1 Centro di Endocrinologia e Medicina della Riproduzione, 2 Consultorio di Genetica, 3 Università `Tor Vergata' and 4 II Clinica Ostetrica e Ginecologica, Università `La Sapienza' Rome, Italy
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Abstract |
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Key words: aneuploidy/FISH/ICSI/sex chromosomes
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Introduction |
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The possibility of visualizing the chromosomes by techniques such as fluorescence in-situ hybridization (FISH) has focused attention on the frequency of numerical chromosomal anomalies in spermatozoa of assisted reproduction patients (Trask, 1991).
An evaluation of the sperm aneuploidy rate has been obtained with the use of multi-colour FISH (Martin et al., 1993). Several studies have shown that the frequency of disomy and nullisomy for the sex chromosome is up to 0.6% (Pfeffer et al., 1999
) whereas the total aneuploidy rate is up to 7.7% (Pang et al., 1999
) in normozoospermic samples. However, the wide range of results reported in the literature is probably due to the number of chromosome probes tested, the number of sperm analysed and the decondensation technique used (Downie et al., 1997
). All data seem to show an increased rate of chromosomal aneuploidy, nullisomy, disomy and diploidy in the sperm of oligozoospermic men who are candidates for ICSI, but with a wide variation in the figures, from 538% depending on the author (Moosani et al., 1995
; Yurov et al., 1996
; Guttenbach et al., 1997
; Storeng et al., 1998
; Aran et al., 1999
; Colombero et al., 1999
; Van Dyck et al., 2000
; Vegetti et al., 2000
; Ohashi et al., 2001
).
Recently, it has been shown that sex chromosomes in sperm are preferentially located in the sub-acrosomal region (Luetjens et al., 1999). It has been suggested that in the case of ICSI, the introduction of sperm which did not undergo acrosomal reaction in oocyte cytoplasm, and with an intact sperm perinuclear theca, may lead to an impaired decondensation of chromatin located in the sub-acrosomal region, especially for sex chromosomes, located there more frequently (Terada et al., 2000
). This may explain the increased incidence of sex chromosomal abnormalities observed in babies conceived with the ICSI procedure.
In order to determine whether the increased rate of sex chromosome abnormalities in ICSI babies may be due to the location of sex chromosomes in the sub-acrosomal region or to an increased rate of sex chromosomes aneuploidy in sperm, we evaluated, with triple-colour FISH, the semen samples of oligozoospermic patients who were candidates for ICSI.
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Materials and methods |
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Decondensation treatment
The slides were washed in 2x standard saline citrate solution (SSC) and incubated for 5 min in 1 mol/l Tris buffer, pH 9.5, containing 25mmol/l dithiothreitol (DTT) (Martini et al., 1995). After decondensation, the slides were washed once in 2xSSC, once in 1xPBS and finally dehydrated through an ethanol series (709090100100%) and air-dried. In order to validate the technique used for decondensation, two other systems of nuclear decondensation, incubation in 3 mol/l NaOH for 5 min or 6 mmol/l EDTA plus 2 mmol/l DTT for 45 min, were also used in five sperm samples. No differences in sex chromosome localization were observed as a result of the variation of decondensation techniques (data not shown).
Triple-colour FISH
Three-colour FISH was performed to determine the frequency of disomy/nullisomy for sex chromosomes and their position compared with an autosome, chromosome 18, using three direct-labelled probes: CEP 18 SpectrumAquaTM, CEP X SpectrumGreenTM, and CEP Y SpectrumOrangeTM (Vysis, Downers Grove, IL, USA).
The hybridization solution, 10 µl of each probe mixture, was applied to a glass slide containing the fixed sperm and covered with a coverslip. The slide was sealed with rubber cement and placed in the Hybrita machine. The Hybrita hybridization system consists of a programmed hot plate where the slides can be co-denatured with the DNA probes and hybridized: the slides are placed on the plate and denatured for 3 min at 75°C, followed by hybridization at 37°C overnight. After hybridization, each slide was washed individually with a solution of 0.4xSSC/0.3% NP40 (Vysis) at room temperature for 2 min. The slides were then mounted with 10 µl of 4',6-diamino-2-phenylindole (DAPI; Vysis Downers Grove, IL, USA) counterstained in anti-fade solution.
Scoring of sperm nuclei
Only slides showing hybridization efficiencies >95% were evaluated. Sperm slides were scored according to previous recommendations (Williams et al., 1993). Sperm nuclei were scored when morphologically preserved, not clumping or overlapping, with a well-defined outline tail and the sperm head decondensed to no more than twice the size of the normal non-decondensed spermatozoa. The presence of the tail was considered essential for a reliable evaluation. FISH preparations were evaluated with a fluorescent microscope (Leica DM interfaced with a computer using the Leica Q-FISH package) at x1000 magnification. The filter used was the VysisTM Aqua, Green, Orange single filter and the VysisTM DAPI/Green/Orange triple band-pass filter set. A minimum of 1000 sperm nuclei per patient for a total of 36 768 cells were examined (24 234 from ICSI men and 12 634 from normozoospermic controls). We evaluated the cells with disomy, two distinct signals for the same chromosome each equal in intensity and size to the single signal found in normal monosomic nuclei, for each chromosome tested 18, X and Y. Spermatozoa were scored as nullisomic for a chromosome if they did not show any signal for that chromosome while a signal for a second tested chromosome was present. A spermatozoon was considered diploid if it exhibited two signals for each tested chromosome and if the tail was evident as well as the normal oval shape of the head. To determine the chromosome position in the sperm nuclei, the nucleus was divided into three regions, one sub-acrosomal, one equatorial and one basal or tail zone, and, in each sperm scored, the position of sex and chromosome 18 was analysed.
Statistical analysis was performed using Student's t-test for continuous variables and 2-test or Fisher's exact test for discontinuous variables and
2 for trend test to determine the correlation between the position of chromosomes. Statistical significance was defined as P < 0.05. The data of the frequency of chromosomal abnormalities are reported as percentage and 95% confidence interval (CI).
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Results |
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Discussion |
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In our study, a statistically significant increase of sperm with sex chromosome abnormalities was found, whereas no statistically significant difference in the chromosome 18 aneuploidy was observed. Many studies have shown that oligozoospermic samples have an increase of aneuploidy in sperm both for sex and autosomic chromosomes (Moosani et al., 1995; In't Veld et al., 1997
; Lahdetie et al., 1997
; Storeng et al., 1998
; Aran et al., 1999
; Colombero et al., 1999
; Pang et al., 1999
; Pfeffer et al., 1999
; Vegetti et al., 2000
; Ohashi et al., 2001
). This discrepancy may be due to different patient selection, different methods of nucleus fixation and DNA decondensation and different chromosomal probes or hybridization techniques used (Downie et al., 1997
). The presence in the semen samples of oligozoospermic men of 15% disomic or nullisomic sperm for sex chromosomes, which in turn may be randomly injected in the cytoplasm of oocytes, and originate embryos with sex chromosome aneuploidy, may explain the higher incidence of sex chromosome abnormalities in offspring born after ICSI procedures (Bonduelle et al., 1998
). The embryos with sex chromosome abnormalities are potentially viable, since most sex chromosome numerical abnormalities are compatible with life. The rate of sperm with sex chromosome numerical abnormalities may well resume the expected risk of having babies with sex chromosome abnormalities. This seems a more convincing explanation for the increased rate in sex chromosome aneuploidy in ICSI infants, than the lack of sex chromosome decondensation in intact acrosome sperm injected inside the oocyte.
The examination of sperm with FISH may be recommended in order to substantiate the risk for sex aneuploidy in embryos. The non-random localization of sex chromosomes in sperm nuclei may also have a role in the determination of sex chromosome aneuploidy, but this theory needs to be scrutinized with further studies, even though it seems to play a marginal role.
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Acknowledgements |
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Notes |
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References |
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accepted on October 3, 2001.