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Original Article |
The Reproductive Molecular Research Group, Department of Obstetrics and Gynecology, University of Cambridge, The Rosie Maternity Hospital, Cambridge, CB2 2SW, United Kingdom
Address all correspondence and requests for reprints to: Dr. R. Sherwin, Department of Obstetrics and Gynecology, University of Cambridge, Box 223, The Rosie Hospital, Cambridge, CB2 2SW, United Kingdom. E-mail: . jras100{at}cam.ac.uk
Abstract
Members of the IL-6 family of cytokines, which includes leukemia inhibitory factor (LIF) and IL-11, play important roles in implantation. The activity of these cytokines is modified by soluble receptors such as the IL-6 receptor (sIL-6R). gp130 is a signal transduction molecule common to the receptor complexes of this family, and its soluble form (sgp130) antagonizes their actions. The purpose of this study was to determine whether secretion of IL-6, LIF, sIL-6R, and sgp130 was different in the endometrium of women with primary unexplained infertility compared with normal fertile women. Endometrial biopsies were taken between d LH+6 and +13 and cultured in serum-free medium for 4 h. Secretion of IL-6, LIF, sIL-6R, and sgp130 was measured in the supernatant by ELISA. We also measured the secretion of IL-6, sIL-6R, and sgp130 by endometrial biopsies taken throughout the menstrual cycle in normal fertile women. Secretion of sgp130 increased 20-fold between d 20 and 26 of the cycle, coinciding with the implantation window (proliferative phase, median, 27.0 pg/ml·mg; range, 23–36; d 20–26, median, 501.5 pg/ml·mg; range, 26.1–1344; P = 0.03). RT-PCR showed that none of the known splice variants of gp130 were present in endometrium, indicating that sgp130 is produced by proteolytic cleavage of the membrane-bound form. IL-6 secretion varied considerably between patients and was greatest during the secretory phase and at menstruation. No significant change was seen in sIL-6R during the cycle. Between LH+6 and +13, secretion of sgp130 was significantly reduced in the infertile group (median, 93.1 pg/ml·mg; range, 28.5–256; compared with the fertile group, median, 223 pg/ml·mg; range, 63–534; U-statistic = 37; P = 0.017). Secretion of IL-6, LIF, and sIL-6R did not differ between the two groups. Immunolocalization of gp130, IL-6R, and the LIF receptor showed that the glandular epithelium and also endothelial cells are targets for IL-6 and LIF. These findings show that during a normal menstrual cycle, sgp130 secretion is greatly increased between d LH+6 and +13, due to proteolytic cleavage of membrane-bound gp130. Infertile patients show reduced secretion of sgp130 compared with fertile controls during this period, which coincides with the implantation window.
OVARIAN STEROIDS INDUCE human endometrium to proliferate and differentiate during the menstrual cycle and to produce a receptive surface for blastocyst implantation (1, 2). Members of the IL-6 family of cytokines are key regulators of implantation. This family includes IL-6, leukemia inhibitory factor (LIF), IL-11, cardiotrophin, oncostatin-M, and ciliary neurotrophic factor (3). These molecules share a common three-dimensional structure in the form of an L-
-helix bundle (4). They signal through specific receptors on the cell surface that all share gp130 as a common accessory signal transduction molecule (5).
In the C57BL6/J strain of mice, LIF is essential for implantation, where it is secreted by the glandular epithelium of the endometrium at the time of embryo attachment (6, 7). Animals that lack the LIF gene produce normal blastocysts, which fail to implant but which can do so upon transfer to wild-type mice (7). Administration of LIF to LIF -/- animals by intrauterine or intraperitoneal injection restores implantation (8). Similarly, deletion of the receptor for IL-11 by homologous recombination also results in mice unable to support implantation (9). In these animals, implantation begins, but the decidualization response is reduced and implantation fails.
Data suggesting a possible role for IL-6 type cytokines in human implantation is only available for LIF and IL-6. LIF mRNA and protein are maximal in the endometrium during the mid-late secretory phase of the menstrual cycle, and uterine flushings show LIF secretion is maximal at this time (10, 11, 12). In situ hybridization and immunohistochemistry indicate that the primary site of synthesis is the glandular epithelium, although some LIF immunoreactivity is present in the stroma (10, 13, 14). mRNA species encoding the receptor components LIFR-ß and gp130 are confined to epithelial cells (13). Uterine flushings taken from infertile women during the secretory phase contain lower levels of LIF protein, when compared with fertile controls (12). Similarly, endometrial explants taken from infertile women and cultured in vitro secrete less LIF than biopsies from fertile patients (15, 16). However, other workers report no difference in LIF mRNA levels in secretory endometrium between fertile and infertile women (13).
IL-6 shows a similar pattern of expression to LIF. IL-6 mRNA and protein secretion are maximal in the mid secretory phase (17, 18). Immunohistochemistry shows weak staining for IL-6 in the proliferative phase, with increased glandular staining around d 19. However, unlike LIF, there is significant IL-6 immunoreactivity in scattered cells in the stroma (17). The IL-6 receptor (IL-6R) is localized to glandular epithelium throughout the cycle (17). Levels of IL-6 in serum and cervical mucus taken from infertile women during the early secretory phase are higher than in fertile controls (19).
The transmembrane protein gp130 is a signal transducer for cytokines of the IL-6 family. Binding of LIF or IL-6 to their receptors promotes formation of a receptor complex with gp130. Signal transduction involves activation of members of the Janus family and phosphorylation of members of the STAT family of transcription factors (20). The mRNA for gp130 has been localized to luminal and glandular epithelium by in situ hybridization (13). Soluble forms of the IL-6R (sIL-6R), the LIF receptor (sLIFR), and gp130 (sgp130) are generated by proteolytic cleavage or by alternative splicing (21, 22, 23). sLIFR antagonizes LIF, whereas soluble forms of the IL-6R act as agonists for IL-6, increasing its bioactivity (22, 23). Soluble gp130 antagonizes signaling of both LIF and IL-6 by competing with membrane-bound gp130 for LIF/LIFR and IL-6/IL-6R complexes (23, 25). Soluble gp130 is synthesized from alternate transcripts (23, 26, 27, 28) as well as by membrane cleavage (24). The activity of the IL-6 family of cytokines therefore depends not only on the level of each cytokine, but also on that of the soluble receptors
The purpose of this study was to establish the secretion pattern of IL-6, LIF, and the soluble receptors sIL-6R and sgp130 throughout the normal menstrual cycle. We also tested the hypothesis that altered levels of immunoreactive IL-6, LIF, or their soluble receptors are secreted from endometrium of infertile patients during the implantation window, when compared with fertile controls.
Materials and Methods
Ethical Committee approval for the collection of endometrial biopsies was obtained from Addenbrooke’s Hospital NHS Trust. Written informed consent was obtained from all patients before their operations. Reagents for tissue culture and other experiments were obtained from Sigma (Poole, UK), unless otherwise stated.
Tissue samples
Endometrial biopsies were obtained from infertile patients (n = 14) who were undergoing laparoscopy, hysteroscopy, and dye insufflation of tubes, as part of their investigations for infertility. All these used a urinary LH surge detection kit (Clearplan, Unipath, Bedford, UK). Biopsies were taken on d LH+6 to +13, and no abnormalities were found at laparoscopy. Patients were aged between 25 and 40 yr (median, 33.9 yr) and had a regular menstrual cycle of between 26 and 32 d. All patients were nulliparous and had experienced at least 2 yr of infertility. Semen analyses were normal in the partners of all patients, as defined by the World Health Organization criteria. Endometrial biopsies were also taken from patients of proven fertility undergoing laparoscopic sterilization in the secretory phase of the cycle (n = 12). These patients again performed home urinary LH surge tests, and endometrial biopsies were taken between LH+6 and +13. These patients were recruited from Addenbrooke’s Hospital (Cambridge, UK) and three additional centers (see Acknowledgments). Fertile patients were aged between 25 and 40 yr (median, 34.1 yr) and had a regular menstrual cycle. Endometrial biopsies were taken from another group of fertile patients who were undergoing sterilization in the menstrual or proliferative phase of the menstrual cycle (n = 7) or diagnostic laparoscopy in the secretory phase (n = 7). These patients did not perform LH surge tests but were found to have no abnormalities at laparoscopy. Their biopsies were dated from the last menstrual period, and the dating was confirmed by histology. Data from these 14 fertile patients was combined with that of the LH-timed fertile controls (LH+6 to +13) to determine the pattern of cytokine secretion throughout the menstrual cycle. Fertile patients had no intrauterine contraceptive device in place and had not used oral contraceptives or other hormonal preparations for at least 3 months before surgery.
Endometrial culture
Endometrial biopsies were obtained from infertile patients by sharp curretage after hysteroscopy and dye insufflation of tubes. This was to prevent the flushing of endometrium into the peritoneal cavity. Biopsies were taken in the same manner from fertile patients after instrumentation of the uterus, which is performed as part of laparoscopic sterilization. Endometrial biopsies were rinsed in a mixture of DMEM and Ham’s F12. Two small pieces were removed, one was fixed in formalin for histology, and one was snap-frozen in liquid nitrogen for the preparation of frozen sections. Most of the biopsy was used for short-term culture. This was rinsed twice in DMEM/F12, prewarmed to 37 C, weighed, cut into approximately 2- to 3-mm pieces, and placed in 6 ml of DMEM/F12 (penicillin 50 IU/ml, streptomycin 50 µg/ml, and 2 mM L-glutamine) in a 6-well plate (Becton Dickinson and Co., Plymouth, UK). This was then cultured at 37 C in a 5% CO2 humidified incubator. At 30-min and 1-, 2-, 3-, and 4-h intervals, 1.2-ml aliquots of culture fluid were removed. Cellular debris was removed by centrifugation. The supernatants were subdivided into 5 x 220-µl aliquots and stored at -70 C until assayed by ELISA. After culture, the explants were blotted dry, and the wet weight was measured.
Enzyme-linked immunosorbent assays for IL-6, sIL-6R, and sgp130
Levels of IL-6, sIL-6R, and sgp130 were measured in the conditioned medium sampled at the 4-h time point using Sandwich quantikine ELISA kits (R&D Systems Inc., Minneapolis, MN) according to the manufacturer’s instructions. Fresh culture medium (DMEM/F12) was used to dilute the standards and used as a zero standard. The characteristics of the ELISAs as reprted by the manufacturer are: IL-6 (D6050, R&D Systems), range, 3–300 pg/ml; sensitivity, 0.7 pg/ml; intra-assay coefficient of variation, 1.6–4.2%; interassay variation, 3.3–6.4%; sIL-6R (DR600, R&D Systems), range, 30–2000 pg/ml; sensitivity, 7 pg/ml, intra-assay coefficient of variation, 2.3–8.6%; interassay variation, 4.2–6.4%; sgp130 (DGP00, R&D Systems), range, 125-8000 pg/ml; sensitivity, 80 pg/ml; intra-assay coefficient of variation, 3.7–5.5%; interassay variation, 3.7–4.5%. The LIF ELISA was from Bender MedSystems (BMS 242; Vienna, Austria); range, 3–200 pg/ml; intra-assay coefficient of variation, 5.5%; interassay variation, 7.0%. The effect of contaminating blood leukocytes in the tissue biopsies on cytokine levels was assessed by culturing aliquots of peripheral blood for up to 4 h under the same culture conditions. No significant secretion of LIF, IL-6, or either soluble receptor was detected in these pilot experiments (data not shown). The cytokine levels in the 4-h supernatant are expressed as picograms per milliliter per milligram of wet weight.
Immunohistochemistry
Small pieces of endometrium were fixed in 10% neutral buffered formalin for 6 h at room temperature (RT) on a rocking platform. Then, the samples were washed in PBS and stored in 70% ethanol until they were embedded in paraffin. Duplicate samples were snap-frozen in liquid nitrogen. Cryosections of 5 µm thickness were cut onto 2% aminopropyltriethoxysilane-coated slides, fixed for 5 min in ice-cold acetone, and air-dried for 2 h before storage at -70 C. Endometrial dating was confirmed on the formalin-fixed portion of the biopsy according to the criteria of Noyes et al. (29). All biopsies used in this study were histologically normal.
Immunostaining for IL-6R and LIFR
Formalin-fixed, paraffin-embedded sections were dewaxed in xylene, pretreated by pressure cooking in 10 mM sodium citrate buffer (pH 6.6) for 1 min, and rinsed in PBS. Endogenous peroxidase activity was blocked using 3% H2O2 in PBS for 10 min. Primary antibody (rabbit antihuman IL-6R; catalog no. sc-661, supplied as 100 µg/ml rabbit IgG; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was diluted 1:200 in 1% BSA in PBS (BSA-PBS) and incubated with the sections for 60 min at 37 C in a humidified chamber. Rabbit IgG (20 mg/ml, DAKO Corp., High Wycombe, UK) diluted to the same IgG concentration was used as a negative control. Unstimulated peripheral blood lymphocytes were used as a positive control for IL-6R staining. The sections were rinsed for 10 min in three changes of PBS and then incubated for 10 min at RT with the secondary antibody (biotinylated, goat antirabbit IgG; Zymed Laboratories, Inc., San Francisco, CA) diluted 1:100 in PBS. The slides were rinsed again for 10 min in three changes of PBS and incubated in streptavidin-horseradish peroxidase (43-4323; Zymed Laboratories, Inc.) diluted 1:400 in PBS for 10 min at RT. This was followed by detection using diaminobenzidine substrate chromogen mix (Sigma). Sections were counterstained with Meyer’s hematoxylin, dehydrated, and mounted. Staining for LIFR was similar except that the primary antibody was a goat polyclonal anti-LIFR antibody (BAF 249, R&D Systems), used at 5 µg/ml with goat IgG as control.
Immunostaining for IL-6
Formalin-fixed, paraffin-embedded sections were dewaxed in xylene, and endogenous peroxidase activity was blocked using 3% H2O2 in PBS for 10 min. Primary antibody (rabbit antihuman IL-6; catalog no. CXH1–066LS, supplied as 20 µg/ml rabbit IgG; Cambridge Bioscience, Cambridge, UK) was diluted 1:250 in 1% BSA-PBS and added to the sections. These were incubated for 60 min at 37 C in a humidified chamber. Rabbit IgG (20 mg/ml, DAKO Corp.) diluted to the same IgG concentration was used as a negative control. The sections were processed identically to the IL-6R immunostaining described above.
Immunostaining for gp130
Frozen sections were allowed to thaw to RT before fixation for 5 min in ice-cold acetone. After washing in PBS, the sections were blocked with 20% rabbit serum (R 9133, Sigma) in 1% BSA-PBS for 30min at RT. The primary antibody (BAF 228, biotinylated, affinity purified, goat polyclonal IgG, anti-gp130, supplied at 50 µg/ml; R&D Systems) was added for 1 h at RT at 1:20 dilution in 5% rabbit serum/1% BSA-PBS. Control antibody (I5256, goat IgG, Sigma) was added at the same protein concentration to control sections. After washing in PBS, the secondary antibody (E466, biotinylated rabbit anti-goat immunoglobulins, supplied at 0.8 mg/ml, DAKO Corp.) was added at 1:200 dilution in 1% BSA-PBS/5% rabbit serum for 1 h at RT. Endogenous peroxidase activity was quenched by incubation with 0.3% H2O2 diluted in methanol for 10 min at RT. Streptavidin-horseradish peroxidase at a dilution of 1:400 in PBS (catalog no. 43-4323, Zymed Laboratories, Inc.) was bound for 10 min at RT. The peroxidase was developed using the diaminobenzidine substrate kit (Sigma) before counterstaining with hematoxylin, dehydration, and mounting.
Analysis of sgp130 splice variants in endometrium
RNA was isolated from endometrium that had been frozen immediately after biopsy by homogenization in Trizol (Life Technologies, Inc. Paisley, UK) according to the manufacturer’s instructions. Endometrial cDNA from the proliferative and secretory phases of the menstrual cycle was synthesized using AMV reverse transcriptase (Super RT from HT Biotechnology Ltd., Cambridge, UK). Total RNA (3 µg) was primed with oligo dT for 60 min at 42 C as described previously (30). One fiftieth of each cDNA was then amplified with primer sets designed to amplify all the known splice variants of gp130. The primers used in this study are shown in Table 1
. The cDNA was amplified using 1 U of Taq polymerase in the manufacturer’s buffer (Bioline, London, UK) with 1.5 mM MgCl2. For primer pair DF5 and DF3, denaturation at 95 C for 1 min was followed by 30 cycles of PCR consisting of 95 C for 30 sec, 57 C for 30 sec, and 72 C for 60 sec. PCR for the primer pair gp130 C and D was performed in the same way except that the primer annealing step was 50 C. The PCR products were analyzed on either a 2% Tris Acetate EDTA agarose gel or a 4% polyacrylamide gel run in Tris-borate EDTA buffer.
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Levels of secreted cytokines and soluble receptors in culture supernatants were measured by ELISA. Data for each patient group is expressed as median and range in picograms per milliliter per milligram of biopsy wet weight. The Mann-Whitney U test was used to determine significant differences in cytokine secretion between the infertile and fertile patient groups in the secretory phase of the cycle. Multiple pairwise comparisons were performed using the Mann-Whitney U test to compare levels of cytokine secretion between biopsies taken from fertile patients at different stages of the menstrual cycle. A two-tailed P value of less than 0.05 was accepted as statistically significant.
Results
Secretion of IL-6, sIL-6R, and sgp130 during the menstrual cycle
Secretion of IL-6 and its soluble receptors sIL-6R and sgp130 was measured from biopsies taken from fertile women (n = 24) throughout the menstrual cycle, after 4-h culture in vitro. IL-6 secretion, shown in Fig. 1A, was low in the proliferative phase and increased between d 20 and 26 of the cycle (proliferative phase, median, 2.2 pg/ml·mg; range, 1.7–3.6; vs. d 20–26, median, 4.2 pg/ml·mg; range, 0.0–15.5). IL-6 secretion remained high during menstruation itself. However, there was considerable variation between biopsies from the same stage in the cycle, and these changes did not reach statistical significance. sIL-6R secretion did not show any significant change during the cycle (Fig. 1B). The median level of secretion between d 20 and 26 was 3.4 pg/ml·mg; range, 2.1–5.4. Secretion of sgp130 was one or two orders of magnitude higher than that of IL-6 or sIL-6R throughout the cycle. A large rise in secretion occurred between d 20 and 26 (proliferative phase, median, 27.0 pg/ml·mg; range, 23–36; d 20–26, median, 501.5 pg/ml·mg; range, 26.1–1344; U-statistic =3; P = 0.03). During d 20 to 26, the median level of sgp130 was approximately 100-fold higher than that of IL-6 in picograms per milliliter per milligram. LIF secretion throughout the cycle was not measured due to limited quantities of culture supernatant.
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The endometrium becomes receptive to the embryo at around LH+5 or +6 and remains receptive for several days. We compared secretion of LIF, IL-6, sIL-6R, and sgp130 by endometrial biopsies taken between LH+6 and +13 from fertile (n = 12) and infertile (n = 14) women. This corresponds to the time when sgp130 secretion is elevated (Fig. 1
). No significant difference in secretion of either LIF, IL-6, or sIL-6R was detected (Table 2). However, sgp130 secretion was significantly reduced in the infertile group (infertile group median, 93.1 pg/ml·mg; range, 28–256; compared with fertile group, median, 223 pg/ml·mg; range, 63–524.4; U-statistic = 37; P = 0.017; Table 2 and Fig. 2). The duration of the implantation window is approximately LH+6 to +11 (32). When sgp130 secretion was compared between biopsies sampled between LH+6 and +11, a similar result was obtained. Median sgp130 levels in supernatants from infertile biopsies (n = 13) were 84 pg/ml·mg (range, 28.5–256) compared with 150 pg/ml·mg (range, 64–401) for fertile biopsies (n = 11, U-statistic = 32, P = 0.02). There was no significant difference in the time of biopsy sampling in the two groups between these time points.
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Immunohistochemistry was used to localize IL-6, IL-6R, LIFR, and gp130 in endometrial biopsies taken from fertile women throughout the menstrual cycle. There was little gp130 immunoreactivity in proliferative phase endometrium (Fig. 3, A and B); however, by d 20 strong staining for gp130 was present in the glandular and luminal epithleium and in endothelial cells (Fig. 3, C and D). There was very weak staining in stromal cells during the secretory phase. Strong IL-6R immunoreactivity was detected in the glandular and luminal epithelium with no apparent change during the cycle. A subset of cells in the endometrial stroma as well as endothelial cells also stained for IL-6R (Fig. 3, G and H). Immunostaining for IL-6 was detected in both glandular epithelium and isolated cells in the stroma throughout the cycle. The epithelial staining increased during the mid-late secretory phase. This IL-6 staining is similar to that reported previously (17). LIFR immunoreactivity was weakly detected in glandular epithelium throughout the menstrual cycle (Fig. 3, E and F). However, endothelial cells stained strongly.
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Soluble gp130 can be produced by either proteolytic cleavage or alternative splicing. Primers were designed to amplify the three known alternatively spliced isoforms of gp130 mRNA that encode sgp130. The isoforms and the location of the primers used are shown in Fig. 4A. RT-PCR of human endometrium using primer pairs C and D or DF5 and DF3 produced DNA products corresponding to the normal full length cDNA described by Hibi et al. (31). None of the gp130 cDNA isoforms known to encode sgp130 were detected in endometrium (Fig. 4, B and C).
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This study measured the secretion of IL-6, LIF, and their soluble receptors from endometrial biopsies because these cytokines play an important part in the preparation of the endometrium for implantation (32). Soluble gp130 was by far the most abundant member of the IL-6 cytokine/receptor family secreted by endometrium at the time of implantation. Significantly lower levels of sgp130 were secreted by endometrial biopsies taken between d LH+6 and +13 in the infertile patient group compared with the fertile controls (93.1 vs. 223 pg/ml·mg; P = 0.017). This period corresponds to the time of maximal secretion of gp130 during the menstrual cycle (Fig. 1). However, implantation is normally believed to occur between LH+6 and +11 (33). When secretion of sgp130 was compared from biopsies sampled between LH+6 and +11, biopsies from infertile women were again found to secrete significantly less sgp130 than fertile biopsies (median, 84 pg/ml·mg; range, 28.5–256) compared with 150 pg/ml·mg (range, 64–401; P = 0.02). The reduced secretion of sgp130 during the window of implantation points to a functional difference in the endometrium at this time.
The biological activity of LIF and IL-6 is affected by the levels of their soluble receptors sIL-6R, sLIFR, and sgp130 (21, 25, 34). The IL-6/sIL6R complex is able to bind and signal through membrane-bound gp130. Soluble gp130 acts as an antagonist preventing the cytokine/receptor complex from initiating signaling through membrane-bound gp130 (20). Two forms of sgp130 have been described in human urine; one contains most of the extracellular region, whereas the other contains only the hematopoietin domain (23). We have not identified which soluble forms of gp130 are secreted by human endometrium. Soluble forms of the LIF receptor (sLIFR) also occur, and both sLIFR and sgp130 block the biological activity of LIF (23, 35). Women in the infertile group secreted reduced levels of sgp130 during the implantation period, whereas secretion of IL-6, LIF, and sIL-6R was similar to fertile patients. Culture supernatants from infertile women contained a mean level of sgp130 of 14 ng/ml corresponding to a concentration of 0.2 nM. The mean level of LIF in the same supernatants was 0.2 ng/ml, giving a concentration of 10 pM and a sgp130/LIF molar ratio of 20. Soluble gp130 is able to bind LIF and IL-6/sIL-6R and has a significant antagonistic effect in vitro on LIF and IL-6 bioactivity at molar ratios of 10–30 (ratio of sgp130 to cytokine; Ref. 23). The large excess of sgp130 compared with LIF and IL-6 would lead us to predict that sgp130 will bind LIF and IL-6. Low concentrations of sgp130 could serve to stabilize the cytokines or significantly antagonize their bioactivity in human endometrium at higher concentrations.
Secretion of sgp130 was low in the proliferative phase and increased to very high levels between d 20 and 26 of the cycle, suggesting up-regulation by progesterone. The mRNA for gp130 has previously been localized to endometrial epithelium, and expression was reported to increase in the secretory phase (13). We found gp130 immunoreactivity was low in the glandular epithelium during the proliferative phase and increased dramatically in the secretory phase. Thus, the mRNA, secretion, and immunostaining data correlate, indicating that the primary source of increased sgp130 secretion between d 20 and 26 of the cycle is the glandular epithelium. Soluble gp130 can be produced by proteolytic cleavage or alternative splicing (26, 27, 28). RT-PCR using primers specific for gp130 cDNA showed that none of the known splice variants that encode soluble forms of gp130 were present in endometrium (Fig. 4). It is therefore likely that sgp130 is produced primarily by proteolytic cleavage of the membrane-bound form (35). A previous study reported gp130 immunoreactivity in the glandular epithelium but indicated that there was no change during the cycle (17). Increased gp130 immunoreactivity on glandular epithelium during the secretory phase has also been reported in the rhesus monkey (36). We have also identified gp130, IL-6R, and LIFR immunostaining on endothelial cells, indicating that they may be a target for the IL-6 type cytokines (Fig. 3, C, E, and G). Both LIF and IL-6 have been reported to act on endothelial cells and may therefore regulate blood vessel development in the endometrium (37, 38).
Levels of LIF secretion between LH+6 and +13 were relatively low when compared with the other molecules assayed, and no significant difference was detected between fertile and infertile women. Three studies have previously reported significant differences in LIF secretion from endometrium when comparing fertile and infertile patients. Delage et al. (15) and Hambartsoumian (16) reported decreased LIF secretion from infertile endometrial biopsies cultured for 24 h. However, these explants were cultured in serum that has been reported to alter LIF secretion (11). The long culture period is also a concern because LIF secretion alters dramatically after explant culture for longer than 4 h (39). Decreased levels of LIF in uterine flushings from infertile women compared with fertile controls on d LH+10 have been reported (12). However, in that study, three of seven fertile patients and seven of seven infertile patients had levels of LIF below the limit of detection of the assay. The results should therefore be interpreted with caution. In this study, in which biopsies were subjected to serum-free short-term culture, there was no significant difference in LIF secretion between fertile and infertile patients.
No significant difference in IL-6 secretion was detected between the infertile and fertile patient groups. However, the level of secretion varied enormously from patient to patient when measured between d LH+6 and +13. This probably reflects real differences in IL-6 secretion. Patients from the same time in the cycle (i.e. LH+8) whose IL-6 secretion varied by 50-fold (187 vs. 4.4 pg/ml·mg), were found to secrete very similar levels of sIL-6R and sgp130, indicating that the differences in IL-6 are not due to abnormal or damaged endometrial biopsies. The functional significance of such variation is unclear. The pattern of IL-6 secretion by endometrium during the menstrual cycle showed increased secretion in the luteal phase compared with the proliferative phase. However, the high patient-to-patient variation meant that this did not reach statistical significance (Fig. 1). Similar results have been reported for endometrial biopsies cultured over 18 h (18). Studies of IL-6 expression in endometrium using Northern and Western blotting showed increased IL-6 mRNA and protein levels in the secretory phase (17). The correlation between these methods and the IL-6 secretion data from this study suggests that the short-term explant culture method provides a useful measure of IL-6 secretion by the endometrium in vivo. IL-6 immunoreactivity was localized in both epithelium and cells in the stroma, many of which appear to be leukocytes. These results are in agreement with previous studies and suggest that both epithelial and stromal compartments are significant sources of secreted IL-6 (17).
IL-6 signals by binding to a membrane receptor consisting of an 80-kDa ligand-binding subunit (IL-6R), and this complex then binds to the 130-kDa signal transduction molecule gp130. Soluble forms of the receptor (sIL-6R) are found in both urine and serum, and sIL-6R is able to form a complex with IL-6 that shows enhanced biological activity in vivo and in vitro (5, 22). sIL-6R can be produced by either alternative splicing or proteolytic cleavage of the membrane-bound form (22, 26). Significant levels of sIL-6R were found to be secreted by endometrial biopsies in this study, but it is not known whether these result from proteolytic cleavage or alternative splicing. The levels of secretion did not change during the cycle and were not altered between fertile and infertile patients. Immunostaining showed that the epithelium as well as leukocytes and endothelial cells within the stroma showed IL-6R immunoreactivity. It is not clear therefore which cell type is responsible for sIL-6R secretion, but we suggest that it is most likely to be glandular epithelium based on the intensity of immunostaining.
The levels of LIF and IL-6 showed no significant difference between fertile and infertile patients. The endometrium from infertile women should therefore experience an increased biological effect of LIF and IL-6 due to the reduced secretion of the antagonist sgp130 in these patients. Dysregulation of other secreted endometrial proteins has been reported in the endometrium of infertile patients. An increase in the secretion of endometrial bleeding associated factor (ebaf) has been reported in infertile women (40). This secreted peptide is a member of the TGFß superfamily and is expressed in the endometrium of fertile women as part of the molecular repertoire of proteins that precede menses (41). In some infertile women, ebaf is aberrantly secreted from the endometrium during the implantation window. Therefore, sgp130 secretion is reduced in the implantation window, whereas ebaf secretion is increased. Aberrant expression of the integrin vß3 and also of the leptin receptor has also been reported to be associated with unexplained infertility at this time during the cycle (42, 43). However the functional consequences of these alterations on the process of implantation is unknown.
In summary, we have used short-term endometrial biopsies to measure secretion of LIF, IL-6, sIL-6R, and sgp130 by endometrium during the cycle. Secretion of sgp130 was substantially higher than that of IL-6 and LIF and increased dramatically between LH+6 and +13 in normal fertile women. Soluble gp130 is produced by proteolytic cleavage of membrane-bound gp130 because none of the known splice variants of gp130 that result in soluble forms of gp130 were detected in endometrium. Secretion of sgp130 by endometrial biopsies taken from women with primary unexplained infertility between LH+6 and +11 was significantly reduced compared with fertile controls from the same time period. This corresponds to the normal time of implantation. In contrast, no difference in secretion of LIF, IL-6, and sIL-6R was seen between the two groups. In consequence, the ratio of LIF/sgp130 and IL-6/sgp130 is altered in endometrium of infertile women. Infertility appears to be associated with a failure by endometrium to express the normal molecular repertoire characteristic of the receptive period. Reduced sgp130 secretion may be a new molecular marker of such dysregulation in women with unexplained infertility.
Acknowledgments
We thank the Consultant and theater staff of Addenbrooke’s Hospital (Cambridge, UK) for their help with this study and, in particular, Dr. A. King for assistance with the endometrial dating. Our special thanks go to the staff of Hinchingbrooke Hospital, Huntingdon; The West Suffolk Hospital, Bury St. Edmunds; and The Queen Elisabeth Hospital, King’s Lynn, who gave enormous help with patient recruitment and without whom the study would not have been possible.
Footnotes
A.M.S. was supported by a Wellcome Trust Research Training Fellowship in Reproductive Biology. R.S. and A.W. were supported by a grant from Ares Serono (Geneva, Switzerland).
Abbreviations: ebaf, Endometrial bleeding associated factor; IL-6R, IL-6 receptor; LIF, leukemia inhibitory factor; LIFR, LIF receptor; RT, room temperature; s, soluble.
Received February 12, 2002.
Accepted May 10, 2002.
References
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  M. Marwood, K. Visser, L. A. Salamonsen, and E. Dimitriadis Interleukin-11 and Leukemia Inhibitory Factor Regulate the Adhesion of Endometrial Epithelial Cells: Implications in Fertility Regulation Endocrinology, June 1, 2009; 150(6): 2915 - 2923. [Abstract] [Full Text] [PDF]
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 A.A Fouladi-Nashta, L Mohamet, J.K Heath, and S.J Kimber Interleukin 1 Signaling Is Regulated by Leukemia Inhibitory Factor (LIF) and Is Aberrant in Lif-/- Mouse Uterus Biol Reprod, July 1, 2008; 79(1): 142 - 153. [Abstract] [Full Text] [PDF]
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 J. S. Fitzgerald, T. G. Poehlmann, E. Schleussner, and U. R. Markert Trophoblast invasion: the role of intracellular cytokine signalling via signal transducer and activator of transcription 3 (STAT3) Hum. Reprod. Update, April 17, 2008; (2008) dmn010v1. [Abstract] [Full Text] [PDF]
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 K. Wanggren, P.G. Lalitkumar, F. Hambiliki, B. Stabi, K. Gemzell-Danielsson, and A. Stavreus-Evers Leukaemia inhibitory factor receptor and gp130 in the human Fallopian tube and endometrium before and after mifepristone treatment and in the human preimplantation embryo Mol. Hum. Reprod., June 1, 2007; 13(6): 391 - 397. [Abstract] [Full Text] [PDF]
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R. M. Popovici, N. K. Betzler, M. S. Krause, M. Luo, J. Jauckus, A. Germeyer, S. Bloethner, A. Schlotterer, R. Kumar, T. Strowitzki, et al. Gene Expression Profiling of Human Endometrial-Trophoblast Interaction in a Coculture Model Endocrinology, December 1, 2006; 147(12): 5662 - 5675. [Abstract] [Full Text] [PDF]
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H. Achache and A. Revel Endometrial receptivity markers, the journey to successful embryo implantation Hum. Reprod. Update, November 1, 2006; 12(6): 731 - 746. [Abstract] [Full Text] [PDF]
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 G. Makkar, E. H. Y. Ng, W. S. B. Yeung, and P. C. Ho Reduced Expression of Interleukin-11 and Interleukin-6 in the Periimplantation Endometrium of Excessive Ovarian Responders during in Vitro Fertilization Treatment J. Clin. Endocrinol. Metab., August 1, 2006; 91(8): 3181 - 3188. [Abstract] [Full Text] [PDF]
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 H. Song and H. Lim Evidence for heterodimeric association of leukemia inhibitory factor (LIF) receptor and gp130 in the mouse uterus for LIF signaling during blastocyst implantation Reproduction, February 1, 2006; 131(2): 341 - 349. [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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 G. X. Rosario, G. Sachdeva, D. D. Manjramkar, D. N. Modi, P. K. Meherji, and C. P. Puri Endometrial expression of immunomodulatory cytokines and their regulators during early pregnancy in bonnet monkeys (Macaca radiata) Hum. Reprod., November 1, 2005; 20(11): 3039 - 3046. [Abstract] [Full Text] [PDF]
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S. J Kimber Leukaemia inhibitory factor in implantation and uterine biology Reproduction, August 1, 2005; 130(2): 131 - 145. [Abstract] [Full Text] [PDF]
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 S Hombach-Klonisch, A Kehlen, P A Fowler, B Huppertz, J F Jugert, G Bischoff, E Schluter, J Buchmann, and T Klonisch Regulation of functional steroid receptors and ligand-induced responses in telomerase-immortalized human endometrial epithelial cells J. Mol. Endocrinol., April 1, 2005; 34(2): 517 - 534. [Abstract] [Full Text] [PDF]
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I. Classen-Linke, G. Muller-Newen, P.C. Heinrich, H.M. Beier, and U. von Rango The cytokine receptor gp130 and its soluble form are under hormonal control in human endometrium and decidua Mol. Hum. Reprod., July 1, 2004; 10(7): 495 - 504. [Abstract] [Full Text] [PDF]
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E. Tuckerman, S.M. Laird, R. Stewart, M. Wells, and T.C. Li Markers of endometrial function in women with unexplained recurrent pregnancy loss: a comparison between morphologically normal and retarded endometrium Hum. Reprod., January 1, 2004; 19(1): 196 - 205. [Abstract] [Full Text] [PDF]
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