PREIMPLANTATION
GENETIC DIAGNOSIS (PGD) FOR ANEUPLOIDY
Preimplantation
genetic diagnosis (PGD) is a state of the art procedure in which
the embryo is tested for certain conditions prior to being placed
in the womb of the woman. The PGD team at Reprogenetics is a world
leader in this technique. This information provides one with an
overview of the PGD process as well as information specific to our
team here at the Institute.
CHROMOSOME ANEUPLOIDY
Chromosomes are string-like structures found
in the center of the cell, the nucleus. Chromosomes contain genes
that are made of DNA. Therefore, our inherited information is housed
on the chromosomes. Normal human cells (embryo, fetus, baby or adult)
contain 46 chromosomes or 23 pairs. We receive 23 chromosomes from
each parent. The first 22 pairs of chromosomes are the same for
men and women and labeled largest to smallest 1 through 22. The
23rd pair determines our sex. A female has 2 “X” chromosomes
whereas a male has an “X” and a “Y”. As
such, the woman can only pass an X to her child in her egg. The
man passes either the X or the Y in the sperm therefore determining
the sex of the child. If an error occurs leading to the egg or sperm
having an extra or missing chromosome, the embryo created by that
egg or sperm would have an extra or missing chromosome. This situation
is called aneuploidy. Having an extra chromosome is known as trisomy
(tri = three of the chromosome) and having a chromosome missing
is known as monosomy (mono = one of the chromosome). If the aneuploidy
involves the larger chromosomes, the embryo may not attach to the
wall of the uterus or may stop developing soon after attaching and
miscarry. However, if the aneuploidy involves chromosomes such as
the 13, 18, 21, X or Y, the pregnancy may still carry on until birth,
even though the pregnancy has a chromosomal disorder. The most common
of these is an extra number 21, known as Down syndrome or trisomy
21 (three 21 chromosomes). Other common aneuploidies are Klinefelter
syndrome (XXY), trisomy 13 and trisomy 18. The features of the chromosome
condition depend upon which chromosome is extra or missing, but
can include physical differences and mental retardation.
RISK OF ANEUPLOIDY AND MATERNAL
AGE
As a woman advances in age, the chance of aneuploidy in her pregnancies
increases. This association is because a woman’s eggs are
as old as she. Females have all of their eggs in the fetal stage
therefore they are born with all the eggs they will have in their
lifetime. In males, sperm is made every 65-75 days therefore the
sperm is not as old as the man. Therefore, the theory regarding
aneuploidy risk and advancing maternal age is that over time the
chromosomes in the egg are less likely to divide properly leading
to the egg having an extra or missing chromosome. The risk of aneuploidy
increases with maternal age. The chances to deliver an affected
child are 1/385 at 30, 1/179 at 35, 1/63 at 40 and 1/19 at the age
of 45. However, the frequency of aneuploidy in embryos is much higher
than what would be expected looking only at affected live borns.
More than 20% of embryos from women in the age range from 35 to
39 are affected. Almost 40% of embryos from women 40 or older are
affected. This difference in percentages in embryos versus live
borns is due to the fact that a pregnancy with aneuploidy is less
likely to attach to the uterus or go to term. Most will be miscarried.
As such, the percentage of affected pregnancies is reduced over
the course of the pregnancy due to the affected pregnancies that
are lost. Any embryo with a missing chromosome (monosomy) will cease
to grow before implantation (except monosomy X and 21), and only
few of those carrying an extra chromosome (trisomy) will go to term.
The lack of implantation and loss rate of aneuploid embryos are
believed to be the main reasons why the pregnancy rate in women
over 40 is so low. The purpose of preimplantation genetic diagnosis
for aneuploidy therefore is to select for transfer only chromosomally
normal embryos so as to achieve more pregnancies, reduce the number
of pregnancy losses, and reduce the number of affected offspring.
PREIMPLANTATION GENETIC DIAGNOSIS
(PGD)
To analyze an egg or embryo, we first have
to biopsy it. Two procedures allow this to be done. The PGD team
of doctors, geneticists and embryologists will decide which procedure
to use.
BIOPSY OF POLAR BODIES
The ripening egg produces two small cells
called polar bodies that degenerate after fertilization. The chromosomal
content of these cells allows us to infer the chromosomal content
of the egg. If one is testing the polar body, an opening is made
in the covering of the egg and the polar body is removed with a
pipette. The polar body is then analyzed while the egg is placed
in an incubator. By analyzing polar bodies, we obtain information
from only the mother. Chromosome abnormalities that may occur after
fertilization, when the sperm meets the egg, will not be detected.
BIOPSY OF BLASTOMERES
A blastomere is a cell from an embryo. To
test the blastomere, an opening is made in the covering of the embryo
during its third day of development when the embryo has 8-10 cells.
A blastomere is removed via aspiration with a pipette. The embryo
is placed in an incubator while the cell is analyzed.
ANALYSIS
The biopsied cells are analyzed using a technique
called fluorescence in-situ hybridization or FISH. This technique
uses probes, small pieces of DNA that are a match for the chromosomes
we want to analyze, to count the chromosomes present. Each probe
is labeled with a different fluorescent dye. These fluorescent probes
are applied to the biopsied cell and attach to the chromosomes.
Under a fluorescent microscope, we then count the number of chromosomes
of each type (color) there are in that cell. The geneticist therefore
can distinguish normal cells from cells with aneuploidy. Testing
of the cells destroys them because they must be glued to a glass
slide and repeatedly heated and cooled. As such, one cannot use
them for another purpose or return them to the embryo. The slides
are kept for future reference. This analysis causes no extra inconvenience
to the patient as it is accomplished in one day.
ADVANTAGES
OF PREIMPLANTATION GENETIC DIAGNOSIS
REDUCTION IN THE CHANCE OF HAVING A CHILD WITH
ANEUPLOIDY
According to current figures, the chance for
a woman delivering a baby with aneuploidy is on average 1% if she
is 35-39 years of age and ~3.5% if she is 40-45. So far, in 427
conceptions obtained through PGD, two (0.5%) were chromosomally
abnormal compared to 2.8% expected for that group of patients according
to their maternal ages (data collected up to 8/2003). PGD does significantly
lower the chance of having an affected baby. However, we are unable
to test all of the chromosomes at present. We therefore recommend
that prenatal testing be performed in the resultant pregnancy via
chorionic villous sampling or amniocentesis in order to confirm
our diagnosis from PGD and to rule out other aneuploidies for which
we do not test.
INCREASED IMPLANTATION RATE
It is well known that the pregnancy rate after
in-vitro fertilization decreases dramatically with maternal age.
Even in IVF centers with the highest pregnancy rates, there is a
decrease from approximately 28% per embryo transferred in women
20-33 years old to 9% in women over 39. Aneuploid embryos have much
lower survival rates than normal embryos, and half of them (the
ones missing a chromosome) seldom implant. It appears likely that
the decrease in pregnancy rates with maternal age is mostly caused
by a corresponding increase in aneuploid embryos.
By performing PGD
for aneuploidy and transferring only chromosomally normal embryos,
we may be able to increase the pregnancy rates noticeably. In three
studies, we have demonstrated an increase in implantation rates
after PGD (Munné et al. 1999, Gianaroli et al. 1999b, Munné
et al. 2003, in reference list). We have also found that the implantation
rate doubled from 12% in controls to 24% in PGD patients when we
analyzed 8 chromosomes but not when only 5 chromosomes were analyzed.
Thus is very important that the PGD test analyzes at least 8 chromosome
pairs, as it is done by Reprogenetics.
REDUCTION IN PREGNANCY LOSSES
In women 35 and older, approximately 35% of
pregnancies are miscarried. Aneuploidy is the cause in 50% or more
of these losses. By transferring only chromosomally normal embryos,
the number of pregnancies going to term should increase. In one
of our studies, we detected a significant reduction in pregnancy
losses after PGD, from 23% to 9%. The increase in implantation rate
and the significant decrease in pregnancy loss rate resulted in
a significant increase in ongoing pregnancies and delivered babies.
ISSUES OF PREIMPLANTATION GENETIC
DIAGNOSIS
PGD is not void of risks but these appear
to be outweighed by the benefits described above.
THE RISK OF EMBRYO BIOPSY
While PGD is a relatively new procedure in
IVF, the micromanipulation or biopsy techniques required to perform
the procedure have been in use for many years. The risk of accidental
damage to an embryo during removal of the cell(s) is very low --
0.6%. Procedures such as intra-cytoplasmic sperm injection (ICSI),
fragment removal and assisted hatching are all performed by making
openings in the covering of the egg and none have been found to
have other than mostly positive effects on embryo development and
implantation.
REMOVAL OF CELLS FROM THE EMBRYO
No part of the future fetus will be lacking
because one or two cells are removed from the embryo approximately
two days after fertilization. All the cells of the embryo remain
totipotent until about the fourth day. Totipotent means having “all
potential”. These cells have not differentiated yet meaning
that each cell by itself can grow into a whole and perfect fetus.
The procedure merely delays continued cell division for a few hours,
after which the embryo reaches the same number of cells as before
and continues its normal development. Normal development has been
seen many times in humans and other mammals after cell loss due
to embryo freezing. One or more cells may fail to survive thawing,
yet the embryos from such develop into normal offspring. An unanswered
question is whether biopsied embryos implant less than untouched
ones. Data regarding such is incomplete. Embryo biopsy may lower
implantation rates slightly while selection of chromosomally normal
embryos via PGD may increase it. The balance between potential biopsy
damage and beneficial effects of PGD seems to be positive.
MISDIAGNOSIS
The accuracy of PGD for aneuploidy is approximately
90%. This means that the error rate is 10%. Within this chance of
misdiagnosis, there is a false negative rate, a false positive rate,
the chance for no result and the chance for mosaicism. A mosaicism
is defined as the embryo having cells with different chromosome
make-up. Typically, all cells of the embryo have the same chromosomal
make-up as they originate from the same fertilized egg. However,
it is possible for cells of the same embryo to have differing numbers
of chromosomes. If we analyze a cell that has normal chromosomal
content, but another cell has an extra chromosome, we erroneously
diagnosed that embryo as being chromosomally normal. Due to the
chance of misdiagnosis as well as the presence of anueploidy for
which we do not test, we recommend prenatal testing as stated earlier.
CASES PERFORMED TO DATE
The preimplantation genetic diagnosis team
of Reprogenetics has been involved in 5500 cases of PGD for aneuploidy
up to February, 2006. These procedures were performed from samples sent to Reprogenetics from over 150 in vitro fertilization clinics in the US.
COST OF THE PROCEDURE
Please inquire at your Fertility Center as
to the current fees for PGD. The PGD fees are in addition to the
cost of in-vitro fertilization (IVF) and embryo transfer. The PGD
fees include the cost of the DNA probes, FISH analysis and the biopsy
procedure. Insurance companies seldom cover the cost of PGD.
Patients may have their fee waived if they enroll in one of the
ongoing randomized trials (see studies), but only 50% of those patients
will have PGD performed, while the others will be controls.
PERFORMANCE OF THE PGD PROCEDURE
Dr. Santiago Munné,
PGD Program Director, and Dr. Jacques Cohen, Scientific Director, have been involved in PGD since the start of the technique. Their work on PGD for aneuploidy has been published in >150 scientific articles, a partial list of which is provided (see publications). They have obtained multiple awards for their research, including the Prize Paper of the Society for Assisted Reproductive Technology in the 1994, 1995, 1998, and 2005 annual Meetings, the General Program Prize at the American Society for Reproductive Medicine (ASRM) in 1996. Samples for PGD are processed in our Laboratories in NJ and CA.
PGD FOLLOW-UP PROGRAM
All patients who achieve pregnancy after IVF
with PGD are asked to participate in our follow-up program. Information
regarding pregnancy, pregnancy outcome and child development will
be gathered.
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