DIPARTIMENTO DI MEDICINA MOLECOLARE...Carlo Foresta DIPARTIMENTO DI MEDICINA MOLECOLARE Servizio per...

Ipogonadismo e Osteoporosi non solo Testosterone

Carlo Foresta

DIPARTIMENTO DI MEDICINA MOLECOLARE Servizio per la Patologia della Riproduzione Umana

40% of cases: idiopathic

Higher peak bone mass, bone size and bone strength with respect to women

The association between 25(OH)D and total and free testosterone is linear at lower levels of 25(OH)D, reaching a plateau at higher levels.

1,362 male subjects Range: 40 and 75 years

3,369 male subjects Range: 40 and 79 years

Controls (41) Bilateral orchiectomized

patients (15)

25OH D (nmol/L) 74.9 ±38.8 30.2 ±16.3 *

*P < 0.05 vs controls

Clinical features of patients:

• Age matched with controls (34.8±6.4 vs 35.8±6.2 years)

• Radical orchiectomy for bilateral testicular cancer without chemo- or radiotherapy

• No nutritional derangements

• Properly compensated with testosterone-replacement therapy

CYP27A1

Minor Role

Nutritional (~10%)

Sun Exposure (~90%)

Sun Rays

Vitamin D

Chole (Ergo)-calciferol

CYP27B1

25OH D 1,25(OH)2 D

Other

Key Enzyme/Organ(s) for 25OH D Production

Vitamin D metabolism

Vitamin D is biologically inactive and need a two-step

hydroxylation at carbons 25 and 1 for activation.

25-hydroxylation

Takeyama et al. Science 1997

CYP2R1 CYP27A1

mitochondrial microsomal

CYP27A1

Vitamin D 25OH D

Inactivation of CYP27A1: Evidences from Human Models

?

CYP2R1

Vitamin D

25OH D

Role of CYP2R1 in Vit D Metabolism

!

CYP2R1 Genetic variants and risk of vitamin D insufficiency

Wang et al. Lancet 2010

CYP2R1

Vitamin D receptor and vitamin D metabolizing enzymes

are expressed in the human male reproductive tract.

Choudhary et al. Arch Biochem Biophys 2005

Blomberg Jensen M et al. Hum Reprod 2010

Foresta et al., JCEM 2011

Controls Severe Hypospermatogenesis

SCOS

OsteoPENIA 0/41 (0%) 3/36 (22.2%) 8/21 (38.1%)

OsteoPOROSIS 0/41 (0%) 2/36 (5.6%) 4/21 (19.0%)

Bone Disorders (Osteopenia + Osteoporosis)

0/41 (0%) 10/36 (27.8%)* 12/21 (57.1%)*

*P< 0.0001 vs controls

Testiculopathic men show low 25OH Vit D (due to low CYP2R1 activity) and high risk of osteoporosis/penia,

despite normal T levels (compensated hypogonadism)

Foresta et al., JCEM 2011

U.O. Patients Controls P

Subjects 125 41 n.s.

Age (Yr) 34.1 ± 6.1 35.8 ± 6.2 n.s.

Weight (Kg) 72.3 ± 7.1 70.2 ± 6.9 n.s.

Height (cm) 175.6 ± 6.9 174.9 ± 6.1 n.s.

Total Testosterone (nmol/L) 17.6 ± 4.9 16.6 ± 5.7 n.s.

17-b-Estradiol (pmol/L) 95.4 ± 33.9 89 ± 32 n.s.

0

2

4

6

8

10

12

14

16

18

20

Patients ControlsControls

*

*

FSH (IU/L)

LH (IU/L)

*P<0.00001

U.O. Patients

Unilateral Orchidectomy (U.O.)

Foresta et al., JEI 2012

Patients (125)

Controls (41)

P

25(OH)D3 (nmol/L) (n,v. 50–125 nmol/L) 41.6 ± 20.6 74.9 ± 38 < 0.00001

1-25(OH)2D3 (nmol/L) (n,v. 43–148 pmol/mL) 99.7 ± 35.1 95.3 ± 31.8 n.s.

PTH (ng/L) (n.v. 17–73 ng/L) 72.8 ± 28.6 49.5 ± 14.2 < 0.00001

BAP (μg/L) (n.v. 2.7–20.1 μg/L) 13.9 ± 4.7 11.6 ± 4.5 <0.01

ICTP (pg/mL) (n.v. <704 pg/mL) 392.4 ± 144.1 317.9 ± 95.7 <0.01

Table 2: Results of bone markers in 125 patients who underwent unilateral orchiectomy compared to healthy controls

Normal DEXA

Osteopenia T-score

< -1 and > 2.5 SD

Osteoporosis T-score < -2.5 SD

DXA Lumbar spine L1-L4

BMD (gr/cm2)

DXA femur

BMD (gr/cm2)

Patients n. 105 (%)

57/105 (54.3 %)

38/105 * (36.2%)

10/105# (9.5%)

1.003 ± 0.146* 0.981 ± 0.115*

Controls n. 41 (%)

41/41 (100%)

0/41 (0%)

0/41 (0%)

1.179 ± 0.119 1.151 ± 0.128

*P< 0.00001; # P< 0.05

Table 3: BMD values and number of subjects with bone disorders (osteopenia or osteporosis) in patient group compared to healthy controls.

Foresta et al., JEI 2012

48h

hCG (ng/mL) 0 2 10 50

50kDa

LH hCG

CYP2R1 Immunoblotting

0

2

4

6

8

10

12

0 2 10 50

2

6

4

12

8

10 M

ediu

m T

(n

M) *

0

5

10

15

0 2 10 50

120

80

40

Ban

d In

ten

sity

(A

.U)

*

hCG increases CYP2R1 expression in mouse Leydig cells

Foresta et al., JEI 2012

*P<0.05

0

20

40

60

80

100

120

140

HH Patients (basaline) HH PatientsBaseline 3 month-therapy

25OH VitD (nmol/L)

1,25(OH)2 VitD (nmol/L)

PTH (ng/L)

HH Patients (basaline)

HH Patients after treatment

(3 months)

P

T (nM) 5.6 ± 3.6 14.4 ± 4.1 <0.001

E2(pM) 66.2 ± 23.2 87.2 ± 28.4 <0.001

LH (UI/L) 0.6 ± 0.2 4.3 ± 3.5 <0.005

FSH (UI/L) 0.6 ± 0.3 5.3 ± 3.2 <0.001

*

*: P<0.05

10 HH patients: - 7 newly diagnosed - 3 free of therapy since at least 6 months

Low 25OH Vit D is associated with true and

compensated hypogonadism and its

levels might be increased by hCG treatment

HH patients pre and post 3 month-therapy with gonadotrophins

Foresta et al., JEI 2012

INSL3

Insulin like-factor 3

• INSL3 is a peptide hormone produced in a differentiation-dependent manner by the Leydig cells under the long-term Leydig cell-differentiation effect of LH

• INSL3 is a marker of Leydig cell function and differentiation status (T reflects the steroidogenic function)

• INSL3 is a better marker of Leydig cell function than T: low INSL3 is observed in true and compensated hypogonadism

Ferlin et al., 2006, 2008, 2009; Foresta et al., 2004; Bay et al., 2005, 2007; Wikström et al., 2006

Tes

tis

MG

63

Ost

eob

last

NC

139 bp

269 bp INSL3-Ex1-2

RXFP2-Ex2-3

Bone

bio

psy

Tes

tis

NC

Control RXFP2

Primary osteoblasts

199 bp

278 bp

Insl3-Ex1-2

Rxfp2-Ex1-3

Tes

tis

NC

Ost

eobla

sts

10d

Ost

eobla

sts

2d

Expression

MicroCT of the distal femur

3D reconstruction of distal femur

*P<0.01 vs WT and P<0.05 vs Rxfp2+/-

*

0

5

10

15

20

25

WT Rxfp2+/- Rxfp2-/-

BV

/TV

/%)

*

Histomorphometric analysis Lumbar spine L3-L4

Rxfp2-/- mouse

Rxfp2-/- mouse

Functional osteoblast impairment causing little bone formation, little mineralizing surface, and

osteoclast alterations

Negative balance between bone formation and bone resorption

Proliferation and differentiation Deposition of collagen Deposition of non collagenous protein Osteoclastogenesis

Alizarin red-S staining for mineralization

Femoral and L1-L4 DEXA

25 young adults (20-35 y) with RXFP2 mutations

10/25 pts <-1 SD (osteopenia) 6/25 pts <-2.5 SD (osteoporosis) 16/25 (64.0%) 0/51 age-matched ctrls

t-score:

P=0.0001

Ferlin et al., 2008

25 (OH) Vitamin D

CYP2R1 25α-hydroxylase

E2

DHT

T ER

AR

RXFP2

1,25(OH) 2 Vitamin D

Osteoblast apoptosis

Osteoblast proliferation and differentiation

Osteoclast activity

Osteoclast apoptosis

Osteoclast activity

Renal Ca++ excretion

Intestinal Ca ++ resorption Cholecalciferol

1α-hydroxylase

Leydig Cell

LH

+

+

+

Ferlin et al., submitted

L

Testosterone

CYP2R1 25OH Vit D

INSL3

Osteocalcin LH

L

Studies on testis-bone interactions identified new

markers of Leydig cell function

Normal T

Normal 25OH Vit D

Normal INSL3

Normal LH

L

Studies on testis-bone interactions identified new

markers of Leydig cell function

Low T

Low 25OH Vit D

Low INSL3

High LH (primary Hypogonadism)

or low LH (secondary Hypogonadism)

L

Studies on testis-bone interactions identified new

markers of Leydig cell function

Normal T

Low 25OH Vit D

Low INSL3

High LH

Higher LH levels are able to maintain T levels (compensated

hypogonadism) through its effect on steroidogenesis but not

25OH vit D and INSL3 levels

DIPARTIMENTO DI MEDICINA MOLECOLARE Sezione di Patologia Clinica &

Servizio per la Patologia della Riproduzione Umana

Alberto Ferlin

Riccardo Selice

Luca De Toni

Giacomo Strapazzon

Antonella Di Mambro

Sabina Magagna

Andrea Garolla

Lisa Gianesello

Lisa Perilli

Arianna Facciolli

Anastasia Pepe

Alexander Agoulnik (FIU, USA)

Roy Morello (UAMS, USA)

Manuela Zaccolo (Univ. Glasgow, UK)

Barbara Muciaccia (UniRoma1)

Sandro Giannini (DIMED-UniPD)

Leonardo Sartori (DIMED-UniPD)

Giuseppe Taglialavoro (DiSCOG-UniPD)

DIPARTIMENTO DI MEDICINA MOLECOLARE Servizio per la Patologia della Riproduzione Umana