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 molecu lar mechanisms underlying uterine dysfunction remain poorly understood. This study investi gated the impact of HS on the microRNA (miRNA) cargo of extracel lular vesicles (EVs) derived from bovine uterine fluid (UF) and its association with reproductive performance. Methods: Environmental conditions were classified according to the tem perature–humidity index (THI) recorded during the study periods: thermoneu tral 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 pro filing. 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, concep tion rates were markedly reduced. Under HS, concep tion rates were markedly reduced, and uterine artery resistance was increased, suggesting impaired uter ine perfusion. EVs isolated from UF showed comparable size and concentration across seasons; however, their miRNA cargo was signifi cantly altered. Small RNA sequencing identified 93 differentially expressed miRNAs, with a predominance of down regulated miRNAs under HS. Functional enrich ment analysis indicated that these miRNAs target genes involved in interleukin6 (IL6) signaling, lipid metabolism, and cellular communication. Notably, HS was associated with the accumulation of miRNAs potentially suppressing IL6 related pathways and lipid oxidation. Moreover, comparison with previously published data showed that a subset of miRNAs was shared with follicular fluid EVs, suggest ing coordinated regulation between ovarian and uterine compartments. Discussion: Overall, HS reshapes the molecular cargo of uterine EVs and may disrupt endometrium–embryo commu nication, contributing to reduced fertility. These find ings provide novel insights into uterine responses to HS and highlight EVassociated miRNAs as potential biomarkers of reproductive dysfunction in dairy cattle.