Human leukocyte antigen (HLA)-G during pregnancy part I: Correlations between maternal soluble HLA-G at midterm,at term,and umbilical cord blood soluble HLA-G at term

Human leukocyte antigen (HLA)-G is a class Ib molecule with restricted tissue distribution expressed on trophoblast cells and has been proposed to have immunomodulatory functions during pregnancy. Soluble HLA-G1 (sHLA-G1) can be generated by the shedding of membrane-bound HLA-G molecules; however, three soluble isoforms also exist (HLA-G5 to -G6). During pregnancy, it is unknown whether there is a correlation between sHLA-G levels in maternal and fetal blood. In 246 pregnancies, we have measured the levels of sHLA-G1/-G5 in maternal blood plasma samples from gestational week 20 (GW20) and at term, as well as in umbilical cord blood samples. Soluble HLA-G levels declined by 38.4% in maternal blood from GW20 to term, and sHLA-G levels were significantly lower in maternal blood at term than in GW20 (P < 0.001). At term, the sHLA-G levels were significantly higher in maternal blood than in umbilical blood (P < 0.001). HLA-G levels in maternal blood in GW20 and at term, and in maternal blood at term and umbilical cord blood, were correlated (P < 0.001 and P < 0.01, respectively). This is the first large study simultaneously measuring sHLA-G in both maternal and umbilical cord blood. The finding that sHLA-G levels are significantly lower in fetal compared with maternal blood at term documents for the first time that sHLA-G is not freely transferred over the placental barrier. Soluble HLA-G levels in maternal and fetal blood were found to be correlated, which may be due to shared genetic factors of importance for production of sHLA-G in the mother and child, or it may support the theory that sHLA-G in the pregnant woman and the fetus is partly derived from a “shared organ”, the placenta.     

Maternal antigen presenting cells are a source of plasmatic HLA-G during pregnancy: longitudinal study during pregnancy

The aim of this work was to investigate the longitudinal evolution of plasmatic soluble HLA-G (sHLA-G: shed HLA-G1 plus HLA-G5) during pregnancy, and if peripheral maternal antigen presenting cells (APC) can be a source of sHLA-G. Blood samples were obtained from 45 volunteers during normal pregnancy, 8 of them monthly; from 8 pregnant volunteers in the first weeks of pregnancy who had later a miscarriage, and from 14 healthy nonpregnant control women. Monocytes obtained during pregnancy showed a moderately HLA-G cell surface expression and stimulation with interferon (IFN)-gamma increased this expression. Monocytes-derived dendritic cells obtained from pregnant women during the first and third trimester of pregnancy secreted more sHLA-G than those obtained from nonpregnant women. Plasmatic sHLA-G concentration in pregnant women was significatively higher than in nonpregnant women, with a peak in the third month. We can conclude that maternal APC are a source of sHLA-G. Women who experienced miscarriage had previously very low or undetectable plasmatic sHLA-G levels in the second month of pregnancy. Data suggest that undetectable sHLA-G could be a risk of complications.     

Anti-fetal immune response mechanisms may be involved in the pathogenesis of placental abruption

Placental abruption is an unpredictable severe complication in pregnancy. In order to investigate the possibility that the activation of the fetal nonadaptive immune system may be involved in the pathogenesis of this disease, IL-6 release from cord blood monocytes was examined by intracellular cytokine staining and flow cytometric analysis. Our results demonstrate that preterm placental abruption (n = 15) in contrast to uncontrollable preterm labor (n = 33) is associated with significantly (P < 0.001) increased release of IL-6 from the fetal monocytes. The same holds true for rhesus disease (n = 9, P < 0.001) that is characterized by a maternal production of antibodies against the rhesus-D antigen expressed by the fetal erythrocytes. This suggests that during rhesus disease, IL-6 release of monocytes is induced by antibody-mediated cross-linking of these cells to the erythrocytes in the fetal circulation. Hence, this assumption favors the idea that also in case of placental abruption, an increased maternal antibody production against paternal antigens leads to an elevated IL-6 release by the fetal monocytes. To elucidate this potential mechanism, the presence of anti-HLA-antibodies was assessed in the maternal circulation of patients with placental abruption (n = 17) and patients with uncontrollable preterm labor (n = 29). The percentage of women producing anti-paternal HLA-antibodies was significantly (P < 0.01) increased in the group of women with preterm placental abruption (47%) in comparison to women with uncontrollable preterm labor (14%). Therefore, our results suggest that an increased humoral immune response of the mother against the fetus may be decisively involved in the pathogenesis of placental abruption.     

Early detection of decreased soluble HLA-G levels in the maternal circulation predicts the occurrence of preeclampsia and intrauterine growth retardation during further course of pregnancy

PROBLEM: Soluble (s) HLA-G1/G5 molecules may potentially affect immune homeostasis during pregnancy. The aim of this study was to determine changes of sHLA-G1/G5 plasma levels throughout normal pregnancy and to assess its predictive value for the occurrence of characteristic gestation-associated diseases during further course of pregnancy. METHOD OF STUDY: sHLA-G1/G5 levels were estimated in plasma samples of 40 non-pregnant women, 291 women throughout normal pregnancy and 236 women affected by different complications. RESULTS: In comparison with non-pregnant women sHLA-G1/G5 levels strongly increased during the first trimenon and then decreased continuously toward term. Non-parametric discriminant analysis showed that women with significantly decreased sHLA-G1/G5 levels in the second trimenon had an increased risk of developing preeclampsia and/or intrauterine growth retardation (IUGR) during further course of pregnancy. However, in the third trimenon, sHLA-G1/G5 levels in affected women did not deviate significantly from those of non-affected women. Surprisingly, significantly increased sHLA-G1/G5 levels were detected in third trimenon women with uncontrollable preterm labor, but not in women with prolonged preterm rupture of fetal membranes. CONCLUSION: For the identification of women with an increased risk of IUGR and/or preeclampsia, measurement of sHLA-G1/G5 plasma levels may be a powerful new tool in prenatal diagnostics.     

Allergic Women have Reduced sHLA-G Plasma Levels at Delivery

Problem  HLA-G antigen maintains a tolerogenic condition at the foeto-maternal interface, counteracts inflammation in autoimmune diseases and soluble HLA-G (sHLA-G) levels decrease in allergic-asthmatics. Taking into consideration these findings, we analyzed if sHLA-G and interleukin-10 (IL-10) could be influenced by pregnancy and labour in allergic and non-allergic women.
Method of study  sHLA-G isoforms and IL-10 levels were determined in the plasma samples of 43 women (15 non-allergic, 28 allergic) during third trimester, at delivery and 2 years after pregnancy by immunoenzymatic assays.
Results  A significant increase in sHLA-G and IL-10 levels was documented at delivery in both allergic and non-allergic women. Allergic women showed lower sHLA-G concentrations. sHLA-G1 was evidenced as the predominant plasma isoform.
Conclusion  The data showed increased sHLA-G and IL-10 concentrations at delivery, regardless of the allergic status. The sHLA-G1 isoform is mainly responsible for the increased sHLA-G levels at delivery.     

Human leukocyte antigen (HLA)-G during pregnancy part II: Associations between maternal and fetal HLA-G genotypes and soluble HLA-G

Human leukocyte antigen (HLA)-G is a class Ib molecule with restricted tissue distribution expressed on the extra-villous trophoblast and seems to have immunomodulatory functions during pregnancy. Studies have linked HLA-G polymorphisms to pregnancy complications such as preeclampsia and recurrent miscarriage. Levels of soluble HLA-G (sHLA-G) in blood plasma from non-pregnant donors seem to be associated with these polymorphisms. In the current study, we have genotyped 246 mothers and their offspring for HLA-G polymorphisms in the 3′-untranslated region (3′UTR) and measured sHLA-G in maternal blood plasma samples from gestational week 20 and at term, as well as in fetal umbilical cord blood samples. This is the first large study simultaneously performing HLA-G genotyping of mother and offspring and measuring sHLA-G in both maternal and umbilical cord blood. The results showed that increasing numbers of 14 bp ins (rs66554220) alleles in the mother–child genotype combinations were associated with higher maternal sHLA-G levels at term when restricting the analysis to 14 bp ins/del heterozygous mothers (p = 0.015). Furthermore, increasing numbers of 14InsG haplotypes (14 bp ins/del and +3142C/G (rs1063320) polymorphism) in mother–child genotype combinations were associated with higher levels of sHLA-G at term in heterozygous 14DelC/14InsG mothers (p = 0.005). In conclusion, the results indicate that there is an association between combined feto-maternal HLA-G genotypes and sHLA-G levels in maternal blood plasma.     

凝血指标测定对诊断胎盘早剥的价值分析

目的探讨血清D-二聚体尧纤维蛋白原测定对胎盘早剥的诊断价值遥方法选取本院2015 年1 月~2017 年4月确诊为 胎盘早剥产妇36 例为观察组,选取正常产妇40 例为对照组,分别测定胎盘早剥产妇尧正常产妇术前血清D-二聚体尧纤维蛋白 原尧部分凝血活酶时间,比较观察组与对照组以上指标是否有差异遥胎盘早剥组产妇术前依据B超诊断24 例,B 超与凝血指标 联合诊断32 例,比较两种诊断方法是否有差异遥结果胎盘早剥产妇术前血清D-二聚体水平高于正常产妇（约0.05）,纤维蛋 白原水平低于正常产妇（约0.05）遥部分凝血活酶时间两组之间无差异（跃0.05）遥凝血指标联合B 超诊断胎盘早剥确诊率高于单 一B 超诊断,差异具有统计学意义（约0.05）遥结论胎盘早剥前期血清D-二聚体水平升高,纤维蛋白原水平下降,上述两个指 标可作为胎盘早剥诊断的依据,联合B 超诊断可提高胎盘早剥的诊断率遥     

Soluble HLA levels in early pregnancy after in vitro fertilization

Intact pregnancy can be interpreted as a state of maternal immunotolerance toward an haploidentical fetus. Soluble HLA (sHLA) molecules increase during episodes of allograft rejection and are discussed as candidates to modulate immune responses. We questioned whether after in vitro fertilization (IVF) the subsequent intact pregnancy, early abortion, or tubal pregnancy influence the courses sHLA serum levels. Therefore, serum samples of 65 IVF patients were assayed by ELISA for sHLA-I, sHLA-G, and sHLA-DR concentrations preovulatorily and after a positive HCG test weekly until the 9th gestational week (GW). In 20 patients experiencing an early abortion the preovulatory sHLA-G mean level of 25.9 +/- 3.9 SEM ng/ml and the share of 4.2 +/- 0.8 SEM % on total sHLA-I were significantly (p < 0.05) reduced compared to women with intact pregnancy. The same differences (p < 0.0001) were seen during the monitoring of sHLA-G and sHLA-I levels in intact pregnancy versus early abortion until 9th GW. Twin pregnancy revealed a drastically increase of sHLA-G levels from the 8th GW compared to singleton pregnancies. Further, individual sHLA-DR levels increased during intact pregnancy but decreased in the group of early abortion. With regard to sensitivity and specificity for pregnancy outcome sHLA quantitation reached similar weight as routine HCG determinations at GW 5. Especially women with preovulatory low sHLA-G levels appear to be on risk for early abortion after IVF.     

Rapid evaluation of soluble HLA-G levels in supernatants of in vitro fertilized embryos

Human leukocyte antigen G (HLA-G) molecules are crucial for the maternal tolerance against the fetus during pregnancy. Thus, the presence of soluble HLA-G (sHLA-G) in embryo cultures is thought to be correlated to a successful pregnancy after assisted reproductive techniques (ART). Here, we established a rapid detection assay based on Luminex technology, which can be integrated into ART proceedings, allowing sHLA-G quantification in sample volumes of only 10 microl within 1.5 hours. Using this method, sHLA-G levels of 526 single-embryo cultures, 47 two-embryo cultures, and 15 three-embryo cultures were analyzed corresponding to 313 ART cycles. In 117 embryo cultures, sHLA-G was detectable. In single-embryo cultures, the sHLA-G levels were positively correlated to embryo quality (p = 0.048, r = 0.20, n = 100). The presence of sHLA-G in embryo cultures was significantly (p < 0.0001) associated with clinical pregnancy after intracytoplasmatic sperm injections (ICSI), especially in couples with male factor infertility, but not after in vitro fertilization (IVF) or in couples with female infertility. Importantly, in sHLA-G negative embryos, the abortion rate was increased threefold (p = 0.04). In conclusion, the results obtained by our novel method support strongly the diagnostic relevance of sHLA-G for predicting pregnancy outcome after ART. The ultimate conditions for this prediction have to be further investigated in a multicenter study.     

HLA-G polymorphisms and soluble HLA-G protein levels in women with recurrent pregnancy loss from Basrah province in Iraq

HLA-G is a nonclassical, class I major histocompatibility complex (MHC) gene that exhibits immunomodulatory properties and likely plays a role in the maintenance of successful pregnancy. In this study, we investigated the role of HLA-G polymorphisms on risk for recurrent pregnancy loss (RPL) and on circulating levels of soluble (s)HLA-G in Iraqi women. DNA and plasma were obtained from blood samples collected at 9-12 weeks gestation from 50 women with RPL and 50 healthy pregnant women in Basrah province, Iraq. As measured by ELISA, median sHLA-G levels were significantly lower in the RPL cases compared to healthy controls (21.4 vs. 38.8 U/ml, respectively; P=0.025), and decreased with increasing maternal age (P=0.0051). However, HLA-G allele and haplotype frequencies did not differ significantly between cases and controls (P values ≥0.12 for all tests). In contrast, homozygosity for the C allele (CC) at a tri-allelic promoter polymorphism, -725C/G/T, was associated with lower concentrations of sHLA-G compared to the CG or CT genotypes (median levels 21.1 vs. 40.1 vs. 42.6 U/ml, respectively; P=0.0089). These results demonstrate that HLA-G genotype influences circulating sHLA-G levels during pregnancy but is not significantly associated with risk of RPL.     

Analyzing HLA-G polymorphisms in children from women with scleroderma

Embryos during pregnancy and organs during transplantation, express high levels of soluble HLA-G (sHLA-G) molecules for successful implantation and protection against maternal immune cells or recipient’s cells. We and others have shown that women with scleroderma (SSc) carry cells/DNA arising from pregnancy, so-called fetal microchimerism (Mc) more often and in higher quantities than healthy women decades after delivery.     

HLA-G in reproduction: studies on the maternal-fetal interface

For more than a decade, investigators have known that membrane-bound and soluble isoforms of the HLA class Ib molecule, HLA-G, are present at the maternal-fetal interface. Although it is clear that extravillous cytotrophoblast cells are major producers, other cells may also contribute. Recent studies in our laboratory raised the question of whether soluble isoforms might reach the maternal and/or fetal blood circulation. A capture enzyme-linked immunoabsorbent assay (ELISA) identified soluble HLA-G (sHLA-G) in maternal blood throughout pregnancy but failed to detect sHLA-G in cord sera. Further studies suggested that the circulating proteins may be either free heavy chain (sHLA-G1 and/or sHLA-G2) or exclusively sHLA-G2. To study the potential function(s) of the soluble isoforms to modulate local or systemic immunity in mothers, we generated recombinant sHLA-G1 and -G2 in both prokaryotic and eukaryotic systems. Preliminary experiments conducted using DNA microarray analysis suggest that sHLA-G is capable of modulating gene expression in blood mononuclear leukocytes. Potential local targets were also identified; decidual and placental macrophages but not trophoblast cells contained mRNA encoding two of the known receptors for HLA-G, ILT2 and ILT4. Collectively, the studies are consistent with the hypothesis that sHLA-G produced at the maternal-fetal interface targets to the cells of the monocyte/macrophage lineage and modulates their functions for the benefit of pregnancy.     

妊娠期高血压疾病的孕期干预与妊娠结局的关系

目的:探讨妊娠期高血压疾病的孕期干预与妊娠结局的关系。方法:回顾性分析1093例孕产妇临床资料,包括妊娠期高血压疾病患者规律产检组(干预组)389例,不规律产检组(对照组)204例,同期分娩的正常妊娠孕产妇(正常组)500例,观察孕期干预对妊娠期高血压疾病孕产妇并发症如胎盘早剥、早产、产后出血、眼底出血、中重度贫血、HELLP综合征等的发生率,以及新生儿窒息、胎儿宫内生长受限(FGR)、胎儿窘迫等围产儿不良结局的发生率。结果:正常组孕产妇并发症、围产儿不良结局及剖宫产率均低于两妊娠期高血压疾病组,差异有统计学意义(P均<0.05);干预组孕产妇并发症发生率(27.25%)和围产儿不良结局发生率(42.42%)均低于对照组,差异有统计学意义(P均<0.05)。结论:妊娠期高血压疾病的孕期监测及保健治疗干预可以降低孕产妇及围产儿不良事件的发生率,改善母婴预后。     

A role for mannose-binding lectin, a component of the innate immune system in pre-eclampsia

Problem Mannose‐binding lectin (MBL) is a pattern‐recognition receptor that activates complement and modulates inflammation. Homozygosity for the most common allele of the MBL2 gene that is associated with high MBL serum concentrations is more prevalent among patients with pre‐eclampsia. The objective of this study was to determine maternal plasma MBL concentrations in normal pregnant women and patients with pre‐eclampsia. Method of study This cross‐sectional study included normal pregnant women (n = 187) and patients with pre‐eclampsia (n = 99). Maternal plasma MBL concentrations were determined by ELISA. Results Women with pre‐eclampsia had a higher median maternal plasma MBL concentration than normal pregnant women. MBL concentration distribution curves were three‐modal, the subintervals in normal pregnancy were low (<143.7), intermediate (143.7–1898.9) and high (>1898.9 ng/mL). The proportion of normal pregnant women was larger in the low subinterval, while the proportion of patients with pre‐eclampsia was larger in the high subinterval (P = 0.02). Normal pregnant women in the high subinterval had a larger rate of placental underperfusion than those in the low and intermediate subintervals (P = 0.02). Conclusions The median maternal plasma MBL concentration is elevated in patients with pre‐eclampsia and a larger proportion of these patients are in the high subinterval than normal pregnant women, suggesting that this component of the innate immune system is involved in the mechanisms of disease in pre‐eclampsia.     

妊娠合并妊娠期高血压疾病晚期妊娠258例临床分析

目的探讨妊娠期高血压疾病对晚期妊娠的影响。方法选取我院3年来孕28w以上住院分娩病例,以妊娠合并妊娠期高血压疾病258例作观察组,随机抽取正常妊娠孕妇480例作对照组,对比分析胎盘早剥、早产、胎儿窘迫、胎儿宫内发育迟缓、死产、产后出血发生率、新生儿窒息率、剖宫产率和对孕妇的影响。结果观察组的胎盘早剥、早产、胎儿窘迫、胎儿宫内发育迟缓、死胎、新生儿窒息率、产后出血发生率、剖宫产率等母婴并发症均明显高于对照组（P〈0．05）。结论妊娠期高血压疾病对晚期妊娠的母婴结局有许多不良影响。     

Placental Transport of Maternal Immunoglobulin G in Pregnancies at Risk of Rh (D) Hemolytic Disease of the Newborn

PROBLEM: The following questions were addressed: Is the placental transport of immunoglobulin (Ig)G, IgG1, and IgG3 diminished in pregnancies at risk of hemolytic disease of the newborn? Is the placental transport of IgG, IgG1, and IgG3 correlated with the hemoglobin concentration in the fetus and AutoAnalyzer (AA) quantitations of maternal anti-D? METHOD OF STUDY: IgG concentrations were determined retrospectively in 41 paired fetal/maternal (f/m) samples in 31 Rh (D) alloimmunized pregnancies. IgG1 and IgG3 concentrations were determined in those 23 cases in which the results of fetal hemoglobin concentration and quantitations of maternal anti-D were available. The results were compared with values found in normal pregnancy and correlated with maternal anti-D AA quantitations and fetal hemoglobin concentrations. RESULTS: Fetal IgG, IgG1, and IgG3 concentrations, and the corresponding fetomaternal ratios in Rh (D) alloimmunized pregnancies, increased with gestational age according to the following formulas (obtained by simple regression): Fetal IgG = ?8.846 + 0.491.gestational age (GA), (R² = 0.544); fetal IgG1 = ?10.021 + 0.46GA, (R² = 0.463); fetal IgG3 = ?0.865 + 0.039GA, (R² = 0.327); f/m IgG = ?1.006 + 0.054?GA, (R² = 0.557); f/m IgG1 = ?1.876 + 0.085GA, (R² = 0.654); f/m IgG3 = ?0.199 + 0.026GA, (R² = 0.146). CONCLUSIONS: The placental transport of IgG, IgG1, and IgG3 in women with Rh (D) immunizations is not diminished compared with normal pregnancy. However, AA quantitations of anti-D are inversely correlated with f/m IgG ratio, f/m IgG1 ratio, and fetal IgG and IgG1 concentrations (P = 0.002, P = 0.004, P = 0.02, and P = 0.02 respectively). The placental transport of IgG3 is significantly higher in pregnancies at risk of hemolytic disease of the newborn compared with IgG3 concentrations in normal pregnancy.     

Associations between maternal and fetal serum levels of immune activation markers and fetal growth

During pregnancy, signs of maternal immunologic sensitization to fetal HLA and other fetoplacental alloantigens are often detectable in peripheral blood. Presumbly, this in part reflects immune activity at the maternal-fetal interface. This response may involve activation of maternal T cells, which stimulate placental growth via lymphokine production. To shed light on this mechanism, data on placental weight, neonatal anthropometry, gestational age, fetomaternal HLA relationships (reflecting a potential for HLA allosensitization), and serum levels of three immune activation markers in maternal and cord blood were collected in a sample of 61 primiparous women and their neonates. The activation markers were soluble CD8 antigen (sCD8), interleukin-2 receptor (sIL-2R), and beta-2 microglobulin (β₂m). Mean fetal and maternal sCD8 and β₂m levels, and mean fetal sIL-2R levels were significantly higher than published norms. Fetal means for all three markers exceeded maternal means, and both sIL-2R and β₂m were highly correlated between mother and fetus. This suggests that fetal sIL-2R and β₂m levels result in part from transport or diffusion from the maternal compartment. No associations were found between fetomaternal HLA relationships, activation markers, and placental weight. The difference between the fetal and maternal β₂m value was significantly correlated with birth weight, controlling for chest circumference and crown-heel length. Associations between birth weight and fetomaternal HLA relationships could not be interpreted with certainty. These findings suggest that maternal immune activation and diffusion or transport of β₂m into fetal compartment enhances fetal growth in fat-free body mass. © 1995 Wiley-Liss, Inc.     

Circulating ficolin-2 and ficolin-3 in normal pregnancy and pre-eclampsia

Ficolins are soluble molecules of the innate immune system that recognize carbohydrate molecules on microbial pathogens, apoptotic and necrotic cells. They act through two distinct routes: initiating the lectin pathway of complement activation and mediating a primitive opsonophagocytosis. In this study, we measured plasma levels of ficolin-2 and ficolin-3 in 60 pre-eclamptic patients, 60 healthy pregnant women and 59 healthy non-pregnant women by enzyme-linked immunosorbent assay (ELISA). Circulating levels of complement activation products (C4d, C3a, SC5b9), angiogenic factors (soluble fms-like tyrosine kinase-1, placental growth factor) and markers of endothelial activation (von Willebrand factor antigen), endothelial injury (fibronectin) and trophoblast debris (cell-free fetal DNA) were also determined. Plasma levels of ficolin-2 were significantly lower in healthy pregnant than in healthy non-pregnant women, while ficolin-3 levels did not differ significantly between the two groups. Furthermore, pre-eclamptic patients had significantly lower ficolin-2 and ficolin-3 concentrations than healthy non-pregnant and pregnant women. In the pre-eclamptic group, plasma ficolin-2 levels showed a significant positive correlation with serum placental growth factor (PlGF) concentrations and significant inverse correlations with serum levels of soluble fms-like tyrosine kinase-1 (sFlt-1), blood urea nitrogen and creatinine, serum lactate dehydrogenase activities, as well as with plasma VWF:antigen, fibronectin and cell-free fetal DNA concentrations. In conclusion, circulating levels of ficolin-2 are decreased in the third trimester of normal pregnancy. There is a further decrease in plasma ficolin-2 concentrations in pre-eclampsia, which might contribute to the development of the maternal syndrome of the disease through impaired removal of the trophoblast-derived material released into the maternal circulation by the hypoxic and oxidatively stressed pre-eclamptic placenta.