Variation in Stability of Endogenous Reference Genes in Fallopian Tubes and Endometrium from Healthy and Ectopic Pregnant Women

RT-qPCR is commonly employed in gene expression studies in ectopic pregnancy. Most use RN18S1, β-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

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

Impact of reference gene selection for type 2 cannabinoid receptor gene expression studies in human spermatozoa

Quantitative real-time polymerase chain reaction (qRT-PCR) has been employed to study the gene expression profiles in human spermatozoa, but accurate analysis is dependent upon normalisation of data against an endogenous control. -Actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are the most commonly used reference genes for normalisation of gene expression in human spermatozoa, but the expression of these genes in many tissues has considerable variation under different physiological, pathological and experimental conditions which limits their effectiveness in normalisation. The expression stability of a panel of 12 reference genes was studied in normal and pathological human spermatozoa using geNorm and NormFinder software. Although there were some discrepancies in the ranking of reference gene stability, each software program ranked B2M, ACTB, CYC1 and 18S RNA within the top 5 and recommended the combined use of at least two reference genes. Normalisation of qRT-PCR data for the cannabinoid receptor type 2 in spermatozoa using the different housekeeping genes demonstrated how, without validation, conflicting results are obtained. We recommend that the arbitrary use of reference genes should be avoided and the validation of reference gene stability should be undertaken prior to every study. For normalisation of CB2 expression, we would recommend using the geometric mean of B2M and ACTB

Decreased circulating anandamide levels in preeclampsia.

The endocannabinoid system has a key role in female reproduction, including implantation, decidualization and placentation. A growing number of studies indicate that placental and peripheral blood anandamide levels correlate closely with both spontaneous miscarriage and ectopic pregnancy. Anandamide has also been implicated in blood pressure regulation. In this study, we aimed to determine circulating anandamide levels in preeclampsia for the first time in the literature. Forty-three preeclamptic patients and 71 healthy pregnant women were involved in this case-control study. Serum anandamide concentrations were determined by high-performance liquid chromatography-mass spectrometry technique. Serum total soluble fms-like tyrosine kinase-1 (sFlt-1) and biologically active placental growth factor (PlGF) levels were measured by electrochemiluminescence immunoassay. For statistical analyses, nonparametric methods were applied. Serum levels of anandamide were significantly lower in preeclamptic patients than in healthy pregnant women (0.75 (0.44-1.03) ng ml-1 vs. 1.30 (0.76-2.0) ng ml-1, P<0.001). Preeclamptic patients had significantly higher sFlt-1 levels (12 121 (7963-18 316) pg ml-1 vs. 2299 (1393-3179) pg ml-1, P<0.001) and significantly lower PlGF concentrations (71.2 (39.2-86.4) pg ml-1 vs. 256.8 (181.1-421.0) pg ml-1, P<0.001) as compared with healthy pregnant women. Serum anandamide concentrations did not correlate with serum levels of sFlt-1 and PlGF in our healthy pregnant and preeclamptic groups. In conclusion, we demonstrated for the first time in the literature that serum anandamide concentrations are decreased in women with preeclampsia. However, the cause and consequence of this observation remain to be determined.Hypertension Research advance online publication, 26 February 2015; doi:10.1038/hr.2015.20