RNA transcripts that were up-regulated in the SPB placentas, relative to the STB placentas, according to the microarray experiment (Mann-Whitney test, adjusted by the Benjamini and Hochberg method, adjusted p<0.05).

a<p>The RNA transcript interrogated by each probeset is shown as the gene symbol and name approved by the HUGO Gene Nomenclature Committee at the European Bioinformatics Institute (<a href="http://www.genenames.org" target="_blank">http://www.genenames.org</a>). Certain probesets interrogating the same gene may be listed as different GenBank accession numbers. This may reflect the different isoforms being interrogated. Other details on the probesets are freely accessible at <a href="http://www.netaffx.com" target="_blank">http://www.netaffx.com</a>.</p>b<p>Only 15 transcripts with the greatest fold-change values are shown. The remaining 225 transcripts are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034328#pone.0034328.s006" target="_blank">Table S5</a>.</p>c<p>Up-regulated transcripts with higher expression levels than the <i>PLAC4</i> mRNA in the placenta, according to the microarray data.</p><p>SPB, spontaneous preterm birth; STB, spontaneous term birth.</p

Gene ontology (GO) terms over-represented in the list of genes that were up-regulated in the SPB placentas, compared with the STB placentas.

<p>Terms with the highest fold of over-representation from each category are shown. The remaining terms are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034328#pone.0034328.s008" target="_blank">Table S7</a>.</p

Gene ontology (GO) terms over-represented in the list of genes that were down-regulated in the SPB placentas, compared with the STB placentas.

<p>Terms with the highest fold of over-representation from each category are shown. The remaining terms are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034328#pone.0034328.s009" target="_blank">Table S8</a>.</p

Systematic identification of SPB-associated placental transcripts in maternal plasma.

<p>Shown in the Venn diagram are the number of microarray probesets representing transcripts which are highly expressed (pale yellow) and aberrantly expressed (red) in the SPB placentas. Transcripts fulfilling both criteria (orange) are potentially detectable in maternal plasma samples obtained from women eventually resulting in SPB. These probesets represent 36 transcripts and are shown in the box, along with the fold-change up-regulated (up arrow) or down-regulated (down arrow) in the SPB placentas, compared with the STB placentas. For transcript represented by more than one probesets, its average fold-change is shown. Transcripts with no official gene symbols are shown in their GenBank accession numbers. Transcripts chosen for RT-qPCR are shown in bold. SPB, spontaneous preterm birth. STB, spontaneous term birth.</p

RNA transcripts that were down-regulated in the SPB placentas, relative to the STB placentas, according to the microarray experiment (Mann-Whitney test, adjusted by the Benjamini and Hochberg method, adjusted p<0.05).

a<p>If a probeset interrogates a RNA transcript with no gene symbol (“—”) approved by the HUGO Gene Nomenclature Committee at the European Bioinformatics Institute (<a href="http://www.genenames.org" target="_blank">http://www.genenames.org</a>), it is shown as a “transcribed locus”. Other details on the probesets are freely accessible at <a href="http://www.netaffx.com" target="_blank">http://www.netaffx.com</a>.</p>b<p>Only 15 transcripts with the greatest fold-change values are shown. The remaining 171 transcripts are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034328#pone.0034328.s007" target="_blank">Table S6</a>.</p><p>SPB, spontaneous preterm birth; STB, spontaneous term birth.</p

Characteristics of participants in the analysis of maternal plasma samples.

a<p>Mann-Whitney rank sum test for continuous variables. Fisher exact test for nominal variables.</p><p>SPB, spontaneous preterm birth.</p

Computer simulation for T21 detection for fractional fetal DNA concentrations of 5% and 15%.

<p>In order to obtain a specificity of greater than 99%, the cutoffs for T21 differentiation were chosen at 3 standard deviations above and below the mean F-S ratio of the euploid group. The sensitivity for paternally- and maternally-derived T21 detection was investigated on different numbers of informative allelic counts on chr21 and chrRef, respectively, for a fractional fetal DNA concentration of 15% (A). Similar analysis was performed for a fractional fetal DNA concentration of 5% (B). (Ma-T21: maternally-derived T21. Pa-T21: paternally-derived T21. Sen=sensitivity. Fe%=fractional fetal DNA concentration. Info AC=informative allelic counts on each of chr21 and chrRef. Info SNP=informative SNPs on each of chr21 and chrRef. Seq depth=sequencing depth. Info AC=Info SNP×Seq depth).</p

Schematic of T21 detection by F-S ratio calculation.

<p>Assuming the fractional fetal DNA concentration in chrRef is f, the F-S ratio would be f/(2-f) on chrRef irrespective of the aneuploidy status of the fetus. On the other hand, the F-S ratio on chr21 would be f/(2-f) if the mother is carrying a euploid fetus, 2f/(2-f) if the mother is carrying a paternally-derived T21 fetus, and f/2 if the mother is carrying a maternally-derived T21 fetus. Therefore, the would be 1 if the mother is carrying a euploid fetus, would become 2 if the mother is carrying a paternally-derived T21 fetus, and would become (1-f/2) if the mother is carrying a maternally-derived T21 fetus.</p

T21 detection by F-S ratio in non-targeted and targeted sequencing data.

<p> values were calculated to differentiate the paternally- and maternally-derived T21 from the euploid fetuses in non-targeted (A) and targeted (B) sequencing data. (Ma-T21: maternally-derived T21. Pa-T21: paternally-derived T21.).</p

Computer simulation to investigate the minimal number of informative allelic counts for T21 detection.

<p>The solid curve represents the minimal number of informative allelic counts required on each of chr21 and chrRef (Y axis), in order to achieve a reliable detection in maternally-derived T21 (sensitivity >99%, specificity >99%) according to a given fractional fetal DNA concentration (X axis). The dash curve represents the paternally-derived T21 model.</p