Sequential FISH analysis using competitive displacement of labelled peptide nucleic acid probes for eight chromosomes in human blastomeres

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1 Human Reproduction Vol.20, No.4 pp , 2005 Advance Access publication January 21, 2005 doi: /humrep/deh735 Sequential FISH analysis using competitive displacement of labelled peptide nucleic acid probes for eight chromosomes in human blastomeres I.E.Agerholm 1,7, S.Ziebe 2, B.Williams 3, C.Berg 4, D.G.Crüger 4, G.Bruun Petersen 4 and S.Kølvraa 5,6 1 The Fertility Clinic, Braedstrup Hospital, DK 8740 Braedstrup, 2 The Fertility Clinic, Rigshospitalet, DK 2100 Copenhagen, Denmark, 3 Applied Biosystem, 35 Wiggins Avenue, Bedford, MA 01730, USA, 4 Department of Clinical Genetics, Vejle Hospital, DK 7100 Vejle, 5 Institute of Human Genetics, University of Aarhus, DK 8000 Aarhus and 6 Department of Clinical Genetics, University Hospital of Aarhus, 8000 Aarhus C, Denmark 7 To whom correspondence should be addressed. iag@bs.vejleamt.dk BACKGROUND: The aim was to introduce a new strategy based on peptide nucleic acid (PNA) probes and competitive displacement for using fluorescence in-situ hybridization (FISH) analysis on human blatomeres. METHODS: Sequential FISH analysis with PNA probes and competitive displacement was performed using three different probe sets. The first set consisted of labelled probe only. The second and third sets included labelled as well as unlabelled probe, corresponding to the labelled probes in the previous cycles. The probes for enumeration were for chromosome 1, 13, 16, 17, 18, 21, X and Y. RESULTS: The performance of PNA probes was similar to the established DNA probes. The strategy of competitive displacement resulted in a destabilization of already bound probe before the next FISH cycle at only 50 8C, which allowed for up to five sequential FISH cycles without loss of signal. CONCLUSIONS: PNA probes are a good alternative to DNA probes in the present set-up, since the low temperature required both for binding and destabilization of PNA probes minimizes the loss of signal, and several FISH cycles can therefore be carried out before FISH errors occur. Key words: aneuploidy screening/blastomeres/competitive displacement/fish/pna probes Introduction The use of preimplantation genetic diagnosis (PGD) to screen embryos for aneuploidy is growing. Most protocols used to determine the chromosome content of a single blastomere are based on fluorescence in-situ hybridization (FISH) (Munne et al., 1993). The optimal method would be a FISH design that screens for the whole chromosome complement. Comparative genome hybridization (CGH) allows for all chromosomes to be analysed, and CGH has already been tried in a PGD setting (Wells and Delhanty, 2000; Voullaire et al., 2000; Wilton et al., 2001; 2003; Wells et al., 2002). However, the process is time consuming and therefore not optimal for analysis of embryos used for assisted reproduction. A more widely used FISH technique for aneuploidy screening is interphase FISH with chromosome-specific probes; however, this method has, in its simple form, the drawback that only a limited number of chromosomes can be analysed in one FISH procedure (Wilton, 2002). To compensate for this, it has been demonstrated that sequential FISH can be applied to blastomeres performing several rounds of FISH with different DNA probes (Benadiva et al., 1996; Gianaroli et al., 1999; Abdelhadi et al., 2003). Paulasova et al. (2004) have also demonstrated that FISH on blastomeres using peptide nucleic acid (PNA) probes are suitable for at least two cycles. One problem when performing repeated FISH cycles using DNA probes is the high temperatures needed to remove annealed probes after each cycle. The high temperature can compromise the integrity of the fixed DNA, and thus increases the risk of false results in subsequent cycles. In the present study we introduce a new strategy for screening human blastomeres. The strategy is based on sequential cycles of interphase FISH with chromosomespecific PNA probes instead of DNA probes, combined with competitive displacement of labelled probe. In PNA oligomers, the deoxyribose phosphate backbone of DNA oligomers is replaced by amino ethylglycerine (Nielsen et al.,1991). The resulting oligomers retain base-specific hybridization and have advantage over regular DNA and RNA oligonucleotide probe in terms of stability and specificity for complementary nucleotide target sequences (Chen et al., 2000). Since the backbone is not charged there is no electrostatic repulsion when PNA hybridizes to its target nucleic acid 1072 q The Author Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.

2 Sequential FISH PNA probes in human blastomeres sequences, which gives a higher stability (Pellestor et al., 2004). An additional consequence of the deoxyribose backbone is that PNA oligonucleotide probes hybridize virtually independently of the salt concentration (Pellestor et al., 2004). Thus the melting temperature of PNA DNA duplex is barely affected by the low ionic strength, which means that low ionic strength hybridization conditions can be used, thus inhibiting reannealing of complementary genomic DNA strand (Taneja et al., 2001). Another important aspect in relation to PGD is the fast kinetics of the PNA hybridization, which means that more diagnostic tests can be performed in less time (Pellestor et al., 2004). In addition to the PNA probes, we introduce a new concept by applying competitive displacement of the bound and labelled probe by presence of unlabelled probe in the following FISH cycle. This means that the displacement of annealed probe between two FISH cycles can be accomplished at a reduced temperature. The aim of the study was to compare PNA probes with the commonly used cloned DNA probes for aneuploidy detection and to use PNA probes for enumeration of chromosomes in single blastomeres from human embryos. Additionally, we wished to use the concept of competitive displacement to lower the denaturation temperature necessary for dissociation of already bound probe from the previous cycle and thereby optimize the sequential FISH procedure to allow more FISH cycles to be performed before FISH errors occur. Materials and methods Lymphocytes Interphase nuclei and metaphase spreads were obtained from peripheral blood lymphocytes of individuals with normal karyotype. From each individual standard lymphocyte metaphase slides were generated as described by Crüger et al. (1996). Patients Patients in our regular IVF program donated the surplus embryos, which had then been cultured for 48 h under standard conditions in four-well dishes (Nunc, Roskilde, Denmark) in Universal IVF medium (MediCult, Jyllinge, Denmark). After donation the embryos were cultured another 24 h before fixation. Blastomere fixation All nuclei from all blastomeres in the donated embryos were fixed individually. The fixations were performed as described in Coonen et al. (1994). Briefly, the embryos were incubated in pronase (5 mg/ml) (Sigma, St Louis, MO, USA) until the zona pellucida was dissolved, and then placed in Ca 2þ,Mg 2þ -free medium (Biopsymedium, MediCult) until the blastomeres were separated. All nuclei were then fixed on poly-l-lysine-coated glass slides (Menzel-Glaser, Braunschweig, Germany) using 0.01 mol/l HCL þ 0.1 % Tween 20. The location of the nuclei was registered and marked by a diamond objective and the slides were dehydrated in phosphate-buffered saline (PBS) followed by 70% 90% 100% ethanol series, and stored at 220 8C until the FISH analysis was performed. Before FISH analysis the slides were incubated with 0.1 mg/ml RNase solution for 30 min at 37 8C, washed in 2 SSC and incubated with 0.005% pepsin for 3 min at 37 8C. Post-fixation was carried out in a formaldehyde solution for 2 min at room temperature and the slides were rinsed in PBS and dehydrated through an ethanol series. Probes for FISH PNA probes. PNA probe mixtures specific for chromosomes 1, 16, 17, 18, X and Y were obtained from Applied Biosystems (Bedford, USA), together with a PNA probe mixture with specificity towards both chromosome 13 and chromosome 21. Each chromosome probe mixture is composed of one to several oligomers with sequence complementarity to chromosome-specific sequences of the relevant alpha monomer. The probe mixtures were available both as labelled and as unlabelled mixtures, and the concept of competitive displacement means that the second and third probe sets used in this study were composed of a combination of labelled probes for the chromosomes analysed in the cycle and unlabelled probes for the chromosomes in the previous cycle. As a consequence the different probe sets must be applied in a fixed order when performing sequential FISH. Probe set A had labelled probes for chromosomes 13 and 21 and no unlabelled probe. Probe set B had labelled probes for chromosome 1, 16 and 17 and unlabelled probes for chromosomes 13 and 21. Probe set C had labelled probes for chromosome 18, X and Y and unlabelled probes for chromosome 1, 16 and 17. The fluorochrome labelling, number of oligomers and concentration of the various probes and the composition of the probe mixtures are shown in Table I. DNA probes. DNA probes were all supplied from Vysis (Abbott Laboratories, Abbott Park, IL, USA) (chromosomes 1, 13, 16, 17, 18, 21, X and Y), except for the first study on lymphocytes and blastomere nuclei where the probes for chromosomes 16 and 17 were from Oncor (Q-biogene, SA, Illkirch, France). FISH procedures Basic PNA FISH. All the basic PNA FISH procedures were carried out at a denaturation temperature of 70 8C. The procedures were as described by Taneja et al. (2001). On slides containing fixed lymphocyte or blastomere nuclei, 2 ml of probe mixture consisting of 20 mmol/l Tris HCL (ph 7.5), 70% formamide (Invitrogen, Carlsbad, CA, USA), 1 Denhart s solution (USB, Cleveland, OH, USA), 10 mmol/l NaCl, 100 mg/ml trna (Sigma), 100 mg/ml salmon sperm DNA (Sigma) (ph ) and different concentrations of PNA oligomers (see Table I) was applied to the slide. After applying a coverslip the slide was denatured and subsequently hybridized under conditions as listed in Table II. Following the hybridization, the coverslip was removed and the slide washed in 50% formamide, 2 SCC as listed in Table II and then washed in 4 SCC/Tween 20 at RT. The slide was finally mounted and microscopically evaluated as described below. Microscopy and signal analysis After each FISH cycle the slides were stained with 4 0,6-diamidino- 2-phenylidole (DAPI) (Vysis DAPI II antifade) and mounted with coverslip. The microscope used for FISH analysis was an Axioplan 2 from Zeiss fitted with a Sensys CCD camera and software from Vysis. The filters used were Texas Red single, FITC single, Aqua single, FITC/Texas Red dual and DAPI/ FITC/Rhodamin triple from Vysis. In order for two signals to be scored as two we employed the criterion that the distance between the two signals should be at least equal to the diameter of the signals. 1073

3 I.E.Agerholm et al. Table I. Fluorochromes of the labelled probes and number of oligomers and concentration of probe for hybridization for both the labelled and unlabelled probes Probe set Labelled probes Unlabelled probes Probes for chromosomes Fluorochromes Number of oligomers Concentration of probe (nmol/l) Probes for chromosomes Number of oligomers Concentration of probe (nmol/l) A 13/21 FITC 9 20 B 1 Deac / Cy FITC 7 30 C 18 Deac X Cy Y FLU Table II. Denaturation, hybridization and washing time and temperature for the probe sets in basic and sequential PNA FISH Probe set Denaturation time (min) Denaturation temp. ( 8C) Basic FISH Sequential FISH PNA and DNA FISH on lymphocytes Both the PNA and the DNA FISH on lymphocytes was performed by scoring 100 nuclei from each of five individuals. The PNA FISH was carried out as described in Basic PNA FISH above. The procedure for the DNA probes was according to the protocol described by Vysis and Oncor. PNA and DNA FISH comparison on blastomere nuclei This procedure involved initial PNA FISH analysis as described above in the Basic PNA FISH section. Following inspection of the nuclei and registration of the number of PNA signals in the different colours, the coverslip was removed and FISH, with DNA probes from Vysis or Oncor, was subsequently performed according to the protocol described by the manufacturers. The DNA probes were in different colours from the PNA probes and the numbers of signals for both probe types were registered in 50 individual blastomere nuclei for each probe set, resulting in 150 nuclei analysed in total in this procedure. In an initial series it was ensured that the denaturation performed before DNA FISH removed all annealed PNA probe. Kinetics of the labelled PNA displacement in the presence of unlabelled PNA probe These experiments were carried out on lymphocyte nuclei and metaphases with either probe set A or B. The first PNA FISH analysis was performed as described above in the Basic PNA FISH section. After inspection and measurement of signals, a subsequent FISH cycle was performed where the slides were denatured at either 20, 30, 40, 50, 60 or 70 8C in the presence or absence of unlabelled probe with same composition as in the first FISH cycle. After the procedure nuclei and metaphases were re-inspected and signals measured again in order to estimate the amount of labelled probe from the first cycle that was displaced at each temperature. In this series the signal intensity were measured using the IPlab software (Scanalytics, Fairfax, VA, USA) Hybridization time (min) Hybridization temp. Washing time (min) A RT 3 42 B RT 3 42 C RT 4 44 RT ¼ room temperature. Washing temp. ( 8C) Sequential FISH with PNA probes at 55 8C As a consequence of the results from the kinetic experiments, the temperature used to dissociate labelled probe in the presence of unlabelled probe was set at 55 8C. Using this dissociation temperature it was investigated whether nuclei from blastomere could withstand up to five cycles without loss of signal. This was done as follows. A first FISH cycle using PNA probes was performed and the number of signals registered. Two additional cycles were then performed by repeating the entire procedure including denaturation, hybridization, washing, mounting, microscopy and removal of coverslip, but without probe in the hybridization mixtures. In the fourth cycle, FISH was done now using the same PNA probe set as in the first cycle, and signals were again registered. Finally, a fifth FISH cycle was performed with DNA probes as previously described, again followed by registration of signals. To simplify the evaluation the DNA probes used were in colours different from the PNA probes for the respective chromosomes. The five cycles were performed on 25 individual blastomere nuclei. Sequential FISH using DNA probes Sequential FISH with the PGD multicolour probe set from Vysis including probes for enumeration of chromosomes 13, 21, 18, X and Y was carried out according to the protocol described by Vysis, resulting in denaturation of the slides at 73 8C. The sequential FISH procedure was carried out as for the PNA probes, with a first cycle including probes followed by three cycles without probe in the hybridization mixture and finally a fifth cycle including probes for the five chromosomes again. The sequential FISH was performed on 29 blastomere nuclei. Ethical approval The ethics committee for Vejle and Fyns counties approved this study.

4 Sequential FISH PNA probes in human blastomeres Table III. FISH on nuclei from blastomeres Chromosomes 13/ X 100 Y 99 Same number of signals for PNA and DNA (%) The percentage represents the frequency of nuclei where DNA and PNA FISH probes gave the same number of signals. At least 50 nuclei were analysed for each probe set. Figure 1. Correlation between denaturation temperature and signal intensity in melting experiments with and without presence of unlabelled probe. The experiments shown here were performed with probe set A followed by probe set B. Results Comparison of PNA and DNA probes for aneuploidy screening Initially it was investigated on 500 diploid lymphocyte nuclei whether the PNA probes for chromosomes 1, 16, 17, 18 13/21, X and Y performed as well as DNA probes. It was evident in this series that the performance of the two types of probes was very similar (data not shown). The only drawback of the PNA probes was that the 13/21 probe set sometimes gave a slightly weaker signal on one of the chromosomes, probably as a result of a centromeric polymorphism. In the vast majority of cases, however, correct scoring was unproblematic. Also, a series of 50 nuclei from human blastomere was analysed for each probe set. Since we do not know the number of chromosomes in each nucleus a priori, the nuclei were subsequently hybridized with DNA probes, and the numbers of signals obtained with the two types of probes were then compared. In this series we found only minor differences in the number of signals between the PNA and the DNA probes (Table III). Effect of competitive displacement and denaturation temperature on the dissociation of labelled PNA probes For all probe sets, presence of unlabelled probe in the denaturation mixture increased the dissociation of bound, labelled probe at a range of temperatures. A typical example using the 13/21 probe set is shown in Figure 1. Although the individual curves varied somewhat, FISH signals were removed from all chromosomes at a denaturation temperature of 50 8C, when unlabelled probe was present. In a separate series it was ensured that denaturation at 55 8C with unlabelled probe did not have a negative effect on the subsequent FISH signals (Figure 2). Number of sequential FISH cycles that blastomere nuclei can withstand In a series of sequential FISH cycles we found no loss of signals after four cycles with PNA probe and a fifth cycle using DNA probes, when the denaturation temperature was 55 8C for the PNA cycles and 70 8C for the final DNA FISH cycle. The findings apply to all chromosomes, including chromosome 13/21 (Table IV). A parallel series of sequential FISH using DNA probes and denaturation temperature at 73 8C resulted in a loss of signal between the first and the fifth cycle ranging from 19% to 37% for the five different chromosomes analysed (data not shown). Discussion In the present study a new principle for FISH-based detection of a substantial number of chromosomes on single blastomere Figure 2. Result of three cycles using the probe sets listed in Table I on a male blastomere at a denaturation temperature of 55 8C. The figure clearly shows that signals from the previous cycle are not present, and that the annealing of probe in the next cycle is not compromised. (A) Chromosomes 13 and 21, green. (B) Chromosome 1, blue; chromosome 16, red; chromosome 17, green. (C) Chromosome 18, blue; chromosome X, red; chromosome Y, green. 1075

5 I.E.Agerholm et al. Table IV. Sequential FISH with four cycle using PNA probes and a fifth cycle using DNA probes Chromosome Fourth cycle, PNA Fifth cycle, DNA Number of signals relative to first cycle (%) Number of signals relative to first cycle (%) 21 0 þ þ1 13 þ X Y The number of signals in the first, fourth and fifth FISH cycle were registered and the number of signals found in the fourth and the fifth cycles were expressed relative to number of signals after first cycle. The percentage of nuclei with the same (0), one less (21) or one more (þ1) signal than in the first cycle are listed for all tested chromosomes. For each chromosome at least 25 nuclei were evaluated. nuclei is presented. The method is based on FISH with synthetic PNA oligomer mixtures. PNA oligomers have hybridization-related advantages over DNA oligomers which mean that aneuploidies can be detected by interphase FISH with synthetic PNA oligomers, which is difficult with synthetic DNA oligomers. In addition, a new concept using excess unlabelled synthetic PNA oligomers with the same composition in the following FISH cycle is introduced. In this context it is of importance that the probes used are synthetic, since it is then unproblematic to synthesize unlabelled oligomers of exactly the same composition as the labelled probe. In the set-up presented here we had access to PNA oligomer mixtures for only eight chromosomes, but our experiments clearly demonstrate that access to further PNA mixtures will make it possible to score single blastomeres for a much higher number of chromosomes. With the PNA oligomer mixtures available at present, however, we were able to demonstrate that the performance of PNA probes was similar to the established DNA probes both on lymphocytes nuclei and blastomere nuclei (Table III), and therefore PNA probes should be well suited for PGD purposes. Only the PNA probe mixture for chromosomes 13 and 21 had performance characteristics slightly inferior to the cloned DNA probes, namely in the form of rare occurrence of a weaker signal from one chromosome. Another problem with the alpha-satellite probes specific for chromosome 13 and 21 is that they share identical alphoid DNA sequences. Therefore, when the 13/21 PNA probe set is hybridized to blastomere nuclei, four signals are usually obtained, which in certain situations can give reduced accuracy in the scoring of spots compared with the scoring of 2 2 signals (Munne and Weier, 1996). Therefore, the scoring of chromosome 13 and 21 is probably slightly less reliable, but if this turns out to be a major problem, a precise scoring of chromosomes 13 and 21 can instead be achieved with a final cycle using cloned DNA probes specific for chromosomes 13 and 21. This strategy is realistic, since we found during the development of the method that four cycles of PNA FISH can be followed by a DNA FISH cycle with a very low error rate One of the benefits of using PNA FISH on blastomeres is that the melting characteristic of PNA DNA hybrids in the presence of unlabelled PNA is very favourable for sequential FISH. To examine the temperature needed for binding and displacement of PNA probes in the presence of unlabelled probe, FISH was initially performed on lymphocyte metaphases with either the first or the second probe set. Our experiments concerning the denaturation temperature necessary for destabilization of the already bound probe before the next FISH cycle thus demonstrated that unlabelled PNA probe does destabilize the base pairing and results in easy displacement of bound PNA probe from a previous cycle. The destabilization depends equally on the temperature and the presence of unlabelled probe with the same composition as the bound and labelled probe (Figure 1). In the presence of unlabelled probe the melting temperature was for all chromosomes around 50 8C, while without unlabelled probe in the following cycle the destabilization was already compromised at 60 8C. In a separate series we found that the displacement was not increased by higher concentrations of unlabelled probe in the displacement. Thus an increase in concentration of unlabelled probe from 5 to 100 nmol/l did not alter the temperature profiles of displacement (data not shown). The fact that a lower temperature is sufficient to remove bound PNA probes is important. Repeated FISH cycles are necessary for the enumeration of several chromosomes, and such a procedure will always require a denaturation step in each cycle. Since the increased temperature is damaging to nuclei and thereby results in FISH errors, it is important that these repeated denaturations can be performed at the lowest possible temperature. Paulasova et al. (2004) have presented a series of two cycles with PNA probes, but due to the fact that they did not use unlabelled probe they had to denature at 73 8C, thereby running a risk of damaging the nucleus. Our concept with presence of unlabelled probe in the following FISH cycle favours denaturation at lower temperature, thereby minimizing FISH errors at least up to four PNA FISH cycles followed by a DNA FISH cycle. Another advantage when using PNA probes for enumeration of human blastomeres is the time frame. Owing to the higher affinity of the DNA PNA duplex the hybridization can be performed at room temperature over a short period, e.g. 30 min. As a consequence of the fast hybridization kinetic of the PNA probes, five cycles of PNA FISH will require only 5 6 h, and the pre-embryos can be transferred at the same day as the biopsy is performed. This is important in the context of PGD, where the results from the preimplantation genetic analyses should be available as fast as possible. Furthermore, in order to make the use of PNA probes clinically relevant the cost of the procedure must be comparable to the cost of using probes that are already commercially available. In conclusion, PNA probes for FISH are well suited for enumeration of chromosomes in single blastomere nuclei and, on the assumption that PNA FISH probes are available for all chromosomes and with five possible cycles utilizing up to five fluorescence colours, this method could in time be developed to screen for all human chromosomes.

6 Sequential FISH PNA probes in human blastomeres Acknowledgements This work was supported by Serono Nordic, Danish Medical Council and Vejle Council. References Abdelhadi I, Colls P, Sandalinas M, Escudero T and Munne (2003) Preimplantation genetic diagnosis of numerical abnormalities for 13 chromosomes. RBM Online 6, Benadiva C, Kligman I and Munné S (1996) Aneuploidy 16 in human embryos increases significantly with maternal age. Fertil Steril 66, Chen C, Wu BL, Wei T, Egholm M and Strauss WM (2000) Unique chromosome identification and sequence-specific structural analysis with short PNA oligomers. Mamm Genome 11, Coonen E, Dumoulin CM, Ramaekers FCS and Hopman AHN (1994) Optimal preparation of preimplantation embryo interphase nuclei for analysis by fluorescence in-situ hybridization. Human Reprod 9, Crüger DG, Bruun-Petersen G and Kølvraa S (1996) Early prenatal diagnosis: standard cytogenetic analysis of coelomic cells obtained by coelocentesis. Prenat Diagn 16, Gianaroli L, Magli C, Ferraretti A and Munné S (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, Munne S and Weier H-UG (1996) Simultaneous enumeration of chromosomes 13, 18, 21, X and Y in interphase cells for preimplantation genetic diagnosis of aneuploidy. Cytogenet Cell Genet 75, Munne S, Lee A, Rosenwaks Z, Grifo J and Cohen J (1993) Diagnosis of major chromosome aneuploidies in human preimplantation embryos. Hum Reprod 8, Nielsen P, Egholm M, Berg RH and Buchardt O (1991) Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 254, Paulasova P, Andreo B, Diblik J, Macek M and Pellestor F (2004) The peptide nucleic acids as probes for chromosomal analysis: application to human oocytes, polar bodies and preimplantation embryos. Mol Hum Reprod 10, Pellestor F, Paulasova P, Macek M and Hamamah S (2004) The peptide nucleic acids: a new way for chromosomal investigation on isolated cells? Hum Reprod 19, Taneja KL, Chavez EA, Coull J and Lansdorp PM (2001) Multicolor fluorescence in situ hybridization with peptide nucleic acid probes for enumeration of specific chromosomes in human cells. Genes Chromosomes Cancer 30, Voullaire L, Slater H, Williamson R and Wilton L (2000) Chromosome analysis of blastomeres from human embryos by using comparative genomic hybridization. Hum Genet 106, Wells D and Delhanty JDA (2000) Comprehensive chromosomal analysis of human preimplantation embryos using whole genome amplification and single cell comparative hybridization. Mol Hum Reprod 6, Wells D, Escudero T, Lavy B, Hirschhorn K, Delhanty JDA and Munné S (2002) First clinical application of comparative genomic hybridization and polar body testing for preimplantation genetic diagnosis of aneuploidy. Fertil Steril 78, Wilton L (2002) Preimplantation genetic diagnosis for aneuploidy screening in early human embryos: a review. Prenat Diagn 22, Wilton L, Williamson B, Mc Bain J and Voullaire L (2001) Determination of aneuploidy in human embryos using comparative genomic hybridization. Fertil Steril 75 (Suppl 1),S6. Wilton L, Voullaire L, Sargeant P, Williamson R and McBain J (2003) Preimplantation aneuploidy screening using comparative genomic hybridization or fluorescence in situ hybridization of embryos from patients with recurrent implantation failure. Fertil Steril 80, Submitted on June 6, 2004; resubmitted on October 10, 2004; accepted on December 9,