Abstract Epigenetic changes are involved in a wide range of common human diseases. Although DNA methylation defects are known to be associated with male infertility in mice, their impact on human deficiency of sperm production has yet to be determined. We have assessed the global genomic DNA methylation profiles in human infertile male patients with spermatogenic disorders by using the Infinium Human Methylation27 BeadChip. Three populations were studied: conserved spermatogenesis, spermatogenic failure due to germ cell maturation defects, and Sertoli cell-only syndrome samples. A disease-associated DNA methylation profile, characterized by targeting members of the PIWI-associated RNA (piRNA) processing machinery, was obtained. Bisulfite genomic sequencing and pyrosequencing in a large cohort (n = 46) of samples validated the altered DNA methylation patterns observed in piRNA-processing genes. In particular, male infertility was associated with the promoter hypermethylation-associated silencing of PIWIL2 and TDRD1. The downstream effects mediated by the epigenetic inactivation of the PIWI pathway genes were a defective production of piRNAs and a hypomethylation of the LINE-1 repetitive sequence in the affected patients. Overall, our data suggest that DNA methylation, at least that affecting PIWIL2/TDRD1, has a role in the control of gene expression in spermatogenesis and its imbalance contributes to an unsuccessful germ cell development that might explain a group of male infertility disorders. Introduction Genetic etiologies have only been able to explain about 15% of cases of male infertility [37][1]. Approximately 4% of men suffer from infertility, with 70% of cases of testicular origin being a consequence of a spermatogenic failure. At least 30% of infertile men suffer from idiopathic infertility of unknown underlying pathophysiology. Mammalian spermatogenesis is a complex and highly regulated developmental process in which mitosis, meiosis and differentiation interact to coordinate the development of a haploid gamete for sexual reproduction. These processes are unique in male germ cell differentiation and depend on precise developmental stage-specific and germ cell type-specific gene expression. Changes in testicular gene expression have been found in spermatogenic failure [38][2]–[39][7]. However, the regulatory network that controls germline transcription in mammals is not properly understood. In this context, it has been suggested that DNA methylation may contribute to the control of gene expression programs essential for successful gametogenesis [40][8]. DNA methylation is an epigenetic process that plays a crucial role in determining the time point and magnitude of gene expression. Unlike the genetic code, the epigenetic code is dynamic and tissue-specific [41][9]. While the genetic code defines a permanent blueprint of information determining phenotypes and specific traits, the epigenetic code provides a dynamic signalling that is capable of modifying phenotypes according to environmental impacts. Epigenetic regulation is a crucial mechanism for cell fate and survival [42][10], [43][11]. In particular, DNA methylation is involved in a wide range of common human diseases [44][12]–[45][15]. Within male germ cells, changes in the epigenetic state are critical for silencing transposable elements, imprinting paternal genes, several aspects of meiosis, post-meiotic gene silencing and DNA compaction. Recombinant mouse models identified a profound impact of DNA methylation processing enzymes (DNA methyltransferases, DNMTs) on sperm production. Expression changes of DNMTs in germline stem cells lead to aberrant survival and differentiation [46][16]. Particularly, a defective DNMT3L results in meiotic failure and impaired spermatogenesis [47][17]. In addition, DNMT3b mutants reveal a delayed entry into meiosis, resulting in a greatly reduced number of spermatocytes [48][18]. Abnormal sperm DNA methylation of imprinted genes is associated with spermatogenic impairment [49][19]–[50][21], and DNA methylation abnormalities may also involve non-imprinted genes [51][22]. In this context, it is tempting to speculate that male infertility could be linked to epigenetic alterations, such as abnormal DNA methylation patterns. It is currently unclear whether DNA methylomes of men with impaired sperm production significantly differ from those presenting a complete and efficient spermatogenic process. To address this matter, we analyzed genome-wide DNA methylation in infertile men with spermatogenic failure. Using the Infinium Human Methylation27 BeadChip technology [52][23], [53][24], we obtained an insight into the impact of DNA methylation in secretory male infertility. Among the nearly 600 genes differentially methylated in testis with impaired spermatogenesis compared with tissue with a conserved spermatogenic pattern, we focused on those coding for proteins directly involved in piRNA processing [PIWIL1 [54][25]; PIWIL2 [55][26]] and associated molecules [TDRD1 [56][27], [57][28], TDRD9 [58][29]], due to their potential role in spermatogenic control. Materials and Methods Subjects of Study Our study recruited thirty-two infertile patients (aged 30–49 years) due to severe spermatogenic failure (SpF), with a phenotype consistent with non-obstructive (secretory) azoospermia or severe oligozoospermia (<5 million sperm/ml). Only testicular samples with homogeneous phenotypes were selected on the basis of the histological pattern of >20 tubules from the same testicular section; samples with mixed histological patterns were excluded from the study. In addition, five patients with a Sertoli cell-only syndrome (SCO) phenotype were studied as methylation/gene expression controls of somatic cells and nine infertile patients (aged 32–50 years), who were diagnosed with obstructive azoospermia (as a consequence of congenital absence of the vas deferens or a previous vasectomy) and showed conserved spermatogenesis (CS) were studied as methylation/gene expression controls of a complete spermatogenic process ([59]Table 1). Infertile individuals were selected from men referred for couple infertility to the Andrology Service of the Fundació Puigvert. The study was approved by the Institutional Review Board of the Center, and all the participants gave their informed written consent to the procedures of the study. Table 1. Phenotypical and histological description of the testicular samples included in the study.[60]^(a). Patient Diagnosis Histology Semen sperm conc. (million/mL) Tubular diameter(µm) Spgonia Spcyte I Round Sptid Elongated Sptid Sertoli Cells Johnsen Score 1 OA CS 0 202.5 25.7 43.6 33.2 26.5 15.3 9.85 2 OA CS 0 186.7 21.4 28.1 31.3 24.8 9.1 9.70 3 OA CS 0 200.0 23.6 28.8 30.5 28.3 15.9 9.62 4 OA CS 0 190.0 20.7 33.7 38.5 33.1 13.5 9.55 5 OA CS 0 200.0 26.7 28.2 20.3 21.1 13.9 9.30 6 OA CS 0.02 206.0 25.8 31.8 29.6 21.4 13.1 9.25 7 OA CS 0.1 229.6 22.8 28.5 16.6 16.0 14.2 9.00 8 OA CS 0 195.0 20.7 30.5 31.9 12.8 11.5 8.70 9 OA CS 0 184.0 14.8 29.8 12.5 22.3 8.4 8.65 Mean 199.3 22.5 31.4 27.2 22.9 12.8 10 SA SpF (rsMF) 0 200.0 23.4 36.0 19.7 10.1 12.2 8.40 11 SSO SpF (rsMF) 5 151.7 15.0 27.6 15.5 9.8 11.3 8.40 12 SSO SpF (rsMF) 0.01 188.0 18.5 33.5 20.4 6.7 19.0 8.30 13 SA SpF (rsMF) 0.004 191.5 25.1 36.2 19.6 0.7 18.6 7.10 14 SSO + OA SpF (rsMF) 0 175.0 17.0 24.0 31.0 1.5 6.0 7.00 15 SSO SpF (rsMF) 3.5 170.0 13.0 20.0 15.0 1.5 11.0 7.00 16 SSO SpF (rsMF) 5 205.0 20.2 28.6 15.7 0.1 23.3 6.60 17 SSO SpF (rsMF) 3 148.8 19.7 22.3 15.4 0.3 18.4 6.50 Mean 178.8 19.0 28.5 19.0 3.8 15.0 18 SA + OA SpF (scMF) 0 200.6 19.5 22.9 5.6 6.9 15.7 7.10 19 SA SpF (scMF) 0 168.3 20.4 18.2 5.3 3.1 13.4 7.30 20 SSO SpF (scMF) 5 181.9 17.1 18.1 10.8 4.1 9.2 7.65 21 SSO SpF (scMF) 9 216.9 20.8 25.0 4.1 3.0 17.4 6.71 22 SSO SpF (scMF) 0.8 193.1 13.1 17.2 5.8 2.1 16.7 6.90 23 SA SpF (scMF) 0 170.0 19.5 17.6 4.0 2.1 14.1 6.70 24 SA SpF (scMF) 0.005 156.9 17.9 21.6 4.2 1.2 8.6 5.80 25 SSO SpF (scMF) 0.4 196.8 24.8 21.1 1.9 0.5 11.8 5.20 26 SA SpF (scMF) 0 190.0 23.0 30.0 0.0 0.0 11.0 5.00 27 SA SpF (scMF) 0 190.0 17.6 18.2 0.0 0.0 20.0 5.00 28 SA SpF (scMF) 0 190.0 22.5 39.6 0.0 0.0 16.0 5.00 29 SA SpF (scMF) 0 168.3 15.1 12.5 2.3 1.7 12.2 6.90 30 SA SpF (scMF) 0 175.0 15.9 10.8 0.0 0.0 10.1 4.73 31 SSO SpF (scMF) 0.5 191.0 18.8 6.6 2.0 1.3 12.8 5.75 32 SSO SpF (scMF) 0.015 190.0 23.0 6.0 5.0 5.0 11.0 4.40 Mean 185.3 19.3 19.0 3.4 2.1 13.3 33 SA SpF (sgMF ) 0.009 166.7 12.7 23.1 18.7 10.6 15.3 8.10 34 SSO SpF (sgMF ) 0.02 148.3 10.8 17.8 7.4 10.2 7.4 6.80 35 SSO SpF (sgMF ) 0.1 164.2 12.0 14.0 9.1 8.0 7.6 6.75 36 SA SpF (sgMF ) 0 171.9 13.1 15.7 10.7 10.9 16.0 5.40 37 SSO SpF (sgMF ) 0.6 136.3 6.0 6.7 1.7 0.2 6.5 4.50 38 SA SpF (sgMF ) 0 153.1 8.3 12.6 0.0 0.0 6.6 3.45 39 SA SpF (sgMF ) 0 97.5 8.5 0.2 0.0 0.0 5.2 2.80 40 SA SpF (sgMF ) 0 115.0 2.8 2.3 0.2 0.0 7.7 3.80 41 SSO SpF (sgMF ) 0.8 156.2 1.1 1.2 1.8 1.8 28.1 2.40 Mean 145.5 8.3 10.4 5.5 4.6 11.1 42 SA SCO 0 163.3 0.0 0.0 0.0 0.0 28.5 2.00 43 SA + OA SCO 0 170.0 0.0 0.0 0.0 0.0 17.0 2.00 44 SA SCO 0 165.0 0.0 0.0 0.0 0.0 19.5 2.00 45 SA SCO 0 165.0 0.0 0.0 0.0 0.0 20.7 2.00 46 SA SCO 0 165.0 0.0 0.0 0.0 0.0 19.1 2.00 Mean 165.6 0.0 0.0 0.0 0.0 21.0 [61]Open in a new tab Abbreviations: conc., concentration; CS, conserved spermatogenesis; OA, obstructive azoospermia; SA, secretory azoospermia; SCO, Sertoli. cell only syndrome; Spcyte, spermatocyte; SpF, spermatogenic failure; Spgonia, spermatogonia; Sptid, spermatid; SSO, severe secretory. oligozoospermia; sgMF, maturation failure at spermatogonia level; scMF, maturation failure at spermatocyte level; rsMF, maturation failure at round. spermatid level. ^(a) The mean number of the different type of cells per tubule is given in each group. The clinical procedures for infertile patients included medical history, physical examination, semen analyses (performed in accordance with World Health Organization guidelines [62][30]) and hormonal study. Concentrations of FSH generally reflected the findings of the testicular histology, although some patients showing primary spermatocyte arrest or hypospermatogenesis had normal FSH (data not shown). Spermiograms included volume, pH, sperm concentration, motility, vitality, morphology and fructose and citrate levels in seminal plasma. The testicular biopsy was obtained when necessary to confirm the clinical diagnosis and for sperm retrieval (TESE) and cryopreservation purposes. The routine genetic study for all non-obstructive samples included karyotype and analysis of chromosome Y microdeletions, the latter performed according to the European guidelines [63][31], [64][32]. Men with a chromosomal aberration or a Y-chromosome microdeletion were not included in the study. Testicular Samples Testicular biopsies of infertile men were obtained under local anesthesia through a small incision. Each specimen was divided into three aliquots, one piece (≈10–20 mg) was fixed in Bouin’s solution and reserved for histological analysis, a second aliquot (≈100–200 mg) processed for sperm extraction, and the third (≈10 mg) was immediately transferred to liquid nitrogen and then stored at –80°C until used for molecular analysis. Histological Analysis Fixed testicular biopsies were cut into 5-µm sections and stained with hematoxylin–eosin. Germ cells of the different levels of maturation (spermatogoniae, spermatocytes I, round spermatids and elongated spermatids) and Sertoli cells were quantified, and the average number per tubule was calculated after analysis of at least 15–20 cross-sectioned tubules/testis. Assessment of the spermatogenic status and the severity of the alteration is shown by a modified Johnsen score (JS) [65][33], calculated on the basis of the number of different cell types per tubule. Using this strategy we confirmed the diagnosis of SCO and CS phenotypes. With respect to SpF patients, eight of them presented maturation failure at the round spermatid level (rsMF), fifteen at the spermatocyte level (scMF) and nine at the spermatogonia level (sgMF), due to the presence of a diminished number of this specific stage and the subsequent germ cell stages in their tubules compared with CS samples ([66]Table 1). Spermatozoa Isolation and DNA Extraction Semen samples obtained from normozoospermic men were collected and allowed to liquefy for 30 min. Before the standard swim-up separation technique, whole semen was centrifuged on a 25% Percoll gradient (20 minutes) to discard somatic cell contamination, further ensuring the purity of the sperm population. The swim-up procedure results in selection of spermatozoa with good motility. Sperm DNA was extracted with an user-developed version of the QIAamp® DNeasy&Tissue Kit purification protocol (Qiagen). Fresh washed (in PBS) sperm was incubated 1∶1 with a lysis buffer containing 20 mM TrisCl (pH 8), 20 mM EDTA, 200 mM NaCl and 4% SDS, supplemented prior to use with 100 mM DTT and 250 ug/ml Proteinase K. Incubation was performed for 4 hours at 55°C with frequent vortexing. Prior to processing in the columns, 200 ul of absolute ethanol and 200 ul of the kit-provided lysis buffer were added to the samples. Then, purification was performed according to kit instructions. DNA Methylation-specific Array Genomic DNA was extracted from testicular biopsies by using the Wizard Genomic DNA Purification kit (Promega, Madison, USA). DNA methylation profile was assessed using the Infinium Human Methylation27 BeadChip (Illumina, San Diego, USA), which assays DNA methylation levels at 27,578 CpG sites. Briefly, DNA was quantified by Quant-iT™ PicoGreen dsDNA Reagent (Invitrogen, Carlsbad, USA) and the integrity was analyzed in a 1.3% agarose gel. Bisulfite conversion of 600 ng of each sample, which results in unmethylated cytosines being converted to uracils, whereas methylated cytosines are not converted, was performed according to the manufacturer’s recommendation for the Illumina Infinium Assay. Effective bisulfite conversion was checked for three controls that were converted simultaneously with the samples. The intensities of the images were extracted and normalized using GenomeStudio (V2010.3, Illumina) software. The methylation score of each CpG was represented as a beta (β) value. The threshold for concluding differential methylation of probes was set at an average delta β value >0.1. Bisulfite Sequencing Genomic DNA was bisulfite-modified using the EZ DNA Methylation-Gold Kit (Zymo Research, Orange, USA) according to the manufacturer’s protocol. The methylation status of selected regions was analyzed by bisulfite genomic sequencing. Bisulfite-converted DNA was amplified ([67]Table S1) and subsequently cloned using the pGEM-t easy kit (Promega, Madison, USA). At least eight independent clones were analyzed in an automated ABI Prism 3700 sequencer (Applied Biosystems, Carlsbad, USA). Pyrosequencing Amplification primers ([68]Table S1) and sequencing settings were designed using a PyroMark assay design (V2.0.01.15; Qiagen). LINE-1 was quantified using the PyroMark Q96 LINE-1 assay (Qiagen). PCR was performed with primers biotinylated to convert the PCR product to single-stranded DNA templates. The Vacuum Prep Tool (Biotage, Uppsala, Sweden) was used to prepare single-stranded PCR products according to the manufacturer’s instructions. Pyrosequencing reactions and methylation quantification were performed using the PyroMark Q96 System (Qiagen). Gene Expression Quantification Total RNA was obtained from the testicular biopsy using the Absolutely RNA Miniprep Kit (Stratagene, La Jolla, CA), according to the instructions provided by the manufacturer. Furthermore, small RNA-containing total RNA was additionally obtained with a mirVana miRNA Isolation Kit (Ambion) from an extra portion of the testicular biopsy whenever this was possible. The quality of RNA was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). Testicular RNA samples included in the study had a 28S/18S ratio >1.3 and an RIN value >7.5. Single-stranded cDNA was obtained by reverse transcription (RT) of 500 ng of RNA using random hexamer primers and the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative real-time PCR (qPCR) reactions were performed on an ABI 7300 real-time PCR system (Applied Biosystems) using gene-specific TaqMan Assays (PIWIL2: Hs00216263_m1; TDRD1: Hs00229805_m1; PGM1: Hs00160062_m1) and custom-designed small RNA TaqMan Assays (Applied Biosystems). Negative controls without template were included in each set of PCR assays as well as a calibrator sample to compare the change in expression of a nucleic acid sequence against the expression in all samples in the same study. PGM1 was previously selected as an appropriate reference gene among ten candidate genes tested (data not shown) for PIWIL2 and TDRD1 data normalization in our study, showing similar Ct values to the ones obtained from target genes, no statistical differences in expression among groups_(Kruskal-Wallis test) and low M-value (GeNorm software; [69][34]) indicating stable expression among samples. For piRNA expression analysis the arithmetic mean value of Ct values of RNU48, RNU19 and RNU6B was used for normalization. Patient and control group samples were always analyzed as paired samples in the same analytical run in order to exclude between-run variations. Real-time qPCR data were pre-processed using the 2^−ΔΔCt strategy and stored in SDS 2.1 software (Applied Biosystems). Expression levels are shown as relative quantification (RQ) values. Statistical Analysis Statistical analyses were performed using SPSS 12.0 software (SPSS Inc, Chicago, Illinois). The nonparametric Mann-Whitney U test was used to analyze differences in absolute expression and methylation level in SpF patient groups compared with controls. Pearson product-moment correlation coefficients were calculated to determine the correlation between the methylation status, expression ratios of the target genes and the various histological parameters in patient groups and controls. A value of p<0.05 was considered significant. Gene Ontology (GO), pathways enrichment analysis was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID; v6.7). piRNA Target Identification The complete set of piRNA sequences was obtained from piRNA bank ([70]http://pirnabank.ibab.ac.in) and aligned (BLAT) to the reference genome. Subsequently, the promoter regions (transcription start site +/−2 kb) of the 580 differentially methylated genes were analyzed for the presence of piRNA complementarity. Promoters with sequence identity of 100% to any piRNA in the data set were regarded as potential regulative target. Results DNA Methylation Profiles Distinguish Male Infertility Disorders from Physiological Germ Cell Development In order to identify the genome-wide DNA methylation changes associated with severe germ cell development deficiencies in the testis we used a DNA methylation bead-assay covering 27,578 CpG sites in the genome [71][23], [72][24], [73][35]. As the probes are almost exclusively located in promoter regions, the array gives a comprehensive overview of 14,495 individual genes. The reproducibility and sensitivity of the array has been described elsewhere [74][23], [75][24], [76][35]. Using this platform, we analyzed the methylation profile of testis with conserved spermatogenesis (CS) (n = 2), spermatogenic failure (SpF) samples (two of them -sample no. 25 and 28- presented maturation failure at the spermatocyte level [scMF] and two -sample nos. 10 and 13- at the round spermatid level [rsMF]) and Sertoli cell-only syndrome patients (SCO) (n = 2), the latter completely lacking germ cells in the testicular tubules. CS and SpF samples show similar numbers of spermatogoniae and spermatocytes in the tubule ([77]Table 1). Comparing all CG sites interrogated by the array platform, SpF samples had a highly similar profile to the CScontrol (r^2 = 0.99). However, unsupervised hierarchical clustering revealed a distinct methylation profile of the three SpF patient samples that entirely lacked elongated spermatids, unlike the CS controls (samples no. 13, 25 and 28; [78]Table 1). Furthermore, the methylation profile of the SpF sample no. 10 was clustered with the CS control samples ([79]Fig. 1A). The SCO specimens had highly variable methylation levels compared with the other groups, reflecting the somatic origin of the Sertoli cells and the specific methylation patterns of germline and somatic tissues. We were also able to identify 633 differentially methylated sites (DMSs; average delta β value >0.1) (327 hypomethylated and 306 hypermethylated) in the three clustered SpF samples relative to the CS control tissue, representing 580 different genes (comprises 4% of the tested genes) ([80]Fig. 1B, [81]Table S2). Interestingly, while most of the probes present on the array were located in CpG islands (73%), the DMSs identified were significantly enriched in CpG-poor promoters (64%; Chi-square test, p<0.01). From a biological ontology point of view, hypermethylated genes are enriched in functions directly related to germline processes, such as germline stem-cell maintenance (Fisher’s exact test, p = 1.6×10^−4), reproductive cellular process (Fisher’s exact test, p = 0.018) and gamete generation (Fisher’s exact test, p = 0.03) ([82]Table S3). In particular, hypermethylation of PIWIL1, PIWIL2, SPATA16, MSH4, INSL3, CNGA1, FANCG and HIST1H1T contributed to the enrichment in the biological process of male gamete generation. Furthermore, other germline-specific genes such as PAGE1 and XAGE3/5 were found to be differentially methylated in SpF patient samples relative to CS ([83]Table S2). Figure 1. DNA methylation microarray analysis determined disease-associated profiles. [84]Figure 1 [85]Open in a new tab (A) Unsupervised hierarchical clustering separated testis with a complete absence of germ cells (SCO) from those with the presence of germ cell lineage, and testis with conserved spermatogenesis (CS) from those with spermatogenic failure (SpF) human samples. (B) Hierarchical clustering of CS, SpF and SCO samples, displaying the 633 CpG sites differentially methylated between CS and SpF samples. (C) Hierarchical clustering of PIWIL1/2 and TDRD1/9 involved in the piRNA processing machinery. Sample number corresponding to that in [86]Table 1 is also indicated. It is of particular note that genes involved in the biogenesis of PIWI-associated RNAs (piRNAs), such as the differentially methylated in the array PIWIL1/2 and their associated molecules TDRD1/9, were able to cluster normal tissue and patient samples (including sample no.10) separately, suggesting that hypermethylation of these genes is a disease-associated event ([87]Fig. 1C). Promoter Hypermethylation-associated Transcriptional Silencing of PIWIL2 and TDRD1 in Infertile Males with Spermatogenic Failure The disruption of genes associated with the piRNA processing machinery has been directly related to spermatogenic failure due to maturation arrest, resulting in male sterility in mouse models. As genome-wide analysis of patient samples with spermatogenic failure identified deregulation of genes involved in piRNA production, we aimed to validate these genes in a larger cohort of samples. To assess the impact of DNA methylation on genes involved in the piRNA processing machinery, we performed bisulfite genomic sequencing of the promoter regions of PIWIL1/2 and TDRD1/9 in three CS samples, six SpF samples (three rsMF and three scMF) and three SCO samples ([88]Fig. 2 and [89]Fig. S1 and [90]S2). Differences in DNA methylation between SpF and CS samples were observed in all the genes analyzed, the magnitude being most striking for PIWIL2 and TDRD1. Both genes displayed minimal or no promoter methylation in CS normal tissue and a great increase in rsMF (Student’s t test <0.05) samples. TDRD9 also exhibited an elevated level of methylation, although we had already detected increased levels in normal tissue. This was even more evident for PIWIL1, for which half of the CpG sites analyzed were found to be methylated in CS samples. As expected, we observed striking differences between CS testis tissue and SCO samples, which is consistent with the germline-specific expression associated with the analyzed genes. Here, hypermethylation was detected in all five genes, probably due to the total absence of germ cells, in SCO specimens. Figure 2. PIWIL2 and TDRD1 become more methylated in human infertility syndromes. [91]Figure 2 [92]Open in a new tab Bisulfite sequencing of the piRNA processing genes PIWIL1 (A), PIWIL2 (B), TDRD1 (C) and TDRD9 (D). Black and white squares indicate CpG methylation and unmethylated sites, respectively. One representative sample of a large difference between testis with conserved spermatogenesis (CS), maturation failure at the spermatocyte (scMF) or at the round spermatid (rsMF) stage, and in Sertoli cell-only syndrome (SCO) are displayed. Sample number corresponding to that in [93]Table 1 is also indicated. (E) Methylation level of gene promoters of PIWIL1, PIWIL2, TDRD1 and TDRD9 in testis with conserved spermatogenesis (CS), maturation failure at the spermatocyte (scMF) or at the round spermatid (rsMF) stage, and in Sertoli cell-only syndrome (SCO) samples. Independent data are also shown in [94]Figure S1 and [95]S2. Significant differences compared to CS samples are indicated (*). To confirm the hypermethylation of PIWIL2 and TDRD1 in SpF-affected patients, locus-specific pyrosequencing was performed in a larger validation patient cohort. In detail, nine normal CS control, thirty-two SpF (rsMF, scMF, sgMF) affected patient samples and five SCO specimens were analyzed to detect the CpG site immediately upstream of the transcription start site. To gain a better insight into the tissue specificity of PIWIL2 and TDRD1, we also included five samples from mature swim up-selected spermatozoa and six somatic tissues from colon, breast, blood, skin, lung and brain in the study. As expected, spermatozoa exhibited an absence of DNA methylation in both genes, whereas both promoters were heavily methylated in all somatic tissues ([96]Fig. 3A and B). In SpF samples, we were able to validate a significant increase in promoter methylation (Mann-Whitney test, p<0.01) of PIWIL2 and TDRD1 ([97]Fig. 3A and B). It is of note that SCO samples and somatic tissues had similar TDRD1 DNA methylation levels, whereas PIWIL2 methylation is reduced in SCO when compared to somatic tissues (Mann-Whitney test, p = 0.015; [98]Fig. 3A and 3B). Furthermore, to have a better insight into methylation level related to the testicular sample cell composition, PIWIL2 and TDRD1 methylation data was first divided by the proportion of somatic cells per tubule ([99]Fig. 3C and D). The number of somatic cells was inferred from the number of Sertoli cells quantified per tubule in each testicular sample, as the Sertoli cells represent the 30% of somatic cells related to one testicular tubule. As no SpF sample was observed to present hyperplasia of Leydig cells it is assumed that the somatic cell number was constant among CS and SpF samples. We observed no statistical difference in methylation per somatic cell among the different subgroups of the study. Additionally PIWIL2 and TDRD1 methylation data was divided by the proportion of germ cells per tubule obtaining a hypermethylation profile, being more considerable as the maturation failure affects an earlier germline stage (CS