Patient demographics and clinical information of cases used for microarray and confirmation analyses.

<p>*, median (range).</p><p>**, The number of term in labor cases is 11 because 6 additional cases were used for qRT-PCR analysis.</p><p>NS, not significant.</p

Effects of miR-143 on PTGS2 expression in AECs and AMCs.

<p>A, qRT-PCR analysis shows that 50 nM of miR-143 mimic transfection significantly increases miR-143 expression, while transfection with 200 nM of miR-143 inhibitor has no effect in AECs. In AMCs, miR-143 mimic (50 nM) and hairpin inhibitor (200 nM) transfections significantly increased and decreased the expression of miR-143, respectively. miR-143 and miR-145 expression levels were normalized with 5S rRNA expression. B, Immunoblot analysis of PTGS2 expression in AECs and AMCs transfected with mimic negative control (lane 1), mimic-miR-143 (lane 2), hairpin inhibitor negative control (lane 3), and inhibitor-miR-143 (lane 4). In AECs, PTGS2 protein expression was 1.5-fold decreased in transfection with miR-143 mimic (p = 0.014), but no changes in transfection with miR-143 inhibitor. In AMC, miR-143 mimic and hairpin inhibitor transfections 1.8-fold increased and 1.3-fold decreased the expression of PTGS2 protein (p = 0.014 for each), respectively. PTGS2 protein level was normalized to that of β-actin. C, qRT-PCR analysis of PTGS2 mRNA in transfected AEC and AMC shows there are no significant differences. The PTGS2 mRNA expression was normalized on the content of RPLPO. n = 4 for each experiments; the graphs show means and SE. *, <i>p</i><0.05.</p

Characterization of isolated amnion epithelial cells (AECs) and amnion mesenchymal cells (AMCs).

<p>A, Morphological and immunophenotypic characteristics of AECs and AMCs on hematoxylin & eosin staining (H&E) and immunofluorescent staining of cytokeratin-7 (red), type I procollagen (green). AECs are positive for cytokeratin-7, while AMCs are positive for type I procollagen. B, qRT-PCR analysis of miR-143 expression which was normalized to 5S rRNA shows significantly higher expression in AMCs than AECs. C, Densitometric analysis of PTGS2 expression level was normalized to β-actin. PTGS2 protein is less abundant in AMCs than in AECs. D, PTGS2 mRNA expression is not different between AECs and AMCs. AECs and AMCs obtained from five women at term not in labor (TNL) were used for all experiments (B–D). *, <i>p</i><0.05.</p

miR-143 binding to the 3′ UTR of PTGS2 mRNA.

<p>A, Putative miR-143-binding site in 333 bp of PTGS2 3′ UTR construct cloned into pMIR-REPORT™ for luciferase reporter assay. B, Luciferase reporter assay of PTGS2 mRNA 3′ UTR in AECs and AMCs. Luciferase activities from AECs and AMCs transfected with 200 ng (AEC) or 500 ng (AMC) of luciferase reporter plasmid containing PTGS2 3′ UTR (pMIR-REPORT_PTGS2), 10 ng of Renilla luciferase reporter pSV40-RL, and miRIDIAN miR-143 mimic (100 nM) or miR-143 hairpin inhibitor (50 nM) or equal amounts of negative controls were measured using Dual-Lucifrerase Reporter Assay System (Promega). pMIR-REPORT was used as a control. In AECs, there is a 58.5% of decrease in luciferase activity following transfection with miR-143 mimic but transfection of miR-143 hairpin inhibitor does not alter luciferase activity. In AMCs, miR-143 mimic transfection decreased luciferase activity by 25.9% compared to the control, while inhibition of miR-143 increased luciferase activity by 46.2% compared to control. Renilla luciferase activity was used for normalization of firefly luciferase activity (n = 5). The graphs show means and SE. *, <i>p</i><0.05.</p

Demographics of study populations.

<p>*Mean±SEM.</p

Correlation between maternal and fetal anti-HLA class I and class II antibodies.

<p>The panel reactivity of maternal anti-HLA antibodies is associated with that of fetal anti-HLA antibodies (for anti-HLA class I antibodies: r = 0.578, p<0.001; for anti-HLA class II antibodies: r = 0.358, p<0.001). <i>PRA</i>, panel-reactive anti-HLA antibodies.</p

Histological features of chronic chorioamnionitis.

<p>(A) A case of spontaneous preterm birth showing destruction of the chorionic trophoblast layer by infiltrating lymphocytes. (B) In another case of spontaneous preterm birth, lymphocytic infiltration extended to the chorioamniotic connective tissue layer. Original magnification X200.</p

C4d deposition on fetal umbilical vein endothelium by immunohistochemistry.

<p>C4d immunoreactivity on fetal umbilical vein endothelium is found in 54 out of 70 cases with preterm labor and intact membranes followed by preterm birth. However, there are only eight C4d positive cases among 70 cases with normal term labor (77.1% vs. 11.4%; p<0.001). <i>PTL</i>, preterm labor and intact membranes and preterm birth; <i>TIL</i>, term labor and normal term birth.</p

Panel-reactive anti-HLA seropositivity in maternal and fetal sera.

<p>(A) A representative case showing the presence of anti-HLA class I antibodies and the absence of anti-HLA class II antibodies in maternal serum with FlowPRA®-I and -II screening tests, followed by flow cytometry. (B) Spontaneous preterm birth cases have a higher maternal anti-HLA class I seropositivity than normal term birth cases (p<0.01). <i>TB</i>, normal term birth; <i>sPTB</i>, spontaneous preterm birth.</p

Dynamics of mtDNA mutations during mouse aging.

<p>Dynamics of mtDNA mutations during mouse aging.</p