Faculty Bibliography

OBJECTIVE:Feeding neonates orally while on nasal continuous positive airway pressure (nCPAP) is a common practice. We hypothesize that pressurized airflow provided by nCPAP will alter the swallowing mechanism in neonates, increasing the risk of aspiration during oral feeding. STUDY DESIGN/METHODS:Infants receiving nCPAP with a RAM cannula and tolerating at least 50% of their feeding orally were included in the study (one term; six preterm infants). Each participant underwent a videofluoroscopic swallow study while on nCPAP and off nCPAP. A non-parametric signed-rank test was used for paired data. RESULT/RESULTS:The incidence of deep penetration (P=0.03) and aspiration (P=0.01) decreased significantly off-nCPAP compared with on-nCPAP. However, the incidence of mild penetration (P=0.65) and nasopharyngeal reflux (P=0.87) remained the same under both conditions. CONCLUSION/CONCLUSIONS:Oral feeding while on-nCPAP significantly increases the risk of laryngeal penetration and tracheal aspiration events. We recommend caution when initiating oral feedings on nCPAP.

OBJECTIVE: The prenatal detection rate of isolated fetal limb abnormalities ranges from 4 to 29.5%. Our aim was to determine the accuracy of a detailed ultrasound protocol in detecting isolated fetal limb abnormalities Methods: This is a retrospective study of infants born at our institution with isolated limb defects from 2009 to 2014. Antepartum and postpartum records were reviewed for genetic testing results. We routinely image both upper and lower extremities, including all long bones, hands, feet, fingers and toes. Posturing, muscular tone and movement are also noted. RESULTS: During the study period, there were 52 neonates born with isolated fetal limb abnormalities who had received a fetal anatomic survey in our ultrasound unit and 15 930 sonograms performed with normal findings; 36 out of the 52 had been prenatally diagnosed (detection rate 69%). The specificity of the protocol was 100% as there were no false positive cases, the positive predictive value was 100% and negative predictive value 99.8%. Forty-three of 52 neonates had normal genetic testing either prenatally or postnatally; 9 neonates did not undergo genetic testing. The average additional time required for this detailed protocol was <5 min for second trimester sonogram. CONCLUSION: A minimal investment in time for detailed evaluation of fetal limbs more than doubles the previously reported prenatal detection rate.

Problem: Intrauterine infections activate a proinflammatory cascade involving cytokines and other mediators that lead to preterm labor. Endotoxin tolerance (ET) is a phenomenon in which exposure to a dose of endotoxin renders tissues less responsive to subsequent exposures. The mechanism underlying ET is not fully understood. To our knowledge, no previous studies have elucidated the role of ET in human placenta. Using placental explants, we examined this phenomenon and whether exosomes play part in it. Method of Study: Placental explants from term and second-trimester pregnancies were cultured and exposed to low dose LPS for three days. Media were collected daily, and the explants were re-exposed to LPS. Cytochalasin-D (inhibitor of exosomes release and uptake) was added with LPS in some groups. TNF-aalpha and IL-10 in placental explants media were determined by ELISA. Exosomes were isolated from media by Total Exosome Isolation Kit, and miRNAs inside exosomes were analyzed by RT-PCR. Results: LPS treatment for 24 hours stimulated the secretion of placental pro-inflammatory cytokines. However, repeated treatment of the placental explants with LPS significantly reduced the subsequent pro-inflammatory effect, indicating ET. The anti-inflammatory cytokine, IL-10, was also induced by LPS; however, its levels were not affected on repeated LPS treatments. Cytochalasin-D treatment resulted in the loss of ET; nevertheless, it did not change IL-10 secretion. We observed that LPS increased exosomes secretion from placental explants. Moreover, miR-146a and others, which negatively modulate inflammatory response, were found higher in the LPS treated exosomes. Taking together, these findings suggest that ET is mediated by exosomes. Conclusion: This study illustrates, for the first time, that LPS induces ET in human placenta, and that exosomes mediate this phenomenon. We speculate that dysregulation of placental exosomes production, and thus tolerance to infection, might be linked to the exaggerated inflammatory response that leads to preterm labor

OBJECTIVE:Gentamicin is a common antibiotic used to treat sepsis in neonates. We hypothesize that obtaining routine gentamicin trough levels may not be necessary in low-risk, term infants. STUDY DESIGN:We performed a retrospective cohort study of term infants (n=346) treated with gentamicin in a single level III neonatal intensive care unit (NICU). The results of gentamicin trough levels and the correlation with risk factors and potential side effects were recorded. In addition, we conducted a survey of 75 academic NICUs across the United States regarding their gentamicin monitoring practice. RESULTS:Routine trough levels did not predict potential gentamicin toxicity in neonates with low risk factors. Regression analysis demonstrated a positive correlation between gentamicin trough levels and serum creatinine. The survey of the NICUs in the United States demonstrated significant inconsistency in gentamicin monitoring practice. CONCLUSION:Obtaining gentamicin trough levels guided by risk factors is more appropriate than obtaining routine trough levels in low-risk term neonates.

BACKGROUND: Midtrimester ultrasound is a valuable method for identifying asymptomatic women at risk for spontaneous preterm delivery (PTD). However, response to various treatments (cerclage, progestogen) has been variable in the clinical setting. It remains unclear how other biomarkers may be used to guide intervention strategies. OBJECTIVE: We applied an amniotic fluid inflammatory scoring system to determine if the degree of inflammation is associated with intervention efficacy in patients with midtrimester short cervix. STUDY DESIGN: Women carrying a singleton fetus between 16-24 weeks' gestation with a short cervix (/=8) or low (score <8) risk for inflammation. Gestational age at delivery was compared for each intervention and risk score status. Risk of delivering as a function of the remaining gestation was evaluated using modified Cox proportional hazards models with incorporation of methods to account for both left and right truncation bias. RESULTS: Ninety patients were included: 24 were in the nonintervention control group, 51 received cerclage, and 15 received 17OHP-C. Inflammation status at time of sampling influenced the efficacy of the treatment (P < .001). Compared to the nonintervention control group, in patients with low inflammation (score < 8), both cerclage (adjusted hazard ratio [HR], 2.86; 95% confidence interval [CI], 1.28-6.37) and 17OHP-C (HR, 3.11; 95% CI, 1.04-9.30) were associated with increased hazard of PTD. In contrast, in patients with high inflammation (score >/=8) both cerclage (HR, 0.22; 95% CI, 0.08-0.65) and 17OHP-C (HR, 0.20; 95% CI, 0.05-0.81) were associated with lower hazard of delivering preterm. CONCLUSION: Cerclage placement or administration of 17OHP-C therapy for midtrimester short cervix for PTD prevention appears beneficial only in the subset of patients with high inflammation. Knowledge of the amniotic fluid inflammatory status may aid in guiding the appropriate therapy for women presenting with midtrimester short cervix who are at increased risk of PTD.

Preterm birth (PTB), defined as any delivery occurring prior to the completion of 37 weeks' gestation, currently accounts for 11-12% of all births in the United States. Maternal genito-urinary infections account for up to 40% of all PTBS and induce a pro-inflammatory state in the host. The potent vasoconstrictor Endothelin-1 (ET-1) is known to be upregulated in the setting of infection, and elicits its effect by binding to the ETA receptor. We have previously shown that antagonism of the ETA receptor with BQ-123 is capable of preventing LPS-induced PTB in mice. We hypothesize that the administration of BQ-123 post LPS exposure will dismantle a positive feedback loop observed with pro-inflammatory cytokines upstream of ET-1. On GD 15.5, pregnant C57BL/6 mice were injected with PBS, LPS, BQ-123, or LPS+BQ-123. Changes at both the level of transcription and translation were observed in uterus and placenta in the ET-1 axis and in pro- and anti-inflammatory cytokines over the course of 12h. We discovered that BQ-123, when administered 10h post LPS, is capable of increasing production of uterine and placental Interleukin-10, causing a shift away from the pro-inflammatory state. We also observed that antagonism of the ETA receptor decreased IL-1β and TNFα in the placenta while also decreasing transcription of ET-1 in the uterus. Our results reinforce the role of ET-1 at the maternal fetal interface and highlight the potential benefit of ETA receptor blockade via the suppression of ET-1, and induction of a Th2 cytokine dominant state.

INTRODUCTION: The pro-inflammatory mediator and potent vasoconstrictor Endothelin-1 (ET-1) is known to be expressed in the placenta. We have recently demonstrated that very low, non-toxic doses of carbon monoxide (CO), prevented infection-induced preterm birth in mice. However the effect(s) of CO on human gestational tissues is yet to be fully explored. We hypothesize that CO will have a protective role against inflammation-induced E. coli by down-regulating the ET axis in placental explants. METHODS: Twenty placentas from elective termination of pregnancy in the second trimester were analyzed with or without exposure to heat killed E. coli over the course of 30 h. Placental ET-1, along with its biologically inactive precursor Big ET-1, and Endothelin Converting Enzyme-1 (ECE-1, responsible for the cleavage of Big ET-1 to ET-1), were analyzed by ELISA. Gene expression for ET-1 (EDN1), ECE-1 and the ETA receptor (EDNRA) were analyzed using qPCR. Localization of ET-1 expression was also demonstrated using immunohistochemistry. RESULTS: E. coli significantly increased ET-1 transcription and secretion of BIG ET-1 and ET-1 in a time dependant manner which was ameliorated when exposed to CO at later time points. In the presence of CO, mRNA levels of ECE-1 were significantly reduced at 3 and 24 h, while EDNRA was significantly reduced at 6 and 18 h. CONCLUSIONS: Up-regulation of ET-1 production in human placenta in the setting of infection can be attenuated by low doses of CO. Our results further explore the anti-inflammatory and regulatory mechanism(s) of CO on the ET axis components at the maternal fetal interface.

BACKGROUND:Although histologic chorioamnionitis (HCA) is known to be associated with poor outcomes in preterm infants, its clinical significance among term infants is not clearly known. OBJECTIVES/OBJECTIVE:To investigate the utility of HCA in determining early onset clinical sepsis (EOCS) among term newborns. METHODS:The incidence of HCA and EOCS in term infants born during 2008-2009 was evaluated in a single center retrospective study (n = 3417). The predictive value of HCA for determining EOCS in term infants admitted to the neonatal intensive care unit (NICU) for suspected sepsis (n = 388) was quantified. Outcome of otherwise healthy term infants in the nursery with HCA was also investigated. RESULTS:Overall, 11% of term infants with HCA also had EOCS. HCA was associated with increased risk for EOCS (OR 2.6, 95% confidence interval 1.6-4.2, P < 0.001) among term infants admitted to the NICU for suspected sepsis. No cases of EOCS were found among otherwise well-appearing infants in the nursery with HCA. Multiple logistic regression analysis indicated that addition of HCA does not increase the power of a model combining C-reactive protein (CRP) and immature to total neutrophil ratio in determining EOCS. CONCLUSION/CONCLUSIONS:Although HCA in term infants is associated with EOCS, it did not improve the ability of CRP and immature to total neutrophil ratio to predict EOCS. Routine placental examination may not contribute to the diagnosis of EOCS in term infants.

AIM/OBJECTIVE:Infection-induced inflammation is a common cause of preterm birth. Pharmacologic inhibition of proinflammatory cytokines improves pregnancy outcome in animal models but there are no universally effective therapies for preterm birth in women. Carbon monoxide (CO) has anti-inflammatory properties at low concentrations but its effects on reproductive tissues is unclear. Therefore, we studied the effect of supplemental CO on the production of cytokines associated with preterm birth by fetal membranes. METHODS:Cross-sections of whole fetal membranes, isolated choriodecidua, and isolated amnion were prepared using tissues collected from women who had normal vaginal deliveries at term. Tissues were placed in an organ explant culture system and stimulated with up to 10(8) CFU/mL Escherichia coli. Cultures were incubated under room air or room air+250 ppm CO for 18 h and cytokine concentrations in conditioned medium were quantified by ELISA. RESULTS:CO inhibited IL-1β and TNF-α (P≤0.001) production by cultures stimulated with 10(7) CFU/mL bacteria but had no detectable effect on IL-10 by full-thickness membranes. Although CO also tended to reduce TNF-α production (P=0.053), no effect of CO was detected for IL-10 or IL-1β for membranes stimulated with 10(8) CFU/mL E. coli. TNF-α, but not IL-1β or IL-10 production, was inhibited by CO for choriodecidual cultures stimulated with 10(7) or 10(8) CFU/mL E. coli (P<0.001). IL-1β production was significantly inhibited by CO for amnion cultures stimulated with 10(7) (P=0.002) and 10(8) (P=0.017) CFU/mL E. coli. Exposure to bacteria had no effect on TNF-α or IL-10 production but CO tended to increase IL-10 production by amnion cultures stimulated with 10(8) CFU/mL E. coli (P=0.037). CONCLUSIONS:These results suggest that CO may help promote an anti-inflammatory environment during intrauterine infections by inhibiting TNF-α and IL-1β production.

AIM/OBJECTIVE:Tissue culture studies indicate that bacterial products stimulate the production of proinflammatory cytokines by reproductive tissues. However, most of these studies have been performed under room air conditions, supplemented with 5% CO₂. In this study, we tested whether O₂ tension affects bacteria-stimulated cytokine production by extra-placental fetal membranes. METHODS:Cultures of full-thickness membranes, isolated choriodecidua, and isolated amnion were exposed to bacteria and incubated under 21% (room air) or 5% O₂ for 18 h. Cytokine concentrations in conditioned medium was quantified by immunoassay. RESULTS:Culture under 5% O₂ increased production of interleukin (IL)-1β and tumor necrosis factor (TNF)-α, but reduced IL-10 and IL-6 production by full membranes. Isolated choriodecidua responded to 5% O₂ with increased IL-1β production and reduced IL-6 production, but had no effect on TNF-α and IL-10 production was not detected. No effect of O₂ tension on IL-1β or IL-6 production by isolated amnion was detected, however, Escherichia coli-stimulated IL-10, TNF-α and IL-8 production was enhanced by culture under 5% O₂. CONCLUSIONS:Increased oxygen tension reduces the pro-inflammatory responsiveness of cell cultures to E. coli and promotes an anti-inflammatory cytokine profile. Differential effects of O₂ tension on choriodecidua and amnion suggests a network of paracrine factors that regulate cytokine levels in response to changes in O₂ tension.