HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) An erratum has been published Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Galán, A. Articles by Simón, C. Search for Related Content PubMed PubMed Citation Articles by Galán, A. Articles by Simón, C. Agricola Articles by Galán, A. Articles by Simón, C.

The Human Blastocyst Regulates Endometrial Epithelial Apoptosis in Embryonic Adhesion¹

^a Instituto Valenciano de Infertilidad, 46020 Valencia, Spain ^b Department of Pediatrics, Obstetrics, and Gynecology and ^c Department of Biochemistry and Molecular Biology, University of Valencia, 46010 Valencia, Spain ^d OBST 5330 Research Unit, Université de Louvain Medical School, Brussels, Belgium

ABSTRACT

The implanting blastocyst must appose and adhere to the endometrial epithelium and, subsequently, invade it. Locally regulated uterine epithelial apoptosis induced by the embryo is a crucial step of the epithelial invasion in rodents. To address the physiological relevance of this process in humans, we investigated the effect of single human blastocysts on the regulation of apoptosis in cultured human endometrial epithelial cells (hEEC) in both apposition and adhesion phases of implantation. Here, we report a co-ordinated embryonic regulation of hEEC apoptosis. In the apposition phase, the presence of a blastocyst rescues hEEC from the apoptotic pathway. However, when the human blastocyst adheres to the hEEC monolayer, it induces a paracrine apoptotic reaction. Fas ligand (Fas-L) was present at the embryonic trophoectoderm. Fas was localized at the apical cell surface of hEEC, and flow cytometry revealed that 60% of hEEC express Fas. Neutralizing adhesion assays revealed that the Fas/Fas-L death system may be an important mechanism to cross the epithelial barrier, which is crucial for embryonic adhesion, and the manipulation of this system could have potential clinical implications as an interceptive mechanism.

apoptosis, implantation/early development

INTRODUCTION

Apoptosis, or the highly orchestrated form of programmed cell death in which cells neatly commit suicide without triggering an inflammatory response in the tissue, is becoming a relevant event in the study of reproductive physiology [1]. Programmed cell death has been implicated in gonadal function [2], human endometrial physiology [3], preimplantation embryo development [4], embryonic implantation [5], and placenta formation [6].

In the human endometrium, proliferation and apoptosis appear in opposing poles of the menstrual cycle, working as a homeostatic mechanism. The proliferative phase is characterized by a low number of apoptotic cells, whereas the secretory phase is characterized by the number increasing significantly, then peaking in the menstrual phase [7, 8]. Most apoptotic cells are present in the epithelial compartment, and the endometrial stroma is less affected [9]. Artificial withdrawal of ovarian hormones is also followed by apoptosis of the uterine epithelial cells; in fact, the predominant type of cell death observed in the endometrial epithelium is apoptosis (97.5%) as opposed to necrosis (2.5%) [10]. At the molecular level, Fas (also called CD95) and Fas ligand (Fas-L, or CD95L) are coexpressed on the surface of the human glandular endometrium [11] throughout the menstrual cycle [12].

Several lines of evidence suggest that in addition to vital functions in maintaining homeostasis of the endometrium, locally regulated apoptosis is important during tissue remodeling in decidualization [13] and blastocyst implantation in animals [5, 14, 15, 16]. Embryonic implantation consists of three related and consecutive phases: apposition, adhesion between trophoectoderm and endometrial epithelium, and invasion. During invasion, the embryonic trophoblast penetrates the endometrial epithelium, destroys the basal membrane, and introduces itself into the stroma (invading up to the uterine vessels). In rodents, ultrastructural studies have demonstrated that uterine epithelial cells in the attachment site undergo apoptotic changes [5]. Recently, biochemical evidence of apoptosis and quantitative assessment of DNA fragmentation in the uterine epithelial cells of a mouse implantation model have been demonstrated. Indeed, transforming growth factor-ß mediates the autocrine/paracrine regulation of apoptosis induced by the embryo, as previously shown in mice [14]. Rat pregnancy during the stromal invasion phase is characterized by a progressive, continuous induction of apoptosis in the maternal tissues surrounding the conceptus [17].

In experimental animals, endometrial apoptosis clearly is hormonally regulated, and the embryo can influence this process in an autocrine/paracrine fashion. Likewise, the endometrium can improve embryonic development [18], modifying the apoptotic embryonic status. However, information regarding the role and the mechanisms by which the human blastocyst may initiate and regulate apoptosis in human endometrial epithelial cells as a crucial mechanism for the embryo to cross the epithelial barrier and to continue the implantation process is lacking.

The purpose of our study was to investigate the effect of single human blastocysts on the regulation of endometrial epithelial apoptosis using an in vitro model that mimics the apposition and adhesion phases of human implantation. Our study also investigated the possible molecular mechanisms involved.

MATERIALS AND METHODS

Institutional Approval and Informed Consent

This project was approved by the institutional review board on the use of human subjects in research at the Instituto Valenciano de Infertilidad, complied with the Spanish Law of Assisted Reproductive Technologies (35/1988), and conformed to guidelines established by the Ethics Committee of the American Society for Reproductive Medicine on human embryo research. Endometrial samples and surplus embryos donated for research were obtained after written consent from patients. The clinical and laboratory work was performed at the Instituto Valenciano de Infertilidad.

Experimental Design

Based on our own previous work [19], we developed an in vitro model to study interactions between the human embryo and endometrial epithelial cells (EEC) [20]. This model has resulted in a clinical program in which embryos are cocultured with EEC until the blastocyst stage and then transferred back to the mother [18]. Embryos for this study were obtained after ovarian superovulation and insemination employing routine in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) procedures. The EEC were isolated from luteal phase endometria and cultured until confluence. Individual human embryos were cocultured with EEC for 5 days (from Day 2 until Day 6 of embryonic development). After embryo transfer, EEC wells were divided according to the embryonic status: EEC with embryos that reached the blastocyst stage, EEC with embryos arrested at any stage of development, and EEC without embryos. Bidimensional monolayers were used for the apposition studies, whereas polarized three-dimensional EEC monolayers were used for the adhesion assay.

Several approaches were followed to investigate the effect of single human embryos in the regulation of early and late apoptosis on human EEC during the apposition phase. First, to detect and quantify early apoptotic events [21], the expression of phosphatidylserine at the cell surface was performed using flow cytometry (FCM) with Annexin-V-fluorescein isothiocyanate (FITC) and propidium iodide (PI). To analyze late apoptotic events [21], DNA strand breaks were detected by the terminal transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) assay using fluorescence and PI or 4,6-diamidino-2-phenylindole (DAPI) nuclear staining. To clarify discrepancies between early and late apoptosis, further experiments measuring cellular DNA content and TUNEL assays on detached EEC were performed.

To analyze the paracrine embryonic effect on the adhesion phase, TUNEL assays and confocal microscopy were performed on the EEC monolayer with attached human blastocysts using a double-labeling technique (ß-hCG and dUTP-FITC). The mechanism and functional significance of the endometrial epithelial apoptosis induced by the embryo was investigated by studying the Fas/Fas-L system at the human endometrial-embryonic interface. The Fas-L in the human blastocysts was assessed by immunohistochemistry, and the Fas was investigated in cultured human EEC by confocal microscopy and FCM. The functional relevance of this system was tested in an adhesion assay with blocking anti-Fas antibody.

Clinical IVF Protocol

The ovarian stimulation protocol using GnRH- and gonadotropins has been described elsewhere [22]. Briefly, a long protocol was used for pituitary desensitization, with administration of leuprolide acetate, 1 mg/day s.c. (Procrin; Abbot S.A., Madrid, Spain), starting in the luteal phase of the previous cycle. After ovarian quiescence, human menopausal gonadotropins were administered (Pergonal and Neo-Fertinorm; Serono, Madrid, Spain) for ovarian stimulation and monitored by serum E₂levels and transvaginal ovarian ultrasound scans. Oocyte retrieval was performed 36 to 38 h after hCG administration (10 000 IU; Profasi; Serono). The standard IVF/ICSI procedure has been described elsewhere [22]. Oocyte-cumulus complexes (OCC) were evaluated under the dissecting microscope and classified [23]. The OCC were incubated at 37°C under 5% CO₂ in atmospheric air.

Endometrial Cell Cultures

Samples of endometria were obtained in the midluteal phase (Days 19–23) from fertile patients undergoing endometrial biopsy (age range, 23–39 yr). Endometrium donors were screened as being negative for human immunodeficiency virus, hepatitis C and B, VDRL, and Mycoplasma. A portion of each specimen was stained with hematoxylin-and-eosin for dating according to the method described by Noyes et al. [24]. Endometrial samples were minced into small pieces (size, <1 mm) and then subjected to mild collagenase digestion. The EEC were grown from isolated endometrial glands purified as described elsewhere [25, 26]. These primary cultures were grown to confluence in steroid-depleted medium: 75% Dulbecco's Modified Eagle's Medium (Sigma, St. Louis, MO), and 25% MCDB-105 (Sigma) containing antibiotics. The medium was supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Grand Island, NY) and 5 µg/ml of insulin (Sigma) as described elsewhere [25, 26]. The homogeneity of cultures was determined by morphological characteristics and verified by immunocytochemical localization of cytokeratin, vimentin, and CD68 antigen as described elsewhere [25]. Functionality of EEC monolayers was demonstrated by the production of prostaglandin E₂ in response to interleukin 1 [26], and morphological features were displayed by scanning electron microscopy [18, 20]. After confluence, growth media were replaced by a 1:1 v:v mixture of IVF:S2 (Scandinavian IVF Science AB, Gotheburg, Sweden), and the endometrial cells were cocultured with single human embryos.

Embryo Coculture

For embryo coculture, individual human embryos were cocultured with EEC for 1 day in IVF:S2 v:v and 4 days in 1 ml of S2 starting at Day 2 after insemination, when they were at two- to four-cell stage. Conditioned media were removed every 24 h as described elsewhere [18, 20]. In each experiment, EEC cultured with the same culture media without any human embryo were used as controls. Embryos achieving the blastocyst stage were transferred back to the mother, and the EEC monolayers were used for the analytical determinations.

Analysis by FCM of Phosphatidylserine Exposure in EEC

To determine Annexin-V⁺ (apoptotic) cells by FCM [27], EEC cells were mechanically detached from plastic wells after incubation with trypsin/EDTA (1:10) in PBS (10 min, 37°C) by pipetting. Cells were collected by centrifugation, washed with PBS, and then resuspended in 490 µl of 1:10 v:v diluted binding buffer (Annexin-V-FITC commercial kit; Immunotech, Coulter, Marseille, France). After this, cells were stained with 5 µl of Annexin V-FITC and 5 µl of PI, as recommended by the kit manufacturer. Cell suspensions were fixed with 4% paraformaldehyde (PFA) in PBS for 30 min at 4°C, resuspended in PBS, and then analyzed by FCM. Live, apoptotic, and necrotic cells were differentiated according to their Annexin-V and PI fluorescence pattern (live cells: Annexin-V^-/PI^-; apoptotic cells: Annexin-V⁺/PI^dim; necrotic cells: Annexin-V⁺/PI^bright).

All FCM determinations were performed in an Epics XL flow cytometer (Beckmann-Coulter, Brea, CA) using an argon-ion laser tuned at 488 nm and 15 mW. FITC-fluorescence was collected by 575 dichroich long pass (DL) + 525 band pass (BP) and PI by 600DL + 575BP filters. Data were collected in four-decade logarithmic amplification. Debris was excluded by analysis of scatter properties. At least 10 000 events per sample were stored in list-mode files. Data were expressed as the percentage of stained cells.

Detection of DNA Strand Breaks by TUNEL

In situ detection of apoptotic cells was performed in EEC cocultured cells plated in chamber-slides (NUNC, Roskilde, Denmark). The EEC monolayers were washed with PBS, fixed with 4% PFA (30 min, 4°C), permeabilized with 0.2% Triton X-100 (5 min, 4°C), and washed again with PBS. Apoptotic cells were detected using a Cell Death Detection Kit (Promega, Madison, WI). Cells were washed for 5 min in equilibration buffer. Incubation with TdT-enzyme and FITC-dUTP was done for 60 min at 37°C in the dark. After the washing steps, cells were counterstained with PI (1 µg/ml; Sigma) or DAPI. Negative controls were made by omitting the TdT enzyme. Slides were mounted with glycerin:distilled water (1:1) and viewed with a fluorescence microscope (Labophot-2; Nikon, Tokyo, Japan).

TUNEL was performed as follows: EEC were fixed in 4% PFA for 10 min, washed in PBS, exposed to 0.3% hydrogen peroxide in methanol for 30 min, washed in PBS, permeabilized in 0.1% Triton X-100 for 10 min on ice, washed twice in PBS, and then prestained in 25 mg/ml of bisbenzimide for 25 min at 37°C. Cell cultures were incubated with 50 U/ml of terminal transferase and 15 mM of fluorescein-dUTP for 60 min at 37°C, washed four times in PBS, and then exposed to a sheep antifluorescein antibody conjugated to peroxidase for 30 min at 37°C. After four washes in PBS, TUNEL staining was developed in a solution of diaminobenzidine and nickel chloride, and after four washes in PBS, the preparations were mounted and observed under combined ultraviolet and visible light.

TUNEL on Detached EEC (FCM Analysis)

The DNA strand breaks were also assessed by FCM on detached EEC as follows: EEC monolayers were washed with PBS and mechanically detached from plastic wells after incubation with trypsin/EDTA (1:10; Boehringer Mannheim, Barcelona, Spain) in PBS (10 min, 37°C) by pipetting. Cells were collected by centrifugation, fixed with 4% PFA (30 min, 4°C), permeabilized with 0.2% Triton X-100 (5 min, 4°C), and washed with PBS. Apoptotic cells were detected using the same Cell Death Detection Kit (Promega). Cells were resuspended in equilibration buffer (5 min, room temperature) and then incubated with TdT-enzyme and FITC-dUTP for 60 min at 37°C in the dark. After the washing steps, cells were counterstained with PI (1 µg/ml; Sigma). Negative controls were made omitting the TdT enzyme. Cell suspensions were analyzed by FCM.

Measurement of Cellular DNA Content

Immediately after transfer of embryos, EEC monolayers were covered with 1 ml of a hypotonic PI solution (50 µg/ml PI, 0.1% trisodium citrate, and 0.1% Triton X-100) and then incubated overnight at 4°C in the dark. With this procedure, a suspension of single nuclei was obtained from the wells by gentle hand pipetting [28]. To extract low-molecular-weight fragments of DNA from apoptotic nuclei, suspensions were washed with PBS and then incubated for 30 min at 37°C before FCM.

In Vitro Attachment Assay

Three-dimensional cultures were prepared for the in vitro embryo attachment assays. Epithelial and stromal cells were obtained from endometrial biopsies and processed as described earlier, with slight modifications. Epithelial cells grew polarized on inserts with extracellular matrix (ECM-gel; Sigma), and stromal cells were seeded on plastic culture dishes beneath them.

Spare blastocysts were also cultured on these endometrial epithelial cells, and blastocysts were allowed to attach to the epithelial surface. After blastocyst attachment (48–72 h), cells were fixed (4% PFA in PBS, 30 min, 4°C), permeabilized (0.2% Triton X-100 in PBS, 5 min, 4°C), and washed with PBS. Attached blastocysts were immunologically localized with an anti-ß-hCG monoclonal antibody from Sigma (1:50, 90 min, room temperature), rabbit antimouse biotin-conjugated secondary antibody from Dako (1:400, 60 min, 37°C), and Extravidin-HRP (1:40, 30 min, room temperature; Sigma). After washing cells were incubated for 10 min with working 3-amino-9-ethyl-carbazole (AEC) substrate solution (0.2 ml stock AEC solution, 3.8 ml of 0.05 M acetate buffer [pH 5.0], and immediately before use, 20 µl of 3% H₂O₂). Reaction was terminated with distilled water [29]. Afterward, apoptotic cells were detected with fluorescent TUNEL-labeling (as described earlier) by confocal microscopy.

Confocal Microscopy

Confocal analysis was performed with NRC 1024 (Bio-Rad, Hempstead, UK). Excitation lines used were 488 (FITC, phycoeritrin [PE]) and 514 (tetramethyl-rodamine B isothiocyanate [TRITC]). The following filters were used: for FITC signals, a HQ515/10 filter; for PE, a BP 580/20 filter, and for TRITC, a BP 605/20 filter. Transmission signals were acquired in every image.

Fas-L Immunohistochemistry in Human and Mouse Blastocysts

Spare human blastocysts and mouse blastocysts were fixed with 2% PFA in PBS for 30 min at 4°C and then permeabilized with 0.2% Triton X-100 for 5 min at 4°C as previously described [19]. To reduce the nonspecific binding, 1% albumin in PBS was applied for 30 min at room temperature and then incubated for 5 min with 3% H₂O₂. After that, blastocysts were incubated in 20 µl of monoclonal biotinylated mouse anti-human Fas-L (Pharmigen, San Diego, CA) for 60 min at 4°C.

To detect and amplify the signal, blastocysts were rinsed with PBS-0.05% Tween and then incubated with Extravidin-HRP (1:40; Sigma) for 30 min at room temperature. Finally, they were incubated in alkaline phosphatase substrate solution (as described) for approximately 5 min until color became evident, then reaction was stopped by immersing the blastocysts in distilled water. Blastocysts were visualized and photographed using an Olympus 35-mm camera attached to an inverted microscope Nikon Diaphot 200. Control incubations included deletion of the antibody.

Fas Confocal Microscopy and FCM in EEC

For confocal microscopy, EEC monolayers were washed with PBS, fixed with 4% PFA, and then incubated with 20 µl of anti-Fas monoclonal antibody labeled with PE (Immunotech) for 1 h at 4°C and diluted in 130 µl of PBS and 1% BSA per well.

For FCM, EEC monolayers were detached as previously described. Cell suspensions were incubated with 20 µl of the same monoclonal antibody and then fixed (30 min, 4% PFA, 4°C) and washed with PBS before FCM. In both techniques, negative controls were made incubating the cells with a PE-labeled nonspecific antibody.

Neutralizing Adhesion Assay with Mouse Blastocysts and Human EEC

Blastocysts were flushed from the uteri on Day 3.5 of pregnancy from eCG/hCG-stimulated, 8-wk-old Swiss female mice (CFLP; Interfauna, Barcelona, Spain) that were mated and plugged with males of the same strain and age. Mouse blastocyst thereby retrieved (n = 243) were cultured in monolayers of polarized human EEC (n = 163) or nonpolarized human EEC (n = 62) or extracellular matrix-coated plates (n = 18) for 48 h in the absence or presence of blocking concentrations of mouse antihuman anti-CD95 immunoglobulin (Ig) G₁(50 ng/ml; Immunotech) at 37°C in a 5% CO₂:95% air-humidified incubator. The percentage of blastocysts attached to EEC monolayer was recorded after a 48-h incubation period. Blastocyst attachment and trophoblast outgrowth were assessed as previously described [20].

Statistical Analysis

The percentage of cells stained were expressed as the mean ± SEM. For statistical comparison among groups, ANOVA followed by Bonferroni correction was applied. Embryonic adhesion is expressed as the percentage of blastocysts from the total. Comparison between groups was performed with Pearson's chi-square test. A P value of less than 0.05 was considered to be statistically significant.

RESULTS

Apposition Phase

The presence of a human blastocyst induces a reduction of EEC apoptotic cells (35.2%) compared to EEC cultured without blastocyst (48.8%) (P < 0.05). Interestingly, EEC monolayers cultured in the presence of an embryo arrested during their development also have a decreased number of apoptotic cells (39.2%) compared to EEC without embryos (48.8%), suggesting an antiapoptotic effect exerted by a soluble factor (or factors) secreted by the human embryo, even for a few days. This antiapoptotic embryonic effect is detected by Annexin-V-FITC FCM (Fig. 1).

View larger version (43K): [in this window] [in a new window]   FIG. 1. Results of Annexin-V-FITC and PI FCM presented as characteristic histograms of an apoptotic cell population (A), live cells (B), and necrotic cells (C). A bar chart (D) shows the percentage of apoptotic, alive, and necrotic cells in control EEC, EEC with arrested embryos, and EEC with blastocyst. Asterisks and daggers indicate P < 0.05 versus control by ANOVA (Bonferroni correction). All data are expressed as the mean ± SEM of four separate experiments.

Late apoptosis events (DNA strand breaks) in EEC monolayers were not detected (Fig. 2). To understand this discrepancy, TUNEL on detached EEC and measurement of cellular DNA content were performed (Fig. 3). The TUNEL experiments demonstrated that unlike Annexin-V FCM on detached cells, only approximately 10% of cells on detached EEC were TUNEL positive cells and showed a similar pattern, regardless of the presence or absence of the human embryo (Fig. 3, A–C). This suggests that cell detachment is a stress-related situation that may be inducing apoptosis in those cells not protected by the antiapoptotic effect exerted by the embryo (as demonstrated by phosphatidylserine externalization). The EEC monolayers showed no Annexin-V staining (data not shown). The different timing for the fixation (for Annexin-V, cells must be stained before fixation; for TUNEL, cells must be stained after fixation) could be why we did not see TUNEL-positive cells. In addition, study of the DNA content revealed that the EEC cell cycle was normal, and that DNA was not fragmented or degraded (Fig. 3D).

View larger version (96K): [in this window] [in a new window]   FIG. 2. TUNEL and DAPI analysis of EEC cocultured with human blastocysts. A) Optical microscopy the TUNEL-negative EEC monolayer (x200). B) DAPI DNA-staining showing the existence of one apoptotic nucleus (x400), which is a normal feature of a healthy monolayer. C) A higher magnification detail of B. x400

View larger version (21K): [in this window] [in a new window]   FIG. 3. Flow cytometric analysis of DNA-strand breaks (TUNEL) on detached EEC. Upper panel illustrates a negative control (A), EEC without blastocyst (B), and EEC with blastocyst (C). Lower histogram (D) shows the DNA cell content (cell cycle) of the EEC used in our model

Adhesion Phase

Unlike the apposition phase, when a human blastocyst adheres to polarized human EEC, we found a massive induction of apoptosis in EEC beneath and around the embryo attachment site compared with cells away from the blastocyst and control cultures (Figs. 4, C and D, and 5B). This is an embryonic proapoptotic effect, because it can be detected late in the apoptotic pathway by TUNEL with conventional fluorescence (Fig. 4, C and D) and confocal microscopy (Fig. 5B). This embryonic proapoptotic effect seems to be mediated by a direct contact between the trophoectoderm of the blastocyst and EEC.

View larger version (97K): [in this window] [in a new window]   FIG. 4. Double labeling (ß-hCG and TUNEL) at the attachment site. A) The ß-hCG staining of the implanting human blastocyst (*). B) The TUNEL staining in the control EEC monolayer without blastocysts. C and D) An implantation site, with apoptotic (yellow-green) cells around the embryo and live (red) cells away from it. x200

View larger version (49K): [in this window] [in a new window]   FIG. 5. Confocal microscopy with TUNEL of an attachment site. A) Control EEC without blastocyst. B) The absence of EEC beneath the trophoectoderm and apoptotic cells close to the blastocyst (asterisk)

Fas/Fas-L at the Blastocyst-EEC Interface

To investigate the potential implication of Fas/Fas-L in this mechanism, we first localized the presence of Fas in the EEC monolayer. Confocal microscopic studies revealed the presence of Fas in the polarized EEC monolayer located at the apical cell surface (Fig. 6A). To quantify the percentage of EEC Fas-positive cells, FCM was performed on EEC monolayers during the apposition phase, revealing that 60% of cells contained Fas, the death receptor, regardless of the presence or absence of human blastocyst (Fig. 6D). Finally, Fas-L was searched for in human (n = 6) and mouse (n = 20) blastocysts using an antihuman antibody. Immunostaining for Fas-L was present in the trophoectoderm of the spare human blastocysts (Fig. 7B) and mouse blastocyst (Fig. 7D). No cytoplasmic labeling was observed in control experiments (Fig. 7, A and C).

View larger version (67K): [in this window] [in a new window]   FIG. 6. Confocal microscopic immunolocalization and FCM quantification of Fas in EEC. A) Confocal microscopy of Fas at the EEC cell membrane surface. B) Negative control. C) Representative FCM histogram of Fas in EEC monolayers. D) Sixty percent of the EEC express this death receptor, regardless of the presence or absence of the human embryo

View larger version (151K): [in this window] [in a new window]   FIG. 7. Inmunohistochemical detection of Fas-L in the human and mouse blastocyst. Red staining was present at the trophoectoderm in both human (B) and mouse (D) blastocysts. Negative controls (A and C) were created by deletion of the antibody. x600

Neutralizing Adhesion Assay

To explore the functional relevance of the Fas/Fas-L system in the adhesion phase of embryonic implantation using mouse blastocysts and human EEC, neutralization experiments were performed that blocked Fas at the endometrial-embryonic interface. To assess the incubation time of anti-Fas necessary to block Fas, a time-course study was performed, revealing that at 24 h, 50% of Fas-bearing cells were stained (data shown to the reviewers). Embryonic adhesion and outgrowth (rupture of the epithelial monolayer) were significantly reduced in EEC cultured in the presence of anti-Fas compared to EEC cultured in the absence of anti-Fas (40% vs. 82.8%, respectively; Fig. 8 and Table 1). The inhibition of embryonic adhesion induced by anti-Fas was consistent in nonpolarized two-dimensional EEC (20.6% vs. 82.4%) and polarized EEC, cultured on ECM (47.7% vs. 83.1%). The specificity of this effect was tested by attachment and outgrowth of blastocysts to ECM coated plates in the presence of same concentrations of anti-Fas (Fig. 8).

View larger version (150K): [in this window] [in a new window]   FIG. 8. Effect of antihuman-CD95 (Anti-Fas; 50 ng/ml) on embryonic adhesion and outgrowth to EEC and matrigel. A) Attachment of a blastocyst to matrigel in the presence of anti-Fas. B) Blastocyst attached to EEC monolayer in the absence of anti-Fas. C and D) Nonattached blastocysts to EEC monolayers in the presence of anti-Fas are presented. x600

DISCUSSION

Blastocyst implantation is a robust phenomenon that has evolved during the course of evolution in eutherian mammals. Nevertheless, adhesion between the embryonic trophoectoderm and EEC plasma membrane and rupture of the endometrial epithelial barrier, which are the prerequisites for embryonic implantation and placental development, seem to be conserved from rodents to primates. Some exceptions exist, however, such as the epitheliochorial placentation in the porcine. Here, we show that EEC apoptosis occurs in implantation sites not only in the mouse [15], rat [5], and hamster [30] but also in the human. Furthermore, this in vitro work demonstrates, to our knowledge for the first time in humans, a co-ordinated regulation of embryonic induction of EEC apoptosis crucial for the embryo to breach the epithelial barrier. In the apposition phase, the presence of a blastocyst rescues EEC from the apoptotic pathway, maintaining more EEC prepared for the initial contact. However, when the blastocyst adheres to the EEC monolayer, it induces a paracrine apoptotic reaction that is maximal in those EEC in close contact with the blastocyst.

Because this embryo-induced apoptotic mechanism was triggered by a direct contact between the blastocyst and EEC, we searched for death receptors with direct access to the apoptotic machinery at the endometrial site and death ligands at the trophoectoderm part. Death receptors transmit apoptosis signals initiated by specific "death ligands." Known death receptors belong to the tumor necrosis factor-receptor gene superfamily, and one of the best characterized is Fas (also called CD95 or APO-1). The ligand that activates this receptor is Fas-L (also called CD95L). The receptor-ligand complex can activate death caspases within seconds, causing an apoptotic demise of the cell within hours [31].

The Fas-L was present in the trophoectoderm, and 60% of human EEC contained Fas located at the apical cell surface. In fact, experiments inhibiting the Fas/Fas-L system in an adhesion model with polarized human EEC and mouse blastocysts (also expressing Fas-L) revealed a disruption of the embryonic adhesion mechanism in EEC cultured in the presence of anti-Fas compared to EEC cultured in the absence of anti-Fas. In addition, this effect was specific (EEC Fas-mediated), because no alteration in adhesion was observed in matrigel-coated plates cultured with anti-Fas.

The Fas and Fas-L play an important role in different types of physiological apoptosis, such as peripheral deletion of T cells at the end of an immune response, killing virus-infected cells or cancer cells by cytotoxic T cells, and killing inflammatory cells at "immune privileged" sites (e.g., the eye). The effect described in the present work, however, is a novel role for this death receptor-ligand system. Signaling of this death system occurs via an adapter protein called Fas-associated death domain (FADD) [32]. The FADD also contains a death-effector domain that binds to an analogous domain repeated in tandem within caspase-8 (also called FLICE) [33]. Caspase-8 then activates downstream effector caspases, such as caspase-9, committing the cell to apoptosis. These mechanisms require further investigation in embryonic-induced apoptosis. Because of their crucial role in the immune response, mouse strains with defective Fas/Fas-L underwent a lethal accumulation of peripheral lymphoid cells [34], and FADD transgenic animals did not survive beyond Day 11.5 of embryogenesis [35]. Therefore, the contribution of this system during implantation in experimental animals has not been tested as yet. Nevertheless, we do not believe that this death system was unique. Nature must provide redundant mechanisms to such a fundamental process, and other death systems likely play a role in this embryonic-induced apoptosis.

Blastocysts can attach to and invade a multitude of sites outside the uterus [36], even to artificial culture dishes without hormonal conditioning [37], whereas they can only attach to the uterus during a window of receptivity under specific hormonal conditions. The relevance of the endometrial epithelium as the "epithelial barrier" for embryonic implantation was documented in the classical experiment of Cowell, with the finding that mouse blastocysts could implant at any stage provided that the endometrial epithelium was damaged [38]. This study suggests that apoptotic death of EEC induced by the embryo is an important mechanism for invading the luminal epithelium and breaching the epithelial barrier, and that the immediate consequence is that the trophoectoderm come in direct contact with the basement membrane and, therefore, stromal invasion can proceed. The basic findings reported herein have obvious clinical implications regarding infertility as well as contraception.

In vitro studies are limited by their own conditions. In this model, the origin of cultured EEC is from both luminal and glandular EEC, and the contribution of each cell population to our cultures cannot be determined. Therefore, the characteristics of this tissue may differ from those of the luminal epithelium.

In human infertility, the term early pregnancy loss (EPL) [39] has been introduced to define the existence of rises in hCG occurring because of the initiation of embryonic implantation but that remain undetected because these women have regular menses in due course. Thus, EPL represents an important event in natural cycles. Using different ultrasensitive hCG assays during the entire luteal phase in women wishing to conceive, it has been established that the chance of a woman having an EPL is as high as 22% to 57% [39–43]. In a recent study [44], we showed a high implantation rate in assisted reproductive techniques, in opposition to what is currently believed. More than 67% of patients had implantation in IVF and oocyte donation, as ascertained by positive urine ß-hCG titers. However, the frequency of EPL in artificial reproduction techniques was approximately 40% (47.7% in IVF and 37.5% in oocyte donation) [44]. Therefore, EPL is linked to an alteration of the epithelial barrier, and this work provides insight into the mechanisms underlying this clinical condition and possible improvements.

In contraception, manipulation of this "kiss of death" induced by the human embryo on the endometrial epithelium provides a unique opportunity to control a crucial and indispensable mechanism for embryonic implantation. Therefore, use of local strategies could have potential clinical implications as an interceptive mechanism.

In summary, we have described in humans a co-ordinated regulation of embryonic induction of EEC apoptosis crucial for the embryo to breach the epithelial barrier as a basic physiological mechanism in embryonic implantation. Furthermore, we have demonstrated that the Fas/Fas-L death system mediates this mechanism, at least in part, and that its manipulation may have potential clinical implications at interception.

FOOTNOTES

First decision: 16 December 1999.

¹ Supported by IVI Foundation and FISss 00/0643 grant from the Spanish Government, Ministerio de Sanidad y Consumo, Madrid.

² Correspondence: Carlos Simón, Instituto Valenciano de Infertilidad. Guardia Civil 23, 46020 Valencia, Spain. FAX: 34 963694735; csimon{at}interbook.net

Accepted: March 9, 2000.

Received: November 4, 1999.

REFERENCES

1. Tilly JL, Strauss III JF, Tenniswood M. Cell Death in the Reproductive Physiology. In: Serono Symposia USA. Norwell, MA: Springer-Verlag; 1997: 1–7.
2. Piquette GN, Tilly JL, Prichard LE, Simón C, Polan ML. Detection of apoptosis in human and rat ovarian follicles. J Soc Gynecol Invest 1994; 1:297–301.[Medline]
3. Tabibzadeh S. Signals and molecular pathways involved in apoptosis, with special emphasis on human endometrium. Hum Reprod Update 1995; 1:303–323.[Abstract/Free Full Text]
4. Jurisicova A, Varmuza S, Casper RF. Involvement of programmed cell death in preimplantation embryo demise. Hum Reprod Update 1995; 1:558–566.[Abstract/Free Full Text]
5. Parr EL, Tung HN, Parr MB. Apoptosis as the mode of uterine epithelial cell death during embryo implantation in mice and rats. Biol Reprod 1987; 36:211–225.[Abstract]
6. Welsh AO, Enders AC. Chorioallontoic placenta formation in the rat. III. Granulated cells invade the uterine luminal epithelium at the time of epithelial cell death. Biol Reprod 1993; 49:38–57.[Abstract]
7. Tao XJ, Tilly KI, Maravei DV, Shifren JL, Kajewski S, Reed JC, Tilly JL, Isaacson KB. Differential expression of members of the bcl-2 gene family in proliferative and secretory human endometrium: glandular epithelial cell apoptosis is associated with increased expression of bax. J Clin Endocrinol Metab 1997; 82:2739–2746.
8. Toki T, Mori A, Shimizu M, Nikaido T, Fujii S. Localization of apoptotic cells within the human endometrium and correlation between apoptosis and p21 expression. Mol Hum Reprod 1998; 4:1157–1164.[Abstract/Free Full Text]
9. Rango UV, Classen-Linke I, Krusche CA, Beier H. The receptive endometrium is characterized by apoptosis in the glands. Hum Reprod 1998; 13:3177–3189.[Abstract/Free Full Text]
10. Nawaz S, Lynch MP, Galand P, Gerschenson LE. Hormonal regulation of cell death in rabbit uterine epithelium. Am J Pathol 1987; 127:51–59.[Abstract]
11. Yamashita H, Otsuki Y, Matsumoto K, Ueki K, Ueki M. Fas ligand, Fas antigen and Bcl-2 expression in human endometrium during the menstrual cycle. Mol Hum Reprod 1999; 5:358–364.[Abstract/Free Full Text]
12. Watanabe H, Kanzaki H, Narukawa S, Inoue T, Katsuragawa H, Kaneko Y, Mori T. Bcl-2 and Fas expression in eutopic human endometrium during the menstrual cycle in relation to endometrial cell apoptosis. Am J Obstet Gynecol 1997; 176:360–368.[CrossRef][Medline]
13. Akcali KC, Kahn SA, Moulton BC. Effect of decidualization on the expression of bax and bcl-2 in the rat uterine endometrium. Endocrinology 1997; 137:3123–3130.[Abstract]
14. Kamijo T, Rajabi MR, Mizunuma H, Ibuki Y. Biochemical evidence for autocrine/paracrine regulation of apoptosis in cultured uterine epithelial cells during mouse embryo implantation in vitro. Mol Hum Reprod 1998; 4:990–998.[Abstract/Free Full Text]
15. Pampfer S, Donnay I. Apoptosis at the time of embryo implantation in mouse and rat. Cell Death Differ 1999; 6:533–545.[CrossRef][Medline]
16. Schlafke S, Enders AC. Cellular basis of interaction between trophoblast and uterus at implantation. Biol Reprod 1975;12:41–65.
17. Piacentini M, Autuori F. Immunohistochemical localization of tissue transglutaminase and Bcl-2 in rat uterine tissues during embryo implantation and post-partum involution. Differentiation 1994; 57:51–56.[CrossRef][Medline]
18. Simón C, Mercader A, García Velasco J, Nikas G, Moreno C, Remohí J, Pellicer A. Co-culture of human embryos with autologous human endometrial epithelial cells in patients with repeated implantation failures. J Clin Endocrinol Metab 1999; 84:2638–2646.[Abstract/Free Full Text]
19. De los Santos MJ, Mercader A, Francés A, Portolés E, Remohí J, Pellicer A, Simón C. Immunoreactive human embryonic interleukin-1 system and endometrial factors regulating their secretion during embryonic development. Biol Reprod 1996; 54:563–574.[Abstract]
20. Simón C, Gimeno MJ, Mercader A, O'Connor JE, Remohí J, Polan ML, Pellicer A. Embryonic regulation of integrins ß₃, ₄ and ₁ in human endometrial epithelial cells in vitro. J Clin Endocrinol Metab 1997; 82:2607–2616.[Abstract/Free Full Text]
21. Darzynkiewicz Z, Juan G, Li X, Murakami T, Traganos F. Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis). Cytometry 1997; 27:1–20.[CrossRef][Medline]
22. Pellicer A, Simón C, Miró T, Castellví RM, Ruiz A, Ruiz M, Perez M, Bonilla-Musoles F. Ovarian response and outcome of in vitro fertilization in patients treated with gonadotrophin-releasing hormone analogues in different phases of the menstrual cycle. Hum Reprod 1989; 4:285–289.[Abstract/Free Full Text]
23. Laufer N, DeCherney AH, Haseltine FP, Polan ML, Mezer HC, Dlugi AM, Sweeney D, Nero F, Naftolin F. The use of high dose menopausal gonadotropin in an in vitro fertilization program. Fertil Steril 1989; 40:734–741.
24. Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Fertil Steril 1950; 1:3–25.
25. Simón C, Piquette G, Francés A, Polan ML. Localization of interleukin-1 type I receptor and interleukin-1 beta in human endometrium throughout the menstrual cycle. J Clin Endocrinol Metab 1993; 77:549–555.[Abstract]
26. Simón C, Piquette GN, Francés A, El-Danasouri I, Polan ML. Effect of interleukin-1 beta (IL-1ß) on the regulation of IL-1 receptor type I and IL-1 beta messenger ribonucleic acid (mRNA) levels and protein expression in cultured human endometrial stromal and glandular cells. J Clin Endocrinol Metab 1994; 78:675–682.[Abstract]
27. Koopman G, Reutelingsperger CPM, Kuijten GAM, Keehnen RMJ, Pals ST, van Oers MHJ. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 1994; 84:1415–1420.[Abstract/Free Full Text]
28. Renau-Piqueras J, O'Connor JE, Báguena-Cervellera R, Grisolía S. Ammonium-chloride induced alterations in growth kinetics and ultrastructure of murine neuroblastoma cells. A flow cytometric and stereological study. Virchows Arch Cell Pathol 1986; 50:271–283.
29. Simón C, Francés A, Piquette G, Hendrikson M, Milki A, Polan ML. Interleukin-1 system in the materno-trophoblast unit in human implantation: immunohistochemical evidence for autocrine/paracrine function. J Clin Endocrinol Metab 1994; 78:847–854.[Abstract]
30. Parkening TA. An ultrastructural study of implantation in the golden hamster II. Trophoblastic invasion and removal of the uterine epithelium. J Anat 1976; 122:211–230.[Medline]
31. Ashkenazi A, Dixit VM. Death receptors: signalling and modulation: Science 1998; 281:1305–1308.[Abstract/Free Full Text]
32. Chinnaiyan AM, O'Rourke K, Tewari M, Dixit VM. FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell 1995; 81:505–512.[CrossRef][Medline]
33. Boldin MP, Gocharov TM, Goltsev YV, Wallach D. Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/Apo-1- and TNF-receptor induced cell death. Cell 1996; 85:803–815.[CrossRef][Medline]
34. Nagata S. Apoptosis by death factor. Cell 1997; 88:335–365.
35. Yeh W-C, de la Pompa JL, McCurrach ME, Shu HB, Elia AJ, Shainian A, Ng M, Wakeham A, Khoo W, Mitchell K, El-Deiry WS, Lowe SW, Goeddel DV, Mak TW. FADD: essential for embryo development and signalling from some, but not all, inducers of apoptosis. Science 1998; 279:1954–1958.[Abstract/Free Full Text]
36. Kirby DRS. The development of mouse blastocysts transplanted to the scrotal and cryptorchid testis. J Anat 1963; 97:119–130.
37. Glasser SR, Mulholland J, Mani SK, Julian J, Munir J, Lampelo S, Soares MJ. Blastocyst-endometrial relationships: reciprocal interactions between uterine epithelial and stromal cells and blastocysts. Troph Res 1991; 5:229–280.
38. Cowell TP. Implantation and development of mouse eggs transferred to the uterus of no-progestational mice. J Reprod Fertil 1969; 19:239–245.[Medline]
39. Wilcox AJ, Weinberg CR, O'Connor JF, Baird DD, Schlattere JP, Canfield RF, Armstrong EG, Nisula BC. Incidence of early loss of pregnancy. N Engl J Med 1988; 319:189–194.[Abstract]
40. Zinaman MJ, Clegg ED, Brown CHC, O'Connor J, Selevan SG. Estimates of human fertility and pregnancy loss. Fertil Steril 1996; 65:503–509.[Medline]
41. Miller JF, Williamson F, Glue J, Gordon YB, Grudzinskas JG, Sykes A. Fetal loss after implantation. Lancet 1980; i:554–556.
42. Edmons DK, Lindsay KS, Miller JF, Williamson E, Wood PJ. Early embryonic mortality in women. Fertil Steril 1982; 38:447–453.[Medline]
43. Hakim RB, Gray RH, Zacur H. Infertility and early pregnancy loss. Am J Obstet Gynecol 1982; 172:1510–1517.
44. Simón C, Landeras J, Zuzuarregui JL, Martín JL, Remohí J, Pellicer A. Early pregnancy losses in in-vitro fertilization and oocyte donation. Fertil Steril 1999; 72:1061–1065.[CrossRef][Medline]

This article has been cited by other articles:

				F. Dominguez, B. Gadea, F. J. Esteban, J. A. Horcajadas, A. Pellicer, and C. Simon Comparative protein-profile analysis of implanted versus non-implanted human blastocysts Hum. Reprod., September 1, 2008; 23(9): 1993 - 2000. [Abstract] [Full Text] [PDF]

				H. Fluhr, S. Krenzer, G. M. Stein, B. Stork, M. Deperschmidt, D. Wallwiener, S. Wesselborg, M. Zygmunt, and P. Licht Interferon-{gamma} and tumor necrosis factor-{alpha} sensitize primarily resistant human endometrial stromal cells to Fas-mediated apoptosis J. Cell Sci., December 1, 2007; 120(23): 4126 - 4133. [Abstract] [Full Text] [PDF]

				K. Diedrich, B.C.J.M. Fauser, P. Devroey, G. Griesinger, and on behalf of the Evian Annual Reproduction (EVAR) The role of the endometrium and embryo in human implantation Hum. Reprod. Update, July 1, 2007; 13(4): 365 - 377. [Abstract] [Full Text] [PDF]

				Y. Hirota, Y. Osuga, K. Koga, O. Yoshino, T. Hirata, C. Morimoto, M. Harada, Y. Takemura, E. Nose, T. Yano, et al. The Expression and Possible Roles of Chemokine CXCL11 and Its Receptor CXCR3 in the Human Endometrium J. Immunol., December 15, 2006; 177(12): 8813 - 8821. [Abstract] [Full Text] [PDF]

				K. Ushizawa, T. Takahashi, K. Kaneyama, M. Hosoe, and K. Hashizume Cloning of the Bovine Antiapoptotic Regulator, BCL2-Related Protein A1, and Its Expression in Trophoblastic Binucleate Cells of Bovine Placenta Biol Reprod, February 1, 2006; 74(2): 344 - 351. [Abstract] [Full Text] [PDF]

				S. L. Straszewski-Chavez, V. M. Abrahams, and G. Mor The Role of Apoptosis in the Regulation of Trophoblast Survival and Differentiation during Pregnancy Endocr. Rev., December 1, 2005; 26(7): 877 - 897. [Abstract] [Full Text] [PDF]

				A. C. Enders, S. Meyers, C. A. VandeVoort, and G. C. Douglas Interactions of macaque blastocysts with epithelial cells in vitro Hum. Reprod., November 1, 2005; 20(11): 3026 - 3032. [Abstract] [Full Text] [PDF]

				R. Matorras, F. Matorras, R. Mendoza, M. Rodriguez, J. Remohi, F. J. Rodriguez-Escudero, and C. Simon The implantation of every embryo facilitates the chances of the remaining embryos to implant in an IVF programme: a mathematical model to predict pregnancy and multiple pregnancy rates Hum. Reprod., October 1, 2005; 20(10): 2923 - 2931. [Abstract] [Full Text] [PDF]

				J. K. Riley, J. M. Heeley, A. H. Wyman, E. L. Schlichting, and K. H. Moley TRAIL and KILLER Are Expressed and Induce Apoptosis in the Murine Preimplantation Embryo Biol Reprod, September 1, 2004; 71(3): 871 - 877. [Abstract] [Full Text] [PDF]

				A. Cervero, J. A. Horcajadas, J. MartIn, A. Pellicer, and C. Simon The Leptin System during Human Endometrial Receptivity and Preimplantation Development J. Clin. Endocrinol. Metab., May 1, 2004; 89(5): 2442 - 2451. [Abstract] [Full Text] [PDF]

				S. L. Straszewski-Chavez, V. M. Abrahams, E. F. Funai, and G. Mor X-linked inhibitor of apoptosis (XIAP) confers human trophoblast cell resistance to Fas-mediated apoptosis Mol. Hum. Reprod., January 1, 2004; 10(1): 33 - 41. [Abstract] [Full Text] [PDF]

				H.-Y. Li, S.-P. Chang, C.-C. Yuan, H.-T. Chao, H.-T. Ng, and Y.-J. Sung Induction of p38 Mitogen-Activated Protein Kinase-Mediated Apoptosis Is Involved in Outgrowth of Trophoblast Cells on Endometrial Epithelial Cells in a Model of Human Trophoblast-Endometrial Interactions Biol Reprod, November 1, 2003; 69(5): 1515 - 1524. [Abstract] [Full Text] [PDF]

				F. Dominguez, A. Galan, J. J. L. Martin, J. Remohi, A. Pellicer, and C. Simon Hormonal and embryonic regulation of chemokine receptors CXCR1, CXCR4, CCR5 and CCR2B in the human endometrium and the human blastocyst Mol. Hum. Reprod., April 1, 2003; 9(4): 189 - 198. [Abstract] [Full Text] [PDF]

				J. Carver, K. Martin, I. Spyropoulou, D. Barlow, I. Sargent, and H. Mardon An in-vitro model for stromal invasion during implantation of the human blastocyst Hum. Reprod., February 1, 2003; 18(2): 283 - 290. [Abstract] [Full Text] [PDF]

				P. Caballero-Campo, F. Dominguez, J. Coloma, M. Meseguer, J. Remohi, A. Pellicer, and C. Simon Hormonal and embryonic regulation of chemokines IL-8, MCP-1 and RANTES in the human endometrium during the window of implantation Mol. Hum. Reprod., April 1, 2002; 8(4): 375 - 384. [Abstract] [Full Text] [PDF]

				B. Selam, U. A. Kayisli, N. Mulayim, and A. Arici Regulation of Fas Ligand Expression by Estradiol and Progesterone in Human Endometrium Biol Reprod, October 1, 2001; 65(4): 979 - 985. [Abstract] [Full Text] [PDF]

				M. Meseguer, J. D. Aplin, P. Caballero-Campo, J. E. O'Connor, J. C. Martín, J. Remohí, A. Pellicer, and C. Simón Human Endometrial Mucin MUC1 Is Up-Regulated by Progesterone and Down-Regulated In Vitro by the Human Blastocyst Biol Reprod, February 1, 2001; 64(2): 590 - 601. [Abstract] [Full Text]

				J. C. Martín, M. J. Jasper, D. Valbuena, M. Meseguer, J. Remohí, A. Pellicer, and C. Simón Increased Adhesiveness in Cultured Endometrial-Derived Cells Is Related to the Absence of Moesin Expression Biol Reprod, November 1, 2000; 63(5): 1370 - 1376. [Abstract] [Full Text]

This Article Abstract Full Text (PDF) An erratum has been published Alert me when this article is cited Alert me if a correction is posted Citation Map Services