Menstrual cycle-dependent expression of tRNA-derived fragments in healthy human endometrium and uterine fluid-derived extracellular vesicles.

The human endometrium undergoes dynamic cyclical remodeling across the menstrual cycle to establish receptivity for embryo implantation. While microRNAs are established regulators of endometrial function and embryo–maternal communication, the contribution of tRNA-derived fragments (tRFs) remains largely unexplored. In this study, we performed comprehensive small RNA sequencing of endometrial biopsy (EB) samples and paired uterine fluid–derived extracellular vesicles (UF-EVs) collected from healthy fertile women across consecutive menstrual cycle phases: mid-proliferative (MP), early secretory (ES), mid-secretory (MS), and late secretory (LS). Using the tRAX analytical framework, we characterized the landscape and compartmentalization of tRF populations. tRFs were detected from all 21 cytosolic tRNA isotypes in both EB and UF-EVs, displaying marked phase- and tissue-specific variation. In EB, tRFs were predominantly derived from tRNA-Lys and tRNA-Val, whereas UF-EVs exhibited greater isotype diversity, including substantial contributions from tRNA-Asp and tRNA-Gly. Fragment-type profiling revealed detection of 5′, 3′-, and internal tRF classes across all phases, supporting broad capture of diverse tRF subtypes. Notably, tRFs derived from tRNA-Val showed the most pronounced menstrual cycle–dependent regulation. Multiple 5′-tRF-Val species, including transcript- and isodecoder-specific fragments from tRNA-Val-CAC and tRNA-Val-AAC, were strongly upregulated during the ES and MS phases compared with the MP phase in both EB and UF-EVs. These coordinated increases suggest selective accumulation and extracellular export of tRNA-Val fragments during the secretory phase, potentially reflecting hormonally driven regulation of tRF biogenesis and vesicle-mediated signaling. Together, these findings provide the first comprehensive characterization of menstrual cycle–dependent tRF expression in the human endometrium and UF-EVs. Our results identify tRNA-Val–derived 5′-tRFs as prominent secretory-phase–associated small RNAs and support a potential role for tRFs as novel regulators and extracellular indicators of endometrial receptivity.