Cardiovascular Disease-Associated MicroRNAs as Novel Biomarkers of First-Trimester Screening for Gestational Diabetes Mellitus in the Absence of Other Pregnancy-Related Complications

Figure 1. MicroRNA gene expression profile in early stages of gestation in pregnancies destinated to develop GDM and term pregnancies with normal course of gestation. MicroRNA gene expression data (2−∆∆Ct) are visualised using the heatmap. In this setting, each row represents a sample (GDM1–GDM121, NP1–NP80) and each column represents a microRNA gene. The colour and intensity of the boxes are used to represent changes of gene expression (2−∆∆Ct). Green colour indicates upregulation, and red colour indicates downregulation. GDM; gestational diabetes mellitus, NP; normal pregnancies.

Figure 2. ROC analysis—the combination of microRNA biomarkers (miR-1-3p, miR-20a-5p, miR-20b-5p, miR-23a-3p, miR-100-5p, miR-125b-5p, miR-126-3p, miR-181a-5p, miR-195-5p, miR-499a-5p, and miR-574-3p). A total of 47.93% pregnancies destinated to develop GDM had an aberrant microRNA expression profile in the whole peripheral venous blood during the first trimester of gestation at a 10.0% FPR. This represents 58 out of 121 pregnancies correctly predicted to develop GDM and 8 out of 80 normal pregnancies predicted false positively to develop GDM.

Figure 3. ROC analysis—the combination of 3 basic clinical characteristics (maternal age and BMI values at early stages of gestation and an infertility treatment by assisted reproductive technology) and 11 dysregulated microRNA biomarkers (miR-1-3p, miR-20a-5p, miR-20b-5p, miR-23a-3p, miR-100-5p, miR-125b-5p, miR-126-3p, miR-181a-5p, miR-195-5p, miR-499a-5p, and miR-574-3p). At a 10.0% FPR, 69.17% of pregnancies destinated to develop GDM were identified during the first trimester of gestation. This represents 84 out of 121 pregnancies correctly predicted to develop GDM and 8 out of 80 normal pregnancies predicted false positively to develop GDM.

Figure 4. ROC analysis—the combination of 7 clinical characteristics (maternal age and BMI at early stages of gestation, an infertility treatment by assisted reproductive technology, history of miscarriage, the presence of trombophilic gene mutations, positive first-trimester screening for PE and/or FGR by FMF algorithm, and family history of diabetes mellitus in first-degree relatives) and 11 dysregulated microRNA biomarkers (miR-1-3p, miR-20a-5p, miR-20b-5p, miR-23a-3p, miR-100-5p, miR-125b-5p, miR-126-3p, miR-181a-5p, miR-195-5p, miR-499a-5p, and miR-574-3p). At a 10.0% FPR, 72.50% of pregnancies destinated to develop GDM were identified during the first trimester of gestation. This represents 88 out of 121 pregnancies correctly predicted to develop GDM and 8 out of 80 normal pregnancies predicted false positively to develop GDM.

Figure 5. ROC analysis—the combination of microRNA biomarkers (miR-20a-5p, miR-20b-5p and miR-195-5p). A total of 30.0% pregnancies destinated to develop GDM requiring a combination of diet and administration of appropriate therapy had aberrant microRNA expression profile in the whole peripheral venous blood during the first trimester of gestation at a 10.0% FPR. This represents 6 out of 20 pregnancies correctly predicted to develop GDM and 8 out of 80 normal pregnancies predicted false positively to develop GDM.

Figure 6. ROC analysis—the combination of 3 basic clinical characteristics (maternal age and BMI values at early stages of gestation and an infertility treatment by assisted reproductive technology) and 3 dysregulated microRNA biomarkers (miR-20a-5p, miR-20b-5p, and miR-195-5p). At a 10.0% FPR, 78.95% pregnancies destinated to develop GDM requiring a combination of diet and administration of appropriate therapy were identified during the first trimester of gestation. This represents 16 out of 20 pregnancies correctly predicted to develop GDM and 8 out of 80 normal pregnancies predicted false positively to develop GDM.

Figure 7. ROC analysis—the combination of 7 clinical characteristics (maternal age and BMI at early stages of gestation, an infertility treatment by assisted reproductive technology, history of miscarriage, the presence of trombophilic gene mutations, positive first-trimester screening for PE and/or FGR by FMF algorithm, and a family history of diabetes mellitus in first-degree relatives) and 3 dysregulated microRNA biomarkers (miR-20a-5p, miR-20b-5p, and miR-195-5p). At a 10.0% FPR, 89.47% pregnancies destinated to develop GDM requiring a combination of diet and administration of appropriate therapy were identified during the first trimester of gestation. This represents 18 out of 20 pregnancies correctly predicted to develop GDM and 8 out of 80 normal pregnancies predicted false positively to develop GDM.

Figure 8. ROC analysis—the combination of microRNA biomarkers (miR-1-3p, miR-20a-5p, miR-20b-5p, miR-100-5p, miR-125b-5p, miR-195-5p, miR-499a-5p, and miR-574-3p). A total of 34.65% pregnancies destinated to develop GDM on diet only had an aberrant microRNA expression profile in the whole peripheral venous blood during the first trimester of gestation at a 10.0% FPR. This represents 35 out of 101 pregnancies correctly predicted to develop GDM and 8 out of 80 normal pregnancies predicted false positively to develop GDM.

Figure 9. ROC analysis—the combination of 3 basic clinical characteristics (maternal age and BMI values at early stages of gestation and an infertility treatment by assisted reproductive technology) and 8 dysregulated microRNA biomarkers (miR-1-3p, miR-20a-5p, miR-20b-5p, miR-100-5p, miR-125b-5p, miR-195-5p, miR-499a-5p, and miR-574-3p). At a 10.0% FPR, 50.50% pregnancies destinated to develop GDM managed by diet only were identified during the first trimester of gestation. This represents 51 out of 101 pregnancies correctly predicted to develop GDM and 8 out of 80 normal pregnancies predicted false positively to develop GDM.

Figure 10. ROC analysis—the combination of 7 clinical characteristics (maternal age and BMI at early stages of gestation, an infertility treatment by assisted reproductive technology, history of miscarriage, the presence of trombophilic gene mutations, positive first-trimester screening for PE and/or FGR by FMF algorithm, and family history of diabetes mellitus in first-degree relatives) and 8 dysregulated microRNA biomarkers (miR-1-3p, miR-20a-5p, miR-20b-5p, miR-100-5p, miR-125b-5p, miR-195-5p, miR-499a-5p, and miR-574-3p). At a 10.0% FPR, 56.44% of pregnancies destinated to develop GDM managed by diet only were identified during the first trimester of gestation. This represents 57 out of 101 pregnancies correctly predicted to develop GDM and 8 out of 80 normal pregnancies predicted false positively to develop GDM.

Figure 11. The KEGG pathway enrichment analysis of 11 microRNAs dysregulated in early pregnancies destinated to develop GDM. The analysis revealed a total of 62 pathways, where at least 18 (29.03%) pathways were cancer related. The results were expressed as –ln of the p-value (−ln(p-value)).

Figure 12. The KEGG pathway enrichment analysis of 11 microRNAs dysregulated in early pregnancies destinated to develop GDM. The analysis revealed a total of 62 various pathways, where a majority of pathways have been shown to play a role in physiological processes and besides to the pathogenesis of cancer. The results were expressed as –ln of the p-value (−ln(p-value)).