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Many couples with genetic disorders face fertility problems and they are at high risk of having a child with a significant phenotype abnormality or experiencing miscarriages. There are some options that are suggested to such couples in order to help them overcome their fertility problem, such as i) prenatal diagnosis and if an embryo is diagnosed with a genetic disease the pregnancy can be terminated ii) gamete donation or adoption or iii) have no children. In the past 20 years another solution is suggested: the preimplantation genetic diagnosis (PGD). It was developed in 1990 and as it is described by Handyside et al., during that first successful procedure Y-chromosome-specific sequences were amplified using PCR in order to detect the sex of embryos retrieved from couples at high risk of X-linked diseases. Only the female embryos were placed to the uterus and many healthy girls were born. (Hardy and Handyside, 1992).
Preimplantation genetic diagnosis is a technique, which was at first introduced as an alternative to prenatal diagnosis for couples at high risk of transmitting a genetic defect to their offspring. PGD procedure is a combination of assisted reproductive (in vitro fertilization) and molecular genetic techniques that allows the detection of genetic disorders and chromosomal abnormalities in the embryo. Polymerase Chain Reaction (PCR) and Fluorescent in situ hybridization (FISH) are the two genetic methods, which are used. Therefore, it is possible to diagnose a specific genetic disease in embryos, obtained through IVF, and only the not effected embryos are selected and transferred into the uterus. PGD reduces the number of repeated spontaneous abortions or therapeutic termination of affected pregnancy and increases the possibility of a successful pregnancy. Of course, the disposal of affected embryos and the termination of affected pregnancies raise ethical dilemmas among the society.
The range of genetic defects that can be diagnosed includes monogenic diseases, numerical and chromosomal abnormalities. In cases of X-linked diseases, scientists use PGD in order to determine the sex of the fetus. It is a really useful technique not only for couples who are carriers of a genetic disease but also for couples at low risk of transmitting that kind of diseases but with poor prognosis in ART treatment because of repeated abortions or advanced age. For those couples the enumeration of chromosomes is required.
In addition, many parents with a child suffering from a disease, which can only be cured by transplantation of haematopoietic stem cells choose to have another child, which can be the matching donor of haematopoietic stem cells or other tissue for the sick sibling. In those cases HLA typing of the embryo by PGD is required in order to ensure that the donor child is the suitable matching donor. Moreover, PGD can be used for sex selection of the embryo and a more recent application of that method is to diagnose predisposition syndromes, including some types of cancer, and late-on set diseases (appear in adulthood), for instance Huntington disease or hereditary forms of Alzheimer's.
Many patients who carry a reciprocal or a Robertsonian translocation are infertile or experience repeated miscarriages. Therefore, the only way for those patients to avoid such problems (e.g. many abortions) is PGD. That is why the number of PGD applications for diagnosis of chromosomes rearrangements has been on rise. Robertsonian translocations can cause infertility depending on the sex of the carrier and the chromosomes involved.
The first step in a PGD procedure is to obtain nuclear material from the embryo for genetic analysis. This process is called biopsy and two major methods are used ; the aspiration of one or two polar bodies from the oocytes ( polar body biopsy) and the removal of one or two blastomeres from early embryos (cleavage stage biopsy), which is the most widely used technique (Shenfield et al., 2003; Thornhill et al., 2005). There is, also, a third approach the blastocyst biopsy, which is not as common as the other two.
Polar body biopsy: The two polar bodies are by-products of meiosis. The first polar body is extruded at the first meiotic division, whereas the other is expelled at the second meiosis. The oogonia enter the first meiosis at the early stages of oogenesis. During this process the chromosomes are replicated and chromosomal material from the mother and the father is exchanged (diploid oocyte). The first polar body that is produced at this stage contains the genetic complement of the oocyte as the whole process occurs inside the follicle, before the ovulation. Then, the oocyte enters the second meiosis, where the duplicated chromatides separate and the second polar body is extruded (Ethrikat, 2003). It is important to analyse both polar bodies by consecutive biopsy in order to minimize the risk of misdiagnosis caused by phenomena such as non-detected allele drop-out and events of recombination that lead to heterozygotous first polar body (Verlinsky et al., 1996; Storm et al., 1997).
The main advantage of the use of polar bodies in PGD is that they are useless by-products and they are not needed for successful fertilization and normal embryonic development. Therefore, they can be safely removed without causing damage to the embryo. Moreover, the procedure is completed at a very early stage of fertilization when syngamy has not yet occurred. It is a minimally invasive method.
On the other hand, even if the polar bodies can be analysed at the chromosomal and monogenic level there are cases, in which this technique is not suitable. It can only detect maternally transmitted genetic or chromosomal abnormalities, while the paternal contribution to the embryo cannot be analysed. Therefore, autosomal dominant diseases and translocations transmitted through the father cannot be diagnosed.
Cleavage stage biopsy: It is usually performed on the third day after fertilization, when embryos normally reach the third division. At this point embryos are at the eight- cell stage, which is proved to be the most suitable for biopsy. It is really important that at this stage all the human cells are immature and they do not have to follow a specific developmental path. The number of individual blastomeres that could be removed from the embryo - one or two - depends on the embryo cell number and the reliability of the diagnostic test used. One blastomere is removed from embryos with <7 cells and two blastomeres from embryos with >7 cells (Thornhill et al., 2005). It must be mentioned that during the diagnosis, the embryos continue dividing in vitro until it is safe to transfer the healthy embryo.
Using the forthmentioned method, it is possible to test disorders carried by both parents and also those that originate after fertilization. Moreover, there is enough time in order the embryos to be transferred to the uterus after the completion of the diagnosis. Unfortunately, a high rate of chromosomal mosaicism is reported in cleavage stage embryos and a small amount of material (tissue) is available for genetic testing (Ziebe et al., 2003; Staessen et al., 2004).
Blastocyst biopsy: The last stage in the embryo's development at which can be biopsied is the blastocyst stage. This technique can be performed five-six days after fertilization in the human and at that point embryos consist of inner mass cells and trophectoderm cells (approximately 150 cells in total). Embryos remain largely intact as only the trophectoderm cells are retrieved and biopsied. There is no loss of inner cell mass for the embryos. After PGD embryos can be replaced during the same cycle or cryopreserved and transferred in a next one cycle.
The major advantage of blastocyst biopsy is that larger amount of cells can be obtained for analysis than by the other two methods and that the possibility of inducing embryo damage is minimal. The drawbacks being that almost half of the embryos in vitro reach the blastocyst stage and that the time available for the genetic diagnosis is very limited as embryos should be transferred to the uterus before hatching on day 6. It is estimated that 21% of started PGD cycles have no suitable embryo for that kind of biopsy (McArthur et al., 2004). Furthermore, trophoblast cells are often multinucleated or even in syncitium. It is possible to overcome this problem by the improvement of media specific for blastocyst culture (Gardner et al., 2000).
Overview of the molecular basis of single gene and chromosomal disease diagnosis
The next step after the biopsy stage is the testing of the obtained genetic material. As it is mentioned beforehand, two analysing methods are generally used in PGD: PCR, used for the analysis of genes in order to detect single-gene (monogenic) diseases and FISH, used for the analysis of chromosomes in cases of chromosomal aberrations.
Both methods can detect pathogenic mutations in DNA sequence, which usually cause a genetic disorder. There are many classes of mutations that may occur:
a) Base substitutions involve replacement of a single base, but there are also some cases in which several bases may be replaced as a result of a form of gene conversion. Transitions - substitution of a pyrimidine by a pyrimidine (C or T) - and transversions - substitution of a purine by a purine (A or G) - are included in this category.
b) Insertions: one or a few nucleotides are inserted into a sequence.
c) Deletions: one or more nucleotides are eliminated from a sequence.
d) Chromosome abnormalities, involve breakage and rejoining of chromatids (structural) or loss or gain of chromosomes (numerical).
PCR: It is a technique, which allows the amplification of a specific region of DNA (gene) in vitro using a thermostable DNA polymerase and synthetic oligonucleotides as primers that anneal at sites which flank the region to be amplified. Simultaneous detection of disease-causing polymorphic alleles or molecular markers linked to the disease mutation (e.g. SNPS and microsatellites) can be performed (Boyle et al., 2004). Molecular markers are commonly used in PGD. They are DNA sequences that lie on the chromosomes so close to the genes that the marker and the gene are inherited together. Therefore, markers are identifiable heritable spots on the chromosomes. Markers can be genes or segments of DNA with no - known coding function. They are present to everyone and they show polymorphism concerning the size and the nucleotide sequence. In terms of genetic disease diagnosis, marker can be linked to the mutated sequence that causes the disease (linkage analysis) or it can be the disease gene. There are few types of markers: Restriction fragment length polymorphisms (RFLPs), Single nucleotide polymorphisms (SNPs), Minisatellites and Microsatellites.
Type of marker
2 allele markers. Initially requierd Southern blotting, now PCR
Easy physical localization
Less informative than microsatellites, can be typed without
DNA VNTRs (minisatellites)
Many alleles, high informative typed by Southern blotting
Easy physical localization. Tend to cluster near ends of chromosomes
DNA VNTRs (microsatellites)
Many alleles, high informative typed by multiplex PCR
(di-, thri- and tetranucleotide
Easy physical localization.Distributed throughout the genome
Table 1: Type of human genetic markers (Strachan and Read, 2001)
Tests for known mutations are mostly based on PCR. Small deletions can be recognised by amplifying a sequence of 100 nucleotides that spans the site of mutation. The deletion is detected as a band shift in the product during electrophoresis. PCR primers in which one or both primers anneal with the sequence deleted are used in order large deletions to be recognised. Mutations that include both base substitution and deletions or insertions for only one or two nucleotides are diagnosed by using allele-specific oligonucleotides as primers, which anneal to a mutant or to wild-type sequence but not both.
Except from the conventional PCR, single-cell PCR, multiplex PCR is also in use. This method reduces problems such as contamination and allele drop- out phenomenon (ADO), where the random not ampflication of one of the alleles in a heterozygotous embryo can lead to a misdiagnosis depending on which allele failed to amplify. Furthermore it allows the stimulus ampflication of two or more DNA sequences (mutation and linked and unlinked markers) and it is useful in genetic diagnosis of more frequent diseases such as cystic fibrosis. There is also the fluorescent PCR, in which the products are fluorescently labelled through the incorporation of fluorescently labelled primers (Ray et al., 2001). PCR results are checked by electrophoresis analysis in order the size differences between the normal and the disease-mutant sample to be detected.
Monogenic diseases that are tested by PCR are traceable to a defect in a single gene and they are classified as below.
a) Autosomal recessive (e.g. Tay- Sachs) with 1:4 risk. In such cases the affected gene is located on one of the 22 autosomes and the inheritance of two defective alleles is required. Therefore, there are no functioning copies of the gene and both parents must be carriers or heterozygous at the locus concerned. The most common indications for PGD are Cystic fibrosis, Î²- thalassamia and spinal muscular atrophy.
b) Autosomal dominant (e.g. Huntington's disease, Charlot-Marie- Tooth disease) with a 1:2 risk. The mutation is located on one of the 22 autosomes and the inheritance of only one mutated allele is required. Usually the function of a protein is affected and all the heterozygous individuals are affected.
c) X- linked recessive diseases (e.g. haemophilia A and B, fragile X, Duchenne disease) with a 1:2 risk in males. In these diseases the affected genes are located on the X- chromosome. Females have to be homozygous for an X- linked mutation in order to suffer from the disease, while for males one copy of the affected allele is enough because they have only one copy of X- chromosome. There is no risk for daughters from carrier mothers to develop the disease. On the other hand, there is a risk of 50 % of having an affected boy in each pregnancy. X- linked dominant disorders such as hypophosphataemic occur rarely.
Although, the most common and accurate method for detecting X- linked disorders is FISH.
Hemophilia A, B
Tyrosine hydroxylase deficiency
Spinal muscular atrophy
1 and 2
Polycystic kidney disease
Table 2: Indications for monogenic PGD (European Journal of Human Genetics, 2008)
FISH: In this technique, embryo cells are broken up and their chromosomes are spread out on microscope slides and hybridised in situ with labelled DNA probes. Each of these probes is specific for a part of a chromosome and is labelled with a fluorophore of a different colour. In fact, the probe hybridises to the sites of homologous sequences in situ within the chromosomal DNA and the region to which it is binding can be visualised by using a fluorescence microscope (bright spot).
The major application of FISH in PGD is to determine the gender of the embryo in order to prevent various sex- linked diseases (e.g. haemophilia). Probes for X and Y chromosomes are used in such cases. Moreover, aneuploidies, haploidies and polyploidies can be diagnosed by using a set of probes such as probes specific for chromosomes 13, 14, 15, 16, 18, 21 and 22. Also, chromosome rearrangements - translocations and inversions - can be located. In Robertsonian translocations a whole chromosome is translocated to another one through centromeric fusion, while in reciprocal translocations there is an exchange of fragments between chromosomes. Carriers of balanced translocations are usually phenotypically normal as no genetic information is missing or is in excess. But, the unbalanced offspring of carriers can be abnormal causing births of children with congenital anomalies (e.g. Down's syndrome). In addition, such carriers may suffer from secondary infertility because of recurrent miscarriages. The risk of normal or balanced offspring, unbalanced offspring or recurrent miscarriage can be approximately estimated according to the chromosomes involved and the size of exchanged fragments ( Scriven et al., 1998).
45 X : Turner's syndrome
1 in 5000
47 XXY: Klinefelter's syndrome
1 in 1000
Trisome 21: Down's sybdrome
1 in 800 (maternal age dependent)
Trisome 18: Edward's sybdrome
1 in 10000
1 in 500
Table 3: Some examples of chromosomal mutations ( Peter Sudbery, 1998)
Ethics and regulations
Although PGD is a technique that increases many couples probability to have a healthy child, ethical issues are raised. Attitudes towards PGD vary worldwide and there are regulations ensuring that PGD is used only for medical reasons and not for trait selection or in a way that it could cause eugenic outcomes (HGC, 2001). There are two main approaches to the regulation of PGD:
a) Statutory legislation as in Austria, Switzerland and Italy, where PGD is banned and in Germany, where only PGD based on polar body biopsies is permitted.
b) Guidelines by scientific societies and ethics committees as in Greece, USA, Portugal and the Republic of Ireland ( Jones H.W. et al., 2004; Krones et al., 2004).
In UK a regulatory body called Human Fertilisation and Embryology Authority (HFEA) was created, which supervise all fertility treatment and embryo research. It is responsible for giving the needed permission to fertility centres wishing to carry out the diagnostics tests.
There are many cases, in which ethical dilemmas are raised. Sex selection -using PGD- is permitted for medical reasons in order to prevent the birth of a child with an X- linked disease. However, sex selection for social or family-balancing reasons is unacceptable as such attitudes can lead to discriminations (sexism, racism). Moreover, testing for late-on set diseases could be considered as "unethical", since children born with such conditions may have long and fulfilling lives without ever developing the disease. It is also possible to develop the disease after many decades of healthy life. Generally the conditions that are licensed for PGD are serious or untreatable genetic disorders which are expressed from birth or early childhood.
Choosing an embryo, which may provide stem cells for an existing sick sibling(saviour siblings') leads to ethical concerns regarding instrumentalization of the embryo and the welfare of the future child. For many it is morally unacceptable to have children for a certain reason because they may perhaps feel that they are not valued for their own existence. They might think that the only reason that they are born is to save their sibling and if the transplant does not work they will grow up in the shadow of the failure.
People with a disability such as deafness or dwarfism tend to prefer to have children, who are in the same condition with them in order to share the same lifestyle and because they think that family life would be better by this way. However, the functioning of this child within society at large would be severely impaired due to the imposed disability. Therefore, such deliberate restriction of the autonomy of the child is not considered justifiable and PGD should not be done.
As the aim of PGD is the birth of a healthy baby, there is a dilemma if this goal should include accepting embryos, which carry a disease. These embryos would not be expected to develop into children with significant symptoms themselves, but the children could potentially pass on the relevant genes to their own offspring. Thus, selecting only completely unaffected embryos and not carriers could be seen as extending the goals of PGD beyond the health of the immediate child in question.
A combination of ART and molecular methods is required in order to achieve a successful PGD. It is really important not only to complete the diagnostic part successfully but also to achieve a normal pregnancy as the goal of the whole procedure is the birth of a healthy child. As it is a recently developed method, some improvements are required in order to reduce the rate of misdiagnosis caused by ADO phenomenon and contamination of the sample. However, PGD is a controversial technology because sometimes is not used for medical reasons. This so useful method that gives to many couples the opportunity to become parents and avoid the disappointment of recurrent miscarriages should strictly be focused on preventing the birth of diseased children. It is not proper to create embryos with specific characteristics that fit to parents or society demands. Every birth is a unique and random phenomenon that preserves the variety of the human population.