1 s2.0-s0015028213027441-main

24-chromosome copy number
analysis: a comparison of
available technologies
Alan H. Handyside, M.A., Ph.D.
Bluegnome, Fulbourn, Cambridge; and Institute of Integrative and Comparative Biology, University of Leeds, Leeds, United
Kingdom
Chromosome aneuploidy, an abnormal number of chromosomes, in human gametes and embryos is a major cause of IVF failure and
miscarriage and can result in affected live births. To avoid these outcomes and improve implantation and live birth rates, preimplantation
genetic screening aims to identify euploid embryos before transfer but has been restricted to analysis of a limited number of chromosomes.
Over the past 15 years, various technologies have been developed that allow copy number analysis of all 23 pairs of chromosomes,
22 autosomes, and the sex chromosomes, or ‘‘24-chromosome’’ copy number analysis in single or small numbers of cells. Herein the
pros and cons of these technologies are reviewed and evaluated for their potential as screening
or diagnostic tests when used in combination with oocyte or embryo biopsy at different stages.
(Fertil Steril 2013;100:595–602. 2013 by American Society for Reproductive Medicine.)
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From the earliest years of IVF, it
had been suspected that a high
incidence of chromosome aneu-ploidy
in human oocytes and embryos
might contribute to low implantation
and pregnancy rates, and the first
attempt to karyotype embryos was re-ported
30 years ago (1). Only three 8-
cell stage embryos were successfully
karyotyped out of eleven analyzed,
and two were identified as aneuploid.
This high incidence of aneuploidy,
albeit in a very small sample, clearly
alarmed the authors and prompted
them to try to reassure clinicians and
patients with the statement: ‘‘It must
be emphasised that over 100 babies
have been born following in vitro fertil-ization
without any apparent chromo-some
abnormality. Chromosome
abnormalities of the kind we have
found clearly result in early embryonic
loss, and probably contribute to the
high failure rate after embryo transfer.’’
Today, with the development of a
range of molecular genetic technolo-gies
that allow copy number analysis
for all 23 pairs of chromosomes, 22
pairs of autosomes, and the sex chro-mosomes,
or ‘‘24 chromosomes,’’ in sin-gle
or small numbers of cells, there is
now definitive evidence for the high
incidence of abnormal chromosome
copy number, or aneuploidy, in both
gametes and all stages of preimplanta-tion
development. Furthermore, these
aneuploidies can arise through gonadal
mosaicism, during meiosis (predomi-nantly
female meiosis), and in the
mitotic cleavage divisions following
fertilization up to and including the
blastocyst stage (2).
The challenge for embryologists
and clinicians remains how to use
this knowledge to improve clinical
practice. No one would knowingly
transfer an aneuploid embryo or, for
example, continue with multiple IVF
cycles in a patient with a very high
incidence of aneuploidy and conse-quently
a low or zero chance of
achieving a pregnancy with her own
oocytes. On the other hand, any strat-egy
to avoid these scenarios with the
use of the available technologies for
aneuploidy testing has to balance the
benefits of identifying euploid em-bryos
for transfer with the potential
costs to the embryo of any invasive
biopsy or any false positive and nega-tive
test results. The pros and cons of
different biopsy methods are reviewed
elsewhere in this section. Here, I
present an overview of available and
emerging technologies for 24-
chromosome copy number analysis.
Received June 12, 2013; revised July 8, 2013; accepted July 15, 2013.
A.H.H. is Head of Preimplantation Genetics at Bluegnome, Cambridge, United Kingdom, with which
he holds patents and which is owned by Illumina Corp., San Diego, California, in which he holds
stock.
Reprint requests: Alan H. Handyside, M.A., Ph.D., Bluegnome, Capital Park CPC4, Fulbourn, Cam-bridge
CB21 5XE, United Kingdom (E-mail: alan.handyside@cambridgebluegnome.com).
Fertility and Sterility® Vol. 100, No. 3, September 2013 0015-0282/$36.00
Copyright ©2013 American Society for Reproductive Medicine, Published by Elsevier Inc.
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VOL. 100 NO. 3 / SEPTEMBER 2013 595

VIEWS AND REVIEWS
SCREENING VERSUS DIAGNOSIS
The testing of oocytes and preimplantation embryos for aneu-ploidy
with the aim of improving IVF outcomes, particularly
reducing miscarriage rates and increasing live birth rates, is
now widely referred to as preimplantation genetic screening
(PGS). However, before comparing the different technologies,
it is instructive to examine the different expectations for a
screening versus a diagnostic test, in the stricter sense of those
terms (Table 1).
Quintessentially, a screening test is noninvasive, rapid,
and sufficiently low cost for application to all patients to pri-oritise
embryos for transfer. Furthermore, the requirements
for accuracy are likely to be less stringent, although false pos-itive
results, which may exclude embryos with normal copy
number, are arguably more undesirable than false negative
results. A good example of such a test is counting the number
of pronuclei formed after insemination. Although useful as an
early indication of fertilization rate, it was originally intended
to avoid the transfer of triploid embryos arising from disper-mic
fertilization, which is one of the commonest causes of
early miscarriage. However, it is well known that in some
cases the formation of pronuclei is asynchronous and
apparent third pronuclei may simply be empty vesicles.
Furthermore, molecular genetic analysis by karyomapping
(see later section) has revealed that among embryos identified
as normally fertilized with two pronuclei, it is relatively com-mon
to find unfertilized parthenogenetically activated
haploid or triploid fertilized embryos (unpublished observa-tions).
So a routine test, which is universally applied to all
IVF cycles, is accepted because of the advantages of moni-toring
the fertilization rate and the low cost of making the
observations, despite the accuracy not being 100%.
Another example of a noninvasive method for embryo se-lection,
which could potentially be used to identify aneuploid
embryos, is the use of incubators fitted with time-lapse micro-scopy
allowing detailed morphokinetic analysis of each
embryo (3). There have now been several reports of an associ-ation
of different parameters with aneuploidy (4). However,
the effectiveness and accuracy of morphokinetic analysis
for identifying aneuploid embryos with only a single aneu-ploidy
versus those with multiple aneuploidies and aneu-ploidies
of different origins has not been established. In
principle, it seems unlikely that all aneuploidies could be
identified in this way, because many implant and cease devel-opment
only at later stages of pregnancy.
With a diagnostic test, in contrast, the costs, both finan-cial
and to the viability of embryo, of the necessary invasive
testing, are still important but secondary to the paramount
objective of diagnostic accuracy (Table 1). The requirement
of a diagnostic test is a high sensitivity and specificity and
in particular a very low incidence of false negative results.
So, for example, preimplantation genetic diagnosis (PGD) of
a severe single gene defect typically requires the use of mul-tiple
highly polymorphic markers specific for the parental
chromosomes in the region of the gene combined with muta-tion
detection. Here the aim is to identify two, and only two,
chromosomes, one from each parent, with any appropriate
combination of unaffected and affected chromosomes. Using
this strategy theoretically reduces the chance of misdiagnos-ing
an unaffected embryo to 1 in 1,000. However, any par-tial
or ambiguous results may result in an unaffected embryo
not being transferred.
For PGS and 24-chromosome copy number analysis, if
the aim is simply to improve IVF rates and reduce miscarriage
rates, a noninvasive test with moderate accuracy may be
effective. On the other hand, for a patient who has experi-enced
repeated pregnancy loss with karyotypically abnormal
conceptuses, the aim is to avoid miscarriage or fetal abnor-mality
and an invasive test with a low false negative rate
may be more appropriate. Furthermore, whereas the efficacy
of any screening test needs to be evaluated by a randomized
controlled trial (RCT) and analysis of clinical pregnancy and
live birth rates, the efficacy of a diagnostic test needs to be
established by validation of the methodology, follow-up
analysis of tested embryos, and monitoring of the pregnancy
outcome at birth.
24-CHROMOSOME COPY NUMBER ANALYSIS
The simplest and least expensive method for identifying ab-normalities
of chromosome number is to spread and count
stained metaphase chromosomes on glass microscope slides.
However, as the original study by Angell et al. (1) demon-strated,
the proportion of embryo cells that can be arrested
in metaphase by microtubule inhibitors is relatively low and
the chromosomes often overlap or are scattered across the
slide and can be lost. Furthermore, because the chromosomes
are generally short and can not be banded by standard stain-ing
methods, the accuracy is reduced further as the pairs of
chromosomes cannot be identified. Although there have
been many studies of human gametes and embryos with the
use of karyotyping, the low efficiency per cell prevents its
use for screening purposes. This has led to the search for mo-lecular
cytogenetic technologies applicable at the level of sin-gle
or small numbers of cells, which ideally would avoid the
need to arrest cells in metaphase. A range of technologies
have been investigated over the past 15 years, including
methods which simply aim to count the overall number of
TABLE 1
Screening versus diagnostic testing of chromosome copy number in
preimplantation embryos.
Screening Diagnosis
All patients Specific indications
Minimally invasive Invasive
All embryos Good-quality embryos only
Rapid with fresh transfer Rapid with fresh transfer, or not time
limited with vitrification
High efficiency Moderate efficiency
Direct or indirect Direct
Accurate Highly accurate
Low false negatives
acceptable
Tolerate false positives
No false negatives
Clinically effective Validation of diagnostic accuracy
Randomized control trials
Low cost Medium to high cost
Handyside. 24-chromosome copy number analysis. Fertil Steril 2013.
596 VOL. 100 NO. 3 / SEPTEMBER 2013

chromosomes, those that identify some or all chromosome
pairs, and, more recently, technologies that quantify the
amount of chromosomal DNA present or identify each indi-vidual
chromosome and its parental origin.
The first molecular cytogenetic technique to be applied
widely to interphase nuclei spread on slides was fluorescence
in situ hybridization (FISH) with a combination of
chromosome-specific probes labeled with different fluoro-chromes.
Although the number of probes which could be
used in the same hybridization was limited to about five,
the first probe set can be washed off the slides and a sequential
hybridization performed so that the number can be increased.
However, the efficiency of hybridization declines rapidly and
the most that can be analyzed with any accuracy is 12–14
chromosomes. Multicolor FISH typically with 5–9 probes in
two sequential hybridizations became the standard methodol-ogy
for PGS for a number of years. However, RCTs demon-strated
a decrease or no improvement in live birth rates per
cycle started in a range of indications. This was attributed
to a number of factors, including FISH errors caused by over-lapping
or split signals in single interphase nuclei and partic-ularly
the limited number of chromosomes analyzed.
Therefore, the focus has now shifted to technologies that
allow all 24 chromosomes to be analyzed. However, attempts
to do this with FISH-based methods, for example, using short
fluorescent oligonucleotide probes in a series of sequential
hybridizations, are not sufficiently accurate at the single-cell
level (5), and 24-color FISH methods require metaphase
chromosomes (6).
COMPARATIVE GENOMIC HYBRIDIZATION
Comparative genomic hybridization (CGH) was originally
developed for karyotyping solid tumors, which are difficult
to karyotype by conventional methods. The method involves
isolating DNA from the test sample and from a karyotypi-cally
normal individual and labeling the DNA with red and
green fluorochromes. The two labeled DNAs are then cohy-bridized
to a normal metaphase chromosome spread and
the intensity of fluorescent labeling with the two probes is
analyzed with a microscope fitted with appropriate filters,
a sensitive camera, and dedicated software. To adapt the
method for use with single cells isolated from cleavage-stage
embryos, Wells et al. (7) used degenerate oligonucleo-tide
priming polymerase chain reaction (PCR) to amplify the
whole genome of both the test cell and control genomic DNA
to avoid artefacts caused by amplification bias. However,
accurate quantification requires a long hybridization of
3 days. Nevertheless, this approach was the first
24-chromosome copy number technology to be used clini-cally,
using a strategy of cleavage-stage biopsy followed
by cryopreservation of the biopsied embryos for transfer in
a later cycle after completion of the CGH analysis (8).
More recently, conventional CGH has been used success-fully
for polar body analysis with fresh transfer (9, 10).
Furthermore, a fast 12-hour protocol has now been developed
which has a resolution high enough to be applicable to not
only whole chromosome aneuploidy but also some transloca-tion
chromosome imbalance (11, 12).
Fertility and Sterility®
ARRAY CGH
The principles of microarray-based CGH (array CGH) are the
same as for conventional CGH, requiring labeled DNA from
both test and control samples, but the labeled DNA is then hy-bridized
to a DNA microarray rather than a metaphase spread.
Analysis is performed by scanning and imaging the array and
measuring the intensity of both hybridization signals relative
to each probe (logR ratio). It was only with the advent of mul-tiple
displacement amplification (MDA) for whole-genome
amplification, which allows micrograms of DNA to be ampli-fied
from single or small numbers of cells, that the use of
microarrays could be considered for preimplantation genetics
(13, 14). For copy number analysis, however, a PCR library–
based whole-genome amplification method is preferable
because of reduced amplification bias, which improves the ac-curacy
of ratio detection and reduces variability between
probes across each chromosome. For single cell analysis, ar-rays
of 3,000 large fragments of human DNA cloned in bac-teria
from loci distributed at 1-Mb intervals across each
chromosome are now available, allowing accurate copy anal-ysis
of whole-chromosome copy number as well as partial-chromosome
copy number abnormalities of chromosome
arms or smaller regions down to a resolution of 10 Mb.
Furthermore, a higher-resolution array with increased
numbers of clones, particularly in the telomeres of each chro-mosome,
is available for use in carriers of structural chromo-some
abnormalities to detect, for example, translocation
chromosome imbalance involving duplication and deletion
of often small segments of these chromosomes. Alternatively,
for whole-chromosome copy number analysis only, it is also
possible to use arrays of chromosome-specific PCR libraries
(15), but these arrays have not yet been extensively validated
for PGS.
Array CGH was the first technology to be widely available
for reliable, accurate, and relatively fast 24-chromosome copy
number analysis and is now used extensively around the world
despite the relatively high cost of testing multiple samples
(Table 2). The first pregnancies and live births following PGS
using array CGH to analyze copy number in the first polar
body were reported in 2010 (16, 17). Subsequently, the
European Society for Human Reproduction and Embryology
(ESHRE) PGS Task Force organized a pilot study testing both
first and second polar bodies for advanced maternal age and
reported a high incidence of copy number abnormalities and
a high concordance for predicted maternal aneuploidies in
the corresponding zygote (18, 19). Detailed analysis of those
pilot study data revealed a high incidence of multiple
meiotic errors in individual oocytes, predominantly caused
by premature predivision of sister chromatids (20). Because
single sister chromatids segregating to the metaphase II
oocyte then segregate randomly, about one-half of all chro-matid
errors in the first meiotic division were balanced in
the second division with the chromatid segregating to either
the second polar body or the zygote. Thus, screening only
the first polar body does not accurately predict the aneuploidy
status of the corresponding zygote.
More recently, separate biopsy of the first and second
polar bodies and array CGH analysis was followed by analysis

VIEWS AND REVIEWS
of the whole embryo at cleavage stages on day 3 after intra-cytoplasmic
sperm microinjection in women of advanced
maternal age (21). This demonstrated that 93% of the aneu-ploidies
detected in a small series of cleavage-stage embryos
were associated with copy number changes in the polar bodies
and therefore of female meiotic origin. However, as in another
study (22), there were false positive predictions, some of
which appeared to be due to amplification bias, particularly
in first polar bodies, and some of which may demonstrate bio-logic
processes such as chromosome lagging.
Several RCTs of the use of array CGH for advanced
maternal age and other indications are ongoing. The first
RCT to be reported involved young good-prognosis patients
who elected to have a single blastocyst transfer to avoid the
complications of multiple pregnancy (23). That study
compared implantation and ongoing clinical pregnancy rates
with single blastocysts selected on the basis of embryo
morphology alone versus single euploid blastocysts of good
morphology and demonstrated a significant improvement in
implantation and ongoing pregnancy rates with array CGH
(42% vs. 69%, respectively, per single blastocyst transfer).
In another retrospective study, cleavage-stage biopsy and
array CGH were used in carriers of reciprocal or Robertsonian
translocations to detect both translocation chromosome
imbalance and aneuploidy and reported higher pregnancy
and live birth rates than previously reported for FISH-based
testing of the translocation chromosomes alone (24).
Although only 61% of cycles had an embryo transfer, clinical
pregnancy rate was 71% per transfer with an implantation
rate per embryo transferred of 64%.
DIGITAL PCR
A novel approach to 24-chromosome copy number analysis
in polar bodies is the use of digital PCR, which was developed
for cancer studies (25). This is a method that counts the pres-ence
of chromosome-specific PCR target DNA by limiting
dilution of the DNA after lysis of each polar body. Thus it
avoids any need for whole-genome amplification and any
associated amplification bias. The polar bodies are simply
lysed and the lysate pipetted into eight separate wells. A
multiplex PCR is then performed, followed by detection of
chromosome-specific products in each of the wells. To control
for amplification failure or allele dropout, multiple target
sequences are ampilifed per chromosome. Under the right
conditions, the number of wells positive for each
chromosome-specific PCR product then reflects the number
of DNA target molecules, i.e., chromatids in the polar body.
This should normally be two in the first polar body and one
in the second, predicting that the fourth chromatid has been
segregated to the zygote. This approach is still being vali-dated,
but initial studies have confirmed that most copy num-ber
errors in the first meiotic division are caused by premature
predivision of sister chromatids resulting in three copies or
only one in the first polar body (Daser, personal communica-tion).
When coupled with robotics and high-throughput plat-forms
for PCR, this technology is both rapid and low cost,
offsetting the cost of analyzing two samples for each fertilized
oocyte. The technique is intended only for polar body analysis
and is particularly relevant in countries such as Germany in
which legal restrictions prevent PGS on embryos beyond
the pronucleate stage of development. Digital PCR could pre-sumably
be used with single blastomeres biopsied from
cleavage-stage embryos, but analysis of cells in S phase
may result in errors.
SNP ARRAY
A single-nucleotide polymorphism (SNP) is a DNA sequence
variant in which, at a particular position or locus, one of
two or more nucleotides may be present on different chromo-somes
within a population. To date, almost 40 million SNPs
TABLE 2
Comparison of available technologies for 24-chromosome copy number analysis.
Method
Duration
of test Complexity
Equipment
cost Reagent cost Resolution Pros and Cons
CGH 12–72 h Medium Medium Low Low Low cost
Skilled
Labor intensive
Array CGH 12–24 h Medium Medium Medium Medium Robust
Scalable
Digital PCR 8 h Medium Medium Low Low Low cost
Scalable
Rapid
Polar body analysis only
Real-time quantitative PCR 4 h Medium Medium Low Low Low cost
Not scalable without additional
equipment
Multiple cell samples only
SNP microarray 16–72 h High High Medium High Genome-wide analysis
Quantitative and marker analysis
Parental origin
Next-generation sequencing 15 h High High Medium Low Scalable with multiplexing
Note: CGH ¼ comparative genomic hybridization; PCR ¼ polymerase chain reaction; SNP ¼ single-nucleotide polymorphism.
Handyside. 24-chromosome copy number analysis. Fertil Steril 2013.

have been validated, spread across the genome but mostly in
noncoding regions. Biallelic SNPs, in which one of two bases
is present, referred to generically as A and B, are valuable
markers, and hundreds of thousands of SNPs can be geno-typed
simultaneously with the use of SNP arrays. Further-more,
for molecular cytogenetics, analysis of the ratio of the
intensity of the B to the A alleles at heterozygous loci allows
the detection of duplications and deletions from whole chro-mosomes
to small regions with high resolution. Normally,
where both chromosomes are present, there should be three
bands representing AA, AB, and BB loci at a ratio of 0, 0.5
and 1. In the duplications, the B-allele ratio at heterozygous
loci splits into two bands representing loci that are either
AAB or ABB. In deletions, loss of heterozygosity (LOH) is de-tected
by the absence of the heterozygous band. SNP arrays
also have the advantage that the parental origin of any abnor-malities
can be investigated by genotyping the parents, al-lowing
the detection of, for example, uniparental disomy.
The use of SNP arrays for chromosome copy number anal-ysis
and PGS has been pioneered by several groups, each of
which used different approaches. Kearns et al. optimized their
lysis and MDA protocol for whole-genome amplification from
single and small numbers of cells to reduce amplification bias
(26). This then allows conventional SNP copy number analysis
simply by examining each chromosome for abnormalities in
the B-allele ratio and for LOH. Treff et al., in contrast, used sta-tistical
methods to examine the intensity and assign a copy
number at each SNP locus across the chromosome (27). The
copy number assignment for the whole chromosome is then
based on the copy number of the majority of loci. By applying
a quality threshold and excluding two results, the accuracy of
this approach was reported to be 98.6% for the 72 cells
analyzed. Furthermore, a prospective blinded nonselection
study of embryo biopsy and retrospective copy number anal-ysis
demonstrated a high predictive value for implantation or
pregnancy failure associated with the transfer of aneuploid
embryos (28). Copy number analysis of trophectoderm sam-ples
with the use of this method, combined with vitrification
of the biopsied blastocysts and thawing and transfer of euploid
blastocysts in a later cycle, resulted in high implantation and
live birth rates: 73% per embryo transfer with an implantation
rate of 65% per blastocyst transferred (29). It was also used
successfully to detect translocation chromosome imbalance
but only at moderate resolution down to10 Mb (30). Finally,
Rabinowitz et al. developed a bioinformatics algorithm using
parental SNP genotypes to improve the accuracy of genotyp-ing
of single cells and used this and a number of other propri-etary
algorithms to analyze chromosome copy number in
blastomeres from cleavage-stage embryos (31).
An alternative approach is to use mendelian analysis of
the SNP genotypes of the parents and single blastomeres or
trophectoderm cells biopsied from each embryo to identify
four sets of informative SNP loci across each chromosome
that represent the four parental chromosomes, and then to
generate a karyomap of the embryo showing the parental
origin of each chromosome or chromosome segment (32).
This requires the phasing of theAand B alleles at heterozygous
loci in each parent, which for chromosome copy number anal-ysis,
can be achieved with the use of an embryo as a reference,
because the embryo would normally only inherit one chromo-some
from each parent. With the use of this approach, tri-somies
of meiotic origin, in which two chromosomes with
different patterns of recombination are present in the embryo,
can be identified by the presence of both chromosomes from
one parent in overlapping segments of the chromosome.
Conversely, monosomies or deletions can be identified with
high resolution simply by the absence of either chromosome
from one parent. Thus, karyomapping is able to identify
copy number abnormalities exclusively based on the genotype
of the embryo and completely avoids the problems associated
with quantification after whole-genome amplification. Of
course, duplications of whole chromosomes or chromosome
segments that are sequence identical can not be identified.
For single-blastomere analysis this could be an advantage,
because mitotic duplication of chromosomes resulting in
chromosome mosaicism during cleavage would not be
detected. However, karyomapping does not exclude quantifi-cation,
and the combination of the two approaches would pro-vide
a powerful method that would identify all types of
chromosome abnormality and their parental origin.
REAL-TIME QUANTITATIVE PCR
Although Treff et al. (33) pioneered the use of SNP arrays for
copy number analysis, the time, cost, and complexity of SNP
analysis, particularly the need to vitrify biopsied blastocysts,
are restrictive, although there is increasing evidence that it
may improve implantation and live birth weights (34). An
alternative method for 24-chromosome copy number analysis
that uses real-time quantitative PCR (qPCR) was therefore
developed and extensively validated (33). With this method,
a preamplification step, followed by a high-order multiplex
PCR reaction in a 384-multiwell plate format, is used to
amplify at least two sequences on each arm of each chromo-some.
Real-time qPCR is then used for the rapid quantificata-tion
of each product, allowing a comparison across the
genome. To avoid amplification bias from whole-genome
amplification, the multiplex PCR is performed on the sample
directly to ensure accurate copy number analysis and there-fore
is only applicable to multiple-cell trophectoderm sam-ples.
However, biopsy and analysis can be completed in
only 4 hours, facilitating the fresh transfer of single euploid
blastocysts in the same cycle (35). The only limitation with
the technology at present is the limited number of samples,
currently two on each plate, which can be run on the available
equipment. However, the use of loading robots and running
the analysis overnight allows higher throughput but extends
the time taken to analyze all of the samples.
NEXT-GENERATION SEQUENCING
The rapid development of next-generation sequencing (NGS)
technologies since James Watson was the first person to have
their genome sequenced and published on the internet in May
2007 is remarkable. Tens of thousands of individuals have
now had their entire genome sequenced, and efforts have
begun to understand all the variants from the reference
sequence that personal genomics identifies (36). It was

VIEWS AND REVIEWS
therefore inevitable that attempts would be made to use NGS
technologies for preimplantation genetics.
Various NGS technologies are available (37). Typically,
however, NGS involves fragmenting the sample DNA into
small 100–200-basepair fragments and ligating linker oligo-nucleotides
to either end of each fragment, one of which can
include a short sequence that effectively ‘‘bar codes’’ the DNA
from that sample. Multiple samples can then be processed
together in a single sequencing cell in which the DNA frag-ments
hybridize via the linker oligonucleotide to complemen-tary
oligonucleotides bound to the surface of the sequencing
cell. Hundreds of thousands of these fragments are sequenced
in parallel by the successive addition and removal of fluores-cent
nucleotides and ultrahigh-resolution imaging. The
sequence of each fragment is then compared with the refer-ence
genome using dedicated software to complete the
sequence. This process is continued until a sufficient ‘‘read
depth,’’ i.e., sequencing of multiple fragments from the
same genomic region, is acquired for accurate sequencing
of the required proportion of the genome.
For PGD, Treff et al. used a targeted NGS strategy and a
multiplex PCR reaction that included both the mutation site
and the chromosome specific target sequences required for
qPCR (38). This strategy reduced the read depth necessary
for accurate sequencing of the mutation site, which reduces
the time required and cost. In parallel, qPCR of the multiplex
PCR products provided rapid analysis of chromosome copy
number.
For chromosome copy number analysis by NGS, the prin-ciple
is straightforward (37). The whole-genome amplification
products from the embryo samples are simply fragmented and
sequenced and the read depth within successive regions of
each chromosome compared across the genome. Because
the number of fragments from a particular chromosome
should be proportional to the copy number, trisomy or mono-somy
will result in greater or less read depth, respectively.
Using this approach with trophectoderm samples from a series
of blastocysts, both whole-chromosome aneuploidy and
translocation-chromosome imbalance has been demonstrated
with an average read depth of only 0.07 and coverage of as
little as 5% of the genome (39).
CONCLUSION
The choice of which of the available technologies reviewed
here for 24-chromosome copy number analysis are selected
by clinics depends on a multiplicity of factors (Table 2). These
include, for example, preferences for biopsy method, fresh
versus frozen transfer, the turnaround time of the test, and
whether or not the clinic wishes to set up an in-house facility
or outsource to a service lab. All of the technologies are highly
accurate. However, particularly at the single-cell level, the
requirement for whole-genome amplification makes them
susceptible to amplification bias and cell-cycle artefacts. On
the other hand, those techniques that use PCR to amplify
from the samples directly, such as digital PCR and real-time
qPCR, are restricted to use on polar bodies and multiple-cell
trophectoderm samples, respectively. A priori, SNP arrays or
NGS-based methods for copy number analysis are likely to
be the most accurate and informative, because they use
sequence data from thousands of loci across each chromo-some
(37). However, the methodologies involved are more
complex and the cost of the equipment is high, so these tests
are probably going to be available only from the larger service
labs. NGS platforms are currently designed for high
throughput and accurate sequencing of whole genomes or
exomes for postnatal and cancer applications. It is likely to
be some time, therefore, before protocols and equipment opti-mized
for flexible low- to medium-throughput applications in
preimplantation genetics are developed and widely adopted.
Another important factor is scalability. If the time, effort,
and cost of a technique increase linearly with the number of
samples to be processed, laboratories can be quickly over-whelmed
and turnaround times compromised. Conventional
CGH is relatively straightforward to set up in house and the
reagent costs are low. However, the interpretation of the
results is highly skilled, and processing large numbers of sam-ples
is time consuming. Array CGH, in contrast, has been
widely adopted because it is a robust technology with a turn-around
time as short as 12 hours. It is also scalable with
decreased cost per sample as increased numbers of samples
are processed together. In addition, the same platform can
be used for detection of translocation-chromosome imbal-ance
and with dedicated prenatal and cancer microarrays.
Real-time qPCR has been extensively validated, it has the
fastest turnaround at 4 hours, allowing fresh transfer of blas-tocysts,
and the cost of the reagents is relatively low. Howev-er,
the equipment used currently allows processing of only
very small numbers of samples and is therefore less scalable,
because it requires multiple platforms.
At present, there is ongoing debate about the optimum
time of biopsy for chromosome copy number analysis (22,
39–43). Clearly, the largest reported increases in
implantation and live birth rates to date have been with
blastocyst biopsy, which is to be expected because there has
been a double selection for normally developing euploid
blastocysts. Blastocyst biopsy is therefore a good choice for
good-prognosis patients and particularly for those wishing
to have elective single-embryo transfer to avoid the complica-tions
associated with multiple pregnancy (23). However, the
cleavage-stage embryos of some poor-prognosis patients
may implant and develop in utero but not develop to the blas-tocyst
stage in vitro. From this point of view, polar body anal-ysis
is applicable to all patients and fertilized embryos and
focuses exclusively on female meiotic errors, which are known
to be the predominant cause of pregnancy loss, abnormal
pregnancy, miscarriage and affected live births. This approach
is, by definition, a screening test in the strict sense, because
it can not provide any information about paternal meiotic
errors or chromosome abnormalities arising after fertilization.
A lower diagnostic accuracy may be tolerated as long as
there is an overall improvement in healthy live birth rates.
ESHRE has organized a large multicenter RCT of polar
body analysis, which is scheduled to be completed in the
next 1–2 years.
Polar body analysis also provides important prognostic
information for couples about the origin of aneuploidies,
the likelihood of pregnancy using their own eggs, and

whether they should consider egg donation as an alternative.
In this respect, the use of SNP genotyping of the couple and
their embryos to identify meiotic trisomies and monosomies
by karyomapping has the advantage that meiotic errors
from either parent can be identified even in single cleavage-stage
blastomeres, providing enhanced prognostic informa-tion,
for example, in cases where there may be an increased
risk of aneuploidy from both partners. Thus, cleavage-stage
biopsy, which is well established and remains widely prac-ticed,
in combination with karyomapping may be another
effective strategy. Particularly if it can be combined with
quantitation, karyomapping should also allow the detection
of uniparental disomy that has been detected at the blastocyst
stage (22).
It is clear from this review of available technologies that
we are some way off from having a small benchtop box in the
IVF lab into which samples can be placed for rapid, accurate,
and low-cost 24-chromosome copy number analysis. Howev-er,
all of the studies involved in developing the various
methods have reinforced the reality that chromosome aneu-ploidy
is common in embryos following IVF, even in younger
women, and is a major factor in IVF failure. Furthermore, we
continue to learn more about their origins and evolution in
preimplantation development. It seems unlikely that we will
ever screen all embryos, particularly if it requires invasive
and time consuming biopsy procedures. However, for high-risk
patients testing for 24-chromosome copy number is
becoming increasingly established as an integral part of
best clinical practice.
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