Background and purpose: Early pregnancy problems, including miscarriage, ectopic pregnancy and pregnancy of unknown location, occur commonly and have significant medical, psychological and economic consequences. Biochemical markers are increasingly being used as an adjunct to ultrasonography and this thesis describes three studies exploring the use of novel biochemical markers in the diagnosis and management of early pregnancy problems. Materials and methods: These are observational studies of women in early pregnancy recruited at Sunderland Royal Hospital and King’s College Hospital. Serum samples were taken from women in early pregnancy and lectin affinity chromatography used to characterise the glycosylation of hCG by gestational age and by pregnancy outcome. Women with a diagnosis of a miscarriage, ectopic pregnancy or pregnancy of unknown location had serum levels of hCG, progesterone, inhibin A, IGFBP-1 and inhibin proαC quantified, and statistical analysis was used to see if spontaneous resolution of the pregnancies could be predicted. Results: Lectin-affinity chromatography reveals five major glyco-isoforms of hCG in early pregnancy, the expression of which changes with gestational age and by pregnancy outcome. The novel markers of the luteo-trophoblastic axis inhibin A, IGFBP-1 and inhibin proαC are found not to be clinically useful in the prediction of spontaneously resolving PULs although when used in the decision trees developed by Elson in 2005, they are useful for predicting spontaneous resolution of miscarriages and failed pregnancies. Conclusions: Novel biochemical markers have the potential to be a useful addition in the management of early pregnancy problems. Further studies are required to explore the physiological basis of these findings and the clinical applicability of these tools.

The rate of preterm birth is 5-9% of all births in Europe, and 12-13% in the United States of America (USA); the rates in both continents are increasing, partly due to the higher number of multiple births associated with assisted conceptions.1 About 30-35% of preterm births are the result of maternal or fetal disease, but 40-45% of premature births result from spontaneous preterm birth (SPL) and 25-30% from preterm rupture of the membranes (PROM). For families struggling to cope with having a baby in special care, this will be one of the most difficult, emotional and stressful times of their lives,2 whatever the longer term outcome. The sequelae of preterm birth also pose significant challenges. Children born preterm are at increased risk of major disabilities, such as cerebral palsy, with the risk increasing with decreasing gestation at birth.3 Many preterm children without disability develop serious behavioural and educational difficulties.4 The prevention of preterm birth and reduction of associated disability are therefore important health priorities, and are a major focus of my work. The principle that health care should be “evidence-based” lies at the heart of today’s National Health Service (NHS) but this has not always been the case. Lessons about the importance of evaluation of treatments can readily be demonstrated within maternity and perinatal care. Perhaps the most famous example is that of thalidomide, which became recommended treatment for pregnant women to relieve morning sickness in the late 1950s.5 By the beginning of the 1960s obstetricians had begun to notice increased numbers of children with severely malformed arms and legs.

Perinatal asphyxia remains a major cause of morbidity and mortality throughout the world. Despite improvements in obstetric and neonatal care there has been little improvement in rates of death and disability. Therapeutic hypothermia has recently been shown to be the only effective treatment following perinatal asphyxia. Selective Head Cooling (SHe) and Whole Body Cooling (WBe) are methods of applying therapeutic hypothermia. Yet the optimum method of cooling for maximum neuroprotection and minimum adverse effects remains unclear. We retrospectively compared four methods of cooling newborns following perinatal asphyxia. One method of SHC and three methods of WBC, using water-filled gloves, a coolant- filled mattress, and a water- filled body wrap. All methods maintained rectal t emperature within narrow target range. We found that WBC using a servo- controlled system minimised initial over-cooling and reduced subsequent fluctuation in rectal t emperature compared with manually-controlled WBC or SHe. We found similar variation in rectal temperature, heart rate, and mean arterial blood pressure using SHC and manually controlled WBe. We developed a technique of applying SHC with minimal systemic hypothermia as a t reatment for perinatal asphyxia in our established experimental model. We conducted a study to assess the neuroprotective and physiological effects of our technique compared to standard care under normothermic conditions. We did not find any difference in brain injury between treatment groups but increased mortality in the hypothermia group. We have.previously used a neuropathology score in our established model of perinatal asphyxia in order to assess treatment effects. Our neuropathology score incorporates subjective assessments of area injure'd and morphological cellular changes in several brain regions. We conducted quantitative cell counting to assess brain injury following perinatal asphyxia. Cell count correlated with neuropathology score in the putamen and hippocampus CA1 and we were able to validate our neuropathology score to assess outcome.

Historically the evaluation and reporting of neurodevelopmental outcomes following perinatal brain injury have been limited to good outcome/poor outcome or disabled/not disabled. More infants are surviving and new treatments to protect the brain require careful testing. Sensitive measures of evaluating developmental outcome in infants with the poorest outcome are required. The aims of this thesis are to describe developmental measures widely used; to examine data arising from a key developmental measure in two groups of children with perinatal brain injury; to use the data to quantify the abilities of even the most severely affected and to compare these findings with neuroimaging in infants with Post Haemorrhagic Ventricular Dilatation (PHVD); and to compare findings of two different editions of the key measure in a group of infants with Neonatal Encephalopathy (NE). Formal assessment at 2 years using the Bayley Scales of Infant Development (Bayley-2) confirmed wide-ranging, but frequently poor outcome, in infants with PHVD. Bayley Developmental Quotients (DQ), used in preference to conventional Bayley-2 Index scores, were found to differentiate between grades of functional ability and disability, even in the most severely delayed infants. Bayley DQ correlated strongly with parenchymal lesion area measured on neonatal cranial ultrasound scans but increasing ventricular size was not associated with reductions in Bayley DQ in infants with preceding grade 3 IVH. Cerebral, thalamic and cerebellar brain volumes from MRI scanning at term age were found to be significantly affected in this cohort. Smaller brain volumes were associated with decreasing Bayley DQ and with motor development in particular. These findings have the potential to enhance the precision of outcome prediction in infants with PVHD which may be of use to clinicians in informing early direction of care and in providing information to families about developmental challenges facing their baby. Comparative study of Bayley-2 scores with revised Bayley Scales of Infant and Toddler Development (Bayley-3) in infants with NE, found higher than expected Bayley-3 scores particularly in those with severe delay. Increased Bayley-3 cut-off thresholds for severe disability are recommended when comparing outcomes using different versions of the test.

Pre-eclampsia is a lead ing cause of maternal and fetal mortality and morbidity. Early-onset pre-eclampsia occurring before 32 weeks gestation affects ~1% of pregnancies. At these gestations conservative management focussing on control of hypertension and seizure prevention, to gain fetal maturity is key. Impaired nitric oxide bioavailability, is thought to play a major role in the maternal manifestations of pre-eclampsia such as hypertension and likewise platelet activation, proteinuria and oedema. There is no current treatment that targets the underlying pathophysiology. Uterine artery Doppler and arterial stiffness were assessed in ninety-nine women, at high a priori risk of pre-eclampsia. The study showed a positive correlation between increased uterine artery impedance and both pulse wave reflection (AI)() and aortic pulse wave velocity (aPWV). Alx was negatively correlated with neonatal birth weight. Infusion studies of S-nitrosoglutathione (GSNO) and labetalol, the standard antihypertensive used in pre-eclampsia, were performed in 12 healthy nonpregnant volunteers to assess the effects on Alx and aPWV and cardiac output. The study showed that GSNO and labetalol have similar effect on blood pressure, however GSI\lO, but not labetalol, reduces Alx. The primary outcome of the GSNO infusion study in preeclampsia was to establish the dose of GSNO at which there was optimal reduction in Alx, without causing a clinically significant fall in blood pressure. Secondary outcomes included the effect on platelet function, soluble biomarkers and proteinuria in the mother and Doppler parameters in both the mother and fetus. Six women underwent infusion and an infusion rate of 1O-30mcg/min GSNO was established as an optimal dose. GSNO significantly reduced platelet activation and trend towards reduction in proteinuria and soluble endoglin was seen. The effects of GSNO in women with preeclampsia and make GSNO a promising candidate for further investigation for the treatment of pre-eclampsia, either as a sole agent or adjunct to current treatments.

Humans may be exposed to cobalt (Co) and chromium (Cr) ions through occupation in the metal industries or through the implantation of surgical devices made from cobalt-chrome alloy. Epidemiological studies have reported a significant excess of respiratory carcinoma in metal industry workers exposed to Cr-containing dusts and vapours. Cr6+ has thus been classified as a Class 1 compound- known to be carcinogenic to humans- by the International Agency for Research on Cancer (IARC). Co2+ has been classified as a Class 2b compound- possibly carcinogenic to humans. The potential teratogenic effects of Co2+ and Cr6+ are largely unknown. Animal experiments have demonstrated an increased risk of miscarriage and foetal malformation following maternal exposure to Co2+ or Cr6+ during pregnancy. Similarly, a recent meta-analysis has shown an association between maternal occupational Cr poisoning and risk of spontaneous abortion in humans. Here, an in vitro strategy was used to investigate the risks to an embryo from Co and Cr ions within the maternal circulation. To do this, human embryonic stem cells (hESCs) were exposed to Co2+ and Cr6+ across a model of the placenta barrier (a confluent bilayer of trophoblast cells) . This indirect exposure of hESCs to Co2+ and Cr6+ induced DNA damage and apoptosis in the hESC colonies. These damaging effects were not caused by the Co and Cr ions passing through the barrier. Instead, they were caused by the release of at least two bystander like signalling molecules (including TNF-a) from the trophoblast barrier. This signalling mechanism between the trophoblast barrier and the hESCs depends on gap junctional intercellular communication within the barrier and the hESC colonies. If translated into an in vivo response, the signalling mechanism uncovered here could imply a sensitive mechanism for miscarriage if the mother is exposed to a low dose of a toxin

This thesis concerns overnight ambulatory monitoring of the human fetal heart rate (FHR) using the transabdominal fetal electrocardiogram (ECG). This technique is non-invasive and provides more information on the FHR than conventional methods. However, the greatest limitation is the variation in recording success with gestational age and the time of recording. Normal FHR variability is a reliable indicator of fetal well being. During hypoxia changes in autonomic nervous system (ANS) activity results in changes in FHR variability. Therefore analysis of heart rate fluctuation enables the study of both sympathetic and parasympathetic branches of ANS. It is known that the fetal ANS matures with advancing gestation. In this thesis, the relationship between FHR parameters with gestational age was explored in fetuses of non-smoking mothers with normal pregnancies, mothers with pre-eclampsia, smokers and growth restricted fetuses. Paired day and night-time recordings were made in a majority of the women, with the aim of describing diurnal patterns of FHR parameters in the different groups and as gestation progressed. The results from overnight FHR recordings in normal recruits support the observation that basal FHR decreased with advancing gestation whilst fetal HRV and number of accelerations increased. For all FHR parameters, except for root mean square of successive difference (RMSSD), significant diurnal variation was observed in normal pregnancies. During night basal FHR decreased, whereas short-and long term FHR variability increased. Moreover, normal fetuses spent significant amount of time in high variation period and exhibited greater number of accelerations at night. On the other hand, growthI restricted fetuses and those of smokers and pre-eclamptic mothers did not exhibit diurnal variation.

Humans are increasingly being exposed to nanoparticles from sources such as CoCr nanoparticles in metal on metal bearing orthopaedic implants, and also due to the increasing use of nanotechnology. The potential for nanoparticle exposures during pregnancy to cause developmental toxicity has been shown in vivo, and most commonly affects fetal neural development. This has generally been presumed to be due to the passage of nanoparticles to the fetus. It has however been shown that nanoparticles can cause DNA damage across placental barriers without crossing them. This 'indirect toxicity' is due to nanoparticles being internalised within the barrier and initiating a signalling cascade that is believed to transverse the barrier via connexin 43 gap junctions. In this thesis BeWo trophoblast cells were used to make in vitro models of the placenta to determine whether indirect nanoparticle toxicity could alter neurodevelopment. BeWo barriers were exposed to CoCr nanoparticles and media was harvested from beneath the barrier. This was applied to human neural progenitor cells that were differentiating into neurons and astrocytes. The viability of these cells was.unaffected although a shift to an astrocytic phenotype did occur. The resultant astrocytes had enlarged nuclear and cytoplasmic areas on immunostaining and were believed to be reactive astrocytes. These astrocytes also exhibited high levels of yH2AX foci on immunostaining, representing increased levels of DNA damage. The developing human cortical neurons, in culture with the astrocytes, were also found to have increased levels of yH2AX foci but to a lesser level than found in astrocytes. Near pure neuronal cultures did not however develop increased levels of yH2AX foci, therefore it was believed that the reactive astrocytes were initiating the DNA damage in the developing neurons. Exposures to differentiating embryoid bodies derived from human embryonic stem cells also found that differentiation into neuroectoderm was not affected. These results indicate that nanoparticies may alter neurodevelopment due to triggering signalling within the placenta, and that this is dependent on the presence of astrocytes, with earlier developmental events not being equally affected. Finally further investigations were performed to delineate the initiation and methods of transfer of this DNA damaging signalling in the BeWo barrier. These found that when nanoparticles come into contact with the barriers, impairment of autophagic flux occurs in the upper layers. Successful knockdown of Cx43 in the BeWo barriers was performed using shRNA lentiviral vectors to investigate the transfer of DNA damaging signalling. This however did not prevent morphological changes from developing in astrocytes, suggesting that Cx43 gap junctions may not be the only method of signalling transport involved in indirect nanoparticle toxicity. These results are discussed in relation to our current knowledge of neurodevelopment and the effects that astrocytes have in health and disease. Based on the results in this thesis I believe that limiting exposure to nanoparticles is important in women of childbearing age.

This thesis presents the QUOTE (Qualitative Understanding of Trial Experience) Study. The study explored women's views and experiences of participating in a large multi-centre randomised controlled trial - The Magpie Trial. The Magpie Trial was designed to test the hypothesis that in women with p're-eclampsia treatment with magnesium sulphate reduces the risk of eclampsia, and so improves outcome. Wqmen recruited to the Magpie Trial all had sufficiently severe pre-eclampsia to warrant consideration of magnesium sulphate for seizure prophylaxis. They were therefore being recruited at a time when they were likely to be having intensive monitoring, and there was often concern about their health and that of the baby. At present little is known about the experiences of pregnant women recruited to trials, especially when in potentially' life-threatening situations. Issues regarding the randomisation of pregnant women to trials have been identified as needing special consideration.

No description available.