{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,6]],"date-time":"2026-01-06T09:11:33Z","timestamp":1767690693070,"version":"3.48.0"},"reference-count":42,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2026,1,6]],"date-time":"2026-01-06T00:00:00Z","timestamp":1767657600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Tunisian Ministry of Higher Education"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["JCM"],"abstract":"<jats:p>Background\/Objectives: Male infertility contributes to nearly half of global infertility cases, yet conventional semen analysis is insufficient to predict assisted reproductive technology (ART) outcomes such as intracytoplasmic sperm injection (ICSI). Sperm DNA fragmentation (SDF) is a promising biomarker of genomic integrity, but clinical implementation remains hindered by methodological heterogeneity. This study compared two SDF assays\u2014TUNEL and Acridine Orange (AO)\u2014regarding their correlations with semen parameters and ICSI outcomes. Methods: Sixty men undergoing ICSI were prospectively enrolled. SDF was analyzed using two flow cytometric assays: TUNEL (detecting DNA strand breaks) and AO (assessing chromatin instability). Semen quality and ICSI outcomes (fertilization, cleavage, blastulation, and embryo utilization rates) were evaluated. Statistical analyses included Spearman\u2019s rank correlation and Mann\u2013Whitney tests. Results: Median SDF levels were significantly higher by TUNEL than AO (17.2% vs. 10.15%; p = 0.0065). Inter-assay correlation was weak (r = 0.299, p = 0.01). AO-derived SDF correlated positively with age (r = 0.311, p = 0.02), while TUNEL showed no such trend. Neither assay correlated significantly with semen parameters or ICSI outcomes, although AO tended to associate with lower motility and slightly reduced top-quality embryo rates. Conclusions: TUNEL and AO capture distinct facets of sperm DNA damage. Their limited correlation and lack of predictive value for ICSI outcomes highlight the need for assay-specific interpretation and standardization. Integration of SDF with additional biomarkers and oocyte factors may enhance its clinical utility.<\/jats:p>","DOI":"10.3390\/jcm15020403","type":"journal-article","created":{"date-parts":[[2026,1,6]],"date-time":"2026-01-06T08:42:58Z","timestamp":1767688978000},"page":"403","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Flow Cytometric Assessment of Sperm DNA Fragmentation by TUNEL and Acridine Orange: Methodological and Clinical Insights"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0984-6083","authenticated-orcid":false,"given":"Mohamed","family":"Abdelkarim","sequence":"first","affiliation":[{"name":"Human Genetics Laboratory, Faculty of Medicine of Tunis, Tunis El Manar University, Tunis 1006, Tunisia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nadine","family":"Ghannem","sequence":"additional","affiliation":[{"name":"Human Genetics Laboratory, Faculty of Medicine of Tunis, Tunis El Manar University, Tunis 1006, Tunisia"},{"name":"Department of Reproductive Biology and Cytogenetics, Aziza Othmana Hospital, Tunis El Manar University, Tunis 1008, Tunisia"},{"name":"Research Laboratory LR16SP01 \u201cInfertility and Oncofertility\u201d, Tunis 1006, Tunisia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Khadija","family":"Kacem-Berjeb","sequence":"additional","affiliation":[{"name":"Department of Reproductive Biology and Cytogenetics, Aziza Othmana Hospital, Tunis El Manar University, Tunis 1008, Tunisia"},{"name":"Research Laboratory LR16SP01 \u201cInfertility and Oncofertility\u201d, Tunis 1006, Tunisia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7572-2495","authenticated-orcid":false,"given":"Sana","family":"Chtourou","sequence":"additional","affiliation":[{"name":"Department of Reproductive Biology and Cytogenetics, Aziza Othmana Hospital, Tunis El Manar University, Tunis 1008, Tunisia"},{"name":"Research Laboratory LR16SP01 \u201cInfertility and Oncofertility\u201d, Tunis 1006, Tunisia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Linda","family":"Debbabi","sequence":"additional","affiliation":[{"name":"Department of Reproductive Biology and Cytogenetics, Aziza Othmana Hospital, Tunis El Manar University, Tunis 1008, Tunisia"},{"name":"Research Laboratory LR16SP01 \u201cInfertility and Oncofertility\u201d, Tunis 1006, Tunisia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anis","family":"Fadhlaoui","sequence":"additional","affiliation":[{"name":"Research Laboratory LR16SP01 \u201cInfertility and Oncofertility\u201d, Tunis 1006, Tunisia"},{"name":"Gynecology and Obstetrics Department, Aziza Othmana Hospital, Tunis 1008, Tunisia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mounir","family":"Ben Mefeteh","sequence":"additional","affiliation":[{"name":"Research Laboratory LR16SP01 \u201cInfertility and Oncofertility\u201d, Tunis 1006, Tunisia"},{"name":"Gynecology and Obstetrics Department, Aziza Othmana Hospital, Tunis 1008, Tunisia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fethi","family":"Zhioua","sequence":"additional","affiliation":[{"name":"Research Laboratory LR16SP01 \u201cInfertility and Oncofertility\u201d, Tunis 1006, Tunisia"},{"name":"Gynecology and Obstetrics Department, Aziza Othmana Hospital, Tunis 1008, Tunisia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8861-3471","authenticated-orcid":false,"given":"Marouen","family":"Braham","sequence":"additional","affiliation":[{"name":"Research Laboratory LR16SP01 \u201cInfertility and Oncofertility\u201d, Tunis 1006, Tunisia"},{"name":"Gynecology and Obstetrics Department, Aziza Othmana Hospital, Tunis 1008, Tunisia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7118-6023","authenticated-orcid":false,"given":"Nozha","family":"Chakroun","sequence":"additional","affiliation":[{"name":"Department of Reproductive Biology and Cytogenetics, Aziza Othmana Hospital, Tunis El Manar University, Tunis 1008, Tunisia"},{"name":"Research Laboratory LR16SP01 \u201cInfertility and Oncofertility\u201d, Tunis 1006, Tunisia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,1,6]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"hoad007","DOI":"10.1093\/hropen\/hoad007","article-title":"Financial Costs of Assisted Reproductive Technology for Patients in Low- and Middle-Income Countries: A Systematic Review","volume":"2023","author":"Njagi","year":"2023","journal-title":"Hum. 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