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What is preimplantation genetic diagnosis (PGD) ? PGD is the genetic
testing of preimplantation stage embryos produced through In-Vitro
Fertilisation for specific single gene disorders or heritable
chromosomal imbalance. PGD experiments were carried out in the UK in the
1980’s and the first PGD baby was born in 1989. Currently, PGD is only
available at a few clinics worldwide. PGD is not a form of
genetic engineering. The genetic material of the embryos is not
modified. In-Vitro fertilization is first carried out to produce the embryos. Step 1: Ovulation The mother is given ovulation-inducing drugs to cause her ovaries to super-ovulate and release several mature ova at the same time. (Normally, only one mature ovum is released from either of a woman’s two ovaries every month, but in super-ovulation, several ova are released all at once.) Drugs that cause super-ovulation contain follicle stimulating hormone and luteinizing hormone. Follicle stimulating hormone stimulates the growth and development of primary follicles each containing a potential egg cell found in the ovaries. This results in the formation of Graafian follicles which contain haploid (containing half the chromosome number i.e.. 23 chromosomes) egg cells. Leutinising hormone causes ovulation. Step 2: Egg retrieval The eggs are retrieved using either of these 2 methods: 1) Sonography, where the doctor places an ultrasound probe in the vagina and guides the aspirating needle through the wall of the vagina into the follicles of the ovary using ultrasound to view the position of the ovary, aspiration needle and follicles. Fluid aspirated out of the follicles in the ovary is checked under a microscope for the presence of ova. This is a more common method.
2) Laparoscopy is used when sonographic egg retrieval is not possible due to difficulties in accessing the ovary, or when a diagnostic assessment of the pelvic organs is required. Here the eggs are retrieved through an incision in the abdomen. A long, thin instrument with a light and a lens at the end allows the doctor to retrieve the ova using an aspiration needle as well as to examine the pelvic organs by viewing images of these internal organs projected on a television screen. Carbon dioxide is put into the abdomen through a special needle that is inserted just below the navel. This gas helps to separate the organs inside the abdominal cavity, making it easier for the physician to see the reproductive organs during laparoscopy. The gas is removed at the end of the procedure. This procedure is usually carried out under general anaesthetic. Step 3: Fertilisation and incubation About 1-2 hours after
egg retrieval, the father is asked to produce a semen sample. The eggs
retrieved are placed in a Petri dish together with sperms from the semen
of the father in the ratio of about 1egg : 50000-75000 sperms and are
incubated together at 37 degrees Celsius (normal body temperature) in
carbon dioxide
incubators for 18 hours in a culture media which nourishes the eggs,
and later, the embryos. Once the embryos are produced, the actual procedure of PGD begins. Step 1: Embryo biopsy 3 days after fertilization has occurred, each successful embryo has divided into about 4 to 12 cells. One or two cells are removed from the early multi-celled embryo so that the genetic material in these cells can be analysed. The removed cell must contain a nucleus with chromosomes present to determine the genetic status of the rest of the embryo. At this point, the cells of the embryo are still distinct from each other. By day four, the embryo begins to compact, a process whereby the individual cells lose their clear outline and they seem to fuse together with the other cells to form the morula stage embryo. On the third day, however, single cells can be individually removed without disrupting the adjacent cells in the embryo. Picture shows a 7 cell embryo fixed in position with a holding pipette on the left. A second pipette on the right is used to drill a hole through the glycoprotein coat of the embryo, known as the zona pellucida and a cell is dislodged from the embryo with a gentle suction. The embryo is held in a warm culture medium that helps to allow the cells to be removed with minimum trauma to the embryo during manipulation. Manipulation is carried out on an inverted microscope. The removal of the cells is not known to be deleterious to the further development of that embryo, as the embryo at this early stage of development can compensate for this loss of material. This is because all cells at this stage are totipotent. (They are fully capable of directing further embryonic development and are capable of forming every type of body cell.) A less
preferred alternative to embryo biopsy is polar body removal. There are
2 polar bodies. The first polar body is produced from the division
of the ovum and can be removed and analysed for its chromosome
complement. Upon penetration of ovum by sperm (i.e. fertilisation)
but before the fusion of their nuclei, the ovum undergoes another cell
division producing 2 cells, one larger than the other. The smaller cell
forms the second polar body while the larger cell's nucleus fuses with
the sperm's nucleus. Polar bodies disintegrate upon implantation and are
not part of the developing foetus. By analysing the genetic material of
the polar bodies, the maternal genetic contribution to the embryo may be
determined. However, results obtained from polar body analysis may not
be conclusive, partly because the paternal genetic contribution will not
be known. In this case embryo biopsy which is more conclusive will have
to be performed. Once a blastomere (a single cell from the embryo) or polar body is removed, it is either fixed on a glass slide for chromosomal analysis, or placed in a small tube of chemical buffer for single gene diagnosis. The cells are then analyzed using techniques called fluorescence in situ hybridization (FISH) or DNA analysis using Polymerase Chain Reaction (PCR) amplification of DNA from single cells.
FISH is used for chromosome analysis to check for sex-linked illnesses, for chromosome abnormalities like translocation (rearrangement of chromosome in which part of a chromosome is detached by breakage and is attached to another chromosome), and for the diagnosis of aneuploidism (the condition of having less or more than the diploid number of chromosomes i.e.. having less or more than 46 chromosomes). It allows the visualisation of specific nucleic acid sequences within a cellular preparation by incubating a fixed, dried cell containing a nucleus in metaphase or interphase stage together with a fluorescently labeled gene probe. The fluorescently labeled DNA probes detect or confirm gene or chromosome abnormalities. The sample DNA is first denatured, a process that separates the complimentary strands within the DNA double helix structure. The fluorescently labeled probe of interest is then added to the denatured sample mixture and hybridizes with the sample DNA at the target site (i.e. it binds to a specific nucleic acid sequence or gene of interest) as it reanneals (or reforms itself) back into a double helix. The probe signal can then be seen through a fluorescent microscope and the sample DNA scored for the presence or absence of the signal. If the gene of interest is present, a signal will be obtained. During the genetic analysis, the embryos are usually grown to the fifth day of development at which time they will be at the morula stage or blastocyst stage. Those embryos found to be free of genetic abnormalities are then placed into the uterine cavity.
Morula stage embryo. The morula contains between 10-30 cells. This is the final stage before the formation of a fluid filled cavity called the blastocoel cavity.
Blastocyst stage. This is attained upon the formation of the blastocoel cavity.
PGD
using FISH on normal male blastomere Polymerase Chain Reaction (PCR) DNA is made up of 4 different bases: adenine, guanine, cytosine and thymine. The DNA is denatured at a temperature of 90-96 degrees Celsius. Oligonucleotides (short nucleotide strands) complementary to the sequence of bases of interest on the DNA are produced by automated chemical synthesis and are used as primers in a special series of DNA-polymerase catalysed reactions. The primers are annealed (i.e.. they bind to their complementary sequences on the single strands of DNA) at 50-60 degrees Celsius. DNA polymerisation by a thermostable DNA polymerase is carried out at 72 degrees Celsius. Starting from the primer, the polymerase can read a template strand and match it with complementary nucleotides very quickly. 2 new helices are thus formed in place of the first, with each new helix comprising of one original strand and one newly assembled complementary strand. The process may be repeated to produce more DNA, and each process take only 1-3 minutes. Once the DNA is amplified, the portions of DNA containing the chromosomes of interest are then analysed for abnormalities. DNA amplification is necessary because a single blastomere will contain only a very small amount of DNA, and more DNA is required for analysis. Mutation analysis is carried out using sequence analysis. refer to table at http://jmd.amjpathol.org/cgi/content/full/4/1/11/T1 for a look at the various analytical methods available and the sort of disorders each method can detect Embryos found to be free from genetic and chromosomal abnormalities through FISH and PCR will then undergo embryo transfer, a 15 minute procedure where a catheter is inserted into the uterine cavity through the cervical canal. This is performed using ultrasound guidance. The mother may be given progesterone, a hormone, to nourish and prepare the wall of the uterus for implantation of the embryo.
Conditions that can be detected by PGD 1)sex-linked disorders Examples of sex-linked disorders:
2)single gene defects This is used to identify conditions where the illness is caused by a single gene that is not functioning normally. These abnormalities may be detected with molecular techniques using PCR amplification of DNA. Examples of single gene defects which can be detected by PGD:
3)chromosomal disorders
Chromosomal disorders are caused by inversions (when a chromosome
is broken in 2 places, it heals such that the internal segment is
flipped over), deletions (The loss of chromosomal material) and
translocations (when the ends of 2 different broken chromosomes heal by
attaching to the wrong partner) in chromosomes. These can be detected
using FISH. Chromosomally unbalanced embryos tend to be miscarried
spontaneously, so couples who have never achieved a viable pregnancy may
want to undergo IVF and use PGD to test potential embryos for such
disorders. 1) Women with a history of miscarriages Foetuses with chromosomal abnormalities tend to be miscarried. PGD will reduce the risk of chromosomal abnormalities in the foetus by testing it in the embryonic stage for certain detectable chromosomal abnormalities. Hence women whose history of miscarriages are due to chromosomal abnormalities of the foetuses will benefit. 2)Carriers of single gene disorders, chromosome translocations or other abnormalities The carrier may be either parent, or both parents. PGD reduces the risk of having a child with the above-mentioned conditions because some of these conditions may be detected at the embryo stage using PGD. 3)Parents with personal histories of prenatal diagnosis followed by termination of foetuses These parents have a higher chance of producing babies with foetal abnormalities. They may not want to have to endure the trauma of another pregnancy termination and may wish to undergo PGD.
4)People who have moral or religious objections to the
termination of pregnancy. 1) Most genetic testing nowadays is done through either amniocentesis (more common) or chronic villus sampling. Amniocentesis is done when the foetus is between 11-15 weeks old. It is an invasive procedure where a needle is inserted into the mother’s uterus in order to draw up some amniotic fluid containing foetal cells so that chromosomal analysis may be performed. Amniocentesis carries with it a small risk of miscarriage. Chronic villus sampling is performed around the third month of pregnancy. Using ultrasound, a thin hollow tube is inserted through the vagina and cervix (transvaginal/transcervical) or through the abdomen (transabdominal) to the edge of the placenta where a tissue sample is taken for chromosomal analysis. Should the foetus be found to have certain defects, the parents then have the choice of terminating the pregnancy. However, with a rather advanced pregnancy, the emotional trauma of undergoing an abortion may be very great. With PGD however, the chromosomal analysis is done before pregnancy, since embryos are tested for abnormalities before being inserted into the uterus. Parents will not be faced with the difficult decision of whether or not to undergo an abortion.
2) Adults who know that they are carriers for certain genetic conditions may undergo PGD to ensure that their children will not suffer from that condition. 3) Will significantly reduce the medical costs because certain diseases require expensive medication and treatment throughout an individual’s lifetime 4) There will be a
reduced risk of miscarriage as only embryos without chromosomal
abnormalities which can currently be detected through PGD are inserted
into the uterus.
1) Instead of using PGD to detect the presence of genetic diseases and chromosomal abnormalities, parents may also use PGD to select the gender of the baby. This may cause gender imbalance, especially in countries like India and China where there is a strong preference for male children. 2) Parents may also use PGD to select embryos having certain genetic traits which they consider desirable. This may be perceived as practicing some form of eugenics. 3)PGD is very expensive. 4)A couple who can conceive naturally will be required to undergo IVF, an expensive procedure with no guarantee of success should they want PGD. 5) If PGD is carried out using PCR, there is a risk of contamination from cellular sources. This contains whole genomic DNA. There is also a risk of carry over contamination from products of former PCR reactions. These will give rise to inaccurate results. 6)During PCR, allele dropout, where an affected allele fails to amplify may occur. This will create problems for the correct diagnosis of autosomal dominant diseases. 7) People of some religions believe that life begins at conception. They have a 2 main concerns regarding PGD:
Some relevant and useful websites
http://www.rgi2.com/pgd.html
http://www.bionews.org.uk/update.lasso?storyid=1639
http://health.allrefer.com
http://jmg.bmjjournals.com/cgi/content/full/39/1/6
http://www.ich.ucd.ac.uk/cmgs/fishpgd.htm
http://www.trisomy.org
http://opbs.okstate.edu/~melcher/MG/MGW4/MG42.html
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