*2.4. Non-Invasive PGT*

Embryo biopsy, performed at every developmental stage, is an invasive process that might condition IVF results. There are two alternatives to invasive biopsy: blastocentesis, consisting in the analysis of the blastocyst fluid (BF), and the examination of the spent culture media. The sampling of BF is performed on the opposite side of the inner cell mass, leaving the embryo fully collapsed [42,43]. Because dynamic collapse and re-expansion of the cavity is a phenomenon routinely observed during laboratory practice, the loss of the BF should not be detrimental to the embryo [44,45]. The aspiration of the BF does not affect embryo architecture, which results in high survival rates of both good and poor morphology embryos [46].

In 2013, Palini et al. [47], using real-time PCR, reported the presence of DNA fragments in BF obtained from day 5 blastocysts. The investigation of these DNA fragments allowed for the identification, with a 95% accuracy, of male embryos, detecting the specific Y-linked protein. Another study, conducted in 2015 by Tobler et al. [48], analyzed BF from 96 embryos: embryonic DNA was recovered and analyzed, using whole genome amplification (WGA), followed by aCGH in 63% of the samples. The results were concordant with those of the matched inner cell mass karyotypes only in 48.3% of the analyzed embryos. This induced the authors to recommend not to use blastocentesis as an alternative approach for PGT. Therefore, the failure of amplification rates after blastocentesis are a lot much higher if compared with those of the traditional TE biopsy [49].

On the contrary, Gianaroli et al. [50] reported the detection of embryonic DNA in 76.5% of the samples, with a diagnosis concordance rate of 97.4%, when compared to the correspondent TE biopsy. Although the analysis of BF seems to be a promising alternative to invasive PGT, further studies must be conducted. It is important to establish whether the loss of the BF could alter cell to cell communication, or the communication of the embryo with its environment. Furthermore, it is still unknown if the DNA material obtained from the blastocentesis is representative of the embryo DNA.

The analysis of spent culture media is another non-invasive alternative to traditional PGT. It consists in the analysis of cell free DNA that can be found in the media, due to its release from the embryo after apoptosis, necrosis or active release as macrovescicle. The DNA detected might have different origins: embryonal, paternal, maternal or extra-DNA. This technique does not require any experienced embryologist, since the embryo remains untouched, but it may be affected by several variables: culture drops volume, group or single embryo culture, the use of sequential or single-step media and fresh or thawed embryos culture [51,52].

A study conducted by Ho et al. [51] in 2018, compared PGT-A on spent culture media before and after blastulation, to determine the best timing for the collection of the media. The media was collected both on day 3 and day 5: DNA was detected in about 97% of samples that were collected on day 3, but only 39% of the samples provided enough material to yield a PGT-A result. On day 5, an amplification was obtained in 97% of the samples, of which 80% produced enough material for a reliable PGT-A. This study suggests the existence of an optimal collection date, to obtain more DNA quantity and a more solid analysis. The main risks that are associated with this kind of PGT-A are gentic contamination (maternal, paternal or lab personnel), the higher rate of apoptosis in aneuploid embryos, and its dependence on laboratory routine.

Many studies have been conducted in the last years to evaluate this technique and many of them reported moderate success rates [51–53]. Although very promising, minimally or non-invasive techniques for analyzing embryo ploidy are still at a premature stage, needing further study and standardization protocols.
