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PREIMPLANTATION GENETIC DIAGNOSIS(PGD)

PREIMPLANTATION GENETIC DIAGNOSIS(PGD)

Preimplantation Genetic Diagnosis (PGD)

The latest developments in genetic science have enabled to genetically analyze IVF embryos. This method is called “embryo screening” (pre-implantation genetic diagnosis). Genetic screening made prior to pregnancy is made by taking 1 or 2 cell samples from an embryo created in laboratory environment.

The special methods used on the cell samples can diagnose numerical and structural chromosomal disorders and single gene disorders that may occur (thalassemia, sickle cell anemia, cystic fibrosis etc.). This screening aims to plant only healthy embryos to the prospective mother’s womb, so that healthy babies can be born.

What is the purpose of PGD?

The finding of genetic diseases of couples or embryos is of vital importance for having healthy offspring. Today’s technological advancement has made it possible to trace many genetic diseases while the baby is still an embryo.

The purpose of PGD is to determine genetic diseases before the pregnancy, during the embryo stage. PGD is used on couples who use IVF techniques because of infertility problems, to trace possible genetic disorders that might occur during the IVF methods.

What are the methods used in PGD?

Approximately 10-12 days after the parents have started the IVF therapy, the egg cells are gathered and each of them is fertilized by a separate sperm cell. The fertile and well developing cells (embryo) are taken 1 or 2 cells in the third day to be genetically analyzed with different techniques.

What are the indications of PGD?

PGD is suggested for couples who carry genetic diseases and want to produce healthy children, couples who use IVF or microinjections to produce children due to infertility and embryos that have a high risk of chromosomal anomalies.

What are the advantages of PGD?

It ensures healthy offspring.
The family is protected from medical or psychological traumas related to ending the pregnancy.
It offers early recognition and cure for children that carry diseases like Beta-Thalassemia.
Pre-pregnancy diagnose is a much easier, cheaper and beneficial diagnosis method compared to the treatment of possible genetic diseases.
The margin of error in these processes in a single cell stage embryo is only about 2%.

What are the diseases that can be identified through PGD?

PGD can be used on almost any type of genetic diseases except some rare special cases. In order to use the PGD method the genetic disease within the family must have been identified.
Chromosomal Diseases Chromosomal diseases can be found in two groups: numerical and structural chromosomal diseases. Couples who have Klinefelter (46, XXY) or mosaic Turner (46, XX / 45, X) syndrome, have numerical chromosome anomalies or structural chromosome anomalies like translocation or inversion will be exposed to an anomaly-specific IVF treatment and thus will have a specific PGD. Chromosomal anomalies that haven’t been rooted yet or translocation type insertional translocation anomalies cannot be diagnosed via PGD. Chromosomal analysis can also be offered to couples with no chromosomal anomalies. In situations like recurrent pregnancy loss or unsuccessful IVFs PGD panels can be used to identify the chromosomal abnormalities in embryos.

Single Gene Diseases

Single gene diseases are harder to trace during the embryo stage via PGD and thus need a more complex technique and more developed tools. The most important requirement to use PGD on a patient is that the DNA change (mutation) that is causing the disease has been previously identified. In order to analyze DNA diseases during the embryo stage, a set-up time of 2 months is needed. The set-up stage is made specifically on the couple according to the genetic area where the mutation has taken place that has caused the disease. In order to minimize the risk of misdiagnosis in single gene tests made on embryos, informative markers will be assigned on the family’s DNA. The usage of informative markers minimizes the risk of misdiagnosis down to 2%. Cases where the disease could not be diagnosed, diseases that are clinically diagnosed but not proven through genetic testing or couples who have normal results on DNA tests cannot be taken into the set-up stage and thus cannot be tested with PGD.

Single Gene Disease

Today many diseases such as Mediterranean Anemia (Beta-Thalassemia), Cystic Fibrosis, Spinal Muscular Atrophy (SMA), Hemophilia, Duchenne Muscular Dystrophy can be identified during the embryo stage through sequencing and fragment analysis.
Families with children who have single gene diseases will first be put into the set-up stage by taking blood sample from the parents and the child, if possible. This stage takes approximately 1-2 months and only then can the parents be taken into IVF treatment. After a 10-12 days hormone-replacement therapy, egg cells are collected from the mother and fertilized with sperm cells collected from the father. The fertile and well developing cells (embryo) are taken 1 cell in the third day to be genetically analyzed with different techniques.

After the analysis cells that do not carry any genetic diseases are chosen to be transferred into the mother’s womb.

Stages of PGD

Set-up
PGD
Embryo transfer

Set-Up

The PGD method used on embryos is a lot more complex and difficult than genetic tests where a simple blood sample suffices. The biggest reason for this is that the process uses a single cell, the blastomere. For this reason a 1-2 month long set-up stage is used to prepare the patients for PGD.
In order to start with the set-up process the genetic disease or mutation in the family must have been identified earlier. It is not technically possible to use the PGD method on families who have only been diagnosed clinically.
Families whose mutations have been identified must additionally also meet some requirements in order to use the PGD method. The most important is the set-up process which is necessary because PGD is used on a single cell. Genetic analysis on embryos is difficult because of the scarce amount of DNA sample. Failure of acquiring results or misdiagnosis is risks that emerge from a single cell analysis. To overcome these problems and to minimize the risks the tools are controlled whether they operate on an optimal level. Further informative markers are specified in order to identify the gene zone closes to the embryo.

The confirmation of the mutation is completed when the disease-specific sequences that will be used for PGD are designed, reproduced and tested on the single cell. In order to minimize the risk of a misdiagnosis informative markers on the parents will be identified, produced and tested.

Stages of the set-up process for single-gene diseases
Taking EDTA blood samples and buccal mucosa samples from the parents and children, if there are any.
If the mutations that cause the disease are not identified yet, the identification of the mutation via DNA analysis from the given blood samples.*

Preimplantation Genetic Diagnosis (PGD)

Parents who have or carry a single gene disease and have applied to an IVF center for PGD can only be started on IVF treatment after the set-up stage. After the set-up stage the potential mother is put on a 10-12 day long hormone-replacement therapy that aims a high rate of egg cell production. Of the egg cells the mature ones will be collected and each of them will be microinjected a sperm cell. After the process the fertile eggs (embryos) are kept in the laboratory for 5-6 days and then transferred to the potential mother. The PGD process is used on developing embryos on the 3rd day (7 to 8 cells) before their transfer and the tests are made on only 1 cell (blastomere).

The most important factor while using PGD on the blastomere cell is to prevent the contamination of the blastomere with another cell. Because of that it is important that the center that takes the blastomere cell is careful and experiences. Every cell taken from the embryo are placed in different sterile pcr tubes that contain the necessary solutions. These tubes are sealed carefully and transferred to the center where the PGD will be made. In cases this method can’t be used, every blastomere is put on a “four well” cup as a separate droplet and transported in that manner. In both methods it is very important to enumerate the blastomere carefully.

If the mutation was previously identified but the set-up center is not working with the disease, the designing and producing of the mutation-specific sequence. These sequences (primer) will be used to confirm the mutation and to test if the sequences work on the single cell. The identification and production of the informative markers close to the gene area of the disease the family has. After all these stages are done the IVF center and family is informed that their set-up is complete.

* The DNA samples are generally sent to a local or foreign center on which genetic location the mutation that causes the disease takes place. These tests can sometimes take up to 3-4 months. If the mutation is identified after the analysis, it means that the target area where the PGD method will be process is determined and the set-up process can begin.

If the mutation was previously identified and is a disease the set-up center is working with, the confirmation of the disease. Predetermined heat and the sequences belonging to the markers will be multiplied. The obtained PCR product will determine the single sequence using the DNA sequence analysis on the sequence machine. The determined sequence will be analyzed for mutations on the previously determined genetic area and will be thus diagnosed.

Since the PGD method uses one single cell, the whole process is made on a single DNA helix obtained from one egg cell and one sperm. A minimum of DNA is used. For this reason, some cells the helixes coming from the parents cannot be multiplied during the PCR and a result cannot be obtained (amplification failure). And sometimes one of the helixes belonging to the mother of the father cannot be amplified. This is called a allel drop-out (ADO). ADO is the most concerned situation during the PGT process since it might cause misdiagnosis. To recognize a possible ADO, a second control mechanism is needed during the analysis. At this point the parent-specific informative markers are used to identify a possible ADO. ADO identified cells will not be evaluated since a healthy analysis cannot be made.

After the evaluation, the healthy cells will be transferred into the potential mother’s womb. Since an analysis made on a single embryo cell has a 2% risk of error, a confirmation must be made during the 11th week with a chorionic tissue sample and an amniocentesis on the 16 th week.
The blastomeres that arrive in the PGD center will first be put through a 45 minute process with a lysis solution. The obtained DNA samples from the blastomeres are added the mutation-specific sequence obtained during the set up and those will be prepared for a polymerase chain reaction (PCR). The prepared mix is processed and analyzed at

HLA (Human Leukocyte Antigen) Typing

Families that have children who have leukemia can make a HLA typing test on the embryos before pregnancy. In families with children who have these types of diseases the mother and father and if possible the child will give blood samples for the set-up process. This process takes approximately 1 month and the parents will after that taken to the IVF treatment.
Approximately 10-12 days after the parents have started the IVF therapy, the egg cells are gathered and each of them is fertilized by a separate sperm cell. The fertile and well developing cells (embryo) are taken 1 cell in the 3rd day to be genetically analyzed. After this analysis embryos compatible to the sick child’s HLA type can be chosen.

Stages of PGD

Set-up
PGD
Embryo transfer

Set-Up

PGD used on families with children who have diseases like leukemia in order to identify the HLA type, is a much more complex and difficult process than HLA typing through blood and tissue samples. The main reasons are that the HLA areas are polymorphic and that the test is used on a single cell (blastomere). For this reason a 1 month long set up must be made before the HLA typing during IVF treatment.
During the set-up stage the tissue types on the HLA area are identified by making DNA tests on the parents and the sick child. With the set up stage the possible HLA types on the embryos can be predicted and the HLA types compatible with the sick child’s can be pre-determined.
Genetic analysis on embryos is difficult because of the scarce amount of DNA sample.
Failures of acquiring results or misdiagnosis are risks that emerge from a single cell analysis.
To overcome these problems and to minimize the risks the tools are controlled whether they operate on an optimal level. After determining the tissue types belonging to the parents and the sick child and the testing of the HLA markers that will be used during the PGT on a single cell, the set-up process will be completed.

Stages of the set-up process for HLA typing

Taking EDTA blood samples and buccal mucosa samples from the parents and the sick child.
The determining of HLA genotypes on blood samples using PCR.
The testing of markers used for HLA genotypes on single cells.
After all these stages are done the IVF center and family is informed that their set-up is complete.

Preimplantation Genetic Diagnosis (PGD)

Parents who have applied to the IVF center for a PGD analysis for the treatment of their leukemic child can be taken in to IVF treatment after the set-up process. After the set-up stage the potential mother is put on a 10-12 day long hormone-replacement therapy that aims a high rate of egg cell production. Of the egg cells the mature ones will be collected and each of them will be microinjected a sperm cell. After the process the fertile eggs (embryos) are kept in the laboratory for 5-6 days and then transferred to the potential mother. The PGD process is used on developing embryos on the 3rd day (7 to 8 cells) before their transfer and the tests are made on only 1 cell (blastomere).

The most important factor while using PGD on the blastomere cell is to prevent the contamination of the blastomere with another cell. Because of that it is important that the center that takes the blastomere cell is careful and experiences. Every cell taken from the embryo are placed in different sterile pcr tubes that contain the necessary solutions. These tubes are sealed carefully and transferred to the center where the PGD will be made. In cases this method can’t be used, every blastomere is put on a “four well” cup as a separate droplet and transported in that manner. In both methods it is very important to enumerate the blastomere carefully.

The blastomeres that arrive in the PGD center will first be put through a 45 minute process with a lysis solution. The obtained DNA samples from the blastomeres are added the HLA marked specific sequences obtained during the set up and those will be prepared for a polymerase chain reaction (PCR). The prepared mix is processed and analyzed at predetermined heat and the sequences belonging to the markers will be multiplied. The obtained PCR product will determine the single sequence using the DNA sequence analysis on the sequence machine. The determined sequence will be analyzed for HLA genotypes on each embryo.

Since the PGD method uses one single cell, the whole process is made on a single DNA helix obtained from one egg cell and one sperm. A minimum of DNA is used. For this reason, some cells the helixes coming from the parents cannot be multiplied during the PCR and a result cannot be obtained (amplification failure). And sometimes one of the helixes belonging to the mother of the father cannot be amplified. This is called a allel drop-out (ADO). ADO is the most concerned situation during the PGT process since it might cause misdiagnosis. To recognize a possible ADO, a second control mechanism is needed during the analysis. At this point the parent-specific informative markers are used to identify a possible ADO. ADO identified cells will not be evaluated since a healthy analysis cannot be made. After the evaluation, the cells that have the same HLA typing as the sick child will be transferred into the potential mother’s womb. Since an analysis made on a single embryo cell has a 2% risk of error, a confirmation must be made during the 11th week with a chorionic tissue sample and an amniocentesis on the 16th week.

Single Gene Disease + HLA

In diseases like thalassemia, sickle cell anemia, leukemia and immunodeficiency disorders (Wiscott-Aldrichsyndrome,Combinedimmunodeficiencydisease, Hypogammaglobulinemia) HLA genotyping can also be used along with pre-pregnancy genetic analysis to determine healthy embryos. In these kinds of families a set-up process is needed for both the parents and the sick child, in order to identify the gene mutations causing the single cell disease and the HLA typing. After that the couple is taken to IVF treatment to acquire embryos. A single cell sample taken during the 3rd day of these embryos will be genetically analyzed for health and compatibility with the sick child’s HLA type. One or two of the fitting embryos will be planted into the potential mother’s womb. Since an analysis made on a single embryo cell has a 2% risk of error, a confirmation must be made during the 11th week with a chorionic tissue sample and an amniocentesis on the 16th week.

Stages of PGD

Set-up
PGD
Embryo transfer

Set-Up

The PGD method used on embryos is a lot more complex and difficult than genetic tests where a simple blood sample suffices. The biggest reason for this is that the process uses a single cell, the blastomere. Further, the determining of the HLA type for in cells that contain diseases like thalassemia makes the processes even more complex. For this reason a 2-3 month long set-up process is needed.

In order to start with the set-up process the genetic disease or mutation in the family must have been identified earlier. It is not technically possible to use the PGD method on families who have only been diagnosed clinically. Families whose mutations have been identified must additionally also meet some requirements in order to use the PGD method. The most important is the set-up process which is necessary because PGD is used on a single cell. Genetic analysis on embryos is difficult because of the scarce amount of DNA sample. Failure of acquiring results or misdiagnosis is risks that emerge from a single cell analysis. To overcome these problems and to minimize the risks the tools are controlled whether they operate on an optimal level. Further informative markers are specified in order to identify the gene zone closes to the embryo. A second important step of the set-up stage is identification of the HLA tissue types by DNA tests on the parents and the sick child. With the set up stage the possible HLA types on the embryos can be predicted and the HLA types compatible with the sick child’s can be pre-determined.

After confirming the mutation, designing, producing and testing of the specific sequences that will be used on the PGD process on embryos and the producing and testing of the informative markers in order to minimize any risk of failure, the typing of HLA tissues and the testing of the HLA markers that will be used during the PGD on single cells, the set-up stage will be completed.

Taking EDTA blood samples and buccal mucosa samples from the parents and the sick child.
If the mutations that cause the disease are not identified yet, the identification of the mutation via DNA analysis from the given blood samples.*
If the mutation was previously identified and is a disease the set-up center is working with, the confirmation of the disease.
If the mutation was previously identified but the set-up center is not working with the disease, the designing and producing of the mutation-specific sequence.
These sequences (primer) will be used to confirm the mutation and to test if the sequences work on the single cell.
The identification and production of the informative markers close to the gene area of the disease the family has.
The testing of these informative markers on the single cell.
The determining of HLA genotypes on blood samples using PCR.
The testing of markers used for HLA genotypes on single cells.
After all these stages are done the IVF center and family is informed that their set-up is complete.
Stages of the set-up process for HLA typing and single gene diseases

* The DNA samples are generally sent to a local or foreign center on which genetic location the mutation that causes the disease takes place. These tests can sometimes take up to 3-4 months. If the mutation is identified after the analysis, it means that the target area where the PGD method will be process is determined and the set-up process can begin.

The most important factor while using PGD on the blastomere cell is to prevent the contamination of the blastomere with another cell. Because of that it is important that the center that takes the blastomere cell is careful and experiences. Every cell taken from the After the set-up stage the potential mother is put on a 10-12 day long hormone-replacement therapy that aims a high rate of egg cell production. Of the egg cells the mature ones will be collected and each of them will be microinjected a sperm cell. After the process the fertile eggs (embryos) are kept in the laboratory for 5-6 days and then transferred to the potential mother. The PGD process is used on developing embryos on the 3rd day (7 to 8 cells) before their transfer and the tests are made on only 1 cell (blastomere). Families with children who carry diseases that can be cured by a bone marrow transplantation, such as thalassemia, sickle cell anemia and immunodeficiency disorders (Wiscott-Aldrich syndrome, Combined immunodeficiency disease, Hypogammaglobulinemia) can apply to an IVF center for a PGD. These families will only be taken into IVF treatment after the completion of the set-up stage.

Preimplantation Genetic Diagnosis (PGD)

Embryo is placed in different sterile pcr tubes that contain the necessary solutions. These tubes are sealed carefully and transferred to the center where the PGD will be made. In cases this method can’t be used, every blastomere is put on a “four well” cup as a separate droplet and transported in that manner. In both methods it is very important to enumerate the blastomere carefully.

The blastomeres that arrive in the PGD center will first be put through a 45 minute process with a lysis solution. The obtained DNA samples from the blastomeres are added the mutation-specific sequence obtained during the set up and those will be prepared for a polymerase chain reaction (PCR). The prepared mix is processed and analyzed at predetermined heat and the sequences belonging to the markers will be multiplied. The obtained PCR product will determine the single sequence using the DNA sequence analysis on the sequence machine. The determined sequence will be analyzed for mutations on the previously determined genetic area and will be thus diagnosed.

Since the PGD method uses one single cell, the whole process is made on a single DNA helix obtained from one egg cell and one sperm. A minimum of DNA is used. For this reason, some cells the helixes coming from the parents cannot be multiplied during the PCR and a result cannot be obtained (amplification failure). And sometimes one of the helixes belonging to the mother of the father cannot be amplified. This is called a allel drop-out (ADO). ADO is the most concerned situation during the PGT process since it might cause misdiagnosis. To recognize a possible ADO, a second control mechanism is needed during the analysis. At this point the parent-specific informative markers are used to identify a possible ADO. ADO identified cells will not be evaluated since a healthy analysis cannot be made. After a mutation analysis the DNA samples of the healthy embryos will be processes for HLA typing. By using the HLA markers predetermined during the set-up stage, the HLA typing of the tissue samples can be determined. This way healthy embryo that has compatible HLA typing with the sick child can be identified.

After the evaluation, the healthy and compatible cells will be transferred into the potential mother’s womb. Since an analysis made on a single embryo cell has a 2% risk of error, a confirmation must be made during the 11th week with a chorionic tissue sample and an amniocentesis on the 16th week.

Reference : http://www.genetiks.com.tr