Overexpression of Endometrial Estrogen Receptor-Alpha in The
Window of Implantation in Women with Unexplained Infertility
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 (
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
Endometrial expression level of
Our findings demonstrate that reduction in the endometrial
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 (
Assuming that unexplained infertility can be due to
disturbances in the molecular and the cellular biomarkers
involved in implantation (15), we hypothesized that
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.
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.
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).
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.
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.
Quantitative real-time polymerase chain reaction analysis
Real-time PCR was performed for relative quantification
|Gene||Primer sequencing (5´→3´)||Accession number|
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
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,).
|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.
Relative expressions of
|- 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.|
|- 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
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
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
Moreover, it seems that any change in the balance between
estrogen and progesterone could disturb the timing
Any inability in the
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
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.
The present study shows the prognostic significance of
The authors wish to thank the Vice Chancellor for Research of Shahid Chamran University of Ahvaz for providing the research grants (No. 867615). The authors declare that there is no conflict of interest.
M.D.; Designed and directed the project. F.M.; Contributed to sample preparation. H.-o-a.G.; Planned the RT-PCR method. N.K.; Performed the experiments. All authors discussed the results and contributed to the writing and read and approved the final manuscript.