Perfusion culture enhanced human endometrial stromal cell growth in alginate-multivalent integrin α5ß1 ligand scaffolds.

A method to functionalize alginate by introducing monomeric or self-assembling (tetrameric) fibronectin (FN) domains is described, leading to a functional scaffold, which is used for three dimensional (3D) culture of human endometrial stromal cells (EnSCs). EnSCs encapsulated in the functional alginate were cultured under perfusion using the TissueFlex® platform, a multiple parallel microbioreactor system for 3D cell culture. The effect of the novel scaffold and the effect of perfusion were examined. Cell viability, proliferation, and extracellular matrix (ECM) deposition were determined and the results compared with those obtained with cells encapsulated in non-functionalized alginate, and also those without perfusion. Staining for focal adhesions and actin showed maximal cell adhesion only for alginate-tetrameric FN scaffolds under perfusion, associated with a significant increase in cell number over 7 days culture; in contrast to poor cell adhesion and a decrease in cell number for non-functionalized alginate scaffolds (irrespective of perfused/static culture) and 3D static culture (irrespective of the scaffold). Conjugation of alginate to FN was an absolute requirement to attenuate the loss of cell metabolic activity over 7 days culture. ECM deposition for blank alginate and alginate-monomeric FN was similar, but increased around 2-fold and 3-fold for alginate-tetrameric FN under static and perfusion culture, respectively. It is concluded that the requirement for EnSC engagement with multivalent integrin α5ß1 ligands and perfused culture are both essential as a first step toward endometrial tissue engineering.

Clustered integrin α5ß1 ligand displays model fibronectin-mediated adhesion of human endometrial stromal cells.

Progress towards endometrial tissue engineering for modelling endometrial diseases and infertility is frustrated by the inability to mimic the fibronectin (FN) extracellular matrix required by human endometrial stromal cells (EnSCs). Here we show that this is because of the requirement to present integrin α5ß1 (the FN receptor) ligands in specifically oriented, polyvalent displays; by engineering controlled self-assembly of the 9th-10th type III FN domain pair (FIII9-10, the minimal integrin α5ß1 ligand) immobilised in a specific orientation to cell culture surfaces. The fraction of adherent EnSCs seen to spread increased significantly for the multimeric ligand surfaces in the order: tetramer>trimer>dimer>monomer. The extent of EnSC spread morphology also increased in the same order, with the tetrameric ligand supporting a morphology most similar to that supported by FN. Our data suggest that only higher-order multimers of FIII9-10 will fully promote cell spreading mediated through integrin α5ß1 binding.

Heparin binding domain in vitronectin is required for oligomerization and thus enhances integrin mediated cell adhesion and spreading.

Vitronectin is a multi-functional protein found predominantly as a monomer in blood and as an oligomer in the extracellular matrix. We have dissected the minimal regions of vitronectin protein needed for effective integrin dependent cell adhesion and spreading. A fragment of vitronectin containing the RGD integrin binding site showed similar binding affinity as that of full vitronectin protein to purified integrin alphavbeta3 but had diminished cell adhesion and spreading function in vivo. We demonstrate that the oligomeric state of the protein is responsible for this effect. We provide compelling evidence for the involvement of the heparin binding domain of vitronectin in the oligomerization process and show that such oligomerization reinforces the activity of vitronectin in cell adhesion and spreading.

Natural selection of human embryos: decidualizing endometrial stromal cells serve as sensors of embryo quality upon implantation.

BACKGROUND: Pregnancy is widely viewed as dependent upon an intimate dialogue, mediated by locally secreted factors between a developmentally competent embryo and a receptive endometrium. Reproductive success in humans is however limited, largely because of the high prevalence of chromosomally abnormal preimplantation embryos. Moreover, the transient period of endometrial receptivity in humans uniquely coincides with differentiation of endometrial stromal cells (ESCs) into highly specialized decidual cells, which in the absence of pregnancy invariably triggers menstruation. The role of cyclic decidualization of the endometrium in the implantation process and the nature of the decidual cytokines and growth factors that mediate the crosstalk with the embryo are unknown. METHODOLOGY/PRINCIPAL FINDINGS: We employed a human co-culture model, consisting of decidualizing ESCs and single hatched blastocysts, to identify the soluble factors involved in implantation. Over the 3-day co-culture period, approximately 75% of embryos arrested whereas the remainder showed normal development. The levels of 14 implantation factors secreted by the stromal cells were determined by multiplex immunoassay. Surprisingly, the presence of a developing embryo had no significant effect on decidual secretions, apart from a modest reduction in IL-5 levels. In contrast, arresting embryos triggered a strong response, characterized by selective inhibition of IL-1beta, -6, -10, -17, -18, eotaxin, and HB-EGF secretion. Co-cultures were repeated with undifferentiated ESCs but none of the secreted cytokines were affected by the presence of a developing or arresting embryo. CONCLUSIONS: Human ESCs become biosensors of embryo quality upon differentiation into decidual cells. In view of the high incidence of gross chromosomal errors in human preimplantation embryos, cyclic decidualization followed by menstrual shedding may represent a mechanism of natural embryo selection that limits maternal investment in developmentally impaired pregnancies.

Natural selection of human embryos: impaired decidualization of endometrium disables embryo-maternal interactions and causes recurrent pregnancy loss.

BACKGROUND: Recurrent pregnancy loss (RPL), defined as 3 or more consecutive miscarriages, is widely attributed either to repeated chromosomal instability in the conceptus or to uterine factors that are poorly defined. We tested the hypothesis that abnormal cyclic differentiation of endometrial stromal cells (ESCs) into specialized decidual cells predisposes to RPL, based on the observation that this process may not only be indispensable for placenta formation in pregnancy but also for embryo recognition and selection at time of implantation. METHODOLOGY/PRINCIPAL FINDINGS: Analysis of mid-secretory endometrial biopsies demonstrated that RPL is associated with decreased expression of the decidual marker prolactin (PRL) but increased levels of prokineticin-1 (PROK1), a cytokine that promotes implantation. These in vivo findings were entirely recapitulated when ESCs were purified from patients with and without a history of RPL and decidualized in culture. In addition to attenuated PRL production and prolonged and enhanced PROK1 expression, RPL was further associated with a complete dysregulation of both markers upon treatment of ESC cultures with human chorionic gonadotropin, a glycoprotein hormone abundantly expressed by the implanting embryo. We postulated that impaired embryo recognition and selection would clinically be associated with increased fecundity, defined by short time-to-pregnancy (TTP) intervals. Woman-based analysis of the mean and mode TTP in a cohort of 560 RPL patients showed that 40% can be considered "superfertile", defined by a mean TTP of 3 months or less. CONCLUSIONS: Impaired cyclic decidualization of the endometrium facilitates implantation yet predisposes to subsequent pregnancy failure by disabling natural embryo selection and by disrupting the maternal responses to embryonic signals. These findings suggest a novel pathological pathway that unifies maternal and embryonic causes of RPL.

Human endometrial stromal cell rho GTPases have opposing roles in regulating focal adhesion turnover and embryo invasion in vitro.

Implantation of the embryo into the uterine compartment is a multistep event involving attachment of the embryo to the endometrial epithelia, followed by invasion of the embryo through the endometrial stroma. RHOA, RAC1, and CDC42 are members of the Rho GTPase family of proteins, which control cell functions such as cell migration and cytoskeletal reorganization. Herein, using a heterologous in vitro coculture model, we show that implantation of mouse blastocysts into human endometrial stromal cells (hESCs) is regulated by Rho GTPase activity in hESCs. Whereas iRNA-mediated silencing of RAC1 expression in hESCs led to inhibition of embryo implantation, silencing of either RHOA or CDC42 in hESCs promoted embryo implantation in coculture assays. Analysis of downstream signaling pathways demonstrated that RAC1 silencing was associated with decreased focal adhesion disassembly and resulted in large focal adhesion complexes in hESCs. In contrast, RHOA or CDC42 silencing resulted in perturbed focal adhesion assembly, leading to a decrease in the number of focal adhesions observed. Furthermore, inhibition of Rho signaling using a Rho kinase inhibitor, Y27632, led to decreased activation of protein tyrosine kinase 2 (PTK2, also called focal adhesion kinase) and decreased focal adhesion assembly. Importantly, perturbation of focal adhesion turnover in hESCs, mediated by PTK2 silencing, resulted in inhibition of embryo implantation into hESC monolayers. These findings suggest that Rho GTPase-PTK2-dependent remodeling of the endometrial stromal cell compartment may be critical for successful embryo implantation.

Derivation and characterisation of the human embryonic stem cell line, OxF1.

OxF1 is a human embryonic stem cell line derived from a surplus embryo donated through the Oxford IVF clinic. The cells have a stable 46 XX karyotype and show expression of Oct 4, Nanog and TRA-1-60. Embryoid bodies differentiate into cells that represent all three germ layers as demonstrated by immunohistochemical localisation of beta III tubulin, nestin, desmin, smooth muscle actin, Gata 6 and cytokeratin 18. Directed differentiation through haematopoiesis has been demonstrated.

Effects of gonadotrophin releasing hormone analogues on human endometrial stromal cells and embryo invasion in vitro.

BACKGROUND: Gonadotrophin releasing hormone (GnRH) analogues are widely used in IVF programmes as a method of suppressing the luteinizing hormone (LH) surge prior to ovarian stimulation, but their roles outside the pituitary remain relatively unknown. A 2002 Cochrane review (Al-Inany et al. Gonadotrophin-releasing hormone antagonists for assisted conception. Cochrane Database Syst Rev 2006;3:CD001750) described lower pregnancy rates in women administered with GnRH antagonist, compared with those using an agonist, as part of an IVF programme, despite the fact that GnRH antagonist is a more effective repressor of LH. This study aimed to analyse the in-vitro effects of GnRH analogues on the decidualizing endometrium, blastocyst invasion and GnRH receptor expression in fertile women. METHODS: We analysed the in-vitro decidualization capacity of endometrial stromal cells, derived from fertile women during the implantation window, in the presence of GnRH analogues. The influence of GnRH analogues on GnRH receptor expression and blastocyst invasion was assessed by in-vitro assays of biomedical marker secretion, immunoblots and blastocyst attachment to the stromal extracellular matrix. RESULTS: We demonstrate that, at the concentrations and time periods used, GnRH analogues did not significantly influence the extent of decidualization of endometrial stromal cells. In addition, no adverse effect of GnRH analogues was seen on human blastocyst invasion. CONCLUSIONS: We suggest that GnRH analogues affect neither the capacity of the endometrium to support invasion nor the invasive potential of the blastocyst in the early stages of implantation.

Implantation of the human embryo requires Rac1-dependent endometrial stromal cell migration.

Failure of the human embryo to implant into the uterine wall during the early stages of pregnancy is a major cause of infertility. Implantation involves embryo apposition and adhesion to the endometrial epithelium followed by penetration through the epithelium and invasion of the embryonic trophoblast through the endometrial stroma. Although gene-knockdown studies have highlighted several molecules that are important for implantation in the mouse, the molecular mechanisms controlling implantation in the human are unknown. Here, we demonstrate in an in vitro model for human implantation that the Rho GTPases Rac1 and RhoA in human endometrial stromal cells modulate invasion of the human embryo through the endometrial stroma. We show that knockdown of Rac1 expression in human endometrial stromal cells inhibits human embryonic trophoblast invasion into stromal cell monolayers, whereas inhibition of RhoA activity promotes embryo invasion. Furthermore, we demonstrate that Rac1 is required for human endometrial stromal cell migration and that the motility of the stromal cells increases at implantation sites. This increased motility correlates with a localized increase in Rac1 activation and a reciprocal decrease in RacGAP1 levels. These results reveal embryo-induced and localized endometrial responses that may govern implantation of the human embryo.

Endometrial cells from women with endometriosis have increased adhesion and proliferative capacity in response to extracellular matrix components: towards a mechanistic model for endometriosis progression.

BACKGROUND: Endometriosis, classified as the presence of endometrial cells in ectopic sites, is a debilitating disease causing pain and infertility in approximately 10% of women of reproductive age. It is associated with the aberrant expression of extracellular matrix (ECM) components and their receptors, integrins. METHODS: We analysed the expression of integrins in stromal cells derived from peritoneal, ovarian and deeply infiltrating endometriotic lesions and from endometrium from women with and without endometriosis in vitro, using quantitative immunocytochemistry. The adhesive and proliferative capacity of each of the cell types in response to ECM components was assessed by in vitro assays of cell attachment and DNA synthesis. RESULTS: We demonstrate that eutopic and ectopic endometrial stromal cells from women with endometriosis exhibit an aberrant integrin profile in vitro compared with stromal cells derived from healthy controls. In addition, the former display increased adhesion and proliferative capacity in response to specific ECM components. CONCLUSIONS: We propose that the increased adhesive and proliferative potential of cells from endometriotic lesions may be a key feature in the pathogenesis of endometriosis. Furthermore, the elevated responsiveness of eutopic cells from women with endometriosis may contribute to the predisposition of some women to the disease.

alphaVbeta6 is a novel receptor for human fibrillin-1. Comparative studies of molecular determinants underlying integrin-rgd affinity and specificity.

Human fibrillin-1, the major structural protein of connective tissue 10-12 nm microfibrils, contains multiple calcium binding epidermal growth factor-like domains interspersed with transforming growth factor beta-binding protein-like (TB) domains. TB4 contains a flexible RGD loop that mediates cell adhesion via alphaVbeta3 and alpha5beta1 integrins. This study identifies integrin alphaVbeta6 as a novel cellular receptor for fibrillin-1 with a K(d) of approximately 0.45 mum. Analyses of this interaction by surface plasmon resonance and immunocytochemistry reveal different module requirements for alphaVbeta6 activation compared with those of alphaVbeta3, suggesting that a covalent linkage of an N-terminal calcium binding epidermal growth factor-like domain to TB4 can modulate alphaV integrin binding specificity. Furthermore, our data suggest alpha5beta1 is a low affinity fibrillin-1 receptor (K(d) > 1 mum), thus providing a molecular explanation for the different alpha5beta1 distribution patterns seen when human keratinocytes and fibroblasts are plated on recombinant fibrillin fragments versus those derived from the physiological ligand fibronectin. Non-focal contact distribution of alpha5beta1 suggests that its engagement by fibrillin-1 may elicit a lesser degree and/or different type of intracellular signaling compared with that seen with a high affinity ligand.

Stromal cells from endometriotic lesions and endometrium from women with endometriosis have reduced decidualization capacity.

OBJECTIVE: To evaluate the phenotype, proliferative, and differentiation capacities in vitro of stromal cells derived from peritoneal, ovarian, and deeply infiltrating endometriosis. DESIGN: Experimental study using phase contrast microscopy, immunocytochemistry, and functional bioassays. SETTING: University-based laboratory. PATIENT(S): Women with and without endometriosis undergoing surgery for benign indications. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The stability in vitro of stromal cells derived from peritoneal (n = 18), ovarian (n = 29), and deeply infiltrating (n = 14) endometriotic lesions, as well as endometrium from women with (n = 5) and without endometriosis (n = 5) was evaluated by detection of endometrial markers. The proliferative and differentiation capacity of the cells was assessed by the use of cell doubling estimation and in vitro decidualization assays. RESULT(S): The expression of the progesterone receptor and CD10 in stromal cells derived from the three types of endometriotic lesions is retained in culture up to passage 10. The doubling time of stromal cells from deeply infiltrating lesions is lower than that of endometrial stromal cells. Levels of prolactin and insulin-like growth factor binding protein-1 (IGFBP-1) are reduced in supernatants from stromal cells derived from the three types of lesions and from the endometrium of women with endometriosis. CONCLUSION(S): The peritoneal, ovarian, and deeply infiltrating endometriotic stromal cell lines we describe retain in vivo tissue markers. Loss of differentiation capacity of the endometriotic cell lines and endometrial cells from women with endometriosis may influence the capacity for proliferation and survival of these cells in the ectopic environment.

Heparin-binding epidermal growth factor and its receptors mediate decidualization and potentiate survival of human endometrial stromal cells.

Heparin-binding epidermal growth factor (HB-EGF) has pleiotropic biological functions in many tissues, including those of the female reproductive tract. It facilitates embryo development and mediates implantation and is thought to have a function in endometrial receptivity and maturation. The mature HB-EGF molecule manifests its activity as either a soluble factor (sol-HB-EGF) or a transmembrane precursor (tm-HB-EGF) and can bind two receptors, EGFR and ErbB4/HER4. In this study, we identify factors that modulate expression of HB-EGF, EGFR, and ErbB4 in endometrial stromal cells in vitro. We demonstrate that levels of sol- and tm-HB-EGF, EGFR, and ErbB4 are increased by cAMP, a potent inducer of decidualization of the endometrial stroma. We also show that production of sol- and tm-HB-EGF is differentially modulated by TNF alpha and TGF beta. Our data suggest that HB-EGF has a function in endometrial maturation in mediating decidualization and attenuating TNF alpha- and TGF beta-induced apoptosis of endometrial stromal cells.

The production of interleukin-11 and decidualization are compromised in endometrial stromal cells derived from patients with infertility.

IL-11 signaling is critical for decidualization of the endometrial stroma in early pregnancy in the mouse. In this study, we investigate the function of IL-11 signaling in cAMP-induced decidualization of human endometrial stromal cells. We show that treatment of endometrial stromal cells with 8-bromo-cAMP (8-Br-cAMP) results in an increase in the levels of secreted IL-11, whereas levels of cell surface IL-11 receptor alpha are similar with or without 8-Br-cAMP treatment. The production of IL-11 correlates with the production of molecular markers of decidualization, prolactin and IGF-binding protein-1. The expression of these markers is inhibited when IL-11 signaling is specifically blocked in decidualizing endometrial stromal cells by the IL-11 antagonist W147A. We demonstrate that 8-Br-cAMP-induced endometrial stromal cells derived from patients with primary infertility produce lower levels of prolactin, IGF-binding protein-1, and IL-11 than cells derived from fertile women. Our results suggest that IL-11 expression is critically important during decidualization in the human endometrium, and that aberrant regulation of endometrial IL-11 production may be associated with some types of infertility.

Interdomain tilt angle determines integrin-dependent function of the ninth and tenth FIII domains of human fibronectin.

Integrins are an important family of signaling receptors that mediate diverse cellular processes. The binding of the abundant extracellular matrix ligand fibronectin to integrins alpha(5)beta(1) and alpha(v)beta(3) is known to depend upon the Arg-Gly-Asp (RGD) motif on the tenth fibronectin FIII domain. The adjacent ninth FIII domain provides a synergistic effect on RGD-mediated integrin alpha(5)beta(1) binding and downstream function. The precise molecular basis of this synergy remains elusive. Here we have dissected further the function of FIII9 in integrin binding by analyzing the biological activity of the FIII9-10 interdomain interface variants and by determining their structural and dynamic properties in solution. We demonstrate that the contribution of FIII9 to both alpha(5)beta(1) and alpha(v)beta(3) binding and downstream function critically depends upon the interdomain tilt between the FIII9 and FIII10 domains. Our data suggest that modulation of integrin binding by FIII9 may arise in part from its steric properties that determine accessibility of the RGD motif. These findings have wider implications for mechanisms of integrin-ligand binding in the physiological context.

Structure of the integrin binding fragment from fibrillin-1 gives new insights into microfibril organization.

Human fibrillin-1, the major structural protein of extracellular matrix (ECM) 10-12 nm microfibrils, is dominated by 43 calcium binding epidermal growth factor-like (cbEGF) and 7 transforming growth factor beta binding protein-like (TB) domains. Crystal structures reveal the integrin binding cbEGF22-TB4-cbEGF23 fragment of human fibrillin-1 to be a Ca(2+)-rigidified tetragonal pyramid. We suggest that other cbEGF-TB pairs within the fibrillins may adopt a similar orientation to cbEGF22-TB4. In addition, we have located a flexible RGD integrin binding loop within TB4. Modeling, cell attachment and spreading assays, immunocytochemistry, and surface plasmon resonance indicate that cbEGF22 bound to TB4 is a requirement for integrin activation and provide insight into the molecular basis of the fibrillin-1 interaction with alphaVbeta3. In light of our data, we propose a novel model for the assembly of the fibrillin microfibril and a mechanism to explain its extensibility.

Functional characterization of W147A: a high-affinity interleukin-11 antagonist.

IL-11 is a member of the gp130 family of cytokines, which signal via assembly of multisubunit receptor complexes containing at least one molecule of the transmembrane signaling receptor gp130. IL-11 forms a high-affinity complex, thereby inducing gp130-dependent signaling. Previous studies have identified three distinct receptor binding sites, I, II, and III, crucial for the binding of murine IL-11 (mIL-11) to both the IL-11R and gp130. In this study, we have further characterized the role of the mIL-11 site III mutant W147A. We show that W147A is a high-affinity specific antagonist of mIL-11-mediated signaling in gp130/IL-11R-transfected Ba/F3 cells. The antagonistic action of W147A is due to its ability to competitively disrupt multimeric gp130/IL-11R signaling complex formation. We also show that W147A inhibits IL-11-mediated signaling in primary human endometrial cells, thus demonstrating the potential utility of W147A in suppressing IL-11 responses in vivo.

Expression and function of interleukin-11 and its receptor alpha in the human endometrium.

The interleukin-11 (IL-11) receptor alpha has an important function in decidualization of mouse endometrial stroma but the function of IL-11 and its receptor in the human endometrium remains unknown. The mRNA for IL-11 and its receptor alpha in human endometrial tissue samples were analysed by semi-quantitative RT-PCR and RNase protection assays respectively. The proteins were detected in frozen endometrial tissue samples by immunofluorescence. The effect of heparin-binding epidermal growth factor (HB-EGF) on secretion of IL-11 by cultured endometrial stromal cells was assessed by enzyme-linked immunosorbent assay. The proliferative potential of IL-11 in endometrial stromal cells was assessed by [(3)H]thymidine uptake. IL-11 and its receptor alpha mRNAs and proteins were detected in the endometrium throughout the cycle. Distinct patterns of localization of the ligand and receptor were observed. HB-EGF induced IL-11 secretion by cultured stromal cells, and IL-11 induced [(3)H]thymidine uptake by these cells. Our data suggest that IL-11-receptor interactions may perform different functions in the human endometrium at different stages of the cycle, and that secretion of IL-11 is modulated by local growth factors.

Synergistic activity of the ninth and tenth FIII domains of human fibronectin depends upon structural stability.

The ninth and tenth FIII domains (FIII9-10) of human fibronectin act in synergy to promote cell adhesion via the interaction with integrin receptors. Here we describe the functional and structural properties of a set of recombinant FIII9-10 mutants containing various alanine substitutions within the key synergistic site, DRVPHSRN in FIII9, either alone or in combination with another substitution (Leu(1408) to Pro), on the opposite face of FIII9, that increases stability and the functional capacity of FIII9-10. We show that the introduction of mutations into the synergistic sequence of FIII9-10 has a negative effect on the adhesion of baby hamster kidney fibroblasts and results in reduced ability of these ligands to recognize integrin alpha(5)beta(1). Conformational stability of the FIII9 domain in the synergy site mutants is likewise reduced in comparison with native FIII9. The Leu(1408) to Pro substitution in mutant FIII9-10 proteins carrying substitutions in the synergy site results in a substantial recovery of the adhesive activity of the mutants and affinity to alpha(5)beta(1). In keeping with the enhancement of functional activity, the Leu(1408) to Pro substitution in the FIII9-10 synergy site mutants also causes a significant increase in conformational stability of FIII9. These observations imply a strong positive correlation between the biological activity and conformational stability of the assessed FIII9-10 mutants and suggest that a Leu(1408) to Pro substitution restores the biological activity of the mutants via their ability to restore their conformational stability. We conclude that domain stability may be a major determinant of the synergistic potential of FIII9. Our data underscore the value of using more than one approach in such structure-function studies and the requirement for validating the global structural integrity of protein ligands in which sequences that disrupt function have been perturbed.

The mitogenic potential of heparin-binding epidermal growth factor in the human endometrium is mediated by the epidermal growth factor receptor and is modulated by tumor necrosis factor-alpha.

Heparin-binding epidermal growth factor (HB-EGF), a member of the epidermal growth factor (EGF) family, is implicated in a variety of biological processes, including reproduction. Previous studies describe increased levels of HB-EGF in the human endometrium during the midsecretory stage of the menstrual cycle, suggesting a function for HB-EGF in implantation of the human blastocyst. Here we have investigated the expression and function of the soluble and transmembrane forms of HB-EGF in the human endometrium. We show that the expression of the transmembrane form of HB-EGF in the human endometrium is modulated according to the stage of the menstrual cycle. We present data demonstrating that both the soluble and transmembrane forms of HB-EGF induce DNA synthesis in human endometrial stromal cells. Furthermore, TNFalpha has a cooperative effect on HB-EGF, EGF, TGFalpha, and betacellulin-induced DNA synthesis in stromal cells, suggesting roles for the EGF family and TNFalpha in regeneration and maturation of human endometrium. Induction of DNA synthesis by HB-EGF and its modulation by TNFalpha in endometrial stromal cells are mediated by the EGF receptor and not the HB-EGF receptor ErbB4. Our data suggest key functions for HB-EGF, TNFalpha, and the EGF receptor in endometrial maturation, via autocrine/paracrine and juxtacrine pathways, in preparation for embryo implantation.