Molecular Human Reproduction, Vol. 5, No. 6, 587-593,
June 1999
© 1999 European Society of Human Reproduction and Embryology
Screening for cystic fibrosis transmembrane conductance regulator gene mutations in men included in an intracytoplasmic sperm injection programme
1 Biologie de la Reproduction, CECOS and 2 Laboratoire de Biochimie Médicale – Biologie Moléculaire, CHU, 63000 Clermont-Ferrand, France
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Abstract |
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The present study was undertaken to evaluate the frequency and nature of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in infertile patients undergoing intracytoplasmic sperm injection. A total of 90 patients were screened for a panel of 10 mutations in the CFTR gene frequently involved in congenital absence of the vas deferens (CAVD); the patients included 14 with azoospermia and CAVD, 39 patients with azoospermia without CAVD (n = 39) and 37 patients with severe oligozoospermia. The length of the polymorphic polypyrimidine tract (allele 5T, 7T and 9T) in the intron 8/exon 9 splice-acceptor site was also determined. In 10 out of 14 patients with CAVD, CFTR mutations were found; nine patients had one
ISOdiaF508 mutation and one patient had two CFTR mutations (N1303K/R117H). Allele 5T was present in eight of these patients. In six patients, 5T was the non-ISOdiaF508 allele and in two patients there was no known CFTR mutation. None of the CFTR mutations were observed in patients with azoospermia without CAVD or with severe oligozoospermia and the frequency of allele 5T was 3.6% (three out of 78 alleles) and 1.35% (one out of 74 alleles) respectively. Our observation suggests that the CFTR gene is not involved in either spermatogenesis or in the pathology of the genital tract, except for CAVD. azoospermia/CFTR mutations/congenital absence of vas deferens/ICSI/oligozoospermia
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Introduction |
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TopAbstractIntroductionReferences |
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An important percentage of severe male fertility problems are related to genetic abnormalities. Among them, infertility caused by obstructive azoospermia has been described in >95% of men with cystic fibrosis (CF) (Lissens et al., 1996
). In addition, it has been established that 60–70% of patients with congenital bilateral absence of the vas deferens (CBAVD) have mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, with no other clinical symptoms of CF (De Braekeleer and Ferec, 1996).
Studies in animals, and cytological observations in man, have suggested a direct involvement of the CFTR protein in spermatogenesis and/or sperm maturation. CFTR mRNA was detected in rat testicular tissue, where it was expressed in post-meiotic round spermatids (Trezise et al., 1993b). Histological studies of testicular tissue in men with clinical CF and CBAVD showed possible cytological abnormalities that preferentially occur at the spermatid stages (Kaplan et al., 1968; Gottlieb et al., 1991) and a reduction in the number of mature spermatozoa found in testicular biopsy (Holsclaw et al., 1971).
A correlation between CF mutations and in-vitro fertilizing capability of epididymal spermatozoa retrieved from patients with congenital absence of the vas deferens (CAVD) has been demonstrated. The fertilization rate was significantly reduced in men with CAVD associated with F508 mutation, compared with men with CAVD without known mutations (Patrizio et al., 1993). In contrast, CFTR gene mutations did not seem to affect sperm function during in-vitro fertilization (IVF) with micromanipulation (Silber et al., 1994; Schlegel et al., 1995).
Numerous studies have examined the possible relationship between CFTR mutations and infertility in patients without CAVD. One CFTR mutation was found in seven out of 56 (12.5%) infertile men (Traystman et al., 1994), while Jarvi et al. detected CFTR gene mutations in three out of 17 (18%) men with obstructive azoospermia without CBAVD (Jarvi et al., 1995). Recently, it has been reported that 14/80 (17.5%) healthy men with infertility due to reduced sperm quality and three out of 21 (14.5%) men with azoospermia without CBAVD had at least one CF mutation (Van Der Ven et al., 1996); these frequencies were higher than the expected CF carrier frequency in the general population.
In contrast, in men with spermatogenetic failure, only one out of 18 (6%) carried a CFTR mutation (Jarvi et al., 1995) and the frequency of CFTR mutations in oligoasthenoteratozoospermic male candidates for intracytoplasmic sperm injection (ICSI) did not differ from the frequency found in the general population; a CFTR mutation was found in four out of 75 (5.3%) patients (Tuerlings et al., 1998).
The aim of this study was to evaluate the frequency and nature of mutations in the CFTR gene in men with azoospermia or severe oligozoospermia, presenting no other genetic abnormalities, and who were included in an ICSI programme.
Materials and methods
Patient population
The study population consisted of men attending the centre of Developmental and Reproductive Biology, Hospital of Clermont-Ferrand, France, for infertility treatment.
A full history was obtained from each subject. The majority of the patients had seen an andrologist prior to presenting at our centre and a complete reproductive physical examination had been performed; they had no history of operative sterilization. Men with a chromosomal aberration or a Y chromosome microdeletion were excluded from this study. None of the patients had any history of CF or showed clinical signs or symptoms of CF. However, sweat tests were not performed and it was not possible to obtain objective measurements of pancreatic status or pulmonary function.
Andrological diagnosis was based on at least two semen analyses. Semen was collected by masturbation after 2–5 days of sexual abstinence. Analyses, including volume, pH, sperm concentration, motility, viability and morphology, were performed in our laboratory according to standard World Health Organization (WHO, 1992) criteria. In the majority of samples, fructose and 
-glucosidase concentrations were measured in seminal plasma according to WHO guidelines and an enzymatic assay was used for the determination of L-carnitine (Grizard et al., 1992).
The combination of small ejaculate volumes, acid pH and low or absent fructose and/or epididymal markers can be found in patients with obstructions of the genital tract. Therefore, these markers were measured in all the azoospermic men who had not undergone any surgical exploration, in order to detect patients with an obstructive azoospermia.
Traditionally, serum concentrations of follicle stimulating hormone (FSH) have been considered to be the most sensitive markers of spermatogenesis, so plasma FSH concentrations were measured in the majority of patients. Moreover, because they participated in an ICSI programme, testicular and/or epididymal biopsies were performed in all azoospermic men.
Patients were classified into three groups: group I was composed of 14 men with CAVD (Table I); 12 had CBAVD and one patient had also an unilateral renal agenesis. Two patients had congenital unilateral absence of the vas deferens (CUAVD) with an obstruction on the controlateral side. All the patients were azoospermic, although spermatozoa were present in the epididymal and/or testicular biopsies. They had normal plasma FSH concentrations, small ejaculate volumes (generally <1 ml) and the pH of the ejaculate was 
7.4. When measured, fructose was very low or absent (normal: 
25 µmoles/ejaculate), -glucosidase was 5 mIU/ejaculate (normal 35 mIU/ejaculate) and carnitine was 40 nmoles/ejaculate (normal 260 nmoles/ejaculate) (Table I).
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Group II included 39 men with azoospermia without CAVD (Table I
). In three patients, the level of plasma FSH was increased (>15 mIU/ml). Spermatozoa were found in testicular and/or epididymal biopsies except in three patients. The presence of spermatozoa in biopsies associated with low levels of seminal biochemical markers reveals an obstructive syndrome whereas in other azoospermic patients with biochemical markers in normal range, the presence of spermatozoa suggests either a very reduced spermatogenesis or an abnormality of the epididymal duct close to the testis or even at the level of the rete testis (Table I).
Group III was composed of 37 patients with severe oligozoospermia (Table II). Sperm counts were between 0.01x106 and 4x106 spermatozoa per ml. For four patients, no spermatozoa were motile and for 15 patients, the percentage of spermatozoa with normal morphology was <25%. The values of plasma FSH were elevated for 13 patients. In all cases, seminal biochemical markers were in the normal range except for one patient with a low concentration of -glucosidase (Table II).
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Analysis of CFTR mutations
Genomic DNA was extracted from peripheral blood samples taken from the patients (Bowtell, 1987
). Each patient was tested for the nine most frequent cystic fibrosis-causing CFTR mutations: F508, I507, 1717–1G
A, G542X, G551D, R553X, W1282X, N1303K, 621+1GT and the three most frequent CFTR mutations involved in CBAVD (F508, R117H and the IVS8 polyT).
Each sample was tested for F508 by heteroduplex analyses (Rommens et al., 1990). The other mutations were detected using either heteroduplex analysis (I507), allele specific oligonucleotide (ASO) hybridization (G542X, 1717–1GA, IVS8 polyT) (Kerem et al., 1990), restriction endonuclease analysis (G551D, R553X, W1282X) (Zielenski et al., 1991) or polymerase chain reaction (PCR)-mediated site-directed mutagenesis (621+1GT, R117H, N1303K) (Friedman et al., 1991).
The CFTR intron 8 polypyrimidine tract length variants were detected after PCR amplification using primers from the flanking introns of CFTR exon 9 (Zielenski et al, 1991) and hybridization with allele-specific oligonucleotides for the 5T, 7T or 9T alleles as previously described (Kiesewetter et al, 1993).
PCR amplification
Extracted DNA was amplified using the primers listed in Table III. Reactions were carried out with 250 ng genomic DNA in a total volume of 50 µl containing 10 mM Tris–HCl (pH9), 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 0.2 mg/ml bovine serum albumin, 200 µM of each deoxynucleotide triphosphate, 1.5 IU of Taq DNA polymerase (Appligene-Oncor, Illkirch, France) and 50 pmoles of the appropriate forward and reverse primers.
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Amplification began with a 10 min incubation at 94°C, and proceeded with 28 cycles, each consisting of 1 min of denaturation at 91°C, 45 s of annealing at appropriate temperature and 1 min of extension at 70°C; with a 10 min incubation at 70°C completing the amplification.
Heteroduplex analysis
(Figure 1) I507 and F508 were directly detected after DNA amplification on 12% polyacrylamide gel electrophoresis. Each DNA was amplified in three different reactions: (i) patient DNA; (ii) patient DNA co-amplified with 250 ng of normal control DNA; and (iii) patient DNA co-amplified with 250 ng homozygous F508 control DNA; control DNAs acted as heteroduplex generators.
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Restriction digestion and electrophoresis
(Figures 2 and 3
) Detection of 621+1GT, R117H, G551D, R553X, W1282X and N1303K were performed using appropriate restriction enzymes (New England Biolabs, Ozyme, Saint Quentin Yvelines, France) as described in Table IV. After PCR, 15 µl of amplified product were incubated as directed for 2 h. Products were analysed by electrophoresis on polyacrylamide gel. The gel was then stained with ethidium bromide for 5 min. Results for each mutation are listed in Table IV.
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ASO hybridization
PCR-amplified DNA (6 µl) of exon 9 or exon 11 was denatured and vacuumblotted onto nylon membrane (Hybond N+, Amersham Pharmacia Biotech, Orsay, France) and hybridized with oligonucleotides which recognize the presence of 5T, 7T or 9T for detection of the IVS8 polyT-stretch, normal or mutated allele for 1717–1G
A and G542X.
Results
Eleven mutations (among the 10 most prevalent mutations in the CFTR gene) were detected on 11 chromosomes from the 14 men with CAVD, yielding a CF mutation frequency of 39.3%. The 5T allele was detected in eight out of 14 patients (an allele frequency of 28.5%) (Table I). One patient was a compound heterozygote, R117H/N1303K. Of the nine patients in whom the CF screening test was able to identify a single mutation, all had the F508 mutation and six (66%) had a 5T allele on the other homologue. The 5T allele was observed in two patients in whom no CFTR mutation could be detected. Neither a CF mutation nor a 5T allele was found in the patient with CBAVD and unilateral renal agenesis. One patient with CUAVD had a F508 mutation and 5T allele on the other copy whereas another patient with CUAVD had no CF mutation but had a 5T allele. No CF mutations were observed in either the 39 patients with azoospermia but without CAVD, or in the 37 patients with oligozoospermia.
The 7T variant was the common allele found with a frequency of 90% (70 out of 78 chromosomes) and 88% (65 out of 74 chromosomes) respectively while the frequency of 9T allele was 6.4% in azoospermia and 10.8% in oligozoospermia. The 5T allele was observed in three chromosomes (3.8%) from azoospermic men but with spermatozoa in their testicular biopsies (Table I) and in one chromosome (1.35%) from oligozoospermic patients (Table II).
Discussion
Since the first documented report of the possible implication of CFTR mutations in men with no symptoms of CF other than CBAVD (Dumur et al., 1990), genetic analysis has confirmed that CBAVD is a mild form of CF in otherwise healthy men in whom only reproduction ductal abnormalities are present (Anguiano et al., 1992). Data from several studies, in a total of 420 patients with CBAVD, showed that 19% were compound heterozygotes and 47% were carriers of one CFTR mutation (Lissens et al, 1996). Our results are in accordance with these data, since nine of the 14 patients (64.2%) with CAVD were heterozygous for one CFTR mutation (F508) and one patient with CBAVD (7.1%) was a compound heterozygote (R117H/N1303K) and was 7T/9T. N1303K is classified as a severe mutation with respect to pancreatic status in CF patients. R117H associated with a 5T variant is known to lead to a mild phenotype in CF patients. In CAVD without CF, R117H is probably associated with the 7T allele allowing correct splicing of exon 9 and translation into a CFTR protein which is partially functional (Kiesewetter et al., 1993). One patient of our study with CBAVD with no detectable CF mutation had an unilateral renal agenesis. This association has been described (Donohue and Fauver, 1989; Mickle et al., 1995) essentially in CUAVD. The incidence of renal agenesis in CBAVD is low, but taken together with other results (Augarten et al., 1994; Casals et al., 1995; Dumur et al., 1995), no mutation in the CFTR gene could be detected in 18 patients with CBAVD associated with renal agenesis. In our study, one patient out of the two with CUAVD and occlusion at the controlateral vas deferens had a F508 mutation. Previously, a CFTR mutation has also been found in eight out of nine (89%) of a group of patients with CUAVD and non-iatrogenic occlusion of the controlateral side (Mickle et al., 1995).
In the general population, the frequency of the 5T variants was estimated at 4.4% (Kiesewetter et al., 1993). 5T variants cause a non-functional protein (Chu et al., 1993). The frequency of this 5T allele is ~30% in males with CBAVD (Chillon et al., 1995; Costes et al., 1995; Zielenski et al., 1995; Dumur et al., 1996) and 68% in men carrying a CFTR mutation on one chromosome.
In our study with CAVD, eight patients (57.1%) had a 5T allele; six patients out of eight (75%) with a 5T allele carried the F508 mutation on the other chromosome. This association, like the association of two mutations, explains the occurrence of a CAVD affecting the ducts derived from the Wolffian ducts after 7 weeks gestation. The splicing efficiency of CFTR exon 9 is poor in the vas deferens compared with nasal epithelial cells, independently of the IVS 8-T genotype. Therefore, men with the 5T variant would produce abnormally low amounts of CFTR protein, which may affect the reproductive tract in CAVD specifically; however, there may be sufficient protein to prevent disease in other organs usually affected by CF (Mak et al., 1997; Teng et al., 1997; Wong et al., 1998).
In our study, among the 39 patients with azoospermia without CAVD, no CFTR mutation was detected. Three patients carried a 5T allele (7.7%). These results do not differ from the general population (4.4%) and are in accordance with other authors (Chillon et al., 1995). These results are not in favour of the hypothesis that an alteration in the CFTR gene is associated with azoospermia without CAVD. However, if we amalgamate the data from our study with those others (Jarvi et al., 1995; Van Der Ven et al., 1996; Kanavekis et al., 1998), among 84 patients, nine carried a mutation (10.7%) and eight out of 53 patients examined had a 5T allele (15.1%). In addition, in our study, in two azoospermic patients with 5T alleles, in whom the presence of the vas deferens has been confirmed by clinical exploration, semen analysis showed typical parameters found in a disorder characterized by seminal vesicle anomalies associated with bilateral ejaculatory duct obstruction (BEDO). Similar to CBAVD, the frequency of CF mutations seems to be higher (six out of seven patients) in BEDO than in the general population (Meschede et al., 1997). Overall, these data do not allow us to conclude that the CFTR gene plays a role in the azoospermic patients without CAVD, and further observations are required.
Among our 37 oligozoospermic patients, no mutation of the CFTR gene and only one 5T allele were found. This agrees with other authors (Tuerlings et al., 1998), who did not find any increase in CFTR mutations among patients with oligozoospermia. These observations suggest no effect of CFTR protein on spermatogenesis in man. They are in agreement with observations reporting CFTR gene expression in the epididymis and vas deferens but not in the cells of seminiferous epithelium in human (Trezise et al., 1993a; Tizzano et al., 1994; White et al., 1998). In addition, it is well known that patients heterozygous for CFTR mutations show normal spermatogenesis. In the same way, a F508 mutation associated with a 3849 + 10 KbCT splice mutation does not impair the spermatogenesis nor does it cause CAVD (Stern et al., 1995; Dreyfus et al. 1996). Finally, it is believed that 2–3% of CF males are fertile (Boat et al., 1989). However, some studies have shown that in groups of infertile men showing oligoasthenoteratozoospermia without CAVD, the frequency of CFTR mutations was higher than in the normal population, suggesting that CFTR protein acts in spermatogenesis and/or sperm cell maturation (Traystman et al., 1994; Van Der Ven et al., 1996).
In conclusion, we do not bring any evidence suggesting a role of the CFTR protein in spermatogenesis and/or sperm cell maturation in infertile men without CAVD. This protein may rather act as a contributing factor in a peculiar genetic background or environment. A more extensive study, with an accurate classification of the patients, is still required to assist the genetic counselling of infertile couples who have chosen to be treated by assisted-fertilization methods such as ICSI.
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Acknowledgments |
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The authors thank M.Petit for technical assistance with mutation analyses and C.Cohendy for typing the manuscript. This work was supported by a grant from the Department of Health and Social Security (PHRC: Gène CFTR et infertilité masculine).
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Notes |
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3 To whom correspondence should be addressed at: Service de Biologie du Développement et de la Reproduction, Hôtel-Dieu, Boulevard Léon Malfreyt, 63003 Clermont-Ferrand, France
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References |
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TopAbstractIntroductionReferences |
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Submitted on September 28, 1998; accepted on February 26, 1999.
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