Soledad Henríquez, Ph. D. , Paulina Kohen, B. S. , Xia Xu, Ph. D

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Estrogen metabolites in human corpus luteum physiology: differential effects on angiogenic activity Soledad Henríquez, Ph.D., Paulina Kohen, B.S., Xia Xu, Ph.D., Timothy D. Veenstra, Ph.D., Alex Muñoz, B.Sc., Wilder A. Palomino, M.D., Jerome F. Strauss, M.D., Ph.D., Luigi Devoto, M.D. Fertility and Sterility Volume 106, Issue 1, Pages 230-237.e1 (July 2016) DOI: 10.1016/j.fertnstert.2016.03.003 Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Tissue levels of E2, E1, P, 2-ME2, 2-ME1, 16-ketoE2, and 4-OHE1 in the CL throughout the luteal phase and after administration of hCG. (A) Estradiol in different stages of the luteal phase (P>.05). (B) Mid-luteal E1 vs. late (aP≤.01). (C) Late luteal P vs. early, mid-, and late plus hCG (bP<.01). (D) Late luteal 2-ME2 and 2-ME1 vs. mid- and late plus hCG (d,eP<.05). (E) Late luteal 16-ketoE2 and 4-OHE1 vs. mid- and late plus hCG (c,fP<.05). (F) Ratio of 16-ketoE2 and 4-OHE1 vs. 2-ME2 and 2-ME1 metabolites. Early luteal vs. mid- and late vs. late plus hCG (g,hP<.001). n = 5 per CL stage. Fertility and Sterility 2016 106, 230-237.e1DOI: (10.1016/j.fertnstert.2016.03.003) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Effect of EMs on VEGF production and angiogenic activity by LGC cultures. (A) 2-Methoxyestrone reduced VEGF production by LGCs compared with basal conditions (bP<.05), whereas hCG increased VEGF levels (aP<.05). (B) Human chorionic gonadotropin increased the angiogenic activity (cP<.05), whereas 2-ME1 partially reduced tube forming induced by hCG (dP>.05). (C) 16-Ketoestradiol and 4-OHE1 increased VEGF production by LGCs compared with basal conditions (eP<.05). (D) Both EMs increased tube formation compared with basal conditions (fP<.05). (E) Photomicrograph of the angiogenic assay. Fertility and Sterility 2016 106, 230-237.e1DOI: (10.1016/j.fertnstert.2016.03.003) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Expression of COMT and P4501A1 in CL collected during the luteal phase and after administration of hCG. (A, B) Western blotting for P4501A1 and COMT. Protein expression of P4501A1 and COMT did not differ between CL of different ages. In vivo hCG administration enhanced expression (*P<.05, n = 5). Fertility and Sterility 2016 106, 230-237.e1DOI: (10.1016/j.fertnstert.2016.03.003) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Immunohistochemistry of VEGF throughout the luteal phase and after administration of hCG. (A) Photomicrographs depict luteal tissue collected during the early, mid-, late, and late plus hCG luteal phase stained for VEGF. (B) The immunohistochemistry signal is prominent in luteal cells of the mid-luteal phase CL and after hCG administration compared with the early and late luteal phase CL (a,bP<.05, n = 5). Fertility and Sterility 2016 106, 230-237.e1DOI: (10.1016/j.fertnstert.2016.03.003) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Immunodetection of StAR in cultured LGCs. (A) Immunofluorescence staining for StAR in primary culture of LGCs under basal conditions. The StAR signal (green) is intense in the cytoplasm (mitochondria) of most cultured cells. (B) Negative control (absence of primary antibody). Fertility and Sterility 2016 106, 230-237.e1DOI: (10.1016/j.fertnstert.2016.03.003) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions