Could the sperm epigenome become a diagnostic tool for evaluation of the infertile man?

Although male infertility is currently diagnosed when abnormal sperm parameters are found, the poor predictive ability of sperm parameters on natural fecundity and medically assisted reproduction outcome poses the need for improved diagnostic techniques for male infertility. The accumulating evidence about the role played by the sperm epigenome in modulation of the early phases of embryonic development has led researchers to focus on the epigenetic mechanisms within the sperm epigenome to find new molecular markers of male infertility. Indeed, sperm epigenome abnormalities could explain some cases of unexplained male infertility in men showing normal sperm parameters and were found to be associated with poor embryo development in IVF cycles. The present mini-review summarizes the current knowledge about this interesting topic, starting from a description of the epigenetic mechanisms of gene expression regulation (i.e. DNA methylation, histone modifications, and non-coding RNAs' activity). We also discuss possible mechanisms by which environmental factors might cause epigenetic changes in the human germline and affect embryonic development, as well as subsequent generations' phenotypes. Studies demonstrating sperm epigenome abnormalities in men with male infertility are reviewed, with particular emphasis on those with the more severe form of spermatogenic dysfunction. Observations demonstrate that the diagnostic and prognostic efficacy of sperm epigenome evaluation will help facilitate the management of men with male factor infertility.

Epigenetic inheritance of DNA methylation changes in fish living in hydrogen sulfide-rich springs.

Environmental factors can promote phenotypic variation through alterations in the epigenome and facilitate adaptation of an organism to the environment. Although hydrogen sulfide is toxic to most organisms, the fish Poecilia mexicana has adapted to survive in environments with high levels that exceed toxicity thresholds by orders of magnitude. Epigenetic changes in response to this environmental stressor were examined by assessing DNA methylation alterations in red blood cells, which are nucleated in fish. Males and females were sampled from sulfidic and nonsulfidic natural environments; individuals were also propagated for two generations in a nonsulfidic laboratory environment. We compared epimutations between the sexes as well as field and laboratory populations. For both the wild-caught (F0) and the laboratory-reared (F2) fish, comparing the sulfidic and nonsulfidic populations revealed evidence for significant differential DNA methylation regions (DMRs). More importantly, there was over 80% overlap in DMRs across generations, suggesting that the DMRs have stable generational inheritance in the absence of the sulfidic environment. This is an example of epigenetic generational stability after the removal of an environmental stressor. The DMR-associated genes were related to sulfur toxicity and metabolic processes. These findings suggest that adaptation of P. mexicana to sulfidic environments in southern Mexico may, in part, be promoted through epigenetic DNA methylation alterations that become stable and are inherited by subsequent generations independent of the environment.

Hybrid deep learning approach to improve classification of low-volume high-dimensional data.

BACKGROUND: The performance of machine learning classification methods relies heavily on the choice of features. In many domains, feature generation can be labor-intensive and require domain knowledge, and feature selection methods do not scale well in high-dimensional datasets. Deep learning has shown success in feature generation but requires large datasets to achieve high classification accuracy. Biology domains typically exhibit these challenges with numerous handcrafted features (high-dimensional) and small amounts of training data (low volume). METHOD: A hybrid learning approach is proposed that first trains a deep network on the training data, extracts features from the deep network, and then uses these features to re-express the data for input to a non-deep learning method, which is trained to perform the final classification. RESULTS: The approach is systematically evaluated to determine the best layer of the deep learning network from which to extract features and the threshold on training data volume that prefers this approach. Results from several domains show that this hybrid approach outperforms standalone deep and non-deep learning methods, especially on low-volume, high-dimensional datasets. The diverse collection of datasets further supports the robustness of the approach across different domains. CONCLUSIONS: The hybrid approach combines the strengths of deep and non-deep learning paradigms to achieve high performance on high-dimensional, low volume learning tasks that are typical in biology domains.

Epigenetic Inheritance and Transgenerational Environmental Justice.

Many chemicals and toxicants are released into our ecosystem and environment every day, which can cause harmful effects on human populations. Agricultural compounds are used in most crop production and have been shown to cause negative health impacts, including effects on reproduction and other pathologies. Although these chemicals can be helpful for pest and weed control, the compounds indirectly impact humans. Several compounds have been banned in the European Union but continue to be used in the United States. Recent work has shown most toxicants affect transgenerational generations more than the directly exposed generations through epigenetic inheritance. While some toxicants do not impact the directly exposed generation, the later generations that are transgenerational or ancestrally exposed suffer health impacts. Due to impacts to future generations, exposure becomes an environmental justice concern. The term "environmental justice" denotes the application of fair strategies when resolving unjust environmental contamination. Fair treatment means that no group should bear a disproportionate share of negative environmental consequences resulting from industrial, municipal, and commercial operations. This article illustrates how research on directly exposed generations is often prioritized over studies on transgenerational generations. However, research on the latter generations suggests the need to take environmental justice concerns seriously moving forward, as future generations could be unduly shouldering harms, while not enjoying benefits of production.

Predicting environmentally responsive transgenerational differential DNA methylated regions (epimutations) in the genome using a hybrid deep-machine learning approach.

BACKGROUND: Deep learning is an active bioinformatics artificial intelligence field that is useful in solving many biological problems, including predicting altered epigenetics such as DNA methylation regions. Deep learning (DL) can learn an informative representation that addresses the need for defining relevant features. However, deep learning models are computationally expensive, and they require large training datasets to achieve good classification performance. RESULTS: One approach to addressing these challenges is to use a less complex deep learning network for feature selection and Machine Learning (ML) for classification. In the current study, we introduce a hybrid DL-ML approach that uses a deep neural network for extracting molecular features and a non-DL classifier to predict environmentally responsive transgenerational differential DNA methylated regions (DMRs), termed epimutations, based on the extracted DL-based features. Various environmental toxicant induced epigenetic transgenerational inheritance sperm epimutations were used to train the model on the rat genome DNA sequence and use the model to predict transgenerational DMRs (epimutations) across the entire genome. CONCLUSION: The approach was also used to predict potential DMRs in the human genome. Experimental results show that the hybrid DL-ML approach outperforms deep learning and traditional machine learning methods.

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.

Epigenetic transgenerational inheritance of parent-of-origin allelic transmission of outcross pathology and sperm epimutations.

Epigenetic transgenerational inheritance potentially impacts disease etiology, phenotypic variation, and evolution. An increasing number of environmental factors from nutrition to toxicants have been shown to promote the epigenetic transgenerational inheritance of disease. Previous observations have demonstrated that the agricultural fungicide vinclozolin and pesticide DDT (dichlorodiphenyltrichloroethane) induce transgenerational sperm epimutations involving DNA methylation, ncRNA, and histone modifications or retention. These two environmental toxicants were used to investigate the impacts of parent-of-origin outcross on the epigenetic transgenerational inheritance of disease. Male and female rats were collected from a paternal outcross (POC) or a maternal outcross (MOC) F4 generation control and exposure lineages for pathology and epigenetic analysis. This model allows the parental allelic transmission of disease and epimutations to be investigated. There was increased pathology incidence in the MOC F4 generation male prostate, kidney, obesity, and multiple diseases through a maternal allelic transmission. The POC F4 generation female offspring had increased pathology incidence for kidney, obesity and multiple types of diseases through the paternal allelic transmission. Some disease such as testis or ovarian pathology appear to be transmitted through the combined actions of both male and female alleles. Analysis of the F4 generation sperm epigenomes identified differential DNA methylated regions (DMRs) in a genome-wide analysis. Observations demonstrate that DDT and vinclozolin have the potential to promote the epigenetic transgenerational inheritance of disease and sperm epimutations to the outcross F4 generation in a sex specific and exposure specific manner. The parent-of-origin allelic transmission observed appears similar to the process involved with imprinted-like genes.

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.

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.

Developmental origins of transgenerational sperm DNA methylation epimutations following ancestral DDT exposure.

Epigenetic alterations in the germline can be triggered by a number of different environmental factors from diet to toxicants. These environmentally induced germline changes can promote the epigenetic transgenerational inheritance of disease and phenotypic variation. In previous studies, the pesticide DDT was shown to promote the transgenerational inheritance of sperm differential DNA methylation regions (DMRs), also called epimutations, which can in part mediate this epigenetic inheritance. In the current study, the developmental origins of the transgenerational DMRs during gametogenesis have been investigated. Male control and DDT lineage F3 generation rats were used to isolate embryonic day 16 (E16) prospermatogonia, postnatal day 10 (P10) spermatogonia, adult pachytene spermatocytes, round spermatids, caput epididymal spermatozoa, and caudal sperm. The DMRs between the control versus DDT lineage samples were determined at each developmental stage. The top 100 statistically significant DMRs at each stage were compared and the developmental origins of the caudal epididymal sperm DMRs were assessed. The chromosomal locations and genomic features of the different stage DMRs were analyzed. Although previous studies have demonstrated alterations in the DMRs of primordial germ cells (PGCs), the majority of the DMRs identified in the caudal sperm originated during the spermatogonia stages in the testis. Interestingly, a cascade of epigenetic alterations initiated in the PGCs is required to alter the epigenetic programming during spermatogenesis to obtain the sperm epigenetics involved in the epigenetic transgenerational inheritance phenomenon.

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.

Prenatal influences on temperament development: The role of environmental epigenetics.

This review summarizes current knowledge and outlines future directions relevant to questions concerning environmental epigenetics and the processes that contribute to temperament development. Links between prenatal adversity, epigenetic programming, and early manifestations of temperament are important in their own right, also informing our understanding of biological foundations for social-emotional development. In addition, infant temperament attributes represent key etiological factors in the onset of developmental psychopathology, and studies elucidating their prenatal foundations expand our understanding of developmental origins of health and disease. Prenatal adversity can take many forms, and this overview is focused on the environmental effects of stress, toxicants, substance use/psychotropic medication, and nutrition. Dysregulation associated with attention-deficit/hyperactivity-disruptive disorders was noted in the context of maternal substance use and toxicant exposures during gestation, as well as stress. Although these links can be made based on the existing literature, currently few studies directly connect environmental influences, epigenetic programming, and changes in brain development/behavior. The chain of events starting with environmental inputs and resulting in alterations to gene expression, physiology, and behavior of the organism is driven by epigenetics. Epigenetics provides the molecular mechanism of how environmental factors impact development and subsequent health and disease, including early brain and temperament development.

Epigenetic variation between urban and rural populations of Darwin's finches.

BACKGROUND: The molecular basis of evolutionary change is assumed to be genetic variation. However, growing evidence suggests that epigenetic mechanisms, such as DNA methylation, may also be involved in rapid adaptation to new environments. An important first step in evaluating this hypothesis is to test for the presence of epigenetic variation between natural populations living under different environmental conditions. RESULTS: In the current study we explored variation between populations of Darwin's finches, which comprise one of the best-studied examples of adaptive radiation. We tested for morphological, genetic, and epigenetic differences between adjacent "urban" and "rural" populations of each of two species of ground finches, Geospiza fortis and G. fuliginosa, on Santa Cruz Island in the Galápagos. Using data collected from more than 1000 birds, we found significant morphological differences between populations of G. fortis, but not G. fuliginosa. We did not find large size copy number variation (CNV) genetic differences between populations of either species. However, other genetic variants were not investigated. In contrast, we did find dramatic epigenetic differences between the urban and rural populations of both species, based on DNA methylation analysis. We explored genomic features and gene associations of the differentially DNA methylated regions (DMR), as well as their possible functional significance. CONCLUSIONS: In summary, our study documents local population epigenetic variation within each of two species of Darwin's finches.

Genomic Clustering of differential DNA methylated regions (epimutations) associated with the epigenetic transgenerational inheritance of disease and phenotypic variation.

BACKGROUND: A variety of environmental factors have been shown to promote the epigenetic transgenerational inheritance of disease and phenotypic variation in numerous species. Exposure to environmental factors such as toxicants can promote epigenetic changes (epimutations) involving alterations in DNA methylation to produce specific differential DNA methylation regions (DMRs). The germline (e.g. sperm) transmission of epimutations is associated with epigenetic transgenerational inheritance phenomena. The current study was designed to determine the genomic locations of environmentally induced transgenerational DMRs and assess their potential clustering. RESULTS: The exposure specific DMRs (epimutations) from a number of different studies were used. The clustering approach identified areas of the genome that have statistically significant over represented numbers of epimutations. The location of DMR clusters was compared to the gene clusters of differentially expressed genes found in tissues and cells associated with the transgenerational inheritance of disease. Such gene clusters, termed epigenetic control regions (ECRs), have been previously suggested to regulate gene expression in regions spanning up to 2-5 million bases. DMR clusters were often found to associate with inherent gene clusters within the genome. CONCLUSION: The current study used a number of epigenetic datasets from previous studies to identify novel DMR clusters across the genome. Observations suggest these clustered DMR within an ECR may be susceptible to epigenetic reprogramming and dramatically influence genome activity.

Epigenetic programming alterations in alligators from environmentally contaminated lakes.

Previous studies examining the reproductive health of alligators in Florida lakes indicate that a variety of developmental and health impacts can be attributed to a combination of environmental quality and exposures to environmental contaminants. The majority of these environmental contaminants have been shown to disrupt normal endocrine signaling. The potential that these environmental conditions and contaminants may influence epigenetic status and correlate to the health abnormalities was investigated in the current study. The red blood cell (RBC) (erythrocyte) in the alligator is nucleated so was used as an easily purified marker cell to investigate epigenetic programming. RBCs were collected from adult male alligators captured at three sites in Florida, each characterized by varying degrees of contamination. While Lake Woodruff (WO) has remained relatively pristine, Lake Apopka (AP) and Merritt Island (MI) convey exposures to different suites of contaminants. DNA was isolated and methylated DNA immunoprecipitation (MeDIP) was used to isolate methylated DNA that was then analyzed in a competitive hybridization using a genome-wide alligator tiling array for a MeDIP-Chip analysis. Pairwise comparisons of alligators from AP and MI to WO revealed alterations in the DNA methylome. The AP vs. WO comparison identified 85 differential DNA methylation regions (DMRs) with ⩾3 adjacent oligonucleotide tiling array probes and 15,451 DMRs with a single oligo probe analysis. The MI vs. WO comparison identified 75 DMRs with the ⩾3 oligo probe and 17,411 DMRs with the single oligo probe analysis. There was negligible overlap between the DMRs identified in AP vs. WO and MI vs. WO comparisons. In both comparisons DMRs were primarily associated with CpG deserts which are regions of low CpG density (1-2CpG/100bp). Although the alligator genome is not fully annotated, gene associations were identified and correlated to major gene class functional categories and pathways of endocrine relevance. Observations demonstrate that environmental quality may be associated with epigenetic programming and health status in the alligator. The epigenetic alterations may provide biomarkers to assess the environmental exposures and health impacts on these populations of alligators.

Environmentally Induced Epigenetic Transgenerational Inheritance of Reproductive Disease.

Reproductive disease and fertility issues have dramatically increased in the human population over the last several decades, suggesting environmental impacts. Epigenetics provides a mechanistic link by which an organism can respond to environmental factors. Interestingly, environmentally induced epigenetic alterations in the germ line can promote aberrant gene expression and disease generationally. Environmentally induced epigenetic transgenerational inheritance is defined as germ-line transmission of altered epigenetic information between generations in the absence of continued environmental exposures. This form of nongenetic inheritance has been shown to directly influence fertility and reproductive disease. This review describes the studies in a variety of species that impact reproductive disease and abnormalities. Observations suggest serious attention be paid to the possibility that ancestral exposures to environmental insults promotes transgenerational inheritance of reproductive disease susceptibility. Environmentally induced epigenetic transgenerational inheritance appears to be an important contributing factor to reproductive disease in many organisms, including humans.

Epigenetic transgenerational inheritance of altered stress responses.

Ancestral environmental exposures have previously been shown to promote epigenetic transgenerational inheritance and influence all aspects of an individual's life history. In addition, proximate life events such as chronic stress have documented effects on the development of physiological, neural, and behavioral phenotypes in adulthood. We used a systems biology approach to investigate in male rats the interaction of the ancestral modifications carried transgenerationally in the germ line and the proximate modifications involving chronic restraint stress during adolescence. We find that a single exposure to a common-use fungicide (vinclozolin) three generations removed alters the physiology, behavior, metabolic activity, and transcriptome in discrete brain nuclei in descendant males, causing them to respond differently to chronic restraint stress. This alteration of baseline brain development promotes a change in neural genomic activity that correlates with changes in physiology and behavior, revealing the interaction of genetics, environment, and epigenetic transgenerational inheritance in the shaping of the adult phenotype. This is an important demonstration in an animal that ancestral exposure to an environmental compound modifies how descendants of these progenitor individuals perceive and respond to a stress challenge experienced during their own life history.

Role of CpG deserts in the epigenetic transgenerational inheritance of differential DNA methylation regions.

BACKGROUND: Previously a variety of environmental toxicants were found to promote the epigenetic transgenerational inheritance of disease through differential DNA methylation regions (DMRs), termed epimutations, present in sperm. The transgenerational epimutations in sperm and somatic cells identified in a number of previous studies were further investigated. RESULTS: The epimutations from six different environmental exposures were found to be predominantly exposure specific with negligible overlap. The current report describes a major genomic feature of all the unique epimutations identified (535) as a very low (<10 CpG/100 bp) CpG density in sperm and somatic cells associated with transgenerational disease. The genomic locations of these epimutations were found to contain DMRs with small clusters of CpG within a general region of very low density CpG. The potential role of these epimutations on gene expression is suggested to be important. CONCLUSIONS: Observations suggest a potential regulatory role for lower density CpG regions termed "CpG deserts". The potential evolutionary origins of these regions is also discussed.

Gene bionetworks involved in the epigenetic transgenerational inheritance of altered mate preference: environmental epigenetics and evolutionary biology.

BACKGROUND: Mate preference behavior is an essential first step in sexual selection and is a critical determinant in evolutionary biology. Previously an environmental compound (the fungicide vinclozolin) was found to promote the epigenetic transgenerational inheritance of an altered sperm epigenome and modified mate preference characteristics for three generations after exposure of a gestating female. RESULTS: The current study investigated gene networks involved in various regions of the brain that correlated with the altered mate preference behavior in the male and female. Statistically significant correlations of gene clusters and modules were identified to associate with specific mate preference behaviors. This novel systems biology approach identified gene networks (bionetworks) involved in sex-specific mate preference behavior. Observations demonstrate the ability of environmental factors to promote the epigenetic transgenerational inheritance of this altered evolutionary biology determinant. CONCLUSIONS: Combined observations elucidate the potential molecular control of mate preference behavior and suggests environmental epigenetics can have a role in evolutionary biology.

Environmental stress and epigenetic transgenerational inheritance.

Previous studies have shown a wide variety of environmental toxicants and abnormal nutrition can promote the epigenetic transgenerational inheritance of disease. More recently a number of studies have indicated environmental stress can also promote epigenetic alterations that are transmitted to subsequent generations to induce pathologies. A recent study by Yao and colleagues demonstrated gestational exposure to restraint stress and forced swimming promoted preterm birth risk and adverse newborn outcomes generationally. This ancestral stress promoted the epigenetic transgenerational inheritance of abnormalities in the great-grand offspring of the exposed gestating female. Several studies now support the role of environmental stress in promoting the epigenetic transgenerational inheritance of disease. Observations suggest ancestral environmental stress may be a component of disease etiology in the current population.