title: Proinflammatory cytokine gene expression in endometrial cytobrush samples harvested from cows with and without subclinical endometritis.
abstract: Thirty postpartum cows (28 to 41 days in milk) without signs of clinical endometritis were categorized as inflammation-negative (N = 18) or subclinical endometritis-positive (N = 12) based on endometrial cytobrush cytology (> 18% polymorphonuclear cells; PMNs). Slides for cytology were prepared before the same cytobrush was transferred to a tube containing 1 mL Trizol reagent. Total RNA was extracted from each cytobrush sample and analysis of il6, il8, tnfα, and βactin gene expression was performed using quantitative real-time polymerase chain reaction. Cytobrush sampling provided sufficient material to prepare cytosmears and extract high quality endometrial mRNA (mean = 0.96 μg RNA per sample). Cytokine expression varied between experimental groups with a 20-fold higher tnfα (P = 0.001), a 30-fold higher il6 (P = 0.01), and a greater than 50-fold higher il8 mRNA expression level (P = 0.0001) in subclinical endometritis-positive versus disease-negative cows. Regression analysis of gene expression levels (cycle threshold) versus PMN frequency showed that the frequency of PMNs in the cytosmear decreased by 3.3% (P = 0.000 01), 2.3% (P = 0.015), and 2.4% (P = 0.05) for each additional cycle threshold required to detect il8, il6, and tnfα gene expression, respectively. Expression of the individual cytokines was positively associated: il8 and il6 (P = 0.0001); il8 and tnfα (P = 0.000 01); and il6 and tnfα (P = 0.0002). In conclusion, the endometrial cytobrush technique was successfully used to obtain material for both cytology and RNA extraction, and il8 gene expression may be useful to predict endometrial inflammation.
authors.affiliation: Department of Large Animal Clinical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
authors.forename: F
authors.initials: F
authors.lastname: Ghasemi
authors.forename: P
authors.initials: P
authors.lastname: Gonzalez-Cano
authors.forename: P J
authors.initials: PJ
authors.lastname: Griebel
authors.forename: C
authors.initials: C
authors.lastname: Palmer
mesh_terms: D000199:Actins / Q000235:genetics; D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000378:metabolism*; D016207:Cytokines / Q000235:genetics*; D004716:Endometritis / Q000378:metabolism / Q000662:veterinary*; D004717:Endometrium / Q000737:chemistry* / Q000473:pathology*; D005260:Female; D015870:Gene Expression; D015850:Interleukin-6 / Q000235:genetics; D016209:Interleukin-8 / Q000235:genetics; D009504:Neutrophils / Q000473:pathology; D012333:RNA, Messenger / Q000032:analysis; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
chemical_list: D000199:Actins; D016207:Cytokines; D015850:Interleukin-6; D016209:Interleukin-8; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha