Evdokia Dimitriadis

Models for Study of Human Embryo Implantation: Choice of Cell Lines?

Abstract Implantation failure and inadequate placental development are important contributors to infertility, recurrent miscarriage, and other pregnancy-related problems in women. Better understanding of these processes is hampered by the difficulty in obtaining human tissue from which primary cells can be prepared and by the very limited access worldwide to human blastocysts for experimentation. Therefore, the use of appropriate cell lines, particularly for functional studies of implantation and placentation, is imperative. While a number of cell lines for both endometrium and trophoblast have been developed and are widely used, it is difficult for researchers to decide which of these are most appropriate for studies of particular functions. This brief review summarizes the known phenotypes of the most widely used cell lines and indicates which might be the most appropriate for individual studies.

Society for Reproductive Biology Founders' Lecture 2009. Preparing fertile soil: the importance of endometrial receptivity

The human endometrium is receptive for implantation of a blastocyst for only 4-5 days in each menstrual cycle. Failure of implantation is a major reason for infertility in women and the inability to achieve endometrial receptivity is responsible for much of the failure of reproductive technologies. Endometrial receptivity requires changes in the uterine luminal and glandular cells, particularly in terms of their secretory capacity and altered expression of adhesion molecules. In parallel with these changes, decidualisation (differentiation) of the endometrial stroma is initiated in women during the receptive phase, regardless of the presence of a blastocyst. Increased leucocyte numbers are also important. The microenvironments provided by the endometrium during the receptive phase and that support implantation are highly complex and constantly changing as implantation progresses. The present review provides a comprehensive overview of the cellular and molecular events of human implantation. It also summarises work from our laboratories emphasising the functional importance of proprotein convertase 6, along with key cytokines (interleukin-11, leukaemia inhibitory factor, activin A) and chemokines (including CX3CL1 and CCL14), during implantation. Of particular importance is how these mediators contribute to receptivity and how they are disturbed in infertile women. Factors that are critical for uterine receptivity may also be manipulated to provide new contraceptive strategies for women.

Local regulation of implantation at the human fetal-maternal interface

Embryo implantation and formation of a functional placenta are complex processes that require a plethora of regulatory molecules. In recent years, many of these mediators have been identified, often from studies in experimental animals. Furthermore, their expression patterns at the embryo-maternal interface in women have been characterized and provide clues to their potential actions. What has been missing in most cases is any experimental demonstration of their function. Proteases, cytokines and chemokines are among the molecules identified at the embryo-maternal interface. Functional studies of the protease, proprotein convertase (PC)6, the gp130 cytokines, leukemia inhibitory factor (LIF) and interleukin (IL)11 and the chemokines, CX3CL1 and CCL14 demonstrate potential actions within the uterine cavity. These actions include: enhancing blastocyst development, modifying adhesive properties of the blastocyst and the uterine epithelial surface, and providing chemotactic guidance to the blastocyst. As implantation proceeds, PC6 and IL-11 also act to drive decidualization. The products (proteases, chemokines and cytokines) produced by these decidual cells provide a unique environment. This is important for both directing and restraining trophoblast invasion and for leukocyte trafficking into the decidua until the placenta is fully established.

Interleukin-11 and leukemia inhibitory factor regulate the adhesion of endometrial epithelial cells: implications in fertility regulation.

Embryo implantation requires the closely harmonized processes of apposition, attachment, and adhesion of the conceptus to the maternal endometrial epithelium. IL-11 and leukemia inhibitory factor (LIF), two IL-6 family cytokines, are produced by the endometrium and are absolutely required for implantation in mice. We examined the effect of IL-11 and LIF on human endometrial epithelial cell adhesion. Both cytokines increased adhesion of primary human endometrial epithelial cells to fibronectin and collagen IV. IL-11 stimulated, whereas LIF had no effect on the adhesion of trophoblast to endometrial epithelial cells. Focused oligogene arrays were used to identify extracellular matrix and adhesion molecules mRNAs regulated by endometrial epithelial cells. We demonstrated by real-time RT-PCR and antibody arrays that both cytokines increased integrin-alpha2 mRNA and protein by endometrial epithelial cells. Signal transducers and activators of transcription (STAT)-3 inhibition reduced IL-11- and LIF-mediated epithelial cell adhesion to fibronectin, suggesting both cytokines regulated adhesion via phosphorylation of STAT3. Addition of either IL-11 neutralizing antibody and IL-11 or LIF and LIF antagonist to endometrial epithelial cells abolished cytokine induced phosphorylated STAT3. LIF but not IL-11 induced adhesion to collagen IV was reduced by an integrin-alpha2beta1 neutralizing antibody. This study demonstrated that IL-11 and LIF regulated endometrial epithelial cell adhesion, suggesting that targeting IL-11 and LIF may be useful in regulating fertility by either enhancing or blocking implantation.

Leukemia inhibitory factor promotes human first trimester extravillous trophoblast adhesion to extracellular matrix and secretion of tissue inhibitor of metalloproteinases-1 and -2

BACKGROUND Leukemia inhibitory factor (LIF) is a pleiotropic cytokine that is essential for blastocyst implantation in mice. It has been suggested that LIF may play a role in human first trimester extravillous trophoblast (EVT) invasion. The aim of the present study was to establish whether LIF induces changes in EVT function related to invasiveness. METHODS Primary first trimester human EVT cell cultures were treated with/without LIF and the effects on cell adhesion to fibronectin (FN), vitronectin (VN) and laminin (LN) were assessed. Transcript levels of integrin subunits that mediate cell adhesion to these extracellular matrix (ECM) elements were determined by real-time RT–PCR. Matrix metalloproteinase (MMP)2 and MMP9 secretion was assessed by gelatine zymography and tissue inhibitors matrix metalloproteinase (TIMP) -1 and TIMP-2 secretion by enzyme-linked immunosorbent assay. RESULTS EVT cells showed increased adhesion to FN, VN and LN ECM elements in response to LIF (20, 20 and 29%, respectively, P < 0.05 FN and VN compared to control; and P < 0.001 LN compared to control). Integrin β4 mRNA levels decreased by 50% following LIF treatment (P < 0.001 versus control). MMP2 and MMP9 secretion was not affected by LIF but LIF did increase secretion of TIMP-1 and -2 (P < 0.001 versus control). LIF stimulated the phosphorylation of signal transducer and activator of transcription (STAT) 3 protein while it did not affect STAT3 protein abundance. The addition of a LIF inhibitor attenuated the LIF-induced STAT3 phosphorylation in EVT. CONCLUSION The results suggest that LIF can regulate EVT invasion, suggesting an important role in early placental development.

Blocking LIF action in the uterus by using a PEGylated antagonist prevents implantation: A nonhormonal contraceptive strategy

Blastocyst implantation is a critical stage in the establishment of pregnancy. Leukemia inhibitory factor (LIF) is essential for mouse blastocyst implantation and also plays a role in human pregnancy. We examined the effect of a potent LIF antagonist (LA) on mouse implantation. In mice, LIF expression peaks on day 3.5 of pregnancy (D3.5) (D0.5 = day of mating plug detection) in the uterine glandular epithelium. LA (7 mg/kg per day) administered from D2.5 to D4.5 via four hourly i.p. injections plus continuous administration via miniosmotic pump resulted in complete implantation failure. To improve its pharmacokinetic properties, we conjugated LA to polyethylene glycol (PEG), achieving a significant increase in serum levels. PEGylated LA (PEGLA) (37.5 mg/kg per day) administered via three i.p. injections between D2.5 and D3.5 also resulted in complete implantation failure. PEGLA immunolocalized to the uterine luminal epithelium at the time of blastocyst implantation. Both LA and PEGLA reduced phosphorylation of the downstream signaling molecule STAT3 in luminal epithelial cells on D3.5. The effects of PEGLA were found to be endometrial, with no embryo-lethal effects observed. These data demonstrate that administration of a PEGylated LIF antagonist is an effective method of targeting LIF signaling in the endometrium and a promising novel approach in the development of nonhormonal contraceptives for women.

Human chorionic gonadotrophin regulates FGF2 and other cytokines produced by human endometrial epithelial cells, providing a mechanism for enhancing endometrial receptivity

BACKGROUND Preimplantation cross-talk between a functional blastocyst and the endometrium is critical for successful blastocyst implantation. This interaction is mediated in part by endometrial cytokines/growth factors secreted by glandular epithelium into the uterine cavity. Recent evidence suggests that blastocyst-derived hCG may influence the endometrial milieu in conception cycles thereby enhancing receptivity and implantation success. This study investigated the effect of hCG on the secretory profile of a select cohort of 44 cytokines/growth factors from primary human endometrial epithelial cells (hEECs). These factors included those with both known and unknown roles during receptivity and implantation. The expression of one previously unknown hCG-regulated factor, fibroblast growth factor 2 (FGF2), in human endometrium and its effects on hEEC function were further examined. METHODS hEECs isolated from endometrial biopsies collected from fertile cycling women (n = 15) were treated ± recombinant hCG (0.2-20 IU/ml) for 48 h and conditioned media was quantitatively analysed using Luminex™ multiplex technology. FGF2 was further investigated by immunohistochemistry, western blot and cell-adhesion assays. RESULTS Of 44 cytokines/growth factors examined, 39 were produced by hEECs with a distinct profile. hCG (2 IU/ml) significantly increased the production of six factors, including those with known roles in receptivity and trophoblast function (interleukin-11), blastocyst migration and adhesion (CXCL10), blastocyst development (granulocyte macrophage colony-stimulating factor) and one unknown with respect to receptivity and implantation (FGF2). Up-regulation of known hCG-regulated proteins, vascular endothelial growth factor and leukaemia inhibitory factor, validated this study. Immunoreactive epithelial FGF2 increased across the menstrual cycle, being highest in secretory and first trimester pregnancy endometrium in vivo. FGF2 (100 ng/ml) stimulated phosphorylation of ERK1/2 in hEEC with no effect on ERK1/2 abundance and stimulated hEEC adhesion to fibronectin and collagen IV (components of blastocyst/trophectoderm extracellular matrix). CONCLUSIONS These findings clearly support roles for hCG and FGF2 in the blastocyst-endometrial cross-talk important for endometrial receptivity and blastocyst implantation.

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.

Immunolocalisation of phosphorylated STAT3, interleukin 11 and leukaemia inhibitory factor in endometrium of women with unexplained infertility during the implantation window

BackgroundUterine receptivity and embryo implantation are critical in the establishment of pregnancy. The diagnosis of endometrial fertility requires more precise measurements of endometrial receptivity. Interleukin (IL-11) and leukemia inhibitory factor (LIF) are essential for murine implantation and signal via intracellular phosphorylation (p) of STAT3 in the endometrium. Both cytokines are present in the endometrium of women duiring the receptive window. Endometrial IL-11, IL-11 receptor alpha (IL-11Ralpha), LIF and pSTAT3 in women with primary unexplained infertility was compared to normal fertile women during the implantation window.MethodsLH timed endometrial biopsies (LH+6 to LH+10) were collected from women with unexplained infertility and normal fertility. pSTAT3, IL-11, IL-11Ralpha and LIF production was determined by immunohistochemistry. Staining intensity was determoned by two independent observers blind to the fertility status of the patient from whom the biopsy was taken. Staining intensity and heterogeneity in each of the endometrial compartments (epithelium; stroma, including decidualized stromal cells; and vasculature) was assessed. The Mann-Whitney U test was used to analyze IL-11, pSTAT3, IL-11Ralpha and LIF immunostaining intensities in the samples.ResultsIL-11, IL-11Ralpha and LIF were present predominantly in glandular epithelium, whilst luminal epithelium showed patchy staining. pSTAT3 was present in both glandular epithelium and stroma. IL-11 and pSTAT3 immunostaining was significantly lower in glandular epithelium in infertile women compared to controls (P < 0.05) whilst IL-11Ralpha and LIF staining did not differ.ConclusionThis is the first demonstration of reduced endometrial pSTAT3 and IL-11 in some women with unexplained infertility. This suggests IL-11 and pSTAT3 may be involved in the secretory transformation of glandular epithelium during receptivity. Reduced IL-11 production and STAT3 phosphorylation may contribute to unexplained infertility in some women.

A distinct cohort of the TGFβ superfamily members expressed in human endometrium regulate decidualization

BACKGROUND Successful blastocyst implantation requires the differentiation of human endometrial stromal cells (HESC), a process known as decidualization. Activin A, a transforming growth factor β (TGFβ) superfamily member, enhances HESC decidualization and localizes to decidual cells in human endometrium. Other TGFβ superfamily members, including BMP2, BMP4, BMP7, GDF5, GDF8, GDF11, TGFβs and Nodal, may also play a role during decidualization. This study aimed to identify these TGFβ family members in human endometrium, and to determine whether they are involved in human decidualization. METHODS Protein localization of TGFβ family members was examined in secretory phase human endometrium and first trimester decidua by immunohistochemistry. mRNA expression was examined in HESC. Activin inhibitors (Activin-M108A/SB431542) with differing specificities for the other TGFβ members under consideration were applied during HESC decidualization in vitro. The secretion levels of potential TGFβ superfamily members were measured during decidualization, and recombinant proteins added to examine their effect. RESULTS This study has identified BMP2, BMP4, BMP7, GDF5, GDF8 and GDF11 but not Nodal in secretory phase human endometrium, but only BMP2, GDF5 and TGFβ1 protein were detected in decidual cells. All ligands except Nodal were expressed by cultured HESC. Both inhibitors significantly reduced decidualization validating the role of activin, but potentially also other TGFβ members, during decidualization. BMP2 and TGFβ1 secretion increased during HESC decidualisation and exogenous administration of these proteins significantly enhanced decidualization in vitro. CONCLUSIONS Like activin, BMP2 and TGFβ1 are likely to be involved in HESC decidualization. This is the first study to identify and localize BMP4, BMP7, GDF5, GDF8 and GDF11 in secretory phase human endometrium. Understanding the factors critical for the implantation process is needed for improving fertility and pregnancy outcomes.