Department of Obstetrics and Gynaecology University of Thessaly Endocrinology and metabolic consequences of PCOS Professor Ioannis E. Messinis MD, PhD (Aberdeen, UK), FRCOG Department of Obstetrics and Gynaecology University of Thessaly Larissa, Greece

DISCLOSURE Nothing to disclose

Outline Definition Endocrine changes Follicle maturation - anovulation Co-morbidities

Definition of PCOS: Rotterdam criteria Clinical or biochemical hyperandrogenism Oligomenorrhea (less than 6-9 menses per year) or oligo- ovulation Polycystic ovaries on ultrasound (≥12 antral follicles in one ovary or ovarian volume ≥10 cm3) The Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group, 2004 Hum. Reprod. 19, 41-7 2 out of 3 criteria

PCOS Definitions 1990-2009 Roe & Dokras 2011 Rev. Obstet. Gynecol 4, 45-51

Outline Definition Endocrine changes Follicle maturation - anovulation Co-morbidities

Endocrine changes in PCOS: Role in pathophysiology Insulin resistance - hyperinsulinaemia Hyperandrogenism (T, A, FAI) Elevated LH levels Normal or reduced FSH levels (lack of intercycle-type rise) Increased levels of AMH

Insulin resistance 50-70% of PCOS cases Independent of obesity Is enhanced in obesity

Insulin resistance: a post-receptor defect ●increased serine phosphorylation ●decreased tyrosine phosphorylation IRSs ●decreased IRSs expression PI3-k ●Glucose transport ●Glycogen synthesis ●Protein synthesis De Leo et al., 2003 Endocr. Rev. 24, 633-67

PCOS: Fasting insulin levels 50 ** 45 * 40 35 30 Fasting insulin levels (μU/mL) 25 20 15 10 5 N HIRS O/M HIRS+O/M PCOS Diamanti-Kandarakis et al., 1999; JCEM 84, 4006-11

PCOS pathogenesis: Insulin resistance Genetic predisposition Skeletal muscle TNFα FFA Adipose tissue Insulin resistance Hyperinsulinemia Ovary ↑Androgens Liver Adrenal ↑IGF I ↑Androgens ↓SHBG ↓IGFBP-1 De Leo et al., 2003 Endocr. Rev. 24, 633-67 ↑IGF I

Endocrine changes in PCOS: Role in pathophysiology Insulin resistance - hyperinsulinaemia Hyperandrogenism (T, A, FAI) Elevated LH levels Normal or reduced FSH levels (lack of intercycle-type rise) Increased levels of AMH

Androgen excess in PCOS Increased steroidogenic activity of theca cells Changed enzymatic activity Increased 17α-hydroxylase/17, 20-lyase Increased 3β-hydroxysteroid-dehydrogenase Decreased 17β-hydroxysteroid-dehydrogenase Increased LH

PCOS: Steroidogenesis in the ovary 3β-HSD Pregnenolone 17α-hydroxy- pregnenolone Dehydroepi- androsterone Androstendiole Progesterone 17α-hydroxy- progesterone Δ4-Αndro- stendione Testosterone 3β-HSD 17,20-lyase 3β-HSD↑ 17β-HSD↓ 3β-HSD

Hyperandrogenaemia in PCOS CONTROLS (n=54) P LH (IU/l) 11.0±5.0 4.1±3.4 <0.001 FSH (IU/l) 5.7±1.2 4.9±1.9 0.004 LH/FSH ratio 2.0±0.9 0.8±0.6 <0.001 E2 (pmol/l) 150.6±64.4 118.7±79.1 0.003 Testo- (nmol/l) 1.8±0.7 1.2±1.0 0.001 Androste- (nmol/l) 8.5±2.8 5.1±1.8 <0.001 It is not only basal LH but also the LH to FSH ratio that is increased in PCOS Hendriks et al., 2008 RBMOnline 16, 765-71 Mean±SD

Endocrine changes in PCOS: Role in pathophysiology Insulin resistance - hyperinsulinaemia Hyperandrogenism (T, A, FAI) Elevated LH levels Normal or reduced FSH levels (lack of intercycle-type rise) Increased levels of AMH

ELEVATED LH In 35-90% of PCOS In 94% increased LH/FSH ratio  Rebar et al., 1976; J. Clin. Invest.  Franks, 1989; Clin. Endocrinol.  Balen et al., 1995; Hum. Reprod.  Van santbrink et al., 1997; Fertil. Steril.  Taylor et al., 1997; JCEM That LH is elevated has been shown in several studies with a wide range from 35 to 90%

Serum LH values in PCOS LH (IU/l) 11 * 10 9 *P<0.001 8 7 6 5 Homburg et al., 2013 Hum. Reprod. 28, 1077-83 4 3 N= 89 34 88 Mean (95% CI) Controls PCOM PCOS

PCOS: A E Hypersecretion of LH HYPOTHALAMUS GnRH PITUITARY Estrogen (-) GnRH PITUITARY (-) Estrogen Androgens LH FSH Peripheral conversion A E Theca cell Granulosa cell Insulin Hirsutism

Augmented LH response to GnRH in PCOS 45 Normal PCOS 40 LH mIU/ml 2 μg 10 μg 20 μg 35 30 25 20 15 10 GnRH iv Between-group differences in LH responsiveness disappeared when controlling for serum testosterone (T) levels. And the LH response to GnRH that is augmented. This study from SanDiego shows the absolute increase in LH in response to three consecutive doses of GnRH. 5 600 800 1000 1200 1400 1600 1800 2000 Clock hours Patel et al., 2004 Clin. Endocrinol. 60, 67-74

Enhanced net increase in LH (ΔLH) in PCOS 50 P<0.05 30-min response to 10 μg iv GnRH (ΔLH IU/l) 40 30 20 10 When we calculated the net increase in LH as the 30-min response to 10 μg GnRH that according to earlier studies of Yen’s group represents the pituitary sensitivity to GnRH we found that this sensitivity is enhanced in PCOS as compared to normal controls. Controls PCOS Dafopoulos et al., 2009 Fertil. Steril. 92, 1378-80

LH PULSES IN PCOS Control PCOS 20 16 Serum LH IU/L 12 8 4 2.5 LH 2.5 LH secretory rate IU/L/min 2.0 1.5 1.0 In terms of LH elevation, it is known that GnRH and LH are secreted in a pulsatile fashion which in PCOS in increased both in frequency and magnitude 0.5 0.0 200 400 600 800 200 400 600 800 Time (min) Barontini et al., 2001 Arch. Med. Res. 32, 544-52

LH PULSES IN PCOS 18 16 LH mIU/ml 14 12 10 8 6 4 2 0800 1200 1600 0800 Amp=3.37 mIU/ml 16 LH mIU/ml 14 8.1±0.1 mIU/ml Amp=1.89 mIU/ml 12 10 8 4.3±0.1 mIU/ml Amp=1.54 mIU/ml 6 4 2 ...and the increase is in proportion to the basal values of LH. 0800 1200 1600 0800 1200 1600 0800 1200 1600 Clock hours Patel et al., 2004 Clin. Endocrinol. 60, 67-74 3 PCOS cases

LH VALUES IN PCOS Anovul. PCOS 100 Post-ovul. PCOS Normal Pool LH IU/L LH/FSH ratio Nevertheless, if consider the data by Hall’s group, it is evident that PCOS patients with ovulatory cycles have basal LH and LH to FSH ration within the normal range. 1 0.1 1 10 100 1000 n= 61 PCOS n= 24 controls Taylor et al., 1997 JCEM 82, 2248-56 Days from menses

HORMONE PROFILE IN OVULATORY PCOS 150 50 LH (IU/L) Normal FSH (IU/L) 40 PCOM 100 30 20 50 10 25 400 Estradiol (pg/ml) Progesterone (ng/ml) 20 300 15 200 ...and based also on data from the same group, the hormone profile of ovulatory cycles in PCOS is almost identical to that of normal controls. It clear therefore that only in anovulatory PCOS the LH can be elevated. 10 100 5 -14 -7 7 14 -14 -7 7 14 n= 26 PCOS n= 16 controls DAYS FROM OVULATION Adams et al., 2004 JCEM 89, 4343-50

Aetiology of increased LH Lack of progesterone (Dafopoulos et al., 2009; Fertil. Steril. 92, 1378-800) Hypeandrogenaemia sustains the abnormal GnRH sensitivity to the negative feedback of progesterone (Eagleson et al., 2000; JCEM 85, 4047-52)

LH AND FSH RESPONSE TO GnRH Gonadotroph (+) E2 It is known that GnRH stimulates the secretion of LH and FSH from the pituitary gonadotrophs. This is influenced by E2 which sensitizes the pituitary to GnRH. Besides E2 another substance that we have named GnSAF plays also a role by antagonizing the sensitizing effect of E2. (-) GnSAF LH FSH

RESEARCH PROTOCOL PCOS Progesterone 100 mg x3 p.o. (n=10) Exp-1 Exp-2 6 days 20 days 6 days Experiment Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 0900 0900 0900 0900 0900 0900 2100 To test this hypothesis we did experiments in two groups of women, a group of PCOS and a control group of normally cycling women. In the PCOS group the same experiment was performed twice 20 days apart during which the patients received progesterone. The same experiment was also performed during the first half of the follicular phase of the control group. The experiment was of 6 days and involved the administration of a single dose FSH 450 IU in the morning of day 1 and before that and then every 12 hours initially and every 24 hours subsequently, we injected a submaximal dose GnRH 10 μg and studied the 30 min response. GnRH 10 μg iv FSH 450 IU sc Exp Control 6 days (n=8) Dafopoulos et al., 2009 Fertil. Steril. 92, 1378-80

PCOS: ΔLH response (30-min) to GnRH (10 μg iv) before and after P4 100 ΔLH IU/l 75 50 Before P4 After P4 Control 25 In terms of the LH response to GnRH, this was increased before any treatment and decreased slighlty but not significantly following the injection of FSH. After treatment with P4 and before the administration of FSH, the ΔLH values had become normal and after the administration of FSH they decreased significantly and similarly in PCOS and controls. This suggsests that GnSAF was produced in similar amounts by the polycystic and the control ovaries. It is not therefore that the augmented response to GnRH in PCOS is due to the reduced production of GnSAF but rather to a defect in the action at the pituitary. 1 2 3 4 5 6 Days rFSH 450 IU Days Dafopoulos et al., 2009 Fertil. Steril. 92, 1378-80 *P<0.05; +P<0.05

LH AND FSH RESPONSE TO GnRH (+) Gonadotroph P4 (-) Coming back to the stimulating effect of GnRH on the pituitary, it is assumed that the longterm exposure of the pituitary to the unopposed E2 action overcomes the action of GnSAF. After the administration of P4 or after ovulation, this steroid modulates the action of E2 and re-establishes a balanced action of E2 and GnSAF. (-) GnSAF LH FSH

Endocrine changes in PCOS: Role in pathophysiology Insulin resistance - hyperinsulinaemia Hyperandrogenism (T, A, FAI) Elevated LH levels Normal or reduced FSH levels (lack of intercycle-type rise) Increased levels of AMH

Serum FSH values in PCOS 7 7 *P<0.001 (Controls) 6 FSH (IU/l) * 6 5 Homburg et al., 2013 Hum. Reprod. 28, 1077-83 5 N= 89 34 88 Mean (95% CI) Controls PCOM PCOS

Serum AMH values in PCOS 100 + * 80 *P<0.001 +P<0.05 (PCOM) 60 AMH (pmol/l) 40 20 Homburg et al., 2013 Hum. Reprod. 28, 1077-83 N= 90 35 90 Mean (95% CI) Controls PCOM PCOS

Outline Definition Endocrine changes Follicle maturation - anovulation Co-morbidities

PCOS: anovulation A large pool of antral follicles 2-8 mm Failure of the selection of the dominant follicle FSH levels lower than the threshold for follicle recruitment- selection Franks et al., 2000; Mol Cell Endocrinol 163, 49-52 Hillier, 1994; Hum Reprod 9, 188-91

Inhibition of antral follicle growth Hypersecretion of LH Hyperinsulinaemia Hyperandrogenism AMH

Anovulatory women with PCOS Hyperinsulinaemia Premature response to (elevated) LH of granulosa cells of follicles of 4 mm vs ovulatory (10 mm) Granulosa cells from small follicles cease to divide and undergo terminal differentiation prematurely (LH ceiling) Anovulation Hillier, 1994; Hum Reprod 9, 188-91 Willis et al., 1998; JCEM 83, 3984-91

Hyperandrogenism in PCOS ARREST GROWTH Androgenic milieu Estrogenic milieu PCOS NORMAL ↑5α-reductase 4X Granulosa cells cease to divide ↑5α-reduced A LH FSH ↓aromatase ↑aromatase FOLLICLE GROWTH

AMH in PCOS Increased production from granulosa cells Facilitates early follicular development Slows down atresia Inhibits FSH stimulating action Inhibits the growth of antral follicles Increases the number of small antral follicles

INTERCYCLE RISE OF FSH LUTEAL FOLLICULAR LH IU/l FSH 2 8 6 4 2 It is known that in normally cycling women the intercycle rise of FSH is responsible for the selection of the dominant follicle 4 2 -8 -6 -4 -2 1 3 5 7 DAYS Messinis et al., 1993; Clin. Endocrinol. 38, 159-163

ANOVULATORY PCOS Lack of intercycle rise of FSH Lack of follicle maturation and ovulation (lack of P4) None of these events takes place in anovulatory women with PCOS. They lack the intercycle rise of FSH and therefore they lack follicle maturation and ovulation. As a result P4 concentrations are low leading to an attenuated negative feedback effect on LH secretion. Nevertheless, the negative feedback mechanism is intact and active since Attenuated negative feedback on LH

OVULATION INDUCTION WITH LOW-DOSE FSH (Step-up) 60 40 LH mIU/ml 20 800 E2 pmol/l 600 400 200 25 When we treated PCOS patients with FSH in a low-dose step-up protocol, as E2 levels increased LH values declined and LH decreased further following ovulation. Messinis & Milingos 1997 Hum. Reprod. Update 3, 235-53 20 P4 nmol/l 15 10 3 9 15 21 27 CYCLE DAYS

ENDOGENOUS LH SURGE IN PCOS DURING OVULATION INDUCTION WITH FSH 60 (a) (b) 40 20 LH mIU/ml Messinis & Milingos 1997 Hum. Reprod. Update 3, 235-53 60 (c) (d) 40 That GnSAF is produced by the PCO ovaries is also derived from these data in which we induced ovulation in anovulatory women with PCOS and characterized the endogenous LH surge in 6-hourly blood samples. It is evident that in two of the 4 cases the LH surge was attenuated suggesting the production of GnSAF. 20 -24 24 48 72 -24 24 48 72 HOURS FROM ONSET OF LH SURGE

AMH AND OVARIAN RESPONSE TO HMG DURING OI IN PCOS 120 100% 100 80 Rate of good response to HMG treatment (%) 60 41% 40 20 0% AMH<4.7 (n=11) AMH=4.7-10.2 (n=17) AMH>10.2 (n=6) ng/ml Prospective Observational n=24 women 34 cycles Amer et al., 2013 Reprod. Biol. Endocrinol. Dec 17;11:115

Outline Definition Endocrine changes Follicle maturation - anovulation Co-morbidities

PCOS associated morbidities Obesity Type II diabetes mellitus Cardiovascular disease Infertility Endometrial cancer Psychological disorders

Obesity and PCOS Childhood obesity is a risk factor for PCOS Obese girls are at a higher risk for developing insulin resistance, metabolic syndrome and PCOS Women with PCOS are at a higher risk for developing obesity Exposure to excess prenatal androgens increases the possibility for PCOS-associated obesity Adolescents with PCOS are at risk for metabolic syndrome and impaired glucose tolerance, and the concomitant obesity compounds these risks.

Metabolic syndrome PCOS (n=84): 23.8% Controls (n=87): 8.0% Hudecova et al., 2011 Fertil. Steril. 96, 1271-4 Average age 43 y Long-term follow-up

Prevalence of Metabolic syndrome in women with PCOS Country U.S. White 52/184 (28.3%) U.S. Black 52/100 (52.0%)* India 84/220 (38.2%) Brazil 69/233 (29.6%) Finland 26/94 (27.7%) Norway 106/258 (41.1%)** Prevalence * P<0.001 (vs U.S. White) ** P<0.05 (vs U.S. White Cross-sectional study Chan et al., 2017 Am. J. Obstet. Gynecol. Apr. 8 (Epub ahead of print)

Conclusions There is still inconsistency regarding the definition of PCOS There are endocrine deviations from normal with interactions in the context of a vicious circle Folliculogenesis is disturbed leading to increased number of small antral follicles Various PCOS associated morbidities can lead to long-term sequelae