Molecular Human Reproduction vol.6 no.11 pp. 1055–1062, 2000
Comprehensive chromosomal analysis of human preimplantation
embryos using whole genome amplification and single cell
comparative genomic hybridization
Dagan Wells1 and Joy D.A.Delhanty
Department of Obstetrics & Gynaecology, University College London Medical School, 86–96 Chenies Mews, London
WC1E 6HX, UK
1To
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: [email protected]
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.
Key words: 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
© European Society of Human Reproduction and Embryology
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;
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D.Wells and Joy D.A.Delhanty
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
1056
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), 1⫻ 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, 1⫻ 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, 2⫻ 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/2⫻ SSC (20⫻ saline sodium citrate
is ⫽ 150 mmol/l NaCl; 15 mmol/l sodium citrate, pH 7) lasting 1 h.
Slides were then washed twice with 2⫻ 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/2⫻ 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/2⫻ SSC at 45°C, twice in 2⫻ 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
CGH of human preimplantation embryos
Table I. Summary of comparative genomic hybridization (CGH) data from 64 embryo cells.
Embryo
no.
Proportion of cells
assessed
CGH interpretation
Classification
1
2
3
6/6
4/4
3/4
normal
mosaic
mosaic
4
5
6
7
8
9
6/7
6/8
3/4
6/6
6/7
5/6
10
11
12
5/7
6/6
8/8
46, XX (6)
45, XX, –1 (3); 46, XX (1)
47, XY, ⫹del(2)(:q32.1-qter) (1); 47, XY, del(2)(pter-q32.1:), ⫹del(2)(pter-q32.1),
del(7)(pter-q31.3:) (1) 45, XY, –2, –2, ⫹del(7)(:q31.3-qter) (1)
46, X, ⫹21 (6)
46, XX (6)
46, XY (1); 46, XX (1); 47, XXY (1)
47, XXY (3); 46, XX (3)
46, XX (4); 47, XX, ⫹19 (1); 46, XX, del(1)(q10-qter) (1)
43, XYY, -2, -6, ⫹8, -11, -13, -22 (1); 47, Y, ⫹1, ⫹2, ⫹3, –13, ⫹14, –16 (1); 45, XX, -1, -3, ⫹9,
⫹13, -14, ⫹15, –17, ⫹19, -20 (1); 43, XYY, -7, -15, -16, -22 (1); 45, XXYY, -2, -5, -10, -11, ⫹12, -13,
⫹15, ⫹21, -22 (1)
46, XY (5)
46, XY (5); 45, XY, -20 (1)
46, XY (4); 45, XY, -21 (1); 49, XXYY, -1, -2, -2, ⫹3, ⫹4, ⫹5, ⫹7, ⫹8, -9, ⫹12, ⫹13, -21, -22 (1)
48, XXYY, ⫹18, -20 (1); 45, XY, ⫹4, ⫹10, -11, ⫹13, -19, -21, -22 (1)
aneuploid
normal
mosaic
mosaic
mosaic
chaotic
normal
mosaic
chaotic
A total of 64 blastomeres were successfully analysed by CGH. The karyotypes were predicted based upon changes in red:green fluorescence ratio along the
length of each chromosome after CGH. These analyses allowed the embryos to be classed as normal (no abnormalities detected in any of the cells); mosaic
(more than one cytogenetically distinct cell line present); aneuploid (all cells displayed an identical chromosome error); or chaotic (multiple aneuploid cell
lines due to random segregation of chromosomes). The number of cells having each chromosomal constitution is shown in parentheses. Cells found to display
chromosome breakage are underlined
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.
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
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D.Wells and Joy D.A.Delhanty
Figure 1. Results of comparative genomic hybridization (CGH) on cells from embryo no. 2. (a) Results from a normal metaphase spread
hybridised with oligonucleotide primed polymerase chain reaction (DOP–PCR) amplified normal male DNA (red fluorescence) and amplified
DNA from a single blastomere (green fluorescence). All autosomes are normal (1:1 green:red ratio) except for chromosome 1, which
appears more red than the others (ratio is ⬍1:1.2). Increased green fluorescence on the X chromosome and more red on the Y also indicate
that the cell tested was female. (b) Profiles of green:red ratio along the length of chromosome 1 revealing that cells 2–4 contain the same
aneuploidy, predicted to be monosomy 1, while cell 1 is normal. Ratios are: 1:1 (black line); 1:1.2 (red line); 1.2:1 (green line). The blue
line depicts the mean green:red ratio obtained by analysis of at least five metaphase spreads.
Figure 2. Results of comparative genomic hybridization (CGH) on
cells from embryo no. 3 revealing chromosome breakage and
reciprocal loss/gain of chromosome fragments. Fragments gained
appear relatively more green, while those lost appear more red. Cell
one is predicted to contain an extra piece of the long arm of
chromosome 2 (q32.1-qter) as well as two normal copies of
chromosome 2. The second cell contains two copies of an abnormal
chromosome 2 that lack the region of 2q that is duplicated in cell 1.
This cell also has one normal chromosome 2 and a deleted
chromosome 7 which lacks the terminal portion of the long arm
(q31.3-qter). The third cell lacks any copies of chromosome 2, but
contains an extra fragment of chromosome 7 (q31.3-qter) reciprocal
to that missing in cell 2. All other chromosomes in these cells were
normal.
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CGH of human preimplantation embryos
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 7q31.3qter, 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.
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 welldocumented 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
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D.Wells and Joy D.A.Delhanty
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 intracytoplasmic 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
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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
CGH of human preimplantation embryos
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 metaphaseanaphase 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.
Acknowledgements
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.
Note added in proof
Similar cytogenetic data have recently been obtained by Voullaire
et al. (2000).
1061
D.Wells and Joy D.A.Delhanty
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Received on February 21, 2000; accepted on July 27, 2000