Mutation frequency of cystic fibrosis transmembrane regulator is not increased in oligozoospermic male candidates for intracytoplasmic sperm injection

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1 FERTILITY AND STERILITY VOL. 69, NO. 5, MAY 1998 Copyright 1998 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Mutation frequency of cystic fibrosis transmembrane regulator is not increased in oligozoospermic male candidates for intracytoplasmic sperm injection Joep H.A.M. Tuerlings, M.D.,* Bart Mol, Ph.D., Jan A.M. Kremer, M.D., Ph.D., Maaike Looman, M.Sc., Eric J.H. Meuleman, M.D., Ph.D., Gerard J. te Meerman, Ph.D., Charles H.C.M. Buys, Ph.D., Hans M.W.M. Merkus, M.D., Ph.D., and Hans Scheffer, Ph.D. University Hospital Nijmegen, Nijmegen, and University of Groningen, Groningen, the Netherlands Received September 24, 1997; revised and accepted January 16, Reprint requests: Joep H.A.M. Tuerlings, M.D., Department of Human Genetics, P.O. Box 9101, 6500HB Nijmegen, the Netherlands (FAX: ; AZN.NL). This work was supported in part by grant from The Dutch Prevention Fund to H.S., G.J.t.M., and C.H.C.M.B. * Department of Human Genetics, University Hospital Nijmegen. Department of Medical Genetics, University of Groningen. Department of Gynecology, University Hospital Nijmegen. Department of Urology, University Hospital Nijmegen /98/$19.00 PII S (98) Objective: To examine the frequency of anomalies of the vas deferens and the frequency of mutations of the cystic fibrosis transmembrane regulator (CFTR) gene in male candidates for intracytoplasmic sperm injection (ICSI) who had severe oligoasthenoteratozoospermia. Design: The clinical data for male candidates for ICSI were studied. The three most frequent cystic fibrosis (CF)-causing CFTR mutations in the Dutch population ( F508, A455E, and G542X) and the three most frequent CFTR mutations potentially causing congenital bilateral absence of the vas deferens (CBAVD) in the Dutch population ( F508, R117H, and IVS8-5T) were analyzed. Delta I507 is also detected by the F508 test. Samples of DNA from patients identified as CFTR mutation carriers were subjected to denaturing gradient gel electrophoresis analysis with use of a two-dimensional electrophoretic technique. Setting: University-based center for reproductive medicine and clinical genetics. Patient(s): Male candidates for ICSI who had oligoasthenoteratozoospermia and no history of operative sterilization and refertilization. Males with a chromosomal aberration or a Y-chromosome microdeletion were excluded. Intervention(s): Semen and blood samples were collected from the patients at their first visit to the clinic. Main Outcome Measure(s): Frequency of anomalies of the vas deferens and frequency of mutations of the CFTR gene in male candidates for ICSI who had oligoasthenoteratozoospermia. Result(s): None of the patients had abnormalities of the vas deferens at physical examination. In 4 of the 150 chromosomes (75 patients), a CFTR mutation was found, yielding a CFTR mutation frequency of 2.7% (95% confidence interval, %). None of the patients had two CFTR mutations. Conclusion(s): The frequency of congenital abnormalities of the vas deferens in patients with oligoasthenoteratozoospermia is low. The frequencies of the CFTR mutations identified in this cohort did not differ significantly from the frequencies found in the normal Dutch population. (Fertil Steril 1998;69: by American Society for Reproductive Medicine.) Key Words: CFTR mutations, cystic fibrosis, vas deferens abnormalities, infertility, OAT Cystic fibrosis (CF) is the most common severe autosomal recessive genetic disorder in the western population, with a reported incidence of 1 in 3,600 in the Netherlands (1). On the basis of these data, the regional carrier frequency of CF-causing mutations in the cystic fibrosis transmembrane regulator (CFTR) is approximately 1 in 30. Mutations in the CFTR gene, which encodes a cyclic adenosine monophosphate regulated chloride channel, are responsible for CF (2). The classic symptoms of CF are progressive lung disease, pancreatic insufficiency, elevated sweat electrolyte values, and infertility in males. Clinical symptoms associated with CFTR mutations vary widely in nature and severity and may also lead to congenital bilateral absence of the vas deferens without further symptoms of CF. 899

2 Ninety-seven percent of male patients with CF are infertile (azoospermia) because of bilateral obstruction of the vas deferens. However, fertile males with CF have also been described (3). Apparently, obstruction of their sperm transport is absent or incomplete. To date, little is known about the frequency of incomplete obstruction because of congenital unilateral absence of the vas deferens or cystic changes in the wolffian duct system and about the frequency of CFTR mutations in these men. Mutation analysis of the CFTR gene in 10 infertile males with congenital unilateral absence of the vas deferens and a sperm count ranging from azoospermia to normal values revealed one single CFTR mutation (4). Another study of 12 infertile patients with congenital unilateral absence of the vas deferens and an anatomically complete and patent vas deferens on the contralateral side showed that 6 were azoospermic and 6 had variable deficiencies of sperm concentration or motility (5). None of these 12 men had a detectable CFTR mutation. Because oligospermia might be the only clinical symptom of CFTR mutations in patients with an absent or incomplete obstruction of the vas deferens, a better knowledge of these frequencies would be beneficial. Van der Ven et al. (6) suggested that the CFTR gene could also play a direct role in spermatogenesis or sperm maturation. They based this hypothesis on a histologic analysis of testicular tissues of male patients with congenital bilateral absence of the vas deferens and CF, yielding a spectrum of results that ranged from normal to severely decreased spermatogenesis. This prompted them to perform a CFTR mutation analysis in men who were infertile as a consequence of reduced sperm quality. The results showed that 14 of 80 (17.5%) of such males had at least one CFTR mutation. This was significantly higher than the expected CFTR carrier frequency in their local population (estimated at 4%). It was hypothesized that a second undetected mutation might be present on the other CFTR allele in the remaining patients. This would suggest that oligoasthenoteratozoospermia would represent a variant form of CF, similar to congenital bilateral absence of the vas deferens. Unfortunately, the anatomy of the genitals of these men was not assessed and the presence of a second mutation on the other CFTR allele could not be confirmed or excluded. Until recently, the pregnancy rate (PR) was low for couples in which the male had oligoasthenoteratozoospermia. The introduction of intracytoplasmic sperm injection (ICSI) dramatically improved the PRs for these couples. To date, PRs of up to 35% per cycle have been achieved (7). Because ICSI circumvents a part of the natural selection mechanisms, the possible transmission of genetic defects to the offspring is of major concern. Therefore, it is important to identify carriers of CFTR mutations among infertile men requesting ICSI. We retrospectively studied 75 patients with severe oligoasthenoteratozoospermia who requested ICSI and studied the results of the andrologic examination with special attention to congenital abnormalities of the genital tract. In addition, we studied the frequency and nature of CFTR mutations in these otherwise healthy men. MATERIALS AND METHODS Patient Population The study population consisted of 75 men with severe oligoasthenoteratozoospermia who did not have a history of operative sterilization and refertilization and who requested ICSI in our clinic. Men with a chromosomal aberration or a Y-chromosome microdeletion were excluded. Oligozoospermic men are candidates for ICSI if their ejaculate contains spermatozoa per ejaculate with propulsive motility (World Health Organization a and b). Andrologic history and examination, hormone measurements, chromosome analysis, and screening for microdeletions of the Y chromosome were performed (8). Semen analysis was performed according to the World Health Organization laboratory manual (9). The first sample was used for further analysis. Informed consent for the DNA studies was obtained from all 75 men before the study was initiated. This study was approved by the institutional review board of the University Hospital Nijmegen. Mutation Analysis Genomic DNA was isolated from peripheral blood as described previously (10). The three most frequent CFTR mutations causing CF in the Dutch population ( F508, A455E, and G542X) and the three most frequent CFTR mutations potentially causing congenital bilateral absence of the vas deferens in the Dutch population ( F508, R117H, and IVS8-5T) were analyzed. The F508 test also detects I507. Analysis of F508 or I507 mutations was performed as described previously (11). Detection of R117H, A455E, and G542X mutations was performed with the use of allelespecific amplification tests, as described by Ferrie et al. (12) for G542X and R117H. For the A455E mutation, the following primers were designed (mismatch in lower case): AEC: 5 -GACACTACACCCATACATTCTCCTAATG-3, AEM: 5 -TCAAGATAGAAAGAGGACAGTTGTTGtA-3, and AEN: 5 -TCAAGATAGAAAGAGGACAGTTGTTG- GC-3. An allele-specific test was performed as follows. The allele-specific polymerase chain reaction (PCR) mixture contained 20 pmol of AEC and AEN primers for amplification of the normal allele and AEC and AEM primers for amplification of the mutant allele and 7 nmol of each dntp in 50 L of reaction buffer (10 mm Tris-HCl [ph 8.3], 50 mm KCl, 0.6 mm MgCl 2, 0.01% [w/v] gelatin, and 0.1% [v/v] Triton X-100). Just before PCR, 300 ng of genomic 900 Tuerlings et al. CFTR mutations in oligoasthenoteratozoospermia Vol. 69, No. 5, May 1998

3 TABLE 1 Testis volume and mean indices of the semen analysis in the men without and the four men with a cystic fibrosis transmembrane regulator gene mutation. Volume of the testis (ml) Semen volume (ml) Sperm concentration ( 10 6 /ml) ph of the semen Motility of the semen (%) Abnormal spermatozoa (%) Fructose concentration in the semen (mmol/l) Mean no. ( SEM) of men without a CFTR mutation 15.0 ( 0.6) 3.7 ( 0.3) 2.9 ( 0.6) ( 2.1) 63 ( 2) 16.5 ( 1.4) Patient 1 ( F508 mutation) NT Patient 2 ( F508 mutation) Patient 3 (R117H mutation) Patient 4 (IVS8-5T stretch) NT Note: NT not tested. Fructose was tested only if the sperm concentration was /ml. DNA and 2 U of AmpliTaq polymerase (Perkin-Elmer, Norwalk, CT) were added per reaction. A 28-cycle PCR was performed: 93 C for 1 minute, 60 C for 1 minute, and 72 C for 1 minute (5 minutes in the final cycle) in a thermocycler (Perkin-Elmer). Electrophoresis was performed in 2% agarose gel and subsequently stained by ethidium bromide. The resulting PCR products were visualized with ultraviolet light (300 nm). To amplify the IVS8 poly-t-stretch, we used the following oligonucleotides as primers: IVS8TF: 5 -TGTGTGTGT- GTGTGTTTT-3 within intron 8, and IVS8TR: 5 -GTTTT- GTTTTGCTTTCTC-3 within exon 8. The intron 8TF primer shows overlap with the flanking polymorphic CA repeat. The last four nucleotides are the first four thymidine residues of the poly-t-tract. The length of the amplified product is 65, 67, or 69 nucleotides, depending on the length of the poly-t-tract alleles. For PCR, 17 pmol of each primer was used with 500 ng of genomic DNA and2uofamplitaq polymerase in a total volume of 40 L under paraffin oil. Obviously, such a succession of TG repeat and T tract is fairly unique in the human genome. To synthesize a specific product, a touchdown program was used. Denaturation was at 94 C for 60 seconds, annealing at 58 C for 25 seconds, and extension at 72 C for 45 seconds. The annealing temperature was decreased in six steps to 51 C. The first four steps were three cycles each, and the last two were only one cycle. These were followed by 14 cycles with an annealing temperature of 51 C and a last cycle of 120 seconds for extension. Thus, in total the PCR program consisted of 29 cycles. Electrophoresis was performed in 13% polyacrylamide gel and subsequently stained by ethidium bromide. The resulting PCR products were visualized with ultraviolet light (300 nm). Samples of DNA from patients identified as CFTR mutation carriers were subjected to denaturing gradient gel electrophoresis analysis with use of a two-dimensional electrophoresis technique, as described previously (13). Statistical Analysis Our null hypothesis was that the frequency of CFTR mutations in men with oligoasthenoteratozoospermia is not different from the frequency of CFTR mutations in men in the normal population. The population frequency of the F508 mutation in the Netherlands is per chromosome (254 of 21,544) (14). The R117H and IVS8-5T frequencies per chromosome in the normal population of the Netherlands are approximately (2 of 232 chromosomes of 116 unaffected, unrelated individuals) and (8 of 212 chromosomes of 106 unaffected, unrelated individuals), respectively. For the A455E, G542X, and I507 mutations, the frequency in the normal population was based on the 3.3% carrier frequency and the locally derived relative frequencies of mutations in CF patients (H.S., unpublished data). To compare the frequency of mutations in the men with oligoasthenoteratozoospermia with the mutation frequency in the normal population, we used standard tests between proportions (Fisher s exact test). The effect, as found by Van der Ven et al. (6), amounts to 14 of 80 individuals with a CFTR mutation (95% CI, ). The power to detect an effect of this size, with a probability threshold of 5%, is 95% for a sample size of 60. If the real effect were 10%, the power would be 0.8 to detect this effect with a sample size of 75. RESULTS The andrologic history and examination of this cohort revealed abnormalities in 34 of 75 patients. A varicocele was diagnosed in 12 patients. Nine men had a history of orchidopexy. Five had had an inguinal hernia. Two patients were known to have chronic colitis. Three patients had a history of adnexitis, one had had a testicular malignancy, one had hypospadias, and one had lymphedema. None of the patients had abnormalities of the vas deferens at physical examination. All patients had a normal semen volume and a normal fructose concentration in their semen. The mean indices from the semen analysis are shown in Table 1. In 4 of the 150 chromosomes (75 patients), a CFTR FERTILITY & STERILITY 901

4 TABLE 2 Frequencies of mutations in the cystic fibrosis transmembrane regulator in the general Dutch population, in the investigated male candidates for ICSI who had oligoasthenoteratozoospermia, in patients with congenital bilateral absence of the vas deferens, and in patients with cystic fibrosis. Mutation General population* (95% CI) Patients with OAT (95% CI) Patients with CBAVD Patients with CF F ( ) ( ) R117H ( ) ( ) IVS8-5T ( ) ( ) ND A455E (ND) 0 (ND) G542X (ND) 0 (ND) I (ND) 0 (ND) Total (ND) ( ) Note: OAT oligoasthenoteratozoospermia; CBAVD congenital bilateral absence of the vas deferens; ND not done. * Locally derived frequencies for F508, R117H, and IVS8-5T (n 21,544 chromosomes, n 232 chromosomes, and n 212 chromosomes, respectively); and theoretical estimates for A455E, G542X, and I507 based on a 3.3% carrier frequency and regionally derived mutation frequencies (H.S., unpublished data). This study (n 150 chromosomes). H.S., unpublished data (n 36 chromosomes). H.S., unpublished data (n 726 for F508, A455E, and G542X; n 272 for R117H; IVS8-5T not determined). Because of different sample sizes, 95% CIs were calculated for the different mutations separately. mutation was found, yielding a mutant CFTR frequency of 2.7% (95% CI, %). None of the patients showed two CFTR mutations. The four mutations identified were F508 (twice; 1.3% of the chromosomes analyzed), R117H (once; 0.6% of the chromosomes analyzed), and IVS8-5T (once; 0.6% of the chromosomes analyzed) (Table 2). One of the four men was known to have a history of cryptorchism, whereas 33 of the 71 remaining men without a CFTR mutation revealed some clinical abnormalities in their andrologic history or at examination. The four patients with a single CFTR mutation were all tested for the presence of a second unidentified mutation with use of a two-dimensional electrophoresis approach, including size separation in the first dimension and denaturing gradient gel electrophoresis in the second (13). This comprehensive method for detection of mutations did not reveal any indication of a second CFTR mutation in these four patients. DISCUSSION Physical examination did not reveal any abnormalities of the genitals, except for one male who had hypospadias. No abnormalities of the vas deferens were detected in this selected cohort of 75 candidates for ICSI, although complete exclusion was not possible because transrectal ultrasonography was not performed. The cause of the infertility in these men was unclear. In this study, 4 of 150 chromosomes (2.7%; 95% CI, %) had detectable mutations in the CFTR gene. Two mutations were not identified in any of the four patients. To exclude the presence of a second unidentified mutation, DNA of these four patients was subjected to a sensitive scanning method for CFTR mutations, including denaturing gradient gel electrophoresis (13). Again, no indications for other CFTR mutations were observed. Obviously, mutations outside the coding region of the CFTR gene or in the promotor region of the gene will not be detected by this test. However, with use of a protocol including this two-dimensional electrophoresis approach, CFTR mutations have been identified in 98% of 700 CFTR alleles from patients with CF in the Netherlands (H.S., unpublished data). Because this test in principle has the same sensitivity for all possible CFTR mutations, we consider the likelihood of overlooking a mutation very small and conclude that the four patients probably represent true heterozygotes for a CFTR mutation. The frequency of CFTR mutations in our severely oligozoospermic population did not differ significantly from the carrier frequencies in the normal population but was significantly different from that found in the study of Van der Ven et al. (6). They detected a frequency of 17.5% in 80 men with a variety of diagnoses varying from oligozoospermia to oligoasthenoteratozoospermia (95% CI, 10 27%). There may be several explanations for the higher frequency of CFTR mutations in that infertile male population (6). One could speculate that males with congenital unilateral absence of the vas deferens or an otherwise incomplete blockage of the vas deferens were included in that study, and/or chance fluctuations may have caused the observed increase in CFTR mutations (4). The investigators failed, however, to present data on the anatomy of the genital tracts of the men who were investigated. In addition, they did not search for the presence of a second CFTR mutation in the carriers identified in their cohort. Physical examination and transrectal ultrasonography of the vas deferens or analysis of the CFTR gene for second CFTR mutations, including the IVS8-5T mutation frequently 902 Tuerlings et al. CFTR mutations in oligoasthenoteratozoospermia Vol. 69, No. 5, May 1998

5 present in congenital bilateral absence of the vas deferens, in the carriers identified by Van der Ven et al. (6) may assist in detecting patients with infertility because of (partial) obstruction of the vas deferens. Although the high carrier frequency of CF-causing CFTR mutations in the western population can be explained by genetic drift (14), it is certainly an argument against the causation of significant male infertility by a single heterozygous CFTR mutation because that would greatly increase the selective pressure on CFTR mutations. However, to date, data are not available on semen analyses of a cohort of men heterozygous for CFTR mutations. Our data strongly suggest that abnormalities of the vas deferens cannot play an important role in the cause of infertility in male ICSI candidates with oligoasthenoteratozoospermia and that the frequencies of the CFTR mutations in such men do not differ significantly from the frequencies found in the normal population. However, to disprove results as significant as those published by Van der Ven et al. (6), a sample that is about four times larger than the original one is required. Therefore, analysis of a larger cohort may still be necessary to substantiate our conclusions. In addition, further studies are needed to elucidate the role and frequency of CFTR mutations in patients with congenital unilateral absence of the vas deferens. Acknowledgments: The authors thank Mrs. Maayke Ariaans for secretarial assistance and for preparing the tables. They also thank Mrs. Margaret Burton for linguistic assistance. References 1. De Vries HG, Collée JM, De Walle HEK, Van Veldhuizen MHR, Smit Sibinga CTh, Scheffer H, et al. Prevalence of deltaf508 cystic fibrosis carriers in The Netherlands: logistic regression on sex, age, region of residence and number of offspring. Hum Genet 1997;99: Kerem BS, Rommens JM, Buchanan JA. Identification of the cystic fibrosis gene: genetic analysis. Science 1989;245: Dodge JH. Male infertility in cystic fibrosis. Lancet 1995;334: Casals T, Bassas L, Ruiz-Romero J, Chillon M, Gimenez J, Ramos MD, et al. Extensive analysis of 40 infertile patients with congenital absence of the vas deferens: in 50% of cases only one CFTR allele could be detected. Hum Genet 1995;95: Mickle J, Milunsky A, Amos JA, Oates RD. 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. Hum Reprod 1995;10: Van der Ven L, Messer L, Van der Ven H, Jeyendran RS, Ober C. Cystic fibrosis mutation screening in healthy men with reduced sperm quality. Hum Reprod 1996;11: Van Steirteghem A, Nagy Z, Joris H, Liu J, Staessen C, Smitz J, et al. High fertilization and implementation rates after intracytoplasmic sperm injection. Hum Reprod 1993;8: Kremer JAM, Tuerlings JHAM, Meuleman EJH, Schoute F, Mariman E, Smeets DFCM, et al. Microdeletions of the Y chromosome and intracytoplasmic sperm injection: from gene to clinic. Hum Reprod 1997;12: World Health Organization. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. Cambridge, UK: Cambridge University Press, Millor SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988; 16: Scheffer H, Verlind E, Penninga D, Te Meerman G, Ten Kate L, Buys C. Rapid screening for deltaf508 deletion in cystic fibrosis. Lancet 1989;2: Ferrie RM, Schwarz MJ, Robertson NH, Vaudin S, Super M, Malone G, et al. Development, multiplexing and application of ARMS tests for common mutations in the CFTR gene. Am J Hum Genet 1992;51: Wu Y, Hofstra R, Scheffer H, Uitterlinden AG, Mullaart E, Buys CHCM, et al. Comprehensive and accurate mutation scanning of the CFTR-gene by two-dimensional DNA electrophoresis. Hum Mutat 1996;8: Thompson EA, Neel JV. Allelic disequilibrium and allele frequency distribution as a function of social and demographic history. Am J Hum Genet 1997;60: FERTILITY & STERILITY 903