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STUDY QUESTION Does sperm DNA recover from damage in all men after 2 years from the end of cytotoxic treatments? SUMMARY ANSWER The current indication of 2 years waiting time for seeking natural pregnancy after cytotoxic treatment may not be adequate for all men, since severe sperm DNA damage is present in a proportion of subjects even after this timeframe. WHAT IS KNOWN ALREADY Data in the literature on sperm DNA fragmentation (SDF) in lymphoma patients after cytotoxic treatments are scarce. The largest longitudinal study evaluated paired pre- and post-therapy (up to 24 months) semen samples from 34 patients while one study performed a longer follow-up (36 months) in 10 patients. The median/mean SDF values >24 months after therapy did not show significant differences but the studies did not explore the proportion of patients with severe DNA damage and the analysis was done on frozen-thawed samples. STUDY DESIGN, SIZE, DURATION In this study, 53 Hodgkin lymphoma (HL) and 25 non-Hodgkin lymphoma (NHL) post-pubertal patients were included over a recruitment period of 10 years (2012–2022). Among them, 18 subjects provided paired semen samples for SDF analysis at the three time points. SDF was evaluated in patients before (T0) and after 2 (T2) and 3 years (T3) from the end of, cytotoxic treatments (chemotherapy alone or in combination with radiotherapy). A cohort of 79 healthy, fertile, and normozoospermic men >18 years old served as controls (recruited between 2016 and 2019). PARTICIPANTS/MATERIALS, SETTING, METHODS SDF was evaluated on fresh semen samples (i.e. spermatozoa potentially involved in natural conception) from patients and controls using TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay coupled with flow cytometry. SDF median values were compared between groups: (i) HL and NHL patients versus controls at the three time points; (ii) HL versus NHL patients at baseline; and (iii) patients at T0 versus T2 and T3. Severe DNA damage (SDD) was defined for SDF levels above the 95th percentile of controls (50%) and the proportion of patients with SDD at all time points was established. MAIN RESULTS AND THE ROLE OF CHANCE At T0, patients displayed higher median SDF than controls, reaching statistical significance in the NHL group: 40.5% [IQR: 31.3–52.6%] versus 28% [IQR: 22–38%], P  < 0.05. Comparing SDF pre-treatment to that post-treatment, HL patients exhibited similar median values at the three time points, whereas NHL showed significantly lower values at T3 compared to T0: 29.2% [IQR: 22–38%] versus 40.5% [IQR: 31.3–52.6%], P  < 0.05. The proportion with SDD in the entire cohort at T2 was 11.6% and 13.3% among HL and NHL patients, respectively. At T3, only one in 16 NHL patients presented SDD. LIMITATIONS, REASONS FOR CAUTION TUNEL assay requires at least 5 million spermatozoa to be performed; hence, severe oligozoospermic men were not included in the study. Although our cohort represents the largest one in the literature, the relatively small number of patients does not allow us to establish precisely the frequency of SDD at T2 which in our study reached 11–13% of patients. WIDER IMPLICATIONS OF THE FINDINGS Our data provide further insights into the long-term effects of cytotoxic treatments on the sperm genome. The persistent severe DNA damage after 2 years post-treatment observed in some patients suggests that there is an interindividual variation in restoring DNA integrity. We propose the use of SDF as a biomarker to monitor the treatment-induced genotoxic effects on sperm DNA in order to better personalize pre-conceptional counseling on whether to use fresh or cryopreserved spermatozoa. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by grants from the Istituto Toscano Tumori (ITT), Fondazione Ente Cassa di Risparmio di Firenze, the European Commission—Reproductive Biology Early Research Training (REPROTRAIN). C.K. G.F. V.R. and A.R.-E. belong to COST Action CA20119 (ANDRONET) which is supported by the European Cooperation in Science and Technology (www.cost.eu). The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]

Introduction: Testicular germ cell tumor is the most frequent neoplasia in men of reproductive age, with a 5‐year survival rate of 95%. Antineoplastic treatments induce sperm DNA fragmentation, especially within the first year post‐therapy. Data in the literature are heterogeneous concerning longer follow‐up periods, and the large majority is limited to 2 years. Objective: To define the timing for the recovery of sperm DNA damage and the proportion of patients with severe DNA damage at 2 and 3 years from the end of therapy. Materials and methods: Sperm DNA fragmentation was evaluated in 115 testicular germ cell tumor patients using terminal deoxynucleotidyl transferase dUTP nick end labeling assay coupled with flow cytometry before (T0) and 2 (T2) and 3 (T3) years post‐treatment. Patients were divided based on the type of treatment: carboplatin, bleomycin–etoposide–cisplatin, and radiotherapy. For 24 patients, paired sperm DNA fragmentation data were available at all time‐points (T0–T2–T3). Seventy‐nine cancer‐free, fertile normozoospermic men served as controls. Severe DNA damage was defined as the 95th percentile in controls (sperm DNA fragmentation = 50%). Results: Comparing patients versus controls, we observed: (i) no differences at T0 and T3 and (ii) significantly higher sperm DNA fragmentation levels (p < 0.05) at T2 in all treatment groups. Comparing pre‐ and post‐therapy in the 115 patients, the median sperm DNA fragmentation values were higher in all groups at T2, reaching significance (p < 0.05) only in the carboplatin group. While the median sperm DNA fragmentation values were also higher in the strictly paired cohort at T2, about 50% of patients returned to baseline. The proportion of severe DNA damage in the entire cohort was 23.4% and 4.8% of patients at T2 and T3, respectively. Discussion: Currently, testicular germ cell tumor patients are advised to wait 2 years post‐therapy before seeking natural pregnancy. Our results suggest that this period may not be sufficient for all patients. Conclusion: The analysis of sperm DNA fragmentation may represent a useful biomarker for pre‐conception counseling following cancer treatment. [ABSTRACT FROM AUTHOR]