In this study, we investigated the clinical data and inflammatory parameters in dogs with pyometra at presentation, before and after surgical correction. Increases in the WBC counts, ALT and ALP activities, and concentrations of cholesterol and amylase and decreases in the RBC counts, haematocrit, hyperglobulinaemia, and hypoalbuminaemia were statistically significant in the dogs with pyometra compared to healthy dogs. Furthermore, significantly increased CRP, SAA, and cfDNA concentrations were found in dogs with pyometra compared to healthy dogs. After surgical correction of pyometra via OHE, the WBC and RBC counts, cholesterol, albumin, globulin concentrations, and ALP activity, as well as the IL-6, HMGB1, and PCT levels significantly decreased compared to pre-OHE.
In the dogs with pyometra, leucocytes and especially neutrophils and monocytes were significantly increased, whereas the RBC counts and haematocrit were decreased. Myeloid hyperplasia, especially neutrophilia, contributes to leucocytosis, and decreased iron utilisation in myeloid cells of the bone marrow by the APPs lactoferrin and hepcidin in response to systemic inflammation can reduce erythropoiesis [13–15]. Hyperproteinaemia, hyperglobulinaemia, and hypoalbuminaemia were also statistically significant in the dogs with pyometra vs. healthy controls, owing to an increased synthesis of APPs and antibodies in response to bacterial infection and inflammation. Decreased albumin levels may suggest a negative APP function  and are commonly associated with an inflammatory disease.
Organ damage is common during systemic inflammation in dogs . There were significant increases in ALT, ALP activities, and cholesterol concentration in the subjects with pyometra in this study. Increased liver enzyme activities and cholesterol concentrations were also observed in intrahepatic cholestasis, which is associated with endotoxaemia and hepatocellular damage caused by diminished circulation and cellular hypoxia . Renal injuries could be found in canine endotoxemia , and increases in creatinine concentrations, although not statistically significant, were found in this study. A decreased glomerular filtration rate due to endotoxaemia, may contribute to increases in amylase and lipase levels. Endotoxaemia may contribute to renal injury, but disease severity may affect the results. All dogs with pyometra survived in this study, which may indicate a lower disease severity, and therefore renal damage that might be statistically insignificant. Dogs with vaginal discharge were selected in this study, which could help with an early diagnosis before septic insult. Surgical correction decreased the RBC indices, total protein, albumin, and globulin levels, while increasing the sodium and chloride levels. Fluid administration during a surgical procedure may result in haemodilution and contribute to changes in electrolytes.
The CRP and SAA concentrations were significantly higher in the dogs with pyometra than in the healthy ones. CRP and SAA are the major APPs in dogs, and these results are consistent with those of previous reports on dogs with SIRS and pyometra  or chronic inflammation [19–21]. We also found a strong positive correlation between CRP and SAA. Thus, combined monitoring of the CRP and SAA biomarkers may be clinically advantageous for diagnosing inflammation. However, cfDNA was the most discriminative among the inflammatory parameters tested, based on the ROC curve analysis. Recent studies have suggested that the presence of cfDNA is due to necrosis, apoptosis, and the release of neutrophil extracellular traps (NETs), which are networks of extracellular fibres, primarily composed of DNA from neutrophils and binding pathogens. These fibres are released as a result of an innate immune mechanism initiated by neutrophils [22, 23], and thus, higher concentrations of cfDNA in dogs with pyometra may be due to tissue damage, resulting from systemic inflammation and NET formation. Besides, we found that the cfDNA concentrations, unlike the CRP and SAA levels, were correlated with the WBC counts, one of the SIRS criteria. The elevation of WBCs, especially neutrophils, is a typical sign of inflammation, suggesting that serum cfDNA concentrations may reflect inflammatory processes in dogs with sepsis.
In the dogs with pyometra, abnormal clinical parameters were strongly correlated with the cfDNA, CRP, and SAA concentrations. Correlations between the concentrations of these parameters and disease severity have been previously reported for CRP, SAA , and cfDNA ; however, there were no significant differences in these parameters before and after surgical correction. Moreover, the CRP level was the only inflammatory parameter that increased after surgery in healthy dogs, consistently with previous studies [12, 19, 26]. Surgical insult such as a laparotomy could affect the CRP concentration, even in healthy dogs, making CRP an unspecific biomarker for therapeutic monitoring. A more representative controlled study should be performed in the future to clarify the significance of this parameter.
When the infection source is removed, the concentrations of several biomarkers should decrease relative to those of endotoxins. Thus, we considered changes in the concentrations of inflammatory parameters as biomarkers for therapeutic monitoring. The study showed that most dogs with pyometra had lower concentrations of all cytokines following surgical treatment. These findings agree with the results of the comparison between dogs with pyometra and healthy ones. Although not all inflammatory parameters were fully accounted for by the treatment effects, IL-6, HMGB1, and PCT emerged as potentially relevant biomarkers for therapeutic monitoring. The baseline concentrations of these molecules were not significantly higher in the dogs with pyometra than in the healthy controls but decreased following the removal of the inflamed uterus, thus indicating their usefulness in the postoperative period in dogs with pyometra .
IL-6 and HMGB1 levels usually increase in canine endotoxaemia and systemic inflammation [5, 7]; however, changes in their levels were not statistically significant in our study. IL-6 is a major proinflammatory cytokine released during an early phase of endotoxaemia , whereas HMGB1 is a late mediator, released by damaged or killed cells . Because the analysed pyometra cases did not develop severe sepsis or organ damage, the peak concentrations of these inflammatory parameters might not have been persistent, or no peak levels were reached. However, the concentrations did significantly drop after surgical correction, and thus, IL-6 and HMGB1 could be useful biomarkers for therapeutic monitoring.
Both human and animal studies have suggested that an increase in the PCT concentration may be associated with serious infections and endotoxaemia [28, 29] and demonstrated that PCT acted as an APP in dogs. In humans, PCT is used for monitoring during treatment and as a prognostic marker . Although PCT did not discriminate subjects with pyometra from healthy dogs, its concentration was significantly lower after surgical correction. Moreover, there were significant correlations between PCT levels and several types of clinical data, such as haematocrit, monocyte counts, BUN, total calcium, albumin, globulin, and ALP concentrations. Thus, application of PCT in clinical practice as a relevant surrogate biomarker for organ dysfunction and the severity of disease may be suggested.
Our study has several potential limitations, one of which is that the identification and assessment of the severity of pyometra were subjective. Additionally, the diagnosis of pyometra was confirmed based on gross findings, making it difficult to distinguish sepsis and SIRS, as gross confirmation of infection may or may not correspond to systemic illness . However, the high frequency of clinical signs, abnormalities in blood tests, and persistent suppurative purulence suggested a systemic inflammatory response as a possible explanation of abnormal clinical criteria. Finally, all subjects survived until discharge, and thus, we could not compare parameters between the survival and non-survival groups. Further studies are necessary to validate the prognostic indicators.