Journal of Postgraduate Medicine October 2010 Vol 56 Issue 4 317

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Review Article
Preimplantation diagnosis of genetic diseases
Adiga SK, Kalthur G, Kumar P, Girisha KM
1
ABSTRACT
One of the landmarks in clinical genetics is prenatal diagnosis of genetic disorders. The recent advances in
the field have made it possible to diagnose the genetic conditions in the embryos before implantation in a
setting of in vitro fertilization. Polymerase chain reaction and fluorescence in situ hybridization are the two
common techniques employed on a single or two cells obtained via embryo biopsy. The couple who seek
in vitro fertilization may screen their embryos for aneuploidy and the couple at risk for a monogenic disorder
but averse to abortion of the affected fetuses after prenatal diagnosis, are likely to be the best candidates
to undergo this procedure. This article reviews the technique, indications, benefits, and limitations of pre-
implantation genetic testing in clinical practice.
KEY WORDS: Pre-implantation diagnosis, genetics, prenatal diagnosis
Introduction
D
iagnosis of genetic disorders early in pregnancy refers to
prenatal diagnosis. A step further, preimplantation genetic
testing is an early form of prenatal diagnosis, where genetic defects
in embryos created in vitro are analyzed before implantation in
the uterus.
[1]
This offers couples at risk of a genetic disease the
chance to have an unaffected child, without facing termination
of pregnancy. Two decades have elapsed since the first description
of pregnancy, after application of this technique in sex selection
of human embryos at risk of an X-linked disease.
[2]
To date, many
studies have addressed the impact of preimplantation genetic
screening in different groups of patients, however, its effectiveness
has not been consistently proven.
[3]
Despite this, according to the
recent European Society of Human Reproduction and Embryology
(ESHRE) preimplantation genetic diagnosis (PGD) consortium
data collection, the number of PGD cycles performed for aneuploidy
screening, pregnancies, and babies reported annually have increased
considerably.
[4]
Here we aim to briefly outline the scope of PGD and
its applications in a clinical setting.
Who Benefits from Preimplantation Testing?
Indications for preimplantation testing can be viewed from two
perspectives. One, the couple is already known to be at risk of
a genetic disease, usually a monogenic disease or chromosomal
abnormality in the offspring, due to a balanced chromosomal
rearrangement in one of the partners. Here the couple is averse
to termination of the affected conceptions after an invasive
prenatal diagnostic test. Although most of them can conceive
naturally, they undergo in vitro fertilization (IVF) so that the
embryos are selected for implantation only if they are found to
be free of the genetic defect. Second, the couples undergo in
vitro fertilization in view of infertility to achieve conception.
This provides the couples a window of opportunity to test the
embryos for common chromosomal aneuploidies. This group
may also be at a higher risk of aneuploidy (especially trisomy 21)
in view of advanced maternal age (> 35 years at the expected
time of confinement). Often the term preimplantation genetic
diagnosis (PGD) is used for the first group and preimplantation
genetic screening (PGS) for the latter.
Mendelian Disorders (Monogenic Disorders)
Single gene disorders, also known as Mendelian disorders are
diagnosed by PCR-based techniques. If one of the couple
carries a mutation for an autosomal dominant disease, they
carry a 50% risk of transmitting the disease to the offspring. The
couple is at 25% risk for an autosomal recessive disease in the
child if both of them are carriers of the disease. In an X-linked
recessive condition, with the female partner being carrier of the
mutation, the risk to a male offspring is 50%. It may be noted
that the occurrence and severity of the disease in the child
are also determined by the penetrance and expressivity of the
condition. Preimplantation genetic testing is being reported for
many more single gene disorders. Expectedly, for all common
diseases like beta thalassemia, cystic fibrosis, spinal muscular
atrophy, myotonic dystrophy, Huntington disease, Marfan
Division of Reproductive
Medicine,
1
Genetics
Clinic, Kasturba Medical
College, Manipal
University, Manipal, India
Address for correspondence:
Dr. Girisha KM,
E-mail: girishkatta@gmail.
com
Received : 09-04-10
Review completed : 22-05-10
Accepted : 28-06-10
PubMed ID : ***
DOI: 10.4103/0022-3859.70943
J Postgrad Med 2010;56:317-20
318 Journal of Postgraduate Medicine October 2010 Vol 56 Issue 4
syndrome, and so on, preimplantation genetic diagnosis has
been successfully done. Technically many of the monogenic
diseases must be amenable to PGD. An option for the X-linked
recessive disease is selection of only female embryos that are
unlikely to be affected by the disease. Sexing of the embryo can
be done by fluorescence in situ hybridization (FISH) or even
polymerase chain reaction (PCR). This is a particularly useful
alternative for specific diagnosis in X-linked mental retardation
and X-linked retinitis pigmentosa, where a specific molecular
defect is not identified or almost impossible to identify. Linkage
analysis is an alternative to identify the unaffected males in
some specific situations.
Aneuploidies and Structural Chromosomal Abnormalities
It is well known that chromosomal aneuploidies are an
important cause of abnormal embryos and reproductive
failures. Using probes for five chromosomes (X, Y, 13, 18,
and 21), it is estimated that 70% of the embryos derived
from IVF were abnormal for at least one of the chromosomal
numbers.
[5]
Hence, it can be expected that incorporation of
aneuploidy screening in embryos can improve the success of
IVF. Additionally, as female partners of most of the couples
seeking IVF have advanced age, they are at an increased risk
of aneuploidy, specifically trisomy 21. Aneuploidy screening
can also be an investigation to look into the causes of poor
implantations and recurrent abortions of unidentified cause.
Aneuploidy screening is by far the most common indication
for preimplantation genetic testing. Second, the couple
where one is a carrier of a balanced structural chromosomal
abnormality may face the risk of abortion, stillbirth, or birth
of a child with malformations and / or mental retardation.
Usually these abnormalities are balanced translocations or
inversions and rarely other rearrangements. FISH probes
for the specific region of interest need to be deployed in
these settings and may not be available at all the centers. If
the female is a carrier, the polar bodies may be analyzed for
chromosomal abnormality.
Obtaining Cells from Early Embryo
Three methods have been developed to carry out PGD.
The most widely used approach is testing the individual
embryonic cells (blastomeres) obtained on the third day
after IVF. One or two blastomeres are removed through
a hole created in the zona pellucida, and the cells are
analyzed.
[6]
Another approach is to use the non-embryo
forming cells (trophoectoderm cells) of the embryos on day
5 of development. In both the cases, healthy embryos are
subsequently transferred to the uterus on days 4, 5 or 6, after
IVF. An alternative method for carrying out PGD examines
the genetic material within the first and second polar bodies,
the by-products of meiosis I and meiosis II, respectively. This
method can be used in case of maternally derived dominant
mutations, translocations, and aneuploidy. It cannot be
used when paternally derived genetic information is critical
for the diagnosis, such as, paternally derived dominant
mutations, translocations, and aneuploidy.
Blastomere Biopsy
Embryo biopsy is the removal of one or two blastomeres
from the cleavage stage embryo. It is the most widely used
biopsy procedure for PGD at present. Ideally a six-to-eight
cell staged embryo is preferred for biopsy. By this time the
embryo completes at least three mitotic divisions. Biopsy of
one or two cells is performed on day 3 and the embryos are
transferred on day 4 or 5. This allows enough time for the
genetic investigations to be performed and for the transfer
of genetically normal embryos, which have progressed till
the blastocyst stage.
Embryos are generally incubated for a brief period in a
medium deficient in divalent cation-like calcium, to disrupt
the tight junctions and cell-to-cell interaction between the
blastomeres. An opening in the zona (~20 m) is created
using the mechanical, enzymatic or laser drilling method.
After creating the opening, the blastomeres are removed
by aspiration, which is the most widely used method. An
aspiration pipette with an inner diameter of 35 40 m is
introduced into the perivitelline space through the hole in
the zona and the cells are completely aspirated or partially
aspirated and pulled out from other blastomeres.
Blastocyst (Trophoectoderm) Biopsy
The use of trophoectoderm (TE) cells as a source of biopsy
material was first suggested two decades ago by Dokras et
al.
[7]
The main advantage of this method is that a large
number of cells are available for genetic analysis unlike in the
blastomere biopsy, where only one or two cells are available.
There are two approaches for collecting the TE cells from
the blastocyst. In the first approach, the zona drilling is
done in the blastocyst, exactly opposite the inner cell mass
(ICM). After four to ten hours of culture, few TE cells
herniate through the zona on which the biopsy can be
performed. In the second approach, the hole is made in early
embryos on the third (6 8 cell stage embryos) or fourth
day (morula stage embryo) of development. The rationale
of this approach is to drill a hole in the zona with minimum
damage to the adjacent cells, as at the blastocyst stage the
perivitelline space is almost non-existent. In this approach
the zona drilling is done on all the available embryos and
they are cultured till they reach the balstocyst stage. The
TE cells, which are closer to the hole on the zona pellucida
approach, can be used for biopsy. In both cases, the biopsies
are obtained by securing the blastocyst with a holding
pipette, with the herniated cells located in the 3 oclock
position. Usually two to nine TE cells are gently aspirated
with a biopsy pipette (internal diameter 30 m) and three
to five laser pulses are then applied in order to detach them
from the blastocyst. The cells obtained are finally ejected
from the biopsy pipette and are ready to be processed. The
major limitation of this approach is the requirement of
sufficient number of embryos reaching up to the blastocyst
stage, the very short time available for genetic assessment,
and that the TE cells may not reflect the true genetic feature
Adiga, et al.: Preimplantation genetic diagnosis
Journal of Postgraduate Medicine October 2010 Vol 56 Issue 4 319
of cells of the ICM.
Polar Body Biopsy
The polar bodies are considered as waste nuclear material
after the meiotic division and do not have any physiological
role to play in the development of embryos. By establishing
whether there is an abnormal gene or chromosome
arrangement in the polar bodies, it is possible to infer the
maternal genetic contribution to the embryo. Therefore,
they are considered excellent material for genetic analysis of
the oocyte. The polar bodies are located in the perivitelline
space enclosed by the zona pellucida, and to gain access to
these materials, an opening in the zona has to be created
either by chemical or laser drilling.
The polar body biopsy technique is advantageous because it
maintains the embryo's integrity, as only meiotic by-products
and not blastomeres are used to assess the genetic integrity
of the developing embryo. Hence, any possible effect of the
procedure on the developmental potential of the embryo
and its polarity is thought to be the least. Nevertheless, it
is hampered by the impossibility of diagnosing paternally-
derived defects, and those originating after fertilization
or first cleavage events. In recent times, the accuracy and
reliability of the results obtained from the genetic analysis
of the polar body is enhanced by investigating both the
polar bodies. The use of this strategy permits chromosomal
abnormalities associated with the second meiotic division
to be identified, and in case of single-gene mutations,
the occurrence of crossing-over between homologous
chromosomes can be verified.
Genetic Testing of Biopsied Cells
In addition to accuracy, the important attributes of the
techniques for preimplantation genetic testing include
robustness and rapidity. Usually, preimplantation genetic
testing uses either a PCR-based technique or FISH for the
detection of genetic defects.
Polymerase Chain Reaction
The polymerase chain reaction amplifies the region of
interest in the DNA of a biopsied cell. It may be noted that
a polar body contains a single copy of the DNA (haploid)
and the cell biopsied at the cleavage stage has double
copies of the DNA (diploid). This is a very demanding
PCR, as only a single cell is the source of DNA. The cell
is lysed and the region of interest in the DNA strand is
amplified using specific primers. The amplified fragment
may be analyzed subsequently for the size, digestibility by
a restriction enzyme or sequencing, to infer the presence
of the defect (mutation). This technique demands that
the defect has already been identified in the couple or their
previous child, and is normally applied for the diagnosis
of single gene disorders. Multiplex PCR, real time PCR,
and quantitative fluorescent PCR have been added to the
technical armamentarium recently.
Fluorescence In situ Hybridization
Fluorescence in situ hybridization involves the use of fluorescent
labeled probes that bind to the target DNA in the biopsied cell.
Both probe and target DNA are denatured and complementary
sequences are allowed to hybridize with each other under
optimal chemical and temperature conditions. When an
excess probe is washed, the number of fluorescent signals
corresponds to the presence and number of target sequences.
In other words, if the probe used is specific for a unique region
on chromosome 21, two signals indicate the presence of two
normal copies of chromosome 21 and three signals imply
trisomy 21 in the biopsied embryo. The common probes used
are: X, Y, 13, 18, and 21. The number of chromosomes tested
in a single FISH is limited by the number of fluorochromes
available. However, mixing of colors, several rounds of FISH on
the same cell, and comparative genomic hybridization (CGH),
enhance the possibility of detecting the abnormalities in several
chromosomes / regions. Obviously, FISH is the technique
employed for detection of aneuploidies of autosomes, and sex
chromosomes and chromosomal rearrangements.
Limitations of Laboratory Techniques and Challenges
The major challenges of PGD are relatively short interval of
time available for analysis and few numbers of cells available
for analysis, compared to the hundreds of cells obtained via
amniocentesis or chorionic villi sampling. Technical difficulties
that may arise during PCR amplification of target DNA
sequences include failure of amplification, contamination with
external DNA, and more specifically the allele drop-out. This
refers to the situation where one of the alleles in a diploid cell
(either normal or abnormal) fails to amplify and the results are
interpreted on the basis of the other allele that is amplified.
This can result in both false positive and false negative results.
It is estimated that the risk of transferring an affected embryo
mistakenly identified as normal by PGD is approximately 2% for
recessive disorders and 11% for dominant disorders.
[8]
Follow-up
testing by prenatal or postnatal sampling can confirm the exact
incidence of such errors.
When FISH is the technique of screening for aneuploidy in
the embryo, failure to hybridize, poor signal intensity, split
or fused signals may give rise to false results. As only a single
cell is analyzed, mosaicism is not accurately picked up by this
technique. This is especially important, as there may be a
trisomy rescue and embryos that are diagnosed to have a trisomy
may eventually develop into a fetus with normal chromosomal
content. It is estimated that misdiagnosis in aneuploidy
screening after biopsy of one blastomere is 7%, with 6% due
to mosaicism.
[9]
Counseling for Preimplantation Genetic Testing
The couples have to undergo an informed counseling addressing
the risk of a genetic disease, IVF, issues related to the technique
and the cost of IVF and PGD. They have to understand the
Adiga, et al.: Preimplantation genetic diagnosis
320 Journal of Postgraduate Medicine October 2010 Vol 56 Issue 4
risk of a genetic disease in their offspring (may be a monogenic
disorder or a chromosomal abnormality), the natural history of
the condition, and the options available to treat or prevent the
condition. They are are informed about the advantages and
disadvantages of prenatal diagnostic options as compared to
preimplantation testing. The options must include choosing not
to proceed with IVF and PGD and sometimes accepting donor
ovum / sperm. Accuracy and error rate of the techniques also
need to be discussed before the couples opt for prevention of
the genetic disease by selecting the method that suits them best.
Pregnancy Outcome
Although there are no published data providing the number
of centers performing PGD, in Europe, according to the
European Society of Human Reproduction and Embryology
(ESHRE) PGD consortium data collection for the year 2004,
where 45 centers participated, 3358 egg collections have been
performed: 1192 in cycles with PGD, 2087 with PGS, and 79
cycles with social sexing. The indications were chromosomal
abnormalities for 559 cycles, X-linked disorders for 113 cycles,
and monogenic diseases for 520. In cycles with PGD for
chromosomal abnormality indications, 76% embryos were
biopsied; among them, 93% provided a diagnosis, of which 25%
were transferable. In cycles with PGD for monogenic diseases,
71% embryos were biopsied; 88% provided a diagnosis, of which
52% were transferable. In total, 69.6% of the cycles led to embryo
transfer.
[10]
In France, 70% of the biopsied embryos provided a
diagnosis, of which 60% were transferable. It has been shown
that the PGD implantation rate is 17%, which is less than the
implantation rate observed in non-PGD ART cycles.
[10]
The
clinical pregnancy rate is reported as 18% per oocyte retrieval
and 25% per embryo transferred, leading to 679 pregnancies
and 528 babies born. The pediatric follow-up of PGD babies
has shown a similar mental and psychomotor developmental
outcome at age two, when compared with children conceived
after IVF-ICSI.
[11]
Embryo biopsy does not seem to affect the
course of pregnancy, the babys characteristics at birth (birth
weight, length, gestational age at delivery), or the rate of
malformations at birth.
[12-14]
Ethical Issues
Many a time parents may opt to select the sex of the fetus by
opting for preimplantation genetic testing, for social reasons.
As per the Preconception and Prenatal Diagnosis Act of 1994,
this is illegal in India and many other countries. Couples may
select a human leukocyte antigen (HLA)-matched embryo for a
previous child with a genetic disease treatable by bone marrow
transplantation. In case of adult onset diseases like Huntington
disease and cancer predisposition syndromes, the possibility of
a new therapy becoming available and the burden of preventive
measures need to be taken into account, when preimplantation
genetic testing is considered. Ethical issues in preimplantation
testing are not completely the same as in prenatal diagnosis, as
the former concerns the selection of unaffected embryos and
the latter, the termination of affected pregnancies.
Conclusions
Preimplantation genetic diagnosis is only offered at a few select
centers worldwide compared to prenatal diagnosis, but the
number of centers is growing steadily along with the number of
diseases that can be diagnosed. PGD needs a close collaboration
between obstetricians, fertility specialists, IVF laboratories, and
human geneticists. It has gained a place among the choices
offered to couples at risk of serious disease transmission. PGD
is an important alternative to standard prenatal diagnosis, for
genetic disorders. Low pregnancy and birth rates and the high
cost of the procedure, however, are important considerations.
References
1. Sermon K. Current concepts in Preimplantation genetic diagnosis
(PGD): a molecular biologists view. Hum Reprod Update
2002;22:312-8.
2. Handyside A, Kontogianni E, Hardy K, Winston R. Pregnancies from
biopsied human Preimplantation embryos sexed by Y-specific DNA
amplification. Nature 1990;344:768-70.
3. Shahine LK, Cedars MI. Preimplantation genetic diagnosis does
not increase pregnancy rates in patients at risk for aneuploidy Fertil
Steril 2006;85:51-6.
4. Goossens V, Harton G, Moutou C, Traeger-Synodinos J, Van Rij M,
Harper JC. ESHRE PGD Consortium data collection IX: cycles from
January to December 2006 with pregnancy follow-up to October
2007. Hum Reprod 2009;24:1786-810.
5. Munne S, Lee A, Rosenwaks Z, Grifo J, Cohen J. Diagnosis of major
chromosome aneuploidies in human preimplantation embryos. Hum
Reprod 1993;8:2185-91.
6. De Vos A, Van Steirteghem A. Aspects of biopsy procedures prior
to preimplantation genetic diagnosis. Prenat Diagn 2001;21:76780.
7. Dokras A, Sargent IL, Ross C, Gardner RL, Barlow DH. Trophectoderm
biopsy in human blastocysts. Hum Reprod 1990;5:821-5.
8. Lewis CM, Pinel T, Whittaker JC, Handyside AH. Controlling
misdiagnosis errors in preimplantation genetic diagnosis: a
comprehensive model encompassing extrinsic and intrinsic sources
of error. Hum Reprod 2001;16:43-50.
9. Munn S, Magli C, Bahe M, Fung J, Legator M, Morrison L, et al.
Preimplantation diagnosis of the aneuploidies most commonly found
in spontaneous abortions and live births: X, Y, 13, 14, 15, 16, 18, 21,
22. Prenat Diagn 1998;18:1459-66.
10. Harper JC, de Die-Smulders C, Goossens V, Harton G, Moutou C,
Repping S, et al. ESHRE PGD consortium data collection VII: cycles
from January to December 2004 with pregnancy follow up to October
2005. Hum Reprod 2008;23:741-55.
11. Nekkebroeck J, Bonduelle M, Desmyttere S, Van den Broeck W,
Ponjaert-Kristoffersen. Mental and psychomotor development of
2-year-old children born after preimplantation genetic diagnosis/
screening. Hum Reprod 2008;23:1560-6.
12. ESHRE PGD Consortium Steering Committee. ESHRE Preimplantation
Genetic Diagnosis Consortium: data collection III (May 2001). Hum
Reprod 2002;17:233-46.
13. Anon. Report of the 11th annual meeting of International Working
Group on Preimplantation Genetics: preimplantation genetic
diagnosisexperience of 3000 clinical cycles. Reprod BioMed
Online 2001;3:49-53.
14. Strom C, Levin R, Strom S, Masciangelo C, Kuliev A, Verlinsky Y.
Neonatal outcome of preimplantation genetic diagnosis by polar
body removal: the first 109 infants. Pediatrics 2000;106:65053.
Source of Support: Nil, Confict of Interest: None declared.
Adiga, et al.: Preimplantation genetic diagnosis
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