Department of Biology, Faculty of Science, Science and Research Branch Islamic Azad University, Tehran, Iran
Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
Department of Endocrinology and Female Infertility, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine,
ACECR, Tehran, Iran
P.O. BOX: 16635-148
Department of Genetics
Reproductive Biomedicine Research Center
Royan Institute for
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Zari Moradi Shabnam,
Mohseni Meybodi Anahita.
Epigenetic Aberration of FMR1 Gene in Infertile Women with
Diminished Ovarian Reserve.
The diminished ovarian reserve (DOR) is a condition characterized by a reduction in the number and/or
quality of oocytes. This primary infertility disorder is usually accompanied with an increase in the follicle-stimulating
hormone (FSH) levels and regular menses. Although there are many factors contributing to the DOR situation, it is likely
that many of idiopathic cases have genetic/epigenetic bases. The association between the FMR1 premutation (50-200
CGG repeats) and the premature ovarian failure (POF) suggests that epigenetic disorders of FMR1 can act as a risk
factor for the DOR as well. The aim of this study was to analyze the mRNA expression and epigenetic alteration (histone
acetylation/methylation) of the FMR1 gene in blood and granulosa cells of 20 infertile women.
Materials and Methods
In this case-control study, we analyzed the mRNA expression and epigenetic altration of the
FMR1 gene in blood and granulosa cells of 20 infertile women. These women were referred to the Royan Institute,
having been clinically diagnosed as DOR patients. Our control group consisted of 20 women with normal antral follicle
numbers and serum FSH level. All these women had normal karyotype and no history of genetic disorders. The number
of CGG triplet repeats in the exon 1 of the FMR1 gene was analyzed in all samples.
Results clearly demonstrated significantly higher expression of the FMR1 gene in blood and granulosa cells of
the DOR patients with the FMR1 premutation compared to the control group. In addition, epigenetic marks of histone 3
lysine 9 acetylation (H3K9ac) and di-metylation (H3K9me2) showed significantly higher incorporations in the regulatory
regions of the FMR1 gene, including the promoter and the exon 1, whereas tri-metylation (H3K9me3) mark showed no
significant difference between two groups.
Our data demonstrates, for the first time, the dynamicity of gene expression and histone modification pattern
in regulation of FMR1 gene, and implies the key role played by epigenetics in the development of the ovarian function.
One of the well-known causes of female infertility is the
diminished ovarian reserve (DOR). DOR is characterized
by a reduction in the number and/or quality of oocytes,
low likelihood of establishing a pregnancy, increased
miscarriage rates, and poor response to ovarian stimulation
in in vitro fertilization (IVF) (1, 2). The prevalence
of DOR has been estimated to be approximately 10%
among young women (3, 4). Despite its prevalence, its
etiology remains a mystery. Aging is the most common
cause of diminished ovarian reserve. Other reasons for
the diminished ovarian reserve include chemotherapy,
radiation therapy, autoimmune diseases, and certain
genetic conditions (5).
The fragile X mental retardation 1 gene (FMR1) is
located at Xq27.3 and is responsible for the fragile X
syndrome, a form of X-linked mental retardation. This
disorder is caused by the expansion of a polymorphic
CGG trinucleotide repeat in the promoter of the FMR1
gene, consisting of more than 200 repeats (full mutation),
instead of the usual 6-54 CGG repeats (6, 7). This trinucleotide
expansion induces methylation of cytosines
within the CpG islands inside the repeat tract and in the
flanking sequence, including the FMR1 gene promoter,
resulting in the epigenetic inactivation of the gene,
which in turn switches off the production of the FMR1
protein (FMRP) (8-10). Premutation alleles (55-200 CGG
repeats) have been associated with premature ovarian
It has been reported that the rearrangements of the X
chromosome are associated with the POF (11). Two main
critical regions in the long arm of X chromosome are
identified which contain putative POF candidate genes:
POF1 (Xq26-q28) (12) and POF2 (Xq13.3-q22) (13). In
POF-1, the FMR1 gene is the most prominent candidate
gene. The relationship between FMR1 premutation status
and POF disease suggests that the FMR1 gene increases
the risk of the POF (14, 15), and, based on the recent
studies, the DOR pathogenesis (16, 17). Besides, the
impact of shorter repeats (45-54 repeats), which are only
slightly longer than normal, is less clear (17, 18).
Epigenetics is the study of heritable changes in gene
activity and expression that occur without change in
DNA sequence (19, 20). Two of the most well-known
epigenetic modifications are chemical modifications
on cytosine residues of DNA (DNA methylation) and
post-translational modification of histones associated
with DNA (histone modifications) (19). Functionally,
the patterns of epigenetic modifications can serve as
epigenetic markers to represent the dynamic level of gene
activity and expression, based on the chromatin state
(21-23). These modifications play an important role in
regulating gene expression by modulating the packaging
of DNA in the nucleus as chromatin domains (23-25).
DNA methylation can suppress transcription through
several mechanisms, including direct inhibition
binding of transcription factor to gene promoters and
indirect inhibition, through the induction of changes
in local chromatin structure at the site of methylation.
As such, methyl-CpG binding proteins (e.g., MeCP2
and MBDs) recognize methylated CpG regions, where
they can acts as mediators of transcriptional repression
through the association with histone deacetylases
(HDACs) in repressor complexes (26-28). Histone
modification is another epigenetic mechanism that is
mostly known by acetylation and methylation of lysine
(K) residues in N-terminal tails of histone proteins (22,
29). Histone methylation can result in the activation
or the inhibition of gene expression, depending on the
localization of the covalently modified lysine residue
(30). For example, tri-methylation of histone 3 at
lysine 4 and di/tri-methylation at lysine 9 (H3K4me
and H3K9me) are particularly correlated with
transcriptional activation and repression, respectively
(31, 32). On the other hnad, acetylation of histones is
commonly linked to active transcription (26, 27).
Several histone modifications are reported for the
FMR1 gene. In cells with the full mutation of FMR1,
CGG repeats are hypermethylated at H3K9 and
hypomethylated at H3K4, and low levels of acetylation
of histones are detected, while normally, histones H3
and H4 are hyperacetylated, H3K4 is hypermethylated,
and H3K9 is hypomethylated (33-35). Previous
studies have shown that the treatment of fragile X
lymphoblastoid cells with the DNA methylation
inhibitor 5-aza-2-deoxycytidine (5-azadC) leads to the
transcriptional reactivation of the FMR1 gene (36).
In addition, the treatment of these cells with HDAC
inhibitors (i.e., butyrate and tricostatin A) resulted in a
modest reactivation of the FMR1 gene. The reactivation
were enhanced when the HDAC inhibitors were used
synergistically with 5-azadC (37). According to our
knowledge, this is the first report of the analysis and
comparison of the expression levels of the FMR1 gene
in blood and granulosa cells and the evaluation of
above mentioned histone modification changes of the
FMR1 gene based on the analysis of the blood cells of
infertile women with DOR.
Materials and Methods
In this study case-control study, samples for
epigenetic changes and gene expression analysis were
categorized into two groups: DOR patients and control
groups, based on follicles number, FSH levels, and the
number of CGG repeats. A total of 20 infertile women
with clinically confirmed DOR conditions and the
FMR1 premutation were recruited at Department of
Genetics of the Royan Institute. Any member of the
DOR group had to satisfy the following conditions:
Patients with 3 oocytes with a conventional stimulation
protocol, antral follicle counts (AFC)<5-7 (2-10 mm
in diameter, measured using the standardized two-
dimensional technique), follicle-stimulating hormone
(FSH) levels>11 IU/l at day 3 of the follicular cycle,
<40 years of old, and regular menstrual cycles for the
past 6 months (Table 1,). Among the DOR patients,
only patients with the FMR1 gene premutation (CGG
repeats >55) were enrolled as the case group. Also, 20
women with normal antral follicle numbers and serum
FSH level were selected as the controls (age 37.38
± 1.32) (Table 1,). Women with abnormal karyotypes
and X chromosomal mosaics were excluded from the
study. All samples were collected during a one-year
period (2013-2014). All patients and control subjects
were Iranian, living in different places in Iran. This
study was approved by the Ethics Committee for
clinical research at the Royan institute and informed
consent was obtained from all participants.
Genomic DNA was isolated from peripheral blood
cells using the standard salting out method (described
in (38). The 5’ UTR of the FMR1 gene containing
the CGG repeats was amplified using the polymerase
chain reaction (PCR) technique by a reverse and
forward primer set following Tassone et al. (39). The
PCR products were separated on a 3% NuSieve 3:1
agarose gel by electrophoresis (Lonza, USA) at 33
v for 4 hours. Each DNA band were purified from
the gel by High Pure PCR Product Purification Kit
(Roche Applied Science, USA) and amplified by the
PCR program described above. As the betaine-PCR
(39) is unable to distinguish between heterozygotes of
full mutation and normal homozygotes, samples that
resulted in the primary PCR products with a single
band were subjected to a secondary PCR screen with
the R primer and the CCG-chimeric primer, instead
of the F primer. Consequently, we used a chimeric
CGGprimer in conjunction with betaine-PCR. The
amplified product will generate a smear on the gel
when there is an expanded allele present, whereas in
the absence of an expanded allele no large smear will
be detected. The numbers of trinucleotide repeats were
confirmed by Sanger sequencing method using ABI
3730XL Capillary Sequencer. Sequencing results were
compared with the sequence of a normal FMR1 gene.
RNA extraction and quantitative real-time polymerase
chain reaction analysis
The blood and granulosa cells of 20 Iranian DOR
patients (the case group) were used for RNA extraction, in
order to study mRNA gene expression. Besides, patients
with normal blood FSH level and more than three follicles
were used as the control group (n=20). Total RNA was
extracted from patient’s blood and granulosa cells using
the Absolutely RNA Nanoprep kit (Aligent, USA).
The integrity of total RNA was checked by denaturing
formaldehyde/MOPS/1% agarose electrophoresis and
then checking its purity via UV-spectrophotometry in
10 mM Na2HPO4/NaH2PO4-buffer (pH=7.0). The A260/
A280-ratio was >2.0. Two distinct ribosomal RNA bands
were identified in each sample examined. To remove
genomic DNA, a DNase treatment was carried out using
the RNase-Free DNase Set (Qiagen, USA). We reverse
transcribed RNA by QuantiTect Whole Transcriptome kit
(Qiagen, USA). To exclude genomic amplification, PCR
was performed with the same total RNA samples without
reverse transcriptase. Products were analyzed on 4%
One Step Quantitative RT-PCR was performed by
the 7500 Real time PCR system (Applied Bio System,
USA), using Power SYBR Green PCR master mix
(Applied Bio System, USA) in triplicate reaction to
ensure consistency. Temperature profile of the real
time-PCR consists of 95°C for 4 minutes, 40 cycles
of 95°C for 10 seconds and 60°C for 30 seconds.
The FMR1 amplicon was an 89 bp product, spanning
between the exons 13 and 14 of the gene. GAPDH was
used to verify the quality of cDNA synthesis and PCR
reaction (Table 2,). The 2-ΔΔCt was calculated for the
obtained data. REST384-ß (2006) software was used
to compare means between groups.
Chromatin immunoprecipitation coupled with real-
time polymerase chain reaction
Chromatin immunoprecipitation (ChIP) experiments
were performed on the regulatory regions of FMR1
gene [described in (38)] using Low Cell ChIP Kit
(Diagenode, Belgium) and antibodies (anti histone
H3 acetyl K9 antibody, anti histone H3 di-methyl
K9 and anti histone H3 tri-methyl K9 (all by Abcam,
UK), following the manufacturer’s instructions.
Chromatin from 1×104 cells was used for each
immunoprecipitation reaction. Quantitative real-time
PCR amplification was performed on DNA recovered
from the ChIP and the total chromatin input. Five
microliters of immunoprecipitated DNA (from a total
50 µl) was quantified in triplicate by real-time PCR,
using Power SYBR Green PCR Master Mix (AB
Applied Biosystems, USA) on a 7500 Real-Time PCR
System (Applied Biosystems, USA). The primers used
for PCR analysis were designed to amplify two different
regions of the FMR1 gene: the promoter region and the
exon 1 near the CGG repeat. The primer pairs for ChIP
experiment are listed in Table 2. Temperature profile
of the real time PCR consists of 95°C 10 minutes,
40 cycles of 95°C 15 seconds and 60°C 1 minute.
Data is presented as the fold enrichment of different
immunoprecipitated DNA relative to a 1/100 dilution
of input chromatin.
RT-PCR; Reverse transcription-polymerase chain reaction and ChIP; Chromatin immunoprecipitation.
Statistical analysis of real-time polymerase chain
Values were expressed as means SEM. All data were
analyzed using the independent sample t test. Differences
were considered statistically significant if P<0.05.
Premutation analysis of FMR1 gene
The results of CGG trinucleotide expansion in the DOR
patients compared with normal individuals, has been
previously reported (17). The frequency of premutation
alleles was statistically higher in the DOR patients in
comparison with the controls (P<0.05), but the difference
in the incidence of intermediate alleles was not statistically
significant between these groups.
Expression analysis of FMR1 gene
Relative mRNA expression of FMR1 gene in granulosa
and blood cells of the control group and the DOR patients
with FMR1 premutation was performed using quantitative
real time-PCR method. The results clearly demonstrate
that the expression of FMR1 gene in both sample types
of DOR patients was about 2 fold higher than that of the
control group. This increase in gene expression level
was statistically significant in both types of cell samples
(P<0.05, Fig .1,).
- Quantitative real time polymerase chain reaction (PCR) analysis of
FMR1 mRNA levels in blood and granulose cells. The results are presented
as 2-ΔΔCt (mean ± SEM) relative to the GAPDH as the endogenous control.
*; Significant difference of FMR1 gene in the DOR patients vs. the controlgroup in P<0.05 and DOR; Diminished ovarian reserve.
Epigenetic profile of FMR1 gene regulatory regions
In order to evaluate the probable epigenetic alterationsoccurred in the regulatory region of the FMR1 gene, and
the level of incorporated histone marks, we focused on
known epigenetic marks of lysine 9 residue of long tailedhistone 3. Evaluated histone marks in this study were
H3K9ac (an euchromatin associated mark) and H3K9me2/
me3 (heterochromatin associated marks). Data analysis
in the regulatory region of FMR1 gene demonstrated that
the incorporation (presence) of H3K9ac and H3K9me2
in the promoter and the exon 1 region were significantly
higher in the DOR patient in comparison with the control
group (P<0.05), whereas the incorporation of H3K9me3
in the regions showed no significant difference (P>0.05,
- Chromation immunoprecipitation (ChIP) analysis of histone
modifications in the promoter region of the FMR1 gene in blood cells. The
results are expressed relative to a 1/100 dilution of the input chromatin
(mean ± SEM).
*; Significant difference of incorporated histone marks in the DOR patients
vs. the control group in P<0.05 and DOR; Diminished ovarian reserve.
- Chromation immunoprecipitation analysis (ChIP) of histone
modifications in the exon1 region of the FMR1 gene in blood cells. The
results are expressed relative to a 1/100 dilution of input chromatin
(mean ± SEM).
*; Significant difference of incorporated histone marks in DOR patients vs.
control group in P<0.05 and DOR; Diminished ovarian reserve.
The FMR1 gene is transcribed in many tissues including
the leukocytes. The previous studies suggested that the
FMR1 gene has a direct effect on the follicular recruitment
and the ovarian reserve, implying that it has an important
role in ovarian physiology and female fecundity. We
investigated the epigenetic marks of methylation and
acetylation of H3K9 on the regulatory region of FMR1
gene and the resulting transcriptional activity of the gene
in blood cells of patients with diminished ovarian reserve.
The CGG repeat lies in the 5’-UTR of the first exon
of the FMR1 gene. Detailed analysis of the FMR1 gene
has revealed that the transcriptional regulation of the
FMR1 gene is influenced by the methylation boundary at
approximately 600-800 nucleotides upstream of the CGG
repeat (40, 41). The epigenetic modifications of the full
mutation alleles include histone modifications, which
consist of deacetylation of histones H3 and H4, low levels
of lysine 4 (H3K4) methylation, and high levels of lysine
9 (H3K9) methylation. All of these changes are associated
with a transcriptionally inactive heterochromatic
configuration (33, 34, 42).
Several studies investigated the epigenetic modifications
of the FMR1 gene in the full mutation alleles associated
with fragile X syndrome. These studies demonstrated that
the transcription and the translation of a methylated full
mutation can be relatively restored by treating fragile
X cells with the DNA demethylating drug 5-azadC
(36), whereas treatment with the inhibitors of histone
deacetylases (TSA and 4-phenylbutyrate) was found to
enhance the effect of 5-azadC, leading to changes in the
epigenetic code of histones H3 and H4 (37, 42).
In our study, epigenetic change of the FMR1 gene
consist of H3K9ac, H3K9me2, and H3K9me3, which
were examined in the promoter and the exon 1 region.
Our results showed that the incorporation of H3K9ac
and H3K9me2 were significantly higher in the regulatory
region of FMR1 in the DOR patient in comparison with the
control groups, whereas the incorporation of H3K9me3
showed no significant difference. Based on the epigenetic
profile data, it can be interpreted that although the
presence of CGG repeats causes an increase in H3K9me2
level, but this hypermethylation is not a permanent state
of heterochromatination. On the other hand, the dominant
hyperacetylation mark observed in this region is strongly
correlated with over expression of FMR1 gene in the
DOR patients rather than the control group.
According to the finding obtained in this study, we
propose that an increase in the number of CGG repeats to
55-200 results in the changes in the chromatin structure,
which itself leads to the recruitment of histone modifier
elements to this part of the genome. These epigenetic
alterations cause the different expression of FMR1 gene
observed in the diminished ovarian failure.
This project was financially supported by the Royan
Institute (Grant No. 91000018). The authors state that
there are no conflicts of interest in this study and would
like to dedicate this paper to the memory of Dr. Saeid
H.E., A.E.; Carried out the experiment, analysed the
data and wrote the manuscript with support from R.F.,
in technical performance, data analysis and drafting the
manuscript. U.A., Sh.Z.M.; Contributed in technical
performance of experiment. P.E.-Y., T.M.; Helped
in sample collection. M.Sh., A.M.M.; Conceived of
the idea and gave final approval of the version to be
published. All authors read and approved the final
te Velde ER,
Female reproductive ageing: current knowledge and future trends.
Trends Endocrinol Metab.
Early ovarian ageing: a hypothesis.Detection and clinical relevance.
A prospective evaluation of clomiphene citrate challenge test screening of the general infertility population.
82(4 Pt 1):
Chronic psychosocial stressors are detrimental to ovarian reserve: a study of infertile women.
J Psychosom Obstet Gynaecol.
FMR1 knockout mice are impaired in a leverpress escape/avoidance task.
Genes Brain Behav.
Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox.
Translational suppression by trinucleotide repeat expansion at FMR1.
High resolution methylation analysis of the FMR1 gene trinucleotide repeat region in fragile X syndrome.
Hum Mol Genet.
Absence of expression of the FMR-1 gene in fragile X syndrome.
Zari Moradi S,
Cytogenetic analysis of 179 Iranian women with premature ovarian failure.
Familial premature ovarian failure due to an interstitial deletion of the long arm of the X chromosome.
N Engl J Med.
Molecular and cytogenetic studies of an X;autosome translocation in a patient with premature ovarian failure and review of the literature.
Am J Med Genet.
Ovarian dysfunction and FMR1 alleles in a large Italian family with POF and FRAXA disorders: case report.
BMC Med Genet.
Premature ovarian failure and the FMR1 gene.
Semin Reprod Med.
The FMR1 gene as regulator of ovarian recruitment and ovarian reserve.
Obstet Gynecol Surv.
Zari Moradi S.
FMR1 premutation: not only important in premature ovarian failure but also in diminished ovarian reserve.
Hum Fertil (Camb).
Diminished ovarian reserve is not observed in infertility patients with high normal CGG repeats on the fragile X mental retardation 1 (FMR1) gene.
Perceptions of epigenetics.
An operational definition of epigenetics.
The complex language of chromatin regulation during transcription.
Chromatin modifications and their function.
Genomic patterns of DNA methylation: targets and function of an epigenetic mark.
Curr Opin Cell Biol.
Gene silencing quantitatively controls the function of a developmental trans-activator.
Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription.
Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex.
The language of covalent histone modifications.
Histone modification patterns and epigenetic codes.
Biochim Biophys Acta.
Heterochromatin and epigenetic control of gene expression.
An epigenetic road map for histone lysine methylation.
J Cell Sci.
Histone modifications depict an aberrantly heterochromatinized FMR1 gene in fragile x syndrome.
Am J Hum Genet.
Acetylated histones are associated with FMR1 in normal but not fragile X-syndrome cells.
Molecular dissection of the events leading to inactivation of the FMR1 gene.
Hum Mol Genet.
In vitro reactivation of the FMR1 gene involved in fragile X syndrome.
Hum Mol Genet.
Synergistic effect of histone hyperacetylation and DNA demethylation in the reactivation of the FMR1 gene.
Hum Mol Genet.
A simple salting out procedure for extracting DNA from human nucleated cells.
Nucleic Acids Res.
A rapid polymerase chain reaction-based screening method for identification of all expanded alleles of the fragile X (FMR1) gene in newborn and high-risk populations.
J Mol Diagn.
A distinct DNA-methylation boundary in the 5’upstream sequence of the FMR1 promoter binds nuclear proteins and is lost in fragile X syndrome.
Am J Hum Genet.
Epigenetic modifications of the FMR1 gene.
Methods Mol Biol.
Differential epigenetic modifications in the FMR1 gene of the fragile X syndrome after reactivating pharmacological treatments.
Eur J Hum Genet.