Читать книгу Genetic Disorders and the Fetus - Группа авторов - Страница 83

As seen from the previous discussion, without the detection and avoidance of the transfer of chromosomally abnormal embryos, there is at least 50 percent chance of loss during implantation or postimplantation development. In addition to the clear benefit of avoiding the transfer of aneuploid embryos, which contributes to improvement of pregnancy outcome of poor‐prognosis IVF patients, this should improve the overall standard of medical practice, upgrading the current selection of embryos by morphologic criteria into aneuploidy testing. This explains a widespread application of PGT‐A aimed at the preselection of embryos with the highest developmental potential demonstrating a clinical benefit, in terms of the improved IVF outcome through improved implantation and pregnancy rates, reduction of spontaneous abortions and improved take‐home baby rates in IVF patients of advanced reproductive age, repeated IVF failures, and recurrent spontaneous abortions.^{24, 25, 114–116, 116, 120}

The failure to detect a positive effect of aneuploidy testing on reproductive outcome in a few studies may be due to possible methodologic deficiencies.^122–124 Despite these methodological shortcomings, which have been heavily criticized in the literature,^125–127 the American Society for Reproductive Medicine Practice Committee did not favor transferring embryos without aneuploidy testing.¹²⁸ This may mean the alternative of incidental transfer of chromosomally abnormal embryos, as every second oocyte or embryo obtained from poor‐prognosis IVF patients is chromosomally abnormal and destined to be lost before or after implantation. In fact, only one in ten of chromosomally abnormal embryos may survive to recognized clinical pregnancy, 5 percent may survive to the second trimester, and only 0.5 percent reach birth. Thus, the majority of chromosomal abnormalities are eliminated before or during implantation, reflecting a poor implantation rate in poor‐prognosis IVF patients, and explaining a high fetal loss rate in those patients without PGT‐A. This has actually been demonstrated by testing products of conception from poor‐prognosis non‐PGT IVF patients, confirming the high prevalence of chromosomal aneuploidy in the absence of PGT‐A. Of 273 cases tested, 64.8 percent had chromosomal abnormalities, up to 79 percent of which could have been detected and not transferred using PGT.¹²⁹

Given these data, the current IVF practice of selecting embryos for transfer based on morphologic criteria may hardly be an acceptable procedure for poor‐prognosis IVF patients. In addition to giving an extremely high risk of establishing an affected pregnancy from the onset, this will significantly compromise the very poor chances of these patients to become pregnant,^{130, 131} especially with the current tendency of limiting the number of transferred embryos to only a few or even one. Although culturing embryos to day 5 (blastocyst) before transfer may allow, to some extent, the preselection of developmentally more suitable embryos compared with day 3, some aneuploid embryos will still be capable of developing to the blastocyst stage.^{132, 133} These abnormal embryos will not be eliminated in the current shift to blastocyst transfer, and may implant and lead to fetal loss, compromising the outcome of pregnancies resulting from the implanted normal embryos in multiple pregnancies. In fact, multiple pregnancies represent a severe complication of IVF, which is currently avoided by preselection and transfer of a single aneuploidy‐free blastocyst with the greatest developmental potential to result in a healthy pregnancy.

However, contrary views also exist about safety, outcome, and efficacy.^{122–124, 134–137} Randomized controlled studies performed with introduction of next‐generation technologies were able to quantify the clinical impact of preselection of aneuploidy‐free zygotes, demonstrating the obvious benefit approximating a 15–20 percent increase in pregnancy rates, compared to embryos transferred solely based on morphological criteria, although this was not universal in all age groups.

The first randomized controlled trial (RCT) using 24‐chromosome analysis was performed in a series of 112 women randomized into two groups:¹³⁸ transfer of a PGT‐A embryo versus transfer of a morphologically normal embryo not biopsied or tested. Of 425 blastocysts tested, 45 percent (191/425) were with aneuploidy, resulting in a 71 percent pregnancy rate compared with 46 percent in the nonbiopsied control group of 389 blastocysts with normal morphology. In the other RCT, involving 72 cases, the transfer of euploid blastocysts resulted in 66 percent implantation and 85 percent delivery rates, compared to 48 percent and 68 percent, respectively, in the control group of 83 morphologically normal embryos but not tested for PGT‐A.¹³⁹ Another RCT did not find differences in pregnancy rates between single euploid embryo transfer and the transfer of two morphologically normal but untested embryos, but a 48 percent twin rate was observed in the latter compared to 0 percent in the single embryo transfer tested group.¹⁴⁰ Significant differences between a PGT‐A group and control groups were also demonstrated in an RCT performed using a cleavage‐stage embryo biopsy.¹⁴¹ Thus, results of RCTs involving 24‐chromosome platforms suggest that it is reasonable to inform assisted reproductive technology (ART) patients of advanced maternal age about the utility of PGT‐A. The precise age range at which women would benefit is still under study, although the optimal outcome seems to be for the 35–39 age group, as suggested by trials conducted by the Society for Assisted Reproductive Technology (SART)¹⁴² and the STAR Study Group.¹⁴³

The switch of aneuploidy testing from FISH to the next‐generation technologies for 24‐chromosome testing,^144–163 allowing improved detection of chromosomally abnormal oocytes and embryos, has, therefore, further confirmed the positive 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}