis (Freije et al, 1992; Kval?y et al, 1994). Data are most consistent that X-Y pairing and recombination are necessary for a spermatocyte to successfully complete meiosis (McIlree et al, 1966; Chandley and Edmond, 1971; Gabriel-Robez et al, 1990; Hale, 1994).
The fluorescence in situ hybridization (FISH) technique can be used for bivalent evaluation with a certain advantage over other techniques. This is mainly because there is no need for microspreading as a precondition (Scherthan et al, 1996). Thus, it can facilitate the evaluation of hundreds of meiotic cells from each specimen, even in pathological cases with an extremely low number of spermatocytes, reflecting serious testicular damage. In contrast, an alternative method, such as microspreading followed by synaptonemal complex protein staining, which is the most common technique for the study of meiotic pairing, is less applicable in such extreme cases (Metzler-Guillemain and Guichaoua, 2000).
In a recent study using testicular biopsies of azoospermic men, a significantly higher rate of some homologous chromosome bivalents was found whenever spermatozoa were detected. Bivalent formation of all 4 pairs of chromosomes that were evaluated was highly correlated, but the rate of the bivalent X-Y was found to be the most sensitive predictor for detection of spermatozoa, with a cutoff value of 47% (Yogev et al, 2000; Yogev et al, 2002).
The purpose of the present study was to observe the X and Y alignment in spermatocytes of men carrying Y chromosome microdeletions. This was performed to evaluate whether the AZF-deleted segment may influence the ability of the Y chromosome to pair. Multicolor FISH probes for both centromere and telomere regions were used. Evaluation of XY bivalent in specimens with AZF microdeletion has not been reported previously.
Materials and Methods
A total of 11 azoospermic men undergoing testicular sperm extraction (TESE) were enrolled in the study. In all cases, azoospermia was reconfirmed before the TESE procedure. The etiology of four men was AZFc deletion (between direct repeats b2/b4): two had AZFb (between palindromes P5/proximal P1) and two others had both AZFb and AZFc deletions [between palindromes P5/distal P1 and P5/del(qter), Table 1]. Three men with obstructive azoospermia due to congenital absence of vas deferens (carriers of cystic fibrosis mutations) served as a control group. Partial evaluation of three cases (nos. 1, 2, 9) was previously published (Yogev et al, 2002).
Table 1 Extent of Y chromosome deletion in each specimen
Testicular volume was between 8 and 25 mL. Four men (nos. 1, 3, 4, 5) had elevated follicle-stimulating hormone levels (>11 mIU/mL). All patients provided written informed consent to undergo genetic evaluation. The study was approved by the local Institutional Review Board Committee in accordance with the Helsinki Declaration of 1975. The evaluation included karyotyping, Y-chromosome microdeletion test, and the percentage of homologous chromosome pairing in spermatocytes. Chromosome karyotyping was performed on peripheral lymphocytes with G-banding. The Y-chromosome microdeletion test was performed by the multiplex polymerase chain reaction with genomic DNA isolation from peripheral blood samples, as previously described (Kleiman et al, 1999a). Presence of the Yq-tip was verified with marker sY1201 (Kuroda-Kawaguchi et al, 2001). The deletion boundaries were determined using additional markers (sY1207; sY1228; sY118; sY1224; sY1192; sY1291; sY639; sY1257).
Biopsy Evaluation
Details of the TESE procedure have been described previously (Hauser et al, 1998; Yogev et al, 2000). Briefly, one biopsy specimen obtained from each testis was divided into two: one small section (<20mg) was utilized for routine Bouins (Sigma Chemical Company, St. Louis, Mo) fixation and histopathologic assessment, counting of testicular cells, and pairing evaluation by FISH. The other section (approximately 50 mg) was minced for spermatozoa extraction to be used in the ICSI procedure. Two additional biopsy specimens were procured from each testis for spermatozoa extraction, with the exception of 2 clearly defined, obstructive, azoospermic patients (Table 1, nos. 9, 11).
Histological analysis of spermatogenesis was performed on hematoxylin- and eosin-stained sections. At least 20 seminiferous tubules were scored in each testis. The number of Sertoli and germ cells at different stages was counted for each tubule section, and the mean value was calculated. The same technician viewed all the slides.
Specimen Preparation and Detection of Chromosome Pairing
Testicular biopsy specimens were prepared as previously described (Yogev et al, 2000). Briefly, mechanical minced tissue was fixed with cold fresh methanol/acetic acid (3/1) solution, dropped onto 2 slides (Super Frost/plus; Menzel-Glaser, Braun-schweig, Germany), and air dried. All the different testicular nuclei, including spermatozoa, were counted. The percentage of spermatocytes and spermatozoa in the sample was calculated from approximately 1600 testicular nuclei per sample.
For detection of chromosome pairing by FISH, the slides (two for each sample) were treated according to the manufacturer's instructions. Triple-color FISH with CEP centromeric DNA probes (Vysis, Downers Grove, Ill) for chromosomes X (spectrum green), Y (spectrum orange), and 18 (spectrum aqua blue), and double-color FISH with TelVysion telomeric probes Xq/Yq (spectrum orange) and Xp/Yp (spectrum green) were used. Two independent observers viewed each slide with an Olympus AX70 fluorescence microscope (Olympus, Tokyo, Japan) equipped with a single band for 4',6-diamidino-2-phenylindole (DAPI), triple-band pass filter for DAPI/fluorescein isothiocyanate (FITC)/TRIC, and a single-band pass filter for FITC and aqua blue.
Nuclei of primary spermatocytes were identified by the size of the nucleus in comparison to the other nuclei, and by the typical granular or threadlike chromatin appearance of the DAPI counterstain. Generally, at least 350 spermatocytes were analyzed in each slide. Cells were scored according to the number and proximity of the signals. The scored nuclei were intact and did not overlap other nuclei. For X and Y chromosomes, only centromeric signals with a maximum of two-signal diameters apart were recognized as close bivalents and marked as X-Y. For the autosomal 18 chromosome bivalent, when one signal, or two signals of similar size, were located at a distance of less than one diameter of the signal domain, a paired bivalent was recorded. For the telomere signals, pairing of the short and long arms of X and Y chromosomes was determined by the same criteria as those for the autosomal 18 chromosome bivalent. The percentage of pairing was calculated by dividing the number of nuclei with paired bivalents by the number of spermatocytes that were evaluated in each slide. The reproducibility between the score of the 2 technicians was <5% of variance (r = 0.95 by Spearman test).
Statistical Analysis
Variances between pairing of p and q X and Y chromosomes were analyzed using the two-tailed Wilcoxon signed rank test. Correlation was evaluated using the Spearman correlation test. All statistical analyses were performed using SPSS for Windows 9.0 (SPSS Inc., Chicago, Ill).
Results
In all 4 patients with AZFc deletion (Table 1), spermatozoa were identified in at least 1 of the 6 locations that had been evaluated (Figure 1b). In the testes of the 2 patients with AZFb deletion and the 2 with AZFb-c deletion, no spermatozoa could be found (Figure 1c and d). In the histological section, which represents only 1 location in each testis, mature spermatids were found in only 2 patients with AZFc deletion (Table 2, nos. 3 and 4).
In the suspension of minced testicular tissue stained by the FISH technique, no spermatozoa were found in any of the samples of AZF-deleted Y chromosomes (Table 2, nos. 1 through 4, and 7 and 8). The percentage of spermatocytes (calculated from the testicular nuclei) varied between 0% and 3% and 0.1% and 9% in men with AZFc and AZFb-c deletions, respectively. The absence of spermatocytes in specimen no. 4 and specimens for FISH in patients 5 and 6 prevented their meiosis evaluation.
All patients had normal karyotype, except for patient no. 8 who was 46,XYqh-/45,X (82%/18%). In this specimen, telomere pairing was not shown because of being not informative.
A total of 7623 spermatocytes were evaluated. Pairing rate of chromosome 18 (Figure 1e and f) was high in all samples (Table 3). Despite this high rate of pairing, previously found to correlate with the presence of spermatozoa (Yogev et al, 2000; Yogev et al, 2002), only spermatocytes were found as the most advanced stage in the biopsies of patients with AZFb-c deletion. Nevertheless, using the X-Y centromeric probes, the rate of bivalent X-Y (X and Y in proximity, Figure 1e and g) was under the cutoff value of 47%, indicating the lack of spermatozoa in patients with the AZFb-c deletions (nos. 7 and 8). The rate of bivalent X-Y was above the cutoff value in biopsies of patients with AZFc deletion (Table 3).
Interestingly, the rate of pairing of chromosomes X and Y in the p arms (Figure 1i through k) was higher than the percentage of spermatocytes with adjacent centromeres for all specimens (P = 0.0225). However, when a high percentage of spermatocytes with the X and Y q-arms far apart was recognized (as was in no. 7, Figure 1k and l), a distance was also found between the centromeric signals. No significant correlation was found between pand q-arms rate of pairing, whereas the highest value of correlation was found between the centromeres of X-Y in proximity and paired q-arms (Table 4).
Discussion
It is well accepted that the region and extent of the Y chromosome microdeletion play a major role in the magnitude of spermatogenesis impairment (Kleiman et al, 2001; Luetjens et al, 2002). It is also known that failure of adjacency between the X and Y chromosomes generates complete breakdown of meiosis (Hale, 1994). In the present study, as was shown also by Krausz et al, no mature sperm cells were detected when the deletions included the entire AZFb or AZFb-c regions (Krausz et al, 2000).
Pairing impairment was found despite the fact that the deletion extended away from the pseudoautosomal region of the q-arm, to the AZFb domain. This finding hints at 2 probable theories: The first is that the extent of the deletion is responsible for the pairing impairment, by generating a spatial disturbance. The second possibility is that the gene/s in AZFb region are accountable for the process of X-Y bivalent formation during meiosis (18-18 pairing was close to normal).
AZFb deletion, encompassing up to 6.2 Mb, eliminates 32 genes and transcripts and AZFb-c deletion comprehends 7-7.7 Mb. This is in contrast to the 3.5 Mb of the AZFc region. The deletions exclude members of testis-specific families of genes that were proposed to be involved in the spermatogenic process (Repping et al, 2002). Only the heterochromatic region, which is variable in its size (about 40 Mb in a study of one man) is in between the AZFc and the pseudoautosomal region (Skaletsky et al, 2003). Obviously, further studies of additional patients with isolated AZFb deletion may verify the correct hypothesis for the spermatogenesis impairment. Since only one autosomal bivalent formation (chromosome 18) was explored, pairing status of some other autosomes may help to confirm the difference between the X-Y bivalent in contrast with the autosome rate of pairing.
It is commonly acclaimed that large microdeletions that include the heterochromatic Yq tip ('terminal' deletion) may cause chromosomal instability (Siffroi et al, 2000). This phenomenon can explain the karyotype of patient no. 8, who was found to be mosaic 46,XYqh-/45,X (82%/18%). Surprisingly, the rate of X-Y bivalents was similar for the 2 men with AZFb-c deletion, although specimen no. 8 had a larger deletion that included the Yq tip. Nevertheless, the number of germ cells in the testicular biopsy of patient no. 8 (Table 2) was lower compared with those of patient no 7.
In normal pairing of X and Y chromosomes, there was clustering of their 4 telomeres in such a way that in 90% of the spermatocytes, only 2 juxtaposed signals were detected (Metzler-Guillemain et al, 2000). In our study, 95%-96% of the spermatocytes in the control specimens were found with all signals close to each other. Whereas AZFc deletion was found to be associated with a moderate decrease in the frequency of this phenomenon (70%-94%), the specimen with AZFb-c deletion showed a dramatic impairment (29%). Therefore, the low rate of clustering of short and long arms of both X and Y telomeres may indicate for the dramatic impairment of the meiosis.
As shown previously, when no spermatozoa or mature spermatids were found in some biopsies, the relatively high rate of the bivalents indicated the possibility of finding some foci of spermatogenesis in other biopsies. Failure in X-Y bivalent formation appears to lead to breakdown of spermatogenesis at the time of the meiotic division (Yogev et al, 2002). Interestingly, in the patient with AZFb-c deletion (no. 7), the short arms paired in 51% of the spermatocytes. However, despite quite a high level of X-Y short-arm pairing, the typical XY body chromatin probably was not formed and consequently, the rate of the detected X-Y bivalent that was recorded by centromeres in proximity was 29% only. A different finding was reported for the short arm of the X chromosome in 2 men with a karyotype of 46,Y,der(x),t(X; Y)(p22.3:q11). Analysis of synaptonemal complexes at pachytene showed that deletion of the X short-arm pseudoautosomal region in these men impaired sex-chromosome pairing even though a "sex vesicle" was formed (Gabriel-Robez et al, 1990).
It can therefore be suggested that, in contrast to the AZFc deletion, the AZFb- and AZFb-c-deleted regions may cause a meiotic impairment by disturbing the X and Y chromosome alignment. Obviously this finding has to be confirmed by increasing the number of patients. The evaluation of the XY body, using monoclonal antibodies against its specific proteins, may assist in clarifying the nature of the meiosis impairment. Our results support the concept that the analysis of Y chromosome microdeletion is of clinical importance not only in terms of defining the etiology of the spermatogenesis impairment, but also because of its clinical prognostic value.
Acknowledgments
We thank Mrs Bella Gore, Marina Ilatov, and Jenny Yerushalmi for their excellent technical assistance. The support of the entire staff of the Institute for the Study of Fertility is much appreciated.
Footnotes
Supported by grants from the Chief Scientific Office, Ministry of Health, and by the Hirsch and Genia Wassermann Memorial Fund for Medical Research, Tel Aviv University.
References
Chandley A, Goetz P, Hargreave TB, Joseph AM, Speed RM. On the nature and extent of XY pairing at meiosis prophase in man. Cytogenet Cell Genet. 1984; 38:241-247.
Chandley AC, Edmond P. Meiotic studies on a subfertile patient with a ring Y chromosome. Cytogenetics. 1971; 10:295-304.
Foote S, Vollrath D, Hilton A, Page DC. The human Y chromosome: overlapping DNA clones spanning the euchromatic region. Science. 1992;258:60-66.
Foresta C, Moro E, Ferlin A. Prognostic value of Y deletion analysis. Hum Reprod. 2001; 16:1543-1547.
Freije D, Helms C, Watson MS, Donis-Keller H. Identification of a second pseudoautosomal region near the Xq and Yq telomeres. Science. 1992;258:1784-1787.
Gabriel-Robez O, Rumpler Y, Ratomponirina C, Petit C, Levilliers J, Croquette MF, Couturier J. Deletion of the pseudoautosomal region and lack of sex-chromosome pairing at pachytene in two infertile men carrying an X; Y translocation. Cytogenet Cell Genet. 1990; 54:38-42.
Hale DW. Is X-Y recombination necessary for spermatocyte survival during mammalian spermatogenesis? Cytogenet Cell Genet. 1994;65:278-282.
Handel MA, Hunt PA. Sex-chromosome pairing and activity during mammalian meiosis. BioEssays. 1992; 14:817-822.
Hauser R, Botchan A, Amit A, et al. Multiple testicular sampling in nonobstructive azoospermia—is it necessary? Hum Reprod. 1998;13:3081-3085.
Kamp C, Hirschmann P, Voss H, Huellen K, Vogt H. Two long homologous retroviral sequence blocks in proximal Yq11 cause AZFa microdeletions as a result of intrachromosomal recombination events. Hum Molec Genet. 2000; 9:2563-2572.
Kent-First MG, Kol S, Muallem A, Ofir R, Manor D, Blazer S, First N, Itskovitz-Elder J. The incidence and possible relevance of Y-linked microdeletions in babies born after intracytoplasmic sperm injection and their infertile fathers. Mol Hum Reprod. 1996; 2:943-950.
Kleiman S, Bar-Shira Maymon B, Yogev L, Paz G, Yavetz H. The prognostic role of the extent of Y-microdeletion on spermatogenesis and maturity of Sertoli cells. Hum Reprod. 2001; 16:399-402.
Kleiman S, Yogev L, Gamzu R, Hauser R, Botchan A, Lessing JB, Paz G, Yavetz H. Genetic evaluation of infertile men. Hum Reprod. 1999a;14:33-38.
Kleiman S, Yogev L, Gamzu R, et al. Three-generation evaluation of Y-chromosome microdeletion. J Androl. 1999b; 20:394-398.
Krausz C, Quintana-Murci S, McElreavey K. Prognostic value of Y deletion analysis: What is the clinical prognostic value of Y chromosome microdeletion analysis? Hum Reprod. 2000; 15:1431-1434.
Kuroda-Kawaguchi T, Skaletsky H, Brown LG, et al. The AZFc region of the Y chromosome features massive palindromes and uniform recurrent deletions in infertile men. Nat Genet. 2001; 29:279-286.
Kval?y K, Galvagni F, Brown WRA. The sequence organization of the long arm pseudoautosomal region of the human sex chromosome. Hum Mol Genet. 1994; 3:771-778.
Luetjens C, Gromoll J, Engelhardt M, von Eckardstein S, Bergmann M, Nieschlag E, Simon M. Manifestation of Y-chromosomal deletions in the human testis: a morphometrical and immunohistochemical evaluation. Hum Reprod. 2002;17:2258-2266.
McIlree ME, Price WH, Brown WMC, Tulloch WS, Newsam JE, Maclean N. Chromosome studies on testicular cells from 50 subfertile men. Lancet. 1966;2; 69 -71.
Mckee BD, Handel MA. Sex chromosomes, recombination, and chromatin conformation. Chromosoma 1993; 102:71-80.
Metzler-Guillemain C, Guichaoua MRC. A simple and reliable method for meiotic studies on testicular samples used for intracytoplasmic sperm injection. Fertil Steril. 2000; 74:916-919.
Metzler-Guillemain C, Usson Y, Mignon C, Depetris D, Dubreuil G, Guichaoua MR, Mattei MG. Organization of the X and Y chromosomes in human, chimpanzee and mouse pachytene nuclei using molecular cytogenetics and three-dimensional confocal analyses. Chromosome Res. 2000; 8:571-584.
Pathak P, Elder FFB. Silver-stained accessory structures on human sex chromosomes. Hum Genet. 1980; 54:171-175.
Reijo R, Alagappan RK, Patrizio P, Page DC. Severe oligozoospermia resulting from deletions of azoospermia factor gene on Y chromosome. Lancet. 1996;347:1290-1293.
Reijo R, Lee TY, Salo P, et al. Diverse spermatogenic defects in human caused by Y chromosome deletions encompassing a novel RNA-binding protein gene. Nat Genet. 1995; 10:383-392.
Repping S, Skaletsky H, Lange J, Silber S, van der Veen F, Oates RD, Page DC, Rozen S. Recombination between palindromes P5 and P1 on the human Y chromosome causes massive deletions and spermatogenic failure. Am J Hum Genet. 2002;71:916-922.
Scherthan H, Weich S, Schwegler H, Heyting C, H?rle M, Cremer T. Centromere and telomere movements during early meiotic prophase of mouse and man are associated with the onset of chromosome pairing. J Cell Biol. 1996;134:1109-1125.
Siffroi JP, Le Bourhis C, Krausz C, et al. Sex chromosome mosaicism in males carrying Y chromosome long arm deletion. Hum Reprod. 2000;15:2559-2562.
Skaletsky H, Kuroda-Kawaguchi T, Minx PJ, et al. The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes. Nature. 2003;423:825-838.
Solari AJ. Synaptonemal complex analysis in human male infertility. Eur J Histochem. 1999; 43:265-276.
Sun C, Skaletsky H, Rozen S, Gromoll J, Nieschlag E, Oates R, Page DC. Deletion of azoospermia factor a (AZFa) region of human Y chromosome caused by recombination between HERV15 provirus. Hum Mol Genet. 2000;9:2291-2296.
Tiepolo L, Zuffardi O. Localization of factors controlling spermatogenesis in the nonfluorescent portion of the Y chromosome long arm. Hum Genet. 1976; 34:119-124.
Vogt PH, Edelmann A, Kirsch S, et al. Human Y chromosome azoospermia factors (AZF) mapped to different subregions in Yq11. Hum Mol Genet. 1996; 5:933-943.
Yogev L, Gamzu R, Kleiman S, Botchan A, Hauser R, Yavetz H. Evaluation of meiotic impairment of azoospermic men by fluorescence in situ hybridization. Fertil Steril. 2000; 74:228-233.
Yogev L, Gamzu R, Paz G, Kleiman S, Botchan A, Hauser R, Yavetz H. Rate of homologous chromosome bivalents in spermatocytes may predict completion of spermatogenesis in azoospermic men. Hum Genet. 2002;110:30-35.(LEAH YOGEV, SHMUEL SEGAL,)
Abstract
Deletions in the q arm of the Y chromosome result in spermatogenesis impairment. The aim of the present study was to observe the X and Y chromosome alignment in the spermatocytes of men with Y chromosome microdeletion of the azoospermia factor (AZF) region. This was performed by multicolor fluorescence in situ hybridization probes for the centromere and telomere regions. Testicular biopsies were performed in a testicular sperm extraction-intracytoplasmic sperm injection set-up in 11 azoospermic men: 8 (nonobstructive) with AZF deletions and 3 (obstructive) controls. Histological sections, cytology preparations of the testicular biopsies, and evaluation of the meiosis according to the percentage of XY and 18 bivalents formation were assessed. Spermatozoa were identified in at least one location in controls and specimens with AZFc-deleted Y chromosomes. Complete spermatocyte arrest was found in those with a deletion that included the entire AZFb region. Bivalent formation rate of chromosome 18 was high in all samples (81%-99%). In contrast, the rate of bivalent X-Y as determined by centromeric probes was lower but in the range favorable with spermatozoa findings in controls and patients with the AZFc deletion (56%-90%), but not in those with AZFb-c deletions (28%-29%). A dramatic impairment in the normal alignment of X and Y telomeres in the specimen with AZFb-c deletion was shown (29%), compared to the specimens with AZFc deletion (70%-94%). It is suggested that the absence of sperm cells in specimens with the entire AZFb and with AZFb-c deletions is accompanied by meiosis impairment, perhaps as a result of the extent of the deletion or because of the absence of genes that are involved in the X and Y chromosome alignment.
Key words: Azoospermia, chromosome pairing, FISH, Y chromosome microdeletion
Deletions of the long arm of the human Y chromosome resulted in cessation of spermatogenesis, and subsequently, infertility (Chandley and Edmond, 1971; Tiepolo and Zuffardi, 1976). Further study led to the proposal of the existence of 3 azoospermia factor (AZF) subregions termed AZFa,b,c (Vogt et al, 1996).
Deletion of the entire AZFa region (0.8 Mb; Sun et al, 2000) is associated with lack of germ cells (Sertoli-cell-only syndrome), and complete deletion of the AZFb region (6.23 Mb; Repping et al, 2002) is generally associated with arrest of spermatogenesis (Krausz et al, 2000; Foresta et al, 2001; Kleiman et al, 2001; Luetjens et al, 2002). Deletion of AZFc is the most common, and is associated either with severe oligozoospermia, or azoospermia, accompanied by a wide spectrum of histological defects (Reijo et al, 1995, 1996). It is remarkably uniform, spanning a 3.5-Mb segment (Kuroda-Kawaguchi et al, 2001).
Both AZFc and AZFa deletions, as well as most AZFb deletions, appear to ensue from homologous recombination between direct repeats on the Y chromosome (Kamp et al, 2000; Sun et al, 2000; Kuroda-Kawaguchi et al, 2001; Repping et al, 2002). Yq microdeletions may be associated with Y chromosomal instability, leading to the formation of the 45,X0 cell in lymphocytes and sperm cell nullisomic for the Y chromosome (Siffroi et al, 2000). The use of sperm retrieval from testes of infertile men to achieve fertilization through intracytoplasmic sperm injection (ICSI) raises the possibility of transmission of Y-related infertility to the male offspring (Kent-First et al, 1996; Vogt et al, 1996; Kleiman et al, 1999b).
During late zygotene/early pachytene stages of the first meiotic division, X and Y chromosomes form a special structure, known as the sex vesicle (Pathak and Elder, 1980) or the XY body (Solari, 1999), which is characterized by a particular chromatin condensation and transcriptional inactivation. The special chromatin condensation of the XY body during meiotic prophase is thought to serve to prevent accidental recombination events between nonhomologous regions of the X and Y chromosomes (Handel and Hunt, 1992; Mckee and Handel, 1993). In the XY body structure chromosomes X and Y are organized into 2 completely separate chromosomal domains (Metzler-Guillemain et al, 2000). Despite the separation, the ends of the X and Y chromosome domains are joined by their pseudoautosomal regions, as was demonstrated by short synaptonemal complex formation (Pathak and Elder, 1980) and by the clustering of their 4 telomeres (Metzler-Guillemain et al, 2000).
The length of the synaptic region varies from cell to cell (Pathak and Elder, 1980). It is not limited to the pseudoautosomal region, and can include a significant portion of the Y chromosome, leading to apparent nonhomologous pairing (Chandley et al, 1984). However, normal sequence exchange occurs exclusively within two stretches of homology, and is most prevalent within the 2.5 Mb of DNA, adjacent to the short arm telomeres of both the X and Y chromosomes.
Crossing within the pseudoautosomal region is functionally significant in that it generates the chiasma that is required to hold the sex chromosomes together during the first metaphase of meiosis. Frequency of recombination within the long arm pseudoautosomal region (320 kb) is high; however, it is neither necessary nor sufficient for successful meiosis (Freije et al, 1992; Kval?y et al, 1994). Data are most consistent that X-Y pairing and recombination are necessary for a spermatocyte to successfully complete meiosis (McIlree et al, 1966; Chandley and Edmond, 1971; Gabriel-Robez et al, 1990; Hale, 1994).
The fluorescence in situ hybridization (FISH) technique can be used for bivalent evaluation with a certain advantage over other techniques. This is mainly because there is no need for microspreading as a precondition (Scherthan et al, 1996). Thus, it can facilitate the evaluation of hundreds of meiotic cells from each specimen, even in pathological cases with an extremely low number of spermatocytes, reflecting serious testicular damage. In contrast, an alternative method, such as microspreading followed by synaptonemal complex protein staining, which is the most common technique for the study of meiotic pairing, is less applicable in such extreme cases (Metzler-Guillemain and Guichaoua, 2000).
In a recent study using testicular biopsies of azoospermic men, a significantly higher rate of some homologous chromosome bivalents was found whenever spermatozoa were detected. Bivalent formation of all 4 pairs of chromosomes that were evaluated was highly correlated, but the rate of the bivalent X-Y was found to be the most sensitive predictor for detection of spermatozoa, with a cutoff value of 47% (Yogev et al, 2000; Yogev et al, 2002).
The purpose of the present study was to observe the X and Y alignment in spermatocytes of men carrying Y chromosome microdeletions. This was performed to evaluate whether the AZF-deleted segment may influence the ability of the Y chromosome to pair. Multicolor FISH probes for both centromere and telomere regions were used. Evaluation of XY bivalent in specimens with AZF microdeletion has not been reported previously.
Materials and Methods
A total of 11 azoospermic men undergoing testicular sperm extraction (TESE) were enrolled in the study. In all cases, azoospermia was reconfirmed before the TESE procedure. The etiology of four men was AZFc deletion (between direct repeats b2/b4): two had AZFb (between palindromes P5/proximal P1) and two others had both AZFb and AZFc deletions [between palindromes P5/distal P1 and P5/del(qter), Table 1]. Three men with obstructive azoospermia due to congenital absence of vas deferens (carriers of cystic fibrosis mutations) served as a control group. Partial evaluation of three cases (nos. 1, 2, 9) was previously published (Yogev et al, 2002).
Table 1 Extent of Y chromosome deletion in each specimen
Testicular volume was between 8 and 25 mL. Four men (nos. 1, 3, 4, 5) had elevated follicle-stimulating hormone levels (>11 mIU/mL). All patients provided written informed consent to undergo genetic evaluation. The study was approved by the local Institutional Review Board Committee in accordance with the Helsinki Declaration of 1975. The evaluation included karyotyping, Y-chromosome microdeletion test, and the percentage of homologous chromosome pairing in spermatocytes. Chromosome karyotyping was performed on peripheral lymphocytes with G-banding. The Y-chromosome microdeletion test was performed by the multiplex polymerase chain reaction with genomic DNA isolation from peripheral blood samples, as previously described (Kleiman et al, 1999a). Presence of the Yq-tip was verified with marker sY1201 (Kuroda-Kawaguchi et al, 2001). The deletion boundaries were determined using additional markers (sY1207; sY1228; sY118; sY1224; sY1192; sY1291; sY639; sY1257).
Biopsy Evaluation
Details of the TESE procedure have been described previously (Hauser et al, 1998; Yogev et al, 2000). Briefly, one biopsy specimen obtained from each testis was divided into two: one small section (<20mg) was utilized for routine Bouins (Sigma Chemical Company, St. Louis, Mo) fixation and histopathologic assessment, counting of testicular cells, and pairing evaluation by FISH. The other section (approximately 50 mg) was minced for spermatozoa extraction to be used in the ICSI procedure. Two additional biopsy specimens were procured from each testis for spermatozoa extraction, with the exception of 2 clearly defined, obstructive, azoospermic patients (Table 1, nos. 9, 11).
Histological analysis of spermatogenesis was performed on hematoxylin- and eosin-stained sections. At least 20 seminiferous tubules were scored in each testis. The number of Sertoli and germ cells at different stages was counted for each tubule section, and the mean value was calculated. The same technician viewed all the slides.
Specimen Preparation and Detection of Chromosome Pairing
Testicular biopsy specimens were prepared as previously described (Yogev et al, 2000). Briefly, mechanical minced tissue was fixed with cold fresh methanol/acetic acid (3/1) solution, dropped onto 2 slides (Super Frost/plus; Menzel-Glaser, Braun-schweig, Germany), and air dried. All the different testicular nuclei, including spermatozoa, were counted. The percentage of spermatocytes and spermatozoa in the sample was calculated from approximately 1600 testicular nuclei per sample.
For detection of chromosome pairing by FISH, the slides (two for each sample) were treated according to the manufacturer's instructions. Triple-color FISH with CEP centromeric DNA probes (Vysis, Downers Grove, Ill) for chromosomes X (spectrum green), Y (spectrum orange), and 18 (spectrum aqua blue), and double-color FISH with TelVysion telomeric probes Xq/Yq (spectrum orange) and Xp/Yp (spectrum green) were used. Two independent observers viewed each slide with an Olympus AX70 fluorescence microscope (Olympus, Tokyo, Japan) equipped with a single band for 4',6-diamidino-2-phenylindole (DAPI), triple-band pass filter for DAPI/fluorescein isothiocyanate (FITC)/TRIC, and a single-band pass filter for FITC and aqua blue.
Nuclei of primary spermatocytes were identified by the size of the nucleus in comparison to the other nuclei, and by the typical granular or threadlike chromatin appearance of the DAPI counterstain. Generally, at least 350 spermatocytes were analyzed in each slide. Cells were scored according to the number and proximity of the signals. The scored nuclei were intact and did not overlap other nuclei. For X and Y chromosomes, only centromeric signals with a maximum of two-signal diameters apart were recognized as close bivalents and marked as X-Y. For the autosomal 18 chromosome bivalent, when one signal, or two signals of similar size, were located at a distance of less than one diameter of the signal domain, a paired bivalent was recorded. For the telomere signals, pairing of the short and long arms of X and Y chromosomes was determined by the same criteria as those for the autosomal 18 chromosome bivalent. The percentage of pairing was calculated by dividing the number of nuclei with paired bivalents by the number of spermatocytes that were evaluated in each slide. The reproducibility between the score of the 2 technicians was <5% of variance (r = 0.95 by Spearman test).
Statistical Analysis
Variances between pairing of p and q X and Y chromosomes were analyzed using the two-tailed Wilcoxon signed rank test. Correlation was evaluated using the Spearman correlation test. All statistical analyses were performed using SPSS for Windows 9.0 (SPSS Inc., Chicago, Ill).
Results
In all 4 patients with AZFc deletion (Table 1), spermatozoa were identified in at least 1 of the 6 locations that had been evaluated (Figure 1b). In the testes of the 2 patients with AZFb deletion and the 2 with AZFb-c deletion, no spermatozoa could be found (Figure 1c and d). In the histological section, which represents only 1 location in each testis, mature spermatids were found in only 2 patients with AZFc deletion (Table 2, nos. 3 and 4).
In the suspension of minced testicular tissue stained by the FISH technique, no spermatozoa were found in any of the samples of AZF-deleted Y chromosomes (Table 2, nos. 1 through 4, and 7 and 8). The percentage of spermatocytes (calculated from the testicular nuclei) varied between 0% and 3% and 0.1% and 9% in men with AZFc and AZFb-c deletions, respectively. The absence of spermatocytes in specimen no. 4 and specimens for FISH in patients 5 and 6 prevented their meiosis evaluation.
All patients had normal karyotype, except for patient no. 8 who was 46,XYqh-/45,X (82%/18%). In this specimen, telomere pairing was not shown because of being not informative.
A total of 7623 spermatocytes were evaluated. Pairing rate of chromosome 18 (Figure 1e and f) was high in all samples (Table 3). Despite this high rate of pairing, previously found to correlate with the presence of spermatozoa (Yogev et al, 2000; Yogev et al, 2002), only spermatocytes were found as the most advanced stage in the biopsies of patients with AZFb-c deletion. Nevertheless, using the X-Y centromeric probes, the rate of bivalent X-Y (X and Y in proximity, Figure 1e and g) was under the cutoff value of 47%, indicating the lack of spermatozoa in patients with the AZFb-c deletions (nos. 7 and 8). The rate of bivalent X-Y was above the cutoff value in biopsies of patients with AZFc deletion (Table 3).
Interestingly, the rate of pairing of chromosomes X and Y in the p arms (Figure 1i through k) was higher than the percentage of spermatocytes with adjacent centromeres for all specimens (P = 0.0225). However, when a high percentage of spermatocytes with the X and Y q-arms far apart was recognized (as was in no. 7, Figure 1k and l), a distance was also found between the centromeric signals. No significant correlation was found between pand q-arms rate of pairing, whereas the highest value of correlation was found between the centromeres of X-Y in proximity and paired q-arms (Table 4).
Discussion
It is well accepted that the region and extent of the Y chromosome microdeletion play a major role in the magnitude of spermatogenesis impairment (Kleiman et al, 2001; Luetjens et al, 2002). It is also known that failure of adjacency between the X and Y chromosomes generates complete breakdown of meiosis (Hale, 1994). In the present study, as was shown also by Krausz et al, no mature sperm cells were detected when the deletions included the entire AZFb or AZFb-c regions (Krausz et al, 2000).
Pairing impairment was found despite the fact that the deletion extended away from the pseudoautosomal region of the q-arm, to the AZFb domain. This finding hints at 2 probable theories: The first is that the extent of the deletion is responsible for the pairing impairment, by generating a spatial disturbance. The second possibility is that the gene/s in AZFb region are accountable for the process of X-Y bivalent formation during meiosis (18-18 pairing was close to normal).
AZFb deletion, encompassing up to 6.2 Mb, eliminates 32 genes and transcripts and AZFb-c deletion comprehends 7-7.7 Mb. This is in contrast to the 3.5 Mb of the AZFc region. The deletions exclude members of testis-specific families of genes that were proposed to be involved in the spermatogenic process (Repping et al, 2002). Only the heterochromatic region, which is variable in its size (about 40 Mb in a study of one man) is in between the AZFc and the pseudoautosomal region (Skaletsky et al, 2003). Obviously, further studies of additional patients with isolated AZFb deletion may verify the correct hypothesis for the spermatogenesis impairment. Since only one autosomal bivalent formation (chromosome 18) was explored, pairing status of some other autosomes may help to confirm the difference between the X-Y bivalent in contrast with the autosome rate of pairing.
It is commonly acclaimed that large microdeletions that include the heterochromatic Yq tip ('terminal' deletion) may cause chromosomal instability (Siffroi et al, 2000). This phenomenon can explain the karyotype of patient no. 8, who was found to be mosaic 46,XYqh-/45,X (82%/18%). Surprisingly, the rate of X-Y bivalents was similar for the 2 men with AZFb-c deletion, although specimen no. 8 had a larger deletion that included the Yq tip. Nevertheless, the number of germ cells in the testicular biopsy of patient no. 8 (Table 2) was lower compared with those of patient no 7.
In normal pairing of X and Y chromosomes, there was clustering of their 4 telomeres in such a way that in 90% of the spermatocytes, only 2 juxtaposed signals were detected (Metzler-Guillemain et al, 2000). In our study, 95%-96% of the spermatocytes in the control specimens were found with all signals close to each other. Whereas AZFc deletion was found to be associated with a moderate decrease in the frequency of this phenomenon (70%-94%), the specimen with AZFb-c deletion showed a dramatic impairment (29%). Therefore, the low rate of clustering of short and long arms of both X and Y telomeres may indicate for the dramatic impairment of the meiosis.
As shown previously, when no spermatozoa or mature spermatids were found in some biopsies, the relatively high rate of the bivalents indicated the possibility of finding some foci of spermatogenesis in other biopsies. Failure in X-Y bivalent formation appears to lead to breakdown of spermatogenesis at the time of the meiotic division (Yogev et al, 2002). Interestingly, in the patient with AZFb-c deletion (no. 7), the short arms paired in 51% of the spermatocytes. However, despite quite a high level of X-Y short-arm pairing, the typical XY body chromatin probably was not formed and consequently, the rate of the detected X-Y bivalent that was recorded by centromeres in proximity was 29% only. A different finding was reported for the short arm of the X chromosome in 2 men with a karyotype of 46,Y,der(x),t(X; Y)(p22.3:q11). Analysis of synaptonemal complexes at pachytene showed that deletion of the X short-arm pseudoautosomal region in these men impaired sex-chromosome pairing even though a "sex vesicle" was formed (Gabriel-Robez et al, 1990).
It can therefore be suggested that, in contrast to the AZFc deletion, the AZFb- and AZFb-c-deleted regions may cause a meiotic impairment by disturbing the X and Y chromosome alignment. Obviously this finding has to be confirmed by increasing the number of patients. The evaluation of the XY body, using monoclonal antibodies against its specific proteins, may assist in clarifying the nature of the meiosis impairment. Our results support the concept that the analysis of Y chromosome microdeletion is of clinical importance not only in terms of defining the etiology of the spermatogenesis impairment, but also because of its clinical prognostic value.
Acknowledgments
We thank Mrs Bella Gore, Marina Ilatov, and Jenny Yerushalmi for their excellent technical assistance. The support of the entire staff of the Institute for the Study of Fertility is much appreciated.
Footnotes
Supported by grants from the Chief Scientific Office, Ministry of Health, and by the Hirsch and Genia Wassermann Memorial Fund for Medical Research, Tel Aviv University.
References
Chandley A, Goetz P, Hargreave TB, Joseph AM, Speed RM. On the nature and extent of XY pairing at meiosis prophase in man. Cytogenet Cell Genet. 1984; 38:241-247.
Chandley AC, Edmond P. Meiotic studies on a subfertile patient with a ring Y chromosome. Cytogenetics. 1971; 10:295-304.
Foote S, Vollrath D, Hilton A, Page DC. The human Y chromosome: overlapping DNA clones spanning the euchromatic region. Science. 1992;258:60-66.
Foresta C, Moro E, Ferlin A. Prognostic value of Y deletion analysis. Hum Reprod. 2001; 16:1543-1547.
Freije D, Helms C, Watson MS, Donis-Keller H. Identification of a second pseudoautosomal region near the Xq and Yq telomeres. Science. 1992;258:1784-1787.
Gabriel-Robez O, Rumpler Y, Ratomponirina C, Petit C, Levilliers J, Croquette MF, Couturier J. Deletion of the pseudoautosomal region and lack of sex-chromosome pairing at pachytene in two infertile men carrying an X; Y translocation. Cytogenet Cell Genet. 1990; 54:38-42.
Hale DW. Is X-Y recombination necessary for spermatocyte survival during mammalian spermatogenesis? Cytogenet Cell Genet. 1994;65:278-282.
Handel MA, Hunt PA. Sex-chromosome pairing and activity during mammalian meiosis. BioEssays. 1992; 14:817-822.
Hauser R, Botchan A, Amit A, et al. Multiple testicular sampling in nonobstructive azoospermia—is it necessary? Hum Reprod. 1998;13:3081-3085.
Kamp C, Hirschmann P, Voss H, Huellen K, Vogt H. Two long homologous retroviral sequence blocks in proximal Yq11 cause AZFa microdeletions as a result of intrachromosomal recombination events. Hum Molec Genet. 2000; 9:2563-2572.
Kent-First MG, Kol S, Muallem A, Ofir R, Manor D, Blazer S, First N, Itskovitz-Elder J. The incidence and possible relevance of Y-linked microdeletions in babies born after intracytoplasmic sperm injection and their infertile fathers. Mol Hum Reprod. 1996; 2:943-950.
Kleiman S, Bar-Shira Maymon B, Yogev L, Paz G, Yavetz H. The prognostic role of the extent of Y-microdeletion on spermatogenesis and maturity of Sertoli cells. Hum Reprod. 2001; 16:399-402.
Kleiman S, Yogev L, Gamzu R, Hauser R, Botchan A, Lessing JB, Paz G, Yavetz H. Genetic evaluation of infertile men. Hum Reprod. 1999a;14:33-38.
Kleiman S, Yogev L, Gamzu R, et al. Three-generation evaluation of Y-chromosome microdeletion. J Androl. 1999b; 20:394-398.
Krausz C, Quintana-Murci S, McElreavey K. Prognostic value of Y deletion analysis: What is the clinical prognostic value of Y chromosome microdeletion analysis? Hum Reprod. 2000; 15:1431-1434.
Kuroda-Kawaguchi T, Skaletsky H, Brown LG, et al. The AZFc region of the Y chromosome features massive palindromes and uniform recurrent deletions in infertile men. Nat Genet. 2001; 29:279-286.
Kval?y K, Galvagni F, Brown WRA. The sequence organization of the long arm pseudoautosomal region of the human sex chromosome. Hum Mol Genet. 1994; 3:771-778.
Luetjens C, Gromoll J, Engelhardt M, von Eckardstein S, Bergmann M, Nieschlag E, Simon M. Manifestation of Y-chromosomal deletions in the human testis: a morphometrical and immunohistochemical evaluation. Hum Reprod. 2002;17:2258-2266.
McIlree ME, Price WH, Brown WMC, Tulloch WS, Newsam JE, Maclean N. Chromosome studies on testicular cells from 50 subfertile men. Lancet. 1966;2; 69 -71.
Mckee BD, Handel MA. Sex chromosomes, recombination, and chromatin conformation. Chromosoma 1993; 102:71-80.
Metzler-Guillemain C, Guichaoua MRC. A simple and reliable method for meiotic studies on testicular samples used for intracytoplasmic sperm injection. Fertil Steril. 2000; 74:916-919.
Metzler-Guillemain C, Usson Y, Mignon C, Depetris D, Dubreuil G, Guichaoua MR, Mattei MG. Organization of the X and Y chromosomes in human, chimpanzee and mouse pachytene nuclei using molecular cytogenetics and three-dimensional confocal analyses. Chromosome Res. 2000; 8:571-584.
Pathak P, Elder FFB. Silver-stained accessory structures on human sex chromosomes. Hum Genet. 1980; 54:171-175.
Reijo R, Alagappan RK, Patrizio P, Page DC. Severe oligozoospermia resulting from deletions of azoospermia factor gene on Y chromosome. Lancet. 1996;347:1290-1293.
Reijo R, Lee TY, Salo P, et al. Diverse spermatogenic defects in human caused by Y chromosome deletions encompassing a novel RNA-binding protein gene. Nat Genet. 1995; 10:383-392.
Repping S, Skaletsky H, Lange J, Silber S, van der Veen F, Oates RD, Page DC, Rozen S. Recombination between palindromes P5 and P1 on the human Y chromosome causes massive deletions and spermatogenic failure. Am J Hum Genet. 2002;71:916-922.
Scherthan H, Weich S, Schwegler H, Heyting C, H?rle M, Cremer T. Centromere and telomere movements during early meiotic prophase of mouse and man are associated with the onset of chromosome pairing. J Cell Biol. 1996;134:1109-1125.
Siffroi JP, Le Bourhis C, Krausz C, et al. Sex chromosome mosaicism in males carrying Y chromosome long arm deletion. Hum Reprod. 2000;15:2559-2562.
Skaletsky H, Kuroda-Kawaguchi T, Minx PJ, et al. The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes. Nature. 2003;423:825-838.
Solari AJ. Synaptonemal complex analysis in human male infertility. Eur J Histochem. 1999; 43:265-276.
Sun C, Skaletsky H, Rozen S, Gromoll J, Nieschlag E, Oates R, Page DC. Deletion of azoospermia factor a (AZFa) region of human Y chromosome caused by recombination between HERV15 provirus. Hum Mol Genet. 2000;9:2291-2296.
Tiepolo L, Zuffardi O. Localization of factors controlling spermatogenesis in the nonfluorescent portion of the Y chromosome long arm. Hum Genet. 1976; 34:119-124.
Vogt PH, Edelmann A, Kirsch S, et al. Human Y chromosome azoospermia factors (AZF) mapped to different subregions in Yq11. Hum Mol Genet. 1996; 5:933-943.
Yogev L, Gamzu R, Kleiman S, Botchan A, Hauser R, Yavetz H. Evaluation of meiotic impairment of azoospermic men by fluorescence in situ hybridization. Fertil Steril. 2000; 74:228-233.
Yogev L, Gamzu R, Paz G, Kleiman S, Botchan A, Hauser R, Yavetz H. Rate of homologous chromosome bivalents in spermatocytes may predict completion of spermatogenesis in azoospermic men. Hum Genet. 2002;110:30-35.(LEAH YOGEV, SHMUEL SEGAL,)