High‐throughput analysis of amniotic fluid proteins associated with histological chorioamnionitis in preterm premature rupture of membranes using an antibody‐based microarray

Abstract

Problem To identify potential proteins in the amniotic fluid (AF) that may be associated with histologic chorioamnionitis (HCA) in patients with preterm premature rupture of membranes (PPROM) using antibody‐based microarray analysis. Method of study This was a retrospective cohort study involving 100 singleton pregnant women with PPROM at 24–34 weeks who underwent amniocentesis and delivered within 120 h of amniocentesis. First, the AF proteomes of 15 patients with PPROM and HCA were compared with those of 15 gestational age‐matched patients without HCA using a protein microarray. Next, 12 candidate proteins associated with HCA were further validated in 100 consecutive patients with PPROM by ELISA. Results Of 507 proteins assessed in the microarray analysis, 46 showed significant intergroup differences. Further quantification confirmed that the levels of EN‐RAGE, IL‐6, MMP‐9, TNFR2, SPARC, TSP2, and uPA were higher in the AF of PPROM patients with HCA than in those without. Multivariate analyses also showed that elevated AF EN‐RAGE, IL‐6, MMP‐9, and TNFR2 levels were independently associated with HCA when adjusted for baseline variables. The frequency of the highest quartile of the aforementioned proteins significantly increased as the total grade of HCA increased; the risk of HCA significantly increased with increasing AF levels of each protein (P for trend < .001). Conclusions Using protein–antibody microarray technology, we discovered several potential AF proteins (EN‐RAGE, IL‐6, MMP‐9, and TNFR2) independently associated with HCA in patients with PPROM. Furthermore, we demonstrated a direct correlation between the gradation of the intra‐amniotic inflammatory response and HCA severity.

Expression of inflammatory, angiogenic, and extracellular matrix‐related mediators in the cervicovaginal fluid of women with preterm premature rupture of membranes: Relationship with acute histological chorioamnionitis

“…50,51 A recent study from our group also showed that TNFR2 levels are significantly elevated in the AF of PPROM women with acute HCA and microbial-associated HCA compared with those without these conditions. 17 Overall, these observations agree with the herein reported TNFR2 data in CVF samples, given the reported association between SPTD risk and acute HCA, and that CVF in PPROM cases may also contain proteins that originate from the AF. 52 In the context of PPROM, the presence of microbial-associated HCA (both HCA and MIAC) represents the worse-case scenario compared with HCA or MIAC alone concerning pregnancy and neonatal outcomes, owing to its strong fetal and intraamniotic inflammatory responses.10-12 Therefore, it is of particular clinical importance to identify patients with PPROM complicated by microbial-associated HCA, preferably based on non-invasive biomarkers, to allow tailoring suitable and timely treatment strategies.…”

“…It is worth noting that, despite these significant relationships, the AUC values of the aforementioned CVF biomarkers ranged within 0.61−0.77, thereby demonstrating poor to fair diagnostic potential for predicting each corresponding endpoint related to acute HCA 46 . Furthermore, some important inflammatory proteins (APRIL, DKK‐3, lipocalin‐2, MIP‐1α, S100A8A9, MMP‐9, VDBP, uPA) were not found to be altered in the CVF of patients with acute HCA or microbial‐associated HCA, whereas previous reports showed that these proteins are elevated in the CVF of women with MIAC/IAI or in the AF of women with acute HCA complicated by PPROM 17,22,23 . These discrepancies can be explained as follows: (i) primarily, HCA‐related inflammation/infection can develop in the interval between CVF sampling and placenta delivery, particularly by an ascending route through the ruptured membranes from the vagina and cervix; (ii) CVF specimens could have been contaminated with vaginal or cervical microbes; thus, inflammatory protein levels measured in the CVF, although it was mixed with AF leaked into vagina, may not completely reflect the inflammatory/infectious state of the amniotic cavity; (iii) early stage ascending intra‐uterine inflammation/infection might be limited to the chorio‐decidua before eliciting inflammatory responses in the amniotic cavity; and (iv) non‐infectious factors, including danger signals, may also contribute to the development of placental inflammation 5 .…”

“…Previous studies on women with PPROM reported rates of funisitis within 17.7%−55.6%, 8,59–61 which are similar to that observed in the present study (GA: 23+0 to 34+0 weeks of gestation), although this rate varied according to inclusion criteria, such as GA and time interval from PPROM to birth. In the setting of PPROM alone, previous analyses of AF proteins showed that IL‐6, IL‐10, and TNFR2 levels are significantly elevated in patients with funisitis than in those without this complication 8,17 . Furthermore, previous studies reported increased serum C‐reactive protein and IL‐6 levels in PPROM patients with funisitis 60,61 .…”

“…Analysis of the amniotic fluid (AF) for molecules that are implicated in the inflammatory/infectious process in the amniotic cavity is considered to be the most precise approach for detecting subclinical HCA complicated by PPROM 8,15,17 . However, the utility of AF‐based testing is limited in clinical practice owing to its invasive nature and the technical difficulty of the amniocentesis procedure, especially in patients with PPROM experiencing severe oligohydramnios after rupture.…”

“…Extracellular matrix (ECM)‐related proteins, such as MMP‐8/9, tissue inhibitor of metalloproteinase (TIMP)−1, transforming growth factor beta‐induced (TGFBI), and urokinase‐type plasminogen activator (uPA) are significantly involved in the regulation of inflammation/membrane rupture 28–30 . Furthermore, evaluation of these biomarkers in AF proved to be useful in diagnosing acute HCA/MIAC/IAI 17,31–34 . In addition, significant associations were previously reported between AF/plasma levels of angiogenesis‐related proteins (e.g., insulin‐like growth factor‐binding protein [IGFBP]−1/2) and acute HCA, as well as tight crosstalk between inflammation and angiogenesis 35–38 .…”