Genome-wide DNA methylation changes in human spermatogenesis.

Sperm production and function require the correct establishment of DNA methylation patterns in the germline. Here, we examined the genome-wide DNA methylation changes during human spermatogenesis and its alterations in disturbed spermatogenesis. We found that spermatogenesis is associated with remodeling of the methylome, comprising a global decline in DNA methylation in primary spermatocytes followed by selective remethylation, resulting in a spermatids/sperm-specific methylome. Hypomethylated regions in spermatids/sperm were enriched in specific transcription factor binding sites for DMRT and SOX family members and spermatid-specific genes. Intriguingly, while SINEs displayed differential methylation throughout spermatogenesis, LINEs appeared to be protected from changes in DNA methylation. In disturbed spermatogenesis, germ cells exhibited considerable DNA methylation changes, which were significantly enriched at transposable elements and genes involved in spermatogenesis. We detected hypomethylation in SVA and L1HS in disturbed spermatogenesis, suggesting an association between the abnormal programming of these regions and failure of germ cells progressing beyond meiosis.

OGRE: calculate, visualize, and analyze overlap between genomic input regions and public annotations.

BACKGROUND: Modern genome sequencing leads to an ever-growing collection of genomic annotations. Combining these elements with a set of input regions (e.g. genes) would yield new insights in genomic associations, such as those involved in gene regulation. The required data are scattered across different databases making a manual approach tiresome, unpractical, and prone to error. Semi-automatic approaches require programming skills in data parsing, processing, overlap calculation, and visualization, which most biomedical researchers lack. Our aim was to develop an automated tool providing all necessary algorithms, benefiting both bioinformaticians and researchers without bioinformatic training. RESULTS: We developed overlapping annotated genomic regions (OGRE) as a comprehensive tool to associate and visualize input regions with genomic annotations. It does so by parsing regions of interest, mining publicly available annotations, and calculating possible overlaps between them. The user can thus identify location, type, and number of associated regulatory elements. Results are presented as easy to understand visualizations and result tables. We applied OGRE to recent studies and could show high reproducibility and potential new insights. To demonstrate OGRE's performance in terms of running time and output, we have conducted a benchmark and compared its features with similar tools. CONCLUSIONS: OGRE's functions and built-in annotations can be applied as a downstream overlap association step, which is compatible with most genomic sequencing outputs, and can thus enrich pre-existing analyses pipelines. Compared to similar tools, OGRE shows competitive performance, offers additional features, and has been successfully applied to two recent studies. Overall, OGRE addresses the lack of tools for automatic analysis, local genomic overlap calculation, and visualization by providing an easy to use, end-to-end solution for both biologists and computational scientists.

WWC2 expression in the testis: Implications for spermatogenesis and male fertility.

The family of WWC proteins is known to regulate cell proliferation and organ growth control via the Hippo signaling pathway. As WWC proteins share a similar domain structure and a common set of interacting proteins, they are supposed to fulfill compensatory functions in cells and tissues. While all three WWC family members WWC1, WWC2, and WWC3 are found co-expressed in most human organs including lung, brain, kidney, and liver, in the testis only WWC2 displays a relatively high expression. In this study, we investigated the testicular WWC2 expression in spermatogenesis and male fertility. We show that the Wwc2 mRNA expression level in mouse testes is increased during development in parallel with germ cell proliferation and differentiation. The cellular expression of each individual WWC family member was evaluated in published single-cell mRNA datasets of murine and human testes demonstrating a high WWC2 expression predominantly in early spermatocytes. In line with this, immunohistochemistry revealed cytosolic WWC2 protein expression in primary spermatocytes from human testes displaying full spermatogenesis. In accordance with these findings, markedly lower WWC2 expression levels were detected in testicular tissues from mice and men lacking germ cells. Finally, analysis of whole-exome sequencing data of male patients affected by infertility and unexplained severe spermatogenic failure revealed several heterozygous, rare WWC2 gene variants with a proposed damaging function and putative impact on WWC2 protein structure. Taken together, our findings provide novel insights into the testicular expression of WWC2 and show its cell-specific expression in spermatocytes. As rare WWC2 variants were identified in the background of disturbed spermatogenesis, WWC2 may be a novel candidate gene for male infertility.

Testicular Architecture of Men with 46,XX Testicular Disorders of Sex Development.

BACKGROUND: A subtype of disorders of sex development (DSD) in individuals with a 46,XX karyotype who are phenotypically male is classified as testicular DSD (46,XX TDSD). These individuals develop testes but are infertile due to germ cell loss. However, little is known about their testicular architecture. METHODS: We analyzed biopsies of four SRY positive 46,XX TDSD men for testicular architecture, Sertoli (SCs) and Leydig cells (LCs). These were compared with biopsies of men with normal spermatogenesis (NS, n = 4), men with Klinefelter syndrome, 47 XXY (KS, n = 4), and men with AZF deletions (AZF, n = 5). Testicular architecture was evaluated and SCs and LCs were analyzed for specific markers (SC: SOX9, DMRT1; LC: INSL3). RESULTS: A smaller number of tubules, more SOX9-negative but similar proportions of DMRT1-negative SCs were found in 46,XX TDSD compared to NS. The lower number of tubules and severe LC hyperplasia observed in 46,XX TDSD were similar to KS. CONCLUSION: Testicular architecture and marker expression of SCs and LCs in 46,XX TDSD men display unique patterns, which are discernable from chromosomal aneuploidies. Given the reduced Y-chromosomal gene content in 46,XX TDSD, the supernumerary X chromosome effects may be decisive regarding the damage on testicular composition and endocrine function.

Transcriptome analyses in infertile men reveal germ cell-specific expression and splicing patterns.

The process of spermatogenesis-when germ cells differentiate into sperm-is tightly regulated, and misregulation in gene expression is likely to be involved in the physiopathology of male infertility. The testis is one of the most transcriptionally rich tissues; nevertheless, the specific gene expression changes occurring during spermatogenesis are not fully understood. To better understand gene expression during spermatogenesis, we generated germ cell-specific whole transcriptome profiles by systematically comparing testicular transcriptomes from tissues in which spermatogenesis is arrested at successive steps of germ cell differentiation. In these comparisons, we found thousands of differentially expressed genes between successive germ cell types of infertility patients. We demonstrate our analyses' potential to identify novel highly germ cell-specific markers (TSPY4 and LUZP4 for spermatogonia; HMGB4 for round spermatids) and identified putatively misregulated genes in male infertility (RWDD2A, CCDC183, CNNM1, SERF1B). Apart from these, we found thousands of genes showing germ cell-specific isoforms (including SOX15, SPATA4, SYCP3, MKI67). Our approach and dataset can help elucidate genetic and transcriptional causes for male infertility.

Single-cell RNA-seq unravels alterations of the human spermatogonial stem cell compartment in patients with impaired spermatogenesis.

Despite the high incidence of male infertility, only 30% of infertile men receive a causative diagnosis. To explore the regulatory mechanisms governing human germ cell function in normal and impaired spermatogenesis (crypto), we performed single-cell RNA sequencing (>30,000 cells). We find major alterations in the crypto spermatogonial compartment with increased numbers of the most undifferentiated spermatogonia (PIWIL4+). We also observe a transcriptional switch within the spermatogonial compartment driven by increased and prolonged expression of the transcription factor EGR4. Intriguingly, the EGR4-regulated chromatin-associated transcriptional repressor UTF1 is downregulated at transcriptional and protein levels. This is associated with changes in spermatogonial chromatin structure and fewer Adark spermatogonia, characterized by tightly compacted chromatin and serving as reserve stem cells. These findings suggest that crypto patients are disadvantaged, as fewer cells safeguard their germline's genetic integrity. These identified spermatogonial regulators will be highly interesting targets to uncover genetic causes of male infertility.

Whole-genome methylation analysis of testicular germ cells from cryptozoospermic men points to recurrent and functionally relevant DNA methylation changes.

BACKGROUND: Several studies have reported an association between male infertility and aberrant sperm DNA methylation patterns, in particular in imprinted genes. In a recent investigation based on whole methylome and deep bisulfite sequencing, we have not found any evidence for such an association, but have demonstrated that somatic DNA contamination and genetic variation confound methylation studies in sperm of severely oligozoospermic men. To find out whether testicular germ cells (TGCs) of such patients might carry aberrant DNA methylation, we compared the TGC methylomes of four men with cryptozoospermia (CZ) and four men with obstructive azoospermia, who had normal spermatogenesis and served as controls (CTR). RESULTS: There was no difference in DNA methylation at the whole genome level or at imprinted regions between CZ and CTR samples. However, using stringent filters to identify group-specific methylation differences, we detected 271 differentially methylated regions (DMRs), 238 of which were hypermethylated in CZ (binominal test, p < 2.2 × 10-16). The DMRs were enriched for distal regulatory elements (p = 1.0 × 10-6) and associated with 132 genes, 61 of which are differentially expressed at various stages of spermatogenesis. Almost all of the 67 DMRs associated with the 61 genes (94%) are hypermethylated in CZ (63/67, p = 1.107 × 10-14). As judged by single-cell RNA sequencing, 13 DMR-associated genes, which are mainly expressed during meiosis and spermiogenesis, show a significantly different pattern of expression in CZ patients. In four of these genes, the promoter is hypermethylated in CZ men, which correlates with a lower expression level in these patients. In the other nine genes, eight of which downregulated in CZ, germ cell-specific enhancers may be affected. CONCLUSIONS: We found that impaired spermatogenesis is associated with DNA methylation changes in testicular germ cells at functionally relevant regions of the genome. We hypothesize that the described DNA methylation changes may reflect or contribute to premature abortion of spermatogenesis and therefore not appear in the mature, motile sperm.

Healthy ageing and spermatogenesis.

Delayed family planning and increased parental age increase the risk for infertility and impaired offspring health. While the impact of ageing on oogenesis is well studied, this is less understood on spermatogenesis. Assessing ageing effects on the male germline presents a challenge in differentiating between the effects of ageing-associated morbidities, infertility and 'pure' ageing. However, understanding the impact of ageing on male germ cells requires the separation of age from other factors. In this review, we therefore discuss the current knowledge on healthy ageing and spermatogenesis. Male ageing has been previously associated with declining sperm parameters, disrupted hormone secretion and increased time-to-pregnancy, among others. However, recent data show that healthy ageing does not deteriorate testicular function in terms of hormone production and spermatogenic output. In addition, intrinsic, age-dependent, highly specific processes occur in ageing germ cells that are clearly distinct from somatic ageing. Changes in spermatogonial stem cell populations indicate compensation for stem cell exhaustion. Alterations in the stem cell niche and molecular ageing signatures in sperm can be observed in ageing fertile men. DNA fragmentation rates as well as changes in DNA methylation patterns and increased telomere length are hallmarks of ageing sperm. Taken together, we propose a putative link between the re-activation of quiescent Adark spermatogonia and molecular changes in aged sperm descending from these activated spermatogonia. We suggest a baseline of 'pure' age effects in male germ cells which can be used for subsequent studies in which the impact of infertility or co-morbidities will be studied.

The role of gonadotropins in testicular and adrenal androgen biosynthesis pathways-Insights from males with congenital hypogonadotropic hypogonadism on hCG/rFSH and on testosterone replacement.

OBJECTIVE: To delineate the role of gonadotropins in male androgen biosynthesis pathways. DESIGN: Case-control study. PATIENTS AND MEASUREMENTS: Twenty five males with congenital hypogonadotropic hypogonadism (CHH) underwent hCG/rFSH and testosterone treatment sequentially. Serum steroid hormone profiles (testosterone precursors and metabolites) on both replacement regimens were analysed, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and compared to those of healthy controls, matched by age, BMI and serum testosterone. RESULTS: On testosterone replacement, serum concentrations of the classic Δ4 pathway hormones progesterone and 17-hydroxy-progesterone (17-OHP), and the marker steroid of an alternative pathway of testosterone synthesis (androstenediol) were decreased, compared to controls. Androstanediol, a marker of the backdoor pathway of dihydrotestosterone (DHT) synthesis, was increased. 17-OH-pregnenolone, androstenedione and DHEAS (Δ5 pathway), three 11-oxygenated C19 androgens (11-keto-A4, 11-keto-T and 11-keto-DHT) and the testosterone (T) metabolites DHT and 17ß-oestradiol (E2) were similar to controls. On gonadotropin replacement, 17-OHP, 17-OH-pregnenolone, DHEAS and androstenedione, as well as DHT, androstenediol, and all 11-oxygenated C19 androgens were normal. Progesterone (Δ4 pathway) was slightly decreased, and androstanediol (backdoor DHT pathway) and E2 (T metabolite) were increased. CONCLUSIONS: In males with CHH, serum steroid hormone profiles resemble those of healthy men, if hCG/rFSH is used for substitution. Gonadotropins contribute to steroid hormone production along the classic Δ4 pathway and co-activate an alternative pathway of testosterone biosynthesis via androstenediol. Backdoor DHT biosynthesis, Δ5 17-OH-pregnenolone, DHEA(S) and androstenedione synthesis and 11-oxygenated C19 androgen production are activated independently of gonadotropins. The androgen replacement modality used for treatment of hypogonadal males with absent or reduced endogenous LH/FSH secretion may impact on long-term health and quality of life.

A germ cell-specific ageing pattern in otherwise healthy men.

Life-long sperm production leads to the assumption that male fecundity remains unchanged throughout life. However, recently it was shown that paternal age has profound consequences for male fertility and offspring health. Paternal age effects are caused by an accumulation of germ cell mutations over time, causing severe congenital diseases. Apart from these well-described cases, molecular patterns of ageing in germ cells and their impact on DNA integrity have not been studied in detail. In this study, we aimed to assess the effects of 'pure' ageing on male reproductive health and germ cell quality. We assembled a cohort of 198 healthy men (18-84 years) for which end points such as semen and hormone profiles, sexual health and well-being, and sperm DNA parameters were evaluated. Sperm production and hormonal profiles were maintained at physiological levels over a period of six decades. In contrast, we identified a germ cell-specific ageing pattern characterized by a steady increase of telomere length in sperm and a sharp increase in sperm DNA instability, particularly after the sixth decade. Importantly, we found sperm DNA methylation changes in 236 regions, mostly nearby genes associated with neuronal development. By in silico analysis, we found that 10 of these regions are located in loci which can potentially escape the first wave of genome-wide demethylation after fertilization. In conclusion, human male germ cells present a unique germline-specific ageing process, which likely results in diminished fecundity in elderly men and poorer health prognosis for their offspring.

Molecular Aging Markers in Patients with Klinefelter Syndrome.

Molecular aging markers provide the opportunity for biological age determination in humans and to study factors, such as genetic determinants, affecting the ageing process. In males with Klinefelter syndrome (KS, non-mosaic karyotype 47, XXY), which is the most common sex chromosome aneuploidy, age-related morbidity and mortality are increased, and a significantly reduced life span has been observed. The aim of this study was to investigate whether Klinefelter patients exhibit molecular signs of premature ageing. We studied, specifically, age-associated DNA methylation patterns (by pyrosequencing) and relative telomere length (TL; by quantitative polymerase chain reaction) in blood in a cohort of Klinefelter patients (n=178 and 266 for DNA methylation and TL, respectively) aged 18-71 years and compared them to the data of age-matched healthy male (n = 184 and 196 for DNA methylation and TL, respectively) and female controls (n = 50). Age-associated DNA methylation patterns were not indicative of accelerated ageing in Klinefelter men. Significantly longer telomeres were found in the young Klinefelter subjects aged 18-24 years (mean=1.51 vs. 1.09 and 1.26 in female and male controls, respectively). However, telomere length in subsequent age groups showed no difference to controls. Gonosomal aneuploidy in Klinefelter syndrome is associated with higher baseline TL at adolescent age, but comparable TL with progressive age in other age groups.

The sperm epigenome does not display recurrent epimutations in patients with severely impaired spermatogenesis.

BACKGROUND: In the past 15 years, numerous studies have described aberrant DNA methylation of imprinted genes (e.g. MEST and H19) in sperm of oligozoospermic men, but the prevalence and genomic extent of abnormal methylation patterns have remained unknown. RESULTS: Using deep bisulfite sequencing (DBS), we screened swim-up sperm samples from 40 normozoospermic and 93 patients diagnosed as oligoasthenoteratozoospermic, oligoteratozoospermic or oligozoospermic, which are termed OATs throughout the manuscript, for H19 and MEST methylation. Based on this screening, we defined three patient groups: normal controls (NC), abnormally methylated oligozoospermic (AMO; n = 7) and normally methylated oligozoospermic (NMO; n = 86). Whole-genome bisulfite sequencing (WGBS) of five NC and five AMO samples revealed abnormal methylation levels of all 50 imprinting control regions in each AMO sample. To investigate whether this finding reflected epigenetic germline mosaicism or the presence of residual somatic DNA, we made a genome-wide inventory of soma-germ cell-specific DNA methylation. We found that > 2000 germ cell-specific genes are promoter-methylated in blood and that AMO samples had abnormal methylation levels at these genes, consistent with the presence of somatic cell DNA. The comparison between the five NC and six NMO samples revealed 19 differentially methylated regions (DMRs), none of which could be validated in an independent cohort of 40 men. Previous studies reported a higher incidence of epimutations at single CpG sites in the CTCF-binding region 6 of H19 in infertile patients. DBS analysis of this locus, however, revealed an association between DNA methylation levels and genotype (rs2071094), but not fertility phenotype. CONCLUSIONS: Our results suggest that somatic DNA contamination and genetic variation confound methylation studies in sperm of infertile men. While we cannot exclude the existence of rare patients with slightly abnormal sperm methylation at non-recurrent CpG sites, the prevalence of aberrant methylation in swim-up purified sperm of infertile men has likely been overestimated, which is reassuring for patients undergoing assisted reproduction.

wg-blimp: an end-to-end analysis pipeline for whole genome bisulfite sequencing data.

BACKGROUND: Analysing whole genome bisulfite sequencing datasets is a data-intensive task that requires comprehensive and reproducible workflows to generate valid results. While many algorithms have been developed for tasks such as alignment, comprehensive end-to-end pipelines are still sparse. Furthermore, previous pipelines lack features or show technical deficiencies, thus impeding analyses. RESULTS: We developed wg-blimp (whole genome bisulfite sequencing methylation analysis pipeline) as an end-to-end pipeline to ease whole genome bisulfite sequencing data analysis. It integrates established algorithms for alignment, quality control, methylation calling, detection of differentially methylated regions, and methylome segmentation, requiring only a reference genome and raw sequencing data as input. Comparing wg-blimp to previous end-to-end pipelines reveals similar setups for common sequence processing tasks, but shows differences for post-alignment analyses. We improve on previous pipelines by providing a more comprehensive analysis workflow as well as an interactive user interface. To demonstrate wg-blimp's ability to produce correct results we used it to call differentially methylated regions for two publicly available datasets. We were able to replicate 112 of 114 previously published regions, and found results to be consistent with previous findings. We further applied wg-blimp to a publicly available sample of embryonic stem cells to showcase methylome segmentation. As expected, unmethylated regions were in close proximity of transcription start sites. Segmentation results were consistent with previous analyses, despite different reference genomes and sequencing techniques. CONCLUSIONS: wg-blimp provides a comprehensive analysis pipeline for whole genome bisulfite sequencing data as well as a user interface for simplified result inspection. We demonstrated its applicability by analysing multiple publicly available datasets. Thus, wg-blimp is a relevant alternative to previous analysis pipelines and may facilitate future epigenetic research.

High-resolution analysis of germ cells from men with sex chromosomal aneuploidies reveals normal transcriptome but impaired imprinting.

BACKGROUND: The most common sex chromosomal aneuploidy in males is Klinefelter syndrome, which is characterized by at least one supernumerary X chromosome. While these men have long been considered infertile, focal spermatogenesis can be observed in some patients, and sperm can be surgically retrieved and used for artificial reproductive techniques. Although these gametes can be used for fertility treatments, little is known about the molecular biology of the germline in Klinefelter men. Specifically, it is unclear if germ cells in Klinefelter syndrome correctly establish the androgenetic DNA methylation profile and transcriptome. This is due to the low number of germ cells in the Klinefelter testes available for analysis. RESULTS: Here, we overcame these difficulties and successfully investigated the epigenetic and transcriptional profiles of germ cells in Klinefelter patients employing deep bisulfite sequencing and single-cell RNA sequencing. On the transcriptional level, the germ cells from Klinefelter men clustered together with the differentiation stages of normal spermatogenesis. Klinefelter germ cells showed a normal DNA methylation profile of selected germ cell-specific markers compared with spermatogonia and sperm from men with normal spermatogenesis. However, germ cells from Klinefelter patients showed variations in the DNA methylation of imprinted regions. CONCLUSIONS: These data indicate that Klinefelter germ cells have a normal transcriptome but might present aberrant imprinting, showing impairment in germ cell development that goes beyond mere germ cell loss.

De novo methylation in male germ cells of the common marmoset monkey occurs during postnatal development and is maintained in vitro.

The timing of de novo DNA methylation in male germ cells during human testicular development is yet unsolved. Apart from that, the stability of established imprinting patterns in vitro is controversially discussed. This study aimed at determining the timing of DNA de novo methylation and at assessing the stability of the methylation status in vitro. We employed the marmoset monkey (Callithrix jacchus) as it is considered the best non-human primate model for human testicular development. We selected neonatal, pre-pubertal, pubertal, and adult animals (n = 3, each) and assessed germ cell global DNA methylation levels by 5-methyl cytosine staining, and Alu elements and gene-specific methylation (H19, LIT1, SNRPN, MEST, OCT4, MAGE-A4, and DDX-4) by pyrosequencing. De novo methylation is progressively established during postnatal primate development and continues until adulthood, a process that is different in most other species. Importantly, once established, methylation patterns remained stable, as demonstrated using in vitro cultures. Thus, the marmoset monkey is a unique model for the study of postnatal DNA methylation mechanisms in germ cells and for the identification of epimutations and their causes.

On the origin of sperm epigenetic heterogeneity.

The influence of epigenetic modifications on reproduction and on the function of male germ cells has been thoroughly demonstrated. In particular, aberrant DNA methylation levels in sperm have been associated with abnormal sperm parameters, lower fertilization rates and impaired embryo development. Recent reports have indicated that human sperm might be epigenetically heterogeneous and that abnormal DNA methylation levels found in the sperm of infertile men could be due to the presence of sperm populations with different epigenetic quality. However, the origin and the contribution of different germ cell types to this suspected heterogeneity remain unclear. In this review, we focus on sperm epigenetics at the DNA methylation level and its importance in reproduction. We take into account the latest developments and hypotheses concerning the functional significance of epigenetic heterogeneity coming from the field of stem cell and cancer biology and discuss the potential importance and consequences of sperm epigenetic heterogeneity for reproduction, male (in)fertility and assisted reproductive technologies (ART). Based on the current information, we propose a model in which spermatogonial stem cell variability, either intrinsic or due to external factors (such as endocrine action and environmental stimuli), can lead to epigenetic sperm heterogeneity, sperm epimutations and male infertility. The elucidation of the precise causes for epimutations, the conception of adequate therapeutic options and the development of sperm selection technologies based on epigenetic quality should be regarded as crucial to the improvement of ART outcome in the near future.

The human RHOX gene cluster: target genes and functional analysis of gene variants in infertile men.

The X-linked reproductive homeobox (RHOX) gene cluster encodes transcription factors preferentially expressed in reproductive tissues. This gene cluster has important roles in male fertility based on phenotypic defects of Rhox-mutant mice and the finding that aberrant RHOX promoter methylation is strongly associated with abnormal human sperm parameters. However, little is known about the molecular mechanism of RHOX function in humans. Using gene expression profiling, we identified genes regulated by members of the human RHOX gene cluster. Some genes were uniquely regulated by RHOXF1 or RHOXF2/2B, while others were regulated by both of these transcription factors. Several of these regulated genes encode proteins involved in processes relevant to spermatogenesis; e.g. stress protection and cell survival. One of the target genes of RHOXF2/2B is RHOXF1, suggesting cross-regulation to enhance transcriptional responses. The potential role of RHOX in human infertility was addressed by sequencing all RHOX exons in a group of 250 patients with severe oligozoospermia. This revealed two mutations in RHOXF1 (c.515G > A and c.522C > T) and four in RHOXF2/2B (-73C > G, c.202G > A, c.411C > T and c.679G > A), of which only one (c.202G > A) was found in a control group of men with normal sperm concentration. Functional analysis demonstrated that c.202G > A and c.679G > A significantly impaired the ability of RHOXF2/2B to regulate downstream genes. Molecular modelling suggested that these mutations alter RHOXF2/F2B protein conformation. By combining clinical data with in vitro functional analysis, we demonstrate how the X-linked RHOX gene cluster may function in normal human spermatogenesis and we provide evidence that it is impaired in human male fertility.

Epigenetic germline mosaicism in infertile men.

Imprinted genes are expressed either from the paternal or the maternal allele, because the other allele has been silenced in the mother's or father's germline. Imprints are characterized by DNA methylation at cytosine phosphate guanine sites. Recently, abnormal sperm parameters and male infertility have been linked to aberrant methylation patterns of imprinted genes in sperm DNA. However, these studies did not account for possible epigenetic heterogeneity in sperm. We have investigated whether spermatozoa are a homogeneous cell population regarding DNA methylation of imprinted genes. Swim-up sperm was obtained from 45 men with normal (n = 19) and abnormal (n = 26) sperm parameters. DNA methylation of the imprinted gene KCNQ1OT1 was measured in multiple pools of 10 spermatozoa by a highly sensitive pyrosequencing-based oligo-sperm methylation assay (OSMA). DNA methylation of four imprinted genes (KCNQ1OT1, MEST, H19 and MEG3) was further analysed by deep bisulfite sequencing, which allows analysis at the single-cell level. Using OSMA, we found a significantly increased variation in the DNA methylation values of the maternally methylated gene KCNQ1OT1 in samples with abnormal sperm parameters. DBS showed that normozoospermic samples had a homogenous pattern of DNA methylation, whereas oligoasthenozoospermic samples contained discrete populations of spermatozoa with either normal or abnormal methylation patterns. Aberrant methylation of H19 appears to occur preferentially on the maternally inherited allele. Our results demonstrate the presence of epigenetic mosaicism in the semen of oligoasthenozoospermic men, which probably results from errors in imprint erasure.