PGD - Reproductive Biology Associates

Each of the 100 trillion cells in the human body (except for red blood cells) contains the entire human genome. Chromosomes are the string-like elements within the nucleus (center) of every cell of your body. They contain genetic information made of DNA. A gene occupies a specific location on a chromosome. Normally, there are 23 identical pairs of chromosomes (6 feet of DNA) in each cell, with a total of 46 chromosomes. Each partner normally provides 23 chromosomes during fertilization. If an egg or sperm have an abnormal chromosome package, the embryo that they create will have a chromosomal abnormality as well. This is sometimes due to a rearrangement of chromosomes, or a missing piece of a chromosome. In some cases there is a missing chromosome, or an extra chromosome (aneuploidy) leading to an inheritable disorder. Any embryo with a missing chromosome (monosomy) will stop growing before implantation (fatal anomaly). If the aneuploidy involves chromosomes including 13, 18, 21, X or Y, the pregnancy may go to term. The most common of these non-fatal anomalies is trisomy 21, or Downs syndrome, in which an extra chromosome 21 is present. Others include Turner’s syndrome in females and Klinefelter’s Syndrome in males.

The first live births following PGD were reported in London in 1989. Two sets of twin girls were born to five couples at risk of passing on an X-linked disorder. About 90% of abnormal embryos can now be detected using PGD techniques. Not all chromosomal or genetic abnormalities can be determined with these procedures, since only a restricted number of chromosomes can be diagnosed at one time during the course of a single procedure. Numerous animal studies and some human studies show that the microsurgery of the embryo (biopsy) needed to remove the cells does not affect the normal development of the baby. This procedure, however, has only been performed in less than 500 patients worldwide, therefore, the precise negative effects, if any, are unknown. Even though there have been more than 200 live births after PGD for aneuploidy world-wide to date (May 2001), this procedure is still relatively new. In animal studies there have been no apparent problems and preliminary evidence with human embryos suggests that this is also true. In a study at the University College of London, researchers recently examined 12 preimplantation embryos with a new technique that combines whole genome amplification (WGA) and comparative geomic hybridization (CGH). Results were that 8 in 12 embryos studied were found to have significant chromosomal abnormalities. This may explain why humans have, at best, a 25% chance of achieving a viable pregnancy per month.

In the diagrams below, D or d represents the defective gene, and N or n represents the normal gene. A mutation does not always result in disease.

Dominant Disorders:

One of the parents has a single defective gene, which dominates it’s normal counterpart. Since offspring inherit half of their genetic material from each parent, there is a 50% risk of inheriting the faulty gene, and therefore the disorder.

Recessive Disorders:

Both parents carry a single defective gene, but also carry a normal gene counterpart. Two defective copies of the gene are necessary to cause inheritance of the disease. Each offspring has a 50% chance of being a carrier, and a 25% chance of inheriting the disorder.

X-Linked Disorders:

Normal females have are XX, and normal males are XY. Women who have a normal gene on one of their X chromosomes are protected from the defective gene on their other X chromosome. However, males lack this protection due to the presence of only one X chromosome. Each male offspring of a mother who carries a defect has a 50% chance of inheriting the defective gene and the disorder. Each female offspring has a 50% chance of being a carrier like her mother. (in the diagram below the X represents the normal gene and the X represents the defective gene)

Preimplantation genetic diagnosis allows the selection and transfer of unaffected (chromosomally normal) embryos which may result in a higher implantation rate per embryo, a reduction in pregnancy loss, and the birth of a higher number of healthy babies. PGD offers a couple an alternative to agonizing over whether to terminate an affected pregnancy after prenatal diagnosis is made following amniocentesis or Chorionic villa sampling (CVS) at later stages of gestation. Almost all genetically linked diseases that can be diagnosed in the prenatal period by either amniocentesis or CVS can also be detected by PGD. The procedure should reduce the psychological trauma experienced by couples who carry an increased risk for an offspring with a genetic disease. Benefits of PGD may include:

At best, about 90% of abnormal embryos can now be detected using PGD techniques.

A relatively large number of eggs or embryos may be found to be abnormal and therefore leave few embryos for replacement. In some cases (11%), no eggs or embryos may be normal. In these cases, embryo replacement will not be recommended. Though this is a disappointing outcome, it is likely that without PGD the IVF cycle would not have resulted in a pregnancy or that an abnormal fetus would have resulted.

The cells to be removed are studied with specialized new techniques. Such procedures may fail due to technical malfunctions.

Not all chromosomal or genetic abnormalities can be determined with these procedures, since only a restricted number of chromosomes can be diagnosed at one time during the course of a single procedure.

It is possible that a normal embryo may be incorrectly identified as being abnormal and not be replaced, or that an abnormal embryo is incorrectly identified as being normal and replaced into the uterus. (PGD is not currently considered a replacement for prenatal testing. Prenatal diagnosis is recommended to confirm the prognosis).

Genetic and developmental damage (0.1%) to the embryo may accidentally occur during removal of the cell(s).

Unknown technical circumstances in the laboratory may cause failure of the testing process, making results unavailable. Failure of the testing process has no effect whatsoever on your embryos. In this case, embryos will be selected for transfer based on existing criteria.

Analysis of a single cell has limitations. Occasionally, chromosome anomalies are present in one cell, but not in other cells of the same embryo, or vice-versa, resulting in mosaicism. This may lead to transfer of an abnormal embryo, or discard of a normal embryo.

PGD for translocation testing can determine the presence or absence of a certain chromosomal disorder, but cannot detect genetic disease nor predict genetic malformation.

Even with a successful PGD procedure, pregnancy may not occur.

Candidates for Embryo Biopsy and PGD

Candidates for Embryo Biopsy and PGD include:

Women over 34 years of age: Women are born with all the eggs they will ever have, and as a woman ages, her eggs are exposed to this ageing process as well. Therefore, the chance of conceiving a chromosomally abnormal offspring increases with age. Overall, the risk of aneuploidy increases from 1 in 385 at age 30, to 1 in 179 at age 35, to 1 in 63 at age 40, and at the age of 45 the chance of delivering an affected child is 1 in 19. By utilizing PGD with IVF it has been learned that in fact as many as 20% of embryos from women aged 35 to 39 are affected, and almost 40% of embryos from women over 40 are affected. Most of these embryos, if replaced into the uterus, will either not implant or will miscarry. These are considered to be the main reasons why the pregnancy and birth rates in women 40 years of age and over are so low. Prior to PGD, larger numbers of embryos were replaced into the uterus in order to increase the chances of conception. Prenatal testing after the IVF cycle is still strongly advised, since this would confirm the prognosis for a normal offspring. It is also possible that an abnormal embryo may be incorrectly identified as normal and replaced into the uterus.

Woman with recurrent pregnancy loss: A male or female partner may contribute an abnormal chromosome package which can cause a fatal anomaly in some pregnancies, and not in others.

Couples with a translocation: A translocation is a change in chromosome configuration in which chromosomes are attached to each other (Robertsonian) or pieces of different chromosomes have been interchanged (reciprocal). Approximately 1 in 900 individuals have a Robertsonian translocation, involving chromosomes 13, 14, 15, 21, 22. Approximately 1 in 625 individuals have a reciprocal translocation. A karyotype of both partners may be done to identify the presence of a translocation. Couples with a translocation may experience recurrent pregnancy loss, or have an offspring with mental of physical problems. In a balanced translocation, when there is no extra or missing chromosome material, and the break in the chromosome does not disrupt gene function, the individual is unaffected. Carriers of balanced translocations may be affected by cryptic congenital malformations, which may or may not be related to the inherited condition. With an unbalanced translocation, one in which there is missing or extra chromosome material, individuals will typically be unaffected, though some do have reduced fertility. However, there is a risk that the egg or sperm of that individual can have an unbalanced translocation, resulting in an embryo being unbalanced. This may cause failure of implantation, recurrent miscarriage, or an offspring with mental or physical problems.

Couples with autosomal dominant diseases in which 50% of embryos would be affected. Couples who have a family history of, or are carriers of, or affected by an inheritable disease.

Couples with repeated IVF failure.

Couples with a history of infertility may be able to identify an etiology, and therefore choose the appropriate treatment.

Couples at risk for offspring inheriting a life-threatening disease, a disease of late onset (Huntington’s), may be better able to plan, choose appropriate treatment options, or accelerate the screening process (such as early screening for breast cancer)

Couples desiring an offspring with stem cells that are an HLA match for an affected offspring with a fatal disease.

The Techniques Utilized

To analyze for the presence of a genetic defect of an embryo, it is necessary to remove either the first polar body of an unfertilized egg and/or 1 or 2 cells from each embryo. This is called an egg or embryo biopsy and is usually done before insemination occurs, or 3 days after fertilization. Biopsy of at the 6-10 cell stage does not adversely affect preimplantation development. At this stage each cell has a full complement of chromosomes. Normally only a single cell is removed from each embryo, since it is expected to be identical to all of the other cells in the embryo. Occasionally, it is necessary to remove a second cell from an embryo, if a signal is not found in the first, for instance. FISH analysis has been used for preconception diagnosis by using the first and second polar bodies as an indicator of the genetic status of the oocyte. A disadvantage of polar body analysis alone is that it does not take into account paternal aneuploidies.

The analysis of the biopsied cell(s) utilizes one of two techniques:

Fluorescence in–situ hybridization (FISH): The biopsied cell(s) are fixed to a glass slide and heated and cooled and their DNA is “labeled” with colored fluorescent dyes called probes (little pieces of DNA that are a match for the chromosomes being tested), one for each chromosome to be identified. At present 8 of the 23 chromosomes can be identified. Once complete, the embryologist counts the colors under a high-powered microscope and is able, in most cases, to distinguish normal from abnormal cells. The process takes about a day. Normal embryos will either be transferred into the uterus on day 4 following egg retrieval, or left in extended culture and transferred on day 5 as blastocysts. The cells utilized for PCR are no longer viable, and will not be replaced into the embryo(s), but may then be stored for future investigation.

Polymerase chain reaction (PCR): A technique which amplifies the number of copies of specific regions of DNA in order to produce enough DNA to be sufficiently tested. DNA is double-stranded (except in some viruses), and the two stands pair up in a very specific way. A gene’s “building-block sequence” is the specific order of appearance of 4 different deoxyribonucleotides within a segment of DNA. These 4 components are: adenine (A), thymadine (T), cytosine (C), and guanine (G). The arrangement of this 4-letter alphabet generates a gene sequence. In this technique DNA is heated (denaturization) in order to separate the 2 strands. Primers are added and then the DNA is cooled in order to allow double-strands to form again. An enzyme is then added in cycles, which can “read” the gene sequence, and result in multiplication of DNA. PCR is utilized to diagnose gene-specific diseases, as well as disease-causing viruses and/or bacteria, or to link a criminal suspect to a crime.

What disorders are RBA currently capable of examining embryos for?

RBA is currently capable of examining embryos from in vitro fertilization utilizing either in-house FISH analysis or PCR in collaboration with other centers for the following disorders:

Age related aneuploidies

Cystic fibrosis

Tay-Sachs

Hemophilia A&B

Retinitis pigmentosa

Sickle cell disease

Thalassemia

Alport’s syndrome

Alpha 1 antitrypsin deficiency

Fragile X

Duchenne muscular dystrophy

Myotonic dystrophy

Lesch-Nyhan syndrome

Rhesus (RhD)

Marfan’s syndrome

Familial adenamatous polyposis coli

Huntington’s disease

X-linked disease by sex determination:

Translocations

In addition, other IVF centers capable of performing embryo biopsy have the option of sending cells to the RBA laboratory for FISH analysis.