A novel test for annexin A5 M2 haplotyping in in vitro fertilization patients and preimplantation embryos

Abstract

Objective: To develop a test for evaluating the annexin A5 M2 haplotype in in vitro fertilization patients and preimplantation embryos. Design: Test performance was measured by comparing Sanger sequencing of parental blood DNA and quantitative real-time polymerase chain reaction (qPCR) of saliva DNA, 3 fibroblast cell line 7-cell aliquots and their corresponding purified DNA, 123 trophectoderm biopsy samples, and DNA isolated from 1 embryonic stem cell line along with the Mendelian inheritance expectations, embryo Sanger sequencing, and single-nucleotide polymorphism (SNP) microarray-based linkage analysis. Setting: Preimplantation genetic testing laboratory research on IVF patient and embryo DNA. Patient(s): An assay was developed for the detection of the M2 haplotype on saliva samples of 6 in vitro fertilization patients. In addition, 13 patients who underwent preimplantation genetic testing with data on parental and embryo biopsy DNA available for research use were evaluated. Intervention(s): None. Main Outcome Measure(s): The concordance rates between Sanger sequencing, SNP array-based linkage analysis, and Mendelian inheritance expectations with qPCR. Result(s): The concordance rate between Sanger sequencing and qPCR was 100% on parental blood DNA and saliva DNA. The sample concordance rate between all replicates of 7-cell aliquots was 100%. The sample concordance rate between 3 cell lines used to prepare 7cell aliquots and purified genomic DNA was 100%. The concordance rate between qPCR and Sanger sequencing results from a single trophectoderm biopsy and isolated embryonic stem cell line was 100%. The concordance rate of trophectoderm biopsy qPCR results and expectations from Mendelian inheritance rules was 97%; however, when SNP array-based linkage analysis was included, the concordance rate reached 100%. Conclusion(s):This study resulted in the development of a convenient saliva collection method and qPCR-based genotyping method to screen for the M2 haplotype. In addition, a novel method for testing preimplantation embryos has been established, providing an alternative to the use of low molecular weight heparin, through selection of embryos without the M2 haplotype.