Ellen Menkhorst

Leukemia inhibitory factor and interleukin-11: Critical regulators in the establishment of pregnancy

Blastocyst implantation into a receptive endometrium is critical to the establishment of pregnancy and is tightly regulated by factors within the blastocyst-endometrial micro-environment. Leukemia inhibitory factor (LIF) and interleukin-11 (IL11) have key roles during implantation. Female mice with a null mutation in the LIF or IL11RA gene are infertile due to a complete failure of implantation or a defective differentiation/decidualization response to the implanting blastocyst, respectively. LIF and IL11 deficiency during pregnancy is associated with infertility and miscarriage in women. Numerous cell populations at the maternal-fetal interface are regulated by LIF/IL11 including the endometrial epithelium, decidualizing stroma, placental trophoblasts and leukocytes. This review focuses on the roles of LIF/IL11 during early pregnancy and highlights their potential as contraceptive targets and therapeutic agents for infertility.

Review: LIF and IL11 in trophoblast-endometrial interactions during the establishment of pregnancy.

Blastocyst implantation into the endometrium is critical for the establishment of pregnancy and is tightly regulated by factors within the blastocyst-endometrial micro-environment. Implantation is a continuum involving blastocyst adhesion to the endometrial epithelium followed by trophoblast penetration of the epithelium. The trophoblast proliferates and invades through the endometrium, with a subpopulation acting to remodel the spiral arteries. Trophoblast-endometrial interactions in humans involve carefully orchestrated temporal and spatial alterations in factors that are critical for pregnancy success. Emerging evidence suggests important roles for locally produced cytokines including interleukin 11 and leukemia inhibitory factor in the various stages of implantation. This review focuses on the role of these cytokines in trophoblast-endometrial interactions during the establishment of human pregnancy.

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

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.

Preimplantation human blastocysts release factors that differentially alter human endometrial epithelial cell adhesion and gene expression relative to IVF success

STUDY QUESTIONnDo human blastocysts which subsequently implant release factors that regulate endometrial epithelial cell gene expression and adhesion to facilitate endometrial receptivity?nnnSUMMARY ANSWERnBlastocysts which subsequently implanted released factors that altered endometrial epithelial gene expression and facilitated endometrial adhesion while blastocysts that failed to implant did not.nnnWHAT IS KNOWN ALREADYnHuman preimplantation blastocysts are thought to interact with the endometrium to facilitate implantation. Very little is known of the mechanisms by which this occurs and to our knowledge there is no information on whether human blastocysts facilitate blastocyst attachment to the endometrium.nnnSTUDY DESIGN, SIZE, DURATIONnWe used blastocyst-conditioned medium (BCM) from blastocysts that implanted (n = 28) and blastocysts that did not implant (n = 28) following IVF. Primary human endometrial epithelial cells (HEECs) (n = 3 experiments) were treated with BCM and the effect on gene expression and adhesion to trophoblast cells determined. We compared the protein production of selected genes in the endometrium of women with normal fertility (n = 40) and infertility (n = 6) during the receptive phase.nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnWe used real-time RT-PCR arrays containing 84 genes associated with the epithelial to mesenchymal transition. We validated selected genes by real-time RT-PCR (n = 3) and immunohistochemistry in the human endometrium (n = 46). Adhesion assays were performed using HEECs and a trophoblast cell line (n = 3).nnnMAIN RESULTS AND THE ROLE OF CHANCEnBlastocysts that implanted released factors that differentially altered mRNA levels for six genes (>1.5 fold) compared with blastocysts that did not implant. A cohort of genes was validated at the protein level: SPARC and Jagged1 were down-regulated (P < 0.01), while SNAI2 and TGF-B1 were up-regulated (P < 0.05) by implanted compared with non-implanted BCM. Jagged-1 (P < 0.05) and Snai-2 protein (P < 0.01) showed cyclical changes in the endometrium across the cycle, and Jagged-1 staining differed in women with normal fertility versus infertility (only) (P < 0.01). HEEC adhesion to a trophoblast cell line was increased after treatment with implanted BCM compared with untreated control (P < 0.05).nnnLIMITATIONS, REASONS FOR CAUTIONnThis is an in vitro study and it would be beneficial to validate our findings using a physiological model, such as mouse.nnnWIDER IMPLICATIONS OF THE FINDINGSnThis new strategy has identified novel pathways that may be important for human preimplantation blastocyst-endometrial interactions and opens the possibility of examining and manipulating specific pathways to improve implantation and pregnancy success.nnnSTUDY FUNDING/COMPETING INTERESTnThis study was supported by the National Health and Medical Research Council of Australia (Fellowship support #550905, #611827) and project grants by Monash IVF, Australia. There are no conflicts of interest to be declared.

Preimplantation Human Blastocyst-Endometrial Interactions: The Role of Inflammatory Mediators

Immune factors such as cytokines, chemokines, and growth factors are known to play important roles in the preimplantation interactions and communication between the blastocyst and receptive endometrium. This crucial dialog occurs during the stages when the blastocyst is in the uterine cavity immediately preceding implantation and the establishment of pregnancy. Human preimplantation processes are difficult to study due to restrictions on tissue availability. This review focuses on the expression and role of immune factors in human blastocyst‐endometrial dialog during the very early stages of implantation. It highlights the importance of immune regulators and the need to develop new models to study human implantation.

Interleukin (IL)11 mediates protein secretion and modification in human extravillous trophoblasts

BACKGROUNDnHuman trophoblast invasion and differentiation are essential for a successful pregnancy outcome. Dysregulation of these processes can lead to placental pathologies such as pre-eclampsia. The molecular mechanisms; however, are poorly understood. Interleukin (IL)11--a cytokine that regulates endometrial epithelial cell adhesion, trophoblast motility and invasion during implantation--may be involved in some of these processes.nnnMETHODS AND RESULTSnThe effect of IL11 on protein expression was investigated in trophoblastic HTR8/SVneo cells and primary extravillous trophoblasts (EVTs) purified from first- trimester placentas. Two-dimension (2D)-differential in-gel electrophoresis analyses revealed that 731 spots were significantly differentially regulated by IL11 in HTR8/SVneo cells: seven spots were analyzed by liquid chromatography-tandem mass spectrometry and 14 unique proteins identified. Protein disulfide isomerase family A, member 3 (PDIA3; endoplasmic reticulum p57) and glucose-regulated protein 78 (GRP78) were further validated to be regulated by IL11 in HTR8/SVneo and primary EVT. One dimension western blot analysis confirmed that PDIA3 was down-regulated in EVT. 2D western blot analysis revealed that GRP78 was post-translationally modified following IL11 treatment. Moreover, IL11 stimulated the secretion of GRP78 in EVT.nnnCONCLUSIONSnData suggest that IL11, possibly via signal transducers and activators of transcription 3 signaling pathway, regulates PDIA3 protein expression and modification/secretion of GRP78. This is the first study to identify PDIA3 and GRP78 as IL11 targets in invasive trophoblasts and identifies a possible mechanism by which IL11 regulates trophoblast function.

Interleukin-11 alters placentation and causes preeclampsia features in mice

Significance Preeclampsia is an insidious disease, unique to humans, affecting ∼8% of pregnancies. There are no early detection tests or pharmacological treatments. Impaired placentation is widely accepted to contribute to the pathogenesis. However, the mechanisms remain elusive, given the complications of studying first-trimester placental development in women. A major limitation for the study of new treatments is the lack of available animal models that recapitulate the full spectrum of preeclampsia features. We have developed a mouse model characterized by elevated levels of the cytokine Interleukin-11 (IL11). This study provides evidence of a novel pathway causative of preeclampsia features in vivo. It also provides a novel in vivo mouse model that is useful for preclinical studies to test potential therapeutics. Preeclampsia (PE) is a pregnancy-specific disorder characterized by hypertension and proteinuria after 20 wk gestation. Abnormal extravillous trophoblast (EVT) invasion and remodeling of uterine spiral arterioles is thought to contribute to PE development. Interleukin-11 (IL11) impedes human EVT invasion in vitro and is elevated in PE decidua in women. We demonstrate that IL11 administered to mice causes development of PE features. Immunohistochemistry shows IL11 compromises trophoblast invasion, spiral artery remodeling, and placentation, leading to increased systolic blood pressure (SBP), proteinuria, and intrauterine growth restriction, although nonpregnant mice were unaffected. Real-time PCR array analysis identified pregnancy-associated plasma protein A2 (PAPPA2), associated with PE in women, as an IL11 regulated target. IL11 increased PAPPA2 serum and placental tissue levels in mice. In vitro, IL11 compromised primary human EVT invasion, whereas siRNA knockdown of PAPPA2 alleviated the effect. Genes regulating uterine natural killer (uNK) recruitment and differentiation were down-regulated and uNK cells were reduced after IL11 treatment in mice. IL11 withdrawal in mice at onset of PE features reduced SBP and proteinuria to control levels and alleviated placental labyrinth defects. In women, placental IL11 immunostaining levels increased in PE pregnancies and in serum collected from women before development of early-onset PE, shown by ELISA. These results indicate that elevated IL11 levels result in physiological changes at the maternal–fetal interface, contribute to abnormal placentation, and lead to the development of PE. Targeting placental IL11 may provide a new treatment option for PE.

Dynamic changes in hyperglycosylated human chorionic gonadotrophin throughout the first trimester of pregnancy and its role in early placentation

STUDY QUESTIONnWhat is the in situ localization and function of hyperglycosylated hCG (hCG-H) in first trimester pregnancy tissues?nnnSUMMARY ANSWERnHCG-H localizes to the syncytiotrophoblast, cytotrophoblast and invasive extravillous trophoblast within the maternal decidua and promotes invasion during the first trimester of pregnancy.nnnWHAT IS KNOWN ALREADYnSerum levels of hCG-H decline dramatically throughout the first trimester of pregnancy. As hCG-H is produced by choriocarcinoma cells, it is proposed to regulate trophoblast invasion.nnnSTUDY DESIGN, SIZE, DURATIONnTissues were collected from elective first trimester pregnancy terminations. Placental villous and decidua basalis were collected from Week 6 to Week 12 of gestation (n = 49).nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnTissues were collected from elective first trimester surgical pregnancy terminations to determine localization, abundance and function of hCG-H. Placental villous outgrowth studies determined the impact of neutralizing endogenous hCG-H on trophoblast function. Real-time proliferation, migration and invasion assays using JEG-3 choriocarcinoma cells further elucidated the role of hCG-H in trophoblast function.nnnMAIN RESULTS AND THE ROLE OF CHANCEnHCG-H localized to syncytiotrophoblast layer of the placental villous from gestational weeks 6-9; thereafter hCG-H localized as a discrete layer between syncytio- and cyto-trophoblast layers. Immunoreactive hCG-H was also observed within the cytotrophoblast layer in Week 7-8 of gestation. HCG-H abundance decreased within placental villous from Weeks 6-12 of gestation (n = 3 placentas per gestational weeks 6-12). HCG-H also localized to anchoring villi within maternal decidua, extravillous trophoblasts invading into the maternal decidua and endovascular trophoblasts remodeling maternal blood vessels. Treatment of primary first trimester villous explants with hCG-H neutralizing antibody reduced trophoblast outgrowth (n = 3 placentas, P < 0.05). Treatment of a trophoblast cell line with neutralizing antibody reduced trophoblast invasion (n = 4, P < 0.05) but did not affect migration or proliferation.nnnLIMITATIONS, REASONS FOR CAUTIONnFunctional invasion and migration assays performed using cell lines. Not possible to perform such assays with primary human material.nnnWIDER IMPLICATIONS OF THE FINDINGSnHCG-H is an important autocrine factor facilitating trophoblast invasion in the first trimester of pregnancy. Targeting hCG-H may prove useful in the treatment of pathologic pregnancies, such as ectopic pregnancies, or pregnancy complications including pre-eclampsia and gestational trophoblast diseases.nnnSTUDY FUNDING/COMPETING INTERESTSnThis work was supported by the Victorian Government Operational Infrastructure Support Program. J.E. is supported by NHMRC project grant #1047756, L.A.S. and E.D. by NHMRC Fellowships #1002018 and #550905 respectively and E.M. by an NHMRC Early Career Fellowship #611827. The authors have no conflicts of interest relating to this work.

The role of leukemia inhibitory factor in tubal ectopic pregnancy.

INTRODUCTIONnEctopic pregnancy is unique to humans and a leading cause of maternal morbidity and mortality. The etiology remains unknown however factors regulating embryo implantation likely contribute. Leukemia inhibitory factor (LIF) has roles in extravillous trophoblast adhesion and invasion and is present in ectopic implantation sites. We hypothesised that LIF facilitates blastocyst adhesion/invasion in the Fallopian tube, contributing to ectopic pregnancy.nnnMETHODSnWe immunolocalised LIF receptor (R) in tubal ectopic pregnancy (N = 5). We used an oviduct cell line (OE-E6/E7) to model Fallopian tube epithelial cells and a trophoblast spheroid co-culture model (HTR-8/SVneo cell line formed spheroids) to model blastocyst attachment to the Fallopian tube. We examined LIF signaling pathways in OE-E6/E7 cells by Western blot. The effect of LIF and LIF inhibition (using a novel LIF inhibitor, PEGLA) on first-trimester placental outgrowth was determined.nnnRESULTSnLIFR localised to villous and extravillous trophoblast and Fallopian tube epithelium in ectopic pregnancy. LIF activated STAT3 but not the ERK pathway in OE-E6/E7 cells. LIF stimulated HTR-8/SVneo spheroid adhesion to OE-E6/E7 cells which was significantly reduced after PEGLA treatment. LIF promoted placental explants outgrowth, while co-treatment with PEGLA blocked outgrowth.nnnDISCUSSIONnOur data suggests LIF facilitates the development of ectopic pregnancy by stimulating blastocyst adhesion and trophoblast outgrowth from placental explants. Ectopic pregnancy is usually diagnosed after 6 weeks of pregnancy, therefore PEGLA may be useful in targeting trophoblast growth/invasion.nnnCONCLUSIONnLIF may contribute to the development of ectopic pregnancies and that pharmacologically targeting LIF-mediated trophoblast outgrowth may be useful as a treatment for ectopic pregnancy.

Interleukin 11 and activin A synergise to regulate progesterone-induced but not cAMP-induced decidualization

Blastocyst implantation, placentation and the establishment of pregnancy in the human are dependent on the adequate decidualization of endometrial stromal cells. Locally produced and temporally regulated products such as interleukin 11 (IL11), and activin A are involved in this process; however, the molecular interactions that regulate decidualization are largely unknown. Here, we investigated whether IL11 and activin A interact to promote human endometrial stromal cell (HESC) decidualization using an in vitro model. HESCs, induced to decidualize by cAMP or progesterone, were treated with IL11, activin A or IL11 plus activin A combined and decidual progress was examined using prolactin as a decidual marker. Treatment with combined IL11 plus activin A enhanced progesterone-induced decidualization above control or treatment with IL11 or activin A alone. Treatment had no effect on cAMP-decidualized HESC. Investigation of IL11 and activin A stimulation of (respectively) activin A and IL11 expression in undifferentiated HESC was by real-time PCR and ELISA. Activin A treatment induced IL11 secretion and phosphorylation of the activin A signalling component, SMAD2 (measured by ELISA). Inclusion of the TGFbeta 1 receptor inhibitor, SB431542, in the activin A treatment, reduced pSMAD2 and IL11 secretion. This study suggests that activin A is an early inducer of decidualization, regulating the secretion of IL11. This data provides insight into the processes underlying decidualization, which are important for understanding implantation and placentation and have potential clinical applications for the regulation of fertility.