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PGD Preimplantation Genetic Diagnosis- The Process
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PGD was first employed in 1989 with
the subsequent birth of normal females to couples at risk
of various X-linked recessive diseases. The number of
diseases potentially diagnosable by genetic screening is vast.
Examples of such genetic disorders that can be screen using PGD include:
chromosomal translocations, Down syndrome, Turner syndrome,
DiGeorge syndrome, alfa-1- antitrypsin deficiency, beta-
thalassemia, Charcot-Marie-Tooth disease, cystic fibrosis,
Fancony anemia, fragile X syndrome, hemophilia A, Huntington
disease, Lesch-Nyhan disease, Marfan's syndrome, myotonic
dystrophy, sickle cell anemia, and Tay-Sachs disease.
PGD has resulted in hundreds of normal
births from parents at risk for transmitting genetic diseases.
Selective implantation of embryos with normal chromosome
compliments has been shown to result in high pregnancy
rates with decreased spontaneous miscarriage rates.
PGD- Detailed Description
PGD involves several steps: genetic counseling,
reproductive counseling, in vitro fertilization (for more detail about IVF visit our IVF Web site ),
and a genetic laboratory with preimplantation genetic diagnosis capabilities. The laboratory personnel must be familiar with DNA technologies such
as fluorescence in-situ hybridization (FISH) for sex determination
and screening for chromosomal abnormalities and performing the polymerase
chain reaction (PCR) for single gene diseases.
Preimplantation genetic diagnosis often involves the following:
- Intracytoplasmic sperm injection
(IVF/ICSI) if indicated
- Microsurgical removal of one or two blastomeres
(embryos) at the six- to eight- cell stage usually three
days after fertilization
- Molecular (by PCR), in case
of single gene diseases, or molecular cytogenetic analysis
- FISH ( fluorescence in situ hybridization) in cases
of chromosome abnormalities
- Studies of the biopsied
cells
- Uterine transfer of unaffected embryos
Blastomere Biopsy
Single cells from a preimplantation
embryo can be removed and genetically tested in a procedure
called blastomere biopsy. Typically embryos are biopsied
on day 3 following egg retrieval following the first
three cleavage divisions (containing from 6 to 8 cells
or blastomeres), although some researchers have performed
biopsies of blastocysts containing 120 cells.
At both
of these stages, the cells of the embryo have not differentiated
into particular body tissues and
there is no damage to the resulting embryo. Biopsies of
embryos, or blastocysts, may be analyzed in a variety
of ways that can detect genetic abnormalities arising
from the maternal or paternal chromosomes.
PGD requires genetic material from the embryo or the polar body when the disease is transmitted by the mother. The polar
body is a small section of an egg that contains the complementary
set of chromosomes present in the oocyte. Therefore,
the genotype (chromosome number, arrangement, etc. ) of the oocyte can be determined by examining
the polar body.
The first polar body of an egg is extruded prior to egg retrieval and thus
before fertilization. This polar body is not necessary
for complete embryonic development and is available
for analysis. A second polar body is extruded at the
time of oocyte fertilization by a sperm. These polar
bodies can be a valuable
source of genetic information.
By using PGD FISH,
with fluorescent-tagged genetic
probes, we can examine the polar body, thus allowing the chromosomal make-up
of the oocyte to be inferred. Studies have shown that
the majority of embryo aneuploids (85%) are due to the
female oocyte. The remainder is of sperm origin.
Large chromosomal abnormalities,
such as extra or missing chromosomes (aneuploidies),
gender determination, and unbalanced chromosomal translocations,
resulting from a parental balanced translocation, can
be detected by a laboratory procedure called fluorescence
in situ hybridization (FISH).
Using this technique, DNA
probes are labeled with colored fluorescent tags that
light up so one can see specific chromosomes, or genes,
under a microscope. The reagents are optimized for use
with imaging software for probe-signal enumeration.
This software allows the simultaneous analysis of up
to 12 different target-specific fluorophores in a single
cell. However, up until now only 9 chromosomes can be
accurately assessed during one analysis using FISH with
up to a 10% error rate.
In cases involving more subtle abnormalities,
on the scale of single genes or even DNA bases or single
gene diseases, highly specialized techniques such as
PCR are required. Such methods rely on the fundamental
principles of the genetic code, and specifically on
the cell's ability to generate a matching, or complementary
segment of DNA.
Structurally, DNA is composed of two
single strands attached to each other to form a double
helix. The bases of one strand always bind to the bases
(A, T, G &C) of the other in a specific fashion:
A pairs with T, and G with C. If one knows the sequence
of the bases in one strand, one can deduce the complementary
sequence of bases in the other strand. Based on a known
sequence of DNA, a synthetic copy of the matching strand
called a DNA probe is created, it will then bind, or
hybridize to that specific gene within a chromosome.
The mutation in the carrier parent(s) needs to be characterized
before preimplantation genetic diagnosis is applied.
PGD Results
PGD results are usually available within 48 hours after blastomere
biopsy, which corresponds to day 5 following egg retrieval.
Depending on their original quality, embryos may, or
may not, reach the blastocyst stage, which is the final stage
of in vitro development. Usually on day 5, embryos
free of genetic defects are transferred into
the patient.
Both the FISH and PCR procedures typically
take 24-48 hours to complete. However, since diagnostic
tests are performed on a single cell, the possibility
of misdiagnosis has to be considered. There are limitations
of the test procedures, e.g. allele dropout in PCR,
either non- specific or inefficient hybridization in
FISH. New techniques like comparative genomic hybridization
(CGH) offer the possibility to analyze all 23 pairs
of chromosomes simultaneously for aneuploidy, translocations
and single gene defects.
Unfortunately, this technology is
not clinically useful due to the time it takes to generate
the results. It currently takes 4-5 days for the results
to be obtained using CGH. This requires the biopsied
embryos to be cryopreserved after biopsy to allow time
for the analysis. There is a single report in the literature
that has accomplished this approach successfully. Another
technique that is emerging, that may have application
to preimplantation genetic diagnosis is Gene Chip technology where literally thousands
of DNA sequences are analyzed simultaneously.
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