Placenta-derived Extracellular Vesicles in Maternal Plasma of Hb Bart’s Fetuses

Placenta-derived Extracellular Vesicles in Maternal Plasma of Hb Bart’s Fetuses | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Placenta-derived Extracellular Vesicles in Maternal Plasma of Hb Bart’s Fetuses Piya Chaemsaithong, Puntabut Warintaksa, Pornpimol Ruangvutilert, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4163008/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Introduction: Alpha-thalassemia is the most common cause of hydrops fetalis among Southeast Asians (also called “Bart’s hydrops fetalis). This condition is considered a fatal disorder; therefore, prenatal screening and diagnosis are extremely important. Changes in soluble analytes in the maternal circulation, such as hormones and angiogenic factors are not predictive of Hb Bart’s fetalis condition. This condition is associated with placental hypoxia, which may trigger the release of placenta-derived extracellular vesicles (EVs) in maternal circulation. Therefore, the determination of changes in placenta-derived exosome and its protein content could provide additional insight into the disease pathways of this disorder. Objectives : We aim to characterize: 1) maternal plasma levels of placenta-derived EVs; and 2) proteomics profiles of maternal plasma EVs in women with Bart’s fetalis fetuses. Methods: This prospective cohort study included women with the following groups: 1) normal pregnancy or control group: women with couple at risk for Hb Bart’s fetuses but subsequently proven as non-Hb Bart’s group and delivered at term without maternal or neonatal complications (Group 1) (n=7); 2) Hb Bart’s with hydropic features group (Group 2) (n=4); 3) Hb Bart’s without hydropic features group (Group 3) (n=7); 4) a disease control group which consisted of women who subsequently delivered with placental associated conditions (Group 4) (n=4); and 5) women with hydrops fetalis from non-Bart’s causes (n=5) (Group 5). Maternal plasma EVs were isolated by the combination of stepwise centrifugation, ultrafiltration and qEV size exclusion chromatography. The EVs were characterized by the particle size, morphology and protein markers. Isolated EVs and their plasma were measured placental alkaline phosphatase (PLAP) level using ELISA. Mass spectrometry was used to determine EV proteomics profile. A p-value of <0.05 was used to infer significance, unless multiple testing was involved, with the false discovery rate controlled at the 10% level (q<0.1). Results: 1) EV particle size in Bart fetuses with hydropic features was significantly smaller than that in normal pregnancy and disease control groups; 2) Bart fetuses with hydropic features had higher maternal placenta-derived PLAP-contained EVs (PLAP-EVs) than that in normal pregnancy; although, PLAP-EV level was highest in the placental associated complications group; 3) Bart fetuses without hydropic features tended to have higher maternal PLAP-EV level compared to normal pregnancy group; 4) hydropic fetuses due to non-Bart’s causes had similar PLAP level compared to normal pregnancy group; 5) among the 16/106 differentially expressed EV proteins, hnRNPA2B1 protein was the highest in Bart fetuses with hydropic features group compared to placental associated complication or normal pregnancy groups; 6) sixteen differentially expressed EV proteins were involved in fibrinogen complex, fibrin clot formation and integrin signaling pathway. Conclusions: This is the first study of placenta-derived EVs in women with Hb Bart’s fetalis. EV particle size is significantly smaller but maternal PLAP-EV level is significantly higher in women with Hb Bart’s fetalis fetuses compared to normal pregnancy. In addition, several EV proteins were differential expressed in women with Bart’s fetuses and these proteins involve in aberrant immune response, pro-inflammatory cytokine regulation mediated by TLRs-MyD88-IRAK4-MAPK axis and vascular injuries as part of pathophysiology of placental edema and hypoxia in Bart’s hydrops. The result of this study supports future research to determine whether placenta-derived EVs in maternal circulation can be used as a liquid biopsy or non-invasive prenatal test for the early identification of Bart’s fetuses. This will ultimately reduce the rate of unnecessary invasive prenatal diagnosis testing. Biological sciences/Molecular biology/Proteomics Health sciences/Medical research/Translational research Bart exosomes extracellular vesicle hydrops fetalis placenta-derived exosome proteomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 What are the novel findings of this work? We reported herein the first study of placenta-derived extracellular vesicle (EVs) in women with Hb Bart’s fetalis. Placental alkaline phosphatase (PLAP)-EV level is significantly higher in women with Hb Bart’s fetalis fetuses compared to normal pregnancy. In addition, several EV proteins are differential expressed in women with Bart’s fetuses and these proteins involve in zymogen activation, fibrinogen complex, vesicles in cellular component, fibrin clot formation and integrin signaling. The result of this study supports future research to determine whether placenta-derived EVs in maternal circulation can be used as a liquid biopsy or non-invasive prenatal test for the early identification of Bart’s fetuses. What are the clinical implications of this work? Our study has demonstrated a profile of extracellular vesicle (EVs) in women with Hb Bart’s fetalis fetuses. Specifically, women with Bart’s fetuses have smaller EVs particle size but higher placental alkaline phosphatase (PLAP)-EV level than those with normal pregnancies. Several proteomics EV proteins are differentially expressed in women with Bart’s fetuses and these proteins involves in zymogen activation, fibrinogen complex, vesicles in cellular component, fibrin clot formation and integrin signaling. We envision that the non-invasive liquid biopsy could provide information in relation to placental health in women with Bart’s fetuses. In this report, the liquid biopsy based on an examination of placenta-derived EVs in maternal blood for the identification of Hb Bart’s fetuses is feasible and this may ultimately reduce the rate of unnecessary invasive prenatal diagnosis testing in the future. Introduction Alpha-thalassemia major (“homozygous alpha-thalassemia-1”) or hemoglobin Bart’s (Hb Bart) disease is the most severe form of thalassemia disease. Such disease is commonly found in Asia, especially Southeast Asia [1, 2, 3, 4, 5] , with an approximate prevalence of 0.23 % [6, 7] . The gene mutation frequency of Southeast Asia is as high as 4.5%-5.0%, leading to a high prevalence of the homozygous mutation (-SEA/-SEA) [8] . Homozygous mutation causes hydrops fetalis leading to fetal death or stillbirth. Additionally, fetal hydrops can lead to a syndrome called “Ballantyne or mirror syndrome”. Such syndrome reflects the simultaneously edematous state of the mother, fetus, and placenta (also called “triple edema) [9] . The mother in mirror syndrome develops preeclampsia in about 60% and this usually occurs in early gestation [9] . Therefore, early identification, diagnosis, and management is a key for prevention of subsequent complications. Currently, prenatal screening of this condition is possibly performed by the determination of the mean corpuscular volume (MCV), dichlorophenolindophenol (DCIP), osmotic fragility (OF), or Hb typing [5, 10, 11] . Any couple at risk of Hb Bart’s hydrops fetalis is counseled to perform an invasive diagnostic test (chorionic villus sampling, amniocentesis, or cordocentesis) or to follow up with a non-invasive approach by performing ultrasonography to evaluate the cardiac diameter/thoracic ratio (CTR), middle cerebral artery peak systolic velocity (MCA-PSV), or placental thickness [5, 11] . In the late first trimester (12-15 weeks’ gestation), CTR of more than 0.5 has the highest predictive performance for the identification of fetal Bart’s hydrops with a sensitivity of 90%, 97.2% specificity, 90% positive predictive value (PPV) and 97.4% negative predictive value (NPV) [5] . The detection rates of MCA-PSV [>1.5 multiple of median (MoM)] and placental thickness (>18 mm) in the first trimester are limited for the prediction of Bart’s hydrops fetalis [MCA-PSV: sensitivity 17.6%, specificity 96.7%, PPV 66.7%, and NPV 76.1%; placental thickness: sensitivity 72.9% at 31.2% false positive rate] [5] . The use of maternal blood biochemical markers (i.e., pregnancy as pregnancy-associated plasma protein A, placental growth factor) including prenatal cell-free fetal DNA for the identification of Hb Bart’s fetalis are poor predictors, thus, they are not routinely used in a clinical setting [5, 12, 13, 14, 15, 16, 17] . Since ultrasound measurement is operator and equipment-dependent, searching for other non-invasive biomarkers at earlier gestation is continuing. The identification of biomarkers for the accurate and early prediction or diagnosis of pregnancy complications is important for the improvement of obstetrical care. It has been shown that proteins in maternal plasma play a role in the characterization and pathogenesis of the great obstetrical syndromes such as preeclampsia or fetal death [18, 19, 20, 21, 22, 23, 24, 25] . Until recently, studies showed these proteins mediated regulatory activities by soluble autocrine, paracrine, and endocrine, signaling pathways through the direct binding of cell surface receptors [26, 27, 28, 29, 30, 31] . However, the maternal-fetal dialogue is now recognized to be a more complex phenomenon in which extracellular vesicles (EVs) are also considered to be mediators in the cross-talk between the feto-placental unit and the mother [26, 27, 28, 29, 30, 31] . EVs are lipid bilayer-enclosed nanoscale particles secreted by cells into the extracellular space [32, 33] carried proteins and nucleic acids [34] , which can have angiogenic/anti-angiogenic, immune-regulatory, growth regulatory and other properties [35, 36] . EVs represent a cargo system that delivers packaged intercellular messengers to specific cells more efficiently than by the release of free molecules into the extracellular space. Being delivered intracellularly these messengers can alter cell physiology. Also, the release of vesicle cargos in the close vicinity of target cells would create a high surface concentration of messengers even with a small number of released molecules [37] . Therefore, deciphering the protein content of EVs could provide further insight into the pathophysiology of pregnancy related complications [38] . In pregnancy, the discovery of circulating fetal genetic material in the maternal plasma has enhanced the non-invasive prenatal diagnosis [39, 40, 41, 42, 43, 44, 45, 46, 47] . The placenta secretes a large number of EVs into maternal circulation since they are shed from the syncytiotrophoblast into the intervillous space and then flushed via the uterine veins into the maternal circulation [48] . The placental EVs were then confirmed the placental origin by detecting placental alkaline phosphatase (PLAP) protein marker. In Hb Bart’s fetalis, the placenta exhibits obvious vascular alterations, i.e., increased villous vessels, thickened vascular endothelium and more branching pattern of vessels [49] , serving as a model for vascular changes in placental hypoxia. Such changes are attributed from multifactorial hypoxia, including placentomegaly, which compromises blood flow from uterine distention. In addition, hydropic villi cause a generalized reduced intervillous space, therefore leading to placental and fetal hypoxia [49, 50, 51] . Consequently, we hypothesized that placental hypoxia in Bart’s fetuses could stimulate the release of placenta-derived EVs. This study aimed to characterize: 1) maternal plasma levels of placenta-derived EVs; and 2) proteomics profiles of maternal plasma EVs in women with Bart’s fetalis fetuses. The findings of this study will serve as a foundation to apply placenta-derived EVs in maternal circulation as a liquid biopsy platform for non-invasive detection of Bart’s fetuses. Results Table 1 demonstrated characteristics of the study populations. Median (IQR) of gestational age at blood sampling (weeks of gestation) of women with Hb Bart’s fetuses with and without hydropic features were 27.93 (22.75-33.75) and 12.86 (12.29-18.29), respectively. There was no difference in gestational age at blood sampling between women with Bart’s fetus with hydropic features and those with normal pregnancy and those with placental associated complications (p>0.05). All women with Bart’s fetuses underwent termination of pregnancy. All hydropic fetuses due to non-Bart’ causes subsequently had fetal death. Causes of hydrops in these fetuses are due to cardiac disease (n=1), Down’s syndrome (n=1; no antenatal care); and unknown causes (n=3). In cases with unknown causes, the evaluation of infection, chromosome and fetal anomaly was performed and the results were normal. Women with placental associated complications group (Disease control group; group 4) were diagnosed with fetal growth restriction (n=2), superimposed preeclampsia (n=1) and preeclampsia (n=2). Plasma EV validation After isolation from maternal plasma, EVs were validated by NTA, TEM, and EV markers using Western blot analysis following the International Society for Extracellular Vesicles guidelines [52] . The representative of particle size distribution and image captured by ViewSizer were showed in Figure 1A and 1B . EV particle size in Bart fetuses with hydropic features (group 2) was significantly smaller than that in the disease control (group 4) and that in normal pregnancy group (group 1) ( Figure 1C ). While, EV particle number have no difference among the groups ( Figure 1D ). The representative TEM images confirmed the intact EV morphology as the cup-shaped nanoscaled vesicles with the diameter <200 nm ( Figure 2 ). For protein evidence, three EV markers including Alix, CD9, and HSP70 were detected in all groups ( Figure 3 ). Therefore, both particle and protein evidence confirm the presence of EVs in the isolates as per the ISEV guidelines [52] . This study could be verified for use in further experiments. Placental-type alkaline phosphatase (PLAP) containing EVs (PLAP-EVs) PLAP is a membrane-bound glycoprotein primarily expressed in the placenta during pregnancy. Since EVs share plasma membranes with their original cells and tissues, we measured PLAP levels in the EV isolates (PLAP-EVs) as the indicator of placental pathology and injury ( Figure 4 ). PLAP-EVs in maternal plasma with Bart hydropic features (group 2) were significantly higher than that in normal pregnancy group (group 1). The disease control group (group 5) had the highest PLAP-EVs comparing to other groups, suggesting that placental pathologies of the disease control group (2 preeclampsia and 2 fetal growth restriction; Table 1 ) released higher amounts of tissue leakage proteins than that of placental edema and hypoxia in Bart’s hydrops. Patients with hydrops fetalis from non-Bart’s causes (group 5) tend to have higher PLAP-EV levels compared to those with normal pregnancy. Proteomics profile of plasma exosomes Plasma EV proteins in the 5 study groups [normal pregnancy group (n=6), Bart fetuses with hydropic features (n=3), Bart fetuses without hydropic features (n=4), hydrops due to non-Bart’s causes (n=2) and disease comparator (n=3)] with technical duplication were subjected to targeted label-free quantification using SWATH proteomics. All 106 EVs proteins were identified and quantified ( Supplementary table 1 ), in which 16 out of 106 proteins were significantly different among groups as shown in Figure 5 , Tables 2 and 3 for multiple comparison and fold change, respectively. Sixteen significantly altered proteins were predicted by Reactome pathway analysis and STRING protein-protein interaction to predict the relevant functional pathways associated with Bart’s hydrops fetalis. Reactome analysis showed that 16 altered EV proteins, as the consequences of hemoglobin Bart’s hydrops, associated with alterations in innate immune system and Toll-Like Receptors (TLRs), Myeloid differentiation factor 88 (MyD88), Interleukin-1 receptor–associated kinase 4 (IRAK4) and mitogen-activated protein kinase (MAPK) axis [53, 54] which is known to regulate inflammatory cytokine production ( Figure 6A ). Protein-protein interaction analysis by STRING revealed that 16 altered EV proteins involved in zymogen activation, aberrant immune response, abnormal coagulation and cellular apoptosis, which may be the consequences of vascular injuries and placental hypoxia in Bart’s hydrops fetalis ( Figure 6B and 6C ). Discussion Principal findings of this study: This is the first study of PLAP-EVs and proteomics of maternal plasma EVs for the identification of women with Hb Bart’s fetuses. We demonstrated that: 1) EV particle size in Bart fetuses with hydropic features was significantly smaller than that in normal pregnancy and disease control groups; 2) Bart fetuses with hydropic features had higher PLAP exosome number than that in normal pregnancy; although, PLAP exosome level was highest in the placental associated complications groups; 3) Bart fetuses without hydropic features tended to have higher PLAP-EV level compared to normal pregnancy group; 4) hydropic fetuses due to non-Bart’s causes had similar PLAP level compared to normal pregnancy group; 5) EV proteomic analysis revealed aberrant immune response, pro-inflammatory cytokine regulation mediated by TLRs-MyD88-IRAK4-MAPK axis and vascular injuries as part of pathophysiology of placental edema and hypoxia in Bart’s hydrops. Prenatal identification program for Hb Bart’s fetalis fetuses Previous studies have shown that there are morphological and functional changes in the responses to persistent fetal anemia [55, 56, 57, 58, 59, 60, 61, 62, 63] . Prior to the fetal hydropics stage, hemodynamic function shows a normal and good compensatory adaptation, by increasing turnover blood volume, or cardiac work and distribution of blood flow into essential organs such as brain and spleen as detected by cardiomegaly, increased middle cerebral artery peak systolic velocity (MCA-PSV) or splenic-PSV [5] . Subsequently, at the high-output hydropic stage, there are fluid accumulations in the body space such as pleural/pericardial effusion or ascites, and these features are considered late ultrasonographic signs usually detected in the second or third trimester. Lastly, persistent fetal anemia results in low cardiac output heart failure as demonstrated by low cardiac output, poor contractility and a markedly increased preload, which usually occur in the last trimester [5] . Therefore, the determination of biomarkers according to disease pathophysiology prior to hydropic state is the key. Currently, sonographic and maternal serum biomarkers have been extensively evaluated as screening tools to identify Hb Bart’s fetuses in pregnancy at risk for this disease. Common sonographic markers include the measurement of cardiothoracic diameter and MCA-PSV [5, 51] . Thus far, only cardiothoracic diameter (≥0.50) can accurately predict fetal Hb Bart’s disease as early as the late first trimester (12–15 weeks of gestation), with a detection rate of 75-100% at 90-100% specificity [5, 51] . The detection rate of MCA-PSV (≥1.5 MoM) was approximately 64%-85% at 16-22 weeks of gestation, at 98-100% specificity [5, 51] . Other sonographic signs including nuchal translucency, placental thickness, liver length, splenic circumference had limited values for early identification of Hb Bart’s fetuses [5, 51] . Recently, Harn-A-Morn et al. demonstrated that serial ultrasound screening (using cardiothoracic diameter and MCA-PSV) for the identification of Hb Bart’s disease during pregnancy, beginning in the first trimester and continuing every 2–4 weeks until 24 weeks, has a sensitivity of 100%, at 10.9% false positive rate, in detecting pre-hydropic signs [11] . The mean gestational age at Hb Bart diagnosis was 15.5 ± 2.6 weeks of gestation. Therefore, our national standard prenatal care program implements such sonographic markers for the routine screening of fetal Hb Bart’s disease in all pregnancies at risk for Hb Bart’s fetuses. The main limitations of serial ultrasound are that it requires specific equipment, and it is operator-dependent, which needs additional training. Several maternal serum biomarkers such as free beta-human chorionic gonadotropin (β-hCG), inhibin-A, pregnancy-associated plasma protein-A (PAPP-A), alpha-fetoprotein (MAFP), unconjugated estriol (uE3) or angiogenic factors (soluble fms-like tyrosine kinase or placental growth factor) have poor predictive values for the detection of Hb Bart’s fetuses [5, 51] . Placenta-derived EVs in Hb Bart’s fetuses In this study, we isolated EVs using the combinatory methods of stepwise centrifugation, ultrafiltration and qEV size exclusion chromatography [64, 65, 66, 67] and then validated the presence of EVs in the isolates using NTA, TEM and Western blot analysis ( Figures 1-3 ) following the ISEV guidelines [52] . Then, placental-specific protein PLAP containing EVs (PLAP-EVs) were measured by ELISA, while other EV proteins were detected by SWATH proteomics compared between Bart’s hydrops group and other comparators as detailed in the methods section. Accordingly, we would like to highlight that this is the first study reporting PLAP-EVs in Bart’s hydrops fetalis. PLAP is a plasma membrane enzyme isoform of alkaline phosphatase specifically produced by the syncytiotrophoblast [68] . PLAP-EVs are specific for pregnancy and are not found in the circulation of non-pregnant women [69, 70] . Herein, we are reporting that the PLAP-EVs was significantly higher in Bart’s fetuses with hydropic features compared to the couple at risk with normal pregnancy outcome ( Figure 4 ). The increment of PLAP-EVs has demonstrated only in Bart’ fetuses’ group since women with hydropic fetuses due to non-Bart’s causes had similar levels of PLAP-EVs as those with normal pregnancy. However, PLAP-EVs in Bart’s fetuses with hydropic feature were lower than pregnant women with placental-associated complications (e.g., preeclampsia and fetal growth restriction). This result suggested that PLAP-EV levels may depend on the spectrum of placental pathologies. Further studies in a larger cohort are warranted to investigate the usability of PLAP-EVs in various pregnancy complications. There are very few studies about EVs in patients with thalassemia and none of these studies evaluated the role of EVs in pregnant women or Bart disease [71, 72, 73] . EVs obtained from patients with β-thalassemia/HbE induced platelet activation, platelet aggregation and platelet-neutrophil aggregation, which partly contributes to the hypercoagulable state [74] . In addition, such microparticles have an effect on endothelial cell activation that leads to the increased adhesion of leukocytes to endothelial cells resulting in thrombosis and vascular dysfunction especially in patients with splenectomized β-thalassemia/HbE disease [71] . Within the maternal circulation, different EV populations are released from several cell types including erythrocytes [75] , endothelial cells [76] , lymphocytes, dendritic cells, and placenta during gestation. The roles of EVs are thought to be involved in cell-to-cell communication between the placenta and maternal immune system [77] . Placenta-derived EVs are thought to promote maternal immune tolerance towards the fetal allograft as they can suppress maternal T-cell signaling [69] . The local immune privilege at the feto-maternal interface has been attributed to the expression of placental-derived EV-associated functional Fas ligand (FasL), programmed death ligand 1 (PD-L1), and TNF-related apoptosis inducing ligand (TRAIL) [69, 78] . In addition, NK cell activity has been shown to be down-regulated, and this is mediated by the expression of NKG2D receptor ligands, UL-16 binding proteins (ULBP) and MHC class I chain-related (MIC) proteins on placenta-derived EVs. The NK cell activity down-regulation leads to maternal cytotoxic activity suppression, thereby, promoting fetal allograft survival [79] . In normal pregnancies, placenta-derived EVs have been shown to modulate the function of maternal endothelium to promote trophoblast migration, angiogenesis and spiral artery remodeling [80] . Yet, under inflammatory or pathological conditions (i.e., obesity, hypoxia and high blood sugar), high number of circulating EVs can induce the release of pro-inflammatory cytokines from endothelial cells which lead to perturbation of physiologic function [80, 81, 82] . Altogether, these suggest that the content and effects of EVs depend on the physiological or pathological of the pregnant women. Evidence of placental hypoxia in Hb Bart’s fetuses includes: 1) the placenta in Hb Bart’s hydrops demonstrated an increased number of immature intermediate villi, which persist despite advancing in gestational age, and this finding is referred to as “generalized delayed villous maturation” [49, 83] ; 2) the presence of villous edema as well as numerous and cytotrophoblastic cells that cover the villous stroma [83] ; and 3) increased number of stromal cells at the periphery beneath the trophoblast layer, also called “peripheral villous stromal hypercellularity (PVSH)” [83] , a sign of placental adaptation to chronic hypoxia. The immature intermediate villi have a large diameter, and their predominance in the Hb Bart’s placenta leads to the narrowing of the intervillous space, impeding fetoplacental oxygen and nutrition transfer. Contractions of the myofibroblastic cells in PVSH lead to the reduction the villous size, thereby widening the intervillous space for an increase in the maternal blood flow [49, 83] . In addition, morphometric studies have shown a branching vascular pattern, which is associated with placental hypoxia. This change is thought to be responsible to the marked placental enlargement, which compromised the blood flow, from the uterine distention and the generally diminished intervillous space due to the numerous intermediate types of villi. Lastly, the dramatic reduction capacity of Hb Bart’s to extract oxygen from the intervillous space also contributes to placental hypoxia. Altogether, the presence of placental hypoxia can stimulate the release of EVs from the placenta into the maternal circulation. The observations that PLAP-EV levels were highest in women with placental associated complication group were consistent with previous studies [82, 84, 85, 86] . We hypothesized that the degree of hemodynamic changes or placental hypoxia is less in women with Bart’s fetuses without hydropic changes as demonstrated by a non-significant trend of high PLAP-EVs. Interestingly, women with hydropic fetuses due to non-Bart’s causes had a comparable PLAP-EV level as the normal pregnancy. Causes of hydrops in these fetuses are due to non-immune causes: cardiac disease (n=1), Down’s syndrome (n=1), and unknown non-immune causes (n=3). These cases had no fetal anemia as shown by normal MCV-PSV. Pathophysiology of hydrops fetalis is not completely understood; however, mainly due to the developmental defects in the microcirculation and lymphatic system, decreased ventricular filling or increased central venous pressure resulting from increased right heart pressure or obstructed lymphatic drainage [87] . The presence and degree of placental hypoxia in these fetuses is currently unknown. Proteomics profile of plasma EVs in Hb Bart’s fetuses We performed proteomic analysis of the plasma EVs of pregnant women who had Hb Bart’s fetuses, demonstrating 16 proteins in the plasma EVs were different from normal pregnancy and women with placental associated complication group ( Figure 5 ). Fibrinogen complex, fibrin clot formation and integrin signaling were functions and processes related to the significantly different proteins. Among differentially expressed proteins, hnRNPA2B1 was highest in Bart fetuses with hydropic features compared to placental associated complications (10 times higher) or normal pregnancy group (2 times higher). hnRNPA2B1 (A2B1), a member of the hnRNPABs subfamily, consists of two structural homologous proteins (hnRNPA2 and hnRNPB1) characterized by a tight sequence correlation and conserved domain structure, with B1 having 12 additional amino acids at N terminus compared with A2. A2B1 is an RNA-binding protein that affects the localization, shearing, stability, translation and other biochemical functions of RNA [88] . The hnRNPs are RNA binding proteins and they complex with heterogeneous nuclear RNA (hnRNA). These proteins are associated with pre-mRNAs in the nucleus and appear to influence pre-mRNA processing and other aspects of mRNA metabolism and transport. hnRNPs is expressed in several tissues such as placenta, ovary, musculoskeletal, endocrine organs and gastrointestinal tracts [89] . In pregnancy, these proteins were differentially expressed in the placenta of women with gestational diabetes mellitus and may play a role in regulating the occurrence and development of gestational diabetes [89] . It is possible that the presence of placental hypoxia in Bart fetuses triggers the release of hnRNPA2B1 since early gestation and this is specific to Hb Bart fetuses. However, a larger trial is required to further validate these findings. Clinical and research implications Ultrasound parameters and some soluble maternal blood biomarkers have been proposed to be used for the prediction of Hb Bart’s fetuses. However, we still lack sensitive methods for effective early screening. In addition, ultrasound parameters require standardization and proper training. Biomarkers from plasma EVs could improve the prediction of Hb Bart’s fetuses. Indeed, this study revealed that the PLAP-EV level in maternal circulation is higher in Bart’s fetuses and 16 EV proteins as well as hnRNPA2B protein in maternal plasma exosome have different concentrations in cases of Hb Bart’s fetuses compared to normal pregnancy and those with placental associated complications. Measuring PLAP-EVs together with differentially expressed EV proteins, might provide independent information to improve the ability to identify affected fetuses with Hb Bart disease. We envision that soluble markers secreted from the placenta can be assessed non-invasively in ongoing pregnancies through a “liquid biopsy”. A term “liquid biopsy” refers to a test performed on any biofluid specimens such as plasma, urine amniotic fluid or cervico-vaginal fluid [90, 91, 92] in which the result can be used to infer pathologic changes in distant tissues (e.g., cancer) or other pathologic processes (e.g., atherosclerosis) [93, 94, 95] . We believe that the non-invasive liquid biopsy could provide information in relation to placental health. In this report, the liquid biopsy based on an examination of placenta-derived EVs in maternal blood for the identification of Hb Bart’s fetuses is feasible. For the strengths and limitations, this is the first study to evaluate PLAP-EVs and proteomics profile of maternal plasma EVs in women with Bart’s fetuses. The strengths of this study are that we have included several groups such as Hb Bart’s group with or without hydropic features, hydropic fetuses due to other causes, or placental associated complication group. The latter group is considered the disease comparator group since compelling evidence suggests abnormal profile of placenta-derived EVs in women with placental associated complications. In addition, we confirmed by definite invasive prenatal diagnosis test that all couple at risks with normal pregnancy group had non-Bart’s fetuses and delivered without maternal or neonatal complications. The separation of EVs in the current study was confirmed by published methods including NTA, TEM and EV markers by Western blotting analysis following the international standard. The main limitation of our study is related to a small sample size. Future research is needed to confirm our findings in a larger number of populations. In the conclusions, EV particle size is smaller, while placental-derived EV level is significantly higher in women with Hb Bart’s fetalis fetuses compared to normal pregnancy. In addition, several EV proteins were differential expressed in women with Bart’s fetuses and these proteins may play roles in innate immunity, TLRs-MyD88-IRAK4-MAPK axis, and vascular injury as the consequences of placental hypoxia in Bart’s hydrops. The result of this study support in future investigations to determine whether placenta-derived EVs in maternal circulation can be used as a liquid biopsy or non-invasive prenatal test for the early identification of Bart’s fetuses. This will ultimately reduce the rate of unnecessary invasive prenatal diagnosis testing. Materials And Methods Study population This was a prospective cohort study in pregnant women at 11-13 weeks’ gestation onwards who attended prenatal care clinics from November 2021 to November 2023. Participants were recruited at: 1) Ramathibodi Hospital, Bangkok, Thailand; and 2) Siriraj Hospital, Bangkok, Thailand. In Thailand, all pregnant women (and their couples if available) are required to perform thalassemia screening at the first prenatal visit. Clinical standard management for thalassemia screening includes the examination of mean corpuscular volume (MCV), Dichlorophenolindophenol (DCIP), osmotic fragility (OF) test, Hb typing, and alpha or beta-thalassemia mutations in some cases. Couples at risk for Hb Bart’s fetuses were counseled to: 1) perform diagnostic test; or 2) follow up with non-invasive approach such as ultrasonography. The diagnostic tests depend on gestational age of the patients, for example, chorionic villous sampling and amniocentesis for the determination of fetal DNA was performed at 11-13 and 16-18 weeks of gestation, respectively, while cordocentesis was performed after 18 weeks of gestation for Hb typing [5, 10, 11] . Inclusion criteria were women at aged ≥18 years and 11-13 weeks of gestation onward. Exclusion criteria were women who were unable to give informed consent or those who had severe major fetal abnormality and those who were couple at risk of Bart’s fetuses without prenatal diagnostic test confirmation. All patients provided written informed consent prior to the collection of samples. The use of clinical databases and biological samples was approved by the Human Research Ethics Committee of Mahidol University, Thailand, with IRB No. COA.MURA2021/439 (Ramathibodi Hospital) and COA.No.SI 840/2021 (Siriraj Hospital). The study was approved by an appropriate institution and we confirmed that all methods were performed in accordance with the relevant guidelines and regulations by including a statement in the methods section. A total of 27 women were included in the study. We stratified participants into the following groups: 1) normal pregnancy or control group: women with couple at risk for Hb Bart’s fetuses but subsequently proven as normal pregnancy (non-Bart affected fetuses) (Group 1) (n=7); 2) Hb Bart’s with hydropic features (Group 2) (n=4); 3) Hb Bart’s without hydropic features (Group 3) (n=7); 4) a disease comparator group which consisted of women who subsequently delivered with placental associated conditions (such as preeclampsia, fetal growth restriction, unexplained fetal death, spontaneous preterm labor or preterm pre-labor rupture of membranes) (Group 4) (n=4); and 5) women with hydrops fetalis from non-Bart’s causes (n=5) (Group 5). All women in Group 1 delivered at term gestation without complication and had appropriate weight for gestational age fetuses. Cases in Group 2 and 3 were matched with patients classified in a disease comparator group (Group 4) based on gestational age at venipuncture and maternal characteristics (age, race, parity, body mass index). Clinical definitions Obstetric and delivery outcomes, including mode of delivery, maternal and neonatal outcomes, were obtained from the maternity computerized records and such information were reviewed by dedicated researchers. Hydrops fetalis is characterized by the abnormal interstitial fluid collection in two or more compartments of the fetal body (peritoneal cavity, pleura, and pericardium) or fluid accumulation in one site and anasarca [96, 97, 98, 99, 100, 101, 102, 103] . Preeclampsia was defined as systolic blood pressure (BP) ≥140 mmHg and/or diastolic BP ≥90 mmHg on at least two occasions measured 4 hours apart in previously normotensive women, accompanied by one or more of the following new-onset conditions at or after 20 weeks of gestation: proteinuria, evidence of other maternal organ dysfunction such as acute kidney injury, liver involvement, elevated liver enzymes, neurological complications, hematological complications, or uteroplacental dysfunction [104] . Fetal growth restriction (FGR) was defined as estimated fetal weight (EFW) or abdominal circumference (AC) <3 rd percentile or EFW or AC <10 th percentile combined with abnormal Doppler findings or a decrease in growth centiles [105] . In all FGR fetuses, we confirmed that neonatal birth weight was below 3 rd percentile at birth. Preterm birth was defined as delivery at <37 completed weeks of pregnancy, and spontaneous preterm delivery included spontaneous onset of labor with intact membranes, pre-labor rupture of membranes (PPROM), and cervical insufficiency [106] . Uncomplicated pregnancy or normal pregnancy group was defined as a live birth at or after 37 weeks of gestation with appropriate weight for gestational age fetuses without any complications. EV isolation Maternal blood was collected in EDTA tube and transported to the laboratory immediately. EVs were isolated from the plasma biofluid by a combination of stepwise centrifugation, ultrafiltration and qEV size exclusion chromatography as described in our previous studies [64, 65, 66, 67] . Briefly, 500 µl of plasma was diluted in filtered PBS (1:1) to 1 mL, centrifuged at 3000 g for 15 min at 4°C to remove cell debris, and centrifuged at 12,000 x g for 20 min at 4°C to remove large particles and protein aggregates. The supernatant was filtered through a 100-kDa cut-off centrifugal filter to remove soluble proteins (with the molecular weight less than 100 kDa) and concentrated the sample volume (from 1 ml to 500 µl) before passing through the qEV size exclusion chromatography (IZON). The EV fractions were collected, pooled, and concentrated by a 3-kDa cut-off centrifugal filter to the final volume of 100 µl of the EV isolate. EV validation Nanoparticle tracking analysis EV size and number were measured by View Sizer 3000 nanoparticle size analyzer (Horiba). One µl of the isolated EVs was diluted in the filtered PBS (1:2000) and then transferred 1 ml into the cuvette with magnetic stirrer to capture 25 images in a video. Acquisition parameter was set as blue laser (445 nm) 210 mW, green laser (520 nm) 12 mW, red laser (635 nm) 8 mW, pulse duration (B/G/R) 15/15/15 ms, 5 s stirring time, 1400 rpm stirring speed, 3s wait time, 300 frames/video and 25 video count. The software calculated particle size and number with subtraction to blank as the filtered PBS alone. Transmission electron microscope (TEM) Intact EV morphology was imaged by TEM. Briefly, ten microliters of the isolated EVs were dotted on parafilm and inverted grid face on the sample for 10 min at room temperature. The grid face was washed in PBS for 1 min twice. After fixing with 2.5% (v/v) glutaraldehyde for 5 min, the grid face was washed in PBS for 1 min twice. The grid face was stained with 2% (w/v) uranyl acetate for 1 min and then air dried for 10 min. The EV on the grid face was captured under TEM. Western blotting of EV protein markers Specific EV markers were detected by Western blotting analysis. Briefly, twenty microliters of the isolated EVs were lysed in 1× reducing buffer containing 62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, and 2.5% beta-mercaptoethanol combination with sonication. After heating at 95°C for 5 min, the EV proteins were measured by Bradford’s assay. Equal 10 µg total protein amount was resolved on 10% SDS-PAGE and then blotted on a nitrocellulose membrane. Non-specific binding sites on the membrane were blocked with 5%skim milk/PBS at room temperature for 30 min, and then probed with primary antibody i.e., rabbit polyclonal anti-CD9 (ab223052, Abcam), rabbit polyclonal anti-HSP70 (ab79852, Abcam), and mouse monoclonal anti-CD63 (ab193349, Abcam) antibodies at dilution 1:1000 in 5%BSA/PBS at 4°C overnight. The membrane was washed with 0.5% tween20 in PBS (PBST) for 3 times, 5 min each. The membrane was probed with secondary antibodies conjugated horseradish peroxidase at dilution 1:2000 in 3% skim milk/PBST at room temperature for 1 h. After washing with PBST, the peroxidase substrate for enhanced chemiluminescence (ECL) was reacted on specific bands. The chemiluminescence signal was observed under chemiluminescence imaging detector (G:BOX, Syngene). Placental alkaline phosphatase (PLAP) ELISA Isolated EVs were measured PLAP level using Human Alkaline phosphatase, placental type sandwich ELISA kit (MBS289869, MyBioSource). Briefly, the highest PLAP concentration (1000 pg/ml) was diluted two-fold to set standard curve (1000, 500, 250, 125, 62.5, 31.25, 15.625, 0 pg/ml). An equal volume of 100 µl recombinant protein standard, 100 µl plasma and 20 µl EVs (add 80 µl PBS to make 100 µl final volume) was added into each well (2 technical replication, 7 biological replications per group) and incubated at 37°C for 2 h. All solution in each well was removed and then 100 μL of detection reagent A working solution to each well. After incubation at 37°C for 1 h, the liquid in each well was removed and washed 3 times by filling each well with 300 µl wash buffer and aspirating completely. 100 µl of detection reagent B working solution was added into each well and then incubated the plate at 37°C for 1 h. After completely wash 5 times, 90 µl of substrate solution was added in each well and then incubated at 37°C for 15 min in dark. Thereafter, 50 µl of stop solution was added and then measured the optical density at 450 nm. The OD of samples and standards was subtracted with blank (0 pg/ml PLAP) before calculation. SWATH proteomics SWATH proteomics was performed as our previous studies [107, 108, 109] . Briefly, 50 µl of EVs were lysed in Laemmli’s buffer (62.5 mM Tris-HCl, pH 6.8, 10% (v/v) glycerol, 2% (w/v) SDS and 2.5% (v/v) beta-mercaptoethanol). Total protein was quantified using the Bradford protein assay (Bio-Rad, Hercules, USA). Fifty micrograms of proteins were reduced, alkylated, and then digested by trypsin enzyme. After stop reaction with 5% formic acid in acetronitrile, the solution containing peptides were dried using a speedvac concentrator. The dried peptides were reconstituted in 0.1% formic acid before desalting by C18 stage tip. The 2 µg in 2 µl equal amount and volume of the desalted peptides were injected into an Eksigent nanoLC ultra nanoflow high performance liquid chromatography coupled with a TripleTOF 6600+ mass spectrometer (ABSciex, Toronto, Canada) installed at the proteomic core facility of Ramathibodi Hospital set for information-dependent acquisition (IDA) and data-independent acquisition (DIA) modes. The peptides were loaded onto a C18 column trap (Nano Trap RP-1, 3 μm 120 Å, 10 mm × 0.075 mm; Phenomenex, CA, USA) at a flow rate of 3 μl/min of 0.1% formic acid in water for 10 min to desalt and concentrate the sample, which was then separated on a C18 analytical column (bioZen Peptide Polar C18 nanocolumn, 75 μm × 15 cm, particle size 3 μm, 120 Å; Phenomenex) with mobile phase gradients at a flow rate of 300 nL/min of 3-30% acetonitrile/0.1% formic acid for 60 min, 30-40% acetonitrile/0.1% formic acid for 10 min, 40-80% acetonitrile/0.1% formic acid for 2 min, 80% acetonitrile/0.1% formic acid for 6 min, 80-3% acetonitrile/0.1% formic acid for 2 min, and 3% acetonitrile/0.1% formic acid for 25 min. The eluate was ionized and sprayed into the mass spectrometer using OptiFlow Turbo V Source (Sciex). Ion source gas 1 (GS1), ion source gas 2 (GS2), and curtain gas were set at 19, 0, and 25 vendor arbitrary units, respectively. The interface heater temperature was 150°C and ion spray voltage was 3.3 kV. Mass spectrometry was operated in the positive ion mode set for 3,500 cycles per 105 min gradient elution. Each cycle performed 1 time of flight (TOF) scan (250 ms accumulation time, 350–1250 m/z window with a charge state of +2) followed by IDA of the 30 most intense ions, while the minimum MS signal was set to 150 counts. The MS/MS scan was operated in high sensitivity mode with 50 ms accumulation time and 100 ppm mass tolerance. Former MS/MS candidate ions were excluded for a period of 12 sec after their first occurrence to reduce the redundancy of identified peptides. DIA mode was performed in a range of 350 to 1500 m/z using a predefined mass window of 7-m/z with an overlap of 1-m/z for 157 transmissible windows. MS scan was set at 2,044 cycles, where each cycle performs 1 TOF-MS scan type (50 ms accumulation time across 100–1500 precursor mass range) acquired in every cycle for a total cycle time of 3.08 sec. MS spectra of 100–1500 m/z were collected with an accumulation time of 96 ms per SWATH window width. Resolution for MS1 was 35,000 and SWATH-MS2 scan was 30,000. Rolling collision energy mode with collision energy spread of 15 eV was applied. The IDA and DIA data (. wiff ) were recorded by Analyst-TF v.1.8 software (ABSciex). A total of 36 wiff files of IDA experiments (5 groups; 6 patients with normal pregnancy group, 3 patients with Hb Bart’s with hydropic features, 4 patients with Hb Bart’s without hydropic features, 3 patients with disease control (disease comparator), and 2 patients with hydrops fetalis from non-Bart’s causes; 2 technical replicates per biological sample) were combined and searched using Protein Pilot v.5.0.2.0 software (ABSciex) against the Swiss-Prot database (UniProtKB 2022_01) Homo sapiens (20,385 proteins in database) with the searching parameters as follows; alkylation on cysteine by iodoacetamide, trypsin enzymatic digestion, 1 missed cleavage allowed, monoisotopic mass, and 1% false discovery rate. The group file (Protein Pilot search result) was loaded into SWATH Acquisition MicroApp v.2.0.1.2133 in PeakView software v.2.2 (Sciex) to generate a spectral library. The maximum number of proteins was set as the number of proteins identified at 1% global FDR from fit. RT alignment was performed by the high abundance endogenous peptides covering the chromatographic range. SWATH data extraction of 36 DIA files (5 groups; 2-6 biological replicates per group; 2 technical replicates per biological sample) was performed by SWATH Acquisition MicroApp (Sciex) using the following parameters; 5-min extraction window, 25 peptides/protein, 6 transitions/peptide, excluding shared peptides, peptide confidence >99%, FDR <1%, and XIC width of 20 ppm. SWATH extraction data, including the identities and quantities of peptides and proteins, was normalized using multiple linear regression by Marker View software and then exported into an Excel file for further analysis. The protein area in each group were analyzed using ANOVA and multiple comparison (Tukey) by SPSS software. The significantly differentially expressed proteins were predicted their functional involvements by STRING protein-protein interaction pathway analysis (https://string-db.org/) and reactome pathway analysis (https://reactome.org/) where the predicted pathways with false discovery rate (FDR) less than 0.05 was considered statistically significant. Statistical analysis Demographics data analysis Demographic and clinical variables were summarized in median [interquartile range (IQR)] for numerical variables and count (percentage) for categorical variables. The comparison of categorical data, and continuous data was assessed by χ2 test, and Mann-Whitney U test, respectively. A probability value (P value) of less than 0.05 was considered statistically significant. The statistical software STATA V17 (California, USA), IBM SPSS version 18 (Armonk, N.Y., USA), and MedCalC version 20.218 (Mariakerke, Belgium) were used for statistical analysis. Calculation of protein levels from ELISA Average the duplicate readings for each standard and samples were subtracted the average zero standard optical density. A best fit curve through the points to establish standard curve for each test could be determined by regression analysis (r 2 >0.99). The protein concentration of each sample was calculated from an equation. Statistical analysis used Mann-Whitney U test at p<0.05. Declarations Funding: This research was funded by The Royal Thai College of Obstetricians and Gynecologists (RTCOG) and Faculty of Medicine Ramathibodi Hospital (RF-65027) to P.C. The funders had no role in study design, analysis and interpretation of data, decision to publish, or preparation of the manuscript. Acknowledgments: We would like to thank nurses and physicians at the antenatal care clinic and labor and delivery unit at Ramathibodi and Siriraj Hospital, Mahidol University. Moreover, we would like to thank the teams for research assistance and facility at Central Laboratory of Pediatrics Department and Research Center at Faculty of Medicine Ramathibodi Hospital, Mahidol University. Contributions Conceptualization: P.C., W.C., S.C. Specimen collection: P.C., P.W., P.R., C.P., T.P.B., M.P. Methodology: P.C., W.C., S.C. Investigation: P.C., P.W., S.L., C.P., K.R., N.C., P.R., T.P.B., T.K., J.P., T.K., W.C. Formal analysis: P.C., P.W., S.L., C.P., K.R., N.C., P.R., T.P.B., T.K., J.P., T.K., W.C. Validation: P.C., W.C., S.C. Data curation: P.C., P.W., S.L., C.P., K.R., N.C., P.R., T.P.B., T.K., W.C. Writing – Original Draft Preparation: P.C. Writing – Review & Editing: P.C., P.W., S.L., C.P., K.R., N.C., P.R., T.P.B., T.K., J.P., T.K., W.C., S.C. Visualization: P.C., W.C. Funding acquisition: P.C. Project administration: P.C. Supervision: P.C., W.C., S.C. All authors have read and agreed to the published version of the manuscript. Data Availability The datasets generated and/or analysed during the current study are available in the ProteomeXchange with identifier PXD051236. [ http://www.ebi.ac.uk/pride ] Submission details: Project Name: Placenta-derived Extracellular Vesicles in Maternal Plasma of Hb Bart’s Fetuses Project accession: PXD051236 Reviewer account details: Username: [email protected] Password: N5riJ8S0 Conflicts of Interest: None References Flint J , et al. High frequencies of alpha-thalassaemia are the result of natural selection by malaria. Nature. 1986; 321 (6072):744-750. Wanapirak C, Muninthorn W, Sanguansermsri T, Dhananjayanonda P, Tongsong T. Prevalence of thalassemia in pregnant women at Maharaj Nakorn Chiang Mai Hospital. J Med Assoc Thai. 2004; 87 (12):1415-1418. Chui DH. Alpha-thalassaemia and population health in Southeast Asia. Ann Hum Biol. 2005; 32 (2):123-130. Modell B, Darlison M. Global epidemiology of haemoglobin disorders and derived service indicators. 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Glob Chall. 2023; 7 (3):2200213. Tables Table 1: Characteristics of participants according to the study groups Normal pregnancy group (Group 1) (n=7) Hb Bart’s fetuses with hydropic features group (Group 2) (n=4) Hb Bart’s fetuses without hydropic features group (Group 3) (n=7) Disease comparator (disease control) group (Group 4) (n=4) Hydropic fetuses with non-Bart’ causes (Group 5) (n=5) P value Maternal age (year) 24.3 (22.24-27.30) 31.8 (29.54-33.77) 37.1 (29.54-33.77) 33.0 (25.53-38.39) 22.9 (20.71-35.11) p=0.51 Maternal weight (kilogram) 60.5 (49.0-70.8) 63.0 (47.0-53.0) 68.8 (62.0-70.5) 66.50 (53.50-76.50) 69.5 (49.5-85.0) p=0.69 Maternal underlying diseases DM Chronic hypertension NA NA NA 50% (2) 50% (2) NA NA GA at blood sampling (weeks) 12.9 (12.00-18.00) 27.9 (22.75-33.75) 12.9 (12.29-18.29) 34.9 (24.43-37.14) 22.0 (21.14-29.50) p<0.05 # GA at delivery or termination of pregnancy (weeks) 38.4 (37.21-39.00) 27.9 (22.75-33.75) 17.4 (14.75-25.14) 36.0 (28.93-38.36) 21.7 (21.43-32.29) p<0.05 ## Route of delivery Vagina 86% (6/7) NA NA 0 (0/7) 100% (5) Indication for Cesarean delivery CPD NA NA Non-reassuring fetal status NA Baby weight (gram) 3,035 (2,787-3,252) NA NA 1,910 (813.75-2,732.50) 1,564.0 (481.0-2,050.0) >0.05 Pregnancy complications* NA NA NA Fetal growth restriction (n=2) Superimpose preeclampsia (n=1) Preeclampsia (n=1) NA Causes of non-Bart’s hydrops ** NA NA NA NA Cardiac disease (n=1) Down’s syndrome (n=1) Unknown non-immune causes (n=3) NA BMI: body mass index; CPD: cephalopelvic disproportion; GA: gestational age; NA: not applicable *: only in disease control group; **: only in Group 5 Data presented as % (n) or median (interquartile) P values determined by Kruskal-Wallis test #p>0.05 for group 1 and 2; group 2 and 4; p=0.006 for group 2 and 4 ## p=0.029 for group 2 and 4 Table 2. Multiple comparison of significantly differential proteins among 5 groups. SwissProt ID Gene name Protein name ANOVA Multiple comparison (Tukey) Normal pregnancy vs Bart hydrops Normal pregnancy vs Bart no hydrops Normal pregnancy vs Disease control Normal pregnancy vs Hydrops not Bart Bart hydrops vs Bart no hydrops Bart hydrops vs Disease control Bart hydrops vs Hydrops not Bart Bart no hydrops vs Disease control Bart no hydrops vs Hydrops not Bart Disease control vs Hydrops not Bart B9A064 IGLL5 Immunoglobulin lambda-like polypeptide 5 0.0051 0.8952 0.9038 0.0220 0.9925 1.0000 0.0093 0.9978 0.0062 0.9991 0.0497 P00736 C1R Complement C1r subcomponent 0.0252 1.0000 0.9988 0.0182 0.9972 0.9997 0.0572 0.9989 0.0556 1.0000 0.1806 P00738 HP Haptoglobin 0.0373 0.9771 0.0769 0.9789 0.9713 0.4227 0.8564 0.8530 0.0609 0.0963 1.0000 P02671 FGA Fibrinogen alpha chain 0.0045 0.9999 0.9395 0.0038 1.0000 0.9400 0.0113 1.0000 0.0400 0.9647 0.0294 P02675 FGB Fibrinogen beta chain 0.0001 0.6147 0.8512 0.0000 0.1194 0.9892 0.0066 0.7952 0.0009 0.5102 0.2032 P02679 FGG Fibrinogen gamma chain 0.0318 0.9983 0.9921 0.0183 0.9243 1.0000 0.0914 0.9868 0.0762 0.9916 0.3686 P02745 C1QA Complement C1q subcomponent subunit A 0.0288 0.9986 0.9999 0.0271 0.9965 0.9999 0.1187 0.9834 0.0618 0.9919 0.0676 P04114 APOB Apolipoprotein B-100 0.0112 0.9995 1.0000 0.0094 0.9998 0.9993 0.0478 1.0000 0.0169 0.9997 0.0890 P04259 KRT6B Keratin, type II cytoskeletal 6B 0.0096 0.0137 0.9997 0.9934 0.9937 0.0374 0.0175 0.0367 0.9817 0.9837 1.0000 P06312 IGKV4-1 Immunoglobulin kappa variable 4-1 0.0481 0.9420 0.2506 0.3387 0.9954 0.1260 0.1730 0.9992 1.0000 0.3315 0.3859 P08519 LPA Apolipoprotein(a) 0.0108 0.9999 0.9757 0.0180 0.9983 0.9686 0.0685 0.9956 0.0095 0.9998 0.0569 P0DOX2 IGA2 Immunoglobulin alpha-2 heavy chain 0.0120 0.0089 0.8261 0.8486 0.9932 0.1304 0.1894 0.0264 1.0000 0.7605 0.7749 P11142 HSPA8 Heat shock cognate 71 kDa protein 0.0464 0.1694 0.8827 0.9002 0.9858 0.0468 0.0667 0.1909 1.0000 0.9995 0.9993 P22626 HNRNPA2B1 Heterogeneous nuclear ribonucleoproteins A2_B1 0.0017 0.0057 0.9564 0.8896 1.0000 0.0025 0.0027 0.0519 0.9989 0.9792 0.9419 P81605 DCD Dermcidin 0.0212 0.0543 0.4212 0.3169 0.9537 0.7686 0.9309 0.0560 0.9975 0.3049 0.2309 Q86YZ3 HRNR Hornerin 0.0080 0.0163 0.9784 0.9813 0.9908 0.0090 0.0153 0.2061 1.0000 0.9113 0.9187 Red is significant difference at p <0.05 Table 3. Fold change of comparison of significantly differential proteins among the 5 sample groups SwissProt ID Gene name Protein name ANOVA Fold change Normal pregnancy/Bart hydrops Normal pregnancy/Bart no hydrops Normal pregnancy/Disease control Normal pregnancy/Hydrops not Bart Bart hydrops/Bart no hydrops Bart hydrops/Disease control Bart hydrops/Hydrops not Bart Bart no hydrops/Disease control Bart no hydrops/Hydrops not Bart Disease control/Hydrops not Bart B9A064 IGLL5 Immunoglobulin lambda-like polypeptide 5 0.0051 6.51 4.04 0.25 1.88 0.62 0.04 0.29 0.06 0.46 7.61 P00736 C1R Complement C1r subcomponent 0.0252 0.98 0.89 0.37 0.84 0.91 0.38 0.86 0.42 0.94 2.25 P00738 HP Haptoglobin 0.0373 0.73 0.39 1.55 1.80 0.54 2.11 2.46 3.94 4.58 1.16 P02671 FGA Fibrinogen alpha chain 0.0045 1.35 0.41 0.11 1.27 0.30 0.08 0.94 0.27 3.10 11.60 P02675 FGB Fibrinogen beta chain 0.0001 0.53 0.64 0.22 0.36 1.20 0.42 0.68 0.35 0.57 1.63 P02679 FGG Fibrinogen gamma chain 0.0318 0.78 0.73 0.24 0.53 0.93 0.31 0.68 0.33 0.73 2.19 P02745 C1QA Complement C1q subcomponent subunit A 0.0288 0.73 0.85 0.19 2.15 1.17 0.27 2.94 0.23 2.52 11.06 P04114 APOB Apolipoprotein B-100 0.0112 0.89 1.02 0.33 0.90 1.15 0.37 1.01 0.32 0.88 2.74 P04259 KRT6B Keratin, type II cytoskeletal 6B 0.0096 0.38 0.92 1.25 1.29 2.44 3.30 3.42 1.35 1.40 1.04 P06312 IGKV4-1 Immunoglobulin kappa variable 4-1 0.0481 2.24 0.42 0.42 1.48 0.19 0.19 0.66 1.00 3.55 3.54 P08519 LPA Apolipoprotein(a) 0.0108 0.93 1.38 0.37 1.21 1.49 0.40 1.30 0.27 0.88 3.28 P0DOX2 IGA2 Immunoglobulin alpha-2 heavy chain 0.0120 0.26 0.57 0.56 1.61 2.18 2.14 6.18 0.98 2.83 2.89 P11142 HSPA8 Heat shock cognate 71 kDa protein 0.0464 0.32 4.89 5.84 2.22 15.46 18.47 7.03 1.19 0.45 0.38 P22626 HNRNPA2B1 Heterogeneous nuclear ribonucleoproteins A2_B1 0.0017 0.32 1.54 2.02 0.97 4.74 6.21 2.99 1.31 0.63 0.48 P81605 DCD Dermcidin 0.0212 0.26 0.39 0.34 5.18 1.48 1.31 19.86 0.88 13.38 15.17 Q86YZ3 HRNR Hornerin 0.0080 0.31 1.53 1.57 0.74 4.94 5.09 2.40 1.03 0.49 0.47 Bold is fold change >2 or <0.5. Additional Declarations No competing interests reported. Supplementary Files Suppltable1final240120.xlsx Supplfigure1final240120.pptx Bartexosomedata.xlsx Cite Share Download PDF Status: Published Journal Publication published 20 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 04 Jun, 2025 Reviews received at journal 03 Jun, 2025 Reviewers agreed at journal 18 May, 2025 Reviewers agreed at journal 27 Dec, 2024 Reviews received at journal 14 Nov, 2024 Reviewers agreed at journal 07 Nov, 2024 Reviewers invited by journal 11 Apr, 2024 Editor assigned by journal 11 Apr, 2024 Editor invited by journal 10 Apr, 2024 Submission checks completed at journal 09 Apr, 2024 First submitted to journal 25 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Chutipongtanate","email":"","orcid":"","institution":"Division of Epidemiology, Department of Environmental and Public Health Sciences, University of Cincinnati College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Somchai","middleName":"","lastName":"Chutipongtanate","suffix":""}],"badges":[],"createdAt":"2024-03-25 11:50:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4163008/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4163008/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-24900-0","type":"published","date":"2025-11-20T15:58:29+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":54865888,"identity":"4bd3ee27-7a2d-4a94-a586-63ea93353f7d","added_by":"auto","created_at":"2024-04-17 20:49:04","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":567973,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlasma EV measurement in size and number using nanoparticle tracking analysis.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) EV size distribution and (\u003cstrong\u003eB\u003c/strong\u003e) image capture by ViewSizer. Plasma exosomes derived from 5 groups of samples were measured particle size (nm) (\u003cstrong\u003eC\u003c/strong\u003e) and particle number (\u003cstrong\u003eD\u003c/strong\u003e). *p\u0026lt;0.05 comparing to disease comparator. EV: extracellular vesicles\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4163008/v1/86a3bd332d44fccaaf91ae65.jpg"},{"id":54865885,"identity":"d3e5b872-3b19-4eda-a4fa-90f0034fe154","added_by":"auto","created_at":"2024-04-17 20:49:03","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1117282,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlasma EV morphology using Transmission electron microscopy (TEM).\u003c/strong\u003e TEM with negative staining demonstrated the presence of intact cup-shaped nanoscale vesicles in the EV isolates from five groups of patients. EV: extracellular vesicles\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4163008/v1/73ad5fd695a424bae45f4400.jpg"},{"id":54865889,"identity":"f41e234b-64c4-4d6c-9679-273dcf7ec148","added_by":"auto","created_at":"2024-04-17 20:49:05","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":222084,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEV marker detection using Western blotting analysis.\u003c/strong\u003e Plasma EVs from all 5 groups including normal pregnancy group, Bart with hydropic features group, Bart not hydropic features group, Hydropic features without Bart, and disease control were detected for the EV markers including HSP70 at 70 kDa, CD63 at 50 and 70 kDa, and CD9 at 17 kDa by Western blotting analysis. The full-length blots of the cropped images were provided in \u003cstrong\u003eSupplementary figure 1\u003c/strong\u003e) EV: extracellular vesicles.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4163008/v1/9cfa0037a7a7b4fe75773172.jpg"},{"id":54865884,"identity":"8a3f5a2b-a085-42a4-b147-512583502d2c","added_by":"auto","created_at":"2024-04-17 20:49:03","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":439635,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePLAP level in plasma EVs.\u003c/strong\u003ePlasma EVs derived from patients in 5 groups i.e., a normal pregnancy group (group 1, n=7), Bart fetuses with hydropic features (group 2, n=4), Bart fetuses without hydropic features (group 3, n=7), hydrops fetalis due to non-Bart’s causes (group 4, n=5), a disease control group (group 5, n=4), were measured PLAP level by using ELISA. Data were presented as the total PLAP detected in plasma EVs (left panel) and PLAP level per EV particle (right panel). EV: extracellular vesicles. PLAP: placental alkaline phosphatase * Compared to normal pregnancy group.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4163008/v1/5928b38fd3c946badcca01e4.jpg"},{"id":54865886,"identity":"7bef90dc-21a3-4734-b3cf-c6c573d64444","added_by":"auto","created_at":"2024-04-17 20:49:03","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":566494,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSixteen altered plasma EV proteins detections by SWATH-proteomics\u003c/strong\u003e(details in \u003cstrong\u003eTables 2\u003c/strong\u003e and \u003cstrong\u003e3\u003c/strong\u003e). *, p\u0026lt;0.05; **, p\u0026lt;0.01; ***, p\u0026lt;0.005. EV: extracellular vesicles\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4163008/v1/ec2b50e118de143442abd039.jpg"},{"id":54865890,"identity":"56a18db1-fa4d-4d16-a1f1-26645060e4bc","added_by":"auto","created_at":"2024-04-17 20:49:05","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":704230,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePathway analysis of 16 significantly altered EV proteins.\u003c/strong\u003e Reactome pathway analysis (\u003cstrong\u003eA\u003c/strong\u003e), STRING protein-protein interaction (\u003cstrong\u003eB\u003c/strong\u003e) and subsequent functional enrichment analysis using Gene Ontology biological process (\u003cstrong\u003eC\u003c/strong\u003e). EV: extracellular vesicles.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4163008/v1/f0961a5f1c44ad3502975d6c.jpg"},{"id":96650930,"identity":"dbb3064b-edf8-4b0d-9535-241e245ec83f","added_by":"auto","created_at":"2025-11-24 16:12:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5471645,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4163008/v1/2bda9786-ed6e-44a9-9d42-1eebbe94c02a.pdf"},{"id":54865893,"identity":"3955ca24-caa3-44a2-81bb-f3e222755139","added_by":"auto","created_at":"2024-04-17 20:49:05","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":40594,"visible":true,"origin":"","legend":"","description":"","filename":"Suppltable1final240120.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4163008/v1/3d2cde8a36bd6405a13e28e7.xlsx"},{"id":54865891,"identity":"230c0dc7-10a1-45de-b14b-4656ff3c65a2","added_by":"auto","created_at":"2024-04-17 20:49:05","extension":"pptx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":7087712,"visible":true,"origin":"","legend":"","description":"","filename":"Supplfigure1final240120.pptx","url":"https://assets-eu.researchsquare.com/files/rs-4163008/v1/943487d59cf29a8fdbfd76a6.pptx"},{"id":54865892,"identity":"9d224a7f-ad93-433a-93f6-ef1c81a652ca","added_by":"auto","created_at":"2024-04-17 20:49:05","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":15871,"visible":true,"origin":"","legend":"","description":"","filename":"Bartexosomedata.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4163008/v1/5549fbe1213462528b0366cb.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Placenta-derived Extracellular Vesicles in Maternal Plasma of Hb Bart’s Fetuses","fulltext":[{"header":"What are the novel findings of this work? ","content":"\u003cp\u003eWe reported herein the first study of placenta-derived extracellular vesicle (EVs) in women with Hb Bart\u0026rsquo;s fetalis. Placental alkaline phosphatase (PLAP)-EV level is significantly higher in women with Hb Bart\u0026rsquo;s fetalis fetuses compared to normal pregnancy. In addition, several EV proteins are differential expressed in women with Bart\u0026rsquo;s fetuses and these proteins involve in zymogen activation, fibrinogen complex, vesicles in cellular component, fibrin clot formation and integrin signaling. The result of this study supports future research to determine whether placenta-derived EVs in maternal circulation can be used as a liquid biopsy or non-invasive prenatal test for the early identification of Bart\u0026rsquo;s fetuses.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eWhat are the clinical implications of this work?\u0026nbsp;\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eOur study has demonstrated a profile of\u0026nbsp;\u003c/em\u003eextracellular vesicle (EVs) \u003cem\u003ein women with Hb Bart\u0026rsquo;s fetalis fetuses. Specifically, women with Bart\u0026rsquo;s fetuses have smaller EVs particle size but higher placental alkaline phosphatase (PLAP)-EV level than those with normal pregnancies. Several proteomics EV proteins are differentially expressed in women with Bart\u0026rsquo;s fetuses and these proteins\u0026nbsp;\u003c/em\u003einvolves in zymogen activation, fibrinogen complex, vesicles in cellular component, fibrin clot formation and integrin signaling. We envision that the non-invasive liquid biopsy could provide information in relation to placental health in women with Bart\u0026rsquo;s fetuses. In this report, the liquid biopsy based on an examination of placenta-derived EVs in maternal blood for the identification of Hb Bart\u0026rsquo;s fetuses is feasible and this may ultimately reduce the rate of unnecessary invasive prenatal diagnosis testing in the future.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eAlpha-thalassemia major (\u0026ldquo;homozygous alpha-thalassemia-1\u0026rdquo;) or hemoglobin Bart\u0026rsquo;s (Hb Bart) disease is the most severe form of thalassemia disease. Such disease is commonly found in Asia, especially Southeast Asia\u003csup\u003e[1, 2, 3, 4, 5]\u003c/sup\u003e, with an approximate prevalence of 0.23 %\u003csup\u003e[6, 7]\u003c/sup\u003e. The gene mutation frequency of Southeast Asia is as high as 4.5%-5.0%, leading to a high prevalence of the homozygous mutation (-SEA/-SEA)\u003csup\u003e[8]\u003c/sup\u003e. Homozygous mutation causes hydrops fetalis leading to fetal death or stillbirth. Additionally, fetal hydrops can lead to a syndrome called \u0026ldquo;Ballantyne or mirror syndrome\u0026rdquo;. Such syndrome reflects the simultaneously edematous state of the mother, fetus, and placenta (also called \u0026ldquo;triple edema)\u003csup\u003e[9]\u003c/sup\u003e. The mother in mirror syndrome develops preeclampsia in about 60% and this usually occurs in early gestation\u003csup\u003e[9]\u003c/sup\u003e. Therefore, early identification, diagnosis, and management is a key for prevention of subsequent complications.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCurrently, prenatal screening of this condition is possibly performed by the determination of the mean corpuscular volume (MCV), dichlorophenolindophenol (DCIP), osmotic fragility (OF), or Hb typing\u003csup\u003e[5, 10, 11]\u003c/sup\u003e. Any couple at risk of Hb Bart\u0026rsquo;s hydrops fetalis is counseled to perform an invasive diagnostic test (chorionic villus sampling, amniocentesis, or cordocentesis) or to follow up with a non-invasive approach by performing ultrasonography to evaluate the cardiac diameter/thoracic ratio (CTR), middle cerebral artery peak systolic velocity (MCA-PSV), or placental thickness\u003csup\u003e[5, 11]\u003c/sup\u003e. In the late first trimester (12-15 weeks\u0026rsquo; gestation), CTR of more than 0.5 has the highest predictive performance for the identification of fetal Bart\u0026rsquo;s hydrops with a sensitivity of 90%, 97.2% specificity, 90% positive predictive value (PPV) and 97.4% negative predictive value (NPV)\u003csup\u003e[5]\u003c/sup\u003e. The detection rates of MCA-PSV [\u0026gt;1.5 multiple of median (MoM)] and placental thickness (\u0026gt;18 mm) in the first trimester are limited for the prediction of Bart\u0026rsquo;s hydrops fetalis [MCA-PSV: sensitivity 17.6%, specificity 96.7%, PPV 66.7%, and NPV 76.1%; placental thickness: sensitivity 72.9% at 31.2% false positive rate]\u003csup\u003e[5]\u003c/sup\u003e.\u003csup\u003e\u0026nbsp;\u003c/sup\u003eThe use of maternal blood biochemical markers (i.e., pregnancy as pregnancy-associated plasma protein A, placental growth factor) including prenatal cell-free fetal DNA for the identification of Hb Bart\u0026rsquo;s fetalis are poor predictors, thus, they are not routinely used in a clinical setting\u003csup\u003e[5, 12, 13, 14, 15, 16, 17]\u003c/sup\u003e. Since ultrasound measurement is operator and equipment-dependent, searching for other non-invasive biomarkers at earlier gestation is continuing.\u003c/p\u003e\n\u003cp\u003eThe identification of biomarkers for the accurate and early prediction or diagnosis of pregnancy complications is important for the improvement of obstetrical care. It has been shown that proteins in maternal plasma play a role in the characterization and pathogenesis of the great obstetrical syndromes such as preeclampsia or fetal death\u003csup\u003e[18, 19, 20, 21, 22, 23, 24, 25]\u003c/sup\u003e. Until recently, studies showed these proteins mediated regulatory activities by soluble autocrine, paracrine, and endocrine, signaling pathways through the direct binding of cell surface receptors\u003csup\u003e[26, 27, 28, 29, 30, 31]\u003c/sup\u003e. However, the maternal-fetal dialogue is now recognized to be a more complex phenomenon in which extracellular vesicles (EVs) are also considered to be mediators in the cross-talk between the feto-placental unit and the mother\u003csup\u003e[26, 27, 28, 29, 30, 31]\u003c/sup\u003e. EVs are lipid bilayer-enclosed nanoscale particles secreted by cells into the extracellular space\u003csup\u003e[32, 33]\u003c/sup\u003e carried proteins and nucleic acids\u003csup\u003e[34]\u003c/sup\u003e, which can have angiogenic/anti-angiogenic, immune-regulatory, growth regulatory and other properties\u003csup\u003e[35, 36]\u003c/sup\u003e. EVs represent a cargo system that delivers packaged intercellular messengers to specific cells more efficiently than by the release of free molecules into the extracellular space. Being delivered intracellularly these messengers can alter cell physiology. Also, the release of vesicle cargos in the close vicinity of target cells would create a high surface concentration of messengers even with a small number of released molecules\u003csup\u003e[37]\u003c/sup\u003e. Therefore, deciphering the protein content of EVs could provide further insight into the pathophysiology of pregnancy related complications\u003csup\u003e[38]\u003c/sup\u003e. In pregnancy, the discovery of circulating fetal genetic material in the maternal plasma has enhanced the non-invasive prenatal diagnosis\u003csup\u003e[39, 40, 41, 42, 43, 44, 45, 46, 47]\u003c/sup\u003e. The placenta secretes a large number of EVs into maternal circulation since they are shed from the syncytiotrophoblast into the intervillous space and then flushed via the uterine veins into the maternal circulation\u003csup\u003e[48]\u003c/sup\u003e. The placental EVs were then confirmed the placental origin by detecting placental alkaline phosphatase (PLAP) protein marker.\u003c/p\u003e\n\u003cp\u003eIn Hb Bart\u0026rsquo;s fetalis, the placenta exhibits obvious vascular alterations, i.e., increased villous vessels, thickened vascular endothelium and more branching pattern of vessels\u003csup\u003e[49]\u003c/sup\u003e, serving as a model for vascular changes in placental hypoxia. Such changes are attributed from multifactorial hypoxia, including placentomegaly, which compromises blood flow from uterine distention. In addition, hydropic villi cause a generalized reduced intervillous space, therefore leading to placental and fetal hypoxia\u003csup\u003e[49, 50, 51]\u003c/sup\u003e. Consequently, we hypothesized that placental hypoxia in Bart\u0026rsquo;s fetuses could stimulate the release of placenta-derived EVs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study aimed to characterize: 1) maternal plasma levels of placenta-derived EVs; and 2) proteomics profiles of maternal plasma EVs in women with Bart\u0026rsquo;s fetalis fetuses. The findings of this study will serve as a foundation to apply placenta-derived EVs in maternal circulation as a liquid biopsy platform for non-invasive detection of Bart\u0026rsquo;s fetuses.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e demonstrated characteristics of the study populations. Median (IQR) of gestational age at blood sampling (weeks of gestation) of women with Hb Bart\u0026rsquo;s fetuses with and without hydropic features were 27.93 (22.75-33.75) and 12.86 (12.29-18.29), respectively. There was no difference in gestational age at blood sampling between women with Bart\u0026rsquo;s fetus with hydropic features and those with normal pregnancy and those with placental associated complications (p\u0026gt;0.05). All women with Bart\u0026rsquo;s fetuses underwent termination of pregnancy. All hydropic fetuses due to non-Bart\u0026rsquo; causes subsequently had fetal death. Causes of hydrops in these fetuses are due to cardiac disease (n=1), Down\u0026rsquo;s syndrome (n=1; no antenatal care); and unknown causes (n=3). In cases with unknown causes, the evaluation of infection, chromosome and fetal anomaly was performed and the results were normal. Women with placental associated complications group (Disease control group; group 4) were diagnosed with fetal growth restriction (n=2), superimposed preeclampsia (n=1) and preeclampsia (n=2). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePlasma EV validation\u003c/p\u003e\n\u003cp\u003eAfter isolation from maternal plasma, EVs were validated by NTA, TEM, and EV markers using Western blot analysis following the International Society for Extracellular Vesicles guidelines\u003csup\u003e[52]\u003c/sup\u003e. The representative of particle size distribution and image captured by ViewSizer were showed in \u003cstrong\u003eFigure 1A\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;1B\u003c/strong\u003e. EV particle size in Bart fetuses with hydropic features (group 2) was significantly smaller than that in the disease control (group 4) and that in normal pregnancy group (group 1) (\u003cstrong\u003eFigure 1C\u003c/strong\u003e). While, EV particle number have no difference among the groups (\u003cstrong\u003eFigure 1D\u003c/strong\u003e). The representative TEM images confirmed the intact EV morphology as the cup-shaped nanoscaled vesicles with the diameter \u0026lt;200 nm (\u003cstrong\u003eFigure 2\u003c/strong\u003e). For protein evidence, three EV markers including Alix, CD9, and HSP70 were detected in all groups (\u003cstrong\u003eFigure 3\u003c/strong\u003e). Therefore, both particle and protein evidence confirm the presence of EVs in the isolates as per the ISEV guidelines\u003csup\u003e[52]\u003c/sup\u003e. This study could be verified for use in further experiments. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePlacental-type alkaline phosphatase (PLAP) containing EVs (PLAP-EVs)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePLAP is a membrane-bound glycoprotein primarily expressed in the placenta during pregnancy. Since EVs share plasma membranes with their original cells and tissues, we measured PLAP levels in the EV isolates (PLAP-EVs) as the indicator of placental pathology and injury\u0026nbsp;(\u003cstrong\u003eFigure 4\u003c/strong\u003e). PLAP-EVs in maternal plasma with Bart hydropic features (group 2) were significantly higher than that in normal pregnancy group (group 1). The disease control group (group 5) had the highest PLAP-EVs comparing to other groups, suggesting that placental pathologies of the disease control group (2 preeclampsia and 2 fetal growth restriction; \u003cstrong\u003eTable 1\u003c/strong\u003e) released higher amounts of tissue leakage proteins than that of placental edema and hypoxia in Bart\u0026rsquo;s hydrops. Patients with hydrops fetalis from non-Bart\u0026rsquo;s causes (group 5) tend to have higher PLAP-EV levels compared to those with normal pregnancy.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eProteomics profile of plasma exosomes\u003c/p\u003e\n\u003cp\u003ePlasma EV proteins in the 5\u0026nbsp;study\u0026nbsp;groups [normal pregnancy group (n=6), Bart\u0026nbsp;fetuses\u0026nbsp;with hydropic features (n=3), Bart\u0026nbsp;fetuses\u0026nbsp;without hydropic features (n=4), hydrops due to non-Bart\u0026rsquo;s causes (n=2) and disease comparator (n=3)] with technical duplication were subjected to targeted label-free quantification using SWATH proteomics. All 106 EVs proteins were identified and quantified (\u003cstrong\u003eSupplementary table 1\u003c/strong\u003e), in which 16 out of 106 proteins were significantly different among groups as shown in \u003cstrong\u003eFigure 5\u003c/strong\u003e, \u003cstrong\u003eTables 2\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;3\u003c/strong\u003e for multiple comparison and fold change, respectively. Sixteen significantly altered proteins were predicted by Reactome pathway analysis and STRING protein-protein interaction to predict the relevant functional pathways associated with Bart\u0026rsquo;s hydrops fetalis. Reactome analysis showed that 16 altered EV proteins, as the consequences of hemoglobin Bart\u0026rsquo;s hydrops, associated with alterations in innate immune system and Toll-Like Receptors (TLRs), Myeloid differentiation factor 88 (MyD88), Interleukin-1 receptor\u0026ndash;associated kinase 4 (IRAK4) and mitogen-activated protein kinase (MAPK) axis\u0026nbsp;\u003csup\u003e[53, 54]\u003c/sup\u003e which is known to regulate inflammatory cytokine production (\u003cstrong\u003eFigure 6A\u003c/strong\u003e). Protein-protein interaction analysis by STRING revealed that 16 altered EV proteins involved in zymogen activation, aberrant immune response, abnormal coagulation and cellular apoptosis, which may be the consequences of vascular injuries and placental hypoxia in Bart\u0026rsquo;s hydrops fetalis (\u003cstrong\u003eFigure 6B\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;6C\u003c/strong\u003e). \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePrincipal findings of this study: This is the first study of PLAP-EVs and proteomics of maternal plasma EVs for the identification of women with Hb Bart\u0026rsquo;s fetuses. We demonstrated that: 1) EV particle size in Bart fetuses with hydropic features was significantly smaller than that in normal pregnancy and disease control groups; 2) Bart fetuses with hydropic features had higher PLAP exosome number than that in normal pregnancy; although, PLAP exosome level was highest in the placental associated complications groups; 3) Bart fetuses without hydropic features tended to have higher PLAP-EV level compared to normal pregnancy group; 4) hydropic fetuses due to non-Bart\u0026rsquo;s causes had similar PLAP level compared to normal pregnancy group; 5) EV proteomic analysis revealed aberrant immune response, pro-inflammatory cytokine regulation mediated by TLRs-MyD88-IRAK4-MAPK axis and vascular injuries as part of pathophysiology of placental edema and hypoxia in Bart\u0026rsquo;s hydrops.\u003c/p\u003e\n\u003cp\u003ePrenatal identification program for Hb Bart\u0026rsquo;s fetalis fetuses\u003c/p\u003e\n\u003cp\u003ePrevious studies have shown that there are morphological and functional changes in the responses to persistent fetal anemia\u003csup\u003e[55, 56, 57, 58, 59, 60, 61, 62, 63]\u003c/sup\u003e. Prior to the fetal hydropics stage, hemodynamic function shows a normal and good compensatory adaptation, by increasing turnover blood volume, or cardiac work and distribution of blood flow into essential organs such as brain and spleen as detected by cardiomegaly, increased middle cerebral artery peak systolic velocity (MCA-PSV) or splenic-PSV\u003csup\u003e[5]\u003c/sup\u003e. Subsequently, at the high-output hydropic stage, there are fluid accumulations in the body space such as pleural/pericardial effusion or ascites, and these features are considered late ultrasonographic signs usually detected in the second or third trimester. Lastly, persistent fetal anemia results in low cardiac output heart failure as demonstrated by low cardiac output, poor contractility and a markedly increased preload, which usually occur in the last trimester\u003csup\u003e[5]\u003c/sup\u003e. Therefore, the determination of biomarkers according to disease pathophysiology prior to hydropic state is the key.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCurrently, sonographic and maternal serum biomarkers have been extensively evaluated as screening tools to identify Hb Bart\u0026rsquo;s fetuses in pregnancy at risk for this disease. Common sonographic markers include the measurement of cardiothoracic diameter and MCA-PSV\u003csup\u003e[5, 51]\u003c/sup\u003e. Thus far, only cardiothoracic diameter (\u0026ge;0.50) can accurately predict fetal Hb Bart\u0026rsquo;s disease as early as the late first trimester (12\u0026ndash;15 weeks of gestation), with a detection rate of 75-100% at 90-100% specificity\u003csup\u003e[5, 51]\u003c/sup\u003e. The detection rate of MCA-PSV (\u0026ge;1.5 MoM) was approximately 64%-85% at 16-22 weeks of gestation, at 98-100% specificity\u003csup\u003e[5, 51]\u003c/sup\u003e. Other sonographic signs including nuchal translucency, placental thickness, liver length, splenic circumference had limited values for early identification of Hb Bart\u0026rsquo;s fetuses\u003csup\u003e[5, 51]\u003c/sup\u003e. Recently, Harn-A-Morn \u003cem\u003eet al.\u003c/em\u003e demonstrated that serial ultrasound screening (using cardiothoracic diameter and MCA-PSV) for the identification of Hb Bart\u0026rsquo;s disease during pregnancy, beginning in the first trimester and continuing every 2\u0026ndash;4 weeks until 24 weeks, has a sensitivity of 100%, at 10.9% false positive rate, in detecting pre-hydropic signs\u003csup\u003e[11]\u003c/sup\u003e. The mean gestational age at Hb Bart diagnosis was 15.5 \u0026plusmn; 2.6 weeks of gestation. Therefore, our national standard prenatal care program implements such sonographic markers for the routine screening of fetal Hb Bart\u0026rsquo;s disease in all pregnancies at risk for Hb Bart\u0026rsquo;s fetuses. The main limitations of serial ultrasound are that it requires specific equipment, and it is operator-dependent, which needs additional training. Several maternal serum biomarkers such as free beta-human chorionic gonadotropin (\u0026beta;-hCG), inhibin-A, pregnancy-associated plasma protein-A (PAPP-A), alpha-fetoprotein (MAFP), unconjugated estriol (uE3) or angiogenic factors (soluble fms-like tyrosine kinase or placental growth factor) have poor predictive values for the detection of Hb Bart\u0026rsquo;s fetuses\u003csup\u003e[5, 51]\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePlacenta-derived EVs\u0026nbsp;in Hb Bart\u0026rsquo;s fetuses\u003c/p\u003e\n\u003cp\u003eIn this study, we isolated EVs using the combinatory methods of stepwise centrifugation, ultrafiltration and qEV size exclusion chromatography\u003csup\u003e[64, 65, 66, 67]\u003c/sup\u003e\u0026nbsp; and then validated the presence of EVs in the isolates using NTA, TEM and Western blot analysis (\u003cstrong\u003eFigures 1-3\u003c/strong\u003e) following the ISEV guidelines\u003csup\u003e[52]\u003c/sup\u003e. Then, placental-specific protein PLAP containing EVs (PLAP-EVs) were measured by ELISA, while other EV proteins were detected by SWATH proteomics compared between Bart\u0026rsquo;s hydrops group and other comparators as detailed in the methods section. Accordingly, we would like to highlight that this is the first study reporting PLAP-EVs in Bart\u0026rsquo;s hydrops fetalis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePLAP is a plasma membrane enzyme isoform of alkaline phosphatase specifically produced by the syncytiotrophoblast\u003csup\u003e[68]\u003c/sup\u003e. PLAP-EVs are specific for pregnancy and are not found in the circulation of non-pregnant women\u003csup\u003e[69, 70]\u003c/sup\u003e. Herein, we are reporting that the PLAP-EVs was significantly higher in Bart\u0026rsquo;s fetuses with hydropic features compared to the couple at risk with normal pregnancy outcome (\u003cstrong\u003eFigure 4\u003c/strong\u003e). The increment of PLAP-EVs has demonstrated only in Bart\u0026rsquo; fetuses\u0026rsquo; group since women with hydropic fetuses due to non-Bart\u0026rsquo;s causes had similar levels of PLAP-EVs as those with normal pregnancy. However, PLAP-EVs in Bart\u0026rsquo;s fetuses with hydropic feature were lower than pregnant women with placental-associated complications (e.g., preeclampsia and fetal growth restriction). This result suggested that PLAP-EV levels may depend on the spectrum of placental pathologies. Further studies in a larger cohort are warranted to investigate the usability of PLAP-EVs in various pregnancy complications.\u003c/p\u003e\n\u003cp\u003eThere are very few studies about EVs in patients with thalassemia and none of these studies evaluated the role of EVs in pregnant women or Bart disease\u003csup\u003e[71, 72, 73]\u003c/sup\u003e. EVs obtained from patients with \u0026beta;-thalassemia/HbE induced platelet activation, platelet aggregation and platelet-neutrophil aggregation, which partly contributes to the hypercoagulable state\u003csup\u003e[74]\u003c/sup\u003e. In addition, such microparticles have an effect on endothelial cell activation that leads to the increased adhesion of leukocytes to endothelial cells resulting in thrombosis and vascular dysfunction especially in patients with splenectomized \u0026beta;-thalassemia/HbE disease\u003csup\u003e[71]\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWithin the maternal circulation, different EV populations are\u0026nbsp;released\u0026nbsp;from several cell types including erythrocytes\u003csup\u003e[75]\u003c/sup\u003e, endothelial cells\u003csup\u003e[76]\u003c/sup\u003e, lymphocytes, dendritic cells, and placenta during gestation. The roles of EVs are thought to be involved in cell-to-cell communication between the placenta and maternal immune system\u003csup\u003e[77]\u003c/sup\u003e. Placenta-derived EVs are thought to promote maternal immune tolerance towards the fetal allograft as they can suppress maternal T-cell signaling\u003csup\u003e[69]\u003c/sup\u003e. The local immune privilege at the feto-maternal interface has been attributed to the expression of placental-derived EV-associated functional Fas ligand (FasL), programmed death ligand 1 (PD-L1), and TNF-related apoptosis inducing ligand (TRAIL)\u003csup\u003e[69, 78]\u003c/sup\u003e. In addition, NK cell activity has been shown to be down-regulated, and this is mediated by the expression of NKG2D receptor ligands, UL-16 binding proteins (ULBP) and MHC class I chain-related (MIC) proteins on placenta-derived EVs. The NK cell activity down-regulation leads to maternal cytotoxic activity suppression, thereby, promoting fetal allograft survival\u003csup\u003e[79]\u003c/sup\u003e. In normal pregnancies, placenta-derived EVs have been shown to modulate the function of maternal endothelium to promote trophoblast migration, angiogenesis and spiral artery remodeling\u003csup\u003e[80]\u003c/sup\u003e. Yet, under inflammatory or pathological conditions (i.e., obesity, hypoxia and high blood sugar), high number of circulating EVs can induce the release of pro-inflammatory cytokines from endothelial cells which lead to perturbation of physiologic function\u003csup\u003e[80, 81, 82]\u003c/sup\u003e. Altogether, these suggest that the content and effects of EVs depend on the physiological or pathological of the pregnant women.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEvidence of placental hypoxia in Hb Bart\u0026rsquo;s fetuses includes: 1) the placenta in Hb Bart\u0026rsquo;s hydrops demonstrated an increased number of immature intermediate villi, which persist despite advancing in gestational age, and this finding is referred to as \u0026ldquo;generalized delayed villous maturation\u0026rdquo;\u003csup\u003e[49, 83]\u003c/sup\u003e; 2) the presence of villous edema as well as numerous and cytotrophoblastic cells that cover the villous stroma\u003csup\u003e[83]\u003c/sup\u003e; and 3) increased number of stromal cells at the periphery beneath the trophoblast layer, also called \u0026ldquo;peripheral villous stromal hypercellularity (PVSH)\u0026rdquo;\u003csup\u003e[83]\u003c/sup\u003e, a sign of placental adaptation to chronic hypoxia. The immature intermediate villi have a large diameter, and their predominance in the Hb Bart\u0026rsquo;s placenta leads to the narrowing of the intervillous space, impeding fetoplacental oxygen and nutrition transfer. Contractions of the myofibroblastic cells in PVSH lead to the reduction the villous size, thereby widening the intervillous space for an increase in the maternal blood flow\u003csup\u003e[49, 83]\u003c/sup\u003e. In addition, morphometric studies have shown a branching vascular pattern, which is associated with placental hypoxia. This change is thought to be responsible to the marked placental enlargement, which compromised the blood flow, from the uterine distention and the generally diminished intervillous space due to the numerous intermediate types of villi. Lastly, the dramatic reduction capacity of Hb Bart\u0026rsquo;s to extract oxygen from the intervillous space also contributes to placental hypoxia. Altogether, the presence of placental hypoxia can stimulate the release of EVs from the placenta into the maternal circulation. The observations that PLAP-EV levels were highest in women with placental associated complication group were consistent with previous studies\u003csup\u003e[82, 84, 85, 86]\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe hypothesized that the degree of hemodynamic changes or placental hypoxia is less in women with Bart\u0026rsquo;s fetuses without hydropic changes as demonstrated by a non-significant trend of high PLAP-EVs. Interestingly, women with hydropic fetuses due to non-Bart\u0026rsquo;s causes had a comparable PLAP-EV level as the normal pregnancy. Causes of hydrops in these fetuses are due to non-immune causes: cardiac disease (n=1), Down\u0026rsquo;s syndrome (n=1), and unknown non-immune causes (n=3). These cases had no fetal anemia\u0026nbsp;as shown by normal MCV-PSV. Pathophysiology of hydrops fetalis is not completely understood; however, mainly due to the developmental defects in the microcirculation and lymphatic system, decreased ventricular filling or increased central venous pressure resulting from increased right heart pressure or obstructed lymphatic drainage\u003csup\u003e[87]\u003c/sup\u003e. The presence and degree of placental hypoxia in these fetuses is currently unknown.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eProteomics profile of plasma EVs in Hb Bart\u0026rsquo;s fetuses\u003c/p\u003e\n\u003cp\u003eWe performed proteomic analysis of the\u0026nbsp;plasma EVs\u0026nbsp;of pregnant\u0026nbsp;women who had Hb Bart\u0026rsquo;s fetuses, demonstrating 16 proteins in the plasma EVs were different from normal pregnancy and women with placental associated complication group (\u003cstrong\u003eFigure 5\u003c/strong\u003e). Fibrinogen complex, fibrin clot formation and integrin signaling were functions and processes related to the significantly different proteins. Among differentially expressed proteins, hnRNPA2B1 was highest in Bart fetuses with hydropic features compared to placental associated complications (10 times higher) or normal pregnancy group (2 times higher).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ehnRNPA2B1 (A2B1), a member of the hnRNPABs subfamily, consists of two structural homologous proteins (hnRNPA2 and hnRNPB1) characterized by a tight sequence correlation and conserved domain structure, with B1 having 12 additional amino acids at N terminus compared with A2. A2B1 is an RNA-binding protein that affects the localization, shearing, stability, translation and other biochemical functions of RNA\u003csup\u003e[88]\u003c/sup\u003e.\u0026nbsp;The hnRNPs are RNA binding proteins and they complex with heterogeneous nuclear RNA (hnRNA). These proteins are associated with pre-mRNAs in the nucleus and appear to influence pre-mRNA processing and other aspects of mRNA metabolism and transport. hnRNPs is expressed in several tissues such as placenta, ovary, musculoskeletal,\u0026nbsp;endocrine organs and gastrointestinal tracts\u003csup\u003e[89]\u003c/sup\u003e. In pregnancy, these proteins were differentially expressed in the placenta of women with gestational diabetes mellitus and may play a role in regulating the occurrence and development of gestational diabetes\u003csup\u003e[89]\u003c/sup\u003e. It is possible that the presence of placental hypoxia in Bart fetuses triggers the release of hnRNPA2B1 since early gestation and this is specific to Hb Bart fetuses. However, a larger trial is required to further validate these findings.\u003c/p\u003e\n\u003cp\u003eClinical and research implications\u003c/p\u003e\n\u003cp\u003eUltrasound parameters and some soluble maternal blood biomarkers have been proposed to be used for the prediction of Hb Bart\u0026rsquo;s fetuses. However, we still lack sensitive methods for effective early screening. In addition, ultrasound parameters require standardization and proper training. Biomarkers from plasma EVs could improve the prediction of Hb Bart\u0026rsquo;s fetuses. Indeed, this study revealed that the PLAP-EV level in maternal circulation is higher in Bart\u0026rsquo;s fetuses and 16 EV proteins as well as hnRNPA2B protein in maternal plasma exosome have different concentrations in cases of Hb Bart\u0026rsquo;s fetuses compared to normal pregnancy and those with placental associated complications. Measuring PLAP-EVs together with differentially expressed EV proteins, might provide independent information to improve the ability to identify affected fetuses with Hb Bart disease.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe envision that soluble markers secreted from the placenta can be assessed non-invasively in ongoing pregnancies through a \u0026ldquo;liquid biopsy\u0026rdquo;. A term \u0026ldquo;liquid biopsy\u0026rdquo; refers to a test performed\u0026nbsp;on any biofluid specimens such as plasma, urine amniotic fluid or cervico-vaginal fluid\u003csup\u003e[90, 91, 92]\u003c/sup\u003e in which the result can be used to infer pathologic changes in distant tissues (e.g., cancer) or other pathologic processes (e.g., atherosclerosis)\u003csup\u003e[93, 94, 95]\u003c/sup\u003e. We believe that the non-invasive liquid biopsy could provide information in relation to placental health. In this report, the liquid biopsy based on an examination of placenta-derived EVs in maternal blood for the identification of Hb Bart\u0026rsquo;s fetuses is feasible.\u003c/p\u003e\n\u003cp\u003eFor the strengths and limitations, this is the first study to evaluate PLAP-EVs and proteomics profile of maternal plasma EVs in women with Bart\u0026rsquo;s fetuses. The strengths of this study are that we have included several groups such as Hb Bart\u0026rsquo;s group with or without hydropic features, hydropic fetuses due to other causes, or placental associated complication group. The latter group is considered the disease comparator group since compelling evidence suggests abnormal profile of placenta-derived EVs in women with placental associated complications. In addition, we confirmed by definite invasive prenatal diagnosis test that all couple at risks with normal pregnancy group had non-Bart\u0026rsquo;s fetuses and delivered without maternal or neonatal complications. The separation of EVs in the current study was confirmed by published methods including NTA, TEM and EV markers by Western blotting analysis following the international standard. The main limitation of our study is related to a small sample size. Future research is needed to confirm our findings in a larger number of populations.\u003c/p\u003e\n\u003cp\u003eIn the conclusions, EV particle size is smaller, while placental-derived EV level is significantly higher in women with Hb Bart\u0026rsquo;s fetalis fetuses compared to normal pregnancy. In addition, several EV proteins were differential expressed in women with Bart\u0026rsquo;s fetuses and these proteins may play roles in innate immunity, TLRs-MyD88-IRAK4-MAPK axis, and vascular injury as the consequences of placental hypoxia in Bart\u0026rsquo;s hydrops. The result of this study support in future investigations to determine whether placenta-derived EVs in maternal circulation can be used as a liquid biopsy or non-invasive prenatal test for the early identification of Bart\u0026rsquo;s fetuses. This will ultimately reduce the rate of unnecessary invasive prenatal diagnosis testing.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eStudy population\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis was a prospective cohort\u0026nbsp;study in pregnant women at 11-13 weeks\u0026rsquo; gestation onwards who attended prenatal care clinics from November 2021 to\u0026nbsp;November 2023. Participants were recruited at: 1) Ramathibodi Hospital, Bangkok, Thailand; and 2) Siriraj Hospital, Bangkok, Thailand.\u0026nbsp;In Thailand, all pregnant women (and their couples if available) are required to perform thalassemia screening at the first prenatal visit. Clinical standard management for thalassemia screening includes the examination of mean corpuscular volume (MCV), Dichlorophenolindophenol (DCIP), osmotic fragility (OF) test, Hb typing, and alpha or beta-thalassemia mutations in some cases. Couples at risk for Hb Bart\u0026rsquo;s fetuses were counseled to: 1) perform diagnostic test; or 2) follow up with non-invasive approach such as ultrasonography. The diagnostic tests depend on gestational age of the patients, for example, chorionic villous sampling and amniocentesis for the determination of fetal DNA was performed at 11-13 and 16-18\u0026nbsp;weeks of gestation, respectively, while cordocentesis was performed after 18 weeks of gestation for Hb typing\u003csup\u003e[5, 10, 11]\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInclusion criteria were women\u0026nbsp;at aged \u0026ge;18 years and 11-13 weeks of gestation onward.\u0026nbsp;Exclusion criteria were women who were unable to give informed consent or those who had severe major fetal abnormality and those who were couple at risk of Bart\u0026rsquo;s fetuses without prenatal diagnostic test confirmation.\u0026nbsp;All patients provided written informed consent prior to the collection of samples. The use of clinical databases and biological samples was approved by the Human Research Ethics Committee of Mahidol University, Thailand, with IRB No. COA.MURA2021/439 (Ramathibodi Hospital) and COA.No.SI 840/2021 (Siriraj Hospital).\u0026nbsp;The study was approved by an appropriate institution and we confirmed that all methods were performed in accordance with the relevant guidelines and regulations by including a statement in the methods section.\u003c/p\u003e\n\u003cp\u003eA total\u0026nbsp;of 27 women\u0026nbsp;were included in the study. We stratified participants into the following groups: 1) normal pregnancy or control group:\u0026nbsp;women with\u0026nbsp;couple at risk for Hb Bart\u0026rsquo;s fetuses but subsequently proven as normal pregnancy (non-Bart affected fetuses) (Group 1) (n=7); 2) Hb Bart\u0026rsquo;s with hydropic features (Group 2) (n=4); 3) Hb Bart\u0026rsquo;s without hydropic features (Group 3) (n=7); 4) a disease comparator group which consisted of\u0026nbsp;women who subsequently delivered with placental associated conditions (such as preeclampsia, fetal growth restriction, unexplained fetal death, spontaneous preterm labor or preterm pre-labor rupture of membranes) (Group 4) (n=4); and 5) women with\u0026nbsp;hydrops fetalis from non-Bart\u0026rsquo;s causes (n=5) (Group 5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll women in Group 1 delivered at term gestation without complication and had appropriate weight for gestational age fetuses. Cases in Group 2 and 3 were matched with patients classified in a disease comparator group (Group 4)\u0026nbsp;based on gestational age at venipuncture and maternal characteristics (age, race, parity, body mass index).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical definitions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eObstetric and delivery outcomes, including mode of delivery, maternal and neonatal outcomes, were obtained from the maternity computerized records and such information were reviewed by dedicated researchers.\u0026nbsp;\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eHydrops fetalis is characterized by the abnormal interstitial fluid collection in two or more compartments of the fetal body (peritoneal cavity, pleura, and pericardium) or fluid accumulation in one site and anasarca\u003csup\u003e[96, 97, 98, 99, 100, 101, 102, 103]\u003c/sup\u003e.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003ePreeclampsia was defined as systolic blood pressure (BP) \u0026ge;140 mmHg and/or diastolic BP \u0026ge;90 mmHg on at least two occasions measured 4 hours apart in previously normotensive women, accompanied by one or more of the following new-onset conditions at or after 20 weeks of gestation: proteinuria, evidence of other maternal organ dysfunction such as acute kidney injury, liver involvement, elevated liver enzymes, neurological complications, hematological complications, or uteroplacental dysfunction\u003csup\u003e[104]\u003c/sup\u003e.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eFetal growth restriction (FGR) was defined as\u0026nbsp;estimated fetal weight (EFW)\u0026nbsp;or abdominal circumference (AC) \u0026lt;3\u003csup\u003erd\u003c/sup\u003e percentile or EFW or AC \u0026lt;10\u003csup\u003eth\u003c/sup\u003e percentile combined with abnormal Doppler findings or a decrease in growth centiles\u003csup\u003e[105]\u003c/sup\u003e.\u0026nbsp;In all FGR fetuses, we confirmed that neonatal birth weight was below 3\u003csup\u003erd\u003c/sup\u003e percentile at birth.\u003c/li\u003e\n \u003cli\u003ePreterm birth was defined as delivery at \u0026lt;37 completed weeks of pregnancy, and spontaneous preterm delivery included spontaneous onset of labor with intact membranes, pre-labor rupture of membranes (PPROM), and cervical insufficiency\u003csup\u003e[106]\u003c/sup\u003e.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eUncomplicated pregnancy or normal pregnancy group was defined as a live birth at or after 37 weeks of gestation with appropriate weight for gestational age fetuses without any complications.\u0026nbsp;\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cstrong\u003eEV isolation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMaternal blood was collected in EDTA tube and transported to the laboratory immediately. EVs were isolated from the plasma biofluid by a combination of stepwise centrifugation, ultrafiltration and qEV size exclusion chromatography as described in our previous studies\u003csup\u003e[64, 65, 66, 67]\u003c/sup\u003e. Briefly, 500 \u0026micro;l of plasma was diluted in filtered PBS (1:1) to 1 mL, centrifuged at 3000 g for 15 min at 4\u0026deg;C to remove cell debris, and centrifuged at 12,000 x g for 20 min at 4\u0026deg;C to remove large particles and protein aggregates. The supernatant was filtered through a 100-kDa cut-off centrifugal filter to remove soluble proteins (with the molecular weight less than 100 kDa) and concentrated the sample volume (from 1 ml to 500 \u0026micro;l) before passing through the qEV size exclusion chromatography (IZON). The EV fractions were collected, pooled, and concentrated by a 3-kDa cut-off centrifugal filter to the final volume of 100 \u0026micro;l of the EV isolate.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEV validation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNanoparticle tracking analysis\u003c/p\u003e\n\u003cp\u003eEV size and number were measured by View Sizer 3000 nanoparticle size analyzer (Horiba). One\u0026nbsp;\u0026micro;l of the isolated EVs was diluted in the filtered PBS (1:2000) and then transferred 1 ml into the cuvette with magnetic stirrer to capture 25 images in a video. Acquisition parameter was set as blue laser (445 nm) 210 mW, green laser (520 nm) 12 mW, red laser (635 nm) 8 mW, pulse duration (B/G/R) 15/15/15 ms, 5 s stirring time, 1400 rpm stirring speed, 3s wait time, 300 frames/video and 25 video count. The software calculated particle size and number with subtraction to blank as the filtered PBS alone.\u003c/p\u003e\n\u003cp\u003eTransmission electron microscope (TEM)\u003c/p\u003e\n\u003cp\u003eIntact EV morphology was imaged by TEM. Briefly, ten microliters of the isolated EVs were dotted on parafilm and inverted grid face on the sample for 10 min at room temperature. The grid face was washed in PBS for 1 min twice. After fixing with 2.5% (v/v) glutaraldehyde for 5 min, the grid face was washed in PBS for 1 min twice. The grid face was stained with 2% (w/v) uranyl acetate for 1 min and then air dried for 10 min. The EV on the grid face was captured under TEM.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWestern blotting of EV protein markers\u003c/p\u003e\n\u003cp\u003eSpecific EV markers were detected by Western blotting analysis. Briefly, twenty microliters of the isolated EVs were lysed in 1\u0026times; reducing buffer containing 62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, and 2.5% beta-mercaptoethanol combination with sonication. After heating at 95\u0026deg;C for 5 min, the EV proteins were measured by Bradford\u0026rsquo;s assay. Equal 10\u0026nbsp;\u0026micro;g total protein amount was resolved on 10% SDS-PAGE and then blotted on a nitrocellulose membrane. Non-specific binding sites on the membrane were blocked with 5%skim milk/PBS at room temperature for 30 min, and then probed with primary antibody i.e., rabbit polyclonal anti-CD9 (ab223052, Abcam), rabbit polyclonal anti-HSP70 (ab79852, Abcam), and mouse monoclonal anti-CD63 (ab193349, Abcam) antibodies at dilution 1:1000 in 5%BSA/PBS at 4\u0026deg;C overnight. The membrane was washed with 0.5% tween20 in PBS (PBST) for 3 times, 5 min each. The membrane was probed with secondary antibodies conjugated horseradish peroxidase at dilution 1:2000 in 3% skim milk/PBST at room temperature for 1 h. After washing with PBST, the peroxidase substrate for enhanced chemiluminescence (ECL) was reacted on specific bands. The chemiluminescence signal was observed under chemiluminescence imaging detector (G:BOX, Syngene).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlacental alkaline phosphatase (PLAP) ELISA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIsolated EVs were measured PLAP level using Human Alkaline phosphatase, placental type sandwich ELISA kit (MBS289869, MyBioSource). Briefly, the highest PLAP concentration (1000 pg/ml) was diluted two-fold to set standard curve (1000, 500, 250, 125, 62.5, 31.25, 15.625, 0 pg/ml). An equal volume of 100 \u0026micro;l recombinant protein standard, 100 \u0026micro;l plasma and 20 \u0026micro;l EVs (add 80 \u0026micro;l PBS to make 100 \u0026micro;l final volume) was added into each well (2 technical replication, 7 biological replications per group) and incubated at 37\u0026deg;C for 2 h. All solution in each well was removed and then 100 \u0026mu;L of detection reagent A working solution to each well. After incubation at 37\u0026deg;C for 1 h, the liquid in each well was removed and washed 3 times by filling each well with 300 \u0026micro;l wash buffer and aspirating completely. 100 \u0026micro;l of detection reagent B working solution was added into each well and then incubated the plate at 37\u0026deg;C for 1 h. After completely wash 5 times, 90 \u0026micro;l of substrate solution was added in each well and then incubated at 37\u0026deg;C for 15 min in dark. Thereafter, 50 \u0026micro;l of stop solution was added and then measured the optical density at 450 nm. The OD of samples and standards was subtracted with blank (0 pg/ml PLAP) before calculation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSWATH proteomics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSWATH proteomics was performed as our previous studies\u003csup\u003e[107, 108, 109]\u003c/sup\u003e. Briefly, 50 \u0026micro;l of EVs were lysed in Laemmli\u0026rsquo;s buffer (62.5 mM Tris-HCl, pH 6.8, 10% (v/v) glycerol, 2% (w/v) SDS and 2.5% (v/v) beta-mercaptoethanol). Total protein was quantified using the Bradford protein assay (Bio-Rad, Hercules, USA). Fifty\u0026nbsp;micrograms of proteins were reduced, alkylated, and then digested by trypsin enzyme. After stop reaction with 5% formic acid in acetronitrile, the solution containing peptides were dried using a speedvac concentrator. The dried peptides were reconstituted in 0.1% formic acid before desalting by C18 stage tip. The 2 \u0026micro;g in 2 \u0026micro;l equal amount and volume of the desalted peptides were injected into an Eksigent nanoLC ultra nanoflow high performance liquid chromatography coupled with a TripleTOF 6600+ mass spectrometer (ABSciex, Toronto, Canada) installed at the proteomic core facility of Ramathibodi Hospital set for information-dependent acquisition (IDA) and data-independent acquisition (DIA) modes. The peptides were loaded onto a C18 column trap (Nano Trap RP-1, 3 \u0026mu;m 120 \u0026Aring;, 10 mm \u0026times; 0.075 mm; Phenomenex, CA, USA) at a flow rate of 3 \u0026mu;l/min of 0.1% formic acid in water for 10 min to desalt and concentrate the sample, which was then separated on a C18 analytical column (bioZen Peptide Polar C18 nanocolumn, 75 \u0026mu;m \u0026times; 15 cm, particle size 3 \u0026mu;m, 120 \u0026Aring;; Phenomenex) with mobile phase gradients at a flow rate of 300 nL/min of 3-30% acetonitrile/0.1% formic acid for 60 min, 30-40% acetonitrile/0.1% formic acid for 10 min, 40-80% acetonitrile/0.1% formic acid for 2 min, 80% acetonitrile/0.1% formic acid for 6 min, 80-3% acetonitrile/0.1% formic acid for 2 min, and 3% acetonitrile/0.1% formic acid for 25 min. The eluate was ionized and sprayed into the mass spectrometer using OptiFlow Turbo V Source (Sciex). Ion source gas 1 (GS1), ion source gas 2 (GS2), and curtain gas were set at 19, 0, and 25 vendor arbitrary units, respectively. The interface heater temperature was 150\u0026deg;C and ion spray voltage was 3.3 kV.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMass spectrometry was operated in the positive ion mode set for 3,500 cycles per 105 min gradient elution. Each cycle performed 1 time of flight (TOF) scan (250 ms accumulation time, 350\u0026ndash;1250 m/z window with a charge state of +2) followed by IDA of the 30 most intense ions, while the minimum MS signal was set to 150 counts. The MS/MS scan was operated in high sensitivity mode with 50 ms accumulation time and 100 ppm mass tolerance. Former MS/MS candidate ions were excluded for a period of 12 sec after their first occurrence to reduce the redundancy of identified peptides. DIA mode was performed in a range of 350 to 1500\u0026thinsp;m/z using a predefined mass window of 7-m/z with an overlap of 1-m/z for 157 transmissible windows. MS scan was set at 2,044 cycles, where each cycle performs 1 TOF-MS scan type (50 ms accumulation time across 100\u0026ndash;1500 precursor mass range) acquired in every cycle for a total cycle time of 3.08 sec. MS spectra of 100\u0026ndash;1500 m/z were collected with an accumulation time of 96 ms per SWATH window width. Resolution for MS1 was 35,000 and SWATH-MS2 scan was 30,000. Rolling collision energy mode with collision energy spread of 15 eV was applied. The IDA and DIA data (.\u003cem\u003ewiff\u003c/em\u003e) were recorded by Analyst-TF v.1.8 software (ABSciex).\u003c/p\u003e\n\u003cp\u003eA total of 36 wiff files of IDA experiments (5 groups; 6 patients with normal pregnancy group, 3 patients with Hb Bart\u0026rsquo;s with hydropic features, 4 patients with Hb Bart\u0026rsquo;s without hydropic features, 3 patients with disease control (disease comparator), and 2 patients with hydrops fetalis from non-Bart\u0026rsquo;s causes; 2 technical replicates per biological sample) were combined and searched using Protein Pilot v.5.0.2.0 software (ABSciex) against the Swiss-Prot database (UniProtKB 2022_01) Homo sapiens (20,385 proteins in database) with the searching parameters as follows; alkylation on cysteine by iodoacetamide, trypsin enzymatic digestion, 1 missed cleavage allowed, monoisotopic mass, and 1% false discovery rate. The group file (Protein Pilot search result) was loaded into SWATH Acquisition MicroApp v.2.0.1.2133 in PeakView software v.2.2 (Sciex) to generate a spectral library. The maximum number of proteins was set as the number of proteins identified at 1% global FDR from fit. RT alignment was performed by the high abundance endogenous peptides covering the chromatographic range. SWATH data extraction of 36 DIA files (5 groups; 2-6 biological replicates per group; 2 technical replicates per biological sample) was performed by SWATH Acquisition MicroApp (Sciex) using the following parameters; 5-min extraction window, 25 peptides/protein, 6 transitions/peptide, excluding shared peptides, peptide confidence \u0026gt;99%, FDR \u0026lt;1%, and XIC width of 20 ppm. SWATH extraction data, including the identities and quantities of peptides and proteins, was normalized using multiple linear regression by Marker View software and then exported into an Excel file for further analysis. The protein area in each group were analyzed using ANOVA and multiple comparison (Tukey) by SPSS software. The significantly differentially expressed proteins were predicted their functional involvements by STRING protein-protein interaction pathway analysis (https://string-db.org/) and reactome pathway analysis (https://reactome.org/) where the predicted pathways with false discovery rate (FDR) less than 0.05 was considered statistically significant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDemographics data analysis\u003c/p\u003e\n\u003cp\u003eDemographic and clinical variables were summarized in median [interquartile range (IQR)] for numerical variables and count (percentage) for categorical variables. The comparison of categorical data, and continuous data was assessed by \u0026chi;2 test, and Mann-Whitney U test, respectively. A probability value (P value) of less than 0.05 was considered statistically significant. The statistical software STATA V17 (California, USA), IBM SPSS version 18 (Armonk, N.Y., USA), and MedCalC version 20.218 (Mariakerke, Belgium) were used for statistical analysis.\u003c/p\u003e\n\u003cp\u003eCalculation of protein levels from ELISA\u003c/p\u003e\n\u003cp\u003eAverage the duplicate readings for each standard and samples were subtracted the average zero standard optical density. A best fit curve through the points to establish standard curve for each test could be determined by regression analysis (r\u003csup\u003e2\u003c/sup\u003e\u0026gt;0.99). The protein concentration of each sample was calculated from an equation. Statistical analysis used Mann-Whitney U test at p\u0026lt;0.05. \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This research was funded by The Royal Thai College of Obstetricians and Gynecologists (RTCOG) and Faculty of Medicine Ramathibodi Hospital (RF-65027) to P.C.\u0026nbsp;The funders had no role in study design, analysis and interpretation of data, decision to publish, or preparation of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e We would like to thank nurses and physicians at the antenatal care clinic and labor and delivery unit at Ramathibodi and Siriraj Hospital,\u0026nbsp;Mahidol University. Moreover, we would like to thank the teams for research assistance and facility at Central Laboratory of Pediatrics Department and Research Center at Faculty of Medicine Ramathibodi Hospital, Mahidol University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: P.C., W.C., S.C.\u003c/p\u003e\n\u003cp\u003eSpecimen collection: P.C., P.W., P.R., C.P., T.P.B., M.P.\u003c/p\u003e\n\u003cp\u003eMethodology: P.C., W.C., S.C.\u003c/p\u003e\n\u003cp\u003eInvestigation: P.C., P.W., S.L., C.P., K.R., N.C., P.R., T.P.B., T.K., J.P., T.K., W.C.\u003c/p\u003e\n\u003cp\u003eFormal analysis: P.C., P.W., S.L., C.P., K.R., N.C., P.R., T.P.B., T.K., J.P., T.K., W.C.\u003c/p\u003e\n\u003cp\u003eValidation: P.C., W.C., S.C.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eData curation: P.C., P.W., S.L., C.P., K.R., N.C., P.R., T.P.B., T.K., W.C.\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; Original Draft Preparation: P.C.\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; Review \u0026amp; Editing: P.C., P.W., S.L., C.P., K.R., N.C., P.R., T.P.B., T.K., J.P., T.K., W.C., S.C.\u003c/p\u003e\n\u003cp\u003eVisualization: P.C., W.C.\u003c/p\u003e\n\u003cp\u003eFunding acquisition: P.C.\u003c/p\u003e\n\u003cp\u003eProject administration: P.C.\u003c/p\u003e\n\u003cp\u003eSupervision: P.C., W.C., S.C.\u003c/p\u003e\n\u003cp\u003eAll authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analysed during the current study are available in the ProteomeXchange with identifier PXD051236. [ http://www.ebi.ac.uk/pride ]\u003c/p\u003e\n\u003cp\u003eSubmission details:\u003c/p\u003e\n\u003cp\u003eProject Name: Placenta-derived Extracellular Vesicles in Maternal Plasma of Hb Bart\u0026rsquo;s Fetuses\u003c/p\u003e\n\u003cp\u003eProject accession: PXD051236\u003c/p\u003e\n\u003cp\u003eReviewer account details:\u003c/p\u003e\n\u003cp\u003eUsername: [email protected]\u003c/p\u003e\n\u003cp\u003ePassword: N5riJ8S0\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003eNone\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFlint J\u003cem\u003e, et al.\u003c/em\u003e High frequencies of alpha-thalassaemia are the result of natural selection by malaria. Nature. 1986;\u003cstrong\u003e321\u003c/strong\u003e(6072):744-750.\u003c/li\u003e\n\u003cli\u003eWanapirak C, Muninthorn W, Sanguansermsri T, Dhananjayanonda P, Tongsong T. Prevalence of thalassemia in pregnant women at Maharaj Nakorn Chiang Mai Hospital. 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Glob Chall. 2023;\u003cstrong\u003e7\u003c/strong\u003e(3):2200213.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1:\u003c/strong\u003e Characteristics of participants according to the study groups\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"738\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.32520325203252%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.788617886178862%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNormal pregnancy group\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(Group 1)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n=7)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.246612466124661%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eHb Bart\u0026rsquo;s fetuses with hydropic features group\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(Group 2)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n=4)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.517615176151761%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eHb Bart\u0026rsquo;s fetuses without hydropic features group\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(Group 3)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n=7)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.91869918699187%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eDisease comparator\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(disease control) group\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(Group 4)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n=4)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.634146341463415%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eHydropic fetuses with non-Bart\u0026rsquo; causes\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(Group 5)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n=5)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.56910569105691%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eP value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.32520325203252%\" valign=\"top\"\u003e\n \u003cp\u003eMaternal age (year)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.788617886178862%\"\u003e\n \u003cp\u003e24.3\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(22.24-27.30)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.246612466124661%\"\u003e\n \u003cp\u003e31.8\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(29.54-33.77)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.517615176151761%\"\u003e\n \u003cp\u003e37.1\u003c/p\u003e\n \u003cp\u003e(29.54-33.77)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.91869918699187%\"\u003e\n \u003cp\u003e33.0\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(25.53-38.39)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.634146341463415%\"\u003e\n \u003cp\u003e22.9\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(20.71-35.11)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.56910569105691%\"\u003e\n \u003cp\u003ep=0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.32520325203252%\" valign=\"top\"\u003e\n \u003cp\u003eMaternal weight (kilogram)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.788617886178862%\"\u003e\n \u003cp\u003e60.5\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;(49.0-70.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.246612466124661%\"\u003e\n \u003cp\u003e63.0\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(47.0-53.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.517615176151761%\"\u003e\n \u003cp\u003e68.8\u003c/p\u003e\n \u003cp\u003e(62.0-70.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.91869918699187%\"\u003e\n \u003cp\u003e66.50\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(53.50-76.50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.634146341463415%\"\u003e\n \u003cp\u003e69.5\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(49.5-85.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.56910569105691%\"\u003e\n \u003cp\u003ep=0.69\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.32520325203252%\" valign=\"top\"\u003e\n \u003cp\u003eMaternal underlying diseases\u003c/p\u003e\n \u003cul\u003e\n \u003cli\u003eDM\u003c/li\u003e\n \u003cli\u003eChronic hypertension\u003c/li\u003e\n \u003c/ul\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.788617886178862%\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.246612466124661%\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.517615176151761%\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.91869918699187%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e50% (2)\u003c/p\u003e\n \u003cp\u003e50% (2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.634146341463415%\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.56910569105691%\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.32520325203252%\" valign=\"top\"\u003e\n \u003cp\u003eGA at blood sampling (weeks)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.788617886178862%\"\u003e\n \u003cp\u003e12.9\u003c/p\u003e\n \u003cp\u003e(12.00-18.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.246612466124661%\"\u003e\n \u003cp\u003e27.9\u003c/p\u003e\n \u003cp\u003e(22.75-33.75)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.517615176151761%\"\u003e\n \u003cp\u003e12.9\u003c/p\u003e\n \u003cp\u003e(12.29-18.29)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.91869918699187%\"\u003e\n \u003cp\u003e34.9\u003c/p\u003e\n \u003cp\u003e(24.43-37.14)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.634146341463415%\"\u003e\n \u003cp\u003e22.0\u003c/p\u003e\n \u003cp\u003e(21.14-29.50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.56910569105691%\"\u003e\n \u003cp\u003ep\u0026lt;0.05\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.32520325203252%\" valign=\"top\"\u003e\n \u003cp\u003eGA at delivery or termination of pregnancy (weeks)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.788617886178862%\"\u003e\n \u003cp\u003e38.4\u003c/p\u003e\n \u003cp\u003e(37.21-39.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.246612466124661%\"\u003e\n \u003cp\u003e27.9\u003c/p\u003e\n \u003cp\u003e(22.75-33.75)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.517615176151761%\"\u003e\n \u003cp\u003e17.4\u003c/p\u003e\n \u003cp\u003e(14.75-25.14)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.91869918699187%\"\u003e\n \u003cp\u003e36.0\u003c/p\u003e\n \u003cp\u003e(28.93-38.36)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.634146341463415%\"\u003e\n \u003cp\u003e21.7\u003c/p\u003e\n \u003cp\u003e(21.43-32.29)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.56910569105691%\"\u003e\n \u003cp\u003ep\u0026lt;0.05\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.32520325203252%\" valign=\"top\"\u003e\n \u003cp\u003eRoute of delivery\u003c/p\u003e\n \u003cul\u003e\n \u003cli\u003eVagina\u003c/li\u003e\n \u003c/ul\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.788617886178862%\"\u003e\n \u003cp\u003e86% (6/7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.246612466124661%\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.517615176151761%\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.91869918699187%\"\u003e\n \u003cp\u003e0 (0/7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.634146341463415%\"\u003e\n \u003cp\u003e100% (5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.56910569105691%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.32520325203252%\" valign=\"top\"\u003e\n \u003cp\u003eIndication for Cesarean delivery\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.788617886178862%\"\u003e\n \u003cp\u003eCPD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.246612466124661%\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.517615176151761%\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.91869918699187%\" valign=\"top\"\u003e\n \u003cp\u003eNon-reassuring fetal status\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.634146341463415%\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.56910569105691%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.32520325203252%\" valign=\"top\"\u003e\n \u003cp\u003eBaby weight (gram)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.788617886178862%\"\u003e\n \u003cp\u003e3,035\u003c/p\u003e\n \u003cp\u003e(2,787-3,252)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.246612466124661%\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.517615176151761%\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.91869918699187%\" valign=\"top\"\u003e\n \u003cp\u003e1,910\u003c/p\u003e\n \u003cp\u003e(813.75-2,732.50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.634146341463415%\"\u003e\n \u003cp\u003e1,564.0\u003c/p\u003e\n \u003cp\u003e(481.0-2,050.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.56910569105691%\"\u003e\n \u003cp\u003e\u0026gt;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.32520325203252%\" valign=\"top\"\u003e\n \u003cp\u003ePregnancy complications*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.788617886178862%\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.246612466124661%\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.517615176151761%\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.91869918699187%\"\u003e\n \u003cul\u003e\n \u003cli\u003eFetal growth restriction (n=2)\u003c/li\u003e\n \u003cli\u003eSuperimpose preeclampsia (n=1)\u003c/li\u003e\n \u003cli\u003ePreeclampsia (n=1)\u003c/li\u003e\n \u003c/ul\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.634146341463415%\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.56910569105691%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.32520325203252%\" valign=\"top\"\u003e\n \u003cp\u003eCauses of non-Bart\u0026rsquo;s hydrops **\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.788617886178862%\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.246612466124661%\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.517615176151761%\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.91869918699187%\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.634146341463415%\"\u003e\n \u003cul\u003e\n \u003cli\u003eCardiac disease (n=1)\u003c/li\u003e\n \u003cli\u003eDown\u0026rsquo;s syndrome (n=1)\u003c/li\u003e\n \u003cli\u003eUnknown non-immune causes (n=3)\u003c/li\u003e\n \u003c/ul\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.56910569105691%\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eBMI: body mass index; CPD: cephalopelvic disproportion; GA: gestational age; NA: not applicable\u003c/p\u003e\n\u003cp\u003e*: only in disease control group; **: only in Group 5\u003c/p\u003e\n\u003cp\u003eData presented as % (n) or median (interquartile)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eP values determined by Kruskal-Wallis test\u003c/p\u003e\n\u003cp\u003e#p\u0026gt;0.05 for group 1 and 2; group 2 and 4; p=0.006 for group 2 and 4\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e##\u003c/sup\u003ep=0.029 for group 2 and 4\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e Multiple comparison of significantly differential proteins among 5 groups.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.208333333333333%\" rowspan=\"2\" style=\"width: 5.2212%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSwissProt ID\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.291666666666667%\" rowspan=\"2\" style=\"width: 7.0639%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGene name\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.916666666666668%\" rowspan=\"2\" style=\"width: 9.2906%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eProtein name\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.208333333333333%\" rowspan=\"2\" style=\"width: 4.4534%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eANOVA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"58.333333333333336%\" colspan=\"10\" valign=\"bottom\" style=\"width: 49.2173%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMultiple comparison (Tukey)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.909090909090908%\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNormal pregnancy vs Bart hydrops\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.909090909090908%\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNormal pregnancy vs Bart no hydrops\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.909090909090908%\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNormal pregnancy vs Disease control\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.909090909090908%\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNormal pregnancy vs Hydrops not Bart\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.090909090909092%\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBart hydrops vs Bart no hydrops\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.090909090909092%\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBart hydrops vs Disease control\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.090909090909092%\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBart hydrops vs Hydrops not Bart\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.090909090909092%\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBart no hydrops vs Disease control\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.090909090909092%\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBart no hydrops vs Hydrops not Bart\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.090909090909092%\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDisease control vs Hydrops not Bart\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"bottom\" style=\"width: 5.2212%;\"\u003e\n \u003cp\u003eB9A064\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.446808510638298%\" valign=\"bottom\" style=\"width: 7.0639%;\"\u003e\n \u003cp\u003eIGLL5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.404255319148938%\" valign=\"bottom\" style=\"width: 9.2906%;\"\u003e\n \u003cp\u003eImmunoglobulin lambda-like polypeptide 5\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.4534%;\"\u003e\n \u003cp\u003e0.0051\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.8952\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.9038\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.0220\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.9925\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e1.0000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.0093\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.9978\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.0062\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.9991\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.0497\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"bottom\" style=\"width: 5.2212%;\"\u003e\n \u003cp\u003eP00736\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.446808510638298%\" valign=\"bottom\" style=\"width: 7.0639%;\"\u003e\n \u003cp\u003eC1R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.404255319148938%\" valign=\"bottom\" style=\"width: 9.2906%;\"\u003e\n \u003cp\u003eComplement C1r subcomponent\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.4534%;\"\u003e\n \u003cp\u003e0.0252\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e1.0000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n 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valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.0769\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.9789\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.9713\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.4227\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.8564\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.8530\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.0609\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.0963\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e1.0000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"bottom\" style=\"width: 5.2212%;\"\u003e\n \u003cp\u003eP02671\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.446808510638298%\" valign=\"bottom\" style=\"width: 7.0639%;\"\u003e\n \u003cp\u003eFGA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.404255319148938%\" valign=\"bottom\" style=\"width: 9.2906%;\"\u003e\n \u003cp\u003eFibrinogen alpha chain\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.4534%;\"\u003e\n \u003cp\u003e0.0045\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.9999\u003c/p\u003e\n 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5.6051%;\"\u003e\n \u003cp\u003e0.6147\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.8512\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.0000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.1194\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.9892\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.0066\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.7952\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.0009\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.5102\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.2032\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"bottom\" style=\"width: 5.2212%;\"\u003e\n \u003cp\u003eP02679\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.446808510638298%\" valign=\"bottom\" style=\"width: 7.0639%;\"\u003e\n \u003cp\u003eFGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.404255319148938%\" valign=\"bottom\" style=\"width: 9.2906%;\"\u003e\n \u003cp\u003eFibrinogen gamma chain\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.4534%;\"\u003e\n \u003cp\u003e0.0318\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.9983\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.9921\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.0183\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.9243\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e1.0000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.0914\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.9868\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.0762\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.9916\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.3686\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"bottom\" style=\"width: 5.2212%;\"\u003e\n \u003cp\u003eP02745\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.446808510638298%\" valign=\"bottom\" style=\"width: 7.0639%;\"\u003e\n \u003cp\u003eC1QA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.404255319148938%\" valign=\"bottom\" style=\"width: 9.2906%;\"\u003e\n \u003cp\u003eComplement C1q subcomponent subunit A\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.4534%;\"\u003e\n \u003cp\u003e0.0288\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.9986\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.9999\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.0271\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.9965\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.9999\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.1187\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.9834\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.0618\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.9919\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.0676\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"bottom\" style=\"width: 5.2212%;\"\u003e\n \u003cp\u003eP04114\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.446808510638298%\" valign=\"bottom\" style=\"width: 7.0639%;\"\u003e\n \u003cp\u003eAPOB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.404255319148938%\" valign=\"bottom\" style=\"width: 9.2906%;\"\u003e\n \u003cp\u003eApolipoprotein B-100\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.4534%;\"\u003e\n \u003cp\u003e0.0112\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.9995\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e1.0000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.0094\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.9998\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.9993\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.0478\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e1.0000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.0169\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.9997\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.0890\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"bottom\" style=\"width: 5.2212%;\"\u003e\n \u003cp\u003eP04259\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.446808510638298%\" valign=\"bottom\" style=\"width: 7.0639%;\"\u003e\n \u003cp\u003eKRT6B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.404255319148938%\" valign=\"bottom\" style=\"width: 9.2906%;\"\u003e\n \u003cp\u003eKeratin, type II cytoskeletal 6B\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.4534%;\"\u003e\n \u003cp\u003e0.0096\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.0137\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.9997\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.9934\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.9937\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.0374\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.0175\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.0367\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.9817\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.9837\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e1.0000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"bottom\" style=\"width: 5.2212%;\"\u003e\n \u003cp\u003eP06312\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.446808510638298%\" valign=\"bottom\" style=\"width: 7.0639%;\"\u003e\n \u003cp\u003eIGKV4-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.404255319148938%\" valign=\"bottom\" style=\"width: 9.2906%;\"\u003e\n \u003cp\u003eImmunoglobulin kappa variable 4-1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.4534%;\"\u003e\n \u003cp\u003e0.0481\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.9420\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.2506\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.3387\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.9954\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.1260\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.1730\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.9992\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e1.0000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.3315\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.3859\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"bottom\" style=\"width: 5.2212%;\"\u003e\n \u003cp\u003eP08519\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.446808510638298%\" valign=\"bottom\" style=\"width: 7.0639%;\"\u003e\n \u003cp\u003eLPA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.404255319148938%\" valign=\"bottom\" style=\"width: 9.2906%;\"\u003e\n \u003cp\u003eApolipoprotein(a)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.4534%;\"\u003e\n \u003cp\u003e0.0108\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.9999\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.9757\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.0180\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.9983\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.9686\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.0685\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.9956\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.0095\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.9998\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.0569\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"bottom\" style=\"width: 5.2212%;\"\u003e\n \u003cp\u003eP0DOX2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.446808510638298%\" valign=\"bottom\" style=\"width: 7.0639%;\"\u003e\n \u003cp\u003eIGA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.404255319148938%\" valign=\"bottom\" style=\"width: 9.2906%;\"\u003e\n \u003cp\u003eImmunoglobulin alpha-2 heavy chain\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.4534%;\"\u003e\n \u003cp\u003e0.0120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.0089\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.8261\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.8486\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.9932\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.1304\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.1894\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.0264\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e1.0000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.7605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.7749\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"bottom\" style=\"width: 5.2212%;\"\u003e\n \u003cp\u003eP11142\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.446808510638298%\" valign=\"bottom\" style=\"width: 7.0639%;\"\u003e\n \u003cp\u003eHSPA8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.404255319148938%\" valign=\"bottom\" style=\"width: 9.2906%;\"\u003e\n \u003cp\u003eHeat shock cognate 71 kDa protein\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.4534%;\"\u003e\n \u003cp\u003e0.0464\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.1694\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.8827\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.9002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.9858\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.0468\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.0667\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.1909\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e1.0000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.9995\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.9993\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"bottom\" style=\"width: 5.2212%;\"\u003e\n \u003cp\u003eP22626\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.446808510638298%\" valign=\"bottom\" style=\"width: 7.0639%;\"\u003e\n \u003cp\u003eHNRNPA2B1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.404255319148938%\" valign=\"bottom\" style=\"width: 9.2906%;\"\u003e\n \u003cp\u003eHeterogeneous nuclear ribonucleoproteins A2_B1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.4534%;\"\u003e\n \u003cp\u003e0.0017\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.0057\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.9564\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.8896\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e1.0000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.0025\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.0027\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.0519\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.9989\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.9792\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.9419\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"bottom\" style=\"width: 5.2212%;\"\u003e\n \u003cp\u003eP81605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.446808510638298%\" valign=\"bottom\" style=\"width: 7.0639%;\"\u003e\n \u003cp\u003eDCD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.404255319148938%\" valign=\"bottom\" style=\"width: 9.2906%;\"\u003e\n \u003cp\u003eDermcidin\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.4534%;\"\u003e\n \u003cp\u003e0.0212\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.0543\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.4212\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.3169\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.9537\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.7686\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.9309\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.0560\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.9975\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.3049\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.2309\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"bottom\" style=\"width: 5.2212%;\"\u003e\n \u003cp\u003eQ86YZ3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.446808510638298%\" valign=\"bottom\" style=\"width: 7.0639%;\"\u003e\n \u003cp\u003eHRNR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.404255319148938%\" valign=\"bottom\" style=\"width: 9.2906%;\"\u003e\n \u003cp\u003eHornerin\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.4534%;\"\u003e\n \u003cp\u003e0.0080\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.0163\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.9784\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.9813\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.382978723404255%\" valign=\"top\" style=\"width: 5.6051%;\"\u003e\n \u003cp\u003e0.9908\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.0090\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e0.0153\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.2061\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.3766%;\"\u003e\n \u003cp\u003e1.0000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.9113\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.319148936170213%\" valign=\"top\" style=\"width: 4.6069%;\"\u003e\n \u003cp\u003e0.9187\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eRed is significant difference at \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u003c/strong\u003e Fold change of comparison of significantly differential proteins among the 5 sample groups\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"4.166666666666667%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eSwissProt ID\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.208333333333333%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eGene name\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.666666666666668%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eProtein name\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.125%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eANOVA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"70.83333333333333%\" colspan=\"10\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eFold change\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.375%\"\u003e\n \u003cp\u003e\u003cstrong\u003eNormal pregnancy/Bart hydrops\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\"\u003e\n \u003cp\u003e\u003cstrong\u003eNormal pregnancy/Bart no hydrops\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.9375%\"\u003e\n \u003cp\u003e\u003cstrong\u003eNormal pregnancy/Disease control\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.5%\"\u003e\n \u003cp\u003e\u003cstrong\u003eNormal pregnancy/Hydrops not Bart\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.8125%\"\u003e\n \u003cp\u003e\u003cstrong\u003eBart hydrops/Bart no hydrops\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\"\u003e\n \u003cp\u003e\u003cstrong\u003eBart hydrops/Disease control\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.9375%\"\u003e\n \u003cp\u003e\u003cstrong\u003eBart hydrops/Hydrops not Bart\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\"\u003e\n \u003cp\u003e\u003cstrong\u003eBart no hydrops/Disease control\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.9375%\"\u003e\n \u003cp\u003e\u003cstrong\u003eBart no hydrops/Hydrops not Bart\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDisease control/Hydrops not Bart\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"4.3478260869565215%\" valign=\"bottom\"\u003e\n \u003cp\u003eB9A064\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.434782608695652%\" valign=\"bottom\"\u003e\n \u003cp\u003eIGLL5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.391304347826086%\" valign=\"bottom\"\u003e\n \u003cp\u003eImmunoglobulin lambda-like polypeptide 5\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.260869565217391%\" valign=\"top\"\u003e\n \u003cp\u003e0.0051\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e6.51\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e4.04\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.608695652173913%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.25\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\" valign=\"top\"\u003e\n \u003cp\u003e1.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.434782608695652%\" valign=\"top\"\u003e\n \u003cp\u003e0.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.04\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.608695652173913%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.29\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.06\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.608695652173913%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.46\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e7.61\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"4.3478260869565215%\" valign=\"bottom\"\u003e\n \u003cp\u003eP00736\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.434782608695652%\" valign=\"bottom\"\u003e\n \u003cp\u003eC1R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.391304347826086%\" valign=\"bottom\"\u003e\n \u003cp\u003eComplement C1r subcomponent\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.260869565217391%\" valign=\"top\"\u003e\n \u003cp\u003e0.0252\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e0.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e0.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.608695652173913%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.37\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\" valign=\"top\"\u003e\n \u003cp\u003e0.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.434782608695652%\" valign=\"top\"\u003e\n \u003cp\u003e0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.38\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.608695652173913%\" valign=\"top\"\u003e\n \u003cp\u003e0.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.42\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.608695652173913%\" valign=\"top\"\u003e\n \u003cp\u003e0.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.25\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"4.3478260869565215%\" valign=\"bottom\"\u003e\n \u003cp\u003eP00738\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.434782608695652%\" valign=\"bottom\"\u003e\n \u003cp\u003eHP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.391304347826086%\" valign=\"bottom\"\u003e\n \u003cp\u003eHaptoglobin\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.260869565217391%\" valign=\"top\"\u003e\n \u003cp\u003e0.0373\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e0.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e0.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.608695652173913%\" valign=\"top\"\u003e\n \u003cp\u003e1.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\" valign=\"top\"\u003e\n \u003cp\u003e1.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.434782608695652%\" valign=\"top\"\u003e\n \u003cp\u003e0.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.11\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.608695652173913%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.46\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.94\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.608695652173913%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e4.58\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e1.16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"4.3478260869565215%\" valign=\"bottom\"\u003e\n \u003cp\u003eP02671\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.434782608695652%\" valign=\"bottom\"\u003e\n \u003cp\u003eFGA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.391304347826086%\" valign=\"bottom\"\u003e\n \u003cp\u003eFibrinogen alpha chain\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.260869565217391%\" valign=\"top\"\u003e\n \u003cp\u003e0.0045\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e1.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.41\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.608695652173913%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.11\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\" valign=\"top\"\u003e\n \u003cp\u003e1.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.434782608695652%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.30\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.08\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.608695652173913%\" valign=\"top\"\u003e\n 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\u003cp\u003e0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e0.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.608695652173913%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.22\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.36\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.434782608695652%\" valign=\"top\"\u003e\n \u003cp\u003e1.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.42\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.608695652173913%\" valign=\"top\"\u003e\n \u003cp\u003e0.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.35\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.608695652173913%\" valign=\"top\"\u003e\n \u003cp\u003e0.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e1.63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"4.3478260869565215%\" valign=\"bottom\"\u003e\n \u003cp\u003eP02679\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.434782608695652%\" valign=\"bottom\"\u003e\n \u003cp\u003eFGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.391304347826086%\" valign=\"bottom\"\u003e\n \u003cp\u003eFibrinogen gamma chain\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.260869565217391%\" valign=\"top\"\u003e\n \u003cp\u003e0.0318\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e0.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e0.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.608695652173913%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.24\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\" valign=\"top\"\u003e\n \u003cp\u003e0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.434782608695652%\" valign=\"top\"\u003e\n \u003cp\u003e0.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.31\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.608695652173913%\" valign=\"top\"\u003e\n \u003cp\u003e0.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.33\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.608695652173913%\" valign=\"top\"\u003e\n \u003cp\u003e0.73\u003c/p\u003e\n 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valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e11.06\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"4.3478260869565215%\" valign=\"bottom\"\u003e\n \u003cp\u003eP04114\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.434782608695652%\" valign=\"bottom\"\u003e\n \u003cp\u003eAPOB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.391304347826086%\" valign=\"bottom\"\u003e\n \u003cp\u003eApolipoprotein B-100\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.260869565217391%\" valign=\"top\"\u003e\n \u003cp\u003e0.0112\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e0.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e1.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.608695652173913%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.33\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\" valign=\"top\"\u003e\n \u003cp\u003e0.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.434782608695652%\" valign=\"top\"\u003e\n \u003cp\u003e1.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.37\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.608695652173913%\" valign=\"top\"\u003e\n \u003cp\u003e1.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.32\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.608695652173913%\" valign=\"top\"\u003e\n \u003cp\u003e0.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.74\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"4.3478260869565215%\" valign=\"bottom\"\u003e\n \u003cp\u003eP04259\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.434782608695652%\" valign=\"bottom\"\u003e\n \u003cp\u003eKRT6B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.391304347826086%\" valign=\"bottom\"\u003e\n \u003cp\u003eKeratin, type II cytoskeletal 6B\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.260869565217391%\" valign=\"top\"\u003e\n \u003cp\u003e0.0096\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.38\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e0.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.608695652173913%\" valign=\"top\"\u003e\n \u003cp\u003e1.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.695652173913043%\" valign=\"top\"\u003e\n \u003cp\u003e1.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.434782608695652%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.44\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.30\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.608695652173913%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.42\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e1.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.608695652173913%\" valign=\"top\"\u003e\n \u003cp\u003e1.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e1.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"4.3478260869565215%\" valign=\"bottom\"\u003e\n \u003cp\u003eP06312\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.434782608695652%\" valign=\"bottom\"\u003e\n 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valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.99\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e1.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.608695652173913%\" valign=\"top\"\u003e\n \u003cp\u003e0.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.48\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"4.3478260869565215%\" valign=\"bottom\"\u003e\n \u003cp\u003eP81605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.434782608695652%\" valign=\"bottom\"\u003e\n \u003cp\u003eDCD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.391304347826086%\" valign=\"bottom\"\u003e\n \u003cp\u003eDermcidin\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.260869565217391%\" valign=\"top\"\u003e\n \u003cp\u003e0.0212\u003c/p\u003e\n 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width=\"7.608695652173913%\" valign=\"top\"\u003e\n \u003cp\u003e0.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.521739130434782%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.47\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eBold\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eis fold change \u0026gt;2 or \u0026lt;0.5.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Bart, exosomes, extracellular vesicle, hydrops fetalis, placenta-derived exosome, proteomics","lastPublishedDoi":"10.21203/rs.3.rs-4163008/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4163008/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntroduction:\u003c/strong\u003e Alpha-thalassemia is the most common cause of hydrops fetalis among Southeast Asians (also called “Bart’s hydrops fetalis). This condition is considered a fatal disorder; therefore, prenatal screening and diagnosis are extremely important. Changes in soluble analytes in the maternal circulation, such as hormones and angiogenic factors are not predictive of Hb Bart’s fetalis condition. This condition is associated with placental hypoxia, which may trigger the release of placenta-derived extracellular vesicles (EVs) in maternal circulation. Therefore, the determination of changes in placenta-derived exosome and its protein content could provide additional insight into the disease pathways of this disorder.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObjectives\u003c/strong\u003e: We aim to characterize: 1) maternal plasma levels of placenta-derived EVs; and 2) proteomics profiles of maternal plasma EVs in women with Bart’s fetalis fetuses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e This prospective cohort study included women with the following groups: 1) normal pregnancy or control group: women with couple at risk for Hb Bart’s fetuses but subsequently proven as non-Hb Bart’s group and delivered at term without maternal or neonatal complications (Group 1) (n=7); 2) Hb Bart’s with hydropic features group (Group 2) (n=4); 3) Hb Bart’s without hydropic features group (Group 3) (n=7); 4) a disease control group which consisted of women who subsequently delivered with placental associated conditions (Group 4) (n=4); and 5) women with hydrops fetalis from non-Bart’s causes (n=5) (Group 5). Maternal plasma EVs were isolated by the combination of stepwise centrifugation, ultrafiltration and qEV size exclusion chromatography. The EVs were characterized by the particle size, morphology and protein markers. Isolated EVs and their plasma were measured placental alkaline phosphatase (PLAP) level using ELISA. Mass spectrometry was used to determine EV proteomics profile. A p-value of \u0026lt;0.05 was used to infer significance, unless multiple testing was involved, with the false discovery rate controlled at the 10% level (q\u0026lt;0.1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003e1) EV particle size in Bart fetuses with hydropic features was significantly smaller than that in normal pregnancy and disease control groups; 2) Bart fetuses with hydropic features had higher maternal placenta-derived PLAP-contained EVs (PLAP-EVs) than that in normal pregnancy; although, PLAP-EV level was highest in the placental associated complications group; 3) Bart fetuses without hydropic features tended to have higher maternal PLAP-EV level compared to normal pregnancy group; 4) hydropic fetuses due to non-Bart’s causes had similar PLAP level compared to normal pregnancy group; 5) among the 16/106 differentially \u0026nbsp;expressed EV proteins, hnRNPA2B1 protein was the highest in Bart fetuses with hydropic features group compared to placental associated complication or normal pregnancy groups; 6) sixteen differentially expressed EV proteins were involved in fibrinogen complex, fibrin clot formation and integrin signaling pathway.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eThis is the first study of placenta-derived EVs in women with Hb Bart’s fetalis. EV particle size is significantly smaller but maternal PLAP-EV level is significantly higher in women with Hb Bart’s fetalis fetuses compared to normal pregnancy. In addition, several EV proteins were differential expressed in women with Bart’s fetuses and these proteins involve in aberrant immune response, pro-inflammatory cytokine regulation mediated by TLRs-MyD88-IRAK4-MAPK axis and vascular injuries as part of pathophysiology of placental edema and hypoxia in Bart’s hydrops. The result of this study supports future research to determine whether placenta-derived EVs in maternal circulation can be used as a liquid biopsy or non-invasive prenatal test for the early identification of Bart’s fetuses. This will ultimately reduce the rate of unnecessary invasive prenatal diagnosis testing.\u003c/p\u003e","manuscriptTitle":"Placenta-derived Extracellular Vesicles in Maternal Plasma of Hb Bart’s Fetuses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-17 20:48:57","doi":"10.21203/rs.3.rs-4163008/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-04T07:09:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-03T11:03:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"125541087092481806375204603890332755021","date":"2025-05-18T17:26:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"243682691722656857447714094029832852644","date":"2024-12-27T09:38:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-14T14:05:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"259398107536104403999402542361034718291","date":"2024-11-07T09:43:48+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-11T15:32:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-11T15:27:04+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-04-10T12:44:02+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-09T14:39:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-03-25T11:47:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5b3f01aa-7b96-4d4b-9bf3-7d1cca7d6d4d","owner":[],"postedDate":"April 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":30565019,"name":"Biological sciences/Molecular biology/Proteomics"},{"id":30565020,"name":"Health sciences/Medical research/Translational research"}],"tags":[],"updatedAt":"2025-11-24T16:09:16+00:00","versionOfRecord":{"articleIdentity":"rs-4163008","link":"https://doi.org/10.1038/s41598-025-24900-0","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-11-20 15:58:29","publishedOnDateReadable":"November 20th, 2025"},"versionCreatedAt":"2024-04-17 20:48:57","video":"","vorDoi":"10.1038/s41598-025-24900-0","vorDoiUrl":"https://doi.org/10.1038/s41598-025-24900-0","workflowStages":[]},"version":"v1","identity":"rs-4163008","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4163008","identity":"rs-4163008","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}