1 Institute of Reproductive Medicine and 2 Institute of Legal Medicine, University of Münster, D-48149 Münster, Germany
3 To whom correspondence should be addressed. Email: nieschl{at}uni-muenster.de
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: b2/b3 deletion/gr/gr deletion/male infertility/microdeletion/Y chromosome
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
With knowledge of the sequence of the human Y chromosome, the molecular mechanism of Y chromosomal microdeletions has now been recognized to derive from the homologous recombination between identical parts within palindromic sequences (Kuroda-Kawaguchi et al., 2001; Repping et al., 2003
; Skaletsky et al., 2003
). This results in rearrangements of the Y chromosome and deletions according to specific patterns. In addition to the large deletions deriving from b2/b4 recombination and resulting in the AZFc pattern, partial deletions within the AZFc region have also been described (de Vries et al., 2002
; Ferlin et al., 2002
; Fernandes et al., 2002
, 2004
; Repping et al., 2003
, 2004
) and cause the loss of a lower number of genes and transcription units.
Whether and to what extent these deletions, which are smaller than the AZFc microdeletion, affect spermatogenesis is still controversial. The gr/gr deletion, described by Repping et al. (2003), comprises more than half of the AZFc region and is thought to be a risk factor for spermatogenic failure, but its penetrance is far lower than that of complete AZFc microdeletions. It removes nine transcription units with testis-specific expression, and the reduction of the copy number of the AZFc genes could explain reduced sperm production (Repping et al., 2003
). It is interesting that this deletion can be transmitted from father to son for several generations, suggesting that it can be compatible with spermatogenesis (Repping et al., 2003
). There are at least two other microdeletion patterns removing only part of the AZFc, i.e. the b1/b3 and the b2/b3 deletion (Repping et al., 2003
, 2004
; Fernandes et al., 2004
). The influence of the b1/b3 deletion on spermatogenesis has not been identified yet, but its frequency in the general population was found to be far lower than that of the gr/gr deletion. b2/b3 deletions probably originate from a gr/rg inversion and subsequent deletion between the amplicons b2 and b3 (Fernandes et al., 2004
; Repping et al., 2004
). gr/gr and b2/b3 deletions were found to be related to particular Y chromosome haplogroups (Repping et al., 2003
, 2004
; Fernandes et al., 2004
).
These studies showed that partial deletions of the AZFc region and rearrangements of the Y chromosome are common in infertile men, but it is not clear yet if this analysis should be implemented in the clinical routine. The identification of new sY markers specific for partial deletions now permits screening of patients without resorting to cumbersome methods to assess the possible clinical relevance of partial AZFc deletions. The aim of this study is to assess the occurrence and frequency of partial AZFc microdeletions in patients suffering from spermatogenic failure compared with controls with normal spermatogenesis.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Clinical data
Serum hormone values were measured by routine methods. These include FSH, LH and testosterone. Blood was drawn by venipuncture between 08:00 h and 11:00 h. After sampling, blood had been immediately chilled on ice and centrifuged, and serum aliquots were frozen at 20°C until assayed. Serum testosterone levels were determined using a commercial enzyme-linked immunosorbent assay (ELISA; DRG Instruments, Marburg, Germany). The normal range for serum testosterone is 1235 nmol/l. LH and FSH were measured by immunofluorometric assay (Autodelfia, Perkin Elmer, Freiburg, Germany). Testis volume was measured by ultrasonography. Semen analysis was performed according to the World Health Organization guidelines (1999). All analytical methods were executed and documented in accordance with the principles of Good Laboratory Practice.
Molecular analysis
Genomic DNA was extracted from peripheral leukocytes collected from a venous blood sample. The DNA was extracted using the Quiagen kit (Hilden, Germany) according to the manufacturer's instructions. A two-step approach was employed to identify the partial AZFc deletions. First screening was performed using five pairs of primers specific for partial deletions of the AZFc region: sY1291, sY1161, sY1191, sY1206 and sY1201 (Repping et al., 2003). Two different multiplex reactions were performed, one including primers sY1291, sY1191 and sY1161, and the other including sY1201 and sY1206. In the case of deletions, the PCR was repeated with single primers (simplex reaction) to confirm the deletion. In a second step, four additional pairs of primers were used to characterize the deletion further: sY143, sY142, sY1258 and sY1197 (Repping et al., 2003
). A 2 µl aliquot of the genomic DNA was amplified by PCR. Other components for the PCR were: 2.5 µl of buffer solution, 1 µl of 1 mmol/l of each dNTP, 1 µl of each primer (20 pmol/µl) and 0.4 µl of Taq polymerase in a total volume of 25 µl. PCR was carried out according to the following protocol: amplification by 35 cycles at 94°C for 60 s, 57°C for 60 s and 72°C for 60 s. The programme was preceded by a 3 min denaturation step at 94°C and followed by a final extension step at 72°C for 10 min. The PCR result was made visible on a 2% agarose gel with ethidium bromide including a 1 kb DNA ladder as a marker. In all PCRs, a female DNA and a water sample (no template) were included as negative controls.
Y chromosome haplogroup typing
Haplogroup typing of three samples from the control group carrying the gr/gr mutation (cases 15, 16 and 17) was performed using nine binary markers on the Y chromosome: an Alu polymorphism (YAP; Hammer and Horai, 1995) and eight single nucleotide polymorphisms (SNPs): SRY 1532, Tat, M9, M74, M170, M172, M173 and M213 (Zerjal et al., 1997
; Santos et al., 1999
; Underhill et al., 2001
). PCR was performed in a total volume of 25 µl using 12 mmol/l MgCl2, 510 µg of bovine serum albumin (BSA), 2.5 µl of 10x PCR buffer (Eurogentec, Köln, Germany), 0.40.8 mmol/l each dNTP, 0.40.6 mmol/l each primer and 0.25 U of Taq polymerase (Eurogentec). Each PCR was started by an initial denaturation step (95°C for 3 min) and followed by a final elongation (72°C for 10 min; YAP, 72°C for 30 min). All enzymes were obtained from New England Biolabs (Frankfurt am Main, Germany), except for RsaI that was purchased from Genecraft (Münster, Germany). Digestion of PCR products was performed in a total volume of 20 µl using 8 µl of PCR product, 1 U of the enzyme, buffer and BSA according to the manufacturer's recommendations. Samples were electrophoresed on native polyacrylamide gels (812%) and bands were visualized by silver staining. Additionally, the SNPs M9 and Tat were sequenced using the Big Dye Terminator cycle sequencing kit and the ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Darmstadt, Germany).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
These data show that the frequency of the gr/gr deletion tends to rise with decreasing sperm concentrations (1.8% in the control group versus 4.0% in the group of patients with impaired spermatogenesis) but the difference is not statistically significant (2 test). The haplogroup analysis of the three subjects with a gr/gr deletion belonging to the control group shows that two of them (cases 15 and 16) are members of the R1* family according to the nomenclature recommended by the Y-Chromosome Consortium (YCC; Jobling and Tyler-Smith, 2003
). This haplogroup is the most common group in Westphalia (36.1%; Brión et al., 2004
). The third sample with a gr/gr mutation (case 17) belongs to haplogroup F* (x I, K), which is the second most frequent group in our area (33.25%; Brión et al., 2004
), (Figure 4).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
From our data, it cannot be concluded whether gr/gr deletions play any pathogenic role in spermatogenetic failure. In fact, the frequency of gr/gr deletions was only slightly, but not significantly more elevated in the oligo-/azoospermic group. This suggests that if gr/gr deletions are a risk factor predisposing to spermatogenic failure, they are not sufficient alone. Environmental factors or mild genetic and epigenetic modifications, such as polymorphisms or methylation patterns, might contribute to the phenotypic expression of spermatogenesis in the presence of gr/gr deletions.
Since gr/gr deletions are commonly found in certain chromosome haplogroups (Repping et al., 2003), we verified whether the Y chromosome haplogroup of our control subjects with gr/gr deletions corresponds to any of the rearranged Y chromosome types described earlier (Repping et al., 2003
, 2004
). Several Y chromosomal haplogroups have been described, whose members show a relatively high frequency of gr/gr deletions (Repping et al., 2003
). The gr/gr-deleted men in our control group belong to haplogroups R1* and F* (x I, K), respectively, which represent the two most frequent groups in Westphalia (Brión et al., 2004
). Haplogroup R1* was also the most frequently represented in the sample population studied by Repping et al. (2003)
, who found gr/gr deletions in 12 out of 106 samples (11.3%) with the R1* haplogroup and in three out of 14 samples (21.4%) with the F* haplogroup, respectively.
We found only one b1/b3 deletion in oligozoospermic subjects as well as in the control group. These results confirm that this type of chromosomal rearrangement is less common than gr/gr deletions but, again, do not yet allow a genotypephenotype correlation.
Concerning b2/b3 deletions, which possibly derive from a gr/rg inversion and subsequent deletion (Fernandes et al., 2004; Repping et al., 2004
), we observed a significantly higher frequency in the control subjects, indicating that this type of rearrangement is polymorphic in nature and does not have any detrimental effect on spermatogenesis. On the contrary, its higher incidence in the control group might indicate some sort of protective role against possible negative effects on spermatogenesis by other environmental, genetic and epigenetic factors of this type of chromosomal organization compared with the reference Y chromosome sequence. To clarify this issue, future studies should analyse the correlation between Y chromosomal rearrangements, haplogroups and spermatogenesis in well characterized populations.
In addition to the partial deletions known, we found some new type of deletions not described in the literature so far. Although DNA blot analysis was not performed, the absence of multiple STS markers in individual subjects suggests that these Y chromosomes underwent complex rearrangements resulting in loss of genetic material. Fluorescence in situ hybridization (FISH) analysis will be necessary to characterize such rearrangements. Most importantly, these three microdeletion patterns (Figure 3) could be found only in subjects with impaired spermatogenesis. Extending the analysis to larger groups of men with normal spermatogenesis will indicate whether these novel deletions play any role in the pathogenesis of spermatogenic failure. The exact structure of the deletion in these patients cannot be predicted on the basis of current knowledge of the reference Y chromosome sequence but, since multiple STS markers are deleted, it is quite possible that large portions of the Y chromosome are lost in these subjects.
In summary, our data show that gr/gr deletions tend to be more frequent in men with oligo- or azoospermia than in men with normal spermatogenesis, but their incidence is not significantly different. Therefore, gr/gr deletions are not sufficient to cause spematogenic failure. On the other hand, other types of rearrangements are significantly more frequent in control subjects, confirming that the reference Y chromosome sequence is only one among possible different polymorphic variants compatible with normal spermatogenesis. More clinical data in different patient populations should be collected to clarify the role of partial AZFc deletions and Y chromosomal rearrangements in spermatogenesis to answer the question of whether partial AZFc deletions are significantly associated with spermatogenetic failure and to give a more detailed view of the phenotypes that partial AZFc deletions can produce. In particular, it will be important to perform similar studies in populations of different ethnic origin, since the prevalence and the phenotypic expression of partial AZFc deletions might differ depending on the Y chromosome haplogroups which are not homogeneously distributed throughout the world. In addition, as suggested by the present data, such analysis might reveal novel, still unrecognized deletion patterns of possible relevance for male reproductive fitness. Finally, the well-known variable phenotype of complete AZFc deletions which can be compatible with natural fertility (Stuppia et al., 1996; Chang et al., 1999
; Calogero et al., 2002
; Gatta et al., 2002
; Kühnert et al., 2004
), together with the even broader range of phenotypes observed in subjects with partial AZFc deletions, show the functional redundancy of the genes in the AZFc region. It remains to be established whether and to what extent each gene affects spermatogenesis and whether some genes could compensate for one another in cases of deletion.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Calogero AE, Garofalo MR, Barone N, Longo GA, De Palma A, Fichera M, Rappazzo G, D'Agata R and Vicari E (2002) Spontaneous transmission from a father to his son of a Y chromosome microdeletion involving the deleted in azoospermia (DAZ) gene. J Endocrinol Invest 25, 631634.[ISI][Medline]
Chang PL, Sauer MV and Brown S (1999) Y chromosome microdeletion in a father and his four infertile sons. Hum Reprod 14, 26892694.
de Vries JW, Repping S, van Daalen SK, Korver CM, Leschot NJ and van der Veen F (2002) Clinical relevance of partial AZFc deletions. Fertil Steril 78, 12091214.[CrossRef][ISI][Medline]
Ferlin A, Moro E, Rossi A and Foresta C (2002) A novel approach for the analysis of DAZ gene copy number in severely idiopathic infertile men. J Endocrinol Invest 25, RC1RC3.[ISI][Medline]
Fernandes S, Huellen K, Goncalves J, Dukai H, Zeisler J, Rajpert De Meyts E, Skakkebaek NE, Habermann B, Krause W, Sousa M et al. (2002) High frequency of DAZ1/DAZ2 gene deletions in patients with oligozoospermia. Mol Hum Reprod 8, 286298.
Fernandes S, Paracchini S, Meyer LH, Floridia G, Tyler-Smith C and Vogt PH (2004) A large AZFc deletion removes DAZ3/DAZ4 and nearby genes from men in haplogroup N. Am J Hum Genet 74, 180187.[CrossRef][ISI][Medline]
Foresta C, Moro E and Ferlin A (2001) Y chromosome mcrodeletions and alterations of spermatogenesis. Endocr Rev 22, 226239.
Gatta V, Stuppia L, Calabrese G, Morizio E, Guanciali-Franchi P and Palka G (2002) A new case of Yq microdeletion transmitted from a normal father to two infertile sons. J Med Genet 39, E27.[CrossRef][Medline]
Hammer MF and Horai S (1995) Y chromosomal DNA variation and the peopling of Japan. Am J Hum Genet 56, 951962.[ISI][Medline]
Jobling MA and Tyler-Smith C (2003) The human Y chromosome: an evolutionary marker comes of age. Nat Rev Genet 4, 598612.[CrossRef][ISI][Medline]
Krausz C, Quintana-Murci L and McElreavey K (2000) Prognostic value of Y deletion analysis: what is the clinical prognostic value of Y chromosome microdeletion analysis. Hum Reprod 15, 14311434.
Krausz C, Forti G and McElreavey K (2003) The Y chromosome and male fertility and infertility. Int J Androl 26, 7075.[ISI][Medline]
Kühnert B, Gromoll J, Kostova E, Tschanter P, Luetjens CM, Simoni M and Nieschlag E (2004) Case report: natural transmission of an AZFc Y-chromosomal microdeletion from a father to his sons. Hum Reprod 19, 886888.
Kuroda-Kawaguchi T, Skaletsky H, Brown LG, Minx PJ, Cordum HS, Waterstrom RH, Wilson RK, Silber S, Oates R, Rozen S and Page DC (2001) The AZFc region of the Y chromosome features massive palindromes and uniform recurrent deletions in infertile men. Nat Genet 29, 243244.[CrossRef][ISI][Medline]
Lanfranco F, Kamischke A, Zitzmann M and Nieschlag E (2004) Klinefelter's syndrome. Lancet 364, 273283.[CrossRef][ISI][Medline]
Maurer B and Simoni M (2000) Y chromosome microdeletion screening in infertile men. J Endocrinol Invest 23, 664670.[ISI][Medline]
Maurer B, Gromoll J, Simoni M and Nieschlag E (2001) Prevalence of Y chromosome microdeletions in infertile men who consulted a tertiary care medical centre: the Münster experience. Andrologia 33, 2733.[CrossRef][ISI][Medline]
Repping S, Skaletsky H, Brown L, van Daalen SK, Korver CM, Pyntikova T, Kuroda-Kawaguchi T, de Vries JW, Oates RD, Silber S et al. (2003) Polymorphism for a 1.6-Mb deletion of the human Y chromosome persists through balance between recurrent mutation and haploid selection. Nat Genet 35, 247251.[CrossRef][ISI][Medline]
Repping S, van Daalen S, Korver CM, Brown LG, Marszalek JD, Gianotten J, Oates RD, Silber S, van der Veen F, Page DC and Rozen S (2004) A family of human Y chromosomes has dispersed throughout northern Eurasia despite a 1.8-Mb deletion in the azoospermia factor c region. Genomics 83, 10461052.[CrossRef][ISI][Medline]
Santos FR, Pandya A, Tyler-Smith C, Pena SD, Schanfield M, Leonard WR, Osipova L, Crawford MH and Mitchell RJ (1999) The central Siberian origin for native American Y chromosomes. Am J Hum Genet 64, 619628.[CrossRef][ISI][Medline]
Simoni M, Gromoll J, Dworniczak B, Rolf C, Abshagen K, Kamischke A, Carani C, Meschede D, Behre HM, Horst J and Nieschlag E (1997) Screening for deletions of the Y chromosome involving the DAZ (deleted in azoospermia) gene in azoospermia and severe oligozoospermia. Fertil Steril 67, 542547.[CrossRef][ISI][Medline]
Simoni M, Kamischke A and Nieschlag E (1998) Current status of the molecular diagnosis of Y-chromosomal microdeletions in the work-up of male infertility. Hum Reprod 13, 17641768.
Simoni M, Bakker E, Eurlings MCM, Matthijs G, Moro E, Müller CR and Vogt PH (1999) Laboratory guidelines for molecular diagnosis of Y-chromosomal microdeletions. Int J Androl 22, 225266.
Simoni M, Bakker E and Krausz C (2004) EAA/EMQN best practice guidelines for molecular diagnosis of Y-chromosomal microdeletions. State of the art 2004. Int J Androl 27, 240249.[CrossRef][ISI][Medline]
Skaletsky H, Kuroda-Kawaguchi T, Minx PJ, Cordum HS, Hillier L, Brown LG, Repping S, Pyntikova T, Ali J, Bieri T et al. (2003) The male specific region of the human Y chromosome is a mosaic of discrete sequence classes. Nature 423, 825837.[CrossRef][ISI][Medline]
Stuppia L, Calabrese G, Guanciali Franchi P, Mingarelli R, Gatta V, Palka G and Dallapiccola B (1996) Widening of a Y-chromosome interval-6 deletion transmitted from a father to his infertile sons accounts for an oligozoospermia critical region distal to the RBM1 and DAZ genes. Am J Hum Genet 59, 13931395.[ISI][Medline]
Underhill PA, Passarino G, Lin AA, Shen P, Mirazon Lahr M, Foley RA, Oefner PJ and Cavalli-Sforza LL (2001) The phylogeography of Y chromosome binary haplotypes and the origins of modern human populations. Ann Hum Genet 65, 4362.[CrossRef][ISI][Medline]
World Health Organization (1999) Laboratory Manual for the Examination of Human Semen and SpermCervical Mucus Interaction, 4th edn. Cambridge University Press,.
Zerjal T, Dashnyam B, Pandya A, Kayser M, Roewer L, Santos FR, Schiefenhovel W, Fretwell N, Jobling MA, Harihara S et al. (1997) Genetic relationships of Asians and Northern Europeans, revealed by Y-chromosomal DNA analysis. Am J Hum Genet 60, 11741183.[ISI][Medline]
Submitted on July 21, 2004; accepted on September 17, 2004.