Cytogenetic and Y chromosome microdeletion screening of a random group of infertile males

FERTILITY AND STERILITY VOL. 79, NO. 2, FEBRUARY 2003 Copyright 2003 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Cytogenetic and Y chromosome microdeletion screening of a random group of infertile males Claire R. Quilter, Ph.D., a,g Elizabeth C. Svennevik, Ph.D., a Paul Serhal, M.R.C.O.G., b David Ralph, FRCS(Urol), c Gulam Bahadur, Ph.D., d Richard Stanhope, M.D., e Marc Sütterlin, M.D., f Joy D. A. Delhanty, Ph.D., d and Kay E. Taylor, Ph.D. a The Galton Laboratory, University College London, London, United Kingdom Received February 21, 2002; revised and accepted June 26, 2002. Supported by The Clinical Cytogenetics Unit, the Galton Laboratory, University College London Hospital (UCLH), London, United Kingdom. Reprint requests: Claire Quilter, Ph.D., Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP, United Kingdom (FAX: 01-223-333346; E-mail: crq20@cam.ac.uk). a The Galton Laboratory. b The Assisted Conception Unit, University College London Hospitals Trust. c Department of Uronephrology, University College London Hospitals Trust. d Department of Obstetrics and Gynecology, University College London Hospitals Trust. e Department of Endocrinology, University College London Hospitals Trust. f Department of Obstetrics and Gynecology, University of Würzburg, Würzburg, Germany. g Present address: Department of Pathology, University of Cambridge, Cambridge, United Kingdom. 0015-0282/03/$30.00 doi:10.1016/s0015-0282(02) 04692-7 Objective: To assess whether to perform routine cytogenetic and Y chromosome microdeletion screening on all infertile male patients. Design: A cytogenetic and Y microdeletion study of a random group of infertile men. Setting: University department. Patient(s): In total, 40 patients had azoospermia (21 nonidiopathic), 27 had severe oligozoospermia/ oligoasthenozoospermia ( 5 10 6 /ml) (5 nonidiopathic), 20 had oligozoospermia/oligoasthenozoospermia (5 20 10 6 /ml) (6 nonidiopathic), and 16 had asthenozoospermia (5 nonidiopathic). Many were candidates for intracytoplasmic sperm injection (ICSI). Intervention(s): Collection of blood samples from all patients and buccal cells from one patient. Main Outcome Measure(s): Karyotype analysis, polymerase chain reaction (PCR) screening for Y chromosome microdeletions, and fluorescence in situ hybridization of abnormal chromosomes. Result(s): Ten (9.7%) subjects, including one nonidiopathic patient, were found to have an abnormal karyotype. Two idiopathic azoospermic patients were missing large portions of Y chromosome euchromatin, confirmed by PCR analysis and an additional idiopathic azoospermic patient had a Y chromosome microdeletion. Conclusion(s): Routine cytogenetic analysis of all infertile male patients is required but it may be advisable to limit routine Y chromosome microdeletion screening to patients with severe male factor infertility ( 5 10 6 /ml). (Fertil Steril 2003;79:301 7. 2003 by American Society for Reproductive Medicine.) Key Words: Chromosome abnormalities, ICSI, male factor infertility, random patients, Y chromosome deletions Infertility is due to a male factor in approximately 50% of cases. More than 90% of male factor infertility is characterized by low numbers of sperm in the semen or production of spermatozoa of poor quality. Contributing factors include infection, genital injury, and environmental factors. However, in 60% of cases male factor infertility can be the result of genetic factors, including cytogenetic abnormalities and microdeletions of the Y chromosome (1). It has been found that 13.7% of men with azoospermia and 4.6% of men with oligozoospermia have chromosomal abnormalities affecting approximately 5.1% of the total number of infertile men (2). Sex chromosome abnormalities are predominant in azoospermic patients and abnormalities of the autosomes are more frequent in patients with oligozoospermia. An excess of reciprocal translocations, of Robertsonian translocations, and an excess of inversions have also been observed in infertile men compared to the newborn population (3). From these studies, however, it is not clear what proportion of chromosome abnormalities are present in patients with moderate infertility ( 5 10 6 /ml). Because many of these patients consider assisted conception it would be of interest to assess the value of routine cytogenetic screening of all patients. Four regions of the Y chromosome long arm are important in male factor fertility (4, 5). It 301

TABLE 1 Clinical abnormalities recorded from nonidiopathic infertile men. Category of male infertility Clinical A SO O AT Varicocoele 2 1 Mumps 2 2 1 Orchidectomy (unilateral) 2 1 Undescended testes 2 1 2 Obstruction 4 Tube abnormalities a 9 1 1 1 Infection 1 Sperm morphology defect 2 Surgery 2 Total 21 5 6 5 A azoospermia; SO severe oligozoospermia/oligoasthenozoospermia ( 5 million/ml); O oligozoospermia/oligoasthenozoospermia (5 20 million/ ml); AT asthenozoospermia ( 20 million/ml). a Unilateral or bilateral absence of vas deferens. has been found that deletions of one of these regions (AZFa) are more commonly associated with Sertoli cell-only syndrome and less frequently with oligozoospermia (4, 6); Sertoli cell-only syndrome to mild oligozoospermia, with deletions of AZFb and AZFc (4, 7); and mild oligozoospermia to normozoospermia associated with abnormal sperm morphology with deletions of AZFd (5). Numerous Y chromosome microdeletion studies have been carried out on infertile men and the overall frequency of microdeletions has been estimated to be 12.2% in azoospermic men and 3.4% in oligozoospermic men (8). The majority of studies tested idiopathic patients with severe male factor infertility ( 5 10 6 /ml). However, a few studies have screened a more random group of men, but such studies are limited (9, 10). A few nonpolymorphic deletions have been found in individuals with higher sperm counts (5 20 10 6 /ml) and even normozoospermia ( 20 10 6 /ml) and also in a few nonidiopathic infertile men with abnormalities such as undescended testes, obstruction of vas deferens, and varicocoele (5, 7, 8). As a result it is unclear whether a wider selection of patients attending a fertility clinic should undergo genetic screening. More studies need to be carried out on patients with a range of phenotypes to clarify the value of routine screening. In this study a group of infertile men whose infertility ranged from azoospermia to a normal sperm count with poor motility (asthenozoospermia) were screened for cytogenetically visible chromosome abnormalities and for microdeletions of the Y chromosome. Many of these patients were referred from fertility clinics as likely candidates for treatment by intracytoplasmic sperm injection (ICSI). On the basis of our results and of other studies the most appropriate genetic screening and assisted conception strategies for such patients are discussed. MATERIALS AND METHODS Subjects A total of 103 patients were analyzed cytogenetically and of these 98 were screened for Y chromosome microdeletions by polymerase chain reaction (PCR) analysis. Nine patients were sent from the Department of Obstetrics and Gynaecology, the University of Würzburg. All other patients were referred to the Clinical Cytogenetics Unit, University College London Hospital either from the Departments of Endocrinology, Uro-nephrology or the Assisted Conception Unit, University College London Hospital. Peripheral lymphocytes were obtained from all patients and from the buccal mucosa of one patient. The patient cohort consisted of 66 individuals with unexplained infertility and 37 patients who were nonidiopathic. In total, 40 patients had azoospermia (21 nonidiopathic), 27 had severe oligozoospermia/oligoasthenozoospermia ( 5 10 6 /ml) (5 nonidiopathic), 20 had oligozoospermia/oligoasthenozoospermia (5 20 10 6 /ml) (6 nonidiopathic), and 16 had asthenozoospermia (5 nonidiopathic). Details of nonidiopathic patients are summarized in Table 1. Cytogenetic Analysis Metaphase cells were obtained from phytohemagglutinin (PHA)-stimulated blood lymphocytes from all patients and stained by a conventional Giemsa//Trypsin//Leishman s (GTL) banding method. Thirty metaphase spreads were routinely analyzed from each subject, which should detect 10% mosaicism with 95% confidence (11). If a second cell line was found, up to 100 metaphases were analyzed to establish the level of mosaicism. A conventional C-banding method was used to confirm the number of centromeres in the dicentric Y chromosomes. 302 Quilter et al. Genetic screening of male infertility Vol. 79, No. 2, February 2003

TABLE 2 Locus, deletion interval, size of product, and annealing temperature of each STS from PCR multiplexes I IV. PCR STS Locus Deletion interval Size of product/bp Ta/ C Reference MI SRY SRY 1A1A 422 62 15 sy238 ZFY 1A1B 350 17 sy276 AMELY 3 215 17 MII sy283 DAZ 6D 496 62 17 sy277 DAZ 6D 311 17 sy78 DYZ3 4B 170 16 MIII sy274 RPS4Y 1A1B 350 58 17 sy154 DYS238 6D 245 16 sy142 DYS230 6B 190 16 MIV sy254 DAZ 6D 379 62 17 sy143 DYS231 6B 311 16 sy160 DYZ1 7 236 16 Fluorescence In Situ Hybridization Fluorescence in situ hybridization (FISH) investigations were carried out on metaphases obtained from the peripheral lymphocytes of some infertile men with structurally abnormal chromosomes to clarify their structure. A minimum of five metaphases containing the structurally abnormal chromosome were examined for each probe. Indirect FISH using a biotin-labeled cosmid that hybridizes to the sex-determining gene SRY and the proximal part of the pseudoautosomal region of the X and Y chromosomes (378E from a cosmid library prepared at the Lawrence Livermore National Laboratory [12]), was carried out according to published methods (13). Commercial probes used in this study consisted of an X/Y subtelomeric probe (Cytocell, Oxford, UK), a probe specific for the Di George region at 22q11.2 (and control region at 22q13.3) (Oncor, Nottingham, UK), and satellite mixes for chromosomes 14/22 (Oncor) and 1/5/19 (Oncor). Experiments were carried out according to manufacturer s instructions. The Oncor probes were set up in dual color reactions, with each satellite mix being hybridized with the Di George probe. Slides were visualized under a fluorescence microscope equipped with a cooled CCD camera (Photometrics, Marpenden, UK) and images were captured using Smart-Capture software (Digital Scientific, Cambridge, UK). PCR Blood was available for DNA extraction from 98 patients with male factor infertility, 93 of whom had a normal male karyotype and 5 with chromosome abnormalities (M33, M52, M75, M103, and M107) using a previously described method (14) or a commercial kit (QIAmp, QUIGEN, Crawley, UK). DNA was also extracted from one buccal cell sample using a commercial kit (QIAmp). In this study, 12 sets of oligonucleotide primers, which amplify 12 different loci distributed along the length of the Y chromosome, were used to screen for Y chromosome microdeletions. The sequences of these primers have been described elsewhere (15 17). The first 50 patients were screened by single primer PCR but for the rest of the patients a series of four multiplex reactions (I IV) were developed to improve the efficiency of analysis. Details of primers are summarized in Table 2. The PCR method was carried out as described elsewhere (18). Female genomic DNA was used as a control. If a sequence tagged site (STS) from a particular individual failed to amplify it was repeated three times before it was considered to be a deletion. For patients in which a deletion was found, DNA was extracted from a second blood sample and in one subject, a buccal cell sample was used to confirm the result. In other studies these primers have been used to screen infertile men and no polymorphic deletions were found (17). In this study primers were also tested with DNAs from 10 fertile sperm donors and all gave normal results. RESULTS Cytogenetic Analysis Ten patients were found to have an abnormal karyotype (summarized in Table 3). Five of them had azoospermia; M95 and M96 had previously undetected Klinefelter syndrome and M75, M99, and M103 were mosaic for a cell line containing a structural rearrangement of the Y chromosome and a 45,X cell line. The C-banding indicated that M75 and M103 had idic(yp) chromosomes and that M99 had an idic(yq) chromosome. Four patients with chromosome abnormalities had oligozoospermia; M33 and M97 each had a Robertsonian translocation between chromosomes 13 and 14, which was paternally inherited in the case of M33, M52 had a pericentric inversion of chromosome 9, and M98 had a derivative 19, FERTILITY & STERILITY 303

TABLE 3 Cytogenetic abnormalities found from G-banded analysis of peripheral lymphocytes. Patient Referral reason Karyotype M33 Severe oligozoospermia 45,XY,der(13;14)(q10; q10)pat M52 Oligoasthenozoospermia 46,XY,inv(9)(p11q12) M75 Azoospermia 45,X [64]/46,X,idic(Y)(q11.2) {36} M95 Azoospermia 47,XXY M96 Azoospermia 47,XXY M97 Severe oligozoospermia 45,XY,der(13;14)(q10; q10) M98 Oligoasthenozoospermia 45,XY,der(19)t(19;22)(q13.4;q11.21) M99 Azoospermia 45,X [5]/46,X,idic(Y)(p11.3) {95} M103 Azoospermia 45,X [6]/46,X,idic(Y)(q11.2) [94] M107 Asthenozoospermia 46,X,inv(Y)(p11q12) which required FISH for clarification. Patient M107, with a pericentric inversion of his Y chromosome, had asthenozoospermia. All patients had idiopathic infertility with the exception of M98 who had a history of torsion of the testes and a left orchidectomy. M75 had a history of undescended testes, but other features of his phenotype strongly suggested a sex chromosome abnormality. The other 93 patients were found to have a normal male karyotype, 46,XY. FISH Three patients required FISH to clarify the structure of their abnormal chromosomes. Patients M75 and M103 were indicated to have idic(yp) chromosomes after cytogenetic analysis. Biotin-labeled cosmid 378E, specific for SRY and the proximal part of pseudoautosomal region-1, was detected with avidin conjugated fluorescein isothiocyanate conjugate (FITC). Hybridization of this probe to the Y chromosome from patient M75 showed a signal at either end of the Y chromosome confirming that it was an idic(yp). An X/Y subtelomeric specific (CY29/c8.2/1; Cytocell) was hybridized to the Y chromosome from patient M103 and a Yp signal was seen at either end confirming that he also had an idic(yp) chromosome (results not shown). Patient M98, who had oligoasthenozoospermia, was found to have an abnormal karyotype after cytogenetic analysis. Both a chromosome 19 and a chromosome 22 were missing and in their place was a derivative chromosome, which appeared to be a translocation between the 19 and 22. Dual color FISH with the Di George probe and the 14/22 satellite probe revealed that the derivative chromosome has both the Di George region and the control region on chromosome 22 translocated to the distal end of 19q. The chromosome 22 breakpoint appears to occur proximal to 22q11.2 but below the 22 centromere, as the 14/22 satellite probe did not hybridize to the derivative chromosome. Dual color FISH was also carried out with the Di George probe and a 1/5/19 satellite probe. The Di George probe identified the derivative chromosome, which was assumed to have a chromosome 19 centromere based on hybridization by the 1/5/19 satellite probe (results not shown). PCR The cytogenetic and FISH analysis indicated an isodicentric Yp chromosome in both azoospermic patients M75 and M103. On PCR analysis both patients were found to be missing all STSs tested from the Yq arm (sy142, sy143, sy154, sy254, sy277, sy283, and sy160). These primers amplify sequences from Y chromosome intervals 6B, 6D, and 7, which represent the AZFb and the AZFc regions, as well as the Yq heterochromatin (4, 19, 20). The STSs amplified from the Yp arm and the centromere were all present. Patient M106 had azoospermia and was the only patient with a cytogenetically normal Y chromosome found to have a microdeletion of the Y chromosome. The PCR analysis indicated that STSs sy142 and sy143 were deleted, which are from Y chromosome deletion interval 6B and form part of the AZFb region (4, 19, 20). The STSs from the Yp arm, the centromere, the AZFc region, and the Y heterochromatin were all found to be present. Additional primers were used to define the proximal breakpoint to deletion interval 5 of the Y chromosome between STSs sy106 (5K2) and sy113 (5M1) and the distal breakpoint to interval 6 between STSs sy143 (6B) and sy154 (6D). The deletions in all three patients were confirmed on DNA extracted from a second blood sample and also from buccal cells in the case of M106. None of the Y chromosome STSs tested were missing from patient M107 (pericentric inversion of Y chromosome). Figure 1 shows the results of DNA from infertile male patients M75, M103, M106, and M107 amplified with multiplexes I (a), II (b), III (c), and IV (d). DISCUSSION In this study, 9.7% (10 of 103) patients were found to have cytogenetic abnormalities. Of these, 6.7% (7 of 103) 304 Quilter et al. Genetic screening of male infertility Vol. 79, No. 2, February 2003

FIGURE 1 DNA from patients M75, M103, M106, and M107, a male and female control, and blank containing water amplified with multiplexes I IV (A D). DNA from patients M75 and M103 were deleted for STSs sy142, sy143, sy154, sy160, sy277, sy283, and sy254 and DNA from patient M106 was deleted for sy142 and sy143. L 100-bp ladder, F female control, M fertile male control, H 2 O. carried cytogenetic abnormalities considered likely to be causative to their infertility and all seven had severe idiopathic infertility. Patients M95 and M96 with Klinefelter syndrome and patients M75, M99, and M103 mosaic for a cell line containing an isodicentric Y chromosome and a 45,X cell line had azoospermia. Patients M33 and M97 with Robertsonian translocations between chromosomes 13 and 14 had severe oligozoospermia ( 5 10 6 /ml). Such abnormal chromosome constitutions are commonly associated with these infertility categories. The presence of the paternally inherited translocation in patient M33 may have contributed to his infertility owing to its being present in a different genetic background. The three other patients had higher sperm counts ranging from 7 22 10 6 /ml and abnormal sperm motility. Two had idiopathic infertility and a pericentric chromosome inversion, chromosome 9 in patient M52 and the Y chromosome FERTILITY & STERILITY 305

in patient M107. Some reports include such chromosome abnormalities in their infertility studies (2, 3, 21); however, evidence suggests that both are benign chromosome variants (22, 23). Unfortunately parental blood was not available to confirm that these variants had been inherited, therefore their role in infertility could not be totally excluded. The third patient (M98) with a chromosome abnormality and higher sperm count was the only nonidiopathic case of a total of 37 patients. Fluorescence in situ hybridization was necessary to confirm that he carried a derivative chromosome 19 with chromosome 22q material attached to it. The terminal portion of the 19q telomere and the short arm and centromeric region of chromosome 22 may have formed a small marker, which was lost early on in cell division. Alternatively, if this rearrangement was present in one of the parents a 3:1 segregation could have resulted in this chromosome constitution. Unfortunately, parental samples were not available for analysis. Although M98 had an unbalanced chromosome constitution, no other clinical features were documented, suggesting that very little euchromatin was missing. Another explanation for the mild phenotype is that the missing material may have been present in other tissues. However, there is a small possibility that hemizygosity of genes located in the missing euchromatin may have contributed to his subfertility. Although it is likely that the chromosome translocation in patient M98 contributed to his phenotype and could also increase the risk of miscarriage in his partner, it should be taken into account that the history of torsion of both testes and a left orchidectomy may solely account for his sperm count of 7 10 6 /ml with 100% poor sperm motility and 100% poor progression. In this study, Y chromosome deletion screening by PCR analysis with Y-specific primers identified 3% (3 of 98) patients with Y deletions and all three had idiopathic nonobstructive azoospermia. Patients M75 and M103 were found to have isodicentric Yp chromosomes from our cytogenetic and FISH studies. The PCR analysis was useful to clarify that euchromatin was deleted, with the AZFc and part of the AZFb regions missing, representing approximately 11 Mb. The PCR analysis identified the presence of an interstitial Yq microdeletion of approximately 5 Mb in the third patient (M106), which was not visible cytogenetically. The AZFa region (5C) was present, but the AZFb region (5O-6B) and possibly the proximal part of the AZFc region (6C-6E) were missing. Microdeletions of proximal AZFc have previously been associated with patients with azoospermia (4, 7, 19) as have deletions of AZFb (4, 5, 7, 19). In our study, Y microdeletions were not found in other categories of infertility. The low number detected may be explained by the fact that a third of patients screened with idiopathic infertility had a sperm count 5 10 6 /ml, including patients with normozoospermia and poor sperm motility. In addition, Y microdeletions were not detected in the 37 patients with nonidiopathic infertility. This suggests that routine screening of such patients may not be unnecessary. Some of the patients with chromosome abnormalities found by this study were suitable candidates for assisted conception. Sperm retrieval from Klinefelter patients resulting in pregnancy by ICSI has been documented (24), but the risk of abnormal pregnancies is still undetermined (25). No evidence of a normal cell line was found in the azoospermic patients with dicentric Y chromosomes or the interstitial Yq microdeletion, which was confirmed by PCR, suggesting that if a Y chromosome were present in any retrievable sperm suitable for ICSI it would be structurally abnormal. Preimplantation genetic diagnosis and the replacement of only female embryos would be an option for assisted conception. However, it has been suggested that patients who are deleted for the entire AZFb region, which was the case for M106, are unlikely to have any mature sperm (26). The translocation patients, including the nonidiopathic ones, were at risk of producing unbalanced gametes and they could also be a recommended preimplantation genetic diagnosis as a form of assisted conception, where only chromosomally balanced embryos would be replaced. Overall our results suggest that cytogenetic screening of infertile men is valuable, with 9.7% of patients in this study having visible chromosome abnormalities. The significance of the chromosome rearrangements in three patients was unclear and may be coincidental, but these results do suggest that all infertile men, especially those seeking assisted conception, should be screened cytogenetically so that steps can be taken to prevent the replacement of unbalanced or potentially infertile offspring. We also found the number of Y microdeletions detected in a random group of male infertility patients to be low. Microdeletions were only present in three patients with idiopathic azoospermia, but were not found in other categories of patients. However, many other studies have identified microdeletions in patients with nonidiopathic severe oligozoospermia ( 5 10 6 /ml). Because our numbers may have been too small to detect any deletions, we cannot rule out the value of screening this group of patients for microdeletions. We did not find microdeletions in the remaining groups of patients with sperm counts ( 5 10 6 /ml) or with nonidiopathic infertility, which suggests that routine screening of such patients may not be required. We conclude from this and other studies that Y chromosome microdeletion screening of male patients with idiopathic severe infertility, including severe oligozoospermia ( 5 10 6 /ml), is worthwhile. However, it would be valuable to carry out further studies on patients with higher sperm counts or idiopathic infertility to support our conclu- 306 Quilter et al. Genetic screening of male infertility Vol. 79, No. 2, February 2003

sion that routine screening of such patients is not appropriate. Acknowledgments: The authors thank the patients for participating in this research and their clinicians at the University College London Hospital for cooperating and making this study possible. The authors would also like to thank staff from the Assisted Conception Unit, University College London Hospital, for help with patient details. The authors are also grateful to all members of Clinical Cytogenetics, formerly of the Galton Laboratory, University College London, for their help with the processing of samples. The experiments were undertaken with the approval of the ethics committee of the University College London Hospitals Trust. References 1. Lilford R, Jones AM, Bishop DT, Thornton J, Mueller R. Case control study of whether subfertility in men is familial. BMJ 1994;309:570 3. 2. Van Assche E, Boudelle M, Tournaye H, Joris H, Verheyen G, Devroey P, et al. Cytogenetics of infertile men. 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