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Division of Reproductive Endocrinology and Infertility (H.-F.C., K.-H.C., M.-Y.W., Y.-S.Y., H.-N.H.), Department of Obstetrics and Gynecology, College of Medicine and the Hospital, National Taiwan University; and Graduate Institute of Immunology (C.-Y.L., H.-N.H.), College of Medicine, National Taiwan University, Taipei 100, Taiwan
Address all correspondence and requests for reprints to: Hong-Nerng Ho, M.D., National Taiwan University Hospital, Department of Obstetrics and Gynecology, 7 Chung-Shan Street Road, Taipei 100, Taiwan. E-mail: . hnho{at}ha.mc.ntu.edu.tw
Abstract
Previous study demonstrated that IL-11 receptor 
knockout female mice (IL-11R-/-) were phenotypically normal but infertile due to defective decidualization. However, the role of IL-11 signaling in human reproduction remains unclear. This study examined the expression of IL-11, IL-11R, and signal transduction factor glycoprotein 130 in different phases of endometrium (six in proliferative phase and four in secretory phase), and the decidua and villi of normal pregnancy (NP; n = 25) and anembryonic pregnancy (AP; n = 25) in the first trimester (gestational week 7–9). RT-PCR showed IL-11, IL-11R, and glycoprotein 130 mRNA expression in all samples, except the absence of IL-11 signal in the unstimulated MRC-5 cell and the proliferative phase endometrium. Real-time quantitative PCR showed that the relative level of IL-11R mRNA was not significantly different among proliferative phase endometrium (relative level; mean ± SEM, 1.4 ± 0.5), secretory phase endometrium (1.3 ± 0.1), or decidua from NP or AP (1.7 ± 0.3 and 1.9 ± 0.4, respectively), but was significantly greater in chorionic villi either from NP or AP (7.6 ± 1.3 and 10.6 ± 1.9, respectively; both P < 0.05, compared with decidua or endometrium). No difference of IL-11R mRNA level was found between NP and AP (1.7 ± 0.3 vs. 1.9 ± 0.4 in deciduas; 7.6 ± 1.3 vs. 10.6 ± 1.9 in villi; both P > 0.05). In situ hybridization localized IL-11R mRNA expression in proliferative phase endometrium (stroma only), secretory phase endometrium (stroma and gland), decidua (stroma and gland), and villi (trophoblast and stroma). The staining intensities were not distinctly different between different groups of samples or between different cell types in a sample. No difference in IL-11R expression was found between NP and AP when either decidua or chorionic villi was analyzed. IL-11 mRNA level was not detected in the proliferative phase (relative level, 0.0 ± 0.0), was barely detectable in the secretory phase (0.03 ± 0.02), and was significantly increased in decidua (1.7 ± 0.2 and 0.1 ± 0.1, respectively, for NP and AP) and chorionic villi (13.0 ± 2.2 and 0.2 ± 0.1). In addition, IL-11 mRNA level was higher in NP than in AP both in decidua (1.7 ± 0.2 vs. 0.1 ± 0.1; P = 0.03) and in villi (13.0 ± 2.2 vs. 0.2 ± 0.1; P < 0.001). Immunohistochemistry study showed that IL-11 was nearly absent in endometrium in both phases, but clearly detectable in decidua and villi. Consistent with the results of quantitative PCR, the staining intensity was stronger in villi and decidua from NP than those from AP. The spatial and temporal changes in IL-11 and its receptor observed in this study suggest that IL-11 may be produced both by the embryo (predominantly) and the decidual cells and exerts its action on chorionic villi and decidua in an autocrine or paracrine manner. In the presence of a baseline level of IL-11R, IL-11 may subsequently regulate placentation and decidualization for the maintenance of a NP. The finding of decreased IL-11 expression in the absence of any change in IL-11R in AP suggests that defective expression of IL-11 but not IL-11R may account for certain cases of AP.
IMPLANTATION AND PLACENTATION are major steps in the development of mammalian embryos (1). The mechanisms underlying failures of implantation or placental development remain mostly unknown. Recently, a number of growth factors and cytokines, including colony-stimulating factor, leukemia inhibitory factor, and IL-11, have been suggested to play a role in these processes (2, 3, 4, 5, 6, 7). A temporal analysis revealed that IL-11 expression was maximal in the normal pregnant uterus at the time of decidualization in mice (4). Subsequently, mating of female mice with a null mutation of the IL-11 receptor (IL-11R-/-) with fertile males of any genotype for periods up to 12 months never resulted in clinical pregnancy (4). Histological study showed that these female mice were infertile due to defective decidualization (4). This observation identified a previously unrecognized critical role of IL-11 in female reproduction of mice during the period of decidualization. However, the role of IL-11 signaling in humans has not been established. Further studies are therefore needed to evaluate the expression of IL-11 and IL-11R in pathological conditions associated with human implantation failure or recurrent abortion.
IL-11 was first isolated as a soluble factor produced by the PU-34 primate bone marrow stromal cell line (8), and subsequently human IL-11 was cloned (9, 10). The actions of IL-11 are mediated through a receptor complex composed of IL-11R and signal transduction factor, glycoprotein 130 (gp130) (11). Recently, IL-11 has been shown to have pleiotropic effects in many tissues, including the hematopoietic system, skin, bone, central nervous system, thymus, and lung (12, 13, 14). Studies have also suggested that IL-11 plays various roles in the urogenital system (4, 12, 14, 15). For example, immunoreactive IL-11 was detected in human follicular fluid, although its action in the ovary has not been established (15).
The present study was designed to evaluate the role of the IL-11 system in early human pregnancy, especially in decidualization and placental formation. We hypothesized that gestational tissues from patients with spontaneous abortion might be characterized by differences in traits of IL-11 expression compared with those in normal pregnancy (NP). To test this hypothesis, we analyzed and compared IL-11, IL-11R, and gp130 expression in NP and anembryonic pregnancy (AP; blighted ovum).
Subjects and Methods
Subjects and materials
After confirmation by ultrasonography, 25 patients with the diagnosis of AP at the gestational age of 7–10 wk were recruited into the study group. Twenty-five age-matched patients with NP of gestational age 7–10 wk who had requested pregnancy termination due to multiparity were recruited as control. The maternal age (mean ± SEM, 32.3 ± 1.2 vs. 33.6 ± 2.1 yr for AP and NP, respectively) and gestational age (mean ± SEM, 8.8 ± 0.5 vs. 8.2 ± 0.6 wk) were comparable. Four of the 25 patients in the AP group had history of repeated spontaneous abortion of three or more times, whereas one in the NP group had this kind of history. Fetal heart motion was documented by ultrasound. Samples of decidua and chorionic villi were obtained during dilatation and curettage. Endometrial tissues were obtained from 10 patients with normal menstruation (6 in the proliferative phase and 4 in the secretory phase) who had undergone hysterectomy due to uterine fibroids. Peripheral blood mononuclear cells (PBMC) were obtained from nine normal, nonpregnant women by venous puncture. Cell lines for use as positive controls, including MRC-5, a human embryonal lung fibroblast cell line (served as a positive control for IL-11 expression); K562, a human chorionic myelogenous leukemia lymphoblast cell line (served as a positive control for IL-11R expression); and HeLa, a human cervical epitheloid carcinoma cell line, were obtained from the Food Industry Research and Development Institute of Taiwan. Informed consent was obtained from each patient who participated in this study. This study was approved by the Committee for Human Body Experiments of National Taiwan University Hospital in 1998.
Tissue and cell processing
The endometrial and gestational tissues were collected aseptically, put into test tubes containing normal saline, and immediately taken to the laboratory for further processing. The preparation of decidual and villus tissues was performed as previously described (16). Briefly, tissue mixtures were macroscopically separated into decidua and chorionic villi, washed twice with HBSS, and divided into portions for further use. After collection, the endometrial tissues were prepared using methods similar to those used for gestational tissues. Small pieces of endometrium, decidua, and villi were also stained with hematoxylin and eosin and examined by a pathologist to exclude any possible pathology. Venous blood was collected into a syringe prerinsed with heparin and sent to the laboratory for immediate preparation of PBMC. The blood samples were diluted 1:1 in HBSS and centrifuged on a 100% Ficol-Hypaque gradient at 1800 rpm for 20 min without the use of brake at completion. Cells at the interface were aspirated, diluted 1:9 in HBSS, and centrifuged twice at 1400 rpm for 5 min. Finally, the cell pellet was collected for further study. Cell lines were thawed, washed, and cultured in T75 tissue culture dishes (Corning, Inc., Corning, NY) using respective medium (-MEM plus 10% FCS for MRC-5; RPMI-1640 plus 10% FCS for K562 and HeLa cells). When cells covered approximately 90% of the dish floor, the culture medium was removed, and the cells (except K562 cells, which are floating cells) were washed with HBSS and trypsinized. The cell suspension was collected, washed, and centrifuged, and the cell pellet was harvested for subsequent subculture or further experiments.
RT-PCR
Portions of the tissues (endometrium, decidua, and chorionic villi) subjected to RT-PCR were first cut into small pieces, washed with PBS (diethyl pyrocarbonate-treated), and homogenized in liquid nitrogen. Subsequently, RNA was extracted using the Trizol reagent (Life Technologies, Inc., Grand Island, NY), chloroform-isopropanol-ethanol method as previously described (17). The concentration of RNA was analyzed by UV spectrophotometer to measure the absorbance at wavelengths of 260 and 280 nm (OD260 and OD280). Purity of RNA was considered adequate when an OD260/OD280 ratio of greater than 1.6 was reached. The integrity of the RNA sample was further verified both by agarose gel electrophoresis and examination of the RT-PCR product of ß-actin, a housekeeping gene. RNA samples (5 µg) were subsequently subjected to first strand cDNA synthesis using oligo(dT) primers and Superscript II reverse transcriptase following the manufacturer’s protocol (First Strand cDNA Synthesis kit, Pharmacia Biotech, Uppsala, Sweden). In a 50-µl reaction mixture, PCR was performed in the presence of 25 µl Taq Master mix (QIAGEN, Valencia, CA), 3 µl first strand cDNA template, 200 ng/µl 5'-primer, 200 ng/µl 3'-primer, and 20 µl double distilled H2O. The primer sets were obtained or designed according to either the published oligonucleotide primer sets or the published cDNA sequences for IL-11, IL-11R, gp130, and ß-actin (18, 19, 20, 21) (Table 1
). All of the primers were designed to span at least two exons to exclude possible genomic DNA contamination. The ß-actin mRNA level was also measured to control for the mRNA level in the sample and for possible semiquantitation. A number of preliminary experiments were performed to determine the PCR conditions and the cycle numbers that could ensure the PCR production in an exponential phase. The PCR was carried out in a thermal cycler (GeneAmp PCR System 2400; Perkin-Elmer Corp., Wellesley, MA) in the following sequence: denaturation at 94 C for 30–60 sec, annealing at 62–65 C for 30–60 sec, extension at 72 C for 1–2 min, and a final extension at 72 C for 10 min. The PCR was repeated for 30–38 cycles according to the gene of interest, and the PCR product was examined using 1.5% agarose gel electrophoresis and stained with ethidium bromide. Several positive and negative controls were used in the PCR. MRC-5 and K562 cells were used as positive controls for IL-11 and IL-11R, respectively. A reverse transcription reaction was carried out without the addition of reverse transcriptase, and the resulting product was subjected to PCR to exclude the possibility of genomic DNA contamination. PCR was also performed without the presence of template DNA to test for cross-contamination of samples.
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To accurately quantify the mRNA levels in the samples, a real-time quantitative PCR system (ABI PRISM 7700 Sequence Detection System, PE Applied Biosystems, Foster City, CA) was used as previously described (22) and with the manufacturer’s manual. Briefly, RNA was prepared, and cDNA was obtained by random hexamer priming. The primers and probes used in the PCR were designed according to the TaqMan primer and probe design system (PE Applied Biosystems), and their sequences are listed in Table 2. FAM (6-carboxyfluorescein) and VIC were the reporter dyes, and TAMRA (6-carboxy-tetramethyl- rhodamine) was the quencher dye. The probes were labeled with reporter dye and quencher dye on the 5' and 3' ends, respectively. During PCR, the reporter dye was released, and the resulting fluorescence was detected and quantified. A number of preliminary experiments were performed to determine the PCR conditions. The PCR was carried out in a thermal cycler (ABI PRISM 7700 Sequence Detection System) in the following sequence: reaction at 50 C for 2 min and 95 C for 10 min; subsequently, the PCR was repeated for 40 cycles at denaturation at 94 C for 15 sec and annealing and extension at 60 C for 1 min. The authenticity of PCR products was verified by 2% agarose gel electrophoresis and by sequencing. The relative concentration of each mRNA was subsequently calculated according to the manufacturer’s user manual. Briefly, the threshold cycle (CT) values of the target gene mRNA (IL-11 or IL-11R) and the internal control (ß-actin) in the studied sample were first measured. The 
CT value of the studied sample was calculated by the following formula: CT = CT of target gene - CT of ß-actin (this CT is designated as sample CT). Similarly, the CT values of the target gene and its respective ß-actin of positive control (proliferative phase endometrium) were also obtained, and the CT was calculated (designated as calibrator CT). CT was then calculated using the following formula: CT = CT (sample) - CT (calibrator); and finally the relative value of each mRNA was calculated by the formula: 2-CT. PCR without template was used as a negative control (called no template control) to verify experimental results.
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Protocols for the synthesis of riboprobes and in situ hybridization were modified according to a previous report (23). Briefly, antisense and sense digoxigenin (DIG)-labeled (by DIG RNA labeling kit, Boehringer Mannheim Biochemica, Mannheim, Germany) riboprobes against IL-11R were synthesized by in vitro transcription using a plasmid (an expression vector, pED, Genetics Institute; kindly supplied by Y.-C. Yang, Indiana University, Indianapolis, IN) containing IL-11R cDNA as the template. The specificity of this probe was verified by Northern blot analysis, and an expected 1.7-kb band indicating the hybridization of the probe with IL-11R mRNA was found (19). Further examination of the endometrium, decidua, and chorionic villi with Northern blot analysis also confirmed the efficiency of this probe (data not shown).
In situ hybridization was carried out as follows. After cleansing, tissue samples were fixed overnight at 4 C with 4% paraformaldehyde, incubated overnight at 4 C with 30% sucrose, and then embedded in OCT (Sakura Finetek, Torrance, CA). During or before experiments, tissues embedded in OCT were sectioned at 6-µm thickness and dried. These cryosections were treated with RNase-free proteinase K (1 µg/ml) at 37 C for 30 min, postfixed with 4% paraformaldehyde/0.2% glutaraldehyde at 4 C for 5 min, subjected to prehybridization at 37 C for at least 10 min, and then hybridized overnight at 55 C in hybridization buffer (40% deionized formamide, 10% dextran sulfate, 1x Denhardt’s solution, 4x SSC, 10 mM dithiothreitol, 1 mg/ml yeast tRNA, 1 mg/ml denatured and sheared salmon sperm DNA, and 5–10 ng/30 µl DIG-labeled riboprobes). On the next day, the tissue sections were washed sequentially with 2x SSC and 1x SSC, incubated at 37 C for 30 min in NTE buffer [500 mM NaCl, 10 mM Tris, and 1 mM EDTA (pH 8.0), containing 20 µg/ml RNase A], and then incubated with 0.1x SSC twice for 30 min each time. Then the sections were blocked in blocking solution (containing 2% normal sheep serum and 0.1% Triton X-100) at room temperature (RT) for 1 h, followed by incubation with sheep anti-DIG antibody (1:300 dilution)-alkaline phosphatase (Fab fragment, Boehringer Mannheim) (in Tri buffer with 2% normal sheep serum and 0.1% Triton X-100) overnight at 4 C in a humidified chamber. Finally, the color reaction was developed with 5-bromo-4-chloro-3-indoxyl-phosphate (BCIP)/nitro-blue tetrazolium chloride (NBT) (DAKO Corp., Carpinteria, CA) for 2–24 h in the dark in a humidified chamber, and counterstained with methyl green (Vector Laboratories, Burlingame, CA). Two negative controls were also used, one using tissue sections hybridized with sense probes and the other using pretreatment with RNase before hybridization with antisense probes. The staining intensities were read and graded anonymously by the same pathologist.
Immunohistochemistry
The immunohistochemistry study was performed according to previous reports (24, 25) and using the Streptavidin-Biotin Universal Detection System according to the protocol provided by the manufacturer (Immunotech, Marseille, France). Briefly, formalin-fixed, paraffin- embedded tissue sections were deparaffinized with xylene and hydrated with step-down concentrations of ethanol. The sections were then incubated with 30% H2O2/70% methanol solution at RT for 5 min, treated with protein blocking agent (goat serum) at RT for 5 min, and incubated overnight at 4 C with anti-IL-11 antibody (1:20 dilution; R&D Systems Inc., Minneapolis, MN). The next day, the sections were washed with PBS, treated with biotinylated secondary antibody at RT for 10 min, and treated with streptavidin-peroxidase reagent at RT for 10 min. The color was developed using 3,3'diaminobenzidine chromogen at RT for 15 min and counterstained with hematoxylin for 1 min.
Statistical analysis
Because the number of cases in each study group is relatively lower (25 cases in each group), a nonparametric testing would be more appropriate for the statistical analysis. Therefore, the difference between groups was analyzed by the Mann-Whitney U test. A P value of less than 0.05 was considered statistically significant.
Results
The expression of IL-11, IL-11Rd, and gp130 genes
PCR revealed that all of the samples except unstimulated MRC-5 cells and human endometrium in the proliferative phase (data not shown) expressed IL-11 mRNA. These included K562 cells, MRC-5 cells stimulated by IL-1 (1 ng/ml), and phorbol 12-myristate 13-acetate (10 ng/ml) (served as positive control), HeLa cells, human PBMC, endometrium in the secretory phase, and decidua and chorionic villi samples from all patients (Fig. 1). In addition, IL-11R and gp130 mRNA were detectable in all samples (Fig. 1).
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mRNA by quantitative PCR
Real-time quantitative PCR was performed to compare the levels of expression of IL-11 system genes in different samples. Using the proliferative phase endometrium as a reference tissue, it was found that the relative levels of IL-11R mRNA were 1.4 ± 0.5, 1.3 ± 0.1, 1.7 ± 0.3, 1.9 ± 0.4, 7.6 ± 1.3, and 10.6 ± 1.9 (mean ± SEM), respectively, in proliferative phase endometrium, secretory phase endometrium, decidua (from NP), decidua (from AP), chorionic villi (from NP), and chorionic villi (from AP) (Fig. 2). These findings indicate that IL-11R mRNA levels did not change in the endometrium during each phase of the menstrual cycle and in the decidua after pregnancy was established. However, chorionic villi contained a distinctly higher level of IL-11R mRNA than decidua or other phases of endometrium (P < 0.05 comparing villi from NP or AP with all other tissues) (Fig. 2). There was, however, no difference in IL-11R mRNA level between NP and AP in either decidua or chorionic villi (in decidua, 1.7 ± 0.3 vs. 1.9 ± 0.4, respectively, for NP and AP, P > 0.05; in villi, 7.6 ± 1.3 vs. 10.6 ± 1.9, P > 0.05).
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). These data indicate that the IL-11 mRNA level was low in endometrium during the proliferative phase and increased during the secretory phase (relative level, 0.0 ± 0.0 vs. 0.03 ± 0.02, respectively, for proliferative and secretory phases; P = 0.004) (Fig. 3) and after NP (1.7 ± 0.2 and 13.0 ± 2.2 in decidua and chorionic villi, respectively; P < 0.05 compared with endometrium during the proliferative phase or the secretory phase). In AP, the IL-11 mRNA level also increased (0.1 ± 0.1 and 0.2 ± 0.1 in decidua and chorionic villi, respectively) compared with that in endometrium of both phases, although it was not as high as in NP. The IL-11 mRNA levels were significantly higher in NP than in AP, both in decidua and chorionic villi (in decidua, 1.7 ± 0.2 vs. 0.1 ± 0.1, respectively, for NP and AP, P < 0.0001; in chorionic villi, 13.0 ± 2.2 vs. 0.2 ± 0.1, P = 0.03) (Fig. 3).
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Immunohistochemical study for IL-11 production found that the immunoreactive IL-11 was barely detectable in the glandular epithelium of secretory phase endometrium and was undetectable in proliferative phase endometrium (representative photographs are shown in Fig. 4, C and D, and the relative staining intensities in different tissues are listed in Table 3). After pregnancy, however, IL-11 immunoreactivity became detectable both in the stromal and glandular cells of decidua and syncytiotrophoblast and cytotrophoblast cells of the chorionic villi (Fig. 4, E–H, and Table 3). Stronger immunoreactivity intensity was found in the glandular epithelium than in the stromal cells of decidua (Table 3). In contrast, the intensity was stronger in trophoblast than in stroma of chorionic villi (Table 3). Comparison of tissue samples from different groups of patients showed that chorionic villi from NP produced a higher level of IL-11 than that from AP (Table 3). Similarly, decidua from NP expressed stronger IL-11 immunoreactivity than those from AP.
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mRNA
Because IL-11R monoclonal antibody was not available at the time of study, in situ hybridization technique was used to examine the expression of IL-11R gene in the endometrium and gestational tissues. Proliferative phase endometrium expressed IL-11R mRNA in its stromal cells but not glandular cells (Fig. 5 and Table 3
). In the secretory phase, however, IL-11R expression was evident in both stromal and glandular cells (Fig. 5). During pregnancy, both stromal and glandular cells of the decidua and trophoblasts of the chorionic villi continuously expressed this transcript (Fig. 6). However, no distinct difference of staining intensity was found among samples from secretory phase endometrium, decidua, or chorionic villi (Figs. 5 and 6 and Table 3). There was no significant difference of IL-11R mRNA levels between NP and AP in either decidua or villi (Table 3).
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Implantation and placentation are complex processes that require the synchronized growth of an embryo and a continuous and adequate decidual development in the endometrium (1). It is conceivable that these processes are susceptible to endogenous or exogenous insults. Among them, defective IL-11R action has been shown to be the mechanism for abnormal decidualization in mice (4). That study reasonably suggests a potential role of the IL-11 system genes in abnormal decidualization in humans. The present study demonstrated the expression of IL-11R mRNA in the endometrium throughout the menstrual cycle and in the decidua of early pregnancy. Although the level of IL-11R mRNA in chorionic villi was higher than in decidua, no significant difference in IL-11R mRNA levels was found between decidua and chorionic villi from NP and AP. In contrast, the IL-11 mRNA level in endometrium progressively increased from the secretory phase and reached peak levels in the decidua and chorionic villi after pregnancy. IL-11 mRNA levels in both decidua and villi of AP were significantly lower than those of NP. Gp130 mRNA was also detectable in all of the samples examined. These data support the hypothesis that IL-11 signaling in human endometrium and gestational tissues may be critical in the maintenance of a NP.
A previous study by Dimitriadis et al. (26) showed that IL-11 expression varied with different phases of the menstrual cycle. The results of our study support their findings and also showed that both IL-11 and IL-11R mRNA were present in the endometrium throughout the menstrual cycle and during early pregnancy. In addition, our study clearly demonstrated that IL-11R levels remained stable during these stages with minimal variation between levels in the endometrium and the decidua. These data are basically consistent with those reported in mice, in which constant levels of IL-11R and gp130 were detected since before pregnancy until 9.5 d post coitum (4). However, the real-time quantitative PCR results of our study also showed that IL-11R and IL-11 expression were augmented in the chorionic villi compared with the decidua. This suggests that IL-11 may be produced both in the embryo and in decidual cells, although the former may be the predominant source in humans due to its greater level. Subsequently, through binding to IL-11R and gp130, IL-11 may exert its action on the embryo and the decidua to regulate chorionic villus development and decidualization, respectively. This potential dual action of IL-11, however, was not clearly demonstrated in a previous study in mice by Robb et al. (4). In that IL-11R knockout model, the number of secondary trophoblast giant cells present near the defective decidua was markedly increased, and the size was enlarged. However, they found that IL-11R-/- embryos could still survive normally in wild-type foster female but that wild-type embryos were unable to survive in the IL-11R-/- female, which does not unanimously support the role of IL-11 action in trophoblast development. Therefore, further study is needed to explain whether the role of IL-11 differs between species. Irrespective of all these unanswered issues, the results of the present study suggest that IL-11 may play a crucial role in the development of human chorionic villi and decidua.
There are inconsistent results obtained by different experimental methods that need to be noted. As detailed previously in Results, in situ hybridization did not show a distinct difference of staining intensity between samples from secretory phase endometrium, decidua, or chorionic villi (Table 3). These data are not consistent with the result of real-time quantitative PCR, which showed that chorionic villi expressed a significantly higher level of IL-11R mRNA than decidua (Fig. 2). The reason for the divergence of these results is not clear, but may involve varied sensitivity of each of the experimental methods.
The presence of IL-11R mRNA with minimal variation in amount despite changing IL-11 levels suggests divergent actions between IL-11 and IL-11R. One possible explanation for this finding is that the level of IL-11 rather than IL-11R may regulate normal placental formation and decidualization. Although previous data indicate that IL-11R knockout leads to infertility in mice (4), complete IL-11R depletion in humans has not been reported. Therefore, a baseline IL-11R level most likely is more than enough to fulfill its role in IL-11 function. This argument is further supported by the lack of difference of IL-11R expression between NP and AP. However, the potential for defects in IL-11R action due to either mutation or deletion in the IL-11R gene cannot be excluded at present. It would therefore be interesting to investigate IL-11 and IL-11R at the genomic level in patients with recurrent spontaneous abortion to identify their possible defects in action.
A significant decrease of IL-11 mRNA was found in AP compared with NP. In the presence of IL-11R, the level of which might only be rarely under the threshold level in humans, the resulting decreased level of IL-11 might lead to defective placental formation and subsequently result in a blighted ovum (AP). However, we cannot completely rule out the possibility that decreased IL-11 level could be secondary to fetal demise, rather than itself being the primary phenomenon. Consequently, more work is needed to clarify this cause-and-effect relationship.
IL-11, IL-6, ciliary neurotrophic factor, leukemia inhibitory factor, and oncostatin M belong to a specific group of cytokines that share the common signal-transduction subunit gp130 (12). Overlapping of the biological function of these cytokines may at least partly be explained by the sharing of this common signal-transduction component in formation of their high-affinity receptor complexes (20). As expected, this study identified the expression of gp130 mRNA in phases of endometrium and in gestational tissues. These data provide an indirect evidence of the role of gp130 in the complete action of IL-11R. However, further detailed spatial and temporal analyses of this gene will be needed to clarify its role in human disease states.
In conclusion, the present study suggests that IL-11 may play a crucial role in the maintenance of early pregnancy. It is likely that IL-11 has dual actions, both on placentation and decidualization, although in humans the major focus might be on the former. Decreased expression of IL-11 during or after implantation probably can account for some cases with implantation failure or recurrent abortion in early pregnancy. This conclusion however needs further confirmation because the cause vs. effect relation cannot be determined at present. In addition to the evident academic interest, further studies of IL-11 action will have important clinical implications. Medical use of IL-11 has been shown to effectively enhance a number of biological functions including bone marrow recovery and platelet formation (13). Extrapolation from these experiences provides strong support for its potential use in treating diseases of the genital organs, where IL-11 action has previously been identified. In addition, the medical use of IL-11 is relatively nontoxic compared with other cytokines. Therefore, after further confirmation, it is likely that IL-11 may eventually be shown to have clinical applications in the treatment of repeated implantation failure and/or recurrent spontaneous abortion.
Acknowledgments
We thank Dr. Yu-Chung Yang (Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN) for her generosity in providing the cDNA clones for this study.
Footnotes
This work was supported by grants from the National Science Council of the Republic of China (NSC89-2314-B-002-582 and NSC89-2314-B-002-289) and the National Health Research Institute (DOH89-DC-1005).
Abbreviations: AP, Anembryonic pregnancy; BCIP, 5-bromo-4-chloro-3-indoxyl-phosphate; CT, threshold cycle; DIG, digoxigenin; gp130, glycoprotein 130; IL-11R, IL-11 receptor; NP, normal pregnancy; NBT, nitro-blue tetrazolium chloride; PBMC, peripheral blood mononuclear cells; RT, room temperature.
Received November 19, 2001.
Accepted February 5, 2002.
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E. Dimitriadis, C. Stoikos, Y.-L. Tan, and L. A. Salamonsen Interleukin 11 Signaling Components Signal Transducer and Activator of Transcription 3 (STAT3) and Suppressor of Cytokine Signaling 3 (SOCS3) Regulate Human Endometrial Stromal Cell Differentiation Endocrinology, August 1, 2006; 147(8): 3809 - 3817. [Abstract] [Full Text] [PDF]
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 E. Dimitriadis, C.A. White, R.L. Jones, and L.A. Salamonsen Cytokines, chemokines and growth factors in endometrium related to implantation Hum. Reprod. Update, November 1, 2005; 11(6): 613 - 630. [Abstract] [Full Text] [PDF]
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H. Cakmak, F. Schatz, S.-T. J. Huang, L. Buchwalder, M. Rahman, A. Arici, and C. J. Lockwood Progestin Suppresses Thrombin- and Interleukin-1{beta}-Induced Interleukin-11 Production in Term Decidual Cells: Implications for Preterm Delivery J. Clin. Endocrinol. Metab., September 1, 2005; 90(9): 5279 - 5286. [Abstract] [Full Text] [PDF]
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E. Dimitriadis, C. Stoikos, M. Baca, W. D. Fairlie, J. E. McCoubrie, and L. A. Salamonsen Relaxin and Prostaglandin E2 Regulate Interleukin 11 during Human Endometrial Stromal Cell Decidualization J. Clin. Endocrinol. Metab., June 1, 2005; 90(6): 3458 - 3465. [Abstract] [Full Text] [PDF]
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N. Karpovich, P. Klemmt, J. H. Hwang, J. E. McVeigh, J. K. Heath, D. H. Barlow, and H. J. Mardon The Production of Interleukin-11 and Decidualization Are Compromised in Endometrial Stromal Cells Derived from Patients with Infertility J. Clin. Endocrinol. Metab., March 1, 2005; 90(3): 1607 - 1612. [Abstract] [Full Text] [PDF]
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 U. von Rango, J. Alfer, S. Kertschanska, B. Kemp, G. Muller-Newen, P.C. Heinrich, H.M. Beier, and I. Classen-Linke Interleukin-11 expression: its significance in eutopic and ectopic human implantation Mol. Hum. Reprod., November 1, 2004; 10(11): 783 - 792. [Abstract] [Full Text] [PDF]
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H.-F. Chen, K.-H. Chao, J.-Y. Shew, Y.-S. Yang, and H.-N. Ho Expression of leukemia inhibitory factor and its receptor is not altered in the decidua and chorionic villi of human anembryonic pregnancy Hum. Reprod., July 1, 2004; 19(7): 1647 - 1654. [Abstract] [Full Text] [PDF]
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N. Karpovich, K. Chobotova, J. Carver, J. K. Heath, D. H. Barlow, and H. J. Mardon Expression and function of interleukin-11 and its receptor {alpha} in the human endometrium Mol. Hum. Reprod., February 1, 2003; 9(2): 75 - 80. [Abstract] [Full Text] [PDF]
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