SpF samples show a <i>PIWIL2</i> and <i>TDRD1</i> hypermethylation pattern.

<p>Methylation levels of <i>PIWIL2</i> (A) and <i>TDRD1</i> (B) in mature spermatozoa (sperm), testis with a conserved spermatogenic pattern (CS), maturation failure at the round spermatid (rsMF), the spermatocyte (scMF) and the spermatogonia (sgMF) stages, Sertoli cell-only syndrome (SCO) and somatic tissue measured by pyrosequencing. The black bar indicates the mean methylation level. Methylation per cell profiling of <i>PIWIL2</i> and <i>TDRD1</i>, displayed as methylation per somatic cell (x100) (C, D) and methylation per germ cell (x100) (E, F) in testis with conserved spermatogenesis (CS), maturation failure at the round spermatid (rsMF), the spermatocyte (scMF) and the spermatogonia (sgMF) stages and Sertoli cell-only syndrome (SCO). The horizontal bar displays the mean cellular expression level. Significant differences from the control are indicated: *p<0.05; **p<0.01.</p

Pearson correlation coefficients and adjusted p values (r;p) between the molecular and the histological parameters for all the samples analysed.

<p>Significant differences are indicated in bold.</p

DNA methylation microarray analysis determined disease-associated profiles.

<p>(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 <i>PIWIL1/2</i> and <i>TDRD1/9</i> involved in the piRNA processing machinery. Sample number corresponding to that in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047892#pone-0047892-t001" target="_blank">Table 1</a> is also indicated.</p

<i>PIWIL2</i> and <i>TDRD1</i> become more methylated in human infertility syndromes.

<p>Bisulfite sequencing of the piRNA processing genes <i>PIWIL1</i> (A), <i>PIWIL2</i> (B), <i>TDRD1</i> (C) and <i>TDRD9</i> (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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047892#pone-0047892-t001" target="_blank">Table 1</a> is also indicated. (E) Methylation level of gene promoters of <i>PIWIL1, PIWIL2</i>, <i>TDRD1</i> and <i>TDRD9</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047892#pone.0047892.s001" target="_blank">Figure S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047892#pone.0047892.s002" target="_blank">S2</a>. Significant differences compared to CS samples are indicated (*).</p

Phenotypical and histological description of the testicular samples included in the study.^(a).

<p>Abbreviations: conc., concentration; CS, conserved spermatogenesis; OA, obstructive azoospermia; SA, secretory azoospermia; SCO, Sertoli.</p><p>cell only syndrome; Spcyte, spermatocyte; SpF, spermatogenic failure; Spgonia, spermatogonia; Sptid, spermatid; SSO, severe secretory.</p><p>oligozoospermia; sgMF, maturation failure at spermatogonia level; scMF, maturation failure at spermatocyte level; rsMF, maturation failure at round.</p><p>spermatid level.</p>(a)<p>The mean number of the different type of cells per tubule is given in each group.</p

Downregulation of <i>PIWIL2</i> and <i>TDRD1</i> is associated with piRNA reduction and LINE-1 hypomethylation.

<p>(A–E) Expression levels of selected piRNAs in testis with conserved spermatogenesis (CS), spermatogenic failure (SpF), Sertoli cell-only syndrome (SCO) phenotypes, measured by qPCR. Expression levels relative to <i>RNU48</i>, <i>RNU19</i> and <i>RNU6B</i> are shown. (F) Methylation profiling of LINE-1 in mature spermatozoa (sperm), testis with conserved spermatogenesis (CS), spermatogenic failure (SpF), Sertoli cell-only syndrome (SCO) phenotypes and somatic tissue, measured by pyrosequencing. Significant differences from the control are indicated: *p<0.05; **p<0.01. Mean expression levels are depicted by horizontal bars.</p

<i>PIWIL2</i> and <i>TDRD1</i> hypermethylation is negatively associated with <i>PIWIL2</i> and <i>TDRD1</i> transcript levels in SpF samples.

<p>Tissular expression profiling of <i>PIWIL2</i> (A) and <i>TDRD1</i> (B) by quantitative real-time qPCR in testis with conserved spermatogenesis (CS), maturation failure at the round spermatid (rsMF), the spermatocyte (scMF) and the spermatogonia (sgMF) stages and Sertoli cell-only syndrome (SCO). Expression levels relative to <i>PGM1</i> are shown. Expression per cell profiling of <i>PIWIL2</i>, displayed as expression ratio per spermatogonia/spermatocyte (x1000) (C) and expression per cell of <i>TDRD1</i>, displayed as expression ratio per spermatocyte (X1000) (D) in testis with conserved spermatogenesis (CS), maturation failure at the spermatocyte (scMF), the round spermatid (rsMF), and the spermatogonia (sgMF) stages. The horizontal bar displays the mean cellular expression level. Significant differences from the control are indicated: *p<0.05; **p<0.01.</p

DataSheet1_Contribution of TEX15 genetic variants to the risk of developing severe non-obstructive oligozoospermia.PDF

Background: Severe spermatogenic failure (SPGF) represents one of the most relevant causes of male infertility. This pathological condition can lead to extreme abnormalities in the seminal sperm count, such as severe oligozoospermia (SO) or non-obstructive azoospermia (NOA). Most cases of SPGF have an unknown aetiology, and it is known that this idiopathic form of male infertility represents a complex condition. In this study, we aimed to evaluate whether common genetic variation in TEX15, which encodes a key player in spermatogenesis, is involved in the susceptibility to idiopathic SPGF.Materials and Methods: We designed a genetic association study comprising a total of 727 SPGF cases (including 527 NOA and 200 SO) and 1,058 unaffected men from the Iberian Peninsula. Following a tagging strategy, three tag single-nucleotide polymorphisms (SNPs) of TEX15 (rs1362912, rs323342, and rs323346) were selected for genotyping using TaqMan probes. Case-control association tests were then performed by logistic regression models. In silico analyses were also carried out to shed light into the putative functional implications of the studied variants.Results: A significant increase in TEX15-rs1362912 minor allele frequency (MAF) was observed in the group of SO patients (MAF = 0.0842) compared to either the control cohort (MAF = 0.0468, OR = 1.90, p = 7.47E-03) or the NOA group (MAF = 0.0472, OR = 1.83, p = 1.23E-02). The genotype distribution of the SO population was also different from those of both control (p = 1.14E-02) and NOA groups (p = 4.33–02). The analysis of functional annotations of the human genome suggested that the effect of the SO-associated TEX15 variants is likely exerted by alteration of the binding affinity of crucial transcription factors for spermatogenesis.Conclusion: Our results suggest that common variation in TEX15 is involved in the genetic predisposition to SO, thus supporting the notion of idiopathic SPGF as a complex trait.</p

Table1_Contribution of TEX15 genetic variants to the risk of developing severe non-obstructive oligozoospermia.XLSX

Background: Severe spermatogenic failure (SPGF) represents one of the most relevant causes of male infertility. This pathological condition can lead to extreme abnormalities in the seminal sperm count, such as severe oligozoospermia (SO) or non-obstructive azoospermia (NOA). Most cases of SPGF have an unknown aetiology, and it is known that this idiopathic form of male infertility represents a complex condition. In this study, we aimed to evaluate whether common genetic variation in TEX15, which encodes a key player in spermatogenesis, is involved in the susceptibility to idiopathic SPGF.Materials and Methods: We designed a genetic association study comprising a total of 727 SPGF cases (including 527 NOA and 200 SO) and 1,058 unaffected men from the Iberian Peninsula. Following a tagging strategy, three tag single-nucleotide polymorphisms (SNPs) of TEX15 (rs1362912, rs323342, and rs323346) were selected for genotyping using TaqMan probes. Case-control association tests were then performed by logistic regression models. In silico analyses were also carried out to shed light into the putative functional implications of the studied variants.Results: A significant increase in TEX15-rs1362912 minor allele frequency (MAF) was observed in the group of SO patients (MAF = 0.0842) compared to either the control cohort (MAF = 0.0468, OR = 1.90, p = 7.47E-03) or the NOA group (MAF = 0.0472, OR = 1.83, p = 1.23E-02). The genotype distribution of the SO population was also different from those of both control (p = 1.14E-02) and NOA groups (p = 4.33–02). The analysis of functional annotations of the human genome suggested that the effect of the SO-associated TEX15 variants is likely exerted by alteration of the binding affinity of crucial transcription factors for spermatogenesis.Conclusion: Our results suggest that common variation in TEX15 is involved in the genetic predisposition to SO, thus supporting the notion of idiopathic SPGF as a complex trait.</p