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In recent times, additional pathways involved in myometrial regulation and also in the action of oxytocin and-adrenergic agonists on the myometrium, other than what has been known previously has been suggested. Knowledge of other pathways will prove useful in designing better therapies for myometrial pathologies. This study was therefore aimed at investigating the possibility of other pathways involved in the activity of either oxytocin or a-adrenergic agonists (ritodrine was used in this study) in myometrial activity with the aid of metabolomics and bioinformatics. High resolution Fourier transform mass spectrometry (HRFTMS), and nuclear magnetic resonance (NMR) spectroscopy coupled with functional uterine assays were combined for an innovative assessment. In vitro pharmacological assay of oxytocin (1 nM) and ritodrine (0.1 nM) on isolated mice uteri mounted in 3 ml organ baths were performed. Mice uteri, treated with oxytocin (OT) or ritodrine (RIT), as well as the physiological buffer in which the uterine tissues were immersed, were rapidly collected and analysed using HRFTMS, 1HNMR and bioinformatics. Resulting data were analyzed via pair-wise chemometric comparison models with p≤ 0.05 considered statistically significant. In addition to previously known metabolites, anandamide, nicotinamide adenine dinucleotide, gamma aminobutyric acid and sphingosine were significantly associated with OT’s activity while a downstream involvement of prostaglandin F1 and phosphatidylinositol signalling was observed with RIT’s activity. These findings adds evidence to the reports on additional regulation of myometrial activity by these …