Toward clinical exomes in diagnostics and management of male infertility.

Infertility, affecting ∼10% of men, is predominantly caused by primary spermatogenic failure (SPGF). We screened likely pathogenic and pathogenic (LP/P) variants in 638 candidate genes for male infertility in 521 individuals presenting idiopathic SPGF and 323 normozoospermic men in the ESTAND cohort. Molecular diagnosis was reached for 64 men with SPGF (12%), with findings in 39 genes (6%). The yield did not differ significantly between the subgroups with azoospermia (20/185, 11%), oligozoospermia (18/181, 10%), and primary cryptorchidism with SPGF (26/155, 17%). Notably, 19 of 64 LP/P variants (30%) identified in 28 subjects represented recurrent findings in this study and/or with other male infertility cohorts. NR5A1 was the most frequently affected gene, with seven LP/P variants in six SPGF-affected men and two normozoospermic men. The link to SPGF was validated for recently proposed candidate genes ACTRT1, ASZ1, GLUD2, GREB1L, LEO1, RBM5, ROS1, and TGIF2LY. Heterozygous truncating variants in BNC1, reported in female infertility, emerged as plausible causes of severe oligozoospermia. Data suggested that several infertile men may present congenital conditions with less pronounced or pleiotropic phenotypes affecting the development and function of the reproductive system. Genes regulating the hypothalamic-pituitary-gonadal axis were affected in >30% of subjects with LP/P variants. Six individuals had more than one LP/P variant, including five with two findings from the gene panel. A 4-fold increased prevalence of cancer was observed in men with genetic infertility compared to the general male population (8% vs. 2%; p = 4.4 × 10-3). Expanding genetic testing in andrology will contribute to the multidisciplinary management of SPGF.

Severe sperm DNA fragmentation may persist for up to 3 years after cytotoxic therapy in patients affected by Hodgkin lymphoma and non-Hodgkin lymphoma.

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.

Testicular and Haematological Cancer Induce Very High Levels of Sperm Oxidative Stress.

Cancer impairs spermatogenesis, whereas results on sperm DNA integrity are controversial and no data are available about sperm oxidative stress. In cancer patients, we detected sperm DNA fragmentation (sDF) and both viable (ROS production in viable sperm fraction/viable spermatozoa) and total (ROS production in viable sperm fraction/total spermatozoa) oxidative stress. We found that cancer (22.50 (17.00-26.75)%, n = 85) increased sDF with respect to the control groups in both normozoospermic subfertile patients (NSP) (12.75 (8.63-14.88)%, n = 52, p < 0.001) and in healthy donors (HD) (8.50 (7.00-14.00)%, n = 19, p < 0.001). The induction of viable oxidative stress (n = 96) with cancer was even higher: 36.60 (24.05-58.65)% versus 11.10 (8.63-14.90)% in NSP (p < 0.001) and 9.60 (8.00-14.03)% in HD (p < 0.001). Similar, albeit lower, differences were found for total oxidative stress. SDF sharply correlated to viable oxidative stress when we considered all subjects (cancer patients and controls) (r = 0.591, p < 0.001, n = 134), but no correlation was found when only cancer patients were studied (r = 0.200; p > 0.05, n = 63). In conclusion, cancer significantly increases sDF and sperm oxidative stress levels. Additional mechanisms to oxidative attack might be responsible for increased sDF in cancer patients. Because sperm oxidative stress might affect the outcomes of sperm cryopreservation, of cancer treatments and of sperm epigenoma, the detection of oxidative stress could be of help in managing the reproductive issues of cancer patients.

Long-term effect of cytotoxic treatments on sperm DNA fragmentation in patients affected by testicular germ cell tumor.

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.

Large-scale analyses of the X chromosome in 2,354 infertile men discover recurrently affected genes associated with spermatogenic failure.

Although the evolutionary history of the X chromosome indicates its specialization in male fitness, its role in spermatogenesis has largely been unexplored. Currently only three X chromosome genes are considered of moderate-definitive diagnostic value. We aimed to provide a comprehensive analysis of all X chromosome-linked protein-coding genes in 2,354 azoospermic/cryptozoospermic men from four independent cohorts. Genomic data were analyzed and compared with data in normozoospermic control individuals and gnomAD. While updating the clinical significance of known genes, we propose 21 recurrently mutated genes strongly associated with and 34 moderately associated with azoospermia/cryptozoospermia not previously linked to male infertility (novel). The most frequently affected prioritized gene, RBBP7, was found mutated in ten men across all cohorts, and our functional studies in Drosophila support its role in germ stem cell maintenance. Collectively, our study represents a significant step towards the definition of the missing genetic etiology in idiopathic severe spermatogenic failure and significantly reduces the knowledge gap of X-linked genetic causes of azoospermia/cryptozoospermia contributing to the development of future diagnostic gene panels.