Currently, the evaluation of male infertility relies upon physical exam and semen and hormone analyses; although quick and relatively inexpensive, these physiologic measurements often do not explain the underlying cause of infertility nor predict the usefulness of various therapeutic interventions. Therefore, new approaches are needed to identify the etiologies of male infertility. Recent data suggest that sperm DNA methylation abnormalities and alterations in sperm mRNA content are found in infertile men 
. Here we extend these studies by performing integrative analysis of sperm DNA methylation and mRNA content using genome-wide approaches to identify significant associations among these profiles and semen parameters.
Due to the unreliable nature of classifying men into abnormal and normal groups during a semen analysis, we used a data driven approach to first qualitatively assess associations among sperm DNA methylation and our patient population. Unsupervised clustering indicated that there was an association between DNA methylation and motility status. This was true both for all of the CpGs on the array and the imprint-only subset.
RPMM separated the 21 men into four classes based on similarity of DNA methylation array data. The median motility values were calculated for each class and the results suggested that the methylation profiles were associated with motility. Comparing the DNA methylation heatmap to the class versus motility boxplot indicates that the low motility class has the most aberrantly methylated CpGs. Overall, these data suggest that low motility sperm have increased hypomethylation relative to high motility sperm. We used LIMMA to identify the significantly altered CpGs conditioned on changes in motility for all CpGs on the array: over one-third of the CpGs (and almost half of the genes represented on the array) were significantly differentially methylated in the low motility samples and the majority of these were hypomethylated. The high prevalence of aberrantly methylated CpGs suggests a genome-wide DNA methylation defect in the low motility sperm. It has been previously hypothesized that the aberrant sperm DNA methylation could be due to abnormal chromatin compaction, inefficient DNA methyltransferases, and/or failure to maintain or acquire the correct methylation marks during spermatogenesis and our results are consistent with this literature 
We initially focused on CpGs mapping to imprinted genes because of their plasticity during spermatogenesis, biological relevance following conception and development, and because previous studies have identified imprinted loci as aberrantly methylated in abnormal sperm 
. In our data, the distribution of significantly hyper- and hypomethylated imprinted loci was nearly equal. Expanding the imprinting analysis to the gene level identified 92 genes with altered CpG methylation, seven of which (DIRAS3
(NCBI 9077), H19
(NCBI 283120), IGF2
(NCBI 3481), MEST/PEG1
(NCBI 4232), PLAGL1/ZAC
(NCBI 5325), MEG3/GTL2
(NCBI 55384), and SNRPN
) have already been noted as aberrantly methylated in abnormal sperm 
. The methylation status of two genes, (PEG3
(NCBI 5178) and LIT1/KCNQ1OT1
(NCBI 10984)) has been inconsistently reported in the literature 
. We observed no statistical differences for these genes between the low and high motility sperm, which is consistent with the results published by Sato, et al. 
. In fact, our study confirmed all of the DNA methylation results reported in the aforementioned study.
To further clarify the potential functional alterations to imprinted genes and critical epigenetic regulatory genes, we evaluated sperm mRNA content of 177 imprinted genes and 99 other transcripts where an a priori
hypothesis for association with male subfertility or epigenetic regulation exists. Twenty genes were identified as demonstrating significantly altered transcript levels in low motility sperm. All of the mRNAs except HDAC1
, and FAS
were present in decreased amounts in low motility sperm, and we did not observe altered mRNA content for BRDT
, which was previously reported to have increased expression in subfertile patients 
Integration of epigenetic and expression data revealed a relationship between transcript content of three epigenetic regulatory genes (HDAC1
, and DNMT3A
) and methylation class. HDAC1
is the predominant histone deacetylase (HDAC) during spermatogenesis. Histone hyperacetlyation is required for the histone to protamine exchange and is facilitated by the degradation of HDAC1 in elongated spermatids 
. If HDAC1 is in excess, one could hypothesize that the histones are not being replaced by protamines, leading to an “immature” sperm chromatin structure, with less compact DNA. Therefore, incomplete or incorrect nuclear compaction may influence overall sperm maturation and be reflected in the physiological endpoint of motility.
is a class III histone deacetylase and this HDAC family is similar to the yeast Sir2 protein which has been associated with chromatin silencing and also plays roles in cellular metabolism and aging 
. In mammals, however, SIRT3 is targeted to the mitochondria and functions to induce the expression of the antioxidant MnSOD to eliminate reactive oxygen species (ROS) generated during oxidative phosphorylation 
. Recent studies have found that increased ROS in sperm have deleterious effects on sperm motility parameters which ultimately have adverse effects on fertility 
. Therefore, the decrease in SIRT3
mRNA in the low motility sperm may reflect reduced MnSOD and increased intracellular ROS during spermatogenesis, leading to a diminished fertility potential.
The literature also suggests that oxidative stress itself can impede the process of DNA methylation, resulting in a hypomethylated phenotype 
. Interestingly, we observed global hypomethylation in the low motility sperm even though we saw increased DNMT3A transcript presence in the low motility sperm. Because DNMT3A is the DNA methyltransferase responsible for de novo
methylation, our data suggests a failure of the low motility sperm to acquire the proper methylation patterns.
Although we were limited by sample size, we used a powerful integrative approach to simultaneously examine sperm DNA methylation and mRNA content utilizing two high density array techniques. We found that: (1) low motility sperm have genome-wide DNA hypomethylation that may be due to a failure of the sperm to complete chromatin compaction properly because of increased HDAC1 presence; (2) low motility sperm have reduced SIRT3 mRNA content which might be related to increased subcellular ROS during spermatogenesis leading to the abnormal motility phenotype; and (3) this oxidative stress may be impeding the ability of DNMT3A to set the correct methylation marks which would also contribute to the hypomethylated phenotype. Our results suggest that additional integrative studies including larger sample sizes as well as prospective studies of fertility following these integrated molecular assessments have great potential to advance our understanding of the molecular features of sperm associated with fertility status.