impact of avoiding aneuploid embryos from transfer. In addition to testing all 24 chromosomes, the switch from blastomere sampling to blastocyst biopsy has also contributed to the positive reproductive PGT‐A outcome, as only established anomalies are tested. With progress in vitrification procedures, blastocyst biopsy coupled with transfer after freezing in a subsequent cycle has become the major approach for PGT, as it also involves a much higher uterine receptivity than in stimulated cycles. Blastocyst biopsy has also improved PGT accuracy, because instead of using single cells, a number of cells are used for analysis. Blastocyst biopsy and vitrification, coupled with 24‐chromosome testing, also simplified the organizational aspects of PGT, because the samples can be processed without the time limits for genetic analysis, also allowing samples to be shipped to specialized centers for more sophisticated testing, if required. Present standards of PGT‐A are presented in Figure 2.3.
Figure 2.3 Present standards of preimplantation genetic analysis for aneuploidies (PGT‐A). Twenty‐four‐chromosome aneuploidy testing by measurements of DNA content – not number of cells. DNA content may include damaged cells and cells still undergoing DNA replication, so the results per embryo derive from proportion of normal (euploid) and abnormal (aneuploid) DNA.
As seen from Figure 2.3, the gold standard for PGT‐A is presently NGS, which is also the initial step in PGT‐M, also using WGA.160–163 Compared with other PGT‐A methods, NGS provides more accurate copy number variations for each chromosome and is, therefore, better able to identify the presence of mosaic aneuploidy within the blastocyst. The detection of mosaicism requires much higher resolution than that provided by array comparative genomic hybridization (array‐CGH). This is achieved by NGS, which has improved accuracy of testing, especially in detecting copy number variations that contribute to the mosaicism detection rate. Different labs use different cut‐off rates, but PGDIS recommendations currently recommend a 20 percent cut‐off: embryos are considered nonmosaic euploid if nonmodel DNA proportions are below 20 percent. Nonmodel DNA over 80 percent is considered nonmosaic aneuploid. Between 20 and 80 percent are considered mosaic.164, 165 Figure 2.4 presents an example of mosaicism detected by NGS. The applicable commercially available kit for NGS is VeriSeq™ PGT Kit (Illumina). Karyomapping supplied by Illumina may also be used for PGT‐A, but requires different equipment and reagents than NGS. The primary application of karyomapping is PGT‐M.65 The other alternative NGS platform is Personal Genome Machine (PGM), developed by Thermo‐Fisher Scientific. The commercially available kit for this platform is Ion ReproSeq™ PGS Kit (Thermo‐Fisher Scientific).
Figure 2.4 Mosaicism for monosomy 3 detected by next‐generation sequencing (NGS). NGS results show a 50 percent mosaicism for monosomy 3, with all other chromosomes showing a normal pattern.
The major concern with NGS is that it is prone to ADO, because WGA must be performed as a first step to generate an adequate amount of DNA for analysis, which, as mentioned earlier, is still extremely inefficient in recovering all genomic sequences. So although NGS allows concomitant PGT‐A and PGT‐M, without simultaneous testing of a sufficient number of linked markers false‐negative results cannot be excluded, which may then lead to misdiagnosis, especially in PGT for dominant diseases. It can therefore be predicted that the technique should be performed with the use of SNP analysis for this purpose, or to work out the level of deep sequencing that can overcome the problem of ADO or develop more efficient WGA.61, 159
Structural rearrangements
The impact of PGT is even higher in translocation patients, with considerable reduction in the spontaneous abortion rate after preimplantation testing, resulting in a corresponding increase in the take‐home baby rate.130, 131 Although previous experience with PGT for chromosome structural rearrangements (PGT‐SR) was based on the use of the FISH technique, which is still applicable in some specific cases, the current standard is the utilization of array‐CGH or NGS technologies, which improves accuracy of testing and also allows a combined PGT‐A. An example of PGT‐SR by NGS is shown in Figure 2.5.
Figure 2.5 Next‐generation sequence‐based testing for translocation 46,XX, t(6;18)(p21.3;p11.2) (derivative chromosome indicated by red arrows).
In our experience of 940 PGT‐SR cycles, the comparison of reproductive outcomes of 609 cycles performed by FISH and 331 performed by array‐CGH and NGS showed significant improvement of the application of next‐generation technologies, resulting in almost doubling pregnancy rate, from 38.8 percent in FISH cycles to 66.5 percent with application of next‐generation technologies, and twofold reduction of spontaneous abortion rate, from 18.1 percent to 8.9 percent.48
A few sophisticated approaches based on next‐generation technologies have been developed for distinguishing noncarrier balanced embryos from normal ones. One such technology involved the use of an SNP microarray.166, 167 However, this method requires the availability of a lot of the unbalanced embryo, as well as parental DNA necessary to serve as a reference for distinguishing balanced translocation from normal blastocysts. The more universal approach is a specially designed NGS technology called mate‐pair sequencing (MPS). This involves high‐depth MPS to identify breakpoint regions and Sanger sequencing to define the exact breakpoint needed for designing specific primers required to identify normal and carrier embryos.168 A similar approach, termed nanopore long‐read sequencing, also discriminates carrier from noncarrier embryos through high‐resolution breakpoint mapping followed by breakpoint PCR.169 Thus, following application of breakpoint PCR, carrier embryos can be discriminated from noncarrier embryos. Both these approaches enable accurate high‐resolution breakpoint mapping directly on balanced reciprocal translocation carriers, providing the option of transferring euploid noncarrier embryos. Thus, the current technologies not only insure an acceptable pregnancy outcome for carriers of structural rearrangement, but also enable avoidance of balanced offspring and continuation of the problem in the next generation.
The presented data provide strong evidence that PGT is currently an important alternative to prenatal diagnosis, as it widens the options available for couples wishing to avoid the birth of an affected child, and provides the possibility of having children for those who would remain childless because of their objection to termination of pregnancy following prenatal diagnosis. At the same time, PGT is also becoming an integral part of assisted reproduction, by avoiding transfer of chromosomally abnormal and potentially nonviable embryos, thereby contributing to a significant increase in implantation and pregnancy rates in IVF, and to a general improvement in the standards of assisted
