Molecular Human Reproduction, Vol. 8, No. 11, 1035-1041,
November 2002
© 2002 European Society of Human Reproduction and Embryology
Reproductive genetics |
Chromosome abnormalities identified by comparative genomic hybridization in embryos from women with repeated implantation failure
1 Murdoch Children’s Research Institute, Royal Children’s Hospital, Parkville, Victoria 3052 and 2 Melbourne IVF, 320 Victoria Parade, East Melbourne, Victoria 3002, Australia
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Abstract |
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Using comparative genomic hybridization, we have detected chromosome abnormality in 76/126 (60%) single blastomeres biopsied prior to implantation from embryos from 20 women with repeated implantation failure following IVF. The abnormalities detected included aneuploidy for one or two chromosomes [32/126 (25%)] and complex chromosomal abnormality [37/126 (29%)]. Most of the chromosomes involved in single aneuploidy were those commonly found in live births or spontaneously aborted fetuses, whereas a greater range of chromosomes were involved in double aneuploidy. In blastomeres with complex abnormality, random and extensive loss and gain of all the chromosomes was observed. Further blastomeres from 25 embryos with single or double aneuploidy and 11 embryos with complex abnormality were analysed following embryo disaggregation. The specific abnormality was confirmed in the majority of cases and in some cases could be assigned as errors in meiotic or mitotic segregation. Complex abnormalities, suggestive of errors in cell cycle regulation, were present in a slightly higher proportion of these embryos than were seen in our previously studied cohort of surplus embryos. The disruption of the normal sequence of chromosome replication and segregation in early human embryos, caused either by maternal cytoplasmic factors or mutations in cell cycle control genes, may be a common cause of repeated implantation failure.
chromosome abnormality/comparative genomic hybridization/human blastomeres/infertility/non-disjunction
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAppendixReferences |
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There is now considerable evidence that a major cause of implantation failure following both in-vivo and in-vitro fertilization is a high incidence of chromosome abnormality in early human embryos. A young fertile couple trying to conceive has a 20–25% chance of being successful per monthly cycle (Short, 1979
). The implantation rate after IVF, defined as the ratio of the number of fetal hearts detected to the number of cleavage stage embryos transferred, is somewhat lower, at 15–20%, even in experienced centres. Investigations using fluorescent in-situ hybridization (FISH) and chromosome-specific probes have shown that chromosome abnormalities in early human embryos can be derived either meiotically or post-zygotically (Harper et al., 1995).
Meiotically derived chromosome abnormality increases with advancing maternal age, leading to an increased risk of a liveborn aneuploid baby, an increased risk of miscarriage and a decreased probability of becoming pregnant following either in-vivo or in-vitro fertilization. Patients who have had three or more IVF cycles (or 
10 transfers) without becoming pregnant are regarded as having recurrent IVF failure and have a very low probability of achieving a pregnancy with further cycles. This suggests that there are factors affecting human fertility occurring at the level of embryo implantation. A high level of chromosome abnormality is present in arrested embryos (Munné et al., 1994; Almeida and Bolton, 1998), and also in morphologically normal high-grade embryos (Iwarsson et al., 1999). Repeated implantation failure is not only distressing for patients who require multiple cycles of treatment, but also adds greatly to the cost of the procedure.
A number of centres now perform preimplantation genetic diagnosis for chromosomal aneuploidy (PGD-AS) on single cells biopsied from cleavage stage embryos using FISH with chromosome-specific probes (Gianaroli et al., 1999; Munné et al., 1999; Kahraman et al., 2000). Screening embryos for chromosome abnormality has been implemented in an attempt to improve the implantation rate in poor prognosis groups by selecting embryos more likely to have a normal karyotype and hence a higher probability of implantation. The selection of chromosome-specific probes used in FISH analysis of preimplantation embryos has been based on their more frequent representation in meiotically derived aneuploidy in live births and in spontaneously aborted fetuses. Current FISH protocols utilize five to nine chromosome probes. Based on data from aborted fetuses and assuming that monosomy occurs as frequently as trisomy at conception, it has been predicted that PGD using FISH with nine chromosome-specific probes (13, 14, 15, 16, 21, 22, X and Y) would detect 70% of chromosomally abnormal conceptuses (Boue et al., 1985; Munné et al., 1998a). PGD-AS using FISH has been shown to be effective in increasing the pregnancy rate in women of advanced maternal age or with recurrent abortion (Munné et al., 1999; Kahraman et al., 2000). This confirms that in these two groups fertility is significantly reduced by the occurrence of chromosomal abnormality in the embryo. The use of PGD-AS, however, has only had a slight impact on increasing the pregnancy rate for women with repeated implantation failure (ESHRE PGD Consortium Steering Committee, 2002). Infertility amongst this group of patients is likely to be multi-factorial and it may be that chromosomal abnormality is not involved or that chromosomal abnormality does occur but does not principally involve meiotic aneuploidy or the chromosomes generally included in the FISH probe sets.
Comparative genomic hybridization (CGH) is a molecular cytogenetic technique that can be applied to single cells in interphase to allow simultaneous analysis of every chromosome (Voullaire et al., 1999; Wells et al., 1999). We have previously reported the application of CGH to single blastomeres from disaggregated human embryos (Voullaire et al., 2000). This study, and one other (Wells and Delhanty, 2000), provided evidence for a high level of chromosome abnormality involving all chromosomes and demonstrated meiotic and post-zygotic mis-segregation, and chromosome breakage.
We present here the chromosome abnormality detected in the application of CGH in a large series of preimplantation diagnoses of chromosomal aneuploidy (PGD-AS/CGH) using single blastomeres biopsied from 141 cleavage stage embryos. The 20 patients selected for this study had a history of repeated implantation failure following IVF. Chromosome abnormality which would be expected to lead to loss of viability was detected in the majority of embryos. As we have reported elsewhere, this has enabled the selection of embryos with a normal karyotype as suitable for transfer (Wilton et al., 2001). We also report our investigation of further blastomeres from 36 of the embryos diagnosed as abnormal. This has permitted an analysis of the nature of the errors that lead to abnormal karyotypes.
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Materials and methods |
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Patient selection and IVF procedure
All couples underwent counselling, which is mandatory in the State of Victoria prior to IVF. Women considering PGD attend an additional session with a genetics counsellor before consenting to embryo biopsy and preimplantation screening for aneuploidy by CGH as approved by our institutional review board. Twenty women, mean age 34 (range 26–41) years, elected to have CGH performed on some or all of their embryos. The women had been examined for other causes of infertility prior to commencing IVF and in none of these cases were significant confounding factors found. All women had a history of repeated implantation failure with
10 (mean 16) embryos transferred previously in multiple embryo transfers (maximum of three embryos per transfer) without achieving a pregnancy. Multiple ovarian follicular development was achieved by administration of exogenous gonadotrophins. After collection, oocytes were inseminated by ICSI and embryos were cultured in human tubal fluid (HTF; Irvine Scientific, Irvine, CA, USA). On day 2 after insemination, the morphology of the embryos was assessed and graded on the basis of cytoplasmic fragmentation (grade 1: <5 segments; grade 2: 5–10 segments; grade 3: 10–30 segments). Early on day 3 of development, the number of blastomeres per embryo was determined and morphology reassessed. Two to five hours later a single blastomere was biopsied from embryos that were grade 1–3 and contained at least six cells. Two 5-cell embryos from one patient were also biopsied. The full details of the clinical IVF procedures and embryo biopsy are described elsewhere (Wilton et al., 2001; L.Wilton et al., unpublished data).
Cryopreservation and thawing of biopsied embryos
Single cell CGH takes 4 days to complete, necessitating the freezing of the embryos for transfer at a later cycle. Initially biopsied embryos were cryopreserved and thawed using methods found to be successful for intact cleavage stage embryos (Edgar et al., 2000). Modifications to the method were implemented to improve the survival rate of biopsied embryos (Wilton et al., 2001).
Embryo disaggregation
Where patient permission had been given, embryos that had been diagnosed as abnormal by CGH were thawed for further analysis. This work was permissible in the State of Victoria because the embryos were allowed to succumb by being left at room temperature for 24 h before being examined. Permission to use embryos that had succumbed in this type of investigation was given by the Infertility Treatment Authority and the work was carried out with the knowledge of the Research and Ethics Committee of the Freemason’s Hospital. Embryos were briefly washed in acidified HTF to remove the zona pellucida and gently pipetted to separate the blastomeres. Individual blastomeres were placed into 5 µl of lysis solution (200 mmol/l KOH).
Comparative genomic hybridization
Blastomeres were analysed using CGH as previously described by us (Voullaire et al., 1999, 2000). Individual blastomeres underwent alkaline lysis followed by whole genome amplification using degenerative oligonucleotide primed (DOP)–PCR. The DOP–PCR product (test DNA) was labelled with Spectrum Green (Vysis Inc., Downers Grove, IL, USA) by nick translation. Genomic DNA extracted from lymphocytes from a normal male was amplified using DOP–PCR and the PCR product labelled with Spectrum Red (Vysis Inc., Downers Grove IL, USA) by nick translation to provide a normal reference DNA. Test and reference DNA were simultaneously hybridized to normal male metaphase template slides and the fluorescent images captured and analysed using Cytovision CGH software (Applied Imaging, Santa Clara, CA, USA) to determine the average green:red fluorescence ratio profile for each chromosome. When the relative DNA copy number in the test and reference DNA is the same, the green:red fluorescence ratio is 1.0. When the relative DNA copy number is less in the test DNA, the ratio is <1.0, and when it is in excess, the ratio is >1.0. Thresholds for significant deviation were set at 0.75 and 1.25.
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Results |
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Preimplantation diagnosis using CGH
A single blastomere was biopsied from a total of 141 embryos from 20 women with repeated implantation failure. Results were obtained from 126 (89%) of the embryos (Table I
, for complete data see Appendix I). Fifteen blastomeres failed to amplify including 11 blastomeres from one patient cycle which was apparently due to inhibition of the PCR. The four other blastomeres gave bands on PCR but not the characteristic smear and failed to show any signal on hybridization. All other cells gave analysable hybridization. A normal result was found in 50 (40%) of the blastomeres. Thirty-two (25%) of the blastomeres were found to have aneuploidy for one or two chromosomes.
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In 11 blastomeres, partial chromosomal imbalance was detected. Partial aneuploidy appears to result from post-zygotic chromosome breakage. It was associated with aneuploidy in two cells, with complex abnormality in at least two cells, and as the sole abnormality in seven cells. This is likely to be an under-detection of the occurrence of chromosome breakage leading to partial aneuploidy, as the resolution of the single cell CGH is estimated to be ~40 Mb (Voullaire et al., 1999
), so chromosome breakage resulting in smaller imbalances would not have been detected.
Thirty-seven (29%) blastomeres showed complex abnormality which we categorized as total chromosome imbalance involving at least three chromosomes. In some cells partial aneuploidy was also identified. Of the 37 blastomeres, seven had aneuploidy for three to six chromosomes where the monosomy or trisomy of specific chromosomes could be determined from the profile. In the majority of blastomeres with complex abnormality, the abnormality was extensive and could not be defined by the CGH profile (Figure 1b). The FISH images obtained from capture of the test and reference hybridizations showed that in many cells there was extensive abnormality including nullisomy, monosomy, disomy, trisomy and/or tetrasomy for various chromosomes in a single cell (Figure 1a). Each of the 24 chromosomes was observed to be involved in complex aneuploidy in at least one blastomere with complex abnormality.
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Follow-up of abnormal embryos
Further investigation was carried out of blastomeres from 36 embryos with an abnormal result on PGD-AS/CGH where patient permission had been given. Twenty-five of the embryos had single or double aneuploidy and 11 embryos had complex abnormality (Table II
). All embryos diagnosed with single or double aneuploidy, for which patient permission for further investigation had been given and that survived thawing, were investigated. In 12 embryos originally diagnosed with single or double aneuploidy, confirmation of the aneuploidy was found in the majority of analysable cells examined, suggesting a meiotically derived chromosome abnormality. This involved a single chromosome in nine cases and two chromosomes in three cases. A discrepancy between the PGD-AS/CGH finding and the follow-up result obtained from the study of other blastomeres in the embryo could be due to post-zygotic mitotic errors resulting in an embryo with chromosome mosaicism or due to an error in diagnosis. A discrepant result was obtained in 13 embryos originally diagnosed as being aneuploid for one or two chromosomes. In one case, the discrepancy was due to post-zygotic non-disjunction (case 9, embryo 1) and in five cases the embryos could be interpreted as mosaic as the specific abnormality could be confirmed in at least one further cell examined. In seven embryos, the specific abnormality could not be identified but the remaining cells showed complex abnormality or other abnormalities including partial aneuploidy of the same chromosome (case 10, embryo 5). The finding of different abnormalities in the investigation of further blastomeres from an embryo has been termed a type 2 error (Munné et al., 1998a). In one embryo (case 3, embryo 9) the remaining cells all showed a normal karyotype and this has been termed a type 1 error (Munné et al., 1998a).
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Eleven embryos diagnosed as having complex abnormality with PGD-AS/CGH were examined further. Confirmation of complex chromosomal abnormality was obtained in nine of the 11 cases. In one embryo (case 4, embryo 13) only normal cells were found and in another embryo (case 5, embryo 6), the remaining cells were normal or had a monosomy for chromosome 6. It is unlikely that complex abnormality would be misdiagnosed with CGH, so it can be assumed that these embryos are mosaic. Six embryos found on follow-up to have further cells with complex abnormality were also found to have some normal cells. This suggests that the formation of the complex abnormality is post-zygotic and not related to an aneuploid conception. In eight embryos originally diagnosed with single or double aneuploidies, complex abnormality was found in one to five other blastomeres in the embryo.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAppendixReferences |
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The data presented here describe the abnormalities found using CGH to obtain a complete karyotype in blastomeres from human embryos prior to implantation. The embryos investigated were from a cohort of women referred because of recurrent implantation failure following IVF. The results confirm the high frequency of chromosome abnormality found in pre-implantation embryos. The ability of CGH to identify all chromosomal aneuploidy minimizes the transfer of chromosomally abnormal embryos that are unlikely to establish a successful pregnancy. Our results show that 40% of the embryos could be considered suitable for transfer, a value similar to that found using FISH for PGD-AS (ESHRE PGD Consortium Steering Committee, 2002
).
Abnormalities found on PGD-AS/CGH included embryos with single and double aneuploidies, and complex abnormalities, as well as partial aneuploidy resulting from chromosome breakage. Assuming that all blastomeres with complex abnormality would be detected using FISH, we estimate that analysis using FISH and a five probe set for chromosomes 13, 18, 21, 16, 22 would have correctly diagnosed 77% of the blastomeres in total, but would have incorrectly diagnosed 38% of the abnormal embryos as normal. Using FISH and a nine probe set for chromosomes XY, 13, 14, 15, 16, 18, 21 and 22 (Munné et al. 1998b), 85% of the blastomeres would have been diagnosed correctly, but 25% of the abnormal embryos would have been diagnosed as normal.
Of the abnormal blastomeres detected on PGD-AS/CGH, 32 (42%) were aneuploid for one (22 embryos) or two (10 embryos) chromosomes. The distribution of chromosomes in single and double aneuploidy is given in Table III. Eleven different chromosomes were involved in aneuploidy in the embryos with single aneuploidy. In five of the 22 single aneuploidies, the chromosomes (4, 7, 9 and 20) were ones not commonly seen in live births or in spontaneously aborted fetuses (and hence not included in FISH probe sets). Double aneuploidy, on the other hand, involved 15 different chromosomes. In these cases, 12 out of the 20 aneuploidies involved chromosomes (1, 3, 4, 6, 8, 9, 10, 11 and 12) which are not commonly seen in live births or in spontaneously aborted fetuses. This different pattern of chromosome involvement is most probably due to single and double aneuploidy having a different likelihood of arising from meiotic or mitotic non-disjunction, and there being a non-random involvement of specific chromosomes in these events (Wolstenholme, 1996).
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There was a small excess (25:17) of chromosomes showing monosomy over those with trisomy. An excess of monosomy has also been observed in blastomeres diagnosed using FISH, and this was attributed to technical artefact including poor probe hybridization and overlapping signals and loss of micronuclei during spreading (Munné et al., 1998b
). Loss of micronuclei would not be expected with CGH, as the whole blastomere is transferred to the tube for DNA amplification. Monosomy, which generates a 1:2 ratio between the test DNA and the reference DNA, gives a more obvious profile shift in CGH than trisomy, which gives a 3:2 ratio. It should be noted that in some of our analyses chromosome 22 was excluded due to apparent artefactual amplification of test DNA. As aneuploidy for chromosome 22 is a relatively common meiotically derived abnormality, it is possible that some cases of trisomy 22 were missed. These factors may explain the small excess seen of monosomy compared with trisomy.
Chromosomal abnormality detected by CGH but not by PGDAS/FISH includes aneuploidy for chromosomes not frequently seen in aborted fetuses, and partial aneuploidy due to chromosome breakage which is likely to result in an unstable karyotype through the formation of acentric and dicentric chromosomes. Both these types of abnormality are likely to be lethal early in development, seen as implantation failure or early embryo failure. Partial aneuploidy resulting from chromosome breakage was found to be the sole abnormality in seven cells in this investigation, and, amongst the single and double aneuploidies, chromosomes other than those generally represented in the probe sets (i.e. XY, 13, 14, 15, 16, 18, 21 or 22) were involved in 13 of the 42 aneuploidies. If these abnormalities are more prevalent among women with repeated implantation failure, it would explain why PGD using FISH with five to nine probes has had only a minor impact in increasing the implantation rate in women who have experienced recurrent implantation failure after IVF (Gianaroli et al., 1999; Munné et al., 1999; ESHRE PGD Consortium Steering Committee, 2002). If, however, mosaicism is a significant cause of implantation failure in patients with recurrent implantation failure, then neither FISH nor CGH might have an effect on improving the implantation rate.
Differentiation of the patients into two groups on the basis of maternal age (Table I
) (i.e. <37 years of age or 37 years of age) showed that in both groups the level of complex abnormality detected by PGD-AS/CGH was similar whereas the level of aneuploidy was higher in the advanced maternal age group. The numbers are small but confirm the association of aneuploidy with increased maternal age and suggest that the incidence of complex abnormality is age independent in this cohort.
Follow-up analysis of abnormal embryos has shown that the specific abnormality could be identified in at least one cell in the embryo in 25/36 (69%) of cases (Table II). Of the 25 embryos diagnosed as aneuploid for one or two chromosomes, an abnormality resulting from meiotic non-disjunction, was found in 12 of which eight embryos were from the older maternal age group and four were from the younger group. This gives an incidence of meiotic aneuploidy in this cohort of 10% but with a higher incidence in the older women. This total incidence is low compared with that found in other studies of human preimplantation embryos using CGH (Voullaire et al., 2000; Wells and Delhanty, 2000) but may not be low compared with the meiotic aneuploidy rate derived from FISH investigations of polar bodies and oocytes and sperm (Munné et al., 1995; Mahmood et al., 2000; Vidal et al., 2001; Delhanty et al., 2002). However, the data do suggest that repeated implantation failure is not associated with an increased level of meiotic aneuploidy.
In addition to those chromosomes that are commonly investigated using FISH, we also found involvement of chromosomes 8, 9 and 11 in meiotic aneuploidy. In the 16 blastomeres with a single aneuploidy that were followed up, nine aneuploidies had an apparently meiotic origin and four had a mitotic origin. Of nine blastomeres with double aneuploidy that were followed up, seven of the 18 aneuploidies involved (4/9 embryos) had an apparently meiotic origin for the aneuploidy whereas three of the 18 aneuploidies had a mitotic origin.
One of the embryos (case 10, embryo 5) contained cells with partial and full aneuploidy for the same chromosome. Detection using single cell CGH of mosaicism for partial and total aneuploidy of the same chromosome has been observed previously (Voullaire et al., 2000; Wells and Delhanty, 2000). Chromosome breakage might be expected to lead to misdivision of both breakage products.
We found a high incidence of complex chromosomal abnormality where karyotypes displayed extensive loss and gain of chromosomes (Figure 1). We have previously described the pattern of complex abnormality observed using CGH analysis and have shown that it can occur in single cells from an embryo, or involve every blastomere (Voullaire et al., 2000). FISH analysis of embryos has identified chromosome aneuploidy that varies between cells in an embryo. This inconsistent pattern of embryo mosaicism has been termed ‘chaotic’ (Delhanty et al., 1997). CGH analysis allows a distinction to be made between an abnormality that involves one or a few chromosomes and an abnormality involving extensive disturbance to the karyotype. Complex abnormality involves the chromosomes randomly in nullisomy through to tetrasomy. Evidence for a clonal or reciprocal type segregation pattern is evident in only some embryos. The observations suggest that complex abnormality is a category of chromosomal abnormality associated with a general disturbance of replication and/or cell division.
In a previous study (Voullaire et al., 2000), the embryos selected for investigation were good quality embryos that were surplus to the requirements of couples many of whom had successfully achieved a pregnancy. In that investigation of 12 disaggregated embryos, only one embryo (8%) had complex abnormality in every cell and three embryos (25%) had complex abnormality in one or two cells, giving a total of 33% of embryos. In this cohort of women with implantation failure, 37 (29%) had complex abnormality at PGD-AS/CGH. Further investigation of embryos diagnosed as having single or double aneuploidy showed that 36% had complex abnormality in at least one cell (range 1–5 cells). If the development of complex abnormality in blastomeres is independent of the occurrence of meiotic aneuploidy, and the data presented here suggest that it may be, then it is possible that a similar incidence of complex abnormality occurs in the embryos diagnosed as normal at PGD-AS/CGH. While the presence of one or two cells with complex abnormality in an 8-cell embryo may not preclude viability, a higher level might be expected to lead to implantation failure. These findings also suggest that mosaicism involving complex abnormality is a frequent occurrence in these patients and that embryos diagnosed as normal at PGD-AS might have a high level of mosaicism that would result in implantation failure.
The data presented here suggest that complex abnormality occurs more frequently in the embryos of women with a history of recurrent implantation failure. The occurrence of complex abnormality may be a significant factor in the infertility of these couples, and the inability of these women to become pregnant even after IVF. Previous observations from FISH investigations of aneuploidy in human embryos (Delhanty et al., 1997; Harrison et al., 2000) have led to the suggestion that some individuals are more prone to chaotic embryos than others. This study supports that observation.
Our data confirm the high level of chromosome abnormality found in early human embryos that appear morphologically normal. In somatic cells the fidelity of chromosome segregation is dependent on the operation of mitotic checkpoints. These delay the transition from metaphase to anaphase until all chromosomes are fully aligned on the spindle. The complex abnormality seen in morphologically normal and actively dividing embryos supports the idea that mitotic checkpoints may not function in the early cleavage embryo (Delhanty and Handyside, 1995; Wells and Delhanty, 2000). The high level of complex abnormality seen in the embryos of this cohort (and particularly in some individuals) suggests that disturbance of the normal early embryonic cell cycle might be a pathology associated with infertility and implantation failure.
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Appendix |
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For Appendix, showing results obtained from preimplantation genetic diagnosis screening for aneuploidy (PGD–AS) using comparative genomic hybridization (CGH) of 141 blastomeres from 20 women, see the Oxford University Press supplementary data site or MCRI website.
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Notes |
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3 To whom correspondence should be addressed. E-mail: voullal{at}cryptic.rch.unimelb.edu.au
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References |
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Submitted on June 2, 2002; accepted on July 30, 2002.
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 A. Obradors, E. Fernandez, M. Oliver-Bonet, M. Rius, A. de la Fuente, D. Wells, J. Benet, and J. Navarro Birth of a healthy boy after a double factor PGD in a couple carrying a genetic disease and at risk for aneuploidy: Case Report Hum. Reprod., August 1, 2008; 23(8): 1949 - 1956. [Abstract] [Full Text] [PDF]
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D.D. Daphnis, E. Fragouli, K. Economou, S. Jerkovic, I.L. Craft, J.D.A. Delhanty, and J.C. Harper Analysis of the evolution of chromosome abnormalities in human embryos from Day 3 to 5 using CGH and FISH Mol. Hum. Reprod., February 1, 2008; 14(2): 117 - 125. [Abstract] [Full Text] [PDF]
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A. Mantzouratou, A. Mania, E. Fragouli, L. Xanthopoulou, S. Tashkandi, K. Fordham, D.M. Ranieri, A. Doshi, S. Nuttall, J.C. Harper, et al. Variable aneuploidy mechanisms in embryos from couples with poor reproductive histories undergoing preimplantation genetic screening Hum. Reprod., July 1, 2007; 22(7): 1844 - 1853. [Abstract] [Full Text] [PDF]
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E.J. Margalioth, A. Ben-Chetrit, M. Gal, and T. Eldar-Geva Investigation and treatment of repeated implantation failure following IVF-ET Hum. Reprod., December 1, 2006; 21(12): 3036 - 3043. [Abstract] [Full Text] [PDF]
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 C. Le Caignec, C. Spits, K. Sermon, M. De Rycke, B. Thienpont, S. Debrock, C. Staessen, Y. Moreau, J.-P. Fryns, A. Van Steirteghem, et al. Single-cell chromosomal imbalances detection by array CGH. Nucleic Acids Res., January 1, 2006; 34(9): e68 - e68. [Abstract] [Full Text] [PDF]
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E.B. Baart, E. Martini, I. van den Berg, N.S. Macklon, R-J.H. Galjaard, B.C.J.M. Fauser, and D. Van Opstal Preimplantation genetic screening reveals a high incidence of aneuploidy and mosaicism in embryos from young women undergoing IVF Hum. Reprod., January 1, 2006; 21(1): 223 - 233. [Abstract] [Full Text] [PDF]
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 F. M. Petit, N. Frydman, M. Benkhalifa, A. Le Du, A. Aboura, R. Fanchin, R. Frydman, and G. Tachdjian Could Sperm Aneuploidy Rate Determination Be Used as a Predictive Test Before Intracytoplasmic Sperm Injection? J Androl, March 1, 2005; 26(2): 235 - 241. [Abstract] [Full Text] [PDF]
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A.R. Thornhill, C.E. deDie-Smulders, J.P. Geraedts, J.C. Harper, G.L. Harton, S.A. Lavery, C. Moutou, M.D. Robinson, A.G. Schmutzler, P.N. Scriven, et al. ESHRE PGD Consortium 'Best practice guidelines for clinical preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS)' Hum. Reprod., January 1, 2005; 20(1): 35 - 48. [Abstract] [Full Text] [PDF]
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 L. Wilton Preimplantation genetic diagnosis and chromosome analysis of blastomeres using comparative genomic hybridization Hum. Reprod. Update, January 1, 2005; 11(1): 33 - 41. [Abstract] [Full Text] [PDF]
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D.D. Daphnis, J.D.A. Delhanty, S. Jerkovic, J. Geyer, I. Craft, and J.C. Harper Detailed FISH analysis of day 5 human embryos reveals the mechanisms leading to mosaic aneuploidy Hum. Reprod., January 1, 2005; 20(1): 129 - 137. [Abstract] [Full Text] [PDF]
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C. Gutierrez-Mateo, D. Wells, J. Benet, J. F. Sanchez-Garcia, M. G. Bermudez, I. Belil, J. Egozcue, S. Munne, and J. Navarro Reliability of comparative genomic hybridization to detect chromosome abnormalities in first polar bodies and metaphase II oocytes Hum. Reprod., September 1, 2004; 19(9): 2118 - 2125. [Abstract] [Full Text] [PDF]
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ESHRE Capri Workshop Group Diagnosis and management of the infertile couple: missing information Hum. Reprod. Update, July 1, 2004; 10(4): 295 - 307. [Abstract] [Full Text] [PDF]
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E.B. Baart, D. Van Opstal, F.J. Los, B.C.J.M. Fauser, and E. Martini Fluorescence in situ hybridization analysis of two blastomeres from day 3 frozen-thawed embryos followed by analysis of the remaining embryo on day 5 Hum. Reprod., March 1, 2004; 19(3): 685 - 693. [Abstract] [Full Text] [PDF]
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F. J. Los, D. Van Opstal, and C. van den Berg The development of cytogenetically normal, abnormal and mosaic embryos: a theoretical model Hum. Reprod. Update, January 1, 2004; 10(1): 79 - 94. [Abstract] [Full Text] [PDF]
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