There are several potential biological mechanisms underlying the associations observed between caffeine and testosterone. After ingestion, caffeine is known to exert various pharmacological effects at the cellular level [
5]. Caffeine’s primary mechanism of action is antagonism of adenosine receptors, and acts on all four adenosine receptor subtypes in the brain (A1, A2a, A2b, A3) [
34,
35]. In addition to those found in the brain, adenosine receptors have also been described in the testes [
36]. Adenosine receptors observed in the testes are mainly localized within the Leydig and Sertoli cells of the seminiferous tubules, and these receptors are associated with an inhibition of cellular responses following activation. Following activation of these receptors, cAMP/protein kinase pathways, which are usually activated in mediation of testosterone production, are downregulated and may lead to lower testosterone production [
37–
39]. It is possible that caffeine affects testosterone production through these adenosine-dependent pathways.
The findings of this study may also be relevant for early life exposure to caffeine, and long-term effects exposure may have on reproductive outcomes. Reproductive studies have begun to investigate the association between caffeine exposure and parameters of reproduction such as sperm quality, semen volume, and egg maturation. A previous by Dlugosz et. al found that doses of caffeine higher than 400 mg/day might decrease sperm motility and/or increase the percentage of dead spermatozoa, but not sufficiently to affect the male fertility in an adverse manner [
40].Rats exposed to high doses of caffeine in utero developed smaller testes compared to controls [
41]. This study also found that stimulated-testosterone ex vivo production was reduced in Leydig cells retrieved from the high-dose caffeine rats. A Danish pregnancy cohort study found that men who were born to mothers drinking 4–7 cups/day of coffee had lower testosterone levels than sons of mothers drinking 0–3 cups/day [
42,
43]. Furthermore, there was a significant, positive association between high caffeine intake and testosterone levels in the adult males. In addition to direct effects on the testes, caffeine has been shown in vitro to induce aberrant DNA methylation and histone acetylation of the steroidogenic factor-1 (SF-1) promoter in the rat fetal adrenals, which acts to reduce transcription of the SF-1 gene. SF-1 is a key transcription factor involved in transcription of genes related to steroidogenesis and testosterone biosynthesis in males, and reduced expression of SF-1 may be a mechanism of low testosterone related to caffeine.
In addition to caffeine, the metabolically active products of caffeine metabolism including theophylline and theobromine of the xanthine class have been shown to have direct effects on gonadotropin-induced steroidogenesis [
44]. Mechanistically, theophylline and theobromine act as a phosphodiesterase inhibitors, adenosine receptor blockers, and histone deacetylase activators [
45,
46]. In one study, various concentrations of theophylline and 1-methyl 3-isobutyl xanthine (MIX) significantly inhibited steroidogenesis. Additionally, higher concentrations of MIX and theophylline also significantly inhibited precursor incorporation into RNA and protein. In another study, Osborne-Mendel rats who were fed varying doses of caffeine, theophylline, and theobromine exhibited significant testicular atrophy and impaired steroidogenesis [
47].Thus, in addition to direct effects of the parent compound caffeine, caffeine’s biologically active metabolites may also act through various pathways to affect testosterone production and half-life.
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