During the last decades, evidence has accumulated suggesting that environmental exposures to adverse life events might critically alter the developmental trajectories of infants’ development and mental health by contributing to the epigenetic regulation of stress-related genes [1, 2]. Pregnancy is a sensitive period for women’s mental health [3, 4] as well as for the embedding of environmental exposures into the developmental phenotype of infants [5]. Concerning this latter, previous studies showed that timing and severity of the exposure during gestation are likely to influence the nature and degree of these effects [6], providing further support for the notion that fetal brain development is characterized by a sequence of sensitive time windows, during which specific biological structures and systems, usually undergoing rapid developmental change at that given point, are particularly vulnerable to environmental influences. Thus, for example, studies on the risk for schizophrenia indicated that the most sensitive period for prenatal exposure to adversity was the first trimester [7] when the processes of neuronal migration occur. In contrast, strongest effects of antenatal adverse exposure on cognitive outcomes [8], stress regulation [9, 10][9, 10] and offspring’s emotional problems [11] have been more frequently reported in the late pregnancy when rapid fetal brain development, including synaptic migration and the beginning of synaptic differentiation, takes place.
Early stressful or traumatic events that occur during the first thousand days – and especially during pregnancy – might leave stable signatures in the epigenome of mothers and infants [12, 13]. Changes in the DNA methylation status of specific portion of stress-related genes are among the most studied epigenetic signatures of the early exposures to adverse life events. DNA methylation is fostered by DNA methyltransferase enzymes (DNMTs) that are responsible for the binding of the methyl group from donor S-adenosyl-methionine onto the 5’ position of the CpG dinucleotide. DNA methylation occurring in regions relevant for gene regulation and expression, characterize by high density of CpG sites (i.e., CpG islands) are of great concern for researchers and clinicians. These regions include exons, promoter regions, and enhancers. Nonetheless, methylation occurring in other regions is thought to interact with other epigenetic mechanisms and to contribute to the emergence of increased or reduced transcriptional sensitivity. For instance, higher methylation in both exonic and intronic regions is effective in recruiting histone deacetylases, leading to gene silencing. DNA methylation usually alter the accessibility of a gene coding region to the molecular transcriptional agents and could lead to altered expression and a consequently altered availability of proteins that are necessary for the adequate development of individuals. The methylation dynamics of genes encoding for the glucocorticoid receptor (NR3C1) and the serotonin transporter (SLC6A4) are key targets of behavioral epigenetics, as they are deeply involved in stress regulation and mental health.
The hypothalamic-pituitary-adrenal (HPA) axis is key in regulating the mobilization of energy in the organism [14]. It is involved in stress reactivity and regulation and it supports the development of behavioral, cognitive, and socio-emotional domains as well as mental health. The HPA axis response to challenging and stressful conditions involves a cascade of hormonal activations that finally leads to secreting glucocorticoids (cortisol in humans) from the adrenal glands. Cortisol secretion is regulated by feedback mechanisms that involve the activation and binding of the hormone to specific glucocorticoid receptors (GRs) in the brain. The NR3C1 gene encodes for specific GRs in the mammalian brain and is epigenetically regulated by environmental exposures. The epigenetic regulation of the NR3C1 gene is highly sensitive to environmental adverse and protective conditions during sensitive periods, including pregnancy. Available evidence shows that the methylation status of NR3C1 gene in cord blood was predicted by maternal psychological distress [15–17], experiences of war-related stress [18, 19] and partner violence [20] during pregnancy.
The serotonergic neurotransmission is also known to affect a wide range of developmental outcomes in infants and mental health in adults. In humans, serotonin is located both in the central nervous system and in peripheral tissues. In the brain, it is located in the neurons of the median and dorsal raphe nuclei, in the cortex, and in the hippocampus. The amygdala, hypothalamus, and the pituitary adrenal gland, which are deeply involved in stress regulation mechanisms, are densely innervated by serotonin neurons. The serotonin transporter is a crucial regulator of the serotonergic system: it removes the serotonin released in the synaptic cleft and it is synthesized by a specific gene, namely the SLC6A4. The expression of this gene is regulated by polymorphic allelic variants and epigenetic mechanisms. A systematic review has recently reported on the potentials of the SLC6A4 gene’s methylation as a biomarker of exposure to life adversity which also associates with less-than-optimal outcomes in children development and adults’ mental health. However, few studies have investigated the link between antenatal exposure to adversity and SCL6A4 gene methylation and these have yielded inconsistent findings. Either negative [21], positive [22] or null [23] associations between maternal antenatal psychological distress and infant SLC6A4 methylation patterns have been reported.
The COVID-19 pandemic dramatically hit northern Italy at the beginning of 2020 [24]. With the increasing number of patients requiring intensive care and with unprecedented mortality rates associated with this clinical condition, the COVID-19 pandemic rapidly grew as a potential collective trauma. The effects of pandemic-related stress have been already highlighted in healthcare professionals working at the forefront of the pandemic emergency [25, 26] as well as in the general population [27]. Moreover, individuals who are experiencing periods of heightened neuroplasticity might be especially sensitive to environmental stress and life adversity. Not surprisingly, a large number of studies are documenting detrimental associations of pandemic-related stress with both infants’ developmental outcomes and maternal mental health indexes. Epigenetic pathways are likely to play a role in mediating the impact of exposure to the pandemic during gestation on maternal and fetal phenotypes, yet they are still largely unknown. It is important to obtain information on the potential epigenetic pathways underlining these associations in order to increase our knowledge of the neurobiology of stress. Furthermore, major stressful events, such as the pandemic or natural disasters, provide the unique opportunity to test for timing effects [28], which are rarely addressed in studies examining the epigenetic underpinnings of antenatal stress exposures. Obtaining information on time periods during pregnancy differentially sensitive to epigenetic regulation by life adversities is key to provide innovative evidence basis to the programming of preventive and care actions during and after the pandemic time.
In the present study, we report on NR3C1 and SLC6A4 methylation in mothers and infants who were exposed to the initial stage of COVID-19 pandemic in Northern Italy – the first lockdown period – between March and May 2020. We focused on the first lockdown period as this constituted an acute major stressful event for the Italian population with a clear and sudden onset and characterized by features of uncertainty, fear and lack of social support [29]. This provides greater leverage for exploring timing effects of pandemic stress-related exposure across gestation on maternal and infant epigenome. More specifically, we assessed the presence of significant differences in NR3C1 and SLC6A4 methylations among those individuals – both mothers and infants – who experienced the lockdown period during different trimesters of pregnancy.