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.
Highlights
The rare inherited disorders are observed throughout the world, especially in the societies with high rates of consanguinity
Sensitive digital technologies are capable of reliably identifying cell-free fetal DNA (cfDNA) within a high background of maternal DNA in the maternal plasma [10, 17,19]. The capabilities of these technologies need to be carefully evaluated in the clinical settings with appropriate controls. In this proof of principle study, we evaluated a digital PCR technology called BEAMing, for detecting male cfDNA from the maternal plasma by targeting Y chromosome-specific sequences of the amelogenin gene (AMELY)
This study was approved by the Research Ethics Committee (REC) of the King Faisal Specialist Hospital and Research Centre (KFSH&RC) and all clinical investigation have been conducted according to the principles expressed in the Declaration of Helsinki
Summary
This study was approved by the Research Ethics Committee (REC) of the King Faisal Specialist Hospital and Research Centre (KFSH&RC) and all clinical investigation have been conducted according to the principles expressed in the Declaration of Helsinki. Women who visited the KFSH&RC In Vitro Fertilization (IVF) Clinic seeking treatment were recruited. A total of 16 blood samples from pregnant women at 2 to 6 weeks following the embryo transfer (i.e., 4 to 8 weeks of pregnancy) were collected. Another set of blood samples (n = 10) from non-pregnant nulliparous women (n = 10) was obtained using them as plasma negative controls for the study
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.