Polymorphic Rearrangements of Human Chromosome 9 and Male Infertility: New Evidence and Impact on Spermatogenesis.

Chromosomal polymorphisms are structural variations in chromosomes that define the genomic variance of a species. These alterations are recurrent in the general population, and some of them appear to be more recurrent in the infertile population. Human chromosome 9 is highly heteromorphic, and how its rearrangement affects male fertility remains to be fully investigated. In this study, we aimed to investigate the association between the polymorphic rearrangements of chromosome 9 and male infertility via an Italian cohort of male infertile patients. Cytogenetic analysis was carried out, along with Y microdeletion screening, semen analysis, fluorescence in situ hybridization, and TUNEL assays using spermatic cells. Chromosome 9 rearrangements were observed in six patients: three of them showed a pericentric inversion, while the others showed a polymorphic heterochromatin variant 9qh. Of these, four patients exhibited oligozoospermia associated with teratozoospermia, along with a percentage of aneuploidy in the sperm of above 9%, in particular, an increase in XY disomy. Additionally, high values for sperm DNA fragmentation (≥30%) were observed in two patients. None of them had microdeletions to the AZF loci on chromosome Y. Our results suggest that polymorphic rearrangements of chromosome 9 might be associated with abnormalities in sperm quality due to incorrect spermatogenesis regulation.

In Vitro Combination of Ascorbic and Ellagic Acids in Sperm Oxidative Damage Inhibition.

It is known that an altered redox balance interferes with normal spermatic functions. Exposure to genotoxic substances capable of producing oxidative stress (OS) can cause infertility in humans. The use of antioxidants to reduce oxidative stress contributes to the improvement in reproductive function. This study focused on an antigenotoxic evaluation of ellagic acid (EA) and ascorbic acid (AA) in combination against benzene genotoxic action on human spermatozoa in vitro. In addition to the evaluation of sperm parameters, damage in sperm genetic material and intracellular ROS quantification were assessed after AA, EA and benzene co-exposure using the TUNEL technique and DCF assay. The results showed that the combination of the two antioxidants generates a greater time-dependent antigenotoxic action, reducing both the sperm DNA fragmentation index and the oxidative stress. The genoprotective effect of AA and EA association in sperm cells lays the foundations for a more in-depth clinical study on the use of antioxidants as a therapy for male infertility.

Impact of Escherichia coli Outer Membrane Vesicles on Sperm Function.

Reproductive tract infections account for approximately 15% of male infertility cases. Escherichia coli (E. coli) represents the most frequently isolated bacterial strain in the semen of infertile men. All Gram-negative bacteria constitutively produce outer membrane vesicles (OMVs). The present study proved, for the first time, the involvement of OMVs in human sperm function. E. coli OMVs were isolated by ultracentrifugation and characterized via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transmission electron microscopy (TEM) and dynamic light scattering (DLS) analysis. Human sperm was exposed to OMVs (8 µg/mL) for different times (30, 45, 60 and 90 min). The vitality, motility, morphology, ROS level and DNA fragmentation of spermatozoa were evaluated. OMVs reduced the progressive motility and increased the immobile spermatozoa amount after 30 min of treatment. In addition, a significant increase in the percentage of intracellular ROS and sperm DNA fragmentation was recorded for each vesicular exposure time. These preliminary findings prove that OMVs contribute to altering human sperm function via two mechanisms: (i) impaired motility and (ii) DNA fragmentation.

Evaluation of Zebrafish DNA Integrity after Individual and Combined Exposure to TiO2 Nanoparticles and Lincomycin.

Environmental contamination by nanoparticles (NPs) and drugs represents one of the most debated issues of the last years. The aquatic biome and, indirectly, human health are strongly influenced by the negative effects induced by the widespread presence of pharmaceutical products in wastewater, mainly due to the massive use of antibiotics and inefficient treatment of the waters. The present study aimed to evaluate the harmful consequences due to exposure to antibiotics and NPs, alone and in combination, in the aquatic environment. By exploiting some of their peculiar characteristics, such as small size and ability to bind different types of substances, NPs can carry drugs into the body, showing potential genotoxic effects. The research was conducted on zebrafish (Danio rerio) exposed in vivo to lincomycin (100 mg/L) and titanium dioxide nanoparticles (TiO2 NPs) (10 µg/L) for 7 and 14 exposure days. The effects on zebrafish were evaluated in terms of cell viability, DNA fragmentation, and genomic template stability (GTS%) investigated using Trypan blue staining, TUNEL assay, and the random amplification of polymorphic DNA PCR (RAPD PCR) technique, respectively. Our results show that after TiO2 NPs exposure, as well as after TiO2 NPs and lincomycin co-exposure, the percentage of damaged DNA significantly increased and cell viability decreased. On the contrary, exposure to lincomycin alone caused only a GTS% reduction after 14 exposure days. Therefore, the results allow us to assert that genotoxic effect in target cells could be through a synergistic effect, also potentially mediated by the establishment of intermolecular interactions between lincomycin and TiO2 NPs.

Comprehensive Analysis of Global Research on Human Varicocele: A Scientometric Approach.

PURPOSE: This study provides a comprehensive analysis of research trends on the etiology, mechanisms, potential risk factors, diagnosis, prognosis, surgical and non-surgical treatment of varicocele, and clinical outcomes before and after varicocele repair. MATERIALS AND METHODS: Varicocele studies published between 1988 and 2020 were retrieved from the Scopus database on April 5, 2021. Original studies on human varicocele were included, irrespective of language. Retrieved articles were manually screened for inclusion in various sub-categories. Bibliometric data was subjected to scientometric analysis using descriptive statistics. Network, heat and geographic mapping were generated using relevant software. RESULTS: In total, 1,943 original human studies on varicocele were published. These were predominantly from the northern hemisphere and developed countries, and published in journals from the United States and Germany. Network map analysis for countries showed several interconnected nodal points, with the USA being the largest, and Agarwal A. from Cleveland Clinic, USA, being a center point of worldwide varicocele research collaborations. Studies of adolescents were underrepresented compared with studies of adults. Studies on diagnostic and prognostic aspects of varicocele were more numerous than studies on varicocele prevalence, mechanistic studies and studies focusing on etiological and risk factors. Varicocele surgery was more investigated than non-surgical approaches. To evaluate the impact of varicocele and its treatment, researchers mainly analyzed basic semen parameters, although markers of seminal oxidative stress are being increasingly investigated in the last decade, while reproductive outcomes such as live birth rate were under-reported in the literature. CONCLUSIONS: This study analyzes the publication trends in original research on human varicocele spanning over the last three decades. Our analysis emphasizes areas for further exploration to better understand varicocele's impact on men's health and male fertility.

TiO2-NPs and cadmium co-exposure: in vitro assessment of genetic and genomic DNA damage on Dicentrarchus labrax embryonic cells.

The increased titanium dioxide nanoparticles (TiO2-NPs) spread and their interaction with organic and inorganic pollutants arouses concern for the potential hazards for organisms and environment. This study tested in vitro the genotoxic effects of TiO2-NPs (1 µg/mL) and cadmium (Cd) (0.1 µg/mL) co-exposure using Dicentrarchus labrax embryonic cells (DLEC) as experimental model. The genotoxicity tests (Comet assay, Diffusion Assay and Random Amplification of Polymorphic DNA (RAPD-PCR) were conducted after 3, 24 and 48 hours of exposure to TiO2-NPs and Cd alone and in combination. The results showed that the percentage of DNA damage and apoptotic cells increases following 48 hours TiO2-NPs exposure, while DNA instability was detected for all the times tested. Cd induced genotoxic effects starting from 3 hour-exposure and for all the treatment times. Cd + TiO2-NPs co-exposure did not cause any genomic damage or apoptosis for all the exposure times. The possibility that Cd and TiO2-NPs form aggregates no longer able of penetrating the nucleus and damaging the genetic material is discussed.

DNA Damage in Human Amniotic Cells: Antigenotoxic Potential of Curcumin and α-Lipoic Acid.

Oxidative imbalances in the gestational phase are responsible for certain complications during pregnancy and for foetal and neonatal genetic disorders. In this work, using human amniocytes, we aimed to evaluate the protection provided to foetal DNA by two concentrations of antioxidant molecules, α-lipoic acid (LA) and curcumin (Cur), against hydrogen peroxide (H2O2)-induced damage. Genotoxicity tests, performed by the random amplification of polymorphic DNA (RAPD-PCR) technique and TUNEL tests, showed that the lowest concentration of LA-protected cells and DNA from H2O2 insults. However, a greater ability to protect the amniocytes' DNA against H2O2 was observed following co-treatment with the highest concentration of Cur with H2O2. In fact, a genomic template stability (GTS%) similar to that of the negative control and a statistically significant reduction in the DNA fragmentation index (DFI) were revealed. Moreover, following a combined treatment with both antioxidants and H2O2, no statistical difference from controls was observed, in terms of both induced mutations and DNA breaks. Furthermore, no effect on morphology or cell viability was observed. The results demonstrate the ability of LA and Cur to protect the genetic material of amniocytes against genotoxic insults, suggesting their beneficial effects in pathologies related to oxidative stress.

Protective Effects of Curcumin on the Outcome of Cryopreservation in Human Sperm.

Cryopreservation causes decreased sperm fertility potential due to reactive oxygen species (ROS) production and physical-chemical damage, resulting in reduced sperm viability and motility. The addition of antioxidants to freezing media could protect sperm from cryo-damage, counteracting the harmful effects of ROS. The aim of this study was to assess the effects of curcumin supplementation in freezing medium on preventing cryo-damage in human semen. Semen samples collected from fertile men were cryopreserved in freezing medium supplemented with different concentrations of curcumin (2.5, 5, 10, and 20 µM). After freezing-thawing, sperm parameters, DNA fragmentation, intracellular ROS, and glutathione peroxidase 4 (GPX4) gene expression were evaluated. Supplementation with 20 µM curcumin in freezing medium caused increases in progressive and nonprogressive motility and significant reductions in intracellular ROS and DNA fragmentation in frozen-thawed sperm cells. Following cryopreservation, GPX4 mRNA expression was significantly upregulated in thawed semen supplemented with 20 µM curcumin compared to the control. The results showed that curcumin supplementation in freezing medium was protective against human sperm parameters and sperm DNA, counteracting oxidative damage induced by the freeze-thaw process.

In vitro ameliorative effects of ellagic acid on vitality, motility and DNA quality in human spermatozoa.

Oxidative stress (OS) plays a significant role in the etiology of male infertility, resulting in the impairment of male reproduction. This condition, characterized by an imbalance in the levels of oxidizing and antioxidant species in the seminal fluid, has a harmful impact on sperm functions and DNA integrity. The present study aimed to evaluate the anti-genotoxic action of ellagic acid, a polyphenolic molecule of natural origin having a powerful antigenotoxic, anti-inflammatory and antiproliferative role. An OS condition was induced in vitro by incubating normozoospermic human semen samples in benzene for 45, 60 and 90 min. DNA integrity was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling assay, RAPD-PCR was performed to calculate the genome template stability, while the percentage of intracellular reactive oxygen species (ROS) was assessed by the 2', 7'-dichlorofluorescein assay. Our results showed that ellagic acid has a consistent protective effect on DNA integrity, as well as on sperm vitality and motility, by counteracting generation of intracellular ROS. The results of this study suggest ellagic acid as a suitable molecule to protect sperm DNA from oxidative stress, with a potentially significant translational impact on the management of the male infertility.

Adsorption of Cd to TiO2-NPs Forms Low Genotoxic AGGREGATES in Zebrafish Cells.

The aquatic environment is involved in the pollutants spreading mechanisms, including nanomaterials and heavy metals. The aims of this study were to assess the in vivo genotoxicity of Cd (1 mg/L) and to investigate the genomic effects generated by its co-exposure with TiO2-NPs (10 µg/L). The study was performed using zebrafish as a model for 5, 7, 14, 21, and 28 days of exposure. The genotoxic potential was assessed by three experimental approaches: DNA integrity, degree of apoptosis, and molecular alterations at the genomic level by genomic template stability (% GTS) calculation. Results showed an increased in DNA damage after Cd exposure with a decrease in % GTS. The co-exposure (TiO2-NPs + Cd) induced a no statistically significant loss of DNA integrity, a reduction of the apoptotic cell percentage and the recovery of genome stability for prolonged exposure days. Characterization and analytical determinations data showed Cd adsorption to TiO2-NPs, which reduced free TiO2-NPs levels. The results of our study suggest that TiO2-NPs could be used for the development of controlled heavy metal bioremediation systems.

In Vitro Effects of Titanium Dioxide Nanoparticles (TiO2NPs) on Cadmium Chloride (CdCl2) Genotoxicity in Human Sperm Cells.

The environmental release of titanium dioxide nanoparticles (TiO2NPs) associated with their intensive use has been reported to have a genotoxic effect on male fertility. TiO2NP is able to bind and transport environmental pollutants, such as cadmium (Cd), modifying their availability and/or toxicity. The aim of this work is to assess the in vitro effect of TiO2NPs and cadmium interaction in human sperm cells. Semen parameters, apoptotic cells, sperm DNA fragmentation, genomic stability and oxidative stress were investigated after sperm incubation in cadmium alone and in combination with TiO2NPs at different times (15, 30, 45 and 90 min). Our results showed that cadmium reduced sperm DNA integrity, and increased sperm DNA fragmentation and oxidative stress. The genotoxicity induced by TiO2NPs-cadmium co-exposure was lower compared to single cadmium exposure, suggesting an interaction of the substances to modulate their reactivity. The Quantitative Structure-Activity Relationship (QSAR) computational method showed that the interaction between TiO2NPs and cadmium leads to the formation of a sandwich-like structure, with cadmium in the middle, which results in the inhibition of its genotoxicity by TiO2NPs in human sperm cells.

Protective activity of ellagic acid in counteract oxidative stress damage in zebrafish embryonic development.

During its development, embryo is easily susceptible to reactive oxygen species (ROS). Evidence demonstrate protective role of the antioxidants, improving both cellular growth and embryonic development. Among these, ellagic acid (EA) is a natural antioxidant with anti-inflammatory and anti-carcinogen properties. The aim of this work was to assess in vitro the protective and anti-genotoxic role of EA during Danio rerio (zebrafish) embryonic development. For the study, zebrafish embryos were treated with H2O2 (15 µM, 30 µM and 45 µM) to simulate an oxidative damage, and with EA (2.5 mM, 5 mM and 10 mM) for 8, 20, 24, 48, 96 hpf (hours post fertilization). Vitality rate, alterations in the morphology and behavior of the larvae and the genomic stability were analyzed. The exposure to H2O2 caused genotoxicity for all exposure times. The incubation in 45 µM H2O2 and 30 µM H2O2 resulted in increased mortality rate of the larvae, as well as 10 mM EA. The co-exposure was performed using to 15 µM H2O2 and 2.5 mM and 5 mM EA and it demonstrated the EA capacity to protect the embryo DNA and development from the oxidative insult. Particularly, the co-exposure to 15 mM H2O2 and 5 mM EA showed an increase in the embryo survival rate and absence of alterations in morphology and behavior at 96 hpf. Interestingly, we observed a higher genomic stability at 8h and 20h co-exposure (15 mM H2O2 and 5 mM EA) time. The decline observed in ROS concentration for both exposure times confirmed the observation. In conclusion, EA protects the zebrafish embryonic development from DNA oxidative damage increasing the embryo survival rate and improving morphological parameters of the larvae.

NPs-TiO2 and Lincomycin Coexposure Induces DNA Damage in Cultured Human Amniotic Cells.

Titanium dioxide nanoparticles (NPs-TiO2 or TiO2-NPs) have been employed in many commercial products such as medicines, foods and cosmetics. TiO2-NPs are able to carry antibiotics to target cells enhancing the antimicrobial efficiency; so that these nanoparticles are generally used in antibiotic capsules, like lincomycin, added as a dye. Lincomycin is usually used to treat pregnancy bacterial vaginosis and its combination with TiO2-NPs arises questions on the potential effects on fetus health. This study investigated the potential impact of TiO2-NPs and lincomycin co-exposure on human amniocytes in vitro. Cytotoxicity was evaluated with trypan blue vitality test, while genotoxic damage was performed by Comet Test, Diffusion Assay and RAPD-PCR for 48 and 72 exposure hours. Lincomycin exposure produced no genotoxic effects on amniotic cells, instead, the TiO2-NPs exposure induced genotoxicity. TiO2-NPs and lincomycin co-exposure caused significant increase of DNA fragmentation, apoptosis and DNA damage in amniocytes starting from 48 exposure hours. These results contribute to monitor the use of TiO2-NPs combined with drugs in medical application. The potential impact of antibiotics with TiO2-NPs during pregnancy could be associated with adverse effects on embryo DNA. The use of nanomaterials in drugs formulation should be strictly controlled in order to minimize risks.

In vitro genotoxic effects of titanium dioxide nanoparticles (n-TiO2 ) in human sperm cells.

Titanium dioxide nanoparticles (TiO2 -NPs) are one of the most widely engineered nanoparticles used. The study has been focused on TiO 2 -NPs genotoxic effects on human spermatozoa in vitro. TiO 2 -NPs are able to cross the blood-testis barrier induced inflammation, cytotoxicity, and gene expression changes that lead to impairment of the male reproductive system. This study presents new data about DNA damage in human sperms exposed in vitro to two n-TiO 2 concentrations (1 µg/L and 10 µg/L) for different times and the putative role of reactive oxygen species (ROS) as mediators of n-TiO 2 genotoxicity. Primary n-TiO 2 characterization was performed by transmission electron microscopy. The dispersed state of the n-TiO 2 in media was spectrophotometrically determined at 0, 24, 48, and 72 hr from the initial exposure. The genotoxicity has been highlighted by different experimental approaches (comet assay, terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL] test, DCF assay, random amplification of polymorphic DNA polymerase chain reaction [RAPD-PCR]). The comet assay showed a statistically significant loss of sperm DNA integrity after 30 min of exposure. Increased threshold of sperm DNA fragmentation was highlighted after 30 min of exposure by the TUNEL Test. Also, the RAPD-PCR analysis showed a variation in the polymorphic profiles of the sperm DNA exposed to n-TiO 2 . The evidence from the DCF assay showed a statistically significant increase in intracellular ROS linked to n-TiO 2 exposure. This research provides the evaluation of n-TiO 2 potential genotoxicity on human sperm that probably occurs through the production of intracellular ROS.

Dioxin-like compounds bioavailability and genotoxicity assessment in the Gulf of Follonica, Tuscany (Northern Tyrrhenian Sea).

The Gulf of Follonica (Italy) is impacted by the chemical pollution from ancient mining activity and present industrial processes. This study was aimed to determine the bioavailability of dioxin-like compounds (DLCs) in coastal marine environment and to assess the genotoxic potential of waste waters entering the sea from an industrial canal. Moderately high levels of DCLs compounds (∑ PCDDs + PCDFs 2.18­29.00 pg/g dry wt) were detected in Mytilus galloprovincialis transplanted near the waste waters canal and their corresponding Toxic Equivalents (TEQs) calculated. In situ exposed mussels did not show any genotoxic effect (by Comet and Micronucleus assay). Otherwise, laboratory exposure to canal waters exhibited a reduced genomic template stability (by RAPD-PCR assay) but not DNA or chromosomal damage. Our data reveal the need to focus on the levels and distribution of DLCs in edible species from the study area considering their potential transfer to humans through the consumption of sea food.

Correction: Metabolomic profiling and biochemical evaluation of the follicular fluid of endometriosis patients.

Correction for 'Metabolomic profiling and biochemical evaluation of the follicular fluid of endometriosis patients' by Marianna Santonastaso et al., Mol. BioSyst., 2017, DOI: 10.1039/c7mb00181a.

Anti-genotoxic ability of α-tocopherol and Anthocyanin to counteract fish DNA damage induced by musk xylene.

Many compounds released into the environment are able to interact with genetic material. The main purpose of genetic toxicology is to investigate the adverse effects of genotoxic molecules such as reduced fitness, changes in gene frequencies and their impact on genetic diversity in populations following genotoxic exposure. However, the ecological effects of many genotoxic compounds remain poorly understood. The aim of this research was to evaluate the genotoxic activity of an artificial musk (musk xylene, MX) and the potential anti-genotoxicity against this chemical compound of two antioxidant substances (α-tocopherol and an anthocyanins enriched extract). The studies were performed both in vivo and in vitro, using the teleost Danio rerio and the DLEC (Dicentrarchus labrax embryonic cells) cell line. We carried out the exposure to these substances at different times. DNA and cell damage and their possible repair were detected by various experimental approaches: DNA strand breaks (Comet Assay), degree of apoptosis (Diffusion Assay) and molecular alterations at the genomic level (RAPD-PCR technique). Data were collected and analyzed for statistical significance using the Student's t test. The results of this study showed that MX exhibited a genotoxic activity even after short exposure times. The anti-genotoxicity experiments evidenced that both α-tocopherol and Anthocyanin were able to contrast the genotoxic effects induced by MX, both in vivo and in vitro.

Genotoxicity assessment of TiO2 nanoparticles in the teleost Danio rerio.

Titanium dioxide nanoparticles (TiO2 NPs), widely used in paints, pharmaceutical preparations and in many consumer products, have been shown to induce cytotoxicity, genotoxicity and carcinogenic responses both in vitro and in vivo. Numerous studies have shown the potential impact of nanoparticles on a series of aquatic organisms and their toxicity has been linked to their dissolution, surface properties and size. In vitro studies have raised concerns about the toxicity of TiO2 NPs, but there are very limited data on ecotoxicity to aquatic life. This in vivo study aimed to describe the genotoxicity of TiO2 NPs in the zebrafish Danio rerio. After 2 weeks of adaptation, groups of zebrafish were exposed to TiO2 NPs (1 and 10µg/L) for 5, 7, 14, 21 and 28 days. The genotoxic potential of TiO2 NPs was assessed by the Comet assay, the Diffusion assay and RAPD-PCR technique. The use of multi-biomarkers has become an important aspect of ecotoxicology to evaluate environmental quality through a wide panel of biological responses triggered by contaminants. The highest genotoxic effect was observed at the maximum concentrations of nanoparticles (10µg/L) with all three tests at 14 and 21 days of exposure. The results suggests the presence of mechanisms that can reduce the n-TiO2 genotoxicity. Future studies are necessary to analyze the DNA repairing capacity in zebrafish cells and so verify the role of the antioxidant defence system in modulating the response to exposure to n-TiO2 in fish.

Influence of titanium dioxide nanoparticles on 2,3,7,8-tetrachlorodibenzo-p-dioxin bioconcentration and toxicity in the marine fish European sea bass (Dicentrarchus labrax).

The present study investigated the influence of nano-TiO(2) (1 mg L(-1)) on 2,3,7,8-tetrachlorodibenzo-p-dioxin(2,3,7,8-TCDD) (46 pg L(-1)) bioconcentration and toxicity in the European sea bass (Dicentrarchus labrax) during 7 days in vivo exposure. A multimarkers approach was applied in different organs: detoxification in liver; innate immunity and pro-inflammatory response and adaptive immunity in gills and spleen; genotoxicity in peripheral erythrocytes and muscle. Bioconcentration of 2,3,7,8-TCDD in presence of nano-TiO2 was investigated in liver, skin and muscle as well as interaction between nano-TiO2 and organic pollutants in artificial sea water (ASW). Nano-TiO2 negatively influenced immune response induced by 2,3,7,8-TCDD in spleen but not in gills and reduced the DNA damage induced by 2,3,7,8-TCDD in erythrocytes. nano-TiO2 did not interfere with 2,3,7,8-TCDD detoxification and bioconcentration according to the observed no interaction of the nano-TiO2 with organic pollutants in ASW.