Background Failure in the endometrial receptivity may account for a significant number of infertility cases including unexplained infertility in women. Reduction in the endometrial estrogen receptor-alpha (ER-α) expression during implantation may be a critical event that coincides with the expression of specific genes and the formation of a receptive endometrium. The aim of the present study was to assess the expression of ER-α in the mid-secretory phase in the endometrium of women with unexplained infertility. Materials and Methods This case-control study was carried out on randomly selected fertile (n=10) and infertile (n=16) women whose source of infertility remained unexplained. We evaluated the expression of ER-α and glycode-lin-A (GdA) through mRNA level measurement with real-time polymerase chain reaction (PCR) in the endometrium of fertile women and patients suffering from unexplained infertility and fertile women. Endometrial biopsies of each subject were collected during a single menstrual cycle 7 days after the peak of luteinizing hormone (LH+7). Results Endometrial expression level of ER-α was significantly (P < 0.05) higher in the patients with unexplained infertility compared to the control. Significantly (P < 0.05) lower levels of GdA expression were seen in women with unexplained infertility. A statistically non-significant negative correlation was observed between ER-α and GdA mRNA expression. Conclusion Our findings demonstrate that reduction in the endometrial GdA expression is associated with elevated expression of ER-α in mid-luteal phase. Disruption in the endometrial ER-α expression, which leads to defects in uterine receptivity, may contribute to unexplained infertility.
Endometrial receptivity plays a key role in the establishment
of a successful implantation and its impairment
may contribute to infertility in women (1). A variety of
molecules such as hormones, receptors, adhesion molecules,
growth factors and cytokines mediate the embryomaternal
crosstalk and facilitate the reception of a blastocyst
and the establishment of implantation (2). During
the menstrual cycle uterine receptivity is regulated by the
secretion of the ovarian steroids. Endometrial proliferation
is induced by estrogen during the preovulatory phase,
whereas progesterone causes secretory changes in the estrogen-
primed endometrium (3).
Ligand-specific intracellular receptors located in stro.
mal and epithelial endometrial cells mediate the actions of
estrogen and progesterone (4). It is thought that the pres.
ence of progesterone after appropriate estrogen priming is
required to stimulate key implantation-specific events in
the mid-secretory phase of the menstrual cycle (5).
Estrogen receptor-alpha (ER-α) increases during the
proliferative phase in response to estrogen and is downregulated
during the window of implantation in response
to progesterone (6). The disappearance of ER-α at the
time of implantation has been reported in most mammalian
species (7). The decline in ER-α coincides with endometrial
gene expression in the mid-luteal phase, and is a
critical event in the establishment of endometrial receptivity
(8). High levels of ER-α during implantation were
observed in women with polycystic ovarian syndrome
(PCOS) and endometriosis. Elevated expression of ER-α
in both groups of patients was associated with the reduction
in beta 3 integrin expression, a marker of endometrial
receptivity (9). It has been suggested that the disappearance
of ER-α at the time of implantation may disturb the
expression pattern of proteins that regulate the endometrial
receptivity.
Glycodelin-A (GdA) is a progesterone-regulated glycoprotein
with immunosuppressive properties that is highly
upregulated in glandular epithelium at implantation and
plays a role in the formation a receptive endometrium
(10). GdA expression is concurrent with pinopode formation
in the receptive endometrium (11), indicating that it
can potentially be seen as a diagnostic marker of morphological
differentiation of human endometrium (12). A
lower glycodelin expression in secretory phase was found
in eutopic endometrium of endometriosis patients and in
uterine flushings from women with unexplained infertility
when compared to the healthy controls (13, 14).
Assuming that unexplained infertility can be due to
disturbances in the molecular and the cellular biomarkers
involved in implantation (15), we hypothesized that
continued ER-α expression may be detrimental to the
development of endometrial receptivity. In present study
expression of GdA, as a particular marker of endometrial
receptivity, was assessed at the time of implantation.
Materials and Methods
This case-control study was approved by the Research
Ethics Committee of Shahid Chamran University of Ahvaz,
Iran. The study was performed in the Laboratory of
Embryology, Department of Biology. Written informed
consent was obtained from each participant.
Sample collection
Endometrial biopsy samples were collected using a
Novak curette in the mid-luteal phase at day luteinizing
hormone (LH)+7 from healthy volunteers women with
proven fertility (n=10, age 32.5 ± 3.2 Y) and women
with unexplained infertility (n=16, age 31.6 ± 3.0 Y) that
showed primary infertility for more than 2 years (30.5
± 4.7 months). The unfertile females were randomly selected
from a population of such females listed in Imam
Khomeini hospital medical records. Endometrial samples
were divided into two parts. One sample was fixed
in 10% formalin and embedded in paraffin. After tissue
processing, 5-6 µm sections were stained with haematoxylin-
eosin, evaluated histologically to correspond all
samples to the assumed time in the cycle according to
the Noyes et al. (16) criteria. The other sample was immediately
stored in RNA later at -80°C for later use in
real-time polymerase chain reaction (RT-PCR). Sample
size was determined based on previous studies (17, 18).
Sample size was smaller in the fertile group due to the low
collaboration. The concentration of LH in morning urine
(ACON Laboratories, Inc., USA) was used to determine
the day of the surge.
All women included in this study had normal ovarian
function and regular menstrual cycles, confirmed based
on their menstrual histories, and none of them had used
steroid hormones, (for at least 6 months prior to study),
and intra-uterine contraceptives. Women with unexplained
infertility showed normal ovulatory cycles and
mid-luteal serum progesterone levels, normal tubal patency
and no recognizable endometriosis based on symptoms
and clinical examination in transvaginal ultrasonography
or diagnostic laparoscopy. Moreover, unexplained infertile
women had partners with normal semen according
to WHO criteria. Patients with history of pelvic inflammatory
diseases, pelvic surgery including cesarean section,
unilateral tubal patency, ovarian hyperstimulation
syndrome, diminished ovarian response, endometriosis or
multiple female factor were excluded from this study.
Hormone assay
Blood samples were obtained in the fasted state on the
same day as endometrial sampling and serum levels of
LH, follicle stimulating hormone (FSH), estradiol (E2),
and progesterone (P4) were measured using commercially
available kits (Abcam plc, UK).
RNA extraction
Total RNA was extracted from the endometrial tissues
(approximately 50-100 mg) using Tripure (Roche Diagnostics,
Germany), according to the recommended protocol
by the manufacturer. RNA integrity was analyzed
via electrophoresis and total RNA concentration was obtained
using a spectrophotometer at an optical density of
260 nm. The RNA was stored at -70°C for future procedures.
cDNA synthesis
Synthesis of cDNA was carried out using 1 mg of total
RNA from each sample with random hexamer primers using
prime Script™ RT reagent Kit (Takara Bio Inc., Japan)
according to the manufacturer’s instructions.
Real-time PCR was performed for relative quantification
of the ER-α and GdA genes expression using ABI
StepOne plus™ System (Applied Biosystems, Germany).
Hypoxanthine phosphoribosyltransferase (HPRT)
gene was used as the housekeeping gene. Forward and
reverse primer sequences for each gene are presented
in Table 1. The specificity of primers for each gene was
analyzed in the BLAST database. The reaction mixture
consisted of 10 µl Master mix SYBR Green, 2 µl
cDNA, 1 µl of each primer (10 pmol/µl), and 7 µl dH2O
(Qiagen, Germany). The standard cycling protocol
used for all genes consisted of DNA denaturation and
enzyme activation at 95°C for 10 minutes, denaturation
95°C for 15 seconds, annealing at 62°C for 15 seconds
and extension and florescence acquiring at 72°C for 15
seconds. The RT-PCR procedure was carried out 40 cycles.
Melting curve analysis was performed by bringing
the temperature from 95°C to 60°C for 60 seconds
at the transition rate of 1 degree per second. As Livak
and Schmittgen (2001) described, for sample analysis
the threshold was set based on the exponential phase of
products and the 2-ΔΔCT method was performed to analyze
the data (19).
Primer sequences used in real-time polymerase chain reaction
Gene
Primer sequencing (5´→3´)
Accession number
ER-α
F: TGCTTCAGGCTACCATTATGGA
NM-001122742
R: TGGCTGGACACATATAGTCGTT
GdA
F: GAGATCGTTCTGCACAGATGG
NM-001018049
R: CGTTCGCCACCGTATAGTTGAT
HPRT
F: TGGACAGGACTGAACGTCTTG
NM-000194
R: CCAGCAGGTCAGCAAAGAATTTA
ER-α; Estrogen receptor-alpha, GdA; Glycodelin-A, and HPRT; Hypoxanthine hosphoribosyltransferase.
Statistical analysis
Data was analyzed by SPSS version 16 software
(SPSS Inc., USA). Independent samples t test was
performed to compare characteristics and hormonal
profile of the fertile and the infertile women. Results
are expressed as mean ± SD. Comparison of ER-α
and GdA expression in studied groups was done using
Mann-Whitney U-test. Spearman correlation
analysis was carried out to investigate the relationship
between variables. The level of significance
was set at P<0.05.
Results
Of the 54 couples with unexplained infertility, 8 couples
were excluded based on their medical records. Among 25
randomly-selected eligible patients with unexplained infertility,
9 couples refused participation. As a result, 16
infertile couples were included in the study. In addition,
10 fertile women (16.1%) out of the 62 eligible couples
were included in the study. The mean age, body mass
index (BMI), cycle length, duration of menses and hormonal
profile in women of both groups are presented in
Table 2. There were no differences in age, BMI, cycle
length, duration of menses and serum LH, FSH, estradiol
and progestrone concentrations between the two groups.
Microscopic analysis of the endometrial biopsies showed
that all samples corresponded histologically to the mid-
luteal phase of endometrial cycle (Fig .1).
Characteristics and hormonal profile of the fertile and infertile women in the mid-luteal phase
Parameter
Fertile women n=10
Infertile women n=16
P value
Age (Y)
31.7 ± 5.9
32.2 ± 5.5
NS
BMI (kg/m2)
23.7 ± 2.8
23.4 ± 2.6
NS
Cycle length (days)
28.2 ± 1.3
28.5 ± 1.5
NS
Menses duration (days)
4.2 ± 0.5
4.5 ± 0.6
NS
LH (mIU/mL)
12.54 ± 6.85
13.27 ± 7.13
NS
FSH (mIU/mL)
5.90 ± 2.62
6.58 ± 2.50
NS
Estradiol (pg/ml)
139.3 ± 55.4
142.9 ± 61.6
NS
Progestrone (ng/mL)
10.93 ± 3.21
11.48 ± 4.86
NS
Independent samples t test was done as the test of significant. Results expressed as mean ± SD. The level of significance was set at P<0.05. BMI; Body mass index, LH; Luteinizing hormone, FSH; Follicle stimulating hormone, and NS; Non significant.
Microscopic structure of endometrium at the mid-luteal phase. A. Scale
bar=200 µm and B. Scale bar=100 µm, H&E. Stromal edema and coiled endometrial
glands that contain secretions with sub-nuclear vacuolization (red
arrows) in their epithelium exhibit endometrium in the mid-luteal phase.
Relative expressions of ER-α and GdA in the mid-luteal
endometrium of the patients with unexplained infertility and
healthy fertile women are shown in Figures 2 and 3. Expression
levels of ER-α and GdA mRNA are given relative to the
expression levels of the reference gene, HPRT. Levels of ER-α
mRNA expression in the endometrium of the patients with unexplained
infertility were significantly higher than those in the
fertile women (P=0.007, Mann-Whitney U-test, Fig .2).
Relative expression of ER-α in the mid-luteal endometrium of patients
with unexplained infertility (n=16) was significantly higher than those in
healthy fertile women (n=10, P=0.007, Mann-Whitney U-test). *; P<0.05.
GdA mRNA levels were significantly lower in the infertile
women compared to the healthy fertile group
(P=0.045, Mann-Whitney U-test, Fig .3).
Relative expression of GdA in the mid-luteal endometrium of patients
with unexplained infertility (n=16) was significantly lower than
those in healthy fertile women (n=10, P=0.045, Mann-Whitney U-test).
*; P<0.05.
A statistically non-significant negative correlation was
observed between ER-α and GdA mRNA expression levels
in the fertile women (r=-0.047, P=0.845) and in the patients
with unexplained infertility (r=-0.205, P=0.316, Fig .4).
Correlation between ER-α and GdA mRNA expressions in the mid-
luteal endometrium of the healthy fertile women (r=-0.047, P=0.845) and
the patients with unexplained infertility (r=-0.205, P=0.316).
Discussion
Implantation failure is believed to be a major cause of
infertility (20). Successful embryo implantation depends
on the development of an endometrium that is receptive to
the embryo (21). Coordinated interactions between estrogen
and progesterone resulting in a series of synchronized
molecular events during menstrual cycle ultimately lead
to the preparation of a receptive endometrium (22).
The present study showed that a lack of appropriate levels
of ER-α downregulation in the mid-luteal phase in the
patients with unexplained infertility relative to the control
group. During implantation ER-α is being downregulated
in response to progesterone. Downregulation of ER-α during
the mid-secretory phase is one of the primary actions
of progesterone. The combination of estrogen withdrawal
and progesterone action is required to stimulate the endometrial
gene expression in the mid-luteal phase (8). Disappearance
of ER-α in the mid-luteal phase provides the
opportunity for progesterone to act alone specifically on
the stroma (6). Paracrine activity of stroma in response
to progesterone results in epithelial gene expression (7).
Similar findings have been reported in patients with endometriosis
and in women with PCOS (9).
Inadequate progesterone levels, defects in the progesterone
receptor, hypersensitivity to estrogen, inappropriate
expression of aromatase and progesterone resistance
are among the reasons that can cause this failure to downregulate
ER-α in the mid-luteal phase. Insufficient serum
level of progesterone in the luteal phase defect (LPD) may
delay the timing of ER-α downregulation during implantation
(23). Resistance to progesterone due to aberrant expression
or activity of receptor results in estrogenicity in
endometrial tissue (24). The loss of progesterone activity
caused by defect in the progesterone receptor (25) and/
or an increase in the local estrogen production due to inappropriate
expression of aromatase (26) may cause the
persistence of ER-α in endometriosis patients. A failure
in ER-α downregulation has been reported in ovarian and
peritoneal endometriosis (27). Increased production of
estrogen contributes to the pathophysiology of the endometriosis
as a mitogen causing aberrant proliferation (28)
and inhibition of apoptosis (29). Overexpression of steroid
receptor co-activators in PCOS patients which marks
the hypersensitivity to estrogen may explain elevated endometrial
ER-α expression (9).
Moreover, it seems that any change in the balance between
estrogen and progesterone could disturb the timing
of ER-α downregulation in mid-luteal phase. Endocrine
disrupting chemicals (EDCs) or xenoestrogens are natural
or synthetic chemicals in the diet or the environment
that mimic the endogenous estrogens functions or interfere
with estrogen signaling pathways (30). Lower levels
of progesterone metabolite have been found during the
luteal phase with higher concentration of Dichlorodiphenyldichloroethylene
(DDE) (31). Impaired implantation
has been reported in patients with an increase in serum
17ß-estradiol (E2) levels during the pre-implantation period,
while reducing E2 levels during the pre-implantation
period by a step-down protocol increases implantation
and pregnancy rates (32). Accordingly, the possibility
of manipulating the receptivity window with the use of
different doses of E2 has been suggested (33). Aberrant
uterine expression of implantation-related genes has been
found at high estrogen levels (34), suggesting that in in
vitro fertilization (IVF) programs estrogen levels regulation
is important for improvement of women fertility.
Any inability in the ER-α downregulation may lead
to failure to express essential proteins associated with
uterine receptivity, in turn resulting in either infertility
or pregnancy loss (35). The present study showes that
ER-α overexpression is accompanied by downregulation
of GdA in the mid-luteal endometrium of the patients
with unexplained infertility. GdA, a potential diagnostic
marker of the endometrial receptivity, is the major progesterone-
regulated glycoprotein and has been demonstrated
in the pinopodes of receptive-phase human endometrium
(11). Lower levels of GdA has been reported in
the secretory phase of the menstrual cycle in the eutopic
tissue of patients with endometriosis (13). In addition,
lower levels of GdA were detected in the uterine flushings
on days LH+10 and LH+12 in women with unexplained
infertility (14) and recurrent miscarriage (36).
A negative but statistically non-significant correlation
was found between ER-α and GdA in fertile women and
in patients with unexplained infertility. Although transcription,
synthesis, and secretion of endometrial GdA
are regulated by progesterone, according to our findings
one can assume that the overexpression of endometrial
ER-α disturbs the expression of special genes during the
implantation, which is detrimental to the development of
uterine receptivity.
Inadequate uterine receptivity is responsible for approximately
two-thirds of implantation failures (37). A range
of cellular and molecular endometrial defects has been
associated with unexplained infertility (38). Microarray
analysis demonstrated that endometrial gene expression
at the time of embryo implantation is considerably different
in the unexplained infertile patients compared to the
fertile women (39).
Therefore, the failure in ER-α downregulation and the
observed disturbance in GdA expression in the patients
with unexplained infertility may elucidate the causes of
unexplained infertility. Our observations suggest that endometrial
ER-α expression may participate in the cascade
of molecular events leading to successful implantation.
The random inclusion of all cases diagnosed with unexplained
infertility is the main strength of this study.
Furthermore, real-time PCR based assay of endometrial
markers, an extremely sensitive technique that allows the
precise measurement of gene expression (40), increases
the accuracy and external validity of our results. However,
data was collected from a single randomized center
and subjects represent only a fraction of the population,
thus reducing the population validity. Moreover, unexplained
infertile women with secondary infertility were
excluded, so its external validity is restricted to women
with primary infertility.
Conclusion
The present study shows the prognostic significance of
ER-α expression in patients with unexplained infertility.
Disruption in the endometrial ER-α expression, which
leads to defects in the uterine receptivity may contribute
to unexplained infertility. In addition, our findings demonstrate
that reduction in endometrial GdA expression
was associated with elevated expression of ER-α in the
mid-luteal phase. However, our study has some limitations
including the low number of cases of unexplained
infertile women with primary infertility. Studies including
more tissue samples and protein-based assays such as
immunohistochemistry and western blot analysis are also
needed to further determine the role of endometrial ER-α.
Understanding of biomarkers involved in the implantation
and the mechanisms governing their relationships
in endometrial receptivity could provide new therapeutic
strategies for unexplained infertility. Whether such defects
of uterine receptivity could be treated by the therapeutic
blockage of ER-α activity or by dealing with the related
causes of ER-α overexpression, e.g., using progestins or
aromatase inhibitors to normalize the expression pattern
of endometrial biomarkers associated with implantation,
requires further investigation.
Dorostghoal, M., Ghaffari, H., Moramezi, F., & Keikhah, N. (2018). Overexpression of Endometrial Estrogen Receptor-Alpha in The Window of Implantation in Women with Unexplained Infertility. International Journal of Fertility and Sterility, 12(1), 37-42. doi: 10.22074/ijfs.2018.5118
MLA
Mehran Dorostghoal; Hamid-o-allah Ghaffari; Farideh Moramezi; Narjes Keikhah. "Overexpression of Endometrial Estrogen Receptor-Alpha in The Window of Implantation in Women with Unexplained Infertility". International Journal of Fertility and Sterility, 12, 1, 2018, 37-42. doi: 10.22074/ijfs.2018.5118
HARVARD
Dorostghoal, M., Ghaffari, H., Moramezi, F., Keikhah, N. (2018). 'Overexpression of Endometrial Estrogen Receptor-Alpha in The Window of Implantation in Women with Unexplained Infertility', International Journal of Fertility and Sterility, 12(1), pp. 37-42. doi: 10.22074/ijfs.2018.5118
VANCOUVER
Dorostghoal, M., Ghaffari, H., Moramezi, F., Keikhah, N. Overexpression of Endometrial Estrogen Receptor-Alpha in The Window of Implantation in Women with Unexplained Infertility. International Journal of Fertility and Sterility, 2018; 12(1): 37-42. doi: 10.22074/ijfs.2018.5118