Equine Cervical Remodelling during Prepartum Period and Placentitis: A Transcriptomic Approach (Mucosa)

Placentitis was induced in five mares at approximately 290d of gestation (placentitis group), four mares with gestationally age-matched (290 d) pregnancies did not receive any treatment (control group), and the remaining three mares were maintained until approximately 330 d of gestation (prepartum group). For induction of placentitis in the former group, Streptococcus equi subsp. zooepidemicus was introduced intracervically. Overall design: Cervical mucosa samples were collected from mares with experimentally induced placentitis (290 d GA, n =5), normal pregnant mares (290 d GA, n=4), and prepartum mares (330d GA, n=3). Total RNA was isolated from all samples using RNeasy Mini Kit (#74104; Qiagen), and DNA digestion was performed on-column using RNase-free DNase I (#79254: Qiagen), followed by cleanup procedures. Paired-end reads with 150 nucleotides in length were produced. A TruSeq Stranded mRNA library prep kit (Illumina, San Diego, CA) was used to prepare libraries for mRNA sequencing. Libraries were loaded onto an Agilent DNA 1000 chip and validated on an Agilent 2100 Bioanalyzer (Agilent). Quantitation was performed with the Illumina Library Quantification Kit, ABI Prism qPCR Mix from Kapa Biosystems. Libraries were run on a NextSeq 500 v2 (Illumina) 300cycles High Output kit in a 2x150 base pairs with paired-end reads. The Fastq files were evaluated for read quality using FastQC 0.11.4. Subsequently, Trim Galore 0.4.1 was used for the adapter and read quality trimming (Phred score threshold of 30). Reads were mapped to the Equus caballus reference genome (EquCab 3.0) using the software STAR 2.5.3a, then annotated with the equine reference annotation from NCBI using Cufflinks 2.2.1. Fragments per kilobase per million (FPKM) was used to determine the expression level of genes. Lastly, we used Cuffdiff 2.2.1 to calculate differentially expressed genes (DEG) between samples from the control and urea groups. The significance level was set at the FDR-adjusted p-value of the test statistic < 0.05 using the Benjamini-Hochberg correction.