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Early prediction of pre-eclampsia using circulating placental exosomes: Newer insights
For correspondence: Dr Taruna Madan, Department of Innate Immunity, ICMR-National Institute for Research in Reproductive & Child Health, Jehangir Merwanji St., Opp. KEM Hospital, Parel, Mumbai 400 012, Maharashtra, India e-mail: taruna_m@hotmail.com
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Received: ,
This article was originally published by Wolters Kluwer - Medknow and was migrated to Scientific Scholar after the change of Publisher.
Abstract
Pre-eclampsia (PE), a multifactorial de novo hypertensive pregnancy disorder, is one of the leading causes of foeto-maternal morbidity and mortality. Currently, antihypertensive drugs are the first-line therapy for PE and evidence suggests that low-dose aspirin initiated early in high risk pregnancies may reduce the risk of development or severity of PE. However, an early prediction of this disorder remains an unmet clinical challenge. Several potential serum biomarkers associated with maternal immunoregulation and placental angiogenesis have been evaluated but are ineffective and inconsistent for early prediction. Although placental biomarkers would be more specific and sensitive in predicting the risk of PE, accessing the placenta during pregnancy is not feasible. Circulating placental exosomes (pEXO), originating from foeto-maternal interface, are being evaluated as the placenta’s surrogate and the best source of non-invasive placental biomarkers. pEXO appear in the maternal circulation starting from six weeks of gestation and its dynamic biological cargo across pregnancy is associated with successful pregnancy outcomes. Therefore, monitoring changes in pEXO expression profiles could provide new insights into the prediction, diagnosis and treatment of PE. This narrative review comprehensively summarizes the available literature on the candidate predictive circulating biomarkers evaluated for PE to date. In particular, the review elucidates the current knowledge of distinct molecular signatures emanating from pEXO in pre-eclamptic women to support the discovery of novel early predictive biomarkers for effective intervention and management of the disease.
Keywords
Early prediction
exosomes
placenta
placental exosomes
pre-eclampsia
pregnancy
serum biomarkers
Hippocrates (460-370 BC), the father of clinical medicine, listed headaches, drowsiness, malaise and convulsions as dangers during pregnancy in his maxim XXXI 507. These spasms noted by Hippocrates were later called ‘Eclampsia’ and the severe hypertensive pregnancy state prior to these convulsions was known as ‘Pre-eclampsia’ (PE)1. At present, the global prevalence of PE is ~4.6 per cent2. A secondary analysis of a randomized controlled clinical Community-Level Interventions for Pre-eclampsia trial (NCT01911494) involving 27 geographical clusters across India, Pakistan, Nigeria and Mozambique studied between 2013-2017 estimated 3.8 per cent incidence rate of PE in Indian pregnant women3. The American College of Obstetricians and Gynecologists (ACOG) Practice June 2020 bulletin enlists the clinical features (Box) of PE, a de novo complication of hypertension during pregnancy that occurs after 20 wk of gestation (WOG)4. Risk factors associated with PE include multiple pregnancies, kidney disease, antiphospholipid (aPL) syndrome, type 1 or type 2 diabetes and history of chronic hypertension, maternal age ≥35 yr, first pregnancy, body mass index >30, family history of PE, sociodemographic characteristics, and medical history of PE5. PE pregnancies can ultimately lead to intrauterine growth restriction and are small for gestational age infants, and in severe cases – pre-term labour with adverse foetal outcomes. The disease manifests early during pregnancy (~10-16 WOG), but the clinical signs and systemic effects become apparent quite late, i.e., by the end of the second trimester. This is one of the major reasons for its poor early prediction, ultimately hampering effective treatment and intervention. Thus, understanding the PE pathogenesis is the need of the hour.
| New-onset hypertension with elevated systolic blood pressure (≤ = 140 mm Hg) or diastolic blood pressure of >90 mm Hg at two different time points at least four hours apart Proteinuria (≥=300 mg/24 h urine), oliguria, renal function compromise (serum creatinine concentration ≥=1.1 mg/dl) Headache (untreatable via painkillers), cerebral disturbances, hypertensive retinopathy |
| HELLP syndrome . Haemolysis (elevated LDH in serum; from destruction of blood cells), elevated liver enzymes (LDH, AST, ALT to double the normal value), low platelet count (<150,000/μl) |
| Shortness of breath and pulmonary oedema (fluid accumulation in lungs) |
LDH, lactate dehydrogenase; AST, aspartate aminotransferases; ALT, alanine aminotransferase; HELLP, haemolysis, elevated liver enzymes and low platelet
The International Society for the Study of Hypertension in Pregnancy and ACOG guidelines classify PE into 2 sub-types, early-onset PE (EOPE) and late-onset PE (LOPE)4,6. EOPE commonly occurs before 34 WOG and comprises ~5-20 per cent of all PE cases whereas LOPE generally develops after 34 WOG and comprising ~75-80 per cent of all PE cases. Despite the presenting symptoms being similar, these are linked to different maternal and foetal outcomes, heredity, biochemical markers and clinical features. Leavey et al7 analyzed placental gene expression of clinically confirmed PE cases using microarray and reported three clinically significant possible aetiologies of PE - maternal, placental and immunological. Although sub-categorization is beneficial for understanding the pathophysiology and prognosis, clinically, the demarcation is not clear and the patients may reflect overlapping pathology.
Early prediction can prevent pre-eclampsia (PE)
ACOG and the Society of Maternal and Fetal Medicine, along with the World Health Organization and the American Heart Association’s American Stroke Association recommend a daily low dose of aspirin (81 mg/day) between 12 and 28 WOG (optimally before 16 WOG) till delivery for pregnant women at high risk for developing PE7. A study involving 1776 high risk women found a significant reduction in pre-term PE incidence when administered 150 mg of aspirin from 11 to 14 WOG to 36 WOG compared to placebo8. Similarly, a meta-analysis of 45 trials showed that aspirin dosing at <16 wk reduced severe PE and foetal growth restriction, suggesting that aspirin could be an effective therapy for reducing the severity and risk of developing PE in high risk pregnancies if started during early gestation9. Early prediction of PE is therefore of great importance, as optimal treatment with aspirin from early pregnancy could reduce the prevalence and severity of PE in high risk pregnant women.
Hypertension, a hallmark of PE as monitored via blood pressure measurements is useful only when PE has already developed with inefficient prediction for LOPE. In many clinical settings, two screening tests are done- first, early in pregnancy to predict pre-term PE and another test later in pregnancy to check whether PE has developed or is likely to develop. The present guidelines, although easy to apply to all pregnant women with no additional cost or test, have a major drawback of low sensitivity10 with only 41 per cent of women (who test positive) developing pre-term PE.
The fact that the cause of PE is multifactorial (genetic, environmental, immune dysregulation, angiogenesis) and there is no single hypothesis that could explain its pathophysiology, the identification of one or more highly sensitive and specific predictive biomarker(s) becomes even more challenging11. It is plausible that a combination of several dysregulated factors originating from injured tissues including the liver and kidneys as well as the primary affected organ, the placenta, may aid in the discovery of a precise yet sensitive biomarker based blood test for prediction of PE due to the significant heterogeneity of this disease.
Recently, dysregulated proteins, miRNAs, metabolites, associated with PE have been found within a class of extracellular vesicles (EVs) termed exosomes isolated from mother’s biological fluids (serum/plasma/urine)12. Exosomes derived from pre-eclamptic placenta showed high levels of PE-specific cargo and can aid in early prediction of the disease13. These circulating nanovesicles could accurately mirror the physiological state of the trophoblasts that are involved in spiral artery remodelling. Thus, factors derived from maternal sera, along with circulating placental exosomes (pEXO), that may have the potential to predict and prevent PE14 are reviewed here.
Search strategy and selection
This narrative review was conceptualised as a ‘State-of-the-art review’15 addressing the current state of knowledge and priorities for future investigations of serum and circulating placental exosomal biomarkers for early prediction of PE. The MEDLINE, PubMed database and Google Scholar were searched using the following keywords: (i) PE AND early prediction AND serum AND biomarkers and (ii) PE AND exosomes. Only articles published in English language over the last century and this millennium were considered without any date restriction with most recent published in 2022. Based on the in-depth reading of the retrieved articles (original publications) a synthesis has been presented in this manuscript.
Current status of various serum biomarkers for early prediction of pre-eclampsia
Numerous research groups have investigated the prognostic value of various individual circulating (serum/plasma derived) biochemical and biophysical indicators and potential combinations of them for predicting PE.
It is adequately reported that circulating levels of soluble fms-like tyrosine kinase 1 (sFlt-1) and placental growth factor (PlGF), which are anti-angiogenic and pro-angiogenic factors, respectively, are dysregulated in PE women16. sFlt-1/PlGF ratio demonstrated the highest significe (7.33) in the PE group in a meta-analysis, suggesting it to be most useful combination of biomarkers for screening with reduction in the admissions particularly for women with suspected PE. However, this ratio has better predictive performance during the third trimester of pregnancy only. On the contrary, the ratio is only moderately accurate in predicting who will develop PE within a month, with 36.7 per cent positive predictive value and ~66 per cent sensitivity. Moreover, another drawback of using this combination biomarker is its inability to distinguish between mild and severe PE16.
In addition to the sFlt-1/PlGF ratio, PlGF alone has also been previously evaluated for PE screening in women with suspected early PE. Plasma PlGF concentrations <100 pg/ml in women with suspected PE at <35 weeks gestation, performed with a sensitivity of 96 per cent and a negative predictive value of 98 per cent to predict whether PE will develop in next 14 days or not. This test has a high negative predictive value compared to other common clinical tests such as blood pressure measurement, liver function tests and proteinuria. It suggestively also reduces time to diagnosis, adverse maternal outcomes, and health service costs. However, its effectiveness is limited to cases of suspected PE at <35 WOG and is not applicable to the general pregnant population without obvious clinical manifestations of the disease17. Furthermore, the sFlt-1/PlGF ratio and PlGF alone, when compared, showed similar results in predicting delivery within two weeks in cases of suspected early PE. Furthermore, sFlt-1/PlGF ratio and PlGF alone, when compared, performed similarly in predicting delivery within two weeks in cases of suspected pre-term PE.
To fill this gap, a new early pregnancy screening algorithm has been developed and validated to predict early PE, combining maternal characteristics with mean arterial blood pressure, uterine arterial resistance, and pulmonary vascular resistance. This test is good at predicting EOPE compared to clinical risk factors alone and significantly reduced the incidence of pre-term PE18. It showed greater sensitivity in predicting early or pre-term PE (~82%) developing within two weeks; however, it does not predict term PE with high sensitivity with an overall detection of approximately 42 per cent of all cases of PE19. However, due to the high cost of tests measuring PlGF levels and ultrasonography, implementation is not yet widespread.
Another vaso-active factor, VEGF (Vascular endothelial growth factor), is a key regulator of vascular function and angiogenesis, known to be dysregulated in PE with association of its gene polymorphisms to an elevated risk of PE18. Further, maternal circulating factors N-terminal prohormone of brain natriuretic peptide20, soluble endoglin (sEng)21, aspartate aminotransferase (AST)22 and serum alanine aminotransferase (ALT)22, cell-free foetal DNA (cffDNA)23, vasoactive factors24, have been highlighted as prospective predictive biomarkers for PE. Women with PE show significantly elevated liver enzymes - AST, ALT and total cholesterol compared to healthy pregnant mothers25. Arterial stretch-sensitive baroreceptors are also impaired in PE which can predict PE with 71 per cent accuracy along with Doppler sonography26.
A study aimed to predict early-onset PE in 327 pregnant women participants during first-trimester screening with a combination of serum histidine-rich glycoprotein level (an angiogenesis regulator known to be lower in PE) and transabdominal uterine artery pulsatility index (PI)27 showed low sensitivity (25%) and positive predictive value (10%). A prospective observational study evaluated serum levels of split and hairy-related protein 1 (SHARP1) and uterine artery Doppler at 11-13 wk28. SHARP1 has a role in the adaptation to oxygen concentration29. SHARP1 serum levels were significantly lower in PE (3.6 ng/ml) compared to healthy pregnancies (4.7 ng/ml, P<0.01). Thirty five women (8.6%) later developed PE and 6 out of them (1.5%) developed EOPE28. A recent study of the same group in the same gestational age group examined serum levels of hypoxia-inducible factor 1-alpha (HIF-1α), uterine artery Doppler ultrasound and final foetal outcome30. They observed significantly higher levels of HIF-1α (P<0.001) in the serum of pre-eclamptic pregnant women (mean 1315.2 pg/ml) compared to controls (mean 699.5 pg/ml) but no change in uterine artery pulsation (PI)30. However, it is important to note that the predictive value of these serum biomarkers is only nine per cent for PE and relatively lower (~1.5%) for EOPE. Pentraxin 3 (PTX3), an inflammatory marker, is elevated at 11-14 wk in pregnant women who developed PE subsequently31. Similarly, asymmetric dimethylarginine is a metabolite (a competitive inhibitor of nitric oxide synthase with a key role in the pathophysiology of endothelial dysfunction), which increases in the first trimester in pregnant women who eventually developed EOPE32. Pro-inflammatory protein, high mobility group Box expressed in trophoblasts, is also increased in PE33. Three serial serum samples from the pregnant women (n=922) demonstrated significantly decreased levels of SP-A (Surfactant protein-A), SP-D (Surfactant protein-D) along with P4/E2 ratio at 10-14 WOG in women with subsequent severe EOPE, implicating their potential to act as a predictive biomarker with a synergistic 90 per cent diagnostic accuracy34. However, more studies with larger cohorts are needed to validate these findings.
Apart from circulating proteins, miRNAs highly expressed in the placenta - C19MC miRNAs such as miR-517-5p, miR-518b and miR-520-h are upregulated in the first trimester maternal serum of women who eventually developed PE. However, a sensitivity of 42.9 per cent and specificity of 86.2 per cent were seen for miR-517-5p35. Furthermore, circulating placenta-derived cffDNA has paved a new direction for non-invasive prenatal testing. The circulating levels of cffDNA increase during PE pregnancy at an early stage, reflecting the placental apoptosis and necrosis associated with the disease. The principle of cffDNA analysis is based on the promoter, the RASSF1A gene (Ras binding domain family 1 isoform A). A study reported by Saraswathy and group showed a three-fold increase in hypermethylated RASSF1A levels in high risk PE pregnancies before clinical manifestations36. Apart from RASSF1A, sex-determining region Y, β-globin and DYS (DNA Y-chromosome segment) have shown potential for PE prediction37. The cutoff value of cffDNA concentration at 22.54 GE/ml can predict PE with a sensitivity of 85 per cent and specificity of 81.8 per cent38, making cffDNA concentration a valuable predictive biomarker. Furthermore, additional studies are needed to minimize population heterogeneity and outcome assessments. Circulating cell-free RNA (cfRNA) has shown increased levels during early PE pregnancy39. Therefore, cfRNA can monitor pregnancy and determine the risk of PE several months prior to appearance of clinical symptoms39. cfRNA changes are reportedly enriched in neuromuscular, endothelial, immune, and tissue-specific genes, reflecting the correlation between PE progression and maternal organ health40. According to Moufarrej et al39, the independent identification and validation of a panel of 18 genes, measured from five to 16 wk of gestation, can form the basis for liquid biopsy tests to predict PE in high risk pregnancies. cfRNA profiling is therefore considered a novel, efficient and non-invasive tool to predict PE and uncover signalling pathways underlying its pathogenesis.
Although the past decade has seen exciting progress in the introduction of new candidate predictive biomarkers, there remains a large unmet clinical need highlighting the need for more specific placenta-derived biomarkers.
Serum exosomes in healthy pregnancy & pre-eclampsia
The new focus of disease-based biomarker discovery - ‘exosomes’ are a subset of EVs approximately 30-150 nm in size, with densities (1.13-1.19 g/ml) and is produced inside the endothelial compartments of almost all eukaryotic cells. They have a bilayer phospholipid shell and are exclusively enriched in tetraspanins (endothelial markers such as CD9, CD63 and CD81), the endothelial sorting complex required for transport and its interacting proteins, ALIX and TSG-10141. Originally, thought as cellular waste disposal units, but now known to play an important role in cell-to-cell communication. These nanovesicles reflect the molecular signature (miRNAs, mRNA, DNA, proteins, lipids, etc.) of their parent cells and upon exocytosis transfer the biological signal to the target cells via paracrine signalling41. However, the process of sorting specific cargo inside each exosome is not fully elucidated. Varied metabolic cargo including enzymes ATPase, pyruvate kinase type M2, glyceraldehyde-phosphate dehydrogenase, enolase 1 and phosphoglycerate kinase 1 and other heat shock proteins (like HSP70 and HSP90) and immunologically important MHC molecules are also found in exosomes. As a source of candidate biomarkers, exosomes have incredible potential as they are naturally stable in the circulation and can easily cross biological barriers such as the blood–brain barrier12.
Maternal serum exosomes include the exosomes of placental origin and appear promising as various studies report their involvement in immune tolerance, foeto-maternal cross-talk, embryo implantation, angiogenesis during a healthy pregnancy and endothelial dysfunction - a key pathological feature of PE. In vitro studies reveal that circulating maternal and umbilical cord-derived exosomes enhance migration and proliferation of endothelial cells, essential for spiral artery remodelling during pregnancy42,43. Wang et al reported that cord blood mesenchymal stem cell (MSC) (hUC-MSCs)-exosome-derived miRNA-133b promotes trophoblast proliferation, invasion and migration by targeting serum/glucocorticoid-regulated kinase 1 (SGK1)44. Previous studies have shown that umbilical cord plasma-derived exosomes induced vascular dysfunction by inhibiting HMGCS1 (3-hydroxy-3-methylglutaryl-CoA synthase 1, involved in cellular invasion and proliferation) in human umbilical vein endothelial cells45. An ex vivo model of placental explant culture showed that the proteome of exosomes is associated with vesicle transport and inflammation, supporting the view that exosomes are involved in immune regulation during early pregnancy46. Human placental ERV-W protein (endogenous retrovirus group W envelope member 1 or syncytin-1) is present in the serum-derived exosomes and is important for foeto-maternal immune tolerance47. STB-EVs (syncytiotrophoblast-derived EVs), which include exosomes, interact with maternal immune cells to stimulate the production of pro-inflammatory cytokines48.
Greening et al49 have reported that the cargo of endometrial receptive phase epithelial cell-derived exosomes consists mainly of proteins for adhesion, migration, invasion and extracellular matrix remodelling essential for embryo implantation and hormonal profile can alter their contents. Accumulated evidence suggests that exosomes release from a cell depends on oxygen tension and glucose. The bioactivity of exosomes gets reduced with gestation depicting that the content, as well as the concentration of exosomes, gets altered under different physiology50,51.
Shedding of the syncytiotrophoblast-derived microparticles (STBM; size range: 20 to 3000 nm) in the maternal circulation increased significantly in PE compared to healthy controls52. Circulating exosomes are also reported to increase in pre-eclamptic women and contain altered molecular cargo making them pro-inflammatory, anti-angiogenic and pro-coagulant, thereby generating maternal systemic inflammation, endothelial vascular dysfunction and clotting system activation, the defining features of PE53. EVs, which include exosomes from pre-eclamptic pregnancies, cause inflammasome activation in trophoblasts via accumulation of activated platelets, leading to PE-like symptoms in pregnant mice54.
Recently, specific proteins, miRNAs associated with PE cases have been found within exosomes isolated from the maternal sera55,56. Evaluation of the profile of these circulating exosomes derived from PE patients could be beneficial as they are present since early gestation with their concentration and cargo altering under a pathological state. Comparative proteomic analysis of human umbilical cord-derived exosomes revealed major coagulation and complement proteins to be differentially regulated in pre-eclamptic pregnancies57. A recent study reported 289 differentially regulated long non-coding RNAs in plasma-derived exosomes of pre-eclamptic pregnant women, of which 159 were enriched in cancer, metabolic and PI3K-Akt signalling pathways. These pathways are reportedly related to PE development58. Table I59,60 tabulates the differentially expressed factors in the circulating exosomes during early pregnancy as potential predictive biomarkers for PE. Table II59-69 summarizes the exosomal miRNAs reported to be dysregulated in PE and their plausible biological effects. Further studies are needed to evaluate the potential of these exosomal-derived factors including the exo-miRNAs to predict PE.
| Candidate predictive biomarker | Detection stage | Role in pathogenesis | Source | Regulation | References |
|---|---|---|---|---|---|
| miR-134, miR-196b, miR-376c, miR-486-3p, miR-590-5p | First trimester | Inhibits trophoblast cell invasion by targeting ITGB-1 | Plasma | Upregulated | 59 |
| hsa-miR-486-1-5p hsa-miR-486-2-5p | 11-14 wk | Inhibition of cell migration, proliferation and invasion | Plasma | Upregulated | 60 |
ITGB-1, integrin subunit beta 1
| Identified exo-miRNAs | Biological process | References |
|---|---|---|
| Upregulated | ||
| miR-23a-3p | Targets ATG12-mediated autophagy | 61 |
| miR-370-3p | Inhibitory towards proliferation, migration and invasion, promotive of apoptosis of HTR-8/SVneo cells | 62 |
| miR-210 | Plausible role in impairment of placental mitochondria and oxidative stress through inhibition of ISCU | 63,64 |
| miR-486 | Inhibits trophoblast invasion, migration and proliferation | 60 |
| miR-155 | eNOS expression inhibited in HUVECs | 65 |
| miR-302c | cyclin D1 and AKT1 genes are targetted which increases anti-angiogenic sENG levels in the circulation | 59 |
| miR-618 | Regulates IRF-8, which regulates the inflammatory immune response | 59 |
| miR-153-3p | Inhibits cell proliferation and invasion and induces apoptosis | 66 |
| Downregulated | ||
| miR-125a-5p | Modulates proliferation, migration and angiogenesis of HTR8/SVneo cells by regulating VEGF-A | 67 |
| miR-517-5p, miR-520a-5p and miR-525-5p | Regulates cell proliferation and invasion | 68 |
| miR-548c-5p | Anti-inflammatory factor which regulates PTPRO involved in the NF-κB signalling pathway | 69 |
| miR-196b | Regulation of expression of proliferation-related genes | 59 |
miRNAs, microRNAs; sENG, soluble endoglin; IRF-8, interferon regulatory factor-8, PTPRO, protein tyrosine phosphatase receptor type O factor; ATG12, autophagy related 12; ISCU, iron-sulphur cluster assembly enzyme; eNOS, endothelial nitric oxide synthase; AKT1, AKT serine/threonine kinase 1; VEGF-A, vascular endothelial growth factor A; NF-κB, nuclear factor kappa B; HUVECs, human umbilical vein endothelial cells; HTR-8/SVneo, placental cell lines
These literary evidences indicate that circulating exosomes are effectively involved in interactions between the foetal, placental and maternal trinity by the exchange of biological cargoes to regulate key physiological processes required for immune tolerance, angiogenesis, embryo implantation, migration and/or cellular proliferation. Therefore, the authors feel that the changes in exosome content are likely to directly influence these physiological processes based on the differential effects induced by exosomes of normotensive and pre-eclamptic origin. Importantly, the maternal exosomes though comprehensive may not be selective or specific as a source of biomarkers in comparison with the isolated circulating pEXO.
Isolation of circulating placental exosomes (pEXO)
Placental vesicles were initially identified as membrane fragments in the maternal circulation of third-trimester normal pregnancies by Taylor’s group and were positive for placental alkaline phosphatase (PLAP), a commonly used marker for the placenta70. They can be isolated through immunoprecipitation using monoclonal anti-PLAP conjugated magnetic beads, ultracentrifugation at >100,000 g, gradient centrifugation or size exclusion chromatography. Although all the isolation methods are limited either in terms of yield and/or purity of exosomes. The concentration of PLAP+ve pEXO positively correlated with the placental weight at delivery indicating that their amount and cargo can be a useful non-invasive tool for detecting placental physiology71. The presence of PLAP in other tissues apart from the placenta may lead to contamination with the non-pEXO. Therefore, more specific markers for the placenta are needed to get a population enriched with pEXO. Some of the proteins which are highly enriched in the placenta are hCG, PAPP-A and HLA-G. A schema depicting the commonly observed markers in the pEXO is represented in Figure. Attempts are being made to investigate the composition and biological functions of these exosomes during different phases of pregnancy. As blood is the preferred biological fluid used for isolating exosomes, it is important to note some of the factors including the choice of anticoagulant, venipuncture, blood sample processing, circadian rhythms, fasting status, haemolysis and platelet activation may affect exosomal composition and characterization during sample processing.
- A schematic illustration of the cargo typically carried by placental exosomes. The molecular cargo of a placental exosome mainly consists of tetraspanins - CD63, CD9, PLAP, cytoskeletal proteins, syncytin-1/2, apoptotic proteins Fas ligand, TNF-related apoptosis-inducing ligand, ULBP-1, ESCRTs, nucleic acids (DNA, mRNA, shRNA, lncRNA and miRNAs) of the originating trophoblasts47,71-74(Part of the figure created with BioRender.com).
Circulating pEXO in healthy pregnancy
The pEXO have a list of functions to their merit for maintenance of a healthy pregnancy via regulating maternal immune tolerance and angiogenesis.
Maternal immune tolerance: pEXO transformed macrophages into a M2-like phenotype with an upregulation of M2 markers, specifically CD163, CD206, CD209, IL-10 and IDO-1, with a significant reduction in the proliferation of helper and cytotoxic T cells and increased regulatory T cells75. The phagocytic CD14++CD16- classical monocytes were transmuted to the CD14+CD16+ intermediate monocytes with enhanced migratory capacity76. With the cargo of proapoptotic proteins FasL, TRAIL and programmed death-ligand 1 (PD-L1), they are involved in the in vitro T-cell-specific apoptosis with loss of CD3-zeta72. The pEXO downregulated the NKG2D receptor on NK cells by bearing MHC-class 1 chain-related molecules (MIC-A and B) and UL16 binding protein, inhibited the cytolytic activity of NK cells, CD8+ along with gamma delta T (γς T) cells and contributed to the immune escape for the foetus during early pregnancy77. The pEXO are also involved in transforming growth factor-β mediated immune suppression and PD-L1-mediated regulatory T-cell (Tregs) activation78. Trophoblasts are known to show elevated levels of HSPE1 (heat shock protein family E) expression which is known to suppress T-cell activation. Kovács et al79 showed that HSPE1 present in trophoblast-derived EVs induced Tregs differentiation (immune suppression) by binding human CD4+ T-cells and differentiating them into Tregs in vitro80. The pEXO induced the release of miR-499 and inhibited the pro-inflammatory NF-κB signalling pathway by targeting the Lin28B/let-7-ras signalling pathway81.
Angiogenesis: The pEXO have been shown to promote EVT (extravascular trophoblast) invasion in vitro with increased invasion under hypoxia and are rich in EVT invasion and angiogenesis-promoting factors such as serine proteases, metalloproteases (MMP-12 and MMP-9), MAPK, MAPKp38 and VEGF51. Human placenta derived mesenchymal stem cell exosomes got incorporated into the endothelial cells, induced angiogenesis-related genes, namely VEGF, MPC-1, tie-2/TEK, IGF, VEGFR2 and angiopoietin-2, and stimulated angiogenesis by endothelial tube formation and migration82. Umbilical cord-MSC-derived exosomes were enriched in miRNA-150 that is pro-angiogenic in utero and aids in the upregulation of VEGF and Notch183. The miR486-1-5p and miR486-2-5p contained in the pEXO are involved in migration, placental development, placental perfusion and vascularization84.
Circulating pEXO in pre-eclampsia
The pioneering study by Pillay et al71 reported that the ratio of the PLAP+pEXO (estimated as amount of PLAP) to the total exosomes was significantly decreased in the third trimester in both, the EOPE and LOPE women compared to the respective gestational age-matched healthy normotensive controls. Placenta-derived EVs derived from an injured placenta induced symptoms of PE in a mouse model85. A mouse model study in which human PE-pEXO containing high levels of sFlt-1 and sEng-induced PE symptoms (impaired angiogenesis) in mice showed that these bilayered nanoscale human exosomes evaded mouse macrophages and overcame the barriers to reach amnion via circulation86. A recent study showed the presence of ferroptosis-related genes, known to be involved in pathogenesis of PE, in the placental EVs derived from the syncytiotrophoblast87. Differential levels of non-coding RNAs (miR-155, miR-152, miR-18a and miR-221-3p) were reported in the circulating pEXO of pre-eclamptic women. The pre-eclamptic pEXO showed increased levels of neprilysin, a metalloproteinase that cleaves vasodilators and is associated with hypertension88. Decreased nitric oxide synthase levels were observed in the pre-eclamptic pEXO compared to the normotensive group89. The miR-155 was highly enriched in the placenta-associated serum exosomes of PE women that was shown to downregulate endothelial nitric oxide synthase and NO production66. Salomon et al60 identified ~300 miRNAs in exosomes throughout gestation, and found two candidate miRNAs- hsa-miR-486-1-5p and hsa-miR-486-2-5p in PE pregnancies.
Elevated levels of sFlt-1 have been observed in pEXO derived from pre-eclamptic placental tissue; these pEXO can thus function as a ‘sequester’ of PlGF and VEGF in the circulation, to prevent downstream interactions between angiogenic factors and the endothelial cells90.
aPL antibodies are known to increase the risk of PE by damaging mitochondria in the syncytiotrophoblast and in turn releasing mitochondrial DNA, a damage-associated molecular pattern (DAMP). The DAMP gets recognized by TLR9 leading to endothelial cell activation, the classic feature of PE pathogenesis. The pEXO derived from syncytiotrophoblast exposed to aPL exhibited increased mitochondrial DNA (mt-DNA) levels91.
Existing studies that identified candidate early predictive biomarkers of PE in the pEXO are represented in Table III90-92.
| Candidate predictive biomarker | Detection stage | Role in pathogenesis | Source | Regulation | References |
|---|---|---|---|---|---|
| sFlt-1 | First trimester | Injures endothelial cell health | Placenta | Upregulated | 90 |
| MMP9 MAPK | 8-12 wk | EVT invasion and angiogenesis- promoting factors | Cytotrophoblasts | Upregulated | 92 |
| mtDNA | First trimester | Activates endothelial cells | Placenta | Upregulated | 91 |
MMP9, matrix metalloproteinase peptide 9; EVT, extravascular trophoblast; MAPK, mitogen-activated protein kinase; mtDNA, mitochondrial DNA
Inference and future directions
To conclude, pEXO represent a liquid biopsy to predict the metabolic and functional state of the placenta in early pregnancy and are promising alternative to the placental tissues inaccessible for sampling during pregnancy. The research on pEXO is still at a preliminary stage, though studies have confirmed that differentially abundant proteins, lipids, metabolites and genetic material (miRNAs and DNA) in the circulating pEXO may reflect the placental physiology/pathophysiology. Procedures of isolation of pEXO need to be standardized for diagnostic purposes as in vitro manipulations (such as perfusion and tissue culture) during exosome isolation can perturb their molecular signature. The need of the hour is multi-omics analyses of the pEXO derived from first-trimester pregnant women that subsequently develop PE in large prospective cohort studies to facilitate the identification of a catalogue of early and specific predictive biomarkers. In view of the promising high sensitivity and specificity of biomarkers derived from pEXO, future multicentric cohort studies are warranted to validate their relevance in clinical application.
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Conflicts of interest
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Supplementary Figure
Supplementary Figure A schematic diagram illustrating production and known functions of placental exosomes (pEXO). The release of pEXO in the maternal circulation and isolation of these placental nanovesicles for omics analysis. The figure describes the known functions of pEXO in a healthy pregnancy. They are involved in immune suppression and spiral artery remodelling during a healthy pregnancy. However, their altered molecular signatures can contribute to the pathogenesis of placental disorders including PE by disrupting the physiological maternal immune tolerance and spiral arteries remodelling93,94 (Part of the figure created with BioRender.com & Servier Medical Art). (Servier is licensed under a Creative Commons Attribution 3.0 Unported License https://creativecommons.org/licenses/by/3.0/).References
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