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Published in final edited form as: Andrology. 2017 Dec 7;6(1):127–135. doi: 10.1111/andr.12450

Congenital Bilateral Absence of the Vas Deferens as an Atypical Form of Cystic Fibrosis: Reproductive Implications and Genetic Counseling

Denise Andréa Silva de Souza a,b, Fábio Rueda Faucz a,c, Lilian Pereira-Ferrari d, Vanessa Santos Sotomaior a, Salmo Raskin a,*
PMCID: PMC5745269  NIHMSID: NIHMS920433  PMID: 29216686

Abstract

Congenital bilateral absence of the vas deferens (CBAVD) is found in 1% to 2% of males with infertility and is present in 6% of obstructive azoospermia cases. Nearly 95% of men with cystic fibrosis (CF, an autosomal recessive disorder) have CBAVD. There are genetic links between CBAVD and CF. Some mutations in the gene encoding Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) can lead to CBAVD as a monosymptomatic form of CF. With the use of assisted reproductive techniques (ART), especially testicular or epididymal sperm aspiration, intracytoplasmic sperm injection and in vitro fertilization, it is possible that men with CBAVD can produce offspring. Therefore, genetic counseling should be offered to couples undergoing ART to discuss the probability of having offspring that carry CFTR gene mutations. The aim of this review is to present the main cause of CBAVD, to call attention to its implications for assisted reproduction and to show the importance of genetic counseling for couples where men have CBAVD, as they can have offspring with a lethal disease.

Keywords: Congenital bilateral absence of the vas deferens, infertility, Cystic Fibrosis, CFTR, assisted reproductive techniques, genetic counseling

INTRODUCTION

About 1 in every 6–10 couples has fertility problems. Sub-fertility originates from males in 20–25% of the cases, from females in 30–40% of the cases and from both in 30% of the cases. The causes of sub-fertility remains unknown in 15% of the cases (World Health Organization, 1997). Among the 20–25% of males with sub-fertility, CBAVD accounts for 1–2% (Hussein et al., 2011). Diagnosis of CBAVD is generally based on these criteria: presence of normal to slightly small sized testicles, non-palpable vas deferens, normal plasma levels of FSH (Follicle-Stimulating Hormone), and reduced ejaculate volume (<1 mL). Semen characteristics are: azoospermic, acidic pH, undetectable or low fructose concentrations (normal: >25 μM) (Boucher et al., 1999), α-glucosidase less than or equal to 5 mIU/ejaculate (normal: greater than or equal to 35 mIU/ejaculate) and carnitine less than or equal to 40 nM/ejaculate (normal: more than 260 nM/ejaculate) (Boucher et al., 1999) and production of spermatozoa in the testicles.

When CBAVD is the only manifestation in a patient who harbors at least one mutation in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, this condition is known as the genital form of Cystic Fibrosis (CF) (Anguiano et al., 1992; Chillón et al., 1995) and can be named CF-CBAVD. CF is an autosomal recessive genetic disease frequent in Euro-descendant populations, occurring in 1 out of 2,000 newborns (Wainwright et al., 1985; Boat et al., 1989). Its incidence varies among different ethnic groups, with lower incidences in non-Euro-descendant populations. In Afro-descendants, the incidence varies from 1 in 14,000 to 1 in 17,000 newborns (Boat et al., 1989; Fitzsimmons, 1993; Hamosh et al., 1998), while in Asian populations, the frequency is 1 in 90,000 newborns (Boat et al., 1989). The frequency of disease-causing mutation carriers is 1 in 20 in certain populations (Wainwright et al., 1985).

Clinically, typical CF is characterized by chronic pulmonary obstruction, pancreatic insufficiency, high electrolyte concentration in sweat (“salty sweat”) (White et al., 1985), male infertility (Mickle et al., 2000) and other abnormalities. Kerem et al. (1989) state that 85% of CF patients have pancreatic insufficiency, although Noone & Knowles (2001) indicate that this prevalence is as high as 95%. Lung problems are the major cause of mortality and can be responsible for 95% of CF mortality (Boat et al., 1989).

In 1985, Knowlton et al., White et al. and Wainwright et al. identified the location of the gene responsible for CF on the long arm of chromosome 7. Subsequent detailed studies have mapped the gene on 7q31.2. The main CF molecular defect was finally determined in 1989, when the CFTR gene was identified and cloned (Kerem et al., 1989; Riordan et al., 1989; Rommens et al., 1989). Riordan et al. (1989) showed that it has a length of 250 kb and a total of 27 exons, numbered from 1 to 24, including exons 6a, 6b, 14a, 14b, 17a and 17b.

The CFTR gene encodes a protein product of 1,480 amino acids and molecular weight of 168,138 daltons (Riordan et al., 1989). It is structured in five domains (Welsh & Smith, 1993): two membrane-spanning domains (MSD-1 and MSD-2) forming the channel and three cytoplasmic domains, from which two domains that bind to ATP (NBD-1 and NBD-2, Nucleotide-binding Domain) are connected by a regulatory domain R. These characteristics defined the name of the gene as CFTR (Cystic Fibrosis Transmembrane Conductance Regulator).

Since its identification in 1989, more than 2,000 mutations have been described in the CFTR gene (Cystic Fibrosis Mutation Database, CFMD, 2017). The most frequent mutation is p.F508del (c.1521_1523delCTT; rs113993960), with a worldwide frequency of 66% (CFMD, 2017), varying among populations. This mutation consists of a three-nucleotide deletion in codon 508, resulting in loss of phenylalanine amino acid, which prevents the protein migration to the top of a plasma membrane (Kerem et al., 1989).

Welsh & Smith (1993) propose four mutation classes in the CFTR gene: class I consists of protein synthesis blocking mutations; class II represents changes in the protein processing; class III comprises changes in protein regulation and class IV represents protein conductivity alteration. Wilschanski et al. (1995) added another class of mutation to the Welsh and Smith system, namely class V, leading to reduced protein synthesis. Mainly due to these different mutation classes, a broad range of phenotypes is seen in CF, varying from the typical manifestation to atypical forms including mild lung disease, idiopathic chronic pancreatitis, asthma, allergic bronchopulmonary aspergillosis, sinusitis, and the congenital bilateral absence of the vas deferens (CBAVD) (Noone & Knowles, 2001), which is the focus of this review.

Assisted reproductive techniques (ART) have enabled men with CBAVD to reproduce and therefore there is a need to identify and discuss the consequences of possible mutations in the CFTR gene in infertile couples.

Cystic Fibrosis-Related Congenital Bilateral Absence of the Vas Deferens (CF-CBAVD)

CBAVD occurs in about 1% to 2% of infertile men (Hussein et al., 2011), but in CF male patients, 95% have CBAVD as the result of mutations in the CFTR gene (Chillón et al, 1995). There are cases of CBAVD that are not associated with CF, among them are cases related to kidney malformations (Lane et al., 2014). However, approximately 80%–97% of patients that present with isolated CBAVD have a mutation in the CFTR gene (Casals et al., 1995; Chillón et al., 1995). Among these, 63%–83% carry mutations in both alleles (Claustres et al., 2000; Jézéquel et al., 2000; Taulán et al., 2007). Polymorphisms in other genes may increase the penetrance of CBAVD-related mutations. These include polymorphisms in the CFTR gene, such as certain polymorphisms in Tr2GFB1 (Transforming Growth Factor) and EDNRA (Endothelin Receptor Type A) genes (Havasi et al., 2010).

New or improved techniques for CFTR mutation screening have identified different mechanisms of mutations leading to CBAVD, as revealed by the identification of large rearrangements and deletions in the CFTR gene of CBAVD patients that could not be detected by previously (Ratbi et al., 2007; Taulán et al., 2007; Trujillano et al., 2013).

In general, mild phenotypes of CF (such as pancreatic insufficiency, mild lung problems, or atypical forms, such as CBAVD - Table 1) are caused by compound heterozygous genotypes with one severe mutation in one allele and one mild mutation in the other or, in some cases, one mild mutation in each allele (Chillón et al., 1995; Cuppens et al., 1998; Noone & Knowles, 2001). According to Uzun et al. (2005), one mild mutation in homozygous or two mild different mutations can cause atypical forms of CF or male infertility without any other clinical manifestation.

Table 1.

Atypical (non-CF) diseases associated with the CFTR gene

Disease Common manifestations shared with CF Fraction of patients with at least one CFTR mutation (%) Reference
Allergic bronchopulmonary aspergillosis Asthma, pulmonary infiltrates Meta-analysis (26%) Agarwal, 2012
CBAVD Absence of vas deferens (bilateral) Meta-analysis (78%) Yu, 2012
CUAVD Absence of vas deferens (unilateral) 9/24 (37.5%) Casals, 2000
Chronic pancreatitis Abnormal pancreatic function 17/48 (35.4%) De Cid, 2010
Diffuse Bronchiectasis (DB) Abnormal dilation of bronchi 37/122 (30%) Bienvenu, 2010
Nasal polyposis Nasal polyps 5/44 (11.4%) Kostuch, 2005
Neonatal transitory hypertrypsinaemia High levels of immunoreactive trypsine 32/47 (62%) Castellani, 2001
Rheumatoid arthritis (RA)
RA and DB		 Joint pain
Joint pain and abnormal dilatation of bronchi		 5/24 (21%)
18/30 (60%)		 Puéchal, 2011
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The reason the majority men with CBAVD with two mutations in the CFTR gene do not present with lung problems is related to differences in the alternative mRNA splicing in different tissues (Cuppens & Cassiman, 2004). Studies by Mak et al. (1997) revealed that mRNA splicing was less efficient in the vas deferens epithelia than in respiratory epithelia, an indication that the dysfunction of CFTR protein is more sensitive in the reproductive system than in other tissues. For example, in a patient homozygous for the IVS9-5T (c.1210-7_1210-6delTT variant, formerly known as IVS8-5T), a sequence of 5 thymines in intron 9 of the CFTR gene that results in loss of exon 10, leads to the formation of only 10% of the normal protein and causes malformation of the vas deferens. Although this is sufficient to prevent pathologies in other organs normally affected by CF (Chillón et al., 1995; Mak et al., 1997). Cuppens & Cassiman (2004) reported that the proportion of transcripts lacking CFTR exon 10 differs between vas deferens and nasal epithelium due to alternative splicing and to the presence of a mild mutation in the CFTR gene, with partial chloride channel activity, which causes dysfunction only in the vas deferens and not in the respiratory epithelium.

Mutations in the CFTR gene disrupt the function of the chloride channels, preventing them from regulating the flow of chloride ions and water across cell membranes. As a result, cells in the male genital tract produce mucus that is abnormally thick and sticky. This mucus clogs the vas deferens as it is forming, causing it to deteriorate before birth. The pathogenicity of CBAVD in CF may occur during development in utero, possibly by the obstruction of the genital tract due to accumulation of thick secretions that lead to degeneration of the vas deferens (Cuppens & Cassiman, 2004). Gaillard et al. (1997) observed the presence of vas deferens in 12–18 weeks aborted fetuses carrying a CFTR mutation, indicating that degeneration may occur later in embryonic development.

Although there are still several factors that remain unexplained in the etiology of CF-CBAVD, the main difference between typical CF and CF-CBAVD is the identification of different and rare CFTR mutations and variants in high frequency in individuals with CF-CBAVD as compared to the typical CF forms, such as the IVS9-5T (polymorfism Tn) variant, the (TG)m variant, the M470V (c.1408A>G, p.Met470Val) variant, and the high frequency of class IV and V CFTR mutations in CF-CBAVD cases.

IVS9-5T [c.1210-7_1210-6delTT; rs562195055; polymorphisms Tn; formerly known as IVS8-5T]

The best characterized CBAVD specific variant is the polymorphic polythymidine tract (Tn) in CFTR intron 9. The presence or absence of exon 10 in CFTR mRNA depends on the size of the sequence of thymines in intron 9 of the CFTR gene. This sequence, called poli-T, may contain 5, 7 or 9 thymines (T5, T7 or T9) and is generically known as c.1210_12T(5_9). The efficiency with which the splice acceptor site is used decreases in parallel with the size of poli-T chain (Chu et al., 1993), which increases the probability of exon 10 loss during splicing and reduces the quantity of normal protein.

mRNA lacking exon 10 translates into an immature protein with no channel activity (Delaney et al., 1993). A rare T3 allele (poli-T chain with 3 thymines) (Claustres, 2005) and recently a T2 allele (poli-T chain with 2 thymines) (Radpour et al., 2009) have been associated with large losses of exon 10 during the splicing and can be considered mutations associated with CBAVD. Another example is the TGm allele (T-G repeats immediately adjacent to the thymines in intron 9) that can alter the penetrance of Tn allele, more specifically the IVS9-5T (sequence of 5 thymines), being directly related to CF and CBAVD (Cuppens et al., 1998).

About 10% of the world population carries the IVS9-5T (Kiesewetter et al., 1993) allele. It presents as a pathogenic variant of incomplete penetrance (Cuppens et al., 1998) with penetrance of 0.6, according to Zielenski et al. (1995), and is frequently encountered in men with CBAVD (a frequency of 40% was reported by Chillón et al., 1995, and 25% by Mak et al., 1999). The presence of the IVS9-5T variant in homozygote conditions produces about 95% of mRNA without exon 10 in the respiratory epithelium, resulting in an alteration in the NBD-1 domain of CFTR protein (Chu et al., 1992).

A particular combination of two alleles (genotype) results in a certain level of mRNA normally produced and yields specific clinical phenotypes. A quantity of normally produced mRNA lower than 1% to 3% leads to a severe phenotype of CF. Levels of normal mRNA between 8% and 12% lead to a normal phenotype, and levels between 4% and 7% lead to atypical or mild forms of CF (Chillón et al., 1995). Carriers of a typical CFTR mutation and an IVS9-5T allele may have low levels of normal mRNA, which is the most common cause of CBAVD (Chillón et al., 1995). Osborne et al. (1994) reported that individuals with CBAVD may be homozygous or heterozygous for IVS9-5T, but must have a second CFTR mutation in trans.

In the work by Chillón et al. (1995), from 102 men with CBAVD, 33.3% had a CFTR mutation in one chromosome and a IVS9-5T allele in the other; 18.6% had two CFTR mutations that did not include the IVS9-5T; 19.6% had no IVS9-5T in both alleles but had a CFTR mutation in one chromosome; 6.9% had no CFTR mutation but one IVS9-5T allele; and 21.6% had no mutation detected (including IVS9-5T), indicating that another gene or genes may be related to CBAVD. Radpour et al. (2007) studied 112 Iranians with CBAVD and found 28.57% IVS9-5T alleles associated in trans with other mutations in the CFTR gene. The IVS9-5T allele and a p.F508del mutation were the most frequent causes of CBAVD in these patients, corresponding to more than 1/3 of the identified alleles.

Bernardino et al. (2003) in a study with 20 Brazilian patients (17 with CBAVD and 3 with another type of obstructive azoospermia) found a frequency of the IVS9-5T allele in 23.5% of the men with CBAVD, similar to the one found in other studies (Chillón et al., 1995; Casals et al., 2000), which points out its relation with the CBAVD phenotype. Although individuals with an IVS9-5T allele in trans with a severe mutation in the CFTR gene show fertility problems (CBAVD) or other atypical forms of CF, approximately 40% are healthy and fertile due to the incomplete penetrance of this allele (Chillón et al., 1995; Zielenski et al., 1995).

In other populations, the frequency of the IVS9-5T in CBAVD patients varies between 3.1% in Mexico, suggesting that this mutation does not play a significant role in CF-CBAVD in that country (Saldanã-Alvarez et al., 2012) and 45.6% in Italy (Giuliani et al., 2010). In Algerian/Tunisian CBAVD patients, the IVS9-5T was found in 12.5% of the alleles (Boudaya et al., 2012), but in China IVS9-5T was found in 44.5% of the CBAVD alleles (Ni et al., 2012). Similar frequencies for the IVS9-5T were found in Chinese (32.02%, Du et al., 2014), Portuguese (31%; Grangeia et al., 2007), Egyptian (30%; Hussein et al., 2011) and Indian CBAVD men (27.1%; Sachdeva et al., 2011).

(TG)m polymorphism

Repeats of 9 to 13 thymine-guanine (TG) downstream to the poly-T (Tn) sequence influence the exon 10 loss (Cuppens et al., 1998; Niksic et al., 1999). Unlike the Tn allele, which influences the efficiency of the splice acceptor site, the TGm alleles change the position of the splicing branch, as a larger number of TG repetitions increase the penetrance of the IVS9-5T allele and, consequently, the frequency with which the exon 10 is removed during splicing (Cuppens et al., 1998). Jézéquel et al. (2000) found a frequency of 36.2% of IVS9-5T alleles in men with alterations in the vas deferens (including CBAVD). Among these, 52.9% were associated to a TG12 chain, 29.4% to a TG13 chain and 17.7% associated to a TG11 chain.

Groman et al. (2004), in a study of 98 men with CBAVD, found 9 men with other atypical forms of CF and 27 fertile men. They found the IVS9-5T allele in cis with three different TG repetitions: TG11-5T, TG12-5T, TG13-5T. Among these, TG12-5T presented the stronger association with the pathogenesis (76% of the affected group). TG13-5T was found only in affected individuals. TG11-5T was considered generally benign since it was detected in 78% of the unaffected group.

Groman et al. (2004) concluded that when the IVS9-5T allele is in trans with a severe mutation, the pathogenicity is 28 and 34 times higher for TG12-5T and TG13-5T, respectively, than for TG11-5T. This allele combination implies a risk of 0.10 for TG11-5T, 0.78 for TG12-5T and 1.0 for TG13-5T (Groman et al., 2004). Radpour et al. (2007), in a study with 12 men with CBAVD, also found the TG12 and TG13 alleles associated in cis with the IVS9-5T allele.

Ni et al., 2012, found TG13 allele in a frequency 19 fold higher in Chinese CBAVD men (9.17%) than in controls (0.48%). The TG12 was significantly higher (55.05% CBAVD versus 44.23% controls) and TG11 lower (35.78% CBAVD versus 55.29% controls). The comparison of TG-T haplotypes revealed a significant 2.5 fold increase of the TG12-5T haplotype in men with CBAVD (33.94% versus 13.46% in controls). TG11-5T and TG13-5T haplotypes were found 1.38% and 9.17%, respectively, in the CBAVD patients and were not found in the control group. However, significant increases of TG11-7T (55.29% controls versus 34.4% CBAVD) and TG12-7T (30.29% controls versus 21.1% CBAVD) haplotypes were observed in the control group. One case with the TG13-7T and TG12-9T genotype was found in the control group, which had not been reported previously. In summary, the IVS9-5T linked to either 12 or 13 TG repeats exhibits a high prevalence among the Chinese CBAVD patients tested. Therefore, the characterization of the TG chain size may indicate part of the penetrance of the IVS9-5T allele.

M470V (c.1408A>G; p.Met470Val; rs213950) variant

Cuppens et al. (1998) noticed an influence of the M470V allele in the penetrance of IVS9-5T. The polymorphic locus 470 (methyonine or valine in the 470 codon) is located in exon 11 and codes part of the first NBD domain. Both 470 methyonine (M470) and 470 valine (V470) lead to production of a CFTR completely glycosylated protein. Although M470 protein maturates more slowly than V470, M470 has a two-fold increased chloride channel activity compared to V470 (Cuppens et al., 1998).

Groman et al. (2004) reported that M470 is always associated to TG11-5T, and V470 to TG12-5T. The TG13-5T is exclusively found in those individuals affected by atypical CF forms (including CBAVD) and occurs only with M470. Du et al., 2014, and Ni et al., 2012, found no statistically significant difference between CBAVD and fertile men with regard to M470V genotype or allele frequencies. However, when the haplotype TG-T-M470V was considered, statistical analysis showed that the TG12-5T-V470 genotype was significantly associated with CBAVD (52.63%) as compared to normal controls (Ni et al., 2012). Similarly, Stuppia et al. (2005) found a frequency of 84.6% of the haplotype TG12-5T-V470 in patients with CBAVD. According to Sun et al. (2006), 10 among 12 men affected with CBAVD carry this haplotype that has 80% penetrance in males. Pompei et al. (2006) showed that M470 allele presents higher variability in its adjacent areas and many of these variations are mutations changing the constitution/function of the CFTR protein.

In another study, Ciminelli et al. (2007) analyzed the M470V locus in Italian couples requiring genetic counseling, and found that in 39% of them, both partners had at least one M470 allele and 89% of these couples had an increased risk of having a child affected with CF. Based on that, a different screening for CF mutations should be performed in this subgroup. However, in a recent meta-analysis, Xu et al. (2014) found the variant M470V was a CBAVD protective factor among French, Chinese, Italian and Iranian populations if this mutation is analyzed separately. This demonstrates the clinical and technical complexity needed to evaluate the relevance of a variant of a specific phenotype.

Other rare CFTR variants related to CBAVD

Only a few typical CF mutations, such as the p.F508del, are found in individuals with CF-CBAVD (Chillón et al., 1995; Uzun et al., 2005). Mak et al. (1999) reported that IVS9-5T is the most common variant among men with obstructive azoospermia followed by p.F508del, a finding supported by the analysis of these mutations in Portuguese men with CBAVD (31% IVS9-5T versus 23.8% p.F508del) (Grangeia et al., 2007).

The most frequent CFTR mutation is p.F508del, classified as class II, and therefore generally associated with the severe form of CF when in homozygosity or compound heterozygosity with a second “severe” class I, II or III allele. However, when associated with other “mild mutations” (classes IV and V), or to specific variants such as the IVS9-5T (polymorphism Tn), the (TG)m and the M470V, it can lead to atypical forms of CF, like CF-CBAVD.

Classes IV and V CFTR mutations are strongly associated with the mild phenotypes of CF (Wilschanski et al., 1995; Mak et al., 1999). Among the class IV mutations, the R117H (p.Arg117His, c.350G>A) in combination with certain Tn alleles leads to different phenotypes: if associated with a IVS9-5T allele, generally it leads to CF; if in combination with a IVS9-7T, the allele can lead to mild forms of CF or to CBAVD (Kiesewetter et al., 1993; Mak et al., 1997; Noone & Knowles, 2001; Cuppens & Cassiman, 2004).

Jézéquel et al. (2000) reported that 19.1% of patients with vas deferens alterations had the 117 Arginine (R117) variation. Among these, 62.5% had the R117H_TG10_7T haplotype and 37.5% the R117H_TG11_7T haplotype. This is in agreement with the observation of Kiesewetter et al. (1993) who found men with malformation of the vas deferens and R117H mutation associated with allele IVS9-7T.

Recently, Thauvin-Robinet et al. (2013) reviewed the data from 179 non-newborn French individuals carrying R117H and a second CFTR variation. Among those, 76% were referred due to CBAVD. They concluded that patients with CBAVD carrying R117H and a severe CF variation should benefit from a clinical evaluation and follow-up, and that depending on their genotype, a CFTR analysis should be considered in their partners in order to identify CF carrier couples and offer prenatal (PND) or preimplantation (PGD) diagnoses.

Other CFTR variants that have been found in CBAVD patients are shown in Table 2. The number of mutant alleles found in men with CAVD (congenital absence of vas deferens) are summarized in Table 3.

Table 2.

CFTR mutations found in men with CBAVD from different nationalities/ancestries

Nationality/Ancestry Mutation Name cDNA Name Protein Name Reference
Algerian 711+1G>T c.579+1G>T NA Boudaya et al., 2012
E1104X c.3310G>T p.Glu1104X
Asian V201M c.601G>A p.Val201Met Danziger et al., 2004
V520I c.1558G>A p.Val520Ile
Q1352H c.4056G>C or c.4056G>T p.Gln1352His
Asian-Indian V456A c.1367T>C p.Val456Ala
Chinese 1001+5G>A NA NA Goh et al. 2007
870-1G-C NA NA Li et al., 2012
1209+1G-C NA NA
1209+2T-G NA NA
3635delT NA NA
NA NA p.Ala357Thr
NA NA p.Thr388Lys
NA NA p.Arg419Ile
Chinese NA NA p.Gly451Lys Li et al., 2012
NA NA p.Cys592Phe
M469V c.1405A>G p.Met469Val Lu et al., 2013
S485C c.1453A>T p.Ser485Cys
E527N NA NA
I556V c.1666A>G p.Ile556Val
R553X c.1657C>T p.Arg553X Lu et al., 2014
T501N NA NA
I507N NA NA
L558S c.1673T>C p.Leu558Ser
French L1227S c.3680T>C p.Leu1227Ser Jézéquel et al., 2000
R117H c.350G>A p.Arg117His Thauvin-Robinet et al., 2013
Hispanic W1098C NA NA Danziger et al., 2004
Indian NA c.650_659delAGTTGTTACA p.Glu217Glyfs*11 Sachdeva et al., 2011
NA c.3854 C>T p.Ala1285Val
Iranian K536X c.1606A>T p.Lys536X Radpour et al., 2006
Y122H c.364T>C p.Tyr122His
Iranian T338A c.1012A>G p.Thr338Ala Radpour et al., 2006
Italian P499A c.1495C>G p.Pro499Ala Arduino et al., 1998
D614G NA p.Asp614Gly Tomaiuolo et al., 2011
Japanese A399D c.1196C>A p.Ala399Asp Bernardino et al., 2003
Jew D1152H NA p.Asp1152His Peleg et al., 2011
Mexican W1089X NA p.Trp1089X Saldanã-Alvarez et al., 2012
G85E NA p.Gly85Glu
Northern European Caucasian P750L c.2249C>T p.Pro750Leu Danziger et al., 2004
Portuguese P439S c.1315C>T p.Pro439Ser Grangeia et al., 2007
V1108L c.3322G>C p.Val1108Leu
P1290S c.3868C>T p.Pro1290Ser
E1401K c.4201G>A p.Glu1401Lys
Spanish DeltaE115 c.343_345del p.Glu115del Casals et al., 1995
K1060T c.3179A>C p.Lys1060Thr
Taiwanese N287K NA NA Wu et al., 2005
M469I NA NA
S895N NA NA
United States R766M c.2297G>T p.Arg766Met Ravnik-Glavac et al., 2000
R792G c.2374C>G p.Arg792Gly
G542X c.1624G>T p.Gly542X Wang et al., 2002
G551D c.1652G>A p.Gly551Asp
R334W c.1000C>T p.Arg334Trp
W1282X c.3846G>A p.Trp1282X
N1303K c.3909C>G p.Asn1303Lys
Various A800G c.2399C>G p.Ala800Gly Mercier et al., 1995
G149R c.445G>A p.Gly149Arg
R258G c.772A>G p.Arg258Gly
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NA: not attributed.

Table 3.

Percentage of abnormal alleles detected in men with CAVD

Number of mutant CFTR alleles
Other than 5T 5T %
2 0 26%
0 2 2%
1 1 26%
1 0 17%
0 1 8%
0 0 22%
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Assisted reproductive techniques and genetic counselling

Most men with CBAVD are diagnosed with a mild form of CF only after the genetic cause of their infertility is identified (Martin et al., 1992). Treatment of men with obstructive azoospermia (OA) as well as with CBAVD has not been available until the last three decades. Silber et al. (1990) was the first to report successful fertilization using epididymal spermatocytes, offering the possibility of men with CBAVD to have children. The technique was named MESA (Microsurgical Epididymal Sperm Aspiration) and consists of spermatocyte aspiration from the epididymis followed by fertilization in vitro. More recently there are other techniques to obtain spermatocytes from individuals with OA, such as PESA (Percutaneous Epididymal Sperm Aspiration), FNA (Fine Needle Sperm Aspiration) and TESA (Testicular Sperm Aspiration). The fertilization is made by ICSI (Intracytoplasmic Sperm Injection) (De Kretser & Baker, 1999).

Kamal et al., 2010, in an in vitro fertilization program using ICSI found similar rates of fertilization, clinical pregnancy, and miscarriage between men with CBAVD and patients having other causes of OA. Attardo et al. (2001) found a pregnancy rate of 30% (and fertilization rate around 50.7%) for men with CBAVD, a rate similar to the one obtained for non-CBAVD infertile men. This suggests that mutations on the CFTR gene do not alter the potential of spermatozoa fertilization. However, recent studies have demonstrated that CFTR protein is involved in a number of processes. These include spermatogenesis and sperm capacitation, acting not only as a simple ion-conducting channel but also as a regulator of other channels/transporters through protein–protein interactions and mediating the activation or inhibition of different signaling pathways, including sAC/cAMP/PKA and NF-kB/COX-2/PGE2, leading to alterations in transcriptional activities important for various reproductive processes (Chen et al., 2012). These are possible molecular mechanisms underlying the clinically observed link between CFTR mutations and male infertility other than CBAVD (Chen et al., 2012).

In accordance with Attardo et al. (2001), Lu et al., 2014, found similar rates of fertilization (70.1% and 68.2%, respectively), embryo quality (51.1% and 52.1%), clinical pregnancy (49.7% and 48.8%) and ectopic pregnancy (5.7% and 2.6%) between CBAVD and non-CBAVD patients who had PESA followed by ICSI. However, the rate of miscarriage/stillbirth (death before or after 20 weeks of gestation, respectively) was higher in men with CBAVD (23.9%) than in those with non-CBAVD obstruction (12.5%, p<0.001). The rate of live births was lower in men with CBAVD (70.5%) than in those with non-CBAVD obstruction (84.9%, p<0.001). Thus, patients with CBAVD presented a significantly increased risk of miscarriage or stillbirth. This risk is possibly a result of CFTR mutations, since the frequency of CFTR mutations was three fold higher in the CBAVD group (13.0%) than in the non-CBAVD group (4.1%, p<0.001) (Lu et al., 2014).

Besides the fertilization, miscarriage/stillbirth rates, and the role of the CFTR gene in male infertility, CFTR mutations from CBAVD patients submitted to ART can be transmitted to offspring. Mak et al. (1999) discusses the difficulty to predict phenotypic characteristics for a child carrier of one or other inherited allele, until the genotypic-phenotypic correlations of CFTR mutations are fully understood. Mak et al. (1999), Danziger et al. (2004) and Wong et al. (2004) underscore the importance of complete sequencing of the CFTR gene in men with CBAVD who desire to have children with ART.

The large number of CFTR variants already detected and the fact that only a few of them have been proven to be pathogenic, suggest that genetic counseling and testing in CF should be done by specialized reference centers. Recently, the CFTR2 Consortium showed that the M470V variant cannot be considered a pathogenic mutation (The Clinical and Functional Translation of CFTR, CFTR2, 2015).

Data from the CFTR2 Consortium also indicated that children diagnosed with CF (through newborn screening) but carrying non-CF-causing variants in one allele and one CF-causing variant in the other allele have significantly higher birth weight and first year growth rate and lower immunoreactive trypsinogen and sweat chloride values, as well as lower rate of persistent Pseudomonas aeruginosa colonization when compared to children with two CF-causing variants (Salinas et al., 2015).

De Kretser & Baker (1999) suggest that genetic analysis should start with the female partner of men with CBAVD. If a comprehensive CFTR analysis is done and no mutations in the CFTR gene is found, then the risk that a child develops any pathology due to mutations in this gene is reduced and the genetic analysis in the male partner is not 100% required, thereby reducing the costs of genetic analysis.

Stuppia et al. (2005) reported that the common mutations in CFTR gene in patients who undergo ART are not found at a rate higher than those expected for the general population, and the risk for a couple of having a child with CF is considerably reduced, as long as there is no previous family history of the disease. This is in accordance to the suggestion from De Kretser and Baker (1999) that the genetic analysis has to be performed for one of the parents, preferably in the infertile one, and if a mutation is found in the CFTR gene or an IVS9-5T allele, then genetic analysis should be performed in the other parent. Additionally, they consider that the analysis of the association between loci TG-5T-M470V can help with the risk calculation of having a child affected by the mild form of CF or CBAVD in couples in which one partner carries mutation in the CFTR gene and the other is a carrier of IVS9-5T allele.

Conversely, Mak et al. (1999) emphasize the importance of performing genetic analysis in both male and female partners, since the relation between genotype and phenotype is not well established and the consequences of inheriting at least one mutation is unknown. Attardo et al. (2001), suggest that men with CBAVD should be considered carriers of at least one mutation in the CFTR gene, unless the entire gene is analyzed and mutations are ruled out.

Cuppens & Cassiman (2004) explain that a man with CBAVD who carries one severe mutation in the CFTR gene has a 50% probability of transmitting this mutation to offspring. The probability that his female partner is a carrier of a mutation in the CFTR gene is 1 in 20 and the transmission of this mutation is 50%. For this couple, the risk of having a child with CF is 1 in 100 – a risk 25 times higher than for the general population (1 in 2,500). When no mutation is detected in the female partner, the risk for the couple is 1 in 1,000 (2.5 times higher than for the general population) (Cuppens & Cassiman, 2004).

For Euro-Brazilians the frequency of mutation within CFTR gene is 1 in 44 (Raskin et al., 2008). If the male partner has CBAVD due to a CFTR gene mutation, the risk for a couple to have a CF child is approximately 5.7 in 1,000 – a risk 43 times higher than for the general population (1 in 7,576).

Therefore, genetic counseling for couples in which the male partner has CBAVD is very important to estimate the risks and possible genotype-phenotype correlations. In addition to genetic counseling, diagnosis for embryo implantation in the uterus (Preimplantation Genetic Diagnosis – PGD) or prenatal diagnostic (Mak et al., 1999; Crosignani & Rubin, 2000; Viville et al., 2000; Allen et al. 2006) can be performed routinely. An informed consent should be obtained from the couple before initiating any ART.

CONCLUSION

CBAVD can be a form of atypical CF, and leads to infertility in the majority of male carriers of CFTR gene mutations. With ART widely available, men with CBAVD are able to reproduce. This increases the risk of passing on deleterious genes to descendants. Thus, every ART specialist should investigate if azoospermia is due to CBAVD. If so, and CBAVD is due to CFTR mutations, the infertile couple should be informed about the reproduction consequences before ART. Couples at risk should be offered comprehensive CFTR genetic testing, taking into account the ethnic group of the patient and subsequent counseling. When a couple seeks ART, their obvious and main goal is to achieve pregnancy and eventually have a child. However, the first goal of the physician should be to identify the cause of infertility, if possible, and also to offer every available technique, such as pre-implantation genetic diagnosis, to minimize the risk of having an affected child with chronic or severe disease.

Acknowledgments

We thank Diane Cooper, MSLS, NIH Library, for providing assistance in writing this manuscript. This research was supported in part by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health (NIH); and in part, by a grant from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Process: 311166/2011-3 - PQ-2 (to F.R.F.).

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