Transcript 1 - M5zn

Preimplantation genetic diagnosis
Maha M. El-hallaq
Master of Biotechnology
Tube Babies
1_ Intra - vaginal Culture
• Summed up this way that after collecting the eggs
are placed with sperm in a tube containing a special
material for the growth of embryos Culture Media,
then this tube is placed in the vagina and hold it in
place by a special payment. Still repayment and the
tube from the vagina after 24-48 hours, and examine
the components inside the tube to make sure
conceive. The fertilized embryo is transferred to the
2_Intra cytoplasmic sperm injection
• This method is ideal and favorite now a very
successful especially for men who complain of the
lack of sperm significantly. As well as when the type
of sperm is good. The main enter the sperm directly
into the cytoplasm of the cell and using sperm and
only one other than the way IVF that are put
thousands of sperm on the egg and are in this way
injection egg after removing cells prosecution her
fine needle very inject sperm into the egg any inside
Used in:
When there are too few sperm in the semen.
When fertilization fails in a way that IVF.
when the movement of sperm is weak .
Statistics have shown that the rate of cases in which
they conducted the operation successfully about
70% as statistics have shown that the eggs fertilized
in this way and transferred to the mother be a
success rate of live births in the same way IVF rate
and possibly more in some .
3_ In Vitro Fertilization
• Is to fertilize the egg with sperm in a test tube after
taking mature eggs from the ovary to be placed with
good sperm only after fertilization until it gets
washed. The fertilized egg then returned (embryos)
to the mother. This process takes from two - five days
and this method gives the best option to choose the
best embryos to be transferred to the mother after
fertilized outside the womb.
Who are the right couples are treated
in a manner IVF ??
Wife is capable of producing eggs and the pair
product of sperm.
Women who are Fallopian tubes have closed or
damaged so as not to allow the sperm to reach the
egg fertilized.
 The men who are infertile due to a lack of or low
sperm movement .
Women between 35-40 years to be able to get a
child where the probationary period in front of them
 Cases of infertility is not known why.
Men who have generated antibodies to the sperm
“Anti-sperm Antibody”.
 When one of the parents are carriers of a genetically
transmitted disease .
Preimplantation genetic diagnosis
It is the beginning of the end of genetic disease…
Dr. Perry Phillips
• Preimplantation genetic diagnosis is a technique
used to identify genetic defects in embryos created
through in vitro fertilization (IVF) before pregnancy.
• (PGD) refers specifically to when one or both genetic
parents has a known genetic abnormality and testing
is performed on an embryo to determine if it also
carries a genetic abnormality.
• Edwards and Gardner successfully performed the first
known embryo biopsy on rabbit embryos in 1968. In
humans, PGD was developed in the United Kingdom in
the mid 1980s as an alternative to current prenatal
diagnoses.Initially, PGD revolved around determination
of gender as an indirect means of avoiding an X-linked
disorder. In 1989 in London, Handyside and colleagues
reported the first unaffected child born following PGD
performed for an X-linked disorder.
• As of 2006, more than 15,000 PGD cycles have been
reported. PGD is currently available for most known
genetic mutations.
Primary candidates for PGD
• Couples with a family history of X-linked disorders (Couples
with a family history of X-linked disease have a 25% risk of
having an affected embryo [half of male embryos].)
• Couples with chromosome translocations, which can cause
implantation failure, recurrent pregnancy loss, or mental or
physical problems in offspring
• Carriers of autosomal recessive diseases (For carriers of
autosomal recessive diseases, the risk an embryo may be
affected is 25%.)
• Carriers of autosomal dominant diseases (For carriers of
autosomal dominant disease, the risk an embryo may be
affected is 50%.)
Advantages for the Procedure
• The Pre-Implantation Genetic Diagnosis technique is
performed before pregnancy begins. This avoids "the
stress, emotional trauma and subsequent moral
dilemma" of amniocentesis.
• Some adults who know that they are carriers of a
genetically transmitted disease decide to not have
children. The Pre-Implantation Genetic Diagnosis
procedure allows them to have a healthy child with
full assurance that it would not be carrying that
• If the procedure became widespread, the incidence
of many diseases and a great deal of human suffering
would be reduced. Many genetic diseases cause a
great deal of suffering in children and enormous
family stress.
Conditions diagnosed using PGD
• PGD should be offered for 3 major groups of
• (1) sex-linked disorders
• (2) single gene defects
• (3) chromosomal disorders
* Sex-linked disorders
• X-linked diseases are passed to the child through a
mother who is a carrier or affected fathers .
• Sex-linked recessive disorders include hemophilia,
fragile X syndrome, most neuromuscular dystrophies
(currently, >900 neuromuscular dystrophies are
known), and hundreds of other diseases. Sex-linked
dominant disorders include Rett
syndrom, incontinentia pigmenti,
pseudohyperparathyroidism, and vitamin D–resistant
**Single gene defects
• PGD is used to identify single gene defects such as cystic
fibrosis, Tay-Sachs disease, sickle cell anemia, and Huntington
disease. Although progress has been made, some single gene
defects, such as cystic fibrosis, have multiple known
mutations. In cystic fibrosis, only 25 mutations are currently
routinely tested. Because most of these rare mutations are
not routinely tested, a parent without any clinical
manifestations of cystic fibrosis could still be a carrier. This
allows the possibility for a parent carrying a rare mutation
gene to be tested as negative but still have the ability to pass
on the mutant cystic fibrosis gene.
• PGD can also be used to identify genetic mutations like BRCA 1, which does not cause a specific disease but increases the
risk of a set of diseases.
***Chromosomal disorders
• The last group includes chromosomal disorders in
which a variety of chromosomal rearrangements,
including translocations, inversions, and deletions,
can be detected using fluorescent in situ
hybridization (FISH). FISH uses telomeric probes
specific to the loci site of interest. Some parents may
have never achieved a viable pregnancy without
using PGD because previous conceptions resulted in
chromosomally unbalanced embryos and were
spontaneously miscarried.
What Are The Concerns Of PGD?
The following are considered concerns or disadvantages
associated with the use of PGD:
• Many people believe that because life begins at conception
and that the destruction of an embryo is the destruction of a
person. In practice, the PGD procedure usually results in a
small number of discarded embryos.
• While PGD helps reduce the chance of conceiving a child with
a genetic factor, it can not completely eliminate this risk. In
some cases, further testing done during pregnancy is needed
to ascertain if a genetic factor is still possible.
• Although genetically present, some resulting diseases only
generate symptoms when carriers reach middle age.
preimplantation genetic screening
Preimplantation genetic screening (PGS) refers to
techniques where embryos from presumed
chromosomally normal genetic parents are screened
for aneuploidy.
Indications for Preimplantation
Genetic Screening
Most early pregnancy losses can be attributed to aneuploidy.
Women of advanced maternal age
Couples with history of recurrent pregnancy loss
Couples with repeated IVF failure
These patient populations are at risk of failure with IVF because
of a high proportion of aneuploid embryos. PGD is believed to
decrease this risk by selecting chromosomally normal
embryos that have a higher chance of implantation.
How Is The PGD/PGS Performed?
How Is The PGD/PGS Performed?
1_ Ovarian stimulation is needed in order to produce multiple
eggs. During the 8- to 14-day hormonal stimulation period,
frequent ultrasonographic examinations and laboratory tests
are performed to monitor the development and maturation of
follicles (egg-containing ovarian cysts).
2_ The eggs are then cultured for a few hours after their retrieval
to allow for final maturation to occur. For the PGD/PGS
procedure at a later stage of embryonic development,
intracytoplasmic sperm injection (ICSI) is preferred. In this
manner, ICSI prevents the chance of polyspermy and the
accidental acquisition of “extra” chromosomal material from
the sperm
3_ Sperm for purposes of egg fertilization are typically obtained
from the male partner by masturbation on the day of egg
4_ The morning after ICSI, the eggs are examined for signs of
fertilization, which is determined by the presence of 2
pronuclei, representing the male and female contribution to
the embryo.
5_ Embryos continue to divide into multicellular entities. Three
days after egg retrieval, when the embryo is normally at the 610 cell stage, the embryos can be prepared for a cleavagestage biopsy. Normal development includes progression to the
2-4 cell stage two days after egg retrieval, and, after three
days, usually 6-10 cells.
Biopsy Techniques
Most clinics perform a cleavage-stage embryo biopsy. However,
one of the following 3 techniques can be used for PGD:
1_Polar body biopsy
• Polar body biopsy works only for female chromosomal
disorders. The mature metaphase II egg extrudes a single
polar body. This polar body can be removed and tested,
providing information on only the chromosomal content of
the egg.
• Because only information about the mother can be obtained
by analyzing polar bodies, chromosomal abnormalities
occurring after fertilization (when the sperm meets the egg)
are not detected.
• This technique is infrequently used .
2_Cleavage-stage embryo biopsy
• The most common approach for PGD/PGS is to biopsy a single
blastomere from day 3 embryos; this allows extraction of a
single blastomere from a developing embryo. The removal of
the blastomere is a technically challenging procedure.
• Removal of blastomere from an 8-cell embryo (cleavage-stage
• Before extracting the single cell from a 6-10 cell embryo, the
embryo is incubated in calcium- and magnesium-free medium
for approximately 20 minutes in order to reduce blastomereto-blastomere adherence.
cell embryo, on Day 3 after IVF, ready
to be biopsied. It is held in place on a
with a holding pipette.
2. The zona drilling pipette is used to drill a
through the shell of the embryo ( the zona)
using acid
The acid Tyrode has breached the zona
4. The embryo biopsy pipette is introduced
this opening, and gentle suction is applied to
dislodge a
single cell ( a blastomere ) from the embryo
The blastomere is being removed. You can see
the nucleus of the blastomere ( the clear area
in the
center of the cell) clearly. This contains the
DNA of
the cell.
6. The embryo biopsy has been completed
successfully !
The biopsied embryo is intact after the biopsy
has been completed. This procedure is
repeated for
all the embryos
8. The biopsied embryo is now returned to the
incubator for further culture. The blastomere will now be
analyzed to find out whether it is XX or XY (for sexual
selection), has normal chromosomes (for fertility), or has
a genetic abnormality (to eliminate a genetic disease
predisposition). Once the results are known, which takes
about 4-6 hours, the desired embryos will then be replaced
into the mother's uterus.
3_Blastocyst biopsy
• Blastocyst formation begins on day 5 post-egg retrieval and is
defined by the presence of an inner cell mass and the outer cell
mass or trophectoderm. At this stage of development, the embryo
is formed of more than 100 cells. A hole is breached in the zona
pellucida in a similar manner as described for a cleavage-stage
embryo biopsy, and cells are removed from the trophectoderm
using a fine biopsy pipette. The inner cell mass is left undisturbed.
• A limitation of this procedure is the potential acquisition of cells
from the trophectoderm that are not representative of the
developing embryo (inner cell mass) due to mosaicism (having
multiple different types of cell lines). In addition,
genetic/aneuploidy testing is completed approximately 24-48 hours
of the embryo biopsy; due to the limited viability of embryos in the
laboratory (≤6 d after egg retrieval), many embryos do not survive
until the time of embryo transfer. Therefore, biopsied blastocysts
must be frozen.
Genetic Testing
Polymerase Chain Reaction ( PCR)
 Fluorescence in situ hybridization (FISH)
Polymerase chain reaction
• PCR is a relatively fast and convenient way to test DNA. The
method has been used in a variety of preimplantation genetic
testing protocols
• Only one cell should be amplified; however, if another cell or
piece of DNA enters the tube, it is also amplified. ICSI must be
used to minimize this problem and to ensure that no excess
sperm are present (paternal contamination) and that all the
cumulus cells have been removed (maternal contamination).
• Errors in PCR can result in misdiagnoses leading to an affected
embryo being transferred or the discarding of a normal
embryo. One error is caused by a phenomenon known as
allele dropout. This refers to the preferential amplification of
one allele over another during the PCR process and is mainly a
problem for PGD of dominant disorders or when 2 different
mutations are carried for a recessive disorder and only one
mutation is being analyzed. In autosomal dominant diseases,
the risk of transferring an affected embryo is 11% and 2% for
recessive disorders.
Fluorescence in situ hybridization
• FISH is used for the determination of sex for X-linked diseases,
chromosomal abnormalities, and aneuploidy screening. FISH
is used more commonly in PGS secondary due to its utility as
an aneuploidy screen. Probes bind to a particular
• Each probe is labeled with a different fluorescent dye. These
fluorescent probes are applied to the cell biopsy sample and
are expected to attach to the specific chromosomes.
• They can be visualized under a fluorescent microscope. The
number of chromosomes of each type (color) present in that
cell is counted. The geneticist can thus distinguish normal cells
from abnormal cells, such as those with aneuploidy
• Chromosomes that can be analyzed with FISH probes include
X, Y, 1, 13, 16, 18, and 21.
A summary of PGD applications
categorized by PCR or FISH
Polymerase chain reaction categorizes the following:
Single gene defects in autosomal disease
Single gene defects in male infertility
Identification of sex in X-linked diseases
Fluorescence in situ hybridization (preferred because PCR
bears the risk of misdiagnosis caused by contamination)
categorizes the following:
Aneuploidy screening in women of advanced maternal age
Aneuploidy screening for male infertility
Identification of sex in X-linked diseases
Recurrent miscarriages caused by parental translocations