Molecular Genetic Analysis of Single Cells

268
Molecular Genetic Analysis of Single Cells
Francesco Fiorentino, Ph.D. 1
1 GENOMA - Molecular Genetics Laboratory, Rome, Italy
Semin Reprod Med 2012;30:268–283
Abstract
Keywords
►
►
►
►
►
PGD
PGS
minisequencing
array-CGH
WGA
Address for correspondence and reprint requests Francesco
Fiorentino, Ph.D., GENOMA - Molecular Genetics Laboratory, Via Po,
102 00198 Rome – Italy (e-mail: fi[email protected]).
Preimplantation genetic diagnosis (PGD) has experienced a considerable technical
evolution since its first application in the early 1990s. The technology for single-cell
genetic analysis has reached an extremely high level of accuracy and enabled the
possibility of performing multiple diagnoses from one cell. Diagnosis of a monogenic
disease can now be combined with aneuploidy screening, human leukocyte antigen
typing, and DNA fingerprinting. New technologies such as microarrays are opening the
way for an increasing number of serious genetic defects to be detected in preimplantation embryos. The new PGD techniques will empower patients and clinicians to screen
for almost any kind of genetic problem in embryos, with the potential to change
completely the manner in which parents approach and manage genetic disease.
Preimplantation genetic diagnosis (PGD) has been introduced
as an alternative to prenatal diagnosis to increase the options
available for fertile couples who have a known genetically
transmittable disease. Its intended goal is to reduce a couple's
risk of transmitting a genetic disorder significantly by diagnosing a specific genetic disease in oocytes or early human
embryos that have been cultured in vitro before a clinical
pregnancy has been established.1,2
Following its first application in 1990,3 PGD has become an
important complement to the presently available approaches
for the prevention of genetic disorders and an established
clinical option in reproductive medicine. The range of genetic
defects that can be diagnosed includes not only single-gene
disorders4–7 but also structural chromosomal abnormalities
such as reciprocal or Robertsonian translocations.8 The scope
of PGD has also been extended to improve in vitro fertilization
(IVF) success for infertile couples, by screening embryos for
common or age-related chromosomal aneuploidies in patients who are deemed to be at increased risk, such as patients
of advanced maternal age and those having a history of
repeated miscarriage.9,10 More recently, PGD has been used
not only to diagnose and avoid genetic disorders, but also to
screen out embryos carrying a mutation predisposing to
cancer or to a late-onset disease. Additionally, PGD permits
selection for certain characteristics, such as human leukocyte
antigen (HLA) matching for tissue type with the ultimate aim
of recovering compatible stem cells from cord blood at birth
for transplantation to an existing sick sibling.11–14
Issue Theme Preimplantation Genetic
Diagnosis; Guest Editors, Alan H.
Handyside, Ph.D., and Kangpu Xu, Ph.D.
Stages for Single-Cell Testing
There are several stages during preimplantation development
at which genetic testing can be performed. PGD is usually
performed by testing single blastomeres removed from day 3
cleavage stage embryos (six to eight cells).
An alternative approach is represented by testing the first
polar body (1PB) before oocyte fertilization (so-called preconception genetic diagnosis)15 or sequential analysis of both
first and second (2PB) PBs,16 which are by-products of female
meiosis as oocytes complete maturation upon fertilization. In
women who are carriers for a genetic disease, genetic analysis
of 1PB and 2PB allows the identification of oocytes that
contain the maternal unaffected gene. Analysis of PBs might
be considered an ethically preferable way to perform PGD for
couples with moral objections to any micromanipulation and
the potential discard of affected embryos.15,17 It may also be
an acceptable alternative for countries in which genetic
testing of embryos is prohibited.15,18 However, PB analysis
is labor intensive because all oocytes must be tested despite
the fact that a significant number will not fertilize or will fail
to form normal embryos suitable for transfer. Furthermore, it
cannot be used for conditions in which the male partner
carries the genetic disorder because only the maternal genetic contribution can be studied.
Single cells for genetic analysis may also be obtained from
the embryo at the blastocyst stage of development on day 5 or
6 after fertilization.19 Biopsy at this stage has the advantage of
Copyright © 2012 by Thieme Medical
Publishers, Inc., 333 Seventh Avenue,
New York, NY 10001, USA.
Tel: +1(212) 584-4662.
DOI http://dx.doi.org/
10.1055/s-0032-1313906.
ISSN 1526-8004.
Molecular Genetic Analysis of Single Cells
allowing more cells to be sampled (5 to 10 cells), making
genetic tests more robust.
Diagnostic Methods
PGD tests have largely focused on two methodologies: fluorescent in situ hybridization (FISH) and polymerase chain
reaction (PCR).
Methods Based on Polymerase Chain Reaction
PCR-based methodologies has been mainly used to diagnose
single-gene disorders in single cells biopsied from embryos of
couples at risk of transmitting a monogenic disease (autosomal
recessive, autosomal dominant, or X-linked) to their offspring.
Almost all genetically inherited conditions diagnosed prenatally can also be detected with this approach. It can
theoretically be performed for any genetic disease with an
identifiable gene. Diagnostic protocols now exist for >200
monogenic disorders.4,7
Originally, X-linked diseases were avoided by selection of
female embryos, by using the FISH procedure to identify
embryos with two X chromosomes.20 A disadvantage of
this approach is that half of the discarded male embryos
will be healthy, a fact that gives rise to ethical criticism and
reduces the chances of pregnancy by depleting the number of
embryos suitable for transfer. In addition, half of the female
embryos transferred are carriers of the condition. In several
X-linked dominant disorders (e.g., fragile X syndrome), there
is also the possibility that, to a varying degree, carrier females
may manifest the disease. For many X-linked diseases, the
specific genetic defect has now been identified allowing a
specific DNA diagnosis. Therefore, there is now a consensus
that it is preferable to use PCR-based tests for sex-linked
disorders for which the causative gene is known, instead of
performing sex selection.21
In PGD for single-gene defects, the disease-linked locus is
amplified from blastomere DNA using targeted primers designed specifically for the mutation of interest.1,2,7,22–24
Amplified fragments of DNA can be then analyzed with
different strategies to detect the disease-causing mutation(s).
Numerous variants of PCR-based protocols have been
assayed at the single-cell level: procedures such as restriction
enzyme digestion,25–27 single-stranded conformational polymorphism,28,29 denaturing gradient gel electrophoresis,30
and allele-specific amplification.31,32 Real-time PCR33,34 has
been used to detect specific mutations.
The strategies just cited have been gradually replaced by
minisequencing,23 a technique based on a single dideoxynucleotide primer extension that permits a quick and accurate
detection of point mutations, as well as small deletions or
duplications. The minisequencing primer is designed to
anneal one base before the target site, and it will be elongated
with only one dideoxynucleotide. The four different dideoxynucleotides are labeled with different fluorochromes, and
the products can be distinguished on an automated DNA
sequencing system. This technique is very versatile and has
been broadly used in mutation and single nucleotide polymorphism (SNP) detection7,12,23(►Fig. 1).
Fiorentino
Multiplex Fluorescent Polymerase Chain Reaction
The pioneering early PGD studies used nonfluorescent nested
PCR and gel electrophoresis to look for the causative mutation
(s). A major step forward came with the introduction of
fluorescent PCR (F-PCR),24 which has become the gold standard in PGD for monogenic disorders, permitting the development of sophisticated and sensitive PGD assays. F-PCR is
based on the 5′ labeling of one of the primers with a fluorochrome and the analysis of the PCR products on an automated
sequencer. This system provides a very accurate molecular
weight determination that is >1000 times more sensitive
than the analysis on ethidium bromide–stained gels. In
addition, F-PCR opened the way to semiautomated analysis
using capillary electrophoresis systems and made possible
the multiplexing of different primer pairs in a single PCR
reaction (multiplex fluorescent PCR[MF-PCR]), enabling the
simultaneous assessment of numerous loci.
The introduction of MF-PCR24 allowed testing of a panel of
short tandem repeat (STR) polymorphic-linked markers in
addition to direct mutation analysis. A MF-PCR protocol
includes at least two polymorphic markers located very close
to, or within (i.e., linked), the gene region surrounding the
disease causing the mutation(s). The study of the DNA of the
patients provides a priori knowledge of which alleles are to be
expected in the embryos, as well as on which alleles the
markers co-segregate with the mutation (►Fig. 2).
This approach substantially improved the robustness of
the PGD protocols and decreased the possibility of misdiagnosis enabling allele dropout (ADO)35 and contamination
detection.
The ADO phenomenon consists of the random nonamplification of one of the alleles present in a heterozygous sample.
ADO seriously compromises the reliability of PGD for singlegene disorders (SGDs) because a heterozygous embryo could
potentially be diagnosed as either homozygous affected (in
which case it would be lost from the cohort of available
embryos) or homozygous normal (and therefore suitable for
replacement) depending on which allele would fail to amplify.
This is particularly concerning in PGD for autosomal dominant disorders, where ADO of the affected allele could lead to
the transfer of an affected embryo.
MF-PCR obviates the ADO problem. In fact, determination
of the specific STR haplotype associated with the mutation
acts both as a diagnostic tool for indirect mutation analysis,
providing an additional confirmation of the results obtained
with the direct genotyping procedure, and as a control of
misdiagnosis due to undetected ADO. Diagnosis is assigned
only when haplotype profiles obtained from linked STR
markers and mutation analysis profiles are concordant.
The multiplex STR markers system also provides an additional control for contamination with exogenous DNA because other alleles, differing in size from those of the parents,
would be detected. The partial “fingerprint” of the embryo
obtained with the MF-PCR strategy provides an added assurance, confirming that the amplified fragment is of embryonic
origin. Furthermore, the polymorphic nature of STR markers
also permits the detection of haploidy and uniparental disomy (UPD).
Seminars in Reproductive Medicine
Vol. 30
No. 4/2012
269
270
Molecular Genetic Analysis of Single Cells
Fiorentino
Figure 1 Minisequencing results of a β-thalassemia preimplantation genetic diagnosis case, in which the mutations of interest were IVSI-110
G > A and IVSI-6 T > C, analyzed by a multiplex reaction. A color is assigned to individual ddNTPs as follows: green/A, black/C, blue/G, and red/T.
The minisequencing reaction produces one (homozygote) or two (heterozygote) peaks depending on the genotype at this locus. The result
obtained for the mutation IVSI-6 T > C (right side) shows the presence of two different-colored peaks, one of which refers to the normal allele (red
peak) and the other to the mutated allele (black peak, mutant base C), consistent with a heterozygosity for this mutation. The result obtained for
the mutation IVSI-110 G > A del-AA (left side) shows the presence of two different-colored peaks, one coming from the normal allele (blue peak)
and the other from the mutated allele (green peak, mutant base A). (A) Heterozygote blastomere for IVSI-6 T > C. (B) Normal blastomere.
(C) Compound heterozygote blastomere for the two mutations. (D) Heterozygote blastomere for IVSI-110 G > A.
The experience of a large series of PGD cycles7 strongly
suggests that PGD protocols for a SGD are not appropriate for
clinical practice without including a set of linked STR
markers. Consequently, this strategy is currently used by
most PGD laboratories.
Indirect Testing by Linkage Analysis
A further advantage of MF-PCR is the possibility of its use as a
tool for indirect mutation analysis in linkage-based protocols.
In fact, any informative polymorphism that lies in close
proximity to the disease locus can be used as a tool to indicate
the presence or absence of the mutation without its direct
detection.
For disorders that are mostly caused by private mutations,
the development of multiplex PCRs exclusively for linked
markers has the advantage that they can be used for several
couples because it omits the detection of the (private) mutation. However, the ability to use such indirect testing with
linked markers depends on the availability of appropriate
family samples to determine which chromosome carries the
disease-causing mutation (s) (determination of phase). Nevertheless, the candidate couples must be informative for at
Seminars in Reproductive Medicine
Vol. 30
No. 4/2012
least two of the markers included in the PCR. Moreover, the
markers should be flanking each side of the mutation to be
able to detect recombination.
For some couples, the disease-causing mutation may have
arisen de novo in the individual.36 In this cases, the couple
should at least have had a child or a pregnancy from which
DNA of the fetus is available. However, it is possible for the
phase of paternal alleles to be determined by performing
single sperm analysis; for maternal alleles, PB analysis can be
performed, although this requires a cycle to be initiated prior
to the determination of phase.37,38
Nevertheless, the advantages of this approach largely
compensate for the possible drawbacks, and the use of
multiplex PCR for markers has become widespread in PGD
for monogenic disorders,7,15,39,40 HLA typing,13,14 or aneuploidy screening.41
Exclusion Testing
Linkage analysis has also been used for exclusion testing for
late-onset Huntington's disease (HD), where someone with a
family history of HD does not wish to know their HD status
but wishes to ensure that their children are not at risk.42
Molecular Genetic Analysis of Single Cells
Fiorentino
Figure 2 Preimplantation genetic diagnosis for β-thalassemia. Pedigree of a couple carrying β-thalassemia mutations and examples of different
results of the HBB gene mutation analysis. Informative short tandem repeat (STR) markers are ordered from telomere (top) to centromere
(bottom). The numbers in STR markers represent the size of polymerase chain reaction products in base pair. STR alleles linked to the paternal and
maternal mutations are colored in red. Embryo 1 is a carrier for IVSI-110 G > A mutation; embryos 2 and 7 are carriers for IVSII-745 C > G
mutation; embryos 3 and 6 are normal; Embryo 8 is a compound heterozygote for the two mutations. Embryo 5 is also affected, although the
mutation analysis result shows a heterozygosity for mutation IVSI-110 G-A. In fact, linked STR markers highlight an allele dropout of the IVSII-745
C > G mutation.
Exclusion testing is based on a linkage analysis PGD
protocol involving the use of polymorphic markers flanking
the HD gene. This strategy allows tracking of the parental and
grandparental origin of the chromosomes and can subsequently be used to identify the low-risk (grandparental) allele
in derived offspring (►Fig. 3). In this way, only embryos are
replaced that do not contain the chromosome 4 derived from
the affected grandparent because inheritance of this chromosome confers a 50% risk of HD. This method can be used to
classify embryos as at low or high risk for developing the
disease (29 Jasper), avoiding the need to detect the mutation
itself.42,43
PGD for HD is also possible by direct testing (i.e., evaluating
the presence or absence of the expanded repeat) or by
linkage-based indirect testing (i.e., ascertaining the presence
or absence of the haplotype associated with triplet expansion). Another way of testing for HD is the so-called nondisclosure PGD that entails direct testing of the expanded repeat
on the embryos without knowing the disease status. It is
applied in those cases in which the prospective parent at risk
does not wish to be informed about his or her own carrier
status but wants to have offspring free of the disease.44
Embryos can be tested for the presence of the mutation
without revealing any of the details of the cycle or diagnosis
to the prospective parents. Nondisclosure testing is controversial and professional generally do not approve it42,45
because it puts practitioners in an ethically difficult position,
that is, when no embryos are available for transfer and a mock
transfer has to be performed to avoid the patient suspecting
that he or she is a carrier or having to undertake PGD cycles
even when the results of previous cycles preclude the patient
being a carrier. The European Society of Human Reproduction
and Embryology (ESHRE) ethics task force21 currently discourages nondisclosure testing, recommending the use of
exclusion testing instead.
Preimplantation Human Leukocyte Antigen Matching
Linkage-based approach has also been used for preimplantation HLA matching.11–14 This strategy has revealed a useful
tool for couples at risk of transmitting a genetic disease,
allowing selection of unaffected embryos that are HLA tissue
type compatible with those of an existing affected child. In
such cases, PGD is used not only to avoid the birth of affected
children but also to conceive healthy children who may also
be potential HLA-identical donors of hematopoietic stem cells
for transplantation to siblings with a life-threatening
disorder.
From a technical point of view, PGD for HLA typing is a
difficult procedure due to the extreme polymorphism of the
HLA region. Taking into account the complexity of the region
(presence of a large number of loci and alleles), the use of a
direct HLA typing approach would require standardization of
a PCR protocol specific for each family, presenting different
HLA allele combinations and making it time consuming and
unfeasible. The use of a preimplantation HLA matching
protocol irrespective of the specific genotypes involved
makes the procedure more straightforward.
Currently, PGD laboratories use a strategy based on a
flexible indirect HLA typing protocol applicable to a wide
spectrum of possible HLA genotypes.11–14 The approach
involves the testing of single blastomeres by fluorescent
multiplex PCR analysis of polymorphic STR markers,
Seminars in Reproductive Medicine
Vol. 30
No. 4/2012
271
272
Molecular Genetic Analysis of Single Cells
Fiorentino
Figure 3 Preimplantation genetic diagnosis (PGD) for Huntington disease (HD) by exclusion testing. Familial pedigree from a couple at risk for
HD. Informative short tandem repeat (STR) markers, linked to the HD gene, are ordered from telomere (top) to centromere (bottom). The
numbers in STR markers represent the size of polymerase chain reaction products in base pair. A and B are at-risk haplotypes, inherited from the
grandparent affected by HD. Embryos 2 and 4 are normal; embryos 1 and 3 are at risk for HD.
scattered throughout the HLA complex of chromosome 6,
obtaining a “fingerprint” of the entire HLA region (►Fig. 4).
STR haplotyping for family members (father, mother, and
affected child) is performed prior to preimplantation HLA
typing to identify the most informative STR markers of the
HLA complex to be used in the following clinical PGD cycles. A
panel of STR markers is studied during the setup phase to
ensure sufficient informativity in all families. For each family,
only heterozygous markers presenting alleles not shared by
the parents are selected, so that segregation of each allele and
discrimination of the four parental HLA haplotypes can be
clearly determined. Informativity is also evaluated for STR
markers linked to the gene regions involved by mutation and
is thus used to avoid possible misdiagnosis due to the wellknown ADO phenomena.
By selecting a consistent number of STR markers evenly
spaced throughout the HLA complex, an accurate mapping of
the whole region can be achieved. Because genes in the HLA
complex are tightly linked and usually inherited in a block,
profiles obtained from such markers in father, mother, and
affected child allow the determination of specific haplotypes.
Thus the HLA region can be indirectly typed by segregation
analysis of the STR alleles, and the HLA identity of the
embryos matching the affected sibling can be ascertained
by evaluating the inheritance of the matching haplotypes.
Because segregation of the STR alleles fully corresponds to the
direct HLA genotyping, STR haplotyping can be used as a
reliable diagnostic tool for indirect HLA matching evaluation.
The use of microsatellite markers for this purpose is very
useful. They may provide information on identity over a
greater distance within the HLA region compared with classical HLA genes alone, making haplotyping more accurate in
Seminars in Reproductive Medicine
Vol. 30
No. 4/2012
predicting compatibility. Another important advantage of
using STR markers in preimplantation HLA matching is that
the whole HLA complex can be covered, which allows the
detection of recombination events between HLA genes
(►Fig. 4).
Preimplantation Genetic Diagnosis Using Whole Genome
Amplification
One of the most exciting development in single-cell analysis
has been the introduction of protocols designed to amplify
the entire genome from a single cell (whole genome amplification [WGA]). The idea was using WGA as a universal first
step to enable secondary analysis of a range of sequences
without the need to optimize primers and reaction conditions for multiplexing, also overcoming the difficulties of
testing single cells.
A recent important evolution of these WGA protocols is
multiple displacement amplification (MDA), a technique that
may offer greater accuracy and more rapid throughput in the
future.46 Amplification of DNA for a single biopsied blastomere gives 106-fold amplification. Aliquots of MDA products can be taken and used as a source of templates for
subsequent locus-specific PCRs, allowing many individual
DNA sequences or genes to be analyzed in the same cell.
The principal advantage of MDA for PGD is that sufficient
amplified DNA is produced to allow extensive parallel genetic
testing and accurate mutation detection by conventional
relatively low sensitivity methods. A further advantage is
that WGA provides a sufficient supply of sample DNA available for other applications, simplifying the combination of
different indications in one PGD cycle, such as combining
mutation with aneuploidy screening by array CGH technique
Molecular Genetic Analysis of Single Cells
Fiorentino
Figure 4 Preimplantation human leukocyte antigen (HLA) matching. Specific haplotypes are determined by genomic DNA analysis of HLA short
tandem repeat (STR) markers from father, mother (upper panel), and affected child (lower panel, left side, black square). The numbers in STR
markers represent the size of polymerase chain reaction products in base pair. Paternally and maternally derived haplotypes are shown on the left
and the right, respectively. The HLA identity of the embryos with the affected sibling has been ascertained evaluating the inheritance of the
matching haplotypes. On the middle of the lower panel, an HLA-matched embryo is shown. The left of the lower panel represents the HLA profile
of an embryo with a single recombination occurrence in maternal haplotypes, between the markers HLA-BC and MOG-CA.
or HLA haplotyping.46 It could therefore be envisaged that in
the future every embryo tested for a monogenic disorder
would also be routinely screened for aneuploidy, investigating all chromosomes.
The main limitations of using this method on single cells are
the high ADO and preferential amplification rates compared
with direct PCR on DNA from a single cell.46,47 In fact, PCR on
MDA products gives ADO rates of between 5% and 31%, which is
unacceptable for direct mutation testing, due to the likelihood
of exclusion of embryos from transfer following inconclusive
results.46,48 Nevertheless, MDA has been used clinically in PGD
of cystic fibrosis, β-thalassemia,49 and fragile X syndrome.50
However, this approach has not been taken further owing to
the effect of high ADO on the reliability of results.
Limitations of the PCR-Based Preimplantation Genetic
Diagnosis Protocols
Genetic diagnosis of single cells is technically demanding, and
protocols have to be stringently standardized before clinical
application.
To establish a diagnostic PGD protocol, extensive preclinical experiments are performed on single cells (lymphocytes,
fibroblasts, cheek cells, or spare blastomeres from research
embryos) to evaluate the efficiency and reliability of the
procedure.
Many genetic disorders can now be diagnosed using DNA
from single cells. However, when using PCR in PGD, one is faced
with a problem that is nonexistent in routine genetic analysis,
namely the minute amounts of available genomic DNA. In fact,
because PGD is performed on single cells, PCR has to be
adapted and pushed to its physical limits. This entails a long
process of fine-tuning the PCR conditions to optimize and
validate the PGD protocol before clinical application.
Three main difficulties are inherently associated with
single-cell DNA amplification. The limited amount of template makes single-cell PCR very sensitive to contamination.
The presence of extraneous DNA can easily lead to a misdiagnosis in clinical PGD. Cellular DNA from excess sperm or
maternal cumulus cells that surround the oocyte is a potential
source of contamination. These cells can be sampled accidentally during the biopsy procedure. For these reasons oocytes
used for PGD of single-gene defects should always be stripped
of their cumulus cells and fertilized by the use of intracytoplasmic sperm injection (ICSI) in which only a single
sperm is inserted into the oocyte. Furthermore, any biopsied
cells should be washed through a series of droplets of medium
before transfer to the PCR tube, and the wash drop should be
tested for contamination.
Other sources of contamination include skin cells from the
operators performing the IVF/PGD procedure or so-called
carryover contamination of previous PCR products, but this
type of contamination can be minimized or eliminated by
following the strict guidelines prescribed by the ESHRE PGD
Consortium.51
Another problem specific to single-cell PCR is represented
by the previously mentioned ADO phenomenon, which is
Seminars in Reproductive Medicine
Vol. 30
No. 4/2012
273
274
Molecular Genetic Analysis of Single Cells
Fiorentino
Figure 5 Results obtained from a reimplantation genetic diagnosis (PGD) case for Robertsonian translocation (13,14). Right side: Capillary
electrophoresis of fluorescent polymerase chain reaction (PCR) products, after multiplex amplification of a set of polymorphic short tandem
repeat (STR) markers located along chromosome 14. On top of the electropherogram, the marker name is located above the corresponding alleles
(peaks). A normal diploid embryo (C) has the normal complement of each parental chromosome; thus two alleles of a chromosome-specific STR
are determined as two peaks. Embryos with a normal copy number for a given chromosome will show a heterozygous pattern for all the STRs used.
The observation of an extra STR allele as a three-peak pattern is diagnostic of the presence of an additional sequence that represents an additional
chromosome, as in the case of a trisomy. Trisomic embryos will produce trisomic patterns for all markers on the same chromosome (B). The
observation of only one STR allele as a one peak pattern is diagnostic of the missing sequence from one chromosome, as in the case of a
monosomy. Monosomic embryos will show a homozygote pattern for all the STRs used for a given chromosome (A). Left side: Fluorescence in situ
hybridization results from the same PGD case as above. Center: Graphical representation of chromosomes 13,14, and STR markers used.
particularly concerning in PGD for autosomal dominant disorders, where ADO of the affected allele could lead to the
transfer of an affected embryo.
Fluorescent in Situ Hybridization Methods
FISH is a cytogenetic technique that uses a set of DNA probes
labeled with distinctly colored fluorochromes that bind to
specific DNA sequences unique to each chromosome.52,53
Imaging systems enable the fluorescent probe signals to be
identified and counted to detect missing or excess chromosomal material (►Fig. 5).
Fish-Based Preimplantation Genetic Diagnosis for
STructural Chromosomal Abnormalities
FISH, to date, is the most widely used method for detecting
unbalanced chromosome rearrangements in embryos produced from carriers of structural chromosomal abnormalities,
Seminars in Reproductive Medicine
Vol. 30
No. 4/2012
such as reciprocal and Robertsonian translocations,4 chromosomal deletions, or duplications. This method requires the PBs
or biopsied cells to be spread on a microscope slide and then
hybridized to chromosome-specific DNA probes.
A commonly used FISH strategy for detection of the
abnormal segregation in reciprocal and Robertsonian translocations involves the use of commercially available centromeric, locus-specific, and subtelomere probes, allowing for
a simplified approach that laboratories can apply
routinely.54–56
FISH strategies for the assessment of reciprocal translocations use three differentially labeled probes: two probes
specific for the subtelomeric regions of the translocated
segments combined with a centromeric probe.8,54 Analysis
of Robertsonian translocations is simpler, involving the use of
specific probes chosen to bind at any point on the long arm of
each chromosome involved in the translocation (►Fig. 5).
Molecular Genetic Analysis of Single Cells
This combination of probes allows embryos that carry an
unbalanced chromosome complement to be distinguished
from healthy ones. However, both strategies do not discriminate between noncarrier embryos and those that carry the
balanced form of the translocation.
Although relatively successful, the FISH procedure is
technically demanding and harbors several technical limitations that are well documented57,58 and include hybridization failure (lack of FISH signals), signal overlap, signal
splitting, and poor probe hybridization, as well as problems
related with the fixation process, such as cell loss and
variable cell fixation. Since FISH was first introduced in
clinical diagnosis, improvements have been established to
diminish the error rate of the technique. 59–61 Technical skill
and sound laboratory practices can minimize most of the
limitations of FISH-based methods, but certain shortcomings remain. Interpretation errors due to the technical
issues described previously can affect the accuracy of the
interpretation of the results, leading to a misdiagnosis of
embryos both in eliminating suitable (normal/balanced)
embryos for transfer, or worse, including abnormal embryos in the transfer cohort errantly.62 Error rates of FISH
protocols for translocation have been reported in some
studies to range from 0% to 10%, with an average error
rate of 6%.57,58,63
Detection of Chromosomal Aneuploidy by Fish
FISH is also able to detect aneuploidy in single blastomeres,
and it was the most common method for aneuploidy screening of embryos derived from subfertile patients undergoing
IVF (preimplantation genetic screening [PGS]). PGS enables
the assessment of the numerical chromosomal constitution of
embryos. It aims to identify and select for transfer only
chromosomally normal (euploid) embryos to increase the
implantation and pregnancy rate for IVF patients, lower the
risk for miscarriage, and reduce the risk of having an infant
with an aneuploidy condition.64
Essentially, FISH probes to detect those aneuploidies most
commonly observed after birth or in miscarriages (involving
detection of chromosomes X, Y, 13, 16, 18, 21, and 22) were
used for the purpose of PGS. This panel of probes has the
potential of detecting >70% of the aneuploidies found in
spontaneous abortions.65 Aneuploidy conditions (involving
chromosomes 8, 9, 15, and 17) that cause lack of implantation
or can result in a miscarriage early in pregnancy have also
been tested.66,67
However, FISH-based PGS allows identification of aneuploidies only for a limited number of chromosomes (5, 9, or 12
chromosomes).68–70 Commercial FISH probe panels are available that will identify five to six chromosomes at a time by
different colors (fluorochromes). The number of chromosomes that can be tested at any one time is limited by the
range of fluorochromes available and the eye's ability to
distinguish between different colors. The numbers of chromosomes investigated may be increased by washing off the
first panel of probes and rehybridizing with an additional
panel of probes, but this increases the time taken to arrive at a
diagnosis. Rehybridization can only be performed with any
Fiorentino
clinical accuracy once, which therefore limits the number of
chromosomes that can be analyzed.
Several studies have demonstrated that aneuploidies may
involve all 24 chromosomes.71–75 Therefore FISH analysis
may result in the transfer of reproductively incompetent
embryos with aneuploidy present in the chromosomes not
analyzed.
PGS currently has several further disadvantages that limit
its clinical value. The main concern is the elevated mosaicism
rate observed in the human cleavage-stage embryo. Mosaicism is defined as the embryo having cells with different
chromosome makeup, and it has been found in up to 57% of
day 3 biopsied embryos.76,77 Mosaicism may represent a
major source of misdiagnosis (60%) because of both falsepositive and false-negative results.
Besides mosaicism, several technical limitations inherent
to the FISH technique have been described. FISH is considered
to have an error rate between 5% and 10%.57,58,63 Overlapping
signals may be a source of misdiagnosis resulting in the false
diagnosis of monosomies. Signal splitting has also been
described, resulting in the detection of false trisomies. Finally,
evidence suggests that up to half of all embryos identified as
aneuploid at the cleavage stage and survive to the blastocyst
stage will “self-correct.”78,79 Therefore an abnormal result
may not necessarily indicate that the embryo is abnormal and
ill-fated.
As a consequence of the limitations just described, it has
been questioned whether some normal embryos might be
excluded from the cohort that is considered suitable for
embryo transfer, which, especially in older women who
might have small embryo cohorts, could result in the failure
to reach embryo transfer.
Since the publication of the first articles on PGS using
cleavage-stage embryos80 and PBs,81–83 there have been
several nonrandomized comparative studies of IVF/ICSI
with or without PGS for advanced maternal age or repeated
implantation failure. Most of these studies reported that PGS
increases the implantation rate,9,10,84,85 decreases the abortion rate, and reduces trisomic conceptions.33,86,87
There are now 11 randomized controlled trials (RCTs)
published on PGS, 10 using cleavage-stage biopsy88–97 and
one using blastocyst biopsy.69 All have used FISH testing of a
limited number of chromosomes, and none have shown an
improvement in delivery rates, with some showing a significant decrease in delivery rates after PGS. Most of the RCTs
have been for patients of advanced maternal
age.88,90,91,93,95,97
These RCTs have shown that PGS, as it was practiced, has
not provided the expected benefits. There are many possible
reasons why these clinical studies failed to deliver the expected improvements in IVF outcome. Putative explanations
for this poor clinical performance could be attributed to an
incomplete understanding of important aspects of embryo
biology, such as embryonic chromosomal mosaicism, which
is present on day 3 of development (i.e., the tested blastomere
is not representative of the whole embryo),98 or self-correction of aneuploidy within the embryo, which may decrease
the chances of a live birth by prematurely labeling an embryo
Seminars in Reproductive Medicine
Vol. 30
No. 4/2012
275
276
Molecular Genetic Analysis of Single Cells
Fiorentino
as abnormal. Indeed, high levels of chromosomal mosaicism
have been observed in blastomeres from cleavage-stage
embryos.74,81,82,99
Alternatively, it has been argued that inadequate cytogenetic methods may have led to reduced diagnostic accuracy and elimination of any potential benefit of
screening.100 In fact, all prior clinical trials using PGS for
IVF have used FISH, which screens for a minority of chromosomes (up to 12 chromosomes).87 Most FISH-based
methods focus on the chromosomes most often found to
be aneuploid in prenatal samples or material from miscarriages. However, these chromosomes are not necessarily the
most relevant for early embryos. Thus the limitations of the
FISH technology could have compromised the results of
these studies.101,102
Emerging Technologies
Increasingly, new techniques for chromosome analysis in
embryos are being sought in an attempt to improve on
current FISH test method performance and now are suitable
for PGD/PGS use.
MF-PCR in Preimplantation Genetic Diagnosis for
Chromosomal Translocation
Recently, a MF-PCR–based PGD approach for detection of
chromosomal imbalances on embryos derived from both
reciprocal and Robertsonian translocation carriers was proposed as a valuable alternative to the FISH-based PGD protocols.103 For reciprocal translocations, the strategy consisted
of a MF-PCR using STR markers flanking the translocation
breakpoints on both chromosomes involved, which tracks all
meiotic segregations. For Robertsonian translocations, STR
markers located at any point along the chromosomes involved allowing for differentiation between aneuploid embryos and normal/balanced embryos by simply enumerating
peak signals (►Fig. 5).
This approach aimed to overcome several of the previously
listed limitations related to the FISH technique while providing significant improvements in terms of test performance,
automation, turnaround time, cost effectiveness, sensitivity,
and reliability of the information obtained.
STR genotyping for both partners of each couple is performed to identify the most informative STR markers to be
used in the clinical PGD cycles. For each couple, only fully
informative heterozygous markers presenting nonshared alleles (i.e., four different alleles, male partner a/b and female
partner c/d; or three different alleles, translocation carrier a/
b, other partner c/c) are selected so that segregation of each
allele could be clearly determined.
Embryos are diagnosed as “normal/balanced” if PCR results clearly indicated two signals (peaks) for each chromosome tested. Embryos are, instead, diagnosed as
“unbalanced” if the PCR results showed a clear and consistent
deviation from the “normal/balanced” signal pattern, such as
(partial or full) trisomies (three peaks), (partial or full)
monosomies (one peak), and nullisomies (no PCR signals)
(►Fig. 5).
Seminars in Reproductive Medicine
Vol. 30
No. 4/2012
Using STR markers simultaneously for each arm involved
in the translocation, one can detect all abnormal segregations
for any translocation. The co-amplification of at least three
fully informative STR markers lying on either side of the
translocation breakpoints increases the accuracy of the test,
avoiding misdiagnosis due to possible ADO occurrences. In
fact, ADO in any one marker is not likely to be repeated in the
others; it would lead to misdiagnosis only if all markers tested
are affected simultaneously. The multiplex STR marker system also provides an additional control for contamination
with exogenous DNA because other alleles, differing in size
from those of the parents, would be detected.7 However, the
approach does not discriminate between noncarrier embryos
and those that carry the balanced form of the translocation.
The MF-PCR-based approach not only diagnoses unbalanced inheritance of chromosomes in translocation carriers,
it also allows for tracking of inheritance of each chromosome.
This tracking of parental origin allows for the diagnosis of
UPD where both chromosomes are inherited from one parent
and no chromosomes are inherited from the other.104,105
FISH-based techniques cannot discern the origin of the chromosome, which can lead to chromosomally balanced embryos for transfer with from UPD.
Finally, the molecular approach is also amenable to automation and allows for easy data interpretation. It may also
make transport PGD easier because placing a cell in a tube is
far easier to train and monitor than teaching any of the
current spreading methods.
Detection of Chromosomal Abnormalities by
Microarrays
Comprehensive chromosome screening techniques75,106–108
have also been recently introduced into current routine PGD
laboratory practices. Some of the most promising progress
toward developing a comprehensive 24-chromosome analysis method has been made possible through the combination
of WGA, a protocol able to amplify the entire genome from a
single cell, and array comparative genomic hybridization
(array-CGH).106,108–110
Array-CGH involves a competitive hybridization of two
differentially labeled DNA samples, one derived from the
embryo (test DNA) and the other from a euploid DNA sample
(reference DNA). The two samples are competitively hybridized to genomic probes of known sequence, immobilized on a
glass microarray, each corresponding to a specific part of a
chromosome. Software analysis of the scanned microarray is
used to estimate the amount of test DNA and control DNA
bound to each probe location, the copy number of the
genomic sequence(s) represented by that probe, and hence
the ploidy of the chromosomal region. This method has a
high resolution, the analysis is fully automated, and the
whole procedure can be performed within 24 hours. For
day 3 or day 5 biopsy, it is possible to perform the embryo
transfer within day 5 or day 6 of embryo development, in a
fresh cycle.
A major advantage of the array-CGH approach over FISH is
that it does not depend on cell fixation onto a microscope
slide, a critical step that requires skill and experience. Because
Molecular Genetic Analysis of Single Cells
the reliability of FISH analysis is linked to the quality of cell
fixation, poorly fixed single cells will affect probe hybridization. The removal of a requirement for the more technically
demanding cell fixation step notably simplifies the sample
preparation procedure, with the potential to increase the
percentage of embryos with a positive result. It may also make
transport PGD easier, enabling broader use of PGD in IVF
programs, because placing single cells into PCR tubes is far
easier to train and monitor than teaching any of the current
spreading methods.72
In addition, data analysis is performed by computational
analysis of signal intensities and not by subjective signal
scoring, as occurs with FISH analysis, allowing for easier
and more reliable data interpretation. Finally, array-CGH is
also amenable to automation for high throughput processing.
The use of automated workstations for all manipulation
greatly increases the number of samples a laboratory can
process and also reduces the risk of mishandling the samples.
Beyond the technical advantages just described lies another important advantage. The WGA plus array-CGH approach
not only diagnoses chromosomal abnormalities, it also enables the simultaneous screening of the embryos for singlegene disorders by PCR-based methods (such as for cases with
combined indications for the couple, e.g., translocation plus
carrier status for a SGD), using the same DNA produced after
the WGA reaction. It could therefore be envisaged that in the
future every embryo tested for a monogenic disorder would
also be routinely screened for aneuploidy.
Preimplantation Genetic Diagnosis for Chromosomal
Translocation Using Array-CGH
Recently, the array-CGH technique was clinically applied for
the detection of chromosomal imbalances in embryos derived
from translocation carriers.114 This approach was proposed
as a valuable alternative to the FISH-based and PCR-based
PGD protocols.
Array-CGH has several advantages compared with the
other methods. In contrast to FISH and PCR, array-CGH allows
screening for all aneuploidies in addition to the unbalanced
derivatives associated with the specific translocation
(►Fig. 6). It is a relatively common for PGD of chromosomal
translocations to be combined with aneuploidy screening to
assess aneuploidies for patients of advanced maternal
age.67,111 With FISH, aneuploidy screening is typically done
by sequential rounds of hybridization followed by chemical
stripping of bound FISH probes. However, it is well recognized
that the accuracy of the FISH analysis decreases with each
additional round of hybridization because the DNA degenerates and hybridization efficiency is reduced.112 Furthermore,
the persistence of signals from the first round of FISH could
result in an incorrect interpretation of signals in the second
round, thereby leading to a higher risk of misdiagnosis or a
loss of normal embryos to false-positive errors. However,
FISH allows the identification of aneuploidies only for a
limited number of chromosomes (5, 9, or 12 chromosomes),
most often found to be aneuploid in prenatal samples or
material from miscarriages. Several studies have demonstrated that aneuploidies may involve all 24 chromo-
Fiorentino
somes.71–75,94,114 This may result in the transfer of
reproductively incompetent embryos with aneuploidy for
chromosomes not analyzed.
Another advantage on the use of array-CGH technology is
that it does not require preclinical validation before each IVF
cycle, which is required for FISH or PCR-based methods of
translocation screening. This spares couples the cost of
workup testing, also allowing a rapid starting of the IVF
treatments, In fact, a PGD cycle can be scheduled directly on
the day of biopsy, based on the number of embryos available
for biopsy.
Finally, array-CGH can also be used for more complex
karyotypes, with multiple rearrangements, whereas FISH
testing is generally very complicated, although the chances
of getting euploid normal/balanced embryos from these cases
is rare.
Although these results indicate that the array-CGH procedure is reliable and suitable for routine clinical application,
the limitations must be considered. Array-CGH cannot detect
haploidy and some polyploidies, such as 69,XXX, 92,XXXX, or
92,XXYY, as well as balanced translocations, because there is
no imbalance in the total DNA content. Additionally, the
occurrence of a contamination event with exogenous DNA
may reduce the number of embryos with a conclusive diagnosis using this technique.
Another potential limitation for routine application of this
approach is that DNA amplification protocols require a minimum level of molecular biology experience as well as a
laboratory environment where DNA contamination is
avoided.
Unlike PCR and SNP microarrays, array-CGH does not allow
for tracking of inheritance of each chromosome, so it cannot
detect UPD.104,105
Finally, microarrays represent at this time an expensive
option for embryo testing compared with the other conventional methods, although the cost of array-based testing will
probably continue to drop as most new technologies do. The
initial setup costs as well as the expertise levels required may
mean that FISH- or PCR-based protocols are still appropriate
for some PGD laboratories.
24-Chromosome Preimplantation Genetic Screening by
Array-CGH
Recently, microarray-based approaches for 24-chromosome
PGS has been proposed to overcome the technical difficulties
that beset earlier PGS studies, allowing screening of the entire
chromosome complement, rather than the limited chromosome assessment (►Fig. 7).72,73,75 Data from comprehensive
aneuploidy screening showed that aneuploidies may occur in
preimplantation embryos in any of the 24 chromosomes,
indicating that aneuploidy screening of all chromosomes is
necessary to determine whether an embryo is chromosomally normal.72,75,114
However, screening for all 24 chromosomes in a single
blastomere biopsied from a cleavage stage embryo may still
be compromised by the degree of mosaicism that has been
observed in such embryos or the possibility of self-correction
of aneuploidy within the embryo. Although a follow-up study
Seminars in Reproductive Medicine
Vol. 30
No. 4/2012
277
278
Molecular Genetic Analysis of Single Cells
Fiorentino
Figure 6 (A) Examples of array-CGH–based preimplantation genetic diagnosis results from a chromosomally unbalanced embryo derived from a
patient carrying a balanced translocation 46,XY,t(3;18)(p26.3;q12), consistent with meiotic adjacent-1 segregation. The imbalances include a
2.5-Mb size loss of a chromosome 3p26.3-pter segment, detected as a shift of the clones located in the above region toward the red line (loss) and
a partial trisomy 18q12-qter, identified with a shift of the specific clones toward the green line (gain). (B) Chromosomal details for these segmental
imbalances.
of nontransferred chromosomically unbalanced embryos derived from translocation carriers revealed that all embryos
that were classified as aneuploid after day 3 diagnosis were
diagnosed again as abnormal after reanalysis, confirming at
the end the previous results regardless of the actual abnormal
genotype.
Because mosaicism is not present at the zygote stage,
aneuploidy screening in PB1 and PB2 has been proposed to
detect most of the aneuploidies that arise from the maternal
genome, which is particularly relevant for patients of advanced maternal age. However this technique ignores the
possible contribution from the paternal genome and any
aneuploidies that may arise during aberrant mitotic division.
Trophectoderm biopsy at the blastocyst stage of development is another alternative, but too few studies have been
undertaken to evaluate if this is a more accurate reflection of
the chromosomal status of the inner cell mass and ultimately
the developing fetus.
Seminars in Reproductive Medicine
Vol. 30
No. 4/2012
Several RCTs performed by array-CGH technique, at different cell biopsy stage and categories of patients, are currently
ongoing with the aim to establish whether PGS results in an
enhanced live-birth rate and, if this is the case, to identify
which patients may benefit from the procedure,
24-Chromosome Preimplantation Genetic Screening
Combined with Preimplantation Genetic Diagnosis for
Single-Gene Disorders
In the last few years, the use of SNP microarrays for PGD has
been introduced.113,114 SNPs are common polymorphic DNA
sequences found throughout the genome.
The microarray platforms typically involve oligonucleotide
probes that are attached directly to the surface of solid
supports, for example on slides or on beads, so each probe
has a specific known location.
SNP arrays rely on a WGA step to amplify the single cell or
small number of cells removed from a developing embryo.
Molecular Genetic Analysis of Single Cells
Fiorentino
Figure 7 Examples of array-CGH–based preimplantation genetic screening (PGS) results from day 3 biopsy. (A, B) Embryo with a complex
aneuploidy involving chromosomes 15, 21 (loss) and 18 (gain).
After amplification, the DNA is fluorescently labeled and
hybridized to the probes attached to the surface of the
microarrays. This DNA is then assessed with automated
fluorescent readers that analyze the probes to determine
hundreds of thousands to millions of genotypes, and then
they are compared with a control population using powerful
computer software to allow for diagnosis of inheritance.
SNP-based arrays offer other options for testing that are
not available on CGH-based systems. SNP-based arrays allow
for simultaneous testing of specific genetic diseases and
aneuploidy in each embryo. This will enable the selective
transfer of genetically and chromosomally normal embryos
for patients undergoing IVF with PGD for monogenic diseases.
Simple haplotyping of SNPs surrounding and embedded in
disease-causing genes allows for selection of embryos that
have not inherited the affected chromosome. In addition,
SNPs genotyping produces a unique DNA fingerprint for each
embryo tested, enabling determination of which embryo was
implanted in any given assisted reproductive technique cycle.
SNP arrays are just beginning to be used clinically in PGD
and await clinical validation studies before they become more
commonplace at IVF clinics. However, encouraging data from
preclinical studies have begun to emerge.115
Karyomapping
Karyomapping is a universal method for the combined detection of chromosome aneuploidy and linkage-based PGD of
single-gene defects that uses genome-wide, high-density SNP
Seminars in Reproductive Medicine
Vol. 30
No. 4/2012
279
280
Molecular Genetic Analysis of Single Cells
Fiorentino
genotyping.116 By genotyping the parents and appropriate
family member(s) of known disease status, the parental
origin of chromosomes and chromosome segments is identified in each embryo by Mendelian analysis of informative SNP
loci. Informative loci are mainly those in which one parent is
homozygous and the other parent heterozygous for the two
SNP alleles. Using the genotype of an affected child, for
example, as a reference, the linkage of the two alleles at
each of these loci to one of the chromosomes in the heterozygous parent can then be established and compared with the
alleles inherited in an embryo. Thus four sets of SNP markers
for the four parental chromosomes are identified and by
linkage can be used to diagnose embryos inheriting the
same parental chromosome haplotypes in the region of the
affected gene. At the same time, the inheritance of two
chromosome haplotypes from one parent, with different
patterns of recombination, trisomy, are identified by overlapping regions in which both haplotypes are present. Conversely, the absence of either parental chromosome
haplotype enables the detection of monosomy or partial
deletions.
Karyomapping is broadly applicable to any single-gene
defect within the region covered by the SNP genotyping
without the need for family- or disease-specific test development. Although diagnosis of SGDs is limited to those cases in
which linkage can be established within the family, even with
de novo mutations, it can still provide linkage information to
confirm the results of mutation detection. Other applications
include combined HLA typing and diagnosis of SGDs and the
diagnosis of translocation or other structural chromosome
imbalances including distinguishing normal and balanced
embryos by identifying the parental haplotypes at the
breakpoints.
Conclusion
3 Handyside AH, Kontogianni EH, Hardy K, Winston RM. Pregnan-
4
5
6
7
8
9
10
11
12
13
14
15
The technology for single-cell genetic analysis has reached an
extremely high level of accuracy and has enabled the possibility of performing multiple diagnoses from one cell. Diagnosis of a monogenic disease can now be combined with HLA
typing, DNA fingerprinting, and array-based CGH for
karyotyping.
New technologies such as microarrays are opening the way
for an increasing number of serious genetic defects to be
detected in preimplantation embryos, and the number of
cases performed and the new indications tested increases
each year.
The new PGD techniques will empower patients and
clinicians to screen for almost any kind of genetic problem
in embryos, with the potential to completely change the way
in which parents approach and manage genetic disease.
References
1 Braude P, Pickering S, Flinter F, Ogilvie CM. Preimplantation
16
17
18
19
20
21
genetic diagnosis. Nat Rev Genet 2002;3(12):941–953
2 Sermon K, Van Steirteghem A, Liebaers I. Preimplantation genetic
diagnosis. Lancet 2004;363(9421):1633–1641
Seminars in Reproductive Medicine
Vol. 30
No. 4/2012
22
cies from biopsied human preimplantation embryos sexed by Yspecific DNA amplification. Nature 1990;344(6268):768–770
Harper JC, Coonen E, De Rycke M, et al. ESHRE PGD Consortium
data collection X: cycles from January to December 2007 with
pregnancy follow-up to October 2008. Hum Reprod 2010;25
(11):2685–2707
Pickering S, Polidoropoulos N, Caller J, Scriven P, Ogilvie CM,
Braude P; Preimplantation Genetic Diagnosis Study Group. Strategies and outcomes of the first 100 cycles of preimplantation
genetic diagnosis at the Guy's and St. Thomas' Center. Fertil Steril
2003;79(1):81–90
Vandervors M, Staessen C, Sermon K, et al. The Brussels' experience of more than 5 years of clinical preimplantation genetic
diagnosis. Hum Reprod Update 2000;6(4):364–373
Fiorentino F, Biricik A, Nuccitelli A, et al. Strategies and clinical
outcome of 250 cycles of preimplantation genetic diagnosis for
single gene disorders. Hum Reprod 2006;21(3):670–684
Munné S, Sandalinas M, Escudero T, Fung J, Gianaroli L, Cohen J.
Outcome of preimplantation genetic diagnosis of translocations.
Fertil Steril 2000;73(6):1209–1218
Munné S, Cohen J, Sable D. Preimplantation genetic diagnosis for
advanced maternal age and other indications. Fertil Steril 2002;
78(2):234–236
Rubio C, Simón C, Vidal F, et al. Chromosomal abnormalities and
embryo development in recurrent miscarriage couples. Hum
Reprod 2003;18(1):182–188
Verlinsky Y, Rechitsky S, Schoolcraft W, Strom C, Kuliev A.
Preimplantation diagnosis for Fanconi anemia combined with
HLA matching. JAMA 2001;285(24):3130–3133
Fiorentino F, Biricik A, Karadayi H, et al. Development and clinical
application of a strategy for preimplantation genetic diagnosis of
single gene disorders combined with HLA matching. Mol Hum
Reprod 2004;10(6):445–460
Fiorentino F, Kahraman S, Karadayi H, et al. Short tandem repeats
haplotyping of the HLA region in preimplantation HLA matching.
Eur J Hum Genet 2005;13(8):953–958
Van de Velde H, De Rycke M, De Man C, et al. The experience of
two European preimplantation genetic diagnosis centres on
human leukocyte antigen typing. Hum Reprod 2009;24(3):
732–740
Fiorentino F, Biricik A, Nuccitelli A, et al. Rapid protocol for preconception genetic diagnosis of single gene mutations by first
polar body analysis: a possible solution for the Italian patients.
Prenat Diagn 2008;28(1):62–64
Verlinsky Y, Rechitsky S, Cieslak J, et al. Preimplantation diagnosis
of single gene disorders by two-step oocyte genetic analysis using
first and second polar body. Biochem Mol Med 1997;62(2):
182–187
Kuliev A, Rechitsky S, Laziuk K, Verlinsky O, Tur-Kaspa I, Verlinsky
Y. Pre-embryonic diagnosis for Sandhoff disease. Reprod Biomed
Online 2006;12(3):328–333
Tomi D, Griesinger G, Schultze-Mosgau A, et al. Polar body
diagnosis for hemophilia a using multiplex PCR for linked polymorphic markers. J Histochem Cytochem 2005;53(3):277–280
Veiga A, Sandalinas M, Benkhalifa M, et al. Laser blastocyst biopsy
for preimplantation diagnosis in the human. Zygote 1997;5
(4):351–354
Harper JC, Coonen E, Ramaekers FC, et al. Identification of the sex
of human preimplantation embryos in two hours using an
improved spreading method and fluorescent in-situ hybridization (FISH) using directly labelled probes. Hum Reprod 1994;9
(4):721–724
Shenfield F, Pennings G, Devroey P, Sureau C, Tarlatzis B, Cohen J;
ESHRE Ethics Task Force . Taskforce 5: preimplantation genetic
diagnosis. Hum Reprod 2003;18(3):649–651
Handyside AH, Lesko JG, Tarín JJ, Winston RM, Hughes MR. Birth
of a normal girl after in vitro fertilization and preimplantation
Molecular Genetic Analysis of Single Cells
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
diagnostic testing for cystic fibrosis. N Engl J Med 1992;327
(13):905–909
Fiorentino F, Magli MC, Podini D, et al. The minisequencing
method: an alternative strategy for preimplantation genetic
diagnosis of single gene disorders. Mol Hum Reprod 2003;9
(7):399–410
Findlay I, Quirke P, Hall J, Rutherford A. Fluorescent PCR: a new
technique for PGD of sex and single-gene defects. J Assist Reprod
Genet 1996;13(2):96–103
Goossens V, Sermon K, Lissens W, et al. Clinical application of
preimplantation genetic diagnosis for cystic fibrosis. Prenat
Diagn 2000;20(7):571–581
De Rycke M, Van de Velde H, Sermon K, et al. Preimplantation
genetic diagnosis for sickle-cell anemia and for beta-thalassemia.
Prenat Diagn 2001;21(3):214–222
Spits C, De Rycke M, Verpoest W, et al. Preimplantation genetic
diagnosis for Marfan syndrome. Fertil Steril 2006;86(2):310–320
el-Hashemite N, Wells D, Delhanty JD. Single cell detection of
beta-thalassaemia mutations using silver stained SSCP analysis:
an application for preimplantation diagnosis. Mol Hum Reprod
1997;3(8):693–698
Harper JC, Wells D, Piyamongkol W, et al. Preimplantation genetic
diagnosis for single gene disorders: experience with five single
gene disorders. Prenat Diagn 2002;22(6):525–533
Vrettou C, Palmer G, Kanavakis E, et al. A widely applicable
strategy for single cell genotyping of β-thalassaemia mutations
using DGGE analysis: application to preimplantation genetic
diagnosis. Prenat Diagn 1999;19(13):1209–1216
Moutou C, Gardes N, Rongières C, et al. Allele-specific amplification for preimplantation genetic diagnosis (PGD) of spinal muscular atrophy. Prenat Diagn 2001;21(6):498–503
Moutou C, Gardes N, Nicod JC, Viville S. Strategies and outcomes
of PGD of familial adenomatous polyposis. Mol Hum Reprod
2007;13(2):95–101
Pierce KE, Rice JE, Sanchez JA, Wangh LJ. Detection of cystic
fibrosis alleles from single cells using molecular beacons and a
novel method of asymmetric real-time PCR. Mol Hum Reprod
2003;9(12):815–820
Vrettou C, Traeger-Synodinos J, Tzetis M, Palmer G, Sofocleous C,
Kanavakis E. Real-time PCR for single-cell genotyping in sickle cell
and thalassemia syndromes as a rapid, accurate, reliable, and
widely applicable protocol for preimplantation genetic diagnosis.
Hum Mutat 2004;23(5):513–521
Findlay I, Ray P, Quirke P, Rutherford A, Lilford R. Allelic drop-out
and preferential amplification in single cells and human blastomeres: implications for preimplantation diagnosis of sex and
cystic fibrosis. Hum Reprod 1995;10(6):1609–1618
Altarescu G, Brooks B, Kaplan Y, et al. Single-sperm analysis for
haplotype construction of de-novo paternal mutations: application to PGD for neurofibromatosis type 1. Hum Reprod 2006;21
(8):2047–2051
Verlinsky Y, Rechitsky S, Verlinsky O, et al. Polar body-based
preimplantation diagnosis for X-linked disorders. Reprod Biomed
Online 2002;4(1):38–42
Spits C, Le Caignec C, De Rycke M, et al. Optimization and
evaluation of single-cell whole-genome multiple displacement
amplification. Hum Mutat 2006;27(5):496–503
Rechitsky S, Strom C, Verlinsky O, et al. Allele dropout in polar
bodies and blastomeres. J Assist Reprod Genet 1998;15(5):
253–257
Dreesen JC, Jacobs LJ, Bras M, et al. Multiplex PCR of polymorphic
markers flanking the CFTR gene; a general approach for preimplantation genetic diagnosis of cystic fibrosis. Mol Hum Reprod
2000;6(5):391–396
Kuliev A, Rechitsky S, Verlinsky O, et al. Preimplantation diagnosis
and HLA typing for haemoglobin disorders. Reprod Biomed
Online 2005;11(3):362–370
Fiorentino
42 Sermon K, De Rijcke M, Lissens W, et al. Preimplantation genetic
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
diagnosis for Huntington's disease with exclusion testing. Eur J
Hum Genet 2002;10(10):591–598
Moutou C, Gardes N, Viville S. New tools for preimplantation
genetic diagnosis of Huntington's disease and their clinical
applications. Eur J Hum Genet 2004;12(12):1007–1014
Stern HJ, Harton GL, Sisson ME, et al. Non-disclosing preimplantation genetic diagnosis for Huntington disease. Prenat Diagn
2002;22(6):503–507
Braude PR, De Wert GMWR, Evers-Kiebooms G, Pettigrew RA,
Geraedts JP. Non-disclosure preimplantation genetic diagnosis
for Huntington's disease: practical and ethical dilemmas. Prenat
Diagn 1998;18(13):1422–1426
Handyside AH, Robinson MD, Simpson RJ, et al. Isothermal whole
genome amplification from single and small numbers of cells: a
new era for preimplantation genetic diagnosis of inherited
disease. Mol Hum Reprod 2004;10(10):767–772
Spits C, Le Caignec C, De Rycke M, et al. Optimization and
evaluation of single-cell whole-genome multiple displacement
amplification. Hum Mutat 2006;27(5):496–503
Hellani A, Coskun S, Benkhalifa M, et al. Multiple displacement
amplification on single cell and possible PGD applications. Mol
Hum Reprod 2004;10(11):847–852
Hellani A, Coskun S, Tbakhi A, Al-Hassan S. Clinical application of
multiple displacement amplification in preimplantation genetic
diagnosis. Reprod Biomed Online 2005;10(3):376–380
Burlet P, Frydman N, Gigarel N, et al. Multiple displacement
amplification improves PGD for fragile X syndrome. Mol Hum
Reprod 2006;12(10):647–652
Harton GL, De Rycke M, Fiorentino F, et al. ESHRE PGD consortium
best practice guidelines for amplification-based PGD. Hum Reprod 2011;26(1):33–40
Delhanty JD, Griffin DK, Handyside AH, et al. Detection of
aneuploidy and chromosomal mosaicism in human embryos
during preimplantation sex determination by fluorescent in
situ hybridisation (FISH). Hum Mol Genet 1993;2(8):1183–1185
Munné S, Lee A, Rosenwaks Z, Grifo J, Cohen J. Diagnosis of major
chromosome aneuploidies in human preimplantation embryos.
Hum Reprod 1993;8(12):2185–2191
Scriven PN, Handyside AH, Ogilvie CM. Chromosome translocations: segregation modes and strategies for preimplantation
genetic diagnosis. Prenat Diagn 1998;18(13):1437–1449
Scriven PN, Flinter FA, Braude PR, Ogilvie CM. Robertsonian
translocations—reproductive risks and indications for preimplantation genetic diagnosis. Hum Reprod 2001;16(11):2267–
2273
Munné S, Sandalinas M, Escudero T, Fung J, Gianaroli L, Cohen J.
Outcome of preimplantation genetic diagnosis of translocations.
Fertil Steril 2000;73(6):1209–1218
Munné S. Preimplantation genetic diagnosis of numerical and
structural chromosome abnormalities. Reprod Biomed Online
2002;4(2):183–196
Velilla E, Escudero T, Munné S. Blastomere fixation techniques
and risk of misdiagnosis for preimplantation genetic diagnosis of
aneuploidy. Reprod Biomed Online 2002;4(3):210–217
Cohen J, Wells D, Munné S. Removal of 2 cells from cleavage stage
embryos is likely to reduce the efficacy of chromosomal tests that
are used to enhance implantation rates. Fertil Steril 2007;87
(3):496–503
Colls P, Escudero T, Cekleniak N, Sadowy S, Cohen J, Munné S.
Increased efficiency of preimplantation genetic diagnosis for
infertility using “no result rescue”. Fertil Steril 2007;88(1):
53–61
Goossens V, De Rycke M, De Vos A, et al. Diagnostic efficiency,
embryonic development and clinical outcome after the biopsy of
one or two blastomeres for preimplantation genetic diagnosis.
Hum Reprod 2008;23(3):481–492
Seminars in Reproductive Medicine
Vol. 30
No. 4/2012
281
282
Molecular Genetic Analysis of Single Cells
Fiorentino
62 Wilton L, Thornhill A, Traeger-Synodinos J, Sermon KD, Harper JC.
80 Gianaroli L, Magli MC, Munné S, Fiorentino A, Montanaro N,
The causes of misdiagnosis and adverse outcomes in PGD. Hum
Reprod 2009;24(5):1221–1228
Li M, DeUgarte CM, Surrey M, Danzer H, DeCherney A, Hill DL.
Fluorescence in situ hybridization reanalysis of day-6 human
blastocysts diagnosed with aneuploidy on day 3. Fertil Steril
2005;84(5):1395–1400
Wilton L. Preimplantation genetic diagnosis for aneuploidy
screening in early human embryos: a review. Prenat Diagn
2002;22(6):512–518
Simpson JL, Bombard AT. Chromosomal abnormalities in spontaneous abortion: Frequency, pathology and genetic counselling.
In: Edmonds K, Bennett MJ, eds. Spontaneous Abortion. London,
UK: Blackwell; 1987:51
Munné S, Magli C, Bahçe M, et al. Preimplantation diagnosis of the
aneuploidies most commonly found in spontaneous abortions
and live births: XY, 13, 14, 15, 16, 18, 21, 22. Prenat Diagn 1998;18
(13):1459–1466
Munné S, Chen S, Fischer J, et al. Preimplantation genetic diagnosis reduces pregnancy loss in women aged 35 years and older
with a history of recurrent miscarriages. Fertil Steril 2005;84
(2):331–335
Colls P, Goodall N, Zheng X, Munné S. Increased efficiency
of preimplantation genetic diagnosis for aneuploidy by testing 12 chromosomes. Reprod Biomed Online 2009;19(4):
532–538
Jansen RP, Bowman MC, de Boer KA, Leigh DA, Lieberman DB,
McArthur SJ. What next for preimplantation genetic screening
(PGS)? Experience with blastocyst biopsy and testing for aneuploidy. Hum Reprod 2008;23(7):1476–1478
Kuliev A, Cieslak J, Ilkevitch Y, et al. Chromosomal abnormalities
in a series of 6733 human oocytes in preimplantation diagnosis
for age-related aneuploidies. Reprod Biomed Online 2003;6:
54–59
Fragouli E, Jaffe-Katz M, Alfarawati M, et al. Comprehensive
chromosome screening of polar bodies and blastocysts from
couples experiencing repeated implantation failure. Fertil Steril
2010;94(3):875–887
Treff NR, Su J, Tao X, Levy B, Scott RT Jr. Accurate single cell 24
chromosome aneuploidy screening using whole genome amplification and single nucleotide polymorphism microarrays. Fertil
Steril 2010;94(6):2017–2021
Treff NR, Northrop LE, Kasabwala K, Su J, Levy B, Scott RT Jr. Single
nucleotide polymorphism microarray-based concurrent screening of 24-chromosome aneuploidy and unbalanced translocations in preimplantation human embryos. Fertil Steril 2011;95
(5):1606–1612, e1–e2
Vanneste E, Voet T, Le Caignec C, et al. Chromosome instability is
common in human cleavage-stage embryos. Nat Med 2009;15
(5):577–583
Gutiérrez-Mateo C, Colls P, Sánchez-García J, et al. Validation of
microarray comparative genomic hybridization for comprehensive chromosome analysis of embryos. Fertil Steril 2011;95
(3):953–958
Baart EB, Van Opstal D, Los FJ, Fauser BC, Martini E. Fluorescence
in situ hybridization analysis of two blastomeres from day 3
frozen-thawed embryos followed by analysis of the remaining
embryo on day 5. Hum Reprod 2004;19(3):685–693
Coonen E, Derhaag JG, Dumoulin JC, et al. Anaphase lagging
mainly explains chromosomal mosaicism in human preimplantation embryos. Hum Reprod 2004;19(2):316–324
Li M, DeUgarte CM, Surrey M, Danzer H, DeCherney A, Hill DL.
Fluorescence in situ hybridization reanalysis of day-6 human
blastocysts diagnosed with aneuploidy on day 3. Fertil Steril
2005;84(5):1395–1400
Munné S, Velilla E, Colls P, et al. Self-correction of chromosomally
abnormal embryos in culture and implications for stem cell
production. Fertil Steril 2005;84(5):1328–1334
Ferraretti AP. Will preimplantation genetic diagnosis assist patients with a poor prognosis to achieve pregnancy? Hum Reprod
1997;12(8):1762–1767
Munné S, Dailey T, Sultan KM, et al. The use of first polar bodies
for preimpantation diagnosis of aneuploidy. Hum Reprod
1995;10(4):1014–1020
Munné S, Sultan KM, Weier HU, Grifo JA, Cohen J, Rosenwaks Z.
Assessment of numeric abnormalities of X, Y, 18, and 16 chromosomes in preimplantation human embryos before transfer.
Am J Obstet Gynecol 1995;172(4 Pt 1):1191–1199; discussion
1199–1201
Verlinsky Y, Cieslak J, Freidine M, et al. Pregnancies following preconception diagnosis of common aneuploidies by fluorescent insitu hybridization. Hum Reprod 1995;10(7):1923–1927
Gianaroli L, Magli MC, Ferraretti AP, Munné S. Preimplantation
diagnosis for aneuploidies in patients undergoing in vitro fertilization with a poor prognosis: identification of the categories for
which it should be proposed. Fertil Steril 1999;72(5):837–844
Munné S, Magli C, Cohen J, et al. Positive outcome after preimplantation diagnosis of aneuploidy in human embryos. Hum
Reprod 1999;14(9):2191–2199
Munné S, Fischer J, Warner A, Chen S, Zouves C, Cohen J; ;
Referring Centers PGD Group. Preimplantation genetic diagnosis
significantly reduces pregnancy loss in infertile couples: a multicenter study. Fertil Steril 2006;85(2):326–332
Colls P, Escudero T, Cekleniak N, Sadowy S, Cohen J, Munné S.
Increased efficiency of preimplantation genetic diagnosis for
infertility using “no result rescue.”. Fertil Steril 2007;88(1):53–61
Staessen C, Platteau P, Van Assche E, et al. Comparison of blastocyst transfer with or without preimplantation genetic diagnosis
for aneuploidy screening in couples with advanced maternal age:
a prospective randomized controlled trial. Hum Reprod 2004;19
(12):2849–2858
Staessen C, Verpoest W, Donoso P, et al. Preimplantation genetic
screening does not improve delivery rate in women under the age
of 36 following single-embryo transfer. Hum Reprod 2008;23
(12):2818–2825
Stevens J, Wale P, Surrey ES, et al. Is aneuploidy screening for
patients aged 35 or over beneficial? A prospective randomized
trial. Fertil Steril 2004;82:S249
Mastenbroek S, Twisk M, van Echten-Arends J, et al. In vitro
fertilization with preimplantation genetic screening. N Engl J
Med 2007;357(1):9–17
Blockeel C, Schutyser V, De Vos A, et al. Prospectively randomized
controlled trial of PGS in IVF/ICSI patients with poor implantation. Reprod Biomed Online 2008;17(6):848–854
Hardarson T, Hanson C, Lundin K, et al. Preimplantation genetic
screening in women of advanced maternal age caused a decrease
in clinical pregnancy rate: a randomized controlled trial. Hum
Reprod 2008;23(12):2806–2812
Mersereau JE, Pergament E, Zhang X, Milad MP. Preimplantation
genetic screening to improve in vitro fertilization pregnancy
rates: a prospective randomized controlled trial. Fertil Steril
2008;90(4):1287–1289
Debrock S, Melotte C, Spiessens C, et al. Preimplantation genetic
screening for aneuploidy of embryos after in vitro fertilization in
women aged at least 35 years: a prospective randomized trial.
Fertil Steril 2010;93(2):364–373
Meyer LR, Klipstein S, Hazlett WD, Nasta T, Mangan P, Karande VC.
A prospective randomized controlled trial of preimplantation
genetic screening in the “good prognosis” patient. Fertil Steril
2009;91(5):1731–1738
Schoolcraft WB, Katz-Jaffe MG, Stevens J, Rawlins M, Munne S.
Preimplantation aneuploidy testing for infertile patients of advanced maternal age: a randomized prospective trial. Fertil Steril
2009;92(1):157–162
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
Seminars in Reproductive Medicine
Vol. 30
No. 4/2012
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
Molecular Genetic Analysis of Single Cells
Fiorentino
98 Vanneste E, Voet T, Melotte C, et al. What next for preimplantation
107 Wells D, Alfarawati S, Fragouli E. Use of comprehensive chromo-
genetic screening? High mitotic chromosome instability rate
provides the biological basis for the low success rate. Hum Reprod
2009;24(11):2679–2682
Harper JC, Coonen E, Handyside AH, Winston RM, Hopman AH,
Delhanty JD. Mosaicism of autosomes and sex chromosomes in
morphologically normal, monospermic preimplantation human
embryos. Prenat Diagn 1995;15(1):41–49
Munne S, Wells D, Cohen J. Technology requirements for preimplantation genetic diagnosis to improve art outcome. Fertil Steril
2010;94:408–430
Harper J, Coonen E, De Rycke M, et al. What next for preimplantation genetic screening (PGS)? A position statement from the
ESHRE PGD Consortium Steering Committee. Hum Reprod
2010;25(4):821–823
Harper J, Sermon K, Geraedts J, et al. What next for preimplantation genetic screening? Hum Reprod 2008;23(3):478–480
Fiorentino F, Kokkali G, Biricik A, et al. Polymerase chain reaction-based detection of chromosomal imbalances on embryos:
the evolution of preimplantation genetic diagnosis for chromosomal translocations. Fertil Steril 2010;94(6):2001–2011, 2011,
e1–e6
Kuwano A, Mutirangura A, Dittrich B, et al. Molecular dissection
of the Prader-Willi/Angelman syndrome region (15q11-13) by
YAC cloning and FISH analysis. Hum Mol Genet 1992;1(6):
417–425
Tomkins DJ, Roux AF, Waye J, Freeman VC, Cox DW, Whelan DT.
Maternal uniparental isodisomy of human chromosome 14 associated with a paternal t(13q14q) and precocious puberty. Eur J
Hum Genet 1996;4(3):153–159
Le Caignec C, Spits C, Sermon K, et al. Single-cell chromosomal
imbalances detection by array CGH. Nucleic Acids Res 2006;34
(9):e68
somal screening for embryo assessment: microarrays and CGH.
Mol Hum Reprod 2008;14(12):703–710
Fiorentino F, Spizzichino L, Bono S, et al. PGD for reciprocal and
Robertsonian translocations using array comparative genomic
hybridization. Hum Reprod 2011;26(7):1925–1935
Hu DG, Webb G, Hussey N. Aneuploidy detection in single cells
using DNA array-based comparative genomic hybridization. Mol
Hum Reprod 2004;10(4):283–289
Fiegler H, Geigl JB, Langer S, et al. High resolution array-CGH
analysis of single cells. Nucleic Acids Res 2007;35(3):e15
Gianaroli L, Magli MC, Ferraretti AP, et al. Possible interchromosomal effect in embryos generated by gametes from translocation
carriers. Hum Reprod 2002;17(12):3201–3207
Harrison RH, Kuo HC, Scriven PN, Handyside AH, Ogilvie CM. Lack
of cell cycle checkpoints in human cleavage stage embryos
revealed by a clonal pattern of chromosomal mosaicism analysed
by sequential multicolour FISH. Zygote 2000;8(3):217–224
Johnson DS, Gemelos G, Baner J, et al. Preclinical validation of a
microarray method for full molecular karyotyping of blastomeres
in a 24-h protocol. Hum Reprod 2010;25(4):1066–1075
Treff NR, Su J, Mavrianos J, et al. Accurate 23 chromosome
aneuploidy screening in human blastomeres using single nucleotide polymorphism (SNP) microarrays. Fertil Steril 2007;88:S1
Brezina PR, Benner A, Rechitsky S, et al. Single-gene testing
combined with single nucleotide polymorphism microarray preimplantation genetic diagnosis for aneuploidy: a novel approach
in optimizing pregnancy outcome. Fertil Steril 2011;95(5):1786,
e5–e8
Handyside AH, Harton GL, Mariani B, et al. Karyomapping: a
universal method for genome wide analysis of genetic disease
based on mapping crossovers between parental haplotypes. J
Med Genet 2010;47(10):651–658
99
100
101
102
103
104
105
106
108
109
110
111
112
113
114
115
116
Seminars in Reproductive Medicine
Vol. 30
No. 4/2012
283