Mutations and polymorphisms in CFTR genes in infertile men with oligospermia or azoospermia

Abstract

Impaired infertility of the male partner is causative or contributOry to in up to one half of all couples unable to conceive spontaneously. A considerable number of genes are now known that have an essential function in human reproduction and which, when deleted or mutated, can cause pathologic changes in the male reproductive system. Congenital bilateral absence of the vas deferens (CBAVD) is an important cause of obstructive azoospermia in otherwise healthy men. It is also present in 95% of men with an autosomal recessive systematic disease--cystic fibrosis. However, clinically affected CF patients present a spectrum of genital phenotypes ranging from normal fertility to severely impaired spermatogenesis and CBAVD. Cystic fibrosis and most cases of CBAVD are caused by mutations in CFTR (cystic fibrosis transmembrane conductance regulator) gene. The aim of this study was to test the possible involvement of the CFTR gene in the aetiology of male infertility other than CBAVD. Twenty one infertile men with oligo or azoospermia were analysed for the presence of mutations and polymorphisms in the CFTR gene. Patients were divided in two groups according to the spermatogram: 1) patients with obstructive azoospermia (V < 2 mL, pH < 7.2, low level of a-glucosidase and fructose and absence of spermatozoa; 2) patients with impaired spermatogenesis or sperm maturation. We performed direct detection for the following mutations: delta F508 and delta 1507 (heteroduplex analysis), 621 + 1 G-->T, and N1303K (PSM--PCRmediated site-specific mutagenesis), A455E, 1717-1 G-->A, S549N, R560T, W1282X, R334W, R347P, R117H, 3849 + 10 kb C-->T and Tn, F508C, 1507V, 1506V polymorphisms (reverse dot blot method). G542X, R553X and GSS1D mutations were tested by SSCP (Single Strand Conformation Polymorphism). We also performed indirect detection of mutations and polymorphisms in 3, 5, 6a, 8, 9, 11, 12, 14a, 14b, 15, 17b, 18, 20, 21 and 23 exons by DGGE (Denaturant Gradient Gel Electrophoresis). Differences between frequencies were tested by chi-square statistic, p values of less than 0.05 were considered statistically significant. Among 42 chromosomes from infertile men with oligo or azoospermia we detected 7 mutations in CFTR gene (16.7%), which was significantly (p = 0.0319) more frequent than in general population (2%). Frequency of 5T allele in analysed group was high (11.9%) compared to general population (5%), but not statistically significant (0.0938). The most common mutation in the group of 10 men with obstructive azoospermia was delta F508. It was detected on one chromosome in five patients. In three of these patients with 4F508 mutation on the other chromosome we found 5T allele on polymorphic Tn locus. In one patient, heterozygous for delta F508 mutation, 711 + 3 A-->G mutation on the other chromosome was detected. In the group of 11 infertile men with impaired spermatogenesis or sperm maturation we detected one mutation--delta F508. Two patients from this group had 5T variant on one chromosome. We analysed 21 infertile men with oligo or azoospermia not caused by endocrine or inflammatory character, or chromosome mutations. Within this group frequency of CFTR mutations was increased compared to general population (p = 0.0319), suggesting that CFTR gene may be involved in the aetiology of infertility in men with oligo or azOospermia. In the group of patients with obstructive azoospermia 50% had at least one mutation, but only 10% had mutations in both chromosomes. One of the possible explanations would be that mutations are in the promoter region, introns or exons that were not included in analyses. The second explanation could be that some cases of obstructive azoospermia are only partially (or not) related to CFTR gene. In the group of patients with impaired spermatogenesis or sperm maturation, the frequencies of CFTR mutations and 5T allele were also increased compared to general population, but lower than in the group with obstructive azoospermia. This fact could mean that the influence of some other genes is higher in this condition than in the case of obstructive azoospermia. We concluded that CFTR gene plays a role in the aetiology of obstructive azoospermia and that is also could be involved in some cases of impaired spermatogenesis and sperm maturation. Due to the high incidence of CFTR mutations in patients with obstructive azoospermia we suggest screening of CFTR mutations before assisted reproduction.