|Preimplantation Genetic Diagnosis (PGD)|
Genetic Diagnosis (PGD)
With recent advances in genetics, there are several inherited disorders
which can now be diagnosed at a molecular level. For couples who are carriers or
affected by any of these conditions and are at high risk for transmitting it to
their offspring, it is currently possible to detect the disorder during
pregnancy. This is done by one of two approaches: amniocentesis or chorionic
villus sampling. However the couples have the dilemma of whether or not to
terminate the pregnancy if the genetic abnormality is present. In some cases
this may also not be a viable option for religious or moral reasons.
An alternative would then be to diagnose the condition in embryos before the
pregnancy is established. Only the unaffected embryos would then be transferred
to the uterus. This technique is referred to as Preimplantation Genetic
Genetic Diagnosis (PGD) is, presently, a valid alternative for families
at a high risk for producing offspring with genetic disorders and to those who
wish to avoid elective pregnancy termination or to prevent the birth of an
affected child following prenatal diagnosis. PGD allows
genetic analysis to be performed on early embryos prior to implantation
and pregnancy. This provides couples at risk the opportunity to know that any
pregnancy they achieve should be unaffected and obviates the need for screening
during a pregnancy and hence prevent the physical and psychological trauma, and
ethical-moral problems associated with possible termination.
Technical advances in molecular genetics now enable physicians and scientists to be able to diagnose some inherited genetic or chromosomal disorders from a single cell of an early embryo. The information gained by PGD is used to select for replacement in the uterus only those embryos considered unlikely to be affected by the specific genetic disorder for which testing is performed.
who have PGD will undergo an in vitro fertilization (IVF) cycle for the purpose of creating
embryos from the woman's eggs and man's sperm which will have genetic testing
prior to replacement into the woman's uterus. The genetic material of the
embryos (which is derived from both parents) is not altered in any way during a
PGD cycle, and early embryological development is similar to natural conception,
except that it occurs in the laboratory.
that show normal development are biopsied with micromanipulation techniques to
obtain sufficient cells (blastomeres) for analysis. The cells removed from each
individual embryo are analyzed by genetic testing using PCR-based DNA
amplification. Those embryos considered to be unaffected on the basis of this
testing will then be available to be transferred into the woman's uterus.
intrinsic difficulties of this diagnostic approach (single cell DNA analysis,
response time of 24 hours, the possibility of Allele Drop-Out - ADO),
require the use of extremely sensitive and automated techniques that provide
unambiguous and reliable results. In performing of PGD cases, “Genoma”
Laboratory uses the most modern and technologically advanced instruments
presently available and employs sophisticated and innovative analytical
procedures. Avant-garde technologies and robotic solutions applied to the entire
analytic process contribute to raise the quality of the analysis, guaranteeing
maximum reliability of the results.
characteristics have enabled “Genoma” to become one of the most highly
qualified European laboratory in the field of preimplantation genetic diagnosis,
a reference center for various national and international IVF centers.
date, “Genoma” Laboratory has performed over 80 PGD cases, collaborating
intensely with prestigious international institutes.
activity of the center results in the publication of different manuscript in
prestigious specialized journals as well as in oral communications in different
meeting, among which the 3rd and 4th International
Symposiums on Preimplantation Genetics.
Genetic Diagnostic procedures have been attempted by GENOMA for various single
gene disorders including:
and Development Section
of “Genoma” Laboratory is able to set-up and perform PGD for any genetic
disorders with an identifiable mutation.
the pathologies that can be diagnosed by PGD DNA analysis?
all genetically inherited conditions that are diagnosed in the prenatal period
can also be detected in the preimplantation period. Diseases which have a high
risk of transmission (25-50%) and are usually associated with significant
morbidity and mortality can be screened for by this technique.
is possible to perform PGD for any genetic disorders, autosomal dominant,
recessive or X-linked, with an identifiable mutation. For pathologies caused by
expansions of nucleotidic triplettes (such as Fragile X, Huntington's disease,
Myotonic Dystrophy, etc.) it is possible to obtain only information on the
absence of triplette expansion. Whether or not to undergo examination must
therefore be evaluated case-by-case. Below
is a table listing of the most frequent genetic diseases that can be diagnosed
Genetic diseases transmittable to offspring that can be analyzed by genetic
diagnosis after biopsy of the ovocytes and embryos:
is PGD carried out?
involves genetic testing of the oocytes and/or embryos obtained by undergoing
IVF. IVF is an assisted reproductive procedure where fertilization
of the egg occurs outside of the body in a controlled setting.
The oocyte (egg) is removed from the woman’s ovary and is placed with
the male’s sperm. If the sperm
fertilizes the egg, the fertilized egg (zygote) begins to divide. The genetic
status of the embryo(s) can be determined before the embryo(s) is/are
transferred into the uterus.
following procedures are necessary when undergoing PGD:
step involves stimulation of the ovaries for ovulation. Ovulation is the
release of a mature egg from its follicle in the ovary. Most patients who
undergo cycles of assisted conception do not have ovulation problems. The aim
of stimulation is to induce, in the chosen cycle of assisted reproduction, the
simultaneous maturation of several follicles in order to have many ovocytes
available and possibly many embryos to transfer. In the IVF process, mature
eggs must be retrieved before they are ovulated.
Medication is used during a woman’s cycle in order to stimulate the
eggs to mature at the correct time. Ultrasound control of the ovary enables us
to know when the eggs are fully mature.
the oocytes are ready to be retrieved, another vaginal ultrasound similar to
those performed to monitor follicular development is performed. A small needle
inserted in a guide attached to the ultrasonic probe is directed through the
vaginal wall into the ovarian follicles. The needle is connected to a suction
pump and the fluid from each accessible ovarian follicle within the ovary is
Fertilization and Embryo Culture
A semen sample is provided to
the laboratory on the day of the egg retrieval. The sample is then processed in order to obtain an optimum sample for
fertilization. In most cases, a
single sperm is injected into the egg by a procedure known as intracytoplasmic
sperm injection or ICSI. At
this time, a second polar body is released from the egg.
The eggs will be fertilized using ICSI to maximize the rate of
fertilization and to monitor the exact timing of polar body removal.
Occasionally, fertilization does not occur, or occurs abnormally (which occurs
to fertile couples as well).After fertilization has
taken place, the embryo is transferred to a special growth medium.
Polar body removal and/or blastomere biopsy
biopsy (also known as embryo biopsy) is a technique that is performed by removal
of one or two cells (blastomeres) from the 4 to 8 cell pre-embryo stage for the
purpose of preimplantation analysis. The egg will typically be fertilized by
using intracytoplasmic sperm injection or ICSI. Following fertilization, the
zygote begins to divide. On the
third day following the egg retrieval, the embryo is at the blastomere stage,
and a cell may be carefully removed for genetic analysis.
With the embryo maintained in position by a pipette with rounded margins,
an opening is performed in the
embryo by using a laser device or thyroid acid: Once realized the hole, a new
micropipette having a greater diameter than the first is positioned: this will
consent, by means of aspiration, the removal of a cell(s) that will be then be
released by applying negative pressure. At this early point of embryo
development, all of the cells are equivalent and thus, removal of a cell from
the embryo at this stage does not remove anything critical for normal
development. The embryo compensates
for the removed cell and should continue to divide following blastomere biopsy.
Therefore, if the technique is carried out correctly, there are no risks for the
removal of the cell(s) from the blastomere, the developing embryo is placed back
into the culture dish and the removed cell(s) is inserted into a test tube for
subsequent genetic analysis.
Polar Body Removal
indicated above, the first polar body is produced from the division of
the egg and can be removed and tested for its chromosome complement or to
identify whether it contains the abnormal gene of concern.
Upon penetration of the egg by the sperm (fertilization), but
prior to the joining of the
sperm’s genetic material with the egg’s genetic material, the egg undergoes
another cell division, producing two unequally sized cells.
The larger cell will join with the sperm’s genetic material to create
the pre-embryo, and the smaller cell is called the second polar body.
The polar bodies have no known function except to assist in cell division.
They are simply “by-products” of the egg’s division. Once
implantation occurs, the polar bodies disintegrate and are not part of the
testing the first and second polar bodies, the genetic make-up of the egg, and
maternal genetic contribution in the resultant embryo, can be determined.
Removal and genetic analysis of the polar bodies occurs on the first and second
day after aspiration. In some instances, it is necessary to confirm a diagnosis
made on polar body analysis by performing blastomere biopsy.
It is also possible that one or more polar bodies fail to provide a
conclusive result. In these
situations, it may be possible to perform blastomere biopsy (embryo
biopsy) for further genetic analysis.
Preimplantation Diagnosis of Single Gene Disorders in blastomeres
PGD for single gene disorders can be accomplished by either polar body removal
or blastomere biopsy. Which method
may be used is determined on a case-by-case basis.
This procedure can be used to select for embryos that do not have a
specific genetic disease by testing the polar bodies or blastomeres for the
the test tube containing the blastomere, a solution is added that permits cell
lysis and thus the liberation of DNA from the cell nucleus. Subsequently, by
means of in vitro enymatic
ampification known as the Polymerase Chain Reaction (PCR), the genetic region of
interest, involved in the mutations that is being searched for, is amplified
millions of times. The genic amplification product then undergoes mutation
analysis to search for genic mutions present in the couple. The analysis of
mutations is the most important and delicate phase of PGD. To guarantee maximum
interpretative reliability, it is indispensable that it be carried out with
methods and instruments that permit the univocal identification of the mutations
that are being searched for. Automated sequence analysis is presently the best
method of genetic analysis, inasmuch as it permits the exact determination and
direct visualization of a specific mutation. The application of this technique
to PGD is done by the use of completely automated state of the art equipment.
centers worldwide are concerned about the possibility of misdiagnosis, which can
occur as a result of failure of allele specific amplification, or allele dropout
(ADO). In basic terms,
this is the failure of one of the genes (allele) to show up in the analysis (it
“drops out” of the picture.) ADO is of concern primarily in blastomere biopsy
when each parent carries a different mutation for a recessive condition.
To minimize the potential for diagnostic error by PGD, it is preferable
to offer polar body removal or to perform a combined analysis of the specific
mutation and specific linked markers that are inherited along with the gene.
This dual amplification allows for higher accuracy and for the detection of ADO,
thus enhancing diagnostic accuracy and reducing the risk for misdiagnosis.
transfer and implantation
mutation analysis of the blastomeres has been completed, once an embryo is
predicted to be free from the genetic disease for which testing was performed,
embryos resulted without genetic anomalies are transferred to the patient.
Embryo transfer is a 15 minute procedure accomplished by inserting a catheter (preloaded with embryos) into the uterine cavity through the cervical canal. This procedure is often performed under ultrasound guidance.
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