Читать книгу Genetic Disorders and the Fetus - Группа авторов - Страница 74

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 ago^41–43 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 mechanical^44–46 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 randomized controlled study.⁴⁷ The procedure is performed as follows: On day 5 of embryo development, when the blastocyst begins to herniate through the zona pellucida, several herniated trophectoderm cells are removed by smooth aspiration into a biopsy pipette with an internal diameter of 30 μm through the zona pellucida, which is opposite the inner cell mass. To break down the tight junctions between trophectoderm cells, three laser shots are applied (with a duration of 0.7 ms pulse for each shot). In the selection of embryos for blastocyst biopsy, poor‐quality blastocysts and those with early stage herniation are avoided.

It is also possible to perform blastocyst biopsy without the application of laser in order to avoid potential damage to the cells. The technique depends on the use of the force of surface tension on the boundary between the biopsy drop and the mineral oil culture medium, instead of laser pulses. The method can be applied only on grades 2 to 2–3 of the day 5 blastocysts, because the tight junction between trophectoderm cells of the early blastocyst (grades 2 and 2–3) is not as strong as the cell connection in the more advanced blastocyst (grades 3–4, 4, and higher). Grade 2 and 2–3 blastocysts are placed into 5 μL equilibrated culture media drops covered by 2 mL of equilibrated mineral oil, held by holding the pipette on the left side, while the biopsy pipette (interior diameter 25 μm) on the right side is used to suck 5–10 extruded trophectoderm cells into the pipette. The embryo is then moved onto the right edge of the biopsy drop with the biopsy pipette, moving slowly toward the inside of the oil environment, making a cytoplasm bridge between blastocyst and sample thinner, until it is finally removed and placed into a separate 5 μL culture media drop in the same dish. This procedure results in nonsticky trophectoderm samples with no evidence of damaged nuclei, with improved amplification efficiency.⁴⁸

As the time for analysis is limited by the implantation window, which is less than 24 hours, the technique of vitrification of biopsied blastocysts is now routine, allowing sufficient time before transfer of the tested embryos in a subsequent freeze–thaw cycle. It appears that this approach has also improved implantation and pregnancy rates, which could also be explained by the better receptivity of the uterus in unstimulated cycles. The other advantage of the method is that the embryo has already passed the natural self‐correction mechanisms, overcoming the natural errors of the cleavage stage, thus enabling the diagnosis of only persisting abnormalities.