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Increasing evidence highlights that term labour is an inflammatory event associated with increased production of pro-­‐inflammatory mediators and leukocyte influx into the intrauterine tissues. Preterm labour (PTL), defined as labour before 37 weeks gestation, is a major clinical problem, and preterm birth is the leading cause of neonatal mortality and morbidity worldwide. The causes of PTL are poorly understood, but intrauterine infection and inflammation have been shown to be important factors. Therefore, there is growing interest in the hypothesis that preterm labour may occur as a result of the premature activation of the inflammatory pathways normally initiated with labour at term, either idiopathically, or in response to a pathological intrauterine infection. The aim of this thesis was to use a mouse model of infection-­induced PTL to: characterise the local inflammatory and immune response to an intrauterine infection; investigate the potential of anti‐inflammatory agents to delay delivery of pups and to improve their survival; and to investigate the role of specific immune cell populations in infection-­induced preterm labour. To characterise the inflammatory and immune response to intrauterine infection, CD1 mice received an intrauterine injection of PBS vehicle or increasing doses of bacterial-­derived lipopolysaccharide (LPS) on day 17 of gestation. Time to delivery, and the number of live born pups were determined. Intrauterine administration of increasing doses of LPS dose-­dependently induced preterm labour and reduced the proportion of live born pups. Analysis of tissues harvested six hours post-­surgery demonstrated that in response to intrauterine LPS administration, there was increased expression of inflammatory cytokines and chemokines within the utero-­placental tissues, amniotic fluid and maternal serum; and an influx of neutrophils into the decidua, compared to mice receiving PBS. Given these results, the potential of anti‐inflammatory agents to delay LPS-­induced preterm delivery and improve pup survival was then investigated using the same mouse model. Prior to intrauterine LPS administration, mice were pre-­‐treated with epi-­lipoxin, BML-­111 (a stable lipoxin analogue), or IL-­10. Time to delivery was unaffected by pre-­treatment with the anti-­inflammatory agents, however epi-­lipoxin significantly increased the proportion of live born pups in mice delivering preterm, compared to mice receiving only LPS. To further investigate the role of immune cells in infection-­induced PTL, antibody-­based depletion strategies were used to selectively deplete specific immune cell populations to determine whether they played a causative role in LPS­‐induced preterm delivery. Despite successful depletion of macrophages or neutrophils, it was not found to significantly affect LPS-­induced preterm delivery, suggesting these immune cells are not required for the induction of preterm labour in response to intrauterine infection. However, it is likely that they contribute to the intrauterine inflammatory response as depletion resulted in altered inflammatory signalling within the intrauterine tissues. Collectively, this work has demonstrated that the presence of intrauterine bacterial LPS, as a surrogate model of infection, induces a robust inflammatory and immune response within the utero‐placental tissues that involves the increased production of inflammatory mediators and the influx of immune cells into the decidua, which ultimately leads to PTL. Whilst the anti-­inflammatory treatments tested here did not delay LPS-­induced PTL, epi-­lipoxin attenuated LPS-­induced mortality in pups born preterm, suggesting this anti‐inflammatory agent may be useful in protecting the fetus from the adverse effects of infection-­induced preterm birth. Using models such as the one described here, are vital to improving our understanding of the events regulating the induction of PTL and will ultimately aid the search for novel therapeutic options for the treatment of PTL.

Although inflammation is a crucial mechanism in response to injury and pathogen clearance, inappropriate or excessive induction of the inflammatory response in pregnancy can cause initiation of the labour cascade and subsequent preterm delivery. Host Defence Peptides (HDPs) have important anti-microbial properties but are also implicated as multifunctional modulators of immunity and infection. They are predominantly secreted by mucosal epithelial cells and released by leukocytes. The specific HDPs that are the focus of this thesis are Human beta-defensin 3 (hBD3) and Human Cathelicidin (hCAP-18/LL-37). The immunomodulatory effect of HDPs in reproductive tissues in response to infection/inflammation has not been well studied. In a pregnant state, the hypothesis of this thesis is that HDPs have a dual role in preventing ascending infection, but also preventing an exacerbated inflammatory response that can cause preterm birth by initiation of the labour cascade. To explore this I determine whether bacterial stimuli can regulate HDPs expression in pregnancy tissues. I also explore what interactions HDPs have on the production/induction of important cytokines that are vital to the inflammatory response. With the aid of HDP knockout mice, the role of these peptides in infection/inflammation and continuation of pregnancy is investigated in a mouse-model of induced preterm-labour. To understand how ascending infection might be controlled by HDPs in pregnancy, I explore how HDPs regulate commensal and pathogenic bacteria. This is achieved by interrogating the maternal microbiome at mucosal sites in HDP knockout animals, utilising the bacterial 16S rRNA gene and next generation sequencing. Results Placental explants respond to Lipopolysaccharide (LPS) challenge by increasing production of pro-inflammatory cytokines. LL-37 but not hBD3 peptide was able to modulate this inflammation by inhibiting the release of these pro-inflammatory cytokines. To establish whether HDPs are critical in the continuation of pregnancy I use a LPS induced mouse–model of preterm labour in animals lacking the genes for the HDPs, Defb14 (Defb14-/-), or Camp (Camp-/-). Intrauterine injection of LPS induced preterm labour in wildtype mice. However, the Defb14-/- and Camp-/- mice do not have an increased rate of preterm labour. Key inflammatory mediators are increased in response to LPS-induced PTL. Camp-/- animals have a similar inflammatory response to wildtype mice when given LPS during pregnancy. To understand how ascending infection might be controlled by HDPs, I interrogated the maternal microbiome at mucosal sites in HDP knockout animals, utilising the bacterial 16S rRNA gene. I established a workflow for 16S rRNA gene sequencing on next-generation sequencing platforms and a bioinformatic pipeline for data analysis. Using this approach I was able to show the mucosal microbiome of Camp-/- animals were significantly different to that of wildtype controls, showing increased diversity in the microbes present. In murine pregnancy, there were very little global cumulative or progressive shifts in bacteria, with the exception of Candidatus arthromitus, which significantly increases with gestation compared to non-pregnancy This thesis has demonstrated that Host Defence Peptides are expressed in pregnancy tissues and have anti-inflammatory properties in response to bacterial stimuli. It is not clear whether the HDPs, hBD3 and LL-37 are fundamental to the immune defence in pregnancy by preventing excessive inflammation, Although, I have shown LL-37 may have a role in modulation of the maternal microbiota.

Physiological inflammation occurs in the female reproductive tract, but pathological inflammation is implicated in reproductive pathologies such as preterm labour and endometrial cancer. Preterm labour (PTL, before 37 weeks of gestation) is the leading cause of preterm birth, neonatal mortality and perinatal morbidities. Endometrial cancer is the commonest gynaecological cancer, and its pathogenesis is characterised by chronic inflammation. The overall aims of this thesis were (i) to develop an in vitro model of myometrial-monocyte interactions to replicate the events occurring in the myometrium in preterm labour (ii) to determine the effects of potential therapeutics such as lipoxins, IL-10 and progesterone, on inflammation, and (iii) to characterise the lipoxin pathway in endometrial adenocarcinoma. Macrophages infiltrate the pregnant myometrium during labour; however the role of these cells is unclear. A myometrial-monocyte coculture model was developed either using non-pregnant primary myometrial smooth muscle cells (UtSMCs), or immortalised pregnant human myometrial cells (PHM1-41), with primary monocytes from term (38-41 weeks of gestation), non-labouring pregnant women. Cultures were stimulated with the toll-like receptor 4 agonist lipopolysaccharide (LPS), in the presence or absence of each of lipoxins, IL-10 and progesterone. A significant and synergistic increase in IL-6 and IL-8 secretion was found in the UtSMC/monocyte coculture after stimulation with LPS for 24 hours, compared to LPS-treated UtSMCs, or monocytes alone, but the increase in IL-6 and IL-8 secretion was not inhibited by lipoxin, epi-lipoxin or benzo-lipoxin. The PHM1-41/monocyte coculture both alone and in response to LPS treatment generated significantly increased IL-6 and IL-8 secretion, compared to vehicle treatment in the coculture and compared to the culture of either cell type alone. IL-1β and TNFα secretion were only detected from the PHM1/monocyte coculture, and monocytes alone. Use of a TNFα blocking antibody partially suppressed LPS-induced IL-6 and IL-8 secretion in the coculture. Coculture of PHM1/monocytes resulted in increased secretion of multiple mediators including pro-inflammatory cytokines, chemokines and growth factors compared to culture of either PHM1 cells or primary monocytes separately, both with vehicle and with LPS. IL-10 inhibited LPS-induced IL-6 and IL-8 secretion from the coculture, as did progesterone, which also inhibited GM-CSF, MCP-1 and CXCL5 secretion. Myocyte contraction, measured by PHM1-41 cells embedded in collagen was increased by primary monocyte treatment. This suggests that not only do infiltrating monocytes increase myometrial inflammation but they can induce myometrial smooth muscle contraction. In endometrial adenocarcinoma, the lipoxin synthesis enzymes, ALOX-5 and -15 and FPR2 mRNA expression were upregulated compared to proliferative phase endometrium, with FPR2, a reported lipoxin receptor, immunolocalised in endometrial adenocarcinoma tissue. Additionally, TNFα treatment of Ishikawa endometrial adenocarcinoma cells increased FPR2 mRNA expression, and upregulation of FPR2 mRNA also occurred in xenograft tumours from CD1 nude mice, compared to the Ishikawa cells from which they originated. These findings highlight FPR2 expression in endometrial adenocarcinoma, and suggest this receptor could mediate inflammatory signals, and lipoxins could be produced by ALOX-5 and ALOX-15. Collectively, these data describe the novel effects of monocytes in the regulation of myometrial smooth muscle cell inflammation, and demonstrate a mechanism by which myometrial inflammation during both term and preterm labour is triggered by infiltrating macrophages. This myocyte/monocyte inflammation is regulated in part by TNFα, and can be suppressed by both IL-10 and progesterone co-treatment. Components of the lipoxin pathway are present in endometrial adenocarcinoma, but their role in regulation of inflammation is still to be elucidated. Future research to clarify the processes, by which leukocyte recruitment is regulated at labour and the role of monocyte/macrophages in altering myocyte properties, could help to elucidate the mechanisms coupling inflammation to labour and provide more appropriate targets for the treatment of PTL.

Studies indicate an association between both term labour (TL) and preterm labour (PTL) and the presence of uterine inflammatory cytokines and leukocyte infiltration. We hypothesized that peripheral leukocytes are recruited to uterine tissues by locally produced cytokines where they contribute to the initiation of TL and PTL. The cytokine expression profile was analyzed using an in vivo mouse model of gestation and two PTL models (Lipopolysaccharide- and RU486-induced). Myometrial neutrophil and macrophage infiltration was also studied. My results demonstrate that macrophage infiltration precedes neutrophil infiltration during late gestation and that both leukocyte subsets increase during PTL and further increase post partum. These changes in leukocyte numbers are associated with significant changes in multiple myometrial cytokines with TL and RU486-induced PTL showing similar cytokine profiles. Importantly, post partum involution, the process by which the uterus completes the reproductive cycle and returns to its pre-pregnant state, appears similar in all three models.

Studies indicate an association between both term labour (TL) and preterm labour (PTL) and the presence of uterine inflammatory cytokines and leukocyte infiltration. We hypothesized that peripheral leukocytes are recruited to uterine tissues by locally produced cytokines where they contribute to the initiation of TL and PTL. The cytokine expression profile was analyzed using an in vivo mouse model of gestation and two PTL models (Lipopolysaccharide- and RU486-induced). Myometrial neutrophil and macrophage infiltration was also studied. My results demonstrate that macrophage infiltration precedes neutrophil infiltration during late gestation and that both leukocyte subsets increase during PTL and further increase post partum. These changes in leukocyte numbers are associated with significant changes in multiple myometrial cytokines with TL and RU486-induced PTL showing similar cytokine profiles. Importantly, post partum involution, the process by which the uterus completes the reproductive cycle and returns to its pre-pregnant state, appears similar in all three models.

Preterm labour (PTL) is defined as labour before 37 completed weeks of gestation. Despite advances in medical research, PTL remains a major clinical problem. Preterm birth (PTB) rates range from approximately 5-18% worldwide. Importantly, PTB is the leading cause of childhood morbidity and mortality. PTL is difficult to predict and the aetiology is poorly understood but infection and inflammation are believed to be major factors. It has been suggested that the presence of intrauterine infection or inflammation may initiate the pathological, preterm activation of the inflammatory cascade associated with term labour. Therefore, PTL therapeutics should aim to inhibit these inflammatory pathways. Statins, 5-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, are potent inhibitors of cholesterol biosynthesis, which act on the mevalonate pathway. In addition to their lipid-lowering effects, statins also have anti-inflammatory and anti-contraction properties. The hypothesis of this thesis was that statins will prevent PTB by reducing inflammation. The aims of this thesis were firstly to investigate the effect of the statins, simvastatin and pravastatin, on inflammation and contractility in a pregnant human myometrial cell line. Secondly, to determine whether simvastatin and/or pravastatin can prevent PTB or improve neonatal outcome in a lipopolysaccharide (LPS)-induced mouse model of PTB. Myometrial cells were either co-treated with LPS and simvastatin/pravastatin, pretreated with simvastatin/pravastatin or treated with simvastatin/pravastatin post-LPS stimulation. The effect of statin treatment on the mRNA expression and the release of inflammatory mediators was then investigated. Simvastatin treatment reduced LPS-induced inflammation by both lowering the expression of pro-inflammatory mediators and increasing the expression of anti-inflammatory mediators. Pravastatin treatment did not alter the expression of inflammatory mediators following LPS stimulation. The effect of simvastatin on the contraction of myometrial cells was investigated by embedding the cells in rat tail collagen to form gels. As these are smooth muscle cells, basal contraction was observed causing the gel size to reduce. When LPS was introduced, this caused the gels to contract further than the vehicle treated gels. Simvastatin attenuated the contraction of the myometrial cells, both alone and in the presence of LPS. These effects were reversed by the addition of mevalonate pathway metabolites, mevalonate and geranylgeranyl pyrophosphate (GG-PP) but not by farnesyl pyrophosphate (F-PP). Simvastatin also lowered levels of phosphorylated myosin light chain (pMLC) in the myometrial cells, which is essential for smooth muscle contraction. Again, this effect was abolished by mevalonate and GG-PP but not F-PP. It is hypothesised that simvastatin attenuated myometrial cell contraction by inhibiting Rho isoprenylation by GG-PP, preventing Rho-associated kinase (ROCK) activation, which then prevented the phosphorylation of MLC. A mouse model of intrauterine LPS-induced PTB was utilised to investigate the effect of statin treatment on PTB and fetal survival. Mice received an intraperitoneal injection of pravastatin (10μg) or simvastatin (20μg or 40μg) on gestational day (D)16. This was followed by ultrasound-guided intrauterine injection of LPS (1μg) on D17 and another pravastatin/simvastatin treatment two hours later. When mice were treated with LPS, 77.8% of mice delivered preterm. When mice received LPS and 20μg simvastatin, 50% delivered preterm. However, when mice were treated with LPS and 40μg simvastatin, 40% delivered preterm, more pups were born alive and uterine pro-inflammatory mRNA expression was downregulated. Conversely, pravastatin did not prevent PTB or improve the percentage of live born pups. In summary, simvastatin treatment exerted anti-inflammatory and anti-contraction effects on human myometrial cells in vitro. The anti-contractile properties were likely due to the inhibition of the Rho/ROCK pathway. Furthermore, in our LPS-induced mouse model of PTB, fewer mice delivered preterm with simvastatin treatment, simvastatin attenuated LPS-induced pup mortality and reduced uterine inflammatory gene expression. These results suggest that statin therapy may be a novel treatment for PTL.

Thesis (M. (Nursing)) -- University of Limpopo, 2022 / Background Despite all the interventions that have been developed previously preterm labour remain to be the leading cause of perinatal morbidity and mortality worldwide. Preterm labour occurs before 37 weeks of gestation under two obstetric circumstances namely, ‗spontaneous preterm labour‘ and ‗indicated preterm labour‘. The aim of the study was to develop the strategies to enhance prevention of preterm labour in selected hospitals in Capricorn District, Limpopo Province. Research Method A sequential explanatory mixed method was adopted, the study was conducted in three phases, namely, quantitative study, qualitative study, and development of strategies. Self-developed questionnaires with 50 item questions each for mothers and registered midwives were administered. Both questionnaires were pre-tested prior to being administered to the respondents of the main study. The sample size of mothers was 77 mothers and 62 registered midwives. Data collected from the respondents were analysed using Statistical Package for Social Science (SPSS) version 25 with the help of the statistician. Tables, pie charts and bar graphs were drawn to present the results. The results of quantitative phase were utilized to formulate the Interview Guides that were used to explore the knowledge and practise of registered midwives and obstetricians regarding preterm labour. Interviews were conducted with 20 mothers, 6 registered midwives and 4 obstetricians until data saturation was reached. Data were analysed qualitatively using Tesch‘s Open-Coding method. v Quantitative Results The quantitative results for midwives revealed that about 60% of the mothers who had preterm labour were teenagers. Most of the respondents had ‗spontaneous preterm labour‘ and did not have any comorbidities while few had ‗indicated preterm labour‘ and were induced. All 62(100%) of the midwives showed that the facility does not offer an Outreach Programme on the prevention of preterm labour. Themes and Sub-Themes Results Themes and sub-themes were coded manually. Results that emerged from the integration and comparison of quantitative and qualitative results revealed that the mothers who went into preterm labour spontaneously had little information or no health education regarding preterm labour. Developed strategies The following strategies were developed based on the identified factors that might hinders the prevention of preterm labour and after exploring the knowledge and practice of midwives and obstetricians in the selected hospitals, Capricorn District, Limpopo Province. Strategies includes strengthening of BANC Plus, staff establishment, laboratory turnaround time, outreach programmes and improvement of counselling and support services. Recommendations of the Study The recommendation of the study is divided into three groups which includes recommendations for midwives at the PHC and hospital, recommendations for the obstetricians at the hospital and the recommendations for the Department of Health. The midwives to visit schools and community centres at least twice in a month to give information to the woman of childbearing age and reinforce health education on each Antenatal Care visit. The obstetricians to screen all mothers who are at risk of preterm labour for infections, follow up the results and treat the mothers accordingly. The Department of Health should hire enough staff so that quality care can be vi rendered to the pregnant woman at the PHC and hospital. The Department of Health should develop the electronic database, to register all mothers who have a history of preterm labour, so that when they are pregnant the database system will also help to identify them at the PHC, and they would then be referred to the hospital in time. If the developed strategies to enhance prevention of preterm labour may be adopted and adhered to by the midwives and obstetricians, these may help in reduction of high figures of preterm labour in the selected hospitals.

In this study, the use of hypnotherapy in high-risk pregnancy is explored from an ecological systems perspective through two case studies. Each case study is described in detail. They explore the experiences of two women during their pregnancy and giving birth, with specific reference to the pregnancy risks and their use of hypnotherapy. The study provides the reader with an in-depth understanding of the use of hypnotherapy before, during and after birth. / Psychology / M.A. (Psychology)

In this study, the use of hypnotherapy in high-risk pregnancy is explored from an ecological systems perspective through two case studies. Each case study is described in detail. They explore the experiences of two women during their pregnancy and giving birth, with specific reference to the pregnancy risks and their use of hypnotherapy. The study provides the reader with an in-depth understanding of the use of hypnotherapy before, during and after birth. / Psychology / M. A. (Psychology)