Transgenerational impacts of early life adversity: from health determinants, implications to epigenetic consequences

Exposure to different environmental factors, social and socioeconomic factors promotes development of the early-life adversity (ELA) phenotype. The persistence of this phenotype across generations is an interesting phenomenon that remains unexplored. Of late many studies have focused on disease-associated outcomes of ELA following exposure during childhood but the persistence of epigenetic imprints transmitted by ELA exposed parents to their offspring remains poorly described. It is possible that both parents are able to transmit ELA-associated genetic imprints to their offspring via transgenerational inheritance mechanisms. Here, we high-light the role of the mother and father in the biological process of conception, from epigenetic reprogramming cycles to later environmental exposures. We explain some of the known determinants of ELA (pollution, socio-economic challenges, infections, etc.) and their disease-associated outcomes. Finally, we highlight the role of epigenetics, mitochondria and ncRNAs as mechanisms mediating transgenerational inheritance. Whether these transgenerational inheritance mechanisms occur in the human context remains unclear but there is a large body of suggestive evidence in non-human models that points out to its existence.


Introduction
Early-life adversity (ELA) is associated with a higher risk for chronic diseases later in life such as autoimmune diseases, allergy or asthma (Elwenspoek et al., 2017a).Additionally, mental disorders such as depression and anxiety are associated with ELA (Malave et al., 2022).It is known that early-life adversities play a role in the onset of disease later in life, and therefore influence the entire health trajectory.The persistence of certain disease-associated traits in the germ line and their inheritance across generations after exposure to ELA suggests that the phenotype can be transmitted over several generations.Over the last decade, transgenerational inheritance has gained prominence as more studies are starting to investigate its role in transmitting the ELA phenotype across generations.Transgenerational inheritance represents Abbreviations: DNAm, DNAmethylation; ELA, Early life adversity; 8OHdG, 8-hydroxydeoxyguanosine; IGF2, Foetal insulin-like growth factor; NcRNAs, Noncoding RNAs; PGCs, Primordial germ cells; HP1, Heterochromatin protein 1; MeH3, Methylated histones HE; DMNT1, DNA methyltransferase 1; SES, Socioeconomic factors; Dnmt3a, DNA methyltransferase 3 alpha; CM, Childhood maltreatment; CSA, Childhood sexual abuse; HPA, Hypothalamic-pituitary-axis; CRP, C-reactive protein; ATP, Adenosine triphosphate; ELM, Early life microbiome; HMOs, Human milk oligosaccharides; SCFAs, Short-chain fatty acids; STAT1, Signal transducer and activator of transcription 1; ELI, Early life infection; ESR, Erythrocyte sedimentation rate; Th, T-helper; AEDs, Anti-epileptic drugs; VPA, Valproate acid; ASD, Autism spectrum disorder; PAHs, Polycyclic aromatic hydrocarbons; EDCs, Endocrine disrupting chemicals; ALSPAC, Avon longitudinal study of parents and children; WHO, World health organization; CGIs, CpG islands; ROS, Reactive oxygen species; MtDNA, Mitochondrial DNA; OP, Oxidative phosphorylation; ICRs, Imprinting control regions; McRNA, Non coding RNAs; SiRNA, Interfering RNA; MiRNAs, MicroRNAs; PiRNA, Piwi-interacting RNA; MEI, meiotic epigenetic inheritance; CRE, cis-regulatory element.a series of altered phenotypes occurring during the second or third generation without re-exposure to environmental stressors (Perez and Lehner, 2019).Conversely, when the altered phenotypes are only observed for a shorter timescale in the first generation, we refer to them as intergenerational transmissions.Intergenerational transmissions frequently occur when a generation is re-exposed to the same environmental conditions as its preceding generation (Kleeman et al., 2022).
Transgenerational inheritance depends of the epigenetic reprogramming which occurs during embryogenesis as the fertilised oocyte develops into a zygote and later becomes a foetus.During this process, both maternal and paternal genomes undergo two rounds of DNA methylation reprogramming (Potabattula et al., 2018).However, epi-mutations can also occur during this step leading to disease development.The first reprogramming round corresponds to the demethylation of genomic DNA within primordial germ cells during early embryogenesis, followed by the restoration of methylation patterns during somatic development.
Embryonic epigenetic reprogramming allows for the removal of parental epigenetic marks or signatures acquired during their developmental exposure to environmental stressors.However, if the germline reprogramming fails, these marks can be conserved and transmitted from one generation to the next (Heard and Martienssen, 2014).Even though the process remains poorly described, there are a handful of studies that highlight the potential role of biological processes involved in transgenerational inheritance.Indeed, Xavier et al. suggested that oxidative stress could affect the transgenerational-inherited information (Xavier et al., 2019).Oxidative stress results in the chemical modification of guanine bases leading to the formation of the mutagenic base, 8-hydroxydeoxyguanosine (8OHdG).In spermatozoa cells exposed to oxidative stress, the formation of 8OHdG is associated with birth defects and miscarriages (Chabory et al., 2009).This DNA damage associated DNA modification, in addition to incomplete or inefficient DNA repair by the oocyte, directly impacts the health of the next generation.Oxidative stress also plays a pivotal role in mediating epigenetic gene silencing by prompting hypermethylation of promoter regions (Campos et al., 2007).From these previous studies, it is indeed evident that oxidative stress occurring in the germline induces "variability" which can potentially be transmitted across generations (Xavier et al., 2019).
As opposed to the general notion that the maternal germline forms the bedrock of transmittable traits across generations, evidence from previous studies suggests that differential methylation in the paternal germline is also associated with in utero malnutrition or obesity in mice models (Radford et al., 2014).Furthermore, it has been demonstrated that after exposure to a specific diet, metabolic changes are induced in the offspring via the paternal germline (Daxinger and Whitelaw, 2012).Indeed, in murine models paternal low-protein or high fat diet leads to a differential gene regulation and metabolic disorders in the offspring (Carone et al., 2010).Moreover, an epigenetic asymmetry between parental genomes have been observed.For example, the foetal insulin-like growth factor (IGF2) is transmitted by the paternal genome and the offspring phenotype shows growth deficiency.However, when transmitted by the maternal genome, the phenotype of the offspring remains normal (DeChiara et al., 1991).
The presence of transgenerational epigenetic inheritance (TEI) in man has been convincingly demonstrated in descendants of both holocaust and Dutch hunger winter survivors.The children of the Dutch hunger winter survivors that were exposed in-utero to famine conditions during their third trimester of development were not only lighter and, smaller at birth than those immediately before the famine, but these babies and their children went on to have a higher risk of cardiovascular and, metabolic diseases, with notably, impaired glucose tolerance and a propensity towards obesity and type 2 diabetes when they were adults (Heijmans et al., 2008).Similarly, offspring of parents exposed to the holocaust had altered methylation levels within the FKBP5 gene which correlated with their parents, showing evidence of possible transgenerational inheritance mechanisms at play (Yehuda et al., 2016).In essence, the Dutch Hunger winter and the holocaust presented an opportunity to study the occurrence and persistence of epigenetic traits throughout life from the pre-/periconceptual period up to adult life.
The underlying mechanisms that drive transgenerational inheritance remain obscure but there are suggestive studies where different mechanisms have been explored in human and non-human models.These mechanisms, which include, DNA methylation, histone posttranslational modifications and non-coding RNAs have been extensively reviewed (Crisóstomo et al., 2023;Lismer and Kimmins, 2023;Jawaid et al., 2021;Champroux et al., 2018).In the current summative review, we focus on how environmental factors (i.e.pollutant exposure), social and socioeconomic factors (i.e.early infection, nutrition and microbiome contribution) during the early-life phase contribute to the development of chronic diseases later in life.In addition to the other mechanisms that have been previously reviewed, we introduce mitochondrial (DNA) mediated imprinting and explore maternal zygotic factor mediated epigenetic imprinting as potential mechanisms to consider in the context of ELA.

Connecting the transgenerational bridge
The maintenance of the "molecular memory" over generations does not only depend on alterations or depletions of DNA sequences but also on epigenetic mechanisms and the persistence of "yet-unknown" factors.Unlike DNA sequence depletions, epigenetic mechanisms are versatile and they allow dynamic gene expression transmission across generations, which is vital for adaptation (Surani, 2001).In addition, every genome possesses inherited maternal and paternal imprints after the fertilization step, which are conserved into adulthood (Reik and Walter, 2001;Ferguson-Smith and Surani, 2001).

Epigenetic reprogramming cycles
Epigenetic information is transmitted by patterns of DNA methylation and histones modifications.DNA methylation is the covalent attachment of a methyl group on CpG site.It has been associated with regulation of gene expression.DNA methylation within gene promoters can inhibit transcriptional activity (i.e. gene silencing) (Aravin et al., 2007).Histones are epigenetic regulators responsible of chromatin structure and gene expression.As DNA methylation, histones can also play a role modifying gene expression levels and patterns (Molina--Serrano et al., 2019).Histone acetylation leads to a better access to DNA for transcription while histone methylation influences transcriptional repression/activation of amino residues (Xavier et al., 2019).
Genomic reprogramming occurs at different stages within germline cells.This process starts with the demethylation of female and male primordial germ cells (PGCs) which erase the parental genomic imprints (Tada et al., 1998).The reactivation of the inactive X chromosome in female germ cell follows the demethylation step (Tam et al., 1994).This step allows the removal/erasing of any aberrant epigenetic modifications, avoiding the transmission of epimutations (Morgan et al., 1999).Following germ line reprogramming, the zygote-reprogramming step includes the interaction of oocyte cytoplasmic factors with both parental genomes (Allen et al., 1990).Maternal genome will be de novo methylated while paternal genome remains unmethylated (Monk et al., 1987).Specific maternal oocyte cytoplasmic factor as the heterochromatin protein 1 (HP1) can interact/ bind with methylated histones HE (meH3) leading to potential de novo DNA methylation (Bannister et al., 2001).HP1 plays a crucial role in nuclear organization, chromatin assembly and gene regulation (Hiragami and Festenstein, 2005).DNA methyltransferase 1 (DMNT1) is a large protein with multiple domains and responsible of intramolecular regulations (Kikuchi et al., 2022).DNMT1-dependant regions conserve specific human genome sequence and enriched genes playing a major role in cell to cell interaction.An enhanced expression of those genes will lead to neurological dysfunction due to environmental stress exposure (Freeman et al., 2020).In addition, Li et al. demonstrated that DNMT1 is essential in the maintenance of methylation imprints (Li et al., 1993).Lastly, research has shown that accurate histone methylation during spermatogenesis is crucial for the development and survival of offspring across several generations.These findings illustrate the significance of histone methylation as a molecular mechanism responsible for paternal epigenetic inheritance.Environmental factors that modify this process could impact embryo development and the transmission of complex diseases across generations (Siklenka et al., 2015).

Environmental pressures and factors associated with ELA
Early-life adversity is a somewhat "catch-all" term for negative aspects of the early-life exposome.It is all-encompassing and includes the social, psychological, financial, physical and emotional environment, categories that are often intertwined.Although it isn't a definitive categorisation, we have previously found it interesting to divide ELA into four categories that we maintain here: the socioeconomic and psychosocial environment, the microbial environment (microbiome and infection), the nutritional and metabolic environment, and the physical environment (e.g.pollution or pharmaceutical exposure) (Grova et al., 2019).However, any form of classification is limited by the overlap between the categories as observed during the COVID-19 pandemic period.Indeed, a lack of social interaction, financial burdens and overall additional societal stress that further affect primary medical, nutritional and educational needs (Holuka et al., 2020;Gøtzsche et al., 1988;Bang, 2023;Jaramillo and Felix, 2023), together with changes in the exposure to pathogens, medication use and the richness of early life nutrition significantly altered the establishment of the microbiome in this early life period (EbioMedicine, 2021;Herzog and Schmahl, 2018).

Socio-economic factors and stress
ELA is a series of environmental experiences and stressful events from conception to adulthood influencing development and health.ELA exposure induces long-term endocrine and pathologies risk later in life (Elwenspoek et al., 2020).Indeed, ELA can lead to gene expression alteration and can be associated with an increased risk for alcohol disorders, suicide and mental health conditions (Shonkoff, 2016;Hughes et al., 2017).ELA is also associated with morbidity and poor socioeconomic outcomes in adulthood (Merrick et al., 2019).Socioeconomic factors (SES) such as education, incomes or occupation events are also considered as crucial determinants in human health and well-being (Phelan et al., 2010).SES regroup different factors as stressful jobs, pollution that can influence human health (Khalatbari-Soltani et al., 2020).They are well known to be associated with cardiovascular, respiratory or intestinal diseases (Adler and Ostrove, 1999).Additionally, literature has shown that maternal behaviour can play a crucial role in determining the health outcomes of children later in life (Barclay et al., 2023).Studies demonstrated that maternal behaviour induces stable alterations of DNA methylation and chromatin structure (Weaver et al., 2004) demonstrating the long-term care maternal effects on gene expression (Weihrauch-Blüher et al., 2018).More recently, economic hardships such as material deprivation or major financial problem are now considered as predictors of low socio-economic status.When occurring during the developmental period of life (pregnancy of the mother (F0)), these factors can directly affect DNA methylation levels.Recently, Clark et al. demonstrated that mothers who experienced maternal economic hardship during pregnancy, gave birth to children with smaller head circumference (Clark et al., 2021a).Here, parents represent generation F0 while their children are generation F1.It is now well recognized that financial issues induce worse cognitive (i.e.memory problem or fatigue) and non-cognitive outcomes (i.e.behaviour or intellectual abilities) during offspring's adulthood (Clark et al., 2021b).Studies demonstrated that developmental periods (i.e.early postnatal) in the F1 generation are the most sensitive periods in term of exposure to ELA in addition to the F0 generation exposure to ELA (Smith et al., 2016).This is unsurprising, as during the perinatal period of children, neurogenesis and neuronal differentiation processes continue together with the establishment of synapses and neural circuits (Agarwal et al., 2005), processes that are sensitive to environmental and epigenetic changes (Bock et al., 2015).Maternal stress can directly or indirectly Fig. 1.DNA methylation is a conduit for transgenerational inheritance.During development, cells undergo epigenetic reprogramming cycles to establish lineage-specific gene expression patterns.These cycles involve the erasure of existing DNA methylation marks, followed by the establishment of new marks that are specific to the cell type or developmental stage.After fertilization, the zygote undergoes a series of reprogramming events to remove DNA methylation inherited from the spermatozoa and oocyte.This erasure of epigenetic marks is necessary for embryonic cells to regain pluripotency.Subsequent reprogramming events then establish new methylation marks that direct the cells to follow specific developmental pathways.
influence foetal development through placenta (Bale, 2016).The detailed underlying mechanism of how maternal stress can alter children development remains unclear.However, as germ cells are sensitive to stress, we can now potentially consider that it is the result of transgenerational transmission (Chan et al., 2018).Indeed we suggest that the maternal environment influenced by adversity exposure play a role in the epigenetic remodelling of the children epigenome such as DNA marks.As the conservation of those marks, supposedly through stem cell, can persist over years it is possible to speculate that without re exposition to additional adversity exposure, the F1 generation can transmit those mark to the generation F2.This mechanism of DNAm marks transmission corresponds to the transgenerational mechanism we highlighted above.So far, studies confirmed that traumatic events often occurring a long time before pregnancy such as childhood maltreatment (CM) lead to neuropsychiatric disorders in descendants (Dubowitz et al., 2001).Childhood sexual abuse (CSA) is one of the most common CM leading to offspring developmental alterations.Indeed, CSA is mostly associated with mental health diseases such as schizophrenia, suicide, depression, anxiety, etc (Browne and Finkelhor, 1986).CSA trauma affects the hypothalamic-pituitary-axis (HPA) leading to an unbalanced stress process (D'Elia et al., 2018).Additionally, when mother experienced trauma during their pregnancies, pro-inflammatory cytokines (i.e.IL-6) and C-reactive protein (CRP) levels were increased in comparison with non-exposed mothers.In a similar manner, SES play a crucial role in terms of health determinant exposure, however, many others external factors such as nutrition need to be considered.Chronic inflammation during pregnancy, triggered by high levels of pro-inflammatory cytokines, can harm the developing foetus, potentially leading to issues with brain development, cognitive function, and even future mental health (Zhou et al., 2023).Maternal stress studies are well described in the literature; however, paternal stress consequences remains unclear.In 2014, McGhee K.E et al. demonstrated that paternal care could also reduce DNA methyltransferase 3a (Dnmt3a) expression in the brain of their descendants (Agarwal et al., 2005).The DNMT3 family plays a crucial role in the establishment of de novo DNA methylation marks during differentiation and embryogenesis (Kato et al., 2007).

Early-life microbiome and nutrition
The ELA exposome includes the developing Early Life Microbiome (ELM) and the nutritional intake that the new-born receives.Individuals that spend up to their first 24 months of life within an institutional setting end up having taxonomic imprints of ELA experiences in their microbial profiles (Charalambous et al., 2021;Reid et al., 2021).The composition of the first ELM depends on the mother`s microbiota and the mode of birth, defining the primary colonisers (Fernandes et al., 2023;Wang et al., 2021).Subsequently, the establishment of this first microbial community depends on the nutrients available and in particular the diversity of human milk oligosaccharides (HMOs) and short-chain fatty acids (SCFAs), received during the first hours and months (Zijlmans et al., 2015;Alcon-Giner et al., 2020).Simultaneously other physiological systems including nervous and immune systems are further developing.Evidence on microbiome-immune interaction exist since the prenatal period between maternal microbial metabolites and foetal thymus (Hu et al., 2019).Whilst in early post-natal period the ELM aides in immune tolerance and maturation where the microbial metabolites are key moderators of immune signalling, differentiation and maturation (Hitch et al., 2022;Campbell et al., 2023).The microbiome-immune axis is closely related to the direct activation and differentiation of immune cell progenitors demonstrating the existence of the microbiome-bone marrow axis (Yoshida et al., 2018;Yan et al., 2022).The underlying mechanism is believed to act through direct methylation-demethylation on bone marrow derived immune progenitors and/or depended on INF-I Interferon type I and STAT1 signalling (Woo and Alenghat, 2022;Burgess et al., 2020).There is now clear evidence for gut-brain, gut-liver, oral-gut-liver, oral-gut-brain and oral-brain axes as well as the gut-lung, and gut-bone marrow axis (Bajaj, 2019;Liu et al., 2021;Pushpass et al., 2019).All of these interactions dependent to a certain extent on communication through nerves such as vagus nerve, which the gut-brain axis uses.Some other understudied routes are via the trigeminal nerve, or arising evidence from SARS-CoV-2 viral infection that suggest taste and olfactory receptors and their nerves (Pushpass et al., 2019;Dong et al., 2022).At the centre of this unintelligible net of interaction axes lays the immune system.Either as a reaction to a "threat" stimulus or as part of the typical physiological process for immune maturation, the microbiome-immune crosstalk is somehow always involved in health trajectories.
Epigenetic modifications are the core strings of how the early-life microbiome and its metabolic competency are able to influence developmental trajectories of health and disease (Wang et al., 2022;Romano and Rey, 2018).Microbial metabolites are essential for proper epigenetic processes, and their deficiency can disrupt and alter normal epigenetic programming (Wang et al., 2022).Nonetheless, all the above examples highlight how this close interaction of the early life exposome the ELM and the host imprint the epigenome and define the health trajectory.What is worth noting is the ELM is given by the mother and its influence is likely adjusted to the mother`s physiology and including the maternal epigenome.The aforementioned interactions or crosstalk's and how they unfold during the early life phase could generate potent epigenetic imprints.Whether these imprints can be transferred onto the next generations remains an open question.

Early life-infections
Early-life infection (ELI) can have devastating short-and long-term effects.For example neonatal bacterial or viral sepsis increases the risk of death within the first 12 days of life threefold.While short-term risks are obvious, serious long-term complications often arise.For example, neurodevelopment in survivors of neonatal sepsis is significantly poorer than usual (Alshaikh et al., 2013), and longitudinal birth cohort analyses have shown that early-life infections associate with both measures of chronic inflammation and overall morbidity and mortality from cardiovascular disease later in life (Barker et al., 1991;Vasto et al., 2007).Early-life respiratory infections, particularly of viral origin, increase the subsequent risk of developing asthma and/or allergies in childhood (Martinez, 2000;Townsi et al., 2018;Malinczak et al., 2020) and hint to a long-term viral-induced type 1 diabetes (Beyerlein et al., 2016).However, although ELI may represent a specific type of ELA, its' intimate link to social behaviour and the social environment means that there may be many other actors in play (Merz and Turner, 2021).Initially, inflammatory elements of the ELI immune responses were thought to be responsible for later-life disease (Hayward et al., 2016).In particular, dysregulated inflammation was proposed to increase the risk of mortality from cardiovascular disease and stroke due to their association with atherosclerosis (Vasto et al., 2007;Hayward et al., 2016).Indeed, chronic exposure to infection, particularly in early life, induces lifelong increased CRP levels (Gurven et al., 2008) as well as eosinophilia, and increased immunoglobulin E, interleukin-6, and erythrocyte sedimentation rate (ESR) (Vasunilashorn et al., 2010).Recent studies in male mice exposed to polyinosinic:polycytidylic acid (Poly I:C), a viral infection mimetic and liposaccharide (LPS), a bacterial infection mimetic have shown that the F2 offspring display behavioural changes which are mediated by miRNAs derived from spermatozoa (Kleeman et al., 2024;Liao et al., 2024).While this phenomenon is evident in mice models of viral and bacterial infection, it remains to be seen whether similar mechanisms are also present in humans.
In the human context ELI occurs in a specific context: the new-born immune system is immature and functional populations of both adaptive and innate immune cells have not been fully established (Merz and Turner, 2021), and the naïve T-helper (Th) cells present are epigenetically biased toward a Th2 phenotype (Dowling and Levy, 2014).This "blank slate" was proposed to be a period of plasticity during which the immune system can adapt and prepare for life in an environment similar to that in which the individual was born (Kollmann et al., 2017(Kollmann et al., , 2012;;Danese and S, 2017), shaping the long-term immune trajectory (Merz and Turner, 2021).This adaptation of the immune system is exemplified by the hygiene hypothesis.At its most simple level, the hygiene hypothesis states that contact with as broad a range of non-pathogenic microorganisms as possible during childhood is a pre-requisite to successfully establishing immune tolerance, and reduced or absent exposure increases the risk of immune-mediated diseases such as auto-immune disease and allergy (Strachan, 1989;Okada et al., 2010;Alexandre-Silva et al., 2018).However, exposure to pathogens, allergens, and air pollution have the opposite effect, increasing the risk of allergy (Merz and Turner, 2021;Gaffin and Phipatanakul, 2009;Burbank et al., 2017).It remains to be investigated whether this increased risk of allergy persists in the subsequent generations via transgenerational inheritance mechanisms.
One of the key intermediates linking ELI to the eventual phenotype may be stress reactivity and the stress-cytokine axis (Bilbo and Schwarz, 2009).ELI, while the immune system is not fully developped, leads to exaggerated cytokine responses, principally IL-1β, IL-6, and TNFα, all of which pass rapidly from the blood to the cerebrospinal fluid (Gutierrez et al., 1993), impairing long-term memory and brain plasticity (Bourgognon and Cavanagh, 2020).In a zebrafish model of ELI infection timing was the key element determining increased baseline pro-inflammatory gene expression in adult animals (Cornet et al., 2020).In human ELA paradigms such as institutionalisation-adoption where increased rates of infections are an integral element of the stressor, we and others have reported a blunted cortisol stress response that is accompanied by immune disturbances including higher numbers of exhausted Natural killer cells and both cytotoxic and helper T-lymphocytes (Elwenspoek et al., 2017b;Fernandes et al., 2021;Reid et al., 2019;Hengesch et al., 2018).
While the molecular mechanisms underlying the long-term pathophysiological effects of ELI remain unclear, a recent observation gave a glimpse into how the phenotype may be maintained over many years and potentially across generations.Immune cells are perpetually renewed from a reservoirs of hematopoietic stem cells, and epigenetic modifications in this compartment led to long-term changes in levels of inflammation and immunometabolism after ELI, that induced functional difference upon re-infection later in life (Fonseca et al., 2020).It is possible, however, that the mechanism may be somewhat simpler.ELA is associated with a higher rate of infection from Herpesviridae such as CMV, EBV and HSV.These chronic viral infections are regularly re-activated once acquired.Chronic CMV infection is associated with a reduction in naïve T-cells (Wertheimer et al., 2014), and specific memory T-cell expansion (Klenerman and Oxenius, 2016;Weltevrede et al., 2016) reminiscent of the ELI and ageing phenotype (Koch et al., 2008).

Early-life drug and pollutant exposure
Direct exposure to medications, drugs and/or environmental toxicants may lead to epigenetic alterations and later contribute to the development of diseases in subsequent generations (Grova et al., 2019).This concept is in line with the classic paradigm of genetic determinism, which emphasizes the role of genetics in the development of disease (Van Cauwenbergh et al., 2020).Therefore, by integrating environmental epigenetics into disease aetiology, we assume that xenobiotics not only impact the generation initially exposed but could also be transmitted to future generations through the germline.Their effects can manifest as a wide spectrum of health issues, including developmental abnormalities (Martin et al., 2022), reproductive disorders (Baratta et al., 2021), increased susceptibility to neurodevelopmental and neurodegenerative diseases (Grova et al., 2019).Anti-epileptic drugs (AEDs) have been widely prescribed to pregnant women in developed countries (Gedzelman and Meador, 2012;Veroniki et al., 2017).Several studies have suggested that children exposed in utero to AEDs present an increased risk of malformations and neurodevelopmental disorders (Blotière et al., 2020).This is particularly the case with valproate acid (VPA), whereas the level of evidence varies for other anti-epileptic drugs such as Topiramate, Carbamazépine, Phénobarbital, Primidone, (fos) phénytoïne (Blotière et al., 2020).In addition to the highest risk of childhood malformations, VPA also entails a high risk of neurodevelopmental disorders.An increased risk, by a factor of 4-10, of developing autism spectrum disorders (ASD) in children has been clearly established in women treated with VPA during pregnancy (Christensen et al., 2013).VPA promotes histone acetylation, which in turn may impact DNA and histone methylation patterns, resulting in modifications in the expression of transcription factors.All these epigenetic modifications have been associated with chromatin remodelling effects (Choi et al., 2016).It has been shown that the ASD phenotype induced by VPA in rodents is transmitted epigenetically, at least up to the third generation (Choi et al., 2016).Tsuji et al. (2022) recently investigated, in rodent model, behavioural deficit triggered by VPA on postnatal day 5 of the F2 generation to evaluate the onset of the ASD phenotype.The results pointed out that impaired locomotion as well as social deficit could be inherited by the subsequent generation and were apparent early in life (Tsuji et al., 2022).Epigenetically-inherited adverse effects of valproate acid were recently demonstrated in 90 families comprising 85 women and 23 men, who experienced complications due to VPA exposure in utero and became parents in their turn.Among their children (n=187), 44 % were suffering from neurodevelopmental disorders, 23 % presented malformation(s), only 47 % had neither developmental disorders nor malformation (Martin et al., 2022).Although there have been reports of multigenerational inheritance across different types of AEDs, there is a scarcity of studies investigating transgenerational inheritance of traits following parental exposure to addictive substances (Yohn et al., 2015).To date, transgenerational phenotypes have also been observed following parental exposure to morphine, alcohol, and nicotine (Baratta et al., 2021;Yohn et al., 2015).It has been conclusively demonstrated that prenatal drug exposure has a significant influence on future generations, leading to consistent alterations in various aspects such as drug-taking behaviour, stress response and dopamine signalling in offspring (Yohn et al., 2015).Lastly, research demonstrates that exposing pregnant female mice to bisphenol A (BPA) leads to obesity in their F2 progeny, a trait that persists for up to the F6 generation even without further exposure.This obesity is linked to increased food intake and is inherited, with its mechanism associated with a chromatin-accessible site within a cis-regulatory element (CRE) located in an intron of the Fto gene.Importantly, exposure to BPA results in demethylation of this CRE (Jung et al., 2022).
A large number of environmental chemicals, such as dioxin, heavy metals, Polycyclic Aromatic Hydrocarbons (PAHs), pesticides, and even some plastics, are also likely to have transgenerational effects.In this context, Van Cauwenbergh et al. recently studied whether persistent epigenetic changes could occur in the male germline following exposure to synthetic endocrine disrupting chemicals (EDCs) (Van Cauwenbergh et al., 2020).To answer this question, they carried out a systematic search that resulted in the inclusion of 43 articles addressing the effects in mammals of commonly-used synthetic endocrine disruptors, including plasticizers (bisphenol A and phthalates), pesticides (methoxychlor, atrazine, dichlorodiphenyltrichloroethane and vinclozine), dioxins and PAHs.In most of these studies performed on animal models, transgenerational epigenetic effects have been highlighted, often associated with the appearance of metabolic disorders, behavioural testicular or prostatic abnormalities as well as tumour development (Van Cauwenbergh et al., 2020;Beck et al., 2022).To date, there is a lack of evidence concerning the mechanisms of action of EDCs on the germline epigenome and the associated risk of disease in human offspring.In this context, longitudinal observational studies such as the Avon Longitudinal Study of Parents and Children (ALSPAC) have provided compelling evidence.One such study pointed out that adolescents C. Holuka et al. whose fathers started smoking before puberty were at increased risk of developing obesity (Northstone et al., 2014).Although the specific biological mechanisms were not investigated by the authors, which finding led the authors to assume that the chemicals (such as PAH, heavy metal, nicotine) present in cigarette smoke could potentially induce epigenetic alterations in the production of spermatogonia in the testes before puberty, which would have an impact on the next generation (Van Cauwenbergh et al., 2020;Soubry et al., 2014).In parallel, World health Organisation (WHO) data highlight that almost the entire world population (99 %) breathes air that exceeds WHO guidelines and contains high levels of environmental pollutants, with low-and middle-income countries being the most exposed.Once it has been demonstrated that airborne particles impair mitochondrial machinery and cause significant damage to the epigenome, the transgenerational effects of air pollution in human were the next logical area to investigate (Shukla et al., 2019).Thus, Shukla et al. recently pointed out that the alteration of DNA methyltransferases activity which in turn triggers changes in DNA methylation in human offspring that could be handed down from one generation to the next and therefore lead again to transgenerational epigenomic inheritance.

Molecular mechanisms of inheritance
Exposure to certain environmental pressures during the early life phase can have long lasting health implications which span generations.In our view, this can only be achieved through the activation of robust mechanisms of inheritance which remain active in the offspring and their subsequent descendants.Several studies have proposed mechanisms to explain how transgenerational inheritance occurs in different species (reviewed in (Xavier et al., 2019); Skvortsova et al., 2018a;Guerrero-Bosagna et al., 2018).Using non-human models has made it possible to identify some overarching interspecies similarities, which provide potential mechanistic insights into the human situation with respect to transgenerational inheritance.It is important to note that these mechanisms do not necessarily occur in splendid isolation of each other but may occur sequentially or concurrently depending on the timing and severity of exposure to ELA.

Maternal zygotic factors mediated genomic imprinting
Genomic imprinting is a heritable epigenetic process, which results in the expression of a subset of autosomal genes in a parent of origin specific manner (Ishida and Moore, 2013).Basing on the evolutionary context, maintenance of genetic imprints across generations from parent to offspring is important because over time it enables species to pass down certain advantageous traits to their offspring.On the contrary, detrimental or diseases associated traits are also passed down in a similar fashion.Genomic reprogramming is an essential step during embryonic development because in resets the epigenetic landscape allowing for a "fresh start."This reprogramming differs for the male and female germ lines in that the male genome undergoes reprogramming after fertilization, while reprogramming of the female genome occurs gradually during foetal development.During embryogenesis, maternal zygotic factors are known to facilitate demethylation of paternal alleles (Gse), protect maternal imprints from demethylation (Pgc7 and stella), maintain these imprints (Zfp57), and regulate epigenetic stability (Trim28) (Keverne, 2015) (Fig. 2).In essence, by providing protection for developing zygotic development of the next generation, the female Fig. 2. Genomic imprinting.DNA methylation, to specific regions of the DNA during gametogenesis results in epigenetic marks that are inherited by the offspring and persist throughout their development.This phenomena leads to parent-specific gene expression patterns in the offspring.Genomic imprinting primarily occurs in imprinted gene clusters in the spermatozoa and oocyte before fertilization.After fertilisation when both the spermatozoa and oocyte undergo sequential demethylation cycles, maternal zygotic factors play a critical role in preserving and maintaining genetic imprints.The offspring inherits two copies of each imprinted gene, that is, one from the mother and one from the father but only one copy is actively expressed, while the other copy is silenced or inactivated.During embryonic development some imprints are erased while some remain in place (Adapted from Surani, 2001) (Created with BioRender.com).
C. Holuka et al. germ line plays a pivotal role in maintaining transgenerational imprints.
In a recent study in mice, it was observed that even when methylation within CpG islands (CGIs) is erased in the parental germ line, the unmethylated gametes can still transmit DNA methylation memory of the CGIs to the zygote (Takahashi et al., 2023).The same study suggests that DNA methylation of the CGIs in these zygotes occurs at the early post-implantation stage in the next generation.This brings up new and interesting perspectives with regard to genomic imprinting where other unknown factors are responsible for maintaining DNA methylation memory.The obvious question is, what is this epigenetic memory?Possibly there is another layer of regulation above the epigenome which enables imprinted genes to be maintained across multiple generations.

Mitochondrial mediated maternal imprinting
The mitochondria are largely considered as the powerhouse of the cell.Nevertheless, they are involved in many other cellular activities and processes besides energy production.The mitochondria produce reactive oxygen species (ROS), which serve as the main currency for intracellular signalling.Mitochondria are also involved in determining cell fate via ROS mediated signal transduction processes.The discovery of mitochondrial DNA (mtDNA) in the 1960s transformed our understanding of the role of mitochondria from being a mere energy factory.MtDNA is a circular double-stranded DNA molecule consisting of 13 protein-coding genes that encode some of the subunits involved in oxidative phosphorylation (OXPHOS).Each cell contains several copies of mtDNA, for example, mtDNA copy number is extremely high in oocytes (≥10 5 copies) as compared to the spermatozoa (≤10 2 copies) (Wai et al., 2010).This phenomenon is particularly interesting when we consider the maternal inheritance of mtDNA.MtDNA is non-recombinant DNA and passes down virtually 'unchanged' through the direct maternal line over successive generations.In this regard, it is evident that mitochondria play an important role in transgenerational inheritance and are an interesting target for accessing the ELA across generations.From previous research, it is evident that the mtDNA undergoes epigenetic changes that potentially influence mitochondrial function (Mposhi et al., 2022(Mposhi et al., , 2023)).As described earlier in this review, there is a growing realisation that mitochondrial dysfunction may be one of the hallmark features characterising the ELA phenotype.
During embryogenesis, the nuclear genome undergoes extensive reprogramming which involves large-scale demethylation as mentioned earlier.After the spermatozoa enters the oocyte, both the maternal and paternal origin chromosomes undergo a sequential demethylation process.Paternal DNA undergoes TET3 mediated demethylation, which spares only imprinting control regions (ICRs) followed by a progressive demethylation of the maternal genome.Previous studies have shown that the maternally inherited factor, Stella, which is encoded by DPPA3 protects the maternal genome and paternal ICRs from active demethylation.Stella is highly expressed in primordial germ cells and its expression is maintained throughout oocyte maturation right up to preimplantation of the embryo (Zhang et al., 2021).In essence, this indicates that the maternal genome, both nuclear and potentially mitochondrial are protected from active demethylation and that not all epigenetic imprints are erased during embryogenesis.
While it may appear logical to assume that all DNA in the cell including mtDNA undergoes demethylation, no studies that have been done to prove otherwise.It therefore, becomes a question of whether mtDNA undergoes similar reprogramming or maintains its epigenetic signature, which is then passed on to the next generation.On the other hand, if it undergoes demethylation, it remains elusive whether the mitochondria have efficient epigenetic machinery to reintroduce methylation.Moreover, it is plausible that mtDNA escapes the demethylation phase during embryogenesis, which enables mitochondrial information to pass down to the next generation unchanged.A recent study (Sirard, 2019) demonstrated that bovine mtDNA methylation patterns were more highly conserved between oocytes and blastocysts compared to somatic (granulosa cells).The maintenance of mtDNA methylation during embryogenesis suggests that maternal mtDNA does not undergo extensive epigenetic changes (demethylation) and therefore mtDNA may play an important role in genomic programming as well as genomic imprinting.Overall, it is evident that mitochondria play a critical role in biological embedding of ELA and provide potential mechanisms to explain transgenerational inheritance.

ncRNA mediated imprinting
The role of noncoding RNAs (ncRNA) such as small interfering RNA (siRNA), microRNAs (miRNA) and piwi-interacting RNA (piRNA) in transgenerational inheritance has been extensively studied in nonhuman models.The transmission of information by these ncRNAs across generations is another potential mechanism for transgenerational inheritance.Certain environmental conditions can evoke transgenerational gene regulation by endogenous siRNAs lasting several generations.For instance, in C. elegans, starvation conditions were shown to induce expression of a pool of endogenous siRNAs targeting several nutritional genes (Rechavi et al., 2014).Expression of these siRNAs was maintained for at least three generations after returning the worms to nutritionally rich conditions, potentially transmitting an epigenetic memory for coping with food shortage.Consistent with this idea, these descendants had increased life span compared to control worms.Heat stress has similarly been found to alter gene expression through ncRNAs, and these expression patterns lasted for two to three generations after a return to normal temperature conditions (Ni et al., 2016).All these lines of evidence point to an ncRNA-mediated mechanism that transfers epigenetic memory to the subsequent generations.Additionally, mouse models demonstrated the involvement of RNA-dependent mechanisms in the inheritance of acquired traits (Table 1).They emphasize the significance of small non-coding RNAs (sncRNAs) in germ cells and shed light on their susceptibility to early traumatic stress.Furthermore, they reveal the repercussions of exposure to such traumatic experiences in early life across multiple generations.Early exposure to toxicants such as DDT play a role in the epigenetic transgenerational inheritance of disease (e.g., obesity) through the germline.DDT induces sperm epigenetic alterations (Gapp et al., 2014;Skinner et al., 2018).
However, the question still remains how these siRNA molecules manage to cross the transgenerational bridge from parent to offspring without encountering the reprogramming phase.Furthermore, it is debatable whether ncRNA mediated transgenerational inheritance occurs in humans as there have been no studies to date that prove the existence of such a mechanism of inheritance.However, over the decades through the use of mice models, our understanding of these versatile ncRNA molecules has greatly improved.Previous studies have pointed out to the role of spermatozoa derived ncRNAs in murine transgenerational inheritance (Beck et al., 2021;Lopes et al., 2023).The role of ncRNAs in regulating gene expression via chromatin remodelling, mediating epigenetic modification, transcriptional and post-transcriptional regulation has been extensively reviewed elsewhere (Kaikkonen et al., 2011).Here, we hypothesize, that gametic ncRNAs could play a role in epigenetic regulation and therefore provide a potential conduit for transgenerational inheritance in humans.

Discussion / Perspectives
Over the past decades, many studies have become more focused on understanding the mechanism that drive epigenetic inheritance.Most of these studies have used animal models due the ethical complexity of carrying out such studies in humans.However, there are many gaps in knowledge as none of the animal studies has managed to reveal the "holy grail" mechanism of transgenerational inheritance that mimics the human situation.In this review, we highlighted and explored potential mechanisms that may drive transgenerational inheritance in humans following exposure to ELA.To describe the underlying mechanism of epigenetic transmission process we first considered the role of genetic and cellular components (i.e.germline cells) as a starting point transmitted by both parents.
Lately, many studies have pointed out the importance of maternal influence in the inheritance mechanism (Perez and Lehner, 2019;Lester et al., 2018;Buss et al., 2017).However, there is now growing evidence suggesting that not only maternal stress or maltreatment can directly influence offspring development.Indeed, paternal stress elicits different mechanisms to induce epigenetic changes such as DNA methylation over time.It is now clear that parental pressures also lead the specific phenotype of offspring later in life "forcing" them to adapt to environmental pressures.Basing on all the studies we have highlighted, it becomes a question of whether maternal exposure to ELA is more important than paternal exposure in creating genomic imprints that have long lasting effects that transcends across generations.Indeed, from our previous studies we have demonstrated that maternal deprivation can induce epigenetic imprints on the offspring's genomes but no studies have been carried out to confirm the persistence of these imprints in succeeding generations.Nevertheless, the question remains whether, maternal imprints persist longer than paternal imprints.Another aspect to consider when looking at the transgenerational inheritance of the ELA phenotype is the biological decline associated with ageing.Indeed, over time, diseases development becomes entangled with biological age and also with epigenetic age.Lately, the use of epigenetic clocks to measure epigenetic age demonstrated that ELA exposure occurring during the first stages of life left a specific reversible epigenetic signature on the genome.It therefore becomes a question of whether ageing is a determinant of ELA associated diseases or rather a parallel event whose characteristic features overlap with the ELA phenotype.Previous studies have suggested that ELA promotes accelerated ageing and also consequently promotes the development of agerelated diseases such as diabetes.It is more likely to develop chronic diseases when ageing, however ELA can play a major part in accelerating their development.ELA exposure results in the genome modification by addition of specifics epigenetic marks.Some of these marks can persist over time while some will disappear few years after exposure.As demonstrated by Thorson et al., phenotypic and epigenetic consequences arise from ancestral exposure to a commonly encountered mixture of plastic-derived chemicals, even when administered at or near the no observable adverse effect level (NOAEL).Phenotypic effects assessed encompassed disease incidence within the transgenerational lineage, focusing on conditions such as testis and kidney diseases, as well as various other ailments (Thorson et al., 2021).The reasons of the DNA methylation conservation remains unknown.What are the mechanisms that determine which traits are conserved and which traits are removed?We hypothesised that age-associated canonical epigenetic marks will be erased while ELA-associated non-canonical epigenetic marks are carried over into the next generation.The conservation process is considered as a major element of the inheritance mechanism.Indeed, it appears that the position on the genome of methylation marks driven by ELA influence the time they remain present over time and brings into question whether cells have developed competent epigenetic machinery to effect erasure of these non-canonical epigenetic marks.Furthermore, studies with fish models showed that exposure to cadmium (Cd) in one generation, along with elevated temperatures in subsequent generations, correlated with changes in the methylation levels of specific genes in female gonads and alterations in population sex ratios (Pierron et al., 2021).
As previously alluded to in this review, another main aspect to consider is particular environmental factors and lifestyles enabling the development of imprints on the brain and immune system.Epigenetics marks such as DNA methylation are known to alter gene expression and by consequence affect the overall biological processes.Therefore, bringing together environmental and genetic factors might be the key point to elucidate the mechanisms behind the transgenerational inheritance process.So far, we have only scratched the surface in terms of understanding the roles of epigenetics, mitochondria and ncRNAs in transgenerational inheritance.We have highlighted these potential mechanisms because of the compelling evidence supporting their role in transgenerational inheritance in human and non-human models.As previously mentioned in this review, the study of transgenerational inheritance in murine models as well as other non-human models brings about the question of whether the same mechanisms of inheritance are present in humans.Although there is compelling evidence supporting meiotic epigenetic inheritance (MEI) in murine models, attributing the same mechanism to other animal models remains challenging due to the absence of clear causality (Bohacek and Mansuy, 2017;Skvortsova et al., 2018b).Given, the relative technical and ethical complexity of undertaking these type of studies in humans, animal models still provide us with the best platform to investigate potential mechanisms.It is evident that different types of adversity during the early life period elicit different inheritance mechanisms.The role of epigenetics in transmitting phenotypes across generations has long been a subject of debate.However, with the accumulation of examples of transgenerational epigenetic inheritance, its significance is becoming increasingly apparent, alongside a growing understanding of the underlying mechanisms.Common features include similar epigenetic signals and transmission methods, but variations exist both between and within organisms.The complex landscape of primary and secondary epigenetic signals reveals diverse regulatory pathways for TEI (Fitz-James and Cavalli, 2022).Here in, we further suggested that the magnitude of exposure determines which mechanisms will be activated.Overall, there is a rising need to construct well designed transgenerational cohorts that would allow to explore transgenerational inheritance in a multi-system approach.A promising starting example is the Lifelines NEXT cohort (Warmink-Perdijk et al., 2020).
To conclude, the mechanisms by which transgenerational inheritance occur are still being studied.Based on our previous studies it is obvious that they involve epigenetic changes, alterations in gene expression or disruption of developmental processes (such as neurodevelopmental impairments or malformations).These changes may lead to lasting effects that are inherited by subsequent generations, even in the absence of continuous exposure to environmental pollutants, infection, stress and negative socio-economic factors.Mice, fish, and more recently, bird models have provided robust evidence of transgenerational inheritance through epigenetic modifications.While these animal models offer valuable insights into the mechanisms of transgenerational epigenetic inheritance, it remains to be seen whether these findings can be directly applied to the human context (Leroux et al., 2017).

Fig. 3 .
Fig. 3. : Potential developmental mechanisms of transgenerational inheritance.Diagram shows DNA methylation dynamics and spermatozoa derived ncRNA activity during embryogenesis.Hydroxymethylation of the paternal genome (blue) takes place soon after fertilisation followed by a passive demethylation of the maternal genome (red).De novo methylation of the fetal genome takes place at the blastocyst stage.We hypotheses that maternal mitochondrial DNA (mtDNA) (green) methylation remains relatively unchanged during embryogenesis and basically involves a transfer of the entire mitochondrial population from mother to offspring.We also hypothesise that spermatozoa-derived ncRNAs (yellow dashed) are present during the pre-implantation stage and they influence critical processes during fetal development.It is also during this stage that certain ELA traits associated with expression of these ncRNAs can be passed on to the F1 generation.Abbreviations: GV, germinal vesicle; MII, Metaphase II; mtDNA, mitochondrial DNA; ncRNA, non-coding RNA (Created with BioRender.com).