Variation of sperm quality and circular RNA content in men exposed to environmental contamination with heavy metals in ‘Land of Fires’, Italy
Authors: Grazia Mele Vincenza , Chioccarelli Teresa , Diano Nadia , Cappetta Donato , Ferraro Bruno , Telesca Marialucia , Moggio Martina , Porreca Veronica , De Angelis Antonella , Berrino Liberato , Fasano Silvia , Cobellis Gilda , Chianese Rosanna , Manfrevola Francesco 
Published: June 2024

Abstract

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 &lt; 0.05 versus CTRL) in the percentage of spermatozoa characterized by structural defects in sperm head and tail; and a high percentage (P &lt; 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 &lt; 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 &lt; 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.