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

2. HPA axis
HPG axis
Stressors sympathoadrenal system (locus coeruleus)

3. 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

4. Functional hypothalamic hypogonadism
stress
GnRH
GnRH

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

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

7. 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:

8. Negative feedback - +
Negative feedback
Negative feedback

9. 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:

10. 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

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

12. 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

13. 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

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

16. 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

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

18. 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-E

19. [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-E

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

23. 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

24. 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

25. CRF
receptors
Urocortins

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

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

28. 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:

29. 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?

30. 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]

31. 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)

32. Extended amygdala

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

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

35. Locus Coeruleus

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

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

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

40. 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 :199

41. 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 :199

42. 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

43. 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

44. 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

45. 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:

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

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

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

49. Distribution of GFP expression with kisspeptin immunoreactiviy in Kiss-GFP mice.
de Croft S et al. Endocrinology 2012;153:
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).

50. GnRH and Kisspeptin neurones in the rhesus monkey3V
VHT
ARC ME
GnRH KP
Scale bar µm
Ramaswamy S et al., Endocrinology, 2008

51. 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 pmol x 3
Peptide pmol x 3
aCSF 500nl x 3 A B C D
Medial Preoptic area
Time (min) 60
120
180
240
300 2 4 6
Peptide pmol x 3
LH ng/ml
Peptide pmol 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

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

53. stress CRF Kisspeptin + Indirect BNST-Amygdala Locus Coeruleus GnRH

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

55. 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

56. 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

57. 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 Kiss mRNA:28S rRNA
Ratio Kiss1r mRNA:28S rRNA A B *
Kinsey-Jones. J. et al. J Neuroendocrinology 2009

58. Effect of acute corticosterone on LH secretion

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

60. 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

61. Ratio Kiss1r mRNA:28S rRNAB
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

62. 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., Neuroscience Letters. 531,

63. 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., Neuroscience Letters. 531,