Non-invasive preimplantation genetic testing for aneuploidy (NI PGT-A)

Abstract

Since the initial report by Stigliani et al. (2013) about the presence of mitochondrial and nuclear DNA in human embryo culture medium, spent media (SM) has been extensively explored with the ultimate aim to develop non-invasive genetic testing (NI PGT) methods. Even after the first clinical application of NI PGT-A by Xu and colleagues (2016) using SM, the efficacy of the method for clinical utility remains under much debate as there are conflicting reports on its success rates as well concerns about non- embryonic DNA contamination. There are two key aspects determining the success of an NI PGT assay; 1) embryology parameters of culture conditions and 2) molecular methods used for downstream processing of SM samples. Most of the studies on NI PGT thus far have used SM samples derived from a single IVF centre and hence differences in choice of culture media, culture volumes, type of culture (fresh vs vitrified-thawed), and timing of sampling (Day3-Day5/6 or Day4-Day5/6) would have, in part, contributed to the differences in success rates obtained by different groups ( Shamonki et al., 2016 , Xu et al., 2016 , Feichtinger et al., 2017 , Lane et al., 2017 , Kuznyetsov et al., 2017 ). Hence, it is pertinent to develop a robust and versatile NI PGT method, functional under multiple embryo culture parameters. The choice of whole genome amplification (WGA) method is another major factor which might impact the success of NI PGT assay. As the embryonic DNA in spent media is most likely fragmented in nature, shorter DNA template lengths can pose challenge to chemistries of certain WGA methods ( Wang et al., 2004 ). Secondly, certain components of embryo culture media might impede the function of buffer and/or enzyme of the WGA system, thereby resulting in suboptimal or no amplification. We have tested two commercially available WGA methods on SM samples namely Sureplex and MDA in addition to a modified MDA approach (suitable for fragmented DNA) and found that the DNA amplification rates were significantly higher in the samples amplified with the modified MDA compared to those amplified by Sureplex and MDA methods. The origin of DNA present in SM can be a confounding factor for NI PGT assay. Microbial contamination has been reported to occur at Kastrop et al., 2007 ). More recently, Vera-Rodriguez et al. (2018) reported very high levels of maternal DNA contamination with only ∼8% of DNA fraction in the SM beingembryonic in origin. However with appropriate culture conditions and amplification methods, several other groups ( Xu et al., 2016 , Kuznyetsov et al., 2017 , Lane et al., 2017 ), including ours, have obtaineda high sex chromosomes as well as overall ploidy concordance rates (range: 72% - 95%) between SM and corresponding TE biopsies. Thisindicatesthat any potential contamination present in these samples was minimal and did not impede the end results; thereby highlighting the importance of adequate embryology culture practice as well appropriate downstream processing steps for a successful NI PGT-A assay. While a few NI PGT-A clinical trials have been initiated over the last two years, many crucial questions regarding the broader clinical efficacy of the method still remain to be answered. Nonetheless, it seems that we are one step closer to the dream of a completely non-invasive method for genetic testing of preimplantation embryos than before.