Developmental programming of

reproductive dysfunction-

contribution 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

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

Developmental Programming

Programming agents

Sex steroids

Nutrition

Drugs of abuse

Stress

Environmental pollutants

Concerns

Not easy to avoid

Difficult to detect

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.

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.

Attributes of Women with PCOS

* Revised Rotterdam Criteria: 2 out of 3.

Attributes

Anovulation / Oligoovulation*

Hyperandrogenism*

Reduced sensitivity to E2 / P4 neg.feedback

Altered insulin sensitivity / Insulin resistance

Hypergonadotropism

Reduced sensitivity to E2 pos. feedback

Women

with PCOS

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Hypertension Risk

Polycystic ovaries*

Increased sensitivity to GnRH Yes

Severity with obesity Yes

PCOS

Consequence of

Prenatal

Steroid

Excess?

Animal Model

Prenatal Steroid-treated Female

Sheep

Prenatal Programming

Days of Gestation

0 30 60 90 147

Control

Testosterone propionate

Testosterone propionate

Dihydrotestosterone

30-90 vs. 60-90: Critical period

T vs. DHT: Quality of Steroid

(androgenic vs. estrogenic)

T30-90 Sheep Exhibit Progressive

Loss of Cyclicity

0

5

10

0

5

10

0

5

10

Control

D60-90

D30-90

Progesterone profiles

(ng/mL)

Dec Dec Apr Apr Aug Aug

1999 1998 2000

100

71

0

Birch et al., Endocrinology 144:1426, 2003

Dec Dec Apr Apr Aug Apr Aug 1999 1998 2000

Percentage of

ewes cycling

0

100

100

0

0

100

100

86

71

2 year old Control and T-treated Ewes

0

10

20

LH

(n

g/m

l)

0

10

20

270 240 276

0 2 4 0 2 4 0 2 4

Time (h)

194 224 247

Prenatal T Produces Adult

Hypergonadotropism (LH Excess)

Manikkam et al., BOR 2008

Prenatal T not DHT excess disrupts ovarian morphology

(estrogenic)

Control

Testosterone

DHT

West et al., Mol Cell Endocrinol., 2001

Prenatal T on follicular recruitment/depletion

D90 D140 10 months

0

40,000

80,000

120,000

Num

ber

of prim

ord

ial fo

llicle

s

*

D140 10 months

0

1,000

2,000

3,000

4,000

5,000

Num

ber

of gro

win

g f

olli

cle

s *

*

*

D90

Control DHT T

Smith et al., BOR 2009

Prenatal T not DHT excess induces

follicular persistence (estrogenic)

Manikkam et al., Endo., 2006

Folli

cle

Dia

mete

r (m

m)

0

5

10

15

C

Days Scanned

D1 D2 D3 D4 D5 D6 D7 D8

T

0

5

10

Nu

mbe

r of

7 m

m a

ntr

al fo

llicle

s

*

Zero

Zero

Nu

mbe

r of

>8 m

m a

ntr

al fo

llicle

s

0

2

3

1

0

2

3

1

Year 1 Year 2

Control DHT T

Steckler et al., Endo., 2007

Fertility / Reproductive Behavior

Year 3 Estrus/Breeding Results

Control n=12; T-treated n=11 aTwo injections of PGF2 11 d apart; 2 T-treated ewes/ram bBased on progesterone

0

40

80

Perc

enta

ge

Ram

marked

Estrus-

synchronizeda

0

40

80

Perc

enta

ge

Pregnantb

Estrus-

synchronizeda

0

40

80

Perc

enta

ge

First Service

Pregnancy Rate

Breeding

Herd

Steckler et al., Thereogenology 2007

Sites of Reproductive Disruption

BRAIN GnRH

e a r l y f o l l i c u l a r d e p l e t i o n o r a r r e s t

p r e m a t u r e o v a r i a n f a i l u r e advance

testosterone

androgen

E2

gonadotropins

OVARY androgen/E2

intra-follicular androgen

altered gene expression

primordial endowment

recruitment

incidences of atresia

Postnatal

E2

Other endocrine &

metabolic changes

PITUITARY

PCOS vs. Prenatal T-treated Sheep

Yes Yes Increased follicular recruitment

Yes Yes Polycystic ovaries

Yes Yes Obesity amplification

Yes

(observational) Yes Visceral adiposity

Yes2 Yes Hypertension

Yes Yes Altered behavior

Yes1 Yes Fetal growth retardation

Yes Yes Insulin resistance

Yes Yes Altered insulin sensitivity

Yes Yes sensitivity to steroid feedback

Yes Yes Hypergonadotropism

Yes Yes Hyperandrogenism

Yes Yes Oligo / anovulation

Prenatal T-treated

sheep

Women with

PCOS Attributes

1Spanish cohort, 2Risk factor in PCOS

Selective Steroid Receptor Modulators (SRMs)

Environmental ‘Endocrine Disruptors’ (EDs)

Contraceptives

Isoflavones

Mycotoxins (Fumonisin B1)

Dioxins

Siloxanes

Lignans

Bisphenol A

PCBs

Bisphenol A

Methoxychlor

Prenatal Programming

Days of Gestation

0 30 90 147

Cottonseed oil

MXC (5 mg/kg/day i.m.)

BPA (5 mg/kg/day i.m.)

MXC: 156 ng/g lipid in adipose fat of Spanish population Botella et al. 2004, Env Res 96: 34

BPA: up to 18.9 ng in maternal and 9.2 ng in fetal blood Schonfelder et al 2002 Environ Health Perspect 110:A703

BPA / MXC Levels Achieved

Sex

0

2

4

6

8

10

BMI Age

BP

A c

on

ce

ntr

atio

n (

ng

/mL

)

<35 >35 <30 >30 M F

Circulating BPA levels in

maternal blood of U.S. women

Range: <0.5 to 22.3 ng/mL

Prenatal BPA Exposure Leads

to Growth Retardation

Control MXC BPA

0

35

40

45

*

0

35

40

45

* c

m

cm

Chest

Circumference Height Weight

0

5

4

6

*

LH

(ng

/mL

)

Prenatal Exposure to BPA

Leads to Early Hypergonadotropism

Control MXC BPA

0

5

10

2 weeks

*

MXC BPA Control

Time from PGF2 (hours)

0 30 60 90 120

LH

(ng

/ml)

0 30 60 90 120 0 30 60 90 120

250

0

50

100

150

200

0

50

100

150

200

0

50

100

150

200

299

244

268

233

273

274

265

234

262

Differential effects of prenatal MXC /BPA

on LH surge

Control MXC BPA

GnR

H labele

d a

rea

* *

Prenatal MXC / BPA reduces

hypothalamic GnRH Expression

Control MXC BPA

ER

-α s

ignal +

SE

M

ac

mPOA

oc

3V

*

Prenatal MXC / BPA effects on

hypothalamic ER expression

Control MXC BPA

ER

-β s

ign

al +

SE

M

Prenatal MXC / BPA effects on

hypothalamic ERb Expression

*

*

mPOA

3V

oc

ac

Prenatal exposure to BPA culminates

in maternal hyperinsulinemia

Control BPA

Insulin

/ g

lucose r

atio

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

D60 D80 D130

*

*

Prenatal T vs. EDC-treated Sheep

BPA = Bisphenol-A, a plasticizer & estrogen mimic; MXC = Methoxychlor, a pesticide & estrogen mimic

Attributes

Hypergonadotropism

Cycle disruption

Dampened LH surge

Fetal growth retardation No

Prenatal

T-treated

Yes

Yes Yes Yes

Yes No

Yes Yes

Yes Yes

No

Prenatal

BPA-treated

Prenatal

MXC-treated

Delayed LH surge onset Yes No Yes

Increased amplitude of E2 No Yes Yes

Organizational Program

Macro-environmental

Micro-environmental

Epigenetic

Adult

Phenotype

Elements of the Primary Organizational Palette

ADULT PHENOTYPE

Toxicants/

Insults

Organizational Program

Adult

Phenotype

Farm Support

Douglas Doop

Participants

Mohan Manikkam

Hirendranath Sarma

Teresa Steckler

Almudena Veiga-Lopez

Christine West

Carol Herkimer

James Lee

National Institute of Health

Collaborators

• Neuroendocrine Douglas L. Foster Jane Robinson

• Ovarian

Keith Inskeep Peter Smith

• Fetal measures

P.S. MohanKumar

• Behavior Theresa Lee

• Insulin sensitivity Sergio Recabarren David Abbott

• Cardiac

Gregory Fink

Animal Models

Insuring

Human Health

Clinical translation

Daniel Dumesic

Teresa Sir-Petermann

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

0

0.5

1

1.5

D65

Maternal

Fetal

D90 D140

T (ng/ml) E2 (pg/ml)

0

20

40

D65 D90 D140 0

5

10

0

20

P=

0.0

7

*

Plasma levels after prenatal

exposure to T

*

* *

*

*

Control T-treated

~ 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

Critical Periods of Reproductive Organ

Development and Differentiation F = Follicle

Primordial follicle differentiation complete

Conception

Implantation

Gonadal differentiation

Development of hypophyseal portal vasculature

FSH in pituitary

Appearance of primary F

Birth (full complement of F)

Ovary clearly distinguishable with mitotically active oogonia

LH and FSH in circulation and pituitary

Appearance of FSH R & antral F

Gestation day

0 147 75 30 14 50 110 55 135 90 40 100

Human

Rhesus Monkey

Mice

Sheep

0 9

9

14

42-63

40

30

60 100

130

125

90 135 110

I GD

I GD

I GD

SM

SM

SM

55 75

1 2 3

1 2 3

1 2 3

I

GD

SM

1 2 3

Gestational age (days)

112 90 230

I: Implantation

GD: Gonadal differentiation

SM: Start of meiosis

1: Primordial follicles

2: Primary follicles

3: Antral follicles

: Birth

150

170

270

4 6 13 20 2-5 17d

Species Comparison of Critical Periods

Prenatal T/DHT on E2 and LH

Control

DHT

T

LH

(n

g/m

L)

Estr

adio

l (p

g/m

L)

-100 0 100

Time relative to LH surge peak in controls

100

200

0

2

4

100

200

0

2

4

0

0

0

100

200

0

2

4

300 6

Veiga-Lopez et al., BOR 2009

Neuroendocrine defects underlying LH defects

Modified from Foster et al.

sensitivity to E2 negative feedback ( T/DHT, androgenic)

sensitivity to P4 negative feedback

sensitivity to GnRH (T/DHT, androgenic)

LH

Excess

sensitivity to E2 positive feedback (T, estrogenic)

LH surge

defect

Impact of of Excess Weight Gain on

Severity of Reproductive Disruptions in

Prenatal T-treated Sheep.

0

60

120

Weig

ht

(Kg)

Age (weeks)

1 6 12 18 24 30 36 42 84 66 48 72 60 54 90 78

C C-Ob T T-Ob

Body Weight

Puberty

Steckler et al. Endocrinology, 2009

6 12 0

535

502

520

525

582

527

T-Treated

6 12 0 18

514

518

521

551

546

512 T-Obese

Luteal Progesterone Rise

Days from PGF2

Pro

geste

rone (

ng/m

L) 0

9

18

0

9

0

9

0

9

0

9

0

9

6 12 0 6 12 0

565

508

533

572

562

Control

604

542

602

553

555

611

C-Obese

Steckler et al. Endocrinology, 2009

2-step Programming

Prenatal

Testosterone

excess

Step 1

Severity of

Reproductive

phenotype

Step 2

Postnatal weight gain

Early life

reprogramming

Second Step