Study on epigenetic modifications in human placenta and buccal mucosa cells of mothers and newborns

This work was inspired by the hypothesis of David Barker, who first supposed the potential role of early life environmental exposure on the risk of developing later in life complex diseases. About 20 years later, his considerations are now a widely validated theory known as DOHaD theory. Indeed, this intriguing and novel research field has been explored in a plethora of different scientific works aimed at investigating the variety of exposure factors and of environmental cues that can influence mother health and consequently the life in the womb. Undoubtedly, placenta reveals its central role in receiving and mediating the adaptative responses to these external stimuli. Thus, placental fisology allow the correct fetal programming based on the conditions in which the new life will grow. AIM: Coherently with this perspective, the present thesis aimed at investigating how methylation - at both global and gene specific levels - can be influenced by the environment during gestation. METHODS: Gene-specific methylation levels of 13 candidate genes involved in different cellular pathways were established using MS- HRM technique, while the total percentage of global 5mC and 5hmC contents were investigated using ELISA assays. The epigenetic analyses were performed on a cohort of 26 pregnant women recruited in Tuscany, one of the Italian regions involved in a multicentric project funded by the Italian Ministry of Health. Two different tissues were collected: buccal epithelial cells both from the mothers and their neonates, as well as placental tissues, allowing to obtain information from both the peripheral tissues and from the organ tissue directly involved in fetal development. Data concerning maternal lifestyle, stressful events and exposure occurred during the pregnancy period were obtained after the administration of a questionnaire. Additionally, the presence of heavy metals, dioxins and PCB were detected in placenta samples by the IZSLER Institute of Bologna. RESULTS: Our data suggest that maternal age can influence both gene-specific methylation levels – maternal LEP gene positively and maternal H19 gene negatively – and global 5methyl cytosine content in maternal buccal cells, that decreases with maternal age. Maternal BMI also emerges in this work as a negative modulator of placental MTHFR gene methylation, while gestational age was found positively related with maternal BDNF promoter methylation. The contribution of the sex of the babies on methylation of CYP1A1 and MECP2 genes also emerged as girls show higher methylation levels than boys in buccal cells (p= 0,0000 and p=0,0299 respectively). High levels of BDNF methylation were also detected in women who experienced stressful events during pregnancy (p = 0,0416) as well as an hypomethylation of HSD11B2 gene in response to passive smoke exposure. On the other hand, placenta shows a higher global methylation in women who contracted influenza or presented fever during gestation (p = 0,0002). From the analyses of the heavy metals (Mn, Pb, Cr, As, Hg, Cd and Ni; mg/Kg, wet weight) and dioxin detection on placental tissues, methylation levels of several genes including OXTR, DNMT3B,CYP1A, LEP and H19 were found responsive to exposures in all the tissues considered. CONCLUSION: The first appreciable data within this project reveal the role of genes involved in cellular pathways capable of perceiving and responding to different stressors. The gene expression remodelling is critical in the early stages of neuronal development. This can contribute to the onset of pathologies related to neurodevelopment - and often characterized by a different incidence between the two sexes - or predisposing the unborn child to them. Our results suggest interesting responses of the methylation patterns to the maternal and uterine environment, as well as to the exposure to toxic compounds. Undoubtedly, the limitation of our study concerns the sample size obtained so far. However the grouping of the different cohorts, which will be done at the end of the whole recruitment will be fundamental and will allow corroborating the findings obtained until now. Moreover, data concerning the analyses from the other Units will better contextualize all the exposures observed. It will allow also an overview of the pathways involved, that are responsive to different cues and exposure observed. Additionally, the follow-up of the babies likely will give us the first information on how these early exposures can affect the early fetal programming.