Heat stress modulates extracellular vesicles miRNA cargo in bovine uterine fluid and uterine function

Introduction: Heat stress (HS) impairs fertility in dairy cattle, yet the molecular mechanisms underlying uterine dysfunction remain poorly understood. This study investigated the impact of HS on the microRNA (miRNA) cargo of extracellular vesicles (EVs) derived from bovine uterine fluid (UF) and its association with reproductive performance. Methods: Environmental conditions were classified according to the temperature–humidity index (THI) recorded during the study periods: thermoneutral conditions in winter (THI: 57.7 ± 0.9; THI < 72) and heat-stress conditions in summer (THI: 76.6 ± 1.8; THI > 72). On the day of estrus (day 0), a total of 20 Holstein cows were enrolled, 10 during winter and 10 during summer. On the same day, Doppler ultrasonography was performed to assess uterine artery hemodynamics, and uterine fluid was collected for EV isolation and miRNA profiling. The conception rate was determined 30 days after artificial insemination (AI) by ultrasound. Pregnancy was defined as the visualization of an embryo with heartbeat by transrectal ultrasonography 30 days after AI. Results: Under HS, conception rates were markedly reduced. Under HS, conception rates were markedly reduced, and uterine artery resistance was increased, suggesting impaired uterine perfusion. EVs isolated from UF showed comparable size and concentration across seasons; however, their miRNA cargo was significantly altered. Small RNA sequencing identified 93 differentially expressed miRNAs, with a predominance of down-regulated miRNAs under HS. Functional enrichment analysis indicated that these miRNAs target genes involved in interleukin-6 (IL-6) signaling, lipid metabolism, and cellular communication. Notably, HS was associated with the accumulation of miRNAs potentially suppressing IL-6-related pathways and lipid oxidation. Moreover, comparison with previously published data showed that a subset of miRNAs was shared with follicular fluid EVs, suggesting coordinated regulation between ovarian and uterine compartments. Discussion: Overall, HS reshapes the molecular cargo of uterine EVs and may disrupt endometrium–embryo communication, contributing to reduced fertility. These findings provide novel insights into uterine responses to HS and highlight EV-associated miRNAs as potential biomarkers of reproductive dysfunction in dairy cattle.