MicroRNAome in decidua: a new approach to assess the maintenance of pregnancy  Yu Wang, M.D., Ph.D., Yang Lv, Ph.D., Liyan Wang, M.D., Chunling Gong, M.S., Jiajia Sun, M.S., Xiujuan Chen, M.D., Yan Chen, M.S., Lei Yang, Ph.D., Yan Zhang, Ph.D., Xukui Yang, B.S., Chunling Bai, Ph.D., Zhuying Wei, M.S., Guangpeng Li, Ph.D.  Fertility and Sterility  Volume 103, Issue 4, Pages 980-989.e6 (April 2015) DOI: 10.1016/j.fertnstert.2015.01.003 Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Comparison of qPCR data and deep-sequencing data for seven known miRNAs. Values shown are fold changes (miscarriage/pregnancy expression levels). The qPCR results were normalized to U6 snRNA (small nuclear RNA) expression levels. Error bars represent standard error. Fertility and Sterility 2015 103, 980-989.e6DOI: (10.1016/j.fertnstert.2015.01.003) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 2 (A) MicroRNA-199b-5p was predicted to bind to a potential site on SGK1. (B) Comparison of SGK1 mRNA expression between aborted and pregnancy deciduas. Error bars indicate standard error for all the patients with technical triplicate. (C) SGK1 expression levels based on Western blot results (n = 6). (D) Immunohistochemistry location in pregnancy (C1, ×200), (C2, ×400), and aborted deciduas (C3, ×200), (C4, ×400); positive staining is indicated with arrows. (*** P values < .001). Fertility and Sterility 2015 103, 980-989.e6DOI: (10.1016/j.fertnstert.2015.01.003) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 3 (A) MicroRNA transfection to overexpress hsa-microRNA-199b-5p. HEK293T was transfected with microRNA-199b-5p mimic and negative control. Expression of SGK1 mRNA was detected 48 hours after transfection. (B) miRNA transfection to knock down hsa-microRNA-199b-5p. Cells were cultured and incubated with microRNA-199b-5p inhibitor and negative control. SGK1 levels were analyzed by qPCR. Experiments were replicated three times, and error bars represent standard error. (C) Expression of SGK1 protein was detected 48 hours after transfection with microRNA-199b-5p mimic and negative control in HEK293T. (D) Expression of SGK1 protein was detected 48 hours after transfection with microRNA-199b-5p inhibitor and negative control in HEK293T. (E) The Ishikawa cell line was transfected with microRNA-199b-5p mimic and negative control. SGK1 mRNA levels were detected 48 hours after transfection. (F) The Ishikawa cell line was transfected with microRNA-199b-5p inhibitor and negative control. SGK1 mRNA levels were detected 48 hours after transfection. (G–I) SGK1 protein levels in Ishikawa cells were analyzed by Western blot 48 hours after transfection. (J) MicroRNA infection to knock down hsa-microRNA-199b-5p. Human endometrial stromal cells were cultured and incubated with lentiviral miRNA-199b-5p inhibitor vector and negative control. Levels of SGK1 were analyzed by qRT-PCR 96 hours after infection. (K, L) Levels of SGK1 were analyzed by Western blot 96 hours after infection. ∗ P < .05; ∗∗ P < .01. Fertility and Sterility 2015 103, 980-989.e6DOI: (10.1016/j.fertnstert.2015.01.003) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 4 (A) Comparison of miRNA-199b-5p expression between a miscarriage mouse model and normal deciduas. (B) Comparison of SGK1 mRNA expression between a miscarriage mouse model and normal deciduas. (C) Comparison of SGK1 protein expression between a miscarriage mouse model and normal deciduas. (D) Comparison of miRNA-199b-5p expression in the uterine tissues of transgenic mice and controls. (E) Comparison of SGK1 mRNA expression in the uterine tissues of transgenic mice and controls. (F) Comparison of SGK1 protein expression in the uterine tissues of transgenic mice and controls. Error bars indicate SE for all the deciduas, with technical triplicate. Fertility and Sterility 2015 103, 980-989.e6DOI: (10.1016/j.fertnstert.2015.01.003) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Pearson correlation coefficients in (A) group A and (B) group P. Fertility and Sterility 2015 103, 980-989.e6DOI: (10.1016/j.fertnstert.2015.01.003) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions