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Context: Perfluoroalkyl-substances (PFAS) are chemical additives considered harmful for humans. We recently showed that accumulation of perfluoro-octanoic acid (PFOA) in human semen of exposed subjects was associated with altered motility parameters of sperm cells, suggesting direct toxicity. Objectives: To determine whether direct exposure of human spermatozoa to PFOA was associated to impairment of cell function. Patients and methods: Spermatozoa isolated from semen samples of ten normozoospermic healthy donors were exposed up to 2 h to PFOA, at concentrations from 0.1 to 10 ng/mL. Viability and motility parameters were evaluated by Sperm Class Analyser. Cell respiratory function was assessed by both mitochondrial probe JC-1 and respiratory control ratio (RCR) determination. Sperm accumulation of PFOA was quantified by liquid chromatography–mass spectrometry. Expression of organic ion-transporters OATP1 and SLCO1B2 was assessed by immunofluorescence and respective role in PFOA accumulation was evaluated by either blockade with probenecid or membrane scavenging through β-cyclodextrin (β-CD). Plasma membrane fluidity and electrochemical potential (ΔΨp) were evaluated, respectively, with Merocyanine-540 and Di-3-ANEPPDHQ fluorescent probes. Results: Compared to untreated controls, a threefold increase of the percentage of non-motile sperms was observed after 2 h of exposure to PFOA regardless of the concentration of PFOA, whilst RCR was significantly reduced. Only scavenging with β-CD was effective in reducing PFOA accumulation, suggesting membrane involvement. Altered membrane fluidity, reduced ΔΨp and sperm motility loss associated with exposure to PFOA were reverted by β-CD treatment. Conclusion: PFOA alters human sperm motility through plasma-membrane disruption, an effect recovered by incubation with β-CD.

Purpose: Undercarboxylated-Osteocalcin (ucOCN), acting on its putative receptor GPRC6A, was shown to stimulate testosterone (T) production by Leydig cells in rodents, in parallel with the hypothalamus-pituitary–gonadal axis (HPG) mediated by luteinizing hormone (LH). The aim of this cross-sectional study was to evaluate the association among serum ucOCN, rs2247911polymorphism of GPRC6A gene and the endocrine/semen pattern in a cohort of infertile males, possibly identifying an involvement of the ucOCN-GPRC6A axis on testis function. Methods: 190 males, including 74 oligozoospermic subjects, 58 azoosperminc patients and 58 normozoospermic controls, were prospectively recruited at the Orient Hospital for Infertility, Assisted Reproduction and Genetics in Syria (Study N. 18FP), from July 2018 to June 2020. Outpatient evaluation included the clinical history, anthropometrics and a fasting blood sampling for hormonals, serum OCN (both carboxylated and undercarboxylated), glycemic and lipid profile and screening for rs2247911GPRC6A gene polymorphism. Results: Higher serum ucOCN associated with higher T and HDL-cholesterol (respectively: r= 0.309, P< 0.001 and r= 0.248, P= 0.001), and with lower FSH (r=  – 0.327, P< 0.001) and LDL-cholesterol (r=  – 0.171; P= 0.018). Patients bearing the GG genotype of rs2247911had higher sperm count compared to GA genotype (P= 0.043) and, compared to both AG and AA genotypes, had higher serum T (P= 0.004, P= 0.001) and lower triglycerides levels (P= 0.002, P< 0.001). Upon normalization for LH levels and body mass index, rs2274911and ucOCN were significantly associated with higher serum T at linear stepwise regression analysis (P= 0.013, P= 0.007). Conclusions: Our data suggest the involvement of ucOCN-GPRC6A axis in the regulation of T production by the testis, subsidiary to HPG.