Association between periodontal disease and preterm prelabour rupture of membranes.

OBJECTIVE: Periodontal disease is a possible contributing factor to preterm delivery. The aim of this study was to compare the periodontal status of women with preterm prelabour rupture of membranes (PPROM) and women with uncomplicated singleton pregnancies. PATIENTS AND METHODS: Seventy-eight women with PPROM at gestational ages between 24 + 0 and 36 + 6 weeks and 77 healthy women with uncomplicated pregnancies, matched for gestational age at sampling without preterm birth, were included in this study. All women underwent evaluation of periodontal and oral hygiene status. RESULTS: Women with PPROM had higher gingival and plaque indexes in crude analysis (gingival index: median 0.80 versus 0.20; p < 0.0001; plaque index: median 0.80 versus 0.10; p < 0.0001), even after adjustment for smoking status (p < 0.0001 and p < 0.0001). Mean clinical attachment loss (CAL) and probing pocket depth (PPD) values were higher in women with PPROM in the crude analysis (CAL: median 2.3 mm versus 1.8 mm; p < 0.0001; PPD: median 2.3 mm versus 1.8; p < 0.0001), as well as after adjustment for smoking status (p < 0.0001 and p < 0.0001). CONCLUSIONS: Pregnant women with PPROM residing in central Europe had worse periodontal status than women with uncomplicated pregnancies.

Testing of drugs using human feto-maternal interface organ-on-chips provide insights into pharmacokinetics and efficacy.

Objectives: To improve preclinical drug testing during pregnancy, we developed multiple microfluidic organ-on-chip (OOC) devices that represent the structure, functions, and responses of the two feto-maternal interfaces (FMis) in humans (fetal membrane [FMi-OOC] and placenta [PLA-OOC]). This study utilized feto-maternal interface OOCs to test the kinetics and efficacy of drugs during pregnancy. Study design: The FMi-OOC contained amnion epithelial, mesenchymal, chorion trophoblast, and decidual cells. The PLA-OOC contained cytotrophoblasts (BeWo), syncytiotrophoblasts (BeWo + forskolin), and human umbilical vein endothelial cell lines. Therapeutic concentrations of either pravastatin or rosuvastatin (200 ng mL-1), a model drug for these experiments, were applied to either decidua (in FMi-OOC) and syncytiotrophoblasts (in PLA-OOC) chambers under normal and oxidative stress conditions (induced by cigarette smoke extract [CSE 1 : 25]) to evaluate maternal drug exposure during normal pregnancy or oxidative stress (OS) associated pathologies, respectively. We determined statin pharmacokinetics and metabolism (LC-MS/MS), drug-induced cytotoxicity (LDH assay), and efficacy to reduce OS-induced inflammation (multiplex cytokine assay). Results: Both OOCs mimicked two distinct human feto-maternal interfaces. The drugs tested permeated the maternal-fetal cell layers of the FMi-OOC and PLA-OOC within 4 hours and generated cell and time-specific statin metabolites from various cell types without causing any cytotoxicity. OS-induced pro-inflammatory cytokines were effectively reduced by statins by increasing anti-inflammatory cytokine response across the FMi-OOC and PLA-OOC. Conclusion: Two distinct feto-maternal interface OOCs were developed, tested, and validated for their utility to conduct preclinical trials during pregnancy. We demonstrated that the placenta and fetal membranes-decidual interface both are able to transport and metabolize drugs and that the safety and efficacy of a drug can be determined using the anatomical structures recreated on OOCs.

Salivary proteinase activity: a potential biomarker for preterm premature rupture of the membranes.

OBJECTIVE: The objective of this study was to design a method to identify patients at risk for preterm premature rupture of the membranes using a simple assay of salivary proteinase activity. STUDY DESIGN: Saliva samples were collected from women in the following groups using Salivette: (1) nonpregnant control; (2) during the second trimester of pregnancy; (3) during active labor at term; (4) women with premature rupture of the membranes before preterm delivery; and (5) postpartum (within 3 hours after delivery at term). Total proteolytic activity in saliva samples was measured by fluorometry using the generic substrate DQ-gelatin in the presence of specific inhibitors to selectively detect matrix metalloproteinase activities. The concentrations of various matrix metalloproteinases in saliva samples were also measured by multiplex bead assay using the Luminex platform. RESULTS: All saliva samples exhibited detectable matrix metalloproteinase activity. Salivary matrix metalloproteinase activity is low in nonpregnant females (0.27 +/- 0.15) and increases in samples taken in the second trimester (0.5 +/- 0.5) and at term during active labor (1.03 +/- 1.2). Samples collected from women with premature rupture of the membranes before preterm delivery had the highest activity (2.5 +/- 3.7) followed by postpartum after normal term delivery (2.1 +/- 1.6). The matrix metalloproteinase activity was higher in premature rupture of the membranes before preterm delivery samples, compared with all other stages of pregnancy. Multiplex matrix metalloproteinase assay documented a significant increase in total matrix metalloproteinase-9 concentration in saliva from premature rupture of the membranes before preterm delivery, compared with any of the other groups. Similarly matrix metalloproteinase-9 activity was also significantly increased in premature rupture of the membranes before preterm delivery group, compared with all others. CONCLUSION: Herein we report a simple test to monitor proteolytic enzyme activity in saliva during pregnancy. The highest matrix metalloproteinase activity is seen in premature rupture of the membranes before preterm delivery and postpartum samples. Ongoing studies aim to further define salivary proteinase activity in patients at high risk for premature rupture of the membranes before preterm delivery and to evaluate its potential as a predictive test for premature rupture of the membranes before preterm delivery.

Proteinase activity in human and murine saliva as a biomarker for proteinase inhibitor efficacy.

As molecularly targeted agents reach the clinic, there is a need for assays to detect their presence and effectiveness against target molecules in vivo. Proteinase inhibitors are one example of a class of therapeutic agent for which satisfactory methods of identifying successful target modulation in vivo are lacking. This is of particular importance while these drugs are in clinical trials because standard maximum-tolerated dose-finding studies often are not suitable due to lack of toxicity. Saliva represents a readily accessible bodily fluid that can be repeatedly sampled and used for assaying in vivo effects of systemic drugs. Here we show the development of a simple assay that can be used to measure proteinase activity in saliva and proteinase inhibition after systemic treatment with three different proteinase inhibitors. A variety of gelatinolytic activities present in human and murine saliva have been assayed with a fluorescent dye-labeled substrate and assigned to different proteinase categories by inclusion of specific classes of inhibitors. Treatment of mice with either matrix metalloproteinase inhibitors or a urokinase inhibitor for a period as short as 48 hours results in levels of the drugs that can be detected in saliva by mass spectrometry and concomitant decreases in salivary proteinase activity, thus demonstrating that these inhibitors successfully modulate their targets in vivo.

Bacterial modulation of human fetal membrane Toll-like receptor expression.

PROBLEM: Preterm premature rupture of fetal membranes (pPROM) occurs in 30-40% of spontaneous preterm births (PTB) and is associated with intra-amniotic infection and inflammation. The membranes may sense and respond to microbes via Toll-like receptors (TLRs); however, little is known about their expression and regulation in this tissue. The objective of this study was to evaluate the expression of TLRs 1-10 in fetal membranes after exposure to pathogens associated with intra-amniotic infection and PTB. METHOD OF STUDY: Normal human term fetal membrane explants were exposed to various bacteria. After 24 hrs, RNA was extracted and quantitative RT-PCR performed for TLRs1-10. RESULTS: Treatment of fetal membranes with Mycoplasma hominis increased expression of TLR4, TLR6, and TLR8 mRNA. Ureaplasma parvum upregulated TLR8 mRNA, and Porphyromonas gingivalis significantly increased fetal membrane TLR7 expression. In contrast, treatment with Gram-negative Escherichia coli (and its cell wall component lipopolysaccharide) downregulated TLR10 mRNA. No effect was detected for Ureaplasma urealyticum, Gardnerella vaginalis, or Group B Streptococcus. CONCLUSION: These findings demonstrate that different types of bacteria have distinct effects on fetal membrane TLR expression patterns. Moreover, these findings highlight the disparity of fetal membrane responses to infection and thus suggest heterogeneity in the mechanisms by which infection-associated pregnancy complications, such as pPROM and PTB, arise.

Fetal membrane biomarker network diversity and disease functions induced by intra-amniotic pathogens.

PROBLEM: Intra-amniotic pathogens and by-products activate innate immune responses encompassing multitudes of signaling molecules and pathways that can result in spontaneous preterm birth (PTB). This study investigates fetal membrane response to bacterial stimulation using a bioinformatics approach. METHOD OF STUDY: Dysregulated biomarker (IL1-ß, IL-2, IL-8, IL-10, and TNF-α) data from fetal membranes at term stimulated with Ureaplasma urealyticum, Ureaplasma parvum, Mycoplasma hominis, E. coli, Group B Streptococci, Polyporhans gingivalis, or Gardnerella vaginalis with 50% (v/v) amniotic fluid (AF) were analyzed by Ingenuity Pathway Analysis. RESULTS: In racially stratified analysis, networks representing late-stage immune inflammation were seen in African-Americans in AF absence. Inflammation was dominant in AF presence as well. In Caucasians, late-stage immune response was dominant with AF, but not in its absence. CONCLUSIONS: Fetal membrane biofunctions in response to bacteria reflect early- and late-stage innate immune defenses that vary based on the presence of AF and subject race.

Amniotic fluid and maternal race influence responsiveness of fetal membranes to bacteria.

Spontaneous preterm birth (PTB) and preterm prelabor rupture of membranes (pPROM) occur more frequently in African-American women than in other racial groups. This may be due to an enhanced inflammatory response to pathogens associated with the condition. It is also possible that amniotic fluid (AF) has different immunomodulatory properties in African-American women that increase their risk of PTB and pPROM. To test this, we cultured fetal membranes from European-American and African-American women with sterile medium (control), Escherichia coli, Gardnerella vaginalis, Group B streptococci (GBS), Polyporphorans gingivalis, Mycoplasma hominis, Ureaplasma urealyticum or Ureaplasma parvum in the presence and absence of 50% autologous AF. Cytokine concentrations were quantified in the conditioned medium. All bacterial species increased IL-8 production. IL-1ß and TNF-α production were stimulated by LPS, E. coli, and G. vaginalis compared with control, but responses to Group B streptococci and P. gingivalis were limited to IL-1ß and TNF-α respectively. Genital mycoplasmas stimulated TNF-α and IL-10 but had no effect on IL-1ß production. African-Americans had twice the IL-1ß response to E. coli as European-Americans (P=0.031). Conversely, European-Americans produced more IL-8 in response to LPS than African-Americans (P=0.026). AF had both pro- and anti-inflammatory properties that varied between races and pathogens. These results suggest that the host response to fetal membrane infections is complex and not generalizable. Interventions to prevent PTB and pPROM may need to be customized based on a patient's race, type of bacterial infection and factors in her AF.