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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