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Cystic fibrosis transmembrane conductance regulator mutations in azoospermic and oligospermic men and their partners
RBMOnline - Vol 19. No 5. 2009 685–694 Reproductive BioMedicine Online; www.rbmonline.com/Article/4035 on web 22 September 2009
Article Cystic fibrosis transmembrane conductance regulator mutations in azoospermic and oligospermic men and their partners Sabina Gallati graduated in Biology and Genetics from the University of Berne. She has undertaken postdoctoral research and training in molecular genetics and forensics in London (UK), Wu¨rzburg (Germany), Boston (USA) and Quantico (USA) and has specialised in Medical Genetic Analysis. In 2003, she became Professor of Human Genetics and Head of the Division of Human Genetics at the Medical Faculty of the University of Berne. Her main research interests are genetic mechanisms in Cystic Fibrosis and CFTR-related disorders, hereditary haemochromatosis and mitochondriopathies.
Dr Sabina Gallati Sabina Gallati1,5, Simone Hess1, Dorothea Galie´-Wunder2, Elisabeth Berger-Menz3, Dominik Bo¨hlen4 Division of Human Genetics, Departments of Paediatrics and Clinical Research, Inselspital, University of Berne, CH-3010 Berne, Switzerland; 2Division of Gynaecological Endocrinology and Reproductive Medicine, Inselspital, University of Berne, CH-3010 Berne, Switzerland; 3Division of Obstetrics and Gynaecology, Lindenhofspital, CH-3001 Berne, Switzerland; 4Division of Urology, Clinic Beau-Site, CH-3013 Berne, Switzerland 5 Correspondence: e-mail: [email protected] 1
Abstract The objective of this study was to investigate the contribution of cystic fibrosis transmembrane conductance regulator (CFTR) to human infertility and to define screening and counselling procedures for couples asking for assisted reproduction treatment. Extended CFTR mutation screening was performed in 310 infertile men (25 with congenital absence of the vas deferens (CAVD), 116 with non-CAVD azoospermia, 169 with severe oligospermia), 70 female partners and 96 healthy controls. CFTR mutations were detected in the majority (68%) of CAVD patients and in significant proportions in azoospermic (31%) and oligospermic (22%) men. Carrier frequency among partners of infertile men was 16/70, exceeding that of controls (6/96) significantly (P = 0.0005). Thus, in 23% of infertile couples both partners were carriers, increasing the risk for their offspring to inherit two mutations to 25% or 50%. This study emphasizes the necessity to offer extended CFTR mutation screening and counselling not only to patients with CAVD but also to azoospermic and oligozoospermic men and their partners before undergoing assisted reproduction techniques. The identification of rare and/or mild mutations will not be a reason to abstain from parenthood, but will allow adequate treatment in children at risk for atypical or mild cystic fibrosis as soon as they develop any symptoms. Keywords: assisted reproduction, cystic fibrosis transmembrane conductance regulator, mutation spectrum, polyvariant mutants, primary infertility, risk for offspring
Introduction Infertility is a major health problem affecting about 15% of all couples in industrially developed countries (Healy et al., 1994; Mau-Holzmann, 2005). Accumulating evidence suggests that in 15% of male and 10% of female infertile individuals genetic factors may represent the underlying aetiology (Foresta et al., 2002). Since assisted reproduction techniques overcome the protective mechanism of natural selection, the risk of transmitting genetic defects and
decreased fertility to the offspring increases considerably (Faddy et al., 2001; Burrello et al., 2002) requiring careful diagnosis of the infertility status and evaluation of the genetic risk for the child before any technique is performed (Foresta et al., 2002). Male infertility due to severe oligozoospermia and azoospermia has been associated with a number of genetic abnormalities, including numerical and structural chromosomal aberrations (Burrello et al., 2002; Douet-Guilbert
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cussed, including the potential contribution of mutant alleles to female infertility as well as the complexity of genetic counselling and risk assessment.
et al., 2005), deletions of the azoospermia factor region of the Y chromosome (Kihaile et al., 2005; Hellani et al., 2006) and mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (Cuppens and Cassiman, 2004; Claustres, 2005). Most numerical chromosome aberrations and azoospermia factor region deletions are de-novo events in the parental germ cells, whereas mutations in the CFTR gene are inherited, posing a plurivalent genetic risk factor for siblings and offspring.
Materials and methods
Apart from classic cystic fibrosis (CF), CFTR mutations are involved in the development of atypical, CF-like and monosymptomatic phenotypes including male infertility due to congenital absence of the vas deferens (CAVD), either bilateral or unilateral (Anguiano et al., 1992; Chillon et al., 1995). Since the discovery of the CFTR gene in 1989 (Riordan et al., 1989), more than 1600 sequence alterations have been reported to the Cystic Fibrosis Mutation Database in Toronto (http://www.genet.sickkids.on.ca/cftr, accessed September, 2009). The extent to which CFTR mutations contribute to the phenotype and to clinical variation depends on their nature and localization as well as on the overall genetic background, epigenetic factors and environmental influences. A common CFTR mutation conferring a mild phenotype is the 5T allele at the polymorphic Tn locus in intron 8, which is known to affect the splicing efficiency resulting in exon 9 skipping. The original classification of the 5T variant as a bilateral CAVD mutation with incomplete penetrance was published in 1995 (Chillon et al., 1995). Further independent studies confirmed that the frequency of the 5T allele in individuals with bilateral CAVD was significantly higher than that of the general population (Groman et al., 2004; Claustres, 2005). In addition, a higher than expected frequency of CFTR mutations has been observed in unilateral CAVD (Mickle et al., 1995), obstructive azoospermia with intact vas deferens (Jarvi et al., 1995; Mak et al., 1999) and non-obstructive azoospermia and oligozoospermia (van der Ven et al., 1996; Dohle et al., 2002), indicating that the CFTR protein may be involved in the process of spermatogenesis or sperm maturation apart from playing a critical role in the development of the epididymal glands and the vas deferens. However, these findings were not confirmed in other studies (Ravnik-Glavac et al., 2001; Larriba et al., 2005). Thus, the contribution of CFTR to other forms of male infertility than bilateral CAVD/ unilateral CAVD is still not clear and needs further investigation.
The diagnosis of primary infertility was based on physical examination, ultrasonography and semen analysis. Additional studies, such as semen cultures and testicular biopsies, were performed when appropriate. All patients were investigated for testicular volumes and pathological findings such as hypotrophic testes, varicoceles, inguinal hernia, CAVD and anomalies of the seminal vesicle and epididymis as well as for prostatitis and epididymitis and were interviewed concerning former operations for undescended testes and nicotine consumption. Semen analysis was performed twice according to the World Health Organization 1999 criteria and the 2004 guidelines on infertility of the European Association of Urology. Spermiograms included volume (ml), pH, total sperm count (million/ejaculate), sperm concentration (million/ml), motility within 60 min after ejaculate (%), morphology (% normal), viability (% live) and fructose (mol/ejaculate) and neutral alpha-glucosidase (mIU/ejaculate) in seminal plasma. Severe oligozoospermia (<5 million spermatozoa per ml) was attributed to 54.5% (169/310) and azoospermia to 45.5% (141/310) of the patients. CAVD was diagnosed in 25 (17.7%) of the azoospermic men. None of the infertile patients and their partners had any clinical manifestations or family history suggestive of CF. Subjects presenting with chromosomal aberrations or Y chromosome microdeletions were excluded from the study.
The major objective of this study was therefore to determine the nature, frequency and distribution of CFTR mutations and genotypes in infertile men and their partners compared with the mutation pattern of classic CF patients in Switzerland on the one hand and healthy controls on the other hand. As commercial kits for routine screening are known to cover around 80% of mutations causing classic CF but only approximately 62% of mutant alleles in the CFTR genes of infertile individuals (Claustres et al., 2000), this study performed extensive screening (LiechtiGallati et al., 1999) of the entire coding CFTR sequence. A further aim was to define a rational approach to CFTR testing in infertile couples. Finally, the significance of extensive CFTR mutation testing for infertile couples is dis-
Subjects and clinical evaluation A total of 310 selected men aged between 27 and 57 years who had consulted for primary couple infertility, 70 of their partners (aged 23–45 years) and 96 healthy and fertile control individuals (47 male aged 26–49 years, 49 female aged 24–46 years) were investigated. Patients and controls were unrelated and mainly of Swiss origin.
Seventy female partners of infertile men diagnosed with one or two CFTR mutations were referred for extended CFTR analysis before entering an intracytoplasmic sperm injection (ICSI) programme and after exclusion of ovulation failure, physical problems, infectious or hormonal diseases. In addition, 96 healthy individuals from the general population referred for molecular genetic analyses in the context of family testing and having at least one healthy child (paternity confirmed) were used as controls.
Analysis of CFTR mutations Informed consent for molecular genetic analyses was obtained from all individuals included in this study. Genomic DNA was extracted from peripheral blood cells according to standard procedures. Screening of the entire coding sequence of the CFTR gene including intron/exon boundaries and the promoter region was performed by single-strand conformation polymorphism/heteroduplex RBMOnlineÒ
Article - CFTR mutations in azoospermic and oligospermic men and their partners - S Gallati et al.
technique with a sensitivity of 97–98% (Liechti-Gallati et al., 1999) followed by direct sequencing of the variants using an ABI 377 sequencing system (Applied Biosystems, USA).
with infertile men without CFTR mutations. No other parameter of semen analysis discriminated between patients with and without CFTR mutations.
Distribution and frequencies of CFTR mutations identified in this study were compared with mutation data generated from 582 Swiss patients with classic CF using the same extended scanning method (Liechti-Gallati et al., 1999).
Based on linear combinations of the clinical and semen characteristics, discriminant analysis provided significant discrimination (P = 0.002) between CFTR mutation carriers and non-carriers for the predictor variables ejaculate volumes and structural abnormalities of the male genital tract. That is, infertile men presenting with ejaculate volumes less than 3 ml and CAVD and/or anomalies of the seminal vesicle and/or inguinal hernia and/or hypotrophic testes and/or cryptorchidism have an increased risk of carrying at least one CFTR mutation.
Statistical analysis The chi-squared statistic, Fisher’s exact test and unpaired ttest were used where appropriate. All P-values were based on two-sided comparisons and those <0.05 were considered to indicate statistical significance. Discriminant analysis was performed to test for diagnostic criteria predicting the presence of CFTR mutations. The analyses were performed using Statistical Package for Social Sciences (SPSS) Advanced Statistical 11.0 software (SPSS, Chicago, USA).
Results Genetic analyses Genetic testing was performed on 310 infertile men referred to the study centre’s laboratory for CFTR mutation screening with a diagnosis of either severe oligozoospermia (169/ 310) or azoospermia (141/310) with (25/141) or without CAVD (116/141). A total of 123 CFTR mutations were identified in 620 chromosomes (20%). Two mutations were found in 60% of the CAVD patients, in 12% of the nonobstructive azoospermic men and in 1% of oligozoospermic men, whereas the percentage of patients with one was 8% (CAVD), 19% (azoospermia) and 21% (oligospermia) and with no mutation was 32% (CAVD), 69% (azoospermia) and 78% (oligozoospermia).
Clinical characterization of patients In comparison with individuals without mutations in the CFTR gene, analysis of the clinical data showed that patients with CFTR mutations presented significantly more frequently (P = 0.005) either with CAVD (68% versus 32%) or with inguinal hernia (11.8% versus 1.5%) and/or cryptorchism (17.6% versus 9.1%) and/or testicular hypotrophy (23.5% versus 7.6%). Semen analysis according to WHO standards demonstrated pathological values for ejaculate volumes (<2.0 ml) in 21%, for semen pH (<7.0 or >8.0) in 11%, for sperm concentrations (<20 106/ml) in 99% and for total sperm count (<40 106/ejaculate) in 94% of the patients. Vitality of the spermatozoa was found to be pathologically reduced (<50%) in 94% of the infertile individuals. Not surprisingly, statistical calculations demonstrated significantly (P < 0.027) reduced ejaculate volumes (1.9 ml), fructose values (16.15 lmol) and pH (pH 6.9) in CAVD patients compared with infertile men without CAVD. However, non-CAVD patients with CFTR mutations also showed the tendency for smaller ejaculate volumes (2.67 versus 3.53 ml) and fructose values (37 versus 45 lmol) compared RBMOnlineÒ
CFTR mutation patterns and frequencies Extensive screening of the CFTR gene identified 39 different mutations (29 missense, four alternative splice-site, two splice-site, two nonsense, one frameshift, one in-frame deletion) in 90 (29%) out of 310 infertile males carrying one or two mutations (Table 1). CAVD patients showed a CFTR mutation frequency of 66% (33/50), azoospermic men without CAVD presented with a frequency of 21.6% (50/232) and oligospermic men with a frequency of 11.8% (40/338). Twenty-one (53.8%) of these mutations were detected in infertile men and/or their partners only, but neither in 1164 chromosomes of CF patients nor in 192 chromosomes of control individuals. Table 1 illustrates the heterogeneity of CFTR mutations and their different distribution in infertile men, CF patients and controls. For instance, the most common mutation worldwide, F508del, decreases from 65.0% in CF patients to 18.0% in CAVD patients, 7.3% in azoospermic men without CAVD, 1.2% in oligospermic patients, 2.1% in female partners and 0.5% in control persons. Whereas the 5T allele, well known to produce variable amounts of exon 9 skipping, increases from lowest frequencies of 1% in classic CF patients and controls to approximately 3% in female partners and oligospermic men up to 6% and 16% in azoospermic patients without and with CAVD, respectively. In CAVD patients and azoospermic men without CAVD, respectively, the most common mutation was F508del (18.0% and 7.3%) followed by the mild mutations 5T (16.0% and 6.0%) and R117H (8.0% and 1.3%). Oligospermic men as well as female partners, however, carried 5T alleles most frequently (3.55% and 2.86%, respectively), followed by F508del (1.18% and 2.14%, respectively). The missense mutation S1235R was not detected in 50 alleles of CAVD patients, and was also very rarely found in classic CF patients (1/ 1164 alleles) and controls (1/192 alleles), but turned out to be the third most common mutation in azoospermic men without CAVD (3/232), in oligospermic men (3/338) and in female partners (3/140). In summary, 26% of CAVD alleles (versus 79% of classic CF alleles) presented with severe CFTR mutations, 38% with mild CFTR mutations and 2% with mutations with uncertain role, whereas azoospermic men without CAVD and oligospermic men, respectively, carried severe CFTR mutations on only 9.1% and 1.8% and mild mutations on 11.6% and 10.1% of their chromosomes. In azoospermic men, an additional 1.1% of
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Table 1. Distribution and frequencies of 39 different CFTR mutations detected in 92 out of 310 infertile men and comparison with female partners of infertile men, classic cystic fibrosis patients and control population.
Values are number of alleles (percentage of all alleles tested). Bold italic = mutations found on more than two alleles and in more than 1% of alleles; fields in dark grey = CFTR mutations associated with classic/severe CF; fields in light grey = CFTR mutations associated with a mild or uncertain, unpredictable phenotype; CAVD = congenital absence of the vas deferens. a Novel mutation identified in this study with unpredictable phenotype.
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mutations were newly detected and, thus, have an unpredictable phenotype. As expected, statistical calculations showed significant differences (P < 0.0001) in the mutation pattern between infertile men and classic CF patients. But
distribution of mutations (mild/severe) also differed significantly between CAVD patients and oligospermic men (P = 0.0307) as well as between azoospermic men without CAVD and oligospermic men (P = 0.0060). Frequencies
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Article - CFTR mutations in azoospermic and oligospermic men and their partners - S Gallati et al.
Table 2. Distribution and frequencies of IVS8-TGm-Tn haplotypes. Haplotype
Infertile males (n = 303)
Partners (n = 65)
Control males (n = 47)
Control females (n = 49)
11TG5T 12TG5T 13TG5T 10TG7T 11TG7T 12TG7T 11TG8T 9TG9T 10TG9T 11TG9T
16 (2.64) 13 (2.15) 4 (0.70) 90 (14.85) 326 (53.80) 68 (11.22) 1 (0.17) 1 (0.17) 70 (11.55) 17 (2.81)
3 (2.31) 1 (0.77) 0 (0.00) 22 (16.92) 68 (52.31) 27 (20.77) 0 (0.00) 0 (0.00) 5 (3.85) 4 (3.08)
1 (1.06) 0 (0.00) 0 (0.00) 20 (21.28) 53 (56.38) 5 (5.32) 1 (1.06) 0 (0.00) 9 (9.57) 5 (5.32)
1 (1.02) 0 (0.00) 0 (0.00) 23 (23.47) 56 (57.14) 10 (10.20) 0 (0.00) 0 (0.00) 5 (5.10) 3 (3.06)
Total alleles
606
130
94
98
of CFTR mutations were highest in CAVD patients (P < 0.0001) and higher in azoospermic men without CAVD than in oligospermic men (P = 0.0023). However, oligospermic men definitely more frequently carried CFTR mutations than individuals of the control group (P = 0.0004). In 70 women whose partners had tested positive for either CFTR mutations or 5T alleles, extended screening of the CFTR gene was also performed revealing a mutation spectrum similar to that of oligospermic men including four 5T alleles, three S1235R, three F508del and one I148T, V754M, V920M, D1152H, 3905insT and Q1352H each (Table 1). The carrier frequency was 22.9% (16/70) exceeding significantly (P = 0.0005) the carrier frequency of the control group analysed in this study presenting with six carriers (6.3%; 3/47 male, 3/49 female).
Polyvariant mutants According to the literature, both the polymorphic loci TGm and Tn in intron 8 determine the proportion of full-length and mis-spliced CFTR transcripts lacking exon 9 in a polyvariant mutant manner. The IVS8-TGmTn haplotypes in infertile men as well as in 65 of their partners were defined and compared distribution and frequencies with those of 96 healthy controls (Table 2). Ten different haplotypes were found in a total of 464 individuals (928 alleles), within which 11TG7T was the most common in all four subgroups, whereas 9TG9T and 11TG8T were identified in only one and two subjects, respectively. Haplotype 11TG9T also showed low frequencies in all categories ranging from 2.81% to 5.32%. The 5T allele was present on a 11TG, 12TG and 13TG background, however, the combination 12TG5T and 13TG5T appeared only in association with infertility and the frequency of 5T did not substantially differ between infertile men and their partners, but was significantly higher (P < 0.013) in infertile couples (5.03%) than in healthy controls (1.04%). Additional significant differences were noticed for haplotype 10TG7T, demonstrating association (P < 0.022) with a healthy and fertile RBMOnlineÒ
condition, for haplotype 10TG9T with an obvious gender-sensitive link (female = 4.39%, male = 11.29%; P = 0.002) and for haplotype 12TG7T with distinct dependency on both infertility and gender. 12TG7T alleles occurred more frequently in infertile couples (12.91%) than in controls (7.81%) and even more frequently (P = 0.024) in women (16.23%) than in men (10.43%). Highest significance was reached between infertile men and their partners (P = 0.005). No association was found with 5T and the valine at the polymorphic locus M470V (c.1540A/G), in that most of the 5T haplotypes appeared on a M470 background and alleles with the V470 variant were not haplotype specific, carrying in 11 cases the constellation 11TG5T, in four cases 12TG5T and in one case 13TG5T.
Single nucleotide polymorphisms A total of 19 common sequence variants were found at similar frequencies in the infertile and control cohorts and their subgroups (c.125G>C, c.356G>A, c.875+40A>G, c.1001+ 11C>T, c.152561A>G, c.1540A>G, c.1898+152T>A, c.2694T>G, c.3030G>A, c.304192G>A, c.304171G>C, c.327293T>C, c.3500140A>C, c.360165C>A, c.4002A>G, c.4006200G>A, c.4096283C>T, c.4374+ 13A>G and c.4521G>A). Four additional nucleotide changes (c.405+46G>T, c.2377C>T, c.275215C>G and c.3417A>T) were identified only in infertile individuals and one novel sequence variant, c.4042T>C (p.Leu1304Leu) in exon 21, was detected at a frequency of 5.0%, 1.9% and 3.0% in infertile males, their partners and male controls, respectively. The mean number of polymorphisms per individual was 2.6–3.3, with a range from 0 to 10 and no differences between groups.
Risk of CF or CFTR-opathies for offspring For couples with infertility associated with CFTR mutations that are planning to have their own children by assisted reproduction, the risk for offspring to be affected by CF or related diseases depends on whether or not the
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Table 3. Genotype and risk calculation for offspring of infertile couples with both partners carrying CFTR mutations. Couple no.
Infertile male CFTR mutation
Female partner CFTR mutation
Offspring genotype
Risk for genotype (%)
01
F508del/wt azoospermia
F508del/wt
F508del/ F508del F508del/wt wt/wt F508del/ F508del F508del/T5 F508del/wt T5/wt F508del/T5 S13Y/T5 F508del/wt S13Y/wt F508del/ I148T I148T/wt F508del/wt wt/wt 17171G>A/ T5 17171G>A/ wt T5/wt wt/wt 3905insT/T5 3905insT/wt T5/wt wt/wt D1152H/T5 D1152H/wt T5/wt wt/wt F1052V/ S1235R S1235R/T5 F1052V/wt T5/wt S1235R/T5 S1235R/wt T5/wt wt/wt S1235R/T5 S1235R/wt T5/wt wt/wt V754M/T5 V754M/wt T5/wt wt/wt Q1352H/T5 Q1352H/wt T5/wt wt/wt
25
02
F508del/T5 CAVD
F508del/wt
03
F508del/S13Ya azoospermia
T5/wt
04
I148T/wt oligospermia
F508del/wt
05
17171G>A/wt oligospermia
T5/wt
06
T5/wt oligospermia
3905insT/wt
07
T5/wt azoospermia
D1152H/wt
08
T5/F1052V oligospermia
S1235R/wt
09
S1235R/wt oligospermia
T5/wt
10, 11
T5/wt oligospermia
S1235R/wt
12
V754M/wt oligospermia
T5/wt
13
T5/wt oligospermia
Q1352H/wt
50 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25
(continued) (continued on next page)
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Table 3 (continued)
Couple no.
Infertile male CFTR mutation
Female partner CFTR mutation
Offspring genotype
Risk for genotype (%)
14
R31C/wt oligospermia
V920M/wt
15
R31C/wt azoospermia
I148T/wt
16
V754M/wt oligospermia
V754M/wt
R31C/V920M R31C/wt V920M/wt wt/wt R31C/I148T R31C/wt I148T/wt wt/wt V754M/V754M V754M/wt wt/wt
25 25 25 25 25 25 25 25 25 50 25
Bold = mutations associated with classic cystic fibrosis; italic = mutations associated with a mild or uncertain, unpredictable phenotype; CAVD = congenital absence of the vas deferens; wt = wildtype allele. a
Novel mutation, detected in this study.
female partner is a carrier. For female partners testing negative by the screening method, the residual risk of being a carrier of a missed mutation is 0.1%. Thus, the maximal risk for a couple with a male partner carrying one or two CFTR mutations and a female partner testing negative to have a CF child is 1/4000 and 1/2000, respectively, and lies below or at least within the risk of a couple from the general Caucasian population. In 16 out of 70 tested couples, both partners were found to be carriers, increasing the risk for their offspring to inherit two mutations to 25% in 13 cases and to even 50% in three cases. The different genotype combinations and estimated risks are given in Table 3. If both parents carry a severe mutation (couples 1 and 2), then the risk to have a child with classic CF is 25%. However, for combinations of one severe CFTR mutation with a 5T allele or a mild missense mutation (couples 2–6) prognosis of clinical outcome is much more difficult considering the wide range of manifestations from monosymptomatic male infertility to CF-like and even typical, but rather mild, CF phenotypes. A still more complex situation is given in couples 3 and 7–13 with a 5T/missense mutation genotype and in couples 8 and 14–16 with a combination of two rare missense mutations whose deleterious effect is questionable.
Discussion As the involvement of CFTR gene mutations in other forms of infertility than bilateral CAVD and the strategy of CFTR testing in couples undergoing assisted reproduction techniques is not yet clear and controversially discussed (Cuppens and Cassiman, 2004; Claustres, 2005; Stuppia et al., 2005), the present study focused on extended CFTR mutation screening in association with primary infertility to get more insight into the diverse contribution of CFTR to human diseases and to be able to offer specific screening and counselling procedures for couples concerned. In 20% of 620 chromosomes from infertile men, 39 different CFTR mutations were identified and, as expected and described by other authors (Claustres et al., 2000; Grangeia RBMOnlineÒ
et al., 2007; Ratbi et al., 2007), the mutation spectrum was markedly different (P < 0.0001) from that found in CF patients. Corresponding to the literature (Claustres et al., 2000; Cuppens and Cassiman, 2004; Mennicke et al., 2005), a majority of the CAVD patients (68%) were found to carry CFTR mutations. In accordance with Dohle et al. (2002) and the recently published data by Tamburino et al. (2008) in patients with non-CAVD azoospermia, this study also detected a significantly higher frequency of CFTR mutations (21.6%) than in the control group (3.1%). However, in contrast to other studies (Pallares-Ruiz et al., 1999; Ravnik-Glavac et al., 2001; Dohle et al., 2002; Larriba et al., 2005; Tamburino et al., 2008), a significant portion of mutations (11.8%) was additionally identified in oligospermic men supporting the recommendation that CFTR genotyping is also required in other forms of primary male infertility than CAVD prior to fertilization treatment (Dohle et al., 2002; Tamburino et al., 2008). Discordant findings of different studies may mainly be explained by differences in the extent and sensitivity of the screening methods, sample size and ethnic background and clinical investigations. The three most common mutations are F508del (65.00%), 3905insT (4.81%) and R553X (3.78%) in the CF patient cohort, F508del (18.00%), 5T (16.00%) and R117H (8.00%) in CAVD patients and 5T (4.56%), F508del (3.68%) and S1235R (1.05%) in infertile nonCAVD men, exemplifying the disease specificity of the mutation patterns illustrated in Table 1. From these data, it can be calculated that commercial kits for routine screening cover around 87% of mutant alleles in the CF population studied here. In infertile individuals, the detection rate of kit analysis is approximately 40% (without 5T) and 67% (including 5T), respectively. Thus, a large proportion of CF alleles will be missed without extensive screening of the CFTR gene. Based on discriminant analysis, this study predicts a high probability for the presence of CFTR mutations especially in patients with reduced ejaculate volumes (<3 ml) and structural abnormalities such as CAVD, inguinal hernia, hypotrophic testes or cryptorchidism, confirming former findings reported by Casals et al. (2000) and representing symptoms that are also frequently observed in
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male CF patients. However, if the condition of heterozygosity for one mild CFTR mutation (heterozygosity for classic CFTR mutations is well known to produce no phenotypic manifestation) is associated with infertility and thus with reduced reproductive fitness, how can such a high prevalence of this condition in the general population be explained? Speculations are that heterozygosity per se may not affect reproductive fitness, but needs a second event such as: (i) a somatic mutation in the reproductive system; (ii) tissue specific preferential expression of the mutated allele; (iii) additional mutation in an other gene involved in spermatogenesis and/or determination of male fertility (digenic effect); and/or (iv) environmental influences. Moreover, it has to be considered that CFTR mutations, and therefore associated infertility, are artificially transmitted as a consequence of assisted reproduction techniques.
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For a man with CFTR-associated infertility, the risk for offspring to have CF or related diseases depends on whether or not the female partner is a carrier. This study analysed 70 women whose partners had tested positive for one or two CFTR mutations and revealed a mutation spectrum similar to that of oligospermic males. Unexpectedly, this study found a significantly increased prevalence of CFTR mutation carriers (22.9%; P = 0.0005) compared with the control group (6.3%), leading to the speculation that in couples with primary infertility both male and female factors may contribute to the unintended childlessness. This assumption is furthermore supported be the fact that 69% (11/16) female carriers were partners of oligospermic men. The carrier frequency in partners of oligospermic patients was 29.7% (11/37) compared with 16.7% (4/24) in partners of azoospermic men and 11.1% (1/9) in partners of CAVD patients. Infertility due to CAVD is a Wolffian duct anomaly that may result either from a morphogenic defect during organogenesis or, in the presence of CFTR mutations, from degenerative lesions secondary to luminal obstruction by abnormal secretions (Patrizio and Salameh, 1998; Blau et al., 2002). Spermatogenic failure resulting in azoospermia and oligozoospermia, however, is a multifactorial disease, explained by the involvement of different gene products including CFTR and the complexity of the spermatogenic process. Since it seems that the male reproductive tract is the most sensitive system of all CFTR-affected tissues, it has been suggested that not only mild mutations but also polyvariant mutants and even single nucleotide polymorphisms may predispose to oligospermia and testicular failure (Cuppens et al., 1998; Boucher et al., 1999; PallaresRuiz et al., 1999). Moreover, experimental studies in humans and rodents have shown developmentally regulated expression of CFTR in both testes and epididymis, indicating an involvement of the CFTR chloride channel in the cytoplasmic volume reduction during spermiogenesis (Trezise et al., 1993; Patrizio and Salameh, 1998). Very recently, Xu et al. (2007) demonstrated that CFTR in spermatozoa is involved in the transport of bicarbonate, an important factor for sperm capacitation, postulating that CFTR mutations with impaired CFTR function may lead to reduced sperm fertilizing capacity and male infertility other than CAVD. Moreover, infertility in women with CF may not be caused only by the failure of spermatozoa to penetrate
cervical mucus, but also from an inability of spermatozoa to capacitate within the uterus and oviduct because of defective CFTR-mediated bicarbonate secretion (Wang et al., 2003). Therefore, it is imaginable that couples with primary infertility due to oligospermia and with both partners being carriers of CFTR mutations from the ‘oligospermic’ spectrum (Table 1) may contribute mutually to the infertile status. This speculation, however, needs more support by further investigations. Although several studies have shown that CFTR exon 9 skipping is modulated by multiple exonic and intronic cisacting elements (Pagani et al., 2000; Buratti et al., 2001; Hefferon et al., 2002), the extent to which transcripts lacking exon 9 are found is predominantly determined by the TGmTn haplotypes in that a higher number of TG repeats combined with five or less T repeats favours the exclusion of exon 9 and the manifestation of an abnormal phenotype (Groman et al., 2004; Disset et al., 2005). This study identified three haplotypes (13TG5T, 12TG5T, 11TG5T) to be highly associated with infertility. 5T haplotypes were found in 5.45% of infertile alleles and in 3.07% of the chromosomes of their partners, whereas the control group presented with only two 5T alleles (1.04%) on a 11TG background. While the frequency of the 5T allele in the group of infertile men was significantly (P = 0.0075) higher than that in controls, it seems in general to be lower in this study’s population compared with the reported frequency of approximately 5% (Mak et al., 2000; Maeda et al., 2002; Groman et al., 2004; Tamburino et al., 2008). In contrast to other authors (de Meeus et al., 1998; Casals et al., 2000), no correlation with the M470V locus was detectable excluding this variant as a major determinant of penetrance for the 5T allele (Claustres, 2005). Furthermore, haplotype 10TG7T seems to be more frequently present in healthy and fertile individuals, predicting rather a normal phenotype. The most startling observation, however, was the association of haplotype 12TG7T with both infertility and gender, increasing the risk for sterility or fertility problems in women. Hence, analyses of TGmTn haplotypes in a larger and well-described cohort of patients and controls is required to elucidate the specific role of these polyvariant mutants in primary infertility. Although it cannot be excluded that partial penetrance could be a feature not only of the TGmTn polymorphic tract but also of other CFTR variants, no evidence of any influences by single nucleotide polymorphisms was obtained. Albeit the fact that the understanding of the basic mechanisms causing human infertility is still poor, it is crucial to determine possible genetic causes and therefore to make the association with CFTR as clear as possible before any assisted reproduction treatment. In 23% of tested couples, both partners were found to be CFTR mutation carriers, increasing the risk for their offspring to inherit two mutations to 25% or even 50%. However, counselling of couples with CFTR-associated infertility is much more complex than in classic CF (Table 3). Although using a sensitive scanning method provides more precise risk estimations, it may introduce new dilemmas for genetic counselling by the identification of novel sequence alterations with unknown clinical significance. Additionally, many combinations of RBMOnlineÒ
Article - CFTR mutations in azoospermic and oligospermic men and their partners - S Gallati et al.
CFTR mutations imply an unpredictable phenotype, e.g. a rare missense mutation combined with a severe mutation may result in either isolated infertility, CF-like disease or mild to classic CF. Despite these difficulties, nondirective genetic counselling providing accurate, full and unbiased information that helps couples make their own decisions and avoid unnecessary fears but also false optimism is an ethical and medical precondition before any application of an assisted reproduction technique. Couples may want medical assistance such as prenatal or preimplantation diagnosis in order to avoid a severely affected child, but may be more reluctant to this in case of milder phenotypes. Thus, the identification of rare and/or mild mutations will not be a reason to abstain from parenthood, but will allow duly and adequate treatment in children at risk for atypical or mild CF as soon as they develop any symptoms, providing them with the best possible benefit from the effects of early therapy. In the case of preimplantation diagnosis, however, embryos carrying one classic and one mild/rare mutation may not be transferred in order to afford optimal conditions for a successful pregnancy (Keymolen et al., 2007). In conclusion, this study emphasizes the necessity to offer extended CFTR mutation screening and counselling not only to patients with CAVD but also to infertile men with azoospermia or severe oligozoospermia and their partners before assisted reproduction is applied. Moreover, the intriguing findings in partners of oligospermic men raise the question if there is also a female factor that may contribute to CFTR-associated infertility. Finally, specific TGmTn haplotypes are not only predictors for the penetrance of 5T and 12TG, but also for a healthy fertile status underscoring the clinical relevance of TGmTn repeats testing.
Acknowledgements The authors thank the many patients and their partners for their contribution to this study and are especially grateful to Dr. H.R. Linder for providing additional information. This study was supported by the Swiss National Foundation, Grant Nos. 3200-066767.01 and 310000-112652, to S.G.
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Larriba S, Bonache S, Sarquella J et al. 2005 Molecular evaluation of CFTR sequence variants in male infertility of testicular origin. International Journal of Andrology 28, 284–290. Liechti-Gallati S, Schneider V, Neeser D et al. 1999 Two buffer PAGE system-based SSCP/HD analysis: a general protocol for rapid and sensitive mutation screening in cystic fibrosis and any other human genetic disease. European Journal of Human Genetics 7, 590–598. Maeda Y, Shiratsuchi A, Namiki M et al. 2002 Inhibition of sperm production in mice by annexin V microinjected into seminiferous tubules: possible etiology of phagocytic clearance of apoptotic spermatogenic cells and male infertility. Cell Death and Differentiation 9, 742–749. Mak V, Zielenski J, Tsui LC et al. 2000 Cystic fibrosis gene mutations and infertile men with primary testicular failure. Human Reproduction 15, 436–439. Mak V, Zielenski J, Tsui LC et al. 1999 Proportion of cystic fibrosis gene mutations not detected by routine testing in men with obstructive azoospermia. Journal of the American Medical Association 281, 2217–2224. Mau-Holzmann UA 2005 Somatic chromosomal abnormalities in infertile men and women. Cytogenetic and Genome Research 111, 317–336. Mennicke K, Klingenberg RD, Bals-Pratsch M et al. 2005 Rational approach to genetic testing of cystic fibrosis (CF) in infertile men. Andrologia 37, 1–9. Mickle J, Milunsky A, Amos JA et al. 1995 Congenital unilateral absence of the vas deferens: a heterogeneous disorder with two distinct subpopulations based upon aetiology and mutational status of the cystic fibrosis gene. Human Reproduction 10, 1728–1735. Pagani F, Buratti E, Stuani C et al. 2000 Splicing factors induce cystic fibrosis transmembrane regulator exon 9 skipping through a nonevolutionary conserved intronic element. Journal of Biological Chemistry 275, 21041–21047. Pallares-Ruiz N, Carles S, Des Georges M et al. 1999 Complete mutational screening of the cystic fibrosis transmembrane conductance regulator gene: cystic fibrosis mutations are not involved in healthy men with reduced sperm quality. Human Reproduction 14, 3035–3040. Patrizio P, Salameh WA 1998 Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) mRNA in
normal and pathological adult human epididymis. Journal of Reproduction and Fertility Supplement 53, 261–270. Ratbi I, Legendre M, Niel F et al. 2007 Detection of cystic fibrosis transmembrane conductance regulator (CFTR) gene rearrangements enriches the mutation spectrum in congenital bilateral absence of the vas deferens and impacts on genetic counselling. Human Reproduction 22, 1285–1291. Ravnik-Glavac M, Svetina N, Zorn B et al. 2001 Involvement of CFTR gene alterations in obstructive and nonobstructive infertility in men. Genetic Testing 5, 243–247. Riordan JR, Rommens JM, Kerem B et al. 1989 Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245, 1066–1073. Stuppia L, Antonucci I, Binni F et al. 2005 Screening of mutations in the CFTR gene in 1195 couples entering assisted reproduction technique programs. European Journal of Human Genetics 13, 959–964. Tamburino L, Guglielmino A, Venti E et al. 2008 Molecular analysis of mutations and polymorphisms in the CFTR gene in male infertility. Reproductive BioMedicine Online 17, 27–35. Trezise AE, Linder CC, Grieger D et al. 1993 CFTR expression is regulated during both the cycle of the seminiferous epithelium and the oestrous cycle of rodents. Nature Genetics 3, 157–164. van der Ven K, Messer L, van der Ven H et al. 1996 Cystic fibrosis mutation screening in healthy men with reduced sperm quality. Human Reproduction 11, 513–517. Wang X, Sharma RK, Sikka SC et al. 2003 Oxidative stress is associated with increased apoptosis leading to spermatozoa DNA damage in patients with male factor infertility. Fertility and Sterility 80, 531–535. Xu WM, Shi QX, Chen WY et al. 2007 Cystic fibrosis transmembrane conductance regulator is vital to sperm fertilizing capacity and male fertility. Proceedings of the National Academy of Sciences of the United States of America 104, 9816–9821.
Declaration: The authors report no financial or commercial conflicts of interest. Received 3 November 2008; refereed 20 January 2009; accepted 1 June 2009.
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Article Cystic fibrosis transmembrane conductance regulator mutations in azoospermic and oligospermic men and their partners Sabina Gallati graduated in Biology and Genetics from the University of Berne. She has undertaken postdoctoral research and training in molecular genetics and forensics in London (UK), Wu¨rzburg (Germany), Boston (USA) and Quantico (USA) and has specialised in Medical Genetic Analysis. In 2003, she became Professor of Human Genetics and Head of the Division of Human Genetics at the Medical Faculty of the University of Berne. Her main research interests are genetic mechanisms in Cystic Fibrosis and CFTR-related disorders, hereditary haemochromatosis and mitochondriopathies.
Dr Sabina Gallati Sabina Gallati1,5, Simone Hess1, Dorothea Galie´-Wunder2, Elisabeth Berger-Menz3, Dominik Bo¨hlen4 Division of Human Genetics, Departments of Paediatrics and Clinical Research, Inselspital, University of Berne, CH-3010 Berne, Switzerland; 2Division of Gynaecological Endocrinology and Reproductive Medicine, Inselspital, University of Berne, CH-3010 Berne, Switzerland; 3Division of Obstetrics and Gynaecology, Lindenhofspital, CH-3001 Berne, Switzerland; 4Division of Urology, Clinic Beau-Site, CH-3013 Berne, Switzerland 5 Correspondence: e-mail: [email protected] 1
Abstract The objective of this study was to investigate the contribution of cystic fibrosis transmembrane conductance regulator (CFTR) to human infertility and to define screening and counselling procedures for couples asking for assisted reproduction treatment. Extended CFTR mutation screening was performed in 310 infertile men (25 with congenital absence of the vas deferens (CAVD), 116 with non-CAVD azoospermia, 169 with severe oligospermia), 70 female partners and 96 healthy controls. CFTR mutations were detected in the majority (68%) of CAVD patients and in significant proportions in azoospermic (31%) and oligospermic (22%) men. Carrier frequency among partners of infertile men was 16/70, exceeding that of controls (6/96) significantly (P = 0.0005). Thus, in 23% of infertile couples both partners were carriers, increasing the risk for their offspring to inherit two mutations to 25% or 50%. This study emphasizes the necessity to offer extended CFTR mutation screening and counselling not only to patients with CAVD but also to azoospermic and oligozoospermic men and their partners before undergoing assisted reproduction techniques. The identification of rare and/or mild mutations will not be a reason to abstain from parenthood, but will allow adequate treatment in children at risk for atypical or mild cystic fibrosis as soon as they develop any symptoms. Keywords: assisted reproduction, cystic fibrosis transmembrane conductance regulator, mutation spectrum, polyvariant mutants, primary infertility, risk for offspring
Introduction Infertility is a major health problem affecting about 15% of all couples in industrially developed countries (Healy et al., 1994; Mau-Holzmann, 2005). Accumulating evidence suggests that in 15% of male and 10% of female infertile individuals genetic factors may represent the underlying aetiology (Foresta et al., 2002). Since assisted reproduction techniques overcome the protective mechanism of natural selection, the risk of transmitting genetic defects and
decreased fertility to the offspring increases considerably (Faddy et al., 2001; Burrello et al., 2002) requiring careful diagnosis of the infertility status and evaluation of the genetic risk for the child before any technique is performed (Foresta et al., 2002). Male infertility due to severe oligozoospermia and azoospermia has been associated with a number of genetic abnormalities, including numerical and structural chromosomal aberrations (Burrello et al., 2002; Douet-Guilbert
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cussed, including the potential contribution of mutant alleles to female infertility as well as the complexity of genetic counselling and risk assessment.
et al., 2005), deletions of the azoospermia factor region of the Y chromosome (Kihaile et al., 2005; Hellani et al., 2006) and mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (Cuppens and Cassiman, 2004; Claustres, 2005). Most numerical chromosome aberrations and azoospermia factor region deletions are de-novo events in the parental germ cells, whereas mutations in the CFTR gene are inherited, posing a plurivalent genetic risk factor for siblings and offspring.
Materials and methods
Apart from classic cystic fibrosis (CF), CFTR mutations are involved in the development of atypical, CF-like and monosymptomatic phenotypes including male infertility due to congenital absence of the vas deferens (CAVD), either bilateral or unilateral (Anguiano et al., 1992; Chillon et al., 1995). Since the discovery of the CFTR gene in 1989 (Riordan et al., 1989), more than 1600 sequence alterations have been reported to the Cystic Fibrosis Mutation Database in Toronto (http://www.genet.sickkids.on.ca/cftr, accessed September, 2009). The extent to which CFTR mutations contribute to the phenotype and to clinical variation depends on their nature and localization as well as on the overall genetic background, epigenetic factors and environmental influences. A common CFTR mutation conferring a mild phenotype is the 5T allele at the polymorphic Tn locus in intron 8, which is known to affect the splicing efficiency resulting in exon 9 skipping. The original classification of the 5T variant as a bilateral CAVD mutation with incomplete penetrance was published in 1995 (Chillon et al., 1995). Further independent studies confirmed that the frequency of the 5T allele in individuals with bilateral CAVD was significantly higher than that of the general population (Groman et al., 2004; Claustres, 2005). In addition, a higher than expected frequency of CFTR mutations has been observed in unilateral CAVD (Mickle et al., 1995), obstructive azoospermia with intact vas deferens (Jarvi et al., 1995; Mak et al., 1999) and non-obstructive azoospermia and oligozoospermia (van der Ven et al., 1996; Dohle et al., 2002), indicating that the CFTR protein may be involved in the process of spermatogenesis or sperm maturation apart from playing a critical role in the development of the epididymal glands and the vas deferens. However, these findings were not confirmed in other studies (Ravnik-Glavac et al., 2001; Larriba et al., 2005). Thus, the contribution of CFTR to other forms of male infertility than bilateral CAVD/ unilateral CAVD is still not clear and needs further investigation.
The diagnosis of primary infertility was based on physical examination, ultrasonography and semen analysis. Additional studies, such as semen cultures and testicular biopsies, were performed when appropriate. All patients were investigated for testicular volumes and pathological findings such as hypotrophic testes, varicoceles, inguinal hernia, CAVD and anomalies of the seminal vesicle and epididymis as well as for prostatitis and epididymitis and were interviewed concerning former operations for undescended testes and nicotine consumption. Semen analysis was performed twice according to the World Health Organization 1999 criteria and the 2004 guidelines on infertility of the European Association of Urology. Spermiograms included volume (ml), pH, total sperm count (million/ejaculate), sperm concentration (million/ml), motility within 60 min after ejaculate (%), morphology (% normal), viability (% live) and fructose (mol/ejaculate) and neutral alpha-glucosidase (mIU/ejaculate) in seminal plasma. Severe oligozoospermia (<5 million spermatozoa per ml) was attributed to 54.5% (169/310) and azoospermia to 45.5% (141/310) of the patients. CAVD was diagnosed in 25 (17.7%) of the azoospermic men. None of the infertile patients and their partners had any clinical manifestations or family history suggestive of CF. Subjects presenting with chromosomal aberrations or Y chromosome microdeletions were excluded from the study.
The major objective of this study was therefore to determine the nature, frequency and distribution of CFTR mutations and genotypes in infertile men and their partners compared with the mutation pattern of classic CF patients in Switzerland on the one hand and healthy controls on the other hand. As commercial kits for routine screening are known to cover around 80% of mutations causing classic CF but only approximately 62% of mutant alleles in the CFTR genes of infertile individuals (Claustres et al., 2000), this study performed extensive screening (LiechtiGallati et al., 1999) of the entire coding CFTR sequence. A further aim was to define a rational approach to CFTR testing in infertile couples. Finally, the significance of extensive CFTR mutation testing for infertile couples is dis-
Subjects and clinical evaluation A total of 310 selected men aged between 27 and 57 years who had consulted for primary couple infertility, 70 of their partners (aged 23–45 years) and 96 healthy and fertile control individuals (47 male aged 26–49 years, 49 female aged 24–46 years) were investigated. Patients and controls were unrelated and mainly of Swiss origin.
Seventy female partners of infertile men diagnosed with one or two CFTR mutations were referred for extended CFTR analysis before entering an intracytoplasmic sperm injection (ICSI) programme and after exclusion of ovulation failure, physical problems, infectious or hormonal diseases. In addition, 96 healthy individuals from the general population referred for molecular genetic analyses in the context of family testing and having at least one healthy child (paternity confirmed) were used as controls.
Analysis of CFTR mutations Informed consent for molecular genetic analyses was obtained from all individuals included in this study. Genomic DNA was extracted from peripheral blood cells according to standard procedures. Screening of the entire coding sequence of the CFTR gene including intron/exon boundaries and the promoter region was performed by single-strand conformation polymorphism/heteroduplex RBMOnlineÒ
Article - CFTR mutations in azoospermic and oligospermic men and their partners - S Gallati et al.
technique with a sensitivity of 97–98% (Liechti-Gallati et al., 1999) followed by direct sequencing of the variants using an ABI 377 sequencing system (Applied Biosystems, USA).
with infertile men without CFTR mutations. No other parameter of semen analysis discriminated between patients with and without CFTR mutations.
Distribution and frequencies of CFTR mutations identified in this study were compared with mutation data generated from 582 Swiss patients with classic CF using the same extended scanning method (Liechti-Gallati et al., 1999).
Based on linear combinations of the clinical and semen characteristics, discriminant analysis provided significant discrimination (P = 0.002) between CFTR mutation carriers and non-carriers for the predictor variables ejaculate volumes and structural abnormalities of the male genital tract. That is, infertile men presenting with ejaculate volumes less than 3 ml and CAVD and/or anomalies of the seminal vesicle and/or inguinal hernia and/or hypotrophic testes and/or cryptorchidism have an increased risk of carrying at least one CFTR mutation.
Statistical analysis The chi-squared statistic, Fisher’s exact test and unpaired ttest were used where appropriate. All P-values were based on two-sided comparisons and those <0.05 were considered to indicate statistical significance. Discriminant analysis was performed to test for diagnostic criteria predicting the presence of CFTR mutations. The analyses were performed using Statistical Package for Social Sciences (SPSS) Advanced Statistical 11.0 software (SPSS, Chicago, USA).
Results Genetic analyses Genetic testing was performed on 310 infertile men referred to the study centre’s laboratory for CFTR mutation screening with a diagnosis of either severe oligozoospermia (169/ 310) or azoospermia (141/310) with (25/141) or without CAVD (116/141). A total of 123 CFTR mutations were identified in 620 chromosomes (20%). Two mutations were found in 60% of the CAVD patients, in 12% of the nonobstructive azoospermic men and in 1% of oligozoospermic men, whereas the percentage of patients with one was 8% (CAVD), 19% (azoospermia) and 21% (oligospermia) and with no mutation was 32% (CAVD), 69% (azoospermia) and 78% (oligozoospermia).
Clinical characterization of patients In comparison with individuals without mutations in the CFTR gene, analysis of the clinical data showed that patients with CFTR mutations presented significantly more frequently (P = 0.005) either with CAVD (68% versus 32%) or with inguinal hernia (11.8% versus 1.5%) and/or cryptorchism (17.6% versus 9.1%) and/or testicular hypotrophy (23.5% versus 7.6%). Semen analysis according to WHO standards demonstrated pathological values for ejaculate volumes (<2.0 ml) in 21%, for semen pH (<7.0 or >8.0) in 11%, for sperm concentrations (<20 106/ml) in 99% and for total sperm count (<40 106/ejaculate) in 94% of the patients. Vitality of the spermatozoa was found to be pathologically reduced (<50%) in 94% of the infertile individuals. Not surprisingly, statistical calculations demonstrated significantly (P < 0.027) reduced ejaculate volumes (1.9 ml), fructose values (16.15 lmol) and pH (pH 6.9) in CAVD patients compared with infertile men without CAVD. However, non-CAVD patients with CFTR mutations also showed the tendency for smaller ejaculate volumes (2.67 versus 3.53 ml) and fructose values (37 versus 45 lmol) compared RBMOnlineÒ
CFTR mutation patterns and frequencies Extensive screening of the CFTR gene identified 39 different mutations (29 missense, four alternative splice-site, two splice-site, two nonsense, one frameshift, one in-frame deletion) in 90 (29%) out of 310 infertile males carrying one or two mutations (Table 1). CAVD patients showed a CFTR mutation frequency of 66% (33/50), azoospermic men without CAVD presented with a frequency of 21.6% (50/232) and oligospermic men with a frequency of 11.8% (40/338). Twenty-one (53.8%) of these mutations were detected in infertile men and/or their partners only, but neither in 1164 chromosomes of CF patients nor in 192 chromosomes of control individuals. Table 1 illustrates the heterogeneity of CFTR mutations and their different distribution in infertile men, CF patients and controls. For instance, the most common mutation worldwide, F508del, decreases from 65.0% in CF patients to 18.0% in CAVD patients, 7.3% in azoospermic men without CAVD, 1.2% in oligospermic patients, 2.1% in female partners and 0.5% in control persons. Whereas the 5T allele, well known to produce variable amounts of exon 9 skipping, increases from lowest frequencies of 1% in classic CF patients and controls to approximately 3% in female partners and oligospermic men up to 6% and 16% in azoospermic patients without and with CAVD, respectively. In CAVD patients and azoospermic men without CAVD, respectively, the most common mutation was F508del (18.0% and 7.3%) followed by the mild mutations 5T (16.0% and 6.0%) and R117H (8.0% and 1.3%). Oligospermic men as well as female partners, however, carried 5T alleles most frequently (3.55% and 2.86%, respectively), followed by F508del (1.18% and 2.14%, respectively). The missense mutation S1235R was not detected in 50 alleles of CAVD patients, and was also very rarely found in classic CF patients (1/ 1164 alleles) and controls (1/192 alleles), but turned out to be the third most common mutation in azoospermic men without CAVD (3/232), in oligospermic men (3/338) and in female partners (3/140). In summary, 26% of CAVD alleles (versus 79% of classic CF alleles) presented with severe CFTR mutations, 38% with mild CFTR mutations and 2% with mutations with uncertain role, whereas azoospermic men without CAVD and oligospermic men, respectively, carried severe CFTR mutations on only 9.1% and 1.8% and mild mutations on 11.6% and 10.1% of their chromosomes. In azoospermic men, an additional 1.1% of
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Table 1. Distribution and frequencies of 39 different CFTR mutations detected in 92 out of 310 infertile men and comparison with female partners of infertile men, classic cystic fibrosis patients and control population.
Values are number of alleles (percentage of all alleles tested). Bold italic = mutations found on more than two alleles and in more than 1% of alleles; fields in dark grey = CFTR mutations associated with classic/severe CF; fields in light grey = CFTR mutations associated with a mild or uncertain, unpredictable phenotype; CAVD = congenital absence of the vas deferens. a Novel mutation identified in this study with unpredictable phenotype.
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mutations were newly detected and, thus, have an unpredictable phenotype. As expected, statistical calculations showed significant differences (P < 0.0001) in the mutation pattern between infertile men and classic CF patients. But
distribution of mutations (mild/severe) also differed significantly between CAVD patients and oligospermic men (P = 0.0307) as well as between azoospermic men without CAVD and oligospermic men (P = 0.0060). Frequencies
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Article - CFTR mutations in azoospermic and oligospermic men and their partners - S Gallati et al.
Table 2. Distribution and frequencies of IVS8-TGm-Tn haplotypes. Haplotype
Infertile males (n = 303)
Partners (n = 65)
Control males (n = 47)
Control females (n = 49)
11TG5T 12TG5T 13TG5T 10TG7T 11TG7T 12TG7T 11TG8T 9TG9T 10TG9T 11TG9T
16 (2.64) 13 (2.15) 4 (0.70) 90 (14.85) 326 (53.80) 68 (11.22) 1 (0.17) 1 (0.17) 70 (11.55) 17 (2.81)
3 (2.31) 1 (0.77) 0 (0.00) 22 (16.92) 68 (52.31) 27 (20.77) 0 (0.00) 0 (0.00) 5 (3.85) 4 (3.08)
1 (1.06) 0 (0.00) 0 (0.00) 20 (21.28) 53 (56.38) 5 (5.32) 1 (1.06) 0 (0.00) 9 (9.57) 5 (5.32)
1 (1.02) 0 (0.00) 0 (0.00) 23 (23.47) 56 (57.14) 10 (10.20) 0 (0.00) 0 (0.00) 5 (5.10) 3 (3.06)
Total alleles
606
130
94
98
of CFTR mutations were highest in CAVD patients (P < 0.0001) and higher in azoospermic men without CAVD than in oligospermic men (P = 0.0023). However, oligospermic men definitely more frequently carried CFTR mutations than individuals of the control group (P = 0.0004). In 70 women whose partners had tested positive for either CFTR mutations or 5T alleles, extended screening of the CFTR gene was also performed revealing a mutation spectrum similar to that of oligospermic men including four 5T alleles, three S1235R, three F508del and one I148T, V754M, V920M, D1152H, 3905insT and Q1352H each (Table 1). The carrier frequency was 22.9% (16/70) exceeding significantly (P = 0.0005) the carrier frequency of the control group analysed in this study presenting with six carriers (6.3%; 3/47 male, 3/49 female).
Polyvariant mutants According to the literature, both the polymorphic loci TGm and Tn in intron 8 determine the proportion of full-length and mis-spliced CFTR transcripts lacking exon 9 in a polyvariant mutant manner. The IVS8-TGmTn haplotypes in infertile men as well as in 65 of their partners were defined and compared distribution and frequencies with those of 96 healthy controls (Table 2). Ten different haplotypes were found in a total of 464 individuals (928 alleles), within which 11TG7T was the most common in all four subgroups, whereas 9TG9T and 11TG8T were identified in only one and two subjects, respectively. Haplotype 11TG9T also showed low frequencies in all categories ranging from 2.81% to 5.32%. The 5T allele was present on a 11TG, 12TG and 13TG background, however, the combination 12TG5T and 13TG5T appeared only in association with infertility and the frequency of 5T did not substantially differ between infertile men and their partners, but was significantly higher (P < 0.013) in infertile couples (5.03%) than in healthy controls (1.04%). Additional significant differences were noticed for haplotype 10TG7T, demonstrating association (P < 0.022) with a healthy and fertile RBMOnlineÒ
condition, for haplotype 10TG9T with an obvious gender-sensitive link (female = 4.39%, male = 11.29%; P = 0.002) and for haplotype 12TG7T with distinct dependency on both infertility and gender. 12TG7T alleles occurred more frequently in infertile couples (12.91%) than in controls (7.81%) and even more frequently (P = 0.024) in women (16.23%) than in men (10.43%). Highest significance was reached between infertile men and their partners (P = 0.005). No association was found with 5T and the valine at the polymorphic locus M470V (c.1540A/G), in that most of the 5T haplotypes appeared on a M470 background and alleles with the V470 variant were not haplotype specific, carrying in 11 cases the constellation 11TG5T, in four cases 12TG5T and in one case 13TG5T.
Single nucleotide polymorphisms A total of 19 common sequence variants were found at similar frequencies in the infertile and control cohorts and their subgroups (c.125G>C, c.356G>A, c.875+40A>G, c.1001+ 11C>T, c.152561A>G, c.1540A>G, c.1898+152T>A, c.2694T>G, c.3030G>A, c.304192G>A, c.304171G>C, c.327293T>C, c.3500140A>C, c.360165C>A, c.4002A>G, c.4006200G>A, c.4096283C>T, c.4374+ 13A>G and c.4521G>A). Four additional nucleotide changes (c.405+46G>T, c.2377C>T, c.275215C>G and c.3417A>T) were identified only in infertile individuals and one novel sequence variant, c.4042T>C (p.Leu1304Leu) in exon 21, was detected at a frequency of 5.0%, 1.9% and 3.0% in infertile males, their partners and male controls, respectively. The mean number of polymorphisms per individual was 2.6–3.3, with a range from 0 to 10 and no differences between groups.
Risk of CF or CFTR-opathies for offspring For couples with infertility associated with CFTR mutations that are planning to have their own children by assisted reproduction, the risk for offspring to be affected by CF or related diseases depends on whether or not the
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Table 3. Genotype and risk calculation for offspring of infertile couples with both partners carrying CFTR mutations. Couple no.
Infertile male CFTR mutation
Female partner CFTR mutation
Offspring genotype
Risk for genotype (%)
01
F508del/wt azoospermia
F508del/wt
F508del/ F508del F508del/wt wt/wt F508del/ F508del F508del/T5 F508del/wt T5/wt F508del/T5 S13Y/T5 F508del/wt S13Y/wt F508del/ I148T I148T/wt F508del/wt wt/wt 17171G>A/ T5 17171G>A/ wt T5/wt wt/wt 3905insT/T5 3905insT/wt T5/wt wt/wt D1152H/T5 D1152H/wt T5/wt wt/wt F1052V/ S1235R S1235R/T5 F1052V/wt T5/wt S1235R/T5 S1235R/wt T5/wt wt/wt S1235R/T5 S1235R/wt T5/wt wt/wt V754M/T5 V754M/wt T5/wt wt/wt Q1352H/T5 Q1352H/wt T5/wt wt/wt
25
02
F508del/T5 CAVD
F508del/wt
03
F508del/S13Ya azoospermia
T5/wt
04
I148T/wt oligospermia
F508del/wt
05
17171G>A/wt oligospermia
T5/wt
06
T5/wt oligospermia
3905insT/wt
07
T5/wt azoospermia
D1152H/wt
08
T5/F1052V oligospermia
S1235R/wt
09
S1235R/wt oligospermia
T5/wt
10, 11
T5/wt oligospermia
S1235R/wt
12
V754M/wt oligospermia
T5/wt
13
T5/wt oligospermia
Q1352H/wt
50 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25
(continued) (continued on next page)
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Table 3 (continued)
Couple no.
Infertile male CFTR mutation
Female partner CFTR mutation
Offspring genotype
Risk for genotype (%)
14
R31C/wt oligospermia
V920M/wt
15
R31C/wt azoospermia
I148T/wt
16
V754M/wt oligospermia
V754M/wt
R31C/V920M R31C/wt V920M/wt wt/wt R31C/I148T R31C/wt I148T/wt wt/wt V754M/V754M V754M/wt wt/wt
25 25 25 25 25 25 25 25 25 50 25
Bold = mutations associated with classic cystic fibrosis; italic = mutations associated with a mild or uncertain, unpredictable phenotype; CAVD = congenital absence of the vas deferens; wt = wildtype allele. a
Novel mutation, detected in this study.
female partner is a carrier. For female partners testing negative by the screening method, the residual risk of being a carrier of a missed mutation is 0.1%. Thus, the maximal risk for a couple with a male partner carrying one or two CFTR mutations and a female partner testing negative to have a CF child is 1/4000 and 1/2000, respectively, and lies below or at least within the risk of a couple from the general Caucasian population. In 16 out of 70 tested couples, both partners were found to be carriers, increasing the risk for their offspring to inherit two mutations to 25% in 13 cases and to even 50% in three cases. The different genotype combinations and estimated risks are given in Table 3. If both parents carry a severe mutation (couples 1 and 2), then the risk to have a child with classic CF is 25%. However, for combinations of one severe CFTR mutation with a 5T allele or a mild missense mutation (couples 2–6) prognosis of clinical outcome is much more difficult considering the wide range of manifestations from monosymptomatic male infertility to CF-like and even typical, but rather mild, CF phenotypes. A still more complex situation is given in couples 3 and 7–13 with a 5T/missense mutation genotype and in couples 8 and 14–16 with a combination of two rare missense mutations whose deleterious effect is questionable.
Discussion As the involvement of CFTR gene mutations in other forms of infertility than bilateral CAVD and the strategy of CFTR testing in couples undergoing assisted reproduction techniques is not yet clear and controversially discussed (Cuppens and Cassiman, 2004; Claustres, 2005; Stuppia et al., 2005), the present study focused on extended CFTR mutation screening in association with primary infertility to get more insight into the diverse contribution of CFTR to human diseases and to be able to offer specific screening and counselling procedures for couples concerned. In 20% of 620 chromosomes from infertile men, 39 different CFTR mutations were identified and, as expected and described by other authors (Claustres et al., 2000; Grangeia RBMOnlineÒ
et al., 2007; Ratbi et al., 2007), the mutation spectrum was markedly different (P < 0.0001) from that found in CF patients. Corresponding to the literature (Claustres et al., 2000; Cuppens and Cassiman, 2004; Mennicke et al., 2005), a majority of the CAVD patients (68%) were found to carry CFTR mutations. In accordance with Dohle et al. (2002) and the recently published data by Tamburino et al. (2008) in patients with non-CAVD azoospermia, this study also detected a significantly higher frequency of CFTR mutations (21.6%) than in the control group (3.1%). However, in contrast to other studies (Pallares-Ruiz et al., 1999; Ravnik-Glavac et al., 2001; Dohle et al., 2002; Larriba et al., 2005; Tamburino et al., 2008), a significant portion of mutations (11.8%) was additionally identified in oligospermic men supporting the recommendation that CFTR genotyping is also required in other forms of primary male infertility than CAVD prior to fertilization treatment (Dohle et al., 2002; Tamburino et al., 2008). Discordant findings of different studies may mainly be explained by differences in the extent and sensitivity of the screening methods, sample size and ethnic background and clinical investigations. The three most common mutations are F508del (65.00%), 3905insT (4.81%) and R553X (3.78%) in the CF patient cohort, F508del (18.00%), 5T (16.00%) and R117H (8.00%) in CAVD patients and 5T (4.56%), F508del (3.68%) and S1235R (1.05%) in infertile nonCAVD men, exemplifying the disease specificity of the mutation patterns illustrated in Table 1. From these data, it can be calculated that commercial kits for routine screening cover around 87% of mutant alleles in the CF population studied here. In infertile individuals, the detection rate of kit analysis is approximately 40% (without 5T) and 67% (including 5T), respectively. Thus, a large proportion of CF alleles will be missed without extensive screening of the CFTR gene. Based on discriminant analysis, this study predicts a high probability for the presence of CFTR mutations especially in patients with reduced ejaculate volumes (<3 ml) and structural abnormalities such as CAVD, inguinal hernia, hypotrophic testes or cryptorchidism, confirming former findings reported by Casals et al. (2000) and representing symptoms that are also frequently observed in
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male CF patients. However, if the condition of heterozygosity for one mild CFTR mutation (heterozygosity for classic CFTR mutations is well known to produce no phenotypic manifestation) is associated with infertility and thus with reduced reproductive fitness, how can such a high prevalence of this condition in the general population be explained? Speculations are that heterozygosity per se may not affect reproductive fitness, but needs a second event such as: (i) a somatic mutation in the reproductive system; (ii) tissue specific preferential expression of the mutated allele; (iii) additional mutation in an other gene involved in spermatogenesis and/or determination of male fertility (digenic effect); and/or (iv) environmental influences. Moreover, it has to be considered that CFTR mutations, and therefore associated infertility, are artificially transmitted as a consequence of assisted reproduction techniques.
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For a man with CFTR-associated infertility, the risk for offspring to have CF or related diseases depends on whether or not the female partner is a carrier. This study analysed 70 women whose partners had tested positive for one or two CFTR mutations and revealed a mutation spectrum similar to that of oligospermic males. Unexpectedly, this study found a significantly increased prevalence of CFTR mutation carriers (22.9%; P = 0.0005) compared with the control group (6.3%), leading to the speculation that in couples with primary infertility both male and female factors may contribute to the unintended childlessness. This assumption is furthermore supported be the fact that 69% (11/16) female carriers were partners of oligospermic men. The carrier frequency in partners of oligospermic patients was 29.7% (11/37) compared with 16.7% (4/24) in partners of azoospermic men and 11.1% (1/9) in partners of CAVD patients. Infertility due to CAVD is a Wolffian duct anomaly that may result either from a morphogenic defect during organogenesis or, in the presence of CFTR mutations, from degenerative lesions secondary to luminal obstruction by abnormal secretions (Patrizio and Salameh, 1998; Blau et al., 2002). Spermatogenic failure resulting in azoospermia and oligozoospermia, however, is a multifactorial disease, explained by the involvement of different gene products including CFTR and the complexity of the spermatogenic process. Since it seems that the male reproductive tract is the most sensitive system of all CFTR-affected tissues, it has been suggested that not only mild mutations but also polyvariant mutants and even single nucleotide polymorphisms may predispose to oligospermia and testicular failure (Cuppens et al., 1998; Boucher et al., 1999; PallaresRuiz et al., 1999). Moreover, experimental studies in humans and rodents have shown developmentally regulated expression of CFTR in both testes and epididymis, indicating an involvement of the CFTR chloride channel in the cytoplasmic volume reduction during spermiogenesis (Trezise et al., 1993; Patrizio and Salameh, 1998). Very recently, Xu et al. (2007) demonstrated that CFTR in spermatozoa is involved in the transport of bicarbonate, an important factor for sperm capacitation, postulating that CFTR mutations with impaired CFTR function may lead to reduced sperm fertilizing capacity and male infertility other than CAVD. Moreover, infertility in women with CF may not be caused only by the failure of spermatozoa to penetrate
cervical mucus, but also from an inability of spermatozoa to capacitate within the uterus and oviduct because of defective CFTR-mediated bicarbonate secretion (Wang et al., 2003). Therefore, it is imaginable that couples with primary infertility due to oligospermia and with both partners being carriers of CFTR mutations from the ‘oligospermic’ spectrum (Table 1) may contribute mutually to the infertile status. This speculation, however, needs more support by further investigations. Although several studies have shown that CFTR exon 9 skipping is modulated by multiple exonic and intronic cisacting elements (Pagani et al., 2000; Buratti et al., 2001; Hefferon et al., 2002), the extent to which transcripts lacking exon 9 are found is predominantly determined by the TGmTn haplotypes in that a higher number of TG repeats combined with five or less T repeats favours the exclusion of exon 9 and the manifestation of an abnormal phenotype (Groman et al., 2004; Disset et al., 2005). This study identified three haplotypes (13TG5T, 12TG5T, 11TG5T) to be highly associated with infertility. 5T haplotypes were found in 5.45% of infertile alleles and in 3.07% of the chromosomes of their partners, whereas the control group presented with only two 5T alleles (1.04%) on a 11TG background. While the frequency of the 5T allele in the group of infertile men was significantly (P = 0.0075) higher than that in controls, it seems in general to be lower in this study’s population compared with the reported frequency of approximately 5% (Mak et al., 2000; Maeda et al., 2002; Groman et al., 2004; Tamburino et al., 2008). In contrast to other authors (de Meeus et al., 1998; Casals et al., 2000), no correlation with the M470V locus was detectable excluding this variant as a major determinant of penetrance for the 5T allele (Claustres, 2005). Furthermore, haplotype 10TG7T seems to be more frequently present in healthy and fertile individuals, predicting rather a normal phenotype. The most startling observation, however, was the association of haplotype 12TG7T with both infertility and gender, increasing the risk for sterility or fertility problems in women. Hence, analyses of TGmTn haplotypes in a larger and well-described cohort of patients and controls is required to elucidate the specific role of these polyvariant mutants in primary infertility. Although it cannot be excluded that partial penetrance could be a feature not only of the TGmTn polymorphic tract but also of other CFTR variants, no evidence of any influences by single nucleotide polymorphisms was obtained. Albeit the fact that the understanding of the basic mechanisms causing human infertility is still poor, it is crucial to determine possible genetic causes and therefore to make the association with CFTR as clear as possible before any assisted reproduction treatment. In 23% of tested couples, both partners were found to be CFTR mutation carriers, increasing the risk for their offspring to inherit two mutations to 25% or even 50%. However, counselling of couples with CFTR-associated infertility is much more complex than in classic CF (Table 3). Although using a sensitive scanning method provides more precise risk estimations, it may introduce new dilemmas for genetic counselling by the identification of novel sequence alterations with unknown clinical significance. Additionally, many combinations of RBMOnlineÒ
Article - CFTR mutations in azoospermic and oligospermic men and their partners - S Gallati et al.
CFTR mutations imply an unpredictable phenotype, e.g. a rare missense mutation combined with a severe mutation may result in either isolated infertility, CF-like disease or mild to classic CF. Despite these difficulties, nondirective genetic counselling providing accurate, full and unbiased information that helps couples make their own decisions and avoid unnecessary fears but also false optimism is an ethical and medical precondition before any application of an assisted reproduction technique. Couples may want medical assistance such as prenatal or preimplantation diagnosis in order to avoid a severely affected child, but may be more reluctant to this in case of milder phenotypes. Thus, the identification of rare and/or mild mutations will not be a reason to abstain from parenthood, but will allow duly and adequate treatment in children at risk for atypical or mild CF as soon as they develop any symptoms, providing them with the best possible benefit from the effects of early therapy. In the case of preimplantation diagnosis, however, embryos carrying one classic and one mild/rare mutation may not be transferred in order to afford optimal conditions for a successful pregnancy (Keymolen et al., 2007). In conclusion, this study emphasizes the necessity to offer extended CFTR mutation screening and counselling not only to patients with CAVD but also to infertile men with azoospermia or severe oligozoospermia and their partners before assisted reproduction is applied. Moreover, the intriguing findings in partners of oligospermic men raise the question if there is also a female factor that may contribute to CFTR-associated infertility. Finally, specific TGmTn haplotypes are not only predictors for the penetrance of 5T and 12TG, but also for a healthy fertile status underscoring the clinical relevance of TGmTn repeats testing.
Acknowledgements The authors thank the many patients and their partners for their contribution to this study and are especially grateful to Dr. H.R. Linder for providing additional information. This study was supported by the Swiss National Foundation, Grant Nos. 3200-066767.01 and 310000-112652, to S.G.
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Declaration: The authors report no financial or commercial conflicts of interest. Received 3 November 2008; refereed 20 January 2009; accepted 1 June 2009.
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