1 Andrology Unit, Department of Urology, 2 Department of Clinical Genetics and 3 Department of Obstetrics and Gynaecology, Erasmus University Medical Centre, Rotterdam, The Netherlands
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Abstract |
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Key words: gene mutations/chromosomal abnormalities/ICSI/male infertility/Y chromosome microdeletions
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Introduction |
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ICSI is the most significant recent development in the treatment of male infertility, enabling couples who were previously deemed infertile to produce offspring, however with the risk of passing on genetic abnormalities, and possibly decreased fertility. To assess the couple's risk of transmitting a genetic abnormality we analysed the results of a combined andrological, cytogenetic and molecular genetic screening of 150 men with oligozoospermia and azoospermia. In addition, we discuss the necessity of genetic counselling for these infertile couples.
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Materials and methods |
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Semen analysis was performed twice according to the World Health Organization (WHO) guidelines for semen analysis (WHO, 1992) with an interval of at least one month. Laboratory investigations included serum gonadotrophins, testosterone, prolactin and sex hormone binding globulin concentrations.
Cytogenetic analysis was performed on metaphase spreads of cultured lymphocytes. Fluorescence in-situ hybridization (FISH) was applied in case 10 (Kievits et al., 1990) with the probes cp9-23.1 and pDP105, specific for the X and Y chromosome short arm telomere regions and the testis determining region of the Y chromosome respectively.
DNA was extracted according to standard procedure and the presence of sub-microscopic deletions of the AZF region on the Y chromosome long arm was analysed using a multiplex PCR for Y-specific markers, including DYS148 and DYS273 (AZFa), DYS218 and DYS224 (AZFb), SY245 and SY255 (DAZ gene, AZFc region). These sequence-tagged sites (STS) were chosen based on the laboratory guidelines described by Simoni et al. (Simoni et al., 1999).
Twelve common mutations of the CFTR gene were tested (F508, A445E, G542X, 17171G
A, R553X, R117H, R1162X, N1303K, W1282X, 3659delC, E60X and S1251N). The mutations tested comprise about 85% of the mutations identified in the Dutch Caucasian cystic fibrosis population. The length of the T-stretch in intron 8 was tested as previously described (Dohle et al., 1999
).
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Results |
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CFTR gene mutations were identified in 14/150 (9.3.%) patients, associated with congenital abnormalities of the vas deferens in 4 cases. All congenital bilateral absence of the vas deferens (CBAVD) patients showed low volume (<1.0 ml) and low pH (<7.0) semen analysis and azoospermia and had normal abdominal ultrasound investigations. In 2 patients the scrotal vas deferens was present, but on vasography a blind ending was visualized in the distal part of the vas deferens. Both men also had low semen volume, low pH and azoospermia. A single CFTR gene mutation was identified in one. Six (16%) men with azoospermia but without an apparent congenital abnormality of the vas deferens showed a single CFTR mutation. In 5/113 (4.4%) patients with severe oligozoospermia a single CFTR mutation was identified.
A genetic abnormality was present in 36/150 (24.0%) men with severe oligozoospermia and azoospermia. Excluding those with a congenital abnormality of the vas deferens a genetic risk factor was present in 11/32 (34%) of men with azoospermia. In men with extreme oligozoospermia a genetic abnormality was identified in 20/113 (17.6%). Two patients carried two risk factors: 1 Klinefelter patient (47,XXY) also carried a R117H mutation in the CFTR gene; another man was found to have both an AZFc deletion of the Y chromosome and a F508 CFTR gene mutation.
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Discussion |
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An abnormal karyotype was found in 10.6% of the men with severe oligozoospermia or azoospermia. The frequency of karyotypic abnormalities in 1791 males with infertility was found to be as high as 12.67% in azoospermia and 4.6% in oligozoospermia (van Assche et al., 1996; Nakamura et al., 2001). Numerical sex chromosomal abnormalities, present in 63% of all cytogenetic abnormalities in infertile males, were commonly found together with azoospermia. Autosomal anomalies, like Robertsonian and reciprocal translocations and inversions were usually present in men with oligozoospermia. In the Klinefelter syndrome the extra X chromosome was associated with germ cell atresia, whereas in the other cytogenetic abnormalities no specific relation to meiotic disturbances was found as these abnormalities also occurred in men with normal spermatogenesis.
In this study DNA analysis of the AZF-a, -b and -c regions on the Y chromosome showed deletions in 5.3% of the patients. All deletions identified comprised the AZFc region, containing the Deleted in Azoospermia (DAZ) gene (Reijo et al., 1995). The Y chromosomal microdeletions are the most common genetic causes of male infertility due to spermatogenic failure and have been reported in 321% of infertile men (van der Ven et al., 1997
; Simoni, 2001). These microdeletions were associated with severe oligozoospermia and non-obstructive azoospermia, normal physical findings and normal concentrations of gonadotrophins. Histological studies of testicular biopsies from men carrying an AZF deletion exhibit a wide spectrum of spermatogenetic defects from complete absence of germ cells (Sertoli-cell-only syndrome) to maturation arrest with occasional production of mature, condensed spermatids (Reijo et al., 1995
).
Men with defects of the Wolffian duct, presenting as CBAVD, were found to carry different mutations of the CFTR gene (Jaffe and Oates, 1994; Oates and Amos, 1994
). CBAVD, previously described as a genital form of cystic fibrosis, appears to be a heterogeneous clinical and genetic condition. We have shown that some patients with CBAVD also have non-genital symptoms of cystic fibrosis, like defective cellular chloride excretion and disturbed pancreatic function (Dohle et al., 1999
). It has been suggested that CBAVD patients are compound heterozygotes for a severe mutation in one allele in combination with a mild CFTR gene mutation in the other. Alterations in the non-coding regions of the gene, such as the polypyrimidine stretch in intron 8, in combination with a mutation in the other allele, were found to cause abnormal levels of CFTR protein, due to exon 9 skipping translation (Chu et al., 1993
). CFTR intron 8 DNA variants may alter the splicing efficiency of the CFTR mRNA in exon 9 (Kiesewetter et al., 1993
), thus causing reduced concentrations of CFTR protein (Chillon et al, 1995
). Impaired CFTR protein function may cause defective, but not absent chloride excretion resulting in absence of the vas deferens, but not in pulmonary or pancreatic insufficiency (Anguiano et al., 1992
). Mutations of the CFTR gene are commonly associated with obstructions of the male genital tract (Mak et al, 2000
), and not with spermatogenic failure (Pallares-Ruiz et al., 1999
). We detected at least one CFTR mutation in 4/6 men with CBAVD and in 10/144 men without a vas deferens-related problem. This seems to confirm the association of mutations in the CFTR gene with obstruction of the male genital tract rather than with primary testicular failure, although recently some men with CBAVD were found to have a defective spermatogenesis (Meng et al., 2001). However, the rate of mutations detected in this study in the non-obstructed group does seem elevated compared with the carrier risk in the Dutch population of 1:30 (De Vries et al., 1997
). This prompted us to initiate an extended study with a larger cohort of patients. Since we tested previously for mutations that are common among cystic fibrosis patients, we are now using extensive screening methods to evaluate the true rate of CFTR mutations/variants in this group of infertile men.
In this study 38 genetic abnormalities were identified in 36 men from a population of 150 men with severe male infertilty. The application of ICSI in these couples may lead to offspring with an enhanced risk of unbalanced chromosome complement, male infertility due to transmission of an AZF deletion, and cystic fibrosis in the case of related genital abnormalities. We conclude that all men with severe oligozoospermia and azoospermia should be offered genetic testing and counselling before assisted reproduction is applied.
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Notes |
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References |
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Submitted on May 30, 2001; accepted on September 7, 2001.