Prediction of preterm labour from a single blood test: The role of the endocannabinoid system in predicting preterm birth in high-risk women

Objective: To determine if plasma concentrations of the N-acylethanolamines (NAEs) N-arachidonoylethanolamine (AEA), N-oleoylethanolamide (OEA) and N-palmitoylethanolamide (PEA) increase in women at high risk for preterm birth (PTB) and whether these could be used to predict preterm delivery and if so, how they compare with current methods. Design: Prospective cohort study. Setting: A large UK teaching hospital. Population: 217 pregnant women were recruited between 24 and 34 gestational weeks at ‘high-risk’ for PTB, recruited from a prematurity prevention clinic or antenatal wards. Methods: Plasma AEA, OEA, and PEA concentrations were measured using ultra-high performance liquid chromatography-tandem mass spectrometry whilst FAAH enzyme activity was measured by fluorometric radiometric assay and CL by ultrasound scan. The clinical usefulness of these measurements were determined by ROC and multivariate analyses. Results: AEA and PEA concentrations were significantly higher in women who delivered prematurely. An AEA concentration >1.095 nM predicted PTB, the gestational age at delivery and the recruitment to delivery interval (RTDI). A PEA concentration >17.50 nM only predicted PTB; FAAH enzyme activity was not related to these changes. Multivariate analysis (all variables) generated an equation to accurately predict the RTDI. Conclusions: A single plasma AEA or PEA measurement can predict PTB. A single AEA measurement predicts the gestational age of delivery and the remaining period of pregnancy with reasonable accuracy and better than existing conventional tests thus offering a better window for primary prevention of PTB

Expression and Function of the Endocannabinoid Modulating Enzymes Fatty Acid Amide Hydrolase and N-Acylphosphatidylethanolamine-Specific Phospholipase D in Endometrial Carcinoma

Background: The concentrations of three N-acylethanolamines (NAEs), anandamide (AEA), N-oleoylethanolamide (OEA), and N-palmitylethanolamide (PEA) are increased in the endometria of women with endometrial cancer (EC). It is widely accepted that plasma levels of these three NAEs are regulated by the actions of the rate-limiting enzymes N-acylphoshatidylethanolamine-specific phospholipase D (NAPE-PLD) and fatty acid amide hydrolase (FAAH), which are synthesizing and degradative, respectively. The expression and activity of these enzymes have not previously been studied in EC. Methods: FAAH activity in peripheral blood lymphocytes, and transcript and protein expression for FAAH and NAPE-PLD in EC tissues were measured using enzyme, quantitative RT-PCR, and histomorphometry (of immunoreactive tissue sections), respectively. Samples were from 6 post-menopausal women with atrophic endometria (controls) and 34 women with histologically diagnosed EC. Concentrations of the three NAEs also measured in plasma and tissues were correlated with lymphocytic FAAH activity and the NAPE-PLD and FAAH transcript and protein levels. Results: Peripheral lymphocyte FAAH activity was unaffected in women with EC compared to controls. The FAAH transcript expression level was significantly (p < 0.0001) 75% lower in EC whilst NAPE-PLD levels were not significantly (p = 0.798) increased. In line with the transcript data, a significant (p < 0.0001) tumor type-dependent 70–90% decrease in FAAH protein and significant 4- to 14-fold increase in NAPE-PLD protein (p < 0.0001) was observed in the malignant tissue with more advanced disease having lower FAAH and higher NAPE-PLD expression than less advanced disease. Correlation analyses also confirmed that tissue NAE concentrations were inversely related to FAAH expression and directly correlated to NAPE-PLD expression and the NAPE-PLD/FAAH ratio. Conclusion: These data support our previous observation of tissue levels of AEA, OEA, and PEA and a role for NAE metabolism in the pathogenesis of EC.</p

Description of study sample.

<p>Data are represented as means ± SEM. Mini-Mental-State Examination (MMSE) normal range: 0–30. Activities of Daily Living (ADL) normal range: 0–6.</p

ECS gene expression levels.

<p>ECS genes (panel a: receptors; panel b: metabolic enzymes) expression in PBMCs from LOAD subjects. Bars represent 2^−DDCt values calculated by Delta–Delta Ct (DDCt) method. Expression was normalized to GAPDH, and data are represented as means ± SEM. *p<0.05 <i>vs</i> control.</p

<i>faah</i> DNA methylation levels <i>vs</i> MMSE score.

<p>a: Amount of methylated DNA at <i>faah</i> gene in LOAD subjects subgrouped on the basis of MMSE score; b: Correlation between changes in DNA methylation at <i>faah</i> gene and LOAD subjects with severe AD, based on MMSE score. Data were compared by Pearson's rank correlation coefficient (p<0.05, r = −0.6240).</p

Genetic and epigenetic regulation of <i>faah</i> gene.

<p>a: Levels of FAAH mRNA in PBMCs from LOAD patients (n = 13) and controls (n = 12); b: Amount of methylated DNA at <i>faah</i> gene in controls (n = 33 ) and LOAD subjects (n = 33); c: Correlation between <i>faah</i> gene expression and % change of DNA methylation in LOAD subjects. Data were compared by Spearman's rank correlation coefficient (p<0.05, r = −0.5326). Scatter dots represent 2^−DDCt values calculated by Delta-Delta Ct (DDCt) method, as described in the Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039186#s2" target="_blank">Methods</a> section.</p

Primers used for gene expression and DNA methylation analysis.

<p>Primers used for gene expression and DNA methylation analysis.</p

Levels of FAAH protein and activity.

<p>a: Analysis of FAAH protein levels in PBMCs from LOAD and control (CT) subjects. Values represent means ± SEM, *p<0.05 <i>vs</i> CT. Representative immunoblots of PBMC lysates reacted with specific anti-FAAH or anti-actin antibodies are shown, as well as FAAH immunoreactivity in rat liver extracts and HeLa cell lysates, used as positive and negative controls respectively. Molecular mass markers and the positions of FAAH and actin are indicated to the right. b: FAAH activity in LOAD and CT subjects, expressed as pmol/min per mg of protein (means ± SEM).</p

Interaction between the <i>CNR1</i> genotype and T2-weighted lesion loads on regional GM volumes.

<p>In yellow, there are shown those areas (i.e., left frontal and cingulate cortex, and right temporal cortex) in which an inverse correlation exists between T2-lesion load and local grey matter volumes in individuals with long AAT repeats, but that is absent in those with short AAT repeats. The plot on the top right shows this effect of interaction in the left frontal and cingulate area. This same effect is also confirmed, on a subject by subject basis, by the sctatterplot on the bottom right. Statistical threshold: p values FWE-corrected <0.05. Spatial coordinates (x,y,z) in the figure are in MNI space. Abbreviations: LL = T2-weighted lesion load; GM = grey matter.</p

<i>CNR1</i> (AAT)n regulates cognitive abilities in MS patients.

<p>A, B. Plot of interaction analysis between CNR1 genotype and performance at WLG test (A) or ST (B), analyzing the GM volume. These data confirm the relative preservation of neuronal structures after inflammatory events in subjects with short AAT repeats. C. Cognitive impairment was more frequent in MS subjects homozygous for long (AAT)n repeats in CNR1 gene than short AAT repeats group. D. CII was higher among subjects of long AAT group. E–H. Correlation plots between CII (E), WLG (F), ST (G), PASAT (H) and the total number of AAT repeats on the two genes are shown to better demonstrate the association between the number of AAT repeats and cognitive performance. I. Subjects carrying short AAT repeats scored better at D-KEFS, an executive function test.</p