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Plasma Anandamide Concentration and Pregnancy Outcome in Women With Threatened Miscarriage
To the Editor: Approximately 40% to 50% of all human conceptions are lost before 20 weeks of gestation.1 Recent animal studies suggest that the endocannabinoid anandamide (N-arachidonoyl-ethanolamine) is critical for both the synchronous development of the blastocyst and the endometrium in preparation for implantation, with low anandamide levels essential for successful implantation.2 Plasma anandamide levels are regulated by fatty acid amide hydrolase (FAAH), the enzyme (up-regulated by progesterone) that metabolizes anandamide into arachidonic acid and ethanolamine.3 Increased FAAH expression and lower anandamide levels have been demonstrated at the implantation site and low FAAH expression and high anandamide levels at the interimplantation site prior to successful implantation.2 Levels of FAAH in peripheral blood mononuclear cells from uncomplicated early pregnancies were significantly lower in women who subsequently miscarried.4 In women undergoing in vitro fertilization and embryo transfer, high plasma anandamide level at 6 weeks after embryo transfer was associated with failure to achieve an ongoing pregnancy.5 We therefore investigated whether plasma anandamide levels could predict outcome in women presenting with threatened miscarriage.
Methods
Anandamide levels were measured in plasma from 45 healthy pregnant nonsmokers (Table) presenting to the early pregnancy assessment unit at 6 to 12 weeks' gestation with a threatened miscarriage (painless vaginal bleeding associated with a viable pregnancy). The assay was conducted with a high-performance liquid chromatography–mass spectrometry isotope dilution method (Waters Micromass Quattro Premier Mass Spectromer; Waters Corporation, Milford, Massachusetts).6 The extraction and quantification were undertaken within 2 hours of blood collection. Patients, clinicians, and researchers were blinded to the results during the follow-up period.
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Table. Characteristics of the Study Groups
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Based on published anandamide data,5-6 a minimum of 6 spontaneous miscarriages and 6 live births would allow a clinically significant difference of 40% in anandamide concentration to be observed with 80% power, assuming 2-sided  = .05. Ethics committee approval for the study and written informed consent from each volunteer were obtained; participants did not receive financial compensation. Groups were compared using the Mann-Whitney U test; significance was set at 2-sided P = .05. Data were analyzed using InStat Version 3.01 (GraphPad Software Inc, San Diego, California), and the area under the receiver operating characteristic curve was calculated using Stata 9 (StataCorp, College Station, Texas).
Results
Of the 45 women, 9 subsequently miscarried and 36 had live births. Both groups had similar characteristics (Table) and underwent similar treatment. The median plasma anandamide concentration in the miscarriage group (3.47 nM; interquartile range, 2.83-3.86) was approximately 3-fold that in the live birth group (1.07 nM; interquartile range, 0.81-1.45; P < .001) (Figure). All women who miscarried had anandamide values greater than 2.0 nM; 34 of the 36 in the live birth group (94.4%) had anandamide values less than 2.0 nM. One of the 2 live-birth patients with values greater than 2.0 nM developed severe preeclampsia and delivered a 1.85-kg growth-restricted infant at 33 weeks; the other had an uncomplicated delivery of a 3.7-kg full-term infant. There were no distinguishing characteristics of outliers.
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Figure. Plasma Anandamide Levels in Women With Threatened Miscarriage
The data are grouped according to those who proceeded to have a live birth and those who miscarried and are shown as individual cases. The median for each group is indicated by a horizontal solid line. The plasma anandamide levels were significantly different (P < .001; 2-tailed Mann-Whitney U test). Square indicates patient who developed severe preeclampsia and delivered at 33 weeks' gestation (birth weight of newborn, 1.85 kg).
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Using an anandamide level of 2.0 nM as an arbitrary cut-off point, a single plasma anandamide measurement provided a sensitivity of 100% (95% confidence interval [CI], 66.4%-100%) and a specificity of 94.4% (95% CI, 81.3%-99.3%) with a negative predictive value of 100% (95% CI, 66.4%-100%) and a positive predictive value of 82% (95% CI, 48.2%-97.7%) for subsequent miscarriage. The area under the receiver operating characteristic curve was 0.972 (95% CI, 0.882-0.999).
Comment
In this pilot study of women with threatened miscarriage, plasma anandamide level was associated with presence or absence of subsequent miscarriage. The study is limited by a small number of participants (with resultant wide confidence intervals) and requires replication in larger and more diverse populations.
Compared with tests based on peripheral blood mononuclear cells, anandamide-level measurement has an advantage of being based on whole blood and not requiring separation. If established as valid and clinically practical, anandamide measurement has the potential for improving the prediction and counseling of women presenting with threatened miscarriages.
Author Contributions: Dr Konje had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study concept and design: Habayeb, Konje.
Acquisition of data: Habayeb, Finney, Evans.
Analysis and interpretation of data: Habayeb, Taylor, Evans, Konje.
Drafting of the manuscript: Habayeb, Taylor, Finney, Konje.
Critical revision of the manuscript for important intellectual content: Taylor, Evans, Konje.
Statistical analysis: Habayeb, Taylor.
Obtained funding: Konje.
Administrative, technical, or material support: Habayeb, Finney, Konje.
Study supervision: Taylor, Evans, Konje.
Financial Disclosures: Dr Konje reported having a patent (No. PCT/GB2005/050028) for the use of anandamide as a predictive test for preterm labor. PerkinElmer markets radiolabelled anandamide and produces a biochemical diagnostics test for chromosomal abnormalities in the first and second trimesters of pregnancy. PerkinElmer has first right of refusal for any patentable property emanating from Dr Konje's laboratory. No other disclosures were reported.
Funding/Support: This study was funded by income from the University Hospitals of Leicester NHS Trust by paying for the salaries of Drs Habayeb and Finney and by PerkinElmer through a grant to support the Endocannabinoid Research Laboratory of Dr Konje. The British United Provident Association (BUPA) Foundation funded some of the consumables used for the laboratory analysis.
Role of the Sponsor: The funding organizations had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation, review, or approval of the manuscript.
Additional Contributions: David J. Taylor, MD, University of Leicester, provided intellectual input into the study and comments on the manuscript. Stephen Bell, PhD, University of Leicester, provided assistance with study concept and design. Marcus Cooke, PhD, University of Leicester, provided advice on the laboratory measurements and his comments on the manuscript. None of these persons received compensation for his role in this study.
Osama M. H. Habayeb, MBBS;
Anthony H. Taylor, PhD;
Mark Finney, MBchB
Reproductive Sciences Section
Mark D. Evans, PhD
Radiation and Oxidative Stress Group
Justin C. Konje, MD
jck4{at}le.ac.uk
Endocannabinoid Research Group
Reproductive Sciences Section
Department of Cancer Studies and Molecular Medicine
University of Leicester
Leicester Royal Infirmary
Leicester, Leicestershire, United Kingdom
1. Wilcox AJ, Weinberg CR, O’Connor JF; et al. Incidence of early loss of pregnancy. N Engl J Med. 1988;319(4):189-194.
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2. Wang H, Xie H, Guo Y; et al. Fatty acid amide hydrolase deficiency limits early pregnancy events. J Clin Invest. 2006;116(8):2122-2131.
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3. Di Marzo V, Fontana A, Cadas H; et al. Formation and inactivation of endogenous cannabinoid anandamide in central neurons. Nature. 1994;372(6507):686-691.
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4. Maccarrone M, Valensise H, Bari M, Lazzarin N, Romanini C, Finazzi-Agro A. Relation between decreased anandamide hydrolase concentrations in human lymphocytes and miscarriage. Lancet. 2000;355(9212):1326-1329.
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5. Maccarrone M, Bisogno T, Valensise H; et al. Low fatty acid amide hydrolase and high anandamide levels are associated with failure to achieve an ongoing pregnancy after IVF and embryo transfer. Mol Hum Reprod. 2002;8(2):188-195.
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6. Habayeb OM, Taylor AH, Evans MD; et al. Plasma levels of the endocannabinoid anandamide in women: a potential role in pregnancy maintenance and labor? J Clin Endocrinol Metab. 2004;89(11):5482-5487.
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Letters Section Editor: Robert M. Golub, MD, Senior Editor.
JAMA. 2008;299(10):1135-1136.
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