INTRODUCTION

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Congenital bilateral absence of the vas deferens (CBAVD) accounts for 1 to 2% of cases of male infertility (1). It has been suggested (2, 3) that most patients with CBAVD can be considered as having a primarily genital mild variant of cystic fibrosis (CF). Actually the majority of those suffering from this condition carry two or at least one mutation of the CF gene, which codifies for the cystic fibrosis transmembrane conductance regulator protein (CFTR), and a few show abnormal sweat electrolytes concentrations as well as symptoms of mild CF (4). As being a CF carrier is not believed to affect phenotype, patients in whom one mutation was found could carry another, undetected, mutation or DNA variant, possibly in the promoter region or in noncoding regions of the CF gene.

In CF patients, the CFTR defect results in impaired transport of electrolytes across the respiratory epithelia (5), which can be studied in vivo by means of measurement of nasal potential difference (NPD) (6). Basal NPD values are more negative in CF patients than in control subjects, and superinfusion of nasal mucosa with a sodium (Na⁺) channel blocker (amiloride) produces a two- to threefold depolarization in CF patients with respect to normal subjects. Moreover in CF no hyperpolarization can be observed when the epithelium is superinfused with a chloride (Cl)-free solution and isoproterenol, thus confirming an unequivocal defect in CFTR function.

For these reasons this method can be considered useful both for research studies evaluating CFTR function, such as in the attempt to normalize ion transport with gene therapy (12, 13), and for clinical purposes, namely the diagnosis of CF in equivocal cases (14, 15). In this connection NPD measurements can also be actually useful to study CFTR function in the airways of patients with CBAVD. Osborne and coworkers (16) studied nine CBAVD patients, and found normal basal NPD values, normal response to amiloride, and a response to low Cl solution which was intermediate between control subjects and CF patients. They concluded that in CBAVD Na⁺ transport is normal and Cl conductance is altered. However, to our knowledge no further studies were published on this subject, and, as recently pointed out by Stern (17), the observation that the nasal epithelium of these patients has abnormal bioelectric properties still needs to be adequately documented.

In view of these considerations we evaluated NPD in a group of patients with CBAVD in order to assess whether their nasal epithelium bioelectric properties are impaired. Gathered data were compared with those of normal subjects and patients with CF.

	METHODS

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Subjects

Informed consent was obtained from all patients. The subjects evaluated in the present study were divided in three groups:

Group 1: 12 men diagnosed as having CBAVD (mean age 35 yr, range 24 to 44 yr). All of them originally requested consultation in Departments specialized in diagnosing and treating sterility, where the diagnosis was established on the basis of azoospermia with normal-sized testes, nonpalpable vas deferens, and small volume and low pH of the ejaculate.

Group 2: 11 non-CF control subjects (4 males, 7 females, mean age 29 yr, range 20 to 38 yr). These subjects were selected among both patients admitted to our center for other well-documented diseases, and healthy volunteers.

Group 3: 10 patients with fully expressed CF (CF controls, 6 males, 4 females, mean age 28 yr, range 19 to 39 yr). Diagnosis was confirmed by at least two abnormal sweat tests, as well as compatible clinical features and, when possible, mutation analysis.

Clinical Evaluation and Mutation Analysis in Patients with CBAVD

All subjects had a detailed history taken that included questions about respiratory, gastrointestinal, pancreatic, and hepatobiliary disease. They were meticulously examined by one registrar skilled in CF clinic (C.C.). Sweat electrolyte analysis was performed at least twice by the classic Gibson and Cooke method (18). At least 75 mg of sweat had to be collected on a 4 × 4 cm filter paper in order to consider the sweat test reliable. Respiratory evaluation included spirometry, chest and paranasal sinuses X-ray, and bacteriological cultures of deep sputum samples or hypopharyngeal secretions obtained after physiotherapy and coughing. Blood samples were taken for testing of glutamic- oxaloacetic transaminase, glutamic-pyruvic transaminase, -glutamyl transferase, bilirubin, pancreatic amylase and lipase, erythrocyte sedimentation rate, and immunoreactive trypsin. Chymotrypsin levels in stools were also measured.

F508, R117H, R1162X, 2183AA G, N1303K, 3849 + 10KbC T, G542X, 1717-1G A, R553X, Q552X, G85E, 711 + 5G A, 3132delTG and 2789 + 5G A were tested using for R117H two specifically designed primers which create a CFoI restriction site when the mutation is absent, and for all the other mutations a reverse dot blot assay (19). The intron 8 polyT sequence was analyzed with the nested-polymerase chain reaction (PCR) method according to Chillon (20).

Electrophysiology

Exclusion criteria for measurement of NPD were acute upper airway infection and previous nasal surgery. NPD was studied as follows: first basal NPD was measured, according to the method proposed by Alton and coworkers (9). After that, the response of NPD to ion channel blockers and activators was studied according to the method previously described by Knowles and coworkers (7).

Briefly, NPD was measured by means of two calomel electrodes (Fisher Scientific Company, Springfield, NJ) connected to a high impedance voltmeter (WPI Inc., Sarasota, FL). The reference electrode was connected to a subcutaneous needle filled with 4% agar/Ringer's solution. The exploring catheter perfused with Ringer's solution was connected to the second calomel electrode. A second catheter allowed perfusion of different drugs and ion solutions and was connected in parallel with the exploring catheter. Calibration was performed previously, considering as acceptable offset values ± 5 mV. The protocol included measurement of NPD under the following different conditions: (1) the basal value was measured by recording NPD for at least 5 s (or until a stable value was observed) at anatomically defined sites underneath the inferior turbinate (0.5, 1, 1.5, 2, and 3 cm posterior to the anterior tip of the turbinate, which is used as an anatomic and physiologic reference point) in order to detect the maximal NPD value; (2) inhibition of NPD during perfusion with amiloride (10⁴ M); (3) change in Cl conductance during perfusion with chloride-free (0Cl) solution (replaced with gluconate) in the presence of amiloride; (4) change in polarization during perfusion with isoproterenol (10⁵ M) added to the perfusate; (5) change in NPD in response to perfusion with chloride-free solution and isoproterenol in the presence of amiloride, i.e., the cumulative effect in NPD change after the two previous conditions.

NPD measurements were performed in both nares. For data analysis the most technically acceptable test was subsequently used, i.e., the tracing with the most stable voltage. Data were sampled at 100 Hz and stored on a personal computer through an A/D converter (DT2801-A; Data Translation, Marlboro, MA) for data analysis with appropriate software (Anadat 5.1; RHT Infodat Inc., Montreal, PQ, Canada). For each condition 3 min of tracing were collected and the last 30 s were used for statistical analysis. NPD values were considered both as absolute values and as absolute (mV) change after each solution. For comparisons among groups the change in polarization was considered, according to the suggestions by Knowles and coworkers (7).

Data are presented as mean ± standard deviation. Comparison of groups was performed using one-way analysis of variance (ANOVA) followed by a post hoc multiple comparison procedure (Tukey-Kramer multiple comparisons test) when a significant difference was found. A p value of less than 0.05 was considered as significant.

	RESULTS

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Individual values of sweat electrolytes, genotype, basal NPD, and anamnestical and clinical data of the 12 patients with CBAVD are reported in Table 1. Mean values of sweat Cl were under 40 mmol/L in seven subjects, under 60 mmol/L in two, and above 60 and 70 mmol/L in respectively two and one patients. In the group of CF control subjects sweat chloride concentrations ranged from 71 to 117 mmol/L. In the CBAVD subjects, history, physical examination, and respiratory evaluation (including pulmonary function testing) were unremarkable in all patients except those specifically reported in Table 1. All the 12 subjects were pancreatic sufficient and no evidence of liver disease could be found in any of them. In order to detect associated genitourinary tract anomalies, abdominal ultrasonography was added to our study protocol when part of the patients had already been examined: among the patients tested, one was excluded from the study because of monolateral renal agenesis. CFTR gene mutations were found in eight patients (seven F508 and one G542X), the 5T variant in two. Only one mutation was found in 7 of 8 patients. Patient 11 was a compound heterozygote for F508/R117H.

Electrophysiology

Basal values. When measured by means of the method proposed by Alton (9), NPD values in the CBAVD group resulted in 17.6 ± 0.5 mV. However two patients (numbers 11 and 12) showed NPD values more negative than 28 mV, which we consider the normal limit for basal NPD, on the basis of previously reported data (14). These two subjects had also abnormal NPD values during superinfusion with 0Cl solution and isoproterenol (see below).

Basal values obtained in the CBAVD group before superinfusion with drugs/solutions were comparable with those obtained with the previous method (14.3 ± 9.0 mV). In control subjects NPD values (13.2 ± 4.6 mV) were not different from CBAVD patients (p > 0.05). Both groups had NPD values significantly less negative than those obtained in the CF group (26.6 ± 12.1 mV, p < 0.01)

Change in polarization during superinfusion with the Na⁺ blocker, amiloride. Patients with CBAVD and normal control subjects showed a mean depolarization of 4.7 ± 4.4 and 4.5 ± 2.5 mV (p > 0.05), respectively, whereas CF patients showed a significantly higher depolarization in comparison to both the other groups (13.6 ± 9.7 mV, p < 0.01). Figure 1 shows individual values and mean ± SD of changes in NPD during this maneuver in the three groups.

View larger version (12K): [in this window] [in a new window]   Figure 1.   Change in NPD (mV) in response to amiloride in subjects with CBAVD, normal control subjects (CONTROLS), and CF patients (CF). Solid horizontal bar indicates the mean change in NPD. Dotted horizontal bars indicate ± 1 SD. *p < 0.01 with respect to CF patients.

Change in NPD during superinfusion with chloride-free solution in the presence of amiloride. In control subjects and CBAVD patients a hyperpolarization was observed (7.8 ± 7.5 mV and 5.5 ± 5.6 mV, respectively, p > 0.05), in contrast with a further slight depolarization in CF patients (1.9 ± 1.8 mV). Data obtained in CF patients were statistically different from those obtained in the CBAVD (p < 0.05) and control groups (p < 0.01). In Figure 2 individual values and mean ± SD measured under these circumstances are reported. In the CBAVD group three subjects (Patients 3, 11, and 12 in Table 1) did not show hyperpolarization during these maneuvers. Patients 3 and 12 had abnormal sweat electrolyte concentrations. Patient 11 was a compound heterozygote for F508/R117H. In addition, Patients 11 and 12 had abnormal basal NPD values.

View larger version (11K): [in this window] [in a new window]   Figure 2.   Change in NPD in response to perfusion with chloride-free solution in the presence of amiloride in the three groups of subjects. Symbols as in Figure 1, except that *p < 0.05 and **p < 0.01 with respect to CF patients.

Change in NPD during perfusion with isoproterenol in the presence of chloride-free solution containing amiloride. In the CF group there was no change in NPD (0.5 ± 1.3 mV), whereas normal control subjects and CBAVD patients showed further hyperpolarization (5.2 ± 2.8 mV and 6.0 ± 5.7 mV, p > 0.05). These data were statistically different in CBAVD (p < 0.01) and normal control subjects (p < 0.05) when compared with CF patients.

Cumulative change in NPD response after perfusion with chloride-free solution plus isoproterenol in the presence of amiloride. As before, the group of CBAVD patients as a whole showed changes in NPD not different from those obtained in normal control subjects (11.5 ± 10.3 mV in CBAVD patients, 13.0 ± 8.4 mV in control subjects, p > 0.05). Both groups differed significantly from data obtained in CF patients (1.4 ± 1.4 mV, p < 0.001 and p < 0.01 when compared with control subjects and CBAVD, respectively). In Figure 3 individual data and mean ± SD are reported. Again, Patients 3, 11, and 12 showed no hyperpolarization, similarly to patients with CF. In Figure 4 sample NPD recordings are reported for the following situations: normal control subject, CF patient, CBAVD patient with normal PD phenotype, and CBAVD patient with CF-like NPD tracing.

View larger version (12K): [in this window] [in a new window]   Figure 3.   Change in NPD in response to the combined effect of chloride-free solution plus isoproterenol in the presence of amiloride. Symbols as in Figure 1, except that *p < 0.01 and **p < 0.001 with respect to CF patients.

View larger version (26K): [in this window] [in a new window]   Figure 4.   Left panels: NPD tracings obtained in a normal control subject (top) and in a patient with CF (bottom). Right panels: two tracings obtained in CBAVD patients are reported; Patient 8 showed normal NPD phenotype (top), while Patient 12 presented abnormal inhibitor/agonist activated NPD (bottom). 0Cl solution = chloride-free solution.

	DISCUSSION

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This study shows normal electrophyiologic properties of nasal epithelium in our group of patients with CBAVD. Actually, basal values of NPD and change in NPD during superinfusion with ion channel blockers and activators are significantly different from data obtained in patients with CF and similar to control subjects. In line with previous studies by others (7) we found an increased rate of Na⁺ transport and a defective Cl secretion in CF patients, as demonstrated by elevated basal NPD and abnormal response to amiloride, chloride-free solution, and isoproterenol. In agreement with previous data by Knowles (7), we also found that there is some overlap between CF patients and normal control subjects after superinfusion with amiloride, and that the higher discrimination between CF patients and control subjects during Cl transport studies is obtained by considering the net change in PD with the combined response to chloride-free solution plus isoproterenol.

Measurement of basal NPD, which can be considered as an estimate of Na⁺ transport, showed increased Na⁺ permeability only in the group of patients with CF. An abnormal Na⁺ transport in CF patients was confirmed by the increased NPD inhibition by the Na⁺ channel blocker, amiloride, that in this group of patients was more than twofold greater than in the control and CBAVD groups. According to such results, Na⁺ transport is to be considered normal in the airway epithelium of almost all patients with CBAVD.

Our studies on Cl transport showed similar results. Basal Cl conductance was studied during superinfusion of the nasal epithelium with a solution with 0Cl in the presence of amiloride. CBAVD patients responded to this maneuver with a hyperpolarization which was on average not different from that obtained in control subjects, whereas CF patients showed a further slight depolarization, thus suggesting altered basal Cl transport. The cyclic adenosine monophosphate (cAMP)- dependent Cl permeability was assessed by means of the -agonist isoproterenol, and the hyperpolarization observed in CBAVD patients confirmed effective cAMP-mediated (CFTR) Cl secretion in this group. Once again these data were similar to those obtained in control subjects and different from those in the CF group, where a defective CFTR Cl secretion was confirmed by no change in NPD. The combined response to chloride-free solution plus isoproterenol was also computed, and again there was a significant difference between CBAVD and CF patients, the former showing normal hyperpolarization, the latter a slight depolarization. These data strongly suggest that basal and cAMP-mediated Cl secretion are normal in almost all patients with CBAVD.

In our CBAVD group, three patients had a response to ion channel blockers and activators in the pathological range of CF patients (Patients 3, 11, and 12). In these subjects CF was confirmed by elevated sweat Cl concentrations together with symptoms compatible with mild CF (Patients 3 and 12) or positive DNA testing (Patient 11). Two of three of these subjects (Patients 11 and 12) had raised basal NPD, a peculiarity which we have previously shown being able to detect patients with CF despite nonpathological sweat test (14). Patient 3 had normal basal NPD values, perhaps because of mild inflammation of nasal mucosa. Data from these three subjects explain the slight, not statistically significant difference in the change of polarization during superinfusion with different solutions in the CBAVD group compared with normal control subjects.

Evidence of unaltered ion transport in nasal epithelium of CBAVD patients is not in contrast with the role of CFTR in the pathogenesis of the vas deferens absence. The percentage of normal CFTR function seems to affect evidence of the disease in various organs. The deferent ductal system of the male genital tract is estimated to need a CFTR activity of more than 10% of normal for correct embryological development (21). Different organs have different sensitivity to CFTR function, such that progressive reduction in CFTR activity is associated with progressive organ involvement, depicting the wide range of phenotype variability of CF (17, 21). In this connection, as airway cells are less sensitive to CFTR activity than the vas deferens, the finding of normal NPD in CBAVD patients could be explained by the fact that in these patients the reduction of CFTR function was not sufficient to alter ion transport in airway epithelia.

Our results are in some disagreement with those of Osborne and coworkers (16), who suggested that basal Cl permeability is impaired in CBAVD patients. They found in nine subjects normal basal NPD values, normal response to amiloride, but a response to low-chloride solution which was intermediate between CF patients and control subjects. Also their population included some patients (two) with positive sweat test. However, as they gave their results only in terms of Na⁺ concentrations, it is not clear whether other patients had an abnormal sweat test on the basis of Cl concentrations. Moreover, they did not assay cAMP-mediated Cl secretion, a most important test in a CFTR-related disorder.

A high frequency of CFTR gene mutation (in 8 of 12 patients), sweat Cl concentrations higher than 60 mmol/L in a minor proportion of our CBAVD patients (4 of 12), and clinical characteristics compatible with CF (8/12) are in agreement with previously reported experiences (4, 20, 22).

Whether or not patients with CBAVD bearing CFTR gene mutations on both chromosomes and/or positive sweat test can be diagnosed as having a mild form of CF is still controversial, mainly because of the possible psychological and social impact related to a diagnosis of CF. Long-term studies aimed at evaluating whether this particular subgroup of CBAVD patients will later in life develop into more fully expressed CF are still lacking. Some investigators (24) suggested clinical follow-up for potential late complications of CF in asymptomatic CBAVD patients with compound heterozygosity and positive sweat test (17). We think that as NPD can highly discriminate between normal subjects and patients with CF (10, 25), also in presence of nonpathological sweat electrolyte concentrations (14, 26), this technique should be included in routine investigations for patients with CBAVD. It is therefore reasonable to suggest follow-up clinical evaluation in CBAVD subjects presenting abnormal ion transport in respiratory epithelia.

In the present study CBAVD men were compared with CF patients and normal control subjects (two groups with subjects of both sexes and with different ages). This could be a potential confounding factor with respect to NPD measurements. However, in previously reported experiences, NPD was not shown to be affected both by age and gender (29), and therefore the results of our study were probably not influenced by these physiologic states.

In conclusion, the results of our study and the subsequent clinical implications can be summarized as follows. First, in our group of patients with CBAVD as a whole, measurement of NPD revealed normal bioelectric properties of nasal epithelium. Therefore, despite the possible role of CFTR in the pathogenesis of the disease, Na⁺ transport, and basal as well as cAMP (CFTR)-dependent chloride permeability were shown to be normal in the airways. Second, in a small proportion of our subjects NPD measurements were abnormal. In these subjects CF was confirmed by elevated sweat Cl concentrations or positive DNA testing. We suggest NPD measurement as a routine evaluation in CBAVD patients, together with sweat test and DNA analysis. In particular, a diagnosis of CF should be confirmed if sweat electrolytes are found to be abnormal in at least two determinations; if sweat testing is not conclusive then DNA analysis should be performed; if two recognized CF mutations are not found then NPD needs to be studied. Subjects with abnormal ion transport in airway epithelia should undergo further clinical follow-up in order to detect possible late complications of CF.