Variation of sperm quality and circular RNA content in men exposed to environmental contamination with heavy metals in 'Land of Fires', Italy.

STUDY QUESTION: Can illegal discharge of toxic waste into the environment induce a new condition of morpho-epigenetic pathozoospermia in normozoospermic young men? SUMMARY ANSWER: Toxic environmental contaminants promote the onset of a new pathozoospermic condition in young normozoospermic men, consisting of morpho-functional defects and a sperm increase of low-quality circular RNA (circRNA) cargo, tightly linked to contaminant bioaccumulation in seminal plasma. WHAT IS KNOWN ALREADY: Epidemiological findings have reported several reproductive anomalies depending on exposure to contaminants discharged into the environment, such as germ cell apoptosis, steroidogenesis defects, oxidative stress induction, blood-testis barrier dysfunctions, and poor sperm quality onset. In this scenario, a vast geographical area located in Campania, Italy, called the 'Land of Fires', has been associated with an excessive illegal discharge of toxic waste into the environment, negatively impacting human health, including male reproductive functions. STUDY DESIGN, SIZE, DURATION: Semen samples were obtained from healthy normozoospermic men divided into two experimental groups, consisting of men living in the 'Land of Fires' (LF; n = 80) or not (CTRL; n = 80), with age ranging from 25 to 40 years. The study was carried out following World Health Organization guidelines. PARTICIPANTS/MATERIALS, SETTING, METHODS: Quality parameters of semen from CTRL- and LF-normozoospermic men were evaluated by computer-assisted semen analysis; high-quality spermatozoa from CTRL and LF groups (n = 80 for each experimental group) were obtained using a 80-40% discontinuous centrifugation gradient. Seminal plasma was collected following centrifugation and used for the dosage of chemical elements, dioxins and steroid hormones by liquid chromatography with tandem mass spectrometry. Sperm morpho-functional investigations (cellular morphology, acrosome maturation, IZUMO1 fertility marker analysis, plasma membrane lipid state, oxidative stress) were assessed on the purified high-quality spermatozoa fraction by immunochemistry/immunofluorescence and western blot analyses. Sperm circRNA cargo was evaluated by quantitative RT-PCR, and the physical interaction among circRNAs and fused in sarcoma (FUS) protein was detected using an RNA-binding protein immunoprecipitation assay. Protein immunoprecipitation experiments were carried out to demonstrate FUS/p-300 protein interaction in sperm cells. Lastly, in vitro lead (Pb) treatment of high-quality spermatozoa collected from normozoospermic controls was used to investigate a correlation between Pb accumulation and onset of the morpho-epigenetic pathozoospermic phenotype. MAIN RESULTS AND THE ROLE OF CHANCE: Several morphological defects were identified in LF-spermatozoa, including: a significant increase (P < 0.05 versus CTRL) in the percentage of spermatozoa characterized by structural defects in sperm head and tail; and a high percentage (P < 0.01) of peanut agglutinin and IZUMO1 null signal cells. In agreement with these data, abnormal steroid hormone levels in LF seminal plasma suggest a premature acrosome reaction onset in LF-spermatozoa. The abnormal immunofluorescence signals of plasma membrane cholesterol complexes/lipid rafts organization (Filipin III and Flotillin-1) and of oxidative stress markers [3-nitrotyrosine and 3-nitrotyrosine and 4-hydroxy-2-nonenal] observed in LF-spermatozoa and associated with a sperm motility reduction (P < 0.01), demonstrated an affected membrane fluidity, potentially impacting sperm motility. Bioaccumulation of heavy metals and dioxins occurring in LF seminal plasma and a direct correlation between Pb and deregulated circRNAs related to high- and low-sperm quality was also revealed. In molecular terms, we demonstrated that Pb bioaccumulation promoted FUS hyperacetylation via physical interaction with p-300 and, in turn, its shuttling from sperm head to tail, significantly enhancing (P < 0.01 versus CTRL) the endogenous backsplicing of sperm low-quality circRNAs in LF-spermatozoa. LIMITATIONS, REASONS FOR CAUTION: Participants were interviewed to better understand their area of origin, their eating habits as well as their lifestyles, however any information incorrectly communicated or voluntarily omitted that could potentially compromise experimental group determination cannot be excluded. A possible association between seminal Pb content and other heavy metals in modulating sperm quality should be explored further. Future investigations will be performed in order to identify potential synergistic or anti-synergistic effects of heavy metals on male reproduction. WIDER IMPLICATIONS OF THE FINDINGS: Our study provides new findings regarding the effects of environmental contaminants on male reproduction, highlighting how a sperm phenotype classified as normozoospermic may potentially not match with a healthy morpho-functional and epigenetic one. Overall, our results improve the knowledge to allow a proper assessment of sperm quality through circRNAs as biomarkers to select spermatozoa with high morpho-epigenetic quality to use for ART. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by 'Convenzione Azienda Sanitaria Locale (ASL) Caserta, Regione Campania' (ASL CE Prot. N. 1217885/DIR. GE). The authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

CRISP2, CATSPER1 and PATE1 Expression in Human Asthenozoospermic Semen.

The etiology of human asthenozoospermia is multifactorial. The need to unveil molecular mechanisms underlying this state of infertility is, thus, impelling. Circular RNAs (circRNAs) are involved in microRNA (miRNA) inhibition by a sponge activity to protect mRNA targets. All together they form the competitive endogenous RNA network (ceRNET). Recently, we have identified differentially expressed circRNAs (DE-circRNAs) in normozoospermic and asthenozoospermic patients, associated with high-quality (A-spermatozoa) and low-quality (B-spermatozoa) sperm. Here, we carried out a differential analysis of CRISP2, CATSPER1 and PATE1 mRNA expression in good quality (A-spermatozoa) and low quality (B-spermatozoa) sperm fractions collected from both normozoospermic volunteers and asthenozoospermic patients. These sperm fractions are usually separated on the basis of morphology and motility parameters by a density gradient centrifugation. B-spermatozoa showed low levels of mRNAs. Thus, we identified the possible ceRNET responsible for regulating their expression by focusing on circTRIM2, circEPS15 and circRERE. With the idea that motility perturbations could be rooted in quantitative changes of transcripts in sperm, we evaluated circRNA and mRNA modulation in A-spermatozoa and B-spermatozoa after an oral amino acid supplementation known to improve sperm motility. The profiles of CRISP2, CATSPER1 and PATE1 proteins in the same fractions of sperm well matched with the transcript levels. Our data may strengthen the role of circRNAs in asthenozoospermia and shed light on the molecular pathways linked to sperm motility regulation.

CircRNA Role and circRNA-Dependent Network (ceRNET) in Asthenozoospermia.

The role of circRNA in reproduction is under investigation. CircRNAs are expressed in human testis, spermatozoa (SPZ), and seminal plasma. Their involvement in embryo development has also been suggested. Asthenozoospermia, a common cause of male infertility, is characterized by reduced or absent sperm motility in fresh ejaculate. While abnormal mitochondrial function, altered sperm tail, and genomic causes have been deeply investigated, the epigenetic signature of asthenozoospermic derived SPZ still remains unexplored. CircRNAs may take part in the repertoire of differentially expressed molecules in infertile men. Considering this background, we carried out a circRNA microarray, identifying a total of 9,138 transcripts, 22% of them novel based and 83.5% with an exonic structure. Using KEGG analysis, we evaluated the circRNA contribution in pathways related to mitochondrial function and sperm motility. In order to discriminate circRNAs with a differential expression in SPZ with differential morphological parameters, we separated sperm cells by Percoll gradient and analyzed their differential circRNA payload. A bioinformatic approach was then utilized to build a circRNA/miRNA/mRNA network. With the aim to demonstrate a dynamic contribution of circRNAs to the sperm epigenetic signature, we verified their modulation as a consequence of an oral amino acid supplementation, efficacious in improving SPZ motility.

Enhancing self-resources in patients with chronic diseases: development and initial validation of the Disease and Care Management Score.

INTRODUCTION: Despite the importance of the assessment in the primary care of the self-resources among patients with chronic diseases, there is not available a measurement that allows this kind of comprehensive assessment. For this reason, the aim of this study was to develop a multi-dimensional score to determine the level of self-resources in chronic patients, describing its initial validation through face and content validity. The developed score was labelled as Disease and Care Management Score. METHODS: We performed a methodological study, encompassing two main phases. The first phase was aimed to develop the Disease and Care Management score, choosing the most suitable measurement to assess each pre-identified determinant of wellbeing in chronic patients. The second phase was aimed to determine the Disease and Care Management score face and content validity through the views of 20 experts. RESULTS: Disease and Care Management score shows evidence of face and content validity. All the obtained quantitative content validity indices (i.e. Content Validity Ratio, Content Validity Indices) were higher than 0,70, showing the pertinence and the adequacy of each pre-identified measure to compute Disease and Care Management score. CONCLUSION: Disease and Care Management score has the potential of addressing the health coaching interventions in primary care for chronic patients. Future research should show its predictive performance, as well as the cut-off to discriminate patients.

Expression Patterns of Circular RNAs in High Quality and Poor Quality Human Spermatozoa.

Circular RNAs (circRNAs) are expressed in human testis and seminal plasma. Until today, there is missing information about a possible payload of circRNAs in human spermatozoa (SPZ). With this in mind, we carried out a circRNA microarray identifying a total of 10.726 transcripts, 28% novel based and 84.6% with exonic structure; their potential contribution in molecular pathways was evaluated by KEGG analysis. Whether circRNAs may be related to SPZ quality was speculated evaluating two different populations of SPZ (A SPZ = good quality, B SPZ = low quality), separated on the basis of morphology and motility parameters, by Percoll gradient. Thus, 148 differentially expressed (DE)-circRNAs were identified and the expression of selected specific SPZ-derived circRNAs was evaluated in SPZ head/tail-enriched preparations, to check the preservation of these molecules during SPZ maturation and their transfer into oocyte during fertilization. Lastly, circRNA/miRNA/mRNA network was built by bioinformatics approach.

CircNAPEPLD is expressed in human and murine spermatozoa and physically interacts with oocyte miRNAs.

Circular RNAs (circRNAs) have a critical role in the control of gene expression. Their function in spermatozoa (SPZ) is unknown to date. Twenty-eight genes, involved in SPZ/testicular and epididymal physiology, were given in circBase database to find which of them may generate circular transcripts. We focused on circNAPEPLDiso1, one of the circular RNA isoforms of NAPEPLD transcript, because expressed in human and murine SPZ. In order to functionally characterize circNAPEPLDiso1 as potential microRNA (miRNA) sponge, we performed circNAPEPLDiso1-miR-CATCH and then profiled the expression of 754 miRNAs, by using TaqMan® Low Density Arrays. Among them, miRNAs 146a-5p, 203a-3p, 302c-3p, 766-3p and 1260a (some of them previously shown to be expressed in the oocyte), resulted enriched in circNAPEPLDiso1-miR-CATCHed cell lysate: the network of interactions generated from their validated targets was centred on a core of genes involved in the control of cell cycle. Moreover, computational analysis of circNAPEPLDiso1 sequence also showed its potential translation in a short form of NAPEPLD protein. Interestingly, the expression analysis in murine-unfertilized oocytes revealed low and high levels of circNAPEPLDiso1 and circNAPEPLDiso2, respectively. After fertilization, circNAPEPLDiso1 expression significantly increased, instead circNAPEPLDiso2 expression appeared constant. Based on these data, we suggest that SPZ-derived circNAPEPLDiso1 physically interacts with miRNAs primarily involved in the control of cell cycle; we hypothesize that it may represent a paternal cytoplasmic contribution to the zygote and function as a miRNA decoy inside the fertilized oocytes to regulate the first stages of embryo development. This role is proposed here for the first time.

A Prospective Study for Introducing Insulin Pens and Safety Needles in a Hospital Setting. The SANITHY Study.

BACKGROUND: to assess costs and safety of insulin pen devices and safety needles as compared to vial/syringes in hospitalized patients requiring insulin therapy in a General Hospital in Northern Italy. MATERIALS AND METHODS: in a prospective 9-month study, consecutive patients admitted to three Hospital Units received insulin therapy through either a traditional disposable syringe method, or pen/safety needles with dual-ended protection, or disposable safety syringes. We compared the median direct (insulin and devices) and indirect (insulin supply at discharge, insulin wastage) costs of a 10-day in-hospital insulin treatment in the 3 study groups, additionally accounting for the costs related to the observed needlestick injury rate. Patients' safety during in-hospital stay (hypo- and hyperglycemia episodes) and satisfaction were also assessed. RESULTS: N=360 patients (55% men, mean age 75.6 years, 57% with DM since ≥10 years) were recruited in the study. Insulin pens had higher median direct cost than both traditional syringes (43 vs. 18 ε/patient, p<.0001) and safety syringes (21.5 ε/patient, p<.0001). However, when also indirect and injuries costs were taken into account, the estimated savings for using pens over traditional syringes were as high as 32 ε/patient (45.8 vs. 77.6 ε/patient, p-value <.0001). No differences in patients' safety were observed. 74% and 12% of patients using pens and syringes would like to continue the method at home, respectively (p<0.0001). DISCUSSION: A selective use of individual pre-filled pens/safety needles for patients who are likely to continue insulin therapy at home may strongly reduce hospital diabetes treatment related costs.