This study investigated the association between a single nucleotide polymorphism (G-29A) in the promoter region of the follicle-stimulating hormone receptor (FSHR) gene and FSHR expression levels in granulosa cells. The researchers found that 72% of subjects with the AA genotype were poor ovarian responders to FSH stimulation during in vitro fertilization treatment. Subjects with the AA genotype also had significantly reduced FSHR mRNA and protein expression in granulosa cells compared to those with the GG genotype. This suggests that poor ovarian response in subjects with the AA genotype is due to reduced FSHR expression at both the transcriptional and translational levels.

1. Follicle-Stimulating Hormone Receptor Polymorphism
(G؊29
A) Is Associated with Altered Level of Receptor
Expression in Granulosa Cells
Swapna S. Desai, Swati K. Achrekar, Bhakti R. Pathak, Sadhana K. Desai,
Vijay S. Mangoli, Ranjana V. Mangoli, and Smita D. Mahale
National Institute for Research in Reproductive Health (S.S.D., S.K.A., B.R.P., S.D.M.), Mumbai 400 012,
India; and Fertility Clinic and IVF Center (S.K.D., V.S.M., R.V.M.), Mumbai 400 007, India
Context: Polymorphisms of the FSHR gene are associated with variable ovarian response to FSH
stimulation in subjects undergoing in vitro fertilization (IVF) treatment. The type of ovarian re-
sponse is correlated with the level of FSH receptor (FSHR) expression on granulosa cells.
Objective:WeinvestigatedwhetherthepolymorphismatpositionϪ29inthepromoteroftheFSHR
gene may contribute in altered receptor expression.
Design and patients: FSHR polymorphism at position Ϫ29 was studied in 100 subjects undergoing IVF
treatment. Association of this polymorphism with level of FSHR expression was retrospectively analyzed.
Setting: The study was conducted at an academic research institute and private IVF clinic.
Methods: The genotype at position Ϫ29 of the FSHR gene was studied in IVF subjects by PCR-
restriction fragment length polymorphism. Total RNA and protein was extracted from granulosa
cells. The relative FSHR mRNA expression was carried out by real-time PCR. The receptor protein
expression was evaluated by Western blot and confocal microscopy.
Results:Theclinicalandendocrinologicalparametersrevealedthatalmost72%ofsubjectswiththe
AA genotype at position Ϫ29 of FSHR gene were poor ovarian responders (odds ratio 8.63, 95%
confidential interval 1.84–45.79; P ϭ 0.001). The lower cleavage intensity predicted by in silico
analysis for A allele as compared with the G allele suggest the difference in the DNA-protein
binding affinity. The relative expression of FSHR at mRNA and protein level was significantly
reduced in subjects with AA genotype as compared with the GG genotype.
Conclusion: Poor ovarian response observed in subjects with the AA genotype at position Ϫ29
of the FSHR gene is due to reduced receptor expression. (J Clin Endocrinol Metab 96: 2805–2812,
2011)
In in vitro fertilization (IVF) treatment, FSH is adminis-
tered for superovulation. Several studies have shown in-
terindividual variability in the type of ovarian response to
FSH stimulation during IVF treatment. Approximately
9–24% of women undergoing IVF treatment respond
poorlytogonadotropinstimulation(1).Suchsubjectsmay
have high basal FSH levels, require high exogenous FSH
dose for ovulation induction, demonstrate low serum es-
tradiol levels, and fewer mature oocytes during IVF treat-
ment. The basis of low response to gonadotropin admin-
istration during ovarian stimulation still remains an
enigma. Several endocrine and ultrasound parameters
such as d 3 serum FSH concentration (2, 3), poor follicular
blood flow (4), age and diminished ovarian reserve (5),
presence of ovarian antibody (6), and serum anti-Mulle-
rian hormone levels (7) have been used as indicators to
ISSN Print 0021-972X ISSN Online 1945-7197
Printed in U.S.A.
Copyright © 2011 by The Endocrine Society
doi: 10.1210/jc.2011-1064 Received March 22, 2011. Accepted June 24, 2011.
First Published Online July 13, 2011
Abbreviations: cETS-1, Cellular homolog to the viral E26 transformation specific sequence;
Ct, threshold cycle; 3D, three-dimensional; FSHR, FSH receptor; hCG, human chorionic
gonadotropin; IVF, in vitro fertilization.
O R I G I N A L A R T I C L E
E n d o c r i n e R e s e a r c h
J Clin Endocrinol Metab, September 2011, 96(9):2805–2812 jcem.endojournals.org 2805

2. adjust the FSH dosage required in poor ovarian respond-
ers. Although a variety of strategies have been used to
predict ovarian response, the treatment of poor ovarian
responders is one of the ongoing challenges in the field of
infertility management (8).
FSH receptor (FSHR), which is present exclusively on
the membrane of granulosa cells, plays an important role
inmediatingFSHaction,therebyinducingfolliculogenesis
(9, 10). It has been observed that reduced expression of
FSHR on granulosa cells may account for poor ovarian
response to FSH stimulation in women undergoing IVF
treatment. The findings suggest that increasing the dose of
exogenous FSH does not improve oocyte development
probablyduetoinsufficiencyofFSHRexpressionongran-
ulosa cells (11).
A number of naturally occurring inactivating muta-
tions in the FSHR gene have been reported in subjects with
infertility. The phenotype of the infertile subjects has been
well correlated with the extent of FSHR inactivation (9,
12). Besides inactivating mutations, several single-nucle-
otide polymorphisms have been identified in the FSHR
coding region. The significance of FSHR gene polymor-
phisms in ovarian response has been reported in subjects
undergoing IVF treatment (13–15). The single-nucleotide
polymorphisms at amino acid positions 307 (Thr307
Ala)
and 680 (Asp680
Ser) were observed to be associated with
altered response to FSH during IVF treatment, and both
variants were reported to be in near complete linkage dis-
equilibrium (13, 16, 17). The evidence of a polymorphism
at position Ϫ29 in the core promoter region of the FSHR
gene has been reported in subjects undergoing IVF treat-
ment (18). Previous studies carried out by our group in-
dicatedtheassociationoftheAAgenotypeatpositionϪ29
with poor ovarian response (19). Interestingly, in a study
conducted in women with hypertension, it was observed
that the AA genotype at position Ϫ29 is associated with
lower serum estradiol levels. Moreover, in vitro functional
studies carried out in Chinese hamster ovary cells with the
promoter constructs of the FSHR gene revealed reduced
transcriptional activity in the case of the A allele compared
with the G allele (20). It has also been observed that the
polymorphism at position Ϫ29 is located in a consensus
sequence(GGAA)forthecellularhomologtotheviralE26
transformation specific sequence (cETS-1) transcription
factor and the A allele at position Ϫ29 might lead to the
loss of the transcription factor binding site (18, 20).
These observations prompted us to investigate whether
the poor ovarian response observed in subjects with the
AA genotype at position Ϫ29 of the FSHR gene is due to
reduced expression of the receptor on granulosa cells. The
aim of present study was to compare the level of the re-
ceptor expression and ovarian response among subjects
with different genotypes at position Ϫ29 of FSHR gene.
Subjects and Methods
Subject selection
The present study was approved by the Ethics Committee for
Clinical Research at the National Institute for Research in Re-
productiveHealthandtheFertilityClinicandIVFCenter.Atotal
of 100 normogonadotropic ovulatory women (menstrual cycle
length25–35d)withinfertilityduetomaleortubalfactororwith
unexplained infertility were retrospectively analyzed. All sub-
jects were of Indian ethnicity. Informed consent was obtained
from all subjects. All recruited subjects were in the age group of
25–46 yr, and their basal serum FSH levels were in the normal
range (2–10 IU/liter). The basal FSH level, FSH amount admin-
istered, estradiol levels before and on the day of the human cho-
rionic gonadotropin (hCG) administration, and the number of
oocytes retrieved were considered for segregating subjects as hy-
perresponders, normal responders, and poor responders. Women
with polycystic ovarian syndrome, endometriosis, fibroids, and hy-
perprolactinemia were excluded from this study.
Ovulation induction
Astandardprotocoldescribedpreviously(19)wasusedforovu-
lationinductionduringIVFtreatment.StimulationwithFSH(150–
600 IU/d) was monitored by measuring the serum estradiol levels
and follicle growth. hCG (10,000 IU) was administered for trigger-
ing ovulation. Oocytes were retrieved after 36 h under transvaginal
ultrasound guidance and mature oocytes (Ͼ14 mm in diameter)
were collected. The number of preovulatory follicles and retrieved
oocytes were recorded for each subject. Serum levels of FSH, LH,
estradiol, and progesterone were measured by chemiluminescence
microparticle assay (Abbott Architect, Abbott Park, IL).
Genotyping for the polymorphism at position ؊29
of the FSHR gene
Genomic DNA was extracted from 250 ␮l of whole blood
obtained from each of the subjects (n ϭ 100) using a commercial
kit (Genexy, New Delhi, India) according to the manufacturer’s
instructions. The GϪ29
A polymorphism was screened by PCR
and restriction fragment length polymorphism as described pre-
viously (19). The findings from restriction fragment length poly-
morphism for all three genotypes were confirmed by direct se-
quencing for 30 samples (10 samples of each genotype) at the
DNA sequencing core facility of the institute.
DNA topography
The three-dimensional (3D) molecular structure of DNA is af-
fectedbynucleotidechanges,whichcancausedifferencesinprotein
binding and affinity. The level of change in the DNA shape that
arises due to the polymorphism (rs1394205) at position GϪ29
A of
the FSHR gene was compared by ranking the structure-change
value as determined by the predicted hydroxyl radical cleavage pat-
tern for DNA sequence of the core promoter region from Ϫ33 to
Ϫ14 bp using the OH Radical Cleavage Intensity Database (21).
The effect of this polymorphism on the structural profile was quan-
titativelymeasuredintermsoftheEuclideandistance,whichisused
as a metric to calculate average structure change (22).
2806 Desai et al. Polymorphism in FSHR Gene and Receptor Expression J Clin Endocrinol Metab, September 2011, 96(9):2805–2812

3. Quantitative real-time PCR
Granulosacellsinthefollicularfluid(nϭ100)wereseparated
by centrifugation at 2000 ϫ g for 10 min at 4 C, and cells were
used to extract total RNA by TRIZOL (Invitrogen, Carlsbad,
CA). The first-strand cDNA was synthesized (4 ␮g of total RNA)
using Superscript III reverse transcriptase enzyme and oligo de-
oxythymidine (dT) as primers (Invitrogen) as per the manufac-
turer’s instructions. The predesigned primers and probes for hu-
man FSHR and ␤-actin (TaqMan Assay-on-Demand gene
expression product; Applied Biosystems, Foster City, CA) were
used for amplification of cDNA by real-time PCR in duplicate
reactions. Because it was not possible to get granulosa cells from
proven fertile women, cDNA was pooled from three IVF subjects
of each genotype and used as a calibrator. The relative mRNA
expression of FSHR in IVF subjects was calculated by ⌬⌬ thresh-
old cycle (Ct) method, where the ⌬Ct was calculated as the dif-
ference between the Ct of FSHR and Ct of ␤-actin in each sample.
The ⌬⌬Ct of each sample was then calculated as the difference
between the ⌬Ct of the sample with ⌬Ct of the calibrator sample,
which was normalized with ␤-actin. The level of FSHR expres-
sion was compared within three genotypes at position Ϫ29 by
ANOVA.
Western blot analysis
To study the association of the FSHR gene polymorphism at
position Ϫ29 with protein expression, total protein was ex-
tracted from granulosa cells collected from 30 subjects (10 sub-
jects of each genotype) by TRIZOL (Invitrogen) as per the man-
ufacturer’s instructions. The protein concentration of the lysate
was determined by Folin-Lowry’s assay and 50 ␮g of protein was
loaded in each lane of a 7.5% SDS-PAGE gel. After electropho-
resis, the protein was transferred on to a 0.45-␮m nitrocellulose
membrane (Hybond C Extra; GE Healthcare, Buckinghamshire,
UK)usingasemidrytransferapparatus(Bio-Rad,Hercules,CA).
Immunoblotting was performed using the monoclonal antibody
to FSHR 106.105 (0.5 ␮g/␮l) (a gift from Dr. J. A. Dias, Wads-
worth Center, David Axelrod Institute for Public Health, Al-
bany, NY) or the monoclonal antibody for ␤-actin (1:1000;
Santa Cruz Biotechnology, Santa Cruz, CA) as the primary an-
tibody and horseradish peroxidase-conjugated goat antimouse
IgG antibody (1:5000; Dako, Copenhagen, Denmark) as the sec-
ondary antibody. The bands were visualized with enhanced
chemiluminescence reagent (ECL-Plus) (GE Healthcare) on x-
ray film. The protein expression of FSHR was normalized to
␤-actin for each sample and compared among subjects with three
genotypes at position Ϫ29 by ANOVA.
In a separate experiment, monoclonal antibody preincubated
with 20 ␮g of synthetic peptide (corresponding to the epitope
297–310 amino acid of human FSHR) was used for probing the
blot (23). This served as a negative control.
Immunofluorescence and confocal microscopy
SurfacelocalizationofFSHRoncumuluscellswascarriedout
using direct immunofluorescence microscopy using a modified
protocol adapted from Beau et al. (24). The cells obtained from
five subjects of each genotype at position Ϫ29 were treated with
anti-FSHR monoclonal antibody 106.105 conjugated to Alexa
fluor 568 (0.5 ␮g per 100 ␮l). The cells were preincubated with
100-fold higher concentration of unlabeled anti-FSHR mono-
clonal antibody prior probing with labeled antibody, which
served as a negative control. Cells were then fixed with 2% para-
formaldehyde. 4Ј-6-Diamidino-2-phenylindole was used as nu-
clear stain, and cells were smeared on a glass slide. Cellular dis-
tributionofthereceptorswasexaminedusingtheLSM510-Meta
confocal system (Carl Zeiss, Jena, Germany). Z stacks were
taken and 3D composite images were generated. The level of
surface expression was quantified using ImageJ software (Na-
tional Institutes of Health, Bethesda, MD). To normalize the
data, the percentage of FSHR expression in subjects with GA and
AAgenotypewascalculatedwithrespecttotheGGgenotypeand
compared by ANOVA.
Statistical analysis
Conformance with the Hardy-Weinberg equilibrium was
computed by a ␹2
test (25). The clinical parameters and the level
ofFSHRexpressionwerecomparedbetweenthestudygroupson
the basis of genotypes at position Ϫ29 and type of ovarian re-
sponse using one-way ANOVA and a least significant difference
post hoc multiple comparisons test. Statistical analysis was per-
formed with Statistics Package for Social Sciences (SPSS) for
Windows, version 16 (SPSS Inc., Chicago, IL). The odds ratio
was calculated using ␹2
analysis to study the association of the
FSHR genotype at position Ϫ29 with poor ovarian response
using Epi Info version 6 (World Health Organization, Geneva,
Switzerland). P Յ 0.05 was considered statistically significant.
Results
Polymorphism at position ؊29 of the FSHR gene
The frequency distribution of the polymorphism at
position Ϫ29 was 47% for the GG genotype, 42% for
the GA genotype, and 11% for the AA genotype in 100
IVF subjects. The ␹2
analysis revealed that the frequency
of genotypes at position Ϫ29 was in Hardy-Weinberg’s
equilibrium.
Clinical and endocrinological parameters
To analyze the potential association between the FSHR
gene polymorphism at position Ϫ29 and the ovarian re-
sponse during gonadotropin stimulation, the clinical, en-
docrine, and ultrasonographic parameters were recorded
for all the subjects recruited in this study (Table 1). Sub-
jects were independently segregated on the basis of geno-
types (GG, GA, or AA) at position Ϫ29. The age, basal
FSH and LH levels, peak estradiol, and progesterone con-
centration before and on the day of hCG treatment
showed no statistically significant difference among the
three genotypes. In contrast to the above parameters, the
amount of exogenous FSH required for ovulation induc-
tion was significantly different in all three genotypes (P ϭ
0.003). Maximum amount of exogenous FSH (4563 Ϯ
271 IU) was required by the subjects with AA genotype,
whereas subjects with the GG genotype required a mini-
mum amount of FSH (2492 Ϯ 154 IU) for ovarian stim-
ulation. The amount of FSH (3265 Ϯ 239 IU) required in
subjects with the GA genotype was lower than that re-
J Clin Endocrinol Metab, September 2011, 96(9):2805–2812 jcem.endojournals.org 2807

4. quired in subjects with the AA genotype. The ultrasound
findings revealed that the number of preovulatory follicles
and retrieved oocytes were significantly low in subjects
with the AA genotype (9 Ϯ 0.8, 9.18 Ϯ 0.9, respectively)
compared with the GG genotype (13.21 Ϯ 0.6, 13.91 Ϯ 1,
respectively) and the GA genotype (12.62 Ϯ 0.7, 12.57 Ϯ
1.1, respectively) (P Ͻ 0.05). Moreover, the number of
mature oocytes (in M II phase) retrieved in case of subjects
with the AA genotype (7 Ϯ 0.8) were significantly lower
comparedwiththeGGgenotype(11.62Ϯ0.8;Pϭ0.022).
Furthermore, the clinical and endocrine parameters
were used to segregate the subjects as hyperresponders,
normal responders, and poor responders and were ob-
served to be significantly different among the three groups
(Table 1). In the study group of 100 subjects, 29 subjects
were poor ovarian responders. When we further segre-
gated these subjects (n ϭ 29) on the basis of genotype at
position Ϫ29, it was observed that only 21.27% of sub-
jects with the GG genotype and 26.19% of the subjects
with the GA genotype were poor ovarian responders. In-
terestingly, 72.72% of the subjects with the AA genotype
were poor ovarian responders. The ␹2
test was used to
study the association between the polymorphism at posi-
tion Ϫ29 with poor ovarian response. The odds ratio for
the GG genotype was 0.48 [95% confidence interval (CI)
0.18–1.29; P ϭ 0.166], for the GA genotype was 0.79
(95% CI 0.30–2.08; P ϭ 0.761), and for the AA genotype
was 8.63 (95% CI 1.84–45.79; P ϭ 0.001) (Table 2).
DNA topography of FSHR gene
We investigated whether the polymorphism at position
Ϫ29 might alter the 3D molecular structure of the DNA,
which could potentially influence protein binding affinity
and phenotype. The polymorphism at position Ϫ29 re-
sulted in a DNA structural change value greater than 0.8.
The magnitude of this structural change value revealed
that the DNA with the A allele (0.12) is less accessible for
binding of transcription factors compared with the G al-
lele (0.94) (Fig. 1A).
FSHR expression at mRNA level
The relative mRNA expression was monitored by
quantitativereal-timePCRandcomparedamongthethree
genotypes at position Ϫ29. The level of FSHR mRNA
TABLE 1. Clinical and endocrinological parameters of the subjects undergoing IVF treatment based on the
genotypes at position Ϫ29 of the FSHR gene and based on the type of ovarian response to FSH stimulation
Parameters
Genotypes at position ؊29 of FSHR gene Type of ovarian response to FSH stimulation
GG
(n ‫؍‬ 47)
GA
(n ‫؍‬ 42)
AA
(n ‫؍‬ 11)
Hyper
(n ‫؍‬ 11)
Normal
(n ‫؍‬ 60)
Poor
(n ‫؍‬ 29)
Age (yr) 32.30 Ϯ 0.62 32.43 Ϯ 0.64 34.61 Ϯ 1.19 30.55 Ϯ 0.91a
31.93 Ϯ 0.47b
34.79 Ϯ 0.91a,b
Basal FSH levels (IU/liter) 6.32 Ϯ 0.32 6.46 Ϯ 0.29 6.93 Ϯ 1.24 5.29 Ϯ 0.45a
6.21 Ϯ 0.26b
7.24 Ϯ 0.42a,b
Basal LH levels (IU/liter) 4.40 Ϯ 0.35 5.18 Ϯ 0.39 5.25 Ϯ 1.80 6.08 Ϯ 0.53 4.55 Ϯ 0.36 4.88 Ϯ 0.54
Total amount of exogenous
FSH administered (IU)
2492.55 Ϯ 154.30a
3265.48 Ϯ 239.23a
4563.64 Ϯ 271.53a
1729.55 Ϯ 136.00a
2673.33 Ϯ 137.63a
4312.93 Ϯ 264.61a
Estradiol levels before hCG
administration (pg/ml)
1688.70 Ϯ 110.00 1855.33 Ϯ 149.00 1640.73 Ϯ 243.00 3094.18 Ϯ 165.67a
1761.30 Ϯ 87.08a
1228.52 Ϯ 133.69a
Estradiol levels on the day
of hCG administration
(pg/ml)
1954.67 Ϯ 141.00 2148.36 Ϯ 180.00 1890.50 Ϯ 286.00 3806.36 Ϯ 163.77a
1988.27 Ϯ 101.85a
1447.07 Ϯ 161.59a
Progesterone levels before
hCG administration
(ng/ml)
0.54 Ϯ 0.03 0.91 Ϯ 0.33 0.62 Ϯ 0.06 0.75 Ϯ 0.08 0.80 Ϯ 0.23 0.49 Ϯ 0.04
Progesterone levels on the day
of hCG administration
(ng/ml)
4.15 Ϯ 0.40 4.99 Ϯ 0.53 4.64 Ϯ 0.52 8.48 Ϯ 1.20a,b
4.61 Ϯ 0.34a
3.73 Ϯ 0.64b
Preovulatory follicles, n 13.21 Ϯ 0.67a
12.62 Ϯ 0.75b
9.00 Ϯ 0.86a,b
19.73 Ϯ 0.99a
13.58 Ϯ 0.35a
7.52 Ϯ 0.56a
Retrieved oocytes, n 13.91 Ϯ 1.02a
12.57 Ϯ 1.16 9.18 Ϯ 0.90a
26.91 Ϯ 1.69a
13.53 Ϯ 0.48a
6.03 Ϯ 0.53a
Mature oocytes, n 11.62 Ϯ 0.86a
9.86 Ϯ 0.99 7.00 Ϯ 0.89a
21.82 Ϯ 1.87a
10.98 Ϯ 0.44a
4.76 Ϯ 0.45a
Values are presented as mean Ϯ SEM. One-way ANOVA tests and LSD post hoc multiple comparisons were used for ANOVA. Same letters (a or b)
for a given parameter indicate statistically significant difference at P Ͻ 0.05.
TABLE 2. Genotype frequencies at the position Ϫ29 of the FSHR gene in subjects undergoing IVF treatment and its
correlation with the occurrence of poor ovarian response
Genotype at
position ؊29
IVF subjects
screened, n
Poor ovarian
responders, n
Poor ovarian
responders (%)
Odds ratio
(95% CI) P
GG 47 10 21.27 0.48 (0.18–1.29) 0.166
GA 42 11 26.19 0.79 (0.30–2.08) 0.761
AA 11 8 72.72 8.63 (1.84–45.79) 0.001a
a
P Ͻ 0.05 by ␹2
test.
2808 Desai et al. Polymorphism in FSHR Gene and Receptor Expression J Clin Endocrinol Metab, September 2011, 96(9):2805–2812

5. expression was observed to be variable. The FSHR ex-
pression at transcript level was 1.3-fold lower in case of
subjectswiththeAAgenotype(0.15Ϯ0.5)comparedwith
the GG genotype (1.49 Ϯ 0.3), and the difference was
observed to be statistically significant (P ϭ 0.039). The
level of expression was intermediate in the case of subjects
with GA genotype (0.90 Ϯ 0.21) (Fig. 1B).
We also compared the relative FSHR mRNA expression
amongsubjectswithvariabletypeofovarianresponse.Itwas
observed that mRNA expression was similar in three groups
(Fig. 2A). Therefore, to investigate whether all the poor re-
sponders (n ϭ 29) in the present study show altered FSHR
expression,wesegregatedthesesubjects(nϭ29)onthebasis
of genotype at position Ϫ29. It was observed that the FSHR
mRNA expression in poor ovarian responders was signifi-
cantly lower in subjects with AA genotype compared with
the GG genotype (P ϭ 0.027) (Fig. 2B).
FSHR expression at protein level
FSHR protein expression in 10 subjects of each genotype
atpositionϪ29wasanalyzedbyWesternblotandquantified
using densitometry. The specificity of the FSHR monoclonal
antibody was confirmed, and it was observed that FSHR
expression at protein level was variable among the three
genotypes (Fig. 3A). It was evident that the relative level of
FSHR protein expression was significantly reduced in the
case of subjects with the AA genotype (0.49 Ϯ 0.08) com-
pared with the GG (0.76 Ϯ 0.05) and GA (0.75 Ϯ 0.08)
genotype (P Ͻ 0.05, Fig. 3B).
The cell surface expression of FSHR was assessed by
direct immunofluorescence technique, and specificity
was confirmed using the negative control (Fig. 4A). Dif-
ferential expression of membrane receptor among three
genotypes at position Ϫ29 was observed (Fig. 4, B–D).
Significantly reduced level of membrane receptor ex-
pression were observed on cumulus cells obtained from
subjects with the AA genotypes (6.6 Ϯ 0.4) compared
with the GG genotype (16.3 Ϯ 1.4) and the GA genotype
(15.2 Ϯ 1.2). The fluoresceins intensity of GG genotype
is considered as 100% and compared with GA and AA
genotype (P Ͻ 0.002, Fig. 4E).
Discussion
In our earlier study, we had recruited 50 women un-
dergoing ovarian stimulation protocol, and the results
indicated that the polymorphism at position Ϫ29 of the
FSHR gene is associated with poor ovarian response
(19). Therefore, we speculated that this polymor-
phism of the FSHR gene might influence the level of
receptor expression. We recruited an additional 100
subjects undergoing IVF treatment and determined the
frequency distribution of the polymorphism at position
Ϫ29 of the FSHR gene in these subjects. The frequency
distribution observed in the present study for 100 sub-
jects was comparable with our previous report for 50
subjects (19). The frequency distribution of this poly-
morphism in subjects undergoing IVF treatment has
also been reported for Indonesian and German popu-
lations (18).
The potential association between the polymorphism
at position Ϫ29 and the clinical parameters was ana-
lyzed in these subjects. It was observed that the subjects
with the AA genotype required significantly higher
amounts of exogenous FSH for ovulation induction
compared with subjects with the GG and GA genotypes.
FIG. 1. A, Comparative predicted hydroxyl radical cleavage intensity
corresponding to the nucleotide sequence of wild-type and
polymorphic FSHR gene. B, The comparison of relative FSHR mRNA
expression among three genotypes at position Ϫ29 of the FSHR gene
in IVF subjects (n ϭ 100). Values presented in bar are mean Ϯ SEM and
compared by one-way ANOVA. ૽, P Ͻ 0.05 is considered statistically
significant.
J Clin Endocrinol Metab, September 2011, 96(9):2805–2812 jcem.endojournals.org 2809

6. These results indicate that the AA genotype imparts
higher resistance to FSH stimulation (Table 1). We also
observed that in subjects with the AA genotype, the
number of preovulatory follicles and mature oocytes
were significantly lower compared with the GG geno-
type (Table 1). These observations suggest that the AA
genotype at position Ϫ29 might be associated with
poor ovarian response, which correlates with our
earlier study (19). Wunsch et al. (18) have
reported that the polymorphism at position
Ϫ29 does not seem to influence the clinical
parameters observed in subjects undergoing
ovarian stimulation substantially, when the
basal FSH and peak estradiol levels were
analyzed.
Identification of the poor responders is clin-
ically relevant to determine individualized
ovarian stimulation protocol during IVF treat-
ment. In the present study, almost 72% of the
subjects with the AA genotype were poor ovar-
ian responders. We calculated the relative risk
of poor ovarian response associated with the
genotype at position Ϫ29 in IVF subjects (n ϭ
100). The ␹2
analysis revealed that subjects
with the AA genotype have a higher risk of
giving poor ovarian response to gonadotropin
stimulation during IVF treatment (Table 2). This suggests
the significance of AA genotype as a biomarker to predict
poorovarianresponders,whichmightimprovetheclinical
outcome during IVF treatment.
To identify the probable reason for the association of
poor ovarian response observed in the AA genotype, we
determined the FSHR mRNA expression in granulosa
cells. Interestingly, we noted that subjects with the AA
genotype displayed significantly lower FSHR mRNA
expression compared with the GG genotype (Fig. 1B).
Our observation strengthens the earlier report in which
the promoter activity of the FSHR gene was found to be
significantly reduced in the case of the A allele compared
with the G allele at position Ϫ29 by in vitro analysis
(20). The reduced expression of the FSHR gene in sub-
jects with the AA genotype might be polymorphism-
associated steric changes in the DNA structure, which
may affect the binding of transcription factors because
this polymorphism is located in the c-ETS-1 transcrip-
tion factor binding site (Fig. 1A) (18). Lower cleavage
intensity observed in the case of the A allele compared
with the G allele suggests its importance in the promoter
efficiency of the FSHR gene causing a decrease in FSHR
expression in subjects with the AA genotype. However,
the mechanism by which the cETS-1 transcription fac-
tor influences human FSHR gene expression is not clear
and needs to be investigated further.
Findings from our previous (19) and present study con-
sistently demonstrate a correlation of the AA genotype at
Ϫ29 position of the FSHR gene with poor ovarian re-
sponse. Although FSHR expression in granulosa cells has
been shown to be associated with individual response to
FSH stimulation (11), the observation was not the same in
the study reported by Thiruppathi et al. (26). Poor ovarian
FIG. 2. A, The comparison of relative FSHR mRNA expression A, in different type of
ovarian responders (n ϭ 100). B, In poor ovarian responders among three genotypes
at position Ϫ29 of the FSHR gene (n ϭ 29). Statistical analysis was carried out by
one-way ANOVA. ૽, P Ͻ 0.05 is considered statistically significant.
FIG. 3. A, The representative image for the protein expression of FSHR
and ␤-actin in granulosa cells in three genotypes at position Ϫ29 of
the FSHR gene. B, The box plot showing the comparison of FSHR
protein (mean Ϯ SEM) among three groups, median (middle bar), and
quartiles (boxes). The statistical analysis was carried out by one-way
ANOVA, and the same letters (a or b) indicate statistically significant
difference at P Ͻ 0.05.
2810 Desai et al. Polymorphism in FSHR Gene and Receptor Expression J Clin Endocrinol Metab, September 2011, 96(9):2805–2812

7. response observed in different individuals during IVF
treatment could be due to other parameters apart from
FSHR expression. This is evident from our observation
that the relative expression of FSHR mRNA levels did not
vary when we segregated the subjects based on ovarian
response(Fig.2A).However,itwasinterestingtonotethat
within poor responders, relative mRNA expression of
FSHR was significantly decreased in the AA genotype
compared with the GG genotype (P ϭ 0.027) (Fig. 2B).
Furthermore, we noted that none of the subjects with the
AA genotype were hyperresponders. These results suggest
that the level of receptor expression is closely associated
with genotype at position Ϫ29 of FSHR gene in poor ovar-
ian responders.
More interestingly, when we compared the FSHR
expression at protein level on the basis of genotypes at
position Ϫ29, we observed that subjects with the AA
genotype expressed significantly lower amounts of re-
ceptor protein compared with the GG and GA geno-
types (Fig. 3). Our observation thus suggests that the
reduced FSHR expression at the transcript level is in
concurrence with the expression of FSHR at the protein
level in subjects with the AA genotype. It has been re-
ported that FSH directly exerts its action on the oocyte
through its receptor, expressed on the membrane of cu-
mulus cells surrounding the oocyte (27). Therefore, in
the present study, we evaluated the
cell surface expression of FSHR by
confocal microscopy using cumulus
cells obtained from recruited subjects.
The results revealed that the receptor
expression on cumulus cells obtained
from subjects with the AA genotype
was significantly lower compared
with the GG and GA genotypes (Fig.
4). Thus, our findings clearly suggest
that reduced FSHR expression in the
AA genotype could be the possible
reason for poor ovarian response to
FSH stimulation observed in these
subjects.
One of the major challenges for
the clinicians during ovarian sti-
mulation protocol is nonavailability
of reliable predictive indicators to
identify women who are poor re-
sponders. The present study clearly
shows that the AA genotype at posi-
tion Ϫ29 of the FSHR gene is associ-
ated with poor ovarian response and
relatively lower expression of recep-
tor on the granulosa cells. Further-
more, large multicentric studies are
required to elucidate the relative contribution of this
polymorphism to be used as a marker for predicting
poor ovarian response during IVF treatment.
Acknowledgments
We are thankful to the participants of the study. The authors
acknowledge Dr. D. Balaiah and Mr. Prashant Tapse (Division
of Biostatistics, National Institute for Research in Reproductive
Health) for their help in statistical analysis. We also thank Dr.
Anurupa Maitra and Ms. Nanda Ugale (DNA sequencing core
facility, National Institute for Research in Reproductive Health)
for their assistance in the DNA sequencing. Technical help pro-
vided by Ms. Savita (Fertility Clinic and the IVF Center) is also
acknowledged. We also thank Dr. Nafisa Balasinor, Ms. Shobha
Sonawane, and Ms. Reshma Goankar in facilitating confocal
microscopy analysis.
Address all correspondence and requests for reprints to:
Smita D. Mahale, Division of Structural Biology, National
Institute for Research in Reproductive Health, Mumbai 400
012, India. E-mail: smitamahale@hotmail.com.
This work (NIRRH/MS/03/2011) was supported by grants
from the Indian Council of Medical Research, New Delhi, India
(P and I/BIC/1/1/2009) and the Board of Research in Nuclear
Sciences, Department of Atomic Energy.
Disclosure Summary: The authors have nothing to disclose.
FIG. 4. The representative image for surface expression of FSHR (green signal) on cumulus
cells by confocal microscopy using monoclonal FSHR antibody conjugated with Alexa Fluor
568 and nuclear staining with 4Ј-6-diamidino-2-phenylindole (red signal). A–D, The 3D
structure of cumulus cells for A, negative control and B–D, three genotypes at position Ϫ29
of the FSHR gene. Bar, 10 ␮m. E, Intensity of fluorescence in each cell was calculated using
ImageJ software. The comparison is of percent FSHR cell surface expression (mean Ϯ SEM)
among the three genotypes by one-way ANOVA, and the same letters (a or b) indicate
statistically significant difference at P Ͻ 0.05. Data analyzed are from six to eight cells from
each subject and a total of 35 cells for each genotype.
J Clin Endocrinol Metab, September 2011, 96(9):2805–2812 jcem.endojournals.org 2811

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