Hum. Reprod. Advance Access originally published online on October 28, 2004
Human Reproduction 2005 20(1):216-220; doi:10.1093/humrep/deh582
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High frequency of gr/gr chromosome Y deletions in consecutive oligospermic ICSI candidates
1 Grup de Genètica Humana, Departament de Ciències Fisiològiques I, Faculty of Medicine, University of Barcelona, 2 Servei de Genètica, Hospital Clínic i Provincial de Barcelona, IDIBAPS, 08036 Barcelona and 3 Obstretics and Ginecology Unit, Hospital Clínic i Provincial of Barcelona, IDIBAPS, 08036 Barcelona, Spain
4 To whom correspondence should be addressed at: Grup de Genètica Humana, Departament de Ciències Fisiològiques I, Facultat de Medicina, Casanova 143, 08036 Barcelona, Spain. Email: roliva{at}ub.edu
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Abstract |
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BACKGROUND: The Y chromosome gr/gr microdeletion eliminates two copies of the DAZ gene and several additional transcriptional units and has been associated as a risk factor for infertility. Our objective was to study the presence of the gr/gr deletion in ICSI candidates in our population and to determine whether the laboratory, clinical and ICSI outcome were different in the gr/gr deleted patients. METHODS: Two hundred and eighty-three ICSI candidates were studied. Semen analysis, serum FSH, LH, testosterone, inhibin B, karyotype and detection of sequence tagged sites in the Y chromosome were performed. RESULTS: gr/gr deletions were detected in 11 (5.07%) of 217 oligospermic and in one (1.52%) of 66 azoospermic consecutive ICSI candidates, but in none of 232 controls (P=0.002). The fertility rate was not different in the four patients of the gr/gr deleted group treated by ICSI (64.38%; 47/73) as compared to average results at our center (65.49%; 2393/3654). CONCLUSIONS: gr/gr deletions are a risk factor for spermatogenic failure at our population, but the prognosis of the four patients of the gr/gr deleted group treated by ICSI is not different from that of other ICSI patients.
Key words: azoospermia/gr/gr deletion/ICSI treatment/oligospermia/Y chromosome
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Introduction |
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Infertility in men affects 5–10% of the whole population. Spermatogenic failure is the most common form, of which
10% are Yq microdeletions (Vollrath et al., 1992
; Reijo et al., 1995; Vogt et al., 1996; Oliva et al., 1998; Moro et al., 2000; Foresta et al., 2001; Yen, 2001; Tyler-Smith and McVean, 2003). The most common Yq microdeletion is that involving the AZFc region in distal Yq11, a region that contains genes expressed in testis and long direct and indirect repeats called amplicons (Yen, 2001; Skaletsky et al., 2003). The amplicons are organized into palindromic structures and often have >99.9% identity. Homologous recombination between amplicons often causes deletions and duplications that explain 12% of all azoospermias and 6% of severe oligospermias (Oliva et al., 1998; Kuroda et al., 2001; Fernandes et al., 2004). However, the recently described 1.6 Mb deletion polymorphism turned out to be the most frequent large Y chromosome deletion (Repping et al., 2003). This deletion, named gr/gr, affects the AZFc region of the Y chromosome and removes two copies of the DAZ gene and several additional transcriptional units. Deletion of the DAZ gene copies, DAZ1 and DAZ2, has been previously reported as causing a decrease in male's sperm number (de Vries et al., 2002; Fernandes et al., 2002; Repping et al., 2003). Thus, rather than causing severe spermatogenic failure, this deletion turned out to be only a genetic risk factor for spermatogenic failure. We initiated the present study in order to determine whether the presence of the gr/gr deletion was also a risk factor for spermatogenic failure at our population and to determine whether the clinical, laboratory and ICSI outcomes of the gr/gr deleted patients were different or comparable to those of the average ICSI patients at our center. |
Methods |
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Studied subjects
The presence of the gr/gr chromosome Y deletion was studied in different groups: 232 controls of which 34 were semen donors, 75 were fertile men with demonstrated paternity and 123 were patients with conditions unrelated to infertility, and 283 consecutive patients with spermatogenic failure, who were ICSI candidates at our infertility clinic. Sixty-six of the patients with spermatogenic failure were azoospermic and 217 patients were severe oligozoospermic (<5million sperm/ml). All the DNA samples were isolated from venous blood at the Unitat de Genètica, Hospital Clínic de Barcelona, Barcelona, using standard extraction procedures. All azoospermic and severe oligozoospermic patients were ICSI candidates at the Assisted Reproduction Unit at the Hospital Clinic de Barcelona. A G-banded karyotype analysis and a Y chromosome microdeletion analysis were performed routinely in most cases. Y chromosome microdeletions were determined through the analysis of the following markers in multiplex reactions: sY254, sY134, sY143 and sY84. We excluded from the present study all patients with an AZFc complete microdeletion removing all copies of the DAZ gene.
Clinical analysis
Semen analysis was performed according to the World Health Organization guidelines and under internal and external quality control. Serum FSH was measured by an immunoenzymatic assay (Immuno1; Technicion, Bayer, Tarrytown, NY). Dimeric inhibin B was measured by a solid phase sandwich enzyme-linked immunosorbent assay (Serotec, Oxford, UK). Ovarian stimulative and ICSI treatments were performed according to protocols previously reported (Balasch et al., 1996; Ballesca et al., 2000). Oocyte aspiration was performed with vaginal ultrasonography 35–36 h after HCG administration. ICSI procedure was performed according to the method previously described (Palermo et al., 1992).
Molecular analysis of the gr/gr deletion
The chromosome Y gr/gr deletions were identified as described (Repping et al., 2003) by the following results: sY1291 negative, sY1161, sY1191, sY1206 and sY1201 all positive. Initially a multiplex reaction including sY1291 and sY1191 was performed on all samples. The conditions for this PCR reaction were the following: 50 ng of DNA, 150 µM dNTPs (each one), 1 µM each primer, 1x buffer and 0.1 units/ml Taq pol (Roche) in a total volume of 10 µl. The primers for sY1191, yielding a PCR product 385 bp long, were forward 5'-CCAGACGTTCTACCCTTTCG-3' and reverse 5'-GAGCCGAGATCCAGTTACCA-3', and the primers for sY1291, resulting in a 527 bp long PCR product, were forward 5'-TAAAAGGCAGAACTGCCAGG-3' and reverse 5'-GGGAGAAAAGTTCTGCAACG-3' (Repping et al., 2003; Gene Bank accession numbers G72340 and G73809, respectively). Female negative controls were included in all cases. After the PCR reaction, the samples were electrophoresed in a 2% agarose gel including a 1 kb DNA ladder as a marker (Invitrogen; Invitrogen Corporation Life technologies, Inc.; Figure 1). All samples in which the sY1191 STS was present but the sY1291 STS was absent, were subsequently studied for the presence of the STSs sY142, sY1201, sY1258, sY1161, sY1197 and sY1206 to confirm that they were gr/gr deletions and not b1/b3 deletions or longer deletions of the AZFc region. Statistical analyses were performed using the SPSS 10.0 statistical package (SPSS Inc., Chicago, IL).
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Results |
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A total of 12 gr/gr Y chromosome deletions were found in the spermatogenic failure population of 283 individuals. This represented the 4.24% of all patients with spermatogenic failure (<5 million sperm/ml; Table I). The difference as compared to the controls (0 gr/gr deleted from 232 controls) was statistically significant [
2 (Pearson)=0.002, Fisher = 0.001]. If the spermatogenic failure group was stratified into azoospermic and oligospermic patients it turned out that a total of 11 out of 217 individuals (5.07%) of the oligospermic group were positive for the gr/gr deletion while only one of the 66 individuals (1.52%) of the azoospermic population was positive for the gr/gr deletion. The sperm count of the gr/gr deleted patients (average 1.38 million/ml) was significantly lower (P<0.001) to the sperm count of the semen donor controls (average 77.19 million/ml).
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The outcome of ICSI treatment was also compared to average outcome of ICSI treatments available for 2003 at our assisted reproduction unit. The rate at which ICSI-treated oocytes proceeded to zygotes was not different between the gr/gr deleted group and the average treatments performed at our center (64.38% or 47/73 in the gr/gr deleted group compared to 65.49% or 2393/3654 in the complete series; Table II).
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Discussion |
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In this paper we report the gr/gr Y chromosome deletion screening of 283 consecutive ICSI candidates and the laboratory data and clinical outcomes after ICSI treatment. A total of 12 gr/gr deletions were detected in the 283 ICSI candidates (4.24%) while no gr/gr deletions were detected in the control groups (n=232; Table I). The differences between the frequency of deletions between the spermatogenic failure and control groups were highly significant (P=0.001; Fisher) consistently with the previous reports (Repping et al., 2003
). Of the 12 gr/gr deletions, only one corresponded to an azoospermic patient (1.52%; 1/66) and 11 out of 217 (5.07%) corresponded to the oligospermic group. This frequency of gr/gr deletion at our population is higher than that reported by Repping et al. (2003) (3.18%; 15/471 in the initial screening, and 3.66%; 9/246 in the association study). However these differences are not significant and, in addition, could be due to slight differences in the type of patients included in the respective studies and/or to differences in the respective populations. The high frequency of gr/gr deletions detected in the oligospermic group (5.07%) contrasts with the low frequency detected in the azoospermic group (1.52%; 1/66). This situation is opposite to that present in the detection of full Y chromosome microdeletions previously performed at our center (16% microdeletions; 8/50 in azoospermia patients as compared to 1.5%; 2/136 in oligospermic patients; Oliva et al., 1998) as well as by other groups (Vollrath et al., 1992; Reijo et al., 1995; Vogt et al., 1996; Foresta et al., 2001; Repping et al., 2003; Fernandes et al., 2004). These results are consistent with the idea that Y chromosome microdeletions cause severe spermatogenic failure while the gr/gr deletion is only a risk factor (rather than a cause) for spermatogenic failure. The fact that the AZFc Y chromosome microdeletion removes all copies of the DAZ gene and 21 transcriptional units of the Y chromosome, while the gr/gr deletion removes only two copies (half) of the DAZ gene and nine transcriptional units is also consistent with this idea (Repping et al., 2003).
We have also determined the ICSI outcomes in the four patients (numbers 3, 4, 8 and 12) of the gr/gr deleted group treated by ICSI as compared to the average ICSI outcomes obtained at our assisted reproduction unit (Table II). The rate at which ICSI-treated oocytes proceeded to zygotes was not different to that derived from the 2003 activity at our center (65.49%; 2393/3654 vs 64.38%; 47/73). No significant differences were detected either at the rate at which pregnancies were achieved in both groups (Table II). These results are also consistent with those obtained using ICSI sperm from AZFc microdeleted patients (Oates et al., 2002). Thus, prognosis of the three patients with a gr/gr deletion and treated by ICSI is not different to that of the average ICSI patients at our center. This result fits well with the known expression of the transcriptional units deleted in the gr/gr deleted patients and its lack of function in the initial stages of development.
In the vast majority of cases of spermatogenic failure in man a clear cause cannot be identified. The gr/gr deletions reported present in 3–5% of oligospermics (Repping et al. 2003; and present paper) cannot be considered as a cause, but rather a risk factor for spermatogenic failure. In addition to this gr/gr deletion there are now an increasing number of risk factors for spermatogenic failure being considered (Rovio et al., 2001; Mengual et al., 2003a). It must also be taken into account that spermatogenesis is a complex differentiation process resulting in profound biochemical, morphology and functional changes sensitive to environmental factors (Hecht, 1990; Oliva and Dixon, 1991; de Yebra et al., 1993; Mengual et al., 2003b). Thus, the opportunity is now open to look for the potential interaction between the presence of gr/gr deletions as a genetic risk factor and other genetic or environmental factors.
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Supported by grants from Ministerio de Ciencia y Tecnologia BMC2003-03937, fondos FEDER, Ministerio de Sanidad y Consumo V-2003-REDC07A-O and by Generalitat de Catalunya 2001SGR00382.
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References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionAcknowledgementsReferences |
|---|
Balasch J, Fabregues F, Creus M, Moreno V, Puerto B, Penarrubia J, Carmona F and Vanrell JA (1996) Pure and highly purified follicle-stimulating hormone alone or in combination with human menopausal gonadotrophin for ovarian stimulation after pituitary suppression in in-vitro fertilization. Hum Reprod 11, 2400–2404.
Ballesca JL, Balasch J, Calafell JM, Alvarez R, Fabregues F, de Osaba MJ, Ascaso C and Vanrell JA (2000) Serum inhibin B determination is predictive of successful testicular sperm extraction in men with non-obstructive azoospermia. Hum Reprod 15, 1734–1738.
de Vries JW, Hoffer MJ, Repping S, Hoovers JM, Leschot NJ and van der Veen F (2002) Reduced copy number of DAZ genes in subfertile and infertile men. Fertil Steril 77, 68–75.[Web of Science][Medline]
de Yebra L, Ballesca JL, Vanrell JA, Bassas L and Oliva R (1993) Complete selective absence of protamine P2 in humans. J Biol Chem 268, 10553–10557.
Fernandes S, Huellen K, Goncalves J et al. (2002) High frequency of DAZ1/DAZ2 gene deletions in patients with severe oligozoospermia. Mol Hum Reprod 8, 286–298.
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 Y haplogroup. Am J Hum Genet 74, 180–187.[CrossRef][Web of Science][Medline]
Foresta C, Moro E and Ferlin A (2001) Y chromosome microdeletions and alterations of spermatogenesis. Endocr Rev 22, 226–239.
Hecht NB (1990) Regulation of haploid expressed genes in male germ cells. J Reprod Fertil 88, 679–693.
Kuroda-Kawaguchi T, Skaletsky H, Brown LG et al. (2001) The AZFc region of the Y chromosome features massive palindromes and uniform recurrent deletions in infertile men. Nat Genet 29, 279–286.[CrossRef][Web of Science][Medline]
Mengual L, Oriola J, Ascaso C, Ballesca JL and Oliva R (2003a) An increased CAG repeat length in the androgen receptor gene in azoospermic ICSI candidates. J Androl 24, 279–284.
Mengual L, Ballesca JL, Ascaso C and Oliva R (2003b) Marked differences in protamine content and P1/P2 ratios in sperm cells from percoll fractions between patients and controls. J Androl 24, 438–447.
Moro E, Ferlin A, Yen PH, Franchi PG, Palka G and 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.
Oates RD, Silber S, Brown LG and 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.
Oliva R and Dixon GH (1991) Vertebrate protamine genes and the histone-to-protamine replacement reaction. Prog Nucleic Acids Res Mol Biol 40, 25–94.[Web of Science][Medline]
Oliva R, Margarit E, Ballesca JL, Carrio A, Sanchez A, Mila M, Jimenez L, Alvarez-Vijande JR and Ballesta F (1998) Prevalence of Y chromosome microdeletions in oligospermic and azoospermic candidates for intracytoplasmic sperm injection. Fertil Steril 70, 506–510.[CrossRef][Web of Science][Medline]
Palermo G, Joris H, Devroey P and Van Steirteghem AC (1992) Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 340, 17–18.[CrossRef][Web of Science][Medline]
Reijo R, Lee TY, Salo P et al. (1995) Diverse spermatogenic defects in humans caused by Y chromosome deletions encompassing a novel RNA-binding protein gene. Nat Genet 10, 383–393.[CrossRef][Web of Science][Medline]
Repping S, Skaletsky H, Brown L 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, 247–251.[CrossRef][Web of Science][Medline]
Rovio AT, Marchington DR, Donat S et al. (2001) Mutations at the mitochondrial DNA polymerase (POLG) locus associated with male infertility. Nat Genet 29, 261–262.[CrossRef][Web of Science][Medline]
Skaletsky H, Kuroda-Kawaguchi T, Minx PJ et al. (2003) The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes. Nature 423, 825–837.[CrossRef][Medline]
Tyler-Smith C and McVean G (2003) The comings and goings of a Y polymorphism. Nat Genet 35, 201–202.[CrossRef][Web of Science][Medline]
Vogt PH, Edelmann A, Kirsch S et al. (1996) Human Y chromosome azoospermia factors (AZF) mapped to different subregions in Yq11. Hum Mol Genet 5, 933–943.
Vollrath D, Foote S, Hilton A, Brown LG, Beer-Romero P, Bogan JS and Page DC (1992) The human Y chromosome: a 43-interval map based on naturally occurring deletions. Science 258, 52–59.
Yen P (2001) The fragility of fertility. Nat Genet 29, 243–244.[CrossRef][Web of Science][Medline]
Submitted on April 5, 2004; resubmitted on August 6, 2004; accepted on September 30, 2004.
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