Molecular techniques used in non-invasive pre-implantation genetic testing: current approaches, applications and challenges

Abstract

Preimplantation genetic testing (PGT) represents an important diagnostic method in assisted reproductive technology (ART) for the screening of chromosomal and genetic aberrations prior to embryo transfer. Non-invasive PGT (niPGT) is a new technology that analyzes cell-free embryonic DNA (cfDNA) released into spent culture media. This is a safer method than conventional PGT that uses invasive biopsies of embryos that can damage their viability. Advanced molecular techniques support the diagnostic accuracy of NiPGT: array comparative genomic hybridization (aCGH) allows genome-wide detection of whole-chromosome aneuploidies and copy number variations through comparative hybridization. Next-generation sequencing (NGS) allows high-throughput deep-coverage analysis with extended dynamic range and allows detection of whole-chromosome aneuploidies, segmental defects and intermediate mosaic thresholds on all 24 chromosomes. Because embryonic cfDNA is so fragmented and present only in trace picogram quantities, whole genome amplification (WGA) is needed to amplify the genetic material for downstream analysis, and other targeted approaches such as quantitative PCR (qPCR) and high-density single nucleotide polymorphism (SNP) arrays are also important, as they enable copy count calculation and provide vital haplotype-specific information on parental inheritance patterns. niPGT has high embryo concordance rates, ranging from 76% to 91%, when compared to conventional biopsies, but it presents significant technical challenges, including maternal DNA contamination from cumulus cells or maternal shedding, which can mask true embryonic aneuploidies and lead to false-negative results, as well as unequal amplification biases and artifacts generated during WGA. Continued refinement of sequencing and amplification protocols, together with more robust clinical validation, will be needed before niPGT can be widely adopted in routine practice.