Lynne Sykes

Vitamin D deficiency and supplementation during pregnancy

Objective  Vitamin D is essential for skeletal health and prolonged deficiency results in infantile rickets and adult osteomalacia. The aim of this study is to determine the vitamin D status in pregnancy and to evaluate the effects of daily and of single‐dose vitamin D supplementation.

The Th1:Th2 Dichotomy of Pregnancy and Preterm Labour

Pregnancy is a unique immunological state in which a balance of immune tolerance and suppression is needed to protect the fetus without compromising the mother. It has long been established that a bias from the T helper 1 cytokine profile towards the T helper 2 profile contributes towards successful pregnancy maintenance. The majority of publications that report on aberrant Th1:Th2 balance focus on early pregnancy loss and preeclampsia. Over the last few decades, there has been an increased awareness of the role of infection and inflammation in preterm labour, and the search for new biomarkers to predict preterm labour continues. In this paper, we explore the evidence for an aberrant Th1:Th2 profile associated with preterm labour. We also consider the potential for its use in screening women at high risk of preterm labour and for prophylactic therapeutic measures for the prevention of preterm labour and associated neonatal adverse outcomes.

Prevention of preterm labour via the modulation of inflammatory pathways

Pregnancy is characterized by a complex interplay of inflammatory events regulated by both the innate and acquired immune systems. Similarly, parturition can be viewed as the activation of “pro-labour” inflammatory pathways, which drive cervical ripening and myometrial activation. Premature activation of these pathways, for example, by infection, can lead to preterm labour and birth. Nuclear factor κβ is a key modulator of these pathways and functions by regulating the expression of prostaglandins, chemokines and pro-inflammatory cytokines involved in both term and preterm labour. Future design of therapeutics that target key mediators of inflammation and immune activation would therefore be a rational approach for preventing preterm labour and immune-mediated neonatal brain damage.

Changes in the Th1:Th2 cytokine bias in pregnancy and the effects of the anti-inflammatory cyclopentenone prostaglandin 15-deoxy-Δ(12,14)-prostaglandin J2.

Pregnancy is a complex immunological state in which a bias towards T helper 2 (Th2) protects the fetus. Evidence suggests that proinflammatory cytokines increase the risk of poor neonatal outcome, independently of the direct effect of preterm labour. The anti-inflammatory prostaglandin 15-deoxy-Δ12,14-Prostaglandin J2 (15dPGJ2) inhibits nuclear factor Kappa B (NF-κB) in amniocytes and myocytes in vitro and is a ligand for the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) receptor. Here we examine the Th1:Th2 cytokine bias in pregnancy and whether 15dPGJ2 could be used to inhibit the production of the proinflammatory cytokines through inhibition of NF-κB while simultaneously promoting Th2 interleukin 4 (IL-4) synthesis via CRTH2 in T helper cells. Peripheral blood mononuclear cells (PBMCs) from women at 28 weeks, term pre-labour, term labour as well as non-pregnant female controls were cultured with 15dPGJ2 or vehicle control and stimulated with phorbol myristyl acetate (PMA)/ionomycin. The percentage of CD4+ cells producing interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) in response to PMA/ionomycin was significantly reduced in pregnancy. 15dPGJ2 reduced IFN-γ and TNF-α production in stimulated T helper cells, but did not alter IL-4 production in CRTH2+ve cells. 15dPGJ2 also reduced phospho-p65 in stimulated PBMCs. In summary, 15dPGJ2 suppresses the Th1 response of PBMCs during pregnancy and active labour whilst maintaining the Th2 response suggesting a therapeutic benefit in reducing neonatal morbidity in inflammation-induced PTL.

Anti-inflammatory prostaglandins for the prevention of preterm labour

Preterm birth occurs in 10-12% of pregnancies and is the primary cause of neonatal mortality and morbidity. Tocolytic therapies have long been the focus for the prevention of preterm labour, yet they do not significantly improve neonatal outcome. A direct causal link exists between infection-induced inflammation and preterm labour. As inflammation and infection are independent risk factors for poor neonatal outcome, recent research focus has been shifted towards exploring the potential for anti-inflammatory strategies. Nuclear factor kappa B (NFκB) is a transcription factor that controls the expression of many labour-associated genes including PTGS2 (COX2), prostaglandins (PGs) and the oxytocin receptor (OXTR) as well as key inflammatory genes. Targeting the inhibition of NFκB is therefore an attractive therapeutic approach for both the prevention of preterm labour and for reducing neonatal exposure to inflammation. While PGs are considered to be pro-labour and pro-inflammatory, the cyclopentenone PG 15-deoxy-Δ(12,14)PGJ2 (15d-PGJ2) exhibits anti-inflammatory properties via the inhibition of NFκB in human amniocytes, myocytes and peripheral blood mononuclear cells in vitro. 15d-PGJ2 also delays inflammation-induced preterm labour in the mouse and significantly increases pup survival. This review examines the current understanding of inflammation in the context of labour and discusses how anti-inflammatory PGs may hold promise for the prevention of preterm labour and improved neonatal outcome.

Specific Lipopolysaccharide Serotypes Induce Differential Maternal and Neonatal Inflammatory Responses in a Murine Model of Preterm Labor.

Intrauterine inflammation is recognized as a key mediator of both normal and preterm birth but is also associated with neonatal neurological injury. Lipopolysaccharide (LPS) is often used to stimulate inflammatory pathways in animal models of infection/inflammation-induced preterm labor; however, inconsistencies in maternal and neonatal responses to LPS are frequently reported. We hypothesized that LPS serotype-specific responses may account for a portion of these inconsistencies. Four different Escherichia coli LPS serotypes (O111:B4, O55:B5, O127:B8, and O128:B12) were administered to CD1 mice via intrauterine injection at gestational day 16. Although control animals delivered at term 60 ± 15 hours postinjection (p.i.), those administered with O111:B4 delivered 7 ± 2 hours p.i., O55:B5 delivered 10 ± 3 hours p.i., O127:B8 delivered 16 ± 10 hours p.i., and O128:B12 delivered 17 ± 2 hours p.i. (means ± SD). A correlation between the onset of preterm labor and myometrial activation of the inflammatory transcription factor, activator protein 1, but not NF-κB was observed. Specific LPS serotypes induced differential activation of downstream contractile and inflammatory pathways in myometrium and neonatal pup brain. Our findings demonstrate functional disparity in inflammatory pathway activation in response to differing LPS serotypes. Selective use of LPS serotypes may represent a useful tool for targeting specific inflammatory response mechanisms in these models.

Chemoattractant receptor homologous to the T helper 2 cell (CRTH2) is not expressed in human amniocytes and myocytes.

Background 15-deoxy-Δ 12,14- Prostaglandin J2 (15dPGJ2) inhibits Nuclear factor kappa B (NF-κB) in human myocytes and amniocytes and delays inflammation induced preterm labour in the mouse. 15dPGJ2 is a ligand for the Chemoattractant Receptor Homologous to the T helper 2 cell (CRTH2), a G protein-coupled receptor, present on a subset of T helper 2 (Th2) cells, eosinophils and basophils. It is the second receptor for Prostaglandin D2, whose activation leads to chemotaxis and the production of Th2-type interleukins. The cellular distribution of CRTH2 in non-immune cells has not been extensively researched, and its identification at the protein level has been limited by the lack of specific antibodies. In this study we explored the possibility that CRTH2 plays a role in 15dPGJ2-mediated inhibition of NF-κB and would therefore represent a novel small molecule therapeutic target for the prevention of inflammation induced preterm labour. Methods The effect of a small molecule CRTH2 agonist on NF-κB activity in human cultured amniocytes and myocytes was assessed by detection of p65 and phospho-p65 by immunoblot. Endogenous CRTH2 expression in amniocytes, myocytes and peripheral blood mononuclear cells (PBMCs) was examined by PCR, western analysis and flow cytometry, with amniocytes and myocytes transfected with CRTH2 acting as a positive control in flow cytometry studies. Results The CRTH2 agonist had no effect on NF-κB activity in amniocytes and myocytes. Although CRTH2 mRNA was detected in amniocytes and myocytes, CRTH2 was not detectable at the protein level, as demonstrated by western analysis and flow cytometry. 15dPGJ2 inhibited phospho-65 in PBMC’S, however the CRTH2 antagonist was not able to attenuate this effect. In conclusion, CRTH2 is not expressed on human amniocytes or myocytes and plays no role in the mechanism of 15dPGJ2-mediated inhibition of NF-κB.

Time interval from elective removal of cervical cerclage to onset of spontaneous labour

OBJECTIVE To determine the time interval between elective removal of a cervical cerclage to the onset of spontaneous labour in women who had either a history- or ultrasound-indicated cervical cerclage. STUDY DESIGN A retrospective cohort study of women with a singleton pregnancy that had either a modified Shirodkar or McDonald cervical cerclage inserted were evaluated for the time interval between elective cerclage removal and onset of spontaneous labour and also spontaneous labour with 72 h of cervical cerclage removal. RESULTS Two hundred and sixty-nine singleton pregnancies with either a modified Shirodkar or McDonald cervical cerclage were analysed. The mean gestational age at cerclage removal was 36.7 ± 1.10 weeks and gestational age at spontaneous labour was 39.0 ± 1.94 weeks (mean ± SD). The median interval between cerclage removal and spontaneous labour was 14 days. Only 18% of women laboured spontaneously within 72 h. Women with ultrasound-indicated cerclage were more likely to deliver within 72 h, compared with women with a history-indicated cervical cerclage (odds ratio, 3.68; 95% confidence interval, 1.31-10.85, p=0.01). CONCLUSION Independent of the indication or technique used for cervical cerclage, the rate of early spontaneous labour following elective removal of cervical cerclage is sufficiently low to justify outpatient management.

Sulfasalazine augments a pro-inflammatory response in interleukin-1β-stimulated amniocytes and myocytes.

Preterm birth occurs in 10% of pregnancies and is a major cause of neonatal morbidity and mortality. The majority of cases of early preterm labour are associated with infection/inflammation, which places the fetal central nervous system at risk. Targeting immune activation is therefore an appealing therapeutic strategy for the prevention of preterm labour and neonatal brain injury. The expression of many labour‐associated and inflammatory‐response genes is controlled by the transcription factors nuclear factor‐κB (NF‐κB) and activator protein‐1 (AP‐1), which makes them therapeutic targets of interest. Sulfasalazine (SASP) has been shown to inhibit NF‐κB and reduce lipopolysaccharide‐induced cytokine concentrations in fetal membrane explants and reduce the rate of Escherichia coli‐induced preterm labour in mice. Its effects upon AP‐1 in the context of pregnancy are unknown. In this study the effect of SASP on interleukin‐1β (IL‐1β) ‐induced NF‐κB and AP‐1 activity, cytokine production and cyclo‐oxygenase‐2 (COX‐2) expression was examined in amniocytes and myocytes. A supra‐therapeutic concentration (5 mm) was required to inhibit IL‐1β‐induced NF‐κB (P < 0·0001) in amniocytes and IL‐1β‐induced NF‐κB (P < 0·01), AP‐1 (P < 0·01) and COX‐2 (P < 0·05) in myocytes. Despite inhibiting IL‐1β‐induced cytokines, a basal increase in IL‐6 (P < 0·01), IL‐8 (P < 0·0001) and tumour necrosis factor‐α (TNF‐α) (P < 0·001) was seen with 5 mm SASP in amniocytes, and significant cytotoxic effects were seen in myocytes. The therapeutic concentration of 0·015 mm had no inhibitory effects on pro‐inflammatory mediators, but led to an augmented response to IL‐1β‐induced IL‐6 (P < 0·01), IL‐8 (P < 0·05) and TNF‐α (P < 0·05) in amniocytes and IL‐8 (P < 0·05) in myocytes. SASP is therefore an unlikely therapeutic candidate for the prevention of inflammation‐induced preterm labour.

The CRTH2 agonist Pyl A prevents lipopolysaccharide-induced fetal death but induces preterm labour.

We have previously demonstrated that the anti‐inflammatory prostaglandin 15‐deoxy‐Δ 12,14‐prostaglandin J2 (15dPGJ2) delays inflammation‐induced preterm labour in the mouse and improves pup survival through the inhibition of nuclear factor‐κB (NF‐κB) by a mechanism yet to be elucidated. 15dPGJ2 is an agonist of the second prostaglandin D2 receptor, chemoattractant receptor homologous to the T helper 2 cell (CRTH2). In human T helper cells CRTH2 agonists induce the production of the anti‐inflammatory interleukins IL‐10 and IL‐4. We hypothesized that CRTH2 is involved in the protective effect of 15dPGJ2 in inflammation‐induced preterm labour in the murine model. We therefore studied the effects of a specific small molecule CRTH2 agonist on preterm labour and pup survival. An intrauterine injection of lipopolysaccharide (LPS) was administered to CD1 mice at embryonic day 16, ± CRTH2 agonist/vehicle controls. Mice were killed at 4.5 hr to assess fetal wellbeing and to harvest myometrium and pup brain for analysis of NF‐κB, and T helper type 1/2 interleukins. To examine the effects of the CRTH2 agonist on LPS‐induced preterm labour, mice were allowed to labour spontaneously. Direct effects of the CRTH2 agonist on uterine contractility were examined ex vivo on contracting myometrial strips. The CRTH2 agonist increased fetal survival from 20 to 100% in LPS‐treated mice, and inhibited circular muscle contractility ex vivo. However, it augmented LPS‐induced labour and significantly increased myometrial NF‐κB, IL‐1β, KC‐GRO, interferon‐γ and tumour necrosis factor‐α. This suggests that the action of 15dPGJ2 is not via CRTH2 and therefore small molecule CRTH2 agonists are not likely to be beneficial for the prevention of inflammation‐induced preterm labour.