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Objective: Injection of methylene blue to testis has been shown to have adverse effect in animal studies but it is still being used frequently as lymphatic mapping agent during lymphatic sparing varicocelectomy in adolescent varicoceles. We aim to report postoperative ultrasound changes after subaortic injection of methylene blue in human testes., Study Design: A retrospective observational study of consecutive patients under 18 years old undergoing laparoscopic varicocelectomy from August 2017 to August 2021 was performed. Demographics such as age, symptoms, pre-operative testicular volume was collected. Primary outcome was change on testicular ultrasound at 3, 6, 12 months after the operation. Secondary outcome was testicular volume difference between affected and unaffected testes, and growth rate of affected testis at 1 year after the operation., Results: Fifty-eight patients were included, with median age of 15 years old (IQR 11-18 years). Thirty-one patients had non-lymphatic sparing varicocelectomy (Group A) and 27 patients had lymphatic sparing varicocelectomy (Group B). There was no statistical difference between the mean testicular volume difference between the two groups, but hydrocele rate was significantly higher in Group A (16% vs 0%, p = 0.03). In group B, 6 patients (22.2%, p = 0.005) developed testicular change detectable by ultrasound. No statistical difference could be demonstrated for median testicular size difference (between affected and unaffected testes) at 1 year between group with and without ultrasound change (-23% vs 0%, p = 0.36). Median follow up time was 20.4 months (IQR 5-32 months)., Discussion: Varicocele treatments improve testicular volume and increase total sperm concentration and lymphatic sparing surgery significantly decreased post-operative hydrocele rates [2]. Different agents have been used to delineate lymphatic vessels and one commonly used agent is methylene blue [10,15-16]. However it has been shown in other animal studies that intraparenchymal injection of methylene blue to rat testis result in degenerative changes [18]. Our study is the first to describe post-operative changes of human testes on imaging after lymphatic sparing varicocelectomy with subdartoic injection of methylene blue. Six patients (22.2%) demonstrated new onset testicular changes on post-operative ultrasound. Although no statistically significant testicular volume reduction was seen in the group with change on imaging, these observations may serve as a surrogate marker for testicular injury or reduced testicular function. It will be desirable for future studies if we can investigate it further with hormonal markers or semen analysis after puberty has been reached., Conclusion: Lymphatic sparing procedure reduced post-operative hydrocele in adolescent varicocele, however long-lasting effect on testis is evident with subdartoic injection of methylene blue. This agent must be used with caution and long term follow up of these patients are needed., (Copyright © 2022 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.)