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.

中文翻译：

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霍奇金淋巴瘤和非霍奇金淋巴瘤患者接受细胞毒治疗后，严重的精子 DNA 碎片可能持续长达 3 年

研究问题 细胞毒性治疗结束 2 年后，所有男性的精子 DNA 是否都能从损伤中恢复？摘要：目前的指示是细胞毒治疗后寻求自然怀孕需要 2 年的等待时间，这可能不足以适合所有男性，因为即使在此时间范围之后，仍有部分受试者存在严重的精子 DNA 损伤。已知的信息 文献中有关细胞毒治疗后淋巴瘤患者精子 DNA 碎片 (SDF) 的数据很少。最大的纵向研究评估了 34 名患者的配对治疗前和治疗后（长达 24 个月）精液样本，而一项研究对 10 名患者进行了更长的随访（36 个月）。治疗后>24个月的中位/平均SDF值没有显示出显着差异，但研究没有探讨具有严重DNA损伤的患者比例，并且分析是对冻融样本进行的。研究设计、规模、持续时间 本研究纳入了 53 名霍奇金淋巴瘤 (HL) 和 25 名青春期后非霍奇金淋巴瘤 (NHL) 患者，招募期为 10 年（2012 年至 2022 年）。其中，18名受试者在三个时间点提供了配对精液样本进行SDF分析。在细胞毒性治疗（单独化疗或联合放疗）结束前（T0）、2年（T2）和3年（T3）后对患者的SDF进行评估。79 名 18 岁以上健康、生育能力正常、精子正常的男性作为对照组（2016 年至 2019 年招募）。参与者/材料、设置、方法 使用 TUNEL（末端脱氧核苷酸转移酶 dUTP 缺口末端标记）测定结合流式细胞术对患者和对照的新鲜精液样本（即可能参与自然受孕的精子）进行 SDF 评估。比较各组之间的 SDF 中值：(i) HL 和 NHL 患者与对照组在三个时间点的比较；(ii) 基线时 HL 与 NHL 患者的比较；(iii) T0 与 T2 和 T3 的患者。严重 DNA 损伤 (SDD) 的定义是 SDF 水平高于对照的第 95 个百分位数 (50%)，并确定了所有时间点患有 SDD 的患者比例。主要结果和机会的作用 在 T0 时，患者表现出比对照组更高的中位 SDF，在 NHL 组中达到统计学显着性：40.5% [IQR: 31.3–52.6%] 对比 28% [IQR: 22–38%]，P < ; 0.05。将 SDF 治疗前与治疗后进行比较，HL 患者在三个时间点表现出相似的中位值，而 NHL 在 T3 时的中位值显着低于 T0：29.2% [IQR：22–38%] 对比 40.5% [IQR] : 31.3–52.6%], P < 0.05。T2 时整个队列中 HL 和 NHL 患者中 SDD 的比例分别为 11.6% 和 13.3%。在 T3 时，只有十分之一的 NHL 患者出现 SDD。局限性、注意事项 TUNEL 检测需要至少 500 万个精子才能进行；因此，严重少精症男性不包括在该研究中。尽管我们的队列代表了文献中最大的队列，但患者数量相对较少，因此我们无法准确确定 T2 时 SDD 的频率，在我们的研究中，SDD 的频率达到了 11-13% 的患者。研究结果的更广泛影响我们的数据进一步深入了解细胞毒性治疗对精子基因组的长期影响。一些患者在治疗后 2 年后观察到持续严重的 DNA 损伤，这表明在恢复 DNA 完整性方面存在个体差异。我们建议使用 SDF 作为生物标志物来监测治疗引起的对精子 DNA 的遗传毒性影响，以便更好地个性化关于是否使用新鲜精子或冷冻保存精子的孕前咨询。研究经费/竞争利益 这项工作得到了 Istituto Toscano Tumori (ITT)、Fondazione Ente Cassa di Risparmio di Firenze、欧盟委员会生殖生物学早期研究培训 (REPROTRAIN) 的资助。CK、GF、VR 和 AR-E。属于 COST Action CA20119 (ANDRONET)，该行动由欧洲科学技术合作组织 (www.cost.eu) 支持。作者宣称没有利益冲突。试用注册号 不适用。