Protease inhibitor-based antiretroviral therapy in pregnancy: effects on hormones, placenta, and decidua.

The use of antiretroviral therapy (ART) in pregnancy is important for maternal health, and has been successful in reducing vertical transmission rates to almost zero in those taking effective ART regimens with good adherence. However, there are reports of higher rates of low birthweight and preterm births in women with HIV, which can be further exacerbated by ART usage in pregnancy. Protease inhibitors, and ritonavir-boosted lopinavir in particular, might directly contribute to placental and uteroplacental pathology in part by altering plasma concentrations of the essential steroid hormones of pregnancy, progesterone and oestradiol. In this Review, we collate the increasing evidence of dysregulated maternal endocrinology, reproductive physiology, and placental compromise associated with protease inhibitors. Based on findings of placental and decidual effects, we recommend that ritonavir-boosted lopinavir should be avoided in pregnancy, in line with US and European guidelines. Long-term follow-up of children exposed to protease inhibitors in utero is also recommended.

Functional Evaluation of STOX1 (STORKHEAD-BOX PROTEIN 1) in Placentation, Preeclampsia, and Preterm Birth.

Revaluation of the association of the STOX1 (STORKHEAD_BOX1 PROTEIN 1) transcription factor mutation (Y153H, C allele) with the early utero-vascular origins of placental pathology is warranted. To investigate if placental STOX1 Y153H genotype affects utero-vascular remodeling-compromised in both preterm birth and preeclampsia-we utilized extravillous trophoblast (EVT) explant and placental decidual coculture models, transfection of STOX1 wild-type and mutant plasmids into EVT-like trophoblast cell lines, and a cohort of 75 placentas from obstetric pathologies. Primary EVT and HTR8/SVneo cells carrying STOX1 Y153H secreted lower levels of IL (interleukin) 6, and IL-8, and higher CXCL16 (chemokine [C-X-C motif] ligand 16) and TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) than wild-type EVT and Swan71 cells. Media from wild-type EVT or Swan71 cells transfected with wild-type STOX1 stimulated: endothelial chemokine expression, angiogenesis, and decidual natural killer cell and monocyte migration. In contrast, Y153H EVT conditioned medium, Swan71 transfected with the Y153H plasmid, or HTR8/SVneo media had no effect. Genotyping of placental decidual cocultures demonstrated association of the placental STOX1 CC allele with failed vascular remodeling. Decidual GG NODAL R165H increased in failed cocultures carrying the placental CC alleles of STOX1. Multivariate analysis of the placental cohort showed that the STOX1 C allele correlated with premature birth, with or without severe early-onset preeclampsia, and small for gestational age babies. In conclusion, placental STOX1 Y153H is a precipitating factor in preterm birth and placental preeclampsia due to defects in early utero-placental development.

Periconceptional exposure to lopinavir, but not darunavir, impairs decidualization: a potential mechanism leading to poor birth outcomes in HIV-positive pregnancies.

STUDY QUESTION: Does HIV protease inhibitor (PI)-based combination antiretroviral therapy (cART) initiated at periconception affect key events in early pregnancy, i.e. decidualization and spiral artery remodeling? SUMMARY ANSWER: Two PIs, lopinavir and darunavir, currently offered as cART options in HIV-positive pregnancies were evaluated, and we found that lopinavir-based cART, but not darunavir-based cART, impaired uterine decidualization and spiral artery remodeling in both human ex vivo and mouse in vivo experimental models. WHAT IS KNOWN ALREADY: Early initiation of cART is recommended for pregnant women living with HIV. However, poor birth outcomes are frequently observed in HIV-positive pregnancies exposed to PI-based cART, especially when it is initiated prior to conception. The correlation between early initiation of PI-cART and adverse birth outcomes is poorly understood, due to lack of data on the specific effects of PI-cART on the early stages of pregnancy involving uterine decidualization and spiral artery remodeling. STUDY DESIGN, SIZE, DURATION: Lopinavir and darunavir were evaluated in clinically relevant combinations using an ex vivo human first-trimester placenta-decidua explant model, an in vitro human primary decidual cell culture system, and an in vivo mouse pregnancy model. The first-trimester (gestational age, 6-8 weeks) human placenta-decidua tissue was obtained from 11 to 15 healthy women undergoing elective termination of pregnancy. C57Bl/6 female mice (four/treatment group) were administered either lopinavir-cART, darunavir-cART or water by oral gavage once daily starting on the day of plug detection until sacrifice. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human: Spiral artery remodeling was assessed by immunohistochemical analysis of first-trimester placenta-decidua explant co-culture system. Trophoblast migration was measured using a placental explant culture. A primary decidual cell culture was used to evaluate the viability of immune cell populations by flow cytometry. Soluble factors, including biomarkers of decidualization and angiogenesis, were quantified by ELISA and Luminex assay using decidua-conditioned media. Mouse: In the mouse pregnancy model, gestational day 6.5 or 9.5 implantation sites were used to assess decidualization, spiral artery remodeling and uterine natural killer (uNK) cell numbers by immunohistochemistry. Transcription factor STAT3 was assayed by immunohistochemistry in both human decidua and mouse implantation sites. MAIN RESULTS AND THE ROLE OF CHANCE: Lopinavir-cART, but not darunavir-cART, impaired uterine decidualization and spiral artery remodeling in both experimental models. Lopinavir-cART treatment was also associated with selective depletion of uNK cells, reduced trophoblast migration and defective placentation. The lopinavir-associated decidualization defects were attributed to a decrease in expression of transcription factor STAT3, known to regulate decidualization. Our results suggest that periconceptional initiation of lopinavir-cART, but not darunavir-cART, causes defective maturation of the uterine endometrium, leading to impairments in spiral artery remodeling and placentation, thus contributing to the poor birth outcomes. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The human first-trimester placenta/decidua samples could only be obtained from healthy females undergoing elective termination of pregnancy. As biopsy is the only way to obtain first-trimester decidua from pregnant women living with HIV on PI-cART, ethics approval and participant consent are difficult to obtain. Furthermore, our animal model is limited to the study of cART and does not include HIV. HIV infection is also associated with immune dysregulation, inflammation, alterations in angiogenic factors and complement activation, all of which could influence decidual and placental vascular remodeling and modify any cART effects. WIDER IMPLICATIONS OF THE FINDINGS: Our findings provide mechanistic insight with direct clinical implications, rationalizing why the highest adverse birth outcomes are reported in HIV-positive pregnancies exposed to lopinavir-cART from conception. We demonstrate that dysregulation of decidualization is the mechanism through which lopinavir-cART, but not darunavir-cART, use in early pregnancy leads to poor birth outcomes. Although lopinavir is no longer a first-line regimen in pregnancy, it remains an alternate regimen and is often the only PI available in low resource settings. Our results highlight the need for reconsidering current guidelines recommending lopinavir use in pregnancy and indicate that lopinavir should be avoided especially in the first trimester, whereas darunavir is safe to use and should be the preferred PI in pregnancy.Further, in current times of the COVID-19 pandemic, lopinavir is among the top drug candidates which are being repurposed for inclusion in clinical trials world-over, to assess their therapeutic potential against the dangerous respiratory disease. Current trials are also testing the efficacy of lopinavir given prophylactically to protect health care workers and people with potential exposures. Given the current extraordinary numbers, these might include women with early pregnancies, who may or may not be cognizant of their gestational status. This is a matter of concern as it could mean that women with early pregnancies might be exposed to this drug, which can cause decidualization defects. Our findings provide evidence of safety concerns surrounding lopinavir use in pregnancy, that women of reproductive age considering participation in such trials should be made aware of, so they can make a fully informed decision. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by funding from the Canadian Institutes of Health Research (CIHR) (PJT-148684 and MOP-130398 to L.S.). C.D. received support from CIHR Foundation (FDN143262 to Stephen Lye). S.K. received a TGHRI postdoctoral fellowship. The authors declare that there are no conflicts of interest. L.S. reports personal fees from ViiV Healthcare for participation in a Women and Transgender Think Tank.

Myometrial activation: Novel concepts underlying labor.

Term labour is a state of physiological inflammation orchestrated by multiple uterine tissues (both fetal and maternal). This physiological inflammation preceding and accompanying labour onset is characterized by an increase in cytokine and chemokine secretion by the fetal membranes, as well as uterine tissues (i.e., decidua and myometrium). Pro-inflammatory cytokines and chemokines activate circulating maternal peripheral leukocytes as well as the uterine vascular endothelium to permit leukocyte infiltration into the uterus. This inflammatory milieu, in the absence of infection, is required for the initiation of labour as the uterine-infiltrated leukocytes secrete matrix metalloproteinases to induce fetal membrane rupture and cervical ripening as well as various labour mediators, which promote contractions of the myometrium. Myometrial activation at term and the onset of labour contractions are directly related to the changes in the ovarian/placental hormone progesterone and its downstream mediators (i.e., the progesterone receptors, PRA/B), which are also critical for maintenance of pregnancy. Our recent data provides direct evidence in support of local and functional P4 withdrawal in the uterine muscle (myometrium) via the activator protein-1 (AP-1) mediated pathway. This review outlines known mechanisms regulating activation of human labour, including progesterone and cytokine signaling. Understanding of the molecular mechanism of myometrial activation and labour onset could facilitate the development of new therapeutics for high-risk pregnant women to prevent premature uterine activation and preterm birth.

Gestational age-dependent gene expression profiling of ATP-binding cassette transporters in the healthy human placenta.

The ATP-binding cassette (ABC) transporters control placental transfer of several nutrients, steroids, immunological factors, chemicals, and drugs at the maternal-fetal interface. We and others have demonstrated a gestational age-dependent expression pattern of two ABC transporters, P-glycoprotein and breast cancer resistance protein throughout pregnancy. However, no reports have comprehensively elucidated the expression pattern of all 50 ABC proteins, comparing first trimester and term human placentae. We hypothesized that placental ABC transporters are expressed in a gestational-age dependent manner in normal human pregnancy. Using the TaqMan® Human ABC Transporter Array, we assessed the mRNA expression of all 50 ABC transporters in first (first trimester, n = 8) and third trimester (term, n = 12) human placentae and validated the resulting expression of selected ABC transporters using qPCR, Western blot and immunohistochemistry. A distinct gene expression profile of 30 ABC transporters was observed comparing first trimester vs. term placentae. Using individual qPCR in selected genes, we validated the increased expression of ABCA1 (P < 0.01), ABCA6 (P < 0.001), ABCA9 (P < 0.001) and ABCC3 (P < 0.001), as well as the decreased expression of ABCB11 (P < 0.001) and ABCG4 (P < 0.01) with advancing gestation. One important lipid transporter, ABCA6, was selected to correlate protein abundance and characterize tissue localization. ABCA6 exhibited increased protein expression towards term and was predominantly localized to syncytiotrophoblast cells. In conclusion, expression patterns of placental ABC transporters change as a function of gestational age. These changes are likely fundamental to a healthy pregnancy given the critical role that these transporters play in the regulation of steroidogenesis, immunological responses, and placental barrier function and integrity.

Decidual leucocytes infiltrating human spiral arterioles are rich source of matrix metalloproteinases and degrade extracellular matrix in vitro and in situ.

PROBLEM: During pregnancy, the decidual spiral arterioles (SpAs) that supply maternal blood to the placenta undergo a series of changes to optimise the transfer of nutrients and oxygen to the developing foetus. Recent studies have shown that initiation of SpA transformation coincides with decidual leucocyte infiltration. Leucocytes are known to be a source of matrix metalloproteinases (MMPs); however, the complete profile of MMPs expressed by decidual NK cells (dNK) and macrophages has not been characterised. We hypothesised that leucocyte-derived MMPs contribute to SpA remodelling. METHODS: Decidual NK cells and macrophages were isolated from first trimester decidua and their MMP repertoire profiled by qRT-PCR (n = 10; 5-11 weeks). Dual immunofluorescence was used to localise MMP expression in situ (n = 3; 5-12 weeks). Gelatin zymography was carried out to assess whether leucocyte-derived MMPs can degrade ECM. In situ zymography and immunofluorescence identified MMP activity in tissue-resident dNK and macrophages. RESULTS: Decidual NK cells cells and macrophages expressed MMP2, -7, -9, -11, -16, -19 and tissue inhibitors of metalloproteinase-1, -2, and -3. Both cell types degraded gelatin using MMP2 and MMP9 and broke down collagen in an in vitro model of the SpA. Extravillous trophoblasts (EVTs) expressed a similar repertoire of MMPs. CONCLUSION: We suggest that matrix remodelling in SpA is initiated by infiltrating leucocytes, while EVTs become involved at later stages.

MicroRNA-218-5p Promotes Endovascular Trophoblast Differentiation and Spiral Artery Remodeling.

Preeclampsia (PE) is the leading cause of maternal and neonatal morbidity and mortality. Defects in trophoblast invasion, differentiation of endovascular extravillous trophoblasts (enEVTs), and spiral artery remodeling are key factors in PE development. There are no markers clinically available to predict PE, leaving expedited delivery as the only effective therapy. Dysregulation of miRNA in clinical tissues and maternal circulation have opened a new avenue for biomarker discovery. In this study, we investigated the role of miR-218-5p in PE development. miR-218-5p was highly expressed in EVTs and significantly downregulated in PE placentas. Using first-trimester trophoblast cell lines and human placental explants, we found that miR-218-5p overexpression promoted, whereas anti-miR-218-5p suppressed, trophoblast invasion, EVT outgrowth, and enEVT differentiation. Furthermore, miR-218-5p accelerated spiral artery remodeling in a decidua-placenta co-culture. The effect of miR-218-5p was mediated by the suppression of transforming growth factor (TGF)-ß2 signaling. Silencing of TGFB2 mimicked, whereas treatment with TGF-ß2 partially reversed, the effects of miR-218-5p. Taken together, these findings demonstrate that miR-218-5p promotes trophoblast invasion and enEVT differentiation through a novel miR-218-5p-TGF-ß2 pathway. This study elucidates the role of an miRNA in enEVT differentiation and spiral artery remodeling and suggests that downregulation of miR-218-5p contributes to PE development.

HIV antiretroviral exposure in pregnancy induces detrimental placenta vascular changes that are rescued by progesterone supplementation.

Adverse birth outcomes are common in HIV-positive pregnant women receiving combination antiretroviral therapy (cART), especially when cART is initiated in early pregnancy. The mechanisms remain poorly understood. Using a mouse model we demonstrate that protease inhibitor based-cART exposure beginning on day 1 of pregnancy was associated with a pro-angiogenic/pro-branching shift in the placenta driven by lower Flt-1 levels and higher Gcm-1 expression. Micro-CT imaging revealed an increase in the number of arterioles in cART-treated placentas, which correlated with fetal growth restriction. Delaying initiation of cART, or supplementing cART-treated mice with progesterone, prevented the pro-angiogenic/pro-branching shift and the associated placenta vascular changes. In agreement with our mouse findings, we observed an increase in the number of terminal-villi capillaries in placentas from HIV-positive cART-exposed women compared to HIV-negative controls. Capillary number was inversely correlated to maternal progesterone levels. Our study provides evidence that cART exposure during pregnancy influences placenta vascular formation that may in turn contribute to fetal growth restriction. Our findings highlight the need for closer investigation of the placenta in HIV-positive pregnancies, particularly for pregnancies exposed to cART from conception, and suggest that progesterone supplementation could be investigated as a possible intervention to improve placenta function in HIV-positive pregnant women.

Chorioamnionitis Induces a Specific Signature of Placental ABC Transporters Associated with an Increase of miR-331-5p in the Human Preterm Placenta.

BACKGROUND/AIMS: The ATP-binding cassette (ABC) transporters mediate drug biodisposition and immunological responses in the placental barrier. In vitro infective challenges alter expression of specific placental ABC transporters. We hypothesized that chorioamnionitis induces a distinct pattern of ABC transporter expression. METHODS: Gene expression of 50 ABC transporters was assessed using TaqMan® Human ABC Transporter Array, in preterm human placentas without (PTD; n=6) or with histological chorioamnionitis (PTDC; n=6). Validation was performed using qPCR, immunohistochemistry and Western blot. MicroRNAs known to regulate P-glycoprotein (P-gp) were examined by qPCR. RESULTS: Up-regulation of ABCB9, ABCC2 and ABCF2 mRNA was detected in chorioamnionitis (p<0.05), whereas placental ABCB1 (P-gp; p=0.051) and ABCG2 (breast cancer resistance protein-BCRP) mRNA levels (p=0.055) approached near significant up-regulation. In most cases, the magnitude of the effect significantly correlated to the severity of inflammation. Upon validation, increased placental ABCB1 and ABCG2 mRNA levels (p<0.05) were observed. At the level of immunohistochemistry, while BCRP was increased (p<0.05), P-gp staining intensity was significantly decreased (p<0.05) in PTDC. miR-331-5p, involved in P-gp suppression, was upregulated in PTDC (p<0.01) and correlated to the grade of chorioamnionitis (p<0.01). CONCLUSIONS: Alterations in the expression of ABC transporters will likely lead to modified transport of clinically relevant compounds at the inflamed placenta. A better understanding of the potential role of these transporters in the events surrounding PTD may also enable new strategies to be developed for prevention and treatment of PTD.

Extravillous Trophoblast and Endothelial Cell Crosstalk Mediates Leukocyte Infiltration to the Early Remodeling Decidual Spiral Arteriole Wall.

Decidual spiral arteriole (SpA) remodeling is essential to ensure optimal uteroplacental blood flow during human pregnancy, yet very little is known about the regulatory mechanisms. Uterine decidual NK (dNK) cells and macrophages infiltrate the SpAs and are proposed to initiate remodeling before colonization by extravillous trophoblasts (EVTs); however, the trigger for their infiltration is unknown. Using human first trimester placenta, decidua, primary dNK cells, and macrophages, we tested the hypothesis that EVTs activate SpA endothelial cells to secrete chemokines that have the potential to recruit maternal immune cells into SpAs. Gene array, real-time PCR, and ELISA analyses showed that treatment of endothelial cells with EVT conditioned medium significantly increased production of two chemokines, CCL14 and CXCL6. CCL14 induced chemotaxis of both dNK cells and decidual macrophages, whereas CXCL6 also induced dNK cell migration. Analysis of the decidua basalis from early pregnancy demonstrated expression of CCL14 and CXCL6 by endothelial cells in remodeling SpAs, and their cognate receptors are present in both dNK cells and macrophages. Neutralization studies identified IL-6 and CXCL8 as factors secreted by EVTs that induce endothelial cell CCL14 and CXCL6 expression. This study has identified intricate crosstalk between EVTs, SpA cells, and decidual immune cells that governs their recruitment to SpAs in the early stages of remodeling and has identified potential key candidate factors involved. This provides a new understanding of the interactions between maternal and fetal cells during early placentation and highlights novel avenues for research to understand defective SpA remodeling and consequent pregnancy pathology.

Human dNK cell function is differentially regulated by extrinsic cellular engagement and intrinsic activating receptors in first and second trimester pregnancy.

Decidual natural killer (dNK) cells express an array of activation receptors to regulate placental immunity and development during early pregnancy. We investigated the functional character of human dNK cells during the first and second trimester of gestation and the interaction between dNK and trophoblast cells. Although the frequency of CD56+CD16- dNK among the total CD45+ leukocytes did not change over this period, the expression of the activating receptors, NKp80 and NKG2D, was greatly upregulated. We observed a significantly higher number of extravillous trophoblast cells in proximity to the dNK cells in the first trimester in comparison with the second trimester decidua. NKG2D expression by first trimester dNK cells was decreased when co-cultured with the HTR-8 trophoblast cell line. In the second trimester, functional markers of dNK activation, i.e., angiogenic factor production (e.g., vascular endothelial growth factor, interleukin-8, interferon-gamma), remained stable despite an increase in NKp80 or NKG2D surface expression. Furthermore, the degranulation capacity of dNK cells, as assessed by CD107a, was decreased in the second trimester. We suggest that in the first trimester, trophoblast-dNK interactions generate a population of dNK cells with a suppressed activating phenotype. In the second trimester, the loss of trophoblast-dNK interactions led to the inhibition of dNK cell function, although their activating receptor expression was increased. We speculate that during pregnancy, two mechanisms operate to modulate the dNK cell activation:suppression of activating receptor levels in the first trimester by trophoblasts and disengagement of receptor-ligand coupling in the second trimester.

Identification of a novel neutrophil population: proangiogenic granulocytes in second-trimester human decidua.

The maternal leukocytes of the first-trimester decidua play a fundamental role in implantation and early development of the fetus and placenta, yet little is known regarding the second-trimester decidual environment. Our multicolor flow cytometric analyses of human decidual leukocytes detected an elevation in tissue resident neutrophils in the second trimester. These cells in both human and murine samples were spatially restricted to decidua basalis. In comparison with peripheral blood neutrophils (PMNs), the decidual neutrophils expressed high levels of neutrophil activation markers and the angiogenesis-related proteins: vascular endothelial growth factor-A, Arginase-1, and CCL2, similarly shown in tumor-associated neutrophils. Functional in vitro assays showed that second-trimester human decidua conditioned medium stimulated transendothelial PMN invasion, upregulated VEGFA, ARG1, CCL2, and ICAM1 mRNA levels, and increased PMN-driven in vitro angiogenesis in a CXCL8-dependent manner. This study identified a novel neutrophil population with a physiological, angiogenic role in human decidua.

Preeclampsia is associated with alterations in the p53-pathway in villous trophoblast.

BACKGROUND: Preeclampsia (PE) is characterized by exaggerated apoptosis of the villous trophoblast of placental villi. Since p53 is a critical regulator of apoptosis we hypothesized that excessive apoptosis in PE is mediated by abnormal expression of proteins participating in the p53 pathway and that modulation of the p53 pathway alters trophoblast apoptosis in vitro. METHODS: Fresh placental villous tissue was collected from normal pregnancies and pregnancies complicated by PE; Western blotting and real-time PCR were performed on tissue lysate for protein and mRNA expression of p53 and downstream effector proteins, p21, Bax and caspases 3 and 8. To further assess the ability of p53 to modulate apoptosis within trophoblast, BeWo cells and placental villous tissue were exposed to the p53-activator, Nutlin-3, alone or in combination with the p53-inhibitor, Pifithrin-α (PFT-α). Equally, Mdm2 was knocked-down with siRNA. RESULTS: Protein expression of p53, p21 and Bax was significantly increased in pregnancies complicated by PE. Conversely, Mdm2 protein levels were significantly depleted in PE; immunohistochemistry showed these changes to be confined to trophoblast. Reduction in the negative feedback of p53 by Mdm2, using siRNA and Nutlin-3, caused an imbalance between p53 and Mdm2 that triggered apoptosis in term villous explants. In the case of Nutlin, this was attenuated by Pifithrin-α. CONCLUSIONS: These data illustrate the potential for an imbalance in p53 and Mdm2 expression to promote excessive apoptosis in villous trophoblast. The upstream regulation of p53 and Mdm2, with regard to exaggerated apoptosis and autophagy in PE, merits further investigation.

Sphingosine signalling regulates decidual NK cell angiogenic phenotype and trophoblast migration.

STUDY QUESTION: Is sphingosine-1-phosphate (S1P) signalling involved in the regulation of the angiogenic function of decidual (d)NK cells during human pregnancy? SUMMARY ANSWER: Human dNK cells, characterized by S1P receptor 5 (S1PR5) expression, are reactive to microenvironmental S1P to modify their VEGF expression and to regulate trophoblast migration and endothelial angiogenesis. WHAT IS KNOWN ALREADY: S1P signalling can modulate peripheral (p)NK cells migration and function. As a unique NK population, human dNK can produce multiple cytokines and angiogenic growth factors to mediate extravillous trophoblast (EVT) invasion and spiral artery remodelling during pregnancy. STUDY DESIGN, SIZE, DURATION: The study was designed to examine S1PR expression and function by freshly isolated human dNK cells in response to different S1P scenarios, created by FTY720, an S1P analogue and S1PR modulator. Ex vivo and in vitro experiments were performed to evaluate the functions of dNK cells. The study was performed between September 2011 and June 2013. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human peripheral blood and decidual samples were collected and the S1PR expression by the decidual leukocytes population was examined. FTY720-induced dNK phenotypic and functional changes (including VEGF and IL-8 expression) were evaluated by multi-colour flow cytometric assays and transwell migration studies. Human placental explant culture and wound healing assays were performed to investigate whether S1P-activated dNK mediated trophoblast migration while angiogenesis was assessed by human umbilical vein endothelial cells (HUVEC) tube formation assays. Both first and second trimester dNK cells were studied to compare the difference in S1PR expression over time at the fetal-maternal interface. MAIN RESULTS AND THE ROLE OF CHANCE: Freshly isolated NK cells (CD45(+)CD56(+)CD16(-)) from blood (pNK) and decidua (dNK) had low S1PR1 reactivity while S1PR5 was prominently expressed by dNK (40%) and, to a lesser extent, by pNK (18%; P < 0.05) cells. S1PR5 expression by dNK was significantly down-regulated by FTY720 treatment, which also impaired decidual leukocyte mobility and cellular contact with invasive EVT. FTY720 significantly reduced VEGF expression by dNK, both in the numbers of VEGF(+) cells and in fluorescence intensity (P < 0.05). IL-8 expression by dNK was not changed by FTY720 and remained low at 8% positivity. Trophoblast migration and HUVEC tube formation were stimulated by control leukocytes, enriched CD56(+) dNK or their conditioned medium, respectively, but this effect was markedly abrogated once they were pretreated with FTY720 (P < 0.05). There was a significant decrease in S1PR5 expression in second trimester dNK cells, compared with those from first trimester (P < 0.05). No significant differences in the levels of angiogenic factors (VEGF or IL-8) were detected between first and second trimester dNK cells. LIMITATIONS, REASONS FOR CAUTION: Our ex vivo and in vitro experimental samples were from healthy women undergoing elective pregnancy termination. FTY720 is a chemical ligand for the S1PRs; little is known regarding the levels or actions of the naturally occurring ligand S1P in human gestational tissues. The in vivo function of S1PR5(+) dNK may be further investigated by using a genetically modified animal model. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to investigate the role of S1PR and S1P interaction on dNK cell physiology and their downstream effects on trophoblast migration. We suggest that S1PR5 may represent a potential target for cellular targeted treatments for gestational diseases such as pre-eclampsia and intrauterine growth restriction that are characterized by inadequate dNK/trophoblast-coordinated uterine spiral artery transformation. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by Canadian Institutes of Health Research (CIHR), MOP82811 to Dr S.J.L.

The molecular role of connexin 43 in human trophoblast cell fusion.

Connexin expression and gap junctional intercellular communication (GJIC) mediated by connexin 43 (Cx43)/gap junction A1 (GJA1) are required for cytotrophoblast fusion into the syncytium, the outer functional layer of the human placenta. Cx43 also impacts intracellular signaling through protein-protein interactions. The transcription factor GCM1 and its downstream target ERVW-1/SYNCYTIN-1 are key players in trophoblast fusion and exert their actions through the ERVW-1 receptor SLC1A5/ASCT-2/RDR/ATB(0). To investigate the molecular role of the Cx43 protein and its interaction with this fusogenic pathway, we utilized stable Cx43-transfected cell lines established from the choriocarcinoma cell line Jeg3: wild-type Jeg3, alphahCG/Cx43 (constitutive Cx43 expression), JpUHD/Cx43 (doxycyclin-inducible Cx43 expression), or JpUHD/trCx43 (doxycyclin-inducible Cx43 carboxyterminal deleted). We hypothesized that truncation of Cx43 at its C-terminus would inhibit trophoblast fusion and protein interaction with either ERVW-1 or SLC1A5. In the alphahCG/Cx43 and JpUHD/Cx43 lines, stimulation with cAMP caused 1) increase in GJA1 mRNA levels, 2) increase in percentage of fused cells, and 3) downregulation of SLC1A5 expression. Cell fusion was inhibited by GJIC blockade using carbenoxylone. Neither Jeg3, which express low levels of Cx43, nor the JpUHD/trCx43 cell line demonstrated cell fusion or downregulation of SLC1A5. However, GCM1 and ERVW-1 mRNAs were upregulated by cAMP treatment in both Jeg3 and all Cx43 cell lines. Silencing of GCM1 prevented the induction of GJA1 mRNA by forskolin in BeWo choriocarcinoma cells, demonstrating that GCM1 is upstream of Cx43. All cell lines and first-trimester villous explants also demonstrated coimmunoprecipitation of SLC1A5 and phosphorylated Cx43. Importantly, SLC1A5 and Cx43 gap junction plaques colocalized in situ to areas of fusing cytotrophoblast, as demonstrated by the loss of E-cadherin staining in the plasma membrane in first-trimester placenta. We conclude that Cx43-mediated GJIC and SLC1A5 interaction play important functional roles in trophoblast cell fusion.

Vascular-leukocyte interactions: mechanisms of human decidual spiral artery remodeling in vitro.

Transformation of uterine spiral arteries is critical for healthy human pregnancy. We recently proposed a role for maternal leukocytes in decidual spiral artery remodeling and suggested that matrix metalloprotease (MMP) activity contributed to the destruction of the arterial wall. In the current study we used our first trimester placental-decidual co-culture (PDC) model to define the temporal relationship and test the mechanistic aspects of this process. PDC experiments were assessed by image analysis over a six-day time-course for degree of vascular transformation and leukocyte distribution around progressively remodeled arterioles. We observed rapid transformation in PDCs associated with loss of vascular smooth muscle cells, widening of the vessel lumen, and significant accumulation of uterine Natural Killer cells and macrophages within the vascular wall (P < 0.001) before trophoblast presence in the vessel lumens. These events did not occur in decidua-only cultures. Active MMP-9 was detected in leukocytes and vascular cells of remodeling arterioles, and inhibition of MMP-2/9 activity in PDC resulted in failure of decidual vascular remodeling compared with vehicle-treated PDCs. Apoptosis of vascular cells, macrophage-mediated phagocytosis, and vascular smooth muscle cell dedifferentiation contributed to the remodeling observed. The PDC model indicates that placental presence is required to initiate decidual spiral artery remodeling but that uterine Natural Killer cells and macrophages mediate the early stages of this process at the cellular level.

The STOX1 genotype associated with pre-eclampsia leads to a reduction of trophoblast invasion by alpha-T-catenin upregulation.

By using complementary in vitro and ex vivo approaches, we show that the risk allele (Y153H) of the pre-eclampsia susceptibility gene STOX1 negatively regulates trophoblast invasion by upregulation of the cell-cell adhesion protein alpha-T-catenin (CTNNA3). This is effectuated at the crucial epithelial-mesenchymal transition of proliferative into invasive extravillous trophoblast. This STOX1-CTNNA3 interaction is direct and includes Akt-mediated phosphorylated control of nucleo-cytoplasmic shuttling and ubiquitin-mediated degradation as shared with the FOX multigene family. This, to our knowledge, is the first time a genotype associated with pre-eclampsia has been shown to directly limit first trimester extravillous trophoblast invasion, the earliest hallmark of pre-eclampsia.

Regulation of the matricellular proteins CYR61 (CCN1) and NOV (CCN3) by hypoxia-inducible factor-1{alpha} and transforming-growth factor-{beta}3 in the human trophoblast.

It is known that a hypoxic environment is critical for trophoblast migration and invasion and is fundamental for appropriate placental perfusion. Because cysteine-rich 61 (CYR61, CCN1) and nephroblastoma overexpressed (NOV, CCN3) are expressed in the extravillous trophoblast and expression levels are deregulated in preeclampsia, we investigated their regulation properties in first-trimester placental explants and in JEG3 choriocarcinoma cells upon a physiological low oxygen tension of 1-3%. In placental explants, both proteins were expressed in the extravillous trophoblast cells and were increased upon hypoxia. JEG3 cells revealed a significant up-regulation of CYR61 and NOV intracellular as well as secreted protein upon hypoxic treatment accompanied by the stabilization of the hypoxia-inducible factor-1alpha (HIF-1alpha). Treatment with dimethyloxalylglycine to mimic hypoxia and silencing of HIF-1alpha using small interfering RNA revealed that only the increase in intracellular protein expression seems to be dependent on HIF-1alpha but obviously not the secretion process. Moreover, recombinant TGF-beta3 was able to further enhance the amount of intracellular CCN proteins as well as secreted CYR61 levels under hypoxia. These results indicate that low oxygen levels trigger elevation of intracellular as well as secreted CYR61 and NOV protein probably in two independent pathways. Addition of recombinant CYR61 and NOV proteins increases migration as well as invasion properties of JEG3 trophoblast cells, which strengthen their role in supporting trophoblast migration invasion properties. In summary, CYR61 and NOV are regulated by HIF-1alpha and TGF-beta3 in the trophoblast cell line JEG3, and their enhanced secretion could be implicated in appropriate placental invasion.

Gonadotropin-releasing hormone-regulated chemokine expression in human placentation.

Placental expression of gonadotropin-releasing hormone (GnRH)-I and II, as well as their cognate receptor, coincides with a period of extensive remodeling of the maternal-fetal interface, near the end of the first trimester of pregnancy. To further define the role of GnRH in human placentation, we performed a microarray screen of HTR-8/SVneo trophoblasts to identify GnRH-regulated genes and their roles in placentation. This screen revealed that GnRH regulates the expression of four angiogenic chemokines: CXCL2, CXCL3, CXCL6, and CXCL8. The microarray data were subsequently confirmed by an extensive Q-PCR time-course analysis. CXCL8, a representative chemokine, was selected for further analysis and shown to be strongly expressed by trophoblasts at the maternal-fetal interface of the human placenta, as well as to accumulate in a GnRH-dependent manner in trophoblast-conditioned media in culture. Trophoblasts were subsequently shown to recruit lymphocytes (Jurkat T cells and primary peripheral blood T and uterine natural killer cells) in chemotaxis assays and this was shown to be GnRH dependent. Furthermore, this recruitment was shown to occur via the release of CXCR1/CXCR2 interacting chemokines, such as the CXCLs investigated in this study. This novel regulation of chemokines by GnRH signaling demonstrates the role of GnRH in regulating the recruitment of lymphocytes to the decidua and the possibility of a direct effect on spiral artery remodeling via the release of proangiogenic chemokines and secondary effects via release of angiogenic factors by recruited lymphocytes.

Fusion assays and models for the trophoblast.

A healthy syncytium in the placenta is vital to a successful pregnancy. The trophoblast builds up the natural barrier between the mother and the developing fetus and is the site of gas, nutrition, and waste exchange. An inadequate formation of this tissue leads to several pathologies of pregnancy, which may result in fetal death during the second trimester or iatrogenic preterm delivery due to intrauterine growth restriction, preeclampsia, or abruption.Cytotrophoblastic cells fuse constantly with the overlying syncytiotrophoblast/syncytium to maintain the function of the trophoblast. Syncytin-1 is the only molecule known to directly induce fusion in the placental trophoblast. Many other proteins, such as gap junctions (e.g., connexin 40) and transcription factors, play a role in the molecular pathways directing the trophoblast turn over. Despite the significance of this process for successful placentation, the mechanisms regulating its activity remain poorly understood.In this chapter we present several different model systems that can be utilized to investigate the regulation of the cell fusion process in the trophoblast. We describe cell-based assays as well as tissue-related protocols. We show how fusion can be monitored in (1) BeWo cells as a trophoblast cell line model, (2) HEK239 using syncytin-1 as a fusion molecule, and (3) a floating villi explant model. Furthermore, we will present strategies to inhibit fusion in the different models. These techniques represent powerful tools to study the molecular mediators of cell fusion in the trophoblast.