RESUMO
RT-qPCR is commonly employed in gene expression studies in ectopic pregnancy. Most use RN18S1, ß-actin or GAPDH as internal controls without validation of their suitability as reference genes. A systematic study of the suitability of endogenous reference genes for gene expression studies in ectopic pregnancy is lacking. The aims of this study were therefore to evaluate the stability of 12 reference genes and suggest those that are stable for use as internal control genes in fallopian tubes and endometrium from ectopic pregnancy and healthy non-pregnant controls. Analysis of the results showed that the genes consistently ranked in the top six by geNorm and NormFinder algorithms, were UBC, GAPDH, CYC1 and EIF4A2 (fallopian tubes) and UBC and ATP5B (endometrium). mRNA expression of NAPE-PLD as a test gene of interest varied between the groups depending on which of the 12 reference genes was used as internal controls. This study demonstrates that arbitrary selection of reference genes for normalisation in RT-qPCR studies in ectopic pregnancy without validation, risk producing inaccurate data and should therefore be discouraged.
Assuntos
Endométrio/patologia , Tubas Uterinas/patologia , Genes , Gravidez Ectópica/genética , Adulto , Algoritmos , Estudos de Casos e Controles , Endométrio/metabolismo , Tubas Uterinas/metabolismo , Feminino , Regulação da Expressão Gênica , Estudos de Associação Genética , Humanos , Fosfolipase D/genética , Fosfolipase D/metabolismo , Gravidez , Gravidez Ectópica/patologia , Padrões de ReferênciaRESUMO
OBJECTIVES: Cannabinoids are effective antiemetics and the "endogenous cannabinoids" (endocannabinoids) are thought to modulate emesis in both humans and animal models. Endocannabinoids, their receptors and their metabolising enzymes are present in peripheral blood and a reduction in blood endocannabinoid concentration has been observed in individuals with excessive nausea and vomiting following parabolic flight manoeuvres. We tested the hypothesis that plasma endocannabinoid levels are similarly perturbed in women with hyperemesis gravidarum (HG), a condition where the aetiopathogenesis is still unknown, compared to normal pregnant controls. METHODS: Plasma N-arachidonoylethanolamine (anandamide), N-oleoylethanolamide and N-palmitoylethanolamide were quantified in women with HG (n = 15) and matched normal pregnant controls (n = 30) using UHPLC-ESI-MS/MS utilising an isotope dilution method and selective ion monitoring. RESULTS: No significant differences in anandamide, oleoylethanolamide and palmitoylethanolamide levels were observed between the two groups. There were no significant correlations between these endocannabinoids and plasma haematocrit and serum urea or sodium concentrations. CONCLUSIONS: These results would suggest that either the circulating endocannabinoids quantified may not be key modulating factors in HG or that the expected endocannabinoid system response to the stress induced by nausea and vomiting of early pregnancy remain unchanged in women with HG.
Assuntos
Ácidos Araquidônicos/sangue , Endocanabinoides/sangue , Etanolaminas/sangue , Hiperêmese Gravídica/sangue , Ácidos Oleicos/sangue , Ácidos Palmíticos/sangue , Alcamidas Poli-Insaturadas/sangue , Adulto , Amidas , Estudos de Casos e Controles , Feminino , Hematócrito , Humanos , Gravidez , Cloreto de Sódio/sangue , Ureia/sangue , Adulto JovemRESUMO
A 31-year-old woman presented with a 7-week history of irregular vaginal bleeding without abdominal pain. She had been using the intrauterine contraceptive device (IUD) for the last 3 years. A pregnancy test was positive and subsequent serum beta human chorionic gonadotropin (ß-HCG) was 4992 mIU/mL. A transvaginal ultrasound scan demonstrated an empty uterus with an associated adnexal mass but no free fluid. A right primary ovarian ectopic pregnancy was diagnosed a laparoscopy. This was managed laparoscopically using monopolar diathermy hook with conservation of the ovary and minimal blood loss. Ovarian pregnancy is rare, especially in women without the classical risk factors for tubal pregnancy, and efforts should be made to exclude ectopic pregnancy in the absence of abdominal pain in a woman of reproductive age presenting with prolonged and irregular vaginal bleeding. Methods to conserve the ovary are also encouraged in cases of ovarian pregnancy.
RESUMO
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.
Assuntos
Amidoidrolases/metabolismo , Ácidos Araquidônicos/sangue , Endocanabinoides/sangue , Etanolaminas/sangue , Tubas Uterinas/enzimologia , Alcamidas Poli-Insaturadas/sangue , Gravidez Ectópica/sangue , Adulto , Amidas , Células Cultivadas , Gonadotropina Coriônica Humana Subunidade beta/metabolismo , Cílios/fisiologia , Implantação do Embrião/fisiologia , Tubas Uterinas/fisiopatologia , Feminino , Humanos , Fase Luteal/fisiologia , Ácidos Oleicos/sangue , Ácidos Palmíticos/sangue , Fosfolipase D/metabolismo , Gravidez , Gravidez Ectópica/fisiopatologia , Adulto JovemRESUMO
CONTEXT: Ectopic pregnancy is associated with significant morbidity and mortality, but the molecular mechanisms underlying this condition remain unclear. Although the endocannabinoids, N-arachidonoylethanolamine (anandamide), N-oleoylethanolamine, and N-palmitoylethanolamine, are thought to play a negative role in ectopic pregnancy, their precise role(s) within the fallopian tube remains unclear. Anandamide activates cannabinoid receptors (CB1 and CB2) and, together with its degrading [e.g. fatty acid amide hydrolase (FAAH)] and synthesizing enzymes (e.g. N-acyl-phosphatidylethanolamine-specific phospholipase D), forms the endocannabinoid system. High anandamide levels are associated with tubal arrest of embryos in mice and may have a similar role in women. OBJECTIVE: The aims were to quantify the levels of the endocannabinoids and evaluate the expression of the modulating enzymes and the cannabinoid receptors in fallopian tubes of women with ectopic pregnancy compared to those of nonpregnant women. DESIGN AND SETTING: We conducted a prospective study at the University Hospitals of the Leicester National Health Service Trust. PARTICIPANTS AND METHODS: Fallopian tubes collected from women with ectopic pregnancy and nonpregnant women with regular menstrual cycles were used for quantification of endocannabinoids by ultra-HPLC tandem mass spectrometry, were fixed in formalin for immunohistochemistry, and had RNA extracted for RT-quantitative PCR or protein extracted for immunoblotting. RESULTS: Anandamide, but not N-oleoylethanolamine and N-palmitoylethanolamine, levels were significantly higher in ectopic fallopian tubes. Endocannabinoid levels from isthmus to ampulla were not significantly different. Cannabinoid receptors and endocannabinoid modulating enzymes were localized in fallopian tube epithelium by immunohistochemistry and showed reduced CB1 and FAAH expression in ectopic pregnancy. CONCLUSION: High anandamide levels and reduced expression of CB1 and FAAH may play a role in ectopic implantation.