Investigating endometrial metabolic characteristics in patients with adenomyosis using gas chromatography/mass spectrometry.

Background: The aetiology and pathogenesis of adenomyosis remain unclear. This study utilised untargeted metabolomics to explore the aberrant amino acid metabolism in adenomyosis. Among the studied metabolites, hydroxyproline is known to promote the proliferation and invasion of tumour cells. Due to detection limitations, hydroxyproline levels were inferred via the expression of prolyl 4-hydroxylase subunit alpha 1 (P4HA1), a crucial enzyme that catalyses the conversion of proline to hydroxyproline.

Methods: Endometrial metabolomic analysis was performed using gas chromatography-mass spectrometry (GC-MS) in 15 and 20 patients with and without adenomyosis. The expression levels of P4HA1 mRNA and protein were detected using quantitative PCR (qPCR), western blotting, and immunohistochemistry. In vitro, Cell Counting Kit-8, and 5-Ethynyl-2'-deoxyuridine (EdU) assays were used to investigate the effect of hydroxyproline on the proliferation of human endometrial stromal cells (hESCs). Additionally, a wound-healing assay was conducted to examine the effect of hydroxyproline on hESCs migration.

Results: In total, 22 differentially expressed metabolites were identified in the adenomyosis group compared to the control group. qPCR results demonstrated that P4HA1 mRNA expression levels were significantly higher in the adenomyosis group than in the control group (p < 0.01), which was further validated at the protein level by western blotting and immunohistochemistry. In vitro, functional assays revealed that hydroxyproline promoted hESCs proliferation and migration in a dose-dependent manner. EdU assays showed a significant increase in the number of EdU-positive hESCs in the 5 mM hydroxyproline treatment group than that in the control group (p < 0.01). Additionally, wound healing assays demonstrated enhanced migration of hESCs after treatment with 5 mM hydroxyproline (p = 0.002).

Conclusion: Hydroxyproline levels were significantly elevated in the endometrial tissues of patients with adenomyosis. Furthermore, hydroxyproline promotes the proliferation and migration of hESCs. These findings provide new insights into the pathogenesis of adenomyosis and suggest potential therapeutic strategies.