Complete mutational screening of the cystic fibrosis transmembrane conductance regulator gene: cystic fibrosis mutations are not involved in healthy men with reduced sperm quality

Nathalie Pallares-Ruiz1, Soukeyna Carles1, Marie Des Georges1, Caroline Guittard1, Françoise Arnal2, Claude Humeau2 and Mireille Claustres1,3

1 Laboratoire de Génétique Moléculaire, Institut de Biologie, CHU, CNRS IGH UPR 1142, 2 Hôpital Arnaud de Villeneuve, Laboratoire de Biologie de la Reproduction, 34060 Montpellier Cedex, France


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Based on the analysis of the most frequent mutations responsible for cystic fibrosis (CF), a higher than expected frequency of CF mutations was recently reported in men with infertility due to reduced sperm quality. To further document whether this condition is associated with severe or mild abnormalities of cystic fibrosis transmembrane conductance regulator (CFTR) functions, we carried out a complete scanning of CFTR sequences using a strategy that detects almost all 850 mutations and 150 polymorphisms reported to date in the CFTR gene. We have investigated a cohort of 56 patients with severe oligoasthenoteratozoospermia (OAT) and 50 controls from southern France for CFTR gene mutations and variations. The frequencies of CF-causing mutations and CFTR variations identified in this OAT sample did not differ significantly from the frequencies found in the normal population. However, we observed a 1.7-fold increase in the proportion of homozygotes for a specific CFTR haplotype (TG11-T7-G1540) in the OAT group (P = 0.025). Our results do not confirm a link between CF mutations and reduced sperm quality. Further studies are needed to substantiate the hypothesis that a combination of variants affecting expression and function of the CFTR protein is associated with male infertility.

Key words: cystic fibrosis mutations/CFTR mutant polyvariants/oligoasthenoteratozoospermia


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Male infertility as a result of isolated congenital bilateral absence of the vas deferens (CBAVD) is a recognized primary genital form of cystic fibrosis (CF). When CBAVD is not associated with renal malformations, extensive analysis of the CFTR (cystic fibrosis transmembrane conductance regulator) gene allows mutations to be identified in up to 85% of men (for review, see Meschede et al., 1998). CFTR genotypes responsible for CBAVD commonly consist of a severe, `classic CF' mutation on a gene and a `mild' mutation retaining some residual CFTR activity on the other gene, such as the `5T allele' (Chillon et al., 1995Go; Costes et al., 1995Go; De Meeus et al., 1997Go; Dork et al., 1997Go). Length variants of a polypyrimidine tract within the splice acceptor site at the end of intron 8 of the CFTR gene, named IVS8(T)n, lead to alternative splicing which results in two types of mRNA transcripts, one with and the other without exon 9. Of the three IVS8(T)n alleles (9T, 7T, 5T), the shortest (5T) is associated with the highest rate of incomplete transcripts. mRNA without exon 9 result in CFTR proteins that will not mature, and will therefore not function as chloride channels in the apical membrane of epithelial cells. Among CBAVD patients of European descent, the frequency of the 5T allele is 4- to 6-fold higher than in the general population (Chillon et al., 1995Go; Zielenski et al., 1995Go; Dork et al., 1997Go). The 5T allele is therefore classified as a CBAVD mutation, although with incomplete penetrance since identical genotypes can be found both in CBAVD and in non-CBAVD individuals.

The analysis of CFTR gene mutations has been extended to other forms of sterility such as bilateral ejaculatory duct obstruction (BEDO), isolated anomalies of the seminal vesicles (IASV), congenital unilateral absence of the vas deferens (CUAVD) (Meschede et al., 1997Go) or Young syndrome (Le Lannou et al., 1995Go). Most men with BEDO and a proportion of men with CUAVD without renal anomalies are carriers of CFTR mutations; however, IASV and Young syndrome do not seem to be associated with alterations in the CFTR gene (Meschede et al., 1998Go).

Moreover, studies suggested that CFTR protein, in addition to its role in the development of Wolffian duct-derived structures, could also be involved in sperm maturation (Trezise et al., 1993Go). First, CFTR is expressed in the post-natal human epididymis, and the CFTR protein is localized in the luminal border of the human cauda epididymal epithelium (for review, see Wong, 1998). Second, it has been shown that the CFTR protein contributes to the secretion of electrolytes and water by the epididymal epithelium, a process which is important in the formation of an optimal fluid microenvironment for sperm maturation and transport (Wong, 1998Go). A recent paper reported that CF mutations are present at higher than expected frequency (up to 17.5%) in healthy non-CBAVD men with reduced sperm quality (van der Ven et al., 1996Go). However, these findings were not confirmed by a recent study (Tuerlings et al., 1998Go). As only the most common CF mutations had been searched in both investigations, we undertook the complete analysis of coding and flanking sequences in patients with severe oligoasthenoteratozoospermia (OAT) in order to obtain further insight into a putative involvement of CFTR dysfunction in male sterility. As it has been shown recently (Cuppens et al., 1998aGo) that some of the more common polymorphisms in the CFTR gene affect expression and function of the CFTR protein, we also analysed the distribution of three intragenic markers [TGn and Tn in intron 8, and 1540A/G (M470V) in exon 10] in men with OAT and in controls. The main objective of this study was to compare the complete CFTR genotypes of 56 men with OAT and 50 controls from the same population background, in order to evaluate a putative involvement of CFTR severe or mild mutants and/or variants in infertility due to altered spermatogenesis.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Population sample composition
The study population consisted of 56 unrelated men with OAT (with <2x106/ml spermatozoa) who requested intracytoplasmic sperm injection (ICSI) at the Department of Reproduction, Hospital University Center of Montpellier. After the men had provided written, informed consent for DNA studies, blood samples were collected between May and August 1998. Only men with no detectable chromosomal aberration, no abnormalities of the vas deferens and normal hormone values were included in the study. Fifty healthy individuals (25 males and 25 females) from the general population living in the same geographical area as the OAT patients were studied as controls. None of the persons analysed had any clinical manifestations or family history suggestive of CF. The study was approved by the Clinical Research Committee of the University Hospital Montpellier.

CFTR gene analysis
All 27 exons and flanking regions of the CFTR gene were screened for mutations and polymorphisms by using denaturing gel gradient electrophoresis (DGGE) assay on genomic DNA amplified by polymerase chain reaction (PCR), a procedure performed in our laboratory since 1992 (Claustres et al., 1993Go; Culard et al., 1994Go). Each PCR product with an abnormal DGGE pattern was directly sequenced in order to identify the mutation or the sequence variation that caused abnormal migration. In addition, we searched for mutations 1811+1.6kbA/G in intron 11 and 3849+10kb in intron 19 by restriction analysis after amplification with appropriate primers, as they have significant frequencies in our population (Des Georges et al., 1998Go). The IVS8(T)n acceptor site polymorphism was analysed using an improved procedure derived from a previously described method (Chillon et al., 1995Go). The alleles at intron 8 (TG)n preceding the (T)n were determined using an appropriate DGGE procedure and/or by direct DNA sequencing.

Statistical analysis
{chi}2 test was used to compare differences between proportions. The statistical analyses were performed with SAS statistical software. P-values < 0.05 were considered to indicate statistical significance.


    Results
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
A total length of 7890 nucleotides, covering most of the exonic and flanking CFTR sequences, was examined in each of 56 men with OAT and 50 controls. This extensive analysis resulted in the identification of 25 different sequence alterations (Table IGo).


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Table I. Characterization of CFTR genotypes in 56 patients with oligoasthenoteratozoospermia (OAT) and in 50 controls
 
Confirmed and potentially disease-causing CFTR gene mutations
No patient had a mutation on both copies of the CFTR gene. Four OAT men had a missense mutation on one chromosome, although only mutation D1152H is commonly accepted as a disease-causing mutation and has been demonstrated to reduce chloride currents in vitro (Vankeerberghen et al., 1998Go). F1052V (Mercier et al., 1993Go) and M952I (Girodon et al., 1996Go) have been previously reported in CF and CBAVD patients to the CF Genetics Analysis Consortium (CFGAC, http://www.genet.sickkids.on.ca/CFTR). I1230T, reported here for the first time, results from the nucleotide change 3821T/C in a highly conserved region in exon 19. It could be a deleterious mutation on the basis of the combination of several criteria. This substitution involves a change of a hydrophobic residue (isoleucine) for a polar residue (threonine) whose side chain has an -OH group that can interact with water by forming hydrogen bonds. Therefore, the replacement of an isoleucine by a threonine could lead to the disturbance of a ß-sheet in the second nucleotide-binding fold domain of the CFTR protein and impair the function of the ATP-binding cassette. The change I1230T has not been detected in 960 CF and 300 CBAVD chromosomes that have been genotyped in our laboratory, nor in 200 non-CF chromosomes that we have examined.

In the control group of 50 individuals from the general population, we detected the deletion {Delta}F508 twice, and two possibly but as yet unconfirmed disease-causing missense mutations. G622D has been reported previously (Zielenski et al., 1996Go) in a patient with oligozoospermia, and V562L was identified in a CF patient (Hughes et al., 1995Go).

Other CFTR sequence variations
A total of 18 different sequence changes have been identified in this study, including 11 missense mutations in the coding sequence, six intronic variations and one nucleotide change in the 5' untranslated region (5'UTR) of CFTR (Table IIGo). We detected two double mutant alleles, 1859G/C associated in cis (on the same gene) with 2134C/T, and 3041-71G/C associated in cis with 4002A/G. We did not find significant differences in the frequencies of variants between controls, OAT, and a group of 50 CBAVD patients whose genotypes had been previously analysed in our laboratory. Six variants present on OAT alleles, 1655T/G (F508C), 1716G/A (E528E), 2377C/T (L749L), 405+46G/T, 3499+37G/A and 4374+13A/G were not found in the control group. Variation 3419T/G in exon 17b is a novel sequence change identified in only one subject from the general population, that changes a non-polar (leucine) for a positively charged (arginine) amino acid residue (L1096R) in a highly conserved cytoplasmic loop of the CFTR protein.


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Table II. Frequency distribution of CFTR gene variants in populations from southern France
 
Polyvariants IVS8(TG)n, (T)n and 1540A/G (M470V)
Study of the polypyrimidine tract in front of exon 9 did not reveal significant differences in the distribution of polyT genotypes between OAT and control groups (Table IIIGo). The 5T allele was not detected in this OAT sample, whereas two of 50 controls (both females) were heterozygous for this allele. In contrast, among 50 CBAVD patients selected at random from our series for comparison, the proportion of those carrying at least one 5T allele (38%) was significantly higher than that of the control group (8%) (P < 0.001).


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Table III. Distribution of IVS8(T)n genotypes in males with oligoasthenoteratozoospermia (OAT), congenital absence of the vas deferens (CBAVD) and in a southern French population (controls)
 
Table IVGo shows that the distribution of genotypes at locus 1540 (M470V) in exon 10 was significantly different between the control and OAT (P = 0.046) or CBAVD groups (P = 0.008), and between the OAT and CBAVD groups (P = 0.001). We observed a higher proportion of GG genotypes in men with OAT (55%) than in controls (36%), and a lower proportion in men with CBAVD (18%).


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Table IV. Distribution of genotypes at locus 1540A/G in males with oligoasthenoteratozoospermia (OAT), congenital absence of the vas deferens (CBAVD) and in a southern French population (controls)
 
The distribution of haplotypes at the three loci IVS8(TG)n-(T)n-1540A/G was not significantly different between controls and OAT, excepted for the combination TG11-T7-G1540 (Table VGo). We found a significantly increased proportion of homozygotes for haplotype 11-7-G in men with OAT (54%) compared with the control group (32%) (P < 0.02).


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Table V. Distribution of IVS8(T)n-1540A/G-IVS8(TG)n haplotypes in infertile men with OAT and in controls
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Despite extensive investigation of the CFTR gene, the frequency of mutations identified in this cohort of men with OAT (3.57%) did not differ significantly from the frequency found in the normal southern French population (4%). These frequencies are maximum figures, as several sequence alterations are not definitely proven disease-causing mutations. Our results strongly differ from those published previously (Van der Ven et al., 1996Go), in which a CF mutation frequency of 17.5% was detected in 80 infertile men with reduced sperm quality. It is possible that this discrepancy may result from the inclusion of undetected CBAVD and other types of obstructive azoospermia in the sample studied by others (Van der Ven et al., 1996Go). Our findings are in accordance with those of Tuerlings and co-workers (Tuerlings et al., 1998Go), who studied the three most frequent CF-causing and the three most frequent CBAVD-causing mutations in male candidates for ICSI from the Dutch population and did not find increased mutation frequencies in 75 men with OAT.

Several missense variations identified in this study (R31C, R75Q, F508C, G576A, R668C, or E528E) have previously been described as potentially disease-causing mutations in attenuated CF-related phenotypes such as bronchiectasis (Bombieri et al., 1998Go), non-CBAVD obstructive azoospermia (Kanavakis et al., 1998Go) or CBAVD (Meschede et al., 1993Go; Chillon et al., 1995Go; Dork et al., 1997Go). However, we considered them as neutral variants on the basis of several criteria. First, they are still classified as polymorphisms in the CFGAC. Second, some variants can been found on chromosomes carrying `true' CFTR mutations (for instance, R668C on CBAVD alleles carrying D443Y). Third, some variants can be found in the normal allele of healthy parents of CF children (for instance F508C associated in trans with {Delta}F508 (Desgeorges et al., 1994Go). Fourth, even when they have been demonstrated to reduce CFTR activity in vitro, their involvement in disease remains controversial. For instance, quantitative and qualitative studies in nasal epithelial cells revealed that E528E, which involves the last nucleotide of exon 10 (1716G/A), results in exon 10 skipping (Cuppens et al., 1998bGo). The resulting transcripts without exon 10 fail to mature to fully glycosylated proteins and therefore do not contribute to apical chloride transport activity. However, a role in disease is unlikely as the rate of exon skipping induced by E528E is only 10% (Cuppens et al., 1998bGo).

Analysis of three polymorphic loci with frequent alleles in the general population showed: (i) an increase in the proportion of both the TG11-T7 haplotype and the 1540G allele; and (ii) a 1.7-fold increase of homozygotes for haplotype TG11-T7-1540G in men with OAT compared with the controls. Our data confirm those reported for Canadian populations (Zielenski et al., 1998Go), in which a 2.8-fold increase of homozygotes for TG11-T7-1540G was found compared with the controls, in a cohort of infertile men with oligozoospermia. These observations are interesting in consideration of recent findings on the involvement of specific alleles at the three loci in the modulation of CFTR expression. It was also demonstrated (Cuppens et al., 1998aGo) that, in addition to the known effects of the (T)n locus, the quantity and quality of CFTR transcripts and/or proteins was affected by two other polymorphic loci, (TG)n and 1540A/G. Longer (TG)n and/or shorter (T)n repeats are less favourable for the efficiency of exon 9 splicing, leading to higher proportions of CFTR transcripts lacking exon 9 and non-functional resulting proteins. This explains why, on a TG12 background, a CFTR gene carrying the 5T allele will be fully penetrant as a CBAVD mutation, whereas shorter (TG)n backgrounds will not be deleterious (Cuppens et al., 1998aGo). The 1540A/G locus in exon 10 is the most polymorphic two-allele locus of the CFTR gene and, in contrast with intron 8 (TG)n and (T)n, is polymorphic at the amino acid level, a G allele encoding valine instead of methionine at codon 470 (M470V). By in-vitro studies, it was shown that the V470 allele yielded a lower functional CFTR protein rate than the M470 allele, independently of the intron 8 genotypes (Cuppens et al., 1998aGo). This finding may explain our previous observation of strong linkage disequilibrium between the 5T and the V470 alleles in the CBAVD population, but not in the normal population (de Meeus et al., 1998Go). Thus, although a particular allele may not by itself have deleterious consequences, the combination of specific alleles at several polymorphic loci might result in less functional or even insufficient CFTR protein. It was postulated (Cuppens et al., 1998aGo) that such `polyvariant mutant' genes might be involved in the partial penetrance of CFTR gene mutations (such as the 5T allele), might be responsible for variations in the phenotype of CFTR mutations, and could explain why apparently normal CFTR genes cause disease.

CFTR expression in testis has been determined to occur in round spermatids in rodents, and to begin in the human testis at puberty, suggesting that the CFTR gene could be involved in later steps of the spermatogenesis process (Trezise et al., 1993Go). Recently, a significant reduction in mature spermatids per tubule was observed in CBAVD patients carrying the 5T allele, but not in CBAVD patients carrying one or two CFTR mutations in the absence of the 5T allele (Larriba et al., 1998Go). This observation suggested that CFTR dysfunctions due to mutations may not be commonly implicated in the efficiency of spermatogenesis in CBAVD patients, whereas the severe reduction in normal CFTR mRNA transcripts in the testis resulting from a gene carrying the 5T allele could alter spermatogenesis in CBAVD.

Our data show that neither the 5T allele nor other CFTR mutations are likely to play an important role in the cause of OAT. The factors associated with reduced sperm quality, including other genetic or chromosomal defects (for review, see Meschede and Horst, 1997) remain to be elucidated. Is homozygosity for haplotype TG11-T7-V470 a sufficient condition to alter CFTR function in spermatogenesis or to contribute to OAT in combination with mutations in other genes? In nasal epithelial cells, it has been shown that, on a T7 background, the (TG)11 allele gave a 2.8-fold increase in the proportion of CFTR transcripts that lacked exon 9, compared with the (TG)10 allele, and a V470 protein has a 1.7-fold less efficient chloride channel activity than M470 (Cuppens et al., 1998aGo). The rates of CFTR functional transcripts and proteins derived from haplotype TG11-T7-V470 in sperm cells are unknown.

In conclusion, the results of an extensive analysis of CFTR coding/flanking sequences do not support the involvement of CF or CBAVD mutations in a sample of 56 infertile men with non-CBAVD-reduced sperm quality. If confirmed by further analysis in other populations, these findings will have consequences with regard to the genetic counselling provided to infertile patients treated by in-vitro fertilization. Analysis of haplotype TG11-T7-V470 in a larger cohort will be necessary to substantiate the hypothesis of a putative link between a particular combination of CFTR polymorphisms and male infertility.


    Acknowledgments
 
We thank all the people and families who agreed to participate in this study. We are indebted to our clinician colleagues, Pr. H.Navratil, Dr D.Lauton and Dr A.Faix (Service d'andrologie, CHU de Montpellier) for their contribution. Special thanks are due to Francioise Seguret (Département d'Information Médicale, CHU de Montpellier) for statistical analysis of our data. This research was supported by a grant from the Direction de la Recherche Clinique, CHU, Montpellier, France, UF7533.


    Notes
 
3 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bombieri, C., Benetazzo, M., Saccomani, A. et al. (1998) Complete mutational screening of the CFTR gene in the 120 patients with pulmonary disease. Hum. Genet., 103, 718–722.[ISI][Medline]

Chillon, M., Casals, T., Mercier, B. et al. (1995) Mutations in the cystic fibrosis gene in patients with congenital absence of the vas deferens. N. Engl. J. Med., 332, 1475–1480.[Abstract/Free Full Text]

Claustres, M., Laussel, M., Desgeorges, M. et al. (1993) Analysis of the 27 exons and flanking regions of the cystic fibrosis gene: 40 different mutations account for 91.2% of the mutant alleles in Southern France. Hum. Mol. Genet., 2, 1209–1213.[Abstract]

Costes, B., Girodon, E., Ghanem, N. et al. (1995) Frequent occurrence of the CFTR intron 8 (TG)n 5T allele in men with congenital bilateral absence of the vas deferens. Eur. J. Hum. Genet., 3, 285–293.[ISI][Medline]

Culard, J.F., Desgeorges, M., Costa, P. et al. (1994) Analysis of the whole coding region and splice junctions in azoospermic men with congenital bilateral aplasia of epididymis or vas deferens. Hum. Genet., 93, 467–470.[ISI][Medline]

Cuppens, H., Lin, W., Jaspers, M. et al. (1998a) Polyvariant mutant cystic fibrosis transmembrane conductance regulator genes. The polymorphic (TG)m locus explains the partial penetrance of the T5 polymorphism as a disease mutation. J. Clin. Invest., 101, 487–496.[Abstract/Free Full Text]

Cuppens, H., Jaspers, M., Lin, W. et al. (1998b) Exon 10 skipping resulting from the E528E polymorphism. Ped. Pulmonol., S17, 302.

De Meeus, A., Guittard, C., Desgeorges, M. et al. (1997) Genetic findings in congenital bilateral aplasia of vas deferens patients and identification of six novel mutations. Hum. Mutat., 138, 234–238.

De Meeus, A., Guittard, C., Desgeorges, M. et al. (1998) Linkage disequilibrium between the M470V variant and the IVS8 polyT alleles of the CFTR gene in CBAVD. J. Med. Genet., 35, 594–596.[Abstract]

Desgeorges, M., Kjelleberg, P., Demaille, J. et al. (1994) A healthy male with compound and double heterozygosities for {Delta}F508, F508C, and M470V in exon 10 of the cystic fibrosis gene. Am. J. Hum. Genet., 54, 384–385.[ISI][Medline]

Des Georges, M., Guittard, C., Bozon, D. et al. (1998) Les bases moléculaires de la mucoviscidose en France: plus de 300 mutations et 506 génotypes différents sont en cause. Médecine-Sciences, 14, 1413–1421.

Dork, T., Dworniczak, B., Aulehla-Scholz, C. et al. (1997) Distinct spectrum of CFTR gene mutations in congenital absence of vas deferens. Hum. Genet., 100, 365–377.[ISI][Medline]

Girodon, E. et al. (1996) Newsletter 67. Cystic Fibrosis Genetic Analysis Consortium (http://www.genet.sickkids.on.ca/CFTR).

Hughes, D. et al. (1995) Newsletter 65. Cystic Fibrosis Genetic Analysis Consortium (http://www.genet.sickkids.on.ca/CFTR).

Kanavakis, E., Tzetis, M., Antoniadi, T. et al. (1998) Cystic fibrosis mutation screening in CBAVD patients and men with obstructive azoospermia or severe oligozoospermia. Mol. Hum. Reprod., 4, 333–337.[Abstract]

Larriba, L., Bassas, L., Gimenez, J. et al. (1998) Testicular CFTR splice variants in patients with congenital absence of the vas deferens. Hum. Mol. Genet., 11, 1739–1744.

Le Lannou, D., Jezequel, P., Blayau, M. et al. (1995) Obstructive azoospermia with agenesis of vas deferens or with bronchiectasia (Young's syndrome): a genetic approach. Hum. Reprod., 10, 338–341.[Abstract]

Mercier, B., Lissens, W., Novelli, G. et al. (1993) Identification of eight novel mutations in a collaborative analysis of a part of the second transmembrane domain of the CFTR gene. Genomics, 16, 296–297.[ISI][Medline]

Meschede, D. and Horst, J. (1997) The molecular genetics of male infertility. Mol. Hum. Reprod., 3, 419–430.[Abstract]

Meschede, D., Eigel, A., Horst, J. et al. (1993) Compound heterozygosity for the {Delta}F508 and F508C cystic fibrosis transmembrane conductance regulator (CFTR) mutations in a patient with congenital bilateral aplasia of the vas deferens. Am. J. Hum. Genet., 53, 292–293.[Medline]

Meschede, D., Dworniczak, B., Behre, H.M. et al. (1997) CFTR gene mutations in men with bilateral ejaculatory-duct obstruction and anomalies of the seminal vesicles. Am. J. Hum. Genet., 61, 1200–1202.[ISI][Medline]

Meschede, D., Dworniczak, B., Nieschlag, E. and Horst, J. (1998) Genetic diseases of the seminal ducts. Biomed. Pharmacother., 52, 197–203.[ISI][Medline]

Trezise, A.E., Linder, C.C., Grieger, D. et al. (1993) CFTR expression is regulated during both the cycle of the seminiferous epithelium and the oestrus cycles of rodents. Nature Genet., 3, 157–164.[ISI][Medline]

Tuerlings, J.H., Mol, B., Kremer, J.A. et al. (1998) Mutation frequency of cystic fibrosis transmembrane regulator is not increased in oligozoospermic male candidates for intracytoplasmic sperm injection. Fertil. Steril., 69, 899–903.[ISI][Medline]

Van der Ven, K., Messer, L., van der Ven, H. et al. (1996) Cystic fibrosis mutation screening in healthy men with reduced sperm quality. Hum. Reprod., 11, 513–517.[Abstract]

Vankeerberghen, A., Wei, L., Teng, H. et al. (1998) Characterization of mutations located in exon 18 of the CFTR gene. FEBS Lett., 437, 1–4.[ISI][Medline]

Wong, P.Y.D. (1998) CFTR gene and male fertility. Mol. Hum. Rep., 4, 107–110.[Abstract]

Zielenski, J., Patrizio, P., Corey, M. et al. (1995) CFTR gene variant for patients with congenital absence of vas deferens. Am. J. Hum. Genet., 57, 958–960.[ISI][Medline]

Zielenski, J. et al. (1996) Newsletter 68. Cystic Fibrosis Genetic Analysis Consortium (http://www.genet.sickkids.on.ca/CFTR).

Zielenski, J., Jarvi, K., Ray, P. et al. (1998) Increased risk of infertility associated with DNA specific DNA marker haplotypes in the CFTR locus. Ped. Pulmonol., S17, 338.

Submitted on June 9, 1999; accepted on September 7, 1999.