Interferon stimulated genes as peripheral diagnostic markers of early pregnancy in sheep: a critical assessment

Abstract

We investigated the diagnostic reliability of pregnancy detection using changes in interferon stimulated gene (ISG) messenger RNA (mRNA) levels in circulating immune cells in ewes. Two different groups of ewes (an experimental group, experiment 1 and a farm group, experiment 2) were oestrus-synchronized and blood sampled on day 14 (D0 = day of insemination in control animals, experiment 1) and day 15 (experiment 2). Real-time PCR were performed to evaluate the abundance of different ISG mRNAs. In the experimental group, peripheral blood mononuclear cells of 29 ewes born and bred in experimental facilities were isolated using a Percoll gradient method. Gene expression for Chemokine (C-X-C motif) ligand 10 ( CXCL10), Myxovirus (influenza virus) resistance 1 ( MX1) and Signal transducer and activator of transcription 1 (STAT1) mRNA were, respectively, 8.3-fold, 6.1-fold and 2.7-fold higher ( P < 0.001) in pregnant compared with non-pregnant ewes. The receiver operating characteristic (ROC) curves generated from the real-time PCR data demonstrated that a reliable cut-off could be established for CXCL10, MX1 and STAT1. In the farm group of animals, peripheral blood leucocytes of 37 cross-bred multiparous ewes bought from several herds were isolated using the PAXgene ® procedure. This blood sampling procedure is achievable in farms, whereas the Percoll method is not. No significant differences ( P > 0.10) in CXCL10, STAT1, MX1, Myxovirus (influenza virus) resistance 2 ( MX2) and ISG15 ubiquitin-like modifier ( ISG15) mRNA expression were found between pregnant and non-pregnant ewes. The ROC curves and the hierarchical classification generated from the real-time PCR data failed to discriminate between pregnant and non-pregnant animals. In this group of animals, our results show a strong variability in ISG expression patterns: 17% of animals identified as non-pregnant by the five tests were in fact pregnant, only 52% of pregnant animals had at least two positive results (two genes above threshold), whereas up to five positive results (five genes above threshold) were needed to avoid misclassification. In conclusion, this study illustrates the high variability in ISG expression levels in immune circulating cells during early pregnancy and, therefore, highlights the limits of using ISG expression levels in blood samples, collected on PAXgene ® tubes on farms, for early pregnancy detection in sheep.Keywords: interferon stimulated genes, pregnancy diagnosis, early pregnancy, implantation, sheep ImplicationsThis study aimed to investigate the accuracy of gene transcript quantification in blood (interferon stimulated genes (ISG)) as a method of early pregnancy diagnosis in sheep. This method of pregnancy detection is based on a single blood sample, whereas routine pregnancy diagnosis, namely ultrasonography, requires specific equipment and a skilled operator. Our results show a higher variability in ISG expression patterns in animals from commercial herds than from a controlled research station. Consequently, this prevents th...

Koyunların Erken Gebelik Döneminde Kemik iliğinde ISG15 Ekspresyonu

“…2). These findings were similar to previous results that there were changed expressions of ISG15 in endometrium [3,24] , blood cells [25,26] , CL [6,27] during early pregnancy in sheep and cattle. It was indicated that the embryonic signal (IFNT) reached the blood cells, CL and BM to up-regulate ISG15 expression through blood or/and immune cells during early pregnancy in sheep.…”

Exploring the concentration‐dependent actions of interferon‐τ on bovine neutrophils to understand the process of implantation

“…The increase in ISG15 in neutrophils with IFNT supplementation is in agreement with the earlier findings obtained from blood cells in sheep, 16 bone marrow of ewes, 17 and liver biopsies 18 of HF cattle in early pregnancy. The increase in ISG15 in neutrophils with IFNT supplementation is in agreement with the earlier findings obtained from blood cells in sheep, 16 bone marrow of ewes, 17 and liver biopsies 18 of HF cattle in early pregnancy.…”

Changes in expression of interferon-stimulated genes and ubiquitin activating enzyme E1-like in ovine thymus during early pregnancy

“…Early pregnancy induces expression of IP-10 mRNA in PBLs, and IFNT treatment also does it in in vitro cultured PBLs, suggesting that upregulation of IP-10 is a pregnancy-dependent event in cows (Sakumoto et al, 2018). There is an upregulation of IP-10 gene in PBMCs during early pregnancy, which is related with early pregnancy recognition in sheep (Mauffré et al, 2016). Therefore, the upregulation of IP-10 may participate in the regulation of maternal thymic functions, which is related with early pregnancy in sheep.…”