1. Effects of dydrogesterone and of its stable metabolite, 20-α-dihydrodydrogesterone, on nitric oxide synthesis in human endothelial cells 
Tommaso Simoncini, M.D., Ph.D., Antonella Caruso, M.D., Maria Silvia Giretti, M.D., Camila Scorticati, Ph.D., Xiao-Dong Fu, M.D., Silvia Garibaldi, M.D., Chiara Baldacci, M.D., Paolo Mannella, M.D., Letizia Fornari, M.D., Andrea R. Genazzani, M.D., Ph.D. 
Fertility and Sterility 
Volume 86, Issue 4, Pages (October 2006)
DOI: /j.fertnstert
Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

2. FIGURE 1
Divergent transcriptional regulation of eNOS expression by DYD, DHD, P, and MPA in human endothelial cells. Steroid-deprived HUVECs were treated for 48 hours with 10 nmol/L P, 10 nmol/L MPA, 10 nmol/L DYD, or 10 nmol/L DHD in the presence or absence of the PR and GR antagonist RU486 (RU, 1 μmol/L). Alternatively, HUVECs were treated for 48 hours with 1 nmol/L E2 in the presence or absence of the previous compounds. (A and C): Nitrites released in the cell culture medium were assayed (yellow bars), and eNOS activity was tested in whole cell lysates (blue bars). (B and D): Endothelial nitric oxide synthase (eNOS) and von Willebrand (as loading control [vWF]) protein amounts were tested on whole cell protein extracts. (A): *P<.05 versus control (CON). (C): *P<.05 versus E2 alone. The experiments were repeated three times in quadruplicate (nitrites measurement) or in triplicate (eNOS activity) with comparable results, Western blot analyses were repeated three times, and representative blots are shown.
Simoncini. Endothelial effects of dydrogesterone. Fertil Steril 2006.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

3. FIGURE 2
Concentration and time dependency of eNOS induction by DHD. Steroid-deprived HUVECs were treated for 48 hours (A–C) with increasing concentrations of DYD or DHD. Steroid-deprived HUVECs were treated for different times with 10 nmol/L DHD (D–E). (A and D): Nitrites released in the cell culture medium were assayed (yellow bars), and eNOS activity was tested in whole cell lysates (blue bars). (B, C, and E): Endothelial nitric oxide synthase (eNOS) and von Willebrand (as loading control [vWF]) protein amount were tested on whole cell protein extracts. *P<.05 versus control (CON). The experiments were repeated three times in quadruplicate (nitrites measurement) or in triplicate (eNOS activity) with comparable results, Western blot analyses were repeated three times, and representative blots are shown.
Simoncini. Endothelial effects of dydrogesterone. Fertil Steril 2006.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

4. FIGURE 3
Rapid eNOS activation by P and DHD (but not MPA) in endothelial cells. Steroid-deprived and serum-starved HUVECs were treated for 30 minutes with 10 nmol/L P, 10 nmol/L MPA, 10 nmol/L DYD, or 10 nmol/L DHD in the presence or absence of the PR and GR antagonist RU486 (RU, 1 μmol/L) (A–B). Steroid-deprived HUVECs were treated for 30 minutes with increasing concentrations of DHD (C). (A and C): Nitrites released in the cell culture medium were assayed (yellow bars) and eNOS activity was tested (blue bars) in whole cell lysates. (B): Endothelial nitric oxide synthase (eNOS) and von Willebrand (as loading control) (vWF) protein amounts were tested on whole cell protein extracts. *P<.05 versus control (CON). The experiments were repeated three times in quadruplicate (nitrites measurement) or in triplicate (eNOS activity) with comparable results, Western blot analyses were repeated three times, and representative blots are shown.
Simoncini. Endothelial effects of dydrogesterone. Fertil Steril 2006.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

5. FIGURE 4
Differential recruitment of MAPK nongenomic signaling pathways by P, DYD, and DHD. Steroid-deprived and serum-starved HUVECs were treated for 30 minutes (A) or 5 minutes (B) with 10 nmol/L P, 10 nmol/L DYD, or 10 nmol/L DHD in the presence or absence of the MEK 1/2 inhibitor PD98059 (PD, 5 μmol/L) or of the PR and GR antagonist RU486 (RU, 1 μmol/L). Alternatively, HUVECs were treated for 30 minutes (A) or 5 minutes (B) with 1 nmol/L E2, in the presence or absence of the previous compounds. (A and C): Nitrites released in the cell culture medium were assayed (yellow bars) and eNOS activity was tested (blue bars) in whole cell lysates. (B and D): Wild-type or phosphorylated ERK 1/2 (P-ERK) was assayed. (A): *P<.05 versus control. (B): *P<.05 versus E2 alone. The experiments were repeated three times in quadruplicates (nitrites measurement) or in triplicates (eNOS activity) with comparable results, Western blot analyses were repeated three times, and representative blots are shown.
Simoncini. Endothelial effects of dydrogesterone. Fertil Steril 2006.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions