Microchip electrophoresis separation of a panel of preterm birth biomarkers.

Preterm birth (PTB) is responsible for over one million infant deaths annually worldwide. Often, the first and only indication of PTB risk is the onset of early labor. Thus, there is an urgent need for an early PTB risk diagnostic that is inexpensive, reliable, and robust. Here, we describe the development of a microchip electrophoresis (µCE) method for separating a mixture of six PTB protein and peptide biomarkers present in maternal blood serum. µCE devices were photografted with a poly(ethylene glycol) diacrylate surface coating to regulate EOF and reduce nonspecific analyte adsorption. Separation conditions including buffer pH, buffer concentration, and applied electric field were varied to improve biomarker peak resolution while minimizing deleterious effects like Joule heating. In this way, it was possible to separate six PTB biomarkers, the first µCE separation of this biomarker panel. LODs were also measured for each of the six PTB biomarkers. In the future, this µCE separation can be integrated with upstream maternal blood serum sample preparation steps to yield a complete PTB risk diagnosis microdevice.

Periodontal Disease, Inflammatory Cytokines, and PGE2 in Pregnant Patients at Risk of Preterm Delivery: A Pilot Study.

Periodontal disease is an infection that, in pregnant women, can act as a risk factor for preterm delivery by increasing local and systemic inflammatory responses. Objective. To analyze the presence of periodontal disease, proinflammatory cytokines, and prostaglandin E 2 (PGE2) in pregnant patients at high risk for preterm delivery. Materials and Methods. Pilot study for a case-control study. We included 46 pregnant patients (23 patients at risk of preterm delivery as cases and 23 patients without risk of preterm delivery as controls). We excluded patients who received periodontal treatment, antibiotics, or antimicrobials over the last 3 months as well as those with infections or diseases such as diabetes or hypercholesterolemia. The patients underwent a periodontal assessment, and their levels of cytokines (interleukin- [IL-] 2, IL-6, IL-10, and tumor necrosis factor- [TNF-] α) and prostaglandin E2 (PGE2) were quantified. Results. Patients with periodontal disease showed higher levels of cytokines (IL-2, IL-6, IL-10, and TNF-α) and PGE 2 . Patients at high risk for preterm birth showed higher IL levels compared with patients at low risk for preterm delivery. PGE 2 increased with the severity of periodontal disease. PGE 2 was higher in patients at low risk for preterm delivery, although this difference was not significant. Conclusion. Periodontal disease can increase the systemic inflammatory response as well as the levels of PGE 2 and inflammatory cytokines in pregnant patients.

Integrated electrokinetically driven microfluidic devices with pH-mediated solid-phase extraction coupled to microchip electrophoresis for preterm birth biomarkers.

Integration in microfluidics is important for achieving automation. Sample preconcentration integrated with separation in a microfluidic setup can have a substantial impact on rapid analysis of low-abundance disease biomarkers. Here, we have developed a microfluidic device that uses pH-mediated solid-phase extraction (SPE) for the enrichment and elution of preterm birth (PTB) biomarkers. Furthermore, this SPE module was integrated with microchip electrophoresis for combined enrichment and separation of multiple analytes, including a PTB peptide biomarker (P1). A reversed-phase octyl methacrylate monolith was polymerized as the SPE medium in polyethylene glycol diacrylate modified cyclic olefin copolymer microfluidic channels. Eluent for pH-mediated SPE of PTB biomarkers on the monolith was optimized using different pH values and ionic concentrations. Nearly 50-fold enrichment was observed in single channel SPE devices for a low nanomolar solution of P1, with great elution time reproducibility (<7% RSD). The monolith binding capacity was determined to be 400 pg (0.2 pmol). A mixture of a model peptide (FA) and a PTB biomarker (P1) was extracted, eluted, injected, and then separated by microchip electrophoresis in our integrated device with ∼15-fold enrichment. This device shows important progress towards an integrated electrokinetically operated platform for preconcentration and separation of biomarkers.

Effect of nonsurgical periodontal therapy on serum and gingival crevicular fluid cytokine levels during pregnancy and postpartum.

BACKGROUND AND OBJECTIVE: A low-grade systemic inflammatory status originating from periodontal infection has been proposed to explain the association between periodontal disease and systemic conditions, including adverse obstetric outcomes. The aim of this study was to evaluate the effect of periodontal therapy during pregnancy on the gingival crevicular fluid and serum levels of six cytokines associated with periodontal disease and preterm birth. MATERIAL AND METHODS: A subsample of 60 women (18-35 years of age) up to 20 gestational weeks, previously enrolled in a larger randomized clinical trial, was recruited for the present study. Participants were randomly allocated to receive either comprehensive nonsurgical periodontal therapy before 24 gestational weeks (n = 30, test group) or only one appointment for supragingival calculus removal (n = 30, control group). Clinical data, and samples of blood and gingival crevicular fluid, were collected at baseline, at 26-28 gestational weeks and 30 d after delivery. The levels of interleukin (IL)-1ß, IL-6, IL-8, IL-10, IL-12p70 and tumor necrosis factor-α were analyzed by flow cytometry. RESULTS: After treatment, a major reduction in periodontal inflammation was observed in the test group, with bleeding on probing decreasing from 49.62% of sites to 11.66% of sites (p < 0.001). Periodontal therapy significantly reduced the levels of IL-1ß and IL-8 in gingival crevicular fluid (p < 0.001). However, no significant effect of therapy was observed on serum cytokine levels. After delivery, the levels of IL-1ß in the gingival crevicular fluid of the test group were significantly lower than were those in the control group (p < 0.001), but there were no significant differences between test and control groups regarding serum cytokine levels. CONCLUSION: Although periodontal therapy during pregnancy successfully reduced periodontal inflammation and gingival crevicular fluid cytokine levels, it did not have a significant impact on serum biomarkers.

The relationship of salivary and cord blood cortisol in preterm infants.

Recent studies reveal that salivary cortisol measurements accurately reflect blood cortisol levels in older children and adults; yet, the relationship between the two values in premature infants has not been established. This study explores the use of salivary cortisol as an accurate measure of adrenal steroid concentrations in premature infants to provide a reliable and less invasive tool for investigating hormonal stress response. Premature infants (n=51) were recruited, with saliva and blood collected immediately after birth, and cortisol levels measured by radioimmunoassay. A linear relationship emerged between cord plasma and salivary cortisol values in the 102 paired samples [(salivary cortisol) = 0.546 +/- 0.192 x (plasma cortisol), r = 0.481 and p = 0.0003]. Findings demonstrated that salivary and plasma cortisol levels were correlated in premature infants. This information will be useful in future studies that assess use of salivary cortisol to evaluate neonatal stress axis function.

Persistently high levels of periodontal pathogens associated with preterm pregnancy outcome.

BACKGROUND: Few studies examining the association between periodontal diseases and preterm birth have explored the underlying microbial and antibody responses associated with oral infection. METHODS: A nested case-control study was performed using data from a recent interventional trial following the delayed-treatment control group of 31 subjects with periodontal diseases. The levels of eight oral bacteria and the maternal immunoglobulin G (IgG) responses in serum to these bacteria were measured at antepartum and postpartum visits to determine the relationship to cases (preterm delivery <37 weeks' gestation) and controls (term delivery). RESULTS: Antepartum, the levels of periodontal pathogens tended to be higher in the preterm (case group) deliveries compared to the term deliveries (control group). Maternal anti-Porphyromonas gingivalis IgG was significantly lower in the preterm group compared to the term group (P = 0.028). Postpartum, levels of P. gingivalis, Tannerella forsythia, Prevotella intermedia, and Prevotella nigrescens were statistically significantly higher in preterm births compared to term deliveries, adjusting for baseline levels. The joint effects of red and orange microbial clusters were significantly higher in the preterm group compared to the term group. CONCLUSIONS: High levels of periodontal pathogens and low maternal IgG antibody response to periodontal bacteria during pregnancy are associated with an increased risk for preterm delivery. Further studies elucidating the role of the microbial load and maternal immune response as related to pregnancy outcome seem merited.

Effects of periodontal therapy during pregnancy on periodontal status, biologic parameters, and pregnancy outcomes: a pilot study.

BACKGROUND: Few studies have examined the potential effects of periodontal treatment during pregnancy on pregnancy outcomes, periodontal status, and inflammatory biomarkers. METHODS: A randomized, delayed-treatment, controlled pilot trial was conducted to evaluate the effects of second-trimester scaling and root planing and the use of a sonic toothbrush on the rate of preterm delivery (<37 weeks gestation). Secondary outcome measures included changes in periodontal status, levels of eight oral pathogens, levels of gingival crevicular fluid (GCF) interleukin-1beta (IL-1beta), prostaglandin E(2) (PGE(2)), 8-isoprostane (8-iso), and IL-6, and serum levels of IL-6, soluble intercellular adhesion molecule 1 (sICAM1), 8-isoprostane, soluble glycoprotein 130 (sGP130), IL-6 soluble receptor (IL-6sr), and C-reactive protein (CRP). Logistic regression models were used to test for effects of treatment on preterm delivery. Secondary outcomes were analyzed by analysis of covariance adjusting for subject baseline values. RESULTS: Periodontal intervention resulted in a significantly decreased incidence odds ratio (OR) for preterm delivery (OR = 0.26; 95% confidence interval = 0.08 to 0.85), adjusting for baseline periodontal status which was unbalanced after randomization. Pregnancy without periodontal treatment was associated with significant increases in probing depths, plaque scores, GCF IL-1beta, and GCF IL-6 levels. Intervention resulted in significant improvements in clinical status (attachment level, probing depth, plaque, gingivitis, and bleeding on probing scores) and significant decreases in levels of Prevotella nigrescens and Prevotella intermedia, serum IL-6sr, and GCF IL-1beta. CONCLUSIONS: Results from this pilot study (67 subjects) provide further evidence supporting the potential benefits of periodontal treatment on pregnancy outcomes. Treatment was safe, improved periodontal health, and prevented periodontal disease progression. Preliminary data show a 3.8-fold reduction in the rate of preterm delivery, a decrease in periodontal pathogen load, and a decrease in both GCF IL-1beta and serum markers of IL-6 response. However, further studies will be needed to substantiate these early findings.

Co-bedding between preterm twins attenuates stress response after heel lance: results of a randomized trial.

OBJECTIVE: To examine the effect of co-bedding between preterm twins on stress response after heel lance. METHODS: Using a multisite randomized controlled trial design, stable preterm twins (67 sets, N=134 infants) between 28 and 36 completed weeks' gestational age, stratified by gestational age (≤ and >32 weeks) and site, were randomly assigned to a co-bedding group, cared for continuously in the same incubator or crib or a standard care group, cared for in a separate incubator or crib, and underwent a medically indicated heel lance after at least 24 hours and no greater than 10 days of group allocation. The reported outcome was cortisol from saliva samples from 89 twins (n=49 co-bedding, n=40 standard care) collected immediately before the heel lance (baseline levels) and 113 twins (n=58 co-bedding, n=55 standard care) collected 20 minutes after heel lance (stress levels) as an index of stress response. RESULTS: No group differences were noted in baseline salivary cortisol levels: 0.36 µg/dL (SD 0.25) in the co-bedding group and 0.43 µg/dL (SD 0.50) in the standard care group. Twenty minutes after lance, levels were significantly lower in the co-bedding group, 0.28 µg/dL (SD 0.25) versus 0.50 µg/dL (SD 0.73) in the standard group (P=0.04). Similarly, the mean of paired changes in salivary cortisol from baseline was lower in the co-bedding group (-0.06 µg/dL) compared with the standard care group (0.14 µg/dL, P<0.05). DISCUSSION: Co-bedding attenuates the stress response of preterm twins undergoing heel lance. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT009176.

Gingival crevicular fluid prostaglandin E2 level as a predictor of preterm low birth weight: a pilot investigation.

Periodontal infections, which serve as a reservoir of inflammatory mediators such as prostaglandin E(2) (PGE(2)), may pose a threat to the fetal-placental unit and cause preterm delivery. This study was conducted to estimate the levels of PGE(2) in gingival crevicular fluid (GCF) and serum to explore the possible use of the GCF-PGE(2) level as a risk predictor of preterm low birth weight (PLBW). Twenty-two pregnant female patients were selected for the study. Samples of GCF and serum were collected from each patient, and sampling was repeated at one month after parturition. The level of PGE(2) in GCF and serum was estimated using a commercially available ELISA kit (NeogenTM). The mean serum PGE(2) level was 4.4 ng/ml and 1.64 ng/ml before and after parturition, respectively, and the difference was statistically significant (P < 0.001). The mean GCF-PGE(2) level was 5.8 ng/ml and 5.5 ng/ml before and after parturition, respectively, but the difference was not significant. There was positive correlation between the serumPGE(2) and GCF-PGE(2) levels, and there was a negative correlation between PGE(2) level and gestational age. The present findings suggest that there is a weak correlation between maternal GCF-PGE(2) level and birth outcome. Further clinical trials with a larger sample size are warranted for further investigation of the association between GCF-PGE(2) level and PLBW.

Serum inflammatory mediators in pregnancy: changes after periodontal treatment and association with pregnancy outcomes.

BACKGROUND: The purposes of this study were to determine: 1) if periodontal treatment in pregnant women before 21 weeks of gestation alters levels of inflammatory mediators in serum; and 2) if changes in these mediators are associated with birth outcomes. METHODS: A total of 823 pregnant women with periodontitis were randomly assigned to receive scaling and root planing before 21 weeks of gestation or after delivery. Serum obtained between 13 and 16 weeks, 6 days (study baseline) and 29 to 32 weeks of gestation was analyzed for C-reactive protein; prostaglandin E(2); matrix metalloproteinase-9; fibrinogen; endotoxin; interleukin (IL)-1 beta, -6, and -8, and tumor necrosis factor-alpha. Cox regression, multiple linear regression, and the t, chi(2), and Fisher exact tests were used to examine associations among the biomarkers, periodontal treatment, and gestational age at delivery and birth weight. RESULTS: A total of 796 women had baseline serum data, and 620 women had baseline and follow-up serum and birth data. Periodontal treatment did not significantly alter the level of any biomarker (P >0.05). Neither baseline levels nor the change from baseline in any biomarker were significantly associated with preterm birth or infant birth weight (P >0.05). In treatment subjects, the change in endotoxin was negatively associated with the change in probing depth (P <0.05). CONCLUSIONS: Non-surgical mechanical periodontal treatment in pregnant women, delivered before 21 weeks of gestation, did not reduce systemic (serum) markers of inflammation. In pregnant women with periodontitis, levels of these markers at 13 to 17 weeks and 29 to 32 weeks of gestation were not associated with infant birth weight or a risk for preterm birth.

Reliability of third molar probing measures and the systemic impact of third molar periodontal pathology.

PURPOSE: This study examined the reliability of assessing clinical periodontal measures on third molars, and the association between oral inflammation with periodontal pathology including third molars, and systemic inflammation including negative obstetric outcomes. PATIENTS AND METHODS: Reliability of third molar probing depth (PD) was assessed for 41 patients by trained examiners. The data for the association between oral inflammation with periodontal pathology and systemic outcomes were derived from an IRB-approved study, "Oral Conditions and Pregnancy." Full mouth periodontal exams including third molars were conducted at less than 24 weeks of pregnancy. Periodontal status, moderate/severe periodontal disease (15 or more sites PD > or =4 mm) was considered as a possible predictor of systemic inflammation and pre-term birth. The upper quartile of the extent of PD for third molars alone (PD > or =4 mm) also was considered as a possible exposure variable for the same outcomes. Chi-square and t tests were used to determine statistical significance (0.05). Significant predictor variables were included in multivariate models. Unconditional logistic multivariate models were used to derive odds ratios (OR) and 95% confidence intervals (CI). RESULTS: Reliability of PD within 1 mm was excellent, and similar for third molars and non-third molars. Data from 1,020 obstetric patients were available for analysis. Eighteen percent of the patients delivered preterm, at less than 37 weeks. Having moderate/severe periodontal disease excluding third molars, was significantly associated with preterm birth (P = .008). Results were more significant if third molars were included (P = .0005). With multivariate models moderate/severe periodontal disease at enrollment including third molar PD, was associated with preterm birth (OR, 1.7; 95% CI, 1.1, 2.6). If only the extent of third molar PD was considered, odds also were increased for preterm birth (OR, 2.4; 95% CI, 1.1, 5.2). If only the extent of third molar PD was considered at enrollment, odds were increased for serum markers of systemic inflammation, elevated serum CRP, and oxidative stress, 8-isoPGF(2alpha). CONCLUSIONS: Dental examiners could reliably assess clinical periodontal measures on third molars. Third molars should be included in studies of systemic outcomes associated with oral inflammation. Women of child-bearing age should be made aware of the systemic risks of oral inflammation with third molar periodontal pathology.