Human Placenta and Evolving Insights into Pathological Changes of Preeclampsia: A Comprehensive Review of the Last Decade.

The placenta, the foremost and multifaceted organ in fetal and maternal biology, is pivotal in facilitating optimal intrauterine fetal development. Remarkably, despite its paramount significance, the placenta remains enigmatic, meriting greater comprehension given its central influence on the health trajectories of both the fetus and the mother. Preeclampsia (PE) and intrauterine fetal growth restriction (IUGR), prevailing disorders of pregnancy, stem from compromised placental development. PE, characterized by heightened mortality and morbidity risks, afflicts 5-7% of global pregnancies, its etiology shrouded in ambiguity. Pertinent pathogenic hallmarks of PE encompass inadequate restructuring of uteroplacental spiral arteries, placental ischemia, and elevated levels of vascular endothelial growth factor receptor-1 (VEGFR-1), also recognized as soluble FMS-like tyrosine kinase-1 (sFlt-1). During gestation, the placental derivation of sFlt-1 accentuates its role as an inhibitory receptor binding to VEGF-A and placental growth factor (PlGF), curtailing target cell accessibility. This review expounds upon the placenta's defining cellular component of the trophoblast, elucidates the intricacies of PE pathogenesis, underscores the pivotal contribution of sFlt-1 to maternal pathology and fetal safeguarding, and surveys recent therapeutic strides witnessed in the past decade.

Expert review: preeclampsia Type I and Type II.

Pregnancy involves an interplay between maternal and fetal factors affecting changes to maternal anatomy and physiology to support the developing fetus and ensure the well-being of both the mother and offspring. A century of research has provided evidence of the imperative role of the placenta in the development of preeclampsia. Recently, a growing body of evidence has supported the adaptations of the maternal cardiovascular system during normal pregnancy and its maladaptation in preeclampsia. Debate surrounds the roles of the placenta vs the maternal cardiovascular system in the pathophysiology of preeclampsia. We proposed an integrated model of the maternal cardiac-placental-fetal array and the development of preeclampsia, which reconciles the disease phenotypes and their proposed origins, whether placenta-dominant or maternal cardiovascular system-dominant. These phenotypes are sufficiently diverse to define 2 distinct types: preeclampsia Type I and Type II. Type I preeclampsia may present earlier, characterized by placental dysfunction or malperfusion, shallow trophoblast invasion, inadequate spiral artery conversion, profound syncytiotrophoblast stress, elevated soluble fms-like tyrosine kinase-1 levels, reduced placental growth factor levels, high peripheral vascular resistance, and low cardiac output. Type I is more often accompanied by fetal growth restriction, and low placental growth factor levels have a measurable impact on maternal cardiac remodeling and function. Type II preeclampsia typically occurs in the later stages of pregnancy and entails an evolving maternal cardiovascular intolerance to the demands of pregnancy, with a moderately dysfunctional placenta and inadequate blood supply. The soluble fms-like tyrosine kinase-1-placental growth factor ratio may be normal or slightly disturbed, peripheral vascular resistance is low, and cardiac output is high, but these adaptations still fail to meet demand. Emergent placental dysfunction, coupled with an increasing inability to meet demand, more often appears with fetal macrosomia, multiple pregnancies, or prolonged pregnancy. Support for the notion of 2 types of preeclampsia observable on the molecular level is provided by single-cell transcriptomic survey of gene expression patterns across different cell classes. This revealed widespread dysregulation of gene expression across all cell types, and significant imbalance in fms-like tyrosine kinase-1 (FLT1) and placental growth factor, particularly marked in the syncytium of early preeclampsia cases. Classification of preeclampsia into Type I and Type II can inform future research to develop targeted screening, prevention, and treatment approaches.

Natriuretic Peptide Signaling in Uterine Biology and Preeclampsia.

Endometrial decidualization is a uterine process essential for spiral artery remodeling, embryo implantation, and trophoblast invasion. Defects in endometrial decidualization and spiral artery remodeling are important contributing factors in preeclampsia, a major disorder in pregnancy. Atrial natriuretic peptide (ANP) is a cardiac hormone that regulates blood volume and pressure. ANP is also generated in non-cardiac tissues, such as the uterus and placenta. In recent human genome-wide association studies, multiple loci with genes involved in natriuretic peptide signaling are associated with gestational hypertension and preeclampsia. In cellular experiments and mouse models, uterine ANP has been shown to stimulate endometrial decidualization, increase TNF-related apoptosis-inducing ligand expression and secretion, and enhance apoptosis in arterial smooth muscle cells and endothelial cells. In placental trophoblasts, ANP stimulates adenosine 5'-monophosphate-activated protein kinase and the mammalian target of rapamycin complex 1 signaling, leading to autophagy inhibition and protein kinase N3 upregulation, thereby increasing trophoblast invasiveness. ANP deficiency impairs endometrial decidualization and spiral artery remodeling, causing a preeclampsia-like phenotype in mice. These findings indicate the importance of natriuretic peptide signaling in pregnancy. This review discusses the role of ANP in uterine biology and potential implications of impaired ANP signaling in preeclampsia.

Alpha-2-macroglobulin is involved in the occurrence of early-onset pre-eclampsia via its negative impact on uterine spiral artery remodeling and placental angiogenesis.

BACKGROUND: Pre-eclampsia (PE) is one of the leading causes of maternal and fetal morbidity/mortality during pregnancy, and alpha-2-macroglobulin (A2M) is associated with inflammatory signaling; however, the pathophysiological mechanism by which A2M is involved in PE development is not yet understood. METHODS: Human placenta samples, serum, and corresponding clinical data of the participants were collected to study the pathophysiologic mechanism underlying PE. Pregnant Sprague-Dawley rats were intravenously injected with an adenovirus vector carrying A2M via the tail vein on gestational day (GD) 8.5. Human umbilical artery smooth muscle cells (HUASMCs), human umbilical vein endothelial cells (HUVECs), and HTR-8/SVneo cells were transfected with A2M-expressing adenovirus vectors. RESULTS: In this study, we demonstrated that A2M levels were significantly increased in PE patient serum, uterine spiral arteries, and feto-placental vasculature. The A2M-overexpression rat model closely mimicked the characteristics of PE (i.e., hypertension in mid-to-late gestation, histological and ultrastructural signs of renal damage, proteinuria, and fetal growth restriction). Compared to the normal group, A2M overexpression significantly enhanced uterine artery vascular resistance and impaired uterine spiral artery remodeling in both pregnant women with early-onset PE and in pregnant rats. We found that A2M overexpression was positively associated with HUASMC proliferation and negatively correlated with cell apoptosis. In addition, the results demonstrated that transforming growth factor beta 1 (TGFß1) signaling regulated the effects of A2M on vascular muscle cell proliferation described above. Meanwhile, A2M overexpression regressed rat placental vascularization and reduced the expression of angiogenesis-related genes. In addition, A2M overexpression reduced HUVEC migration, filopodia number/length, and tube formation. Furthermore, HIF-1α expression was positively related to A2M, and the secretion of sFLT-1 and PIGF of placental origin was closely related to PE during pregnancy or A2M overexpression in rats. CONCLUSIONS: Our data showed that gestational A2M overexpression can be considered a contributing factor leading to PE, causing detective uterine spiral artery remodeling and aberrant placental vascularization.

Estrogen Actions in Placental Vascular Morphogenesis and Spiral Artery Remodeling: A Comparative View between Humans and Mice.

Estrogens, mainly 17ß-estradiol (E2), play a critical role in reproductive organogenesis, ovulation, and fertility via estrogen receptors. E2 is also a well-known regulator of utero-placental vascular development and blood-flow dynamics throughout gestation. Mouse and human placentas possess strikingly different morphological configurations that confer important reproductive advantages. However, the functional interplay between fetal and maternal vasculature remains similar in both species. In this review, we briefly describe the structural and functional characteristics, as well as the development, of mouse and human placentas. In addition, we summarize the current knowledge regarding estrogen actions during utero-placental vascular morphogenesis, which includes uterine angiogenesis, the control of trophoblast behavior, spiral artery remodeling, and hemodynamic adaptation throughout pregnancy, in both mice and humans. Finally, the estrogens that are present in abnormal placentation are also mentioned. Overall, this review highlights the importance of the actions of estrogens in the physiology and pathophysiology of placental vascular development.

PVT1/miR-145-5p/HK2 modulates vascular smooth muscle cells phenotype switch via glycolysis: The new perspective on the spiral artery remodeling.

INTRODUCTION: Vascular smooth muscle cells (VSMC) switched from a contractile phenotype to a synthetic phenotype during the decidual spiral artery (SPAs) remodeling process. The lncRNA plasmacytoma variant translocation 1 (PVT1) and glucose metabolism have been found to regulate the VSMC phenotype switch. This study aimed to analyze the dynamic expression of PVT1 and glycolytic key enzymes hexokinase2 (HK2) at different remodeling stages in early human pregnancy and elucidate the underlying mechanism of the PVT1/miR-145-5p/HK2 axis involved in the spiral artery remodeling. METHODS: qRT-PCR, Western blot (WB) analysis, Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) were used to detect the expression and localization of PVT1 and HK2 in decidual tissue. HA-VSMCs were transfected with specific siRNA, shRNA and plasmids to regulate corresponding genes. Extracellular lactate, cellular ATP, ROS, and intracellular NADPH levels were measured using the corresponding assay kits. Migration was measured by wound-healing and Transwell assays. Contractile phenotypic markers α-SMA, MYH11 with calponin and synthetic phenotypic markers OPN and vimentin were detected by WB. The PDC model was used to detect the degree of spiral arterial remodeling. RESULTS: PVT1 and HK2 were upregulated with gestational age (GA) increasing in decidual tissue during the early pregnancy. HK2 regulated the glycolytic activity and VSMC phenotype switch in vitro. PVT1 regulated the glycolytic activity and VSMC phenotype switch through HK2. PVT1 played a ceRNA role in regulating HK2 expression by sponging miR-145-5p. PVT1 and HK2 influenced spiral artery remodeling in the PDC model. DISCUSSION: PVT1 and HK2 were upregulated, and miR-145-5p was downregulated in decidua with the GA increasing. Meanwhile, the PVT1/miR-145-5p/HK2 axis may be involved in regulating the phenotypic switch and migratory capacity of VSMCs by affecting glycolysis in decidual SPAs remodeling.

Low-Dose Tacrolimus Promotes the Migration and Invasion and Nitric Oxide Production in the Human-Derived First Trimester Extravillous Trophoblast Cells In Vitro.

Placentation is one of the most important determinants for a successful pregnancy, and this is dependent on the process of trophoblast migration and invasion. Progesterone receptors (PGR) are critical effectors of progesterone (P4) signaling that is required for trophoblast migration and invasion conducive to a successful gestation. In immune complicated pregnancies, evidence has shown that abnormal placentation occurs because of aberrant expression of PGR. Therapeutic intervention with tacrolimus (FK506) was able to restore PGR expression and improve pregnancy outcomes in immune-complicated gestations; however, the exact mode of action of tacrolimus in assisting placentation is not clear. Here, we attempt to uncover the mode of action of tacrolimus by examining its effects on trophoblast invasion and migration in the human-derived extravillous trophoblast (EVT) cell line, the HTR-8/SVneo cells. Using a variety of functional assays, we demonstrated that low-dose tacrolimus (10 ng/mL) was sufficient to significantly (p < 0.001) stimulate the migration and invasion of the HTR-8/SVneo cells, inducing their cytosolic/nuclear progesterone receptor expression and activation, and modulating their Nitric Oxide (NO) production. Moreover, tacrolimus abrogated the suppressive effect of the NOS inhibitor Nω- Nitro-L-Arginine Methyl Ester (L-NAME) on these vital processes critically involved in the establishment of human pregnancy. Collectively, our data suggest an immune-independent mode of action of tacrolimus in positively influencing placentation in complicated gestations, at least in part, through promoting the migration and invasion of the first trimester extravillous trophoblast cells by modulating their NO production and activating their cytosolic/nuclear progesterone-receptors. To our knowledge, this is the first report to show that the mode of action of tacrolimus as a monotherapy for implantation failure is plausibly PGR-dependent.

Placental sFlt-1 Gene Delivery in Early Primate Pregnancy Suppresses Uterine Spiral Artery Remodeling.

Uterine spiral artery remodeling (SAR) is essential for promoting placental perfusion and fetal development. A defect in SAR results in placental ischemia and increase in placental expression and serum levels of the soluble fms-like tyrosine kinase-1 (sFlt-1) receptor that binds to and suppresses vascular endothelial growth factor (VEGF) bioavailability, thereby leading to maternal vascular dysfunction. We have established a nonhuman primate model of impaired SAR and maternal vascular dysfunction by prematurely elevating estradiol levels in early baboon pregnancy. However, it is unknown whether this primate model of defective SAR involves an increase in placental expression of sFlt-1, which may suppress VEGF bioavailability and thus SAR in the first trimester. Therefore, to establish the role of sFlt-1 in early pregnancy, SAR was quantified in baboons treated on days 25 through 59 of gestation (term = 184 days) with estradiol or with the sFlt-1 gene targeted selectively to the placental basal plate by ultrasound-mediated/microbubble-facilitated gene delivery technology. Placental basal plate sFlt-1 protein expression was 2-fold higher (P < 0.038) and the level of SAR for vessels > 25 µm in diameter was 72% and 63% lower (P < 0.01), respectively, in estradiol-treated and sFlt-1 gene-treated baboons than in untreated animals. In summary, prematurely elevating estradiol levels or sFlt-1 gene delivery increased placental basal plate sFlt-1 protein expression and suppressed SAR in early baboon pregnancy. This study makes the novel discovery that in elevated levels sFlt-1 has a role both in suppressing SAR in early primate pregnancy and maternal vascular endothelial function in late gestation.

Decidual macrophage derived MMP3 contributes to extracellular matrix breakdown in spiral artery remodeling in early human pregnancy.

Remodeling of the uterine spiral arteries is required for a successful pregnancy. This process requires the co-ordinated activity of a number of different cell types including uterine natural killer cells, decidual macrophages, extravillous trophoblast cells, vascular smooth muscle cells and endothelial cells. We have previously demonstrated that decidual macrophages facilitate breakdown of fibronectin and laminin in a model of spiral artery remodeling. The aim of the current study was to determine which matrix metalloproteinases (MMPs) decidual macrophages express and play roles in extracellular matrix (ECM) breakdown in vascular remodeling. Decidual macrophages were isolated from first trimester decidua and cultured for 24 h to obtain conditioned medium. MMP secretion was assessed by a membrane based array and immunohistochemistry of decidual sections. In addition, the chorionic plate artery (CPA) model was used with decidual macrophage conditioned medium, with and without a MMP3 inhibitor and ECM protein expression assessed using quickscore. The decidual macrophages secreted a wide range of MMPs, with MMP3 being the most predominant. Co-localization of MMP3 to decidual macrophages was confirmed by immunohistochemistry. Decidual macrophage conditioned medium facilitated breakdown of laminin and fibronectin in the CPA model, an effect that was abrogated by the MMP3 inhibitor. These data further support the role of decidual macrophages in tissue remodeling in the first trimester of pregnancy. An alteration in their numbers or phenotype would impact spiral artery remodeling and contribute to the etiology of a number of complications of pregnancy.

Imbalances in circulating angiogenic factors in the pathophysiology of preeclampsia and related disorders.

Preeclampsia is a devastating medical complication of pregnancy that can lead to significant maternal and fetal morbidity and mortality. It is currently believed that there is abnormal placentation in as early as the first trimester in women destined to develop preeclampsia. Although the etiology of the abnormal placentation is being debated, numerous epidemiologic and experimental studies suggest that imbalances in circulating angiogenic factors released from the placenta are responsible for the maternal signs and symptoms of preeclampsia. In particular, circulating levels of soluble fms-like tyrosine kinase 1, an antiangiogenic factor, are markedly increased in women with preeclampsia, whereas free levels of its ligand, placental, growth factor are markedly diminished. Alterations in these angiogenic factors precede the onset of clinical signs of preeclampsia and correlate with disease severity. Recently, the availability of automated assays for the measurement of angiogenic biomarkers in the plasma, serum, and urine has helped investigators worldwide to demonstrate a key role for these factors in the clinical diagnosis and prediction of preeclampsia. Numerous studies have reported that circulating angiogenic biomarkers have a very high negative predictive value to rule out clinical disease among women with suspected preeclampsia. These blood-based biomarkers have provided a valuable tool to clinicians to accelerate the time to clinical diagnosis and minimize maternal adverse outcomes in women with preeclampsia. Angiogenic biomarkers have also been useful to elucidate the pathogenesis of related disorders of abnormal placentation such as intrauterine growth restriction, intrauterine fetal death, twin-to-twin transfusion syndrome, and fetal hydrops. In summary, the discovery and characterization of angiogenic proteins of placental origin have provided clinicians a noninvasive blood-based tool to monitor placental function and health and for early detection of disorders of placentation. Uncovering the mechanisms of altered angiogenic factors in preeclampsia and related disorders of placentation may provide insights into novel preventive and therapeutic options.

Novel Technologies for Target Delivery of Therapeutics to the Placenta during Pregnancy: A Review.

Uterine spiral artery remodeling is essential for placental perfusion and fetal growth and, when impaired, results in placental ischemia and pregnancy complications, e.g., fetal growth restriction, preeclampsia, premature birth. Despite the high incidence of adverse pregnancies, current treatment options are limited. Accordingly, research has shifted to the development of gene therapy technologies that provide targeted delivery of "payloads" to the placenta while limiting maternal and fetal exposure. This review describes the current strategies, including placental targeting peptide-bound liposomes, nanoparticle or adenovirus constructs decorated with specific peptide sequences and placental gene promoters delivered via maternal IV injection, directly into the placenta or the uterine artery, as well as noninvasive site-selective targeting of regulating genes conjugated with microbubbles via contrast-enhanced ultrasound. The review also provides a perspective on the effectiveness of these technologies in various animal models and their practicability and potential use for targeted placental delivery of therapeutics and genes in adverse human pregnancies affected by placental dysfunction.

Steroid hormones and first trimester vascular remodeling.

Successful implantation and placentation require neoangiogenesis and the remodeling of the uterine spiral arteries. Progesterone and estradiol control various of the placental functions, but their role in vascular remodeling remains controversial. Therefore, this chapter aims to summarize the current knowledge regarding the role of steroid hormones in the uteroplacental vascular remodeling during the first trimester of gestation.

Roles of HLA-G in the Maternal-Fetal Immune Microenvironment.

During pregnancy, the maternal uterus and fetus form a special microenvironment at the maternal-fetal interface to support fetal development. Extravillous trophoblasts (EVTs), differentiated from the fetus, invade into the decidua and interact with maternal cells. Human leukocyte antigen (HLA)-G is a non-classical MHC-I molecule that is expressed abundantly and specifically on EVTs in physiological conditions. Soluble HLA-G (sHLA-G) is also found in maternal blood, amniotic fluid, and cord blood. The abnormal expression and polymorphisms of HLA-G are related to adverse pregnancy outcomes such as preeclampsia (PE) and recurrent spontaneous abortion (RSA). Here we summarize current findings about three main roles of HLA-G during pregnancy, namely its promotion of spiral artery remodeling, immune tolerance, and fetal growth, all resulting from its interaction with immune cells. These findings are not only of great significance for the treatment of pregnancy-related diseases but also provide clues to tumor immunology research since HLA-G functions as a checkpoint in tumors.

Heme oxygenase-1 gene modified human placental mesenchymal stem cells promote placental angiogenesis and spiral artery remodeling by improving the balance of angiogenic factors in vitro.

INTRODUCTION: Abnormal placental vascular development is a possible cause of preeclampsia. Mesenchymal stem cell (MSC)-based therapy is a promising approach for tissue repair and angiogenesis. Further, heme oxygenase-1 (HO-1) has beneficial effects on the angiogenic balance during pregnancy. We explored the effects of HO-1 overexpression on placental vascularization using human placenta-derived MSCs (hPMSCs). METHODS: hPMSCs were isolated from term placenta, and the HO-1 gene was transfected with a lentivirus. Proliferation, migration, and apoptosis of hPMSCs and HO-hPMSCs were examined using CCK8 assay, trans-well assay, and flow cytometry, respectively. Paracrine secretion of the angiogenesis factors VEGF and PlGF, as well as the anti-angiogenesis factors sFlt-1 and sEng, from hPMSC/HO-hPMSCs was measured by qRT-PCR and ELISA. Human umbilical cord endothelial cells and a villus-decidua co-culture were treated with conditioned media to study the effect of HO-1-hPMSCs on tube formation and villus vascular remodeling. RESULTS: HO-1 significantly improved the proliferation and migration of hPMSCs. Additionally, HO-1 reduced hPMSCs apoptosis. The levels of VEGF were increased in HO-1-hPMSCs, whereas those of sFlt-1 decreased. Tube formation assays showed that the conditioned media from HO-1-hPMSCs resulted in more branching points than those from the controls. The villus-decidua co-culture system confirmed that HO-1-hPMSCs are conducive to angiogenesis and vascular remodeling. DISCUSSION: HO-1-modified hPMSCs improve placental vascularization by promoting a balance of pro- and anti- angiogenesis factors, which is worthy of further study as an alternative treatment for preeclampsia.

Dysregulation of hypoxia-inducible factor-1α (Hif1α) expression in the Hmox1-deficient placenta.

INTRODUCTION: Severe hypoxia exists in placentas during early pregnancy, with reoxygenation during mid-gestation. Hypoxia-inducible factor-1α (Hif1α), an oxygen sensor, initiates placental vascular development. We have shown that the placental vasculature in Hmox1-deficient (Hmox1+/-, Het) pregnancies is impaired, with morphological defects similar to Hif1α-deficient placentas. MATERIALS AND METHODS: Whole wild-type (WT) and Het mouse placentas were collected at E8.5 (1%-3% O2) and E9.5-15.5 (8%-10% O2). mRNA levels were determined using real-time RT-PCR or PCR arrays and protein levels using Western blot. Bone marrow-derived macrophages (BMDMs) from WT, Het, and Hmox1 knockout (KO) mice, representing different Hmox1 cellular levels, were generated to study the role of Hmox1 on Hif1α 's response to hypoxia-reoxygenation and gestational age-specific placental lysates. RESULTS: Hif1α was expressed in WT and Het placentas throughout gestation, with protein levels peaking at E8.5 and mRNA levels significantly upregulated from E9.5-E13.5, but significantly lower in Het placentas. Genes associated with angiogenesis (Vegfa, Vegfr1, Mmp2, Cxcl12, Angpt1, Nos3), antioxidants (Sod1, Gpx1), and transcription factors (Ap2, Bach1, Nrf2) were significantly different in Het placentas. In response to in vitro hypoxia-reoxygenation and to WT or Het placental lysates, Hif1α transcription was lower in Het and Hmox1 KO BMDMs compared with WT BMDMs. DISCUSSION: These findings suggest that deficiencies in Hmox1 underlie the insufficient placental Hif1α response to hypoxia-reoxygenation during gestation and subsequently impair downstream placental vascular formation. Therefore, a dysregulation of Hif1α expression caused by any genetic defect or environmental influence in early pregnancy could be the root cause of pregnancy disorders.

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.

Obesogenic diet exposure alters uterine natural killer cell biology and impairs vasculature remodeling in mice†.

Prepregnancy obesity associates with adverse reproductive outcomes that impact maternal and fetal health. While obesity-driven mechanisms underlying adverse pregnancy outcomes remain unclear, local uterine immune cells are strong but poorly studied candidates. Uterine immune cells, particularly uterine natural killer cells (uNKs), play central roles in orchestrating developmental events in pregnancy. However, the effect of obesity on uNK biology is poorly understood. Using an obesogenic high-fat/high-sugar diet (HFD) mouse model, we set out to examine the effects of maternal obesity on uNK composition and establishment of the maternal-fetal interface. HFD exposure resulted in weight gain-dependent increases in systemic inflammation and rates of fetal resorption. While HFD did not affect total uNK frequencies, HFD exposure did lead to an increase in natural cytotoxicity receptor-1 expressing uNKs as well as overall uNK activity. Importantly, HFD-associated changes in uNK coincided with impairments in uterine artery remodeling in mid but not late pregnancy. Comparison of uNK mRNA transcripts from control and HFD mice identified HFD-directed changes in genes that play roles in promoting activity/cytotoxicity and vascular biology. Together, this work provides new insight into how obesity may impact uNK processes central to the establishment of the maternal-fetal interface in early and mid pregnancy. Moreover, these findings shed light on the cellular processes affected by maternal obesity that may relate to overall pregnancy health.

SIRT1 negatively regulates invasive and angiogenic activities of the extravillous trophoblast.

PROBLEM: Dysregulation of extravillous trophoblast (EVT) invasion leads to pregnancy complications, such as pre-eclampsia, fetal growth restriction, and placenta accreta. The aim of this study was to explore the role of SIRT1 in EVT invasion and its underlying mechanism. METHOD OF STUDY: SIRT1-specific siRNA was transfected into Swan 71 cells, an immortalized first trimester trophoblast cell line. The Boyden chamber invasion assay, the scratch wound healing assay, and cell proliferation assay were performed. The expression levels of epithelial-to-mesenchymal transition (EMT) markers, matrix metalloproteinase-2 (MMP-2), MMP-9, p-Akt, Akt, p-p38MAPK, p38MAPK, p-ERK, ERK, p-JNK, JNK, Fas, and Fas ligand (FasL) were examined by western blot. Tube formation assay was conducted by using Matrigel. RESULTS: SIRT1 knockdown by siRNA significantly enhanced invasion and migration as well as the expression of MMP-2, MMP-9, and EMT markers in Swan 71 cells, but reduced proliferation. The effects of SIRT1 knockdown on invasion, migration, proliferation, and endothelial-like tube formation in Swan 71 cells were reversely regulated by blockade of Akt and p38MAPK signaling. In addition, SIRT1 knockdown markedly promoted colocalization of Swan 71 cells to human umbilical vein endothelial cell (HUVEC) networks and induced reduction in Fas and enhancement of FasL. Conditioned media of SIRT1 knockdown-Swan 71 cells caused reduction in cell proliferation and augmentation of cytotoxicity along with increased Fas expression in HUVECs. CONCLUSION: Our results suggest that SIRT1 may be associated with placental development by controlling EVT invasion and spiral artery remodeling via modulation of EMT, MMP-2, MMP-9, Akt/p38MAPK signaling, and Fas/FasL.

uNK cell-derived TGF-ß1 regulates the long noncoding RNA MEG3 to control vascular smooth muscle cell migration and apoptosis in spiral artery remodeling.

Successful pregnancy depends on correct spiral artery (SpA) remodeling, and thus, on normal patterns of the vascular smooth muscle cell (VSMC) apoptosis and migration. Uterine natural killer (uNK) cells-derived transforming growth factor ß1 (TGF-ß1) is known to mediate the separation of VSMC layers via as yet unknown mechanisms. Likewise, the long noncoding RNA maternally expressed gene 3 (MEG3) is a tumor suppressor that has been shown to regulate cancer cell apoptosis and migration; however, its role in VSMC loss is unclear. Thus, the aim of the present study was to assess the effects of uNK-derived TGF-ß1 and MEG3 on VSMC function during SpA. Analyses were conducted to assess the effects of downregulating MEG3 expression, and/or administering treatments to increase or block TGF-ß1 signaling on VSMC survival and behavior. The results of these analyses showed that treating the VSMC with uNK cell-derived supernatant or recombinant human TGF-ß1 promoted MEG3 and matrix metalloprotease 2 expression and VSMC apoptosis and migration, and suppressed VSMC proliferation. Conversely, MEG3 silencing promoted VSMC proliferation and inhibited VSMC apoptosis and migration. Notably, TGF-ß1 signaling induction had no significant effect on the proliferation, apoptosis, nor migration of the MEG3-silenced VSMC. Together, these findings suggest that MEG3 is regulated by uNK-derived TGF-ß1, and itself mediates VSMC apoptosis and migration; thus, it may be an important positive regulator of VSMCs separation during maternal SpA remodeling.

LncRNA MEG3 inhibits trophoblast invasion and trophoblast-mediated VSMC loss in uterine spiral artery remodeling.

Extravillous trophoblasts (EVTs) migrate into uterine decidua and induce vascular smooth muscle cell (VSMC) loss through mechanisms thought to involve migration and apoptosis, achieving complete spiral artery remodeling. Long noncoding RNA maternally expressed gene 3 (MEG3) can regulate diverse cellular processes, such as proliferation and migration, and has been discovered highly expressed in human placenta tissues. However, little is known about the role of MEG3 in modulating EVT functions and EVT-induced VSMC loss. In this study, we first examined the location of MEG3 in human first-trimester placenta by in situ hybridization. Then, exogenous upregulation of MEG3 in HTR-8/SVneo cells was performed to investigate the effects of MEG3 on EVT motility and EVT capacity to displace VSMCs. Meanwhile, the molecules mediating EVT-induced VSMC loss, such as tumor necrosis factor-α (TNF-α), Fas ligand (FasL), and tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) were detected at transcriptional and translational levels. Finally, VSMCs were cocultured with MEG3-upregulated HTR-8/SVneo to explore the role of MEG3 on EVT-mediated VSMC migration and apoptosis. Results showed that MEG3 was expressed in trophoblasts in placental villi and decidua, and MEG3 enhancement inhibited HTR-8/SVneo migration and invasion. Meanwhile, the displacement of VSMCs by HTR-8/SVneo and the expression of TNF-α, FasL and TRAIL in HTR-8/SVneo were reduced following MEG3 overexpression in HTR-8/SVneo. Furthermore, HTR-8/SVneo with MEG3 upregulation impaired VSMC migration and apoptosis. The PI3K/Akt pathway, which is possibly downstream, was inactivated in MEG3-upregulated HTR-8/SVneo. These findings suggest that MEG3 might be a negative regulator of spiral artery remodeling via suppressing EVT invasion and EVT-mediated VSMC loss.