Molecular Human Reproduction, Vol. 6, No. 11, 1055-1062,
November 2000
© 2000 European Society of Human Reproduction and Embryology
Genetic diagnosis |
Comprehensive chromosomal analysis of human preimplantation embryos using whole genome amplification and single cell comparative genomic hybridization
Department of Obstetrics & Gynaecology, University College London Medical School, 86–96 Chenies Mews, London WC1E 6HX, UK
Abstract
Analysis of small numbers of chromosomes using interphase fluorescent in-situ hybridization (FISH) probes has revealed that 50% of human preimplantation embryos contain abnormal cells. Detection of high levels of mosaicism with so few probes has led some researchers to extrapolate that a full analysis of all 23 pairs of chromosomes would reveal that all human embryos contain a proportion of abnormal cells. However, existing cytogenetic protocols cannot achieve such an analysis due to technical limitations. We have developed a novel technique based on whole genome amplification and comparative genomic hybridization (CGH), which for the first time allows the copy number of every chromosome to be assessed in almost every cell of a cleavage-stage embryo. We have successfully analysed 64 cells (blastomeres) derived from 12 embryos and have detected unusual forms of aneuploidy, high levels of chromosomal mosaicism, non-mosaic aneuploidy and chromosome breakage. This is the first report of a comprehensive assessment of chromosome copy number in human embryos and indicates that, despite high levels of mosaicism, some embryos do have normal chromosome numbers in every cell. Such embryos may have a superior developmental potential, and their low frequency may explain correspondingly low success rates of natural and assisted conception in humans.
aneuploidy/CGH/mosaicism/preimplantation genetic diagnosis/single cell
Introduction
Compared with other species for which data is available, humans display a low fecundity. Couples of proven fertility that are trying to have another child have only a 25% chance of achieving a viable pregnancy per menstrual cycle. Measurements of chorionic gonadotrophin concentrations in urine indicate that fertilization may actually occur in ~60% of cycles; however 52% of all women that conceive experience early miscarriage (Short, 1979
; Edmonds et al., 1982). The success rate for couples receiving IVF treatment is also low compared with other species (~20–30% per cycle), and early pregnancy tests carried out in the first 2 weeks following IVF have revealed that 30% of positive tests do not ultimately result in a live birth (Seppala, 1985; Sharma et al., 1986). This high level of early embryonic death must contribute significantly to the observed low fecundity. Various factors may cause these early pregnancy failures, but chromosome abnormality is likely to be the most important. The lethality of many aneuploidies is highlighted by cytogenetic analysis, which reveals that >60% of spontaneous abortions at <12 weeks are aneuploid (Boue et al., 1975, 1985; Hassold et al., 1980). It is thought that the true level of abnormality is greater still, as most abnormal embryos probably fail to survive to implantation. Such conceptions are not usually detected and no material can be recovered for cytogenetic analysis. Since most pregnancy failure is likely to occur during the preimplantation phase of development, the chromosomes of these embryos are of particular interest. Furthermore, cytogenetic analysis of embryos provides evidence for the origins of chromosomal abnormalities detected later in gestation and at birth.
Preimplantation genetic diagnosis, in which the genetic status of an embryo is inferred from a single cell biopsied 3 days after fertilization, provided the first opportunity for chromosomal analysis of embryos produced by fertile couples. At this stage of development embryos are usually composed of 6–10 cells (blastomeres). Ideally, cytogenetic investigation would involve analysis of metaphase chromosomes from these cells. However, efforts to karyotype embryos using the conventional techniques of culture synchronization, disruption of the mitotic spindle and G-banding have only produced analysable metaphase chromosomes in a low proportion of cells (Angell et al., 1986; Plachot et al., 1987; Papadopoulos et al., 1989; Clouston et al., 1997). Most of the reliable data that has been collected thus far has been obtained using fluorescence in-situ hybridization (FISH) to detect specific chromosomes in interphase nuclei. Interphase FISH detection of the X and Y chromosomes first indicated that mosaicism is a common feature of human preimplantation embryos (Delhanty et al., 1993). Although there was some resistance to these findings at first, subsequent studies incorporating autosomal probes and testing between three and nine different chromosomes have detected aneuploidy or mosaicism in more than half of the embryos investigated (Munné et al., 1993, 1998; Harper et al., 1995; Delhanty et al., 1997; Laverge et al., 1997; Iwarsson et al., 1999). These figures have led some researchers to extrapolate that a full analysis of all 23 pairs of chromosome would reveal all embryos to be mosaic. This suggestion has important implications for our understanding of human development as well as the origin of clinically significant phenomena such as confined placental mosaicism (CPM) and uniparental disomy (UPD). Unfortunately interphase FISH is subject to technical difficulties that limit the number of chromosomes that may be simultaneously assessed. The accuracy per probe per cell has been determined to be ~91–96% for euploid samples and somewhat less for trisomic samples (Ruangvutilert et al., 2000). Consequently as more probes are applied the probability of experimental artefacts causing discordant results between cells of the same embryo, leading embryos to be incorrectly classed as mosaic, grows rapidly. As a result few studies have employed more than five chromosomal probes.
Comparative genomic hybridization (CGH) is an alternative method to interphase FISH or G-banding (Kallioniemi et al., 1992). This method has been widely applied to the detection of chromosome copy number changes in tumours and has successfully identified a range of unbalanced chromosomal complements in prenatal samples. CGH does not require the preparation of chromosomes from the sample and in a single experiment reveals the copy number of every chromosome segment >10 Mb in size. Such a method would be extremely useful for gauging levels of aneuploidy and mosaicism in preimplantation embryos; however published CGH protocols require 0.2–1.0 µg of sample DNA whereas a single cell contains only 6 pg. After extensive work-up we have overcome this problem by employing degenerate oligonucleotide primed polymerase chain reaction (DOP–PCR) to amplify the whole genome prior to CGH analysis (Wells et al., 1999). The application of single cell CGH to each cell of a human preimplantation embryo provides the first opportunity to assess the copy number of all chromosomes and thus the genuine abnormality and mosaicism level at this stage of development. We report here the application of this method to single blastomeres from cleavage-stage human embryos.
Materials and methods
Sample collection
All research involving human embryonic material was done with the approval of the ethics committee of University College London Hospitals Trust in a laboratory licensed by the Human Fertilisation and Embryology Authority (HFEA). Surplus embryos were donated by patients undergoing treatment at the Assisted Conception Unit of University College Hospital. Only normally fertilized, normally developing embryos with very little cellular fragmentation were chosen for this study. After fertilization, embryos were cultured according to standard protocols for 3 days. The zona pellucida encapsulating the embryo was then removed using acid Tyrode's treatment, and individual cells were separated by mechanical disaggregation in phosphate-buffered saline (PBS) (calcium and magnesium free) + 5% bovine serum albumin (BSA). Individual cells were washed three times in PBS, transferred to a microfuge tube containing 3 µl of proteinase K (125 µg/ml), and overlaid with oil. Cell lysis was achieved by incubation at 37°C for 45 min followed by 15 min at 99°C.
Whole genome amplification
Degenerate oligonucleotide primed PCR was employed for whole genome amplification (WGA). A 50 µl reaction mixture contained 0.2 mmol/l dNTPs, 2.0 µmol/l DOP primer CCGACTCGAGNNNNNNATGTGG (Telenius et al., 1992), 1x PCR buffer and 2.5 IU Taq polymerase (HT Biotechnology, Cambridge, UK). Thermal cycling conditions were as follows: 94°C for 9 min; eight cycles of 94°C for 1 min, 30°C for 1.5 min, 72°C for 3 min; 25 cycles of 94°C for 1 min, 62°C for 1 min, 72°C for 1.5 min; and finally 72°C for 8 min.
Stringent precautions against contamination (Wells and Sherlock, 1998), were observed throughout single cell isolation, lysis and amplification procedures. The incidence of contamination was assessed regularly using control blanks that were subjected to the entire DOP–PCR and CGH procedure.
DNA labelling and probe preparation
Immediately after completion of the first reaction a 1/10 volume of WGA product was removed and further amplified in a 50 µl reaction containing 0.2 mmol/l nucleotides (dATP, dCTP, dGTP), 0.1 mmol/l dTTP, 1x PCR buffer, 2 IU Taq polymerase, DOP primer (2.0 µmol/l) and 2.5 µl of fluorescein-11-dUTP or rhodamine-4-dUTP. Incubations were as follows: 94°C for 4 min; 94°C for 1 min, 62°C for 1 min, 72°C for 1.5 min (25 cycles); and finally 72°C for 8 min. DOP–PCR amplified 46 XY (control) buccal cells and amplified blastomeres, labelled with fluorochromes of different colour, were co-precipitated with 30 µg of Cot-1 DNA and dissolved in 10 µl of hybridization mixture (50% formamide, 10% dextran sulphate, 2x SSC). Labelled DNA samples dissolved in hybridization mixture were denatured at 75°C for 10 min then cooled to 37°C for ~45 min before being applied to denatured normal chromosome spreads as described below.
Comparative genomic hybridization
Metaphase spreads were prepared according to standard protocols and the slides aged for 1–3 days at room temperature prior to use. The slides were then dehydrated through an alcohol series (70, 90, and 100% ethanol for 5 min each) and air-dried. This preceded an incubation in 100 µg/ml RNAseA/2x SSC (20x saline sodium citrate is = 150 mmol/l NaCl; 15 mmol/l sodium citrate, pH 7) lasting 1 h. Slides were then washed twice with 2x SSC, each wash lasting 5 min, and then immersed in proteinase K buffer (2 mmol/l calcium chloride (CaCl2); 20 mmol/l Tris–HCl, pH 7.5) at 37°C for 5 min. This was followed by a 7 min treatment at 37°C with proteinase K (50 ng/ml in proteinase K buffer). Following a brief wash in PBS:1% w/v MgCl2 the slides were fixed with paraformaldehyde (1% paraformaldehyde; 1% MgCl2 in PBS) for 10 min at room temperature and then washed in PBS, sent through an alcohol series, and air-dried.
Denaturation of the slides was achieved by applying 70% deionized formamide/2x SSC under a coverslip and heating the slides in an oven at 75°C for 5 min. Immediately after denaturation the coverslips were removed and the slides washed in 70% ethanol chilled to –20°C. Slides were then put through an alcohol series and dried before the denatured probe was finally added. A coverslip was placed on top and sealed with rubber cement. Hybridization of the probe proceeded over 72 h during which time the slides were kept in a humidified chamber at 37°C.
After hybridization the slides were washed three times in 50% formamide/2x SSC at 45°C, twice in 2x SSC at 45°C and once at room temperature, each wash lasting 10 min. The slides were then washed at room temperature with TN (0.15 mol/l NaCl; 0.1 mmol/l Tris–HCl, pH 7.5) with 0.1% Tween 20 detergent for 10 min, followed by distilled water for a further 10 min. Finally the slides were put through an alcohol series, air-dried, and were mounted in anti-fade medium (Vector Labs, Peterborough, UK) containing diamidinophenylindole (DAPI) to counterstain the chromosomes and nuclei.
Microscopy and image analysis
Metaphase chromosome preparations were photographed using a Zeiss Axioskop microscope equipped with a Photometrics KAF1400 CCD camera, and SmartCapture software supplied by Vysis, Richmond, UK. Image analysis was performed using Vysis Quips CGH software. Green:red fluorescence ratios of >1.2:1 was indicative of gain of material, while ratios of <0.8:1 indicated loss.
Results
Extensive optimization and testing revealed that the optimal WGA method for use in conjunction with single cell CGH was DOP–PCR (Wells et al., 1999). In preliminary experiments this method yielded greater quantities of DNA than rival methods (>20 µg), sufficient for over 100 separate PCR experiments, and provided 100% accurate aneuploidy detection.
During this study we investigated 73 cells from 12 normally developing embryos (grade 1 or 2) and a full CGH analysis was obtained from 64 (88%). Only good quality embryos were chosen because they are considered to have a good potential for further development and are preferentially selected for transfer by embryologists during IVF treatment. However, we have found that CGH works with similar efficiency when applied to nucleated cells from arrested embryos and poor quality (grade 3) embryos (Wells et al., unpublished data). Of the 12 embryos, nine were found to contain aneuploid cells (Table I). Indeed three (nos. 3, 4 and 9) had no normal cells. One embryo (no. 4) displayed the same abnormality in all cells tested and was predicted to have a 46, X,+21 karyotype as a result of two meiotic errors. Eight embryos were mosaic each containing more than one chromosomally distinct cell line. By definition such mosaicism can only arise from a mitotic error occurring after fertilization. Various chromosomes were involved in these abnormalities giving rise to a range of unusual cell lines. Some of the abnormalities observed were of varieties never detected during later stages of human development. Autosomal monosomies, which when complete are associated with very early fetal lethality, were detected in three embryos (nos. 2, 9 and 12); of these embryo no. 2 probably originated from a meiotic error as monosomy occurs in a majority of cells (Figure 1). Additionally three cases of nullisomy (loss of both copies of a chromosome) were identified. This involved the total loss of chromosome 2 material in one cell from embryos nos. 3 and 12 and loss of the single X chromosome in one cell of embryo no. 9, a male embryo. Embryos nos. 9 and 12 were highly abnormal. Each cell examined contained a different array of chromosome abnormalities, suggesting that chromosomes had been segregated in a random `chaotic' manner. Cells from embryo no. 9 contained between 5 and 11 aneuploid chromosomes, and 22 out of the 24 different types of chromosome (22 autosomes, X and Y) were abnormal in one or more cells. However, embryo no. 12 contained four normal cells in addition to four cells that showed multiple (between one and 14) aneuploid chromosomes. Although chaotic embryos were known to be aneuploid for multiple chromosomes, this is the first time the true extent of their aneuploidy has been demonstrated.
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Embryos nos. 6 and 7 were probably derived from 47, XXY zygotes; in which case a third of the embryos investigated (nos. 2, 4, 6, 7) contained aneuploidy of meiotic origin. At least nine embryos contained some normal cells and three embryos displayed a balanced karyotype in all the cells assessed. The finding of entirely normal embryos is highly significant, as it is likely that they have a superior potential for post-implantation development. The normal embryos could not be distinguished from the aneuploid, mosaic or chaotic embryos on the basis of morphological criteria.
In addition to loss and gain of whole chromosomes, partial gains and losses were also identified in two embryos. It is unlikely that these abnormalities would have been detected using interphase FISH as this method only allows a small region of each chromosome to be visualized. Embryo no. 3 contained several errors involving chromosome 2 (Figure 2). One cell had an excess of chromosome 2q32.1-qter, while another cell appeared to contain the reciprocal – an excess of 2pter-q32.1, as well as a deficiency of 2q32.1-qter. A third cell from the same embryo contained no chromosome 2 material at all. The same embryo also had an apparent breakage of chromosome 7, with one cell having a deficiency of 7q31.3-qter, while another had an excess of the same region. These errors occurred on an otherwise normal chromosomal background. Embryo no. 8 also displayed a structural rearrangement that resulted in a deficiency of the entire short arm of chromosome 1 in one blastomere; again the cell was otherwise normal. It is not possible to define the precise nature of these structural rearrangements using CGH.
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The nine cells that failed to give a result either lysed during isolation or were observed to be anucleate prior to amplification. A number of chromosomal regions (1p34-pter, 19 and 22) sometimes appeared to be lost from the test samples (i.e. an increase in red relative to green fluorescence). This was also seen in control CGH experiments in which normal (46, XY) DNA was used as both the test (green) and reference (red) samples. These apparent losses of chromosomes are well-documented artefacts of the CGH procedure, and can be excluded by repeating the experiment with the colours used for labelling test and reference DNAs reversed. No human DNA was detected in any DOP–PCR negative controls.
Discussion
Aneuploidy at the preimplantation stage is an order of magnitude more common in humans than in other mammals for which data is available, and usually results in early embryonic lethality. The loss of large numbers of embryos in this way is likely to be a major factor in the observed low fecundity and poor IVF success rates in humans. If the incidence of mosaicism and aneuploidy in human embryos is to be accurately assessed it is essential that the copy number of all 24 types of chromosome be determined in every cell. However, analyses of small numbers of cells using traditional cytogenetic methods are plagued by low efficiency of metaphase production and technical difficulties that preclude a full analysis of all chromosomes (Angell et al., 1986; Plachot et al., 1987; Papadopoulos et al., 1989; Clouston et al., 1997). Recently developed methods involving cell fusion can force an increased proportion of cells into metaphase, and spectral karyotyping can make the resultant chromosomes more amenable to analysis (Willadsen et al., 1999); yet, despite these advances, a complete examination of all cells remains impossible. As an alternative to analysis of metaphase chromosomes interphase FISH has been widely employed and usually produces results from almost all of the cells assessed (~95%). However, less than half the chromosome complement can be analysed in any one cell (e.g. Munné et al., 1993, 1998; Harper et al., 1995; Delhanty et al., 1997; Iwarsson et al., 1999). We have achieved a full analysis of all the chromosomes in every cell of a preimplantation embryo by the novel application to single cells of WGA and CGH. The efficacy of the WGA–CGH method performed on minute DNA samples has been demonstrated by our work on microdissected tumour specimens (Wells et al., 1999) and by work on single cells (Voullaire et al., 1999).
The proportion of embryos that are aneuploid declines throughout pregnancy, presumably due to strong selection against those with unbalanced chromosome constitutions. At 3 weeks 9% of embryos are aneuploid, this falls to 5% at 10 weeks and to only 0.5% at full term (Lubs and Ruddle, 1970; Boue et al., 1985). During the first few days after conception, however, it is clear that much higher rates of abnormality are tolerated and there is evidence that selection against the abnormal cells only begins at the morula–blastocyst transition (day 4 or 5) (Evsikov and Verlinsky, 1998). Three quarters of the embryos in this study were found to contain aneuploid cells, a greater proportion than the 50–58% previously identified using interphase FISH analysis (Munné et al., 1993, 1998; Harper et al., 1995; Delhanty et al., 1997), but less than some researchers had anticipated. Interphase FISH analysis of as few as four chromosomes has detected abnormal cells in more than half of the embryos tested; extrapolation from this data suggested that a full analysis of all chromosomes would reveal that every embryo contains a proportion of aneuploid cells. However, our data indicate that in cells where aneuploidy occurs, several chromosomes are often involved. This increases the probability that an abnormal embryo will be detected with small numbers of FISH probes, and may lead to artificially high calculations of total aneuploidy rate. For example, our group has extensively studied chromosomes X, Y and 1 in embryos (Delhanty et al., 1997). The use of this small number of probes would have allowed identification of more than half of the abnormal embryos in this study, despite detecting only one eighth of all chromosomes.
Varieties of aneuploidy rarely (or never) seen during later stages of gestation were observed during this study; these included errors involving the largest chromosomes as well as monosomy and even nullisomy. Three quarters of the cells from embryo no. 2 contained only one copy of chromosome 1 (Figure 1
), while embryos nos. 3 and 12 each contained a single blastomere with no copies of chromosome 2 and embryo no. 9 contained a cell nullisomic for the X chromosome. Abnormalities of these types are not only absent from newborns, but also from prenatal tests taken at 12–16 weeks, and are not seen in first trimester spontaneous abortions with the rare exception of monosomy 21. Thus it seems likely that embryos with such aneuploidy have a very limited developmental potential and are probably incapable of implantation and the formation of a clinical pregnancy. The lethality of such chromosomal errors is not unexpected as they result in imbalance or deletion of hundreds of genes. This will inevitably include essential housekeeping genes that perform vital cellular functions.
It is possible that not all of the nine embryos containing abnormal cells would have arrested or aborted. Eight of these nine embryos were mosaic as a result of post-zygotic errors. Two of these embryos were mostly normal in composition, and a further four had at least one normal cell. Experimental evidence from diploid/tetraploid chimeric mouse embryos indicates that abnormal cells may be preferentially allocated to the trophectoderm leaving a majority of normal cells in the embryo proper (James and West, 1994). Whether preferential allocation occurs in humans is unknown, but comparisons of day 3 embryos and blastocysts (day 5 or 6) have failed to demonstrate an increase in aneuploid cells in the trophectoderm relative to the inner cell mass that will form the fetus. However, there does appear to be a decrease in the proportion of abnormal cells in the embryo as a whole (Evsikov and Verlinsky, 1998). This suggests that in some mosaic embryos, euploid cells may come to form the majority of the embryo by virtue of a growth advantage over aneuploid cells.
Even embryos with non-mosaic aneuploidy due to a meiotic error can potentially give rise to a normal fetus if further nondisjunction restores a normal karyotype (e.g. loss of one copy of a trisomic chromosome or doubling of a monosomic chromosome). The euploid cells may then outgrow the original aneuploid cells, or be preferentially allocated to the inner cell mass. In most cases such correction events would be considered highly unlikely, but with the increased rate of mitotic loss/duplication of chromosomes apparent in human preimplantation embryos and strong selection for euploidy it may be more common than otherwise expected. Embryo no. 2 may have undergone this process, beginning as monosomy 1 due to an error in meiosis, but becoming `normal' in one of the four cells. In this case, the balanced cell may have a substantial advantage over the monosomy 1 cells for future growth and development. Rescue of an aneuploid fetus by loss or gain of chromosomes will frequently result in uniparental disomy, particularly in the case of an originally monosomic conception (Engel and DeLozier-Blanchet, 1991). This too may have clinical consequences if the chromosome in question carries imprinted regions or recessive mutations.
Embryo no. 4 was aneuploid as a consequence of a double meiotic error. This embryo was comprised entirely of cells that had lost a sex chromosome and gained a copy of chromosome 21 (46, X,+21). Trisomy (particularly involving chromosome 21) and monosomy for the X chromosome are the most common chromosomal abnormalities detected in human pregnancies (Boue et al., 1985). However, it is unusual for both these errors to occur in the same conception. Over 95% of Turner's syndrome (45, X) pregnancies and 75% of those with 47, XX or XY +21 fail to go to term and thus the probability of an embryo such as this producing a child is predicted to be very low (Hassold and Jacobs, 1984).
Two embryos (nos. 6 and 7) displayed mosaicism involving only the sex chromosomes and were most likely to have been derived from 47, XXY zygotes. Interestingly both of these embryos were from the same couple who were receiving intra-cytoplasmic sperm injection (ICSI) treatment. A significant increase in the incidence of sex chromosome abnormality has been reported in children conceived using ICSI, affecting at least 1% (Martin, 1996; Bonduelle et al., 1999). These abnormalities are thought to be related to the father's infertility rather than the ICSI procedure. In this case the presence of XXY cells in both of the embryos suggests that the father may have been mosaic for Klinefelter's syndrome, with abnormal cells present in his gonadal tissue leading to the production of sperm with both X and Y chromosomes. Gonadal mosaics that produce a preponderance of abnormal gametes have been previously reported (Cozzi et al., 1994). The detection of XX cells in both of these embryos is also significant for preimplantation genetic diagnosis (PGD) for the avoidance of X-linked disease. In this case a single cell biopsied from an embryo is usually subjected to X and Y chromosome FISH to determine the sex of the embryo as a whole. The presence of XX and XY cell lines within the same embryo could lead to a misdiagnosis, although XX/XY mosaic embryos have not been previously reported and are likely to be rare. It has been suggested that all men undergoing ICSI be karyotyped to determine whether they carry a chromosome abnormality.
The most bizarre chromosomal arrangements detected in this study were found in embryos nos. 9 and 12. Embryo no. 9 displayed a complete breakdown of normal chromosome segregation, such that no two cells had the same chromosomal complement. Virtually all chromosomes were involved, with cells containing between five and 11 aneuploid chromosomes each. Embryos with apparently random allocation of chromosomes to daughter cells have been previously detected using interphase FISH and given the classification `chaotic' (Harper and Delhanty, 1996; Conn et al., 1998), but this is the first time the true extent of their aneuploidy has been determined. Embryo no. 12 contained four normal cells, but the remaining cells contained numerous aneuploidies and resembled the cells of a chaotic embryo. There was no pattern to the aneuploidy observed; chromosome losses and gains happened with similar frequency and there was no evidence that any particular chromosome was involved more often than any other. Surprisingly embryos with chaotic chromosome segregation do survive to the blastocyst stage, but it is unlikely that they progress much further and probably fail to implant (Evsikov and Verlinsky, 1998; and our unpublished observations).
Simple copy number changes involving whole chromosomes in human preimplantation embryos have been well documented by investigators using FISH, but the use of CGH in this study has also allowed the detection of an additional type of error involving structural alteration of chromosomes. Embryo no. 3 displayed breakage of chromosomes 2 and 7. In each case different cells contained the reciprocal products of the breakages, confirming that the losses were not experimental artefacts. Some of the fragments were acentric and would not be stably transmitted to daughter cells unless they became fused to another chromosome. The resultant loss of material would leave the embryo with a potentially lethal monosomy for that chromosomal region. Loss of a chromosome fragment was also observed in a cell from embryo no. 8, which was deficient for the entire short arm of chromosome 1. It seems that chromosome breakage is usually the sole defect in affected cells, suggesting that the phenomena of whole chromosome aneuploidy and breakage could be caused by different factors. Both sets of breakpoints seen in embryo no. 3 map to well defined chromosomal fragile sites, regions prone to breakage. Fragile sites can be induced by depletion of certain nutrients from the culture medium (Martin et al., 1990), and are frequently involved in de-novo chromosome rearrangements (Warburton, 1991). Similar structural anomalies to those reported here were detected by Clouston et al. in 40% of human blastocysts analysed by G-banding. The greater incidence of breakage in their study may reflect the increased duration in culture of the 6–8-day-old blastocysts that they investigated (Clouston et al., 1997). Other G-banding studies have also detected chromosome damage, but generally <50% of the embryos analysed provide any analysable metaphases (Angell et al., 1986; Plachot et al., 1987; Papadopoulos et al., 1989).
One of the few limitations of CGH is that it only detects relative alterations in chromosome copy number and cannot detect changes that involve the entire set of chromosomes (i.e. change in ploidy). This may have some significance for our analysis as some 10–15% of blastomeres are said to be haploid or tetraploid (Plachot et al., 1987; Harper et al., 1995). While tetraploid cells may be a normal feature of trophectoderm development haploid cells are generally considered to be abnormal and may correlate with a low probability of further embryonic development.
In most dividing cells, a series of checkpoints act to ensure that each phase of the cell cycle is completed before progression to the next. It is possible that deficiency of the metaphase-anaphase checkpoint, which prevents anaphase from occurring until all chromosomes are properly attached to a bipolar mitotic spindle, could also be responsible for the chromosome malsegregation seen in human embryos. Absence of this checkpoint has been demonstrated in murine oocytes (Le Maire-Adkins et al., 1997) and a similar situation may exist in humans. If so the checkpoint may remain non-functional until the expression of the embryonic genome begins. This is not thought to occur until the 4-cell stage or possibly later (Braude et al., 1988), giving a number of divisions during which errors could accumulate. If the cell cycle and other essential metabolic functions are largely driven by maternal factors during the preimplantation stage then this would also explain the survival of embryos with monosomy, nullisomy or multiple aneuploidy during this phase of development. It seems likely that cells containing such abnormalities would die soon after the production of their own mRNA and proteins became necessary.
Whether embryos generated using IVF techniques can truly reflect that which occurs following natural fertilization is unknown. It may be that certain ovarian stimulation or embryo culture protocols used for IVF exacerbate problems that exist in vivo. Various cellular stresses may be caused by inappropriate culture media and can result in chromosome damage (Martin et al., 1990). Culture induced errors have been shown to affect meiotic chromosome segregation and may also influence mitotic divisions (Almeida and Bolton, 1995; Dumoulin et al., 1995). It may be significant that many of the traditional embryo culture media used are relatively simple and do not necessarily mimic conditions within the Fallopian tube. Evidence that hormonal stimulation protocols and/or culture conditions do influence aneuploidy rates has also been found (Munné et al., 1997). This may have additional significance for a subset of IVF/PGD patients that produce a disproportionately high number of chaotic embryos (Delhanty et al., 1997). Despite these possible influences the high frequency of aneuploidy in spontaneous abortions and the generally low fecundity in humans is suggestive of high levels of aneuploidy in natural conceptions. Furthermore, the available data indicates no increase in chromosomal abnormality for babies conceived using IVF procedures (Seppala, 1985; SART & ASRM, 1995).
In most IVF units each cycle provides <30% of couples with a child. Several embryos are usually available for transfer and those chosen are selected on morphological grounds. However, even the most highly aneuploid embryos in this study were of good morphology. These embryos have little chance of forming a viable pregnancy and their exclusion using techniques such as single cell CGH might increase IVF pregnancy rates. Miscarriage rates have been reduced by the selection of euploid embryos after analysis of just six chromosomes and if all 24 types of chromosome were assessed an even more significant improvement would be expected (Munné et al., 1999). We are currently working to reduce the length of the procedure so that it can be applied to PGD, and initial results have been promising. With just 24 h hybridization, we are able to detect trisomy for chromosomes 13, 14 and 18. Alternatively transfer at the blastocyst stage or embryo freezing could be considered, giving extra time for diagnosis.
The high rates of aneuploidy and mosaicism that have been detected in this study may explain the relatively poor fecundity rate of humans as a species and the low success rates of assisted conception techniques. Our single cell CGH technique has overcome the limitations of earlier cytogenetic methods and for the first time has allowed the copy number of every chromosomal region over 10 Mb to be assessed in virtually all cells. Although the 12 embryos studied here represent too small a sample to establish the absolute level of aneuploidy and mosaicism they nonetheless provide extremely interesting and important data on the nature of chromosomal abnormality within human embryos. The extent of aneuploidy within chaotic embryos was finally revealed as were aneuploidies not previously recorded in human conceptions. Chromosome breakage with loss and gain of reciprocal fragments in daughter cells was also observed, and importantly entirely euploid embryos, that may have a greater probability of producing a child, were detected. It is our contention that the apparent breakdown in normal chromosome segregation seen in these embryos is a consequence of absent or deficient cell cycle checkpoints.
Note added in proof
Similar cytogenetic data have recently been obtained by Voullaire et al. (2000).
Acknowledgments
We thank Mr Paul Serhal, Director, and all the staff and patients of the Assisted Conception Unit of University College Hospital. This work was supported initially by the Wellcome Trust (grants O46416 and O39938) and later by the Medical Research Council in the form of a fellowship awarded to D.W.
Notes
1 To whom correspondence should be addressed at: The Institute for Reproductive Medicine and Science, St Barnabas Medical Centre, 101 Old Short Hills Road, Suite 501, Livingston, NJ 07052, USA. E-mail: daganwells{at}hotmail.com 
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Submitted on February 21, 2000; accepted on July 27, 2000.
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