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Biol Reprod. 2009 May; 80(5): 920–927.
Prepublished online 2009 January 14. doi:  10.1095/biolreprod.108.073601
PMCID: PMC2849829

IL11 Antagonist Inhibits Uterine Stromal Differentiation, Causing Pregnancy Failure in Mice1

Abstract

Hormonal contraceptives are unsuitable for many women; thus, the development of new, nonhormonal contraceptives is of great interest. In women, uterine epithelial expression of interleukin 11 (IL11) and its receptor (IL11RA) suggests IL11 is critical for blastocyst attachment during implantation. Il11ra-deficient mice are infertile due to a defective decidualization response to the blastocyst, leading to total pregnancy loss. We examined the effect of administering a PEGylated IL11 antagonist, PEGIL11A (where PEG is polyethylene glycol), on pregnancy outcomes in mice and IL11 signaling in human endometrial epithelial cells (HES). PEGIL11A was detected in sera up to 72 h after intraperitoneal (IP) injection versus up to 2 h for the non-PEGylated antagonist. Following IP injection, PEGIL11A localized to uterine decidual cells and reduced immunoreactive cyclin D3 (IL11 decidual target). To inhibit IL11 action during early decidualization, PEGIL11A or control were administered IP on Days 3–6 (beginning just prior to maximal decidual Il11 expression). On Day 6, mesometrial decidualization was disturbed in PEGIL11A-treated animals with regions of hemorrhage visible in the mesometrial decidua. On Day 10, severe decidual destruction was visible: implantation sites contained significant hemorrhage, and the uterine luminal epithelium had reformed, suggesting a return to estrous cycling. These results demonstrate that PEGIL11A blocked IL11 action in the decidua during early decidualization, which totally abolished pregnancy and which is equivalent to the Il11ra−/− mouse. PEGIL11A significantly diminished STAT3 phosphorylation in HES cells in vitro (P ≤ 0.05). This study provides valuable information for PEGIL11A that could lead to the development of this protein as a nonhormonal contraceptive.

Keywords: contraception, cytokines, decidua, interleukin 11, uterus

INTRODUCTION

Steroidal contraceptives are unsuitable for many women, but current alternatives are limited. The development of nonsteroidal contraceptives is therefore of great interest. This paper describes a proof-of-concept study to evaluate the in vivo uterine efficacy and in vitro human endometrial epithelial activity, and, thus, contraceptive potential, of an interleukin 11 (IL11) antagonist.

IL11, a pleiotropic cytokine in the IL6 superfamily [1], is expressed in a wide variety of adult tissues [2], including the uterus [37]. In women, uterine IL11 is predominantly expressed during the receptive period (when the uterus is receptive to an implanting blastocyst) by the luminal and glandular endometrial epithelium and by decidualized stromal cells [37]. Epithelial IL11 and its receptor (IL11RA) are dysregulated in the endometrium of women with unexplained/idiopathic infertility or infertility and endometriosis compared with that of fertile controls [8, 9]. There is a positive correlation between IL11 levels in uterine flushings (collected during receptivity) and the responsiveness of women to ovarian stimulation prior to in vitro fertilization (IVF) [10], suggesting that reduced IL11 production may be responsible for the reduced implantation and/or pregnancy rates in excessive ovarian responders during IVF treatment. These studies suggest that IL11 secreted by the uterine epithelium into the uterine lumen acts on the endometrial luminal epithelium and blastocyst to facilitate blastocyst attachment and implantation. Thus, inhibition of uterine IL11 action during the period of uterine receptivity may prevent attachment of the blastocyst to the luminal epithelium and block implantation, resulting in pregnancy failure.

In mice, uterine expression of Il11 and its specific receptor Il11ra only occurs postimplantation by decidual cells and not by the luminal or glandular endometrial epithelium [11]. Thus, in mice, IL11 action is not required for initial blastocyst attachment to the luminal epithelium.

In mice, initial embryo attachment occurs on Day (D) 3.5 of pregnancy (D0 = day of vaginal plug detection) [12]. Following the initiation of implantation, uterine stromal cells adjacent to the embryo proliferate and differentiate into decidual cells (decidualization). The murine embryo is initially encapsulated by the avascular primary decidual zone before stromal cell differentiation radiates, forming the well vascularized mesometrial decidual zone by D6 [13]. In primates and rodents, the decidua acts to inhibit trophoblast migration during implantation [13].

Uterine Il11 is expressed between D4.5 and D10.5, with the highest expression found between D5.5 and D7.5, during the period of secondary decidual differentiation and mesometrial decidual expansion [11]. Il11ra-deficient female mice show normal oestrous cycling, ovulation, fertilization, and blastocyst formation and attachment to the luminal epithelium but are infertile due to a defective decidualization response to the implanting blastocyst [11, 14]. Tissue-specific Il11ra-deficient mice show that disrupted IL11 signaling in the ovary or embryo do not contribute to these decidual defects [11, 14]. Il11ra−/− mice are infertile due to a defective decidual response to the implanting blastocyst [11, 14]: the decidua basalis does not form; networks of giant trophoblast cells form [11, 14]; and the uterine lumen becomes filled with red blood cells and neutrophils [11].

IL11 signals via a heterodimeric receptor that binds initially to the specific IL11 receptor chain, IL11RA, and then recruits the IL11 receptor complex signaling component IL6ST (also known as gp130), followed primarily by activation of the janus kinase and signal transducers and activators of transcription (JAK/STAT) pathway [2]. In mice, Il11ra and Il6st transcript expression levels in the uterus are stable throughout the estrous cycle and pregnancy [11] and are identified in the whole embryo only after D10 of gestation [15]. Studies in Il11ra−/− mice show that decidual IL11 regulates the expression of extracellular matrix [16, 17] and cell cycle molecules, including cyclin D3 [18], supporting a role for IL11 in stromal cell remodeling during early decidualization.

In this study, we examined how inhibition of IL11 action during early pregnancy affected implantation and pregnancy outcomes in the mouse. We identified the period of sera retention for a non-PEGylated IL11 antagonist (IL11A; where PEG is polyethylene glycol) and PEGylated IL11 antagonist (PEGIL11A), uterine localization of PEGIL11A, and the result of PEGIL11A inhibition of IL11 action in the decidua in mice and in human endometrial epithelial cells in vitro.

MATERIALS AND METHODS

Animals

Female (virgin 8- to 12-wk-old) and male C57BL/6J mice (Monash Animal Services, Clayton, Australia) were housed under conventional conditions, with food and water available ad libitum and a 12L:12D cycle. Mice deficient in Il11ra were generated by gene targeting as previously described [19].

All procedures were approved by the Monash Medical Centre (B) Animal Ethics Committee or the Royal Melbourne Hospital Animal Ethics Committee, and this study followed the NHMRC Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.

IL11 Antagonists

IL11A, PEGylated IL11A (PEGIL11A), and PEG control were kindly donated by CSL Limited (Parkville, Victoria, Australia) [20]. IL11A binds to the IL11 receptor but inhibits binding of the IL11 receptor complex signaling component, IL6ST, preventing the initiation of downstream gene transcription [20]. IL11A was covalently bound to PEG to increase the period of sera retention [21].

Serum Retention of IL11A and PEGIL11A

Cycling female mice (n = 4/group) were administered 1 mg/kg IL11A or 3.2 mg/kg PEGIL11A (equivalent molarity = 10 μM) in saline by intraperitoneal (IP) injection (time 0) before being killed by CO2 asphyxiation and cervical dislocation at various time points (IL11A or calcium- and magnesium-free phosphate buffered saline (PBS) control = 5, 10, 30 min, 1, 2, and 5 h; PEGIL11A or PEG control = 10 min, 1, 2, 6, 24, 48, and 72 h). Blood was collected by cardiac puncture and stored at 4°C overnight before serum isolation by centrifugation.

The concentration of IL11A in sera was determined by ELISA. Briefly, Maxisorp 96-well immunoplates (Nunc; In Vitro Technologies) were coated (50 μl/well) with recombinant mouse Il11ra chimera (2 μg/ml in PBS; R&D systems, BioScientific Pty. Ltd.) and incubated overnight at 4°C before blocking with 5% skim milk powder in PBS (200 μl/well) for 2 h at room temperature (RT). Standards (IL11A and PEGIL11A) and serum samples were serially diluted to obtain full titration curves. Standards and sera were plated in duplicate (100 μl/well) and incubated overnight at 4°C. Wells were washed (five times; 0.05% Tween-20 in PBS) before the addition of biotinylated IL11 (50 μl/well; 0.3 μg/ml in 1% BSA, 0.05% Tween-20 in Tris-buffered saline [TBS]; R&D Systems) and incubated for 1 h at RT. Wells were again washed before the addition of strepavidin horseradish peroxidase (HRP, 50 μl/well; 1:1000 in TBS, Sigma-Aldrich) and incubation for 30 min at RT. Tetramethylbenzidine substrate (100 μl/well; Millipore/Chemicon) was added to the well and incubated for 30 min at RT before the reaction was stopped by the addition of 50 μl/well of 2 M phosphoric acid and the absorbance read at 450 nm. The EC50 of the standards and samples were calculated using the Kaleidagraph software. The concentration of IL11A and PEGIL11A in serum was converted to nanomoles/liter (nM) of mutein, and this was averaged for the samples and plotted against time.

Inhibition of IL11 Activity During Early Pregnancy

To inhibit IL11 action during early pregnancy, mated female mice (D0) were injected IP with 100 μl PEGIL11A or PEG control. Four postovulatory injection regimes were trialled (Table 1) beginning at 1000 h (i. and ii.) or 1700 h (iii. and iv.) on D3. In the regimes that began at 1000 h (i. and ii.), two IP injections of PEGIL11A were given (1000 h and 1700 h) before implantation was initiated (2200–2300 h on D3, as reviewed in [12]). In the regimes that began at 1700 h (iii. and iv.), only one IP injection of PEGIL11A was given before the initiation of implantation. Mice were killed on D6 or D10 (regimes i. and ii. only), and the number of corpora lutea on each ovary and implantation sites in the uterus were counted before fixation in 10% neutral buffered formalin.

TABLE 1.
PEGIL11A IP injection regimes used to inhibit decidualization in pregnant mice.

Mated Il11ra−/− mice were killed on D6 or D10, and the number of corpora lutea on each ovary and implantation sites in the uterus were counted before fixation in 10% neutral buffered formalin.

Immunohistochemistry

Uteri were collected from (1) (for PEG immunohistochemistry only) pregnant animals administered 50 mg/kg PEGIL11A by IP injection on D5 and collected after 3 h; and (2) (for PEG and cyclin D3 immunohistochemistry) pregnant animals treated with PEGIL11A following regime ii. (described above) and collected on D6. Formalin fixed, paraffin-embedded implantation sites (n = 4/treatment group) were sectioned at 5 μm, placed onto SuperFrost slides, dried, deparaffinized, and rehydrated.

PEG Immunolocalization

Uterine localization of PEGIL11A was identified by PEG immunohistochemistry. PEG immunolocalization was performed using the Animal Research Kit Peroxidase (DAKO) as per the manufacturer's instructions except that a microwave antigen retrieval step was included (10 mM sodium citrate, pH 6.0 for 5 min) and the primary antibody (40 μg/ml) was incubated for 30 min at RT. The primary antibody was mouse anti-PEG monoclonal IgG1 (E11 [22]; a gift from Steve Roffler, Institute of Biomedical Sciences, Academia Sinica, Taiwan) and a negative control of mouse IgG1 (DAKO) was applied to control sections at the same concentration as the primary antibody.

Cyclin D3 Immunolocalization

Inhibition of IL11 action in the decidua following IP injection of the IL11 antagonist was confirmed using immunohistochemistry for cyclin D3, an IL11 target gene that is down-regulated in the mesometrial decidua of the Il11ra−/− mouse [18]. Mice were IP injected with PEGIL11A or PEG control using regime ii. (described below) and uteri were collected on D6.

Cyclin D3 was identified in decidua using a modification of the method described in Li et al. [18]. Sections were microwave irradiated (10 mM sodium citrate, pH 6.0 for 5 min) before incubation with 3% (v/v) hydrogen peroxide for 10-min blocked endogenous peroxidase activity and sections were washed in 0.025% Triton X-100. Nonspecific binding was blocked with 10% normal goat serum for 1 h at RT prior to overnight incubation at 4°C with the cyclin D3 polyclonal antibody (2 μg/ml; Santa Cruz Biotechnology; cat. no. sc-182). Incubation with a biotinylated swine anti-rabbit secondary antibody for 30 min at RT was followed by a 30 min incubation with streptavidin-biotin complex/HRP (DAKO). Finally, sections were stained with 3,3′-diaminobenzidine (DAB) (DAKO) and counterstained with methyl blue (Sigma; CI 42780). Sections were washed in 0.6% Tween-20 in TBS for 5 min three times between each step. Negative controls consisted of sections incubated with rabbit IgG (2 μg/ml) instead of primary antibody.

Immunostaining was analyzed semiquantitively by two independent observers. The proportion of cells showing positive nuclear staining for cyclin D3 in each of the endometrial compartments (decidua, epithelium, and myometrium) was assessed and allocated a score between 0 and 3 where 0 = no stain; 1 = occasional cells stained; 2 = 50% cells stained; and 3 = majority of cells stained, relative to positive and negative controls.

Western Blot for Phosphorylated STAT3 and STAT3 in Human Endometrial Epithelial Cells (HES)

The endometrial epithelial cell line, HES (a gift from Dr. D.A Kniss, Department of Obstetrics and Gynaecology, The Ohio State University, Ohio), which models the endometrial epithelium phenotype [23], was cultured in a 6-well tissue culture plate (n = 3 experiments, n = 5 replicates/treatment except PEG control where n = 2) in RPMI-1640 (Sigma-Aldrich) containing 10% fetal calf serum, 1% antibiotics (penicillin and streptomycin), and 1% l-glutamine in a 5% CO2 incubator at 37°C. When the cells reached confluence, they were cultured overnight in serum-free media to inhibit IL11 production prior to experimentation. The cells were incubated for 15 min at 37°C with 100 ng/ml recombinant human IL11 (Genway Biotech Inc., San Diego, California) ± 1000 ng/ml PEGIL11A or 1000 ng/ml PEG control.

Following the treatment, cells were lysed and homogenized in ice-cold lysis buffer (50 mM Trizma base pH 7.4, 150 mM NaCl, 2 mM EDTA, 2 mM ethylene glycol tetraacetic acid (EGTA), 25 mM NaF, 0.2% Triton X-100, 0.3% Nonidet P-40) containing Protease Inhibitor Mixture Set III (1:500; Calbiochem, Merck Pty. Ltd.). Lysates were centrifuged, and supernatants were assayed for total protein using the BCA Protein Assay Kit (Pierce, Quantum Scientific). Proteins (60 μg per sample) were resolved on a 11% SDS/PAGE gel, transferred to Hybond-P PVDF membranes (GE Healthcare), and blocked in 10% skim milk powder in TBS for 1 h at 25°C. Membranes were incubated overnight at 4°C with rabbit anti-mouse pStat3 (Tyr705, Cell Signaling Technology; 1:1000 in 5% skim milk/TBS). After washes in TBS and 1% Tween-20 TBS, HRP goat anti-rabbit secondary antibody (DAKO; 1:1500 in 5% skim milk/TBS) and the ECL Plus Detection System (GE Healthcare) were applied. Membranes were exposed to autoradiography film (Hyperfilm ECL; GE Healthcare) for 5 min. Membranes were then washed in stripping buffer (Re Blot Plus, Chemicon International) before blocking again using 10% skim milk/TBS, incubated overnight with rabbit anti-mouse Stat3 (Cell Signaling Technology; 1:800 in 5% skim milk/TBS), and detected as described above except the films were exposed for 30 sec. The films were scanned, and the densitometry was performed with Quantity One 1-D Analysis Software (Bio-Rad Laboratories Pty., Ltd.). Background-corrected density OD/mm2 for pSTAT3/STAT3 (phosphorylated STAT3/STAT3) was obtained, and the ratios for PEGIL11A-treated and untreated cells were calculated.

Statistics

GraphPad Prism 5.0 for Windows (GraphPad Software, San Diego, CA) was used for all the statistical analyses. An unpaired t-test was used to compare PEGIL11A and IL11A serum concentration at the 10-min and 2-h time points. A Wilcoxon signed rank test (nonparametric, paired t-test;) was used to compare the number of implantation sites to corpora lutea in each animal and implantation site number between treatment groups [24]. The Mann-Whitney U-test (nonparametric t-test) was used to analyze cyclin D3 immunostaining between the control and PEGIL11A-treated samples. One-way ANOVA was used to compare the pSTAT3/STAT3 ratio between treatment groups of HES cells cultured in vitro. A P value of less than 0.05 was considered to be significant.

RESULTS

Serum Retention of IL11A and PEGIL11A

IL11A was detected in serum from 5 min to 2 h after IP injection but was undetectable at 5 h following injection (Fig. 1). In contrast, PEGIL11A was detected in serum from 10 min to 72 h after injection but was undetectable at 96 h following injection (Fig. 1). IL11A and PEGIL11A were not detected in serum from mice injected with either PBS or PEG controls at any time points targeted (data not shown). PEGIL11A serum concentration was significantly lower than IL11A at 10 min (IL11A, 31.7 ± 8.6 nM; PEGIL11A, 8.9 ± 3.3 nM; t = 2.495, df = 6, P < 0.05) but significantly higher at 2 h (PEGIL11A, 53.8 ± 14.3 nM; IL11A, 7.6 ± 0.8 nM; t = 3.219, df = 6, P < 0.05).

FIG. 1.
IL11A and PEGIL11A serum levels following IP injection. IL11A (black; 1 mg/kg) or PEGIL11A (white; 3.2 mg/kg) were injected IP into nonpregnant mice (n = 4/treatment/time point). Sera were collected at the time points shown, and IL11A was detected by ...

Uterine Localization of PEGIL11A

PEGIL11A localized to specific cells in the decidualized stroma 3 h following 50 mg/kg IP injection (Fig. 2A). In animals treated with PEGIL11A following injection regime ii., strong localization of PEGIL11A was observed in all decidual cells on D6 48 h following the last IP injection of PEGIL11A (Fig. 2B). These data suggest that although some PEGIL11A localized to decidual cells within 3 h of IP injection, the majority of PEGIL11A localized to decidual cells after this time.

FIG. 2.
Uterine localization of PEGIL11A following IP injection in mated mice identified by PEG immunohistochemistry. As a negative control, sections were incubated with IgG at the same concentration as the primary antibody (inset). A) Implantation site showing ...

PEGIL11A Reduced Cyclin D3 Immunoreactivity

Cyclin D3 staining was found in decidual cells in all control implantation sites examined (n = 4; Fig. 3A). In PEGIL11A-treated implantation sites, cyclin D3 staining was reduced, with 2 of 4 implantation sites having no staining and 2 of 4 implantation sites having only occasional cells in the remaining decidua containing immunoreactive cyclin D3 (Fig. 3B). Mean cyclin D3 in decidual cells was consistently more intense in implantation sites from control-treated animals compared to PEGIL11A-treated animals (2.6 ± 0.2 vs. 0.3 ± 0.1, respectively, P < 0.05; Fig. 3C). Cyclin D3 staining was never detected in the glandular epithelium or the myometrium (data not shown).

FIG. 3.
PEGIL11A reduced immunoreactive cyclin D3 protein in decidual cells of D6 implantation sites. As a negative control, sections were incubated with IgG at the same concentration as the primary antibody (inset). A) PEG control (IP injections followed regime ...

PEGIL11A Inhibits Decidual Transformation

Postovulatory treatment with PEGIL11A did not affect the ability of the blastocyst to initially implant into the uterus (Fig. 4, A–C), with the number of corpora lutea visible on the ovary not significantly different from the number of implantation sites found in the uterus on D6 and D10 following any treatment regime (Table 2) or in Il11ra−/− mice [11].

FIG. 4.
PEGIL11A inhibited decidual transformation resulting in pregnancy failure. Shown are representative uteri (AC) and implantation sites (DO) collected from mated mice treated with control (PEG; A, D, H, and L) and PEGIL11A (B, C, E, F ...
TABLE 2.
Corpora lutea and implantation site numbers following PEGIL11A or PEG control administration during early pregnancy.

On D6, control-treated animals had well-developed implantation sites in the uterus (Fig. 4A), and the implantation sites of those treated with PEGIL11A (Fig. 4, B and C) or the Il11ra−/− mice [11] were smaller than the control. The ex vivo uteri of regime i. and ii. PEGIL11A-treated animals (Table 1; Fig. 4C) were very similar in appearance to those of Il11ra−/− mice [11], with very small implantation sites (Fig. 4C) and hemorrhage visible in the uterine lumen. Morphological analysis of implantation site cross-sections (Fig. 4, D–K) indicated that while the decidua of control animals had differentiated into clear antimesometrial and mesometrial decidual zones (Fig. 4D) and the mesometrial decidual zone was highly vascularized (Fig. 4H), decidual formation, particularly formation of the mesometrial decidual zone, was considerably retarded in PEGIL11A-treated (Fig. 4, E, F, I, and J) and Il11ra−/− (Fig. 4, G and K) animals. When the initial injection was given at 1000 h on D3 with a second injection at 1700 h on D3 (regimes i. and ii; Fig. 4F), inhibition of mesometrial decidual formation was further reduced (compared to regimes iii. and iv, Fig. 4E) and regions of hemorrhage were visible in the mesometrial decidual zone (Fig. 4J). Implantation sites from regimes i. and ii (Fig. 4, F and J) most closely resembled those in Il11ra−/− implantation sites (Fig. 4, G and K).

On D10, while clear decidual, placental, and embryonic components were apparent in implantation sites of control animals (Fig. 4L), in PEGIL11A-treated mice (regimes i. and ii.), severe decidual breakdown had occurred (Fig. 4, M and N). Most implantation sites treated with PEGIL11A contained only hemorrhagic debris and giant trophoblast cells (Fig. 4M) and 8/13 implantation sites examined on D10 had no decidual tissue visible (Fig. 4M). When decidual tissue remained (Fig. 4N), there was clearly dysregulated trophoblast invasion and regions of hemorrhage. Any conceptus present (3/13 implantation sites) was necrotic at D10. The uterine luminal epithelium had reformed in 10/13 implantation sites by D10 (Fig. 4M). Loss of decidual tissue, dysregulated trophoblast invasion, and hemorrhages were also found in Il11ra−/− implantation sites (Fig. 4O).

PEGIL11A Reduced IL11 Induction of STAT3 Phosphorylation in Human Endometrial Epithelial Cells

The effect of IL11 on pSTAT3 and STAT3 in HES cells was examined by Western blot (Fig. 5). Phosphorylated STAT3 was not detectable in HES cultured under serum-free conditions (Fig. 5A). Addition of IL11 to HES stimulated pSTAT3 (Fig. 5A). Coincubation of cells with PEGIL11A significantly diminished IL11-induced pSTAT3 compared to cells treated with IL11 (F2, 11 6.606, P = 0.0130; Fig. 5B). STAT3 protein abundance was not affected when the different treatments were added to HES (Fig. 5A).

FIG. 5.
PEGIL11A diminished IL11-induced pSTAT3 in human endometrial epithelial cells (HES) in vitro. A) Immunoblot for pSTAT3 and total STAT3 in cell lysates from HES cells. B) Graphical representation of pSTAT3/total STAT3 ratios calculated from densitometry ...

DISCUSSION

In this study, administration of a novel PEGylated IL11 antagonist to mice during early pregnancy (prior to implantation), totally disrupted the establishment of pregnancy by disturbing the process of decidualization of endometrial stromal cells. This decidual phenotype mimicked that of the Il11ra−/− mouse [11, 14], thus confirming a critical role for IL11 action in the very early stages of implantation in this species. This study therefore demonstrated the in vivo efficacy of this new inhibitor and that it can effectively reach and act in the uterus following IP injection. PEGIL11A also reduced IL11-induced pSTAT3 in human endometrial epithelial cells, confirming its ability to block IL11 action in human cells. Given that IL11 is implicated in the very earliest stages of implantation in humans and other species, this antagonist has potential for contraceptive purposes in both humans and animals.

Blockage of IL11 action by PEGIL11A administration during early pregnancy impaired mesometrial decidual formation. The exact role of IL11 in implantation and decidualization is not well understood, however, IL11 regulates the expression of a number of cell cycle and extracellular matrix components, supporting a role for IL11 in stromal cell remodelling during implantation [1618]. Gene-targeting studies cannot pinpoint the precise time that the targeted factor acts, in this case, exactly at what time in the implantation process IL11 becomes critical for stromal cell decidualization [11, 14]. Here, a more severe decidual defect was found when injection of PEGIL11A began early on D3 (1000 h) compared to when injection of PEGIL11A began later on D3 (1700 h), despite both injection regimes beginning prior to the initiation of blastocyst attachment. In vivo, Il11 uterine mRNA expression does not begin until D4 (although Il11ra mRNA expression occurs throughout the oestrous cycle) [11, 14], and hence administration of antagonist well before endogenous IL11 appears would have provided sufficient time for the antagonist to localize and bind to stromal IL11RA before endogenous IL11 production began. The observation that only some decidual cells had immunoreactive PEGIL11A 3 h post-IP injection, but all decidual cells stained positively for PEGIL11A 48 h after the final IP injection, supports this. The results presented here indicate that IL11 action is critical during the initiation of decidualization at the time of embryo implantation (D4).

In Il11ra−/− mice [11, 14] and also in this study, trophoblast cell invasion into the decidua was dysregulated, suggesting that IL11 may play a role in inhibiting trophoblast invasion. The mesometrial decidua is the site of trophoblast cell invasion in rodents [13] and inhibition of IL11 action, shown here and also in the Il11ra−/− mouse [11, 14], disrupts mesometirial decidual formation. It is interesting to note that in pseudopregnant Il11ra−/− mice induced to decidualize by oil (no trophoblast present), all mesometrial decidual formation is not completely inhibited [11], suggesting that dysregulated trophoblast invasion may contribute to loss of the mesometrial decidua, rather than occur as a consequence of this loss [18].

We examined the in vivo effect of PEGIL11A on the production of a known IL11 decidual target, cyclin D3 [18]. Cyclin D3 expression is reduced in the mesometrial decidua of the Il11ra−/− mouse on D8 [18]. In the control implantation sites on D6 in this study, cyclin D3 localized to decidual cells, particularly in the mesometrial decidual zone (Fig. 3A), but in the treated implantation sites, positive staining was reduced to very low levels. As in [18], inhibition of IL11 action did not block all immunoreactive decidual cyclin D3, and it is not possible to determine from this study whether IL11 inhibits cyclin D3 expression directly or indirectly via its action on decidualization.

The reepithelialization that occurs around the failed implantation site following PEGIL11A treatment (Fig. 4M) resembles very closely that seen following endometrial breakdown in a mouse model for menstruation [25]. In that model, complete endometrial repair and restoration of full thickness endometrium (epithelium plus stromal compartments) occurs very rapidly [26], and therefore it is anticipated that the PEGIL11A-treated animals will return to estrous cycling once the failed implantation site has been shed.

Enhanced plasma retention of drugs compensates for the loss of biological activity caused by PEGylation, by increasing pharmacological activity [27]. Previously, we used an inhibitor of leukaemia inhibitory factor (LIF), similar to that used here for IL11, to block implantation in the mouse [24]: PEGylation of this LIF inhibitor increased the period of sera retention such that pharmacological activity in the uterus was sufficient to block implantation [24]. PEGIL11A was detected in serum up to 72 h after IP injection compared with only 2 h for IL11A, suggesting that PEGIL11A was retained in serum for longer than IL11A, as is found following PEGylation of other cytokines [28], including IL11 [27]. PEGIL11A was clearly identified in the decidualized stroma of implantation sites 48 h following IP injection. It could be anticipated that PEGIL11A would still be present (and presumably still effective) in the uterus for at least 48 h after IP injection.

In women, IL11 distribution during implantation differs from that in the mouse. In mice, IL11 and its receptor immunolocalize only to stromal cells [11], and attachment of the blastocyst to the luminal epithelium is normal in the Il11ra1−/− mouse [11, 14], confirming that in mice the attachment phase of implantation does not require IL11. Importantly however, in women, IL11 immunolocalizes particularly to epithelial cells during the receptive phase of the menstrual cycle and also to decidualizing stromal cells even in nonconception cycles [37]. Further, IL11 and IL11RA are down-regulated in the endometrial epithelium during the receptive phase in infertile women (with or without endometriosis) [3, 8, 9, 29], supporting a role for IL11 in early blastocyst implantation. IL11 is also present in the uterine lumen during the period of uterine receptivity [10], suggesting that it could act on the initial attachment of the blastocyst to the luminal epithelium. However, to date, it is not known whether the IL11RA is present on the blastocyst epithelium. This study therefore provides proof-of-concept for the development of a novel, nonhormonal contraceptive for women, as inhibition of IL11 action during the period of uterine receptivity in women should inhibit implantation, preventing the establishment of pregnancy. PEGIL11A should now be trialed in a primate model species to demonstrate the efficacy of this antagonist in blocking epithelial IL11 action during blastocyst implantation.

This paper demonstrated for the first time that administration of a long-acting IL11 antagonist blocked IL11 action in the uterus during early pregnancy and in vitro in human endometrial epithelial cells. In the mouse, its action resulted in a severe decidual deficiency leading to total pregnancy loss, as is found in the Il11ra−/− mouse. Given that there is strong evidence that in women epithelial IL11 is also functionally important at implantation and that PEGIL11A reduced IL11 action in human endometrial epithelial cells in vitro, it is proposed that PEGIL11A has potential as a new nonhormonal contraceptive agent for women.

Acknowledgments

We are grateful to Melinda Marwood, Joanne Yap, Jun Gu, and Dr. Neil Borg (Prince Henry's Institute) for their excellent technical assistance; Drs. Felicity Dunlop, Pierre Scotney, Manuel Baca, and Andrew Nash (CSL Limited) for providing the IL11 antagonists and technical assistance; Dr. D.A. Kniss for the gift of the HES cell line; and Dr. Steve Roffler for his donation of PEG antibody.

Footnotes

1Supported by the Consortium for Industrial Collaboration in Contraceptive Research Program of the Contraceptive Research and Development Program, Eastern Virginia Medical School (Sub-project CIG-07-116) and by the NH&MRC of Australia (#388901, #388916, and #461219).

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