can be developed analogous to noninvasive prenatal testing (NIPT), because these DNA may originate from breakdown of nuclear DNA derived from cells damaged at biopsy or undergoing apoptosis during cell division. Available reports suggest that the technique may in future be applicable for preselection of euploid embryos for transfer.49–58 Although the usefulness of blastocoele fluid for PGT by different groups lacks consensus, its use has been proposed to identify at‐risk embryos from younger patients who otherwise have no accessible indication for PGT‐A.49, 50 The other approach, based on the use of cell‐free DNA in culture media, represents the genuine noninvasive approach analogous to monitoring cell‐free DNA in maternal plasma during pregnancy,51–58 with the concordance studies showing progress. In one of these studies, the test was offered to infertility patients presenting for PGT‐A in the format of a clinical trial. Enough DNA for testing was detected in 88% of cases, with 80% concordance to biopsy results.55 In another prospective study, blastocoele samples and spent culture medium samples were compared to diagnostic biopsy samples that were processed for PGT‐M and PGT‐A. Overall results demonstrated that neither blastocoele samples nor spent medium were sufficiently robust approaches for aneuploidy or single‐gene disorders and cannot be applied clinically until the risk of maternal contamination can be excluded. This risk is particularly high in spent culture medium samples due to maternal cumulus DNA contamination.56 In the other study DNA in spent medium was shown to be detectable on day 3, but more reproducibly on day 5, with concordances of 65 and 70 percent with biopsy samples, which are not high enough for practical application.57
Although the origin of the DNA in spent culture medium is not clear, it was postulated that results of the test may have a prognostic utility and better prediction of reproductive outcome if euploid embryos with euploid spent media results are transferred. This is in contrast to the euploid embryo transfer outcome with spent media showing imbalanced results.58 This is also in agreement with the blastocoele data, which suggested a significantly improved embryo transfer outcome for those euploid embryos whose blastocoele fluid has a higher quantity of DNA based on WGA.50 Thus, clearly more research is needed to validate the usefulness of spent culture medium and blastocoele fluid for PGT.
Preimplantation genetic analysis
Initially, PGT was justified only for high‐risk pregnancies. Maternal age was not expected to be an indication for such early testing, initially being considered even a contraindication to PGT. However, the development and improvement of the methods for sampling and genetic analysis have made use of PGT for chromosomal disorders a reality. Despite continuing discussion of the impact of PGT‐A, it is used routinely worldwide as a tool in assisted reproduction technologies, especially for improving the effectiveness of IVF in poor‐prognosis patients, particularly in carriers of chromosomal rearrangements. As a result, the majority of PGT cycles are still done for PGT‐A.21–25
Single‐gene disorders
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.