the polymorphism of fsh receptor gene in indonesian women undergoing assisted reproduction dwi anita...
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THE POLYMORPHISM OF FSH RECEPTOR GENE
IN INDONESIAN WOMEN UNDERGOING ASSISTED REPRODUCTION
DWI ANITA SURYANDARI
Structure of FSH reseptor (FSHR) moleculemember of the family of G-protein coupled receptors
The FSHR molecule consist of 3 domains
a. Extracellular domain
b. Transmembrane domain
c. Intracellular domain
a
b
c
Single gene of 2p21, 54 kbp, consisting 10 exons and 9 introns
Thre307Ala Asn680Ser
FSH Receptor gene
Objective:
1). To assess the incidence of different FSHR genotypes in Indonesian women undergoing assisted reproduction (anovulatory patients) and those with normo ovulatory controls
2). To correlate the FSHR genotypes with baseline serum FSH levels. and their response to exogenous FSH prior to super ovulation induction in anovulatory patients.
Design : Cross sectional study
Samples : 130 normoovulatory controls and 187 normogonadotropic anovulatory women who participated in assisted reproduction in Bunda Hospital, Jakarta.
Main outcome Measure(s): Prevalence of FSHR polymorphisms, Baseline serum FSH levels and dosage of FSH administered.
DNA ISOLATIONDNA AMPLIFICATION
PCR (Sampel DNA; PCR buffer 1X; 1,5 mM MgCl2; 0,2 mM dNTP; 0,4 pmol/µl
primer L8211 dan H8310; 0,25 unit enzim DNA polimerase Taq; dan ddH2O steril )
total volume 25 µl
95o C 1’
58o C 1’
72o C 5’
DenaturasiEkstensi
30 siklus
95o C 5’
4o C
72o C 1’30’’
Annealing
Denaturasi awal
Ekstensi akhir
ELECTROPHORESIS
agarosa /acrylamide
Loading:PCR produk (6 l produk PCR + 2 l Loading Buffer)
- DNA Marker
X174RF/HaeIII
Electrophoresis 80 V, 1,5 H
Visualisasi of DNA gel by Gel-Doc 1000 [Bio Rad]
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Determination of FSHR gene polymorphisms in 307 position by SSCP using 20% acrylamide gel.
Ala/Thre Ala/Ala Thre/Thre
0
10
20
30
40
50
60
70
AA AT TT
genotip gen FSHR(posisi307)
Pe
rse
nta
se
kontrol
WHO-2
0
10
20
30
40
50
60
SS AS AA
genotip FSHR (posisi 680)
pe
rse
nta
se
kontrol
WHO-2
The distribution of FSHR genotypes in anovulatory patients (n= 187) and normo-ovulatory controls (n=130) was significantly different for both genotypes (p< 0,05; Chi-square test).
The distribution of FSHR gene Polymorphisms in normoovulatory controls (n= 130) and in anovulatory
patients (n = 187)
44,24,44,64,8
55,25,4
Ala/Ala Ala/Thre Thre/Thre
Genotip FSHR (posisi 307)
Kada
r bas
al FS
H (IU
/L)
44,24,44,64,8
55,25,4
Ser/Ser Ser/Asn Asn/Asn
Genotip FSHR (posisi 680)
Kada
r FSH
bas
al (I
U/L)
The baseline serum FSH level in ser/ser genotypes was significantly different from Ser/Asn or Asn/Asn genotypes (p< 0,01; Anova).
The baseline serum FSH levels in normoovulatory controls
435024N =
genotip FSHR (307)
Thre ThreAla ThreAla Ala
kada
r FSH
(IU/
L)
16
14
12
10
8
6
4
2
0
The baseline serum FSH levels and the dosage of FSH stimulation was not significantly different among women with 307 FSHR genotypes (p> 0,05; Kruskal-Wallis test).
435024N =
genotip FSHR (posisi 307)
Thre ThreAla ThreAla Ala
jum
lah
ampu
l FSH
100
80
60
40
20
0
The baseline serum FSH levels and the dose of FSH stimulation in anovulatory patients with 307 FSHR genotypes
315036N =
genotip 680
Asn AsnSer AsnSer Ser
jum
lah
ampu
l FSH
100
80
60
40
20
0315036N =
Genotip FSHR (posisi 680)
Asn AsnSer AsnSer Ser
Kad
ar F
SH
IU/L
16
14
12
10
8
6
4
2
0
The baseline serum FSH levels and the dosage of FSH stimulation in women with Ser/Ser genotype was significantly higher than those in Ser/Asn and Asn/Asn genotypes in 680 position (p< 0,01; Kruskal-Wallis test).
The baseline serum FSH levels and the dosage of FSH stimulation in anovulatory patients with 680 FSHR genotypes
The ovarian response to FSH stimulation is dependent to the FSH receptor genotypes
• Each FSHR isoform may show different expression on the cell surface, determining the variation modes of interaction with the hormone.
• Each receptor genotypes may show different turnover or down regulation rate.
• Each FSHR isoform may have different response to FSH stimulation.
Gene expression test ?
CONCLUTIONS
1.There is no linkage of polymorphic nucleotide 307 with 680 of FSHR gene detectable in Indonesian women.
2. The FSHR genotypes of 680 position determine ovarium response to FSH stimulation.
* Polymorphism of FSHR gene not linkage between position 307 and 680, making 9 isoform.
01020304050
AS-AS
AS-AN
AS-TS
AS-TN
AN-AN
AN-TN
TS-TS
TS-TN
TN-TN
Kombin asi alel FSHR (posisi 307 & 680)
Pers
en
tase (
%)
Indonesia
As-Teng
Arab
Suriname
Belanda
Polymorphism of FSHR gene in Indonesian, South of Asia, Arabian, Surinames, and Holland women