Transgenerational sperm DMRs escape DNA methylation erasure during embryonic development and epigenetic inheritance.

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.

Role of epigenetics in the etiology of hypospadias through penile foreskin DNA methylation alterations.

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.

Examination of generational impacts of adolescent chemotherapy: Ifosfamide and potential for epigenetic transgenerational inheritance.

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.

Environmental induced transgenerational inheritance impacts systems epigenetics in disease etiology.

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.

Role of epigenetic transgenerational inheritance in generational toxicology.

Many environmental toxicants have been shown to be associated with the transgenerational inheritance of increased disease susceptibility. This review describes the generational toxicity of some of these chemicals and their role in the induction of epigenetic transgenerational inheritance of disease. Epigenetic factors include DNA methylation, histone modifications, retention of histones in sperm, changes to chromatin structure, and expression of non-coding RNAs. For toxicant-induced epigenetic transgenerational inheritance to occur, exposure to a toxicant must result in epigenetic changes to germ cells (sperm or eggs) since it is the germ cells that carry molecular information to subsequent generations. In addition, the epigenetic changes induced in transgenerational generation animals must cause alterations in gene expression in these animals' somatic cells. In some cases of generational toxicology, negligible changes are seen in the directly exposed generations, but increased disease rates are seen in transgenerational descendants. Governmental policies regulating toxicant exposure should take generational effects into account. A new approach that takes into consideration generational toxicity will be needed to protect our future populations.

Developmental alterations in DNA methylation during gametogenesis from primordial germ cells to sperm.

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.

Genome-wide CpG density and DNA methylation analysis method (MeDIP, RRBS, and WGBS) comparisons.

Genome-wide DNA methylation analysis is one of the most common epigenetic processes analysed for genome characterization and differential DNA methylation assessment. Previous genome-wide analysis has suggested an important variable in DNA methylation methods involves CpG density. The current study was designed to investigate the CpG density in a variety of different species genomes and correlate this to various DNA methylation analysis data sets. The majority of all genomes had >90% of the genome in the low density 1-3 CpG/100 bp category, while <10% of the genome was in the higher density >5 CpG/100 bp category. Similar observations with human, rat, bird, and fish genomes were observed. The methylated DNA immunoprecipitation (MeDIP) procedure uses the anti-5-methylcytosine antibody immunoprecipitation followed by next-generation sequencing (MeDIP-Seq). The MeDIP procedure is biased to lower CpG density of <5 CpG/100 bp, which corresponds to >95% of the genome. The reduced representation bisulphite (RRBS) protocol generally identifies DMRs in higher CpG density regions of ≥3 CpG/100 bp which corresponds to approximately 20% of the genome. The whole-genome bisulphite (WGBS) analyses resulted in higher CpG densities, often greater than 10 CpG/100bp. WGBS generally identifies ≥2 CpG/100bp, which corresponds to approximately 50% of the genome. Limitations and potential optimization approaches for each method are discussed. None of the procedures can provide complete genome-wide assessment of the genome, but MeDIP-Seq provides coverage of the highest percentage. Observations demonstrate that CpG density is a critical variable in DNA methylation analysis, and different molecular techniques focus on distinct genomic regions.

Ancestral plastics exposure induces transgenerational disease-specific sperm epigenome-wide association biomarkers.

Plastic-derived compounds are one of the most frequent daily worldwide exposures. Previously a mixture of plastic-derived toxicants composed of bisphenol A, bis(2-ethylhexyl) phthalate, and dibutyl phthalate at low-dose exposures of a gestating female rats was found to promote the epigenetic transgenerational inheritance of disease to the offspring (F1 generation), grand-offspring (F2 generation), and great-grand-offspring (F3 generation). Epigenetic analysis of the male sperm was found to result in differential DNA methylation regions (DMRs) in the transgenerational F3 generation male sperm. The current study is distinct and was designed to use an epigenome-wide association study to identify potential sperm DNA methylation biomarkers for specific transgenerational diseases. Observations indicate disease-specific DMRs called epimutations in the transgenerational F3 generation great-grand-offspring of rats ancestrally exposed to plastics. The epigenetic DMR biomarkers were identified for testis disease, kidney disease, and multiple (≥2) diseases. These disease sperm epimutation biomarkers were found to be predominantly disease-specific. The genomic locations and features of these DMRs were identified. Interestingly, the disease-specific DMR-associated genes were previously shown to be linked with each of the specific diseases. Therefore, the germline has ancestrally derived epimutations that potentially transmit transgenerational disease susceptibilities. Epigenetic biomarkers for specific diseases could be used as diagnostics to facilitate clinical management of disease and preventative medicine.

Epigenetic transgenerational inheritance, gametogenesis and germline development†.

One of the most important developing cell types in any biological system is the gamete (sperm and egg). The transmission of phenotypes and optimally adapted physiology to subsequent generations is in large part controlled by gametogenesis. In contrast to genetics, the environment actively regulates epigenetics to impact the physiology and phenotype of cellular and biological systems. The integration of epigenetics and genetics is critical for all developmental biology systems at the cellular and organism level. The current review is focused on the role of epigenetics during gametogenesis for both the spermatogenesis system in the male and oogenesis system in the female. The developmental stages from the initial primordial germ cell through gametogenesis to the mature sperm and egg are presented. How environmental factors can influence the epigenetics of gametogenesis to impact the epigenetic transgenerational inheritance of phenotypic and physiological change in subsequent generations is reviewed.

Sperm DNA methylation epimutation biomarker for paternal offspring autism susceptibility.

BACKGROUND: Autism spectrum disorder (ASD) has increased over tenfold over the past several decades and appears predominantly associated with paternal transmission. Although genetics is anticipated to be a component of ASD etiology, environmental epigenetics is now also thought to be an important factor. Epigenetic alterations, such as DNA methylation, have been correlated with ASD. The current study was designed to identify a DNA methylation signature in sperm as a potential biomarker to identify paternal offspring autism susceptibility. METHODS AND RESULTS: Sperm samples were obtained from fathers that have children with or without autism, and the sperm then assessed for alterations in DNA methylation. A genome-wide analysis (> 90%) for differential DNA methylation regions (DMRs) was used to identify DMRs in the sperm of fathers (n = 13) with autistic children in comparison with those (n = 13) without ASD children. The 805 DMR genomic features such as chromosomal location, CpG density and length of the DMRs were characterized. Genes associated with the DMRs were identified and found to be linked to previously known ASD genes, as well as other neurobiology-related genes. The potential sperm DMR biomarkers/diagnostic was validated with blinded test sets (n = 8-10) of individuals with an approximately 90% accuracy. CONCLUSIONS: Observations demonstrate a highly significant set of 805 DMRs in sperm that can potentially act as a biomarker for paternal offspring autism susceptibility. Ancestral or early-life paternal exposures that alter germline epigenetics are anticipated to be a molecular component of ASD etiology.

Integration of sperm ncRNA-directed DNA methylation and DNA methylation-directed histone retention in epigenetic transgenerational inheritance.

BACKGROUND: Environmentally induced epigenetic transgenerational inheritance of pathology and phenotypic variation has been demonstrated in all organisms investigated from plants to humans. This non-genetic form of inheritance is mediated through epigenetic alterations in the sperm and/or egg to subsequent generations. Although the combined regulation of differential DNA methylated regions (DMR), non-coding RNA (ncRNA), and differential histone retention (DHR) have been shown to occur, the integration of these different epigenetic processes remains to be elucidated. The current study was designed to examine the integration of the different epigenetic processes. RESULTS: A rat model of transiently exposed F0 generation gestating females to the agricultural fungicide vinclozolin or pesticide DDT (dichloro-diphenyl-trichloroethane) was used to acquire the sperm from adult males in the subsequent F1 generation offspring, F2 generation grand offspring, and F3 generation great-grand offspring. The F1 generation sperm ncRNA had substantial overlap with the F1, F2 and F3 generation DMRs, suggesting a potential role for RNA-directed DNA methylation. The DMRs also had significant overlap with the DHRs, suggesting potential DNA methylation-directed histone retention. In addition, a high percentage of DMRs induced in the F1 generation sperm were maintained in subsequent generations. CONCLUSIONS: Many of the DMRs, ncRNA, and DHRs were colocalized to the same chromosomal location regions. Observations suggest an integration of DMRs, ncRNA, and DHRs in part involve RNA-directed DNA methylation and DNA methylation-directed histone retention in epigenetic transgenerational inheritance.

Epigenome-wide association study for glyphosate induced transgenerational sperm DNA methylation and histone retention epigenetic biomarkers for disease.

The herbicide glyphosate has been shown to promote the epigenetic transgenerational inheritance of pathology and disease in subsequent great-grand offspring (F3 generation). This generational toxicology suggests the impacts of environmental exposures need to assess subsequent generations. The current study was designed to identify epigenetic biomarkers for glyphosate-induced transgenerational diseases using an epigenome-wide association study (EWAS). Following transient glyphosate exposure of gestating female rats (F0 generation), during the developmental period of gonadal sex determination, the subsequent transgenerational F3 generation, with no direct exposure, were aged to 1 year and animals with specific pathologies identified. The pathologies investigated included prostate disease, kidney disease, obesity, and presence of multiple disease. The sperm were collected from the glyphosate lineage males with only an individual disease and used to identify specific differential DNA methylation regions (DMRs) and the differential histone retention sites (DHRs) associated with that pathology. Unique signatures of DMRs and DHRs for each pathology were identified for the specific diseases. Interestingly, at a lower statistical threshold overlapping sets of DMRs and DHRs were identified that were common for all the pathologies. This is one of the first observations that sperm histone retention can potentially act as a biomarker for specific diseases. The DMR and DHR associated genes were identified and correlated with known pathology specific-associated genes. Observations indicate transgenerational epigenetic biomarkers of disease pathology can be identified in the sperm that appear to assess disease susceptibility. These biomarkers suggest epigenetic diagnostics could potentially be used to facilitate preventative medicine.

Genome-Wide Mapping of DNA Methylation 5mC by Methylated DNA Immunoprecipitation (MeDIP)-Sequencing.

Methylated DNA immunoprecipitation is a large scale purification technique. It enables the isolation of methylated DNA fragments for subsequent locus-specific or genome-wide analysis. Here we describe an immunoprecipitation protocol using a monoclonal mouse anti 5-methyl-cytidine antibody followed by next-generation sequencing (MeDIP-Seq).

Transgenerational disease specific epigenetic sperm biomarkers after ancestral exposure to dioxin.

Dioxin was historically one of the most common industrial contaminants with several major industry accidents, as well as governmental actions involving military service, having exposed large numbers of the worldwide population over the past century. Previous rat studies have demonstrated the ability of dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)) exposure to promote the epigenetic transgenerational inheritance of disease susceptibility in subsequent generations. The types of disease previously observed include puberty abnormalities, testis, ovary, kidney, prostate and obesity pathologies. The current study was designed to use an epigenome-wide association study (EWAS) to identify potential sperm DNA methylation biomarkers for specific transgenerational diseases. Therefore, the transgenerational F3 generation dioxin lineage male rats with and without a specific disease were compared to identify differential DNA methylation regions (DMRs) as biomarkers for disease. The genomic features of the disease-specific DMRs were characterized. Observations demonstrate that disease-specific epimutation DMRs exist for the transgenerational dioxin lineage rats that can potentially be used as epigenetic biomarkers for testis, kidney, prostate and obesity diseases. These disease-specific DMRs were associated with genes that have previously been shown to be linked with the specific diseases. This EWAS for transgenerational disease identified potential epigenetic biomarkers and provides the proof of concept of the potential to develop similar biomarkers for humans to diagnose disease susceptibilities and facilitate preventative medicine.

Environmental impacts on sperm and oocyte epigenetics affect embryo cell epigenetics and transcription to promote the epigenetic inheritance of pathology and phenotypic variation.

Previous studies have demonstrated that exposure to environmental factors can cause epigenetic modifications to germ cells, particularly sperm, to promote epigenetic and transcriptome changes in the embryo. These germ cell and embryo cell epigenetic alterations are associated with phenotypic changes in offspring. Epigenetic inheritance requires epigenetic changes (i.e. epimutations) in germ cells that promote epigenetic and gene expression changes in embryos. The objective of this perspective is to examine the evidence that germ cell epigenome modifications are associated with embryo cell epigenetic and transcriptome changes that affect the subsequent development of all developing somatic cells to promote phenotype change. Various epigenetic changes in sperm, including changes to histone methylation, histone retention, non-coding RNA expression and DNA methylation, have been associated with alterations in embryo cell epigenetics and gene expression. Few studies have investigated this link for oocytes. The studies reviewed herein support the idea that environmentally induced epigenetic changes in germ cells affect alterations in embryo cell epigenetics and transcriptomes that have an important role in the epigenetic inheritance of pathology and phenotypic change.

Epigenome-wide association study for atrazine induced transgenerational DNA methylation and histone retention sperm epigenetic biomarkers for disease.

Atrazine is a common agricultural herbicide previously shown to promote epigenetic transgenerational inheritance of disease to subsequent generations. The current study was designed as an epigenome-wide association study (EWAS) to identify transgenerational sperm disease associated differential DNA methylation regions (DMRs) and differential histone retention regions (DHRs). Gestating female F0 generation rats were transiently exposed to atrazine during the period of embryonic gonadal sex determination, and then subsequent F1, F2, and F3 generations obtained in the absence of any continued exposure. The transgenerational F3 generation males were assessed for disease and sperm collected for epigenetic analysis. Pathology was observed in pubertal onset and for testis disease, prostate disease, kidney disease, lean pathology, and multiple disease. For these pathologies, sufficient numbers of individual males with only a single specific disease were identified. The sperm DNA and chromatin were isolated from adult one-year animals with the specific diseases and analyzed for DMRs with methylated DNA immunoprecipitation (MeDIP) sequencing and DHRs with histone chromatin immunoprecipitation (ChIP) sequencing. Transgenerational F3 generation males with or without disease were compared to identify the disease specific epimutation biomarkers. All pathologies were found to have disease specific DMRs and DHRs which were found to predominantly be distinct for each disease. No common DMRs or DHRs were found among all the pathologies. Epimutation gene associations were identified and found to correlate to previously known disease linked genes. This is one of the first observations of potential sperm disease biomarkers for histone retention sites. Although further studies with expanded animal numbers are required, the current study provides evidence the EWAS analysis is effective for the identification of potential pathology epimutation biomarkers for disease susceptibility.

Epigenome-wide association study for pesticide (Permethrin and DEET) induced DNA methylation epimutation biomarkers for specific transgenerational disease.

BACKGROUND: Permethrin and N,N-diethyl-meta-toluamide (DEET) are the pesticides and insect repellent most commonly used by humans. These pesticides have been shown to promote the epigenetic transgenerational inheritance of disease in rats. The current study was designed as an epigenome-wide association study (EWAS) to identify potential sperm DNA methylation epimutation biomarkers for specific transgenerational disease. METHODS: Outbred Sprague Dawley gestating female rats (F0) were transiently exposed during fetal gonadal sex determination to the pesticide combination including Permethrin and DEET. The F3 generation great-grand offspring within the pesticide lineage were aged to 1 year. The transgenerational adult male rat sperm were collected from individuals with single and multiple diseases and compared to non-diseased animals to identify differential DNA methylation regions (DMRs) as biomarkers for specific transgenerational disease. RESULTS: The exposure of gestating female rats to a permethrin and DEET pesticide combination promoted transgenerational testis disease, prostate disease, kidney disease, and the presence of multiple disease in the subsequent F3 generation great-grand offspring. The disease DMRs were found to be disease specific with negligible overlap between different diseases. The genomic features of CpG density, DMR length, and chromosomal locations of the disease specific DMRs were investigated. Interestingly, the majority of the disease specific sperm DMR associated genes have been previously found to be linked to relevant disease specific genes. CONCLUSIONS: Observations demonstrate the EWAS approach identified disease specific biomarkers that can be potentially used to assess transgenerational disease susceptibility and facilitate the clinical management of environmentally induced pathology.

Epigenome-wide association study for transgenerational disease sperm epimutation biomarkers following ancestral exposure to jet fuel hydrocarbons.

Jet fuel hydrocarbons is the generic name for aviation fuels used in gas-turbine engine powered aircraft. The Deepwater Horizon oil rig explosion created the largest environmental disaster in U.S. history, and the second largest oil spill in human history with over 800 million liters of hydrocarbons released into the Gulf of Mexico over a period of 3 months. Due to the widespread use of jet fuel hydrocarbons, this compound mixture has been recognized as the single largest chemical exposure for military personnel. Previous animal studies have demonstrated the ability of jet fuel (JP-8) exposure to promote the epigenetic transgenerational inheritance of disease susceptibility in subsequent generations. The diseases observed include late puberty, kidney, obesity and multiple disease pathologies. The current study is distinct and was designed to identify potential sperm DNA methylation biomarkers for specific transgenerational diseases. Observations show disease specific differential DNA methylation regions (DMRs) called epimutations in the transgenerational F3 generation great-grand-offspring male rats ancestrally exposed to jet fuel. The potential epigenetic DMR biomarkers were identified for late puberty, kidney, obesity, and multiple diseases, and found to be predominantly disease specific. These disease specific DMRs have associated genes that were previously shown to be linked with each of these specific diseases. Therefore, the germline (i.e. sperm) has environmentally induced ancestrally derived epimutations that have the potential to transgenerationally transmit disease susceptibilities to subsequent generations. Epigenetic biomarkers for specific diseases could be developed as medical diagnostics to facilitate clinical management of disease, and allow preventative medicine therapeutics.

Developmental origins of transgenerational sperm histone retention following ancestral exposures.

Numerous environmental toxicants have been shown to induce the epigenetic transgenerational inheritance of disease and phenotypic variation. Alterations in the germline epigenome are necessary to transmit transgenerational phenotypes. In previous studies, the pesticide DDT (dichlorodiphenyltrichloroethane) and the agricultural fungicide vinclozolin were shown to promote the transgenerational inheritance of sperm differential DNA methylation regions, non-coding RNAs and histone retention, which are termed epimutations. These epimutations are able to mediate this epigenetic inheritance of disease and phenotypic variation. The current study was designed to investigate the developmental origins of the transgenerational differential histone retention sites (called DHRs) during gametogenesis of the sperm. Vinclozolin and DDT were independently used to promote the epigenetic transgenerational inheritance of these DHRs. Male control lineage, DDT lineage and vinclozolin lineage F3 generation rats were used to isolate round spermatids, caput epididymal spermatozoa, and caudal sperm. The DHRs distinguishing the control versus DDT lineage or vinclozolin lineage samples were determined at these three developmental stages. DHRs and a reproducible core of histone H3 retention sites were observed using an H3 chromatin immunoprecipitation-sequencing (ChIP-Seq) analysis in each of the germ cell populations. The chromosomal locations and genomic features of the DHRs were analyzed. A cascade of epigenetic histone retention site alterations was found to be initiated in the round spermatids and then further modified during epididymal sperm maturation. Observations show that in addition to alterations in sperm DNA methylation and ncRNA expression previously identified, the induction of differential histone retention sites (DHRs) in the later stages of spermatogenesis also occurs. This novel component of epigenetic programming during spermatogenesis can be environmentally altered and transmitted to subsequent generations through epigenetic transgenerational inheritance.

Epigenome-wide association study (EWAS) for potential transgenerational disease epigenetic biomarkers in sperm following ancestral exposure to the pesticide methoxychlor.

Environmental exposures such as chemical toxicants can alter gene expression and disease susceptibility through epigenetic processes. Epigenetic changes can be passed to future generations through germ cells through epigenetic transgenerational inheritance of increased disease susceptibility. The current study used an epigenome-wide association study (EWAS) to investigate whether specific transgenerational epigenetic signatures of differential DNA methylation regions (DMRs) exist that are associated with particular disease states in the F3 generation great-grand offspring of F0 generation rats exposed during gestation to the agricultural pesticide methoxychlor. The transgenerational epigenetic profiles of sperm from F3 generation methoxychlor lineage rats that have only one disease state were compared to those that have no disease. Observations identify disease specific patterns of DMRs for these transgenerational rats that can potentially serve as epigenetic biomarkers for prostate disease, kidney disease, obesity, and the presence of multiple diseases. The chromosomal locations, genomic features, and gene associations of the DMRs are characterized. Disease specific DMR sets contained DMR-associated genes that have previously been shown to be associated with that specific disease. Future epigenetic biomarkers could potentially be developed and validated for humans as a disease susceptibility diagnostic tool to facilitate preventative medicine and management of disease.