1 Centre for Early Human Development, Monash Institute of Reproduction and Development, Monash University, Clayton, Victoria and 2 Monash IVF, Melbourne, Australia
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Abstract |
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Key words: aneuploidy/DNA fingerprinting/embryonic blastomeres/single cell PCR
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Introduction |
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To improve outcomes of IVF patients with a poor prognosis for pregnancy due to advanced maternal age (>35 years), a history of unexplained recurrent miscarriages and repeated failed IVF (more than three cycles), fluorescent in-situ hybridization (FISH) has been performed to identify euploidy in oocyte polar bodies or embryonic blastomeres using up to nine different chromosomal probes on one fixed nucleus (Gianaroli et al., 1997a,b
; Magli et al., 1998
; Munné et al., 1998
; Verlinsky et al., 1998
). FISH analysis of embryos from these patient groups consistently reveals aneuploidy and mosaicism rates of up to 50% (Munné et al., 1994
; Harper et al., 1995
; Kuo et al., 1998
; Magli et al., 1998
; Verlinsky et al., 1998
; Gianaroli et al., 1999
; Bielanska et al., 2000
). In most clinical preimplantation genetic diagnosis (PGD) programmes, the selection of euploid embryos for transfer has resulted in higher implantation and ongoing pregnancy rates for poor prognosis patients, in particular for women of advanced maternal age (Magli et al., 1998
; Damario et al., 1999
; Giarnaroli et al., 1999; Rubio et al., 2000
). More recently, efforts have focused on development of new methods to determine the numeracy of all 23 pairs of human chromosomes, including comparative genomic hybridization (CGH) (Wells and Delhanty, 2000
; Vouillaire et al., 2000
), spectral karyotyping (Marquez et al., 1998
) and nuclear conversion (Verlinsky and Evsikov, 1999
). However, any added clinical benefit of these methods over FISH has yet to be assessed in a large randomized trial.
Conventional and fluorescent polymerase chain reaction (FL-PCR) incorporating various microsatellite markers (Mansfield, 1993; Muggleton-Harris et al., 1993
; Pickering et al., 1994
; Pickering and Muggleton-Harris, 1995
; Pertl et al., 1994
) has been investigated for potential application in PGD. It has been shown that single cell multiplex FL-PCR can simultaneously provide information on multiple loci including identity of sex, an individual DNA fingerprint and genetic status for inherited conditions such as cystic fibrosis (Findlay et al., 1995
; Findlay and Quirke, 1996
). FL-PCR DNA fingerprinting using chromosome-specific microsatellite markers has also been used in prenatal diagnosis for the detection of chromosomal aneuploidies (Mansfield, 1993
; Pertl et al., 1994
, 1996
) and on single cells (Sherlock et al., 1998
; Findlay et al., 1999
). Theoretically, the amount of DNA produced in FL-PCR amplification is proportional to the quantity of the initial target sequence when strict experimental conditions are adhered to, and thus allelic ratios for any particular locus can be calculated from the final fluorescent yield (Ferre, 1992
; Wells and Sherlock, 1998
). Accordingly, disomy can be defined by an allelic ratio of 1:1, whereas a trisomy can either be defined as a tri-allelic pattern with an allelic ratio of 1:1:1 or a double dosage di-allelic pattern with an allelic ratio of 2:1. Previously published DNA fingerprinting systems developed for single cells, where the target DNA is in the order of 6 pg, have been plagued with several problems, including high rates of either preferential allelic amplification (PA) or allelic drop-out (ADO) (Sherlock et al., 1998
; Findlay et al., 1998
, 1999
). ADO is defined as the total amplification failure of one allele at a heterozygous locus so that only one allele is detectable after the analysis of the PCR product, whereas PA is the under-representation of one of the two heterozygous alleles resulting in a distortion from the expected 1:1 allelic ratio. The effect of ADO, PA or both reduces the degree of reliability for quantitation of FL-PCR products at the single cell level.
A reliable single cell PCR DNA fingerprinting system would be a very powerful tool in PGD for unique identification of a DNA sample and a means to simultaneously detect specific gene defects and chromosomal aneuploidy. No other current technique has the potential for such a multitude of diagnoses. To this end, we developed a new single cell PCR DNA fingerprinting system based on multiplex FL-PCR amplification of five highly polymorphic microsatellite markers located on four different chromosomes and evaluated its performance on both buccal cells and blastomeres from cleavage stage IVF embryos.
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Materials and methods |
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Embryo biopsy and FISH
Couples on the PGD programme at Monash IVF underwent standard IVF treatment including ovulation induction, surgical aspiration of the oocytes and sperm collection. Oocytes were fertilized by ICSI. On day 3 of embryonic development, cleavage stage embryos with 58 cells were considered suitable for biopsy. Embryos were incubated in Ca2+/Mg2+ free medium prior to zona drilling using acid Tyrode's solution and one or two cells were biopsied.
Isolation of human blastomeres from aneuploid embryos
Aneuploid embryos diagnosed by FISH at day 3 are regarded as genetically abnormal. Nine aneuploid embryos were obtained from patients with either advanced maternal age (>36 years) or repeated IVF failure (>3 cycles) who had a history of infertility. Under guidelines established by the Infertility Treatment Authority in Victoria, aneuploid embryos deemed to be unsuitable for transfer must be left to `succumb' on the bench for 24 h before being available for research. Succumbed embryos were treated with pronase (2 mg/ml in HEPES buffered human tubal fluid culture medium) for 1 min to dissolve the zona pellucida and transferred into Ca2+/Mg2+-free medium to dissociate the blastomeres. Single blastomeres were carefully washed through three 5 µl drops of PBS buffer and transferred together with 12 µl of PBS buffer into a sterile 0.2 ml PCR tube on ice.
Microsatellite markers
Five microsatellite markers were used in the pentaplex single cell DNA fingerprinting: D21S1413 (Findlay et al., 1998), D18S51 (Straub et al., 1993
), D13S258 (Toth et al., 1998
), D13S631 (Sherlock et al., 1998
) and DXS8377 (Hu et al., 1996
). Each microsatellite marker was selected for high heterozygosity (average 0.91). Based on available published allelic size ranges, appropriate fluorochrome tags (6-FAM, HEX and TET) were selected, where possible, to avoid overlapping profiles. Primers were synthesized and fluorescently labelled by Applied Biosystems, Australia. All primer pairs were diluted in molecular biology grade H2O (Sigma, Melbourne, Australia) to 200 pmol/µl stock solutions under sterile conditions and stored in aliquots of 100 pmol/µl at 20°C until use. For development of an octaplex single cell DNA fingerprinting system, primers for an additional three microsatellite markers were added to the pentaplex system.
Single cell multiplex FL-PCR
The optimized single cell multiplex FL-PCR developed for the five microsatellite markers consisted of the following: 2.5 µl of 10xTaq PCR Buffer (500 mmol/l KCl, 100 mmol/l TrisHCl, pH 9.0 and 15 mmol/l MgCl2), 0.5 µl of 10 mmol/l dNTP (200 µmol/l), 0.3 µl of Taq polymerase (5 U/µl) (Amersham Pharmacia Biotech, Sydney, Australia), 11.20 µl molecular biology grade H2O and 10.5 µl of primer mix making a final volume of 25 µl. Multiplex FL-PCR was performed using a Hotstart on the 9700 Thermocycler PCR machine (Applied Biosystems). Reactions were subjected to 35 thermal cycles consisting of denaturation for 45 s at 94°C, annealing for 45 s at 60°C, and extension for 1 min at 72°C. With each single cell multiplex FL-PCR, positive and negative controls were always included to ensure that the PCR reaction mix was functional and none of the reagents were contaminated. Positive control tubes contained 1020 cells in 12 µl of PBS buffer, whereas negative control tubes contained either 12 µl of PBS buffer from the last wash droplet and no cell.
Genescan analysis of DNA fingerprints
All PCR products were analysed using the ABI Prism 377 DNA Sequencer and associated Genescan 672 software (Applied Biosystems). PCR product (0.51.0 µl) was mixed with 1.54 µl of formamide, 0.15 µl loading buffer and 0.31 µl of Genescan TAMRA internal standard (Applied Biosystems). Samples were denatured at 95°C for 3 min, placed on ice and 2.5 µl loaded into the pre-formed wells of a 6% denaturing polyacrylamide gel. Samples were electrophoresed in 1xTris/borate/EDTA (TBE) buffer for 3.5 h at 3000 V and fragments automatically sized by Genescan software using the internal standard and a local Southern sizing alogrithm. Fluorescent product yield was calculated from integration of the peak area. Genescan profiles were generated showing the PCR products as coloured peaks dependent on the fluorescent dye used: TET (green), HEX (black) and 6-FAM (blue).
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Results |
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DNA fingerprinting of blastomeres from aneuploid embryos
To determine if this system could also reliably and accurately DNA fingerprint embryonic cells, blastomeres from aneuploid embryos identified by FISH were subjected to PCR DNA fingerprinting. Nine slow developing aneuploid embryos were obtained, and following dissociation using pronase, a total of 21 blastomeres were isolated intact and transferred into PCR tubes (some additional blastomeres lysed or fragmented during dissociation). From all embryos, at least two sister blastomeres were available for PCR DNA fingerprinting. The aneuploid status of these embryos determined by independent FISH analysis (Table I) was unknown prior to fingerprinting analysis. Sixteen of the 21 blastomeres produced informative DNA fingerprints (amplification of at least three microsatelite markers) with 13 of the 16 informative DNA fingerprints being fully comprehensive (amplification of all five microsatellite markers). The remaining five blastomeres produced unacceptable DNA fingerprints (amplification of two or less microsatellite markers). Due to the aneuploid status of these embryos, calculating the number of possible allelic amplifications would be misleading. Consequently, the total number of microsatellite marker amplifications was employed to establish the rate of reliability. Using these five microsatellite markers on 21 blastomeres, a total of 81 microsatellite marker amplifications were observed out of a possible 105 (Table I
), establishing a 77% reliability rate. There were five incidences of ADO (6.5%) and five incidences of PA (6.5%). Based on the percentage of correct microsatellite marker amplifications (taking into account the incidence of ADO), an overall accuracy rate for this pentaplex DNA fingerprinting system on blastomeres was calculated at 94%.
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Discussion |
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A diploidy status at any particular locus was determined by the presence of two alleles with an expected allelic ratio of 1:1, a monosomy by the presence of only one allele and a trisomy by the presence of three alleles with the expected ratio of 1:1:1 (tri-allelic) or two alleles with an expected allelic ratio of 2:1 (double dosage di-allelic). There were five incidences of double dosage di-allelic patterns observed in the DNA fingerprints of blastomeres in this study. Even though it is not possible to differentiate between double dosage di-allelic and PA, this single cell DNA fingerprinting system displayed a PA rate of only 6.5% and therefore it was more than likely that a third copy of the chromosome was present. There were also five incidences of discordance between sister blastomeres (Table I). This could either be explained by embryonic mosaicism resulting from mitotic cell division errors, or by the occurrence of PA at some of these loci, distorting the allelic ratios. Overall, due to the possibility of parental homozygosity, ADO and PA, it is essential that three or more microsatellite markers per chromosome under analysis are included in a single cell DNA fingerprinting system for confident aneuploidy detection.
PCR DNA fingerprinting has many unique advantages including confirmation of parental allelic contribution to the embryo, the identification of extraneous DNA contamination that could cause a misdiagnosis and detection of uniparental disomy in an embryo if parental DNA fingerprints were available. The newly developed pentaplex DNA fingerprinting system has high discriminating power for identification with a 1 in 630 probability that any two siblings will share an identical DNA fingerprint. Using this pentaplex DNA fingerprinting system it was possible to separate each sibling embryo in the cohorts by their unique allelic fingerprints. Thus, this system could potentially be used to track IVF embryos from the time of transfer to term and identify the viable embryo that produced the pregnancy in a multiple transfer. It is also possible to combine mutation detection for PGD for single gene disorders with the amplification of microsatellite markers for aneuploidy detection (Blake et al., 1999). Many couples presenting for PGD for single gene disorders have either experienced the birth of an affected child or undergone prenatal testing resulting in termination of pregnancy or several pregnancies. Hence, these women are usually of advanced maternal age and have a greater chance of producing embryos with chromosomal aneuploidies. It would be most unfortunate if a single gene disorder-free pregnancy resulted in a chromosomally aneuploid fetus, for example Down's syndrome. Consequently, it would be possible to combine a pentaplex chromosome 21 specific DNA fingerprinting system with mutation detection and offer simultaneous mutation and chromosome 21 screening for couples of advanced maternal age presenting primarily for PGD of a single gene disorder.
The incorporation of additional microsatellite markers on clinically relevant chromosomes to this pentaplex DNA fingerprinting system is a feasible prospect and is currently under development. To date, we have successfully added a further three microsatellite marker primers to produce an octaplex DNA fingerprinting system (Figure 4). Initial studies on 15 single buccal cells showed high reliability and accuracy (>90%) and low ADO and PA (<10%) and produced DNA fingerprints that were easily interpretable with virtually no background interference or non-specific amplification. This octaplex DNA fingerprinting system (two markers for chromosomes 13, 18, 21 and X) is therefore exceptionally robust. The physical limits of single cell DNA fingerprinting are unknown, although the further addition of four microsatellite markers could be possible in view of the successful development of this octaplex system. A more complex and comprehensive single cell PCR DNA fingerprinting system based on compatible microsatellite marker primers for clinically relevant chromosomes (three microsatellite markers per chromosome) could potentially offer PGD couples the possibility of a combined detection system for single gene disorders and chromosomal aneuploidy. The availability of more comprehensive fingerprinting systems would also allow a larger study to be undertaken to assess the origin, nature and incidence of chromosomal mosaicism in IVF embryos from different patient groups.
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Acknowledgements |
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Notes |
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References |
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Bielanska, M.M., Tan, S.L. and Ao, A. (2000) Significance of mosaicism in the human preimplantation embryo. Hum. Reprod., 15 (Abstract Book 1), 12.
Blake, D., Tan, S.L. and Ao, A. (1999) Assessment of multiplex fluorescent PCR for screening single cells for trisomy 21 and single gene defects. Mol. Hum. Reprod., 5, 11661175.
Boué, A. and Boué, J. (1976) Chromosomal anomalies in early spontaneous abortion (their consequences on early embryogenesis and invitro growth of embryonic cells). Curr. Top. Pathol., 62, 193208.[ISI][Medline]
Boué, A., Boué, J. and Grapp, A. (1985) Cytogenetics of pregnancy wastage. Adv. Hum. Genet., 14, 157.[ISI][Medline]
Cui, K. and Matthews, C.D. (1996) Nuclear structural conditions and PCR amplification in human preimplantation diagnosis. Mol. Hum. Reprod., 2, 6371.[Abstract]
Damario, M.A., Davis, O.K. and Rosenwaks, Z. (1999) The role of maternal age in the assisted reproductive technologies. Reprod. Med. Rev., 7, 4160.
Delhanty, J.D. and Handyside, A.H. (1995) The origin of genetic defects in the human and their detection in the preimplantation embryo. Hum. Reprod. Update, 1, 201215.[ISI][Medline]
Eiben, B., Bahr-Porsch, S., Borgmann, S. et al. (1990) Cytogenetic analysis of 750 spontaneous abortions with the direct-preparation method of chorionic villi and its implications for studying genetic causes of pregnancy wastage. Am. J. Hum. Genet., 47, 656663.[ISI][Medline]
Evsikov, S. and Verlinsky, Y. (1998) Mosaicism in the inner cell mass of the human blastocysts. Hum. Reprod., 13, 31513155.[Abstract]
Ferre, F. (1992) Quantitative or semi-quantitative PCR: reality versus myth. PCR Methods Applic., 2, 19.[Medline]
Findlay, I. and Quirke, P. (1996) Fluorscent polymerase chain reaction: Part 1. A new method allowing genetic diagnosos and DNA fingerprinting of single cells. Hum. Reprod. Update, 2, 137152.
Findlay, I., Urquhart, A., Quirke, P. et al. (1995) Simultaneous DNA `fingerprinting', diagnosis of sex and single-gene defect status from single cells. Mol. Hum. Reprod., 10, 10051013.
Findlay, I., Toth, T., Matthews, P. et al. (1998) Rapid trisomy diagnosis (21, 18 and13) using fluorescent PCR and short tandem repeats: applications for prenatal diagnosis and preimplantation genetic diagnosis. J. Assist. Reprod. Genet., 15, 266275.[ISI][Medline]
Findlay, I., Matthews, P. and Quirke, P. (1999) Preimplantation genetic diagnosis using fluorescent polymerase chain reaction: results and future developments. J. Assist. Reprod. Genet., 16, 199206.[ISI][Medline]
Gianaroli, L., Magli, M.C., Munné, S. et al. (1997a) Will preimplantation genetic diagnosis assist patients with a poor prognosis to achieve pregnancy? Hum. Reprod., 12, 17621767[Abstract]
Gianaroli, L., Magli, M.C., Ferraretti, A.P. et al. (1997b) Preimplantation genetic diagnosis increases the implantation rate in human in vitro fertilization by avoiding the transfer of chromosomally abnormal embryos. Fertil. Steril., 68, 11291131.
Gianaroli, L., Magli, M.C., Ferraretti, A.P. et al. (1999) Preimplantation diagnosis for aneuploidies in patients undergoing in vitro fertilization with a poor prognosis: identification of the categories for which it should be proposed. Fertil. Steril., 72, 837844.[ISI][Medline]
Harper, J.C., Coonen, E., Handyside, A.H. et al. (1995) Mosaicism of autosomes and sex chromosomes in morphologically normal monospermic preimplantation human embryos. Prenat. Diagn., 15, 4149.[ISI][Medline]
Hu, L.J., Laporte, J., Kioschis, P. et al. (1996) X-linked myotubular myopathy: refinement of the gene to a 280-kb region with new and highly informative microsatellite markers. Hum. Genet., 98, 178181.[ISI][Medline]
Kuo, H.C., Ogilvie, C.M. and Handyside, A.H. (1998) Chromosomal mosaicism in cleavage-stage human embryos and the accuracy of single-cell genetic analysis. J. Assist. Reprod. Genet., 15, 276280.[ISI][Medline]
Magli, M.C., Gianaroli, L., Munné, S. et al. (1998) Incidence of chromosomal abnormalities from a morphologically normal cohort of embryos in poor-prognosis patients. J. Assist. Reprod. Genet., 15, 297301.[ISI][Medline]
Magli, M.C., Jones, G.M., Gras, L. et al. (2000) Chromosome mosaicism in day 3 aneuploid embryos that develop to morphologically normal blastocysts in vitro. Hum. Reprod., 15, 101106.[Medline]
Mansfield, E.S. (1993) Diagnosis of Down syndrome and other aneuploidies using quantitative polymerase chain reaction and small tandem repeat polymorphisms. Hum. Mol. Genet., 2, 4350.[Abstract]
Marquez, J., Cohen, J. and Munné, S. (1998) Chromosome identification in human oocytes and polar bodies by spectral karyotyping. Cytogenet. Cell Genet., 81, 254258.[ISI][Medline]
Muggleton-Harris, A.L., Glazier, A.M. and Pickering, S.J. (1993) Biopsy of the human blastocyst and polymerase chain reaction (PCR) amplification of the beta-globin gene and a dinucleotide repeat motif from 26 trophectoderm cells. Hum. Reprod., 8, 21972205.[Abstract]
Munné, S., Lee, A., Rosenwaks, Z. et al. (1993) Diagnosis of major chromosome aneuploidies in human preimplantation embryos. Hum. Reprod., 8, 21852191.[Abstract]
Munné, S., Weier, H., Grifo, J. et al. (1994) Chromosome mosaicism in human embryos. Biol. Reprod., 51, 373379.[Abstract]
Munné, S., Sultan, K.M., Weier, H.U. et al. (1995) Assessment of numeric abnormalities of X, Y, 18 and 16 chromosomes in preimplantation human embryos before transfer. Am. J. Obstet. Gynecol., 172, 1191201.[ISI][Medline]
Munné, S., Bahce, M., Fung, J. et al. (1998) Preimplantation diagnosis of the aneuploidies most commonly found in spontaneous abortions and live births: XY, 13, 14, 15, 16, 18, 21, 22. Prenat. Diagn., 18, 14591466.[ISI][Medline]
Pertl, B., Yau, S.C., Sherlock, J. et al. (1994) Rapid molecular method for prenatal detection of Down's syndrome. Lancet, 14, 11971198.
Pertl, B., Wietgasser, U., Kopp, S. et al. (1996) Rapid detection of trisomies 21 and 18 and sexing by quantitative fluorescent multiplex PCR. Hum Genet., 98, 5559.[ISI][Medline]
Pickering, S.J., McConnell, J.M., Johnson, M.H. et al. (1994) Use of a polymorphic dinucleotide repeat sequence to detect non-blastomeric contamination of the polymerase chain reaction in biopsy samples for preimplantation diagnosis. Hum. Reprod., 9, 15391545.[Abstract]
Pickering, S.J. and Muggleton-Harris, A.L. (1995) Reliability and accuracy of polymerase chain reaction amplification of two unique target sequences from biopsies of cleavage stage and blastocyst stage human embryos. Hum. Reprod., 10, 10211029.[Abstract]
Ray, P.F., Ao, A., Taylor, D.M. et al. (1998) Assessment of the reliability of single blastomere analysis for preimplantation diagnosis of the F508 deletion causing cystic fibrosis in clinical practice. Prenat. Diagn., 18, 14021412.[ISI][Medline]
Rubio, C., Vidal, F., Minguez, Y. et al. (2000) High incidence of chromosomal abnormalities in preimplantation embryos from recurrent spntaneous abortion patients. Hum. Reprod., 15 (Abstract Book), 8182.[Medline]
Sandalinas, M., Sadowy, S., Calderon, G. et al. (2000) Survival of chromosome abnormalities to blastocyst stage. Hum. Reprod., 15 (Abstract Book 1), 11.
Sherlock, J., Cirigliano, V., Petrou, M. et al. (1998) Assessment of diagnostic quantitative fluorescent multiplex polymerase chain reaction assays performed on single cells. Ann. Hum. Genet., 62, 923.[ISI][Medline]
Straub, R.E., Speer, M.C., Luo, Y. et al. (1993) A microsatellite genetic linkage map of human chromosome 18. Genomics, 15, 4856.[ISI][Medline]
Toth, T., Findlay, I., Papp, C. et al. (1998) Prenatal detection of trisomy 13 from amniotic fluid by quantitative fluorescent polymerase chain reaction. Prenat. Diagn., 18, 669674.[ISI][Medline]
Verlinsky, Y. and Evsikov, S. (1999) A simplified and efficient method for obtaining metaphase chromosomes from individual human blastomeres. Fertil. Steril., 72, 11271133.[ISI][Medline]
Verlinsky, Y., Cieslak, J., Ivakhnenko, V. et al. (1998) Implantation diagnosis of common aneuploidies by the first and second-polar body FISH analysis. J. Assist. Reprod. Genet., 15, 285289.[ISI][Medline]
Vidal, F., Gimenez, C., Rubio, C. et al. (1998) FISH preimplantation diagnosis of chromosome aneuploidy in recurrent pregnancy wastage. J. Assist. Reprod. Genet., 15, 310313.[ISI][Medline]
Vouillaire, L., Slater, H., Williamson, R. et al. (2000) Chromosome analysis of blastomeres from human embryos by using comparative genomic hybridization. Hum. Genet., 106, 210217.[ISI][Medline]
Wells, D. and Delhanty, J.D. (2000) Comprehensive chromosomal analysis of human preimplantation embryos using whole genome amplification and single cell comparative genomic hybridization. Mol. Hum. Reprod., 6, 10551062.
Wells, D. and Sherlock, J.K. (1998) Strategies for preimplantation genetic diagnosis of single gene disorders by DNA amplification. Prenat. Diagn., 18, 13891401.[ISI][Medline]
accepted on October 24, 2001.