Clinical chorioamnionitis at term VII: the amniotic fluid cellular immune response

Abstract

Objective 1) To characterize the cellular composition of the amniotic fluid of patients diagnosed with clinical chorioamnionitis at term, as a function of the presence or absence of microorganisms determined by cultivation techniques, and 2) to characterize the cytokine production by white blood cells present in the amniotic fluid using flow cytometry-based techniques. Material and Methods Amniotic fluid samples from 20 women with the diagnosis of clinical chorioamnionitis at term were analyzed using cultivation techniques (for aerobic and anaerobic bacteria, as well as genital Mycoplasmas). Amniotic fluid IL-6 concentrations were determined by an enzyme-linked immunosorbent assay. Amniotic fluid leukocytes were visualized by using hematoxylin and eosin staining and immunofluorescence. Immunophenotyping of surface markers and cytokines was performed in amniotic fluid leukocytes using flow cytometry. Results 1) Neutrophils (CD45+ CD15+ cells) were the most common leukocyte subset found in the amniotic fluid, followed by monocytes (CD45+ CD14+ cells); other white blood cells (such as lymphocytes and natural killer cells) were scarce in the amniotic fluid; 2) the absolute counts of neutrophils and monocytes were significantly higher in patients with microorganisms found in the amniotic fluid than in those without detectable microorganisms, using cultivation techniques; 3) there was a significant correlation between the absolute counts of neutrophils and monocytes determined by flow cytometry (Spearman’s correlation = 0.97; p <0.001); 4) there was a significant correlation between the absolute white blood cell count determined with a hemocytometer chamber and flow cytometric analysis (Spearman’s correlation = 0.88; p<0.001); and 5) the profile of cytokine expression differed between monocytes and neutrophils; while neutrophils predominantly produced TNF-α and MIP-1β, monocytes expressed higher levels of IL-1β and IL-1α. Conclusion Flow cytometry analysis of the amniotic fluid of patients with intra-amniotic infection and clinical chorioamnionitis at term demonstrated that neutrophils and monocytes are the most common cells participating in the inflammatory process. We have characterized for the first time the differential cytokine expression by these cells in this important complication of pregnancy.

The immunophenotype of amniotic fluid leukocytes in normal and complicated pregnancies

“…Regardless of their origin, amniotic fluid neutrophils are a part of the innate immune host defense mechanisms that take place in the amniotic cavity of women with intra-amniotic infection 77, 163, 164 . This concept is supported by evidence demonstrating that amniotic fluid neutrophils 1) are a source of anti-microbial products 90, 165–168 and cytokines 69 , 2) can trap and kill bacteria invading the amniotic cavity by forming neutrophil extracellular traps (NETs) 68 , and 3) can phagocytize microorganisms commonly found in the lower genital tract, e.g., Streptococcus agalactiae (also known as Group B Streptococcus or GBS), Ureaplasma urealyticum , Gardnerella vaginalis , and Escherichia coli 132 . Together, these findings show that even in the absence of microbial invasion, neutrophils are present in the amniotic fluid throughout gestation and ready to participate in the host defense mechanisms taking place in the amniotic cavity.…”

“…Previous studies have shown that intra-amniotic infection is characterized by the infiltration of neutrophils and monocytes/macrophages 23, 27, 67–69, 129–132 . It was recently suggested that amniotic fluid ILCs were also implicated in regulating intra-amniotic infection and inflammation in preterm gestations 119 .…”

“…The cellular components of the amniotic fluid include different cell types derived from exfoliating surfaces of the developing fetus, including the skin, respiratory system, urinary tract, and gastrointestinal tract, as well as stem cells 107–115 . Some cytological studies have shown that, in the absence of infection, the amniotic fluid also includes a low number of innate immune cells including macrophages 110, 111, 116–118 , neutrophils 23, 69 , and the recently described innate lymphoid cells or ILCs 119 . Yet, the number of macrophages and/or ILCs is increased in pathological conditions in which fetal organs are exposed to the amniotic fluid (e.g.…”

“…neural tube defects 118, 120–125 and gastroschisis 118, 126–128 ). The number of amniotic fluid neutrophils, on the other hand, is a useful marker for intra-amniotic inflammation 23, 27, 67–69, 129–132 . However, in the absence of infection and/or inflammation or birth defects, the immune cellular composition of the amniotic fluid is still poorly understood.…”

Twenty-four percent of patients with clinical chorioamnionitis in preterm gestations have no evidence of either culture-proven intraamniotic infection or intraamniotic inflammation

“…Clinical chorioamnionitis is the most frequent pregnancy-related infection diagnosed in labor and delivery units worldwide [1]. The prevalence of this condition in term gestations ranges from 5% to 12% [2], while in preterm gestations, it is diagnosed in 20% of patients with preterm premature rupture of the membranes (PROM) [3–5] and in about 10% of patients with preterm labor and intact membranes [6–11].…”