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48: Amniotic fluid (AF) soluble myeloid differentiation (MD)-2 factor as regulator of intraamniotic inflammation in infection-induced preterm birth

      Objective

      Activation of TLR-2 and TLR-4 by bacteria requires involvement of the adaptor protein MD2. A soluble form of MD2 (sMD2) has the potential to alter the intensity of the inflammatory response. The functional role of sMD2 is heterogenous with either a stimulatory or an inhibitory effect based on cellular specificity. Our objectives were: 1) to determine the relationship between the expression level of AF sMD2 and intensity of the inflammatory response to infection and 2) to test in-vitro the ability of recombinant sMD2 to augment the release of amniochorion pro-inflammatory cytokines.

      Study Design

      AF sMD2 levels were analyzed in 152 samples from 52 singleton women with normal pregnancy (2nd trimester genetic karyotyping, n=26; 3rd trimester lung maturity, n=26) and 100 patients with preterm labor symptoms, who underwent amniocentesis to rule-out infection. Microbial cultures returned positive in 51 cases [Gram (−) bacteria (n=24); Gram (+) bacteria (n=27)]. An amniochorion explant system was employed (term cesarean, n=3). Tissues were incubated for 6h and 18h in the presence of recombinant sMD2 (0.1, 1 and 10 g/mL). Expression level of sMD2 was investigated by Western-blotting. The levels of AF and explant medium of IL-6 and TNF-α were measured by ELISA.

      Results

      1) AF sMD2 immunoreactivity was GA regulated with lower levels in the 3rd trimester (2W-ANOVA, P<0.001; 2) AF levels of sMD2 were significantly upregulated in women with infection, independent of bacterial type or GA (P=0.005); 3) There was a significant correlation between AF sMD2 immunoreactivity and concentration of IL-6 (R=0.320, P=0.001) and TNFα (R=0.267, P=0.007); 4) In-vitro, recombinant sMD2 (10g/mL) heightened the production of TNF-α at 6h (P=0.047) and of IL-6 at 6h (P=0.013) and 18h (P=0.020).

      Conclusion

      Intra-amniotic infection is characterized by a significant upregulation in the level of sMD2 which appears to enhance amniochorion production of pro-inflammatory cytokines.