Detection and analysis of DNA material in human blastocoel fluid

Abstract

Preimplantation genetic diagnosis (PGD) is becoming a widely-accepted technique during in vitro fertilization (IVF). However, a disadvantage of PGD is the invasive biopsy methods used to sample embryonic cells or polar bodies. Recent studies have found that genetic material can be detected in blastocoel fluid (BF) and culture medium. In our study, BF and trophectoderm (TE) cells were simultaneously collected from the same donated human blastula. To generate enough DNA for analysis, we used multiple displacement amplification (MDA) based whole genome amplification (WGA). MDA-WGA samples were probed with primers designed to identify spinal muscular atrophy (SMA) and phenylpropionate ketoneuria (PKU), plus the Y chromosome sex-determining region (SRY). This demonstrated that DNA fragments were present in each of the TE and BF samples (7/7). The positive PCR amplification rates for SMA, PKU, SRY and β-actin in BF were 42.9% (3/7), 60% (3/5), 42.9% (3/7) and 71.4% (5/7) respectively, but the positive rate of amplification with TE samples was 100% (7/7), 100% (7/7), 71.43% (5/7) and 100% (7/7). After sequencing, identical alleles were found in matched BF and TE samples. In summary, BF-DNA could be detected using MDA-WGA and PCR, and sequences in PCR positive samples were identical in matched BF-DNA and TE samples.