Abstract
Azoospermia, the complete absence of sperm in the ejaculate, presents a major barrier to male fertility. Oxidative stress and impaired cellular homeostasis are key contributors to germ cell loss, particularly in chemotherapy-induced azoospermia. Sulforaphane (SFN), a potent activator of the Nrf2 pathway, offers antioxidant benefits, but its systemic delivery is limited by bioavailability and potential reductive stress. This study aimed to evaluate the regenerative potential of SFN-loaded exosomes (SFN + EXO) in a rat model of azoospermia. Human serum-derived exosomes were isolated, characterized and engineered to encapsulate SFN. Azoospermia was induced in Wistar rats via intratesticular busulfan injection. Animals were assigned to five groups: healthy control, azoospermic control, SFN, exosomes (EXO) and SFN + EXO. Spermatogenesis parameters, histopathology, testosterone levels, oxidative stress markers and gene expression of Nrf2, autophagy and germ cell markers were evaluated. SFN + EXO treatment significantly improved sperm count, motility, morphology and testis weight index compared to controls and monotherapy groups. Histological recovery of spermatogenic lineages was superior in SFN + EXO rats, accompanied by reduced fibrosis and normalized testicular architecture. Expression of DAZL and VASA was fully restored, while aberrant upregulation of Nrf2 and autophagy genes (LC3, Beclin1, p62) in azoospermic testes was normalized only by SFN + EXO. Antioxidant enzyme activity (GPx, TAC) was significantly enhanced, suggesting redox balance recovery. Local delivery of sulforaphane via exosomes effectively reverses chemotherapy-induced spermatogenic failure through modulation of oxidative stress and autophagy, promoting germ cell regeneration. This exosome-based platform offers a promising therapeutic avenue for male infertility.
Data availability
No datasets were generated or analysed during the current study.
Abbreviations
- SFN:
-
Sulforaphane
- EXO:
-
Exosome
- SFN+EXO:
-
Sulforaphane-loaded exosome
- Nrf2:
-
Nuclear factor erythroid 2-related factor 2
- ROS:
-
Reactive oxygen species
- SOD:
-
Superoxide Dismutase
- GPx:
-
Glutathione Peroxidase
- MDA:
-
Malondialdehyde
- TAC:
-
Total antioxidant capacity
- SSCs:
-
Spermatogonial stem cells
- SPCs:
-
Spermatocytes
- SPTs:
-
Elongated spermatids
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Acknowledgements
The authors would like to thank the Stem Cell Research Center and Core Research Laboratory, Tabriz University of Medical Sciences, for supporting this work. The graphical abstract was created with BioRender (www.biorender.com). The authors used an AI-assisted tool solely for language editing, grammar checking and writing style enhancement. No AI-generated content, data, or images were used in the preparation of this manuscript and the authors take full responsibility for the final text.
Funding
This study was supported by grants from Tabriz University of Medical Sciences and the Stem Cell Research Center (Grant Numbers: 70877 and 71978). It was also conducted as part of the Ph.D. research project of Shahin Ahmadian’s dissertation titled “Studying the regenerative effects of engineered exosomes loaded with Nrf2 activator in the rat model of azoospermia”. The project received ethical approval on February 13, 2023, from the Research Ethics Committee for Laboratory Animals at Tabriz University of Medical Sciences (Ethical Code: IR.TBZMED.AEC.1401.084).
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S.A. performed all experiments, data analysis, graphical abstract design, data visualization and wrote the initial draft of the manuscript. M.F. participated in animal husbandry, treatments and H&E sperm counting. R.R performed pathological studies on H&E and Masson’s trichrome slides. B.R, A.A., M.T. and R.R reviewed and revised the initial draft of the manuscript. M.M. designed and conceptualized the project and the manuscript.
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Ethics approval and consent to participate
All experimental protocols, under the Ph.D. project of Shahin Ahmadian title “Study of the restorative effects of engineered exosomes containing Nrf2 activator in a rat model with azoospermia,” were approved by the Animal Ethics Committee of Tabriz University of Medical Sciences (approval number IR.TBZMED.AEC.1401.084; approval date: February 13, 2023). All animal procedures were performed in accordance with the ARRIVE 2.0 guidelines, relevant institutional regulations and national guidelines for the care and use of laboratory animals. The human serum samples utilized in this project were remnants from routine screenings at the Iranian Blood Transfusion Organization. These samples were delivered to the laboratory in a completely anonymous manner, with no personal identifiers provided to the research team before the exosome extraction process. The ethics committee waived the requirement for informed consent for the use of these anonymous residual samples.
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Ahmadian, S., Fajri, M., Roelen, B.A. et al. Targeted activation of Nrf2 via sulforaphane-loaded exosomes attenuated azoospermic condition in the rat model. Sci Rep (2026). https://doi.org/10.1038/s41598-026-40709-x
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DOI: https://doi.org/10.1038/s41598-026-40709-x
