Can inflammatory biomarkers based on first trimester complete blood count parameters predict placental abruption ?: A case-control study.

OBJECTIVES: Placental abruption (PA) is associated with adverse maternal and neonatal outcomes and has an etiological mechanism that is not yet fully understood. The prediction of PA, which has been the subject of numerous studies, remains a challenge. In particular, there is evidence that PA can be considered a chronic process. So, this study aimed to show inflammatory biomarkers based on complete blood count parameters may be used to predict PA. STUDY DESIGN: A sample of 110 cases (pregnant women with PA) and 110 controls (healthy pregnant women with spontaneous labor) were required the study. The present case-control study included a total of 220 pregnant women. Inflammatory makers were used to evaluate the PA prediction RESULTS: Increases in body mass index, mean corpuscular volume and paletelet lymphocyte ratio are considered protective factors, while increases in neutrophil, the systemic inflammatory response index, neutrophil lymphocyte ratio and the pan-immune inflammation score are considered risk factors. Each 1 unit increase in neutrophil count increases the risk of a PA diagnosis by 1.81 times. CONCLUSION: Recent studies indicate a strong heterogeneity of clinical courses leading to PA in premature and term births. In the present study, our results showed that, inflammation is associated with PA.

Immune rebalancing at the maternal-fetal interface of maternal SARS-CoV-2 infection during early pregnancy.

The current coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2) remains a threat to pregnant women. However, the impact of early pregnancy SARS-CoV-2 infection on the maternal-fetal interface remains poorly understood. Here, we present a comprehensive analysis of single-cell transcriptomics and metabolomics in placental samples infected with SARS-CoV-2 during early pregnancy. Compared to control placentas, SARS-CoV-2 infection elicited immune responses at the maternal-fetal interface and induced metabolic alterations in amino acid and phospholipid profiles during the initial weeks post-infection. However, subsequent immune cell activation and heightened immune tolerance in trophoblast cells established a novel dynamic equilibrium that mitigated the impact on the maternal-fetal interface. Notably, the immune response and metabolic alterations at the maternal-fetal interface exhibited a gradual decline during the second trimester. Our study underscores the adaptive immune tolerance mechanisms and establishment of immunological balance during the first two trimesters following maternal SARS-CoV-2 infection.

Can System Inflammation Response Index or Systemic Immune Inflammation Index predict gestational diabetes mellitus in the first trimester? A prospective observational study.

OBJECTIVE: To evaluate System Inflammation Response Index (SIRI) and Systemic Immune Inflammation Index (SII), which are the inflammatory indices, for the prediction of gestational diabetes mellitus (GDM) in the first trimester. METHODS: This was a prospective observational study conducted in a tertiary center from April 2023 to September 2023. Ninety-four pregnant women with gestational diabetes and 107 healthy pregnant women were included. The two groups were compared according to first-trimester SIRI and SII values. A receiver operating characteristic (ROC) curve analysis was performed to determine the optimal cut-off levels of SII and SIRI in predicting GDM. RESULTS: Significantly higher first-trimester SII and SIRI values were present in the gestational diabetes group (P < 0.001). Optimal cut-off values in the prediction of gestational diabetes were found to be 1.58 (area under the curve [AUC] 0.71, 67% sensitivity, 65% specificity, 95% confidence interval [CI] 0.64-0.78, P < 0.001) and 875 (AUC 0.70, 66% sensitivity, 65% specificity, 95% CI 0.63-0.77, P < 0.001) for SIRI and SII, respectively. Neutrophil counts, mean platelet volume (MPW), neutrophil to lymphocyte ratio (NLR), and red cell distribution width (RDW) were significantly higher in the GDM group (P < 0.001, P = 0.02, P = 0.01, P < 0.01, respectively). CONCLUSION: Novel inflammatory indices SII and SIRI may be useful in the prediction of GDM in the first trimester, but their utility in the prediction of insulin requirement is questionable. They may be used as additional tools in routine clinical practice.

Induced Abortion and the Risk of Rh Sensitization.

Importance: While population-level data suggest Rh immunoglobulin is unnecessary before 12 weeks' gestation, clinical evidence is limited. Thus, guidelines vary, creating confusion surrounding risks and benefits of Rh testing and treatment. As abortion care in traditional clinical settings becomes harder to access, many people are choosing to self-manage and need to know if ancillary blood type testing is necessary. Objective: To determine how frequently maternal exposure to fetal red blood cells (fRBCs) exceeds the most conservative published threshold for Rh sensitization in induced first-trimester abortion. Design, Setting, and Participants: Multicenter, observational, prospective cohort study using high-throughput flow cytometry to detect circulating fRBCs in paired maternal blood samples before and after induced first-trimester abortion (medication or procedural). Individuals undergoing induced first-trimester abortion before 12 weeks 0 days' gestation were included. Paired blood samples were available from 506 participants who underwent either medical (n = 319 [63.0%]) or procedural (n = 187 [37.0%]) abortion. Exposure: Induced first-trimester abortion. Main Outcomes and Measures: The primary outcome was the proportion of participants with fRBC counts above the sensitization threshold (125 fRBCs/5 million total RBCs) after induced first-trimester abortion. Results: Among the 506 participants, the mean (SD) age was 27.4 (5.5) years, 313 (61.9%) were Black, and 123 (24.3%) were White. Three of the 506 participants had elevated fRBC counts at baseline; 1 of these patients had an elevated fRBC count following the abortion (0.2% [95% CI, 0%-0.93%]). No other participants had elevated fRBC counts above the sensitization threshold after induced first-trimester abortion. The median change from baseline was 0 fRBCs, with upper 95th and 99th percentiles of 24 and 35.6 fRBCs, respectively. Although there was a strong association between the preabortion and postabortion fRBC counts, no other baseline characteristic was significantly associated with postabortion fRBC count. Conclusions and Relevance: Induced first-trimester abortion is not a risk factor for Rh sensitization, indicating that Rh testing and treatment are unnecessary before 12 weeks' gestation. This evidence may be used to inform international guidelines for Rh immunoglobulin administration following first-trimester induced abortion.

Cytokine Patterns in Maternal Serum From First Trimester to Term and Beyond.

Pregnancy implies delicate immunological balance between two individuals, with constant changes and adaptions in response to maternal capacity and fetal demands. We performed cytokine profiling of 1149 longitudinal serum samples from 707 pregnant women to map immunological changes from first trimester to term and beyond. The serum levels of 22 cytokines and C-reactive protein (CRP) followed diverse but characteristic trajectories throughout pregnancy, consistent with staged immunological adaptions. Eotaxin showed a particularly robust decrease throughout pregnancy. A strong surge in cytokine levels developed when pregnancies progressed beyond term and the increase was amplified as labor approached. Maternal obesity, smoking and pregnancies with large fetuses showed sustained increase in distinct cytokines throughout pregnancy. Multiparous women had increased cytokine levels in the first trimester compared to nulliparous women with higher cytokine levels in the third trimester. Fetal sex affected first trimester cytokine levels with increased levels in pregnancies with a female fetus. These findings unravel important immunological dynamics of pregnancy, demonstrate how both maternal and fetal factors influence maternal systemic cytokines, and serve as a comprehensive reference for cytokine profiles in normal pregnancies.

White blood cells in pregnancy: reference intervals for before and after delivery.

BACKGROUND: White blood cells (WBC) are commonly measured to investigate suspected infection and inflammation in pregnant women, but the pregnancy-specific reference interval is variably reported, increasing diagnostic uncertainty in this high-risk population. It is essential that clinicians can interpret WBC results in the context of normal pregnant physiology, given the huge global burden of infection on maternal mortality. METHODS: We performed a longitudinal, repeated measures population study of 24,318 pregnant women in Oxford, UK, to map the trajectory of WBC between 8-40 weeks of gestation. We defined 95% reference intervals (RI) for total WBC, neutrophils, lymphocytes, eosinophils, basophils, and monocytes for the antenatal and postnatal periods. FINDINGS: WBC were measured 80,637 times over five years. The upper reference limit for total WBC was elevated by 36% in pregnancy (RI 5.7-15.0×109/L), driven by a 55% increase in neutrophils (3.7-11.6×109/L) and 38% increase in monocytes (0.3-1.1×109/L), which remained stable between 8-40 weeks. Lymphocytes were reduced by 36% (1.0-2.9×109/L), while eosinophils and basophils were unchanged. Total WBC was elevated significantly further from the first day after birth (similar regardless of the mode of delivery), which resolved to pre-delivery levels by an average of seven days, and to pre-pregnancy levels by day 21. INTERPRETATION: There are marked changes in WBC in pregnancy, with substantial differences between cell subtypes. WBC are measured frequently in pregnant women in obstetric and non-obstetric settings, and results should be interpreted using a pregnancy-specific RI until delivery, and between days 7-21 after childbirth. FUNDING: None.

Frequency, timing and risk factors for primary maternal cytomegalovirus infection during pregnancy in Quebec.

INTRODUCTION: Maternal Cytomegalovirus (CMV) infection in the first trimester (T1) of pregnancy is a public health concern, as it increases the risk of severe neurodevelopmental outcomes associated with congenital infection compared to infections occurring later during pregnancy. OBJECTIVES: To determine CMV seroprevalence in T1 of pregnancy, its trend, risk factors and the incidence rate of primary infection during pregnancy. METHODS: Using the biobank of the prospective cohort "Grossesse en Santé de Québec" collected between April 2005 and March 2010 at the Québec-Laval Hospital, Québec, Canada, maternal CMV serology was determined using Abbott Architect Chemiluminescence microparticle immunoassays for immunoglobulin G(IgG), immunoglobulin M(IgM) titration and IgG avidity testing. Changepoint detection analysis was used to assess temporal trends. Risk factors associated with seropositivity were determined by multivariable logistic regression. RESULTS: CMV seroprevalence in T1 of pregnancy was 23.4% (965/4111, 95% CI, 22.1-24.7%). The incidence rate for CMV primary infection during pregnancy was 1.8 (95% CI, 1.2-2.6) per 100 person-years. No changepoint was identified in the maternal CMV-seroprevalence trend. Multivariable analyses showed that T1 maternal CMV seropositivity was associated with having one child OR 1.3 (95% CI, 1.10-1.73) or two or more children OR 1.5 (95%CI, 1.1-2.1), ethnicity other than Caucasian OR 2.1 (95% CI, 1.1-3.8) and country of birth other than Canada and the USA OR 2.8 (95% CI, 1.5-4.9). CONCLUSIONS: In this cohort, maternal seroprevalence in T1 of pregnancy and seroconversion rate were low. This information and identified risk factors could help guide the development and implementation of preventive actions and evidence-based health policies to prevent CMV infection during pregnancy.

Decidual stromal cells support tolerance at the human foetal-maternal interface by inducing regulatory M2 macrophages and regulatory T-cells.

During pregnancy, the semi-allogeneic nature of the foetus requires maternal immune adaption and acquisition of tolerance at the foetal-maternal interface. Macrophages with regulatory properties and regulatory T (Treg) cells are central in promoting foetal tolerance and are enriched in the decidua (the uterine endometrium during pregnancy). Although tissue-resident decidual stromal cells (DSC) have been implicated in regulatory functions, it is not known if they are able to induce the regulatory phenotype of macrophages and T-cells. In this study we report that maternally derived DSC are able to induce homeostatic M2 macrophages and Treg cells. CD14+ monocytes and CD4+ T-cells from healthy non-pregnant women were cultured in the presence or absence of conditioned medium (CM) from DSC isolated from 1st trimester and term placentas. DSC-CM alone was able to promote the survival of macrophages and to induce a regulatory CD14brightCD163+CD209+CD86dim phenotype, typical for decidual macrophages and similar to that induced by M-CSF. Interestingly, DSC-CM was also able to overrule the pro-inflammatory effects of GM-CSF by upregulating CD14, CD163 and CD209. Protein-profiling showed that M-CSF was secreted by DSC, and blocking of M-CSF partially reversed the M2 phenotype and reduced viability. DSC-CM also expanded CD25brightFoxp3+ Treg cells, an expansion that was abolished by a SMAD3-inhibitor, indicating the contribution of TGF-ß signaling. In conclusion, our findings collectively emphasize the role of tissue-resident stromal cells in shaping the tolerogenic environment at the foetal-maternal interface.

The Role of TNF-α and Anti-TNF-α Agents during Preconception, Pregnancy, and Breastfeeding.

Tumor necrosis factor-alpha (TNF-α) is a multifunctional Th1 cytokine and one of the most important inflammatory cytokines. In pregnancy, TNF-α influences hormone synthesis, placental architecture, and embryonic development. It was also shown that increased levels of TNF-α are associated with pregnancy loss and preeclampsia. Increased TNF-α levels in complicated pregnancy draw attention to trophoblast biology, especially migratory activity, syncytialisation, and endocrine function. Additionally, elevated TNF-α levels may affect the maternal-fetal relationship by altering the secretory profile of placental immunomodulatory factors, which in turn affects maternal immune cells. There is growing evidence that metabolic/pro-inflammatory cytokines can program early placental functions and growth in the first trimester of pregnancy. Furthermore, early pregnancy placenta has a direct impact on fetal development and maternal immune system diseases that release inflammatory (e.g., TNF-α) and immunomodulatory factors, such as chronic inflammatory rheumatic, gastroenterological, or dermatological diseases, and may result in an abnormal release of cytokines and chemokines in syncytiotrophoblasts. Pregnancy poses a challenge in the treatment of chronic disease in patients who plan to have children. The activity of the disease, the impact of pregnancy on the course of the disease, and the safety of pharmacotherapy, including anti-rheumatic agents, in pregnancy should be considered.

Maternal platelets pass interstices of trophoblast columns and are not activated by HLA-G in early human pregnancy.

In early human gestation, maternal arterial blood flow into the intervillous space of the developing placenta is obstructed by invaded trophoblasts, which form cellular plugs in uterine spiral arteries. These trophoblast plugs have recently been described to be loosely cohesive with clear capillary-sized channels into the intervillous space by 7 weeks of gestation. Here, we analysed localisation of maternal platelets at the maternal-foetal interface of human first trimester pregnancy, and tested the hypothesis whether HLA-G, which is primarily expressed by extravillous trophoblasts, affects aggregation and adhesion of isolated platelets. Immunohistochemistry of first trimester placental sections localised maternal platelets in vessel-like channels and adjacent intercellular gaps of extravillous trophoblasts in distal parts of columns. Furthermore, this localisation was confirmed by transmission electron microscopy. Neither co-incubation of HLA-G overexpressing JAR cells with isolated platelets, nor incubation with cell-derived soluble HLA-G or recombinant HLA-G affected platelet adhesion and aggregation. Our study suggests that maternal platelets flow through vessel-like channels of distal trophoblast columns and spread into adjacent lateral intercellular gaps, where platelet-derived factors could contribute to trophoblast differentiation into the invasive phenotype.

Eomesodermin regulate decidual CD4+T cell function during human early pregnancy.

Decidual CD4+T (dCD4+T) cells play pivotal roles in inducing and maintaining maternal-fetal tolerance. Dysfunctional dCD4+T cells are associated with miscarriage. In the present study, we demonstrated that the T-box transcription factor protein eomesodermin (Eomes) was involved in the functional regulation of dCD4+T cells during early pregnancy. We concluded the higher Eomes expression dCD4+T cells during normal pregnancy, and the Eomes+dCD4+T cells displayed an active status and produced more Th2- and Treg type cytokines. Decreased number and altered function of Eomes+dCD4+T cells were observed in miscarriage. Progesterone, the traditional treatment for miscarriage, had no effect on Eomes expression by dCD4+T cells from normal pregnancy, but increased Eomes expression by dCD4+T cells from miscarriage. We also found the higher frequency of Eomes+dCD4+T cells from miscarriage in response to cyclosporine, tacrolimus, Trophoblasts, and HTR8/SVneo cell line, might provide new strategy for therapy to promote maternal-fetal tolerance and prevent pregnancy loss. These results indicated that Eomes might be promising early warming targets of miscarriage, though further studies are required to determine that the altered number and function of Eomes+dCD4+T cells are the cause or consequence of miscarriage.

Outcome of pregnancies with recent primary cytomegalovirus infection in first trimester treated with hyperimmunoglobulin: observational study.

OBJECTIVE: To examine the efficacy of hyperimmunoglobulin (HIG) treatment in women with a recent primary cytomegalovirus (CMV) infection up to 14 weeks' gestation. METHODS: This is an ongoing observational study conducted at the prenatal medicine departments of the University Hospitals of Tübingen, Bonn, Cologne and Erlangen, Germany, as well as at the Laboratory Prof. Gisela Enders and Colleagues in Stuttgart, Germany and the Institute for Medical Virology at the University of Tübingen, Tübingen, Germany. Enrolment criteria were the presence of confirmed recent primary CMV infection in the first trimester and a gestational age at first HIG administration of ≤ 14 weeks. The following inclusion criteria indicated a recent primary infection: low anti-immunoglobulin (Ig)-G levels, low anti-CMV-IgG avidity in the presence of a positive CMV-IgM test and no positive reactivity or just seroconversion anti-gB2-IgG-reactivity. HIG administration was started as soon as possible within a few days after the first visit. HIG was administered intravenously at a dose of 200 IU/kg maternal body weight and repeated every 2 weeks until about 18 weeks' gestation. The primary outcome was maternal-fetal transmission at the time of amniocentesis. Multivariate logistic regression analysis was used to determine significant covariates that could predict maternal-fetal transmission. RESULTS: We included 149 pregnancies (153 fetuses) that completed the treatment. Median maternal age and weight were 32.0 years and 65.0 kg, respectively. Median gestational age at the time of first referral to one of the four centers was 9.4 weeks. Median anti-CMV-IgG level, anti-CMV-IgM index and CMV-IgG avidity were 5.7 U/mL, 2.5 and 22.3%, respectively. HIG treatment was started at a median gestational age of 10.6 weeks and ended at a median of 17.9 weeks. Within this time frame, HIG was administered on average four times in each patient. Amniocentesis was carried out at a median gestational age of 20.4 weeks. In 143 (93.5%) of the 153 cases, the fetus was not infected. Maternal-fetal transmission occurred in 10 cases (6.5% (95% CI, 3.2-11.7%)). On uni- and multivariate logistic regression analysis, the level of anti-IgM index was the only factor associated significantly with maternal-fetal transmission at amniocentesis. However, only four (40.0%) of the 10 cases with maternal-fetal transmission had an anti-IgM index above 11.4, which corresponds to the 95th centile of pregnancies without transmission. CONCLUSIONS: HIG is a treatment option to prevent maternal-fetal transmission in pregnancy with a primary CMV infection. However, HIG treatment seems to be beneficial primarily in women with a recent primary infection in the first trimester or during the periconceptional period, and when it is administered at a biweekly dose of 200 IU/kg. © 2021 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Pregnancy Zone Protein (PZP) is significantly upregulated in the decidua of recurrent and spontaneous miscarriage and negatively correlated to Glycodelin A (GdA).

BACKGROUND: Pregnancy Zone Protein (PZP) is an immunosuppressive protein that is expressed by the placenta and has also been identified in immune cells. When PZP and Glycodelin A (GdA) are combined, they act synergistically to inhibit Th-1 immune response. Little is known about its combined expression and role in normal and disturbed first trimester pregnancy. PATIENTS AND METHODS: We investigated the expression of PZP and GdA in placental tissue obtained from spontaneous miscarriage (SM) (n = 19) and recurrent miscarriage (RM) (n = 17) at gestational weeks 6-13 by immunohistochemistry and on mRNA-level by either TaqMan PCR or in situ hybridization. Placental tissue from legal terminations of healthy pregnancies (n = 15) served as control group. Immunofluorescence double staining was used to analyse the combined expression of PZP and GdA in decidual tissue. RESULTS: The protein level of PZP was significantly increased in decidual stroma of SM samples compared to the decidua of control specimens and also significantly upregulated in the decidual stroma cells in the RM group. Concerning GdA, the decidual stroma revealed a significantly decreased protein level in the group with spontaneous abortions than in the group with healthy pregnancies. There was also a significant downregulation of GdA in the decidual stroma of RM samples compared to the control group. We observed a significant negative correlation of PZP and GdA in decidual stromal tissue of recurrent abortion. We could confirm the staining results for PZP as well as for GdA on mRNA level. Both proteins are co-localized in decidual stroma as analysed by immunofluorescence double staining. CONCLUSION: A balanced expression of GdA and its carrier protein PZP in the decidua seems crucial for a successful ongoing pregnancy. According to our data, these immunosuppressive proteins are co-localized in the decidual tissue and show a negative correlation only in patients suffering from recurrent abortion.

Phenotypic and functional characterization of first-trimester human placental macrophages, Hofbauer cells.

Hofbauer cells (HBCs) are a population of macrophages found in high abundance within the stroma of the first-trimester human placenta. HBCs are the only fetal immune cell population within the stroma of healthy placenta. However, the functional properties of these cells are poorly described. Aligning with their predicted origin via primitive hematopoiesis, we find that HBCs are transcriptionally similar to yolk sac macrophages. Phenotypically, HBCs can be identified as HLA-DR-FOLR2+ macrophages. We identify a number of factors that HBCs secrete (including OPN and MMP-9) that could affect placental angiogenesis and remodeling. We determine that HBCs have the capacity to play a defensive role, where they are responsive to Toll-like receptor stimulation and are microbicidal. Finally, we also identify a population of placenta-associated maternal macrophages (PAMM1a) that adhere to the placental surface and express factors, such as fibronectin, that may aid in repair.

The O-Linked N-Acetylglucosamine Containing Epitope H (O-GlcNAcH) is Upregulated in the Trophoblastic and Downregulated in the Fibroblastic Cells in Missed Miscarriage Human Chorionic Villi With Simple Hydropic Degeneration.

Epitope H contains an O-linked N-acetylglucosamine (O-GlcNAcH) residue in a specific conformation and/or environment recognized by the mouse monoclonal antibody H. O-GlcNAcH is present in several types of cells and in several polypeptides, including cytokeratin 8 and vimentin, on the latter in cells under stress. In the present work, we examined the expression of the O-GlcNAcH in 60 cases of endometrial curettings from missed miscarriage cases containing normal and simple hydropic degenerated chorionic villi in each case, using monoclonal antibody H and indirect immunoperoxidase and Western blot immunoblot. In all cases examined the expression of the O-GlcNAcH was cytoplasmic as follows: (1) syncytiotrophoblastic cells showed very low expression in chorionic villi (CV) with nonhydropic degeneration (NHD) and high expression in hydropic degenerated (HD) CV; (2) cytotrophoblastic cells showed low expression in CV with NHD and high expression in HD CV; (3) fibroblastic cells showed high expression in CV with NHD and very low expression in HD CV; (4) histiocytes showed very low expression in both types of CV; (5) endothelial cells showed high expression in both types of CV. An immunoblot of CV from one case of a legal abortion from a normal first-trimester pregnancy showed 5 polypeptides with 118.5, 106.3, 85, 53, and 36.7 kD bearing the epitope H and the 53 kD corresponded to cytokeratin 8. The expression of the O-GlcNAcH is upregulated in the trophoblastic cells and downregulated in the fibroblastic cells in the HD CV in comparison to the NHD CV.

The uteroplacental contact zone cytokine influence on NK cell cytotoxicity to trophoblasts.

OBJECTIVE: The present study was to estimate the role of cytokines for trophoblast death in NK cells presence. METHODS: This study involves assessment of NK-92 line NK cell cytotoxic activity against JEG-3 line cells, in presence of cytokines. We also assessed the effect of secretory placenta products on NK cell cytotoxic activity toward JEG-3 line cells. RESULTS: Uteroplacental contact zone cytokines are able to enhance trophoblast mortality both by themselves in case of IL-1ß, IL-6, IFNγ, IL-4, TGFß, bFGF, and also through increasing the cytotoxic potential of NK cells in case of IL-1ß, IFNγ, IL-8, TGFß, and GM-CSF. PLGF decreases NK cell cytotoxicity for trophoblasts. Secretory products of first trimester placenta enhance NK cell cytotoxic potential for trophoblasts. CONCLUSIONS: Cytokines of the uteroplacental contact zone can appear a mechanism ensuring trophoblast mortality dynamics throughout pregnancy.

Bacterial lipopolysaccharide induces the intracellular expression of trophoblastic specific CD74 isoform in human first trimester trophoblast cells: Correlation with unsuccessful early pregnancy.

OBJECTIVE: During first trimester of human pregnancy, the maternal system develops immunity against infection and to provide protection of allogeneic foetus from abortion. This study was undertaken to determine the role of trophoblast specific CD74 isoforms in first trimester trophoblast derived cells under normal and lipopolysaccharide (LPS) stimulated conditions. METHODS: Gene and protein of CD74 were determined in first trimester trophoblast derived cells, JEG-3 and ACH-3 P and also in human placenta by PCR, western blotting and immunoprecipitation. Effect of LPS mediated infection on the regulation of CD74 isoforms was studied intracellularly and also on the cells surface by flow cytometry. RESULTS: Data demonstrated that JEG-3 and ACH-3 P cells under normal conditions have not expressed CD74 isoforms neither intracellularly or nor on the surface. These results were further validated directly in human placenta. However, treatment of these trophoblast cells with a bacterial LPS, significantly upregulated CD74 mRNA expression (p < 0.05). Furthermore, expression of CD74 on the surface was not detected even after stimulation with LPS. Interestingly, CD74 isoform at 35 kDa was significantly detected intracellularly upon stimulation with LPS (p < 0.05). These results were further confirmed by western blotting followed by immunoprecipitation. CONCLUSIONS: To the best of our knowledge, this is the first study concluded that the bacterial LPS induce infection in the first trimester trophoblasts via intracellular upregulation of CD74. Data indicated that the lack of cell surface expression of trophoblastic specific isoforms of CD74 may provide protection for human pregnancy in the first trimester.

Differential Secretion of Inflammatory Cytokines by Human Trophoblasts in the Presence of Escherichia coli, Lactobacillus crispatus, and Lactobacillus jensenii.

INTRODUCTION: The existence of a placental microbiome would require a non-antagonistic relationship between potentially colonizing bacteria and trophoblasts. OBJECTIVE: The immunologic response of trophoblasts to specific potentially invading bacteria needs further analysis. METHODOLOGY: Immortalized first trimester human trophoblasts Swan 71 (Sw.71) were coincubated with Escherichia coli, Lactobacillus jensenii, Lactobacillus crispatus, and incubated alone (i.e., control group; 4 conditions with n = 6 for each condition). Chemokines and cytokines were measured. ANOVA with post hoc pairwise analysis was used to compare cytokines/chemokines concentrations in the 4 culture media. RESULTS: Sw.71 co-incubated with E. coli, L. jensenii or L. crispatus resulted in differential secretion of 11 of the 26 assayed cytokines/chemokines. Sw.71 co-incubated with any of the 3 bacteria responded with significant increased secretion of interleukin (IL)-8 and granulocyte macrophage colony-stimulating factor. All bacteria elicited the secretion of IL-6 and interferon (IFN) α2, 2 proinflammatory cytokines. In addition, Lactobacillus species resulted in increased secretion of IL-12p40 and IFNγ. While E. coli did not modify secretion of anti-inflammatory cytokines, Sw.71 cells responded to co-incubation with Lactobacillus species by secreting increased levels of IL-10 and IL-1ra. Both Lactobacillus species led to a decreased secretion of IL-4. CONCLUSION: All 3 bacterial species triggered significant release of chemokines and inflammatory cytokines, suggesting that a commensal relationship with trophoblasts may not be feasible.

An Anti-inflammatory microRNA Signature Distinguishes Group 3 Innate Lymphoid Cells From Natural Killer Cells in Human Decidua.

Innate lymphoid cells (ILCs) are a heterogeneous subset of lymphocytes deeply implicated in the innate immune responses to different pathogens, in lymphoid organogenesis and in the maintenance of tissue homeostasis. Group 3 innate lymphoid cells (ILC3) have been detected in human decidua, where they play a role in the early inflammatory phase favoring implantation and tissue remodeling as well as in the subsequent regulatory phase preventing fetal rejection and supporting neoangiogenesis. A balance between inflammation and immune tolerance is required to maintain pregnancy, thus maternal immune system must be controlled by finely tuned mechanisms. microRNAs (miRNAs) are small non-coding RNAs with important regulatory roles in immune cells, but their function in decidual ILC3 (dILC3) and in decidual NK (dNK) cells is still undefined. Here, we examined the miRNome by microarray in these cells during the first trimester of pregnancy and compared with miRNA profiles of peripheral blood NK (pbNK) cells from pregnant women. We show that distinct miRNA profiles could clearly distinguish dILC3 from NK cells. Correlation analyses revealed that dNK and pbNK miRNome profiles are more similar to each other as compared to dILC3. In particular, we identified 302 and 279 mature miRNAs differentially expressed in dILC3 as compared to dNK and pbNK, respectively. The expression of miR-574-3p and the miR-99b/let-7e/miR-125a miRNA cluster resulted the most increased in dILC3. Remarkably, gene ontology analysis and pathway enrichments of miRNA targets revealed an involvement of these miRNAs in the promotion of anti-inflammatory responses. In agreement to this finding, we also found a higher expression of the anti-inflammatory miR-146a-5p in dILC3 with respect to NK cells. Overall, our data identified specific miRNA signatures distinguishing dILC3, dNK, and pbNK cells. Our data suggest the existence of a tight epigenetic control mediated by miRNAs in dILC3, potentially acting as a brake to prevent exaggerated inflammatory responses and to maintain the immune homeostasis in the early phases of pregnancy.

Multimodal immune phenotyping of maternal peripheral blood in normal human pregnancy.

Changes in maternal immunity during pregnancy can result in an altered immune state, and as a natural perturbation, this provides an opportunity to understand functional interactions of the immune system in vivo. We report characterization of maternal peripheral immune phenotypes for 33 longitudinally sampled normal pregnancies, using clinical measurements of complete blood counts and major immune cell populations, as well as high parameter flow cytometry for 30 leukocyte antigens characterizing 79 cell populations, and monitoring of 1305 serum proteins using the SomaLogic platform. Cellular analyses characterized transient changes in T cell polarization and more persistent alterations in T and B cell subset frequencies and activation. Serum proteomic analysis identified a potentially novel set of 7 proteins that are predictive of gestational age: DDR1, PLAU, MRC1, ACP5, ROBO2, IGF2R, and GNS. We further show that gestational age can be predicted from the parameters obtained by complete blood count tests and clinical flow cytometry characterizing 5 major immune cell populations. Inferring gestational age from this routine clinical phenotyping data could be useful in resource-limited settings that lack obstetric ultrasound. Overall, both the cellular and proteomic analyses validate previously reported phenotypic immunological changes of pregnancy and uncover potentially new alterations and predictive markers.