Pre-Implantation Genetic Diagnosis (PGD)

Pre-implantation genetic diagnosis (PGD) is an alternative to prenatal diagnosis, in which genetic testing is performed on embryos produced by In Vitro Fertilization (IVF) before a clinical pregnancy is established. PGD has been mostly applied to woman of advanced maternal age undergoing IVF in order to increase implantation rates, to reduce spontaneous abortions and to reduce trisomies such as Downs Syndrome. This latter type of PGD is called aneuploidy screening. PGD has also been applied to chromosomal disorders, such translocations, in which it has proven to decrease the number of spontaneous abortions while preventing the conception of affected babies. The number of single gene disorders that have been diagnosed by PGD continues to grow. The most common ones include Cystic fibrosis, fragile X, Myotonic Dystrophy, Thalasaemia and Tay Sachs.

PGD usually requires that the couple undergo In Vitro Fertilization (IVF) treatment. This involves hormonal treatments that allow the collection of multiple eggs from the mother. The eggs are then fertilized using the father’s sperm and the resulting embryos are transferred to an incubator. After three days the embryos usually consist of a tiny ball of eight cells, known as blastomeres. One to two embryo cells (blastomeres) are then removed (biopsied) from each embryo and subjected to genetic testing. If the blastomere is found to be unaffected by the inherited disease then the embryo that it was removed from should also be unaffected. Embryos that are revealed to be healthy can be transferred to the uterus, ultimately producing unaffected babies.

Preimplantation genetic diagnosis (PGD) is an alternative to prenatal diagnosis for people at risk of passing on an inherited disease to their children. The main benefit of PGD is that it maximizes the chance that a couple will have an unaffected pregnancy, greatly reducing the possibility that they will have to contemplate pregnancy termination.

I. SINGLE GENE DISORDERS (MONOGENIC DISEASES)

Genes are chemical messages that instruct cells how to grow and perform the different chemical reactions necessary for life. There are more than 30,000 different genes and virtually every cell in the human body contains two copies of each. One copy of each gene is inherited from the Father and the other copy is inherited from the Mother. It is very important for good health that every gene functions correctly; just one defective gene can result in serious disease. Because children acquire their genes from their parents it is possible for a defective gene to be passed from one generation to the next. This is why some diseases are said to ‘run in families’ affecting generation after generation.

Disorders caused by the inheritance of a single defective gene are known as monogenic diseases or single gene disorders. Monogenic diseases fall into two main categories. First there are ‘recessive’ diseases, which do not produce any symptoms unless a defective copy of the gene is passed on by both the Mother and the Father. The second category is comprised of disorders that are said to be ‘dominant’, which only require one defective copy of the gene to be inherited in order to occur. Hundreds of different monogenic diseases, caused by errors in hundreds of different genes, have been discovered. Most of these disorders are very rare; however a few are relatively common. Well known monogenic diseases include cystic fibrosis, sickle cell anemia and Tay Sachs disease, which are recessive diseases, and myotonic dystrophy and Marfan syndrome, which are dominant.

A. PGD Analysis with PCR

The biopsied blastomeres (cells) are analyzed using a technique called the polymerase chain reaction (PCR). Each cell contains a minute amount of DNA (the material from which the genes are made). PCR is used to amplify the DNA to a detectable level. Once amplification has been accomplished scientists can use a variety of techniques to screen an individual gene for abnormalities. Only embryos with cells that are unaffected by the inherited disease are transferred to the mother’s uterus and consequently only unaffected babies should be born. Using PGD techniques it is possible to detect changes in the genetic code that cause inherited diseases. This is possible even if the defects (mutations) affect just one letter of the 3,000,000,000 letter genetic code.

Testing of the biopsied cells destroys them because their membranes must be broken open to release the DNA. As such, one cannot use them for any other purpose or return them to the embryo.

B. The Risk Of Embryo Biopsy

While PGD is a relatively new procedure in IVF, the micromanipulation or biopsy techniques required to perform the procedure have been in use for many years. The risk of accidental damage to an embryo during removal of the cell(s) is very low, around 0.6%. Procedures such as intracytoplasmic sperm injection (ICSI), fragment removal and assisted hatching are all performed by making openings in the covering of the egg just like with PGD and none have been found to have adverse effects on embryo development and implantation.

No part of the future fetus will be lacking because of the removal of one or two cells from the embryo on the third day after fertilization. All the cells at this stage are said to be totipotent, literally meaning “all potential”. These cells have not differentiated yet and can form any part of the resulting fetus. The cells that are removed are simply replaced by the embryo. The procedure merely delays continued cell division for a few hours, after which the embryo reaches the same number of cells as before and continues its normal development. An unanswered question is whether biopsy affects the frequency with which embryos implant in the uterus. The existing data is incomplete; however, any reduction in embryo implantation due to the effects of embryo biopsy seems to minor.

The accuracy of PGD for monogenic disorders is approximately 95%. This means that the error rate is 5%. This figure includes normal embryos incorrectly diagnosed as affected and abnormal embryos wrongly diagnosed unaffected. Additionally, approximately 10% of embryos tested cannot be diagnosed due to inconclusive results. In most cases inconclusive results are due to the failure of PCR to amplify the DNA to a sufficient level for the disease gene to be detected. Due to the small chance of misdiagnosis as well as the presence of unrelated problems such as chromosome abnormality (for example Downs Syndrome), which we do not test for, we recommend prenatal testing be carried out after a pregnancy is established.

C. Specific Disease Information

1. Myotonic Dystrophy

Myotonic Dystrophy, also known as Steinert's disease or Dystrophia Myotonica (DM) is the most common form of muscular dystrophy, affecting roughly 1 in 8,000 people. The disease has a variety of symptoms including an inability of muscles to relax after contraction, respiratory problems, adverse reactions to anesthesia, cardiac disease, difficulty in swallowing, digestive problems, excessive sleeping and mental disorders. People with DM are more likely to develop diabetes and cataracts later in life. The extent to which these symptoms are manifest varies between individuals. Some individuals remain undiagnosed because their symptoms are so mild. However, at the opposite end of the spectrum infants with the most severe form of myotonic dystrophy often die shortly after birth. In many cases the disease displays an effect known as "anticipation", which means that the symptoms become progressively worse with each generation.

Myotonic dystrophy is a monogenic disease, caused by the inheritance of a single defective gene. Everybody inherits two copies of the myotonic dystrophy gene (one copy from each parent). The inheritance of one defective copy of the gene is sufficient to cause myotonic dystrophy, in other words it is inherited in a dominant fashion. This means that if you are at risk of transmitting a defective myotonic dystrophy gene on average 50% of your children will have the disease.

It is possible to test the myotonic dystrophy gene during pregnancy, thus revealing whether the fetus is affected with the disease. If the fetus is affected then the parents face the difficult decision of whether to continue with the pregnancy or have a termination. An alternative to prenatal diagnosis is to use preimplantation genetic diagnosis (PGD), a method that allows detection of myotonic dystrophy in embryos before they implant in the uterus. The main purpose of this test is to allow patients to have children unaffected by a specific inherited disease, without having to contemplate termination of an affected pregnancy. PGD tests for myotonic dystrophy have been successfully applied resulting in the birth of unaffected babies.

To perform the PGD test it is first necessary for the parents to undergo in vitro fertilization (IVF). Using IVF a number of embryos are usually produced. The embryos are grown in an incubator for three days, by which time they consist of a small ball of about eight cells. At this point a single cell can be removed without harming the embryo. The cell can then be subjected to genetic analysis to determine whether it carries a defective copy of the myotonic dystrophy gene. If no defective myotonic dystrophy gene is detected then the embryo is diagnosed as unaffected. Unaffected embryos can be transferred to the mother’s uterus and any resulting pregnancy should be unaffected.

2. Cystic Fibrosis

Cystic fibrosis is the most common form of inherited defect affecting Caucasians (people of European descent) and currently affects approximately 30,000 people in the United States of America . Approximately one in 25 Caucasians carries a defective copy of the cystic fibrosis gene. Cystic fibrosis is a recessive disease, in other words the inheritance of two defective copies of the gene (one from each parent) is necessary to cause the disease. People with one defective cystic fibrosis gene and one normal cystic fibrosis gene are not affected by the disease, but are said to be ‘carriers’. If a man and a woman who are both carriers of cystic fibrosis have children then on average one child in four will inherit a defective gene from each parent and will therefore be affected by the disease.

Cystic fibrosis affects the mucus and sweat glands of the body resulting in the production of thick mucus in the breathing passages of the lungs. This leads to chronic lung infections. Additionally, mucus obstructs the pancreas, preventing enzymes from reaching the intestines to help break down and digest food. In males cystic fibrosis is also frequently associated with infertility.

It is possible to test the cystic fibrosis gene during pregnancy, thus revealing whether the fetus is affected with the disease. If the fetus is affected then the parents face the difficult decision of whether to continue with the pregnancy or have a termination. An alternative to prenatal diagnosis is to use preimplantation genetic diagnosis (PGD), a method that allows detection of affected embryos before they implant in the uterus. The main purpose of this test is to allow patients to have children unaffected by a specific inherited disease, without having to contemplate termination of an affected pregnancy.

To perform the PGD test it is first necessary for the parents to undergo in vitro fertilization (IVF). Using IVF a number of embryos are usually produced. The embryos are grown in an incubator for three days, by which time they consist of a small ball of about eight cells. At this point a single cell can be removed without harming the embryo. The cell can then be subjected to genetic analysis to determine whether it carries a defective copy of the cystic fibrosis gene. If no defective cystic fibrosis gene is detected then the embryo is diagnosed as unaffected. Unaffected embryos can be transferred to the mother’s uterus and any resulting pregnancy should be unaffected.

3. Tay Sachs Disease

In its classical form Tay Sachs disease causes progressive destruction of the central nervous system and is fatal during childhood. The first symptoms of Tay Sachs usually begin to appear at about six months of age. Development slows, there may be a loss of peripheral vision, and the child exhibits an abnormal startle response. By about two years of age, most children experience seizures and diminishing mental function. As the disease progresses the affected child begins to lose physical and mental abilities and may experience difficulties swallowing and breathing. Ultimately, affected children become blind, mentally retarded, paralyzed, and unresponsive to their surroundings. In cases of classical Tay Sachs survival does not usually exceed five years.

Tay Sachs is a recessive disease, in other words the inheritance of two defective copies of the gene (one from each parent) is necessary to cause the disease. People with one defective Tay Sachs gene and one normal Tay Sachs gene are not affected by the disease, but are said to be ‘carriers’. If a man and a woman who are both carriers of Tay Sachs have children then on average one child in four will be affected with the disease, having inherited a defective gene from each parent. A person's chances of being a Tay Sachs carrier are significantly higher if he or she is of eastern European (Ashkenazi) Jewish descent. Approximately one in every 27 Jews in the United States is a carrier of the TSD gene.

It is possible to test the Tay Sachs gene during pregnancy, thus revealing whether the fetus is affected with the disease. If the fetus is affected then the parents must decide whether to continue with the pregnancy or have a termination. An alternative to prenatal diagnosis is the use of preimplantation genetic diagnosis (PGD), a method that allows detection of affected embryos before they implant in the uterus. The main purpose of this test is to allow patients to have children unaffected by a specific inherited disease, without having to contemplate termination of an affected pregnancy.

To perform the PGD test it is first necessary for the parents to undergo in vitro fertilization (IVF). Using IVF a number of embryos are usually produced. The embryos are grown in an incubator for three days, by which time they consist of a small ball of about eight cells. At this point a single cell can be removed without harming the embryo. The cell can then be tested to determine whether it carries any defective copies of the Tay Sachs gene. If a normal copy of the Tay Sachs gene is detected then the embryo is diagnosed as unaffected. Unaffected embryos can be transferred to the mother’s uterus and any resulting pregnancy should be unaffected.

II. PGD FOR TRANSLOCATION 

Chromosomes are string-like structures found in the center of the cell, the nucleus. They contain genes that are made of DNA. Therefore, our inherited information is housed on the chromosomes. Normal human cells (embryo, fetus, baby or adult) contain 46 chromosomes, or 23 pairs. We receive 23 chromosomes from each parent.

The first 22 pairs of chromosomes are the same for men and women and are labeled largest to smallest from 1 through 22. The 23rd pair determines our sex. A female has two "X" chromosomes whereas a male has an "X" and a "Y." As such, the woman can only pass an X to her child in her egg. The man passes either the X or the Y in the sperm therefore determining the sex of the child. 

A. Chromosome Translocations

A translocation is a change in chromosome structure in which chromosomes are attached to each other or pieces of different chromosomes have been interchanged. An individual with a translocation is unaffected if there is no extra or missing chromosome material and if the break in the chromosome did not disrupt gene function. If there is no additional or missing chromosome material, the translocation is considered to be "balanced." A translocation is "unbalanced" if there is extra or missing material.

Individuals with balanced translocations typically have no medical issues though some do have fertility concerns, such as reduced fertility. The concern regarding having a balanced translocation is that, though the individual is healthy, the egg or sperm of that individual can have an unbalanced chromosome make-up that leads to the resultant embryo or pregnancy being unbalanced. The presence of an unbalanced translocation can lead to an embryo not implanting, a pregnancy being lost or a child being born with mental and physical problems. Individuals with a translocation may, therefore, experience multiple pregnancy losses or have a child affected with physical and mental problems that may be lethal.

B. Reciprocal Translocations

Approximately one in 625 individuals has a reciprocal translocation. These translocations involve any of the chromosomes. Reciprocal defines the translocation as one in which chromosomes have swapped material. Breaks occur anywhere in the chromosomes allowing for pieces to be interchanged between them.

C. Robertsonian Translocations

Approximately one in 900 individuals has a Robertsonian translocation. These translocations involve chromosomes 13, 14, 15, 21 or 22. These chromosomes have a unique structure in that they are primarily made of a bottom half. This translocation results from fusion of two of these chromosomes such that the two bottoms are attached.

D. PGD – The Procedure

1. Biopsy of Polar Bodies

When the person with the translocation is female, we may be able to analyze the polar body. The ripening egg produces two small cells called polar bodies that degenerate after fertilization. The chromosomal content of these cells allows us to infer the chromosomal content of the egg. If one is testing the polar body, an opening is made in the covering of the egg and the polar body is removed with a pipette. The polar body is then analyzed while the egg is placed in an incubator. By analyzing polar bodies, we obtain information from only the mother. Chromosome abnormalities that may occur after fertilization, when the sperm meets the egg, will not be detected.

2. Biopsy of Blastomeres

We analyze blastomeres when the male has the translocation and, in certain cases, when the female has the translocation. A blastomere is a cell from an embryo. To test the blastomere, an opening is made in the covering of the embryo during its third day of development when the embryo has 8-10 cells. A blastomere is removed via aspiration with a pipette. The embryo is placed in an incubator while the cell is analyzed.

3. Analysis

The biopsied cells are analyzed using a technique called fluorescence in-situ hybridization or FISH. This technique uses probes, small pieces of DNA that are a match for the chromosomes we want to analyze, to study the chromosomes present. Each probe is labeled with a different fluorescent dye. These fluorescent probes are applied to the biopsied cell and attach to the chromosomes. Probes attach to specific areas of the chromosome or can be used to color the whole chromosome. Under a fluorescent microscope, balanced and unbalanced chromosomal make-up can be identified in that cell. The geneticist, therefore, can distinguish normal cells from cells with an unbalanced translocation.

Testing of the cells destroys them because they must be glued to a glass slide and repeatedly heated and cooled. As such, one cannot use them for another purpose or return them to the embryo. The slides are kept for future reference. This analysis causes no extra inconvenience to the patient as it is accomplished in one day.

E. Advantages of PGD

1. Reduction in the Chance of Having a Child with the Translocation

If your child has a translocation, this can cause reproductive problems for them as well.

2. Reduction in Pregnancy Losses

The PGD procedure significantly reduces the chance of pregnancy loss. The patients who achieved pregnancy after PGD had experienced miscarriage in the majority (>90%) of their previous pregnancies. When these same patients underwent PGD, fewer than 10% of pregnancies were miscarried. This is a significant reduction in pregnancy losses.

F. Misdiagnosis

The accuracy of PGD for translocation is approximately 90%. This means that the error rate is 10%. Within this chance of misdiagnosis, there is a false negative rate, a false positive rate, the chance for no result and the chance for mosaicism. A mosaicism is defined as the embryo having cells with different chromosome make-up. Typically, all cells of the embryo have the same chromosomal make-up as they originate from the same fertilized egg. However, it is possible for cells of the same embryo to have differing numbers of chromosomes.

If we analyze a cell that has normal chromosomal content, but another cell has an extra chromosome, we erroneously diagnosed that embryo as being chromosomally normal. Due to the chance of misdiagnosis as well as the presence of other chromosome conditions for which we do not test, we recommend prenatal testing via chorionic villious sampling or amniocentesis.

G. Few Eggs Produced or No Normal Embryos for Transfer

Overall, translocation patients produced an average of 9.5 mature eggs compared to 13 in non-translocation patients. The proportion of abnormal embryos found from translocation carriers has ranged from 0 to 100%, with an average of 65% abnormal embryos. In approximately 22% of cycles, all the embryos were chromosomally abnormal. Therefore, it is possible that less than three embryos (or even none) will be available for transfer, which may lead to lower pregnancy rates than for non-translocation patients.

H. Preliminary Analyses

If the individual with the translocation is a male, we recommend FISH analyses of sperm prior to undergoing PGD. Determination of the percentage of unbalanced sperm will allow for estimates of the percentage of embryos that will be unbalanced, and therefore, determination of whether PGD is the best option.

III. PGD For Aneuploidy

Chromosomes are string-like structures found in the center of the cell, the nucleus. Chromosomes contain genes that are made of DNA. Therefore, our inherited information is housed on the chromosomes. Normal human cells (embryo, fetus, baby or adult) contain 46 chromosomes or 23 pairs. We receive 23 chromosomes from each parent. The first 22 pairs of chromosomes are the same for men and women and labeled largest to smallest 1 through 22. The 23rd pair determines our sex. A female has 2 “X” chromosomes whereas a male has an “X” and a “Y”. As such, the woman can only pass an X to her child in her egg. The man passes either the X or the Y in the sperm therefore determining the sex of the child. If an error occurs leading to the egg or sperm having an extra or missing chromosome, the embryo created by that egg or sperm would have an extra or missing chromosome. This situation is called aneuploidy. Having an extra chromosome is known as trisomy (tri = three of the chromosome) and having a chromosome missing is known as monosomy (mono = one of the chromosome). If the aneuploidy involves the larger chromosomes, the embryo may not attach to the wall of the uterus or may stop developing soon after attaching and miscarry. However, if the aneuploidy involves chromosomes such as the 13, 18, 21, X or Y, the pregnancy may still carry on until birth, even though the pregnancy has a chromosomal disorder. The most common of these is an extra number 21, known as Down syndrome or trisomy 21 (three 21 chromosomes). Other common aneuploidies are Klinefelter syndrome (XXY), trisomy 13 and trisomy 18. The features of the chromosome condition depend upon which chromosome is extra or missing, but can include physical differences and mental retardation.

A. Risk of Aneuploidy and Maternal Age

As a woman advances in age, the chance of aneuploidy in her pregnancies increases. This association is because a woman’s eggs are as old as she. Females have all of their eggs in the fetal stage therefore they are born with all the eggs they will have in their lifetime. In males, sperm is made every 65-75 days therefore the sperm is not as old as the man. Therefore, the theory regarding aneuploidy risk and advancing maternal age is that over time the chromosomes in the egg are less likely to divide properly leading to the egg having an extra or missing chromosome. The risk of aneuploidy increases with maternal age. The chances to deliver an affected child are 1/385 at 30, 1/179 at 35, 1/63 at 40 and 1/19 at the age of 45. However, the frequency of aneuploidy in embryos is much higher than what would be expected looking only at affected live borns. More than 20% of embryos from women in the age range from 35 to 39 are affected. Almost 40% of embryos from women 40 or older are affected. This difference in percentages in embryos versus live borns is due to the fact that a pregnancy with aneuploidy is less likely to attach to the uterus or go to term. Most will be miscarried. As such, the percentage of affected pregnancies is reduced over the course of the pregnancy due to the affected pregnancies that are lost. Any embryo with a missing chromosome (monosomy) will cease to grow before implantation (except monosomy X and 21), and only few of those carrying an extra chromosome (trisomy) will go to term. The lack of implantation and loss rate of aneuploid embryos are believed to be the main reasons why the pregnancy rate in women over 40 is so low. The purpose of preimplantation genetic diagnosis for aneuploidy therefore is to select for transfer only chromosomally normal embryos so as to achieve more pregnancies, reduce the number of pregnancy losses, and reduce the number of affected offspring.

B. Biopsy of Polar Bodies

The ripening egg produces two small cells called polar bodies that degenerate after fertilization. The chromosomal content of these cells allows us to infer the chromosomal content of the egg. If one is testing the polar body, an opening is made in the covering of the egg and the polar body is removed with a pipette. The polar body is then analyzed while the egg is placed in an incubator. By analyzing polar bodies, we obtain information from only the mother. Chromosome abnormalities that may occur after fertilization, when the sperm meets the egg, will not be detected.

C. Biopsy of Blastomeres

A blastomere is a cell from an embryo. To test the blastomere, an opening is made in the covering of the embryo during its third day of development when the embryo has 8-10 cells. A blastomere is removed via aspiration with a pipette. The embryo is placed in an incubator while the cell is analyzed.

D. Analysis

The biopsied cells are analyzed using a technique called fluorescence in-situ hybridization or FISH. This technique uses probes, small pieces of DNA that are a match for the chromosomes we want to analyze, to count the chromosomes present. Each probe is labeled with a different fluorescent dye. These fluorescent probes are applied to the biopsied cell and attach to the chromosomes. Under a fluorescent microscope, we then count the number of chromosomes of each type (color) there are in that cell. The geneticist therefore can distinguish normal cells from cells with aneuploidy. Testing of the cells destroys them because they must be glued to a glass slide and repeatedly heated and cooled. As such, one cannot use them for another purpose or return them to the embryo. The slides are kept for future reference. This analysis causes no extra inconvenience to the patient as it is accomplished in one day.

E. Reduction in the Chance for Aneuploidy

According to current figures, the chance for a woman delivering a baby with aneuploidy is on average 1% if she is 35-39 years of age and ~3.5% if she is 40-45. So far, in 427 conceptions obtained through PGD, two (0.5%) were chromosomally abnormal compared to 2.8% expected for that group of patients according to their maternal ages (data collected up to 8/2003). PGD does significantly lower the chance of having an affected baby. However, we are unable to test all of the chromosomes at present. We therefore recommend that prenatal testing be performed in the resultant pregnancy via chorionic villous sampling or amniocentesis in order to confirm our diagnosis from PGD and to rule out other aneuploidies for which we do not test.

F. Increased Implantation Rate

It is well known that the pregnancy rate after in-vitro fertilization decreases dramatically with maternal age. Even in IVF centers with the highest pregnancy rates, there is a decrease from approximately 28% per embryo transferred in women 20-33 years old to 9% in women over 39. Aneuploid embryos have much lower survival rates than normal embryos, and half of them (the ones missing a chromosome) seldom implant. It appears likely that the decrease in pregnancy rates with maternal age is mostly caused by a corresponding increase in aneuploid embryos.

By performing PGD for aneuploidy and transferring only chromosomally normal embryos, we may be able to increase the pregnancy rates noticeably. In three studies, a demonstrated increase in implantation rates after PGD was seen (Munné et al. 1999, Gianaroli et al. 1999b, Munné et al. 2003). The implantation rate also doubles from 12% in controls to 24% in PGD patients when 8 chromosomes were analyzed but not when only 5 chromosomes were analyzed. Thus is very important that the PGD test analyzes at least 8 chromosome pairs.

G. Reduction in Pregnancy Losses

In women 35 and older, approximately 35% of pregnancies are miscarried. Aneuploidy is the cause in 50% or more of these losses. By transferring only chromosomally normal embryos, the number of pregnancies going to term should increase. In one study, a significant reduction in pregnancy losses after PGD, from 23% to 9% was demonstrated. The increase in implantation rate and the significant decrease in pregnancy loss rate resulted in a significant increase in ongoing pregnancies and delivered babies.

H. Misdiagnosis

The accuracy of PGD for aneuploidy is approximately 90%. This means that the error rate is 10%. Within this chance of misdiagnosis, there is a false negative rate, a false positive rate, the chance for no result and the chance for mosaicism. A mosaicism is defined as the embryo having cells with different chromosome make-up. Typically, all cells of the embryo have the same chromosomal make-up as they originate from the same fertilized egg. However, it is possible for cells of the same embryo to have differing numbers of chromosomes. If we analyze a cell that has normal chromosomal content, but another cell has an extra chromosome, we erroneously diagnosed that embryo as being chromosomally normal. Due to the chance of misdiagnosis as well as the presence of anueploidy for which we do not test, we recommend prenatal testing as stated earlier.