for at‐risk couples.
HLA typing
Preimplantation HLA typing is an attractive PGT indication. The first case of preimplantation HLA typing was performed in combination with PGT for Fanconi anemia complementation group C (FA‐C), which resulted in a successful hematopoietic reconstitution in the affected sibling by transplantation of stem cells obtained from the HLA‐matched offspring resulting from PGT.5 To improve access to the HLA‐identical bone marrow transplantation in sporadic bone marrow failures, this approach was then applied with the sole purpose of ensuring the birth of an HLA‐identical offspring, not involving PGT, which also resulted in radical treatment of a sibling with a sporadic Diamond–Blackfan anemia (DBA) by stem cell transplantation from an HLA‐identical child born following preimplantation HLA typing.79 Preimplantation HLA typing has become one of the most useful indications for PGT, performed currently with or without testing for the causative gene.79–88
Despite the ethical issues involved,80 preimplantation HLA typing procedures have so far been performed in hundreds of cases with affected children requiring HLA‐compatible stem cell transplantation, including thalassemia, Fanconi anemia, Wiskott–Aldrich syndrome, X‐linked adrenoleukodystrophy, X‐linked hyper‐IgM syndrome, X‐linked hypohidrotic ectodermal dysplasia with immune deficiency, X‐linked chronic granulomatous disease, cancer syndromes, incontinentia pigmenti, leukemias, and inherited and sporadic forms of DBA.81–91
We applied PGT‐HLA in 485 cycles, including HLA typing alone, or combined with PGT‐M for 35 different conditions. Overall, 424 HLA‐matched unaffected embryos were detected and transferred in 291 cycles, resulting in 125 clinical pregnancies and birth of 117 HLA‐matched children, as potential donors for their siblings.25, 48, 92 Although the majority of cases were performed for thalassemia, this approach has a great life‐saving potential for affected siblings with congenital immunodeficiency. We performed 135 PGT cycles for 18 different immunodeficiency conditions, resulting in the birth of 54 children free of immunodeficiency, stem cell donors for transplantation treatment of affected siblings, with a total cure, such as Fanconi anemia and hyper‐IgM syndrome.48
Similar experience has been reported from other large series, such as from Istanbul: 626 PGT‐HLA cycles for 312 couples were performed (122 HLA only and 504 with PGT‐M), resulting in the birth of 128 thalassemia‐free children. Stem cells of 66 of these children were used for cord blood or bone marrow transplantation, which resulted in successful bone marrow reconstitution in all but two of them (transplantation treatment of the remaining 57 sibling pending).93, 94
Chromosomal disorders
The theoretical rate of chromosomally abnormal embryos at fertilization is approximately 40 percent, taking into account both the rate of aneuploidies in oocytes and sperm and fertilization‐related abnormalities.95, 96 Mouse data show that most aneuploidies, although compatible with cleavage, are lost during implantation.97, 98 Additional losses of chromosomally abnormal embryos occur after implantation, clinically recognized as spontaneous abortions, more than half of which are caused by chromosomal abnormalities. As a result of this selection against chromosomal abnormalities before and after implantation, only 0.65 percent of newborns have chromosomal disorders, many of which lead to serious disability and early death (see also Chapter 1).
Prevalence and origin of chromosomal errors
A wide range in the frequency of chromosomal aneuploidy in human oocytes has been reported (17–70 percent), but most of these studies have been performed on poor‐quality oocytes left over after the failure of IVF attempts. A high rate of hypohaploidy observed in oocytes was considered to be artificially induced by spreading techniques, so aneuploidy rate was calculated by doubling the number of hyperhaploid oocytes. This ignores chromatid malsegregation and/or chromosome lagging events, contradicting the results of the observation that the rate of hypohaploidy is higher than the rate of hyperhaploidy.99 Cytogenetic analysis of unfertilized oocytes was also improved by parthenogenetic activation of human oocytes.100
As mentioned, an attempt at noninvasive cytogenetic analysis of oocytes was undertaken in the early 1980s through visualization of the second polar body chromosomes by transplanting the polar body into a fertilized egg.33 The success rate of visualization of polar body chromosomes was then improved by different methods, demonstrating the practical implication of polar body analysis for chromosomal errors originating from maternal meiosis,101–103 in contrast to the report on the uselessness of the first polar body for this purpose.104 Various approaches were explored in the attempt to visualize the chromosomes of the first and second polar bodies, as well as of the biopsied blastomeres, including nuclear transfer, electrofusion, and chemical methods.102, 105, 106 However, the major progress in chromosome analysis of oocytes and embryos was achieved with introduction of the fluorescence in situ hybridization (FISH) technique,107–113 microarray technology, and NGS. Our meiosis data based on the analysis of 22,986 oocytes detected 9,812 aneuploid oocytes (46.8 percent), originating comparably from the first and second meiotic divisions. Overall, meiotic division errors were observed in 33.1 percent of oocytes in meiosis I, 38.1 percent in meiosis II, and 28.8 percent in both.
Although the aneuploidy rate in embryos is comparable to that in oocytes, the types of anomalies in the oocytes and embryos were significantly different,114–116 also showing inconsistency between the expected and observed frequency of some types of aneuploidies. In our current practice of PGT‐A, the analysis of 2,922 blastocysts, 56.0 percent of embryos were aneuploid, comprising 13.0 percent monosomy, 13.0 percent trisomy, 8.0 percent numerical mosaic, 14.0 percent segmental mosaic, and 8.0 percent complex errors.48 It is of interest that no age dependence was revealed for monosomies in embryos, suggesting that the rate of monosomies detected in embryo by PGT‐A may be of artefactual nature.
A possible explanation for this discordance is that the majority of monosomies detected in embryos are derived from mitotic errors, assuming technical causes are excluded. In fact, a significant proportion of the cleavage‐stage monosomies appeared to be euploid after their reanalysis.117, 118 No monosomies, except monosomy 21, are detected after implantation, so either they are eliminated before implantation or have no biological significance, reflecting the poor viability of the monosomic embryos and their degenerative changes. In one relevant study the progression and survival of different types of chromosome abnormalities were followed up in 2,204 fertilized oocytes.119 A variety of chromosome abnormalities was detected, including many types of errors not recorded later in development. However, these appeared to be tolerated until activation of the embryonic genome, after which there were declines in frequency. Nevertheless, many aneuploid embryos still successfully reach the blastocyst stage, even if some chromosome errors present during preimplantation development are not seen in later pregnancy.
Aneuploidies
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