Molecular Human Reproduction, Vol. 8, No. 5, 502-510,
May 2002
© 2002 European Society of Human Reproduction and Embryology
Reproductive genetics |
Single cell CGH analysis reveals a high degree of mosaicism in human embryos from patients with balanced structural chromosome aberrations
1 Department of Molecular Medicine, Karolinska Institutet, Karolinska Hospital, S-171 76, 2 Clinical Research Center, Huddinge University Hospital, Karolinska Institutet, S-141 86, 3 Department of Clinical Sciences, Obstetrics and Gynaecology Unit, Huddinge Hospital, S-141 86 Stockholm, Sweden
Abstract
We have performed comparative genomic hybridization (CGH) analysis of single blastomeres from human preimplantation embryos of patients undergoing preimplantation genetic diagnosis (PGD) for inherited structural chromosome aberrations and from embryos of IVF couples without known chromosomal aberrations. The aim was to verify the PGD results for the specific translocation, reveal the overall genetic balance in each cell and visualize the degree of mosaicism regarding all the chromosomes within the embryo. We successfully analysed 94 blastomeres from 28 human embryos generated from 13 couples. The single cell CGH could verify most of the unbalanced translocations detected by PGD. Some of the embryos exhibited a mosaic pattern regarding the chromosomes involved in the translocation, and different segregation could be seen within an embryo. In addition to the translocations, we found a high degree of numerical aberrations including monosomies, trisomies and duplications or deletions of parts of chromosomes. All of the embryos (100%) were mosaic, containing more than one chromosomally uniform cell line, or even chaotic with a different chromosomal content in each blastomere.
embryo/mosaicism/PGD/single cell CGH
Introduction
Carriers of structural chromosome aberrations are at risk of having unbalanced offspring, possibly resulting in children born with severe malformations and mental retardation. In addition, these couples often suffer from repeated miscarriages and infertility problems. Preimplantation genetic diagnosis (PGD), is an alternative to prenatal diagnosis and termination of affected pregnancies. PGD, including IVF, embryo biopsy and genetic diagnosis using interphase fluorescence in-situ hybridization (FISH) with different probe combinations, provides a possibility to detect and select balanced embryos for transfer. We have applied PGD in 55 treatment cycles for 24 couples carrying chromosome aberrations, resulting in nine pregnancies with eight babies born and one ongoing pregnancy. All babies, including the ongoing pregnancy, are balanced carriers of the respective translocation.
In a previous study, we analysed 64 spare embryos from PGD treatment cycles of couples with balanced structural chromosome aberrations. The embryos were analysed by interphase FISH analysis using 4–5 probes located on different chromosomes. The results showed that 73% of the embryos were mosaic or chaotic (Iwarsson et al., 2000
). This is in agreement with other interphase FISH studies, showing that normal developing embryos exhibit a high number of numerical aberrations and a high degree of mosaicism at the 8-cell stage (Munne et al., 1994, 1995; Harper et al., 1995; Delhanty et al., 1997; Iwarsson et al., 1999). However, due to technical limitations, only a few chromosomes may be analysed using interphase FISH. Comparative genomic hybridization (CGH) is a method that gives an overview of the whole genome and allows the detection of DNA copy number changes. In previous single cell CGH analyses of normal IVF embryos, non-mosaic aneuploidies, chromosome breakage and a high degree of mosaicism were detected; 75% of the IVF embryos were mosaic or even chaotic (Voullaire et al., 2000; Wells and Delhanty, 2000).
We have performed CGH analysis of single blastomeres from human preimplantation embryos from patients who underwent PGD due to inherited structural chromosome aberrations. The aim was to verify the PGD results of the embryo analysis regarding the specific translocation, detect the overall genetic balance in each cell and visualize the degree of mosaicism regarding all the chromosomes.
Materials and methods
The human preimplantation embryos used in this study were donated from patients with structural chromosome aberrations who were undergoing PGD treatments. Normal developing embryos from cases with Robertsonian translocations, two patients with a der(13;14)(q10;q10) and one patient with a der(14;15)(q10;q10), and four cases with the reciprocal translocations t(6;7)(p25;q11.2), t(10;11)(p11.2;q23.3), t(9;13)(q12;p13) and t(10;11)(q12;p13) were investigated (Tables I and II). The embryos were spare embryos that were considered unbalanced and unsuitable for transfer after PGD. In addition, we analysed six embryos from six IVF patients without known structural aberrations as a reference material. The morphology of the embryos was evaluated according to a scoring system derived by Mohr et al. and described in detail by Fridström et al. (Mohr and Trounson 1985; Fridstrom et al., 1999). The embryos included in this study exhibited scores of 1.5–3.0. The average maternal age was 34 years (range 29–41).
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On day 4 after IVF the embryos were segregated and the blastomeres were transferred to individual PCR tubes. Each blastomere was analysed by single cell PCR amplification and subsequent CGH analysis using a previously developed protocol (Klein et al., 1999
). In brief, the cells were lysed by proteinase K treatment, and the DNA was digested with MseI. Linkers were ligated to the generated restriction fragments and PCR was conducted using primers complementary to the linker sequence. The PCR products were re-amplified and labelled with digoxigenin and detected with fluorescein isothiocyanate-antibody. The reference DNA (normal female) labelled with Spectrum Red was purchased from Vysis (Downers Grove, IL, USA). The test DNA and the reference DNA were co-hybridized to male metaphase slides (Vysis). After CGH hybridization in a moist chamber for 48 h, the slides were washed and analysed using Quips CGH karyotyping Imaging Software (Vysis). A total of 10–15 metaphases were captured and evaluated for each blastomere. Red/green ratios of 1:>1.25 were indicative of amplified regions and ratios 0.75:<1 were indicative of deleted regions. Some regions were excluded from analysis (centromeres, telomeric regions, 1q, 16p, chromosome 19 and 22) as these regions are difficult to interpret by CGH (Kallioniemi et al., 1994). An analysis was considered successful if the PCR products from the single cell were in large amounts and in the expected size range and if the CGH resulted in strong, even, red/green balanced hybridization to the metaphases. Results
In order to evaluate the method, we analysed single lymphocytes from 10 different normal individuals. No genetic change could be detected in any of these samples and the sex of the donor could be verified. In addition, lymphocytes from a patient with trisomy 18 and a patient with trisomy 21 were analysed and the CGH analysis could detect the imbalance. The success rate for the single lymphocyte analysis was 95%.
The single cell CGH analysis was then applied to blastomeres from embryos. A total of 94 blastomeres from 28 human embryos were successfully analysed. These embryos included 12 embryos from four reciprocal translocation carrier couples, 10 embryos from three couples with Robertsonian translocations and six embryos from IVF patients without known structural aberrations (Tables I and II
). The success rate for the blastomere CGH analysis was 70%.
Detection of unbalanced translocations
PGD results were available for 15 of the 22 embryos from the PGD patients. Regarding the remaining seven embryos, the biopsied blastomeres lacked nuclei or the FISH analysis was incomplete. For 10 out of 15 embryos with both PGD and CGH results, the single cell CGH analyses supported the PGD results (Table II). For three of the embryos, the PGD results were in agreement with the CGH results of at least one blastomere within the embryo, and for one embryo the CGH and PGD were not in agreement at all. However, these embryos were highly mosaic or chaotic, which explains the different chromosomal content in blastomeres within these embryos. One embryo showed a haploid chromosomal content in the biopsied blastomeres, a type of abnormality that cannot be detected by CGH.
PGD analysis of embryo 9 from couple 18 was carried out by FISH using three probes (Table I), and showed two 11p signals, one 10 centromere signal and three 11 centromere signals. This implies an adjacent 2 segregation, which results in a chromosome 10 and 11 content in agreement with the CGH results (Figure 1).
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However, small deletions or amplifications of the telomeric regions were difficult to interpret. Therefore, imbalances involving a translocation with a very distal breakpoint could be missed as the resolution of CGH is limited and unreliable ratio changes may appear at the telomeric regions (Kallioniemi et al., 1994
). Figure 2 outlines CGH analysis of embryo 1 from couple 14. Eight blastomeres from embryo 1 were analysed by single cell CGH and all showed an excess of 7q11-7qtel. This implies an adjacent 1 segregation, and a main karyotype of the embryo of 47,XXYder(6)t(6;7) (p25;q11.2) (see also Figure 5). However, this segregation gives, in addition to the extra 7q material, a lack of the most distal part of 6p. This deficiency is not detected by CGH (Figure 2).
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Another example is embryo 2 from couple 18 for which the single cell CGH analysis revealed the same chromosomal content regarding the translocation chromosomes in all eight blastomeres, a lack of 10q12-10qtel and a normal copy number of chromosome 11. The PGD results (original FISH data not shown) imply an adjacent 1 segregation that results in the lack of 10q12-10qtel and a gain of 11p13-11ptel (Figure 3B
). However, the gain of 11p13-11ptel was not detected by the CGH analysis (Figure 3).
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Some of the embryos exhibited a mosaic pattern regarding the chromosomes involved in the translocation, and different distributions of these chromosomes could be seen within an embryo. Embryo 4 from couple 14 showed a mosaic pattern regarding the chromosomes involved in the translocation. A different distribution of chromosomes 6, 7 and a derivative 7 is implicated by the single cell CGH (Figure 4
). The different contents may be the result of mitotic malsegregation and one may speculate that one of the blastomeres containing the der(7) chromosome could be the result of conception and the others are products of mitotic malsegregation.
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Mosaicism
In addition to the unbalanced translocations, we found a high degree of numerical aberrations including monosomies and trisomies for whole chromosomes or chromosome arms. All of the embryos (100%) were mosaic, containing more than one chromosomally uniform cell line, or even chaotic with a different chromosomal content in each blastomere (Figure 5
). We classified the embryos from the PGD couples with chromosomal aberrations into three different groups (Table II). (i) Balanced and mosaic. Three embryos were balanced regarding the translocation, but contained some blastomeres with other aberrations. Two of these embryos were derived from carriers of Robertsonian translocations and one embryo from a carrier of a reciprocal translocation. These embryos had not been chosen for transfer because of PGD failure (two embryos) and the third was not transferred due to the presence of two other balanced embryos in the same treatment cycle. (ii) Uniformly unbalanced and mosaic. Eleven embryos were uniformly unbalanced regarding the translocation, i.e. each blastomere showed the same segregation pattern of the translocation. In addition, they all contained blastomeres with other aberrations except for one embryo where only one cell was analysed. (iii) Chaotic. Eight of the 22 embryos were chaotic, exhibiting a different chromosomal content within each blastomere, both regarding the translocation chromosomes as well as other chromosomes. In addition to those described above, 17 blastomeres from six embryos donated from patients without known structural chromosome aberrations were analysed. All of these embryos showed numerical aberrations as well as mosaicism.
In order to verify the aneuploidies and mosaicism discovered by CGH, the nuclei obtained at the PGD biopsy were reanalysed in some cases. In five of the embryos, a specific aneuploidy was present in all blastomeres or in a majority of the blastomeres, and for these embryos the biopsied nuclei were rehybridized with FISH probes detecting the specific aneuploidy. The rehybridization analysis supported the CGH results for all five embryos (not shown). For embryo 2 (couple 18), single cell CGH revealed an extra chromosome 2 in all eight blastomeres, and the copy number of chromosome 18 varied between blastomeres, five blastomeres with a normal copy number of 18, three blastomeres with monosomy 18 and two blastomeres with trisomy 18. The biopsied blastomeres from this embryo were rehybridized with centromere 2 and 18 probes. The trisomy 2 was also present in these two biopsied blastomeres and chromosome 18 was present in two copies (Figure 3C and 5).
Discussion
About 70–80% of all human conceptions fail to develop to term (Jacobs, 1990) and a majority of these embryos are lost either before implantation or early post-implantation. The success rate for patients going through IVF or PGD is similar at ~25%. Interphase FISH investigations of spare embryos from IVF and PGD patients have shown that these embryos exhibit a high degree of aneuploidies and mosaicism (Munne et al., 1994, 1995; Harper et al., 1995; Delhanty et al., 1997; Iwarsson et al., 1999). This may be one explanation for the high rate of developmental failure.
The interphase studies were performed using probes located on different chromosomes. In general, the more probes included in an interphase analysis, the more unreliable interpretation of the results will be. This means that only a limited part of the genome can be analysed using interphase FISH. In the present study, we have analysed blastomeres from human pre-embryos using CGH, a method that gives an overview of the whole genome and allows the detection of DNA copy number changes. Single cell CGH analysis was performed using the protocol that was developed and published by Klein et al. (Klein et al., 1999).
We successfully analysed a total of 94 blastomeres from 28 human embryos generated from seven couples going through PGD treatments for structural chromosome aberrations and from six IVF patients without known structural aberrations (Table II). The CGH analysis success rate was 70% for the blastomeres. This is a low success rate as compared with that for the single lymphocytes (95%). One explanation is the fact that many blastomeres within an embryo are lacking nuclei. It has been shown that 5% of good quality embryos and 12% of poor quality embryos are anucleated (Hardy et al., 1993). The embryos analysed in this study were imbalanced and highly mosaic and the degree of anucleated cells might be high. However, anucleated blastomeres might not explain all of the 30% analyses that failed. We have used a CGH protocol that differs from those used in other studies with higher success rates of 97 and 88% respectively (Voullaire et al., 2000; Wells and Delhanty, 2000). However, only normal IVF embryos were analysed in these studies. In our hands, the protocol by Klein et al. was the one that gave the most accurate results. However, improvements might be made.
CGH analysis is an attractive alternative to interphase FISH analysis regarding the detection of imbalances and aneuploidies in single cells. CGH displays all the chromosomes in one analysis and detects copy number changes. CGH for PGD has been applied clinically for determination of aneuploidy, and resulted in the birth of a healthy baby (Wilton et al., 2001). Single cell CGH was more recently developed and different protocols have been published (Voullaire et al., 1999, 2000; Wells et al., 1999; Wells and Delhanty, 2000). All present protocols have a time requirement that is impossible to fit into the PGD situation with day 4 transfer. However, by freezing the embryos after biopsy it becomes possible to await the results. Another possibility is to perform blastocyst transfer. In this study, we found a resolution limit of 10–20 Mb for CGH, and smaller imbalances might be missed. This is in agreement with another single cell CGH analysis that estimated the resolution to be 10–40 Mb (Voullaire et al., 1999). However, if the resolution of CGH can be refined it may be an alternative to interphase FISH in PGD treatment for patients with structural chromosomal aberrations. In this study, the CGH results supported the PGD results in 11 out of 15 embryos for which both PGD and CGH results were available (Figure 1).
In three embryos the CGH analysis revealed a highly mosaic or chaotic embryo, but at least one blastomere was in agreement with the PGD results (Table II). However, small deletions or amplifications of the telomeric regions were difficult to interpret by the CGH analysis. Therefore, imbalances involving a translocation with a very distal breakpoint could be missed as the resolution of CGH is limited and unreliable ratio changes may appear at the telomeric regions (Kallioniemi et al., 1994) (Figures 2 and 3). In addition, translocations involving regions of the karyotype that are subjected to CGH artefacts cannot be diagnosed by this method.
Some of the embryos exhibited a mosaic pattern regarding the chromosomes involved in the translocation, and different distributions of these chromosomes could be seen within an embryo (Figure 4). In addition, we found a high degree of numerical aberrations including monosomies, trisomies and duplications or deletions of parts of chromosomes. All of the embryos (100%) were mosaic, containing more than one chromosomally uniform cell type, or even chaotic with different chromosomal content in each blastomere. The detected degree of mosaicism is very high as compared with previous interphase FISH studies (Munne et al., 1994, 1995; Harper et al., 1995; Delhanty et al., 1997; Iwarsson et al., 1999, 2000). In a single cell CGH analysis of normal IVF pre-embryos, non-mosaic aneuploidies, chromosome breakage and a high degree of mosaicism were detected; 75% of the IVF embryos were mosaic or even chaotic (Wells and Delhanty, 2000). In the present study, we found an even higher prevalence of mosaic/chaotic embryos. However, the embryos analysed in this study were previously diagnosed as unbalanced regarding the chromosomes involved in the translocation or were considered unsuitable for transfer for other reasons, and may therefore show a higher degree of mosaicism as compared with normal or balanced embryos. We could not detect any difference in the degree of mosaicism between embryos from Robertsonian translocation carriers versus embryos from carriers of reciprocal translocations.
The mosaicism in the embryos has to be the result of mitotic errors occurring after fertilization. Some studies have shown that the IVF procedure influences the development of the embryo (Shaw et al., 1991; Pickering et al., 1995; Munne et al., 1997), and one may speculate that mitotic segregation may be disturbed by environmental conditions. However, the low success rate observed in normal conceptions suggests that mosaicism and aneuploidies are frequent in vivo as well as in IVF treatments. All the embryos derived from normal IVF patients were mosaic. However, the number of analysed IVF embryos is too small to evaluate the degree of mosaicism in these embryos.
In this study we observed a tendency for some couples to be more prone to generate chaotic embryos than others and this observation has been described previously (Delhanty et al., 1997). Couple 18 became pregnant at the third PGD treatment cycle after a two-embryo transfer. They donated three spare embryos for this study, one of which was balanced and the other two unbalanced. Some mosaicism was detected within these embryos. However, the degree of mosaicism was not as pronounced as in the four embryos from couple 14, who had a history of four miscarriages and three unsuccessful PGD cycles (Figure 5). One may speculate that some couples could have a disposition to generate aneuploid/mosaic embryos and that this might be one cause of repeated miscarriages.
The chaotic embryos were not scored lower than non-chaotic regarding morphology. Thus, at this developmental stage there is no correlation between the chromosomal content and the appearance of the embryo. Can mosaic embryos or even chaotic embryos continue to divide and differentiate? What is the lowest number of normal cells required at the 8-cell stage in order to give rise to a vital embryo? One may speculate about different scenarios. The first one involves preferential segregation of balanced cells to the inner cell mass, leaving the abnormal cells in the trophectoderm (James and West, 1994). This could also explain the phenomenon of confined placental mosaicism (Kalousek and Dill, 1983; Kalousek and Vekemans, 1996). However, this has not been verified in human embryos (Evsikov and Verlinsky, 1998). The second scenario is programmed cell death (apoptosis) of abnormal blastomeres and/or selective survival (growth advantage) of normal cells. This would lead to a successive accumulation of normal cells within an embryo during development. A third scenario is that the aneuploid/mosaic embryos fail to develop beyond a point and are eliminated prior to blastocyst formation, or at later developmental stages. The developmental potential of embryos with different aneuploidies was studied by Sandalinas et al. Extensive mosaicism was detected in blastocysts and trisomic embryos reached the blastocyst stage. However, monosomies, with the exception of monosomy X and 21, haploidies and aneuploidies combined with extensive mosaicism never developed to blastocysts (Sandalinas et al., 2001). They showed the existence of a strong selection against chromosomally abnormal embryos, but not all abnormalities were removed at the blastocyst stage.
In conclusion, we have performed CGH analysis of single blastomeres from human preimplantation embryos from patients going through PGD for inherited structural chromosome aberrations. The CGH analysis of the embryos revealed the unbalanced translocations in most cases, but small deletions or amplifications of the telomeric regions were difficult to interpret. Unbalances involving a translocation with a very distal breakpoint could be missed as the resolution of CGH is limited and unreliable ratio changes may appear at the telomeric regions (Kallioniemi et al., 1994). However, if the resolution of the CGH can be refined and if the time requirement for the CGH analysis can be limited or if the time for transfer is altered, CGH may be an alternative to interphase FISH in the PGD treatment for patients with structural chromosomal aberrations. The embryos in this study contained a high degree of numerical aberrations and all embryos (100%) were mosaic or even chaotic with a different chromosomal content in each blastomere. These findings ought to be considered in the clinical PGD situation, and the analysis of two blastomeres is to be preferred as this reduces the risk for misdiagnosis.
Acknowledgements
This work was supported by the Swedish Medical Research Council, the Magnus Bergvall Stiftelse and the Swedish Society of Medicine.
Notes
4 To whom correspondence should be addressed. E-mail: helena.malmgren{at}cmm.ki.se 
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Submitted on October 2, 2001; accepted on February 13, 2002.
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