Screening for microdeletions of Y chromosome genes in patients undergoing intracytoplasmic sperm injection

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1 Human Reproduction vol.14 no.7 pp , 1999 Screening for microdeletions of Y chromosome genes in patients undergoing intracytoplasmic sperm injection C.Krausz 1,3,4, C.Bussani-Mastellone 2, S.Granchi 1, K.McElreavey 3, G.Scarselli 2 and G.Forti 1 1 Andrology Unit, Department of Clinical Physiopathology, 2 Department of Obstetric and Gynecology, University of Florence, Firenze, Italy and 3 Immunogenetique Humaine, Institut Pasteur, Paris, France 4 To whom correspondence should be addressed at: Immunogenetique Humain, Institut Pasteur, 25, Rue du Docteur Roux, Paris, Cedex 15, France The potential of assisted reproduction techniques to transmit genetic defects causing male infertility raises questions concerning the need for a systematic genetic screen and counselling. Deletions of the long arm of the Y chromosome are frequently associated with a failure of spermatogenesis. The search for Y specific sequences and for the gene families RNA binding motif (RBM) and deleted in azoospermia (DAZ) have been introduced in many laboratories. The incidence of Y microdeletions varies widely between studies, from 1 55%. These differences are mainly related to study design. The highest incidence of microdeletions has been reported in well selected idiopathic azoospermic patients. Since microdeletions have been reported also in nonidiopathic patients, it is important to define what is the deletion frequency in unselected patients. We report Y chromosome microdeletion screening in 134 unselected patients undergoing intracytoplasmic sperm injection (ICSI). In the first part of the study we tested six Y chromosome markers. We found three patients with microdeletions (2.2%). Subdivision of the study population revealed a deletion incidence of 4.7% in azoospermic/ cryptozoospermic patients; an incidence of 7% in idiopathic patients and an incidence of 16% in idiopathic azoospermic/ cryptozoospermic patients. The second part of the study consisted of a screen for the presence of the Y chromosome genes, DBY, CDY, XKRY, eif-1a, DAZ and BPY2. No additional gene-specific deletions were found. Further data on gene specific screening are needed especially for selected idiopathic patients. Key words: infertility/icsi/screening/y chromosome Introduction Male factor infertility accounts for about half the cases of couple infertility. Although the aetiology of reduced testicular function remains unknown in the majority of cases, assisted reproduction techniques such as in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) can apparently overcome the need for an aetiological definition. However, the potential of these techniques to transmit genetic defects causing male infertility raises questions concerning the need for a systematic genetic screen and relevant genetic counselling of affected patients. Deletions of the long arm of the Y chromosome are frequently associated with a failure of spermatogenesis (Simoni et al., 1998). Three non-overlapping regions of the Y chromosome have been defined in patients with severe oligozoospermia or azoospermia (Vogt et al., 1996). These regions have been termed azoospermia factor (AZF) a, b and c. In each region, candidate genes have been proposed (DFFRY for AZFa; RBM for AZFb and DAZ for AZFc) (Ma et al., 1993; Reijo et al., 1995; Chai et al., 1997; Brown et al., 1998) and are widely included as part of Y chromosome microdeletion screens. Other genes and gene families have been identified on the long arm of Y chromosome, some of which are located within AZF deletion intervals (Lahn and Page, 1998). Absence of one or more of these genes could be a cause of infertility. Since 1994 several combined clinical and molecular studies have been performed in order to determine the incidence of microdeletions and attempt to correlate the type of deletion with the infertile phenotype (Reijo et al., 1995, 1996; Vogt et al., 1996; Quereshi et al., 1996; Stuppia et al. 1996, 1998; Najmabadi et al., 1996; Nakahori et al., 1996; Pryor et al., 1997; Foresta et al., 1997, 1998; Vereb et al., 1997; Kremer et al., 1997; Mulhall et al., 1997; Simoni et al., 1997; Van der Ven et al., 1997; Girardi et al., 1997; Selva et al., 1997; Oliva et al., 1998). However, the incidence of Y microdeletions varies widely between studies, from 1% (Van der Ven et al., 1997) to 55% (Foresta et al., 1998). Differences in deletion frequency and, in some studies the position of the deletions, are probably related to study design but they may also reflect genuine population variances or environmental influences. The majority of the studies have included patients presenting idiopathic azoo- or oligozoospermia (Reijo et al., 1995, 1996; Vogt et al., 1996; Quereshi et al., 1996; Stuppia et al. 1996, 1998; Najmabadi et al., 1996; Nakahori et al., 1996; Vereb et al., 1997; Mulhall et al., 1997; Simoni et al., 1997; Van der Ven et al., 1997; Girardi et al., 1997; Selva et al., 1997; Foresta et al., 1998) and there are only limited data available on unselected patients with or without abnormal andrological findings (Kremer et al., 1997; Pryor et al., 1997; Oliva et al., 1998). Since microdeletions are present in some non-idiopathic patients (Pryor et al., 1997; Kremer et al., 1997) it is important to define the deletion frequency in unselected patients undergoing assisted reproduction techniques. The purpose of the current study was to define the position, extent and frequency of Y microdeletions in a group of European Society of Human Reproduction and Embryology 1717

2 C.Krausz et al. Table I. Microdeletion frequency in the 134 patients. Subclassification on the basis of semen parameters Number of Classification Sperm count Deletion patients (10 6 /ml) frequency (%) 22 Azoospermia none 1/22 (4.5) 42 Cryptozoospermia 1 2/42 (4.7) 26 Severe oligozoospermia 1 5 0/26 27 Oligozoospermia /27 13 Pure asthenozoospermia 20 0/13 4 Normozoospermia 20 0/4 unselected patients of Italian origin undergoing an ICSI programme using a series of anonymous sequence-tagged site (STS) markers, and secondly to screen a subgroup of this population (affected by idiopathic and non-idiopathic azooand severe oligozoospermia) for deletions of specific Y chromosome gene or gene families, such as dead box Y (DBY), chromodomain Y (CDY), XK related Y (XKRY), eukaryotic translation initiation factor 1AY (eif1ay), basic protein Y2 (BPY2) and deleted in azoospermia (DAZ). The latter approach was adopted to determine if the deletion frequency increased using gene-specific markers. Materials and methods Patients A total of 134 unselected, consecutive patients undergoing ICSI treatment (in 1 year) were analysed, without any exclusion criteria. The indications for ICSI and micro epididymal surgical aspiration (MESA)/ICSI or testicular sperm aspiration (TESA)/ICSI in our centre are azoospermia (in combination with surgical retrieval), oligozoospermia or asthenozoospermia with of progressively motile spermatozoa after swim-up or gradient selection, and pregnancy failure in three previous IVF treatments. On the basis of at least two semen analyses the patients were classified as azoospermic (absence of spermatozoa in the ejaculate), cryptozoospermic ( spermatozoa/ml), oligozoospermic ( spermatozoa/ml), pure asthenozoospermic ( spermatozoa/ ml with motility a b 50% according to the WHO manual) (WHO Manual, 1992) and normozoospermic ( spermatozoa/ml with motility a b 50%) patients. The patients cohort consisted of 22 azoospermic, 42 cryptozoospermic, 53 oligozoospermic and four normozoospermic/13 asthenozoospermic patients. A subclassification of the oligozoospermic patients is given in Table I. Each man was questioned about his medical and surgical history and underwent a thorough andrological examination. Karyotype, serum follicle-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone levels were measured for all patients while prolactin (PRL) was determined only in selected cases. In most of the patients ultrasonography of testes was performed. Bacteriological examination of the seminal fluid, seminal markers of duct patency (alpha glucosidase, fructose) and screening for mutations in the cystic fibrosis gene (CFTR) was performed in patients suspected to have urogenital tract infection or obstructive azoospermia respectively. Bilateral testicular biopsy or fine needle sperm aspiration in order to cryopreserve retrieved testicular or epididymal spermatozoa are ongoing procedures for azoospermic and cryptozoospermic patients. Patients who had Y microdeletions were counselled and the father 1718 and/or brother of the men were asked to give blood for DNA analysis in order to determine if the deletion was de novo. Molecular analysis of the Y chromosome Genomic DNA was obtained from peripheral blood leukocytes. Routine polymerase chain reaction (PCR) amplification of genomic DNA was performed. STS screening for Y microdeletions Each man was analysed for the presence of six STS spanning the three AZF regions: sy84 (AZFa), sy134, sy131 (AZFb), sy152, sy157, sy158 (AZFc). In a subgroup of patients [affected by azoospermia and severe oligozoospermia ( spermatozoa/ml)] an additional screening for the genes DBY, CDY, XKRY, eif1ay, BPY2 and DAZ (sy254, sy255) was performed. Oligonucleotide primer sequences for each of the genes are presented in Table II. Female genomic DNA was used as a negative control. A patient sample was considered positive for the given STS/gene when the PCR product of the expected size was present and was considered negative if a product of the expected size was not obtained after three PCR attempts. In all men amplification of the sex-determining gene, SRY, was used as a positive control. Patients that had Yq deletions were also analysed with the STS markers SY114, sy116, sy125, sy 129, sy139 in order to further define the extent of the deletions. A group of 10 fertile men with normal sperm parameters were tested with all the markers. Southern blotting was performed in order to confirm the presence of Yq deletions. A total of 10 µg of genomic DNA was digested with EcoRI and TaqI, run on a 0.7% agarose gel, transferred to a nylon membrane, and hybridized with 32 P-labelled probes. Results Primary Y chromosome screen A total of 134 men were screened for the presence of submicroscopic Y chromosome deletions. Three patients (2.2%) were found deleted following the routine screening programme for six STS markers. All three patients (A1, A21, A50) were deleted for the AZFc regions, while patient A21 presented a contiguous deletion in AZFb and patient A50 an additional deletion in AZFa and proximal AZFb (Table III). In order to define the break points of these deletions a complementary set of primers was used. Deletions in patients A50 and A21 were confirmed by Southern blot analysis using the DAZ gene as a probe (data not shown). Analysis of the father and normozoospermic brother of A1 and the father of A21 were tested for Y markers, all of which were present. All markers/genes were present in a control group of 10 fertile men. Secondary Yq gene-specific screen Screening for the presence of the Y chromosome genes, DBY, CDY, XKRY, eif1ay, DAZ and BPY2, was carried out in the 90 patients who presented with either azoospermia, cryptozoospermia or severe oligozoospermia. Lack of amplification with the gene specific primers was observed in the three patients, A1, A21, A50, described above. All other individuals were positive for these genes by PCR amplification. Patient A1 was deleted for the markers sy152 sy158 (AZFc) including the DAZ and BPY2 gene families (Figure 1). Patient A21 was deleted for the region defined by the markers sy125 sy158

3 Microdeletions of Y chromosome in ICSI patients Table II. Oligonucleotide sequences for the detection of the genes DBY, CDY, XKRY, eif-1ay and BPY2 Gene name Left primers Right primers Size (bp) DBY AGT TTA TTC TAA CCT AGG CAA ACG TCC AAC CAG GCC TGT AGT GAG GCC 164 CDY TCA TAC AAT CCA ATT GTA CTG G TTC TAT CCC TCG GGC TGA GCT C 132 XKRY GTT GTG GAC TCA ATA ATT GCC TCT GGA TGA TTT TCA GTA TCT CC 136 eif-1ay GCA AAC TGA TTT ATT TTC ATT GTT T CAG CAA ATA TTA TGG TCT TTT ATC C 138 BPY2 CAG CGT ATC ATA GAA AAT GT AGT ACT TTA TTT GCA GGT TCT G 142 Table III. Summary of DNA analysis of the three patients with Y microdeletions: (a) STS PCR data; (b) gene-specific screen data Patient sy84 DBY CDY XKRY sy114 sy116 sy125 eif-1ay sy129 sy131 sy134 sy139 sy152 DAZ BPY2 sy158 A1 A21 A50 PCR product is present; PCR product is not detected. (AZFb and AZFc) including the genes eif1a, DAZ and BPY2. Patient A50 was deleted for the markers sy84 sy125 and sy139 sy158. These two deletions included the genes DBY, CDY, XKRY, eif1ay, BPY2 and DAZ (Figure 1; Table III). Microdeletions versus semen analysis Table I describes the deletion frequency for each category of semen profile. Deletions were found in patients presenting with cryptozoospermia 2/42 (4.7%) and azoospermia 1/22 (4.5%) while no deletions were found in oligo- or normozoospermic individuals. Microdeletions versus aetiology The results of the andrological history and physical examination are summarized in Table IV. Abnormal andrological findings were detected in 102 patients. Abnormal chromosome complements were found in six patients, two Robertsonian translocations, one Klinefelter syndrome and one patient with 46,XY(nf), one with 46,X,inv(Y)(q11.23), one patient with chromosomal mosaicism 45,X/46,X,idic(Y)(q11.2)/47,X,idic(Ynf)(q11.2)/ 47,X,idic (Ynf)(q11.2) 21 where nf non-fluorescent and was due to the deletion of heterochromatin. The two patients with Robertsonian translocations had a mild infertile phenotype (pure asthenozoospermia) with associated abnormal andrological findings (recurrent urogenital infections). A total of 28 patients with no andrological and cytogenetical abnormalities were defined as idiopathic. In this study, deletions were not detected in patients with abnormal andrological findings while one patient with abnormal chromosome complement (mosaicism) was found to be deleted. The incidence of Y chromosome microdeletions in men defined as having idiopathic infertility in this study is 7% (2/28). Two patients with Y chromosome deletions are included in the group of azoo-/ cryptozoospermic men with no abnormal finding at andrological and cytogenetic examination. The incidence of Y microdeletion for this combined group is 2/12 (16%), indicating that patients presenting idiopathic azoo- or cryptozoospermia are at a high risk for this genetic anomaly. Figure 1. Representative PCR analysis for patient A1 and A50. (A) Amplification of DBY, CDY, XKRY. (B) Amplification of eif-1ay, DAZ, BPY2. Lane 1: negative control; lane 2: female control; lane 3: male control; lane 4: patient A1; lane 5: patient A50. Molecular weight marker (100 bp ladder) is shown. Discussion The development of an STS-based mapping strategy (Vollrath et al., 1992) has permitted the rapid screening of a large number of infertile patients for Y chromosome microdeletions (Reijo et al., 1995, 1996; Vogt et al., 1996; Quereshi et al., 1996; Stuppia et al., 1996, 1998; Najmabadi et al., 1996; Nakahori et al., 1996; Vereb et al., 1997; Mulhall et al., 1997; Simoni et al., 1997; Van der Ven et al., 1997; Girardi et al., 1997; Selva et al., 1997; Foresta et al., 1998; Oliva et al., 1998). Several studies have been published on this subject but it is still under question which category of men should be routinely tested. The majority of microdeletions described in the literature has been found in idiopathic azoo- or severe oligozoospermic patients (Reijo et al., 1995, 1996; Vogt et al., 1719

4 C.Krausz et al. Table IV. Summary of findings during andrological history, examination and cytogenetic analysis in 134 men undergoing ICSI procedure Varicocele Cryptorchidism Infection Obstructive Inguinal Endocrine Systemic Karyotype Miscellaneous a No azoospermia surgery disease disease abnormality findings a Including testicular trauma (3), toxic agents (1), immunological (1), lift testis (4), familiar infertility (2), radio/chemotherapy (3). 1996; Quereshi et al., 1996; Stuppia et al. 1996, 1998; Najmabadi et al., 1996; Nakahori et al., 1996; Vereb et al., 1997; Mulhall et al., 1997; Simoni et al., 1997; Van der Ven et al., 1997; Girardi et al., 1997; Selva et al., 1997; Foresta et al., 1998; Oliva et al., 1998). It is important to note that the majority of the studies used as inclusion criteria idiopathic azoospermia and/or severe oligozoospermia (Vogt et al., 1996; Stuppia et al., 1996; Reijo et al., 1996; Najmabadi et al., 1996; Nakahori et al., 1996; Foresta et al., 1997; Vereb et al., 1997; Simoni et al., 1997). There are relatively few published studies of patients presenting mild forms of oligozoospermia (Quereshi et al., 1996; Mulhall et al., 1997; Van der Ven et al., 1997) and non-idiopathic azoo- or oligozoospermia (Pryor et al., 1997; Kremer et al., 1997; Oliva et al., 1998). In order to address these clinical questions we studied 134 consecutive, unselected infertile patients with semen profiles ranging from azoospermia to normozoospermia. To date, Y screening in consecutive ICSI patients has been performed in three countries, the USA (Pryor et al., 1997), the Netherlands (Kremer et al., 1997) and in Spain (Oliva et al., 1998). Surprisingly, in our study a high proportion of patients (80%) presented abnormal andrological findings, which is apparently in contrast with the general prevalence of idiopathic infertility among infertile male patients (50 70%). This difference may be related to the extent of the medical work-up. On the other hand, for many of the abnormal andrological findings it is difficult to establish a direct cause effect relationship, i.e. their actual contribution to impaired spermatogenesis. Abnormal findings such as varicocele, recurrent urogenital infections or monolateral cryptorchidism show considerable variability of spermatogenic impairment therefore they have been defined as idiopathic by some authors. The overall incidence of Y microdeletions was 2.2%. This figure accords with other studies on unselected patients. Further analysis of the data indicated a subgroup of patients who have a significantly higher risk of Y deletions (16%). However some caution is recommended in interpreting these data as the study group is relatively small. This subgroup of patients are idiopathic azoo-/cryptozoospermic individuals. No deletions were found in severe oligozoospermic patients which may be due to the high prevalence of non-idiopathic patients in our study population. The shift in deletion incidence from 2.2% in the unselected population to 16% in a defined subgroup indicates that patient selection criteria plays a fundamental role in the determination of deletion frequency. The position of deletions and the associated phenotypes of the three deleted patients follow the general genotype/phenotype tendencies reported in the literature. Patient A50 has a double deletion in AZFa and AZFc regions and has a more severe phenotype (azoospermia) than patients A1 and A21 (both cryptozoospermic), who have deletions in 1720 AZFc and AZFc b respectively. AZFa deletions have rarely been found in the literature, most of the cases have been described by another Italian group in patients with Sertoli cell-only syndrome (SCOS) type I. Unfortunately testicular histology was not available for patient A50 therefore a precise genotype/phenotype correlation is not possible. Another unanswered clinical question is how many and which loci should be included in routine Y chromosome screening programmes. It has been reported that the number of STSs included in a screen does not significantly influence the frequency of Y microdeletions (Simoni et al., 1998). While a high number of STS can protect against inaccuracy, it can also lead to the detection of clinically irrelevant polymorphic variants (Simoni et al., 1998). Therefore the selection of STS markers or gene(s) to be screened is important to increase the sensitivity and the specificity of the analysis. In an attempt to resolve this question we used gene-specific markers which included in the AZFa region DBY, in the AZFb region CDY, XKRY and eif1ay and in the AZFc region BPY2 and DAZ. The precise biological functions of these genes are not clear, however their localization on the Y chromosome and, for some of them, testis-specific expression (DAZ, CDY, XKRY and BPY2) and/or involvement in RNA metabolism (DAZ, DBY and eif1ay) suggest a potential role in spermatogenesis. A subgroup of 90 patients affected by azoo-, crypto- and severe oligozoospermia was screened for microdeletions of these genes. This group of patients is known to be at higher risk for Y chromosome microdeletions than moderate oligo- or normozoospermic infertile men (Simoni et al., 1998). Apart from the three patients in whom the Y deletion was already determined by STS markers, no gene-specific deletions were detected. This result could be explained by the relatively low number of men with idiopathic infertility included in the current study, or by the fact that some of these genes are present in multicopies on the Y chromosome; therefore it is possible that a critical copy of these gene families could be missing even though a product is amplified. Alternatively, it is possible that gene-specific deletions are rare events and only large Y deletions, removing several genes, are associated with male infertility. Although in our study we found deletions only in a subgroup of patients, the pre-icsi screening of all our male patients will continue. Increasing the number of idiopathic patients should provide in the future a more accurate evaluation of the efficiency of a gene-based screening. Acknowledgements The authors thank the medical staff of the Andrology Unit (Drs M.Maggi, A.Magini and M.Mancini) and of the Reproductive Physi-

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