INTRODUCTION
Preimplantation
Genetic Diagnosis (PGD) is an adjunct and possible alternative to
prenatal diagnosis consisting of analyzing embryos obtained
through IVF before they are transferred to the womb of the female.
As such, it has the advantage of not transferring abnormal
embryos. Abnormal pregnancies will not be conceived and the trauma
of having to decide to continue or end an abnormal pregnancy will
be eliminated.
Approximately 80%
of the PGD procedures performed are performed for the indication
of advanced maternal age (35 years or older). There is a decrease
in pregnancy rates with advancing maternal age with the decrease
being more dramatic as the age increases. Pregnancy rates
decrease from 45% at age 28 to 35% by age 35, to 20% by age 40,
and to close to 0% by age 44. As this trend can be reversed with
donation of eggs from a young woman, the problem is not a uterine
problem but an egg problem. Normally, humans have 46 or 23 pairs
of chromosomes. Chromosomes are the string-like structures of
genetic information in our cells. Any number of chromosomes other
than 46 is abnormal. Most genetic studies have detected a very
high rate of chromosome abnormality such as aneuploidy, an extra
or missing chromosome, in eggs and embryos. These rates can be
ten or more times greater than what is found in first trimester
pregnancies. These rates of aneuploidy increase with maternal age
at the same rate as the pregnancy rate decreases. Though some
chromosome conditions are compatible with life, such as Down
syndrome, many others are not. Many cause the embryo to stop
growing before it can even implant. This is why patients have
lower pregnancy rates as they age. In a given IVF cycle, a woman
30-34 years old will have aneuploidy in at least 30% of embryos, a
woman 35-39 will have at least 35%, and a woman 40-44 will have
aneuploidy in over 50% of embryos. This means that if a patient
produces 6 embryos in a cycle, only 4 or less may survive if she
is 30-39, and less than 3 if she is 40-44 years old. PGD may help
select the embryos that are normal thus giving better chances of
surviving and producing a pregnancy, thus ameliorating the effect
of female age in reproduction.
THE PROCEDURE
The most common
method of PGD is by the biopsy of a cell on the third day of
embryo culture, when it usually has 6-10 cells. An opening is made
in the zona pellucida, the shell of the embryo, and a cell is
gently removed. The cell is fixed to a glass slide in a process
that eliminates all but the chromosomes of the cell. Thus, the
cell cannot be used for anything else but to analyze the
chromosomes. Because we have only one cell to analyze, methods
usually applied to cells from procedures such as chorionic villous
sampling (CVS) or amniocentesis cannot be used. Instead, a
technique called fluorescence in-situ hybridization (FISH) is
utilized. 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. For instance the
probe for chromosome 21 is labeled in green. When the probes bind
to the chromosomes in the nucleus and we see three green dots
instead of two (normal), the cell, and that embryo, have trisomy
21 (Down syndrome). Unfortunately this technique cannot analyze
all chromosomes simultaneously. Therefore, we have chosen to test
for chromosomes that are more commonly seen in aneuploidy which
lead to lack of implantation, pregnancy loss or delivery of a
child with a condition like Down syndrome (chromosomes XY, 13, 15,
16, 17, 18, 21, 22).
The analysis of the
fixed cell takes about 8 hours. When the diagnosis is obtained,
those embryos classified as normal are transferred to the womb.
The major risk of this process is the embryo biopsy. Although
very few embryos (about 2 in a thousand) are damaged by the
biopsy, it is not known if it affects survival of the embryo. If
there is any detrimental effect of the biopsy, it is compensated
in excess by the positive effect of selection and transfer of
those embryos that are normal as implantation rates increase.
An alternative to
embryo biopsy is polar body biopsy. The polar bodies are sister
cells of the egg. By analyzing them we can infer the chromosome
content of the egg, without affecting the egg. A problem with
polar body biopsy is that 30% of the abnormalities detected in
embryos occur after fertilization and cannot be detected in polar
bodies.
The accuracy of PGD
is not 100%. This is because we can analyze only one cell per
embryo. We could analyze two cells from the embryo, but current
data indicate that this is detrimental for the embryo as the
implantation rate is reduced by half when this is done. Current
available information indicates that about 10% of embryos
diagnosed using either embryo biopsy or polar body biopsy, are
misdiagnosed. Still, this is a great improvement over transferring
embryos without any diagnosis knowing that one third to over half
of them are abnormal.
PGD is not a
procedure that every IVF center can perform. It requires a highly
trained team of personnel, with many PGD cycles of experience, and
expensive and sophisticated equipment. When choosing an IVF center
with a PGD program, choose one that has performed hundreds of
cycles or that uses a reference PGD center with this experience.
RESULTS
Increase in implantation rates
Several studies using embryo biopsy have shown an increase in
implantation after PGD. The first study by Gianaroli et al. (1999,
Fertility and Sterility) observed a significant two-fold increase
in implantation, from 10.2% to 22.5% in a group of patients with
an average maternal age of 36. In another study (Munne et al,
2003, Reproductive Biomedicine Online) a group of patients with
average maternal age of 40 years old, we detected a 20%
implantation rate in the PGD group compared to a 10% in the
control group. No reports on implantation rates comparing PGD and
controls have been published so far regarding polar body analysis.
Preliminary data from our group in a third study indicate that not
only the implantation rate increases, but also the pregnancy rate,
which jumped from 22% to 41% in a group of patients with an
average maternal age of 40.
Decrease in Spontaneous Abortions
The
objective of PGD is not only to improve pregnancy rates but also
to make sure that the pregnancy goes to term. Unfortunately, in
addition to lower implantation rates, women are more lose their
pregnancy the older they are, again, because most of these
pregnancies are chromosomally abnormal. For instance, women
younger than 35 will lose 13% of their pregnancies, but that
increases to 24% by age 38 and 40% by age 41.
By
using PGD we observed a decrease in spontaneous abortions from 23%
in the control group to 9% in the PGD group. Thus, PGD not only
helps increasing the chances of conceiving but also of keeping the
pregnancy.
Reduction in Percent of Aneuploid Offspring
So far, more than
2000 cases of PGD of aneuploidy (extra or missing chromosome) have
been performed, either using embryo biopsy or polar body biopsy.
Large numbers are needed to demonstrate a decrease in aneuploid
offspring, from the 2.7% aneuploidy for chromosomes 13, 18 or 21
detected in CVS in women 39 years old, to 0.3% after PGD (assuming
a 10% error rate). Indeed, misdiagnoses have already occurred
after PGD.
In spite of
these misdiagnoses, the rate of offspring with aneuploidy detected
after PGD is lower than expected without PGD. For instance, two of
427(0.5%) fetuses were found with aneuploidies for chromosomes XY,
13, 15, 16, 18, 21, and 22 after PGD compared to a 2.7% rate
expected in a population of the same age range. That is a
four-fold decrease of chromosomally abnormal conceptions after
PGD.
Reduction of
Multiples
Many infertile
patients think that having twins or multiples would be a bonus,
getting two or more babies for the price of one. Unfortunately,
multiple pregnancies have ten times more risk of miscarrying, the
babies have higher rates of congenital malformations than
singletons, and the rate of pregnancy complications is increased.
Without knowing the chromosomal make-up of embryos, a greater
number of embryos may be transferred to try to increase the
implantation rate. This can lead to a higher multiples rate. By
selecting embryos via PGD, less embryos are replaced on average.
In our latest study, an average of 2 embryos were replaced in the
in the PGD group compared to 4 in the non-PGD group. This resulted
in 46% rate of twin and multiple pregnancies in the non-PGD group
and only 17% in the PGD group.
CONCLUSION
PGD is an option
available to women of advanced maternal age who are seen in
various IVF centers worldwide. PGD of chromosome abnormalities
has been shown to reduce the risk of trisomic offspring, increase
implantation rates, and decrease spontaneous abortions. For the
purpose of increasing implantation, PGD is a selection tool to
identify embryos that are chromosomally normal. Though not
perfect, PGD may be a wonderful option to some couples.
