1 Department of Obstetrics and Gynaecology, National University Hospital, Lower Kent Ridge Road, Singapore 119074
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Abstract |
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Key words: AZF/azoospermia/male infertility/prognosis/Y microdeletions
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Introduction |
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Mapping studies on the Y chromosome have been greatly facilitated by the use of polymerase chain reaction (PCR) techniques to amplify sequence-tagged sites (STS) spanning the deleted regions (Foote et al., 1992; Vollrath et al., 1992
). Yq11.23 also known as deletion interval 6 (DI6) is rich in Y-specific repetitive sequences that may be responsible for its frequent deletion (Yen, 1998
). Using these STS, the presence of submicroscopic deletions in Yq11.23 has been detected in a number of azoospermic and severely oligozoospermic men. However, these deletions were variable both in extent and location, and did not seem to fall into any easily recognizable pattern. For convenience of identification of pattern and analysis, it has been recommended that the AZF locus is divided into three non-overlapping deleted subregions, AZFa, AZFb and AZFc, located in the proximal, central and distal segments of Yq11 respectively (Vogt et al., 1996
).
Recent genetic studies of male infertility have identified at least 15 novel genes or families in the human Y chromosome (Ma et al., 1993; Reijo et al., 1995
; Lahn and Page, 1997
). Stronger candidate genes for the AZF belong to two gene families that encode testis-specific, RNA-binding proteins; the RNA-binding-motif (RBMY) gene family from the AZFb, and the deleted-in-azoospermia (DAZ) from the AZFc subregions (Ma et al., 1993
; Reijo et al., 1995
). The DFFRY gene (Y-linked homologue of the DFFRX Drosophila fat facets related X gene), a potential candidate gene for fertility (Jones et al., 1996
), has recently been mapped to the AZFa subregion but its significance as an AZF candidate is uncertain since it has an active homologue in the X chromosome and the gene product is expressed ubiquitously in adult and embryonic tissues (Brown et al., 1998
). The issue is further complicated very recently by the description of the DBY gene (DEAD/H box polypeptide, Y chromosome) in the AZFa subregion. DBY gene (the human homologue of DEAD box proteins of the mouse PL10 that concerns spermatogenesis) produces two transcripts in which the shorter transcript is expressed exclusively in the testis (Lahn and Page, 1997
; Foresta et al., 2000
). Recently another AZF subregion has been described, AZFd, located between AZFb and AZFc but a candidate gene has not yet been found (Kent-First et al., 1999
).
Since the establishment of a rapid molecular screening programme for the detection of microdeletions in Yq11 and the identification of DAZ gene in the AZF locus as a cause of azoospermia (Henegariu et al., 1994; Reijo et al., 1995
) there has been interest among the geneticists and clinicians to establish a molecular meaning to the causes of idiopathic male infertility and develop a molecular diagnostic tool for male infertility. Over the last few years, there have been numerous publications on microdeletions in the Y chromosome that were determined to establish the distribution of the deletions among different groups of male infertile patients and also characteristics of the deletion. However, there is a large variation among the various studies in reporting the incidence of deletions among their groups of patients. A number of factors have been implicated such as patient selection criteria, experimental designs and ethnic variation (Girardi et al., 1997
; Liow et al., 1998
; Simoni et al., 1998
; Calogero et al., 1999
).
Other authors (Krausz et al., 2000) proposed that a prognosis can be made from the pattern of Y chromosome deletion along the AZF locus. They based their findings on the analysis of their data and a few publications of highly selected patient criteria. Although there is merit in this proposal, some factors need to be resolved before a more conclusive prognosis can be derived from the Y microdeletion analysis.
Firstly, it has been proposed that the deletion of AZFa is associated with lack of germ cells or Sertoli cell-only syndrome (SCOS), and that deletion of AZFb is associated with spermatogenic arrest and deletion of genes within the AZFc would lead to maturation arrest of post-meiotic germ cells (Vogt et al., 1996). Unfortunately, most of these studies do not include histological findings in patients with Y microdeletions. For those few studies that attempt to correlate the type of deletions with phenotype, no definitive conclusions were established (Foresta et al., 1997
, 1998
; Girardi et al., 1997
; Krausz et al., 1999
; Kleiman et al., 1999a
,b
). Therefore, the moment is still not appropriate to derive a prognosis of the phenotype based on the Y chromosome microdeletion analysis. The reason is that this approach is still subjective as it is based on observation from a few patients without the benefit of statistical analysis of significance and in-depth understanding of the role of genes in male infertility. More studies are needed to derive a definitive correlation whose significance can be determined by the odds ratio analysis.
Secondly, the original STS map of Yq11.23 has been revised several times and its accuracy is still questioned (Foote et al, 1992; Jones et al., 1994
; Reijo et al., 1995
; Yen, 1998
). In a PCR analysis of Y chromosome analysis, selected STSs are used that are specific for the Y-specific candidate genes for AZF and those STSs that are mapped adjacent and distal to these genes. Deletion of STS other than those for Y-specific candidate genes could represent clinically irrelevant polymorphism rather than the cause of infertility (Girardi et al., 1997
; Pryor et al., 1997
; Simoni et al., 1998
; Kleiman et al., 1999a
,b
; Krausz et al., 1999
). Although the frequency of microdeletions found is independent of the number of STS used, the higher number of STS would protect against inaccuracy (Simoni et al., 1998
). It has been suggested (van Landuyt et al., 2000
) that only four STS markers representing the three AZF regions and a more distal region in AZFc might be sufficient to detect most of Yq deletions. However, there should be sufficient number of carefully selected STS whose deletions in the AZF subregions would result in defective spermatogenesis. This is to ensure the sensitivity and specificity of the analysis. A number of 2030 STS has been suggested as being sufficient to provide a good coverage of the important regions of the Y chromosome (Pryor and Roberts, 1998
). Recently, the European Academy of Andrology formulated laboratory guidelines for molecular diagnosis of Y chromosomal microdeletions (Simoni et al., 1999
). This is the first attempt to standardize and improve the sensitivity and quality of the diagnosis. Briefly, the guidelines recommend that the diagnosis of Y microdeletions should be carried out by multiplex PCR amplification of genomic DNA, using ZFX/ZFY as an internal PCR control. Autoclaved water and a DNA sample from a fertile male and from a woman were used as a blank, and as external positive and negative controls respectively, that should be run in parallel with each multiplex PCR. The multiplex PCR diagnosis of Y chromosomal deletions is carried out in two steps. In the first step, the primer sets include two STS loci in each AZF subregion that is able to detect >90% of the deletion in the three AZF subregions. Once the deletion has been detected, the subsequent step will include primer sets that can determine the extent of the deletion. STS that are either polymorphic or repetitive should be excluded.
Thirdly, diagnosis of Y chromosomal microdeletion should be simple, reliable, reproducible, time- and cost-effective. Since PCR meets most of the requirements, most laboratories have switched from Southern blot analysis to PCR. PCR analysis of AZF mutations is defined by deletions or absence of the genes. This is in contrast to other PCR-based investigations where detection of mutations is based on the presence of defined amplification products. There are many causes of failure of PCR amplification leading to misintepretation of results. Moreover, genomic DNA that has been stored for a very long time inexplicably becomes less amenable to amplification of sequences of >200 bp (Ma et al., 2000). It is also possible that conventional PCR techniques will not detect mosaicisms where deletion is present in the germ line but where somatic DNA is normal (Simoni et al., 1998
; Bourhis et al., 2000
).
Fourthly, the deletion of the DAZ gene does not always result in azoospermia. Various studies have reported that some DAZ-deleted men can still produce spermatozoa in their ejaculate even though they are severely oligozoospermic (Girardi et al., 1997; Liow et al., 1998
; van Landuyt et al., 2000
). There are multiple copies of the gene which are polymorphic in the DAZ repeat region (Saxena et al., 1996
; Yen et al., 1997
). Partial loss of the DAZ gene copies (dosage effect) may lead to oligozoospermia (rather than azoospermia) because of insufficient quantities of transcripts (Ma et al., 2000
). In most cases of Y microdeletion analysis, the RBMY gene is seldom deleted in both azoospermic and oligozoospermic men. One possible reason is that a critical copy of the RBMY gene families could be missing even though a product is amplified (Krausz et al, 1999
).
Since intracytoplasmic sperm injection (ICSI) has been widely used as an effective tool in overcoming male infertility, it raises the possibility of genetic transmission of infertility from father to son. There have been instances in which subfertile men have similar deletions observed in lymphocyte and testicular DNA (Kleiman et al., 1999a) and male offspring have inherited the Y microdeletions from their father (Kent-First et al., 1996
; Vogt et al., 1996
; Mulhall et al., 1997
; Pryor et al., 1997
; Chang et al., 1999
; Kamischke et al., 1999
; Kleiman et al., 1999b
; Page et al., 1999
). In this instance, Y microdeletion analysis provides a useful diagnostic tool in identifying these male patients so that proper counselling can be given before treatment by ICSI.
Although the functions of the genes present in the AZF locus have yet to be determined the patterns of expression of these genes during spermatogenesis are likely to be the result of multiple processes that involve the developmental phases of spermatogenic cells (mitotic, meiotic and post-meiotic phases) and regulation of gene expression that occur at the transcription, translation and post-translation levels. The expression of these genes is primarily determined by the intrinsic genetic programme of spermatogenetic cells (Eddy, 1998), since azoospermic and severely oligozoospermic men with Y microdeletions are otherwise healthy and extrinsic factors that can affect spermatogenesis have been excluded. The number of spermatozoa in some men with Y microdeletions have been observed to decrease over a period of time (Girardi et al., 1997
; Simoni et al., 1997
). Thus, genes that are present in multiple copies (e.g. DAZ and RBMY) may deplete over time, resulting in oligozoospermia and eventually azoospermia. Early detection of Y microdeletions in men and their ICSI offspring can improve the clinical management of these patients. Sperm cryopreservation and testicular biopsy are two possible options that can be considered before their condition deteriorates.
In conclusion, accurate interpretation of the results of molecular diagnosis of Y chromosomal microdeletions is necessary for clinical management. Phenotypic prognosis based on the pattern of Y microdeletions is rather subjective and inconclusive even though there is a cause-and-effect relationship between Y chromosomal microdeletions and male infertility. A definite prognosis can only be made when a certain genotype is strongly correlated with a phenotype that is based not only on the pattern of Y microdeletions, but also the knowledge of how these genes function and regulate spermatogenesis. This can be achieved when laboratories follow a set of guidelines that have been formulated to improve the sensitivity and accuracy of PCR diagnosis of Y chromosomal microdeletions, so as to prevent the outcome of the diagnosis from being overestimated. In addition, knowledge derived from research to determine the functional role of genes on the AZF locus and their homologues in the regulation of spermatogenesis would enable clinicians to make a sound prognosis and to counsel their patients more effectively.
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Notes |
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This debate was previously published on Webtrack, www.oup.co.uk/humrep/comment on October 10, 2000
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References |
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Brown, G.M., Furlong, R.A., Sargeant, C.A. et al. (1998) Characterisation of the coding sequence and fine mapping of the human DFFRY gene and comparative expression analysis and mapping to the Sxr interval of the mouse Y chromosome of the Dffry gene. Hum. Mol. Genet., 7, 97107.
Calogero, A.E., Garofalo, M.R. and D'Agata, R. (1999) Factors influencing the variable incidence of Y chromosome microdeletions in infertile patients. Current status of the molecular diagnosis of Y-chromosomal microdeletions in the work-up of male infertility. Hum. Reprod., 14, 275.
Chang, P.L., Sauer, M.V. and Brown, S. (1999) Y microdeletion in a father and his four infertile sons. Hum. Reprod., 14, 26892694.
Eddy, E.M. (1998) Regulation of gene expression during spermatogenesis. Sem. Cell,. Dev. Biol., 9, 451457.[ISI][Medline]
Foote, S., Vollrath, D., Hilton, A. et al. (1992) The human Y chromosome: Overlapping DNA clones spanning the euchromatic region. Science, 258, 6066.[ISI][Medline]
Foresta, C., Ferlin, A., Garolla, A. et al. (1997) Y-chromosome deletions in idiopathic severe testiculopathies. J. Clin. Endocrinol. Metab., 82, 10751080.
Foresta, C, Ferlin, A., Garolla, A. et al. (1998) High frequency of well-defined Y-chromosomal deletions in idiopathic Sertoli cell-only syndrome. Hum. Reprod., 13, 302307.[ISI][Medline]
Foresta, C., Ferlin, A. and Moro, E. (2000) Deletion and expression analysis of AZFa genes on the human Y chromosome revealed a major role for DBY in male infertility. Hum. Mol. Genet., 9, 11611169.
Girardi, S.K., Mielnik, A. and Schlegel, P.N. (1997) Submicroscopic deletions in the Y chromosome of infertile men. Hum. Reprod., 12, 16351641.[Abstract]
Henegariu, O., Hirschmann, P., Kilian, K. et al. (1994) Rapid screening of the Y chromosome in idiopathic sterile men, diagnostic for deletions in AZF, a genetic Y factor expressed during spermatogenesis. Andrologia, 26, 97106.[ISI][Medline]
Jones, M.H., Khwaja, O.S.A., Briggs, H. et al. (1994) A set of ninety-seven overlapping yeast artificial chromosome clones spanning the human Y chromosome euchromatin. Genomics, 24, 266275.[ISI][Medline]
Jones, M.H., Furlong, R.A., Burkin, H. et al. (1996) The Drosophila developmental gene fat facets ha a human homologue in Xp11.4 which escapes X-inactivation and has related sequences on Yq11.2. Hum. Mol. Genet., 5, 16951701.
Kamischke, A., Gromoll, J., Simoni, M. et al. (1999) Transmission of a Y chromosomal deletion involving the deleted in azoospermia (DAZ) and chromodomain (CDY1) genes from father to son through intracytoplasmic sperm injection. Hum. Reprod., 14, 23202322.
Kent-First, M.G., Kol, S., Muallem, A. et al. (1996) The incidence and possible relevance of Y-linked microdeletions in babies born after intracytoplasmic sperm injection and their infertile fathers. Mol. Hum. Reprod., 2, 943950.[Abstract]
Kent-First, M., Muallem, A., Shultz, J. et al. (1999) Defining regions of the Y-chromosome responsible for male infertility and identification of a fourth AZF region (AZFd) by Y-chromosome microdeletion detection. Mol. Reprod. Dev., 53, 2741.[ISI][Medline]
Kleiman, S.E., Yogev, L., Gamzu, R. et al. (1999a) Genetic evaluation of infertile men. Hum. Reprod., 14, 3338.
Kleiman, S.E., Yogev, L., Gamzu, R. et al. (1999b) Three-generation evaluation of Y-chromosome microdeletion. J. Androl., 20, 394398.
Krausz, C., Bussani-Mastellone, C., Granchi, S. et al. (1999) Screening for microdeletion of Y chromosome genes in patients undergoing intracytoplasmic sperm injection. Hum. Reprod., 14, 17171721.
Krausz, C., Quintana-Murci, L. and McElreavey, K. (2000) What is the clinical prognostic value of Y chromosome microdeletion analysis? Hum. Reprod., 15, 14311434.
Lahn, B.T. and Page, D.C. (1997) Functional coherence of the human Y chromosome. Science, 278, 675680.
Liow, S.L. Ghadessy, F.J., Ng, S.C. et al. (1998) Y chromosome microdeletions, in azoospermic or near-azoospermic subjects, are located in the AZFc (DAZ) subregion. Mol. Hum. Reprod., 4, 763768.[Abstract]
Ma, K., Inglis, J.D., Sharkey, A. et al. (1993) A Y chromosome gene family with RNA-binding protein homology: candidates for the azoospermic factor AZF controlling human spermatogenesis. Cell, 75, 12871295.[ISI][Medline]
Ma, K., Mallidis, C. and Bhasin, S. (2000) The role of Y chromosome deletions in male infertility. Eur. J. Endocrinology, 142, 418430.[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]
Page, D.C., Silber, S. and Brown, L.G. (1999) Men with infertility caused by AZFc deletion can produce sons by intracytoplasmic sperm injection, but are likely to transmit the deletion and infertility. Hum. Reprod., 14, 17221726.
Pryor, J.L. and Roberts, K.P. (1998) Principles of sequence-tagged site selection in screening for Y deletions. Current status of the molecular diagnosis of Y-chromosomal microdeletions in the work-up of male infertility. Hum. Reprod., 13, 1768.[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.
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]
Saxena, R., Brown, L.G., Hawkins, T. et al. (1996) The DAZ gene cluster on the human Y chromosome arose from an autosomal gene that was transposed, repeatedly amplified and pruned. Nature Genet., 14, 292299.[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 oligospermia. Fertil. Steril., 67, 542547.[ISI][Medline]
Simoni, M., Kamischka, A. and Nieschlag, E. (1998) Initiative for international quality control. Current status of the molecular diagnosis of Y-chromosomal microdeletions in the work-up of male infertility. Hum. Reprod., 13, 17641768.
Simoni, M., Bakker, M.C., Eurlings, M.C.M. et al. (1999) Laboratory guidelines for molecular diagnosis of Y chromosomal microdeletions. Int. J. Androl., 22, 292299.[ISI][Medline]
Tiepolo, L. and Zuffardi, O. (1976) Localization of factors controlling spermatogenesis in the nonfluorescent portion of the Y chromosome. Hum. Genet., 34, 119224.[ISI][Medline]
van Landuyt, L., Lissens, W., Stouffs, K. et al. (2000) Validation of a simple Yq deletion screening programme in an ICSI candidate population. Mol. Hum. Reprod., 6, 291297.
Vogt, P.H., Edelmann, A., Kirsch, 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]
Yen, P.H. (1998) A long-range restriction map of deletion interval 6 of the human Y chromosome: A region frequently deleted in azoospermic males. Genomics, 54, 512.[ISI][Medline]
Yen, P.H., Chai, N.N. and Salido, E.C. (1997) The human DAZ genes, a putative male infertility factor on the Y chromosome, are polymorphic in the DAZ repeat regions. Mammal. Genome, 8, 756759.[ISI][Medline]