Trophectoderm DNA fingerprinting by quantitative real-time PCR successfully distinguishes sibling human embryos

Abstract

Purpose To validate a novel and more practical system for trophectoderm DNA fingerprinting which reliably distinguishes sibling embryos from each other. Methods In this prospective and blinded study two-cell and 5-cell samples from commercially available sibling cell lines and excess DNA from trophectoderm biopsies of sibling human blastocysts were evaluated for accurate assignment of relationship using qPCR-based allelic discrimination from 40 single nucleotide polymorphisms (SNPs) with low allele frequency variation and high heterozygosity. Results Cell samples with self relationships averaged 95.1± 5.9 % similarity. Sibling relationships averaged 57.2±5.9 % similarity for all 40 SNPs, and 40.8±8.2 % similarity for the 25 informative SNPs. Assignment of relationships was accomplished with 100 % accuracy for cell lines and embryos. Conclusions These data demonstrate the first trophectoderm qPCR-based DNA fingerprinting technology capable of unequivocal discrimination of sibling human embryos. This methodology will empower research and development of new markers of, and interventions that influence embryonic reproductive potential.

Autologous mitochondrial transfer as a complementary technique to intracytoplasmic sperm injection to improve embryo quality in patients undergoing in vitro fertilization—a randomized pilot study

“…Singleembryo transfer (SET) was strongly recommended, but double-embryo transfer (DET) was also authorized. When DET was performed with embryos from both groups, a fingerprinting analysis was planned to trace the babies or products of conception (35). Fingerprinting consisted of obtaining the DNA of the successfully implanted embryos either from tissue from remains of conception in the case of spontaneous miscarriage or from buccal swabs in the case of live birth when DET had been performed.…”

45,X product of conception after preimplantation genetic diagnosis and euploid embryo transfer: evidence of a spontaneous conception confirmed by DNA fingerprinting

“…Therefore, the additional time and expense of evaluating parental DNA could be avoided with this methodology as also previously reported [15]. …”

“…Furthermore, with this study we confirm that parental DNA information is not necessary since discrimination was achieved based on a clear genotypic heterogeneity among samples, without incorporating selection of informative SNPs from parental genotype. Therefore, the additional time and expense of evaluating parental DNA could be avoided with this methodology as also previously reported [ 15 ].…”

“…The estimated probability that siblings will have identical genotypes is 0.5 raised to the power of the number of SNPs used, since all of these markers have alleles close to 50:50 in the population. This technique has been validated by Scott and colleagues [ 15 ] as the first seamless and inexpensive trophectoderm qPCR-based DNA fingerprinting technology allowing the unequivocal discrimination of sibling human embryos and foetuses. Briefly, for either the multiplex amplification PCR product residual from the TE biopsy or the purified genomic DNA of the POC, real-time PCR for allelic discrimination was performed in duplicate reactions for each of the individual 40 loci.…”

Cumulus cell transcriptome profiling is not predictive of live birth after in vitro fertilization: a paired analysis of euploid sibling blastocysts

“…Live birth was defined as the birth of a live infant at R24 weeks of gestation. DNA fingerprinting of the delivered infant was performed using quantitative polymerase chain reaction (qPCR) to determine the identity of the embryo that resulted in a live birth (32). Patients who were not pregnant or had a twin live birth were not included in the analysis as differences in gene expression between two sibling embryos with the same clinical outcome would not be informative for embryo selection purposes.…”