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
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Abstract |
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Key words: infertility/ICSI/screening/Y chromosome
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Introduction |
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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 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 azoo- and 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.
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Materials and methods |
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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.
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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 (<5x106 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.
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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 32P-labelled probes.
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Results |
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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.
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Discussion |
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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.
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Acknowledgments |
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Notes |
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References |
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Chai, N.N., Salido, E.C. and Yen, P.H. (1997) Multiple functional copies of the RBM gene family, a spermatogenesis candidate on the human Y chromosome. Genomics, 45, 355361.[ISI][Medline]
Foresta, C., Ferlin, A., Garolla, A. et al. (1997) Y-chromosome deletions in idiopathic severe testiculopathies. J. Clin. Endocrinol. Metabol., 82, 10751080.
Foresta, C., Ferlin, A., Garolla, A., et al. (1998) High frequency of well-defined Y-chromosome deletions in idiopathic Sertoli cell-only syndrome. Hum. Reprod., 13, 302307.[ISI][Medline]
Girardi, S.K. Mielnik, A. and Schlegel, P.N. (1997) Submicroscopic deletions in the Y chromosome of infertile men. Hum. Reprod., 12, 16351641.[Abstract]
Kremer, J.A.M., Tuerlings, J.H.A.M., Meuleman, E.J.H. et al. (1997) Microdeletions of the Y chromosome and intracytoplasmic sperm injection: from gene to clinic. Hum. Reprod., 12, 687691.[Abstract]
Lahn, B.T. and Page, D. (1998) Functional coherence of the human Y chromosome. Science, 278, 675680.
Ma, K., Inglis, J., Sharkey, A. et al. (1993) A Y chromosome gene family with RNA-binding protein homology: candidates for the azoospermia factor AZF controlling spermatogenesis. Cell, 75, 12871295.[ISI][Medline]
Mulhall, J.P., Reijo, R., Alagappan, R. et al. (1997) Azoospermic men with deletion of the DAZ gene cluster are capable of completing spermatogenesis: fertilization, normal embryonic development and pregnancy occur when retrieved testicular spermatozoa are used for intracytoplasmic sperm injection. Hum. Reprod., 12, 503508.[ISI][Medline]
Najmabadi, H., Huang, V., Yen, P. et al. (1996) Substantial prevalence of microdeletions of the Y-chromosome in infertile men with idiopathic azoospermia and oligospermia detected using a sequence tagged site based mapping strategy. J. Clin. Endcrinol. Metabol., 81, 13471352.[Abstract]
Nakahori, Y., Kuroki, Y., Komaki, R., et al. (1996) The Y chromosome region essential for spermatogenesis. Horm. Res., 46 (Suppl. 1), 2023.[ISI][Medline]
Oliva, R., Margarit, E., Ballesca, J.L. et al. (1998) Prevalence of Y chromosome microdeletions in oligozoospermic and azoospermic candidates for intracytoplasmic sperm injection. Fertil. Steril., 70, 506510.[ISI][Medline]
Pryor, J.L., Kent-First, M., Muallem, A. et al. (1997) Microdeletions in the Y chromosome of infertile men. N. Engl. J. Med., 336, 534539.
Quereshi, S.J., Ross, A.R., Ma, K. et al. (1996) PCR screening for Y chromosome microdeletions: a first step towards the diagnosis of genetically determined spermatogenic failure in men. Mol. Hum. Reprod., 2, 775779.[Abstract]
Reijo, R., Lee, T.Y., Salo, P. et al. (1995) Diverse spermatogenic defects in humans caused by Y chromosome deletions encompassing a novel RNA-binding protein gene. Nature Genet., 10, 383393.[ISI][Medline]
Reijo, R., Alagappan, R.K., Patrizio, P. et al. (1996) Severe oligospermia resulting from deletions of azoospermia factor gene on Y chromosome. Lancet, 347, 12901293.[ISI][Medline]
Selva, J., Kanafani, S., Prigent, Y. et al. (1997) Incidence of AZF (azoospermia factor) deletions and familial forms of infertility among patients requiring Intracytoplasmic spermatozoa injection (ICSI). J. Assist. Reprod. Genet., 14, 593595.[ISI][Medline]
Simoni, M., Gromoll, J., Dworniczak, B. et al. (1997) Screening for deletions of the Y chromosome involving the DAZ (deleted in azoospermia) gene in azoospermia and severe oligozoospermia. Fertil. Steril., 67, 542547.[ISI][Medline]
Simoni, M., Kamishke, A. and Nieschlag, E. (1998) Current status of the molecular diagnosis of Y-chromosomal microdeletions in the work up of male fertility. Hum. Reprod., 13, 17641768.
Stuppia, L., Mastroprimiano, G., Calabrese, G. et al. (1996) Microdeletions in interval 6 of the Y chromosome detected by STS-PCR in 6 of 33 patients with idiopathic oligo- and azoospermia. Cytogenet. Cell Genet., 72, 155158.[ISI][Medline]
Stuppia, L., Gatta, V., Calabrese, G. et al. (1998) A quarter of men with idiopathic oligo-azoospermia display chromosomal abnormalities and microdeletions of different types in interval 6 of Yq11. Hum. Genet., 102, 566570.[ISI][Medline]
Van der Ven, K., Montag, M., Peshka, B. et al. (1997) Combined cytogenetic and Y chromosome microdeletion screening in males undergoing intracytoplasmic sperm injection. Mol. Hum. Reprod., 3, 699704.[Abstract]
Vereb, M., Agulnik, A.I. Houston, J.T. et al. (1997) Absence of DAZ gene mutations in cases of non-obstructed azoospermia. Mol. Hum. Reprod., 3, 5559.[Abstract]
Vogt, P.H., Edelmann, A., Kirsh, S. et al. (1996) Human Y chromosome azoospermia factors (AZF) mapped to different subregions in Yq11. Hum. Mol. Genet., 5, 933943.
Vollrath, D., Foote, S., Hilton, A. et al. (1992) The human Y chromosome: a 43-interval map based on naturally occurring deletions. Science, 258, 5259.[ISI][Medline]
World Health Organization (1992) Laboratory Manual for the Examination of Human Semen and SemenCervical Mucus Interaction, 3rd edn. Cambridge University Press, Cambridge.
Submitted on December 2, 1998; accepted on March 12, 1999.