{"paper_id":"f5b61c8f-8bf4-4d63-b897-8c309374cc6a","body_text":"Endometrial receptivity plays a key role in the establishment\nof a successful implantation and its impairment\nmay contribute to infertility in women ( 1 ). A variety of\nmolecules such as hormones, receptors, adhesion molecules,\ngrowth factors and cytokines mediate the embryomaternal\ncrosstalk and facilitate the reception of a blastocyst\nand the establishment of implantation ( 2 ). During\nthe menstrual cycle uterine receptivity is regulated by the\nsecretion of the ovarian steroids. Endometrial proliferation\nis induced by estrogen during the preovulatory phase,\nwhereas progesterone causes secretory changes in the estrogen-\nprimed endometrium ( 3 ).\nLigand-specific intracellular receptors located in stro.\nmal and epithelial endometrial cells mediate the actions of \nestrogen and progesterone ( 4 ). It is thought that the pres.\nence of progesterone after appropriate estrogen priming is \nrequired to stimulate key implantation-specific events in\nthe mid-secretory phase of the menstrual cycle ( 5 ).\nEstrogen receptor-alpha ( ER-α ) increases during the\nproliferative phase in response to estrogen and is downregulated\nduring the window of implantation in response\nto progesterone ( 6 ). The disappearance of  ER-α  at the\ntime of implantation has been reported in most mammalian\nspecies ( 7 ). The decline in  ER-α  coincides with endometrial\ngene expression in the mid-luteal phase, and is a\ncritical event in the establishment of endometrial receptivity\n( 8 ). High levels of  ER-α  during implantation were\nobserved in women with polycystic ovarian syndrome\n(PCOS) and endometriosis. Elevated expression of  ER-α \nin both groups of patients was associated with the reduction\nin beta 3 integrin expression, a marker of endometrial\nreceptivity ( 9 ). It has been suggested that the disappearance\nof  ER-α  at the time of implantation may disturb the\nexpression pattern of proteins that regulate the endometrial\nreceptivity.\nGlycodelin-A ( GdA ) is a progesterone-regulated glycoprotein \nwith immunosuppressive properties that is highly \nupregulated in glandular epithelium at implantation and \nplays a role in the formation a receptive endometrium \n( 10 ).  GdA  expression is concurrent with pinopode formation \nin the receptive endometrium ( 11 ), indicating that it \ncan potentially be seen as a diagnostic marker of morphological \ndifferentiation of human endometrium ( 12 ). A \nlower glycodelin expression in secretory phase was found \nin eutopic endometrium of endometriosis patients and in \nuterine flushings from women with unexplained infertility \nwhen compared to the healthy controls ( 13 ,  14 ).\nAssuming that unexplained infertility can be due to \ndisturbances in the molecular and the cellular biomarkers \ninvolved in implantation ( 15 ), we hypothesized that \ncontinued  ER-α  expression may be detrimental to the \ndevelopment of endometrial receptivity. In present study \nexpression of  GdA , as a particular marker of endometrial \nreceptivity, was assessed at the time of implantation.\n\nThis case-control study was approved by the Research \nEthics Committee of Shahid Chamran University of Ahvaz, \nIran. The study was performed in the Laboratory of \nEmbryology, Department of Biology. Written informed \nconsent was obtained from each participant.\n\nEndometrial biopsy samples were collected using a \nNovak curette in the mid-luteal phase at day luteinizing \nhormone (LH)+7 from healthy volunteers women with \nproven fertility (n=10, age 32.5 ± 3.2 Y) and women \nwith unexplained infertility (n=16, age 31.6 ± 3.0 Y) that \nshowed primary infertility for more than 2 years (30.5 \n± 4.7 months). The unfertile females were randomly selected \nfrom a population of such females listed in Imam \nKhomeini hospital medical records. Endometrial samples \nwere divided into two parts. One sample was fixed \nin 10% formalin and embedded in paraffin. After tissue \nprocessing, 5-6 µm sections were stained with haematoxylin-\neosin, evaluated histologically to correspond all \nsamples to the assumed time in the cycle according to \nthe Noyes et al. ( 16 ) criteria. The other sample was immediately \nstored in RNA later at -80°C for later use in \nreal-time polymerase chain reaction (RT-PCR). Sample \nsize was determined based on previous studies ( 17 ,  18 ). \nSample size was smaller in the fertile group due to the low \ncollaboration. The concentration of LH in morning urine \n(ACON Laboratories, Inc., USA) was used to determine \nthe day of the surge.\nAll women included in this study had normal ovarian \nfunction and regular menstrual cycles, confirmed based \non their menstrual histories, and none of them had used \nsteroid hormones, (for at least 6 months prior to study), \nand intra-uterine contraceptives. Women with unexplained \ninfertility showed normal ovulatory cycles and \nmid-luteal serum progesterone levels, normal tubal patency \nand no recognizable endometriosis based on symptoms \nand clinical examination in transvaginal ultrasonography \nor diagnostic laparoscopy. Moreover, unexplained infertile \nwomen had partners with normal semen according \nto WHO criteria. Patients with history of pelvic inflammatory \ndiseases, pelvic surgery including cesarean section, \nunilateral tubal patency, ovarian hyperstimulation \nsyndrome, diminished ovarian response, endometriosis or \nmultiple female factor were excluded from this study.\n\nBlood samples were obtained in the fasted state on the \nsame day as endometrial sampling and serum levels of \nLH, follicle stimulating hormone (FSH), estradiol (E2), \nand progesterone (P4) were measured using commercially \navailable kits (Abcam plc, UK).\n\nTotal RNA was extracted from the endometrial tissues \n(approximately 50-100 mg) using Tripure (Roche Diagnostics, \nGermany), according to the recommended protocol \nby the manufacturer. RNA integrity was analyzed \nvia electrophoresis and total RNA concentration was obtained \nusing a spectrophotometer at an optical density of \n260 nm. The RNA was stored at -70°C for future procedures.\n\nSynthesis of cDNA was carried out using 1 mg of total \nRNA from each sample with random hexamer primers using \nprime Script™ RT reagent Kit (Takara Bio Inc., Japan) \naccording to the manufacturer’s instructions.\n\nReal-time PCR was performed for relative quantification \nof the  ER-α  and  GdA  genes expression using ABI \nStepOne plus™ System (Applied Biosystems, Germany). \nHypoxanthine phosphoribosyltransferase (HPRT) \ngene was used as the housekeeping gene. Forward and \nreverse primer sequences for each gene are presented \nin Table 1. The specificity of primers for each gene was \nanalyzed in the BLAST database. The reaction mixture \nconsisted of 10 µl Master mix SYBR Green, 2 µl \ncDNA, 1 µl of each primer (10 pmol/µl), and 7 µl dH 2 O \n(Qiagen, Germany). The standard cycling protocol \nused for all genes consisted of DNA denaturation and \nenzyme activation at 95°C for 10 minutes, denaturation \n95°C for 15 seconds, annealing at 62°C for 15 seconds \nand extension and florescence acquiring at 72°C for 15 \nseconds. The RT-PCR procedure was carried out 40 cycles. \nMelting curve analysis was performed by bringing \nthe temperature from 95°C to 60°C for 60 seconds \nat the transition rate of 1 degree per second. As Livak \nand Schmittgen (2001) described, for sample analysis \nthe threshold was set based on the exponential phase of \nproducts and the 2 -ΔΔCT  method was performed to analyze \nthe data ( 19 ).\nPrimer sequences used in real-time polymerase chain reaction\nER-α ; Estrogen receptor-alpha,  GdA ; Glycodelin-A, and HPRT; Hypoxanthine hosphoribosyltransferase.\n\nData was analyzed by SPSS version 16 software \n(SPSS Inc., USA). Independent samples t test was \nperformed to compare characteristics and hormonal \nprofile of the fertile and the infertile women. Results \nare expressed as mean ± SD. Comparison of  ER-α  \nand  GdA  expression in studied groups was done using \nMann-Whitney U-test. Spearman correlation \nanalysis was carried out to investigate the relationship \nbetween variables. The level of significance \nwas set at P<0.05.\n\nOf the 54 couples with unexplained infertility, 8 couples \nwere excluded based on their medical records. Among 25 \nrandomly-selected eligible patients with unexplained infertility, \n9 couples refused participation. As a result, 16 \ninfertile couples were included in the study. In addition, \n10 fertile women (16.1%) out of the 62 eligible couples \nwere included in the study. The mean age, body mass \nindex (BMI), cycle length, duration of menses and hormonal \nprofile in women of both groups are presented in \nTable 2. There were no differences in age, BMI, cycle \nlength, duration of menses and serum LH, FSH, estradiol \nand progestrone concentrations between the two groups. \nMicroscopic analysis of the endometrial biopsies showed \nthat all samples corresponded histologically to the mid-\nluteal phase of endometrial cycle ( Fig .1 ).\nCharacteristics and hormonal profile of the fertile and infertile women in the mid-luteal phase\nIndependent 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.\nMicroscopic structure of endometrium at the mid-luteal phase. A. Scale \nbar=200 µm and B. Scale bar=100 µm, H&E. Stromal edema and coiled endometrial \nglands that contain secretions with sub-nuclear vacuolization (red \narrows) in their epithelium exhibit endometrium in the mid-luteal phase.\nRelative expressions of  ER-α  and  GdA  in the mid-luteal \nendometrium of the patients with unexplained infertility and \nhealthy fertile women are shown in Figures 2 and 3. Expression \nlevels of  ER-α  and  GdA  mRNA are given relative to the \nexpression levels of the reference gene, HPRT. Levels of  ER-α  \nmRNA expression in the endometrium of the patients with unexplained \ninfertility were significantly higher than those in the \nfertile women (P=0.007, Mann-Whitney U-test,  Fig .2 ).\nRelative expression of  ER-α  in the mid-luteal endometrium of patients \nwith unexplained infertility (n=16) was significantly higher than those in \nhealthy fertile women (n=10, P=0.007, Mann-Whitney U-test). *; P<0.05.\nGdA  mRNA levels were significantly lower in the infertile \nwomen compared to the healthy fertile group \n(P=0.045, Mann-Whitney U-test,  Fig .3 ).\nRelative expression of  GdA  in the mid-luteal endometrium of patients \nwith unexplained infertility (n=16) was significantly lower than \nthose in healthy fertile women (n=10, P=0.045, Mann-Whitney U-test). \n*; P<0.05.\nA statistically non-significant negative correlation was \nobserved between  ER-α  and  GdA  mRNA expression levels \nin the fertile women (r=-0.047, P=0.845) and in the patients \nwith unexplained infertility (r=-0.205, P=0.316,  Fig .4 ).\nCorrelation between  ER-α  and  GdA  mRNA expressions in the mid-\nluteal endometrium of the healthy fertile women (r=-0.047, P=0.845) and \nthe patients with unexplained infertility (r=-0.205, P=0.316).\n\nImplantation failure is believed to be a major cause of \ninfertility ( 20 ). Successful embryo implantation depends \non the development of an endometrium that is receptive to \nthe embryo ( 21 ). Coordinated interactions between estrogen \nand progesterone resulting in a series of synchronized \nmolecular events during menstrual cycle ultimately lead \nto the preparation of a receptive endometrium ( 22 ).\nThe present study showed that a lack of appropriate levels \nof  ER-α  downregulation in the mid-luteal phase in the \npatients with unexplained infertility relative to the control \ngroup. During implantation  ER-α  is being downregulated \nin response to progesterone. Downregulation of  ER-α  during \nthe mid-secretory phase is one of the primary actions \nof progesterone. The combination of estrogen withdrawal \nand progesterone action is required to stimulate the endometrial \ngene expression in the mid-luteal phase ( 8 ). Disappearance \nof  ER-α  in the mid-luteal phase provides the \nopportunity for progesterone to act alone specifically on \nthe stroma ( 6 ). Paracrine activity of stroma in response \nto progesterone results in epithelial gene expression ( 7 ). \nSimilar findings have been reported in patients with endometriosis \nand in women with PCOS ( 9 ).\nInadequate progesterone levels, defects in the progesterone \nreceptor, hypersensitivity to estrogen, inappropriate \nexpression of aromatase and progesterone resistance \nare among the reasons that can cause this failure to downregulate \n ER-α  in the mid-luteal phase. Insufficient serum \nlevel of progesterone in the luteal phase defect (LPD) may \ndelay the timing of  ER-α  downregulation during implantation \n( 23 ). Resistance to progesterone due to aberrant expression \nor activity of receptor results in estrogenicity in \nendometrial tissue ( 24 ). The loss of progesterone activity \ncaused by defect in the progesterone receptor ( 25 ) and/\nor an increase in the local estrogen production due to inappropriate \nexpression of aromatase ( 26 ) may cause the \npersistence of  ER-α  in endometriosis patients. A failure \nin  ER-α  downregulation has been reported in ovarian and \nperitoneal endometriosis ( 27 ). Increased production of \nestrogen contributes to the pathophysiology of the endometriosis \nas a mitogen causing aberrant proliferation ( 28 ) \nand inhibition of apoptosis ( 29 ). Overexpression of steroid \nreceptor co-activators in PCOS patients which marks \nthe hypersensitivity to estrogen may explain elevated endometrial \n ER-α  expression ( 9 ).\nMoreover, it seems that any change in the balance between \nestrogen and progesterone could disturb the timing \nof  ER-α  downregulation in mid-luteal phase. Endocrine \ndisrupting chemicals (EDCs) or xenoestrogens are natural \nor synthetic chemicals in the diet or the environment \nthat mimic the endogenous estrogens functions or interfere \nwith estrogen signaling pathways ( 30 ). Lower levels \nof progesterone metabolite have been found during the \nluteal phase with higher concentration of Dichlorodiphenyldichloroethylene \n(DDE) ( 31 ). Impaired implantation \nhas been reported in patients with an increase in serum \n17ß-estradiol (E2) levels during the pre-implantation period, \nwhile reducing E2 levels during the pre-implantation \nperiod by a step-down protocol increases implantation \nand pregnancy rates ( 32 ). Accordingly, the possibility \nof manipulating the receptivity window with the use of \ndifferent doses of E2 has been suggested ( 33 ). Aberrant \nuterine expression of implantation-related genes has been \nfound at high estrogen levels ( 34 ), suggesting that in in \nvitro fertilization (IVF) programs estrogen levels regulation \nis important for improvement of women fertility.\nAny inability in the  ER-α  downregulation may lead \nto failure to express essential proteins associated with \nuterine receptivity, in turn resulting in either infertility \nor pregnancy loss ( 35 ). The present study showes that \n ER-α  overexpression is accompanied by downregulation \nof  GdA  in the mid-luteal endometrium of the patients \nwith unexplained infertility.  GdA , a potential diagnostic \nmarker of the endometrial receptivity, is the major progesterone-\nregulated glycoprotein and has been demonstrated \nin the pinopodes of receptive-phase human endometrium \n( 11 ). Lower levels of  GdA  has been reported in \nthe secretory phase of the menstrual cycle in the eutopic \ntissue of patients with endometriosis ( 13 ). In addition, \nlower levels of  GdA  were detected in the uterine flushings \non days LH+10 and LH+12 in women with unexplained \ninfertility ( 14 ) and recurrent miscarriage ( 36 ). \nA negative but statistically non-significant correlation \nwas found between  ER-α  and  GdA  in fertile women and \nin patients with unexplained infertility. Although transcription, \nsynthesis, and secretion of endometrial  GdA  \nare regulated by progesterone, according to our findings \none can assume that the overexpression of endometrial \n ER-α  disturbs the expression of special genes during the \nimplantation, which is detrimental to the development of \nuterine receptivity.\nInadequate uterine receptivity is responsible for approximately \ntwo-thirds of implantation failures ( 37 ). A range \nof cellular and molecular endometrial defects has been \nassociated with unexplained infertility ( 38 ). Microarray \nanalysis demonstrated that endometrial gene expression \nat the time of embryo implantation is considerably different \nin the unexplained infertile patients compared to the \nfertile women ( 39 ).\nTherefore, the failure in  ER-α  downregulation and the \nobserved disturbance in  GdA  expression in the patients \nwith unexplained infertility may elucidate the causes of \nunexplained infertility. Our observations suggest that endometrial \n ER-α  expression may participate in the cascade \nof molecular events leading to successful implantation.\nThe random inclusion of all cases diagnosed with unexplained \ninfertility is the main strength of this study. \nFurthermore, real-time PCR based assay of endometrial \nmarkers, an extremely sensitive technique that allows the \nprecise measurement of gene expression ( 40 ), increases \nthe accuracy and external validity of our results. However, \ndata was collected from a single randomized center \nand subjects represent only a fraction of the population, \nthus reducing the population validity. Moreover, unexplained \ninfertile women with secondary infertility were \nexcluded, so its external validity is restricted to women \nwith primary infertility.\n\nThe present study shows the prognostic significance of \n ER-α  expression in patients with unexplained infertility. \nDisruption in the endometrial  ER-α  expression, which \nleads to defects in the uterine receptivity may contribute \nto unexplained infertility. In addition, our findings demonstrate \nthat reduction in endometrial  GdA  expression \nwas associated with elevated expression of  ER-α  in the \nmid-luteal phase. However, our study has some limitations \nincluding the low number of cases of unexplained \ninfertile women with primary infertility. Studies including \nmore tissue samples and protein-based assays such as \nimmunohistochemistry and western blot analysis are also \nneeded to further determine the role of endometrial  ER-α .\nUnderstanding of biomarkers involved in the implantation \nand the mechanisms governing their relationships \nin endometrial receptivity could provide new therapeutic \nstrategies for unexplained infertility. Whether such defects \nof uterine receptivity could be treated by the therapeutic \nblockage of  ER-α  activity or by dealing with the related \ncauses of  ER-α  overexpression, e.g., using progestins or \naromatase inhibitors to normalize the expression pattern \nof endometrial biomarkers associated with implantation, \nrequires further investigation.","source_license":"CC-BY-4.0","license_restricted":false}