Preimplantation Genetics

PREIMPLANTATION GENETIC DIAGNOSIS

What is Pre-implantation Genetics or PGD?
Genetic diseases can be diagnosed prior to a child being born. The most common way is to diagnose if there is a problem in the unborn child is by doing a Chorion Biopsy at 8-9 weeks or Amniocentesis at around the 4th-5th month of pregnancy (prenatal diagnosis).

In case a baby is found to be abnormal, it can be aborted. However this is a traumatic process- physically and psychologically. Instead of undergoing this trauma, Pre-implantation Diagnosis, popularly called PGD, can be done. In this a standard ICSI cycle is carried out, and a lot of embryos are produced. A small piece of the embryo is then removed and examined for any genetic or chromosomal defects. The defective embryos are discarded and the normal embryos are put back in the womb. Thus with the help of PGD one can have normal babies, without undergoing the trauma of Amniocentesis and abortion.

The detection of genetic disease in the human embryo before implantation gives parents a chance to start a pregnancy, knowing that the baby will be free of inherited disorder that is prevalent in their family.

What are the Indications?
There are several groups of patients in which PGD is the preferred option:

1. PGD is used to avoid conception of abnormal babies in couple who are at high genetic risk: especially in those who have had a previously affected genetically abnormal babies or in patients with age more than 35 years.

In India, the most common abnormalities are that of Downs Syndrome (mentally retarded babies with three chromosomes 21 or 3 chromosomes 18) and Thallasemia (genetic disorder commonly seen in Kutchis, Lohanas, Sindhis and Greeks). Downs Syndrome is commonly seen in babies of elderly women above the age of 35 years.

2. It has been observed that many women with advanced maternal age, which are Undergoing IVF/ICSI may have a higher number of genetically abnormal embryos. PGD can be used in identifying and transferring back normal embryos, thus decreasing the risk of miscarriages and increasing the chance of normal pregnancy.

3.In many couples suffering from repeated miscarriages, chromosomal Aneuploidy and Translocations, may play a role. PGD with multicolor FISH can be used as a screening test in these couples, thus reducing the chances of abortion.

4. Finally there are those patients with moral or religious objection to pregnancy termination such as Jains or Catholics. This group would prefer selection at the pre-implantation stage, giving them a reasonable chance of starting with a normal pregnancy.

How is PGD done?
The patient undergoes the standard ICSI cycle. Following ICSI, the eggs are placed in culture media and kept in the Carbon Dioxide incubators, for 3 days. On the third day numerous 6-8 celled embryos (earliest form of life) are formed. A small hole is made in the cover of the embryo called the zona. This hole is made using a machine called the Micromanipulator. The hole can be made either mechanically using needles or chemically using acid Tyrode solution.

Alternately the hole can be made using a Laser. Babies And Us has had the privilege of having the first Laser machine in the country to be used for cutting cells.(see Assisted Laser hatching link).

At least two cells or Blastomeres are removed from the embryo with the aid of the Micromanipulator These aspirated Blastomeres are sent to the Genetic Laboratory, for analysis by the two techniques of FISH or PCR, depending on the condition, which is being investigated.

Will the removal of Blastomeres damage the embryo?
A frequently asked question is whether the remaining embryos will survive and will have the same normal complement of genetic material as the parent embryo. The embryo at this stage is totipotent; that means each of the individual Blastomere has the potential to grow independently into an embryo and an individual, if implantation takes place. Hence removing two of the eight cells is acceptable.

How are the Blastomeres tested?
There are two kinds of genetic problems. One of these is due to chromosomal defects. Normally there are 46 chromosomes. The other defects are due to abnormalities in the genes. Genes are blocks of DNA that make up the chromosomes. These defects can be diagnosed by either FISH or PCR.

1. Fluorescent In Situ Hybridisation(FISH):
This is used for analyzing chromosome related Aneuploidy such as Downs syndrome and sex chromosome related problems such as Klinefelteror Turners syndrome. It has been extensively used in X linked recessive disorders such as Hemophilia A, Duchennes Muscular Dystrophy, Lesh-Nyhans syndrome, Colour Blindness etc.

In FISH, the Blastomere is fixed on a slide. The fluorescent probe is then added to the cell and allowed to hybridize with the Centromere DNA of the specific chromosomes. The nucleus is then observed under fluorescent microscope, making use of various filters. The chromosomes are seen as orange, blue or green coloured fluorescent dots ,shining against a blue background .By simply counting the dots one can make out the problem or disorder. For example in Tisomy 21 there are 3 orange dots (representing 3 chromosome 21s), as compared to normal, where there are only two dots (representing Chromosome 21).

FISH can be carried out in two hours. If a Embryo biopsy is done on the morning of day 3, then the FISH result is available by afternoon. The normal embryos can then be transferred back by late afternoon of the same day.

2. Polymerase Chain reaction (PCR):
This technique of genetic analysis is performed for diagnosing single gene defects. There are numerous diseases such as Cystic Fibrosis, Sickle Cell Anemia, Thalassemia and Swteinerts Myotonic Dystrophy, which can be diagnosed with this technique. Embryo sexing can also be done with this method. However unlike FISH, PCR cannot differentiate between XX and XO.

Genetic disorders may be caused by several changes in the DNA. These may involve a point mutation or the deletion or insertion of one or a few base pairs. The DNA mutation from a single cell is difficult to analyze. The cyclical process of the PCR makes millions of copies of the small portion of DNA, which is being screened for. In each PCR, the amount is doubled. It becomes easy to detect an error in the amplified portion of the DNA, since millions of copies are present.

The important points in PCR reaction are:
After amplification, it is analyzed by a method called Gel Electrophoresis, wherein the DNA is analyzed on the basis of migration of various fragments on the gel, when an electric current is applied. Though Cystic Fibrosis is very extensively investigated in the West, Thalassemia will be the main disease to contend with in our country. Already few successful PGDs for Thal have been carried out in the Mediterranean. The biggest problem in Thal at present , is the numerous mutations which are present in the country.

What is the scope of PGD in India?
Since the first successful clinical PGD, more than 100 centers worldwide have started performing this technique. More than a thousand successful births following PGD have been recorded. In India, few clinics, including Babies And Us at Lilavati Hospital, have started doing preliminary work in this very difficult area.

Does Babies And Us have the ability to accurately perform the complex task of PGD? Pre-implantation Genetic Diagnosis is a very complex and precise technique. Its successful clinical application is based on the unification of a highly successful ICSI program and a very accurate Genetic Laboratory. At present Clinical PGD has been done on a very limited number of couples worldwide. It has a tremendous potential to develop into a successful clinical procedure, in the future.

We have the latest imported Equipment from Olympus and Vysis to perform this technique. Furthermore we are at present performing PGD in collaboration with the IVF Institute in Naples, Italy. A PGD expert from Naples comes down to Mumbai every 4 months to perform this task.

How accurate is PGD?
PGD is 95-97% accurate. However one can never be sure. All patients who conceive after PGD are requested to undergo prenatal diagnosis to confirm that the pregnancy is normal.

Also the chance of a patient conceiving after PGD is in the region of 20-25%. That means one in five patient who undergoes PGD will become pregnant.