Improved synthesis and application of an alkyne-functionalized isoprenoid analogue to study the prenylomes of motor neurons, astrocytes and their stem cell progenitors.

Protein prenylation is one example of a broad class of post-translational modifications where proteins are covalently linked to various hydrophobic moieties. To globally identify and monitor levels of all prenylated proteins in a cell simultaneously, our laboratory and others have developed chemical proteomic approaches that rely on the metabolic incorporation of isoprenoid analogues bearing bio-orthogonal functionality followed by enrichment and subsequent quantitative proteomic analysis. Here, several improvements in the synthesis of the alkyne-containing isoprenoid analogue C15AlkOPP are reported to improve synthetic efficiency. Next, metabolic labeling with C15AlkOPP was optimized to obtain useful levels of metabolic incorporation of the probe in several types of primary cells. Those conditions were then used to study the prenylomes of motor neurons (ES-MNs), astrocytes (ES-As), and their embryonic stem cell progenitors (ESCs), which allowed for the identification of 54 prenylated proteins from ESCs, 50 from ES-MNs, and 84 from ES-As, representing all types of prenylation. Bioinformatic analysis revealed specific enriched pathways, including nervous system development, chemokine signaling, Rho GTPase signaling, and adhesion. Hierarchical clustering showed that most enriched pathways in all three cell types are related to GTPase activity and vesicular transport. In contrast, STRING analysis showed significant interactions in two populations that appear to be cell type dependent. The data provided herein demonstrates that robust incorporation of C15AlkOPP can be obtained in ES-MNs and related primary cells purified via magnetic-activated cell sorting allowing the identification and quantification of numerous prenylated proteins. These results suggest that metabolic labeling with C15AlkOPP should be an effective approach for investigating the role of prenylated proteins in primary cells in both normal cells and disease pathologies, including ALS.

Embryonic motor neuron programming factors reactivate immature gene expression and suppress ALS pathologies in postnatal motor neurons.

Aging is a major risk factor in amyotrophic lateral sclerosis (ALS) and other adult-onset neurodegenerative disorders. Whereas young neurons are capable of buffering disease-causing stresses, mature neurons lose this ability and degenerate over time. We hypothesized that the resilience of young motor neurons could be restored by re-expression of the embryonic motor neuron selector transcription factors ISL1 and LHX3. We found that viral re-expression of ISL1 and LHX3 reactivates aspects of the youthful gene expression program in mature motor neurons and alleviates key disease-relevant phenotypes in the SOD1G93A mouse model of ALS. Our results suggest that redeployment of lineage-specific neuronal selector transcription factors can be an effective strategy to attenuate age-dependent phenotypes in neurodegenerative disease.

Depletion of Mettl3 in cholinergic neurons causes adult-onset neuromuscular degeneration.

Motor neuron (MN) demise is a hallmark of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Post-transcriptional gene regulation can control RNA's fate, and defects in RNA processing are critical determinants of MN degeneration. N6-methyladenosine (m6A) is a post-transcriptional RNA modification that controls diverse aspects of RNA metabolism. To assess the m6A requirement in MNs, we depleted the m6A methyltransferase-like 3 (METTL3) in cells and mice. METTL3 depletion in embryonic stem cell-derived MNs has profound and selective effects on survival and neurite outgrowth. Mice with cholinergic neuron-specific METTL3 depletion display a progressive decline in motor behavior, accompanied by MN loss and muscle denervation, culminating in paralysis and death. Reader proteins convey m6A effects, and their silencing phenocopies METTL3 depletion. Among the m6A targets, we identified transactive response DNA-binding protein 43 (TDP-43) and discovered that its expression is under epitranscriptomic control. Thus, impaired m6A signaling disrupts MN homeostasis and triggers neurodegeneration conceivably through TDP-43 deregulation.

Improved synthesis and application of an alkyne-functionalized isoprenoid analogue to study the prenylomes of motor neurons, astrocytes and their stem cell progenitors.

Protein prenylation is one example of a broad class of post-translational modifications where proteins are covalently linked to various hydrophobic moieties. To globally identify and monitor levels of all prenylated proteins in a cell simultaneously, our laboratory and others have developed chemical proteomic approaches that rely on the metabolic incorporation of isoprenoid analogues bearing bio-orthogonal functionality followed by enrichment and subsequent quantitative proteomic analysis. Here, several improvements in the synthesis of the alkyne-containing isoprenoid analogue C15AlkOPP are reported to improve synthetic efficiency. Next, metabolic labeling with C15AlkOPP was optimized to obtain useful levels of metabolic incorporation of the probe in several types of primary cells. Those conditions were then used to study the prenylomes of motor neurons (ES-MNs), astrocytes (ES-As), and their embryonic stem cell progenitors (ESCs), which allowed for the identification of 54 prenylated proteins from ESCs, 50 from ES-MNs and 84 from ES-As, representing all types of prenylation. Bioinformatic analysis revealed specific enriched pathways, including nervous system development, chemokine signaling, Rho GTPase signaling, and adhesion. Hierarchical clustering showed that most enriched pathways in all three cell types are related to GTPase activity and vesicular transport. In contrast, STRING analysis showed significant interactions in two populations that appear to be cell type dependent. The data provided herein demonstrates that robust incorporation of C15AlkOPP can be obtained in ES-MNs and related primary cells purified via magnetic-activated cell sorting allowing the identification and quantification of numerous prenylated proteins. These results suggest that metabolic labeling with C15AlkOPP should be an effective approach for investigating the role of prenylated proteins in primary cells in both normal cells and disease pathologies, including ALS.

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

STUDY QUESTION: Does sperm DNA recover from damage in all men after 2 years from the end of cytotoxic treatments? SUMMARY ANSWER: The current indication of 2 years waiting time for seeking natural pregnancy after cytotoxic treatment may not be adequate for all men, since severe sperm DNA damage is present in a proportion of subjects even after this timeframe. WHAT IS KNOWN ALREADY: Data in the literature on sperm DNA fragmentation (SDF) in lymphoma patients after cytotoxic treatments are scarce. The largest longitudinal study evaluated paired pre- and post-therapy (up to 24 months) semen samples from 34 patients while one study performed a longer follow-up (36 months) in 10 patients. The median/mean SDF values >24 months after therapy did not show significant differences but the studies did not explore the proportion of patients with severe DNA damage and the analysis was done on frozen-thawed samples. STUDY DESIGN, SIZE, DURATION: In this study, 53 Hodgkin lymphoma (HL) and 25 non-Hodgkin lymphoma (NHL) post-pubertal patients were included over a recruitment period of 10 years (2012-2022). Among them, 18 subjects provided paired semen samples for SDF analysis at the three time points. SDF was evaluated in patients before (T0) and after 2 (T2) and 3 years (T3) from the end of, cytotoxic treatments (chemotherapy alone or in combination with radiotherapy). A cohort of 79 healthy, fertile, and normozoospermic men >18 years old served as controls (recruited between 2016 and 2019). PARTICIPANTS/MATERIALS, SETTING, METHODS: SDF was evaluated on fresh semen samples (i.e. spermatozoa potentially involved in natural conception) from patients and controls using TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay coupled with flow cytometry. SDF median values were compared between groups: (i) HL and NHL patients versus controls at the three time points; (ii) HL versus NHL patients at baseline; and (iii) patients at T0 versus T2 and T3. Severe DNA damage (SDD) was defined for SDF levels above the 95th percentile of controls (50%) and the proportion of patients with SDD at all time points was established. MAIN RESULTS AND THE ROLE OF CHANCE: At T0, patients displayed higher median SDF than controls, reaching statistical significance in the NHL group: 40.5% [IQR: 31.3-52.6%] versus 28% [IQR: 22-38%], P < 0.05. Comparing SDF pre-treatment to that post-treatment, HL patients exhibited similar median values at the three time points, whereas NHL showed significantly lower values at T3 compared to T0: 29.2% [IQR: 22-38%] versus 40.5% [IQR: 31.3-52.6%], P < 0.05. The proportion with SDD in the entire cohort at T2 was 11.6% and 13.3% among HL and NHL patients, respectively. At T3, only one in 16 NHL patients presented SDD. LIMITATIONS, REASONS FOR CAUTION: TUNEL assay requires at least 5 million spermatozoa to be performed; hence, severe oligozoospermic men were not included in the study. Although our cohort represents the largest one in the literature, the relatively small number of patients does not allow us to establish precisely the frequency of SDD at T2 which in our study reached 11-13% of patients. WIDER IMPLICATIONS OF THE FINDINGS: Our data provide further insights into the long-term effects of cytotoxic treatments on the sperm genome. The persistent severe DNA damage after 2 years post-treatment observed in some patients suggests that there is an interindividual variation in restoring DNA integrity. We propose the use of SDF as a biomarker to monitor the treatment-induced genotoxic effects on sperm DNA in order to better personalize pre-conceptional counseling on whether to use fresh or cryopreserved spermatozoa. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from the Istituto Toscano Tumori (ITT), Fondazione Ente Cassa di Risparmio di Firenze, the European Commission-Reproductive Biology Early Research Training (REPROTRAIN). C.K., G.F., V.R., and A.R.-E. belong to COST Action CA20119 (ANDRONET) which is supported by the European Cooperation in Science and Technology (www.cost.eu). The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Does an increase in adipose tissue 'weight' affect male fertility? A systematic review and meta-analysis based on semen analysis performed using the WHO 2010 criteria.

INTRODUCTION: Obesity negatively impact on the metabolism of sex hormones, leading to reduced testosterone serum levels. However, how the obesity could negatively impact on the overall gonadal function, particularly on male fertility, remained unclear so far. OBJECTIVE: To systematically review evidences regarding the influence of body weight excess on the sperm production. METHODS: A meta-analysis was conducted, searching all prospective and retrospective observational studies reporting male subjects older than 18 years old, with body weight excess from overweight to severe obesity were considered. Only studies using the V edition of the World Health Organization (WHO) manual for semen analysis interpretation were considered. No specific interventions were considered. Search was focused on studies comparing overweight/obese to normal weight subjects. RESULTS: Twenty-eight studies were considered. Total sperm count and sperm progressive motility were significantly lower in overweight compared to normal weight subjects. Meta-regression analyses demonstrated that patients' age impacted on sperm parameters. Similarly, obese men showed lower sperm concentration, total sperm number, progressive and total motilities, and normal morphology lower than normal weight subjects. Reduced sperm concentration in obese men was influenced by age, smoking habit, varicocele, and total testosterone serum levels at meta-regression analyses. CONCLUSIONS: The male potential fertility is reduced in subjects with increased body weight, compared to normal weight men. The higher was the increased body weight, the worst was the sperm quantity/quality. This result comprehensively included obesity among non-communicable risk factor for male infertility, shedding new lights on the negative impact of increased body weight on overall gonadal function.

Multiparametric Ultrasound for Diagnosing Testicular Lesions: Everything You Need to Know in Daily Clinical Practice.

BACKGROUND: Ultrasonography (US) represents the gold standard imaging method for the assessment of testicular lesions (TL). The gray-scale (GSUS) and color-Doppler (CDUS) ultrasound examination allow sonographers to investigate the size, margins, echotexture, and vascular features of TLs with the aim to differentiate benign from malignant lesions. Recently, the use of contrast-enhanced US (CEUS) and sonoelastography (SE) has led to further improvements in the differential diagnosis of TL. Although GSUS and CDUS are often sufficient to suggest the benign or malignant nature of the TL, CEUS can be decisive in the differential diagnosis of unclear findings, while SE can help to strengthen the diagnosis. The contemporary combination of GSUS, CDUS, CEUS, and SE has led to a new diagnostic paradigm named multiparametric US (mp-US), which is able to provide a more detailed characterization of TLs than single techniques alone. This narrative and pictorial review aimed to describe the mp-US appearance of several TLs. METHODS: An extensive Medline search was performed to identify studies in the English language focusing on the mp-US evaluation of TLs. RESULTS: A practical mp-US "identity card" and iconographic characterization of several benign and malignant TLs is provided herein. CONCLUSIONS: The mp-US characterization of TL reported herein can be useful in daily clinical practice.

Semen Cryopreservation for Men Banking for Oligozoospermia, Cancers, and Other Conditions: 24 Years' Experience of an Italian Bank.

BACKGROUND: Sperm cryopreservation is recommended to preserve male fertility for cancer patients or other medical conditions at risk of sperm decline. Whether motility and viability recovery rates vary depending on the medical conditions requiring cryopreservation is poorly known. We report here on the 24-year experience of our semen bank. METHODS: Motility and viability recovery rates were evaluated in 1973 collections from patients with various medical conditions and 67 collections from donors, and the results were related to basal semen quality. RESULTS: Motility and viability recovery were highly related to basal semen quality and varied between cancer and non-cancer conditions, independently of the duration of cryopreservation and patient age. In samples with a sperm number below 2 × 106/mL, recovery rates approximated to zero. The highest recovery rates were found in donor collections. Cut-off values for the recovery of at least 1% motile spermatozoa were established based on initial semen quality. CONCLUSIONS: Our results indicate that the occurrence of any pathological or medical condition resulted in lower recovery rates with respect to donors, indicating that intrinsic sperm characteristics drive susceptibility to cryodamage. Established cut-off values for motility recovery can be useful for patient counseling as well as for ART laboratories to decide the type of procedure.

Brain Calcifications: Genetic, Molecular, and Clinical Aspects.

Many conditions can present with accumulation of calcium in the brain and manifest with a variety of neurological symptoms. Brain calcifications can be primary (idiopathic or genetic) or secondary to various pathological conditions (e.g., calcium-phosphate metabolism derangement, autoimmune disorders and infections, among others). A set of causative genes associated with primary familial brain calcification (PFBC) has now been identified, and include genes such as SLC20A2, PDGFB, PDGFRB, XPR1, MYORG, and JAM2. However, many more genes are known to be linked with complex syndromes characterized by brain calcifications and additional neurologic and systemic manifestations. Of note, many of these genes encode for proteins involved in cerebrovascular and blood-brain barrier functions, which both represent key anatomical structures related to these pathological phenomena. As a growing number of genes associated with brain calcifications is identified, pathways involved in these conditions are beginning to be understood. Our comprehensive review of the genetic, molecular, and clinical aspects of brain calcifications offers a framework for clinicians and researchers in the field.

SMN post-translational modifications in spinal muscular atrophy.

Since its first identification as the gene responsible for spinal muscular atrophy (SMA), the range of survival motor neuron (SMN) protein functions has increasingly expanded. This multimeric complex plays a crucial role in a variety of RNA processing pathways. While its most characterized function is in the biogenesis of ribonucleoproteins, several studies have highlighted the SMN complex as an important contributor to mRNA trafficking and translation, axonal transport, endocytosis, and mitochondria metabolism. All these multiple functions need to be selectively and finely modulated to maintain cellular homeostasis. SMN has distinct functional domains that play a crucial role in complex stability, function, and subcellular distribution. Many different processes were reported as modulators of the SMN complex activities, although their contribution to SMN biology still needs to be elucidated. Recent evidence has identified post-translational modifications (PTMs) as a way to regulate the pleiotropic functions of the SMN complex. These modifications include phosphorylation, methylation, ubiquitination, acetylation, sumoylation, and many other types. PTMs can broaden the range of protein functions by binding chemical moieties to specific amino acids, thus modulating several cellular processes. Here, we provide an overview of the main PTMs involved in the regulation of the SMN complex with a major focus on the functions that have been linked to SMA pathogenesis.

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

INTRODUCTION: Testicular germ cell tumor is the most frequent neoplasia in men of reproductive age, with a 5-year survival rate of 95%. Antineoplastic treatments induce sperm DNA fragmentation, especially within the first year post-therapy. Data in the literature are heterogeneous concerning longer follow-up periods, and the large majority is limited to 2 years. OBJECTIVE: To define the timing for the recovery of sperm DNA damage and the proportion of patients with severe DNA damage at 2 and 3 years from the end of therapy. MATERIALS AND METHODS: Sperm DNA fragmentation was evaluated in 115 testicular germ cell tumor patients using terminal deoxynucleotidyl transferase dUTP nick end labeling assay coupled with flow cytometry before (T0 ) and 2 (T2 ) and 3 (T3 ) years post-treatment. Patients were divided based on the type of treatment: carboplatin, bleomycin-etoposide-cisplatin, and radiotherapy. For 24 patients, paired sperm DNA fragmentation data were available at all time-points (T0 -T2 -T3 ). Seventy-nine cancer-free, fertile normozoospermic men served as controls. Severe DNA damage was defined as the 95th percentile in controls (sperm DNA fragmentation = 50%). RESULTS: Comparing patients versus controls, we observed: (i) no differences at T0 and T3 and (ii) significantly higher sperm DNA fragmentation levels (p < 0.05) at T2 in all treatment groups. Comparing pre- and post-therapy in the 115 patients, the median sperm DNA fragmentation values were higher in all groups at T2 , reaching significance (p < 0.05) only in the carboplatin group. While the median sperm DNA fragmentation values were also higher in the strictly paired cohort at T2 , about 50% of patients returned to baseline. The proportion of severe DNA damage in the entire cohort was 23.4% and 4.8% of patients at T2 and T3 , respectively. DISCUSSION: Currently, testicular germ cell tumor patients are advised to wait 2 years post-therapy before seeking natural pregnancy. Our results suggest that this period may not be sufficient for all patients. CONCLUSION: The analysis of sperm DNA fragmentation may represent a useful biomarker for pre-conception counseling following cancer treatment.

The SMN Complex at the Crossroad between RNA Metabolism and Neurodegeneration.

In the cell, RNA exists and functions in a complex with RNA binding proteins (RBPs) that regulate each step of the RNA life cycle from transcription to degradation. Central to this regulation is the role of several molecular chaperones that ensure the correct interactions between RNA and proteins, while aiding the biogenesis of large RNA-protein complexes (ribonucleoproteins or RNPs). Accurate formation of RNPs is fundamentally important to cellular development and function, and its impairment often leads to disease. The survival motor neuron (SMN) protein exemplifies this biological paradigm. SMN is part of a multi-protein complex essential for the biogenesis of various RNPs that function in RNA metabolism. Mutations leading to SMN deficiency cause the neurodegenerative disease spinal muscular atrophy (SMA). A fundamental question in SMA biology is how selective motor system dysfunction results from reduced levels of the ubiquitously expressed SMN protein. Recent clarification of the central role of the SMN complex in RNA metabolism and a thorough characterization of animal models of SMA have significantly advanced our knowledge of the molecular basis of the disease. Here we review the expanding role of SMN in the regulation of gene expression through its multiple functions in RNP biogenesis. We discuss developments in our understanding of SMN activity as a molecular chaperone of RNPs and how disruption of SMN-dependent RNA pathways can contribute to the SMA phenotype.

Effects of diabetes mellitus on sperm quality and fertility outcomes: Clinical evidence.

BACKGROUND: Diabetes mellitus is a global epidemic characterized by high morbidity and mortality. Diabetes mellitus can lead to acute and chronic systemic complications. Among them, a negative effect of diabetes mellitus on sperm quality and male/couple fertility has been suggested. However, available studies in diabetes mellitus men evaluated relatively small cohorts with discordant results. OBJECTIVES: To evaluate the clinical evidences of the effects of diabetes mellitus on sperm quality and fertility outcomes. METHODS: An extensive Medline search was performed identifying studies in the English language. RESULTS: The prevalence of diabetes mellitus in infertile men ranges from 0.7% to 1.4%, while the prevalence of infertility in diabetes mellitus men, evaluated in a few studies, ranges from 35% to 51%. Male diabetes mellitus seems to play a negative effect on couple fecundity, while being childless or subfertile men might increase the risk of diabetes mellitus. Available cross-sectional studies investigating semen parameters and male sexual hormones in men with diabetes mellitus are heterogeneous, assessed relatively small cohorts, show often discordant results and frequently are not directly comparable to derive robust conclusions. Two meta-analyses support a negative effect of diabetes mellitus on sperm normal morphology and no effect on sperm total count, with contradictory results regarding other semen parameters. Considering only studies on type 1 diabetes mellitus men, meta-analyses support a negative effect of diabetes mellitus on sperm motility and no effect on sperm total count, with contradictory results regarding other semen parameters. The rate of children observed among type 1 diabetes mellitus men was lower than controls, especially in subjects with a longer diabetes mellitus duration. Couples with a diabetes mellitus male partner undergoing assisted reproduction techniques showed lower pregnancy rates than controls. No study evaluated the impact of diabetes mellitus treatment on semen quality and male fertility. CONCLUSIONS: Overall, available data show that diabetes mellitus might impair male reproductive health and couple fertility. However, further larger and full of details studies are needed.

Hypogonadism and sexual function in men affected by adrenocortical carcinoma under mitotane therapy.

Purpose: Adrenocortical carcinoma (ACC) is a rare and aggressive tumor. ACC male patients under adjuvant mitotane therapy (AMT) frequently develop hypogonadism, however sexual function has never been assessed in this setting. The aim of this retrospective study was to evaluate in AMT treated ACC patients the changes in Luteinizing hormone (LH), Sex Hormone Binding Globulin (SHBG), total testosterone (TT) and calculated free testosterone (cFT), the prevalence and type of hypogonadism and sexual function, the latter before and after androgen replacement therapy (ART). Methods: LH, SHBG, TT and cFT were assessed in ten ACC patients at baseline (T0) and six (T1), twelve (T2), and eighteen (T3) months after AMT. At T3, ART was initiated in eight hypogonadal patients, and LH, SHBG, TT and cFT levels were evaluated after six months (T4). In six patients, sexual function was evaluated before (T3) and after (T4) ART using the International Index of Erectile Function-15 (IIEF-15) questionnaire. Results: Under AMT we observed higher SHBG and LH and lower cFT levels at T1-T3 compared to T0 (all p<0.05). At T3, hypergonadotropic hypogonadism and erectile dysfunction (ED) were detected in 80% and 83.3% of cases. At T4, we observed a significant cFT increase in men treated with T gel, and a significant improvement in IIEF-15 total and subdomains scores and ED prevalence (16.7%) in men under ART. Conclusion: AMT was associated with hypergonatropic hypogonadism and ED, while ART led to a significant improvement of cFT levels and sexual function in the hypogonadal ACC patients. Therefore, we suggest to evaluate LH, SHBG, TT and cFT and sexual function during AMT, and start ART in the hypogonadal ACC patients with sexual dysfunction.

Strongly acidic gastroesophageal reflux and esophageal lumen pH before and after esophageal lavage with water or two bicarbonate concentrations in anesthetized dogs.

OBJECTIVE: To increase acidic esophageal lumen pH in dogs that developed gastroesophageal reflux (GER) during anesthesia. We compared water and 2 different bicarbonate concentrations. ANIMALS: 112 healthy, nonbrachycephalic dogs presented for ovariectomy. PROCEDURES: Following standard anesthesia and surgery protocols for ovariectomy in all dogs, esophageal lumen impedance and pH were monitored using a dedicated probe. Esophageal impedance indicates the presence of GER whereas pH indicates the acidity level. Dogs with strongly acidic GER and an esophageal lumen pH value < 4.0 were included in the study, and lavage was performed with either tap water, bicarbonate 1%, or bicarbonate 2% until the pH increased to > 4.0. The effect of lavage on esophageal pH was compared using the Kruskal-Wallis and Wilcoxon 2 sample tests. Associations between lavage and pH changes were determined. RESULTS: Of 48/112 dogs with strongly acidic GER, 33% neutralized their esophageal pH during surgery. For the 32 dogs that maintained an esophageal lumen pH value < 4, esophageal lavage with water increased the lumen pH to > 4 in 78.6% of dogs, whereas both bicarbonate concentrations increased it in 100% of the dogs to a more neutral pH (P < .0001). The dogs in the water group were more likely to regurgitate after anesthesia (36% vs 0% in both bicarbonate groups, P = .028). CLINICAL RELEVANCE: Bicarbonate 1% and 2% increased esophageal lumen pH to more than 4 after strongly acidic GER. Lavage with water was mildly effective, but required large volumes and predisposed to further regurgitation after anesthesia.

Motoneuron Diseases.

Motoneuron diseases (MNDs) represent a heterogeneous group of progressive paralytic disorders, mainly characterized by the loss of upper (corticospinal) motoneurons, lower (spinal) motoneurons or, often both. MNDs can occur from birth to adulthood and have a highly variable clinical presentation, even within gene-positive forms, suggesting the existence of environmental and genetic modifiers. A combination of cell autonomous and non-cell autonomous mechanisms contributes to motoneuron degeneration in MNDs, suggesting multifactorial pathogenic processes.

The European Academy of Andrology (EAA) ultrasound study on healthy, fertile men: An overview on male genital tract ultrasound reference ranges.

BACKGROUND: So far, male genital tract color-Doppler ultrasound (MGT-CDUS) was not standardized. Recently, the European Academy of Andrology (EAA) published the results of a multicenter study assessing the CDUS characteristics of healthy-fertile men (HFM) to obtain normative parameters. OBJECTIVES: To report the EAA US study (i) standard operating procedures (SOPs) for assessing MGT-CDUS, (ii) main MGT-CDUS normative parameters, and (iii) compare the EAA and previously published "normal" CDUS values. METHODS: A cohort of 248 HFM (35.3 ± 5.9 years) was studied, evaluating MGT-CDUS before and after ejaculation following SOPs. RESULTS: SOPs for MGT-CDUS assessment are summarized here. All subjects underwent scrotal CDUS and 188 men underwent transrectal ultrasound before and after ejaculation. The main CDUS reference ranges and characteristics of the HFM-MGT are reported here. The mean testicular volume was ∼17 mL. The lower limit for right and left testis was 12 and 11 mL, defining testicular hypotrophy. The upper limit for epididymal head, body, tail, and vas deferens was 11.5, 5, 6, and 4.5 mm, respectively. Testicular and epididymal arterial reference ranges are reported. The EAA varicocoele classification is reported. CDUS-varicocoele was detected in ∼37% of men. Prostate mean volume was ∼25 mL, while lower and upper limits were 15 and 35 mL, defining hypotrophy and enlargement, respectively. Prostate arterial reference ranges are reported. Prostate calcifications and inhomogeneity were frequent; midline prostatic cysts were rare and small. Ejaculatory duct abnormalities were absent. The upper limit for periprostatic venous plexus was 4.5 mm. Lower and upper limits of seminal vesicles (SV) anterior-posterior diameter were 6 and 16 mm, defining hypotrophy or dilation, respectively. Seminal vesicle volume and ejection fraction reference ranges are reported. SV-US abnormalities were rare. Deferential ampullas upper limit was 6 mm. A discussion on the EAA and previously published "normal" CDUS values is reported here. CONCLUSIONS: The EAA findings will help in reproductive and general male health management.

The European Academy of Andrology (EAA) ultrasound study on healthy, fertile men: Prostate-vesicular transrectal ultrasound reference ranges and associations with clinical, seminal and biochemical characteristics.

BACKGROUND: Transrectal ultrasound (TRUS) parameters are not standardized, especially in men of reproductive age. Hence, the European Academy of Andrology (EAA) promoted a multicenter study to assess the TRUS characteristics of healthy-fertile men (HFM) to establish normative parameters. OBJECTIVES: To report and discuss the prostate and seminal vesicles (SV) reference ranges and characteristics in HFM and their associations with clinical, seminal, biochemical parameters. METHODS: 188 men (35.6 ± 6.0 years) from a cohort of 248 HFM were studied, evaluating, on the same day, clinical, biochemical, seminal, TRUS parameters following Standard Operating Procedures. RESULTS: TRUS reference ranges and characteristics of the prostate and SV of HFM are reported herein. The mean PV was ∼25 ml. PV lower and upper limits were 15 and 35 ml, defining prostate hypotrophy and enlargement, respectively. PV was positively associated with age, waistline, current smoking (but not with T levels), seminal volume (and negatively with seminal pH), prostate inhomogeneity, macrocalcifications, calcification size and prostate arterial parameters, SV volume before and after ejaculation, deferential and epididymal size. Prostate calcifications and inhomogeneity were frequent, while midline prostatic cysts were rare and small. Ejaculatory duct abnormalities were absent. Periprostatic venous plexus size was positively associated with prostate calcifications, SV volume and arterial peak systolic velocity. Lower and upper limits of SV anterior-posterior diameter after ejaculation were 6 and 16 mm, defining SV hypotrophy or dilation, respectively. SV total volume before ejaculation and delta SV total volume (DSTV) positively correlated with ejaculate volume, and DSTV correlated positively with sperm progressive motility. SV total volume after ejaculation was associated negatively with SV ejection fraction and positively with distal ampullas size. SV US abnormalities were rare. No association between TRUS and time to pregnancy, number of children or history of miscarriage was observed. CONCLUSIONS: The present findings will help in better understanding male infertility pathophysiology and the meaning of specific TRUS findings.

Retromer dysfunction in amyotrophic lateral sclerosis.

Retromer is a heteropentameric complex that plays a specialized role in endosomal protein sorting and trafficking. Here, we report a reduction in the retromer proteins-vacuolar protein sorting 35 (VPS35), VPS26A, and VPS29-in patients with amyotrophic lateral sclerosis (ALS) and in the ALS model provided by transgenic (Tg) mice expressing the mutant superoxide dismutase-1 G93A. These changes are accompanied by a reduction of levels of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit GluA1, a proxy of retromer function, in spinal cords from Tg SOD1G93A mice. Correction of the retromer deficit by a viral vector expressing VPS35 exacerbates the paralytic phenotype in Tg SOD1G93A mice. Conversely, lowering Vps35 levels in Tg SOD1G93A mice ameliorates the disease phenotype. In light of these findings, we propose that mild alterations in retromer inversely modulate neurodegeneration propensity in ALS.