Item Open AccessUniversal ultrasound screening in the third trimesterMoraitis, Alexandros; Moraitis, Alexandros [0000-0003-4634-1129]Background Pregnant women are currently offered two ultrasound scans, one at booking (around 12 weeks’ gestation) and one at around 20 weeks’ gestation. No further scans are offered unless there are clinical indications. Ultrasound has an important role in the management of high-risk pregnancies. However, there is no clear evidence that it is effective in screening low risk and unselected women. The majority of complications, such as stillbirth and shoulder dystocia occur in low-risk pregnancies, first because most pregnancies are classified as low-risk and second, possibly due to inadequate screening. An effective ultrasound screening programme in late pregnancy combined with an intervention, like induction of labour, for the screen positives could potentially improve pregnancy outcomes. However, the diagnostic accuracy of many ultrasonic features is unknown in low-risk populations and there is a possibility of iatrogenic harm by intervening when it is not necessary. Objectives 1. To assess the diagnostic effectiveness of late pregnancy ultrasound in nulliparous women based on the existing research literature. 2. To analyse the prospective cohort study, Pregnancy Outcome Prediction Study, for the above ultrasound findings and combine the results with the meta-analyses. 3. Finally, use the results to provide inputs for health economic analyses of the cost-effectiveness of universal ultrasound screening and assess the need, potential design, and acceptability of a future randomised controlled trial. Methods The following key ultrasound measurements were identified which might be used in late pregnancy screening: (i) suspected small for gestational age (SGA), (ii) suspected large for gestational age (LGA), (iii) high resistance pattern of umbilical artery Doppler flow velocimetry, (iv) low cerebro-placental ratio (CPR), (v) severe oligohydramnios, (vi) borderline oligohydramnios. I found that there was an on-going Cochrane Diagnostic Test Accuracy review for SGA, hence I focused on the other five measures. The protocol was registered with the PROSPERO register of systematic reviews (CRD42017064093). Medline, EMBASE, Clinical Trials.gov and the Cochrane library were searched from inception. Studies that performed an ultrasound scan ≥24 weeks of gestational age in unselected, low or mixed risk populations were included, excluding studies which only included high risk pregnancies. The risk of bias in each included study was assessed using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS 2) tool. Meta-analysis was performed using the hierarchal summary receiver operating characteristic curve (HSROC) analysis and bivariate logit-normal models. I also performed new analyses on previously unpublished data of the Pregnancy Outcome Prediction (POP) study which was one of the few studies that blinded ultrasound scan results to the clinicians. Results 41 studies of LGA met our inclusion criteria involving 112,034 patients in total. Ultrasonic suspicion of fetal macrosomia was strongly predictive of the risk of delivering a large baby with the positive LRs (LR+) ranging from 7 to 12. However, it was only weakly predictive of the risk of shoulder dystocia with LR+ around 2. 13 studies of umbilical artery (UA) Doppler that met our inclusion criteria including 67,764 patients in total. UA Doppler had weak/moderate predictive accuracy for detecting SGA and severely SGA (<3rd percentile) infants (LR+ between 2.5 and 3.0). However, it did not predict neonatal morbidity at term. The results were very similar in both the POP study and the meta-analysis (which included the POP study) with the only notable difference being that the association with severe SGA in the POP study was slightly stronger. 16 studies of CPR met the inclusion criteria involving 121,607 patients in total. CPR may be slightly more predictive than UA Doppler in identifying pregnancies at an increased risk of adverse outcome. In the case of SGA, the positive LRs were in the region of 3.5 to 4.0. Moreover, unlike UA Doppler, a low level of CPR was associated with an increased risk of neonatal morbidity. However, the association with morbidity was weaker with positive LRs of <2.0. Furthermore, in both analyses, there was very significant heterogeneity in relation to both SGA and neonatal morbidity. 14 studies of severe oligohydramnios that met our inclusion criteria involving 109,679 patients in total. Diagnosis of severe oligohydramnios was associated with a positive LR for SGA of between 2.5 and 3.0. It was also associated with positive LRs for admission to NICU and emergency caesarean section for fetal distress of between 1.5 and 2.5. However, the study quality was variable and only two studies containing <5% of the patients included in the meta-analysis blinded the results of the scan. 11 studies of borderline oligohydramnios (including the POP study) met our inclusion criteria involving 37,848 patients in total. Borderline oligohydramnios was weakly/moderately predictive of SGA (positive LRs 2.5 to 3.0). This was observed in the meta-analysis of multiple studies of variable quality. There was also a comparable association between borderline oligohydramnios and severe SGA in the only study where the scan result was blinded, the POP study. Finally, by analysing of the POP cohort We identified the 4.6% of women who had a breech presentation, and for more than half of these, it had not previously been clinically suspected. Most of these women were delivered by planned Caesarean section. No woman in the cohort had a vaginal breech delivery or experienced an intrapartum Caesarean for undiagnosed breech. An introduction of a policy of third trimester ultrasound for fetal presentation would prevent about 5000 emergency Caesarean sections and 8 perinatal deaths annually in the UK. The policy would be cost-effective at a cost of £19.80 per scan. Conclusion There is a strong clinical and health economic case for implementing late pregnancy ultrasound screening to assess fetal presentation. Universal ultrasound screening for macrosomia would increase the detection of LGA infants at birth but is unlikely to increase the detection of shoulder dystocia or associated neonatal morbidity in a clinically significant way. Umbilical artery Doppler, CPR, severe oligohydramnios, and borderline oligohydramnios were all weakly predictive of the risk of delivering an SGA infant but either non-predictive or weakly predictive of the risk of neonatal morbidity. They should not be used alone to screen for neonatal morbidity, however a positive result would justify further fetal monitoring due to the association of all above markers with SGA. Item Open AccessThe role of uterine natural killer cell-inhibition in pregnancyShreeve, NormanEducation of natural killer (NK) cells is a genetically determined process that primes NK-cell activity upon the binding of their inhibitory receptors to self-ligands. Although NK-cell education is predictable and measurable in the laboratory, its biological significance is unknown. This thesis shows that the inhibitory NK cell receptor NKG2A protects against the hypertensive disorder of pregnancy pre-eclampsia in individuals genetically programmed to favour the engagement of NKG2A with its ligand HLA-E. Using NKG2A-deficient Klrc1-/- mice, this thesis demonstrates that NKG2A is required to educate uterine NK cells to regulate uterine vascular adaptation to pregnancy, placental function and transcriptome, as well as fetal growth. Immune checkpoint blockade of NKG2A during pregnancy in wild-type mice did not affect fetal growth, suggesting acute ablation of this pathway does not interfere with pregnancy outcome. In addition, generation of a humanised mouse model to test the notion that uterine NK cell inhibition driven by KIR: HLA interactions leads to poor pregnancy outcomes, was attempted. Item Open AccessInnate Lymphoid Cell Diversity in Pregnancy and Ovarian CancerHuhn, OisinMany parallels have been drawn between the mechanisms that support successful pregnancies and those that facilitate tumour expansion. These include an immune profile bearing the hallmarks of immune suppression and reduced cytotoxicity. Innate Lymphoid Cells (ILCs) are a diverse group of lymphocytes which exhibit tissue specific phenotypes and functions. They are found in both the uterine lining during early pregnancy (decidua) and the ascites of ovarian cancer patients. Despite several attempts, there is still no consensus regarding the phenotypic profiles, subset composition and functional roles of ILCs in either tissue. To address this, I have developed a mass cytometry panel and stained resting and stimulated ILCs from peripheral blood, decidua and ascites. Within the decidua, a number of ILC subsets are identifiable including three main subsets of decidual NK cells (dNK), dNK1-3. The most abundant subset, dNK1, express receptors for HLA class I found on invading extravillous trophoblast (EVT) including Killer-cell Ig-like receptors (KIR), LILRB1 and NKG2A. In contrast to dNK2 and dNK3, dNK1 respond poorly to ‘missing self’ and PMA plus ionomycin treatment. However, upon cross-linking of an activating KIR they can degranulate and produce cytokines/chemokines. dNK1 responsiveness to activating KIR increases with the expression of additional KIR which may be a result of increased granularity. This is the first time mass cytometry has been used to characterise the phenotype and function of decidual ILCs. KIR variants are associated with infectious disease and pregnancy outcome. KIR2DL1 is an inhibitory KIR present on both KIR A and KIR B haplotypes. KIR2DL1 alleles typically found on KIR A haplotypes (KIR2DL1A) in Caucasians are associated with increased risk of developing pre-eclampsia. Using a novel antibody combination, I am able to stain for NK cells expressing particular KIR2DL1 allotypes. KIR2DL1A and KIR2DL1B are co-dominantly expressed by NK cells from peripheral blood (pbNK) and decidua but KIR2DL1A is preferentially expressed. Allotypes display similar inhibitory capacities but KIR2DL1A+ pbNK degranulate more in response to ‘missing self’ than KIR2DL1B+ pbNK. This approach is particularly powerful because KIR2DL1 allotypes can be compared within the same individual and have therefore experienced the same HLA class I environment. NK cells are increasingly being targeted in cancer immunotherapies and can exhibit both pro- and anti-tumorigenic properties. They are the major ILC subset within the ascites of ovarian cancer patients but phenotypic and functional descriptions are conflicting. Once again applying the CyTOF methodology, I find that there are three major subsets of ascites derived NK cells (aNK). These include previously described CD56bright and CD56dim aNK subsets and an additional NKG2Ahigh KIRlo subset which expresses tissue resident markers (TR aNK). Upon PMA plus ionomycin treatment, the proportion of TR aNK cells producing XCL1 and IFNg is higher than that of other aNK subsets. TR aNK are also highly responsive to ‘missing self’ and, similar to dNK cells, increased responsiveness is associated with increased granularity. A detailed characterisation of ascites derived ILCs is important to better understand existing cancer treatments and to provide insight for designing new therapeutic strategies. Item Open AccessInvestigating Cerebral Function in Neonates Using Diffuse Optical Tomography(2019-10-26) Lee, Chuen Wai; Lee, Chuen Wai [0000-0001-7785-2963]Developing techniques to monitor cerebral function is becoming increasingly recognised as a way forward to detect and understand brain injury in patients. Diffuse optical tomography (DOT) is a promising imaging technique that is non-invasive, portable and relatively simple to operate. In this thesis, I describe the development and design of DOT headgear for neonates and subsequently apply this in neonatal studies of cerebral function. Firstly, the DOT headgear was applied to healthy neonates to investigate the early stages of vocal specialisation. In this functional activation study, the cerebral haemodynamic changes observed in response to vocal and non-vocal stimuli are presented in Chapter 6. In recent years, the study of spontaneous cerebral activity that gives rise to resting state networks (RSNs) is becoming widely popular. These studies rely on infants to remain asleep to minimise subject motion and optimise data quality. Using DOT combined with electroencephalography (EEG), the effect of sleep state on RSNs was studied for the first time in healthy neonates with compelling results that are presented in Chapter 7. In the context of sleep state and RSNs, combined DOT-EEG was also applied to neurologically compromised patients in the neonatal intensive care unit (NICU). The results of this case-based study are presented in Chapter 8. These studies demonstrate the potential clinical use of DOT in neonates to evaluate cerebral function. In completion of this thesis, the implications of the results and findings from the studies are discussed and future directions explored. Item Open AccessMonitoring of cerebral oxygenation, cerebrovascular reactivity and circulatory function in preterm infants(2018-10-20) Sortica da Costa, CristineMonitoring of cerebral oxygenation, cerebrovascular reactivity and circulatory function in preterm infants Brain injury in the preterm infant is associated with death and lifelong disability. Cerebral hypoxia and fluctuations in cerebral blood flow in the first two days of life have been implicated in the pathophysiology of haemorrhagic and ischaemic brain injury. Monitoring of haemodynamic changes during the early transitional circulation from in-utero to ex-utero life are currently based on standard measurements of systemic oxygenation and mean arterial blood pressure, with no reliable assessment of end-organ perfusion. In this thesis, measurements using near-infrared spectroscopy (NIRS) and functional echocardiography were made to assess cerebral perfusion and systemic blood flow in a cohort of preterm infants undergoing intensive care. This thesis is divided into four sections: i) The feasibility of continuous monitoring of cerebral oxygenation and cerebrovascular reactivity is demonstrated in a series of case reviews, and the association between cerebral oxygenation and cerebrovascular reactivity with outcome of brain injury and mortality is described. ii) Combining measurements of systemic blood flow with end organ perfusion was applied to define MABPOPT in preterm infants based on an index of cerebrovascular reactivity. Deviations below MABPOPT were associated with intraventricular haemorrhage and mortality. iii) The complexity of brain and systemic signals was studied by using multi-scale entropy analysis. Most studies using cerebral NIRS or systemic measurements of blood flow use linear analysis; however, a complex biological system, such as the human brain, includes many regulatory mechanisms that interact in a complex manner, resulting in effects that cannot be understood wholly through the analysis of its individual constituents. Lower complexity of brain signals was observed in infants who developed intraventricular hemorrhage or died. iv) Changes in systemic and cerebral oxygenation in a cohort of preterm infants in the first 48 hours of life was assessed using functional echocardiography. The patterns of changes in cardiac output and cerebral oxygenation in infants who did and did not have intraventricular haemorrhage are discussed. Furthermore, the relationship between the presence of a haemodynamically significant ductus arteriosus and brain injury is assessed. Item Open AccessWomen’s Experiences of Late Pregnancy Ultrasound: Implications for Antenatal Care(2015-02-24) Dacey, AlisonObjective: This study addresses an identified gap in knowledge regarding the expectations and experiences of women undergoing third trimester ultrasound in the United Kingdom. Design: This dissertation is a qualitative study. It is theoretically informed by grounded theory. Methodologically, it draws on data collected through semi-structured interviews (n=50) conducted 18 months – 4 years following participation in the Pregnancy Outcome Prediction study (POPs) Participants were identified from the POPs database and sampled according to three criteria regarding pregnancy outcomes. Setting: Interviews were conducted in participant’s homes in South East England. Findings: The data revealed three core themes: ultrasound as a means of ascribing reality to the pregnancy; the importance of the visual aspect of ultrasound; and the reassuring effect of ultrasound. Ultrasound played a pivotal role in the confirmation of life and the reality of the child. The visual aspect of ‘seeing’ the unborn child provided a level of reassurance beyond other aspects of routine care and for this group of mothers was the significant and defining moment of psychological reassurance. Conclusions: While the underlying themes were analogous there were differences between how women experienced late trimester ultrasound from earlier scans, the data revealed important findings pertaining to the role of ultrasound in preparing for birth and transition to parenthood. It raised questions relating to the environment of scanning, the experiences of research participation during pregnancy, and the implications of these scanning experiences for routine antenatal care. Item Controlled AccessImmunogenetic regulation of Natural Killer cell function in pregnancy(2017-12-01) Gaynor, Louise Michelle; Gaynor, Louise Michelle [0000-0002-6617-3110]Uterine NK (uNK) cells are a distinct subset of NK cells in the decidua of humans and rodents during pregnancy, which are essential for remodelling of the spiral arteries supplying the feto-placental unit. Similarly to peripheral NK cells, uNK cells express Natural Killer receptors (NKRs) that engage MHC class I molecules. Evidence from human genetic association studies suggests that, in the presence of allogeneic cognate paternal MHC class I ligands, inhibitory uterine NKRs are associated with disorders of pregnancy arising from impaired decidual vascular remodelling. Conversely, enhancement of human uNK cell activity through activating NKRs is associated with high birth weight. Evidence from mouse models corroborates that uNK cell activity is modulated by interactions between NKRs and MHC class I, but has largely focussed on the effect of paternal MHC. In this study, the contribution of maternal immunogenetic regulation of NK cell function to reproductive outcome was assessed independently of parental MHC disparity in mice. To evaluate the role of NKR genes in isolation, I used congenic B6.BALB-TC1 (TC1) mice that differ from C57BL/6 (B6) mice only within the region of chromosome six encoding NKRs that recognise MHC class I. Absence of a major inhibitory NKR for self-MHC, Ly49I, in TC1 mice causes a compensatory shift in the NKR repertoire expressed and preserves a majority subpopulation of educated NK cells. B6 and TC1 splenic and uterine NK cells are similarly functionally reactive and mature, and no significant differences could be detected in spiral arterial remodelling or fetal growth between these strains in MHC-syngeneic matings. This supports data from human immunogenetic studies showing that maternal uterine NKRs are not associated with differences in pregnancy outcome in the absence of novel paternal MHC class I ligands, and highlights the importance of maternal and paternal co-regulation of uNK cell activity during pregnancy. No mouse models of uNK cell activation are currently available with which to corroborate human immunogenetic associations between activating uterine NKRs and high birth weight. Male m157-transgenic (m157-Tg) mice, which ubiquitously express viral m157 glycoprotein ligands for the activating NKR Ly49H, were mated with B6 females. Exclusive expression of m157 glycoprotein by trophoblast improved placental efficiency, but did not enhance fetal growth. Some fertility clinics surmise that uNK cell activation initiates the pathogenesis of spontaneous abortion. It has been suggested that this may occur due to reduced expression by human uNK cells of miR-483-3p, which stimulates endogenous insulin-like growth factor (IGF)-1 production and uNK cell cytotoxicity in vitro. It is demonstrated here that neither miR-483-3p nor IGF-1 regulate murine NK cell development, maturation or function. No discernible reproductive phenotype is evident in miR-483 deficient females. It can be inferred that post-transcriptional control by miR-483 is not biologically relevant to murine NK cell function. Although m157-Tg mice may provide an interesting model to further study uNK cell-mediated placental adaptations, it remains important to identify a murine model of enhanced uNK cell function to corroborate human immunogenetic associations with high birth weight and to challenge the supposition that uNK cell activation is harmful to pregnancy.