THE POLYMORPHISM OF FSH RECEPTOR GENE

IN INDONESIAN WOMEN UNDERGOING ASSISTED REPRODUCTION

DWI ANITA SURYANDARI

ROLE

OF

FSH

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

Asn/Asn Ser/Ser Ser/Asn

PCR-RFLP of FSHR gene in 680 position by 2% gel agarose

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