We evaluated the frequency of chromosomal aberrations and microdeletions of the Y chromosome in a sample of 204 patients included in an intracytoplasmic ...
Molecular Human Reproduction vol.3 no.8 pp. 699–704, 1997
Combined cytogenetic and Y chromosome microdeletion screening in males undergoing intracytoplasmic sperm injection
K.van der Ven1,4, M.Montag1, B.Peschka2, J.Leygraaf2, G.Schwanitz2, G.Haidl3, D.Krebs1 and H.van der Ven1 1Section
of Endocrinology and Reproductive Medicine, Department of Obstetrics and Gynecology, 2Department of Human Genetics and 3Department of Dermatology and Andrology, University of Bonn, Sigmund Freudstrasse 25, 53127 Bonn, Germany
4To
whom correspondence should be addressed
We evaluated the frequency of chromosomal aberrations and microdeletions of the Y chromosome in a sample of 204 patients included in an intracytoplasmic sperm injection (ICSI) programme. The prevalence of Y chromosome deletions in males with severely or only moderately reduced sperm counts is mainly unknown, so that patients were chosen with sperm counts ranging from mild oligozoospermia to azoospermia. While six out of 158 (3.8%) patients showed constitutional chromosomal aberrations, only two out of 204 (0.98%) patients were diagnosed with a microdeletion of Yq11. One had a terminal deletion in subinterval 6 of Yq11.23 which included the DAZ gene and a corresponding sperm count ,0.13106 spermatozoa/ml. The second patient had an isolated deletion of marker Y6PH54c, a more proximal site in subinterval 5 on Yq11.23, but repeatedly showed sperm counts of 3–83106 spermatozoa/ml. Thus, of the 158 patients who underwent a combined cytogenetic and Y-microdeletion screening, eight patients (5.1%) showed chromosomal abnormalities, either at the cytogenetic (n 5 6) or the molecular level (n 5 2). In conclusion, although rare in number, microdeletions of the Y chromosome can also be observed in patients with moderately reduced sperm counts. A more proximal site of the deletion breakpoint does not necessarily imply a more severe impairment of spermatogenesis than a distal deletion site. In our sample, the overall frequency of constitutional chromosomal aberrations exceeded the incidence of microdeletions of the Y chromosome even in patients with idiopathic azoo- or severe oligozoospermia. Key words: azoospermia/constitutional chromosome aberrations/genetic risk/oligozoospermia/Y chromosome microdeletions
Introduction The direct injection of a single spermatozoon or spermatid into the cytoplasm of an oocyte (intracytoplasmic sperm injection, ICSI) is the most invasive of all currently available techniques of microassisted reproduction. Due to its high success rates, even in cases of severely impaired spermatogenesis, the ICSI method has been rapidly adopted worldwide. At the same time, the use of ICSI has raised a vivid discussion concerning potential genetic risks associated with this technique. Indeed, ICSI allows reproduction in a group of individuals in whom an increased rate of chromosomal abnormalities is well documented (Chandley, 1979; Retief et al., 1984; De Braekeler and Dao, 1991; Testart et al., 1996). The incidence of constitutional aberrations in azoospermic males has repeatedly been described to be as high as 14–15% (Chandley, 1979; Retief et al., 1984). Furthermore, recent reports indicate that microdeletions of the Y chromosome are detectable in ~10% of men with idiopathic azoospermia and, to a lesser extent, are also associated with severe oligozoospermia (Reijo et al., 1995; Najmabadi et al., 1996; Vogt et al., 1996). Although there is no increased risk of fetal malformations in the latter group, the fact that infertility can be passed on to © European Society for Human Reproduction and Embryology
male offspring of ICSI patients has raised demands that males with azoospermia and severe oligozoospermia should be tested for Y-microdeletions in addition to a cytogenetic evaluation prior to ICSI. It is currently difficult to estimate the genetic risk for patients with andrological infertility who consider ICSI, since studies on cytogenetic abnormalities, as well as screening for Y chromosome microdeletions, have only been carried out on separate groups. Cytogenetic data obtained on infertility patients are difficult to compare because of different ascertainment procedures (Chandley, 1979; Retief et al., 1984; Pandiyan and Jecquier, 1996). With regard to Y chromosome microdeletions, available data have been mainly gathered from small, well selected patient samples with the majority of probands being azoospermic, so that a reliable risk assessment for the subgroup of patients with severe oligozoospermia is not yet possible (Reijo et al., 1995, 1996a; Najamabadi et al., 1996; Stuppia et al., 1996). Additionally, little if any information is currently obtainable concerning the prevalence of Y-microdeletions among males with only moderately reduced semen parameters. Despite the cloning of several potential spermatogenesis loci, e.g. the RBM1 (RNA Binding Motif) gene family (Ma 699
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Table I. Microdeletions of the Y chromosome in 204 patients with reduced semen parameters Mean sperm count (3 106/ml)
No. cases
No. with microdeletions
Y chromosome microdeletions
Azoospermia ,1 1–5 5–10 10–20 .20a
13 32 47 27 52 33
0 1 0 1 0 0
– sY153→sY157 – Y6HP54c – –
with sperm counts .20 3106 spermatozoa/ml were included in the study because of severe teratozoospermia and/or asthenozoospermia and previous in-vitro fertilization failures.
aPatients
et al., 1993), the TSPY (testis-specific protein on the Y) (Arnemann et al., 1991) and the DAZ (Deleted in Azoospermia) genes (Reijo et al., 1995), the pathophysiological relationship between Y deletions and the resulting impairment of spermatogenesis has not yet been established. It thus seems plausible that microdeletions of the Y chromosome might also be detectable in cases of less severe impairment of spermatogenesis, depending on the site of the Y chromosome microdeletion and the putative spermatogenesis gene involved. We report here the results of a combined cytogenetic and molecular-genetic screening programme for microdeletions of the Y chromosome in 204 patients undergoing ICSI. Because the majority of such cases are for oligozoospermia, patient selection was extended to individuals with less severely reduced semen parameters in addition to individuals with idiopathic azoospermia and severe oligozoospermia.
Materials and methods Patients Patients were recruited from the in-vitro fertilization (IVF) programme at the Section of Endocrinology and Reproductive Medicine at the University of Bonn, Germany. The populations consisted of 204 males with azoospermia or severe to moderate oligozoospermia who were planning to undergo ICSI because of male factor infertility with or without fertilization failure in previous IVF attempts. Each patient underwent an extensive andrological evaluation. Patients with known causes of infertility (obstructive azoospermia, testicular maldescent, testicular injury and testicular malignancy, cancer chemo- or radiotherapy) were excluded from the study. The participants’ semen analyses showed azoospermia (n 5 13) or variable degrees of oligozoospermia which were subclassified according to sperm count as follows: ,1, 1–5, 5–10 and 10–203106 spermatozoa/ml (n 5 158; Table I). A further 33 patients who had sperm counts .203 106/ml were recruited because of severe asthenozoospermia and/or teratozoospermia and repeated fertilization failures in at least two previous IVF attempts; 50 normozoospermic males of proven fertility served as controls. All participants had given informed consent following a protocol approved by the Ethic’s committee of the University of Bonn. Polymerase chain reaction screening for Y chromosome microdeletions Isolation of the DNA from blood samples was performed by a modified salting-out procedure (Miller et al., 1988) or using the QIAamp Blood Kit (Quiagen Inc. Chatsworth, CA, USA).
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Figure 1. Microdeletions of Yq11 in two oligozoospermic men. (A) Y-chromosome.(B) Y-chromosome subintervals. (C) Sequence tagged sites (STS). (D) Deletion patterns of patients. Solid boxes indicate presence of an STS; blank boxes indicate absence of an STS (modified after Qureshi et al. 1996). A series of Y chromosome-specific sequence-tagged sites (STS) had been characterized previously and the primer sequences described (Foote et al., 1992). A total of 30 loci, covering the euchromatic region between the loci SRY on Yp (corresponding STS: sY14) and DYS 240 (sY157) in distal Yq11.23 was examined (Figure 1). The majority of markers were concentrated in subintervals 5 and 6 of Yq11, where microdeletions have already been reported. 28 specific markers were amplified in five separate multiplex polymerase chain reactions (PCR) as described by Henegariu et al. (1994), and PCR products were separated by agarose gel electrophoresis. Two additional loci (sY254 and sY255) covering the region of the DAZ gene in subinterval 6 of Yq11 were amplified separately as described by Reijo et al. (1995). Amplification products were again visualized by
Cytogenetic abnormalities in males undergoing ICSI ethidium bromide staining after agarose gel electrophoresis. Failure of amplification of a specific marker in three subsequent PCR setups was interpreted as deletion of the corresponding Y locus. In addition to the 204 probands with azoo- or severe to moderate oligozoospermia, 50 normozoospermic males of proven fertility as well as seven normal females served as controls for the STS analysis. Female probands were included as controls in each PCR run to ensure Y-specific amplification of the STS used in this study. Cytogenetic evaluation Between 9/1995 and 11/1996, 158 males with pathological semen parameters underwent a cytogenetic investigation prior to initiation of an ICSI treatment. Since cytogenetic evaluation was restricted to patients newly entering the programme, a further 46 males who underwent repeat ICSI attempts during that time span were not karyotyped. Chromosome analysis was performed on peripheral lymphocytes. On average, 20 metaphases (450–550 bands per genome) were analysed per patient. In the case of numerical mosaics, 50–100 mitotic cells were examined. When structural chromosomal aberrations were present, the evaluation was extended to prometaphase stages (with an average of 850 bands per genome). In cases of complex structural rearrangements, additional molecular-cytogenetic analyses by fluorescence in-situ hybridization (FISH) were performed. In all individuals with structural chromosomal aberrations, additional investigations in close relatives were recommended to determine the origin of the abnormality (familial or de novo). Detailed data about the cytogenetic evaluation of the study sample will be published in the near future (Leygraaf et al., 1997). Patients who were diagnosed as having a chromosomal abnormality underwent genetic counselling, where the potential implications of their findings for themselves, as well as for future pregnancies, were discussed. Prenatal diagnosis was recommended in all cases of structural rearrangements.
Results Y chromosome microdeletion screening Among 204 individuals examined, two males with microdeletions of the Y chromosome long arm were identified (0.98%). Both patients had undergone a cytogenetic investigation prior to the Y-microdeletion screening and were cytogenetically normal. One patient had a terminal deletion in subinterval 6 of Yq11.23, which extended from marker sY153 to marker sY157 and thus included the region of the putative spermatogenesis gene DAZ. Semen parameters were: sperm count ,0.13106 spermatozoa/ml, ,10% motile spermatozoa (progression WHO type c-d), ,5% morphologically normal spermatozoa. The second patient exhibited a more unusual deletion pattern with an isolated deletion of marker Y6HP54c, which was located in the distal portion of subinterval 5 of Yq11.23 (Figure 1). In this patient the semen parameters were: sperm count 3–83106 spermatozoa/ml, 10–50% motile spermatozoa (progression WHO type c), ,10 % morphologically normal spermatozoa. A testicular biopsy was not performed in either of these patients. No microdeletions were identified in the subgroups with azoospermia or with sperm counts .103106 spermatozoa/ml.
Table II. Cytogenetic abnormalities in 158 patients with reduced semen parametersa Mean sperm count (3 106/ml)
No. cases
No. with abnormal karyotypes
Abnormal karyotypesb
Azoospermia ,1
11 24
1 2
1–5 5–10 10–20 .20c
39 40 34 10
1 1 0 1
46, 47, 46, 46, 46, – 46,
XY del (Y) XXY/ 46, XY/ 48, XXXY XY t1(1;21) t2(1;9;21) del (9) XY t (1;5) (p32; q31) XY t (4;5) (q21; p11.2) XY t (3;12) (p24; p12)
aOnly
158 of the 204 patients who were screened for Y-microdeletions were examined cytogenetically. bOne patient in each category. cPatients with sperm counts .203106 spermatozoa/ml were included in the study because of severe teratozoospermia and/or asthenozoospermia and previous in-vitro fertilization failures.
Cytogenetic evaluation A cytogenetic evaluation could be performed in 158 out of 204 patients in our study group. Constitutional chromosomal aberrations were identified in six out of 158 patients (Table II). Among 11 males with azoospermia, one patient with an isochromosome Y with two Yp arms and deletion of the terminal portion of Yq was identified (karyotype: 46, X del (Y) (q11.23→qter). With STS analysis, the deletion breakpoint could be localized in proximal Yq11.23, with all markers distal from sY86 being deleted. In this case, no family study could be performed. Two out of 24 patients with sperm counts ,13106 spermatozoa/ml were diagnosed as having a constitutional chromosomal aberration; one had Klinefelter’s syndrome (47,XXY, 46,XY, 48,XXXY, relative percentages of karyotypes: 91.8, 6.1, and 2.1%). The diagnosis was confirmed in fibroblast cultures, hair roots and oral mucous cells with the percentage of cytogenetically normal cells never exceeding 23%. The second patient carried a complex translocation involving three chromosomes (46, XY t1(1;21), t2(1; 9; 21), del (9), which had arisen de novo. One constitutional aberration each was identified in the patient groups with sperm counts of 1–5, 5–10 and .203106 spermatozoa/ml. All three cases consisted of reciprocal autosomal translocations and were familial (Table II). The third patient was included in the study because of severe teratozoospermia and previous fertilization failures in IVF. Control subjects A total of 50 normozoospermic men of proven fertility were typed according to the protocol used in the study group. No microdeletions were identified in any of the control subjects. No cytogenetic evaluations were performed in any of the control samples.
Discussion The purpose of this study was the evaluation of the relative frequencies of karyotypic abnormalities and Y-microdeletions in a population of ICSI patients, with the emphasis on 701
K.van der Ven et al.
patients with oligozoospermia. The frequency of constitutional chromosomal aberrations exceeded the incidence of microdeletions of the Y chromosome in our sample. Whereas six out of 158 (3.8%) patients showed constitutional chromosomal aberrations, only two out of 204 (0.98%) patients were diagnosed with a microdeletion of Yq11 (Table III). Of the 158 patients who underwent a combined cytogenetic and Y microdeletion screening, eight patients (5.1%) showed abnormalities, either at the cytogenetic (n 5 6) or the molecular level (n 5 2). Cytogenetic aberrations The frequencies of chromosomal aberration in our patient sample are in agreement with other studies, where an increase in chromosomal abnormalities with decreasing sperm counts has been documented. In azoospermic males, the frequency of chromosomal abnormalities has been shown to be 14.1% (Retief et al., 1984) and 15.4% (Chandley, 1979), compared with 4.1% in patients with oligozoospermia ,103106 spermatozoa/ml (Retief et al., 1984) and 1.76% in probands with sperm counts of 1–203106 spermatozoa/ml (Chandley, 1979). Our overall rate of constitutional chromosomal abnormalities among ICSI patients (3.8%) is comparable with the frequency observed in a large cohort of 1210 men with
Table III. Frequencies of constitutional chromosomal aberrations versus microdeletions of Yq11 in patients with reduced semen parameters Mean sperm count (3106/ml)
Constitutional aberrations
Y chromosome microdeletions
(n 5 158 patients) (%)
(n 5 204 patients) (%)
Azoospermia ,1 1–5 5–10 10–20 .20
1/11 2/24 1/39 1/40 0/34 1/10
(9) (8.3) (2.6) (2.5) (0) (10)
0/13 1/32 0/47 1/27 0/52 0/33
(0) (3.1) (0) (3.7) (0) (0)
Total
6/158
(3.8)
2/204
(0.98)
abnormal semen, where 3.6% of probands carried abnormal karyotypes (Pandiyan and Jequier, 1997). Y chromosome microdeletions Available data on frequencies of Y chromosome microdeletions in oligozoospermic males are still few, with the definition of the term ‘severe oligozoospermia’ underlying some additional variation (Table IV). We identified one individual with a terminal microdeletion in Yq11 among 92 individuals with sperm counts ,53106 spermatozoa/ml. Thus, the incidence of Y chromosome microdeletions, which was ~1%, is considerably lower than previously published frequencies (Tables III and IV). The patient’s deletion pattern and corresponding semen parameters were in agreement with other published cases of terminal deletions of Yq11 (Reijo et al., 1995; Najmabadi et al., 1996; Vogt et al., 1996). Among patients with sperm counts .53106 spermatozoa/ml, we identified one male with a more unusual deletion pattern, an isolated deletion of marker Y6HP54c, which is located in subinterval 5 of Yq11.23 (Figure 1). Again, data concerning frequencies of Y microdeletions in moderately oligozoospermic males are sparse. Qureshi et al. (1996) reported nine patients with sperm counts of 5–203106 spermatozoa/ml and very recently Vereb et al. (1997) reported 125 patients with oligozoospermia, all without detectable Y chromosome microdeletions. For patients with different degrees of oligozoospermia, we suggest that microdeletions of the Y chromosome might be rarer than in azoospermic males, so that screening of a larger number of patients will be necessary to obtain an accurate estimate of the deletion frequency. Microdeletions and the clinical picture The exact relationship between localization and extent of Y chromosome microdeletions and the resulting impairment of spermatogenesis is still a matter of discussion. Isolated deletions of Y chromosomal markers have been described previously, either located more distal in subinterval
Table IV. Incidence of microdeletions of the Y chromosome in males with azoospermia and severe oligozoospermia Author Kobayashi et al., 1994 Reijo et al., 1995 Reijo et al., 1996 Qureshi et al., 1996 Najmabadi et al., 1996 Vogt et al., 1996 Stuppia et al., 1996 Vereb et al., 1997
Number abnormal
Percentage
Diagnosisa
63 89 35
10 12 2
15.8 13.5 5.7
51 47 50 10 370
4 4 10 1 12
7.8 8.5 20.0 10.0 3.2
19 14 43 125
4 2 5 0
21 14.3 11.6 0
azoospermia, severe oligozoospermia azoospermia severe oligozoospermia ,1 3 106 spermatozoa/ml azoospermia oligozoospermia 0.1–20 3 106/ml azoospermia oligozoospermia ,1 3 106/ml azoospermia and oligozoospermia, ,2 3 106/ml azoospermia oligozoospermia azoospermia oligozoospermia ,0.1–20 3 106/ml
Number analysed
aIn cases where no sample sizes or sperm counts are indicated for oligozoospermia, the data are absent in the corresponding publication.
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6 of Yq11 (Qureshi et al., 1996; Stuppia et al. 1996) or with isolated deletions of a member of the YRRM family (Kobayashi et al., 1994). Those cases were associated with azoospermia or severe oligozoospermia. Neither the presence of the RBM1 genes nor the DAZ gene seems to be strictly required for completion of spermatogenesis, because cases of deletions of either gene in the presence of spermatozoa, albeit reduced in number, have been described. Reijo et al. (1996b) reported that deletions in men with oligozoospermia are as large or even larger than those observed in men with azoospermia or Sertoli-cell only syndrome. However, Vogt et al. (1996) proposed that Y chromosome microdeletions follow a certain deletion pattern with three commonly deleted non-overlapping subregions in proximal, middle and distal Yq11, designated AZF a, b, and c. In each subregion, the deletions exhibit a uniform pattern in individual patients and lead to distinct histopathological pictures related to the site of deletion. In general, proximal deletions were postulated to lead to a more severe impairment of spermatogenesis than terminal deletions. Our patient with a deletion of Y6HP54c does not meet those predictions. This individual’s deletion is located in subregion AZFb according to Vogt et al. (1996) and should result in azoospermia due to spermatocyte arrest. The patient’s sperm count was 3–83106 spermatozoa/ml, although sperm morphology and motility were severely reduced. Qureshi et al. (1996) reported another patient with a deletion of four consecutive STS in AZFa but diminished spermatogenesis (sperm count 4.33106 spermatozoa/ml) instead of azoospermia. Those obvious discrepancies raise the question as to how microdeletions of the Y chromosome can be pathognomonic for azoospermia or oligozoospermia in humans. In that context, it must be clarified whether the microdeletion itself or the loss of putative spermatogenesis genes subsequent to a microdeletion causes impairment of germ cell formation. Candidate genes for human spermatogenesis Of the potential spermatogenesis loci which have been cloned on the long arm of the Y chromosome, the potential site of the azoospermia factor (AZF), the DAZ gene (Deleted in Azoospermia) has received much attention. DAZ was first reported to be a single copy gene, as opposed to the loci TSPY and RBM1, and a high proportion of azoospermic males have microdeletions that include DAZ (Reijo et al., 1995, 1996a). However, an autosomal homologue of the human DAZ gene, called DAZH, has been cloned on chromosome 3. Evidently DAZ, which has in the meantime been shown to be a cluster of genes on Yq11, arose from transposition and repeated amplification of the autosomal DAZH (Saxena et al., 1996). Moreover, the DAZ gene is autosomal in all other mammals except the primates (Cooke et al., 1996; Reijo et al., 1996b). The high sequence homology of the autosomal DAZ across species, as opposed to the obvious degenerative forces being active on the Y chromosomal DAZ challenges the theory of a special function of the Y chromosomal DAZ in human spermatogenesis. Indeed, direct sequencing of the 59 end of the DAZ gene in 30 non-deleted azoospermic males failed to detect mutations in any of the patients, which would have
been formal proof that DAZ is AZF (Vereb et al., 1997). In contrast, transcription of DAZ-like sequences was detected in spermatogonia (Menke et al., 1997), although no distinction between DAZ and DAZH could be made in those studies. In summary, the question as to whether the Y chromosomal or the autosomal DAZ plays a major role in human spermatogenesis remains unresolved. The TSPY gene (testis specific protein on the Y) is expressed in spermatogonia and primary spermatocytes in adults, similar to DAZ. Between 30 and 60 copies of TSPY are present on the Y chromosome, some of which are interspersed with RBM sequences in Yq11.23. Thus, microdeletions of Yq11 might also interfere with members of the TSPY gene family, which should be considered additional candidates for AZF (Delbridge et al., 1997). The RBM1 gene family (RNA Binding Motif, formerly YRRM1), originally cloned by Ma et al. (1993) comprises several gene copies clustered in Yq11.23, similarly to TSPY and DAZ. In addition to being expressed in the adult testis, RBM1 may have a role in germ cell development. As opposed to DAZ and TSPY, RBM1 is the only human candidate spermatogenesis gene that has a Y-linked homologue in marsupials which is transcribed in the testis (Delbridge et al., 1997). The conserved Y-location of RBM1 argues strongly for a critical male specific function of this gene, because the marsupial Y represents a minimal Y chromosome containing only essential male specific genes. However, similar to DAZ, RBM1 and TSPY seem to have autosomal relatives (Delbridge et al., 1997). At present, it cannot be decided which, if any, of the candidate genes located on Yq11 has a key role in human spermatogenesis. The fact that each gene family has multiple members within the region thought to be the site of AZF might explain the discrepancies between site and extension of microdeletions in Yq11 and semen parameters or histological findings, respectively. Partial or complete redundancy of the function of autosomal and Y chromosomal loci might add to the problem. The potential existence of germline mosaicism in infertile males could be of importance with regard to the accuracy of a PCR screening approach for microdeletions of Yq11. Mosaicism involving an intact and a deleted Y has been proposed for fathers of ICSI-derived sons, where the son had microdeletions in the AZF-region while the father appeared normal (KentFirst et al., 1996). Germline mosaicism for microdeletions of Yq could also be the reason for some cases of reduced sperm counts, but would not be detectable by a PCR-based detection system. Finally, it should be mentioned that microdeletions of the long arm of the Y chromosome could theoretically inhibit meiosis per se, rather than through the loss of spermatogenesis loci. The fact that the large number of karyotypic abnormalities, which can also interfere with regular meiosis, exceeded that of Y-microdeletions in our study may support this. Based on the relative frequencies of chromosomal abnormalities and microdeletions of the Y chromosome in our patient sample and given the current knowledge about the role of genes on Yq in spermatogenesis, we conclude that it might be premature to recommend a general Y-microdeletion screening of all males considering ICSI. 703
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The identification of the essential genes involved in spermatogenesis, whether located on autosomes or the Y chromosome, will help to find appropriate measures to determine the genetic risk for offspring of patients undergoing ICSI treatment.
Acknowledgements We wish to thank Ms G.Engels for excellent technical assistance.
References Arnemann, J., Jakubiczka, S., Thuring, S. et al. (1991) Cloning and sequence analysis of a human Y-chromosome derived, testicular cDNA, TSPY. Genomics, 11, 108–114. Chandley, A.C. (1979) The chromosomal basis of human infertility. Br. Med. Bull., 35, 181–185. Cooke, H.J., Lee, M.L., Kerr, S. et al. (1996) A murine homologue of the human DAZ gene is autosomal and expressed only in male and female gonads. Hum. Mol. Genet., 5, 513–516. De Braekeleer, M. and Dao, T.M. (1991) Cytogenetic studies in male infertility: a review. Hum. Reprod., 6, 245–250. Delbridge, M.L., Harry, J.L.,Toder, R. et al. (1997) A human candidate spermatogenesis gene, RBM1, is conserved and amplified on the marsupial Y chromosome. Nature Genet., 15, 131–136. Foote, S., Vollrath, D., Hilton, A. et al. (1992) The human Y chromosome: overlapping DNA clones spanning the euchromatic region. Science, 258, 60–66. Henegariu, O., Hirschmann, P., Kilian, K. et al. (1994) Rapid screening of the Y chromosome in idiopathic sterile men, diagnostic for deletions in AZF, a genetic Y factor expressed during spermatogenesis. Andrologia, 26, 97–106. Kent-First, M.G., Kol, S., Muallem, A. et al. (1996) The incidence and possible relevance of Y-linked microdeletions in babies born after intracytoplasmic sperm injection and their infertile fathers. Mol. Hum. Reprod., 2, 943–950. Kobayashi, K., Mizuno, K., Hida, A. et al. (1994) PCR analysis of the Ychromosome long arm in azoospermic patients: evidence for a second locus required for spermatogenesis. Hum. Mol. Genet., 3, 1965–1967. Leygraaf, J., Peschka, B., van der Ven, K. et al. (1977) Die Ha¨ufigkeit chromosomaler Auffa¨lligkeiten bei Ehepaaren vor Behandlung durch intrazytoplasma tische Spermieninjektion (ICSI). Ferlilita`t, in press. Ma, K., Inglis, J.D., Sharkey, A. et al. (1993) A Y-chromosome gene family with RNA-binding protein homology: candidates for the azoospermia factor AZF controlling human spermatogenesis. Cell, 75, 1287–1295. Menke, D.B., Mutter, G.L. and Page, D.C. (1997) Expression of DAZ, an Azoospermia Factor candidate in human spermatogonia. Am. J. Hum. Genet., 60, 237–241. Miller, S.A., Dykes, D.D. and Polesky, H.F. (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res., 16, 1215–1218. Najmabadi, H., Huang, V., Yen, P. et al. (1996) Substantial prevalence of microdeletions of the Y-chromosome in infertile men with idiopathic azoospermia and oligozoospermia detected using a sequence-tagged-sitebased mapping strategy. J. Clin. Endocrinol. Metab., 81, 1347–1352. Pandiyan, N. and Jequier, A.M. (1996) Mitotic chromosomal anomalies among 1210 infertile men. Hum. Reprod., 11, 2604–2608. Qureshi, S.J., Ross, A.R., Ma, K. et al. (1996) Polymerase chain reaction screening for Y chromosome microdeletions: a first step towards trhe diagnosis of genetically-determined spermatogenic failure in men. Mol. Hum. Reprod., 2, 775–779. Reijo, R., Alagappan, R.K., Patrizio, P. and Page, D.C. (1996a) Severe oligozoospermia resulting from deletions of azoospermia factor gene on Ychromosome. Lancet, 347, 1290–1293. Reijo, R., Seligman, J, Dinulos, M.B. et al. (1996b) Mouse autosomal homolog of DAZ, a candidate male sterility gene in humans, is expressed in male germ cells before and after puberty. Genomics, 35, 346–352. Reijo, R., Lee, T.-Y., Salo, P. et al. (1995) Diverse spermatogenic defects in humans caused by Y-chromosome deletions encompassing a novel RNAbinding protein gene. Nature Genet., 10, 383–393.
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Retief, A.E., Van Zyl, J.A., Menkveld, R. et al. (1984) Chromosome studies in 496 infertile males with a sperm count below 10 million/ml. Hum. Genet., 66, 162–164. Saxena, R., Brown, L.M., Hawkins, T.B. et al. (1996) The DAZ gene cluster on the human Y-chromosome arose from an autosomal gene that was transposed, repeatedly amplified and pruned. Nature Genet., 14, 292–299. Stuppia, L., Mastroprimiano, G., Calabrese, G. et al. (1996) Microdeletions in interval 6 of the Y-chromosome detected by STS–PCR in 6 of 33 patients with idiopathic oligo- or azoospermia. Cytogenet. Cell Genet., 72, 155–158. Testart, J., Gautier, E., Brami, C. et al. (1996) Intracytoplasmic sperm injection in infertile patients with structural chromosome abnormalities. Hum. Reprod., 11, 2609–2612. Vereb, M., Agulnik, A.I., Houston, J.T. et al. (1997) Absence of DAZ gene mutations in cases of non-obstructed azoospermia. Mol. Hum. Reprod., 3, 55–59. Vogt, P.H., Edelmann, A., Kirsch, S. et al. (1996) Human Y-chromosome azoospermia factors (AZF) mapped to different subregions in Yq11. Hum. Mol. Genet., 5, 933–943. Received on February 27, 1997; accepted on May 27, 1997