1 Unitat de Biologia Cellular, Facultat de Ciències, Universitat Autònoma Barcelona, 08193-Barcelona, Spain, 2 Genetics and IVF Institute, Fairfax and 3 Medical College of Virginia, Richmond, VA, USA
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Abstract |
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Key words: chromosome 21 disomies/flow cytometry/fluorescence in-situ hybridization/sperm sex selection
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Introduction |
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Levinson et al. reported the first pregnancy after the use of MicroSort FCS for the production of a higher proportion of female embryos in a preimplantation diagnosis case (Levinson et al., 1995). Recently (Fugger et al., 1998
) the first births of normal daughters from the use of MicroSort followed by intrauterine insemination (IUI), in-vitro fertilization (IVF), or intracytoplasmic sperm injection (ICSI) were described.
Sperm samples are flow sorted based on the approximately 3% total DNA content difference between human X- and Y-chromosome bearing sperm cells. Effective enrichment to 100% of the desired population is not attainable and variations in the sorting efficiency between different semen samples are observed.
Different factors associated with the determination of the total DNA content during the sorting procedure (Cran and Johnson, 1996) could lead to the observed interdonor heterogeneity. Furthermore, several characteristics specific to human sperm cells such as physical shape and morphology, variation in the amount of human Y heterochromatin and significant differences in DNA content between individuals and between chromosome pairs (autosomal polymorphisms) may influence the results of the FCS efficiency.
On the other hand, FISH studies in decondensed sperm heads have established that some chromosomes have special tendencies to non-disjunction (reviewed in Downie et al., 1997 and Egozcue et al., 1997). Particularly, it has been reported that chromosome 21 shows a high incidence of disomy in sperm cells (0.38%: Blanco et al., 1996; 0.29%: Spriggs et al., 1996; 0.37: McInnes et al., 1998). Furthermore, Griffin et al. reported an excess of Y-bearing spermatozoa among spermatozoa disomic for chromosome 21 (Griffin et al., 1996) suggesting that the extra chromosome 21 preferentially segregates with the Y chromosome. Any chromosomal imbalance will clearly modify the total DNA content of the cell. Table I
illustrates the differences in total DNA content in haploid X- and haploid Y-bearing spermatozoa and in disomic sperm nuclei for chromosomes 21 and the sex chromosomes.
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Materials and methods |
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Physical limitations prevent the use of the same sperm sample for non-sorting, X-sorting and Y-sorting. There was one control and one sort (either X or Y) for each semen sample processed. In two cases more than one sample was processed in different experiments (see below). From each of the processed samples, an aliquot was fixed onto slides as previously reported (Vidal et al., 1993), labelled for a blind evaluation and shipped to Barcelona (Spain) for FISH analysis. Of the slides received in Barcelona, 25 were taken at random for analysis in this study.
Fluorescent in-situ hybridization
Slides shipped from Fairfax were stored at 20°C until processed for FISH analysis as previously described by Vidal et al. (1993). Multicolour FISH with centromeric DNA probes for chromosome X (Spectrum green; Vysis Inc., Downers Grove, IL, USA), satellite III DNA probes for chromosome Y (Spectrum aqua; Vysis Inc.) and locus specific probes for chromosome 21 (loci q22.14-q22.3, Spectrum orange; Vysis Inc.) was used for the study. FISH incubation and detection were performed according to a standard protocol (Blanco et al., 1997). Analyses were done using an Olympus BX60 epifluorescence microscope equipped with a triple-band pass filter for DAPI/Texas red/FITC and single-band pass filters for FITC, Texas red and aqua.
Data collection and analysis
Sperm nuclei scoring was done according to the strict criteria described by Blanco et al. (1996). Sperm chromosome content for chromosome X, chromosome Y and chromosome 21 was evaluated through the signals displayed. All hybridized scorable nuclei present in a given slide was recorded. At the end of the study, the results obtained were compared with the treatment (X-sort, Y-sort or non-sorted) used in each case. Data were statistically analysed by an InStat® 2.01 program (Graph Pad, San Diego, CA, USA).
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Results |
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In the X-sorted group, five slides from five different donors with a total number of 14 986 spermatozoa were evaluated. The X:Y ratio showed highly significant differences from the expected 1:1 (P < 0.0001) and fully agreed with the sorting treatment used. A mean of 0.16% (0.030.25%) spermatozoa disomic for chromosome 21 and a mean of 0.09% (0.030.16%) of spermatozoa disomic for the sex chromosomes were observed in this group.
In the Y-sorted group, eight slides from seven different donors with a total number of 21 482 spermatozoa were evaluated. The X:Y ratio appeared clearly skewed towards Y-bearing spermatozoa showing also highly significant differences from the expected 1:1 (P < 0.0001). For the chromosome 21, the disomy frequency was 0.14% (0.110.17%) and for the sex chromosomes the disomy frequency was 0.08% (00.23%).
No significant differences were found in the distribution of XX, XY, YY and 21 disomic spermatozoa (Table III) in the total numbers of MicroSort non-sorted and MicroSort sorted samples (0.0630 < P < 0.9147). Finally, it is important to note the lack of diploid spermatozoa in all the MicroSort groups analysed (non-sorted, X-sorted and Y-sorted).
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Discussion |
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It is well documented that MicroSort protocols currently provide an average of 85% enrichment in X-bearing spermatozoa and 65% enrichment for Y-bearing spermatozoa. Fugger et al. (1998), in their recent paper, pointed out that `continued refinement is expected to provide improvements in both sperm purity and recovery'. The present study was designed to evaluate the `purity' of the sorted fractions and analyses were focused on the disomy level for chromosome 21 and for the sex chromosomes in the sorted spermatozoa.
In our study, the number of spermatozoa scored in the flow processed samples (range: 11883961 spermatozoa) may seem somewhat low for an accurate analysis of aneuploidy level in sperm nuclei. In fact, it is well known that it is recommended to score 10 000 spermatozoa per individual (reviewed by Egozcue et al., 1997). However, this figure has been estimated from control samples, with normal sperm counts (~50x106 spermatozoa/ml), and the evaluation of 10 000 sperm cells represents about 0.02% of the sample. After flow sorting the retrieval rate varies between 0.6 and 1.2% of the spermatozoa processed (Fugger et al., 1998) thus normal sperm samples result in an average of 200 000 spermatozoa. Taking this data into consideration, the number of spermatozoa analysed in our study, although apparently low, represents 0.62% of the finally recovered spermatozoa resulting in figures 30-fold to 100-fold of those usually scored.
One of the aims of this study was to evaluate whether the frequency of sex chromosome disomies and chromosome 21 disomies normally existing in an ejaculate (basal level) would be increased in the X- or Y-bearing enriched fractions, given the DNA content variations due to these disomies (Table I). Our study showed similar results for each of the analysed disomies in all of the FCS processed sample groups (Table III
). The observed results may possibly indicate that flow sorting is not associated with any increase in the frequency of disomies for the analysed chromosomes relative to previously published data (Downie et al., 1997
; Egozcue et al., 1997
) (Table III
).
However, despite the straightforward resolution of the system, a small number of spermatozoa carrying disomies for the chromosomes analysed were found to be present in the sorted fractions. The presence of these abnormal spermatozoa may be from the portion of the fraction that was not successfully sorted (15% for the X-sorted samples and 35% for the Y-sorted samples) since sorted fractions are not absolutely `pure' for the desired sex-bearing spermatozoa. In fact, the recovery of a similar percentage of spermatozoa carrying XX disomies (meaning 9% excess in total DNA content difference) and spermatozoa disomic for chromosome 21 (with 1.5% excess in total DNA content difference) in the Y-sorted population support the idea that these spermatozoa appear randomly and probably result from the non-pure fraction.
To date, few cases of carriers of structural and numerical reorganizations have been studied by FISH in decondensed sperm nuclei. However, some studies indicate that apart from the presence of unbalanced gametes resulting from the expected meiotic behaviour of the chromosome abnormality, there is an increase in the frequency of diploid spermatozoa (Han et al., 1994; Rousseaux et al., 1995
; Mercier and Brenson, 1997
; Van Hummelen et al., 1997
; Blanco et al., 1998
). Results obtained so far by FISH studies in decondensed sperm nuclei from infertile men are quite variable, ranging from normality (Miharu et al., 1994
; Guttenbach et al., 1997
) to a significant increase of diploid spermatozoa and/or sex chromosome disomies (Moosani et al., 1995
; Pang et al., 1995
; Bernardini et al., 1997
; Egozcue et al., 1997
; Finkelstein et al., 1998
; In't Veld et al., 1997; Pieters et al., 1998
). Our study showed a total absence of diploid spermatozoa in all groups after MicroSort FCS. These results may indicate that diploid sperm cells are selectively excluded from flow sorted samples as a result of their substantial difference in total DNA content. Reduced disomy rates have been observed when comparing MicroSort processed samples to baseline frequencies published for untreated samples (reviewed by Downie et al., 1997 and Egozcue et al., 1997) (Table III
). MicroSort FCS before ICSI might be useful in selected infertile candidates and in cases of structural reorganizations of some chromosomes but further research is needed in this area.
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Acknowledgments |
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Notes |
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References |
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Submitted on April 12, 1999; accepted on September 10, 1999.