Annual Report From Researcher
Annual Report to Holly Martin Stillbirth Research Fund – December 2012 Dr Alexander Heazell, Clinical Lecturer in Obstetrics, Maternal and Fetal Health Research Centre, University of Manchester. Introduction Stillbirth research at the Maternal and Fetal Health Research Centre has three areas of activity: understanding the underlying causes of stillbirth, stillbirth prevention and improving care for parents who experience a stillbirth. This research programme has been funded by Sands, Tommy’s and Holly Martin Stillbirth Research Fund. This year, money donated from Holly Martin Stillbirth Research Fund (HMSBRF) has been used to pursue research projects to understand the placental causes of stillbirth and to develop alternative markers of fetal hypoxia. In addition, HMSBRF funding has been used to complete two projects which have resulted in peer-reviewed publications. The projects funded by HMSBRF were: 1. Quantitative Assessment of Placental Morphology in Different Causes of Stillbirths 2. Determining Markers of Chronic Fetal Hypoxia in Umbilical Venous Blood 3. Placental Inflammation in Women with Reduced Fetal Movements Funding was used for staff time and laboratory consumables. The funding for laboratory consumables enabled two medical students, Christina Parkinson and Emma Badger, to complete intercalated BSc and MRes degrees respectively. This approach maximises the impact of the funding as the student’s laboratory time is free, but financial support in the form of consumables is required. Holly Martin Stillbirth Research Fund – Annual Report 2012 2 Quantitative Assessment of Placental Morphology in Different Causes of Stillbirths Funding – Technician Salary for Ainslie Garrod, Laboratory Consumables. Background – Stillbirth is often the end result of a pathological process involving the placenta. However, a significant proportion of stillbirths remain unexplained. Current assessment of placental structure and patterns (morphology) is complicated as the microscope slides are assessed subjectively by an expert, the terms and diagnostic criteria are varied and there is uncertainty regarding the origin of features identified on histopathological examination. Hypotheses – We hypothesised that quantitative assessment of placental morphology would identify different morphology in different causes of stillbirth and live births and ultimately lead to a standardised ay of examining the placenta. Methods – We use placental samples from stillbirths of known causes, including: cord accident; diabetes; hypertension; infection; intrauterine growth restriction (IUGR); unknown cause and compared them to live births. Morphology was assessed using immunohistochemistry to identify five features of placental structure that may explain what is going wrong in the placenta. Five areas of each tissue section were analysed by computerised image analysis. This gave a frequency for all of these features, which was then compared to that in normal pregnancies. Results – There were more syncytial nuclear aggregates in cord accident, hypertension, IUGR and stillbirths of unknown cause (Figure 1A). Proliferation was decreased in all stillbirths, but particularly in cord accidents and IUGR (Figure 1B). The number of blood vessels was decreased in IUGR and unknown cause (Figure 1C). The proportion of avascular villi was increased in all stillbirths; the largest increase was in IUGR, cord accident and unknown stillbirths (Figure 1D). [* p<0.05, ** p<0.01, *** p<0.001]. Figure 1 In stillbirths, IUGR has a distinct pattern: increased syncytial nuclear aggregates, reduced proliferation, reduced vascularity and thickened trophoblast. Two out of ten stillbirths of unknown cause had the same placental phenotype as IUGR. Conclusion – Quantitative assessment of placental morphology can distinguish between different causes of stillbirth. When these criteria are applied to stillbirths of unknown cause, 20% had a similar pattern to IUGR. Applying quantitative assessment in addition to expert qualitative assessment may reduce the proportion of stillbirths classified as “unexplained”. A greater proportion of stillbirths may be affected by IUGR than previously thought. To really understand what these changes represent, we need to understand which changes happen before death and which occur after death and before analysis of the placenta. Holly Martin Stillbirth Research Fund – Annual Report 2012 3 Determining Markers of Chronic Fetal Hypoxia in Umbilical Venous Blood Funding – Laboratory consumables (ELISAs for analysis of proteins in umbilical blood) Introduction – The incidence of stillbirth is increased in pregnancies complicated by diabetes mellitus (DM), intrauterine growth restriction (IUGR) and after reduced fetal movements (RFM), when stillbirth is thought to result from fetal hypoxia. The relative infrequency of stillbirth makes carrying out clinical trials focussed on reducing stillbirth challenging, as the studies need to be extremely large. A robust marker of fetal hypoxia would be useful to use as a proxy outcome for stillbirth in clinical trials as this would reduce the sample size. Aim – The objective of this study was to determine the effectiveness of four potential markers of fetal hypoxia in normal pregnancies and complicated pregnancies. Methods – Venous blood was obtained from double clamped umbilical cords immediately after delivery in controls with a normal pregnancy, either after Caesarean section (CS) or vaginal delivery (VD) (n=30), DM (n=10), IUGR (n=7) and RFM (n=21). Endothelin-1, erythropoietin (EPO) and S100B were measured using enzyme linked immunosorbent assay (ELISA), a means to measure substances in serum, and the proportion of nucleated red blood cells (NRBCs) was determined by flow cytometry. These were analysed as they are all linked to hypoxia in other studies. Results – Erythropoietin concentrations were significantly higher in infants with RFM during pregnancy than controls (39.9 vs 15.8 mIU/ml, p<0.019). There was a trend to an increase in Erythropoietin in samples from DM and IUGR (Figure 2A). Endothelin-1 levels were significantly elevated in infants delivered vaginally and those with RFM, compared to controls delivered by Caesarean section (18.5 vs. 14.8 pg/ml, p<0.011; Figure 2B). There was no significant difference in the number of NRBCs or S100B concentrations when pregnancy complications were present compared to controls. Figure 2 Conclusion – None of the markers investigated were elevated in all pregnancy complications. However, erythropoietin has the strongest potential for the detection of fetal hypoxia. The increase in Endothelin-1 in vaginal delivery compared to Caesarean section reflects acute, but not chronic, hypoxia. NRBCs and S100B are not useful in the detection of chronic fetal hypoxia. Further research is required to identify a robust marker of chronic hypoxia. We have recently collaborated with Dr David Gaze at St George’s Hospital Medical School in London to measure two more markers (ischaemia modified albumin and troponin I in these samples). We need to collect more samples from pregnancies complicated by IUGR and DM to confirm the utility of erythropoietin in identifying fetal hypoxia in these pregnancies. A B * * Holly Martin Stillbirth Research Fund – Annual Report 2012 4 Placental Inflammation in Women with Reduced Fetal Movements Funding – Laboratory consumables Introduction – Intrauterine growth restriction (IUGR) and stillbirth are important pregnancy complications affecting 4-7% and 0.5% of births. Reduced fetal movements (RFM) is associated with a 2-3 times increased risk of IUGR and stillbirth. This is associated with abnormal placental function, although we do not fully appreciate the reasons why the placenta does not work normally. Other studies have found that placental inflammation is associated with IUGR and stillbirth. Hypothesis – We hypothesised that there would be increased placental inflammation in pregnancies with RFM, particularly those ending in poor pregnancy outcome. Methods – 56 placentas from women with RFM and 56 placentas from uncomplicated pregnancies were analysed. The presence of inflammation in placental tissue sections was assessed by immunohistochemistry for CD45 a leukocyte (white blood cell) marker. Tissue sections where leukocytes were identified then stained with specific markers for different types of leukocyte: macrophages (CD68), cytotoxic T-lymphocytes (CD8) and neutrophils (neutrophil elastase). Twenty-three placental cytokine levels were measured by Bioplex assay or cytometric bead array. Results – There were more white blood cells present in the placenta in pregnancies with RFM compared to normal pregnancies. White blood cell numbers were even higher in RFM pregnancies with poor pregnancy outcome (PPO), shown in purple in Figure 3 (p<0.0001). This increase was mainly due to cytotoxic T-lymphocytes infiltrating the placenta. Some differences in cytokine levels were detected. IL-10, which is an anti-inflammatory cytokine, was lower in the placenta in RFM. Levels of inflammatory cytokines IL-1α and IL-1RA were higher in the placenta in RFM (p<0.0002), particularly where there was poor outcome (p<0.0003). CCL3, a cytokine that can attract leukocytes into the placenta, were higher in RFM compared to normal pregnancies. Figure 3 Conclusion – Pregnancies affected by RFM, in particular those with a poor outcome, showed greater evidence of placental inflammation. This was associated with differences in the expression of some signals associated with inflammation (IL-10, IL-1α, IL1RA and CCL3). These may be disrupting the function of the placenta, leading to reduced supply of nutrients and oxygen to the fetus. A better appreciation of why leukocytes are present in the placenta and the effects they exert on placental function will improve the understanding of stillbirth and IUGR. White blood cells shown in brown Holly Martin Stillbirth Research Fund – Annual Report 2012 5 Publications 1. Graham N, Garrod A, Bullen P, Heazell AEP. Placental Expression of Anti-angiogenic Proteins in Mirror Syndrome: A Case Report. Placenta 2012;33(6):528-531. 2. Curtis S, Jones CJP, Garrod A, Hulme C, Heazell AEP. Identification of Autophagic Vacuoles and Regulators of Autophagy in Villous Trophoblast from Normal Term Pregnancies and in Fetal Growth Restriction Journal of Maternal Fetal and Neonatal Medicine 2012: In Press. Presentations 1. Smith A, Garrod A, Batra G, Jones RL, Sibley CP, Heazell AEP. Quantitative assessment of placental morphology identifies specific causes of stillbirth and reduces the proportion of stillbirths of “unknown cause. Proceedings of the International Stillbirth Association Meeting, Baltimore, October 2012. 2. Heazell AEP, Smith A, Garrod A, Batra G, Sibley CP, Jones RL. Quantitative Assessment of Placental Morphology Identifies Specific Causes of Stillbirth and Reduces the Proportion of Stillbirths of “Unknown Cause” Fetal Growth, Birmingham, September 2012. 3. Girard S, Badger E, Heazell AEP, Dutton P, Allan SM, Jones RL. Placental Inflammation is Associated with Poor Pregnancy Outcome in Women with Reduced Fetal Movements. Proceedings of the SGI Summit, Brisbane, August 2012. 4. Heazell AEP, Smith A, Garrod A, Batra G, Sibley CP, Jones RL. Quantitative Assessment of Placental Morphology Identifies Specific Causes of Stillbirth and Reduces the Proportion of Stillbirths of “Unknown Cause” Proceedings of the SGI Summit, Brisbane, August 2012. 5. Badger E, Heazell AEP, Dutton P, Jones R. Placental Inflammation is Associated with poor pregnancy outcome in women with reduced fetal movements. Journal of Reproductive Immunology 2012;94(1):84. 6. Graham N, McVey R, Bullen P, Heazell AEP. Placental features of Mirror Syndrome – A Case Report. Arch Dis Child 2012;97(Suppl I): A55. Future Plans The proposed projects for 2013 extend our current research programme; particularly to understand the involvement of the placenta in stillbirth. The first project is to investigate which method of storage after stillbirth best preserves placental structure for analysis. We plan to take samples from ten placentas and store them in different methods, including refrigeration, storage in saline and storage in culture medium. We will then determine which method provides the best quality of tissue for later analysis. This will be carried out by Ainslie Garrod – research technician. Financial support for this project from HMSBRF includes salary and laboratory consumables. The second project will be conducted by Imogen Ptacek (intercalating medical student) as part of her MRes qualification in collaboration with Dr Paul Brownbill at MFHRC. These experiments will assess changes in placental structure occur after death of the baby. We will take placental tissue and tie off the fetal blood vessels so there is no flow in them; then we will perfuse the maternal side of the placenta for as long as possible (aiming about eight hours). We will then look at the placental structure to identify any changes. If present, these will inform us about placental changes after stillbirth. Then we will examine placentas from IUGR pregnancies comparing those which are stillborn and those which are live born using similar methods used in the quantitative analysis presented here. This will provide information to investigate whether cases of IUGR when the baby is stillborn have a more severely damaged placenta compared to those who are live born, or whether both placentas are damaged and it is the fetal response to placental dysfunction that differs.