Catalin S. Buhimschi

Uncultivated Bacteria as Etiologic Agents of Intra-Amniotic Inflammation Leading to Preterm Birth

ABSTRACT Intra-amniotic infection and inflammation are major causes of preterm birth (PTB). However, intra-amniotic inflammation is often detected in the absence of infection. This may partly be due to the culturing methods employed in hospital laboratories, which are unable to detect the uncultivated species. In this study, intra-amniotic microbial infections associated with PTB were examined by both culture and 16S rRNA-based culture-independent methods and were corroborated by the presence of intra-amniotic inflammation. Amniotic fluid (AF) specimens from 46 pregnancies complicated by PTB and 16 asymptomatic women were analyzed. No bacterial DNA was amplified in AF collected from the asymptomatic women. Among the 46 samples associated with PTB, bacterial DNA was amplified from all (16/16) of the culture-positive samples and 17% (5/30) of the culture-negative samples. In the culture-positive group, additional species were detected in more than half (9/16) of the cases by PCR and clone analysis. Altogether, approximately two- thirds of the species detected by the culture-independent methods were not isolated by culture. They included both uncultivated and difficult-to-cultivate species, such as Fusobacterium nucleatum, Leptotrichia (Sneathia) spp., a Bergeyella sp., a Peptostreptococcus sp., Bacteroides spp., and a species of the order Clostridiales. To examine intra-amniotic inflammation, an AF proteomic fingerprint (mass-restricted score) was determined by surface-enhanced laser desorption ionization-time-of-flight mass spectrometry. Inflammation was detected in all five samples which were culture negative but PCR positive. Women who were PCR positive more often had elevated interleukin-6 levels in their AF, histological chorioamnionitis, and funisitis and delivered neonates with early-onset neonatal sepsis. Previously unrecognized, uncultivated, or difficult-to-cultivate species may play a key role in the initiation of PTB.

Proteomic biomarker analysis of amniotic fluid for identification of intra-amniotic inflammation

Background  Intra‐amniotic inflammation is associated with poor neonatal outcome independent of prematurity. We applied proteomic technology (SELDI: surface‐enhanced laser desorption ionisation) to identify the proteomic profile of intra‐amniotic inflammation.

Proteomic Profiling of the Amniotic Fluid to Detect Inflammation, Infection, and Neonatal Sepsis

Background Proteomic analysis of amniotic fluid shows the presence of biomarkers characteristic of intrauterine inflammation. We sought to validate prospectively the clinical utility of one such proteomic profile, the Mass Restricted (MR) score. Methods and Findings We enrolled 169 consecutive women with singleton pregnancies admitted with preterm labor or preterm premature rupture of membranes. All women had a clinically indicated amniocentesis to rule out intra-amniotic infection. A proteomic fingerprint (MR score) was generated from fresh samples of amniotic fluid using surface-enhanced laser desorption ionization (SELDI) mass spectrometry. Presence or absence of the biomarkers of the MR score was interpreted in relationship to the amniocentesis-to-delivery interval, placental inflammation, and early-onset neonatal sepsis for all neonates admitted to the Newborn Special Care Unit (n = 104). Women with “severe” amniotic fluid inflammation (MR score of 3 or 4) had shorter amniocentesis-to-delivery intervals than women with “no” (MR score of 0) inflammation or even “minimal” (MR score of 1 or 2) inflammation (median [range] MR 3–4: 0.4 d [0.0–49.6 d] versus MR 1–2: 3.8 d [0.0–151.2 d] versus MR 0: 17.0 d [0.1–94.3 d], p < 0.001). Nonetheless, a “minimal” degree of inflammation was also associated with preterm birth regardless of membrane status. There was a significant association between the MR score and severity of histological chorioamnionitis (r = 0.599, p < 0.001). Furthermore, neonatal hematological indices and early-onset sepsis significantly correlated with the MR score even after adjusting for gestational age at birth (OR for MR 3–4: 3.3 [95% CI, 1.1 to 9.2], p = 0.03). When compared with other laboratory tests routinely used to diagnose amniotic fluid inflammation and infection, the MR score had the highest accuracy to detect inflammation (white blood cell count > 100 cells/mm3), whereas the combination of Gram stain and MR score was best for rapid prediction of intra-amniotic infection (positive amniotic fluid culture). Conclusions High MR scores are associated with preterm delivery, histological chorioamnionitis, and early-onset neonatal sepsis. In this study, proteomic analysis of amniotic fluid was shown to be the most accurate test for diagnosis of intra-amniotic inflammation, whereas addition of the MR score to the Gram stain provides the best combination of tests to rapidly predict infection.

Impact of a comprehensive patient safety strategy on obstetric adverse events

OBJECTIVE We implemented a comprehensive strategy to track and reduce adverse events. STUDY DESIGN We incrementally introduced multiple patient safety interventions from September 2004 through November 2006 at a university-based obstetrics service. This initiative included outside expert review, protocol standardization, the creation of a patient safety nurse position and patient safety committee, and training in team skills and fetal heart monitoring interpretation. We prospectively tracked 10 obstetrics-specific outcome. The Adverse Outcome Index, an expression of the number of deliveries with at least 1 of the 10 adverse outcomes per total deliveries, was analyzed for trend. RESULTS Our interventions significantly reduced the Adverse Outcome Index (linear regression, r(2) = 0.50; P = .01) (overall mean, 2.50%). Concurrent with these improvements, we saw clinically significant improvements in safety climate as measured by validated safety attitude surveys. CONCLUSION A systematic strategy to decrease obstetric adverse events can have a significant impact on patient safety.

Dysregulation of Hydrogen Sulfide Producing Enzyme Cystathionine γ-lyase Contributes to Maternal Hypertension and Placental Abnormalities in Preeclampsia

Background— The exact etiology of preeclampsia is unknown, but there is growing evidence of an imbalance in angiogenic growth factors and abnormal placentation. Hydrogen sulfide (H2S), a gaseous messenger produced mainly by cystathionine &ggr;-lyase (CSE), is a proangiogenic vasodilator. We hypothesized that a reduction in CSE activity may alter the angiogenic balance in pregnancy and induce abnormal placentation and maternal hypertension. Methods and Results— Plasma levels of H2S were significantly decreased in women with preeclampsia (P<0.01), which was associated with reduced placental CSE expression as determined by real-time polymerase chain reaction and immunohistochemistry. Inhibition of CSE activity by DL-propargylglycine reduced placental growth factorproduction from first-trimester (8–12 weeks gestation) human placental explants and inhibited trophoblast invasion in vitro. Knockdown of CSE in human umbilical vein endothelial cells by small-interfering RNA increased the release of soluble fms-like tyrosine kinase-1 and soluble endoglin, as assessed by enzyme-linked immunosorbent assay, whereas adenoviral-mediated CSE overexpression in human umbilical vein endothelial cells inhibited their release. Administration of DL-propargylglycine to pregnant mice induced hypertension and liver damage, promoted abnormal labyrinth vascularization in the placenta, and decreased fetal growth. Finally, a slow-releasing H2S-generating compound, GYY4137, inhibited circulating soluble fms-like tyrosine kinase-1 and soluble endoglin levels and restored fetal growth in mice that was compromised by DL-propargylglycine treatment, demonstrating that the effect of CSE inhibitor was attributable to inhibition of H2S production. Conclusions— These results imply that endogenous H2S is required for healthy placental vasculature and that a decrease in CSE/H2S activity may contribute to the pathogenesis of preeclampsia. (Circulation. 2013;127:2514-2522.)

Universal cervical-length screening to prevent preterm birth: a cost-effectiveness analysis.

To determine whether routine measurement of second‐trimester transvaginal cervical length by ultrasound in low‐risk singleton pregnancies is a cost‐effective strategy.

Preeclampsia-Related Inflammatory Cytokines Regulate Interleukin-6 Expression in Human Decidual Cells

Preeclampsia, a common pregnancy disorder associated with an increase in systemic inflammation, is the leading cause of maternal and fetal morbidity and mortality throughout the world. It is associated with shallow extravillous trophoblast invasion of the decidua, leading to uteroplacental blood flow that is inadequate for the developing fetal-placental unit. In preeclamptic women, interleukin-6 (IL-6) levels in plasma, but not placenta, are elevated, prompting evaluation of the decidua as a potential source of this excess, circulating IL-6. The current study found significantly higher immunohistochemical staining for IL-6 in decidual cells from preeclamptic versus preterm, gestational age-matched control placentas. Pro-inflammatory cytokines associated with the genesis of preeclampsia (i.e., tumor necrosis factor-alpha and interleukin-1beta) enhanced IL-6 mRNA levels and increased secreted IL-6 levels in first trimester leukocyte-free decidual cell incubations, as measured by real time quantitative RT-PCR, ELISA, and Western blotting. Therefore, decidual cell-derived IL-6 may contribute to excess circulating IL-6 levels that can promote both endothelial cell dysfunction (and subsequent vascular dysfunction) and the pathogenesis of preeclampsia whereas locally elevated IL-6 levels may contribute to an excess of decidual macrophages implicated in shallow extravillous trophoblast invasion of the decidua.

Proteomic Biomarkers of Intra-amniotic Inflammation: Relationship with Funisitis and Early-onset Sepsis in the Premature Neonate

Our goal was to determine the relationship between 4 amniotic fluid (AF) proteomic biomarkers (human neutrophil defensins 2 and 1, calgranulins C and A) characteristic of intra-amniotic inflammation, and funisitis and early-onset sepsis in premature neonates. The mass restricted (MR) score was generated from AF obtained from women in preterm labor (n = 123). The MR score ranged from 0–4 (none to all biomarkers present). Funisitis was graded histologically and interpreted in relation to the MR scores. Neonates (n = 97) were evaluated for early-onset sepsis. There was significant correlation between the severity of AF inflammation and the presence (53/123) and grades of funisitis (p < 0.001). Funisitis occurred independently of the amniocentesis-to-delivery interval or status of the membranes and was best predicted by an MR score 3–4 and an earlier gestational age (GA) at delivery. Neonates born to women with an MR score 3–4 had an increased incidence of suspected/confirmed sepsis, even after adjusting for GA at birth. Calgranulin C had the highest association with clinically significant funisitis, while calgranulin A had the strongest association with early-onset sepsis. To conclude, AF proteomic analysis shows that women with MR scores 3–4 are more likely to have histologic funisitis, and deliver neonates with early-onset sepsis.

Uterine activity during pregnancy and labor assessed by simultaneous recordings from the myometrium and abdominal surface in the rat.

OBJECTIVES The aim of this study was to analyze records of uterine electrical activity made from the abdominal surface of pregnant and laboring rats to examine whether similar quantitative information can be extracted as from direct recording from the uterine muscle. STUDY DESIGN Electrical activity during pregnancy (nonlabor, days 18 to 22), term labor (day 22), and preterm labor (onapristone injected on day 18, delivery on day 19) was measured with use of electrodes attached to the uterine wall and to the abdominal surface. The fast Fourier transform and wavelet transforms were obtained for representative electromyographic bursts. Power spectra were generated. Intrauterine pressure was also measured. RESULTS (1) Several parameters have been identified for use in following up the progressive increase in uterine activity that occurs in preparation for and during labor. Analyses of amplitude, frequency, and percent time active represent a convenient method for objectively determining the efficiency of uterine contraction. (2) Most of the changes in these parameters appear in the last 24 hours before delivery. (3) Although the uterine electromyographic signals on the abdominal surface are attenuated, the abdominal surface signals are generally similar to the signals obtained from the uterine muscle during pregnancy and during preterm and term labor. (4) The characteristics of onapristone-induced preterm labor are generally similar to those of spontaneous term labor. CONCLUSIONS Recording of uterine electromyographic activity from the abdominal surface may be useful in following the progression of pregnancy and in predicting and diagnosing labor.

Proteomic profiling of urine identifies specific fragments of SERPINA1 and albumin as biomarkers of preeclampsia

OBJECTIVE The cause of preeclampsia remains unknown and the diagnosis can be uncertain. We used proteomic-based analysis of urine to improve disease classification and extend the pathophysiologic understanding of preeclampsia. STUDY DESIGN Urine samples from 284 women were analyzed by surface-enhanced laser desorption/ionization. In the exploratory phase, 59 samples were used to extract the proteomic fingerprint characteristic of severe preeclampsia requiring mandated delivery and to develop a diagnostic algorithm. In the challenge phase, we sought to prospectively validate the algorithm in 225 women screened for a variety of high- and low-risk conditions, including preeclampsia. Of these, 19 women were followed longitudinally throughout pregnancy. The presence of biomarkers was interpreted relative to clinical classification, need for delivery, and other urine laboratory measures (ratios of protein to creatinine and soluble fms-like tyrosine kinase-1 to placental growth factor). In the translational phase, biomarker identification by tandem mass spectrometry and validation experiments in urine, serum, and placenta were used to identify, quantify, and localize the biomarkers or related proteins. RESULTS We report that women with preeclampsia appear to present a unique urine proteomic fingerprint that predicts preeclampsia in need of mandated delivery with highest accuracy. This characteristic proteomic profile also has the ability to distinguish preeclampsia from other hypertensive or proteinuric disorders in pregnancy. Pregnant women followed longitudinally who developed preeclampsia displayed abnormal urinary profiles more than 10 weeks before clinical manifestation. Tandem mass spectrometry and de novo sequencing identified the biomarkers as nonrandom cleavage products of SERPINA1 and albumin. Of these, the 21 amino acid C-terminus fragment of SERPINA1 was highly associated with severe forms of preeclampsia requiring early delivery. In preeclampsia, increased and aberrant SERPINA1 immunoreactivity was found in urine, serum, and placenta, in which it localized predominantly to placental villi and placental vascular spaces adherent to the endothelium. In addition, significant perivascular deposits of misfolded SERPINA1 aggregates were exclusively identified in preeclamptic placentae. CONCLUSION Proteomics-based characterization of urine in preeclampsia identified a proteomic fingerprint composed of SERPINA1 and albumin fragments, which can accurately diagnose preeclampsia and shows promise to discriminate it from other hypertensive proteinuric diseases. These findings provide insight into a novel pathophysiological mechanism of preeclampsia related to SERPINA1 misfolding, which may offer new therapeutic opportunities in the future.