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Placenta-specific plasma miR518b is a potential biomarker for preeclampsia
Journal Pre-proofs Letter to the Editor Placenta-specific plasma miR518b is a potential biomarker for preeclampsia Munjas Jelena, Miron Sopić, Ivana Joksić, Ursula Prosenc Zmrzljak, Nataša Karadžov-Orlić, Rok Košir, Amira Egić, Željko Miković, Ana Ninić, Vesna Spasojević-Kalimanovska PII: DOI: Reference:
S0009-9120(19)31218-4 https://doi.org/10.1016/j.clinbiochem.2020.02.012 CLB 10088
To appear in:
Clinical Biochemistry
Received Date: Revised Date: Accepted Date:
9 November 2019 6 February 2020 18 February 2020
Please cite this article as: M. Jelena, M. Sopić, I. Joksić, U. Prosenc Zmrzljak, N. Karadžov-Orlić, R. Košir, A. Egić, Z. Miković, A. Ninić, V. Spasojević-Kalimanovska, Placenta-specific plasma miR518b is a potential biomarker for preeclampsia, Clinical Biochemistry (2020), doi: https://doi.org/10.1016/j.clinbiochem. 2020.02.012
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Placenta-specific plasma miR518b is a potential biomarker for preeclampsia Munjas Jelenaa, Miron Sopića, Ivana Joksićb, Ursula Prosenc Zmrzljakc, Nataša KaradžovOrlićd,e, Rok Koširc, Amira Egićd,e, Željko Mikovićd,e, Ana Ninića; Vesna SpasojevićKalimanovskaa aDepartment
of Medical Biochemistry, Faculty of Pharmacy, University of Belgrade; Street
Vojvode Stepe 450, 11000 Belgrade, Serbia b
Genetic Laboratory Department, Obstetrics and Gynaecology Clinic "Narodni Front"; Street
Kraljice Natalije 62, 11000 Belgrade, Serbia c
BIA Separations CRO, Labena Ltd.; Street Verovškova 64, 1000 Ljubljana, Slovenia
d
High-Risk Pregnancy Department, Obstetrics and Gynaecology Clinic "Narodni Front";
Street Kraljice Natalije 62, 11000 Belgrade, Serbia e
School of Medicine, University of Belgrade; Street Dr Subotica 8, 11000 Belgrade, Serbia
Jelena Munjas: [email protected] Miron Sopić: [email protected] Ivana Joksić: [email protected] Ursula Prosenc Zmrzljak: [email protected] Nataša Karadžov-Orlić: [email protected] Rok Košir: [email protected] Amira Egić: [email protected] Željko Miković: [email protected] Ana Ninić: [email protected] Vesna Spasojević-Kalimanovska: [email protected] Corresponding author: Miron Sopić Email: [email protected]
Address: Vojvode Stepe 450, Department of Medical Biochemistry, Faculty of Pharmacy, University of Belgrade, 11000 Belgrade, Serbia Telephone number: +38111 3951 265 Fax number: +38111 3972840 Word count: 3.447 Number of tables: 3 Number of figures: 3
Abstract Introduction: MicroRNAs have a significant role in the pathogenesis of preeclampsia. Circulating microRNAs could represent a potential biomarker for preeclampsia. The aim of this study was to evaluate plasma miR210-3p and miR518b in preeclampsia and healthy pregnancy for the first time by digital droplet PCR (ddPCR). Methods: Thirty-six pregnant women (seventeen healthy pregnancies, nineteen preeclampsia patients) were involved from the Clinic for Gynaecology and Obstetrics “Narodni front” in Belgrade, Serbia. Plasma miR210-3p, miR518b and cel-miR-39 as a spike-in control were measured by ddPCR. Results: MiR518b was significantly elevated in preeclampsia compared to a healthy pregnancy (P=0.034; 0.302(0.217-0.421) vs. 0.171(0.110-0.266)). MiR210-3p showed no significant difference between the two groups (P=0.951). The adjustment of miR518b was made for a gestational age and smoking status and the difference between the preeclampsia and healthy pregnancy group was more significant (P=0.026; 0.300(0.216-0.419) vs. 0.172(0.121-0.245)). Plasma miR-518b was significantly higher in the group of preeclampsia patients with proteinuria above the 75th percentile for the group (P=0.033), in women who smoked (P=0.039), and was positively related to uric acid in preeclampsia (P=0.018, r=0.536). Plasma miR518b was able to significantly discriminate between preeclampsia and healthy pregnancy, yielding AUC of 0.712 (95%CI:0.539-0.891), P=0.028. Conclusions: In this study plasma microRNA were measured for the first time in preeclampsia and healthy pregnancies with ddPCR. Placenta-specific miR-518b could serve as a potential biomarker for
discriminating preeclampsia and healthy pregnancy, which should be confirmed on a larger study population. This study has failed to confirm the same potential for miR210-3p. Keywords: preeclampsia, miR518b, miR210-3p, digital droplet PCR
Introduction: Preeclampsia (PE) stands out among the hypertensive disorders of pregnancy for its impact on maternal and neonatal health, since it is one of the leading causes of maternal and perinatal mortality and morbidity worldwide (1). Preeclampsia is defined as hypertension developing after 20 weeks of gestation with one or more of the following: proteinuria, maternal organ dysfunction (including renal, hepatic, hematological, or neurological complications), or fetal growth restriction (2). However, the pathogenesis of preeclampsia is only partially understood and it is related to disturbances in placentation at the beginning of pregnancy, followed by generalized inflammation and progressive endothelial damage. Although pathophysiological changes exist from very early stages of the pregnancy, hypertension and proteinuria usually become apparent in the second half of pregnancy and are present in 2%–8% of all pregnancies overall (1). The placenta has always been a central figure in the etiology of preeclampsia because the removal of the placenta is necessary for symptoms to regress (3). Due to its heterogeneity and elusive pathophysiology, preeclampsia still lacks a suitable biomarker for diagnosis and prediction. The diagnosis of preeclampsia is one of the most important goals in maternal-fetal medicine, considering the impact it has on maternal and fetal health. Although a great deal of biomarkers is being assessed, the evidence is not yet consistent and studies assessing new biomarkers are highly encouraged. (1, 2, 4). MicroRNAs (miRNAs) are small, about 22-24 nucleotides long, noncoding RNAs that regulate gene expression at posttranscriptional level, binding to the target mRNA and promoting their degradation or inhibiting translation. In that way miRNAs regulate more than 30% of the human genome (5). Many miRNAs have been identified in human placental tissues, where they have an important role in placental development, trophoblast proliferation, and migration and angiogenesis (6). Altered expression of numerous miRNAs in placenta has been related to pathophysiological processes that occur in preeclampsia (7, 8, 9). Placental miRNAs are
released into the maternal circulation via an exosome mediated pathway where they are found in a very stable form of microparticles (10). A specific placental miRNA pattern dynamically changes during pregnancy and those changes are reflected in maternal plasma (11, 12). Moreover, many studies so far have shown abnormal levels of circulating placenta-derived miRNAs in pregnancies affected by pre-eclampsia. Therefore, circulating placenta-derived microRNAs could be biomarkers with great potential in both prediction and diagnosis of PE. However, many discrepancies exist between studies, probably due to different technology used for miRNA quantification and sample collection (7, 8, 9). MiR518b (chromosome 19 microRNA cluster, C19MC) belongs to a family of placentaspecific microRNAs which are exclusively expressed in the placenta (8, 13). Aberrant upregulation of miR-518b in preeclamptic placentas may contribute to excessive trophoblast proliferation, which is well recognized as the fundamental pathological change in preeclampsia (14). MiR518b has also been shown to be up-regulated in maternal plasma of PE patients with a potential to be validated as a biomarker of PE (15, 16). The most highly studied, and most abundantly expressed miRNA in placental tissue is so called hypoxia-related miR210 (9). MiR210 belongs to a group of placenta-associated miRNAs (8) and is ubiquitously expressed in placenta and many other tissues (17, 18). MiR210 is detected in maternal circulation, and its levels are up-regulated both in placental tissue and maternal circulation in PE. Upregulation of miR210 in placenta results in a negative regulation of trophoblast cell migration and invasion, which are one of the main pathophysiological mechanisms which lead to placental dysfunction (19). Digital droplet PCR (ddPCR) is considered to be a powerful tool for microRNA quantification, especially from low abundant samples (such as serum/plasma), with many potential advantages over real-time PCR, including the capability to obtain absolute quantification without external references and robustness to variations in PCR efficiency (20, 21).
Therefore, the aim of this study was to evaluate for the first time circulating plasma levels of miR210-3p and miR518b in preeclampsia patients and normal pregnancies with ddPCR, and to evaluate their potential role as a biomarker of PE. Material and methods: This study was designed as observational, case-control study. The study included 36 pregnant women who came to the Clinic for Gynaecology and Obstetrics “Narodni front”, Belgrade, Serbia, either for their routine examination of were admitted at the Clinic for further examination, from March to June 2018. For all patients, blood pressure measurement, complete blood count, routine biochemistry, urine analysis and fetal ultrasound with doppler examination were performed. Based on clinical and laboratory features all subjects were classified in two groups: control group (17 healthy pregnant women) and preeclampsia group (19 patients). Preeclampsia was diagnosed according to the established criteria. Severe preeclampsia was considered if blood pressure was >160 mmHg systolic or >110 mmHg diastolic; presence of HELLP syndrome (hemolysis, platelet level <100,000/dl, ALT or AST elevations of twofold the upper limit), impending eclampsia (headache, epigastric pain and visual disturbances), worsening thrombocytopenia, or worsening fetal growth restriction. Proteinuria was not a criteria for the severity of PE, since proteinuria at levels higher than those for the diagnosis of preeclampsia does not predict clinical outcome (22). All women in our study were diagnosed as having mild form of preeclampsia. Demographic data (age, body mass index (BMI), smoking status, medication used), information on medical and obstetrics history and lifestyle factors for each patient were collected by a questionnaire. Smokers were defined as subjects who have smoked ten cigarettes a day for at least two years. Mean arterial pressure was calculated as (systolic pressure+2xdiastolic pressure)/3.
Patient exclusion criteria were: presence of diabetes mellitus, multifetal pregnancy, major fetal anomaly, fetal death, any condition requiring termination of pregnancy, any maternal chronic disease and inability to give informed consent. This study was conducted according to the guidelines laid down in the Declaration of Helsinki. The study was approved by the Ethic Committee of the Clinic for Gynecology and Obstetrics “Narodni front” and informed consents from each patient were obtained at the time of samples collection. Twelve hour fasting whole blood was drawn in EDTA containing tubes. MicroRNAs were isolated from the cell-free plasma samples, since blood cells represent a significant source of circulating microRNAs and can cofound in their measurement (23). Platelets represent a significant source of circulating microRNAs that could lead to sample contamination, especially after freeze/thaw cycle which disturbs the platelet’s membrane and leads to secretion of miRNA into the plasma sample (24). Plasma was isolated in less than one hour following phlebotomy, which is essential in microRNA analysis, in order to minimize contamination of microRNAs originating from blood cells. For obtaining platelet poor plasma, samples were double-centrifuged. Firstly, samples were centrifuged at 2500×g for 15 minutes at room temperature. Plasma was collected in a plastic tube, leaving 1 cm of plasma above the buffy layer and cautiously handled to avoid disturbance. The plasma was then centrifuged a second time at 2500×g for 15 min at room temperature, and collected afterwards into a new plastic tube, leaving approximately 100 µL at the bottom of the plastic tube (25). MicroRNAs were isolated from 200 μL of plasma according to the manufacturer’s protocol (Cat. Number 217184; miRNeasy Serum/Plasma Kit, Qiagen, Düsseldorf, Germany). Platelet poor plasma samples were additionally centrifuged in conical tubes for 10 min at 16,000 x g and 4°C in a fixed-angle rotor for removal of additional cellular nucleic acids attached to cell debris, according to the manufacturer’s protocol and stored at -80°C. In order to adjust for RNA extraction efficiency,
non-human (Caenorhabditis elegans) cel-miR-39 was used as a spike-in control (Qiagen: miRNeasy Serum/Plasma Spike-In, Cat. Number 219610). After addition of QIAzol Lysis Reagent, 3.5 μl spike-in control (1.6 x 108 copies/μl working solution) was added into each sample, and the isolation was continued according to the protocol. Total RNA was eluted from the column by two sequential elutions with 14 μL water to yield approximately 24 μL of eluate. After isolation, samples were stored at -80°C until further use. Reverse transcription (RT) reactions were performed using Taq-Man miRNA Reverse Transcription Kit (Cat. Number: 4366596) and specific TaqMan MicroRNA Assays (Cat. Number: 4427975: hsa-miR-210-3p, 000512; has-miR-518b, 001156; cel-miR-39-3p, 000200; Thermo Fisher Scientific, Foster City, CA, USA) adding 5 μL of total isolated RNA in each reaction. Thermal protocol for RT was as following: 30 minutes at 16°C; 30 minutes at 42°C and 5 minutes at 85°C. After RT, samples were stored at -80°C until further use. QX200 Droplet Digital PCR (ddPCR) System (Bio-Rad) was used for ddPCR analysis. Each 20µl ddPCR reaction consisted of 10µl of ddPCR Supermix for Probes (no dUTP) (Bio-Rad), 1µl of miRNA assay (Applied biosystems) and 1 μl of miRNA-specific transcribed cDNA. For measurement of cel-miR-39-3p the samples were previously diluted 50x. Each reaction supermix was transferred to a cartridge along with 70 μl of Droplet Generation Oil (Bio-Rad) and partitioned into approximately 15,000–20,000 droplets using the QX200 Droplet Generator (Bio-Rad). Droplets were transferred to 96-well plates (Eppendorf) and sealed using the PX-1 plate sealer (Bio-Rad). The PCR was performed in a T100 Thermal Cycler (Bio-Rad). The cycling conditions were as following: 95 °C 10 min, 1 cycle; 95 °C 30 sec, assay specific annealing temperature 1 min, 40 cycles; 98°C 10 min, 4 °C ∞. Annealing temperature for miR-210-3p and miR-518b assays was 60°C, the annealing temperature for cel-miR-39 assay was 57°C. All steps are with a 2 °C/s temperature ramp rate. Following end-point amplification, the individual droplets were measured with the QX200
Droplet Reader (Bio-Rad) and analyzed using QuantaSoft software (Bio-Rad) to determine target miRNA amount. Statistical analysis Data with normal distribution are shown as means with standard deviation. Data with skewed distribution were log-transformed and presented as geometric means with 95% confidence intervals for the mean. Data were tested using Student’s t Test. Data with skewed distribution even after transformation were represented as medians with interquartile ranges and tested with the Mann-Whitney test. Univariate associations were evaluated by Pearson’s linear correlation analysis. Adjustment for confounding factors was performed using analysis of covariance (ANCOVA). Discriminative abilities of the examined parameters were assessed using the receiving operative characteristic (ROC) curve analysis, while the curve was plotted, and the area under the curve (AUC) presented as C statistics from the analysis. Interpretation of the area under ROC curves (AUCs) is done by the recommended guidelines (26). Categorical variables were analysed using Chi-square tests for contingency tables. A P-value of less than 0.05 was considered statistically significant. Results: PE group and CG were age matched. Mean gestational age for PE and CG was not significantly different, ranging between 20-39 weeks of gestation for CG, and 24-38 week of gestation for PE group. PE group had significantly higher BMI compared to CG (P=0.030), while percentage of weight gain was not significantly different between the two groups. PE group had a significantly higher systolic and diastolic blood pressure, as well as mean arterial pressure compared to CG (P<0.001; P<0.001; P<0.001respectively). All the women in PE group, except two patients, had proteinuria ≥300 mg/24h, while serum protein levels were not significantly different between the two groups. PLT count was significantly lower in PE group compared to CG (P=0.016), while uric acid levels were significantly higher in PE group compared to CG
(P=0.001). Hemoglobin, RBC, ALT, AST and creatinine showed no significant difference between the investigated groups (Table 1). The results from this study showed that miR-518b was significantly elevated in plasma of women with PE compared to CG (P=0.034, Figure 1A). Plasma miR-210-3p showed no significant difference between the two groups (P=0.951; Figure 2). PE patients were divided into two groups according to the 75 percentile cut-off for proteinuria for the entire patient distribution (proteinuria75p=4.17 g/24h). Plasma miR-518b was significantly higher in the group of PE patients with proteinuria above the 75th percentile for the group (Figure 1B; P=0.033). Plasma miR-518b in CG correlated positively with gestational age, percentage of weight gain, uric acid levels and plasma miR-210-3p (Table 2). In PE group, miR-518b correlated positively with uric acid and plasma miR-210-3p (Table 2). Plasma miR-518b was up regulated in women with PE who smoked (P=0.039, Table 3). Therefore, the adjustment of plasma miR-518b levels was made for gestational age and smoking status and the difference between the PE group and CG was even more significant (P=0.026; Figure 1C). The potential of plasma miR-518b for discriminating PE patients from the controls was investigated by ROC curve analysis, and comparative analysis of AUCs for miR518b, miR210-3p, mean arterial pressure and uric acid was made. Plasma miR-518b was able to significantly discriminate between PE and CG, yielding AUC of 0.715 (95% CI: 0.539-0.891), P=0.028, Figure 3. The patients were also classified into early (before 34 weeks; 10 women) and late onset (at or after 34 weeks; 9 women) PE (22), and miR-518b and miR210-3p were compared. There were no significant difference between the early and late onset PE for miR518b (P=0.268) and miR210-3p (P=0.682). Discussion In this study for the first time, cell-free plasma levels of placenta-specific miR518b and placenta-associated miR210-3p were measured in PE patients and healthy pregnancy by digital
droplet PCR. Digital droplet PCR (ddPCR) is a breakthrough, high throughput technology that provides ultrasensitive and absolute nucleic acid quantification. It is particularly useful for lowabundance targets, such as miRNA quantification from serum/plasma samples, obtaining highest precision and sensitivity, absolute quantification without the use of external references, with the advantage of removal of PCR efficiency bias. MicroRNA quantification by ddPCR and real-time PCR by Hindson et al. revealed greater precision (coefficients of variation decreased 37–86%) and improved day-to-day reproducibility of ddPCR. More importantly, when applied to microRNA detection in clinical serum samples, ddPCR showed superior diagnostic performance compared to real-time PCR (21). Implementation of this method for microRNA quantification would therefore improve the assessment and the use of microRNAs as biomarkers. In this study, it has been shown that plasma levels of placenta-specific miR518b were significantly higher in PE patients compared to CG, even after adjustment for confounding factors (Figure 1A, 1C). In agreement with our results, Hromadnikova et al. showed that along with up-regulation in the placenta, miR518 levels were increased in plasma of PE patients, even early in pregnancy, and proposed a possible role in the inducement of gestational hypertension and preeclampsia (16). In addition, Miura et al. found increased miR518b levels in severe cases of PE (15). Even though it has been shown that expression of miR518b was up-regulated in placental tissue in severe PE (27) certain studies have pointed to down-regulation of miR518 in placenta of PE (28). However, these discrepancies are most likely caused by different sampling position in the chorionic or basal plate of the placenta (27). Recently, Liu et al have proposed a mechanism by which up-regulation of miR518b in placenta could contribute to the pathophysiology of PE. The authors showed that miR518 directly targets and represses Ras-related protein 1b (Rap1b) expression, which results in excessive proliferation of trophoblast cells, especially the column
ones, trough Rap1b–Ras–MAPK pathway. Excessive proliferation of trophoblasts disables correct differentiation toward invasive cell phenotype, leading to the shallow invasion of decidual stroma and spiral arteries, which represents a hallmark of preeclampsia placentas (14). Throughout gestation, plasma microRNA levels rise until the delivery, and consequently, levels of circulating microRNA are dependent from gestational age (29-31). This study did not investigate plasma microRNA changes throughout gestation, but the positive correlation of plasma miR518b and gestational age was observed in CG (Table 2). Also, we found that plasma miR518b levels were affected by smoking status (Table 3). Although smoking is inversely related to preeclampsia occurrence (32), Maccani et al. have pointed to downregulation of several microRNAs in the placental tissue in mothers who smoked (33). Therefore, plasma miR-518b levels were adjusted for gestational age and smoking status, but the difference between PE and CG was even more significant (Figure 1C). Furthermore, in PE patient, miR518 increase was consistent with proteinuria worsening (Figure 1B), one of the hallmarks for PE pathogenesis (2). Uric acid, besides its utility in diagnosis of PE, is proposed to have direct pathophysiological role in PE development (34). Plasma miR518b levels showed positive correlation with uric acid in both investigated groups (Table 2), which further confirms its involvement in PE. Finally, diagnostic potential for plasma miR518b for PE was shown by ROC curve analysis, where plasma miR-518b was able to significantly discriminate between PE and CG, yielding AUC of 0.715, unlike miR210-3p, which did not show significant diagnostic potential by ROC curve analysis (Figure 3). Therefore, miR-518b could serve as a potential biomarker of PE. Furthermore, it is worth mentioning that in AUC analysis (available only in 27 out of 36 cases, data not shown), mir518b performed much better than uterine pulsatility index measurement (UtPI) (AUCUtPI=0.515 (95%CI: 0.283-0.740), P=0.921), which is one of the parameters used for screening and prediction of PE (35). However, it did not perform better than mean arterial
pressure and uric acid. Mean arterial pressure is considered to be a better predictor of preeclampsia compared to diastolic or systolic pressure. (35, 36). In this study, all preeclampsia cases had distinctive hypertension, therefore, higher ranges of mean arterial pressure than the control group. Considering elevated blood pressure was one of the diagnostic criteria for PE (1, 2), by which PE patients were selected in this study, it is not surprising that mean arterial pressure showed better performance in ROC curve analysis than miR-518b. In addition, it was showed that uric acid is involved in the pathogenesis of PE (34), and thus it could be used as a biomarker for prediction of PE in women at moderate and low risk and is also a predictor of adverse outcomes of PE for both the mother and the newborn (37, 38, 39). Pregnant women with abnormal weight and obesity are subject to the increased risk of developing PE (40). A positive association was found between pre-pregnancy BMI and placental expression of several microRNAs at birth, as well as with gestational weight gain, which was related to fetal gender. (41). Also, a specific placental miRNA profile was identified in maternal obesity (42). In this study, the PE group had significantly higher BMI compared to CG. Plasma miR-518b correlated positively with the percentage of weight gain in CG, however, no correlation with current BMI or percentage of weight gain was found in PE group. On the other hand, plasma levels of miR-210-3p did not differ between PE and CG (Figure 2). MiR210 is one of the so-called master hypoxamiRs (hypoxia-induced miRNAs), since it has been found upregulated by hypoxia in all cells and tissues tested to date (17, 18). It was proposed that induction of miR210 in the placenta could interfere with trophoblast invasion and contribute to PE development (19, 43). Murphy et al. have found that plasma miR-210 levels are increased only in the severe cases of PE, while in mild cases, there was no difference compared to the CG (44). The reason for that could lie in the fact that more severe cases of PE are associated with increased hypoxia, which reflects in increased hypoxia-driven trophoblast cell debris carrying microRNA in the circulation, and further results in higher miR-210 plasma
levels (9). The majority of patients in this study were not diagnosed with severe forms of PE, the most prevalent was a mild form of PE (2), so we can presume that this is the reason why plasma levels of miR210-3p did not differ in our PE and CG. Furthermore, miR210 is expressed by many tissues and cells other than placenta (17, 18), so miR-210-3p plasma levels could be affected by other factors beside PE in patients and controls. MiR210 was elevated in serum samples from PE patients vs. healthy controls (19), but serum and plasma are shown to have different microRNA profiles (45) due to the coagulation process (46) and the release of microRNAs from blood cells which are major contributor to the circulating microRNA pool - platelets, leukocytes and erythrocytes (9, 23, 24). Therefore, for microRNA measurement in blood of pregnant women, Ge et al. proposed plasma as more suitable sampling material over serum (46). It is important to highlight, that in this study both microRNAs were measured in cell-free plasma samples, so it can be presumed that the coagulation process and cells contamination did not confound in this measurement, so this could be the explanation for discrepancy existing between our study and the serum microRNA profiles in PE. However, some limitations within this study should be mentioned. Firstly, study group sizes were small, so these findings should be confirmed using larger study groups. Secondly, all PE patients received some form of therapy which could have influenced the results. Most commonly prescribed antihypertensive drugs in pregnant women with PE are methyldopa and nifedipine. Based on data from Pharmaco-mir database (www.pharmaco-mir.org) (47) both drugs can be influenced by numerous miRNAs. Currently there are no studies showing interaction of mentioned drugs and miRNAs investigated in our study, so further research is needed to better clarify possible effects of antihypertensive therapy on miRNA expression. In conclusion, to the best of our knowledge, this is the first study where plasma microRNAs levels were measured by ddPCR in PE. Circulating levels of miR518 were higher in PE patients
compared to CG, and could serve as a potential diagnostic marker for PE. MiR518b is related to proteinuria and uric acid level in PE patients, which are one of the hallmarks of the disease, confirming its role in PE. Plasma levels of miR210-3p did not differ between the investigated groups. Acknowledgments: This study was financially supported by ddPCR Grant challenge initiative from Labena Ltd., Verovškova 64, 1000 Ljubljana, Slovenia and by a grant from the Ministry of Education, Science and Technological Development, Serbia (project number 175035). We declare that there is no conflict of interest.
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26. Swets JA. Measuring the accuracy of diagnostic systems, Science. 1988;240:1285-93. 27. Xu P, Zhao Y, Liu M, Wang Y, Wang H, Li YX, et al. Variations of microRNAs in human placenta and plasma from preeclamptic pregnancy. Hypertension 2014;63:127684. 28. Hromadnikova I, Kotlabova K, Ondrackova M, Pirkova P, Kestlerova A, Novotna V, et al. Expression profile of C19MC microRNAs in placental tissue in pregnancy-related complications. DNA Cell Biol 2015;34:437-57. 29. Hromadnikova I, Kotlabova K, Doucha J, Dlouha K, Krofta L. Absolute and Relative Quantification of Placenta-Specific MicroRNAs in Maternal Circulation with Placental Insufficiency–Related Complications. J Mol Diagn 2012;14:160-7. 30. Miura K, Miura S, Yamasaki K, Higashijima A, Kinoshita A, Yoshiura KI, et al. Identification of pregnancy-associated microRNAs in maternal plasma. Clin Chem 2010;56:1767-71. 31. Morisaki S, Miura K, Higashijima A, Abe S, Miura S, Hasegawa Y, et al. Effect of labor on plasma concentrations and postpartum clearance of cell‐free, pregnancy‐associated, placenta‐specific microRNAs. Prenat Diagn 2015;35: 44-50. 32. Wei J, Liu CX, Gong TT, Wu QJ, Wu L. Cigarette smoking during pregnancy and preeclampsia risk: a systematic review and meta-analysis of prospective studies. Oncotarget 2015;6:43667. 33. Maccani MA, Avissar-Whiting M, Banister CE, McGonnigal B, Padbury JF, Marsit CJ. Maternal cigarette smoking during pregnancy is associated with downregulation of miR-16, miR-21, and miR-146a in the placenta. Epigenetics 2010;5:583-9. 34. Khaliq OP, Konoshita T, Moodley J, Naicker T. The Role of Uric Acid in Preeclampsia: Is Uric Acid a Causative Factor or a Sign of Preeclampsia? Curr Hypertens Rep 2018;20:80.
35. Walsh CA, Baxi LV. Mean arterial pressure and prediction of pre-eclampsia.2008:10791080. 36. Cnossen JS, Vollebregt KC, De Vrieze N, Ter Riet G, Mol BW, Franx A, Khan KS, Van Der Post JA. Accuracy of mean arterial pressure and blood pressure measurements in predicting pre-eclampsia: systematic review and meta-analysis. Bmj. 2008 May 15;336(7653):1117-20. 37. Rezk M, Gaber W, Shaheen A, Nofal A, Emara M, Gamal A, Badr H. First versus second trimester mean platelet volume and uric acid for prediction of preeclampsia in women at moderate and low risk. Hypertension in pregnancy. 2018 Jul 3;37(3):111-7. 38. Ryu A, Cho NJ, Kim YS, Lee EY. Predictive value of serum uric acid levels for adverse perinatal outcomes in preeclampsia. Medicine. 2019 May;98(18). 39. Damacena AT, Poiati JR, Moreira DA, Reis GS, Oliveira LG, Peraçoli JC, Costa RA, Borges VT. 115 Association between uric acid serum and maternal and perinatal results in preeclampsia: Risk factors, prediction of preeclampsia. Pregnancy Hypertension: An International Journal of Women's Cardiovascular Health. 2016 Jul 1;6(3):235. 40. Motedayen M, Rafiei M, Tavirani MR, Sayehmiri K, Dousti M. The relationship between body mass index and preeclampsia: A systematic review and meta-analysis. International Journal of Reproductive BioMedicine. 2019 Jul;17(7):463. 41. Tsamou M, Martens DS, Winckelmans E, Madhloum N, Cox B, Gyselaers W, Nawrot TS, Vrijens K. Mother's Pre-pregnancy BMI and Placental Candidate miRNAs: Findings from the ENVIRONAGE Birth Cohort (vol 7, 5548, 2017). 42. Carreras-Badosa G, Bonmatí A, Ortega FJ, Mercader JM, Guindo-Martínez M, Torrents D, Prats-Puig A, Martinez-Calcerrada JM, de Zegher F, Ibáñez L, Fernandez-Real JM. Dysregulation of placental miRNA in maternal obesity is associated with pre-and
postnatal growth. The Journal of Clinical Endocrinology & Metabolism. 2017 Jul 1;102(7):2584-94. 43. Adel S, Mansour A, Louka M, Matboli M, Elmekkawi SF, Swelam N. Evaluation of MicroRNA-210 and Protein tyrosine phosphatase, non-receptor type 2 in Pre-eclampsia. Gene 2017;597:105-9. 44. Murphy MS, Casselman RC, Tayade C, Smith GN. Differential expression of plasma microRNA in preeclamptic patients at delivery and 1 year postpartum. Am J Obstet Gynecol 2015;213:367-e1. 45. Wang K, Yuan Y, Cho JH, McClarty S, Baxter D, Galas DJ. Comparing the MicroRNA spectrum between serum and plasma. PloS One 2012;7: e41561. 46. Ge Q, Shen Y, Tian F, Lu J, Bai Y, Lu Z. Profiling circulating microRNAs in maternal serum and plasma. Mol Med Rep 2015;12:3323-30. 47. Rukov JL, Wilentzik R, Jaffe I, Vinther J, Shomron N. Pharmaco-miR: linking microRNAs and drug effects. Briefings in bioinformatics. 2013 Jan 31;15(4):648-59. P=0.034
0.450
A
0.421 0.400 0.350 0.302
miR-518b
0.300 0.250
0.266 0.217
0.200 0.171 0.150 0.100
0.110
0.050 0.000
CG
PE
B
P=0.033
P=0.026 C
0.450 0.419 0.400
adj miR-518b
0.350 0.300
0.300 0.250
0.245 0.216
0.200 0.172 0.150 0.121 0.100 0.050 0.000
CG
Figure 1.
PE
A) Plasma miR-518b in CG and PE B) Plasma miR-518b in PE group according 75% cut-off for proteinuria, values are shown as medians with interquartile ranges
C) Plasma miR-518b in CG and PE after adjustment for gestational age and smoking *values are show as geometric means with 95% confidence intervals for the mean
0.450 0.400
P=0.951 0.397 0.358
miR-210-3p
0.350 0.300 0.250
0.262
0.258
0.200
0.185
0.172 0.150 0.100 0.050 0.000
CG
Figure 2. Plasma miR-210-3p in CG and PE *values are show as geometric means with confidence intervals
PE
AUCmiR518b=0.715 (95% CI 0.539-0.891); P=0.028 AUCmiR210-3p=0.458 (95% CI 0.262-0.655); P=0.669 AUCuric acid=0.797 (95% CI 0.649-0.946); P=0.002 AUCMAP=0.895 (95% CI 0.750-1.000); P<0.001
Figure 3. Comparative ROC curve for the potential of miR518b, miR210-3p, Mean arterial pressure (MAP) and Uric acid in discriminating PE patients from the controls AUC- area under the receiving operative characteristic curve
Figure 4. ROC curve for the potential of uterine pulsatility index (UtPI) in discriminating PE patients from the controls AUC- area under the receiving operative characteristic curve 48. Table 1. Clinical and laboratory characteristics of CG and PE Variable
Control group
Preeclampsia
P
Number of subjects
17
19
/
Week of gestation$
33 (20-39)
33 (24-38)
0.961
Age (years) $
32 (22-40)
34 (20-51)
0.610
BMI, kg/m2
26±3
29±6
0.030
Weight gain, %
16±8
22±12
0.155
Smoking status (smokers/non-smokers)
4/13
4/15
0.858&
Systolic blood pressure (mm Hg)
110±14
138±15
<0.001
Diastolic blood pressure (mm Hg)
71±11
89±13
<0.001
Mean arterial pressure (mm Hg)
84±11
105±14
<0.001
Total proteins, g/L
63±5
69±6
0.068
Proteinuria, mg/24h#
<150
0.500 (0.300-4.170)
/
Hemoglobin, g/L
115±7
115±12
0.898
RBC, x1012/L
3.8±0.3
4.0±0.5
0.129
PLT, x109/L
238±47
192±58
0.016
ALT, IU/L
21±19
35±29
0.100
AST, IU/L
17±7
26±16
0.056
Uric acid, μmol/L
246±47
333±86
0.001
Creatinine, μmol/L
56±7
58±8
0.551
Medication (with/without therapy) Methyldopa
/
19/0
/
Nifedipine
/
8/11
/
Diazepam
/
11/8
/
Progesterone
/
3/16
/
Aspirin
/
3/16
/
BMI- body mass index; RBC- red blood cells; PLT-platelets; ALT- alanine-aminotransferase; ASTaspartate-aminotransferase; Values are shown as means± standard deviations; #-values are shown as medians with interquartile ranges; $-values are shown as mean with minimum-maximum range; &Groups were compared with Chisquare test
Table 2. Correlations of miR-518b in CG and PE group miR-518b, CG
miR-518b, PE
Parameter r
P
r
P
Week of gestation
0.645
0.005
-0.331
0.166
Age (years)
0.101
0.699
0.235
0.332
BMI, kg/m2
-0.017
0.949
0.178
0.467
Weight gain, %
0.527
0.030
0.306
0.203
Systolic blood pressure (mm Hg)
0.297
0.247
0.041
0.867
Diastolic blood pressure (mm Hg)
0.101
0.699
0.132
0.590
Mean arterial pressure (mm Hg)
0.189
0.467
0.100
0.683
Total proteins, g/L
-0.044
0.866
-0.370
0.119
Proteinuria, mg/24h
-0.800
0.410
0.310
0.197
Hemoglobin, g/L
-0.023
0.931
-0.321
0.180
RBC, x1012/L
-0.234
0.366
-0.203
0.405
PLT, x109/L
-0.295
0.250
0.306
0.202
ALT, IU/L
-0.045
0.864
0.233
0.336
AST, IU/L
-0.017
0.949
0.323
0.177
Uric acid, μmol/L
0.527
0.030
0.536
0.018
Creatinine, μmol/L
0.297
0.247
-0.056
0.819
miR-210-3p
0.699
0.002
0.578
0.010
BMI- body mass index; RBC- red blood cells; PLT-platelets; ALT- alanine-aminotransferase; ASTaspartate-aminotransferase
Table 3. miR-518b and miR-210-3p according to smoking status in PE patients
Patients Non-smokers
Smokers
Number of subjects
15
4
miR-518b
0.256 (0.188-0.350)
0.561 (0.147-2.145)
0.039
miR-210-3p
0.225 (0.167-0.303)
0.425 (0.083-2.167)
0.100
Values are shown as geometric means with interquartile ranges
49.
P
S0009-9120(19)31218-4 https://doi.org/10.1016/j.clinbiochem.2020.02.012 CLB 10088
To appear in:
Clinical Biochemistry
Received Date: Revised Date: Accepted Date:
9 November 2019 6 February 2020 18 February 2020
Please cite this article as: M. Jelena, M. Sopić, I. Joksić, U. Prosenc Zmrzljak, N. Karadžov-Orlić, R. Košir, A. Egić, Z. Miković, A. Ninić, V. Spasojević-Kalimanovska, Placenta-specific plasma miR518b is a potential biomarker for preeclampsia, Clinical Biochemistry (2020), doi: https://doi.org/10.1016/j.clinbiochem. 2020.02.012
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Placenta-specific plasma miR518b is a potential biomarker for preeclampsia Munjas Jelenaa, Miron Sopića, Ivana Joksićb, Ursula Prosenc Zmrzljakc, Nataša KaradžovOrlićd,e, Rok Koširc, Amira Egićd,e, Željko Mikovićd,e, Ana Ninića; Vesna SpasojevićKalimanovskaa aDepartment
of Medical Biochemistry, Faculty of Pharmacy, University of Belgrade; Street
Vojvode Stepe 450, 11000 Belgrade, Serbia b
Genetic Laboratory Department, Obstetrics and Gynaecology Clinic "Narodni Front"; Street
Kraljice Natalije 62, 11000 Belgrade, Serbia c
BIA Separations CRO, Labena Ltd.; Street Verovškova 64, 1000 Ljubljana, Slovenia
d
High-Risk Pregnancy Department, Obstetrics and Gynaecology Clinic "Narodni Front";
Street Kraljice Natalije 62, 11000 Belgrade, Serbia e
School of Medicine, University of Belgrade; Street Dr Subotica 8, 11000 Belgrade, Serbia
Jelena Munjas: [email protected] Miron Sopić: [email protected] Ivana Joksić: [email protected] Ursula Prosenc Zmrzljak: [email protected] Nataša Karadžov-Orlić: [email protected] Rok Košir: [email protected] Amira Egić: [email protected] Željko Miković: [email protected] Ana Ninić: [email protected] Vesna Spasojević-Kalimanovska: [email protected] Corresponding author: Miron Sopić Email: [email protected]
Address: Vojvode Stepe 450, Department of Medical Biochemistry, Faculty of Pharmacy, University of Belgrade, 11000 Belgrade, Serbia Telephone number: +38111 3951 265 Fax number: +38111 3972840 Word count: 3.447 Number of tables: 3 Number of figures: 3
Abstract Introduction: MicroRNAs have a significant role in the pathogenesis of preeclampsia. Circulating microRNAs could represent a potential biomarker for preeclampsia. The aim of this study was to evaluate plasma miR210-3p and miR518b in preeclampsia and healthy pregnancy for the first time by digital droplet PCR (ddPCR). Methods: Thirty-six pregnant women (seventeen healthy pregnancies, nineteen preeclampsia patients) were involved from the Clinic for Gynaecology and Obstetrics “Narodni front” in Belgrade, Serbia. Plasma miR210-3p, miR518b and cel-miR-39 as a spike-in control were measured by ddPCR. Results: MiR518b was significantly elevated in preeclampsia compared to a healthy pregnancy (P=0.034; 0.302(0.217-0.421) vs. 0.171(0.110-0.266)). MiR210-3p showed no significant difference between the two groups (P=0.951). The adjustment of miR518b was made for a gestational age and smoking status and the difference between the preeclampsia and healthy pregnancy group was more significant (P=0.026; 0.300(0.216-0.419) vs. 0.172(0.121-0.245)). Plasma miR-518b was significantly higher in the group of preeclampsia patients with proteinuria above the 75th percentile for the group (P=0.033), in women who smoked (P=0.039), and was positively related to uric acid in preeclampsia (P=0.018, r=0.536). Plasma miR518b was able to significantly discriminate between preeclampsia and healthy pregnancy, yielding AUC of 0.712 (95%CI:0.539-0.891), P=0.028. Conclusions: In this study plasma microRNA were measured for the first time in preeclampsia and healthy pregnancies with ddPCR. Placenta-specific miR-518b could serve as a potential biomarker for
discriminating preeclampsia and healthy pregnancy, which should be confirmed on a larger study population. This study has failed to confirm the same potential for miR210-3p. Keywords: preeclampsia, miR518b, miR210-3p, digital droplet PCR
Introduction: Preeclampsia (PE) stands out among the hypertensive disorders of pregnancy for its impact on maternal and neonatal health, since it is one of the leading causes of maternal and perinatal mortality and morbidity worldwide (1). Preeclampsia is defined as hypertension developing after 20 weeks of gestation with one or more of the following: proteinuria, maternal organ dysfunction (including renal, hepatic, hematological, or neurological complications), or fetal growth restriction (2). However, the pathogenesis of preeclampsia is only partially understood and it is related to disturbances in placentation at the beginning of pregnancy, followed by generalized inflammation and progressive endothelial damage. Although pathophysiological changes exist from very early stages of the pregnancy, hypertension and proteinuria usually become apparent in the second half of pregnancy and are present in 2%–8% of all pregnancies overall (1). The placenta has always been a central figure in the etiology of preeclampsia because the removal of the placenta is necessary for symptoms to regress (3). Due to its heterogeneity and elusive pathophysiology, preeclampsia still lacks a suitable biomarker for diagnosis and prediction. The diagnosis of preeclampsia is one of the most important goals in maternal-fetal medicine, considering the impact it has on maternal and fetal health. Although a great deal of biomarkers is being assessed, the evidence is not yet consistent and studies assessing new biomarkers are highly encouraged. (1, 2, 4). MicroRNAs (miRNAs) are small, about 22-24 nucleotides long, noncoding RNAs that regulate gene expression at posttranscriptional level, binding to the target mRNA and promoting their degradation or inhibiting translation. In that way miRNAs regulate more than 30% of the human genome (5). Many miRNAs have been identified in human placental tissues, where they have an important role in placental development, trophoblast proliferation, and migration and angiogenesis (6). Altered expression of numerous miRNAs in placenta has been related to pathophysiological processes that occur in preeclampsia (7, 8, 9). Placental miRNAs are
released into the maternal circulation via an exosome mediated pathway where they are found in a very stable form of microparticles (10). A specific placental miRNA pattern dynamically changes during pregnancy and those changes are reflected in maternal plasma (11, 12). Moreover, many studies so far have shown abnormal levels of circulating placenta-derived miRNAs in pregnancies affected by pre-eclampsia. Therefore, circulating placenta-derived microRNAs could be biomarkers with great potential in both prediction and diagnosis of PE. However, many discrepancies exist between studies, probably due to different technology used for miRNA quantification and sample collection (7, 8, 9). MiR518b (chromosome 19 microRNA cluster, C19MC) belongs to a family of placentaspecific microRNAs which are exclusively expressed in the placenta (8, 13). Aberrant upregulation of miR-518b in preeclamptic placentas may contribute to excessive trophoblast proliferation, which is well recognized as the fundamental pathological change in preeclampsia (14). MiR518b has also been shown to be up-regulated in maternal plasma of PE patients with a potential to be validated as a biomarker of PE (15, 16). The most highly studied, and most abundantly expressed miRNA in placental tissue is so called hypoxia-related miR210 (9). MiR210 belongs to a group of placenta-associated miRNAs (8) and is ubiquitously expressed in placenta and many other tissues (17, 18). MiR210 is detected in maternal circulation, and its levels are up-regulated both in placental tissue and maternal circulation in PE. Upregulation of miR210 in placenta results in a negative regulation of trophoblast cell migration and invasion, which are one of the main pathophysiological mechanisms which lead to placental dysfunction (19). Digital droplet PCR (ddPCR) is considered to be a powerful tool for microRNA quantification, especially from low abundant samples (such as serum/plasma), with many potential advantages over real-time PCR, including the capability to obtain absolute quantification without external references and robustness to variations in PCR efficiency (20, 21).
Therefore, the aim of this study was to evaluate for the first time circulating plasma levels of miR210-3p and miR518b in preeclampsia patients and normal pregnancies with ddPCR, and to evaluate their potential role as a biomarker of PE. Material and methods: This study was designed as observational, case-control study. The study included 36 pregnant women who came to the Clinic for Gynaecology and Obstetrics “Narodni front”, Belgrade, Serbia, either for their routine examination of were admitted at the Clinic for further examination, from March to June 2018. For all patients, blood pressure measurement, complete blood count, routine biochemistry, urine analysis and fetal ultrasound with doppler examination were performed. Based on clinical and laboratory features all subjects were classified in two groups: control group (17 healthy pregnant women) and preeclampsia group (19 patients). Preeclampsia was diagnosed according to the established criteria. Severe preeclampsia was considered if blood pressure was >160 mmHg systolic or >110 mmHg diastolic; presence of HELLP syndrome (hemolysis, platelet level <100,000/dl, ALT or AST elevations of twofold the upper limit), impending eclampsia (headache, epigastric pain and visual disturbances), worsening thrombocytopenia, or worsening fetal growth restriction. Proteinuria was not a criteria for the severity of PE, since proteinuria at levels higher than those for the diagnosis of preeclampsia does not predict clinical outcome (22). All women in our study were diagnosed as having mild form of preeclampsia. Demographic data (age, body mass index (BMI), smoking status, medication used), information on medical and obstetrics history and lifestyle factors for each patient were collected by a questionnaire. Smokers were defined as subjects who have smoked ten cigarettes a day for at least two years. Mean arterial pressure was calculated as (systolic pressure+2xdiastolic pressure)/3.
Patient exclusion criteria were: presence of diabetes mellitus, multifetal pregnancy, major fetal anomaly, fetal death, any condition requiring termination of pregnancy, any maternal chronic disease and inability to give informed consent. This study was conducted according to the guidelines laid down in the Declaration of Helsinki. The study was approved by the Ethic Committee of the Clinic for Gynecology and Obstetrics “Narodni front” and informed consents from each patient were obtained at the time of samples collection. Twelve hour fasting whole blood was drawn in EDTA containing tubes. MicroRNAs were isolated from the cell-free plasma samples, since blood cells represent a significant source of circulating microRNAs and can cofound in their measurement (23). Platelets represent a significant source of circulating microRNAs that could lead to sample contamination, especially after freeze/thaw cycle which disturbs the platelet’s membrane and leads to secretion of miRNA into the plasma sample (24). Plasma was isolated in less than one hour following phlebotomy, which is essential in microRNA analysis, in order to minimize contamination of microRNAs originating from blood cells. For obtaining platelet poor plasma, samples were double-centrifuged. Firstly, samples were centrifuged at 2500×g for 15 minutes at room temperature. Plasma was collected in a plastic tube, leaving 1 cm of plasma above the buffy layer and cautiously handled to avoid disturbance. The plasma was then centrifuged a second time at 2500×g for 15 min at room temperature, and collected afterwards into a new plastic tube, leaving approximately 100 µL at the bottom of the plastic tube (25). MicroRNAs were isolated from 200 μL of plasma according to the manufacturer’s protocol (Cat. Number 217184; miRNeasy Serum/Plasma Kit, Qiagen, Düsseldorf, Germany). Platelet poor plasma samples were additionally centrifuged in conical tubes for 10 min at 16,000 x g and 4°C in a fixed-angle rotor for removal of additional cellular nucleic acids attached to cell debris, according to the manufacturer’s protocol and stored at -80°C. In order to adjust for RNA extraction efficiency,
non-human (Caenorhabditis elegans) cel-miR-39 was used as a spike-in control (Qiagen: miRNeasy Serum/Plasma Spike-In, Cat. Number 219610). After addition of QIAzol Lysis Reagent, 3.5 μl spike-in control (1.6 x 108 copies/μl working solution) was added into each sample, and the isolation was continued according to the protocol. Total RNA was eluted from the column by two sequential elutions with 14 μL water to yield approximately 24 μL of eluate. After isolation, samples were stored at -80°C until further use. Reverse transcription (RT) reactions were performed using Taq-Man miRNA Reverse Transcription Kit (Cat. Number: 4366596) and specific TaqMan MicroRNA Assays (Cat. Number: 4427975: hsa-miR-210-3p, 000512; has-miR-518b, 001156; cel-miR-39-3p, 000200; Thermo Fisher Scientific, Foster City, CA, USA) adding 5 μL of total isolated RNA in each reaction. Thermal protocol for RT was as following: 30 minutes at 16°C; 30 minutes at 42°C and 5 minutes at 85°C. After RT, samples were stored at -80°C until further use. QX200 Droplet Digital PCR (ddPCR) System (Bio-Rad) was used for ddPCR analysis. Each 20µl ddPCR reaction consisted of 10µl of ddPCR Supermix for Probes (no dUTP) (Bio-Rad), 1µl of miRNA assay (Applied biosystems) and 1 μl of miRNA-specific transcribed cDNA. For measurement of cel-miR-39-3p the samples were previously diluted 50x. Each reaction supermix was transferred to a cartridge along with 70 μl of Droplet Generation Oil (Bio-Rad) and partitioned into approximately 15,000–20,000 droplets using the QX200 Droplet Generator (Bio-Rad). Droplets were transferred to 96-well plates (Eppendorf) and sealed using the PX-1 plate sealer (Bio-Rad). The PCR was performed in a T100 Thermal Cycler (Bio-Rad). The cycling conditions were as following: 95 °C 10 min, 1 cycle; 95 °C 30 sec, assay specific annealing temperature 1 min, 40 cycles; 98°C 10 min, 4 °C ∞. Annealing temperature for miR-210-3p and miR-518b assays was 60°C, the annealing temperature for cel-miR-39 assay was 57°C. All steps are with a 2 °C/s temperature ramp rate. Following end-point amplification, the individual droplets were measured with the QX200
Droplet Reader (Bio-Rad) and analyzed using QuantaSoft software (Bio-Rad) to determine target miRNA amount. Statistical analysis Data with normal distribution are shown as means with standard deviation. Data with skewed distribution were log-transformed and presented as geometric means with 95% confidence intervals for the mean. Data were tested using Student’s t Test. Data with skewed distribution even after transformation were represented as medians with interquartile ranges and tested with the Mann-Whitney test. Univariate associations were evaluated by Pearson’s linear correlation analysis. Adjustment for confounding factors was performed using analysis of covariance (ANCOVA). Discriminative abilities of the examined parameters were assessed using the receiving operative characteristic (ROC) curve analysis, while the curve was plotted, and the area under the curve (AUC) presented as C statistics from the analysis. Interpretation of the area under ROC curves (AUCs) is done by the recommended guidelines (26). Categorical variables were analysed using Chi-square tests for contingency tables. A P-value of less than 0.05 was considered statistically significant. Results: PE group and CG were age matched. Mean gestational age for PE and CG was not significantly different, ranging between 20-39 weeks of gestation for CG, and 24-38 week of gestation for PE group. PE group had significantly higher BMI compared to CG (P=0.030), while percentage of weight gain was not significantly different between the two groups. PE group had a significantly higher systolic and diastolic blood pressure, as well as mean arterial pressure compared to CG (P<0.001; P<0.001; P<0.001respectively). All the women in PE group, except two patients, had proteinuria ≥300 mg/24h, while serum protein levels were not significantly different between the two groups. PLT count was significantly lower in PE group compared to CG (P=0.016), while uric acid levels were significantly higher in PE group compared to CG
(P=0.001). Hemoglobin, RBC, ALT, AST and creatinine showed no significant difference between the investigated groups (Table 1). The results from this study showed that miR-518b was significantly elevated in plasma of women with PE compared to CG (P=0.034, Figure 1A). Plasma miR-210-3p showed no significant difference between the two groups (P=0.951; Figure 2). PE patients were divided into two groups according to the 75 percentile cut-off for proteinuria for the entire patient distribution (proteinuria75p=4.17 g/24h). Plasma miR-518b was significantly higher in the group of PE patients with proteinuria above the 75th percentile for the group (Figure 1B; P=0.033). Plasma miR-518b in CG correlated positively with gestational age, percentage of weight gain, uric acid levels and plasma miR-210-3p (Table 2). In PE group, miR-518b correlated positively with uric acid and plasma miR-210-3p (Table 2). Plasma miR-518b was up regulated in women with PE who smoked (P=0.039, Table 3). Therefore, the adjustment of plasma miR-518b levels was made for gestational age and smoking status and the difference between the PE group and CG was even more significant (P=0.026; Figure 1C). The potential of plasma miR-518b for discriminating PE patients from the controls was investigated by ROC curve analysis, and comparative analysis of AUCs for miR518b, miR210-3p, mean arterial pressure and uric acid was made. Plasma miR-518b was able to significantly discriminate between PE and CG, yielding AUC of 0.715 (95% CI: 0.539-0.891), P=0.028, Figure 3. The patients were also classified into early (before 34 weeks; 10 women) and late onset (at or after 34 weeks; 9 women) PE (22), and miR-518b and miR210-3p were compared. There were no significant difference between the early and late onset PE for miR518b (P=0.268) and miR210-3p (P=0.682). Discussion In this study for the first time, cell-free plasma levels of placenta-specific miR518b and placenta-associated miR210-3p were measured in PE patients and healthy pregnancy by digital
droplet PCR. Digital droplet PCR (ddPCR) is a breakthrough, high throughput technology that provides ultrasensitive and absolute nucleic acid quantification. It is particularly useful for lowabundance targets, such as miRNA quantification from serum/plasma samples, obtaining highest precision and sensitivity, absolute quantification without the use of external references, with the advantage of removal of PCR efficiency bias. MicroRNA quantification by ddPCR and real-time PCR by Hindson et al. revealed greater precision (coefficients of variation decreased 37–86%) and improved day-to-day reproducibility of ddPCR. More importantly, when applied to microRNA detection in clinical serum samples, ddPCR showed superior diagnostic performance compared to real-time PCR (21). Implementation of this method for microRNA quantification would therefore improve the assessment and the use of microRNAs as biomarkers. In this study, it has been shown that plasma levels of placenta-specific miR518b were significantly higher in PE patients compared to CG, even after adjustment for confounding factors (Figure 1A, 1C). In agreement with our results, Hromadnikova et al. showed that along with up-regulation in the placenta, miR518 levels were increased in plasma of PE patients, even early in pregnancy, and proposed a possible role in the inducement of gestational hypertension and preeclampsia (16). In addition, Miura et al. found increased miR518b levels in severe cases of PE (15). Even though it has been shown that expression of miR518b was up-regulated in placental tissue in severe PE (27) certain studies have pointed to down-regulation of miR518 in placenta of PE (28). However, these discrepancies are most likely caused by different sampling position in the chorionic or basal plate of the placenta (27). Recently, Liu et al have proposed a mechanism by which up-regulation of miR518b in placenta could contribute to the pathophysiology of PE. The authors showed that miR518 directly targets and represses Ras-related protein 1b (Rap1b) expression, which results in excessive proliferation of trophoblast cells, especially the column
ones, trough Rap1b–Ras–MAPK pathway. Excessive proliferation of trophoblasts disables correct differentiation toward invasive cell phenotype, leading to the shallow invasion of decidual stroma and spiral arteries, which represents a hallmark of preeclampsia placentas (14). Throughout gestation, plasma microRNA levels rise until the delivery, and consequently, levels of circulating microRNA are dependent from gestational age (29-31). This study did not investigate plasma microRNA changes throughout gestation, but the positive correlation of plasma miR518b and gestational age was observed in CG (Table 2). Also, we found that plasma miR518b levels were affected by smoking status (Table 3). Although smoking is inversely related to preeclampsia occurrence (32), Maccani et al. have pointed to downregulation of several microRNAs in the placental tissue in mothers who smoked (33). Therefore, plasma miR-518b levels were adjusted for gestational age and smoking status, but the difference between PE and CG was even more significant (Figure 1C). Furthermore, in PE patient, miR518 increase was consistent with proteinuria worsening (Figure 1B), one of the hallmarks for PE pathogenesis (2). Uric acid, besides its utility in diagnosis of PE, is proposed to have direct pathophysiological role in PE development (34). Plasma miR518b levels showed positive correlation with uric acid in both investigated groups (Table 2), which further confirms its involvement in PE. Finally, diagnostic potential for plasma miR518b for PE was shown by ROC curve analysis, where plasma miR-518b was able to significantly discriminate between PE and CG, yielding AUC of 0.715, unlike miR210-3p, which did not show significant diagnostic potential by ROC curve analysis (Figure 3). Therefore, miR-518b could serve as a potential biomarker of PE. Furthermore, it is worth mentioning that in AUC analysis (available only in 27 out of 36 cases, data not shown), mir518b performed much better than uterine pulsatility index measurement (UtPI) (AUCUtPI=0.515 (95%CI: 0.283-0.740), P=0.921), which is one of the parameters used for screening and prediction of PE (35). However, it did not perform better than mean arterial
pressure and uric acid. Mean arterial pressure is considered to be a better predictor of preeclampsia compared to diastolic or systolic pressure. (35, 36). In this study, all preeclampsia cases had distinctive hypertension, therefore, higher ranges of mean arterial pressure than the control group. Considering elevated blood pressure was one of the diagnostic criteria for PE (1, 2), by which PE patients were selected in this study, it is not surprising that mean arterial pressure showed better performance in ROC curve analysis than miR-518b. In addition, it was showed that uric acid is involved in the pathogenesis of PE (34), and thus it could be used as a biomarker for prediction of PE in women at moderate and low risk and is also a predictor of adverse outcomes of PE for both the mother and the newborn (37, 38, 39). Pregnant women with abnormal weight and obesity are subject to the increased risk of developing PE (40). A positive association was found between pre-pregnancy BMI and placental expression of several microRNAs at birth, as well as with gestational weight gain, which was related to fetal gender. (41). Also, a specific placental miRNA profile was identified in maternal obesity (42). In this study, the PE group had significantly higher BMI compared to CG. Plasma miR-518b correlated positively with the percentage of weight gain in CG, however, no correlation with current BMI or percentage of weight gain was found in PE group. On the other hand, plasma levels of miR-210-3p did not differ between PE and CG (Figure 2). MiR210 is one of the so-called master hypoxamiRs (hypoxia-induced miRNAs), since it has been found upregulated by hypoxia in all cells and tissues tested to date (17, 18). It was proposed that induction of miR210 in the placenta could interfere with trophoblast invasion and contribute to PE development (19, 43). Murphy et al. have found that plasma miR-210 levels are increased only in the severe cases of PE, while in mild cases, there was no difference compared to the CG (44). The reason for that could lie in the fact that more severe cases of PE are associated with increased hypoxia, which reflects in increased hypoxia-driven trophoblast cell debris carrying microRNA in the circulation, and further results in higher miR-210 plasma
levels (9). The majority of patients in this study were not diagnosed with severe forms of PE, the most prevalent was a mild form of PE (2), so we can presume that this is the reason why plasma levels of miR210-3p did not differ in our PE and CG. Furthermore, miR210 is expressed by many tissues and cells other than placenta (17, 18), so miR-210-3p plasma levels could be affected by other factors beside PE in patients and controls. MiR210 was elevated in serum samples from PE patients vs. healthy controls (19), but serum and plasma are shown to have different microRNA profiles (45) due to the coagulation process (46) and the release of microRNAs from blood cells which are major contributor to the circulating microRNA pool - platelets, leukocytes and erythrocytes (9, 23, 24). Therefore, for microRNA measurement in blood of pregnant women, Ge et al. proposed plasma as more suitable sampling material over serum (46). It is important to highlight, that in this study both microRNAs were measured in cell-free plasma samples, so it can be presumed that the coagulation process and cells contamination did not confound in this measurement, so this could be the explanation for discrepancy existing between our study and the serum microRNA profiles in PE. However, some limitations within this study should be mentioned. Firstly, study group sizes were small, so these findings should be confirmed using larger study groups. Secondly, all PE patients received some form of therapy which could have influenced the results. Most commonly prescribed antihypertensive drugs in pregnant women with PE are methyldopa and nifedipine. Based on data from Pharmaco-mir database (www.pharmaco-mir.org) (47) both drugs can be influenced by numerous miRNAs. Currently there are no studies showing interaction of mentioned drugs and miRNAs investigated in our study, so further research is needed to better clarify possible effects of antihypertensive therapy on miRNA expression. In conclusion, to the best of our knowledge, this is the first study where plasma microRNAs levels were measured by ddPCR in PE. Circulating levels of miR518 were higher in PE patients
compared to CG, and could serve as a potential diagnostic marker for PE. MiR518b is related to proteinuria and uric acid level in PE patients, which are one of the hallmarks of the disease, confirming its role in PE. Plasma levels of miR210-3p did not differ between the investigated groups. Acknowledgments: This study was financially supported by ddPCR Grant challenge initiative from Labena Ltd., Verovškova 64, 1000 Ljubljana, Slovenia and by a grant from the Ministry of Education, Science and Technological Development, Serbia (project number 175035). We declare that there is no conflict of interest.
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0.450
A
0.421 0.400 0.350 0.302
miR-518b
0.300 0.250
0.266 0.217
0.200 0.171 0.150 0.100
0.110
0.050 0.000
CG
PE
B
P=0.033
P=0.026 C
0.450 0.419 0.400
adj miR-518b
0.350 0.300
0.300 0.250
0.245 0.216
0.200 0.172 0.150 0.121 0.100 0.050 0.000
CG
Figure 1.
PE
A) Plasma miR-518b in CG and PE B) Plasma miR-518b in PE group according 75% cut-off for proteinuria, values are shown as medians with interquartile ranges
C) Plasma miR-518b in CG and PE after adjustment for gestational age and smoking *values are show as geometric means with 95% confidence intervals for the mean
0.450 0.400
P=0.951 0.397 0.358
miR-210-3p
0.350 0.300 0.250
0.262
0.258
0.200
0.185
0.172 0.150 0.100 0.050 0.000
CG
Figure 2. Plasma miR-210-3p in CG and PE *values are show as geometric means with confidence intervals
PE
AUCmiR518b=0.715 (95% CI 0.539-0.891); P=0.028 AUCmiR210-3p=0.458 (95% CI 0.262-0.655); P=0.669 AUCuric acid=0.797 (95% CI 0.649-0.946); P=0.002 AUCMAP=0.895 (95% CI 0.750-1.000); P<0.001
Figure 3. Comparative ROC curve for the potential of miR518b, miR210-3p, Mean arterial pressure (MAP) and Uric acid in discriminating PE patients from the controls AUC- area under the receiving operative characteristic curve
Figure 4. ROC curve for the potential of uterine pulsatility index (UtPI) in discriminating PE patients from the controls AUC- area under the receiving operative characteristic curve 48. Table 1. Clinical and laboratory characteristics of CG and PE Variable
Control group
Preeclampsia
P
Number of subjects
17
19
/
Week of gestation$
33 (20-39)
33 (24-38)
0.961
Age (years) $
32 (22-40)
34 (20-51)
0.610
BMI, kg/m2
26±3
29±6
0.030
Weight gain, %
16±8
22±12
0.155
Smoking status (smokers/non-smokers)
4/13
4/15
0.858&
Systolic blood pressure (mm Hg)
110±14
138±15
<0.001
Diastolic blood pressure (mm Hg)
71±11
89±13
<0.001
Mean arterial pressure (mm Hg)
84±11
105±14
<0.001
Total proteins, g/L
63±5
69±6
0.068
Proteinuria, mg/24h#
<150
0.500 (0.300-4.170)
/
Hemoglobin, g/L
115±7
115±12
0.898
RBC, x1012/L
3.8±0.3
4.0±0.5
0.129
PLT, x109/L
238±47
192±58
0.016
ALT, IU/L
21±19
35±29
0.100
AST, IU/L
17±7
26±16
0.056
Uric acid, μmol/L
246±47
333±86
0.001
Creatinine, μmol/L
56±7
58±8
0.551
Medication (with/without therapy) Methyldopa
/
19/0
/
Nifedipine
/
8/11
/
Diazepam
/
11/8
/
Progesterone
/
3/16
/
Aspirin
/
3/16
/
BMI- body mass index; RBC- red blood cells; PLT-platelets; ALT- alanine-aminotransferase; ASTaspartate-aminotransferase; Values are shown as means± standard deviations; #-values are shown as medians with interquartile ranges; $-values are shown as mean with minimum-maximum range; &Groups were compared with Chisquare test
Table 2. Correlations of miR-518b in CG and PE group miR-518b, CG
miR-518b, PE
Parameter r
P
r
P
Week of gestation
0.645
0.005
-0.331
0.166
Age (years)
0.101
0.699
0.235
0.332
BMI, kg/m2
-0.017
0.949
0.178
0.467
Weight gain, %
0.527
0.030
0.306
0.203
Systolic blood pressure (mm Hg)
0.297
0.247
0.041
0.867
Diastolic blood pressure (mm Hg)
0.101
0.699
0.132
0.590
Mean arterial pressure (mm Hg)
0.189
0.467
0.100
0.683
Total proteins, g/L
-0.044
0.866
-0.370
0.119
Proteinuria, mg/24h
-0.800
0.410
0.310
0.197
Hemoglobin, g/L
-0.023
0.931
-0.321
0.180
RBC, x1012/L
-0.234
0.366
-0.203
0.405
PLT, x109/L
-0.295
0.250
0.306
0.202
ALT, IU/L
-0.045
0.864
0.233
0.336
AST, IU/L
-0.017
0.949
0.323
0.177
Uric acid, μmol/L
0.527
0.030
0.536
0.018
Creatinine, μmol/L
0.297
0.247
-0.056
0.819
miR-210-3p
0.699
0.002
0.578
0.010
BMI- body mass index; RBC- red blood cells; PLT-platelets; ALT- alanine-aminotransferase; ASTaspartate-aminotransferase
Table 3. miR-518b and miR-210-3p according to smoking status in PE patients
Patients Non-smokers
Smokers
Number of subjects
15
4
miR-518b
0.256 (0.188-0.350)
0.561 (0.147-2.145)
0.039
miR-210-3p
0.225 (0.167-0.303)
0.425 (0.083-2.167)
0.100
Values are shown as geometric means with interquartile ranges
49.
P