RBMOnline - Vol 5. No 3. 294–299 Reproductive BioMedicine Online; www.rbmonline.com/Article/705 on web 5 September 2002
Articles
Current features of preimplantation genetic
diagnosis
Dr Anver Kuliev
Dr Yury Verlinsky
Dr Anver Kuliev received his PhD in Clinical Cytogenetics from
Moscow Research Institute of Human Morphology in 1969. In
1979 he took the responsibility for the World Health Organization
(WHO)’s Hereditary Diseases Program in Geneva, where he
developed the community-based programs for prevention of
genetic disorders and early approaches for prenatal diagnosis.
He moved to Reproductive Genetics Institute in 1990, where he
heads the WHO Collaborating Center for Prevention of Genetic
Disorders, and scientific research in prenatal and
preimplantation genetics. He is an author of more than hundred
papers and nine books in the above areas, including three
books in the field of Preimplantation Genetics.
Dr Yury Verlinsky is a graduate, postgraduate and PhD of Kharkov University of the former USSR. His research
interests include cytogenetics, embryology and prenatal and preimplantation genetics. He introduced polar body
testing for preimplantation genetic diagnosis and developed the methods for karyotyping second polar body and
individual blastomeres. He has published over 100 papers, as well as three books on preimplantation genetics.
Anver Kuliev1, Yury Verlinsky
Reproductive Genetics Institute, 2825 North Halsted Street, Chicago, IL 60657, USA
1Correspondence: Tel: (773) 472 4900; Fax: (773) 871 5221; e-mail: anverkuliev@hotmail.com
Abstract
More than 4000 preimplantation genetic diagnosis (PGD) cycles have been performed, suggesting that PGD may no longer
be considered a research activity. The important present feature of PGD is its expansion to a variety of conditions, which
have never been considered as an indication for prenatal diagnosis, including the late-onset disorders with genetic
predisposition and preimplantation non-disease testing, with the further improvement of the accuracy of PGD for single
gene disorders. PGD has also become a useful tool for the improvement of the effectiveness of IVF, through avoiding the
transfer of chromosomally abnormal embryos, representing more than half of the embryos routinely transferred in IVF
patients of advanced maternal age and other poor prognosis patients. PGD is of particular hope for the carriers of balanced
chromosomal translocations, as it allows accurate pre-selection of a few balanced or normal embryos resulting from the
extremely poor meiotic outcome, especially in reciprocal translocations. With the current progress in polymerase chain
reaction- (PCR-) based detection of chromosomal abnormalities in oocytes and embryos, PGD may soon be performed for
both chromosomal and single gene disorders using the same biopsied polar body or blastomere, frequently required with the
currently expanded PGD application. The available clinical outcome data of more than 3000 PGD embryo transfers further
suggest an acceptable pregnancy rate and safety of the procedure, as demonstrated by the follow-up information available
for more than 500 children born from these PGD transfers.
Keywords: chromosomal abnormalities, clinical outcome, expanding indications, IVF effectiveness, late-onset disorders with
genetic predisposition, preimplantation genetic diagnosis, preimplantation HLA typing
Introduction
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Preimplantation genetic diagnosis (PGD) is no longer a
research activity, although more studies are still required to
further improve the reliability and accuracy of genetic analysis
and to develop methods for preimplantation testing of
cytoplasmic abnormalities and transcriptional disorders, not
available at the present time. One of the important features of
the present PGD is its expansion to a variety of conditions,
which have never been considered to be an indication for
prenatal diagnosis (Simpson, 2002; Rechitsky et al., 2002).
The employment of PGD methods will be useful with the
current progress in construction of human gametes, therapeutic
cloning and the establishment of embryonic stem cells (Tesarik
et al., 2001; Lacham-Kaplan et al., 2001; Tesarik and
Mendoza, 2002; Trounson and Cram, 2002). These
developments have been reviewed by the Fourth International
Symposium on Preimplantation Genetics and the 12th Annual
Meeting of the International Working Group on
Preimplantation Genetics, held in Cyprus on 11–13 April
2002, which also summarized the current PGD experience,
with its new directions and controversies. As in previous years,
the growing implications of PGD for the pre-selection of
aneuploidy-free embryos for assisted reproduction and its
Articles - Current features of PGD - A Kuliev, Y Verlinsky
wider application for the improvement of the effectiveness of
IVF have been reviewed. A special consideration was given to
the currently expanding indications for PGD, with the
relationship to the ethical and social issues involved. These
recent developments are presented below under four major
topics: (i) future directions, summarizing the prospects for
preimplantation testing of cytoplasmic and transcriptionally
relevant abnormalities, the recent developments in
micromanipulation techniques and the progress in
understanding of the origin of aneuploidies; (ii) expanding
indications for PGD; (iii) PGD in assisted reproduction; and
(iv) PGD clinical outcomes.
Future directions
It is well known that the pre-selection of oocytes or embryos
with the highest potential to implant and go to term cannot be
achieved properly by testing only for nuclear abnormalities,
requiring the evaluation of the cytoplasmic maturity of oocytes
and embryos. For example, variation in mitochondrial content
should affect the functionality of the gametes and embryos.
Although significant progress in the analysis of cytoplasmic
abnormalities and the evaluation of the cytoplasmic
competence of oocytes and embryos has been achieved, no test
is presently available to apply in clinical practice (Cohen and
Malter, 2002). The same is true of transcriptional patterns of
preimplantation development, suggesting similarities of
embryonic and cancer cells in a global demethylation of DNA
and genetic deprogramming to a proliferative stem cell state
(Monk et al., 2002). It is obvious that the genetic expression is
highly stage-dependent, so knowledge of the normal
expression patterns may allow the performance of
preimplantation screening for the expression of a range of
imprinted genes, in order to identify the stage-specific
expression anomalies.
Nuclear abnormalities, the testing for which is currently a
routine PGD practice, increase significantly with age,
representing one of the major causes of reproductive failures in
women of advanced maternal age. However, despite advanced
maternal age remaining the only well-documented risk factor
for maternal meiotic non-disjunction, the basic mechanisms of
the maternal-age effect is still unknown. The alterations in
maternal recombination rates in meiosis are the other
identified risk factor for chromosome nondisjunction, although
measurements of meiotic recombination have been made only
indirectly, through the examination of DNA-polymorphism
inheritance in families with trisomic livebirths or spontaneous
abortions. Direct evidence of the relationship between reduced
recombination and non-disjunction has recently been obtained
in the analysis of human spermatozoa (Martin et al., 2002). By
studying recombination in the pseudo-autosomal region in X
and Y chromosomes, the lack of recombination in this region
was established, with association to XY nondisjunction and
production of aneuploid spermatozoa.
The other direct study of meiotic crossovers was performed in
human fetal oocytes, by staining for a mismatch-repair protein
MLH1 (Tease et al., 2002). As the distribution of foci of this
protein at pachytene matches that of chiasmata at metaphase I,
representing an excellent marker for recombination, the
number and distribution of the MLH1 foci were determined for
chromosomes 13, 18, 21 and X. In addition to a sex-specific
difference in control of recombination, it was shown that some
chromosomes 18 and 21 lacked an MLH1 focus, representing
a high risk of nondisjunction in the process of completion of
meiosis, while neither chromosome 13 and X lacked this locus
in any of the oocytes classified as normal. So the detection of
the recombination-deficient oocytes may indicate an increased
risk of meiotic chromosome nondisjunction and production of
aneuploid ova.
One recent development in micromanipulation and nuclear
transfer was work on the construction of human gametes. This
was attempted for creating both female and male gametes,
demonstrating a strong morphological evidence for
haploidization (Galat and Verlinsky, 2002; Lacham-Kaplan,
2001; Tesarik et al., 2001; Tesarik and Mendoza, 2002).
However, no cytogenetic proof for haploidization has been
presented, required also to ensure the normalcy of the resulting
gametes, deriving from the somatic cell transfer into the
matured oocytes. The nuclear transfer technique, by contrast,
has been further used for visualization of blastomere
chromosomes in PGD for translocations, demonstrating its
clinical usefulness (Verlinsky et al., 2002a). The technique
was applied in 52 PGD cycles for translocations, with a
clinically acceptable rate of conversion of interphase nuclei of
the blastomeres into metaphase. Of a total of 437 blastomeres
tested, the technique was successful in 383 (88%), making
possible the pre-selection of normal embryos or those with
balanced chromosomal complement for transfer in 38 PGD
cycles for translocations.
A highly reproducible technique is presently available for the
derivation of human embryonic stem cells and their directed
differentiation into different cell types (Trounson and Pera,
2001; Trounson and Cram, 2002). A success rate in the
establishment of human embryonic stem cells is
approximately 66%. The directed differentiation of these cells,
using factors produced from differentiating or somatic cells,
was achieved for the following cell types: ciliated epithelia of
the lung, secretary epithelia in the gut-like structures, neuronal
cells and muscles, including cardiomyocytes. The aggregates
of muscles were shown to beat 45–60 times per minute,
evident for 11–21 days after initiation of the co-culture. In
addition, a spontaneous differentiation of embryonic stem cells
into insulin-producing beta cells was observed (Assady et al.,
2001), as well as the production of hematopoietic precursor
cells, following a co-culture of embryonic stem cells with a
mouse bone marrow cell line (Kaufman et al., 2001). These
developments provide an obvious potential for the therapeutic
use of embryonic stem cells in clinical practice.
Expanding indications for PGD
Late onset disorders: Among the recent applications, PGD for
inherited predisposition to late-onset disorders included cancer
predisposition, presently offered to patients with
predisposition to familial adenomatous polyposis, Von Hippel
Lindau syndrome, retinoblastoma, Li-Fraumeni syndrome
(determined by p53 tumour suppressor gene mutations),
neurofibromatosis type I and II and familial posterior fossa
brain tumour (Verlinsky et al., 2001a; Rechitsky et al., 2002).
For the latter, overall 20 PGD cycles were performed, resulting
in the pre-selection and transfer of 40 mutation-free embryos,
which yielded five unaffected clinical pregnancies and four
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healthy children born by the present time. Despite the
controversy of PGD use for late-onset disorders, such data
demonstrate the usefulness of this approach as the only
acceptable option for at-risk couples to avoid the birth of
children with an inherited predisposition to cancer and to have a
healthy child of their own.
Perhaps one of the most unexpected applications of PGD was
in the genetic predisposition for Alzheimer’s disease, which is
an autosomal dominant familial predisposition to a presenile
form of dementia (Verlinsky et al., 2002b). The disease is
determined by a nearly completely penetrant autosomal
dominant mutation in the amyloid precursor protein gene, for
which no treatment is available, despite a possible predictive
diagnosis. The results demonstrated the feasibility of PGD for
early-onset Alzheimer’s, providing a non-traditional option for
patients who may wish to avoid the transmission of the mutant
gene predisposing to early Alzheimer’s in their potential
children. For some patients, this may be the only reason for
undertaking pregnancy, as the pregnancy may be established
free from an inherited predisposition to Alzheimer’s from the
very onset.
HLA typing: Preimplantation human leukocyte antigen (HLA)
matching has recently emerged as a tool for the pre-selection of
potential donor progeny for bone marrow transplantation
(Verlinsky et al., 2001b). Previously, this was applied to a case
of Fanconi anaemia, in which the pre-selection and transfer of
unaffected embryos with an HLA match with the affected
sibling yielded a clinical pregnancy and the birth of a healthy
carrier of the Fanconi anaemia gene, whose cord blood was
transplanted to the affected sibling, resulting in a successful
haemopoietic reconstitution. The method has currently been
applied to HLA genotyping in 18 cycles (five for thalassaemia,
six for Fanconi anaemia, one for Wiscott-Aldrich syndrome and
six for leukaemia) from 11 couples overall, involving the testing
of 197 embryos in combination with genetic disease, and to 86
embryos for HLA typing only. This resulted in the pre-selection
of 37 (19%) HLA-matched embryos, including 21 (17.5%) in
combination with pre-selection of unaffected embryos following
mutation testing and 16 (22%) not involving mutation analysis.
These HLA-matched embryos have been transferred back to
patients, resulting in four clinical pregnancies and the birth of
two HLA-matched children, one pending transplantation of the
cord blood stem cells to the affected sibling with leukaemia
(Verlinsky et al., unpublished data).
296
Dynamic mutations: The application of PGD has further
expanded to dynamic mutations, with the largest series of PGD
for these disorders performed as previously in the Brussels
centre (Sermon, 2002). In addition to myotonic dystrophy,
Huntington’s disease, and fragile X syndrome (FRAXA), PGD
was performed for spinal and bulbar muscular atrophy and
spino-cerebellar ataxia 7. Overall, 161 PGD cycles were
performed for 72 patients, resulting in the transfer of
unaffected embryos in approximately 85% of cycles and 20%
clinical pregnancies per transfer. The majority of these cases
was done for myotonic dystrophy and Huntington’s disease,
including 94 cycles for myotonic dystrophy and 44 for
Huntington’s disease, resulting in 80 and 38 transfers, and 18
and 7 clinical pregnancies, respectively. The accuracy of PGD
has been further improved in this work with the application of
fluorescence PCR (FL-PCR) with the expand long template
(ELT) kit, which enabled the reduction of the allele dropout
(ADO) rate from approximately 30% in both conventional
PCR and FL-PCR to as low as 5% in the testing for myotonic
dystrophy. The other attractive approach for improving the
accuracy of PCR analysis in single cells involved the
application of real-time PCR, which was found to reduce the
ADO rate by half, compared with conventional PCR or FLPCR (Rechitsky et al., 2002). The application of these
approaches, together with the simultaneous testing for the
causative mutation and at least one or two linked markers, may
avoid the risk of misdiagnosis completely in PGD for single
gene disorders.
PCR-based testing for chromosomal disorders: With the
expanding PGD application, it is becoming current practice to
test for single gene disorders together with chromosomal
abnormalities, such as in cases of advanced maternal age or
chromosomal translocations. So, ideally, the same single cell
should be utilized for testing both genetic and chromosomal
disorders. As previously demonstrated, one of the possible
approaches for PCR-based testing for chromosomal
aneuploidies is DNA fingerprinting, which is based on the
patterns of alleles that uniquely identify an individual, relying
on a multiplex FL-PCR of low template DNA. Using
tetranucleotide microsatellite markers with a high
heterozygosity index and broad allelic size distribution, the
single cell DNA fingerprinting systems have been developed
for detection of aneuploidies for chromosomes 13, 18 and 21,
which showed 93% reliability, 94% accuracy, 6% ADO rate
and 10% preferential amplification rate (Katz et al., 2002).
This system has been applied for a combined testing of
Down’s syndrome and cystic fibrosis in single cells.
Further progress has been achieved in the development of
comparative genome hybridization (CGH), which is another
feasible PCR-based method for the testing of chromosomal
abnormalities in a single cell. This method may enable the
detection and exclusion from transfer of the aneuploid
embryos, of which a sizable proportion could be misdiagnosed
as normal by the commercially available five-colour probe
(Wilton et al., 2001). The previously reported limitations of
the CGH protocol, involving a 3-day duration of the procedure
incompatible with the current laboratory framework for PGD,
has presently been overcome either by performing the polar
body CGH, or by accelerating the procedure to be completed
in 38 h. The first practical application of CGH using the first
polar body was attempted in a 40-year-old IVF patient and
resulted in the detection of a single aneuploidy-free oocyte
from the 10 available for testing. The transfer of the embryo
resulting from this oocyte, however, yielded no clinical
pregnancy (Wells et al., 2002). The follow-up fluorescence insitu hybridization (FISH) analysis of all the remaining
embryos resulting from the nine aneuploid oocytes has
confirmed a CGH-based diagnosis at day 3, suggesting that the
procedure may be applied in clinical practice. Acceleration of
the CGH procedure to be applied to blastomere biopsy was
also achieved, using a specific protocol for the degenerate
oligonucleotide primed-polymerase chain reaction CGH
(DOP-PCR-CGH), applying different Taq polymerase and
labelling methods (Ozen et al., 2002). In addition to shortening
the procedure, this also yielded a three-fold greater DOP-PCR
product. In the meantime, the standard CGH protocol, which
takes 5 days to complete, has presently been applied in 20
Articles - Current features of PGD - A Kuliev, Y Verlinsky
frozen cycles for poor prognosis IVF patients, resulting in the
pre-selection and transfer of 19 aneuploidy-free embryos in 13
cycles, which yielded three unaffected clinical pregnancies
(Wilton et al., 2002).
For the first time, CGH was also performed for testing
structural chromosomal rearrangements in single blastomeres,
obtained from spare embryos that were diagnosed unbalanced
[t(6;7)(p25;q11.2); t(10;11)(p11.2;q23.3); t(9;13)(q12;p13);
and t(10;11)(q12;p13)] and therefore unsuitable for transfer
(Malmgren et al., 2002). A total of 94 blastomeres from 28
human embryos have been analysed, confirming the diagnosis
of the unbalanced translocations performed by FISH analysis.
In addition, a high degree of numerical aberrations was found,
including monosomies and trisomies for whole chromosomes
or their parts. It is interesting that almost all the embryos
appeared to be mosaic, containing more than one
chromosomally distinct cell line, or even chaotic with different
chromosomal contents in each blastomere.
PGD in assisted reproduction
As already mentioned, the most universal application of PGD
has been the pre-selection of aneuploidy-free embryos for IVF
patients of advanced maternal age (International Working
Group on Preimplantation Genetics, 2001). The usefulness of
PGD in assisted reproduction is obvious from the data on the
prevalence of nuclear abnormalities in the oocytes of women
of 35 years and older, which is further confirmed to be over
50% even by testing of only five chromosomes (chromosomes
13, 16, 18, 21 and 22) (Kuliev et al., 2002). These data are
based on the FISH analysis of 6733 oocytes, obtained in 1297
PGD cycles performed in IVF patients of average age 38.5
years. A total of 3509 (52%) aneuploid oocytes was detected,
originating comparably from the first and second meiotic
divisions. Overall, 41.8% oocytes had errors in meiosis I,
30.7% errors in meiosis II, and 27.6% both meiotic division
errors. In a total of 45.1% of the abnormal oocytes with
complex errors, the same chromosome in both meiotic
divisions was involved in 21.5% of cases, while different
chromosomes were observed in 78.5% of oocytes. Of 3224
detected aneuploidy-free zygotes, 2587 were transferred in
1100 treatment cycles (2.35 embryos per transfer), resulting in
241 (21.9%) clinical pregnancies and 176 healthy children
born, suggesting a positive clinical outcome following
aneuploidy testing of oocytes in a group of IVF patients of
average age 38.5 years.
The above overall rates of nuclear abnormalities in oocytes are
comparable to those detected in preimplantation embryos in
PGD for aneuploidies at the cleavage stage, taking into
consideration additional fertilization-related abnormalities and
paternally-derived meiotic errors, which could also have been
detected at this stage. In approximately the same number of
PGD cycles performed for aneuploidy at the cleavage stage by
the Saint Barnabas and SISMeR centres, the proportion of
embryos with chromosomal abnormalities was as high as 60%
(Munné, 2002; Gianaroli et al., 1999, 2002a). However, the
types of anomalies in oocytes and embryos were significantly
different, which is mainly attributable to a high frequency of
mosaicism, comprising approximately half the chromosomal
abnormalities at the cleavage stage. Although the origin of a
high rate of mosaicism is still unclear, preliminary data
indicate that the mosaicism may be of different nature. Some
mosaic types are increased with maternal age (Munné et al.,
2002b), while others are possibly attributable to the
immaturity of the centrosome structures in spermatozoa,
which may be the case in testicular sperm extraction (TESE)
patients (Silber et al., 2002). It could be that a significant
proportion of mosaic embryos originates from the oocytes that
are aneuploid from the outset, through a process similar to that
known as aneuploidy ‘rescue’. A possible high rate of further
mitotic errors in cleaving embryos deriving from the oocytes
with the complex aneuploidies may also explain a
phenomenon of chaotic embryos, which make up almost half
the embryos with mosaicism.
The overall experience of using PGD for chromosomal
disorders presently exceeds 3000 cases, confirming the positive
impact on clinical outcome (Munné et al., 1999; Gianaroli et
al., 1999), although there is a need to further quantify its impact
in randomized controlled studies, which are currently in
progress. Preliminary data from these studies confirm a
previously suggested positive impact on implantation rate,
which was found to double as a result of the pre-selection of
chromosomally normal embryos for transfer (Gianaroli et al.,
2002a; Staessen et al., 2002). It is obvious, however, that the
improvement of the outcome of PGD may be expected only
when the number of embryos biopsied is equal to or higher than
the number of embryos expected to be replaced without PGD
(Munné et al., 2002c). A clinical impact of aneuploidy testing,
in terms of improved pregnancy and implantation rates, as well
the improved outcome of pregnancies through the reduction of
spontaneous abortions, has been further confirmed not only for
IVF patients with advanced maternal age, but also for other
poor prognosis patients, including those with repeated IVF
failures, repeated spontaneous abortions and altered karyotypes
(Gianaroli et al., 2002a).
The latter group includes the carriers of balanced
translocations, who have an extremely poor chance of having
an unaffected pregnancy. It is therefore not surprising that
PGD appeared to be highly efficient in assisting these couples
to have an unaffected pregnancy and to deliver a child free of
unbalanced translocation. More than 400 clinical cycles have
been performed at the present time, resulting in at least 100
clinical pregnancies and births of unaffected children. The data
are in agreement with previous reports, suggesting a
considerable reduction of spontaneous abortion rate in PGD
patients with balanced translocations (Munné et al., 2000a;
Munné, 2002; Verlinsky et al., 2002a).
Clinical outcome of PGD
Data on clinical outcome are presently available from the
single largest series of 1416 PGD embryo transfer cycles
performed in the Chicago centre (Verlinsky et al., 2002a;
Kuliev et al., 2002; Ginsberg et al., 2002) and from a sample
of 1670 PGD transfer cycles collected from 25 different
centres (ESHRE Preimplantation Genetic Diagnosis
Consortium, 2002). These transfers resulted in 338 (23.9%)
and 309 (18.5%) clinical pregnancies, respectively, and the
birth of 539 unaffected children overall (260 and 269,
respectively), with multiple pregnancies in approximately onethird of the cases. The overall congenital malformation rate
was 5.4% and 6.6%, respectively, which is not different from
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population prevalence, of which only 2.25% and 3.9%,
respectively, were attributable to the major abnormalities.
Overall, more than 4000 PGD cycles has presently been
performed, with further wider application not only in the USA
and Western Europe, but also in the Eastern Mediterranean and
Asian countries. For example, a network of six PGD centres
has recently been established in China and has already
performed 79 PGD cycles for different indications, resulting in
18 clinical pregnancies and the birth of 12 healthy children
(Zhuang, 2002). Also, increasingly PGD is being performed
for conditions that have never been subject to traditional
prenatal diagnosis, such as preimplantation gender
determination for social reasons, performed recently in India
(Malpani et al., 2001) and Jordan (Kilani and Haj Hassan,
2002). However, the majority of PGD cycles are still
performed for chromosomal disorders, with the ratio of PGD
cycles for chromosomal and single gene disorders
approximately 3:1, the latter group presently including lateonset disorders with genetic predisposition, the proportion of
which is gradually increasing. More than 1000 PGD cycles
have currently been performed for single gene disorders, with
the outcome data available for more than 200 clinical
pregnancies and births suggesting the acceptable accuracy of
PGD, which may be further improved by the above-mentioned
new developments in DNA analysis of single cells.
In PGD for chromosomal disorders, the indications are also
expanding, with further obvious interest in PGD for
translocations because of a strong impact of PGD on reducing
the spontaneous abortion rate in the carriers of balanced
translocations. Almost half these cycles has been performed by
the St Barnabas and Chicago centres (Fisher et al., 2002;
Verlinsky et al., 2002a), with an increasing number of other
centres, such as the Brussels (Van Assche et al., 2002),
SISMeR (Gianaroli et al., 2002b), London (Braude, 2002) and
Seoul (Lim et al., 2002) centres, performing a few dozen PGD
cycles for translocations. The available results demonstrate a
clear advantage of PGD over traditional prenatal diagnosis for
translocations, attributable to a poor meiotic outcome,
particularly in reciprocal translocations. As mentioned, the
accuracy of PGD for translocations has recently been
improved by the introduction of blastomere nucleus
conversion to metaphase, which will allow a reliable testing
for any complex chromosomal rearrangement. It is also true
that the testing for translocations has to be considered in
combination with aneuploidy analysis, even for the
chromosomes not involved in the structural rearrangement, as
there is presently evidence for considering chromosomal
translocations as a risk factor for aneuploidy. This may in
future be performed by PCR-based methods, which will soon
provide the opportunity for simultaneous testing for single
gene disorders, translocations and aneuploidies in the same
biopsied cell.
embryos to only two, and even to only one in blastocyst
transfer, in an attempt to avoid complications due to multiple
pregnancies, the pre-selection of the chromosomally normal
embryos will soon become standard IVF practice; there is no
way of deliberately transferring untested embryos, more than
50% of which are aneuploid, definitely compromising the
outcome of IVF in a traditional setting. In the meantime,
information on the availability of such options should be
provided at least to poor prognosis IVF patients, so they can
have the opportunity of improving their chances of becoming
pregnant and avoiding the establishment of a pregnancy
destined to be lost due to chromosomal aneuploidy. The
clinical outcome is presently available in approximately 700
clinical pregnancies resulting from PGD for aneuploidies,
supporting the above-mentioned observations on the positive
impact of PGD on implantation and pregnancy rates and the
improvement of pregnancy outcomes.
In conclusion, PGD has become a practical tool for assisted
reproduction and genetic services, providing an important
option for couples with genetic risk to avoid the birth of an
affected child and have a healthy child of their own. In
addition, the application of the technique has been extended to
non-disease testing, such as gender determination for social
reasons and the pre-selection of HLA-compatible donor
progeny. Although preimplantation testing was never intended
to be applied without genetic diagnosis, the fact that thousands
of individuals in need of haemopoietic stem cell
transplantation still fail to find an HLA-matched donor for
years, with many of them dying before they can find a match,
led to the application of preimplantation HLA matching as a
primary indication. This is highly relevant for those couples
that have an affected child with bone marrow diseases and
cancers, requiring cord blood or bone marrow transplantation.
The strategy would clearly not be clinically acceptable through
traditional prenatal diagnosis because of a possible clinical
pregnancy termination after HLA matching. But these couples
can now initiate an HLA-matched pregnancy from the outset
with the intention of treating their existing child for whom no
other treatment is available. Despite the need for further
ethical considerations, preimplantation non-disease testing is
clearly an important option for couples with an affected child
in need of an HLA-matched bone marrow transplantation. This
is obvious from the current increase of the acceptance of nondisease preimplantation testing by couples who are not at
genetic risk but who would like to combine their planning for
the next child with pre-selection of HLA-compatible progeny
to be able to treat their affected child in need of transplantation
therapy. These future applications will require a careful ethical
consideration (Edwards, 2002).
The next (Fifth) International Symposium on Preimplantation
Genetics will be held in Antalya, Turkey, on June 5–7, 2003.
References
298
Finally, there is further evidence to support the clinical
usefulness of PGD for assisted reproduction, as more than half
the preimplantation embryos are chromosomally abnormal
from the outset and have to be excluded from transfer in IVF
patients of advanced maternal age. It is evident that the further
improvement of IVF efficiency will be unrealistic without the
pre-selection of aneuploidy-free oocytes and embryos. With
the current tendency for limiting the number of transferred
Assady S, Maor G, Amit M et al. 2001 Insulin production by human
embryonic stem cells. Diabetes 50, 1691–1696.
Braude P 2002 Strategies and outcomes of the first 100 cycles of
PGD for serious familial genetic conditions. Reproductive
BioMedicine Online 4 (suppl. 2), 37 (abstr.).
Cohen J, Malter H 2002 Cytoplasmic abnormalities. Reproductive
BioMedicine Online 4 (suppl. 2), 11 (abstr.).
Edwards R 2002 Social and ethical issues on PGD, cloning and gene
Articles - Current features of PGD - A Kuliev, Y Verlinsky
therapy. Reproductive BioMedicine Online 4 (suppl. 2), 36–37
(abstr.).
ESHRE Preimplantation Genetic Diagnosis Consortium. 2002 Data
collection III, May 2002. Human Reproduction 17, 233–246.
Fisher J, Escudero T, Chen S et al. 2002 Obstetric outcome of 100
cycles of PGD of translocations and other structural
abnormalities. Reproductive BioMedicine Online 4 (suppl. 2), 26
(abstr.).
Galat V, Verlinsky Y 2002 Haploidization of somatic cell nuclei by
cytoplasm of human oocytes. Reproductive BioMedicine Online 4
(suppl. 2), 48–49 (abstr.).
Gianaroli L, Magli MC, Ferraretti AP, Munné S 1999
Preimplantation diagnosis for aneuploidies in patients undergoing
in vitro fertilization with poor prognosis: identification of the
categories for which it should be proposed. Fertility and Sterility
72, 837–844.
Gianaroli L, Magli MC, Ferraretti AP 2002a Preimplantation genetic
diagnosis for IVF patients with a poor prognosis. Reproductive
BioMedicine Online 4 (suppl. 2), 25 (abstr.).
Gianaroli L, Magli MC, Ferraretti AP et al. 2002b Robertsonian and
reciprocal translocations. Reproductive BioMedicine Online 4
(suppl. 2), 26–27 (abstr.).
Ginsberg N, Cieslak J, Rechitsky S et al. 2002 Reproductive
BioMedicine Online 4 (suppl. 2), 31 (abstr.).
International Working Group on Preimplantation Genetics 2001
Preimplantation genetic diagnosis – experience of 3000 clinical
cycles. Report of the 11th Annual Meeting of International
Working Group on Preimplantation Genetics, in association with
10th International Congress of Human Genetics, Vienna, May 15,
2001. Reproductive BioMedicine Online 3, 49–53.
Katz MG, Mansfield J, Gras L et al. 2002 Diagnosis of trisomy 21 in
preimplantation embryos by single-cell DNA fingerprinting.
Reproductive BioMedicine Online 4, 43–50.
Kaufman DS, Hanson ET, Lewis RL et al. 2001 Hematopoietic
colony-forming cells derived from human embryonic stem cells.
Proceedings of the National Academy of Sciences of the USA 98,
10716–10721.
Kilani Z, Haj Hassan L 2002 Sex selection and preimplantation
genetic diagnosis at the Farah Hospital. Reproductive
BioMedicine Online 4, 68–70.
Kuliev A, Cieslak J, Ilkevitch Y, Verlinsky Y 2002 Nuclear
abnormalities in series of 6733 human oocytes. Reproductive
BioMedicine Online 4 (suppl. 2), 11 (abstr.).
Lacham-Kaplan O, Daniels R, Trounson A 2001 Fertilization of
mouse oocytes using somatic cells as male germ cells.
Reproductive BioMedicine Online 3, 205–211.
Lim CK, Min JH, Song GJ et al. 2002 Reliability of PGD with FISH
analysis in reciprocal or Robertsonian translocation carriers.
Reproductive BioMedicine Online 4 (suppl. 2), 29 (abstr.).
Malmgren H, Sahlén S, Inzunza J 2002 Single cell CGH analysis of
human preimplantation embryos from PGD patients with
balanced structural chromosome aberrations. Reproductive
BioMedicine Online 4 (suppl. 2), 14–15 (abstr.).
Malpani A, Malpani A, Modi D 2001 The use of preimplantation
genetic diagnosis in sex selection for family balancing in India.
Reproductive BioMedicine Online 4, 16–20.
Martin R, Shil Q, Field LL, Rademaker A 2002 Reduced
recombination and non-disjunction in human spermatozoa.
Reproductive BioMedicine Online 4 (suppl. 2), 12 (abstr.).
Monk M, Holding C, Goto T 2002 Embryo-specific gene expression
in human preimplantation development and cancer. Reproductive
BioMedicine Online 4, (suppl. 2), 12 (abstr.).
Munné S 2002 Preimplantation genetic diagnosis of numerical and
structural chromosome abnormalities. Reproductive BioMedicine
Online 4, 183–196.
Munné S, Magli C, Cohen J et al. 1999 Positive outcome after
preimplantation diagnosis of aneuploidy in human embryos.
Human Reproduction 14, 2191–2199.
Munné S, Sandalinas M, Escudero T et al. 2000a Outcome of
preimplantation genetic diagnosis of translocations. Fertility and
Sterility 73, 1209–1218.
Munné S, Sandalinas M, Escudero T et al. 2002b Some mosaic types
increase with maternal age. Reproductive BioMedicine Online 4
(suppl. 2), 19 (abstr.).
Munné S, Cohen J, Sable D 2002c Preimplantation genetic diagnosis
for advanced maternal age and other indications. Fertility and
Sterility 78, 234–236.
Ozen S, Rechitsky S, Verlinsky Y 2002 Optimization of highresolution single cell comparative genomic hybridization to fit in
PGD framework. Reproductive BioMedicine Online 4 (suppl. 2),
44 (abstr.).
Rechitsky S, Verlinsky O, Chistokhina A et al. 2002 Preimplantation
genetic diagnosis for cancer predisposition. Reproductive
BioMedicine Online 5, 148–155.
Sermon K 2002 Preimplantation genetic diagnosis for dynamic
mutations. Reproductive BioMedicine Online 4 (suppl. 2), 24
(abstr.).
Silber S, Sadowy S, Lehahan K et al. 2002 High rate of chromosome
mosaicism but not aneuploidy in embryos from karyotypically
normal men requiring TESE. Reproductive BioMedicine Online 4
(suppl. 2), 20 (abstr.).
Simpson JL 2002 PGD as part of pre-pregnancy genetic screening.
Reproductive BioMedicine Online 4 (suppl. 2), 10 (abstr.).
Staessen C, Van Assche E, Platteau P et al. 2002 Ongoing
implantation rate after blastocyst transfer with or without PGD
for aneuploidy screening in women over 37 years (a randomized
controlled trial). Reproductive BioMedicine Online 4 (suppl. 2),
18 (abstr.).
Tease C, Hartshorne G, Hulten MA 2002 Patterns of meiotic
recombination in human fetal oocytes. American Journal of
Human Genetics 70, 1469–1479.
Tesarik J, Mendoza C 2002 Reconstruction of oocytes from patient’s
somatic cells. Reproductive BioMedicine Online 4 (suppl. 2), 22
(abstr.).
Tesarik J, Nagy ZP, Sousa M et al. 2001 Fertilizable oocytes
reconstructed from patient’s somatic cell nuclei and donor
ooplasts. Reproductive BioMedicine Online 2, 160–164.
Trounson AO 2002 Embryonic stem cells. Reproductive BioMedicine
Online 4 (suppl. 2), 23 (abstr.).
Trounson A, Pera M 2001 Human embryonic stem cells. Fertility
and Sterility 76, 660–661.
Van Assche E, Staessen C, Ogur G et al. 2002. PGD for reciprocal
and Robertsonian translocations in 41 treatment cycles.
Reproductive BioMedicine Online 4 (suppl. 2), 27 (abstr.).
Verlinsky Y, Rechitsky S, Verlinsky O et al. 2001a Preimplantation
diagnosis for p53 tumour suppressor gene mutations.
Reproductive BioMedicine Online 2, 102–105.
Verlinsky Y, Rechitsky S, Schoolcraft W et al. 2001b Preimplantation
diagnosis for Fanconi anemia combined with HLA matching.
Journal of the American Medical Association 285, 3130–3133.
Verlinsky Y, Cieslak J, Evsikov S et al. 2002a Nuclear transfer for
full karyotyping and preimplantation diagnosis for translocations.
Reproductive BioMedicine Online 5, 300–305.
Verlinsky Y, Rechitsky S, Verlinsky O et al. 2002b Preimplantation
diagnosis for early onset Alzheimer disease caused by V717L
mutation. Journal of the American Medical Association 287,
1018–1021.
Wells D, Escudero T, Levy B et al. 2002 Comprehensive aneuploidy
screening in human IVF embryos using CGH: protocol
development and clinical application. Reproductive BioMedicine
Online 4 (suppl. 2), 14 (abstr.).
Wilton L, Williamson R, McBain J et al. 2001 Birth of healthy infant
after preimplantation confirmation of euploidy by comparative
genomic hybridization. New England Journal of Medicine 345,
1537–1541.
Wilton L, Williamson R, McBain J et al. 2002 Preimplantation
diagnosis of aneuploidy using comparative genomic
hybridization. Reproductive BioMedicine Online 4 (suppl. 2),
13–14 (abstr.).
Zhuang GL 2002 PGD: China’s experience. Reproductive
BioMedicine Online 4 (suppl. 2), 39 (abstr.).
299