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Developmental programming of
reproductive dysfunctioncontribution from environmental
steroid mimics
Vasantha Padmanabhan, ...
Relevance to Human
Reproductive Health
• Risk faced by female fetus whose mother has been
exposed to excess steroids for v...
Developmental Programming
Programming agents
Sex steroids
Nutrition
Drugs of abuse
Stress
Environmental pollutants

Concer...
Core Hypothesis
Exposure to excess native or
environmental sex steroids during
critical periods of development
produces ch...
Polycystic Ovary Syndrome
• PCOS is the most common
endocrinopathy affecting the health
status of reproductive age women.
...
Attributes of Women with PCOS
Attributes
Anovulation / Oligoovulation*

Women
with PCOS
Yes

Hyperandrogenism*
Hypergonado...
PCOS

Consequence of
Prenatal
Steroid
Excess?
Animal Model
Prenatal Steroid-treated Female
Sheep
Prenatal Programming
Control
Testosterone propionate
Testosterone propionate

Dihydrotestosterone
0 30 60
147
90
Days of G...
T30-90 Sheep Exhibit Progressive
Loss of Cyclicity
10

Progesterone profiles
(ng/mL)

Percentage of
ewes cycling
100

Cont...
Prenatal T Produces Adult
Hypergonadotropism (LH Excess)
2 year old Control and T-treated Ewes
194

247

224

270

276

24...
Prenatal T not DHT excess disrupts
ovarian morphology
(estrogenic)

Control

Testosterone

DHT
West et al., Mol Cell Endoc...
Prenatal T on follicular recruitment/depletion
5,000

80,000

40,000

*

Number of growing follicles

Number of primordial...
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

...
Fertility / Reproductive Behavior
Year 3 Estrus/Breeding Results
Estrussynchronizeda

40

80

40

Ram
marked
Control n=12; T-treated n=11

Pregnantb
aTwo

8...
Sites of Reproductive Disruption
testosterone

BRAIN
GnRH

E2
androgen

PITUITARY
gonadotropins

Other endocrine &
metabol...
PCOS vs. Prenatal T-treated Sheep
Women with
PCOS
Yes
Yes
Yes
Yes

Prenatal T-treated
sheep
Yes
Yes
Yes
Yes

Polycystic ov...
Environmental ‘Endocrine Disruptors’ (EDs)
Selective Steroid Receptor Modulators (SRMs)
Contraceptives

Siloxanes
Mycotoxi...
Bisphenol A
Methoxychlor
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
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 ...
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
...
Prenatal BPA Exposure Leads
to Growth Retardation
Control
6

BPA
45

45

*

*

40

*

4

40

cm

cm

5

MXC

35

0

0

Wei...
Prenatal Exposure to BPA
Leads to Early Hypergonadotropism
2 weeks
LH (ng/mL)

10

*

5

0
Control

MXC

BPA
Differential effects of prenatal MXC /BPA
on LH surge
Control

MXC

250

BPA

299

233

265

244

273

234

268

200

274
...
GnRH labeled area

Prenatal MXC / BPA reduces
hypothalamic GnRH Expression

*

Control

MXC

*

BPA
Prenatal MXC / BPA effects on
hypothalamic ER expression

ac

3V
mPOA

oc

ER-α signal +SEM

*

Control

MXC

BPA
Prenatal MXC / BPA effects on
hypothalamic ERb Expression

3V

mPOA

oc

ER-β signal +SEM

ac

*
*

Control

MXC

BPA
Insulin / glucose ratio

Prenatal exposure to BPA culminates
in maternal hyperinsulinemia
0.35

*

0.3
0.25

*

0.2
0.15
0...
Prenatal T vs. EDC-treated Sheep
Prenatal
T-treated

Prenatal
BPA-treated

Prenatal
MXC-treated

Hypergonadotropism

Yes

...
ADULT PHENOTYPE
Elements of the Primary Organizational Palette

Adult

Organizational Program
Phenotype

Epigenetic
Micro-...
Participants
Mohan Manikkam
Hirendranath Sarma
Teresa Steckler
Almudena Veiga-Lopez
Christine West

Farm Support
Douglas D...
Collaborators
• Neuroendocrine
 Douglas L. Foster
 Jane Robinson

• Behavior
 Theresa Lee

• Ovarian
 Keith Inskeep
 ...
Animal Models
Clinical translation
Daniel Dumesic
Teresa Sir-Petermann

Insuring
Human Health
DEVELOPMENTAL PROGRAMMING
Hormonal, nutritional, and metabolic environment
to which the offspring is exposed during
develo...
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
...
~ 40% Female Human Fetuses at Mid-Gestation Have Serum
Free Testosterone Levels in the Fetal Male Range

Beck-Peccoz et al...
Critical Periods of Reproductive Organ
Development and Differentiation
F = Follicle
Conception
Implantation
Gonadal differ...
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 di...
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 (...
Neuroendocrine defects underlying LH defects

Modified from Foster et al.

sensitivity to E2 negative feedback ( T/DHT, an...
Impact of of Excess Weight Gain on
Severity of Reproductive Disruptions in
Prenatal T-treated Sheep.
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

...
Luteal Progesterone Rise
18
508

542

Control

502

C-Obese

512

T-Treated

T-Obese

Progesterone (ng/mL)

9
0
533

553

...
2-step Programming
Prenatal
Testosterone
excess

Early life
reprogramming

Step 1

Postnatal weight gain

Second Step

Ste...
Developmental Programming of Reproductive Dysfunction- Contribution from Environmental Steroid Mimics
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Developmental Programming of Reproductive Dysfunction- Contribution from Environmental Steroid Mimics

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

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Transcript of "Developmental Programming of Reproductive Dysfunction- Contribution from Environmental Steroid Mimics"

  1. 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. 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. 3. Developmental Programming Programming agents Sex steroids Nutrition Drugs of abuse Stress Environmental pollutants Concerns Not easy to avoid Difficult to detect
  4. 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. 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. 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. 7. PCOS Consequence of Prenatal Steroid Excess?
  8. 8. Animal Model Prenatal Steroid-treated Female Sheep
  9. 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. 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. 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. 12. Prenatal T not DHT excess disrupts ovarian morphology (estrogenic) Control Testosterone DHT West et al., Mol Cell Endocrinol., 2001
  13. 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. 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. 15. Fertility / Reproductive Behavior
  16. 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. 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. 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
  19. 19. Environmental ‘Endocrine Disruptors’ (EDs) Selective Steroid Receptor Modulators (SRMs) Contraceptives Siloxanes Mycotoxins (Fumonisin B1) Isoflavones Lignans PCBs Bisphenol A Dioxins
  20. 20. Bisphenol A
  21. 21. Methoxychlor
  22. 22. 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
  23. 23. 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
  24. 24. 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
  25. 25. 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
  26. 26. Prenatal Exposure to BPA Leads to Early Hypergonadotropism 2 weeks LH (ng/mL) 10 * 5 0 Control MXC BPA
  27. 27. 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
  28. 28. GnRH labeled area Prenatal MXC / BPA reduces hypothalamic GnRH Expression * Control MXC * BPA
  29. 29. Prenatal MXC / BPA effects on hypothalamic ER expression ac 3V mPOA oc ER-α signal +SEM * Control MXC BPA
  30. 30. Prenatal MXC / BPA effects on hypothalamic ERb Expression 3V mPOA oc ER-β signal +SEM ac * * Control MXC BPA
  31. 31. 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
  32. 32. 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
  33. 33. ADULT PHENOTYPE Elements of the Primary Organizational Palette Adult Organizational Program Phenotype Epigenetic Micro-environmental Macro-environmental Toxicants/ Insults
  34. 34. Participants Mohan Manikkam Hirendranath Sarma Teresa Steckler Almudena Veiga-Lopez Christine West Farm Support Douglas Doop Carol Herkimer James Lee National Institute of Health
  35. 35. 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
  36. 36. Animal Models Clinical translation Daniel Dumesic Teresa Sir-Petermann Insuring Human Health
  37. 37. 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
  38. 38. 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
  39. 39. ~ 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
  40. 40. 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
  41. 41. 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
  42. 42. 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
  43. 43. 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
  44. 44. Impact of of Excess Weight Gain on Severity of Reproductive Disruptions in Prenatal T-treated Sheep.
  45. 45. 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
  46. 46. 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
  47. 47. 2-step Programming Prenatal Testosterone excess Early life reprogramming Step 1 Postnatal weight gain Second Step Step 2 Severity of Reproductive phenotype
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