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. Email: iag{at}bs.vejleamt.dk
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Abstract |
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Key words: aneuploidy screening/blastomeres/competitive displacement/FISH/PNA probes
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Introduction |
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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 chromosome-specific 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 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 PNADNA 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.
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Materials and methods |
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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 Ca2+,Mg2+-free medium (Biopsy-medium, 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 + 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 20 °C 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 °C, washed in 2x SSC and incubated with 0.005% pepsin for 3 min at 37 °C. 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.
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FISH procedures
Basic PNA FISH. All the basic PNA FISH procedures were carried out at a denaturation temperature of 70 °C. The procedures were as described by Taneja et al. (2001). On slides containing fixed lymphocyte or blastomere nuclei, 2 µl of probe mixture consisting of 20 mmol/l TrisHCL (pH 7.5), 70% formamide (Invitrogen, Carlsbad, CA, USA), 1x Denhart's solution (USB, Cleveland, OH, USA), 10 mmol/l NaCl, 100 µg/ml tRNA (Sigma), 100 µg/ml salmon sperm DNA (Sigma) (pH 7.07.5) 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, 2x SCC as listed in Table II and then washed in 4x SCC/Tween 20 at RT. The slide was finally mounted and microscopically evaluated as described below.
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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 °C 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).
Sequential FISH with PNA probes at 55 °C
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 °C. 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 °C. 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.
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Results |
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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).
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Discussion |
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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 x 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 PNADNA 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 °C, while without unlabelled probe in the following cycle the destabilization was already compromised at 60 °C. 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 °C, 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 DNAPNA 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 56 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.
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Acknowledgements |
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References |
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Submitted on June 6, 2004; resubmitted on October 10, 2004; accepted on December 9, 2004.
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