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

Abstract

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.

Epimutations in human sperm from patients with impaired spermatogenesis

“…Unfortunately, Marques et al have not considered genetic variation as another confounder in this type of studies. As shown by us, a common genetic variant at the H19 locus is associated with DNA hypomethylation, which should not be confused with an infertilityassociated epimutation [4].…”

“…We noticed that in the study by Leitão and collaborators [4], density gradient separation was not performed and direct swim-up was employed, thereby increasing the likelihood of having somatic cells contamination in their sperm samples, as is suggestive from the author´s WGBS data, including for H19 methylation values (ranging from 75 to 82% methylation in normal controls in their study vs 95% in our study [2]. For density gradient centrifugation, we used Puresperm gradients (Nidacon, Gothenburg, Sweden) which contain silane-coated silica particles that enable motile sperm to be separated from non-germinal cells and seminal plasma.…”

“…Most of the previous studies were performed on swim-up samples, and we have found that samples prepared in this way can be contaminated with somatic DNA. Of course, we could not reanalyze the samples that were used by others, but our lack of finding any evidence for an epimutation in 93 sperm samples, which make our study one of the largest in its field, justifies our conclusion that "the prevalence of aberrant methylation in swim-up purified sperm of infertile men has likely been overestimated" [4]. Irrespective of the purification procedure used, the DNA should always be prescreened for somatic DNA contamination by bisulfite sequencing of at least the four genes suggested in our paper (H19, MEST, DDX4, and XIST) or the four genomic loci suggested by Jenkins et al [8].…”

“…Epimutations in human sperm from patients with impaired spermatogenesis Joana Marques 1,2* , Filipa Carvalho 1,2 , Alberto Barros 1,2,3 and Mário Sousa 3,4 In this Letter to the Editor, we comment on the recent publication by Leitão et al The sperm epigenome does not display recurrent epimutations in patients with severely impaired spermatogenesis (Clinical Epigenetics 2020, DOI: 10.1186/s13148-020-00854-0), where concerns about the validity of our studies reporting imprinting errors in human sperm from infertile patients have been raised.…”

No evidence for intervention-associated DNA methylation changes in monocytes of patients with posttraumatic stress disorder or anorexia nervosa

“…The patients for the WGBS were selected based on their responsiveness to the therapy, absence of co-morbidity, and non-smoking status (Supplementary Table 2, Additional File 1). For PTSD samples, WGBS libraries were prepared as previously described [29]. For the AN samples, 5 µg DNA at 50 ng/µl in water was used for library preparation and NGS sequencing on a HiSeq X platform (DKFZ Genomics & Proteomics Core Facility (GPCF) with 1 sample per lane.…”