| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Original Studies |
Department of Medical and Surgical Sciences (C.F., E.M., A.G., A.F.), Clinica Medica 3; Institute of Histology and Embryology (M.O.), University of Padova, 35128 Padova, Italy
Address correspondence and requests for reprints to: Dr. Carlo Foresta, Clinica Medica 3, University of Padova, Via Ospedale 105, 35128 Padua, Italy. E-mail: forestac{at}protec.it
 |
Abstract |
|---|
 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Eleven of 40 (27.5%) unilateral excryptorchid patients affected by bilateral testiculopathy and 28 of 110 (25.4%) patients affected by idiopathic severe primary testiculopathy showed Yq microdeletions, whereas no microdeletions were found in all the other subjects, nor in male relatives of patients with deletions. Microdeletions were located in different parts of Yq, including known regions involved in spermatogenesis (DAZ and RBM, AZFa, b, and c) and other loci still poorly defined. No difference in localization of deletions was evident between cryptorchid and idiopathic patients.
Microdeletions in Yq may be responsible for severe bilateral testicular damage that could be phenotypically expressed by unilateral cryptorchidism, as well as by idiopathic infertility.
|
Introduction |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Testicular maldescent occurs bilaterally in 10–15% of cases, and both the bilateral and unilateral forms are well-known causes of altered spermatogenesis (2, 3, 4, 5). In addition to impaired fertility, cryptorchid patients show an increased risk for development of testicular cancer (6, 7, 8). The pathogenesis of these two sequelae remains unclear, as they might be due to an underlying disorder of the testis or to the consequence of the cryptorchid location (9).
The etiology of cryptorchidism is probably multifactorial, related to extrinsic (extragonadal) or intrinsic (gonadal) causes disrupting testicular descent (1, 10). Extrinsic pathogenesis may be considered when hormonal abnormalities (such as androgen deficiency) or defects in testicular descent (mechanical anomalies) exist. In such cases the testicular damage of the cryptorchid testis is related only to its prolonged permanence in the abdomen. In fact, the normal descended testis in these forms sometimes appears normal or even hypertrophic, and these patients exhibit normal sperm production (10). Intrinsic gonadal causes are suspected when unilateral cryptorchidism is associated with a bilateral testicular damage. This hypothesis is further confirmed by the evidence of testicular cancer originating from the contralateral (not retained) testis.
In recent years, a growing body of evidence has demonstrated the existence of a genetic basis for primary testiculopathies related to microdeletions in the euchromatic region of the Y chromosome long arm (Yq11) (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28), where an azoospermia factor (AZF) has been suggested to exist (29). Two gene-family candidates for AZF have been well characterized: RNA-binding motif (RBM) (30, 31, 32, 33, 34) and deleted in azoospermia (DAZ) (11, 35, 36). However, regions of Yq11 outside these genes may also play important roles in the regulation of spermatogenesis (12, 16, 17, 20, 21, 25, 28), and three distinct nonoverlapping subregions, defined as AZFa, AZFb, and AZFc, have been distinguished in Yq11 (16, 37): AFZc includes DAZ (16, 25, 37); AZFb contains functional copies of RBM (33, 37); and only recently, a new putative gene for AZFa has been identified (DFFRY: drosophila fat-facets related Y gene) (28, 38, 39). However, the roles of these three loci in determining the disruption of spermatogenesis have not yet been elucidated, and a clear correlation between genotype and phenotype is lacking. Generally, it is thought that microdeletions in Yq are the cause of 10–15% of idiopathic azoospermia or severe oligozoospermia. However, this prevalence is higher when evaluated in most severe idiopathic primary testiculopathies, characterized by a complete lack of germ cells (Sertoli cell-only syndrome) or by an important depopulation of these cells (severe hypospermatogenesis) (11, 12, 16, 17, 18, 19, 20, 21, 23, 28).
Therefore, to clarify whether cryptorchidism might be the expression of a congenital gonadal abnormality, we investigated the frequency of Y chromosome microdeletions in unilateral excryptorchid subjects manifesting important bilateral testiculopathies.
|
Subjects and Methods |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Our study was approved by the Hospital Ethical Committee, and informed consent was obtained from each patient. Among adult men who underwent orchidopexy because of unilateral cryptorchidism, we selected 60 patients: 40 azoospermic and severely oligozoospermic subjects (sperm count < 5 x 106/mL) showing clinical, hormonal, seminal, and testicular cytological features of bilateral testicular damage (Group 1) and 20 moderately oligozoospermic subjects (sperm count 10–20 x 106/mL) showing a normal testicular cytological pattern in the descended testis (Group 2). All patients were studied with a comprehensive history and general investigation to exclude other possible causes of testicular damage, and in all selected patients cryptorchidism was not associated with other phenotypic anomalies. As comparison, we studied 110 men with azoospermia and severe oligozoospermia (Group 3) and 20 men with moderate oligozoospermia (Group 4), whose infertility was considered idiopathic. Furthermore, we evaluated 50 azoospermic and severely oligozoospermic men with known causes of testiculopathy, such as orchi-epididymitis, testicular trauma or chemoradiotherapy (Group 5), and 100 healthy normozoospermic fertile men (Group 6).
Semen samples were obtained on two different occasions, and complete semen analyses were performed according to guidelines of the World Health Organization (40). All selected patients underwent ultrasound scanning of the testes to evaluate testicular size and to rule out subclinical varicocele and parenchymal lesions compatible with neoplasm (if found, these patients were excluded from the study). FSH, LH, and testosterone plasma concentrations were measured by RIA (Ares-Serono, Milan, Italy and Radim, Rome, Italy). Only patients with an apparently normal 46,XY karyotype were included in this study.
Testicular fine needle aspiration and cytological quantification
The testicular structure was analyzed in Groups 1–4 by means of a bilateral fine needle aspiration cytology (FNAC) as previous described (41), using 23 gauge (0.6 mm) butterfly needles and aspirating with a 20 mL syringe. The cellular material was placed on microscope slides, air-dried for 24 h, stained with May-Grünwald and Giemsa (Merck KGaA, Darmstadt, Germany), and examined under a light microscope at x125, x400, and x1250 magnifications. At least 200 spermatogenic cells were counted per smear, and the following forms were identified and expressed as relative percentages: spermatogonia, primary and secondary spermatocytes, early and late spermatids, and spermatozoa. The interposed Sertoli cells were expressed as the Sertoli index (SEI, the number of Sertoli cells per 100 spermatogenic cells), which has been found to be a reliable index of the tubular germ potential (41, 42, 43).
As described in previous studies (41, 42, 43), cytological analysis in azoospermic and oligozoospermic patients has identified the following: 1) complete absence of germ cells, defined as Sertoli cell-only syndrome (SCOS); 2) quantitative reduction of the germ line with respect to Sertoli cells, indicating different degrees of hypospermatogenesis; 3) spermatogonia or spermatocytes arrest; 4) spermatids arrest; and 5) normal germ line with increased percentage of mature spermatozoa, indicating an obstruction of the efferent ducts. Only patients showing SCOS and various degrees of hypospermatogenesis were included in this study.
The results are given in the text as mean + SD. Statistical comparisons between groups were made by ANOVA. P values less than 0.05 were regarded as statistically significant.
Sequence-tagged site (STS)-PCR and criteria used to define microdeletion
A set of 31 previously described Y-specific STSs (11, 14, 30, 38, 44), spanning the euchromatic region of Yq, was tested in each
patient. The order of STSs and Yq-deletion intervals are shown in Fig. 1
according to Reijo et al. (11, 14); AZFa, b, and c regions
are defined according to Vogt et al. (37).
|
Twenty-five fathers or brothers of 39 patients with Yq deletion were also investigated under the same experimental conditions. None of the male relatives of patients with Yq deletion had histories of cryptorchidism or infertility.
Southern blot analysis
PCR results for the DAZ gene were confirmed in five patients (no. 173, 232, 253, 288, 355). For each patient, 20 µg genomic DNA were digested with HindIII and fractionated by 1% agarose gel electrophoresis. After alkali blotting to nylon membrane (Hybond N+, Amersham Pharmacia Biotech, Milan, Italy), the DNA was prehybridized for 30 min using 12 mL of Rapid-hyb buffer (Amersham Pharmacia Biotech, Milan, Italy). 32P-labeled DNA probes were prepared from PCR product for sY254 by the random primed method (Boerhinger Mannheim, Milan, Italy) and added to the prehybridized membrane at 106 cpm per milliliter of Rapid-hyb Buffer. After 3 hr of hybridization at 60 C, the filter was washed several times in 2 x SSC (3 M NaCl, 0.3 M sodium citrate) and 0.1% SDS for 30 min at room temperature, and in 0.1 x SSC and 0.1% SDS for 30 min at 50 C. Finally, the blot was exposed to Kodak X-Omatic film at -70 C for 48 hr.
|
Results |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
PCR analysis of the Y chromosome performed in 100 normal fertile
men (Group 6) did not detect any abnormalities, while no amplification
was observed in women. Using the criteria listed above, PCR analysis
with this set of Y-DNA markers showed deletions of portions of Yq in 11
of the 40 excryptorchid patients of Group 1 (27.5%) and in 28 out of
the 110 idiopathic patients of Group 3 (25.4%). No deletions were
found in patients of Groups 2, 4, and 5. Table 1 summarizes the classification and Yq
analysis results of subjects recruited in this study.
|
summarizes the PCR results of
the excryptorchid patients with Yq deletions, as discussed below.
Briefly, all deletions were of interstitial portions of the Yq, and 2
or 3 distinct regions were identifiable in interval 5-proximal interval
6 (partially overlapping AZFa and/or AZFb) and in interval 6D
overlapping the DAZ gene in AZFc region. Figure 1B summarizes the
deletions found in patients affected by idiopathic severe
testiculopathy. Two patients had terminal deletions of the Yq
euchromatin, and 1 patient presented with a large deletion involving
AFZb and AZFc, while 25 patients showed interstitial deletions,
distributed in different Yq regions. Sixteen deletions were located in
the AZFc region overlapping the DAZ gene, and 6 of them had concomitant
deletions in interval 5, overlapping AZFa and partially overlapping
AZFb; 2 deletions overlapped the AZFa region; 3 deletions were located
in a region partially including AFZb; 4 deletions overlapped the RBM
gene, either alone or with extension to other intervals.
The father or brothers of 25 patients with Yq deletions (Fig. 1) were
also investigated, and no Yq deletions were found. Figure 2 shows an
example of Southern blot analysis for sY254 (DAZ gene): in fertile men
and in patients without Yq deletions 2 fragments nearly 4 kb in
size are seen, probably reflecting the multiple copies of this
gene, while no bands were observed in patient no. 355 with the
contemporary appearance of a very small band of about 0.2 kb,
confirming the deletion observed by PCR. No fragments were seen in
female DNA. Additional blots with labeled probe and an excess of
unlabeled probe showed fragments of the same weight but at lower
intensity, confirming the identity of such bands (data not shown).
|
reports the seminal pattern,
testicular cytological picture, and hormonal levels of patients
compared with fertile controls. In all patients with severe
testiculopathies significant reductions in testicular volume and
increases in FSH plasma concentrations were evident, without
differences in plasma concentrations of LH and testosterone.
Furthermore, testicular volume and hormonal levels were not different
in patients with and without deletion. Excryptorchid patients
presenting a normal testicular cytological pattern of the descended
testis showed only a reduction in testicular volume of the
excryptorchid testis.
|
|
Discussion |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Testicular FNAC performed on both the excryptorchid and on the normally descended testis allowed us to select two groups of adult unilateral excryptorchid subjects: a group affected by bilateral severe testiculopathy, characterized by great reduction or complete absence of germ cells in both testes, and a group presenting with severe tubular damage in the excryptorchid testis,but with a completely normal spermatogenesis in the scrotal testis. As comparison, we studied a group of infertile patients affected by bilateral severe testiculopathy of unknown origin, a condition that, in recent years, has been associated with Yq microdeletions.
We found a high prevalence of Yq11 microdeletions in unilateral excryptorchid patients affected by severe bilateral testiculopathy (11/40, 27.5%) and in patients affected by severe idiopathic bilateral testiculopathy (28/110, 25.4%). Such microdeletions were not found in fertile controls or in male relatives of patients, allowing us to exclude polymorphisms and to consider them as de novo deletions, probably occurring as mitotic or meiotic error in the germ line of the fertile father. The prevalence of such deletions in cryptorchid subjects was high but not different from that observed in idiopathic severe testiculopathies, in agreement with previous studies reporting that this frequency increases with the severity of the testicular tubular damage (11, 12, 14, 17, 20, 21, 25, 28). Ours is the first study specifically reporting Yq microdeletions in cryptorchid patients; such deletions are probably responsible for a bilateral testicular damage, a consequence of which is cryptorchidism, rather than suggesting a role for the Y chromosome in controlling the testicular descent. In fact, no Yq alteration was found in unilateral excryptorchid patients presenting with moderate oligozoospermia sustained by a normal tubular function in the contralateral descended testis. In these cases the pathogenesis of the maldescent was more likely the result of extragonadal causes (alteration in transabdominal and inguinal testicular descent), and testicular damage may reflect the prolonged cryptorchid location. We have not included a group of bilateral excryptorchid subjects, as the bilateral testiculopathy observed in such patients may be related to the prolonged ectopic location, adding an additional confusing element.
Taken together, Yq microdeletion patterns do not allow us to individuate clear differences in localization and extent of deletions between idiopathic and cryptorchid patients or between azoospermic and severely oligozoospermic men. In cryptorchid patients deletions were distributed in three different Yq regions: the first was localized in interval 6D (AZFc region), including at least the DAZ gene, and it was deleted in 5 out of 11 patients (45.4%); the second, in interval 5D-5M was an overlapping region common to patients no. 59, 71, and 90, including the region between AZFa and AZFb; the third had an overlapping region common to patients no. 71, 90, 317, 46, and 16 in interval 5P-5Q, partially overlapping the proximal part of AZFb region; no specific RBM deletion was observed in any patient. Apart from DAZ, most deletions do not overlap genes with known functions in spermatogenesis; however, new Y-linked genes have been described in these regions (25, 39, 49, 50, 51, 52); therefore, further studies are necessary to clarify the actual role of these genes in spermatogenesis. In addition to these new genes, the deletion presented by patient no. 59 overlaps the AZFa region, for which a new candidate gene (DFFRY) has been identified in interval 5C that seems to be responsible for severe germ cell depopulation (28, 36). The Yq deletions of patients no. 71 and 90 may also involve this new gene, as DFFRY lies between STS markers sY84 and sY87 (28, 38, 39).
Yq deletions were distributed in different regions in idiopathic patients . AZFc deletions overlapping the DAZ gene were the most frequent, and this gene resulted deleted in about 68% of patients. Therefore, according to our observations, the role of the DAZ gene family in disrupting spermatogenesis is strengthened, even if a clear role for this gene in male germ cell development is still lacking. In one third of idiopathic subjects with a DAZ gene deletion there was a concomitant deletion in interval 5 overlapping the AZFa and parts of the AZFb regions. Like cryptorchid subjects, men with idiopathic infertility showed deletions in Yq regions not yet well characterized. In such cases it should be necessary to ascertain the role of new genes recently identified because, for example, patients no. 28, 38, and 111 probably have a deletion involving not only the AZFa-candidate gene DFFRY, but also other genes, such as DBY and UTY (that lie between markers sY87 and sY88 in interval 5D), TB4Y (interval 5D), or BPY1 (interval 5G). Specific deletions in the RBM gene occurred less frequently and in both SCOS and SH, confirming previous data and further supporting a minor role for this gene in determining a severe testiculopathy (11, 12, 13, 15, 16, 17, 18, 21, 22, 23, 28, 52) and not allowing us to correlate testicular phenotype with RBM deletions.
As previously noted (12, 15, 17, 18, 20, 21, 28, 52, 53, 54), PCR analysis frequently showed noncontiguous deletions. The Y chromosome seems to be highly unstable and prone to deletions, probably because it is rich in repetitive elements and repeats (21, 25, 51). Other than reflecting really separated microdeletions, noncontiguous deletions may be explained by other hypotheses, as some STSs may be from repetitive sequences (as demonstrated for sY146, sY153 and sY155) (51), or a complex rearrangement (e.g. inversion with subsequent interstitial deletions) in the father may have occurred.
In conclusion, our findings, even though preliminary, clearly demonstrate for the first time that at least a subgroup of cryptorchid patients shows a deletion in regions of the Y chromosome believed to be important in male germ cell development. The observed Yq deletions seem to be responsible for severe bilateral testicular damage that can be phenotypically expressed by unilateral cryptorchidism, as well as by idiopathic infertility, probably because of altered testicular responses to mechanisms regulating the testicular descent.
|
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received February 8, 1999.
Revised May 14, 1999.
Revised June 30, 1999.
Accepted July 14, 1999.
|
References |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  C. Foresta, D. Zuccarello, A. Garolla, and A. Ferlin Role of Hormones, Genes, and Environment in Human Cryptorchidism Endocr. Rev., August 1, 2008; 29(5): 560 - 580. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Ferlin, B. Arredi, E. Speltra, C. Cazzadore, R. Selice, A. Garolla, A. Lenzi, and C. Foresta Molecular and Clinical Characterization of Y Chromosome Microdeletions in Infertile Men: A 10-Year Experience in Italy J. Clin. Endocrinol. Metab., March 1, 2007; 92(3): 762 - 770. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Hellani, S. Al-Hassan, A. Al-Duraihim, and S. Coskun Y chromosome microdeletions: are they implicated in teratozoospermia? Hum. Reprod., December 1, 2005; 20(12): 3505 - 3509. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Yoshida, M. Fukami, I. Sasagawa, T. Hasegawa, N. Kamatani, and T. Ogata Association of Cryptorchidism with a Specific Haplotype of the Estrogen Receptor {alpha} Gene: Implication for the Susceptibility to Estrogenic Environmental Endocrine Disruptors J. Clin. Endocrinol. Metab., August 1, 2005; 90(8): 4716 - 4721. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Ferlin, A. Garolla, A. Bettella, L. Bartoloni, C. Vinanzi, A. Roverato, and C. Foresta Androgen receptor gene CAG and GGC repeat lengths in cryptorchidism Eur. J. Endocrinol., March 1, 2005; 152(3): 419 - 425. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Foresta, A. Garolla, L. Bartoloni, A. Bettella, and A. Ferlin Genetic Abnormalities among Severely Oligospermic Men Who Are Candidates for Intracytoplasmic Sperm Injection J. Clin. Endocrinol. Metab., January 1, 2005; 90(1): 152 - 156. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Tessari, E. Salata, A. Ferlin, L. Bartoloni, M.L. Slongo, and C. Foresta Characterization of HSFY, a novel AZFb gene on the Y chromosome with a possible role in human spermatogenesis Mol. Hum. Reprod., April 1, 2004; 10(4): 253 - 258. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Ferlin, M. Simonato, L. Bartoloni, G. Rizzo, A. Bettella, T. Dottorini, B. Dallapiccola, and C. Foresta The INSL3-LGR8/GREAT Ligand-Receptor Pair in Human Cryptorchidism J. Clin. Endocrinol. Metab., September 1, 2003; 88(9): 4273 - 4279. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A Ferlin, E Moro, A Rossi, B Dallapiccola, and C Foresta The human Y chromosome's azoospermia factor b (AZFb) region: sequence, structure, and deletion analysis in infertile men J. Med. Genet., January 1, 2003; 40(1): 18 - 24. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. D. Oates, S. Silber, L. G. Brown, and D. C. Page Clinical characterization of 42 oligospermic or azoospermic men with microdeletion of the AZFc region of the Y chromosome, and of 18 children conceived via ICSI Hum. Reprod., November 1, 2002; 17(11): 2813 - 2824. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Peterlin, T. Kunej, J. Sinkovec, N. Gligorievska, and B. Zorn Screening for Y chromosome microdeletions in 226 Slovenian subfertile men Hum. Reprod., January 1, 2002; 17(1): 17 - 24. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Foresta, E. Moro, and A. Ferlin Prognostic value of Y deletion analysis: The role of current methods Hum. Reprod., August 1, 2001; 16(8): 1543 - 1547. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Foresta, A. Bettella, E. Moro, A. Roverato, M. Merico, and A. Ferlin Sertoli Cell Function in Infertile Patients with and without Microdeletions of the Azoospermia Factors on the Y Chromosome Long Arm J. Clin. Endocrinol. Metab., June 1, 2001; 86(6): 2414 - 2419. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Foresta, E. Moro, and A. Ferlin Y Chromosome Microdeletions and Alterations of Spermatogenesis Endocr. Rev., April 1, 2001; 22(2): 226 - 239. [Abstract] [Full Text]
|
|
|
|
|
|
E. Moro, A. Ferlin, P. H. Yen, P. G. Franchi, G. Palka, and C. Foresta Male Infertility Caused by a de Novo Partial Deletion of the DAZ Cluster on the Y Chromosome J. Clin. Endocrinol. Metab., November 1, 2000; 85(11): 4069 - 4073. [Abstract] [Full Text]
|
|
|
|
 |
|
 C. Foresta, A. Ferlin, and E. Moro Deletion and expression analysis of AZFa genes on the human Y chromosome revealed a major role for DBY in male infertility Hum. Mol. Genet., May 1, 2000; 9(8): 1161 - 1169. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
