836: Comprehensive Screening for Presence and Localization of TLR4 Immunoreactivity in Placenta and Fetal Membranes

Toll-like receptor-4 (TLR4) along with the adaptor molecule MD-2 is responsible for mediating the inflammatory response to Gram-negative bacteria. Numerous studies have described the expression of TLR4 in placenta and fetal membranes with particular focus in preterm birth (PTB) associated with infection/inflammation. However, divergent results have been reported regarding TLR4 pattern of expression in reproductive tissues. We sought to investigate whether this lack of consistency may be due to antibody selection and epitope variability. We conducted a systematic evaluation of TLR4 expression in human placenta and fetal membranes using all commercially available anti-human TLR4 antibodies: C-18 (Santa Cruz), H-80 (Santa Cruz), HTA125 (Santa Cruz) and Ab47093 (Abcam). Immunohistochemistry (IHC) for TLR4 using all of the above antibodies was conducted on serial sections of placenta and chorioamnion of singleton pregnancies in the following groups: 1) Idiopathic preterm birth (iPTB, n=5); 2) Preterm birth complicated by histologic chorioamnionitis (HCA, n=5); 3) Term elective Cesarean (n=5). Staining intensity for each antibody was appreciated using the H-scoring method by independent investigators. 1) There was no correlation in H-scores obtained with the 4 antibodies on placenta sections. Although H80 yielded the most intense level of staining, other antibodies (Ab47093 or HTA125) were unable to confirm the cellular location or level of intensity. 2) None of the antibodies determined a significant increase inTLR4 staining in placental villous or extravillous trophoblast of the HCA group. C18 registered a decrease in villous H-score of HCA cases compared to the other groups. 3) The 4 antibodies had a significantly better level of agreement for fetal membranes. The staining pattern noted with H80 in chorioamnion correlated with all 3 other antibodies: C18 (P=0.004), HTA125 (p=0.008) and Ab47093 (P=0.037) but no significant difference among groups was detected. There is a large heterogeneity in the ability of available IHC antibodies to identify TLR4, especially in the placenta. Alternatively, the variability may be related to differential cellular processing of the TLR4 receptor.