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DNA analysis for preconception and preimplantation diagnosis is well established, which enables genetic analysis of minute quantities of DNA obtained from a single or few cells.^{12, 19, 20} Because this also increases the chance of DNA contamination in PGT, decontamination procedures were applied in the initial stages, based on elimination of double‐stranded DNA sequences,⁵⁹ excluding also possible contamination with the embryology and PCR reagents, such as water, salt solutions, oligonucleotides, and Taq polymerase.

The major source of contamination in PGT is still cellular contamination, such as cumulus cells, spermatozoa, or cell fragments, which might be amplified simultaneously with polar bodies or embryo biopsies, creating the possibility for erroneous testing. Because potential misdiagnosis of PGT may be caused by sperm DNA contamination, it is currently a routine IVF practice to perform PGT‐M for single‐gene defects following microsurgical fertilization by intracytoplasmic sperm injection (ICSI).

Nevertheless, the major source of misdiagnosis is preferential amplification or allele‐specific amplification, referred to as allele dropout (ADO), which may happen in single‐cell genetic analysis. As much as 8 percent of ADO was observed in PCR analysis of the first polar bodies, reaching over 20 percent in blastomeres.⁶⁰ False‐negative diagnoses have been observed in PGT for X‐linked disorders, myotonic dystrophy, and cystic fibrosis (CF), at the initial stage of PGT clinical application.^{3, 22, 23, 25, 35, 59} Clearly, the failure to detect one of the mutant alleles in compound heterozygous samples due to ADO will lead to misdiagnosis. However, this is no longer a problem with the application of protocols for simultaneous detection of the causative gene together with highly polymorphic markers closely linked to the gene tested.⁵⁹ With simultaneous testing of a sufficient number of linked markers amplified together with the gene in question, the risk of misdiagnosis may be substantially reduced or even practically eliminated. The protocol involves a multiplex nested PCR analysis, with the initial first‐round PCR reaction containing all the pairs of outside primers, followed by amplification of separate aliquots of the resulting PCR product with the inside primers specific for each site. Following the nested amplification, PCR products are analyzed by restriction digestion, real‐time PCR, direct fragment size analysis, or mini‐sequencing. Depending on the mutation being studied, different primer systems are designed with special emphasis on eliminating false priming to possible pseudogenes, for which purpose the first‐round primers are designed to anneal to the regions of nonidentity with a pseudogene.^{25, 59}

With the introduction of next‐generation technologies and the use of WGA prior to DNA analysis, the risk of ADO is further increasing, presenting even more problems in achieving accurate diagnosis.⁶¹ To improve the reliability of the test, the use of multiple linked markers became even more important, with importance of not only excluding the presence of the mutant gene, but also confirming the presence of the normal allele(s). Although a sufficient number of informative closely linked markers are usually available for multiplex PCR, this might not be the case in performing PGT by conventional PCR analysis in some ethnic groups.⁵⁹ Currently available protocols allow an accurate PGT for complex cases, requiring testing for two, three, and even more different mutations.

PGT generally requires knowledge of sequence information for Mendelian diseases, but may also be performed when the exact mutation is unknown. With the expanded use of single nucleotide polymorphisms (SNPs), linkage analysis allows PGT for any monogenic disease, irrespective of the availability of specific sequence information.^59–64 This is a more universal approach to track the inheritance of the mutation without actual testing for the gene itself, such as in karyomapping.⁶⁵ On the other hand, a specific diagnosis is required for X‐linked disorders, which may be performed by polar body analysis to preselect the embryos deriving from mutation‐free oocytes which may be transferred irrespective of gender or the paternal genetic contribution.⁶⁶

Polar body analysis (see Table 2.2 and Figure 2.2) also provides the prospect of pre‐embryonic diagnosis, which is required in many population groups where objection to the embryo biopsy procedures makes PGT nonapplicable. We performed the first pre‐embryonic genetic diagnosis for Sandhoff disease in a couple with a religious objection to embryo destruction.⁶⁷ Although pre‐embryonic genetic diagnosis was previously attempted by first polar body testing,^68–71 it is not actually sufficient for accurate genotype prediction without second polar body analysis, as shown in Figure 2.1. It is understood that for pre‐embryonic testing the second polar body analysis should be done prior to pronuclei fusion (syngamy), to ensure that only zygotes originating from mutation‐free oocytes are allowed to progress to embryo development and to be transferred, avoiding the formation and possible discard of any unaffected embryo.

A particular challenge is also presented by PGT for mitochondrial diseases, which still cannot be done reliably. A novel approach has been made to transfer a nuclear genome from the pronuclear stage zygote of an affected woman to an enucleated donor zygote, or to transfer the metaphase II spindle from an unfertilized oocyte of an affected woman to an enucleated donor oocyte.⁷²

As seen from Table 2.1, PGT is no longer restricted to conditions presented at birth; it is gradually expanding to include common diseases with genetic predisposition, such as cancers, performed in 10.5 percent of PGT‐M cycles, or nongenetic indications (7.2 percent of cases), such as PGT‐HLA with the purpose of stem cell therapy of affected siblings in the family.⁶²

Here we discuss the application of PGT‐M to a wider range of disorders, including conditions determined by de novo mutations (DNMs), genetic predisposition for late‐onset disorders, and preimplantation HLA matching (Table 2.1).