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

An outbreak of monkeypox in Doedain District, Rivercess County, Liberia, June, 2017

Introduction: Monkeypox is a zoonotic virus disease with symptoms similar to smallpox, although less severe. The last confirmed monkeypox case in Liberia was recorded in 1970 from Grand Gedeh County. On June 23, 2017, Rivercess County Health Team received information from Dodain District, Rivercess County about a suspected monkeypox that reported at the clinic on June 19. We investigated to verify the report, confirm the diagnosis, determine the source and magnitude of the disease, and recommend evidence-based control and prevention measures. Methods: We defined a suspected case as any person who presented with generalized rash with fever, headache, lymphadenopathy, back pain, myalgia, and weakness in Dodain District from June 1 to July 1, 2017. We defined the probable case as a suspected case in whom the clinician suspected monkeypox. A confirmed case was a suspected or probable case with laboratory confirmed monkeypox. We reviewed patient records, and using the case definitions, we conducted active case search and contact tracing in the affected community to identify cases. We interviewed family and community members to identify cases and contacts. We performed laboratory tests on identified cases to confirm the diagnosis. We monitored and followed up contacts for 21 days to see if any developed signs and symptoms. Results: We identified two cases, one confirmed, and one suspected. The confirmed case was an 8-year old male with onset of symptoms on 17 June 2017. He presented with rashes, fever, and headache. His mother (a suspected case/primary case) was a farmer married to a hunter. She had similar symptoms (onset date, 19th April 2017) but recovered two weeks before her son's onset. Although the suspected case's husband was a hunter, there was no clear information that the primary case had been exposed to bush meat. Both the mother and her child had not traveled outside their area of residence. Both cases responded well to symptomatic treatment. None of the 15 contacts developed signs and symptoms in the 21-day follow-up. Conclusion: This was a confirmed outbreak of monkeypox in Dodain District, Rivercess County whose source was not identified. The outbreak was most likely spread through person-to-person transmission. The outbreak was controlled through effective case management, awareness in the community and early reporting

From Fertilisation to Implantation in Mammalian Pregnancy-Modulation of Early Human Reproduction by the Endocannabinoid System.

There is an increasing recognition that the endocannabinoid system is the crucial cytokine-hormone system regulating early human pregnancy. The synchronous development of the fertilized embryo and the endometrium to ensure timely implantation has been shown to be one of the pivotal steps to successful implantation. This development is thought to be regulated by a finely balanced relationship between various components of the endocannabinoid system in the endometrium, the embryo and the Fallopian tube. In addition, this system has also been shown to be involved in the regulation of the development and maturation of the gametes prior to fertilization. In this review, we will examine the evidence from animal and human studies to support the role of the endocannabinoid system in gametogenesis, fertilization, implantation, early pregnancy maintenance, and in immunomodulation of pregnancy. We will discuss the role of the cannabinoid receptors and the enzymes involved in the synthesis and degradation of the key endocannabinoid ligands (e.g., anandamide and 2-arachinoylglycerol) in early reproduction

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

Pathogenesis of Ectopic Pregnancy: Is there a Role for the Endocannabinoid System in Modulating Embryo-Tubal Transport?

Background: The molecular mechanisms of ectopic pregnancy remain unclear. Studies from knockout mice suggest that perturbations in oviductal endocannabinoid levels, endocannabinoid receptors (CB1) or endocannabinoid degrading enzyme (fatty acid amide hydrolase, FAAH) expression result in infertility secondary to physical trapping of embryos in their oviducts. Perturbations in endocannabinoid metabolism and action may therefore underlie ectopic pregnancy. Aims: To (1) quantify plasma and tubal endocannabinoid levels (2) evaluate blood activity of FAAH and the endocannabinoid degrading enzyme N-acylphosphatidyl-ethanolamine phospholipase-D (NAPE-PLD) and relate that to β-hCG levels (3) evaluate the expression of cannabinoid receptors (CB1, CB2), FAAH and NAPE-PLD in Fallopian tubes and (4) examine the effect of endocannabinoids [N-arachidonoylethanolamine (AEA), N-oleoylethanolamide (OEA) and N-palmitoylethanolamide (PEA)] on cilia beat frequency (CBF) in tubal epithelial cells ex-vivo. Methods: Whole blood collected from women with ectopic pregnancy and suitable controls were used for quantification of endocannabinoids by UHPLCMS/MS and FAAH and NAPE-PLD activity by HPLC methods. Fallopian tubes were fixed in formalin for immunohistochemistry and had RNA and protein extracted for RT-qPCR and immunoblotting respectively. Fallopian tube explants were exposed to endocannabinoids and changes in CBF evaluated using highspeed digital camera. Results: Plasma AEA, OEA, PEA and tubal AEA were significantly higher (p < 0.05) in ectopic pregnancy compared to controls. Tubal OEA and PEA showed a similar trend though the results were not statistically significant. Blood FAAH but not NAPE-PLD activity was attenuated (p < 0.05) in ectopic pregnant women consistent with the higher endocannabinoid level observed in plasma. CB1 and FAAH were localised in Fallopian tube and showed significant attenuation (p < 0.05) in ectopic pregnancy compared to luteal phase controls. Exposure of Fallopian tube epithelial cells to OEA unlike methanandamide and PEA resulted in a significant reduction (p < 0.05) in CBF. Conclusion: The results implicate ECS dysfunction in the pathogenesis of ectopic pregnancy

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