Clinical significance of histologic chorioamnionitis with a negative amniotic fluid culture and non-invasive predictors for histologic chorioamnionitis in patients with preterm labor and premature membrane rupture

Abstract

Objectives: (1) To evaluate the effect of histologic chorioamnionitis (HCA) with a negative amniotic fluid (AF) culture on adverse pregnancy and neonatal outcomes and inflammatory status in the AF compartment in women with preterm labor (PTL) or preterm premature rupture of membranes (PPROM) and (2) to identify novel immunoregulatory proteins in maternal plasma associated with HCA by using a membrane-based human cytokine microarray technology in women with PTL and PPROM.

Methods: This study consisted of two phases. (1) In the first phase of the study, 153 consecutive women diagnosed as having a PTL or PPROM (20–34 weeks) who delivered singleton gestations within 48 hours of amniocentesis participated. AF obtained through amniocentesis was cultured, and interleukin (IL)-6, IL-8, and metalloproteinase-9 (MMP-9) levels were determined. The placentas were examined histologically. (2) In the second phase of the study, a nested case-control study was conducted to identify novel plasma biomarkers associated with HCA. The second phase consisted of two stages. Firstly, plasma samples were obtained < 96 h before delivery from 14 cases with HCA and 14 control subjects (without HCA) in women with PTL. Discovery work using by membrane-based protein microarray was performed to compare the profiles of immunoregulatory proteins in the maternal plasma. Secondly, validation of selected candidate biomarkers was done by ELISA in the final cohort (n=74) with additional 46 plasma samples in women with PTL. Membrane-based microarray analysis (n=28) and validation (n=82) with additional 54 plasma samples in women with PPROM was performed in the same manner as in PTL patients. Receiver operating characteristic curves were generated to compare the diagnostic accuracy to predict HCA between serum C-reactive protein which has been in clinical use and the candidate proteins.

Results: (1) In the first phase of the study, the prevalence of HCA with negative AF culture was 23.5% (36/153). The women with HCA but with a negative AF culture (group 2) and those with a positive AF culture (group 3) had a significantly lower mean gestational age at amniocentesis and delivery than those with a negative AF culture and without HCA (group 1). Women in group 3 had the highest levels of AF IL-6, IL-8, and MMP-9, followed by those in group 2, and those in group 1. Composite neonatal morbidity was significantly higher in groups 2 and 3 than in group 1, but this was no longer significant after adjusting for confounders caused mainly by the impact of gestational age. (2) In the second phase of the study, differentially expressed proteins (12 proteins in PTL and 14 proteins in PPROM) were identified by membrane-based protein microarray analysis. In women with PTL (n=74), validation by ELISA confirmed significantly higher levels of S100 A8/A9 in women with HCA, compared with control subjects. However, this significance was not remained after adjusting for gestational age at sampling. In women with PPROM (n=82), ELISA validation found S100 A8/A9, MMP-9, and IL-6 in maternal plasma to have significantly higher levels in women with HCA, compared with those without HCA. After adjusting for gestational age at sampling, use of tocolytics, and corticosteroids administration, increased plasma MMP-9 was significantly associated with HCA in women with PPROM. However, its diagnostic indices were not superior to those of serum C-reactive protein in predicting HCA.

Conclusion: (1) In women who delivered preterm neonates, HCA with a negative AF culture was associated with increased risks of preterm birth, intense intra-amniotic inflammatory response, and prematurity-associated composite neonatal morbidity, and its risks are similar to the risk posed by positive AF culture. (2) The protein expression pattern in the maternal plasma is significantly altered between women with and without HCA. Although not found in women with PTL using by membrane-based protein microarray and ELISA validation, in our cohort of PPROM patients, increased levels of MMP-9 in maternal plasma can be a potentially novel candidate biomarker for predicting HCA non-invasively and antenatally.