Expression patterns of apoptosis- and ferroptosis-associated transcripts in uterine tissues of female dogs diagnosed with open- or closed-cervix pyometra

In dogs diagnosed with pyometra, cervical patency status may influence the predominance of apoptosis or ferroptosis, two mechanistically distinct forms of regulated cell death. We evaluated the expression of selected transcripts related to apoptotic and ferroptotic pathways in the uterine tissuee of female dogs diagnosed with open- or closed-cervix pyometra. Twenty-four bitches were classified as healthy (n = 8), open-cervix pyometra (n = 8), or closed-cervix pyometra (n = 8). Uterine tissue samples from each dog were collected for the quantification of apoptotic- (BAX, BCL-2, TP53, and CASP3) and ferroptosis-related transcripts (GPX4, SLC7A11, and TFRC) via qPCR. Immunohistochemical analysis was conducted using antibodies specific to the proteins encoded by BAX, BCL2, TP53, CASP3, GPX4, SLC7A11, and TFRC genes. Furthermore, protein-protein interaction (PPI) and enrichment analyses for the same genes were conducted. Data were fitted in Kruskal-Wallis tests to fit the effect of healthy, open-cervix pyometra, or closed-cervix pyometra on gene expression profile and immunohistochemistry staining parameters. Compared to control, the expression of BAX, SLC7A11, TFRC, GPX were upregulated, BCL2 was downregulated in closed-cervix pyometra. Mild and strong immunopositivity in BAX, BCL2, TP53, CASP3 antibodies was observed in closed pyometra cases. In closed-cervix pyometra, severe inflammation was observed in the endometrium, with immunopositivity for SLC7A11 and GPX4 highlighted within the cytoplasm of mononuclear inflammatory cells, primarily located in the endometrial stroma. PPI analysis revealed proteins encoded by target genes, such as FADD, FASLG, and GSDME, considering maximum of 20 interactions per protein, resulting network highlights significant roles of proteins in various biological pathways, including apoptosis, ferroptosis, inflammation, cellular immunity. Apoptosis and ferroptosis are regulated in pyometra related cervical patency status and these pathways, more active in closed-cervix pyometra. This is the first study to identify apoptosis and ferroptosis pathways in pyometra, providing new insights into its pathophysiology and emphasizing prognostic relevance of both pathways, particularly in closed-cervix cases.