Preimplantation genetic diagnosis (PGD) was first reported in 1990. Thereafter, more and more indications for PGD, including monogenic diseases (MGD) and translocations, are presently available, and the list of indications of PGD is expanding from early-onset and serious conditions to late-onset diseases. Polymerase chain reaction has been used for PGD of MGD, while newer techniques, including karyomapping and next-generation sequencing, emerge in recent decade. The limitations of various methods for PGD are discussed in this review.
Highlights
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Various indications for PGD are available. The indications for PGD have expanded from early–onset, serious diseases to the more controversial late-onset diseases.
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Limitations of PGD should be addressed properly to reduce the chance of misdiagnosis.
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Technical errors can be reduced by implementing strict laboratory protocols.
Background
The first baby delivered after sex selection by genetic test on embryo for X-linked diseases in a process known as preimplantation genetic diagnosis (PGD) was reported in 1990 . In the report, two pairs of twins with four babies born without severe X-linked diseases were born. Soon after, the first baby diagnosed by PGD for another monogenic disease (MGD), cystic fibrosis, was born free from the parental mutant alleles . The aim of PGD is to provide an alternative reproductive option for couples at risk of giving birth to offspring with severe genetic diseases of childhood onset so as to avoid the physical and psychological trauma related to abortions when an affected pregnancy is diagnosed after spontaneous pregnancy.
Indications of PGD
Potentially fatal genetic conditions are common indications for PGD ( http://guide.hfea.gov.uk/pgd/ ). Cystic fibrosis is the most common, potentially fatal autosomal recessive disease in the Caucasian population. The offspring of a couple in which both partners carry heterozygous pathogenic mutations would have a 25% chance of suffering this debilitating condition that primarily affects the lungs. The disease has no cure and requires multidisciplinary medical care, and the affected individuals would have shortened life expectancy. For example, in the Chinese population, thalassemia is the most common autosomal recessive disease, especially in the southeastern part of China. Babies with thalassemia major (i.e., homozygous for the deletion of alpha-globin genes in alpha-thalassemia or heterozygous mutations in beta-globin genes in beta-thalassemia) do not survive, suffer from hemoglobin Bart’s hydrops fetalis syndrome, or are blood transfusion dependent from early infancy because of Cooley’s anemia. PGD is a reproductive option for couples carrying thalassemia traits . In addition to childhood-onset genetic diseases, the indications of PGD have been extended to adult-onset diseases . Huntington’s disease (HD) and spinocerebellar ataxia are good examples of adult-onset diseases that can be detected by PGD. Individuals with these mutations or trinucleotide repeats in the disease range will develop these debilitating diseases later in their lives, most likely in adulthood, with no cure. Mutations with incomplete penetrance are also indications for PGD. Mutations of the tumor suppressor genes BRCA1 and 2 are good examples, and both can cause cancer predisposing syndrome . Patients with mutation of the BRCA1 or BRCA2 gene will experience substantially elevated risks of cancers of breast, ovarian, colonic, pancreatic, and prostatic origins.
There are some ethical concerns regarding the use of PGD in these adult-onset diseases or conditions with incomplete penetrance because of the “tradition” to offer PGD to early-onset conditions as their phenotypes are expressed early. In conditions with incomplete penetrance, there is a possibility, although very slim, that the individuals carrying the mutations may not develop the genetic conditions. However, there is no consensus on the line drawn to demarcate what conditions are serious enough or what age onset is eligible for PGD. Individuals carrying the mutations suffer from psychological burden to various extents, worrying about the development of the full-blown disease later in their lives. One author concluded that it may be preferable to allow testing for every detectable genetic condition, with parental choice as the sole justification required for embryo selection rather than restricting PGD to serious conditions .
The list of indications for PGD is expanding. The pace of discovery of novel rare disease-causing genes by whole exome sequencing using next-generation sequencing (NGS) has rapidly increased in the past 3-5 years . Increasing number of genetic mutations for congenital syndromes or inherited diseases are being found, and all these mutations can potentially become indications for PGD.
As mentioned before, the aim of PGD is to avoid vertical transmission of the pathogenic gene mutation to the next generation. Occasionally, the at-risk individual may not be prepared to know whether they carry the pathogenic mutation or not, and they are not ready to undertake the pre-symptomatic tests. For example, in HD, the at-risk individual may have a strong family history of HD and clearly understand that there is no cure. This bad news may be too burdensome, and the individual may not prepared to face it yet, but would like to avoid the possibility of vertical transmission. PGD with exclusion test can be an option . Embryos with the at-risk haplotype would be discarded. This approach can avoid disclosure of the status of the at-risk individual against their will and at the same time prevent vertical transmission to the next generation. However, with this approach, there are concerns of unnecessary invasive procedures for the couple, in the case they do not carry the genetic mutations, and embryo wastage with disposal of the unaffected embryos. Therefore, this approach is not allowed even in some developed countries .
Another indication of PGD is tissue typing. For diseases such as beta-thalassemia, cord blood or bone marrow transplantation is a curative treatment option for an affected child and alleviates the need for regular blood transfusion and the complications of iron overload. However, the possibility of finding an unrelated donor compatible for bone marrow donation is rare. The purpose of performing PGD for beta-thalassemia couples together with HLA typing is to select disease-free embryos with a HLA that matches the affected child for transplantation. Successful implantation and birth of the transferred embryos would allow the collection of HLA-matched cord blood stem cells for transplantation. These savior children can offer an opportunity to cure affected siblings in the family, and the family can have another healthy member without the severe diseases . However, this raises ethical concerns , and the National Health Service of England does not include this condition in their clinical commission policy .
Indications of PGD
Potentially fatal genetic conditions are common indications for PGD ( http://guide.hfea.gov.uk/pgd/ ). Cystic fibrosis is the most common, potentially fatal autosomal recessive disease in the Caucasian population. The offspring of a couple in which both partners carry heterozygous pathogenic mutations would have a 25% chance of suffering this debilitating condition that primarily affects the lungs. The disease has no cure and requires multidisciplinary medical care, and the affected individuals would have shortened life expectancy. For example, in the Chinese population, thalassemia is the most common autosomal recessive disease, especially in the southeastern part of China. Babies with thalassemia major (i.e., homozygous for the deletion of alpha-globin genes in alpha-thalassemia or heterozygous mutations in beta-globin genes in beta-thalassemia) do not survive, suffer from hemoglobin Bart’s hydrops fetalis syndrome, or are blood transfusion dependent from early infancy because of Cooley’s anemia. PGD is a reproductive option for couples carrying thalassemia traits . In addition to childhood-onset genetic diseases, the indications of PGD have been extended to adult-onset diseases . Huntington’s disease (HD) and spinocerebellar ataxia are good examples of adult-onset diseases that can be detected by PGD. Individuals with these mutations or trinucleotide repeats in the disease range will develop these debilitating diseases later in their lives, most likely in adulthood, with no cure. Mutations with incomplete penetrance are also indications for PGD. Mutations of the tumor suppressor genes BRCA1 and 2 are good examples, and both can cause cancer predisposing syndrome . Patients with mutation of the BRCA1 or BRCA2 gene will experience substantially elevated risks of cancers of breast, ovarian, colonic, pancreatic, and prostatic origins.
There are some ethical concerns regarding the use of PGD in these adult-onset diseases or conditions with incomplete penetrance because of the “tradition” to offer PGD to early-onset conditions as their phenotypes are expressed early. In conditions with incomplete penetrance, there is a possibility, although very slim, that the individuals carrying the mutations may not develop the genetic conditions. However, there is no consensus on the line drawn to demarcate what conditions are serious enough or what age onset is eligible for PGD. Individuals carrying the mutations suffer from psychological burden to various extents, worrying about the development of the full-blown disease later in their lives. One author concluded that it may be preferable to allow testing for every detectable genetic condition, with parental choice as the sole justification required for embryo selection rather than restricting PGD to serious conditions .
The list of indications for PGD is expanding. The pace of discovery of novel rare disease-causing genes by whole exome sequencing using next-generation sequencing (NGS) has rapidly increased in the past 3-5 years . Increasing number of genetic mutations for congenital syndromes or inherited diseases are being found, and all these mutations can potentially become indications for PGD.
As mentioned before, the aim of PGD is to avoid vertical transmission of the pathogenic gene mutation to the next generation. Occasionally, the at-risk individual may not be prepared to know whether they carry the pathogenic mutation or not, and they are not ready to undertake the pre-symptomatic tests. For example, in HD, the at-risk individual may have a strong family history of HD and clearly understand that there is no cure. This bad news may be too burdensome, and the individual may not prepared to face it yet, but would like to avoid the possibility of vertical transmission. PGD with exclusion test can be an option . Embryos with the at-risk haplotype would be discarded. This approach can avoid disclosure of the status of the at-risk individual against their will and at the same time prevent vertical transmission to the next generation. However, with this approach, there are concerns of unnecessary invasive procedures for the couple, in the case they do not carry the genetic mutations, and embryo wastage with disposal of the unaffected embryos. Therefore, this approach is not allowed even in some developed countries .
Another indication of PGD is tissue typing. For diseases such as beta-thalassemia, cord blood or bone marrow transplantation is a curative treatment option for an affected child and alleviates the need for regular blood transfusion and the complications of iron overload. However, the possibility of finding an unrelated donor compatible for bone marrow donation is rare. The purpose of performing PGD for beta-thalassemia couples together with HLA typing is to select disease-free embryos with a HLA that matches the affected child for transplantation. Successful implantation and birth of the transferred embryos would allow the collection of HLA-matched cord blood stem cells for transplantation. These savior children can offer an opportunity to cure affected siblings in the family, and the family can have another healthy member without the severe diseases . However, this raises ethical concerns , and the National Health Service of England does not include this condition in their clinical commission policy .
Diagnostic methods
The method of choice for the diagnosis of MGD in PGD is usually polymerase chain reaction (PCR) . PCR can be customized for particular mutations or changes in the DNA sequences. However, PCR on single cells entails a number of challenges. First, the amplification efficiency for single-cell samples is generally lower than that of PCR for genomic DNA in routine genetic testing . The lower amplification efficiency can be attributed to the process of collection of samples and the PCR procedure itself. The collection of biopsy samples is operator dependent, and cell loss may occur during the manipulation of single cells or the tubing process. Spontaneous cell lysis and biopsy of anucleated fragment or degenerated cells are other causes of reduced amplification efficiency. Use of different cell lysis protocols would result in some variation in the amplification efficiency .
The timing of biopsy also affects the amplification efficiency. In a polar body biopsy, collection of both the first and the second polar bodies is preferred, while in a cleavage-stage biopsy, collection of one cell instead of two cells is preferred because available evidence strongly suggests that collection of two cells severely jeopardizes the implantation and pregnancy rates . Evidence also shows that the removal of one blastomere from a 6-8-cell cleavage-stage embryo (approximately 10-20% of cell mass) does not detrimentally affect the development of the biopsied embryo into a good-quality blastocyst . With the current techniques, results of genetic tests can be available within 2 days. Therefore, biopsied Day 3 embryos are usually cultured to Day 5 or 6, and the resulting good-quality blastocysts with normal genetic results are placed back into the same cycles. For trophectoderm biopsy, 3-5 cells out of 100-150 cells in a blastocyst (about 2-5% of cell mass) are collected, and the risk of amplification failure is lower than that with single cells . In addition to amplification efficiency, improved implantation rate after trophectoderm biopsy was clearly illustrated by a paired randomized trial in 2013. The sustained implantation rate was not affected by trophectoderm biopsy, while it was reduced by 39% after blastomere biopsy . Although the clinical outcome is better with trophectoderm biopsy, time is usually insufficient to complete the genetic test within the tight schedule of a PGD cycle. Therefore, except in rare circumstances, the biopsied blastocysts require vitrification immediately after the biopsy, and genetically normal blastocysts are cryopreserved to be replaced in subsequent frozen-thawed embryo transfer cycles. A good vitrification program is a prerequisite for a successful PGD program using trophectoderm biopsy. The pregnancy rate is good after warming of the vitrified biopsied blastocysts. A pregnancy rate of 73% was reported in a retrospective analysis , and many studies showed similar efficacy of vitrification for both non-biopsied and biopsied blastocysts .
Another challenge of single-cell PCR is the allele dropout (ADO) phenomenon, which can greatly increase the misdiagnosis rate . ADO occurs in approximately 10-20% of cases . When it occurs, only one of the two alleles is amplified by PCR. In PGD for autosomal recessive genetic diseases, ADO can cause a problem when differentiating the carrier from the affected embryos. In autosomal dominant or X-linked genetic diseases, ADO can also cause misdiagnosis between normal and affected embryos. For this reason, incorporation of linked markers is recommended to identify ADO. The linked markers could be either microsatellite markers or single nucleotide polymorphism (SNP) markers, and ideally, they should be intragenic . Using multiplex PCR, the mutation and the polymorphic markers are amplified together to enhance the diagnostic accuracy.
Only a tiny amount of DNA (approximately 5-10 pg/ml) can be obtained from a single cell . A large number of amplification cycles are needed before a mutation can be detected. The amplification may also amplify contaminants in the samples, causing misdiagnosis. Contamination can be from genomic DNA of the technical operator or patients, either from cumulus cells or sperm sticking on the zona pellucida, or even from carry-over contamination from PCR products amplified previously . Intracytoplasmic sperm injection (ICSI) can reduce the contamination from zona-bound sperm or cumulus cells, and ICSI becomes one of the requirements for PGD using PCR nowadays. Strict aseptic protocols in PGD laboratory, separation of the pre- and post-PCR rooms, and dedicated equipment and reagents for different procedures can further reduce the chances of contamination .
There has been much advancement in recent decades for PGD, which includes the use of comprehensive chromosomal screening by array comparative genomic hybridization (aCGH), SNP array, and NGS for aneuploidy screening. aCGH was established as a commercially available platform. It involves a relatively straightforward and rapid protocol for PCR library-based whole-genome amplification, DNA labeling, hybridization, and array scanning. These procedures can be completed within the tight schedule of PGD and are used worldwide . However, aCGH can only detect copy number variants including duplications or deletions but cannot detect mutations of MGDs. The use of SNP array is mainly limited to laboratories in the USA because of the high cost of the required equipment, length and complexity of its protocol, and need for extensive interpretation of the results. Compared with aCGH, SNP array has some advantages such as higher resolution with an average spacing of approximately 5 kb and the possibility of identifying the origin of genetic aberrations from the genotype information of parents . The combined use of parental SNP genotypes and straightforward Mendelian genetic analysis enables the generation of a karyomap for each chromosome or chromosome segment inherited by each embryo for the diagnosis of MGD or chromosomal abnormalities, which is the basic principle of PGD by karyomapping . Although karyomapping is commercially available and theoretically applicable to all MGDs without the need for prior optimization of the assay for the disease concerned, the cost of the platform is higher than other platforms, and the requirement for genetic information from an affected individual or embryos as reference makes the platform not commonly used for PGD of MGDs.
NGS is a kind of massive parallel sequencing approach that greatly reduced the cost of sequencing the human genome . NGS-based preimplantation genetic aneuploidy testing has been validated in various centers . However, the resolution of the current NGS-based preimplantation genetic screening is not adequate for the detection of genetic mutations in the majority of MGDs. There was only one report of using targeted NGS-based PGD for MGD . This method can offer the diagnosis of both MGD and aneuploidy in a single test, and the cost calculated by the authors in their centers is comparable to those on other platforms used routinely. However, there is no other report following that publication, probably because of the difficulty and cost involved in designing primer sequences for individual MGDs and need of bioinformatics experts in the interpretation of data for each single MGD. Further studies and modifications of the method are needed before its routine clinical application.
