The process is for the determination of the genetic information in question. There are two types of genetic information that are of interest:
1. Chromosomal Composition
The most common test performed is the determination of the chromosomal composition of the embryo. This is referred to as aneuploidy testing. Each normal embryo contains 46 chromosomes, 23 of which are contributed by the sperm and 23 by the oocyte. An embryo that contains 46 normal chromosomes is called a euploid embryo. An embryo that has more or less than 46 chromosomes is called an aneuploid embryo. There are two types of chromosomes, the sex chromosomes (X and Y), which determine the gender of the embryo, and the autosomes (1-22), which determine almost everything else. The autosomes are normally present in pairs. The sperm contributes one sex chromosome (X or Y) and 22 autosomes. The oocyte contributes one sex chromosome (X only) and 22 autosomes. Sometimes microarray is referred to as 24-chromosome microarray: 22 chromosomes, and X and Y are counted as one each, for a total of 24. As previously mentioned there are two techniques for testing the chromosomes of a biopsied embryo.
The procedure that has been used the longest is called Fluorescent In Situ Hybridization (FISH). Currently, cells are tested with probes that detect twelve of the 23 chromosome pairs: 8, 13, 14, 15, 16, 17, 18, 20, 21, 22, X and Y. This means that a cell from a normal embryo would have 24 spots of light detected when tested.
The process of screening embryos for genetic abnormality has become much more useful because of a new technology called 23-chromosome microarray. It is now possible to determine the complete chromosomal composition of an embryo, (that is all 23) from testing a single cell. Each chromosome is composed of two strands of DNA that are chemically attracted to each other in a way that holds them together for their entire length. These two strands of DNA are referred to as complementary strands and it is the chemical attraction between complementary strands that is the basis for testing chromosomes with microarray. 23-chromosome microarray is done by first making many copies of the DNA obtained from the nucleus of the cell that has been removed from the embryo to be tested. That process is known as PCR or polymerase chain reaction. PCR refers to a technique for amplifying or copying minute DNA amounts from a single cell to analyze for a specific genetic defect. The extracted cell is placed in a buffered solution and rapidly heated to break the original DNA strands and then cooled to reassemble them into copies. Multiple "thermal" cycles eventually produce billions of duplications of a single, targeted DNA sequence.
The two strands of DNA are then separated and only one strand from each chromosome is used for testing. The DNA is then broken up into very small pieces and then stained with a dye of a certain color. DNA obtained from a cell, which is known to have normal chromosomes, is then broken up into the exact same small pieces and stained with a dye of a different color.
A glass slide is then coated with the pieces of DNA that are from the complementary strand of the stained pieces of DNA to be tested. These pieces of DNA are from a cell with normal chromosomes and are arranged in a pattern on the slide referred to as a microarray. Because of their ability to attract the complementary strand, pieces of DNA on the microarray will attract the stained pieces of DNA when it is added. The stained pieces of DNA from the test cell and the control cell are then added to the microarray. Since each cell has been stained a different color, it is possible to see how much DNA from each cell has been attracted to the pieces of DNA on the microarray by determining how much of each color is present. The pieces of DNA on the microarray represent all of the chromosomes in a normal cell and the location of all of the pieces that make up each chromosome are known. Therefore it is possible to determine how many copies of each chromosome are present by looking at the amount of each color that is present on all of the pieces of DNA that make up a particular chromosome on the microarray. For example, if the test cell has two copies of a particular chromosome then it is normal. So when the microarray is examined, there would be an equal amount of each color present for that chromosome since the control cell also has two copies of each chromosome. If however, the test cell has three copies of a particular chromosome; then it is abnormal and referred to as a trisomy. This is the case in the condition known as Down's Syndrome where there are three copies of chromosome 21. The microarray from a trisomy would have more of the color from the test cell than the color from the control cell.
2. Single Gene Mutations
23-chromosome microarray can also be used to diagnose single gene mutations (the presence or absence of a specific gene in a given embryo). This type of information is useful when one of the prospective parents is known to be a carrier of a gene responsible for a particular disease. The number of diseases known to be caused by a single gene abnormality is growing as researchers learn more about the composition of the human genome (there are more than 300+ single gene mutation tests available). Currently, the most frequently tested genes are those giving rise to diseases such as Cystic Fibrosis, Tay-Sachs, Hemophilia, Thalassemia, Marfan Syndrome and Sickle Cell Disease among others.
This type of testing is much different than chromosomal testing. Chromosomes are very large compared to single genes. Each gene is composed of a small piece of DNA that in turn is made from combinations of four basic molecules hooked together in a precise sequence. There are many genes present in each chromosome so in order to detect whether or not the gene in question is present, it must be separated from the chromosome and then copied millions of times so that there is enough present to be seen by laboratory detectors. This is done by cutting the gene out of the chromosome by enzymes called restriction endonucleases. These enzymes will dissolve the attachment of the gene to the chromosome in a very precise manner so that only the sequence of DNA that is needed will come out. This piece of DNA corresponding to the gene in question is then put through a process called the Polymerase Chain Reaction (PCR). These amplified replications can then be tested for more than 300 single gene mutations.
Numerical chromosome irregularities (aneuploidies) are common in early human embryos and contribute significantly to implantation failure as well as to early pregnancy loss. Although the sperm certainly contributes, it is primarily the oocyte that is the driving force in the establishment of embryo competence. The karyotype of the mature oocyte (MII) has emerged as being the most important single determinant of oocyte developmental competence with aneuploid oocytes rendered unfertilizable, arresting at various stages after fertilization, producing embryos that fail to implant, resulting in early miscarriages, or producing a variety of chromosomal birth defects. Assessment of embryo morphology with phase-contrast microscopy, although helpful, fails to provide reliable information by which to define the developmental competence of the embryo.
Traditional karyotyping of metaphase chromosomes using commercially available FISH is too inefficient and unreliable at the single cell level to be of practical use in fresh In-Vitro Fertilization (IVF). Although widely used to identify embryo chromosome irregularities, FISH is of limited value because only a few chromosomes can be identified in each cell (usually X, Y, 13, 15, 16, 18, 21, and 22). An attempt to increase the number of chromosomes analyzed by simultaneously adding more FISH probes reduces the accuracy of differentiating between various chromosomal elements and can lead to misdiagnosis. The matter is further complicated by the fact that the chromosome probes used in commercial FISH were designed to detect aneuploidies involved in spontaneous miscarriages, not necessarily the often lethal chromosomal aneuploidies that lead to dysfunctional embryogenesis and failed implantation.
Newer technologies such as 23-chromosome microarray facilitate single cell karyotyping. The technique both enumerates and provides a comprehensive assessment of each chromosome, thereby identifying breakages and even partial aneuploidies.
Egg/Embryo karyotype (chromosome) testing using 23-chromosome microarray, while clearly a major breakthrough in the In-Vitro Fertilization (IVF) arena, is not a panacea. First, an embryo diagnosed to have all its chromosomes (euploid) through egg and/or embryo microarray testing will, in about 15% of cases, turn out to be "incompetent." This is due to the fact that even chromosomally normal cells, upon further division at times can generate some aneuploid cells, leading to a condition known as Mosaicism (the presence of both chromosomally normal and abnormal cells) in the blastocyst. Depending on the percentage of aneuploid cells in the advanced embryo (blastocyst), mosaicism might not be lethal. Second, a competent embryo might fail to continue developing because of poor uterine receptivity rather than embryo aneuploidy. Third, embryo transfer (ET), another rate-limiting factor in In-Vitro Fertilization (IVF), requires a great deal of technical expertise and there is a wide variation in such expertise.
The above serves to explain why the transfer of "competent" (23-chromosome microarray normal) embryos will result in a live birth rate of 60-70% but not in 100% of cases.
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