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.

Sperm DNA Fragmentation and Sperm-Borne miRNAs: Molecular Biomarkers of Embryo Development?

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 profile­we 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.

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.

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.

Polymorphic Cytosine-Adenine-Guanine Repeat Length of Androgen Receptor Gene and Gender Incongruence in Trans Women: A Systematic Review and Meta-Analysis of Case-Control Studies.

INTRODUCTION: It has been hypothesized that gender incongruence in transgender women could result from an antenatal impaired androgen activity on the developing brain. As the length of polymorphic cytosine-adenine-guanine (CAG) repeat sequences in the androgen receptor (AR) gene is inversely correlated with AR transcriptional activity, some studies explored a possible association between long CAG repeats and gender incongruence in trangender women. Yet results remain inconclusive. AIM: To systematically evaluate whether a difference exists in the length of AR CAG repeat sequences between trans women and men without gender incongruence. METHODS: A thorough search of Medline, Scopus, Cochrane Library, Web of Science, and CINAHL databases was carried out to identify suitable case-control studies. Methodological quality of the included articles was assessed using the Newcastle-Ottawa Scale. In the absence of between-studies heterogeneity, as assessed by the Cochrane's Q and I2 tests, standardized mean differences (SMDs) in the length of AR CAG repeats were combined using a fixed effect model. Funnel plot and trim-and-fill analysis were used to assess publication bias. MAIN OUTCOME MEASURE: The association of gender incongruence in transgender women with longer length of AR CAG repeat sequences was evaluated by calculating pooled standardized mean difference with 95% confidence interval (CI). RESULTS: 5 studies included in the quantitative analysis collectively provided information on 795 trans women and 1,355 control men. At the overall estimate, the MtF group exhibited a significantly longer length of AR CAG repeat sequences (pooled standardized mean difference: 0.13, 95% CI: 0.04 to 0.22; P = 0.005; I2 = 0%, Pfor heterogeneity = 0.51). Sensitivity analysis demonstrated the high stability of the result. Funnel plot revealed a possible publication bias, and the trim-and-fill test detected 2 putative missing studies. Nevertheless, the significant association persisted even when pooled estimate was adjusted for publication bias. CLINICAL IMPLICATIONS: These findings could suggest a contribution of a genetically mediated impairment in androgen signaling in development of gender incongruence for transgender women. STRENGTH & LIMITATIONS: This is the first meta-analysis exploring the relationship between AR CAG repeat polymorphism and gender incongruence. However, interactions with other functional genetic variants were not explored, and caution should be exercised when generalizing these results because of the possible variability in the distribution of CAG repeats among different populations and ethnic groups. CONCLUSION: Trans woman population exhibits significantly longer polymorphic CAG repeat sequences in the AR gene. Further studies are warranted to elucidate whether, how and to what extent multiple functional variants in sex hormone signaling genes could be associated with gender incongruence/dysphoria. D'Andrea S, Pallotti F, Senofonte G, et al. Polymorphic Cytosine-Adenine-Guanine Repeat Length of Androgen Receptor Gene and Gender Incongruence in Trans Women: A Systematic Review and Meta-Analysis of Case-Control Studies. J Sex Med 2020;17:543-550.

Seminal plasma miRNAs in Klinefelter syndrome and in obstructive and non-obstructive azoospermia.

MicroRNAs are small, non-coding, single-strand oligonucleotides which regulate gene expression. There is little evidence in the literature about their role in azoospermia and no studies have investigated their presence in the seminal plasma of men with Klinefelter syndrome. This retrospective study investigated if there were any differences in microRNA expression (miR-509-5p, miR-122-5p, miR-34b-3p, miR-34c-5p) in the seminal plasma of patients with obstructive azoospermia, non-obstructive azoospermia and Klinefelter syndrome. Hormone levels were also investigated to identify any correlations with microRNA expression. We analysed 200 subjects (40 Klinefelter syndrome, 60 non-obstructive azoospermia with a normal karyotype, 60 obstructive azoospermia and 40 who were normozoospermic). All subjects underwent semen examination. Total RNA was obtained from seminal plasma and microRNA expression was analysed by RT-qPCR. There was a significant reduction in the expression of all investigated miRNAs in the seminal plasma of all patient categories in comparison with controls. There was a weak negative correlation between FSH values and miR-509-5p expression in non-obstructive azoospermic patients (r = - 0.391; p = 0.014). We hypothesize that in non-obstructive azoospermia and Klinefelter syndrome patients, the downregulation of microRNAs may be caused by damage to the germ cells and aberrant spermatogenesis. In our opinion the identification of seminal plasma microRNAs deriving almost exclusively from the testes could be essential for the development of specific biomarkers for male infertility. The expression of such microRNAs, in combination with hormone values, could comprise testicular markers of abnormal spermatogenesis and failed mature sperm production.

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.

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.

Human sperm motility: a molecular study of mitochondrial DNA, mitochondrial transcription factor A gene and DNA fragmentation.

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.

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.

Sperm DNA fragmentation in Italian couples with recurrent pregnancy loss.

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.

Sperm cryopreservation: effects on chromatin structure.

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. More studies are needed to establish the true importance of such damage, especially to improve the results of ART.

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.

COVID-19 and its treatments: lights and shadows on testicular function.

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.

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.

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.

Higher clusterin immunolabeling and sperm DNA damage levels in hypertensive men compared with controls.

BACKGROUND: Clusterin, a heterodimeric glycoprotein found at several sites in the human male reproductive tract, could be a marker of morphologically abnormal spermatozoa, while TUNEL positivity indicates DNA fragmentation. Metabolic disorders such as diabetes mellitus and obesity may compromise sperm quality and fertility of men; however, little evidence specifically links hypertension with the impairment of male reproductive function. METHODS: By flow cytometric, immunofluorescence (TUNEL assay and clusterin immunolabeling) and immunohistochemical (peroxidase-streptavidin method) analyses, we have compared both clusterin- and TUNEL labeling in ejaculated spermatozoa from healthy normotensive donors and hypertensive subjects with the purpose to reveal possible differences between the two conditions. RESULTS: Data analysis from the normotensive (n=25) and hypertensive subjects (n=25) demonstrate a significant correlation between high levels of clusterin immunolabeling and the presence of sperm DNA damage, which is often associated with abnormal morphology. In the normotensive subjects, a low percentage (15.3±4.5) of spermatozoa positive for high levels of clusterin was detected; however, this percentage significantly increased (30.9±13.0) (P<0.01) in hypertensive subjects. Standard semen evaluations does not reveal any significant differences between the two groups of subjects, except for a reduced forward motility and lower sperm vitality in the hypertensive subjects. CONCLUSIONS: This pilot study strongly suggests a relationship between hypertension and markers indicative of poor sperm quality. In hypertensive subjects, high levels of clusterin immunolabeling identified a consistent fraction of ejaculated spermatozoa carrying both DNA fragmentation and strong morphological alterations, which was not correlated with age or with sperm cell mortality. The alternative possibility that sperm damage observed is due to adverse effects of anti-hypertensive drugs does not find support in the literature nor in the drug data sheets. The relationship observed between hypertension and human semen represents a novel and possibly relevant information to be considered in the study of male fertility.

Comment on Boitrelle et al. The Sixth Edition of the WHO Manual for Human Semen Analysis: A Critical Review and SWOT Analysis. Life 2021, 11, 1368.

We wish to congratulate Boitrelle and colleagues for their comprehensive critical review on the Sixth Edition of the WHO Laboratory Manual for Human semen examination [...].

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.

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.