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This study evaluated the protective effects of L-proline against heat stress-induced damage during sperm preparation incubation, a critical step in assisted reproductive technologies (ART). Semen samples from 30 normozoospermic men were divided into two groups: a control group and a treatment group supplemented with 2 mmol\/L L-proline. Following initial incubation at 37\u00a0\u00b0C for 30\u00a0min, samples were further subdivided and subjected to controlled thermal exposures at 25\u00a0\u00b0C, 37\u00a0\u00b0C, and 42\u00a0\u00b0C for both short-term (3\u00a0h) and long-term (24\u00a0h) incubation periods, simulating physiological and heat-stress conditions. Sperm parameters, including motility, viability, morphology, and biochemical markers of oxidative stress, including catalase (CAT), superoxide dismutase (SOD), total antioxidant capacity (TAC), malondialdehyde (MDA), and nitric oxide (NO), were comprehensively assessed. Results indicated that heat stress exposure significantly impaired sperm function and disrupted redox homeostasis. Supplementation with 2 mmol\/L L-proline significantly preserved sperm motility and viability during heat stress conditions compared to the control group (\n                    <jats:italic>p<\/jats:italic>\n                    \u2009&lt;\u20090.05). However, no significant protective effect on sperm morphology was observed. Additionally, L-proline enhanced antioxidant defenses by improving CAT, SOD, and TAC levels while concurrently modulating MDA and NO levels under heat stress conditions (\n                    <jats:italic>p<\/jats:italic>\n                    \u2009&lt;\u20090.05). In conclusion, these findings highlight the potential of L-proline as a protective supplement to ameliorate the detrimental effects of heat stress on human sperm quality and oxidative status, suggesting that incorporating L-proline into sperm preparation protocols may offer a promising strategy to enhance clinical outcomes.\n                  <\/jats:p>","DOI":"10.1007\/s00726-026-03524-2","type":"journal-article","created":{"date-parts":[[2026,6,12]],"date-time":"2026-06-12T03:54:48Z","timestamp":1781236488000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["L-Proline protects human sperm from heat stress-induced oxidative damage"],"prefix":"10.1007","volume":"58","author":[{"given":"Mojtaba","family":"Moradi","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mohsen","family":"Shayestehyekta","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amir Hossein","family":"Hashemian","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Azita","family":"Faramarzi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,6,12]]},"reference":[{"key":"3524_CR1","doi-asserted-by":"crossref","unstructured":"Abdelnour SA, Khalil WA, Khalifa NE, Khalil FMA, Hassan MA (2024) L-proline: A promising tool for boosting cryotolerance and fertilizing ability of cryopreserved sperm in animals. 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