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Miscarriage and intrauterine growth restriction (IUGR) are devastating complications in fetal/neonatal alloimmune thrombocytopenia (FNAIT). We previously reported the mechanisms for bleeding diatheses, but it is unknown whether placental, decidual immune cells or other abnormalities at the maternal–fetal interface contribute to FNAIT. Here we show that maternal immune responses to fetal platelet antigens cause miscarriage and IUGR that are associated with vascular and immune pathologies in murine FNAIT models. Uterine natural killer (uNK) cell recruitment and survival beyond mid-gestation lead to elevated NKp46 and CD107 expression, perforin release and trophoblast apoptosis. Depletion of NK cells restores normal spiral artery remodeling and placental function, prevents miscarriage, and rescues hemorrhage in neonates. Blockade of NK activation receptors (NKp46, FcɣRIIIa) also rescues pregnancy loss. These findings shed light on uNK antibody-dependent cell-mediated cytotoxicity of invasive trophoblasts as a pathological mechanism in FNAIT, and suggest that anti-NK cell therapies may prevent immune-mediated pregnancy loss and ameliorate FNAIT.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening disease in which intracranial hemorrhage (ICH) is the major risk. Although thrombocytopenia caused by maternal antibodies against β3 integrin and occasionally against other platelet antigens (e.g. GPIbα) has long been assumed to be the cause of bleeding, the mechanism of ICH has never been adequately explored. Utilizing murine models of FNAIT and a high frequency ultrasound imaging system, we found that ICH only occurred in fetuses and neonates with anti-β3 integrin- but not anti-GPIbα-mediated FNAIT, despite similar thrombocytopenia in both groups. Only anti-β3 integrin-mediated FNAIT reduced brain and retina vessel density, impaired angiogenic signalling, and increased endothelial cell apoptosis; which were abrogated by maternal administration of intravenous immunoglobulin (IVIG). ICH and impairment of retinal angiogenesis was further reproduced in neonates by injection of anti-β3 integrin- but not anti-GPIbα-antisera. Utilizing cultured human endothelial cells, we found that cell proliferation, network formation, and Akt phosphorylation were inhibited only by murine anti-β3 integrin-antisera and human anti-HPA-1a IgG purified from mothers with FNAIT children. Our data suggest fetal hemostasis is unique in that impairment of angiogenesis rather than thrombocytopenia is likely the cause of ICH; importantly maternal IVIG therapy can effectively prevent this devastating disorder. Disclosures No relevant conflicts of interest to declare.

Background: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a life threatening disease often leading to severe bleeding diatheses, and/or miscarriage; although the incidence of miscarriage has not been adequately studied. FNAIT is caused by a maternal immune response against fetal platelet antigens, in which >80% reported cases have antibodies against β3 integrin. Maternal antibodies and Natural Killer (NK) cells may target antigen positive trophoblast cells and cause miscarriage, but this hypothesis has never been explored.In this study, we investigated whether platelet antibodies and NK cells impaired trophoblast invasion, and placental angiogenesis and function. Methods: β3 integrin deficient female mice were immunized with wild-type (WT) platelets and bred with WT males. Placental vascularisation and function were investigated by echography and micro-computered tomography (CT). Angiogenic and inflammatory cytokines, placenta growth factor (PlGF) and fms-like tyrosine receptor levels (Flt-1) were detected by ELISA. Placental function such as nutrient transport was detected by maternal intravenous administration of biotin and its perfusion to the fetus. NK cell phenotype was assessed by FACS. Placenta pathology was investigated by hematoxylin & eosin staining and immuno-histochemistry for cytokeratin-7, NK cell perforin and smooth muscle actin. Apoptosis and decidual remodeling were investigated by TUNEL assays. In vitro, NK cells were co-cultured with trophoblast cell lines and cytotoxicity was detected by flow cytometry. Results and discussion: Growth restriction and fetal loss/miscarriage only occurred in immunized mothers around embryo day E14.5. Placenta of affected fetuses had significantly reduced vascularization and materno-placental perfusion as demonstrated by ultrasound. CT scan also confirmed shallow development of placental sponge capillaries. These observations are validated by significantly reduced biotin perfusion to the fetuses which had a high hypoxia level. Immunized mice exhibited enlarged spleens as well as an increased Th1 and Th17 immune responses. These pro-inflammatory responses may contribute to trophoblast Flt-1 over-expression and a decreased plasma PlGF/sFlt-1 ratio. E.14.5, the end of organogenesis, is concomitant with trophoblast invasion into spiral arteries. The remodeling of spiral arteries lowers maternal vascular resistance and increases utero-placental blood flow which is critical for healthy pregnancy. Expression of trophoblast marker, Cytokeratin 7, was decreased in the placentas of immunized mice, suggesting scanty invasion invasion. Histology of affected placenta confirmed ischemic tissues, as revealed by significantly reduced numbers of maternal red blood cells in the labyrinth. Unexpectedly, decidual NK cell number remained elevated by day E14.5 in the placenta of immunized mice. Importantly activated NK cells released significant amount of perforin in the placenta, which may destroy target cells expressing β3 integrin. These observations support an abnormal remodeling process mediated by maternal antibodies and NK cells, since increased apoptosis was found in the decidua of immunized pregnant mice compared to naive control. Interestingly, maternal intravenous immunoglobulin therapy ameliorated survival of FNAIT fetuses.. To investigate the molecular and cellular mechanism involved, human choriocarcinoma cells (BeWo) were incubated with both murine anti-β3 antibodies and human HPA-1a IgG. Our data demonstrated, for the first time, that anti-platelet antibodies induced over-expression of the anti-angiogenic molecule, Flt-1 by BeWo cells. Conclusion: Our data suggest that maternal β3 integrin antibodies and NK cells impaired placental pro-angiogenic signalling and function. Reduced trophoblast invasion may cause poor materno-placental perfusion and fetus loss/miscarriage in FNAIT. Disclosures No relevant conflicts of interest to declare.