Effects of Ureaplasma parvum lipoprotein multiple-banded antigen on pregnancy outcome in mice.

Ureaplasma spp. are members of the family Mycoplasmataceae and have been considered to be associated with chorioamnionitis and preterm delivery. However, it is unclear whether Ureaplasma spp. have virulence factors related to these manifestations. The purpose of the present study was to determine whether the immunogenic protein multiple-banded antigen (MBA) from Ureaplasma parvum is a virulence factor for preterm delivery. We partially purified MBA from a type strain and clinical isolates of U. parvum, and also synthesized a diacylated lipopeptide derived from U. parvum, UPM-1. Using luciferase assays, both MBA-rich fraction MRF and UPM-1 activated the NF-κB pathway via TLR2. UPM-1 upregulated IL-1ß, IL-6, IL-12p35, TNF-α, MIP2, LIX, and iNOS in mouse peritoneal macrophage. MRF or UPM-1 was injected into uteri on day 15 of gestation on pregnant C3H/HeN mice. The intrauterine MRF injection group had a significantly higher incidence of intrauterine fetal death (IUFD; 38.5%) than the control group (14.0%). Interestingly, intrauterine injection of UPM-1 caused preterm deliveries at high concentration (80.0%). In contrast, a low concentration of UPM-1 induced a significantly higher rate of fetal deaths (55.2%) than the control group (14.0%). The placentas of the UPM-1 injection group showed neutrophil infiltration and increased iNOS protein expression. Our data indicate that MBA from the clinical isolate of U. parvum is a potential virulence factor for IUFD and preterm delivery in mice and that the N-terminal diacylated lipopeptide is essential for the initiation of inflammation.

Neuromyelitis optica IgG causes placental inflammation and fetal death.

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the CNS and affects women of childbearing age. Most patients with NMO have circulating Abs, termed NMO-IgG, against the astrocytic water channel protein aquaporin-4. In the CNS, NMO-IgG causes complement-mediated astrocyte damage, inflammatory cell infiltration, and myelin loss. In this study, we show that aquaporin-4 is expressed in the syncytiotrophoblast of human and mouse placenta. Placental aquaporin-4 expression is high during mid-gestation and progressively decreases with advancing pregnancy. Intraperitoneally injected NMO-IgG binds mouse placental aquaporin-4, activates coinjected human complement, and causes inflammatory cell infiltration into the placenta and placental necrosis. There was no damage to maternal organs that express aquaporin-4, including the brain, spinal cord, kidneys, and skeletal muscle. In control experiments, no placentitis was found in mice injected with NMO-IgG without complement, non-NMO-IgG with human complement, or in aquaporin-4 null mice injected with NMO-IgG and human complement. The infiltrating cells were primarily neutrophils with a few scattered eosinophils and macrophages. NMO-IgG and human complement-induced placentitis caused fetal death, but some fetuses were born normal when lower amounts of NMO-IgG and human complement were injected. Sivelestat, a neutrophil elastase inhibitor, and aquaporumab, a nonpathogenic IgG that competes with NMO-IgG for aquaporin-4 binding, significantly reduced NMO-IgG and human complement induced placentitis and fetal death. Our data suggest that NMO-IgG can cause miscarriage, thus challenging the concept that NMO affects only the CNS. These findings have implications for the management of NMO during pregnancy.

Antiphospholipid antibodies in stillbirth.

OBJECTIVE: To compare antiphospholipid antibodies in deliveries with and without stillbirth using a multicenter, population-based case-control study of stillbirths and live births. METHODS: Maternal sera were assayed for immunoglobulin (Ig)G and IgM anticardiolipin and anti-ß2-glycoprotein-I antibodies. Assays were performed in 582 stillbirth deliveries and 1,547 live birth deliveries. RESULTS: Elevated levels of IgG anticardiolipin and IgG anti-ß2-glycoprotein-I antibodies were associated with an approximate threefold increased odds of stillbirth (crude odds ratio [OR] 3.43, 95% confidence interval [CI] 1.79-6.60, 3.8% compared with 1.1% and OR 3.17, 95% CI 1.30-7.72, (1.9% compared with 0.6%, respectively) when all deliveries with stillbirth were compared with all deliveries with live birth. When the subset of stillbirths not associated with fetal anomalies or obstetric complications was compared with term live births, elevated IgG anticardiolipin antibodies were associated with stillbirth (5.0% compared with 1.0%; OR 5.30, 95% CI, 2.39-11.76; IgG anti-ß2-glycoprotein-I antibodies (1.9% compared with 0.6%) had an OR of 3.00 (95% CI 1.01-8.90) and IgM anticardiolipin antibodies (6.0% compared with 3.0%) had an OR of 2.03 (95% CI 1.09-3.76). Elevated levels of anticardiolipin and anti-ß2-glycoprotein-I antibodies were associated with a threefold to fivefold increased odds of stillbirth. CONCLUSIONS: Our data support consideration of testing for antiphospholipid antibodies in cases of otherwise unexplained stillbirth. LEVEL OF EVIDENCE: II.

Maternal floor infarction/massive perivillous fibrin deposition: a manifestation of maternal antifetal rejection?

OBJECTIVE: Massive perivillous fibrin deposition (MPFD) and maternal floor infarction (MFI) are related placental lesions often associated with fetal death and fetal growth restriction. A tendency to recur in subsequent pregnancies has been reported. This study was conducted to determine whether this complication of pregnancy could reflect maternal antifetal rejection. METHODS: Pregnancies with MPFD were identified (n = 10). Controls consisted of women with uncomplicated pregnancies who delivered at term without MPFD (n = 175). Second-trimester maternal plasma was analyzed for panel-reactive anti-HLA class I and class II antibodies. The prevalence of chronic chorioamnionitis, villitis of unknown etiology, and plasma cell deciduitis was compared between cases and controls. Immunohistochemistry was performed on available umbilical vein segments from cases with MPFD (n = 4) to determine whether there was evidence of complement activation (C4d deposition). Specific maternal HLA-antibody and fetal HLA-antigen status were also determined in paired specimens (n = 6). Plasma CXCL-10 concentrations were measured in longitudinal samples of cases (n = 28 specimens) and controls (n = 749 specimens) by ELISA. Linear mixed-effects models were used to test for differences in plasma CXCL-10 concentration. RESULTS: (i) The prevalence of plasma cell deciduitis in the placenta was significantly higher in cases with MPFD than in those with uncomplicated term deliveries (40% versus 8.6%, P = 0.01), (ii) patients with MPFD had a significantly higher frequency of maternal anti-HLA class I positivity during the second trimester than those with uncomplicated term deliveries (80% versus 36%, P = 0.01); (iii) strongly positive C4d deposition was observed on umbilical vein endothelium in cases of MPFD, (iv) a specific maternal antibody against fetal HLA antigen class I or II was identified in all cases of MPFD; and 5) the mean maternal plasma concentration of CXCL-10 was higher in patients with evidence of MPFD than in those without evidence of MFPD (P < 0.001). CONCLUSION: A subset of patients with MPFD has evidence of maternal antifetal rejection.

Post-partum bilateral renal cortical necrosis in antiphospholipid syndrome and systemic lupus erythematosus.

In the presence of systemic lupus erythematosus or related autoimmune disorders, antiphospholipid syndrome (APS) is termed secondary APS. Pregnancy-related renal failure due to SAPS is rarely reported in the literature. We present the case of a young primgravida woman with bilateral renal cortical necrosis due to secondary APS in late pregnancy.

Direct and indirect antigen presentation lead to deletion of donor-specific T cells after in utero hematopoietic cell transplantation in mice.

In utero hematopoietic cell transplantation (IUHCTx) is a promising method to induce donor-specific tolerance but the mechanisms of antigen presentation that educate host T cells and the relative importance of deletion vs regulation in this setting are unknown. We studied the roles of direct and indirect antigen presentation (mediated by donor- and host-derived antigen-presenting cells [APCs], respectively) in a mouse model of IUHCTx. We found that IUHCTx leads to precocious maturation of neonatal host dendritic cells (DCs) and that there is early differentiation of donor-derived DCs, even after transplantation of a stem cell source without mature APCs. We next performed allogeneic IUHCTx into donor-specific T-cell receptor transgenic mice and confirmed that both direct and indirect antigen presentation lead to clonal deletion of effector T cells in chimeras. Deletion did not persist when chimerism was lost. Importantly, although the percentage of regulatory T cells (Tregs) after IUHCTx increased, there was no expansion in Treg numbers. In wild-type mice, there was a similar deletion of effector cells without expansion of donor-specific Tregs. Thus, tolerance induction after IUHCTx depends on both direct and indirect antigen presentation and is secondary to thymic deletion, without de novo Treg induction.

The αIIb p.Leu841Met (Cab3(a+) ) polymorphism results in a new human platelet alloantigen involved in neonatal alloimmune thrombocytopenia.

BACKGROUND: Fetal-neonatal alloimmune thrombocytopenia (FNAIT) diagnosis relies on maternofetal incompatibility and alloantibody identification. Genotyping for rare platelet (PLT) polymorphisms allowed the identification of three families with suspected or confirmed maternofetal incompatibility for the αIIb-c.2614C>A mutation (Halle et al., Transfusion 2008;48:14-15). STUDY DESIGN AND METHODS: A polymerase chain reaction-sequence-specific primers amplification assay was designed to genotype the αIIb-c.2614C>A mutation. HEK293 cells expressing αIIb-Leu841 or αIIb-Met841 αIIbß3 forms were used to probe the reactivity of maternal sera from these families and to study the effects of the substitution on αIIbß3 expression and functions. RESULTS: Tested by flow cytometry (FCM), one serum sample specifically reacted with αIIb-Met841 but not with αIIb-Leu841 αIIbß3. This specificity revealed the αIIb-Leu841 polymorphism as a new alloantigen named Cab3(a+) . Cross-match testing using FCM also showed the Cab3(a+) antigen to be expressed at the PLT surface. As for anti-human PLT alloantigen (HPA)-3a (or -3b) and anti-HPA-9bw, detection of anti-Cab3(a+) alloantibodies appeared difficult and required whole PLT assays when classical monoclonal antibody-specific immobilization of PLT antigen test failed. In our FNAIT set, the immune response to Cab3(a+) maternofetal incompatibility could induce severe thrombocytopenias and life-threatening hemorrhages. The p.Leu841Met substitution has limited effects, if any, on local αIIb structure, preserving both αIIbß3 expression and functions. CONCLUSION: The Cab3(a+) polymorphism is a new rare alloantigen (allelic frequency <1%) carried by αIIb that might result in severe life-threatening thrombocytopenias. In Sub-Saharan African populations, higher Cab3(a+) gene frequencies (up to 8.2%; Halle et al., Transfusion 2008;48:14-15) and homozygous people are observed.

Reproductive parameters following a PRRS outbreak where a whole-herd PRRS MLV vaccination strategy was instituted post-outbreak.

This study assessed the effect of whole-herd porcine reproductive and respiratory syndrome (PRRS) modified-live virus (MLV) vaccination on herd-level reproductive performance, PRRS virus (PRRSV) viremia, and antibody in a subset of females in a 1,200-sow commercial herd in Thailand. Following a PRRSV outbreak, the entire herd was vaccinated with PRRS MLV twice at 3-week intervals and at 3-month intervals, thereafter. Reproductive performance data over a 3-year period were available for analysis. Serum samples were collected before and after vaccination and tested by PRRSV ELISA and reverse transcription-polymerase chain reaction. Vaccination was statistically associated with a lower abortion rate (1.4 vs. 1.6 %), farrowing rate (83.8 vs. 90.0 %), total born (10.6 vs. 11.4 piglets/litter), liveborn (10.0 vs. 10.3 piglets/litter), stillbirths (4.6 vs. 7.0 %), mummies (0.7 vs. 1.6 %), and a higher return rate (11.3 vs. 5.9 %) when compared with the period before the PRRSV outbreak. Pregnant females vaccinated during early gestation farrowed fewer liveborn and more mummies than the comparison group, whereas females vaccinated during late gestation had a lower farrowing rate. In this herd, PRRS whole-herd vaccination had neutral, positive, and negative effects on reproductive performance. Thus, the decision to implement whole-herd vaccination should be balanced between the benefits derived from reproductive performance improvements, e.g., fewer abortions, stillborn piglets, and mummified fetuses, and the effect of vaccination on pregnant females.

Blockage of heme oxygenase-1 abrogates the protective effect of regulatory T cells on murine pregnancy and promotes the maturation of dendritic cells.

Regulatory T cells (Treg) play an important role in fetal protection. They expand during normal pregnancy and protect fetal antigens from maternal effector cells. Their effect is associated with the up-regulation of tolerance-associated molecules at the fetal-maternal interface. Among these, Heme Oxygenase-1 (HO-1, coded by Hmox1) is of special importance as its blockage correlates with increased abortion rates and its up-regulation positively affects pregnancy outcome. Here, we aimed to investigate whether the protective effect of Treg is mediated by HO-1 in a mouse model. HO-1 blockage by Zinc Protoporhyrin (ZnPPIX) abrogated the protective effect of Treg transfer. We found that HO-1 is important in maintaining maternal dendritic cells (DCs) in an immature state, which contributes to the expansion of the peripheral Treg population. This brings to light one essential pathway through which Treg mediates the semi-allogeneic fetus tolerance.

Zinc supplementation during pregnancy protects against lipopolysaccharide-induced fetal growth restriction and demise through its anti-inflammatory effect.

LPS is associated with adverse developmental outcomes, including preterm delivery, fetal death, teratogenicity, and intrauterine growth restriction (IUGR). Previous reports showed that zinc protected against LPS-induced teratogenicity. In the current study, we investigated the effects of zinc supplementation during pregnancy on LPS-induced preterm delivery, fetal death and IUGR. All pregnant mice except controls were i.p. injected with LPS (75 µg/kg) daily from gestational day (GD) 15 to GD17. Some pregnant mice were administered zinc sulfate through drinking water (75 mg elemental Zn per liter) throughout the pregnancy. As expected, an i.p. injection with LPS daily from GD15 to GD17 resulted in 36.4% (4/11) of dams delivered before GD18. In dams that completed the pregnancy, 63.2% of fetuses were dead. Moreover, LPS significantly reduced fetal weight and crown-rump length. Of interest, zinc supplementation during pregnancy protected mice from LPS-induced preterm delivery and fetal death. In addition, zinc supplementation significantly alleviated LPS-induced IUGR and skeletal development retardation. Further experiments showed that zinc supplementation significantly attenuated LPS-induced expression of placental inflammatory cytokines and cyclooxygenase-2. Zinc supplementation also significantly attenuated LPS-induced activation of NF-κB and MAPK signaling in mononuclear sinusoidal trophoblast giant cells of the labyrinth zone. It inhibited LPS-induced placental AKT phosphorylation as well. In conclusion, zinc supplementation during pregnancy protects against LPS-induced fetal growth restriction and demise through its anti-inflammatory effect.

Unilateral uterine ischemia/reperfusion-induced bilateral fetal loss and fetal growth restriction in a murine model require intact complement component 5.

The role of complement in ischemia/reperfusion-induced fetal growth restriction and fetal loss is unknown. C5-deficient or wild type timed-pregnant mice were subjected to unilateral uterine ischemia/reperfusion on gestation day 13, either by (1) partial flow restriction by right ovarian artery clamping for 30 min, or (2) total flow restriction by clamping both ovarian and uterine arteries for 5 min. Ischemia/reperfusion-challenged pregnancy outcomes were compared to sham-operated controls 5 days later. Ischemia/reperfusion-treated wild type mice exhibited significantly increased bilateral fetal loss, which was greater in total flow restriction than in partial flow restriction, and decreased fetal weights, which were the same in total flow restriction and partial flow restriction for the surviving fetuses. Placental weights were unchanged by treatments. Ischemia/reperfusion increased uterine, but not placental, myeloperoxidase activity, which correlated with fetal loss. In contrast, C5-deficient mice were protected from both fetal growth restriction and fetal loss, and exhibited no increase in myeloperoxidase activity. These results demonstrate that unilateral uterine ischemia/reperfusion results in bilateral fetal loss and fetal growth restriction, mediated by a systemic mechanism. In the current model, this pathological process is completely dependent on intact complement component 5.

Female mouse fetal loss mediated by maternal autoantibody.

Systemic lupus erythematosus (SLE), a disease of women during childbearing years, is characterized by the production of double-stranded DNA antibodies. A subset of these antibodies, present in 40% of patients, cross-reacts with the NR2A and NR2B subunits of the N-methyl-d-aspartate receptor (NMDAR). In this study, we show that, in mouse models, these antibodies cause a loss of female fetus viability by inducing apoptosis of NR2A-expressing neurons within the brainstem late in fetal development; gender specificity derives from a time-dependent increased expression of NR2A in female brainstem or increased vulnerability of female fetal neurons to signaling through NR2A-containing NMDARs. This paradigm is consistent with available data on the sex ratio of live births of women with SLE. It represents a novel mechanism by which maternal autoantibodies can severely affect fetal health in a gender-specific fashion and raises the question of how many maternal antibodies affect brain development or exhibit gender-specific fetal effects.

TLR9 provokes inflammation in response to fetal DNA: mechanism for fetal loss in preterm birth and preeclampsia.

Preterm birth, the major cause of neonatal mortality in developed countries, is associated with intrauterine infections and inflammation, although the exact mechanisms underlying this event are unclear. In this study, we show that circulating fetal DNA, which is elevated in pregnancies complicated by preterm labor or preeclampsia, triggers an inflammatory reaction that results in spontaneous preterm birth. Fetal DNA activates NF-κB, shown by IκBα degradation in human PBMCs resulting in production of proinflammatory IL-6. We show that fetal resorption and preterm birth are rapidly induced in mice after i.p. injection of CpG or fetal DNA (300 µg/dam) on gestational day 10-14. In contrast, TLR9(-/-) mice were protected from these effects. Furthermore, this effect was blocked by oral administration of the TLR9 inhibitor chloroquine. Our data therefore provide a novel mechanism for preterm birth and preeclampsia, highlighting TLR9 as a potential therapeutic target for these common disorders of pregnancy.

The role of complement in the antiphospholipid syndrome: a novel mechanism for pregnancy morbidity.

OBJECTIVES: Despite the experimental research data on antiphospholipid syndrome (APS), the pathogenesis of thrombosis and fetal loss remains unknown. The objective of this study was to analyze the major advances in the field of complement activation as a possible thrombosis mechanism in the APS. METHODS: The authors conducted a systemic analysis of the English literature and summarized both animal and human data that indicate the inappropriate complement activation as a mechanism causing thrombosis in the APS. RESULTS: The important role of complement activation in the pathogenesis of fetal loss was established using mice deficient in a complement regulatory protein. Further studies have shown that the infusion of human IgG antiphospholipid antibodies (aPL) induced fetal loss in pregnant mice, an effect that was abrogated by the concurrent administration of a C3 convertase inhibitor. Further studies suggested that C5a and neutrophils were the key components responsible for fetal injury. Moreover, use of F(ab)'2 fragments of aPL suggested the complement activation occurred mainly via the classical pathway. Other studies using models of induced thrombosis suggested that antibodies against ß2GPI required the presence of terminal complement components to induce thrombus formation, and mice deficient in C3 or C5 were found to be resistant to aPL-induced thrombosis. Based on the aforementioned findings, it has been suggested that heparin prevents fetal loss in patients with APS by inhibiting complement activation rather than by its anticoagulant effect. CONCLUSIONS: The studies on complement are significant because they shift the focus of research in APS from thrombosis to inflammation. However, as human data are limited, more clinical research is necessary before the above findings translate in changes in the management of APS.

[Pregnancy and antiphospholipid syndrome]. / Grossesse et syndrome des antiphospholipides.

Antiphospholipid syndrome (APS) is associated with a risk of obstetrical complications, affecting both the mother and the fetus. Obstetrical APS is defined by a history of three consecutive spontaneous miscarriages before 10 weeks of gestation (WG), an intra-uterine fetal death after 10 WG, or a premature birth before 34 WG because of severe pre-eclampsia, eclampsia or placental adverse outcomes (intrauterine growth retardation, oligohydramnios). Pregnancy in women with a diagnosis of obstetric APS is at increased risk for placental abruption, HELLP (Hemolysis, Elevated Liver enzymes, Low Platelet count) syndrome and thrombosis that may be part of a catastrophic antiphospholipid syndrome (CAPS). A previous thrombosis and the presence of a lupus anticoagulant are risk factors for pregnancy failure. A multidisciplinary approach, associating the internist, the anesthesiologist and the obstetrician, is recommended for these high-risk pregnancies. Preconception counseling is proposed to identify pregnancy contraindications, and to define and adapt the treatment prior and during the upcoming pregnancy. Heparin and low-dose aspirin are the main treatments. The choice between therapeutic or prophylactic doses of heparin will depend on the patient's medical history. The anticoagulant therapeutic window for delivery should be as narrow as possible and adapted to maternal thrombotic risk. There is a persistent maternal risk in the postpartum period (thrombosis, HELLP syndrome, CAPS) justifying an antithrombotic coverage during this period. We suggest a monthly clinical and biological monitoring which can be more frequent towards the end of pregnancy. The persistence of notches at the Doppler-ultrasound evaluation seems to be the best predictor for a higher risk of placental vascular complications. Treatment optimization and multidisciplinary antenatal care improve the prognosis of pregnancies in women with obstetric APS, leading to a favorable outcome most of the time.

Anatomical and pathological findings in hearts from fetuses and infants with cardiac manifestations of neonatal lupus.

OBJECTIVE: The autopsy and clinical information on children dying with anti-SSA/Ro-associated cardiac manifestations of neonatal lupus (cardiac NL) were examined to identify patterns of disease, gain insight into pathogenesis and enhance the search for biomarkers and preventive therapies. METHODS: A retrospective analysis evaluating reports from 18 autopsies of cardiac NL cases and clinical data from the Research Registry for Neonatal Lupus was performed. RESULTS: Of the 18 cases with autopsies, 15 had advanced heart block, including 3 who died in the second trimester, 9 in the third trimester and 3 post-natally. Three others died of cardiomyopathy without advanced block, including two dying pre-natally and one after birth. Pathological findings included fibrosis/calcification of the atrioventricular (AV) node, sinoatrial (SA) node and bundle of His, endocardial fibroelastosis (EFE), papillary muscle fibrosis, valvular disease, calcification of the atrial septum and mononuclear pancarditis. There was no association of pathology with the timing of death except that in the third-trimester deaths more valvular disease and/or extensive conduction system abnormalities were observed. Clinical rhythm did not always correlate with pathology of the conduction system, and the pre-mortem echocardiograms did not consistently detect the extent of pathology. CONCLUSION: Fibrosis of the AV node/distal conduction system is the most characteristic histopathological finding. Fibrosis of the SA node and bundle of His, EFE and valve damage are also part of the anti-Ro spectrum of injury. Discordance between echocardiograms and pathology findings should prompt the search for more sensitive methods to accurately study the phenotype of antibody damage.