It is now theoretically possible to diagnose>99% of inherited single-gene disorders by means of preimplantation genetic diagnosis (PGD). The list includes more than 400 genetic diseases and continues to expand.
The technology required for preimplantation genetic diagnosis (PGD) encompasses genetics, IVF, embryo biopsy and single-cell diagnosis. Since separate disciplines and techniques are required for embryo biopsy and diagnosis, preimplantation genetic diagnosis (PGD) must be carried out by a genetics team in collaboration with an IVF team with access to molecular biology technology. The embryo biopsy technique is performed by trained embryologists, whilst the diagnosis is performed by a molecular biology laboratory and confirmed by genetics specialists.
The procedure for preimplantation genetic diagnosis (PGD) involves:
Selection of a viable embryo.
Collecting genetic material (embryo biopsy).
Preparation of the genetic material for analysis (DNA isolation and amplification).
Analysis of results and selection of the embryo for transfer to the recipient.
Genetic analysis of embryos in fertility clinics currently has two different applications:
Preimplantation genetic diagnosis (PGD) for couples known to carry genetic disorders such as single-gene diseases or chromosomal translocations.
Preimplantation genetic selection (PGS or PGD-A), used to identify embryos with a normal karyotype.
Embryo biopsy is performed using micromanipulation equipment used for ICSI. All of the biopsy techniques involve two stages: zona drilling and aspiration. Cell biopsies can be taken from oocytes/embryos at three different stages:
Polar body biopsy in the unfertilized oocyte/zygote.
Cleavage stage biopsy from the 6- to 8-cell embryo.
Blastocyst stage biopsy.
Polar Body Biopsy
Biopsy of the first polar body was developed in order to overcome ethical objections to embryo biopsy, on the basis that the “ethical status” of the unfertilized oocyte differs from that of the embryo. Some individuals opt for preimplantation genetic diagnosis (PGD) in order to avoid termination of pregnancy, and performing the test on a preimplantation embryo may be just as objectionable as termination of pregnancy. Polar body biopsy was first used for the detection of CF. Due to crossing-over events, the second polar body is also required in some situations.
Polar body screening detects only maternal meiotic aneuploidy and will not identify errors that are due to paternal or post-zygotic factors. Biopsy of both polar bodies is recommended for preimplantation genetic diagnosis (PGD).
Cleavage Stage Biopsy
Biopsies performed at the four-cell stage may alter the ratio of inner cell mass to trophectoderm (TE) cells, which may be detrimental to embryo development. Therefore, biopsy at the six- to eight-cell stage, on Day 3 post-insemination, is preferred. Several difficulties arise from cleavage stage embryo biopsy: the first is that human embryonic cells are very fragile and easily lyse. If this occurs during the biopsy procedure, the nucleus may be lost, and another cell will have to be removed.
Compaction occurs between the eight-cell and morula stage, and during compaction the cells of the embryo can no longer be distinguished as they flatten out over each other to maximize intercellular contacts. If the biopsy is performed at this stage, it is very difficult to remove a blastomere, as it has established strong contact with adjacent blastomeres. Trying to remove a cell from a compacted embryo may also result in lysis of the cell.
The thickness and dynamics of the zona pellucida also vary between patients and can lead to some problems during the biopsy procedure. In many cases, numerous sperm is associated with the zona pellucida, and therefore intracytoplasmic sperm injection (ICSI) should always be used with PCR techniques to reduce the risk of sperm contamination.
Studies of both cryopreserved/thawed embryos and those biopsied for preimplantation genetic diagnosis (PGD) have shown that up to 50% of the embryo mass may be lost, and yet lead to a healthy live birth. Almost 97% of embryo biopsies are successful, with more than 90% of the embryos surviving. Pregnancies following embryo biopsy showed no significant developmental differences compared to controls. Deliveries, including infant birth weight and Apgar scores, were considered to be normal.
Embryos that have reached the blastocyst stage are currently preferred for embryo biopsy, performed on Day 5 or 6 post-insemination. Vitrification is accepted as a successful method of cryopreserving blastocysts, and vitrifying blastocysts after biopsy allows more time for the diagnosis. Blastocyst biopsy has the advantage that a larger number of cells can be removed from the outer TE layer without affecting the inner cell mass from which the fetus later develops. Analysis of a larger number of cells is of benefit in diagnosis of monogenic diseases. However, TE cells may have diverged genetically from the inner cell mass (ICM): confined placental mosaicism, where the chromosome status of the embryo is different from the placenta, is observed in at least 1% of conceptions.
Studies indicate that blastocysts may have high levels of chromosomal mosaicism. Preferential allocation of abnormal cells to the TE may also be a mechanism of early human development. In this case the TE would not be representative of the rest of the embryo, and the analysis would complicate and compromise the outcome for the patient.
One of the limitations of blastocyst biopsy is that many embryos will not reach the blastocyst stage. Although improvements in culture conditions have increased the numbers of blastocysts available, a large number of embryos are required for preimplantation genetic diagnosis (PGD) diagnosis to ensure that normal embryos are available for transfer. This may not be the case when embryos are cultured to the blastocyst stage.
Analysis of Biopsied Cells
Tools and technology used to examine cellular chromosomes and genes have evolved considerably over the past decade: the original FISH/PCR methods have now been replaced by more sophisticated, complex and accurate technology to analyze all 24 chromosomes, complemented by computer software and bioinformatics for data analysis in reaching a diagnosis. Modern tests rely on initial DNA amplification in order to supply a template sufficient for comprehensive chromosome screening (CCS).
A variety of “kits” offering different analytical platforms for CCS are commercially available, each with its own specific advantages and disadvantages. External commercial laboratories can now carry out diagnostic tests on cells that have been biopsied and sent from an IVF laboratory. The field continues to evolve rapidly, with new technologies offering increased sensitivity in combination with automation and high throughput.
The increased resolution of new technologies will continue to add to overall data about the health of preimplantation embryos. The biological significance of some of the new information revealed by these technologies is as yet unknown. There is a crucial need for further ongoing basic research directed toward understanding the molecular biology of preimplantation development.
Cells removed from the embryo after biopsy are used for diagnosis. PCR can be used to diagnose single-gene defects, triplet repeat disorders and embryo sex. Karyotyping requires a metaphase spread of chromosomes, and therefore this cannot be used on single embryonic cells, as they do not divide well in culture and it is difficult to obtain metaphase spreads. In cases where a metaphase spread is obtained, the chromosomes are short and difficult to band. FISH was previously used to examine chromosomes in embryos for embryo sexing, chromosome abnormalities and aneuploidy, now replaced by techniques for CCS.
Similar to above, PCR can be used to diagnose single-gene defects, triplet repeat disorders and embryo sex, but it is not a simple procedure. The procedure is complicated by the major problems of contamination and allele dropout. If a diagnosis is available on whole DNA, it should be possible to make such a diagnosis sensitive at the single-cell level. However, some modifications of the procedure may be required.
A common method of making the PCR procedure more sensitive is the use of nested PCR, where an inner set of primers amplifies the original PCR product. Since amplification failure can occur, it is essential that preimplantation genetic diagnosis (PGD) do not rely on a negative result. To ensure that a single-cell PCR method is accurate and sensitive, a preliminary workup is usually performed on single cells, such as buccal cells, from normal, carriers and affected individuals.
PCR products have been analyzed by hetero-duplex analysis, SSCP, ARMS and restriction endonuclease digestion. Fluorescent PCR is a quantitative PCR method that can also be used. For the diagnosis of some diseases, such as fragile X, polymorphic markers may be used to identify which chromosome the embryo has inherited; i.e., the normal or at-risk chromosome.
Single-cell PCR is so sensitive that it will amplify any DNA that may contaminate the PCR reaction, such as a stray cumulus or sperm cell that may have been released from the zona during the biopsy, cells from the atmosphere or DNA found in the air or medium. Steps must be taken to eliminate contamination in order to reduce these problems to a minimum. These include working in a positive pressure PCR room, performing ICSI for all PCR diagnosis and examining PCR products in a separate laboratory.
Misdiagnoses reported after preimplantation genetic diagnosis (PGD) probably arise from contamination. These problems can be reduced with the use of a multiplex PCR with markers that can identify all four parental alleles to ensure that the amplified product is of embryonic origin.
Allele Dropout (ADO)
Allele dropout (ADO), or preferential amplification, refers to the situation where one of the two alleles preferentially amplifies over the other. For example, for a heterozygous cell, the normal allele may preferentially amplify so that the diagnosis would only identify the normal allele. The embryo would be diagnosed as normal instead of heterozygous. This would not cause a problem for recessive conditions where both partners carry the same mutation, but would create problems for dominant disorders, or in cases where the couple carry different mutations for a recessive disorder, as affected embryos could be diagnosed as normal. To reduce this problem, methods can be built into the diagnosis to ensure that both alleles can be identified.
Fluorescent In-Situ Hybridization (FISH)
FISH was the first technique used in preimplantation diagnosis of aneuploidy, and been subsequently applied to detect chromosomal translocations. Fluorescently labeled molecular probes are used to identify chromosomes or their fragments, with the probes binding to specific DNA regions and appearing as fluorescent spots. Three types of FISH probes have been commonly used:
Repeat sequences or alpha satellite probes, which can be used in interphase and metaphase chromosomes. They bind to repeat sequences, usually to the centromeres (with the exception of chromosome 9 and Y) and can be used directly labeled with fluorochromes. They require only 1 hour for hybridization and have been cloned in plasmids and cosmids. Probes for 13/21 and 14/22 cross-hybridize.
Locus-specific probes can be used in interphase or metaphase chromosomes and bind to a unique sequence. They require 6–12 hours for hybridization and have been cloned in cosmids or YACs (yeast artificial chromosomes).
Chromosome paints can only be used in metaphase chromosomes. They paint the entire chromosome.
The FISH technique involves several stages:
Pepsin digestion to remove any protein from around the nuclei. This is especially important for blastomeres.
Paraformaldehyde fixation to ensure that the nuclei adhere to the slide.
Denaturation to make nuclear and probe DNA single-stranded.
Hybridization: probes find and bind to the complementary sequence.
Post-hybridization washes to remove unbound probe.
Detection; for use with indirect probes.
FISH has been used to sex embryos for preimplantation genetic diagnosis (PGD) in cases of X-linked disease. It has advantages over PCR sexing as the copy number are identified: the difference between XO and XX can be determined, and there is no risk of contamination. Probes for chromosomes X, Y and 18 are used and only embryos showing normal female chromosomes are transferred. However, specific diagnosis of the disorder using a molecular method is preferred for diagnosis of X-linked disease, as this will differentiate between affected male, unaffected male, carrier female and non-carrier female.
FISH diagnosis is performed on interphase nuclei. The biopsied blastomere is disrupted in hypotonic solution, digested and fixed. FISH allows every nucleus within an embryo to be examined, but the number of chromosomes that can be analyzed at one time is limited. Using two rounds of FISH on a single nucleus allows a panel of seven to nine chromosomes to be screened (commonly including X, Y, 13, 16, 18, 21, 22), but repeated denaturation leads to DNA degeneration and decreases the efficiency of the procedure.
FISH analysis has many limitations and is associated with a high risk of technical error in the preparation of the material. It is not possible to test all 24 chromosomes simultaneously. Thus, the diagnosis of PGS-FISH is limited to the most common abnormalities, including chromosome 13, 15–18, 21, 22, X and Y. A study showed that patients treated by preimplantation genetic diagnosis (PGD) using FISH on Day 3 embryos had a lower pregnancy rate than the control group, casting doubt on the utility of preimplantation genetic diagnosis (PGD) by this method as an adjunct to IVF.
Comprehensive Chromosome Screening
Advances in molecular biology technology now allow all 24 chromosomes to be analyzed using a variety of techniques that have been validated for clinical application, including micro-array comparative genomic hybridization (aCGH), single-nucleotide polymorphism micro-arrays (SNP arrays), and quantitative polymerase chain reaction (qPCR). aCGH and SNP arrays depend on first amplifying the cell DNA (whole genome amplification), and this creates a risk of allele dropout, which can potentially result in misdiagnosis of monogenic diseases.
A number of different analytical platforms are available, and technology continues to evolve: aCGH has been widely used in recent years, but is now slowly being replaced by Next-Generation/Massive Parallel Sequencing (NGS/MPS).
Comparative Genomic Hybridization (CGH)
Comparative genomic hybridization detects duplications or deletions of chromosome fragments. DNA labeled with a fluorescent dye from a normal control patient is placed on a slide or metaphase plate, alongside fluorescently labeled patient DNA. The two genomes, coded with different colors, are cut enzymatically into small fragments which then reorganize on chromosomes using rules of complementarity and competition for hybridization sites.
All quantitative differences between them are visible as the predominance of one color over another. The main disadvantage of the classical CGH method is its low resolution, which averages 10 Mbp. An improved version, known as array CGH (aCGH), is therefore used in preimplantation diagnosis. Array CGH is considered to be precise and highly specific, but resolution is limited in detecting translocated fragments below 6 Mbp in size.
Single-Nucleotide Polymorphism (SNP) Analysis
Single-nucleotide polymorphisms are sites in a genome where one nucleotide in a specific locus is different from the others in the population. SNP markers utilize platforms/plates that allow thousands or millions of SNPs in a human population to be determined during a single DNA analysis. There are a large number of SNP methods based on hybridization, starter elongation, ligation, or the so-called invasive rupture.
Next-Generation Sequencing (NGS)
Next-Generation Sequencing uses sequencing on reaction vessels with a diameter of 3 μm to allow the parallel processing of a large number of nucleic acid molecules, up to an entire human genome. NGS has revolutionized sequencing: more than 10 NGS platforms are currently available, and technology continues to evolve. NGS can be used in fresh and frozen cycles, and can be used to analyze many types of genetic variability. It is the only method that allows aneuploidy or translocation of all chromosomes and mutations responsible for any single-gene disease to be analyzed using one biopsy: whole chromosome abnormalities, segmental abnormalities, translocations, single-gene disorders and mitochondrial disorders can potentially be diagnosed. A number of samples can be simultaneously sequenced during a single “run,” which reduces the cost per sample.
DNA is first isolated from a single or several cells, and the whole genome is amplified. The DNA is then cut into fragments of 100–200 base pairs and placed on a 2 × 2 cm “chip.” A sequence of each fragment is then compared to a reference sequence, and the results are analyzed using a computer. The sequencing library can be automated, reducing hands-on time, minimizing human errors and enabling higher throughput and consistency.
Copy Number Variation Sequencing (CNV-seq)
CNV-seq is a refinement of NGS technology that can detect and quantify gene duplications or deletions. Around 99% of the variations in human gene copy number are benign, but the remainder is associated with clinically significant chromosome disease syndromes. Around 200 different chromosomal diseases are known to be caused by CNV, and these may be either inherited from parents or due to errors occurring during chromosome replication.
Karyomapping uses SNP micro-array technology to map crossovers between parental haplotypes, using the principles of linkage analysis and chromosomal haploblock inheritance. The mother, father and a reference affected family member or grandparents are compared to map the origin of each chromosome inherited. Karyomap gene chips have been designed that use biomarkers within the genome to assess the probability that an embryo carries a gene variant for single-gene disorders. They can simultaneously analyze nearly 300,000 SNP loci and can be used for preimplantation genetic diagnosis (PGD) of various monogenic diseases, as well as PGS for aneuploidy.