Vitamin D3 and Female Reproduction:

A Role Beyond Bone Health

Genevieve Neal-Perry, MD PhDAssociate Professor Ob/Gyn and Neuroscience

Fellowship Director, Division of Reproductive Endocrinology and Infertility Albert Einstein College of Medicine and Montefiore Medical Center

Objective1) To describe vitamin D3 physiology2) To discuss the differential impact of in utero

+ perinatal vs. pubertal vitamin D3 deficiency on a. pubertyb. reproductive physiology

i. Hypothalamusii. Pituitaryiii. Ovary

1) Fat soluble steroid hormone vitamin D3 steroid

hormone receptor (VDR)

RXR2) Genomic vs. non-genomic

Vitamin D3

classicalnon-classical

Calicidiol ½ life =15 d

Caltriol ½ life =15 h

UV-B 20-30 min 2x wk

Vitamin D3 Requirements Group FDA

Recommend Dose (IU)

Safe High FDA (IU)

Toxic Dose

Best Dose

Infants 0-12 months

400* <1000 1000-1500 400-800

Children 600* 2000 ≥3000- 4000

Pregnant Women

600* 2000

10,000 per day**

≥4000 800-1000 D3 /day

50,000 D2/2 week

50,000 D2/monthLactating Women

600*

Adults <50 600*Adults 51-70

600*

Adults >70 800**RDA and IOM recommendations **safe for at least 5 months

0

500

1000

1500

2000

Inte

rnat

ion

Units

(IU)

Sunshine Cod liver oil (tbsp)

Cooked salmon (3 ½ oz)

Milk(8 oz)

Cereal (8 oz)

Egg Yolk

Swiss cheese ( 1 oz)

Warning Label !To achieve the daily recommendation of 600 IU of vitamin D3 you have to eat:30 eggs = 50 cubes of Swiss cheese =120 oz of cereal = 6 cups milk

Dietary Sources of Vitamin D3

Food Serving Vitamin D (IU)

Cod Liver Oil 1 Tbsp 1360Cooked Salmon 3 ½ oz 360

Cooked Mackerel 3 ½ oz 345Canned Tuna Fish 3 oz 200

Canned Sardines 1 ¾ oz 250Fortified OJ 8 oz 100

Fortified Milk 8 oz 98Fortified cereal 8 oz 40

Egg (yolk) 1 egg 20

Health ImplicationsStatus Serum 25(OH)D3

nmol/liter ng/mlDeficient <30 <12Insufficient 30-49 16-19Adequate 50-124 20-49Toxic >125 >50

1) People with dark skin pigmentation African American > Hispanic > Caucasian

2) Obese individuals3) Elderly4) Reproductive-aged women, neonates,

and adolescents

Who is at Risk for Vitamin D3 Deficiency?

Vitamin D3 deficiencyRacial, Sex and Age

disparities

Reproductive-aged women and neonates represent one of the largest groups with vitamin D3

deficiency

Yetley, Am J Clin Nutr 2008:88(2): 558S National Health and Nutrition Examination Survey 2000-2004

%

<50

nmol

/L v

it D

3

Racial disparities in mean serum 25-OH D3 concentration from the National Health and

Nutrition Examination Survey 2000-2004

Yetley, E. A Am J Clin Nutr 2008;88:558S

Increasing weight and black race widens disparities in vitamin D3 deficiency

Why should Ob/Gyn MDs be concerned about sex and

racial disparities in vitamin D3 deficiency?

“2 out of every 3 pregnant women in the U.S. have suboptimal vitamin D

status, with even higher prevalences reported among African-American and

Hispanic women”[Looker, et al. Am J Clin Nutrition 2008]

Increased medical costs!!

Vitamin D3 Deficiency and Reproductive Health

Vitamin D3 deficiency

PCOS and Obesity

Recurrent pregnancy

loss

Infertility

Small for gestational

age

In the lab……

Overarching Objective

To determine the mechanism (s)by which VD3 regulates female

reproduction

Overarching Hypothesis

Developmental VD3 deficiency disrupts hypothalamic-pituitary-gonadal physiology in females

1) VDR and α-hydroxylase (cyp27b1) is widely distributed in reproductive tissue

2) Vitamin D3 is necessary for neuronal development

3) Vitamin D3 deficiency in humans is associated with reproductive dysfunction

4) Rodent models of vitamin D3 deficiencya. diet induced vitamin D3 deficiency in ratsb. Transgenic mice

Rationale

Objective #1

Determine the effect of developmental VD3 deficiency

on puberty

1) Transgenic cyp27b1 null mice

2) Wild type littermates

Experimental Model

Experimental Design

preconception perinatal

birth weaningon day 21

peripubertalintrauterine

conception 1) Vaginal opening2) 1st estrus3) Estrous cycling

Group 1: Control WT and KO (VD3 sufficient diet throughout)

Group 3: KO in utero and perinatal VD3 deficient diet Group 4: KO peripubertal VD3 deficient diet

Group 2

Group 3 Group 4

Group 2: KO in utero, perinatal and peripubertal VD3 deficient diet

Does in utero, perinatal and peripubertal VD3 deficiency

affect puberty?

Mice exposed to in utero, perinatal and peripubertal VD3 deficiency

have a delayed pubertyVO 1st estrus Days between VO

and 1st estrus

preconception perinatal

birth Weaning D21

peripubertalin utero

conception VO1st Estrus

Group 2

VD3 deficiency restricted to pregnancy and lactation does not

disrupt puberty.

preconception perinatal

birth weaning D21

peripubertalin utero

conception VO1st Estrus

Group 3

Will in utero and perinatal VD3 deficiency disrupt puberty?

VO 1st estrus Days between VO and 1st estrus

Peripubertal VD3 deficiency delays puberty.

Will peripubertal VD3 deficiency disrupt puberty?

preconception perinatal

birth Weaning D21

peripubertalIn utero

conception VO1st estrus

Group 4

VO 1st estrus Days between VO and 1st estrus

Modifiers of the pubertal transition

Modified from Roa et al., Molecular and Cellular Endocrinology 324(2010):87

Genetic background

Is the effect of peripubertal VD3 deficiency on puberty associated with the Cyp27b1 knockout?

VD3 deficiency does not significantly affect peripubertal growth curves

0 1 2 3 40.00

0.02

0.04

0.06WT (D+)KO (D+)KO (D-)

Postnatal growth

Postnatal age (wk)

BM

I (g/

mm

2 )

Objective #2To determine if VD3 deficiency affects estrous cyclicity

The mouse estrous cycle: the rodent equivalent of the menstrual cycle

Estrous Cycle Length

Aver

age

cycl

e le

ngth

(day

s)

WT KO(D-) KO(D+)0

5

10

15

Peripubertal VD3 deficiency extends the estrous cycle

*

Peripubertal VD3 deficiency disrupts the estrous cycle

* p<0.05

P (n

g/m

l)Pr

l (n

g/m

l)E 2

(pg/

ml)

LH (n

g/m

l)FS

H

(ng/

ml)

Estrus Diestrus-1 Diestrus-2 Proestrus Estrus

WT VD3+

Day

estro

us s

tage

5 10 15 20

D2

D1

E

PKO VD3-

Day

estro

us s

tage

5 10 15 20

D2

D1

E

P

KO VD3+

Day

estro

us s

tage

5 10 15 20

D2

D1

E

P

Peripubertal VD3 deficiency disrupts the estrous cycle

LH surge LH surge LH surge

Will VD3 supplementation restore the estrous cycle in adults exposed to peripubertal VD3 deficiency?

a, P<0.01 vs. WT; b, P<0.05 vs. D+

WT KO (D-) KO (D+)0

10

20

30

40

50 Diestrus I

% o

f tim

e sp

ent i

ndi

estr

us I

per 5

day

s

WT KO (D-) KO (D+)0

10

20

30

40

50 Diestrus II

a

% o

f tim

e sp

ent i

nD

iest

rus

II pe

r 5 d

ays

WT KO (D-) KO (D+)0

10

20

30

40

a

Proestrus

% o

f tim

e sp

ent i

npr

oest

rus

per 5

day

s

WT KO (D-) KO (D+)0

10

20

30

40

a

Estrus

% o

f tim

e sp

ent i

nes

trus

per

5 d

ays

(A) (B)

(C) (D)

KO (peripubertal Vit D3-)

Day

estr

ous

stag

e

0 5 10 15

D2

D1

E

P

KO (rescue Vit D3+)

Day

estr

ous

stag

e

0 5 10 15

P

E

D1

D2

(E) (F)

Female reproductive physiology

estradiolovarian egg development

Communication between the brain and the ovaries change with increasing reproductive age.

Progesterone

Objective #3

To determine if VD3 deficiency disrupts ovarian and/or hypothalamic-pituitary

physiology

VD3 deficiency is associated with arrested follicular development

KO (D-) + saline KO (D-) + PMSG + hCG

CL

CLCL

CL

CL

VD3 deficiency does not affect ovarian responsiveness to superovulation

Peripubertal VD3 deficiency does not affect diestrus

LH and FSH release?

N= 3-8

Follicle Stimulating Hormone

Is VDR expressed in GnRH neurons?

Negative ControlGT1-7 KidneyGT1-7 Kidney

VDR

Does peripubertal VD3 deficiency affect GnRH neuron density?

VD3 deficiency does not affect the number of GnRH neurons

GnR

H ir

cells

0

20

40

60

80

in uterolactationperipubertal

D+D+D+

D+D+D-

VD3+

VD3-

Peripubertal VD3 deficiency does not affect GnRH neuron density

1) Delays the puberty2) Reversibly disrupts estrous cyclicity 3) Does not primarily affect ovarian

physiology4) Does not affect GnRH neuron density5) Does not disrupt negative feedback

effects of estradiol during diestrus6) GT1-7 neurons express VDR

Summary of peripubertal VD3 deficiency

1) Changes in dietary habits and reduced sun exposure have resulted in near epidemic levels of VD3 insufficiency & deficiency

2) Vitamin D3 insufficiency & deficiency disproportionately affect reproductive aged women, neonates and adolescents thereby making these groups more susceptible to the adverse consequences of VD3 deficiency on the reproductive axis

Conclusion

AcknowledgmentsCary Dicken, MDJoe Davis, MDYan Sun, MD PhDJun Shu, MDDavelene Israel PhDAnne Etgen, PhDNanette Santoro, MDJohn Hardin MDStreamson Chua, MD PhD

SupportR21HD0663511 Zondek Award for Reproductive ResearchDepartment of Ob/Gyn Einstein College of Medicine

Cary Dicken REI fellow

Jun Shu, technician

Joe DavisREI fellow

Yan Sun Post Doc