Epigenetic processes allow organisms to respond to their environment with changes in gene expression. Epigenetic molecular processes include DNA methylation, histone modifications, non-coding RNAs, RNA methylation and chromatin structure. Epigenetic information and epimutations can be transmitted to subsequent generations through sperm or eggs, and can change gene expression and an organism's phenotype even in transgenerational generations that were themselves never exposed to an inducing factor. All cell types and tissues derived from germ cells carrying epimutations will have the potential for an altered epigenome and altered gene expression. Tissues sensitive to an altered gene expression profile will have a different phenotype and have susceptibility to develop disease or abnormalities as the individual ages. Environmental exposures in past generations could have contributed to the incidence of abnormalities and disease in the current human population.

Environmental factors can influence the epigenetic inheritance of pathology across generations. Prior studies found various toxicants increased pathology in their descendants through epigenetic transgenerational inheritance. Previously, three generations of rats were exposed separately to vinclozolin (F0 generation), a jet fuel hydrocarbon mixture (F1 generation), and dichlorodiphenyltrichloroethane (DDT) (F2 generation), producing a transgenerational F5 generation. The current study extends this study by breeding this transgenerational F5 generation with wild-type rats to create maternal and paternal outcross lineages, labeled MOC and POC, respectively. In the current study, we examined the differential DNA methylated regions (DMRs) in the outcross F6 generation offspring to detect potential parent-of-origin lineage‑specific inheritance impacts. When comparing both outcross MOC and POC lineages of the F6 generation to a control outcross F3 generation from the same lineage, with no toxicant exposure, a higher number of DMRs were observed in the F6 generation lineages. Additionally, while there was some common overlap between the two MOC and POC outcross generations, only the F6 POC generation had overlaps with the F5 multigenerational exposure lineage generation of the previous study, which was also a paternal outcross. Since previous pathology analysis has been extensive, limited pathology (e.g. obesity) was performed in the current study to primarily confirm the transgenerational pathology in the F6 generation outcross lineages. Comparisons between the control same lineage F3 generation and both of the outcross F6 generations identified outcross‑specific results, suggesting sex-specific epigenetic inheritance after ancestral toxicant exposure. Therefore, both the oocyte and sperm facilitate epigenetic transgenerational inheritance.

Previous research on the generational stability of epigenetic transgenerational inheritance was conducted through a ten-generation study of all transgenerational generations in mammals. This study demonstrated both the stability of epigenetic inheritance across generations and demonstrated a generational increase incidence of disease pathology. Building on this research, the present study follows the same lineage of rats with ances-tral vinclozolin exposure through twenty generations. The findings offer important insights into long-term mammalian models of epigenetic transgenerational inheritance. Observations demonstrate an increase in differential DNA methylated regions across multiple generations. This indicates a persistent and stable transmission of epigenetic alterations. Additionally, deoxyuridine triphosphate (dUTP) transferase-mediated nick end labeling apoptosis assays revealed elevated levels of germline apoptosis in the male rats of the maternal and paternal lineages. This suggests a potential consequence of epigenetic dysregulation in spermatogenesis. Ancestrally exposed rats to vinclozolin showed significant parturition abnormalities in both the maternal and paternal line-ages after 16 generations. This included maternal deaths during labor and stillbirths. Pathological assessments revealed abnormalities across multiple tissue types and an increased incidence of disease. This suggests the physiological consequences of the gen-erational stability of epigenetic inheritance. Observations establish the generational sta-bility of epigenetic inheritance over twenty generations in a mammalian model system; however, new pathology in later generations involving parturition abnormalities was also observed. The generational stability of transgenerational effects observed in this study has implications for human health, particularly regarding environmental toxicant exposures, reproductive health disorders, and disease susceptibility.

In traditional models of Drosophila flies and Caenorhabditis elegans worms, epigenetic transgenerational inheritance has persisted for hundreds of generations. Mammalian studies, however, have primarily investigated up to three or four generations. Environmental exposures to toxicants or other stressors have been linked to epigenetic transgenerational inheritance. Changes within the epigenome have the potential to alter gene expression and genetic processes, as well as potentially alter phenotypes and evolution in all organisms. In this study, an outcrossed mammalian colony of outbred rats was bred for 10 successive generations and assessed for epigenetic alterations and incidence of pathological abnormalities. The hypothesis tested was that epigenetic transgenerational inheritance of disease would be maintained for 10 generations. Sperm from male rats were analysed for DNA methylation and pathology abnormalities in the outcross female and male lineage rats in successive generations. Effects on male sperm were observed in differential DNA methylated regions in both lineages. A dramatic increase in apoptosis of male spermatogenic cells in the testes for both male and female lineages was observed. Pathology of different tissue types was assessed by artificial intelligence-based technologies using deep learning histology protocols developed. Observations showed a dramatic increase in disease incidence within the 10th generation (F13) compared to the control lineage for kidney, ovary, prostate, and testis for both outcross lineages. As previously observed, no gross pathologies were observed in other organs. Observations demonstrate the long-term impact of ancestral exposures on epigenetic modifications and health outcomes across multiple generations are stable for 10 generations in mammals.

Environmentally induced epigenetic transgenerational inheritance of phenotypic variation and disease susceptibility requires the germ cell (sperm or egg) transmission of integrated epigenetic mechanisms involving DNA methylation, histone modifcations, and noncoding RNA (ncRNA) actions. Previous studies have demonstrated that transgenerational exposure and disease-specifc differential DNA methylation regions (DMRs) in sperm are observed and that ncRNA-mediated DNA methylation occurs. The current study was designed to determine if transgenerational exposure-specifc ncRNAs exist in sperm. Specifcally, toxicants with distinct mechanisms of action including the fungicide vinclozolin (anti-androgenic), pesticide dichlorodiphenyltrichloroethane (estrogenic), herbicide atrazine (endocrine disruptor at cyclic adenosine monophosphate level), and hydrocarbon mixture jet fuel (JP8) (aryl hydrocarbon receptor disruptor) were used to promote transgenerational disease phenotypes in F3 generation outbred rats. New aliquots of sperm, previously collected and used for DNA methylation analyses, were used in the current study for ncRNA sequencing analyses of nuclear RNA. Signifcant changes in transgenerational sperm ncRNA were observed for each transgenerational exposure lineage. The majority of ncRNA was small noncoding RNAs including piwi-interacting RNA, tRNA-derived small RNAs, microRNAs, rRNA-derived small RNA, as well as long ncRNAs. Although there was some overlap among the different classes of ncRNA across the different exposures, the majority of differentially expressed ncRNAs were exposure-specifc with no overlapping ncRNA between the four different exposure lineages in the transgenerational F3 generation sperm nuclear ncRNAs. The ncRNA chromosomal locations and gene associations were identifed for a small number of differential expressed ncRNA. Interestingly, an overlap analysis between the transgenerational sperm DMRs and ncRNA chromosomal locations demonstrated small populations of overlapping ncRNA, but a large population of nonoverlapping ncRNAs. Observations suggest that transgenerational sperm ncRNAs have both exposure-specifc populations within the different classes of ncRNA, as well as some common populations of ncRNAs among the different exposures. The lack of co-localization of many of the ncRNAs with previously identifed transgenerational DMRs suggests a distal integration of the different epigenetic mechanisms. The potential use of ncRNA analyses for transgenerational toxicant exposure assessment appears feasible

Preterm birth (PTB) has dramatically increased within the population (i.e. >10%) and preeclampsia is a significant sub-category of PTB. Currently, there are no practical clinical parameters or biomarkers which predict preeclampsia induced PTB. The current study investigates the potential use of epigenetic (DNA methylation) alterations as a maternal preeclampsia biomarker. Non-preeclampsia term births were compared to preeclampsia PTBs to identify DNA methylation differences (i.e. potential epigenetic biomarker). Maternal buccal cell cheek swabs were used as a marker cell for systemic epigenetic alterations in the individuals, which are primarily due to environmentally induced early life or previous generations impacts, and minimally impacted or associated with the disease etiology or gestation variables. A total of 389 differential DNA methylation regions (DMRs) were identified and associated with the presence of preeclampsia. The DMRs were genome-wide and were predominantly low CpG density (<2 CpG/100 bp). In comparison with a previous PTB buccal cell epigenetic biomarker there was a 15% (60 DMR) overlap, indicating that the majority of the DMRs are unique for preeclampsia. Few previously identified preeclampsia genes have been identified, however, the DMRs had gene associations in the P13 K-Akt signaling pathway and metabolic gene family, such as phospholipid signaling pathway. Preliminary validation of the DMR use as a potential maternal biomarker used a cross-validation analysis on the samples and provided 78% accuracy. Although prospective expanded clinical trials in first trimester pregnancies and clinical comparisons are required, the current study provides the potential proof of concept a preeclampsia epigenetic biomarker may exist. The availability of a preeclampsia PTB maternal susceptibility biomarker may facilitate clinical management and allow preventative medicine approaches to identify and treat the preeclampsia condition prior to its occurrence.

Deep learning involves an artificial intelligence (AI) approach and has been shown to provide superior performance for automating image recognition tasks, as well as exceeding human capabilities in both time and accuracy. Histopathology diagnostics is one of the more popular challenges at the intersection of artificial intelligence, computer vision, and medicine. Developing methods to automatically detect and identify pathologies in digitized histology slides imposes unique challenges due to the large size of these images and the complexity of the features present in biological tissue. Most methods that are capable of human-level recognition in histopathology are tuned to a specific problem since the computational complexity exceeds that of traditional image classification problems. In the current study, a deep learning approach is developed and presented that can be trained to locate and accurately classify different types of pathologies in gigapixel digitized histology slides along with completing the binary disease classification for the entire image. The approach uses a novel pyramid tiling approach to take advantage of spatial awareness around the area to be classified, while maintaining efficiency and scalability for gigapixel images. The approach is trained and validated on a wide variety of tissue types (i.e., testis, ovary, prostate, kidney) and pathologies taken from an epigenetically altered histology study at Washington State University. The newly developed procedure was optimized and validated along with comparison and validation on public histology datasets. The current developed procedure was found to be optimal and more reproducible when compared to manual procedures, and optimal to previous protocols that used fragmented tissue or slide analysis. Observations demonstrate that the deep learning histopathology analysis is significantly more efficient and accurate than standard manual histopathology analysis.

Treponeme-associated hoof disease (TAHD) is an emerging disease of elk ( Cervus canadensis ) in the U.S. Pacific West. Because environmental epigenetics is the primary molecular process that mediates environmental factor impacts on a host organism and disease, the role of epigenetics in TAHD etiology was examined. The current study was designed to examine potential effects of TAHD on systemic epigenetic modifications in infected elk over a range of TAHD lesion severity. Leg tendons that contain predominantly fibroblast connective tissue cells were used to isolate fibroblast cells for epigenetic analysis in unaffected and TAHD-positive male and female Roosevelt and Rocky Mountain elk. Differential DNA methylation regions (DMRs) between the unaffected and TAHD-positive elk were identified for both female and male elk. The presence of TAHD was associated with alteration of the connective tissue cell epigenetics, and DMR associated genes identified. Therefore, the infected elk were found to have a systemic epigenetic alteration that was associated with the disease, despite pathology being generally limited to feet. If the elk germline epigenetics is altered then generational transmission of susceptibility to TAHD may impact subsequent generations through epigenetic inheritance. This first study of epigenetic changes associated with disease in elk suggests that TAHD promotes a systemic effect on the elk epigenetics which could exert health impacts on the elk.

The current study was designed to use an epigenome-wide association approach (EWAS) to identify potential systemic DNA methylation alterations that are associated with obesity using 22 discordant twin pairs. Buccal cells (from a cheek swab) were used as a non-obesity relevant purified marker cell for the epigenetic analysis. Analysis of differential DNA methylation regions (DMRs) was used to identify epigenetic associations with metabolic and dietary measures related to obesity with discordant twins. An edgeR analysis provided a DMR signature with p < 1e-04, but statistical significance was reduced due to low sample size and known multiple origins of obesity. A weighted gene coexpression network analysis (WGCNA) was performed and identified modules (p < 0.005) of epigenetic sites that correlated with different metabolic and dietary measures. The DMR and WGCNA epigenetic sites were near genes (e.g., CIDEC, SPP1, ZFPG9, and POMC) with previously identified obesity associated pathways (e.g., metabolism, cholesterol, and fat digestion). Observations demonstrate the feasibility of identifying systemic epigenetic biomarkers for obesity, which can be further investigated for clinical relevance in future research with larger sample sizes. The availability of a systemic epigenetic biomarker for obesity susceptibility may facilitate preventative medicine and clinical management of the disease early in life.

Abnormal penile foreskin development in hypospadias is the most frequent genital malformation in male children, which has increased dramatically in recent decades. A number of environmental factors have been shown to be associated with hypospadias development. The current study investigated the role of epigenetics in the etiology of hypospadias and compared mild (distal), moderate (mid shaft), and severe (proximal) hypospadias. Penile foreskin samples were collected from hypospadias and non-hypospadias individuals to identify alterations in DNA methylation associated with hypospadias. Dramatic numbers of differential DNA methylation regions (DMRs) were observed in the mild hypospadias, with reduced numbers in moderate and low numbers in severe hypospadias. Atresia (cell loss) of the principal foreskin fibroblast is suspected to be a component of the disease etiology. A genome-wide (> 95%) epigenetic analysis was used and the genomic features of the DMRs identified. The DMR associated genes identified a number of novel hypospadias associated genes and pathways, as well as genes and networks known to be involved in hypospadias etiology. Observations demonstrate altered DNA methylation sites in penile foreskin is a component of hypospadias etiology. In addition, a potential role of environmental epigenetics and epigenetic inheritance in hypospadias disease etiology is suggested.

Three successive multiple generations of rats were exposed to different toxicants and then bred to the transgenerational F5 generation to assess the impacts of multiple generation different exposures. The current study examines the actions of the agricultural fungicide vinclozolin on the F0 generation, followed by jet fuel hydrocarbon mixture exposure of the F1 generation, and then pesticide dichlorodiphenyltrichloroethane on the F2 generation gestating females. The subsequent F3 and F4 generations and F5 transgenerational generation were obtained and F1-F5 generations examined for male sperm epigenetic alterations and pathology in males and females. Significant impacts on the male sperm differential DNA methylation regions were observed. The F3-F5 generations were similar in similar to 50% of the DNA methylation regions. The pathology of each generation was assessed in the testis, ovary, kidney, and prostate, as well as the presence of obesity and tumors. The pathology used a newly developed Deep Learning, artificial intelligence-based histopathology analysis. Observations demonstrated compounded disease impacts in obesity and metabolic parameters, but other pathologies plateaued with smaller increases at the F5 transgenerational generation. Observations demonstrate that multiple generational exposures, which occur in human populations, appear to increase epigenetic impacts and disease susceptibility.

Germline transmission of epigenetic information is a critical component of epigenetic inheritance. Previous studies have suggested that an erasure of DNA methylation is required to develop stem cells in the morula embryo. An exception involves imprinted genes that escape this DNA methylation erasure. Transgenerational differential DNA methylation regions (DMRs) have been speculated to be imprinted-like and escape this erasure. The current study was designed to assess if morula embryos escape the erasure of dichlorodiphenyltrichloroethane-induced transgenerational sperm DMR methylation. Observations demonstrate that the majority (98%) of transgenerational sperm DMR sites retain DNA methylation and are not erased, so appearing similar to imprinted-like sites. Interestingly, observations also demonstrate that the majority of low-density CpG genomic sites had a significant increase in DNA methylation in the morula embryo compared to sperm. This is in contrast to the previously observed DNA methylation erasure of higher-density CpG sites. The general erasure of DNA methylation during embryogenesis appears applicable to high-density DNA methylation sites (e.g. CpG islands) but neither to transgenerational DMR methylation sites nor to low-density CpG deserts, which constitute the vast majority of the genome's DNA methylation sites. The role of epigenetics during embryogenesis appears more dynamic than the simple erasure of DNA methylation.

The current study was designed to use a rodent model to determine if exposure to the chemotherapy drug ifosfamide during puberty can induce altered phenotypes and disease in the grand-offspring of exposed individuals through epigenetic transgenerational inheritance. Pathologies such as delayed pubertal onset, kidney disease, and multiple pathologies were observed to be significantly more frequent in the F1 generation offspring of ifosfamide lineage females. The F2 generation grand-offspring ifosfamide lineage males had transgenerational pathology phenotypes of early pubertal onset and reduced testis pathology. Reduced levels of anxiety were observed in both males and females in the transgenerational F2 generation grand-offspring. Differential DNA methylated regions (DMRs) in chemotherapy lineage sperm in the F1 and F2 generations were identified. Therefore, chemotherapy exposure impacts pathology susceptibility in subsequent generations. Observations highlight the importance that prior to chemotherapy, individuals need to consider cryopreservation of germ cells as a precautionary measure if they have children.

Preterm birth is the major cause of newborn and infant mortality affecting nearly one in every ten live births. The current study was designed to develop an epigenetic biomarker for susceptibility of preterm birth using buccal cells from the mother, father, and child (triads). An epigenome-wide association study (EWAS) was used to identify differential DNA methylation regions (DMRs) using a comparison of control term birth versus preterm birth triads. Epigenetic DMR associations with preterm birth were identified for both the mother and father that were distinct and suggest potential epigenetic contributions from both parents. The mother (165 DMRs) and female child (136 DMRs) at p < 1e−04 had the highest number of DMRs and were highly similar suggesting potential epigenetic inheritance of the epimutations. The male child had negligible DMR associations. The DMR associated genes for each group involve previously identified preterm birth associated genes. Observations identify a potential paternal germline contribution for preterm birth and identify the potential epigenetic inheritance of preterm birth susceptibility for the female child later in life. Although expanded clinical trials and preconception trials are required to optimize the potential epigenetic biomarkers, such epigenetic biomarkers may allow preventative medicine strategies to reduce the incidence of preterm birth.

Because epigenetics is a critical component for gene expression, the hypothesis was tested that DNA methylation alterations are dynamic and continually change throughout gametogenesis to generate the mature sperm. Developmental alterations and stage-specific DNA methylation during gametogenesis from primordial germ cells (PGCs) to mature sperm are investigated. Individual developmental stage germ cells were isolated and analyzed for differential DNA methylation regions (DMRs). The number of DMRs was highest in the first three comparisons with mature PGCs, prospermatogonia, and spermatogonia. The most statistically significant DMRs were present at all stages of development and had variations involving both increases or decreases in DNA methylation. DMR-associated genes were identified and correlated with gene functional categories, pathways, and cellular processes. Observations identified a dynamic cascade of epigenetic changes during development that is dramatic during the early developmental stages. Complex epigenetic alterations are required to regulate genome biology and gene expression during gametogenesis.

There is mounting evidence that environmental exposures can result in effects on health that can be transmitted across generations, without the need for a direct exposure to the original factor, for example, the effect of grandparental smoking on grandchildren. Hence, an individual's health should be investigated with the knowledge of cross-generational influences. Epigenetic factors are molecular factors or processes that regulate genome activity and may impact cross-generational effects. Epigenetic transgenerational inheritance has been demonstrated in plants and animals, but the presence and extent of this process in humans are currently being investigated. Experimental data in animals support transmission of asthma risk across generations from a single exposure to the deleterious factor and suggest that the nature of this transmission is in part due to changes in DNA methylation, the most studied epigenetic process. The association of father's prepuberty exposure with offspring risk of asthma and lung function deficit may also be mediated by epigenetic processes. Multi-generational birth cohorts are ideal to investigate the presence and impact of transfer of disease susceptibility across generations and underlying mechanisms. However, multi-generational studies require recruitment and assessment of participants over several decades. Investigation of adult multi-generation cohorts is less resource intensive but run the risk of recall bias. Statistical analysis is challenging given varying degrees of longitudinal and hierarchical data but path analyses, structural equation modelling and multilevel modelling can be employed, and directed networks addressing longitudinal effects deserve exploration as an effort to study causal pathways.

Environmental toxicants have been shown to promote the epigenetic transgenerational inheritance of disease through exposure specific epigenetic alterations in the germline. The current study examines the actions of hydrocarbon jet fuel, dioxin, pesticides (permethrin and methoxychlor), plastics, and herbicides (glyphosate and atrazine) in the promotion of transgenerational disease in the great grand-offspring rats that correlates with specific disease associated differential DNA methylation regions (DMRs). The transgenerational disease observed was similar for all exposures and includes pathologies of the kidney, prostate, and testis, pubertal abnormalities, and obesity. The disease specific DMRs in sperm were exposure specific for each pathology with negligible overlap. Therefore, for each disease the DMRs and associated genes were distinct for each exposure generational lineage. Observations suggest a large number of DMRs and associated genes are involved in a specific pathology, and various environmental exposures influence unique subsets of DMRs and genes to promote the transgenerational developmental origins of disease susceptibility later in life. A novel multiscale systems biology basis of disease etiology is proposed involving an integration of environmental epigenetics, genetics and generational toxicology.