Shotgun Proteomics Identifies Serum Fibronectin as a Candidate Diagnostic Biomarker for Inclusion in Future Multiplex Tests for Ectopic Pregnancy

Ectopic pregnancy (EP) is difficult to diagnose early and accurately. Women often present at emergency departments in early pregnancy with a 'pregnancy of unknown location' (PUL), and diagnosis and exclusion of EP is challenging due to a lack of reliable biomarkers. The objective of this study was to identify novel diagnostic biomarkers for EP. Shotgun proteomics, incorporating combinatorial-ligand library pre-fractionation, was used to interrogate pooled sera (n = 40) from women undergoing surgery for EP, termination of viable intrauterine pregnancy and management of non-viable intrauterine pregnancy. Western blot was used to validate results in individual sera. ELISAs were developed to interrogate sera from women with PUL (n = 120). Sera were collected at time of first symptomatic presentation and categorized according to pregnancy outcome. The main outcome measures were differences between groups and area under the receiver operating curve (ROC). Proteomics identified six biomarker candidates. Western blot detected significant differences in levels of two of these candidates. ELISA of sera from second cohort revealed that these differences were only significant for one of these candidates, fibronectin. ROC analysis of ability of fibronectin to discriminate EP from other pregnancy outcomes suggested that fibronectin has diagnostic potential (ROC 0.6439; 95% CI 0.5090 to 0.7788; P>0.05), becoming significant when 'ambiguous' medically managed PUL excluded from analysis (ROC 0.6538; 95% CI 0.5158 to 0.7918; P<0.05). Fibronectin may make a useful adjunct to future multiplex EP diagnostic tests

Laparoscopic treatment of isolated superficial peritoneal endometriosis for managing chronic pelvic pain in women:study protocol for a randomised controlled feasibility trial (ESPriT1)

Background: Endometriosis (where endometrial-like tissue is found outside the uterus) affects ~ 176 million women worldwide and can lead to debilitating pelvic pain. Three subtypes of endometriosis exist, with ~ 80% of women having superficial peritoneal endometriosis (SPE). Endometriosis is diagnosed by laparoscopy and, if SPE is found, gynaecologists usually remove it surgically. However, many women get limited pain relief from surgical removal of SPE. We plan to undertake a future large trial where women who have only SPE found at initial laparoscopy are randomly allocated to have surgical removal (excision or ablation) of SPE, or not. Ultimately, we want to determine whether surgical removal improves overall symptoms and quality of life, or whether surgery is of no benefit, exacerbates symptoms, or even causes harm. The primary objective of this feasibility study is to determine what proportion of women with suspected SPE undergoing diagnostic laparoscopy will agree to randomisation. The secondary objectives are to determine if there are differences in key prognostic parameters between eligible women that agree to be randomised and those that decline; how many women having laparoscopy for investigation of chronic pelvic pain are eligible for the trial; the range of treatment effects and variability in outcomes and the most acceptable methods of recruitment, randomisation and assessment tools. Methods: We will recruit up to 90 women with suspected SPE undergoing diagnostic laparoscopy over a 9-month recruitment period in four Scottish hospitals and randomise them 1:1 to either diagnostic laparoscopy alone (with a sham port to achieve blinding of the allocation) or surgical removal of endometriosis. Baseline characteristics, e.g. age, index of social deprivation, ethnicity, and intensity/duration of pain will be collected. Participants will be followed up by online questionnaires assessing pain, physical and emotional function at baseline, 3 months, 6 months and 12 months. Discussion: Recruitment to a randomised controlled trial to assess the effectiveness of surgery for endometriosis may be challenging because of preconceived ideas about treatment success amongst patients and clinicians. We have designed this study to assess feasibility of recruitment and to inform the design of our future definitive trial. Trial registration: ClincicalTrials.gov, NCT04081532 Status: Recruiting

Systematic review of patient-specific predictors of pain improvement to endometriosis surgery

BACKGROUND: Up to 28% of endometriosis patients do not get pain relief from therapeutic laparoscopy but this subgroup is not defined. OBJECTIVES: To identify any prognostic patient-specific factors (such as but not limited to patients’ type or location of endometriosis, sociodemographics and lifestyle) associated with a clinically meaningful reduction in post-surgical pain response to operative laparoscopic surgery for endometriosis. SEARCH STRATEGY: PubMed, Cochrane and Embase databases were searched from inception to 19th May 2020 without language restrictions. Backward and forward citation tracking was used. SELECTION CRITERIA, DATA COLLECTION AND ANALYSIS: Cohort studies reporting prognostic factors, along with scores for domains of pain associated with endometriosis before and after surgery, were included. Studies that compared surgeries, or laboratory tests, or outcomes without stratification were excluded. Results were synthesised but variation in study designs and inconsistency of outcome reporting precluded us from doing a meta-analysis. MAIN RESULTS: Five studies were included. Quality assessment using the Newcastle Ottawa Scale graded three studies as high, one as moderate and one as having a low risk of bias. Four of five included studies separately reported that a relationship exists between more severe endometriosis and stronger pain relief from laparoscopic surgery CONCLUSION: Currently there are few studies of appropriate quality to answer the research question. We recommend future studies report core outcome sets to enable meta-analysis. FUNDING: NIHR PB-PG-0317-20018 PROSPERO: CRD4201810860

Artificial membrane-binding proteins stimulate oxygenation of stem cells during engineering of large cartilage tissue

Restricted oxygen diffusion can result in central cell necrosis in engineered tissue, a problem that is exacerbated when engineering large tissue constructs for clinical application. Here we show that pre-treating human mesenchymal stem cells (hMSCs) with synthetic membrane-active myoglobin-polymer–surfactant complexes can provide a reservoir of oxygen capable of alleviating necrosis at the centre of hyaline cartilage. This is achieved through the development of a new cell functionalization methodology based on polymer–surfactant conjugation, which allows the delivery of functional proteins to the hMSC membrane. This new approach circumvents the need for cell surface engineering using protein chimerization or genetic transfection, and we demonstrate that the surface-modified hMSCs retain their ability to proliferate and to undergo multilineage differentiation. The functionalization technology is facile, versatile and non-disruptive, and in addition to tissue oxygenation, it should have far-reaching application in a host of tissue engineering and cell-based therapies

The pre-main sequence binary HK Ori : Spectro-astrometry and EXPORT data

In this paper we present multi-epoch observations of the pre-main sequence binary HK Ori. These data have been drawn from the EXPORT database and are complemented by high quality spectro-astrometric data of the system. The spectroscopic data appear to be very well represented by a combination of an A dwarf star spectrum superposed on a (sub-)giant G-type spectrum. The radial velocity of the system is consistent with previous determinations, and does not reveal binary motion, as expected for a wide binary. The spectral, photometric and polarimetric properties and variability of the system indicate that the active object in the system is a T Tauri star with UX Ori characteristics. The spectro-astrometry of HK Ori is sensitive down to milli-arcsecond scales and confirms the speckle interferometric results from Leinert et al. The spectro-astrometry allows with fair certainty the identification of the active star within the binary, which we suggest to be a G-type T Tauri star based on its spectral characteristics.Comment: MNRAS in press 8 pages 7 figure

Gabapentin for the Management of Chronic Pelvic Pain in Women (GaPP1): A Pilot Randomised Controlled Trial

Chronic pelvic pain (CPP) affects 2.1–24% of women. Frequently, no underlying pathology is identified, and the pain is difficult to manage. Gabapentin is prescribed for CPP despite no robust evidence of efficacy. We performed a pilot trial in two UK centres to inform the planning of a future multicentre RCT to evaluate gabapentin in CPP management. Our primary objective was to determine levels of participant recruitment and retention. Secondary objectives included estimating potential effectiveness, acceptability to participants of trial methodology, and cost-effectiveness of gabapentin. Women with CPP and no obvious pelvic pathology were assigned to an increasing regimen of gabapentin (300-2700mg daily) or placebo. We calculated the proportion of eligible women randomised, and of randomised participants who were followed up to six months. The analyses by treatment group were by intention-to-treat. Interviews were conducted to evaluate women’s experiences of the trial. A probabilistic decision analytical model was used to estimate cost-effectiveness. Between September 2012–2013, 47 women (34% of those eligible) were randomised (22 to gabapentin, 25 to placebo), and 25 (53%) completed six-month follow-up. Participants on gabapentin had less pain (BPI difference 1.72 points, 95% CI:0.07–3.36), and an improvement in mood (HADS difference 4.35 points, 95% CI:1.97–6.73) at six months than those allocated placebo. The majority of participants described their trial experience favorably. At the UK threshold for willingness-to-pay, the probabilities of gabapentin or no treatment being cost-effective are similar. A pilot trial assessing gabapentin for CPP was feasible, but uncertainty remains, highlighting the need for a large definitive trial

Combination of gefitinib and methotrexate to treat tubal ectopic pregnancy (GEM3): a multicentre, randomised, double-blind, placebo-controlled trial

BACKGROUND: Tubal ectopic pregnancies can cause substantial morbidity or even death. Current treatment is with methotrexate or surgery. Methotrexate treatment fails in approximately 30% of women who subsequently require rescue surgery. Gefitinib, an epidermal growth factor receptor inhibitor, might improve the effects of methotrexate. We assessed the efficacy of oral gefitinib with methotrexate, versus methotrexate alone, to treat tubal ectopic pregnancy. METHODS: We performed a multicentre, randomised, double-blind, placebo-controlled trial across 50 UK hospitals. Participants diagnosed with tubal ectopic pregnancy were administered a single dose of intramuscular methotrexate (50 mg/m2) and randomised (1:1 ratio) to 7 days of additional oral gefitinib (250 mg daily) or placebo. The primary outcome, analysed by intention to treat, was surgical intervention to resolve the ectopic pregnancy. Secondary outcomes included time to resolution of ectopic pregnancy and serious adverse events. This trial is registered at the ISRCTN registry, ISCRTN 67795930. FINDINGS: Between Nov 2, 2016, and Oct 6, 2021, 328 participants were allocated to methotrexate and gefitinib (n=165) or methotrexate and placebo (n=163). Three participants in the placebo group withdrew. Surgical intervention occurred in 50 (30%) of 165 participants in the gefitinib group and in 47 (29%) of 160 participants in the placebo group (adjusted risk ratio 1·15, 95% CI 0·85 to 1·58; adjusted risk difference -0·01, 95% CI -0·10 to 0·09; p=0·37). Without surgical intervention, median time to resolution was 28·0 days in the gefitinib group and 28·0 days in the placebo group (subdistribution hazard ratio 1·03, 95% CI 0·75 to 1·40). Serious adverse events occurred in five (3%) of 165 participants in the gefitinib group and in six (4%) of 162 participants in the placebo group. Diarrhoea and rash were more common in the gefitinib group. INTERPRETATION: In women with a tubal ectopic pregnancy, adding oral gefitinib to parenteral methotrexate does not offer clinical benefit over methotrexate and increases minor adverse reactions. FUNDING: National Institute of Health Research

Maternal Serum Macrophage Inhibitory Cytokine-1 as a Biomarker for Ectopic Pregnancy in Women with a Pregnancy of Unknown Location

Ectopic pregnancy (EP) occurs in 1-2% of pregnancies, but is over-represented as a leading cause of maternal death in early pregnancy. It remains a challenge to diagnose early and accurately. Women often present in early pregnancy with a 'pregnancy of unknown location' (PUL) and the diagnosis and exclusion of EP is difficult due to a lack of reliable biomarkers. A serum biomarker able to clearly distinguish between EP and other pregnancy outcomes would greatly assist clinicians in diagnosing and safely managing PULs. This study evaluates the ability of maternal serum macrophage inhibitory cytokine-1 (MIC-1) levels to differentiate between EP and other pregnancy outcomes in women with a PUL.Sera were collected from 120 women with a PUL at first clinical presentation and assayed for MIC-1 by ELISA. Results were classified according to ultimate pregnancy outcome and the discriminatory ability of MIC-1 to diagnose EP was assessed.Serum MIC-1 levels were lower in women with histologically confirmed (definite) EP (dEP) (median 552 ng/mL; interquartile range (IQR) 414-693 ng/mL) compared to women with definite viable intra-uterine pregnancies (dVIUPs) (722 ng/mL; IQR 412-1122 ng/mL), and higher when compared to women with definite non-viable intra-uterine pregnancies (dNVIUPs) (465 ng/mL; IQR 341-675 ng/mL). MIC-1 levels were significantly higher in women with dEP compared to women whose PULs resolved without medical intervention (srPUL) (401 ng/mL; IQR 315-475 ng/mL) (p<0.003). There were no women with an ectopic pregnancy where serum MIC-1>1000 ng/mL.Serum MIC-1 levels in PUL were not able to categorically diagnose EP, however, MIC-1 could distinguish women with an EP that required medical intervention and those women whose PULs spontaneously resolved. A single serum MIC-1 measurement also excluded EP at levels above 1000 ng/mL. MIC-1 may play a role in the development of a combined assay of biomarkers for the diagnosis of EP

Ectopic Pregnancy as a Model to Identify Endometrial Genes and Signaling Pathways Important in Decidualization and Regulated by Local Trophoblast

The endometrium in early pregnancy undergoes decidualization and functional changes induced by local trophoblast, which are not fully understood. We hypothesized that endometrium from tubal ectopic pregnancy (EP) could be interrogated to identify novel genes and pathways involved in these processes. Gestation-matched endometrium was collected from women with EP (n = 11) and intrauterine pregnancies (IUP) (n = 13). RNA was extracted from the tissue. In addition, tissues were prepared for histological analysis for degree of decidualization. We compared a) the samples from EP that were decidualized (n = 6) with non-decidualized samples (n = 5), and b) the decidualized EP (n = 6) with decidualization-matched IUP (n = 6) samples using an Affymetrix gene array platform, with Ingenuity Pathway Analysis, combined with quantitative RT-PCR. Expression of PRL and IGFBP1 was used to confirm the degree of decidualization in each group. There were no differences in PRL or IGFBP1 expression in the decidualization-matched samples but a marked reduction (P<0.001) in the non-decidualized samples. Decidualization was associated with increased expression of 428 genes including SCARA5 (181-fold), DKK1 (71-fold) and PROK1 (32-fold), and decreased expression of 230 genes including MMP-7 (35-fold) and SFRP4 (21-fold). The top canonical pathways associated with these differentially expressed genes were Natural Killer Cell and Wnt/b-Catenin signaling. Local trophoblast was associated with much less alteration of endometrial gene expression with an increase in 56 genes, including CSH1 (8-fold), and a reduction in 29 genes including CRISP3 (8-fold). The top associated canonical pathway was Antigen Presentation. The study of endometrium from tubal EP may promote novel insights into genes involved in decidualization and those influenced by factors from neighboring trophoblast. This has afforded unique information not highlighted by previous studies and adds to our understanding of the endometrium in early pregnancy

Association between MUC1 gene polymorphism and expected progeny differences in Nelore cattle (Bos primigenius indicus)

MUC1 is a heavily glycosylated mammalian transmembrane protein expressed by mucosal secretory tissues for both protection against microbial infection and lubrication. An important characteristic of MUC1 is its variable number of tandem repeats (VNTR) containing several sites for O-glycosylation. VNTR length has been associated with many human diseases and with certain economically important traits in domestic ruminants. The aim of the present study was to correlate the length of MUC1 gene VNTR with expected progeny differences (EPDs) obtained for growth, fertility and carcass traits. Five alleles were identified, with alleles containing short VNTRs being more frequent than those with long, thereby demonstrating that Brazilian Nelore cattle are characterized by high frequencies in short MUC1 VNTRs. Statistical analyses revealed there to be no significant association between VNTR length and EPDs for weight at 120 days (W120 ), scrotal circumference at 365 (SC 365 ) and 450 (SC 450 ) days, age at first calving (AFC), and rib eye area (REA)