Maternal plasma microRNAs as potential biomarkers for triaging pregnancies of unknown location and ectopic pregnancy diagnosis

Abstract

Background: Pregnancy of unknown location (PUL) is classified if an early pregnancy is not visualised on transvaginal ultrasonography (TVUS). Biomarkers currently used to triage PUL outcomes have varying accuracy. Delayed or missed diagnosis of ectopic pregnancies (EP) continue to cause significant morbidity and mortality. We investigated whether maternal plasma microRNAs (miRNAs) can predict and differentiate high-risk EP from viable (VIUP) or non-viable (NVIUP) intrauterine pregnancies.

Methods: Plasma was collected from women with PUL/EP (n = 120), mostly between four to eight weeks' gestation, where outcomes of EP (n = 39), VIUP (n = 58) and NVIUP (miscarriage, n = 23) were determined using TVUS. Nanostring nCounter miRNA assay was used to examine the expression of ∼800 miRNAs in 22 women. Differentially expressed miRNAs were validated using RT-qPCR in 98 women.

Results: Nanostring nCounter miRNA assay identified 19 miRNAs which were expressed significantly higher in EP/NVIUP compared with VIUP. Two miRNAs were validated in a second, separate validation cohort using RT-qPCR: hsa-miR-21-5p in EP was 2.8-fold higher than in VIUP (p = 0.03, ROC AUC = 0.64), and hsa-miR-411-5p had 0.2-fold decreased expression (p = 0.02, ROC AUC = 0.66). Combining the divergent miRNAs as a ratio improved discrimination of EP from VIUP (p < 0.001, ROC AUC = 0.74).

Conclusion: Plasma miRNAs are differentially expressed in EP and VIUP and are detectable as early as four gestational weeks. Exploring miRNA targets may further understanding of EP pathophysiology, offering the potential to use miRNA as predictive and diagnostic markers in early pregnancy.

Keywords: Biomarkers; Ectopic pregnancy; MicroRNA; PUL; Plasma.

Fig. 1

Flowchart of patient recruitment and breakdown of data available for discovery, technical and biological validation studies Abbreviations: FPUL: Failed PUL; LTFU: Lost to follow up; PPUL: Persistent PUL; IPUV: Intrauterine pregnancy of unknown viability; PUL: Pregnancy of unknown location; EP: Ectopic pregnancy; NVIUP: Non-viable intrauterine pregnancy; VIUP: Viable intrauterine pregnancy.

Fig. 2

Eight discriminatory miRNAs in early pregnancy plasma when comparing VIUP with EP (N = 15) in discovery study. nCounter probe counts for A: hsa-miR-222-3p, B: hsa-miR-20a-5p + hsa-miR-20b-5p, C: hsa-miR-21-5p, D: hsa-miR-185-5p, E: hsa-miR-148a-3p, F: hsa-miR-19b-3p, G: hsa-miR-1285-5p, and H: hsa-miR-22-3p detected in human plasma in women in early pregnancy who subsequently had a confirmed VIUP (N = 8, green), EP (N = 7, red), or NVIUP (N = 7, purple) ∗Statistical significance of differences between groups was tested using Nanostring nSolver platform. Graphs show geometric mean with geometric standard deviation and p-values, with outcome on x axis, and log(10) counts on y axis. Only statistically significant differences are presented. Abbreviations: VIUP = Viable intrauterine pregnancy; EP = Ectopic pregnancy; NVIUP = Non-viable intrauterine pregnancy.

Fig. 3

Seven discriminatory miRNAs in early pregnancy plasma when comparing VIUP with NVIUP (N = 15) in discovery study. nCounter probe counts for A: hsa-miR-107, B: hsa-miR-223-3p, C: hsa-let-7d-5p, D: hsa-miR-191-5p, E: hsa-miR-29b-3p, F: hsa-miR-374a-5p, and G: hsa-miR-199a-3p + hsa-miR-199b-3p detected in human plasma in women in early pregnancy who subsequently had a confirmed VIUP (N = 8, green), EP (N = 7, red), or NVIUP (N = 7, purple) ∗Statistical significance of differences between groups was tested using Nanostring nSolver platform. Graphs show geometric mean with geometric standard deviation and p-values, with outcome on x axis, and log(10) counts on y axis. Only statistically significant differences are presented. Abbreviations: VIUP = Viable intrauterine pregnancy; EP = Ectopic pregnancy; NVIUP = Non-viable intrauterine pregnancy.

Fig. 4

Four discriminatory miRNAs in early pregnancy plasma when comparing VIUP with EP and NVIUP (N = 22) in discovery study. nCounter probe counts for A: hsa-miR-1246, B: hsa-miR-130a-3p, C: hsa-miR-320e, and D: hsa-miR-30d-5p detected in human plasma in women in early pregnancy who subsequently had a confirmed VIUP (N = 8, green), EP (N = 7, red), or NVIUP (N = 7, purple) ∗Statistical significance of differences between groups was tested using Nanostring nSolver platform. Graphs show geometric mean with geometric standard deviation and p-values, with outcome on x axis, and log(10) counts on y axis. Statistically significant differences are presented. Abbreviations: VIUP = Viable intrauterine pregnancy; EP = Ectopic pregnancy; NVIUP = Non-viable intrauterine pregnancy.

Fig. 5

Two discriminatory miRNAs in early pregnancy plasma when comparing VIUP with EP (N = 82) in biological validation study. Fold changes for A: hsa-miR-21-5p, and B: hsa-miR-411-5p in women in early pregnancy who subsequently had a confirmed VIUP (N = 50, green), or EP (N = 32, red) ∗Statistical significance of differences between groups was tested using Mann-Whitney (non-parametric, continuous). Graphs show geometric mean with geometric standard deviation and p-values, with outcome on x axis, and log(10) fold change on y axis. Statistically significant differences are presented. Abbreviations: VIUP = Viable intrauterine pregnancy; EP = Ectopic pregnancy.

Fig. 6

Two discriminatory miRNAs in early pregnancy plasma when comparing VIUP with EP (N = 82) in biological validation study. Area under the Receiver operating characteristic (ROC) curve (AUC) for A: hsa-miR-21-5p, and B: hsa-miR-411-5p, in women in early pregnancy who subsequently had a confirmed VIUP (N = 50), or EP (N = 32) ROC curve with area, 95 % confidence interval and p-value presented. Statistically significant differences are presented. Abbreviations: ROC = Receiver operating characteristic.

Fig. 7

Two discriminatory miRNAs in early pregnancy plasma when comparing VIUP with EP (N = 82) in biological validation study. A: Fold changes, and B: area under the Receiver operating characteristic (ROC) curve (AUC) for hsa-miR-21-5p/hsa-miR-411-5p ratio, in women in early pregnancy who subsequently had a confirmed VIUP (N = 50, green), or EP (N = 32, red) ∗Statistical significance of differences between groups was tested using Mann-Whitney (non-parametric, continuous). Graph shows geometric mean with geometric standard deviation and p-values, with outcome on x axis, and log(10) fold change on y axis. ROC curve with area, 95 % confidence interval and p-value presented. Statistically significant differences are presented. Abbreviations: VIUP = Viable intrauterine pregnancy; EP = Ectopic pregnancy; ROC = Receiver operating characteristic.