Decoding Myometrial Activation in an Equine Ascending Placentitis Model: Transcriptomic Insights

Rebecca Hutchinson1, Kirsten Scoggin1, Mats Troedsson1, Hossam El-Sheikh Ali<sup>1
1. Department of Veterinary Science, University of Kentucky, Lexington, Kentucky</sup>

Abstract Text:

Equine ascending placentitis is a condition in horses that is orthologous to chorioamnionitis in humans. It involves inflammation of the placenta, most commonly due to ascending infection, and is a major contributor to abortions, stillbirths, and perinatal mortality in horses worldwide.The main event in placentitis-induced preterm labor is the loss of myometrial quiescence, which triggers the onset of labor. However, the molecular mechanisms driving myometrial activation in horses are not well understood. To explore these mechanisms, we examined the transcriptome of the equine myometrium during ascending placentitis and compared it to gestationally matched normal pregnancy. In brief, myometrial samples were collected post-euthanasia from mares with induced placentitis around 290 days of gestation (placentitis group; n=6) and from un-inoculated mares (control group; n=6). RNA sequencing (RNA-Seq) was performed using the Illumina NovaSeq6000 platform, with raw reads processed using TrimGalore 0.4.3 before mapping to EquCab3.0 via STAR 2.4.3a. Mapped reads were quantified using Cufflinks 2.2.1 with NCBI annotation. Differentially expressed genes (DEGs) were identified using Cuffdiff 2.2.1, with a false discovery rate (FDR) < 0.05. Our analysis revealed 263 DEGs in the myometrium during placentitis compared to the control, including seven genes (APOBEC3Z1B, PLA2G2D, CXCL17, MMP8, MMP13, S100A8, and S100A9) that were uniquely upregulated (exclusively expressed) during placentitis. Gene groups related to connexins (GJC2, also known as connexin-47), matrix metalloproteinases (MMP1, MMP8, MMP13, and MMP25), prostaglandin signaling (PTGS2, PTGER3), calcium channel regulation (TRPM2), and calcium ion transport (RAMP2, CACNA1D, PRKCB, COR1A, and DEFB1) point to a clear pathway for myometrial activation during equine placentitis. Additionally, we identified several potential chemoattractants (CCL2, CCL3, CCL4, CCL5, CXCL1, CXCL3, CXCL16, and CXCL17) in the myometrium during placentitis, a finding supported by significant leukocytic infiltration observed in myometrial samples from the placentitis group. Leukocytic infiltration is a well-known hallmark of myometrial activation. To identify potential upstream regulators of myometrial activation, we performed upstream regulator analysis using Ingenuity Pathway Analysis, which revealed 12 potential regulators: TLR2, STAT1, IRF7, SPI1, PTGS2, S100A8, S100A9, ICAM1, NRG1, HIF1A, PRKCB, and TYROBP. Immunolabeling of TLR2, PTGS2, S100A8, S100A9, and HIF1A showed significant positive correlation with their respective mRNA expression. Overall, this study enhances our understanding of key regulators and mechanisms of myometrial activation during equine placentitis, which may inform the development of therapies to prevent preterm labor and treat placentitis. Notably, our findings underscore the central role of TLR2 in initiating inflammatory signaling within the equine myometrium during placentitis, which drives the upregulation of contraction-associated transcripts such as PTGS2, PTGER3, MMP1, MMP8, and GJC2. Consequently, targeting TLR2 and its associated pathways—for example, through TLR antagonists—represents a promising therapeutic strategy to mitigate placentitis-induced preterm birth. Beyond elucidating the mechanisms of myometrial activation described in this study, our work establishes a foundation for generating new hypotheses and guiding future research directions.