Preimplantation Genetic Diagnosis PGD

Definition

Pre-implantation Genetic Diagnosis (PGD) is performed in conjunction with in vitro fertilization (IVF). PGD detects the genetic abnormalities either at gene level or at chromosome level before transferring the embryo into the uterus to avoid the transfer of genetically affected embryos. PGD is offered for three conditions:

GD for Aneuploidy Screening
PGD for single Gene Defect
GD for Chromosome Translocatio

PGD for Aneuploidy Screening (PGD-AS)

PGD-AS is the most common type of PGD analysis. The embryos are tested using a panel of chromosomes that are commonly involved in miscarriages or trisomy pregnancies. Embryos screened this way and reported as normal have a higher rate of implantation, lower spontaneous loss and a reduced risk of trisomy offspring (e.g., Down’s syndrome).

A normal human cell contains 46 chromosomes or 23 pairs of chromosomes. These chromosomes are string like structures that resides in the nucleus and carry the genetic information.
Autosomes (22 pairs of chromosomes) are same in men and women. The 23rd pair of chromosome is called sex chromosomes. Women normally have two of the same sex chromosomes, called the X chromosome, while men normally have 2 different sex chromosomes, known as X and Y chromosomes.
A Single Sperm contains half of the total number of 46 chromosomes or 23 chromosomes each i.e. 22X or 22Y.
An egg contains half of the total number of 46 chromosomes or 23 chromosomes each i.e. 22X.
A normal fertilized embryo is derived by the fusion of 23 chromosomes from an egg (22X) and 23 chromosomes from a single sperm (22X) or (22Y).
Abnormal cell division in sperm or eggs might result in greater or fewer chromosomes than the normal 23. Therefore, any embryo that is derived from these sperm or eggs will carry extra or missing chromosomes, an abnormal condition referred as aneuploidy.
In approximately, 70% of recurrent miscarriages abnormal numbers (aneuploidy) of chromosomes are identified.
Some of the most common chromosome abnormalities found in miscarriages are Trisomy (3 copies of chromosome) 16, 22, 21, 15, 18 or 13; Triploidy (3 copies of all the chromosomes); and abnormalities of the sex chromosomes i.e. additional or missing sex chromosomes.
Chromosomally abnormal embryos usually:

Fail to implant in the uterus
Miscarry
Develop and give birth to trisomy babies e.g. Down’s Syndrome

The percentage of chromosomally abnormally embryos that each couple produces varies depending on their clinical status.
Proportion of embryos that are abnormal is determined by

Advanced Maternal Age (>35 years)
Number of Failed IVF cycles
Number of Miscarriages during normal conception
Quality of Sperm

Any deviation from having 2 copies of each chromosome is considered abnormal. If only one of two chromosomes is identified the embryo is considered monosomy and if 3 chromosomes is identified it is considered trisomy. Both these conditions are abnormal and not suitable for embryo transfer.
For PGD-AS embryos created by IVF are cultured in the laboratory for 3 days to grow ideally to an 8-cell stage. At that point one or two cells are removed by making a hole on the outer shell of the embryo. This procedure is called embryo biopsy. The biopsied embryo is returned back to culture until the result for that cell/cells are obtained usually on 5th day after egg retrieval.
Removal of one or two cells from an eight cell embryo does not compromise the embryonic development.
It is important that the embryo to be biopsied should have at least 5-cell and minimal fragmentation on the 3rd day of embryonic development. If the embryo contains too few cells, a biopsy might jeopardize the viability of the embryo.
The nuclear material from the biopsied cell is tested with Fluorescent In- Situ Hybridization (FISH) to determine the chromosome status of each cell.
A mixture of FISH probes in one or two sequential hybridizations is used. Possible chromosomes to be tested include 13, 15, 16, 18, 21, 22; X and Y. Abnormalities in these chromosomes are found commonly in miscarriages and abnormal live births.
A normal cell should show 2 copies of FISH signals for each of the numbered chromosomes, and either 2X signals for females or 1X and 1Y signals for males.
The PGD test result may not be obtained or is incorrect if:
The cell is lost during fixation or nucleus is ruptured and no result will be available.
The chromosomes in the nucleus fail to spread properly and lye on top of each other underestimating the number of chromosomes and can lead to a false result.
FISH probe fails to bind to a chromosome suggesting a missing chromosome.
Embryo biopsy occurs in the middle of cell division then the result appears to be two nuclei and four sets of chromosomes, FISH analysis from these cells can be difficult to interpret.
PGD cycles are most successful when 8 or more embryos are created by IVF and at least 5 are of good quality based on the number of cells, fragmentation and uniformity in cell size.
PGD usually does not increase the pregnancy rates but it reduces the miscarriages and the incidence of trisomy pregnancies.
PGD is most successful in the patients who have more than 3 embryos that do not show abnormalities and at least two of them are developed to the blastocyst stage.

PGD for Single Gene Mutatio

Indicated when there is increased risk of conceiving a child with severe genetic disorder:
Both partners carry a gene for an autosomal recessive disorder as in
Cytic Fibrosis (CF)
Sickle Cell Anaemia (SCA)
Medium Chain Acyl-CoA Dehydrogenase Deficiency (MCADD)
PhenylKetonUria (PKU)
Beta Thalassaemia
Tay-Sachs Disease

One partner may carry a gene for a dominant disorder as in

Huntington disease,
familial hypercholesterolaemia
Marfan Syndrome
Myotonic Dystrophy

X-Linked Inherited Diseases An X-linked disorder requires just one copy of the mutated gene, inherited from an affected parent (males are affected and females are typically unaffected carriers)

Duchenne’s muscular dystrophy
Hemophilia
Fragile X

This procedure involves prior testing to detect the gene mutation so that a gene probe can be produced.

There are several possible reasons for failure to achieve a successful identification of the gene or to achieve pregnancy. These include:

Possible failure to amplify the gene in question or degraded nuclear material that does not allow for a clear DNA signal.
PGD for single gene mutation can only detect the embryos that are carrying the mutation irrespective of the implantation potential of the embryo.

PGD for Chromosomal Translocation or Structural Abnormalities

Chromosomal translocations involve a rearrangement of the chromosome material so that some of the genetic material from one chromosome is located on another one. This is known as a “balanced” translocation where all of the normal genetic material is present.

If one partner carries a balanced chromosome rearrangement, he or she can produce sperms or eggs that can contain extra or missing segment of a particular chromosome material. These may produce conceptions which contain either excess or deficient total genetic material “unbalanced translocation”. This can result in failed early embryonic development, recurrent pregnancy loss or birth of a child with mental and/or physical defects.
Not all translocations or structural rearrangements can be tested by PGD. The reference lab would need to analyse the karyotype from the blood of both partners prior to start the IVF cycles to check the feasibility of performing the PGD for that anomaly.
Although medical evidence shows that PGD in couples who carry chromosomal translocations helps in reducing the pregnancy loss but it cannot eliminate the miscarriages due to other factors.
PGD for translocation can be carried out in conjunction with aneuploidy screening using the limited number of FISH probes.

IMPORTANT POINTS ABOUT PGD

PGD-AS is a screening, NOT diagnostic test. It allows for screening out of the most common aneuploid birth defects and may reduce miscarriage rate.
PGD of any type may not increase pregnancy rates and in some cases may reduce the chances for pregnancy due to decreased embryos available for transfer.
PGD may reduce the opportunity for cryopreservation of embryos.
PGD may result in NO embryos for transfer.
In cases of low embryo number or poor embryo progression, plan for PGD may be cancelled.