Estefanía Cabello, M. S. , Pablo Garrido, Ph. D. , Javier Morán, M. S

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Effects of resveratrol on ovarian response to controlled ovarian hyperstimulation in ob/ob mice Estefanía Cabello, M.S., Pablo Garrido, Ph.D., Javier Morán, M.S., Carmen González del Rey, M.D., Ph.D., Plácido Llaneza, M.D., Ph.D., David Llaneza-Suárez, Ph.D., Ana Alonso, Ph.D., Celestino González, Ph.D. Fertility and Sterility Volume 103, Issue 2, Pages 570-579.e1 (February 2015) DOI: 10.1016/j.fertnstert.2014.10.034 Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Effects of resveratrol on insulin resistance parameters and T levels in wild-type and ob/ob mice. (A, B) Plasma levels of fasting blood glucose and insulin, respectively. (C) Histogram depicting HOMA-IR, a method for evaluating insulin sensitivity, in control and experimental groups of mice. (D) Testosterone levels in plasma. Values are expressed as mean ± SEM for groups of eight mice in each. a,bSignificant difference (P<.05), aOP vs. NOP, bOR vs. OP. NOP = nonobese placebo; NOR = nonobese resveratrol; OP = obese placebo; OR = obese resveratrol. Fertility and Sterility 2015 103, 570-579.e1DOI: (10.1016/j.fertnstert.2014.10.034) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Effects of resveratrol on reproductive function in wild-type and ob/ob mice. (A) Representative images of the female reproductive system, which includes uterus and ovaries, in obese and nonobese mice treated with resveratrol or placebo. (B) Reproductive system weight relative to body weight among four experimental groups. (C, D) Histograms representing the treatment effects on total oocyte number and percentage of healthy oocytes retrieved, respectively. Values are expressed as mean ± SEM for groups of eight mice in each. a,cSignificant difference (P<.05), aOP vs. NOP, cNOR vs. NOP. RS = reproductive system. Fertility and Sterility 2015 103, 570-579.e1DOI: (10.1016/j.fertnstert.2014.10.034) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 3 (A–D) Representative histologic images from ovaries stained with hematoxylin and eosin for the quantitative assessment of follicles, in the control and experimental groups of mice. Nonobese mice (C57BL/6J; wild-type mice) treated with placebo (NOP), nonobese mice treated with resveratrol 3.75 mg/kg (NOR), obese mice (B6.V-LEP OB/J; ob/ob mice) treated with placebo (OP), and obese mice treated with resveratrol 3.75 mg/kg (OR) daily for 20 days and undergoing COH protocol. Scale bar, 100 μm; original magnification, 10×. (E–I) Effects of resveratrol on follicular development in wild-type and ob/ob mice. Histograms representing the quantitative assessment of follicles and corpus lutea. Follicles were counted and classified as (E) primary follicles, (F) total growing follicles, and (G) preovulatory follicles. (H, I) Number of corpus lutea and atretic follicles, respectively. Values are expressed as mean ± SEM for groups of eight mice in each. a,b,cSignificant difference (P<.05), aOP vs. NOP, bOR vs. OP, cNOR vs. NOP. Fertility and Sterility 2015 103, 570-579.e1DOI: (10.1016/j.fertnstert.2014.10.034) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Effects of resveratrol on the adipose tissue levels of IL-6 and TNF-α in wild-type and ob/ob mice. (A, B) Densitometric analysis of the proinflammatory cytokines IL-6 and TNF-α, respectively, normalized for β-actin as seen in the representative Western blots of one experiment. Values are expressed as mean ± SEM for groups of eight mice in each. a,b,cSignificant difference (P<.05), aOP vs. NOP, bOR vs. OP, cNOR vs. NOP. Fertility and Sterility 2015 103, 570-579.e1DOI: (10.1016/j.fertnstert.2014.10.034) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Effects of resveratrol on body weight and food intake in wild-type and ob/ob mice. (A) Body weight evolution during the 20 days of the treatment period. (B, C) Histograms representing food intake relative to body weight before (day 0) and after (day 20) the treatment period, respectively. Values are expressed as mean ± SEM for groups of eight mice in each. aSignificant difference (P<.05), OP vs. NOP. Fertility and Sterility 2015 103, 570-579.e1DOI: (10.1016/j.fertnstert.2014.10.034) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions