Flow chart of sample inclusion.

<p>Flow chart of sample inclusion.</p

<i>In situ</i> detection of Pg in placental and umbilical cord sections from preterm and term tissues.

<p><b>A.</b> Placental villous section from a preterm specimen demonstrating the distribution of Pg (red, white arrow) within the extracellular matrix. Cytotrophoblasts/syncytiotrophoblasts (green) were detected with cytokeratin-7 specific antibody. <b>B.</b> Corresponding preterm placental section stained with pre-adsorbed Pg-specific antiserum (red) and cytokeratin-7 specific antibody (green). <b>C.</b> Pre-term placental specimen demonstrating Pg (red, white arrow) attached to cytotrophoblasts/syncytiotrophoblasts (green). <b>D.</b> Term placental villus section demonstrating Pg (red, white arrow) in association with syncytiotrophoblast (green). <b>E.</b> Preterm umbilical cord section demonstrating extracellular and internalized Pg (white arrow) within the perivascular stroma, myofibroblast (green) were labelled with vimentin specific antibody. <b>F.</b> Representative umbilical cord section from a term specimen, which were negative for Pg. Cell nuclei (blue) were stained with DAPI. <b>*</b>Indicates autofluorescent red blood cells in maternal or fetal circulation. In all panels, magnified inserts are Pg positive regions demarcated by arrows. All scale bars (bottom left corner) are equivalent to 10 μm, images are 400x or 600x magnification.</p

The distribution of Pg positive placental sections according to gestational age.

<p>The distribution of Pg positive placental sections according to gestational age.</p

Endotoxin induced chorioamnionitis results in migration of gammadelta T-cells to sites of mucosal damage at late GA.

<p>After 2d or 14d of endotoxin exposure, hardly any gammadelta T-cells were detected in the upper mucosa of the preterm intestine (A+B). At 140d GA, gammadelta T-cells were not detected in the lamina propria whereas lymphoid follicles in the basal mucosa became populated with gamma delta T-cells (C+D). 30d after endotoxin injection, increased numbers of gammadelta T-cells were found within areas of mucosal damage (E).</p

Intraamniotic endotoxin induces eNOS expression.

<p>Weak eNOS staining was seen at 2d post endotoxin treatment (A) while expression increased in animals exposed to endotoxin for 14 or days 30d (B-C).</p

Experimental design.

<p>Gestational development and effects of chorioamnionitis were studied at two different gestational ages. At 125d GA, fetuses were comparable to 27 weeks of human gestation. Lambs were almost near term at 140d GA since term gestation is 147d. Chorioamnionitis was induced by a single injection of endotoxin under ultrasound guidance at 111d GA and at 123d GA. Animals were delivered at 125d GA and animals of the control group underwent the same procedure with an injection of saline. Animals of 140d GA had a separate control group to assess gestational changes.</p

At 14 and 30d after intraamniotic endotoxin injection, massive influx of PMNs was detected (A+B).

<p>At 140d GA, some MPO positive cells were detected in control sections (C). For each experimental group mean cell counts of MPO positive cells are depicted per high-power field (D).</p

Ileal TLR4 and MD-2 mRNA was evaluated by ovine specific RT-PCR amplification of cDNA samples from healthy control lambs and lambs subjected to endotoxin induced chorioamnionitis.

<p>Representative DNA fragments are shown for each group (n = 4). cDNAs were standardized for GAPDH content. Quantitative data were obtained by densitometric evaluation of RT-PCR products which were compared to a standard curve obtained by amplification of a serial dilution of highly concentrated cDNA. At 125d GA, TLR4 and MD-2 mRNA levels were significantly (*) lower compared to animals at 140d GA. TLR4 and MD-2 mRNA expression was significantly reduced following intraamniotic endotoxin injection for 2, 14 and 30d.</p

Immunolocalisation of ZO-1 (red) in the fetal intestine.

<p>At 125d GA, a fragmented staining pattern for ZO-1 was seen in control animals (A) or lambs exposed to endotoxin for 2 (B) or 14d (C). At 140d GA, a normal ZO-1 distribution was detected in control animals (D) that became disrupted upon endotoxin exposure for 30d (E). Magnification 200x. For inset, 1000x magnification was used.</p

ROC curve and PPV-NPV plot of clinical prediction rule for histological chorioamnionitis (A+B) and histological chorioamnionitis with fetal involvement (C+D).

<p>Figures in PPV-NPV plots indicate inclusive (≥) cut-off values for positive test scores.</p