Preliminary study of the incidence of disomy in sperm fractions after MicroSort flow cytometry

F. Vidal1,4, J. Blanco1, E.F. Fugger2,3, K. Keyvanfar2, M. Norton2, J.D. Schulman2,3 and J. Egozcue1

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Using triple colour fluorescent in-situ hybridization (FISH) we have evaluated, on a blind basis, the disomy level for chromosome 21 and the sex chromosomes in flow cytometric sorted sperm samples. There were no statistically significant differences in the disomy rates when comparing the sorted samples (either for X- or Y-bearing spermatozoa) with non-sorted samples. There were no diploid spermatozoa observed in any sample group after MicroSort® processing.

Key words: chromosome 21 disomies/flow cytometry/fluorescence in-situ hybridization/sperm sex selection


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The use of flow cytometry sorting (FCS) to separate human X- and Y-bearing spermatozoa, based on the difference in their DNA content, was first described by Johnson et al. (1993). The resulting separation efficiency, evaluated by fluorescent in-situ hybridization (FISH), demonstrated that human spermatozoa can be sorted to an enrichment of 80–90% for X spermatozoa and 60–70% for Y spermatozoa (Johnson et al., 1993Go; Vidal et al., 1998Go).

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., 1995Go). Recently (Fugger et al., 1998Go) 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, 1996Go) 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., 1996Go) 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 IGo 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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Table I. Differences in total DNA content between disomic sperm nuclei and X or Y-bearing haploid spermatozoa (calculated from Mendelsohn et al., 1972)
 
The purpose of this study was to evaluate the resolution of the MicroSort system through analysis of the incidence of disomy for chromosome 21 and for the sex chromosomes in sperm fractions obtained after cytometric flow sorting. In this sense, chromosome 21 is a useful candidate to be used to examine sorting accuracy because, concerning DNA content, it is the smallest chromosome in the human genome (Mendelsohn et al., 1972Go; Morton, 1991Go) and, as previously said, it is one of the chromosomes more prone to non-disjunction. To reach this objective, a collaborative trial between the Genetics and IVF Institute from Fairfax and our centre was undertaken.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Sperm sample processing
Sperm samples from 14 different donors (aged 19–35 years) were obtained and processed in Fairfax using MicroSort sperm separation technology as described (Fugger et al., 1998Go). Briefly, specimens were stained with a 9 µmol/l solution of the vital fluorochrome bisbenzimide (Hoechst 33342; Calbiochem Behring Corp., La Jolla, CA, USA) for 1 h at 35°C. All sperm samples were processed through a modified FACS Vantage (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) with an argon laser operating in the ultra violet (UV) spectrum. Spermatozoa were passed through the flow cytometer and sorted for X-bearing spermatozoa, sorted for Y-bearing spermatozoa, or not sorted (control group) and collected into 0.5 ml tubes (Falcon no. 145440; Research Products International Corporation, Mount Prospect, IL, USA) coated with 1% bovine albumin fraction V. X- or Y-chromosome bearing spermatozoa were preferentially selected based on the intensity of fluorescence emitted by the individual cells and separated electronically into tubes containing the respective enriched fraction. Non-sorted spermatozoa were also passed through the cytometer and collected in a tube without selection based on fluorescence intensity.

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., 1993Go), 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., 1997Go). 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).


    Results
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 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of 68 942 sperm cells from the three sample groups, with an average of 2757 cells scored per slide (range 1188–3961), were analysed for this study. Hybridization was efficient with an overall frequency of hybridization of 98.29% (range 94.24–100%). Percentages of X- and Y-bearing spermatozoa observed in the evaluated samples are shown in Table IIGo and were consistent with the protocol used in each case.


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Table II. Results of triple colour FISH in all slides from non sorted, X-sorted and Y-sorted sperm samples
 
Twelve slides from nine different donors with a total number of 32 474 spermatozoa were evaluated in the non-sorted group (Table IIGo). Three slides were evaluated from one donor and two slides from another donor; in each case the slides originated from different sorting experiments. The mean proportion of spermatozoa disomic for chromosome 21 was 0.18% (0.07–0.33%) and the mean proportion of spermatozoa disomic for the sex chromosomes was 0.10% (0.03–0.21%). No significant differences (P = 0.8437) were found in the distribution of X-bearing spermatozoa disomic for chromosome 21 and Y-bearing spermatozoa disomic for chromosome 21.

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% (0.03–0.25%) spermatozoa disomic for chromosome 21 and a mean of 0.09% (0.03–0.16%) 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% (0.11–0.17%) and for the sex chromosomes the disomy frequency was 0.08% (0–0.23%).

No significant differences were found in the distribution of XX, XY, YY and 21 disomic spermatozoa (Table IIIGo) 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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Table III. Total disomy results from triple-colour FISH analysis in spermatozoa from non-sorted, X-sorted and Y-sorted
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
MicroSort flow cytometric separation of human sperm cells is becoming recognized as the only validated method for separation of X- and Y-bearing human spermatozoa for preconception gender selection. Sperm enrichment of the desired fraction after sorting has been supported by FISH analysis (Johnson et al., 1993Go; Vidal et al., 1998Go). Concerns have been raised and refuted regarding the possible mutagenic risk of Hoechst 33 342 stain and UV irradiation (Ashwood-Smith, 1994Go; Johnson and Schulman, 1994Go); however, animal work (Johnson, 1991Go, 1992Go; Cran et al., 1993Go; Cran and Johnson, 1996Go; Rath et al., 1996Go) has already led to the birth of hundreds of normal offspring in a variety of species and seems to indicate the safety of the procedure. Furthermore, normal births with successful clinical data after the use of MicroSort FCS followed by assisted reproduction protocols have been recently reported (Fugger et al., 1998Go). Current experience indicates that sperm samples after MicroSort FCS result in acceptable pregnancy rates from normal semen samples when used with IUI as well as with oligozoospermic semen samples used with IVF or ICSI (Fugger, unpublished data). Taking these results into consideration, the number of couples enquiring about preconception gender selection in couples at risk for transmitting sex linked diseases or for family balancing may increase considerably.

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: 1188–3961 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., 1998Go) 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 0.6–2% 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 IGo). Our study showed similar results for each of the analysed disomies in all of the FCS processed sample groups (Table IIIGo). 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., 1997Go; Egozcue et al., 1997Go) (Table IIIGo).

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., 1994Go; Rousseaux et al., 1995Go; Mercier and Brenson, 1997Go; Van Hummelen et al., 1997Go; Blanco et al., 1998Go). Results obtained so far by FISH studies in decondensed sperm nuclei from infertile men are quite variable, ranging from normality (Miharu et al., 1994Go; Guttenbach et al., 1997Go) to a significant increase of diploid spermatozoa and/or sex chromosome disomies (Moosani et al., 1995Go; Pang et al., 1995Go; Bernardini et al., 1997Go; Egozcue et al., 1997Go; Finkelstein et al., 1998Go; In't Veld et al., 1997; Pieters et al., 1998Go). 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 IIIGo). 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.


    Acknowledgments
 
This work was partially supported by a research grant from Fundación Salud 2000.


    Notes
 
4 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ashwood-Smith, M.J. (1994) Human sperm–sex selection. Safety of human sperm selection by flow cytometry. Hum. Reprod., 9, 757.[ISI][Medline]

Bernardini, L., Martini, E., Geraedts, J.P.M. et al. (1997) Comparison of gonosomal aneuploidy in spermatozoa of normal fertile men and those with severe male factor detected by in-situ hybridization. Mol. Hum. Reprod., 3, 431–438.[Abstract]

Blanco, J., Egozcue, J. and Vidal F. (1996) Incidence of chromosome 21 disomy in human spermatozoa as determined by fluorescent in-situ hybridization. Hum. Reprod., 11, 722–726.[Abstract]

Blanco, J., Egozcue, J. and Vidal, F. (1997) Increased incidence of disomic sperm nuclei in a 47, XYY male assessed by fluorescent in situ hybridization (FISH). Hum. Genet., 99, 413–416.[ISI][Medline]

Blanco, J., Clusellas, N., Egozcue, J. et al. (1998) FISH in sperm heads allow the analysis of the chromosome segregation and interchromosomal effects in carriers of structural reorganizations. Results in a translocation carrier t(5;8) (q33;q13). Cytogenet. Cell Genet.,

Cran, D.G. and Johnson, L.A. (1996) The predetermination of embryonic sex using flow cytometrically separated X and Y spermatozoa. Hum. Reprod. Update, 2, 355–363.[Abstract/Free Full Text]

Cran, D.G., Johnson, L.A., Miller, N.G.A. et al. (1993) Production of bovine calves following separation of X- and Y-bearing sperm and in vitro fertilisation. Vet. Rec., 132, 40–41.

Downie, S., Flaherty, S. and Matthews, D. (1997) Detection of chromosomes and estimation of aneuploidy in human spermatozoa using fluorescence in-situ hybridization. Mol. Hum. Reprod., 3, 585–598.[Abstract]

Egozcue, J., Blanco, J. and Vidal, F. (1997) Chromosome studies in human sperm nuclei using fluorescence in-situ hybridization (FISH). Hum. Reprod. Update, 3, 441–452.[Abstract/Free Full Text]

Finkelstein, S., Mukamel, E., Yavetz, H. et al. (1998) Increased rate of nondisjunction in sex cells derived from low-quality semen. Hum. Genet., 102, 129–137.[ISI][Medline]

Fugger, E.F., Black, S.H., Keyvanfar, K. et al. (1998) Births of normal daughters after MicroSort sperm separation and intrauterine insemination, in-vitro fertilization, or intracytoplasmic sperm injection. Hum. Reprod., 13, 2367–2370.[Abstract]

Griffin, D.K., Abruzzo, M.A., Millie, E.A. et al. (1996) Sex ratio and disomic sperm: evidence that the extra chromosome 21 preferentially segregates with the Y chromosome. Am. J. Hum. Genet., 59, 1108–1113.[ISI][Medline]

Guttenbach, M., Martínez-Expósito, M.J., Michelmann, H.V. et al. (1997) Incidence of diploid sperm and disomic sperm nuclei in 45 infertile men. Hum. Reprod., 12, 468–473.[ISI][Medline]

Han, T.H., Ford, J.H., Flaherty, S.P. et al. (1994) A fluorescent in situ hybridization analysis of the chromosome constitution of ejaculated sperm in a 47,XYY male. Clin. Genet., 45, 67–70.[ISI][Medline]

In't Veld, P.A., Broekmans, F., de France, H. et al. (1997) Intracytoplasmic sperm injection (ICSI) and chromosomally abnormal spermatozoa. Hum. Reprod., 12, 752–754.[Abstract]

Johnson, L.A. (1991) Sex preselection in swine: altered sex ratios in offsprings following surgical insemination of flow-sorted X- and Y-bearing sperm. Reprod. Dom. Anim., 26, 309–314.[ISI]

Johnson, L.A. (1992) Gender preselection in domestic animals using flow cytometrically sorted sperm. J. Anim. Sci., 70, 8–18.

Johnson, L.A. and Schulman, J.D. (1994) Human sperm–sex selection. The safety of sperm selection by flow cytometry. Hum. Reprod., 9, 758–759.

Johnson, L.A., Welch, G.R., Keyvanfar, K. et al. (1993) Gender preselection in humans? Flow cytometric separation of X and Y spermatozoa for the prevention of X-linked diseases. Hum. Reprod., 8, 1733–1739.[Abstract]

Levinson, G., Keyvanfar, K., Wu, J.C. et al. (1995) DNA-based X-enriched sperm separation as an adjunct to preimplantation genetic testing for the prevention of X-linked disease. Mol. Hum. Reprod. 1, see Hum. Reprod., 10, 979–982.

McInnes, B., Rademaker, A. and Martin, R. (1998) Donor age and the frequency of disomy for chromosomes 1, 13, 21 and structural abnormalities in human spermatozoa using multicolour fluorescence in-situ hybridization. Hum. Reprod., 13, 2489–2494.[Abstract]

Mendelsohn, M.L., Mayall, B.H., Bogart, E. et al. (1972) DNA content and DNA-based centromeric index of the 24 human chromosomes. Science, 179, 1126–1129.[ISI]

Mercier, S. and Brenson, J.L. (1997) Analysis of chromosomal equipment in spermatozoa of a 46,XY/47,XY/+8 male by means of multicolour fluorescent in situ hybridization: confirmation of a mosaicism and evaluation of risk for offspring. Hum. Genet., 99, 42–46.[ISI][Medline]

Miharu, N., Best, R.G. and Young, S.R. (1994) Numerical chromosome abnormalities in spermatozoa of fertile and infertile men detected by fluorescence in situ hybridization. Hum. Genet., 93, 502–506.[ISI][Medline]

Moosani, N., Pattinson, H.A., Carter, M.D. et al. (1995) Chromosomal analysis of sperm from men with idiopathic infertility using sperm karyotyping and fluorescence in situ hybridization. Fertil. Steril., 64, 811–817.[ISI][Medline]

Morton, N.E. (1991) Parameters of the human genome. Proc. Natl Acad. Sci. USA, 88, 7474–7476.[Abstract]

Pang, M.G., Zackowski, J.L., Hoegerman, S.F. et al. (1995) Detection by fluorescence in situ hybridization of chromosome 7, 11, 12, 18, X and Y abnormalities from oligoasthenoteratozoospermic patients of an in vitro fertilization programme. J. Assist. Reprod. Genet., 12 (Suppl.), 53S.

Pieters, M.H., Speed, R.M., de Boer, P. et al. (1998) Evidence of disturbed meiosis in a man referred for intracytoplasmic sperm injection. Lancet, 351, 957.

Rath, D., Johnson, L.A., Dobrinski, J.R. et al. (1996) Birth of piglets following in vitro fertilisation using sperm flow cytometrically sorted for gender. Theriogenology, 45, 256.

Rousseaux, S., Chevret, E., Monteil, M. et al. (1995) Sperm nuclei analysis of a Robertsonian t(14q21q) carrier, by FISH, using three plasmids and two YAC probes. Hum. Genet., 96, 655–660.[ISI][Medline]

Spriggs, E.L., Rademaker, A.W. and Martin, R.H. (1996) Aneuploidy in human sperm: the use of multicolor FISH to test various theories of non-disjunction. Am. J. Hum. Genet., 58, 356–362.[ISI][Medline]

Van Hummelen, P., Manchester, D., Lowe, X. et al. (1997) Meiotic segregation, recombination, and gamete aneuploidy assessed in a t(1;10)(p22.1;q22.3) reciprocal translocation carrier by three- and four probe multicolour FISH in sperm. Am. J. Hum. Genet., 61, 651–659.[ISI][Medline]

Vidal, F., Moragas, M., Català, V. et al. (1993) Sephadex filtration and human serum albumin gradients do not select spermatozoa by sex chromosome: a fluorescent in-situ hybridization study. Hum. Reprod., 8, 1740–1743.[Abstract]

Vidal, F., Fugger, E.F., Blanco, J. et al. (1998). Efficiency of MicroSort flow cytometry for producing sperm populations enriched in X- or Y-chromosome haplotypes: a blind trial assessed by double and triple colour fluorescent in-situ hybridization. Hum. Reprod., 13, 308–312.[Medline]

Submitted on April 12, 1999; accepted on September 10, 1999.