CCR2 mediates the adverse effects of LPS in the pregnant mouse.

In our earlier work, we found that intrauterine (i.u.) and intraperitoneal (i.p.) injection of LPS (10-µg serotype 0111:B4) induced preterm labor (PTL) with high pup mortality, marked systemic inflammatory response and hypotension. Here, we used both i.u. and i.p. LPS models in pregnant wild-type (wt) and CCR2 knockout (CCR2-/-) mice on E16 to investigate the role played by the CCL2/CCR2 system in the response to LPS. Basally, lower numbers of monocytes and macrophages and higher numbers of neutrophils were found in the myometrium, placenta, and blood of CCR2-/- vs. wt mice. After i.u. LPS, parturition occurred at 14 h in both groups of mice. At 7 h post-injection, 70% of wt pups were dead vs. 10% of CCR2-/- pups, but at delivery 100% of wt and 90% of CCR2-/- pups were dead. Myometrial and placental monocytes and macrophages were generally lower in CCR2-/- mice, but this was less consistent in the circulation, lung, and liver. At 7 h post-LPS, myometrial ERK activation was greater and JNK and p65 lower and the mRNA levels of chemokines were higher and of inflammatory cytokines lower in CCR2-/- vs. wt mice. Pup brain and placental inflammation were similar. Using the IP LPS model, we found that all measures of arterial pressure increased in CCR2-/- but declined in wt mice. These data suggest that the CCL2/CCR2 system plays a critical role in the cardiovascular response to LPS and contributes to pup death but does not influence the onset of inflammation-induced PTL.

The Local and Systemic Immune Response to Intrauterine LPS in the Prepartum Mouse.

Inflammation plays a key role in human term and preterm labor (PTL). Intrauterine LPS has been widely used to model inflammation-induced complications of pregnancy, including PTL. It has been shown to induce an intense myometrial inflammatory cell infiltration, but the role of LPS-induced inflammatory cell activation in labor onset and fetal demise is unclear. We investigated this using a mouse model of PTL, where an intrauterine injection of 10 µg of LPS (serotype 0111:B4) was given at E16 of CD1 mouse pregnancy. This dose induced PTL at an average of 12.7 h postinjection in association with 85% fetal demise. Flow cytometry showed that LPS induced a dramatic systemic inflammatory response provoking a rapid and marked leucocyte infiltration into the maternal lung and liver in association with increased cytokine levels. Although there was acute placental inflammatory gene expression, there was no corresponding increase in fetal brain inflammatory gene expression until after fetal demise. There was marked myometrial activation of NFκB and MAPK/AP-1 systems in association with increased chemokine and cytokine levels, both of which peaked with the onset of parturition. Myometrial macrophage and neutrophil numbers were greater in the LPS-injected mice with labor onset only; prior to labor, myometrial neutrophils and monocytes numbers were greater in PBS-injected mice, but this was not associated with an earlier onset of labor. These data suggest that intrauterine LPS induces parturition directly, independent of myometrial inflammatory cell infiltration, and that fetal demise occurs without fetal inflammation. Intrauterine LPS provokes a marked local and systemic inflammatory response but with limited inflammatory cell infiltration into the myometrium or placenta.

The Arabin pessary to prevent preterm birth in women with a twin pregnancy and a short cervix: the STOPPIT 2 RCT.

BACKGROUND: Preterm birth is common in twins and accounts for significant mortality and morbidity. There are no effective preventative treatments. Some studies have suggested that, in twin pregnancy complicated by a short cervix, the Arabin pessary, which fits around the cervix and can be inserted as an outpatient procedure, reduces preterm birth and prevents neonatal morbidity. OBJECTIVE: STOPPIT 2 aimed to evaluate the clinical utility of the Arabin cervical pessary in preventing preterm birth in women with a twin pregnancy and a short cervix. DESIGN: STOPPIT 2 was a pragmatic, open label, multicentre randomised controlled trial with two treatment group - the Arabin pessary plus standard care (intervention) and standard care alone (control). Participants were initially recruited into the screening phase of the study, when cervical length was measured. Women with a measured cervical length of ≤ 35 mm were then recruited into the treatment phase of the study. An economic evaluation considered cost-effectiveness and a qualitative substudy explored the experiences of participants and clinicians. SETTING: Antenatal clinics in the UK and elsewhere in Europe. PARTICIPANTS: Women with twin pregnancy at < 21 weeks' gestation with known chorionicity and gestation established by scan at ≤ 16 weeks' gestation. INTERVENTIONS: Ultrasound scan to establish cervical length. Women with a cervical length of ≤ 35 mm at 18+ 0-20+ 6 weeks' gestation were randomised to standard care or Arabin pessary plus standard care. Randomisation was performed by computer and accessed through a web-based browser. MAIN OUTCOME MEASURES: Obstetric - all births before 34+ 0 weeks' gestation following the spontaneous onset of labour; and neonatal - composite of adverse outcomes, including stillbirth or neonatal death, periventricular leukomalacia, early respiratory morbidity, intraventricular haemorrhage, necrotising enterocolitis or proven sepsis, all measured up to 28 days after the expected date of delivery. RESULTS: A total of 2228 participants were recruited to the screening phase, of whom 2170 received a scan and 503 were randomised: 250 to Arabin pessary and 253 to standard care alone. The rate of the primary obstetric outcome was 18.4% (46/250) in the intervention group and 20.6% (52/253) in the control group (adjusted odds ratio 0.87, 95% confidence interval 0.55 to 1.38; p = 0.54). The rate of the primary neonatal outcome was 13.4% (67/500) and 15.0% (76/506) in the intervention group and control group, respectively (adjusted odds ratio 0.86, 95% confidence interval 0.54 to 1.36; p = 0.52). The pessary was largely well tolerated and clinicians found insertion and removal 'easy' or 'fairly easy' in the majority of instances. The simple costs analysis showed that pessary treatment is no more costly than standard care. LIMITATIONS: There was the possibility of a type II error around smaller than anticipated benefit. CONCLUSIONS: In this study, the Arabin pessary did not reduce preterm birth or adverse neonatal outcomes in women with a twin pregnancy and a short cervix. The pessary either is ineffective at reducing preterm birth or has an effect size of < 0.4. FUTURE WORK: Women with twin pregnancy remain at risk of preterm birth; work is required to find treatments for this. TRIAL REGISTRATION: Current Controlled Trials ISRCTN98835694 and ClinicalTrials.gov NCT02235181. FUNDING: This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 25, No. 44. See the NIHR Journals Library website for further project information.

Women who are pregnant with twins have a much higher risk of going into labour early and having an early (preterm) birth than women who are pregnant with only one baby. For this reason, babies who are twins are much more likely to die or to have serious health complications in the first months of life. Although we know that women with twin pregnancy are at risk, there are no treatments that are recommended to prevent early births. Some studies have suggested that the Arabin pessary can help. The Arabin pessary is a silicone ring that fits around the cervix (neck of the womb). The pessary can be put in place in a clinic without any need for an anaesthetic. Some studies have suggested that the Arabin pessary helps and others have suggested that it does not. It appears to be most helpful when the cervix (neck of the womb) is already shortening. Shortening of the neck of the womb is a sign that early birth is even more likely. We asked women with twin pregnancy to take part in STOPPIT 2. Women who agreed had an ultrasound scan of the neck of the womb, which measured its length. Those with a short cervix were randomised to be offered the Arabin pessary (in addition to standard care) or standard care alone. This allocation was carried out 'at random' by a computer. We followed women up until the end of their pregnancy and collected information on the babies' health after birth. We found that the Arabin pessary did not reduce the risk of an early birth; nor did it reduce the risk of health complications for the baby. We conclude that the Arabin pessary should not be used for this purpose.

Open randomised trial of the (Arabin) pessary to prevent preterm birth in twin pregnancy with health economics and acceptability: STOPPIT-2-a study protocol.

INTRODUCTION: The STOPPIT-2 study aims to determine the clinical utility of the Arabin cervical pessary in preventing preterm birth in women with a twin pregnancy and a short cervix, about which there is current uncertainty. STOPPIT-2 will resolve uncertainty around effectiveness for women with a twin pregnancy and a cervical length of 35 mm or less, define adverse effects, ascertain acceptability and estimate National Health Service costs and savings. METHODS: STOPPIT-2 is a pragmatic multicentre open-label randomised controlled trial. Consenting women with twin pregnancy will have an transvaginal ultrasound scan of their cervical length performed between 18+0 and 20+6 weeks' gestation by an accredited practitioner: women with a cervical length of ≤35 mm will be eligible for inclusion in the treatment phase of the study. The intervention by the insertion of the Arabin cervical pessary will be compared with standard treatment (no pessary).The primary outcomes are (obstetric) spontaneous onset of labour for the mother leading to delivery before 34 weeks' gestation and (neonatal) a composite of specific adverse outcomes or death occurring up to the end of the first 4 weeks after the estimated date of delivery to either or both babies.We plan to recruit 500 women in the treatment phase of the study. Assuming a treatment effect of 0.6, and background rates of 35% and 18%, respectively, for each of the primary outcomes, our study has 85% power to detect a difference between the intervention and the control groups. ANALYSIS: Data will be analysed on the intention-to-treat principle. ETHICS: STOPPIT-2 was approved by the South East Scotland Ethics Committee 02 on 29 August 2014, reference number 14/SS/1031 IRAS ID 159610. DISSEMINATION: Peer reviewed journals, presentations at national and international scientific meetings. TRIAL REGISTRATION NUMBER: ISRCTN98835694 and NCT02235181.

Does progesterone prophylaxis to prevent preterm labour improve outcome? A randomised double-blind placebo-controlled trial (OPPTIMUM).

BACKGROUND: Progesterone prophylaxis is widely used to prevent preterm birth but is not licensed and there is little information on long-term outcome. OBJECTIVE: To determine the effect of progesterone prophylaxis in women at high risk of preterm birth on obstetric, neonatal and childhood outcomes. DESIGN: Double-blind, randomised placebo-controlled trial. SETTING: Obstetric units in the UK and Europe between February 2009 and April 2013. PARTICIPANTS: Women with a singleton pregnancy who are at high risk of preterm birth because of either a positive fibronectin test or a negative fibronectin test, and either previous spontaneous birth at ≤ 34 weeks+0 of gestation or a cervical length of ≤ 25 mm. INTERVENTIONS: Fibronectin test at 18+0 to 23+0 weeks of pregnancy to determine risk of preterm birth. Eligible women were allocated (using a web-based randomisation portal) to 200 mg of progesterone or placebo, taken vaginally daily from 22+0 to 24+0 until 34+0 weeks' gestation. Participants, caregivers and those assessing the outcomes were blinded to group assignment until data collection was complete. MAIN OUTCOME MEASURES: There were three primary outcomes, as follows: (1) obstetric - fetal death or delivery before 34+0 weeks' gestation; (2) neonatal - a composite of death, brain injury on ultrasound scan (according to specific criteria in the protocol) and bronchopulmonary dysplasia; and (3) childhood - the Bayley-III cognitive composite score at 22-26 months of age. RESULTS: In total, 96 out of 600 (16%) women in the progesterone group and 108 out of 597 (18%) women in the placebo group had the primary obstetric outcome [odds ratio (OR) 0.86, 95% confidence interval (CI) 0.61 to 1.22]. Forty-six out of 589 (8%) babies of women in the progesterone group and 62 out of 587 (11%) babies of women in the placebo group experienced the primary neonatal outcome [OR 0.72, 95% CI 0.44 to 1.17]. The mean Bayley-III cognitive composite score of the children at 2 years of age was 97.3 points [standard deviation (SD) 17.9 points; n = 430] in the progesterone group and 97.7 points (SD 17.5 points; n = 439) in the placebo group (difference in means -0.48, 95% CI -2.77 to 1.81). LIMITATIONS: Overall compliance with the intervention was 69%. HARMS: There were no major harms, although there was a trend of more deaths from trial entry to 2 years in the progesterone group (20/600) than in the placebo group (16/598) (OR 1.26, 95% CI 0.65 to 2.42). CONCLUSIONS: In this study, progesterone had no significant beneficial or harmful effects on the primary obstetric, neonatal or childhood outcomes.The OPPTIMUM trial is now complete. We intend to participate in a comprehensive individual patient-level data meta-analysis examining women with a singleton pregnancy with a variety of risk factors for preterm birth. TRIAL REGISTRATION: Current Controlled Trials ISRCTN14568373. FUNDING: This trial was funded by the Medical Research Council (MRC) and managed by the National Institute for Health Research (NIHR) on behalf of the MRC-NIHR partnership.

Prostaglandin inhibitors in preterm labour.

Prematurity accounts for the majority of neonatal morbidity and mortality in the developed world. The process of labour resembles inflammation, with prostaglandin and cytokine production both before and during labour. Anti-inflammatory drugs therefore have the potential to prevent preterm delivery. Indomethacin is the only tocolytic drug proven to delay delivery beyond 37 weeks and to reduce the incidence of low birth weight (<2500 g). There are, however, fetal side-effects such as ductal constriction and impaired renal function associated with its use. It is the type 2 isoform of cyclo-oxygenase (COX-2), which is important for the production of prostaglandins within intrauterine tissues and that up-regulation of COX-2 is associated with labour. Although indomethacin is currently the most common non-steroidal anti-inflammatory drug (NSAID) used in the treatment of preterm labour, it was hoped that COX-2-selective drugs, used as tocolytics, would target COX-2 activity and potentially spare COX-1-specific fetal side-effects. Experience with sulindac and nimesulide has been linked with both constriction of the ductus arteriosus and oligohydramnios. It is unclear whether this is due to COX-2-dependent side-effects, or due to accumulation of drug in the fetal circulation leading to levels that would cause COX-1 inhibition. Currently, the use of COX-2-selective drugs should therefore be confined to randomized controlled trials.

Human labour is associated with a decline in myometrial chemokine receptor expression: the role of prostaglandins, oxytocin and cytokines.

PROBLEM: Human labour is an inflammatory process with a heavy infiltration of immune cells into the myometrium and cervix induced by local chemokine production. Myometrial cells also express chemokine receptors, but there is little information about their behaviour or function during pregnancy and labour. METHOD OF STUDY: We studied the behaviour of the receptors (CCR2, CXCR1 and CXCR2) for the CCL2 and CXCL8 in human myometrium, because both have been shown to be important in labour. RESULTS: We found that there was a significant decline in the mRNA expression of all three receptors in the upper segment and a similar trend in the lower segment with the onset of term labour (TL). Chemokine receptor mRNA expression was increased by stretch, reduced by oxytocin and PGF(2α) acting via phospholipase C (PLC). CXCR2 declined with exposure to CXCL8, consistent with the negative relationship observed in labouring myometrial tissue. The mRNA changes were confirmed by western analysis and flow cytometry. CONCLUSION: These data show that myometrial chemokine receptor expression is reduced with the onset of term labour probably in response to the increased activity of chemokines, oxytocin and PGF(2α) .

Stretch and inflammatory cytokines drive myometrial chemokine expression via NF-κB activation.

Both human preterm labor (PTL) and term labor are consistently associated with a chemokine-induced inflammatory infiltration of the myometrium. However, what regulates myometrial chemokine expression and whether the increase in expression precedes the onset of labor, and so may have a role in its causation, or occurs after, and is simply a consequence of labor, is uncertain. Therefore, we assessed 1) chemokine expression in nonlaboring and laboring myometrial samples obtained at and before term and 2) the factors that regulate myometrial chemokine expression. We found that term labor was characterized by an increase in CXCL8 and CCL2 in both upper and lower segments, whereas PTL was associated with a distinct pattern of chemokine expression, with increases in CCL5, CXCL5, and CCL20 in the lower segment myometrium only. Further, we found that chemokine expression in myometrial cell cultures was increased by stretch and inflammatory cytokines and reduced by prostglandins and oxytocin and that the primary mediator of stretch and cytokine effects was nuclear factor κB (NF-κB) and to a lesser extent MAPK. These data show that PTL appears to be associated with a distinct pattern of chemokine expression, that stretch and cytokines both drive myometrial chemokine expression primarily via activation of NF-κB. These data suggest that the modulation of NF-κB activity may be of potential benefit in the management of PTL.