Differences in Uterine Fluid-Induced Endometrial Cell Responses and Uterine Microbiota in Repeat Breeder Cows

Repeat breeder (RB) cows fail to conceive after three or more breeding attempts, yet the uterine factors contributing to this condition remain unclear. Here, we compared uterine fluid composition, endometrial cell responses to uterine fluid, and uterine microbiota between normal pregnant (Normal) and RB cows. Uterine fluid was collected from multiparous Holstein cows classified as Normal (n = 5; conception within three breeding attempts) or RB (no conception after four attempts); RB cows were classified as RB-Rec (n = 4; conceived after uterine lavage) or RB-NoRec (n = 3; did not conceive after uterine lavage). Uterine fluids were applied to bovine endometrial cells, and microbiota composition was analyzed. Concentrations of lipopolysaccharide in the uterine fluid did not differ between the three groups, and uterine fluid from both RB groups significantly increased C-X-C motif chemokine ligand 8 secretion relative to the Normal group. RNA-seq revealed distinct endometrial cell responses to RB-NoRec uterine fluid, with enrichment of immune-related pathways including phagosome, ferroptosis, and cellular senescence. Uterine microbiota profiles differed among groups, with short-chain fatty acid-producing genera (e.g., Fusicatenibacter and Dorea) predominant in Normal cows and inflammation-associated genera (e.g., Trueperella and Helicobacter) predominant in RB-NoRec cows. These data indicate that uterine fluid properties and microbiota composition vary with reproductive status and may modulate endometrial immune activation associated with fertility outcomes.