1 Division of Urology, Department of Surgery, Mount Sinai Hospital, Toronto, Ontario, 2 Department of Genetics, Research Institute, The Hospital for Sick Children, Toronto, Ontario, 3 Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, 4 Programme in Integrative Biology, Research Institute, The Hospital for Sick Children, Toronto, Ontario, 5 Department of Pediatrics, University of Toronto, Toronto, Ontario, 6 Division of Urology, Department of Surgery, University of Toronto, Toronto, Ontario, and 7 Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada
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Abstract |
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Key words: azoospermia/CFTR mutations/cystic fibrosis/male infertility/testicular failure
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Introduction |
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Three alleles have been identified within the polypyrimidine tract of the CFTR intron 8 splice acceptor site (IVS8-T tract), consisting of nine, seven and five thymidines (9T, 7T and 5T) (Chu et al., 1993). The 5T variant produces a lower level of normal CFTR mRNA transcripts than the 9T and 7T alleles (Chu et al., 1993
; Mak et al., 1997
) and has been demonstrated to be significantly associated with some of the above-mentioned conditions, including disseminated bronchiectasis (Pignatti et al., 1996
), CBAVD (Chillon et al., 1995
) and epididymal obstruction (Jarvi et al., 1995
). A number of studies have suggested that the CFTR gene may play a direct role in spermatogenesis (Trezise and Buchwald, 1991
; Trezise et al., 1993
), and that mutations in the gene, including the 5T variant, may have an adverse effect on this process (van der Ven et al., 1996
; Larriba et al., 1998
). We examined the role of CFTR in spermatogenesis by assessing the frequency of CFTR gene mutations and the 5T variant in infertile males with non-obstructive azoospermia due to primary spermatogenic failure.
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Materials and methods |
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CFTR gene mutation analysis
Peripheral venous blood for CFTR genotype analysis was obtained from each of the 45 patients. Genomic DNA was isolated from blood lymphocytes according to standard protocols (Miller et al., 1988), and then subjected to the following molecular evaluations. First, extensive screening for CFTR gene sequence alterations; all 27 exons of the CFTR gene and their flanking intron sequences as well as the promoter region were amplified by the polymerase chain reaction (PCR), and amplicons were subjected to electrophoresis and heteroduplex shift analysis on Hydrolink gel matrix (MDE; FMC Bioproducts, Rockland, MA, USA) in multiplex fashion, followed by DNA transfer to a nylon membrane and hybridization with exon-specific radiolabelled oligonucleotides (Aznarez et al., 1998
); band shifts and unusual patterns were further characterized by direct sequencing of corresponding CFTR regions. Second, analysis of the IVS8-T tract; exon 9 including the IVS8-T tract was PCR-amplified with primers located in introns flanking exon 9 (Zielenski et al., 1991
), and evaluated by allele-specific oligonucleotide hybridization (Kiesewetter et al., 1993
).
Statistical analysis
The 2 statistic, Fisher's exact test, and unpaired t-test were used where appropriate. All P values were based on two-sided comparisons and those < 0.05 were considered to indicate statistical significance.
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Results |
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Discussion |
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Expression studies of CFTR in rodent testes by in-situ hybridization demonstrated maximal expression in round spermatids (Trezise and Buchwald, 1991; Trezise et al., 1993
), suggesting that dysfunctional CFTR may lead to decreased cytoplasmic volume reduction in early spermatids. Similar experiments in human males, however, failed to confirm these findings (Tizzano et al., 1994
). The direct involvement of CFTR in spermatogenesis has also been suggested by the higher than expected frequency of CFTR mutations in men with oligozoospermia (van der Ven et al., 1996
), but the decreased sperm count in these men with CFTR mutations may be secondary to partial reproductive tract obstruction and not to abnormal spermatogenesis (Silber and Rodriguez-Rigau, 1981
). A description was reported recently (Larriba et al., 1998
) of a paucity of mature spermatids in testicular biopsies of CBAVD men with 5T compared with those of CBAVD men with non-5T CFTR mutations, suggesting that the 5T allele may play a role in spermatogenesis. These investigators also recommended that all azoospermic men presenting with infertility undergo CFTR mutation analysis.
To conclude, we did not find an association between CFTR gene mutations and the non-obstructive azoospermia phenotype. However, it must be emphasized that screening for sequence alterations in all exons and their flanking intron sequences and promoter region, as in the present study, does not preclude the presence of mutations within introns of the CFTR gene. In addition, although our subjects did not undergo karyotypic or Y long-arm microdeletion analyses, it is likely that some of them would have chromosomal abnormalities or azoospermia factor region deletions (Mak and Jarvi, 1996). We also did not find a difference between non-obstructive azoospermic men with and without 5T in terms of important clinical parameters that reflect adequacy of the seminiferous epithelium in supporting normal spermatogenesis such as testicular volume, serum FSH concentration and testicular histology. Therefore, our findings support the contention that CFTR does not contribute pathogenetically to the abnormal gametogenesis in primary testicular failure. Whether CFTR plays a role in the capacitation of spermatozoa, as implicated by the decreased fertilization rate using epididymal spermatozoa for in-vitro fertilization from CBAVD men with CFTR mutations compared with spermatozoa from CBAVD men without mutations (Patrizio et al., 1993
), awaits further studies. Finally, although we concur with the general recommendation that all couples contemplating pregnancy should be offered CFTR mutation analysis (National Institutes of Health, 1997
), based on the low frequency of CFTR gene sequence alterations in our study population, we suggest that routine screening of the male patient with non-obstructive azoospermia is not indicated.
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Acknowledgments |
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Financial disclosures
Professor Tsui founded Ellisis, Toronto, which is primarily concerned with cloning the genes associated with inflammatory bowel disease. Professor Tsui was on the scientific advisory board of Visible Genetics Inc., Toronto, which is concerned with DNA sequencing technology. These activities are not directly related to this study. Dr Durie was a medical advisor to Scandipharm, Birmingham, AL, USA, which manufactures pharmaceuticals for cystic fibrosis, but which does not manufacture diagnostic testing materials.
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Notes |
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References |
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Submitted on June 28, 1999; accepted on October 12, 1999.