Characterization of the fetal blood transcriptome and proteome in maternal anti-fetal rejection: evidence of a distinct and novel type of human fetal systemic inflammatory response

BACKGROUND: The human fetus is able to mount a systemic inflammatory response when exposed to microorganisms. This stereotypic response has been termed the 'fetal inflammatory response syndrome' (FIRS), defined as an elevation of fetal plasma interleukin-6 (IL-6). FIRS is frequently observed in patients whose preterm deliveries are associated with intra-amniotic infection, acute inflammatory lesions of the placenta, and a high rate of neonatal morbidity. Recently, a novel form of fetal systemic inflammation, characterized by an elevation of fetal plasma CXCL10, has been identified in patients with placental lesions consistent with 'maternal anti-fetal rejection'. These lesions include chronic chorioamnionitis, plasma cell deciduitis, and villitis of unknown etiology. In addition, positivity for human leukocyte antigen (HLA) panel-reactive antibodies (PRA) in maternal sera can also be used to increase the index of suspicion for maternal anti-fetal rejection. The purpose of this study was to determine (i) the frequency of pathologic lesions consistent with maternal anti-fetal rejection in term and spontaneous preterm births; (ii) the fetal serum concentration of CXCL10 in patients with and without evidence of maternal anti-fetal rejection; and (iii) the fetal blood transcriptome and proteome in cases with a fetal inflammatory response associated with maternal anti-fetal rejection. METHOD OF STUDY: Maternal and fetal sera were obtained from normal term (n = 150) and spontaneous preterm births (n = 150). A fetal inflammatory response associated with maternal anti-fetal rejection was diagnosed when the patients met two or more of the following criteria: (i) presence of chronic placental inflammation; (ii) ≥80% of maternal HLA class I PRA positivity; and (iii) fetal serum CXCL10 concentration >75th percentile. Maternal HLA PRA was analyzed by flow cytometry. The concentrations of fetal CXCL10 and IL-6 were determined by ELISA. Transcriptome analysis was undertaken after the extraction of total RNA from white blood cells with a whole-genome DASL assay. Proteomic analysis of fetal serum was conducted by two-dimensional difference gel electrophoresis. Differential gene expression was considered significant when there was a P 1.5. RESULTS: (i) The frequency of placental lesions consistent with maternal anti-fetal rejection was higher in patients with preterm deliveries than in those with term deliveries (56% versus 32%; P < 0.001); (ii) patients with spontaneous preterm births had a higher rate of maternal HLA PRA class I positivity than those who delivered at term (50% versus 32%; P = 0.002); (iii) fetuses born to mothers with positive maternal HLA PRA results had a higher median serum CXCL10 concentration than those with negative HLA PRA results (P < 0.001); (iv) the median serum CXCL10 concentration (but not IL-6) was higher in fetuses with placental lesions associated with maternal anti-fetal rejection than those without such lesions (P < 0.001); (v) a whole-genome DASL assay of fetal blood RNA demonstrated differential expression of 128 genes between fetuses with and without lesions associated with maternal anti-fetal rejection; and (vi) comparison of the fetal serum proteome demonstrated 20 proteins whose abundance differed between fetuses with and without lesions associated with maternal anti-fetal rejection. CONCLUSION: We describe a systemic inflammatory response in human fetuses born to mothers with evidence of maternal anti-fetal rejection. The transcriptome and proteome of this novel type of fetal inflammatory response were different from that of FIRS type I (which is associated with acute infection/inflammation).ope

Behçet’s Disease with Deep Vaginal Ulcer Diagnosed for the First Time during Pregnancy

Behçet’s disease is a chronic multisystem inflammatory disease characterized by recurrent oral ulcer, genital ulcer and ocular inflammation. It affects mainly young adults ranging from twenties to forties, so the relationship between Behçet’s disease and pregnancy has been investigated in women during reproductive ages. In pregnant women, genital ulcers arise from infectious and noninfectious causes. We report a case of a Behçet’s disease with deep vaginal ulcer diagnosed for the first time during pregnancy.ope

Glycogen phosphorylase isoenzyme BB plasma concentration is elevated in pregnancy and preterm preeclampsia

Glycogen phosphorylase is a key enzyme in glycogenolysis. Released with myocardial ischemia, blood concentration of glycogen phosphorylase isoenzyme BB (GPBB) is a marker of acute coronary syndromes. Pregnancy imposes metabolic stress, and preeclampsia is associated with cardiac complications. However, plasma GPBB concentration during pregnancy is unknown. This study was conducted to determine maternal plasma GPBB concentration in normal pregnancy and in preeclampsia. Plasma samples from 6 groups (n=396) were studied: nonpregnant and pregnant women with normal term delivery, term and preterm preeclampsia, and term and preterm small-for-gestational-age neonates. GPBB concentration was measured with a specific immunoassay. Placental tissues (n=45) obtained from pregnant women with preterm and term preeclampsia, spontaneous preterm delivery, and normal term delivery were analyzed for potential GPBB expression by immunoblotting. Median plasma GPBB concentration was higher in pregnant women than in nonpregnant women (38.7 versus 9.2 ng/mL; P<0.001), which remained significant after adjusting for age, race, and parity. Maternal plasma GPBB concentrations did not change throughout gestation. Cases of preterm (but not term) preeclampsia had higher median plasma GPBB concentrations than gestational age-matched normal pregnancy cases (72.6 versus 26.0 ng/mL; P=0.001). Small-for-gestational-age neonates did not affect plasma GPBB concentration. GPBB was detected in the placenta and was less abundant in preterm preeclampsia than in preterm delivery cases (P<0.01). There is physiological elevation of plasma GPBB concentration during pregnancy; an increase in maternal plasma GPBB is a novel phenotype of preterm preeclampsia. It is strongly suggested that these changes are attributed to GPBB of placental origin.ope

Chronic chorioamnionitis displays distinct alterations of the amniotic fluid proteome

Acute chorioamnionitis of infectious origin and chronic chorioamnionitis of immunological origin are two major placental lesions of spontaneous preterm birth with elevated amniotic fluid interleukin-6 and CXCL10 concentrations, respectively. The changes in the amniotic fluid proteome associated with intra-amniotic infection and acute chorioamnionitis are well defined, yet alterations unique to chronic chorioamnionitis remain to be elucidated. This study was conducted to determine those amniotic fluid proteins changing specifically in the presence of chronic chorioamnionitis. Amniotic fluid obtained from acute chorioamnionitis, chronic chorioamnionitis and gestational age-matched controls were analysed by two-dimensional (2D) difference in gel electrophoresis and MALDI-TOF analyses. The type of histological inflammation was used to define each condition in preterm labour cases (n = 125) and term not in labour cases (n = 22), and the amniotic fluid concentrations of interleukin-6, CXCL8, CXCL10 and prostaglandin F(2α) were also measured by specific immunoassays. Among preterm labour cases, 31 differentially expressed proteins were identified in chronic chorioamnionitis cases as compared to both acute chorioamnionitis and control cases. Importantly, glycodelin-A, which maintains maternal tolerance against an allogeneic fetus, was decreased in chronic chorioamnionitis, while haptoglobin was increased. We report the amniotic fluid proteome of chronic chorioamnionitis for the first time, and the findings herein strongly suggest that there is a pathophysiological association between the changes of immunomodulatory proteins in the amniotic fluid and chronic chorioamnionitis, a histological manifestation of maternal anti-fetal allograft rejection.ope

The soluble interleukin-6 receptor is a mediator of hematopoietic and skeletal actions of parathyroid hormone

Both PTH and IL-6 signaling play pivotal roles in hematopoiesis and skeletal biology, but their interdependence is unclear. The purpose of this study was to evaluate the effect of IL-6 and soluble IL-6 receptor (sIL-6R) on hematopoietic and skeletal actions of PTH. In the bone microenvironment, PTH stimulated sIL-6R protein levels in primary osteoblast cultures in vitro and bone marrow in vivo in both IL-6(+/+) and IL-6(-/-) mice. PTH-mediated hematopoietic cell expansion was attenuated in IL-6(-/-) compared with IL-6(+/+) bone marrow, whereas sIL-6R treatment amplified PTH actions in IL-6(-/-) earlier than IL-6(+/+) marrow cultures. Blocking sIL-6R signaling with sgp130 (soluble glycoprotein 130 receptor) inhibited PTH-dependent hematopoietic cell expansion in IL-6(-/-) marrow. In the skeletal system, although intermittent PTH administration to IL-6(+/+) and IL-6(-/-) mice resulted in similar anabolic actions, blocking sIL-6R significantly attenuated PTH anabolic actions. sIL-6R showed no direct effects on osteoblast proliferation or differentiation in vitro; however, it up-regulated myeloid cell expansion and production of the mesenchymal stem cell recruiting agent, TGF-β1 in the bone marrow microenvironment. Collectively, sIL-6R demonstrated orphan function and mediated PTH anabolic actions in bone in association with support of myeloid lineage cells in the hematopoietic system.ope

Maternal HLA panel-reactive antibodies in early gestation positively correlate with chronic chorioamnionitis: evidence in support of the chronic nature of maternal anti-fetal rejection

PROBLEM : Maternal tolerance of the fetus is essential for viviparity, yet anti-fetal rejection occurs in several pregnancy complications. Chronic chorioamnionitis is a feature of anti-fetal cellular rejection. There is a robust association between chronic chorioamnionitis and maternal seropositivity for anti-human leukocyte antigen (HLA) panel-reactive antibodies (PRA) at the time of delivery. This longitudinal study was performed to assess maternal HLA PRA status in early gestation and the temporal evolution of maternal HLA PRA in the context of chronic chorioamnionitis and, thereby, to determine whether HLA PRA during the course of pregnancy is useful for the detection of anti-fetal rejection. METHOD OF STUDY : Maternal sera obtained before 16 weeks of gestation and at delivery were analyzed for HLA PRA in cases with (N = 100) and without (N = 150) chronic chorioamnionitis. RESULTS : IgG (but not IgM) HLA class I and II PRA positivity at delivery was higher in cases with chronic chorioamnionitis than in those without chronic chorioamnionitis. IgG HLA class I PRA positivity before 16 weeks of gestation was higher in cases with chronic chorioamnionitis than in those without (30.3 versus 13.3%; P = 0.001). Positive conversion (negative HLA PRA before 16 weeks of gestation but positive at delivery) of IgG HLA class I and II PRA was significantly associated with chronic chorioamnionitis. Fetal HLA class I antigen-specific antibodies were confirmed in 12 of 16 mothers tested who were sensitized to HLA class I antigens before 16 weeks of gestation. CONCLUSION : Positive maternal HLA PRA before 16 weeks of gestation and the temporal evolution of maternal HLA PRA are associated with the presence of chronic chorioamnionitis at the time of delivery. Maternal IgG HLA PRA has the potential to be a monitoring tool of anti-fetal rejection. Furthermore, the findings herein indicate that subsets of fetuses are exposed to alloimmune HLA antibodies for months, especially in cases with chronic chorioamnionitis.ope

The risk of preterm birth in vanishing twin: A multicenter prospective cohort study

Objective: To evaluate not only the risk of total preterm birth (PTB) but also spontaneous preterm birth (sPTB) and indicated preterm birth (iPTB) in vanishing twin (VT). Study design: This is a secondary analysis of a multicenter prospective cohort study. In 12 different healthcare institutions, women with singleton pregnancies were enrolled in early pregnancy and followed up till delivery. Results: A total of 4,746 women were included in the final analysis, and. the frequency of VT was 1.1% (54/4746). VT group had a higher risk for total PTB (PTB<34 weeks, 2.1% vs. 14.8%, p<0.001; PTB<32 weeks, 1.6% vs. 13.0%, p<0.001; PTB<28 weeks, 0.9% vs. 13.0%, p<0.001) than singleton group. The VT group had increased risk for both sPTB and iPTB (<34 weeks, <32 weeks, and <28 weeks), and this increased risk for sPTB and iPTB in VT group remained significant even after controlling for confounders such as maternal age, parity, pre-pregnancy BMI, and mode of conception. Conclusion: Vanishing twin can be an independent risk factor for both sPTB and iPTB when compared with singleton pregnancy.ope

Prenatal Diagnosis of Congenital Heart Diseases and Associations with Serum Biomarkers of Aneuploidy: A Multicenter Prospective Cohort Study

Purpose: We assessed prenatal detection rates of congenital heart disease (CHD) and associations between maternal serum biomarkers and non-chromosomal CHD in singleton pregnancies. Materials and methods: This study was conducted as a secondary analysis of data obtained during a multicenter prospective cohort study that investigated the cost-effectiveness of prenatal testing for fetal aneuploidy. We analyzed the prenatal detection rate and accuracy for CHD screening via ultrasound during the second trimester, as well as associations between serum biomarkers and CHDs, in singleton newborns without chromosomal abnormalities. Results: Among 6715 women, 142 (2.1%) newborns were born with CHDs, of which 67 (1.0%) newborns had major CHDs. The prenatal detection rate for all CHDs and major CHDs were 34.5% and 58.2%, respectively. After excluding isolated ventricular septal defects, the detection rate for critical CHDs was 85.9%. Women with low pregnancy-associated plasma protein A (PAPP-A) (<0.4 multiples of the median, MOM) face increased risks of non-chromosomal CHDs [adjusted odds ratio (aOR) 2.76; 95% confidence interval (CI) 1.36-5.13] and major CHDs (aOR 7.30; 95% CI 3.18-15.59), compared to those without CHDs. A higher inhibin A level (≥2.5 MOM; aOR 4.84; 95% CI 1.42-12.46) was associated with non-chromosomal major CHDs. Conclusion: Ultrasonography performed during the second trimester by obstetricians detected over 85% of critical CHDs. Low maternal serum PAPP-A or high inhibin-A was associated with non-chromosomal CHDs. These results may contribute to an improvement in prenatal diagnosis of CHDs.ope

Unexplained fetal death has a biological signature of maternal anti-fetal rejection: chronic chorioamnionitis and alloimmune anti-human leucocyte antigen antibodies

AIMS : Chronic chorioamnionitis is a histological manifestation of maternal anti-fetal cellular rejection. As failure of graft survival is the most catastrophic event in organ transplantation, we hypothesized that fetal death could be a consequence of maternal rejection. The aim of this study was to assess whether there is evidence of cellular and antibody-mediated rejection in fetal death. METHODS AND RESULTS : Placental histology was reviewed for the presence of chronic chorioamnionitis in unexplained preterm fetal death (n=30) and preterm live birth (n=103). Amniotic fluid CXCL10 concentrations were measured with a specific immunoassay. Chronic chorioamnionitis was more frequent in fetal death than in live birth (60.0% versus 37.9%; P<0.05) and fetal death had a higher median amniotic fluid CXCL10 concentration than live birth (2.0 versus 1.8 ng/ml, P<0.05), after adjusting for gestational age at amniocentesis. Maternal anti-human leucocyte antigen class II panel-reactive seropositivity determined by flow cytometry was higher in fetal death compared to live birth (35.7% versus 10.9%; P<0.05). CONCLUSIONS : Chronic chorioamnionitis is a common pathologic feature in unexplained preterm fetal death. This novel finding suggests that cellular and antibody-mediated anti-fetal rejection of the mother is associated with fetal death (graft failure) in human pregnancy.ope

Methylome of fetal and maternal monocytes and macrophages at the feto-maternal interface

Problem : Decidual macrophages (dMφ) of the mother and placental macrophages (Hofbauer cells, HC) of the fetus are deployed at a critical location: the feto-maternal interface. This study was conducted to compare the DNA methylome of maternal and fetal monocytes, dMφ, and HC and thereby to determine the immunobiological importance of DNA methylation in pregnancy. Method Of Study : Paired samples were obtained from normal pregnant women at term not in labor and their neonates. Maternal monocytes (MMo) and fetal monocytes (FMo) were isolated from the peripheral blood of mothers and fetal cord blood, respectively. dMφ and HC were obtained from the decidua of fetal membranes and placentas, respectively. DNA methylation profiling was performed using the Illumina Infinium Human Methylation27 BeadChip. Quantitative real-time PCR and Western Blot were performed for validation experiments. Results : (i) Significant differences in DNA methylation were found in each comparison (MMo versus FMo, 65 loci; dMφ versus HC, 266 loci; MMo versus dMφ, 199 loci; FMo versus HC, 1030 loci). (ii) Many of the immune response-related genes were hypermethylated in fetal cells (FMo and HC) compared to maternal cells (MMo and dMφ). (iii) Genes encoding markers of classical macrophage activation were hypermethylated, and genes encoding alternative macrophage activation were hypomethylated in dMφ and HC compared to MMo and FMo, respectively. (iv) mRNA expressions of DNMT1, DNMT3A, and DNMT3B were significantly lower in dMφ than in HC. (v) 5-azacytidine treatment increased expression of INCA1 in dMφ. Conclusions : The findings herein indicate that DNA methylation patterns change during monocyte-macrophage differentiation at the feto-maternal interface. It is also suggested that DNA methylation is an important component of the biological machinery conferring an anti-inflammatory phenotype to macrophages at the feto-maternal interface.ope