Epigenetic germline mosaicism in infertile men

Abstract

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.

Interindividual Variation in DNA Methylation at a Putative POMC Metastable Epiallele Is Associated with Obesity

“…The samples were part of the MesyBepo follow-up study (Bobbert et al, 2013 Gromoll). DNA was extracted as described previously (Laurentino et al, 2015).…”

On the origin of sperm epigenetic heterogeneity

“…Nevertheless, when carefully re-analysing the methylation patterns obtained from previous studies that used cloning and sequencing for the analysis of DNA methylation in sperm, it becomes clear that some data already indicated the presence of epigenetic heterogeneity/mosaicism in sperm. The patterns shown by several of the clones sequenced (Flanagan et al 2006, Kobayashi et al 2007, Marques et al 2008, Hammoud et al 2010, Marques et al 2010, Wu et al 2010, Minor et al 2011, Sato et al 2011, Ankolkar et al 2012, Montjean et al 2013) resemble at a small scale the binomial (sequences fully methylated vs fully unmethylated) distribution of sequencing patterns obtained using deep-bisulphite sequencing (DBS; Laurentino et al 2015). Due to the requirement for a high number of individual sequences analysed from each single sample, the detection and measurement of epigenetic heterogeneity in sperm as well as in other tissues only became feasible now, with the advent of high-resolution analysis methodologies such as nextgeneration sequencing/DBS (Mikeska et al 2010, Meaburn & Schulz 2012, Beygo et al 2013, Gries et al 2013, Smallwood et al 2014, Putnik et al 2015.…”

“…It is noteworthy that a high and constant fraction of epimutated sperm cells could be repeatedly detected for all analysed genes in each sample from a cohort of infertile men presenting with oligoasthenoteratozoospermic and abnormal average MEST DNA methylation (Laurentino et al 2015). This pattern is reminiscent, and in accordance with, the spermatogenic system present in human testes, in which a large number of progenitor cells (type A pale spermatogonia), originating from the same original small pool of PGCs, give rise to a relatively small number of sperm (16 from each cell that enters differentiation; ).…”

“…Due to the requirement for a high number of individual sequences analysed from each single sample, the detection and measurement of epigenetic heterogeneity in sperm as well as in other tissues only became feasible now, with the advent of high-resolution analysis methodologies such as nextgeneration sequencing/DBS (Mikeska et al 2010, Meaburn & Schulz 2012, Beygo et al 2013, Gries et al 2013, Smallwood et al 2014, Putnik et al 2015. The use of novel and highly sensitive DNA isolation and pyrosequencing-based sequencing techniques, such as OSMA (oligo-sperm methylation assay; Laurentino et al 2015) and limiting dilution (LD; Kuhtz et al 2014), allowed the detection of DNA methylation heterogeneity in the sperm of infertile men. This heterogeneity could be further confirmed using DBS, which enabled the undoubted detection of epigenetically distinct sperm populations in the semen of a group of infertile men (Fig.…”

“…However, epimutations in sperm and the possible underlying heterogeneity have not been studied mostly due to technology-based limitations. Only recently, new technologies enabled the study of DNA methylation in single sperm, spermatogonia and gonocytes to address potential epigenetic heterogeneity among and within these cell types (Jenkins et al 2014, Kuhtz et al 2014, Laurentino et al 2015.…”