Thousands of different single gene mutations, cancer predisposition genes, and now multi-factorial diseases can be screened in the early human embryo before transfer during an IVF cycle1. Having multiple cells to work with removes some of the potential issues that surrounded early attempts at single cell biopsy including reductions in allele drop-out (ADO) and preferential amplification (PA). Allele drop-out arises in the early stages of amplification by polymerase chain reaction (PCR), where one allele of a heterozygote is not primed and products for this allele are not created. In a heterozygote cell, this means that one allele will be missed which can lead to misdiagnosis as a homozygous embryo2. Preferential amplification is similar to ADO except that some product is made for one allele while more product is made for the other allele. Again, depending on the down-stream method of analysis chosen, this can lead to errant results and potential misdiagnosis. Dreesen’s and colleagues have published a thorough review of misdiagnosis during PGT-M (PGD)3, and Warren and colleagues have shown how using the PG-Seq™ kit with TSE lessens or eliminates the issues of PA and ADO4.
Although a PGT-M result may indicate that an embryo is unaffected by a monogenic disease, standard PGT-M methods are unable to assess aneuploidy. Depending on the age of the female patient, somewhere between 44% and 75% of these transferred embryos may be aneuploid. A robust method combining WGA along with gene-specific primers in a single reaction to allow both aneuploidy detection (PGT-A) and monogenic disease detection (PGT-M) would be a great leap forward for patients undergoing PGT-M.
Combining PGT-A and PGT-M has proved to be difficult without some concessions from either test. Many labs perform combined PGT-A and PGT-M by following their normal WGA methods for PGT-A to a certain point, and then split the WGA into 2 different samples and process each of them separately (personal communication). This system usually creates enough reads for PGT-A analysis and enough PCR product for downstream PGT-M testing, however splitting the WGA products into separate aliquots creates a few obvious issues, including the chance for sample switches/failed amplification, preferential amplification, and increased allele drop out (ADO) rates. In addition, if the lab wants to use next generation sequencing (NGS) technology for all downstream analysis then this splitting typically creates samples with different bar codes so the sample would need to be analyzed separately for PGT-A and PGT-M, which adds to the cost.
The target sequence enrichment (TSE) protocol from PerkinElmer allows for linkage-based analysis of most gene mutations using SNP haplotyping along with detailed analysis of aneuploidy in all 23 chromosomes simultaneously. This method uses whole genome amplification (WGA) to allow low pass sequencing coverage to detect aneuploidy down to approximately 10 MB in size along with TSE to specifically and reliably amplify the SNP set for each specific patient set. This method allows for PGT-M with no allele drop out or preferential amplification for the gene mutation or associated SNPs and PGT-A analysis all in a single tube and in a single economical sequencing run.
- De Rycke M, Goossens V, Kokkali G, Meijer-Hoogeveen M, Coonen E, Moutou C. EHSRE PGD Consortium data collection XIV-XV: cycles from January 2011 to December 2012 with pregnancy follow-up to October 2013. Hum Reprod. 2017 32(10):1974-1994.
- Findlay I, Ray P, Quirke P, Rutherford A, Lilford R. Allelic drop-out and preferential amplification in single cells and human blastomeres: implications for preimplantation diagnosis of sex and cystic fibrosis. Hum Reprod. 1995 Jun:10(6):1609-18.
- Dreesen J, Destouni A, Kourlaba G, Degn B, Mette WC, Carvalho F, Moutou C, Sengupta S, Shanjal S, Renwick P, Davies S, Kanavakis E, Harton G, Traeger-Synodinos J. Evaluation of PCR-based preimplantation genetic diagnosis applied to monogenic diseases: a collaborative ESHRE PGD consortium study. Eur J Hum Genet. 2014 Aug;22(8):1012-8.
- Warren K, Protopsaltis S, Jasper M. DOPlify®, Target Sequence Enrichment and Allele Dropout-is there a benefit? PGD-IS International Society Annual Meeting 2018. Bangkok Thailand.