Mitochondrial activation in the growth-restricted fetus of monochorionic twins  Yao-Lung Chang, M.D., Chao-Nin Wang, M.D., Ph.D., Pei-Cih Wei, B.S., Hsiu-Huei Peng, M.D., An-Shine Chao, M.D., Shuenn-Dyh Chang, M.D., Po-Jen Cheng, M.D., Tzu-Hao Wang, M.D., Ph.D.  Fertility and Sterility  Volume 100, Issue 1, Pages 241-246.e2 (July 2013) DOI: 10.1016/j.fertnstert.2013.03.003 Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 1 The mitochondrial DNA (mtDNA) fold difference (small/large) was positively correlated with the fetal birth weight (FBW) ratio (large/small) between two fetuses in each pair of monochorionic twins. A positive correlation (r = 0.53, P<.001) was identified when the relationship between the small/large mtDNA fold difference and the large/small fetal birth weight ratio was analyzed with the Pearson correlation. Fertility and Sterility 2013 100, 241-246.e2DOI: (10.1016/j.fertnstert.2013.03.003) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Mitochondrial labeling with MitoTracker in amniotic fluid mesenchymal stem cells that were derived from three pairs of monochorionic (MC) twins with selective intrauterine growth restriction. Mitochondria were shown in red fluorescence with MitoTracker reagent and recorded with a laser scanning confocal microscope (Leica). The object lens used was ×64. (A) The MC twins were delivered at the gestational age of 29 weeks, with birth weights of 1,000 g in the small and 1,500 g in the large infant. (B) The MC twins were delivered at the gestational age of 32 weeks, with birth weights of 1,470 g in the small and 1,860 g in the large infant. (C) The MC twins were delivered at the gestational age of 31 weeks, with birth weights of 1,380 g in the small and 1,920 g in the large infant. Fertility and Sterility 2013 100, 241-246.e2DOI: (10.1016/j.fertnstert.2013.03.003) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Mitochondrial staining intensities in the selective intrauterine growth restriction fetuses (small twin) were significantly higher than those in the larger fetuses (large twin). After amniotic fluid mesenchymal stem cell specimens were stained with MitoTracker, mitochondrial fluorescent intensities were quantitatively analyzed with the Image J 1.45s program. Mitochondrial staining intensities in the selective intrauterine growth restriction fetuses (199 ± 31, small twin) were significantly (*P<.05, Mann-Whitney U test) higher than those in the larger fetuses (151 ± 23, large twin). Fluorescence is in arbitrary units. Three nonoverlapping high power fields of each specimen were analyzed. Fertility and Sterility 2013 100, 241-246.e2DOI: (10.1016/j.fertnstert.2013.03.003) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Increased mitochondrial DNA ratio between the smaller/larger fetuses in monochorionic (MC) twins with selective intrauterine growth restriction (sIUGR). The fold differences of the cord blood mitochondrial DNA content between the small and large twins were significantly (*P<.01) higher in the MC twins with sIGUR (2.5 ± 1.2, n = 24 pairs) than in those without sIUGR (1.2 ± 0.3, n = 19 pairs). Fertility and Sterility 2013 100, 241-246.e2DOI: (10.1016/j.fertnstert.2013.03.003) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 Confirmation of cell surface markers of the amniotic fluid mesenchymal stem cells (AFMSCs) using flow cytometry. The human AFMSC cells were analyzed for cell surface markers with flow cytometry using fluorescein isothiocyanate (FITC)-conjugated or phythoerythrin-conjugated antibodies. All of AFMSCs in this study were positive for CD29 and CD44, but negative for CD34 and CD133. Fertility and Sterility 2013 100, 241-246.e2DOI: (10.1016/j.fertnstert.2013.03.003) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions