MHR-Basic Science of Reproductive Medicine Vol.14, No.2 pp. 117–125, 2008
Advance Access publication on January 25, 2008
doi:10.1093/molehr/gam087
Analysis of the evolution of chromosome abnormalities
in human embryos from Day 3 to 5 using CGH and FISH
D.D. Daphnis1,2,3, E. Fragouli1, K. Economou2, S. Jerkovic2, I.L. Craft2, J.D.A. Delhanty1
and J.C. Harper1
1
UCL Centre for Preimplantation Genetic Diagnosis, Department of Obstetrics and Gynaecology, University College London, 86-96
Chenies Mews, London WC1E 6HX, UK; 2London Fertility Centre, Cozen’s House, 112a Harley Street, London W1G 7JH, UK
3
Correspondence address. E-mail: [email protected]
The use of interphase fluorescent in situ hybridization (FISH) has shown that a large number of human embryos exhibit
chromosomal abnormalities in vitro. The most common abnormality is mosaicism which is seen in up to 50% of preimplantation
embryos at all stages of development. In this study, comparative genomic hybridization (CGH) was used to analyse 1 – 2 cells biopsied on Day 3 of development while the rest of the embryo was cultured until Day 5. Embryos were spread on Day 5 and analysed
by FISH using probe combinations that varied depending on the CGH result, to investigate the progress of any abnormalities
detected on Day 3. A total of 37 frozen – thawed embryos were analysed in this study. One gave no CGH or FISH results and
was excluded from analysis. Six embryos failed to give any FISH result as they were degenerating on Day 5. Thirty embryos provided results from both techniques. According to the CGH results, the embryos were divided into two groups; Group 1 had a
normal CGH result (13 embryos) and Group 2 an abnormal CGH result (17 embryos). For Group 1, three embryos showed
normal CGH and FISH results, while 10 embryos were mosaic after FISH analysis, with various levels of abnormalities. For
Group 2, FISH showed that all embryos were mosaic or completely chaotic. The combination of CGH and FISH enabled the
thorough investigation of the evolution of mosaicism and of the mechanisms by which it is generated. The main two mechanisms
identified were whole or partial chromosome loss and gain. These were observed in embryos examined on both Day 3 and 5.
Keywords: blastocyst; chromosome abnormalities; comparative genomic hybridisation; mosaicism; preimplantation embryos
Introduction
Fluorescent in situ hybridization (FISH) has been employed for the
investigation of numerical and structural chromosome abnormalities
both in the clinical PGD setting, but also in a research context
(Harper et al., 1995; Delhanty et al., 1997; Munné et al., 2002;
Simopoulou et al., 2003; Baart et al., 2004; Coonen et al., 2004).
Such studies have shown that at least 40– 50% of human embryos
are chromosomally mosaic, whereas some of them show such high
levels of abnormalities, that they are considered to be completely
chaotic. Mosaicism and chaotic embryos have been observed at all
stages of preimplantation development (Ruangvutilert et al., 2000,
Gonzalez-Merino et al., 2003; Coonen et al., 2004; Bielanska et al.,
2005) but using FISH a limited number of chromosomes can be examined. These findings therefore led to the question of whether any
embryo was uniformly chromosomally normal at this early stage of
human development (Delhanty, 2001).
Comparative genomic hybridization (CGH) is a method closely
related to FISH, which allows the copy number of every chromosome
to be assessed in a single hybridization by reference to a normal DNA
sample. CGH combined with a whole genome amplification (WGA)
approach has been successfully applied for the analysis of single
embryonic blastomeres (Voullaire et al., 2000; Wells and Delhanty,
2000; Wilton et al., 2001, Voullaire et al., 2002; Trussler et al.,
2004), and MII oocyte– polar body pairs (Fragouli et al., 2006) and
has provided an insight into the true incidence of mosaicism and
aneuploidy.
The aim of our study was to investigate the evolution of chromosome abnormalities between the cleavage and blastocyst stages. To
achieve this, we employed CGH to examine the entire genome of
1–2 blastomeres biopsied from spare non-transferred embryos on
Day 3 (cleavage stage). FISH analysis of the remaining embryo followed on Day 5 (blastocyst stage), with the application of specific
probe sets, selected on the basis of the CGH results. The combination
of CGH and FISH enabled us to carry out a detailed investigation into
embryonic mosaicism, its underlying mechanisms, and how these
affect embryos during the various stages of human preimplantation
development.
Materials and Methods
This study was performed on spare frozen– thawed good quality embryos
donated from patients undergoing IVF and ICSI cycles at the London Fertility
Centre. Informed consent was obtained in all cases and the study was licensed
by the Human Fertilization and Embryology Authority.
Patients
Thirteen couples with no known chromosomal abnormalities participated in
this investigation. These couples were referred to the London Fertility Centre
for IVF or ICSI treatment, due to different fertility problems. A total of
# The Author 2008. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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117
Daphnis et al.
37 embryos were donated for the purpose of this study, while the average
maternal age was 33.1 years (age range: 28– 39 years).
Superovulation
Pituitary down regulation was achieved by the administration of gonadotrophin
releasing hormone analogue (GnRHa) depot injection (Prostap, leuprorelin
acetate; Wyeth Laboratories, UK) 3.75 mg or nasal spray (Synarel, nafarelin
acetate; Pharmacia, UK) 2 mg/ml per day starting on cycle Day 2 for 10–13
days. Ovarian stimulation was commenced using recombinant follicle stimulating hormone (FSH) (Puregon; Organon, UK) either 150 IU/day if under 38 or
225 IU/day if 38 or older. The patients were monitored with serum estradiol
levels and vaginal ultrasound regularly starting on Day 6 of stimulation and
the dose of FSH adjusted accordingly. Between Days 12 and 14 of stimulation
patients who had a cohort of follicles of 18–20 mm in diameter were given
10 000 IU of human chorionic gonadotrophin (hCG) (Pregnyl; Organon, UK)
to trigger ovulation. Thirty-six hours later, follicular aspiration was carried
out under transvaginal ultrasound guidance.
Cell isolation and lysis
Each blastomere was washed through three drops of PBS/PVA and collected in
0.2 ml PCR tubes containing 3 ml lysis buffer [consisting of 125 mg/ml Proteinase K (Sigma, UK), 17 mM sodium dodecyl sulphate (SDS; BDH chemicals, UK), Nuclease-Free Water (H2O; Promega, UK) and overlaid with oil.
Cell lysis was achieved by incubating at 378C for 1 h, followed by an incubation at 958C for 15 min to inactivate the PK enzyme.
WGA and CGH protocol
The degenerate oligonucleotide primed–polymerase chain reaction (DOP–
PCR) was employed to amplify the genome of embryonic blastomeres (test
DNA) and single 46,XY buccal cells (reference DNA). The protocol used
was as described in Fragouli et al., (2006). The CGH protocol was performed
as described by Wells et al. (2002) with modifications described in Fragouli
et al. (2006). The test embryonic DNA was labelled in spectrum green
and the control 46,XY buccal cell DNA was labelled in spectrum red. The combination of DOP– PCR and CGH, applied to single cells has been previously
validated by our group (Wells et al., 1999) as well as others (Voullaire et al.,
1999).
Embryo culture
Oocytes were retrieved using Flushing Medium (supplemented with Sodium
Pyruvate, Human Serum Albumin (HSA), Heparin 10 IU/ml, Penicillin
50 000 IU/l, Streptomycin 50 mg/l and HEPES; Medicult Ltd, UK), incubated
in 6% CO2 in air, at 378C, inseminated and cultured in 500 ml of IVF Medium
(Bicarbonate buffered medium containing Human Serum Albumin, Penicillin
and Sodium pyruvate; Vitrolife, Scandinavia). On Day 1, oocytes were assessed
for the number of pronuclei (PN) and transferred into 25 ml microdroplets of
Cleavage Medium (Bicarbonate buffered medium containing Human Serum
Albumin, Penicillin-G, EDTA, Glucose, inorganic salts and amino acids;
G-1, Vitrolife, Scandinavia). On Day 3, the best embryos were selected for
transfer and suitable spare embryos were cryopreserved. Only embryos that
arose from a bipronucleate zygote were included in the study.
Embryo freezing
Microscopy and image analysis
Metaphase spreads were observed using an Olympus BX 40 fluorescent microscope with a cooled charged–coupled device (CCD) system and filters for the
fluorochromes used. Ten metaphases were captured on average per hybridization. Analysis and interpretation of the captured images was feasible by
using Vysis Quips CGH software (Vysis/Abbott, UK) that converted fluorescent intensities into a red– green ratio for each chromosome. Equal sequence
copy number between the test and the reference DNAs was seen as no
fluctuation of the ratio profile from 1:1. Green:red fluorescence ratios of
.1.2:1 indicated gain of chromosome material, whereas ratios of ,0.8:1
indicated loss.
Embryo spreading and FISH
Good quality Day-1 embryos were considered for freezing when two PN and
two polar bodies were visible 16– 18 h after the oocytes were subjected to
sperm either by IVF insemination or ICSI. Day-3 embryos were considered
to be of good quality when they consisted of 6 –8 cells and were of grade 2
and above.
All embryos (either Day 1 or 3) were placed for a 5-min wash in Cryo-PBS
medium (Vitrolife; Scandinavia) and then taken through consecutive washes of
embryo freezing solutions 1 and 2 (EFS1 and EFS2; Vitrolife, Scandinavia) for
10 min each at room temperature. The embryos were loaded into the freezing
straw (maximum two embryos per straw) and placed into a cryobath. The cryobath was linked to a computer programme which controlled temperature until it
reached 21808C.
The embryos were spread on Day 5 as described previously by Harper et al.
(1994).
The FISH protocol was carried out as described by Daphnis et al. (2005). The
probes and conditions used are summarized in Table I. Each FISH experiment
included a control male lymphocyte slide with mapped nuclei in order to assess
efficiency of probe hybridization in the sequential rounds. The first round of
FISH was carried out using the probe cocktail for chromosomes X, Y and
18. This round was performed to confirm the embryonic sex as was identified
during the CGH analysis, and therefore served as an internal control between
the FISH and CGH methods by verifying that both methods would give the
same result for the sex chromosomes and chromosome 18. Probe combinations
which varied depending on the CGH result were used during the second FISH
round. All probes were commercially available (Abbott/Vysis, UK).
Embryo thawing
Embryo classification and scoring of embryos
All thawed embryos (either at PN stage or Day 3 cell stage) were taken through
consecutive washes of embryo thaw solutions 1, 2 and 3 (ETS1, ETS2 and
ETS3; Vitrolife, Scandinivia) for 5, 5 and 10 min, respectively, at room temperature. A final wash was performed through Cryo-PBS medium for 5 min at
room temperature followed by 5 min on a heated stage (388C). PN stage
embryos, after thaw, were initially cultured in G1 (Vitrolife, Scandinavia)
medium until Day 3 and subsequently in G2 (Vitrolife, Scandinavia) medium
until blastocyst stage, while Day 3 thawed-embryos were directly cultured in
G2 medium.
The criteria for signal scoring were as suggested by Hopman et al. (1988).
Probe signals had to be a minimum of a width apart to be scored as being
two or more separate signals. Embryo classification took place according to criteria described in Delhanty et al. (1997). FISH efficiency was measured by
using a control male lymphocyte slide in each FISH experiment.
Embryo biopsy
The embryos were incubated for 1–2 min in Caþ2/Mgþ2-free biopsy medium
(G-PGD; Vitrolife, Scandinavia). The zona pellucida surrounding the embryo
was drilled with a laser system as described by Veiga et al. (1999). One to
two cells were aspirated gently from the embryo and the embryo was subsequently washed in IVF medium. The embryos were then placed in G-2
blastocyst medium (Vitrolife, Scandinavia) from Day 3 to 5.
118
Mosaicism and causative events
Embryos were scored on Days 3 and 5. Classification of chromosomal mosaicism and associated mechanisms were as described in Daphnis et al. (2005).
Diploid mosaic embryos with aneuploid cells were considered to have arisen
through three different mechanisms: (i) when the embryo contained cells
with monosomies, then the mechanism was classed as ‘chromosome loss’
(CL), (ii) when the embryo contained cells with trisomies, then the mechanism
was classed as ‘chromosome gain’ (CG) and (iii) when the embryo had monosomies and trisomies of the same chromosome(s) in different cells, this was
classified as mitotic non-disjunction (MND). Nuclei with multiple abnormalities affecting at least three chromosomes where classed as chaotic and not
Analysis of the evolution of chromosome abnormalities
Table I. FISH probe combinations and stringency conditions.
Chromosomes
Probe combination
Stringency conditions
Probe vol (ml)
X, Y, 18
9, 16, 22
3, 11, 13
10, 14
1, 16
3, 6, 18
Y, 4
X, Y, 16
Xcep/Ycep/18cep
9cep/16cep/22LSI
3cep/11cep/13LSI
10cep/14q
1p/1q/16cep
3cep/6cep/18q
Ycep/4cep
Xcep/Ycep/16q
a
2 ml
0.5 ml/0.5 ml/0.7 ml
0.5 ml/0.5 ml/0.7 ml
0.5 ml/0.6 ml
0.6 ml/0.5 ml/0.5 ml
0.5 ml/0.5 ml/0.6 ml
0.5 ml/0.5 ml
0.6 ml/0.5 ml/0.6 ml
Denaturation
P-H washes
Co, 758 for 5 min
Sep, 758 for 5 min
Sep, 758 for 5 min
Co, 758 for 5 min
Sep, 758 for 5 min
Sep, 738 for 5 min
Co, 758 for 5 min
Co, 758 for 3 min
60% FA
Vysisb
Vysisb
60% FA
50% FA
60% FA
60% FA
50% FA
P-H washes, post-hybridization washes; Co, co, denaturation; Sep, separate denaturation.
a
This was a probe cocktail, where 2 ml from the cocktail were added into the probe mixture.
b
The post-hybridization washes termed ‘long washes’ proposed by Vysis (UK).
included in the analysis of the events. This included nullisomies and
tetrasomies.
During the course of this study, we were also able to accurately identify
chromosome breakage leading to partial mosaicism.
Results
CGH analysis of embryos
Out of a total of 37 embryos examined, CGH and FISH results were
obtained for 30 embryos. Both methods failed for one embryo,
which was considered to be of poor quality and was excluded from
the study. Additionally, there were six embryos which degenerated
prior to being spread for FISH analysis. However, all these embryos
had at least one of their cells biopsied on Day 3 (7 cells in all).
CGH analysis of these blastomeres demonstrated a normal karyotype
in three cases, two were classed as aneuploid and one was considered
to be chaotic. A total of 54 blastomeres were biopsied on Day 3 and 48
(88.9%) of those provided an interpretable CGH result with the
method being successful for at least one cell from each of the 36
embryos.
Samples that provided no analysable CGH results had fluorescent
signals on metaphase chromosomes that were too weak for analysis
and interpretation to be carried out. CGH failure was attributed to
different factors, such as WGA failure due to blastomeres being
anucleate (e.g. 3.1b), incomplete cell lysis, or weak counterstain
chromosome banding (e.g. 1.1a, 1.2b, 2.1b, 2.2b and 7.2b).
During analysis and interpretation of CGH images, known artifacts
(Kallioniemi et al., 1994) were observed for the heterochromatic
regions and the short and/or long arm telomeres of some chromosomes
and were excluded from analysis. Consequently abnormalities involving distal breakpoints could not be confidently detected.
FISH optimization
Overall, 90% (88 –95) of the control 46,XY lymphocyte nuclei
showed diploid signals for all 14 probes used (Table II) with the
highest hybridization efficiencies being observed for the the a-satellite
probes for chromosomes 18cep and Xcep, and the lowest for the subtelomeric and/or locus-specific probes for chromosomes 1p, 13LSI
and 16q. The difference in the efficiency of the Xcep and Ycep
probe (used in the first and second round of FISH) was not statistically
significant (P , 0.05) and can be attributed to the fact that FISH efficiency decreases with sequential rounds of hybridization due to the
degeneration of the DNA. Furthermore, the 5% difference
between the first round overall efficiency (95%) and the various
Table II. Probe efficiencies scored in 200 interphase nuclei of each control
slide whilst carrying out 2-round FISH.
Probe combinations
Efficiency
per probe (%)
Overall
efficiency (%)
Xcep/Ycep/18cep
9cep/16cep/22LSI*
10cep/14q*
3cep/11cep/13LSI
1p/1q/16cep*
3cep/6cep/18q*
Ycep/4cep*
Xcep/Ycep/16q*
97/96/97
92/94/91
90/90
92/92/89
89/90/93
95/91/90
94/92
92/93/88
95
90
88
90
87
90
90
87
*Carried out in the second round.
second round efficiencies has been well documented (Conn et al.,
1998; Munne et al., 1998; Ruangvutilert et al., 2000).
FISH analysis of embryos
FISH results were obtained for 30 embryos (343/359 spread blastomeres—96%). No results were obtained in cases of cell loss due to
spreading, or failure of the first and/or second FISH rounds. The
results obtained for the sex of all the embryos were consistent
between CGH and FISH.
Interpretation of CGH and FISH results
A total of thirty embryos (41 biopsied blastomeres) provided results
from both techniques. Out of these, only three (10%) were identified
to be uniformly normal both after CGH and FISH analysis. The rest
of the embryos demonstrated various levels of abnormality and
mosaicism. Table III shows the embryos with a normal CGH result
(Group 1) while the embryos which were classed as abnormal after
CGH (Group 2) can be seen in Table IV.
Normal CGH result
CGH showed normal chromosome complements in biopsied cells
from thirteen embryos (Group 1) (Table III). Two blastomeres
(11%) failed to give interpretable results (1.1a and 2.1b). For
embryos which had two cells available (embryos 6.2, 13.2 and 13.3)
for analysis, the sex CGH result was in agreement between both
119
Daphnis et al.
Table III. Results from embryos showing normal CGH findings (Group 1) followed by sequential FISH analysis.
Embryo no.
1.1
Cell
CGH Result on Day 3
FISH Result on Day 5b
Cells
Results (no. of cells)
13
Dip (7)/þX,þY(2)/þ22(2)/-18(2)
Mosaic Diploid/Aneuploid
2
4
Dip (1)/þX,þY(1)
Dip (4)
Mosaic Diploid/Aneuploid
Uniformly normal
5
Dip(3)/-22(2)
Mosaic Diploid/Aneuploid
Dip (3)/218(2)/þ18(1)/222(1)/216,-18(1)
Dip (7)
Dip (25)/218(1)/2X(1)/216,þ22(1)/
chaotic(3)
Dip (3)/tet(2)/chaotic(1)
Mosaic Aneuploid/Diploid
Uniformly normal
Mosaic Diploid/
Aneuploid/Chaotic
Mosaic Diploid/Polyploid/
Chaotic
Mosaic Diploid/
Aneuploid/Chaotic
Uniformly normal
Mosaic Diploid/Chaotic
Mosaic Diploid/Polyploid/
Aneuploid/Chaotic
Mosaic Diploid/Polyploid/
Aneuploid/Chaotic
7.1
9.1
9.2
a
b
a
a
b
a
b
a
a
a
No result
rev ish XY
rev ish XY
rev ish XY
No result
rev ish XY
rev ish XY
rev ish XX
rev ish XY, enh(Xp11.2-q22)a
rev ish XY, dim(Yq12)a
8
7
31
9.4
a
rev ish XX, dim(11q23-q25)a
6
9.5
a
rev ish XY
11.1
12.1
13.2
a
a
a
b
a
b
rev ish
rev ish
rev ish
rev ish
rev ish
rev ish
1.3
2.1
6.2
13.3
XY, enh(Yp11.3-11.2)a
XX
XX, dim(19p13.3-p13.2)a
XX
XX
XX
Interpretation
11
Dip(7)/2X(3)/chaotic(1)
10
3
15
Dip (10)
Dip(1)/chaotic(2)
Dip(7)/tet(1)/trip(1)/29,þ22(1)/chaotic(5)
35
Dip(28)/trip(2)/þX(2)/chaotic(3)
a
These findings were considered as artefacts. dip¼diploid, tet¼tetraploid, trip¼triploidy. The 2 indicates loss of chromosome and þ indicates gain of
chromosome, e.g. þ18 is trisomy 18 or 21 is monosomy 1.
b
The 1st round of FISH was always performed with the X/Y/18 probe cocktail. The 2nd round was performed with the 9/16/22 probe combination unless stated
in bold and underlined combinations.
cells. In three embryos (2.1, 9.1 and 11.1) the CGH demonstrated a
normal karyotype, which was confirmed during FISH.
Ten embryos were identified to be mosaic with various levels of
abnormalities upon FISH examination. The probe combination
employed during the first FISH round for all of these embryos was
Xcep/Ycep/18cep, while chromosomes 9, 16 and 22 were scored
during the second round.
For the embryos with a normal karyotype on Day 3, a total of
150 cells were spread on Day 5 and 106 (70.6%) of these were
diploid for the chromosomes tested. Six of the ten embryos (60%)
classed as mosaic contained of cells with abnormal chromosome
complements, such as nullisomies, and tetrasomies and also cells in
which abnormalities were scored for more than three chromosomes.
These were classed as mosaic chaotic. Embryos 9.4, 13.2 and 13.3
were found to contain polyploid cells (4% overall, either triploid or
tetraploid).
Abnormal CGH result
Table IV shows details of the embryos with one or more abnormalities
scored in at least one biopsied cell (Group 2). Therefore, the FISH
probe combinations were tailored according to the chromosome
anomalies that the CGH revealed. Twenty-eight blastomeres were
biopsied from these 17 embryos, with the CGH being successful for
25 (89.3%).
During CGH analysis, one cell displayed a normal karyotype with
the other being characterized as abnormal (4.2a, 7.3b, 8.1a and 9.6b)
in four different embryos. Chromosomes were shown to be affected
either by partial or whole duplications or deletions. Chromosomes
1(x4), 16(x5) and 22(x4) demonstrated the highest incidence of
chromosome deletion (whole or partial), while chromosomes 1(x4),
2(x3) and Y(x5) revealed the highest rate of chromosome duplication
(whole or partial). Chromosomes 13 and 15 were identified to be completely deleted on three different occasions, but no duplication, partial
or whole, was observed.
120
Chromosome 1 was involved in three instances of partial chromosome duplication or deletion with breakage being observed at the
1p36.1 locus (Fig. 1, bold letters in legends). This might be attributed
to the presence of a fragile site at that specific chromosome location.
Similarly, on chromosome 2 breakage was identified at a possible
fragile site between regions 2q21 to q31 (Fig. 1, bold letters in
legends).
On Day 5, FISH analysis revealed that embryos were mosaic or
completely chaotic at the time of spreading. Additionally, out of
183 cells examined on Day 5, only 68 (37.1%) were diploid for the
chromosomes tested. In total, 12/17 (70.5%) embryos consisted of at
least one cell with a chaotic complement, while four of them
(23.5%) were completely chaotic (Table IV). In all, seven embryos
(41%) totally lacked cells diploid for the chromosomes tested and
all were characterized to be mosaic aneuploid or mosaic aneuploid/
chaotic.
The FISH results for embryos 4.2, 7.3, 8.1 and 9.6, for which CGH
analysis revealed one of two blastomeres with a normal chromosome
complement while the other was abnormal, varied. Hence, two
embryos (7.3 and 9.6) consisted of predominantly diploid cell lines,
while the other two (4.2 and 8.1) were in their majority aneuploid.
In 15 out of the 17 embryos the abnormality seen in a biopsied cell
after CGH was confirmed during FISH analysis on Day 5 (Table IV).
We also investigated whether chromosome breakage persisted in later
embryonic development, and could therefore be detected with FISH.
CGH analysis of cell 9.7a identified a gain of the chromosome region
1p36.3-q21, and absence of 1q31-q44 ((rev ish XY, enh(1p36.3-q21),
dim(1q31-q44)). We therefore selected to examine the remainder of
embryo 9.7 with two sub-telomeric probes for the short and long arms
of chromosome 1 (Table IV). Entire loss of 1 was seen in two blastomeres, partial loss of 1p and gain of 1q was scored for one cell, while
another cell showed partial loss of 1q and gain of 1p. Additionally,
CGH detected chromosome breakage for 4p in embryo 12.2, and FISH
was able to confirm the partial CL in 2/12 cells.
Analysis of the evolution of chromosome abnormalities
Table IV. Results from embryos showing abnormal CGH findings (Group 2) followed by sequential FISH analysis (over three pages).
Embryo Cell CGH result on day 3
No.
FISH result on day 5b
Interpretation
Cells Results (no. of cells)
1.2
2.2
3.1
a
a
b
a
4.2
b
a
b
6.3
a
7.2
b
a
7.3
b
a
8.1
b
a
b
8.2
a
9.3
a
9.6
a
9.7
b
a
9.8
b
a
10.1
12.2
b
a
a
12.3
a
14.2
a
b
rev ish XY, dim(22)
rev ish XY, dim(18)
No result
rev ish XY,
enh(5pter, 9qter, 17),
dim(4,19)
No result
rev ish XX
rev ish XX, enh(1,
22), dim(16pter, 18)
rev ish XY,
enh(6p25-p21.1)a
rev ish XY, enh(Y)
rev ish XYY,
enh(1p36.2-36.1,
2q31-p25, 5, 7, 8, 18,
19, 20, 21 and Y),
dim(1p31-q44,
2q32-q37, 3, 6, 11,
13, 14, 15)
No result
rev ish XY,
dim(22q11.1-q13)
rev ish XY
rev ish XX
rev ish XX, enh(4,
6qter, 12p11.2-q24.3,
14q21-q32)
dim(2q31-q37, 10)
rev ish XX, enh(9
and 16)
rev ish XY,
dim(15q15-q26, 16)
rev ish XX,
enh(2q21-q33,
3q11.1-q25),
dim(1p36.1-p31, 16,
19 and 22)
rev ish XX
rev ish XY,
enh(1p36.3-q21),
dim(1q31-q44)
rev ish XY, dim(1)
rev ish XY,
enh(17p13-q11,
18p11.3-q11.1),
dim(3p26-p14)
rev ish XY, enh(6)
rev ish XX, enh(20)
rev ish XY, enh(Y),
dim(4pter)
rev ish XY,
enh(Yq11.1-q12),
dim(16q21-q24)
rev ish XX, enh(1,
10, 16qter), dim(8,
13, 21, 22)
rev ish XX, enh(2,6),
dim(9qter,13, 15,
16qter, 17)
2
3
Chaotic(2)
Chaotic(3)
Chaotic
Chaotic
2
Chaotic(2)
Chaotic
Dip(9)/2X(8)/218(5)/218,222(2)/2X,218(2)/þ18,þ16(1)/trip(1)/
chaotic(5)
Mosaic Aneuploid/Diploid/
Polyploid/Chaotic
8
Dip(2)/þX(2)/216,2X(1)/2X,þ22(1)/2X(1)/222(1)
Mosaic Aneuploid/Diploid
3
3cep/11cep/13LSI bChaotic(3)
Chaotic
Dip(13)/þ22(5)/216,222(2)/29(1)/tet(1)/chaotic (8)
Mosaic Diploid/Aneuploid/
Polyploid/Chaotic
10cep/14q b 210(2)/210,þ14qter(1)/chaotic(1)
Mosaic Aneuploid/Chaotic
Dip(6)/chaotic(4)
Mosaic Diploid/Chaotic
Dip(1)/216, 216(2)/216,þ22(2)
Mosaic Aneuploid/Diploid
Dip(13)/216,222(3)/tet(2)/222(2)/218(1)/þX(1)/chaotic(1)
Mosaic Diploid/Aneuploid/
Polyploid/Chaotic
33
30
4
10
4
23
5
Mosaic Aneuploid/Chaotic
1p/1q/16cep b
1qter,21pter,þX(1)/þ1qter,21pter,þY(1)/þ1qter,21pter(1)/216,2X(1)/
chaotic(1)
17
3cep/6cep/18cep bDip(11)/þ6(3)/26(1)/218,þY(1)/2X,þY(1)
Mosaic Diploid/Aneuploid
19
12
Dip(6)/þX,þX(6)/þX(3)/2X(1)/222(1)/tet(1)/trip(1)/
Ycep/4cep bDip(6)/218(2)/2Y(2)/chaotic(2)
Mosaic Aneuploid/Diploid/Polyploid
Mosaic Diploid/Aneuploid/Chaotic
2
Xcep/Ycep/16q b 216,218(1)/216(1)
Mosaic Aneuploid
6
Dip(1)/216(1)/216,222(1)/þ16,þ22(1)/chaotic(2)
Mosaic Aneuploid/Diploid/Chaotic
a
These findings were considered as artefacts.
The 1st round of FISH was always performed with the X/Y/18 probe cocktail. The 2nd round was performed with the 9/16/22 probe combination unless stated
in bold and underlined combinations.
dip ¼ diploid, tet ¼ tetraploid, trip ¼ triploidy. 2 indicates loss of chromosome and þ indicates gain of chromosome, e.g. þ 18 is trisomy 18 or 21 is
monosomy 1
b
121
Daphnis et al.
Figure 1: CGH and FISH analysis results from embryo 7.2. A. Interpretation of CGH experiment showing the cumulative analysis of ten metaphases from blastomere 7.2a. The test sample (blastomere 7.2a) is rev ish XYY, enh(1p36.2-36.1, 2p25-q31, 5, 7, 8, 18, 19, 20, 21 and Y), dim(1p31-q44, 2q32-q37, 3, 6, 11, 13, 14,
15). The blue arrow indicates the possible fragile site for chromosome 1 and the red arrow the fragile site for chromosome 2. B. Results from the two sequential
rounds of FISH in a cell from embryo 7.2. In B1 the cell was subjected to the X(green)/Y(red)/18(aqua) probe cocktail where it showed female (XX) and monosomy
18. In B2 the cell was subjected to the 3(orange)/11(aqua)/13(green) probe combination which showed monosomy 3, nullisomy 11 and trisomy 13 Overall, the FISH
results confirmed the CL events for chromosomes 3 and 11 and revealed reciprocal loss and gain (MND event) for chromosomes 13, 18 and Y
Cytogenetic events leading to mosaicism
Our previous study involving two probes per chromosome (Daphnis
et al., 2005), concluded that some losses may be attributed to FISH
artifacts when they are confined to just one cell. This was considered
when we were scoring FISH signals in this group of embryos by
reviewing the efficiency shown by the control lymphocyte sample
and the overall results.
In total, only 10% (3/30) of the embryos were uniformly normal.
The rest were either mosaic 76.7% (23/30) or completely chaotic
13.3% (4/30). The mechanisms scored on Day 3 and 5 embryos
were either CL, CG or MND. Interestingly, all events involved
whole chromosomes for the embryos where the CGH results showed
normal karyotypes (Group 1). The main mechanism leading to mosaicism was whole CL (50% of embryos) (Table V). However, whole CG
was frequently observed as well (8/18, 44.5% of embryos), with MND
of whole chromosomes affecting only 5.5% of cases (Table V).
In the embryos where there was at least one abnormal karyotype
(Group 2) revealed during CGH analysis, 57 events led to aneuploid
mosaicism. The main mechanism in these cases was whole CL
(37%) followed by whole CG (19%) and whole MND (16%)
(Table VI). The incidence of breakage events leading to mosaicism
122
involving partial chromosomes was also considerable especially for
partial CL, which accounted for 14%. Chromosomes involved in
breakage events included 1, 2, 3, 4, 9, 14, 15, 16, 17 and 18. All
these chromosomes showed breakage in the short (p) or long (q)
arms. Chromosome 18, however, demonstrated a partial CG of
18p11.3-q11.1 during CGH, and a partial CL in the same region in
one cell during FISH. These findings revealed a reciprocal aneuploidy
attributed to MND for that region of chromosome 18 (Tables V
and VI).
Discussion
The aim of this study was to examine the entire chromosome complement of biopsied cells on Day 3 of embryonic development by
CGH, and then perform FISH analysis of the whole embryo on Day
5 to investigate the progress of any abnormalities detected on Day
3. A total of 30 embryos provided results from both techniques.
None showed clear constitutional aneuploidy due to a meiotic error.
Only three embryos (10%) showed normal results both after CGH
and for all the blastomeres analysed by FISH for the chromosomes
tested.
Analysis of the evolution of chromosome abnormalities
Table V. Mechanisms leading to aneuploid mosaicism detected by FISH
analysis for the embryos which revealed cells with normal karyotypes after
CGH (Group 1).
Table VI. Mechanisms leading to aneuploid mosaicism for the embryos,
which revealed a least one cell with abnormal karyotype after CGH
(Group 2).
Embryo
Classification
Event
Chromosome
Embryo Classification
Event
Chromosome
1.1
Mosaic Diploid/Aneuploid
1.3
2.1
6.2
7.1
Mosaic Diploid/Aneuploid
Uniformly normal
Mosaic Diploid/Aneuploid
Mosaic Aneuploid/Diploid
Uniformly normal
Mosaic Diploid/Aneuploid/
Chaotic
Mosaic Diploid/Polyploid/
Chaotic
Mosaic Diploid/Aneuploid/
Chaotic
Uniformly normal
Mosaic Diploid/Chaotic
Mosaic Diploid/Polyploid/
Aneuploid/Chaotic
Mosaic Diploid/Polyploid/
Aneuploid/Chaotic
18
22, X, Y
X, Y
—
22
16, 22
18
—
16, 18, X
22
—
1.2
2.2
3.1
4.2
9.1
9.2
1CL
3 CG
2CG
—
1CL
2CL
1MND
—
3CL
1CG
—
—
—
—
2CL(w)
1CG(w)
1MND(w)
1CL(w)
1CG(w)
2MND(w)
—
2CL(w)
1MND(w)
—
—
—
22, X
16
18
16
Y
22, X
—
9, 16
22
1CL
X
—
—
1CL
1CG
1CG
—
—
9
22
X
1CL(p)/1CL(w)
1CG(p)/2CG(w)
—
1CL(p)/1CL(w)
1CG(w)
1CL(p)/4CL(w)
2CG(p)/1CG(w)
2qter/10
14qter/4, 12
—
15qter/16
22
3pter/16, 18, 19, 22
2qter, 3qter/X
3CL(w)
1CG(w)
2MND(p)
1CL(p)/1CL(w)
1CG(p)/1CG(w)
1MND(p)/
1MND(w)
1CL(w)
1CG(w)
1MND(w)
1CL(p)/1CL(w)
1MND(w)
1CL(p)/1CL(w)
1CG(w)
2CL(p)/3CL(w)
1, 16, X
Y
1pter, 1qter
3pter/X
17pter/Y
18cep/6
9.4
9.5
11.1
12.1
13.2
13.3
Total
13
6.3
7.2
7.3
8.1
8.2
9.3
9.6
9CL (50%)
8CG (44.5%)
1MND (5.5%)
(All events involved whole chromosomes).
CL, chromosome loss; CG, chromosome
non-disjunction.
gain;
9.7
MND,
Chaotic
Mosaic Diploid/
Aneuploid/Polyploid/
Chaotic
Mosaic Aneuploid/
Chaotic
Mosaic Diploid/Chaotic
Mosaic Aneuploid/
Chaotic
Mosaic Diploid/
Aneuploid/Polyploid/
Chaotic
Mosaic Aneuploid/
Chaotic
9.8
Mosaic Diploid/
Aneuploid
10.1
Mosaic Aneuploid/
Diploid/Polyploid
12.2
Mosaic Diploid/
Aneuploid/Chaotic
Mosaic Aneuploid
mitotic
Compared to other similar studies, the normality rate found in our
study is quite low since Wells and Delhanty (2000), Voullaire et al.
(2000) and Trussler et al. (2004) showed rates of 25, 25 and 42.5%,
respectively. This difference could be attributed to the fact that all
three above mentioned investigations analysed good quality cleavage
stage fresh embryos. Additional, events leading to mosaicism may
occur at Days 4 and 5. Our group of embryos was also considered
of good quality, but the freezing –thawing process could have compromised them. Laverge et al. (1998) after analysing 63 frozen –thawed
embryos by FISH concluded that embryos which do not progress
developmentally after freezing and thawing, carry chromosomal
abnormalities. IVF culture conditions could also be responsible for
the high frequency of mosaicism in this study. It has been previously
documented that a sudden decrease in temperature could in turn affect
cytokinesis, leading to the generation of diploid/polyploid embryos
(Munné and Cohen, 1998). In addition, it has been suggested that
embryos produced by different stimulation protocols and cultured
under different conditions have very diverse mosaicism rates
(Munné et al., 1997).
Follow-up FISH analysis indicated that in Group 1 embryos
(embryos with a normal CGH result) the prevailing type of mosaicism
was diploid/aneuploid. Chaotic complements were present in some
cells from six embryos but were much less prevalent compared to
embryos in Group 2.
Embryos 13.2 and 13.3 displayed triploid cells, the origin of which
is unclear. The underlying mechanism that could lead to diploid/
triploid mosaics may be an incorporation of another gamete into one
of the daughter cells generated after the first mitotic division or later
(Muller et al., 1993). Embryo 13.3 displayed mosaicism involving
the sex chromosomes as well as triploidy. Similar observations have
been reported in a CGH study of human Day 3 embryos where 2/12
embryos displayed mosaicism involving only sex chromosomes
Chaotic
Chaotic
Chaotic
Mosaic Aneuploid/
Diploid/Polyploid/
Chaotic
Mosaic Aneuploid/
Diploid
12.3
14.2
Total
17
Mosaic Aneuploid/
Diploid/Chaotic
2CG(w)
2MND(w)
8CL(p) (14%)
21CL(w) (37%)
5CG(p) (9%)
11CG(w) (19%)
3MND(p) (5%)
9MND(w) (16%)
22
20
X
4pter/18
Y
16qter/18
Y
9qter, 16qter/
13,15,17
2, 6
16, 22
CL, Chromosome loss; CG, Chromosome gain; MND, mitotic
non-disjunction.
‘w’ stands for whole chromosome event (either loss, gain or MND).
‘p’ stands for partial chromosome event (either loss, gain or MND.
which were thought to have arisen from 47, XXY zygotes after ICSI
treatment (Wells and Delhanty, 2000).
In 17 embryos at least one cell showed an abnormal karyotype when
analysed by CGH. Ultimately, all 17 embryos were classified as
mosaic or chaotic (Group 2, Table IV). Four embryos (23.5%) were
completely chaotic with three or more abnormalities affecting different chromosomes in all cells. All four embryos when analysed by
FISH on Day 5 were arrested consisting of only 2–3 blastomeres
each. The latter is consistent with suggestions that this fully chaotic
state is associated with impaired development (Delhanty and Handyside, 1995; Delhanty et al., 1997). Evsikov and Verlinsky (1998) postulated that embryos with chaotic chromosome segregation do survive
to the blastocyst stage but will not progress further and would fail to
implant.
123
Daphnis et al.
Apart from embryos 4.2 and 7.3 all embryos were arrested (,30
blastomeres) and overall had a decreased number of blastomeres
with a diploid cell component compared to Group 1. It has been
implied from blastocyst studies that embryos with a lower proportion
of a diploid cell line (therefore a higher degree of mosaicism) have a
low developmental potential (Ruangvutilert et al., 2000). However,
this selection against embryos with a high degree of mosaicism does
not operate perfectly.
Similar data were obtained during our FISH study on Day 5
embryos for the group of embryos (Group I), which were arrested
(Daphnis et al., 2005). Wells and Delhanty (2000) also found an
increased incidence of chromosome 1, but also chromosome 2 deletions, (partial or whole) on six different occasions. Baart et al.
(2004) also found chromosome 1 abnormalities affecting 9/29 (31%)
embryos when analysed by FISH. In our study chromosome 2 was
mostly involved in duplications (partial or whole). Furthermore, in a
recent CGH study nine instances of trisomy 22 were found (Trussler
et al., 2004), which is in accordance with a FISH study investigating
differences in chromosome susceptibility to aneuploidy that concluded that trisomy 22 is the most common aneuploidy when
meiotic and mitotic events are included (Munne et al., 2003). In our
study, aneuploidy of chromosome 22 was also increased with losses
being more prominent than gains.
FISH analysis was able to confirm the sex status of all 17 embryos
as shown by CGH. Analysis of the CGH and FISH data demonstrated
results ‘in agreement’ for 15/17 embryos (probes were unavailable for
the other two embryos). By using FISH to examine these embryos on
Day 5, we were able to detect either the same abnormalities as those
identified by Day 3 CGH analysis, or the reciprocal ones affecting
the chromosome(s) in question.
The use of CGH to examine human preimplantation embryos has
enabled the detection of chromosome breakage leading to structural
anomalies. Partial aneuploidy due to chromosome breakage is likely
to result in an unstable karyotype through the formation of acentric
and dicentric chromosomes (Voullaire et al., 2002). Chromosome
breakage appears to belong in a separate category from whole chromosome changes. Interestingly, chromosome breakage was not observed
in embryos with a normal CGH result on biopsy.
Mechanisms of aneuploidy mosaicism
The most common mechanism of mosaicism was found to be CL,
closely followed by CG, affecting both Group 1 and Group 2
embryos. However, the difference in frequency between CL, CG
and MND was not statistically significant. Similar results were
obtained in our previous study (Daphnis et al., 2005), adding to the
conclusion that CL, which most likely arises due to anaphase lag, is
the most common mechanism, in line with the findings of others
(Coonen et al., 2004), responsible for the high level of mosaicism
present in embryos.
As far as the mechanisms leading to partial aneuploidy are concerned, partial CL affected 14% of the examined blastomeres, followed by partial CG (9%) and partial MND (5%). It has been
postulated that chromosome breakage and whole chromosome aneuploidy could be caused by different factors (Wells and Delhanty,
2000). In a recent study, 6% of the embryos analysed by CGH were
affected solely by partial aneuploidy (Voullaire et al., 2002). Wells
and Delhanty (2000) proposed that acentric fragments would not be
stably transmitted to daughter cells and the resultant loss of material
would leave the embryo with a potentially lethal monosomy for that
chromosome region. Furthermore, both initial studies of embryos
using CGH observed breakage affecting chromosome locus 2q31
(Wells and Delhanty, 2000; Voullaire et al., 2000), which is similar
124
to this study along with the breakpoint in chromosome 1 (1p31).
Both sets of breakpoints map to defined chromosomal fragile sites
(Sutherland, 2003), which are prone to breakage due to adverse
culture conditions.
During this study, the combination of CGH and FISH clearly
demonstrated that chromosome breakage is rather frequent and can
persist up until the blastocyst stage of preimplantation development.
In conclusion, this study demonstrates that the finding of a chromosomally normal blastomere on Day 3 suggests that the remainder of
the embryo may have a normal chromosome complement on Day 5
of development. In contrast, abnormal cell on Day 3 is a predictor
of a poor fate for that particular embryo.
Funding
We would like to thank Life-Force Research Ltd. for funding this study. Also,
we would like to express gratitude to the whole London Fertility Centre team
and the UCL Centre for PGD.
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Submitted on March 2, 2007; resubmitted on September 3, 2007; accepted on
October 18, 2007
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