Human sperm cells can form paracetamol metabolite AM404 that directly interferes with sperm calcium signalling and function through a CatSper-dependent mechanism.

Do paracetamol (N-acetyl-para-aminophenol (APAP) or acetaminophen) and/or its metabolites affect human sperm Ca2+-signalling and function? While APAP itself does not interact with Ca2+-signalling in human sperm, its metabolite N-arachidonoyl phenolamine (AM404), produced via fatty acid amide hydrolase (FAAH), interferes with human sperm Ca2+-signalling and function through a suggested CatSper channel-dependent action. Studies have shown that adult men with high urinary levels of over-the-counter mild analgesic APAP have impaired sperm motility and increased time-to-pregnancy. This study consists of (i) an in vivo human pharmaceutical APAP exposure experiment to understand to what degree APAP reaches the sperm cells in the seminal fluid; (ii) in vitro calcium imaging and functional experiments in freshly donated human sperm cells to investigate CatSper channel-dependent activation by APAP and its metabolites; and (iii) experiments to understand the in situ capabilities of human sperm cells to form APAP metabolite AM404. Three healthy young males participated in the in vivo human exposure experiment after prior consent. Human semen samples were provided by healthy young volunteer donors after prior consent on the day of the in vitro experiments. Pharmaceutical APAP exposure reaches the seminal plasma in high micromolar concentrations and accumulates in the seminal plasma between 3 and 5 days of exposure (P-value 0.023). APAP and its primary metabolite 4-aminophenol (4AP) do not interact with human sperm Ca2+-signalling. Instead, the APAP metabolite AM404 produced via FAAH interferes with human sperm Ca2+-signalling through a CatSper-dependent action. Also, AM404 significantly increases sperm cell penetration into viscous mucous (P-value of 0.003). FAAH is functionally expressed in human sperm cells in the neck/midpiece region, as evidenced by immunohistochemical staining and the ability of human sperm cells to hydrolyse the fluorogenic FAAH substrate arachidonyl 7-amino, 4-methyl coumarin amide in an FAAH-dependent manner. Importantly, human sperm cells have the capacity to form AM404 in situ after exposure to 4AP (P-value 0.0402 compared to vehicle-treated sperm cells). The experiments were conducted largely in vitro. Future studies are needed to test whether APAP can disrupt human sperm function in vivo through the action of AM404. We hypothesize that these observations could, at least in part, be responsible for the negative association between male urinary APAP concentrations, sperm motility and time-to-pregnancy. D.M.K. is funded by the Lundbeck Foundation, grant number R324-2019-1881, and the Svend Andersen Foundation. A.R. is funded by a BRIDGE-Translational Excellence Programme grant funded by the Novo Nordisk Foundation, grant agreement number: NNF18SA0034956. All authors declare no competing interests. N/A.

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