CD137+ and regulatory T cells as independent prognostic factors of survival in advanced non-oncogene addicted NSCLC patients treated with immunotherapy as first-line.

BACKGROUND: Immune checkpoint inhibitors (ICIs), administered alone or combined with chemotherapy, are the standard of care in advanced non-oncogene addicted Non-Small Cell Lung Cancer (NSCLC). Despite these treatments' success, most long-term survival benefit is restricted to approximately 20% of patients, highlighting the need to identify novel biomarkers to optimize treatment strategies. In several solid tumors, immune soluble factors, the activatory CD137+ Tcells, and the immunosuppressive cell subsets Tregs and MDSCs (PMN(Lox1+)-MDSC and M-MDSCs) correlated with responses to ICIs and clinical outcomes thus becoming appealing predictive and prognostic factors. This study investigated the role of distinct CD137+ Tcell subsets, Tregs, MDSCs, and immune-soluble factors in NSCLC patients as possible biomarkers. METHODS: The levels of T cells, MDSCs and soluble factors were evaluated in 89 metastatic NSCLC patients who underwent ICIs as first- or second-line treatment. T cell analysis was performed by cytoflurimetry evaluating Tregs and different CD137+ Tcell subsets also combined with CD3+, CD8+, PD1+, and Ki67+ markers. Circulating cytokines and immune checkpoints were also evaluated by Luminex analysis. All these parameters were correlated with several clinical factors (age, sex, smoking status, PS and TPS), response to therapy, PFS , and OS . The analyses were conducted in the overall population and in patients treated with ICIs as first-line (naïve patients). RESULTS: In both groups of patients, high levels of circulating CD137+ and CD137+PD1+ T cells (total, CD4 and CD8) and the soluble factor LAG3 positively correlated with response to therapy. In naïve patients, PMN(Lox1+)-MDSCs negatively correlated with clinical response, and a high percentage of Tregs was associated with favorable survival. Moreover, the balance between Treg/CD137+ Tcells or PMN(Lox1+)-MDSC/CD137+ Tcells was higher in non-responding patients and was associated with poor survival. CD137+ Tcells and Tregs resulted as two positive independent prognostic factors. CONCLUSION: High levels of CD137+, CD137+PD1+ Tcells and sLAG3 could predict the response to ICIs in NSCLC patients independently by previous therapy. Combining the evaluation of CD137+ Tcells and Tregs also as Treg/CD137+ T cells ratio it is possible to identify naive patients with longer survival.

Safety of gender affirming treatment in assigned female at birth transgender people and association of androgen and estrogen ß receptor polymorphisms with clinical outcomes.

PURPOSE: Gender affirming hormone treatment (GAHT) with androgens in assigned female at birth (AFAB) people with Gender Incongruence (GI) can induce and maintain variable phenotypical changes, but individual response may be genetically determined. To clarify the role of AR and ERß polymorphisms we prospectively evaluated AFAB subjects undergoing virilizing GAHT. METHODS: Fifty-two AFAB people with confirmed GI were evaluated before (T0) and after 6 (T6) and 12 months (T12) of testosterone enanthate 250 mg i.m. every 28 days. Hormone profile (testosterone, estradiol), biochemical (blood count, glyco-metabolic profile) and clinical parameters (Ferriman-Gallwey score, pelvic organs) were evaluated at each time-point, as well as number of CAG and CA repeats for AR and ERß, respectively. RESULTS: All subjects have successfully achieved testosterone levels within normal male ranges and improved their degree of virilization, in absence of significant side effects. Hemoglobin, hematocrit and red blood cells were significantly increased after treatment, but within normal ranges. Ultrasound monitoring of pelvic organs showed their significant reduction already after 6 months of GATH, in absence of remarkable abnormalities. Furthermore, a lower number of CAG repeats was associated with a higher Ferriman-Gallwey score post treatment and a higher number of CA repeats was associated with uterine volume reduction. CONCLUSION: We confirmed safety and efficacy of testosterone treatment on all measured parameters. This preliminary data hints a future role of genetic polymorphisms to tailor GAHT in GI people, but evaluation on a larger cohort is necessary as the reduced sample size could limit data generalization at this stage.

Bladder Neck Obstruction: Experience and Management in a Sperm Bank.

Primary Bladder Neck Obstruction (PBNO) management provides medical and surgical treatment, such as transurethral incisions that can lead to retrograde ejaculation. The aim of this study was to investigate the maintenance of anterograde ejaculation and semen quality before and after this surgical procedure. A retrospective evaluation was carried out between 2011 and 2020. A total of 73 patients diagnosed with PBNO were recruited. Ejaculatory function, semen quality, and the fertility of recruited subjects were evaluated. Semen parameters-Baseline, 8.2% of patients were oligozoospermic and 12.3% had a semen volume below the WHO 2010 fifth percentile. Post-surgery, 20% of patients were oligozoospermic. We detected a significant decrease in total sperm number, a significant increase in the number of abnormal forms, and a reduction in the leukocyte concentration. Ejaculatory function-A total of 7.7% of patients reported anejaculation after transurethral incision of the bladder neck. Fertility-9.2% of the patients already had children before surgery; 13.8% had naturally conceived children in the years following surgery; 76.9% had no desire for paternity at the time. Our data have important implications for sperm bank management. The alterations in semen parameters and the risk of anejaculation suggest that the use of sperm cryopreservation before surgery for PBNO should be encouraged.

A decade of WHO 2010: total sperm number temporal trend and role of lifestyle factors.

After a huge decline in sperm concentration between 1938 and 1991 was reported, many researchers investigated the possibility of a worsening of human sperm quality. Despite massive efforts, published evidence is still controversial. Similarly, the role of lifestyle factors on semen parameters is debated. We conducted a monocentric Italian study to evaluate the total sperm number trend over the last 10 years (from 2010 to 2019). Additionally, we evaluated the association between lifestyle factors and total sperm number in order to identify possible damaging factors. We performed a retrospective study analyzing subjects aged 18-55 years who had their semen analyzed between 2010 and 2019. A total of 3329 subjects were included: 1655 subjects referred to our department (Department of Experimental Medicine, Sapienza University of Rome, Roma, Italy) for idiopathic infertility and 1674 subjects referred for preconceptional or andrological screening with no confirmed andrological diseases. Semen samples were examined according to World Health Organization (WHO) 2010 criteria by two seminologists with the same training and the same equipment. For statistical evaluations, only total sperm number (×10 6 per ejaculate) was taken into consideration. We detected no significant changes in mean total sperm number during the last decade, in either the entire population or the two subgroups (infertile group and control group). In a multivariate analysis total sperm number was significantly associated with the history of infertility, body mass index (BMI) and cigarette smoking. Our results suggest that infertile men are "vulnerable" subjects, particularly susceptible to several negative factors, many of which still remain unknown. Our study highlights the need for studies addressing men's lifestyle in order to find and reduce deleterious agents.

Environmental disruptors and testicular cancer.

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.

4BNT162b2 mRNA COVID-19 vaccine and semen: What do we know?

BACKGROUND: The effects of an mRNA COVID-19 vaccine on spermatozoa parameters are not known. The aim of this study was to evaluate the effect of the BNT162b2 mRNA COVID-19 vaccine on human semen, comparing spermatozoa parameters before and after vaccine inoculation. MATERIALS AND METHODS: In this single-center prospective study, voluntary subjects who received mRNA vaccines from February to July 2021 were enrolled. The study population included male subjects aged between 18 and 45 years who completed the BNT162b2 mRNA COVID-19 vaccine cycle. All subjects were evaluated before the first dose of vaccine (T0) and after 3 months (T1) with semen analysis and further analysis of seminal plasma, including colorimetric determination of reactive oxygen metabolites (d-ROM test), electrolytes, and interleukin 6 (IL-6) assessment by enzyme-linked immunosorbent assay technology. RESULTS: The experimental sample included 47 subjects (age: 29.3 ± 6.0 years, range 24-32; body mass index: 23.15 ± 2.5 kg/m2 , range 19.2-28.0). All the subjects reported no systemic side effects. No significant differences were observed in any spermatozoa parameter between T0 and T1. A subanalysis was performed in oligoazoospermic and asthenozoospermic subjects, confirming the same results. Electrolyte analysis also showed no significant differences before and after vaccine inoculation. Finally, no significant differences were observed in T0, compared to T1 for the d-ROM test and IL-6. DISCUSSION AND CONCLUSION: In this study, no significant differences in spermatozoa parameters before and after vaccine inoculations were found. Furthermore, oxidative stress analysis,, the activity of the cell membrane, and IL-6, as a marker of inflammation, was not affected by the mRNA COVID-19 vaccine. These results suggest that this vaccine is safe for male semen quality.

Epigenetic Effects of Gender-Affirming Hormone Treatment: A Pilot Study of the ESR2 Promoter's Methylation in AFAB People.

Virilization of gender-incongruent subjects to whom were assigned the female gender at birth (AFAB) is achieved through testosterone administration. Inter-individual differences in the timing and acquisition of phenotypic characteristics, even if the same hormone preparations and regimens are used, are frequently observed. Polymorphisms of sex hormone receptors and methylation of their gene promoters, as well of several imprinted genes as H19, may underlie the differential response to treatment. Thus, the aim of this study was to examine the possible relationship between the CpG methylation profile of the estrogen receptor 2 gene (ESR2) and H19 promoters and their influence on phenotype modifications in a cohort of AFAB people at baseline (T0) and after 6 mo (T6) and 12 mo (T12) of testosterone therapy (testosterone enanthate, 250 mg i.m. every 28 d). A total of 13 AFAB subjects (mean age 29.3 ± 12.6) were recruited. The percentage of methylation of the ESR2 promoter significantly increased at T6 (adj. p = 0.001) and T12 (adj. p = 0.05), while no difference was detected for H19 (p = 0.237). Methylation levels were not associated with androgen receptor (AR)/estrogen receptor beta (ERß) polymorphisms nor hormone levels at baseline and after six months of treatment. On the other hand, total testosterone level and patient age resulted in being significantly associated with ESR2 methylation after twelve months of treatment. Finally, the difference in ESR2 promoter methylation between T6 and baseline was significantly associated with the number of CA repeats of the ERß receptor, adjusted vs. all considered variables (R2 = 0.62, adj. R2 = 0.35). No associations were found with CAG repeats of the AR, age, and estradiol and testosterone levels. Despite the small sample size, we can hypothesize that treatment with exogenous testosterone can modify the ESR2 methylation pattern. Our data also indicated that epigenetic changes may be regulated, suggesting that the modulation of estrogen signaling is relevant shortly after the beginning of the treatment up to T6, with no further significant modification at T12. Furthermore, estrogen receptor methylation appears to be associated with the age of the subjects and exogenous testosterone administration, representing a marker of androgenic treatment. Nonetheless, it will be necessary to increase the number of subjects to evaluate how epigenetic regulation might play a relevant role in the modulation of phenotypical changes after testosterone treatment.

Sperm DNA damage and cytokines in varicocele: A case-control study.

Varicocele is a vascular disease characterised by the abnormal enlargement of the pampiniform plexus veins and a well-known cause of male infertility. The aim of this study was to investigate the relationship between sperm DNA fragmentation (SDF) and inflammation in the pathogenesis of varicocele. We included 84 varicocele patients and 85 normozoospermic healthy controls, further analysed according to the body mass index, the smoking habit (smokers/non-smokers) and the varicocele severity (low/high grade). Semen parameters, SDF (by TUNEL) and inflammatory cytokines (by Luminex xMAP analysis) were evaluated. Varicocele patients showed significantly reduced semen parameters (volume, total sperm number, progressive motility, normal morphology) and increased SDF. Moreover, we observed a significant reduction of IFN-γ, IL-6, TNF-α and an increase of IL-10. No difference was reported according to the smoking habit, body mass index and varicocele severity. The observed cytokines pathway suggests the establishment of a chronic inflammatory condition, which may contribute to the alteration of semen quality. A thorough knowledge of the cytokine network might contribute to better understanding the link between inflammation and semen quality in varicocele and its impact on reproductive health.

Semen quality in non-Hodgkin lymphoma survivors: a monocentric retrospective study.

STUDY QUESTION: How is semen quality affected by treatment in survivors of non-Hodgkin lymphoma (NHL)? SUMMARY ANSWER: Before 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 ALREADY: Testicular 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, DURATION: We 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, METHODS: The 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 CHANCE: Pre-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 < 0.05). Post-treatment analyses showed that standard first-line treatments alone had a more favourable outcome than intensified regimens for semen parameters, with total sperm number returning to near-baseline values at 18 months (T0: 195.0 ± 189.8 versus T18: 113.4 ± 103.1, P = 0.278), and a 7.7% prevalence of azoospermia at 2 years. In this subgroup receiving standard first-line treatments, radiotherapy of the pelvis versus other 'high' sites (mediastinum, latero-cervical and axillary lymph nodes, etc.) was associated with an increased risk of developing post-treatment azoospermia (odds ratio 4.29, 95% CI 1.81-10.14; P = 0.001). Two-thirds of subjects who had relapsed or had disease progression after first-line treatment and then underwent salvage treatment ± haematopoietic stem cell transplant became azoospermic. Fertility data were available for 176 patients: 15.9% already had at least one child prior to the NHL diagnosis and 12.5% (22 patients) desired children after treatment. Fourteen patients achieved fatherhood: 12 through natural conception and two following ART. LIMITATIONS, REASONS FOR CAUTION: The 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 FINDINGS: The 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 NUMBER: N/A.

Effects of endocrine disruptors on fetal testis development, male puberty, and transition age.

PURPOSE: Endocrine disruptors (EDs) are exogenous substances able to impair endocrine system; consequently, they may cause numerous adverse effects. Over the last years, particular focus has been given to their harmful effects on reproductive system, but very little is known, especially in males. The aim of this review is to discuss the detrimental effects of EDs exposure on fetal testis development, male puberty, and transition age. METHODS: A search for the existing literature focusing on the impact of EDs on fetal testis development, male puberty, andrological parameters (anogenital distance, penile length, and testicular volume), and testicular cancer with particular regard to pubertal age provided the most current information available for this review. Human evidence-based reports were given priority over animal and in vitro experimental results. Given the paucity of available articles on this subject, all resources were given careful consideration. RESULTS: Information about the consequences associated with EDs exposure in the current literature is limited and often conflicting, due to the scarcity of human studies and their heterogeneity. CONCLUSIONS: We conclude that current evidence does not clarify the impact of EDs on human male reproductive health, although severe harmful effects had been reported in animals. Despite controversial results, overall conclusion points toward a positive association between exposure to EDs and reproductive system damage. Further long-term studies performed on wide number of subjects are necessary in order to identify damaging compounds and remove them from the environment.

Correction: gr/gr deletion predisposes to testicular germ cell tumour independently from altered spermatogenesis: results from the largest European study.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Disruption of Circadian Rhythms: A Crucial Factor in the Etiology of Infertility.

: Infertility represents a growing health problem in industrialized countries. Thus, a greater understanding of the molecular networks involved in this disease could be critical for the development of new therapies. A recent finding revealed that circadian rhythmicity disruption is one of the main causes of poor reproductive outcome. The circadian clock system beats circadian rhythms and modulates several physiological functions such as the sleep-wake cycle, body temperature, heart rate, and hormones secretion, all of which enable the body to function in response to a 24 h cycle. This intricated machinery is driven by specific genes, called "clock genes" that fine-tune body homeostasis. Stress of modern lifestyle can determine changes in hormone secretion, favoring the onset of infertility-related conditions that might reflect disfunctions within the hypothalamic-pituitary-gonadal axis. Consequently, the loss of rhythmicity in the suprachiasmatic nuclei might affect pulsatile sexual hormones release. Herein, we provide an overview of the recent findings, in both animal models and humans, about how fertility is influenced by circadian rhythm. In addition, we explore the complex interaction among hormones, fertility and the circadian clock. A deeper analysis of these interactions might lead to novel insights that could ameliorate the therapeutic management of infertility and related disorders.

Cryopreservation of Sperm: Effects on Chromatin and Strategies to Prevent Them.

Cryopreservation is a technique that can keep sperm alive indefinitely, enabling the conservation of male fertility. It involves the cooling of semen samples and their storage at -196 °C in liquid nitrogen. At this temperature all metabolic processes are arrested. Sperm cryopreservation is of fundamental importance for patients undergoing medical or surgical treatments that could induce sterility, such as cancer patients about to undergo genotoxic chemotherapy or radiotherapy, as it offers these patients not only the hope of future fertility but also psychological support in dealing with the various stages of the treatment protocols.Despite its importance for assisted reproduction technology (ART) and its success in terms of babies born, this procedure can cause cell damage and impaired sperm function. Various studies have evaluated the impact of cryopreservation on chromatin structure, albeit with contradictory results. Some, but not all, authors found significant sperm DNA damage after cryopreservation. However, studies attempting to explain the mechanisms involved in the aetiology of cryopreservation-induced DNA damage are still limited. Some reported an increase in sperm with activated caspases after cryopreservation, while others found an increase in the percentage of oxidative DNA damage. There is still little and contradictory information on the mechanism of the generation of DNA fragmentation after cryopreservation. A number of defensive strategies against cryoinjuries have been proposed in the last decade. Most studies focused on supplementing cryoprotectant medium with various antioxidant molecules, all aimed at minimising oxidative damage and thus improving sperm recovery. Despite the promising results, identification of the ideal antioxidant treatment method is still hampered by the heterogeneity of the studies, which describe the use of different antioxidant regimens at different concentrations or in different combinations. For this reason, additional studies are needed to further investigate the use of antioxidants, individually and in combination, in the cryopreservation of human sperm, to determine the most beneficial conditions for optimal sperm recovery and preservation of fertility.

gr/gr deletion predisposes to testicular germ cell tumour independently from altered spermatogenesis: results from the largest European study.

The association between impaired spermatogenesis and TGCT has stimulated research on shared genetic factors. Y chromosome-linked partial AZFc deletions predispose to oligozoospermia and were also studied in TGCT patients with controversial results. In the largest study reporting the association between gr/gr deletion and TGCT, sperm parameters were unknown. Hence, it remains to be established whether this genetic defect truly represents a common genetic link between TGCT and impaired sperm production. Our aim was to explore the role of the following Y chromosome-linked factors in the predisposition to TGCT: (i) gr/gr deletion in subjects with known sperm parameters; (ii) other partial AZFc deletions and, for the first time, the role of partial AZFc duplications; (iii) DAZ gene dosage variation. 497 TGCT patients and 2030 controls from two Mediterranean populations with full semen/andrological characterization were analyzed through a series of molecular genetic techniques. Our most interesting finding concerns the gr/gr deletion and DAZ gene dosage variation (i.e., DAZ copy number is different from the reference sequence), both conferring TGCT susceptibility. In particular, the highest risk was observed when normozoospermic TGCT and normozoospermic controls were compared (OR = 3.7; 95% CI = 1.5-9.1; p = 0.006 for gr/gr deletion and OR = 1.8; 95% CI = 1.1-3.0; p = 0.013 for DAZ gene dosage alteration). We report in the largest European study population the predisposing effect of gr/gr deletion to TGCT as an independent risk factor from impaired spermatogenesis. Our finding implies regular tumour screening/follow-up in male family members of TGCT patients with gr/gr deletion and in infertile gr/gr deletion carriers.

Long-Term Follow Up of the Erectile Function of Testicular Cancer Survivors.

The diagnosis of testicular cancer (TC) can have a considerable and persistent impact on a patient's sexuality, especially given its location. The high prevalence of TC in young adults, and the good prognosis, explain the great interest in sexual dysfunction and its influence on post-treatment quality of life. The aim of this study was to evaluate the impact of the diagnosis and treatments (inguinal orchiectomy and chemotherapy) on sex life. For this purpose, we recruited 241 TC patients attending the Laboratory of Seminology-Sperm Bank "Loredana Gandini" for sperm cryopreservation (mean age 31.3 ± 6.9 years) and 223 cancer-free healthy men who were undergoing andrological screening (mean age 32.0 ± 7.7 years). The IIEF-15 questionnaire was administered at the baseline (post-orchiectomy, pre-chemotherapy-T0) and at 6 (T1), 12 (T2), 18 (T3), 24 (T4), 48 months (T5) and >5 years (T6, median 96 months) after chemotherapy to all patients, to evaluate the following domains: erectile function (EF), orgasmic function (OF), sexual desire (SD), intercourse satisfaction (IS) and overall satisfaction (OS). A subgroup of patients also underwent blood sex hormone analysis for further correlations with IIEF scores. At the baseline, 37.7% of patients had erectile dysfunction (EF score <26) and all IIEF domains except OF showed significantly lower scores than in controls (p < 0.001). Long-term follow-up revealed persistently lower scores in TC survivors than in controls for EF, SD, IS, and OS. Furthermore, most IIEF domains did not improve significantly in TC patients during the duration of the follow-up, with the exception of EF, which showed a significant improvement from T2. Finally, no significant correlation was found between hormone levels (gonadotropin and testosterone) and IIEF-15 scores. In conclusion, TC and its treatment have a significant effect on sexuality. The absence of a clear correlation with biochemical hypogonadism suggests that this may to a large extent be due to the surgical procedure itself, or to the psychological impact of a cancer diagnosis.

Inhibin B: are modified ranges needed for orchiectomised testicular cancer patients?

Inhibin B is a gonadal hormone that downregulates the pituitary production of follicle-stimulating hormone (FSH). In recent years, inhibin B has proved to be an excellent marker of spermatogenesis and even a predictive factor for the recovery of fertility in patients undergoing orchiectomy and antineoplastic treatments. We propose to study inhibin B levels in orchiectomised testicular cancer patients, in order to identify a minimum value representative of normal semen quality. This retrospective study evaluates hormonal and semen parameters of 290 normozoospermic patients attending the Laboratory of Seminology - Sperm Bank "Loredana Gandini" (Rome, Italy) for cryopreservation of seminal fluid following a diagnosis of testicular cancer (TC group) and 117 healthy, normozoospermic men as a control group (CTR group). The percentile distribution of gonadotropin and inhibin B values in the TC and CTR groups was analyzed. There was a statistically significant difference between the two groups in the levels of all hormones (P ≤ 0.001) and in all semen parameters (P < 0.05). About 20% of TC patients revealed inhibin B levels below the 5th percentile of CTR group, despite normozoospermia, and 31.4% had normal spermatogenesis in the presence of FSH values >95th percentile of CTR group. Orchiectomised patients for testicular cancer presented inhibin B levels lower than healthy patients, despite normozoospermia. Our study revealed the poor sensitivity of the current inhibin B reference range when applied to monorchidic patients, suggesting the need to establish more representative ranges to enable more appropriate counseling in relation to the patient's new endocrine condition.

Cytological and molecular aspects of the ageing sperm.

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.

Fatherhood and Sperm DNA Damage in Testicular Cancer Patients.

Testicular cancer (TC) is one of the most treatable of all malignancies and the management of the quality of life of these patients is increasingly important, especially with regard to their sexuality and fertility. Survivors must overcome anxiety and fears about reduced fertility and possible pregnancy-related risks as well as health effects in offspring. There is thus a growing awareness of the need for reproductive counseling of cancer survivors. Studies found a high level of sperm DNA damage in TC patients in comparison with healthy, fertile controls, but no significant difference between these patients and infertile patients. Sperm DNA alterations due to cancer treatment persist from 2 to 5 years after the end of the treatment and may be influenced by both the type of therapy and the stage of the disease. Population studies reported a slightly reduced overall fertility of TC survivors and a more frequent use of ART than the general population, with a success rate of around 50%. Paternity after a diagnosis of cancer is an important issue and reproductive potential is becoming a major quality of life factor. Sperm chromatin instability associated with genome instability is the most important reproductive side effect related to the malignancy or its treatment. Studies investigating the magnitude of this damage could have a considerable translational importance in the management of cancer patients, as they could identify the time needed for the germ cell line to repair nuclear damage and thus produce gametes with a reduced risk for the offspring.

Differential expression of miRNAs in the seminal plasma and serum of testicular cancer patients.

Various microRNAs from the miR-371-3 and miR-302a-d clusters have recently been proposed as markers for testicular germ cell tumours. Upregulation of these miRNAs has been found in both the tissue and serum of testicular cancer patients, but they have never been studied in human seminal plasma. The aim of this study was, therefore, to assess the differences in the expression of miR-371-3 and miR-302a-d between the seminal plasma and serum of testicular cancer patients, and to identify new potential testicular cancer markers in seminal plasma. We investigated the serum and seminal plasma of 28 pre-orchiectomy patients subsequently diagnosed with testicular cancer, the seminal plasma of another 20 patients 30 days post-orchiectomy and a control group consisting of 28 cancer-free subjects attending our centre for an andrological check-up. Serum microRNA expression was analysed using RT-qPCR. TaqMan Array Card 3.0 platform was used for microRNA profiling in the seminal plasma of cancer patients. Results for both miR-371-3 and the miR-302 cluster in the serum of testicular cancer patients were in line with literature reports, while miR-371and miR-372 expression in seminal plasma showed the opposite trend to serum. On array analysis, 37 miRNAs were differentially expressed in the seminal plasma of cancer patients, and the upregulated miR-142 and the downregulated miR-34b were validated using RT-qPCR. Our study investigated the expression of miRNAs in the seminal plasma of patients with testicular cancer for the first time. Unlike in serum, miR-371-3 cannot be considered as markers in seminal plasma, whereas miR-142 levels in seminal plasma may be a potential marker for testicular cancer.

Influence of CAG Repeat Polymorphism on the Targets of Testosterone Action.

In the last decade, ample evidence has demonstrated the growing importance of androgen receptor (AR) CAG repeat polymorphism in andrology. This genetic parameter is able to condition the peripheral effects of testosterone and therefore to influence male sexual function and fertility, cardiovascular risk, body composition, bone metabolism, the risk of prostate and testicular cancer, the psychiatric status, and the onset of neurodegenerative disorders. In this review, we extensively discuss the literature data and identify a role for AR CAG repeat polymorphism in conditioning the systemic testosterone effects. In particular, our main purpose was to provide an updated text able to shed light on the many and often contradictory findings reporting an influence of CAG repeat polymorphism on the targets of testosterone action.