Molecular Human Reproduction, Vol. 7, No. 10, 987-994,
October 2001
© 2001 European Society of Human Reproduction and Embryology
Reproductive genetics |
High deletion frequency of the complete AZFa sequence in men with Sertoli-cell-only syndrome
1 Reproduction Genetics, Institute of Human Genetics, University of Heidelberg, Germany, 2 Department of Human Genetics, Faculty of Medicine, University of Porto, 3 Institute of Biomedical Sciences Abel Salazar, University of Porto, Portugal, 4 Department of Urology, New York Presbyterian Hospital–Cornell & The Population Council, New York, USA, 5 Institute for the Study of Fertility, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel, 6 Department of Andrology, University of Marburg, 7 Department of Obstetrics and Gynecology, Medical University of Lübeck, 8 Institute of Pathology, Medical University of Lübeck, 9 Department of Andrology, UKE, Hamburg and 10 Clinic of Urology, University of Giessen, Germany 11 To whom correspondence should be addressed at: Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany. E-mail: peter_vogt{at}med.uni-heidelberg.de
Abstract
We have developed a rapid screening protocol for deletion analysis of the complete AZFa sequence (i.e. 792 kb) on the Y chromosome of patients with idiopathic Sertoli-cell-only (SCO) syndrome. This Y deletion was mapped earlier in proximal Yq11 and first found in the Y chromosome of the SCO patient JOLAR, now designated as the AZFa reference patient. We now show that similar AZFa deletions occur with a frequency of 9% in the SCO patient group. In two multiplex polymerase chain reaction experiments, deletions of the complete AZFa sequence were identified by a typical deletion pattern of four new sequence-tagged sites (STS): AZFa-prox1, positive; AZFa-prox2, negative; AZFa-dist1, negative; AZFa-dist2, positive. The STS were established in the proximal and distal neighbourhoods of the two retroviral sequence blocks (HERV15yq1 and HERV15yq2) which encompass the break-point sites for AZFa deletions of the human Y chromosome. We have found deletions of the complete AZFa sequence always associated with a uniform SCO pattern on testicular biopsies. Patients with other testicular histologies as described in the literature and in this paper have only partial AZFa deletions. The current AZFa screening protocols can therefore be improved by analysing the extension of AZFa deletions. This may provide a valuable prognostic tool for infertility clinics performing testicular sperm extraction, as it would enable the exclusion of AZFa patients with a complete SCO syndrome.
AZFa deletions/AZFa STS multiplex PCR/idiopathic male infertility/human Y chromosome/SCO syndrome
Introduction
Approximately 15% of the men seeking help at infertility clinics suffer from idiopathic azoospermia, i.e. the absence of mature sperm cells (spermatozoa) in seminal fluid. In 5–20% of these patients, the molecular analysis of their Y chromosome revealed a de-novo microdeletion of at least one of the three azoospermia factor regions, AZFa, AZFb and AZFc (Vogt et al., 1996
; Girardi et al., 1997; Vogt, 1998; Kleiman et al., 1999; Krausz et al., 1999). In many infertility clinics, this has led to a broad interest in the molecular analysis of Y chromosomal microdeletions in men with idiopathic azoospermia or severe oligozoospermia and numerous reports have now confirmed the presence of the three AZF loci in Yq11.
However, there is no consensus on the number of sequence-tagged site (STS) loci which have to be analysed in order to identify the detected AZF deletion as a causative agent of the observed male infertility phenotype and many infertility clinics have restricted their AZF deletion analyses to only one or two STS loci in each AZF region to reduce the time and costs involved with the extensive screening programme originally proposed (Vogt et al., 1996). Indeed, the detection of a deletion at only one of the non-polymorphic STS loci mapped to any of the three AZF loci would be sufficient to diagnose an AZF deletion if the molecular extension of the deletion is identical for all patients, as reported for the reference patients published by Vogt et al. (1996). However, deletions may have variable extents in different patients, so single STS analysis is not an adequate evaluation for deletion detection of an AZF region.
Partial AZF deletions have been identified repeatedly in different infertility clinics [e.g., for AZFa: (Ferlin et al., 1999); for AZFb: (Krausz et al., 2000); for AZFc: (Stuppia et al., 1997)] and were not always associated with the testicular pathologies proposed by Vogt et al. (1996) to occur with the AZFa, AZFb, and AZFc deletions, but were represented by variable testicular pathologies. Therefore, AZF screening programmes based on the analysis of single STS loci within an AZF region have only limited clinical value, unless the extent of the observed AZF deletion is analysed in further polymerase chain reaction (PCR) experiments.
Since the complete human proximal Yq11 region has now been sequenced (GenBank accession no. NT_011875), the sequence of a complete AZFa deletion, as defined by the molecular extension of the Y deletion of the AZFa reference patient JOLAR (Vogt et al., 1996), can now be estimated. Both borders of the AZFa deletion for JOLAR were recently mapped to a long identical sequence domain of two recombined retroviral sequence blocks (HERV15yq1 and HERV15yq2) present at a distance of 781.557 kb in proximal Yq11 (Kamp et al., 2000). Similar results have been found for eight further Sertoli-cell-only (SCO) patients with a complete AZFa deletion (Blanco et al., 2000; Kamp et al., 2000; Sun et al., 2000). Therefore, complete AZFa deletions can now be defined as the result of a homologous intrachromosomal recombination event between two HERV15 sequence blocks located in proximal Yq11, intervals D3 and D6 respectively.
Based on this knowledge, we developed a novel AZFa screening protocol based on a series of new STS markers (AZFa-prox1, AZFa-prox2, AZFa-dist1, AZFa-dist2) established around both HERV15 sequence blocks. Analysis of the sY83 and 12f2 STS loci can be included to map the site of the HERV15yq1/HERV15yq2 recombination event (ID1 or ID2). We combined these STS in two PCR multiplex mixes: AZFa-prox and AZFa-dist, and tested this protocol on the Y chromosomes of 100 patients with a histologically proven SCO syndrome. Complete AZFa deletions were found with a frequency of 9% in this SCO patient group.
Materials and methods
Selection of SCO patients
The 100 individuals in our SCO patient group were evaluated clinically and found to have non-obstructive (secretory) azoospermia without evidence of testicular tumours or other abnormalities. A histological diagnosis of complete germ cell aplasia (SCO syndrome) was made after careful examination of bilateral testis biopsies showed that all seminiferous tubules from both testes had no germ cells present. In this context, it is important to note that the complete absence of germ cells was diagnosed by a single biopsy from each testis. Although most of our patients were then expected to have a complete SCO syndrome throughout their testes, the presence of isolated tubuli with germ cells could not be fully excluded. The karyotype in all SCO patients was found to be normal, 46,XY.
Genomic DNA isolation from blood samples and testicular testis biopsies
Genomic DNA was extracted from blood samples (79 patients) collected in EDTA tubes by a published protocol (Vogt et al., 1996) and from frozen/thawed tissue samples of testicular biopsies (21 patients) collected in SpermFreeze medium (FertiPro N.V. cat. no. 3080) using the protocol described in the Ambion kit for simultaneous extraction of RNA and DNA from tissue samples (Technical Bulletin 161; Ambion, Austin, TX, USA). Briefly, 50–100 mg of testicular tissue (normal weight of biopsy samples) was placed in a volume of 500 µl denaturation solution and disrupted by an Ultra Turrax T8 tissue homogenizer using the dispersing tool S8N-5G for mini-volumes in a 2 ml plastic tube. DNA collected in the organic phases was extracted with an equal volume of extraction buffer (0.1 mol/l NaCl, 10 mmol/l Tris–HCl pH 8, 1 mmol/l EDTA, 1% SDS) after the pH value was adjusted to pH 7–8 with 5 N NaOH. The DNA samples, now in the extraction buffer, were precipitated by the addition of 1/15 vol. 7.5 mol/l ammonium acetate and 2 vol. of ice-cold ethanol. DNA pellets were rinsed with 70% ethanol and dissolved in 40 µl of TE buffer (10 mmol/l Tris–HCl, pH 8, 0.1 mmol/l EDTA) by a short heat pulse (5 min at 55°C). For each AZFa multiplex PCR experiment, a 2 µl aliqout of this DNA solution was used.
Multiplex PCR assay for complete AZFa deletions
A series of new Y-specific STS was established to analyse the proximal (AZFa-prox1 and AZFa-prox2) and the distal (AZFa-dist1 and AZFa-dist2) genomic Y regions present and absent in patients with AZFa deletions (Figure 1). AZFa-prox1 and AZFa-prox2 flank the HERV15yq1 sequence block in Yq11 interval D3 recently identified to be the proximal borderline of complete AZFa deletions (Kamp et al., 2000); AZFa-dist1 and AZFa-dist2 flank the HERV15yq2 sequence block in Yq11 interval D6, recently identified to be the distal borderline of complete AZFa deletions (Kamp et al., 2000). In the same protocol, primers for the STS sY83 and 12f2 could be added (sY83 for ID1 in AZFa-prox mix and 12f2 for ID2 in AZFa-dist mix) to identify the recombination site of the fused HERV15yq1/HERV15yq2 sequence blocks. Each primer mix also contained an STS identical to the ZFY and ZFX loci, to serve as a positive control in each screening experiment. The primer concentrations used in the two PCR multiplex mixes are as follows. AZFa-prox primer mix: 0.3 µmol/l AZFa-prox1 for/rev; 0.2 µmol/l AZFa-prox2 for/rev; 0.2 µmol/l sY83 L/R (optional for ID1 recombination site); 0.1 µmol/l ZFY/ZFX for/rev (positive control); AZFa-dist primer mix: 0.4 µmol/l AZFa-dist 1 for/rev; 0.1 µmol/l AZFa-dist2 for/rev; 0.2 µmol/l 12f2 for/rev (optional for ID2 recombination site); 0.1 µmol/l ZFY/ZFX for/rev.
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All primer sequences are given in Table I
and were deposited in the GenBank under the given accession numbers. Both PCR reactions were performed in a 50 µl reaction volume with 1xPCR buffer (Gibco), using 5 mmol/l MgCl2 for AZFa-prox and 2.5 mmol/l MgCl2 for the AZFa-dist reaction, with 0.2 mmol/l dNTP, and 2 IU Taq DNA Polymerase (Gibco) with 200 ng of the genomic patient DNA.
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For lower DNA concentrations (usually when extracted from tissue samples of testis biopsies) both AZFa primer mixes could also be run without including STS sY83 and ZFX/ZFY in AZFa-prox and 12f2 in AZFa-dist, respectively, to ensure a sufficient template concentration for each PCR reaction.
Appropriate positive and negative genomic DNA controls in each multiplex PCR experiment were provided by DNA samples from a fertile man and from a woman respectively. The DNA sample from the fertile man was a control for the sensitivity and specificity of each performed PCR assay, and the female DNA sample controlled for the Y specificity of the primer pairs used and also for putative contamination. In addition, a water sample containing all reaction components with the exception of the genomic DNA sample was run with each set of primers in order to detect possible reagent contamination.
The PCR reaction profile (for Biometra T-Gradient Personal Cycler) involved a presoak for 3 min at 94°C and 33 PCR cycles, performed as follows: denaturation for 1 min at 94°C, annealing for 1 min at 63°C (AZFa-prox mix), or for 1 min at 57°C (AZFa-dist mix), with subsequent polymerization for 1 min at 65°C for both mixes and a final extension step for 2 min at 65°C. The PCR products (20 µl aliquots) were analysed on a 3% agarose gel stained with ethidium bromide. As a length marker we used the GeneRulerTM 100 bp DNA ladder-plus.
Results
Mapping of complete AZFa sequence in BAC sequence contig of proximal Yq11
Originally, the AZFa region in proximal Yq11 was defined by the deletion of the Y chromosomal STS loci: sY83, sY84, sY85, sY86 and sY87 in the SCO patient JOLAR (Vogt et al., 1996). This corresponded to the deletion of Yq11 interval D3–D6 according to the map of Vogt et al. (1996), and to the deletion of Yq11 interval 5C according to the map of Vollrath et al. (Vollrath et al., 1992) respectively, and it has been suggested that the analysis of the STS loci sY82 in Yq11 interval D2 and sY88 in Yq11 interval D7 maps the AZFa borderlines (Vogt et al., 1996; Simoni et al., 1999). We have now mapped these STS loci in the BAC sequence contig in proximal Yq11 (deposited in the GenBank under the accession no. NT_011875) and compared their positions to the location of the major AZFa proximal and distal breakpoints recently found in two retroviral sequence blocks (HERV15yq1, HERV15yq2; Figure 1
). The molecular length of the so-called ‘AZFa sequence’, i.e. the sequence deleted in reference patient JOLAR, now designated as a reference patient for complete AZFa deletions, was estimated to be 792 kb (Kamp et al., 2000). Selection of this reference patient to define the AZFa sequence has been confirmed since a similar AZFa deletion size was estimated in eight additional SCO patients (Blanco et al., 2000; Kamp et al., 2000; Sun et al., 2000).
Based on this BAC sequence map between Yq11 interval D2–D7, sY82, marking Yq11 interval D2, is located at a distance of 109.692 kb proximal to HERV15yq1, and sY88, marking Yq11 interval D7, is located 369.748 kb distal to HERV15yq2 (Figure 1). Therefore, their use as STS loci for marking the AZFa borderlines requires critical revision.
Novel PCR multiplex protocol for analysis of complete AZFa deletions
With the knowledge of the complete AZFa sequence, a novel screening protocol focusing on the direct analysis of complete AZFa deletions was developed. It was based on a series of new STS markers that map around the two homologous retroviral sequence blocks HERV15yq1 (AZFa-prox1 and AZFa-prox2) and HERV15yq2 (AZFa-dist1 and AZFa-dist2) (Figure 1; Table I). The site of the HERV15yq1/HERV15yq2 recombination event (ID1 or ID2) can be analysed with the STS loci sY83 (deleted if ID1 recombination event) and 12f2 (deleted if ID2 recombination event). For further fine-mapping experiments, five novel STS (AZFa-dist3 to AZFa-dist7) were established in Yq11 interval D6, located distal to the HERV15yq2 sequence (Table I). We also established a new STS from the 3' untranslated region (UTR) of the UTY3 (Ubiquitous TPR motif Y) (Lahn and Page, 1997) gene (UTY3pe) to mark the beginning of Yq11, interval D7. The UTY3 gene (GenBank accession no. AF000994) has a 3'–5' polarity towards the Y centromere in Yq11.
For a convenient parallel analysis of the six AZFa STS, we established two PCR multiplex mixes. The proximal AZFa multiplex mix (AZFa-prox) contained the STS for AZFa-prox1, AZFa-prox2 and sY83, and the distal AZFa multiplex mix (AZFa-dist) contained the STS for AZFa-dist1, AZFa-dist2 and 12f2. Using the guidelines of good laboratory practice and internal quality control, and also to allow trouble-shooting in diagnostic PCR amplification techniques (Neumaier et al., 1998), we introduced an additional PCR analysis of the ZFY/ZFX gene as a positive control in each multiplex PCR experiment. The ZFY and ZFX PCR amplification products are identical and therefore always visible in the patients' DNA samples, even when the complete Yq11 and the proximal Yp11 region is deleted (e.g. in XX males) due to the X chromosomal location of the ZFX gene.
We expected the presence of STS AZFa-prox1 and AZFa-dist2 to be linked to the absence of STS AZFa-prox2 and AZFa-dist1 in the event of a deletion of the complete AZFa sequence in the patient's genomic Y DNA (see Figure 1). Additional deletion of sY83 was expected when the observed AZFa deletion was the result of a recombination event in the ID1 domain of the HERV15 sequence blocks; whereas deletion of the 12f2 locus would indicate that the observed AZFa deletion was the result of a recombination event in the ID2 domain of both HERV15 sequence blocks. Presence of both STS, sY83 and 12f2, would identify a partial AZFa deletion. Examples of the typical STS deletion patterns for both types of complete AZFa deletions are given in Figure 2.
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Deletion analysis of the complete AZFa sequence in 100 men with complete SCO syndrome
We previously proposed that deletion of the complete AZFa sequence in the Y chromosome of infertile men is associated histologically with a complete SCO syndrome (Vogt et al., 1996
; Vogt, 1998). Using our novel AZFa PCR multiplex system, we therefore analysed the presence of complete AZFa deletions in 100 patients with a histologically proven complete SCO syndrome.
Of the 100 SCO patients, six (i.e. 6%) showed the STS deletion pattern expected for complete AZFa sequence deletions, i.e. AZFa-prox1 and AZFa-dist2 present but AZFa-prox2 and AZFa-dist1 absent (Table II). Four of them (ANDSCH, ERWTHO, GERKEL, LECGER) had their HERV15yq1 and HERV15yq2 recombination site in the ID1 domain, shown by additional deletion of the sY83 STS. Two of them (JOLAR, HERREI) had their HERV15yq1/HERV15yq2 recombination site in the ID2 domain shown by deletion of the 12f2 STS. Since, in earlier experiments, we were able to bridge the AZFa break-fusion regions of the same six SCO patients by PCR with primers from the recombined HERV15yq1/HERV15yq2 sequence blocks (Kamp et al., 2000), our novel AZFa PCR multiplex system confirmed its reliability for the direct analysis of complete AZFa deletion, and also confirmed that the mean length of a complete AZFa sequence in proximal Yq11 is 792 kb (Figure 1).
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Interestingly, we found one SCO patient (SCO-2) with a complete AZFa deletion, where the distal breakpoint in Yq11 interval D6 was located beyond the HERV15yq2 sequence block. His DNA sample did not amplify the STS AZFa-dist2 marking the distal borderline of the HERV15yq2 sequence and AZFa-dist3, but was positive for AZFa-dist4 located ~5.5 kb distal to HERV15yq2. The deletion of sY83 reflected a recombination event in the ID1 domain of the HERV15yq1/HERV15yq2 sequence blocks as the apparent cause of the AZFa deletion in this SCO patient.
AZFa analysis of the SCO patients LÜB-8 and LÜM-17 indicated only the presence of sY82 in Yq11 interval D2 and the absence of all STS in interval D6 (Table II). We therefore extended the Y-DNA analysis of both patients using the UTY3pe marker for analysis of Yq11 interval D7 and using a series of markers spanning Yq11 interval D8–D25 in the multiplex PCR protocol described in Vogt et al. (1996). We found that all STS distal to sY82 were also absent in the Y chromosome of both patients. This was surprising because it suggests that the long arm of the Y chromosome is deleted completely in both patients, although their karyotypes were reported to be ‘normal, 46,XY’ (W.Küpker, personal communication). Our analysis was further confirmed when it was shown that STS sY160 (Vollrath et al., 1992), a marker for Y chromosome heterochromatin, was also absent on the Y chromosome of both patients. This observation reinforces the value of combining both molecular and traditional cytogenetic analyses of these patients (Rucker et al., 1998.)
Discussion
Complete AZFa deletions are always associated with an SCO syndrome
After having screened more than 1000 patients with idiopathic azoospermia or severe oligozoospermia in our laboratories, the specific testicular phenotype associated with each AZF deletion, as originally suggested by Vogt et al. (1996), has been observed only when the molecular extension of the detected AZF deletion is comparable with those originally described by Vogt et al. (1996). This observation reflects the fact that each AZF region contains multiple spermatogenic genes that are present within each of the AZF deletion intervals (Lahn and Page, 1997; Vogt, 1998).
In the AZFa region, a splice site mutation or complete deletions of the Drosophila Fat Facets Related Y (DFFRY) gene, also named Ubiquitin-specific protease 9 Y (USP9Y), were shown to be associated with severe hypospermatogenesis (Ferlin et al., 1999; Sun et al., 1999) and deletions of the Dead Box Y (DBY) gene were found to be associated with variable testicular pathologies including the SCO syndrome (Foresta et al., 2000). For partial deletions of the AZFa region, the presence of one or both Y genes may still be associated with at least focal preservation of germ cells. When both spermatogenic genes (DFFRY/USP9Y and DBY) are absent, a complete SCO syndrome (the proposed AZFa histopathology) has, in our experience, always been observed.
Since it cannot yet be ruled out that more Y genes are present in the AZFa region (Sargent et al., 1999), it is also possible that determination of complete SCO pathology is predicted by absence of the complete AZFa gene content (deletion of the complete AZFa sequence) now estimated at 792 kb in the AZFa reference patient JOLAR. Until this uncertainty has been resolved, it is advisable to limit prediction of a histologic diagnosis of complete SCO syndrome to those patients who have been verified to have a Y chromosome microdeletion of a molecular length comparable to that of the AZFa-reference patient JOLAR (Figure 1).
For this purpose we established the rapid AZFa screening protocol presented in this paper. It is focused on the deletion analysis of complete AZFa deletions caused by intrachromosomal recombination events of two retroviral sequence blocks in Yq 11, interval D3 (HERV15yq1) and in Yq11, interval D6 (HERV15yq2) (Blanco et al., 2000; Kamp et al., 2000; Sun et al., 2000) Complete AZFa deletion events were revealed by a typical STS deletion pattern in two PCR multiplex mixes: AZFa-prox and AZFa-dist (Figures 1 and 2) and the putative HERV15yq1/HERV15yq2 recombination site (ID1 or ID2) was identified by deletion analysis of sY83 (ID1) and 12f2 (ID2) respectively. We confirmed the reliability of this AZFa screening protocol by its application to 100 DNA samples from patients with a histologically proven SCO syndrome.
Nine SCO patients with a complete AZFa deletion were identified. The distal AZFa breakpoint in the SCO-2 patient was mapped 5.5 kb distal to the HERV15yq2 sequence block. This pointed to the possibility that a primary AZFa deletion event caused by recombination of the HERV15yq1 and HERV15yq2 sequence blocks could be extended by additional small secondary deletion events probably occurring stochastically in the neighbourhood of the first breakpoint fusion region.
The primary sites for the terminal Yq deletions in the Y chromosomes of LÜB-8 and LÜM-17 were difficult to identify but their proximal Yq11 breakpoints were mapped in the neighbourhood of HERV15yq1, in Yq11, interval D2. A HERV15yq1 and HERV15yq2 recombination could therefore not be excluded as the primary site for this deletion event. Both deletions included the complete AZFa sequence and therefore were expected to result in a complete SCO syndrome in the patients' testes tubules. Surprisingly, a normal 46,XY karyotype was reported for both patients, although it should have been possible to detect their large Yq deletions under the microscope. It is possible that in both patients, two Yq- chromosomes fused together during their meiotic divisions to form a dicentric iso(Yp) chromosome. In metaphase plates, iso(Yp) chromosomes have a phenotype similiar to a normal Y chromosome because the kinetochore activity of the second centromere is suppressed (Daniel, 1985). Similiar Yqdel fusion events are frequently found in men with large Yq11 deletions and can be easily identified by fluorescent in-situ hybridization (FISH) analysis with a centromeric DNA probe (Köhler and Vogt, 1994). For practical reasons, it was not possible to perform this FISH analysis on the chromosomes of LÜB-8 and LÜM-17.
Partial AZFa deletions are not always associated with an SCO syndrome
Partial AZFa deletions would be defined by patients with detectable sY83 and 12f2 loci as well as AZFa-dist1 or AZFa-prox2 respectively, and we assume that they are not always associated with the SCO syndrome. One example, patient SAYER (Qureshi et al., 1996), has been proposed to have a complete AZFa deletion. This patient had severe hypospermatogenesis with postmeiotic disruption of the spermatogenic process on histology. A DNA sample from patient SAYER (kindly provided by Prof. Howard Cooke, MRC Human Genetics Unit, Western General Hospital, Edinburgh, UK) was therefore reanalysed in our laboratory using the PCR protocol for complete AZFa deletions as described in this paper. We found that SAYER must have a partial AZFa deletion because both AZFa-dist1 and AZFa-dist2 were present in the SAYER DNA sample (C.Kamp, unpublished results).
Other partial AZFa deletions reported include the DFFRY/USP9Y and DBY deletions (Ferlin et al., 1999; Foresta et al., 2000). As expected, most of these deletions have been associated with severe hypospermatogenesis on diagnostic biopsy but not with SCO syndrome.
High frequencies of AZFa deletions occur only in patients with SCO syndrome
In the routinely performed clinical Y deletion screening programmes on DNA samples of men with idiopathic azoospermia, AZFa deletions are only rarely found. Even after selection of patients with a small testicular volume and high levels of the follicle stimulating hormone (FSH), the frequency of AZFa deletions does not increase (C.Kamp, unpublished results). On the other hand, it has been claimed that the frequency of AZFa deletions is high in patients with a histologically proven SCO syndrome although the numbers published have been variable [50%, (Forresta et al., 1998); 18.2%, (Blagosklonova et al., 2000)]. Of 18 individuals with a bilateral SCO syndrome identified by the fine needle aspiration cytology method, nine patients with a complete deletion of the AZFa region were identified (Foresta et al., 1998). In 22 individuals with complete SCO syndrome, identified in conventional testicular biopsies (Blagosklonova et al., 2000), AZFa deletions were identified in four individuals by a PCR experiment with two nested primer pairs of the DFFRY/USP9Y gene. In our patient group of 100 individuals, all with a histologically proven SCO pathology, we found nine complete AZFa deletions, i.e. a frequency of 9%. A high deletion frequency of the complete AZFa sequence therefore seems to occur only in the SCO patient group. In the large group of men with non-obstructive azoospermia, SCO patients have been found with a frequency of 11–27% (Tournaye et al., 1996; Rucker et al., 1998).
Improved guidelines for analysis of complete AZFa deletions
Although the number of SCO patients with a complete AZFa deletion is still small, we would predict that only complete AZFa deletions, as defined in this paper by the typical STS deletion pattern (AZFa-prox1 and AZF-dist2 present with AZFa-prox2 and AZFa-dist1 absent), will be consistently associated with the occurrence of the severe SCO testicular pathology in the patients' testes tubules. We therefore suggest that the current European Academy of Andrology (EAA) laboratory guidelines for analysis of complete AZFa deletions (Simoni et al., 1999) should be revised by replacing the STS markers sY82, sY83 and sY87, sY88, for the extension analyses of the AZFa deletion, by the four novel STS markers AZFa-prox1, AZFa-prox2 and AZFa-dist1, AZFa-dist2 as presented in this paper. The STS sY82 is located ~100 kb more proximal to the HERV15yq1 than AZFa-prox1 and the STS sY88 is located 370 kb more distal to the HERV15yq2 sequence block than AZFa-dist2. Furthermore, the absence of sY83 and sY87 does not always appear to be linked to the deletion of the complete AZFa sequence. In two patients with deletion of a complete AZFa sequence due to a HERV15yq1/HERV15yq2 recombination event in the ID2 domain, sY83 was present (Figure 2) and deletion of sY87 was also found in patients with hypospermatogenesis like SAYER (Quershi et al., 1996). This is explained by the fact that sY87 is located 235 kb proximal to the major distal AZFa breakpoints in the HERV15yq2 sequence block (Figure 1). Indeed, with our new AZFa screening protocol, we found that STS AZFa-dist1, and also STS GY6 marking the DBY exon 1, were present in the DNA sample of SAYER (C.Kamp, unpublished results). SAYER is therefore a patient with only a partial AZFa deletion.
The STS loci sY84 and sY86 used in the EAA minimal set for the analysis of AZFa deletions (Simoni et al., 1999) are always deleted in patients with complete AZFa deletions. However, both are located proximal to the location of the AZFa candidate genes DFFRY/USPY9 and DBY (Figure 1). The possible deletion of these genes can therefore not be investigated by deletion analyses of sY84 or sY86. Therefore, it might be useful in future to replace sY84 and sY86 in the EAA minimal set by suitable STS markers for deletion analysis of the DFFRY/USPY9 and DBY genes. This work is in progress.
The association of complete AZFa deletions with a complete SCO syndrome in all seminiferous tubules requires further AZFa screening programmes. If this is confirmed, our rapid AZFa multiplex PCR protocol might become a valuable prognostic tool for infertility clinics considering testicular sperm extraction, as it would enable them to exclude AZFa patients with a complete SCO syndrome from procedures that would be predictably unsuccessful (Anniballo et al., 2000).
Acknowledgements
We are grateful to all SCO patients who have provided us with their written consent to use their blood and testicular tissue samples for this AZFa screening programme. We also thank our colleagues, Howard Cooke for having provided us with the genomic DNA sample of SAYER, and Mark Jobling for providing the information on the primer sequences of the 12f2 STS prior to publication in Rosser et al. (Am. J. Hum. Genet., 67, 1526–1543, 2000). S.K. and H.Y. thank Prof. G.Paz for making this collaboration possible. Mrs Kartrin Christophers-Genzmer and Mrs Anne Jordan are thanked for their help in preparing the final version of this manuscript and especially for their critical review of English grammar. This study was supported by a grant to P.H.Vogt from the Deutsche Forschungsgemeinschaft (DFG: Vo403/11.3).
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Submitted on March 16, 2001; accepted on July 17, 2001.
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