Research Doctorate - Doctor of Philosophy (PhD) / Maternal asthma is associated with low birth weight, a risk factor for disease in adult life. To determine the mechanisms involved, the relationships between mother, placenta and fetus were examined in asthmatic and non-asthmatic pregnancies. Maternal asthma and its treatment (no glucocorticoid or glucocorticoid) was monitored throughout pregnancy. Fetal growth was examined during gestation, and at birth, neonatal size and sex were determined. Placental 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) enzyme activity and umbilical vein plasma cortisol and estriol concentrations were measured. Placental cytokine, growth factor and glucocorticoid receptor (GR) mRNA were determined using quantitative RT-PCR. Birth weight of female neonates in the no glucocorticoid asthmatic group only, was significantly reduced compared to females of the non-asthmatic group. Male neonates were unaffected by asthma or its treatment. Asthmatic women pregnant with a female fetus showed a significant increase in circulating monocytes and glucocorticoid treatment as pregnancy progressed, while those pregnant with a male fetus did not, suggesting that maternal asthma worsens in the presence of a female fetus. 11beta-HSD2 activity was significantly reduced in placentae from female neonates of the no glucocorticoid group compared to other female neonates and was associated with a trend towards higher plasma cortisol, reduced fetal adrenal activity demonstrated by lower cord blood estriol, reduced placental GR expression, no alteration in placental or fetal insulin-like growth factors or their binding proteins and a significantly increased Th2:Th1 cytokine mRNA ratio, which was inversely correlated with 11beta-HSD2 activity in all females. Reduced placental 11beta-HSD2 activity may be an important component leading to decreased female fetal growth in pregnancies complicated by asthma. This study provides strong evidence for a fetal sex-specific effect on the maternal immune system which can have adverse effects on the female fetus. The female fetus alters maternal inflammatory pathways, which when not controlled by the use of inhaled glucocorticoids results in reduced placental 11beta-HSD2 activity, contributing to suppressed fetal adrenal function and a late gestation decrease in female fetal growth.

Preterm birth remains a major challenge in obstetrics. It complicates up to 13% of all pregnancies and accounts for approximately 80% of neonatal mortality and morbidity. Bacterial Vaginosis (BV) is associated with a 1.4-fold increased risk of preterm birth. Due to ineffectiveness of antibiotics in preventing preterm labour, probiotics have been proposed to serve as an alternative for treatment of BV and prevention of preterm birth. The objectives of this thesis were to determine 1) the effect of Lactobacillus rhamnosus GR-1 (L. rhamnosus GR-1) supernatant on cytokine profile and prostaglandin (PG)-regulating enzyme expression in lipopolysaccharide (LPS)-stimulated human chorion and placental trophoblast cells from human placentae, 2) the potential signaling pathways through which lactobacilli act and 3) the potential role of immune and placental trophoblast cells in initiating a response to LPS and L. rhamnosus GR-1 treatments. Primary cultures of human placental trophoblast cells were pre-treated with lactobacilli supernatant and then with LPS. In addition, immune cells were removed from cell suspensions using a magnetic purification technique to determine their role in modulating cytokine levels. The expression of pro- and anti-inflammatory cytokines and prostaglandin-regulating enzymes was then determined. We found sex-specific differences in the ability of LPS to increase the output of TNF-α, IL-10, and PTGS2. We also showed that L. rhamnosus GR-1 is able to act through the JAK/STAT and MAPK pathways to increase IL-10 and G-CSF, and independently down-regulates PTGS2 and TNF-α and up-regulates PGDH. The increase in G-CSF and PGDH were only observed in women carrying a female fetus. L. rhamnosus GR-1 may serve as an alternative to antibiotics in preventing some infection/inflammation-mediated cases of preterm birth.

Preterm birth remains a major challenge in obstetrics. It complicates up to 13% of all pregnancies and accounts for approximately 80% of neonatal mortality and morbidity. Bacterial Vaginosis (BV) is associated with a 1.4-fold increased risk of preterm birth. Due to ineffectiveness of antibiotics in preventing preterm labour, probiotics have been proposed to serve as an alternative for treatment of BV and prevention of preterm birth. The objectives of this thesis were to determine 1) the effect of Lactobacillus rhamnosus GR-1 (L. rhamnosus GR-1) supernatant on cytokine profile and prostaglandin (PG)-regulating enzyme expression in lipopolysaccharide (LPS)-stimulated human chorion and placental trophoblast cells from human placentae, 2) the potential signaling pathways through which lactobacilli act and 3) the potential role of immune and placental trophoblast cells in initiating a response to LPS and L. rhamnosus GR-1 treatments. Primary cultures of human placental trophoblast cells were pre-treated with lactobacilli supernatant and then with LPS. In addition, immune cells were removed from cell suspensions using a magnetic purification technique to determine their role in modulating cytokine levels. The expression of pro- and anti-inflammatory cytokines and prostaglandin-regulating enzymes was then determined. We found sex-specific differences in the ability of LPS to increase the output of TNF-α, IL-10, and PTGS2. We also showed that L. rhamnosus GR-1 is able to act through the JAK/STAT and MAPK pathways to increase IL-10 and G-CSF, and independently down-regulates PTGS2 and TNF-α and up-regulates PGDH. The increase in G-CSF and PGDH were only observed in women carrying a female fetus. L. rhamnosus GR-1 may serve as an alternative to antibiotics in preventing some infection/inflammation-mediated cases of preterm birth.

Prenatal assessment of fetal health is routinely offered throughout pregnancy to ensure that the most effective management can be provided to maintain fetal and maternal well-being. Currently, invasive testing is used for definitive diagnosis of fetal aneuploidy, which is associated with a 1% risk of iatrogenic fetal loss. Developing non-invasive prenatal testing (NIPT) is a key area of research and methods to increase the level of cell-free fetal DNA (cffDNA) within the maternal circulation have been discussed to improve accuracy of such tests. In this study, three strategies; co-amplification at lower denaturation temperature polymerase chain reaction (COLD-PCR), inverse-PCR and Pippin Prep™ gel electrophoresis, were analysed to identify a novel approach to selectively enrich shorter cffDNA fragments from larger maternal cell-free DNA (cfDNA). The sensitivity of droplet digital PCR (ddPCR) against real-time PCR (qPCR) was compared for fetal sex and RHD genotyping. In addition RHD zygosity testing was carried out for non-maternal samples. Consequently, Pippin Prep™ gel electrophoresis was combined with ddPCR analysis for the NIPD of Down Syndrome (DS) in pseudo-maternal samples. The results revealed that the Pippin Prep™ gel electrophoresis enrichment approach successfully demonstrated 2-fold to 5-fold increases in the cffDNA fraction. However, further optimisation assays of COLD-PCR and inverse-PCR using actual maternal samples were required. The spike experiments for DS detection revealed that with the present assay IV overrepresentation of the chromosome 21 target could be significantly detected for samples with ≥15% ‘cffDNA fraction’. In conjunction with the Pippin Prep™ enrichment method, this would have enabled assessment of all 10 maternal samples. Alternatively, fetal sex and RHD genotyping results determined that ddPCR provides a more sensitive platform compared to qPCR approaches, particularly for samples that express low cffDNA fractions (<2%). The ddPCR platform also proved to be a rapid and accurate system for the determination of RHD zygosity. This study highlights that ddPCR could be used as opposed to qPCR for accurate determination of fetal sex and RHD status. While sequencing approaches currently provide the most sensitive platforms for NIPT of fetal aneuploidy, high costs (>£400) prevent universal application. The combination of cffDNA enrichment with ddPCR analysis could provide a cheaper and more widely available platform for NIPD. However, further large scale validation studies using actual maternal samples are required.