show a 50 percent mosaicism for monosomy 3, with all other chromosomes showing a normal pattern."/>
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 reproduction practices.
Ethical and legal issues
Considerations on ethical and legal issues are evolving, along with the evolution of the technology for the control of genetic diseases, and have become one of the key subjects in discussing the acceptability of preconception and preimplantation testing for genetic disorders. Ethical and legal issues determine, to a considerable extent, whether these new approaches are promoted to become an integral part of preventive genetics services or are waived on ethical grounds.8 PGT could be regarded as an ethically acceptable procedure in the context of a general objective of genetic service, which, according to the WHO, is to help genetically disadvantaged people live and reproduce as normally and as responsibly as possible.9 Because PGT is heavily based on IVF, it also is relevant that IVF is considered to be ethically acceptable in many countries.170–174 However, complex ethical and legal issues are confronted differently in various countries (see Chapter 36).175 For example, in Germany, the future of PGT depends on an Embryo Protection Law, which has been in effect since 1991.172, 176 This law is very strict and prohibitive of embryo research. However, it prohibits only destructive research that impairs the chances of the embryo becoming a human being up to the eight‐cell stage. In fact, blastocyst biopsy may be possible without any conflict with the law because, together with chorionic villus sampling (CVS), such embryo biopsies are considered beneficial, allowing decisions to be made before replacement. Therefore, there is no conflict about the provision of PGT in Germany before the pronucleate stage, which is currently under way. However, this must be done for diagnostic purposes only, not for research. Even in the case of tripronucleate embryos, only observation is permitted, not experimentation.
This approach may also resolve the ethical issues impeding PGT in Austria, Switzerland, Malta, and other predominantly Catholic countries.177 The same holds for other countries where no preventive measures have been allowed on religious grounds. For example, the law has evolved recently in Switzerland, citizens voting to introduce PGT into clinical practice. In contrast, in France, there seems to be no law at all concerning either PGT or embryo research. However, the attitude of the National Ethical Committee toward PGT is influenced by the fact that the diagnosis is based on genetic analysis of only one or a few cells and that all male embryos are discarded after gender determination, while half of them are completely normal. However, the testing is presently based on specific diagnosis, rather than gender determination. Another concern is that PGT increases the need for IVF, which is provided free of charge in France. Finally, prenatal diagnosis, also provided free of charge, was enough to avoid genetic disorder, so the provision of PGT was considered to be an additional prenatal test, without taking into account the suffering caused by selective abortions after prenatal diagnosis. Nevertheless, there are presently a few well‐established centers in France providing a full range of PGT services.
In Italy, PGT and many aspects of IVF were forbidden by Act of Parliament for almost 7 years. Only three oocytes were allowed to be aspirated for fertilization in vitro, clearly reflecting the opinions of the hierarchy of the Roman Catholic Church.178 Prior to the law, Italy had been among the most active centers involved in the development and application of PGT for genetic and chromosomal disorders. After the laws were enacted, it was difficult to perform PGT, which nevertheless is presently performed without restriction.
In some countries, such as Belgium, the decision on embryo research and PGT