Longitudinal changes in plasma hemopexin and alpha-1-microglobulin concentrations in women with and without clinical risk factors for pre-eclampsia

RESEARCH ARTICLE Longitudinal changes in plasma hemopexin and alpha-1-microglobulin concentrations in women with and without clinical risk factors for pre-eclampsia Katja MurtoniemiID 1,2 *, Grigorios KalapotharakosID 3 , Tero Vahlberg 4 , Katri Ra ¨ikkonen 5 , Eero Kajantie 6,7,8,9 , Esa Ha ¨ma ¨la ¨inen 10 , Bo Åkerstro ¨m 11 , Pia M. Villa 12 , Stefan R. Hansson 3 , Hannele Laivuori 1,13,14 1Medical and Clinical Genetics, University of Helsinki and Helsinki University Hospital, University of Helsinki, Finland, 2Department of Obstetrics and Gynaecology, University of Turku and Turku University Hospital, Turku, Finland, 3Skåne University Hospital, Department of Clinical Sciences Lund, Department of Obstetrics and Gynecology, Lund University, Lund, Sweden, 4Department of Clinical Medicine, Biostatistics, University of Turku and Turku University Hospital, Turku, Finland, 5Department of Psychology and Logopedics, Faculty of Medicine, University of Helsinki, Helsinki, Finland, 6PEDEGO Research Unit, MRC Oulu, Oulu University Hospital and University of Oulu, Oulu, Finland, 7National Institute for Health and Welfare, Helsinki, Finland, 8Children‘s Hospital, University of Helsinki and Helsinki University Hospital, Helsinki, Finland, 9Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway, 10 Department of Clinical Chemistry, University of Helsinki, Helsinki, Finland, 11 Division of Infection Medicine, Department of Clinical Sciences, Lund University, Lund, Sweden, 12 Department of Obstetrics and Gynecology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland, 13 Institute for Molecular Medicine Finland, Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland, 14 Department of Obstetrics and Gynecology, Tampere University Hospital and Tampere University, Faculty of Medicine and Health Technology, Tampere, Finland *[email protected] Abstract Recent studies have shown increased concentration of fetal hemoglobin (HbF) in preeclamptic women. Plasma hemopexin (Hpx) and alpha-1-microglobulin (A1M) are hemoglobin scavenger proteins that protect against toxic effects of free heme released in the hemoglobin degradation process. We used an enzyme-linked immunosorbent assay to analyze maternal plasma Hpx and A1M concentrations at 12–14, 18–20 and 26–28 weeks of gestation in three groups: 1) 51 women with a low risk for pre-eclampsia (LRW), 2) 49 women with a high risk for pre-eclampsia (PE) who did not develop PE (HRW) and 3) 42 women with a high risk for PE who developed PE (HRPE). The study had three aims: 1) to investigate whether longitudinal differences exist between study groups, 2) to examine if Hpx and A1M concentrations develop differently in pre-eclamptic women with small for gestational age (SGA) fetuses vs. pre-eclamptic women with appropriate for gestational age fetuses, and 3) to examine if longitudinal Hpx and A1M profiles differ by PE subtype (early-onset vs. lateonset and severe vs. non-severe PE). Repeated measures analysis of variance was used to analyze differences in Hpx and A1M concentrations between the groups. We found that the differences in longitudinal plasma Hpx and A1M concentrations in HRW compared to HRPE and to LRW may be associated with reduced risk of PE regardless of clinical risk factors. In women who developed PE, a high A1M concentration from midgestation to late second PLOS ONE | https://doi.org/10.1371/journal.pone.0226520 December 16, 2019 1 / 17 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Murtoniemi K, Kalapotharakos G, Vahlberg T, Ra¨ikkonen K, Kajantie E, Ha¨ma¨la¨inen E, et al. (2019) Longitudinal changes in plasma hemopexin and alpha-1-microglobulin concentrations in women with and without clinical risk factors for pre-eclampsia. PLoS ONE 14(12): e0226520. https://doi.org/10.1371/journal. pone.0226520 Editor: Frank T. Spradley, University of Mississippi Medical Center, UNITED STATES Received: August 12, 2019 Accepted: November 26, 2019 Published: December 16, 2019 Copyright: ©2019 Murtoniemi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: The datasets generated during and/or analysed during the current study are not publicly available due to prohibitions by national laws since the data include patient report data. However, the datasets generated during and/or analysed during the current study are available prof. Anu-Katriina Pesonen, e-mail address: anukatriina. [email protected] on reasonable request in completely anonymized form. Due to the sensitive trimester was associated with SGA. There were no differences in longitudinal Hpx and A1M concentrations from first to late second trimester in high-risk women who developed earlyonset or. late-onset PE or in women who developed severe or. non-severe PE. Introduction Pre-eclampsia (PE) is a hypertensive disease that occurs in 2–8% of pregnancies. Globally, PE is a major cause of maternal and fetal morbidity and mortality [1,2] and it indicates an elevated risk for subsequent long-term, non-communicable diseases of the mother and the newborn child [3,4]. The exact cause of PE is unknown, but the understanding of the pathophysiological mechanisms of the disease has increased gradually. Currently, most popular theory is that the disease develops in two stages. The first stage occurs during early placental development, when impaired invasion of extravillous trophoblasts to the maternal spiral arteries causes defective remodeling of the arteries and incomplete vascular adaptation to pregnancy [5]. This causes uneven blood flow in the intervillous space of the placenta [6], intermittent hypoxia and oxidative stress [7]. Damages to the placenta-blood barrier follows and there is increased leakage of placental and fetal products into the maternal blood circulation [8–10]. It is known that poor placentation is associated with early-onset PE and small for gestational age (SGA) neonates, which may or may not be associated with PE [11]. The second stage of PE occurs later in pregnancy, after 20 weeks of gestation (GW) and the typical clinical manifestations are hypertension and proteinuria. This stage is characterized by endothelial dysfunction [12], maternal hypovolemia, vasoconstriction [13] and inflammation [14]. Circulating toxic factors derived from the placenta trigger an inflammatory response and induce general endothelial dysfunction. General organ damage develops, which, in turn, leads to the typical clinical manifestations of PE. Maternal susceptibility and certain maternal characteristics (e.g., obesity, metabolic syndrome, hypertension) play an important role at this stage [11,15]. Earlier studies suggest that the plasma concentrations of free fetal hemoglobin (HbF) are increased in women who will develop PE and that HbF plays an important role bridging stage I and II [16–19]. The production of HbF is increased in PE placentas [20]. Extracellular HbF induces oxidative stress by forming reactive oxygen species (ROS). This, in turn, may result in damage to the placenta-blood barrier and leakage of extracellular HbF into the maternal circulation. Circulating extracellular hemoglobin (Hb) itself is a potent toxin, and free heme released during the degradation process of Hb exerts also oxidative and toxic effects on surrounding cells and tissue [21]. In fact, high levels of cell-free HbF in the umbilical cord correlate with fetal growth restriction (FGR) [22]. Hemopexin (Hpx) and alpha-1-microglobulin (A1M) are proteins involved in the hemoglobin scavenger system that protects against the toxic effects of free heme released during Hb degradation. Haptoglobin binds Hb and is the primary protective scavenger protein. Hemopexin has high affinity to free heme and is regarded as a back-up system when the Hb-binding capacity of haptoglobin is exceeded [23], although it does function independently of haptoglobin and does not require total haptoglobin depletion [24]. Plasma Hpx directs heme primarily to the parenchymal cells of the liver for catabolism, iron storage and redistribution; by binding free heme Hpx protects the endothelium [25]. Hpx has also enzymatic activity which increases during pregnancy and may be lead to reduced angiotensin II sensitivity during normal pregnancy [26]. A1M has heme-binding and heme-degrading [27] as well as enzymatic reductase Longitudinal changes in plasma hemopexin and alfa-1-microglobulin concentrations during pregnancy PLOS ONE | https://doi.org/10.1371/journal.pone.0226520 December 16, 2019 2 / 17 nature of the patient report data, data requests may require further approval by the PREDO Study Board, that also enables collaboration in PREDO data analysis through specific study proposals. Funding: This project has been supported by Erasmus + Program of the European Union (Framework agreement number: 2013-0040), GK, SH, BÅ; The PREDO project has been supported by: EVO research funding (A special Finnish state subsidy for health science research, HUS364/ 2017) HL, PV, EH, KM; Academy of Finland, n:o 127437, 129306, 130326, 134791, 263924 and 315690 EK, 1284859, 12848591, 1312670 KR, 278941, 134957, 121196, HL, (https://www.aka.fi/ en/funding/the); European Commission (Horizon2020 award 733280 RECAP Research on Children and Adults Born Preterm EK), https://ec. europa.eu/info/research-and-innovation/funding/ funding-opportunities/prizes/horizon-prizes_en; the Finnish Foundation for Pediatric Research EK, (https://www.lastentautientutkimussaatio.fi); Finnish Foundation for Cardiovascular Research EK, (http://www.sydantutkimussaatio.fi/en/ foundation); Signe and Ane Gyllenberg Foundation EK, KR, (https://gyllenbergs.fi/en/); Sigrid Juselius Foundation EK, (https://sigridjuselius.fi/en/); University of Helsinki Research Funds, HL, (https:// www.helsinki.fi/en/research/research-environment/ research-funding#section-6424); Faculty of Medicine, University of Helsinki (73456128), KR, (https://www.helsinki.fi/en/faculty-of-medicine); Finnish Medical Foundation HL, KM, (https:// laaketieteensaatio.fi/en/home/); Juho Vainio Foundation EK, (http://juhovainionsaatio.fi/en/juhovainio-foundation/); Novo Nordisk Foundation EK, (https://www.novonordisk.com/about-novonordisk/corporate-governance/foundation.html); Jane and Aatos Erkko Foundation, HL, (https://jaes. fi/en/); Pa¨ivikki and Sakari Sohlberg Foundation, HL, (http://www.pss-saatio.fi/english.htm); Finska la¨karesa¨llskapet, HL, KM, (https://www.fls.fi/ sallskapet/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: SRH and BÅholds patent related to diagnosis and treatment of PE and are cofounders of A1M Pharma and Preelumina Diagnostics (www.a1m.se). The pre-existing intellectual properties involve four patents owned by A1M Pharma; 1) HbF and A1M as early stage markers for preeclampsia-1550535 2) Medical use of A1M-2638915 3) Diagnosis and treatment of preeclampsia-201500335 4) Biomarkers for preeclampsia-PA 2015 70146. We confirm that this properties which counteract heme-induced and ROS-induced cellular oxidation [28]. Both Hpx and A1M are potential biomarkers for predicting PE [19,29] and several reports suggest that A1M may be a therapeutic target for PE [30–32], the most recent one for the mice PE model Storkhead box 1 (STOX-1) [33]. There are studies on plasma Hpx and A1M concentrations during the first trimester of pregnancy [19] and after manifestation of PE in late pregnancy [29]. In a previous study on maternal plasma Hpx and A1M concentrations in late second trimester we found that, during 26–28 GW, women at high risk for PE who did not develop PE had higher A1M concentrations than women at high risk who did develop PE [34]. In the present study we followed the plasma concentrations of these hemoglobin scavenger proteins at three time points during pregnancy in three different groups: women with a low risk for PE, in women with a high risk for PE who did not develop PE and in women with a high risk for PE who did develop PE. The focus in this study is on the longitudinal changes of Hpx and A1M concentrations during pregnancy. The aim was to clarify whether there are differences in Hpx and A1M concentrations longitudinally between the study groups and to increase our understanding of the pathophysiology of PE. A recent study showed that the toxicity of HbF causes pathophysiological processes which lead to a compromised feto-placental circulation and FGR [22]. Since HbF is toxic to the fetus, the role of Hpx and A1M was also evaluated in women with PE who experienced FGR complications and who did not experience FGR complications. For this, we studied pre-eclamptic women with an SGA fetus (PESGA) and high-risk women who developed PE and who delivered an appropriate for gestational age (AGA) neonate (PEAGA). Our third aim was to investigate if there are differences in longitudinal Hpx and A1M concentrations depending on the subtype of the PE, i.e., between early-onset PE vs. late-onset PE and between non-severe vs. severe PE. Materials and methods This nested case-control study is a part of the multidisciplinary Prediction and Prevention of Pre-eclampsia and Intrauterine Growth Restriction (PREDO) project [35]. Women with known risk factors for PE were prospectively recruited between September 2005 and June 2009 at ten maternity clinics in Finland. The ethics Committee of the Helsinki and Uusimaa Hospital District approved the study and written informed consent was obtained from all participants. In total 142 women were included: 42 women with PE (HRPE), 49 randomly selected highrisk women who did not develop PE (HRW) and 51 low-risk women (LRW). Seven women in the HRPE participated also in the acetylsalicylic acid (ASA) trial arm of the PREDO project and were treated with a daily low oral dose of acetylsalicylic acid (LDA 100 mg/d) starting before 14 th GW. The inclusion and exclusion criteria are described in Table 1 and the flowchart in Fig 1. The criteria for PE were systolic blood pressure �140 mmHg and/or a diastolic blood pressure �90 mmHg after 20 GW and a urinary 24-h protein excretion of �0.3 g or dipstick equivalent in two consecutive measurements. Pre-eclampsia superimposed on chronic hypertension were included in the primary outcome. Secondary outcomes were PE combined with SGA infant, early-onset pre-eclampsia (diagnosed before 34 +0 weeks of gestation) and severe pre-eclampsia (systolic blood pressure �160 mmHg and/or diastolic blood pressure �110 mmHg and/or proteinuria �5 g/24 hours). SGA was defined as a birthweight �- 2 SDs [36]. All participants had their first visit at 12–14 GW. Uterine artery blood flow was measured with Doppler sonography. Gestational age was confirmed by crown-rump length measurement. The first-trimester mean arterial pressure (MAP) was calculated with the equation: MAP = diastolic blood pressure + (systolic blood pressure–diastolic blood pressure)/3. Longitudinal changes in plasma hemopexin and alfa-1-microglobulin concentrations during pregnancy PLOS ONE | https://doi.org/10.1371/journal.pone.0226520 December 16, 2019 3 / 17 does not alter our adherence to all PLOS ONE policies on sharing data and materials. The plasma Hpx concentration was measured using a sandwich ELISA kit (Human Hemopexin, Genway Biotech Inc., San Diego, CA, USA) and a multilabel counter (Wallac 1420, Perkin-Elmer, Wallac, Turku, Finland) (absorbance at 450nm). Table 1. Inclusion and exclusion criteria for high-risk women. Inclusion criteria Obesity (body mass index over 30 kg/m 2 ) Chronic hypertension (�140/90 mmHg or antihypertensive medication before GW 20) Sjo¨gren’s syndrome History of gestational diabetes History of pre-eclampsia (blood pressure �140 mmHg systolic or �90 mmHg diastolic and proteinuria �0.3 g/day or dipstick equivalent in two consecutive measurements) History of small for gestational age (birthweight <-2SD) History of fetus mortus (fetal death after 22 weeks of gestation or >500 g weight in a previous pregnancy) Systemic lupus erythematosus Type 1 diabetes mellitus Exclusion criteria Tobacco smoking (during this pregnancy) Multiple pregnancy History of asthma History of peptic ulcer Placental ablation Inflammatory bowel diseases (Crohn’s disease, ulcerative colitis) Rheumatoid arthritis Hemophilia or thrombophilia (previous venous or pulmonary thrombosis or coagulopathy) https://doi.org/10.1371/journal.pone.0226520.t001 Fig 1. Flowchart of study cohort. PE = pre-eclampsia, SGA = small for gestational age, AGA = appropriate for gestational age. https://doi.org/10.1371/journal.pone.0226520.g001 Longitudinal changes in plasma hemopexin and alfa-1-microglobulin concentrations during pregnancy PLOS ONE | https://doi.org/10.1371/journal.pone.0226520 December 16, 2019 4 / 17 Plasma A1M concentration was measured using an in-house ELISA and microtiter plates, which were coated with mouse monoclonal anti-A1M antibodies (clone 35.14). The monoclonal anti-A1M antibodies were produced against human urinary A1M by Agrisera AB (Va¨nna¨s, Sweden). Human urinary A1M was prepared in the laboratory of the Section for Infection Medicine, Department of Clinical Sciences, Lund University, Lund, Sweden, as described earlier [37]. Briefly, the plasma samples were incubated overnight at +4˚C under sealing film in 5 μg/ml solution of phosphate buffered saline (PBS) buffer (+ 0.05% tween-20, 100 μl/well). After washing three times with PBS buffer, 100 μl of human urinary A1M reference standard (1.56–100 ng/ml in PBS buffer) or unknown diluted plasma samples (1:1000 with PBS buffer) were added to the wells and incubated for 1 h at room temperature, in darkness and on a rotational shaker (250–500 rpm). After washing three times with PBS buffer, 100 μl of the detection antibody solution was added (horseradish peroxidase-coupled mouse monoclonal anti-A1M antibody clone 57.10; in PBS) and incubated for 1 h at room temperature, in darkness and on a rotational shaker (250–300 rpm). After washing three times with PBS, 100 μl of 3,3’,5,5’-tetramethylbenzidine (TMB substrate, SureBlue TM TMB Microwell Peroxidase Substrate, KPL cat. no. 50-00-04) was added, sealed and again incubated for 20 min without shaking, and the reaction was stopped by adding 100 μl of 1 M sulfuric acid. Absorbance at 450 nm was read using the same Wallac 1420 Multilabel Counter. Statistical analyses Data was analyzed for normality. The differences of means of normally distributed baseline variables between the groups were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc tests. The Kruskal-Wallis test was used if the data was not normally distributed. Comparisons of medians of non-normally distributed variables were made with Mann-Whitney’s U test. Bonferroni corrections were used in post-hoc comparisons. Categorical variables were compared with Fisher’s exact test and in pairwise comparisons Bonferroni correction was used. Repeated measures analysis of variance (RMANOVA) was used to analyze differences in plasma Hpx and A1M concentrations between the groups. The model included the main effects of time, group and the interaction effect time x group. Log-transformed Hpx and A1M values were used in these analyses due to positively skewed distributions. The best covariance structure was selected by using AICC (Akaike information criterion with a correction for small sample sizes) and compound symmetry covariance structure was chosen. The results of the values for Hpx and A1M are expressed as geometric means with 95% confidence intervals. One-way ANOVA followed by Tukey’s post-hoc tests was used for multiple comparisons of biomarker concentrations at each sampling point, if further analyses were indicated after RMANOVA. In all analyses a p-value <0.05 was considered statistically significant. Statistical analyses were performed using version 25.0 of the SPSS statistic software package. Results Baseline characteristics The baseline characteristics of LRW, HRW and HRPE are shown in Table 2 including the division of HRPE into two groups: 1. PESGA and 2. PEAGA. One woman in the HRW group had type 1 diabetes mellitus and one had Sjo¨gren´s syndrome (Table 2). HRW and HRPE were older (p = 0.007) and had a higher body mass index (BMI) (p<0.001) than LRW. There were more women with chronic disease in the HRW and HRPE groups than in in the LRW group (p<0.001). Due to the inclusion criteria of the study there were, in the HRW and HRPE groups Longitudinal changes in plasma hemopexin and alfa-1-microglobulin concentrations during pregnancy PLOS ONE | https://doi.org/10.1371/journal.pone.0226520 December 16, 2019 5 / 17 compared to the LRW group, less primiparous women (p<0.001) and more women with prior SGA (p = 0.002), BMI �30 kg/m 2 (p<0.001), chronic hypertension (p<0.001), prior PE (p<0.001) and prior gestational diabetes (p = 0.041). Pregnancy characteristics The pregnancy characteristics of LRW, HRW and HRPE and the subgroups of SGAPE and AGAPE are shown in Table 3. LRW gained more weight during pregnancy compared to HRW and HRPE (p = 0.009 and p = 0.028, respectively) and the mean uterine artery pulsatility index in the first trimester was lower (p<0.001 for both comparisons). There was more gestational diabetes in HRW and HRPE compared to LRW (p = 0.014). Women in the HRPE group delivered earlier than in the HRW or LRW groups (p<0.001) and in the HRW group earlier than in the LRW group (p<0.001). There were more deliveries with vacuum extraction among HRW than HRPE and LRW (p = 0.006). Women in the HRPE group delivered smaller infants than in the HRW (p<0.001) or LRW (p = 0.015) groups. There was no difference in birth weight SD score between HRW and LRW (p = 0.146). Women in the PESGA group delivered earlier (median 31.9 GW) than in the other groups (p<0.001 in all pairwise comparisons). Outcomes PE phenotypes are shown in Table 4. Of 42 women who developed PE, 33 (79%) gave birth to an AGA infant and 9 (21%) to an SGA infant. There were 21 women with severe and nonsevere PE, respectively. Eleven women (26%) had early-onset PE and 32 (78%) late-onset PE. Table 2. Baseline characteristics of study groups. Characteristics LRW N = 51 HRW N = 49 HRPE N = 42 PEAGA n = 33 PESGA n = 9 Age, years a 29 (5) 33 (6) 31(6) 31.0(6.5) 31.0(8.0) BMI, pre-pregnancy, kg/m 2 b 22.6 (2.4) 28.5 (9.7) 29.3(10.6) 29.6(11.0) 29.0(14.8) Primiparous, n (%) c 30 (59) 12 (25) 10(24) 6(18.20) 4(44.4) Infertility treatment, n (%) 4 (9) 7 (15) 5(14) 4(14.8) 1(11.1) Chronic disease, n (%) c 10 (17) 27 (46) 22(37) 17(51.5) 5(55.6) BMI �30 kg/m 2 c 1 (3) 21(43) 16(38) 13(38.2) 3(37.5) Prior pre-eclampsia, n (%) c 0 14(29) 21(50) 19(57.6) 2(22.2) SGA in previous pregnancy, n (%) d 0 9(18) 4(10) 2(6.1) 2(22.2) Chronic hypertension c 0 14 (29) 10(24) 8(24.2) 2(22.2) Prior GDM, n (%) e 0 5 (10) 4(10) 4(11.8) 0(0.0) Type 1 DM, n (%) f 0(0.0) 1(2.0) 0(0.0) 0(0.0) 0(0.0) Prior fetus mortus, n (%) 0 2(4) 1(2) 1(2.9) 0(0.0) SLE, n (%) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 0(0.0) Sjo¨gren´s syndrome, n (%) 0(0.0) 1(2.0) 0(0.0) 0(0.0) 0(0.0) aDifference across the groups p <0.001, no difference across the high-risk groups, median and IQR presented bDifference between LRW vs. HRW or HRPE, p<0.001 for both comparisons, median and IQR presented cDifference across the groups (p<0.001), no difference across the high-risk groups dDifference across the groups (p = 0.002), no difference across the high-risk groups eDifference across the groups (p = 0.041), no difference across the high-risk groups LRW = low-risk women, HRW = high-risk women, HRPE = high-risk women who developed pre-eclampsia, PEAGA = pre-eclamptic women with appropriate for gestational age fetuses, PESGA = pre-eclamptic women with small for gestational age fetuses, IQR = interquartile range, BMI = body mass index, SGA = small for gestational age, GDM = gestational diabetes, DM = diabetes mellitus, SLE = systemic lupus erythematosus https://doi.org/10.1371/journal.pone.0226520.t002 Longitudinal changes in plasma hemopexin and alfa-1-microglobulin concentrations during pregnancy PLOS ONE | https://doi.org/10.1371/journal.pone.0226520 December 16, 2019 6 / 17 Table 3. Pregnancy characteristics of study groups. Pregnancy characteristics LRW N = 51 HRW N = 49 HRPE N = 42 PEAGA n = 33 PESGA n = 9 Weight gain during pregnancy, kg(%) a 14.8(5.5) 11.7(9.0) 12.0(6.6) 12.3 (5.4) 11.1(10.4) Gestational diabetes, n(%) b 4 (7.8) 13(26.5) 12(28.6) 10 (30.3) 2(22.2) I trimester mean UTA PI 0.93(0.26) c 1.07(0.40) d 1.25(0.47) e 1.19(0.48) f 1.41(0.42) g I trimester MAP, mmHg h 86.3(7.7) 97.0 (11.4) 101.6(13.3) 100.6 (13.0) 105.2(14.5) Highest MAP, mmHg i 94.0(9.7) 105.0(12.7) 127.8(10.8) 128.3(16.7) 128.7(14.5) Highest proteinuria, g/day j 0 0 1.5(2.3) 1.06(2.18) 2.30(5.20) Gestational weeks at birth k 40.6(1.3) 39.9 (2.1) 38.4(3.2) 38.7(2.4) 31.9(5.6) Mode of delivery, n (%) Vaginal 33 (65.0) 34 (69.4) 26 (61.9) 20 (61.0) 6 (66.7) Vacuum extraction k 8 (16.0) 0 (0.0) 6 (14.3) 5 (15.2) 1 (11.1) Elective cesarean section 1 (2.0) 3 (6.1) 1 (2.4) 0 (0.0) 1 (11.1) Cesarean section during labor 8 (16.0) 12 (24.5) 8 (19.0) 7 (21.2) 1 (11.1) Umbilical artery pH l 7.24 (0.09) 7.27(0.08) 7.27(0.08) 7.24(0.09) 7.22(0.11) Newborn birthweight, g m 3524 (582) 3690(712) 3109(1259) 3370(687) 1358(1125) Newborn birthweight, SD score n -0.11 0.38 -0.78 -0.30 -2.51 Placental weight, g o 594(111) 641(128) 537(158) 582(143) 367(71) LRW = low-risk women, HRW = high-risk women, HRPE = high-risk women who developed pre-eclampsia, PEAGA = women, who developed pre-eclampsia and had appropriate for gestational age fetuses, PESGA = women, who developed pre-eclampsia and had small for gestational age fetuses, UTA PI = uterine artery pulsatility index, MAP = mean arterial pressure, IQR = interquartile range, SD = standard deviation aDifference across the groups (p = 0.015), no difference across the high-risk groups, median and IQR presented bDifference across the groups (p = 0.025), no difference across the high-risk groups cDiffered from dand e, p<0.001 and from f(p = 0.006) and g(p = <0.001), fdiffered from g(p = 0.018), median and IQR presented hDifference across the groups (p<0.001), no difference across the high-risk groups, mean and SD presented iDifference between the groups (p<0.001) except between AGAPE vs. SGAPE, median and IQR presented jNo difference between AGAPE with SGAPE, median and IQR presented khigh-risk controls differed from low-risk women and high-risk women with subsequent PE, p = 0.006 lDifference between the groups (the highest p = 0.018) except between LRW vs. HRW, median and IQR presented mNo difference between the groups, mean and SD presented nHRPE differed from HRW and LRW, p<0.001 and p = 0.003, respectively. SGAPE differed from LRW, HRW and AGASGA (all p-values <0.001) and AGAPE differed from HRW (p = 0.018), median and IQR presented oHRPE differed from LRW and HRW (p = 0.015 and p<0.001, respectively), SGAPE differed from other groups (p<0.001), mean presented pDifference between HRW and HRPE ((p = 0.001), SGAPE differed from other groups (p<0.001), mean and SD presented https://doi.org/10.1371/journal.pone.0226520.t003 Table 4. Distribution of pre-eclampsia phenotypes. Women with SGA fetus n Women with AGA fetus n Severe EOPE 7 Severe EOPE 3 Non-severe EOPE 0 Non-severe EOPE 1 Severe LOPE 1 Severe LOPE 10 Non-severe LOPE 1 Non-severe LOPE 19 Total 9 33 SGA = small for gestational age, AGA = appropriate for gestational age, n = number of women, EOPE = early-onset pre-eclampsia, LOPE = late-onset pre-eclampsia https://doi.org/10.1371/journal.pone.0226520.t004 Longitudinal changes in plasma hemopexin and alfa-1-microglobulin concentrations during pregnancy PLOS ONE | https://doi.org/10.1371/journal.pone.0226520 December 16, 2019 7 / 17 Correlation and covariates The Hpx and A1M concentrations of LRW at 12–14 GW correlated positively, after logarithmic normalization of the data, (Pearson r = 0.33, p = 0.022), as did they in the HRW group at 18–20 GW (Pearson r = 0.30, p = 0.039). Hpx and A1M concentrations did not correlate in other respects. The BMI was a significant covariate for the Hpx (main effect BMI, p = 0.001 in the RMANOVA model, Pearson r = 0.20, p<0.001), but not for the A1M concentration. Therefore, the Hpx concentration was adjusted for BMI. Also other covariates were tested: Hpx and A1M concentrations were adjusted for maternal age, ASA usage, gestational diabetes, MAP in the first trimester and mean uterine artery pulsatility index. None of the tested variables turned out to be a significant covariate for Hpx or for A1M. Hemopexin and alpha-1-microglobulin concentrations At first trimester sampling the mean GW was 13.0 (11.9–15.9), at second sampling 19.4 GW (18.0–21.3) and at third sampling 27.1 GW (25.0–28.7). The geometric mean plasma concentrations of Hpx, Hpx adjusted for BMI and A1M are shown in Tables 5A, 5B and 6, respectively. Hemopexin All pre-eclampsia vs. controls. There was a difference in the plasma Hpx concentration and the change of the Hpx concentration between the three study groups, main effect Group, p = 0.002 and interaction effect Time x group, p = 0.021 (Table 5A,Fig 2A), and the result remained significant after adjustment for BMI, main effect Group, p = 0.004 and interaction effect Time x group, p = 0.010 (Table 5B and Fig 2B). When adjusted for BMI, the plasma concentration of hemopexin in the LRW and HRPE groups changed similarly from 12–14 to 26– 28 weeks of gestation (main effect Group, p = 0.374, interaction effect Time x group, p = 0.063) while the Hpx concentration of HRW was at the same level throughout the study period and therefore differed from the Hpx concentration of LRW and HRPE (interaction effect Time x group, p = 0.006, Fig 2B). The level of plasma Hpx in the HRW group during the first half of the pregnancy (from 12– 14 to 18–20 GW) was lower than in the LRW and HRPE groups (main effect Group, p = 0.001, Table 5A,Fig 2A). This difference persisted after adjustment for BMI (main effect Group, p<0.001, Table 5B,Fig 2B). After midgestation, the mean Hpx concentration decreased in the LRW group (-0.16 mg/ ml) and increased slightly in the HRW group (0.01 mg/ml). These changes differed significantly from each other (p = 0.012) over time (between 18–20 and 26–28 GW). During the same period, the mean Hpx concentrations were higher in the HRPE than the LRW group (main effect Group, p = 0.015, changes in Hpx concentration -0.05 vs. -0.16, respectively, p = 0.056) and the HRW group (main effect Group, p = 0.039). The changes in Hpx concentrations did not differ between HRPE and HRW after midgestation (-0.05 vs. 0.01, respectively, p = 1.000, Table 5A,Fig 2A). After adjustment for BMI there was no difference in the Hpx concentration between LRC and HRPE. Small for gestational age and appropriate for gestational age (Table 5). The mean plasma Hpx concentration in the PESGA group decreased over time from 12–14 to 26–28 GW, but in the PEAGA group it remained at the same level from 12–14 to 18–20 GW and then decreased from 18–20 to 26–28 GW. These differences between PESGA and PEAGA were not statistically significant (main effect Group, p = 0.449, interaction effect Time x group, p = 0.449). Longitudinal changes in plasma hemopexin and alfa-1-microglobulin concentrations during pregnancy PLOS ONE | https://doi.org/10.1371/journal.pone.0226520 December 16, 2019 8 / 17 When analyzed separately at each sampling point, the Hpx concentration in the PEAGA group was higher than in the HRW group (1.27 vs. 1.11 mg/ml, respectively, p = 0.020) at 18– 20 GW. At 26–28 GW PEAGA had a higher Hpx concentration than LRW (1.22 vs. 1.03 mg/ ml, respectively, p = 0.004). Early-onset and late-onset pre-eclampsia (Table 5). Although the Hpx concentrations of high-risk women who developed late-onset PE compared to high-risk women who developed early-onset PE were constantly higher at each sampling point, the difference was significant neither before (main effect Group p = 0.575 and interaction effect Time x group p = 0.998) nor after adjustment for BMI (p = 0.574 and p = 0.996, respectively). When analyzed separately at each sampling point, the Hpx concentrations of women who developed late-onset PE was higher than in HRW at 12–14 GW (1.28 vs. 1.12 mg/ml, respectively, p = 0.034) and at 18–20 GW (1.26 vs. 1.11 mg/ml, respectively, p = 0.041). The Hpx concentrations of women who developed late-onset PE was higher than in the LRW group at 26– 28 GW (1.21 vs. 1.03, respectively, p = 0.011). Table 5. Geometric mean plasma hemopexin concentrations (mg/ml) by subgroups at three different sampling points during pregnancy. A before adjustment for body mass index, B after adjustment for body mass index. Table 5. A Group 12–14 GW 95% CI 18–20 GW 95% CI 26–28�GW 95% CI Hpx mg/ml Lower Bound Upper Bound Hpx mg/ml Lower Bound Upper Bound Hpx mg/ml Lower Bound Upper Bound LRW N = 51 1.20 1.13 1.28 1.19 1.12 1.26 1.03 e 0.97 1.09 HRW N = 49 1.12 a 1.06 1.19 1.11 b 1.05 1.18 1.13 1.06 1.20 HRPE N = 42 1.27 1.19 1.36 1.25 1.17 1.33 1.20 f 1.12 1.29 PESGA n = 9 1.29 1.11 1.49 1.17 1.02 1.34 1.12 0.97 1.31 PEAGA n = 33 1.27 1.18 1.36 1.27 c 1.19 1.36 1.22 g 1.13 1.32 EOPE n = 11 1.23 1.08 1.40 1.22 1.08 1.38 1.17 1.02 1.35 LOPE n = 31 1.28 a 1.19 1.39 1.26 d 1.18 1.36 1.21 h 1.12 1.31 SEV n = 21 1.28 1.17 1.41 1.26 1.16 1.38 1.18 1.07 1.31 MILD n = 21 1.26 1.15 1.38 1.24 1.13 1.35 1.22 i 1.11 1.35 Table 5. B Group 12–14 GW 95% CI 18–20 GW 95% CI 26–28�GW 95% CI Hpx_A mg/ml Lower Bound Upper Bound Hpx_A mg/ml Lower Bound Upper Bound Hpx_A mg/ml Lower Bound Upper Bound LRW N = 51 1.24 1.17 1.32 1.23 1.17 1.31 1.05 0.99 1.12 HRW N = 49 1.11 1.04 1.17 1.10 1.03 1.16 1.11 1.05 1.18 HRPE N = 42 1.24 1.13 1.31 1.23 1.15 1.31 1.18 1.10 1.26 PESGA n = 9 1.26 1.09 1.47 1.15 1.00 1.31 1.10 0.95 1.28 PEAGA n = 33 1.24 1.14 1.33 1.26 1.17 1.34 1.20 1.11 1.30 EOPE n = 11 1.21 1.06 1.38 1.19 1.06 1.35 1.15 1.00 1.33 LOPE n = 31 1.25 1.16 1.36 1.24 1.16 1.34 1.19 1.09 1.29 SEV n = 21 1.28 1.16 1.40 1.26 1.15 1.37 1.17 1.07 1.30 MILD n = 21 1.20 1.09 1.33 1.21 1.10 1.32 1.17 1.06 1.30 �Published earlier as medians [34]. GW = week of gestation, CI = confidence interval, Hpx = mean hemopexin concentration, A_Hpx = mean hemopexin concentration after adjustment for BMI, LRW = low-risk women, controls HRW = high-risk women, controls, HRPE = high-risk women who developed pre-eclampsia, EOPE = women who developed early-onset pre-eclampsia, LOPE = women, who developed late-onset pre-eclampsia, SEV = women, who had severe pre-eclampsia, MILD = women, who had non-severe (mild) pre-eclampsia, PESGA = women who developed pre-eclampsia and gave birth to an small for gestational age infant, PEAGA = women who developed pre-eclampsia and gave birth to an appropriate for gestational age infant ap = 0.034 blower than cp = 0.020 and dp = 0.041, elower than f= 0.003, gp = 0.004, hp = 0.011 and ip = 0.029 https://doi.org/10.1371/journal.pone.0226520.t005 Longitudinal changes in plasma hemopexin and alfa-1-microglobulin concentrations during pregnancy PLOS ONE | https://doi.org/10.1371/journal.pone.0226520 December 16, 2019 9 / 17 22. 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