Perfluoro-alkyl substances (PFAS) are persistent environmental pollutants suggested to impair antibody response. Legacy PFAS, such as perfluoro-octanoic acid (PFOA), are largely investigated but little is known about the role of emerging alternatives such as C6O4. This study investigated the differential effects of PFOA and C6O4 on human B cell differentiation, activation, and immunoglobulin production in vitro. Peripheral blood mononuclear cells and purified B lymphocytes from healthy donors were exposed to increasing concentrations (20-100 ng/mL) of PFOA or C6O4. T cell-dependent interferon-γ release and CD40L expression were unaffected by the exposure to either compounds. However, CD69 mean fluorescence intensity showed a dose-dependent reduction upon PFOA exposure, in addition to a halved IL-4 secretion compared to stimulated controls. PFOA 100 ng/mL also reduced B cell proliferation by approximately 30%, significantly decreased CD69 + and CD83 + B cells by 25-40%, and altered subset composition with a 20% increase in naïve cells and a 15% reduction in marginal zone-memory cells. In long-term B cell cultures, PFOA was associated with delayed and smaller cluster formation, with halved cluster area from day 5 onwards and suppressed IgG secretion by 30-45% at all time points. C6O4 was associated with 20% reductions in IgG release at days 3-6. PFOA seems to impact the immune function by both impairing T cell-dependent IL-4 production and by directly affecting B cell maturation and antibody production, resulting in robust immunosuppression. Differently, C6O4 appears less impactful. These findings underscore the need for compound-specific risk assessment of PFAS, supporting the continuity of monitoring.

Environmental exposure to per- and polyfluoroalkyl substances (PFAS) rises concerns about male reproductive health. Despite a wide body of experimental evidence supporting the biological role of PFAS exposure in male reproductive effects, available epidemiological studies are variably consistent about this association. Four databases were searched up to July 13, 2025 (PROSPERO: CRD420251267798). The systematic review follows Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Studies were included that assessed the association of PFAS on sperm outcomes. The primary effect measure was the Pearson correlation coefficient (r). Outcomes included sperm concentration, semen volume, total sperm count, motility, morphology, pH, and sperm DNA fragmentation, Fourteen studies including 7465 participants were included in this review. The results of meta-analyses indicate heterogeneous patterns of association, with PFAS mixtures showing limited associations with conventional semen parameters but demonstrating consistent inverse relationships with progressive motility (correlation scale [r] = -0.058, 95% confidence interval [CI]: -0.100 to -0.015) and sperm deoxyribonucleic acid (DNA) integrity (r = 0.141, 95% CI: 0.087-0.195). Individual congeners exhibited distinct association profiles, suggesting potential congener-specific effects on male reproductive function. The consistent association between increased DNA fragmentation across PFAS metrics suggest a possible role in unexplained male infertility and prompts further investigation.

Microplastics (MPs) are ubiquitous environmental contaminants that have recently been detected in human semen, raising concerns about potential implications for male reproductive health. However, how MPs are retained, distributed, and interact within seminal fluid remains poorly understood. In this study we investigated the presence, localization, and behaviour of environmental MPs in undigested human semen from five normozoospermic donors, adopting an analytical workflow designed to preserve the native architecture of the ejaculate and maintain biological context. Semen samples (whole semen and spermatozoa-enriched fractions) were analysed by fluorescence microscopy followed by chemical identification using & micro;Raman microspectroscopy. MPs were consistently detected in all donors, with particles predominantly in the microscale range (on average <10 mu m). Polymer composition reflected materials commonly reported in environmental and biological matrices, including polypropylene (PP), polyethylene (PE), and polystyrene (PS). Crucially, despite systematic examination of both whole semen and spermatozoa fractions, no evidence of MP adhesion to, or internalisation within, spermatozoa was observed within the particle size range accessible to the adopted & micro;Raman-based analytical workflow. These findings indicate that, under native conditions, MPs are dispersed within seminal plasma and can co-sediment with sperm cells during processing without establishing detectable direct particle-membrane interactions. To explore a plausible source pathway within the male reproductive tract, prostate tissue from one donor was also analysed. In contrast to semen, prostate specimens contained larger MPs, up to similar to 60 mu m. Although preliminary and observational, this preliminary finding is consistent with the possibility that the prostate may contribute to the differential retention of particles, with smaller MPs potentially reaching the ejaculate more readily. Overall, our findings refine the biological interpretation of MPs in semen as a downstream signature of tissue-level exposure and transport, and support future studies, particularly addressing nanoscale plastics and clinical correlates, to clarify reproductive risk.

Perfluoroalkyl substances (PFASs) raise concerns about their environmental accumulation. Experimental data have suggested that PFASs interfere with bone metabolism from the early stages of life. However, mechanisms underlying this association are unclear. The aim of this study was to evaluate the possible association between environmental exposure to PFAS and vitamin D (VitD), serum calcium and parathyroid hormone (PTH) levels in subjects residing in high-exposure area of the Veneto Region of Italy. In this cross-sectional observational study, 1174 subjects who previously adhered to the 2016-2018 Regional Surveillance Plan for plasma levels of PFASs were recalled in 2023 and evaluated for demographic, anthropometrics and blood analyses. Data on nutritional habits and VitD supplementation were obtained by a dedicated questionnaire. Serum concentrations of PFASs, calcium, 25-hydroxy-vitamin D (25OH-VitD) and PTH were determined from blood sampling. Perfluorooctanoic acid (PFOA), perfluorooctanesulfonate (PFOS) and perfluorohexanesulfonic acid (PFHxS) were the only three PFASs, of 12, quantifiable in at least 90% of the samples and considered for further analyses. Generalized additive models, using linear regression and smoothing thin plate splines, detected a positive association between serum calcium and all considered PFAS (PFOA: β = 0.03; CI 95% 0.01-0.06; PFOS: β = 0.06; CI 95% 0.02-0.09, PFHxS: β = 0.04; CI 95% 0.01-0.06). Estimated degrees of freedom (EDF) analysis showed the approximately linear association between serum calcium with PFOA (EDF = 1.89) and PFHxS (EDF = 1.21), but not for PFOS (EDF = 3.69). Differently, PFAS levels showed no association with either 25-hydroxy-vitamin D or PTH, except for ln-transformed 25OH-D and PFOS (β = 0.04; CI 95% 0.00-0.08). Stratified analyses confirmed the positive association between all considered PFAS and calcium in subjects not taking a VitD supplementation. Results show that high exposure levels to PFAS may interfere with calcium metabolism, independently of lifestyle and dietary factors. Further elucidation on the mechanisms underlying calcium homeostasis disruption, including multiple binding-equilibrium with serum albumin, remains to be addressed.

Supplementary Data from Association Study between Polymorphisms in DNA Methylation–Related Genes and Testicular Germ Cell Tumor Risk

Environmental pollution is a growing global concern, yet its effects on reproductive health remain poorly understood. While numerous epidemiological studies have identified strong associations between exposure to pollutants and adverse reproductive outcomes, the precise biological mechanisms underlying these effects remain elusive. A comprehensive literature search was performed by two reviewers using the US National Library of Medicine (NCBI Pubmed) up to August 15, 2025. Chemical and physical contaminants, including endocrine-disrupting chemicals (EDCs), particulate matter (PM), heavy metals, and heat stress (HS), have been implicated in disrupting of essential reproductive processes, such as gametogenesis, fertilization, implantation, and embryogenesis. Despite accumulating evidence, the complexity of these interactions has hindered the development of targeted interventions and effective regulatory policies. This review argues that investment in reproductive environmental research is not only necessary but urgent. The pervasive nature of pollution and its potential long-term consequences on fertility and pregnancy outcomes warrant an intensified focus on mechanistic studies, improved environmental monitoring, and the integration of toxicological data into reproductive medicine. It is imperative to prioritize research that can provide the necessary insights to mitigate these threats. This paper highlights the knowledge gaps, emphasizing the need for a multidisciplinary approach that combines epidemiology, toxicology, and molecular biology to inform policies and safeguard reproductive health.

Environmental changes are a growing global concern, and their impact on reproductive health remains incompletely understood. In this narrative review, conducted on behalf of the Italian Society of Fertility and Sterility and Reproductive Medicine (SIFES-MR), we examined the impact of the environment on male fertility, considering endocrine-disrupting chemicals (EDCs), air pollution, and global warming, with the aim of identifying strategies to improve reproductive outcomes. Scientific literature demonstrates that all these aspects may contribute to a decline in reproductive health, impairing sperm count, motility, and morphology as well as reducing testicular hormonal function. Future research should focus on the role of environmental factors in male hypogonadism, impaired spermatogenesis, genital abnormalities, and transgenerational effects.

Objectives: The identification of methods to improve the endometrial receptivity (ER) is increasingly of interest. The effect of the electromagnetic field associated with Quantum Molecular Resonance (QMR) on ER was investigated here. Methods: Ishikawa cells were used to evaluate the effects of QMR both on the expression of a group of genes involved in ER, i.e., HOXA10, HOXA11, LIF, ITGB3, and ITGAV, and on cell toxicity. Endometrial samples were obtained from six patients during routine diagnostic procedures, four of which were subsequently used to assess the transcriptional response to QMR through microarray. Results: Compared to unexposed controls, a single exposure of Ishikawa cells to QMR for 20 min was associated with a significant and power-dependent up-regulation of all the selected ER-related genes up to 8 power units (PU). Repeated exposure to QMR, up to three consecutive days, showed a significant up-regulation of all the selected genes at power values of 4 PU, from day two onwards. Negligible cytotoxicity was observed. Gene set enrichment analysis, on microarray data of endometrial biopsies stimulated for three consecutive days at 4 PU, showed a significant enrichment of specific gene sets, related to the proteasome system, the cell adhesion, the glucocorticoid receptor, and cell cycle pathways. Conclusions: Our results suggest a possible favorable impact of QMR on ER.

PURPOSE:Klinefelter syndrome (KS) is the most prevalent sex chromosome disorder among males. The communication of the KS diagnosis holds significant implications for the diagnosis's acceptance. Recently, the increased use of prenatal diagnostic procedures has raised the question of whether, when, and by whom information, once provided to parents, should be communicated to their children/adolescents. Currently, there is limited information on this topic. This study aims to investigate the most suitable timing, content, and healthcare professionals (HCPs) according to KS patients' suggestions for conveying the diagnosis, analyzing the impact of communicating the KS diagnosis on patients and their reception of the communication in real-life situations. Furthermore, research entails a comparison of the actual communication and the patients' preferred mode of communication. METHODS:Self-reported interview data was collected from 196 adults diagnosed with KS. The interview was structured, consisting of 32 multiple-choice questions covering various areas related to diagnosis communication. RESULTS:Most patients with Klinefelter syndrome reported that earlier communication would have been beneficial. Communication before the age of 18 and by parents increased the likelihood of overcoming negative consequences and relying on psychological support. CONCLUSION:To mitigate the adverse effects of poorly timed and inadequately delivered communication, typically by a single person, it is advisable that such communication be carried out at the onset of adolescence by an interdisciplinary team of HCPs (including psychologists, geneticists, endocrinologists) and parents. The information provided should not solely concentrate on hormonal and fertility aspects, but also consider other factors such as psychological variables.

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.

Background: Osteoporosis (OP) is as a systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of the bone tissue, leading to bone fragility and fracture susceptibility[1]. It represents a major cause of fractures in individuals over the age of 50 years potentially leading to complex sequelae and increased mortality. OP is widely considered to be much more prevalent in women, even though approximately 39% of new osteoporotic fractures estimated to have occurred worldwide in the year 2000 were in men[1]. The prevalence of OP in the US has been estimated in 3–6% in men >50 years and in 13–18% in women >50 years[2]. However, a study showed a comparable prevalence of osteoporosis for men aged 70 years or older and women aged 65 years[3]. Male OP is clearly underestimated, under-diagnosed and under-treated. The diagnosis is often made late in the natural history of the pathology or even after a fracture event. While guidelines on screening politics do not agree whether and when men should be considered, clinical evidence indicates that men tend to have more OP-related complications and higher mortality rate. Objectives: At present, there is no universally accepted policy for screening of male population to identify individuals with OP or those at high risk of fracture. To this end, the aim of this study was to provide a screening of male OP in a random population of aging men, independently of risk factors suggested by international guidelines, to provide real-life data on the actual fracture risk in aging men who wouldn’t be otherwise evaluated for bone loss on the basis of clinical guidelines. Methods: The study was approved by the Hospital EC (no. 3389/U16/14), and each participant (age >60 y o) gave written informed consent. A standardized questionnaire was used to collect information about smoking habits, alcohol misuse, and calcium intake. Exclusion criteria were any medical disorder or medications known to affect calcium metabolism, and testosterone replacement therapy. Vitamin D and testosterone were evaluated by chemiluminescence methods. Bone density was evaluated at lumbar vertebrae and proximal femur by Radiofrequency Echographic Multi-Spectrometry (REMS), based on the analysis of ultrasound signal backscattering. Subsequently a subgroup of patients underwent also femoral and lumbar DEXA and Heel quantitative ultrasound (QUS) scan. Results: A total of 300 male patients were enrolled and performed REMS scans. The prevalence of biochemical hypogonadism and vitamin D insufficiency were 18% and 85% respectively. Subjects were grouped on the basis of their REMS T-score: 61% had a T-score > −1, whereas 28% had osteopenia and 11% had OP. The frequencies of low testosterone and vitamin D insufficiency were significantly higher in patients with osteopenia or OP (22% and 100%) than in healthy subjects (15% and 76%; p = 0.029 and p = 0.013 respectively). Calcium intake and the frequency of smokers did not differ between groups. A subgroup of 80 patients, who were eligible, also underwent DEXA examination; showed concordance between DEXA and REMS in 90% of cases. The same subgroup had also performed a QUS scan, showing a 85% concordance with DEXA, as compared to REMS. Conclusion: Based on clinical experience, published guidelines and epidemiology of secondary causes and risk factor for OP in men[4-6], we suggest that OP should be considered in all men when risk factors for low BMD and fractures are present, independently from age. Age per se is sufficient for screening after the age of 60 years. Indeed, if clinicians just better consider bone health in their male patients bearing in mind different conditions, risk factors, lifestyle behaviours, and drugs able to deteriorate bone mass and quality, OP in men could be less ignored than in the past. In addition, non-ionizing approaches such as REMS should be advised for large screenings, given the lack of exposure to radiations, wide availability, lower costs and portability. REFERENCES: [1] DOI: 10.1016/0002-9343(93)90218-e.[2] DOI: 10.1359/jbmr.1997.12.11.1761.[3] DOI: 10.1007/s11657-012-0113-0.[4] DOI: 10.1080/14656566.2018.1428559.[5] DOI: 10.1016/S0140-6736(18)32112-3.[6] DOI: 10.1038/nrendo.2013.171. Acknowledgements: NIL. Disclosure of Interests: None declared.

Non-obstructive azoospermia (NOA) is the most severe form of male factor infertility. It results form from either primary or secondary testicular failure. Here, we report cases of two patients with NOA due to maturation arrest and increased serum FSH, treated with GnRH agonist and gonadotrophins. The two NOA patients underwent a pharmacological treatment consisting of pituitary desensibilization using a GnRH agonist and testicular stimulation using menotropin. Testicular stimulation started one month after the beginning of GnRH agonist treatment. The female partner underwent controlled ovarian stimulation (COS) followed by intracytoplasmic sperm injection (ICSI). On the third day of the cycle, menotropin daily doses was administered. When at least one follicle ≥14 mm was visualized, pituitary blockage was performed using GnRH antagonist ganirelix. When three or more follicles attained a mean diameter of ≥17 mm, triptorelin acetate was administered to trigger final follicular maturation. Oocyte retrieval was performed 35 hours later. After treatment, male partner blood levels of the FSH, LH, decreased and total testosterone were increased. Spermatozoa was observed after semen collection in both cases. After COS, oocytes were retrieved and ICSI was performed. Embryos were biopsied for preimplantation genetic testing (PGT) and those considered euploidy were transferred resulting in positive implantation, ongoing pregnancy, and livebirth on both cases. In this report we present a successful strategy for hypergonadotropic hypogonadism AOA men, as an alternative approach to the surgical testicular sperm recovery. Nevertheless, prospective randomized trials are needed to confirm our findings.

Aims: Follicle-stimulating hormone (FSH) plays a fundamental role in reproduction stimulating ovarian folliculogenesis, Sertoli cells function and spermatogenesis. However, the recent identification of FSH receptor (FSHR) also in extra-gonadal tissues has suggested that FSH activity may not be limited only to fertility regulation, with conflicting results on the possible role of FSH in endothelial cells. The aim of this study was to investigate FSH role on endothelial function in Human Umbilical Vein Endothelial Cells (HUVECs). Results: Endothelial Nitric oxide synthase (eNOS) expression, eNOS phosphorylation and Nitric Oxide (NO) production resulted increased after the stimulation of HUVEC with recombinant human FSH (rhFSH) at 3.6x10(3) ng/ml, with increasing Calcium release from intracellular stores. Furthermore, IP3 production increased after rhFSH stimulation despite PTX treatment and NFAT1 was observed prevalently in nucleus. We observed a statistical difference between untreated cells and cells stimulated with 0.36x10(3) ng/ml and between cells stimulated with 0.36x10(3) ng/ml and cells stimulated with 1.8x10(3) ng/ml at 4 and 8 h by Wound healing assay, respectively. Furthermore, a higher cellular permeability was observed in stimulated cells, with atypical VE-cadherin distribution, as well as filamentous actin. Conclusions: Our findings suggest that FSH at high concentrations elicits a signalling that could compromise the endothelial membrane. Indeed, VE-cadherin anomalies may severely affect the endothelial barrier, resulting in an increased membrane permeability. Although NO is an important vasodilatation factor, probably an excessive production could impact on endothelial functionality, partially explaining the increased risk of cardiovascular diseases in menopausal women and men with hypogonadism.

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 β-hydroxy-butyrate (β-HB). In addition to serum, β-HB is detected in several biofluids and β-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 β-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 β-HB on spermatozoa undergone to capacitation was also assessed. Results Two hours of exposure to β-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 β-HB after capacitation was associated with a significantly higher percentage of sperm cells with progressive motility compared to β-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 β-HB, underpinning a major role for this enzyme. Conclusion Our results suggest a possible physiological role for β-HB that could represent an energy metabolite in support of cell motility on capacitated spermatozoa right before encountering the oocyte.

Purpose Erectile dysfunction (ED) is one of the most prevalent male sexual dysfunctions. ED has been in the past mistakenly considered a purely psycho-sexological symptom by patients and doctors. However, an ever-growing body of evidence supporting the role of several organic factors in the pathophysiological mechanisms underlying ED has been recognized. Methods The Italian Society of Andrology and Sexual Medicine (SIAMS) commissioned an expert task force involving several other National Societies to provide an updated guideline on the diagnosis and management of ED. Derived recommendations were based on the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) system. Results Several evidence-based statements were released providing the necessary up-to-date guidance in the context of ED with organic and psychosexual comorbidities. Many of them were related to incorrect lifestyle habits suggesting how to associate pharmacotherapies and counseling, in a couple-centered approach. Having the oral therapy with phosphodiesterase type 5 inhibitors as the gold standard along with several other medical and surgical therapies, new therapeutic or controversial options were also discussed. Conclusions These are the first guidelines based on a multidisciplinary approach that involves the most important Societies related to the field of sexual medicine. This fruitful discussion allowed for a general agreement on several recommendations and suggestions to be reached, which can support all stakeholders in improving couple sexual satisfaction and overall general health.

Endocrine-disrupting chemicals (EDC) are exogenous compounds interfering with the actions of hormones at several steps. They are part of our environment and come in contact with humans frequently, through different routes. EDCs have shown deleterious effects on the endocrine system, mainly on the thyroid gland and steroid hormones, in the context of female and male reproduction. The hypothalamic–pituitary–adrenal (HPA) axis plays a pivotal role in the regulation of homeostasis, through regulation of adaptation to stress at multiple levels. Alterations of the HPA axis lead to severe metabolic and cardiovascular diseases, making this complex system a central player for human health. The HPA axis is a potential preferential target for disruption due to the chemical properties of EDCs and the specific features of the adrenal glands. Nonetheless, the consequences of EDCs exposure on the HPA axis have been underinvestigated to date in humans. The first part of this chapter provides an extensive summary of the mechanisms of HPA axis adaptation to environmental stimuli. The second part describes the current evidence on the deleterious effects of the main EDCs on the HPA axis.

CONTEXT:Mutations in the androgen receptor (AR) gene might be associated with infertility mainly because they cause various degrees of androgen insensitivity.OBJECTIVE:The aim of the study was to evaluate the frequency and type of AR variants in a large cohort of infertile males.METHODS:A total of 8224 males of Italian idiopathic infertile couples were referred to the University Hospital of Padova. The main outcome measures were mutational screening of AR, computational, and functional analyses.RESULTS:We found 131 patients (1.6%) harboring 45 variants in AR gene, of which 18 were novel missense AR variants. Patients with AR gene variants had lower sperm count (P = .048), higher testosterone (T) concentration (P < .0001), and higher androgen sensitivity index (ASI) (luteinizing hormone × T, P < .001) than patients without variants. Statistical analyses found T ≥ 15.38 nmol/L and ASI ≥ 180 IU × nmol/L2 as the threshold values to discriminate with good accuracy patients with AR variants. Patients with oligozoospermia and T ≥ 15.38 nmol/L had a 9-fold increased risk of harboring mutations compared with patients with normal sperm count and T < 15.38 nmol/L (odds ratio 9.29, 95% CI 5.07-17.02). Using computational and functional approaches, we identified 2 novel variants, L595P and L791I, as potentially pathogenic.CONCLUSION:This is the largest study screening AR gene variants in men of idiopathic infertile couples. We found that the prevalence of variants increased to 3.4% in oligozoospermic subjects with T ≥ 15.38 nmol/L. Conversely, more than 80% of men with AR gene variants had low sperm count and high T levels. Based on our findings, we suggest AR sequencing as a routine genetic test in cases of idiopathic oligozoospermia with T ≥ 15.38 nmol/L.

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, perfluorooctanoic acid (PFOA) and acetic acid, 2,2-difluoro-2-((2,2,4,5-tetrafluoro-5(trifluoromethoxy)-1,3-dioxolan-4yl)oxy)-ammonium salt (1:1) (C6O4) were used as, respectively, legacy-linear and new-generation-cyclic PFASs. Human recombinant SHBG 30-234 domain (SHBG(30-234)), 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 SHBG(30-234) and hSA. ITC revealed the binding of T to SHBG(30-234) with a K-d 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 SHBG(30-234). 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.

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.