Mol. Hum. Reprod. Advance Access originally published online on April 20, 2004
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Molecular Human Reproduction, Vol. 10, No. 6, pp. 467-472, 2004
© European Society of Human Reproduction and Embryology 2004
The peptide nucleic acids as probes for chromosomal analysis: application to human oocytes, polar bodies and preimplantation embryos
1Laboratory of Assisted Reproduction, Motol Hospital, Vuvalu 84, 150 06 Praha 5, Czech Republic and 2CNRS UPR 1142, Institut de Génétique Humaine, 141 rue de la Cardonille, F-34396 Montpellier Cedex 5, France
3 To whom correspondence should be addressed. e-mail: franck.pellestor{at}igh.cnrs.fr
 |
ABSTRACT |
|---|
 Top ABSTRACT IntroductionMaterials and methodsResultsDiscussionREFERENCES |
|---|
Peptide nucleic acids (PNA) are synthetic DNA mimics based on an uncharged polyamide backbone, which hybridize with complementary DNA with high affinity and specificity. PNA have recently become recognized as efficient tools for in situ chromosomal identification. In the present study, this new approach has been tried on isolated human oocytes, polar bodies and blastomeres. Using centromeric PNA probes specific for chromosomes 1, 4, 9, 16, X and Y, we tested multicolour labelling PNA reaction on 27 oocytes and 23 blastomeres. Sequential PNA hybridization was performed on five oocytes and combined PNA and fluorescence in situ hybridization (FISH) reactions on two oocytes. Both the rates and the types of abnormalities observed are in agreement with results from previous FISH studies. This preliminary study indicates that PNA probes allow a reliable chromosomal analysis in isolated human oocytes and blastomeres and consequently might provide an interesting adjunct to FISH for diagnostic analysis.
Key words: aneuploidy/blastomere/oocyte/peptide nucleic acids/polar body
|
Introduction |
|---|
|
TopABSTRACTIntroductionMaterials and methodsResultsDiscussionREFERENCES |
|---|
The advent of molecular cytogenetic techniques and their adaptation to in situ analysis of isolated cells have led to the development of preimplantation chromosomal analysis. The ability to investigate chromosomal content of preimplantation embryos has offered an important option to couples at risk of transmitting chromosomal abnormalities to their offspring (Handyside et al., 1998). The procedure has also been extended to the in situ analysis of mature oocytes and polar bodies prior to IVF (Munné et al., 1995; Dyban et al., 1996; Petit et al., 2000), in order to select chromosomally normal oocytes and then increase the efficiency of the in vitro reproductive procedure. To date, two techniques have been adapted to the chromosomal investigation of isolated cells, i.e. fluorescence in situ hybridization (FISH) and primed in situ labelling (PRINS) (Munné et al., 1993; Pellestor et al., 1996; Delhanty et al., 1997). They constitute two distinct procedures for in situ chromosomal identification and each presents advantages and disadvantages. Because of its relative simplicity and the commercial availability of numerous DNA probes, FISH has been adopted by most preimplantation genetic diagnosis (PGD) centres as the method of choice for chromosomal diagnosis. The FISH methodology has essentially been used for the sexing of embryos, the assessment of trisomy and the detection of unbalanced segregation of translocations (Scriven et al., 1998; Harper and Wells, 1999).
A new technical approach has been recently introduced in cytogenetics, based on the use of peptide nucleic acids (PNA) as probes for the in situ detection of human chromosomes. PNA constitute a new class of synthetic DNA mimics in which the deoxyribose phosphate backbone supporting the nucleic acid bases is replaced by a non-charged peptide backbone, thus conferring more stability and affinity to the PNA probes than to the DNA probes (Nielsen et al., 1991). The efficiency of PNA probes has been demonstrated in the studies of telomeric and centromeric repeat sequences (Lansdorp et al., 1996; Chen et al., 1999), and more recently for the specific in situ identification of human chromosomes on lymphocytes, amniocytes and sperm (Taneja et al., 2001; Pellestor et al., 2003). Because PNA probes present advantages in terms of efficiency and specificity, we have conducted a study to test this new type of probe on isolated human oocytes, polar bodies and blastomeres, in order to assess the possibility of using it for preimplantation diagnosis of aneuploidy. Simple and sequential multicolour PNA labelling procedures were experimented on both human oocytes and blastomeres. PNA probes were also tested in combination with DNA probes. In the present study, we describe these new procedures and report preliminary results of the chromosomal screening performed on 34 oocytes and 23 blastomeres.
|
Materials and methods |
|---|
|
TopABSTRACTIntroductionMaterials and methodsResultsDiscussionREFERENCES |
|---|
Sample preparation
The present research programme was approved by the local Research Ethics Committee at the Motol Hospital. Metaphase chromosomes were freshly prepared from peripheral blood lymphocytes by standard cytogenetic methods, fixed in methanol:glacial acetic acid (3:1) and spread on cleaned slides (Verma,R.S. and Babu,H., 1995). Slides were passed through an ethanol series (70%, 90%, 100%), 2 min each step, and air-dried. Slides were then stored at room temperature until use for PNA labelling reactions.
Human oocytes were obtained from patients participating in the IVF programme. All women involved in this study were counselled about the procedure and gave consent for their unfertilized oocytes to be donated for this analysis. The oocytes used in the present study were those that had failed to fertilize when examined 16–18 h after being incubated with sperm. These oocytes were maintained in culture for a further 24 h. After this additional culture, oocytes were observed in detail for the presence of pronuclei and polar bodies, using a phase contrast microscope. Oocytes showing no fertilization were prepared for chromosomal analysis. Fixation was performed according to the air-drying method of Tarkowski (1966). After fixation and air-drying, the slides were scanned under a phase contrast microscope. The presence of metaphase spreads and polar bodies, their quality and position on the slide were noted. This assisted in locating and photographing the material under the fluorescence microscope. Slides were then stored at room temperature.
Embryos used in the present study were donated embryos, obtained after informed consent from patients participating in IVF programmes. Embryos that had abnormal development (i.e. with unequally sized blastomeres, degenerate blastomeres, fragmentation) and were not involved in IVF procedure, were used for the present study. Embryos were biopsied on day 3. The zona pellucida was opened by laser, and two to four blastomeres were aspirated by micropipette and fixed individually on clean glass slides following the protocol of Harper et al. (1994). After fixation and microscopic scanning, slides were kept at room temperature.
PNA probes
PNA probes specific for chromosomes 1, 4, 9, 16, 18, X and Y were kindly provided by Applied Biosystems (formerly Boston Probes Inc., USA). Each probe consists of a mixture of several short synthetic sequences (18–22 base units) specific for the centromeric repeat sequence of the targeted chromosomes. The probes specific for chromosomes 4, 9 and X were labelled with fluorescein. The probes specific for chromosomes 16, 18 and Y were labelled with rhodamine and the chromosome 1-specific probe was labelled in blue with diethylaminocoumarine. The PNA probes were supplied ready to use in hybridization buffer.
PNA hybridization and post-hybridization washes
Before the PNA hybridization procedure, the fixed oocytes were denaturated by immersion in 70% formamide, 2xstandard saline citrate (SSC) at 73°C for 3 min and then dehydrated in a series of ice-cold ethanol washes (70%, 90%, 100%) before being allowed to air-dry. Probe aliquots of 3 µl were mixed and denaturated for 6 min in a waterbath pre-heated to 73°C. The probe solution was applied to the preparation slide, covered with a 22x22 mm coverslip and placed in a humidified hybridization chamber 60 min at 37°C.
For lymphocyte and blastomere preparations, both formamide and heat denaturation were tested. In heat denaturation, the PNA hybridization mixture was added to the slide under a coverslip, sealed with rubber cement, and the preparation and PNA probes were denaturated simultaneously at 75°C for 3 min and left to hybridize at 37°C for 60 min in a moist chamber. After hybridization, the coverslips were removed by washing of slides in 1xphosphate-buffered saline (PBS), 0.1% Tween 20 for 2 min. The slides were then transferred to 58°C pre-warmed 1xPBS, 0.1% Tween 20 for 10 min and rinsed in 2xSSC, 0.1% Tween 20 for 1 min. The excess fluid was drained from the slides, which were then mounted in Vectashield antifade solution (Vector Laboratories, USA) containing 4,6-diamidino-2-phenylindole (DAPI) (0.3 µg/ml).
For sequential PNA hybridization on oocyte preparations, a first round of hybridization was performed with a mixture of PNA probes specific for chromosomes 1, 4 and 16. Following hybridization as described above, the coverslip was removed and the slide was washed for 3 min in 0.4xSSC at 72°C, and then in 2xSSC–0.05% Tween 20 at room temperature. The slide was counterstained with DAPI mounted in Vectashield antifade solution, and examined under an epifluorescence microscope equipped with specific filters. After recording of the results of the first labelling round, the slide was washed twice in 2xSSC–0.05% Tween 20 for 2x15 min at room temperature in order to remove immersion oil, coverslip and antifade solution. The slide was briefly rinsed in 1xPBS and distilled water before being dehydrated in an ethanol series and incubated for 2.5 min at 73°C in denaturation solution. This short denaturation step allowed removal of previously hybridized probes and gently denaturating of chromosomes. Then the second round of PNA hybridization was performed as described for the first round, using PNA probes for chromosomes 9 and 18. After hybridization, slides were washed as described above, counterstained with DAPI mounted in Vectashield antifade solution, and observed under the epifluorescence microscope.
FISH hybridization
In three embryos, the FISH technique was utilized as a control. In parallel with the PNA hybridizations performed on blastomeres, FISH assays were performed on one blastomere from each embryo, using satellite Vysis probes targeting the same chromosomes as the PNA probes. The FISH procedure was carried out according to the manufacturer’s protocol for hybridization and washing.
Combined PNA and FISH assay
In two oocyte spreads, the two labelling techniques were associated. To proceed, after independent denaturation, a 5 µl PNA probe mixture specific for chromosome 9 was combined with a 5 µl Vysis hybridization mixture for chromosome 16 labelling (CEP 16 satellite II Spectrum Orange). This mixture was applied to the slides, sealed under a coverslip with rubber cement and the slides were incubated at 37°C for 60 min in a humidified box. After hybridization, the slides were washed twice 5 min in 2xSSC, 0.1% Tween 20 at room temperature, counterstained with DAPI and mounted in Vectashield antifade solution.
Microscopic analysis and scoring criteria
Slides were analysed using a Leica DMRB microscope (Leica SARL, France), equipped with a DAPI single band-pass filter (filter A, no. 513804), a fluorescein isothiocyanate (FITC) single band-pass filter (filter I3, no. 513808), a tetramethylrhodamine B isothiocyanate (TRITC) single band-pass filter (filter N2.1, no. 513812), a FITC/TRITC double band-pass filter (filter G/R, no. 513803), and a triple filter (filter B/G/R, no. 513836) for simultaneous observation of DAPI/coumarine, FITC and TRITC signals.
Previously described scoring criteria were used for the interpretation of probe labelling in chromosome spreads and interphasic nuclei (Munné et al., 1994; Anahory et al., 2003). A clearly uniform and distinct spot was considered to be one single signal for one chromosome. On chromosome spreads from oocytes and polar bodies, two homologues from one univalent were considered as separated when their centromeric fluorescent signals were separated by more than the diameter of two centromeric signals. On blastomeres, two signals represented two homologous chromosomes when their distances apart were at least two centromeric signal diameters. A double-splitted signal was considered as one signal for one chromosome when two adjacent signals, smaller than the diameter of the other homologous signal, had no more than one relevant diameter in between the two.
|
Results |
|---|
|
TopABSTRACTIntroductionMaterials and methodsResultsDiscussionREFERENCES |
|---|
PNA probes for chromosomes 1, 4, 9 16, 18, X and Y were used in various combinations for double and triple in situ labelling reactions and sequential labelling procedures. Specificity and intensity of PNA probes were first evaluated on both metaphases and interphase nuclei. Specific localizations of the signals for each probe on each relevant chromosome were visualized. The PNA efficiency was tested by the recording of clearly visible fluorescent signals in 100 lymphocyte nuclei. According to the probes, the efficiency rate ranged from 96 to 100%. The resulting fluorescent signals were bright and well distinct. In double and triple colour experiments, the signals were always easily accountable without cross-hybridization or significant background (Figure 1A).
|
PNA assays on human oocytes were performed on a sample of 34 isolated oocytes (Table I). In 27 of them, simple multicolour PNA assays were done, and a sequential PNA procedure was experimented in five oocytes, by combining two rounds of chromosome labelling (Figure 1E, F). Two other oocytes were utilized to test the combined PNA and FISH protocol, with the PNA probe for chromosome 9 and the FISH probe for chromosome 16 (Figure 1G).
|
In 17 cases (14 in the group of simple PNA assay, two in the group of sequential PNA reaction, and one in a combined PNA–FISH reaction), corresponding polar bodies were obtained through the fixation procedure. Twenty-six oocytes (76.5%) displayed a normal pattern of signal according to the probes used (Figure 1B). These data were confirmed by a similar labelling in 16 corresponding polar bodies (Figure 1C, G). In one case (P13, oocyte no. 2), a discordance between labelling in metaphase II and polar body was observed: signals for chromosomes 4 and 18 were absent in the polar body whereas the associated oocyte metaphase showed distinct spots for these two chromosomes. This discrepancy was blamed on the low quality of the polar body material, which hindered efficient hybridization of probes. Eight oocytes (23.5%) displayed numerical abnormalities for the targeted chromosomes. One case of whole chromosome hyperhaploidy was observed for chromosome 16. The non-disjunction was confirmed by the lack of this chromosome in the corresponding first polar body. Four other cells displayed supernumerary single chromatids, for chromosomes 9 (two cases), 16 and 18 respectively. Balanced chromatid separation was observed in three oocytes, involving chromosomes 4, 16 and X (Figure 1D). Although the implication of balanced chromatid separation in the formation of aneuploidy was controversial, we chose to distinguish these events and involve them in the description of abnormal chromosomal constitution because of the significant separation of the two homologous chromatids in the three cases.
Twenty-seven blastomeres from 10 embryos were processed for PNA experiments (Table II). Four nuclei (two from embryo E5, one from embryo E7 and one from embryo E8) were lost during the fixation procedure. Consequently, successful PNA experiments were achieved on 23 nuclei. In three cases (E8, E9, E10), the FISH technique was used as control in one blastomere.
|
Satisfactory results were obtained with a hybridization timing of PNA probes similar to that for oocytes (i.e. 60 min). The quality of the in situ labelling, in terms of intensity and specificity, remained excellent (Figure 2A–D). Based on the combinations of PNA probes used, three types of chromosome patterns were observed, i.e. diploidy, aneuploidy and mosaicism. Only three embryos (E4, E7 and E10) appeared to be diploid for the chromosomes tested (Figure 2A). Three others displayed single aneuploidy: embryo E1 was found to be trisomic for chromosome 9 in the two nuclei, embryo E3 showed an extra chromosome 16, and embryo E8 displayed a monosomy for chromosome 16, confirmed by the FISH labelling of one nucleus. Embryo E5 was aneuploid for two chromosomes, with an additional chromosome 18 and a missing X chromosome. Embryos E2 and E6 displayed diploid/aneuploid mosaic pattern (Figure 2B–D). In embryo E9, an unbalanced labelling pattern was also observed in the two nuclei analysed by PNA and FISH respectively. The nucleus analysed by PNA showed monosomy 1 whereas the nucleus labelled by FISH exhibited a more complex constitution with trisomy 1 and trisomy 18. This unbalanced pattern could reflect a chaotic chromosomal constitution.
|
|
Discussion |
|---|
|
TopABSTRACTIntroductionMaterials and methodsResultsDiscussionREFERENCES |
|---|
The present study describes the first use of PNA on isolated human oocytes and blastomeres for in situ chromosomal identification. PNA oligomers already function efficiently in many molecular genetic methods (Nielsen, 2001), but their introduction in cytogenetics is relatively recent. PNA technology was first used for telomere in situ detection in cancer and ageing research, using consensus telomeric probes (Lansdorp et al., 1996; Rufer et al., 1998; Boei et al., 2000). Subsequently, PNA has proven successful for in situ identification of several human chromosomes and detection of numerical abnormalities on lymphocytes, fibroblasts, amniocytes and sperm, using specific satellite PNA probes (Chen et al., 2000; Taneja et al., 2001; Pellestor et al., 2003). The present results indicate that PNA probes can also be used for chromosomal screening on human oocytes and blastomeres. However, our study was restricted by the difficulty of obtaining sufficient numbers of oocytes and embryos, and the difficulty of performing experiments in two separate laboratories, while taking into consideration the constraints of an IVF programme.
To validate the efficiency of the PNA procedure, we used the FISH technique as control in three labelling experiments. The FISH assays confirmed the results obtained with PNA. Of course, no technique can guarantee a total labelling efficiency. To date, in FISH studies of human oocytes, the rate of FISH errors has been estimated at 10% (Dailey et al., 1996; Munné et al., 1996), and a review of the various FISH-based procedures applied on human oocytes and blastomeres (CGH, M-FISH, multilocus FISH) shows that all these approaches present pitfalls limiting their efficiency. The results obtained with the PNA method in our small series indicate that PNA probes are at least as efficient as FISH probes. A confirmation of this efficiency is provided by the confirmatory results obtained in polar bodies, since only one case of discordance was observed between signals in metaphase II oocytes and first polar bodies.
To evaluate the suitability of the PNA technique, we have tested various procedures using PNA probes, such as a sequential labelling procedure or in combination with FISH labelling. The sequential labelling procedure constitutes a simple and economical approach for scoring several chromosomes. The elementary hybridization protocol of PNA probes lends itself well to this type of procedure, and the PNA binding does not lose its efficiency. Using the described intermediate washing procedure, no significant residual signals from previous hybridization were seen, and the background signals remained low, probably because of the high binding specificity of the PNA probes which limited the illegitimate binding of probes, and the short time of in situ hybridization.
Also, the possibility of combining PNA and FISH on a same preparation opens up interesting possibilities for multiplex assays, with larger combinations of fluorochromes. The PNA are compatible with a wide range of reporter molecules and fluorochromes. The present data describe the use of PNA combined with the most currently used fluorochromes (fluorescein, rhodamine and coumarine), but the procedure of PNA synthesis allows labelling of the PNA molecules with other fluorochromes such as Cyanine and Alexa dyes, available in a wide variety of colours. Because the introduction of PNA into cytogenetics is recent, the commercial availability of PNA probes is still limited to consensus telomeric and human-specific satellite probes, and their prices remain 20% more expensive than the FISH probes. One may hope that the success of the first generation of centromeric PNA probes will stimulate the future production of an extended variety of PNA probes and a decrease in their cost. Custom-made PNA molecules for unique sequences can already be obtained from manufacturers.
Both rates and types of abnormalities scored in oocytes and blastomeres are in good agreement with the data of previous FISH studies. The incidence of abnormalities observed in our oocyte sample is within the range of the aneuploid rates currently found in FISH studies (Dailey et al., 1996; Mahmood et al., 2000; Pujol et al., 2003), with the identification of chromosome non-disjunction, extra single chromatid and balanced chromatid separation (Table I). In the same way, PNA assays revealed the occurrence of frequent chromosomal abnormalities, such as aneuploidy and mosaicism, in preimplantation embryos, in accordance with data from other molecular cytogenetic analyses (Delhanty et al., 1997; Marquez et al., 2002).
The PNA technology possesses undeniable advantages for in situ recognition of complementary DNA sequences. The unique chemical make-up of these probes confers numerous beneficial properties, including enhanced hybridization rate, in situ resistance to nucleases and proteases and high stability over a wide pH range. Hybridization of DNA probes to DNA is destabilized by electrostatic repulsion between the negatively charged phosphate backbones of complementary strands. On the contrary, the neutral backbone of PNA oligomers provides very strong binding between PNA and DNA strands. Unlike DNA probes, which require high salt concentration to bind, PNA probes can bind to DNA targets under low ionic strength conditions which disfavour reannealing of complementary strands. This advantage is particularly interesting for targeting repetitive sequences, since both their length and their repetitive nature facilitates in situ renaturation over hybridization with probes. These properties mean that it is not necessary to design long PNA probes. PNA oligomers from 15 to 25 units, like those used in the present study, constitute efficient probes for fast in situ detection of specific DNA sequences, without generating significant background.
The present study indicates the feasibility of using PNA probes on isolated cells such as human oocytes and blastomeres. Because of the simplicity and the rapidity of the PNA procedure, these preliminary results shows that PNA provides a new and interesting approach for basic chromosomal analysis, particularly when chromosome identification must be performed on limited amounts of material and in a limited time-period, such as in preimplantation diagnosis. Further studies are needed to validate this approach and assess its limitations before clinical application can be considered. We hope that this preliminary presentation of the emergent PNA technology will encourage larger scale follow-up studies. One can predict that in situ PNA methodology is going to make significant progress, and if future work demonstrates the accuracy of PNA, the procedure may act as a valuable complement to FISH for in situ cytogenetic investigations.
|
Acknowledgements |
|---|
Supported by a European grant COPERNICUS 2 (Contract ICA-CT-2000-10012, proposal ICA2-1999-20007).
|
REFERENCES |
|---|
|
TopABSTRACTIntroductionMaterials and methodsResultsDiscussionREFERENCES |
|---|
Anahory T, Andréo B, Régnier-Vigouroux G, Soulié JP, Baudouin M, Demaille J and Pellestor F (2003) Sequential multiple probe fluorescence in-situ hybridisation analysis of human oocytes and polar bodies by combining centromeric labelling and whole chromosome painting. Mol Hum Reprod 9,577–585.
Boei JJWA, Vermeulen S and Natarajan AT (2000) Analysis of radiation-induced chromosomal aberrations using telomeric and centromeric PNA probes. Int J Rad Biol 76,136–167.
Chen C, Hong YK, Ontiveros SD, Egholm M and Strauss WM (1999) Single base discrimination of CENP-B repeats on mouse and human Chromosomes with PNA-FISH. Mamm Genome 10,13–18.[CrossRef][ISI][Medline]
Chen C, Wu B, Wei T, Egholm M and Strauss WM (2000) Unique chromosome identification and sequence-specific structural analysis with short PNA oligomers. Mamm Genome 11,384–391.[CrossRef][ISI][Medline]
Dailey T, Dale B, Cohen J and Munné S (1996) Association between nondisjunction and maternal age in meiosis-II human oocytes. Am J Hum Genet 59,176–184.[ISI][Medline]
Delhanty JD, Harper JC, Ao A, Handyside AH and Winston RM (1997) Multicolour FISH detects frequent chromosomal mosaicism and chaotic division in normal preimplantation embryos from fertile patients. Hum Genet 99,755–760.[CrossRef][ISI][Medline]
Dyban A, Freidine M, Severova E, Cieslak J, Ivakhnenko V and Verlinsky Y (1996) Detection of aneuploidy in human oocytes and corresponding first polar bodies by fluorescent in situ hybridization. J Assist Reprod Genet 13,73–78.[CrossRef][ISI][Medline]
Handyside AH, Scriven PN and Ogilvie CM (1998) The future of preimplantation genetic diagnosis. Hum Reprod 13(Suppl. 4),249–255.
Harper JC and Wells D (1999) Recent advances and future developments in PGD. Prenat Diagn 19,1193–1199.[CrossRef][ISI][Medline]
Harper JC, Coonen E, Ramaekers FC, Delhanty JD, Handyside AH, Winston RM and Hopman AH (1994) Identification of the sex of human preimplantation embryos in two hours using an improved spreading method and fluorescent in-situ hybridization (FISH) using directly labelled probes. Hum Reprod 9,721–724.
Lansdorp PM, Verwoerd NP, van de Rijke FM, Dragowska V, Little MT, Dirks RW, Raap AK and Tanke HJ (1996) Heterogeneity in telomere length of human chromosomes. Hum Mol Genet 5,685–691.
Mahmood R, Brierley CH, Faed MJ, Mills JA and Delhanty JD (2000) Mechanisms of maternal aneuploidy: FISH analysis of oocytes and polar bodies in patients undergoing assisted conception. Hum Genet 106,620–626.[CrossRef][ISI][Medline]
Marquez C, Sandalinas M, Hahçe M, Alikani M and Munné S (2002) Chromosome abnormlaities in 1255 cleavage-stage human embryos. RBMOnline 1,17–26.
Munné S and Weier HUG (1996) Simultanous enumeration of chromosomes 13, 18, 21, X and Y in interphase cells for preimplantation genetic diagnosis of aneuploidy. Cytogenet Cell Genet 75,263–270.[ISI][Medline]
Munné S, Lee A, Rosenwaks Z, Grifo J and Cohen J (1993) Diagnosis of major chromosome aneuploidies in human preimplantation embryos. Hum Reprod 8,2185–2191.
Munné S, Grifo J, Cohen J and Weier H-UG (1994) Chromosome abnormalities in arrested human preimplantation embryos: a multiple probe fluorescence in situ hybridization (FISH) study. Am J Hum Genet 55,150–159.[ISI][Medline]
Munné S, Dailey T, Sultan KM, Grifo J and Cohen J (1995) The use of first polar bodies for preimplantation diagnosis of aneuploidy. Hum Reprod 10,1014–20.
Nielsen PE (2001) Peptide nucleic acid: a versatile tool in genetic diagnostics and molecular biology. Curr Opin Biotechnol 12,16–20.[CrossRef][ISI][Medline]
Nielsen PE, Egholm M, Berg RH and Buchardt O (1991) Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 254,1497–1500.
Pellestor F, Girardet A, Coignet L andréo B and Charlieu JP (1996) Assessment of aneuploidy for chromosomes 8, 9, 13, 16 and 21 in human sperm by using primed in situ labelling technique. Am J Hum Genet 58,797–802.[ISI][Medline]
Pellestor F, Andréo B, Taneja K and Williams B (2003) PNA on human sperm: a new approach for in situ aneuploidy estimation. Eur J Hum Genet 11,337–341.[CrossRef][ISI][Medline]
Petit C, Martel-Petit V, Fleurentin A, Monnier-Barbarino P, Jonveaux P and Gérard H (2000) Use of PRINS for preconception screening of polar bodies for common aneuploidies. Prenat Diagn 20,1067–1071.[CrossRef][ISI][Medline]
Pujol A, Boiso I, Benet J, Veiga A, Durban M, Campillo M, Egozcue J and Navarro J (2003) Analysis of nine chromosome probes in first polar bodies and metaphase II oocytes for the detection of aneuploidies. Eur J Hum Genet 11,325–336.[CrossRef][ISI][Medline]
Rufer N, Dragowska W, Thornbury G, Roosnek E and Lansdorp PM (1998) Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry. Nat Biotechnol 16,743–747.[CrossRef][ISI][Medline]
Scriven PN, Handyside AH and Ogilvie CM (1998) Chromosome translocations: segregation modes and strategies for preimplantation genetic diagnosis. Prenat Diagn 18,1437–1449.[CrossRef][ISI][Medline]
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 30,57–63.[CrossRef][ISI][Medline]
Tarkowski A (1966) An air-drying method for chromosome preparation from mouse eggs. Cytogenetics 5,394–400.[ISI]
Verma RS and Babu H (1995) Human Chromosomes. Principles and Techniques. 2nd Edition, McGraw-Hill Inc, New York, USA.
Submitted on February 2, 2004; resubmitted on February 18, 2004; accepted on February 25, 2004.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  I.E. Agerholm, S. Ziebe, B. Williams, C. Berg, D.G. Cruger, G. B. Petersen, and S. Kolvraa Sequential FISH analysis using competitive displacement of labelled peptide nucleic acid probes for eight chromosomes in human blastomeres Hum. Reprod., April 1, 2005; 20(4): 1072 - 1077. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Pellestor, P. Paulasova, M. Macek, and S. Hamamah The Use of Peptide Nucleic Acids for In Situ Identification of Human Chromosomes J. Histochem. Cytochem., March 1, 2005; 53(3): 395 - 400. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Pellestor, T. Anahory, and S. Hamamah The chromosomal analysis of human oocytes. An overview of established procedures Hum. Reprod. Update, January 1, 2005; 11(1): 15 - 32. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||