Potential role of circulating microRNAs as a biomarker for unexplained recurrent spontaneous abortion Weibing Qin, M.D., Ph.D., Yunge Tang, M.D., Ning.

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Potential role of circulating microRNAs as a biomarker for unexplained recurrent spontaneous abortion Weibing Qin, M.D., Ph.D., Yunge Tang, M.D., Ning Yang, M.D., Xiangcai Wei, M.D., Jiehua Wu, M.D. Fertility and Sterility Volume 105, Issue 5, Pages 1247-1254.e3 (May 2016) DOI: 10.1016/j.fertnstert.2016.01.028 Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 1 The expression profiles of global miRNAs in the plasma of unexplained recurrent spontaneous abortion (URSA) and normal early pregnancy (NEP) patients. (A) Heat map of global miRNAs in plasma from three URSA patients (1, 2, 3) and three NEP patients (4, 5, 6). (B) Log-log scatter plot comparing global miRNA expression profiles in plasma of URSA patients with those of NEP control subjects. (C) Volcano plot of differentially expressed miRNAs. The vertical lines correspond to 2.0-fold up and 0.5-fold down, and the horizontal line represents a P value of 0.05. The red point in the plot represents the differentially expressed miRNAs with statistical significance. Fertility and Sterility 2016 105, 1247-1254.e3DOI: (10.1016/j.fertnstert.2016.01.028) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 2 qRT-PCR validation of circulating miRNAs. (A) Differential expression results of circulating miRNAs according to microarray. (B) Differential expression results of circulating miRNAs according to quantitative reverse-transcription polymerase chain reaction. Abbreviations as in Figure 1. Fertility and Sterility 2016 105, 1247-1254.e3DOI: (10.1016/j.fertnstert.2016.01.028) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 3 The potential biomarker miRNAs according to quantitative reverse-transcription polymerase chain reaction. Relative miRNA expression levels are normalized against the amount of U6; the results are shown in the graphs. Significant overexpression of miR-320b, miR-146b-5p, miR-221-3p, and miR-559 was found in URSA versus NEP. And significant underexpression of miR-101-3p was found in URSA versus NEP. Horizontal lines represent the median. P value was calculated with the use of the nonparametric Mann-Whitney U test. Abbreviations as in Figure 1. *P<.05. Fertility and Sterility 2016 105, 1247-1254.e3DOI: (10.1016/j.fertnstert.2016.01.028) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Venn diagram of differentially expressed miRNAs. Target gene can be predicted as a target gene by these three databases (Targetscan, miRbase, and miRanda) simultaneously. These differentially expressed miRNAs have 947 target genes. Fertility and Sterility 2016 105, 1247-1254.e3DOI: (10.1016/j.fertnstert.2016.01.028) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 Target genes network of differentially expressed miRNAs. Blue dots are target genes; red squares are miRNAs. Fertility and Sterility 2016 105, 1247-1254.e3DOI: (10.1016/j.fertnstert.2016.01.028) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 3 Result of the gene ontology (GO) and pathway analysis of the target genes predicted by differentially expressed miRNAs. The ontology covers three domains: biologic process, cellular component and molecular function. −Log10 (P) indicates the GO score related to genes with the biologic process by P value. (A) Main biologic process of differentially expressed miRNAs. (B) Main cellular component of differentially expressed miRNAs. (C) Main molecular function of differentially expressed miRNAs. (D) Results of pathway analysis the target genes predicted by differentially expressed miRNAs. Fertility and Sterility 2016 105, 1247-1254.e3DOI: (10.1016/j.fertnstert.2016.01.028) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions