Search

Advanced maternal age (AMA) is defined as childbearing in mothers ≥35 years of age and is becoming increasingly prevalent in high income countries. AMA has been associated with increased risk of adverse pregnancy outcomes, particularly stillbirth. Although AMA mothers have higher rates of chromosomal abnormalities and maternal co-morbidities, AMA remains an independent risk factor for stillbirth. Despite these findings, the etiology behind this increased risk is unknown. We hypothesise that an altered maternal environment, including increased oxidative stress and inflammation, due to ageing causes placental dysfunction which increases AMA mothers’ vulnerability to stillbirth.A holistic approach was applied to investigate placental dysfunction in AMA. Firstly, a systematic review and meta-analysis comprehensively reviewed existing data on AMA and associated adverse pregnancy outcomes. Secondly, Manchester Advanced Maternal Age Study (MAMAS), a multi-centre prospective observational cohort study, was conducted to investigate risk factors for composite adverse pregnancy outcome (CAPO) in AMA. MAMAS utilised both uni- and multivariate analysis on demographic and clinical data, and measuring biomarkers of ageing and placental dysfunction by ELISA in maternal circulation during the third trimester of pregnancy. Utero-placental dysfunction was directly investigated in uncomplicated AMA pregnancies by quantifying placental morphology, placental nutrient transport capabilities and both placental and maternal uterine vascular responses. Finally, a C57BL/6J murine model of AMA was developed and characterised to further investigate maternal age on pregnancy outcome and the role of the placenta. In the meta-analysis, maternal age was linearly associated with increased risk of stillbirth and other adverse outcomes strongly associated with placental dysfunction (fetal growth restriction, preeclampsia and placental abruption). In MAMAS, smoking status and primiparity were predictive of CAPO. After adjustment, AMA mothers had an odd ratio of 2.05-3.43 of CAPO compared to 20-30 year old mothers. AMA mothers showed evidence of increased oxidative stress and pro-inflammatory bias. AMA mothers who suffered CAPO showed reduced placental endocrine capacity seen in placental dysfunction. Placentas from uneventful AMA pregnancies showed evidence of accelerated ageing and placental adaptation with increased nutrient transport, increased placental weight but reduced efficiency, and altered vascular function. AMA mice showed many similar aspects to human AMA with increased fetal loss, fetal growth restriction and increased placental size. These studies provide robust evidence for increased incidence of adverse pregnancy outcome due to placental dysfunction in pregnancies of women of AMA. This finding requires the appropriate recognition in a clinical context, with a greater focus on personalised obstetric care in an attempt to reduce stillbirth rates in this high risk population. By optimising antenatal and obstetric care for AMA mothers, we could reduce stillbirth rates by 4.7% - the population attributable risk due to AMA. These studies highlight key areas of future research that will further understanding into stillbirth risk in AMA pregnancy, test predictive models and test therapies and clinical care interventions an ultimately improve pregnancy outcome in mothers of AMA. Pregnant mothers over the age of 35 years old are classified as advanced maternal age (AMA). AMA is known to be associated with increased risks in pregnancy, including the baby being born too small or dying before birth (stillbirth). This risk of stillbirth in AMA is even true independently of the genetic defects in the eggs and maternal diseases that are more often seen in these women due to their age. However, it is not know why this increased risk of stillbirth occurs in AMA mothers. The placenta is a vital organ in pregnancy that regulates blood and nutrient supply to the baby and is known to not work properly (known a placental dysfunction) in pregnancies where the baby doesn’t grow properly or is stillborn. We propose that changes in the mother due to her age will affect the way that her placenta works and this is the reason why more babies are born small or are stillborn in AMA mothers compared to younger mothers. Firstly, we reviewed existing studies to see how many have found evidence of pregnancy diseases known to be caused by placental dysfunction in AMA pregnancies. We set up a clinical study called Manchester Advanced Maternal Age Study (MAMAS) and recruited pregnant mothers to investigate lifestyle risk factors for having poor outcomes (with placental links) in AMA. We collected mothers’ blood to see if higher levels of markers of ageing were detectable in AMA and if they were related to increased risk of poor outcome. The placentas from these mothers were used to measure their size, whether the cells were dividing or dying and how well they could transport nutrients from mother to baby. Placental and uterine vessels were tested to see how well they can control blood flow to the baby during pregnancy. Finally, a mouse model of AMA was developed and characterised to further investigate maternal age on pregnancy outcome and the role of the placenta.Previous studies showed that AMA was more strongly linked to increased risk of pregnancy outcomes that are associated with placental dysfunction than those that are not. In MAMAS, smoking and carrying a first pregnancy made mothers and being AMA increased risk of suffering a poor outcome. AMA mothers who had normal outcomes showed evidence of increased ageing markers but also increased mechanisms to counteract ageing related damage in their blood. AMA mothers who had poor outcome had higher levels of age related damage and inflammatory responses to that damage. They had lower levels of placental hormones indicating placental dysfunction. Placentas from AMA pregnancies had to work harder to keep the baby healthy by increasing their size, nutrient transport and altering placental and maternal vascular function. However, they still functioned less efficiently than placentas from younger mothers. Our mouse model of AMA showed many similar aspects to human AMA with small pups, high rates of stillbirth and larger placentas. These studies provide robust evidence for placental dysfunction can explain why mothers over the age of 35 years are more at risk of having small babies or stillborn babies. This finding should be recognised by doctors and midwives when deciding how to manage their pregnancies. If so, we could reduce national stillbirth rates by 4.7%. The mouse model offers a way for us to test interventions in AMA pregnancies to improve their outcome.

Scientific abstract of thesis entitled "Effect of Folate Deficiency on Placental Function" submitted by Bernadette Carolyn Baker for the degree of PhD from the University of Manchester, August 2015.Insufficient maternal folate during pregnancy increases the risk of the baby being small for gestational age (SGA). Studies in teenagers, a population vulnerable to folate deficiency and SGA birth, have shown that low maternal folate status is associated with impaired placental cell turnover and reduced transport suggesting placental dysfunction underlies SGA in maternal folate deficiency. Mechanisms through which folate-depletion compromises placental function are currently unknown. In non-placental cells, folate modulates microRNAs (miRs), post-transcriptional regulators of cellular functions. Expression of miRs is altered in placentas of SGA compared to normally grown babies but there are no data on differential miR expression or regulation in placentas from folate deficient women.This PhD investigated the hypothesis that placental dysfunction observed in folate deficient women is mediated by altered miR expression. Three placental preparations were compared (villous tissue in explant culture, BeWo choriocarcinoma cells and isolated cytotrophoblast cells) to determine the optimum in vitro system to study the direct effects of folate deficiency. In cytotrophoblast cells, folate deficiency significantly elevated apoptosis and reduced the activity of the system A amino acid transporter, consistent with observations in the placentas of folate-deficient teenagers. The reduction in system A activity by low folate was not associated with altered mRNA expression for the isoforms of system A, implicating an effect of low folate on post-translational regulation of the nutrient transporter. Targeted examination of villous tissue from teenagers with low folate status identified up-regulation of miR-222-3p a folate-sensitive miR. An unbiased miR array identified up-regulation of a further 16 miRs suggesting that maternal folate deficiency in vivo results in aberrant placental miR expression. Bioinformatic analysis of the folate sensitive miRs predicted gene targets known to be altered in placentas from SGA pregnancy that were likely to alter placental function. Two miRs altered in placentas from women with low folate status, miR-30e-3p and miR-34b-5p, were also significantly altered in folate deficient cytotrophoblasts confirming a direct effect of folate on trophoblast miR expression. Inhibition of these miRs in vitro had no effects on placental functions that are altered in vivo in folate-deficient women. Gene array and in silico analysis identified functional endpoints affected by these folate sensitive miRs, including cell signalling for proliferation and survival and oxidative stress, which might contribute to placental dysfunction in folate deplete women. Overall, this study has demonstrated for the first time that folate deficient conditions can directly alter trophoblast system A transport and cell survival and thus could contribute to the increased susceptibility to SGA births in folate deficient women. It has also contributed to the knowledge that miR expression is differentially altered in placentas exposed to folate-deficient versus sufficient conditions in vivo and that miRs are directly altered by folate depletion in vitro. These studies provide the foundation for future research to define the functional consequences of altered expression of folate-sensitive miRs and their target genes to explain how altered miRs could be affecting placental function resulting in development of SGA.

Pregnancies complicated with diabetes mellitus (DM) are associated with poor maternal and fetal outcomes, such as birth trauma, fetal overgrowth (macrosomia) and programming of the fetus to develop metabolic syndrome in adult life. Maternal hyperglycemia is thought to contribute to fetal macrosomia, however the role of the placenta in these pregnancies is incompletely understood, therefore we aimed to investigate the specific consequences of high glucose on placental metabolism. To achieve this aim an in vitro model of placental exposure to high glucose was developed. This model was used with the aim of analysing how high glucose alters the transcriptome and metabolome of these cells, using a systems biology approach to identify candidate functional pathways which may be altered in placenta as a result of hyperglycemia. These candidate functional pathways were validated in an ex vivo model of placenta exposed to high glucose and in placental tissue from pregnancies complicated by DM. A trophoblast cell line (BeWo) was cultured in low (5 mM) and high (12 mM or 25 mM) D-glucose conditions for 48 hours. Transcriptomic and metabolomic analysis of these cells was performed using microarrays, and gas- and liquid-chromatography-mass spectrometry, respectively. Transcript and metabolite changes were independently analysed and integrated, using network analysis. From the integrated analysis of the ‘omic datasets, β-fatty acid oxidation (β-FAO), purine metabolism, phosphatidylinositol/PI3K phosphate pathway and lipid metabolism, were identified as candidates for further study. Changes within the PI3K pathway and lipid metabolism/β-fatty acid oxidation were validated in an ex vivo placental explant model of high glucose and in placental tissue from women with DM, compared to uncomplicated pregnancies. mRNA, protein expression and protein activation of key molecules within the PI3K pathway were not significantly altered in placenta as a response of high glucose ex vivo or DM in vivo. The second candidate functional pathway, lipid metabolism, has previously been implicated in association with placental dysfunction in pregnancies complicated by DM. Placental fatty acid transporter and lipase protein expression, as well as, relative abundance of different fatty acids were unaltered in response to high glucose or DM. High glucose levels increased triglyceride levels within the placenta, indicating reduced rates of β-FAO. The effect of high glucose could be ameliorated using a PPARα agonist. This may provide a novel therapeutic intervention to prevent excess esterification of fatty acids to triglycerides in maternal diabetes, which may in turn influence fetal growth.This study illustrates how a systems biology approach can be used to identify novel candidate functional pathways that are altered within the trophoblast in response to high glucose. Thus, improving understanding of placental dysfunction in these pregnancies and providing novel candidate pathways for future study, which may represent potential therapeutic targets for intervention of fetal macrosomia in pregnancies complicated by DM. CD of Excel Worksheets (Appendix b)Containing:1.Gene changes from the microarray1a. All microarray data with no cut-offs1b. Gene changes for 5 mM D-glucose following 24 hours of culture compared to 5 mM D-glucose following overnight culture (Time 0); fold change cut-off ±1.3.1c. Gene changes for 12 mM D-glucose following 24 hours of culture compared to 5 mM D-glucose following overnight culture (Time 0); fold change cut-off ±1.3.1d. Gene changes for 25 mM D-glucose following 24 hours of culture compared to 5 mM D-glucose following overnight culture (Time 0); fold change cut-off ±1.3.1e. Gene changes for 12 mM D-glucose following 24 hours of culture compared to 5 mM D-glucose following 24 hours of culture; fold change cut-off ±1.3.1f. Gene changes for 25 mM D-glucose following 24 hours of culture compared to 5 mM D-glucose following 24 hours of culture; fold change cut-off ±1.3.1g. Summary of gene changes for 12 mM and 25 mM D-glucose following 24 hours of culture compared to 5 mM D-glucose following 24 hours of culture; fold change cut-off ±1.3.2. Transcriptome analysis datasets2a. Transcriptome analysis of gene changes for 12 mM D-glucose following 24 hours of culture compared to 5 mM D-glucose following 24 hours of culture. 2a.1. Inferred network analysis 2a.2 Non-inferred network analysis2b. Transcriptome analysis of gene changes for 12 mM D-glucose following 24 hours of culture compared to 5 mM D-glucose following 24 hours of culture 2b.1. Inferred network analysis 2b.2 Non-inferred network analysis2c. Transcriptome analysis of gene changes unique to different levels of high glucose 2c.1. Analysis of genes unique to 12 mM compared to 5 mM D-glucose2c.2. Analysis of genes unique to 25 mM compared to 5 mM D-glucose3.Metabolome analysis datasets3a. Footprint metabolites identified using GC-MS3b. Fingerprint metabolites identified using GC-MS3c. Footprint metabolites identified using LC-MS3d. Fingerprint metabolites identified using LC-MS4.Summary of network analysis from the transcriptome and metabolome datasets4a. Master spreadsheet of all data PDF copy of publication arising from this thesis.Hulme C. H., Westwood M., Myers J.E., Heazell A.E. (2012). A high-throughput colorimetric-assay for monitoring glucose consumption by cultured trophoblast cells and placental tissue. Placenta, 33 (11), 949-51.

Pre-eclampsia and intrauterine growth restriction (IUGR) are common complications of pregnancy with significant implications for mother and fetus. The exact cause is unknown, but current theories suggest that abnormal trophoblast invasion leads to placental under perfusion and hypoxia with the generation of reactive oxygen species (ROS). A result of this insult is increased trophoblast apoptosis. The cell cycle regulator p53 has been identified in increased amounts in the trophoblast in pre-eclampsia and IUGR. It is possible that modulation of this potent apoptotic signal could improve placental function, potentially offering a therapy for these presently untreatable conditions.In these studies, trophoblast apoptosis was induced with Nutlin-3, a synthetic compound which inhibits the binding of p53 to its inhibitor, Mdm2, therefore acting as a p53 activator. Treatment with Nutlin-3 induced apoptotic cell death in BeWo choriocarcinoma cells, primary trophoblast cells and placental villous explants, suggesting that p53 is intrinsic to trophoblast survival. This increase in apoptosis was associated with an increase in p53, Mdm2, p21 and Puma protein expression in BeWo cells and p21 and Puma mRNA in placental villous explants. No effect was observed in Bax expression. Co-treatment with Nutlin-3 and the p53 inhibitor Pifithrin-α reduced apoptosis to the level of control tissue in BeWo cells, primary trophoblast cells and villous explants, suggesting that reducing p53 activity improves trophoblast function.The use of hypoxia and ROS was associated with an increase in apoptosis and p21 and Puma in placental villous explants. Furthermore, the extent of apoptosis and p21 and Puma expression was reduced by co-treatment with Pifithrin-α, suggesting that the ameliorating effects of Pifithrin-α on trophoblast apoptosis are maintained following an insult from a less specific p53 activating mechanism, further supporting the continued investigation of p53 modulation as a therapeutic manipulation of placental function.Pregnant mice were exposed to Nutlin-3 to ascertain the effect of p53 over-activity on pregnancy outcome. The establishment of a model of placental dysfunction in the mouse would have allowed investigation into the effect of p53 inhibition with Pifithrin-α. Despite clear evidence of p53 modulation in maternal tissues, the decidua, no effect was observed upon pregnancy outcome or upon fetal or placental tissue. These findings suggest that the mouse placenta may not be a good model of p53 over-activity, although the exact mechanism of resistance remains unclear.In conclusion, p53 exaggeration is inducible in trophoblast with Nutlin-3, hypoxia and ROS. Furthermore, Pifithrin-α is able to reduce this effect, suggesting that the p53 pathway may be a realistic target for modulation in the placenta. The pregnancy complications pre-eclampsia and intrauterine growth restriction (IUGR) represent significant risks to both mother and fetus. At present, delivery remains the only viable treatment for these conditions. Premature delivery poses significant risks to the baby and predisposes to the development of chronic diseases in adulthood; there are also significant financial costs to the health service. The exact cause of pre-eclampsia and IUGR is unknown. However, it is suggested that poor invasion of maternal tissues and a failure of vessel remodelling by the developing trophoblast (placenta cells) may be of fundamental importance. Poor placental blood flow subsequently promotes trophoblast cell death via a process called apoptosis. A key protein in the regulation of apoptosis is p53, which is increased in pre-eclampsia and IUGR. The use of Nutlin-3, a p53 activator, should theoretically increase apoptosis in trophoblast, whilst the p53 inhibitor Pifithrin-α should reduce apoptosis offering a potential therapy for exaggerated trophoblast apoptosis.Exposure of BeWo choriocarcinoma cells and isolated primary trophoblast cells to Nutlin-3 increased apoptosis and p53 transcription target protein expression, suggesting that p53 activity is inducible in trophoblast. Co-treatment of these cells with the p53 inhibitor Pifithrin-α reduced apoptosis to the level of control suggesting that modulation of p53 within the trophoblast is feasible. Similar findings were also observed in fragments of placental tissue.Exposure of placental tissue to hypoxia or reactive oxygen species (ROS) also induced apoptosis and targets of the p53 pathway. Again, treatment with Pifithrin-α reduced this effect, suggesting that p53 modulation with Pifithrin-α is possible even following a pathological insult.The use of Nutlin-3 in mouse pregnancy did not cause an alteration in pregnancy outcome and had no effect upon the placenta suggesting that the mouse may not be a good model of trophoblast p53 activity in the human.The development of effective ways of reducing excess trophoblast apoptosis by targeting the p53 pathway may prove to be a viable therapy in these common conditions, potentially improving outcomes for affected babies.