Androgen Receptor Expression on Granulosa Cells
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
1000
n o i s s e r 100 p ) x 0 e 0
0 A 1 N X ( R 10 m
r H S F 1 1 10 100 1000 Androgen Receptor mRNA expression (X1000) Correlation between FSHR and AMHr2 mRNA expression levels
n 100 o i s s e r p x e
2 r 10 o t p e c e r - H M
A 1 1 10 100 1000 FSH-receptor mRNA expression 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. 21, 2593, 2006
4/20/2009 Claus Yding Andersen, MSc, DMSc: Molecular Action of LH on the Follicle 30 Androgen priming using aromatase inhibitor and hCG during early follicular phase GnRH antagonist down-reguglation in modified antagonist protocols
Steroid levels in the preovulatory follicular fluid (nmol/l) (Mean SEM) Group 2 Group 3 Group 1 P value + AI + AI + hCG Progesterone 91 8 102 9 101 6 P > 0.10 (mol/l) Testosterone 30 3.2a,b 51 2.8a 46 3.7b P < 0.001
Androstenedione 317 35a 414 21a 379 21 P < 0.001
Estradiol 1902 194 1855 133 2316 239 P > 0.10
Yding Andersen C & Løssl K, Fertil Steril, 2008;89:1760
4/20/2009 Claus Yding Andersen, MSc, DMSc: Molecular Action of LH on the Follicle 31 Androgen priming: Use of hCG and aromatase inhibitor
hCG and letrozole resulted in an improved number of embryos
and perhaps better outcome
However, unwanted effects were also observed
but it do suggest that androgen priming holds some promise requiring further optimisation Conclusion
Androgens seem to enhance FSH sensitivity in primates In human small antral follicles AR expression is highly significantly correlated to FSHR expression Follicular fluid concentrations of testosterone also correlate significantly to FSHR expression Androgen priming prior to COH may be attractive Further characterization of antral follicles is required
Western Blot and immunohistochemistry of the Androgen Receptor Androgen priming with aromatase inhibitor Characteristics of the follicular fluid (Mean SEM)
Group I Group II Group III ANOVA
Oestradiol/Testosterone 65 4a,b 37 2a,c 52 4b,c P<0.001
Oestradiol/Androstenedione 6.2 0.5a 4.5 0.3a,b 6.0 0.4b P<0.003
Fertilization (%) 60 40 60 P<0.03
AMH (ng/ml) 1,6 0,2 1,5 0,4 2,6 0,3 P < 0.001
Inhibin-B (ng/ml) 46 6 27 4 27 3 P < 0.003
Yding Andersen C & Løssl K, Fertil Steril, 2008;89:1760 1000 e n ) o L r / l e t o s m o t n s ( e t n
o d i i
t 100 u a l r f t r n a e l c u n c i o l l c o F 10 1 10 100 1000 FSH-Receptor mRNA levels