Characterization of cytokine gene expression in uterine cytobrush samples of non-endometritic versus endometritic postpartum dairy cows.

To better understand uterine inflammation in postpartum dairy cows we collected sequential cytobrush samples at 29-35 and at 49-55 d in milk (DIM). Based on the uterine cytology, cows were classified as Non-endometritic (n = 23; <18% neutrophils) or Endometritic (n = 12; ≥18% neutrophils) at 29-35 DIM and Non-endometritic (n = 17; <10% neutrophils) or Endometritic (n = 9; ≥10% neutrophils) at 49-55 DIM. Cows defined as Sham Controls (n = 6) were examined by vaginoscopy at 29-35 DIM and identified as Non-endometritic (<10% neutrophils) at 49-55 DIM. Cytokine gene expression in cytobrush samples was assessed using qRT-PCR. Sham Controls did not differ significantly (P > 0.17) from Non-endometritic cows at 49-55 DIM and these data were combined (n = 23). Uterine cytology-based classification using the aforementioned thresholds effectively separated cows into groups with Endometritic cows having significantly higher expression of pro-inflammatory (interleukin (IL)-1α, IL-1ß, IL-6, IL-8, IL-17A CSF-1; P < 0.01) and regulatory (IL-1RA and IL-10; P < 0.03) cytokines, relative to Non-endometritic cows. Furthermore, Non-endometritic cows showed a significant decline (P < 0.03) in the expression of pro-inflammatory (IL-1α, IL-6, IL-8) and regulatory (IL-10) cytokine genes as the postpartum period progressed; whereas Endometritic cows exhibited a sustained elevation in transcript abundance throughout the sample period for both pro-inflammatory and regulatory cytokine genes. Expression of transforming growth factor (TGF) genes was more complex with TGF-ß3 expression significantly (P < 0.01) lower at 29-35 DIM and TGF-ß1 gene expression significantly (P < 0.03) increased at 49-55 DIM in Endometritic versus Non-endometritic cows. Expression of TGF-ß2 gene was 2.7-fold higher (P < 0.01) at 29-35 DIM in cows that remained Endometritic when compared to cows recovering by 49-55 DIM. Some Non-endometritic cows (n = 4) at 29-35 DIM were reclassified as Endometritic at 49-55 DIM. The sampling procedures at 29-35 DIM did not alter either the cellular response (P > 0.43) or cytokine gene expression (P > 0.17) at 49-55 DIM. In conclusion, normal uterine involution is characterized by a progressive decline in pro-inflammatory and regulatory cytokine gene expression, while cows with endometritis show a dysregulated inflammatory process characterized by a sustained elevation in pro-inflammatory and regulatory cytokine gene expression. This analysis also shows that decreased TGF-ß2 gene expression at 29-35 DIM may be an indicator of recovery from endometritis.

Proinflammatory cytokine gene expression in endometrial cytobrush samples harvested from cows with and without subclinical endometritis.

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 ßactin 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.