Pre-implantation genetic diagnosis (PGD) is essentially an alternative to prenatal diagnosis, in which genetic testing is performed on embryos produced by in vitro fertilization (IVF) before a pregnancy is established. PGD has been mostly applied to woman of advanced maternal age undergoing IVF in order to increase implantation rates and to reduce the chances of miscarriage and genetically abnormal offspring. This modality of PGD is known as PGD of aneuploidy. Briefly stated, PGD involves the creation of several embryos in vitro from the eggs and sperm of an interested couple. The embryos are permitted to develop till 6 to 10 cell stage, at which point one of the embryonic cells is removed from each embryo and the cellular DNA is analysed for chromosomal abnormalities or genetic mutations either by polymerase chain reaction [PCR] or Fluorescence Insitu Hybridization [FISH].
The aim of PGD is to prevent embryos that are carrying a genetic abnormality from being transferred into the uterus. At this point, PGD is helpful if a couple has an increased risk of having a child with a specific genetic disorder, such as cystic fibrosis, Down syndrome or Turner’s syndrome.
Who should consider PGD
- Advanced maternal age
Women of advanced maternal age (>35years) are at a higher risk of producing aneuploid embryos, resulting in implantation failure, higher risk of miscarriage or chromosomally abnormal child birth.
- Recurrent miscarriage
Chromosomal abnormality is one of the most common causes of recurrent first trimester abortions. Either one of the couple or both may be a carrier of a balanced translocation or an aneuploid mosaic.
- Repeated IVF/ICSI failure cycles
Couple with repeated unsuccessful IVF cycles should be evaluated for the presence of chromosomal abnormality.
- Unexplained infertility
One of the reason for unexplained infertility in either of the partners may be a carrier of a translocation or an aneuploid mosaicism
- Male Factor infertility
Some of the male factor infertility disorders are due to chromosomal abnormality i.e aneuploidy or a structural chromosomal abnormality. Men carrying a balanced translocation are at risk of producing abnormal sperm.
- Y-Chromosome deletions
Y chromosome deletions are found in ~ 5-20% of men with a very low sperm count,. These deletions do not appear to cause any genetic disease, but decrease the chances of fertility.
- Genetic Causes
- Aneuploidy : Too many or too few chromosomes , it is always associated with physical and/or mental developmental problems. It occurs at the time of fertilization .
- Trisomy : Instead of a pair, 3 chromosomes are present
- Monosomy : Instead of a pair single chromosome is present.
If the extra or missing chromosome is an autosome (chromosomes 1 to 22), the embryo may not implant or may stop normal development soon after implantation and undergo spontaneous abortion.
But chromosomes involving 13,18, 21, X or Y, embryo may implant and carry to term. - Down syndrome – Trisomy -21 Three copies of chromosome
- Patau syndrome – Trisomy – 13
- Edward syndrome – Trisomy – 18
- Klinefelter syndrome – presence of an extra sex chromosome 47, XXY
- Turner Syndrome – missing a sex chromosome 45 , X.
PGD Procedure
- Blastomere Biopsy
- Biopsies done at 6-8 cell stage of embryo a hole is made in the zona with laser just sufficient to delink the blastomere from the developing embryo and gently pulled out and fixed on a slide for genetic study.
- After removal of one blastomere the developing embryo is placed back into culture media and returned to CO2 incubator for further development.
- At this early point of embryo development , all the cells are equivalent and thus, removal of a one blastomere does not remove any thing critical for normal development. The embryo compensates for the removed cell and should continue to divide following blastomere biopsy.
- Removed blastomere can be analysed using a technique called PCR or FISH.
- Results are usually obtained by noon on day 5 of embryo development according to the result normal embryo will be transferred to uterine cavity in hope for implantation.
- Polar Body Biopsy
Polar body from the oocyte can be removed and tested for its chromosome complement or to identify whether it contains the abnormal gene of concern. The polar body have no known function except to assist in cell division. They are simply “by-products” of the oocytes division. Once implantation occurs, the polar body disintegrate and are not part of the developing fetus.
In some instances it is necessary to confirm a diagnosis made on polar body analysis by performing blastomere biopsy. It is also possible that polar body fail to provide a conclusive result. In these situations , it may be possible to perform blastomere biopsy for further genetic analysis. - FISH [Fluorescence Insitu Hybridization] :
FISH is used to determine the proper structure of specific chromosome. A probe or “tag” attached to a specific chromosome by matching the sequence of the probe with the sequence of the DNA being tested. The probe has fluorescence and “lights up” under a microscope. Embryos with too much or too little fluorescence have an abnormality. This technique is most useful in diagnosing recurrent miscarriages, repeated IVF failures, age related chromosome abnormalities, abnormalities of chromosome 13,15, 16,17,18,21,22 , X & Y.
- PCR [Polymerase Chain Reaction] :
PCR is a DNA analysis technique and useful for specific single gene disorders, such as cystic fibrosis or Tay sechs. An enzyme is used to make millions of copies of the cellular DNA. This availability of abundance of the DNA facilitates easy evaluation of specific gene disorders. The sequence of base pairs in DNA helix is compared with normal gene and disorders are identified.
- Mosaicism :
Some embryos may contain blastomeres which are genetically normal, and within the same embryo, other blastomere which are abnormal, is called mosaicism. Misdiagnosis can occur due to mosaicism. This may result in the transfer of an embryo carrying a chromosome abnormality or the failure to transfer a normal embryo.
