Search

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

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.

A tissue-specific protein fraction has been detected in rat osteogenic tissue. Dissociative extraction of adult rat bone matrix with 4 M guanidinium chloride solution was followed sequentially by gel chromatography and polyacrylamide gel electrophoresis. By the latter procedure a prominent protein component of molecular weight 19,000 was isolated from the low molecular weight fraction, and antibodies directed against this protein were raised in rabbits. The antibodies were mainly against antigenic sites on this protein, as shown by protein blotting techniques. By embedding rat tissues in hydrophilic plastic and by using immunohistochemical procedures the presence of this protein was demonstrated specifically in bone matrix in vivo, in osteogenic tissue developing in diffusion chamber culture, and in a malignant osteoblast cell line (UMR 106). Soft tissues (liver, kidney, spleen, gut, skin, thymus, eye) showed no reactivity with the antiserum and in vitro a further malignant osteoblast cell line (ROS 17/2.8) did not synthesize the 19,000 molecular weight protein. This protein appears to be expressed solely by osteogenic tissue and may be used as a biochemical criterion of osteogenic differentiation.

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.

Substrates utilising clustered arginine-glycine-aspartic acid (RGD) ligand displays support greater cell adhesion over random displays. However, cell adhesion to integrin 5β1 requires the synergy site on the 9th type III fibronectin domain (FIII) in addition to RGD on the 10th FIII domain. Here, we have designed and expressed soluble protein chimeras consisting of an N-terminal 9th-10th FIII domain pair, IgG-derived hinge and leucine zipper-derived helix; the latter mutated to yield di-, tri- and tetrameric coiled coils and thus self-assembling, multimeric integrin 5β1 ligands. A unique C-terminal cysteine was appended to the helix to facilitate 'anchoring' of the chimeras with a defined orientation on a surface. Size-exclusion chromatography and circular dichroism demonstrated that the chimeras self-assembled as multimers in solution with defined secondary structures predicted from theoretical calculations. Biotinylation via a thioether bond was used to selectively bind the chimeras to streptavidin-coated surfaces, each of which was then shown to bind integrin 5β1 by surface plasmon resonance. Spreading of fibroblasts to surfaces derivatised with the chimeras was found to proceed in the order: tetramer > trimer > dimer > monomer. Thus, we describe novel polyvalent integrin 5β1 ligands for facile derivatisation of substrates to improve cell adhesion in vitro.

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.

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.

Afa/Dr family of adhesins are produced by pathogenic Escherichia coli strains that are especially prevalent in chronic diarrhoeal and recurrent urinary tract infections. Most notably, they are found in up to 50% of cystitis cases in children and 30% of pyelonephritis in pregnant women. Afa/Dr adhesins are capped surface fibrils that mediate recognition of the host and subsequent bacterial internalization. Using the newly solved three-dimensional structure of the minimal invasive complex (AfaDE) combined with biochemical and cellular assays, we reveal the architecture of the fibrillar cap and identify a novel mode of synergistic integrin recognition.

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.

A number of cytokines and their receptors are abundantly expressed at the materno-fetal interface and are thought to have a function in the regulation of placentation. Granulocyte-colony stimulating factor (G-CSF) is expressed by stromal cells in both placental tissue and maternal decidua throughout placentation. In this study, we examined the expression of placental G-CSF receptor (G-CSFR) mRNA and protein throughout gestation by ribonuclease protection assays, Western blotting, and immunohistochemistry. The major placental form of G-CSFR mRNA, corresponding to a membrane-bound form of the protein, was present in first-trimester placental tissues; levels decreased in second- and were highest in third-trimester placental tissues. Two placental G-CSFR molecules, 120 kDa and 150 kDa, were detected in first- and third-, but not second-, trimester tissues. The level of the 150-kDa G-CSFR was greater in the third- than in first-trimester samples. These differences were irrespective of whether or not the patients had received prostaglandin E1 analogues, prostaglandin E1 analogues and oxytocin, oxytocin alone, or mifepristone before labor. We demonstrated by immunohistochemistry that interstitial cytotrophoblast in first- and second-trimester decidual tissue and cytotrophoblast in term fetal membranes express G-CSFR. These data demonstrate that the expression of specific forms of placental G-CSFR is strictly cell type- and developmental stage-specific, and they suggest that G-CSFR may be important in decidual invasion of cytotrophoblast and in trophoblast function during placentation.

Endometriosis is characterized by the presence of functional endometrial tissue outside the uterine cavity, most commonly on the ovary and peritoneum. The aetiology of endometriosis is not understood, although the adhesion of endometrial cells to the extracellular matrix (ECM) would be expected to play a central role in its pathogenesis. The expression of ECM molecules in endometrium and in endometriosis has been investigated using immunohistochemistry and western blotting techniques. The ECM components collagen IV, laminin, vitronectin, and fibronectin had a similar pattern of expression throughout the menstrual cycle in endometrium and endometriosis. Expression of tenascin was elevated in the stroma of the functionalis region of the endometrium during the proliferative stage of the menstrual cycle and in endometriosis. Tenascin expression in endometriosis was not modulated according to the stage of the menstrual cycle. It is concluded that expression of tenascin is strictly regulated in endometrium and may be important in endometrial regeneration and in the pathogenesis of endometriosis.

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 high affinity interaction of integrin α5β1 with the central cell binding domain (CCBD) of fibronectin requires both the Arg-Gly-Asp (RGD) sequence (in the 10th type III repeat) and a second site (in the adjacent 9th type III repeat) which synergizes with RGD. We have attempted to map the fibronectin binding interface on α5β1 using monoclonal antibodies (mAbs) that inhibit ligand recognition. The binding of two anti-α5 mAbs (P1D6 and JBS5) to α5β1 was strongly inhibited by a tryptic CCBD fragment of fibronectin (containing both synergy sequence and RGD) but not by GRGDS peptide. Using recombinant wild type and mutated fragments of the CCBD, we show that the synergy region of the 9th type III repeat is involved in blocking the binding of P1D6 and JBS5 to α5β1. In contrast, binding of the anti-β1 mAb P4C10 to α5β1 was inhibited to a similar extent by GRGDS peptide, the tryptic CCBD fragment, or recombinant proteins lacking the synergy region, indicating that the RGD sequence is involved in blocking P4C10 binding. P1D6 inhibited the interaction of a wild type CCBD fragment with α5β1 but had no effect on the binding of a mutant fragment that lacked the synergy region. The epitopes of P1D6 and JBS5 mapped to the NH2-terminal repeats of the α5 subunit. Our results indicate that the synergy region is recognized primarily by the α5 subunit (in particular by its NH2-terminal repeats) but that the β1 subunit plays the major role in binding of the RGD sequence. These findings provide new insights into the mechanisms, specificity, and topology of integrin-ligand interactions.

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 function of the endometrium in the implantation of the blastocyst depends on the regulated, cyclical regeneration of endometrial tissue and the expression of a receptive phenotype in response to steroid hormones. Experiments using animal models suggest that heparin-binding epidermal growth factor-like growth factor (HB-EGF) is important for endometrial receptivity, and that it may directly mediate blastocyst implantation. We have investigated the expression of HB-EGF mRNA and protein in pregnant and nonpregnant human endometrium and placenta. Our data demonstrate that HB-EGF mRNA expression is low in the endometrium during the proliferative stage of the menstrual cycle and increases in the secretory stage, with highest expression immediately prior to the implantation window (day 19–21), after which levels decrease. Immunohistochemical detection of HB-EGF shows that it is present in the stroma of proliferative stage endometrium and that it is localized to the apical surface of the luminal epithelium of midsecretory stage endometrium. Levels of HB-EGF mRNA are low in pregnant endometrium and high in placental tissues at an early stage of development. Our data suggest that expression of human endometrial HB-EGF coincides with the expression of a receptive phenotype, and that HB-EGF may have an important function in the implantation of the human blastocyst and early placental development. Dev. Genet. 21:102–108, 1997. © 1997 Wiley-Liss, Inc.

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.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the epidermal growth factor family. In rodents HB-EGF has been found to mediate the mitogenic effects of oestrogen on the endometrial glandular cells and those of progesterone on the stromal cells. HB-EGF has also been found to be expressed at the site of implantation before any other discernible sign of blastocyst attachment in rodents. Our aim was to determine whether HB-EGF mRNA was expressed in the human pregnant and non-pregnant endometrial tissues. Normal endometrial tissues at different stages of the menstrual cycle, first trimester chorionic villi and decidua, second trimester chorionic villi and term placental tissues were snap frozen and stored at -80 degrees C. Reverse transcription-polymerase chain reaction (RT-PCR) was used to analyse the expression of HB-EGF mRNA in these tissues. HB-EGF mRNA was found to be expressed in human endometrium throughout the menstrual cycle. It was also present in first trimester chorionic villi and decidua, second trimester villi and term placenta. These findings suggest that HB-EGF may have a role in the growth or function of the human uterus, and that it may be important at the feto-maternal interface throughout pregnancy.

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.

The secondary structure of the tenth type III module from human fibronectin has been determined using NMR. This type of module appears many times in a wide variety of proteins. The type III module described here contains an Arg-Gly-Asp sequence known to be involved in cell-cell adhesion. The module was expressed in yeast and characterized by amino acid sequencing and mass spectrometry. 2D and 3D NMR spectroscopy of 15N-labeled protein was used to perform sequence-specific assignment of the spectrum. The secondary structure was defined by patterns of nuclear Overhauser effects, 3JNH-alpha CH spin-spin coupling constants, and amide proton solvent exchange rates. The molecule consists of seven beta-strands in two antiparallel beta-sheets with an immunoglobulin-like fold similar to that predicted for homologous modules in the cytokine receptor super family [Bazan, J. F. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6934-6938]. The Arg-Gly-Asp sequence is located on a loop between the beta-strands F and G.

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.

The 9th-10th type III fibronectin domain pair shows promise in tissue engineering and tumour vasculature targeting. Calorimetry and structure-function analysis were used to investigate the effects of solution formulation and lyophilisation of a mutant ((9-10)FNIII-P). A single endothermic transition for (9-10)FNIII-P in solution was observed at pH8, irrespective of addition of sucrose or PEG. The temperature at the maximum heat capacity (T(m)) and enthalpy (deltaH) of the transition increased for increasing sucrose concentrations but decreased for increasing PEG concentrations. The transition was fitted to a single two-state unfolding mechanism (in contrast to unfolding in guanidine. x HCl) and was partially reversible only at pH 4, with increasing concentrations of sucrose causing a marked fall in deltaH between scans. Circular dichroism spectra for the thermal unfolding of (9-10)FNIII-P at pH 4 showed loss of native beta-sheet structure and loss of aromatic contributions to the peak centred around 226 nm yielding an intermediate conformation, which in the presence of sucrose was more disordered. Despite a glass transition (T(g)') for (9-10)FNIII-P(aq) of -70 degrees C, primary drying at -30 degrees C did not perturb its conformation upon reconstitution or its biological activity following lyophilisation; the addition of sucrose or PEG had no influence on structure or activity. The main consideration in the formulation of (9-10)FNIII-P was therefore pH.

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.

Non-ionic surfactants have been employed as alternatives to PVA for the emulsification–encapsulation of a conformationally labile protein (FIII9′-10) into PLGA microspheres. FIII9′-10 was encapsulated using a w/o/w double emulsification–evaporation technique and the microspheres fabricated were characterized by SEM and CLSM. The peptide backbone integrity of FIII9′-10 was assayed by SDS-PAGE and the degree of unfolding of FIII9′-10 following emulsification–encapsulation was assessed using a fibroblast cell-attachment assay. The encapsulation efficiency for FIII9′-10 was 25% when using PVA, compared to 50–60% when using Igepal CA-630 or Triton-X100, with values below for the other surfactants. FIII9′-10 released from microspheres promoted cell attachment in a concentration-dependent manner, only Igepal CA-630 and Triton X-100 maintaining near-maximal cell attachment, indicating that the conformation of the relatively unstable FIII9′ domain was preserved. All non-ionic surfactants reduced microsphere surface porosity, compared to PVA, and an increasing surface rugosity (leading to minor ‘ridges’) could be correlated with decreasing surfactant HLB. Low surface porosities did not effect the diffusion of FIII9′-10 from the microspheres' internal pores in a ‘burst release’, as may have been imagined. In summary, non-ionic surfactants should be considered over PVA for the maintenance of biological activity of conformationally labile proteins during encapsulation.

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.