Pre-implantation genetic diagnosis is used to analyse pre-implantation stage embryos or oocytes for genetic defects, generally for severe Mendelian disorders and chromosome abnormalities. New but controversial indications for pre-implantation genetic diagnosis include identifying human leukocyte antigen compatible embryos suitable as donor, sex selection and adult-onset disorders, particularly cancer. Pre-implantation genetic screening is a variant of pre-implantation genetic diagnosis to improve outcomes of in-vitro fertilisation. Array comparative genomic hybridisation is replacing fluorescence in-situ hybridisation for aneuploidy screening. Besides technical advancement of array platform, the success of pre-implantation genetic screening is strongly related to the embryonic biological nature of chromosomal mosaicism. Having been applied for more than 20 years, pre-implantation genetic diagnosis is recognised as an important alternative to prenatal diagnosis. Diagnosis from a single cell, however, remains a technically challenging procedure, and the risk of misdiagnosis cannot be eliminated.
Introduction
Pre-implantation genetic diagnosis (PGD) is generally defined as a test to identify genetic defects in embryos obtained through in-vitro fertilisation (IVF). It has been applied for couples whose potential offspring are at risk of a known genetic abnormality. In contrast, pre-implantation genetic screening (PGS), a variant of PGD, refers to a test in which embryos undergo aneuploidy screening in order to improve IVF outcomes. Because only disease-free embryos are transferred to the uterus for implantation, pre-implantation genetic testing is an attractive means of preventing heritable genetic diseases, and provides an alternative to current prenatal diagnostic procedures, which are frequently followed by the difficult decision of pregnancy termination if results are unfavourable.
Pre-implantation genetic diagnosis was first accomplished in humans more than 20 years ago. Since then, PGD has been accomplished for many known genetic mutations, and more than 50,000 cycles have been carried out worldwide. Although the indications for PGD are well established, PGS is a relatively new, evolving technique and remains to be evaluated.
Indications and conditions
Indications for pre-implantation genetic diagnosis
Couples undertaking PGD are firstly known to have a past negative reproduction outcome: birth of a child with genetic disease or termination of pregnancy after prenatal diagnosis. Secondly, they are known carriers of a genetic disease, either known from family cases or from abnormality discovered during infertility investigations (e.g. chromosomal imbalances). These people fall into the following categories: (1) couples with a family history of X-linked disorders (such couples have a 25% risk of having an affected embryo or 50% risk for male embryos); (2) carriers of autosomal recessive diseases (the risk of having an affected embryo is 25%); (3) carriers of autosomal dominant diseases (the risk of having an affected embryo is 50%); and (4) carriers of structural chromosome abnormalities, reciprocal and Robertsonian translocations, inversions, deletions, insertions, etc.
Other indications for PGD are rare and controversial:
Sex selection
Pre-implantation genetic diagnosis for sex selection can be motivated by cultural, social, ethnic, psychological, and other reasons, such as the desire for family balancing. The use of PGD for sex selection unrelated to disease is controversial and ethically debatable.
Human leukocyte antigen matching
Human leukocyte antigen typing (HLA), in addition to monogenic testing for a particular condition, could provide a potential donor for stem cell or bone marrow transplantation to an affected sibling or other relative. This has been previously used to avoid the birth of a child with Fanconi anaemia, an autosomal recessive disorder, whose HLA matched cord blood stem cells were successfully transplanted to cure the affected sibling.
Indications for pre-implantation genetic screening
Most early pregnancy losses are believed to be attributed to aneuploidy. Because only chromosomally normal embryos are transferred into the uterus after selection by PGS, pregnancy rate and delivery rate are expected to be increased. At present, most PGD centres offer PGS to couples with one or more of the following indications: (1) advanced maternal age (cut-off varies between 35 and 40 years of age); (2) recurrent pregnancy loss (two occurrences or more); (3) repeated implantation failure (more than three occurrences); and (4) severe male factor infertility.
Indications and conditions
Indications for pre-implantation genetic diagnosis
Couples undertaking PGD are firstly known to have a past negative reproduction outcome: birth of a child with genetic disease or termination of pregnancy after prenatal diagnosis. Secondly, they are known carriers of a genetic disease, either known from family cases or from abnormality discovered during infertility investigations (e.g. chromosomal imbalances). These people fall into the following categories: (1) couples with a family history of X-linked disorders (such couples have a 25% risk of having an affected embryo or 50% risk for male embryos); (2) carriers of autosomal recessive diseases (the risk of having an affected embryo is 25%); (3) carriers of autosomal dominant diseases (the risk of having an affected embryo is 50%); and (4) carriers of structural chromosome abnormalities, reciprocal and Robertsonian translocations, inversions, deletions, insertions, etc.
Other indications for PGD are rare and controversial:
Sex selection
Pre-implantation genetic diagnosis for sex selection can be motivated by cultural, social, ethnic, psychological, and other reasons, such as the desire for family balancing. The use of PGD for sex selection unrelated to disease is controversial and ethically debatable.
Human leukocyte antigen matching
Human leukocyte antigen typing (HLA), in addition to monogenic testing for a particular condition, could provide a potential donor for stem cell or bone marrow transplantation to an affected sibling or other relative. This has been previously used to avoid the birth of a child with Fanconi anaemia, an autosomal recessive disorder, whose HLA matched cord blood stem cells were successfully transplanted to cure the affected sibling.
Indications for pre-implantation genetic screening
Most early pregnancy losses are believed to be attributed to aneuploidy. Because only chromosomally normal embryos are transferred into the uterus after selection by PGS, pregnancy rate and delivery rate are expected to be increased. At present, most PGD centres offer PGS to couples with one or more of the following indications: (1) advanced maternal age (cut-off varies between 35 and 40 years of age); (2) recurrent pregnancy loss (two occurrences or more); (3) repeated implantation failure (more than three occurrences); and (4) severe male factor infertility.
Pre-implantation genetic diagnosis procedure
Before requesting PGD, couples should be offered genetic counselling to evaluate the risk of having an affected offspring. Genetic tests should be carried out to confirm the diagnosis of the affected or carrier parent, to generate a mutation report with the confirmed genotype leading to the condition.
Women undergo IVF before PGD starts. Genetic material is obtained from gametes or developing embryos for genetic testing. Genetic testing is carried out using polymerase chain reaction (PCR), fluorescence in-situ hybridisation (FISH), or comparative genomic hybridisation (CGH). Unaffected embryos are then transferred into the uterus for ensuing implantation and pregnancy.
Embryo biopsy
Embryo biopsy should be carried out before day 6 of conception, when implantation occurs. The three potential approaches include (1) polar body biopsy; (2) blastomere biopsy from the day 3 embryo with six to eight cells; and (3) trophectoderm biopsy from day 5 or day 6 blastocyst.
For certain indications, polar body biopsy can be proposed, as it only allows identification of the maternal genetic material. The first or second polar bodies, or both, are removed after their extrusion from the oocyte and are studied by genetic testing. The advantage of this method is that the use of the discarded daughter cell(s) of the female meiotic division does not affect any functional part of the oocyte. Furthermore, testing the first polar body is ethically acceptable in countries that do not permit testing of embryos.
The most common approach of embryo biopsy is to remove one blastomere, sometimes two, from day 3 cleavage stage embryos. Zona pellucida opening for blastomere removal can be carried out by mechanical or chemical means, or by laser. Blastomeres are removed by aspiration or extrusion. Diagnostic efficiency, embryonic development and clinical outcome have recently been compared between biopsy of one or two blastomeres for PGD. It was concluded that the biopsy of only one cell significantly lowers the efficiency of a PCR-based diagnosis, whereas the effect of one- or two-cell removal on delivery rates per cycle is highly debated.
Blastocyst formation begins on day 5 after fertilisation, and consists of an inner cell mass and an outer cell mass (trophectoderm). At this stage of development, the embryo is made up of more than 100 cells. A hole is breached in the zona pellucida in a similar manner as for the cleavage-stage embryo biopsy, and five to 10 cells are removed from the trophectoderm, resulting in more genetic material to start with. The inner cell mass is left undisturbed. Only 50–60% of embryos survive in-vitro to reach the blastocyst stage. Although this results in less blastocysts available for testing, compared with day 3 embryos, blastocyst biopsy procedure provides a better implantation rate. For blastocyst biopsy, there is insufficient time for analysis as the diagnosed embryos need to be transferred not later than on day 6. Hence, the embryos have to be cryopreserved and transferred at a later date as a freeze–thaw embryo replacement cycle. The freezing and thawing of embryos significantly reduces the chance of a successful pregnancy. The introduction of newly developed vitrification methods (e.g. ultra rapid cooling and warming to prevent ice crystal formation), may overcome this problem and allow sufficient time for genetic testing.
Genetic testing
Fluorescence in-situ hybridisation
Genetic testing is carried out with different techniques, based on the disease that is to be avoided. Fluorescence in-situ hybridisation is used for the determination of sex for X-linked diseases, chromosomal abnormalities, and aneuploidy screening. The technique is based on the hybridisation of specific regions of a chromosome with specific DNA probes, which are labelled with different fluorescent dyes. For X-linked disorders, specific centromeric probes are used for X and Y chromosomes. For translocations and other chromosome abnormalities, triple colour FISH is normally used with three different probes for the chromosomes involved in the translocation. For aneuploidy screening, it is important to examine as many chromosomes as possible, and up to 15 probes have been used. The limits of this technique are the probability of cell-fixation failure, hybridisation failure, and overlap of the fluorescent signals from two chromosomes. These technical difficulties might be associated with high error rate of FISH. As FISH is limited by the number of chromosomes that can be examined and its intrinsic technical ability, many groups are replacing FISH with array-comparative genomic hybridisation (array-CGH).
Polymerase chain reaction
The second widely used genetic testing method is PCR. It involves amplification of a specific DNA fragment millions of times to produce enough material for subsequent analysis. It is used for the diagnosis of single gene defects, including X-linked disorders, dominant and recessive disorders. A growing number of genes have been analysed by PCR-based PGD and a variety of mutation-detection strategies used, such as minisequencing and real-time PCR assay ( Fig. 1 ).
Polymerase chain reaction used for PGD has to be highly efficient and accurate. Polymerase chain reaction optimisation is usually needed as the technique needs 10–100 ng DNA (∼2000–20,000 cells), whereas starting material in PGD is usually only one cell (about 6 pg DNA). The accuracy of the PCR diagnosis can be hampered by exogenous DNA contamination, amplification failure, and allele dropout (ADO). The latter two problems may be caused by imperfect lysis of the cell and inefficient denaturation of DNA. In addition, DNA degradation has been suggested as a possible cause of ADO and preferential amplification, possibly more frequently in embryos of poor morphology and blastomeres with an unclear nucleus.
Co-amplification of the mutation locus, together with flanking linked polymorphic markers, has become the gold standard in PGD for single gene disorders. This multiplex PCR strategy effectively enhances diagnosis by analysing the mutation itself and the polymorphic allele(s) that are inherited with it. Multiplex PCR also reduces the risk of misdiagnosis resulting from ADO, as it is unlikely that ADO will affect all of the markers in the same reaction. The simultaneous amplification of one or more polymorphic markers is also a way to detect possible exogenous DNA contamination.
Whole-genome amplification
The scarcity of DNA is always a severely limiting factor for PGD. One approach to overcome this problem is whole-genome amplification (WGA). With this technique, a single genome from a cell can be amplified numerous times, thus providing sufficient DNA templates for downstream DNA analysis, such as PCR of mutation region and multiple polymorphic markers. Another benefit of WGA is that there is no need to optimise multiplex PCR reactions at the single cell level, thus greatly simplifying the assay development process. A panel of linked markers associated with a mutation can be applied to any individual carrying a similar mutation, as there is no limitation to the number of markers that can be selected. This makes PGD assay more standardised and straightforward.
Whole-genome amplification strategies can be PCR based, such as primer extension pre-amplification and degenerate oligonucleotide primed (DOP) PCR, or non-PCR based, such as multiple displacement amplification. Complete coverage of the genome is rarely achieved by any WGA method, especially when the starting material is from a single cell; another drawback of WGA is its relatively high ADO rate (average ADO rate of 25% for multiple displacement amplification) and preferential amplification of certain alleles. Therefore, PGD protocols using WGA amplified sample should include more short tandem repeat markers than those used in direct single-cell PCR. This enables accurate diagnosis even if some loci fail to amplify or if ADO occurs during WGA. For PGS, currently the most common WGA method is combining primer extension pre-amplification and DOP, which uses degenerate oligonucleotide primers coupled with universal adaptors for library preparation then followed by universal primer PCR. This method is able to generate enough DNA for the following array analysis.
Comparative genomic hybridisation
As previously discussed, the usefulness of FISH is limited, because only a few chromosomes can be screened simultaneously. Complete karyotyping of the whole 23 pairs of chromosomes in a single hybridisation can now be achieved by CGH. In principle, the test DNA is labelled with one fluorescent dye, such as Cy3, whereas a karyotypically normal DNA sample (46, XY, or 46, XX) is labelled with Cy5, and serves as reference. Both test and reference DNAs are mixed in equal proportions and co-hybridised to either metaphase spreads from a normal control cell line (metaphase-CGH) or onto an array containing a number of defined DNA probes (array-CGH). Test and reference DNAs compete for hybridisation sites on each of the 23 pairs of chromosomes. Differences in fluorescence intensities are identified and analysed using computer software, which is able to recognise chromosome areas that are either gains (e.g. trisomy) or losses (e.g. monosomy) in the test DNA sample.
Array-based comparative genomic hybridisation
As metaphase CGH is time-consuming (up to 72 h) and technically challenging, array-CGH has been more commonly applied in PGD in recent years. On array platform, well-defined DNA probes, such as bacterial artificial chromosomes, are spotted onto a slide. Each array contains hundreds to thousands of clones throughout the whole genome. Notably, CGH array platforms for PGD and PGS are of low resolution (2–10 Mb), as the diagnostic target is large chromosomal aneuploidies. Probes are not in regions with copy number variation (CNV) to avoid the problems of interpreting copy number variations of unknown significance. Comprehensive chromosome analysis can be achieved in less than 24 h using array-CGH, within the time-frame of an embryo transfer in a fresh cycle after PGD ( Fig. 2 ).
