Chromosomal Preimplantation Genetic Diagnosis: 25 Years and Counting

Abstract

Preimplantation genetic diagnosis (PGD), first successfully carried out in humans in the early 1990s, initially involved the PCR sexing of embryos by Y- (and later also X-) chromosome specific detection. Because of the problems relating to misdiagnosis and contamination of this technology however the PCR based test was superseded by a FISH-based approach involving X and Y specific probes. Sexing by FISH heralded translocation screening, which was shortly followed by preimplantation genetic screening (PGS) for Aneuploidy. Aneuploidy is widely accepted to be the leading cause of implantation failure in assisted reproductive technology (ART) and a major contributor to miscarriage, especially in women of advanced maternal age. PGS (AKA PGD for aneuploidy PGD-A) has had a chequered history, with conflicting lines of evidence for and against its use. The current practice of trophectoderm biopsy followed by array CGH or next generation sequencing is gaining in popularity however as evidence for its efficacy grows. PGS has the potential to identify viable embryos that can be transferred thereby reducing the chances of traumatic failed IVF cycles, miscarriage or congenital abnormalities and facilitating the quickest time to live birth of chromosomally normal offspring. In parallel to chromosomal diagnoses, technology for PGD has allowed for improvements in accuracy and efficiency of the genetic screening of embryos for monogenic disorders. The number of genetic conditions available for screening has increased since the early days of PGD, with the human fertilization and embryology authority currently licensing 419 conditions in the UK [1]. A novel technique known as karyomapping that involves SNP chip screening and tracing inherited chromosomal haploblocks is now licensed for the PGD detection of monogenic disorders. Its potential for the universal detection of chromosomal and monogenic disorders simultaneously however, has yet to be realized.

Full text

REVIEW ARTICLE
Chromosomal Preimplantation Genetic Diagnosis: 25 Years
and Counting
Kathryn D. Sanders
1,2
•Darren K. Griffin
1
Received: 7 March 2017 / Accepted: 21 March 2017 / Published online: 22 April 2017
ÓThe Author(s) 2017. This article is an open access publication
Abstract Preimplantation genetic diagnosis (PGD), first
successfully carried out in humans in the early 1990s, initially
involved the PCR sexing of embryos by Y- (and later also X-)
chromosome specific detection. Because of the problems
relating to misdiagnosis and contamination of this technology
however the PCR based test was superseded by a FISH-based
approach involving X and Y specific probes. Sexing by FISH
heralded translocation screening, which was shortly followed
by preimplantation genetic screening (PGS) for Aneuploidy.
Aneuploidy is widely accepted to be the leading cause of
implantation failure in assisted reproductive technol-
ogy (ART) and a major contributor to miscarriage, especially
in women of advanced maternal age. PGS (AKA PGD for
aneuploidy PGD-A) has had a chequered history, with con-
flicting lines of evidence for and against its use. The current
practice of trophectoderm biopsy followed by array CGH or
next generation sequencing is gaining in popularity however
as evidence for its efficacy grows. PGS has the potential to
identify viable embryos that can be transferred thereby
reducing the chances of traumatic failed IVF cycles, miscar-
riage or congenital abnormalities and facilitating the quickest
time to live birth of chromosomally normal offspring. In
parallel to chromosomal diagnoses, technology for PGD has
allowed for improvements in accuracy and efficiency of the
genetic screening of embryos for monogenic disorders. The
number of genetic conditions available for screening has
increased since the early days of PGD, with the human
fertilization and embryology authority currently licensing 419
conditions in the UK [1]. A novel technique known as kary-
omapping that involves SNP chip screening and tracing
inherited chromosomal haploblocks is now licensed for the
PGD detection of monogenic disorders. Its potential for the
universal detection of chromosomal and monogenic disorders
simultaneously however, has yet to be realized.
Keywords Preimplantation genetic screening 
Preimplantation genetic diagnosis Aneuploidy 
Fluorescent in situ hybridization Karyomapping Next
generation sequencing
Introduction
Preimplantation Genetic Diagnosis PGD is essentially a
medical intervention designed to minimize the chances of
transfer of genetically abnormal embryos in an IVF setting.
Its primary utility is to help families at risk of transmitting
genetic disorders conceive a normal child and/or to
improve IVF success rates by the selective implantation of
chromosomally normal embryos. Typically, the process
involves referral and genetic counselling for the nature of
the specific problem, standard IVF, embryo biopsy, genetic
diagnosis of the biopsied cells then selective transfer of
embryo(s) thought to be genetically normal.
The First PGD Cases
The first recorded PGD case in model species was a
chromosomal one, performed to control sex ratio in rab-
bits [2]. Gardner and Edwards successfully assessed tro-
phoblast fragments for inactive sex chromatin (Barr body)
&Darren K. Griffin
d.k.griffin@kent.ac.uk
1
School of Biosciences, University of Kent,
Canterbury CT2 7NJ, UK
2
Genesis Genetics Ltd, London Biosciences Innovation
Centre, Royal College Street, London NW1 0NH, UK
123
J. Fetal Med. (June 2017) 4:51–56
DOI 10.1007/s40556-017-0123-5
Article published online: 2023-05-08

in females and thereby accurately determined the sex of
blastocysts. Application of this technology to humans
clearly had the potential to screen for X-linked recessive
diseases before implantation of an embryo, avoiding
invasive prenatal assessments and the possibility of a
difficult decision deciding whether to terminate. Follow-
ing the development of in vitro fertilization (IVF) in 1978
[3], clinical progress in this area became possible and
thus, in 1990, the first human embryos underwent blas-
tomere biopsy and the sex was established by PCR
amplifying a Y-specific repeat sequence. The unaffected
female embryos having two copies of the X chromosome
and thus no amplified signal lacking the Y were trans-
ferred, resulting in successful pregnancy and healthy live
birth free from the X-linked condition [4]. This led the
way to PGD in other monogenic conditions such as cystic
fibrosis, which was successfully achieved in 1992 [5].
Early PGD used polymerase chain reaction (PCR) to
amplify short fragments of the known affected region of
DNA using nested primers; providing confirmation if the
cell and thus embryo possessed the sequence which coded
for the condition in question. Thereafter, for most of the
history of PGD diagnoses were either monogenic, usually
involving increasingly sophisticated variants of PCR, or
chromosomal, initially involving fluorescent in situ
hybridization (FISH), but later involving whole karyotype
screening approaches. The purpose of this review is to
concentrate on the chromosomal side of the diagnoses.
FISH was first introduced clinically in 1992 to sex
embryos using probes specific to X and Y chromosomes
[6,7]. It is thus 25 years since we performed the first
chromosomal PGD cases. Later in 1993, the first aneu-
ploidy screening cases using FISH were carried out,
assessing chromosome copy numbers of the most common
trisomies associated with live birth defects X, Y, 13, 18 and
21 [8,9]. The number of chromosomes that could be
screened simultaneously was limited by the colours of the
probes: red, yellow, green, aqua and blue. PGS most
commonly was used for patients undergoing IVF with the
following indications: advanced maternal age (AMA),
recurrent miscarriage, recurrent implantation failure and
those with severe male factor infertility. By screening
embryos to identify and transfer chromosomally normal
embryos, IVF success rates and pregnancy outcomes
should be improved.
The Trouble with PGS
Initial, retrospective, studies of PGS indicated that there
was an increase in implantation rates and decrease in
pregnancy loss following PGS with FISH [9,10]. Several,
randomized controlled trials (RCTs) challenged this
however, showing either no significant improvement or a
detrimental effect on successful outcomes of IVF [11,12].
There are differing opinions regarding why these studies
had varying outcomes. Firstly, there is concern that the
process of embryo biopsy at the cleavage stage could have
an adverse effect on the embryo, at this stage in embryo
development there are normally 8 cells, removing one of
these could reduce the success of the future development of
that embryo. Related to this, the other remaining cells (and
hence the developmental potential of the embryo) could be
damaged in the biopsy process and this could be operator-
dependent. Therefore, the studies that saw a decrease in
implantation rate when compared to standard IVF without
PGS, have been criticized for inadvertently causing dam-
age during the biopsy process in those embryos which were
later transferred and failed to implant. Secondly, it is
known that embryos can present some degree of chromo-
somal variation between cells or mosaicism. For some
cases of PGS it is possible that the cell that is screened will
present as chromosomally normal, where in fact the other
cells are abnormal, creating a false negative result. Thirdly,
another possible contributor is that with most PGS studies
using FISH, not all chromosomes are analysed. The chro-
mosomes that are screened may have appeared euploid, but
those chromosomes that have not been screened could be
aneuploid, resulting in the transfer of an abnormal embryo.
The practice of PGS in the clinical setting ultimately
declined following the publication of these RCTs. FISH in
most clinical IVF cases is now not the method of choice, in
part due to lack of confidence in the technique but also
from the emergence of advanced technology which was
subsequently applied to PGS.
Trophectoderm Biopsy and Improved Methods
for PGS and PGD
Improved culture conditions leading to a greater number of
embryos reaching the blastocyst stage in regular IVF pre-
sented an opportunity for an improved approach to PGS
protocols. Trophectoderm biopsy on day 5 of embryo
development was now an attractive option over the con-
ventional blastomere biopsy on day 3. The advantage of
trophectoderm biopsy is clear, by day 5 of embryo devel-
opment there are many cells that make up both the inner
cell mass (ICM) and the trophectoderm. Removing a few
cells from the trophectoderm while leaving the ICM
undisturbed in theory has the potential for less adverse
effects on the developing embryo and the advantage of
providing more cells for analysis, than blastomere biopsy.
A study by Kokkali et al. [13] demonstrated an improved
implantation rate with blastocyst biopsy over cleavage
biopsy and subsequent studies support these findings [14].
52 J. Fetal Med. (June 2017) 4:51–56
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More recently, it has been shown that trophectoderm
biopsy can be more consistent and reproducible across
different practitioners and clinics compared to blastomere
biopsy [15].
Perhaps the major technical advance in our ability to
screen biopsied cells for chromosome abnormalities was the
development of whole genome amplification (WGA)
[16,17]. This approach increases the amount of available
DNA where only small amounts are initially available from
single cells. WGA enables multiple tests to be carried out,
for example, PGS and PGD simultaneously, while benefiting
from an increase in accuracy and sensitivity. Another benefit
is that WGA products can be stored for later subsequent
analysis in the instance of test failure or to confirm findings.
WGA enabled array comparative genomic hybridization
(aCGH) for the analysis of all chromosome copy number.
aCGH essentially compares the amplified DNA labelled in
one fluorescent colour with known, normal DNA labelled in
another colour simultaneously hybridized to a genome-wide
microarray. Chromosome-by-chromosome ratio analysis
gives an indication of cytogenetic gain or loss e.g., trisomy
or monosomy. Randomized clinical trials suggest benefits
for screening by aCGH in terms of the usual outcomes used
to measure IVF success [18].
Another application of WGA was that multiplex PCR
was successfully adapted for PGD, this allowed for the
simultaneous analysis of linked markers to screen for
monogenic conditions as well as aneuploidy for selected
chromosomes. This permitted screening of multiple con-
ditions, with greater accuracy as allele dropout (ADO—
where a heterozygous individual was erroneously called as
homozygous due to allele specific amplification) presented
less of an issue with this technique than that seen with
earlier applications of PGD due to the fact that multiple
loci could be screened. Human leukocyte antigen (HLA)
typing, also known as saviour siblings, could also be
combined with aneuploidy and monogenic PGD and was
first successfully performed using PGD in 2000 [19]. This
process establishes a pregnancy and live birth that is a
HLA-match to an existing sibling, by selecting an embryo
which is a HLA-match for transfer who can then be a stem
cell transplant donor for their older brother or sister. As
most couples requiring this form of PGD are of AMA there
is therefore a potential benefit to be able to combine this
with PGS [20].
Is There Still a Problem with PGS?
Despite improvements in technology there is still an
ongoing debate regarding the effectiveness of PGS for
improved implantation and ongoing pregnancy rates. There
have been several studies that have shown an improvement
when PGS is used. A systematic review by Lee et al. [21]
where they combined the findings of 19 articles, which
were comprised of 3 RCTs and 16 observational studies,
showed that PGS overall had improved success rates when
compared to morphology based embryo selection. While,
RCTs are considered the best design for research, the
nature of ART in the clinical setting makes it difficult to
create studies that meet all the criteria of a RCT, for
example patients will always want to be in the group with
the best outcome, they may wish to switch groups during
the study to get what they perceive to be the best outcome,
this can skew results, but would be unethical not to let
patients have a choice. There are also many more
unknowns associated with this area of medicine, such as
the complex interaction of the physiologies of two people
(in order to produce a third). The comparison of different
retrospective studies carried out in different clinics with
varying approaches to ART and differing levels of biopsy
practitioner skill can still play a big part in the variation of
results presented. Ideally, all clinics would be uniform in
their techniques to draw conclusive comparisons however
this is not always practicable. It should also be kept in
mind, in those cases where PGS does improve outcome,
whether the cost implications associated with PGS match
the increase in success rates. PGS techniques remain rel-
atively high cost when added to a conventional IVF cycle.
The effect on the patient however, is difficult to quantify.
Couples undergoing IVF cycles with PGS may potentially
avoid the transfer of an embryo which has a high chance of
miscarriage, meaning that they will be able to progress to
the next possibly successful cycle much quicker than if an
aneuploid embryo is transferred, implanted and miscarried.
Karyomapping and Next Generation Sequencing
(NGS)
Karyomapping, first developed in 2010 [22], is a method
that uses the principles of linkage analysis and the inheri-
tance of chromosomal haploblocks, in which the mother,
father and a reference affected family member or grand-
parents are compared to map the origin of each chromo-
some inherited (and any crossovers between grandparental
chromosomes). Karyomaps allow the tracking of affected
genes that reside on these haploblocks, which can then
subsequently be used for PGD to identify unaffected
embryos before transfer. When applied to screening
embryos this can also give valuable additional information
to detect monosomy, uniparental disomy and meiotic tri-
somies. The karyomaps that are produced offer easy visu-
alization of the chromosomes and present clearly where
there is crossover of genetic material. Karyomapping has
the advantage of allowing for diagnosis of genetic
J. Fetal Med. (June 2017) 4:51–56 53
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conditions, while simultaneously screening for chromoso-
mal imbalances however its potential for the use in aneu-
ploidy screening has yet to be realized [23]. This method
has been made possible through whole genome sequencing,
it is more commonly used with single nucleotide poly-
morphisms (SNPs) chip technology but can also be used
alongside next generation sequencing (NGS). To date
however, NGS has primarily been used for aneuploidy
screening.
NGS is a high resolution whole genome sequencing
technology that allows for the processing of samples at
high throughput with a high level of accuracy. Recent
studies show NGS to have 100% specificity and sensitivity
making it superior to aCGH for PGS [24]. NGS provides
the ability to run samples simultaneously which gives the
potential to make this technology lower cost and quicker
than that seen with aCGH. It has also has the potential to
identify small copy number variations (CNVs), which can
affect embryo development and result in severe birth
defects [25].
The Impact of Cryopreservation and ‘‘Freeze All’’
Strategies on PGS
ART has seen improvement in embryo cryopreservation
techniques. In PGS, there has been a shift towards the
cryopreservation of embryos and transfer later, when the
status of embryos has been confirmed. Increased preg-
nancy success rates when screening for aneuploidy, have
been attributed to the advancement of PGS technologies,
such as aCGH and NGS. However, studies have shown
there to be an improvement associated with frozen
embryo transfer (FET) compared to fresh embryo
transfer. This improvement is thought to be due to
ovarian stimulation having a detrimental effect on the
endometrium, which lowers implantation rate. This
stimulation is not encountered during a frozen embryo
transfer and could therefore lead to higher implantation
rates [26]. Further research is required to determine if
PGS is offering increased pregnancy success rates in
addition to those seen with FET.
What We Have Learnt from Research into PGS?
A greater understanding of meiosis, crossing over and
molecular biology has led to improvements in PGD and
PGS. Similarly however by studying the chromosomal
aspects of PGS we can understand the basic biology of
early human development better. One example is the
incidence of mosaicism, previously it was believed that
mosaicism was very rare and that in the case of trisomies
it would be throughout all the cells in an embryo. Indeed
some studies have suggested that most human embryos
are aneuploid and mosaic. In a recent study by Maxwell
et al. [27], WGA products that had initially been
assessed using aCGH were retested using NGS. Embryos
thathadoriginallybeendeterminedtobeeuploidby
aCGH were found to be mosaic by NGS, some of these
mosaic embryos that had been transferred resulted in
miscarriage, this provided an explanation as to why these
pregnancies subsequently failed. However, other
embryos that had been found to be mosaic through
retesting using NGS were found to have resulted in a
healthy live birth. Two per cent of all normal pregnan-
cies are post zygotically mosaic; therefore, caution is
required when considering mosaic embryos at the time
of transfer. Further research is required to ascertain the
prevalence of mosaic cells in embryos and the implica-
tions on pregnancy outcome.
We have learnt that aneuploidy in embryos is com-
monplace but we are learning more about the effect of
abnormalities on embryo development. It has for
instance been found that aneuploidy rates are lower at
day5thanday3[14], raising the possibility that some
aneuploidies are corrected or selected against between
day 3 and day 5. It has also been suggested that meiotic
abnormality, for example in patients who are Robertso-
nian translocation carriers; can affect the segregation of
other structurally normal chromosomes, this may results
in an interchromosomal effect on the subsequent mitotic
divisions and thus a higher abnormality rate in these
patients than in other unaffected patients [28]. It is
beyond the scope of this review to cover all the bio-
logical implications of PGS findings, however the fact
remains that it is a unique, fundamental system to study
and much further research is still needed to address basic
biological questions. For instance, we are still not
entirely sure of the precise incidence, cell by cell, of
aneuploidy in blastocyst embryos and, indeed, if a small
amount of abnormality is commonplace in most
embryos.
The Future and Conclusions
PGD and PGS have both come a long way since their first
use in the early 1990s. We now believe that most embryos
are aneuploid and that by transferring embryos that are
aneuploid it is likely to result in failure to implant, mis-
carriage or the birth of an affected child. Pregnancy rate
increases are consistently being reported more often when
PGS is used however we still do not know if or when it is
safe to transfer embryos with some level of postzygotic
aneuploidy. Aneuploidy screening is of course only one of
54 J. Fetal Med. (June 2017) 4:51–56
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several selection strategies for assessing and determining
embryos for transfer. All will require further review,
ensuring the highest possible chances of IVF. It is unde-
niable that further research in the future is required to
optimize PGS for clinical use. For instance, we need to
understand mosaicism better; where a nonmosaic euploid
embryo is available this will always be the first choice for
transfer, but where mosaic embryos are all that is available
we need to better understand under what circumstances
these are safe to transfer. We need to understand the origin
of trisomies; embryos with trisomies that are meiotic in
origin should not be transferred, however we need to be
better informed when detecting if a trisomy is postzygotic
and the clinical outcome this will lead to, if transferred.
A final question therefore is: if PGS can be demonstrated to
increase IVF significantly, should it be offered to all IVF
patients? While aneuploidy is more prevalent among patients
of AMA, there are still many embryos that are aneuploid in
younger patients. Moreover, as previously mentioned, CNVs
can affect any age group of patients, PGS optimized for CNV
screening can be used to benefit all patients of any age. Such a
suggestion is likely to be contentious, particularly among the
opponents of PGS. In any event, the debate for and against
PGS is likely to rage for some time yet. A consideration rarely
aired however is the issue of whether it is unethical not to offer
PGS, given its potential benefits.
Open Access This article is distributed under the terms of the
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tivecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
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