Mol. Hum. Reprod. Advance Access originally published online on May 20, 2008
Molecular Human Reproduction 2008 14(7):423-430; doi:10.1093/molehr/gan032
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CXCL10 and IL-6 induce chemotaxis in human trophoblast cell lines
1Fundación Instituto Valenciano de Infertilidad (FIVI), University of Valencia, C/ Guadassuar 1, Bajo, 46015 Valencia, Spain 2Department of Pediatrics, Obstetrics and Gynecology, School of Medicine, University of Valencia, Av. Blasco Ibáñez 17, 46010 Valencia, Spain
3 Correspondence address. E-mail: csimon{at}ivi.es
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The investigation of trophoblast chemoattractive molecules in humans is of high interest for the reproductive field. Current evidence in ruminants demonstrates that CXCL10, formerly the interferon-
-inducible protein 10 (IP-10), is a potent chemotactic molecule implicated in the migration of trophoblast cells during early gestation. The aim of this work was to explore the existence of CXCL10/CXCR3 in the human model. Furthermore, chemotaxis assays were performed to demonstrate CXCL10 chemotactic activity in the human trophoblast cell lines JEG-3 and AC-1M88. Surprisingly, the conditioned media from epithelial endometrial cells (EEC) induced the highest trophoblast migration rate. Cytokine and chemokine membrane protein arrays were used to identify the secreted protein profile of EEC-conditioned media, and IL-6 was found to be the most abundant and CXCL13 the second most abundant molecule. Using a chemotaxis assay on AC-IM88, IL-6 antibody blocked the effect of EEC, indicating IL-6 to be an effective chemoattractive factor for trophoblast cells in the human model. Key words: CXCL10/chemotaxis/implantation/IL-6
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Introduction |
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Specific molecular crosstalk between the embryo and the endometrium during the human implantation process has been extensively reported (Paria et al., 2002; Dominguez et al. 2005). The endometrial epithelium is a key element where molecular interactions between the embryo and the endometrium seem to be initiated (Simón et al., 1997; Meseguer et al., 2001), and a variety of chemokines are produced and secreted by the endometrial epithelium (Caballero-campo et al., 2002).
Chemokines, a family of small secreted polypeptides with a low molecular weight, are specialized in the attraction of specific leukocyte subsets through binding to cell-surface receptors. Many of these molecules have been implicated in reproductive processes like ovulation, menstruation, embryo implantation, parturition, and in pathological processes like preterm delivery, HIV infection, endometriosis and the ovarian hyperstimulation syndrome (Lusso, 2006). During the apposition phase in embryonic implantation and leukocyte adhesion, the blastocyst/endometrium and leukocyte/endothelium dialogs rely on soluble mediators, e.g. cytokines, chemokines and other factors, which are produced and act in a bidirectional fashion (Dominguez et al., 2003a). We also know that other chemokine receptors, such as CCR5 or CCR2b, are expressed on the surface of the human blastocyst (Dominguez et al., 2003b), although direct proof of the chemokine attraction of the human blastocyst has not yet been demonstrated.
CXCL10, formerly the interferon--inducible protein 10 (IP-10), is a soluble 10 kDa chemokine belonging to the CXC chemokine subfamily, and is induced by different factors (IL-1, TNF-
, IFN- and ) in many cell types. It also has chemoattractive properties over Th1-lymphocytes, eosinophils, monocytes and dendritic cells. CXCL10 shows pleiotrophic biological activity, including migration (Taub et al., 1993), stimulation of T-cells adhesion to endothelial cells (Lloyd et al., 1996), modulation of adhesion molecules, inhibition of tumor growth in vivo (Luster and Leder, 1993) and inhibition of angiogenesis (Angiolillo et al., 1995). Recently, CXCL10 has also been found to be secreted by human endometrial stromal cells (Kai et al., 2002). In addition, CXCL10 has been demonstrated to take part in the migration of trophoblast cells, apposition and initial adhesion during early gestation in ruminants (Nagaoka et al., 2003a; Imakawa et al., 2005, 2006). CXCL10 targets the CXCR3 receptor that is also present in both the human endometrium (Kitaya et al., 2004) and trophoblast cells (Hirota et al., 2006).
This study was planned to investigate the expression, immunolocalization and possible chemoattractive function of CXCL10/CXCR3 (formerly IP-10) in the human model. Surprisingly, a functional comparison of this molecule using chemotaxis assays demonstrates that the conditioned media from endometrial epithelial cells (EEC) was superior to CXCL10, where IL-6 was the most abundant molecule with a chemoattractive capability.
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Institutional approval and informed consent
This study was approved by the institutional ethical board on the use of human subjects in research at the Instituto Valenciano de Infertilidad (IVI), and complies with the Spanish Law of Assisted Reproductive Technologies (35/88). All patients participating in this study signed a written statement of consent and received information about the study. This study has been conducted in accordance within the guidelines in the Declaration of Helsinki.
Endometrial samples, cell lines and primary EEC
Human endometrial samples throughout the menstrual cycle from normal fertile cycling women aged 23–39 years (n = 25) were obtained after signing a consent form. A small portion of each specimen was histologically examined and dated accordingly. Endometrial biopsies (n = 25) were distributed into five groups (n = 5 per group): Group I, early-mid proliferative (Days 1–8); II, late proliferate phase (Days 9–14); III, early secretory (Days 15–18); IV, mid-secretory (Days 19–22) and Group V, late secretory phase (Days 23–28). We analyzed the expression pattern of CXCL10 by quantitative fluorescent RT–PCR (QF-PCR) and immunohistochemistry.
AC-1M88, JEG-3 and HEC-1-A human endometrial cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and from the German Collection of Microorganisms and Cell Cultures (DSMZ, Germany), and were grown at 37°C in a 5% CO2 incubator.
To obtain human primary EEC in culture, endometrial biopsies from patients in the luteal phase were minced into small pieces (<1 mm) and digested with a mild collagenase solution (0.1%) at 37°C for 1 h. Endometrial epithelium was isolated and purified as previously described (Simón et al., 1993). EEC were cultured to confluence in a steroid-depleted medium containing a 3:1 mixture of DMEM (Sigma, St Louis, MO) and MCDB-105 (Sigma), 5 mg insulin (Sigma), which was supplemented with 10% charcoal-dextran treated fetal bovine serum (FBS) (Hyclone, Logan, UT). The homogeneity and purity of the EEC cultures were assessed by immunohistochemical markers (Simón et al., 1994) and morphological characteristics (scanning electron microscopy) (Simón et al., 1999). After confluence, the culture media were replaced with CCM medium (Vitrolife, Sweden). Conditioned media and CCM were collected as a control after a 24-h exposure to these cells.
RNA isolation and DNase I digestion
Total RNA was extracted from whole endometrial tissue or cell lines. We used placenta and lymphocyte mRNA and water as the positive and negative controls, respectively. Samples were collected and processed in Trizol (Gibco/BRL, Madrid, Spain) according to the manufacturer's instructions. These were followed by two rounds of phenol/chloroform clean-up, precipitated overnight at –20°C with 0.5 volumes of isopropanol and washed with ethanol 70% (v/v). Total RNA (20 µg) of endometrial biopsies and cell lines was treated with 5 µl of DNase I (Clontech, Palo Alto, CA, USA) at 37°C for 30 min. This was followed by one round of phenol/chloroform, another round with only chloroform, and precipitation overnight at –20°C using a 0.1 volume of 2 M sodium acetate pH 5.2 and a 2.5 volume of 100% ethanol. RNA was washed with 80% ethanol and the pellet was dissolved in 20 µl of RNase-free water. The integrity and quantity of the RNA was assessed using a RNA 6000 Nano Assay kit (Agilent technologies) measured in the Agilent 2100 Bioanalyser (Agilent technologies). The RNA integrity number was >7 and considered to be of good RNA quality in all cases.
Reverse transcription and PCR
Reverse transcription (RT) was carried out using the Advantage RT-for-PCR KIT (Clontech). One microgram of each sample was diluted in diethylpyrocarbonate (DEPC)-treated water with oligo (dT); the mixture was heated at 70°C for 2 min and kept on ice until the master mix was added. For each RT, a blank was prepared using all the reagents except the RNA sample, for which an equivalent volume of DEPC water was substituted. The RT blank was used to prepare the PCR blank. Once all the components were mixed, samples were incubated at 42°C for 1 h, and heated at 94°C for 5 min to stop cDNA synthesis and to destroy DNase activity. The product was diluted to a final volume of 100 µl with DEPC-treated water and stored at –20°C until PCR analysis. The PCR primers for CXCR3 and CXCL10 have the following sequence; CXCR3: forward 5'-ACCACAAGCACCAAAGCA-3' and reverse 5'-GGTAGCGGTCAAAGCTGA-3'. CXCL10: forward 5'-GAACTGTACGCTGTACCTGCA-3' and reverse 5'-TTGATGGCCTTCGATTCTGGA-3'. For the second round of PCR, inner primers for CXCR3 were used: forward 5'-ACACCTTCCTGCTCCACCTA-3' reverse 5'-GTTCAGGTAGCGGTCAAAGC-3'.
Real-time fluorescent PCR
The LightCycler (Roche Diagnostics, GmbH Mannheim, Germany) instrument was used to determine the relative gene expression quantification of CXCL10; GAPDH was chosen as the housekeeping gene control. The SYBR® Green I double-stranded DNA binding dye (Roche Diagnostics) was the chemical of choice for these assays. The oligonucleotide sequences designed for the amplification of CXCL10 were those used for the RT–PCR (see above). All the real-time PCR assays were run using the SYBR® Green PCR Master Mix and the universal thermal cycling parameters indicated by the manufacturer (60°C annealing temperature for all primers). Relative quantification was done by the standard curve method using the SYBR® Green I dye and taking into account the efficacy of the reaction. Data are presented as the relative average value of each gene investigated and then normalized with the average value of the housekeeping gene obtained on different days in each designated phase of the menstrual cycle in three duplicates. Quantification data were analyzed at the beginning of the exponential phase (cycles 30–35) with the Lightcycler analysis software version 3.5. To validate real-time PCR, standard curves with r > 0.95 and slope values between 3.1 and 3.4 were required.
Immunohistochemistry of the human endometrium
Five formalin-fixed and paraffin-embedded endometrial biopsies from each menstrual cycle group (n = 5 in each group) were sectioned and mounted on glass slides coated with Vectabond TM (Vector Lab, Burlingame, CA, USA). Twelve serial sections (6 µm) from each sample were prepared, and the first and last sections were stained with hematoxylin–eosin. After deparaffinization and rehydratation, sections were rinsed three times with phosphate-buffer saline (PBS) for 5 min. Immunohistochemistry was performed on endometrial sections using the DAKO LSAB Peroxidase Kit. Non-specific binding was blocked with non-fat milk (50 mg/ml in PBS). Sections were incubated for 40 min at room temperature with anti-human CXCL10 (R&D systems, Minneapolis, MN). Negative controls were incubated with PBS with BSA 1% and Tween 20 0.1%. Slides were mounted with Entellan (Merck, Darmstadt, Germany).
Immunocytochemistry of human blastocysts
For the immunostaining of human blastocysts, we used a fluorescence staining method with a primary monoclonal antibody against human CXCR3 (R&D systems) at 10 mg/ml. Human embryos were previously fixed with 2% freshly prepared paraformaldehyde in PBS for 30 min at 4°C in micro-drops under mineral oil (Simón et al., 1994). After fixation, blastocysts were treated with 0.2% Triton X-100 (Sigma) in PBS for 10 min at 4°C to permeabilize fixed cells to facilitate the access of the antibody. FITC-labeled goat secondary antibodies (Sigma) were used for 30 min at 37°C. Blastocysts were photographed using a Nikon Eclipse 80I (Nikon, Japan). Six human triploid blastocysts were analyzed for CXCR3, and three used as negative controls. Confocal analysis was performed with an NRC 1024 instrument (Bio-Rad, Hempstead, UK). The excitation line used was 488 (FITC), and the filter used was HQ515/10 (FITC).
Protein array
The conditioned media obtained from cultured EEC were analyzed using chemiarrayTM (Chemicon International, Temecula, CA) containing 120 proteins (See Table I). This system has a range limit detection of 10–250 000 pg/ml, and the variation of duplicates ranges from 0 to 10% in duplicate experiments. A total of 10 EEC-conditioned media (n = 2 pooled in groups of five) and 15 control media (n = 3 pooled in groups of five) were analyzed. Five hundred mircoliters of media from each monolayer of EEC were collected and centrifuged to avoid cell contamination, but only 50 µl were pooled and diluted to 1 ml with blocking buffer (one-fourth dilution). ChemiarrayTM membranes were blocked for 2 h at room temperature prior to media incubation. Each pool of media was incubated in two different membranes (Arrays VI and VII) for 2 h at room temperature. After sample incubation and washing, a diluted biotin-conjugated anti-cytokine primary antibody was applied to each membrane for 2 h at room temperature. After washing, diluted HRP–streptavidin was added to membranes for 2 h at room temperature, treated with the ECL detection system and exposed to Kodak X-omat film.
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Image densitometry analyses of all the membranes were performed using the Image J software (http://rsb.info.nih.gov/ij/). Spot intensity was quantified, and relative expression levels between proteins were calculated. For all the membranes, the background signal was subtracted using Image J tools, and the spot intensity was quantified as densitometry arbitrary units using positive spots to obtain relative values.
Cell migration assay
QCMTM 24-well colorimetric cell migration assay (Chemicon Int.) was used to evaluate the migration rates of the trophoblast cell lines JEG-3 and AC-1M88 cells to CXCL10 and IL-6. Based on the Boyden chamber principle, 0.5 x 106 cells were grown in inserts with an 8 µm pore size using serum-free media. In the lower chamber, different concentrations of CXCL10 (0.025, 0.05, 0.1, 0.5 and 1 µg/ml), IL-6 (1, 2.5 and 5 ng/ml) and IL-6 blocking antibody (1, 5 and 10 µg/ml) were added. Positive controls, which included the addition of 10% FBS to the medium and negative controls, were incubated only with the serum-free medium. At least three wells were determined in each situation analyzed (n = 3). Furthermore, the conditioned media from EEC in culture were added to the lower chamber (n = 6). Migration was analyzed at 24 h incubation by extracting the cells that had migrated thorough the micropore membrane using a colorimetric measure and reading (560 nm) with a microplate reader (Spectramax®, Molecular Devices, Toronto, Canada). The optical density obtained correlated with the number of cells migrated using the FindGraphTM software.
Statistical and bioinformatic analysis
The optical density of each situation in the chemotaxis experiments was correlated with control samples by a linear regression analysis followed by an analysis of variance. Statistical significance was defined as either P < 0.05* or 0.01**. Student's t-tests were employed for the mean comparisons between groups using the Bonferroni correction when the data followed a normal distribution. The statistical analysis was performed using the Statistical Package for Social Sciences (SPSS, Chicago, IL).
Statistical array analysis was carried out using the R software (http://www.r-project.org/) and the appropriate Bioconductor packages (http://www.bioconductor.org/) run under R (see below). In order to remove all the possible sources of variation of a non-biological origin between arrays, densitometry values between arrays were transformed to the logarithmic scale (log2) and normalized using the quantile normalization function implemented in the limma package. Statistically significant differences between groups were identified using the Student's t-test. Moreover, with the aim to correct the raw P-values and to ascertain the false discovery rate (FDR), a multiple hypothesis test [Benjamini and Hochberg (BH) test] was carried out using the Biocondutor multitest package (See Table II).
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Identification and quantification of CXCL10 and CXCR3 mRNA in the human endometrium and human endometrial cell lines
We detected CXCL10 mRNA in endometrial samples and primary cultures of EEC. However, no expression was found in the epithelial cell line HEC-1-A (Fig. 1A1). CXCR3 receptor mRNA expression was detected in both the endometrium and placenta with the first round of PCR (Fig. 1A2). With the second round of PCR, CXCR3 mRNA was evident in the trophoblast cell lines JEG-3 and AC-1M88, and in HEC-1-A epithelial cell line (Fig. 1A3), indicating the low expression of the CXCR3 receptor in these cell lines.
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QF-PCR of CXCL10 mRNA was performed in endometria from the five groups indicated throughout the menstrual cycle. Data obtained from 25 patients (5 in each group) were analyzed, and the mean and SD were obtained. CXCL10 expression peaked (14.7-fold-up) in the late proliferative phase (Group II, Days 9–14) and maintained a plateau in the remaining phases of the menstrual cycle (Fig. 1B).
Immunolocalization of endometrial CXCL10 throughout the menstrual cycle and CXCR3 in the human blastocyst
The data obtained from 25 patients (5 in each group of the menstrual cycle) were analyzed by immunohistochemistry for CXCL10. Weak staining restricted to the endometrial glands was observed in the early proliferative phase (Group I, Days 1–8), (Fig. 2A1). CXCL10 staining clearly peaked in the late proliferative phase with a strong expression not only in the glandular and luminal epithelium, but also in the stroma (Fig. 2A2), thus corroborating the mRNA expression profile. During the early secretory phase, a weak CXCL10 signal was detected in the stroma cells, but moderate staining was still observed in the luminal epithelium. The mid-secretory phase showed moderate staining in all locations, while CXCL10 staining in the late secretory phase decreased in the stroma and epithelium (Fig. 2A4–5). To confirm the presence of the CXCR3 receptor in the human blastocyst, we localized this receptor in three triploid human embryos that reached the blastocyst stage and were donated for research purposes; staining was localized in the trophoectoderm (Fig. 2B).
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Chemotactic effect of CXCL10 and EEC-conditioned media on the migration of trophoblast cell lines
As proof of concept, we investigated the chemotactic effect of CXCL10 in a well-established model using the human trophoblast cell lines JEG-3 and AC-1M88 in inserts with a pore size of 8 µm using serum-free media. In the lower chamber, different concentrations of chemotactic agents were placed and trophoblast attracted cells were counted. At least three wells were studied in each situation analyzed (n = 3). Figure 3 shows the chemotactic effects of CXCL10 and EEC-conditioned media on JEG-3 (Fig. 3A) and AC-1M88 (Fig. 3B). In the JEG-3 cell line, higher concentrations of CXCL10 (0.1, 0.5 and 1 µg/ml) induced a significant increase in cell migration when compared with controls, but EEC-conditioned media was superior. In AC-1M88 cells, CXCL10 (0.025, 0.05, 0.1, 0.5 ng/ml) did not produce any statistical differences in the cell migration compared with controls. Surprisingly, EEC-conditioned media was a superior chemotactic agent in the two cell lines analyzed, and proved to be even better than the positive controls. Therefore, we decided to investigate putative chemotactic agents within the EEC-conditioned media.
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EEC-conditioned media composition
To determine their composition, we analyzed the EEC-conditioned media using a ChemiarrayTM, a protein membrane array containing more than 120 proteins (a complete list of the proteins studied can be consulted in Table I).
Comparison of EEC-conditioned media versus control media (CCMTM) revealed the profile of secreted/consumed proteins by EEC (Table II). Interestingly, the most abundant/secreted protein in EEC-conditioned media was IL-6 with a 5.45-fold increase compared with control media, followed by PlGF (3.74-fold), GCSF, Eotaxin-3, BLC (CXCL13) or IGFBP-2, all of which with 2-fold increase, or more. However, only IL-6, BCL (CXCL13) and bFGF were statistically higher and presented a FDR value of <0.05.
Chemotactic effect of IL-6
We decided to test different IL-6 concentrations (1, 2.5 and 5 µg/ml) in the migratory capability of the trophoblast cell line AC-1M88. At least three wells were studied in each situation analyzed (n = 3). Statistical differences were found in the cell migration rate among controls, 2.5 and 5 ng/ml concentrations of IL-6 (Fig. 4). To further confirm this effect, a blocking IL-6 antibody was added at three different concentrations (1, 5 and 10 µg/ml) to EEC-conditioned media. We observed a clear and significant reduction of the chemotactic effect on AC-1M88 (Fig. 4B) at the 5 µg/ml concentration, confirming that IL-6 is a potent chemotactic agent for the trophoblast cells present in EEC-conditioned media.
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When a human blastocyst contacts with the endometrial epithelium, chemokine receptors CXCR1, CXCR4 and CCR5 increase in number, and the polarization of these receptors becomes evident (Dominguez et al., 2003b). These chemokines, secreted locally by the endometrium in the window of implantation or by the human blastocyst in the apposition phase (Caballero-Campo et al., 2002), may act as a signal for receptor polarization/dimerization, thereby acting as a sensor mechanism to increase the local cell responsiveness in the activation of endometrial adhesion molecules. Could the blastocyst form these complexes in response to endometrial chemokines and, therefore, trigger a trophoblast similar adhesion response? We still do not know, but our initial hypothesis was that CXCL10, or perhaps others, could perform a similar action in humans as it does in ruminants. In humans, the simultaneous presence of Regular upon Activation, Normal T cell Expressed and Secreted (RANTES) and monocyte chemoattractant protein (MCP-1) induces the heterodimeric receptor complex CCR2B-CCR5, which has unique features, including the reduction of the threshold concentration of chemokine required to induce a response and promoting adhesive properties in the immune cells (Mellado et al., 2001). Furthermore, CCR2B and CCR5 receptors have already been localized at the human blastocyst (Dominguez et al., 2003b).
In humans, the oligomerization of CXCL10 is also required for transendothelial migration, an essential step for lymphocyte recruitment in vivo (Campanella et al., 2006). It has been demonstrated that CXCL10/CXCR3 can stimulate the trophoblast migration in caprine (Nagaoka et al., 2003a; Imakawa et al., 2005, 2006), and CXCL10 stimulates the migration of immune cells in the goat uterus (Nagaoka et al., 2003b). Furthermore, adhesive activity of trophoblast cells to EEC was promoted by CXCL10 (Kai et al., 2002), stimulating the expression of integrins 5v as a first step in the adhesion of the blastocyst (Nagaoka et al., 2003a).
In the present work, we have investigated the localization and chemoattractive potential of CXCL10 and its receptor CXCR3 in the human model. CXCR3 was localized at the trophoectoderm of the human blastocyst, and the CXCL10 protein was present in the endometrium throughout the menstrual cycle. Therefore, the complete system is in place at the embryo–endometrial unit. To confirm our hypothesis, we analyzed the CXCL10 chemotactic activity using the well established human trophoblastic cell lines JEG-3 and AC-1M88 (Hannan et al., 2006). Although, a positive chemotactic effect of CXCL10 on human trophoblast cells was observed at different concentrations, EEC-conditioned media present a surprisingly superior chemotactic effect, suggesting that CXCL10 has a limited chemotactic effect on the human trophoblast, possibly due to the limited quantity of the CXCR3 receptor present at the embryo compared with white blood cells.
To further understand which secreted proteins could be implicated in this potent chemotactic effect, we analyzed EEC-conditioned media using a protein array (ChemikineTM) including more than 120 proteins related to the chemokine and cytokine family, and also with other important secreted molecules related to the implantation process. Remarkably, the most abundant protein present in the media was IL-6. We then tested its chemotactic effect on the AC-1M88 trophoblastic cells, and found a consistent maximal chemotactic activity in the dose–response experiments that was abolished when IL-6 blocking antibodies were added to EEC-conditioned media. The effect of IL-6 on human implantation has been extensively studied (Sánchez-Cuenca et al., 1999; Dimitriadis et al., 2005). In fact in mice, the numbers of implantation sites or litter sizes decrease when this cytokine is absent (Salamonsen et al., 2000). Another study suggested that IL-6 could be considered an endometrium–trophoblastic regulator of cytotrophoblastic gelatinases (Meisser et al., 1999), indicating a clear effect on chemotaxis in the invasion process.
The second more abundantly secreted molecule in EEC-conditioned media was CXCL13 (B-cell-attracting homing chemokine). It has been described in the immune system as natural antibody production and mucosal immunity (Ansel et al., 2002). Interestingly, our group has recently demonstrated that this chemokine is the most consumed (and therefore reduced) in the conditioned media from those human embryos that were able to implant when compared with non-implanted ones (Dominguez et al., 2008). We have collected information from two separate studies, this being that an abundant chemokine secreted by the endometrial epithelium in culture is that most needed by the blastocyst that has the capability to implant (Dominguez et al., 2008).
Although CXCL10 and IL-6 display a positive chemotactic activity over trophoblast cell lines on their own, we have demonstrated that the combination of molecules secreted by the human EEC has the maximal chemotactic effect in vitro.
In conclusion, the CXCL10/CXCR3 system is present in the human endometrial–blastocyst interface with a chemoattractive function. However, EEC-conditioned media is the most potent chemoactive force where IL-6 and CXCL13 are the most abundant molecules in the EEC participating in trophoblast migration.
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The participation of FD in this work was partially supported by a grant from Ministerio de Educacion y Ciencia. PTQ05-01-01360 (Torres Quevedo).
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Submitted on April 15, 2008; resubmitted on May 13, 2008; accepted on May 15, 2008.
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