*2.3. Trophoectoderm Biopsy*

The blastocyst is composed of two different cell types: the inner cell mass, which will evolve in fetal tissues, and the trophoectoderm (TE) considered the precursor of future placenta. The advantages correlated to TE biopsy are mainly three: first of all, TE is not involved in fetus formation, as it will form extra-embryonic tissues. The second benefit is that blastocyst stage embryos have already activated their genome, allowing for a more accurate analysis [25]. Finally, a sample of about five-eight cells is needed for the test, determining a loss of about 10% of all of the cells forming the blastocyst (about 100–150). When compared to the cell mass loss determined by the removal of two blastomere, this procedure seems to be much less invasive [33]. Blastocyst biopsy also implicates other practical advantages: embryos vitrified at this stage show a higher survival rate if compared with cleavage stage embryos. Therefore, it allows for postponing the transfer and even adopt a single embryo transfer policy reducing multiple pregnancies [34].

Three main approaches can be followed for TE biopsy: the first consist in opening the zona pellucida at cleavage stage using a laser-assisted drilling and then waiting for the formation of expanded or herniating blastocysts on day 5 [35]. Cleavage-stage zona drilling is performed to obtain a faster biopsy on herniating blastocysts and reduce the chance of sudden collapse. Although being widely adopted, this procedure presents two main limitations; it entails two sessions of embryo manipulation outside the incubator and there is the concrete risk of having the inner cell mass herniating outside the zona. The second approach to TE biopsy is to leave the embryo in culture until blastocyst full expansion and then open the zona immediately before the biopsy, with assisted laser hatching. This strategy requires a single intervention on the embryo and the zona can be opened in a region far from the inner cell mass, reducing its involvement in the biopsy process. The last method takes advantages of both the previous approaches: it consists of opening the zona when the blastocyst is fully expanded and then waiting for the TE herniation. Figure 1 shows the blastocysts biopsy laser assisted steps.

**Figure 1.** In the figure, the sequence of a blastocyst biopsy laser assisted is shown. The blastocyst is initially orientated by mean of the holding pipette in order to keep the inner cell mass as far as possible from the site of biopsy (**A**). Subsequently, the biopsy pipette is introduced through a hole performed with laser in the zona pellucida and a little number of trophectoderm cells are gently aspirated (**B**,**C**). Finally, the removed trophectoderm cells is transferred in a tube for the genetic analysis (**D**). The white narrow indicates the inner cell mass. The black narrow indicates the removed trophectoderm cells.

It has been demonstrated that the biopsy protocol might affect clinical outcomes [36]. The approach entailing sequential hatching and biopsy results in a significantly higher survival rate after thawing, implantation, clinical pregnancy, and LB rate if compared to the cleavage stage hatching approach. However, day 3 pre-hatching, extends the time of exposure outside the incubator and the risk of having a blastocyst herniating from the inner cell mass requiring extra manipulation during the biopsy. Furthermore this procedure allows a better synchronization with the natural expanding process of the blastocysts that could take place on day 5, 6, or 7. This technique is also cost-effective, since leaving the embryo undisturbed from fertilization to blastocyst formation allows for the employment of single-step media and time-lapse incubation protocol.

Another controversial theme regarding TE biopsy is whether day 6 and day 7 blastocysts should be analyzed or not. A study by Piccolomini and co-workers [37] investigated if slow development might reflect embryo ploidy status. This group compared blastocyst biopsy performed on day 5 versus day 6 and reported similar aneuploidy rate (61.4% on day 5 vs. 69.9% on day 6). The study by Taylor et al. [38] evidenced that day 5 blastocysts had a significantly higher chance of being euploid than day 6 blastocysts (54.6% vs. 42.8%). Both of the studies concluded that blastocysts formed on day 6 and have the same chance of resulting in a live birth rate as those formed on day 5. The study by Hernandez-Nieto et al. [39] found that the rate of embryo euploidy was significantly lower in day 7 blastocysts when compared to day 5 or day 6 cohorts (40.5% vs. 54.7% vs. 52.9%, respectively). In his study there was also a significant decrease in the odds of implantation, clinical pregnancy, and LB, but no association with pregnancy loss in patients who transferred day 7 biopsied euploid blastocysts.

Although day 5 blastocysts may have the higher euploid rates, its relationship with embryo development is still unclear [4,40,41]. On the other hand, day 7 blastocysts can be viable, of top morphology, euploid, and result in a healthy live birth. Therefore, culturing embryos an additional day increases the number of embryos useable per IVF cycle and provides further opportunity for patients who have only a few or low-quality blastocysts. These findings underlined the importance of performing biopsy of all blastocysts available independently of their morphology or growth-timing.
