Gary Entrican

The role of infection in miscarriage

BACKGROUND Miscarriage is the spontaneous loss of a pregnancy before 12 weeks (early miscarriage) or from 12 to 24 weeks (late miscarriage) of gestation. Miscarriage occurs in one in five pregnancies and can have considerable physiological and psychological implications for the patient. It is also associated with significant health care costs. There is evidence that potentially preventable infections may account for up to 15% of early miscarriages and up to 66% of late miscarriages. However, the provision of associated screening and management algorithms is inconsistent for newly pregnant women. Here, we review recent population-based studies on infections that have been shown to be associated with miscarriage. METHODS Our aim was to examine where the current scientific focus lies with regards to the role of infection in miscarriage. Papers dating from June 2009 with key words ‘miscarriage’ and ‘infection’ or ‘infections’ were identified in PubMed (292 and 327 papers, respectively, on 2 June 2014). Relevant human studies (meta-analyses, case–control studies, cohort studies or case series) were included. Single case reports were excluded. The studies were scored based on the Newcastle – Ottawa Quality Assessment Scale. RESULTS The association of systemic infections with malaria, brucellosis, cytomegalovirus and human immunodeficiency virus, dengue fever, influenza virus and of vaginal infection with bacterial vaginosis, with increased risk of miscarriage has been demonstrated. Q fever, adeno-associated virus, Bocavirus, Hepatitis C and Mycoplasma genitalium infections do not appear to affect pregnancy outcome. The effects of Chlamydia trachomatis, Toxoplasma gondii, human papillomavirus, herpes simplex virus, parvovirus B19, Hepatitis B and polyomavirus BK infections remain controversial, as some studies indicate increased miscarriage risk and others show no increased risk. The latest data on rubella and syphilis indicate increased antenatal screening worldwide and a decrease in the frequency of their reported associations with pregnancy failure. Though various pathogens have been associated with miscarriage, the mechanism(s) of infection-induced miscarriage are not yet fully elucidated. CONCLUSIONS Further research is required to clarify whether certain infections do increase miscarriage risk and whether screening of newly pregnant women for treatable infections would improve reproductive outcomes.

Chlamydia trachomatis infection increases fallopian tube PROKR2 via TLR2 and NFκB activation resulting in a microenvironment predisposed to ectopic pregnancy

Chlamydia trachomatis and smoking are major risk factors for tubal ectopic pregnancy (EP), but the underlying mechanisms of these associations are not completely understood. Fallopian tube (FT) from women with EP exhibit altered expression of prokineticin receptors 1 and 2 (PROKR1 and PROKR2); smoking increases FT PROKR1, resulting in a microenvironment predisposed to EP. We hypothesize that C. trachomatis also predisposes to EP by altering FT PROKR expression and have investigated this by examining NFκB activation via ligation of the Toll-like receptor (TLR) family of cell-surface pattern recognition receptors. PROKR2 mRNA was higher in FT from women with evidence of past C. trachomatis infection than in those without (P < 0.05), and was also increased in FT explants and in oviductal epithelial cell line OE-E6/E7 infected with C. trachomatis (P < 0.01) or exposed to UV-killed organisms (P < 0.05). The ability of both live and dead organisms to induce this effect suggests ligation of a cell-surface-expressed receptor. FT epithelium and OE-E6/E7 were both found to express TLR2 and TLR4 by immunohistochemistry. Transfection of OE-E6/E7 cells with dominant-negative TLR2 or IκBα abrogated the C. trachomatis–induced PROKR2 expression. We propose that ligation of tubal TLR2 and activation of NFκB by C. trachomatis leads to increased tubal PROKR2, thereby predisposing the tubal microenvironment to ectopic implantation.

Recombinant ovine interferon gamma inhibits the multiplication of Chlamydia psittaci in ovine cells

The local production of interferon-gamma (IFN-gamma) in sheep in response to Chlamydia psittaci was measured by cannulation of the efferent lymph duct draining the site of challenge inoculation. Peak production of IFN-gamma (256 U/ml) was detected 24 h after challenge. Based on these physiological data, functional studies were carried out in vitro to determine the effect of recombinant ovine (rOv) IFN-gamma on the multiplication of C. psittaci in ovine fibroblasts. IFN-gamma inhibited the multiplication of C. psittaci in ovine cells over a range of concentrations (250 U/ml to 2.5 U/ml) in a dose-dependent manner. The inhibition of chlamydial multiplication was most pronounced when cells were treated with rOvIFN-gamma for 24 h before infection. The addition of exogenous L-tryptophan (500 micrograms/ml) to cultures within 48th of infection abrogated the anti-chlamydial effect of rOvIFN-gamma thus suggesting that tryptophan deprivation is an anti-chlamydial mechanism induced by rOvIFN-gamma in these ovine cells.

Development of a sandwich ELISA for ovine granulocyte/macrophage colony-stimulating factor.

Recombinant ovine granulocyte/macrophage colony-stimulating factor (rOv GM-CSF) has been expressed in Chinese hamster ovary cells. A stable, cloned line of these cells has been established which secretes high levels (40 mu g ml(-1)) of rOv GM-CSF. Three murine monoclonal antibodies (mAbs) were produced which reacted with rOv GM-CSF on Western blots. These mAbs also neutralised the activity of both recombinant and native Ov GM-CSF in a bone marrow haemopoietic progenitor cell assay. Two of the mAbs, which recognise mutually exclusive epitopes, were selected for the development of a sandwich enzyme-linked immunosorbent assay (ELISA) to measure GM-CSF in biological samples of ovine origin.

Cotinine Exposure Increases Fallopian Tube PROKR1 Expression via Nicotinic AChRα-7 : A Potential Mechanism Explaining the Link between Smoking and Tubal Ectopic Pregnancy

Tubal ectopic pregnancy (EP) is the most common cause of maternal mortality in the first trimester of pregnancy; however, its etiology is uncertain. In EP, embryo retention within the Fallopian tube (FT) is thought to be due to impaired smooth muscle contractility (SMC) and alterations in the tubal microenvironment. Smoking is a major risk factor for EP. FTs from women with EP exhibit altered prokineticin receptor-1 (PROKR1) expression, the receptor for prokineticins (PROK). PROK1 is angiogenic, regulates SMC, and is involved in intrauterine implantation. We hypothesized that smoking predisposes women to EP by altering tubal PROKR1 expression. Sera/FT were collected at hysterectomy (n=21). Serum levels of the smoking metabolite, cotinine, were measured by enzyme-linked immunosorbent assay. FTs were analyzed by q-RT-PCR, immunohistochemistry, and Western blotting for expression of PROKR1 and the predicted cotinine receptor, nicotinic acetylcholine receptor α-7 (AChRα-7). FT explants (n=4) and oviductal epithelial cells (cell line OE-E6/E7) were treated with cotinine and an nAChRα-7 antagonist. PROKR1 transcription was higher in FTs from smokers (P<0.01). nAChRα-7 expression was demonstrated in FT epithelium. Cotinine treatment of FT explants and OE-E6/E7 cells increased PROKR1 expression (P<0.05), which was negated by cotreatment with nAChRα-7 antagonist. Smoking targets human FTs via nAChRα-7 to increase tubal PROKR1, leading to alterations in the tubal microenvironment that could predispose to EP.

Intranasal Infection with Chlamydia abortus Induces Dose-Dependent Latency and Abortion in Sheep

Background Latency is a key feature of the animal pathogen Chlamydia abortus, where infection remains inapparent in the non-pregnant animal and only becomes evident during a subsequent pregnancy. Often the first sign that an animal is infected is abortion occurring late in gestation. Despite this, little is understood of the underlying mechanisms that control latency or the recrudescence of infection that occurs during subsequent pregnancy. The aim of this study was to develop an experimental model of latency by mimicking the natural route of infection through the intranasal inoculation of non-pregnant sheep with C. abortus. Methodology/Principal Findings Three groups of sheep (groups 1, 2 and 3) were experimentally infected with different doses of C. abortus (5×103, 5×105 and 5×107 inclusion forming units (IFU), respectively) prior to mating and monitored over 2 breeding cycles for clinical, microbiological, pathological, immunological and serological outcomes. Two further groups received either negative control inoculum (group 4a,b) or were inoculated subcutaneously on day 70 of gestation with 2×106 IFU C. abortus (group 5). Animals in groups 1, 2 and 5 experienced an abortion rate of 50–67%, while only one animal aborted in group 3 and none in group 4a,b. Pathological, microbiological, immunological and serological analyses support the view that the maternal protective immune response is influenced by initial exposure to the bacterium. Conclusions/Significance The results show that intranasal administration of non-pregnant sheep with a low/medium dose of C. abortus results in a latent infection that leads in a subsequent pregnancy to infection of the placenta and abortion. In contrast a high dose stimulates protective immunity, resulting in a much lower abortion rate. This model will be useful in understanding the mechanisms of infection underlying latency and onset of disease, as well as in the development of novel therapeutics and vaccines for controlling infection.

Evidence of prokineticin dysregulation in fallopian tube from women with ectopic pregnancy

OBJECTIVEnTo demonstrate expression and regulation of prokineticins (PROKs) and their receptors (PROKRs) in fallopian tube (FT) from women who are not pregnant and women with ectopic pregnancy (EP).nnnDESIGNnTissue analysis.nnnSETTINGnLarge United Kingdom teaching hospital.nnnPATIENT(S)nWomen undergoing hysterectomy for benign gynecological conditions (n = 15) and surgery for EP (n = 16).nnnINTERVENTION(S)nQuantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry were used to determine FT PROK/PROKR messenger RNA (mRNA) expression and protein localization, respectively. The PROK/PROKR levels were measured in tubal explant cultures stimulated with estrogen (E) and progestogen.nnnMAIN OUTCOME MEASURE(S)nDifferential expression of PROK and PROKR.nnnRESULT(S)nThe FT PROK2 and PROKR1 mRNA levels were up-regulated during the P-dominant midluteal phase of the menstrual cycle. Increased PROKR1 expression was observed in tubal explant cultures treated with medroxy-progesterone acetate (MPA). The PROK and PROKR proteins were localized to the epithelium and smooth muscle layers of the FT. The PROKR1 and PROKR2 mRNA levels were lower in FT from women with EP compared with nonpregnant FT from the midluteal phase.nnnCONCLUSION(S)nThese data suggest a potential role for PROKs in FT function. The PROKs are known to affect smooth muscle contraction in the gut. Dysregulated PROK expression in FT could affect FT smooth muscle contractility and embryo-tubal transport, providing a potential cause for EP.

The molecular cloning of the ovine gamma-interferon cDNA using the polymerase chain reaction

The molecular cloning of the ovine gamma-interferon cDNA using the polymerase chain reaction General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim.

Antibody Responses to Recombinant Protein Fragments of the Major Outer Membrane Protein and Polymorphic Outer Membrane Protein POMP90 in Chlamydophila abortus-Infected Pregnant Sheep

ABSTRACT Chlamydophila abortus is one of the major causes of infectious abortion in pregnant sheep (enzootic abortion of ewes or EAE) worldwide. Organisms shed in infected placentas and uterine discharges at lambing time are the main sources of environmental contamination, responsible for transmission to susceptible animals and possible human contacts. In the present study, a recently developed test, based on a recombinant fragment of the polymorphic outer membrane protein POMP90 (rOMP90-4 indirect enzyme-linked immunosorbent assay [iELISA]) and one based on the variable segment 2 (VS2) region of the major outer membrane protein (MOMP) (MOMP VS2 iELISA) were compared using sera from C. abortus-infected ewes at different stages throughout pregnancy. The rOMP90 iELISA detected antibody much earlier in pregnancy than the MOMP iELISA, which, like the complement fixation test, detected antibody only at the time of abortion or lambing. No anti-MOMP antibody response could be detected in three of seven experimentally infected ewes. Furthermore, the rOMP90 iELISA detected antibody in an animal that seroconverted during the course of the study, which the MOMP iELISA failed to detect. Overall, the results show that the rOMP90-4 iELISA is considerably more sensitive than the MOMP VS2 iELISA for identifying animals infected with C. abortus. Earlier detection of infection will allow appropriate control measures to be taken to reduce environmental contamination, thus limiting the spread of infection, financial losses, and the possible risks of zoonotic transmission to humans.

Expression of secretory leukocyte protease inhibitor and elafin in human fallopian tube and in an in-vitro model of Chlamydia trachomatis infection

BACKGROUNDnSecretory leukocyte protease inhibitor (SLPI) and elafin are anti-protease and anti-microbial molecules with a role in innate immune defence. They have been demonstrated at multiple mucosal surfaces including those of the female reproductive tract.nnnMETHODS AND RESULTSnThis study details their expression in human Fallopian tubes (ampullary region) throughout the menstrual cycle (n = 18) and from women with ectopic pregnancy (n = 6), and examined their regulation by infection with Chlamydia trachomatis in an in-vitro model. Quantitative real-time PCR analysis showed that SLPI and elafin were constitutively expressed in the Fallopian tube during the menstrual cycle but were increased in ectopic pregnancy (P < 0.05 versus early-mid luteal phase, P < 0.01 versus all phases, respectively). SLPI and elafin were immunolocalised to the Fallopian tube epithelium in biopsies from non-pregnant women and those with ectopic pregnancy. An in-vitro culture model of C. trachomatis infection of the OE-E6/E7 oviductal epithelial cell line showed that elafin mRNA expression was upregulated in response to chlamydial infection.nnnCONCLUSIONnThese data suggest that SLPI and elafin have a role in the innate immune defence of the Fallopian tube in infection and ectopic pregnancy. Their role is likely to include regulation of protease activity, wound healing and tissue remodelling.