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Background: Currently used methods to estimate fetal size in the third trimester, are suboptimal in detecting fetal growth restriction. Inclusion of a 3D ultrasound fractional thigh volume within an estimated fetal weight model may provide a more accurate estimate of fetal size. Low first trimester maternal serum Pregnancy associated plasma protein-A (PAPP-A < 0.415MoM / 5th centile) is associated with Small for gestational age (SGA) and placental dysfunction (PD) and this group provides an enriched population to assess the value of fractional thigh volume estimation. SGA is currently defined using birthweight and heavily influenced by skeletal growth in EFW models. Hypothesis, Aims and objectives: Longitudinal fractional thigh volume could provide a more reproducible and accurate assessment of fetal size and growth trajectory in pregnancies complicated by low maternal serum PAPP-A. Methods: New Ultrasound Parameters in Pregnancy 2 (NUPs2) was a prospective, longitudinal cohort study of 112 women with low maternal serum PAPP-A. Measurements of 2D fetal biometry and fractional thigh volume (Tvol) were obtained at 6 weekly scan intervals from 15 weeks until 38 weeks' gestation. Automated volume measurements were obtained using 4D view software (GE Healthcare). Reproducibility and accuracy were assessed using intraclass correlation coefficients (ICC) and BlandAltman Plots. Fetal growth, incorporating Tvol as a measure of non-skeletal growth, was compared in pregnancies associated with and without a low PAPP-A. In addition, multi-level linear regression analyses were used to interrogate the impact of low PAPPA on fetal growth, following adjustment for known important covariates. Results: Tvol measurements were highly reproducible (ICC > 0.99) and have a low measurement error especially in the third trimester. Inclusion of Tvol within an estimated fetal weight (EFW) model provided a more accurate in utero-assessment when compared to birthweight than any 2D EFW model (BPD-AC-Tvol ICC 0.96 [0.93 – 0.98] Versus Hadlock HC-AC-FL ICC 0.93 [0.85 – 0.96]). Findings from this thesis suggest that pregnancies affected by low PAPP-A are associated with a reduction in skeletal growth across gestation compared with a non-low PAPP-A. The reduction in skeletal growth remained consistent for low PAPP-A with and without PD. Reduction of skeletal and adipose growth was also shown in those with PD and low PAPP-A. Discussion: The addition of Tvol, especially in the third trimester, improved the accuracy and reproducibility of EFW models. While within the low PAPP-A cohort, a novel growth pattern of reduced skeletal growth was identified. A subgroup of fetuses within the low PAPP-A cohort were identified as SGA; One cohort with reduced skeletal mass without PD, the other with PD, likely leading to a nutritional deficit leading to a smaller overall size. Therefore, women with low PAPP-A without PD may benefit from alternative scan surveillance, antenatal care and timing of delivery from current practice.

Pregnancy associated plasma protein A (PAPP-A) is produced in increasing amounts with advancing pregnancy, a major source being the placenta. It was originally described as a homotetramer with ~ 200,000 M[r] subunits. An alternative structure has since been proposed of a disulphide bridged complex consisting of equimolar amounts of the PAPP-A subunit and the proform of eosinophil major basic protein (pro MBP).The increasing levels of PAPP-A produced during pregnancy and reports of possible proteinase inhibitory activity and immunomodulatory activity suggested that it may be an antiarthritic agent, involved in the amelioration of rheumatoid arthritis (RA) observed during pregnancy. PAPP-A has been purified from late pregnancy plasma using ammonium sulphate precipitation, gel filtration chromatography, ion exchange chromatography and affinity chromatography. The purity has been confirmed by SDS PAGE and western blot analysis. The purified PAPP-A has been used to investigate functions of PAPP-A which may support or refute a role in the amelioration of the symptoms of RA during pregnancy. Potential immunomodulatory actions of PAPP-A may be mediated by the inhibition of cytokine production or action. The effects of PAPP-A on the production of proinflammatory cytokines and also on the production of prostaglandin E[2] (PGE[2]), an inflammatory mediator, were studied using the THP 1 human monocytic cell line and the MG-63 human osteoblast- like cell line. PGE[2] was measured by radioimmunoassay and the cytokines IL-1, IL-6 and TNFalpha using commercial ELISA kits from Biosource International. IL-1alpha stimulated the production of PGE[2] and IL-6 in the MG-63 cells, whereas PAPP-A had no significant effect on basal or IL-1alpha stimulated IL-6 and PGE[2] production. In THP 1 cells, LPS, as expected, stimulated the production of PGE[2], IL-l?, IL-6 and TNFalpha. PAPP-A alone also significantly stimulated their production at higher concentrations, having a much greater effect at higher concentrations than LPS alone. PAPP-A, when incubated with LPS, produced a greater than additive effect on the production of PGE[2], but in combination with LPS had no significant effect on the levels of IL-6 obtained with LPS alone. Binding of PAPP-A to IL-1alpha and TGFbeta was studied using [125]I-cytokines. Formation of PAPP-A cytokine complexes was determined using either native SDS PAGE or gel filtration chromatography. No evidence of cytokine binding was obtained. The binding of PAPP-A to human cartilage was also investigated, since this may facilitate any protective effects of PAPP-A, such as proteinase inhibition, on cartilage. Cartilage binding was studied using immunohistochemical methods. The results indicated that PAPP-A does bind to cartilage, suggesting a possible protective role. The effect of PAPP-A on retinoic acid stimulated resorption of bovine nasal cartilage was studied and cartilage proteoglycan release measured as glycosaminoglycans using the dimethylemethylene blue assay. PAPP-A appeared to stimulate the resorption of cartilage. A key issue was whether PAPP-A functions as a proteinase inhibitor thus preventing connective tissue breakdown. Inhibition of elastase, cathepsin B, plasmin and trypsin was studied using colorimetric and fluorescent peptide substrates. PAPP-A was shown to competitively inhibit elastase, possibly due to the electrostatic interactions between the negatively charged PAPP-A and positively charged elastase. It did not inhibit the other enzymes studied,A monoclonal antibody to PAPP-A was produced by immunization of mice with the purified PAPP-A. This is currently been evaluated as to its potential use in a screening ELISA assay for monitoring PAPP-A levels. Overall the effects of PAPP-A on monocytic cell cytokine and PGE2 production and its stimulation of cartilage proteoglycan breakdown suggest it does not have a major role in the remission of RA during pregnancy. However this work has demonstrated functions of PAPP-A not previously reported and confirmed earlier observations of inhibitory activity towards elastase. Further work is required to determine the key functions of PAPP-A during pregnancy.