The Mac Is Back: The Role of Macrophages in Human Healthy and Complicated Pregnancies.

Pregnancy is a fascinating immunological paradox: the semi-allogeneic fetus generally grows without any complications. In the placenta, fetal trophoblast cells come into contact with maternal immune cells. Inaccurate or inadequate adaptations of the maternal immune system could lead to problems with the functioning of the placenta. Macrophages are important for tissue homeostasis, cleanup, and the repair of damaged tissues. This is crucial for a rapidly developing organ such as the placenta. The consensus on macrophages at the maternal-fetal interface in pregnancy is that a major proportion have an anti-inflammatory, M2-like phenotype, that expresses scavenger receptors and is involved in tissue remodeling and the dampening of the immune reactions. Recent multidimensional analyses have contributed to a more detailed outlook on macrophages. The new view is that this lineage represents a highly diverse phenotype and is more prevalent than previously thought. Spatial-temporal in situ analyses during gestation have identified unique interactions of macrophages both with trophoblasts and with T cells at different trimesters of pregnancy. Here, we elaborate on the role of macrophages during early human pregnancy and at later gestation. Their possible effect is reviewed in the context of HLA incompatibility between mother and fetus, first in naturally conceived pregnancies, but foremost in pregnancies after oocyte donation. The potential functional consequences of macrophages for pregnancy-related immune reactions and the outcome in patients with recurrent pregnancy loss are also discussed.

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.

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.

The Role of Macrophages in Oocyte Donation Pregnancy: A Systematic Review.

The embryo of an oocyte donation (OD) pregnancy is completely allogeneic to the mother, which leads to a more serious challenge for the maternal immune system to tolerize the fetus. It is thought that macrophages are essential in maintaining a healthy pregnancy, by acting in immunomodulation and spiral arterial remodeling. OD pregnancies represent an interesting model to study complex immunologic interactions between the fetus and the pregnant woman since the embryo is totally allogeneic compared to the mother. Here, we describe a narrative review on the role of macrophages and pregnancy and a systematic review was performed on the role of macrophages in OD pregnancies. Searches were made in different databases and the titles and abstracts were evaluated by three independent authors. In total, four articles were included on OD pregnancies and macrophages. Among these articles, some findings are conflicting between studies, indicating that more research is needed in this area. From current research, we could identify that there are multiple subtypes of macrophages, having diverse biological effects, and that the ratio between subtypes is altered during gestation and in aberrant pregnancy. The study of macrophages' phenotypes and their functions in OD pregnancies might be beneficial to better understand the maternal-fetal tolerance system.

Optimization of microRNA Acquirement from Seminal Plasma and Identification of Diminished Seminal microRNA-34b as Indicator of Low Semen Concentration.

About 10-15% of couples who want to conceive suffer from subfertility, while in 30% of these cases, a male factor plays a role. Levels of particular microRNAs in seminal plasma, including those involved in spermatogenesis, may serve as an indicative parameter for subfertility. We first optimized a protocol for acquiring microRNAs from seminal plasma. Next, using a test-validation strategy in a male cohort, we aimed to identify microRNAs of which the levels are related to semen motility and concentration. By qPCR, 742 microRNAs were profiled in three normozoospermic samples, three seminal samples with a low semen motility (asthenozoospermia), and three with a low semen concentration (oligozoospermia). MicroRNAs showing significant differences between groups were further validated in a second cohort consisting of 40 samples with normozoospermia (control group), 47 samples with asthenozoospermia, and 19 samples with oligozoospermia (of which 74% also low motility). Highest microRNA yields were obtained with the Biofluids RNA extraction kit, with inclusion of MS2 RNA carrier and proteinase K treatment to the protocol, and when 50 µL of seminal plasma was used as input. Exosome isolation prior to RNA extraction did not lead to enhanced yields. In the test cohort, 236 microRNAs could be detected, of which 54 microRNAs showed a difference between groups. Five microRNAs were analyzed in the validation cohort. MiR-34b-5p levels in the control group were significantly higher compared to the asthenozoospermia group (p < 0.05) and compared to the oligozoospermia group (p < 0.001). We optimized microRNA acquirement from seminal plasma and identified microRNA levels in relation to semen concentration and motility. As recent human and mouse studies show that the miR-34 family is a marker of low semen concentration and is crucial in spermatogenesis, seminal plasma miR-34b-5p may represent a suitable candidate to study further as a marker of male subfertility.

Reactive Species Interactome Alterations in Oocyte Donation Pregnancies in the Absence and Presence of Pre-Eclampsia.

In pregnancy, maternal physiology is subject to considerable adaptations, including alterations in cardiovascular and metabolic function as well as development of immunological tolerance towards the fetus. In an oocyte donation pregnancy, the fetus is fully allogeneic towards the mother, since it carries both oocyte donor antigens and paternal antigens. Therefore, oocyte donation pregnancies result in an immunologically challenging pregnancy, which is reflected by a higher-than-normal risk to develop pre-eclampsia. Based on the allogeneic conditions in oocyte donation pregnancies, we hypothesized that this situation may translate into alterations in concentration of stable readouts of constituents of the reactive species interactome (RSI) compared to normal pregnancies, especially serum free thiols, nitric oxide (NO) and hydrogen sulfide (H2S) related metabolites. Indeed, total free thiol levels and nitrite (NO2-) concentrations were significantly lower whereas protein-bound NO and sulfate (SO42-) concentrations were significantly higher in both oocyte donation and naturally conceived pregnancies complicated by pre-eclampsia. The increased concentrations of nitrite observed in uncomplicated oocyte donation pregnancies suggest that endothelial NO production is compensatorily enhanced to lower vascular tone. More research is warranted on the role of the RSI and bioenergetic status in uncomplicated oocyte donation pregnancies and oocyte donation pregnancies complicated by pre-eclampsia.

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.

Calcium-Binding Proteins S100A8 and S100A9: Investigation of Their Immune Regulatory Effect in Myeloid Cells.

High expression levels of the calcium-binding proteins S100A8 and S100A9 in myeloid cells in kidney transplant rejections are associated with a favorable outcome. Here we investigated the myeloid cell subset expressing these molecules, and their function in inflammatory reactions. Different monocyte subsets were sorted from buffy coats of healthy donors and investigated for S100A8 and S100A9 expression. To characterize S100A9high and S100A9low subsets within the CD14+ classical monocyte subset, intracellular S100A9 staining was combined with flow cytometry (FACS) and qPCR profiling. Furthermore, S100A8 and S100A9 were overexpressed by transfection in primary monocyte-derived macrophages and the THP-1 macrophage cell line to investigate the functional relevance. Expression of S100A8 and S100A9 was primarily found in classical monocytes and to a much lower extent in intermediate and non-classical monocytes. All S100A9+ cells expressed human leukocyte antigen—antigen D related (HLA-DR) on their surface. A small population (<3%) of CD14+ CD11b+ CD33+ HLA-DR− cells, characterized as myeloid derived suppressor cells (MDSCs), also expressed S100A9 to high extent. Overexpression of S100A8 and S00A9 in macrophages led to enhanced extracellular reactive oxygen species (ROS) production, as well as elevated mRNA expression of anti-inflammatory IL-10. The results suggest that the calcium-binding proteins S100A8 and S100A9 in myeloid cells have an immune regulatory effect.

Blood cell mRNAs and microRNAs: optimized protocols for extraction and preservation.

Assessing messenger RNA (mRNA) and microRNA levels in peripheral blood cells may complement conventional parameters in clinical practice. Working with small, precious samples requires optimal RNA yields and minimal RNA degradation. Several procedures for RNA extraction and complementary DNA (cDNA) synthesis were compared for their efficiency. The effect on RNA quality of freeze-thawing peripheral blood cells and storage in preserving reagents was investigated. In terms of RNA yield and convenience, quality quantitative polymerase chain reaction signals per nanogram of total RNA and using NucleoSpin and mirVana columns is preferable. The SuperScript III protocol results in the highest cDNA yields. During conventional procedures of storing peripheral blood cells at -180°C and thawing them thereafter, RNA integrity is maintained. TRIzol preserves RNA in cells stored at -20°C. Detection of mRNA levels significantly decreases in degraded RNA samples, whereas microRNA molecules remain relatively stable. When standardized to reference targets, mRNA transcripts and microRNAs can be reliably quantified in moderately degraded (quality index 4-7) and severely degraded (quality index <4) RNA samples, respectively. We describe a strategy for obtaining high-quality and quantity RNA from fresh and stored cells from blood. The results serve as a guideline for sensitive mRNA and microRNA expression assessment in clinical material.

Tregs from human blood differentiate into nonlymphoid tissue-resident effector cells upon TNFR2 costimulation.

Tregs can facilitate transplant tolerance and attenuate autoimmune and inflammatory diseases. Therefore, it is clinically relevant to stimulate Treg expansion and function in vivo and to create therapeutic Treg products in vitro. We report that TNF receptor 2 (TNFR2) is a unique costimulus for naive, thymus-derived Tregs (tTregs) from human blood that promotes their differentiation into nonlymphoid tissue-resident (NLT-resident) effector Tregs, without Th-like polarization. In contrast, CD28 costimulation maintains a lymphoid tissue-resident (LT-resident) Treg phenotype. We base this conclusion on transcriptome and proteome analysis of TNFR2- and CD28-costimulated CD4+ tTregs and conventional T cells (Tconvs), followed by bioinformatic comparison with published transcriptomic Treg signatures from NLT and LT in health and disease, including autoimmunity and cancer. These analyses illuminate that TNFR2 costimulation promoted tTreg capacity for survival, migration, immunosuppression, and tissue regeneration. Functional studies confirmed improved migratory ability of TNFR2-costimulated tTregs. Flow cytometry validated the presence of the TNFR2-driven tTreg signature in effector/memory Tregs from the human placenta, as opposed to blood. Thus, TNFR2 can be exploited as a driver of NLT-resident tTreg differentiation for adoptive cell therapy or antibody-based immunomodulation in human disease.

HLA-targeted flow cytometric sorting of blood cells allows separation of pure and viable microchimeric cell populations.

Microchimerism is defined by the presence of low levels of nonhost cells in a person. We developed a reliable method for separating viable microchimeric cells from the host environment. For flow cytometric cell sorting, HLA antigens were targeted with human monoclonal HLA antibodies (mAbs). Optimal separation of microchimeric cells (present at a proportion as low as 0.01% in artificial mixtures) was obtained with 2 different HLA mAbs, one targeting the chimeric cells and the other the background cells. To verify purity of separated cell populations, flow-sorted fractions of 1000 cells were processed for DNA analysis by HLA-allele-specific and Y-chromosome-directed real-time quantitative PCR assays. After sorting, PCR signals of chimeric DNA markers in the positive fractions were significantly enhanced compared with those in the presort samples, and they were similar to those in 100% chimeric control samples. Next, we demonstrate applicability of HLA-targeted FACS sorting after pregnancy by separating chimeric maternal cells from child umbilical cord mononuclear cells. Targeting allelic differences with anti-HLA mAbs with FACS sorting allows maximal enrichment of viable microchimeric cells from a background cell population. The current methodology enables reliable microchimeric cell detection and separation in clinical specimens.

Inflammatory placental lesions are specifically observed in healthy oocyte donation pregnancies with extreme fetal-maternal incompatibility.

INTRODUCTION: Oocyte donation (OD) pregnancy is a risk factor for pre-eclampsia (PE). Due to a higher extent of fetal-maternal human leukocyte antigens (HLA) mismatching in OD pregnancies compared to naturally conceived (NC) and in vitro fertilization (IVF) pregnancies, the immune response in OD placentas is probably divergent and affects clinical outcomes. We hypothesized that placental pathology varies among diverse pregnancy conditions and is related to fetal-maternal HLA incompatibility. METHODS: Placental lesions were scored in four patient groups: OD-PE (n = 16), OD-healthy (n = 37), NC-PE (n = 45), and IVF-healthy (n = 17). All combinations were genotyped for HLA-A, -B, -C, -DR, and -DQ to calculate fetal-maternal HLA mismatches. Placentas showing chronic deciduitis with plasma cells were immunofluorescently stained with CD138 and the anti-inflammatory cytokine interleukin-10 (IL-10). RESULTS: The distribution and severity of placental lesions varied among groups. The OD-healthy group had the highest inflammation score and greatest extent of chronic deciduitis with plasma cells (p < 0.05). However, the majority of CD138+ plasma cells (90%) in OD-healthy group expressed IL-10, in contrast to the OD-PE group (58%). The OD-healthy group was separated into semi-allogeneic (≤5 HLA mismatches) and fully allogeneic (>5 mismatches) subgroups. The elevated inflammatory pathology score and chronic deciduitis with plasma cells were found more often in the HLA-class-I fully allogeneic OD-healthy group than the IVF-healthy group (p < 0.05). DISCUSSION: Placental inflammatory lesions are most often present in uncomplicated OD pregnancies. Immune cells that infiltrate these lesions might play an immunosuppressive role to protect OD pregnancies from complications when facing a higher extent of fetal-maternal HLA mismatching.

Identification of a unique intervillous cellular signature in chronic histiocytic intervillositis.

INTRODUCTION: Chronic histiocytic intervillositis (CHI) is a rare histopathological lesion in the placenta characterized by an infiltrate of CD68+ cells in the intervillous space. CHI is associated with adverse pregnancy outcomes such as miscarriage, fetal growth restriction, and (late) intrauterine fetal death. The adverse pregnancy outcomes and a variable recurrence rate of 25-100% underline its clinical relevance. The pathophysiologic mechanism of CHI is unclear, but it appears to be immunologically driven. The aim of this study was to obtain a better understanding of the phenotype of the cellular infiltrate in CHI. METHOD: We used imaging mass cytometry to achieve in-depth visualization of the intervillous maternal immune cells and investigated their spatial orientation in situ in relation to the fetal syncytiotrophoblast. RESULTS: We found three phenotypically distinct CD68+HLA-DR+CD38+ cell clusters that were unique for CHI. Additionally, syncytiotrophoblast cells in the vicinity of these CD68+HLA-DR+CD38+ cells showed decreased expression of the immunosuppressive enzyme CD39. DISCUSSION: The current results provide novel insight into the phenotype of CD68+ cells in CHI. The identification of unique CD68+ cell clusters will allow more detailed analysis of their function and could result in novel therapeutic targets for CHI.

Increased influx of myeloid dendritic cells during acute rejection is associated with interstitial fibrosis and tubular atrophy and predicts poor outcome.

Dendritic cells are key players in renal allograft rejection and have been identified as an intrinsic part of the kidney. Here we quantified and phenotyped the dendritic cell populations in well-defined biopsies of 102 patients with acute renal allograft rejection in comparison with 78 available pretransplant biopsies. There was a strong increase in BDCA-1(+) and DC-SIGN(+) myeloid, BDCA-2(+) plasmacytoid, and DC-LAMP(+) mature dendritic cells in rejection biopsies compared with the corresponding pretransplant tissue. Mature dendritic cells were mostly found in clusters of lymphoid infiltrate and showed a strong correlation with the Banff infiltrate score. The presence of both myeloid and plasmacytoid dendritic cell subsets in the kidney during acute rejection correlated with interstitial fibrosis and tubular atrophy. Importantly, the myeloid dendritic cell density at the time of acute rejection was an independent risk factor for loss of renal function after the first year. Thus, acute renal allograft rejection is characterized by an influx of myeloid and plasmacytoid dendritic cells, strongly associated with local damage in the graft. Hence, the density of myeloid dendritic cells during acute rejection could be an important risk factor for the long-term development of chronic changes and loss of graft function.

Human decidual tissue contains differentiated CD8+ effector-memory T cells with unique properties.

During pregnancy, maternal lymphocytes at the fetal-maternal interface play a key role in the immune acceptance of the allogeneic fetus. Recently, CD4(+)CD25(bright) regulatory T cells have been shown to be concentrated in decidual tissue, where they are able to suppress fetus-specific and nonspecific immune responses. Decidual CD8(+) T cells are the main candidates to recognize and respond to fetal HLA-C at the fetal-maternal interface, but data on the characteristics of these cells are limited. In this study we examined the decidual and peripheral CD8(+) T cell pool for CD45RA, CCR7, CD28, and CD27 expression, using nine-color flow cytometry. Our data demonstrate that decidual CD8(+) T cells mainly consist of differentiated CD45RA(-)CCR7(-) effector-memory (EM) cells, whereas unprimed CD45RA(+)CCR7(+) naive cells are almost absent. Compared with peripheral blood EM CD8(+) T cells, the decidual EM CD8(+) T cells display a significantly reduced expression of perforin and granzyme B, which was confirmed by immunohistochemistry of decidual tissue sections. Interestingly, quantitative PCR analysis demonstrates an increased perforin and granzyme B mRNA content in decidual EM CD8(+) T cells in comparison with peripheral blood EM CD8(+) T cells. The presence of high levels of perforin and granzyme B mRNA in decidual EM T cells suggests that decidual CD8(+) T cells pursue alternative means of EM cell differentiation that may include a blockade of perforin and granzyme B mRNA translation into functional perforin and granzyme B proteins. Regulation of decidual CD8(+) T cell differentiation may play a crucial role in maternal immune tolerance to the allogeneic fetus.

Uncomplicated oocyte donation pregnancies display an elevated CD163-positive type 2 macrophage load in the decidua, which is associated with fetal-maternal HLA mismatches.

PROBLEM: The embryo of an oocyte donation (OD) pregnancy is completely allogeneic to the mother, which may challenge the maternal immune system to tolerize the fetus. Decidual macrophages are essential in maintaining a healthy pregnancy, and type 2 macrophages may exhibit immune suppressive activity. We hypothesized that the composition of decidual macrophages is different between uncomplicated OD pregnancies and non-OD in vitro fertilization (IVF) pregnancies, and is related to fetal-maternal incompatibility. METHOD OF STUDY: Women with uncomplicated pregnancy were enrolled: 25 singleton OD pregnancies and 17 non-OD IVF pregnancies. The extent of immunohistochemical staining of CD14 (pan-macrophage marker) and CD163 (type 2 macrophage marker) in both decidua basalis and parietalis was quantitated by digital image analysis. Maternal and fetal DNA was typed for human leukocyte antigen (HLA)-A, -B, C, -DRB1, and -DQB1, and fetal-maternal HLA mismatches were calculated. RESULTS: OD pregnancies showed a higher percentage of CD163+ staining (P = .040) and higher CD163/CD14 ratio (P = .032) in the parietalis than non-OD IVF. The OD group was separated into a semi-allogeneic group (≤5 fetal maternal HLA mismatches) and a fully allogeneic group (> 5 mismatches). The HLA-fully-allogeneic OD group, but not the HLA-semi-allogeneic OD group, showed significantly elevated CD163/CD14 ratio in the parietalis compared with the non-OD IVF group (P < .05). CONCLUSIONS: Uncomplicated OD pregnancies display a higher CD163-positive cell fraction in the total decidual macrophage population compared to autologous pregnancies, which may suggest that a local type 2 macrophage-related mechanism is needed to compensate for the higher fetal-maternal HLA mismatch load.

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.

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.

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.