MicroRNAs miR-4535 and miR-1915-5p in Amniotic Fluid As Predictive Biomarkers for Chorioamnionitis

Figures & data

Table 1. Clinical characteristics of the pregnant participants and the microbiome species present in their amniotic fluid, analyzed using comprehensive miRNA array.

Patient code	Chorioamnionitis ^‡	WBC in maternal peripheral blood (cells/μl)	CRP level in maternal peripheral blood (mg/dl)	Microbiome species (relative abundance [%]) detected
				miCAM; 11 species ^†
				Ureaplasma parvum	Streptococcus agalactiae	Gardnerella vaginalis	Streptococcus anginosus	Bacteroides fragilis	Mycoplasma hominis	Others
α1	Stage II	6700	3.0	33.7	–	–	–	–	64.5	–
α2	Stage II	17,900	4.2	44.5	54.5	–	–	–	–	–
β1	Stage III	20,100	2.4	99.9	–	–	–	–	–	–
β2	Stage III	19,600	0.8	100.0	–	–	–	–	–	–
β3	Stage III	13,400	0.7	99.8	–	–	–	–	–	–
γ1	Stage III	19,700	8.6	–	–	99.5	–	–	–	–
γ2	Stage II	14,000	4.8	–	–	99.5	–	–	–	–
γ3	Stage II	7700	0.7	–	–	13.1	9.8	5.9	–	70.9
δ1	Stage II	10,400	3.1	–	–	–	–	–	–	100.0
δ2	Stage II	6400	1.3	–	–	–	–	–	–	100.0
δ3	Stage II	9800	0.1	–	–	–	–	–	–	100.0
δ4	Stage II	7000	0.2	–	–	–	–	–	–	100.0
ε1	Stage 0–1	–	–	–	–	–	–	–	–	100.0
ε2	Stage 0–1	–	–	–	–	–	–	–	–	100.0
ε3	Stage 0–1	–	–	–	–	–	–	–	–	100.0

† Sneathia sanguinegens, Eikenella corrodens, Prevotella bivia, Lactobacillus jensenii and Porphyromonas endodontalis were not detected.

‡ The staging is based on Blanc’s classification. Stage III (chorioamnionitis): neutrophilic infiltrates in subamniotic connective tissues and the amniotic epithelium; stage II (chorionitis): neutrophilic infiltration of the chorionic plate or membranous chorionic connective tissues; stage I (subchorionitis): patchy or diffused accumulation of neutrophils within the subchorionic plate or decidua.

CRP: C-reactive protein; miCAM: Microbiome chorioamnionitis; WBC: White blood cell.

Figure 1. Identification of candidate miRNAs for diagnosing severe chorioamnionitis in pregnant women.

(A) Flowchart of the study design. Predictive biomarkers in the amniotic fluid for the diagnosis of chorioamnionitis were identified using comprehensive miRNA array analysis and ddPCR™. Phases I–IV were sequentially completed. (B) Fifteen pregnant women were divided into five groups. The levels of nine miRNAs were identified using comprehensive miRNA array analysis of the amniotic fluid samples from pregnant women with or without chorioamnionitis. (C) Expression of six miRNAs in the amniotic fluid of patients with microbiomic chorioamnionitis (miCAM). Red, orange and yellow circles indicate the miRNA levels in the amniotic fluid of three pregnant women with Blanc’s classification stage III; violet and blue circles indicate the miRNA levels in the amniotic fluid of two pregnant women with Blanc’s classification stages 0 or I. (D) Red circles indicate the miRNA levels in 10 pregnant women with Blanc’s classification stages II–III plus miCAM. The bars indicate the median.

miCAM: Microbiome chorioamnionitis.

Figure 2. Diagnostic relationship between the level of candidate miRNAs or 16S rDNA and severe chorioamnionitis.

(A) Expression levels of each candidate miRNA and the copy numbers of 16S rDNA in pregnant women divided into three groups: Blanc’s classification stages II–III (group II–III: n = 37, red circles); Blanc’s classification stages 0–I (group 0–I: n = 11, blue circles); and healthy pregnant women between 15 and 17 gestational weeks (control: n = 15, gray circles). Data are expressed as the median ± 95% CI. Thick and thin bars indicate the median and 95% CI, respectively. Statistical differences among the groups were assessed using Dunn’s multiple comparison test. (B) Correlation between miRNA expression and 16S rDNA copy number in amniotic fluid samples from 37 pregnant women with Blanc’s classification stages II–III. r indicates the correlation index. (C) Diagnostic and predictive accuracies of miRNA levels and 16S rDNA copy number for chorioamnionitis. Blue, green, yellow and purple lines indicate receiver operating characteristic curves for 16S rDNA, miR-191-5p, miR-1915-5p and miR-4535, respectively.

*p < 0.01; **p < 0.0001.

Figure 3. Expression of candidate miRNAs and quantification of 16S rDNA in patients with microbiomic chorioamnionitis.

(A) Comparison of miR-4535, miR-1915-5p and miR-191-5p levels and 16S rDNA copy numbers in the amniotic fluid from pregnant women with microbiomic chorioamnionitis (miCAM) (n = 21) and without miCAM (non-miCAM) (n = 16). Data are expressed as the median ± 95% CI. Thick and thin bars indicate the median and 95% CI, respectively. (B) Diagnostic and predictive accuracies of miRNA expression and 16S rDNA copy number for chorioamnionitis and miCAM. Blue, green, yellow and purple lines indicate receiver operating characteristic curves for 16S rDNA, miR-191-5p, miR-1915-5p and miR-4535, respectively.

*p < 0.05.

CRP: C-reactive protein; miCAM: Microbiome chorioamnionitis; WBC: White blood cell.

Figure 4. Relationship between (A) maternal white blood cell counts or (B) serum CRP and miRNA levels or 16S rDNA copy number in the amniotic fluid of 37 pregnant women with Blanc’s classification stages II–III.

Data are expressed as the median ± 95% CI. Thick and thin bars indicate the median and 95% CI, respectively.

*p < 0.05.

CRP: C-reactive protein; WBC: White blood cell.

Figure 5. Relationship between (A) fetal white blood cell counts or (B) serum CRP and miRNA levels or 16S rDNA copy number in the amniotic fluid of pregnant women with Blanc’s classification stages II–III.

Data are expressed as the median ± 95% CI. Thick and thin bars indicate the median and 95% CI, respectively.

*p < 0.05.

CRP: C-reactive protein; WBC: White blood cell.

Table 2. Association of neonatal inflammatory indicators between newborns with the third quartile of miRNA levels in the amniotic fluid.

	High inflammatory indicators ^†	Low inflammatory indicators ^‡	p-value ^§
miR-4535 (copies/μl)  ≥2665.4  <2665.4	15 1	8 7	0.0102
miR-1915-5p (copies/μl)  ≥212.94  <212.94	17 2	6 6	0.0144
miR-191-5p (copies/μl)  ≥1181.4  <1181.4	15 3	8 5	0.1712
16S rDNA (copies/ml)  ≥21,945  <21,945	15 3	8 5	0.1712

n = 31 for pregnant women with Blanc’s classification stages II–III.

† Neonatal CRP level > 0 mg/dl after birth, or white blood cell count <5000 or >25,000/μl after birth.

‡ Neonatal CRP level = 0 mg/dl after birth and white blood cell count 5000–25,000/μl after birth.

§ Statistical analysis was performed using the Pearson’s χ-square test.

Figure 6. Clinical significance of candidate miRNAs in the serum of patients with severe chorioamnionitis.

(A) Levels of miR-4535, miR-1915-5p and miR-191-5p in the serum of pregnant women with Blanc’s classification stages II–III and microbiomic chorioamnionitis (n = 9) and those with Blanc’s classification stages 0–I (n = 5). Data are expressed as the median ± 95% CI. Thick and thin bars indicate the median and 95% CI, respectively. (B) Correlation of the expression of miR-4535, miR-1915-5p and miR-191-5p in the amniotic fluid (x-axis) and serum (y-axis) of pregnant women with Blanc’s classification stages II–III and miCAM (n = 9). r indicates the correlation index.

*p < 0.05.

miCAM: Microbiome chorioamnionitis.