Search

Preterm birth is the largest contributor to neonatal morbidity and is often associated with chorioamnionitis, defined as inflammation/infection of the fetal membranes (FMs). Chorioamnionitis is characterised by neutrophil infiltration of the FMs and is associated with elevated levels of the neutrophil chemoattractant, interleukin (IL)‐8 and the proinflammatory cytokine, IL‐1β. While FMs can respond to infections through innate immune sensors, such as toll‐like receptors (TLRs), the downstream mechanisms by which chorioamnionitis arises are not fully understood. A novel group of non‐classical microRNAs (miR‐21a, miR‐29a, miR‐146a‐3p, Let‐7b) function as endogenous danger signals by activating the ssRNA viral sensors TLR7 and TLR8. In this study, the pro‐inflammatory roles of TLR7/TLR8‐activating miRs were examined as mediators of FM inflammation in response to bacterial lipopolysaccharide (LPS) using an in vitro human FM explant system, an in vivo mouse model of pregnancy, and human clinical samples. Following LPS exposure, miR‐146a‐3p was significantly increased in both human FM explants and wild‐type mouse FMs. Expression of miR‐146a‐3p was also significantly elevated in FMs from women with preterm birth and chorioamnionitis. FM IL‐8 and inflammasome‐mediated IL‐1β production in response to LPS was dependent on miR‐146a‐3p and TLR8 downstream of TLR4 activation. In wild‐type mice, LPS exposure increased FM IL‐8 and IL‐1β production and induced preterm birth. In TLR7−/−/TLR8−/− mice, LPS exposure was able to initiate but not sustain preterm birth, and FM inflammation was reduced. Together, we demonstrate a novel signalling mechanism at the maternal‐fetal interface in which TLR8‐activating miR‐146a‐3p acts as an intermediate danger signal to drive FM inflammasome‐dependent and ‐independent mechanisms of inflammation and, thus, may play a role in chorioamnionitis and subsequent preterm birth. [ABSTRACT FROM AUTHOR]

BACKGROUND The endometrium is a highly dynamic tissue that undergoes dramatic proliferation and differentiation monthly in order to prepare the uterus for implantation and pregnancy. Intrauterine infection and inflammation are being increasingly recognized as potential causes of implantation failure and miscarriage, as well as obstetric complications later in gestation. However, the mechanisms by which the cells of the endometrium respond to infection remain understudied and recent progress is slowed in part owing to similar overlapping studies being performed in different species. OBJECTIVE AND RATIONALE The aim of this scoping review is to systematically summarize all published studies in humans and laboratory animals that have investigated the innate immune sensing and response of the endometrium to bacteria and viruses, and the signaling mechanisms involved. This will enable gaps in our knowledge to be identified to inform future studies. SEARCH METHODS The Cochrane Library, Ovid Embase/Medline, PubMed, Scopus, Google Scholar, and Web of Science databases were searched using a combination of controlled and free text terms for uterus/endometrium, infections, and fertility to March 2022. All primary research papers that have reported on endometrial responses to bacterial and viral infections in the context of reproduction were included. To focus the scope of the current review, studies in domesticated animals, included bovine, porcine, caprine, feline, and canine species were excluded. OUTCOMES This search identified 42 728 studies for screening and 766 full-text studies were assessed for eligibility. Data was extracted from 76 studies. The majority of studies focused on endometrial responses to Escherichia coli and Chlamydia trachomatis , with some studies of Neisseria gonorrhea , Staphylococcus aureus , and the Streptococcus family. Endometrial responses have only been studied in response to three groups of viruses thus far: HIV, Zika virus, and the herpesvirus family. For most infections, both cellular and animal models have been utilized in vitro and in vivo , focusing on endometrial production of cytokines, chemokines, and antiviral/antimicrobial factors, and the expression of innate immune signaling pathway mediators after infection. This review has identified gaps for future research in the field as well as highlighted some recent developments in organoid systems and immune cell co-cultures that offer new avenues for studying endometrial responses to infection in more physiologically relevant models that could accelerate future findings in this area. WIDER IMPLICATIONS This scoping review provides an overarching summary and benchmark of the current state of research on endometrial innate immune responses to bacterial and viral infection. This review also highlights some exciting recent developments that enable future studies to be designed to deepen our understanding of the mechanisms utilized by the endometrium to respond to infection and their downstream effects on uterine function. [ABSTRACT FROM AUTHOR]

Macrophages are important antigen presenting cells which can release extracellular vesicles (EVs) carrying functional cargo including non‐coding RNAs. Macrophages can be broadly classified into M1 'classical' and M2 'alternatively‐activated' macrophages. M1 macrophages have been linked with inflammation‐associated pathologies, whereas a switch towards an M2 phenotype indicates resolution of inflammation and tissue regeneration. Here, we provide the first comprehensive analysis of the small RNA cargo of EVs from human M1 and M2 primary macrophages. Using small RNA sequencing, we identified several types of small non‐coding RNAs in M1 and M2 macrophage EVs including miRNAs, isomiRs, tRNA fragments, piRNA, snRNA, snoRNA and Y‐RNA fragments. Distinct differences were observed between M1 and M2 EVs, with higher relative abundance of miRNAs, and lower abundance of tRNA fragments in M1 compared to M2 EVs. MicroRNA‐target enrichment analysis identified several gene targets involved in gene expression and inflammatory signalling pathways. EVs were also enriched in tRNA fragments, primarily originating from the 5′ end or the internal region of the full length tRNAs, many of which were differentially abundant in M1 and M2 EVs. Similarly, several other small non‐coding RNAs, namely snRNAs, snoRNAs and Y‐RNA fragments, were differentially enriched in M1 and M2 EVs; we discuss their putative roles in macrophage EVs. In conclusion, we show that M1 and M2 macrophages release EVs with distinct RNA cargo, which has the potential to contribute to the unique effect of these cell subsets on their microenvironment. [ABSTRACT FROM AUTHOR]

Objective: Miscarriage affects 1 in 7 pregnancies, and antiphospholipid autoantibodies (aPLs) are one of the biggest risk factors for recurrent pregnancy loss. While aPLs target the endometrial stroma, little is known about their impact. Endometrial stromal cells (EnSCs) undergo decidualization each menstrual cycle, priming the uterus to receive implanting embryos. Thus, appropriate decidualization and EnSC function is key for establishment of a successful pregnancy. This study was undertaken to explore the effects of aPL on EnSC decidualization, senescence, and inflammation. Methods: EnSCs under decidualizing conditions were exposed to aPL or control IgG alone or in the presence of either a Toll‐like receptor 4 (TLR‐4) antagonist, a p38 MAPK inhibitor, a reactive oxygen species (ROS) inhibitor, low molecular weight heparin (LMWH), or acetyl salicylic acid. Secretion of decidualization markers and inflammatory interleukin‐8 were quantified by enzyme‐linked immunosorbent assay, and senescence‐associated β‐galactosidase activity was evaluated. In a mouse model of decidualization, aPL or control IgG was administered, and uterine expression levels of decidualization and inflammatory markers were quantified by real‐time quantitative polymerase chain reaction. Results: Antiphospholipid antibodies increased human EnSC decidualization, senescence, and inflammation. This phenotype was recapitulated in the mouse model. The decidualization and inflammatory responses were partially mediated by TLR‐4 and p38 MAPK, while the decidualization and senescence responses were ROS‐dependent. LMWH, commonly used to treat aPL‐positive women at risk of obstetric complications, reduced the ability of aPL to increase EnSC decidualization and inflammation. Conclusion: These findings shed new light on the pathogenesis of pregnancy complications in women with aPLs and underscore the benefit of heparin in preventing pregnancy loss in this high‐risk population. [ABSTRACT FROM AUTHOR]

Problem: Women with antiphospholipid antibodies (aPL) are at increased risk for pregnancy loss and preeclampsia. aPL target the trophoblast and induce a pro‐inflammatory, anti‐angiogenic and anti‐migratory profile. Since infection during pregnancy can increase the risk for preeclampsia, a viral infection could further increase this in women with aPL. The goal of this study was to characterize the effect of viral components on trophoblast responses to aPL. Method of study: A human first trimester trophoblast cell line was treated with or without aPL or control IgG in the presence of media, viral dsRNA or viral ssRNA. Supernatants were measured for inflammatory IL‐1β and IL‐8; inflammasome‐associated uric acid and caspase‐1 activity; and anti‐angiogenic sFlt‐1. Trophoblast migration was measured using a two‐chamber assay. Results: Viral dsRNA augmented aPL‐induced trophoblast caspase‐1 activity, and IL‐1β and IL‐8 secretion in an additive manner. Viral ssRNA inhibited aPL‐induced uric acid, IL‐1β and sFlt‐1 secretion, and further exacerbated aPL‐inhibition of trophoblast migration. Conclusion: While viral ssRNA may have some protective effects on aPL‐induced inflammation and anti‐angiogenic responses, viral dsRNA exacerbated aPL‐mediated inflammation and viral ssRNA further limited cell migration, which could prove detrimental to placentation. Thus, viral infections may contribute to adverse pregnancy outcomes in women with aPL. [ABSTRACT FROM AUTHOR]

Antiphospholipid syndrome (APS) is an autoimmune disease characterized by thrombosis and pregnancy morbidity (PM) obstetric events together with persistent high titers of circulating antiphospholipid antibodies (aPL). Several mechanisms that explain the development of thrombosis and PM in APS include the association of aPL with alterations in the coagulation cascade and inflammatory events. Other mechanisms disturbing cellular homeostases, such as mitochondrial dysfunction, autophagy, and cell proliferation, have been described in other autoimmune diseases. Therefore, the objective of this study was to investigate the impact of aPL from different patient populations on endothelial cell mitochondrial function, activation of the mammalian target of rapamycin (mTOR) and autophagy pathways, and cellular growth. Using an in vitro model, human umbilical vein endothelial cells (HUVECs) were treated with polyclonal immunoglobulin G (IgG) purified from the serum of women with both PM and vascular thrombosis (PM/VT), with VT only (VT), or with PM and non-criteria aPL (seronegative-obstetric APS, SN-OAPS). We included IgG from women with PM without aPL (PM/aPL-) and healthy women with previous uncomplicated pregnancies (normal human serum, NHS) as control groups. Mitochondrial function, mTOR activation, autophagy, and cell proliferation were evaluated by Western blotting, flow cytometry, and functional assays. IgG from women with PM/VT increased HUVEC mitochondrial hyperpolarization and activation of the mTOR and autophagic pathways, while IgG from patients with VT induced endothelial autophagy and cell proliferation in the absence of elevated mTOR activity or mitochondrial dysfunction. IgG from the SN-OAPS patient group had no effect on any of these HUVEC responses. In conclusion, aPL from women with PM and vascular events induce cellular stress evidenced by mitochondrial hyperpolarization and increased activation of the mTOR and autophagic pathways which may play a role in the pathogenesis of obstetric APS. [ABSTRACT FROM AUTHOR]

Pre-gestational diabetes is a risk factor for preeclampsia, a condition associated with inflammatory markers, a dysregulated angiogenic profile, and impaired placentation. Using an in vitro model, we previously reported that hyperglycemic levels of glucose induced a pro-inflammatory (IL-1β, IL-8, RANTES, GRO-α), anti-angiogenic (sFlt-1) and anti-migratory profile in a human trophoblast cell line. The IL-1β response to excess glucose was mediated by uric acid-induced activation of the NLRP3 inflammasome. Allopurinol is a xanthine oxidase inhibitor that inhibits uric acid and reactive oxygen species (ROS) production. Thus, we sought to test the effects of allopurinol on the IL-1β and other inflammatory, angiogenic and migratory responses that are triggered in the trophoblast by excess glucose. Under excess glucose conditions, allopurinol significantly inhibited trophoblast secretion of inflammatory IL-1β; caspase-1 activity; IL-8; RANTES; and GRO-α. Allopurinol also significantly inhibited excess glucose-induced trophoblast secretion of anti-angiogenic sFlt-1. The presence of IL1Ra significantly inhibited excess glucose-induced trophoblast IL-8 and GRO-α secretion but had no effect on RANTES or sFlt-1. Conversely, DPI, a ROS inhibitor, significantly inhibited excess glucose-induced trophoblast GRO-α and sFlt-1 secretion, but had no effect on IL-8 or RANTES. Together, our findings indicate that the xanthine oxidase inhibitor allopurinol inhibited excess glucose-induced trophoblast IL-1β secretion. Additionally, through its inhibition of both IL-1β and ROS production by the trophoblast, allopurinol reduced the additional pro-inflammatory and anti-angiogenic responses to excess glucose. Thus, allopurinol may be a candidate medication to prevent placental dysfunction and adverse pregnancy outcomes, such as preeclampsia, in pregnant women with diabetes. [ABSTRACT FROM AUTHOR]

The endothelium is a monolayer of cells that covers the inner surface of blood vessels and its integrity is essential for the maintenance of vascular health. Endothelial dysfunction is a key pathological component of antiphospholipid syndrome (APS). Its systemic complications include thrombotic endocarditis, valvular dysfunction, cerebrovascular occlusions, proliferative nephritis, deep vein thrombosis, and pulmonary embolism. In women, APS is also associated with pregnancy complications (obstetric APS). The conventional treatment regimens for APS are ineffective when the clinical symptoms are severe. Therefore, a better understanding of alterations in the endothelium caused by antiphospholipid antibodies (aPL) may lead to more effective therapies in patients with elevated aPL titers and severe clinical symptoms. Currently, while in vivo analyses of endothelial dysfunction in patients with APS have been reported, most research has been performed using in vitro models with endothelial cells exposed to either patient serum/plasma, monoclonal aPL, or IgGs isolated from patients with APS. These studies have described a reduction in endothelial cell nitric oxide synthesis, the induction of inflammatory and procoagulant phenotypes, an increase in endothelial proliferation, and impairments in vascular remodeling and angiogenesis. Despite these lines of evidence, further research is required to better understand the pathophysiology of endothelial dysfunction in patients with APS. In this review, we have compared the current understanding about the mechanisms of endothelial dysfunction induced by patient-derived aPL under the two main clinical manifestations of APS: thrombosis and gestational complications, either alone or in combination. We also discuss gaps in our current knowledge regarding aPL-induced endothelial dysfunction. [ABSTRACT FROM AUTHOR]

Objective: Progesterone supplementation prevents preterm birth (PTB) in some high-risk women, but its mechanism of action is unknown. One-third of PTB is associated with preterm premature rupture of membranes (PPROMs). We have previously shown that progesterone inhibits basal and Tumor Necrosis Factor (TNF) α-induced apoptosis in an explant model of human fetal membranes. This study investigates the molecular mechanisms responsible for progesterone-mediated inhibition of apoptosis in fetal membranes. Methods: Human fetal membranes were collected at elective cesarean at term (no labor, no infection; n = 6), washed, and pretreated with/without progesterone (125 ng/mL) for 24 hours. Thereafter, membranes were treated with/without TNFα (50 ng/mL) and/or progesterone for 48 hours, harvested, and homogenized. Apoptosis was determined by evaluating caspase-3, -8, and -9 activities. Expression of pro- BH3 interacting domain death against, Bc1-2 associated X protein (BID, BAX) and antiapoptotic proteins (X-linked inhibitor of apoptosis protein [XIAP], Bcl-2, FLICE inhibitory protein [FLIP]) were measured by Western blot. Results: TNFα increased apoptosis (measured by caspase-3, -8, and -9 activities) in fetal membranes, and this effect was abrogated by progesterone. Under basal conditions, progesterone suppressed expression of the proapoptotic protein, BID, by 0.45 (0.14)-fold, and increased expression of the antiapoptotic proteins, Bcl-2 and XIAP; no change was seen in BAX or FLIP. In contrast, TNFα increased BID expression by 5.15 (2.92)-fold, which was prevented by pretreatment with progesterone. Conclusions: Progesterone inhibits apoptosis in fetal membranes by suppressing expression of the proapoptotic protein, BID (for both basal and TNFα-induced apoptosis), and upregulating expression of the antiapoptotic proteins, XIAP and Bcl-2 (under basal conditions only). These data provide a mechanism by which progesterone supplementation may prevent PPROM and PTB in some women at high risk. [ABSTRACT FROM AUTHOR]

Abstract: Extracellular vesicles (EVs) extruded by the human placenta are increasingly being recognized as an essential mode of feto‐maternal communication. In the past two decades, there has been an explosion of research into the roles that placental EVs play in modulating the maternal immune and cardiovascular systems during healthy pregnancies, as well as how this communication is altered in obstetric diseases. This review aims to introduce readers to the processes of placental EV formation and the cargos they carry, and also to collate and summarize the published literature that investigates the immunological effects of placental EVs throughout human pregnancy. [ABSTRACT FROM AUTHOR]

Problem: Chorioamnionitis and infection‐associated inflammation are major causes of preterm birth. Magnesium sulfate (MgSO4) is widely used in obstetrics as a tocolytic; however, its mechanism of action is unclear. This study sought to investigate how MgSO4 modulates infection‐associated inflammation in fetal membranes (FMs), and whether the response was time dependent. Method of Study: Human FM explants were treated with or without bacterial lipopolysaccharide (LPS); with or without MgSO4 added either: 1 hour before LPS; at the same time as LPS; 1 hour post‐LPS; or 2 hours post‐LPS. Explants were also treated with or without viral dsRNA and LPS, alone or in combination; and MgSO4 added 1 hour post‐LPS After 24 hours, supernatants were measured for cytokines/chemokines; and tissue lysates measured for caspase‐1 activity. Results: Lipopolysaccharide‐induced FM inflammation by upregulating the secretion of a number of inflammatory cytokines/chemokines. Magnesium sulfate administered 1‐hour post‐LPS inhibited FM secretion of IL‐1β, IL‐6, G‐CSF, RANTES, and TNFα. Magnesium sulfate administered 2 hours post‐LPS augmented FM secretion of these factors as well as IL‐8, IFNγ, VEGF, GROα and IP‐10. Magnesium sulfate delivered 1‐ hour post‐LPS inhibited LPS‐induced caspase‐1 activity, and inhibited the augmented IL‐1β response triggered by combination viral dsRNA and LPS. Conclusion: Magnesium sulfate differentially modulates LPS‐induced FM inflammation in a time‐dependent manner, in part through its modulation of caspase‐1 activity. Thus, the timing of MgSO4 administration may be critical in optimizing its anti‐inflammatory effects in the clinical setting. MgSO4 might also be useful at preventing FM inflammation triggered by a polymicrobial viral‐bacterial infection. [ABSTRACT FROM AUTHOR]

Problem: Inflammasomes are signaling platforms that, upon sensing pathogens and sterile stressors, mediate the release of mature forms of interleukin (IL)‐1β and IL‐18. The aims of this study were to determine (i) the expression of major inflammasome components in the chorioamniotic membranes in spontaneous labor at term, (ii) whether there are changes in the inflammasome components associated with the activation of caspase‐1 and caspase‐4, and (iii) whether these events are associated with the release of the mature forms of IL‐1β and IL‐18. Method of study: Chorioamniotic membranes were collected from women at term with and without spontaneous labor. mRNA abundance and protein concentrations of inflammasome components, nucleotide‐binding oligomerization domain‐containing (NOD)1 and NOD2 proteins, caspase‐1, caspase‐4, IL‐1β, and IL‐18 were quantified by qRT‐PCR (n = 28–29 each), ELISA (n = 10 each) or immunoblotting (n = 8 each), and immunohistochemistry (n = 10 each). Active caspase‐1 and caspase‐4, as well as mature IL‐18, were determined by immunoblotting (n = 4 each), and pro‐ and mature forms of IL‐1β were determined by ELISA (n = 4–7 each). Results: Inflammasome components and NOD proteins were expressed in the chorioamniotic membranes obtained from women at term. The chorioamniotic membranes from women who underwent labor had (i) higher concentrations of NLRP3 (NOD‐like receptor family, pyrin domain‐containing protein 3) and NOD1 protein, (ii) greater immunoreactivity for caspase‐1 and caspase‐4, (iii) a greater quantity of the active form of caspase‐1 (p20), and (iv) higher mRNA abundance and protein concentrations of pro‐ and mature IL‐1β. However, mRNA abundance and protein concentrations of the mature form of IL‐18 were not increased in tissues from women who underwent labor at term. Conclusions: Spontaneous labor at term is characterized by the expression of inflammasome components, which may participate in the activation of caspase‐1 and lead to the cleavage and release of mature IL‐1β by the chorioamniotic membranes. These results support the participation of the inflammasome in the mechanisms responsible for spontaneous parturition at term. [ABSTRACT FROM AUTHOR]

Objective: Women with antiphospholipid antibodies (aPL) are at risk for pregnancy complications associated with poor placentation and placental inflammation. Although these antibodies are heterogeneous, some anti–β2‐glycoprotein I (anti‐β2GPI) antibodies can activate Toll‐like receptor 4 (TLR‐4) and NLRP3 in human first‐trimester trophoblasts. The objective of this study was to determine the role of negative regulators of TLR and inflammasome function in aPL‐induced trophoblast inflammation. Methods: Human trophoblasts were not treated or were treated with anti‐β2GPI aPL or control IgG in the presence or absence of the common TAM (TYRO3, AXL, and Mer tyrosine kinase [MERTK]) receptor ligand growth arrest–specific protein 6 (GAS6) or the autophagy‐inducer rapamycin. The expression and function of the TAM receptor pathway and autophagy were measured by quantitative reverse transcription–polymerase chain reaction (qRT‐PCR), Western blotting, and enzyme‐linked immunosorbent assay (ELISA). Antiphospholipid antibody–induced trophoblast inflammation was measured by qRT‐PCR, activity assays, and ELISA. Results: Anti‐β2GPI aPL inhibited trophoblast TAM receptor function by reducing cellular expression of the receptor tyrosine kinases AXL and MERTK and the ligand GAS6. The addition of GAS6 blocked the effects of aPL on the TLR‐4–mediated interleukin‐8 (IL‐8) response. However, the NLRP3 inflammasome‐mediated IL‐1β response was not affected by GAS6, suggesting that another regulatory pathway was involved. Indeed, anti‐β2GPI aPL inhibited basal trophoblast autophagy, and reversing this with rapamycin inhibited aPL‐induced inflammasome function and IL‐1β secretion. Conclusion: Basal TAM receptor function and autophagy may serve to inhibit trophoblast TLR and inflammasome function, respectively. Impairment of TAM receptor signaling and autophagy by anti‐β2GPI aPL may allow subsequent TLR and inflammasome activity, leading to a robust inflammatory response. [ABSTRACT FROM AUTHOR]

Problem Women with antiphospholipid antibodies ( aPL) are at risk for pregnancy complications despite treatment with low molecular weight heparin ( LMWH) or aspirin ( ASA). aPL recognizing beta2 glycoprotein I can target the uterine endothelium, however, little is known about its response to aPL. This study characterized the effect of aPL on human endometrial endothelial cells ( HEECs), and the influence of LMWH and ASA. Method of study HEECs were exposed to aPL or control IgG, with or without low-dose LMWH and ASA, alone or in combination. Chemokine and angiogenic factor secretion were measured by ELISA. A tube formation assay was used to measure angiogenesis. Results aPL increased HEEC secretion of pro-angiogenic VEGF and Pl GF; increased anti-angiogenic sFlt-1; inhibited basal secretion of the chemokines MCP-1, G- CSF, and GRO-α; and impaired angiogenesis. LMWH and ASA, alone and in combination, exacerbated the aPL-induced changes in the HEEC angiogenic factor and chemokine profile. There was no reversal of the aPL inhibition of HEEC angiogenesis by either single or combination therapy. Conclusion By aPL inhibiting HEEC chemokine secretion and promoting sFlt-1 release, the uterine endothelium may contribute to impaired placentation and vascular transformation. LMWH and ASA may further contribute to endothelium dysfunction in women with obstetric APS. [ABSTRACT FROM AUTHOR]

Inflammasomes are cytosolic multiprotein complexes that orchestrate inflammation in response to pathogens and endogenous danger signals. Herein, we determined whether the chorioamniotic membranes from women in spontaneous preterm labor with acute histologic chorioamnionitis (1) express major inflammasome components; (2) express caspase (CASP)-1 and CASP-4 as well as their active forms; (3) exhibit apoptosis-associated speck-like protein containing a CARD (ASC)/CASP-1 complex formation; and (4) release the mature forms of interleukin (IL)-1β and IL-18. We utilized quantitative reverse transcription polymerase chain reaction, enzyme-linked immunosorbent assay, immunoblotting, and immunohistochemistry to determine the messenger RNA (mRNA) and protein expression of major inflammasome components, nucleotide-binding oligomerization domain (NOD) proteins, and the pro- and mature/active forms of CASP-1, CASP-4, IL-1β, and IL-18. The ASC/CASP-1 complex formation was determined using an in situ proximity ligation assay. When comparing the chorioamniotic membranes from women in spontaneous preterm labor with acute histologic chorioamnionitis to those without this placental lesion, we found that (1) the mRNA of NLR family pyrin domain-containing protein (NLRP)1, NLRP3, NLR family CARD domain-containing protein 4 (NLRC4), and NOD2 were higher; (2) the NLRP3 protein was increased; (3) the mRNA and active form (p10) of CASP-1 were greater; (4) the mRNA and active form of CASP-4 were increased; (5) the mRNA and mature form of IL-1β were higher; (6) the mature form of IL-18 was elevated; and (7) ASC/CASP-1 complex formation was increased. In conclusion, spontaneous preterm labor with acute histologic chorioamnionitis is characterized by an upregulation of NLRP3 and the active form of CASP-4, as well as increased ASC/CASP-1 complex formation, which may participate in the activation of CASP-1 and the maturation of IL-1β and IL-18 in the chorioamniotic membranes. These findings provide the first evidence that supports a role for the inflammasome in the pathological inflammation implicated in spontaneous preterm labor with acute histologic chorioamnionitis. [ABSTRACT FROM AUTHOR]

The article discusses adverse pregnancy outcomes in patients with antiphospholipid syndrome (APS); and therapeutic options to prevent obstetric APS. Topics include abnormal placental development induced by antiphospholipid antibodies (aPL), which can reduce proliferation and invasion of extravillous trophoblasts; mouse models of pathogenic mechanisms of pregnancy complications; biomarkers like antiangiogenic factors for poor pregnancy outcomes; and therapies like antithrombotic treatment.

Inflammasomes are cytosolic signaling platforms that regulate the activation of caspase (CASP)-1, which induces the maturation of interleukin (IL)-1β and IL-18. Herein, we determined whether the chorioamniotic membranes from women in spontaneous labor at term with acute histologic chorioamnionitis express major inflammasome components and whether these changes are associated with the activation of CASP-1 and CASP-4 and the release of mature IL-1β and IL-18. When comparing the chorioamniotic membranes from women in spontaneous labor at term with acute histologic chorioamnionitis to those without this placental lesion, we found that (1) the messenger RNA (mRNA) abundance of NLR family pyrin domain containing 3 (NLRP3), NLR family CARD domain containing 4 (NLRC4), absent in melanoma 2 (AIM2), and nucleotide binding oligomerization domain 2 (NOD2) was higher; (2) the NLRP3 and NLRC4 protein quantities were increased; (3) the mRNA and protein expressions of CASP-1 and its active forms were greater; (4) CASP-4 was increased at the mRNA level only; (5) the mRNA and protein expressions of IL-1β and its mature form were higher; and (6) a modest increase in the total protein concentration and abundance of the mature form of IL-18 was observed. In vitro incubation of the chorioamniotic membranes with the CASP-1 inhibitor, VX765, decreased the release of endotoxin-induced IL-1β and IL-18 (2-fold) but not IL-6 or tumor necrosis factor α. In conclusion, spontaneous labor at term with acute histologic chorioamnionitis is characterized by an upregulation of inflammasome components which, in turn, may participate in the activation of CASP-1 and lead to the release of mature IL-1β by the chorioamniotic membranes. These results support a role for the inflammasome in the mechanisms responsible for spontaneous labor at term with acute histologic chorioamnionitis. [ABSTRACT FROM AUTHOR]

Problem Inflammasome activation requires two steps: priming and assembly of the multimeric complex. The second step includes assembly of the sensor molecule and adaptor protein ASC (an apoptosis-associated speck-like protein containing a CARD), which results in ASC speck formation and the recruitment of caspase ( CASP)-1. Herein, we investigated whether there is inflammasome assembly in the chorioamniotic membranes and choriodecidual leukocytes from women who underwent spontaneous labor at term. Method of Study Using in situ proximity ligation assays, ASC/ CASP-1 complexes were determined in the chorioamniotic membranes from women who delivered at term without labor or underwent spontaneous labor at term with or without acute histologic chorioamnionitis (n=10-11 each). Also, ASC speck formation was determined by flow cytometry in the choriodecidual leukocytes isolated from women who delivered at term with or without spontaneous labor (n=9-12 each). Results (i) ASC/ CASP-1 complexes were detected in the chorioamniotic membranes; (ii) ASC/ CASP-1 complexes were greater in the chorioamniotic membranes from women who underwent spontaneous labor at term than in those without labor; (iii) ASC/ CASP-1 complexes were even more abundant in the chorioamniotic membranes from women who underwent spontaneous labor at term with acute histologic chorioamnionitis than in those without this placental lesion; (iv) ASC speck formation was detected in the choriodecidual leukocytes; and (v) ASC speck formation was greater in the choriodecidual leukocytes isolated from women who underwent spontaneous labor at term than in those without labor. Conclusion There is inflammasome assembly in the chorioamniotic membranes and choriodecidual leukocytes during spontaneous labor at term. [ABSTRACT FROM AUTHOR]

Recent studies have linked antenatal infection with Zika virus ( ZIKV) with major adverse fetal and neonatal outcomes, including microcephaly. There is a growing consensus for the existence of a congenital Zika syndrome ( CZS). Previous studies have indicated that non-placental macrophages play a key role in the replication of dengue virus ( DENV), a closely related flavivirus. As the placenta provides the conduit for vertical transmission of certain viruses, and placental Hofbauer cells ( HBCs) are fetal-placental macrophages located adjacent to fetal capillaries, it is not surprising that several recent studies have examined infection of HBCs by ZIKV. In this review, we describe congenital abnormalities associated with ZIKV infection, the role of HBCs in the placental response to infection, and evidence for the susceptibility of HBCs to ZIKV infection. We conclude that HBCs may contribute to the spread of ZIKV in placenta and promote vertical transmission of ZIKV, ultimately compromising fetal and neonatal development and function. Current evidence strongly suggests that further studies are warranted to dissect the specific molecular mechanism through which ZIKV infects HBCs and its potential impact on the development of CZS. [ABSTRACT FROM AUTHOR]

Problem Risk factors for preterm birth include placental abruption, giving rise to excessive decidual thrombin, and intrauterine bacterial infection. Human endometrial endothelial cells (HEECs) express Toll-like receptors (TLRs), and infection-derived agonists trigger HEECs to generate specific inflammatory responses. As thrombin, in addition to inducing coagulation, can contribute to inflammation, its effect on HEEC inflammatory responses to the TLR4 agonist, bacterial lipopolysaccharide (LPS), was investigated. Method of study HEECs were pre-treated with or without thrombin or specific protease-activated receptor (PAR) agonists, followed by treatment with or without LPS. Supernatants were measured for cytokines and chemokines by ELISA and multiplex analysis. Results Thrombin significantly and synergistically augmented LPS-induced HEEC secretion of interleukin (IL)-6, IL-8, granulocyte colony-stimulating factor (G-CSF), and growth-regulated oncogene-alpha (GRO-α), and significantly augmented monocyte chemotactic protein (MCP)-1, tumor necrosis factor-alpha (TNF-α), and vascular endothelial growth factor (VEGF) secretion additively. Similar to thrombin, a PAR1 agonist synergistically augmented the LPS-induced HEEC secretion of inflammatory IL-6, IL-8, G-CSF, and GRO-α. Conclusion Thrombin, via PAR1 activation, synergistically augments LPS-induced HEEC production of chemokines involved in immune cell recruitment and survival, suggesting a mechanism by which intrauterine abruption and bacterial infection may together be associated with an aggravated uterine inflammatory response. [ABSTRACT FROM AUTHOR]

Anti-phospholipid antibodies (aPL) are autoantibodies that are associated with thrombosis and a range of pregnancy complications including recurrent pregnancy loss and pre-eclampsia. The three clinically relevant, well-characterized aPL are anti-cardiolipin antibodies, lupus anticoagulant and anti-beta-2-glycoprotein I (β2GPI) antibodies. aPL do not bind directly to phospholipids but instead bind to a plasma-binding 'cofactor'. The most extensively studied cofactor is β2GPI, whose role in pregnancy is not fully elucidated. Although the pathogenicity of aPL in recurrent pregnancy loss is well established in humans and animal models, the association of aPL with infertility does not appear to be causative. aPL may exert their detrimental effects during pregnancy by directly binding trophoblast cells of the placenta, altering trophoblast signalling, proliferation, invasion and secretion of hormones and cytokines, and by increasing apoptosis. Heparin is commonly used to treat pregnant women with aPL; however, as thrombotic events do not occur in the placentae of all women with aPL, it may exert a protective effect by preventing the binding of aPL to β2GPI or by acting through non-thrombotic pathways. The aim of this review is to present evidence summarizing the current understanding of this field. [ABSTRACT FROM AUTHOR]

Objective Pregnancies in women with the antiphospholipid syndrome (APS) are frequently complicated by fetal loss and intrauterine growth restriction (IUGR). How circulating antiphospholipid antibodies (aPL) cause pregnancy complications in APS is poorly understood. We sought to determine whether the low-density lipoprotein receptor family member apolipoprotein E receptor 2 (ApoER2) mediates trophoblast dysfunction and pregnancy complications induced by aPL. Methods Placental and trophoblast ApoER2 expression was evaluated by immunohistochemistry and immunoblotting. Normal human IgG and aPL were purified from healthy individuals and APS patients, respectively. The role of ApoER2 in aPL-induced changes in trophoblast proliferation and migration and in kinase activation was assessed using RNA interference in HTR-8/SVneo cells. The participation of ApoER2 in aPL-induced pregnancy loss and IUGR was evaluated in pregnant ApoER2+/+ and ApoER2-/- mice injected with aPL or normal human IgG. Results We found that ApoER2 is abundant in human and mouse placental trophoblasts and in multiple trophoblast-derived cell lines, including HTR-8/SVneo cells. ApoER2 and its interaction with the cell surface protein β2-glycoprotein I were required for aPL-induced inhibition of cultured trophoblast proliferation and migration. In parallel, aPL antagonism of Akt kinase activation by epidermal growth factor in trophoblasts was mediated by ApoER2. Furthermore, in a murine passive-transfer model of pregnancy complications of APS, ApoER2-/- mice were protected from both aPL-induced fetal loss and aPL-induced IUGR. Conclusion ApoER2 plays a major role in the attenuation of trophoblast function by aPL, and the receptor mediates aPL-induced pregnancy complications in vivo in mice. ApoER2-directed interventions can now potentially be developed to combat the pregnancy complications associated with APS. [ABSTRACT FROM AUTHOR]

Problem Thrombophilia is associated with pregnancy complications. Treatment with low molecular weight heparin ( LMWH) improves pregnancy outcome, but the underlying mechanisms are not clear. Methods of study We analyzed Treg frequency in blood from thrombophilic pregnancies treated with LMWH ( n = 32) or untreated ( n = 33) and from healthy pregnancies ( n = 39) at all trimesters. Additionally, we treated pregnant wild-type, heterozygous and homozygous factor- V- Leiden ( FVL) mice with LMWH or PBS and determined Treg frequency, pro-/anti-inflammatory cytokine levels and Caspase-3-activity in placenta and decidua. Results Treg frequencies were increased in second and third trimester in LMWH-treated thrombophilic pregnancies compared to controls. Treg levels were comparable to those of normal pregnancies. Homozygous FVL mice had decreased decidual Tregs compared to wild-type mice. LMWH treatment normalized Tregs and was associated with increased decidual IL-10 m RNA. LMWH diminished Caspase-3-activity in mice of all genotypes. Conclusion We demonstrated anti-apoptotic and anti-inflammatory effects of LMWH in pregnant FVL mice. LMWH increased Treg levels in mice and humans, which suggests benefits of LMWH treatment for thrombophilic women during pregnancy. [ABSTRACT FROM AUTHOR]

Problem Diabetes confers an increased risk of preeclampsia, but its pathogenic role in preeclampsia is poorly understood. The objective of this study was to elucidate the effects of excess glucose on trophoblast function and whether any changes could be reversed by metformin. Method of study The human first trimester trophoblast cell line (Sw.71) was treated with glucose at 5, 10, 25, and 50 m m, in the presence and absence of metformin. Trophoblast migration was quantified and supernatant cytokine, chemokine, and angiogenic factors measured. Results Increasing concentrations of glucose significantly increased trophoblast secretion of the inflammatory cytokines/chemokines: IL-1β, IL-6, IL-8, GRO-α, RANTES, and G-CSF; significantly increased trophoblast secretion of the anti-angiogenic factors sFlt-1 and sEndoglin; and significantly decreased trophoblast migration. Excess glucose-induced trophoblast IL-1β production was inhibited by disabling the Nalp3/ASC inflammasome. Metformin partially reduced the glucose-induced inflammatory response, but had no effect on the anti-angiogenic or antimigratory response. Conclusion Excess glucose induced a pro-inflammatory, anti-angiogenic, and antimigratory state in first trimester trophoblast cells. Glucose-induced trophoblast IL-1β secretion was mediated by the inflammasome. Glucose-induced inflammation was partially reversed by metformin. These findings demonstrate the pleiotropic effects of hyperglycaemia on the trophoblast, providing potential explanations for the strong link between diabetes and preeclampsia. [ABSTRACT FROM AUTHOR]

Objective To investigate whether an elevated interferon-α (IFNα) level early in pregnancy is associated with poor pregnancy outcomes and to examine the relationship of an elevated IFNα level to angiogenic imbalance. Methods Women were enrolled in a longitudinal case-control study of pregnant patients with lupus. Serum samples obtained monthly throughout pregnancy were assayed for IFNα and for the antiangiogenic factor soluble Flt-1 and the proangiogenic factor placenta growth factor (PlGF). Each of 28 patients with systemic lupus erythematosus (SLE) with a poor pregnancy outcome was matched to an SLE patient with an uncomplicated pregnancy and to a pregnant healthy control. The effects of IFNα and/or soluble Flt-1 on human endothelial cells and endothelial cell-trophoblast interactions were assessed. Results Compared to SLE patients with uncomplicated pregnancies, patients with preeclampsia had increased IFNα levels before clinical symptoms. Patients without autoimmune disease who developed preeclampsia did not have increased IFNα levels. In SLE patients with low IFNα levels, marked angiogenic imbalance (higher soluble Flt-1, lower PlGF, and higher soluble Flt-1:PlGF ratios) preceded maternal manifestations of preeclampsia, whereas in SLE patients with high IFNα levels, preeclampsia occurred without evidence of systemic angiogenic imbalance. Treatment of human endothelial cells with soluble Flt-1 induced expression of sFLT1 messenger RNA, and IFNα dramatically amplified responses to soluble Flt-1. In a model of spiral artery transformation, only the combination of IFNα and soluble Flt-1 disrupted the ability of trophoblast cells to remodel endothelial tube structures. Conclusion Our findings identify a new mechanism by which IFNα induces an antiangiogenic milieu and increases the sensitivity of endothelial cells to soluble Flt-1, and suggest that elevated IFNα levels may contribute to the pathogenesis of preeclampsia in some pregnant patients with SLE. [ABSTRACT FROM AUTHOR]

Problem Women with antiphospholipid syndrome ( APS) are at increased risk of recurrent pregnancy loss ( RPL) and preeclampsia. Antiphospholipid antibodies (a PL) directly alter trophoblast function. Treatment with low molecular weight heparin ( LMWH) reduces the risk of RPL but not preeclampsia. Moreover, LMWH stimulates trophoblast sFlt-1 release, an anti-angiogenic factor associated with preeclampsia. Since vitamin D deficiency is associated with APS and preeclampsia, this study sought to determine the effect of vitamin D on trophoblast function in the setting of a PL and LMWH. Method of study A human first trimester trophoblast cell line ( HTR8) and primary trophoblast cultures were treated with or without a PL in the presence and absence of vitamin D, LMWH or both. Trophoblast secretion of inflammatory cytokines and angiogenic factors were measured by ELISA. Results Vitamin D alone or in combination with LMWH attenuated the aPL-induced trophoblast inflammatory response in the HTR8 cells and primary cultures. While vitamin D did not have any impact on a PL-mediated modulation of angiogenic factors in the primary trophoblast, it significantly inhibited LMWH-induced s Flt-1 release. Conclusion LMWH in combination with vitamin D may be more beneficial than single-agent therapy by preventing a PL-induced trophoblast inflammation and reversing LMWH-induced s Flt-1 secretion. [ABSTRACT FROM AUTHOR]

Objective Antiphospholipid antibodies (aPL) interfere with several physiologic functions of human trophoblasts, including reducing their ability to migrate, decreasing their production of angiogenic factors, and inducing an inflammatory response. This may provide the underlying mechanism by which aPL responses lead to recurrent pregnancy loss or preeclampsia in women with obstetric antiphospholipid syndrome (APS). Although treatment with heparin may reduce the rate of recurrent pregnancy loss, the risk of preeclampsia remains high. Therefore, alternative treatments are needed for the management of pregnant patients with APS. Since aspirin-triggered lipoxins (ATLs) have immune and angiogenic modulatory properties, the objective of this study was to determine the effects of the ATL 15-epi-lipoxin A4 on the function of aPL-altered human trophoblasts in the first trimester of pregnancy. Methods A first-trimester human trophoblast cell line (HTR8) was treated with mouse anti-human β2-glycoprotein I monoclonal antibodies (aPL) in the presence or absence of the ATL 15-epi-lipoxin A4. Trophoblast migration and interactions with endometrial endothelial cells were measured using Transwell and coculture assays. Trophoblast secretion of cytokines and angiogenic factors was measured by enzyme-linked immunosorbent assay. Results Treatment of HTR8 cells with ATL reversed the aPL-induced decrease in trophoblast migration, an effect that appeared to be regulated through restoration of interleukin-6 production. Using a model of spiral artery transformation, aPL and sera from APS patients with pregnancy morbidity disrupted trophoblast-endothelial cell interactions, and treatment with ATL restored the stability of the cocultures. In contrast, ATL treatment did not resolve the proinflammatory and antiangiogenic responses of trophoblasts induced by aPL. Conclusion These findings indicate that ATLs may have some benefits in terms of preventing the effects of aPL on trophoblast function, which raises the possibility of the use of ATLs as an adjuvant therapy in women with aPL. [ABSTRACT FROM AUTHOR]

Problem Microbial-driven responses in placenta are linked with adverse pregnancy outcomes. The role of Toll-like receptor ( TLR) function in Hofbauer cells ( HBCs) and fetal macrophages of the placental villous core remains understudied. Method of Study Flow cytometry and immunohistochemistry (IHC) were used to establish the phenotype of HBCs. Regulation of cytokine secretion in response to treatment with TLR agonists and expression levels of TLRs and co-activators were compared in HBCs, placental fibroblasts (FIBs), and human umbilical vein endothelial cells (HUVECs) using ELISA and qPCR. Results Although flow cytometry and IHC revealed HBCs to be M2 (anti-inflammatory) macrophages, LPS and polyinosinic: polycytidylic acid [poly (I:C)] treatments markedly enhanced IL-6 secretion by HBCs, and expression of TLR-2, TLR-3, TLR-4, CD14, and MD-2 was the highest in HBCs. Conclusion These results indicate that although HBCs are M2 macrophages, inflammatory responses are induced through TLR-3 and TLR-4 in this cell type, suggesting a role in microbial-driven placental/fetal inflammation. [ABSTRACT FROM AUTHOR]

Problem Inflammation during pregnancy has devastating consequences for the placenta and fetus. These events are incompletely understood, thereby hampering screening and treatment. Method of study The inflammatory profile of villous tissue was studied in pregnancies at high-risk of placental dysfunction and compared to uncomplicated pregnancies. The systemic inflammatory profile was assessed in matched maternal serum samples in cases of reduced fetal movements ( RFM). Results Placentas from RFM pregnancies had a unique inflammatory profile characterized by increased interleukin ( IL)-1 receptor antagonist and decreased IL-10 expression, concomitant with increased numbers of placental macrophages. This aberrant cytokine profile was evident in maternal serum in RFM, as were increased levels of alarmins (uric acid, HMGB1, cell-free fetal DNA). Conclusion This distinct inflammatory profile at the maternal-fetal interface, mirrored in maternal serum, could represent biomarkers of placental inflammation and could offer novel therapeutic options to protect the placenta and fetus from an adverse maternal environment. [ABSTRACT FROM AUTHOR]

Problem Women with antiphospholipid syndrome ( APS) are at risk for pregnancy complications. Antiphospholipid antibodies (a PL) alter trophoblast function by triggering an inflammatory cytokine response; modulating angiogenic factor secretion; and inhibiting migration. While patients with APS are often treated with hydroxychloroquine ( HCQ), its effect on trophoblast function is poorly understood. Method of study A human first trimester trophoblast cell line was treated with or without antihuman β2 GPI m Abs in the presence or absence of HCQ. Supernatants were analyzed by ELISA. Cell migration was measured using a colormetric assay. Results Antiphospholipid antibodies-induced trophoblast IL-8, IL-1 β, PlGF, and s Endoglin secretion were not altered by HCQ. a PL-induced inhibition of trophoblast migration was partially reversed by HCQ, even though HCQ significantly increased secretion of pro-migratory IL-6 to greater than baseline. a PL-induced upregulation of TIMP2 appears to inhibit trophoblast migration; the inability of HCQ to prevent a PL-induced TIMP2 may explain why migration was only partially restored. Conclusion Hydroxychloroquine reversed the a PL-inhibition of trophoblast IL-6 secretion and partially limited a PL-inhibition of cell migration. Thus, some form of combination therapy that includes HCQ may be beneficial to pregnant APS patients. [ABSTRACT FROM AUTHOR]

Problem Human endometrial endothelial cell ( HEEC) innate immunity remains poorly characterized. Based on their direct contact with the circulation, HEECs are uniquely positioned to be exposed to viral infections. This study evaluated the innate immune response generated by HEECs after exposure to the TLR3 agonist, Poly(I:C) and the TLR8 agonist, viral ss RNA. Method of Study HEECs were treated with or without Poly(I:C) or ss RNA. Culture supernatants were measured for cytokines by multiplex analysis. RNA was analyzed by qRT- PCR for type I interferons and antiviral factors. Results Treatment of HEECs with Poly(I:C) rapidly upregulated the secretion of IL-2, IL-6, IL-8, IFN-γ, G- CSF, GM- CSF, MCP-1, MIP-1β, RANTES, and GRO-α after 12 hr, while ss RNA treatment induced the slower secretion of IL-6, IL-8, IFN-γ, G- CSF, VEGF, and GRO-α after 24 hr. Both viral components induced HEEC IFN-α and IFN-β expression. While treatment with Poly(I:C) induced APOBEC3G and OAS expression, treatment with ssRNA upregulated APOBEC3 G and M×A mRNA. Conclusion Our findings demonstrate that HEECs can differentially sense and respond to viral components by generating distinct inflammatory and antiviral immune responses, indicating that these cells likely play an active role in the immune protection of the uterus toward viral infections. [ABSTRACT FROM AUTHOR]

Women with antiphospholipid syndrome (APS) are at risk of recurrent pregnancy loss and obstetrical disorders, such as preeclampsia and intrauterine growth restriction (IUGR). Antiphospholipid antibodies (aPL) directly target the placenta by binding beta2-glycoprotein I (β2GPI) expressed on the trophoblast. We recently demonstrated in human first trimester trophoblast cells that anti-β2GPI antibodies (Abs) induce the secretion of IL-1β in a Toll-like receptor 4 (TLR4)-dependent manner. IL-1β secretion requires processing of pro-IL-1β and this is mediated by the inflammasome, a complex of Nalp3, apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1. The objective of this study was to determine if aPL induce IL-1β production in trophoblast via the inflammasome. Using a human first trimester trophoblast cell line, we demonstrated that a mouse anti-β2GPI mAb and human polyclonal aPL-IgG induce IL-1β processing and secretion, which was partially blocked upon caspase-1 inhibition. Nalp3 and ASC knockdown also attenuated anti-β2GPI Ab-induced IL-1β secretion. Furthermore, aPL stimulated the production of uric acid in a TLR4-dependent manner; and inhibition of uric acid prevented aPL-induced IL-1β production by the trophoblast. These findings demonstrate that aPL, via TLR4 activation, induce a uric acid response in human trophoblast, which in turn activates the Nalp3/ASC inflammasome leading to IL-1β processing and secretion. This novel mechanism may account for the inflammation at the maternal-fetal interface, which causes placental dysfunction and increases the risk of adverse pregnancy outcome in patients with APS. [ABSTRACT FROM AUTHOR]

Problem There is a strong correlation between intrauterine bacterial infection and preterm labor. While inflammation is a common mechanism, certain pathogens may trigger placental apoptosis. TLR2 activation by gram-positive bacterial peptidoglycan ( PDG) induces first-trimester trophoblast apoptosis and decreased IL-6 secretion. This is dependent upon the presence of TLR1 and the absence of TLR6 and both TLR2 coreceptors. As TLR10 is also a TLR2 coreceptor, the objective of this study was to determine its expression and function in the trophoblast. Method of study First-and third-trimester human placental tissue and isolated trophoblast were evaluated for TLR10 expression. A first-trimester human trophoblast cell line stably transfected with a TLR10 dominant negative ( TLR10- DN) or vector control was treated with or without PDG and analyzed for apoptosis and IL-6. Results TLR10 was expressed by trophoblasts during the first and third trimesters of pregnancy. PDG-induced trophoblast caspase-3 activity was inhibited by the presence of the TLR10- DN. The presence of the TLR10- DN had no effect on PDG reduction in trophoblast IL-6 secretion. Conclusion This study demonstrates that trophoblast TLR10 plays a role in promoting apoptosis triggered by gram-positive bacterial components and suggests that TLR10 may regulate the balance between trophoblast survival and cell death. [ABSTRACT FROM AUTHOR]

Problem Preterm premature rupture of fetal membranes ( pPROM) occurs in 30-40% of spontaneous preterm births ( PTB) and is associated with intra-amniotic infection and inflammation. The membranes may sense and respond to microbes via Toll-like receptors ( TLRs); however, little is known about their expression and regulation in this tissue. The objective of this study was to evaluate the expression of TLRs 1-10 in fetal membranes after exposure to pathogens associated with intra-amniotic infection and PTB. Method of study Normal human term fetal membrane explants were exposed to various bacteria. After 24 hrs, RNA was extracted and quantitative RT- PCR performed for TLRs1-10. Results Treatment of fetal membranes with Mycoplasma hominis increased expression of TLR4, TLR6, and TLR8 mRNA. Ureaplasma parvum upregulated TLR8 mRNA, and Porphyromonas gingivalis significantly increased fetal membrane TLR7 expression. In contrast, treatment with Gram-negative Escherichia coli (and its cell wall component lipopolysaccharide) downregulated TLR10 mRNA. No effect was detected for Ureaplasma urealyticum, Gardnerella vaginalis, or Group B Streptococcus. Conclusion These findings demonstrate that different types of bacteria have distinct effects on fetal membrane TLR expression patterns. Moreover, these findings highlight the disparity of fetal membrane responses to infection and thus suggest heterogeneity in the mechanisms by which infection-associated pregnancy complications, such as pPROM and PTB, arise. [ABSTRACT FROM AUTHOR]

STUDY QUESTION What is the effect of pravastatin on antiphospholipid antibody (aPL) modulation of human first trimester trophoblast function? SUMMARY ANSWER Pravastatin does not prevent the effects of aPL on human first trimester trophoblast cell function. WHAT IS KNOWN ALREADY Antiphospholipid syndrome (APS) is associated with recurrent pregnancy loss and late pregnancy complications, such as pre-eclampsia, owing to direct targeting of the placenta by aPL. While treatment with heparin reduces the rate of pregnancy loss, the risk for severe pre-eclampsia remains high. Thus, there is a need to find alternative treatments for the prenatal management of patients with APS. Statins have recently been shown to prevent aPL-mediated fetal loss in mice but their effects on a human pregnancy model of APS have not yet been studied. DESIGN, DATA COLLECTION, METHODS The human first trimester trophoblast cell line, HTR8, and human first trimester trophoblast primary cultures were incubated with or without a mouse anti-human beta 2 glycoprotein I (β2GPI) monoclonal antibody in the presence or absence of pravastatin. Cytokine and angiogenic factor secretion were measured by enzyme-linked immunosorbent assay and multiplex analysis. Cell migration was measured using a colorimetric two-chamber migration assay. MAIN FINDINGS Using the human first trimester trophoblast cell line, HTR8, pravastatin significantly augmented, compared with no treatment, aPL-dependent secretion of interleukin (IL)-8 (P< 0.05), IL-1β (P< 0.05) and soluble endoglin (P< 0.01) but had no effect on aPL-induced up-regulation of vascular endothelial growth factor, placenta growth factor or growth-related oncogene alpha secretion. Furthermore, pravastatin alone limited basal HTR8 cell migration (P< 0.01), and did not mitigate the adverse effect of aPL on trophoblast migration. Pravastatin also had no impact on the secretion of pro-inflammatory cytokines and angiogenic factors by primary human first trimester trophoblast cells exposed to aPL. LIMITATIONS AND WIDER IMPLICATIONS OF THE FINDINGS While our in vitro findings suggest that pravastatin may not be effective in preventing pregnancy complications in patients with APS, the in vivo condition may be more complex, and thus, more studies are needed to determine the effectiveness of pravastatin in the prevention of aPL-associated pregnancy complications in humans. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the American Heart Association. [ABSTRACT FROM AUTHOR]

Problem Uterine innate immunity remains poorly characterized, and while endometrial endothelial cells are known to express Toll-like receptors ( TLRs), little is known about their function in these cells. The present study evaluated the effect of Gram-negative bacterial lipopolysaccharide ( LPS) on human endometrial endothelial cell ( HEECs) cytokine secretion and tissue factor expression, and the role of TLR-4 in these responses. Methods Human endometrial endothelial cells were treated with or without LPS ± LPS- RS, a TLR-4 antagonist, via the binding of MD-2. After 24 hr, cell-free supernatants were evaluated for cytokines by multiplex analysis and cell lysates were analyzed for tissue factor expression by Western blot. Results Treatment of HEECs with LPS significantly upregulated the secretion of IL-6, IL-8, and G- CSF, and this was prevented by LPS- RS. LPS also induced tissue factor expression by the HEECs; however, this was unaffected by LPS- RS. Conclusion These findings suggest that TLR-4 is functional in HEECs and its activation by bacterial LPS induces a specific cytokine/chemokine response. However, bacterial LPS also induced tissue factor expression in what seemed to be a TLR-4-independent fashion, suggesting that this bacterial component can act on the HEECs through TLR-4-dependent and TLR-4-independent pathways. These findings indicate that endometrial endothelial cells may play an active role in uterine innate immunity. [ABSTRACT FROM AUTHOR]

Problem Toll-like receptor-4 ( TLR-4) protects against Gram-negative bacteria expressed lipopolysaccharide and 'danger signals' from injured or dying cells. Although decidual cells ( DCs) and interstitial trophoblasts ( ITs) are in close contact, TLR-4 has been studied extensively only in ITs. Method of study Formalin-fixed, paraffin-embedded serial sections of endometrium in follicular and luteal phases and deciduas from first and second trimester elective terminations and third trimester normal deliveries were immunostained for TLR-4, trophoblast-specific cytokeratin, and DC-specific vimentin. HSCORE assessed TLR-4 immunostaining in DCs versus ITs. Results TLR-4 HSCORES were significantly higher in: (i) first trimester DCs than luteal phase pre-decidual stromal cells; (ii) first and third versus second trimester DCs, but similar between third trimester deciduas parietalis and basalis; (iii) first versus second trimester ITs; (iv) DCs versus ITs across gestation. Conclusion Higher TLR-4 in DCs than ITs suggests DCs as primary targets for Gram-negative bacteria and/or inflammation-related danger signals. [ABSTRACT FROM AUTHOR]

Envelope glycoproteins of human endogenous retrovirus (HERV), such as syncytin 1 (HERV-W), are highly expressed in the placenta and some family members have immunomodulatory properties. Placental microvesicles (MV), which are shed into the maternal circulation during pregnancy, have been demonstrated to induce immune cell activation. Therefore, the aim of this study was to investigate the immunological properties of the highly expressed placental HERV-W protein, syncytin 1, and its potential involvement in placental MV modulation of immune cell activity. The MV shed from first trimester, normal term and pre-eclamptic term placentas, and from the BeWo trophoblast cell line, all contain syncytin 1. Recombinant syncytin 1 and syncytin 1-positive BeWo trophoblast MV both induced peripheral blood mononuclear cell (PBMC) activation, indicated through production of cytokines and chemokines. Reducing syncytin 1 content in BeWo MV inhibited PBMC activation. Recombinant syncytin 1 and syncytin-1-positive BeWo MV dampened PBMC responses to lipopolysaccharide challenge. Our findings suggest that syncytin 1 is shed from the placenta into the maternal circulation in association with MV, and modulates immune cell activation and the responses of immune cells to subsequent lipopolysaccharide stimulation. These studies implicate placental MV-associated HERV in fetal regulation of the maternal immune system. [ABSTRACT FROM AUTHOR]

Citation Carroll TY, Mulla MJ, Han CS, Brosens JJ, Chamley LW, Giles I, Pericleous C, Rahman A, Sfakianaki AK, Paidas MJ, Abrahams VM. Modulation of trophoblast angiogenic factor secretion by antiphospholipid antibodies is not reversed by heparin. Am J Reprod Immunol 2011; 66: 286-296 Problem Women with antiphospholipid antibodies (aPL) are at risk of miscarriage and pre-eclampsia, obstetrical disorders associated with reduced trophoblast invasion and spiral artery transformation. aPL target the placenta by binding beta2-glycoprotein I (β2GPI) on the trophoblast. In this study, we determined whether aPL alter the trophoblast secretion of angiogenic factors and evaluated the effect of low molecular weight heparin (LMWH) on this response. Method of study First-trimester trophoblast was treated with anti-β2GPI antibodies with or without LMWH. Angiogenic factor secretion was measured by enzyme-linked immunosorbent assay. Results Trophoblast cells produced more vascular endothelial growth factor (VEGF), placenta growth factor (PlGF), and soluble endoglin following exposure to anti-β2GPI Abs, and this occurred in both a MyD88-dependent and MyD88-independent manner. LMWH was unable to reverse the effects of the anti-β2GPI Abs on trophoblast VEGF secretion, but enhanced PlGF. Strikingly, LMWH upregulated soluble fms-like tyrosine kinase receptor-1 (sFlt-1) secretion independently of aPL. Conclusion This study demonstrates that aPL perturb the secretion of trophoblast angiogenic factors. LMWH does not reverse this effect but exacerbates sFlt-1 secretion, a potent anti-angiogenic factor. These findings may help to explain why women with antiphospholipid syndrome, who are treated with heparin to prevent early pregnancy loss, remain at increased risk of developing late obstetrical complications, such as pre-eclampsia. [ABSTRACT FROM AUTHOR]

Citation Tang Z, Tadesse S, Norwitz E, Mor G, Abrahams VM., Guller S. Isolation of Hofbauer cells from human term placentas with high yield and purity. Am J Reprod Immunol 2011; 66: 336-348 Problem Placental villus macrophages (i.e., Hofbauer cells, HBCs) were identified more than 100 years ago. Alterations in their numbers and characteristics are associated with several complications of pregnancy. Although HBCs have previously been isolated and cultured, there is no consensus methodology to obtain these cells with high yield and purity for in vitro studies. Method of study Hofbauer cells were isolated from human term placentas using protocols in which cytotrophoblasts (CTs) and fibroblasts (FIBs), other major villous cell types, were isolated in parallel. Enzymatic digestion, Percoll gradients, and immunoselection were used to isolate the three cell types. Purity was assessed by morphology, flow cytometry, and phagocytosis assays. Results Hofbauer cells were isolated with 98-99% purity and a yield of 130-200 × 106 cells/80-100 g of tissue. HBCs exhibited a pleiomorphic and vacuolated appearance for at least 5 days in culture medium with and without serum. High levels of phagocytosis in HBCs, but not in CTs or FIBs, confirmed macrophage function in HBCs. Phagocytotic activity was maintained across several days in culture. Conclusion Hofbauer cells were isolated from term placenta with high yield and purity using protocols in which CTs and FIBs were also obtained. This methodology will foster future studies that examine the role of HBCs in regulating villus function. [ABSTRACT FROM AUTHOR]

Citation Rose JA, Rabenold JJ, Parast MM, Milstone DS, Abrahams VM, Riley JK. Peptidoglycan induces necrosis and regulates cytokine production in murine trophoblast stem cells. Am J Reprod Immunol 2011; 66: 209-222 Problem Intrauterine bacterial infection during pregnancy may lead to adverse outcome. The objective of this study was to assess whether peptidoglycan (PGN) derived from Gram-positive bacteria induces trophoblast stem (TS) cell death or alters TS cell cytokine production. Method of study Toll-like receptor (TLR) transcript expression was assessed by RT-PCR. Protein expression was determined by confocal microscopy or flow cytometry. 7-Aminoactinomycin D (7-AAD) staining was used to assess TS cell death. Morphological features of cell death were evaluated by transmission electron microscopy. The presence of cleaved caspase-3 and high mobility group box 1 (HMGB1) protein was examined by Western blot. Cytokine levels in cell supernatants were determined using a mouse cytokine 23-plex panel. Results Toll-like receptor 2 and TLR4 protein was expressed from the 1-cell stage through the blastocyst stage of murine embryo development. Murine TS cells expressed TLR2 and TLR6 but not TLR1 or TLR4 RNA. Only TLR2 protein was detected at the plasma membrane of TS cells. PGN induced TS cell death by a caspase-3-independent mechanism. The cell death pathway induced by PGN was morphologically consistent with necrosis. Finally, PGN induced HMGB1 release and increased MIP-1β secretion while inhibiting the constitutive release of RANTES. Conclusion Peptidoglycan-induced TS cell necrosis and the subsequent release of HMGB1 and MIP-1β may regulate an infection-induced inflammatory response at the maternal-fetal interface and thus may play a role in the pathogenesis of infection-associated pregnancy complications. [ABSTRACT FROM AUTHOR]

Mulla MJ, Myrtolli K, Potter J, Boeras C, Kavathas PB, Sfakianaki AK, Tadesse S, Norwitz ER, Guller S, Abrahams VM. Uric acid induces trophoblast IL-1β production via the inflammasome: implications for the pathogenesis of preeclampsia. Am J Reprod Immunol 2010; 65: 542-548 Preeclampsia is associated with hyperuricemia, which correlates with the disease severity. Levels of circulating uric acid increase before the clinical manifestations, suggesting that they may be causally related. Uric acid, or monosodium urate (MSU), activates the Nod-like receptor, Nalp3, leading to inflammasome activation and IL-1β processing. Because preeclampsia is associated with placental immune/inflammatory dysregulation, we sought to determine in the trophoblast, the presence of the Nalp3 inflammasome, and the effect of MSU on its activation. Isolated first- and third-trimester trophoblasts were assessed for expression of the inflammasome components, Nalp1, Nalp3, and ASC. First-trimester trophoblast cells were incubated with or without MSU, and after which, IL-1β secretion and processing and caspase-1 activation were determined. Trophoblast cells expressed Nalp1, Nalp3, and ASC under basal conditions. Following incubation with MSU, first-trimester trophoblast IL-1β secretion was upregulated. This correlated with increased expression levels of active IL-1β and active caspase-1. ASC knockdown reduced MSU-induced IL-1β secretion. These findings demonstrate that uric acid activates the inflammasome in the trophoblast, leading to IL-1β production. This may provide a novel mechanism for the induction of inflammation at the maternal-fetal interface leading to placental dysfunction and adverse pregnancy outcome, including preeclampsia. [ABSTRACT FROM AUTHOR]

Toti P, Arcuri F, Tang Z, Schatz F, Zambrano E, Mor G, Niven-Fairchild T, Abrahams VM, Krikun G, Lockwood CJ, Guller S. Focal increases of fetal macrophages in placentas from pregnancies with histological chorioamnionitis: potential role of fibroblast monocyte chemotactic protein-1. Am J Reprod Immunol 2011; 65: 470-479 Histopathological chorioamnionitis (HCA) is caused by microbial-driven infiltration of leukocytes to the maternal-fetal interface resulting in adverse neonatal outcomes in a subset of pregnancies. The role of placental villus macrophages (i.e. Hofbauer cells, HBCs) in the pathophysiology of HCA is unelucidated. The number of HBCs in human term placental villi in HCA and control groups was compared using immunohistochemistry. Levels of monocyte chemotactic protein (MCP-1) expression were measured in primary cultures of syncytioytrophoblasts (SCTs) and fibroblasts (FIBs) treated with bacterial compounds [lipopolysaccharide (LPS) and peptidoglycan] and pro-inflammatory cytokines (TNF-α and IL-1β) using ELISA and quantitative real-time PCR. Immunohistochemistry revealed a focal increase in HBCs in HCA. Treatment of FIBs with LPS, IL-1β, and TNF-α significantly increased MCP-1 mRNA and protein expression. Conversely, MCP-1 mRNA and protein levels were virtually undetectable in treated and untreated SCTs. These results demonstrate cell-type-specific regulation of MCP-1 expression in human placenta. A model is presented in which bacterial products and inflammatory cytokines initiate a fibroblast-driven cytokine cascade resulting in recruitment of fetal monocytes to placenta which focally increases levels of HBCs in pregnancies complicated by HCA. [ABSTRACT FROM AUTHOR]