Human leukocyte antigen HLA-C, HLA-G, HLA-F, and HLA-E placental profiles are altered in early severe preeclampsia and preterm birth with chorioamnionitis.

BACKGROUND: The extravillous trophoblast expresses each of the nonclassical major histocompatibility complex class I antigens-human leukocyte antigens E, F, and G-and a single classical class I antigen, human leukocyte antigen C. We recently demonstrated dynamic expression patterns of human leukocyte antigens C, G, and F during early extravillous trophoblast invasion and placentation. OBJECTIVE: This study aimed to investigate the hypothesis that the immune inflammatory mediated complications of pregnancy such as early preeclampsia and preterm labor may show altered expression profiles of nonclassical human leukocyte antigens. STUDY DESIGN: Real-time quantitative polymerase chain reaction, western blot, and immunohistochemistry were performed on placental villous tissues and basal plate sections from term nonlaboring deliveries, preterm deliveries, and severe early-onset preeclampsia, both with and without small-for-gestational-age neonates. RESULTS: Human leukocyte antigen G is strongly and exclusively expressed by the extravillous trophoblast within the placental basal plate, and its levels increase in pregnancies complicated by severe early-onset preeclampsia with small-for-gestational-age neonates relative to those of healthy term controls. Human leukocyte antigen C shows a similar profile in the extravillous trophoblast of preeclamptic pregnancies, but significantly decreases in the villous placenta. Human leukocyte antigen F protein levels are decreased in both extravillous trophoblast and villous placenta of severe early-onset preeclamptic pregnancies, both with and without small-for-gestational-age neonates, compared with those found in term and preterm birth deliveries. Human leukocyte antigen E decreases in blood vessels in placentas from preeclamptic pregnancies relative to its levels in term and preterm birth deliveries. Placental levels of human leukocyte antigens F and C are increased in cases of preterm birth with chorioamnionitis relative to those of cases of idiopathic preterm birth. CONCLUSION: Dysregulation of placental human leukocyte antigen expression at the maternal-fetal interface may contribute to compromised maternal tolerance in preterm birth with chorioamnionitis and excessive maternal systemic inflammation associated with severe early-onset preeclampsia.

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.

Interaction between dolutegravir and folate transporters and receptor in human and rodent placenta.

BACKGROUND: Due to the critical role of folates in neurodevelopment, it is important to understand potential interactions between anti-HIV drugs used during pregnancy, and folate delivery pathways in the placenta. This study investigates the effect of dolutegravir (DTG) exposure on the functional expression of the reduced folate carrier (RFC), proton-coupled folate transporter (PCFT), and folate receptor-α (FRα) in the placenta. METHODS: Human placental cell lines, human placental explants, and a pregnant mouse model treated with clinically relevant concentrations of DTG were used. Gene and protein expression were assessed by qPCR, immunoblot and immunohistochemical assays. Folate transport function was measured by applying radioisotope-based transport assays. FINDINGS: In placental cells, clinically relevant DTG exposure for 3h or 6h was associated with a modest but significant reduction in the expression of RFC and PCFT both at the mRNA and protein levels, as well as decreased uptake of RFC and PCFT substrates [3H]-methotrexate and [3H]-folic acid, respectively. In pregnant mice, DTG administration was associated with an increase in both placental RFC and PCFT mRNA expression, accompanied by a decrease in placental FRα mRNA under folate-deficient dietary conditions. INTERPRETATION: These findings demonstrate a potential interaction between DTG and folate transport pathways in the placenta, particularly in vivo, under folate deficient conditions, potentially impacting folate delivery to the foetus in the context of DTG-based ART during pregnancy. FUNDING: Funded by Ontario HIV Treatment Network, grant #506657; and Eunice Kennedy Shriver National Institute of Child Health & Human Development of the National Institutes of Health, award #R01HD104553.

Decidual Inflammation Drives Chemokine-Mediated Immune Infiltration Contributing to Term Labor.

Infiltration of maternal peripheral leukocytes into the uterine tissues is a critical event occurring before, during, and after term labor (TL). In this article, we investigate the contribution of uterine smooth muscle (myometrium) and pregnant endometrium (decidua) to the inflammatory process during human TL. We hypothesize that labor-related physiological inflammation is orchestrated by uterine-secreted cytokines, which dually activate the uterine vascular endothelium and maternal leukocytes to promote their adhesion and infiltration into the uterus. Using Luminex and ELISA assays, we examine a full range of cytokines (45 proteins) in media conditioned by primary decidual and myometrial cells from TL and term not in labor (TNL) women. The effect of conditioned media on the activation of human uterine microvascular endothelial cells was measured by qPCR and on peripheral leukocytes by flow cytometry. Transendothelial migration of calcein-labeled primary leukocytes toward media was assessed by fluorometry. Stromal decidual cells secrete significantly higher levels of multiple cytokines compared with myometrial cells (p < 0.05) and significantly more cytokines during TL than TNL. These cytokines activate uterine microvascular endothelial cells through the upregulation of cell adhesion molecule VCAM-1 and peripheral leukocytes by upregulation of CD11b. Furthermore, multiple cytokines secreted from the TL decidua and myometrium significantly increase migration of granulocytes, monocytes, and lymphocytes compared with TNL (p < 0.05), which was blocked by a broad-spectrum chemokine inhibitor (FX125L). These data reveal the critical role for decidual- and myometrial-secreted cytokines in the activation of inflammatory pathways leading to labor. We suggest that these pathways represent targets for therapeutic intervention during preterm labor.

ACE2 Is Expressed in Immune Cells That Infiltrate the Placenta in Infection-Associated Preterm Birth.

COVID-19 is associated with increased incidence of preterm birth (PTB). We assessed pathways by which SARS-CoV-2 could access the placenta. Placentae, from PTB with or without chorioamnionitis (ChA), or from term pregnancies (n = 12/13/group) were collected. Peripheral blood was collected from healthy pregnant women (n = 6). Second trimester placental explants (16-20 weeks, n = 5/group) were treated with lipopolysaccharide (LPS, to mimic bacterial infection) and ACE2, CCL2, IL-6/8 and TNFα mRNA was assessed. ChA-placentae exhibited increased ACE2 and CCL2 mRNA expression (p < 0.05). LPS increased cytokine and ACE2 mRNA in placental explants. Placental ACE2 protein localized to syncytiotrophoblast, fetal endothelium, extravillous trophoblast and in immune cells-subsets (M1/M2 macrophage and neutrophils) within the villous stroma. Significantly increased numbers of M1 macrophage and neutrophils were present in the ChA-placenta (p < 0.001). Subsets of peripheral immune cells from pregnant women express the ACE2 mRNA and protein. A greater fraction of granulocytes was positive for ACE2 protein expression compared to lymphocytes or monocytes. These data suggest that in pregnancies complicated by ChA, ACE2 positive immune cells in the maternal circulation have the potential to traffic SARS-CoV-2 virus to the placenta and increase the risk of vertical transmission to the placenta/fetus.

Efficient derivation of human trophoblast stem cells from primed pluripotent stem cells.

Human trophoblast stem cells (hTSCs) provide a valuable model to study placental development and function. While primary hTSCs have been derived from embryos/early placenta, and transdifferentiated hTSCs from naïve human pluripotent stem cells (hPSCs), the generation of hTSCs from primed PSCs is problematic. We report the successful generation of TSCs from primed hPSCs and show that BMP4 substantially enhances this process. TSCs derived from primed hPSCs are similar to blastocyst-derived hTSCs in terms of morphology, proliferation, differentiation potential, and gene expression. We define the chromatin accessibility dynamics and histone modifications (H3K4me3/H3K27me3) that specify hPSC-derived TSCs. Consistent with low density of H3K27me3 in primed hPSC-derived hTSCs, we show that knockout of H3K27 methyltransferases (EZH1/2) increases the efficiency of hTSC derivation from primed hPSCs. Efficient derivation of hTSCs from primed hPSCs provides a simple and powerful model to understand human trophoblast development, including the pathogenesis of trophoblast-related disorders, by generating disease-specific hTSCs.

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.

Expression of severe acute respiratory syndrome coronavirus 2 cell entry genes, angiotensin-converting enzyme 2 and transmembrane protease serine 2, in the placenta across gestation and at the maternal-fetal interface in pregnancies complicated by preterm birth or preeclampsia.

BACKGROUND: Although there is some evidence that severe acute respiratory syndrome coronavirus 2 can invade the human placenta, limited data exist on the gestational age-dependent expression profile of the severe acute respiratory syndrome coronavirus 2 cell entry mediators, angiotensin-converting enzyme 2 and transmembrane protease serine 2, at the human maternal-fetal interface. There is also no information as to whether the expression of these mediators is altered in pregnancies complicated by preeclampsia or preterm birth. This is important because the expression of decidual and placental angiotensin-converting enzyme 2 and transmembrane protease serine 2 across gestation may affect the susceptibility of pregnancies to vertical transmission of severe acute respiratory syndrome coronavirus 2. OBJECTIVE: This study aimed to investigate the expression pattern of specific severe acute respiratory syndrome coronavirus 2 cell entry genes, angiotensin-converting enzyme 2 and transmembrane protease serine 2, in the placenta across human pregnancy and in paired samples of decidua and placenta in pregnancies complicated by preterm birth or preeclampsia compared with those in term uncomplicated pregnancies. STUDY DESIGN: In this study, 2 separate cohorts of patients, totaling 87 pregnancies, were included. The first cohort was composed of placentae from first- (7-9 weeks), second- (16-18 weeks), and third-trimester preterm (26-31 weeks) and third-trimester term (38-41 weeks) pregnancies (n=5/group), whereas the second independent cohort included matched decidua and placentae from pregnancies from term uncomplicated pregnancies (37-41 weeks' gestation; n=14) and pregnancies complicated by preterm birth (26-37 weeks' gestation; n=11) or preeclampsia (25-37 weeks' gestation; n=42). Samples were subjected to quantitative polymerase chain reaction and next-generation sequencing or RNA sequencing for next-generation RNA sequencing for angiotensin-converting enzyme 2 and transmembrane protease serine 2 mRNA expression quantification, respectively. RESULTS: In the first cohort, angiotensin-converting enzyme 2 and transmembrane protease serine 2, exhibited a gestational age-dependent expression profile, that is, angiotensin-converting enzyme 2 and transmembrane protease serine 2 mRNA was higher (P<.05) in the first-trimester placenta than in second-trimester, preterm birth, and term placentae (P<.05) and exhibited a negative correlation with gestational age (P<.05). In the second cohort, RNA sequencing demonstrated very low or undetectable expression levels of angiotensin-converting enzyme 2 in preterm birth, preeclampsia, and term decidua and in placentae from late gestation. In contrast, transmembrane protease serine 2 was expressed in both decidual and placental samples but did not change in pregnancies complicated by either preterm birth or preeclampsia. CONCLUSION: The increased expression of these severe acute respiratory syndrome coronavirus 2 cell entry-associated genes in the placenta in the first trimester of pregnancy compared with those in later stages of pregnancy suggests the possibility of differential susceptibility to placental entry to severe acute respiratory syndrome coronavirus 2 across pregnancy. Even though there is some evidence of increased rates of preterm birth associated with severe acute respiratory syndrome coronavirus 2 infection, we found no increase in mRNA expression of angiotensin-converting enzyme 2 or transmembrane protease serine 2 at the maternal-fetal interface.

Exploring the impact of HIV infection and antiretroviral therapy on placenta morphology.

INTRODUCTION: Women living with HIV experience more adverse birth outcomes; the mechanisms are not fully understood. We examined placenta morphology and associations with birth outcomes in a Canadian cohort of women living with HIV (HIV+) on antiretroviral therapy (ART) from conception and HIV-uninfected (HIV-) women. METHODS: Term placentas from 94 women (40 HIV-, 54 HIV+) were studied. Trimmed placenta weight was collected. Placenta digital photos were used to compute morphometric parameters. Regression models investigated associations between log-transformed placenta parameters and birth outcomes. RESULTS: We observed a trend towards lower placenta weight and smaller placenta area in the HIV+ group, both of which were significantly associated with small for gestational age births. HIV+ serostatus was associated with 6-fold (95%CI 2-20) greater odds of having placenta area in the lowest quartile (<236 cm2). Cord marginality (distance from the edge) was significantly lower in the HIV+ group (p = 0.004), with 35% of placenta having an abnormal (marginal or velamentous) cord insertion vs. 12.5% in the HIV- group (p = 0.01). Velamentous cord insertion was seen in 13% of placentas in the HIV+ vs. 0% in HIV- group (p = 0.02). A significant correlation between cord marginality and placenta thickness was observed in the HIV- group, with a more marginal cord being associated with a thicker placenta. This correlation was not observed in the HIV+ group. HIV+ placentas exposed to protease inhibitors were significantly less circular compared to the HIV- group (p = 0.03). CONCLUSION: Our data suggest that HIV/ART exposure affects placenta morphology and is associated with higher rates of abnormal cord insertion.

Palmitic acid induces inflammation in placental trophoblasts and impairs their migration toward smooth muscle cells through plasminogen activator inhibitor-1.

A critical component of early human placental development includes migration of extravillous trophoblasts (EVTs) into the decidua. EVTs migrate toward and displace vascular smooth muscle cells (SMCs) surrounding several uterine structures, including spiral arteries. Shallow trophoblast invasion features in several pregnancy complications including preeclampsia. Maternal obesity is a risk factor for placental dysfunction, suggesting that factors within an obese environment may impair early placental development. Herein, we tested the hypothesis that palmitic acid, a saturated fatty acid circulating at high levels in obese women, induces an inflammatory response in EVTs that hinders their capacity to migrate toward SMCs. We found that SMCs and SMC-conditioned media stimulated migration and invasion of an EVT-like cell line, HTR8/SVneo. Palmitic acid impaired EVT migration and invasion toward SMCs, and induced expression of several vasoactive and inflammatory mediators in EVTs, including endothelin, interleukin (IL)-6, IL-8 and PAI1. PAI1 was increased in plasma of women with early-onset preeclampsia, and PAI1-deficient EVTs were protected from the anti-migratory effects of palmitic acid. Using first trimester placental explants, palmitic acid exposure decreased EVT invasion through Matrigel. Our findings reveal that palmitic acid induces an inflammatory response in EVTs and attenuates their migration through a mechanism involving PAI1. High levels of palmitic acid in pathophysiological situations like obesity may impair early placental development and predispose to placental dysfunction.

Reprint of: 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.

Differential Role of Smad2 and Smad3 in the Acquisition of an Endovascular Trophoblast-Like Phenotype and Preeclampsia.

During placental development, cytotrophoblast progenitor cells differentiate into the syncytiotrophoblast and invasive extravillous trophoblasts (EVTs). Some EVTs further differentiate into endovascular trophoblasts (enEVTs) which exhibit endothelial-like properties. Abnormal placental development, including insufficient enEVT-mediated remodeling of the uterine spiral arteries, is thought to be a precipitating factor in the onset of preeclampsia (PE), a pregnancy-related hypertensive disorder. Several members of the transforming growth factor-ß (TGF-ß) superfamily, such as TGF-ßs, Nodal, and Activin have been reported to either promote or inhibit the invasive EVT pathway. These ligands signal through serine/threonine receptor complexes to activate downstream signaling mediators, Smad2 and Smad3. In this study, we determined Smad2 and Smad3 expression pattern in placenta and their effects on trophoblast invasion and differentiation. Total Smad2/3 levels were relatively constant across gestation while the ratio of active phosphorylated forms to their total levels varied with gestational stages, with a higher pSmad2/total Smad2 in later gestation and a higher pSmad3/total Smad3 in early gestation. Immunofluorescent staining revealed that pSmad3 was localized in nuclei of EVTs in anchoring villi. On the other hand, pSmad2 was mostly absent in this invasive EVT population. In addition, pSmad3/total Smad3, but not pSmad2/total Smad2, was significantly lower in both early onset and late onset PE cases, as compared to gestational age-matched controls. Functional studies carried out using a first trimester trophoblast cell line, HTR-8/SVneo, and first trimester human placental explants showed that Smad2 and Smad3 had differential roles in the invasive pathway. Specifically, siRNA-mediated knockdown of Smad2 resulted in an increase in trophoblast invasion and an upregulation of mRNA levels of enEVT markers while the opposite was observed with Smad3 knockdown. In addition, Smad2 siRNA accelerated the EVT outgrowth in first trimester placental explants while the Smad3 siRNA reduced the outgrowth of EVTs when compared to the control. Furthermore, knockdown of Smad2 enhanced, whereas overexpression of Smad2 suppressed, the ability of trophoblasts to form endothelial-like networks. Conversely, Smad3 had opposite effects as Smad2 on network formation. These findings suggest that Smad2 and Smad3 have opposite functions in the acquisition of an enEVT-like phenotype and defects in Smad3 activation are associated with PE.

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.

Interaction of Pregnancy-Specific Glycoprotein 1 With Integrin Α5ß1 Is a Modulator of Extravillous Trophoblast Functions.

Human pregnancy-specific glycoproteins (PSGs) serve immunomodulatory and pro-angiogenic functions during pregnancy and are mainly expressed by syncytiotrophoblast cells. While PSG mRNA expression in extravillous trophoblasts (EVTs) was reported, the proteins were not previously detected. By immunohistochemistry and immunoblotting, we show that PSGs are expressed by invasive EVTs and co-localize with integrin 5. In addition, we determined that native and recombinant PSG1, the most highly expressed member of the family, binds to 51 and induces the formation of focal adhesion structures resulting in adhesion of primary EVTs and EVT-like cell lines under 21% oxygen and 1% oxygen conditions. Furthermore, we found that PSG1 can simultaneously bind to heparan sulfate in the extracellular matrix and to 51 on the cell membrane. Wound healing assays and single-cell movement tracking showed that immobilized PSG1 enhances EVT migration. Although PSG1 did not affect EVT invasion in the in vitro assays employed, we found that the serum PSG1 concentration is lower in African-American women diagnosed with early-onset and late-onset preeclampsia, a pregnancy pathology characterized by shallow trophoblast invasion, than in their respective healthy controls only when the fetus was a male; therefore, the reduced expression of this molecule should be considered in the context of preeclampsia as a potential therapy.

Tuning FOXD3 expression dose-dependently balances human embryonic stem cells between pluripotency and meso-endoderm fates.

Forkhead box D3 (FOXD3) is a key transcription factor maintaining pluripotency in mouse embryonic stem cells (ESCs). Yet to date studies on its role in human ESCs are quite limited. In this study, we report that deletion of FOXD3 in human ESCs results in loss of pluripotency and spontaneous differentiation toward meso-endoderm. Ectopic overexpression of FOXD3 in hESCs leads to two different phenotypes: Human ESCs expressing high levels of FOXD3 undergo spontaneous meso-endoderm differentiation, whereas those with lower levels of FOXD3 maintain pluripotency. Next we deleted endogenous FOXD3 in the low ectopic expression model and find that addition of exogenous FOXD3 at a low level could rescue FOXD3-deficiency phenotype in hESCs. In summary, our findings suggest that FOXD3 dose-dependently regulates the balance of human ESCs between pluripotency and meso-endoderm fates, which adds to our understanding of the role of FOXD3 in humans.

Failure of Decidualization and Maternal Immune Tolerance Underlies Uterovascular Resistance in Intra Uterine Growth Restriction.

Failure of uterine vascular transformation is associated with pregnancy complications including Intra Uterine Growth Restriction (IUGR). The decidua and its immune cell populations play a key role in the earliest stages of this process. Here we investigate the hypothesis that abnormal decidualization and failure of maternal immune tolerance in the second trimester may underlie the uteroplacental pathology of IUGR. Placental bed biopsies were obtained from women undergoing elective caesarian delivery of a healthy term pregnancy, an IUGR pregnancy or a pregnancy complicated by both IUGR and preeclampsia. Decidual tissues were also collected from second trimester terminations from women with either normal or high uterine artery Doppler pulsatile index (PI). Immunohistochemical image analysis and flow cytometry were used to quantify vascular remodeling, decidual leukocytes and decidual status in cases vs. controls. Biopsies from pregnancies complicated by severe IUGR with a high uterine artery pulsatile index (PI) displayed a lack of: myometrial vascular transformation, interstitial, and endovascular extravillous trophoblast (EVT) invasion, and a lower number of maternal leukocytes. Apoptotic mural EVT were observed in association with mature dendritic cells and T cells in the IUGR samples. Second trimester pregnancies with high uterine artery PI displayed a higher incidence of small for gestational age fetuses; a skewed decidual immunology with higher numbers of; CD8 T cells, mature CD83 dendritic cells and lymphatic vessels that were packed with decidual leukocytes. The decidual stromal cells (DSCs) failed to differentiate into the large secretory DSC in these cases, remaining small and cuboidal and expressing lower levels of the nuclear progesterone receptor isoform B, and DSC markers Insulin Growth Factor Binding protein-1 (IGFBP-1) and CD10 as compared to controls. This study shows that defective progesterone mediated decidualization and a hostile maternal immune response against the invading endovascular EVT contribute to the failure of uterovascular remodeling in IUGR pregnancies.

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.

TGFb1 suppresses the activation of distinct dNK subpopulations in preeclampsia.

BACKGROUND: Decidual natural killer (dNK) cells are the predominant lymphocytes accumulated at the maternal-fetal interface. Regulatory mechanism of dNK cells in preeclampsia, a gestational complication characterized by high blood pressure and increased proteinuria occurring after 20 weeks pregnancy, is not completely understood. METHODS: Multi-parameter flow cytometry is applied to investigate the phenotype and function of dNK cells freshly isolated from decidual samples or conditionally cultured by TGFb stimulation. FINDINGS: In preeclampsia, we documented elevated numbers of CD56+ CD3- dNK cells in close proximity to Foxp3+ regulatory T (Treg) cells within the decidua. In vitro experiments using dNK cells from early gestation showed that dNK activation (IFNG, IL-8 and CD107a) can be downregulated by Treg cells. The expression of these markers by dNK cells was significantly lower in preeclampsia. We also observed a positive correlation between the expression of dNK activation receptors (NKp30 and NKG2D) and the expression of IFNG in specific dNK subsets. TGFb levels are increased in the decidua of preeclamptic pregnancies. We analyzed co-expression of activation (IFNG/IL-8/CD107a) and angiogenic (VEGF) markers in dNK cells. TGFb treatment reduced while blockade of TGFb increased co-expression of these markers. INTERPRETATION: Our findings suggest that elevated decidual TGFb1 supresses the activation of specific subsets of dNK which in turn contributes to the uteroplacental pathology associated with the onset of preeclampsia.