A microplate-based DCFH-DA assay for the evaluation of oxidative stress in whole semen
Aims: The well-documented relationship between sperm oxidation and male infertility strongly encourages the development of assays for reactive oxygen species detection in semen samples. The present study aims to apply the microplate-based 2’,7’-dichlorofluorescein diacetate assay to the evaluation o...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2022-09-01
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| Series: | Heliyon |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2405844022019302 |
| _version_ | 1811180883216957440 |
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| author | Serena Benedetti Simona Catalani Silvia De Stefani Mariangela Primiterra Alessandra Fraternale Francesco Palma Simone Palini |
| author_facet | Serena Benedetti Simona Catalani Silvia De Stefani Mariangela Primiterra Alessandra Fraternale Francesco Palma Simone Palini |
| author_sort | Serena Benedetti |
| collection | DOAJ |
| description | Aims: The well-documented relationship between sperm oxidation and male infertility strongly encourages the development of assays for reactive oxygen species detection in semen samples. The present study aims to apply the microplate-based 2’,7’-dichlorofluorescein diacetate assay to the evaluation of oxidative stress in unprocessed whole semen, thus avoiding sample centrifugations and other manipulations that may cause significant reactive oxygen species increments. Main methods: The fluorescence assay consisted in the quantification of both intracellular and extracellular reactive oxygen species levels in unwashed semen specimens by using the probe 2’,7’-dichlorofluorescein diacetate into a 96-well plate. The method was useful for the preliminary assessment of the oxidation levels of whole semen samples from men undergoing standard sperm analysis as well as to evaluate the effect of some pro-glutathione molecules on semen oxidative status. Key findings: The 2’,7’-dichlorofluorescein diacetate assay was successfully adapted to the evaluation of oxidative stress in whole semen, effectively revealing the perturbation of the redox homeostasis of the sample. Accordingly, specimens with abnormal sperm parameters (n = 10) presented oxidation indexes significantly higher than those with normospermia (n = 10) [7729 (range 3407–12769) vs. 1356 (range 470–2711), p < 0.001]; in addition, semen oxidation indexes negatively correlated to sperm motility and morphology. Noteworthy, whole semen exposure to pro-glutathione compounds led to reduced semen oxidation levels and sperm protection against oxidative damage. Significance: Based on our pilot experimental data, the microplate-based 2’,7’-dichlorofluorescein diacetate assay appears to be a convenient method for the detection of reactive oxygen species levels in whole semen samples, avoiding artifacts due to semen centrifugation steps. At the same time, the test could be a helpful tool for the basic and quick screening of antioxidant molecules able to preserve semen quality. |
| first_indexed | 2024-04-11T09:10:16Z |
| format | Article |
| id | doaj.art-4232fca29c4a4590af99829a97097bb6 |
| institution | Directory Open Access Journal |
| issn | 2405-8440 |
| language | English |
| last_indexed | 2024-04-11T09:10:16Z |
| publishDate | 2022-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Heliyon |
| spelling | doaj.art-4232fca29c4a4590af99829a97097bb62022-12-22T04:32:31ZengElsevierHeliyon2405-84402022-09-0189e10642A microplate-based DCFH-DA assay for the evaluation of oxidative stress in whole semenSerena Benedetti0Simona Catalani1Silvia De Stefani2Mariangela Primiterra3Alessandra Fraternale4Francesco Palma5Simone Palini6Department of Biomolecular Sciences, University of Urbino, Via Saffi 2, Urbino, Italy; Corresponding author.Department of Biomolecular Sciences, University of Urbino, Via Saffi 2, Urbino, ItalyClinica Nuova Ricerca, Viale Luigi Settembrini 17/H, Rimini, ItalyClinica Nuova Ricerca, Viale Luigi Settembrini 17/H, Rimini, ItalyDepartment of Biomolecular Sciences, University of Urbino, Via Saffi 2, Urbino, ItalyDepartment of Biomolecular Sciences, University of Urbino, Via Saffi 2, Urbino, ItalyPhysiopathology of Reproduction Unit, Cervesi Hospital, Via Ludwig Van Beethoven 1, Cattolica, ItalyAims: The well-documented relationship between sperm oxidation and male infertility strongly encourages the development of assays for reactive oxygen species detection in semen samples. The present study aims to apply the microplate-based 2’,7’-dichlorofluorescein diacetate assay to the evaluation of oxidative stress in unprocessed whole semen, thus avoiding sample centrifugations and other manipulations that may cause significant reactive oxygen species increments. Main methods: The fluorescence assay consisted in the quantification of both intracellular and extracellular reactive oxygen species levels in unwashed semen specimens by using the probe 2’,7’-dichlorofluorescein diacetate into a 96-well plate. The method was useful for the preliminary assessment of the oxidation levels of whole semen samples from men undergoing standard sperm analysis as well as to evaluate the effect of some pro-glutathione molecules on semen oxidative status. Key findings: The 2’,7’-dichlorofluorescein diacetate assay was successfully adapted to the evaluation of oxidative stress in whole semen, effectively revealing the perturbation of the redox homeostasis of the sample. Accordingly, specimens with abnormal sperm parameters (n = 10) presented oxidation indexes significantly higher than those with normospermia (n = 10) [7729 (range 3407–12769) vs. 1356 (range 470–2711), p < 0.001]; in addition, semen oxidation indexes negatively correlated to sperm motility and morphology. Noteworthy, whole semen exposure to pro-glutathione compounds led to reduced semen oxidation levels and sperm protection against oxidative damage. Significance: Based on our pilot experimental data, the microplate-based 2’,7’-dichlorofluorescein diacetate assay appears to be a convenient method for the detection of reactive oxygen species levels in whole semen samples, avoiding artifacts due to semen centrifugation steps. At the same time, the test could be a helpful tool for the basic and quick screening of antioxidant molecules able to preserve semen quality.http://www.sciencedirect.com/science/article/pii/S24058440220193022’,7’-dichlorofluorescein diacetateMale infertilityMicroplate fluorescence readerOxidative stressPro-glutathione moleculesReactive oxygen species |
| spellingShingle | Serena Benedetti Simona Catalani Silvia De Stefani Mariangela Primiterra Alessandra Fraternale Francesco Palma Simone Palini A microplate-based DCFH-DA assay for the evaluation of oxidative stress in whole semen Heliyon 2’,7’-dichlorofluorescein diacetate Male infertility Microplate fluorescence reader Oxidative stress Pro-glutathione molecules Reactive oxygen species |
| title | A microplate-based DCFH-DA assay for the evaluation of oxidative stress in whole semen |
| title_full | A microplate-based DCFH-DA assay for the evaluation of oxidative stress in whole semen |
| title_fullStr | A microplate-based DCFH-DA assay for the evaluation of oxidative stress in whole semen |
| title_full_unstemmed | A microplate-based DCFH-DA assay for the evaluation of oxidative stress in whole semen |
| title_short | A microplate-based DCFH-DA assay for the evaluation of oxidative stress in whole semen |
| title_sort | microplate based dcfh da assay for the evaluation of oxidative stress in whole semen |
| topic | 2’,7’-dichlorofluorescein diacetate Male infertility Microplate fluorescence reader Oxidative stress Pro-glutathione molecules Reactive oxygen species |
| url | http://www.sciencedirect.com/science/article/pii/S2405844022019302 |
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