Interleukin-1β regulation of gonadotropin-releasing hormone messenger ribonucleic acid in cultured human endometrial stromal cells

FERTILITY AND STERILITY威 VOL. 79, NO. 2, FEBRUARY 2003 Copyright ©2003 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.

Interleukin-1␤ regulation of gonadotropinreleasing hormone messenger ribonucleic acid in cultured human endometrial stromal cells Hong-Yuan Huang, M.D.,a Francisco Raga, M.D.,b Yan Wen, M.D.,b Jan S. Kruessel, M.D.,b Yung-Kuei Soong, M.D.,a and Mary Lake Polan, M.D., Ph.D.b Stanford University School of Medicine, Stanford, California

Received September 28, 2001; revised and accepted June 27, 2002. Supported in part by NIH Grant No. HD31575 (to M.L.P.) and Chang Gung University Research Grant No. CMRP 897 (to H.Y.H.). Reprint requests: HongYuan Huang, M.D., Department of Obstetrics and Gynecology, Lin-Kou Medical Center, Chang Gung Memorial Hospital, 5, Fu-Hsing Street, KweiShan, Tao-Yuan, Taiwan (FAX: 886-3-3288252; E-mail: hongyuan@adm. cgmh.org.tw). a Department of Obstetrics and Gynecology, Lin-Kou Medical Center, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan. b Department of Gynecology and Obstetrics, Stanford University School of Medicine. 0015-0282/03/$30.00 doi:10.1016/S0015-0282(02) 04690-3

Objective: To investigate the role of interleukin-1␤ (IL-1␤) in regulating GnRH mRNA expression in cultured human endometrial stromal cells using a modified semiquantitative competitive reverse transcription and polymerase chain reaction (PCR). Design: A controlled study. Setting: Clinical and academic research setting in a university medical center. Patient(s): Women undergoing hysterectomy for nonmalignant indications. Intervention(s): Confluent stromal cell cultures treated with steroid hormones were stimulated with IL-1␤ and attenuated by anti-IL-1␤ antibody or IL-1 receptor antagonist. Main Outcome Measure(s): The human endometrial stromal cell expression of GnRH and its receptor were determined by PCR. Interleukin-1␤-mediated regulation of stromal cell GnRH mRNA expression was determined by quantitative competitive PCR. Result(s): The GnRH and GnRH receptor mRNA expression were amplified in cultured stromal cells by PCR and two rounds of nested PCR, respectively. Treatment with IL-1␤ stimulated stromal cell GnRH mRNA expression at concentrations of IL-1␤ above 10 IU/mL. Recombinant IL-1 receptor antagonist and anti-IL-1␤ antibody attenuated the increase of gene expression of GnRH initiated by IL-1␤. Conclusion(s): These results provide indirect evidence that IL-1␤ may play a crucial role at the level of embryo–maternal interaction by regulating stromal cell expression of GnRH and its receptor, both known to be important in mediating trophoblast invasion and placental hormone regulation. (Fertil Steril威 2003;79: 399 – 406. ©2003 by American Society for Reproductive Medicine.) Key Words: Embryo implantation, gonadotropin-releasing hormone, interleukin-1, polymerase chain reaction

Gonadotropin-releasing hormone regulates gonadotropin biosynthesis and release from the anterior pituitary by a specific receptor. Extrapituitary expression and action of GnRH has been demonstrated in several extrapituitary organs (1, 2). Recently, a possible role for GnRH in preimplantation embryonic development, endometrial preparation, and implantation has been suggested. We have shown that both GnRH and its receptor are expressed at the mRNA and protein level in vivo by human endometrium throughout the entire menstrual cycle (3, 4). Moreover, we have also demonstrated that preimplantation embryos (mouse and human) and fallopian tubes also express

this hormone and its receptor, suggesting that GnRH may play a role in the embryonic/endometrial dialogue during early implantation (5, 6). Human endometrium is an active site for cytokine production and action (7, 8). Several studies strongly suggest a critical role for autocrine/paracrine cytokines as major local regulators of steroid hormone action (9) and implicated them in the implantation process (10). The interleukin-1 system has also been implicated as a major factor in these events. Interleukin-1 is a family of polypeptides comprised of two agonists: interleukin-1␣ and 1␤ (IL-1␣ and IL-1␤) and an inhibitor, interleukin-1 re399

ceptor antagonist (IL-1ra) (11). Two receptors (IL-1R) have been identified and characterized as type I (IL-1R tI) (12) and type II (IL-1R tII) (13). Both IL-1 agonist and antagonist are recognized by IL-1R tI, and trigger signal responses in target cells (14, 15). The IL-1R type II (IL-1R t II) is found on many cells, but primarily on neutrophils, monocytes, and B-lymphocytes.

Cells were cultured in 75% Dulbecco’s minimum essential medium (DMEM; GIBCO-BRL, Grand Island, NY) and 25% MCDB-105 (Sigma Chemical Co., St. Louis, MO), containing antibiotics, 5 ␮g/mL insulin (Sigma), and 10% charcoal-stripped fetal bovine serum (⬍5 pg/mL of E2 and ⬍10 ng/dL of P) (Gimmini, Calabasas, CA).

Recent knowledge of IL-1 expression in both human endometrium and the embryo has led to a more detailed outline of implantation events. The presence of the IL-1 system has been documented in human endometrium (16 – 18). Furthermore, the results obtained from immunohistochemical studies have localized the complete IL-1 system in human oocytes and embryos at all developmental stages (19, 20). These results suggest that the entire IL-1 system may play an important role in embryo implantation and decidualization of stromal cells.

Stromal cells (1–5 passages) were plated at 2 ⫻ 105/well in 24-well culture plates (Falcon, Becton Dickinson, Lincoln Park, NJ) and cultured in standard medium. Confluence cell cultures were treated with serum-free standard medium supplemented with 10 ␮g/mL human apo-transferrin (Sigma), 50 ␮g/mL ascorbic acid (Sigma), 1 mol/L P (Sigma), 10 nmol/L E2 (Sigma), 20 ng/mL epidermal growth factor (EGF) (Sigma), and 1 mg/mL bovine serum albumin (BSA) (Irvine Inc., Santa Ana, CA) for 9 consecutive days to mimic the usual in vivo implantation period. Control confluent cells were cultured in the same medium in the absence of E and P. Unless indicated otherwise, standard medium and serum-free medium were renewed every 2–3 days throughout the culture period. Conditioned serum-free standard medium was collected and frozen at ⫺70°C until assayed for endogenous IL-1␤, IL-1ra, and PRL production.

The GnRH agonists are now used routinely in conjunction with exogenous gonadotropins in most ovulation induction protocols for patients undergoing IVF. The benefits of using these analogues are the increase in the overall pregnancy rate by preventing premature LH surge and increasing the number of retrieved oocytes and embryos transferred (21). In addition, inadvertent GnRH administration during early pregnancy has been described, suggesting a positive role for extrapituitary GnRH in human early embryonic implantation (22, 23). Little is known about the possible role of cytokines and extrahypothalamic GnRH in human endometrium during embryonic implantation. We hypothesized that IL-1␤ and GnRH may both play a role in the embryonic/endometrial dialogue during early implantation. The aim of this study was to investigate the role of IL-1␤ in regulating GnRH mRNA expression in human endometrial stromal cells using a modified semiquantitative competitive reverse transcription and polymerase chain reaction (PCR).

MATERIALS AND METHODS Human Endometrial Stromal Cell Isolation and Cell Culture

Human luteal phase endometrium (n ⫽ 8) was obtained from surgical specimens of normally cycling women undergoing hysterectomy for nonmalignant indications including uterine fibroid or urinary incontinence, in accordance with the guidelines of the Declaration of Helsinki and with approval by the Stanford University Institutional Review Board after informed consent. The tissue samples used for this study were histologically normal. Stromal cells were separated from the glandular epithelium after collagenase digestion and cultured using an established in vitro model as previously described (24). Cultures prepared by this method contained ⬍0.1% of endometrial epithelial or vascular cell in the stromal cells culture (25). 400

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Hormonal Treatment

ELISA for IL-1␤, IL-1ra, and PRL Levels in Conditioned Medium Conditioned media were collected before the addition of cytokine for measurement of endogenously produced IL-1 using an ELISA kit (R&D Systems, Minneapolis, MN) with a detection limit of 1 pg/mL and an intra-assay precision of 2.3%–3.4%, and interassay precision of 3.4%–7.1%; and for measurement of IL-1ra with a detection limit of 14 pg/mL and an intra-assay precision of 2.7%– 8.3%, and interassay precision of 4.9%–5.9%. As a marker of decidualization, PRL in conditioned medium derived from stromal cell cultures was measured by ELISA (Diagnostic Systems Laboratories, Webster, TX) with a detection limit of 0.14 ng/mL and intra-assay and interassay coefficients of variation of 5.5%–9.0% and 6.6%–10.4%, respectively. All samples were assayed in triplicate.

Dose–Response Study of Recombinant Human IL-1␤ Confluent stromal cells treated with steroid hormones for 9 days were stimulated with recombinant human IL-1␤ (1 ⫻ 105 IU/␮g; Genzyme, Cambridge, MA) in a dose-dependent study (0 –1,000 IU/mL) for an additional 24 hours. As a control for IL-1␤ specificity, stromal cells were cultured in serum-free medium in the presence of recombinant human IL-1␤ (100 IU/mL) neutralized with increasing concentrations of recombinant IL-1ra (0 –10 ␮g/mL) (R&D Systems) or anti-IL-1␤ monoclonal antibody (0 –3 ␮g/mL) (Genzyme) for an additional 24 hours. As a protein control on cell behavior, stromal cells were cultured with same dose dependent of increasing concentrations of albumin (BSA; Irvine

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TABLE 1 The human oligonucleotide primers for mRNA amplification of stromal cells. mRNA

␤-actin

Primers 5’–3’

Size (bp) 838

Up-stream (5’ end)

ATC TGG CAC CAC ACC TTC TAC AAT GAG CTG CG CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT GC AAA ACT CCT AGC TGG CCT TAT GGA ATA TGT GCA ACT TGG TG GGA ATA TGT GCA ACT TGG TG A GCG TTG GGT TTC TGC GAT TGT CAT GCC ACT GGA TG

Down-stream (3’ end) Up-stream (5’ end)

TTA GAG TCT TCA GCC GTG CTC CAA CAG CAA AGT CGG ACA GT

Down-stream (3’ end)

AGC ATG AAA AGA GGG ATG ATG

Up-stream (5’ end) Down-stream (3’ end)

GnRH

GnRH receptor, 1st PCR GnRH receptor, 2nd PCR

Up-stream (5’ end) Down-stream (3’ end) Competitor (3’ end)

Position on cDNA 294–325 1,131–1,100

399

524

47–67 445–426 445–426 ⫹213–198 300–319

231

823–801 477–496

204

706–687

Huang. IL-1␤ and GnRH in human endometrial stromal cell. Fertil Steril 2003.

Scientific, Irvine, CA). Minimums of three experiments of individual cultures from different specimens were performed.

quenched at 4°C. Products were stored at ⫺20°C until the subsequent PCR.

RNA Analysis Total RNA was extracted from cultured stromal cells using the guanidinium isothiocyanate method (RNAzol, Teltest, Inc., Friendswood, TX). The RNA concentration was quantified by measuring optical density with a Spectronic 601 spectrophotometer (Milton Roy Co., Rochester, NY). RNA was diluted to 1 g/L for reverse transcription-PCR.

Aliquots of the reverse transcription products were subjected to PCR in the PCR master mix containing AmpliTaq DNA polymerase and corresponding paired primers to a total volume of 100 ␮L. The PCR cycles were composed of one cycle of 99°C for 5 minutes to denature all proteins, 30 cycles of 45 seconds at 94°C, 45 seconds at 55°C, and 60 seconds at 72°C. Reaction was terminated at 72°C for 5 minutes and quenched at 4°C.

Primers for Reverse Transcription and PCR

Nested PCR for the GnRH Receptor

Sequences of human cDNA clones for the mRNAs to be detected in human endometrial stromal cells for ␤-actin (26), GnRH (27), and GnRH receptor (28) were obtained from the GenBank Database of the National Center for Biotechnology Information of the National Institutes of Health. The primer sequences, locations on the cDNA, and sizes of the amplified fragments are listed in Table 1. The ␤-actin mRNA expression was used as an internal positive control, being detected in all the samples studied, thus confirming the RNA has not been widely degraded.

Reverse Transcription-PCR For reverse transcription-PCR, the GenAmp RNA PCR kit (Perkin-Elmer, Foster City, CA) was used as in a previously established protocol (24, 29). In brief, reactions were started with 1 ␮g of total RNA extracted from stromal cells in a total volume of 20 ␮L of reverse transcription master mix containing 2.5 ␮M oligo(dT) and the reverse transcription reaction was carried out in the DNA Thermal Cycler 480 (Perkin-Elmer GeneAmp, PCR Instrument System, Branchburg, NJ) using a program with one 15-minute reverse transcription cycle at 42°C, followed by 5 minutes at 99°C, then FERTILITY & STERILITY威

The localization of the expected fragment (524 bp) for the GnRH receptor mRNA was also examined in the cultured stromal cells and amplified signal was detected using one round of PCR (40 cycles) in only 20% of the samples studied. For this reason, the sensitivity was further increased by performing two rounds of nested PCR (30 cycles in the first and the second round, respectively) with corresponding pairs of primers for GnRH receptor developed as our earlier report (4, 30) indicated. Program parameters were identical with those of PCR for GnRH expression. After the second round of PCR was completed, samples were stored at ⫺20°C until agarose gel electrophoresis was carried out.

Agarose Gel Electrophoresis A 2% agarose gel (GIBCO-BRL) electrophoresis was carried out and gels were stained with ethidium bromide (Sigma). Aliquots (25 ␮L) of each PCR product and dye buffer were analyzed in parallel with a 100-bp DNA ladder (GIBCO-BRL) as a standard. After completion of electrophoresis, the gel was analyzed and photocopies of the blot were printed on the UV-densitometry (Gel-Doc 1000 system, Bio-Rad Laboratories, Hercules, CA). 401

FIGURE 1 (A), A representative blot of products of PCR amplification for cultured endometrial stromal cell GnRH (S). Human luteal endometrium (ME, LE) and placenta (P) were used as positive control. ME, human midluteal endometrium; LE, human late luteal endometrium. (B), The products of nested PCR for stromal cell GnRH receptor. L ⫽ 100 bp DNA ladder.

Huang. IL-1␤ and GnRH in human endometrial stromal cell. Fertil Steril 2003.

Construction of the Competitive and Target cDNA Fragments for Standard Curve and Quantitative Competitive PCR for GnRH For the purpose of determining the steady-state level of GnRH mRNA, competitive and native cDNA fragments were constructed for semiquantitative competitive PCR using an established methodology with modification as previously described (31). In summary, the target (native) cDNA (399 bp) was constructed from known human endometrial sequences. A competitive cDNA fragment was constructed by deletion of a 195-bp fragment from the GnRH target cDNA to be detected. The deleted cDNA fragment was synthesized from 1 ␮g human endometrial RNA amplified with the 5⬘-end original primer and 3⬘-end competitive primer and was purified from 2% agarose gel with an agarose gel DNA extraction kit (Boehringer, Mannheim, Germany). The PCR product extracted by DNA gel extraction kit was sequenced by DNA sequencer (Perkin-Elmer). The standard curve is produced with different amounts of standard (native) cDNA amplified with a constant amount of competitor cDNA. To determine the quantitative stromal cell GnRH mRNA expression as a function of IL-1␤ concentration, we coamplified the PCR products for 30 cycles in the presence of defined amounts of internal standard cDNA for GnRH.

Data Analysis The statistical analysis was carried out by using the Statistical Package for Social Science Stat view Package (SPSS Inc., Chicago, IL) with a P value of ⬍.05 considered statis402

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tically significant. Statistical analysis was performed by ANOVA and t test in this study.

RESULTS Level of IL-1␤, IL-1ra, and PRL in Conditioned Media From Cultured Human Endometrial Stromal Cells A total of 17 representative experiments from individual cultures were included in this study. The effect of steroids on PRL production, a known marker of decidualization, by human endometrial stromal cells treated for 9 days with E and P in serum-free medium was 3.9 ⫾ 0.4 ng/106 cells (the mean ⫾ SD of PRL levels [per day] obtained from 17 representative experiments). The low level of PRL suggests that these cultured endometrial cells were not yet decidualized. In addition, there was no detectable IL-1␤ (⬍3.9 pg/ 106 cells/mL) and IL-1ra (⬍31.2 pg/106 cells/mL) in conditioned media from cultured human endometrial stromal cells. There was also no detectable IL-1ra in conditioned media from stromal cells cultured for 24 hours with increasing concentrations of IL-1␤.

Expression Pattern of GnRH and its Receptor mRNA in Human Endometrial Stromal Cells The GnRH and GnRH receptor mRNA in stromal cells was both amplified by PCR (Fig. 1). Figure 1A shows a sequence of 399 bp of GnRH mRNA amplified from stromal cells. Human luteal endometrium and placenta RNA were amplified as positive controls. Figure 1B shows a 231-bp band corresponding to GnRH receptor mRNA transcription after two rounds of nested PCR.

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FIGURE 2 A representative blot shows the standard curve obtained for GnRH mRNA by plotting the logarithmically transformed ratios of the densities of target cDNA to competitive cDNA against the log amount of initially added target cDNA in each PCR.

Huang. IL-1␤ and GnRH in human endometrial stromal cell. Fertil Steril 2003.

Quantitative Analysis of Stromal Cell GnRH mRNA Level Regulated by IL-1␤ To examine semiquantitatively the amount of GnRH mRNA expression in the stromal cells with different concentrations of IL-1␤, we coamplified the PCR products for 30 cycles in the presence of defined amounts of internal standard cDNA for GnRH. Figure 2 shows the standard curve obtained for GnRH mRNA by plotting the logarithmically transformed ratios of the densities of target cDNA (399 bp) to competitive cDNA (204 bp) against the log amount of initially added target cDNA in each PCR. In Figure 3, the semiquantitative PCR of the stromal cell samples showed a progressive increase in the mRNA levels with increasing concentrations of IL-1␤ above 10 IU/mL in comparison to control (P⬍.05) (Fig. 3A). The IL-1␤-mediated increase in GnRH mRNA expression in stromal cells was attenuated by recombinant IL-1ra (Fig. 3B), and antiIL-1␤ antibody (Fig. 3C) in a dose-dependent manner.

DISCUSSION To the best of our knowledge, we have shown for the first time that both GnRH and its receptor are expressed at the mRNA level and regulated by IL-1␤ in cultured human FERTILITY & STERILITY威

endometrial stromal cells. We have previously hypothesized that GnRH may play a role in the embryonic/endometrial dialogue during early implantation (4, 5). To further examine this hypothesis, we assessed GnRH and GnRH receptor mRNA in human endometrial stromal cells, demonstrating that IL-1␤ may play a regulatory effect, increasing GnRH expression. Sex steroids and growth factors have been shown to regulate the stromal cell growth and differentiation in vitro (25, 32). Our results revealed that GnRH and its receptor were expressed in cultured human endometrial stromal cells after steroid stimulation for 9 days, consistent with such expression in luteal phase endometrium of the menstrual cycle. Furthermore, our results demonstrated that the regulatory effect of IL-1␤ on endometrial stromal cell GnRH expression was attenuated by either IL-1 receptor antagonist or anti-IL-1 antibody, suggesting a receptor–ligand relation in human endometrial stromal cells. The IL-1 system is a family of polypeptides comprised of IL-1 agonist, IL-1 receptor antagonist, and IL-1 receptors. Both IL-1 agonist and receptor antagonist bind to the IL-1 receptor. The IL-1ra is a specific inhibitor that competes with IL-1␣ and IL-1␤ for the binding site of IL-1R tI and blocks 403

FIGURE 3 A representative blot shows the quantitative PCR of different endometrial stromal cell samples incubated with increasing concentrations of IL-1␤ (0 –1,000 IU/mL), demonstrating a progressive increase in the mRNA levels (A). As a control of IL-1␤ specificity, recombinant IL-1ra (B) and anti-IL-1␤ antibody (C) attenuated IL-1␤-mediated up-regulation of GnRH mRNA expression in stromal cells in a dose-dependent manner. * Bars with the same symbols showed significant differences in comparison to control group (P⬍.05). Values are mean ⫾ SD of measurements on three representative blots.

Huang. IL-1␤ and GnRH in human endometrial stromal cell. Fertil Steril 2003.

signal transduction by IL-1 (33, 34). Recent knowledge of IL-1 expression in human endometrium and embryo has led to a more detailed outline of implantation events. Our earlier reports documented expression of the IL-1 system in single blastomeres of preimplantation human and mouse embryos (31, 35). Individual mouse embryos expressed peak IL-1ra mRNA expression at the blastocyst stage either cultured alone or cocultured with helper cells (30, 36). Interleukin-1␤ and transforming growth factor (TGF) may play crucial roles at the embryo–maternal interface during trophoblast invasion by regulating stromal cell expression of collagenase and their inhibitors, all of which are known to be important in trophoblast invasion (24). These results suggest that the entire IL-1 system may be relevant for human endometrial and embryo physiology. Furthermore, the presence of GnRH and its receptor mRNA and immunoreactive protein expression was demonstrated in human endometrium and embryo, suggesting that this family of molecules must be considered to display another paracrine language that may be important to endometrial and embryo cross-talk during embryo implantation. The GnRH is the hypothalamic hormone controlling secretion of both FSH and LH from the anterior pituitary (37). It is also one of the paracrine/autocrine regulators of human 404

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trophoblast hCG secretion during early pregnancy (38). Embryo implantation is the result of an embryonic–maternal dialogue, in which the embryo and the endometrium induce changes in each other to promote successful placentation and pregnancy. The role of GnRH in controlling placental hCG production and secretion has been fully demonstrated both in vitro (39) and in vivo (40), especially in first trimester placenta. An increasing body of evidence indicates that, in addition to the central action, a variety of human tissues express extrahypothalamic GnRH, which is immunologically, biologically, and chemically identical to the hypothalamic hormone (41). Recent studies have demonstrated the expression of mRNA for the human GnRH receptor in several extrapituitary organs such as the placenta, myometrium, breast, prostate, and ovary (2, 42– 45). In the present study, the expression of extrahypothalamic GnRH was regulated by IL-1␤—assumed to be of embryonic origin—in cultured human endometrial stromal cells, demonstrating a possible role of this decapeptide in the embryonic– endometrial dialogue necessary for embryo implantation. Our results identified the levels of IL-1␤ at ⬎10 IU/mL (100 pg/mL), which may significantly increase the stromal cell GnRH mRNA expression. This is compatible to

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the physiological level of IL-1␤ during embryo peri-implantation interface (46). These results are concordant with our earlier report that infertile women undergoing IVF have a significantly higher success rate and implantation rate if the administration of GnRH agonist was maintained during the early stages of embryo development and implantation (22, 47). Quantitative analysis of low abundance mRNA derived from cells can be achieved by a modification known as quantitative competitive PCR (48 –50), in which an internal control molecule possessing a small deletion in the amplified portion of the specific molecule is amplified simultaneously with the target sample instead of another control molecule such as ␤-actin or globulin. In addition, because the efficiency of amplification of the internal control molecules is identical to that of the target template, quantitative PCR can avoid the discrepancies associated with tube-to-tube or sample-to-sample variations in the kinetics of the reverse transcription reaction. The use of reverse transcription-PCR with internal RNA competitive standards (competitors) provides a means for measuring absolute amounts of mRNA transcripts in small numbers of cells as shown in our earlier reports (24, 29). This methodology requires analysis of multiple reactions to determine the equimolar point of the products produced from mRNA versus competitor RNA. In the present study, we present a methodology modified by earlier studies (4, 51) to produce one standard curve for each assay with all unknown samples compared directly to this standard curve. The logarithmically transformed ratios of target cDNA to competitive cDNA were plotted against the log amount of initially added target cDNA in each PCR reaction to obtain the standard curve. The number of transcripts in an unknown sample mRNA can be directly determined by reverse transcription-PCR of the sample with the same amount of competitor RNA and comparison of the ratio of products to the standard curve. Thus, stromal cell GnRH mRNA expression is increased with increasing concentrations of IL-1 agonist. This is a principal advantage over other similar techniques, such as Northern blots, which only provide semiquantitative results. In summary, the purpose of this study was to document the presence of GnRH and its receptor in cultured human endometrial stromal cells and to examine the ability of IL-1␤ to regulate GnRH expression. On the basis of these findings, we hypothesize that the embryo may communicate with and influence the endometrial stromal cell through the IL-1 system, suggesting that the IL-1 system may play a paracrine role in embryonic development and communication during the implantation process.

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IL-1␤ and GnRH in human endometrial stromal cell

Vol. 79, No. 2, February 2003