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.
Prospects for noninvasive preimplantation genetic testing
Although there is no evidence of the detrimental effect of biopsy procedures on embryo viability, the potential for damage cannot be excluded, so the development of noninvasive preimplantation genetic testing (NIPGT) is of increasing importance. The fact that DNA fragments of approximately 150 bp are present in cell‐free DNA of blastocoele fluid and spent culture medium, is the basis of the concept that NIPGT can be developed analogous to noninvasive prenatal testing (NIPT), because these DNA may originate from breakdown of nuclear DNA derived from cells damaged at biopsy or undergoing apoptosis during cell division. Available reports suggest that the technique may in future be applicable for preselection of euploid embryos for transfer.49–58 Although the usefulness of blastocoele fluid for PGT by different groups lacks consensus, its use has been proposed to identify at‐risk embryos from younger patients who otherwise have no accessible indication for PGT‐A.49, 50 The other approach, based on the use of cell‐free DNA in culture media, represents the genuine noninvasive approach analogous to monitoring cell‐free DNA in maternal plasma during pregnancy,51–58 with the concordance studies showing progress. In one of these studies, the test was offered to infertility patients presenting for PGT‐A in the format of a clinical trial. Enough DNA for testing was detected in 88% of cases, with 80% concordance to biopsy results.55 In another prospective study, blastocoele samples and spent culture medium samples were compared to diagnostic biopsy samples that were processed for PGT‐M and PGT‐A. Overall results demonstrated that neither blastocoele samples nor spent medium were sufficiently robust approaches for aneuploidy or single‐gene disorders and cannot be applied clinically until the risk of maternal contamination can be excluded. This risk is particularly high in spent culture medium samples due to maternal cumulus DNA contamination.56 In the other study DNA in spent medium was shown to be detectable on day 3, but more reproducibly on day 5, with concordances of 65 and 70 percent with biopsy samples, which are not high enough for practical application.57
Although the origin of the DNA in spent culture medium is not clear, it was postulated that results of the test may have a prognostic utility and better prediction of reproductive outcome if euploid embryos with euploid spent media results are transferred. This is in contrast to the euploid embryo transfer outcome with spent media showing imbalanced results.58 This is also in agreement with the blastocoele data, which suggested a significantly improved embryo transfer outcome for those euploid embryos whose blastocoele fluid has a higher quantity of DNA based on WGA.50 Thus, clearly more research is needed to validate the usefulness of spent culture medium and blastocoele fluid for PGT.
Preimplantation genetic analysis
Initially, PGT was justified only for high‐risk pregnancies. Maternal age was not expected to be an indication for such early testing, initially being considered even a contraindication to PGT. However, the development and improvement of the methods for sampling and genetic analysis have made use of PGT for chromosomal disorders a reality. Despite continuing discussion of the impact of PGT‐A, it is used routinely worldwide as a tool in assisted reproduction technologies, especially for improving the effectiveness of IVF in poor‐prognosis patients, particularly in carriers of chromosomal rearrangements. As a result, the majority of PGT cycles are still done for PGT‐A.21–25
Single‐gene disorders
DNA analysis for preconception and preimplantation diagnosis is well established, which enables genetic analysis of minute quantities of DNA obtained from a single or few cells.12, 19, 20 Because this also increases the chance of DNA contamination in PGT, decontamination procedures were applied in the initial stages, based on elimination of double‐stranded DNA sequences,59 excluding also possible contamination with the embryology and PCR reagents, such as water, salt solutions, oligonucleotides, and Taq polymerase.
The major source of contamination in PGT is still cellular contamination, such as cumulus cells, spermatozoa, or cell fragments, which might be amplified simultaneously with polar bodies or embryo biopsies, creating the possibility for erroneous testing. Because potential misdiagnosis of PGT may be caused by sperm DNA contamination, it is currently a routine IVF practice to perform PGT‐M for single‐gene defects following microsurgical fertilization by intracytoplasmic sperm injection (ICSI).
Nevertheless, the major source of misdiagnosis is preferential amplification or allele‐specific amplification, referred to as allele dropout (ADO), which may happen in single‐cell genetic analysis. As much as 8 percent of ADO was observed in PCR analysis of the first polar bodies, reaching over 20 percent in blastomeres.60 False‐negative diagnoses have been observed in PGT for X‐linked disorders, myotonic dystrophy, and cystic fibrosis (CF), at the initial stage of PGT clinical application.3, 22, 23, 25, 35, 59 Clearly, the failure to detect one of the mutant alleles in compound heterozygous samples due to ADO will lead to misdiagnosis. However, this is no longer a problem with the application of protocols for simultaneous detection of the causative gene together with highly polymorphic markers closely linked to the gene tested.59 With simultaneous testing of a sufficient number of linked markers amplified together with the gene in question, the risk of misdiagnosis may be substantially reduced or even practically eliminated. The protocol involves a multiplex nested PCR analysis, with the initial first‐round PCR reaction containing all the pairs of outside primers, followed by amplification of separate aliquots of the resulting PCR product
