Figure 2.2 Preimplantation genetic testing for de novo tuberous sclerosis* type II deletion (TSC2 gene, exon 7–10 deletion 16p13.3) and preimplantation genetic testing for aneuploidies by next‐generation sequencing (NGS). Of 15 oocytes tested by polar body analysis, ten were affected and five free of deletion. The embryos deriving from deletion‐free oocytes were tested for aneuploidy by NGS; three of these were euploid (embryos 7, 12, and 17) and one (embryo 5) with monosomy 14. Two of the mutation‐free euploid embryos (embryos 7 and 12, the NGS results of which are shown bottom right) were transferred in a frozen cycles, resulting in a twin pregnancy and birth of two unaffected children free from deletion. *Tuberous sclerosis complex is an autosomal dominant multisystem disorder characterized by hamartomas in multiple organ systems, including the brain, skin, heart, kidneys, and lung. Central nervous system manifestations include epilepsy, learning difficulties, behavioral problems, and autism. The affected mother had had epilepsy since 3 months old and lympho‐angio‐leiomyomatosis.
Preimplantation genetic testing based on embryo biopsy
Despite clear advantages, the polar body approach does not provide diagnosis of the paternal alleles and the gender of the embryo and therefore cannot be used to avoid the transfer of male embryos in cases of the X‐linked disorders, unless specific diagnosis can be achieved on oocytes using the polar body approach (see later). The fact that the genotype of the oocyte is inferred from the genotype of the polar body, rather than determined directly is another weakness. In these situations, embryo biopsy becomes the much more comprehensive approach, with blastocyst biopsy currently being a standard procedure.
The first clinical application of embryo biopsy for PGT by Handyside et al.19 was performed at the cleavage stage for X‐linked disorders by gender determination.19 The study of the viability of the biopsied pre‐embryos did not reveal any detrimental effect of these procedures: it was shown that more than 70 percent of the manipulated embryos reached blastocyst stage, with no significant reductions in cell number and energy substance (glucose and pyruvate) uptake.37
Embryo biopsy, initially applied at the cleavage stage, has become a method of choice in most centers, resulting in the birth of thousands of children free of genetic disorders.21–25 Nevertheless, some programs have demonstrated significant detrimental effect of the procedure, especially when instead of one blastomere two were removed, or even a single blastomere removed in inexperienced hands (see later). Also, there were problems due to the high rate of allele dropout and the high frequency of mosaicism at this stage (see later). The switch to blastocyst biopsy largely solved these problems and became standard. In fact, blastocyst biopsy was first introduced in the 1960s in rabbits by Gardner and Edwards.38 As the number of cells in human blastocyst increases up to more than 100, a few cells (approximately five cells) are removed from IVF embryos without affecting viability. Another advantage of this approach is that trophectoderm cells are biopsied without affecting the inner cell mass, from which the embryo is derived. Initially the viability of biopsied blastocysts in vitro was studied using morphologic criteria and the patterns of human chorionic gonadotropin (hCG) secretion. Hatching was observed in 38.5 percent of blastocysts, with hCG detected first on day 8, peaking at day 10, and still detectable in some blastocysts at day 14.39 For the individual blastocysts, the pattern of hCG secretion correlated with the assessment of morphology.40
Blastocyst biopsy for clinical purposes was first attempted over 20 years ago41–43 and is currently a PGT standard.44, 45 This is also the method used in uterine lavage, which may soon appear as a realistic approach for PGT without IVF.46 Both mechanical44–46 and laser techniques were used for blastocyst biopsy, which has become a method of choice in most centers, also resulting in improved pregnancy rates, particularly in frozen PGT cycles. The advantage of blastocyst biopsy over cleavage‐stage sampling was demonstrated by a well‐designed
