**3. PGT Molecular Techniques**

The aCGH technique allows for detecting variations in the number of copies and rearrangements of each of the 24 chromosomes when comparing the biopsied genetic material with a reference sample. After amplification by WGA the sample is labelled with fluorescent probes and hybridized to a DNA microarray. The color adopted by each spot after hybridization allows for identifying chromosomal loss or gain. A laser scanner and a data processing software are used to detect fluorescence and analyse aneuploidy and chromosomal rearrangements [54].

Single Nucleotide Polymorphism Array (SNP) is performed using an array setup consisting in DNA hybridization, fluorescence microscopy, and solid surface DNA capture. SNP found in the analyzed sample are compared with SNP of maternal and paternal derivation to assess the ploidy status [55].

Real Time Quantitative Polymerase Chain Reaction (rtq-PCR) is an assay based on polymerase chain reaction; it can identify the whole chromosome asset detecting the copy number of each chromosome. To determine the copy number, it compares three or four locus-specific amplicons along each chromosome to a reference gene from the same chromosome. It is unable to identify chromosome aberrations and uniparental disomy, but can identify triploidy [56].

Next Generation Sequencing (NGS) is the latest approach for pre-implantation genetic testing. The first step of this protocol consists in WGA as for aCGH. After genome amplification, a bar-coding procedure takes place, in which different samples are labelled with specific sequences. This process allows for combining from 24 to 96 biopsies (depending on the sequencing platform adopted) in a sequencing run and this optimized cost per sequenced embryo. Each sequence is then compared to a reference human genome and a specific software is used to identify copy number variations and large deletions or duplications [57–59].

The availability of different molecular techniques opened a debate about their sensitivity and reliability for PGT. The study by Friedenthal et al. documented that the implantation and ongoing pregnancy/live birth rate were significantly higher in women undergoing NGS (71,6% and 62,0%, respectively) as compared to those evaluated by aCGH (64.6% and 54.4%, respectively). Moreover, the comparison between NGS and aCGH group documented significantly less biochemical pregnancies (8.7% vs. 15.1%) and a similar rate of spontaneous abortion (12.4% vs. 12.7%). Based on these findings, the authors concluded that PGT using NGS significantly improves clinical outcomes with respect to the aCGH technique and suggested that NGS could be more effective in identifying mosaic embryos and those with partial aneuploidies or triploidy [60].

Friedenthal et al. [61] investigated clinical error rates in frozen-thawed embryo transfer (FTET) cycles of single euploid embryo diagnosed by NGS (1151 cases) and aCGH (846 cycles). The clinical error rates in the NGS cohort resulted in being lower with respect aCGH: 0.7% vs. 1.3% per embryo, 1% vs. 2% per pregnancy with gestational sac, 0.1% vs. 0.4% per ongoing pregnancy/live birth rate, and 13.3% vs. 23.3% per spontaneous abortion. Therefore, although NGS and aCGH are highly sensitive methods for PGT, clinicians must still consider the chance of errors occurring.
