Table 1.

Summary of technical characteristics, advantages and disadvantages of methodologies employed in current and future PGT applications.

		Diagnostic employment	Technology characteristics	Advantages	Disadvantages
Biopsy strategy	Polar bodies	Indirect assessment of oocyte's chromosomal content	a) Double biopsy of individual PBs on Day 0 and Day 1	Analysis performed on waste products of female meiosis	Analysis limited to the maternal genome (excludes paternal genome and potential mitotic errors)
			OR	Allowed in countries where embryo biopsy is banned	Biopsy and analysis of both PBs required (time-consuming and non-cost-effective procedure)
			b) Single biopsy of both PBs on Day 1	Compatible with fresh ET after genetic diagnosis	Technical issues associated to single cell-based analysis
					High amplification failure rate (≈10%)
	Blastomere	Direct assessment of embryo's chromosomal status	Single or double blastomere biopsy on Day 3	Vast experience worldwide	High impact on embryo reproductive competence
				Embryos reach the cleavage stage in a synchronous fashion (day 3 of preimplantation development)	Technical issues associated with single cell-based analysis
				Standardized approach	Lowest diagnostic reliability
				Compatible with fresh ET	High amplification failure rate (≈10%)
	Trophectoderm	Direct assessment of embryo's chromosomal status	5–8 cell trophectoderm biopsy on Day 5, 6, or 7, depending on embryo developmental rate	Robust diagnosis (5–10 cells are retrieved and analyzed)	The IVF laboratory must be experienced in extended embryo culture and vitrification
				Selection of TE cells to biopsy. The ICM is not involved	Limited experience worldwide
				Highly cost-effective (biopsy of developmentally-competent embryos only)	Most commonly incompatible with fresh embryo transfer
				No impact on embryo reproductive competence
				Low amplification failure rate (≈1%)
PGT-A	FISH	Assessment of the copy number of a limited set of chromosomes	Fluorescent probes for specific chromosomes are hybridized with single cell DNA. Number of chromosomes is inferred from the number of probe's signals	First genetic methodology employed for chromosomal assessment	Allows screening of some of the chromosomes only
					Labor intense
					Incompatible with multicellular biopsy (TE)
	aCGH	Comprehensive assessment of the copy number of all chromosomes	DNA content extracted from a specimen is amplified and labeled with a fluorochrome and cohybridised with a normal reference DNA labeled with a different color onto a microarray slide carrying DNA fragments representative of a whole karyotype. Color ratios identified through HD scans refer to different chromosomal statuses	Compatible with both multicellular biopsy and single cell analysis Comprehensive testing of all 24 chromosomes	Low sensitivity for mosaicism and segmental aneuploidies Expensive and labor intense
	qPCR	Comprehensive assessment of the copy number of all chromosomes	Unique sequences in each chromosome are selectively amplified using RealTime PCR. Amplification curves for each chromosome are compared across them and with a reference. Steeper curves correlate to higher amounts of starting material (trisomy), slower curves with fewer starting material (monosomy).	Compatible with both multicellular biopsy and single cell analysis Comprehensive testing of all 24 chromosomes Cheap and automatable Compatible with combined PGT-A and PGT-M analysis	Low sensitivity for segmental aneuploidies
	NGS	Comprehensive assessment of the copy number of all chromosomes	DNA content extracted from biopsy specimen is amplified, fragmented and tagged with sample-specific sequences. Multiple samples can be parallely sequenced using different technologies. Sequences generated are searched on genomic database to identify their location on the genome. Comparison between number of reads representing specific regions are used to infer chromosomal copy number.	Compatible with both multicellular biopsy and single cell analysis Comprehensive testing of all 24 chromosomes High sensitivity for segmental aneuploidies and mosaicism Increasingly cheap and automatable Compatible with combined PGT-A and PGT-M analysis
	Nanopore	Comprehensive assessment of the copy number of all chromosomes	Lysated cells are loaded on a Nanopore DNA sequencer where an electrical current feeds single strand DNA through a flow-cell membrane whilst recording voltage changes occurring in the protein nanopores. This information is then translated into DNA sequences that are subsequently aligned to a reference DNA and analyzed for chromosomal copy number variation.	Compatible with both multicellular biopsy and single cell analysis Comprehensive testing of all 24 chromosomes Cheap cost of analytical unit Reduce overall time for analysis and allow fresh embryo transfer also in blastocyst biopsy cases Small device footprint. Potential to be installed within the IVF laboratory (technical personnel and diagnostic accreditation still required)	Unknown sensitivity for mosaicism and segmental aneuploidies Low base calling precision Currently not applicable for PGT-M purposes
PGT-M	Direct + Linkage analysis	Direct and indirect detection of presence of single gene mutation	Custom primers are employed to amplify the specific genetic region containing the mutation to investigate. Product amplification are subjected to mini-sequencing to determine the allelic status.	Robust diagnostic strategy	Requires custom made probes for each couple tested
				Possible to detect de novo mutations	Test set-up and validation is time consuming
				Applicable to almost all cases
				Low implementation costs
	Karyomapping	Indirect assessment of presence of single gene mutation through haplotyping	DNA content extracted from a biopsy specimen is amplified and labeled with a fluorochrome and hybridized onto a microarray slide carrying 300,000 SNP variants. Embryo's haplotype is reconstructed based on the frequencies of informative SNPs.	Applicable with minimal patient-specific custom set up	Not applicable if affected gene is located in a region with insufficient SNP markers
				Short work up time	Requires DNA analysis of an affected person in the family to set up the test
					Inapplicable in cases involving de novo mutation
					High costs of implementation
	NGS	Direct assessment of presence of single gene mutation	Targeted amplification of region of interest followed by sequencing and reads analysis	Multi-gene analysis	High analytical costs (current)
Additional markers for embryo selection	ePGT-M	Parallel assessment of Mendelian and multi-factorial genetic inheritance	Based on SNP array and bioinformatic algorithms.	Simultaneous assessment of pathogenetic and predisposing conditions	Ethical considerations to be expertly evaluated
	mtDNA mutation	Assessment of mutation load in mitochondrial genome	Custom primers are employed to amplify the specific mtDNA region containing the mutation to investigate. Product amplification are subjected to genotyping to determine mutation status.	Diagnosis of pathogenic mitochondrial conditions	Difficult interpretation of results due to heteroplasmy
	mtDNA load	Assessment of amount of mitochondria present in biopsied specimen	Highly conserved mtDNA regions are targeted in Real Time PCR amplification. Amplification curves are compared with internal standards	Additional data on cellular energetic supply and metabolism for embryo selection purposes	Unproven in large RCT studies
	Epigenetics	Assessment of inheritable and induced epigenetic alterations	Alternative approaches possible	Additional data on embryo viability status for selection purposes
	Transcriptomics	Monitoring/assessment of developmental processes ongoing in the embryo	Alternative approaches possible	Additional data on embryo viability status for selection purposes
niPGT-A		Non-invasive assessment of embryo's chromosomal status	Cell-free DNA is amplified and subjected to NGS protocol	Embryonic cells are not removed for diagnostic purposes	Diagnostic accuracy and sensitivity must be proven in large clinical studies
				Potential universal applicability