MIF Increases sFLT1 Expression in Early Uncomplicated Pregnancy and Preeclampsia.

Insufficient immune tolerance during pregnancy is associated with pathological conditions such as preeclampsia (PE). Soluble fms-like tyrosine kinase-1 (sFLT1), which exerts a role in the late stage of PE, has shown its beneficial anti-inflammatory effects in inflammation-associated diseases. Macrophage migration inhibitory factor (MIF) was reported to upregulate sFLT1 production in experimental congenital diaphragmatic hernia. However, the placental sFLT1 expression in early uncomplicated pregnancy and whether MIF can regulate sFLT1 expression in uncomplicated and preeclamptic pregnancy are unclear. We collected first-trimester placentas and term placentas from uncomplicated and preeclamptic pregnancies to investigate sFLT1 and MIF expression in vivo. Primary cytotrophoblasts (CTBs) and a human trophoblast cell line (Bewo) were used to study the regulation of MIF on sFLT1 expression in vitro. In placentas from first-trimester pregnancy, we observed a high expression of sFLT1, specifically in extravillous trophoblasts (EVTs) and syncytiotrophoblast (STB) cells. MIF mRNA levels strongly correlated with sFLT1 expression in term placentas from preeclamptic pregnancies. In in vitro experiments, sFLT1 and MIF levels increased significantly in CTBs during their differentiation to EVTs and STBs, and MIF inhibitor (ISO-1) significantly reduced sFLT1 expression in a dose-dependent manner during this process. sFLT1 showed significant upregulation with increasing doses of MIF in Bewo cells. Our results show that sFLT1 is highly expressed at the maternal-fetal interface during early pregnancy and that MIF can increase sFLT1 expression in early uncomplicated pregnancy and PE, which suggests that sFLT1 plays an essential role in the modulation of inflammation in pregnancy.

Imaging mass cytometry reveals the prominent role of myeloid cells at the maternal-fetal interface.

Although the immunological complexity of the maternal-fetal interface is well appreciated, the actual interaction of maternal immune cells and fetal trophoblasts is insufficiently understood. To comprehend the composition and spatial orientation of maternal immune cells and fetal extravillous trophoblasts, we applied imaging mass cytometry on decidua basalis of the three trimesters of healthy pregnancy. Within all trimesters, we observed considerably higher frequencies of myeloid cells in the decidua than is seen with single-cell suspension techniques. Moreover, they were the most pronounced cell type in the microenvironment of other decidual cells. In first trimester, HLA-DR- macrophages represented the most abundant myeloid subcluster and these cells were frequently observed in the vicinity of trophoblasts. At term, HLA-DR+ macrophage subclusters were abundantly present and frequently observed in the microenvironment of T cells. Taken together, our results highlight the dynamic role of myeloid cells at the human maternal-fetal interface throughout gestation.

Primary Trophoblast Cultures: Characterization of HLA Profiles and Immune Cell Interactions.

Introduction: Trophoblasts are essential in fetal-maternal interaction during pregnancy. The goal was to study HLA profiles of primary trophoblasts derived from placentas, and to investigate their usefulness in studying interaction with immune cells. Methods: After enzymatic digestion of first-trimester placental tissue from seven donors (6-9 weeks gestation) and trophoblast enrichment we cultured cytotrophoblasts (CTB) in stem cell medium. CTB were differentiated into EVT in a Matrigel-containing medium. A subset of CTB/EVT was profiled for microRNA levels. Expression of classical HLA molecules and of HLA-G was studied by flow cytometry, qPCR, and ELISA. Secondary trophoblast cell lines JAR and JEG-3 were studied as controls. Lymphocytes were investigated during co-culturing with EVT. Results: The trophoblasts could be easily maintained for several passages, upregulated classical trophoblast markers (GATA3, TFAP2C, chromosome-19 microRNAs), and upon differentiation to EVT they were selective in expressing HLA-C. EVT showed increasing expression of total HLA-G, an increasing proportion of HLA-G1 over G2- and G3 isoforms, and elevated excretion of soluble HLA-G. These features were distinct from those of the secondary trophoblast cell lines. TNF-α and IL-8 represented the most abundantly secreted cytokines by CTB, but their levels were minimal in EVT cultures. As proof of principle, we showed that EVT affect lymphocytes in three-day co-cultures (n=4) by decreasing activation marker HLA-DR. Conclusion: We verified the possibility culturing trophoblasts from first-term placentas, and their capability of differentiating to HLA-G expressing EVT. This culture model better represents the in-vivo situation than previously studied secondary trophoblast cell lines and enables mechanistic studies of fetal-maternal interactions.

Circulating Levels of Anti-C1q and Anti-Factor H Autoantibodies and Their Targets in Normal Pregnancy and Preeclampsia.

Preeclampsia (PE) generally manifests in the second half of pregnancy with hypertension and proteinuria. The understanding of the origin and mechanism behind PE is incomplete, although there is clearly an immune component to this disorder. The placenta constitutes a complicated immune interface between fetal and maternal cells, where regulation and tolerance are key. Stress factors from placental dysfunction in PE are released to the maternal circulation evoking the maternal response. Several complement factors play a role within this intricate landscape, including C1q in vascular remodeling and Factor H (FH) as the key regulator of alternative pathway complement activation. We hypothesize that decreased levels of C1q or FH, or disturbance of their function by autoantibodies, may be associated with PE. Autoantibodies against C1q and FH and the concentrations of C1q and FH were measured by ELISA in maternal sera from women with preeclamptic and normal pregnancies. Samples originated from cohorts collected in the Netherlands (n=63 PE; n=174 control pregnancies, n=51 nonpregnant), Finland (n=181 PE; n=63 control pregnancies) and Norway (n=59 PE; n=27 control pregnancies). Serum C1q and FH concentrations were higher in control pregnancy than in nonpregnant women. No significant differences were observed for serum C1q between preeclamptic and control pregnancy in any of the three cohorts. Serum levels of FH were lower in preeclamptic pregnancies compared to control pregnancies in two of the cohorts, this effect was driven by the early onset PE cases. Neither anti-C1q autoantibodies nor anti-FH autoantibodies levels differed between women with PE and normal pregnancies. In conclusion, levels of anti-C1q and anti-FH autoantibodies are not increased in PE. C1q and FH are increased in pregnancy, but importantly, a decrease in FH concentration is associated with PE.

Different immunoregulatory components at the decidua basalis of oocyte donation pregnancies.

Oocyte donation (OD) pregnancies are characterized by more fetal-maternal human leukocyte antigen (HLA) mismatches compared with naturally conceived (NC) and in vitro fertilization (IVF) pregnancies. The maternal immune system has to cope with greater immunogenetic dissimilarity, but involved immunoregulation remains poorly understood. We examined whether the amount of regulatory T cells (Tregs) and immunoregulatory cytokines in decidua basalis of OD pregnancies differs from NC and IVF pregnancies. The cohort included 25 OD, 11 IVF and 16 NC placentas, maternal peripheral blood, and umbilical cord blood of uncomplicated pregnancies. Placenta slides were stained for FOXP3, IL-10, IL-6, gal-1, TGF-ß and Flt-1. Semi-quantitative (FOXP3+ Tregs) and computerized analysis (cytokines) were executed. The blood samples were typed for HLA class I and II to calculate fetal-maternal HLA mismatches. The percentage of Tregs was significantly higher in pregnancies with 4-6 HLA class I mismatches (n = 17), compared to 0-3 mismatches (n = 35; p = 0.04). Cytokine analysis showed significant differences between OD, IVF and NC pregnancies. Flt-1 was significantly lower in pregnancies with 4-6 HLA class I mismatches (p = 0.004), and in pregnancies with 6-10 HLA mismatches in total (p = 0.024). This study suggests that immunoregulation at the fetal-maternal interface in OD pregnancies with more fetal-maternal HLA mismatches is altered.

Placental Complement Activation in Fetal and Neonatal Alloimmune Thrombocytopenia: An Observational Study.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a disease that causes thrombocytopenia and a risk of bleeding in the (unborn) child that result from maternal alloantibodies directed against fetal, paternally inherited, human platelet antigens (HPA). It is hypothesized that these alloantibodies can also bind to the placenta, causing placental damage. This study aims to explore signs of antibody-mediated placental damage in FNAIT. We performed a retrospective study that included pregnant women, their newborns, and placentas. It comprised 23 FNAIT cases, of which nine were newly diagnosed (14 samples) and 14 were antenatally treated with intravenous immune globulins (IVIg) (21 samples), and 20 controls, of which 10 had anti-HLA-class I antibodies. Clinical information was collected from medical records. Placental samples were stained for complement activation markers (C1q, C4d, SC5b-9, and mannose-binding lectin) using immunohistochemistry. Histopathology was examined according to the Amsterdam criteria. A higher degree of C4d deposition was present in the newly diagnosed FNAIT cases (10/14 samples), as compared to the IVIg-treated FNAIT cases (2/21 samples, p = 0.002) and anti-HLA-negative controls (3/20 samples, p = 0.006). A histopathological examination showed delayed maturation in four (44%) placentas in the newly diagnosed FNAIT cases, five (36%) in the IVIg-treated FNAIT cases, and one in the controls (NS). C4d deposition at the syncytiotrophoblast was present in combination with low-grade villitis of unknown etiology in three newly diagnosed FNAIT cases that were born SGA. We conclude that a higher degree of classical pathway-induced complement activation is present in placentas from pregnancies with untreated FNAIT. This may affect placental function and fetal growth.

Maternal-Fetal HLA Compatibility in Uncomplicated and Preeclamptic Naturally Conceived Pregnancies.

Introduction: In pregnancy, the mother and fetus differ in HLA antigens, and yet the maternal immune system generally tolerates the fetus. KIR receptors expressed by maternal uterine NK cells at the maternal-fetal interface directly interact with HLA-C on extravillous trophoblast cells for optimal placental development. In this study, we aimed to determine whether there is a preferential selection for HLA compatibility and specific KIR/HLA-C combinations in uncomplicated and preeclamptic naturally conceived pregnancies compared to what would be expected by chance. Methods: Genotyping for maternal and fetal HLA-A, -B, -C, -DR, and -DQ, and maternal KIR was performed for 451 uncomplicated pregnancies and 77 pregnancies complicated with preeclampsia. The number of HLA antigen (mis)matches between mother and fetus was calculated and compared to expected values obtained by randomization of the HLA haplotype, inherited from the father, over the existing maternal haplotype of the fetuses. A similar methodology was executed for analysis of the KIR/HLA-C data (n=309). Results: In uncomplicated pregnancies, the degree of maternal-fetal HLA matching was not different than expected-by-chance values. In preeclamptic pregnancies, the degree of maternal-fetal HLA matching was different in observed compared to expected-by-chance values (p=0.012). More specifically, the degree of maternal-fetal matching of HLA-C was higher in the actual preeclamptic pregnancies than was expected-by-chance (p=0.007). Preeclamptic pregnancies showed an overall tendency towards higher maternal-fetal HLA compatibility, for total HLA matches (p=0.021), HLA class I (p=0.038) and HLA-C (p=0.025) compared to uncomplicated pregnancies. Conclusion: The data suggest that there is no preferential selection of maternal-fetal HLA compatibility in uncomplicated pregnancies. In contrast, increased total HLA, HLA class I and, especially, HLA-C compatibility is associated with preeclampsia, suggestive for a role of HLA mismatches in immune regulation leading to uncomplicated pregnancy.

Visualizing Dynamic Changes at the Maternal-Fetal Interface Throughout Human Pregnancy by Mass Cytometry.

During healthy pregnancy, a balanced microenvironment at the maternal-fetal interface with coordinated interaction between various immune cells is necessary to maintain immunological tolerance. While specific decidual immune cell subsets have been investigated, a system-wide unbiased approach is lacking. Here, mass cytometry was applied for data-driven, in-depth immune profiling of the total leukocyte population isolated from first, second, and third trimester decidua, as well as maternal peripheral blood at time of delivery. The maternal-fetal interface showed a unique composition of immune cells, different from peripheral blood, with significant differences between early and term pregnancy samples. Profiling revealed substantial heterogeneity in the decidual lymphoid and myeloid cell lineages that shape gestational-specific immune networks and putative differentiation trajectories over time during gestation. Uncovering the overall complexity at the maternal-fetal interface throughout pregnancy resulted in a human atlas that may serve as a foundation upon which comprehension of the immune microenvironment and alterations thereof in pregnancy complications can be built.

Soluble HLA-G levels in seminal plasma are associated with HLA-G 3'UTR genotypes and haplotypes.

Soluble HLA-G (sHLA-G) levels in human seminal plasma (SP) can be diverse and may affect the establishment of maternal-fetal tolerance and thereby the outcome of pregnancy. We investigated whether sHLA-G levels in SP are associated with polymorphisms in the 3'-untranslated region (UTR) and UTR haplotypes of the HLA-G gene. Furthermore, we compared the HLA-G genotype distribution and sHLA-G levels between men, whose partner experienced unexplained recurrent miscarriage (RM), and controls. Soluble HLA-G levels (n = 156) and HLA-G genotyping (n = 176) were determined in SP samples. The concentration of sHLA-G was significantly associated with several single-nucleotide polymorphisms (SNPs): the 14 base pair (bp) insertion/deletion (indel), +3010, +3142, +3187, +3196, and + 3509. High levels of sHLA-G were associated with UTR-1 and low levels with UTR-2, UTR-4, and UTR-7 (P < .0001). HLA-G genotype distribution and sHLA-G levels in SP were not significantly different between the RM group (n = 44) and controls (n = 31). In conclusion, seminal sHLA-G levels are associated with both singular SNPs and 3UTR haplotypes. HLA-G genotype and sHLA-G levels in SP are not different between men whose partner experienced RM and controls, indicating that miscarriages are not solely the result of low sHLA-G levels in SP. Instead, it is more likely that these miscarriages are the result of a multifactorial immunologic mechanism, whereby the HLA-G 3'UTR 14 bp ins/ins genotype plays a role in a proportion of the cases. Future studies should look into the functions of sHLA-G in SP and the consequences of low or high levels on the chance to conceive.

Increased HLA-G Expression in Term Placenta of Women with a History of Recurrent Miscarriage Despite Their Genetic Predisposition to Decreased HLA-G Levels.

Human leukocyte antigen (HLA)-G is an immune modulating molecule that is present on fetal extravillous trophoblasts at the fetal-maternal interface. Single nucleotide polymorphisms (SNPs) in the 3 prime untranslated region (3'UTR) of the HLA-G gene can affect the level of HLA-G expression, which may be altered in women with recurrent miscarriages (RM). This case-control study included 23 women with a medical history of three or more consecutive miscarriages who delivered a child after uncomplicated pregnancy, and 46 controls with uncomplicated pregnancy. Genomic DNA was isolated to sequence the 3'UTR of HLA-G. Tissue from term placentas was processed to quantify the HLA-G protein and mRNA levels. The women with a history of RM had a lower frequency of the HLA-G 3'UTR 14-bp del/del genotype as compared to controls (Odds ratio (OR) 0.28; p = 0.039), which has previously been related to higher soluble HLA-G levels. Yet, HLA-G protein (OR 6.67; p = 0.006) and mRNA (OR 6.33; p = 0.010) expression was increased in term placentas of women with a history of RM as compared to controls. In conclusion, during a successful pregnancy, HLA-G expression is elevated in term placentas from women with a history of RM as compared to controls, despite a genetic predisposition that is associated with decreased HLA-G levels. These findings suggest that HLA-G upregulation could be a compensatory mechanism in the occurrence of RM to achieve an ongoing pregnancy.

Stronger T-Cell Alloreactivity and Diminished Suppressive Capacity of Peripheral Regulatory T Cells in Infertile Women Undergoing In Vitro Fertilization.

PROBLEM: Increasing evidence suggests modulation of the maternal immune response to be essential for successful pregnancy. We studied the immunophenotypic profile and function of peripheral blood T lymphocytes in infertile women undergoing in vitro fertilization (IVF) and fertile control population. METHOD: We collected peripheral blood mononuclear cells (PBMC) from infertile patients with recurrent implantation failure (RIF), infertile patients with successful IVF (IVFs), and normal fertile women. Cells were phenotypically analyzed, and the proliferative response and cytokine production were studied in mixed lymphocyte cultures (MLC), using lymphocytes of the own partner, or a third-party male as stimulators cells. To examine the suppressive capacity of regulatory regulatory T cells (Tregs), we performed MLC studies with a CD45(+) fraction depleted for CD4(+) CD25(bright) T cells. RESULTS: No significant differences in proportions of subsets of circulating T lymphocytes were observed. The proliferative allo-immune response of PBMC of IVF women (RIF and IVFs) was significantly higher, with higher production of T-helper cells (Th1) and Th2 cytokines, compared to the fertile women. This difference in proliferation and cytokine production was associated with a diminished suppressive capacity of Tregs in these women. CONCLUSION: The higher allo-immune response of the IVF women compared to fertile women might be the result of a diminished suppressive capacity of Tregs and emphasizes the important role of Tregs during conception.

Differential immunoregulation in successful oocyte donation pregnancies compared with naturally conceived pregnancies.

In oocyte donation (OD) pregnancies, there is a higher level of antigenic dissimilarity between mother and fetus compared with naturally conceived (NC) pregnancies. We hypothesize that a higher degree and/or a different type of immunoregulation is necessary to maintain an uncomplicated OD pregnancy. Different immunological aspects of successful OD pregnancies (n=28) were compared with those of NC pregnancies (n=51), and non-donor IVF (n=20) pregnancies. Maternal peripheral blood mononuclear cells (mPBMCs) were characterized by flow cytometry; the outcome correlated with the number of mother-child HLA mismatches. The fetus-specific alloreactivity of mPBMCs was measured in a mixed lymphocyte reaction. The percentages of CD4(+)CD25(bright) and CD4(+)CD25(dim) cells were higher in mPBMCs of OD and IVF pregnancies compared with NC pregnancies. The percentage of CD4(+)CD25(dim) cells in mPBMCs of OD pregnancies correlated positively with the number of HLA mismatches. Functional studies showed a lower proliferative response to umbilical cord blood by mPBMCs in OD pregnancies. In conclusion, we found a higher degree of peripheral immunoregulation in OD and IVF pregnancies compared with NC pregnancies. A higher number of HLA mismatches in successful OD pregnancies leads to increased percentages of activated T cells in peripheral blood, but not to a higher alloreactivity to the fetus. These studies show that immunoregulation in OD pregnancy is different from that in NC pregnancies. The antigenic dissimilarity in OD pregnancies may play a role in the pathophysiology of preeclampsia.

Changes in cytokine production and composition of peripheral blood leukocytes during pregnancy are not associated with a difference in the proliferative immune response to the fetus.

We analyzed peripheral blood from women at term pregnancy for leukocyte composition, in vitro proliferative responses and cytokine production after nonspecific and fetus-specific stimulation. Maternal peripheral blood mononuclear cells (PBMCs) were collected and stimulated with umbilical cord blood (UCB) of the mother's own child, third-party UCB, nonspecific stimulus phytohemagglutinin, and anti-CD3 antibody, with PBMCs of nonpregnant women (cPBMC) as controls. Nine combinations of patient, child, third party child, and controls were selected on basis of sharing one human leukocyte antigen (HLA)-DR antigen. The response of mPBMC upon specific stimulation with fetal antigens was similar to that of cPBMC. No differences were found when comparing the mother's response upon stimulation to her own child with stimulation to that with a control child. Nonspecific stimulation with phytohemagglutinin and anti-CD3 antibody did not reveal a difference in proliferation rate between mPBMC and cPBMC. However, mPBMC contained a higher percentage of CD14(+) cells (p = 0.001) and activated T cells (CD25(dim), p < 0.0001), but a lower percentage CD16(-)CD56(bright) natural killer (NK) cells (p = 0.001) and CD16(+)CD56(+) NK cells (p = 0.003). mPBMC produced more interleukin (IL)-6, IL-10, and IL-17 compared with cPBMC (p < 0.05). We found differences in lymphocyte composition and cytokine production between mPBMC and cPBMC. These differences did not result in quantitative changes in proliferative responses during pregnancy compared with responses in nonpregnant controls.

Isolation of mesenchymal stem cells of fetal or maternal origin from human placenta.

Recently we reported that second-trimester amniotic fluid (AF) is an abundant source of fetal mesenchymal stem cells (MSCs). In this study, we analyze the origin of these MSCs and the presence of MSCs in human-term AF. In addition, different parts of the human placenta were studied for the presence of either fetal or maternal MSCs. We compared the phenotype and growth characteristics of MSCs derived from AF and placenta. Cells from human second-trimester (mean gestational age, 19(+2) [standard deviation, +/- 1(+3)] weeks, n = 10) and term third-trimester (mean gestational age, 38(+4) [standard deviation, +/- 1] weeks, n = 10) AF, amnion, decidua basalis, and decidua parietalis were cultured in M199 medium supplemented with 10% fetal calf serum and endothelial cell growth factor. Cultured cells were immunophenotypically characterized, the adipogenic and osteogenic differentiation capacity was tested, and the growth kinetics were analyzed. The origin of fetal and maternal cells was determined by molecular human leukocyte antigen typing. We successfully isolated MSCs from second-trimester AF, amnion, and decidua basalis as well as term amnion, decidua parietalis, and decidua basalis. In contrast, MSCs were cultured from only 2 out of 10 term AF samples. The phenotype of MSCs cultured from different fetal and maternal parts of the placenta was comparable. Maternal MSCs from second-trimester and term decidua basalis and parietalis showed a significantly higher expansion capacity than that of MSCs from adult bone marrow (p < .05). Our results indicate that both fetal and maternal MSCs can be isolated from the human placenta. Amnion is a novel source of fetal MSCs, likely contributing to the presence of MSCs in AF. Decidua basalis and decidua parietalis are sources for maternal MSCs. The expansion potency from both fetal and maternal placenta-derived MSCs was higher compared with adult bone marrow-derived MSCs.

Mesenchymal stem cells in human second-trimester bone marrow, liver, lung, and spleen exhibit a similar immunophenotype but a heterogeneous multilineage differentiation potential.

BACKGROUND AND OBJECTIVES: We previously found that human fetal lung is a rich source of mesenchymal stem cells (MSC). Here we characterize and analyze the frequency and function of MSC in other second-trimester fetal tissues. DESIGN AND METHODS: Single cell suspensions of fetal bone marrow (BM), liver, lung, and spleen were made and analyzed by flow cytometry for the expression of CD90, CD105, CD166, SH3, SH4, HLA-ABC, HLA-DR, CD34 and CD45. We assessed the frequency of MSC by limiting dilution assay. RESULTS: The frequency of MSC in BM was significantly higher than in liver, lung, and spleen (p<0.05). On primary non-expanded cells from fetal liver, lung and spleen the number of cells positive for mesenchymal markers was significantly higher within the CD34 positive population than within the CD34 negative population. The phenotype of the culture-expanded MSC was similar for all fetal tissues, i.e. CD90, CD105, CD166, SH3, SH4 and HLA-ABC positive and CD34, CD45 and HLA-DR negative. Culture-expanded cells from all tissues were able to differentiate along adipogenic and osteogenic pathways. However, adipogenic differentiation was less in MSC derived from spleen, and osteogenic differentiation was reduced in liver-derived MSC (p<0.05). INTERPRETATION AND CONCLUSIONS: Our results indicate that culture-expanded MSC derived from second-trimester fetal tissues, although phenotypically similar, exhibit heterogeneity in differentiating potential. We speculate that these differences may be relevant for the clinical application of MSC.