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Study question Does S100P involve in the regulation of lipid metabolism in trophoblast syncytialization during the early stage of pregnancy? Summary answer S100P suppressed lipid droplets overloading in trophoblast syncytialization during the early stage of pregnancy. What is known already S100P is a 95-amino acid protein belongs to the large family of S100 calcium-binding proteins and is exclusively expressed in syncytiotrophoblast layer of placenta, enhancing trophoblast cells proliferation, motility and invasion. Recent studies indicated S100P was aberrantly expressed in various malignant tumors, promoting their proliferation, survival, the formation and invasion of vessels, and has a close relationship with their grade malignancy, hormone dependency and the drug-resistance. Syncytialization is trophoblast cells differentiated from cytotrophoblasts fused into multinucleated cell clusters with cell boundaries deficiency. Study design, size, duration Tissues were collected from elective first trimester pregnancy terminations of healthy people and patients with recurrent spontaneous abortion. Human trophoblast stem cells (hTSCs) were isolated from Week 6 to Week 8 of fresh placental tissues (n = 8) and use forskolin to induce syncytialization. Participants/materials, setting, methods Tissues were collected from elective first trimester surgical pregnancy terminations to determine localization, abundance and function of S100P. The level of S100P protein and gene was measured by Real-time quantitative PCR (RT-qPCR), western blot, ELISA and immunofluorescence in hTSC induced ST(2D)-TSCT. The lipid droplets were assessed by Oil red O stain, BODIPY and electron microscope. Serum S100P concentration in normal and recurrent miscarriage patients were measured by ELISA. Main results and the role of chance S100P was exclusively localized in syncytiotrophoblast layer of the placental villous from gestational weeks 6–8 (n = 4); S100p serum concentration was increased markedly as the gestation progressed (n = 40, p Limitations, reasons for caution The role of S100P on placenta lacks the evidence from animal models. Wider implications of the findings: S100P is an important factor facilitating in trophoblast syncytialization in the first trimester of pregnancy and could be a biomarker of the early pregnancy sustenance. Trial registration number Not applicable

Study question Is the low HLA-G expression a determinant of early reproductive loss? Summary answer Low expression of HLA-G is associated with pregnancy complications and can be one of the reasons of spontaneous abortion (such as RPL). What is known already The dysregulated maternal immune responses to invading embryos may play role in RIF, RPL, and second- and third-trimester obstetrical conditions. HLA-G is a molecule that was first known to confer protection to the fetus from destruction by the immune system of its mother, thus critically contributing to fetal–maternal tolerance due to inducing displacement of pro-inflammatory to Th1 cell-mediated response of Th2, has a positive influence on the process of implantation. HLA-G is mainly restricted to the fetal–maternal interface on the extravillous cytotrophoblast, to placenta, amnion. Study design, size, duration It was a prospective complex cohort study from 2016–2020 years with pathomorphological investigations. The purpose of this study is to investigate the HLA-G and KIR2DL4 expression in chorionic villous among 3 groups (study included 118 cases of abortion material): group 1 – 36 cases after missed abortion with normal karyotype, group 2 – 36 cases after missed abortion with polyploidy and group 3 – 46 cases after induced abortion group (normal pregnancy). Participants/materials, setting, methods Criteria of inclusion: abortive material from 3 groups of women with missed or after induced abortion; 6–12 weeks, singleton pregnancy, cytogenetic of chorionic villous was obligatorily - normal fetal karyotype and polyploidy of fetus. Pathomorophological investigation included H&E stain, IHC and confocal laser scanning microscopy. Immunohistochemical examination included quantitative and qualitative assessment of the expression of Anti-HLA-G (mouse monoclonal) in an extra villous trophoblast and Anti-KIR2DL1+KIR2DL3 + KIR2DL4 + KIR2DS4 (rabbit polyclonal) in chorionic villi. Main results and the role of chance The immunohistochemical study showed homogenous distribution HLA-G expression in extravillous trophoblast cells (EVT) and KIR2DL4 expression in chorion villous both in missed abortion groups and in induced abortion group. HLA-G expression average relative area in 1 and 2 groups was not statistically different (in 1 group with normal karyotype 33,9±3,5 and in 2 group 38,6±2,8). But the expression of HLA-G in 3 group was strictly higher (55,6 ±2,4). The average relative area of KIR2DL4 receptor wasn’t statistically different among 3 groups. However, the histological picture both missed abortion groups (for the genetic\immunological reasons for rejection) is the only one - this is a missed abortion of an early terms of gestation. In a histological study of missed abortion, as our study shows, the histological picture is similar in 1 and 2 groups. Thus, in 1 group with a normal karyotype of the fetus (before conducting the chorion cytogenetic study in the genetics laboratory) in 59.2% the histological examination determined a picture of an impaired early pregnancy with signs of trophoblast chromosomal pathology. Thus, without a cytogenetic study of the chorion, it is impossible to clearly determine whether the chromosomal pathology of the fetus is the cause of missed abortion. Limitations, reasons for caution There is no limitations, reasons for caution. Wider implications of the findings: Thus, HLA-G molecule has a leading role in the onset and successful prolongation of pregnancy, implantation, placentation and fetal development. Trial registration number 98–2019

STUDY QUESTION Does CD147 regulate trophoblast functions in vitro? SUMMARY ANSWER CD147 exists as a receptor complex on human trophoblast and regulates the implantation, invasion and differentiation of trophoblast. WHAT IS KNOWN ALREADY CD147 is a membrane protein implicated in a variety of physiological and pathological conditions due to its regulation of cell-cell recognition, cell differentiation and tissue remodeling. Reduced placental CD147 expression is associated with pre-eclampsia, but the mechanism of actions remains unclear. STUDY DESIGN, SIZE, DURATION A loss of function approach or functional blocking antibody was used to study the function of CD147 in primary human cytotrophoblasts isolated from first trimester termination of pregnancy and/or in the BeWo cell line, which possesses characteristics of human cytotrophoblasts. PARTICIPANTS/MATERIALS, SETTING METHODS CD147 expression was analyzed by immunofluorescence staining and western blotting. CD147-associated protein complex on plasma membrane were separated by blue native gel electrophoresis and identified by reversed-phase liquid chromatography coupled with quadrupole time-of-flight hybrid mass spectrometer. Cell proliferation and invasion were determined by fluorometric cell proliferation assays and transwell invasion assays, respectively. Matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA) activities were measured by gelatin gel zymography and uPA assay kits, respectively. Cell migration was determined by wound-healing assays. Cell fusion was analyzed by immunocytochemistry staining of E-cadherin and 4',6-diamidino-2-phenylindole. The transcripts of matrix proteinases and trophoblast lineage markers were measured by quantitative PCR. Extracellular signal-regulated kinase (ERK) activation was analyzed by western blot using antibodies against ERKs. MAIN RESULTS AND THE ROLE OF CHANCE CD147 exists as protein complexes on the plasma membrane of primary human cytotrophoblasts and BeWo cells. Several known CD147-interacting partners, including integrin β1 and monocarboxylate transporter-1, were identified. Suppression of CD147 by siRNA significantly (P < 0.05) reduced trophoblast-endometrial cell interaction, cell invasion, syncytialization, differentiation and ERK activation of BeWo cells. Consistently, anti-CD147 functional blocking antibody suppressed the invasiveness of primary human cytotrophoblasts. The reduced invasiveness was probably due to the restrained (P < 0.05) enzyme activities of MMP-2, MMP-9 and uPA. LIMITATIONS, REASONS FOR CAUTION Most of the above findings are based on BeWo cell lines. These results need to be confirmed with human first trimester primary cytotrophoblast. WIDER IMPLICATIONS OF THE FINDINGS This is the first study on the role of CD147 in trophoblast function. Further investigation on the function of CD147 and its associated protein complexes will enhance our understanding on human placentation. STUDY FUNDING/COMPETING INTEREST(S) This work was supported in part by the University of Hong Kong Grant 201011159200. The authors have no competing interests to declare.

STUDY QUESTION: What is the nature of cellular Corin expression in human gestational tissues? SUMMARY ANSWER: CORIN is expressed in non-pregnant late secretory phase endometrium, first trimester human implantation sites and is up-regulated with decidualization ex vivo. WHAT IS KNOWN ALREADY: Adequate trophoblast invasion and spiral artery remodeling/transformation is critical for successful implantation. CORIN, best known for its role in activating atrial natruietic peptide (ANP) to regulate blood pressure, has recently been proposed to be centrally involved in trophoblast invasion and spiral artery remodeling. It is postulated that ANP, activated by CORIN, promotes trophoblast invasion and that a deficiency causes pre-eclampsia. Mice deficient in either Corin or ANP displayed poor trophoblast invasion, impaired spiral artery remodeling and phenocopied human pre-eclampsia. However, the precise cellular localization of CORIN within human gestational tissues has not been well characterized. STUDY DESIGN, SIZE, DURATION: We measured CORIN protein localization in a number of human gestational tissues relevant to early embryo/placental implantation: non-pregnant (NP) endometrial biopsies (n = 5 per phase of the menstrual cycle), first trimester placental bed biopsies (n = 12) and pre-term control (n = 10) and severe early onset preeclamptic placentas (n = 15). Endometrial stromal cells were isolated from human endometrial biopsies (n = 5) and induced to decidualize ex vivo. Finally, CORIN concentrations were measured in serum obtained from pregnant women during the first trimester of whom, 56 subsequently ended up with a healthy term delivery (controls), 18 developed fetal growth restriction (FGR) and 21 had a miscarriage. PARTICIPANTS/MATERIALS, SETTING, METHODS: We performed immunohistochemistry to assess CORIN localization. Changes in Corin mRNA expression in human endometrial stromal cells decidualized ex vivo were measured by quantitative RT-PCR, and levels of CORIN within human sera were measured by ELISA. MAIN RESULTS AND THE ROLE OF CHANCE: CORIN was expressed in both NP late secretory phase endometrium and first trimester decidua within placental bed biopsies. Importantly, decidualization of primary human endometrial cells ex vivo significantly increased Corin expression (P < 0.05). CORIN was also detected within the villous cytotrophoblast, but there was no change in mRNA levels in placentas complicated by severe preterm pre-eclampsia when compared with pre-term controls. Although CORIN was detected in first trimester serum, levels did not change across gestation, nor could they predict miscarriage or FGR (other disorders of impaired placental invasion). LIMITATIONS, REASONS FOR CAUTION: Owing to the fact that we utilized early pregnancy human specimens, this is mainly a descriptive study with a limited amount of functional experiments. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to thoroughly characterize Corin mRNA and protein expression in human gestational tissue. Our findings support recent data from murine studies collectively suggesting that CORIN plays a critical role in trophoblast migration and spiral artery remodeling during early pregnancy in humans. Therefore, further studies of CORIN biology in early pregnancy may identify new therapeutic targets to improve implantation quality in early pregnancy and potentially reduce the rates of pregnancy complications caused by inadequate implantation (pre-eclampsia, FGR and miscarriage). STUDY FUNDING/COMPETING INTEREST(S): This study was supported by The National Health and Medical Research Council of Australia (Salary support #490970, #490995). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. The authors declare that no competing interests exist.

BACKGROUND Dendritic cells (DCs), which are biased toward a tolerogenic profile, play a pivotal role in tissue-remodeling processes and angiogenesis at the maternal-fetal interface. Here, we analyzed the effect of trophoblast cells on the functional profile of DCs to gain insight on the tolerogenic mechanisms underlying the human placental-maternal dialog at early stages of gestation. METHODS DCs were differentiated from peripheral blood monocytes obtained from fertile women (n = 21), in the presence of interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor during 5 days in culture. Then, DCs were cultured with trophoblast cells (Swan-71 cell line obtained from normal cytotrophoblast, at 7 weeks) for 24 h and for an additional 24 h in the absence or presence of lipopolysaccharide (LPS) from Escherichia coli. DCs were recovered and used for flow cytometry, enzyme-linked immunosorbent assay, RT-PCR and suppression and migration assays. RESULTS Trophoblast cells significantly prevented the increase in CD83 expression induced by LPS without affecting the expression of CD86, CD40 and human leukocyte antigen-DR (P < 0.05). Trophoblast cells significantly decreased the production of IL-12p70 and tumor necrosis factor-α, while it increased the production of IL-10 (P < 0.05). No changes were observed in the production of IL-6 and monocyte chemotactic protein-1. The culture of DCs with trophoblast cells, also suppressed the stimulation of the allogeneic response triggered by LPS (P < 0.05). Conditioned DCs were able to increase the frequency of CD4 + CD25 + Foxp3 cells and this effect was accompanied by an increase in indoleamine 2, 3-dioxygenase expression in DCs (P < 0.05). CONCLUSIONS The interaction of DCs with trophoblast cells promotes the differentiation of DCs into cells with a predominantly tolerogenic profile that could contribute to a tolerogenic microenvironment at the maternal-fetal interface.

background: Fetal cells are shed from the regressing chorionic villi and it is possible to retrieve extravillous cytotrophoblast cells by transcervical sampling. The abundance of trophoblast cells in transcervical samples suggests that this non-invasive approach could distinguish between normal and abnormal pregnancies, such as an ectopic pregnancy (EP) and blighted ovum (BO). We aim to identify and quantify fetal trophoblast cells in the cervical canal during the first trimester to assess their usefulness to predict an abnormal pregnancy. methods: Patients, age 18–45, presenting with a normal intrauterine pregnancy (IUP; n ¼ 37), diagnosis of EP (n ¼ 10) or BO (n ¼ 5) were enrolled for collection of transcervical specimens using a cytobrush and fixative rinse. Non-pregnant, nulliparous women (n ¼ 7) were included as negative controls. Cells were cleared of mucus by acidification, prepared on microscope slides and labeled with a monoclonal antibody recognizing the trophoblast marker, human leukocyte antigen (HLA)-G. HLA-G positive and negative cells were counted to calculate the ratio of trophoblast cells to total cervical cells. results: Trophoblast cells were observed in 35/37 normal IUP, 6/10 EP and 4/5 BO specimens. The average frequency of HLA-G positive cells in the normal IUP cervical samples was 1 in 2000, which was 4-fold higher than samples from patients with EP or BO (P , 0.001). Receiver operating characteristic analysis showed that EP and BO pregnancies were distinguishable from normal pregnancies with 93% sensitivity, 95% specificity, 97% positive predictive value and 87% negative predictive value. conclusions: This pilot study presents evidence that trophoblast cells can be reliably obtained and identified among cervical cells in the first trimester by immunohistochemical staining for HLA-G, and suggests for the first time that abnormal pregnancies may be predictable based on the abundance of trophoblast cells in the cervical canal.

BACKGROUND: Tubal pregnancy (TP) is twice as common following IVF when compared with natural conception. This is surprising, since embryo transfer is aimed for an accurate area in the uterine cavity. We thus hypothesized that either the embryo or the Fallopian tube actively participates in a pathological process leading to implantation outside the uterine cavity. Since we recently found that E-cadherin expression is a useful marker of endometrial receptivity, we considered that it may have a role in TP following IVF. Therefore, the aim of this study was to compare E-cadherin expression and localization in tubal implantation sites from spontaneous TP and TP post-IVF. METHODS: We compared E-cadherin immunohistochemistry levels on cross-sections of Fallopian tubes in 11 spontaneous (antegrade) versus 13 post-IVF (retrograde) TP. The intensity of immunoreactivity was scored in a semi-qualitative blinded manner. RESULTS: The semi-quantitative intensity score in IVF tubal samples was more than double that observed in spontaneous TP (16.9 versus 7.3, respectively, P< 0.0005). E-cadherin showed the most intense immunostaining in cytotrophoblast cells of chorionic villi in ectopic TP post-IVF compared with negative or weak staining in spontaneous ectopic TP. CONCLUSIONS: E-cadherin can serve as a marker of implantation. Differential expression of this adhesion molecule in TP post-IVF, when compared with natural conception, may reflect a different mechanism of embryo implantation. Moreover, the observation that E-cadherin is mostly expressed in trophoblasts, and not in the tubal wall, suggests that the preimplantation embryo may actively participate in locating a suitable implantation site.

BACKGROUND: An effective embryonic–maternal interaction is crucial for successful human pregnancy. Failure of this process is a major cause of infertility and can lead to placental dysfunction resulting in recurrent miscarriage, fetal retardation and pre-eclampsia. Research is severely constrained by ethical and practical considerations; therefore, we aimed to generate cytotrophoblast stem (CTBS) cell lines from human embryonic stem cells (HESCs). METHOD: beta-HCG was used as a marker of viable trophoblast cells. In defined culture, embryoid bodies were generated from HESCs and selected for trophoblast enrichment by rounds of cellular aggregation and disaggregation. Distinct CTBS cell lines were isolated and characterized. Spheroid cytotrophoblast bodies were generated and their interaction with luteal-phase endometrial stroma was analysed by real-time image analysis. RESULTS: Three CTBS cell lines were derived, which were maintained in the absence of residual HESCs, fibroblast feeder cells or extracellular matrix. CTBS cells displayed typical cytotrophoblast and syncytiotrophoblast characteristics and exhibited further differentiation to invasive endovascular cell phenotype. One cell line was generated with constitutive expression of enhanced green fluorescent protein (eGFP). Spheroid trophoblast bodies mimicked closely the early invasive stages of implantation when incubated with human endometrial stromal preparations in vitro. CONCLUSION: These human CTBS cell lines are a significant new model for investigating human placentation and may have considerable potential in cell therapy applications.

BACKGROUND: Early in vitro studies of blastocyst formation in several primate species have demonstrated the feasibility of such studies. Initial studies of in vitro-fertilized oocytes cultured with buffalo rat liver cells suggested that other epithelial cells might be used to assess blastocyst adherence and penetration in vitro. METHODS: Macaque blastocysts were incubated with different epithelial cell lines or with Matrigel. The interaction was studied using light and transmission electron microscopy. RESULTS: In general, zona-free blastocysts attached 2 days after placing on the substrates. MDCK cells provided optimal conditions for blastocyst development. The best preparations showed some development of an amniotic cavity and distribution of cytotrophoblast and syncytial trophoblast. Distribution of syncytial trophoblast at the margin of the site and cytotrophoblast centrally was similar to that seen at the trophoblastic plate stage in this species. However, there was less syncytial trophoblast than is normally found at this stage, and total time from fertilization to the trophoblastic plate stage was delayed 2 days. CONCLUSIONS: While in vitro studies with blastocysts cannot completely mimic the intrauterine environment, they can illustrate some of the potential interactions and provide a situation in which parameters may be manipulated.

BACKGROUND: The study was designed to investigate the ultrastructural features of the early human feto-maternal interface when generated by in-vitro co-culture, and compare these with findings reported previously from human pregnancies. METHODS: Placental villi and decidua parietalis tissues from 8-12 week pregnancies were co-cultured in vitro over a 4-day period. The co-incubations were ended at 24 h intervals and processed for electron microscopical studies, and for immunocytochemistry using anti-cytokeratin antibody (CAM 5.2) for trophoblast. RESULTS: Loss of the syncytium at points of contact with the decidual stroma, cytotrophoblast column formation, differentiation and invasion of extravillous trophoblast (EVT) cells into the decidual stroma over the 4-day period of co-culture were observed. Cellular components, such as actin filaments, microtubules, glycogen granules and lamellipodic processes found in EVT cells were consistent with active cellular locomotion. CONCLUSIONS: These ultrastructural studies emphasize the usefulness of this model in investigating the formation of the feto-maternal interface of human pregnancy. The recruitment of cytotrophoblast to the syncytium by a process involving fusion of the intervening plasma membranes, and the migration of EVT cells causing little or no damage to the surrounding decidual cells, resemble in-vivo data.

The present study was designed to investigate the effects of immunoglobulin G obtained from patients with antiphospholipid syndrome (APS) on in-vitro models of trophoblast invasiveness and differentiation. We tested the binding of affinity-purified immunoglobulin G to human primary trophoblast cells. These antibodies affected the invasiveness and differentiation of cytotrophoblast cells after binding to the cell surface. In addition, we determined whether the drugs used to treat APS might be able to restore the trophoblast functions. Low-molecular weight heparin, in a dose-dependent manner, significantly reduced the immunoglobulin G binding to trophoblast cells and restored in-vitro placental invasiveness and differentiation. No effect was observed in the presence of acetylsalicylic acid. These observations may help in understanding the role of these treatments in women with APS.

Premature rupture of fetal membranes can have serious clinical implications, especially for the initiation of preterm labour and its consequences. To account for this phenomenon many studies have attempted to identify membrane features that may be uniquely associated with the site of rupture. Our previous work has identified an area of the fetal membrane, following spontaneous term birth which exhibits alterations consistent with structural weakness. The aim of this study was to determine if these changes existed prior to labour. In formalin-fixed paraffin-embedded tissue sections an area of the fetal membrane overlying the cervix, termed the 'cervical membranes', was characterized by an increased thickness of the connective tissue layer (215% increase, P < 0.01) and decreased thickness of both the cytotrophoblast (36% reduction, P < 0.01) and decidual layers (64% reduction, P < 0.01) compared to the rest of the membrane. This resulted in the cervical membranes being significantly thinner (P < 0.05) than the rest of the membrane. Similar changes were also detected in frozen sections of fetal membranes. These regional differences have two important implications in that: (i) the cervical membrane may represent a region of structural weakness susceptible to rupture during labour, and (ii) the paracrine relationships between fetal membranes and the myometrium may be qualitatively affected within different regions of the uterus.

Pregnancy failure in mice has been associated with increased placental concentrations of the pro-inflammatory cytokine tumour necrosis factor-alpha (TNF-alpha). To investigate the role of uterine TNF-alpha in human first trimester miscarriage, we have collected human decidual and trophoblast tissue from women (i) undergoing surgical termination of pregnancy (n = 27), (ii) undergoing a sporadic miscarriage (n = 20) and (iii) with a history of recurrent pregnancy loss [>3 consecutive pregnancy losses (n = 26)] undergoing a further miscarriage. Formalin fixed tissues were examined for TNF-alpha mRNA (in-situ hybridization) and protein (immunohistochemistry). In decidua from all three groups, TNF-alpha protein and mRNA were co-localized to the decidual stroma, the luminal surface of some maternal vessels and to the glandular epithelium. Chorionic villi from the normal pregnancy and the sporadic miscarriage group exhibited co-localized TNF-alpha protein and mRNA in the syncytiotrophoblast and cytotrophoblast. In the recurrent miscarriage group, however, 63.6% of the biopsies showed positive immunostaining in only the cytotrophoblast, compared with 4.0% of women undergoing surgical termination of pregnancy and 0.0% of women with a sporadic failed pregnancy (P < 0.001). TNF-alpha mRNA was also localized exclusively to this layer. This may be a secondary effect caused by a different mechanism of pregnancy loss unique to this subgroup.

A cell line has been established from human placentae at the first trimester of normal pregnancy. The cell line was obtained by culture of purified cytotrophoblast cells in serum-free medium supplemented with epidermal growth factor, insulin, dexamethasone and 0.1% bovine serum albumin. The cells can be subcultured for >30 passages in one to three splits. All the cells were mononuclear epithelial-like cells positive to cytokeratin 18, gonadotrophin-releasing hormone (GnRH), neuropeptide Y, neurotensin, leucine-enkephalin, dopamine and 5-hydroxytryptamine immunocytochemical staining. The cells secreted GnRH, progesterone and oestradiol (in the presence of testosterone) but little human chorionic gonadotrophin and no beta-endorphin. The cell line showed human karyotypes and had a population doubling time of 48 h in serum-free medium. However, the cells would stop growing in the medium containing fetal bovine serum. A normal cytotrophoblast cell line established in serum-free medium will be particularly useful in the study of cytotrophoblast cell proliferation and differentiation.

The aim of our study was to localize oestrogen receptor (ER) and progesterone receptor (PR) in the trophoblast population at the maternofetal interface in early pregnancy. Rat monoclonal antibodies to human ER and PR were used to study 44 cases of chorionic villi and 82 cases of decidua using an immunocytochemical method. The PR-immunoreactive products were localized in the nuclei of syncytiotrophoblast and cytotrophoblast cells of the villi. Specific staining was also present in the cytoplasm. Villous stroma and vessels were stained occasionally. Using cytokeratin staining in an adjacent section or double staining with PR and cytokeratin, the distribution of invading trophoblast cells and their PR expression were examined. In decidual stroma, a type of interstitial cell was identified which simultaneously expressed cytokeratin and PR in the cytoplasm, indicating that the invading trophoblast cells may express PR. All extravillous populations at the interface were positive for PR, including the syncytial lining of the decidual surface, the cytotrophoblast column, the cytotrophoblast shell and the interstitial trophoblast. The immunoreactivity of PR was also localized in the nuclei of vascular endothelial cells, whereas Factor VIII was localized in the cytoplasm of the same cells, thus confirming their endothelial nature. In contrast to PR, little ER could be detected in the trophoblast cell population using anti-ER antibody D75 in our study.

Abnormalities of pregnancy such as pre-eclampsia and intrauterine growth retardation are characterized by shallow trophoblastic invasion of the placental bed, the precise molecular pathophysiology of which remains to be fully elucidated. An in-vitro model involving a co-culture of first trimester placental villi and decidua parietalis explants (of 8-12 weeks gestation) was developed and used to characterize the migration and local invasion of trophoblast cells. Trophoblast proliferation (confirmed by Ki-67 immunostaining), differentiation and loose attachment of placental villi to the underlying decidual epithelium or stroma occurred within the first 24 h of co-culture. This was followed by erosion of the syncytial layer of the placental villi and commencement of a progressive cytotrophoblast invasion after 48 h of co-culture, which continued until 120 h, when the experiments were terminated. E-cadherin was expressed at the interfaces between trophoblast cells within the villi, but expression of this adhesion molecule seemed to be down-regulated in the invasive trophoblast cells. Our results suggest that the model could be useful in investigating the factors that control early human placentation and the feto-maternal interface.

Many features contributing to the success of pregnancy in humans have been identified over the last 20 years. Trophoblast cells (which form the interface of fetal tissue with the mother) have specialized immunological features which may confer unique transplantation protection for the fetus throughout pregnancy. Both syncytiotrophoblast and cytotrophoblast cells do not express classical class I [human leucocyte antigen (HLA)-A or -B] or II (HLA-DP, -DQ or -DR) major histocompatibility complex (MHC) alloantigens, and the regulation of these cell surface glycoproteins appears to be at the transcriptional level. In contrast, extravillous cytotrophoblast cells express the non-classical class I MHC molecule HLA-G. One form of HLA-G (HLA-G1) is potentially capable of presenting a variety of peptide antigens to T cells. Alternatively, HLA-G may act as a cell surface class I MHC molecule, protecting cytotrophoblast from maternal MHC non-restricted natural killer (NK) cell attack; the expression of HLA-G by otherwise HLA-null cell transfectants has been shown to decrease their sensitivity to NK cell-mediated cytolysis. All fetal trophoblast populations throughout gestation express high levels of cell surface complement regulatory proteins, providing protection from complement-mediated damage at the materno-fetal interface. Analyses of trials with allogeneic leucocytes for the treatment of recurrent spontaneous abortion show little evidence of an improved success rate in immunized patients.

We investigated the expression of the tumour-suppressor and cell cycle control protein p53 in human first trimester and term placenta, three choriocarcinoma cell lines (Jeg-3, JAR, BeWo) and human choriocarcinoma. Using monoclonal antibodies against p53 (DO-7, Ab-6, DO-1, PAb 1801), paraffin-embedded sections of first trimester and full-term placentae, human choriocarcinoma and Jeg-3, JAR and BeWo, as well as cytospins, were evaluated immunohistochemically. In addition, Western blots were carried out with the same antibodies on choriocarcinoma cell lines. In placentae, a small number of villous and extravillous cytotrophoblast cells, as well as very few syncytiotrophoblast cells, stained intensively. Also, p53 was visualized in some nuclei of the placental basal plate, whereas stroma and endothelium were negative for p53. Jeg-3, JAR and BeWo also showed a positive nuclear reaction with all applied antibodies. In paraffin-embedded sections of human choriocarcinoma, staining was confined to the nuclei of malignant cells. The results suggest that p53 is overexpressed not only in malignant tumour cells but in certain trophoblast cell populations of the human placenta as well.

Chorionic gonadotrophin (CG) is the first clear embryonic signal during early pregnancy in primates. CG has close structural and functional similarities to pituitary luteinizing hormone (LH) which is regulated by gonadotrophin releasing hormone (GnRH). To study the regulatory mechanism of CG secretion in primate embryos, we examined the production and timing of secretion of GnRH in peri-implantation embryos of the rhesus monkey. In-vivo fertilized/developed morulae and early blastocysts, recovered from non-superovulated, naturally-bred rhesus monkeys by non-surgical uterine flushing, were cultured in vitro to hatched, attached and post-attached blastocyst stages using a well-established culture system. We measured GnRH and CG in media samples from cultured embryos with a sensitive radioimmunoassay and bioassay, respectively. The secretion of GnRH (pg/ml; mean +/- SEM) by embryos (n = 20) commenced from low levels (0.32 +/- 0.05) during the pre-hatching blastocyst stage to 0.70 +/- 0.08 at 6-12 days and 1.30 +/- 0.23 at greater than or equal to 13 days of hatched blastocyst attachment and proliferation of trophoblast cells. GnRH concentrations in culture media obtained from embryos (n = 5) that failed to hatch and attach were mostly undetectable (less than or equal to 0.1). Samples that did not contain detectable GnRH failed to show detectable CG. Immunocytochemical studies, using a specific monoclonal anti-GnRH antibody (HU4H) as well as polyclonal antisera (LR-1), revealed that immunopositive GnRH cells were localized in pre-hatching blastocysts (n = 4), in blastocysts (n = 2) after 5-10 days of attachment and in monolayer cultures (n = 4) of well-established embryonic trophoblast cells. GnRH positive staining was seen only in cytotrophoblasts but not in syncytiotrophoblasts. Similarly, cytotrophoblast, but not syncytiotrophoblast, cells of the rhesus placenta were immunopositive. In controls, either in the absence of antibody or in the presence of antibody pre-absorbed with GnRH, these cells failed to show stain. These observations indicate, for the first time, that an immunoreactive GnRH is produced and secreted by blastocysts during the peri-attachment period and by embryo-derived cytotrophoblast cells in the rhesus monkey.

Parathyroid hormone related protein (PTHrP) is an important humoral factor in hypercalcaemia of malignancy. In addition there is increasing evidence that this peptide has a physiological role in fetal development, especially in cellular growth and differentiation. Both in-situ hybridization and immunohistochemistry were used together and for the first time to identify sites of PTHrP gene expression and peptide in fetal and extra-embryonic tissues of first trimester human pregnancy. PTHrP mRNA and peptide were identified in the avascular amnion and the syncytiotrophoblast while mRNA alone was expressed in the cytotrophoblast. Its expression in these extra-embryonic tissues is consistent with postulated roles for PTHrP in implantation, relaxation of endometrial muscle and regulation of vascular tone. Expression of both mRNA and peptide occurred in endo-, meso- and ectodermal structures of the fetus, consistent with local production of the peptide rather than cellular uptake from amniotic fluid and supporting a role for PTHrP in cellular growth and differentiation.

In the present study, we investigated the biological characteristics of different molecular forms of chorionic gonadotrophin (HCG) secreted by the human cytotrophoblast during its morphological and functional differentiation in culture. Highly purified cytotrophoblasts were prepared from term placentae and cultured for 24 to 96 h in the absence or presence of 8-bromo-3',5'-cAMP. Media were collected at 24 h intervals and the secreted isoforms of HCG were then separated by polyacrylamide gel isoelectric focusing (pH range 8.0-3.0) and quantified by radioimmunoassay. The secretion of HCG was significantly increased by 8-bromo-cAMP (from 23.5 +/- 6.3 ng/ml at 24 h to 1619 +/- 835.8 ng/ml at 96 h; controls, 9.3 +/- 0.1 ng/ml at 24 h and 26.6 +/- 3.5 ng/ml at 96 h, mean +/- SD). Analysis of media concentrates from cAMP-stimulated cultures by isoelectric focusing revealed the presence of several distinct peaks of HCG within the pH range of 7.3-4.8; major peaks consistently exhibited isoelectric points (pI) of 7.3-7.0 (peak 1), 5.6-5.4 (peak 2) and 5.1-4.8 (peak 3). The relative HCG content of the most acidic peak (as % of total on gel) progressively increased with time of exposure to the cAMP analogue (from 19.8 +/- 1.6% at 24 h to 34.4 +/- 4.3% at 96 h, mean +/- SEM, P less than 0.01). HCG recovered from peak 1 exhibited the highest receptor-binding capacity and in-vitro biological potency.(ABSTRACT TRUNCATED AT 250 WORDS)