Genetic testing part two: Micro array assays may deliver on clinical promise

October 24, 2010Carole 1 Comment »

In vitro fertilization (IVF) has delivered steadily improving pregnancy rates since the first live birth from IVF in 1978, but future gains in delivery rates may require genetic analysis of embryos before transfer. Not all embryos are created equal. Some transferred embryos will not implant. Some will implant and fail early in the pregnancy. Some will implant and babies will be born with chromosomal abnormalities like an additional Chromosome 21, causing Downs Syndrome. Some will implant and a healthy baby will be born. Unfortunately, all these embryos look the same to the embryologist and if the embryos are still (apparently) alive and at the right developmental stage on the day of transfer (either day 3 or 5 of culture), back they go into the uterus with fingers crossed.

Because embryos with an abnormal number of chromosomes look normal, more than one embryo is typically transferred, compensating for the expectation that some embryos will fail at some stage. Transferring more embryos increases the pregnancy rate but also increases the  possibility of having multiple-gestation pregnancies (twins or more) which are more likely to have problems during the pregnancy and even after birth, possibly even lasting health problems. Older women tend to have a lower pregnancy rate, in large part due to the fact that older women produce eggs with more chromosome abnormalities and thus embryos with more aneuploidy. Aneuploid (chromosomally abnormal) embryos do not produce healthy pregnancies. Eliminating these from the transfer pool and only transferring chromosomally normal embryos should increase delivery rates.

What the ART field has been looking for is a way to quickly, reliably and cheaply determine which embryos in the transfer pool are chromosomally normal and transferring only those to increase the delivery rate for all patients.

A little background may help. For each of the 22 human non-sex chromosomes, there should be a pair of chromosomes, one inherited from Mom and one from Dad. Aneuploidy is a condition in which an abnormal number of chromosomes (more than or less than two) for a type of chromosome (1-22) exist. Extra or too few copies of sex chromosomes inherited from one parent or the other are also possible. Many aneuploidies are lethal for the embryo and others can cause early or recurrent miscarriages. Some aneuploidies such as an extra Chromosome 21 are compatible with life but they cause Down’s Syndrome, a genetic condition causing various levels of cognitive impairment and physical problems.

The clinical potential of PGS has not be fully realized due to technical limitations of FISH. Fluorescent In Situ Hybridization or FISH was developed to identify aneuploid embryos before transfer, but FISH to detect aneuploid embryos hasn’t increased delivery rates and some studies suggest PGS may actually reduce the rates due to a combination of technical problems and genetic instability in cleavage stage embryos, making test results less accurate.

FISH is performed on chromosomes isolated from a single embryonic cell and  placed on a slide. The chromosomes are tested with fluorescent probes specific for some but not all possible chromosomes.  The FISH assay is not able to test all 24 chromosomes, 22 regular and two sex chromosomes, on one cell because the slide prep can’t hold up to repeated probings, washings and reprobing that would be necessary. So choices are made in advance about which chromosomes will be tested and it is possible to miss abnormalities by not probing for all possible chromosomes. Removing more than one cell at a time from the eight cell embryo is sometimes used to increase the reliability of FISH results, but this is also controversial because some studies suggest that this extra biopsy reduces pregnancy rates by stressing the embryo.

Two new assays, Comparative Genomic Hybridization (CGH) and Single Nucleotide Polymorphism (SNP) arrays may overcome the limitations of FISH, providing a more accurate clinical predictor of which embryos are best to transfer. The paper “Use of comprehensive chromosomal screening for embryo assessment: microarrays and CGH” provides an in-depth technical explanation of how CGH and SNP arrays work. For an overview of the various applications of  chromosome-based genetic testing  on health care, you can read “Copy number variation and genomic alterations in health and disease”.

What is Comparative Genomic Hybridization? CGH, also called molecular karyotyping, is a method to identify copy number variants (CNVs) or changes in the normal number of chromosomes or quantity of genetic material associated with each chromosome. These variations from normal are detected as variations in the number of chromosomes of each type or variations in smaller stretches of DNA of at least 1000 base pairs.  Molecular karyotyping looks at DNA from the entire genome and compares it to control DNA, looking for areas in which there are abnormal quantities or copies of DNA associated with each chromosome.

The principle of the test is based on genetic compare and contrast. The entire genome (all the DNA from a sample of embryonic cells) is amplified creating thousands of copies of the original DNA to work with and this super sized sample is labeled with a colored fluorescent dye- colored either red or green. A control batch of chromosomally normal DNA sample is also amplified and labeled with the other color fluorescent dye. After the DNA batches are labeled, equal amounts of DNA from each batch (control and test) are mixed together and analyzed for their comparative ability to compete for a match (or hybridize) with normal DNA on a test slide. The test slide can contain small pieces of normal cell DNA (microarray CGH assay)  or whole metaphase-stage chromosomes (chromosomal CGH assay).

When  a piece of DNA finds a match to the pieces of DNA on the slide, it will hybridize or stick to the DNA on the slide like the matching side of a zipper, nucleotide base pair matched to nuclear base pair. If there are too many copies of one sequence of test DNA (red colored), it will grab up more of the available matches than the green colored control DNA. Red will then be the dominant color for that hybridization spot on the test slide. The ratio of red to green fluorescence detected along the length of the slide is analyzed. Ratios in favor of either green or red identify regions in the test DNA that are either deficient or have extra copies of the test DNA relative to the slide DNA. CGH can not detect balanced translocations or inversions because the copy number remains the same for these abnormalities, but they are still abnormal because sections of DNA are swapped between chromosomes.

Comparative Genomic Hybridization has several advantages over FISH. Abnormalities can be identified on smaller units of the chromosome in CGH than is possible with FISH. In addition, using CGH, all 24 chromosomes can be analyzed instead of only 10-12 chromosomes that are currently analyzed using FISH.

What is a SNP? A Single Nucleotide Polymorphism can be thought of as naturally occurring variation in the letters used to write genes.  Nucleotides are the DNA alphabet that is used to write the genetic code for a gene. For every gene, there can be variations in how the gene is written and sometimes you have a choice of two alleles or letters in the sequence that will work equally well to write a gene.  For example, Eric can be written as “Eric” or “Erik” or even “Erich”, all acceptable variations of letters used to write the same name. Most SNPs have two possible variations (alleles) at a single nucleotide position or locus. Some genetic variations are harmless, some may be advantageous and others cause disease. By looking at all the SNPs in a person’s DNA, one person can be distinguished from another- a type of genetic fingerprint. These natural variations can be detected at the level of the nucleotide or single base pairs, compared to CGH which finds variations that must be at least 1000 base pairs long.

For SNP microarrays, the technical strategy is a little different than for CGH. The test DNA is  amplified and labeled but it is allowed to find a match on the test microarrray in parallel with the control DNA on a separate part of the slide, but they are not mixed beforehand. The fluorescent intensity of spots for test and control DNA on the two sides of the slide are compared with one another directly. Embryo DNA can be compared to parental DNA as well so it is possible to identify the parental source of each inherited piece of DNA. In addition,  the intensity of fluorescent labeling indicates an excess or deficit of chromosomes relative to the normal number found in the control DNA.

Both of these to tests have advantages over FISH

  • All 24 chromosomes can be analyzed in the embryo, instead of only 10-12 with FISH. Arrays allow simultaneous testing of all chromosomes at once. In FISH analysis, every new probe requires that the test slide is re-used by removing the old probe and reprobing, degrading the FISH signal and test quality with each test cycle.
  • CGH and SNP is more sensitive than FISH because these methods are able to detect variations in not just whole chromosomes but also smaller regions of at least 1000 base pairs for CGH and  single base pair for SNP analysis. Theoretically, a variation of SNP can be used to detect single gene mutations as well, allowing both aneuploidy testing and testing for single gene mutations in the same sample.
  • The use of trophectoderm biopsy (which is preferred for these tests) allows more cells to be analyzed, resulting in more reliable clinical results.
  • Trophectoderm biopsy may have another advantage. Some research has suggested that biopsy of the eight cell embryos may not be the best test source because the cleavage stage embryo may be less genetically stable and have more genetic mosaicism than later stages of embryo development.
  • SNP analysis can be used as a type of fingerprint analysis to compare a child’s DNA profile with the embryos transferred to identify which embryos implanted in a successful pregnancy, which may yield clues to genetic markers that are associated with successful implantation and delivery. This fingerprint analysis may also be used to confirm genetic linkage of the embryo to parents before transfer, possibly providing an extra safeguard against embryo mix ups.

Although promising, the verdict is still out on whether CGH and SNP analysis will improve delivery rates for IVF patients and there are some additional issues (of course) with any new test. SNP is particularly expensive and so costs will probably need to come down significantly for widespread clinical use to be feasible. Secondly, there is some argument as to which stage of embryo development is best for biopsy to get the most reliable results. If trophectoderm biopsy is used, the clinic must also have perfected the technique of vitrification because test results will be returned too late for a fresh transfer and embryos are typically cryopreserved at the blastocyst stage. If excellent PGD techniques are combined with poor freezing techniques, no usable embryos can be recovered so clinics must master cryopreservation techniques. Dr. Joyce Harper elaborates on some of these obstacles in her opinion piece in BioNews, “What next for PGS?”.

PGS using single nucleotide polymorphisms may be problematic because extra information about other diseases may be revealed that are not immediately important to the future of the embryo- raising ethical issues. Will we find out “too much” about the embryo and start to select on the basis of traits that are not lethal or associated with genetic disease? Do we select on other genetic characteristics such as having the BRCA gene which is associated with increased risk of breast cancer in the future?- or a gene that causes heart problems in midlife?  If we find a deafness gene, is it ethically acceptable to select against this embryo? With these technical advances may come new ethical issues as we get more information about embryos prior to transfer, allowing patients ever greater choices about the composition of the families they create.

The data is still out in these new assays and several new random controlled trials are currently underway to look at whether these next generation techniques will finally deliver on the promise of improved delivery rates from pre-transfer selection of chromosomally normal embryos. The potential pay-off is huge- a way to increase the pregnancy rate while simultaneously decreasing the multiple rate, increasing the chances for a single healthy baby for every IVF patient.

© 2010, Carole. All rights reserved.

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