Although, several studies have reported the impact of an aberrant DNA methylation 
on spermatogenesis and fertility in mouse model systems, so far none of this knowledge has been transferred to impaired sperm production in humans. Consequently, we investigated the methylation profiles, especially of piRNA-associated proteins, and their potential consequences in defective human spermatogenesis.
We screened for global DNA methylation changes in diseased specimens using an array technology capable of profiling the DNA methylation level of 14,495 genes. It has been described that although the vast majority of methylation acquisition in male germ cells is completed in primordial germ cells, before birth, changes of DNA methylation continue to occur at a reduced number of CpGs during spermatogenesis before pachytene 
. In order to distinguish SpF-associated differences in DNA methylation from physiological spermatogenic process, we selected samples that had similar number of cells from the earliest stages of the germline. With this strategy, not only did we detect more than 600 differentially methylated CpG sites, which allowed us to separate CS and SpF samples, but also we identified that the affected promoters were enriched in genes involved in germline function and spermatogenesis, suggesting that gene repression by hypermethylation of germline specific genes is probably a driver of infertility. Detecting similar numbers of gains and losses within differentially methylated positions rules out the possibility of unidirectional methylation changes, the converse of the global hypomethylation observed in other germ cell-related pathological diseases such as seminoma 
. Interestingly, in agreement with our results, some of the SpF-hypermethylated genes were previously found hypermethylated in DNA from semen with poor sperm concentration (i.e. SFN
gene and the maternally imprinted genes PLAGL1
. The SCO specimens exhibited a different profile of methylation compared with control and SpF samples. This could reflect the distinct sample compositions, whereby SCO samples almost exclusively contained somatic cells, whereas SpF samples showed impaired spermatogenesis and germ cells still present. However, we need to bear in mind that additional phenotypic changes related to somatic cells are observed in SCO, such as a greater number of Sertoli cells (). Thus, an aberrant pattern of methylation associated with this extremely severe phenotype of secretory infertility could not be ruled out.
Among the genes that are hypermethylated in the SpF phenotype, genes that encode PIWI family members and their associated proteins involved in piRNA processing such as PIWIL1/2
were able to cluster normal tissue and patient samples separately. These results and the fact that impaired expression of these genes leads to sterility in animal mouse models 
encouraged us to analyze this subgroup of genes in more detail. We identified and confirmed that PIWIL2
were hypermethylated in SpF specimens. The increase in methylation in SpF patients could be partially explained by the increase proportion of somatic cells in the samples but interestingly, an additional statistically significant increase in the methylation level of germ cells was observed ( and F) in SpF subphenotypes when compared to CS samples, being more pronounced in the sgMF subphenotype. The gain in methylation was shown to be significantly correlated with lower PIWIL2
expression level analyzing the entire tissue and more importantly the expression per cell. The remarkable correlation coefficient between the PIWIL2
transcript levels per cell and the number of elongated spermatids in the testicular tubule additionally underlines the determinant role of PIWIL2
expression in the progression of the spermatogenic process. Moreover, its potential use as a surrogate marker for the presence of full spermatogenesis in severe non-obstructive infertile patients should be additionally considered.
The abnormal methylation of PIWIL2,
but not of PIWIL1,
in spermatogenic impairment suggests that proper methylation is essential in the early stages of spermatogenesis. PIWIL2 has been described as being expressed in the germline during early spermatogenesis 
. However, PIWIL1 is expressed after birth in pachytene spermatocytes and spermatids and has been posited to act in translational control in the latest stages of spermatogenesis 
. There is further evidence that PIWIL2 has essential roles in the initial phases of spermatogenesis: transposon silencing in fetal gonocytes 
, germline stem cell self-renewal 
and early prophase of meiosis 
in mammalian testis. Furthermore, PIWIL2 has been implicated in translational regulation of many genes during early spermatogenesis since it binds piRNAs and mRNAs 
TDRD1 interacts directly with both PIWIL2 and PIWIL1 
. Although it does not affect the ability of PIWI proteins to associate with piRNAs in embryonic testes, it ensures the entry of correct transcripts into the normal piRNA pool 
The importance of PIWIL2
in the efficient production of mature spermatocytes was previously reported in a model system using homozygous knock-out mice 
. Interestingly, both recombinant mouse models revealed a common phenotype: a defect in early prophase of the first meiosis in the spermatogenesis resulting in sterility. Concordantly, our study reveals a remarkable and significant negative correlation between PIWIL2
methylation and the number of cells from the earliest steps of spermatogenesis, spermatogoniae and spermatocytes. Although still significant, the degree of correlation was lower for postmeiotic germ cells, suggesting a weaker linear relationship between PIWIL2
methylation and the latest stages of the spermatogenic process. The number of germ cells was positively correlated with PIWIL2
expression in the whole tissue and with PIWIL2
expression per cell. Taken together, these results suggest the involvement of PIWIL2
in the human germ cell development process. We suggest that DNA hypermethylation in the promoter regions of PIWIL2
leads to the transcriptional repression of these genes contributing to spermatogenic derangement.
Moreover, as PIWIL2 and TDRD1 physically and functionally interact in the biogenesis of piRNAs, a crucial role of these 26–31 nt small RNAs in spermatogenesis may be suspected. We identified a downregulation of mature piRNAs in SpF samples, similarly to what was described in fetal germ cells of the Mili null model 
. The most immediate functional consequence of piRNA depletion is a derepression of repetitive elements 
. Whether this leads directly to maturation arrest in spermatogenesis or additional functions of piRNAs and whether associated complexes contribute to the severe phenotype is currently being investigated. The repression of PIWIL2
gene expression in the severe spermatogenic defects examined in this study, leads us to speculate that the molecular alterations affecting piRNAs and their machinery are involved in human infertility.
The aberrant methylation and expression of PIWI-family genes has the ability to provoke methylation changes of additional loci. Genetic and molecular characterization identified interactions between methyltransferases and piRNA pathway members. The PIWI/DNMT3L complex targets genomic loci, sequence-guided by small RNAs 
as well as PIWIL2
null models revealed a loss of methylation at LINE-1 and intracisternal A-particle (IAP) transposons, leading to reactivation of repetitive elements that contribute to meiotic arrest and male infertility. Consistently, we detected several SpF samples with hypomethylated LINE-1 sequences, suggesting that reactivation of transposons also participates in the human spermatogenic failure. The activation of retrotransposons affects meiotic and premeiotic germ cells, but not the later stages of spermatogenesis. Interestingly, LINE-1 methylation was only affected in sgMF and scMF samples, but not in rsMF, where the number of spermatocytes was similar to that in normal testis. This is consistent with the assumption that meiotic spermatocytes are protected against retrotransposons.
In addition to repetitive elements, single genomic loci are also targeted by PIWI complexes sequence-guided by piRNAs 
. Here, we identified seven differentially methylated genes in SpF samples with complementary sequences to piRNAs. Taking into account that piRNA-guided binding of the PIWI complex has the ability to alter DNA methylation, we hypothesize that the differentially methylated promoters containing piRNAs binding sites are directly affected by the altered expression of PIWIL2
In summary, we identified not only an aberrant DNA methylation profile at CpG sites in male infertility of testicular origin, but also DNA methylation changes in germline-specific genes, in particular PIWIL2 and TDRD1, with functional consequences such as loss of DNA methylation in repetitive elements and a defective production of piRNAs. Therefore, we propose that DNA methylation, at least that affecting PIWIL2/TDRD1, plays a role in the control of human spermatogenic gene expression, and this process critically contributes to a successful germ cell development.