{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T23:58:34Z","timestamp":1788393514581,"version":"build-2803163510"},"reference-count":44,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2020,5,4]],"date-time":"2020-05-04T00:00:00Z","timestamp":1588550400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003407","name":"Ministero dell\u2019Istruzione, dell\u2019Universit\u00e0 e della Ricerca","doi-asserted-by":"publisher","award":["PRIN2017"],"award-info":[{"award-number":["PRIN2017"]}],"id":[{"id":"10.13039\/501100003407","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["JCM"],"abstract":"<jats:p>Sperm DNA fragmentation (sDF) negatively affects reproduction and is traditionally detected in total sperm population including viable and non-viable spermatozoa. Here, we aimed at exploring the ability of DNA fragmentation to discriminate fertile and subfertile men when detected in viable (viable sDF), non-viable (non-viable sDF), and total spermatozoa (total sDF). We revealed sDF in 91 male partners of infertile couples and 71 fertile men (max 1 year from natural conception) with LiveTUNEL coupled to flow cytometry, able to reveal simultaneously DNA fragmentation and cell viability. We found that the three sDF parameters discriminated fertile and subfertile men with similar accuracy and independently from age and basal semen parameters: AUCs (area under the curves) (95% CI) were: 0.696 (0.615\u20130.776), p &lt; 0.001 for total sDF; 0.718 (0.640\u20130.797), p &lt; 0.001 for viable sDF; 0.760 (0.685\u20130.835), p &lt; 0.001 for non-viable sDF. We also found that total and non-viable but not viable sDF significantly correlated to age and semen quality. In conclusion, the three sDF parameters similarly discriminated fertile and subfertile men. Viable spermatozoa with DNA fragmentation are likely cells able to fertilize the oocyte but failing to properly support subsequent embryo development. Non-viable sDF could be a sign of a subtler damage extended beyond the non-viable cells.<\/jats:p>","DOI":"10.3390\/jcm9051341","type":"journal-article","created":{"date-parts":[[2020,5,5]],"date-time":"2020-05-05T06:41:20Z","timestamp":1588660880000},"page":"1341","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["DNA Fragmentation in Viable and Non-Viable Spermatozoa Discriminates Fertile and Subfertile Subjects with Similar Accuracy"],"prefix":"10.3390","volume":"9","author":[{"given":"Monica","family":"Muratori","sequence":"first","affiliation":[{"name":"Department of Experimental and Clinical Biomedical Sciences \u201cMario Serio\u201d, Center of Excellence DeNothe, University of Florence, 50139 Florence, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giulia","family":"Pellegrino","sequence":"additional","affiliation":[{"name":"Department of Experimental and Clinical Biomedical Sciences \u201cMario Serio\u201d, Center of Excellence DeNothe, University of Florence, 50139 Florence, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giusi","family":"Mangone","sequence":"additional","affiliation":[{"name":"Pediatric Section, Department of Health Sciences, University of Florence and Anna Meyer Children\u2019s University Hospital, 50139 Florence, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chiara","family":"Azzari","sequence":"additional","affiliation":[{"name":"Pediatric Section, Department of Health Sciences, University of Florence and Anna Meyer Children\u2019s University Hospital, 50139 Florence, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francesco","family":"Lotti","sequence":"additional","affiliation":[{"name":"Department of Experimental and Clinical Biomedical Sciences \u201cMario Serio\u201d, Center of Excellence DeNothe, University of Florence, 50139 Florence, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicoletta","family":"Tarozzi","sequence":"additional","affiliation":[{"name":"9.baby, Family and Fertility Center, 40125 Bologna, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Luca","family":"Boni","sequence":"additional","affiliation":[{"name":"Clinical Trials Center, AOU Careggi, 50139 Florence, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9049-0020","authenticated-orcid":false,"given":"Andrea","family":"Borini","sequence":"additional","affiliation":[{"name":"9.baby, Family and Fertility Center, 40125 Bologna, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mario","family":"Maggi","sequence":"additional","affiliation":[{"name":"Department of Experimental and Clinical Biomedical Sciences \u201cMario Serio\u201d, Center of Excellence DeNothe, University of Florence, 50139 Florence, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elisabetta","family":"Baldi","sequence":"additional","affiliation":[{"name":"Department of Experimental and Clinical Medicine, Center of Excellence DeNothe, University of Florence, 50139 Florence, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2020,5,4]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"646","DOI":"10.1093\/humupd\/dmx022","article-title":"Temporal trends in sperm count: A systematic review and meta-regression analysis","volume":"23","author":"Levine","year":"2017","journal-title":"Hum. Reprod. Update"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1152\/physrev.00017.2015","article-title":"Male Reproductive Disorders and Fertility Trends: Influences of Environment and Genetic Susceptibility","volume":"96","author":"Skakkebaek","year":"2016","journal-title":"Physiol. Rev."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"82","DOI":"10.1186\/s12940-017-0291-8","article-title":"Does air pollution play a role in infertility? A systematic review","volume":"16","author":"Gatimel","year":"2017","journal-title":"Environ. Health"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1186\/s12958-018-0436-9","article-title":"Lifestyle and fertility: The influence of stress and quality of life on male fertility","volume":"16","author":"Ilacqua","year":"2018","journal-title":"Reprod. Biol. Endocrinol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"56","DOI":"10.1007\/s11934-018-0805-0","article-title":"The Role of Lifestyle in Male Infertility: Diet, Physical Activity, and Body Habitus","volume":"19","author":"Hayden","year":"2018","journal-title":"Curr. Urol. Rep."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1007\/978-3-030-21664-1_5","article-title":"Sperm DNA Fragmentation: Mechanisms of Origin","volume":"1166","author":"Muratori","year":"2019","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"109","DOI":"10.2119\/molmed.2014.00158","article-title":"Investigation on the Origin of Sperm DNA Fragmentation: Role of Apoptosis, Immaturity and Oxidative Stress","volume":"21","author":"Muratori","year":"2015","journal-title":"Mol. Med."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1039","DOI":"10.1093\/humrep\/14.4.1039","article-title":"Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic","volume":"14","author":"Evenson","year":"1999","journal-title":"Hum. Reprod."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"e221","DOI":"10.1111\/j.1365-2605.2009.00995.x","article-title":"Sperm chromatin structure assay as an independent predictor of fertility in vivo: A case-control study","volume":"33","author":"Giwercman","year":"2010","journal-title":"Int. J. Androl."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"582","DOI":"10.1016\/j.fertnstert.2015.06.005","article-title":"DNA fragmentation in brighter sperm predicts male fertility independently from age and semen parameters","volume":"104","author":"Muratori","year":"2015","journal-title":"Fertil. Steril."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2876","DOI":"10.1093\/humrep\/del251","article-title":"Sperm DNA fragmentation: Paternal effect on early post-implantation embryo development in ART","volume":"21","author":"Borini","year":"2006","journal-title":"Hum. Reprod."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"2908","DOI":"10.1093\/humrep\/des261","article-title":"The effect of sperm DNA fragmentation on miscarriage rates: A systematic review and meta-analysis","volume":"27","author":"Robinson","year":"2012","journal-title":"Hum. Reprod."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"951","DOI":"10.1016\/j.rbmo.2018.12.029","article-title":"Association between sperm DNA fragmentation and idiopathic recurrent pregnancy loss: A systematic review and meta-analysis","volume":"38","author":"Tan","year":"2019","journal-title":"Reprod. Biomed. Online"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Cairo Consensus Workshop Group (2020). The current status and future of andrology: A consensus report from the Cairo workshop group. Andrology, 8, 27\u201352.","DOI":"10.1111\/andr.12720"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2","DOI":"10.1111\/j.1365-2605.2009.01042.x","article-title":"The TUNEL assay consistently underestimates DNA damage in human spermatozoa and is influenced by DNA compaction and cell vitality: Development of an improved methodology","volume":"34","author":"Mitchell","year":"2011","journal-title":"Int. J. Androl."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1007\/978-3-030-21664-1_7","article-title":"Oxidative Damage to Sperm DNA: Attack and Defense","volume":"1166","author":"Drevet","year":"2019","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_17","first-page":"300","article-title":"Male Infertility: The Effect of Natural Antioxidants and Phytocompounds on Seminal Oxidative Stress","volume":"25","author":"Adewoyin","year":"2012","journal-title":"Reprod. Biomed. Online"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"118","DOI":"10.1186\/s12958-018-0431-1","article-title":"Radiations and male fertility","volume":"16","author":"Kesari","year":"2018","journal-title":"Reprod. Biol. Endocrinol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"145","DOI":"10.2164\/jandrol.111.013193","article-title":"Diabetes mellitus and sperm parameters","volume":"33","author":"Condorelli","year":"2012","journal-title":"J. Androl."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"461","DOI":"10.1016\/j.urology.2008.07.053","article-title":"Role of oxidative stress in pathogenesis of varicocele and infertility","volume":"73","author":"Agarwal","year":"2009","journal-title":"Urology"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"22","DOI":"10.1016\/j.maturitas.2014.02.011","article-title":"How to overcome male infertility after 40: Influence of paternal age on fertility","volume":"78","author":"Belloc","year":"2014","journal-title":"Maturitas"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"176","DOI":"10.2164\/jandrol.04152","article-title":"DNA damage in bovine sperm does not block fertilization and early embryonic development but induces apoptosis after the first cleavages","volume":"27","author":"Fatehi","year":"2006","journal-title":"J. Androl."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"483","DOI":"10.1016\/j.fertnstert.2019.04.029","article-title":"Sperm DNA fragmentation is correlated with poor embryo development, lower implantation rate, and higher miscarriage rate in reproductive cycles of non-male factor infertility","volume":"112","author":"Borges","year":"2019","journal-title":"Fertil. Steril."},{"key":"ref_24","first-page":"343","article-title":"Effect of sperm DNA fragmentation on embryo development: Clinical and biological aspects","volume":"21","author":"Bilinski","year":"2017","journal-title":"JBRA Assist. Reprod."},{"key":"ref_25","unstructured":"World Health Organization (2010). Laboratory Manual for the Examination and Processing of Human Semen, WHO Press. [5th ed.]."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"7492","DOI":"10.1038\/s41598-019-43981-2","article-title":"Sperm selection with density gradient centrifugation and swim up: Effect on DNA fragmentation in viable spermatozoa","volume":"9","author":"Muratori","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"336","DOI":"10.2164\/jandrol.109.008508","article-title":"Small variations in crucial steps of TUNEL assay coupled to flow cytometry greatly affect measures of sperm DNA fragmentation","volume":"31","author":"Muratori","year":"2010","journal-title":"J. Androl."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1035","DOI":"10.1093\/humrep\/den058","article-title":"Nuclear staining identifies two populations of human sperm with different DNA fragmentation extent and relationship with semen parameters","volume":"23","author":"Muratori","year":"2008","journal-title":"Hum. Reprod."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"621","DOI":"10.1093\/molehr\/gam046","article-title":"Characterization of M540 bodies in human semen: Evidence that they are apoptotic bodies","volume":"13","author":"Marchiani","year":"2007","journal-title":"Mol. Hum. Reprod."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"2415","DOI":"10.1093\/humrep\/deq214","article-title":"Analysis of the relationships between oxidative stress, DNA damage and sperm vitality in a patient population: Development of diagnostic criteria","volume":"25","author":"Aitken","year":"2010","journal-title":"Hum. Reprod."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"729","DOI":"10.1093\/humrep\/deq370","article-title":"M540 bodies interfere with TUNEL analyses in human semen samples","volume":"26","author":"Muratori","year":"2011","journal-title":"Hum. Reprod."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"e13126","DOI":"10.1111\/and.13126","article-title":"Role of oxidative stress, infection and inflammation in male infertility","volume":"50","author":"Agarwal","year":"2018","journal-title":"Andrologia"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1016\/j.juro.2015.07.089","article-title":"Characterization of Nuclease Activity in Human Seminal Plasma and its relationship to Semen Parameters, Sperm DNA Fragmentation and Male Infertility","volume":"195","author":"Abad","year":"2016","journal-title":"J. Urol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1381","DOI":"10.1007\/s11255-018-1915-9","article-title":"Dynamic assessment of human sperm DNA damage I: The effect of seminal plasma-sperm co-incubation after ejaculation","volume":"50","author":"Arroyo","year":"2018","journal-title":"Int. Urol. Nephrol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"253","DOI":"10.1002\/j.1939-4640.2003.tb02670.x","article-title":"Spontaneous DNA fragmentation in swim-up selected human spermatozoa during long term incubation","volume":"24","author":"Muratori","year":"2003","journal-title":"J. Androl."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"301","DOI":"10.1016\/j.fertnstert.2016.12.018","article-title":"Effects of age on fertility and sexual function","volume":"107","author":"Eisenberg","year":"2017","journal-title":"Fertil. Steril."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1002\/j.1939-4640.2002.tb02599.x","article-title":"Sperm Chromatin Structure Assay: Its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques","volume":"23","author":"Evenson","year":"2002","journal-title":"J. Androl."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"475","DOI":"10.1093\/molehr\/gat025","article-title":"The source and significance of DNA damage in human spermatozoa; a commentary on diagnostic strategies and straw man fallacies","volume":"19","author":"Aitken","year":"2013","journal-title":"Mol. Hum. Reprod."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Ribeiro, S.C., Muratori, M., De Geyter, M., and De Geyter, C. (2017). TUNEL labeling with BrdUTP\/anti-BrdUTP greatly underestimates the level of sperm DNA fragmentation in semen evaluation. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0181802"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"581","DOI":"10.1111\/j.1365-2605.2010.01118.x","article-title":"Sumo1-ylation of human spermatozoa and its relationship with semen quality","volume":"34","author":"Marchiani","year":"2011","journal-title":"Int. J. Androl."},{"key":"ref_41","first-page":"587","article-title":"Apoptosis in human ejaculated sperm cells (notulae seminologicae 9)","volume":"28","author":"Baccetti","year":"1996","journal-title":"J. Submicrosc. Cytol. Pathol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"3446","DOI":"10.1093\/humrep\/dei231","article-title":"Sperm DNA fragmentation: Threshold value in male fertility","volume":"20","author":"Sergerie","year":"2005","journal-title":"Hum. Reprod."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"715","DOI":"10.1111\/j.2047-2927.2013.00111.x","article-title":"Comprehensive analysis of sperm DNA fragmentation by five different assays: TUNEL assay, SCSA, SCD test and alkaline and neutral Comet assay","volume":"1","author":"Abad","year":"2013","journal-title":"Andrology"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"325","DOI":"10.1016\/j.rbmo.2013.06.014","article-title":"The impact of sperm DNA damage in assisted conception and beyond: Recent advances in diagnosis and treatment","volume":"27","author":"Lewis","year":"2013","journal-title":"Reprod. Biomed. Online"}],"container-title":["Journal of Clinical Medicine"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2077-0383\/9\/5\/1341\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,13]],"date-time":"2025-10-13T13:32:37Z","timestamp":1760362357000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2077-0383\/9\/5\/1341"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,5,4]]},"references-count":44,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2020,5]]}},"alternative-id":["jcm9051341"],"URL":"https:\/\/doi.org\/10.3390\/jcm9051341","relation":{},"ISSN":["2077-0383"],"issn-type":[{"value":"2077-0383","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,5,4]]}}}