Identification of a Y chromosome haplogroup associated with reduced sperm counts

Transcription

1 2001 Oxford University Press Human Molecular Genetics, 2001, Vol. 10, No Identification of a Y chromosome haplogroup associated with reduced sperm counts C. Krausz 1,2, L. Quintana-Murci 1, E. Rajpert-De Meyts 3, N. Jørgensen 3, M.A. Jobling 4, Z.H. Rosser 4, N.E. Skakkebaek 3 and K. McElreavey 1, * 1 Immunogénétique Humaine, INSERM E021, Institut Pasteur, Paris, France, 2 Andrology Unit, University of Florence, Florence, Italy, 3 University Department of Growth and Reproduction, Rigshospitalet, 9, Blegdamsvej, DK-2100, Copenhagen, Denmark and 4 Department of Genetics, University of Leicester, Leicester, UK Received March ; Revised and Accepted June 21, 2001 In man, infertility is associated with microdeletions of specific regions of the long arm of the Y chromosome. This indicates that factors encoded by the Y chromosome are necessary for spermatogenesis. However, the majority of men with either idiopathic azoospermia or oligozoospermia have grossly intact Y chromosomes and the underlying causes of their infertility are unknown. We hypothesized that some of these individuals may carry other rearrangements or sequence variants on the non-recombining region of the Y chromosome that may be associated with reduced spermatogenesis. To test this hypothesis, we typed the Y chromosome in a group of Danish men with known sperm counts and compared the haplotype distribution with that of a group of unselected Danish males. We found that one class of Y chromosome, referred to as haplogroup 26+, was significantly overrepresented (27.9%; P < 0.001) in the group of men with either idiopathic oligozoospermia (defined as < sperm/ml) or azoospermia compared to the control Danish male population (4.6%). This study defines, for the first time, a class of Y chromosome that is at risk for infertility in a European population. This observation suggests that selection may be indeed active on the Y chromosome, at least in the Danish population, raising the possibility that it could alter the pattern of Y chromosome haplotype distribution in the general population. INTRODUCTION A number of recent reports have focused upon poor and possibly declining semen quality in industrialized countries (1,2). There are, however, striking geographical differences, illustrated best by a difference in semen quality between Denmark and Finland (3). In Denmark, a high frequency of young men with suboptimal sperm quality has been reported (4). In contrast, sperm counts in Finland appear to remain unchanged (5). In 30 50% of infertile men the cause of infertility is unknown and these individuals are classified as idiopathic (6). Microdeletions of the long arm of the Y chromosome define three regions, AZFa, AZFb and AZFc, which are associated with a failure of spermatogenesis (7). However Y microdeletions are a cause of infertility in 15% of men with azoospermia or severe oligozoospermia (< sperm/ml), which represents only a minor fraction of idiopathic male infertility (8). Epidemiological data suggest that male infertility has an important genetic component (9). Infertile couples have fewer sibs compared with fertile control groups and male infertility shows a distinct pattern of familial aggregation, suggesting that non-mendelian multifactorial inheritance could play a significant role (9). This is further highlighted by data that indicate a recurrence of male infertility in uncle nephew pairs (9). An effect of the Y-chromosomal background in men with idiopathic infertility cannot be excluded, since a major biological function of this chromosome is male germ cell development and/or maintenance (8). The precise biological role or contribution to spermatogenesis of each of the genes or gene families on the Y chromosome has yet to be elucidated. The complex multicopy nature of many candidate fertility genes on the human Y chromosome also makes their molecular analysis difficult. However, a Y chromosome effect on spermatogenesis can be determined indirectly by the definition of Y chromosome haplogroups (hgs; a monophyletic group of Y chromosomes defined by shared allelic states at slowly mutating binary markers such as single nucleotide polymorphisms). This straightforward approach can determine the different male genetic components present in a study population. Here, we haplotyped the Y chromosomes of two groups a sample of the general Danish population and a sample of azoo-/oligozoospermic Danish men. The latter were previously screened for Y chromosome microdeletions and shown not to harbour microdeletions in the AZFa, AZFb or AZFc regions (10). When the hg distributions were compared between the two groups, a highly significant bias was detected, indicating that a particular hg is at higher risk for infertility. RESULTS The binary markers 92R7 (C T), M9 (C G) and SRY-1532 (A G) were used to define four hgs (Fig. 1). These hgs *To whom correspondence should be addressed. Tel: ; Fax: ; kenmce@pasteur.fr

2 1874 Human Molecular Genetics, 2001, Vol. 10, No. 18 Figure 1. Phylogenetic tree (maximum parsimony network) showing the evolutionary relationships between Y chromosome lineages (hgs) defined by three biallelic polymorphisms. Arrowheads indicate the derived states. Areas of circles are proportional to the hg frequency in the unselected control group (A) and the azoo-/oligozoospermic group (B). A filled circle indicates a hg in principle detectable, but not found, in our chromosome set. Note that a back mutation occurs at position SRY-1532 and defines hg3. Table 1. hg distributions in an unselected control Danish population and the study population with < sperm/ml hg1 hg2+ hg3 hg26+ Control population (n = 128) 58 (45.3%) 45 (35.2%) 19 (14.8%) 6 (4.6%) With known sperm count 29 (42.0%) 23 (33.3%) 12 (17.4%) 5 (7.2%) Mean sperm count, mill/ml With unknown sperm count 29 (49.1%) 22 (37.2%) 7 (11.8%) 1 (1.9%) Azoo-/oligozoospermic (n = 43) 13 (30.25%) 13 (30.25%) 5 (11.6%) 12 (27.9%) Mean sperm count, mill/ml 5.2 (n = 13) 4.4 (n = 13) 7.5 (n = 5) 2.4 (n = 12) These individuals do not harbour deletions of the Y chromosome AZF regions. The mean sperm count for each group is indicated. In the control population sperm counts were available for 56 individuals. hg nomenclature is adapted from that used elsewhere, with a + after hg number indicating that the other derived lineages defined by markers we have not used may be contained with hg2+ and hg26+. present a pattern of distribution that varies among populations depending on several factors, including ethnic origin, geographical location and linguistic affiliation (11). A study of 128 unselected Danish men from the general population indicated that hg1 is the most represented (45.3%), followed by hg2+ (35.2%), hg3 (14.8%) and hg26+ (4.6%) (Table 1). This is not significantly different from a sample of 56 Danish Y chromosomes studied by Rosser et al. (11) who further subdivide hg2+ (into hgs 2 and 9) and hg26+ (into hgs 12, 16 and 26). While hg1 is the most frequent hg in Europe, with a cline of increasing frequency towards the west, hg26+ is much less common overall. Semen parameters were available for 69 of the 128 unselected control population. Although the number of samples within each group is low, particularly for hg26+, mean sperm counts are reduced in hg26+ individuals compared with the other hgs, suggesting that this group may be at risk for infertility. To test this hypothesis, we determined the hg distribution in 43 men of Danish origin with azoo- or oligozoospermia (defined using WHO standards as < sperm/ml). Thirty-three of these men sought infertility treatment and were defined as having idiopathic infertility. Known causes of spermatogenic failure were excluded, including a history of cryptorchidism, varicocele (grade II or greater) and microdeletions of the AZF regions (10). Within this group, 12 (27.9%) belong to hg26+ (clinical data are described in Table 2). These men presented with a range of phenotypes from azoospermia to oligozoospermia. The distribution of hgs between the unselected control population and the azoo-/oligozoospermic population was found to be significantly different (heterogeneity G-test G = , P < 0.001). Based on the observed data, the odds ratio (which approximates the relative risk) of developing either azoo- or oligozoospermia was estimated between Danish individuals with Y chromosomes belonging to hg1 compared with hg26+, and found to be 8.92 [ (95% confidence limits)].

3 Human Molecular Genetics, 2001, Vol. 10, No Table 2. Clinical data of men with hg26+ Y chromosomes identified in this study Code FSH (U/l) LH (U/l) T (nmol/l) Inhibin B (pg/ml) Sperm count ( 10 6 /ml) Semen vol. (ml) P /20 P /20 P /15 P /22 P /16 P /20 P /12 P /20 P /18 P /12 P /20 P /18 P /15 P < /8 Testis size (L/R ml) DISCUSSION We have identified a class of Y chromosome, hg26+, that is associated with unexplained reduced sperm counts in individuals from the Danish population. In Europe, hg26+ exhibits its highest frequencies in northern European populations (11). The frequency of hg26+ in Saami is 46%, in Estonian and Lithuanian populations it is 46%, and it is 63% in Finnish populations (11). Although the highest incidence is observed in Finno-Ugric language speakers, the presence of the hg in some non-finno-ugric speakers, such as the Balto-Slavic Lithuanians, suggests that the association is primarily geographic. The high incidence in the northern and eastern Baltic regions contrasts with the rest of Europe. In Germanic speaking populations of northern Europe, to whom the Danish people belong, the frequency of hg26+ is considerably less (6% Germans, 8% northern Swedish, 8% Gotlander and 4% Norwegian). The incidence of hg26+ in our control population is consistent with these data at 4.6%. The high incidence of hg26+ ( 28%) in the group of men with < sperm per ml could be caused by two effects. Either this class of Y chromosome is genuinely associated with a reduction of sperm counts, or the difference in hg frequency is caused by local geographic Y chromosome substructure that could result in significant bias in hg distribution. We sought to eliminate the latter by recruiting individuals from the same geographic region (samples were obtained from greater Copenhagen region and Zealand island). Locally restricted Y modal haplotypes, suggesting geographic substructuring, have been reported in Arab populations of Galilee and in Italian populations (12,13). The latter observation is important in the light of a recent report describing a significant difference in Y chromosome hg distribution that was observed between men with idiopathic infertility compared with the general Italian male population (13). However, in this study, the majority of the infertile men were recruited from central Italy and when the control population was geographically divided to take this into account, no difference was observed in the haplotype distribution (13). Recently a Y chromosome hg associated with reduced sperm counts and an increased risk ( 2) of azoospermia was reported in Japan (14). This hg, termed II by the authors (also known as the YAP+ sublineage 3G), is a YAP+ Y chromosome variant not observed in European and African populations and specific to East Asian populations (15). It is thought that these Asianspecific YAP+ chromosomes migrated to Japan with the Jomon people about years ago (16). This event was followed by additional migrations of YAP individuals which has resulted in a highly geographically structured distribution of the YAP+ Y lineage in Japan, with considerable differences in YAP+ frequency from 0 to 50% in some regions (16,17). Geographic substructuring of the Y chromosome to this extent may offer an alternative explanation for these data (18). Although we cannot formally exclude geographic structuring of the Y chromosome in Denmark we feel that it is unlikely. A considerable body of evidence suggests that sperm counts are declining in certain European countries by 2% per year and that this decline began within the last 30 to 40 years (2). Our data indicate that a class of Y chromosomes is overrepresented in men with reduced sperm counts, and taken together suggest that the selection acting on this Y is a recent phenomenon. Most of the hg26+ men have very low sperm counts. Sperm counts within this range are associated with very poor reproductive success and, in the absence of assisted reproduction, these chromosomes will be rapidly eliminated from the population. Taking into account the hg26+ frequency in the Danish population ( 5%) and assuming a mean selective disadvantage for these chromosomes of 0.5, this Y chromosome lineage would disappear from the Danish population within 12 generations. However, the reproductive fitness of the individuals belonging to this hg is difficult to establish, since this calculation makes a number of assumptions, such as absence of migration of hg26+ chromosomes from

4 1876 Human Molecular Genetics, 2001, Vol. 10, No. 18 surrounding populations and that all hg26+ chromosome sublineages are equally susceptible to this effect. Other genetic factors have been associated with differences in human sperm production. These include the HLA genes (19 21), a polymorphism of cytochrome P450-1A1 (22) and variations in the trinucleotide repeat length of the androgen receptor (23). In the latter case, an increased length of the CAG repeat in exon 1 has been reported to be associated with reduced spermatogenesis and idiopathic azoospermia or oligozoospermia, although this has been subsequently challenged (24). Recently, genetic variation in susceptibility to endocrine disruption by estrogen has been demonstrated in mice, where dose-dependent effects on testes weight and spermatogenesis were detected between strains of mice (25). Environmental pressure by as yet unknown factors that are active in industrialized countries or regions with highly intensive agriculture, such as Denmark, could explain negative selection on a rare haplotype that is associated with decreased reproductive fitness. Within hg26+, negative selective pressures may act on a specific class of Y chromosome that may exhibit structural rearrangements, polymorphisms in Y chromosome-specific gene coding sequences or differences in gene copy numbers, all of which may alter spermatogenic efficiency. Copy numbers of certain Y-specific gene families associated with male fertility, such as deleted in azoospermia (DAZ), are known to vary in different human populations (7). Further studies are necessary to determine the molecular mechanism of this susceptibility and to determine if this trend is also present in circum-geographically close human populations that have differences in semen quality and a different Y chromosome ancestry, such as Finland. MATERIALS AND METHODS Subjects The study population was recruited and studied through a comprehensive andrological examination, including semen analysis and hormonal analysis by the Department of Growth and Reproduction (Rigshospitalet, Copenhagen, Denmark). The complete clinical investigation of these cases is described elsewhere (10). Essentially, the study population consisted of consecutive patients with idiopathic male infertility who sought ICSI treatment and who were referred to the Rigshospitalet for a full andrological work-up. The control group consisted of young military conscripts from the general Danish population (semen analysis was available for some of these male subjects described elsewhere; 10). All subjects gave an informed consent for molecular analysis of their blood samples, and the study was approved by a local ethical committee. Semen and hormonal analysis Semen analysis was performed according to the WHO 1992 guidelines (26). Serum concentrations of follicle-stimulating hormone (FSH), luteinising hormone (LH) and sex hormone binding globulin (SHBG) were measured using a timeresolved immunofluorometric assay from DELFIA, (Wallac, Turku, Finland). Testosterone and oestradiol were measured by radioimmunoassay (Coat-a-Count, Diagnostic products Corp., Los Angeles, CA and Immuno Diagnostic Systems, Boldon, UK), respectively. Serum inhibin B was measured in duplicate in a double antibody enzyme immunometric assay using a monoclonal antibody raised against the inhibin β-subunit in combination with a labelled antibody raised against the inhibin α-subunit as described previously (31). Molecular analysis DNA was prepared from peripheral blood lymphocytes using standard techniques. The markers used to define the Danish Y chromosome hgs were the binary markers 92R7 (C T) (27,28), M9 (C G) (29) and SRY-1532 (A G) (30), and were typed as described by Rosser et al. (11). ACKNOWLEDGEMENTS The authors are grateful to the Italian Telethon (grant no. 281/b) and to l Association pour la Recherche sur le Cancer (ARC). The authors wish to acknowledge the help of the clinical and laboratory staff at the Department of Growth and Reproduction, Rigshospitalet, Copenhagen. M.A.J. is a Wellcome Senior Research Fellow in Basic Biomedical Science (grant no ). The collection and analysis of all control data was supported by a grant from the Danish Medical Research Council. L.Q.-M. is supported by an INSERM poste vert. REFERENCES 1. Carlsen, E., Giwercman, A., Skakkebaek, N.E. and Keiding, N. (1992) Evidence for decreasing quality of semen during past 50 years. Br. Med. J., 305, Auger, J., Kunstmann, J.M., Czyglik, F. and Jouannet, P. (1995) Decline in semen quality among fertile men in Paris during the past 20 years. N. Engl. J. Med., 332, Jensen, T.K., Vierula, M., Hjollund, N.H., Saaranen, M., Scheike, T., Saarikoski, S., Suominen, J., Keiski, A., Toppari, J. and Skakkebaek, N.E. (2000) Semen quality among Danish and Finnish men attempting to conceive. The Danish first pregnancy planner study team. Eur. J. Endocrinol., 142, Andersen, A.G., Jensen, T.K., Carlsen, E., Jorgensen, N., Andersson, A.M., Krarup, T., Keiding, N. and Skakkebaek, N.E. (2000) High frequency of sub-optimal semen quality in an unselected population of young men. Hum. Reprod., 15, Vierula, M., Niemi, M., Keiski, A., Saaranen, M., Saarikoski, S. and Suominen, J. (1996) High and unchanged sperm counts of Finnish men. Int. J. Androl., 19, Skakkebaek, N.E., Giwercman, A. and de Kretser, D. (1994) Pathogenesis and management of male infertility. Lancet, 343, Vogt, P.H., Edelmann, A., Kirsch, S., Henegariu, O., Hirschmann, P., Kiesewetter, F., Kohn, F.M., Schill, W.B., Farah, S., Ramos, C. et al. (1996) Human Y chromosome azoospermia factors (AZF) mapped to different subregions in Yq11. Hum. Mol. Genet., 7, McElreavey, K., Krausz, C. and Bishop, C.E. (2000).The human Y chromosome and male infertility. In McElreavey, K. (ed.) The Genetic Basis of Male Infertility, Springer, Heidelberg, pp Meschede, D., Lemcke, B., Behre, H.M., De Geyter, C., Nieschlag, E. and Horst, J. (2000) Clustering of male infertility in the families of couples treated with intracytoplasmic sperm injection. Hum. Reprod., 15, Krausz, C., Rajpert-De Meyts, E., Frydelund-Larsen, L., Quintana-Murci, L., McElreavey, K. and Skakkebaek, N.E. (2001) Double blind Y chromosome microdeletion analysis in men with known sperm parameters and reproductive hormone profiles: Microdeletions are specific for spermatogenic failure. J. Clin. Endocrinol. Metab., 86, Rosser, Z.H., Zerjal, T., Hurles, M.E., Adojaan, M., Alavantic, D., Amorim, A., Amos, W., Armenteros, M., Arroyo, E., Barbujani, G. et al. (2000) Y-chromosomal diversity in Europe is clinal and influenced primarily

5 Human Molecular Genetics, 2001, Vol. 10, No by geography, rather than by language. Am. J. Hum. Genet., 67, Nebel, A., Filon, D., Weiss, D.A., Weale, M., Faerman, M., Oppenheim, A. and Thomas, M.G. (2000) High-resolution Y chromosome haplotypes of Israeli and Palestinian Arabs reveal geographic substructure and substantial overlap with haplotypes of Jews. Hum. Genet., 107, Previderè, C., Stuppia, L., Gatta, V., Fattorini, P., Palka, G. and Tyler-Smith, C. (1999) Y-chromosomal DNA haplotype differences in control and infertile Italian subpopulations. Eur. J. Hum. Genet., 7, Kuroki, Y., Iwamoto, T., Lee, J., Yoshiike, M., Nozawa, S., Nishida, T., Ewis, A.A., Nakamura, H., Toda, T., Tokunaga, K. et al. (1999) Spermatogenic ability is different among males in different Y chromosome lineage. J. Hum. Genet., 44, Hammer, M.F., Karafet, T., Rasanayagam, A., Wood, E.T., Altheide, T.K., Jenkins, T., Griffiths, R.C., Templeton, A.R. and Zegura, S.L. (1998) Out of Africa and back again: nested cladistic analysis of human Y chromosome variation. Mol. Biol. Evol., 15, Hammer, M.F. and Horai, S. (1995) Y chromosomal DNA variation and the peopling of Japan. Am. J. Hum. Genet., 56, Shinka, T., Tomita, K., Toda, T., Kotliarova, S.E., Lee, J., Kuroki, Y., Jin, D.K., Tokunaga, K., Nakamura, H. and Nakahori, Y. (1999) Genetic variations on the Y chromosome in the Japanese population and implications for modern human Y chromosome lineage. J. Hum. Genet., 44, Jobling, M.A. and Tyler-Smith, C. (2000) New uses for new haplotypes the human Y chromosome, disease and selection. Trends Genet., 16, Tsujimura, A., Takahara, S., Kitamura, M., Miura, H., Koga, M., Sada, M., Tsuji, T., Matsumiya, K. and Okuyama, A. (1999) HLA-DR antigen and HLA-DRB1 genotyping with non-obstructive azoospermia in Japan. J. Androl., 20, Miura, H., Tsujimura, A., Nishimura, K., Kitamura, M., Kondoh, N., Takeyama, M., Fujioka, H., Sada, M., Tsuji, T., Matsumiya, K. et al. (1998) Susceptibility to idiopathic azoospermia in Japanese men is linked to HLA class I antigen. J. Urol., 159, Chan, P.J., Su, B.C., Kalugdan, T.H. and Tredway, D.R. (1994) The relationship between human sperm fertilizing capacity and histocompatibility linked antigen (HLA) alleles gene sequences. Hum. Reprod., 9, Fritsche, E., Schuppe, H.C., Dohr, O., Ruzicka, T., Gleichmann, E. and Abel, J. (1998) Increased frequencies of cytochrome P4501A1 polymorphisms in infertile men. Andrologia, 30, Dowsing, A.T., Yong, E.L., Clark, M., McLachlan, R.I., de Kretser, D.M. and Trounson, A.O. (1993) Linkage between male infertility and trinucleotide repeat expansion in the androgen-receptor gene. Lancet, 354, Dadze, S., Wieland, C., Jakubiczka, S., Funke, K., Schroder, E., Royer-Pokora, B., Willers, R. and Wieacker, P.F. (2000) The size of the CAG repeat in exon 1 of the androgen receptor gene shows no significant relationship to impaired spermatogenesis in an infertile Caucasoid sample of German origin. Mol. Hum. Reprod., 6, Spearow, J.L., Doemeny, P., Sera, R., Leffler, R. and Barkley, M. (1999) Genetic variation in susceptibility to endocrine disruption by estrogen in mice. Science, 285, World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction, 3rd edn. Cambridge University Press, Cambridge, UK. 27. Mathias, N., Bayès, M. and Tyler-Smith, C. (1994) Highly informative compound haplotypes for the human Y chromosome. Hum. Mol. Genet., 3, Hurles, M.E., Veitia, R., Arroyo, E., Armenteros, M., Bertranpetit, J., Pèrez-Lezaun, A., Bosch, E., Shlumukova, M., Cambon-Thomsen, A., McElreavey, K. et al. (1999) Recent male-mediated gene flow over a linguistic barrier in Iberia, suggested by analysis of a Y-chromosomal DNA polymorphism. Am. J. Hum. Genet., 65, Underhill, P.A., Jin, L., Lin, A.A., Mehdi, S.Q., Jenkins, T., Vollrath, D., Davis, R.W., Cavalli-Sforza, L.L. and Oefner, P.J. (1997) Detection of numerous Y chromosome biallelic polymorphisms by denaturing high-performance liquid chromatography. Genome Res., 7, Whitfield, L.S., Sulston, J.E. and Goodfellow, P.N. (1995) Sequence variation of the human Y chromosome. Nature, 378, Illingworth, P.J., Groome, N.P., Byrd, W., Rainey, W.E., McNeilly, A.S., Mather, J.P. and Bremner, W.J. (1996) Inhibin-B: a likely candidate for the physiologically important form of inhibin in men. J. Clin. Endocrinol. Metab., 81,

6 1878 Human Molecular Genetics, 2001, Vol. 10, No. 18