Di Persio et al. Clin Epigenet (2021) 13:160 https://doi.org/10.1186/s13148-021-01144-z RESEARCH Open Access Whole-genome methylation analysis of testicular germ cells from cryptozoospermic men points to recurrent and functionally relevant DNA methylation changes Sara Di Persio1†, Elsa Leitão2†, Marius Wöste3, Tobias Tekath3, Jann‑Frederik Cremers4, Martin Dugas3, Xiaolin Li5, Gerd Meyer zu Hörste5, Sabine Kliesch4, Sandra Laurentino1, Nina Neuhaus1*† and Bernhard Horsthemke2,6† Abstract Background: Several studies have reported an association between male infertility and aberrant sperm DNA meth‑ ylation patterns, in particular in imprinted genes. In a recent investigation based on whole methylome and deep bisulfte 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 fnd 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 diference in DNA methylation at the whole genome level or at imprinted regions between CZ and CTR samples. However, using stringent flters to identify group‑specifc methylation diferences, we detected 271 diferentially 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 diferentially 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‑associ‑ ated genes, which are mainly expressed during= meiosis× and spermiogenesis, show a signifcantly diferent 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‑ specifc enhancers may be afected. 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 refect or contribute to premature abortion of spermatogenesis and therefore not appear in the mature, motile sperm. *Correspondence: [email protected] †Sara Di Persio and Elsa Leitão are shared frst authors †Nina Neuhaus and Bernhard Horsthemke are shared last authors 1 Centre of Reproductive Medicine and Andrology, University Hospital of Münster, 48149 Münster, Germany Full list of author information is available at the end of the article © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Di Persio et al. Clin Epigenet (2021) 13:160 Page 2 of 19 Keywords: Cryptozoospermia, Testicular germ cells, DNA methylation, Diferentially methylated regions, Single cell RNA sequencing, Spermatogenesis, Male infertility Background cells (ESCs). WGBS has also been performed on PGCs Life-long production of sperm through the process of and advanced germ cells (AGCs) of human embryos spermatogenesis is supported by spermatogonia, the highlighting, among other features, the importance of most undiferentiated adult germ cell type. Te pool of DNA methylation at transposons [5]. With regard to the spermatogonia ensures male fertility, as these cells have adult germline, Hammoud et al. [21] have studied murine the potential to self-renew as well as to give rise to dif- spermatogonia but genome-wide analysis of DNA meth- ferentiating daughter cells. Spermatogonia originate ylation in testicular germ cells (TGCs) of adult men and, from primordial germ cells (PGCs), which are specifed in particular, its comparison to TGCs from patients with very early during embryonic development. Tese PGCs impaired spermatogenesis constitutes a research gap. undergo erasure of DNA methylation, which allows the Publications of high-quality single-cell RNA sequenc- establishment of sperm-specifc DNA methylation pro- ing (scRNA-seq) transcriptomes obtained from human fles during later stages of gametogenesis [1]. testicular tissues with intact spermatogenesis [22–25] Erasure of DNA methylation takes place in two sequen- have greatly advanced our knowledge with regard to the tial stages. During the initial stage, a global decrease in transcriptional changes associated with human germ methylated cytosines occurs, whereas in the second stage cell diferentiation. Moreover, comparative analyses of methylation is removed from imprinting control regions scRNA-seq results from men with intact and severely and meiotic genes [2–4]. In human male PGCs, the epi- impaired spermatogenesis have provided insight into genetic ground state of global methylation levels has been the molecular mechanisms associated with failure of found in foetuses between 7 and 11 weeks of age [5–7]. germ cell diferentiation [25, 26]. In order to assess the Currently, it is not known when the de novo DNA meth- presence of aberrant epigenetic patterns in TGCs and ylation rise commences in human fetal germ cells, [8] but the potential association with aberrant transcriptional importantly, this process of de novo global methylation profles, we combined the two powerful approaches of continues until well after birth in primates. [9] WGBS data with scRNA-seq in samples with intact and A number of publications have reported that male impaired spermatogenesis. infertility is associated with aberrant sperm DNA meth- ylation profles, particularly in imprinted genes [10–15]. Results However, we found no recurrent epimutations by deep Isolation and characterization of human testicular germ bisulfte sequencing analysis of sperm from patients with cells severely impaired spermatogenesis (n = 93) and controls To analyse DNA methylation diferences between human (n = 40), combined with whole genome bisulfte sequenc- testicular germ cells (TGCs) in normal and impaired ing (WGBS) of selected samples [16]. Tis study, which spermatogenesis, we isolated germ cells from patients is one of the largest in this feld, rather revealed that with obstructive azoospermia (CTR, n = 24) and cryp- the presence of residual somatic DNA in swim-up puri- tozoospermia (CZ, n = 10) (Fig. 1a, Additional fle 1: fed sperm samples and genetic variation are major con- Table S1). By deep bisulfte sequencing (DBS) of H19, founders of methylation studies in sperm. In line with the MEST, DDX4 and XIST, we identifed 21 CTR (87.5%) recommendations made by Åsenius et al. [17], these two and fve CZ (50%) samples with pure germ cell fractions, confounders need to be considered in prospective stud- of which we selected four from each group for whole ies to clarify if there is indeed an increased prevalence of genome bisulfte sequencing (WGBS) (Table 1, Fig. 1a, aberrant methylation in infertile men. Additional fle 1: Tables S2 and S3, Additional fle 2: Fig. Only few studies have used whole genome bisulfte S1). Tese samples were found to have normal meth- sequencing (WGBS) to investigate DNA methylation of ylation values in the four regions, consistent with the human spermatozoa at base pair resolution [16, 18–20]. absence of somatic DNA, and no signifcant diference Te main fndings of the methylation studies were that was found between the two groups (Fig. 1b, Additional there is no signifcant methylation at non-CpG sites, that fle 1: Table S3). there are large regions of low methylation in a manner Ploidy analyses of the selected samples showed an independent of genomic features such as CpG islands enrichment of haploid cells in the CTR samples com- (CGIs) and promoters, and that CGI shores in sperm are pared to the CZ samples in the initial single-cell sus- more shallow compared to CGI shores in embryonal stem pension (Fig. 1c). Histological analysis of the testicular Di Persio et al. Clin Epigenet (2021) 13:160 Page 3 of 19 A 1N 2N 1N 2N 4N Single cell RNA 4N Normal control sequencing (CTR, n=24) Histological (Di Persio et al., 2021 Ploidy analysis analysis Ploidy analysis bioRxiv) Supernatant Deep bisulfite fraction (SN) sequencing (DBS) Whole genome Cryptozoospermic bisulfite sequencing (CZ, n=10) (WGBS, n=4 per group) Testicular Single cell Culture Attached biopsy suspension fraction (AT) Day 0Day 1Day 3-4 B C D E Most advanced cell type Fig. 1 Testicular germ cell samples selection for whole genome bisulfte sequencing. a Schematic representation of the experimental design. b Box plot showing the methylation values of H19, MEST, DDX4 and XIST measured using deep bisulfte sequencing (DBS) of the supernatant fraction at day 3–4 of culture for the normal controls (CTR, teal, n 4) and the cryptozoospermic (CZ, purple, n 4) samples. No signifcant diference = = was found in the methylation values of the four genes between the two groups.