Human Reproduction, Vol. 17, No. 1, 13-16,
January 2002
© 2002 European Society of Human Reproduction and Embryology
Genetic risk factors in infertile men with severe oligozoospermia and azoospermia
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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BACKGROUND: Male infertility due to severe oligozoospermia and azoospermia has been associated with a number of genetic risk factors. METHODS: In this study 150 men from couples requesting ICSI were investigated for genetic abnormalities, such as constitutive chromosome abnormalities, microdeletions of the Y chromosome (AZF region) and mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. RESULTS: Genetic analysis identified 16/150 (10.6%) abnormal karyotypes, 8/150 (5.3%) AZFc deletions and 14/150 (9.3%) CFTR gene mutations. An abnormal karyotype was found both in men with oligozoospermia and azoospermia: 9 men had a sex-chromosomal aneuploidy, 6 translocations were identified and one marker chromosome was found. Y chromosomal microdeletions were mainly associated with male infertility, due to testicular insufficiency. All deletions identified comprised the AZFc region, containing the Deleted in Azoospermia (DAZ) gene. CFTR gene mutations were commonly seen in men with congenital absence of the vas deferens, but also in 16% of men with azoospermia without any apparent abnormality of the vas deferens. CONCLUSIONS: A genetic abnormality was identified in 36/150 (24%) men with extreme oligozoospermia and azoospermia. Application of ICSI in these couples can result in offspring with an enhanced risk of unbalanced chromosome complement, male infertility due to the transmission of a Y-chromosomal microdeletion, and cystic fibrosis if both partners are CFTR gene mutation carriers. Genetic testing and counselling is clearly indicated for these couples before ICSI is considered.
Key words: gene mutations/chromosomal abnormalities/ICSI/male infertility/Y chromosome microdeletions
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Introduction |
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Male infertility has been associated with several genetic and non-genetic conditions, such as hypogonadotrophic hypogonadism, testicular maldescence, structural abnormalities of the male genital tract, genital infections, previous scrotal or inguinal surgery, varicoceles, chronic illness, medication and exposure to chemicals. In at least 40% of men no cause of the infertility was found (Crosignani et al., 1992). Genetic abnormalities were identified in men with unexplained oligozoospermia and azoospermia, including numerical and structural chromosomal abnormalities (Chandley, 1998
), deletions of the azoospermia factor region (AZF) of the Y chromosome (Reijo et al., 1995; Vogt et al., 1996) and mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, commonly associated with congenital vas deferens abnormalities (Jaffe and Oates, 1994; Dohle et al., 1999). Most numerical chromosome abnormalities and AZF deletions are de-novo events in the parental germ cells. Some abnormalities associated with infertility are inherited, like reciprocal and Robertsonian translocations and CFTR mutations (Mak and Jarvi, 1996). 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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For this study 150 men were selected from couples requesting ICSI who had an infertility duration of at least one year and a semen analysis showing <1x106/ml motile sperm. History taking focused on urogenital development, chronic illness, pulmonary diseases, growth disturbances, medication, male accessory gland infections (MAGI), previous inguinal and scrotal surgery and occupational exposure to heat or chemicals. All men were investigated for genital malformations and ultrasound investigation of the scrotal content was performed. Written, informed consent was obtained from each patient prior to the study commencing.
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, 1717–1G
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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Genetic testing was performed on 37 men with azoospermia (24.7%) and 113 with severe oligozoospermia (75.3%). Table I
shows the results of the andrological evaluation. A testicular tumour was found in 2 cases. In 85 (56.6%) of the andrological evaluations no male infertility associated factor was identified.
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Table II
shows 38 genetic abnormalities identified in 36 (24%) patients. Cytogenetic analysis showed an abnormal result in 16 (10.6 %) patients. A sex-chromosomal aneuploidy was identified in 9 men. The Klinefelter syndrome (47,XXY) was present in 6 patients, of which 3 had azoospermia and 3 had an occasional non-motile sperm in the ejaculate (cryptozoospermia). One azoospermic patient presented a 46,XX karyotype, with Y-short arm DNA sequences present in one of the X chromosome short arms. FSH was high (>7 IU/ml) in all men with a 47,XXY karyotype and in the 46,XX male (33 IU/ml). One Klinefelter patient (case no. 6) also carried a CFTR gene mutation (R117H). Six balanced translocations and one marker chromosome were found in men with oligozoospermia and a normal phenotype. FSH was either normal or slightly elevated, and oligozoospermia was present in all translocations.
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Analysis of the AZF-a, -b and -c regions of the Y chromosome identified 8 (5.3%) men with an AZFc (DAZ) deletion, 5 with severe oligozoospermia and 3 with azoospermia. The deleted markers were sY254 and sY255. A normal testicular volume was present in 5 men and FSH was increased in 3. Six men with a Yq11 microdeletion had unexplained male infertility, one man (case no. 2) also carried a CFTR gene mutation (
F508). 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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Genetic counselling in reproductive medicine starts by obtaining an accurate diagnosis. The family history, physical examination (including evaluation of dysmorphology) and various laboratory tests on both partners are generally required (WHO, 1992
). A substantial number of infertile men, however, present without a history associated with fertility problems and have normal findings on physical examination and endocrine laboratory testing. Only semen analysis is abnormal, often showing a decreased number of sperm cells (oligozoospermia), decreased sperm motility (asthenozoospermia) and many abnormal forms on morphological examination (teratozoospermia). Combinations of these abnormalities occur frequently and are described as the OAT(oligo-astheno-teratozoospermia)-syndrome. These idiopathic forms of male subfertility may be explained by factors like endocrine disruption due to environmental pollution, reactive oxygen species and genetic abnormalities (Crosignani et al., 1992). Although infertility associated with chromosome abnormalities has long been recognized, the introduction of ICSI as a method to overcome severe spermatogenic defects has stimulated many investigators to explore further the genetic basis of male infertility. In this study we confirmed that male infertility, both with and without a detectable cause, was associated with several genetic risk factors such as constitutional chromosome abnormalities, microdeletions of the Y chromosome and mutations in the CFTR gene.
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 3–21% 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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4 To whom correspondence should be addressed at: Department of Urology, University Hospital Rotterdam, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands. E-mail: Dohle{at}urol.azr.nl
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Submitted on May 30, 2001; accepted on September 7, 2001.
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