PGD reveals cause for IVF implantation failure

During in vitro fertilization – IVF, embryos are chosen for transfer based on two separate criteria. In all IVF cycles, the microscopic evaluation of the embryo is carried out at varied stages to identify those with the best overall appearance and development characteristics. In some IVF cycles, preimplantation genetic diagnosis – PGD is performed to also identify those embryos with abnormalities in the number of chromosomes present. These abnormalities are known as aneuploidies. Embryo chromosome abnormalities such as aneuploidies are thought to be responsible for implantation failure, miscarriage and birth defects. For determining abnormalities in chromosome number, two

PGD methods

have been used. The first, which is called FISH analysis, is in general clinical use today. With FISH, or fluorescent in-situ hybridization, a fluorescent "tag" or marker is attached to a molecular probe which recognizes and "sticks" to a portion of a specific chromosome. This enables an observer to count the number of copies of that chromosome within a cell of the embryo. A normal human embryo contains 23 pairs of chromosomes for a total of 46. Aneuploid embryos may be missing one chromosome of a pair and therefore have only 45 chromosomes or have an extra copy and have 47. FISH can be performed on several different chromosomes at once but not all 23. The chromosomes most commonly tested are those that are found to be abnormal most commonly in miscarriage specimens — 13, 16, 18, 21, 22, X, Y and sometimes 15 and 17.

A second type of

PGD analysis

is called comparative genomic hybridization (CGH). During CGH, a labeled identical copy of each chromosome in a cell is created. This DNA copy is then hybridized, or matched up with, a labeled copy of DNA from a cell that is known to be normal. The sample being tested is labeled with one color and the normal DNA is labeled with a different color. The amount of each color is analyzed by computer software. If the cell being tested contains the normal amount of chromosomes, the color intensity is the same as the normal control cell. If there are too many chromosomes, the intensity will be greater and if there are too few chromosomes, the intensity will be lighter.

CGH is a difficult test to perform and takes 5 days to complete. If performed on DNA from a polar body biopsy, then a fresh embryo transfer might still be performed. If performed at the cell cleavage stage on Day 3, then the embryos have to be cryopreserved in order and used later during a frozen embryo transfer cycle. This has been a limitation of the general application of the technique.

In a study published in the May 2007 issue of the medical journal Fertility and Sterility, researchers set out to determine if couples who failed to achieve pregnancy with IVF after multiple transfers had a higher incidence of chromosome abnormalities than other patients.

They looked at data from

PGD screening

using CGH on 176 embryos from 28 women. Twenty-two women had a history of recurrent implantation failure (RIF), and six did not. Women were classified as having RIF when 10 or more embryos had been transferred previously without achieving a pregnancy. The average number of previously transferred embryos in this group was 16.5.

The authors created a category of abnormalities that they referred to as "complex". An embryo was considered to have a complex abnormality if there 3 or more chromosomes that were not present in correct numbers. Seventy-six (45%) embryos tested normally and were suitable for transfer, 46 (27%) showed aneuploidy for one or two chromosomes, 7 (4%) showed partial aneuploidy, and 49 (29%) had complex abnormalities involving three or more chromosomes.

There were several interesting findings

  • The older patients exhibited a greater percentage of embryos that had simple abnormalities involving one or two chromosomes. This is a well known fact: Older women produce a higher percentage of aneuploid embryos.
  • The percentage of embryos with complex abnormalities did not vary with age.
  • The percentage of embryos with complex abnormalities was higher in women who had recurrent implantation failure compared to other types of patients.
  • 25% of the time, chromosome abnormalities found in the embryos with complex abnormalities would be missed if only FISH testing was performed.
  • These results suggest that aneuploidy for one to two chromosomes occurs by a different mechanism than aneuploidies for larger number of chromosomes. Couples with recurrent implantation failure may therefore suffer from a different type of problem. Evidence from other types of studies suggest that the chromosome errors in this situation arise after fertilization when the embryo is dividing. These post fertilization errors are as likely to occur in younger women as in older women. They occur more commonly in couples with recurrent implantation failure and therefore may represent a new diagnostic category of infertility.