Membrane Cholesterol Inhibits Progesterone-Mediated Sperm Function through the Possible Involvement of ABHD2.

Abhydrolase domain containing 2-acylglycerol lipase (ABHD2) was recently claimed as the membrane receptor of progesterone (P4) in sperm cells, mediating cell processes such as sperm chemotaxis and acrosome reaction. Here, we investigated the role of membrane cholesterol (Chol) on ABHD2-mediated human sperm chemotaxis. Human sperm cells were obtained from twelve normozoospemic healthy donors. ABHD2-Chol interaction was modelled by computational molecular-modelling (MM). Sperm membrane Chol content was depleted by incubating cells with cyclodextrin (CD) or augmented by the incubation with the complex between CD and Chol (CD:Chol). Cell Chol levels were quantified by liquid chromatography-mass spectrometry. Sperm migration upon P4 gradient was evaluated through the accumulation assay in a specific migration device. Motility parameters were evaluated by sperm class analyzer, whilst intracellular calcium concentration, acrosome reaction and mitochondrial membrane potential were evaluated with calcium orange, FITC-conjugated anti-CD46 antibody and JC-1 fluorescent probes, respectively. MM analysis showed the possible stable binding Chol to ABHD2, resulting in to major impact on the protein backbone flexibility. The treatment with CD was associated with a dose-dependent increase in sperm migration in a 160 nM P4 gradient, together with increase in sperm motility parameters and levels of acrosome reaction. The treatment with CD:Chol was associated with essentially opposite effects. Chol was, thus, suggested to inhibit P4-mediated sperm function through the possible inhibition of ABHD2.

Lipidomic Profile of Human Sperm Membrane Identifies a Clustering of Lipids Associated with Semen Quality and Function.

Reduced sperm motility and/or count are among the major causes of reduced fertility in men, and sperm membranes play an important role in the spermatogenesis and fertilization processes. However, the impact of sperm lipid composition on male fertility remains under-investigated. The aim of the present study was to perform a lipidomic analysis of human sperm membranes: we performed an untargeted analysis of membrane lipid composition in fertile (N = 33) and infertile subjects (N = 29). In parallel, we evaluated their serum lipid levels. Twenty-one lipids were identified by their mass/charge ratio and post-source decay spectra. Sulfogalactosylglycerolipid (SGG, seminolipid) was the most abundant lipid component in the membranes. In addition, we observed a significant proportion of PUFAs. Important differences have emerged between the fertile and infertile groups, leading to the identification of a lipid cluster that was associated with semen parameters. Among these, cholesterol sulfate, SGG, and PUFAs represented the most important predictors of semen quality. No association was found between the serum and sperm lipids. Dietary PUFAs and SGG have acknowledged antioxidant functions and could, therefore, represent sensitive markers of sperm quality and testicular function. Altogether, these results underline the important role of sperm membrane lipids, which act independently of serum lipids levels and may rather represent an independent marker of reproductive function.

Vitamin D deficiency: a potential risk factor for cancer in obesity?

Obesity is considered an abnormal or excessive accumulation of adipose tissue, due to a prolonged positive energy balance that arises when energy intake is greater than energy expenditure, leading to an increased risk for the individual health and for the development of metabolic chronic diseases including several different types of cancer. Vitamin D deficiency is a metabolic alteration, which is often associated with the obesity condition. Vitamin D is a liposoluble vitamin, which plays a pivotal role in calcium-phosphate metabolism but extraskeletal effects have also been described. Among these, it plays an important role also in adipocyte physiology and glucose metabolism, typically dysregulated in subjects affected by obesity. Moreover, it is now recognized that Vitamin D also influences the processes of cell proliferation, differentiation, adhesion potentially leading to carcinogenesis. Indeed, data indicate a potential link between vitamin D levels and cancer, and higher vitamin D concentrations have been associated with a lower risk of developing different kinds of tumors, including breast, colon, lymphoma, lung, and prostate cancers. Thus, this review will revise the literature regarding this issue investigating and highlighting the potential mechanism of action, which might lead to new therapeutical options.

Investigation of the Interaction between Human Serum Albumin and Branched Short-Chain Perfluoroalkyl Compounds.

The current trend dealing with the production of per- and polyfluoroalkyl substances (PFASs) involves the shifting toward branched short-chain fluorinated compounds known as new-generation PFASs. A key aspect to be clarified, to address the adverse health effects associated with the exposure to PFASs, is their binding mode to human serum albumin (hSA), the most abundant protein in plasma. In this study, we investigated the interaction between hSA and two representative branched short-chain PFASs, namely, HPFO-DA and C6O4. In-solution studies revealed that both compounds bind hSA with affinities and stoichiometries lower than that of the legacy long-chain perfluoroalkyl compound PFOA. Competition experiments using hSA-binding drugs with known site-selectivity revealed that both HPFO-DA and C6O4 bound to pockets located in subdomain IIIA. The crystal structure of hSA in complex with HPFO-DA unveiled the presence of two binding sites. The characterization and direct comparison of hSA interactions with new-generation PFASs may be key elements for the understanding of the toxicological impact of these compounds.

Cutaneous Melanoma and Hormones: Focus on Sex Differences and the Testis.

Cutaneous melanoma, the most aggressive type of skin cancer, remains one the most represented forms of cancer in the United States and European countries, representing, in Australia, the primary cause of cancer-related deaths. Recently, many studies have shown that sex disparities previously observed in most cancers are particularly accentuated in melanoma, where male sex is consistently associated with an increased risk of disease progression and a higher mortality rate. The causes of these sex differences rely on biological mechanisms related to sex hormones, immune homeostasis and oxidative processes. The development of newer therapies, such as immune checkpoint inhibitors (ICIs) (i.e., anti-PD-1 and anti-CTLA-4 monoclonal antibodies) has dramatically changed the treatment landscape of metastatic melanoma patients, though ICIs can interfere with the immune response and lead to inflammatory immune-related adverse events (irAEs). Recently, some studies have shown a potential adverse influence of this immunotherapy treatment also on male fertility and testicular function. However, while many anticancer drugs are known to cause defects in spermatogenesis, the effects of ICIs therapy remain largely unknown. Notwithstanding the scarce and conflicting information available on this topic, the American Society of Clinical Oncology guidelines recommend sperm cryopreservation in males undergoing ICIs. As investigations regarding the long-term outcomes of anticancer immunotherapy on the male reproductive system are still in their infancy, this review aims to support and spur future research in order to understand a potential gonadotoxic effect of ICIs on testicular function, spermatogenesis and male fertility.

E2F1 copy number variations in germline and breast cancer: a retrospective study of 222 Italian women.

BACKGROUND: Breast cancer is the most common neoplasia among women in developed countries. The risk factors of breast cancer can be distinguished in modifiable and unmodifiable factors and, among the latter, genetic factors play a key role. Copy number variations (CNVs) are genetic variants that are classified as rare when present in less than 1% of the healthy population. Since rare CNVs are often cause of diseases, over the last years, their contribution in carcinogenesis has become a relevant matter of study. E2F1 is a transcriptional factor that plays an important role in regulating cell cycle and apoptosis. Its double and conflicting role is the reason why it acts both as oncogene and as tumour suppressor, depending on cell context. Since anomalies in expression or in number of copies of E2F1 have been related to several cancers, we aimed to study number of germline copies of E2F1 in women with breast cancer in order to better elucidate their contribution as predisposing factor to this tumour. METHODS: We performed, hence, a retrospective study on 222 Italian women with breast cancer recruited from October 2002 to December 2007. TaqMan CNV assay and Real-Time PCR were carried out to analyse, respectively, E2F1 CNV and E2F1 expression in the subjects of the study. Chi square test or Fisher's exact test and Student's t-test were used to calculate the frequency of CNVs and differences in continuous variables between groups, respectively. RESULTS: Intriguingly, we found that 10/222 (4.5%) women with breast cancer had more copies than controls (0/200, 0%), furthermore, the number of copies positively correlated with E2F1 gene expression in breast cancer tissue, suggesting that the constitutive gain of the gene could translate into an increased risk of genomic instability. Additionally, we found that altered E2F1 copies were present prevalently in the patients with contralateral breast cancer (20%) and all of them had a positive family history, both typically associated with hereditary cancer. CONCLUSIONS: Our findings suggest that copy number variations of E2F1 might be a susceptibility factor for breast cancer, however, further studies on large cohorts are to be performed in order to better delineate the phenotype linked to the gain of E2F1 copies.

Identification of Rare LRP5 Variants in a Cohort of Males with Impaired Bone Mass.

Osteoporosis is the most common bone disease characterized by reduced bone mass and increased bone fragility. Genetic contribution is one of the main causes of primary osteoporosis; therefore, both genders are affected by this skeletal disorder. Nonetheless, osteoporosis in men has received little attention, thus being underestimated and undertreated. The aim of this study was to identify novel genetic variants in a cohort of 128 males with idiopathic low bone mass using a next-generation sequencing (NGS) panel including genes whose mutations could result in reduced bone mineral density (BMD). Genetic analysis detected in eleven patients ten rare heterozygous variants within the LRP5 gene, which were categorized as VUS (variant of uncertain significance), likely pathogenic and benign variants according to American College of Medical Genetics and Genomics (ACMG) guidelines. Protein structural and Bayesian analysis performed on identified LRP5 variants pointed out p.R1036Q and p.R1135C as pathogenic, therefore suggesting the likely association of these two variants with the low bone mass phenotype. In conclusion, this study expands our understanding on the importance of a functional LRP5 protein in bone formation and highlights the necessity to sequence this gene in subjects with idiopathic low BMD.

Sperm Cholesterol Content Modifies Sperm Function and TRPV1-Mediated Sperm Migration.

Transient receptor potential channels-vanilloid receptor 1 (TRPV1) regulates thermotaxis in sperm-oriented motility. We investigated the role of membrane cholesterol (Chol) on TRPV1-mediated human sperm migration. Semen samples were obtained from five normozoospemic healthy volunteers. Sperm membrane Chol content, quantified by liquid chromatography-mass spectrometry, was modified by incubating cells with 2-hydroxypropyl-ß-cyclodextrin (CD) or the complex between CD and Chol (CD:Chol). The effect on sperm migration on a 10 µM capsaicin gradient (CPS), a TRPV1 agonist, was then investigated. Motility parameters were evaluated by Sperm Class Analyser. Intracellular calcium concentration and acrosome reaction were measured by staining with calcium orange and FITC-conjugated anti-CD46 antibody, respectively. TRPV1-Chol interaction was modelled by computational molecular-modelling (MM). CD and CD:Chol, respectively, reduced and increased membrane Chol content in a dose-dependent manner, resulting in a dose-dependent increase and reduction of sperm migration in a CPS gradient. MM confirmed a specific interaction of Chol with a TRPV1 domain that appeared precluded to the Chol epimer epicholesterol (Epi-Chol). Accordingly, CD:Epi-Chol was significantly less efficient than CD:Chol, in reducing sperm migration under CPS gradient. Chol inhibits TRPV1-mediated sperm function by directly interacting with a consensus sequence of the receptor.

Testosterone is sequestered in dysfunctional adipose tissue, modifying androgen-responsive genes.

BACKGROUND/OBJECTIVE: The recognized association between male hypogonadism and obesity has multifactorial implications on adipose tissue (AT) physiology. The fat solubility of testosterone (T) suggests a sequestration process in fat depots, leading to reduced circulating levels of T in obesity. Several evidence suggest that steroids play a two-sided inhibitory role on adipogenesis by locally decreasing lipid accumulation and by stimulating lipolysis. The current study investigates T trafficking and activity in dysfunctional AT. SUBJECTS/METHODS: Samples of subcutaneous AT (SAT) were obtained from explants from lipoaspirate plastic surgery in six obese and six normal weight male patients. Experimental procedures on both SAT explants and insulin-resistant (IR) 3T3-L1 adipocytes were performed, including real-time PCR and mass-spectrometry quantification. RESULTS: A significant deregulation of gene responsiveness to androgens in IR cells and obese SAT was observed (all p < 0.05), together with reduced T release after adrenergic stimulation (-10% compared with -55% in lean SAT, p = 0.021). Higher concentrations of intracellular T and estradiol in obese SAT were also observed (2.4 vs. 1.3 ng/g, p = 0.013 and 0.075 vs. 0.22 ng/g, p = 0.004, respectively). Testosterone accumulation resulted in even lower expression in androgen-responsive genes involved in lipolytic and anti-adipogenic pathways from both in vitro and ex vivo experiments. CONCLUSIONS: These results suggest an altered response of dysfunctional fat cells to testosterone stimulation, which normally favors lipolysis and induces an anti-adipogenic effect. The considerable reduction of lipolytic T release after adrenergic stimulation in obese SAT contributes to AT dysfunction, in a feedforward loop further reducing T levels in obese hypogonadal males.

Increased Cardiovascular Risk Associated with Chemical Sensitivity to Perfluoro-Octanoic Acid: Role of Impaired Platelet Aggregation.

Perfluoro-alkyl substances (PFAS), particularly perfluoro-octanoic acid (PFOA), are persisting environmental chemicals showing bioaccumulation in human tissues. Recently, exposure to PFAS has been associated with increased prevalence of cardiovascular diseases (CVDs). However, a causal role of PFAS in atherosclerosis pathogenesis is under-investigated. Here, we investigated the effect of PFOA exposure on platelets' function, a key player in atherosclerosis process. PFOA accumulation in platelets was evaluated by liquid chromatography-mass spectrometry. Changes in platelets' membrane fluidity and activation after dose-dependent exposure to PFOA were evaluated by merocyanine 540 (MC540) and anti P-Selectin immune staining at flow cytometry, respectively. Intracellular calcium trafficking was analyzed with Fluo4M probe, time-lapse live imaging. Platelets' aggregation state was also evaluated with Multiplate® aggregometry analyzer in 48 male subjects living in a specific area of the Veneto region with high PFAS environmental pollution, and compared with 30 low-exposure control subjects. Platelets' membrane was the major target of PFOA, whose dose-dependent accumulation was associated in turn with increased membrane fluidity, as expected by a computational model; increased activation at resting condition; and both calcium uptake and aggregation upon activation. Finally, exposed subjects had higher serum and platelets levels of PFOA, together with increased aggregation parameters at Multiplate®, compared with controls. These data help to explain the emerging association between PFAS exposure and CVD.

E2F1 germline copy number variations and melanoma susceptibility.

BACKGROUND: Melanoma is an aggressive type of skin cancer whose aetiology remains elusive as both environmental and genetic factors can contribute to its development. Recent studies have demonstrated the existence of multiple copies of E2F1 gene in melanoma specimens which could explain the deregulated E2F1 activity in this type of cancer. This finding suggests a key role for this transcription factor in the malignant transformation of melanocytes. Therefore, E2F1 has been considered as a potential therapeutic target for this form of skin cancer. Since germline copy number variations (CNVs) have been associated with increased susceptibility to different types of cancer, the aim of our study was to assess germline E2F1 CNV in melanoma patients. However, CNVs not necessarily lead to gene dosage imbalance, hence, further factors, in association with CNVs, could contribute to clinical manifestations. Considering that heat stress has been hypothesised as a contributing factor to skin cancer, we also investigated the effect of heat stress on E2F1 expression. METHODS: E2F1 CNV was measured in genomic DNA isolated from blood of 552 patients diagnosed with melanoma and 520 healthy subjects using TaqMan Copy Number Assays. E2F1 mRNA expression was also evaluated by RT-qPCR in the melanoma cell line, SK MEL 267, before and after exposure to heat stress. RESULTS: We found that patients diagnosed with melanoma (1.6%, 9/552) harboured frequently altered germline E2F1 copies compared to healthy subjects (0%, 0/520). Moreover, the difference among the two groups was statistically significant (p = 0.004). Furthermore, we found that heat exposure alone can significantly induce E2F1 expression. CONCLUSIONS: This is the first study that shows a relation between germline E2F1 CNV and melanoma, suggesting that altered copies of this gene might be a predisposing factor to skin cancer. Our results also suggest that environmental insults, such as heat stress, could contribute to an aberrant E2F1 activity by inducing E2F1 mRNA expression. Therefore, subjects with multiple constitutive copies of E2F1 are at greater risk of developing melanoma when exposed to heat. Altogether our results corroborate with the hypothesis that susceptibility to melanoma depends on both the environment and genetic factors.

Water and soil pollution as determinant of water and food quality/contamination and its impact on male fertility.

Over the past two decades, public health has focused on the identification of environmental chemical factors that are able to adversely affect hormonal function, known as endocrine disruptors (EDs). EDs mimic naturally occurring hormones like estrogens and androgens which can in turn interfere with the endocrine system. As a consequence, EDs affect human reproduction as well as post and pre-natal development. In fact, infants can be affected already at prenatal level due to maternal exposure to EDs. In particular, great attention has been given to those chemicals, or their metabolites, that have estrogenic properties or antagonistic effects on the activity of androgen or even inhibiting their production. These compounds have therefore the potential of interfering with important physiological processes, such as masculinization, morphological development of the urogenital system and secondary sexual traits. Animal and in vitro studies have supported the conclusion that endocrine-disrupting chemicals affect the hormone-dependent pathways responsible for male gonadal development, either through direct interaction with hormone receptors or via epigenetic and cell-cycle regulatory modes of action. In human populations, epidemiological studies have reported an overall decline of male fertility and an increased incidence of diseases or congenital malformations of the male reproductive system. The majority of studies point towards an association between exposure to EDs and male and/or female reproductive system disorders, such as infertility, endometriosis, breast cancer, testicular cancer, poor sperm quality and/or function. Despite promising discoveries, a causal relationship between the reproductive disorders and exposure to specific toxicants has yet to be established, due to the complexity of the clinical protocols used, the degree of occupational or environmental exposure, the determination of the variables measured and the sample size of the subjects examined. Despite the lack of consistency in the results of so many studies investigating endocrine-disrupting properties of many different classes of chemicals, the overall conclusion points toward a positive association between exposure to EDs and reproductive system. Future studies should focus on a uniform systems to examine human populations with regard to the exposure to specific EDs and the direct effect on the reproductive system.

Novel insights on testicular volume and testosterone replacement therapy in Klinefelter patients undergoing testicular sperm extraction. A retrospective clinical study.

STUDY QUESTION: To investigate whether sperm recovery is related to clinical features, hormone parameters and testosterone replacement therapy (TRT) in patients with Klinefelter syndrome (KS). SUMMARY ANSWER: This study provides three interesting insights: (i) the probability to retrieve sperm is not related to testicular volume; (ii) TRT does not affect sperm retrieval rate (SRR); and (iii) reduced levels of LH and FSH represent a negative predictor of sperm retrieval in patients with TRT. WHAT IS KNOWN ALREADY: Classical KS shows a karyotype with one extra X chromosome in all of somatic cells and clinical manifestations characterized by hypergonadotropic hypogonadism and infertility. STUDY DESIGN, SIZE AND DURATION: We performed a retrospective cohort study. Data from 111 consecutive KS azoospermic patients undergoing testicular sperm extraction (TESE) were collected from 2005 to 2016. PARTICIPANTS/MATERIALS, SETTING AND METHODS: Data on anthropometric parameters, reproductive hormones and testicular volumes were collected. SRR was related to clinical characteristics and compared between TRT and untreated patients. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 38 patients (34.2%) had successful sperm recovery. The comparison of clinical characteristics did not differ between patients with and without sperm recovery. Sperm retrieval was successful also in subjects with smaller testes. The comparison of SRR in patients with or without TRT was not different (33.3% vs 34.6%). In TRT group, LH and FSH levels were significantly lower in subjects with no sperm retrieval (P values, respectively, <.05 and <.001). LIMITATIONS AND REASONS FOR CAUTION: Well-designed controlled studies are necessary to confirm these data aimed to set the best therapeutic approach for fertility management in hypogonadal patients with nonmosaic KS. WIDER IMPLICATIONS OF THE FINDINGS: Age at TESE, anthropometric measures, testis volume, sex hormones levels and semen parameters are not predictive parameters of SRR. Among TRT patients, reduced gonadotropin is related to failure in sperm retrieval.

The rs2274911 polymorphism in GPRC6A gene is associated with insulin resistance in normal weight and obese subjects.

OBJECTIVE: Identification of the novel endocrine role of osteocalcin (OC) and its receptor GPRC6A has given rise to a new branch of research in OC/GPRC6A axis related to glucose metabolism. GPRC6A- and OC-deficient mice share features of the metabolic syndrome, in addition to male infertility. Recently, the polymorphism rs2274911 in GPRC6A was shown to be associated with testicular impairment. We aimed to investigate the role of rs2274911 polymorphism in glucose and lipid metabolism in a cohort of normal weight and obese subjects DESIGN, PATIENTS, SETTINGS: A total of 392 male and females, including 218 obese patients and 174 age-matched normal weight controls, were retrospectively selected. RESULTS: The distribution of rs2274911 alleles and genotypes did not differ either between normal weight and obese subjects or sexes (all P > 0·05). Age- and OC-adjusted multivariate analysis revealed that, in the normal weight group, fasting insulin and HOMA-IR increased in GA (P = 0·016 and P = 0·025) and AA genotypes (P = 0·033 and P = 0·040) compared with GG homozygotes. In the obese group, AA homozygotes had increased fasting glucose (P = 0·041 vs GG). Triglycerides, fasting insulin and HOMA-IR increased in both GA (P = 0·020, P < 0·001 and P = 0·001) and AA genotype (P = 0·021, P = 0·013 and P = 0·013). CONCLUSION: In a cohort of normal weight and obese subjects, we found that the nonrare polymorphism rs2274911 in the GPRC6A gene was associated with insulin resistance features, independently of the metabolic phenotype and OC levels.

Ketone Body ß-Hydroxy-Butyrate Sustains Progressive Motility in Capacitated Human Spermatozoa: A Possible Role in Natural Fertility.

Background Calorie restriction is recognized as a useful nutritional approach to improve the endocrine derangements and low fertility profile associated with increased body weight. This is particularly the case for dietary regimens involving ketosis, resulting in increased serum levels of ketone bodies such as ß-hydroxy-butyrate (ß-HB). In addition to serum, ß-HB is detected in several biofluids and ß-HB levels in the follicular fluid are strictly correlated with the reproductive outcome in infertile females. However, a possible direct role of ketone bodies on sperm function has not been addressed so far. Methods Semen samples were obtained from 10 normozoospermic healthy donors attending the University Andrology Unit as participants in an infertility survey programme. The effect of ß-HB on cell motility in vitro was evaluated on isolated spermatozoa according to their migratory activity in a swim-up selection procedure. The effect of ß-HB on spermatozoa undergone to capacitation was also assessed. Results Two hours of exposure to ß-HB, 1 mM or 4 mM, proved to be ineffective in modifying the motility of freshly ejaculated spermatozoa isolated according to the migratory activity in a swim-up procedure (all p values > 0.05). Differently, sperm maintenance in 4 mM ß-HB after capacitation was associated with a significantly higher percentage of sperm cells with progressive motility compared to ß-HB-lacking control (respectively, 67.6 ± 3.5% vs. 55.3 ± 6.5%, p = 0.0158). Succinyl-CoA transferase inhibitor abolished the effect on motility exerted by ß-HB, underpinning a major role for this enzyme. Conclusion Our results suggest a possible physiological role for ß-HB that could represent an energy metabolite in support of cell motility on capacitated spermatozoa right before encountering the oocyte.

Experimental evidence of a limited impact of new-generation perfluoroalkyl substance C6O4 on differentiating human dopaminergic neurons from induced pluripotent stem cells.

Perfluoroalkyl substances (PFASs) are persistent pollutants, raising concerns for human health. Legacy PFAS perfluoro-octanoic acid (PFOA) accumulate in brains of people at high environmental exposure, especially in areas enriched with dopaminergic neurons (DN). In vitro exposure to 10 ng/mL PFOA for 24 h was also associated with an altered molecular and functional phenotype of DN differentiated from human induced pluripotent stem cells (hiPSCs). Acetic acid, 2,2-difluoro-2-((2,2,4,5-tetrafluoro-5(trifluoromethoxy)- 1,3-dioxolan-4-yl)oxy)-ammonium salt (1:1), known as C6O4, is a new generation PFAS proposed to have a safer profile. Here we investigated the effect of C6O4 exposure on the molecular phenotype of hiPSC-derived DN. Cells were exposed to C6O4 for 24 h, at the concentration of 10 ng/mL, at neuronal commitment (DP1), neuronal precursor (DP2) and the mature dopaminergic (DP3) phases of differentiation. Liquid-chromatography/mass-spectrometry showed negligible cell accumulation of C6O4 at each differentiation stage and by staining with Merocyanine-540 we observed unaltered cell membrane fluidity. Immunofluorescence showed that the expression of tyrosine hydroxylase (TH) and ßIII-Tubulin was unaffected by the exposure to C6O4 at each differentiation phase (respectively: DP1, p = 0.332; DP2, p = 0.623; DP3, p = 0.816, with respect to control unexposed conditions). Exposure to C6O4 is presumed to have minor effects on cell molecular/functional phenotype of developing human DN cells, requiring confirm on in vivo models.

Observational Cross-Sectional Study on Mediterranean Diet and Sperm Parameters.

Infertility, affecting 15 to 25% of couples in the most developed countries, is recognized by the World Health Organization as a public health issue at a global level. Different causes are acknowledged to reduce fertility in both sexes. In particular, about 40-50% of cases recognize a male factor. Dietary habits and lifestyle are acknowledged to influence sperm quality and are therefore important modifiable factors in male reproductive health. Conditions such as overweight/obesity, impaired glucose metabolism and determinants of metabolic syndrome, together with unhealthy lifestyle behavior, i.e., smoking cigarettes and physical inactivity, are suggested to have a negative impact on male fertility. While individual elements and characteristics of the Western diet and habits are considered risk factors for male infertility, the Mediterranean diet (MD) seems to promote reproductive potential for improving sperm quality. It is also interesting to note that previous observational studies reported a positive correlation between the consumption of the single food classes of the MD pattern (i.e., vegetables and fruits, poultry, fish and seafood, whole grains, low-fat dairy products) and the quality of several sperm parameters. To evaluate the relationship between sperm parameters and MD adherence, we performed a cross-sectional study on the seminal data of 300 males (mean age 34.6 ± 9.1 years) who spontaneously referred to our center of reproductive medicine. The evaluation of adherence to MD was performed with a validated 14-point Mediterranean Diet Adherence Screener (MEDAS) questionnaire. Our findings showed that sperm parameters such as sperm count, motility, viability and normal morphology are significantly and positively correlated with MEDAS, independently of BMI and age. In addition, the application of an ROC curve on MEDAS value vs. seminal alterations identified 6.25 as the score threshold value below which altered sperm parameters were more likely to occur [AUC = 0.096 (CI: 0.059-0.133; p < 0.00)]. Therefore, adhering to the MD with at least a MEDAS score of 6.26 increases the probability of normozoospermia. Moreover, subjects who had a MEDAS value lower than 6.25 had an Odds Ratio of 6.28 (CI = 3.967-9.945) for having at least one altered sperm parameter compared to those who were more adherent to the MD. In conclusion, our findings show that a higher adherence to the MD is associated with better semen parameters, in particular in relation to sperm count, sperm concentration, typical sperm morphology, and sperm progressive motility.

In vitro binding analysis of legacy-linear and new generation-cyclic perfluoro-alkyl substances on sex hormone binding globulin and albumin, suggests low impact on serum hormone kinetics of testosterone.

In humans, serum testosterone (T) is largely bound to the sex hormone binding globulin (SHBG) and human serum albumin (hSA), resulting in a 2-3 % of unbound or "free" active quote (FT). Endocrine-disrupting chemicals, including perfluoro-alkyl substances (PFAS), are recognized to interfere with the hormonal axes, but the possible impact on the FT quote has not been addressed so far. Here we investigated the possible competition of two acknowledged PFAS molecules on T binding to SHBG and hSA. In particular, perfluoro-octanoic acid (PFOA) and acetic acid, 2,2-difluoro-2-((2,2,4,5-tetrafluoro-5(trifluoromethoxy)-1,3-dioxolan-4-yl)oxy)-ammonium salt (1:1) (C6O4) were used as, respectively, legacy-linear and new-generation-cyclic PFASs. Human recombinant SHBG 30-234 domain (SHBG30-234), produced in HEK293-F cells, and delipidated recombinant hSA were used as in vitro protein models. Isothermal Titration Calorimetry (ITC) and tryptophan fluorescence quencing (TFQ) were used to evaluate the binding modes of T and PFAS to SHBG30-234 and hSA. ITC revealed the binding of T to SHBG30-234 with a Kd of 44 ± 2 nM whilst both PFOA and C6O4 showed no binding activity. Results were confirmed by TFQ, since only T modified the fluorescence profile of SHBG30-234. In hSA, TFQ confirmed the binding of T on FA6 site of the protein. A similar binding mode was observed for PFOA but not for C6O4, as further verified by displacement experiments with T. Although both PFASs were previously shown to bind hSA, only PFOA is predicted to possibly compete with T for the binding to hSA. However, on the base of the binding stoichiometry and affinity of PFOA for hSA, this appears unlikely at the blood concentrations of the chemical documented to date.

Legacy perfluoro-alkyl substances impair LDL-cholesterol uptake independently from PCSK9-function.

Perfluoro-alkyl substances (PFAS) are pollutants, whose exposure was associated with altered levels of low-density lipoproteins (LDL) in humans. Here we investigated this clinical outcome in two groups of young male adults residing in areas of respectively low and high environmental exposure to perfluoro-octanoic-acid (PFOA). From the Regional Authority data on pollution areas, 38 not-exposed and 59 exposed age-matched participants were evaluated for serum levels of total cholesterol (Total-Chol), LDL-Chol, high-density lipoprotein cholesterol (HDL-Chol), triglycerides (Tgl) and chromatography quantified PFOA. Human hepato-carcinoma cell line HepG2 was exposed to PFOA or perfluoro-octane-sulfonate (PFOS), as legacy PFAAs, and C6O4 as new generation compound. Fluorimetry was used to evaluate the cell-uptake of labelled-LDL. Proprotein Convertase Subtilisin/Kexin 9 (PCSK9)-mediated LDL-receptor (LDL-R) degradation and sub-cellular localization of LDL-R were evaluated by western blot analysis. Serum levels of PFOA, were positively and significantly correlated with Total-Chol (ρ = 0.312, P = 0.002), LDL-Chol (ρ = 0.333, P = 0.001) and Tgl (ρ = 0.375, P < 0.001). Participants with high serum LDL-Chol and Tgl levels, according to the cardiovascular risk, were more prevalent in exposed compared to not-exposed subjects (respectively: 23.7% vs 5.3%, P = 0.023 and 18,6% vs 0%, P = 0.006). Exposure of HepG2 cells to PFOA or C6O4 100 ng/mL was associated with a significantly lower LDL uptake than controls but no major impact of any PFAAs on PCSK9-mediated LDL-R degradation was observed. Compared to controls, exposure to PFAS showed an unbalanced LDL-R partition between membrane and cytoplasm. Endocytosis inducer sphingosine restored LDL-R partition only in samples exposed to C6O4. These data suggest a novel endocytosis-based mechanism of altered lipid trafficking associated with the exposure to legacy PFAS.

Seminal cadmium affects human sperm motility through stable binding to the cell membrane.

Environmental pollutants are claimed to be major factors involved in the progressive decline of the fertility rate worldwide. Exposure to the heavy metal Cadmium (Cd) has been associated with reproductive toxicity due to its ionic mimicry. However, the possible direct accumulation of Cd in human sperm cells has been poorly investigated. In this study, we aimed to clarify the possible direct effect of Cd exposure on sperm function through the analysis of its cell accumulation. Semen samples from 30 male subjects residing in high environmental impact areas and adhering to the "Exposoma e Plurifocalità nella Prevenzione Oncologica" campaign for testis cancer prevention were compared with semen samples from 15 males residing in low exposure areas. Semen levels and cell Cd content were quantified by inductively coupled plasma (ICP) spectroscopy. Cell Cd distribution was assessed by scanning electron microscopy coupled with energy dispersive spectroscopy (SEM-EDS). The impact of Cd on sperm function was evaluated by the in vitro exposure to the heavy metal, whilst possible scavenging approaches/agents were assessed. In addition to higher values of semen Cd, exposed subjects showed a reduction in total motile sperm fraction compared to not-exposed controls (59.6% ± 13.6% vs. 66.3% ± 7.3%, p = 0.037). Semen Cd levels were also significantly correlated with SEM-EDS signals of Cd detected on the head and neck of sperm (respectively p = 0.738, p < 0.001 and ρ = 0.465, p < 0.001). A total of 2 h of in vitro exposure to 0.5 µM Cd was associated with a significant reduction of sperm progressive motility. Scavenging approaches with either hypo-osmotic swelling or 10 µM reduced glutathione were ineffective in blunting cell Cd and restoring motility. The reduction of exposure levels appears to be the main approach to reducing the reproductive issues associated with Cd.