Lluís Bassas

Epigenetic disruption of the PIWI pathway in human spermatogenic disorders.

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.

Aberrant DNA methylation patterns of spermatozoa in men with unexplained infertility

STUDY QUESTION Are there DNA methylation alterations in sperm that could explain the reduced biological fertility of male partners from couples with unexplained infertility? SUMMARY ANSWER DNA methylation patterns, not only at specific loci but also at Alu Yb8 repetitive sequences, are altered in infertile individuals compared with fertile controls. WHAT IS KNOWN ALREADY Aberrant DNA methylation of sperm has been associated with human male infertility in patients demonstrating either deficiencies in the process of spermatogenesis or low semen quality. STUDY DESIGN, SIZE, DURATION Case and control prospective study. This study compares 46 sperm samples obtained from 17 normospermic fertile men and 29 normospermic infertile patients. PARTICIPANTS/MATERIALS, SETTING, METHODS Illumina Infinium HD Human Methylation 450K arrays were used to identify genomic regions showing differences in sperm DNA methylation patterns between five fertile and seven infertile individuals. Additionally, global DNA methylation of sperm was measured using the Methylamp Global DNA Methylation Quantification Ultra kit (Epigentek) in 14 samples, and DNA methylation at several repetitive sequences (LINE-1, Alu Yb8, NBL2, D4Z4) measured by bisulfite pyrosequencing in 44 sperm samples. A sperm-specific DNA methylation pattern was obtained by comparing the sperm methylomes with the DNA methylomes of differentiated somatic cells using data obtained from methylation arrays (Illumina 450 K) of blood, neural and glial cells deposited in public databases. MAIN RESULTS AND THE ROLE OF CHANCE In this study we conduct, for the first time, a genome-wide study to identify alterations of sperm DNA methylation in individuals with unexplained infertility that may account for the differences in their biological fertility compared with fertile individuals. We have identified 2752 CpGs showing aberrant DNA methylation patterns, and more importantly, these differentially methylated CpGs were significantly associated with CpG sites which are specifically methylated in sperm when compared with somatic cells. We also found statistically significant (P < 0.001) associations between DNA hypomethylation and regions corresponding to those which, in somatic cells, are enriched in the repressive histone mark H3K9me3, and between DNA hypermethylation and regions enriched in H3K4me1 and CTCF, suggesting that the relationship between chromatin context and aberrant DNA methylation of sperm in infertile men could be locus-dependent. Finally, we also show that DNA methylation patterns, not only at specific loci but also at several repetitive sequences (LINE-1, Alu Yb8, NBL2, D4Z4), were lower in sperm than in somatic cells. Interestingly, sperm samples at Alu Yb8 repetitive sequences of infertile patients showed significantly lower DNA methylation levels than controls. LIMITATIONS, REASONS FOR CAUTION Our results are descriptive and further studies would be needed to elucidate the functional effects of aberrant DNA methylation on male fertility. WIDER IMPLICATIONS OF THE FINDINGS Overall, our data suggest that aberrant sperm DNA methylation might contribute to fertility impairment in couples with unexplained infertility and they provide a promising basis for future research. STUDY FUNDING/COMPETING INTERESTS This work has been financially supported by Fundación Cientifica de la AECC (to R.G.U.); IUOPA (to G.F.B.); FICYT (to E.G.T.); the Spanish National Research Council (CSIC; 200820I172 to M.F.F.); Fundación Ramón Areces (to M.F.F); the Plan Nacional de I+D+I 2008-2011/2013-2016/FEDER (PI11/01728 to AF.F., PI12/01080 to M.F.F. and PI12/00361 to S.L.); the PN de I+D+I 2008-20011 and the Generalitat de Catalunya (2009SGR01490). A.F.F. is sponsored by ISCIII-Subdirección General de Evaluación y Fomento de la Investigación (CP11/00131). S.L. is sponsored by the Researchers Stabilization Program from the Spanish National Health System (CES09/020). The IUOPA is supported by the Obra Social Cajastur, Spain.

Changes in the expression profile of the meiosis-involved mismatch repair genes in impaired human spermatogenesis.

DNA mismatch repair (MMR) genes have been described to participate in crossover events during meiotic recombination, which is, in turn, a key step of spermatogenesis. This evidence suggests that MMR family gene expression may be altered in infertile men with defective sperm production. In order to determine the expression profile of MMR genes in impaired human spermatogenesis, we performed transcript levels analysis of MMR genes (MLH1, MLH3, PMS2, MSH4, and MSH5), and other meiosis-involved genes (ATR, HSPA2, and SYCP3) as controls, by real-time reverse transcription-polymerase chain reaction in testis from 13 patients with spermatogenic failure, 5 patients with primary germ cell tumors, and 10 controls with conserved spermatogenesis. Correlation of the expression values with the histological findings was also performed. The MMR gene expression values, with the exception of PMS2, are significantly decreased in men with spermatogenic failure. The pattern of MMR reduction correlates with the severity of damage, being maximum in maturation arrest. Specifically, expression of the testicular MSH4 gene could be useful as a surrogate marker for the presence of intratesticular elongated spermatid in patients with nonobstructive azoospermia, contributing to predict the viability of assisted reproduction. Interestingly, a reduction in the MSH4 and MSH5 transcript concentration per spermatocyte was also observed. The decreased expression level of other meiosis-specific genes, such as HSPA2 and SYCP3, suggests that the spermatocyte capacity to express meiosis-related genes is markedly reduced in spermatogenic failure, contributing to meiosis impairment and spermatogenic blockade.

Clinical relevance of Y-linked CNV screening in male infertility: new insights based on the 8-year experience of a diagnostic genetic laboratory

AZF microdeletion screening is routinely performed in the diagnostic work-up for male infertility; however, some issues remain debated. In this study, we provide insights into the sperm concentration cutoff value for routine testing, the predictive value of AZFc deletion for testicular sperm retrieval and the Y-background contribution to the interpopulation variability of deletion frequencies. In the Spanish population, partial AZFc rearrangements have been poorly explored and no data exist on partial duplications. In our study, 27/806 (3.3%) patients carried complete AZF deletions. All were azoo/cryptozoospermic, except for one whose sperm concentration was 2 × 106/ml. In AZFc-deleted men, we observed a lower sperm recovery rate upon conventional TESE (9.1%) compared with the literature (60–80% with microTESE). Haplogroup E was the most represented among non-Spanish and hgr P among Spanish AZF deletion carriers. The analysis of AZFc partial rearrangements included 330 idiopathic infertile patients and 385 controls of Spanish origin. Gr/gr deletion, but not AZFc partial duplications, was significantly associated with spermatogenic impairment. Our data integrated with the literature suggest that: (1) routine AZF microdeletion testing could eventually include only men with ≤2 × 106/ml; (2) classical TESE is associated with low sperm recovery rate in azoospermic AZFc-deleted men, and therefore microTESE should be preferred; (3) Y background could partially explain the differences in deletion frequencies among populations. Finally, our data on gr/gr deletion further support the inclusion of this genetic test in the work-up of infertile men, whereas partial AZFc duplications do not represent a risk for spermatogenic failure in the Spanish population.

Sperm gene expression profile is related to pregnancy rate after insemination and is predictive of low fecundity in normozoospermic men

BACKGROUND Assessment of male fertility is traditionally based on microscopic evaluation of semen. However, the classical semen parameters do not adequately reflect sperm function, and their clinical value in predicting fertility is limited. We hypothesize that the sperm expression profile could reflect the fertilizing quality of spermatozoa and could be more informative for predicting the in vivo reproductive fitness of men with normal semen parameters. METHODS Sperm gene expression patterns of 68 normozoospermic donors (43 Phase I and 25 Phase II), used for therapeutic IUI, were analysed via TaqMan Arrays. RESULTS Significant differences in the expression of individual genes were observed between groups of donors with the lowest and highest pregnancy rates (PRs) after IUI. Additionally, we have developed a molecular means to classify the fertility status of semen donors for IUI based on the expression signature of four genes. In the Phase I study, this model had 90% sensitivity and 97% specificity for discriminating donors resulting in low PRs (cut-off value: <13.6%), far better than that obtained from the combination of sperm parameters. The translation of the model was validated in Phase II donors resulting in a sensitivity of 71.5% and a specificity of 78%. CONCLUSIONS Our findings contribute to the search for the most valuable genetic markers which are potentially useful as tools for predicting pregnancy. Our expression model could complement classical semen analysis in order to identify sperm donors with a less favourable IUI reproductive outcome despite having normal semen parameters. It may also be useful for the study of sperm function in couples with unexplained infertility.

Association of cystic fibrosis genetic modifiers with congenital bilateral absence of the vas deferens

OBJECTIVE To investigate whether genetic modifiers of cystic fibrosis (CF) lung disease also predispose to congenital bilateral absence of the vas deferens (CBAVD) in association with cystic fibrosis transmembrane conductance regulator (CFTR) mutations. We tested the hypothesis that polymorphisms of transforming growth factor (TGF)-β1 (rs 1982073, rs 1800471) and endothelin receptor type A (EDNRA) (rs 5335, rs 1801708) are associated with the CBAVD phenotype. DESIGN Genotyping of subjects with clinical CBAVD. SETTING Outpatient and hospital-based clinical evaluation. PATIENT(S) DNA samples from 80 subjects with CBAVD and 51 healthy male controls from various regions of Europe. This is one of the largest genetic studies of this disease to date. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Genotype analysis. RESULT(S) For single nucleotide polymorphism (SNP) rs 5335, we found increased frequency of the CC genotype among subjects with CBAVD. The difference was significant among Turkish patients versus controls (45.2% vs. 19.4%), and between all cases versus controls (36% vs. 15.7%). No associations between CBAVD penetrance and polymorphisms rs 1982073, rs 1800471, or rs 1801708 were observed. CONCLUSION(S) Our findings indicate that endothelin receptor type A polymorphism rs 5335 may be associated with CBAVD penetrance. To our knowledge, this is the first study to investigate genetic modifiers relevant to CBAVD.

Comprehensive investigation in patients affected by sperm macrocephaly and globozoospermia

The aim of this study was to provide a comprehensive genetic/phenotypic characterization of subjects suffering infertility owing to sperm macrocephaly (n = 3) or globozoospermia (n = 9) and to investigate whether the patients’ genetic status was correlated with the alteration of various sperm parameters. AURKC was sequenced in case of sperm macrocephaly while the DPY19L2 status has been analyzed by multiple approaches including a novel qPCR–based copy number assay in case of globozoospermia. Globozoospermic patients were also analyzed for SPACA1, a novel candidate gene herein tested for the first time in humans. The effect of the patients’ genetic status was interrogated by implementing the molecular screening with the characterization of several sperm parameters: (i) routine sperm analysis, integrated with transmission electron microscopy; (ii) sperm fluorescent in situ hybridization (FISH) analysis; (iii) sperm DNA fragmentation (DF) analysis. Moreover, for the first time, we performed microsatellite instability analysis as a marker of genome instability in men with sperm macrocephaly and globozoospermia. Finally, artificial reproductive technology (ART) history has been reported for those patients who underwent the treatment. Macrocephalic patients had an AURKC mutation and >89% tetraploid, highly fragmented spermatozoa. DPY19L2 was mutated in all patients with >80% globozoospermia: the two homozygous deleted men and the compound heterozygous showed the severest phenotype (90–100%). The newly developed qPCR method was fully validated and has the potential of detecting also yet undiscovered deletions. DPY19L2 status is unlikely related to FISH anomalies and DF, although globozoospermic men showed a higher disomy rate and DF compared with internal reference values. No patient was mutated for SPACA1. Our data support the general agreement on the negative correlation between macro/globozoospermia and conventional intracytoplasmic sperm injection outcomes. Microsatellites were stable in all patients analyzed. The comprehensive picture provided on these severe phenotypes causing infertility is of relevance in the management of patients undergoing ART.

Altered miRNA Signature of Developing Germ-cells in Infertile Patients Relates to the Severity of Spermatogenic Failure and Persists in Spermatozoa.

The aim of this study was to assess the cellular miRNA expression behaviour in testes with spermatogenic failure (SpF). We performed a high-throughput screen of 623 mature miRNAs by a quantitative RT-qPCR-based approach in histologically well-defined testicular samples with spermatogenic disruption at different germ-cell stages, which revealed altered patterns of miRNA expression. We focussed on the differentially expressed miRNAs whose expression correlated with the number of testicular mature germ-cells and described the combined expression values of a panel of three miRNAs (miR-449a, miR-34c-5p and miR-122) as a predictive test for the presence of mature germ-cells in testicular biopsy. Additionally, we determined decreased cellular miRNA content in developing germ-cells of SpF testis; this was more noticeable the earlier the stage of germ-cell differentiation was affected by maturation failure. Furthermore, we showed that the miRNA expression profile in mature sperm from mild SpF patients was widely altered. Our results suggest that the cellular miRNA content of developed germ-cells depends heavily on the efficacy of the spermatogenic process. What is more, spermatozoa that have fulfilled the differentiation process still retain the dysregulated miRNA pattern observed in the developing SpF germ-cells. This altered miRNA molecular signature may have functional implications for the male gamete.

Altered gene expression signature of early stages of the germ line supports the pre‐meiotic origin of human spermatogenic failure

The molecular basis of spermatogenic failure (SpF) is still largely unknown. Accumulating evidence suggests that a series of specific events such as meiosis, are determined at the early stage of spermatogenesis. This study aims to assess the expression profile of pre‐meiotic genes of infertile testicular biopsies that might help to define the molecular phenotype associated with human deficiency of sperm production. An accurate quantification of testicular mRNA levels of genes expressed in spermatogonia was carried out by RT‐qPCR in individuals showing SpF owing to germ cell maturation defects, Sertoli cell‐only syndrome or conserved spermatogenesis. In addition, the gene expression profile of SpF was compared with that of testicular tumour, which is considered to be a severe developmental disease of germ cell differentiation. Protein expression from selected genes was evaluated by immunohistochemistry. Our results indicate that SpF is accompanied by differences in expression of certain genes associated with spermatogonia in the absence of any apparent morphological and/or numerical change in this specific cell type. In SpF testicular samples, we observed down‐regulation of genes involved in cell cycle (CCNE1 and POLD1), transcription and post‐transcription regulation (DAZL, RBM15 and DICER1), protein degradation (FBXO32 and TM9SF2) and homologous recombination in meiosis (MRE11A and RAD50) which suggests that the expression of these genes is critical for a proper germ cell development. Interestingly, a decrease in the CCNE1, DAZL, RBM15 and STRA8 cellular transcript levels was also observed, suggesting that the gene expression capacity of spermatogonia is altered in SpF contributing to an unsuccessful sperm production. Altogether, these data point to the spermatogenic derangement being already determined at, or arising in, the initial stages of the germ line.

Association of PIWIL4 genetic variants with germ cell maturation arrest in infertile Spanish men.

and allele frequencies in the patients and controls. Hardy–Weinberg equilibrium was tested by the χ2 test. The significance level was established at P < 0.05. These statistical analyses were performed using the SPSS software version 12 (Lead Technologies, Chicago, USA). Pairwise linkage disequilibrium (LD) between SNPs, haplotype frequencies, and association analyses were performed with SNPassoc, genetics and haplo.stats in R package (http://www.creal.cat/jrgonzalez/ software.htm). Our results show that the SNPs rs7110167, rs57607909, and rs593690 were in LD (Figure 1) in our cohort of individuals (D’ score >0.85), whereas rs508485 is independent of the other three (D’ <0.12). No significant differences in frequencies of allele, allele carrier, and genotypes were observed between patients and controls at the rs7110167, rs57607909, and rs593690 loci. Interestingly, we found that the frequencies of allele T (61.4% vs. 40.2%; P = 0.021) and allele T carrier (CT + TT) (86.4% vs. 60.7%; P = 0.033) in MA patients were higher than they were in the controls at the rs508485 locus. When the genotype frequencies were compared between the groups, a difference in genotype distribution was found in MA versus controls, although it was not statistically significant (P = 0.06) (Table 2). This SNP could have potential consequences for mRNA stability by altering the PIWIL4 3’UTR binding to miRNAs. To further investigate the relationship of the four SNP in PIWIL4 and defects in sperm production, we performed a haplotype analysis of these variants in patient and control groups. A total of six haplogroups were found in a frequency > 0.05 (Table 3). The frequency of haplotype TGCT was higher in patients (29.4% vs. 18.9%; P = 0.036) and in SCO compared with controls (32.5% vs. 18.9%; P = 0.005), whereas a similar frequency of the haplotype TGCT was observed in HS and controls (17.6% vs. 18.9%). MA patients also presented a higher frequency of haplotype TGCT (33.4% vs. 18.9%; P = 0.24) and CGTT (14.3% vs. 7.6%; P = 0.24) than controls although the difference was not statistically significant, which is probably attributable to the low number of MA samples for the haplotype frequency estimation. Haplotypes CCTC (15.3% vs. 9.4%; P = 0.018) and CGTC (15.3% vs. 10.3%; P = 0.027) may also be risk factors for SCO infertility. A higher frequency of haplotype TGCT was also found in patients classified as azoospermic (29.3% vs. 18.9%; P = 0.036) and severely oligozoospermic (33.2% vs. 18.9%; P = 0.14), although the difference was not statistically significant for the oligozoospermic group; probably due to the lower number of samples. Our results bring additional information from different populations about the involvement of PIWIL4 genetic polymorphisms in Dear Editor, The PIWI proteins (originally P-element-induced wimpy testis in Drosophila) are predominantly present in the germ-line in diverse organisms and are involved in the processing of a class of small RNAs known as piRNAs (see Refs.1,2 for review). The human PIWI protein family consists of four members: PIWIL1–4. Of these, PIWIL4 is known to have essential roles in the first phases of spermatogenesis: its expression is restricted to gonocytes and it is required for transposon silencing.3 The lack of this gene in mice causes meiotic arrest in spermatogenesis.4 The goal of our study was to evaluate the frequency of several PIWIL4 genetic variants in our population to better define the relationship between PIWIL4 single nucleotide polymorphisms (SNPs) and both defective spermatogenesis and specific spermatogenic disorders. We have genotyped four PIWIL4 SNPs (rs7110167 C/T, exon 7-synonimous; rs57607909 G/C, exon 9-missense; rs593690 T/C, exon 13-synonimous, and rs508485 C/T, 3’UTR) (Table 1), with a reported minor allele frequency (MAF) of >0.05 in the general Caucasian population (HapMap CEU), in genomic DNA from 79 nonobstructive infertile men (patients) with azoospermia (n = 61) or severe oligozoospermia (sperm counts <5 × 106 ml−1; n = 18), and 56 men diagnosed with obstructive azoospermia (as a consequence of congenital absence of the vas deferens or a previous vasectomy), who showed conserved spermatogenesis (controls). Patients underwent testicular biopsy, which showed a histological pattern with a total absence of germ cells (Sertoli cell-only syndrome, SCO; n = 40), >90% of maturation arrest, either in spermatogonia or in primary spermatocytes (MA; n = 22) and hypospermatogenesis (HS; n = 17). Men with a chromosomal aberration or with a Y-chromosome microdeletion had been previously excluded from the study. All the men included in this study were recruited from the Andrology Service of Fundació Puigvert and gave their informed consent for the study, which was approved by the Institutional Ethical Committee. All of them were Caucasians of Spanish origin. The current method for SNP determination was allelic discrimination using a Real Time PCR System and SNP Genotyping Assays (Applied Biosystems, Foster City, CA, USA). The χ2 test using Fisher exact test to the 5% limit was employed to compare carrier Association of PIWIL4 genetic variants with germ cell maturation arrest in infertile Spanish men