Genes and male infertility: what can go wrong?

Best practice policies for male infertility

Report on evaluation of the azoospermic male

An abnormality of cystic fibrosis transmembrane conductance regulator (CFTR) gene is known to be one of the etiologies of male infertility. CFTR gene mutations are associated with cystic fibrosis (CF-severe phenotype) to congenital bilateral absence of the vas deferens (CBAVD-mild phenotype). CF is the most common autosomal recessive disorders in the Caucasians, characterized by chronic lung disease, pancreatic insufficiency, rise in sweat chloride, and obstructive azoospermia. The milder phenotype is classified as congenital absence of the vas deferens (bi- or unilateral) (CBAVD or CUAVD) or ejaculatory duct obstruction (EDO). Some of these CAVD cases are associated with unilateral renal anomalies (URA). The role of CFTR gene in this subtype of CBAVD-URA is still not understood clearly. The utility of advanced assisted reproductive technologies such as intracytoplasmic sperm injection (ICSI) helps CBAVD males to become biological fathers. If female partner is CF carrier, there is a risk of having a child with CF or CF-related disorders. The currently available CFTR mutation panels cover the most common mutations of Caucasians. Recent studies conducted in South Asian population suggested different spectrum of CFTR mutations than Caucasians. There is a need to develop population-specific CFTR gene mutation panels especially for South Asians where CF or CF-related disorders were once considered rare.

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.

Increased frequency of CFTR gene mutations identified in Indian infertile men with non-CBAVD obstructive azoospermia and spermatogenic failure.

Apart from cystic fibrosis, mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are also involved in congenital bilateral absence of the vas deferens (CBAVD). A mutation is identified in about 80% of the CFTR genes derived from CBAVD patients; the genetic defect in the remainder is yet unknown. In contrast to CF patients, when CFTR is involved, at least one of the mutant CFTR genes of CBAVD patients harbors a mild mutation. A polyvariant mutant CFTR gene is the most frequent CBAVD causing mutant CFTR gene. Here, combinations of particular alleles at several polymorphic loci yield insufficient functional CFTR. The fact that most CBAVD patients, that carry mutations on both CFTR genes, have no lung disease is most probably explained by tissue specific alternative splicing, which is increased in vas deferens compared to bronchial tissue. It has also been reported that CBAVD may be involved in other forms of infertility than CBAVD, however this has not always been confirmed in other studies. Because of techniques such as intracytoplasmic sperm injection, CBAVD patients are now able to father children, however such couples have an increased risk of having a child with cystic fibrosis, and therefore genetic testing and counselling should be provided.

Background & objectives:Due to limited information available on the frequency and spectrum of cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene mutations in congenital bilateral absence of vas deferens (CBAVD) in Indian population, it is difficult to provide accurate genetic counselling to couples. The present study was undertaken to investigate the spectrum and frequency of CFTR gene mutations in Indian men with CBAVD and to determine the female CF carrier status.Methods:Direct DNA sequencing of the CFTR gene was carried out in eighty CBAVD men, their female partners and fifty controls from the general population. Pathological significance of the identified novel CFTR gene variants was carried out using in silico tools. Appropriate genetic counselling was provided to the couples prior to intracytoplasmic sperm injection (ICSI).Results:A significant association was observed for CFTR gene variants in Indian CBAVD men versus controls (odds ratio: 12.1; 95% confidence interval: 4.8-30.4; P<0.0001). A total of 20 CFTR gene variants were identified in 53 CBAVD men. Eight novel missense CFTR gene variants (L214V, A238P, E379V, L578I, F587L, L926W, R1325K and R1453Q); two novel splice-site gene variants (c.1-30C>G and IVS1+2T>G) and ten previously reported mutations (R75Q, c.1210-12[5], F508del, A309G, R334W, I444T, R668C, R709X, A1285V and Q1352H) were detected in CBAVD men. The novel and reported CFTR gene mutations were L926W (2.5%, P=0.26), R1453Q (2.5%, P=0.26), F508del (8.75%, P=0.03) and c.1210-12[5] (42.5%, P=0.002). A total of 13 (16.2%) female partners were found to be a CF carrier. Nine couples had a risk of transmitting mutant CFTR allele to the offspring.Interpretation & conclusions:The heterogeneous spectrum of CFTR gene in Indian population suggests the necessity of screening CBAVD men and female partners for accurate genetic counselling prior to undergoing ICSI.

Congenital bilateral absence of the vas deferens (CBAVD) found in otherwise healthy infertile males, is associated with a high incidence of mutated cystic fibrosis transmembrane conductance regulator (CFTR) alleles, and is considered a genital form of cystic fibrosis (CF). The CF gene may also be involved in the aetiology of male infertility in cases other than CBAVD. The present study was undertaken to test the involvement of CFTR gene mutations in 14 CBAVD males and additionally in cases of male infertility caused by obstructive azoospermia (n = 10) and severe oligozoospermia (n = 3). The entire coding region of the CFTR gene was analysed using denaturing gradient gel electrophoresis (DGGE). The three allele (5T, 7T, 9T) polymorphic tract of thymidines in intron 8 (IVS8-polyT) of which the 5T allele acts as a mild mutation, causing reduced levels of normal CFTR mRNA due to deletion of exon 9, was also analysed. Of the 14 CBAVD cases, four (28.6%) were found to have mutations in both copies of the CFTR gene, six (42.8%) had one CFTR mutation, and in the remaining four (28.6%) no CFTR mutations were found. Of the 10 cases with obstructive azoospermia, three (30%) had one CFTR mutation and in the remaining seven (70%) no mutations were found. None of the three severe oligozoospermia cases carried a CFTR mutation. The frequency of the IVS8(5T) allele was 14.3% (4/28) for the CBAVD cases and 5% (1/20) for the obstructive azoospermia cases, none of the severe oligozoospermia males carried the IVS8-5(5T) allele. The data indicate that while there is a strong association between male infertility caused by CBAVD and mutations in the CFTR gene, cases of obstructive azoospermia without CBAVD also seem to be associated with CFTR gene mutations.

Many studies have shown that congenital absence of the vas deferens (CAVD) is a genital cystic fibrosis transmembrane conductance regulator (CFTR)-mediated phenotype, with a broad spectrum of abnormalities causing male infertility. The genotype of these patients includes mutations in the CFTR gene, e.g. DeltaDeltaF508, R117H and the T5 allele; all of which are commonly found in CAVD. In this study we have screened the entirety of CFTR gene in 47 males with anomalies of the vas deferens: 37 cases of congenital bilateral absence of the vas deferens, three cases of congenital unilateral absence of the vas deferens and seven cases of obstructive azoospermia with hypoplastic vas deferens. Among the 94 chromosomes studied, 65 mutations, of which three are novel (2789+2insA, L1227S, 4428insGA), were identified. The majority of patients (63.8%) had two detectable CFTR gene mutations. Furthermore, high frequencies of the DeltaDeltaF508 mutation (44.7%), the T5 allele (36.2%) and R117H mutation (19.1%) were observed.

Congenital bilateral absence of the vas deferens (CBAVD) is a form of male infertility in which mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been identified. The molecular basis of CBAVD is not completely understood. Although patients with cystic fibrosis have mutations in both copies of the CFTR gene, most patients with CBAVD have mutations in only one copy of the gene. To investigate CBAVD at the molecular level, we have characterized the mutations in the CFTR gene in 102 patients with this condition. None had clinical manifestations of cystic fibrosis. We also analyzed a DNA variant (the 5T allele) in a noncoding region of CFTR that causes reduced levels of the normal CFTR protein. Parents of patients with cystic fibrosis, patients with types of infertility other than CBAVD, and normal subjects were studied as controls. Nineteen of the 102 patients with CBAVD had mutations in both copies of the CFTR gene, and none of them had the 5T allele. Fifty-four patients had a mutation in one copy of CFTR, and 34 of them (63 percent) had the 5T allele in the other CFTR gene. In 29 patients no CFTR mutations were found, but 7 of them (24 percent) had the 5T allele. In contrast, the frequency of this allele in the general population was about 5 percent. Most patients with CBAVD have mutations in the CFTR gene. The combination of the 5T allele in one copy of the CFTR gene with a cystic fibrosis mutation in the other copy is the most common cause of CBAVD: The 5T allele mutation has a wide range of clinical presentations, occurring in patients with CBAVD or moderate forms of cystic fibrosis and in fertile men.

Background. Congenital bilateral absence of the vas deferens (CBAVD) is a form of male infertility in which mutations in the cystic fibrosis transmembrane con- ductance regulator (CFTR) gene have been identified. The molecular basis of CBAVD is not completely under- stood. Although patients with cystic fibrosis have muta- tions in both copies of the CFTR gene, most patients with CBAVD have mutations in only one copy of the gene. Methods. To investigate CBAVD at the molecular lev- el, we have characterized the mutations in the CFTR gene in 102 patients with this condition. None had clini- cal manifestations of cystic fibrosis. We also analyzed a DNA variant (the 5T allele) in a noncoding region of CFTR that causes reduced levels of the normal CFTR protein. Parents of patients with cystic fibrosis, patients with types of infertility other than CBAVD, and normal subjects were studied as controls. Results. Nineteen of the 102 patients with CBAVD had mutations in both copies of the CFTR gene, and none of them had the 5T allele. Fifty-four patients had a mutation in one copy of CFTR, and 34 of them (63 per- cent) had the 5T allele in the other CFTR gene. In 29 patients no CFTR mutations were found, but 7 of them (24 percent) had the 5T allele. In contrast, the fre- quency of this allele in the general population was about 5 percent. Conclusions. Most patients with CBAVD have muta- tions in the CFTR gene. The combination of the 5T allele in one copy of the CFTR gene with a cystic fibrosis mu- tation in the other copy is the most common cause of CBAVD. The 5T allele mutation has a wide range of clin- ical presentations, occurring in patients with CBAVD or moderate forms of cystic fibrosis and in fertile men. (N Engl J Med 1995;332:1475-80.)

Cystic fibrosis (CF) is a common recessive disorder caused by >1600 mutations in the CF transmembrane conductance regulator (CFTR) gene. About 13% of CFTR mutations are classified as "splicing mutations," but for almost 40% of these, their role in affecting the pre-mRNA splicing of the gene is not yet defined. In this work, we describe a new splicing mutation detected in three unrelated Italian CF patients. By DNA analyses and mRNA studies, we identified the c.1002-1110_1113delTAAG mutation localized in intron 6b of the CFTR gene. At the mRNA level, this mutation creates an aberrant inclusion of a sequence of 101 nucleotides between exons 6b and 7. This sequence corresponds to a portion of intron 6b and resembles a cryptic exon because it is characterized by an upstream ag and a downstream gt sequence, which are most probably recognized as 5'- and 3'-splice sites by the spliceosome. Through functional analysis of this splicing defect, we show that this mutation abolishes the interaction of the splicing regulatory protein heterogeneous nuclear ribonucleoprotein A2/B1 with an intronic splicing regulatory element and creates a new recognition motif for the SRp75 splicing factor, causing activation of the cryptic exon. Our results show that the c.1002-1110_1113delTAAG mutation creates a new intronic splicing regulatory element in intron 6b of the CFTR gene exclusively recognized by SRp75.

Letter from the editor

Introduction Congenital Bilateral Absence of the Vas Deferens (CBAVD) is a condition leading to obstructive azoospermia and male infertility. CBAVD is associated with mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Congenital Unilateral Absence of the Vas Deferens (CUAVD), where only one vas deferens is missing, is also associated with mutations in the CFTR gene. However, the genetic causes of both conditions are not fully understood and warrant further investigation. We present two brothers, one with CBAVD and one with CUAVD, who were subjected to whole exome sequencing. Objective To assess genetic similarities using whole exome sequencing between two brothers with differing reproductive capacities. Methods Whole exome sequencing was performed in two brothers. Subject A was 39-year-old-male who had previously fathered a child and was diagnosed with CUAVD during his vasectomy. Subject B was a 37-year-old male diagnosed with CBAVD, and genetic testing significant for one copy of a mutated cystic fibrosis gene (unaffected CF carrier). Whole blood was collected. DNA was extracted and quality assessed. Finally, whole exome sequencing was completed. Results Whole exome sequencing results were subjected to annotation and filtering. Both subjects were noted to share a rare mutation in the CFTR gene (p.R347H). CFTR mutations are the most common cause of CBAVD and have also been linked to CUAVD. However, this is a unique case of two brothers with the same genetic mutation but different phenotypic expressions of the mutation. Conclusions We have identified a rare CFTR variant (p.R347H) in two brothers with different reproductive capacities. We present evidence that absence of the vas deferens (unilateral or bilateral) may be associated with CFTR mutations, even in the absence of diagnosed cystic fibrosis. Understanding the phenotypic spectrum of CFTR gene mutations is pivotal to treating associated male infertility. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Acerus Pharmaceuticals, Boston Scientific, Coloplast, Endo Pharmaceuticals, Empower Pharmacy, Nestle Health, Olympus, Hims Inc.

Cystic fibrosis (CF) is usually considered a rare disease in the Indian population. Two studies have reported on the frequency of cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in Indian males with congenital absence of the vas deferens (CAVD), however, data on the spectrum of CFTR gene mutations are still lacking. Therefore, the present study was designed to identify the spectrum of CFTR gene mutations as well as to investigate an association of CF genetic modifiers in the penetrance of CAVD in infertile Indian men. A total of 60 consecutive infertile males with a diagnosis of CAVD were subjected to CFTR gene analysis which revealed 13 different CFTR gene mutations and 1 intronic variant that led to aberrant splicing. p.Phe508del (n = 16) and p.Arg117His (n = 4) were among the most common severe forms of CFTR mutations identified. The IVS8-T5 allele, which is considered as a mild form of CFTR mutation, was found with an allelic frequency of 28.3%. Eight novel mutations were also identified in the CFTR gene from our patient cohort. It is noteworthy that the spectrum of CFTR gene mutation is heterogeneous, with exon 4 and exon 11 as hot spot regions. Moreover, we also found an association of the CF genetic modifiers, viz., transforming growth factor (TGF)-β1 and endothelial receptor type-A (EDNRA) genes with the CAVD phenotype. The findings are of considerable clinical significance because men suffering from infertility due to CAVD can decide to use artificial reproduction technology. The children of men with CAVD are at risk of carrying CFTR mutations; therefore, genetic counseling is a crucial step for such patients. With special reference to developing countries, such as India, where whole gene sequencing is not feasible, the outcome of our study will make the screening procedure for CFTR gene simpler and more cost-effective as we have identified hot spot regions of the CFTR gene which are more prone to mutation in Indian males with CAVD. Moreover, this is the first study from the Indian population to investigate the association of CF genetic modifiers with penetrance of the CAVD phenotype. The observed association of the genetic modifiers TGF-β1 and EDNRA in the penetrance of CAVD further supports their involvement in genesis of the vas deferens.