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Pregnancy is a unique condition where the maternal immune system is continuously adapting in response to the stages of fetal development and signals from the environment. The placenta is a key mediator of the fetal/maternal interaction by providing signals that regulate the function of the maternal immune system as well as provides protective mechanisms to prevent the exposure of the fetus to dangerous signals. Bacterial and/or viral infection during pregnancy induce a unique immunological response by the placenta, and type I interferon is one of the crucial signaling pathways in the trophoblast cells. Basal expression of type I interferon-β and downstream ISGs harbors physiological functions to maintain the homeostasis of pregnancy, more importantly, provides the placenta with the adequate awareness to respond to infections. The disruption of type I interferon signaling in the placenta will lead to pregnancy complications and can compromise fetal development. In this review, we focus the important role of placenta-derived type I interferon and its downstream ISGs in the regulation of maternal immune homeostasis and protection against viral infection. These studies are helping us to better understand placental immunological functions and provide a new perspective for developing better approaches to protect mother and fetus during infections.

Problem: A successful outcome to pregnancy is critically dependent on the initiation of maternal immune tolerance before embryo implantation. Cells of embryonic origin that come in contact with the uterine microenvironment can exert influence over the phenotype and function of immune cells to facilitate robust implantation; however, what influence they may have on B cells remains unknown. In this study, we investigate the effect of human trophoblast cells on B-cell phenotype and the subsequent effect on peri-implantation events. Method of study: We cultured purified human B cells with the first-trimester human trophoblast cell line Swan 71 to investigate trophoblast-B-cell interactions and utilized trophoblast spheroids in an in vitro implantation model of migration and invasion. Results: Trophoblast-educated B cells or TE-B cells were found to consist of B cells in committed lineages such as plasmablasts and memory B cells, as well as increased proportions in subsets of CD24hiCD27+ regulatory B cells and CD19+IL-10+ B cells. Conditioned media from the TE-B cells showed reduced production of pro-inflammatory cytokines that influenced the T-cell proliferation and cytokine production. Using trophoblast spheroids, we assessed the role of TE-B cells in trophoblast invasion and migration. Our results demonstrate a protective effect of TE-B-conditioned media against deleterious inflammation as evidenced by survival of the trophoblast spheroid in the presence of an immune assault and promotion of a migratory phenotype. Conclusion: We posit that trophoblast-mediated education of B cells leads to their acquisition of properties capable of modulating inflammation in the uterine environment during the peri-implantation period. Refereed/Peer-reviewed

At the end of 2019, a new coronavirus disease, COVID‐19, emerged and quickly spread around the world. Severe acute respiratory syndrome Coronavirus 2 (SARS‐CoV‐2), the causative virus of this disease, belongs to the β‐coronavirus family, together with SARS and middle east respiratory syndrome, and has similar biological characteristics to these viruses. For obstetricians, the susceptibility and prognoses of pregnant women and the effects of the infection on the fetus have been the focus of attention; however, at present, the seriousness of the disease in pregnant women is not apparent, and COVID‐19 does not increase the rate of miscarriage, stillbirth, preterm labor or teratogenicity. Even so, carriers might transmit SARS‐CoV‐2 to pregnant women. Thus, we must keep in mind that all medical personnel must understand and maintain standard precautions in their clinical and laboratory practices.

Caused by a novel type of virus, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), coronavirus disease 2019 (COVID‐19) constitutes a global public health emergency. Pregnant women are considered to have a higher risk of severe morbidity and even mortality due to their susceptibility to respiratory pathogens and their particular immunologic state. Several studies assessing SARS‐CoV‐2 infection during pregnancy reported adverse pregnancy outcomes in patients with severe conditions, including spontaneous abortion, preterm labor, fetal distress, cesarean section, preterm birth, neonatal asphyxia, neonatal pneumonia, stillbirth, and neonatal death. However, whether these complications are causally related to SARS‐CoV‐2 infection is not clear. Here, we reviewed the scientific evidence supporting the contributing role of Treg/Th17 cell imbalance in the uncontrolled systemic inflammation characterizing severe cases of COVID‐19. Based on the recognized harmful effects of these CD4+ T‐cell subset imbalances in pregnancy, we speculated that SARS‐CoV‐2 infection might lead to adverse pregnancy outcomes through the deregulation of otherwise tightly regulated Treg/Th17 ratios, and to subsequent uncontrolled systemic inflammation. Moreover, we discuss the possibility of vertical transmission of COVID‐19 from infected mothers to their infants, which could also explain adverse perinatal outcomes. Rigorous monitoring of pregnancies and appropriate measures should be taken to prevent and treat early eventual maternal and perinatal complications.

PROBLEM Studies assessing the association between vitamin D deficiency and preeclampsia (PE) have not reached a consensus. The study aimed to investigate the role of vitamin D in the occurrence of PE through its immune-modulatory effects on Treg/Th17 cell ratio. METHOD OF STUDY This is a case-control study of third-trimester pregnant women. Peripheral blood 25(OH)D, TGF-β1, IL-6, and Treg/Th17 cells were analyzed. RESULTS One hundred and sixty-three pregnant women were recruited, and 100 women (59 with a normal pregnancy (NP) and 41 with PE) were included in the study. The prevalence of vitamin D deficiency was 69.3%. Vitamin D-deficient pregnant women (25(OH) D

Problem Zika virus (ZIKV) has gained public concern for its association with microcephaly in infants born to ZIKV-infected mothers. To reach the fetus the virus must overcome the defense mechanisms provided by trophoblast cells. Additionally, in the first trimester, the integrity of the placenta is critical for fetal protection as damage to differentiating trophoblast can affect placental formation and function. We sought to investigate the effect of ZIKV infection on trophoblast cells and the factors that might increase the risk for ZIKV infection during pregnancy. Methods First-trimester human trophoblast cells, Swan 7.1, were infected with ZIKV, herpes simplex virus-2 (HSV-2), and yellow fiver (YFV). C57BL/6 pregnant mice were infected with HSV-2, ZIKV, or coinfection. Placental viral titers were determined by RT-PCR. Results ZIKV infection induces apoptosis in first-trimester trophoblasts and prevents differentiation of these cells. Furthermore, HSV-2 infection enhances placental sensitivity to ZIKV by enhancing the expression of TAM receptors, which facilitate ZIKV cell entry. Conclusion These findings may explain the mechanism by which ZIKV breaches the placental barrier to access the fetus. Furthermore, our results suggest that patients with HSV-2 infection are at a higher risk for the teratogenic effects induced by ZIKV.

Problem Quantitative measurement of proteins in bodily fluids or cellular preparations is critical for the evaluation of biomarkers or the study of complex cellular processes. While immunoassays are the most common quantitative approach used so far, they are not practical for the evaluation of multiple proteins. Microfluidic technology allows a fine spatial control in immobilizing proteins and biomolecules inside microchannels, eliminating cross-reactivity between competing analytes, and allowing rapid and sensitive detection of targeted antigens for multiple applications. We report the characterization and validation of the Simple Plex™ platform for the detection and quantification of cytokines and chemokines from human and mouse samples. Method Cytokine and chemokine expression levels were determined using Simple Plex cartridges from ProteinSimple. Serum samples were obtained from the Yale Biorepository. Results Our data demonstrate an excellent correlation between the results obtained with Simple Plex and conventional immunoassays such as ELISA and Luminex. Conclusion We describe the characterization and validation of Simple Plex, a novel multi-analyte, automated microfluidic platform that allows the evaluation of cytokines and chemokines from human and mice biological samples. Simple Plex showed significant advantages over traditional approaches in terms of low sample volume requirements, sensitivity and dynamic range, coefficient of variation, and reproducibility.

Progress in our understanding of the role of the maternal immune system during healthy pregnancy will help us better understand the role of the immune system in adverse pregnancy outcomes. In this review, we discuss our present understanding of the 'immunity of pregnancy' in the context of the response to cervical and placental infections and how these responses affect both the mother and the fetus. We discuss novel and challenging concepts that help explain the immunological aspects of pregnancy and how the mother and fetus respond to infection.

PROBLEM: Approximately half of all human embryo implantations result in failed pregnancy. Multiple factors may contribute to this failure, including genetic or metabolic abnormalities of the embryo. However, many of these spontaneous early abortion cases are attributed to poor uterine receptivity. Furthermore, although many fertility disorders have been overcome by a variety of assisted reproductive techniques, implantation remains the rate-limiting step for the success of the in vitro fertilization (IVF) treatments. RESULTS: We, as well as others, have demonstrated that endometrial biopsies performed either during the spontaneous, preceding cycle, or during the IVF cycle itself, significantly improve the rate of implantation, clinical pregnancies and live births. These observations suggest that mechanical injury of the endometrium may enhance uterine receptivity by provoking the immune system to generate an inflammatory reaction. In strong support of this idea, we recently found that dendritic cells (DCs), an important cellular component of the innate immune system, play a critical role in successful implantation in a mouse model. CONCLUSION: In this review we discuss the hypothesis that the injury-derived inflammation in the biopsy-treated patients generates a focus for uterine DCs and Mac accumulation that, in turn, enhance the endometrial expression of essential molecules, that facilitate the interaction between the embryo and the uterine epithelium.

Toll-like receptors (TLRs) form the major family of pattern recognition receptors (PRRs) that are involved in innate immunity. Innate immune responses against microorganisms at the maternal-fetal interface may have a significant impact on the success of pregnancy, as intrauterine infections have been shown to be strongly associated with certain complications of pregnancy. At the maternal-fetal interface, TLRs are expressed not only in the immune cells but also in non-immune cells such as trophoblasts and decidual cells; moreover, their expression patterns vary according to the stage of pregnancy. Here, we will update potential functions of TLRs in these cells, their recognition and response to microorganisms, and their involvement in the innate immunity. The impact of TLR-mediated innate immune response will be discussed via animal model studies, as well as clinical observations.

Problem Vitamin D has a pivotal role in regulating immune responses in women with recurrent pregnancy loss (RPL), but the underlying mechanism has not been completely clarified. This study aimed to determine the correlation between vitamin D and Treg/Th17 and the effects of vitamin D supplementation on Treg/Th17 balance in RPL patients. Methods of study The level of vitamin D was determined in women with normal pregnancy and RPL by electrochemiluminescence. The percentages of CD4+ Foxp3+ Treg, CD4+ IL-17+ Th17, and CD4+ Foxp3+ IL-17+ T cells were determined by flow cytometry before and after vitamin D supplementation. Changes about Treg/Th17 balance after culturing with active vitamin D in vitro were determined. Vitamin D metabolic activity of peripheral blood mononuclear cells was also detected by RT-PCR. Results Compared with normal pregnancy, both the level of vitamin D and the Treg/Th17 ratio were significantly decreased in women with RPL. There was a positive correlation between the level of vitamin D and the Treg/Th17 ratio in the RPL group. Within the RPL group, those who received 2 months of vitamin D supplementation showed a significantly increased Treg/Th17 ratio compared with those without vitamin D supplementation. In vitro analysis showed that adding different concentrations of active vitamin D increased the Treg/Th17 ratio, also the mRNA levels of the vitamin D receptor and the metabolic enzyme CYP24A1 increased significantly. Conclusion The occurrence of RPL may be related to vitamin D insufficiency and Treg/Th17 imbalance. The Treg/Th17 imbalance seen in women with RPL can be restored by vitamin D supplementation both in vivo and in vitro. The effects of vitamin D on the immune regulation of RPL indicate that vitamin D might be used as an alternative therapy in the future.

Implantation and the establishment of pregnancy in mammals involves an intricate interplay of hormones, cytokines, growth factors, proteins, lipids, ions and the extracellular matrix between the uterine epithelium, stroma, immune cells and the conceptus trophectoderm. The divergent nature of implantation in the mouse, human and pig provides not only an interesting contrast in the establishment of pregnancy and early embryonic development but also intriguing similarities with regard to early endometrial-conceptus signaling. An interesting pro-inflammatory cytokine expressed in a number of mammalian species during the period of implantation is interleukin-1β (IL1B). The presence of IL1B might be involved with immunotolerance at the maternal-placental interface and has been proposed as one of the mediators in placental viviparity. The production of IL1B and other proinflammatory cytokines might play a role in establishing pregnancy through modulation of the nuclear factor kappa-B (NFKB) system in a number of species. A model for the regulation of cellular progesterone receptor expression and NFKB activation for endometrial receptivity and conceptus attachment is continuing to evolve and is discussed in the present review.

Citation Pizzonia J, Holmberg J, Orton S, Alvero A, Viteri O, Mclaughlin W, Feke G, Mor G. Multimodality animal rotation imaging system (MARS) for in vivo detection of intraperitoneal tumors. Am J Reprod Immunol 2012; 67: 84–90 Problem Ovarian cancer stem cells (OCSCs) have been postulated as the potential source of recurrence and chemoresistance. Therefore identification of OvCSC and their complete removal is a pivotal stage for the treatment of ovarian cancer. The objective of the following study was to develop a new in vivo imaging model that allows for the detection and monitoring of OCSCs. Method of Study OCSCs were labeled with X-Sight 761 Nanospheres and injected intra-peritoneally (i.p.) and sub-cutaneously (s.c.) to Athymic nude mice. The Carestream In-Vivo Imaging System FX was used to obtain X-ray and, concurrently, near-infrared fluorescence images. Tumor images in the mouse were observed from different angles by automatic rotation of the mouse. Results X-Sight 761 Nanospheres labeled almost 100% of the cells. No difference on growth rate was observed between labeled and unlabeled cells. Tumors were observed and monitoring revealed strong signaling up to 21 days. Conclusion We describe the use of near-infrared nanoparticle probes for in vivo imaging of metastatic ovarian cancer models. Visualization of multiple sites around the animals was enhanced with the use of the Carestream Multimodal Animal Rotation System.

Problem The presence of immune infiltrates in the tumor does not always correlate with an anti-tumoral immune response. We previously identified two sub-populations of epithelial ovarian cancer (EOC) cells with differential cytokine profile. We hypothesize that these two sub-populations of EOC cells may differentially regulate the immune phenotype in the tumor microenvironment and therefore affect the immune response.

Citation Ramhorst R, Fraccaroli L, Aldo P, Alvero AB, Cardenas I, Leiros CP, Mor G. Modulation and recruitment of inducible regulatory T cells by first trimester trophoblast cells. Am J Reprod Immunol 2012; 67: 17–27 Problem The specialized regulatory T-cells (Treg) population, essential for maternal tolerance of the fetus, performs its suppressive actions in the critical peri-implantation phase of pregnancy. In the present work, we investigated whether trophoblast cells are able to induce Treg recruitment, differentiation, and whether these mechanisms are modified by a bacterial or viral infection. Method of Study Human T-regulatory cells were differentiated from naive CD45RA+ CCR7+ cells obtained from peripheral blood mononuclear cells cultured with IL-2 and TGFβ over 5 days. Induction of iTregs (CD4+ Foxp3+ cells) was evaluated using low serum conditioned media (LSCM), obtained from two first trimester trophoblast cell lines, Swan-71 and HTR8. Coculture experiments were carried out using transwell assays where trophoblast cells were in the absence or presence of PGN, LPS, or Poly [I:C]. Cytokine production was measured by multiplex analysis. Results Trophoblast cells constitutively secrete high levels of TGFβ and induced a significant increase of Foxp3 expression accompanied by a specific T-reg cytokine profile. Moreover, trophoblast cells were able to recruit iTregs in a specific manner. Conclusion We demonstrate that trophoblast cells have an active role on the recruitment and differentiation of iTregs, therefore, contributing to the process of immune regulation at the placental–maternal interface.

Hofbauer cells (HBCs) are placental macrophages that are present in the villus across gestation. Despite their identification more than 100 years ago, their specific role in placental function remains largely unelucidated. We initially review aspects of their history and biology as well as evidence for putative sites of origin. To gain insight into their potential function, we then describe complications of pregnancy including villitis of unknown etiology (VUE) and histological chorioamnionitis (HCA), in which alterations in numbers, gene expression, or other characteristics of HBCs have been documented to occur. We further review methods for isolation of HBCs and in vitro studies that explore their role in relation to other major cell types in the placenta and examine their actions in cytokine-mediated inflammation. We conclude that HBCs play a key role in placental pathophysiology, and future advances in their isolation and culture would enable mechanistic insight into their villus function.

The concept that pregnancy is associated with immune suppression has created a myth of pregnancy as a state of immunological weakness and, therefore, of increased susceptibility to infectious diseases. A challenging question is whether the maternal immune system is a friend or a foe of pregnancy. In this review, we discuss data associated to the role of the immune system during pregnancy. We propose a new paradigm in terms of the fetal-maternal immune interaction as well as the immunological response of the mother to microorganism. Our challenge is to better understand the immunology of pregnancy in order to deliver the appropriate treatment to patients with pregnancy complications as well as to determine public policies for the protection of pregnant women during pandemics.

Citation Hanlon DJ, Aldo PB, Devine L, Alvero AB, Engberg AK, Edelson R, Mor G. Enhanced stimulation of anti-ovarian cancer CD8+ T cells by dendritic cells loaded with nanoparticle encapsulated tumor antigen. Am J Reprod Immunol 2011; 65: 597–609 Problem Dendritic cell (DC)-based cancer therapies are favored approaches to stimulate anti-tumor T-cell responses. Unfortunately, tolerance to tumor antigens is difficult to overcome. Biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP) are effective reagents in the delivery of drugs and tumor-associated antigens (TAA). In this study, we assessed the capacity of a PLGA NP-based delivery system to augment CD8 T-cell responses to ovarian cancer TAA. Method of Study Human DC were generated from blood monocytes by conventional in vitro differentiation and loaded with either soluble tumor lysate or NP/lysate conjugates (NPL). These antigen-loaded DC were then used to stimulate autologous CD8+ T cells. Cytokine production and activation markers were evaluated in the CD8+ T cells. Results DC loading with NPL increased cytokine production by stimulated CD8 T cells and induced T-cell expression of cell surface co-stimulatory molecules, typical of anti-tumor immune responses. In contrast, delivery of naked tumor lysate antigens preferentially induced a T-cell profile characteristic of tolerization/exhaustion. Conclusion These findings indicate that delivery of TAA in NP enables DC to efficiently activate anti-tumor CD8+ T cells. PLGA NP encapsulation of tumor-derived lysate protein antigens is an encouraging new preparative methodology for DC-based vaccination meriting clinical testing.

Citation Atay S, Gercel-Taylor C, Suttles J, Mor G, Taylor DD. Trophoblast-derived exosomes mediate monocyte recruitment and differentiation. Am J Reprod Immunol 2011; 65: 65–77 Introduction Trophoblast cells have been demonstrated to regulate monocyte migration and differentiation, leading to pro-inflammatory profiles. Because trophoblast cells release exosomes with immunoregulatory properties, trophoblast-derived exosomes are proposed to ‘educate’ monocytes, creating a pro-inflammatory environment. Method of study Exosomes were isolated from conditioned media of Swan71 cells by ultrafiltration and ultracentrifugation. Exosome-induced migration was assessed using a two-chamber system. Cytokine profiles were defined using cytokine arrays, and mRNA levels of affected cytokines were examined by qRT-PCR and ELISA. Results Within 20 min, 8–10% of monocytes took up labeled exosomes isolated from Swan71 cells. Trophoblast-derived exosomes increased monocyte migration in a dose-dependent manner and produced significant increases in production of interleukin (IL)-1β, IL-6, Serpin-E1, granulocyte colony-stimulating factor, granulocyte/monocyte colony-stimulating factor, and tumor necrosis factor-α. Conclusion This study presents the initial demonstration that trophoblast-derived exosomes are capable of recruiting and ‘educating’ monocytes to produce pro-inflammatory cytokine/chemokine profiles in a cell-contact-independent manner.

Problem—The NFκB pathway is a major source of pro-inflammatory cytokines, which may contribute to cancer chemoresistance. We showed that constitutive NFκB activity is characteristic of the ovarian cancer stem cells (OCSCs). The aim of this study is to determine if the inhibition of NFκB by Eriocalyxin B (EriB) in the OCSCs may induce cell death in otherwise chemoresistant cells. Methods—OCSCs and mature ovarian cancer cells (mOCCs) were treated with increasing concentrations of EriB. Cell viability was measured using the Celltiter 96 assay and caspase activity was quantified using Caspase-Glo™ assay. Cytokine levels were quantified using xMAP technology. Results—EriB decreased the percent of viable cells in all cultures tested with GI50 of 0.5 −1µM after 48h of treatment. The intracellular changes associated with EriB-induced cell death are: 1) inhibition of NF-κB activity; 2) decreased cytokine production; 3) activation of caspases; and 4) down-regulation of XIAP. In addition, EriB is able to sensitize OCSCs to TNFα and FasLmediated cell death. Conclusions—Inhibition of the NFκB pathway induces cell death in the OCSCs. Since the OCSCs may represent the source of recurrence and chemoresistance, the use of NFκB inhibitors like EriB may prevent recurrence in ovarian cancer patients.

Apoptosis, programmed cell death, is an essential feature of normal placental development but is exaggerated in association with placental disease. Placental development relies upon effective implantation and invasion of the maternal decidua by the placental trophoblast. In normal pregnancy, trophoblast apoptosis increases with placental growth and advancing gestation. However, apoptosis is notably exaggerated in the pregnancy complications, hydatidiform mole, pre-eclampsia, and intrauterine growth restriction (IUGR). Placental apoptosis may be initiated by a variety of stimuli, including hypoxia and oxidative stress. In common with other cell-types, trophoblast apoptosis follows the extrinsic or intrinsic pathways culminating in the activation of caspases. In contrast, the formation of apoptotic bodies is less clearly identified, but postulated by some to involve the clustering of apoptotic nuclei and liberation of this material into the maternal circulation. In addition to promoting a favorable maternal immune response, the release of this placental-derived material is thought to provoke the endothelial dysfunction of pre-eclampsia. Widespread apoptosis of the syncytiotrophoblast may also impair trophoblast function leading to the reduction in nutrient transport seen in IUGR. A clearer understanding of placental apoptosis and its regulation may provide new insights into placental pathologies, potentially suggesting therapeutic targets.

Problem Among pregnant women, acquired viral infections with a concurrent bacterial infection is a detrimental factor associated to poor prognosis. We evaluate the effect of a viral infection that does not lead to pre-term labor on the response to low doses of lipopolysaccharide (LPS). Our objectives were (i) to characterize the effect of a viral infection concurrent with exposure to microbial products on pregnancy outcome and (ii) to characterize the placental and fetal immune responses to the viral sensitization to LPS.

Recurrent spontaneous abortion (RSA) is a growing problem worldwide. In a majority of cases, the cause remains unknown but there is increasing evidence that immunologic factors play an important role. Intravenous immunoglobulin (IVIg) therapy has been proposed to have immune modulatory effects and therefore been applicable for the treatment of patients with RSA. Although its efficacy is still controversial, several recent studies suggest that IVIg treatment may improve pregnancy outcomes. CD4+ T cells and their related cytokines play an important role in maternal-fetal immune regulation, and an imbalance of Th17/Treg cell ratio has been proposed as a cause for RSA. We review the scientific evidence supporting a modulatory effect of IVIg on Th17/Treg cell balance and discuss the potential mechanisms how IVIg might enhance Treg cells function. We propose that correction of Th17/Treg cell dysregulation could be one of the mechanisms that can explain the positive therapeutic effects of IVIg therapy. Consequently, selecting patients with abnormal Th17/Treg cell ratios could increase the success of IVIg therapy.

Placental immune response and its tropism for specific viruses and pathogens affect the outcome of the pregnant woman's susceptibility to and severity of certain infectious diseases. The generalization of pregnancy as a condition of immune suppression or increased risk is misleading and prevents the determination of adequate guidelines for treating pregnant women during pandemics. There is a need to evaluate the interaction of each specific pathogen with the fetal/placental unit and its responses to design the adequate prophylaxis or therapy. The complexity of the immunology of pregnancy and the focus, for many years, on the concept of immunology of pregnancy as an organ transplantation have complicated the field and delayed the development of new guidelines with clinical implications that could help to answer these and other relevant questions. Our challenge as scientists and clinicians interested in the field of reproductive immunology is to evaluate many of the 'classical concepts' to define new approaches for a better understanding of the immunology of pregnancy that will benefit mothers and fetuses in different clinical scenarios.

Toll-like receptors (TLR) form the major family of pattern recognition receptors (PRR) that are involved in innate immunity. Innate immune responses against microorganisms at the maternal-fetal interface may have a significant impact on the success of pregnancy, as intrauterine infections have been shown to be strongly associated with certain disorders of pregnancy. At the maternal-fetal interface, TLRs are expressed not only in the immune cells but also in non-immune cells such as trophoblasts and decidual cells; moreover, their expression patterns vary according to the stage of pregnancy. Here, we will describe potential functions of TLRs in these cells, their recognition and response to microorganisms, and their involvement in the innate immunity. The impact of TLR-mediated innate immune response will be discussed via animal model studies, as well as clinical observations.

Problem Herpes simplex virus type 2 (HSV-2) infection is one of the most prevalent diseases worldwide and is mainly sexually transmitted. When infecting pregnant women, HSV-2 is able to infect the placenta, can reach the fetus, and may affect the fetal development. We sought to determine the prevalence of HSV-2 infection and reactivation in asymptomatic pregnant women, the correlation between IgG in the maternal circulation and cord blood, and the correlation between circulating IgG, placental, and newborn infection (blood cord). Method of study Serum samples and placental tissues from pregnant women and umbilical cord blood samples from their newborns were collected. Anti-HSV-2 antibodies were identified by ELISA, and HSV-2 DNA was detected by nested PCR. Results The seropositivity of IgG in pregnant women was 29.7% and IgM was detected in 1 woman (0.5%). In the umbilical cord of newborns, 33.1% were IgG-positive and IgM was detected in 2 samples (1.5%). A positive correlation between HSV-2 IgG titers in serum from pregnant women and cord blood samples was found (r = .36, P = .001). A difference between the positive and negative placental groups (maternal side) was found in titers of IgG in sera of umbilical cord, which were significantly higher in the positive placental group (P = .004). Conclusion We describe for the first time that newborns from mothers with HSV-2 placental infection have higher IgG titers in sera of umbilical cord, suggesting IgGs antibodies can be indicative of placental viral infection in asymptomatic women.

Systemic inflammation and abnormal/poor placentation represent hallmarks of pre-eclampsia. Accumulating evidence suggests that infectious agents might increase the risk of pre-eclampsia; the innate immune defense mechanisms may interact with pro-inflammatory pathways, and contribute to the development of pre-eclampsia. The evidence for this has been supported by indirect epidemiologic and clinical studies, as well as by some direct support from experimental studies. Recent data directly implicate signaling by Toll-like receptors in the pathogenesis of pre-eclampsia, and establish a crucial link between pre-eclampsia and defense against both foreign pathogens and endogenously generated inflammatory ligands. Here, we review the rapid progress in this field, which has improved our understanding of the interplay between pathogen invasion, innate immune defense mechanisms, and pre-eclampsia.

Approximately half of all human embryo implantations result in failed pregnancy. Multiple factors may contribute to this failure, including genetic or metabolic abnormalities of the embryo. However, many of these spontaneous early abortion cases are attributed to poor uterine receptivity. Furthermore, although many fertility disorders have been overcome by a variety of assisted reproductive techniques, implantation remains the rate-limiting step for the success of the in vitro fertilization (IVF) treatments. It has been demonstrated that endometrial biopsies performed either during the spontaneous, preceding cycle, or during the IVF cycle itself, significantly improve the rate of implantation, clinical pregnancies and live births. These observations suggest that mechanical injury of the endometrium may enhance uterine receptivity by provoking the immune system to generate an inflammatory reaction. In strong support of this idea, we recently found that dendritic cells (DCs), an important cellular component of the innate immune system, play a critical role in successful implantation in a mouse model. In this review, we discuss the hypothesis that the injury-derived inflammation in the biopsy-treated patients generates a focus for uterine DCs accumulation that, in turn, enhances the endometrial expression of essential molecules, which facilitate the interaction between the embryo and the uterine epithelium.

Problem Epidemiological data suggested that pandemic influenza increased the risks of spontaneous abortion and premature labor, while seasonal influenza also increased the risk of schizophrenia in adolescence. However, their pathogenesis is so far unknown. Method of study The first trimester trophoblast cell lines, namely, Swan71 and HTR8 cells were challenged with A/Udorn/72 influenza virus (H3N2). At indicated time points, cells were examined for expression of influenza proteins. Viral replication in culture media, apoptosis and the expression of human leukocyte antigen (HLA)-G were also examined. Results Intracellular localization of viral proteins was observed. Twenty-four hours after inoculation, virus was detected in culture media while most cells fell into apoptosis. During apoptosis, expression of HLA-G was unchanged. Conclusion We revealed replication of low pathogenic influenza virus in the first trimester trophoblast cell lines. Placental damages are likely to be induced by direct cytopathic effects of influenza virus and subsequent apoptosis rather than down regulation of HLA-G expression and subsequent rejection by maternal immune system.

Background—Resistance to apoptosis is one of the key events that confer chemo-resistance and is mediated by over-expression of anti-apoptotic proteins, which inhibit caspase activation. The objective of this study was to evaluate whether the activation of an alternative cell death pathway (caspase-independent) could promote death in chemo-resistant ovarian cancer cells. We report the characterization of NV-128 as an inducer of cell death through a caspase-independent pathway. Methods—Primary cultures of epithelial ovarian cancer cells (EOC) were treated with increasing concentration of NV-128 and GI50 was determined using Cell titer 96 assay. Apoptotic proteins were characterized by western blot analyses, Caspase Glo assays, immunohistochemistry, and flow cytometry. Protein-protein interactions were determined using immunoprecipitation. In vivo activity was measured in a xenograft mice model. Results—NV-128 is able to induce significant cell death in both Paclitaxel- and Carboplatinresistant EOC cells with GI50 between 1 and 5 ug/ml. Cell death was characterized by chromatin condensation but was caspase-independent. The activated pathway involved the down-regulation of phospho-AKT, phospho- mTOR, and phospho-S6 kinase, and the mitochondrial translocation of Beclin-1 followed by nuclear translocation of EndoG. Conclusion—We have characterized a novel compound, which inhibits mTOR and promotes caspase-independent cell death. Our results indicate that inhibition of mTOR may represent a relevant pathway for induction of cell death in cells resistant to the classical caspase-dependent apoptosis. These findings demonstrate the possibility of using therapeutic drugs, such as NV-128, which could have beneficial effects in chemo-resistant ovarian cancer patients.

Toll-like receptors (TLRs) recognize conserved sequences on the surface of pathogens and trigger effector cell functions. Previously, we described the expression of TLR3 by human trophoblast and their ability to respond to (Poly[I:C]). Here we evaluate the effect of Poly[I:C] on mouse pregnancy and characterize the local and systemic response. C57B/6 wild type (wt) and TLR3 knockout (TLR3KO) mice were treated with Poly[I:C] at 16.5 dpc and pregnancy outcome recorded. Morphologic changes, cytokines and chemokines levels in blood and utero-placental tissue were determined. NF-kappa B pathway was evaluated in vivo and in vitro. Poly[I:C] in C57B/6 wt mice caused preterm delivery within 24 hr (4.5 mg/kg). No effect was observed in TLR3KO mice. In addition, we observed local (placenta) and systemic (serum) response characterized by increased production of proinflammatory cytokines and chemokines. The NF-kappa B pathway was activated by Poly[I:C] in human and mice trophoblast cells. We report that Poly[I:C] induces preterm delivery via TLR3-dependent manner. Furthermore, we demonstrate that the trophoblast is able to recognize Poly[I:C] through TLR3 and respond to viral infection, modulating the immune system at the feto-maternal interface.

Preeclampsia is associated with an increased release of factors from the placental syncytium into maternal blood, including the antiangiogenic factors soluble fms-like tyrosine kinase-1 and soluable endoglin, the antifibrinolytic factor plasminogen activator inhibitor-1, prostanoids, lipoperoxides, cytokines, and microparticles. These factors are suggested to promote maternal endothelium dysfunction and are associated with placental damage in pregnancies also complicated with intrauterine growth restriction (IUGR). In this report, we briefly describe the interaction of syncytial factors with hypoxia, reactive oxygen species, and apoptosis in the pathophysiology of preeclampsia and IUGR. Given the critical role of the syncytium in these complications of pregnancy, we also present a novel methodology in which laser capture microdissection followed by Western blotting is used to assess levels of syncytial Fas ligand, a key protein in the apoptotic cascade.

During normal pregnancy, the decidua is populated by a variety of leucocytes; however, cells of the innate immune system seem to dominate this tissue. Their presence suggests that the innate immune system is not indifferent to the fetus and has been associated with a response of the maternal immune system to the "semi-allograft fetus." New evidences, however, indicates that these immune cells are critical for decidual and trophoblast development, rather than induction of tolerance. We hypothesized that, during implantation, an inflammatory environment is necessary for the attachment and invasion of the blastocyst. Therefore, we propose the existence of an "inflammatory-mediated embryo implantation" condition that is dependent on the proper "education" of the innate immune system by the trophoblast. Here we postulate that trophoblast cells successfully orchestrate their inflammatory environment and regulate immune cell differentiation and activation.

The association between chronic inflammation and cancer has long been observed. Furthermore, NF-kappaB activation and the subsequent production of cytokines, chemokines, growth factors, and antiapoptotic proteins has been found to be involved in cancer progression and chemoresistance. However, the signals inducing NF-kappaB in cancer cells are still not well understood. Here, we reviewed the association between chronic inflammation and cancer, the role of NF-kappaB and its inhibitors as potential anticancer drugs, and Toll-like receptors as possible signal initiators for NF-kappaB activation and inflammation-induced carcinogenesis and chemoresistance. Furthermore, we propose that, the stimulation of Toll-like receptors by microbial components and/or endogenous ligands may represent the initial signal promoting a proinflammatory environment that will enhance tumor growth and chemoresistance.

Background: During implantation, apoptosis is critical for the appropriate tissue remodeling of the maternal decidua and invasion of the developing embryo, yet the regulation of apoptosis is also imperative for a successful pregnancy. The quick and effective removal of apoptotic cells by tissue macrophages represents an essential process, which prevents the release of self-antigens, and in the case of pregnancy, paternal alloantigens. Methods of study: Recent studies have shown that the process of apoptotic cell clearance is not a neutral event, but rather an active one that induces macrophage production of anti-inflammatory cytokines and survival factors. Apoptotic cell clearance is, therefore, necessary for the resolution of inflammatory conditions, which during pregnancy could have lethal consequences. Conclusions: The function of the maternal immune system during implantation and throughout pregnancy is, therefore, an important area of investigation. This review will discuss the role of decidual macrophages in apoptotic cell clearance during pregnancy.

Adult sexual dimorphism in neuronal cell number is controlled by estrogen exposure during a tightly defined period of rat brain development. The mechanisms of estrogen's effect are unknown; one possibility is regulation of programmed cell death (apoptosis). In this study we have shown that estradiol can function as a neuroprotective agent or an inducer of apoptosis, depending on the estrogen receptor-subtype present in the cell. Thus, ERalpha has a neuroprotective effect, while ERbeta mediates the induction of apoptosis in neuronal cells. Moreover, we show that estrogen-induced apoptosis through ER-beta requires the expression of Fas- and Fas ligand (FasL) proteins, since the absence of FasL in neurons prevents this effect. Furthermore, we demonstrate that microglia-secreted products induce the expression of FasL necessary to mediate estradiol-ERbeta apoptotic effect. These findings may explain the dichotomous effect of fetal estradiol on the adult neuronal number.

PROBLEM: The low frequency of maternal immune responses to paternally inherited fetal antigens raises the following question: What regulates the immunobiology of pregnancy? Data suggest that this state is the result of peripheral immune-tolerance, an active process of immune-regulation in which activated T cells undergo apoptosis. We studied Fas ligand (FasL) expression and apoptosis in normal and pathologic placentas to find out whether the Fas-FasL-induced apoptosis takes place during implantation. METHOD OF STUDY: FasL expression in paraffin sections was detected using specific antibodies and confirmed with reverse transcriptase-polymerase chain reaction of total RNA from frozen placentas. Apoptosis was detected using the terminal deoxy (d)-UTP nick end-labeling assay. RESULTS: FasL was found in the normal placenta and in gestational trophoblastic disease. Apoptotic leukocytes were localized to the maternal-fetal interface corresponding in localization with the distribution of FasL. CONCLUSIONS: We propose that FasL expression in the placenta is a mechanism responsible for the development of maternal immune tolerance specific for paternal alloantigens and operates in pathologic states characterized by trophoblastic invasion/proliferation.

Citation Aldo PB, Mulla MJ, Romero R, Mor G, Abrahams VM. Viral ssRNA induces first-trimester trophoblast apoptosis through an inflammatory mechanism. Am J Reprod Immunol 2010; 64: 27–37 Problem Infection during pregnancy represents a significant cause of mobility and mortality. While viruses pose a major threat, little is known about their effect on early pregnancy, or the mechanisms involved. The objective of this study was to characterize the trophoblast response following exposure to viral ssRNA. Method of study First trimester trophoblast cells were treated with or without viral ssRNA. Cytokine production was measured using multiplex analysis and ELISA. Apoptosis was determined using Hoechst staining, cell viability, and caspase activity assays. Results Treatment of trophoblasts with viral ssRNA increased their secretion of IL-8, IL-6, and IFNβ. However, the ssRNA also induced trophoblast apoptosis. To test whether the viral ssRNA-induced inflammatory response was responsible for this induction of apoptosis, conditioned media (CM) from trophoblasts were added to a fresh culture of cells. The CM from viral ssRNA-treated induced higher levels of trophoblast apoptosis than the control CM. Moreover, recombinant IFNβ induced trophoblast apoptosis. Conclusion We demonstrate that viral ssRNA induces a pro-inflammatory and type I interferon response in the trophoblast and this inflammatory process may indirectly induce trophoblast apoptosis. These results provide a novel mechanism by which certain viral infections might compromise placental integrity and function, and therefore, pregnancy outcome.

The prevalence of viral infections in the amniotic fluid (AF) has not yet been ascertained. The aim of this study was to determine the prevalence of specific viral nucleic acids in the AF and its relationship to pregnancy outcome.From a cohort of 847 consecutive women undergoing midtrimester amniocentesis, 729 cases were included in this study after exclusion of documented fetal anomalies, chromosomal abnormalities, unavailability of AF specimens and clinical outcomes. AF specimens were tested by quantitative real-time PCR for the presence of genome sequences of the following viruses: adenoviruses, herpes simplex virus (HSV), varicella zoster virus (VZV), human herpesvirus 6 (HHV6), human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), parvovirus B19 and enteroviruses. Viral nucleic acid testing was also performed in maternal blood and cord blood in the population of women in whom AF was positive for viruses and in a control group of 29 women with AF negative for viral nucleic acids. The relationship between the presence of viruses and pregnancy and neonatal outcome was examined. The correlation between the presence of nucleic acids of viruses in the AF and the concentration of the cytokine interleukin-6 (IL-6) and the T cell chemokine CXCL-10 (or IP-10) in AF and maternal blood were analyzed.Viral genome sequences were found in 16 of 729 (2.2%) AF samples. HHV6 was the most commonly detected virus (7 cases, 1.0%), followed by HCMV (6 cases, 0.8%), parvovirus B19 (2 cases, 0.3%) and EBV (1 case, 0.1%), while HSV, VZV, enteroviruses and adenoviruses were not found in this cohort. Corresponding viral DNA was also detected in maternal blood of six out of seven women with HHV6-positive AF and in the umbilical cord plasma, which was available in one case. In contrast, viral DNA was not detected in maternal blood of women with AF positive for parvovirus B19, HCMV, EBV or of women with AF negative for viruses. HHV6 genome copy number in AF and maternal blood was consistent with genomic integration of viral DNA and genetic infection in all women. There was no significant difference in the AF concentration of IL-6 and IP-10 between patients with and without VIAC. However, for HCMV, there was a significant relationship between viral copy number and IP-10 concentration in maternal blood and AF. The group of women with AF positive for viral DNA delivered at term healthy neonates without complications in 14 out of 16 cases. In one case of HHV6 infection in the AF, the patient developed gestational hypertension at term, and in another case of HHV6 infection in the AF, the patient delivered at 33 weeks after preterm premature rupture of membranes (PPROM).Viral nucleic acids are detectable in 2.2% of AF samples obtained from asymptomatic women in the midtrimester. HHV6 was the most frequently detected virus in AF. Adenoviruses were not detected. Vertical transmission of HHV6 was demonstrated in one case.

Background: The presence of immune infiltrate in tumor sites has been thought to represent an immune response against the tumor. However, new evidences indicate that these immune cells may be supporting tumor growth rather than inhibiting it. Previously, we described a sub-group of epithelial ovarian cancer (EOC) cells that express TLR-4 and MyD88 and produce several cytokines and chemokines. We hypothesize that EOC cells could recruit immune cells and induce their differentiation toward tumor-supporting immune cells. The objective of this study is to determine the consequence of an activated TLR-4-MyD88 signaling pathway on migration and differentiation of the monocyte-like THP-1 cells. Materials and Methods: R182 (MyD88-positive) and CP70 (MyD88-negative) EOC cells were incubated in the presence or absence of 10 μg/mL LPS for 48 hr and cell-free conditioned media (CM) was prepared. THP-1 cells were incubated in 50% CM for 24 hr. Cytokine profile (IL-6, IL-8, IL-10, IL-12, MIF, MCP-1, GROα, RANTES, IFNγ, and TNFα) of cell-free supernatants and THP-1 lysates was characterized using Beadlyte Human Multicytokine kit and xMAP technology. Migration assay was performed using QCM 24-well Colorimetric Cell Migration Assay kit. Results: THP-1 cells migrate towards R182- and CP70-CM equally. However, enhanced migration was observed towards CM from LPS-treated-R182 (45%) than towards CM from LPS-treated CP70 (9%). When incubated with CM from LPS-treated-R182, THP-1 cells express a cytokine profile characteristic of M2 macrophages (high levels of MCP-1, MIF and IL-6). This was not observed when THP-1 was incubated with LPS alone or with CM from LPS-treated–CP70. Conclusion: In the present study we demonstrate that EOC cells have the capacity to attract THP-1 cells. Furthermore, we demonstrate that MyD88+ EOC cells are able to differentiate THP-1 cells into growth-promoting macrophages and establish a pro-tumor environment that can induce cancer cell proliferation. Our data suggest that cancer cells have the ability to attract and ‘educate’ immune cells to support its growth.

Introduction: Increased trophoblast apoptosis has been observed in pregnancy complications such as preeclampsia. The factors promoting this cell death are unknown. Trophoblasts express Toll-like receptors (TLR), enabling them to recognize microorganisms. In response to the TLR2 ligand peptidoglycan (PDG), trophoblasts undergo apoptosis suggesting that gram-positive bacteria may promote trophoblast apoptosis. The objective of this study was to characterize the cellular mechanism by which TLR2 induces apoptosis. We report the role of TLR6 in the regulation of TLR2 mediated apoptosis following ligation by PDG. Methods: First trimester trophoblast cell lines express TLR1 and TLR2 but lack TLR6 and are sensitive to PDG-mediated apoptosis. Three stable transfectants were made to express either a TLR1 dominant negative (TLR1-DN), a TLR2 dominant negative (TLR2-DN) or full length TLR6. Cells were treated with PDG (40 μg/mL) after which cell viability, caspase-3 activity and cytokine production were evaluated. Results: Trophoblasts expressing the TLR2-DN showed a 57.4 ± 1.2% inhibition in PDG-induced caspase-3 activity compared with wildtype cells, while the TLR1-DN inhibited PDG-induced caspase-3 activity by 61.6 ± 1.1%. Expression of TLR6 by trophoblasts, reversed PDG-mediated apoptosis as shown by reduced caspase-3 activity (77.6 ± 1.2%) and increased cytokine production. Conclusion: These results suggest that a gram-positive bacterial infection during pregnancy may promote trophoblast apoptosis via TLR2/TLR1 heterodimers. Expression of TLR6, however, may protect the trophoblast from PDG-induced apoptosis and instead promote an inflammatory response. These findings shed new light on the mechanisms by which infection may affect placental function and pregnancy outcome.

Introduction: Preeclampsia and IUGR have been linked to improper trophoblast migration that impairs maternal spiral artery transformation minimizing vessel dilation and therefore restricting blood flow to the developing fetus. Endothelial cells are thought to play an essential role in directing trophoblast migration and transformation, although the mechanism by which this occurs is not fully understood. We have developed a novel in vitro system that evaluates endothelial-trophoblast interaction and signaling in a three-dimensional environment. Methods: An immortalized human endometrial endothelial cell line (HEEC) (Krikun et al., 2004) and first trimester trophoblast cell lines HTR-8 and Swan.71 were used. Endothelial transformation into vessel-like structures occurred in Matrigel Basement Membrane Matrix. Cell membrane linker dye and OpenLab Image Analysis software were used to monitor trophoblast migration and transformation. Cytokine and chemokine production was determined using a multiplex bead assay. Results: Upon differentiation in matrigel, endothelial cells undergo a shift in cytokine production characterized by an increased secretion of chemokines. First trimester trophoblast cells migrate toward the differentiated endothelium, and reach it within 4–8 hr. Complete replacement of the endothelium is seen by 48 hr. The transformed tubes maintain structure and remain functional for several weeks allowing for further co-culture studies. Conclusion: We report the characterization of a novel three-dimensional in vitro system of trophoblast–endothelium cell interaction. We found significant changes in the phenotype (cytokine expression and LPS responses) of endothelial cells upon differentiation in matrigel as compared to the monolayer culture. These changes may be necessary for the endothelium to direct trophoblast migration. Furthermore, this model represents an ideal co-culture system to evaluate the role of additional cell types, e.g. immune cells, involved in the process of spiral artery transformation.