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Maternal exposure to air pollution during pregnancy had been associated with adverse pregnancy outcomes, such as pre-eclampsia and fetal growth restriction (FGR), but the mechanism of this effect is poorly understood. Particulate matter (PM) is a ubiquitous air pollutant that is present both the indoor and outdoor environment and potentially bound with toxic materials such as heavy metals and/or polycyclic aromatic hydrocarbons (PAHs). Placental explant culture offers an in-vitro means to study PM induced changes in placental endocrine function and cellular turnover. This PhD explores the possible routes of air pollutant exposure and their mechanism influences on placental dysfunction that can cause adverse pregnancy outcomes. Placentas were collected following elective caesarean section (ELCS) at-term, between October 2017 and January 2019 at St. Mary's Hospital, Manchester, and 41 villous explants were established, but 12 were excluded, as 4 were contaminated and 8 were non-responsive. Initially, placental explants were exposed to diesel exhaust particles (DEP) for 48-hour from day 5 until 7 of culture, to determine if there was a short term effect of this air pollutant on hCG secretion, and the result showed that 48-hour treatment were not give a significant changed in hCG secretion between treated and control group. Informed with the short term effect, the duration of air pollutants treatment on explants in culture was extended over 6 days. Explants were treated with DEP and house dust (HD), to mimic maternal exposure to PM2.5 bound PAH. DEP treatment (SRM1650b; 24-hours/day; 6-days/week; mean DEP diameter ~166nm) significantly increased hCG secretion, increased 8-OHdG oxidative damage and promoted the formation of villi with an intact syncytiotrophoblast in a dose-dependent manner, whereas HD (SRM2585; 24-hours/day; 6-days/week; mean HD diameter ~166nm) significantly inhibited hCG secretion, increased the number of shed villi without regeneration and increased 8-OHdG oxidative damage. Differences in PAH levels between DEP and HD may be a reason for the difference in hCG secretion response and cellular turnover by placental explants. Analysis of PAH and heavy metal levels in placentas and maternal blood has been conducted to indicate the functional ability of the placenta to act as a protective barrier for the fetus. Placentas and maternal blood from 53 patients were collected between June 2015 and June 2017 at St. Mary's Hospital, Manchester. Heavy metals, as detected by an inductive coupled mass spectrometer (ICP-MS), were present in the majority of samples, except for Cr and Ni which not detected in maternal blood. Cu and Pb levels were higher in maternal blood, whereas Cd and Hg levels were higher in placenta samples. PAH levels, as determined by gas chromatography with mass spectrometric (GC-MS), were low in placental samples, with only 8 out of 12 samples containing detectable levels, and most of the positive samples (n=5/8) contained only one PAH. Amongst 10 PAH compounds, fluorene, fluoranthene and pyrene detected in 8, 3 and 3 samples, respectively, whereas other compounds were detected in only one sample. Low maternal exposure on ambient PM10 and PM2.5 levels throughout the gestation period, with median (IQR) levels of 17.6 (16.6 – 17.9)µg/m3 and 10.1 (9.3 – 10.8)µg/m3 respectively, as determined by air pollution monitoring stations, may be a reason for low heavy metal and PAH levels in the biological samples. The investigation of maternal exposure to air pollution extended with a feasibility study of indoor air particle measurement in 20 residential homes of pregnant women across Greater Manchester (GM), between February and August 2019. The majority (n=17/20) of residential homes contained indoor PM2.5 levels below 20µg/m3 , as measured by an air particle monitor (DYLOS DC1700) for 24-hour. Simultaneously, outdoor PM2.5 levels were estimated from air pollution monitoring stations, and the results showed that the outdoor PM2.5 levels in most residential areas (n=16/20) were < 20µg/m3 . The results suggest that pregnant women in GM were exposed to levels of outdoor PM10 and PM2.5 below WHO standard limits, such that these concentrations may not have a significant effect on placental function and pregnancy outcomes. In conclusion, interesting insights into the potential mechanism of pollution-related adverse pregnancy outcomes have been revealed by in-vitro experiments and the methods used in this study may be applied to future studies to investigate the effect of air pollution on placental function in other different aspects. However, this in-vitro investigation could not be compared with the real exposure to air pollution level, particularly in GM. Maternal exposure to air pollution in GM, at the current time, may not give a significant impact on adverse pregnancy outcomes. Further studies are needed to unravel the biological mechanism that underlies adverse pregnancy outcomes due to exposure to air pollution.

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.

A vital factor for foetal development is the nutrient transport at placental level. This is because any disturbances in the maternal compartments, for example due to maternal stress or nutritional status, which will affect foetal development, will involve the foetal-placental barrier. Moreover, numerous studies have linked other factors such as preterm labour as the leading cause of perinatal morbidity and mortality in the developed world. To this date, despite a numerous epidemiological and clinical studies that identify potential risk factors for the mother as well as the foetus, there is no comprehensive analysis at all these levels taken from the same cohort of patients. Our working hypothesis is that for a successful pregnancy certain events at nutritional, biochemical, genetic and molecular level could be tightly linked. Therefore, in this study we followed a “holistic” approach investigating how maternal stress, nutrition, placental mTOR and glucocorticoid receptor (GR) signalling can influence pregnancy outcome. We have decided to map in detail the components of these two signalling pathways as they appear to cross-talk as well as been implicated in stress responses. The largest part of the questionnaire was focused on the nutritional status with questions targeting the maternal dietary habits before, as well as during, pregnancy. The collection of data took place at the Department of Obstetrics and Gynecology, University of Crete Medical School. With regards to this profile, key findings included the significant reduction in the intake of alcohol, caffeine-containing and sugar-containing refreshments, whereas passive smoking during pregnancy stayed the same. Another major finding of this part of the study was the effects of maternal stress on foetal weight and how pregnancy planning was implicated in this complex relation. In our cohort, women with negative attitudes during pregnancy gave birth to infants with significantly lower birth weights (2.5Kg) than those women showing positive or neutral attitudes towards their pregnancy (2.9Kg). We then assessed how maternal stress might affect this signalling cascade using two placental models (BeWo and JEG-3 cell lines) mimicking a stress milieu in vitro. Treatment of these cell lines with cortisol (100nM and 1000nM) significantly downregulated Deptor and upregulated GAS5 at mRNA level. In an attempt to dissect further a potential gene-environment interaction, we have assessed how 4 well-characterised polymorphisms (ThtIII 1, Bcl I, ER22/23EK, N363S) of the GR gene might affect foetal and placental weight. We have demonstrated that only the maternal ThtIII 1 polymorphism was suggestive of a nature-nurture interaction since only in ThtIII 1 II (CC), maternal stress attitude predicts foetal weight-reduction, but not in ThtIII 1 (GC) independent of confounders such as BMI, pregnancy planning or fast food eating during pregnancy. This is the first time that a gene-environment interaction between a common GR polymorphism and foetal weight was noted. Finally, one of the most important findings of our study came from the preclinical studies using placental tissues. Quantitative PCR revealed that the major transcripts in the human placenta are GRα, GAS5 (decoy for GR DNA binding) and Deptor. We have shown for first time that there are marked differences in the relative mRNA abundance of these components between term and preterm labour as well as colocalisation of GRα with GAS5. With regards to placental regulation these data conclusively demonstrate that: a) there is evidence of gene-environment interaction between maternal stress, pregnancy planning, glucocorticoid receptor polymorphisms and foetal weight and b) potential cross-talk of mTOR and glucocorticoid signalling. We propose that measuring maternal stress levels in addition to circulating cortisol and mapping for known GR polymorphisms could become a useful non-invasive tool of diagnostic and prognostic value, with implications for preterm labour.

Abstract: Introduction: Pregnancy complications associated with prenatal hypoxia have been linked to the development of cardiovascular disease in adult offspring. Prenatal hypoxia (due to maternal or placental hypoxia) has been shown to increase placental oxidative stress and impair placental function in a sex-specific manner, thereby affecting fetal development. Studies suggest that prenatal hypoxia can reduce mitochondrial respiratory capacity, which in turn, could lead to placental dysfunction and impaired fetal organ development. Recently, we, together with our collaborators, have demonstrated that prenatal hypoxia-induced placental oxidative stress can impair development of key fetal organ system (such as the brain) through the release of placenta-derived factors into the fetal circulation. However, the effects of placenta-derived factors on fetal cardiomyocyte development are yet to be determined. Given that oxidative stress is central to placental dysfunction and altered fetal organ development, placental function may be improved by the use of an antioxidant. However, drug use in pregnancy is restricted due to potential adverse effects on the fetus should transplacental passage occur. Thus, the main objective of my PhD studies was to improve placental function without direct drug exposure to the fetus in order to avoid off-target effects during development. I assessed a placenta-targeted innovative treatment strategy using a mitochondrial-targeted antioxidant (MitoQ) encapsulated into biodegradable nanoparticles (nMitoQ) as a delivery system, which diffuses into the placental syncytium to release the antioxidant treatment within the placenta without crossing the placental basal membrane to reach the fetus. I tested whether this placenta-targeted nMitoQ treatment targeted in hypoxic dams improved placental oxidative stress and placental morphology and function. In addition, I assessed whether nMitoQ treatment in hypoxic dams prevents fetal programming of cardiac diseases via the release of placenta-derived factors. Methods: Pregnant Sprague-Dawley rats were intravenously injected with saline or nMitoQ (100µl of 125 μM) on gestational day (GD) 15 and exposed to either normoxia (21% O2) or hypoxia (11% O2) from GD15-21. On GD21, placentae from both sexes were collected for detection of oxygenation, superoxide, nitrotyrosine, nitric oxide, CD31 (endothelial cell marker), and maternal and fetal blood spaces, Vegfa and Igf2 expression in the placental labyrinth zone. In a second set of experiments, on GD21, male and female placental labyrinth zones were collected for mitochondrial functional assessments. To assess the effects of placenta-derived factors on fetal cardiomyocyte development in vitro, on GD21, male and female placentae were harvested, placed in culture, and conditioned media (containing placental-derived factors) was collected after 24h. Male and female cardiomyocytes from control dam fetuses were incubated with same sex placental conditioned media. Results: nMitoQ treatment reduced the prenatal hypoxia-induced increase in placental superoxide levels in both male and female placentae but improved oxygenation in only female placentae. Without altering nitric oxide levels, nMitoQ treatment reduced nitrotyrosine levels in hypoxic female placentae. Prenatal hypoxia reduced placental Vegfa and Igf2 expression along with reduced maternal and fetal blood spaces and fetal capillaries area in the labyrinth zone from both sexes, while nMitoQ increased Vegfa and Igf2 expression only in hypoxic female placentae. Prenatal hypoxia reduced mitochondrial complex IV activity in male placentae, which was improved by nMitoQ treatment. Interestingly, in females, nMitoQ had no effect on placental mitochondrial function, but prenatal hypoxia increased contribution of the N-pathway (through complex I) and decreased contribution of the S-pathway (through complex II). nMitoQ treatment led to increased oxygenation in hearts and livers of hypoxic female fetuses, whereas oxygenation was improved in hearts but not livers of nMitoQ treated prenatally hypoxic male fetuses. Furthermore, factors derived from hypoxic placentae of dams treated with nMitoQ prevented increases in the percentage of binucleated cardiomyocytes (marker of terminal differentiation) and the size of mononucleated and binucleated cardiomyocytes (sign of hypertrophy). Conclusion: In summary, my studies suggests that treatment strategies targeted against placental oxidative stress can improve placental function in complicated pregnancies and may prevent fetal programming of cardiac disease. However, the mechanisms (i.e. changes in placental morphological and mitochondrial functional capacity) were distinct between males and females. Thus, sex differences need to be taken into account when developing placenta-targeted therapeutic strategies to improve fetal development in complicated pregnancies.