{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T23:24:56Z","timestamp":1790378696621,"version":"4.1.0"},"reference-count":45,"publisher":"MDPI AG","issue":"21","license":[{"start":{"date-parts":[[2025,11,1]],"date-time":"2025-11-01T00:00:00Z","timestamp":1761955200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Both absolute and presumably active rDNA (with a hypomethylated promoter region) copy number (CN) in the haploid human sperm genome are highly variable among individuals. Using a combination of droplet digital PCR and deep bisulfite sequencing, we have quantified absolute and presumably active rDNA CN in sperm samples (N = 190) with normal (NSPs) vs. abnormal semen parameters (ASPs), as well as in samples leading or not leading to a clinical pregnancy. ASP samples had a significantly lower presumably active CN (104 \u00b1 31) than normozoospermic samples (115 \u00b1 31). The loss of presumably active rDNA copies is explained by an increased promoter methylation (13.9% in ASP vs. 12.1% in NSP). When correcting for confounding factors, most importantly semen quality, samples not leading to a clinical pregnancy after IVF\/ICSI displayed a significantly lower absolute (225 \u00b1 51) and presumably active CN (103 \u00b1 30) than samples with pregnancy (249 \u00b1 62 and 115 \u00b1 31, respectively). This between-group difference was most noticeable in normozoospermic males: absolute CN 220 \u00b1 54; presumably active CN 107 \u00b1 32 in samples without pregnancy and absolute CN 246 \u00b1 63; presumably active CN 120 \u00b1 28 in samples with pregnancy. We propose that absolute\/active rDNA CN in sperm is a modulating factor contributing to idiopathic male infertility. In NSP samples, presumably active CN increases with absolute CN, which may have a positive impact on fertility and ART outcome. Our results suggest that approximately 60 active sperm rDNA copies are sufficient to establish a pregnancy.<\/jats:p>","DOI":"10.3390\/ijms262110657","type":"journal-article","created":{"date-parts":[[2025,11,3]],"date-time":"2025-11-03T13:15:14Z","timestamp":1762175714000},"page":"10657","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Sperm rDNA Copy Number and Methylation Are Associated with Male-Factor Infertility"],"prefix":"10.3390","volume":"26","author":[{"given":"Alina","family":"Michler","sequence":"first","affiliation":[{"name":"Institute of Human Genetics, Julius Maximilians University, 97074 W\u00fcrzburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sarah","family":"Kie\u00dfling","sequence":"additional","affiliation":[{"name":"Institute of Human Genetics, Julius Maximilians University, 97074 W\u00fcrzburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jana","family":"Durackova","sequence":"additional","affiliation":[{"name":"Institute of Human Genetics, Julius Maximilians University, 97074 W\u00fcrzburg, Germany"},{"name":"Institute of Clinical Genetics and Genomic Medicine, University Hospital, 97080 W\u00fcrzburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas","family":"Hahn","sequence":"additional","affiliation":[{"name":"Fertility Center, 65189 Wiesbaden, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Martin","family":"Schorsch","sequence":"additional","affiliation":[{"name":"Fertility Center, 65189 Wiesbaden, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0737-0763","authenticated-orcid":false,"given":"Thomas","family":"Haaf","sequence":"additional","affiliation":[{"name":"Institute of Human Genetics, Julius Maximilians University, 97074 W\u00fcrzburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,11,1]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1506","DOI":"10.1093\/humrep\/dem046","article-title":"International estimates of infertility prevalence and treatment-seeking: Potential need and demand for infertility medical care","volume":"22","author":"Boivin","year":"2007","journal-title":"Hum. Reprod."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Mascarenhas, M.N., Flaxman, S.R., Boerma, T., Vanderpoel, S., and Stevens, G.A. (2012). National, regional, and global trends in infertility prevalence since 1990: A systematic analysis of 277 health surveys. PLoS Med., 9.","DOI":"10.1371\/journal.pmed.1001356"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1011","DOI":"10.1111\/andr.13728","article-title":"How exome sequencing improves the diagnostics and management of men with non-syndromic infertility","volume":"13","author":"Stallmeyer","year":"2025","journal-title":"Andrology"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"473","DOI":"10.1038\/nature08162","article-title":"Distinctive chromatin in human sperm packages genes for embryo development","volume":"460","author":"Hammoud","year":"2009","journal-title":"Nature"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"e14200","DOI":"10.1111\/acel.14200","article-title":"Age-related and species-specific methylation changes in the protein-coding marmoset sperm epigenome","volume":"23","author":"Dittrich","year":"2024","journal-title":"Aging Cell"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"60","DOI":"10.1159\/000323806","article-title":"Methylation status of imprinted genes and repetitive elements in sperm DNA from infertile males","volume":"5","author":"Zechner","year":"2011","journal-title":"Sex. Dev."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"642","DOI":"10.1111\/j.1365-2605.2009.01000.x","article-title":"Idiopathic male infertility is strongly associated with aberrant methylation of MEST and IGF2\/H19 ICR1","volume":"33","author":"Poplinski","year":"2010","journal-title":"Int. J. Androl."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"129","DOI":"10.1016\/j.fertnstert.2010.06.076","article-title":"Assessing loss of imprint methylation in sperm from subfertile men using novel methylation polymerase chain reaction Luminex analysis","volume":"95","author":"Sato","year":"2011","journal-title":"Fertil. Steril."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"e13242","DOI":"10.1111\/acel.13242","article-title":"A germ cell-specific ageing pattern in otherwise healthy men","volume":"19","author":"Laurentino","year":"2020","journal-title":"Aging Cell"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Oluwayiose, O.A., Wu, H., Saddiki, H., Whitcomb, B.W., Balzer, L.B., Brandon, N., Suvorov, A., Tayyab, R., Sites, C.K., and Hill, L. (2021). Sperm DNA methylation mediates the association of male age on reproductive outcomes among couples undergoing infertility treatment. Sci. Rep., 11.","DOI":"10.1038\/s41598-020-80857-2"},{"key":"ref_11","first-page":"1257","article-title":"Age-related methylation changes in the human sperm epigenome","volume":"15","author":"Bernhardt","year":"2023","journal-title":"Aging"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"966","DOI":"10.1111\/acel.12603","article-title":"Methylation of the ribosomal RNA gene promoter is associated with aging and age-related decline","volume":"16","author":"Montesanto","year":"2017","journal-title":"Aging Cell"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"e14497","DOI":"10.1111\/acel.14497","article-title":"rDNA copy number variation and methylation during normal and premature aging","volume":"24","author":"Geisen","year":"2025","journal-title":"Aging Cell"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"e13181","DOI":"10.1111\/acel.13181","article-title":"Increasing methylation of sperm rDNA and other repetitive elements in the aging male mammalian germline","volume":"19","author":"Potabattula","year":"2020","journal-title":"Aging Cell"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1214","DOI":"10.18632\/aging.203891","article-title":"Ribosomal DNA methylation in human and mouse oocytes increases with age","volume":"14","author":"Potabattula","year":"2022","journal-title":"Aging"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Potabattula, R., Dittrich, M., Hahn, T., Schorsch, M., Ptak, G.E., and Haaf, T. (2025). rDNA copy number variation and methylation in human and mouse sperm. Int. J. Mol. Sci., 26.","DOI":"10.3390\/ijms26094197"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1007\/s10577-018-9587-y","article-title":"Dosage effects of human ribosomal genes (rDNA) in health and disease","volume":"27","author":"Porokhovnik","year":"2019","journal-title":"Chromosome Res."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"305","DOI":"10.1016\/j.schres.2018.01.001","article-title":"Abundance of ribosomal RNA gene copies in the genomes of schizophrenia patients","volume":"197","author":"Chestkov","year":"2018","journal-title":"Schizophr. Res."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"500623","DOI":"10.1038\/s41467-024-49397-5","article-title":"Ribosomal DNA copy number is associated with body mass in humans and other mammals","volume":"15","author":"Law","year":"2024","journal-title":"Nat. Comm."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"100562","DOI":"10.1016\/j.xgen.2024.100562","article-title":"Ribosomal DNA copy number variation associates with hematological profiles and renal function in the UK Biobank","volume":"4","author":"Evans","year":"2024","journal-title":"Cell Genom."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"719","DOI":"10.1016\/S1097-2765(01)00317-3","article-title":"Molecular mechanisms mediating methylation-dependent silencing of ribosomal gene transcription","volume":"8","author":"Santoro","year":"2001","journal-title":"Mol. Cell"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"325","DOI":"10.1101\/gr.241745.118","article-title":"Ribosomal DNA harbors an evolutionarily conserved clock of biological aging","volume":"29","author":"Wang","year":"2019","journal-title":"Genome Res."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1520","DOI":"10.1016\/S0015-0282(03)00366-2","article-title":"Effect of male age on fertility: Evidence for the decline in male fertility with increasing age","volume":"79","author":"Hassan","year":"2003","journal-title":"Fertil. Steril."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2523","DOI":"10.1093\/humrep\/dez223","article-title":"Male ageing is negatively associated with the chance of live birth in IVF\/ICSI cycles for idiopathic infertility","volume":"34","author":"Horta","year":"2019","journal-title":"Hum. Reprod."},{"key":"ref_25","first-page":"449","article-title":"On the chromosomal distribution of DNA complementary to ribosomal and soluble RNA","volume":"23","author":"Ritossa","year":"1966","journal-title":"Natl. Cancer Inst. Monogr."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"509","DOI":"10.1073\/pnas.60.2.509","article-title":"Unstable redundancy of genes for ribosomal RNA","volume":"60","author":"Ritossa","year":"1968","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"211","DOI":"10.1093\/oxfordjournals.jhered.a111437","article-title":"Effects of rRNA gene copy number and nucleolar variation on early development: Inhibition of gastrulation in rDNA-deficient chick embryos","volume":"85","author":"Delany","year":"1994","journal-title":"J. Hered."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1748","DOI":"10.1093\/ps\/77.12.1748","article-title":"Ribosomal RNA gene copy number and nucleolar-size polymorphisms within and among chicken lines selected for enhanced growth","volume":"77","author":"Su","year":"1998","journal-title":"Poult. Sci."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1016\/S0925-4773(02)00181-8","article-title":"Epigenetic reprogramming in mouse primordial germ cells","volume":"117","author":"Hajkova","year":"2002","journal-title":"Mech. Dev."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1354","DOI":"10.4161\/epi.6.11.17993","article-title":"DNA methylation imprinting marks and DNA methyltransferase expression in human spermatogenic cell stages","volume":"6","author":"Marques","year":"2011","journal-title":"Epigenetics"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Jenkins, T.G., Aston, K.I., Pflueger, C., Cairns, B.R., and Carrell, D.T. (2014). Age-associated sperm DNA methylation alterations: Possible implications in offspring disease susceptibility. PLoS Genet., 10.","DOI":"10.1371\/journal.pgen.1004458"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"339","DOI":"10.1038\/nature10960","article-title":"A unique regulatory phase of DNA methylation in the early mammalian embryo","volume":"484","author":"Smith","year":"2012","journal-title":"Nature"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1034","DOI":"10.1101\/gr.201541.115","article-title":"Sperm is epigenetically programmed to regulate gene transcription in embryos","volume":"26","author":"Teperek","year":"2016","journal-title":"Genome Res."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"365","DOI":"10.1016\/j.tcb.2022.08.008","article-title":"The initiation of mammalian embryonic transcription: To begin at the beginning","volume":"33","author":"Perry","year":"2023","journal-title":"Trends Cell Biol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"201","DOI":"10.1111\/j.1768-322X.1991.tb03016.x","article-title":"Nucleolar changes in human embryo during the pre-implantation stage. Activation of ribosomal genes during the nucleologenesis","volume":"72","author":"Grillo","year":"1991","journal-title":"Biol. Cell"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"459","DOI":"10.1038\/332459a0","article-title":"Human gene expression first occurs between the four- and eight-cell stages of preimplantation development","volume":"332","author":"Braude","year":"1988","journal-title":"Nature"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"jcs258798","DOI":"10.1242\/jcs.258798","article-title":"Transcription of rRNA in early mouse embryos promotes chromatin reorganization and expression of major satellite repeats","volume":"135","author":"Chebrout","year":"2022","journal-title":"J. Cell Sci."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"457","DOI":"10.1038\/ng1990","article-title":"Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome","volume":"39","author":"Weber","year":"2007","journal-title":"Nat. Genet."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"545","DOI":"10.1128\/MMBR.00005-16","article-title":"The epigenetic pathways to ribosomal DNA silencing","volume":"80","author":"Srivastava","year":"2016","journal-title":"Microbiol. Mol. Biol. Rev."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1558","DOI":"10.1128\/MCB.23.5.1558-1568.2003","article-title":"In exponentially growing Saccharomyces cerevisiae cells, rRNA synthesis is determined by the summed RNA polymerase I loading rate rather than by the number of active genes","volume":"23","author":"French","year":"2003","journal-title":"Mol. Cell. Biol."},{"key":"ref_41","unstructured":"World Health Organization (2025, September 22). WHO Laboratory Manual for the Examination and Processing of Human Semen, Available online: https:\/\/apps.who.int\/iris\/handle\/10665\/44261."},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Xu, B., Li, H., Perry, J.M., Singh, V.P., Unruh, J., Yu, Z., Zakari, M., McDowell, W., Li, L., and Gerton, J.L. (2017). Ribosomal DNA copy number loss and sequence variation in cancer. PLoS Genet., 13.","DOI":"10.1371\/journal.pgen.1006771"},{"key":"ref_43","first-page":"1511","article-title":"rDNA copy number variation and methylation from birth to sexual maturity","volume":"17","author":"Michler","year":"2025","journal-title":"Aging"},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Babaian, A. (2017). Intra- and inter-individual variation in human ribosomal RNAs. bioRxiv.","DOI":"10.1101\/118760"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"351","DOI":"10.1007\/978-1-4939-7774-1_19","article-title":"Locus-specific DNA methylation analysis by targeted deep bisulfite sequencing","volume":"1767","author":"Leitao","year":"2018","journal-title":"Meth. Mol. Biol."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/21\/10657\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,11,3]],"date-time":"2025-11-03T13:31:28Z","timestamp":1762176688000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/21\/10657"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,11,1]]},"references-count":45,"journal-issue":{"issue":"21","published-online":{"date-parts":[[2025,11]]}},"alternative-id":["ijms262110657"],"URL":"https:\/\/doi.org\/10.3390\/ijms262110657","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,11,1]]}}}