Maternal circulating exosomal miR-185-5p levels as a predictive biomarker in patients with recurrent pregnancy loss

Abstract

Purpose: The aim of this study was to explore the predictive role of microRNAs (miRNAs) from maternal serum exosomes in early recurrent pregnancy loss (RPL) and the related mechanism in early pregnancy.

Methods: Maternal serum was collected from pregnant women with RPL history or women with ongoing pregnancy (OP); serum exosomes were extracted and identified. Differentially expressed (DE) miRNAs in exosomes were screened by RNA sequencing and further validated by qRT-PCR. Next, the predictive value of exosomal miRNA and the clinical indicators for subsequent miscarriage in RPL patients were evaluated. Additionally, we verified the regulatory relationship between miR-185-5p and vascular endothelial growth factor (VEGF) in decidual natural killer (dNK) cells by overloading or inhibiting the exosomal miR-185-5p level in trophoblast cells.

Results: The miRNA sequencing revealed 43 DE miRNAs between OP and RPL patients. The five most significant DE miRNAs (miR-22-3p, miR-185-5p, miR-335-3p, miR-362-5p, and miR-378a-3p) were selected for identification, and miR-185-5p was increased in RPL patients. The area under curve (AUC) of the receiver operating characteristic was 0.925 when using miR-185-5p as a biomarker for subsequent miscarriage in RPL patients. In addition, miR-185-5p in exosomes secreted from HTR-8 cells reduces VEGF expression of dNK cells.

Conclusions: The current study, for the first time, successfully constructed the correlation between maternal circulating exosomal miR-185-5p expression pattern and RPL, which may be involved in the pathogenesis of RPL by downregulating the VEGFA of dNK cells and perturbing angiogenesis at the maternal-fetal interface.

Keywords: Biomarker; Exosomes; MicroRNAs; Recurrent pregnancy loss; Vascular endothelial growth factor.

Fig. 1

Quality control of serum exosomes purified from OP patients and RPL patients. a Serum exosomes were presented as cup-like structures with a diameter of approximately 100 nm under the electron microscope. Scale bar: 100 nm. b Exosomes showed diameters ranging from 30 to 150 nm using nanoscale flow cytometry. c Exosome protein markers (CD9, CD63, and CD81) and pregnancy-related proteins (HLA-G and PLAP) were identified using western blot analysis, exo1 and exo2 were from OP group, and exo3 was from RPL group

Fig. 2

The expressions of serum exosomal miRNAs from RPL patients and OP women. a Volcano plot map of DE miRNAs between RPL group and OP group, and the ten most distinctly altered miRNAs were marked. b Heatmap of 43 DE miRNAs (P<0.05). c Scatter plot showing the correlations of DE miRNAs in the RPL group and OP group, and the ten most DE miRNAs were marked. d The relative expressions of the five DE miRNAs in the validation samples (n=10/each group). Mann–Whitney U test was used. **** P<0.0001

Fig. 3

The predictive value of miR-185-5p and other clinical indicators for RPL in the validation cohort (43 OP patients vs. 13 RPL patients). a The expression level of miR-185-5p was significantly higher in patients with RPL than in those with OP. b The ROC curve showed the predictive value of miR-185-5p for RPL, and the AUC was 0.925. c Expression levels of progesterone, hCG on D28 of pregnancy, germ diameter, gestational sac diameter between RPL group and OP. d The ROC curve showed the predictive value of hCG levels on D28 in pregnant women, gestational sac diameter, progesterone levels, and germ diameter for RPL with AUCs of 0.708, 0.650, 0.605, and 0.782, respectively. e ROC curve showing the predictive value of model 1 (combination of progesterone levels, germ diameter, gestational sac diameter, and hCG levels on D28 in pregnant women) and model 2 (combination of hCG levels on D28 in pregnant women, germ diameter, and miR-185-5p expression) for RPL, with AUCs of 0.795 and 0.945, respectively. Mann–Whitney U test was used for the expression level of miR-185-5p, progesterone, and hCG levels on D28, and Student’s t test was used for germ diameter and gestational sac diameter. * P<0.05, *** P<0.001, and **** P<0.0001

Fig. 4

Enrichment analysis of target genes of miR-185-5p. a Venn diagram of target genes predicted by the four databases. b The top 20 GO terms for the target genes (all predicted) of miR-185-5p. c The top 20 statistically significantly enriched pathways in the KEGG analysis

Fig. 5

Comparison of miR-185-5p and VEGF expression in villi and decidua between the RPL and control groups (n=10/each group). a Expression of miR-185-5p in villi between the two groups. b Expression of miR-185-5p in the decidua between the two groups. c Expression of VEGF in villi between the two groups. d VEGF expression in the decidua between the two groups. Student’s t test was used. ***P< 0.001, ****P< 0.0001, and ns not significant

Fig. 6

Overloading of miR-185-5p reduced VEGF expression in dNK cells and suppressed angiogenesis. a Relative miR-185-5p expression in dNK cells after transfection with the agomir, antagomir, agomir negative control, and antagomir negative control, detected using qRT-PCR. Relative VEGF expression level in dNK cells after transfection with the agomir, antagomir, and negative control (NC) detected using b qRT-PCR and c ELISA. d, e, f Tube formation by HUVECs in vitro in supernatants of conditioned media obtained from cells transfected with the agomir, antagomir, and NC. NC groups were transfected with the control agomir/antagomir. Magnification, 10×. All experiments were repeated at least three times. The data are presented as the means ± SDs. ANOVA and Bonferroni’s post hoc t test was used. *P< 0.05, **P< 0.01, and ****P< 0.0001

Fig. 7

Exosomes from miR-185-5p-overexpressing HTR-8 cells reduced VEGF expression in dNK cells. Quality control of exosomes purified from the HTR-8 cell supernatant using electron microscopy (a), nanoscale flow cytometry (b), and western blot analysis (c). Expression levels of miR-185-5p in HTR-8 cells (d) and supernatant exosomes (e) after transfection with the agomir, antagomir, agomir negative control, and antagomir negative control were detected using qRT-PCR. VEGF expression level in dNK cells after coculture with exosomes from transfected HTR-8 cell supernatants detected using f qRT-PCR and g ELISA. NC groups were transfected with the control agomir/antagomir. All experiments were repeated at least three times. ANOVA and Bonferroni’s post hoc t test was used. *P< 0.05, **P< 0.01, ***P< 0.001, and ****P< 0.0001