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

Karakas B, Qubbaj W, Al-Hassan S, Coskun S - PLoS ONE (2015)

Bottom Line: 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).BEAMing technique can also be used to early detect fetal DNA alterations in other pregnancy-associated disorders.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Oncology, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia.

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.

No MeSH data available.


(A) The mean and standard error (SE) of the AMELY gene fractions from plasma samples of the iparous women (n = 10), the 4-week-pregnant mothers with girl deliveries (n = 4), and the 4-week-pregnant mothers with boy deliveries (n = 5). (B) The mean and standard error (SE) of the AMELY gene fractions of the plasma samples of 4 or 7 week-pregnant women with either girl or boy deliveries. *Statistically significant (P<0.05).
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pone.0126501.g003: (A) The mean and standard error (SE) of the AMELY gene fractions from plasma samples of the iparous women (n = 10), the 4-week-pregnant mothers with girl deliveries (n = 4), and the 4-week-pregnant mothers with boy deliveries (n = 5). (B) The mean and standard error (SE) of the AMELY gene fractions of the plasma samples of 4 or 7 week-pregnant women with either girl or boy deliveries. *Statistically significant (P<0.05).

Mentions: We next determined the plasma AMELY levels of non-pregnant iparous women (n = 10) using them as negative controls for any biological background. We found the mean and standard error of AMELY- specific bead fractions to be 0.020 ± 0.005, indicating a minimal background for the negative plasma control samples (Fig 3A).


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

Karakas B, Qubbaj W, Al-Hassan S, Coskun S - PLoS ONE (2015)

(A) The mean and standard error (SE) of the AMELY gene fractions from plasma samples of the iparous women (n = 10), the 4-week-pregnant mothers with girl deliveries (n = 4), and the 4-week-pregnant mothers with boy deliveries (n = 5). (B) The mean and standard error (SE) of the AMELY gene fractions of the plasma samples of 4 or 7 week-pregnant women with either girl or boy deliveries. *Statistically significant (P<0.05).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4430227&req=5

pone.0126501.g003: (A) The mean and standard error (SE) of the AMELY gene fractions from plasma samples of the iparous women (n = 10), the 4-week-pregnant mothers with girl deliveries (n = 4), and the 4-week-pregnant mothers with boy deliveries (n = 5). (B) The mean and standard error (SE) of the AMELY gene fractions of the plasma samples of 4 or 7 week-pregnant women with either girl or boy deliveries. *Statistically significant (P<0.05).
Mentions: We next determined the plasma AMELY levels of non-pregnant iparous women (n = 10) using them as negative controls for any biological background. We found the mean and standard error of AMELY- specific bead fractions to be 0.020 ± 0.005, indicating a minimal background for the negative plasma control samples (Fig 3A).

Bottom Line: 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).BEAMing technique can also be used to early detect fetal DNA alterations in other pregnancy-associated disorders.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Oncology, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia.

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.

No MeSH data available.