Challenges
Cytogenetics in reproductive
medicine: the contribution of
comparative genomic
hybridization (CGH)
Dagan Wells1,2* and Brynn Levy3
Summary
Cytogenetic research has had a major impact on the
field of reproductive medicine, providing an insight
into the frequency of chromosomal abnormalities that
occur during gametogenesis, embryonic development
and pregnancy. In humans, aneuploidy has been found to
be relatively common during fetal life, necessitating
prenatal screening of high-risk pregnancies. Aneuploidy
rates are higher still during the preimplantation stage of
development. An increasing number of IVF laboratories
have attempted to improve pregnancy rates by using pre-
implantation genetic diagnosis (PGD) to ensure that the
embryos transferred to the mother are chromosomally
normal. This paper reviews some of the techniques that
are key to the detection of aneuploidy in reproductive
samples including comparative genomic hybridization
(CGH). CGH has provided an unparalleled insight into
the nature of chromosome imbalance in human embryos
and polar bodies. The clinical application of CGH for
the purposes of PGD and the future extensions of the
methodology, including DNA microarrays, are discuss-
ed. BioEssays 25:289–300, 2003.
ß 2003 Wiley Periodicals, Inc.
The role of aneuploidy in pregnancy failure
Research suggests that fewer than 50% of naturally conceived
human embryos reach full term, with most lost before or shortly
1
The Institute for Reproductive Medicine and Science, St. Barnabas
Medical Center, New Jersey.
2
Dept. Obstetrics and Gynaecology, University College London,
London, UK.
3
Depts. Human Genetics and Pediatrics, Mount Sinai School of Medicine,
New York City, New York.
*Correspondence to: Dagan Wells, The Institute for Reproductive
Medicine and Science, 101 Old Short Hills Road, Suite 501, New
Jersey 07052. E-mail: dagan.wells@embryos.net
DOI 10.1002/bies.10232
Published online in Wiley InterScience (www.interscience.wiley.com).
Abbreviations: CGH, comparative genomic hybridization; FISH,
fluorescence in situ hybridization; IVF, in vitro fertilization; PCR,
polymerase chain reaction; PGD, preimplantation genetic diagnosis;
SKY, spectral karyotyping; ULM, uterine leiomyoma; ULMS, uterine
leiomyosarcoma; WCP, whole chromosome paint.
BioEssays 25:289–300, ß 2003 Wiley Periodicals, Inc.
after implantation.(1,2) A similar pattern of early embryonic
mortality is seen in in vitro fertilization (IVF), where a significant
proportion of embryos arrest in development during the first
few days following fertilization. Of those that survive and are
transferred to the mother, only a minority result in a live birth.
Although multiple factors may negatively influence embryo
survival, chromosomal abnormality is one of the most signi-
ficant. The development of a single cell zygote into an im-
mensely complex fetus is a highly regulated process requiring
flawless control and precise timing of gene expression.
Chromosome imbalance (aneuploidy) usually has a catastro-
phic effect on the developing embryo by altering the dosage of
hundreds of expressed genes. It is therefore not surprising that
all autosomal monosomies are lethal and that most trisomies
lead to early fetal loss. The lethality of chromosome imbalance
is emphasized by studies of material from spontaneous abor-
tions, which reveal that the majority of these failed pregnancies
are aneuploid.(3,4)
Cytogenetic analyses of human preimplantation embryos,
generated using IVF technology, have revealed extremely
high levels of chromosomal abnormality at this early stage of
development, with more than half of all embryos shown to
contain aneuploid cells.(5–7) Most of the abnormal chromo-
some arrangements detected at this stage are likely to be
lethal and their existence may explain a significant proportion
of failed IVF cycles. It is not possible to determine whether or
not chromosome abnormality is equally common in embryos
produced by natural fertilization, but it is likely that high levels
of aneuploidy also exist in this case.
Prenatal testing for aneuploidy
The high frequency of aneuploid conception in humans has
necessitated the creation of extensive prenatal testing pro-
grams. Such tests usually employ ‘conventional’ forms of
cytogenetic analysis, which focus on the analysis of cells that
have been arrested in the metaphase or prometaphase stage
of mitosis. At this point, the chromosomes appear as distinct
structures and can be identified on the basis of their size,
centromere position, and the pattern of bands induced by
various staining procedures (e.g. trypsin treatment and
Giemsa staining, G-banding). In addition to revealing
BioEssays 25.3 289
Challenges
information regarding chromosome number (ploidy), chromo-
some banding also facilitates the detection of deletions,
duplications, inversions and other structural abnormalities. In
most cases, samples received by clinical cytogenetic labora-
tories require cell culture in order to generate the metaphase
chromosomes necessary for comprehensive cytogenetic
analysis. The time taken for cell culture varies from 3–10
days and depends on the types of tissue under analysis.
Prenatal cytogenetic testing is indicated for a variety of
reasons including anomalous findings during an ultrasound
scan, abnormal maternal serum screening results and ad-
vanced maternal age. Prenatal samples are typically derived
from amniotic fluid (amniocytes), chorionic villi/placental tissue
and, more rarely, percutaneously obtained umbilical cord
blood. Defining the type of chromosomal abnormality, if any is
present, assists in estimation of the fetus’ chances of survival
and likely phenotype. This can aid patients making the difficult
decision of whether or not to terminate the pregnancy. Cyto-
genetic evaluation is also recommended for newborns pre-
senting with congenital abnormalities, where it can assist in
defining the etiology as well as predicting the phenotype.
Postnatal samples are easily obtained from peripheral blood
but may also be derived from most tissue biopsies. It is also
possible to collect and culture tissue derived from the products
of conception following spontaneous abortion, thus providing
an indication of whether chromosomal abnormality was a
factor in the miscarriage.
The impact of aneuploidy on
in vitro fertilization
Although success rates vary widely between different clinics,
worldwide, the probability of achieving a pregnancy using IVF
is generally less than 30% per embryo transferred. In most
cases, multiple embryos are generated in each treatment
cycle and IVF clinics decide which of these to transfer to
the mother based on morphological characteristics that are
indicators of embryo viability. However, morphological as-
sessment is insufficient for the exclusion of chromosome
abnormality, as many aneuploid embryos display normal
morphology during the preimplantation phase of development.
It has been suggested that the efficiency of assisted repro-
ductive techniques could be improved if chromosomally
normal embryos could be detected and preferentially selected
for transfer to the mother. By avoiding the unwitting transfer of
embryos carrying fatal chromosomal anomalies, pregnancy
rates should be increased. In an effort to identify the chromo-
somally normal embryos, some infertility centres have now
introduced tests specifically designed for cytogenetic screen-
ing.(6,8,9) Tests of this type focus on the use of technologies
developed for preimplantation genetic diagnosis (PGD).
Preimplantation genetic diagnosis
Preimplantation genetic diagnosis (PGD) was initially applied
as an alternative to prenatal diagnosis for patients at risk of
290 BioEssays 25.3
transmitting specific genetic diseases to their offspring.(10)
Patients requesting PGD must first undergo IVF treatment, not
necessarily for reasons of infertility, but because this allows
access to multiple oocytes (and thus embryos) within a single
reproductive cycle. Oocyte retrieval and fertilization are follow-
ed by the biopsy of 1–2 cells (blastomeres) derived from 6–
10 cell embryos. Alternatively, polar bodies are removed
from the oocytes (Fig. 1). These biopsied cells then provide the
chromosomal material or DNA required for subsequent
specific genetic tests. If a biopsied cell is found to be free of
the specific mutations or chromosomal errors assessed, then
it is inferred that the rest of the embryo is also unaffected by the
abnormalities in question. Only ‘‘unaffected’’ embryos are
transferred to the mother’s uterus, and consequently, any
pregnancy resulting from the procedure is expected to be
normal with respect to the genetic abnormalities tested. PGD
is therefore a suitable option for ‘‘at-risk’’ couples that are
adverse to pregnancy termination but who none-the-less
desire their own children, unaffected for the disease/condition
that they are at risk for.
To date there are thought to have been over 3,500 cases
of PGD (all indications) conducted worldwide, resulting in over
500 unaffected pregnancies. However, detailed published
data only exists for approximately 2,400 cycles.(11) PGD has
been successfully applied to the diagnosis of more than
30 monogenic disorders(10) and for the detection of chromo-
some imbalance in families carrying structural chromosome
rearrangements, such as translocations.(9,12 –14) The prefer-
ential transfer of chromosomally normal embryos allows trans-
location carriers to avoid repeated aneuploid pregnancies
and has been shown to significantly reduce the incidence of
spontaneous abortion, a traumatic occurrence that frequently
affects these patients.(12) Although significant numbers of
PGD cycles are conducted for inherited disease or chromo-
some rearrangement, infertile patients, undergoing preim-
plantation testing to identify chromosomally normal embryos
and thus improve their chances of successful fertility treat-
ment, now represent the largest PGD patient group. This
group is primarily composed of women of advanced repro-
ductive age (>35), who are at increased risk of producing
oocytes with an abnormal number of chromosomes.(6,8,9)
Methods of cytogenetic testing
One of the great challenges of prenatal and preimplantation
genetic testing has been to find methods that are rapid, allow
accurate analysis, and reveal the maximum cytogenetic
information concerning the sample. Conventional cytogenetic
techniques provide detailed information on every chromo-
some in a cell and as a result have enjoyed great success
and wide application. However, the necessity for cells in
metaphase means that a significant number of living cells must
be sampled and cultured. This requirement, under certain
circumstances, leads to a variety of problems. In most cases,

conventional cytogenetic tests cannot be applied to samples
composed of dead tissue because it is mitotically inactive.
Furthermore, living tissues are not always easy to culture and it
can be difficult to obtain good quality metaphase chromosome
preparations from certain types of sample. This is particularly
true of attempts to culture and produce metaphase chromo-
somes from solid tumors. Additionally, the inherent genetic
instability of tumors can lead to culture-related changes in
the karyotype that do not reflect the true in vivo karyotype of
the tumor.
The need for cell culture also extends the length of time
required for diagnosis. In the case of prenatal, this means that
most patients must endure over a week of anxiety before they
receive their results. The desire to obtain results more quickly
has lead to a proliferation of rapid prenatal tests, requiring no
cell culture. These tests, based on the polymerase chain re-
action (PCR) or fluorescence in situ hybridization (FISH), can
provide results within a few hours of receiving the sample,
but are considerably less comprehensive than conventional
methods, only assessing the copy number of a handful of
chromosomes and providing little or no data concerning more
subtle chromosomal alterations.
In the field of PGD, only one or two cells are available for
analysis and these are usually found to be in the interphase
stage of the cell cycle. During interphase, the chromosomes
are contained within the nucleus and are not readily dis-
tinguishable as separate structures, thus precluding the use of
conventional cytogenetic techniques. For PGD, test results
must usually be obtained within 48 hours to allow transfer of
the embryo(s) to the mother, and consequently there is no
time for cell culture. On the rare occasions when the biopsied
Challenges
Figure 1. Embryo biopsy. To allow access to the
blastomeres, a hole is been made in the encapsulating
zona pellucida using acid Tyrodes solution or a laser. A
single blastomere can then be carefully aspirated. This
image shows the moment at which the cell is removed
from a day-3 embryo composed of approximately 8 cells.
The larger pipette on the left holds the embryo in place by
gentle suction, while the smaller pipette on the right
passes through the hole in the zona pellucida to access
the cells.
cells are found to be in metaphase, it is theoretically possible
for all the chromosomes to be analyzed by conventional
means. However, this is not recommended as chromo-
somes may be lost when the cell is spread onto the slide or
they may overlap one another and thus be impossible to
distinguish. As with rapid prenatal tests, most PGD ane-
uploidy screening protocols tend to rely on the detection of a
limited number of chromosomes using fluorescence in situ
hybridization.
Fluorescence in situ hybridization (FISH)
The combination of traditional cytogenetic techniques with
molecular genetic methodologies has added a new and
powerful dimension to human genetics. Fluorescence in situ
hybridization forms the fundamental basis of most molecular
cytogenetic techniques. FISH utilizes chromosomal probes,
usually composed of cloned fragments of DNA. These probes
will only anneal to their matching complimentary DNA se-
quences, which are present on target chromosomes that are
spread on a microscope slide. The probe DNA is labeled with
fluorescent molecules and consequently any sites at which it
anneals are marked by a discrete fluorescent signal. FISH
signals are visible in interphase nuclei as well as on meta-
phase chromosomes and therefore allow ploidy analysis to be
performed even in the absence of cells in metaphase.(15–17)
FISH analysis is usually employed for the detection of specific
numerical and structural abnormalities using locus-specific
probes, centromere-specific probes and whole/partial chro-
mosome painting probes. The probes are carefully selected to
provide information on specific regions of interest.
BioEssays 25.3 291
Challenges
In reproductive medicine FISH has proven particularly
useful for the direct analysis of chromosomes in uncultured
prenatal samples, allowing a more rapid but limited diagnosis
to be made. For this purpose, it has been successfully applied
to both cultured and uncultured chorionic villus cells,(15,17–19)
amniocytes(16,20) and fetal cells circulating in the maternal
blood.(21–24) Strategies employing FISH are also responsible
for the vast majority of PGD cases performed for chromosomal
indications (translocation carriers and aneuploidy screening
carried out for IVF patients).(25,26) PGD tests have capitalized
on the ability of FISH to rapidly detect chromosomal imbalance
in cells regardless of the phase of the cell cycle.
The use of FISH for aneuploidy detection
in human embryos
The PGD protocol for carriers of structural chromosome re-
arrangements is customized for each case. This is because it
is necessary to use FISH probes specific for the chromosomes
involved in their rearrangement, such that any of the chromo-
some imbalances that may result can be detected. However,
for infertile couples where the woman is of advanced repro-
ductive age the same protocol can be applied for all patients. In
most cases, a panel of chromosomal probes are tested on
the biopsied cell(s).(6,8) The number of probes included in the
panel varies from five to nine depending on the PGD center
conducting the test. The chromosomes usually chosen for
analysis are those most frequently found to be aneuploid in
prenatal samples and in material from spontaneous abortions
(i.e. 13, 16, 18, 21, 22, X and Y).(16) Similar probe mixtures
have also formed the basis of rapid prenatal tests (CVS and
amniocentesis) based on FISH. A wide variety of unusual
aneuploidies, never seen in prenatal samples, are tolerated at
the preimplantation stage and, ultimately fail to form a
successful pregnancy. Such aneuploidies escape detection
by FISH probes currently used or PGD.
Unfortunately the number of chromosomes that can be
assessed in single cells using FISH is limited by the probability
of hybridization failure and the possibility that the fluorescent
signals of two chromosomes will overlap each other, giving
the appearance of a solitary signal. These problems arise
because of the three-dimensional spatial arrangement of
the chromosomes in the interphase nucleus and the limited
number of spectrally distinct fluorochromes available for probe
labeling. Overlapping signals lead to an over estimation of the
incidence of deletions and monosomies. In addition, errors
occasionally arise due to signals appearing to be split, resemb-
ling two chromosomes situated in close proximity.(27) Inter-
phase-FISH studies conducted on cell lines show an error rate
of >5% per probe per cell and consequently PGD laboratories
do not currently assess more than nine probes per cell.(27) If all
forms of aneuploidy are to be detected, a different approach is
needed. Newer multicolor FISH techniques, such as M-FISH
292 BioEssays 25.3
and SKY(28,29) allow all chromosomes in a metaphase spread
to be uniquely painted by hybridization probes. However, as
with chromosome banding techniques these methods require
metaphase chromosomes and cannot be applied to cells in
interphase.
Scientific data from the chromosomal
screening of preimplantation embryos
In addition to the obvious clinical benefits, there is also compel-
ing scientific motivation for complete chromosome screening
of single embryo cells and prenatal samples. Chromosomal
analysis of prenatal samples has revealed important data
concerning the incidence of aneuploidy during the last weeks
of the first trimester and in the later stages of human pre-
gnancy. These data have revealed that aneuploidy in humans
appears to be an order of magnitude higher when compared
with most other mammal species. Data from first trimester
spontaneous abortions hints at even higher levels of chromo-
some imbalance during earlier stages of pregnancy, an
indication that has been confirmed by the use of FISH during
PGD cycles. Preimplantation testing has revealed that, during
the first few days of life, aneuploidy rates are at their highest
affecting a proportion of cells in at least 50% of embryos.(5–7) A
wide range of aneuploidy has been detected at the preim-
plantation stage, including some types that are not observed in
prenatal samples.(5 –7,9) Presumably these chromosome
imbalances become lethal before the time of prenatal testing,
with many affected embryos undergoing developmental arrest
prior to implantation.
Further unexpected data have been obtained from surplus
IVF embryos donated for research. All of the blastomeres from
these embryos can be tested and have revealed that a large
proportion of embryos are mosaic, having more than one
chromosomally distinct cell line present. Early FISH studies,
which were capable of examining three different chromo-
somes per cell, revealed that
30% of embryos were mosaic,
regardless of the chromosomes tested. Later studies, using as
many as nine chromosomal probes, showed that mosaicism
actually affects more than 50% of human embryos.(6,7) The
fact that high levels of mosaicism were detected after analyses
of small numbers of chromosomes, led some researchers to
speculate that a full analysis of all 24 types of chromosome
(22 autosomes, X and Y) would reveal that every embryo
contains at least one aneuploid cell. The suggestion that mo-
saicism might be a normal feature of early human devel-
opment was significant, but could neither be confirmed nor
disproved using FISH. It was therefore important to find a
method capable of providing a full assessment of the copy
number of every chromosome in single cells, not just for
diagnostic application, but also to answer outstanding ques-
tions relating to the prevalence of mosaicism and aneuploidy in
early human development.

Several attempts to produce a full chromosomal analysis
from biopsied cells have focussed on novel methods of forcing
them into metaphase.(30–32) While these techniques have had
some successes, they are relatively time consuming and
sometimes fail to yield metaphase chromosomes of suitable
quality for diagnostic purposes. Additionally, they face the
same problems related to the spreading and analysis of a
single metaphase as discussed earlier. Ultimately, a method
capable of a full chromosome analysis in single cells without
the need for metaphases was devised, based on the technique
known as comparative genomic hybridization (CGH).
Comparative genomic hybridization (CGH)
The development of CGH has provided molecular cytoge-
netics with a versatile technique that answers many of the
deficiencies of FISH and conventional cytogenetic analysis,
allowing the entire genome to be scanned, in a single step,
for imbalances in chromosomal material. Importantly, CGH
achieves comprehensive aneuploidy screening without the
requirement for metaphase chromosomes from the sample
and therefore circumvents the need for cell culture. CGH was
developed in 1992 by Kallioniemi et al.(33) who utilized it to
identify 16 different previously unknown regions of amplifica-
tion in tumor cell lines and primary bladder tumors. CGH
effectively reveals any DNA sequence copy number changes
(i.e. gains, amplifications or losses) in a particular specimen
and maps these changes on normal chromosomes.(33,34)
When used for the analysis of a mosaic sample CGH can
detect changes that are present in as little as 30–50% of the
specimen cells.
CGH is accomplished by labeling the sample DNA with a
green fluorescent molecule and mixing it with an equal amount
of DNA derived from a chromosomally normal control and
labeled with a red fluorochrome. This DNA mixture is then
allowed to hybridize to normal male (46,XY) metaphase
spreads on a microscope slide. Fragments of red and green
DNA compete for the hybridization sites along each chromo-
some. If, for example, the sample (green) were chromosomally
normal then there would be effectively no difference between
the sample and the control (red) DNAs. In this case, every
chromosome would be covered equally with hybridized red
and green DNA fragments and a yellow coloration would be
observed (Fig. 2). However, if the sample were from a trisomy
21 case, there would be relatively more green DNA fragments
from chromosome 21 than red. In this case, by virtue of their
greater numbers, green fragments would tend to out-compete
red fragments for hybridization to chromosome 21 and the
ratio of red to green fluorescence along the length of this
chromosome would become skewed in favor of green. The
converse occurs if the sample is deficient in chromosomal
material, resulting in a more intense red color over the deleted
chromosomal region (Fig. 2). Computer-assisted measure-
ment of the red:green ratio along each chromosome allows
Challenges
all over/under-represented regions to be identified. CGH
has detected deletions and duplications in the range of 3–
5 Mb.(35–37) Since the smallest autosome is in excess of
50 Mb, CGH analysis provides a very powerful and sensitive
method for detecting whole chromosome aneuploidy, as well
as duplications and deletions of significant size.
CGH was primarily developed as a cancer research tool(33)
and was quickly applied to the analysis of solid tumors.
Conventional cytogenetic analysis of solid tumors is difficult
because very few metaphase spreads are obtained after
cell culture and their quality is often inadequate to allow
recognition of banding patterns. CGH allows direct analysis of
genomic DNA obtained from tumor specimens, thus over-
coming the problems associated with cell culturing and poor
metaphase spread quality. The use of CGH for the analysis
of tumors has revealed a number of new recurring chromo-
somal gains, amplifications, losses and deletion sites that
had not previously been detected by traditional cytogenetic
analysis in various tumors, including: breast cancer,(38)
uveal melanomas,(39–41) small-cell lung carcinoma,(42,43)
gliomas,(44) sarcomas,(45,46) and head, neck and pancreatic
carcinomas.(47,48) The prognostic value of the chromosomal
alterations detected by CGH has also been assessed in a
number of neoplasms including node-negative breast can-
cer,(49) renal cell carcinomas,(50) uveal melanoma,(51) cuta-
neous melanoma(52) and bladder cancer.(53)
CGH has also been applied to neoplastic samples that are
directly relevant to reproductive medicine. It has been
employed to assess DNA sequence copy number changes
in cervical carcinoma,(54) prostate cancer,(55,56) testicular
germ cell tumors,(41,57) ovarian tumors(58,59) and endometrial
cancer.(60,61) It has also been used to contrast cytogenetic
alterations in uterine leiomyoma (ULM) and uterine leiomyo-
sarcoma (ULMS).(62,63) ULMs are associated with abnormal
uterine bleeding, infertility and abdominal pain and are a
leading indication for hysterectomy.
CGH in clinical cytogenetics
The identification of the origin of additional or missing
chromosomal material in a newborn or prenatal sample is
crucial for discussing potential genotype–phenotype correla-
tions during genetic counseling. Additionally, the ability to
delineate the regions of imbalance in patients with chromoso-
mal abnormalities, may ultimately lead to the discovery of the
gene(s) that are responsible for their clinical features. FISH
remains the primary molecular cytogenetic technique utilized
when a complete and detailed karyotype cannot be obtained
by conventional methods. The use of multiple whole chromo-
some paints (WCP) and probes is often necessary in order to
resolve the origin of aberrant chromosomal material.(64,65)
This strategy is arduous and, in many cases, if a chromosomal
aberration cannot be defined using three to five FISH probes,
BioEssays 25.3 293
Challenges

Figure 2. Mapping of chromosomal gains and losses detected by CGH. A loss in the short arm, dim (p21p31) and gain in the long arm, enh
(q21q32.1) are illustrated. A: Visual inspection of gains and losses of chromosomal material. Non-aneuploid chromosomal regions show a
yellow colour. Green regions represent a gain of specimen DNA at that region while red areas reflect greater hybridization of reference DNA
at that location due to the depletion of specimen DNA. B: Inverted DAPI image allows for identification of the chromosome. C: Computer-
generated CGH fluorescence ratio profile. The center line in the CGH profile represents the balanced state of the chromosomal copy
number (ratio 1.0). Gains are viewed to the right (>1.20) and losses (<0.80) to the left of the centre line.

the investigation is halted due to financial limitations and
time expense.
The identification of additional/missing cytogenetic materi-
al can also be achieved by utilizing other molecular cytogenetic
techniques like reverse FISH(66,67) and multicolor FISH (SKY
and M-FISH).(28,29) While the information derived using
reverse FISH is highly informative, the procedure is technically
demanding and requires specialized micromanipulation
equipment to microdissect the region of interest from abnormal
chromosomes. The advantage of CGH over conventional
FISH with WCPs and multicolor FISH is its ability to identify the
precise chromosomal region from which the additional
unknown material was derived in a single experiment. CGH
analysis is therefore an ideal way to overcome many of these
shortcomings, providing a genome wide search without any
prior information of the chromosomal aberration in question.
To date, more than 1500 articles have been published on
CGH with approximately 90% reporting the utility of CGH to
delineate cytogenetic changes in cancer specimens (http://
www3.ncbi.nlm.nih.gov/entrez/ query.fcgi). About 6% of CGH
papers have dealt with technical aspects and only a limited
number have described the application of CGH in a clinical
cytogenetics setting. CGH has been particularly useful in
clinical cytogenetics and has facilitated the identification and
characterization of intrachromosomal duplications, deletions,
unbalanced translocations and marker chromosomes(68–71) in
both prenatal and pediatric samples. CGH has also been
useful in revising incorrectly assigned karyotypes. Figure 3
illustrates a case originally identified as an intrachromosomal
duplication of the distal long arm of chromosome 4, but shown
by CGH to be a derivative chromosome 4 with the additional
material being derived from chromosome 13.
The ability of CGH to define more precisely the chromoso-
mal material comprising marker chromosomes and unba-
lanced rearrangements has helped to further define critical
chromosomal regions that are associated with adverse
phenotypic outcomes(71,72) thus providing prognostic informa-
tion for genetic counseling. This information is also beneficial
to prenatally ascertained cases of marker chromosomes as
it may provide couples with a means to make rational and
informed decisions concerning the pregnancy. In pedi-
atric cases, such information may provide the parents with a

294 BioEssays 25.3


realistic prognosis and be important for the clinical manage-
ment of the infant. In addition to being able to identify excess
and/or missing chromosomal material not resolvable by G-
banding, CGH could also be used as a backup method for
aneuploidy analysis of specimens that have failed to grow in
cell culture. This would be particularly useful in the analysis of
non-viable fetal tissue derived from products of conception,
approximately 60% of which are estimated to have a
chromosome abnormality (mainly aneuploidy).(4,73)
The application of CGH to single cells
To use CGH to determine the true incidence of chromosome
abnormality in human embryos and to develop CGH proto-
cols compatible with PGD several modifications to exist-
ing methods were necessary. Routine applications of CGH
require
200 ng of DNA, whereas a single cell contains only
5–10 pg.(74) Consequently, the DNA content of the cell must be
amplified prior to use for CGH. We achieved the
40,000 fold
amplification necessary using a polymerase chain reaction
(PCR) based strategy to enzymatically copy the single cell
genome multiple times. The amplification is so efficient that
sufficient DNA can be generated from a single cell for nume-
rous subsequent PCR analyses, as well as for CGH.(75)
Challenges
Figure 3. A: Partial GTG karyotype of the
proband showing the chromosome 4 homologues.
The normal chromosome 4 is on the left and the
aberrant chromosome 4 with additional material on
the long arm is shown on the right. B: Partial
ideograms and CGH profiles showing a gain on the
long arm of chromosome 13 from 13q33 to 13qter
(orange box). The lines in the CGH profiles
represent green:red ratios of (from left to right)
0.5; 0.75; 1.0; 1.25 and 1.5. A ratio of 1.0 repre-
sents the balanced state of the chromosomal copy
number. C: Confirmation of the CGH finding by
FISH with a chromosome 13 long arm subtelo-
meric probe. Signals are observed on the two
normal chromosome 13 homologues as well as on
the longer derivative chromosome 4.
To date, only a small number of embryos have been studied
using CGH; however, it has already been possible to confirm
and extend many of the cytogenetic findings that were ob-
tained using FISH. Some extremely unusual abnormalities
have been observed including monosomies affecting the
largest chromosomes and also nullisomy.(76,77) Presumably
chromosome imbalances of this type begin to affect embryo
viability negatively soon after the expression of the embryonic
genome is initiated. Mosaicism was found to be extremely
common affecting 16/24 embryos analyzed by CGH.(76,77) Of
particular interest were three embryos that were classed as
‘chaotic’ mosaics. This type of embryo contains cells that have
multiple chromosome imbalances due to a complete break-
down of accurate chromosome segregation. The existence of
chaotic embryos was initially recognized using FISH analysis,
but a complete characterization was not possible. CGH
analyses of single blastomeres have provided a more com-
plete picture, revealing that as many as 14 chromosomes can
display imbalance in individual cells derived from chaotic
embryos.(76) The two published embryo-CGH studies de-
tected eight non-mosaic embryos out of 24 analyzed. One of
the embryos was monosomic for chromosome 4 in every cell
examined, while another displayed a double aneuploidy, 46, X,
BioEssays 25.3 295
Challenges
þ21 (i.e. Down’s syndrome and Turner’s syndrome) in every
cell. Importantly, the other six non-mosaic embryos were
found to be chromosomally normal in every cell examined.
These results demonstrate that attempts to deduce the
incidence of mosaicism by extrapolating from the limited
chromosomal screening afforded by FISH overestimated the
frequency of this phenomenon. Given a full analysis of all
chromosomes in all cells of human embryos, it is clear that
chromosomal mosaicism, although common, is not a universal
feature of human preimplantation development. The over-
estimations from FISH data were probably due to occasional
experimental errors that caused discordance between dif-
ferent cells of the same embryo, artificially increasing the
perceived level of mosaicism. It is presumed that uniformly
normal embryos have the greatest potential for further
development; however, the fate of the mosaic embryos is less
clear. Half of the mosaic embryos detected by CGH contained
a normal number of chromosomes in at least half of their cells.
Whether or not it is possible for such embryos to form a viable
pregnancy is not currently known, but the occasional rescue of
mosaic embryos could explain some incidences of confined
placental mosaicism.
FISH probes currently used in PGD hybridize to a defined
region on the target chromosomes and thus provide definitive
copy number information on these regions alone. The pre-
sence of the rest of the chromosome is assumed, but not
conclusively proven. Consequently, any imbalance outside of
the targeted FISH probe region will seldom be detected using
FISH. During the CGH studies, five instances of chromosome
breakage resulting in imbalance of specific regions, rather
than whole chromosomes, were detected.
A meiotic error in chromosome segregation was consid-
ered a likely cause for abnormalities in 7/22 embryos. This was
judged on the basis that an identical chromosome imbalance
was present in most (if not all) cells of the embryo. Two of the
24 embryos that were investigated using CGH could not be
assessed for meiotic error, as extensive chaotic mosaicism
confused any attempts to estimate their original chromosomal
status. Currently, the most comprehensive FISH analysis in
use for PGD assesses nine chromosomes (13, 15, 16, 17, 18,
21, 22, X, Y). Several of the aneuploid cell types identified by
the CGH studies had chromosomal imbalances that would not
have been detected using this FISH strategy, strengthening
the argument for the adaptation of CGH for clinical screening
of embryos.
Clinical application of CGH for
preimplantation genetic diagnosis
The application of CGH in a clinical PGD setting is not straight-
forward. After embryo biopsy there is only a narrow window of
time for diagnoses to be made. The majority of PGD Centers
employ day-3 blastomere biopsy and aim to transfer embryos
before the end of day 4. However, most published CGH pro-
296 BioEssays 25.3
tocols require in excess of 72 hours to complete and are
therefore incompatible with preimplantation screening. Two
different strategies have been proposed to address this
problem: embryos may be frozen following biopsy and thawed
after the CGH analysis has been completed, or alternatively
an accelerated CGH protocol could be applied to polar bodies
that are available for biopsy on the day of fertilization. Both
of these methodologies have now been applied in a clinical
setting;(78,79) however each has limitations and the most
efficacious method remains to be determined.
Standard cryopreservation protocols cause a small reduc-
tion in embryo viability, an effect that is exacerbated by embryo
biopsy. Thus, any improvement in implantation rates gained
after embryo biopsy and CGH-screening may be counter-
balanced to some extent by a reduction in the number of viable
embryos following cryopreservation. Cryopreservation is not
required for PGD strategies based on CGH analysis of polar
bodies. The main limitation of strategies based on polar body
biopsy is that imbalances arising in male meiosis cannot be
detected, although it should be noted that these are infrequent
compared with those of maternal origin. The most significant
obstacle to utilizing CGH in IVF clinics relates to the technical
complexities of the technique, i.e., currently, all existing CGH
strategies are labor intensive and require expertise with
several cytogenetic and molecular genetic techniques. A less-
involved means of analysis will be essential if CGH or related
technologies are to be applied in a significant number of clinics.
Scientific data from the analysis of polar
bodies using CGH in a clinical setting
Although very few PGD cycles using CGH have been con-
ducted, interesting scientific data concerning the chromo-
somal content of oocytes, polar bodies and embryos has
already begun to accumulate. The use of CGH to analyze polar
bodies promises to confirm the identity of the chromosomes
that are at greatest risk of malsegregation during female
meiosis. Previous chromosomal analyses of oocytes and polar
bodies, using techniques such as G-banding or SKY, have
experienced problems related to chromosome morphology
and spreading. These difficulties have compromized efforts to
determine the rate of aneuploidy, particularly monosomy, in
oocytes. Because CGH is a DNA-based technique, it cir-
cumvents these difficulties.
In a recent study(79) of first polar bodies from a 40-year-old
IVF patient, nine out of ten polar bodies were found to contain
chromosome imbalance. Loss of chromosomal material was
detected in six polar bodies (potentially leading to trisomy in
the corresponding embryos), while a gain of material was
detected in four (indicating a risk of monosomy in the embryo).
One polar body was aneuploid for two different chromo-
somes.(79) Five of the abnormal oocytes were predicted to
have imbalance involving chromosomes 16, 22 or X, which are
frequently aneuploid in spontaneous abortions. This high

frequency of presumably lethal aneuploidy further illustrates
the reasoning behind preimplantation testing during IVF of
women of advanced reproductive age. Interestingly, seven of
the ten unbalanced chromosomes in this set of polar bodies
were equal or smaller in size than chromosome 14. A similar
correlation between aneuploidy rate and chromosomal size
has recently been observed using spectral karyotyping (SKY)
analysis of oocytes.(80) Small chromosomes tend to form
fewer chiasmata and it has been suggested that reduced
recombination could predispose to nondisjunction.(81)
Microarrays
Comparative genomic hybridization has already had a signi-
ficant impact in the field of reproductive genetics; however the
technique remains time consuming and requires knowledge of
multiple molecular genetic and cytogenetic methods, particu-
larly when applied to single cells. If CGH is ever to be applied to
large numbers of PGD cases it will be essential to develop a
simplified method that can be used by infertility clinics, most of
which have relatively little cytogenetic expertise. Thus, any
modified method should preferably avoid the need to perform
karyotyping. Several methods currently under development
might fulfill this requirement; however, the most promising is
the use of microarrays.
The DNA targets for hybridization in conventional CGH
analysis are metaphase chromosomes. The resolution achiev-
able by conventional CGH is restricted to about 2 to 10 Mb for
low copy number losses and gains such as partial mono-
somies and trisomies.(36,82,83) To overcome this limitation, an
innovative strategy, called matrix or microarray-CGH (M-
CGH), has been devised. In M-CGH, chromosomal targets are
replaced by arrays consisting of well-defined genomic clones
(typically BAC, PAC or YAC clones), which are spotted onto a
glass microscope slide using automatic robotic devices. The
array may consist of hundreds or thousands of clones, each
corresponding to a small region of the genome. This allows for
genomic imbalances to be elucidated with much higher
resolution
100–200 kb.(84–89)
Most of the major steps in conventional CGH are also
features of M-CGH, with the obvious difference being the
hybridization of differentially labeled specimen (test) and refe-
rence DNA onto slides with DNA arrays instead of meta-
phase spreads. For M-CGH, image acquisition is facilitated by
laser scanning rather than fluorescence microscopy and
quality control, normalization and statistic array evaluation
are performed with dedicated software tools. Results are
summed up as separate logarithmic signal ratio values for
each of the tested clones on a given array. M-CGH has
recently proven successful for the detection of whole chromo-
some aneuploidy and also microdeletions.(84,87,90,91)
Aneuploidy screening using microarrays circumvents the
need for karyotyping and thus lends itself to automation. If
M-CGH can be adapted to the analysis of single cells it will offer
Challenges
the possibility of wide application in infertility clinics that wish to
identify the most viable embryos.
Conclusions
Methods for chromosomal analysis have become increasingly
powerful, benefiting enormously from the fusion of traditional
cytogenetic techniques and molecular genetics. Fluorescence
in situ hybridization and comparative genomic hybridization
have been amongst the most significant methodological ad-
vances. CGH has overcome many of the technical limitations
that beset earlier cytogenetic methods, allowing detailed
chromosomal data to be obtained from a variety of tissues
that were previously considered problematic. In the field of
reproductive medicine, as in other fields, CGH has been
employed for the ascertainment of chromosomal duplications,
amplifications and deletions that contribute to neoplastic
transformation. This has revealed the chromosomal location
of tumor suppressor genes and oncogenes that play a role in
neoplasia affecting tissues of the reproductive system. The
application of CGH to prenatal and pediatric samples has also
proven extremely beneficial, allowing the delineation of
complex or cryptic chromosomal rearrangements that could
not be defined using classical cytogenetic techniques. CGH
has also been applied to the analysis of mitotically inactive
cells derived from products of conception, shedding light on
the spectrum of chromosomal abnormalities causing miscar-
riage. Finally, the use of CGH to analyze human preimplanta-
tion embryos has provided unique scientific data concerning
the variety and rate of aneuploidy at this early developmental
stage. Most recently, this has led to methods for screening IVF
embryos, assisting in the identification of those with the
greatest potential for further development. In the future, CGH
or related techniques such as M-CGH, will allow IVF clinics to
screen embryos for any form of aneuploidy. It is hoped that this
will enable the preferential transfer of the embryos most likely
to form a viable pregnancy and thus lead to improvements in
the outcome of assisted reproductive procedures.
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