Department of Medical and Surgical Sciences, Clinica Medica 3, University of Padova, Via Ospedale 105, 35128 Padova, Italy
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Abstract |
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Key words: DAZ/DFFRY/male infertility/RBM/Y-chromosome
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Introduction |
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Candidate genes for these regions have been isolated in recent years: (i) the RBM (RNA-binding motif, formerly YRRM) family (Ma et al., 1993), consists of about 30 copies on both arms of the Y chromosome belonging to at least six subfamilies (Chai et al., 1997
). Only RBM-I is actively transcribed, the most functional copies of this gene are located on interval 6B (Elliott et al., 1997
), thus making it the best candidate for the AZFb region (Vogt et al., 1997
); (ii) the DAZ (deleted in azoospermia) family (Reijo et al., 1995
; Saxena et al., 1996
), consisting of multiple (at least three) functional genes clustered on interval 6D (Gläser et al., 1997
; Yen et al., 1997
, 1998
) and representing the AZFc candidate; (iii) DFFRY (Drosophila fat-facets related Y) (Brown et al., 1998
), a recently characterized gene located in interval 5C and supposed to represent the AZFa candidate.
At present, AZFc represents the most frequently deleted region in infertile men. Such deletions appear to remove the entirety of the DAZ gene cluster and have been associated with a variety of spermatogenic alterations, ranging from azoospermia due to Sertoli cell-only to oligozoospermia with different testicular phenotype (Reijo et al., 1995, 1996
; Najmabadi et al., 1996a
; Stuppia et al., 1996
; Vogt et al., 1996
; Foresta et al., 1997
, 1998
; Girardi et al., 1997
; Pryor et al., 1997
; Simoni et al., 1997
; Liow et al., 1998
). Deletions in AZFb overlapping the RBM gene and deletions in AZFa occur less frequently, and in such cases they have been detected in different abnormalities of spermatogenesis (Reijo et al., 1995
; Najmabadi et al., 1996a
; Vogt et al., 1996
; Foresta et al., 1997
, 1998
; Girardi et al., 1997
; Pryor et al., 1997
; Liow et al., 1998
). DFFRY is the least studied gene, as until now it was found to be absent only in three subjects previously that were deleted in the AZFa interval, and presenting with Sertoli cell-only syndrome or severe hypospermatogenesis (Brown et al., 1998
). Therefore, a relationship between Yq microdeletions and testicular phenotype is still lacking, and the roles of DAZ, RBM and DFFRY in determining the disruption of spermatogenesis have not yet been elucidated. Such difficulties in genotypephenotype associations could arise from the different patient selection criteria utilized in these studies, that may be based only on clinical (infertility, history, hormonal concentrations, testicular volume), and/or seminological data (normo-, oligo-, azoospermia) and/or testicular structure (Sertoli cell-only syndrome, hypospermatogenesis, spermatogenic arrest, obstructive forms).
In this study, we performed a polymerase chain reaction (PCR)-based Yq analysis including DAZ, RBM and DFFRY genes in a large group of infertile patients affected by azoospermia or oligozoospermia and characterized by well-defined testicular alterations, in order to determine the importance of AZF regions in disrupting spermatogenesis.
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Materials and methods |
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Semen samples were obtained on two different occasions, separated by a 3-week interval, following a 3-day period of sexual abstinence, and complete semen analyses were performed according to WHO guidelines (WHO, 1992). The diagnosis of azoospermia was established by pellet analysis after semen centrifugation (1000 g, 20 min). Plasma concentrations of follicle stimulating hormone (FSH) and luteinizing hormone (LH) were measured in each subject by radioimmunoassay using 125I-labelled FSH and LH (Ares-Serono, Milan, Italy). Plasma concentrations of testosterone were determined by radioimmunoassay using 3H-labelled testosterone (Radim, Rome, Italy). Only patients with an apparently normal 46,XY karyotype were included in this study. All patients were studied with a comprehensive history and general investigation for exclusion of possible causes of testicular damage, such as cryptorchidism, varicocele, seminal tract infections, drug use, endocrinopathies, post-mumps orchitis, testicular trauma or torsion.
As a comparison, we have studied 50 azoospermic and severely oligozoospermic men (sperm count <5x106/ml) with known causes of testiculopathy (group 5), such as orchi-epididymitis, testicular trauma or chemoradiotherapy, 30 obstructive azoospermic men (congenital or acquired obstruction of the seminal tract) (group 6), and 100 healthy normozoospermic fertile men (group 7).
Testicular fine needle aspiration and cytological quantification
The testicular structure was analysed by means of bilateral FNAC, as described previously (Foresta and Varotto, 1992; Foresta et al., 1992
, 1995
). Briefly, bilateral fine needle aspiration was performed using 23-gauge (0.6 mm) butterfly needles and aspirating with a 20-ml syringe. The cellular material was stained with May-Grünwald and Giemsa, examined under a light microscope and at least 200 spermatogenic cells (spermatogonia, primary spermatocytes, secondary spermatocytes, early and late spermatids and spermatozoa) were counted per smear. Spermatogenic cells were expressed as relative percentages, while the interposed Sertoli cells were expressed as the Sertoli index (SEI, the number of Sertoli cells/100 spermatogenic cells), which has been found to be a reliable index of the tubular germ potential (Foresta and Varotto, 1992
; Foresta et al., 1992
, 1995
).
As described in previous studies (Foresta and Varotto, 1992; Foresta et al., 1992
, 1995
), this cytological analysis allows us to classify azoo/oligozoospermic subjects as follows: (i) Sertoli cell-only syndrome, defined as the complete absence of germ cells; (ii) hypospermatogenesis, defined as quantitative reduction of the germ line with respect to Sertoli cells; different degrees of hypospermatogenesis were distinguished on the basis of the SEI (see Results); (iii) spermatogonia or spermatocytes arrest; (iv) spermatids arrest; and (v) obstructive forms, presenting with a normal germ line with increased percentage of mature spermatozoa. Only patients showing Sertoli cell-only syndrome, various degrees of hypospermatogenesis and, as controls, obstructive azoospermia were included in this study, therefore excluding qualitative spermatogenesis alterations (maturation arrest).
Sequence-tagged sitePCR amplification
A set of 38 Y-specific sequence-tagged sites (STS) (Vollrath et al., 1992; Ma et al., 1993
; Kobayashi et al., 1995
; Reijo et al., 1995
, 1996
; Brown et al., 1998
) spanning the euchromatic region of Yq was tested in each patient. The order of STS and Yq deletion intervals as described previously are shown in Figure 1
(Reijo et al., 1995
, 1996
); AZF regions are defined as previously reported (Vogt et al., 1997
). AZF-candidate genes studied were as follows: DFFRY-5', DFFRY 4.1, DFFRY J/D and DFFRY 3.1 (Brown et al., 1998
) amplified the regions 20152136, 57536206, 88539157 and 92979451 respectively of the coding region of the DFFRY gene: sY277, sY254, sY279, sY283 and sY255 (Reijo et al., 1995
, 1996
) amplified the regions 12771588 (from intron 12 to intron 23), 14091789 (from exon 2 to exon 3), 16632497 (from intron 23 to intron 45), 25063002 (from intron 45 to intron 56) and 30423165 (within exon 6) respectively of the genomic DNA of the DAZ gene (clone 63C9); F19/E355 (Ma et al., 1993
; Kobayashi et al., 1995
) amplified the region 12921733 of the RBM coding sequence, corresponding to the distal part of exon 11 to the most part of exon 12.
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All primers were analysed in 100 normal healthy men of proven fertility (positive controls) before their application in patients, in order to ascertain that each of them produced a single amplification product of the expected size. The Y-specificity was determined in 10 normal women (negative controls). Patients were considered normal if the PCR product was of the expected size and negative only after three amplification failures. The repeated experiments were performed on new samples of DNA extracted from a different blood collection. Furthermore, only DNA extracts which gave a normal PCR amplification of the SRY locus on Yp (sY14, Vollrath et al., 1992) were considered.
Twenty-four fathers or brothers of 40 deleted patients were also investigated under the same experimental conditions, while no male relatives were available for the other 16 patients. None of the male relatives of patients with Yq deletion had a history of infertility.
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Results |
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Each of the 38 STS produced an amplification product of the expected size in all normal fertile men and failed to amplify in normal women. In particular, primer pairs sY277, sY254, sY279, sY283 and sY255 gave an amplification product of 311, 380, 834, 496 and 123 bp respectively, corresponding to the DAZ genomic DNA sequence; F19/E355 amplified 800 bp, according to previous reports (Ma et al., 1993; Kobayashi et al., 1995
). Only the cDNA sequence is available for the DFFRY gene (Brown et al., 1998
) and we obtained PCR products of approximately 1.5 kb for DFFRY-5', 2 kb for DFFRY 4.1, 1.2 kb for DFFRY J/D and 200 bp for DFFRY 3.1, which represent the normal bands (Nabeel A.Affara, personal communication).
Deletions of Yq were observed in 19 out of 55 patients affected by idiopathic Sertoli cell-only syndrome (group 1, 34.5%) and in 21 out of 85 patients affected by idiopathic bilateral severe hypospermatogenesis (group 2, 24.7%). No deletions were found in patients presenting with idiopathic moderate and mild oligozoospermia (groups 3 and 4), or in patients affected by severe testiculopathies of known aetiology (group 5) and in obstructive azoospermic patients (group 6) (Table I).
Figure 1 summarizes the PCR results of patients with deletions, that will be discussed below. Briefly, three patients had a terminal deletion of Yq, while the others showed interstitial deletions. A specific deletion only in the AZFc region involving the DAZ gene was present in 17 out of 40 patients (42.5%), and it was more frequent in patients with severe hypospermatogenesis (13/21, 61.9%) than in patients with Sertoli cell-only syndrome (4/19, 21.1%); among these, deletions confined to the DAZ gene, not including other flanking STS, were present in five out of 17 cases and they were associated both with severe hypospermatogenesis and Sertoli cell-only syndrome. A deletion involving only the RBM gene with or without some flanking regions was present in six out of 40 subjects (15.0%) and was equally frequent in both groups, with a prevalence of 15.8% in Sertoli cell-only syndrome (3/19) and of 14.3% in severe hypospermatogenesis (3/21). More proximal deletions confined to regions overlapping the DFFRY gene were present in five out of 40 patients (12.5%), with a slightly lower percentage in Sertoli cell-only syndrome (2/19, 10.5%) than in severe hypospermatogenesis (3/21, 14.3%). Among these, a deletion involving only the DFFRY gene was present only in one patient (no. 327) and it was associated with severe hypospermatogenesis. There was a higher prevalence of deletions involving more than one AZF-candidate gene in Sertoli cell-only syndrome than in severe hypospermatogenesis: seven of 19 (36.8%) patients of the first group and only two of 21 (9.5%) of the second group had a deletion of two genes; three patients (15.8%) with Sertoli cell-only syndrome and none with severe hypospermatogenesis had a large Yq deletion involving all three genes.
The father or brothers of 24 deleted patients were also investigated and no deletions were found. No male relatives were available for the remaining patients, but on the basis of these and previous results it is possible to conclude that these represent de novo deletions and may be considered the aetiological factor of the spermatogenic defect.
Testicular volumes were significantly reduced and FSH plasma concentrations significantly increased compared with controls (10.8 ± 3.7 ml and 16.4 ± 5.3 IU/l respectively, P <0.05), without significant differences in plasma concentrations of LH and testosterone in subjects affected by a severe testiculopathy (groups 1, 2 and 5), confirming the primary testiculopathy involving only the spermatogenic system. All these parameters were not different from controls in the other groups of patients (groups 3, 4 and 6). Furthermore, testicular volume and hormone concentrations were not different in patients with and without deletion (data not shown).
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Discussion |
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In the present study we have looked for the relative importance in spermatogenesis of Yq regions with particular interest to AZF-candidate genes, screening different populations of infertile men affected by azoospermia and oligozoospermia and selected on the basis of their testicular phenotype and clinical history. We found a high prevalence of Yq deletions in idiopathic severe testiculopathies characterized by the complete absence (Sertoli cell-only syndrome) or a strong reduction (severe hypospermatogenesis) of germ cells (40 out of 140, 28.6%), while milder forms of idiopathic testiculopathy were not associated with Yq alterations. No Yq region was deleted in patients whose infertility was ascribed to known aetiologies, further supporting the hypothesis that Yq deletions are responsible for the testicular damage observed in `idiopathic' forms. The confirmation of the pathogenic role for such deletions derives also from the analysis of fertile normozoospermic men and male relatives of patients with deletions, that allowed us to exclude normal polymorphisms and to consider them as de novo deletions. The prevalence of Yq deletions found in this study is undoubtedly high but in agreement with previous papers reporting that this frequency increases with the severity of testicular tubular damage (Reijo et al., 1995; Najmabadi et al., 1996a
; Vogt et al., 1996
; Foresta et al., 1997
, 1998
; Girardi et al., 1997
; Simoni et al., 1997
; Liow et al., 1998
). In fact, idiopathic Sertoli cell-only syndrome that represents the worst condition of male infertility shows the highest prevalence of deletion (19/55, 34.5%); furthermore, when such genetic anomalies are associated with residual spermatogenesis, the final sperm output is compromised, and no Yq deletion is found when the sperm concentration is >5x106/ml. The quite small number of patients with mild and moderate oligozoospermia may hide a very low rate of deletion, as only isolated cases have been described in these groups of patients (Pryor et al., 1997
).
Our results confirm the predominant role of deletions in the AZFc region overlapping the DAZ gene family among other Yq regions in determining a severe tubular damage. A deletion confined to this region was the most frequent finding, as it was present overall in more than 40% of patients with deletions, and more often found in severe hypospermatogenesis rather than in Sertoli cell-only syndrome, since 13 out of 17 (76.5%) patients deleted for this gene belong to this group. However, only five out of 17 patients presented a deletion of the DAZ gene not including other flanking regions, and they were affected either by severe hypospermatogenesis or Sertoli cell-only syndrome. Therefore, a non-unique phenotype is associated with DAZ deletions, even if the absence of DAZ appears not sufficient to determine the complete loss of the spermatogenic line, but rather seems to produce a reduction in number of these cells. The multicopy nature of this gene has previously made it difficult to detect deletions of some copies or intragenic mutations. The mechanisms by which DAZ gene deletions cause this spermatogenic impairment remain unclear, since little is known about the functions of this RNA-binding protein in human spermatogenesis, except that it is expressed in different phases of the spermatogenetic cycle (Menke et al., 1997; Habermann et al., 1998
) and it may regulate splicing events or translation (Elliott et al., 1997
, 1998
).
Deletions in AZFb involving the RBM gene are less frequently detected, both in Sertoli cell-only syndrome and in severe hypospermatogenesis. These data confirm previous reports on the role of this gene family in male infertility and do not allow us to correlate testicular phenotype with RBM deletions. Like DAZ, RBM is in multiple copies on the Y chromosome and this has complicated attempts to prove its role in human spermatogenesis, as detrimental mutations have not yet been identified. Furthermore, the exact copy number of functional RBM genes is uncertain, even if the critical region for RBM expression has been mapped to interval 6B (Elliott et al., 1997). RBM is a nuclear protein with dynamic modulations in its spatial location in the different spermatogenic cells, suggesting that it possesses different functions related to pre-mRNA splicing (Elliott et al., 1998
), though how it functions during male germ cell development is not known.
The most intriguing feature of the present study regards the analysis and role of the DFFRY gene. Our results seem to suggest this gene as the AZFa candidate, since it was absent in a fraction of patients and one of them (no. 327) presented a normal PCR amplification of other STS contained in the AZFa region. Deletions of this gene seem to be more frequently associated with a depopulation of germ cells rather than with their complete absence. However, the mechanism by which a spermatogenic impairment occurs is unknown. Mutations or microdeletions in DFFRY, if found, will confirm that this gene is responsible for the AZFa phenotype. It must be noted, however, that deletions overlapping DFFRY frequently include also neighbouring regions, in which new genes have been mapped (Lahn and Page, 1997). In particular, interval 5C/D seems to harbour DBY and UTY genes (Lahn and Page, 1997
; Mazeyrat et al., 1998
) that may be absent in some patients and may contribute with DFFRY to the AZFa phenotype. However, DBY and UTY seem to lie between markers sY87 and sY88 (Mazeyrat et al., 1998
) and therefore should be normally present in patient no. 327, whose phenotype should be due only to the absence of DFFRY.
The microdeletion pattern observed in this study differs from the AZF classification and genotypephenotype relation proposed previously (Vogt et al., 1996, 1997
), but suggests that larger deletions are associated with more severe spermatogenic phenotype. This hypothesis is supported by the evidence that in patients with Sertoli cell-only syndrome Yq deletions frequently involved large regions of interval 5 and 6 and that, in a high proportion of cases, two or even three AZF-candidate genes were absent, while these associations were extremely rare in the group of patients affected by hypospermatogenesis. Even if a correlation between Yq deletions and testicular phenotype is lacking, it could be argued that larger deletions may show the additional effects of each single gene deletion.
As previously noted by us and other groups (Najmabadi et al., 1996a; Stuppia et al., 1996
; Foresta et al., 1997
, 1998
; Girardi et al., 1997
; Pryor et al., 1997
; Duell et al., 1998
; Rossato et al., 1998
), PCR analysis frequently showed non-contiguous deletions. The Y chromosome seems to be highly unstable and prone to deletions, probably since it is rich in repetitive elements and repeats (Girardi et al., 1997
; Yen et al., 1998
), and interstitial double deletions may be explained by different hypotheses: (i) the PCR observations may reflect really separated microdeletions; (ii) some STS may be from repetitive sequences (as demonstrated for example for sY146, sY153 and sY155) (Yen et al., 1998
); and (iii) a complex rearrangement (e.g. an inversion with subsequent interstitial deletion) in the father may be the cause. We do not think that these observations reflect a population effect, as reported patients are not from a single geographical area but come from different regions of Italy.
Finally, the determination of Yq deletions in infertile men has important clinical and ethical implications, especially in severely oligozoospermic patients, as it has been clearly demonstrated that spermatozoa carrying a Yq deletion are able to fertilize and give rise to pregnancies by means of assisted reproduction techniques (Mulhall et al., 1997; Rossato et al., 1998
). Therefore, such analysis provides a careful diagnosis and allows the andrologist to forego empirical and often expensive treatments; at the same time it dictates that infertile men undergoing such procedures should be informed about the possibility of passing on a Yq deletion to male offspring and a screening for microdeletions of the Y chromosome should be offered to them.
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Acknowledgments |
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Notes |
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References |
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Submitted on November 30, 1998; accepted on March 18, 1999.