Investigating the role of CD44 and hyaluronate in embryo-epithelial interaction using an in vitro model.

Implantation failure is an important impediment to increasing success rates in assisted reproductive technologies. Knowledge of the cascade of morphological and molecular events at implantation remains limited. Cell surface CD44 and hyaluronate (HA) have been reported in the uterus, but a role in intercellular interaction at implantation remains to be evaluated. Mouse embryos were co-cultured with human Ishikawa endometrial epithelial monolayers over 2 days. Attachment was tenuous during the first 24 h, after which it became stable, leading to breaching of the monolayer. The effects of enzymatically reducing the density of HA, or introducing a function-blocking antibody to CD44, were monitored during progression from weak to stable embryonic attachment. Hyaluronidase-mediated removal of surface HA from the epithelial cells enhanced the speed of attachment, while a similar treatment of embryos had no effect. The antibody to CD44 caused retardation of initial attachment. These results suggest that CD44-HA binding could be employed by embryos during initial docking, but the persistence of HA in epithelial cells might be detrimental to later stages of implantation by retarding attainment of stable attachment.

Studies of the dynamics of nuclear clustering in human syncytiotrophoblast.

Syncytial nuclear aggregates (SNAs), clusters of nuclei in the syncytiotrophoblast of the human placenta, are increased as gestation advances and in pregnancy pathologies. The origins of increased SNAs are unclear; however, a better appreciation of the mechanism may give insight into placental ageing and factors underpinning dysfunction. We developed three models to investigate whether SNA formation results from a dynamic process of nuclear movement and to generate alternative hypotheses. SNA count and size were measured in placental explants cultured over 16 days and particles released into culture medium were quantified. Primary trophoblasts were cultured for 6 days. Explants and trophoblasts were cultured with and without cytoskeletal inhibitors. An in silico model was developed to examine the effects of modulating nuclear behaviour on clustering. In explants, neither median SNA number (108 SNA/mm(2) villous area) nor size (283 µm(2)) changed over time. Subcellular particles from conditioned culture medium showed a wide range of sizes that overlapped with those of SNAs. Nuclei in primary trophoblasts did not change position relative to other nuclei; apparent movement was associated with positional changes of the syncytial cell membrane. In both models, SNAs and nuclear clusters were stable despite pharmacological disruption of cytoskeletal activity. In silico, increased nuclear movement, adhesiveness and sites of cytotrophoblast fusion were related to nuclear clustering. The prominence of SNAs in pregnancy disorders may not result from an active process involving cytoskeleton-mediated rearrangement of syncytial nuclei. Further insights into the mechanism(s) of SNA formation will aid understanding of their increased presence in pregnancy pathologies.

Tracking nutrient transfer at the human maternofetal interface from 4 weeks to term.

INTRODUCTION: In this study we have tracked glycogen and glycoprotein flux associated with nutrient uptake into trophoblast in early deciduochorial and later haemochorial placenta. METHODS: α-amylase, glycogen synthase and glycogen phosphorylase were immunohistochemically localised in 6-14 week and term placenta and first trimester decidua. Placentae of 4-18 weeks' gestation and term were also stained with 22 biotinylated lectins. RESULTS: Histochemical data were consistent with glycogenolysis in decidual gland epithelium and placental cyto- and syncytiotrophoblast; α-amylase was present in decidual secretions but absent in placenta. Glycogen and glycogen synthase were both apparent in villous cytotrophoblast cells and columns. Profound changes were observed in placental glycosylation during gestation. Syncytial microvilli were richly glycosylated as were first trimester vacuoles but, by term, syncytiotrophoblast showed little lectin binding except in microvillous and basal membranes. Cytotrophoblast Golgi bodies were active in the first trimester; at term the cells were generally more glycosylated than syncytiotrophoblast. DISCUSSION: We deduce that decidual cell glycogen is broken down for transport into the placenta where the products may be reassembled into glycogen or used for metabolic processes. First trimester histiotrophe is internalised by syncytiotrophoblast, then broken down in apical vacuoles containing lysosomal markers. This process declines after haemotrophic nutrition commences. Transition from histiotrophic to haemotrophic nutrition involves reduced amounts of uterine secretory derivatives reaching the placenta, and reduction in internalisation of glycoprotein by syncytiotrophoblast, presumably reflecting the shift to low molecular weight nutrients. Glycogen accumulates in cytotrophoblast from early pregnancy and is mobilised for utilisation by fetoplacental tissues.

Endometrial apical glycoproteomic analysis reveals roles for cadherin 6, desmoglein-2 and plexin b2 in epithelial integrity.

Implantation failure is one of the major causes of infertility and remains a major barrier to assisted reproduction success. Initial receptivity to implantation is regulated by the endometrial luminal epithelium under maternal hormonal control. Identification of epithelial cell surface components involved in embryo attachment will have translational applications in early pregnancy failure, infertility and contraception. In this study, vectorial biotinylation has been used to characterize the apical glycoproteome of Ishikawa cells, a polarized cell line that serves as a model of the implantation-receptive human endometrial luminal epithelium. Of 46 surface-associated glycoproteins detected by mass spectrometry, half are newly reported in this cell type; a subgroup of these were chosen for evaluation in tissue, and all were shown to be expressed apically in vivo in the mid-secretory (implantation) phase of the menstrual cycle, thus validating the model. Eleven adhesion molecules were detected, some already known to be involved in implantation, others novel. Cadherin 6, desmoglein 2 and plexin b2 were surprisingly found in the apical as well as the lateral membrane domain; their knock-down compromised epithelial integrity. This method of targeting glycosylated apical surface moieties in a polarized epithelial culture model shows excellent selectivity and identifies candidate cell adhesion molecules that are also present in vivo in secretory phase endometrial epithelium.

Fetoplacental vascular alterations associated with fetal growth restriction.

INTRODUCTION: Placental functional impairment in pregnancies with fetal growth restriction (FGR) can arise from fetoplacental vascular abnormalities. We aimed to compare the micro and macrovasculature of placentas from normal pregnancies with those showing late onset FGR. METHODS: Placental arterial casts (n = 12 normal, 6 FGR) were prepared. Chorionic arterial number and inter-branch length were examined. Microvascular features were quantified in CD34-stained tissue sections obtained by systematic (n = 12 normal, 12 FGR) and targeted (n = 6 normal, 6 FGR) sampling from the placental periphery and centre. RESULTS: Adjusted for the weight of the placenta or the surface area of the chorionic plate, the number of chorionic arteries was similar in normal and FGR arterial casts. Inter-branch length per unit placental weight was greater in the first generation of arterial branches in FGR (p < 0.05). Villi in FGR placentas were more poorly vascularised, particularly at the periphery and in grossly visible hypovascular regions. Intermediate and terminal FGR villi in these areas exhibited reduced vessel lumens, loss of CD34, and infilling with CD34-negative cells of what appeared to be previously existing vascular spaces. CONCLUSION: Differences in chorionic arterial branching patterns between normal and FGR placentas arise from differences in placental size. FGR placentas show microvascular regression and extreme hypovascularity in peripheral areas. These features may well limit the ability of the placenta to meet fetal nutrient requirements late in gestation. Targeted sampling is more effective than systematic random sampling in revealing vascular defects.

Placental alkaline phosphatase de-phosphorylates insulin-like growth factor (IGF)-binding protein-1.

BACKGROUND: Insulin-like growth factors (IGF) regulate fetal growth through their effects on placenta. Their actions are influenced by IGF binding protein-1. Phosphorylated IGFBP-1 (pIGFBP-1) has high affinity for IGF-I and usually inhibits IGF-I activity but during pregnancy, it is de-phosphorylated to generate lower affinity isoforms and consequently, increased IGF bioavailability. Here we investigate the role of placenta in this process. RESULTS: Our data show that term human placental explants, but not their conditioned medium, can de-phosphorylate IGFBP-1 through the action of placental alkaline phosphatase (PLAP). DISCUSSION: PLAP-mediated de-phosphorylation of IGFBP-1 may provide a mechanism for controlling IGF-I bioavailability and action at the maternal/fetal interface.

Trophoblast specialisations during pregnancy in the tammar wallaby, Macropus eugenii: a morphological and lectin histochemical study.

INTRODUCTION: The tammar wallaby has a short gestation (26.5 days) and vascular modifications to expedite transport during that brief pregnancy. Here we examine trophoblast structural attributes that would facilitate materno-fetal exchange. MATERIALS AND METHODS: Four specimens of Macropus eugenii between days 23 and 26 gestation were examined using electron microscopy and 24 lectins to characterise glycosylated secretions and their internalisation. RESULTS: Two trophoblast phenotypes were found, flattened cells generally in contact with the underlying uterine epithelium and giant cells associated with histiotrophe. The latter appeared to penetrate uterine clefts, occasionally detach and become necrotic. Lectin histochemistry and ultrastructure indicated the presence of many lysosomes and residual bodies especially in trophoblast giant cells; these contained glycans, mainly apically, which were also detected in secretions and cell debris. Trophoblast basal membranes bore extensive filopodia. Giant cells were less common in vascular trilaminar areas and here the trophoblast barrier became thinner near term. DISCUSSION: Loss of Maackia amurensis agglutinin binding suggested cleavage of terminal sialic acid residues as an early post-internalisation event in the trophoblast. Lectin staining indicated degradation occurred in an apical-basal direction, and the heavily glycosylated basal membrane appeared specialised for transport out of the cell. CONCLUSION: Granules seen ultrastructurally and histochemically, particularly in giant trophoblast cells of the bilaminar area, suggest that internalised histiotrophe is broken down here and nutrients transferred to the embryo via the specialised basal plasma membrane. The trilaminar vascular area contained mostly flattened trophoblast cells, supporting the suggestion that gaseous exchange is its primary function.

Compliance effects in a randomised controlled trial of yoga for chronic low back pain: a methodological study.

STUDY DESIGN: Methodological study nested within a multicentre randomised controlled trial (RCT) of yoga plus usual general practitioner (GP) care vs usual GP care for chronic low back pain. OBJECTIVE: To explore the treatment effects of non-compliance using three approaches in an RCT evaluating yoga for low back pain. SUMMARY OF BACKGROUND DATA: A large multicentre RCT using intention-to-treat (ITT) analysis found that participants with chronic low back pain who were offered a 12-week progressive programme of yoga plus usual GP care had better back function than those offered usual GP care alone. However, ITT analysis can underestimate the effect of treatment in those who comply with treatment. As such, the data were analysed using other approaches to assess the problem of non-compliance. The main outcome measure was the self-reported Roland Morris Disability Questionnaire (RMDQ). METHODS: Complier average causal effect (CACE) analysis, per-protocol analysis and on-treatment analysis were conducted on the data of participants who were fully compliant, predefined as attendance of at least three of the first six sessions and at least three other sessions. The analysis was repeated for participants who had attended at least one yoga session (i.e. any compliance), which included participants who were fully compliant. Each approach was described, including strengths and weaknesses, and the results of the different approaches were compared with those of the ITT analysis. RESULTS: For the participants who were fully compliant (n=93, 60%), a larger beneficial treatment effect was seen using CACE analysis compared with per-protocol, on-treatment and ITT analyses at 3 and 12 months. The difference in mean change in RMDQ score between randomised groups was -3.30 [95% confidence interval (CI) -4.90 to -1.70, P<0.001] at 3 months and -2.23 (95% CI -3.93 to -0.53, P=0.010) at 12 months for CACE analysis, -3.12 (95% CI -4.26 to -1.98, P<0.001) at 3 months and -2.11 (95% CI -3.33 to -0.89, P=0.001) at 12 months for per-protocol analysis, and -2.91 (95% CI -4.06 to -1.76, P<0.001) at 3 months and -2.10 (95% CI -3.31 to -0.89, P=0.001) at 12 months for on-treatment analysis. For the participants who demonstrated any compliance (n=133, 85%), the results were generally consistent with the fully compliant group at 3 months, but the treatment effect was smaller. The difference in mean change in RMDQ score between randomised groups was -2.45 (95% CI -3.67 to -1.24) for CACE analysis, -2.30 (95% CI -3.43 to 1.17) for per-protocol analysis and -2.15 (95% CI -3.25 to -1.06) for on-treatment analysis, which was slightly less than that for ITT analysis. In contrast, at 12 months, per-protocol and on-treatment analyses showed a larger treatment effect compared with CACE and ITT analyses: per protocol analysis -1.86 (95% CI -3.02 to -0.71), on-treatment analysis -1.99 (95% CI -3.13 to -0.86) and CACE analysis -1.67 (95% CI -2.95 to -0.40). CONCLUSION: ITT analysis estimated a slightly smaller treatment effect in participants who complied with treatment. When examining compliance, CACE analysis is more rigorous than per-protocol and on-treatment analyses. Using CACE analysis, the treatment effect was larger in participants who complied with treatment compared with participants who were allocated to treatment, and the difference between ITT and CACE analyses for the fully compliant group at 3 months was small but clinically important. Per-protocol and on-treatment analyses may produce unreliable estimates when the effect of treatment is small. INTERNATIONAL STANDARD RANDOMISED TRIAL NUMBER REGISTER: ISRCTN 81079604.

Analysis of syncytial nuclear aggregates in preeclampsia shows increased sectioning artefacts and decreased inter-villous bridges compared to healthy placentas.

Syncytial nuclear aggregates (SNAs) are increased in pregnancy complications and include 'true' syncytial knots and inter-villous bridges. Apparent nuclear overlay caused by sectioning artefacts are frequently counted from single sections. Haematoxylin and eosin stained serial sections were assessed for frequency of SNA subtypes in placentas from normal, preeclamptic and fetal growth restricted (FGR) pregnancies. There were more sectioning artefacts and syncytial knots and fewer bridges in samples from preeclampsia compared to controls. There were no significant differences between FGR and control samples. This suggests the villous tree in preeclampsia has less inherent structural support and trophoblast cell dynamics are different.

The fetomaternal interface shows vascular hypoglycosylation in the tammar wallaby Macropus eugenii: comparison with a range of non-mammalian and eutherian placentae.

INTRODUCTION: Blood vessel glycosylation at the fetomaternal interface of four near-term specimens of tammar wallaby, Macropus eugenii, has been examined at days 23-26 of the 26.5 day pregnancy and compared with that of other species. METHODS: A panel of 23 lectins was used to compare vasculature in tammar with non-mammalian (shark, skink) and eutherian species at early and late gestation (camel, horse and alpaca), and term/near-term (cat, lion, dog, mink and elephant). RESULTS: Strikingly low levels of all the glycans tested, apart from sialic acids, were found in capillary endothelium of both the trilaminar omphalopleure and underlying surface endometrium of the tammar, though deeper endometrial vessels showed normally high levels of glycosylation. Only maternal vasculature of the mink placenta showed a comparable lack of expression. DISCUSSION: One reason for a reduced endothelial glycocalyx may be to facilitate diffusion of gases and nutrients as the tammar trophoblast lacks the indentation by overlying vessels that is seen in the other near-term placentae. Early epitheliochorial placentae of other species with equal diffusion distances to the tammar, showed normal vascular glycosylation. However, their pregnancies are much longer. CONCLUSION: The hypoglycosylation of tammar vessels at the fetomaternal interface may allow continued transfer of nutrients and gaseous exchange during the extremely rapid period of organogenesis which occurs during the short 26.5 day pregnancy of this marsupial. Given the short gestation period of the tammar, we suggest that a thinner endothelial glycocalyx has evolved to facilitate diffusion of gases and nutrients between the maternal and fetal compartments.

Human placental multipotent mesenchymal stromal cells modulate trophoblast migration via Rap1 activation.

INTRODUCTION: Little is known about the interaction between human placental multipotent mesenchymal stromal cell (hPMSC) and trophoblast. We hypothesize that hPMSCs produce hepatocyte growth factor (HGF) which may interact with trophoblasts and regulate their migration during placentation. METHODS: hPMSCs were isolated from term placentas and conditioned medium was collected after 2 days of culture in hypoxic (<1% O2) or control (20% O2) conditions. Selective agonist and inhibitor or siRNA for protein kinase A (PKA) or Rap1 were combined with Rap1-GTP pull down assays, flow cytometry, integrin ß1 activation assays and adhesion and migration studies to investigate HGF signaling effects in trophoblasts. The hPMSC abundance and HGF level in preeclamptic placentas were compared with gestational age-matched controls. RESULTS: HGF was expressed by hPMSCs and was decreased in hypoxia. HGF induced cAMP production and Rap1 activation in trophoblasts, which in turn activated integrin ß1. The HGF and PKA activator 6-Bnz-cAMP induced Rap1 activation with increased trophoblast adhesion and migration. The alterations were inhibited by PKA inhibitor H89 or Rap1 siRNA. HGF and cAMP expression were reduced in preeclamptic placentas. hPMSC number was decreased in preeclamptic placenta compared to controls (0.68 ± 0.1% vs. 1.32 ± 0.5%; P = 0.026). hPMSC conditioned medium enhanced trophoblast migration which was inhibited by c-Met blocking antibody, but migration was reduced by conditioned medium from hPMSC cultured in hypoxia. CONCLUSIONS: hPMSCs secrete HGF and increase trophoblast cAMP production. The cAMP effector PKA modulates adhesion and migration of trophoblast via signaling to Rap1 and integrin ß1.

Syncytial nuclear aggregates in normal placenta show increased nuclear condensation, but apoptosis and cytoskeletal redistribution are uncommon.

INTRODUCTION: Syncytial nuclear aggregates (SNAs) are increased in pregnancy complications; however, little is known about their origin or function. This study aimed to characterise SNAs in more detail than has been reported previously. METHODS: Immunohistochemistry and morphological examination at the light and ultrastructural level were used to determine the nature and structure of SNAs. RESULTS: SNAs comprising bridges and syncytial knots had similar frequency with 974 per mm3 of villous tissue (IQR 717-1193) and 833 per mm3 (IQR 766-1190), respectively while there were approximately four times as many sectioning artefacts than knots and bridges combined. SNAs had increased proportions of condensed nuclei compared to the remaining syncytiotrophoblast (33.3% vs. 8.9%) and decreased proportions of euchromatic nuclei (0.0% vs. 16.2%), as assessed by examination of an electron micrograph archive. SNAs showed little evidence of apoptosis, with weak positivity for the apoptosis markers M30-neoepitope at 16.6% and TUNEL at 10.0%; strong staining was rarely seen for either marker. Immunofluorescence demonstrated rare association of actin (α, ß or γ) with SNAs, whereas tubulin was in close proximity to SNAs and cytokeratin was seen within and surrounding SNAs. DISCUSSION: M30-positive SNAs traced through serial sections were significantly more likely to be syncytial knots or sectioning artefacts than bridges. Nuclei within SNAs showed signs consistent with degeneration; however, this is unlikely to be an apoptotic process. There are few changes in configuration of cytoskeletal proteins around SNAs. CONCLUSIONS: These data suggest that the biogenesis and functional significance of SNAs still require resolution.

Dicer-dependent miRNAs provide an endogenous restraint on cytotrophoblast proliferation.

Mature microRNAs (miRNAs) are processed from non-functional (pre)-miRNAs by the enzyme Dicer. In this study, manipulation of Dicer level was used to explore the influence of miRNAs on cytotrophoblast proliferation in human placenta. Immunohistochemistry (IHC) showed Dicer in cytotrophoblast, but not in terminally differentiated syncytiotrophoblast. siRNA-mediated knockdown of Dicer was used to effect a global reduction in miRNA in first trimester placental explants, as a result of which cytotrophoblast proliferation was significantly enhanced. QPCR and IHC analysis following Dicer knockdown revealed that the expression of two nodal pro-mitogenic signalling molecules expressed within cytotrophoblast, ERK and SHP-2, was significantly enhanced. Studies are now required to identify individual miRNAs involved in regulating trophoblast proliferation.

Syncytin 1 in the human placenta.

This study characterises HERV-W (syncytin 1) expression in normal and pathologic placenta and in BeWo cells. HERV-W mRNA levels were higher in the first trimester than at term, and similar patterns were observed with another retrovirally-derived mRNA species, ERV-3. N-glycosylated syncytin 1 precursor (73 kDa) is cleaved to surface-associated (SU) and transmembrane (TM) subunits. Both were evident in villous trophoblast, where perinuclear and punctate cytoplasmic deposits were observed, and linear TM subunit immunoreactivity was seen at the syncytial microvillous membrane. Punctate immunoreactivity was seen in BeWo cells with antibodies to SU and TM, and the two were co-localised. SU immunoreactivity was observed in association with fetal endothelium, and this effect was increased in tissue from pre-eclamptic placentas, which also showed a higher level of total SU protein. Absence of the TM subunit from endothelium suggests it is not a biosynthetic source. We suggest that SU is released from trophoblast into fetal circulation where it may bind vascular endothelium.

Signalling of DNA damage and cytokines across cell barriers exposed to nanoparticles depends on barrier thickness.

The use of nanoparticles in medicine is ever increasing, and it is important to understand their targeted and non-targeted effects. We have previously shown that nanoparticles can cause DNA damage to cells cultured below a cellular barrier without crossing this barrier. Here, we show that this indirect DNA damage depends on the thickness of the cellular barrier, and it is mediated by signalling through gap junction proteins following the generation of mitochondrial free radicals. Indirect damage was seen across both trophoblast and corneal barriers. Signalling, including cytokine release, occurred only across bilayer and multilayer barriers, but not across monolayer barriers. Indirect toxicity was also observed in mice and using ex vivo explants of the human placenta. If the importance of barrier thickness in signalling is a general feature for all types of barriers, our results may offer a principle with which to limit the adverse effects of nanoparticle exposure and offer new therapeutic approaches.

Differential expression of angiotensin II type 1 and type 2 receptors at the maternal-fetal interface: potential roles in early placental development.

Angiotensin II (Ang II) is locally generated in the placenta and regulates syncytial transport, vascular contractility and trophoblast invasion. It acts through two receptor subtypes, AGTR1 and AGTR2 (AT1 and AT2), which typically mediate antagonising actions. The objectives of this study are to characterise the cellular distribution of AGTR1 and AGTR2 at the maternal-fetal interface and explore the effects on cytotrophoblast turnover. Low levels of AGTR2 mRNA were detected in first trimester placental homogenates using real-time PCR. Immunohistochemistry using polyclonal antibodies against AGTR1 and AGTR2 detected the receptors in first trimester placenta, decidua basalis and villous tip outgrowths in culture. Serial staining with cytokeratin-7 was used to identify extravillous trophoblasts (EVTs). AGTR1 was found in the syncytiotrophoblast microvillous membrane, in a subpopulation of villous cytotrophoblasts, and in Hofbauer cells. AGTR1 was strongly upregulated in cytotrophoblasts in cell columns and villous tip outgrowths, but was absent in interstitial and endovascular EVTs within the decidua. AGTR2 immunostaining was present in Hofbauer cells and villous cytotrophoblasts, but was absent from syncytiotrophoblast. Faint staining was detected in cell column cytotrophoblasts and villous outgrowths, but not in EVTs within the decidua. Both receptors were detected in placental homogenates by western blotting. Ang II significantly increased proliferation of cytotrophoblasts in both villous explants and villous tip outgrowths, but did not affect apoptosis. Blockade of AGTR1 and AGTR2 together abrogated this effect. This study shows specific expression patterns for AGTR1 and AGTR2 in distinct trophoblast populations at the maternal-fetal interface and suggests that Ang II plays a role in placental development and generation of EVTs.

First trimester histiotrophe shows altered sialylation compared with secretory phase glycoconjugates in human endometrium.

OBJECTIVES: Histiotrophe is now recognized as being an important feature of early human pregnancy, providing nutrients and growth factors to the developing embryo. Our aim was to examine the glycan composition of histiotrophe from first trimester decidua and to compare it with secretions present in endometrial glands from the late secretory phase of the menstrual cycle. STUDY DESIGN: Twenty samples of decidua from pregnancies between 8 and 11 weeks were processed into epoxy resin and sections stained with a panel of 22 lectins, together with six late secretory phase endometrial biopsies. MAIN OUTCOME MEASURES: Specimens were analysed using a semi-quantitative ranking system and the density of lectin binding to the glandular secretions and the epithelium assessed. RESULTS: With the onset of pregnancy, beta-galactose, alpha-N-acetyl galactosamine and N-Acetyl lactosamine bound by Arachis hypogaea, Glycine max, Helix pomatia and Erythrina crystagalli agglutinins appeared in terminal positions on oligosaccharide chains, suggesting loss of the capping sialic acid residues present in the non-pregnant state. CONCLUSIONS: Suppression of terminal sialylation is evident during early pregnancy, suggesting that modifications to endometrial glandular activity occur in response to placental signals. The changes may facilitate absorption of histiotrophe by the trophoblast and enhance availability of substrates for degradation.

Characterisation of Hofbauer cells in first and second trimester placenta: incidence, phenotype, survival in vitro and motility.

Macrophages, known as Hofbauer cells, are most abundant in placental villous stroma in the first and second trimesters. Their functions are not well defined. We have used a combination of in situ and in vitro methods to characterise these cells. Lectin histochemistry and immunohistochemistry were used to identify macrophages in situ. The lectin from Maclura pomifera (MPA) was found to mark cells bearing the CD68 antigen with optimal specificity and selectivity. MPA staining was used to show that they increase in number from mid first to mid second trimester, becoming much less abundant at term. The cells are absent from mesenchymal villi, being associated primarily with villous stroma containing the prominent interstitial channels characteristic of immature intermediate villi. A mixed stromal cell isolate was studied in monolayer culture, including the use of time-lapse microscopy. Cells from first or second trimester tissue contained a subpopulation of about 14-17% of cells that exhibited a macrophage-like morphology and expressed CD68 as well as MPA-binding glycans. These cells were short-lived in monoculture, but could persist in vitro in association with a fibroblast layer for several weeks. They could switch rapidly from a macrophage-like to a fibroblastic morphology, were highly motile and associated in clusters that rapidly formed and dissipated over periods of a few hours. These data suggest that Hofbauer cells play a role in the maturation of mesenchymal into immature intermediate-type stroma. They may be important in the excavation of stromal channels. Their prolonged viability in mixed cultures suggests a paracrine relationship with resident fibroblasts. Their location and migratory behaviour predict an ability to move rapidly around the villous stroma, perhaps within the channel system, and to make transient contacts both with other macrophages and stromal cells.

Early stages of implantation as revealed by an in vitro model.

Our limited understanding of the processes underlying steroid hormonal control of human endometrial receptivity is largely due to the lack of a relevant model system. To overcome scarcity of material, we have developed a model in which mouse embryos attach to human Ishikawa cells, which express functional steroid hormone receptors. Blastocysts flushed from day 4 pregnant superovulated mice were transferred to confluent Ishikawa cell monolayers. After 48 h of co-culture, 85% of the blastocysts had attached loosely, but only 40% attached stably to the epithelial cell surface. In contrast, 95% of the embryos attached stably to tissue culture plastic. Thus, weak attachment of a majority of the embryos was followed by stronger adhesion of a smaller proportion. Seventeen percent of the transferred blastocysts modified the epithelial cell surface with loss of MUC1 at the attachment site, extending variably to adjacent epithelial cells. Initially, stable attachment occurred without disruption to the integrity of the epithelial monolayer, but at later stages after the embryo had spread laterally, displacement of subjacent cells was observed. A modest increase in stable attachment, but no changes to MUC1 clearance, was observed after assisted hatching. After 24 h priming of Ishikawa cells by 17beta-oestradiol (OE(2)) followed by 72-h incubation with medroxyprogesterone acetate and OE(2), stable attachment increased from 40 to 70%. Initial attachment is efficient either in the presence or in the absence of hormone; steroid treatment increased the incidence of stable attachment. Implantation failure is predicted to occur in this model when embryos fail to progress from initial to stable attachment.

Altered glycosylation in peri-implantation phase endometrium in women with stages III and IV endometriosis.

BACKGROUND: Endometriosis is a common cause of infertility and pelvic pain. Lectin histochemistry has shown that glycan expression is a sensitive marker of differentiation in the normal endometrium. Endometrial biopsies were taken during the implantation window from women with subfertility and advanced (stage III and IV) endometriosis to evaluate specific glycans bound by lectins from Dolichos biflorus agglutinin (DBA) and Vicia villosa agglutinin (VVA), which detect related but distinct glycan sequences regulated by progesterone action. METHODS: Endometrial tissue from 12 women with subfertility and advanced endometriosis and 11 healthy controls were taken on days 19-24 of the menstrual cycle and processed into either epoxy resin or paraffin wax. Lectin histochemistry was analysed using light microscopy to quantify the amount of glandular reaction product. RESULTS: There was a significant (P = 0.011) reduction in DBA binding to endometrium from patients with endometriosis compared with controls, which was not seen with VVA (P = 0.135). Three stage IV biopsies and one stage III biopsy completely failed to bind DBA and, of these, three showed moderate glandular binding of VVA. DBA and VVA binding differed significantly (P= 0.0039) in the endometriosis specimens whereas in controls no significant difference was detected (P = 0.812). CONCLUSION: Secretory phase glycosylation in women with advanced endometriosis differs from that in healthy women with a reduction in fucosylated N-acetylgalactosamine sequences bound by DBA. Shorter VVA-binding glycans are not significantly affected. In addition to indicating abnormalities of epithelial differentiation, these findings may be directly relevant to implantation failure, as blastocyst attachment requires a critical interaction with the epithelial glycocalyx.