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

Embryo Biology

VOLUME 2, ISSUE 3, P278-286, AUGUST 01, 2021


Bhavini Rana, B.A., Raymond Zimmerman, M.S., Diego Marin, Ph.D., Jia Xu, Ph.D., Edward Messick, M.S., Simon Fishel, Ph.D., Nathan Treff, Ph.D.



To develop a test for evaluating the annexin A5 M2 haplotype in in vitro fertilization patients and preimplantation embryos.


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.


Preimplantation genetic testing laboratory research on IVF patient and embryo DNA.


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.



Main Outcome Measure(s)

The concordance rates between Sanger sequencing, SNP array-based linkage analysis, and Mendelian inheritance expectations with qPCR.


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 7-cell 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%.


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.