Noninvasive Digital Detection of Fetal DNA in Plasma of 4-Week-Pregnant Women following In Vitro Fertilization and Embryo Transfer

PLoS One. 2015 May 13;10(5):e0126501. doi: 10.1371/journal.pone.0126501. eCollection 2015.

Abstract

The discovery of cell-free fetal DNA (cfDNA) circulating in the maternal blood has provided new opportunities for noninvasive prenatal diagnosis (NIPD). However, the extremely low levels of cfDNA within a high background of the maternal DNA in maternal circulation necessitate highly sensitive molecular techniques for its reliable use in NIPD. In this proof of principle study, we evaluated the earliest possible detection of cfDNA in the maternal plasma by a bead-based emulsion PCR technology known as BEAMing (beads, emulsion, amplification, magnetics). Blood samples were collected from in vitro fertilization (IVF) patients at 2 to 6 weeks following embryo transfer (i.e., 4 to 8 week pregnancies) and plasma DNA was extracted. The genomic regions of both X and Y chromosome-specific sequences (AMELX and AMELY) were concurrently amplified in two sequential PCRs; first by conventional PCR then by BEAMing. The positive beads either for AMELX or AMELY gene sequences were counted by a flow cytometer. Our results showed that the pregnancies yielding boys had significantly higher plasma AMELY gene fractions (0.512 ± 0.221) than the ones yielding girls (0.028 ± 0.003) or non-pregnant women (0.020 ± 0.005, P= 0.0059). Here, we clearly demonstrated that the BEAMing technique is capable of reliably detecting cfDNA in the blood circulation of 4-week-pregnant women, which is only two weeks after the embryo transfer. BEAMing technique can also be used to early detect fetal DNA alterations in other pregnancy-associated disorders.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromosomes, Human, Y / genetics
  • DNA / blood*
  • Embryo Transfer
  • Female
  • Fertilization in Vitro
  • Fetus / physiology
  • Humans
  • Male
  • Polymerase Chain Reaction
  • Pregnancy
  • Sensitivity and Specificity
  • Sex Determination Analysis / methods*
  • Treatment Outcome

Substances

  • DNA

Grants and funding

This project was supported by the Kingdom of Saudi Arabia National Science, Technology and Innovation Plan (NSTIP) strategic technologies in the Kingdom-Award Nos. (10-BIO951-20 and 10-BIO1432-20). (http://www.kacst.edu.sa/en/research/pages/biotechnology.aspx) The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.