NRF2 loss recapitulates heritable impacts of paternal cigarette smoke exposure.

Paternal cigarette smoke (CS) exposure is associated with increased risk of behavioral disorders and cancer in offspring, but the mechanism has not been identified. Here we use mouse models to investigate mechanisms and impacts of paternal CS exposure. We demonstrate that CS exposure induces sperm DNAme changes that are partially corrected within 28 days of removal from CS exposure. Additionally, paternal smoking is associated with changes in prefrontal cortex DNAme and gene expression patterns in offspring. Remarkably, the epigenetic and transcriptional effects of CS exposure that we observed in wild type mice are partially recapitulated in Nrf2-/- mice and their offspring, independent of smoking status. Nrf2 is a central regulator of antioxidant gene transcription, and mice lacking Nrf2 consequently display elevated oxidative stress, suggesting that oxidative stress may underlie CS-induced heritable epigenetic changes. Importantly, paternal sperm DNAme changes do not overlap with DNAme changes measured in offspring prefrontal cortex, indicating that the observed DNAme changes in sperm are not directly inherited. Additionally, the changes in sperm DNAme associated with CS exposure were not observed in sperm of unexposed offspring, suggesting the effects are likely not maintained across multiple generations.

High efficiency rare sperm separation from biopsy samples in an inertial focusing device.

Immotile and rare sperm isolation from a complex cell background is an essential process for infertility treatment. The traditional sperm collection process from a biopsy sample requires long, tedious searches, yet still results in low sperm retrieval. In this work, a high recovery, high throughput sperm separation process is proposed for the clinical biopsy sperm retrieval process. It is found that sperm have different focusing positions compared with non-sperm cells in the inertial flow, which is explained by a sperm alignment phenomenon. Separation in the spiral channel device results in a 95.6% sperm recovery in which 87.4% of non-sperm cells get removed. Rare sperm isolation from a clinical biopsy sample is performed with the current approach. The chance of finding sperm is shown to increase 8.2 fold in the treated samples. The achieved results highly support this method being used for the development of a rapid biopsy sperm sorting process. In addition, the mechanism was proposed and can be applied for the high-efficiency separation of non-spherical particles in general.

Young women with poor ovarian response exhibit epigenetic age acceleration based on evaluation of white blood cells using a DNA methylation-derived age prediction model.

STUDY QUESTION: Is poor ovarian response associated with a change in predicted age based on a DNA methylation-derived age prediction model (the Horvath algorithm) in white blood cells (WBCs) or cumulus cells (CCs)? SUMMARY ANSWER: In young women, poor ovarian response is associated with epigenetic age acceleration within WBC samples but is not associated with age-related changes in CC. WHAT IS KNOWN ALREADY: The majority of human tissues follow predictable patterns of methylation which can be assessed throughout a person's lifetime. DNA methylation patterns may serve as informative biomarkers of aging within various tissues. Horvath's 'epigenetic clock', which is a DNA methylation-derived age prediction model, accurately predicts a subject's true chronologic age when applied to WBC but not to CC. STUDY DESIGN, SIZE, DURATION: A prospective cohort study was carried out involving 175 women undergoing ovarian stimulation between February 2017 and December 2018. Women were grouped according to a poor (≤5 oocytes retrieved) or good (>5 oocytes) response to ovarian stimulation. Those with polycystic ovary syndrome (PCOS) (n = 35) were placed in the good responder group. PARTICIPANTS/MATERIALS, SETTING, METHODS: DNA methylation patterns from WBC and CC were assessed for infertile patients undergoing ovarian stimulation at a university-affiliated private practice. DNA was isolated from peripheral blood samples and CC. Bisulfite conversion was then performed and a DNA methylation array was utilized to measure DNA methylation levels throughout the genome. Likelihood ratio tests were utilized to assess the relationship between predicted age, chronologic age and ovarian response. MAIN RESULTS AND THE ROLE OF CHANCE: The Horvath-predicted age for WBC samples was consistent with patients' chronologic age. However, predicted age from analysis of CC was younger than chronologic age. In subgroup analysis of women less than 38 years of age, poor ovarian response was associated with an accelerated predicted age in WBC (P = 0.017). Poor ovarian response did not affect the Horvath-predicted age based on CC samples (P = 0.502). No alternative methylation-based calculation was identified to be predictive of age for CC. LIMITATIONS, REASONS FOR CAUTION: To date, analyses of CC have failed to identify epigenetic changes that are predictive of the aging process within the ovary. Despite the poor predictive nature of both the Horvath model and the novel methylation-based age prediction model described here, it is possible that our efforts failed to identify appropriate sites which would result in a successful age-prediction model derived from the CC epigenome. Additionally, lower DNA input for CC samples compared to WBC samples was a methodological limitation. We acknowledge that a universally accepted definition of poor ovarian response is lacking. Furthermore, women with PCOS were included and therefore the group of good responders in the current study may not represent a population with entirely normal methylation profiles. WIDER IMPLICATIONS OF THE FINDINGS: The process of ovarian and CC aging continues to be poorly understood. Women who demonstrate poor ovarian response to stimulation represent a common clinical challenge, so clarifying the exact biological changes that occur within the ovary over time is a worthwhile endeavor. The data from CC support a view that hormonally responsive tissues may possess distinct epigenetic aging patterns when compared with other tissue types. Future studies may be able to determine whether alternative DNA methylation sites can accurately predict chronologic age or ovarian response to stimulation from CC samples. Going forward, associations between epigenetic age acceleration and reproductive and general health consequences must also be clearly defined. STUDY FUNDING/COMPETING INTEREST(S): No external funding was obtained for the study and there are no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

The Role of the Epididymis and the Contribution of Epididymosomes to Mammalian Reproduction.

It is well-established that testicular spermatozoa are immature and acquire motility and fertilization capabilities during transit throughout the epididymis. The epididymis is a duct-like organ that connects the testis to the vas deferens and is comprised of four anatomical regions: the initial segment, caput, corpus, and cauda. Sperm maturation occurs during epididymal transit by the interaction of sperm cells with the unique luminal environment of each epididymal region. In this review we discuss the epididymis as an essential reproductive organ responsible for sperm concentration, maturation (including sperm motility acquisition and fertilizing ability), protection and storage. Importantly, we also discuss specific characteristics and roles of epididymal-derived exosomes (epididymosomes) in establishing sperm competency within the intricate process of reproduction. This review suggests that an increasing body of evidence is working to develop a complete picture of the role of the epididymis in male reproduction, offspring health, and disease susceptibility.

Paternal germ line aging: DNA methylation age prediction from human sperm.

BACKGROUND: The relationship between aging and epigenetic profiles has been highlighted in many recent studies. Models using somatic cell methylomes to predict age have been successfully constructed. However, gamete aging is quite distinct and as such age prediction using sperm methylomes is ineffective with current techniques. RESULTS: We have produced a model that utilizes human sperm DNA methylation signatures to predict chronological age by utilizing methylation array data from a total of 329 samples. The dataset used for model construction includes infertile patients, sperm donors, and individuals from the general population. Our model is capable predicting age with an R2 of 0.89, a mean absolute error (MAE) of 2.04 years, and a mean absolute percent error (MAPE) of 6.28% in our data set. We additionally investigated the reproducibility of prediction with our model in an independent cohort where 6 technical replicates of 10 individual samples were tested on different arrays. We found very similar age prediction accuracy (MAE = 2.37 years; MAPE = 7.05%) with a high degree of precision between replicates (standard deviation of only 0.877 years). Additionally, we found that smokers trended toward increased age profiles when compared to 'never smokers' though this pattern was only striking in a portion of the samples screened. CONCLUSIONS: The predictive model described herein was built to offer researchers the ability to assess "germ line age" by accessing sperm DNA methylation signatures at genomic regions affected by age. Our data suggest that this model can predict an individual's chronological age with a high degree of accuracy regardless of fertility status and with a high degree of repeatability. Additionally, our data suggest that the aging process in sperm may be impacted by environmental factors, though this effect appears to be quite subtle and future work is needed to establish this relationship.

Age-associated sperm DNA methylation alterations: possible implications in offspring disease susceptibility.

Recent evidence demonstrates a role for paternal aging on offspring disease susceptibility. It is well established that various neuropsychiatric disorders (schizophrenia, autism, etc.), trinucleotide expansion associated diseases (myotonic dystrophy, Huntington's, etc.) and even some forms of cancer have increased incidence in the offspring of older fathers. Despite strong epidemiological evidence that these alterations are more common in offspring sired by older fathers, in most cases the mechanisms that drive these processes are unclear. However, it is commonly believed that epigenetics, and specifically DNA methylation alterations, likely play a role. In this study we have investigated the impact of aging on DNA methylation in mature human sperm. Using a methylation array approach we evaluated changes to sperm DNA methylation patterns in 17 fertile donors by comparing the sperm methylome of 2 samples collected from each individual 9-19 years apart. With this design we have identified 139 regions that are significantly and consistently hypomethylated with age and 8 regions that are significantly hypermethylated with age. A representative subset of these alterations have been confirmed in an independent cohort. A total of 117 genes are associated with these regions of methylation alterations (promoter or gene body). Intriguingly, a portion of the age-related changes in sperm DNA methylation are located at genes previously associated with schizophrenia and bipolar disorder. While our data does not establish a causative relationship, it does raise the possibility that the age-associated methylation of the candidate genes that we observe in sperm might contribute to the increased incidence of neuropsychiatric and other disorders in the offspring of older males. However, further study is required to determine whether, and to what extent, a causative relationship exists.

Intra-sample heterogeneity of sperm DNA methylation.

The study of sperm epigenetics is challenging and limited largely to sperm population estimates rather than individual spermatozoa. While this type of approach is likely sufficient for most somatic cell lines, it is problematic in a tissue where a single cell is disproportionality influential. Furthermore, we know very little about the epigenetic variability between different sperm from the same ejaculate. Thus, it is essential that we better understand the heterogeneity of sperm epigenetic marks within an ejaculate. In this study, we have performed sperm genome-wide DNA methylation analyses on single ejaculates from 20 individuals. Sperm samples were subjected to gradient separation, following which the 90% layer ('high-quality sperm') and the 35% layer ('low-quality sperm') were isolated and analyzed separately using the Illumina 450K methylation array. We did not identify any single CpG that was differentially methylated between the two fractions. In contrast, we did identify 772 significant regional methylation alterations between the two layers. Coefficient of variance analysis also revealed that, in addition to having multiple sites that appear to be differentially methylated, the 35% layer sperm population, as a whole, displayed significantly higher variability in DNA methylation than did the 90% layer. In conclusion, while the two sperm populations analyzed here do not appear to be entirely distinct, those sperm that are generally considered to be of 'poor-quality' display some consistent regions of alteration and, more strikingly, demonstrate more heterogeneity than sperm considered to be of more normal quality.

The Sperm Epigenome, Male Aging, and Potential Effects on the Embryo.

The effect of paternal aging on fertility, embryo quality, and offspring health is an important area of study that has received far less attention than the age effect in women. This is, in part, due to the fact that in females there are dramatic alterations to fertility and pregnancy outcomes that abruptly occur as a female ages. Such abrupt alterations to pregnancy success and/or embryonic and offspring health are not seen in males. Instead, there are subtle alterations to pregnancy success and offspring phenotypes that occur as a man ages. It is believed that, at least in part, these alterations can be explained by perturbations to the sperm epigenome that occur over time. This chapter will explore the effect of aging on the sperm epigenome and the potential impacts these perturbations may have on embryonic development and ultimately offspring health.

The sperm epigenome: implications for the embryo.

Recent advances, including the human genome project and numerous studies of cancer and other diseases, have shown that the genetic code is not simply limited to the sequence of the four bases of DNA but also includes epigenetic programming, heritable changes that affect gene expression [Riggs A, Martinssen R, Russo V (2007) Introduction. In: Riggs A, Martinssen R, Russo V (eds) Epigenetics mechanisms of gene regulation. Cold Spring Harbor Press, New York]. The science of epigenetics is important in understanding many diseases and biological processes, including in identifying the causes of disease and better understanding the mechanisms by which the environment can affect gene expression [Carrell Fertil Steril 97 (2):267-274, 2012]. This chapter will focus on the epigenome of sperm and particularly highlight the potential role of the sperm epigenome in embryogenesis.The sperm epigenome is unique and highly specialized because of the unique nature and function of sperm and because of the diverse requirements for successful fertilization. Due to the need for motility, sperm chromatin must be compacted and highly organized. During spermiogenesis the chromatin is packaged tightly into the sperm head by the replacement of most histones with protamines. This allows for protection of the DNA from the hostile environment in the female reproductive tract. Remaining histones can have chemical modifications to the tails of the protein that either facilitate or repress gene transcription. Sperm, like embryonic stem cells, have a unique pattern of histone modifications that includes both activating and silencing marks in the promoters of genes associated with development. These bivalent marks, along with DNA hypomethylation, comprise a unique state in which the key genes are "poised" for possible activation in embryogenesis. Sperm epigenetic abnormalities have been linked with multiple diseases including male factor infertility and poor embryogenesis.

An assessment of alterations to human sperm methylation patterns in coronavirus disease 2019 infected and healthy control males.

OBJECTIVE: To determine whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection affects male reproductive health, considering the many potential factors that contribute to declines in male fertility on a semiglobal scale. DESIGN: In total, 64 human semen samples-32 treatment and 32 control-were laboratory processed and bioinformatically analyzed to assess differences in DNA methylation patterns. Implementing multiple bioinformatic tools, the analyses conducted will elicit between-group differences with respect to epigenetic age, epigenetic instability, semiglobal, and regional methylation, in addition to methylation patterns as a function of time since infection. SETTING: University hospital. PATIENTS: The study cohort of 64 individuals was drawn from a larger population of 94 volunteer participants recruited at the Human Reproduction Center at the Clinical Hospital of the Ribeirao Preto Medical School-University of São Paulo between June 2021 and January 2022 as well as in accordance with the ethical guidelines established by the Declaration of Helsinki. INTERVENTION: Exposure to SARS-CoV-2. MAIN OUTCOME MEASURE(S): Effects on male reproductive health were reported as differences in DNA methylation measured using an array. Mean ß values at key regulatory loci for human spermatocytes were analyzed and compared between groups. Further analysis of ß values using epigenetic age, instability, semiglobal, and regional methylation tools provided an analysis with substantial breadth and depth. RESULTS: In all analyses, there were no differences between groups. Considering these results, it can be inferred that infection with SARS-CoV-2 does not alter the epigenome of human spermatocytes in significant and/or persistent ways. Tangentially, these data also suggest that human male reproductive health is minimally altered by the virus, or that it is altered in a way that is independent of epigenetic programming. CONCLUSION: Infection with SARS-CoV-2 has been reportedly associated with alterations in male fertility. This study asserts that such alterations do not have an epigenetic basis but are likely a result of concomitant symptomatology, i.e., fever and inflammation. Across the multiple bioinformatic analyses conducted, the results of this test did not detect any differences in DNA methylation patterns between coronavirus disease 2019 and noncoronavirus disease semen donor groups.

Global effects of identity and aging on the human sperm methylome.

BACKGROUND: As the average age of fatherhood increases worldwide, so too does the need for understanding effects of aging in male germline cells. Molecular change, including epigenomic alterations, may impact offspring. Age-associated change to DNA cytosine methylation in the cytosine-guanine (CpG) context is a hallmark of aging tissues, including sperm. Prior studies have led to accurate models that predict a man's age based on specific methylation features in the DNA of sperm, but the relationship between aging and global DNA methylation in sperm remains opaque. Further clarification requires a more complete survey of the methylome with assessment of variability within and between individuals. RESULTS: We collected sperm methylome data in a longitudinal study of ten healthy fertile men. We used whole-genome bisulfite sequencing of samples collected 10 to 18 years apart from each donor. We found that, overall, variability between donors far exceeds age-associated variation. After controlling for donor identity, we see significant age-dependent genome-wide change to the methylome. Notably, trends of change with age depend on genomic location or annotation, with contrasting signatures that correlate with gene density and proximity to centromeres and promoter regions. CONCLUSIONS: We uncovered epigenetic signatures that reflect a stable process which begins in early adulthood, progressing steadily through most of the male lifespan, and warrants consideration in any future study of the aging sperm epigenome.

The sperm epigenome and potential implications for the developing embryo.

Recent work in the field of male fertility has yielded significant increases in our understanding of the sperm epigenome and its potential role in embryonic development. These new findings have enabled a broad classification of a normal epigenetic state in the male gamete and have provided insight into the possible etiologies of some idiopathic male infertility cases. Histone retention and modification, protamine incorporation into the chromatin, DNA methylation, and spermatozoal RNA transcripts appear to play important roles in the epigenetic state of mature sperm. These epigenetic factors may reveal a historical record of spermatogenesis, portend future functions in embryogenesis, and help to elucidate mechanism of pluripotency. In contrast to the once held dogma regarding the importance of the paternal epigenome, the unique epigenetic landscape in sperm appears to serve more than the gamete itself and is likely influential in the developing embryo. In fact, growing evidence suggests that mature sperm provide appropriate epigenetic marks that drive specific genes toward activation and contribute to the pluripotent state of the embryonic cells. Although not definitive, the current literature provides evidence for the role of the sperm epigenome in the embryo. Future work must be focused on the characterization of epigenetic abnormalities commonly found in individuals with compromised fertility to further establish this role. Additionally, studies should target the effects of environment and aging on the sperm epigenetic program and subsequent fertility loss to determine the etiology of aberrant epigenetic profiles.

Diverse monogenic subforms of human spermatogenic failure.

Non-obstructive azoospermia (NOA) is the most severe form of male infertility and typically incurable. Defining the genetic basis of NOA has proven challenging, and the most advanced classification of NOA subforms is not based on genetics, but simple description of testis histology. In this study, we exome-sequenced over 1000 clinically diagnosed NOA cases and identified a plausible recessive Mendelian cause in 20%. We find further support for 21 genes in a 2-stage burden test with 2072 cases and 11,587 fertile controls. The disrupted genes are primarily on the autosomes, enriched for undescribed human "knockouts", and, for the most part, have yet to be linked to a Mendelian trait. Integration with single-cell RNA sequencing data shows that azoospermia genes can be grouped into molecular subforms with synchronized expression patterns, and analogs of these subforms exist in mice. This analysis framework identifies groups of genes with known roles in spermatogenesis but also reveals unrecognized subforms, such as a set of genes expressed across mitotic divisions of differentiating spermatogonia. Our findings highlight NOA as an understudied Mendelian disorder and provide a conceptual structure for organizing the complex genetics of male infertility, which may provide a rational basis for disease classification.

Simulated Wildfire Smoke Significantly Alters Sperm DNA Methylation Patterns in a Murine Model.

Wildfires are now a common feature of the western US, increasing in both intensity and number of acres burned over the last three decades. The effects of this changing wildfire and smoke landscape are a critical public and occupational health issue. While respiratory morbidity due to smoke exposure is a priority, evaluating the molecular underpinnings that explain recent extrapulmonary observations is necessary. Here, we use an Apoe-/- mouse model to investigate the epigenetic impact of paternal exposure to simulated wildfire smoke. We demonstrate that 40 days of exposure to smoke from Douglas fir needles induces sperm DNA methylation changes in adult mice. DNA methylation was measured by reduced representation bisulfite sequencing and varied significantly in 3353 differentially methylated regions, which were subsequently annotated to 2117 genes. The differentially methylated regions were broadly distributed across the mouse genome, but the vast majority (nearly 80%) were hypermethylated. Pathway analyses, using gene-derived and differentially methylated region-derived gene ontology terms, point to a number of developmental processes that may warrant future investigation. Overall, this study of simulated wildfire smoke exposure suggests paternal reproductive risks are possible with prolonged exposure.

Sperm DNA methylation changes after short-term nut supplementation in healthy men consuming a Western-style diet.

BACKGROUND: Many environmental and lifestyle factors have been implicated in the decline of sperm quality, with diet being one of the most plausible factors identified in recent years. Moreover, several studies have reported a close association between the alteration of specific sperm DNA methylation signatures and semen quality. OBJECTIVES: To evaluate the effect of tree nut consumption on sperm DNA methylation patterns in healthy individuals reporting eating a Western-style diet. MATERIAL AND METHODS: This is a post hoc analysis conducted in a subset of participants (healthy, non-smoking, and young) from the FERTINUTS 14-wk randomized-controlled, parallel trial, recruited between December 2015 and February 2017. The participants included in the current study (n = 72) were randomly selected in a proportion 2:1 from the original FERTINUTS trial between the 98 participants that completed the entire dietary intervention (nut group, n = 48; control group, n = 24). Sperm DNA methylation patterns were examined at baseline and after 14 weeks in 48 individuals consuming 60 g/d of mixed nuts (nut group) and in 24 individuals following the usual Western-style diet avoiding consumption of nuts (control group). RESULTS: Over the course of the trial, no significant changes in global methylation were observed between groups. However, in the nut group, we identified 36 genomic regions that were significantly differentially methylated between the baseline and the end of the trial and 97.2% of the regions displayed hypermethylation. We identified no such change in the control group over the same period of time. We also utilized the recently developed germ line age calculator to determine if nut consumption resulted in alterations to the epigenetic age of cells and no significant differences were found. DISCUSSION AND CONCLUSION: Adding nuts to a regular Western-style diet subtly impacts sperm DNA methylation in specific regions, demonstrating that there are some sperm epigenome regions that could respond to diet.

Male adiposity, sperm parameters and reproductive hormones: An updated systematic review and collaborative meta-analysis.

The present updated systematic review and meta-analysis aims to summarize the evidence from published studies with low risk for any important bias (based on methodological quality assessment) investigating the potential associations of adiposity with sperm quality and reproductive hormones. We conducted a systematic search of the literature published in MEDLINE-PubMed and EMBASE through June 2019. Based on the criteria in our review, 169 eligible publications were used for data abstraction. Finally, 60 articles were included in the qualitative analysis and 28 in the quantitative analysis. Our systematic review results indicated that overweight and/or obesity were associated with low semen quality parameters (i.e., semen volume, sperm count and concentration, sperm vitality and normal morphology) and some specific reproductive hormones (e.g., inhibin B, total testosterone and sex hormone-binding globulin). Overweight and/or obesity were also positively associated with high estradiol concentrations. Meta-analysis indicated that overweight and/or obesity categories were associated with lower sperm quality (i.e., semen volume, sperm count and concentration, sperm vitality, total motility and normal morphology), and underweight category was likewise associated with low sperm normal morphology. In conclusion, our results suggest that maintaining a healthy body weight is important for increasing sperm quality parameters and potentially male fertility.

Transgenerational Epigenetics: A Window into Paternal Health Influences on Offspring.

Transgenerational epigenetic inheritance provides a mechanism by which environmental exposures and lifestyle decisions can affect the offspring directly through the gamete. It is this pattern of inheritance that has shed light on the fact that preconception lifestyle decisions that a father makes are significant because they can significantly impact the offspring. Understanding the epigenetic alterations in gametes and the potential implications of these changes is key to the health of future generations.

Microfluidic System for Rapid Isolation of Sperm From Microdissection TESE Specimens.

OBJECTIVES: To demonstrate a novel prototype microfluidic system for rapid isolation of sperm from real and simulated microdissection testicular sperm extraction samples. METHODS: The novel microfluidic system was tested using minced testicular biopsies from patients with nonobstructive azoospermia. The samples were split into 2 portions, conventional processing vs microfluidic. The embryologists were blinded to the processing protocol and searched the specimens for sperm after processing. We recorded the number of sperm found and the time to sperm identification and compared the sperm retrieval rates. RESULTS: When compared to conventional methods, samples processed through the microfluidic system were cleaner (decreased somatic cells/debris), with the average number of sperm identified per minute improving from 1.52 sperm per minute for the control and 13.5 sperm per minute with the device yielding an 8.88 fold improvement in the sperm found per minute for the device as compared to the control. Preliminary viability and morphology tests show a minimal impact on sperm processed through the microfluidic system. CONCLUSION: The presented microfluidic system can facilitate rapid and efficient isolation of sperm from microdissection testicular sperm extraction samples. A prospective clinical trial to verify these results is needed to confirm this preliminary data.

The combined effect of obesity and aging on human sperm DNA methylation signatures: inclusion of BMI in the paternal germ line age prediction model.

Male aging and obesity have both been shown to contribute to declines in fertility in men. Recent work in aging has shown consistent epigenetic changes to sperm as a man ages. In fact, our lab has built a tool that utilizes DNA methylation signatures from sperm to effectively predict an individual's age. Herein, we performed this preliminary cohort study to determine if increased BMI accelerates the epigenetic aging in sperm. A total of 96 participants were divided into four age groups (22-24, 30, 40-41, and > 48 years of age) and additionally parsed into two BMI sub-categories (normal and high/obese). We found no statistically significant epigenetic age acceleration. However, it is important to note that within each age category, high BMI individuals were predicted to be older on average than their actual age (~ 1.4 years), which was not observed in the normal BMI group. To further investigate this, we re-trained a model using only the present data with and without BMI as a feature. We found a modest but non-significant improvement in prediction with BMI [r2 = 0.8814, mean absolute error (MAE) = 3.2913] compared to prediction without BMI (r2 = 0.8739, MAE = 3.3567). Future studies with higher numbers of age-matched individuals are needed to definitively understand the impact of BMI on epigenetic aging in sperm.

Serum dioxin levels and sperm DNA methylation age: Findings in Vietnam war veterans exposed to Agent Orange.

Exposure to dioxin, a known endocrine disruptor and carcinogen, is associated with poor reproductive outcomes. Yet, few studies have explored the role of DNA methylation in these relationships. Utilizing a publicly available dataset from 37 male Air Force Health Study participants exposed to dioxin-contaminated Agent Orange during the Vietnam war, we cross-sectionally examined the relationship of serum dioxin levels with a novel DNA methylation-based measure of sperm age (DNAm-agesperm). DNAm-agesperm was calculated using CpG sites on the Illumina HumanMethylation450 BeadChip. We estimated associations of dioxin levels with DNAm-agesperm using linear regression models adjusted for chronological age, body mass index, and smoking status. Chronological age was highly correlated with DNAmagesperm (r = 0.80). In fully-adjusted linear models, a one percent increase in serum dioxin levels was significantly associated with a 0.0126-year (i.e. 4.6-day) increase in DNAm-agesperm (95%CI: 0.003, 0.022, p = 0.01). Further analyses demonstrated significant negative associations of dioxin levels (ß = -0.0005, 95%CI: -0.0010, 0.00004, P = 0.03) and DNAm-agesperm (ß = -0.02, 95%CI: -0.04, -0.001, P = 0.03) with methylation levels of FOXK2 - a gene previously reported to be hypomethylated in infertile men. In sum, we demonstrate associations of dioxin with increased methylation aging of sperm. DNAm-agesperm may provide utility for understanding how dioxin levels impact sperm health and potentially male reproductive capacity in human population studies. Moreover, our pilot study contributes further evidence that some environmental toxicants are associated with methylation aging. Additional studies are necessary to confirm these findings, and better characterize dioxin and sperm methylation relationships with male reproductive health.