Intracellular pathways: from stimula to final effects Claus Yding Andersen, MSc, DMSc Professor Laboratory of Reproductive Biology University Hospital Copenhagen, University of Copenhagen, Copenhagen, Denmark E-mail: [email protected] ”ART: Experimental bases for improving outcomes for the benefit of patients” SERONO SYMPOSIA, February 19-20, 2010 Istanbul, Turkey In order to increase the efficacy of ovarian stimulation and possibly enhance outcome of infertility treatment one approach is to understand the starting point better: the human small antral follicle and ask whether it is possible to improve follicular heath and FSH sensitivity prior to the application of supra physiological FSH levels Content Gonadotropins: mode of action Effect of androgens on follicles Human small antral follicles: characterization Androgen Receptor and FSHR expression Clinical trial to study androgen priming ”ART: Experimental bases for improving outcomes for the benefit of patients” SERONO SYMPOSIA, February 19-20, 2010 Istanbul, Turkey Classification of follicles in the human ovary Gougeon A. Endocr Rev. 1996;17:121. FSH and LH interacts with its receptor on the theca and 4/20/2009 Claus Ydinggranulosa Andersen, MSc, DMSc: Molecular cells Action of LH on the Follicle 5 Signalling pathways activated by the FSHR and the LHR Extracelluar activation of the receptor leads to intracellular activation of : Adenylyl cyclase cAMP pathway PKC inositol phosphate pathway MAPK cascade FSHR govern the induction of LHR formation, but the control of FSHR formation is not well characterized but at what follicular stage LHR expressed on granulsa cells Effect of FSH and LH on steroidogenesis Granulosa cells Theca cells LH Oestradiol FSH Androgens LH Androgen has a dual role in folliculogenesis During the early stage of follicular development androgens acts as an enhancer of FSH-stimulated follicular differentiation Androgens inhibit later stages of follicular development Serve as substrate for oestrogen production Tetsuka M & Hillier SG, J Steroid Biochem Molec Biol, 1997, 61, 233 Androgens augments follicular FSH receptor expression in primate ovaries Weil S et al., JCEM, 2951, 84, 1999 Androgen Receptor gene expression in the primate ovary Weil S et al., JCEM, 1998, 83, 2479 Androgens augments follicular FSH receptor expression in primate ovaries Weil S et al., JCEM, 2951, 84, 1999 Small antralHalf follicles a human ovary Corpus Luteum Follicles Corpus luteum Human ovaries for cryopreservation Collection of follicular fluid and granulosa cells of small human antral follicles Analysis mRNA expression in GC and measurement of follicular fluid hormones in a total of 64 follicles randomly collected mRNA results from a total of 55 follicles Follicular fluids collected in a total of 24 women: Number of follicles per women Number of patients 1 5 2 8 3 7 4 2 6 1 8 1 Does Androgen Receptor expression and testosterone levels in the follicle correlate with the FSH-receptor expression in naive human small antral follicles? mRNA levels measured in the granulosa cells: AR, FSHR, LHR, CYP19a1, AMHR2 Hormones measured in the follicular fluid: AMH, Inhibin-B, oestradiol, progesterone, testosterone and androstenedione Western Blot and immunohistochemistry of the Androgen Receptor Gene Expression in Human Granulosa Cells from Follicles 3 -9 mM in Diameter in Relation to the Hormonal Profiles of the Associated Follicle Fluids Androgen Receptor mRNA expression (x1000) (Mean SEM) < 20 20 – 45 > 45 –130 > 130 Spearman Number 11 16 14 14 FSH-R (x1000) 18 8 56 13 133 31 181 49 P <0.0001 LH-R (x1000) 0,63 0,27 0,53 0,19 0,87 0,22 1,76 0,69 P <0.02 AMHR-2 (x1000) 4,6 1,4 21,2 7,8 16,2 3,8 23,2 3,1 P <0.0008 CYP-19 (x1000) 38 21 33 16 19 7 48 15 P >0.10 No significant relation to Follicular Fluid hormone parameters AMH, Inhibin-B, Oestradiol, Progesterone, Androstenedione and Testosterone 4/20/2009 Claus Yding Andersen, MSc, DMSc: Molecular Action of LH on the Follicle 16 Correlation between FSHR and AR mRNA expression levels F S H r m R N A e x p re s s io n (X 1 0 0 0 ) 1000 100 10 1 1 Androgen Receptor mRNA expression (X1000) 10 100 1000 AMH-receptor2 expression Correlation between FSHR and AMHr2 mRNA 100 10 1 1 FSH-receptor mRNA expression expression levels 10 100 1000 Follicular fluid concentrations of testosterone in relation to mRNA expression of gonadotropin receptors, CYP19 and AMH-r2 genes in the corresponding granulosa cells Testosterone concentration (nmol/L) (Mean SEM) < 145 146 – 210 211 - 350 >350 Spearman Number 15 13 13 13 FSH-R (x1000) 52 17 49 16 122 35 185 51 P < 0.003 LH-R (x1000) 1,0 0,3 0,7 0,2 1,2 0,6 0,8 0,3 P >0.10 AMHR-2 (x1000) 10 2 25 10 17 4 20 5 P >0.10 CYP-19 (x1000) 38 16 46 22 36 13 19 9 P >0.10 Significant relation to Follicular Fluid inhibin-B and androstenedione concentrations, whereas the other hormone parameters including AMH, Oestradiol and Progesterone did not correlate. 4/20/2009 Claus Yding Andersen, MSc, DMSc: Molecular Action of LH on the Follicle 19 Gene Expression in Human Granulosa Cells from Follicles 3- 9 mM in Diameter in Relation to the Hormonal Profiles of the Associated Follicle Fluids LH-receptor (x1000) (Mean SEM) 0.12 0.12 – 0.25 0.25 – 1.0 > 1.0 Spearman Number 18 12 11 14 FSH-R (x1000) 62 16 59 19 100 21 182 52 P <0.05 Progesterone(nM) 246 32 308 75 435 99 576 112 P <0.05 Oestradiol (nM) 158 75 120 82 102 50 418 260 P >0.10 CYP-19 (x1000) 12 6 34 21 33 17 123 67 P >0.10 No significant relation to AMH-FF, AMH-R2, AR and FF testosterone 4/20/2009 Claus Yding Andersen, MSc, DMSc: Molecular Action of LH on the Follicle 20 Human small antral follicles are characterized by Confirming results from primates Androgen Receptor expression on granulosa cells A significant correlation between mRNA levels of AR and FSHR A significant correlation between FF-testosterone and FSHR expression mRNA levels of FSHR is significantly correlated to those of LHR PCO women are sensitive to FSH stimulation They often hyper-respond to exogenous FSH stimulation Granulosa cells are hyper-responsive to FSH in vitro They use less exogenous FSH and less FSH per oocyte retrieved PCO women are characterized by Enhanced LH activity Enhanced androgen action Without the action of FSH Could some of the mechanisms that make PCO women sensitive to FSH stimulation be used to enhance follicular sensitivity in low or moderate responder patients? Aim: Temporary reversible PCO-like condition prior to ovarian stimulation Androgen Priming: Clinical study Prospective, Non-randomized Pilot Study 45 patients with 15 in each of 3 different groups Inclusion criteria – IVF/ICSI treatment – age between 20 and 40 – regular menstrual cycle – BMI between 18 and 30 kg/m² – FSH and LH < 12 IU/l cd 2-3 Stimulation protocol – Androgen priming hCG, 1250 IE Aromatase inhibitor, letrozole 2,5 mg daily Group 3 Group 2 GnRH antagonist, cetrorelix 3 mg Group 1 menotropin 150/225 Individual dose Downregulation and priming Flexible antagonist protocol cd2 cd5 cd8 cd14 stim.1 stim.7 Løssl K et al., Hum Reprod. 21, 2593, 2006 Androgen priming using aromatase inhibitor and hCG during early follicular phase GnRH antagonist down-reguglation in modified antagonist protocols Baseline Characteristics Group 2 Group 3 Group 1 P value + AI + AI + hCG No. patient 15 15 15 Age (mean) 32,4 ± 1 32,6 ± 1 32,8 ± 1 NS IVF/ICSI (n) 5/10 5/10 5/10 Earlier treatments (n) 0,7 ± 0,3 0,9 ± 0,3 0,8 ± 0,3 NS Pretreatment FSH, LH, oestradiol and testosterone levels similar between groups Løssl K et al., Hum Reprod. 21, 2593, 2006 4/20/2009 Claus Yding Andersen, MSc, DMSc: Molecular Action of LH on the Follicle 27 Androgen priming using aromatase inhibitor and hCG during early follicular phase GnRH antagonist down-reguglation in modified antagonist protocols Outcome of stimulation Group 2 Group 3 Group 1 P value + AI + AI + hCG Days of stimulation 9,4 9,6 10,3 NS Total FSH dose (IE) 2030 1917 2110 NS Endometrium (mm) 9,9 9,1 8,5 NS Follicles (>12 mm) 6,1 ± 0,7 9,5 ± 1,5 8,4 ± 1,3 NS Equal number of follicles on stimulation day 1 Løssl K et al., Hum Reprod. 21, 2593, 2006 4/20/2009 Claus Yding Andersen, MSc, DMSc: Molecular Action of LH on the Follicle 28 Androgen priming using aromatase inhibitor and hCG during early follicular phase GnRH antagonist down-regulation in modified antagonist protocols Outcome of IVF stimulation Group 2 Group 3 Group 1 P value + AI + AI + hCG Aspirations (n) 13 15 13 Oocytes (n) 5,7 ±0,6 8,4 ±1,2 8,4 ±1,1 NS Fertilizationrate 61 ± 6,1 40 ± 5,2* 60 ± 7,4 *P=0,03 Embryos (n) 3,1 ±0,4 2,7 ±0,6 5,2 ±0,7* *P=0,01 Cryopreserved 1,0 ±0,5 1,0 ±0,5 2,6 ±0,7 NS embryos (n) Løssl K et al., Hum Reprod. 21, 2593, 2006 4/20/2009 Claus Yding Andersen, MSc, DMSc: Molecular Action of LH on the Follicle 29 Androgen priming using aromatase inhibitor and hCG during early follicular phase GnRH antagonist down-regulation in modified antagonist protocols Reproductive Outcome Group 2 Group 3 Group 1 P value + AI + AI + hCG Cycles 15 15 15 NS Pos. hCG 47% 40% 47% NS Implantation rate 39 % 20% 26% NS Ongoing/cycle 27 % 27% 33% NS Ongoing/transfer 33 % 31% 42% NS Løssl K et al., Hum Reprod.