The uterine immune profile: A method for individualizing the management of women who have failed to implant an embryo after IVF/ICSI.

A unique endometrial immune reaction should occur to promote the human embryo implantation. We postulated that an immune disequilibrium may impact the initial dialogue between the mother and her embryo. In 2012, we set a method of uterine immune profiling for patients with unexplained repeated implantation failures (RIF). The method documents the local Th-1/ Th-2 equilibrium and the recruitment and state of maturation/activation of uNK cells. In function of the disequilibrium observed, personalization of assisted reproductive treatments was suggested. As the concept of personalization in function of the uterine immune profile had never been proposed, a large cohort study and a controlled cohort study were first conducted in RIF patients. 80 % of the RIF patients showed a local disequilibrium if compared to fertile controls. The local disequilibrium was identified in 3 categories: over-immune activation in 45 %, low- local immune activation in 25 % and mixed profile in 10 %. Personalization of treatments in function of the immune profile allowed to restore a live birth rate by 40 % at the following embryo transfer. RIF patients with endometriosis show some particularities regarding their immune profiles. We also suggested that immunotherapy (corticoids, intralipids) may have targeted indications based on a better understanding of the immune type of disequilibrium documented. Personalization of treatments for RIF patients seems to be essential to promote the subsequent live birth rate. The endometrial immune profiling is an innovative method aiming to detect a local immune disequilibrium and, if present, to test preventively its correction under treatment.

Sera from early-onset, severely preeclamptic women directly modulate HLA-E expression in the EA.hy296 endothelial cell line.

The expression of endothelial HLA-E in the context of the systemic inflammatory response observed in preeclampsia has not been established. An experimental study was designed to determine the effect of the sera of pregnant women on the expression of HLA-E in EA.hy296 endothelial cells. First, measurements of protein fractions were performed in sera from early-onset, severely preeclamptic women without HELLP syndrome, in which there was no significant difference in total proteins between the groups, but a reduced level of plasma albumin and an increase in α1-globulin were observed in both groups of pregnant women compared with non-pregnant women. Measurements of colloid osmotic pressure (COP) using a recalculated albumin/globulin ratio formula determined only a significant decrease in COP in all pregnant groups compared with non-pregnant women. The expression of membrane HLA-E was increased in EA.hy296 endothelial cells stimulated with sera of early-onset, severely preeclamptic women, while recombinant interferon-γ (IFN-γ) significantly reduced the expression of membrane HLA-E. Pro-inflammatory cytokines were measured by Luminex in the serum samples, and increased levels of tumor necrosis factor (TNF) and decreased levels of IFN-γ were observed in early-onset, severe preeclampsia compared with normal pregnancy. Moreover, soluble HLA-E was detected in these serum samples by Western blot and ELISA, but no significant difference was found. This raises the possibility that a systemic inflammatory response promotes a compensatory mechanism of COP balance in severe preeclampsia by release of inflammation-induced factors, including endothelial HLA-E. Evidence is now provided regarding HLA-E expression by EA.hy296 cells.

New pre-conception immune biomarkers for clinical practice: interleukin-18, interleukin-15 and TWEAK on the endometrial side, G-CSF on the follicular side.

Identification of biomarkers of optimal uterine receptivity to the implanting embryo as well as biomarkers of oocyte competence would undoubtedly improve the efficiency of assisted reproductive technology (ART). Expression of IL-15 and IL-18 has been shown to be different in patients with failed implantation after IVF/ICSI compared with fertile controls and both correlate with local uNK (CD56+) recruitment and angiogenesis. Tumor necrosis factor weak inducer of apoptosis (TWEAK) has been described in mice as a potent early immune regulator able to protect the conceptus. The results of our studies in human suggest that TWEAK modulates the IL-18 related cytotoxicity of uNK cells. Quantification of IL-18, TWEAK and IL-15 mRNA expression by real-time PCR in endometrial tissue collected in mid-luteal phase of non-conception cycles allowed documentation of physiological events that occur at the time of uterine receptivity. Such information may be useful for the physician especially in patients where embryos fail to implant. Cytokine quantification may assist in understanding the mechanisms leading to repeated IVF/ICSI failure: either depletion of cytokines necessary for the apposition-adhesion, or an excess of cytokines leading to local cytotoxicity, may impair the implantation of the embryo. Other new data suggest that a pre-conception dialogue mediated by the oocyte and the follicular fluid and the oocyte may contribute to later implantation success. Follicular concentration of G-CSF appears as a useful biomarker of oocyte competence before fertilization. Moreover both in human and animal models, evidence of a role of the endometrium as a biosensor of the embryo is emerging.

Characterization of the main placental cytokine profiles from HIV-1-infected pregnant women treated with anti-retroviral drugs in France.

Cytokines are involved in regulating HIV-1 infection. They are also placental environment major components. We assessed the potential impact of HIV-1 infection and/or anti-retroviral drugs on the placental cytokine profiles that may be involved in controlling HIV-1 placental dissemination. Placental explants were obtained after elective caesarean section from anti-retroviral-treated HIV-1-infected pregnant women and from HIV-1 non-infected pregnant women. The main placental cytokines were assessed for protein secretion in the supernatants of 24-h placental culture explants and/or in uncultured placental explants for mRNA expression levels. The cytokine profiles were different between the HIV-1-infected and the non-infected groups. Higher medians of leukaemia inhibiting factor (LIF), tumour necrosis factor (TNF)-alpha and interleukin (IL)-8 secretion were found in the 24-h culture supernatant of term placenta from HIV-1-infected women. High median levels of IL-16 and regulated upon activation normal T cell expressed and secreted (RANTES) levels were found in both groups. The mRNA expression medians were lower for TNF-alpha and IL-8 and higher for stromal cell-derived factor-1 (SDF-1) in uncultured placental explants from HIV-1-infected women. In the HIV-1-infected group, but not in the non-infected group, the secretion levels of TNF-alpha and IL-8, as well as their mRNA expression levels, were highly positively correlated; furthermore, their secretion levels were correlated positively with LIF and IL-10 secretion levels. We found no correlation between the cytokine levels and the immunovirological status of the HIV-1-infected mothers or the type or duration of treatment. These results highlight the potential impact of HIV-1 and of the anti-retroviral treatments on the placental cytokines pattern, independently of their anti-viral activity.

Down modulation of TNF-alpha mRNA placental expression by AZT used for the prevention of HIV-1 mother-to-child transmission.

Mechanisms of HIV-1 in utero mother-to-child transmission (MTCT) protection provided by AZT are not completely understood. The placental cytokine network is involved in the control of HIV-1 in utero transmission but the effect of AZT on this network is unknown. To evaluate the effects of AZT on placental cytokine expression, the chorionic villi from HIV-1 uninfected women term placentae were cultured with 0, 100, and 2,000 ng/ml AZT. Tissue fragments were harvested at days 1, 4, and 7 to determine the level of cytokine mRNA by real-time RT-PCR. The viability and morphology of the placental histocultures were monitored by the expression of beta-human chorionic gonadotropin (beta-hCG) gene, lipopolysaccharide (LPS) activation, and microscopic examination. AZT at 2,000 ng/ml significantly down-regulated TNF-alpha mRNA expression at day 1 and day 4, but had no effect on beta-hCG, stromal cell-derived factor 1 (SDF-1), and IL-10 gene expression. AZT did not induce any deleterious impact on placental tissue structure. Furthermore, activation of chorionic villi by LPS for 24 h up-regulated IL-10 and TNF-alpha mRNA expression. Down-regulation of TNF-alpha mRNA could represent a mechanism through which AZT can decrease the risk of HIV-1 MTCT, in addition to its direct effect on HIV-1 replication.

Cell-free HIV type 1 infection is restricted in the human trophoblast choriocarcinoma BeWo cell line, even with expression of CD4, CXCR4 and CCR5.

The restriction of cell-free HIV-1 infection has been demonstrated in placental trophoblast choriocarcinoma BeWo cells. We tried to determine the level of the viral replication cycle at which this restriction occurs. BeWo cells produce infectious viruses after transfection with HIV-1 plasmids, independently of viral tropism. CCR5 and CXCR4, but not the CD4 molecule, were detected at the cell surface. We therefore derived CD4-expressing clones from transfected BeWo cells. Cell-free virus infection of these clones resulted in neither virus production nor viral sequence integration, indicating that the restriction occurs before integration of the virus. If we used luciferase reporter viruses pseudotyped with HIV-1 Env R5 and X4 for infection, no luciferase activity was detected, even in the BeWo-CD4+ clone, in contrast to what was observed in VSV-G pseudotyped virus infection. Our results show that infection of trophoblast-derived cells with cell-free virus is at least restricted at the level of entry. Thus, BeWo is an interesting human placental cell line that is resistant to HIV-1, even if CD4, CXCR4, and CCR5 are expressed.

Combined treatment by pentoxifylline and tocopherol for recipient women with a thin endometrium enrolled in an oocyte donation programme.

BACKGROUND: To evaluate the effect of an antifibrotic treatment by a combination of pentoxifylline (PTX) and tocopherol (vitamin E) in patients with a thin endometrium who were enrolled in an oocyte donation programme. METHODS: Eighteen oocyte recipients who failed to develop a pre-ovulatory endometrial thickness of at least 6 mm after receiving vaginal micronized estradiol were enrolled in the study. The patients received a combination of PTX (800 mg/day) and vitamin E (1000 IU/day) for 6 months. The main outcome measurements were the change in endometrial thickness and the pregnancy and delivery rates after treatment. RESULTS: Endometrial thickness increased significantly (P <0.001), with a mean of (+/-SD) 4.9 +/-0.6 mm before and 6.2 +/- 1.4 mm after treatment, with 72% (13/18) of patients being good responders. Five patients either did not respond to the treatment or responded only slightly. Three patients, of which two had received previous radiotherapy, became spontaneously pregnant, and two became pregnant after embryo transfer. Three patients did not have embryo transfer. A total of four babies were delivered. The pregnancy rate was thus 33% and the delivery rate 27%. CONCLUSION: Treatment by combination of PTX and vitamin E appears to improve the pregnancy rate in patients with a thin endometrium by increasing the endometrial thickness and improving ovarian function. This was especially noticeable in patients who had previously received total body irradiation.

Concentration of leukaemia inhibitory factor (LIF) in uterine flushing fluid is highly predictive of embryo implantation.

BACKGROUND: There is strong evidence that locally secreted cytokines control the implantation process and can cause implantation failure. Uterine flushing fluids were analysed to determine their concentrations of leukaemia inhibitory factor (LIF) and tumour necrosis factor (TNF). METHODS AND RESULTS: We began by flushing the uterine cavities of 33 infertile patients on day 26 of two consecutive cycles. The concentrations of LIF (by enzyme-linked immunosorbent assay) and TNF (by bioassay) were significantly correlated during these cycles (r = 0.762, P = 0.0001 and r = 0.822, P = 0.001 respectively) and hence reliable. Then, after a reference flushing of 30 infertile patients, we followed the outcome of their first consecutive cycle of ovarian stimulation, which preceded either IVF or intrauterine insemination. A total of 10 patients became pregnant. The median concentration of LIF was 0 pg/ml (range: 0-177) and of TNF was 0 U/ml (range: 0-6.17) among those who became pregnant, and 203 pg/ml (range: 0-1620) and 2.14 U/ml (range: 0-16) respectively among those who did not. The LIF concentration was significantly lower in the pregnant group (P = 0.0013). CONCLUSION: A low concentration of LIF in the uterine flushing fluid at day 26 was predictive of subsequent implantation. Use of this procedure should increase the number of IVF attempts yielding successful pregnancies and also lead to corrective therapies.

Biological activity of the suppressor cells inducer factor secreted by the Jeg-3 choriocarcinoma cell line.

PROBLEM: We wanted to further study the mechanisms of immune suppression and suppression inducing capacities by choriocarcinoma products, e.g. both crude human choriocarcinoma supernatant (HCS) and especially an active fraction obtained by high performance liquid chromatography (HPLC) from the culture supernatant of the Jeg-3 human choriocarcinoma cell line, since it appeared by weight and charge criteria to be a different molecular species than the low molecular weight fraction previously isolated from mouse and human term placenta. It was important to know whether the purified material was active in vivo as it was in vitro. Therefore, we tested the effects of HCS in vivo in three systems: prevention of fetal demise in the CBA/J x DBA/2 abortion prone murine mating combination, where the effects of the HPLC purified fraction were also monitored as well as by a cell transfer system, where the suppression is revealed by a local GVH/HVG assay, and finally enhancement the survival of a mildly immunogenic tumor allograft. METHODS: An active fraction was isolated from HCS by ion exchange HPLC. Female CBA/J were mated with DBA/2 and the influence of 3 intraperitoneal injections of both crude HCS and the active fraction was evaluated by monitoring the percentage of fetal resorptions. Simultaneously, on the day when resorptions were counted, maternal splenocytes from these females were harvested and were injected by the subcutaneous way in C3H/HEJ hind feet. The lymph node reactivity (HVG + GVH) was assessed by [3H]thymidine intake by cells harvested from the draining popliteal lymph nodes. For assessment of influence of HCS on allograft rejection, BALB/b (H-2b) mice received a subcutaneous injection of allogeneic P815 tumor cells (H-2d). The influence of HCS injections on tumor survival was analyzed by regular measurements of the mean tumor diameter. RESULTS: Intraperitoneal injection of HCS reduced fetal resorptions from 24.7 to 13%. Injection of the in vitro active fraction induced the same rate of reduction. The mean intensity of HvG/GvH reaction was 13400 cpm per lymph node when splenocytes from the control group were injected compared to 2900 cpm when splenocytes from treated mice were used. P815 tumor allografts were completely rejected in all cases after 21 days. Weekly subcutaneous injections of HCS prolonged tumor survival in all cases up to at least 30 days. CONCLUSION: The fraction isolated from HCS increased very efficiently the survival of allografts as well as those of allogeneic fetuses in a resorption prone murine mating. The choriocarcinoma cell line might prove to be a useful source of immunosuppressive materials, which could otherwise be important for the fetal-maternal tolerance and a successful pregnancy.

Follicular fluid concentration of leukaemia inhibitory factor is decreased among women with polycystic ovarian syndrome during assisted reproduction cycles.

BACKGROUND: The possibility that a specific cytokine profile could be detected in the ovaries of patients with polycystic ovarian syndrome (PCOS) was investigated. METHOD: Enzyme-linked immunosorbent assay (ELISA) or bioassays were used to assess the concentrations of leukaemia inhibitory factor (LIF), tumour necrosis factor, interleukin 11, gamma interferon, progesterone and oestradiol in follicular fluids from preovulatory follicles collected after ovarian stimulation from 15 PCOS patients, 15 infertile control patients with regular cycles, and 8 oocyte donors. RESULTS: LIF and progesterone concentrations were significantly lower in the follicular fluid of PCOS patients (LIF median: 265 pg/ml) compared with controls (LIF median: 816 pg/ml); LIF and progesterone follicular fluid concentrations were correlated (r = 0.720, P = 0.0001). The LH/FSH ratio was negatively correlated with LIF concentrations (r = - 0.714, P = 0.0075). Although the PCOS and control patients did not differ significantly in age, ovarian reserve or IVF indication, the implantation rate was significantly lower among the women with PCOS (IR = 9 versus 21%, P = < 0.01). CONCLUSION: The specific cytokine profile of the PCOS patients is probably related to the lower implantation rate since follicular fluid LIF appears to function as an embryotrophic agent.

Cytokines and anorexia nervosa.

OBJECTIVE: Recent studies have indicated that the inflammatory cytokines could be implicated in anorexia nervosa and in its complications. To determinate the potential role of interleukins (IL-1, IL-2, IL-4, IL-6, IL-10), interferon (IFN gamma), tumor necrosis factor (TNF-alpha), and transforming growth factor (TGF-beta2) in anorexia nervosa, serum concentrations of these cytokines were measured in patients suffering from anorexia nervosa in comparison to healthy subjects. METHOD: Twenty-nine anorexic women according to DSM-IV criteria participated in the study. The control group consisted of 20 healthy women without eating disorders, mood disorders, and immunological disorders. RESULTS: We find that serum IL-2 and TGF-beta2 concentrations were both significantly decreased in anorexic patients, although the other cytokines did not differ significantly between the two groups. CONCLUSION: Our results show that in patients with anorexia nervosa, there are lower levels of specific cytokines (especially IL-2 and TGF-beta2). These levels may reflect the combination of impaired nutrition and weight loss, therefore, the dysregulation of these cytokines may contribute in anorexia's complications. Follow-up studies should examine the effects of parameters such as starvation, psychopathologic factors, and psychoneuroendocrinological perturbation which could affect interplay between cytokines, neuropeptides, and neurotransmitters.

Cell-to-cell contact results in a selective translocation of maternal human immunodeficiency virus type 1 quasispecies across a trophoblastic barrier by both transcytosis and infection.

Mother-to-child transmission can occur in utero, mainly intrapartum and postpartum in case of breastfeeding. In utero transmission is highly restricted and results in selection of viral variant from the mother to the child. We have developed an in vitro system that mimics the interaction between viruses, infected cells present in maternal blood, and the trophoblast, the first barrier protecting the fetus. Trophoblastic BeWo cells were grown as a tight polarized monolayer in a two-chamber system. Cell-free virions applied to the apical pole neither crossed the barrier nor productively infected BeWo cells. In contrast, apical contact with human immunodeficiency virus (HIV)-infected peripheral blood mononuclear cells (PBMCs) resulted in transcytosis of infectious virus across the trophoblastic monolayer and in productive infection correlating with the fusion of HIV-infected PBMCs with trophoblasts. We showed that viral variants are selected during these two steps and that in one case of in utero transmission, the predominant maternal viral variant characterized after transcytosis was phylogenetically indistinguishable from the predominant child's virus. Hence, the first steps of transmission of HIV-1 in utero appear to involve the interaction between HIV type 1-infected cells and the trophoblastic layer, resulting in the passage of infectious HIV by transcytosis and by fusion/infection, both leading to a selection of virus quasispecies.

Plasmodium falciparum induces a Th1/Th2 disequilibrium, favoring the Th1-type pathway, in the human placenta.

During pregnancy, a local and systemic Th2 bias of maternal immunity favors Th1-dependent infections such as malaria. This study measured cytokines secreted in cultures of chorionic villi, placental blood cells (PBC), and serum in term placentas from 88 malaria-infected and -noninfected Cameroon women. Interleukin (IL)--2 and --4 were consistently low; IL-1 beta, IL-6, granulocyte-macrophage colony-stimulating factor, and transforming growth factor (TGF)--beta 2 were highest in villi cultures. Tumor necrosis factor (TNF)--alpha, interferon (IFN)--gamma, and IL-10 were highest in PBC cultures. Malaria placental infection increased Th1-type cytokines, whereas Th2-type cytokines and TGF-beta 2 were unchanged. Addition of lipopolysaccharide or infected erythrocytes to cultures increased TNF-alpha, IL-1 beta, IL-6, and IL-10 secretions but not those of IFN-gamma and IL-4. Overall, Plasmodium falciparum induced a placental immune response involving both Th1- and Th2-type cell activation. Although the Th1 pathway was favored, IL-10 secretion was also increased, and this increase should be effective in protecting the placenta by controlling the negative effects of Th1 cytokines on pregnancy.

FAS/FAS ligand interaction at the placental interface is not required for the success of allogeneic pregnancy in anti-paternal MHC preimmunized mice.

PROBLEM: FAS ligand (FASL) induces apoptosis in FAS+ T cells. FAS-FASL interaction can explain tolerance of some types of allografts. Do similar interactions prevent rejection of the fetal allograft and transplacental passage of maternal T cells capable of causing GvH leading to runting? METHOD OF STUDY: We examined growth of subcutaneous EL-4 (H-2b) tumor cells in syngeneic MRL.lpr/lpr mice mated with syngeneic MRL.lpr/lpr or allogeneic C57/B1/6.lpr/lpr males. Lpr/lpr mice lack FAS and their T cells should remain effective at the fetomaternal interface. In some studies, the mice were preimmunized against C57B1/6 to enhance the likelihood of fetal rejection and/or runting. Cytotoxic lymphocyte (CTL) activity in the spleens was assessed using a 51Cr-release assay. Birth weights and weights at weaning were measured. RESULTS: Tumor rejection was delayed by about 3 days in allopregnant lpr/lpr females compared to syngeneic pregnant females occurred, but tumors were rejected and in a secondary fashion with minimal delay allopregnant females preimmunized to H-2b. CTL activity was present more in decidua than in spleen in preimmunized mice, and allopregnancy failed to reduce this activity. No neonates born to preimmunized or control lpr/lpr mothers developed runt disease. Indeed, the birth weight was greater for immunized females, but at 10 weeks, the difference compared to non-immunized females disappeared. CONCLUSIONS: FAS/FASL interaction between FAS+ T cells and FASL+ fetal trophoblasts at the fetomaternal interface is not mandatory for successful allopregnancy. The potential roles of other apoptosis-induction mechanisms at the fetomaternal interface, such as TNF/TRAIL, require critical testing before enthusiasm is merited. It maybe necessary to eliminate all such mechanisms to show a deleterious effect on pregnancy. Spontaneous deletion of all pathways seems a priori unlikely to occur, and hence, of dubious relevance to fetal loss.

Arthroscopic treatment of pigmented villonodular synovitis of the shoulder.

Pigmented villonodular synovitis (PVNS) rarely affects the shoulder. Fewer than 30 cases have been reported in the English and French language literature. In those patients, PVNS was treated by open surgery involving total or local synovectomy, sometimes associated with total shoulder replacement, cuff tear repair, or arthroplastic head resection. The authors report 2 cases of PVNS of the shoulder treated arthroscopically and discuss the advantages and limitations of this technique in the treatment of shoulder PVNS.

Comparative reactivity of different HLA-G monoclonal antibodies to soluble HLA-G molecules.

Different HLA-G monoclonal antibodies (mAbs) were first evaluated for their capability to identify soluble HLA-G (sHLA-G) in ELISA. Three of them, namely 87G, BFL.1 and MEM-G/9, when used as coating mAbs together with W6/32 capture mAb, identified beta2-microglobulin (beta2m)-associated-sHLA-G but not soluble HLA-B7 (sHLA-B7) in cell culture supernatants from transfected cells. By comparison, the anti-HLA class I mAb 90 did recognize both sHLA-G and sHLA-B7. By using these HLA-G mAbs, sHLA-G was identified in amniotic fluids as well as in culture supernatants of first trimester and term placental explants but not in cord blood. Intron 4-retaining sHLA-G isoforms were identified in some amniotic fluids by the use of an intron 4-specific mAb (16G1). Reactivity of these different HLA-G mAbs was then compared to determine their respective binding sites on soluble and membrane-bound HLA-G. Using both ELISA and flow cytometry analysis, we showed that they did not compete with each other, which suggested that they did not recognize the same determinants. Finally, we report that two mAbs directed against the alpha1 domain of HLA class I heavy chain (mAb 90 and YTH 862) did compete with 87G, therefore demonstrating that this latter mAb recognized an epitope localized on this external domain of HLA-G.

Localization of the Th2 cytokines IL-3, IL-4, IL-10 at the fetomaternal interface during human and murine pregnancy and lack of requirement for Fas/Fas ligand interaction for a successful allogeneic pregnancy.

PROBLEM: Th2 cytokines and Fas/Fas ligand interactions are proposed to be part of the placental barrier that contribute to the success of allogeneic pregnancy. To fully understand the role regulation of Th2 cytokines, we must isolate and identify the cells that produce them. We also need to assess the requirement for Fas/Fas ligand interaction in facilitating a successful allogeneic pregnancy. METHOD OF STUDY: To assess the site of production of Th2 cytokines, we used immunohistochemistry sections from placental and decidual tissue obtained at various stages of gestation in mice and humans. We used mice that are genetically deficient in Fas/Fas ligand interactions and raised specific anti-paternal CTLs by anti-paternal immunization of the mother before mating. RESULTS: The detailed results show that in both species the bulk of Th2 production may come from non-lymphoid tissues in the placenta and decidua, with a major role for trophoblasts. This raises questions about the mechanism(s) by which alloimmunization enhances local Th2 cytokine production. This issue is discussed. CONCLUSIONS: The success of allopregnancy in mice with circulating anti-paternal CTLs and deficient Fas/Fas ligand interactions rules out a mandatory role for such a mechanism in ensuring the success of allogeneic pregnancy.

The emerging role of immunoregulation of fibrinogen-related procoagulant Fgl2 in the success or spontaneous abortion of early pregnancy in mice and humans.

PROBLEM: Abortion of chromosomally normal embryos in the CBA X DBA/2 mating combination is triggered by release of Th1 cytokines (tumor necrosis factor [TNF]-alpha, interferon [IFN]-gamma, and interleukin [IL]-1), which cause abortion via a novel prothrombinase, Fgl2, and polymorphonuclear leukocytes. The site of activation may be maternal vascular endothelium on arteries and veins nourishing the placenta. Activation of coagulation is also prominent in spontaneous abortion of chromosomally normal human embryos. We asked where is Fgl2 up-regulated in the uterus in murine abortions, and if similar Fgl2 expression occurs in human pregnancy failure. METHODS: Control CBA X DBA/2 pregnant mice, or from mice injected with TNF-alpha + IFN-gamma on day 7.5 of gestation, were removed on day 8.5, fixed, sectioned, and subject to in situ hybridization for Fgl2. Sections were also stained for fibrin. Elective first trimester termination samples or biopsies taken early in the course of a recurrent miscarriage were similarly fixed, sectioned, and analyzed by in situ hybridization. Control and cytokine-treated mice were anticoagulated with heparin, an activator of antithrombin III, and/or the direct anti-thrombin inhibitor hirudin. RESULTS: Low level Fgl2 expression localized to basal decidua remote from the embryo was noted in control mice; cytokine treatment, which causes greater than 80% of abortions, produced a striking up-regulation in this area as well as in a band at the junction of decidua and myometrium. Trophoblast also became strikingly positive. Fgl2 expression was associated with increased fibrin staining. Anticoagulation significantly protected against abortions, but doses were limited by the complication of retroplacental hemorrhage. In tissue from normal first trimester pregnancy, minimal Fgl2 positivity was seen in some villous syncytiotrophoblast, in villous stroma, cytotrophoblast, and in some cells in decidua. In spontaneous abortion of normal embryo, striking Fgl2 positivity was seen in syncytiotrophoblast and extravillous cytotrophoblast, in association with areas of thrombus formation. CONCLUSIONS: Fgl2 appears to be physiologically expressed and may protect against the internal danger of maternal and/or fetal bleeding during pregnancy and at parturition; a role in inhibiting transplacental traffic is also possible. External dangers in the form of stress, endotoxin, and antigens eliciting Th1 cytokine responses upregulate Fgl2 prothrombinase in trophoblast as well as in decidua, which results in spontaneous abortion of immunogenetically "weaker" embryos.

Stromal cell-derived factor 1 (SDF-1) and antenatal human B cell lymphopoiesis: expression of SDF-1 by mesothelial cells and biliary ductal plate epithelial cells.

The chemokine stromal cell-derived factor 1 (SDF-1) stimulates the growth of pre-B cells in vitro, and mice with a disrupted SDF-1 gene have abnormal fetal liver B cell lymphopoiesis. The origin of SDF-1 production has not been determined yet. Using an anti-SDF-1 mAb, we performed immunohistochemical studies in four human embryos and five fetuses to define which cells express the SDF-1 protein at sites of antenatal B cell lymphopoiesis. All mesothelial cells contained SDF-1 at all stages of development, including in the intraembryonic splanchnopleuric mesoderm early into gestation. In fetal lungs and kidneys, SDF-1 was expressed by epithelial cells, and a few B lymphoid precursors, expressing V pre-B chains, were also detected. In the fetal liver, in addition to mesothelial cells, biliary epithelial cells were the only cells to contain SDF-1. Pre-B cells expressing V chains were abundant and exclusively located around the edge of portal spaces, in close contact with biliary ductal plate epithelial cells. They did not colocalize with biliary collecting ducts. Biliary ductal plate epithelial cells and liver B cell lymphopoiesis display a parallel development and disappearance during fetal life. These results indicate that early B cell lymphopoiesis in the splanchnopleura may be triggered by mesothelial cells producing SDF-1. Later into gestation, biliary ductal plate epithelial cells may support B cell lymphopoiesis, thus playing a role similar to that of epithelial cells in the avian bursa of Fabricius, and of thymic epithelial cells for T cell lymphopoiesis.

Interleukin-6 levels in co-culture of human in vitro fertilization embryos with Vero cells are not predictive of future successful development.

PROBLEM: In an attempt to predict successful embryo transfer and implantation, we measured interleukin (IL)-6 levels in culture supernatants of co-cultured preimplantation human embryos. We tested whether all in vitro fertilized human embryos in co-cultures do secrete IL-6, and whether there was any difference in such production between embryos that successfully reached the blastocyst stage and blocked embryos. We also addressed the question of IL-6 secretion by co-culture support cells, namely Vero cells themselves. METHOD OF STUDY: Each fertilized oocyte was cultured individually and transferred in culture wells supplemented with a feeder layer of Vero cells at day 2. In vitro IL-6 production was measured by bioassay of the culture media. RESULTS: Because Vero cells themselves secrete IL-6, it became impossible, in co-culture, to quantify production of IL-6 by the sole embryos. On the other hand, the co-culture technique has shown us that embryos are likely to consume IL-6. There was no difference between blastocysts and blocked embryos. CONCLUSIONS: IL-6 levels in human embryo co-cultures do not correlate with future successful embryo transfer.