The Effects of Cetrorelix and Triptorelin on the Viability and Steroidogenesis of Cultured Human Granulosa Luteinized Cells

in vivo 26: 835-840 (2012)

CHRYSSA METALLINOU1, FRANK KÖSTER2, KLAUS DIEDRICH2, NIKOS NIKOLETTOS3 and BYRON ASIMAKOPOULOS1

1Laboratory of Physiology, School of Medicine, Democritus University of Thrace, Alexadroupolis, Greece; 2Department of Gynecology and Obstetrics, University of Lübeck, Lübeck, Germany; 3IVF Unit General University Hospital, School of Medicine, Democritus University of Thrace, Alexadroupolis, Greece

Abstract. Background: We investigated the effects of the In addition to its central action, it is suggested that GnRH gonadotropin-releasing hormone (GnRH) agonist triptorelin exerts peripheral actions since the presence of GnRHR has as well the GnRH antagonist cetrorelix those of on the been demonstrated in many extrapituitary tissues, including viability and steroidogenesis in human granulosa luteinized the human ovary. In particular, GnRHR mRNA was detected (hGL) cell cultures. Materials and Methods: The hGL cells in human granulosa-lutein cells (hGL) and in ovarian surface were obtained from 34 women undergoing ovarian epithelial cells, as well as in ovarian cancer cells, where it stimulation for IVF treatment. The cells were cultured for 48 has been suggested that locally-produced GnRH plays an h with or without 1 nM or 3 nM of cetrorelix or triptorelin in autocrine/paracrine role (1). serum-free media. The cell viability was evaluated by the After the discovery of the chemical structure of the GnRH MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium molecule (2), a plethora of GnRH analogues were prompthy bromide] assay. The concentrations of estradiol and developed, initially agonists, and later, antagonists. GnRH progesterone in culture supernatants were measured by analogues are widely used by patients undergoing assisted ELISA. Results: Treatment with triptorelin slightly increased reproductive techniques (ART) in ovarian stimulation cell viability, whereas treatment with 3 nM cetrorelix led to a protocols in order to achieve lower cancellation rates and significant decrease. Estradiol concentrations were reduced higher pregnancy rates. They suppress the endogenous with 3 nM triptorelin. Cultures treated with high-dose of gonadotropin release and prevent the premature luteinizing either cetrorelix or triptorelin tended to secrete less hormone (LH) surges, which can cause premature ovulation. progesterone than controls. Conclusion: Cetrorelix GnRH agonists, upon binding to the GnRHR cause significantly reduces the viability of hGL cells. Triptorelin gonadotropin release (flare-up effect) and their further and cetrorelix may have minor effects on steroidogenesis. administration leads to the cessation of secretion through a These results suggest that GnRH analogues may influence desensitization mechanism (3-5). The antagonistic analogues ovarian functions. directly suppress gonadotropin release by a competitive blocking of the GnRHR (4, 6). The use of GnRH antagonists Gonadotropin-releasing hormone (GnRH) via pituitary high in ART has been associated with the absence of the flare-up affinity receptors (GnRHR) regulates the biosynthesis and effect, shorter stimulation protocols and a low incidence of secretion of gonadotropins which, in turn, act directly on the ovarian hyperstimulation syndrome (6). However, it has been ovary by stimulating steroidogenesis and gametogenesis (1). suggested that GnRH antagonist protocols are associated with slightly lower clinical pregnancy and embryo implantation rates, and with lower estradiol (E2) levels on the day of ovulation induction, as compared to GnRH Correspondence to: C. Metallinou, Laboratory of Physiology, agonist protocols (7, 8). School of Medicine, Democritus University of Thrace, Dragana, The finding of putative functional GnRHR in the human 68100 Alexandroupolis, Greece. Tel: +30 2551030641, Fax: +30 2551030358, e-mail: [email protected] ovary raised concerns as to the possible effects of GnRH analogues on ovarian functions during ovarian stimulation. Key Words: GnRH analogues, viability, granulosa luteinized cells, Thus many studies were conducted to evaluate the actions of estradiol, progesterone. GnRH agonists on ovarian steroidogenesis, mainly in hGL

0258-851X/2012 $2.00+.40 835 in vivo 26: 835-840 (2012) cells, but with highly contradictory results, suggesting either separated and counted with trypan blue exclusion test. They were inhibition, stimulation, or no effects. Such controversy might then seeded at a density of 20,000 live cells/well into 96-well plates result from differences in the methodologies utilized and in and treated for a further 48 h with GnRH analogues. During this period, the culture medium contained no FCS to avoid potential the type of GnRH agonists used (9-19). On the other hand, interactions with growth factors present in serum. limited studies have been performed to examine the effects of GnRH antagonists on ovarian steroidogenesis (18-22). Experimental design. The cultures derived from each patient were Thus there is some controversy about the direct effects of divided into four treatment groups and a control group. Cultures were GnRH analogues on the human ovary and hGL cells. treated with the GnRH antagonist cetrorelix (Cetrotide; Serono Furthermore many researches have investigated the direct Europe Ltd., Frankfurt, Germany) and with the GnRH agonist effects of GnRH analogues on apoptosis and proliferation (Gonapeptyl; Ferring, Kiel, Germany) each at concentrations of 1 nM predominantly in human ovarian cancer cells, where it is and 3 nM. In the control group, no GnRH agonist or antagonist was added. Each group contained culture wells in such way that at least suggested to mediate antiproliferative and proapoptotic two cultures from each patient were included in all five groups. actions (23, 24). Accordingly comparisons were made between cultures belonging to The aim of this study was to investigate in vitro the effects different groups but obtained from the same woman. of the GnRH agonist triptorelin, as well as those of cetrorelix After treatment, the viability was evaluated using the [3-(4, 5- on the viability of hGL cells. Furthermore, we examined dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] (MTT) their effects on the steroidogenesis of hGL cells. assay. In addition, the supernatants of each group were collected and stored at –20˚C for determination of estradiol (E2) and progesterone Materials and Methods concentrations by enzyme-linked immunosorbent assay (ELISA).

Granulosa cell collection. The study was conducted at the Cell viability count by MTT colorimetric assay. The MTT test was Laboratory of Physiology, Democritus University of Thrace, originally described by Mosmann (25) and evaluates the viability of Alexandroupolis, Greece with the assistance of the Department of cells. This method is based on the ability of the yellow water- Gynecology and Obstetrics, University Clinic of Schleswig-Holstein, soluble tetrazolium salt MTT to be reduced from the succinate- Campus Lübeck, Germany. Granulosa cells were collected at the tetrazolium reductase system belonging to the mitochondrial time of oocyte retrieval from 34 women (27 to 40 years old) who respiratory chain of metabolic active cells, to the purple, water- participated in IVF cycles because of male factor infertility. insoluble formazan crystals. The quantity of formazan produced is Controlled ovarian hyperstimulation (COH) followed a GnRH directly proportional to the number of metabolically active cells. antagonist/recombinant follicle-stimulating hormone (recFSH) and a The concentration of the created formazan can be determined by human menopausal gonadotropins (hMG) regimen (Lübeck measuring the optical density (OD) at 595 nm (26-28). Protocol), as described elsewhere (19). The study protocol was The procedure used for the MTT assay was as follows: MTT approved by the Scientific Committee of the University Hospital of (Sigma, Deisenhofen, Germany) was dissolved at a concentration of Alexandroupolis. 5 mg/ml in sterile PBS and filtered. Then 10 μl of MTT were added to each well with 100 μl of Dulbecco’s medium without phenol red Cell preparation. After collection of oocytes, the follicular fluids (Sigma-Aldrich, Deisenhofen, Germany) and the plates were were centrifuged at 200 ×g for 10 min to separate the granulosa cells. incubated at 37˚C for 4 h. Subsequently, 100 μl of solvent buffer The pellet was washed twice with phosphate-buffered saline (PBS) containing 10% Sodium dodecyl sulphate (SDS) and 50% N,N- without Ca2+ and Mg2+. The cell pellet was resuspended in PBS and dimethylformamide (pH=4.7) were added. The plate remained for 10- transferred to a Ficoll gradient column (Sigma-Aldrich, Deisenhofen, 20 h in the dark at room temperature. The OD was measured using an Germany) for 20 min at 300 ×g centrifugation. hGL cells were ELISA plate reader (MRX; Dynatech Labs., Chantilly, VA, USA) at collected from the interphase and transferred to a 15 ml centrifuge a wavelength of 595 nm and with a reference filter of 650 nm. tube (Falcon; BD-Biosciences, Heidelberg, Germany). The remaining erythrocytes were removed by incubation in haemolysis buffer (150 Steroid hormone assays. E2 and progesterone levels in the media from hGL cell cultures were assayed by commercial ELISA kits mM NH4Cl, 10 mM NaHCO3, 0.1 mM EDTA) for 5 min at room temperature. The cells were then pelleted by centrifugation (200 ×g according to the manufacturer’s instructions. for 10 min) and resuspended in culture medium (18). E2: Diametra DKO003 (Diametra, Foligno, Italy); sensitivity= 10 pg/ml; intra-assay coefficient of variation (CV)=3.69%; inter- Cell culture. The collected hGL cells were cultured for 48 h in RPMI- assay CV=3.87%. Progesterone: DRG ELISA EIA 1561 (DRG 1640 with glutamine (Invitrogen, Karlsruhe, Germany) enriched with Instruments GmbH, Marburg, Germany); sensitivity=0.045 ng/ml; 6% fetal calf serum (FCS; Invitrogen), 2% Ultroser G (CytoGen GmBH, intra-assay CV=5.4%-6.86%; inter-assay CV=5.59%-9.96%. Sinn, Germany), 100 IU/ml penicillin and 100 ng/ml streptomycin (Invitrogen), at 37˚C in a humidified atmosphere of 5% CO2. Data analysis, statistical methods. At least two cultures from every Considerable attention has been attributed to the fact that high woman were included in every experimental group. Thus the amounts of exogenous gonadotropins are used in COH, which might experimental groups were interdependent, and the comparisons affect the GnRHR expression. Therefore the cells were cultured in between them were performed with the Wilcoxon’s matched pairs their initial state for 48 h to allow recovery from any effect of the in test. p-Value <0.05 was considered statistically significant. All vivo exposure to gonadotropins, and thus theoretically the effects of calculations were performed using the Statistica 7.1 software IVF hormones were minimised. After this period, the cells were (StatSoft Inc., Tulsa, OK, USA).

836 Metallinou et al: GnRH Analogues and Granulosa Luteinized Cells

Table I. Effects of Gonadotropin-releasing hormone (GnRH) analogues Table II. Estradiol (pg/ml) and progesterone (ng/ml) concentrations in on the cell viability of human granulosa luteinized (hGL) cells as it was granulosa cell culture supernatants after 48-h treatments. evaluated with the MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] assay. Values are the mean±SD and represent the Experimental group Estradiol (pg/ml) Progesterone (ng/ml) optical density at 595 nm with reference filter at 650 nm. Control 17.552±11.259 261.6±267.2 Group N Mean±SD p-Value Cetrorelix, 1 nM 17.833±12.555 282.7±259.3 (compared to control) Cetrorelix, 3 nM 18.133±13.914 212.2±192.5b,c Triptorelin, 1 nM 16.295±10.586 269.3±261.2 Control 34 0.215±0.154 - Triptorelin, 3 nM 13.532±11.200a 236.3±197.3 Cetrorelix, 1 nM 34 0.229±0.184 n.s. Cetrorelix, 3 nM 34 0.155±0.060 ** Values are the mean±SD; ap=0.002 vs. 1 nM cetrorelix; bp=0.017 vs. 1 nM Triptorelin, 1 nM 34 0.224±0.143 * cetrorelix; cp=0.008 vs. 1 nM triptorelin. Triptorelin, 3 nM 34 0.234 ±0.164 *

*p<0.05; **p<0.005; n.s.=not significant. Discussion

Results The results of the present study show that cetrorelix significantly reduces and triptorelin slightly increases the Effects of triptorelin and cetrorelix on cell viability. The cell viability of hGL cells. High amounts of triptorelin reduced viability measurements are presented in Table I. Treatment of the E2 production of hGL cells compared to cells treated cultured cells with 3 nM or 1 nM triptorelin induced a slight with low doses of cetrorelix. Furthermore, cultures treated increase of 10% or 4% respectively, of cell viability compared with 3 nM cetrorelix or triptorelin tended to secrete lower to untreated cells. Treatment with 3 nM cetrorelix for 48 h led levels of progesterone. to a 28% decrease of cell viability in comparison with the In a previous study comparing granulosa luteinized cell control group (p<0.005). Exposure to 1 nM cetrorelix had no cultures treated either with cetrorelix or with the GnRH significant effects compared to the untreated control group agonist leuprorelide, we found that the GnRH analogues did (p>0.05). In addition the differences that were found among not significantly affect steroidogenesis (19). It is worth the experimental groups were statistically significant (p<0.05). noting that the present results derived from hGL cell cultures In particular the decrease of the cell viability induced by the under basal conditions. Special care was given in order to high dose of cetrorelix (3 nM) compared with the slight avoid the presence of growth factors in the culture media. increase induced by triptorelin (3 nM, 1 nM) or the low dose With the presence of gonadotropins or other growth factors of cetrorelix (1 nM) was statistically significant. The increase which might be present in cultures media or supplement as in viability between the low dose of cetrorelix (1 nM) and FCS, the effects of GnRH analogues on the function of triptorelin (1 nM) was also statistically significant. granulosa luteinized cells could be different. It has been reported that GnRHR causes transactivation of tyrosine Effects of triptorelin and cetrorelix on steroidogenesis. kinase receptors (RTKs), such as epidermal growth factor Exposing hGL cells either to triptorelin or to cetrorelix for receptor (EGFR), insulin-like growth factor receptor (IGFR) 48 h did not affect significantly the E2 secretion, compared and fibroblast growth factor receptor (FGFR) (29). In cases to control values, although E2 levels from cultures treated where ligands for RTKs are present, the cross-talk between with 3 nM of triptorelin were lower than controls. Statistical GnRHR and RTKs might be responsible for effects much significant lower E2 levels were observed in cultures treated different from those observed in the present study. with 3 nM triptorelin compared to 1 nM cetrorelix-treated The observed reduction in viability by a high dose of cells (Table II). cetrorelix was considerable and in line with the observed Regarding the progesterone production, no statistically reduction of progesterone production in cultures treated with significant differences were found between cells treated with a high dose of cetrorelix. Regarding their antiproliferative GnRH analogues and cells incubated with the culture medium effects, GnRH antagonists can induce apoptosis in certain only. Nevertheless, treatment with 3 nM of cetrorelix or ovarian cancer lines by mediated GnRHR activation of Gi triptorelin reduced the progesterone levels by 19% and 10% protein (30, 31). respectively, but not in a statistically significant way. Cell Can the observed effects be attributed to the whole spectrum cultures treated with 3 nM of cetrorelix produced statistically of GnRH agonists and GnRH antagonists? In our opinion, it is significant lower amounts of progesterone than cultures not self-evident that other GnRH agonists or antagonists will treated with 1 nM of cetrorelix and those treated with 1 nM necessarily give similar results. It has been suggested that of triptorelin (Table II). “different GnRH ligands, both agonists and antagonists, can

837 in vivo 26: 835-840 (2012) determine preferential interactions with different intracellular Acknowledgements protein complexes through stabilization of the GnRHR in different conformations, leading to the activation of specific The study was supported by the German Akademic Exchange signal transduction pathways” (30). The classical intracellular Service (DAAD) and the Greek Scholarships Foundation (IKY) through the programme IKYDA 2005. GnRH signalling pathway, which is also predominant in gonadotrope cells, involves the activation of Gq protein, which References leads to the activation of phospholipase C β (PLCβ), generation of inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) as 1 Metallinou C, Asimakopoulos B, Schröer A and Nikolettos N: 2+ well as to the downstream mobilization of intracellular Ca and Gonadotropin-releasing hormone in the ovary. Reprod Sci 14: protein kinase C (PKC). The other possible signal transduction 737-749, 2007. pathway of GnRHR is via Gs, to activate adenylate cyclase and 2 Schally AV, Arimura A, Baba Y, Nair RM, Matsuo H, Redding elevate cAMP (30, 31). Moreover, binding of GnRH agonists TW and Debeljuk L: Isolation and properties of the FSH and or antagonists to GnRHR may stabilize it in a conformation LH-releasing hormone. Biochem Biophys Res Commun 43: 393- which activates the G protein, leading to cell cycle arrest and to 399, 1971. i 3 Karten MJ and Rivier JE: Gonadotropin-releasing hormone analog induction of apoptosis (30, 31). design. Structure-function studies toward the development of Furthermore, it has been reported that GnRHR signal agonists and antagonists: Rationale and perspective. Endocr Rev transduction interacts with other receptors such as EGFR and 7: 44-46, 1986. FGFR (30, 31). Thus, it is reasonable to presume that the 4 Chillik C and Acosta A: The role of LHRH agonists and cross-talk between GnRHR and EGFR or FGFR could be antagonists. Repro Biomed 2: 120-128, 2001. different according to the type of agonistic or antagonistic 5 Ortmann O, Weiss JM and Diedrich K: Gonadotropin-releasing ligand binding the GnRHR. In other words, not all GnRH hormone (GnRH) and GnRH agonists: Mechanisms of action. Reprod Biomed 5: 1-7, 2002. agonists or antagonists will generate exactly the same response 6 Shapiro DB: An overview of GnRH antagonists in infertility by binding GnRHR of hGL cells. In the study of Bussenot et treatments. Introduction: Fertil Steril 80(Suppl 1): S1-7, al. (12), five GnRH agonists were compared regarding their 2003. effect on E2 production from hGL cell cultures, with results 7 Al-Inany H and Aboulghar M: GnRH antagonist in assisted reproduction: A Cochrane review. Hum Reprod 17: 874-885, showing that some GnRH agonists increased E2 production, whereas others did not. Much of the discrepancy on the 2002. reported controversial effects of GnRH analogues on hGL 8 Al-Inany H, Abou-Setta AM and Aboulghar M: Gonadotrophin- releasing hormone antagonists for assisted conception. Cochrane cells may lie on the use of different GnRH analogues. Database Syst Rev 19: 3, 2006. Another interesting point is the dose of GnRH analogues. It 9 Parinaud J, Bourreau E, Vicitez G and Pontonnier G: Effect of a has been suggested that the action of GnRH and GnRH agonists luteinizing hormone-releasing hormone (Buserelin) on on cancer cell lines is dual and biphasic: low concentrations steroidogenesis of cultured human preovulatory granulosa cells. stimulate cellular growth, migration and invasion, whereas high Fertil Steril 50: 597-602, 1988. concentrations inhibit these functions (29). It is not clear if this 10 Pellicer A and Miro F: Steroidogenesis in vitro of human dual and biphasic action is valid for hGL cells regarding their granulosa luteal cells pretreated in vivo with gonadotropin- releasing hormone analogues. Fertil Steril 54: 590-596, 1990. metabolic and steroidogenic activity. Previous studies with 11 Miro F, Sampaio MC, Tarin JJ and Pellicer A: Steroidogenesis unstimulated hGL cell cultures have provided indications for a in vitro of human granulosa-luteal cells pre-treated in vitro with dose-dependent effect on steroidogenesis (9, 13). However, two gonadotropin-releasing hormone analogues employing results from other studies do not support this (32-34). The different protocols. Gyn Endocr 6: 77-84, 1992. present study provides indications for a dose-dependent effect 12 Bussenot I, Azoulay-Barjonet C and Parinaud J: Modulation of of the GnRH antagonist cetrorelix on cell viability and the steroidogenesis of cultured human granulosa-lutein cells by progesterone production. Apparently the finding of reduced gonadotropin-releasing hormone analogs. J Clin Endocrinol Metab 76: 1376-1379, 1993. viability is new and interesting but on its own it cannot provide 13 Guerrero HE, Stein P, Asch RH, de Fried EP and Tesone M: a strong argument for modification of current IVF protocols. Effect of a gonadotropin-releasing hormone agonist on This result can be translated as a thought for the optimization luteinizing hormone receptors a steroidogenesis in ovarian cells. of IVF outcomes with stronger luteal support. On the other Fertil Steril 59: 803-808, 1993. hand, the GnRH agonist triptorelin seemed to have a dose- 14 Uemura T, Namiki T, Kimura A, Yanagisawa T and Minaguchi H: Direct effects of gonadotropin-releasing hormone on the dependant negative effect on E2 production of hGL cell cultures. In conclusion, the present results support the notion that ovary in rats and humans. Horm Res 41: 7-13, 1994. 15 Dor J, Bider D, Shulmann A, Levron JL, Shine S, Mashiach S GnRH analogues influence the viability and steroidogenesis and Rabinovivi J: Effects of gonadotrophin-releasing hormone of hGL cells suggesting that they may have direct ovarian agonists on human ovarian steroid secretion in vivo and in vitro effects. Further experiments are required in order to elucidate – results of a prospective randomized in vitro fertilization study. the underlying mechanisms. Hum Reprod 15: 1225-1230, 2000.

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16 Silva ALA, Abreu LGA, Rosa-e-Silva ACJS, Ferriani RA and 27 Ferrari M, Fornasiero MC and Isetta AM: MTT colorimetric Silva-de-Sa MF: Leuprolide acetate reduces both in vivo and in assay for testing macrophage cytotoxic activity in vitro. J vitro ovarian steroidogenesis in infertile women undergoing Immunol Methods 131: 165-172, 1990. assisted reproduction. Steroids 73: 1475-1484, 2008. 28 Van de Loosdrecht AA, Beeleen RHJ, Ossankoppele GJ, 17 Vitale AM, Abramovich D, Peluffo MC, Meresman G and Broekhoven MG, and Langenheijsen MMAC: A tetrazolium- Tesone M: Effect of gonadotropin-releasing hormone agonist and based colorimetric MTT assay to quantitate human monocyte antagonist on proliferation and apoptosis of human luteinized mediated cytotoxicity against leukemic cells from cell lines and granulosa cells. Fertil Steril 85: 1064-1067, 2006. patients with acute myeloid leukemia. J Immunol Methods 174: 18 Weiss JM, Oltmanns K, Gürke EM, Polack S, Eick F, Felberbaum 311–320, 1994. R, Diedrich K and Ortmann O: Actions of gonadotropin-releasing 29 Cheung Lydia WT and Wong Alice ST: Gonadotropin-releasing hormone antagonists on steroidogenesis in human granulosa hormone: GnRH receptor signalling in extrapituitary tissues. lutein cells. Eur J Endocr 144: 677-685, 2001. FEBS 275: 5479-5495, 2008. 19 Asimakopoulos B, Nikolettos N, Nehls B, Diedrich K, Al-Hasani 30 Gründker C, Naor Z, Millar RP, Seger R, Leung PC and Emons S and Metzen E: Gonadotropin-releasing hormone antagonists do G: Mechanisms of action of GnRH. In: GnRH Analogs in not influence the secretion of steroid hormones but affect the Human Reproduction. B. Lunenfeld (ed.). London Taylor & secretion of vascular endothelial growth factor from human Francis., pp. 1-34, 2005. granulosa luteinized cell cultures. Fertil Steril 86: 636-641, 2006. 31 Millar R, Pawson A, Lu ZL, Morgan K, Davidson, R, Lopez de 20 Minaretzis D, Alpen MM and Oskowitz SP: Gonadotrophin- Maturana R, Naor Z, Brown P and Maudsley S: New concepts in releasing hormone antagonist versus agonist administration in GnRH receptor function. In: M. Updates in Infertility Treatment women undergoing controlled ovarian hyperstimulation: cycle 2004. M Filicori (ed.). Bologna, Italy, Medimond Srl., pp. 51- performance and in vitro steroidogenesis of granulosa-lutein 61, 2004. cells. Am J Obstr Gynec 172: 1518-1525, 1995. 32 Dodson W, Myers T, Morton PC and Conn PM: Leuprolide 21 Lin Y, Kahn J and Hillensjo T: Is there a difference in the acetace: serum and follicular fluid concentrations and effects on function of granulosa-luteal cells in patients undergoing in vitro human fertilization, embryo growth, and granulosa-lutein cell fertilization either with gonadotrophin-releasing hormone agonist progesterone accumulation in vitro. Fertil Steril 50: 612-617, 1988. or gonadotrophin-releasing hormone antagonist? Hum Reprod 33 Frederick J, Hickey MJ, Francis MM, Sauer MV and Paulson 14: 885-888, 1999. RJ: The effect of leuprolide acetate on steroidogenesis by 22 Winkler N, Bukulmez O, Hardy DB and Carr BR: Gonadotropin- granulosa and theca cells in vitro. J In Vitro Fertil Embryo releasing hormone antagonists suppress aromatase and anti- Transfer 8: 230-234, 1991. Mullerian hormone expression in human granulosa cells. Fertil 34 Fabbri R, Porcu E, Pession A, Sereni E, Marsella T, Seracchioli Steril 94: 1832-1839, 2010. R and Flamigni C: The effect of leuprorelin on steroidogenesis 23 Kang SK, Choi KC, Yang HS and Leung PC: Potential role of of human preovulatory granulosa cells in vitro. J Assist Reprod gonadotrophin-releasing hormone (GnRH)-I and GnRH-II in the Genet 13: 287-292, 1996. ovary and ovarian cancer. Endocr Relat Cancer 10: 169-177, 2003. 24 Cheng CK and Leung PC: Molecular Biology of Gonadotropin- Releasing Hormone (GnRH)-I, GnRH-II, and Their Receptors in Humans. Endocr Rev 26: 283-306, 2005. 25 Mosmann T: Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65: 55-63, 1983. Received April 20, 2012 26 Gerlier D and Thomasset N: Use of MTT colorimetric assay to Revised July 4, 2012 measure cell activation. J. Immunol Methods 94: 57-63, 1986. Accepted July 5, 2012

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