Diagnostic methods in PGD are based on DNA technology. PCR is used for the detection of single gene mutations related to monogenic disorders while FISH is used to screen for aneuploidy or structural chromosomal abnormalities Sophie will give an in depth talk on the methods of analysis.
PRE-IMPLANTATION GENETICDIAGNOSIS (PGD) What is PGD? First reported in 1990, PGD combines advances in molecular genetics and in assisted reproductive technology and is conducted before the embryo is placed in the womb of the woman . PGD is a procedure used in conjunction with In Vitro Fertilization (IVF) in which genetic analysis is done of a single cell from an eight-cell embryo. PGD is conducted with an intention to improve the chances of a “normal” pregnancy. WHY?
PRE-IMPLANTATION GENETICDIAGNOSIS (PGD) Why PGD? Reproductive failure (i.e. failed implantation, miscarriages and major birth anomalies) are far more likely to be due to embryo incompetence (70-75%) than to a lack of uterine receptivity (25-30%). The number of chromosomes in a cell is referred to as its ploidy. A cell with a normal number of chromosomes is referred to, as euploid, while one with an irregular chromosome number is aneuploid. Itappears that it is the ploidy of the mature egg (rather than the sperm) that determines the post-fertilization chromosome configuration of the embryo. The embryos ploidy, in turn, determines its competence.
ANEUPLOIDY Aneuploidy = abnormal number of chromosomes, and is a type of chromosome abnormality. An extra or missing chromosome is a common cause of genetic disorders (birth defects). Aneuploidy occurs during cell division when the chromosomes do not separate properly between the 2 cells. Aneuploidy =most frequent cause of spontaneous abortions. Aneuploidy outcome- termination of developing fetus, Most common extra chromosomes among live births are 21, 18 and 13.
WHY CONSIDER PGD IN ADDITION TOIVF? Chromosome abnormalities (aneuploidies) are associated with failed implantation, pregnancy loss, and the birth of children with multiple congenital anomalies. Preimplantation genetic diagnosis (PGD) provides a means of testing for these chromosome abnormalities and selecting the best embryos for transfer. So, what is aneuploidy?
WHEN TO CONSIDER PGD IN ADDITIONTO IVF? Recurrent miscarriages One child already affected with a genetic disease Family history of inherited disease Maternal age older than 38 Couples with >3 IVF failures Epididymal or Testicular sperm aspiration with >1 IVF failures Family “balancing” for sex
INDICATIONS FOR PGD When there is suspicion of Chromosomal Disorders Chromosomal rearrangements Inversions Translocations Chromosome Deletions Severe monogenic diseases cystic fibrosis, ß thalassaemia, sickle cell anemia, fragile X syndrome, myopathies
STAGES IN PGD1. Monitor egg maturation in the ovary by the use of Ultrasound & Hormone levels2. Collect eggs (mother’s own or from donor) in 2 steps - A. Injection of human chorionic gonadotropin (hCG) and follicle stimulating hormone (FSH) to time egg ripening B. Transvaginal aspiration using hollow needle
STAGES IN PGD3. Obtain sperm from father/donor) & assess quality4. Combine eggs and sperm in vitro, using intracytoplasmic sperm injection (ICSI), if sperm is low quality5. Nurture embryo growth by incubating in medium containing various nutrients and hormones6. For doing PGD, remove one cell called blastomere from the 6-8-cell embryo after 2-3 days (6-8 cell stage) for testing.7. If fine on PDG, incubate until embryo is 5-6 days old (blastocyst) and transfer embryos (usually 3-6) to uterus, artifically removing zona pellucida.
METHOD OF PGD In skilled hands, this generally does not harm the developing embryo and this is done using a fine glass needle to puncture the zona pellucida and aspirate the cell. Blastomere Biopsy on Day 3 Genetic Analysis
PROBLEMS WITH PGD The single blastomere cell cells contains too little DNA to do extensive testing. So “modified multiple displacement amplification” is done to allow researchers to amplify or make carbon enough copies of the DNA they obtain from an embryo obtained by in vitro fertilization, to do multiple tests. The amplified DNA from the couple’s embryos are then sent for testing for aneuploidy using a test called 23- chromosome microarray.
BENEFITS OF PGD Increases implantation success rate Reduction in the chance of having a child with aneuploidy Reduces the possibility of having to choose to terminate the pregnancy following a diagnosis of a probable genetic disorder. Reduction in pregnancy losses
PGD PROCESS 2 types of PGD assessment techniques are common: 1. Genetic testing for specific disease loci by Polymerase Chain Reaction (PCR) or gene chips) amplification of DNA specific to a gene of interest (family history guides choice of genes). 2. Chromosome “painting” or Fluorescence In Situ Hybridization (FISH)
PCR Molecular genetic disorders, i.e single gene disorders - diagnosed by PCR Appears to be available in India in some clinics Needs to be adapted to single-cell PCR due to scarcity of embryonic material available
PCR - GENETIC DISORDERSDETECTABLE Tay Sachs (autosomal recessive; ~98% accuracy) Cystic fibrosis (autosomal recessive; ~85% for common allele mutation) Huntington’s disease (autosomal dominant) Thalassemias (autosomal recessive blood disorder) Duchenne muscular dystrophy (X-linked recessive) Spinal muscular atrophy As more genetic tests are developed, more will be available for predictive purposes in PDG.
FISH Appears to be the most widely performed PGD test in India & involves chromosome “painting” or karyotyping, using fluorescent probes specific for each chromosome. This procedure destroys the tested cell but permits number and size of each chromosome to be checked. Useful for identifying aneuploidies (incorrect chromosome numbers) and translocations. Conventional FISH cannot fully access all the chromosomes - in fact, only about 12 of them.
FISH Thus, even when FISH reveals that all the accessed chromosomes are normal, there still remains more than a 40% chance of chromosomal aneuploidy involving those chromosomes not targeted by the test. FISH testing is able to detect the most common chromosome abnormalities in order to reduce the risk of having an affected pregnancy or child., including Down syndrome, Trisomy 18, Trisomy 13, and sex chromosome anomalies.
CGH / CMA Comparative genomic hybridization (CGH) is a molecular-cytogenetic method for the analysis of copy number changes (gains/losses) in the DNA content of a given subjects DNA This technique compares the amount of DNA present for each chromosome in a single cell, and compares it to that of a normal standard. Chromosomal Microarray Analysis (CMA) allows for evaluation of all 23 chromosome pairs in a single cell. Research shows, however, that abnormalities involving any chromosome can increase the risk of miscarriage and reduce the effectiveness of IVF.
CGH / CMA This test is valuable at looking for previously unknown mutations that can lead to children with dysmorphic features, developmental delays, mental retardation, and autism. Microarray can be performed on three different sample types: polar bodies (from fertilized eggs), blastomeres (from Day 3 embryos), or blastocyst/trophectoderm (from Day 5 embryos). 23-chromosome microarray will replace FISH for this reason .
CGH / CMA Relatively new technique Does not appear to be available in India. By the CMA chromosome testing, one is able to analyze the entire chromosome complement of a single cell, reducing the risk of failed implantation and miscarriage, and increasing the chance of having a healthy baby
TRANSLOCATION TESTING Accuracy of the PGD for translocation is 90% and may be of 2 types: 1. “Balanced” if chromosome material merely switches locations with no net loss or gain; or 2. “Unbalanced” if switch is accompanied by a net loss or net gain of genetic material
PGD - ADVANTAGES PGD can help eliminate some genetic diseases cures for which are not likely to be found soon (eg, Tay-Sachs disease, cystic fibrosis, Huntington disease, X-linked dystrophies. genetic disease testing is currently performed through amniocentesis or chorionic villus testing (CVS) with fetus aged 10-16 weeks. If positive for genetic defect, the option is to have a child with a genetic disease or to undergo MTP. Difficult and often traumatic decision that PGD can eliminate.
PGD - LIMITATIONS PGD is expensive, time and labor intensive to develop and work with single-cell diagnostic techniques. PGD can only detect a specific genetic disease in an embryo. Itcannot detect many genetic disorders at a time and cannot guarantee that the fetus will not have an unrelated birth defect. Removal of a single cell without breaking it or causing serious damage is technically difficult and requires skill and experience. Damage to the embryo (projected to be 0.1%) may accidentally occur during removal of the cell.
PGD - LIMITATIONS For aneuploidy screening, not all chromosomal or genetic abnormalities can be diagnosed with PGD because only a restricted number of chromosomes can be examined at one time during the course of a single procedure. Currently, FISH offers evaluation of less than half of the 23 chromosomes; usually 9-11 are analyzed. Studies using comparative genetic hybridization (CGH) and FISH demonstrate that as many as 25% of aneuploid embryos are characterized as normal because the abnormal chromosomes were not analyzed.
PGD - LIMITATIONS Current recommendations from the Society for Assisted Reproductive Technology (SART) and American Society for Reproductive Medicine (ASRM) state that Available evidence does not support the use of PGS to improve live-birth rates for advanced maternal age, recurrent pregnancy loss, or implantation failure Also recommends that patients be counseled about the limitations of the technique and should not make future treatment decisions based solely on PGD result.
PGD - POTENTIAL In the future, genetic links to common diseases (eg, diabetes, hypertension, cardiovascular diseases, endometriosis, cancers) may be identified, and PGD will become available to control the transmission of these diseases to future generations.
PGD –EQUIPMENT/CONSUMABLES/MANPOWER Equipment cost is negligible and consists of: Thermal cyclers, fluorescence microscopes The problem is with: High consumable cost – for PCR, FISH and CGH Manpower cost – also very high. Need highly trained personnel
PGD - LOCATION A PGD test center would presumably need to be attached to an IVF clinic – time between testing and implantation is 1-2 days. Transport of genetic material/embryos does not seem feasible. Hospitals – some offer PGD (*), others just have IVF clinics Dozens of IVF clinics across India Many claim to offer PGD (primarily FISH) The Centre for DNA Fingerprinting and Diagnostics (CDFD) – currently not offering PGD but a VERY wide range of pre-natal tests offered. http://www.cdfd.org.in/index.html