Genetic Abnormalities among Severely Oligospermic Men Who Are Candidates for Intracytoplasmic Sperm Injection
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《临床内分泌与代谢杂志》
Abstract
Recent reports suggest that children born after intracytoplasmic sperm injection performed for male factor infertility are at increased risk of congenital malformations and chromosome aberrations. To explain these observations, we hypothesized that infertile men may be more likely than fertile men to have genetic abnormalities. We studied 750 severely oligozoospermic men (sperm count <5 million/ml) who were candidates for intracytoplasmic sperm injection, and 303 fertile men. We analyzed the peripheral blood karyotype, the Y chromosome long arm for detection of microdeletions in the azoospermia factors, and mutations in the cystic fibrosis gene and the androgen receptor gene. We also analyzed sperm for chromosome aneuploidies among the 421 men who subsequently entered the in vitro fertilization program. A total of 104 genetic abnormalities were diagnosed, corresponding to a frequency of 13.9% (104 of 750). Chromosomal aberrations were present in 5.6% (42 of 750) of infertile men and 0.3% of controls (one of 295), and they were in most cases alterations of the sex chromosomes. Y chromosome long-arm microdeletions were detected in 6.0% (45 of 750) of infertile men and most frequently included the azoospermia factor c, whereas no cases were found in controls (zero of 210). Mutations in the cystic fibrosis gene were diagnosed in 1.2% (nine of 750) of infertile men and 1.0% of controls (three of 303), and mutations in the androgen receptor gene were found in 1.1% (eight of 750) of infertile men and none of the 188 controls. Sperm sex chromosome aneuploidies were increased in men with karyotype anomalies and Y chromosome microdeletions as well as in subjects without constitutional genetic abnormalities. This study shows that the frequency of genetic alterations is increased among men with severe spermatogenic impairment. Genetic tests and genetic counseling should therefore be considered in oligozoospermic men who are candidates for intracytoplasmic sperm injection.
Introduction
INFERTILITY AFFECTS ABOUT 15% of couples trying to conceive in Western countries (1). Accumulating evidence suggests that genetic abnormalities may be present in a large proportion of infertile couples. Intracytoplasmic sperm injection (2) allows some men with a very low sperm count or even no sperm in the ejaculate to become fathers, using sperm retrieved from the epididymis or the testis. Concerns have been raised regarding the potential of intracytoplasmic sperm injection to facilitate the transmission of genetic diseases, because this technique bypasses many of the steps of normal fertilization; a single spermatozoon is injected into the oocyte, eliminating the usual process of natural selection. Early studies suggested that intracytoplasmic sperm injection was safe (3), but more recent data have indicated a higher risk of congenital malformation (4, 5), chromosome aneuploidy (6, 7, 8), low birth weight (9), and imprinting defects such as Angelman syndrome (10) for children conceived by intracytoplasmic sperm injection than for children conceived naturally.
Many constitutional genetic anomalies affecting spermatogenesis can be analyzed during the diagnostic evaluation. Members of our group and others have recommended specific genetic tests in male and female infertile subjects (11), but these recommendations have been based on limited data, and comprehensive studies involving a large number of infertile patients have been lacking. We thus conducted a study to assess the prevalence of several genetic abnormalities among 750 severely oligozoospermic men who were candidates for intracytoplasmic sperm injection.
Subjects and Methods
The study was approved by the hospital ethical committee, and participants gave written informed consent. Seven hundred and fifty consecutive, infertile men, affected by severe oligozoospermia (sperm count <5 million/ml) and candidates for intracytoplasmic sperm injection, attended our university center from 1996–2001, and were screened for genetic anomalies. All patients were of Caucasian origin and came from different Italian regions. No other selection apart from severe oligozoospermia was applied. Subjects presenting with azoospermia were excluded from the study.
Karyotype analysis was conducted by the classical cytogenetic technique with the application of GTG and QFQ banding techniques on at least 25 metaphases from peripheral blood lymphocyte culture. Sex chromosome mosaics occurring at a level of less of 5% were not considered. Pericentric inversions of chromosome 9 or other structural chromosomal variants and polymorphisms (such as familial inversion of the Y chromosome or large Y chromosome heterochromatin) were considered as normal cytogenetic events.
Routine diagnostic Y chromosome long-arm microdeletion analysis was performed by three multiplex PCR analyses on genomic DNA extracted from lymphocytes. A total of nine sequence-tagged sites (STSs) spanning the azoospermia factor (AZF) regions (AZFa, -b, and -c) (12) were used, as follows: sY86, DF3.1 for the USP9Y gene, DBY1 for the DBY gene, and sY95 in the AZFa region; sY117, sY125, and sY127 in the AZFb region; and sY254 and sY255 for the DAZ gene in the AZFc region. Each multiplex set contained the control marker for the SRY gene (sY14). Negative results (no amplifications) were considered only after three amplification failures, repeating the experiments on new DNA extracted from a second blood collection, as previously reported (13, 14). Furthermore, before assuming a deletion, each locus was analyzed separately in a single PCR. Analyses were always performed with a male control sample, a female sample, and a blank sample. Additional STSs were analyzed when a deletion was found, to obtain both confirmation of the deletion and better deletion breakpoints (15, 16, 17), as follows: sY1197 and sY1192 for the proximal breakpoint of AZFc deletions; sY1264 and sY1224 for the proximal breakpoint of AZFb deletions; sY143 for the distal breakpoint of AZFb; and sY1190, sY1125, and sY1201 for the distal breakpoint of AZFc deletions. Extensive analysis of AZFa deletion breakpoints was previously performed (14). Confirmation of deletions was eventually performed by Southern blotting (13, 18, 19). Details of PCR amplification and STSs have been reported previously (13, 14, 18, 19, 20). Y chromosome microdeletion analysis was performed only in patients with normal karyotypes.
The analysis of CFTR gene mutations was performed by a standard reverse dot blot technique for 49 different mutations. Analysis of these mutations allows us to identify about 90% of cystic fibrosis gene carriers from our region (northeast Italy) and about 70% of carriers from other regions of Italy. Analysis of the poly-T allelic variants at the intron 8-exon 9 junction of the CFTR gene was also performed.
Analysis of mutations in the androgen receptor gene was conducted by PCR and subsequent direct sequencing on an ABI PRISM sequencer (Applied Biosystems, Foster City, CA), using a set of 11 oligonucleotide primers covering exons 1–8 (21).
Among subjects attending our center for semen analysis, we recruited 303 consecutive men with normozoospermia as controls. All of these men had peripheral blood analyzed for CFTR gene mutations. Other tests performed in some, but not all, of these men (based on availability of blood) included karyotype analysis (in 295 men), analysis for Y chromosome microdeletions (in 210 men), and analysis for androgen receptor gene mutations (in 188 men).
Of the 750 oligospermic patients, 421 entered an intracytoplasmic sperm injection program. These 421 men were analyzed for numerical alterations of sperm sex chromosomes by means of fluorescence in situ hybridization, as previously reported (22). Among the 303 normozoospermic fertile men, 103 were used as controls for this analysis. At least 5000 sperm were analyzed for each subject, and percentages of sperm with the Y chromosome, the X chromosome, nullisomies (absence of sex chromosomes), and XX, XY, and YY disomies (abnormal presence of the two sex chromosomes) were calculated.
Statistical analysis
Comparison of proportions (prevalence of genetic anomalies in patients and controls) were carried out using Fisher’s exact test. Comparisons of means of aneuploidy rates among groups were analyzed by Wilcoxon rank sum test. A value of P < 0.05 (two-sided) was considered to indicate statistical significance.
Results
Constitutional genetic abnormalities
A total of 104 genetic abnormalities were diagnosed (Table 1), corresponding to a frequency of 13.9% (104 of 750); none of the men had more than one defect. Chromosomal aberrations were present in 5.6% (42 of 750), Y chromosome microdeletions in 6.0% (45 of 750), CFTR gene mutations in 1.2% (nine of 750), and androgen receptor gene mutations in 1.1% (eight of 750). The frequencies of chromosomal anomalies and Y chromosome microdeletions were significantly higher than those among normospermic controls (P < 0.001 for both), whereas the frequencies of CFTR and AR gene mutation were not significantly different.
Most of the chromosomal abnormalities involved the sex chromosomes (32 of 42, 76%), especially in the form of numerical abnormalities (Table 2). The majority of abnormal karyotype were of the Klinefelter’s type, either in the classic 47,XXY form or in the mosaic 47,XXY/46,XY form (in total 29 of 42, 69%). Reciprocal translocations (exchange of material between two nonhomologous chromosomes) and Robertsonian translocations (joining of two acrocentric chromosomes at the centromeres with loss of their short arms to form a single abnormal chromosome) accounted for 24% (10 of 42) of cases, and they involved different autosomes.
Forty-five Y chromosome microdeletions were found by standard diagnostic screening, and they were all confirmed by additional detailed STS analysis. Thirty-three men (33 of 45, 73%) had a deletion in AZFc. Less frequently we found a deletion in the AZFb (eight of 45, 18%). In all cases they represented partial AZFb deletion, resembling previously described deletions (20). Four patients had deletions in the AZFa region (four of 45, 9%). They have been previously described and analyzed in detail (14); three of them had deletion of the DBY gene only, and one of the USP9Y gene only.
Nine patients had mutations in the CFTR gene. Three of them had the classical F508 mutation, four cases had other less frequent mutations (R553X, D579G, and R1158X), and two subjects had the 5T allele. All of these mutations were heterozygous. Four of the nine patients with CFTR gene abnormalities were subsequently found to have unilateral absence or atresia of the vas deferens, but in the remaining five subjects no apparent abnormality was observed.
Point mutations in the androgen receptor gene were found in eight subjects; all subjects had different mutations. Seven of these have been previously reported (Y571H, R607X, R615H, D695N, M780I, R855A, and V866M) and shown to be associated with different degrees of androgen insensitivity, whereas one mutation represents a novel finding. This is a T to A transition at nucleotide 3120 determining substitution of a phenylalanine in isoleucine at amino acid 747 (F747I). Five patients with androgen receptor gene mutations had no phenotypic abnormality other than oligozoospermia, two had a history of isolated bilateral cryptorchidism, and one reported bilateral cryptorchidism associated with hypospadias and inguinal hernia.
Sperm chromosomal abnormalities
Results are shown in Table 3. Compared with normospermic controls, patients with classic Klinefelter’s syndrome had a significantly lower percentage of normal Y-bearing sperm and a higher percentage of XX and XY disomies; patients with Y chromosome microdeletions also had a significantly lower percentage of normal Y-bearing spermatozoa compared with normal controls and a concomitant increase in nullisomic sperm and XY disomy. Sperm sex chromosome analysis in subjects with CFTR gene mutations showed no alterations, confirming that spermatogenesis proceeds regularly in these patients. The analysis in patients who had no evident germline abnormalities revealed a normal proportion of X- and Y-bearing normal spermatozoa, but an increased percentage of genetically abnormal sperm, with a significant increase in disomies.
Discussion
We found that 13.9% of 750 severely oligozoospermic men who were candidates for intracytoplasmic sperm injection had a genetic abnormality, including chromosomal aberrations, Y chromosome long-arm microdeletions, CFTR, and androgen receptor gene mutations. Furthermore, the percentage of sperm with aneuploidies was increased not only in men with chromosomal aberrations or Y chromosome microdeletions, but also among oligospermic men without genetic abnormalities detectable in peripheral blood.
Constitutional genetic abnormalities
The increased prevalence of chromosomal abnormalities observed among men in our study is consistent with previous findings among infertile men (23) and among male partners of couples seeking intracytoplasmic sperm injection (24, 25, 26); this figure is inversely related to the sperm count. These findings may account for the observation that children conceived by intracytoplasmic sperm injection have an increased risk of an abnormal karyotype compared with children conceived naturally (5, 6, 7). Although the majority of sperm from men with classic or mosaic Klinefelter’s syndrome or with translocations are chromosomically normal, a higher than normal rate of sex chromosome aneuploidies is found. Therefore, intracytoplasmic sperm injection can be successfully used in these men (27, 28), but careful genetic counseling is warranted.
Our analysis of Y chromosome long-arm microdeletions confirmed that they represent the most common genetic abnormality in severe male infertility, affecting 6% of men in our cohort (29). Microdeletions result in severe primary testiculopathy with azoospermia or severe oligozoospermia, and they can involve one or more AZF regions, with loss of different spermatogenesis genes (14, 29, 30, 31, 32, 33, 34, 35). Although complete AZFa and complete AZFb deletions invariably result in azoospermia (29), partial deletions of these regions may cause also severe oligozoospermia. However, the far most frequent finding in oligozoospermic men is deletion of AZFc. Men with Y chromosome microdeletions were more likely than normospermic men to produce sperm with sex chromosomal anomalies, in particular nullisomy and XY disomy.
The association between mutations of the CFTR gene and male infertility has been previously recognized, and congenital absence of the vas deferens is in most cases regarded as a mild or incomplete form of cystic fibrosis (36, 37, 38, 39). CFTR mutations may also present with unilateral congenital absence of vas deferens (39, 40, 41) or without any apparent abnormality of the vas deferens (42). The prevalence of CFTR gene mutations in our population did not differ significantly from that in controls (1.2% vs. 1.0%, respectively). However, we did not select patients on the basis of absence of the vas deferens. Spermatogenesis in patients with CFTR mutations is normal, and aneuploidy is not increased in the sperm of affected patients. These subjects are good candidates for intracytoplasmic sperm injection, using sperm retrieved from the ejaculate, testis, or epididymis (43). Because of the risk of cystic fibrosis in the offspring of couples in which the female partner is heterozygous for a CFTR mutation (44), screening for CFTR mutations should be considered and is recommended when alterations of the vas deferens are present.
We found a low (1.1%) prevalence of androgen receptor gene mutations in severely oligospermic men, confirming a previous smaller study (45). The prevalence of these mutations was not significantly different from that in controls; this may be explained by the relatively small sample size (no mutations among 188 controls). Mutations in the androgen receptor have been invariably associated with infertility and have not been reported in normal men (http://www.androgendb.mcgill.ca). Our results suggest that screening for androgen receptor gene mutations should be considered in severely oligozoospermic men regardless of the presence of additional phenotypic features suggesting androgen deficiency (such as cryptorchidism or hypospadia), but that such mutations are uncommon.
Sperm chromosomal abnormalities
Sperm of severely infertile men might carry genetic alterations, either because a constitutional abnormality is present in all tissues, including germ cells, or because only sperm are altered as a result of mitotic or meiotic errors due to impaired spermatogenesis. We found an increased percentage of chromosomally abnormal sperm among oligozoospermic men who had no detectable constitutional abnormalities compared with normospermic men (3.7% vs. 1.6%).
Implications for intracytoplasmic sperm injection
Although our study did not assess outcomes of intracytoplasmic sperm injection or genetic abnormalities in offspring, our findings may explain the greater prevalence of congenital anomalies in children conceived by this technique (4, 5) and, in particular, in children conceived by oligozoospermic men (5). Our results combined with prior reports (5) suggest that some congenital anomalies (such as chromosomal alterations) in children born from intracytoplasmic sperm injection are likely to be related not to the technique, but to the underlying genetic cause of the oligozoospermia.
These findings support recent recommendations to screen oligospermic men who are candidates for intracytoplasmic sperm injection for genetic abnormalities. All such men should be asked about family history of infertility and genetic disorders and be carefully examined, counseled, karyotyped, and systematically screened for Y chromosome microdeletions and androgen receptor gene mutations. Because identical mutations in the androgen receptor gene may be associated with different phenotypes (http://www.androgendb.mcgill.ca), the consequences of these mutations in offspring conceived by the use of assisted reproduction techniques cannot be reliably predicted. Y Chromosome microdeletions, and therefore infertility, are invariably transmitted to any male offspring, because the anomaly is also present in spermatozoa (35). In contrast, the presence in these subjects of gametes lacking the sex chromosomes or carrying XY disomy would suggest an increased risk of birth of 45,X and 47,XXY children (46, 47). Although these findings need additional studies, the possible selective use of X-bearing sperm or transfer of XX embryos during in vitro fertilizing techniques should be discussed during the genetic counseling.
Finally, because aneuploidy is more common among oligospermic than normospermic men even in the absence of detectable constitutional abnormalities, and these abnormalities could also be transmitted to offspring, counseling is warranted even for men whose peripheral blood reveals no genetic abnormalities.
Footnotes
This work was supported by the Italian Ministry of University (to C.F.) and the University of Padova (to A.F.).
First Published Online October 27, 2004
Abbreviations: AZF, Azoospermia factor; STS, sequence-tagged site.
Received July 26, 2004.
Accepted October 20, 2004.
References
de Kretser DM 1997 Male infertility. Lancet 349:787–790
Palermo G, Joris H, Devroey P, Van Steirtheghem AC 1992 Pregnancies after intracytoplasmic sperm injection of a single spermatozoon into an oocyte. Lancet 340:17–18
Koulischer L, Verloes A, Lesenfants S, Jamar M, Herens C 1997 Genetic risk in natural and medically assisted procreation. Early Pregnancy 3:164–171
Hansen M, Kurinczuk JJ, Bower C, Webb S 2002 The risk of major birth defects after intracytoplasmic sperm injection and in vitro fertilization. N Engl J Med 346:725–730
Sutcliffe AG, Taylor B, Saunders K, Thornton S, Lieberman BA, Grudzinskas JG 2001 Outcome in the second year of life after in-vitro fertilisation by intracytoplasmic sperm injection: a UK case-control study. Lancet 357:2080–2084
In’t Veld P, Branderburg H, Verhoeff A, Dhont M, Los F 1995 Sex chromosomal anomalies and intracytoplasmic sperm injection. Lancet 346:773
Liebaers I, Bonduelle M, Van Assche E, Devroey P, Van Steirteghem A 1995 Sex chromosome abnormalities after intracytoplasmic sperm injection. Lancet 346:1095
Loft A, Petersen K, Erb K Mikkelsen AL, Grinsted J, Hald F, Hindkjaer J, Nielsen KM, Lundstrom P, Gabrielsen A, Lenz S, Hornnes P, Ziebe S, Ejdrup HB, Lindhard A, Zhou Y, Nyboe Andersen A 1999 Danish cohort of 730 infants born after intracytoplasmic sperm injection (ICSI) 1994–1997. Hum Reprod 14:2143–2148
Schieve LA, Meikle SF, Ferre C, Peterson HB, Jeng G, Wilcox LS 2002 Low and very low birth weight in infants conceived with use of assisted reproductive technology. N Engl J Med 346:731–737
Cox GF, Burger J, Lip V Mau UA, Sperling K, Wu BL, Horsthemke B 2002 Intracytoplasmic sperm injection may increase the risk of imprinting defects. Am J Hum Genet 71:162–164
Foresta C, Ferlin A, Gianaroli L, Dallapiccola B 2002 Guidelines for the appropriate use of genetic tests in infertile couple. Eur J Hum Genet 10:303–312
Vogt PH, Edelmann A, Kirsch S Henegariu O, Hirschmann P, Kiesewetter F, Kohn FM, Schill WB, Farah S, Ramos C, Hartmann M, Hartschuh W, Meschede D, Behre HM, Castel A, Nieschlag E, Weidner W, Grone HJ, Jung A, Engel W, Haidl G 1996 Human Y chromosome azoospermia factors (AZF) mapped to different subregions in Yq11. Hum Mol Genet 5:933–943
Ferlin A, Moro E, Garolla A, Foresta C 1999 Human male infertility and Y chromosome deletions: role of the AZF-candidate genes DAZ, RBM and DFFRY. Hum Reprod 14:1710–1716
Foresta C, Ferlin A, Moro E 2000 Deletion and expression analysis of AZFa-genes on the human Y chromosome revealed a major role for DBY in male infertility. Hum Mol Genet 9:1161–1169
Kuroda-Kawaguchi T, Skaletsky H, Brown LG, Minx PJ, Cordum HS, Waterston RH, Wilson RK, Silber S, Oates R, Rozen S, Page DC 2001 The AZFc region of the Y chromosome features massive palindromes and uniform recurrent deletions in infertile men. Nat Genet 29:279–286
Oates RD, Silber S, Brown LG, Page DC 2002 Clinical characterization of 42 oligospermic or azoospermic men with microdeletion of the AZFc region of the Y chromosome, and of 18 children conceived via ICSI. Hum Reprod 17:2813–2824
Repping S, Skaletsky H, Lange G, Silber S, Van Der Veen F, Oates RD, Page DC, Rozen S 2002 Recombination between palindromes P5 and P1 on the human Y chromosome causes massive deletions and spermatogenic failure. Am J Hum Genet 71:906–922
Foresta C, Moro E, Garolla A, Onisto M, Ferlin A 1999 Y chromosome microdeletions in cryptorchidism and idiopathic male infertility. J Clin Endocrinol Metab 84:3660–3665
Moro E, Ferlin A, Yen PH, Franchi PG, Palka G, Foresta C 2000 Male infertility caused by a de novo partial deletion of the DAZ cluster on the Y chromosome. J Clin Endocrinol Metab 85:4069–4073
Ferlin A, Moro E, Rossi A, Dallapiccola B, Foresta C 2003 The human Y chromosome’s azoospermia factor b (AZFb) region: sequence, structure, and deletion analysis in infertile men. J Med Genet 40:18–24
Lubahn DB, Brown TR, Simental JA, Higgs HN, Migeon CJ, Wilson EM, French FS 1989 Sequence of the intron/exon junctions of the coding region of the human androgen receptor gene and identification of a point mutation in a family with complete androgen insensitivity. Proc Natl Acad Sci USA 86:9534–9538
Foresta C, Galeazzi C, Bettella A, Stella M, Scandellari C 1998 High incidence of sperm sex chromosomes aneuploidies in two patients with Klinefelter’s syndrome. J Clin Endocrinol Metab 83:203–205
Chandley AC 1979 The chromosomal basis of human infertility. Br Med Bull 35:181–186
Peschka B, Leygraaf J, van der Ven K Montag M, Schartmann B, Schubert R, van der Ven H, Schwanitz G 1999 Type and frequency of chromosome aberrations in 781 couples undergoing intracytoplasmic sperm injection. Hum Reprod 14:2257–2263
Gekas J, Thepot F, Turleau C, Siffroi JP, Dadoune JP, Briault S, Rio M, Bourouillou G, Carre-Pigeon F, Wasels R, Benzacken B 2001 Chromosomal factors on infertility in candidate couples for ICSI: an equal risk of constitutional aberrations in women and men. Hum Reprod 16:82–90
Meschede D, Lemcke B, Exeler JR, De Geyter C, Behre HM, Nieschlag E, Horst J 1998 Chromosome abnormalities in 447 couples undergoing intracytoplasmic sperm injection: prevalence, types, sex distribution and reproductive relevance. Hum Reprod 13:576–582
Hinney B, Guttembach M, Schmid M, Engel W, Michelmann HW 1997 Pregnancy after intracytoplasmic sperm injection with sperm form a man with a 47,XXY Klinefelter’s syndrome. Hum Reprod 12:2447–2450
Palermo GD, Schlegel PN, Sillas ES, Veeck LL, Zaninovic N, Menendez S, Rosenwaks Z 1998 Births after intracytoplasmic injection of sperm obtained by testicualr extraction from men with nonmosaic Klinefelter’s syndrome. N Engl J Med 338:588–590
Foresta C, Moro E, Ferlin A 2001 Y chromosome microdeletions and alterations of spermatogenesis. Endocr Rev 22:226–239
Lahn BT, Page DC 1997 Functional coherence of the human Y chromosome. Science 278:675–680
Tilford CA, Kuroda-Kawaguchi T, Skaletski H, Rozen S, Brown LG, Rosenberg M, McPherson JD, Wylie K, Sekhon M, Kucaba TA, Waterston RH, Page DC 2001 A physical map of the human Y chromosome. Nature 409:943–945
Foresta C, Moro E, Rossi A, Rossato M, Garolla A, Ferlin A 2000 Role of AZFa candidate genes in male infertility. J Endocrinol Invest 23:646–651
Elliott DJ, Millar MR, Oghene K, Ross A, Kiesewetter F, Pryor J, McIntyre M, Hargreave TB, Saunders PT, Vogt PH, Chandley AC, Cooke H 1997 Expression of RBM in the nuclei of human germ cells is dependent on a critical region of the Y chromosome long arm. Proc Natl Acad Sci USA 94:3848–3853
Reijo R, Lee TY, Salo P, Alagappan R, Brown LG, Rosenberg M, Rozen S, Jaffe T, Straus D, Hovatta O, de la Chapelle A, Silber S, Page DC 1995 Diverse spermatogenic defects in humans caused by Y chromosome deletions encompassing a novel RNA-binding protein gene. Nat Genet 14:292–299
Reijo R, Alagappan RK, Patrizio P, Page DC 1996 Severe oligozoospermia resulting from deletions of azoospermia factor gene on Y chromosome. Lancet 347:1290–1293
Claustres M, Guittard C, Bozon D, Chevalier F, Verlingue C, Ferec C, Girodon E, Cazeneuve C, Bienvenu T, Lalau G, Dumur V, Feldmann D, Bieth E, Blayau M, Clavel C, Creveaux I, Malinge MC, Monnier N, Malzac P, Mittre H, Chomel JC, Bonnefont JP, Iron A, Chery M, Georges MD 2000 Spectrum of CFTR mutations in cystic fibrosis and in congenital absence of the vas deferens in France. Hum Mutat 16:143–156
Quinzii C, Castellani C 2000 The cystic fibrosis transmembrane regulator gene and male infertility. J Endocrinol Invest 23:684–689
Attardo T, Vicari E, Mollica F, Grazioso C, Burrello N, Garofalo MR, Lizzio MN, Garigali G, Cannizzaro M, Ruvolo G, D’Agata R, Calogero AE 2001 Genetic, andrological and clinical characteristics of patients with congenital bilateral absence of the vas deferens. Int J Androl 24:73–79
Casals T, Bassas L, Egozcue S, Ramos MD, Gimenez J, Segura A, Garcia F, Carrera M, Larriba S, Sarquella J, Estivill X 2000 Heterogeneity for mutations in the CFTR gene and clinical correlations in patients with congenital absence of the vas deferens. Hum Reprod 15:1476–1483
Mickle J, Milunsky A, Amos JA, Oates RD 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. Hum Reprod 10:1728–1735
Dork T, Dworniczak B, Aulehla-Scholz C, Wieczorek D, Bohm I, Mayerova A, Seydewitz HH, Nieschlag E, Meschede D, Horst J, Pander HJ, Sperling H, Ratjen F, Passarge E, Schmidtke J, Stuhrmann M 1997 Distinct spectrum of CFTR gene mutations in congenital absence of vas deferens. Hum Genet 100:365–377
Dohle GR, Halley DJ, Van Hemel JO, van den Ouwel AM, Pieters MH, Weber RF, Govaerts LC 2002 Genetic risk factors in infertile men with severe oligozoospermia and azoospermia. Hum Reprod 17:13–16
Silber SJ, Nagy Z, Liu J, Tournaye H, Lissens W, Ferec C, Liebaers I, Devroey P, Van Steirteghem AC 1995 The use of epididymal and testicular spermatozoa for intracytoplasmic sperm injection: the genetic implications for male infertility. Hum Reprod 10:2031–2043
Hargreave TB 2000 Genetically determined male infertility and assisted reproduction techniques. J Endocrinol Invest 23:697–710
Hiort O, Holterhus PM, Horter T, Schulze W, Kremke B, Bals-Pratsch M, Sinnecker GH, Kruse K 2000 Significance of mutations in the androgen receptor gene in males with idiopathic infertility. J Clin Endocrinol Metab 85:2810–2815
Siffroi JP, Le Bourhis C, Krausz C, Barbaux S, Quintana-Murci L, Kanafani S, Rouba H, Bujan L, Bourrouillou G, Seifer I, Boucher D, Fellous M, McElreavey K, Dadoune JP 2000 Sex chromosome mosaicism in males carrying Y chromosome long arm deletions. Hum Reprod 15:2559–2562
Patsalis PC, Sismani C, Quintana-Murci L, Taleb-Bekkouche F, Krausz C, McElreavey K 2002 Effects of transmission of Y chromosome AZFc deletions. Lancet 360:1222–1224(Carlo Foresta, Andrea Gar)
Recent reports suggest that children born after intracytoplasmic sperm injection performed for male factor infertility are at increased risk of congenital malformations and chromosome aberrations. To explain these observations, we hypothesized that infertile men may be more likely than fertile men to have genetic abnormalities. We studied 750 severely oligozoospermic men (sperm count <5 million/ml) who were candidates for intracytoplasmic sperm injection, and 303 fertile men. We analyzed the peripheral blood karyotype, the Y chromosome long arm for detection of microdeletions in the azoospermia factors, and mutations in the cystic fibrosis gene and the androgen receptor gene. We also analyzed sperm for chromosome aneuploidies among the 421 men who subsequently entered the in vitro fertilization program. A total of 104 genetic abnormalities were diagnosed, corresponding to a frequency of 13.9% (104 of 750). Chromosomal aberrations were present in 5.6% (42 of 750) of infertile men and 0.3% of controls (one of 295), and they were in most cases alterations of the sex chromosomes. Y chromosome long-arm microdeletions were detected in 6.0% (45 of 750) of infertile men and most frequently included the azoospermia factor c, whereas no cases were found in controls (zero of 210). Mutations in the cystic fibrosis gene were diagnosed in 1.2% (nine of 750) of infertile men and 1.0% of controls (three of 303), and mutations in the androgen receptor gene were found in 1.1% (eight of 750) of infertile men and none of the 188 controls. Sperm sex chromosome aneuploidies were increased in men with karyotype anomalies and Y chromosome microdeletions as well as in subjects without constitutional genetic abnormalities. This study shows that the frequency of genetic alterations is increased among men with severe spermatogenic impairment. Genetic tests and genetic counseling should therefore be considered in oligozoospermic men who are candidates for intracytoplasmic sperm injection.
Introduction
INFERTILITY AFFECTS ABOUT 15% of couples trying to conceive in Western countries (1). Accumulating evidence suggests that genetic abnormalities may be present in a large proportion of infertile couples. Intracytoplasmic sperm injection (2) allows some men with a very low sperm count or even no sperm in the ejaculate to become fathers, using sperm retrieved from the epididymis or the testis. Concerns have been raised regarding the potential of intracytoplasmic sperm injection to facilitate the transmission of genetic diseases, because this technique bypasses many of the steps of normal fertilization; a single spermatozoon is injected into the oocyte, eliminating the usual process of natural selection. Early studies suggested that intracytoplasmic sperm injection was safe (3), but more recent data have indicated a higher risk of congenital malformation (4, 5), chromosome aneuploidy (6, 7, 8), low birth weight (9), and imprinting defects such as Angelman syndrome (10) for children conceived by intracytoplasmic sperm injection than for children conceived naturally.
Many constitutional genetic anomalies affecting spermatogenesis can be analyzed during the diagnostic evaluation. Members of our group and others have recommended specific genetic tests in male and female infertile subjects (11), but these recommendations have been based on limited data, and comprehensive studies involving a large number of infertile patients have been lacking. We thus conducted a study to assess the prevalence of several genetic abnormalities among 750 severely oligozoospermic men who were candidates for intracytoplasmic sperm injection.
Subjects and Methods
The study was approved by the hospital ethical committee, and participants gave written informed consent. Seven hundred and fifty consecutive, infertile men, affected by severe oligozoospermia (sperm count <5 million/ml) and candidates for intracytoplasmic sperm injection, attended our university center from 1996–2001, and were screened for genetic anomalies. All patients were of Caucasian origin and came from different Italian regions. No other selection apart from severe oligozoospermia was applied. Subjects presenting with azoospermia were excluded from the study.
Karyotype analysis was conducted by the classical cytogenetic technique with the application of GTG and QFQ banding techniques on at least 25 metaphases from peripheral blood lymphocyte culture. Sex chromosome mosaics occurring at a level of less of 5% were not considered. Pericentric inversions of chromosome 9 or other structural chromosomal variants and polymorphisms (such as familial inversion of the Y chromosome or large Y chromosome heterochromatin) were considered as normal cytogenetic events.
Routine diagnostic Y chromosome long-arm microdeletion analysis was performed by three multiplex PCR analyses on genomic DNA extracted from lymphocytes. A total of nine sequence-tagged sites (STSs) spanning the azoospermia factor (AZF) regions (AZFa, -b, and -c) (12) were used, as follows: sY86, DF3.1 for the USP9Y gene, DBY1 for the DBY gene, and sY95 in the AZFa region; sY117, sY125, and sY127 in the AZFb region; and sY254 and sY255 for the DAZ gene in the AZFc region. Each multiplex set contained the control marker for the SRY gene (sY14). Negative results (no amplifications) were considered only after three amplification failures, repeating the experiments on new DNA extracted from a second blood collection, as previously reported (13, 14). Furthermore, before assuming a deletion, each locus was analyzed separately in a single PCR. Analyses were always performed with a male control sample, a female sample, and a blank sample. Additional STSs were analyzed when a deletion was found, to obtain both confirmation of the deletion and better deletion breakpoints (15, 16, 17), as follows: sY1197 and sY1192 for the proximal breakpoint of AZFc deletions; sY1264 and sY1224 for the proximal breakpoint of AZFb deletions; sY143 for the distal breakpoint of AZFb; and sY1190, sY1125, and sY1201 for the distal breakpoint of AZFc deletions. Extensive analysis of AZFa deletion breakpoints was previously performed (14). Confirmation of deletions was eventually performed by Southern blotting (13, 18, 19). Details of PCR amplification and STSs have been reported previously (13, 14, 18, 19, 20). Y chromosome microdeletion analysis was performed only in patients with normal karyotypes.
The analysis of CFTR gene mutations was performed by a standard reverse dot blot technique for 49 different mutations. Analysis of these mutations allows us to identify about 90% of cystic fibrosis gene carriers from our region (northeast Italy) and about 70% of carriers from other regions of Italy. Analysis of the poly-T allelic variants at the intron 8-exon 9 junction of the CFTR gene was also performed.
Analysis of mutations in the androgen receptor gene was conducted by PCR and subsequent direct sequencing on an ABI PRISM sequencer (Applied Biosystems, Foster City, CA), using a set of 11 oligonucleotide primers covering exons 1–8 (21).
Among subjects attending our center for semen analysis, we recruited 303 consecutive men with normozoospermia as controls. All of these men had peripheral blood analyzed for CFTR gene mutations. Other tests performed in some, but not all, of these men (based on availability of blood) included karyotype analysis (in 295 men), analysis for Y chromosome microdeletions (in 210 men), and analysis for androgen receptor gene mutations (in 188 men).
Of the 750 oligospermic patients, 421 entered an intracytoplasmic sperm injection program. These 421 men were analyzed for numerical alterations of sperm sex chromosomes by means of fluorescence in situ hybridization, as previously reported (22). Among the 303 normozoospermic fertile men, 103 were used as controls for this analysis. At least 5000 sperm were analyzed for each subject, and percentages of sperm with the Y chromosome, the X chromosome, nullisomies (absence of sex chromosomes), and XX, XY, and YY disomies (abnormal presence of the two sex chromosomes) were calculated.
Statistical analysis
Comparison of proportions (prevalence of genetic anomalies in patients and controls) were carried out using Fisher’s exact test. Comparisons of means of aneuploidy rates among groups were analyzed by Wilcoxon rank sum test. A value of P < 0.05 (two-sided) was considered to indicate statistical significance.
Results
Constitutional genetic abnormalities
A total of 104 genetic abnormalities were diagnosed (Table 1), corresponding to a frequency of 13.9% (104 of 750); none of the men had more than one defect. Chromosomal aberrations were present in 5.6% (42 of 750), Y chromosome microdeletions in 6.0% (45 of 750), CFTR gene mutations in 1.2% (nine of 750), and androgen receptor gene mutations in 1.1% (eight of 750). The frequencies of chromosomal anomalies and Y chromosome microdeletions were significantly higher than those among normospermic controls (P < 0.001 for both), whereas the frequencies of CFTR and AR gene mutation were not significantly different.
Most of the chromosomal abnormalities involved the sex chromosomes (32 of 42, 76%), especially in the form of numerical abnormalities (Table 2). The majority of abnormal karyotype were of the Klinefelter’s type, either in the classic 47,XXY form or in the mosaic 47,XXY/46,XY form (in total 29 of 42, 69%). Reciprocal translocations (exchange of material between two nonhomologous chromosomes) and Robertsonian translocations (joining of two acrocentric chromosomes at the centromeres with loss of their short arms to form a single abnormal chromosome) accounted for 24% (10 of 42) of cases, and they involved different autosomes.
Forty-five Y chromosome microdeletions were found by standard diagnostic screening, and they were all confirmed by additional detailed STS analysis. Thirty-three men (33 of 45, 73%) had a deletion in AZFc. Less frequently we found a deletion in the AZFb (eight of 45, 18%). In all cases they represented partial AZFb deletion, resembling previously described deletions (20). Four patients had deletions in the AZFa region (four of 45, 9%). They have been previously described and analyzed in detail (14); three of them had deletion of the DBY gene only, and one of the USP9Y gene only.
Nine patients had mutations in the CFTR gene. Three of them had the classical F508 mutation, four cases had other less frequent mutations (R553X, D579G, and R1158X), and two subjects had the 5T allele. All of these mutations were heterozygous. Four of the nine patients with CFTR gene abnormalities were subsequently found to have unilateral absence or atresia of the vas deferens, but in the remaining five subjects no apparent abnormality was observed.
Point mutations in the androgen receptor gene were found in eight subjects; all subjects had different mutations. Seven of these have been previously reported (Y571H, R607X, R615H, D695N, M780I, R855A, and V866M) and shown to be associated with different degrees of androgen insensitivity, whereas one mutation represents a novel finding. This is a T to A transition at nucleotide 3120 determining substitution of a phenylalanine in isoleucine at amino acid 747 (F747I). Five patients with androgen receptor gene mutations had no phenotypic abnormality other than oligozoospermia, two had a history of isolated bilateral cryptorchidism, and one reported bilateral cryptorchidism associated with hypospadias and inguinal hernia.
Sperm chromosomal abnormalities
Results are shown in Table 3. Compared with normospermic controls, patients with classic Klinefelter’s syndrome had a significantly lower percentage of normal Y-bearing sperm and a higher percentage of XX and XY disomies; patients with Y chromosome microdeletions also had a significantly lower percentage of normal Y-bearing spermatozoa compared with normal controls and a concomitant increase in nullisomic sperm and XY disomy. Sperm sex chromosome analysis in subjects with CFTR gene mutations showed no alterations, confirming that spermatogenesis proceeds regularly in these patients. The analysis in patients who had no evident germline abnormalities revealed a normal proportion of X- and Y-bearing normal spermatozoa, but an increased percentage of genetically abnormal sperm, with a significant increase in disomies.
Discussion
We found that 13.9% of 750 severely oligozoospermic men who were candidates for intracytoplasmic sperm injection had a genetic abnormality, including chromosomal aberrations, Y chromosome long-arm microdeletions, CFTR, and androgen receptor gene mutations. Furthermore, the percentage of sperm with aneuploidies was increased not only in men with chromosomal aberrations or Y chromosome microdeletions, but also among oligospermic men without genetic abnormalities detectable in peripheral blood.
Constitutional genetic abnormalities
The increased prevalence of chromosomal abnormalities observed among men in our study is consistent with previous findings among infertile men (23) and among male partners of couples seeking intracytoplasmic sperm injection (24, 25, 26); this figure is inversely related to the sperm count. These findings may account for the observation that children conceived by intracytoplasmic sperm injection have an increased risk of an abnormal karyotype compared with children conceived naturally (5, 6, 7). Although the majority of sperm from men with classic or mosaic Klinefelter’s syndrome or with translocations are chromosomically normal, a higher than normal rate of sex chromosome aneuploidies is found. Therefore, intracytoplasmic sperm injection can be successfully used in these men (27, 28), but careful genetic counseling is warranted.
Our analysis of Y chromosome long-arm microdeletions confirmed that they represent the most common genetic abnormality in severe male infertility, affecting 6% of men in our cohort (29). Microdeletions result in severe primary testiculopathy with azoospermia or severe oligozoospermia, and they can involve one or more AZF regions, with loss of different spermatogenesis genes (14, 29, 30, 31, 32, 33, 34, 35). Although complete AZFa and complete AZFb deletions invariably result in azoospermia (29), partial deletions of these regions may cause also severe oligozoospermia. However, the far most frequent finding in oligozoospermic men is deletion of AZFc. Men with Y chromosome microdeletions were more likely than normospermic men to produce sperm with sex chromosomal anomalies, in particular nullisomy and XY disomy.
The association between mutations of the CFTR gene and male infertility has been previously recognized, and congenital absence of the vas deferens is in most cases regarded as a mild or incomplete form of cystic fibrosis (36, 37, 38, 39). CFTR mutations may also present with unilateral congenital absence of vas deferens (39, 40, 41) or without any apparent abnormality of the vas deferens (42). The prevalence of CFTR gene mutations in our population did not differ significantly from that in controls (1.2% vs. 1.0%, respectively). However, we did not select patients on the basis of absence of the vas deferens. Spermatogenesis in patients with CFTR mutations is normal, and aneuploidy is not increased in the sperm of affected patients. These subjects are good candidates for intracytoplasmic sperm injection, using sperm retrieved from the ejaculate, testis, or epididymis (43). Because of the risk of cystic fibrosis in the offspring of couples in which the female partner is heterozygous for a CFTR mutation (44), screening for CFTR mutations should be considered and is recommended when alterations of the vas deferens are present.
We found a low (1.1%) prevalence of androgen receptor gene mutations in severely oligospermic men, confirming a previous smaller study (45). The prevalence of these mutations was not significantly different from that in controls; this may be explained by the relatively small sample size (no mutations among 188 controls). Mutations in the androgen receptor have been invariably associated with infertility and have not been reported in normal men (http://www.androgendb.mcgill.ca). Our results suggest that screening for androgen receptor gene mutations should be considered in severely oligozoospermic men regardless of the presence of additional phenotypic features suggesting androgen deficiency (such as cryptorchidism or hypospadia), but that such mutations are uncommon.
Sperm chromosomal abnormalities
Sperm of severely infertile men might carry genetic alterations, either because a constitutional abnormality is present in all tissues, including germ cells, or because only sperm are altered as a result of mitotic or meiotic errors due to impaired spermatogenesis. We found an increased percentage of chromosomally abnormal sperm among oligozoospermic men who had no detectable constitutional abnormalities compared with normospermic men (3.7% vs. 1.6%).
Implications for intracytoplasmic sperm injection
Although our study did not assess outcomes of intracytoplasmic sperm injection or genetic abnormalities in offspring, our findings may explain the greater prevalence of congenital anomalies in children conceived by this technique (4, 5) and, in particular, in children conceived by oligozoospermic men (5). Our results combined with prior reports (5) suggest that some congenital anomalies (such as chromosomal alterations) in children born from intracytoplasmic sperm injection are likely to be related not to the technique, but to the underlying genetic cause of the oligozoospermia.
These findings support recent recommendations to screen oligospermic men who are candidates for intracytoplasmic sperm injection for genetic abnormalities. All such men should be asked about family history of infertility and genetic disorders and be carefully examined, counseled, karyotyped, and systematically screened for Y chromosome microdeletions and androgen receptor gene mutations. Because identical mutations in the androgen receptor gene may be associated with different phenotypes (http://www.androgendb.mcgill.ca), the consequences of these mutations in offspring conceived by the use of assisted reproduction techniques cannot be reliably predicted. Y Chromosome microdeletions, and therefore infertility, are invariably transmitted to any male offspring, because the anomaly is also present in spermatozoa (35). In contrast, the presence in these subjects of gametes lacking the sex chromosomes or carrying XY disomy would suggest an increased risk of birth of 45,X and 47,XXY children (46, 47). Although these findings need additional studies, the possible selective use of X-bearing sperm or transfer of XX embryos during in vitro fertilizing techniques should be discussed during the genetic counseling.
Finally, because aneuploidy is more common among oligospermic than normospermic men even in the absence of detectable constitutional abnormalities, and these abnormalities could also be transmitted to offspring, counseling is warranted even for men whose peripheral blood reveals no genetic abnormalities.
Footnotes
This work was supported by the Italian Ministry of University (to C.F.) and the University of Padova (to A.F.).
First Published Online October 27, 2004
Abbreviations: AZF, Azoospermia factor; STS, sequence-tagged site.
Received July 26, 2004.
Accepted October 20, 2004.
References
de Kretser DM 1997 Male infertility. Lancet 349:787–790
Palermo G, Joris H, Devroey P, Van Steirtheghem AC 1992 Pregnancies after intracytoplasmic sperm injection of a single spermatozoon into an oocyte. Lancet 340:17–18
Koulischer L, Verloes A, Lesenfants S, Jamar M, Herens C 1997 Genetic risk in natural and medically assisted procreation. Early Pregnancy 3:164–171
Hansen M, Kurinczuk JJ, Bower C, Webb S 2002 The risk of major birth defects after intracytoplasmic sperm injection and in vitro fertilization. N Engl J Med 346:725–730
Sutcliffe AG, Taylor B, Saunders K, Thornton S, Lieberman BA, Grudzinskas JG 2001 Outcome in the second year of life after in-vitro fertilisation by intracytoplasmic sperm injection: a UK case-control study. Lancet 357:2080–2084
In’t Veld P, Branderburg H, Verhoeff A, Dhont M, Los F 1995 Sex chromosomal anomalies and intracytoplasmic sperm injection. Lancet 346:773
Liebaers I, Bonduelle M, Van Assche E, Devroey P, Van Steirteghem A 1995 Sex chromosome abnormalities after intracytoplasmic sperm injection. Lancet 346:1095
Loft A, Petersen K, Erb K Mikkelsen AL, Grinsted J, Hald F, Hindkjaer J, Nielsen KM, Lundstrom P, Gabrielsen A, Lenz S, Hornnes P, Ziebe S, Ejdrup HB, Lindhard A, Zhou Y, Nyboe Andersen A 1999 Danish cohort of 730 infants born after intracytoplasmic sperm injection (ICSI) 1994–1997. Hum Reprod 14:2143–2148
Schieve LA, Meikle SF, Ferre C, Peterson HB, Jeng G, Wilcox LS 2002 Low and very low birth weight in infants conceived with use of assisted reproductive technology. N Engl J Med 346:731–737
Cox GF, Burger J, Lip V Mau UA, Sperling K, Wu BL, Horsthemke B 2002 Intracytoplasmic sperm injection may increase the risk of imprinting defects. Am J Hum Genet 71:162–164
Foresta C, Ferlin A, Gianaroli L, Dallapiccola B 2002 Guidelines for the appropriate use of genetic tests in infertile couple. Eur J Hum Genet 10:303–312
Vogt PH, Edelmann A, Kirsch S Henegariu O, Hirschmann P, Kiesewetter F, Kohn FM, Schill WB, Farah S, Ramos C, Hartmann M, Hartschuh W, Meschede D, Behre HM, Castel A, Nieschlag E, Weidner W, Grone HJ, Jung A, Engel W, Haidl G 1996 Human Y chromosome azoospermia factors (AZF) mapped to different subregions in Yq11. Hum Mol Genet 5:933–943
Ferlin A, Moro E, Garolla A, Foresta C 1999 Human male infertility and Y chromosome deletions: role of the AZF-candidate genes DAZ, RBM and DFFRY. Hum Reprod 14:1710–1716
Foresta C, Ferlin A, Moro E 2000 Deletion and expression analysis of AZFa-genes on the human Y chromosome revealed a major role for DBY in male infertility. Hum Mol Genet 9:1161–1169
Kuroda-Kawaguchi T, Skaletsky H, Brown LG, Minx PJ, Cordum HS, Waterston RH, Wilson RK, Silber S, Oates R, Rozen S, Page DC 2001 The AZFc region of the Y chromosome features massive palindromes and uniform recurrent deletions in infertile men. Nat Genet 29:279–286
Oates RD, Silber S, Brown LG, Page DC 2002 Clinical characterization of 42 oligospermic or azoospermic men with microdeletion of the AZFc region of the Y chromosome, and of 18 children conceived via ICSI. Hum Reprod 17:2813–2824
Repping S, Skaletsky H, Lange G, Silber S, Van Der Veen F, Oates RD, Page DC, Rozen S 2002 Recombination between palindromes P5 and P1 on the human Y chromosome causes massive deletions and spermatogenic failure. Am J Hum Genet 71:906–922
Foresta C, Moro E, Garolla A, Onisto M, Ferlin A 1999 Y chromosome microdeletions in cryptorchidism and idiopathic male infertility. J Clin Endocrinol Metab 84:3660–3665
Moro E, Ferlin A, Yen PH, Franchi PG, Palka G, Foresta C 2000 Male infertility caused by a de novo partial deletion of the DAZ cluster on the Y chromosome. J Clin Endocrinol Metab 85:4069–4073
Ferlin A, Moro E, Rossi A, Dallapiccola B, Foresta C 2003 The human Y chromosome’s azoospermia factor b (AZFb) region: sequence, structure, and deletion analysis in infertile men. J Med Genet 40:18–24
Lubahn DB, Brown TR, Simental JA, Higgs HN, Migeon CJ, Wilson EM, French FS 1989 Sequence of the intron/exon junctions of the coding region of the human androgen receptor gene and identification of a point mutation in a family with complete androgen insensitivity. Proc Natl Acad Sci USA 86:9534–9538
Foresta C, Galeazzi C, Bettella A, Stella M, Scandellari C 1998 High incidence of sperm sex chromosomes aneuploidies in two patients with Klinefelter’s syndrome. J Clin Endocrinol Metab 83:203–205
Chandley AC 1979 The chromosomal basis of human infertility. Br Med Bull 35:181–186
Peschka B, Leygraaf J, van der Ven K Montag M, Schartmann B, Schubert R, van der Ven H, Schwanitz G 1999 Type and frequency of chromosome aberrations in 781 couples undergoing intracytoplasmic sperm injection. Hum Reprod 14:2257–2263
Gekas J, Thepot F, Turleau C, Siffroi JP, Dadoune JP, Briault S, Rio M, Bourouillou G, Carre-Pigeon F, Wasels R, Benzacken B 2001 Chromosomal factors on infertility in candidate couples for ICSI: an equal risk of constitutional aberrations in women and men. Hum Reprod 16:82–90
Meschede D, Lemcke B, Exeler JR, De Geyter C, Behre HM, Nieschlag E, Horst J 1998 Chromosome abnormalities in 447 couples undergoing intracytoplasmic sperm injection: prevalence, types, sex distribution and reproductive relevance. Hum Reprod 13:576–582
Hinney B, Guttembach M, Schmid M, Engel W, Michelmann HW 1997 Pregnancy after intracytoplasmic sperm injection with sperm form a man with a 47,XXY Klinefelter’s syndrome. Hum Reprod 12:2447–2450
Palermo GD, Schlegel PN, Sillas ES, Veeck LL, Zaninovic N, Menendez S, Rosenwaks Z 1998 Births after intracytoplasmic injection of sperm obtained by testicualr extraction from men with nonmosaic Klinefelter’s syndrome. N Engl J Med 338:588–590
Foresta C, Moro E, Ferlin A 2001 Y chromosome microdeletions and alterations of spermatogenesis. Endocr Rev 22:226–239
Lahn BT, Page DC 1997 Functional coherence of the human Y chromosome. Science 278:675–680
Tilford CA, Kuroda-Kawaguchi T, Skaletski H, Rozen S, Brown LG, Rosenberg M, McPherson JD, Wylie K, Sekhon M, Kucaba TA, Waterston RH, Page DC 2001 A physical map of the human Y chromosome. Nature 409:943–945
Foresta C, Moro E, Rossi A, Rossato M, Garolla A, Ferlin A 2000 Role of AZFa candidate genes in male infertility. J Endocrinol Invest 23:646–651
Elliott DJ, Millar MR, Oghene K, Ross A, Kiesewetter F, Pryor J, McIntyre M, Hargreave TB, Saunders PT, Vogt PH, Chandley AC, Cooke H 1997 Expression of RBM in the nuclei of human germ cells is dependent on a critical region of the Y chromosome long arm. Proc Natl Acad Sci USA 94:3848–3853
Reijo R, Lee TY, Salo P, Alagappan R, Brown LG, Rosenberg M, Rozen S, Jaffe T, Straus D, Hovatta O, de la Chapelle A, Silber S, Page DC 1995 Diverse spermatogenic defects in humans caused by Y chromosome deletions encompassing a novel RNA-binding protein gene. Nat Genet 14:292–299
Reijo R, Alagappan RK, Patrizio P, Page DC 1996 Severe oligozoospermia resulting from deletions of azoospermia factor gene on Y chromosome. Lancet 347:1290–1293
Claustres M, Guittard C, Bozon D, Chevalier F, Verlingue C, Ferec C, Girodon E, Cazeneuve C, Bienvenu T, Lalau G, Dumur V, Feldmann D, Bieth E, Blayau M, Clavel C, Creveaux I, Malinge MC, Monnier N, Malzac P, Mittre H, Chomel JC, Bonnefont JP, Iron A, Chery M, Georges MD 2000 Spectrum of CFTR mutations in cystic fibrosis and in congenital absence of the vas deferens in France. Hum Mutat 16:143–156
Quinzii C, Castellani C 2000 The cystic fibrosis transmembrane regulator gene and male infertility. J Endocrinol Invest 23:684–689
Attardo T, Vicari E, Mollica F, Grazioso C, Burrello N, Garofalo MR, Lizzio MN, Garigali G, Cannizzaro M, Ruvolo G, D’Agata R, Calogero AE 2001 Genetic, andrological and clinical characteristics of patients with congenital bilateral absence of the vas deferens. Int J Androl 24:73–79
Casals T, Bassas L, Egozcue S, Ramos MD, Gimenez J, Segura A, Garcia F, Carrera M, Larriba S, Sarquella J, Estivill X 2000 Heterogeneity for mutations in the CFTR gene and clinical correlations in patients with congenital absence of the vas deferens. Hum Reprod 15:1476–1483
Mickle J, Milunsky A, Amos JA, Oates RD 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. Hum Reprod 10:1728–1735
Dork T, Dworniczak B, Aulehla-Scholz C, Wieczorek D, Bohm I, Mayerova A, Seydewitz HH, Nieschlag E, Meschede D, Horst J, Pander HJ, Sperling H, Ratjen F, Passarge E, Schmidtke J, Stuhrmann M 1997 Distinct spectrum of CFTR gene mutations in congenital absence of vas deferens. Hum Genet 100:365–377
Dohle GR, Halley DJ, Van Hemel JO, van den Ouwel AM, Pieters MH, Weber RF, Govaerts LC 2002 Genetic risk factors in infertile men with severe oligozoospermia and azoospermia. Hum Reprod 17:13–16
Silber SJ, Nagy Z, Liu J, Tournaye H, Lissens W, Ferec C, Liebaers I, Devroey P, Van Steirteghem AC 1995 The use of epididymal and testicular spermatozoa for intracytoplasmic sperm injection: the genetic implications for male infertility. Hum Reprod 10:2031–2043
Hargreave TB 2000 Genetically determined male infertility and assisted reproduction techniques. J Endocrinol Invest 23:697–710
Hiort O, Holterhus PM, Horter T, Schulze W, Kremke B, Bals-Pratsch M, Sinnecker GH, Kruse K 2000 Significance of mutations in the androgen receptor gene in males with idiopathic infertility. J Clin Endocrinol Metab 85:2810–2815
Siffroi JP, Le Bourhis C, Krausz C, Barbaux S, Quintana-Murci L, Kanafani S, Rouba H, Bujan L, Bourrouillou G, Seifer I, Boucher D, Fellous M, McElreavey K, Dadoune JP 2000 Sex chromosome mosaicism in males carrying Y chromosome long arm deletions. Hum Reprod 15:2559–2562
Patsalis PC, Sismani C, Quintana-Murci L, Taleb-Bekkouche F, Krausz C, McElreavey K 2002 Effects of transmission of Y chromosome AZFc deletions. Lancet 360:1222–1224(Carlo Foresta, Andrea Gar)