Most studies on epigenetic effects of obesity are conducted in mother-child dyads, and the effects of the paternal germ line are rarely examined. We undertook an analysis of the association between paternal overweight/obesity status and sperm methylation percentage at the regulatory regions of imprinted genes MEG3-IG, MEG3, PEG1/MEST, IGF2, H19, GRB10, NNAT, NDN, SNRPN, SGCE/PEG10, PLAGL1, and PEG3. We found significant differences in DNA methylation patterns between overweight/obese men and men of normal BMI at multiple DMRs. An important finding was that a fraction of the mature haploid spermatozoa in each sample had not acquired their fully methylated or unmethylated status at imprinted DMRs, as is expected for paternally or maternally methylated DMRs, respectively. Intriguingly, we found that after adjusting for potential confounding by age and patient status, DMRs that should be unmethylated in sperm (maternally methylated DMRs) were closer to 0 % methylation if men were overweight or obese. These genes included GRB10, NDN, SNRPN, and SGCE/PEG10. In addition, overweight/obese men had a higher fraction of sperm with complete DNA methylation at regions where paternal methylation (closer to 100 %) is expected, as compared to normal weight men. This was the case at the IGF2 and H19 DMRs. The MEG3-IG and MEG3 DMRs are separated by about 20 kb in humans and are unusual in terms of establishment of paternal imprint marks. According to studies in mice [31], methylation at these two DMRs is acquired at different developmental time points: the MEG3-IG DMR methylation is established during maturation of sperm, while the MEG3 DMR acquires methylation only after fertilization. Both regions play an essential role in expression regulation of MEGs and PEGs in the human body [32]. In semen of overweight/obese men, we observed that a higher fraction of sperm cells were fully methylated at the DMR of MEG3-IG as compared to semen from normal weight men, while in the same subgroup of overweight/obese men, DNA methylation at the DMR of MEG3 was closer to 0 %. Hence, in overweight or obese men, both regions had a tendency to be closer to the theoretically expected levels for methylation in these regions.

Interestingly, analyses in the Newborn Epigenetic STudy (NEST) cohort showed that newborns of obese fathers had comparable methylation patterns at some imprinted gene DMRs. A matching positive trend between high BMI and methylation at H19 and MEG3-IG DMRs was observed in the NEST cohort, but results did not reach significance. Similar paradoxical results regarding expected theoretical levels of methylation were found. Lower DNA methylation was seen at DMRs of genes expected to be unmethylated on the paternal allele (e.g. PEG3, NNAT, and PEG1/MEST) [25]. In the current study, sperm from overweight/obese men also had a lower, albeit not significant methylation profile at PEG3 and NNAT. Although the results—comparing the same DMRs in this study to those children born to obese fathers in the NEST cohort study—were not all significant, the overall directions of associations are consistent with the notion that methylation differences detected in the sperm of overweight/obese men in this study could persist in their offspring. Taken together, the results of the NEST and TIEGER studies suggest that linkages between sperm and offspring epigenetic modifications merit further investigation. Human data on paternal obesity and its potential effects on sperm and offspring epigenetics are limited. To our knowledge, only one recent study investigated a potential effect of obesity on the sperm epigenome. Donkin et al. performed a comprehensive epigenetic mapping of sperm from obese versus normal weight men [33]. Despite their limited sample size (10 obese men), potential for selection bias, and the fact that different CpG sites were examined, the Donkin et al. study corroborates our findings that methylation differences exist in the sperm methylome for obese men versus lean men. Some studies indicate that the effects of epigenetic modifications through sperm can be substantial for the offspring. Feinberg et al. found that within a group of men who had already fathered a child with autism, differences in methylation in the sperm genome were present at genes associated with autism spectrum disorder phenotypes. These results suggest that epigenetic differences in sperm may have profound offspring phenotypic consequences [34]. Methylation profiles of sperm also may impact offspring viability. In a study of patients undergoing IVF, sperm DNA methylation patterns were found to be predictive of IVF embryo quality [35]. In animal models, deregulated methylation at imprinted genes has been observed in sperm from obese parents, which was associated with altered developmental capacity of the embryo and reduced pluripotency and metabolic function [36, 37]. Although the relationship between BMI and clinical sperm parameters remains controversial in humans, there is growing evidence that male obesity is related to decreased fertility [38], which may be related to impaired DNA methylation in sperm [39]. Epigenetic effects from obese mothers on their son’s sperm have also been reported. Ge et al. investigated DNA methylation patterns in a mouse model at imprinted genes in sperm of offspring born to obese and/or diabetic dams. DNA methylation was increased at DMRs of the H19 and Peg3 genes [40]. In the current study, similar regions are differentially methylated by obesity status. However, only DNA methylation at H19 was significant in men. From these data, it can be concluded that molecular mechanisms, hormonal imbalances, diet, or other obesity-related factors could be the cause of epigenetic changes in sperm. Several animal studies have demonstrated that the sperm epigenome may be responsive to dietary factors. Effects were measured through negative pregnancy outcomes, altered gene expression patterns in the offspring, and/or diminished reproductive health in subsequent generations [10, 11, 22]. However, a recent study in mice by Shea et al. reported conflicting results demonstrating that the sperm methylome was much more strongly correlated with epigenetic variations occurring randomly or due to other unknown factors rather than diet [41]. Their findings suggest that obesity and body composition are more important than diet composition, or that diet may have an influence via modified body composition. For instance, obesity is associated with elevated estrogen levels, and results from animal studies suggest that increased exposure to estrogen in the testes may lead to abnormal methylation patterns, providing a possible mechanism for how body fat can impact DNA methylation [38, 42]. Further investigation is needed to disentangle the independent contributions of diet versus body composition or its metabolic/hormonal status in regard to sperm epigenetics. Another environmental factor that could also induce epigenetic modifications in sperm DNA is testis hyperthermia in obese subjects. Animal data provides evidence that heat can perturb the dynamics of DNA methylation programming in the paternal genome; this was shown in bulls by Rahman et al. [43]. A recent report in humans provides evidence that the state of varicocele, a condition related to heat affecting sperm, is related to DNA methylation in spermatozoa [44]. Hence, we cannot exclude the possibility that obesity-related genital heat could have influenced our results.

Other exposures, such as to the endocrine-disrupting factor BPA or its metabolites, may also be involved. These environmental factors could play a role in influencing both weight gain and spermatogenesis, and thus, it cannot be completely ruled out that both increased BMI and methylation changes are parallel results of environmental exposures [45]. Future studies should strive to elucidate the underlying mechanisms by which overweight/obesity may affect methylation differences, and if a third factor, such as an environmental exposure or nutritional profiles, may be influencing both outcomes.

The methylation changes we found in this study were subtle. However, these small changes in sperm may still have an impact on offspring phenotypes. Murphy et al. reported that a 1 % change in methylation at the IGF2 DMR leads to either a doubling or halving of IGF2 transcription, depending on the direction of methylation. This degree of change is equivalent to complete loss of imprinting, which is particularly significant because this loss of imprinting is often observed in cancers [46]. The modest methylation changes in sperm associated with obesity found here are also consistent with methylation changes induced in humans by other early-life environmental exposures and lifestyle factors, including prenatal exposure to famine [47], maternal nutrition supplementation [48], use of medicines during pregnancy [49, 50], and maternal exposure to cadmium [51] and lead (in press by Nye and Hoyo in Environmental Epigenetics). Large changes in methylation may in fact be less meaningful in regard to intergenerational impact because significant methylation aberrations may not result in viable offspring.

Although others have done a similar analysis in smaller populations [33], a possible weakness of this study remains the limited statistical power to detect significant differences. Furthermore, one third of our population was recruited at the Duke Fertility Center. Although we adjusted for Fertility Center patient status, there may be residual confounding resulting from inherent differences in characteristics of sperm DNA between (obese) men with potential fertility problems attending the clinic and those not attending the clinic. Hence, our results on sperm epigenetic differences may partly reflect the effects in patients seeking care at a fertility clinic, rather than the general population. Additionally, we focused on self-reported Caucasian men only, while North Carolina also includes a substantial population from other ethnicities or origins, with a higher prevalence of obesity, e.g., African Americans [52]. Given the increasing prevalence of obesity in males, our provocative findings require replication in a larger multiethnic study.

Strengths of our study include that our study population was comprised of men of reproductive age. We further did not sub select our obese and non-obese groups by any criteria. No exclusions were made based on health conditions (other than those related to fertility), as was done in the Donkin et al. study [33]. We focused on a group of regulatory elements that are established during spermatogenesis (or early post-fertilization in the case of the MEG3 DMR) and that are critical in the programming of the correct expression patterns of imprinted genes. Because imprinted genes are generally spared the global reprograming post-fertilization, methylation patterns established during spermatogenesis are persistent, and thus transferred to the offspring.

A remarkable finding in our descriptive analysis was the fact that the epigenetic imprinting is not as “complete” as theoretically expected, and that some paternally imprinted genes (e.g., MEG3) are not fully methylated until after fertilization. There are currently no comparable epigenetic studies on human (maturing) sperm in the literature for us to compare our results against, highlighting the lack of research on gametic epigenetic profiles at various stages of maturation and development. Hence, we can only suggest hypotheses for why men with normal BMI have a larger fraction of epigenetically “incomplete” sperm cells. One possible explanation is that a larger deviation from the expected methylation pattern in sperm reflects normal epigenetic variation at the DMRs on a population level, allowing for adaptive phenotypic plasticity in response to environmental changes [5]. A chronic exposure to an obesity-related microenvironment in the testes during spermatogenesis may alter the sperm epigenome. If this exposure persists through generations, it may influence long-term evolutionary trends [12]. Despite the observed associations between paternal obesity, epigenetic modification, and offspring phenotype, the timing and nature of paternal obesity’s influence on sperm and consequently offspring methylation profiles is potentially important. Persistent changes in epigenetic profiles may vary if the exposure starts during early development of the male gametes, before puberty, or at later age [3]. Overall, our results point to a need for investigation of the impact of the environment on the human sperm epigenome, as well as its normal variation.