Screening and Identification of mRNA Candidate Immunobiomarkers during Pregnancy in Sheep

Animal immunity refers to the ability of an animal to resist invasion by pathogens, such as bacteria, viruses, parasites and fungi, as well as to cope with other harmful substances, which was provided by the animal’s immune system. The immune system of a female animal faces a great challenge during pregnancy. Part of the immune function must be suppressed to protect the embryo from being excessively rejected in the parent body, which could lead to abortion or restricted growth and development. Meanwhile, the parent body also needs to maintain a certain level of immune function to protect itself against invasion by foreign pathogens and excessive invasion during embryo implantation. Interleukin 6 (IL-6) plays a crucial role in angiogenesis and induced parturition, while granulocyte-macrophage colony-stimulating factor plays a facilitating role in embryo implantation (Gan et al., 2002). The interferon-g (IFN-g) and tumor necrosis factor-a secreted by uterine natural killer (NK) cells are believed to have a general regulatory effect on the pregnancy process. Wu et al., (2010) found that there was IFN-g expression in the placenta during embryo implantation and late pregnancy. In non-pregnant animals, uterine NK cells expressed low levels of IFN-g and as pregnancy progressed, the expression level of IFN-g gradually increased, but no significant changes in the level of IFN-g were observed in the peripheral blood circulation and the spleen. This indicates that the immunity of the parent body during pregnancy has a significant influence on the maternal-fetal.
       
Biomarkers are biochemical indicators that can mark changes or possible changes in the structure or function of systems, organs, tissues, cells and subcellular components, which have a wide range of clinical applications. The measurement of biomarkers provides insight into the ongoing biological processes within an organism and the examination of one or multiple specific biomarkers helps in the identification of diseases, early diagnosis and physiological monitoring during treatment (Prasanta et al., 2023). The emergence of new methods, such as transcriptome sequencing, metabolome sequencing, small RNA sequencing and DNA methylation sequencing, has provided scientists with additional options for in-depth research in life sciences. During pregnancy, the immunity of female animals undergoes certain changes and effective monitoring of immune biomarkers can provide a reference to improve the management of pregnant animals. In the study, we performed transcriptome sequencing to examine mRNAs and analysis of differential expression data, GO, KEGG pathway studies showed that seven mRNAs were significantly upregulated in ovarian tissues during pregnancy, all of which are involved in processes such as bacterial and viral defense, immune regulation and so. QRT-PCR was used to detect the expression of seven different mRNAs in the blood of pregnant ewes and the result confirmed that the expression was significantly elevated in the blood of pregnant ewes. The experiment indicates that the seven mRNAs are promising candidate biomarkers for immune monitoring, which is of great significance for the screening and early intervention of diseases during animal pregnancy.

Experimental animals and collection of samples
 
The study was conducted at Xichang University between January 2023 and June 2024. Experimental animals and collection of samples were as same as Chang et al., (2024).
 
RNA extraction
 
Total RNA was extracted as same as Chang et al., (2024).

Library synthesis and high-throughput sequencing
 
Library construction, transcriptome sequencing were as same as Chang et al., (2024).
       
Sequencing data were filtered as same as Chang et al., (2024). The filtered data were mapped to the reference genome (https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_002742125.1/) using HISAT2 (v2.0.4), followed by detection of differentially spliced genes using rMATS (v3.2.5). The filtered data were aligned to the database established by BGI using Bowtie 2 (v2.2.5), which includes known and novel, coding and non-coding transcripts. Gene expression levels were then calculated using RSEM (v1.2.12) and differential expression analysis was performed using DESeq 2.0 (v1.4.5) with a Q value of ≤0.05. To take insight to the change of phenotype, GO and KEGG enrichment analysis of annotated different gene expression was performed based on Hypergeometric test.
 
Clinical detection of mRNA
 
The study used approximately 0.1 mg of each RNA sample and reverse transcribed it into cDNA using an RT reagent. For the qRT-PCR reaction system (20 mL), 10 mL of 2xSYBR qPCR mix, 0.4 mL of the upstream and downstream primers (2 mmol/L each), 1.2 mL of cDNA and 8 mL of RNase-free dd H₂O were used, making up a total volume of 20 mL. Which was performed at 95^oC for 10 min, then 95^oC for 15 s, 60^oC for 60 s for 42 cycles and 72^oC for 20 s. All primers used in the qRT-PCR are shown in Table 1. Each qPCR experiment was performed in triplicate and the relative RNA expression values were calculated using the 2-DDCT method. Differences were deemed statistically significant if P<0.05. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference.


 
Statistical analysis
 
One-way ANOVA was used to determine the significance of differences in expression of both genes during different physiological stages of sheep bloods.

Library construction and high-throughput sequencing
 
Sequencing was performed on 12 samples in four time periods (FP, LP, P30 and P45) using the DNBSEQ platform, yielding an average output of 11.50 Gb per sample. The average alignment rate of the samples to the reference genome was 93.88%. In the experiment, a total of 23,219 genes were detected, of which 467 were found to be differentially expressed. Compared to the LP sample, 26 genes were upregulated in the FP, 24 genes were upregulated in the P30. Compared to the FP,82 genes were upregulated in the P45. Compared to the P30, 47 genes were upregulated in the P45 (Fig 1). Compared to LP and FP, gene MX2 was extremely significantly upregulated at P45; compared to FP, gene DDX58, IRF7 and PLA2G1B were extremely significantly upregulated at P45; IFIT5, ISG15 and RSAD2 were significantly upregulated at P45 (Table 2).




 
Stacked histogram of expression levels
 
To visually represent the number of genes in different fragments per kilobase of transcript per million mapped reads (FPKM) ranges for each sample, gene counts were tabulated for three FPKM conditions (FPKM≤1, FPKM 1-10, FPKM≥10). The experimental data analysis showed that the sample genes were predominantly expressed at high, moderate and low levels, with genes at extremely low expression levels accounting for only 30.57% to 33.97% (Fig 2).


 
Alternative splicing
 
The experiment detected five types of alternative splicing events: skipped exon (SE), alternative 5’ splicing site (A5SS), alternative 3’ splicing site (A3SS), mutually exclusive exons (MXE) and retained introns. Among the alternative splicing and differential alternative splicing events, the SE category held the highest proportion, constituting 72.51%-74.64% and 37.04%-66.67%, respectively (Fig 3).


 
GO annotation and KEGG pathway analysis
 
The study performed GO and KEGG pathway analyses for 467 differentially expressed genes. In terms of biological processes, the 467 genes were enriched in 261 entries of biological processes, 258 entries of cellular components and 260 entries of molecular function. The top five ranked GO terms are listed in (Table 3). 467 genes primarily classified into 231 pathways, with the top 10 KEGG entries being signal transduction, immune system, infectious diseases (viral), signaling molecules and interactions, cancers (overview), endocrine system, global and overview maps, cell growth and death, transport and catabolism and cellular community-eukaryotes.


       
Through GO and KEGG pathway analysis, seven mRNAs were found to be involved in processes related to defense against bacterial and viral infections and regulation of body’s immunity (Table 4).


 
QRT-PCR
 
The results indicated a significant increase in the expression of seven mRNAs in the blood of pregnant ewes, with very low expression levels in the blood of non-pregnant ewes (p<0.01) (Fig 4).


       
Finding and discovering valuable biomarkers has become an important focus of current research. High-throughput sequencing technologies, such as transcriptomics and proteomics, have emerged as an important means of screening small-molecule biomarkers Mann  et al. (2021). The study successfully constructed RNA libraries for sheep in the early stages of pregnancy, yielding an average output of 11.50 G of data per sample. The average alignment rate of the samples to the reference genome was 93.88%, exceeding the sequencing yield of 10 G with an average alignment rate of 81.22% as reported by Wu et al., (2020). The data output rate of this study was higher than the sequencing results of the study mentioned above, demonstrating the reliability of the sequencing data and ensuring a solid foundation for further in-depth analysis in subsequent experiments. The experiment detected 467 differential genes, comprising 418 differential mRNAs. Differential expression analysis revealed markedly different gene expression patterns in different physiological periods in animals. These findings further confirmed the involvement of genes in the regulation of animal physiological activities, which was consistent with the reports in the existing literature (Chang et al., 2017; Chang et al., 2022).
       
GO and KEGG pathway analysis combined with differential expression analysis revealed that the expression of gene MX2 was significantly elevated at P30 and P45. Additionally, it was involved in biological processes such as immune system functions, antiviral responses, interferon responses, innate immune  responses and defense responses against viruses. The study by Fang et al., (2023) confirmed an association between MX2 expression levels and complement C3 and C4, which was consistent with the findings of this study. The expression level of gene DDX58 was extremely significantly elevated in P45, which was involved in the regulation of influenza A virus pathway, positive regulation of the host defense response to viruses, viral response, positive regulation induced by interleukin-6/8, positive regulation of interferon secretion and innate immune responses. Lu et al., (2022) demonstrated that the cytoplasmic DNA-sensing pathway may play an important role in the pathogenesis of sepsis, with DDX58 being one of its key functional targets. The analysis in this study indicates that DDX58 plays an important role in the regulation of animal immunity, consistent with the aforementioned literature. The expression level of gene IRF7 was significantly elevated in P45; it was involved in the regulation of adaptive immune responses, immunoglobulin-mediated immune responses,  positive regulation of type I interferon secretion, interferon-a secretion, positive regulation of interferon-a/b secretion, regulation of MyD88-dependent/non-independent toll-like receptor signaling pathways, type I interferon biosynthetic process, antiviral defense responses and type I interferon signaling pathways. Studies have confirmed that the IRF7 gene plays an important role in host resistance, body defense, maintenance of maternal animal health and its regulation. As a key regulator factor for type I interferon, it is regulated by multiple pathways (Yu et al., 2018) and plays a crucial role in the host’s antiviral innate immune response (Chen et al., 2019). Studies have reported the absence of the transcription factor IRF3 in poultry and the production of type I interferon is mainly induced by IRF7 (Adam et al., 2011; Diwakar et al., 2017), demonstrating that IRF7 is an important regulator of interferons, proinflammatory cytokines and interferon-stimulated genes, highlighting its significant contribution to innate immunity. The expression level of gene IFIT5 was extremely significantly elevated at P45 and was involved in biological processes such as positive regulation of I-kappaB kinase/NF-kappaB signaling, negative regulation of viral genome replication and response to viruses, playing an important role in host resistance, body defense, maintenance of maternal animal health and its regulation. The research demonstrated through transcriptomic studies that the mRNA transcription levels of the IFIT5 gene in lung and intestinal tissues of 6-week-old ducks increased more than 1000-fold 24 h after avian influenza infection (Hillary et al., 2012). The expression level of gene ISG15 was significantly elevated at P45 and it was involved in response reactions to bacteria and viruses, IL-10 secretion and interferon secretion, among other processes. It was related to the body’s defense against viruses and bacteria, as well as improved resistance. The research have shown that ISG15 inhibits encephalomyocarditis virus (EMCV) replication in vitro and in vivo and knockout of the ISG15 gene can increase susceptibility to EMCV (He et al., 2023). The gene RSAD2, also known as Viperin, is an interferon-induced antiviral protein. The analysis of this experiment showed that the expression level of RSAD2 was significantly elevated at P45 and it was involved in response to viruses, negative regulation of protein secretion and positive regulation of Th2 cell cytokine secretion, contributing to increased viral resistance and immune responses within the body. The research suggested that IL-6 and RSAD2 are synergistically involved in the immune response of the host to the Orf virus (Jiang et al., 2023). The biological functions of RSAD2 analyzed in this experiment were consistent with those reported in the literature (Jiang et al., 2023). The expression level of gene PLA2G1B was extremely significantly elevated at P45 and the analysis revealed that it was involved in mucosal innate immune responses, antimicrobial reaction, defense against Gram-positive bacteria and antimicrobial peptide-mediated humoral immune response, which were related to the defense against bacteria and immunity of the body. It was found that patients with lung adenocarcinoma with low expression of PLA2G1B exhibited a poorer prognosis and this result was related to the fact that patients with low expression presented stronger immune rejection and less immune cell infiltration, thus affecting immune cytotoxic function and consequently impacting overall patient prognosis (Zhu et al., 2022).
       
The qRT-PCR results showed that the seven mRNAs were expressed at low levels in the blood of non-pregnant ewes, while their expression increased significantly in the blood of pregnant ewes. Seven mRNAs were involved in processes such as immune response, secretion of immune factors and defense against bacteria/viruses. Combined with differential expression patterns in pregnant and non-pregnant ewes, it is conceivable that these seven mRNAs, including MX2, DDX58, IRF7, IFIT5, ISG15, RSAD2 and PLA2G1B, could serve as feasible immunobiomarkers. In infectious diseases, C-reactive protein and procalcitonin are commonly used inflammatory markers (Li et al., 2024). The research demonstrated that ABCA3 expression was associated with a prognosis and immune cell infiltration in LUAD patients, making it a potentially valuable biomarker to predict the prognosis of non-small cell lung cancer Zhang et al., (2024). MicroRNA sequencing analysis revealed a significant upregulation of miR-26a in bovine plasma during early pregnancy, suggesting its potential as a biomarker for early pregnancy (Jason et al., 2017). However, its application in pregnancy diagnosis needs to be further investigated. By detecting and analyzing immunobiomarkers, it is possible to better understand the immune status of diseased or healthy animals, enabling the development of individualized treatment or healthcare plans, which are of significant importance for diagnosis, treatment and physiological monitoring. Seven mRNAs, including MX2, DDX58, IRF7, IFIT5, ISG15, RSAD2 and PLA2G1B, emerged as crucial small molecules for immune monitoring, identified by high-throughput sequencing, bioinformatic analysis and validation of quantitative expression patterns in clinical blood samples. However, due to the differences in sample types, sample collection and processing, detection methods, animal breed and sex and personnel operations, more in-depth research is warranted to assess the specificity and accuracy of the seven mRNAs as immunobiomarkers for immune monitoring.

The RNA library for sheep was successfully constructed using high-throughput sequencing technology and in combination with bioinformatics analysis, a total of 467 differentially expressed genes were identified. Among them, MX2, DDX58, IRF7, IFIT5, ISG15, RSAD2 and PLA2G1B exhibited significantly elevated expression during pregnancy. GO and KEGG pathway analyses revealed that these candidate genes are functionally associated with antibacterial and antiviral defense mechanisms as well as immune system regulation. QRT-PCR validation further indicated that these genes exhibit threshold-dependent expression patterns, suggesting their potential utility as immunobiomarkers for pregnancy monitoring. These findings have important implications for developing screening protocols and early intervention strategies for pregnancy-associated disorders in livestock.

The present study was supported by National Natural Science Foundation of China (grant nos. 32360905) , China Agricultural University Joint Fund for supporting Xichang University (grant nos. ZXL202403) and Doctoral Fund of Xichang University (grant nos. YBZ202334 and YBZ202307).
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal procedures for experiments were approved by the Laboratory Animal Welfare and Ethics Committee of Xichang University(No.xcc2023001) and in accordance with the “Guidelines for Use of Experimental Animals” of the Ministry of Agriculture.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish or preparation of the manuscript.