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BACKGROUND: Implantation failure is likely to be a major cause of infertility. Studies in mice have identified a number of molecules that are involved in implantation, but the mechanisms of implantation in the human remain unclear, largely due to the lack of models for implantation in the human that provide functional information. METHODS: Human hatched blastocysts were co-cultured with human endometrial stromal cell monolayers. Time-lapse photography of implanting blastocysts, immunostaining for cytokeratin and actin, and measurement of hCG secreted into the culture supernatants were performed. RESULTS: Blastocysts attached to and implanted into the stromal cell layer. Trophoblast outgrowth onto, and invasion into, the stromal cell layer occurred largely at two opposite poles, the orientation of which was aligned to that of the stromal cell fibroblasts. High-resolution image analysis demonstrated that the trophoblast completely penetrated the stromal cell layer. Immunostaining of whole-mounts of implantation sites revealed distinctive actin and cytokeratin-positive anchoring structures adjacent to the basal surface of the trophoblast. Blastocysts implanting into stromal cells secreted higher levels of hCG compared with those cultured on plastic. CONCLUSIONS: A robust model for the study of mechanisms of implantation of the human embryo into the endometrial stroma has been established.

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.

Binding of the extracellular matrix molecule fibronectin to the integrin receptor alpha(5)beta(1) elicits downstream signaling pathways that modulate cell function. Fibronectin-alpha(5)beta(1) interaction occurs via the conserved RGD sequence in the tenth FIII (FIII10) domain of fibronectin. A synergistic site containing the sequence PHSRN in the adjacent FIII9 domain has also been identified. Here we investigate the function of the eighth FIII domain in integrin-mediated cell adhesion using a wide range of methods, including biochemical, biological, and biophysical assays of integrin binding, cell adhesion, and protein denaturation. Mutation of the FIII9 synergistic site (PHSRN to PHAAA) in FIII9-10 reduced the binding activity for integrin alpha(5)beta(1) to levels observed for FIII10 alone, but the corresponding mutant in FIII8-9-10 showed no loss of binding activity. Cell adhesion assays also demonstrated enhanced functional activity of constructs containing FIII8. Equilibrium chemical denaturation studies indicated that FIII8 confers conformational stability upon FIII9, but only if the exposed loops, PHSRN and VKNEED on FIII9 and FIII8, respectively, are intact. These results demonstrate that the loss of integrin binding activity, observed upon alteration of the PHSRN synergistic site of FIII9-10, results partly from a loss of conformational stability of FIII9. Our data suggest a mechanism for integrin alpha(5)beta(1)-fibronectin interaction, which in addition to the primary RGD binding event, involves a conformation-sensitive scanning by the integrin for accessible sites on the ligand, whereupon full activation of downstream signaling occurs.

Human fibrillin-1 is the major structural protein of extracellular matrix 10–12 nm microfibrils. It has a disulfide-rich modular organization which consists primarily of cbEGF (Ca2+-binding epidermal growth factor-like) domains and TB (transforming growth factor β-binding protein-like) domains. TB4 contains an RGD (Arg-Gly-Asp) integrin-binding motif. The atomic structure of this region has been solved by X-ray crystallography and shows the TB4 and flanking cbEGF domains to be arranged as a tetragonal pyramid with N- and C-termini exposed at opposite ends of the fragment. The RGD integrin-binding motif is located within a flexible loop. We have used a variety of biophysical, biochemical and cell biology methods to investigate the molecular properties of integrin–fibrillin-1 interactions and have demonstrated that recombinant fibrillin-1 domain fragments mediate binding to integrins αVβ3, α5β1 and αVβ6. Integrin αVβ3 is a high-affinity fibrillin-1 receptor ( K d ∼40 nM), whereas integrins αVβ6 and α5β1 show moderate-affinity ( K d ∼450 nM) and low-affinity ( K d >1 μM) binding respectively. Different patterns of α5β1 distribution are seen when human keratinocytes and fibroblasts are plated on to fibrillin domain fragments compared with those seen for fibronectin, suggesting that fibrillin may cause a lesser degree or different type of intracellular signalling. A number of disease-causing mutations which affect the TB4 domain have been identified. These are being investigated for their effects on integrin binding and/or changes in intramolecular structure. Abbreviations: BHK, baby-hamster kidney; cbEGF, Ca2+-binding epidermal growth factor-like; EGF, epidermal growth factor; EM, electron microscopy; LTBP, latent transforming growth factor β-binding protein; SPR, surface plasmon resonance; TB, transforming growth factor β-binding protein-like

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.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.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.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.

BACKGROUND: Endometriosis, classified as the presence of endometrial cells in ectopic sites, is a debilitating disease causing pain and infertility in 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.

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.

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.

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.

The mechanisms that mediate implantation of the human embryo remain poorly understood and represent a fundamental problem in reproductive biology. Candidate molecules that mediate and facilitate implantation have been identified in animal studies, and include heparin binding epidermal growth factor. Here we demonstrate a potential function for the transmembrane form of heparin-binding epidermal growth factor in mediating blastocyst attachment to the endometrium, in two different novel in vitro models for human implantation. Furthermore, we demonstrate specific localisation of the heparin-binding epidermal growth factor receptor ErbB4, on the surface of the trophectoderm in peri-implantation human blastocysts. Our data lead the way for further dissection of the molecular mechanisms of implantation of the human embryo, and have implications for infertility, in vitro fertilization and contraception.

The ninth and tenth type III domains (FIII9–10) in the central cell binding domain of human fibronectin contain integrin receptor binding sites, including RGD in FIII10 and a synergy site, PHSRN, in FIII9. The specific amino acids that contribute to cell binding have been identified by the use of wild-type and mutant fragments of human fibronectin containing the FIII9–10 domain pair. At high concentrations FIII9–10 mimics, to a large extent, the biological activity of the full-length fibronectin molecule. However, FIII9 is conformationally unstable, even in the context of the FIII9–10 pair. Here we report the construction of a series of hybrid mouse–human FIII9–10 pairs that confer varying degrees of conformational stability to FIII9. The conformational stability of the human FIII9 module was increased 2–3-fold by substitution of Leu1408 with Pro. We demonstrate that the biological activity of this mutant is enhanced. The resulting FIII9–10 mutant has good solution properties and will provide a template into which further mutations can be incorporated in order to probe the structure–function relationship of the cell binding module of fibronectin.

Integrins are heterodimeric glycoproteins that have been found to undergo dynamic temporal and spatial changes in the endometrium during the menstrual cycle and in early pregnancy. Specificity of integrins is known to be different in human endometrial stromal cells and decidual cells. These shifts of integrins suggested to play an important role in embryo implantation and can be modulated by progesterone, cAMP derivatives, and cytokines. The mechanisms of decidualization and its precise physiological role are still not clearly understood and in vitro systems could provide an alternative that overcomes limitations of studying such complex biological phenomena in vivo at the time of implantation. This study was undertaken to establish an in vitro model system for human decidualization using 8-bromo-cAMP and to investigate the characteristics of stromal integrin expression in vitro by 8-Br-cAMP. Endometrial stromal cells were isolated and cultured, and then were induced to decidualize by 0.5 mM 8-Br-cAMP for 15 days. Immunofluorescence staining and flow cytometric analyses of the integrin subunits (alpha1, alpha4, alpha5, alpha6, beta1 and alphavbeta3) were performed at day 9. In the presence of 8-Br-cAMP, the staining intensity of alphavbeta3 was significantly higher than control and measurements for alpha1, alpha4, alpha5, alpha6, and beta1 were similar. Immunofluorescent localization of the integrins reflected the differences obtained from the flow cytometric analyses described above. In summary, the expression of alphavbeta3 integrin increased in stromal cells in vitro decidualized by 8-Br-cAMP and this up-regulation of alphavbeta3 integrin expression during decidualization might influence on human implantation.

The N-acetylation of arylamines and hydrazines used as drugs may alter their pharmacological or toxicological activity. Arylamine N-acetyltransferases (NATs) are involved in drug metabolism, as they catalyse the N-acetylation of arylamine and mono-substituted hydrazine substrates. Placental metabolism regulates the nature of the chemicals which reach the developing fetus. The study of drug metabolism during pregnancy is important in determining the effect on the fetus of drugs administered to the mother and the maternal drug dose required, important if the treatment is to be effective. There are two forms of NAT in humans, NAT1 and NAT2, which are encoded at multi-allelic loci. There is inter-individual variation in both NAT1 and NAT2 activity, which has implications in drug dosage. Using a combination of enzyme activity measurements and Western blotting, this study has characterised the arylamine N-acetylation capabilities of placenta and cord blood. NAT1 activity in placenta and cord blood demonstrated inter-individual variation and the variation was in the range expected for adult NAT1 activity. The genotypes of bothNAT1 * andNAT2 * were determined using DNA prepared using placental blood clots (maternal DNA) and cord blood (fetal DNA). The results indicate that placental NAT activity is an important factor when consideringN-acetylation during pregnancy.

The ninth and tenth type III domains of fibronectin each contain specific cell binding sequences, RGD in FIII10 and PHSRN in FIII9, that act synergistically in mediating cell adhesion. We investigated the relationship between domain-domain orientation and synergistic adhesive activity of the FIII9 and FIII10 pair of domains. The interdomain interaction of the FIII9-10 pair was perturbed by introduction of short flexible linkers between the FIII9 and FIII10 domains. Incremental extensions of the interdomain link between FIII9 and FIII10 reduced the initial cell attachment, but had a much more pronounced effect on the downstream cell adhesion events of spreading and phosphorylation of focal adhesion kinase. The extent of disruption of cell adhesion depended upon the length of the interdomain linker. Nuclear magnetic resonance spectroscopy of the wild type and mutant FIII9-10 proteins demonstrated that the structure of the RGD-containing loop is unaffected by domain-domain interactions. We conclude that integrin-mediated cell adhesion to the central cell binding domain of fibronectin depends not only upon specific interaction sites, but also on the relative orientation of these sites. These data have implications for the molecular mechanisms by which integrin-ligand interactions are achieved.

The structure of mosaic proteins depends on the nature and strength of interactions between individual modules. Here we investigated the structural significance of module-module interactions in the RGD-depen dent cell binding region of human fibronectin, comprising the ninth and tenth fibronectin type III. A combination of protein engineering, thermodynamics and nuclear magnetic resonance methods was employed to establish a relationship between intermodular protein - protein interactions and the structural properties of the module pair. A poly(glycine) peptide link connecting the C terminus of the ninth and the N terminus of the tenth module was introduced to probe the range of the interaction. NMR studies (Chemical shifts and 15N relaxation) together with equilibrium and kinetic unfolding experiments were carried out on five different single and double module constructs. The results show that non-specific protein - protein interactions provide the bulk of the thermodynamic stabilization and the motional constraint of the two modules. Specific interactions between the two modules are restricted to the wild-type module pair and decline very rapidly with the insertion of additional linker residues. This low level of specificity is nonetheless sufficient to fine-tune the precise module-module orientation and to provide the full biological activity of the wild-type pair. This suggests that individual modules in mosaic proteins can achieve a high degree of motional constraint and mutual stabilization without the requirement for intricate and specific interactions in the module-module interfaces.

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 α5β1 (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 α5β1 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 α5β1 binding.

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 α5β1 ligands and perfused culture are both essential as a first step toward endometrial tissue engineering. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2011.

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 αvβ3 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.Structured summaryMINT-7905703: Vn (uniprotkb:P04004) and Vn (uniprotkb:P04004) bind (MI:0407) by molecular sieving (MI:0071)

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.

BackgroundPregnancy 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 FindingsWe 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-1?, -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.ConclusionsHuman 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.

BackgroundRecurrent 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 FindingsAnalysis 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.ConclusionsImpaired 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.

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.

Recently, significant advances in our understanding of embryo implantation have been made using animal models, notably in the mouse and nonhuman primates. However, the determination of the molecular and cellular events that underpin the early stages of implantation in the human remains an intractable problem, in part due to the inaccessibility of early human implantation sites. In the absence of in vivo implantation sites, several experimental in vitro model systems have been developed recently that mimic the different stages of human embryo implantation that occur in vivo during the first few weeks of pregnancy. These include solid-phase assays of blastocyst attachment and trophoblast invasion, and two- and three-dimensional blastocyst-endometrial cell cocultures. An important feature of such models is that they allow functional studies to be performed and are not restricted to generating descriptive data. These models have the potential to make an important contribution to the development of new therapeutic and diagnostic strategies for implantation failure in the future, as well as toxicity testing. We describe the strengths and weakness of the models and some of the advances that have been made by the use of these in vitro models.

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.

'To whom correspondence should be addressed We investigated the length of time between the onset of pain symptoms and the surgical diagnosis of endometriosis hi women from the UK and the USA. A total of 218 women with surgically confirmed disease, recruited through endometriosis self-help groups, completed a postal questionnaire. The mean ± SD delay hi diagnosis for women from the USA was 11.73 ± 9.05 years, significantly higher than the equivalent delay of 7.96 ± 7.92 years for women from the UK (P < 0.01). The stage of disease did not affect the length of time between the onset of symptoms and diagnosis. Therefore there is considerable delay hi the diagnosis of endometriosis for women from both the UK and the USA. Efforts to reduce this delay are required to minimize the suffering of women with this disease.

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.

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.

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.

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.

The relationship between endometriosis and polymorphisms in the N-acetyl transferase 2 (NAT 2) gene was investigated in a UK population, as this gene has been previously implicated in the aetiology of the disease. Point mutations in the gene result in the variant alleles NAT 2 *5, *6 and *7 from the wild-type NAT 2 *4 allele. Homozygotes for the NAT 2 *4 wild type allele are fast NAT acetylators, while heterozygotes with one wild-type allele and a variant NAT 2 *5, *6 or *7 allele have reduced enzyme activity, and individuals with two variant alleles are slow acetylators. The NAT 2 *4/*6 genotype was significantly more common among affected women (35.2%) than population controls (8.1%; P = 0.0001) or unaffected women (4.2%; P = 0.02). Significantly more affected women (57.4%) were fast acetylators than were population controls (32.3%; P < 0.01) or unaffected women (33.3%; P < 0.05). These data suggest that altered NAT 2 enzyme activity may be a predisposition factor in endometriosis, or that NAT 2 alleles may be in linkage disequilibrium with a susceptibility allele in the same chromosomal region.

An association between endometriosis and the glutathione S-transferase (GST) M1 null mutation has been reported in French and Slavic populations. We aimed to replicate this association of endometriosis in a UK population, and to test for association with the GSTT1 null mutation or the cytochrome P450 (CYP) 1A1 MspI polymorphism. We genotyped 148 women each with endometriosis (sporadic cases, n = 91; familial cases, n = 57), a population control of 95 male blood donors, and a control group of 53 women with a normal pelvis at hysterectomy. No significant differences were found between cases and controls in the frequencies of the GSTM1 and GSTT1 null mutations, or the CYP1A1 MspI polymorphism. However, the combination of the GSTM1 null genotype and the CYP1A1 MspI polymorphism was associated with a small increased risk of endometriosis, and this warrants further investigation. We also tested for linkage to the chromosome 1p13 region, to which GSTM1 has been mapped, in 52 sister-pairs with stage III-IV disease using three highly polymorphic microsatellite markers. However, there was no evidence of linkage, suggesting that this region may not be implicated in disease susceptibility.

Human arylamine N-acetyltransferase type 1 (NAT1), better known as a drug-metabolising enzyme, has been proposed to acetylate the folate catabolite p-aminobenzoylglutamate (p-abaglu) to N-acetamidobenzoylglutamate (ap-abaglu) which is a major urinary folate catabolite. Using mass spectroscopic analysis, we demonstrate the formation of ap-abaglu by recombinant human NAT1 and human placental homogenates. Using density gradient centrifugation the placental enzymic activity which acetylates p-aba and the placental enzymic activity acetylating p-abaglu both have an S(20,w) value of 3.25 S. This is the expected value for a monomer of human NAT1 (33 kDa). The specific NAT1 inhibitor 5-iodosalicylate inhibits acetylation of both p-aba and p-abaglu catalysed by either recombinant human NAT1 or placental samples as the source of enzyme. These data demonstrate that NAT1 is the major placental enzyme involved in acetylating p-abaglu.

An association between the N314D polymorphism of galactose-1-phosphate uridyl transferase and endometriosis has recently been reported in a North American population. To determine whether such an association exists in the UK population, we genotyped 148 women with sporadic (n = 91) or familial (n = 57) endometriosis, a control population of 95 male blood donors and a control group of 53 women with a normal pelvis at hysterectomy. Heterozygosity for the polymorphism was found in 14.9% (22/148) of affected women, 13.7% (13/95) of male blood donors and 11.3% (6/53) of women with a normal pelvis. There was no statistically significant difference in the frequency of the polymorphism between cases and controls in the UK population, even when the cases were divided into groups of moderate-severe disease, sporadic cases or familial cases. We conclude that the galactose-1-phosphate uridyl transferase N314D polymorphism is unlikely to be associated with endometriosis in the UK population.

The study of placental xenobiotic metabolism is important for the determination of foetal exposure to environmental chemicals as placental metabolism influences the nature of chemicals reaching the foetus from its mother's blood. Arylamine N-acetyltransferases are drug metabolizing enzymes which N-acetylate hydrazines and arylamines, including carcinogenic arylamines and sulphonamide drugs. The two human arylamine N-acetyltransferase isoenzymes, NAT1 and NAT2, are encoded at multi-allelic loci. Here, we have determined N-acetyltransferase (NAT) activity in term placentas from normal, uncomplicated pregnancies. Both NAT1 and NAT2 enzyme activities were detectable. Placental NAT1 activity was at least 1000 fold greater than NAT2 activity. There was a 6 fold inter-placental variation in NAT1 activity. Mean placental NAT1 specific activity was 1.42 nmoles para-aminobenzoic acid N-acetylated.min-1.mg protein-1, which is comparable to NAT1 specific activities which have been measured in adult tissues. The NAT1, but not the NAT2, protein was detectable in placentas by Western blotting. Maternal and foetal NAT genotypes were determined from placenta, using placental blood clots and cord blood respectively, allowing NAT haplotype determination. There appeared to be linkage disequilbrium between NAT1* and NAT2* alleles such that the combination NAT1*10/NAT2*4 was found 3.5 times more frequently than would be expected.

We have shown previously that expression of 46 and 54 kDa human myometrial Gαs protein isoforms is increased during gestation and then subsequently decreased during labour. These proteins appear to be coded for by Gαs-Small (with a serine residue at position 72) and Gαs-Large (with a serine residue at position 87) mRNA splice variants respectively. In the study presented here we have used a Gαs cDNA template to generate [32P]cytidine cRNA ribo-probes for use in RNase protection assays, so as to measure total myometrial Gαs mRNA levels in relation to the pattern of expression of Gαs mRNA splice variants during pregnancy and labour. We report that total levels of human myometrial Gαs mRNA remain similar in non-pregnant and pregnant women but are substantially reduced during parturition. Our data also provide strong evidence that alternative splicing of Gαs precursor mRNA has a primary role in regulating expression of Gαs protein isoforms during pregnancy and labour. The inclusion of an additional serine codon in Gαs mRNAs during pregnancy involves a switch in alternative splicing pathways. We speculate that this switch may be due to a change in specificity of splicing factors that are modulated during pregnancy and labour.

Granulosa cells are essential for follicular development and corpus luteum formation and their functions are regulated by gonadotrophins through G protein-coupled receptors. The dominant second messenger pathway involves the stimulation of cyclic AMP formation by Gαs-linked receptors. In this paper we have investigated the expression of Gαs mRNA splice variants in relation to expression of Gαs protein isoforms in granulosa cells obtained from patients undergoing in vitro fertilization. We have carried out ribonuclease protection assays using cRNA riboprobes which are capable of detecting all Gαs mRNA isoforms as well as quantifying total amounts of Gαs mRNA. Granulosa cells express the message for Gαs-Large and Gαs-Small and the presence of two distinct protein products was confirmed by immunoblotting using the antibody RM/1. Moreover, the data show that a significant fraction of Gαs-Large and Gαs-Small mRNAs contain an extra CAG codon. This should generate proteins with an extra serine residue, resulting in Gαs variants with the consensus sequence of a protein kinase C phosphorylation site. These results highlight the possible interaction between different signalling pathways in the control of cAMP production and the need to investigate the relationship between Gαs variants and different adenylyl cyclase isozymes in patients with normal and abnormal ovarian function.

The expression of integrin subunits has been investigated in the stroma and epithelium of eutopic and ectopic endometrial tissues, using immunohistochemistry and fluorescently activated cell-sorting techniques. Integrin subunits exhibited tissue-specific expression in both eutopic and ectopic endometrium. Integrin alpha 3 subunit was up-regulated in ectopic endometrium compared with the eutopic counterpart, whereas alpha 6 integrin subunit was down-regulated in the ectopic tissues. Cycle stage-dependent expression of alpha v and beta 3, observed in eutopic endometrium, was absent in the ectopic counterpart. It is concluded that the development and regeneration of the endometrium involve complex integrin-ligand interactions and that regulation of specific adhesive events is lost in endometriosis.

In previous studies using specific G alpha s antibodies we have identified several human myometrial G alpha s protein isoforms with molecular masses of 45, 46, 47, 54, and 58 kDa, respectively. During pregnancy, levels of the 46- and 54-kDa proteins are significantly increased compared to those in nonpregnant myometrium and then decreased at the onset of labor. In this study we investigated the expression of G alpha s messenger ribonucleic acid (mRNA) splice variants, which are generated as a result of alternative splicing of a single mRNA precursor, in term pregnancy and parturition to determine whether there was any correlation with the observed changes in G alpha s protein isoforms. A myometrial G alpha s complementary DNA was synthesized using RT-PCR and cloned into pCRtmII suitable for preparation of riboprobes for use in ribonuclease protection assays. Using this technique, we identified at least three myometrial G alpha s mRNAs, including two forms of G alpha s-Large (with or without the serine at amino acid 87) and one form of G alpha s-Small (with the serine at amino acid 72). G alpha s Small (with the serine) and G alpha s-Large (with the serine) mRNAs encode for the 46- and 54-kDa G alpha s protein isoforms, respectively, and were increased in term pregnancy and then subsequently decreased after the onset of labor. Our data suggest that posttranscriptional regulation of G alpha s mRNAs may be important in the differential expression of G alpha s protein isoforms during pregnancy and labor.

Analysis of the age of onset of pain symptoms in 83 sister-pairs concordant for endometriosis supports the suggestionthat endometriosis has a genetic basis.Key words: endometriosis/geneticsIntroductionThere is increasing evidence that genetic and environmentalfactors interact in the development of endometriosis, althoughthe precise aetiology of the disease remains uncertain. Agenetic susceptibility is likely as there is a familial tendency(Kennedy et al., 1995) and a 6-9 times increased prevalenceamong the first-degree relatives of affected women comparedto that of the general population (Simpson et al, 1980; Moenand Magnus, 1993). The major environmental factor that hasbeen implicated is exposure to pollutants such as dioxins andpolychlorodiphenyl compounds (PCBs). The incidence andseverity of spontaneous endometriosis in monkeys have bothbeen correlated with chronic dioxin exposure (Rier et al,1993), and some women with endometriosis have elevatedPCB concentrations in their blood (Gerhard and Runnebaum,1992). It has also been suggested that dioxin pollution couldbe an explanation for the higher prevalence of endometriosisin industrialized countries compared to the developing world(Koninckx et al, 1994).The aim of this study was to test two hypothetical modelsfor the aetiology of the disease by determining the age andyear of onset of symptoms in a cohort of non-twin sistersconcordant for endometriosis. The models were (i) commonexposure: sister-pairs would be expected to develop symptomsin the same year if they were commonly exposed to a causativeenvironmental agent with a fixed incubation time, and (ii)genetic: the age of onset of symptoms would tend to be thesame in both sisters if the disease had a purely genetic basis.We also analysed the age and year of surgical diagnosisand the interval between the onset of symptoms and theestablishment of the diagnosis.Materials and methods

Fibronectins (FN) contain sites, in addition to the cell recognition site RGD in the tenth type III domain (FIII10), that are required for adhesive activity. The role of FIII10 and the adjacent FIII9 was analysed in functional cell adhesion assays recombinant FIII domains in which the domain boundaries were strictly conserved. FIII9 had no adhesive activity. FIII10, and FIII9 plus FIII10 had less activity than FN, whereas the activity of FIII9-10 was similar to FN. We conclude that FIII9 acts synergistically with FIII10 in cell adhesion, and that this synergy is dependent upon the structural integrity of the FIII9-10 pair of domains.

Recent reports have described transformation- and tumour-specific expression of fibronectin isoforms generated by alternative splicing of the fibronectin pre-mRNA. We have investigated the expression and distribution of EDIIIA+ and EDIIIB+ fibronectin splice variants in tumorigenic and non-tumorigenic somatic cell hybrids made by fusing fibrosarcoma-derived cells (HT1080) and normal fibroblasts (GM00097). Alternative splicing of EDIIIA and EDIIIB was assessed quantitatively by S1 nuclease analyses. The levels of EDIIIA+ and EDIIIB+ fibronectin mRNAs were similar in the parental and hybrid cells. Domain-specific monoclonal antibodies were used in immunohistochemical studies to identify EDIIIA+ and EDIIIB+ fibronectins in fixed cells. GM00097 and the non-tumorigenic hybrid (clone G3) showed high levels of both EDIIIA+ and EDIIIB+ fibronectin staining. The tumorigenic hybrid (clone C1) showed reduced amounts of EDIIIA+ fibronectin, but no detectable EDIIIB+ fibronectin. No fibronectin was detected on the surface of HT1080 cells. Western blots of protein extracted from culture supernatants and extracellular matrices revealed that GM00097 and G3 cells incorporated most of the EDIIIA+ and EDIIIB+ fibronectin into the extracellular matrix whereas C1 cells released a large proportion of the EDIIIA+ fibronectin, and almost all of the EDIIIB+ fibronectin, into the supernatant. We conclude that there are differences in the presence of EDIIIA+ and EDIIIB+ FNs on the surface of tumorigenic and non-tumorigenic cells and that these differences are due to differential incorporation of FN variants into the ECM.

The cell binding sites CS1 and CS5 in the IIICS region of human fibronectin (FN) mediate the adhesion of specific cell types by interacting with the integrin alpha 4 beta 1. IIICS pre-mRNA is alternatively spliced via the use of three alternative splice acceptor sites and one alternative splice donor site. These alternative splicing pathways can potentially give rise to variant FN molecules which are CS1+,CS5+; CS1+,CS5-; CS1-,CS5+ or CS1-,CS5-. Here we show that selection of the acceptor site which incorporates mRNA encoding CS1 and CS5 is more frequent in foetal tissues compared to adult liver, whereas an alternative acceptor site and the alternative donor site, which exclude CS1 and CS5, are used at a higher level in adult liver compared to foetal tissue. All possible splice junctions were accurately processed, and selected at different levels in mRNA expressed from a IIICS minigene transiently transfected into a HeLa cell line which does not express FN, suggesting that all the cellular factors required for alternative processing of IIICS are present in this system. Furthermore, pre-mRNA expressed from a mutant construct lacking IIICS-1 intron sequence, was correctly processed in HeLa cells via selection of all possible splice sites. On the basis of our results we propose that regulation of splice site selection in IIICS and thus expression of CS1 and CS5 is achieved by subtle tuning of splicing systems involving the interaction of local cis elements and cellular factors which are not necessarily restricted developmentally or tissue-specifically, and that expression of CS1 and CS5 is independently regulated.

Objective: To describe the occurrence of endometriosis in monozygotic twins. Design: Postal questionnaire plus confirmation of disease status. Setting: Twins were recruited via the American Endometriosis Association and the National Endometriosis Society of Great Britain and via British gynecologists. Result(s): Fourteen twin pairs were concordant for endometriosis, and two were discordant. Nine pairs of twins had moderate-severe endometriosis. Conclusion(s): These findings contribute to the growing body of literature that suggests endometriosis has a genetic basis.

Exon EDIIIA of the fibronectin (Fn) gene is alternatively spliced via pathways which either skip or include the whole exon in the messenger RNA (mRNA). We have investigated the role of EDIIIA exon sequences in the human Fn gene in determining alternative splicing of this exon during transient expression of alpha globin/Fn minigene hybrids in HeLa cells. We demonstrate that a DNA sequence of 81bp within the central region of exon EDIIIA is required for alternative splicing during processing of the primary transcript to generate both EDIIIA+ and EDIIIA- mRNA's. Furthermore, alternative splicing of EDIIIA only occurs when this sequence is present in the correct orientation since when it is in antisense orientation splicing always occurs via exon-skipping generating EDIIIA- mRNA.