The adenosine deaminase family acting on RNA 1 can be a useful diagnostic biomarker in chorioamnionitis

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The adenosine deaminase family acting on RNA 1 can be a useful diagnostic biomarker in chorioamnionitis

https://doi.org/10.21203/rs.3.rs-2994863/v1

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published 01 May, 2024

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Chorioamnionitis (CAM) is an acute infection leading to preterm birth. However, diagnosis of CAM before delivery is challenging because of its complicated diagnostic criteria. On the other hand, RNA editing is a phenomenon that changes the phenotype by replacing bases at the RNA level and is triggered by inflammation. We investigated the correlation between RNA editing enzyme Adenosine deaminase family acting on RNA 1 (ADAR1) and CAM in clinical specimens derived fromnon-CAM, CAM (pathologically diagnosed), and clinical CAM (clinically diagnosed) patients. ADAR1 was upregulated in the placenta of CAM and clinical CAM patients (p<0.001 and p=0.005). ADAR1 had a significantly higher area under the curve (AUC) (0.735 and 0.828) than other genetic characteristics in diagnosing CAM and clinical CAM patients. ADAR1 also had significantly higher AUC (0.701 and 0.837) than other clinical characteristics for CAM and clinical CAM patients. ADAR1 can be a useful diagnostic biomarker in CAM patients before delivery.

Health sciences/Biomarkers

Health sciences/Medical research

ADAR1

chorioamnionitis

diagnosticmarker

Intrauterine infection/inflammation (IUI) is mainly an ascending infection in which vaginal and cervical pathogens ascend to the uterus and can affect the fetus before delivery begins. Premature rupture of the membranes and bacterial vaginosis often lead to ascending infections such as chorioamnionitis (CAM). CAM is a condition involving infection and inflammation of the chorion and amnion and is the most common cause of preterm birth, although it complicates up to 4% of term births as well.¹ CAM exposure can also have long-term consequences for exposed infants, including fetal inflammatory reaction syndrome (FIRS), such as neurodevelopment impairment, cerebral palsy, and the development of immune-mediated diseases later in life, including wheezing and asthma.^2–5 Chorion and amniotic membrane are crucial mediators of the fetal/maternal interaction by providing signals that regulate the function of the maternal immune system and protective mechanisms to prevent the exposure of the fetus to dangerous signals. Bacterial or viral infection during pregnancy induces a unique immunological response in the placenta, and type I interferon (IFN) is the crucial signaling pathways in the trophoblast cells, leading to CAM.⁶

CAM is currently diagnosed by postpartum histopathology. Therefore, CAM diagnosis before delivery has yet to be achieved, and clinical CAM is widely used in clinical practice. The diagnostic criteria for clinical CAM are defined as maternal fever of 38.0°C or higher and one or more items (1) maternal tachycardia ≥ 100 beats/min, (2) uterine tenderness, (3) foul odor of vaginal secretions/amniotic fluid, and (4) maternal white blood cell count ≥ 15,000/µL. Otherwise, it is defined as the case where all of (1) to (4) is observed even if the maternal body temperature is less than 38.0°C.⁷

For a long time, attempts have been made to diagnose CAM before delivery. ^7–14 As there is still no biomarker effective for CAM diagnosis, we investigated whether epigenetics could be a biomarker for CAM. We focused on RNA editing in CAM. In recent years, the phenomenon of RNA editing, in which specific bases are converted into other bases in transcribed RNA, has been focused in the research field of epigenetics. RNA editing is a recently identified epigenetic mechanism that regulates the post-transcriptional activity of essential genes by altering their amino acid sequence, leading to changes in their function. ¹⁵ One such RNA editing process, wherein the conversion of adenosine (A) to inosine (I) in primary RNA transcripts (A-to-I editing) is mediated by a family of Adenosine Deaminases Acting on RNA (ADAR), leads to the diversification of the transcriptome in human cells. ¹⁶ ADAR1 is a protein that is induced by IFN and is a good indicator of inflammation. Since CAM is also an inflammation induced by IFN, we thought that if ADAR1 could be easily examined in the placenta before delivery, it would be possible to diagnose CAM.

ADAR1 expression is positively correlated with CAM and clinical CAM

The expression of ADAR1 was examined in their relationship with clinical parameters on ruptured membranes that underwent delivery. The expression of ADAR1 was associated with pre-partum body mass index (BMI) (p = 0.016), pregnancy numbers (p = 0.007), CAM patients (p < 0.001), and clinical CAM patients (p = 0.005). As expected, these results indicate that ADAR1 expression is higher in patients with positive parameters associated with CAM and clinical CAM (Table 1).

CAMs patients and clinical CAMs patients were associated with ADAR1 and cytokines

Next, we analyzed the expression of inflammatory-related gene and ADAR1 in clinical samples (Fig. 1). First, we compared CAM and non-CAM. ADAR1 and IL-8 of CAM patients were significantly higher than those of non-CAMs patients (P = 0.044 and p = 0.002). Next, we compared clinical CAM and non-CAM. ADAR1, NF-κB, IL-6, and IL-8 of clinical CAMs patients were significantly higher than those of non-CAMs patients (P = 0.002, p = 0.016, p = 0.030, and p = 0.016). These results indicated that ADAR1 was highly relevant to both CAM and clinical CAM patients.

Receiver operating characteristic curve analyses to predict the interferon pathway and cytokines with CAM patients or clinical CAM patients.

We investigated correlations among each genetic characteristic, i.e., ADAR1, MDA-5, RIG-I, NF-κB, IRF3, IRF7, IFN-\({\alpha }\), IFN-β, IL-1β, IL-6, IL-8, TNF-\({\alpha }\), and CAM or clinical CAM patients. We used ROC curve analyses to predict CAM patients and clinical CAM patients. ADAR1 and IL-8 had a significantly higher area under the curve (AUC) (0.735 and 0.752) than other genetic characteristics in CAM patients. Additionally, ADAR1 had a significantly higher AUC (0.828) than other genetic characteristics in terms of CAM patients. Therefore, ADAR1 was found to be more closely associated with CAM patients and clinical CAM patients than other cytokines (Table 2).

Receiver operating characteristic curve analyses to predict CAM or clinical CAM patients using ADAR1 or clinical factors

We investigated correlations among ADAR1 and each clinical characteristic, i.e., age, pre-partum BMI, meconium-stained liquor, group B streptococcus, primiparous woman, fetal weight, weeks of pregnancy, water breaking times, lactobacillus species, intestinal bacteria, and CAM patients and clinical CAM patients. We used ROC curve analyses to predict CAM patients and clinical CAM patients. ADAR1 had a significantly higher AUC (0.701) than other clinical characteristics for the diagnosis of CAM patients. Additionally, ADAR1 had a significantly higher AUC (0.837) than other clinical characteristics for the diagnosis of clinical CAM patients (Table 3).

These results indicate that ADAR1 can predict CAM and clinical CAM patients. CAM can be easily diagnosed by examining the expression of ADAR1 in the placenta before delivery. This technology can be operated at any facility and will contribute to protecting the health of pregnant women and their fetuses.

In this study, we aimed to establish a diagnostic technique for preconception CAM. The analysis of ADAR1 expression in the placenta proved to be a minimally invasive and simple CAM diagnostic technique.

CAM is modified by stimulation of type I IFN associated with infection. Type I IFNs are polypeptides able to induce an anti-microbial state, modulate innate immune responses, and activate the adaptive immune system. ^{18, 19} Type both viral and bacterial pathogens can induce type I IFN responses. These pathogens activate TLRs, NLRs, RIG-I-like receptors (RLRs), and cGAS, which promote inflammation-related signaling through type I IFN genes (STING). ^{20, 21} Bacterial or viral infection during pregnancy induces a unique immunological response by the placenta, and IFN is one of the crucial signaling pathways in the trophoblast cells. Basal expression of IFN harbors physiological functions to maintain the homeostasis of pregnancy, more importantly, provides the placenta with adequate awareness to respond to infections.⁶

On the other hand, ADAR1, an RNA-editing enzyme, is attracting attention in the area of inflammation research. RNA editing is a recently identified epigenetic mechanism that regulates the posttranscriptional activity of essential genes by altering their amino acid sequences, leading to changes in gene expression.²² A-to-I RNA editing is catalyzed by adenosine deaminase ADAR enzymes that bind to and edit dsRNA. The A-to-I RNA editing of messenger RNA can alter its characteristics to promote a more aggressive phenotype. ADAR1 is known to be induced by type I INF. The present study aimed to clarify the ADAR1 expression in CAM and clinical CAM patients.

ADAR1 expression was significantly higher in CAM clinical specimens than in non-CAM specimens. Furthermore, CAM and clinical CAM specimens contained more ADAR1 than non-CAM patient specimens. Furthermore, we examined the correlations of CAM and clinical CAM patients with interferon-related pathways. ADAR1 is significantly associated with CAM and clinical CAM patients, which could serve as a desirable diagnostic target for CAM.

We acknowledge that our study has some limitations. The number of patients was small, and the examination was performed at a single facility. Further prospective studies involving more patients and facilities would provide more definitive data to clarify the significance of our findings.

In conclusion, ADAR1 has proven to be a potential diagnostic target for CAM.

Patients and tissue specimens

The study was approved by the institutional ethics committee of the Okayama University (approval number: 2302-023). Informed consent was obtained from all the participants. All procedures were performed in accordance with relevant ethical standards and institutional ethics committee regulations. Seventy-three female patients. of admitted with ruptured membranes, who underwent delivery at the Okayama University Hospital between April 2014 and October 2022, were recruited into this study after providing informed consent. CAM lesions were confirmed by histopathology. These CAM stages was performed according to the Blanc classification. The Blanc classification included 3 patients in stage 0, 27 patients i n stage 1, 13 patients in stage 2, and 30 patientsi n stage 3. Patients Moreover, Clinical CAM cases included 1 patient in stage 2 and 12 patients in stage 3. In this time, the CAM-negative group was divided into Blanc classification stages 0 and 1, and the CAM-positive group was divided into Blanc classification stages 2 and 3.

RNA isolation and real-time quantitative PCR analyses

On human sample, RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE (Thermo Fisher Scientific, Waltham, MA, USA) was isolation and purification of total RNA from formalin-fixed paraffin-embedded (FFPE) tissue samples. These cell line and tissue samples of RNA were assessed using real-time quantitative PCR analyses analysis. The iTaq Universal SYBR Green OneStep Kit and the MiniOpticon Real-Time PCR System (Bio-Rad, CA, USA) were used for gene expression analysis by real-time quantitative PCR. GAPDH was used as a normalization control. Primer sequences for ADAR1,melanoma differentiation-associated protein5 (MDA5), retinoic acid-inducible gene I(RIG-I), nuclear factor-kappa B (NF-κB), interferon regulatory factor (IRF)-3, IRF-7, interferon (IFN)-\({\alpha }\), IFN-β, Interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)-\({\alpha }\) and GAPDH genes are shown in Supplementary Table 1.

Statistical analysis

Statistical analyses were performed using the Mann–Whitney U-test for comparisons with controls and one-factor ANOVA followed by Fisher's protected least significance difference test for all pairwise comparisons. A receiver operating characteristic (ROC) curve was generated, and the area under the curve (AUC) was calculated to evaluate the discriminatory ability of each scoring system. We also examined the data in cross-tabulated form to explore CAM and clinical CAM. The analyses were performed with the software package StatView version 25.0 (Abacus Concepts, Berkeley, CA). Differences were considered significant at P < 0.05.

Ethics approval and consent to participate. Informed consent was obtained from each patient, and the institutional review board (The Ethics Committee of the Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences and Okayama University Hospital) approved the study (2302-023). x

Data availability. All data generated or analyzed during this study are included in this published article.

ACKNOWLEDGMENTS

None

AUTHOR CONTRIBUTIONS

Conceived and designed experiments: K.N., K.T.; performed experiments: K.N., K.S., J. M., E.E., H.M.; analyzed data: K.N., K.T., J. M., E.E.; contributed reagents, materials, and other analytical tools: K.N., K.S., K.T., J.M., E.E., H.M.; wrote the manuscript: K.N., K.S., K.T., J.M., E.E., H.M. All authors have read and approved the final manuscript.

FUNDING

This study was supported by grants from JSPS KAKENHI (19K09753 and 22K09619) to KN.

COMPETING INTERESTS

The authors declare no competing interests.

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Tables 1 to 3 are available in the Supplementary Files section.

No competing interests reported.

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Journal Publication

published 01 May, 2024

Read the published version in Placenta →

Version 1

posted

You are reading this latest preprint version

The adenosine deaminase family acting on RNA 1 can be a useful diagnostic biomarker in chorioamnionitis

Status:

Journal Publication

Version 1

Abstract

Figures

INTRODUCTION

RESULTS

ADAR1 expression is positively correlated with CAM and clinical CAM

CAMs patients and clinical CAMs patients were associated with ADAR1 and cytokines

DISCUSSION

MATERIALS AND METHODS

Patients and tissue specimens

RNA isolation and real-time quantitative PCR analyses

Statistical analysis

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1