Immunohistochemical localization of inducible nitric oxide synthase on human fetal amnion in intra-amniotic infection.

Abstract

Amniotic fluid levels of nitric oxide metabolites are significantly elevated in intra-amniotic infection. We hypothesized that fetal amnion is a possible site for the production of nitric oxide. Because inducible nitric oxide synthase is the key enzyme responsible for the generation of nitric oxide in patients with intra-amniotic infection, we used immunohistochemistry to localize it on human fetal amnion. Human fetal amnions were obtained from patients with and without intra-amniotic infection (n = 5, respectively). Intra-amniotic infection was diagnosed by positive amniotic fluid cultures and placental pathologic features. Human fetal amniotic membranes were processed into tissue blocks and embedded in paraffin. A rabbit polyclonal antibody against human inducible nitric oxide synthase was used as the primary antibody, followed by avidin-biotin immunoperoxidase localization. Normal rabbit serum was used as a negative control and ovarian carcinoma cells were used as the positive control. Anti-inducible nitric oxide synthase labeling of human fetal amniotic membranes in patients with intra-amniotic infection showed positive immunostaining of epithelial cells, specifically in the cytoplasm of the perinuclear area. In contrast, no anti-inducible nitric oxide synthase immunostaining on human fetal amniotic membranes could be identified in patients without intra-amniotic infection. Our data provide important evidence that inducible nitric oxide synthase can be induced on human fetal amnion in intra-amniotic infection. These findings strongly support our hypothesis that human fetal amnion may be a possible site for the synthesis of nitric oxide after inducible nitric oxide synthase is induced in response to infectious products in intra-amniotic infection.