Trophectoderm biopsy for preimplantation genetic test and technical tips: A review

Abstract Background Recently, the Japan Society of Obstetrics and Gynecology initiated a clinical study of preimplantation genetic test for aneuploidy. There will be a great need for a standardized embryo biopsy technique in Japan. However, the gold standard trophectoderm (TE) biopsy procedure has not been established, and this review outlines the clinical use of TE biopsy. Methods Based on literature, the method and associated techniques for TE biopsy, a dissection method of TE cells from blastocysts, were investigated. Main findings Two TE biopsy methods are used, namely assisted hatching (herniating) and non‐assisted hatching (direct suction); however, it is not clear which of these methods is superior. It is critical to understand whether the flicking or pulling method is beneficial. Conclusion Non‐assisted hatching biopsy method may cause blastocyst collapse with a higher probability, and it may extend the biopsy time. The biopsy procedure should be performed within 3 minutes, and thus direct TE suction may have greater disadvantages. It is a fact that pulling method of TE dissection with laser pulse is simple; however, excess laser shots may induce a higher frequency of mosaicism. It is important to understand that each technique of TE biopsy has benefits and disadvantages.


| Biopsy method; Hatching or non-assisted hatching?
The first clinical report of preimplantation genetic diagnosis (PGD) using cleavage-stage embryo biopsy was published by Handyside in 1990. 6 At the same time, researchers began studying trophectoderm biopsy and demonstrated the feasibility of this method for PGD. 7,8 Initially, cleavage-stage embryo biopsy predominated, [9][10][11][12][13] particularly that using FISH protocols. [14][15][16][17][18][19][20][21][22][23] The first live birth case following blastocyst biopsy for PGD was reported in 2002. 24 McArthur et al 25 described the details of trophectoderm biopsy procedure and reproductive outcomes of PGD using this technique with FISH or real-time quantitative polymerase chain reaction (qPCR) in 2005. Notably, in their approach, zona breaching was performed on day 3 or 4, and 2-9 cells from the herniated trophectoderm were teased free from the remaining trophectoderm with laser pulse. 25,26 This protocol was frequently applied for clinical trials; however, researchers also proposed another approach, that is, non-assisted hatching method. Capalbo et al 27 reported a direct aspiration technique for biopsy of the trophectoderm without assisted hatching in 2014, demonstrating the benefits of this protocol in daily laboratory work. Because the timing of biopsy for hatching blastocyst is critical, excess herniation of trophectoderm may induce incarceration ( Figure 1A). There is no evidence for the association between embryo incarceration and embryo development. However, practitioner should still avoid causing excess trophectoderm herniation. Continuous observation using time-lapse monitoring technology and direct TE aspiration with non-assisted hatching are helpful for detecting and preventing such early incarceration. Non-assisted hatching has disadvantages. For example, if blastocyst collapse occurs during TE cell aspiration, the biopsy procedure may be difficult and be associated with a higher risk of losing the inner cell mass (ICM) area. Furthermore, blastocyst collapse may extend the biopsy time, despite recommendations that the biopsy procedure should be performed within 3 minutes. 28 No published clinical data have shown which method is better for biopsy.

| Optimal timing of zona drilling
When establishing standard approaches for trophectoderm biopsy, 25,26 it is essential to consider the timing of zona breaching during the cleavage or blastocyst stage ( Figure 1B) This is because it has been hypothesized that ICM splitting may generate monozygotic twining during the repeat of collapse and expansion in the process of blastulation in a clinical study with time lapse, 32 or in an 8-shaped hatching animal experiment. 33 However, not only the number of cell compensation, but also the chorionicity and amnionicity are also very important for normal fetal development after embryo splitting. 34 Moreover, cell fate has already been determined at the blastocyst stage 35 and ICM is formed by two cell lines, which are epiblasto and primitive endoderm in early blastocyst stage. 36 Therefore, full attention should be paid to TE biopsy, and the double laser zona drilling may be effective to avoid impact on ICM.

| An appropriate number of biopsied TE cells is required to maintain implantation potential
The number of biopsied cells is one of the most critical factors affecting TE biopsy, 40 the general consensus among researchers is that this number may be 5 cells. 26,41 However, it is difficult to apply this in the clinical setting, and attempts have been shown to result in poor-grade TE and fragmentation. Fewer cell biopsies may be less invasive but result in a higher risk of amplification failure, whereas increased cell biopsies may lead to a lower risk of amplification failure but increase blastocyst disruption. Several clinical reports have described the incidence of DNA amplification and inconclusive results using a CCS device, at rates of 12.5% and 4.5-8.2% by polar body biopsy, 42,43 2.9%-12.3% and 0%-18.6% by single blastomere isolation, [44][45][46][47][48] and 0%-4.0% and 0%-4.6% by TE, 28,49-55 respectively (Table 1). Blastocyst biopsy results in lower amplification failure rates because of the superior cell numbers obtained from biopsy comparing with that of polar body or blastomere biopsy. Cimadomo et al showed that 8.0 trophectoderm cells were required for successful DNA amplification and conclusive diagnostic results; thus, the most suitable cell number for biopsy may be 5-10 cells rather than 1-5 cells. Overall, the use of these invasive techniques for TE biopsy may reduce the rate of live births by 5% live births. 56

| When should we perform TE biopsy?
No conclusion has yet been reached regarding the best timing of TE biopsy, and we interpret that a point of contention may be the num- On the other hand, from the results of our clinical study, blastocysts, which have earlier developmental speed, showed better reproductive outcomes such as clinical pregnancy and live birth rate 58 ; meanwhile, smaller size blastocysts (<160 µm) until day 7 had little chance to achieve clinical pregnancy. We hypothesize that biologically high-potential embryos reaching full-size blastocyst until 130 hours after insemination have a higher chance to be euploidy, similar to the report that day 7 blastocysts showed a lower euploidy rate. 59,60 Finally, the timing of biopsy may be better in the developed or developing blastocyst, which have larger cell numbers.
Additionally, if an embryo has an earlier developmental speed, such as developed blastocyst in day 5, favorable result may be obtained.

| Size of hatching point and time length to perform the biopsy
In general, single TE biopsy should be performed within 3 minutes. 28 This can be achieved using a simple biopsy. However, thick TE samples may be difficult to dissect from blastocysts. Thinning the TE area of the cut plane makes biopsy less difficult; thus, we suggest that smaller zona holes may produce less constriction of the hatching site for micro-cutting ( Figure 1C). However, this process may be more complicated. Indeed, if a practitioner ignores the optimal timing of biopsy, a large 8-shaped hatching site may be generated, resulting in incarceration ( Figure 1A). To prevent this, a time-lapse system may be useful. Furthermore, the size of the suction pipette for TE may be altered to minimize the area of the cut plane; several suction pipettes with inner diameters of 20-40 μm have been developed and are available from commercial sources.

| Trophectoderm biopsy technique using flicking or pulling
The first solid-state laser to be used for ART, which was applied to trap spermatozoa, was neodymium:yttrium-aluminum-garnet  may influence the results. Further investigation is needed to assess this mechanism, and it is necessary to conclude whether flicking method is superior to pulling method ( Figure 1D).

| Timing of cryopreservation after TE biopsy
There are two transfer strategies for euploidy embryos currently in clinical practice: vitrified-warmed or fresh embryo transfer. Biopsy of blastomeres on day 3 permits analysis of diagnostic outcomes before day 5 using advanced molecular biology techniques. Several reports have described PGT-A using the fresh transfer strategy. 47,84,85 However, the use of TE biopsy and vitrified-thawed blastocyst transfer protocols is increasing worldwide. [86][87][88][89][90] On the other hand, the optimal timing of vitrification after TE biopsy has not been thoroughly discussed. In one study, the optimal time was reported to be 10-15 minutes, 91 whereas another study reported that 30 minutes or less was the optimal. 92 In contrast, another report showed that the optimal time was >3 hours to enable blastocysts to reach re-expansion. 93

ACK N OWLED G EM ENTS
None.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.