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The biopsy of gametes opened an intriguing possibility of preconception diagnosis of inherited diseases, because genetic analysis of biopsied gamete material made it realistic to select gametes containing an unaffected allele for fertilization and subsequent transfer.²⁸ In this way, not only was the selective abortion of an affected fetus avoided but also fertilization involving affected gametes, as an option for couples at risk of conceiving a genetically abnormal fetus.

Although preconception genetic testing could be achieved by genotyping either oocytes or sperm, the latter approach is still not realistic. Development of methods for culture of primary spermatocytes and spermatogonia followed by genetic analysis of matured spermatides is theoretically possible, but this still remains a subject for future research, such as in the framework of the current attempts at haploidization.^{29, 30} The technique of sperm duplication has been introduced, which may allow testing of the sperm duplicate. However, errors may arise in the reduplication procedure, making the technique of sperm duplication inapplicable for clinical practice.^{31, 32}

The only approach for preconception diagnosis at present, therefore, is genotyping oocytes by biopsy and subsequent genetic analysis of polar bodies. The first attempt to obtain oocyte karyotypes was undertaken in the mouse model by testing the second polar body in the early 1980s, but the technique required much improvement to be considered for clinical application.³³ Polar bodies were then used to test the possibility of amplification of β‐globin sequences, again in the mouse model.³⁴ The first clinical application of the polar body approach was introduced in 1990.²⁰ It was demonstrated that, in the absence of crossover, the first polar body will be homozygous for the allele not contained in the oocyte and second polar body. However, the first polar body approach will not predict the eventual genotype of the oocytes if crossover occurs, because the primary oocyte in this case will be heterozygous for the abnormal gene. The frequency of crossover varies with the distance between the locus and the centromere, approaching as much as 50 percent for telomeric genes, for which the first polar body approach would be of only limited value, unless the oocytes can be tested further (Figure 2.1). Therefore, the second polar body analysis is required to detect hemizygous normal oocytes resulting after the second meiotic division. In fact, the accumulated experience shows that the most accurate diagnosis can be achieved in cases where the first polar body is heterozygous, so the detection of the normal or mutant gene in the second polar body predicts the opposite mutant or normal genotype of the resulting maternal contribution to the embryo after fertilization.⁴

Figure 2.1 Scheme demonstrating the principle of preimplantation genetic analysis by sequential DNA analysis of the first and second polar body, using the cystic fibrosis (CF) locus as an example.

Source: Verlinsky Y, Kuliev AMA. Preimplantation genetic diagnosis. In: Milunsky A, Milunsky JM, eds. Genetic disorders and the fetus: diagnosis, prevention and treatment, 6th edn. Oxford, UK: John Wiley & Sons, 2010.

To study a possible detrimental effect of the procedure, micromanipulated oocytes were followed and evaluated at different stages of development.^{3, 4, 35} The absence of any deleterious effect of polar body removal on fertilization, preimplantation, and, possibly, postimplantation development made it possible to consider the polar body approach as a nondestructive test for genotyping the oocytes before fertilization and implantation. In another study, to assess the effect of the second polar body sampling on the viability and developmental potential of the resulting embryo, 343 biopsied and 445 nonbiopsied mouse embryos were compared for the percentage of embryos reaching the blastocyst stage.³⁶

The results of PGT‐M performed by polar body biopsy, representing the world's largest series, is shown in Table 2.2. A total of 1,016 PGT‐M cycles were performed, for 538 autosomal recessive, 191 autosomal dominant, and 287 X‐linked disorders. Of 1,016 cycles initiated, 838 (82.5 percent) resulted in transfer of 1,656 embryos (1.98 embryos per transfer on the average), 349 (41.6 percent) clinical pregnancies, and 385 babies born. Only two misdiagnoses were observed in the case of PGT for fragile‐X syndrome and muscular dystrophy, which were due to consented transfer of additional embryo with insufficient marker analysis to exclude the probability of allele dropout (ADO) (see later). The example of PGT‐M by polar body sampling is shown in Figure 2.2.

Table 2.2 Clinical outcome of PGT‐M performed by polar body approach.

Conditions/mode of inheritance/sampling type	Patient	Cycles	Embryo transfer	No. embryos	Pregnancy	Spontaneous abortions	Baby
Autosomal recessive
Polar bodies	76	131	99	204	36	10	36
Polar bodies + blastomere/blastocyst	254	407	344	701	143	21	168
Subtotal	330	538	443	905	179	31	204
Autosomal dominant
Polar bodies	29	52	40	84	22	4	21
Polar bodies + blastomere/blastocyst	79	139	122	233	49	7	61
Subtotal	108	191	162	317	71	11	82
X‐linked
Polar bodies	39	86	63	110	22	4	20
Polar bodies + blastomere/blastocyst	116	201	170	324	77	12	79
Subtotal	155	287	233	434	99	16	99
Total	593	1016	838	1656	349 (41.6%)	58 (17%)	385

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.