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Purpose Testicular cancer (TC) is the most common malignancy among young adult males. The etiology is multifactorial, and both environmental and genetic factors play an essential role in the origin and development of this tumor. In particular, exposure to environmental endocrine disruptors (EEDs), resulting from industrialization and urbanization, seems crucial both in pre-and postnatal life. However, the lack of long-term studies on a wide caseload and the difficulty in evaluating their toxic effects in vivo make it challenging to establish a causal link. This review aims to discuss the main human epidemiological studies currently available in the literature to define a possible association between these chemicals and TC. Methods A comprehensive Medline/PubMed and Embase search was performed, selecting all relevant, peer-reviewed papers in English published from 2002 to January 2022. Other relevant papers were selected from the reference lists. Results To date, literature evidence is limited due to the scarcity and heterogeneity of human studies and shows controversial data, highlighting the complexity of the topic. However, most human epidemiological studies seem to point toward a correlation between EEDs exposure and TC. Conclusion Although the molecular mechanisms are not yet fully understood, the role of EEDs in TC onset is plausible, but several factors, such as the individual genetic background, the exposure time, and the complex mechanism of action of these chemicals, do not allow defining the causal link with certainty and make further studies necessary to investigate this complex topic.

Purpose Testicular germ cell tumours (TGCTs) is the most common malignancy among young adult males. The etiology is multifactorial and both environmental and genetic factors play an important role in the origin and development of TGCT. Genetic susceptibility may result from the interaction of multiple common and low-penetrance genetic variants and one of the main candidate genes is PDE11A. Many PDE11A polymorphisms were found responsible for a reduced PDE activity in TGCT patients, who often also display impaired hormone and sperm profile. The aim of this study was to investigate testicular function and PDE11A sequence in testicular cancer cases. Methods Semen analysis was performed in 116 patients with unilateral and bilateral sporadic TGCTs and in 120 cancer-free controls. We also investigated hormone profile and PDE11A polymorphisms using peripheral blood samples. Results Our data revealed that TGCT patients showed lower testosterone levels, higher gonadotropins levels and worse semen quality than controls, although the mean and the medians of sperm parameters are within the reference limits. PDE11A sequencing detected ten polymorphisms not yet associated with TGCTs before. Among these, G223A in homozygosity and A288G in heterozygosity were significantly associated with a lower risk of testicular tumour and they displayed a positive correlation with total sperm number. Conclusions Our findings highlight the key role of PDE11A in testis and suggest the presence of an underlying complex and fine molecular mechanism which controls testis-specific gene expression and susceptibility to testicular cancer.

Purpose The SARS-CoV-2 pandemic has rapidly spread worldwide and, among the others, the male gender was quickly recognized as an independent risk factor for both the disease and its consequences. Since the possibility of long-term hormonal axis changes and male gamete impairment have been hypothesized but a relatively low levels of evidence has been reached, we focused this narrative mini-review on summarizing key state-of-the-art knowledge on male reproductive effects of COVID-19 as a quick reference for reproductive health specialists. Methods A comprehensive Medline/PubMed and Embase search was performed selecting all relevant, peer-reviewed papers in English published from 2020. Other relevant papers were selected from the reference lists. Results Available evidence indicates that the likelihood of direct testicular damage from SARS-CoV-2 is somewhat low, but there are many indirect ways (fever, cytokine imbalance, and drugs) through which the pituitary-gonadal axis and spermatogenesis may be disrupted. These alterations are probably transient, but as available evidence is low quality, it cannot be excluded that previous pathologies or comorbidities might modulate the risk of their persistence. On the other hand, available evidence shows high safety regarding andrological health for available vaccines, although studies are mainly focused on mRNA vaccines. Conclusion A careful andrological evaluation of men recovering from COVID-19 is highly recommended. Since available evidence is relatively scarce, a careful andrological follow-up and counseling of these patients are mandatory.

The evaluation of morpho-functional sperm characteristics alone is not enough to explain infertility or to predict the outcome of Assisted Reproductive Technologies (ART): more sensitive diagnostic tools are needed in clinical practice. The aim of the present study was to analyze Sperm DNA Fragmentation (SDF) and sperm-borne miR-34c-5p and miR-449b-5p levels in men of couples undergoing ART, in order to investigate any correlations with fertilization rate, embryo quality and development. Male partners (n = 106) were recruited. Semen analysis, SDF evaluation and molecular profiling analysis of miR-34c-5p and miR-449b-5p (in 38 subjects) were performed. Sperm DNA Fragmentation evaluation- a positive correlation between SDF post sperm selection and the percentage of low-quality embryos and a negative correlation with viable embryo were found. SDF > 2.9% increased the risk of obtaining a non-viable embryo by almost 4-fold. Sperm miRNAs profile—we found an association with both miRNAs and sperm concentration, while miR-449b-5p is positively associated with SDF. Moreover, the two miRNAs are positively correlated. Higher levels of miR-34c-5p compared to miR-449b-5p increases by 14-fold the probability of obtaining viable embryos. This study shows that SDF, sperm miR-34c-5p, and miR-449b-5p have a promising role as biomarkers of semen quality and ART outcome.

Alterations affecting the mitochondrial genome and chromatin integrity of spermatozoa impair male reproductive potential. This study aimed to evaluate the impact of mitochondrial DNA (mtDNA) copy number alterations on sperm motility and on the molecular mechanism regulating the number of mtDNA copies, through analysis of mitochondrial transcription factor A (TFAM) gene expression. It also investigated any correlation between mtDNA copy number and sperm DNA fragmentation (SDF). Sixty-three asthenozoospermic semen samples (Group A) and 63 normokinetic semen samples (Group N) were analysed according to WHO (WHO laboratory manual for the examination and processing of human semen, World Health Organization, Geneva, 2010). Sperm mtDNA copy number and TFAM gene expression were quantified by real time quantitative polymerase chain reaction. SDF was evaluated using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay. The mtDNA copy number was higher in asthenozoospermic semen samples and was negatively correlated with sperm concentration, total sperm number and total motile spermatozoa. The caseload showed a global negative correlation of TFAM gene expression with total motile sperm and a positive correlation with abnormal forms, SDF and mtDNA copy number, but this was not confirmed within each subgroup. SDF was significantly increased in asthenozoospermic samples and correlated with abnormal forms. No correlation was found between SDF and mtDNA copy number. Our results suggest a potential role of mtDNA content as an indicator of semen quality and support the hypothesis that dysregulation of TFAM expression is accompanied by a qualitative impairment of spermatogenesis. Since mtDNA copy number alterations and impaired chromatin integrity could affect reproductive success, these aspects should be evaluated in relation to assisted reproductive techniques.

This study aims to evaluate genetic contribution and sperm DNA fragmentation (SDF) in a cohort of 18 unrelated globozoospermic Italian men (Group G). Semen samples were assessed according to the WHO 2010 Laboratory Manual and compared with 31 fertile controls. We focused our genetic analysis on the exons of the main globozoospermia-associated genes, performing qualitative PCR to assess deletion of DPY19L2 and sequencing to detect mutations of SPATA16 and PICK1. SDF was evaluated using the TUNEL assay. In Group G, 10 patients had a complete form of globozoospermia, whereas 8 patients had a partial form. Molecular analysis revealed deletion of DPY19L2 in six of the patients, all of them with complete globozoospermia, while no mutations were found in the examined exons of PICK1 and SPATA16. TUNEL analysis showed a higher SDF% in Group G. Our findings confirm DPY19L2 defects as the most frequent genetic alteration in Italian patients contributing to globozoospermic phenotypes. Furthermore, spermatozoa with acrosomal defects could also display high levels of SDF as a possible consequence of abnormally remodeled chromatin. The possible effect on offspring of chromatin structure abnormalities and altered DNA integrity should be carefully evaluated by clinicians, especially regarding the feasibility and safety of artificial reproductive techniques, which represent the only treatment that allows these patients to conceive.

This study aims to evaluate genetic contribution and sperm DNA fragmentation (SDF) in a cohort of 18 unrelated globozoospermic Italian men (Group G). Semen samples were assessed according to the WHO 2010 Laboratory Manual and compared with 31 fertile controls. We focused our genetic analysis on the exons of the main globozoospermia-associated genes, performing qualitative PCR to assess deletion of DPY19L2 and sequencing to detect mutations of SPATA16 and PICK1. SDF was evaluated using the TUNEL assay. In Group G, 10 patients had a complete form of globozoospermia, whereas 8 patients had a partial form. Molecular analysis revealed deletion of DPY19L2 in six of the patients, all of them with complete globozoospermia, while no mutations were found in the examined exons of PICK1 and SPATA16. TUNEL analysis showed a higher SDF% in Group G. Our findings confirm DPY19L2 defects as the most frequent genetic alteration in Italian patients contributing to globozoospermic phenotypes. Furthermore, spermatozoa with acrosomal defects could also display high levels of SDF as a possible consequence of abnormally remodeled chromatin. The possible effect on offspring of chromatin structure abnormalities and altered DNA integrity should be carefully evaluated by clinicians, especially regarding the feasibility and safety of artificial reproductive techniques, which represent the only treatment that allows these patients to conceive.

STUDY QUESTIONHow is semen quality affected by treatment in survivors of non-Hodgkin lymphoma (NHL)?SUMMARY ANSWERBefore cancer treatment, most NHL subjects were normozoospermic and, while standard first-line treatments seemed compatible with post-treatment recovery after 18 months, salvage therapy followed by haematopoietic stem cell transplant caused permanent damage to spermatogenesis in many cases, with 66% azoospermic subjects in the long term.WHAT IS KNOWN ALREADYTesticular function has been widely investigated in relation to the most common malignancies in men of reproductive age, such as testicular cancer and Hodgkin lymphoma, but NHL has been somewhat under-investigated. The available reports generally show a post-treatment worsening of semen parameters in NHL survivors, but they involved small caseloads or a subgroup of broader caseloads, and their results are not comparable.STUDY DESIGN, SIZE, DURATIONWe conducted a retrospective analysis of 222 subjects who attended our University Hospital Sperm Bank between 2002 and 2017 for sperm cryopreservation after a diagnosis of NHL.PARTICIPANTS/MATERIALS, SETTING, METHODSThe study included 222 patients with NHL who underwent sperm cryopreservation before any antineoplastic treatment. Subjects with any comorbidity and/or other conditions interfering with sperm parameters were excluded. All patients underwent a careful medical history and physical examination at the time of sperm cryopreservation (T0) and had at least one follow-up visit at 6 (T6), 12 (T12), 18 (T18) and/or 24 months (T24) or more than 24 months (T > 24), with a median follow-up of 47.5 months (range 28–140 months). Fertility information was collected through the administration of a questionnaire.MAIN RESULTS AND THE ROLE OF CHANCEPre-treatment, more than 80% of NHL patients were normozoospermic and in 15.9% of cases had already fathered a child. Aggressive lymphomas were associated with worse baseline semen volume and total sperm number compared to indolent subtypes (P LIMITATIONS, REASONS FOR CAUTIONThe main limitations of the study are the lack of data on blood hormones for evaluation of testicular function as a whole and the non-compliance of several patients in attending follow-up visits at all time points, resulting in a reduced sample size for the treatment subgroup analyses. Furthermore, despite a good fertility questionnaire response rate (>80%), the low number of NHL survivors actively seeking fatherhood limits the generalization of results.WIDER IMPLICATIONS OF THE FINDINGSThe increased survival of NHL patients of reproductive age makes it essential to focus on the testicular toxicity of the treatment. Sperm cryopreservation must be suggested before any treatment. Two years after first-line treatments, sperm number showed signs of recovery: this finding is of the utmost importance for oncofertility counselling, as it indicates that only a standard first-line chemotherapy in many patients may be compatible with at least a partial spermatogenesis recovery in the long term. Nonetheless, it is expected that up to 30% of subjects will require treatment intensification, which could result in permanent testicular damage; in such cases the use of banked semen might represent the patient’s best chance for future fertility.STUDY FUNDING/COMPETING INTEREST(S)This work was supported by a grant from the Italian Ministry of Education and Research (MIUR-PRIN 2015-2015XSNA83-002) and the ‘Sapienza’ University of Rome, Faculty of Medicine. The authors report no conflicts of interest.TRIAL REGISTRATION NUMBERN/A.

STUDY QUESTION Is ageing associated with a decline in semen quality and molecular changes to human sperm? SUMMARY ANSWER Semen quality declines with advancing age and characteristic molecular changes take place during the ageing process, including increased sperm DNA damage, altered sperm protamination and altered seminal plasma miRNA profile. WHAT IS KNOWN ALREADY During ageing, the reproductive system is exposed to physiological changes and potentially damaging factors that may impair testicular function. Reactive oxygen species (ROS) can induce errors during DNA replication, transcription or post-transcriptional events (fragmentation, chromatin condensation abnormalities and protamine expression defects). STUDY DESIGN, SIZE, DURATION Semen parameters from 2626 healthy men aged 20-81 years were evaluated retrospectively from those attending our University Laboratory between 2011 and 2016 for andrological screening or as part of an andrological work-up. Subjects were divided into six groups by age (20-32, 33-37, 38-40, 41-44, 45-50, 51-81 years). From these subjects, semen samples from 40 elderly men (50-81 years) and 40 young men (20-40 years) (control group), all non-smokers of normal weight, were selected for the evaluation of sperm chromatin integrity, PRM1, PRM2, TNP1 and TNP2 gene expression, and microRNA expression profile in seminal plasma. PARTICIPANTS/MATERIALS, SETTING, METHODS Semen was analysed according to WHO 2010. Sperm DNA fragmentation (SDF) was evaluated using TUNEL assay; sperm PRM1, PRM2, TNP1 and TNP2 gene expression was evaluated by quantitative RT-PCR amplification; miRNA expression profiles were analysed by TaqMan Array Cards and validated by RT-PCR amplification. MAIN RESULTS AND THE ROLE OF CHANCE Cytological analysis - Semen volume, progressive motility and number of progressively motile sperm were significantly lower in elderly than in younger subjects (sextiles 51-81 versus 20-32 years; P < 0.001), while the percentage of abnormal forms in these subjects was significantly higher than in the 20-32 age group (P = 0.002). Binomial logistic regression models revealed an association between age and semen parameters: age 51-81 was associated with changes in total sperm number (OR 2.47; 95% CI 1.52-4.02; P < 0.001), progressive motility (OR 3.63; 95% CI 2.49-5.30; P < 0.001), and abnormal forms (OR 3.89; 95% CI 2.71-7.26; P < 0.001). Obesity was associated with reduced progressive motility (OR 1.58; 95% CI 1.14-2.19; P = 0.006) and an increase in abnormal forms (OR 1.87; 95% CI 1.02-3.57; P = 0.021). In contrast, smoking did not contribute significantly to changes in semen parameters. Molecular analysis - Elderly men showed a significantly higher percentage of SDF (23.1 ± 8.7 versus 9.8 ± 2.6%; P < 0.001) and a significantly lower expression of PRM1 (mean fold change 2.2; P = 0.016) and PRM2 (mean fold change 4.6; P < 0.001), compared to younger controls. Furthermore, miR-146a showed a 3-fold lower expression (P < 0.001), miR-371 a 14-fold lower expression (P < 0.001), and miR-122 a 5-fold lower expression (P = 0.01) in the elderly men. LIMITATIONS, REASONS FOR CAUTION While typical chronic age-related conditions (cardiovascular, respiratory diseases) were excluded, the presence of subclinical underlying diseases cannot be excluded in the elderly population. Subjects referred to our clinic might not be fully representative of the general population. Although a careful medical history and physical examination excluded most andrological conditions that might affect spermatogenesis, we cannot exclude the presence of possible asymptomatic or idiopathic conditions. Furthermore, TUNEL, in common with other SDF detection methods (with the exception of the alkaline comet assay), does not distinguish between single and double strand breaks. WIDER IMPLICATIONS OF THE FINDINGS The role of obesity suggests that conditions related to lifestyle factors may further worsen age-related sperm parameter impairment. Increased SDF and altered protamine expression suggest the genomic fragility of sperm in advanced age. Changes in the miRNA expression pattern with age could contribute to the identification of a characteristic molecular signature of the ageing process, a potential new biomarker for male reproductive function during the physiological ageing process. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by a grant from the Italian Ministry of Education and Research (MIUR-PRIN 2015- 2015XSNA83-002) and 'Sapienza' University of Rome Faculty of Medicine. The authors have no conflicts of interest. TRIAL REGISTRATION NUMBER N/A.

Il progresso della Procreazione Medicalmente Assistita ha incrementato lo sviluppo e l’utilizzo dei test di valutazione del danno al DNA nemaspermico. Tali test rappresentano un utile strumento nello studio molecolare del gamete maschile ma attualmente il loro impiego nella clinica presenta delle limitazioni. L’assenza di una metodica gold standard e della standardizzazione delle diverse tecniche diagnostiche rendono difficile l’introduzione di tali metodiche nella diagnosi di infertilita maschile.

The aim of this study was to study the incidence of Y chromosome microdeletions in a Caucasian population of Klinefelter syndrome (KS) patients and to investigate the possible association between Y chromosome microdeletions and KS. We conducted a retrospective study on 118 KS patients, 429 patients with non-obstructive azoospermia (NOA), and 155 normozoospermic men. Eight of the 118 KS patients had undergone testicular sperm extraction (TESE). All patients underwent semen examination and Y chromosome microdeletions evaluated by PCR, using specific sequence tagged site (STS) primer sets, which spanned the azoospermia factor AZFa, AZFb, and AZFc regions of the Y chromosome. Semen analysis of the KS group revealed: 1 patient with oligozoospermia, 1 with severe oligoasthenoteratozoospermia, 2 with cryptozoospermia, and 114 with azoospermia. Eight of the 114 azoospermic KS patients underwent TESE, and spermatozoa were recovered from three of these, all of whom had non-mosaic karyotype 47, XXY. 10.7% of the NOA patients presented AZF microdeletions. In 429 cases with NOA, 8 cases had AZFa + b + c deletion, 6 cases had AZF b + c deletion, 4 cases had AZFa microdeletion, 8 cases had AZFb microdeletion, and 20 cases had AZFc microdeletion. Just one KS patient (0.8%) presented microdeletion in the AZFc region. The percentage of microdeletions in KS patients was lower than in NOA patients, suggesting that AZF microdeletions and KS do not have a causal relationship and that Y chromosome microdeletions are not a genetic factor linked to KS.

The aetiopathogenesis of recurrent pregnancy loss (RPL) is heterogeneous. The aim of this study was to investigate the male factor in Italian couples experiencing RPL following natural conception. The study investigated 112 men from RPL couples and two control groups: 114 infertile men with one or more impaired semen parameters and 114 fertile men with high-quality semen parameters. Semen parameters were examined according to WHO criteria. Sperm DNA fragmentation (SDF) was evaluated using TdT-mediated dUDP nick-end labelling (TUNEL) assay. With the exception of ejaculate volume, the seminal profile of patients with RPL was similar to that of fertile patients and better than the infertile ones. Despite good spermatogenesis, however, sperm DNA integrity was impaired in the RPL group, with SDF values significantly higher than in fertile controls (18.8 ± 7.0 versus 12.8 ± 5.3, P < 0.001) and similar to those of infertile patients. SDF also showed a positive correlation with the age of patients with RPL and number of miscarriages. The results suggest a correlation between increased SDF and impaired reproductive capacity in terms of both fertilization and pregnancies carried to term, but high SDF cannot yet be considered a predictive factor for the risk of RPL.