Placental expression profiling in preeclampsia: local overproduction of hemoglobin may drive pathological changes  Magnus Centlow, M.Sc., Piero Carninci, Ph.D., Krisztian Nemeth, M.D., Eva Mezey, M.D., Ph.D., Michael Brownstein, M.D., Ph.D., Stefan R. Hansson, M.D., Ph.D.  Fertility and Sterility  Volume 90, Issue 5, Pages 1834-1843 (November 2008) DOI: 10.1016/j.fertnstert.2007.09.030 Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Heatmap of significant genes. Expression profile of genes found significantly changed in at least one group comparison. After filtering genes for signal-to-noise ratio above 2.0 and fold change above 1.4, a total of 30 unique genes were obtained. Genes are sorted on mean expression ratio from high to low in the PE group. A cut off q-value at 0.005 was selected. Fertility and Sterility 2008 90, 1834-1843DOI: (10.1016/j.fertnstert.2007.09.030) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Real-time PCR quantification of Hbα2, Hbγ, Hbβ, and HLA-DPA1 in placenta and blood samples. All values are normalized against the amount of β-actin and presented as scatter plots. (A) Hbα mRNA expression in the placenta. Significant changes were found between PE versus controls (P=.004) and between PE/Notch (PE with notching) versus controls (P=.03). (B) Hbγ relative mRNA values showing significant changes between PE versus controls (P=.003) and between PE/Notch versus controls (P=.03). (C) Hbβ showed significant overexpression in PE versus controls (P=.02) and in PE/Notch versus controls (P=.04). (D) HLA-DPA1 mRNA was overexpressed in the notch group versus all other groups versus controls (P=.01), versus PE (P=.01), and versus PE with notching (P=.02). (E) Hbα mRNA expression in blood samples. No changes were seen between the groups. (F) Hbγ mRNA expression was very low in blood samples. No changes were seen between PE and controls. See text for abbreviations. Fertility and Sterility 2008 90, 1834-1843DOI: (10.1016/j.fertnstert.2007.09.030) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 3 In situ hybridizations from placenta and placenta bed samples. Schematic image of the human placenta showing the villous section (v) of the placenta and below a placenta bed section (m) with spiral arteries (s) in between. (A) A light field image of Hbα mRNA expression in a representative PE placenta sample. Hbα expression was especially seen in and around blood vessels. However, several scattered cells in the villous section are also seen. (B) Dark field image of the same section. (C) A dark field image of Hbα mRNA expression from a representative control placenta. Compared with PE placentas, the control placentas show fewer Hbα-expressing cells in the villous section. (D) Light field image from a representative myometrial sample from a PE patient. Hbα expression is only seen in the spiral arteries, no expression is seen in the myometrial tissue. (E) The same myometrial section in dark field. (F) A myometrial sample from a control placenta. Hbα mRNA expression is similar to the expression seen in PE myometrium. mRNA expression for Hbγ and Hbβ were similar to that for Hbα (data not shown). Bar = 100 μm. See text for abbreviations. Fertility and Sterility 2008 90, 1834-1843DOI: (10.1016/j.fertnstert.2007.09.030) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 4 A representative image of Hbγ protein expression in the placenta. Protein expression is shown with a red fluorescent marker. In the PE placenta there is a strong expression of Hbγ in the vascular lumen (lu), but Hbγ is also expressed in the vascular endothelium (arrow) as well as in the extravascular section of the villous stroma (A). The placenta from normotensive controls, however, showed no expression of Hbγ in the vascular lumen (B), but Hbγ is expressed in the vascular endothelium (arrow). Bar = 25 μm. See text for abbreviations. Fertility and Sterility 2008 90, 1834-1843DOI: (10.1016/j.fertnstert.2007.09.030) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions