1 Service de Biologie de la Reproduction SIHCUS-CMCO, 19, rue Louis Pasteur BP120, 67303 Schiltigheim, France and 2 Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, 1, rue Laurent Fries, BP 163, 67404 Illkirch Cedex, C.U. de Strasbourg, France
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Abstract |
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Key words: chromosomal aneuploidy/ICSI/infertility/morphological anomaly/teratozoospermia
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Introduction |
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Materials and methods |
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Probes included satellite probes for chromosome X (pBam X5) and 1 (pUC1.77), and a probe recognising the heterochromatic region of chromosome Y (pCY98). Probes were directly labelled using a nick translation kit (Boehringer, Mannheim, Germany) with: (i) rhodamine-4-dUTP (Amersham, Les Ullis, France) for the chromosome X probe; (ii) fluorescein isothiocyanate (FITC)-12-dUTP (Boehringer) for the chromosome Y probe; and (iii) a mixture (1:1) of FITC-12dUTP and rhodamine-4-dUTP for the chromosome 1 probe, as described previously (Viville et al., 1998). In order to study spermatozoa used for ICSI, FISH analysis was performed on one semen specimen from each patient after the spermatozoa had been purified through a three-layer Percoll gradient.
Sperm nuclei decondensation was obtained by a 4 min incubation in 0.1 mol/l TrisHCl buffer (pH 8) containing 25 mmol/l dithiothreitol (DTT). Hybridization was performed in 60% formamide/2x sodium chloride/sodium citrate (SSC)/10% dextran sulphate for 2 h following a 1 min denaturation at 75°C. Post-hybridization washes included 5 min in 60% formamide/2x SSC and 5 min in 2x SSC at 42°C, followed by two 5 min washes in 4x SSC/0.05% Tween 20 at room temperature. After dehydration through an ethanol series, slides were counterstained with 4',6-diaminidino-2-phenylindol (DAPI) in antifading solution (Vector, Burlingame, CA, USA). Signal analysis was performed on a Zeiss microscope according to published scoring criteria (Hopman et al., 1988). Results were compared with two controls with normal sperm characteristics and proven fertility. Hybridization efficiencies ranged between 92.6 and 99.6%, while interpretable signals ranged between 68.3 and 95.3% (Table II
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Results |
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The diploidy rate of patient 1 (0.4%) was significantly greater than that of the two controls (0.1% and 0.02%) (P < 0.05; see Discussion). Patient 4, who presented 64% macrocephalic spermatozoa, showed the highest aneuploidy rate (66.9%; Table II). In addition, the sex ratio was 4.1 in favour of Y,1, and the diploidy rate was 22.3%. In total, 89.2% of the spermatozoa presented an abnormal chromosomal constitution. For this patient, therefore, only 10.8% of the spermatozoa presented a normal haploid FISH signal for the three chromosomes analysed.
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Discussion |
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In the case of patient 4, ~90% of the spermatozoa analysed were aneuploid or diploid. A sex ratio distortion in favour of the Y-bearing spermatozoa was also found (In't Veld et al., 1997); no explanation was apparent for this phenomenon.
Other workers (Martin and Rademaker, 1988), using the human spermatozoonhamster oocyte fusion karyotype method, found that there was no significant correlation between the frequencies of chromosomally and morphologically abnormal spermatozoa. Karyotyping of human spermatozoa by injection into mouse oocytes showed that some morphological sperm head abnormalities are associated with an elevated aneuploidy rate (Lee et al., 1996
); however, no increase was seen with macrocephalic spermatozoa. This latter result contradicts the present data and all other known FISH analyses reported on patients with macrocephalic spermatozoa. In these cases, as reported here, virtually all spermatozoa were chromosomally abnormal, a finding also supported by others (Kahraman et al., 1999
), who studied the fertility and ICSI outcome of 17 patients with macrocephalic spermatozoa. Although chromosomal content was not assessed by the latter group, the observed low fertility rate may be explained by a high frequency of sperm aneuploidy.
In addition to the case of macrocephaly, the chromosomal content of spermatozoa from three patients affected with other types of absolute teratozoospermia was studied. To our knowledge, the present study provides the first report of a three-colour FISH analysis of spermatozoa from cases of globozoospermia, shortened flagella syndrome and spermatozoa displaying abnormal acrosomes. The absence of any significant increase in aneuploidy rate suggests that there is no significant relationship between the frequency of numerical chromosomal abnormalities and these specific morphological abnormalities. Our results thus suggest that the chance of conceiving a child with a numerical chromosomal abnormality following ICSI does not appear to be increased for these patients. However, it must be considered reasonable that such causes of infertility may be transmitted to any resulting offspring. For example, globozoospermia has been characterized in mice lacking the casein kinase II ' catalytic subunit (Xu et al., 1999
). It appears likely, therefore, that some cases of globozoospermia in humansas was suggested previouslymay represent a genetic trait (Florke-Gerloff et al., 1984
). Even if the chance of conceiving a chromosomally normal child appears reasonable, there remains the genetic risk of transmitting the paternal phenotype to a male child. In such cases ICSI may introduce a new notion in genetics, the `heridity of infertility'.
In conclusion, on the basis of the results presented here and from other studies, ICSI should not be recommended to patients presenting macrocephalic spermatozoa. For the other cases, additional studies are needed to estimate the genetic risk involved and the chances of conception. Further studies should also be performed to establish specific aneuploidy rates associated with other predominant morphological abnormalities.
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Acknowledgments |
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Notes |
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References |
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Submitted on June 5, 2000; accepted on August 24, 2000.