Stress and Infertility Kevin O’Byrne Division of Women’s Health Faculty of Life Sciences and Medicine

HPA axis HPG axis Stressors sympathoadrenal system (locus coeruleus)

Republican senate hopeful, Todd Akin: “If it’s legitimate rape, the female body has ways to try to shut that whole thing down, and does not cause pregnancies” X BBC News, 20th August 2012

Functional hypothalamic hypogonadism stress GnRH GnRH

LH pulses and Functional hypothalamic amenorrhoea Sam Yen 1989 LH (mIU/ml)

FHA: [Exclude: depression, eating/psychiatric disorders or excessive exercise] (Menses) (88%) (25%) Sarah Berga et al. Fertility & Sterility 2003

Serum cortisol, serum free cortisol index (total cortisol/CBG), CSF cortisol, and CSF CRH in 15 women with FHA and 14 EW [cort pulse amp ↑] Brundu, B. et al. J Clin Endocrinol Metab 2006;91:1561-1565

Negative feedback - + Negative feedback Negative feedback

Intravenous injection of corticosterone results in brief elevation of circulating corticosterone and differential nuclear retention patterns of hippocampal GR and MR Corticosterone pulses throughout the active phase of the circadian cycle in the rat Conway-Campbell, B. L. et al. Endocrinology 2007;148:5470-5477

Model of pulsatile corticosterone regulation of GRs in the hippocampus Rapid proteasome-dependent clearance of activated GRs, but not MRs, provides a novel mechanism to allow dynamic interaction with rapidly changing physiological and environmental conditions. Conway-Campbell, B. L. et al. Endocrinology 2007;148:5470

Ultradian hormone stimulation induces glucocorticoid receptor-mediated pulses of gene transcription Stavreva DA et al 2009, Nat Cell Biol, 11:1093-102

Clinically recognised forms of stress- induced reproductive dysfunction: Functional hypothalamic amenorrhoea:- psychosocial Anorexia (bulimic) nervosa:- nutritional compromise Exercise-associated:- excessive exercise Growing recognition that each of these syndromes develops in response to exposure to combinations of psychogenic and metabolic stressors

Functional hypothalamic amenorrhoea Mild indices of “psychological stress” Dysfunctional attitudes Unrealistic expectations Higher levels of perfectionism Higher need for social approval Difficulty coping with daily hassles Mild metabolic deficits Higher incidence of subclinical eating abnormalities exercising

Agonistic interaction between female conspecifics O’Byrne KT et al. Physiol Behav. 1989, 45:1077-88

Schreihofer DA et al., Endocrinology 1996, 137:3770 Feeding-induced stimulation of pulsatile LH secretion (48 h) Schreihofer DA et al., Endocrinology 1996, 137:3770

Exercise-induced amenorrhoea Cynomolgus monkey Williams, N. I. et al. Endocrinology 2001

Schematic diagram of the experimental design (monkey model of FHA) (Psychosocial stress) Group 1 Group 2 Group 3 Williams, N. I. et al. Am J Physiol Endocrinol Metab 293: E270-E276 2007

[Subthreshold stressors synergise to compromise reproductive function] Percentage of monkeys showing abnormal menstrual cycles (i.e., either cycles >44 days in length or anovulatory cycles) in E3+4 for each of the 3 experimental groups [Subthreshold stressors synergise to compromise reproductive function] Williams, N. I. et al. Am J Physiol Endocrinol Metab 293: E270-E276 2007

Variation of serum cortisol during ovarian stimulation for in vitro fertilization Tica VI et al., Gynaecol Endo 2008, 24:12-17

Kate Silverton conceived 'miracle baby' naturally just weeks after stopping IVF Kate spent two years undergoing IVF treatment, and after four failed attempts thought she had lost all hope of becoming a mother. So it’s little wonder Kate looks overjoyed as she shows off the baby she never thought she would have. Dailymail: 23rd January, 2012

CRF neurones and fibres BNST Amygdala BNST = Bed Nucleus of the Stria Terminalis LC = Locus Coeruleus Swanson LW et al 1983 MPO = Medial Preoptic Area

CRF receptors Urocortins

Effect of CRF on Multi-Unit Activity (MUA) volleys and LH pulses in the monkey Williams , C.L. et al. Neuroendocrinology 1990

Effect of icv urocortin-II on pulsatile LH secretion in the rat Li, X. F. et al. Endocrinology 2005

Effects of the selective type 2 CRF receptor antagonist, astressin2-B, on restraint stress-induced suppression of LH pulsatile secretion in the rat Li, X. F. et al. Endocrinology 2005;146:318-322

Differential role for CRF receptors in stress-induced suppression of LH Psychological stress (eg. restraint): CRF-R1 and CRF-R2 Metabolic stress (eg. insulin-induced hypoglycaemia): CRF-R2 Immunological stress (eg. lipopolysaccharide): CRF-R2 Site of action?

Effects of intra-medial preoptic area CRF antagonist (AST) Treatment groups AST aCSF LH pulse interval (min) 10 20 30 40 50 60 70 Baseline (2 h) Restraint (1 h) Post restraint (3 h) # * LH (ng/ml) Time (h) 2 4 6 [Restraint stress]

LH pulse interval (min) Intra-mPOA Urocortin II or CRH Treatment groups aCSF 0.01µg 0.1µg 1µg 0.2µg CRH LH pulse interval (min) 10 20 30 40 50 * UCN II base line (2 h) Post treatment (1st 2 h) Post treatment (2nd 2 h)

Extended amygdala

Effect of intra-BNST CRF antagonist Li XF et al. 2011 J Neuroendocrinology 23, 3-11

Effect of neurotoxic lesion of medial amygdala on stress-induced suppression of LH pluses Lin Y etal 2011 Endocrinology

Locus Coeruleus

Effects of intra-locus coeruleus CRF on LH pulses Jackie Mitchell et al. Endocrinology 2005

Restraint Hypoglycaemia Mitchell, J. C. et al. Endocrinology 2005

Increased CRF to locus coeruleus in macaque model of functional hypothalamic amenorrhoea Bethea CL et al 2013, Neurobiology of Disease. 50, 96-106 High stress resilience (HSR) Stress sensitive (SS)

Bethea CL et al., Mol Neurobiol 2008 38:199 Individual stress-sensitivity & reproductive dysfunction in female macaques Schematic diagram of experimental design High stress resilience (HSR) Medium stress resilience (MSR) Stress sensitive (SS) Bethea CL et al., Mol Neurobiol 2008 38:199

Bethea CL et al., Mol Neurobiol 2008 38:199 GnRH expression – hypothalamus HSR>MSR>SS Serotonin gene expression - Raphae: HSR>MSR>SS Number of serotonin neurones - Raphae: HSR>MSR>SS GABA expression – hypothalamus SS>MSR>HSR CRH expression – hypothalamus/amygdala SS>MSR>HSR Bethea CL et al., Mol Neurobiol 2008 38:199

Representative LH pulse patterns in the early follicular phase of a non-stressed, control menstrual cycle. highly stress-resilient stress-sensitive Representative LH pulse patterns in 1 highly stress-resilient (HSR; A) and 1 stress-sensitive (SS; B) monkey over an 8-h period in the early follicular phase of a nonstressed, control menstrual cycle. *Pulses defined by PULSAR algorithm. C: mean LH pulse frequency measured in the early follicular phase of a nonstressed, control menstrual cycle in HSR, medium stress-resilient (MSR), and SS monkeys. Herod S M et al. Am J Physiol Endocrinol Metab 2011;300:E19-E27 ©2010 by American Physiological Society

Reverse with CRF-R1 antagonist Non-stressed (open bar) Mild combined stress: move + diet (black bar) (ACUTE) Reverse with CRF-R1 antagonist LH pulse frequency measured during daytime hours (1000–1800) both in the early follicular phase of a control, nonstressed menstrual cycle (open bars) and on the same day in a cycle with mild combined stress (move + diet) in HSR, MSR, and SS monkeys (A) and in HSR vs. animals that became anovulatory in response to mild combined stress (MSR + SS; B). *P < 0.05. Herod S M et al. Am J Physiol Endocrinol Metab 2011;300:E19-E27 ©2010 by American Physiological Society

stress - CRF GnRH Amygdala-BNST CRF-R? Locus Coeruleus CRF-R2 (Both Psychol, but NOT metabolic) stress - CRF-R1 (Psychol) Medial Pre-optic Area GnRH

Putative circuitry leading to the suppression of pulsatile GnRH/gonadotropin secretion (shown in green) by immunologic stress (iv injection of bacterial LPS; blue lines), psychogenic stress (confining the animal inside a restraint tube; red lines), or metabolic stress (insulin induced hypoglycemia; yellow lines). Putative circuitry leading to the suppression of pulsatile GnRH/gonadotropin secretion (shown in green) by immunologic stress (iv injection of bacterial LPS; blue lines), psychogenic stress (confining the animal inside a restraint tube; red lines), or metabolic stress (insulin induced hypoglycemia; yellow lines). Circuits at the top of the diagram, above the horizontal dotted line, are those directly inferred from the experiments of Lin et al. (1) in which either the medial or the central nucleus of the amygdala was chemically lesioned or the effects of stress on Fos expression in these nuclei were measured. The circuits leading to GnRH suppression by hypoglycemia are unaffected by lesions in either amygdalar location, and the data supporting their routings are derived from other studies as cited in the text. The graph in the lower right corner shows the temporary interruption of episodic LH secretion after LPS injection in an MeA lesioned animal [Fig. 2G in Lin et al (1)]. Redrawn from several sources, including Ulrich-Lai and Herman (2) and Feder et al. (3). Merriam G R , Koenig J I Endocrinology 2011;152:340-342

Kiss & Reproduce “I’d rather remain a frog thanks”

Kisspeptin neurones may act as central processors for relaying signals to GnRH neurones 2003 Dungan, H. M. et al. Endocrinology 2006, modified

Kisspeptin signalling and the reproductive neuroendocrine axis Medial Preoptic area Dungan, H. M. et al. Endocrinology 2006

Distribution of GFP expression with kisspeptin immunoreactiviy in Kiss-GFP mice. de Croft S et al. Endocrinology 2012;153:5384-5393 AVPV ARC GFP Kiss & Kiss Distribution and correlation of GFP expression with kisspeptin immunoreactiviy in Kiss-GFP mice. A–C, Distribution of GFP (A) and kisspeptin-immunoreactive (B) cells in the AVPV of adult female mouse with overlay in C. 3V, Third ventricle. D, Distribution of GFP cells in the preoptic area (POA) of an adult male mouse. E, Distribution of GFP cells in the posterior hypothalamus (PH) and adjoining brain regions including the periaqueductal gray (PAG) and premammillary nuclei (PMN) in an adult female mouse. FR, Fasciculus retroflexus. F and G, Distribution of GFP cells (F) and kisspeptin immunoreactivity (G) in the ARN of an adult female mouse, with overlay in H. Scale bar, 50 μm (A, D, and F) and 150 μm (E).

GnRH and Kisspeptin neurones in the rhesus monkey 3V VHT ARC ME GnRH KP Scale bar - 100 µm Ramaswamy S et al., Endocrinology, 2008

Medial Preoptic area Arcuate nucleus Kisspeptin antagonist & GnRH pulse generator frequency LH pulse intervals (min) 20 40 60 80 Controls Peptide 234 (pmol) 50 X 3 10 X 3 2h Baseline 1st h after Peptide 234 / aCSF 2nd + 3rd h post Peptide 234 / aCSF 2 4 6 Time (min) 120 180 240 300 LH ng/ml Peptide 234 10pmol x 3 Peptide 234 50pmol x 3 aCSF 500nl x 3 A B C D Medial Preoptic area Time (min) 60 120 180 240 300 2 4 6 Peptide 234 50pmol x 3 LH ng/ml Peptide 234 10pmol x 3 aCSF 500nl x3 LH pulse intervals (min) 20 40 80 Controls Peptide 234 (pmol) 50 X 3 10 X 3 * 2h Baseline 1st h after Peptide 234 / aCSF 2nd + 3rd h post Peptide 234 / aCSF # A B C D Arcuate nucleus Li XF. et al. PlosOne 2009, 4:e8334

Kisspeptin neurones may act as central processors for relaying signals to GnRH neurones STRESS / CRF? Dungan, H. M. et al. Endocrinology 2006, modified

stress CRF Kisspeptin + Indirect BNST-Amygdala Locus Coeruleus GnRH

? Kisspeptin signalling and the reproductive neuroendocrine axis Dungan, H. M. et al. Endocrinology 2006 Kisspeptin signalling and the reproductive neuroendocrine axis Stress CRF Corticosterone ?

LH pulse interval (min) 10 A Saline B Restraint 8 2 h baseline 6 1st h post-stress or corresponding control period 2nd + 3rd h post-stress or corresponding control period 4 2 G LH pulse interval (min) * 10 20 30 40 50 Control Restraint Insulin LPS 10 C Saline D Insulin 8 LH (ng/ml) 6 4 2 10 E Saline F LPS 8 6 4 2 1 2 3 4 5 1 2 3 4 5 Time (h) Kinsey-Jones. J. et al. J Neuroendocrinology 2009

Ratio Kiss1r mRNA:28S rRNA mPOA ARC 0.030 1.2 * * * * 0.025 1.0 0.020 0.8 P=0.055 Ratio Kiss1 mRNA:28S rRNA 0.015 0.6 # 0.010 # 0.4 0.005 0.2 0.000 * 0.0 * * * * 0.16 0.10 0.08 0.12 Ratio Kiss1r mRNA:28S rRNA 0.06 0.08 0.04 0.04 0.02 0.00 0.00 Control Control Control Restraint IIH LPS Control Control Control Restraint IIH LPS Kinsey-Jones. J. et al. J Neuroendocrinology 2009

Ratio Kiss1r mRNA:28S rRNA 0.00 0.01 0.02 0.03 0.04 0.05 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.06 Control CRF mPOA ARC Ratio Kiss1 mRNA:28S rRNA Ratio Kiss1r mRNA:28S rRNA A B * Kinsey-Jones. J. et al. J Neuroendocrinology 2009

Effect of acute corticosterone on LH secretion

Hypoglycaemic stress & GnRH pulse generator in rhesus monkey Chen MD etal Neuroendocrinology, 1992,56,666-73

Effect of chronic corticosterone on LH secretion amplitude (ng/ml) LH pulse 4 1 2 3 10 20 Control CORT LH (ng/ml) 200 400 600 30 GnRH Time (h) 5 6 (ng/ml.min) LH interval (min) 60 120 180 240 300 360 8 12 CORT 225 ng/ml CORT 51 ng/ml Effect of chronic corticosterone on LH secretion

Ratio Kiss1r mRNA:28S rRNA B 0.005 0.010 0.015 0.020 0.025 0.030 0.035 0.040 0.045 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 mPOA 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.09 ARC Control Chronic CORT Acute CORT C D * Ratio Kiss1 mRNA:28S rRNA Ratio Kiss1r mRNA:28S rRNA Kinsey-Jones. J. et al. J Neuroendocrinology 2009

mPOA ARC mPOA (most) ARC (most) Colocalisation of CRF receptor in kisspeptin neurones in the medial preoptic area (mPOA) and arcuate (ARC) of rats mPOA ARC mPOA (most) ARC (most) Takumi K etal., 2012. Neuroscience Letters. 531, 40 - 45

mPOA ARC mPOA most ARC few Double fluorescence immunolocalization of GR and kisspeptin. Low-power views of AVPV/PeN (A) and ARC (B) showing kisspeptin (red) and GR immunoreactivity (green) with DAPI cell nuclear staining (blue). mPOA ARC mPOA most ARC few Takumi K etal., 2012. Neuroscience Letters. 531, 40 - 45