Identification of Potentially Novel Molecular Targets of Endometrial Cancer Using a Non-Biased Proteomic Approach.

The present study was aimed at identifying novel proteins in endometrial cancer (EC), employing proteomic analysis of tissues obtained after surgery. A differential MS-based proteomic analysis was conducted from whole tissues dissected from biopsies from post-menopausal women, histologically confirmed as endometrial cancer (two endometrioid and two serous; n = 4) or normal atrophic endometrium (n = 4), providing 888 differentially expressed proteins with 246 of these previously documented elsewhere as expressed in EC and 372 proteins not previously demonstrated to be expressed in EC but associated with other types of cancer. Additionally, 33 proteins not recorded previously in PubMed as being expressed in any forms of cancer were also identified, with only 26 of these proteins having a publication associated with their expression patterns or putative functions. The putative functions of the 26 proteins (GRN, APP, HEXA, CST3, CAD, QARS, SIAE, WARS, MYH8, CLTB, GOLIM4, SCARB2, BOD1L1, C14orf142, C9orf142, CCDC13, CNPY4, FAM169A, HN1L, PIGT, PLCL1, PMFBP1, SARS2, SCPEP1, SLC25A24 and ZC3H4) in other tissues point towards and provide a basis for further investigation of these previously unrecognised novel EC proteins. The developmental biology, disease, extracellular matrix, homeostatic, immune, metabolic (both RNA and protein), programmed cell death, signal transduction, molecular transport, transcriptional networks and as yet uncharacterised pathways indicate that these proteins are potentially involved in endometrial carcinogenesis and thus may be important in EC diagnosis, prognostication and treatment and thus are worthy of further investigation.

Expression of the putative cannabinoid receptor GPR55 is increased in endometrial carcinoma.

Although the expression of the putative cannabinoid receptor GPR55 has been shown to be involved in the growth of various tumours and is increased in a number of cancers, its expression has not been examined in patients with endometrial cancer (EC). Quantitative RT-PCR (for mRNA levels) and immunohistochemistry (for protein levels) were used to measure GPR55 expression in patients with Type 1 and Type 2 EC and correlated against cannabinoid receptor (CB1 and CB2) protein levels using non-cancerous endometrium as the control tissue. The data indicated that GPR55 transcript and GPR55 protein levels were significantly (p < 0.002 and p < 0.0001, respectively) higher in EC tissues than in control tissues. The levels of immunoreactive GPR55 protein were correlated with GPR55 transcript levels, but not with the expression of CB1 receptor protein, and were inversely correlated with CB2 protein expression, which was significantly decreased. It can be concluded that GPR55 expression is elevated in women with EC, and thus could provide a potential novel biomarker and therapeutic target for this disease.

A Narrative Review of the Role of Diet and Lifestyle Factors in the Development and Prevention of Endometrial Cancer.

Endometrial cancer is the most common cancer affecting the reproductive organs of women living in higher-income countries. Apart from hormonal influences and genetic predisposition, obesity and metabolic syndrome are increasingly recognised as major factors in endometrial cancer risk, due to changes in lifestyle and diet, whereby high glycaemic index and lipid deposition are prevalent. This is especially true in countries where micronutrients, such as vitamins and minerals are exchanged for high calorific diets and a sedentary lifestyle. In this review, we will survey the currently known lifestyle factors, dietary requirements and hormonal changes that increase an individual's risk for endometrial cancer and discuss their relevance for clinical management. We also examine the evidence that everyday factors and clinical interventions have on reducing that risk, such that informed healthy choices can be made. In this narrative review, we thus summarise the dietary and lifestyle factors that promote and prevent the incidence of endometrial cancer.

Nitric oxide positively affects endometrial receptivity via FAAH and NAPE-PLD in vitro.

OBJECTIVE: To determine if models of human 'receptive' and 'non-receptive endometrium' differ in their responses to nitric oxide (NO) supplementation by measuring the levels of the enzymes of the endocannabinoid system (ECS) (fatty acid amide hydrolase (FAAH) and N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD)), which control the 'anandamide tone' essential for successful pregnancy. DESIGN: A study of FAAH and NAPE-PLD expression (using human endometrium) through the menstrual cycle and an in vitro using a model of 'receptive' (Ishikawa) and 'non-receptive' (HEC-1A) human endometrial cell lines treated with the NO-donating compound S-nitroso-N-acetylpenicillamine (SNAP). RESULTS: Immunoreactivity measured by optimised H-score for both FAAH and NAPE-PLD was reduced in secretory (receptive) endometrium compared to proliferative (non-receptive) endometrium (P = 0.0009 and <0.0001, respectively). FAAH and NAPE transcript levels were significantly higher in untreated Ishikawa cells than in HEC-1A cells (P = 0.0228 and 0.0001, respectively). Treatment of cultures with SNAP resulted in an increase in the amount of FAAH mRNA produced by Ishikawa cells and a decrease in NAPE-PLD mRNA. No effect of SNAP was observed in HEC-1A cells. Similarly, FAAH protein was significantly decreased in endometria representative of the receptive endometrium. CONCLUSION: These data suggest that NO most likely affects the expression of ECS enzymes in the implantation site of a receptive endometrium; a phenomenon not seen in a non-receptive endometrium. These effects are most marked with FAAH expression, suggesting that FAAH may play the more critical role in ensuring the correct 'anandamide tone' for successful embryo implantation than NAPE-PLD. LAY SUMMARY: Embryo implantation into the wall of the uterus is only successful when the inner wall of the uterus (the endometrium) is 'receptive', because if it is 'non-receptive', implantation will fail. Previous work showed that enzymes of the 'endocannabinoid system' are critical for implantation by maintaining the correct level of a fat called anandamide. This is by balancing its synthesis (by N-acylphosphatidylethanolamine specific phospholipase D, NAPE-PLD) and degradation (by fatty acid amide hydrolase, FAAH). Using immortalised cell lines as models of 'receptive' and 'non-receptive' human endometrium, we demonstrate a key stimulator of implantation, nitric oxide, has a positive effect on implantation by both increasing the mRNA levels of the degrading enzyme (FAAH) and decreasing the expression of the synthesising enzyme (NAPE-PLD). These effects are most marked with the degrading enzyme, suggesting that FAAH plays a more critical role than NAPE-PLD in ensuring the correct 'anandamide tone' for successful embryo implantation.

(Endo)Cannabinoids and Gynaecological Cancers.

Gynaecological cancers can be primary neoplasms, originating either from the reproductive tract or the products of conception, or secondary neoplasms, representative of metastatic disease. For some of these cancers, the exact causes are unknown; however, it is recognised that the precise aetiopathogeneses for most are multifactorial and include exogenous (such as diet) and endogenous factors (such as genetic predisposition), which mutually interact in a complex manner. One factor that has been recognised to be involved in the pathogenesis and progression of gynaecological cancers is the endocannabinoid system (ECS). The ECS consists of endocannabinoids (bioactive lipids), their receptors, and metabolic enzymes responsible for their synthesis and degradation. In this review, the impact of plant-derived (Cannabis species) cannabinoids and endocannabinoids on gynaecological cancers will be discussed within the context of the complexity of the proteins that bind, transport, and metabolise these compounds in reproductive and other tissues. In particular, the potential of endocannabinoids, their receptors, and metabolic enzymes as biomarkers of specific cancers, such as those of the endometrium, will be addressed. Additionally, the therapeutic potential of targeting selected elements of the ECS as new action points for the development of innovative drugs will be presented.

Selection of Endogenous Control Reference Genes for Studies on Type 1 or Type 2 Endometrial Cancer.

A panel of 32 candidate reference genes was used to identify the most stable genes for gene normalisation in quantitative RT-PCR studies using endometrial biopsies obtained from women with endometrial cancer (type 1 or type 2) and without cancer (controls). RNA from the biopsies was isolated, examined for purity and quality, and then reverse transcribed into cDNA before being subjected to real-time qRT-PCR analysis in triplicate within the TaqMan gene Expression Assay kit. The most 'stable' endogenous control genes were then identified using the geNorm qbase + 2 and NormFinder software packages. PSMC4, PUM1 and IPO8 were identified as the best reference genes combination for type 1 endometrial cancer (grades 1, 2 and 3), whereas for type 2 endometrial cancer (serous and carcinosarcoma), UBC, MRPL19, PGK1 and PPIA were the best reference genes combination. We conclude that the use of these normaliser combinations should provide accurate interpretation of gene expression at the transcript level in endometrial cancer studies especially for types 1 and 2 cancers.

The Impact of Focality and Centricity on Vulvar Intraepithelial Neoplasia on Disease Progression in HIV+ Patients: A 10-Year Retrospective Study.

BACKGROUND: The impact of lesion focality and centricity in relation to patient outcome and disease recurrence of vulvar intraepithelial neoplasia (VIN) is an understudied area of research, especially in immunocompromised women. The prevalence and incidence of VIN have increased steadily since the 1980s because of the co-existence of human papillomavirus (HPV) and human immunodeficiency virus (HIV). In this study, we retrospectively examined the records of VIN patients to determine the effect of lesion focality and centricity with respect to the interval to disease recurrence. MATERIALS AND METHODS: All women diagnosed with VIN and managed between January 2002 and December 2011 were included (n = 90) and followed up until December 2017. Symptoms at the time of presentation, including HIV positivity (n = 75), were collated, including the influences of multifocality and multicentricity on time to disease recurrence. RESULTS: Multicentricity caused a more rapid recurrence of disease than unicentricity (p = 0.006), whereas multifocality increased the risk of recurrence more than unifocality (p < 0.0001). Viral load in the HIV+ patients was not associated with time to disease recurrence, but the reduced number of CD4+ lymphocytes present in HIV+ patients was. Treatment modalities had no effect on disease recurrence. CONCLUSION: Both focality and centricity have effects on interval to recurrence and final patient outcome, with multifocal disease having a poorer prognosis. Centricity and focality should be recorded at the time of diagnosis and act as a warning for disease recurrence. HIV+ VIN patients with multifocal disease and/or known immunosuppression (low CD4+ lymphocyte counts) should be regarded as "high-risk" patients and treated accordingly.

Identification of Novel Predictive Biomarkers for Endometrial Malignancies: N-Acylethanolamines.

Objective: To identify new biochemical markers for endometrial cancer (EC). Recent evidence suggests that members of the endocannabinoid system (N-acylethanolamines) that bind to and activate receptors that are dysregulated in EC are involved in this tumour's biology. These observations suggest increased N-acylethanolamine levels in the tissue that might appear in plasma and could be used as disease biomarkers. Methods: N-arachidonoylethanolamine (anandamide, AEA) and the N-acylethanolamine substances, N-oleoylethanolamine (OEA), and N-palmitoylethanolamine (PEA) were quantified in plasma and endometrial tissue collected from 31 EC and seven atrophic controls using UHPLC-MS/MS. Receiver-operating characteristics (ROC) and logistic regression were used to determine diagnostic accuracy. Cannabinoid receptor 1 (CB1) and 2 (CB2) protein levels were determined by specific immunohistochemistry and histomorphometric analyses. Correlations between plasma and tissue levels of the three N-acylethanolamines and tissue levels of the three N-acylethanolamines and CB1 and CB2 receptor expression levels were determined using correlation analysis. Results: Plasma and tissue AEA and PEA levels were significantly (p < 0.05) higher in EC than controls whilst OEA levels were significantly elevated in type 1 EC tissues but not in plasma. There were significant positive correlations between plasma and tissue levels of AEA (R 2 = 0.302, p = 0.008) and PEA (R 2 = 0.182, p = 0.047), but not for OEA (R 2 = 0.022, p = 0.506). The diagnostic accuracies for EC were: sensitivity of 53.3%, specificity of 100% for plasma AEA (>1.36 nM); sensitivity of 73.3%, specificity of 100% for plasma PEA (>27.5 nM); and sensitivity of 93.3%, specificity of 28.6% for plasma OEA (>4.97 nM). Logistic regression increased the area under the ROC curve (AUC) from 0.781 for AEA, 0.857 for PEA, and 0.543 for OEA to a combined AUC of 0.933 for EC diagnosis. Significant inverse correlations between tissue AEA (R 2 = 0.343, p = 0.003) and PEA (R 2 = 0.384, p < 0.0001) levels and CB1 expression were observed. No correlation between tissue levels of OEA and CB1 and tissue levels of any of the three N-acylethanolamines and CB2 protein expression were observed, except in the type 1 EC patients. Conclusion: Since plasma AEA and PEA are significantly elevated in patients with EC and a reflection of production by the endometrial tumour, then these lipids have the potential to be useful biomarkers for the early diagnosis of EC.

The levonorgestrel-releasing intrauterine device induces endometrial decidualisation in women on tamoxifen.

There is conflicting literature on whether the levonorgestrel-releasing intrauterine system (LNG-IUS; Mirena®) induces decidualisation in the tamoxifen-treated endometrium. The expression of the decidualisation marker IGFBP-1 was measured using immunohistochemistry in endometrial biopsies and in serum (using ELISA) of 20 postmenopausal women at the start of tamoxifen-treatment for breast cancer. Ten women were then fitted with LNG-IUS and the other ten received tamoxifen-treatment only and acted as controls. Samples were taken at baseline and after 12 months. At baseline, all endometrial samples were negative for IGFBP-1 and at 12 months, IGFBP-1 was only expressed in the endometria of women fitted with the LNG-IUS, confirming the observed histological features of decidualisation. By contrast, serum IGFBP-1 concentrations were increased by tamoxifen, but not in the group receiving LNG-IUS. In conclusion, tamoxifen induces a rise in serum IGFBP-1 suggesting a systemic, possibly hepatic effect, whilst LNG abrogates this in both the liver and endometrium. Impact statement What is already known on this subject? Previous reports of the use of LNG-IUS in women on tamoxifen have provided conflicting evidence as to whether the endometrium exhibited decidualisation or not. These reports were however based solely on histological examination and lacked supporting biochemical data. What do the results of this study add? After 12 months of treatment with LNG-IUS, the endometria of women on tamoxifen show histological features of decidualisation and the presence of the decidualisation marker IGFBP-1, suggesting that levonorgestrel protects the tamoxifen-treated uterus from additional pathology by causing decidualisation. Serum levels of IGFBP-1 were expected to be a reflection of uterine production, but contrary to expectations, higher levels were identified in women on tamoxifen alone. These data suggest that an inhibition of tamoxifen-induced serum IGFBP-1 production (possibly from a hepatic source) by LNG-IUS occurred and indicates independent systemic effects of both drugs in post menopausal breast cancer patients. What are the implications of these findings for clinical practice and/or further research? This research demonstrated a mechanism for endometrial protection in women on tamoxifen. It also alerts clinicians to the fact that both tamoxifen and LNG-IUS exert systemic effects in this patient group.

New Insights of Uterine Leiomyoma Pathogenesis: Endocannabinoid System.

BACKGROUND The aim of this study was to determine if components of the endocannabinoid system are modulated in uterine leiomyomas (fibroids). Components studied included cannabinoid receptors 1 (CB1) and 2 (CB2); the G protein-coupled receptor GPR55; transient potential vanilloid receptor 1 (TRPV1) and the endocannabinoid modulating enzymes N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD) and fatty acid amide hydrolase (FAAH), and their N-acylethanolamine (NAE) ligands: N-arachidonylethanolamine (AEA), N-oleoylethanolamine (OEA), and N-palmityolethanaolamine (PEA). MATERIAL AND METHODS Transcript levels of CB1, CB2, TRPV1, GPR55, NAPE-PLD, and FAAH were measured using RT-PCR and correlated with the tissue levels of the 3 NAEs in myometrial tissues. The tissues studied were: 1) fibroids, 2) myometrium adjacent/juxtaposed to the fibroid lesions, and 3) normal myometrium. Thirty-seven samples were processed for NAE measurements and 28 samples were used for RT-PCR analyses. RESULTS FAAH expression was significantly lower in fibroids, resulting in a NAPE-PLD: FAAH ratio that favors higher AEA levels in pre-menopausal tissues, whilst PEA levels were significantly lower, particularly in post-menopausal women, suggesting PEA protects against fibroid pathogenesis. The CB1: CB2 ratio was lower in fibroids, suggesting that loss of CB1 expression affects the fibroid cell phenotype. Significant correlations between reduced FAAH, CB1, and GPR55 expression and PEA in fibroids indicate that the loss of these endocannabinoid system components are biomarkers of leiomyomata. CONCLUSIONS Loss of expression of CB1, FAAH, GPR55, and PEA production are linked to the pathogenesis of uterine fibroids and further understanding of this might eventually lead to better disease indicators or the development of therapeutic potentials that might eventually be used in the management of uterine fibroids.

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.

Cannabinoid receptor expression in estrogen-dependent and estrogen-independent endometrial cancer.

The lack of good diagnostic/prognostic biomarkers and the often late presentation of endometrial cancer (EC) hinders the amelioration of the morbidity and mortality rates associated with this primarily estrogen-driven disease, a disease that is becoming more prevalent in the population. Previous studies on the expression of the classical cannabinoid receptors, CB1 and CB2, suggest these could provide good diagnostic/prognostic biomarkers for EC but those observations have been contradictory. In this study, we sought to resolve the inconsistency of CB1 and CB2 expression levels in different EC studies. To that end, we used qRT-PCR and immunohistochemistry (IHC) for CB1 and CB2 in endometrial biopsies from women with or without EC and found that transcript levels for both CB1 and CB2 were significantly decreased by 90 and 80%, respectively in EC. These observations were supported by histomorphometric studies where CB1 and CB2 staining intensity was decreased in all types of EC. These data suggest that the loss of both types of CB receptors is potentially involved in the development of or progression of EC and that CB1 and CB2 receptor expression could serve as useful histological markers and therapeutic targets in the treatment of or prevention of EC.

Validation of endogenous control reference genes for normalizing gene expression studies in endometrial carcinoma.

Real-time quantitative RT-PCR (qRT-PCR) is a powerful technique used for the relative quantification of target genes, using reference (housekeeping) genes for normalization to ensure the generation of accurate and robust data. A systematic examination of the suitability of endogenous reference genes for gene expression studies in endometrial cancer tissues is absent. The aims of this study were therefore to identify and evaluate from the thirty-two possible reference genes from a TaqMan(®) array panel their suitability as an internal control gene. The mathematical software packages geNorm qBasePLUS identified Pumilio homolog 1 (Drosophila) (PUM1), ubiquitin C (UBC), phosphoglycerate kinase (PGK1), mitochondrial ribosomal protein L19 (MRPL19) and peptidylpropyl isomerase A (cyclophilin A) (PPIA) as the best reference gene combination, whilst NormFinder identified MRPL19 as the best single reference gene, with importin 8 (IPO8) and PPIA being the best combination of two reference genes. BestKeeper ranked MRPL19 as the most stably expressed gene. In addition, the study was validated by examining the relative expression of a test gene, which encodes the cannabinoid receptor 1 (CB1). A significant difference in CB1 mRNA expression between malignant and normal endometrium using MRPL19, PPIA, and IP08 in combination was observed. The use of MRPL19, IPO8 and PPIA was identified as the best reference gene combination for the normalization of gene expression levels in endometrial carcinoma. This study demonstrates that the arbitrary selection of endogenous control reference genes for normalization in qRT-PCR studies of endometrial carcinoma, without validation, risks the production of inaccurate data and should therefore be discouraged.

The evolving role of the endocannabinoid system in gynaecological cancer.

BACKGROUND: The 'endocannabinoid system' (ECS), comprising endogenous ligands (endocannabinoids) and their regulating enzymes, together with the cannabinoid receptors, has attracted a great deal of attention because it affects not only all facets of human reproduction, from gametogenesis through to parturition and beyond, but also targets key mechanisms affecting some hallmarks of cancer. Recent evidence showing that cannabinoid receptors play a very important role in the development of malignancies outside of the reproductive organs suggests a similar role for the ECS in the establishment or continued development of gynaecological malignancy. METHODS: Primary papers and review articles, and primary sources within these papers, up to December 2014, on the evolving role of the ECS in cancer, with a special focus on gynaecological cancers, were obtained by Medline and PubMed searches using the search terms: 'cancer', 'cannabinoid', 'endocannabinoid', 'gynaecology' and 'malignancy'. Non-English manuscripts were excluded. RESULTS: More than 2100 sources were obtained from which only 112 were specifically important to the topic. Analysis of those articles supports a role of the ECS in gynaecological cancers but leaves many gaps in our knowledge that need to be filled. How some of the relevant receptors are activated and cause changes in cell phenotypes that progress to malignancy remains undiscovered and an area for future research. Increasing evidence suggests that malignant transformation within the female genital tract could be accompanied by deregulation of components of the ECS, acting through rather complex cannabinoid receptor-dependent and receptor-independent mechanisms. CONCLUSIONS: The paucity of studies in this area suggests that research using animal models is needed to evaluate endocannabinoid signalling in cancer networks. Future randomized clinical studies should reveal whether endocannabinoids or their derivatives prove to be useful therapeutic targets for gynaecological and other cancers.

The endocannabinoid system and sex steroid hormone-dependent cancers.

The "endocannabinoid system (ECS)" comprises the endocannabinoids, the enzymes that regulate their synthesis and degradation, the prototypical cannabinoid receptors (CB1 and CB2), some noncannabinoid receptors, and an, as yet, uncharacterised transport system. Recent evidence suggests that both cannabinoid receptors are present in sex steroid hormone-dependent cancer tissues and potentially play an important role in those malignancies. Sex steroid hormones regulate the endocannabinoid system and the endocannabinoids prevent tumour development through putative protective mechanisms that prevent cell growth and migration, suggesting an important role for endocannabinoids in the regulation of sex hormone-dependent tumours and metastasis. Here, the role of the endocannabinoid system in sex steroid hormone-dependent cancers is described and the potential for novel therapies assessed.

Elevated anandamide and related N-acylethanolamine levels occur in the peripheral blood of women with ectopic pregnancy and are mirrored by changes in peripheral fatty acid amide hydrolase activity.

BACKGROUND: Studies from knockout mice suggest that perturbations in oviductal endocannabinoid levels, endocannabinoid receptors, or endocannabinoid degrading enzyme [fatty acid amide hydrolase (FAAH)] expression result in infertility secondary to physical trapping of embryos. Similar observations have been made in ectopic pregnant women together with a suggestion that the endocannabinoid receptor gene polymorphism 1359G/A (rs1049353) is associated with ectopic pregnancy. These observations led to the hypothesis that ectopic pregnancy is associated with a perturbation in levels of endocannabinoids and FAAH activity and that such changes are associated with impaired tubal function. AIMS: The objective of the study was to quantify the plasma levels of endocannabinoids (anandamide, oleoylethanolamide, and palmitoylethanolamide) and evaluate blood endocannabinoid metabolizing enzyme activities FAAH and N-acyl-phosphatidyl-ethanolamine phospholipase D (NAPE-PLD) in ectopic pregnancy and normal pregnant controls and relate that to ß-human chorionic gonadotropin (ß-hCG) levels. Additionally, we wanted to examine the effect of endocannabinoids on cilia beat frequency in Fallopian tube epithelial cells ex vivo. PARTICIPANTS AND METHODS: Whole blood collected from ectopic and normal pregnancies was used for quantification of plasma endocannabinoid levels by ultra-HPLC-tandem mass spectrometry of FAAH and NAPE-PLD enzyme activities by radiometric assays, and ß-hCG by immunoassay. Fallopian tube epithelial cells from healthy volunteers were treated with endocannabinoids and cilia beat frequency analyzed using a high-speed digital camera and CiliaFA software. RESULTS: FAAH activity (P < .05) but not NAPE-PLD activity was significantly reduced in ectopic pregnancies. All 3 endocannabinoids levels were significantly higher (P < .05) in ectopic pregnancy. There was no correlation between endocannabinoids, enzyme activity, and ß-hCG levels. Oleoylethanolamide (P < .05), but not methanandamide or palmitoylethanolamide, significantly decreased cilia beat frequency in Fallopian tube epithelial cells. CONCLUSION: Elevated endocannabinoid levels and reduced FAAH activity are associated with ectopic pregnancy and may modulate tubal function, suggesting dysfunctional endocannabinoid action in ectopic implantation. Oleoylethanolamide may play a critical role in embryo-tubal transport.

Modulation of the endocannabinoid system in viable and non-viable first trimester pregnancies by pregnancy-related hormones.

BACKGROUND: In early pregnancy, increased plasma levels of the endocannabinoid anandamide (AEA) are associated with miscarriage through mechanisms that might affect the developing placenta or maternal decidua. METHODS: In this study, we compare AEA levels in failed and viable pregnancies with the levels of the trophoblastic hormones (beta-human chorionic gonadotrophin (beta-hCG), progesterone (P4) and (pregnancy-associated placental protein-A (PAPP-A)) essential for early pregnancy success and relate that to the expression of the cannabinoid receptors and enzymes that modulate AEA levels. RESULTS: The median plasma AEA level in non-viable pregnancies (1.48 nM; n = 20) was higher than in viable pregnancies (1.21 nM; n = 25; P = 0.013), as were progesterone and beta-hCG levels (41.0 vs 51.5 ng/mL; P = 0.052 for P4 and 28,650 vs 6,560 mIU/L; P = 0.144 for beta-hCG, respectively, but were not statistically significant). Serum PAPP-A levels in the viable group were approximately 6.8 times lower than those in the non-viable group (1.82 vs 12.25 mg/L; P = 0.071), but again these differences were statistically insignificant. In the spontaneous miscarriage group, significant correlations between P4 and beta-hCG, P4 and PAPP-A and AEA and PAPP-A levels were observed. Simultaneously, immunohistochemical distributions of the two main cannabinoid receptors and the AEA-modifying enzymes, fatty acid amide hydrolase (FAAH) and N-acylphosphatidylethanolamine-phospholipase D (NAPE-PLD), changed within both the decidua and trophoblast. CONCLUSIONS: The association of higher AEA levels with early pregnancy failure and with beta-hCG and PAPP-A, but not with progesterone concentrations suggest that plasma AEA levels and pregnancy failure are linked via a mechanism that may involve trophoblastic beta-hCG, and PAPP-A, but not, progesterone production. Although the trophoblast, decidua and embryo contain receptors for AEA, the main AEA target in early pregnancy failure remains unknown.

Changes in glandular and stromal estrogen and progesterone receptor isoform expression in eutopic and ectopic endometrium following treatment with the levonorgestrel-releasing intrauterine system.

OBJECTIVES: The levonorgestrel (LNG) intrauterine system (LNG-IUS) has been shown to improve symptoms in women with minimal to moderate endometriosis. The precise mechanism for this is unknown. We hypothesized that this involves alteration in the expression of estrogen receptors (ER) and progesterone receptors (PR). STUDY DESIGN: A prospective study of tissues obtained prospectively from 28 women with laparoscopically confirmed minimal to moderate endometriosis treated with LNG-IUS for 6 months. Endometrial and endometriotic biopsies obtained before and 6 months after treatment were processed and stained for ER-α, ER-ß and PR expression by immunohistochemistry. Photographs were obtained and the receptors quantified. RESULTS: The mean (±SD) age of the 28 women was 31±7.2 (range 18-42) years. Eight of them at initial biopsy were in the proliferative phase and 20 in the secretory phase. ER-α, ER-ß and PR expression decreased significantly in the glandular (P<0.0001) and stromal (P<0.0001) compartments of the eutopic endometrium after treatment with LNG-IUS. Similarly, ER-α, ER-ß and PR were significantly decreased in the stromal compartment of ectopic endometrium (P<0.0001), and significantly decreased in the ectopic glands of ER-α (P<0.0001), ER-ß (P=0.0002) and PR (P=0.0064) expression. CONCLUSION: The ameliorative effect of LNG-IUS on the symptoms of minimal to moderate endometriosis is likely modulated through a decrease in the expression of glandular and stromal ER-α, ER-ß and PR in the ectopic endometrium.

Histomorphometric evaluation of cannabinoid receptor and anandamide modulating enzyme expression in the human endometrium through the menstrual cycle.

Plasma anandamide (AEA) levels fluctuate throughout the menstrual cycle and in early pregnancy in a pattern suggesting its involvement in implantation and early pregnancy maintenance through mechanisms that might involve its binding to cannabinoid receptors CB1 and CB2. Plasma AEA levels are maintained by the actions of the enzymes fatty acid amide hydrolase (FAAH) and N-acylphosphatidylethanolamine-phospholipase D (NAPE-PLD). All of these component parts of the 'endocannabinoid system' have been demonstrated in rodent but not in human uteri. This study aimed to demonstrate the presence of the endocannabinoid system in the human uterus and catalogue its modulation. Immunohistochemical techniques were employed to localise and determine the distribution of immunoreactive CB1, CB2, FAAH, and NAPE-PLD in well-characterised menstrual cycle biopsy samples. Immunoreactive CB1 and CB2 were widely distributed throughout the uterine tissue. In the myometrium and endometrium, smooth muscle cells were immunoreactive, although the vascular smooth muscle cells in both tissues were more so. In the endometrium, CB1 and CB2 immunoreactivity was primarily restricted to the glandular epithelium and expression was unrelated to the phase of the cycle. FAAH immunoreactivity in the endometrium was highest in the mid-proliferative gland and mid-secretory stroma, whilst NAPE-PLD immunoreactivity was down-regulated in the secretory epithelial gland compared to the proliferative epithelial gland and unaffected in the stroma. These data indicate that elements of the 'endocannabinoid system' coexist in many cell types within the uterus and may provide insight into the sites of action of endogenous and exogenous cannabinoids during endometrial transformation.

The relationship between plasma levels of the endocannabinoid, anandamide, sex steroids, and gonadotrophins during the menstrual cycle.

OBJECTIVE: To further investigate the relationship between plasma anandamide (AEA), sex steroids, and gonadotrophins to improve our understanding of how AEA may be involved in human fertility. DESIGN: Cross-sectional and longitudinal study. SETTING: University Hospital of Leicester NHS Trust, Leicester Royal Infirmary. PATIENT(S): Healthy premenopausal and postmenopausal volunteers. INTERVENTION(S): UPLC-MS/MS-measured plasma AEA and ELISA-measured serum FSH, LH, estradiol, and progesterone levels at five different phases of the menstrual cycle and postmenopause. MAIN OUTCOME MEASURE(S): Plasma AEA, serum steroids and gonadotrophins. RESULT(S): Changes in AEA levels were similar in the two cohorts. The mean +/- SEM levels in the early follicular phase (0.89 +/- 0.06) for the cross-sectional cohort and the longitudinal cohort (0.73 +/- 0.03) were higher than those in the late follicular phase (0.77 +/- 0.09 cross-sectional; 0.63 +/- 0.08 longitudinal). The highest AEA levels were measured at ovulation (1.38 +/- 0.14 and 1.33 +/- 0.16) and the lowest level was measured in the late luteal phase (0.66 +/- 0.07 and 0.56 +/- 0.06). There was a statistically significant positive correlation between AEA, estradiol (P=0.0015), LH (P<0.0001) and FSH levels but not progesterone (P=0.022). CONCLUSION(S): Peak plasma AEA occurred at ovulation and positively correlated with estradiol and gonadotrophin levels suggesting that these may be involved in the regulation of AEA levels.