Estrogens and androgens affect human luteal cell function Anna Tropea, M.D., Antonio Lanzone, M.D., Federica Tiberi, B.S., Federica Romani, M.D., Stefania Catino, M.L.T., Rosanna Apa, M.D.  Fertility and Sterility  Volume 94, Issue 6, Pages 2257-2263 (November 2010) DOI: 10.1016/j.fertnstert.2010.02.009 Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 1 (A) Effect of estrogens (Es) on basal P release by human luteal cells (LC). Human LC were cultured for 24 hours in medium alone (C, control) or with increasing concentrations of estrone (E1), E2, or estriol (E3) (from 3 × 10−7 M–3 × 10−5 M). Each value represents the mean ± SEM of 12 independent experiments, each done in triplicate. Results are expressed as a percentage of control set equal to 100. ***P<.001, **P<.01, *P<.05 versus control values. Different superscript letters indicate statistical differences (P<.05) among the different concentrations within each treatment (E1, E2, or E3); two columns with the same letter do not differ. (B) Effect of androgens on basal P release by human LC. Human LC were cultured for 24 hours in medium alone (control) or with of T, androstendione (A), or dihydrotestosterone (DHT) (from 10−11 M–10−7 M). The figure shows increasing concentrations of androgens starting from the highest ineffective dose. Each value represents the mean ± SEM of 12 independent experiments, each done in triplicate. Results are expressed as a percentage of control set equal to 100. ***P<.001, **P<.01, *P<.05 versus control values. Different superscript letters indicate statistical differences (P<.05) among the different concentrations within each treatment (T, A, or DHT); two columns with the same letter do not differ. (C) Effect of estrogens (Es) on hCG-stimulated P release by human LC. Human LC were cultured for 24 hours in medium alone (control) or with hCG (100 ng/mL) in combination or not with E1, E2, or E3 (3 ×10−5 M). Each value represents the mean ± SEM of 12 independent experiments, each done in triplicate. Results are expressed as a percentage of control set equal to 100. °°°P<.001 versus control values; ***P<.01 versus hCG values. (D) Effect of androgens on hCG-stimulated P release by human LC. Human LC were cultured for 24 hours in medium alone (control) or with hCG (100 ng/mL) in combination or not with T, A, or DHT (10−7 M). Each value represents the mean ± SEM of 12 independent experiments, each done in triplicate. Results are expressed as a percentage of control set equal to 100. °°°P<.001 versus control values; ***P<.001, **P<.01 versus hCG values. Fertility and Sterility 2010 94, 2257-2263DOI: (10.1016/j.fertnstert.2010.02.009) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 2 (A) Effects of estrogens (Es) on prostaglandin (PG) F2α release by human luteal cells (LC). Human LC were cultured for 24 hours in medium alone (C, control) or with estrone (E1), E2, or estriol (E3) (from 3 × 10−7 M–3 × 10−5 M). Each value represents the mean ± SEM of 12 independent experiments, each done in triplicate. Results are expressed as a percentage of control set equal to 100. ***P<.001, *P<.05 versus control values. Different superscript letters indicate statistical differences (P<.05) among the different concentrations within each treatment (E1, E2, or E3); two columns with the same letter do not differ. (B) Effects of androgens on PGF2α release by human LC. Human LC were cultured for 24 hours in medium alone (control) or with T, androstendione (A), or dihydrotestosterone (DHT) (from 10−11 M–10−7 M). The figure shows increasing concentrations of androgens starting from the highest ineffective dose. Each value represents the mean ± SEM of 12 independent experiments, each done in triplicate. Results are expressed as a percentage of control set equal to 100. ***P<.001, **P<.01, *P<.05 versus control values. Different superscript letters indicate statistical differences among the different concentrations within each treatment (T, A, or DHT); two columns with the same letter do not differ (P<.05). (C) Effect of Es on PGE2 release in human LC. Human LC were cultured for 24 hours in medium alone (control) or with increasing concentration of E1, E2, or E3 (from 3 × 10−7 M–3 × 10−5 M). Each value represents the mean ± SEM of 12 independent experiments, each done in triplicate. Results are expressed as a percentage of control set equal to 100. ***P<.001 versus control values. Different superscript letters indicate statistical differences (P<.05) among the different concentrations within each treatment (E1, E2, or E3); two columns with the same letter do not differ. Fertility and Sterility 2010 94, 2257-2263DOI: (10.1016/j.fertnstert.2010.02.009) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 3 (A) Effect of estrogens (Es) on vascular endothelial growth factor (VEGF) release in human luteal cells (LC). Human LC were cultured for 24 hours in medium alone (C, control), with CoCl2 (10 μM), or in the presence of estrone (E1), E2, or estriol (E3) (from 3 × 10−7 M–3 × 10−5 M). Each value represents the mean ± SEM of 12 independent experiments, each done in triplicate. Results are expressed as a percentage of control set equal to 100. ***P<.001, **P<.01 versus control values. Different superscript letters indicate statistical differences (P<.05) among the different concentrations within each treatment (E1, E2, or E3); two columns with the same letter do not differ. (B) Effect of androgens on VEGF release in human LC. Human LC were cultured for 24 hours in medium alone (control), with CoCl2 (10 μM), or in presence of T, androstendione (A), or dihydrotestosterone (DHT) (from 10−11 M–10−7 M). Each value represents the mean ± SEM of 12 independent experiments, each done in triplicate. Results are expressed as a percentage of control set equal to 100. ***P<.001, **P<.01, *P<.05 versus control values. Different superscript letters indicate statistical differences (P<.05) among the different concentrations within each treatment (T, A, or DHT); two columns with the same letter do not differ. Fertility and Sterility 2010 94, 2257-2263DOI: (10.1016/j.fertnstert.2010.02.009) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 4 (A) Effect of aromatase inhibitor (AI) alone or in presence of androgens on P release by human luteal cells (LC). Human LC were cultured for 24 hours in medium alone (C, control) or in presence of AI (4 nM) with or without T or androstendione (A) (10−7 M). Each value represents the mean ± SEM of 12 independent experiments, each done in triplicate. Results are expressed as a percentage of control set equal to 100. ***P<.001, **P<.01 vs. control values. (B) Effects of AI alone or in presence of androgens on prostaglandin (PG)F2α release by human LC. Human LC were cultured for 24 hours in medium alone (control) or in presence of AI (4 nM) with or without T or A (10−7 M). Each value represents the mean ± SEM of 12 independent experiments, each done in triplicate. Results are expressed as a percentage of control set equal to 100. ***P<.001, **P<.01 versus control values. (C) Effect of AI alone or in the presence of androgens on vascular endothelial growth factor (VEGF) release in human LC. Human LC were cultured for 24 hours in medium alone (control) or in presence of AI (4 nM) with or without T or A (10−7 M). Each value represents the mean ± SEM of 12 independent experiments, each done in triplicate. Results are expressed as a percentage of control set equal to 100. ***P<.001 versus control values. (D) Effect of AI on PGE2 release in human LC. Human LC were cultured for 24 hours in medium alone (control) or in the presence of AI (4 nM). Each value represents the mean ± SEM of 12 independent experiments, each done in triplicate. Results are expressed as a percentage of control set equal to 100. ***P<.001 versus control values. Fertility and Sterility 2010 94, 2257-2263DOI: (10.1016/j.fertnstert.2010.02.009) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions