GRF 2nd One Health Summit 2013: Presentation by Vasantha PADMANABHAN, University of Michigan, United States of America

1. Developmental programming of
reproductive dysfunctioncontribution from environmental
steroid mimics
Vasantha Padmanabhan, PhD.
Departments of Pediatrics, Obstetrics
and Gynecology, & Molecular and
Integrative Physiology and the
Reproductive Sciences Program
University of Michigan

2. Relevance to Human
Reproductive Health
• Risk faced by female fetus whose mother has been
exposed to excess steroids for variety of reasons:
 failed contraception and continued exposure
to contraceptive steroids
 use of anabolic steroids
 Industrial pollutants with androgenic/estrogenic activity - xeno
estrogens
 dietary estrogens – phytoestrogens
 disease (children of PCOS women)
• Small for gestational babies

3. Developmental Programming
Programming agents
Sex steroids
Nutrition
Drugs of abuse
Stress
Environmental pollutants
Concerns
Not easy to avoid
Difficult to detect

4. Core Hypothesis
Exposure to excess native or
environmental sex steroids during
critical periods of development
produces changes in postnatal
neuroendocrine, ovarian and
metabolic sites culminating in
reproductive /metabolic
dysfunctions in adult life.

5. Polycystic Ovary Syndrome
• PCOS is the most common
endocrinopathy affecting the health
status of reproductive age women.
• PCOS contributes towards
early-onset type II
diabetes, obesity,
atherosclerosis and
endometrial cancer.

6. Attributes of Women with PCOS
Attributes
Anovulation / Oligoovulation*
Women
with PCOS
Yes
Hyperandrogenism*
Hypergonadotropism
Reduced sensitivity to E2 / P4 neg.feedback
Yes
Reduced sensitivity to E2 pos. feedback
Yes
Increased sensitivity to GnRH
Yes
Polycystic ovaries*
Altered insulin sensitivity / Insulin resistance
Yes
Hypertension
Risk
Severity with obesity
*
Yes
Yes
Revised Rotterdam Criteria: 2 out of 3.
Yes
Yes

7. PCOS
Consequence of
Prenatal
Steroid
Excess?

8. Animal Model
Prenatal Steroid-treated Female
Sheep

9. Prenatal Programming
Control
Testosterone propionate
Testosterone propionate
Dihydrotestosterone
0 30 60
147
90
Days of Gestation
30-90 vs. 60-90:
Critical period
T vs. DHT:
Quality of Steroid
(androgenic vs. estrogenic)

10. T30-90 Sheep Exhibit Progressive
Loss of Cyclicity
10
Progesterone profiles
(ng/mL)
Percentage of
ewes cycling
100
Control
5
100
86
D60-90
71
71
0
10
100
0
0
100
5
0
10
0
100
D30-90
5
0
0
Aug Dec
1998
Apr Aug
1999
Dec Apr
2000
Apr Aug Dec Apr Aug Dec Apr
2000
1998
1999
Birch et al., Endocrinology 144:1426, 2003

11. Prenatal T Produces Adult
Hypergonadotropism (LH Excess)
2 year old Control and T-treated Ewes
194
247
224
270
276
240
20
LH (ng/ml)
10
0
20
10
0
0
2
4
0
2
Time (h)
4
0
2
4
Manikkam et al., BOR 2008

12. Prenatal T not DHT excess disrupts
ovarian morphology
(estrogenic)
Control
Testosterone
DHT
West et al., Mol Cell Endocrinol., 2001

13. Prenatal T on follicular recruitment/depletion
5,000
80,000
40,000
*
Number of growing follicles
Number of primordial follicles
120,000
*
*
4,000
3,000
2,000
1,000
*
0
0
D90
D140
10 months
Control
D90
T
D140
10 months
DHT
Smith et al., BOR 2009

14. Follicle Diameter (mm)
10
5
0
T
10
5
3
*
2
1
Zero
Number of 7 mm antral follicles
C
0
Year 2
3
2
1
0
Zero
Year 1
15
Number of >8 mm antral follicles
Prenatal T not DHT excess induces
follicular persistence (estrogenic)
0
D1
D2
D3
D4
D5
Days Scanned
D6
Manikkam et al., Endo., 2006
D7
D8
Control
T
Steckler et al., Endo., 2007
DHT

15. Fertility / Reproductive Behavior

16. Year 3 Estrus/Breeding Results
Estrussynchronizeda
40
80
40
Ram
marked
Control n=12; T-treated n=11
Pregnantb
aTwo
80
40
0
0
0
Breeding
Herd
Percentage
80
Percentage
Percentage
Estrussynchronizeda
First Service
Pregnancy Rate
injections of PGF2 11 d apart; 2 T-treated ewes/ram
on progesterone
Steckler et al., Thereogenology 2007
bBased

17. Sites of Reproductive Disruption
testosterone
BRAIN
GnRH
E2
androgen
PITUITARY
gonadotropins
Other endocrine &
metabolic changes
OVARY
androgen/E2
intra-follicular androgen
altered gene expression
primordial endowment
recruitment
incidences of atresia
Postnatal
E2
early f ollicular depletion
or ar rest
advance
prem ature ovarian fail ure

18. PCOS vs. Prenatal T-treated Sheep
Women with
PCOS
Yes
Yes
Yes
Yes
Prenatal T-treated
sheep
Yes
Yes
Yes
Yes
Polycystic ovaries
Yes
Yes
Increased follicular recruitment
Yes
Yes
Altered insulin sensitivity
Insulin resistance
Fetal growth retardation
Altered behavior
Hypertension
Yes
Yes
Yes
Yes
Yes
Visceral adiposity
Yes
Obesity amplification
Yes
Yes
Yes
Yes1
Yes
Yes2
Yes
(observational)
Yes
Attributes
Oligo / anovulation
Hyperandrogenism
Hypergonadotropism
 sensitivity to steroid feedback
1Spanish
cohort, 2Risk factor in PCOS

20. Environmental ‘Endocrine Disruptors’ (EDs)
Selective Steroid Receptor Modulators (SRMs)
Contraceptives
Siloxanes
Mycotoxins
(Fumonisin B1)
Isoflavones
Lignans
PCBs
Bisphenol A
Dioxins

21. Bisphenol A

22. Methoxychlor

23. Prenatal Programming
Cottonseed oil
MXC
(5 mg/kg/day i.m.)
BPA
(5 mg/kg/day i.m.)
0 30
147
90
Days of Gestation

24. BPA / MXC Levels Achieved
BPA: up to 18.9 ng in maternal and 9.2 ng in fetal blood
Schonfelder et al 2002 Environ Health Perspect 110:A703
MXC: 156 ng/g lipid in adipose fat of Spanish population
Botella et al. 2004, Env Res 96: 34

25. Circulating BPA levels in
maternal blood of U.S. women
BPA concentration (ng/mL)
10
8
6
4
2
0
<35 >35
<30 >30
Age
BMI
Range: <0.5 to 22.3 ng/mL
M
Sex
F

26. Prenatal BPA Exposure Leads
to Growth Retardation
Control
6
BPA
45
45
*
*
40
*
4
40
cm
cm
5
MXC
35
0
0
Weight
35
0
Height
Chest
Circumference

27. Prenatal Exposure to BPA
Leads to Early Hypergonadotropism
2 weeks
LH (ng/mL)
10
*
5
0
Control
MXC
BPA

28. Differential effects of prenatal MXC /BPA
on LH surge
Control
MXC
250
BPA
299
233
265
244
273
234
268
200
274
262
150
100
50
LH (ng/ml)
0
200
150
100
50
0
200
150
100
50
0
0
30
60
90
120 0
30
60
90
120 0
Time from PGF2 (hours)
30
60
90
120

29. GnRH labeled area
Prenatal MXC / BPA reduces
hypothalamic GnRH Expression
*
Control
MXC
*
BPA

30. Prenatal MXC / BPA effects on
hypothalamic ER expression
ac
3V
mPOA
oc
ER-α signal +SEM
*
Control
MXC
BPA

31. Prenatal MXC / BPA effects on
hypothalamic ERb Expression
3V
mPOA
oc
ER-β signal +SEM
ac
*
*
Control
MXC
BPA

32. Insulin / glucose ratio
Prenatal exposure to BPA culminates
in maternal hyperinsulinemia
0.35
*
0.3
0.25
*
0.2
0.15
0.1
0.05
0
D60
D80
Control
D130
BPA

33. Prenatal T vs. EDC-treated Sheep
Prenatal
T-treated
Prenatal
BPA-treated
Prenatal
MXC-treated
Hypergonadotropism
Yes
Yes
No
Cycle disruption
Yes
Yes
Yes
Dampened LH surge
Yes
Yes
No
Increased amplitude of E2
Yes
Yes
No
Delayed LH surge onset
Yes
No
Yes
Fetal growth retardation
Yes
Yes
No
Attributes
BPA = Bisphenol-A, a plasticizer & estrogen mimic; MXC = Methoxychlor, a pesticide & estrogen mimic

34. ADULT PHENOTYPE
Elements of the Primary Organizational Palette
Adult
Organizational Program
Phenotype
Epigenetic
Micro-environmental
Macro-environmental
Toxicants/
Insults

35. Participants
Mohan Manikkam
Hirendranath Sarma
Teresa Steckler
Almudena Veiga-Lopez
Christine West
Farm Support
Douglas Doop
Carol Herkimer
James Lee
National Institute of Health

36. Collaborators
• Neuroendocrine
 Douglas L. Foster
 Jane Robinson
• Behavior
 Theresa Lee
• Ovarian
 Keith Inskeep
 Peter Smith
• Insulin sensitivity
 Sergio Recabarren
 David Abbott
• Fetal measures
 P.S. MohanKumar
• Cardiac
 Gregory Fink

37. Animal Models
Clinical translation
Daniel Dumesic
Teresa Sir-Petermann
Insuring
Human Health

38. DEVELOPMENTAL PROGRAMMING
Hormonal, nutritional, and metabolic environment
to which the offspring is exposed during
development permanently "programs" many
aspects of development and subsequent
expression of physiology during adulthood.
Barker’s Hypothesis
FETAL ORIGIN OF ADULT DISEASE
Evolutionary terms
reflects benefits of plasticicty
in development

39. Plasma levels after prenatal
exposure to T
T (ng/ml)
1
Fetal
P=0.07
1.5
E2 (pg/ml)
40
*
*
0.5
0
*
20
*
0
*
10
Maternal
*
5
0
20
0
D65
D90
D140
Control
D65
D90
D140
T-treated

40. ~ 40% Female Human Fetuses at Mid-Gestation Have Serum
Free Testosterone Levels in the Fetal Male Range
Beck-Peccoz et al., J Clin Endocrinol Metab. 1991; 73:525
Cole et al., J Clin Endocrinol Metab. 2006; 91:3654

41. Critical Periods of Reproductive Organ
Development and Differentiation
F = Follicle
Conception
Implantation
Gonadal differentiation
Ovary clearly distinguishable with mitotically active oogonia
Development of hypophyseal portal vasculature
LH and FSH in circulation and pituitary
FSH in pituitary
Primordial follicle differentiation complete
Appearance of primary F
Appearance of FSH R & antral F
Birth (full complement of F)
0 14 30 40 50 55 75 90 100 110
Gestation day
135 147

42. Species Comparison of Critical Periods
I:
GD:
SM:
1:
2:
3:
:
SM
GD
12
I
4 6 13 20 2-5
3
17d
Implantation
Gonadal differentiation
Start of meiosis
Primordial follicles
Primary follicles
Antral follicles
Birth
Mice
I
GD
SM
1
14
30
55
75
2
110
90
3
135
150
Sheep
GD
I
9
SM
1
40
60
100
2
3
125
170
Rhesus Monkey
I
0
GD
SM
1
2
3
9
42-63
90
112
130
230
Human
Gestational age (days)
270

43. Prenatal T/DHT on E2 and LH
6
4
100
2
0
0
200
4
100
2
0
0
200
4
100
2
0
-100
0
Control
T
DHT
Estradiol (pg/mL)
200
LH (ng/mL)
300
0
100
Time relative to LH surge peak in controls
Veiga-Lopez et al., BOR 2009

44. Neuroendocrine defects underlying LH defects
Modified from Foster et al.
sensitivity to E2 negative feedback ( T/DHT, androgenic)
sensitivity to P4 negative feedback
LH
Excess
sensitivity to GnRH (T/DHT, androgenic)
sensitivity to E2 positive feedback (T, estrogenic)
LH surge
defect

45. Impact of of Excess Weight Gain on
Severity of Reproductive Disruptions in
Prenatal T-treated Sheep.

46. Body Weight
120
Weight (Kg)
C
C-Ob
T
T-Ob
60
Puberty
0
1
6
12
18
24
30
36
42
48
54
Age (weeks)
60
66
72
78
84
90
Steckler et al. Endocrinology, 2009

47. Luteal Progesterone Rise
18
508
542
Control
502
C-Obese
512
T-Treated
T-Obese
Progesterone (ng/mL)
9
0
533
553
520
514
572
555
525
518
562
604
582
546
565
602
535
551
611
527
521
9
0
9
0
9
0
9
0
9
0
0
6
12
0
6
12
0
6
Days from PGF2
12
0
6
12
18
Steckler et al. Endocrinology, 2009

48. 2-step Programming
Prenatal
Testosterone
excess
Early life
reprogramming
Step 1
Postnatal weight gain
Second Step
Step 2
Severity of
Reproductive
phenotype