GnRH agonist and GnRH antagonist protocols in ovarian stimulation: differential regulation pathway of aromatase expression in human granulosa cells  Mohamad Khalaf, Hervé Mittre, Jérôme Levallet, Vincent Hanoux, Christine Denoual, Michel Herlicoviez, Pierre-Jacques Bonnamy, Annie Benhaim  Reproductive BioMedicine Online  Volume 21, Issue 1, Pages 56-65 (July 2010) DOI: 10.1016/j.rbmo.2010.03.017 Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 Concentrations of oestradiol in (A) serum and (B) follicular fluid (follicles >14mm) in women undergoing gonadotrophin-releasing hormone (GnRH) agonist (n=28) or antagonist (n=22). Oestradiol was determined by radioimmunoassay. Values are mean±SD. ∗P<0.05. Reproductive BioMedicine Online 2010 21, 56-65DOI: (10.1016/j.rbmo.2010.03.017) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 (A) Aromatase activity and (B) aromatase gene expression in human granulosa lutein cells treated with gonadotrophin-releasing hormone (GnRH) agonist or antagonist. Aromatase activity was quantified by 3H2O method. mRNA aromatase were quantified by real-time reverse-transcription PCR and values were normalized to mRNA GAPDH. Values are means±SD. ∗P<0.05. Reproductive BioMedicine Online 2010 21, 56-65DOI: (10.1016/j.rbmo.2010.03.017) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 (A) FSH receptor gene and (B) progesterone receptor gene expression in human granulosa lutein cells treated with gonadotrophin-releasing hormone (GnRH) agonist or antagonist. mRNA FSH receptors were quantified by semi-quantitative reverse-transcription PCR and values were normalized to mRNA β-actin. mRNA progesterone receptors were quantified by real-time reverse-transcription PCR and values were normalized to mRNA glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Values are means±SD of five separate experiments using total RNA preparations from follicles >14mm from five patients. ∗P<0.05. Reproductive BioMedicine Online 2010 21, 56-65DOI: (10.1016/j.rbmo.2010.03.017) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 PKC activity in granulosa lutein cells treated with gonadotrophin-releasing hormone (GnRH) agonist or antagonist. Total cell lysates were prepared and subjected to the PepTag assay. Bands visualized on the agarose gel were quantified by densitometry. The values were expressed as a percentage of the value measured in cells from the GnRH agonist protocol. Values are means±SD of three separate experiments using granulosa lutein cells proteins from follicles >14mm from three patients. ∗P<0.05. Reproductive BioMedicine Online 2010 21, 56-65DOI: (10.1016/j.rbmo.2010.03.017) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 5 Effect of various treatments on the oestradiol production by human granulosa lutein cells: (A) control or (B) previously desensitized with gonadotrophin-releasing hormone (GnRH) antagonist (10−5mol/l) during 24h. (A) Cells cultured in the presence of androstenedione (10−7mol/l) were treated for 24h with recombinant FSH alone (rFSH; 500mIU/ml), rFSH (500mIU/ml) and GnRH agonist (Ga; 10−7mol/l), rFSH (500mIU/ml)+phorbol myristate acetate (PMA; 10ng/ml), rFSH (500mIU/ml)+GnRHa (10−7mol/l)+GnRH antagonist (Gant; 10−5mol/l) and rFSH (500mIU/ml)+Gant (10−5mol/l)+PMA (10ng/ml). (B) GnRH-desensitized cells were treated for 24h with androstenedione (10−7mol/l) and with rFSH (500mIU/ml) alone and in combination with PMA (10ng/ml). At the end of the culture, media were frozen at −20°C until oestradiol determination by radioimmunoassay. Data are expressed as a percentage of oestradiol production in presence of FSH. Values are means±SD of three experiments performed in triplicate. ∗P<0.05. Reproductive BioMedicine Online 2010 21, 56-65DOI: (10.1016/j.rbmo.2010.03.017) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions