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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Key words: azoospermia/gr/gr deletion/ICSI treatment/oligospermia/Y chromosome
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Introduction |
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Methods |
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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 3536 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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Discussion |
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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 35% 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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Acknowledgements |
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References |
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Submitted on April 5, 2004; resubmitted on August 6, 2004; accepted on September 30, 2004.