Advertisement

Serum amyloid A1 and pregnancy zone protein in pregnancy complications and correlation with markers of placental dysfunction

  • Ingrid K. Fosheim
    Correspondence
    Corresponding author: Ingrid Knutsdotter Fosheim, MD. Preferred.
    Affiliations
    Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway (Drs Fosheim, Jacobsen, Sugulle, Alnaes-Katjavivi, Fjeldstad, Ueland, and Staff)

    Division of Obstetrics and Gynaecology, Oslo University Hospital, Oslo, Norway (Drs Fosheim, Jacobsen, Sugulle, Alnaes-Katjavivi, Fjeldstad, and Staff)
    Search for articles by this author
  • Daniel P. Jacobsen
    Affiliations
    Division of Obstetrics and Gynaecology, Oslo University Hospital, Oslo, Norway (Drs Fosheim, Jacobsen, Sugulle, Alnaes-Katjavivi, Fjeldstad, and Staff)
    Search for articles by this author
  • Meryam Sugulle
    Affiliations
    Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway (Drs Fosheim, Jacobsen, Sugulle, Alnaes-Katjavivi, Fjeldstad, Ueland, and Staff)

    Division of Obstetrics and Gynaecology, Oslo University Hospital, Oslo, Norway (Drs Fosheim, Jacobsen, Sugulle, Alnaes-Katjavivi, Fjeldstad, and Staff)
    Search for articles by this author
  • Patji Alnaes-Katjavivi
    Affiliations
    Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway (Drs Fosheim, Jacobsen, Sugulle, Alnaes-Katjavivi, Fjeldstad, Ueland, and Staff)

    Division of Obstetrics and Gynaecology, Oslo University Hospital, Oslo, Norway (Drs Fosheim, Jacobsen, Sugulle, Alnaes-Katjavivi, Fjeldstad, and Staff)
    Search for articles by this author
  • Heidi E.S. Fjeldstad
    Affiliations
    Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway (Drs Fosheim, Jacobsen, Sugulle, Alnaes-Katjavivi, Fjeldstad, Ueland, and Staff)

    Division of Obstetrics and Gynaecology, Oslo University Hospital, Oslo, Norway (Drs Fosheim, Jacobsen, Sugulle, Alnaes-Katjavivi, Fjeldstad, and Staff)
    Search for articles by this author
  • Thor Ueland
    Affiliations
    Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway (Drs Fosheim, Jacobsen, Sugulle, Alnaes-Katjavivi, Fjeldstad, Ueland, and Staff)

    Research Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway (Drs Ueland and Lekva)

    K.G. Jebsen Thrombosis Research and Expertise Center, University of Tromsø, Tromsø, Norway (Dr Ueland)
    Search for articles by this author
  • Tove Lekva
    Affiliations
    Research Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway (Drs Ueland and Lekva)
    Search for articles by this author
  • Anne C. Staff
    Affiliations
    Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway (Drs Fosheim, Jacobsen, Sugulle, Alnaes-Katjavivi, Fjeldstad, Ueland, and Staff)

    Division of Obstetrics and Gynaecology, Oslo University Hospital, Oslo, Norway (Drs Fosheim, Jacobsen, Sugulle, Alnaes-Katjavivi, Fjeldstad, and Staff)
    Search for articles by this author
Open AccessPublished:November 02, 2022DOI:https://doi.org/10.1016/j.ajogmf.2022.100794

      Highlights

      • Protein misfolding markers are for the first time assessed in placental dysfunction
      • Protein misfolding markers are dysregulated in early-onset preeclampsia
      • Protein misfolding markers correlate with proxies for placental dysfunction
      • The results implicate protein misfolding and amyloid in placental dysfunction

      BACKGROUND

      Hypertensive disorders of pregnancy (preeclampsia, gestational hypertension, and chronic hypertension), diabetes mellitus, and placental dysfunction confer an increased risk of long-term maternal cardiovascular disease. Preeclampsia is also associated with acute atherosis that involves lesions of uteroplacental spiral arteries, resembling early stages of atherosclerosis. Serum amyloid A1 is involved in hypercoagulability and atherosclerosis and may aggregate into amyloid—aggregations of misfolded proteins. Pregnancy zone protein may inhibit amyloid aggregation. Amyloid is involved in Alzheimer's disease and cardiovascular disease; it has been identified in preeclampsia, but its role in preeclampsia pathophysiology is unclear.

      OBJECTIVE

      We hypothesized that serum amyloid A1 would be increased and pregnancy zone protein decreased in hypertensive disorders of pregnancy and diabetic pregnancies and that serum amyloid A1 and pregnancy zone protein would correlate with placental dysfunction markers (fetal growth restriction and dysregulated angiogenic biomarkers) and acute atherosis.

      STUDY DESIGN

      Serum amyloid A1 is measurable in both the serum and plasma. In our study, plasma from 549 pregnancies (normotensive, euglycemic controls: 258; early-onset preeclampsia: 71; late-onset preeclampsia: 98; gestational hypertension: 30; chronic hypertension: 9; diabetes mellitus: 83) was assayed for serum amyloid A1 and pregnancy zone protein. The serum levels of angiogenic biomarkers soluble fms-like tyrosine kinase-1 and placental growth factor were available for 547 pregnancies, and the results of acute atherosis evaluation were available for 313 pregnancies. The clinical characteristics and circulating biomarkers were compared between the pregnancy groups using the Mann-Whitney U, chi-squared, or Fisher exact test as appropriate. Spearman's rho was calculated for assessing correlations.

      RESULTS

      In early-onset preeclampsia, serum amyloid A1 was increased compared with controls (17.1 vs 5.1 µg/mL, P<.001), whereas pregnancy zone protein was decreased (590 vs 892 µg/mL, P=.002). Pregnancy zone protein was also decreased in diabetes compared with controls (683 vs 892 µg/mL, P=.01). Serum amyloid A1 was associated with placental dysfunction (fetal growth restriction, elevated soluble fms-like tyrosine kinase-1 to placental growth factor ratio). Pregnancy zone protein correlated negatively with soluble fms-like tyrosine kinase-1 to placental growth factor ratio in all study groups. Acute atherosis was not associated with serum amyloid A1 or pregnancy zone protein.

      CONCLUSION

      Proteins involved in atherosclerosis, hypercoagulability, and protein misfolding are dysregulated in early-onset preeclampsia and placental dysfunction, which links them and potentially contributes to future maternal cardiovascular disease.

      Key words

      Why was this study conducted?

      Preeclampsia and placental dysfunction place women at risk of future cardiovascular disease, but the mechanisms are largely unknown. This study aimed at assessing biomarkers that may contribute to this risk.

      Key findings

      Circulating serum amyloid A1 and pregnancy zone protein—markers of protein misfolding— are dysregulated in early-onset preeclampsia, and their levels correlate with antiangiogenic biomarkers and fetal growth restriction, which are proxies for placental dysfunction.

      What does this add to what is known?

      Our finding of a correlation between protein misfolding markers and proxies for placental dysfunction is novel. Markers of protein misfolding are implicated in early-onset preeclampsia and placental dysfunction and may provide a pathway linking these pregnancy complications to the epidemiologic increased risk of maternal cardiovascular disease.

      Introduction

      Preeclampsia is a potentially severe pregnancy complication, defined as new-onset hypertension and at least 1 other preeclampsia-associated sign of organ dysfunction (eg, proteinuria, fetal growth restriction, and elevated liver transaminases) in the second half of pregnancy.
      • Brown MA
      • Magee LA
      • Kenny LC
      • et al.
      Hypertensive disorders of pregnancy: ISSHP classification, diagnosis, and management recommendations for international practice.
      Hypertensive disorders of pregnancy, including preeclampsia and other pregnancy complications associated with placental dysfunction (eg, fetal growth restriction, preterm birth, and gestational diabetes mellitus [GDM]) are associated with a 2–8-fold increased risk of future maternal cardiovascular disease.
      • Staff AC
      • Redman CW
      • Williams D
      • et al.
      Pregnancy and long-term maternal cardiovascular health: progress through harmonization of research cohorts and biobanks.
      • Irgens HU
      • Reisaeter L
      • Irgens LM
      • Lie RT.
      Long term mortality of mothers and fathers after pre-eclampsia: population based cohort study.
      • Wu P
      • Haththotuwa R
      • Kwok CS
      • et al.
      Preeclampsia and future cardiovascular health: a systematic review and meta-analysis.
      Pregnancy can thus be regarded as a sex-specific stress test for predicting maternal risk of cardiovascular disease. Cardiovascular diseases remain a major cause of death for both men and women, but female-specific pathophysiological mechanisms have so far been understudied.
      • Vogel B
      • Acevedo M
      • Appelman Y
      • et al.
      The Lancet Women and Cardiovascular Disease Commission: reducing the global burden by 2030.
      The maternal features of preeclampsia result from excessive systemic vascular inflammation secondary to increased shedding of proinflammatory factors from a malperfused and dysfunctional placenta.
      • Staff AC.
      The two-stage placental model of preeclampsia: an update.
      ,
      • Redman CWG
      • Staff AC
      • Roberts JM.
      Syncytiotrophoblast stress in preeclampsia: the convergence point for multiple pathways.
      Placental malperfusion leads to oxidative and endoplasmic reticulum stress, eliciting the release of proinflammatory and antiangiogenic factors.
      • Burton GJ
      • Yung HW
      • Cindrova-Davies T
      • Charnock-Jones DS
      Placental endoplasmic reticulum stress and oxidative stress in the pathophysiology of unexplained intrauterine growth restriction and early onset preeclampsia.
      Placental dysfunction in early-onset preeclampsia is associated with insufficient physiological spiral artery transformation.
      • Staff AC.
      The two-stage placental model of preeclampsia: an update.
      ,
      • Brosens IA
      • Robertson WB
      • Dixon HG.
      The role of the spiral arteries in the pathogenesis of preeclampsia.
      Both early- and late-onset preeclampsia are associated with excessive placental senescence and high rates of uteroplacental acute atherosis.
      • Staff AC.
      The two-stage placental model of preeclampsia: an update.
      ,
      • Kwiatkowski S
      • Dołęgowska B
      • Kwiatkowska E
      • et al.
      Do the physiological aging of the placenta and the changes in angiogenesis marker sFlt-1 and PlGF concentrations predispose patients to late-onset preeclampsia?.
      ,
      • Mayne BT
      • Leemaqz SY
      • Smith AK
      • Breen J
      • Roberts CT
      • Bianco-Miotto T.
      Accelerated placental aging in early onset preeclampsia pregnancies identified by DNA methylation.
      Acute atherosis is a spiral artery wall lesion with some morphologic similarities to the early stages of atherosclerosis.
      • Staff AC
      • Fjeldstad HE
      • Fosheim IK
      • et al.
      Failure of physiological transformation and spiral artery atherosis: their roles in preeclampsia.
      ,
      • Pitz Jacobsen DP
      • Fjeldstad HE
      • Johnsen GM
      • et al.
      Acute atherosis lesions at the fetal-maternal border: current knowledge and implications for maternal cardiovascular health.
      It is characterized by subintimal foam cells, artery wall necrosis, and inflammation and is associated with thrombosis and a risk of downstream placental infarcts.
      • Robertson WB
      • Brosens I
      • Dixon HG.
      The pathological response of the vessels of the placental bed to hypertensive pregnancy.
      Circulating proangiogenic placental growth factor (PlGF) and antiangiogenic soluble fms-like tyrosine kinase-1 (sFlt-1) are mainly of placental origin during pregnancy and are dysregulated in preeclampsia and other placental dysfunction syndromes such as fetal growth restriction.
      • Maynard SE
      • Min JY
      • Merchan J
      • et al.
      Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia.
      ,
      • Shinohara S
      • Uchida Y
      • Kasai M
      • Sunami R.
      Association between the high soluble fms-like tyrosine kinase-1 to placental growth factor ratio and adverse outcomes in asymptomatic women with early-onset fetal growth restriction.
      Consequently, we have argued that dysregulated angiogenic biomarkers (eg, low PlGF, high sFlt-1/PlGF ratio) may be utilized as proxy markers for placental dysfunction and syncytiotrophoblast stress.
      • Redman CWG
      • Staff AC
      • Roberts JM.
      Syncytiotrophoblast stress in preeclampsia: the convergence point for multiple pathways.
      Amyloid consists of fibrous aggregates of misfolded proteins, has slow spontaneous dissociation,
      • Sack Jr., GH
      Serum amyloid A (SAA) proteins.
      and is implicated in several chronic, progressive diseases, including Alzheimer's disease, type 2 diabetes mellitus (DM), and atherosclerosis.
      • Chiti F
      • Dobson CM.
      Protein misfolding, amyloid formation, and human disease: a summary of progress over the last decade.
      ,
      • Teoh CL
      • Griffin MD
      • Howlett GJ.
      Apolipoproteins and amyloid fibril formation in atherosclerosis.
      Amyloid also accumulates in the urine and placenta of women with preeclampsia.
      • Buhimschi IA
      • Nayeri UA
      • Zhao G
      • et al.
      Protein misfolding, congophilia, oligomerization, and defective amyloid processing in preeclampsia.
      ,
      • Kalkunte SS
      • Neubeck S
      • Norris WE
      • et al.
      Transthyretin is dysregulated in preeclampsia, and its native form prevents the onset of disease in a preclinical mouse model.
      Circulating amyloid precursors transthyretin and amyloid precursor protein are dysregulated in preeclampsia.
      • Buhimschi IA
      • Nayeri UA
      • Zhao G
      • et al.
      Protein misfolding, congophilia, oligomerization, and defective amyloid processing in preeclampsia.
      • Kalkunte SS
      • Neubeck S
      • Norris WE
      • et al.
      Transthyretin is dysregulated in preeclampsia, and its native form prevents the onset of disease in a preclinical mouse model.
      • Cheng S
      • Banerjee S
      • Daiello LA
      • et al.
      Novel blood test for early biomarkers of preeclampsia and Alzheimer's disease.
      Urine testing for amyloid has been suggested as a test for preeclampsia severity.
      • Valtanen RS
      • Buhimschi CS
      • Bahtiyar MO
      • et al.
      Conformation-dependent anti-Aβ monoclonal antibody signatures of disease status and severity in urine of women with preeclampsia.
      Serum amyloid A1 (SAA1) is an acute-phase protein mainly produced in the liver and is one of many proteins able to aggregate into amyloid.
      • Sack Jr., GH
      Serum amyloid A (SAA) proteins.
      Placental expression of SAA1 may play a role in initiating parturition, though its expression throughout pregnancy is uncertain.
      • Gan XW
      • Wang WS
      • Lu JW
      • et al.
      De novo synthesis of SAA1 in the Placenta Participates in parturition.
      SAA1 is also implicated in hypercoagulability by promoting amyloid formation in fibrin(ogen) and driving platelets into a prothrombotic state.
      • Page MJ
      • Thomson GJA
      • Nunes JM
      • et al.
      Serum amyloid A binds to fibrin(ogen), promoting fibrin amyloid formation.
      Pregnancy zone protein (PZP) is a plasma protein that is highly up-regulated during pregnancy,
      • Ekelund L
      • Laurell CB.
      The pregnancy zone protein response during gestation: a metabolic challenge.
      and it may play a progestational role through immunomodulation.
      • Wyatt AR
      • Cater JH
      • Ranson M.
      PZP and PAI-2: structurally diverse, functionally similar pregnancy proteins?.
      Further, low circulating levels of the protein have been linked to pregnancy loss.
      • Than GN
      • Csaba IF
      • Szabó DG
      • Karg NJ
      • Novák PF.
      Quantitative immunological study of pregnancy-associated alpha2-globulin antigen.
      In vitro, PZP stabilizes and inhibits misfolded amyloid-β from aggregating into amyloid fibrils.
      • Cater JH
      • Kumita JR
      • Zeineddine Abdallah R
      • et al.
      Human pregnancy zone protein stabilizes misfolded proteins including preeclampsia- and Alzheimer's-associated amyloid beta peptide.
      Whether PZP inhibits SAA1 aggregation into amyloid-A—the amyloid type derived from SAA1—is unknown.
      Circulating SAA1 has been shown to be increased in GDM
      • Eren MA
      • Vural M
      • Cece H
      • et al.
      Association of serum amyloid A with subclinical atherosclerosis in women with gestational diabetes.
      and has also been studied in preeclampsia, though results from these predominantly small studies are conflicting.
      • Engin-Ustün Y
      • Ustün Y
      • Karabulut AB
      • Ozkaplan E
      • Meydanli MM
      • Kafkasli A.
      Serum amyloid A levels are increased in pre-eclampsia.
      • Kristensen K
      • Wide-Swensson D
      • Lindstrom V
      • Schmidt C
      • Grubb A
      • Strevens H.
      Serum amyloid A protein and C-reactive protein in normal pregnancy and preeclampsia.
      • Can M
      • Sancar E
      • Harma M
      • Guven B
      • Mungan G
      • Acikgoz S.
      Inflammatory markers in preeclamptic patients.
      • Reimer T
      • Rohrmann H
      • Stubert J
      • et al.
      Angiogenic factors and acute-phase proteins in serum samples of preeclampsia and HELLP patients: a matched-pair analysis.
      • Güngör ZB
      • Tüten A
      • Ekmekçi H
      • et al.
      Possible effects of lipoprotein-associated phospholipase A2 single-nucleotide polymorphisms on cardiovascular risk in patients with preeclampsia.
      • Uckan K
      • Sahin HG.
      Serum amyloid A, procalcitonin, highly sensitive C reactive protein and tumor necrosis factor alpha levels and acute inflammatory response in patients with hemolysis, elevated liver enzymes, low platelet count (HELLP) and eclampsia.
      Derived PZP levels were decreased in women with preeclampsia in 2 small studies.
      • Horne CH
      • Briggs JD
      • Howie PW
      • Kennedy AC.
      Serum -macroglobulins in renal disease and preeclampsia.
      ,
      • Griffin JF.
      Pregnancy-associated plasma protein levels at term in normal pregnancy, preeclampsia and essential hypertension.
      These previous SAA1 and PZP studies from pregnancy have not assessed biomarker concentrations in relation to placental dysfunction.
      As SAA1 and PZP have opposite roles in augmenting and attenuating amyloid aggregation, we hypothesized that circulating SAA1 is increased and PZP is decreased in preeclampsia, particularly in early-onset disease and in pregnancies complicated by diabetes. We further hypothesized that SAA1 and PZP levels correlate with proxies for placental dysfunction (eg, dysregulated angiogenic biomarkers, fetal growth restriction, or acute atherosis) as an indication of amyloid deposition in placental dysfunction.

      Materials and Methods

      Patient recruitment and sample collection

      Data and biological samples from 549 singleton pregnant women recruited between 2001 and 2017 to the Oslo Pregnancy Biobank at Oslo University Hospital were included in this study. All had extensive clinical data from pregnancy and delivery and blood samples available for biomarker analyses. All women gave informed written consent. The study was approved by the Regional committee for Medical and Health Research Ethics in South-Eastern Norway and conducted in accordance with the principles of the Helsinki Declaration.
      Hypertensive disorders of pregnancy (HDP) were defined according to the 2018 Guidelines of the International Society for the Study of Hypertension in Pregnancy.
      • Brown MA
      • Magee LA
      • Kenny LC
      • et al.
      Hypertensive disorders of pregnancy: ISSHP classification, diagnosis, and management recommendations for international practice.
      Gestational hypertension (GH) was defined as new-onset hypertension (blood pressure ≥140 mm Hg systolic and/or ≥90 mm Hg diastolic on ≥2 occasions ≥6 hours apart) at ≥20 weeks’ gestation. Preeclampsia was defined as GH accompanied by ≥1 other new-onset sign(s) of maternal organ dysfunction
      • Brown MA
      • Magee LA
      • Kenny LC
      • et al.
      Hypertensive disorders of pregnancy: ISSHP classification, diagnosis, and management recommendations for international practice.
      (eg, proteinuria, acute kidney injury, liver involvement, eclampsia, hemolysis, and fetal growth restriction) at ≥20 weeks’ gestation. Early-onset preeclampsia (EO-PE) and late-onset preeclampsia (LO-PE) were defined as preeclampsia delivered <34 vs ≥34 weeks’ gestation, respectively.
      • Staff AC.
      The two-stage placental model of preeclampsia: an update.
      Chronic hypertension (CHT) was defined as hypertension occurring prepregnancy or diagnosed <20 weeks’ gestation. Superimposed preeclampsia was defined as CHT and ≥1 new-onset preeclampsia-associated feature(s) at ≥20 weeks’ gestation. DM diagnoses (type 1 DM [DM1], type 2 DM [DM2], gestational DM [GDM]) were based on diagnoses identified in the clinical setting by endocrinologists according to current guidelines.

      World Health Organization. (1999). Definition, diagnosis and classification of diabetes mellitus and its complications: report of a WHO consultation. Part 1, Diagnosis and classification of diabetes mellitus. World Health Organization.

      ,

      Friis CM, Roum EMS, Holm HO, Toft JH, Roland MCP, Thordarson HB. Svangerskapsdiabetes. Norsk gynekologisk forening Veileder i fødselshjelp. 2020. Available at:https://www.legeforeningen.no/foreningsledd/fagmed/norsk-gynekologisk-forening/veiledere/veileder-i-fodselshjelp/svangerskapsdiabetes/. Accessed March 24, 2022.

      Women with both an HDP and DM diagnosis were classified as HDP+DM. Controls were women who remained normotensive and euglycemic throughout pregnancy.
      None of the included women had regular uterine contractions, ruptured fetal membranes, or signs of infection at the time of blood sampling. None had known chronic diseases apart from women with CHT (n=25), pregestational DM (n=79), or hypothyroidism (n=33). No controls had CHT or DM. Gestational age at inclusion was determined by routine ultrasound screening at gestational week 17 to 20, except in 25 women, where gestational age was determined by embryo transfer date (in vitro fertilization) and in 7 women by first day of the last menstrual period before pregnancy. Sex-specific newborn weight percentiles were calculated according to Norwegian fetal growth curves.
      • Johnsen SL
      • Rasmussen S
      • Wilsgaard T
      • Sollien R
      • Kiserud T.
      Longitudinal reference ranges for estimated fetal weight.
      Fetal growth restriction was defined as sex-specific newborn weight ≤third percentile. No newborns had structural or apparent chromosomal abnormalities.
      The presence or absence of uteroplacental acute atherosis was assessed in decidua basalis vacuum suction biopsies collected after delivery of the placenta and evaluated as described previously.
      • Harsem NK
      • Staff AC
      • He L
      • Roald B.
      The decidual suction method: a new way of collecting decidual tissue for functional and morphological studies.
      Acute atherosis was defined as spiral arteries with ≥2 adjacent CD68-positive vacuolated subintimal cells.
      • Alnaes-Katjavivi P
      • Lyall F
      • Roald B
      • Redman CW
      • Staff AC.
      Acute atherosis in vacuum suction biopsies of decidua basalis: an evidence based research definition.
      For most women, blood samples were obtained immediately before elective cesarean delivery and centrifuged within 120 minutes of sampling at 4°C for 10 minutes at 1800 g. Ethylenediaminetetraacetic acid (EDTA) plasma was stored at −80°C until immunoassay. For some of the included women (n=81), a blood sample was collected 1–31 days before delivery instead of at delivery. Blood was sampled from an antecubital vein or intravenous cannula without ongoing infusion or from an arterial catheter (n=20). Maternal serum samples were also collected at the same time point as EDTA plasma as described previously.
      • Moe K
      • Heidecke H
      • Dechend R
      • Staff AC.
      Dysregulation of circulating autoantibodies against VEGF-A, VEGFR-1 and PlGF in preeclampsia - a role in placental and vascular health?.

      Biochemistry and immunoassays

      The SAA1 and PZP concentrations were measured by enzyme-linked immunosorbent assay (ELISA) in maternal EDTA plasma in duplicates. The mean value of the duplicates was used for analysis. All reagents were obtained from R&D Systems (Minneapolis, MN) catalog numbers DY3019-05 (SAA1) and DY8280-05 (PZP). Assays were performed in accordance with the manufacturer's instructions. Optical density was determined at 450 nm and corrected at 540 nm. The coefficients of variation were 6.4% for SAA1 and 2.4% for PZP.
      Serum levels of sFlt-1 and PlGF were determined for 547 pregnancies as previously described
      • Moe K
      • Heidecke H
      • Dechend R
      • Staff AC.
      Dysregulation of circulating autoantibodies against VEGF-A, VEGFR-1 and PlGF in preeclampsia - a role in placental and vascular health?.
      using Elecsys immunoassays (Roche Diagnostics, Switzerland) with a fully automated electrochemiluminescence immunoassay platform (Cobas E analyzer, Roche Diagnostics).
      Serum levels of acute-phase reactant high-sensitivity C-reactive protein (hsCRP) in either fresh (n=219) or thawed (n=161) samples were analyzed as described previously
      • Moe K
      • Alnaes-Katjavivi P
      • Størvold GL
      • et al.
      Classical cardiovascular risk markers in pregnancy and associations to uteroplacental acute atherosis.
      using a particle-enhanced turbidimetric method (Cobas 8000 c702; Roche Diagnostics, Rotkreuz, Switzerland). hsCRP was included in this biomarker study owing to its known association to cardiovascular diseases
      • Koenig W.
      High-sensitivity C-reactive protein and atherosclerotic disease: from improved risk prediction to risk-guided therapy.
      and preeclampsia.
      • Braekke K
      • Holthe MR
      • Harsem NK
      • Fagerhol MK
      • Staff AC.
      Calprotectin, a marker of inflammation, is elevated in the maternal but not in the fetal circulation in preeclampsia.
      CRP correlates with SAA outside pregnancy,
      • Ridker PM
      • Rifai N
      • Pfeffer MA
      • et al.
      Inflammation, pravastatin, and the risk of coronary events after myocardial infarction in patients with average cholesterol levels. Cholesterol and recurrent events (CARE) Investigators.
      whereas a correlation in pregnancy has been less studied.
      • Can M
      • Sancar E
      • Harma M
      • Guven B
      • Mungan G
      • Acikgoz S.
      Inflammatory markers in preeclamptic patients.

      Statistics

      The data were analyzed using SPSS Statistics 26.0 (IBM). The Mann-Whitney U test was used for continuous variables. The chi-squared or Fisher exact test was used for categorical variables, as appropriate. Spearman rho (rS) was calculated for analyzing correlations between continuous variables, as data were skewed. We did not correct for multiple comparisons. A P value <.05 was considered significant.

      Results

      Table lists the clinical characteristics of the main pregnancy groups (controls, n=258; HDP, n=163; DM, n=83; HDP+DM, n=45), acute atherosis rate, and circulating biomarker concentrations. Prepregnancy body mass index (BMI), mean blood pressures in the first half of pregnancy and at inclusion, rates of acute atherosis, hsCRP, and sFlt-1/PlGF ratio were increased, and gestational age and newborn weight percentiles were lower in HDP and HDP+DM than in controls. Supplemental Tables 1 and 2 list the number of women in each HDP and DM subgroup, respectively, and their clinical characteristics.
      There were no significant differences in SAA1 or PZP levels or clinical characteristics when comparing the group with samples collected 1 to 31 days before delivery with the group with samples collected at cesarean delivery (data not shown).

      Serum amyloid A1 and pregnancy zone protein concentrations in hypertensive and diabetic pregnancies

      As shown in Table 1, the plasma SAA1 levels were markedly higher in HDP than in controls (normotensive and euglycemic, P<.001), whereas SAA1 did not differ between DM or HDP+DM compared with controls. Of the HDP subgroups, the EO-PE group had increased levels of SAA1 than the controls (P<.001, Supplemental Table 1) and than all remaining subgroups of HDP (Figure 1, A). SAA1 did not differ between any DM subgroup and controls (normotensive and euglycemic, Supplemental Table 2).
      Table 1Clinical characteristics and circulating biomarkers by pregnancy inclusion groups (total cohort, n=549)
      Fosheim. Markers of protein misfolding in pregnancy complications. Am J Obstet Gynecol MFM 2022.
      VariableControls (n=258)HDP (n=163)P valueDM (n=83)P valueHDP+DM (n=45)P value
      Maternal age at inclusion (y)33.5 (20.1–45.5)33.1 (19.0–52.9).2533.4 (24.1–45.9).6433.0 (21.1–44.0).22
      Prepregnancy body mass index (kg/m2)22.4 (17.6–39.5)24.0 (18.3–41.1)<.00124.5 (19.1–39.4)<.00127.3 (19.6–41.1)<.001
      Mean SBP <20 wk (mm Hg)110 (85–140)120 (90–158)<.001114 (92–138)<.001120 (95–164)<.001
      Mean DBP <20 wk (mm Hg)68 (40–88)75 (55–103)<.00169 (50–84).2173 (50–90)<.001
      First pregnancy24%45%<.00122%.6142%.01
      First delivery39%62%<.00141%.7753%.07
      Previous preeclampsia, n (%)a12 (8%)26 (43%)<.0017 (14%).179 (43%)<.001
      SBP at inclusion (mm Hg)121 (86–156)151 (117–220)<.001120 (90–155).77150 (120–189)<.001
      DBP at inclusion (mm Hg)75 (48–110)95 (63–130)<.00174 (50–90).9492 (66–110)<.001
      Gestational age at inclusion (wk)39.0 (33.7–42.1)34.4 (24.3–41.1)<.00138.0 (28.7–40.4)<.00136.9 (29.0–40.0)<.001
      Preterm delivery (<37 wk gestation)3%65%<.00110%.0140%<.001
      Early-onset preeclampsia (delivery <34 wk gestation)48%16%
      Newborn weight (g)3483 (1325–5130)2087 (500–5040)<.0013802 (988–5857)<.0013574 (620–5424).70
      Newborn weight percentile62.6 (0–100)5.0 (0–99.9)<.00186.1 (0–100)<.00190.9 (0–100).01
      Fetal growth restriction2%45%<.0015%.104%.22
      Acute atherosisb9% (14/160)39% (37/96)<.0017% (2/31)1.0027% (7/26).01
      Serum-sFlt-1 (pg/mL)c3683 (1081–17,185)11,204 (1429–61,348)<.0014629 (1260–18,857).0037597 (2003–21,430)<.001
      Serum-PlGF (pg/mL)c172 (23–2074)58 (6–624)<.001166 (35–1027).82104 (29–1852)<.001
      sFlt-1/PlGF ratioc22.3 (1.1–615.1)208.6 (2.3–3264.3)<.00124.9 (2.7–209.0).2478.9 (1.4–470.8)<.001
      Serum-hsCRP (mg/L) (ref.: 0.0–4.0)d3.1 (0.6–76.6), n=2014.4 (0.6–107.0), n=117.0023.9 (0.6–32.7), n=36.264.8 (0.8–15.9), n=26.04
      Plasma-SAA1 (µg/mL)5.08 (0.41–120)9.90 (0.80–120)<.0014.06 (0.37–120).066.07 (0.22–115.95).22
      Plasma-PZP (µg/mL)892 (32–3240)818 (12–2992).15683 (40–3406).01548 (25–3280).01
      For continuous variables, the median value and range are shown; for categorical variables, percentages are shown. Statistical differences were calculated by comparing each pregnancy group with the control group (normotensive and euglycemic pregnant women).
      DBP, diastolic blood pressure; DM, diabetes mellitus (pregestational or gestational); HDP, hypertensive disorders of pregnancy; HDP+DM, women with both HDP and DM; hsCRP, high-sensitivity C-reactive protein; PlGF, placental growth factor; PZP, pregnancy zone protein; SAA1, serum amyloid A1; SBP, systolic blood pressure; sFlt-1, soluble fms-like tyrosine kinase-1.
      aNulliparous women were excluded when analyzing the rate of previous preeclampsia
      bDecidua basalis acute atherosis was assessed in 313 women (all had spiral arteries present)
      csFlt-1 and PlGF concentrations were available for all except 2 women with HDP
      dhsCRP concentration was available for 380 women.
      Figure 1
      Figure 1Scatter plots of circulating biomarkers
      Fosheim. Markers of protein misfolding in pregnancy complications. Am J Obstet Gynecol MFM 2022.
      Concentrations of circulating biomarkers by subgroup of hypertensive disorder of pregnancy (irrespective of diabetes mellitus status) and controls (normotensive and euglycemic) (n=466). A, serum amyloid A1 (SAA1) concentrations; B, PZP concentrations. The Mann-Whitney U test was used to calculate statistical differences between groups. Line at median. Triple asterisks denote P<.001. Double asterisks denote P<.01. Single asterisk denotes P<.05
      CHT, chronic hypertension; EO-PE, early-onset preeclampsia (delivery <34 weeks gestation); GH, gestational hypertension; LO-PE, late-onset preeclampsia (delivery ≥34 weeks gestation); PZP, pregnancy zone protein.
      The PZP concentration was lower in both DM and in HDP+DM than in controls (both P=.01). In contrast, PZP was similar in HDP and controls. The levels of PZP were decreased in EO-PE than in controls (P=.002, Supplemental Table 1) and CHT (Figure 1, B). PZP was decreased in women with DM1 and DM2 than in controls (P=.02 and.003, respectively, Supplemental Table 2).

      Correlation between serum amyloid A1 and glycated hemoglobin in diabetic pregnancies

      In DM1, SAA1 (at sampling in late pregnancy) correlated negatively with glycated hemoglobin (HbA1c) measured in the first (rS=−0.43; P=.002), second (rS=−0.27; P=.04), and third (rS=−0.33; P=.01) trimesters. First trimester HbA1c ≥53 mmol/mol was associated with lower SAA1 (2.9 vs 5.0 µg/mL; P=.03). PZP did not correlate with HbA1c (data not shown).

      Serum amyloid A1 and pregnancy zone protein concentrations and their relationship with proxies for placental dysfunction

      We compared SAA1 and PZP levels with proxies for placental dysfunction. Women with acute atherosis did not differ in SAA1 or PZP levels compared with women without acute atherosis within any of the pregnancy groups (Supplemental Table 3). However, increased SAA1 levels were seen in pregnancies with fetal growth restriction, and they correlated positively with the antiangiogenic sFlt-1/PlGF ratio in the total cohort (13.3 vs 5.1 µg/mL, rS=0.24; both P<.001) and in HDP (13.7 vs 6.1 µg/mL, rS=0.40; both P<.001). PZP was not associated with fetal growth restriction but correlated negatively with sFlt-1/PlGF in the total cohort (rS=−0.13, P=.002), HDP (rS=−0.19; P=.015), and HDP+DM (rS=−0.30; P=.049).

      Correlations between serum amyloid A1, pregnancy zone protein, and high-sensitivity C-reactive protein

      Finally, we compared SAA1 and PZP with the general inflammation marker hsCRP. SAA1 correlated positively with hsCRP in the total cohort (rS=0.46; P<.001) and in all pregnancy groups: controls (rS=0.35; P<.001), HDP (rS=0.58; P<.001), DM (rS=0.35; P=.04), and HDP+DM (rS=0.62; P=.001). Moreover, SAA1 was increased in women with hsCRP above an upper reference limit of 4 mg/L (9.4 vs 4.3 µg/mL; P<.001). We found no correlation between PZP and hsCRP (data not shown). We found no correlation between SAA1 and PZP for the total cohort or for any pregnancy groups except for HDP+DM (rS=-0.37; P=.01).
      SAA1 and PZP concentrations did not consistently correlate with prepregnancy BMI, early pregnancy blood pressure, gestational age at sampling, or fetal sex in the total cohort or in any subgroups; the few exceptions are listed in Supplemental Results.

      Discussion

      Principal findings

      In line with our hypothesis, we found increased SAA1 and decreased PZP in pregnancies complicated by EO-PE. SAA1 and PZP correlated significantly with signs of placental dysfunction, as determined by fetal growth restriction and an antiangiogenic biomarker pattern. In contrast to our hypothesis, we did not find an association between acute atherosis and SAA1 or PZP.

      Results

      Our finding of increased circulating SAA1 in EO-PE may indicate the presence of amyloid-A in these women. Amyloid-A deposits are typically found in chronic inflammatory diseases,
      • Westermark GT
      • Fändrich M
      • Westermark P.
      AA amyloidosis: pathogenesis and targeted therapy.
      as prolonged inflammation (demonstrated by CRP elevation) causing persistently elevated SAA1 may lead to amyloid-A deposits.
      • Sack Jr., GH
      Serum amyloid A (SAA) proteins.
      Other types of amyloid (amyloid-β and transthyretin) have been identified by immunohistochemistry in placentae from preeclamptic pregnancies.
      • Buhimschi IA
      • Nayeri UA
      • Zhao G
      • et al.
      Protein misfolding, congophilia, oligomerization, and defective amyloid processing in preeclampsia.
      ,
      • Kalkunte SS
      • Neubeck S
      • Norris WE
      • et al.
      Transthyretin is dysregulated in preeclampsia, and its native form prevents the onset of disease in a preclinical mouse model.
      Whether amyloid-A is deposited in the placenta in response to increased SAA1 is unknown. We speculate that this is the case and that placental amyloid-A might exacerbate existing placental dysfunction, particularly for women with EO-PE, by disturbing cell-cell interactions and tissue structure.
      • Sack Jr., GH
      Serum amyloid A (SAA) proteins.
      Furthermore, our findings support our hypothesis that poor placentation and placental dysfunction, as evidenced by an antiangiogenic shift in the sFlt-1/PlGF ratio and fetal growth restriction, correlates with increasing SAA1 and decreasing PZP. PZP has been shown in vitro to form stable compounds with amyloid-β-peptide, thus inhibiting amyloid-β aggregation.
      • Cater JH
      • Kumita JR
      • Zeineddine Abdallah R
      • et al.
      Human pregnancy zone protein stabilizes misfolded proteins including preeclampsia- and Alzheimer's-associated amyloid beta peptide.
      We hypothesize that PZP may similarly inhibit SAA1 misfolding. Although we did not explore any mechanistic interaction between them in this study, we hypothesize that low PZP in the setting of increased SAA1 may facilitate the formation of amyloid-A and other forms of amyloid associated with preeclampsia, such as amyloid-β and transthyretin.
      Previous studies have found increased circulating levels of acute-phase proteins in preeclampsia,
      • Can M
      • Sancar E
      • Harma M
      • Guven B
      • Mungan G
      • Acikgoz S.
      Inflammatory markers in preeclamptic patients.
      ,
      • Uckan K
      • Sahin HG.
      Serum amyloid A, procalcitonin, highly sensitive C reactive protein and tumor necrosis factor alpha levels and acute inflammatory response in patients with hemolysis, elevated liver enzymes, low platelet count (HELLP) and eclampsia.
      ,
      • Braekke K
      • Holthe MR
      • Harsem NK
      • Fagerhol MK
      • Staff AC.
      Calprotectin, a marker of inflammation, is elevated in the maternal but not in the fetal circulation in preeclampsia.
      in line with our findings of increased hsCRP in EO-PE (Supplemental Table 1). Also in line with previous observations,
      • Can M
      • Sancar E
      • Harma M
      • Guven B
      • Mungan G
      • Acikgoz S.
      Inflammatory markers in preeclamptic patients.
      SAA1 correlated positively with hsCRP levels in our cohort, confirming a role for excessive inflammation in SAA1 dysregulation.
      Preeclampsia is characterized by hypercoagulability and a risk of thromboembolism.
      • Brown MA
      • Magee LA
      • Kenny LC
      • et al.
      Hypertensive disorders of pregnancy: ISSHP classification, diagnosis, and management recommendations for international practice.
      ,
      • Lalic-Cosic S
      • Dopsaj V
      • Kovac M
      • et al.
      Evaluation of global haemostatic assays and fibrin structure in patients with pre-eclampsia.
      SAA1 increases the thrombotic ability of platelets,
      • Page MJ
      • Thomson GJA
      • Nunes JM
      • et al.
      Serum amyloid A binds to fibrin(ogen), promoting fibrin amyloid formation.
      and as SAA1 is increased in EO-PE, we speculate that SAA1 participates in mediating hypercoagulability in EO-PE. Further studies are needed to explore this hypothesis.
      Unexpectedly, SAA1 correlated negatively with HbA1c—a marker of blood glucose control—in women with DM1. Poor glucose control is associated with other inflammatory markers such as hsCRP and with increased cardiovascular risk.
      • Eren MA
      • Vural M
      • Cece H
      • et al.
      Association of serum amyloid A with subclinical atherosclerosis in women with gestational diabetes.
      ,
      • Gustavsson CG
      • Agardh CD.
      Inflammatory activity increases with haemoglobin A1c in patients with acute coronary syndrome.
      ,
      • McEneny J
      • Daniels JA
      • McGowan A
      • et al.
      A cross-sectional study demonstrating increased serum amyloid A related inflammation in high-density lipoproteins from subjects with type 1 diabetes mellitus and how this association was augmented by poor glycaemic control.
      A previous study found a positive correlation between SAA and HbA1c in women with GDM.
      • Eren MA
      • Vural M
      • Cece H
      • et al.
      Association of serum amyloid A with subclinical atherosclerosis in women with gestational diabetes.
      Assays detecting SAA measure both SAA1 and SAA2—nearly identical SAA isoforms. Hence, we expected a positive correlation between SAA1 and HbA1c in our DM1 group also. BMI likely did not confound our finding, as it was not associated with SAA1 in DM1 or other study groups apart from GDM (Supplemental Results). As seen from Supplemental Table 2, the median prepregnancy BMI of women with DM1 was within the normal range (24.2 kg/m2). To the best of our knowledge, we are among the first to address SAA1 and glycemic control in DM1 during pregnancy. The negative correlation between SAA1 and HbA1c in DM1 may reflect the complex regulation of inflammation in pregnancy and merits further investigation.

      Clinical and research implications

      Low circulating PZP levels have been linked to pregnancy loss.
      • Than GN
      • Csaba IF
      • Szabó DG
      • Karg NJ
      • Novák PF.
      Quantitative immunological study of pregnancy-associated alpha2-globulin antigen.
      Women with pregestational DM and those who develop EO-PE may harbor an unfavorable preconception endometrial environment, resulting in inadequate placentation and clinical manifestations of preeclampsia during pregnancy.
      • Ng SW
      • Norwitz GA
      • Pavlicev M
      • Tilburgs T
      • Simón C
      • Norwitz ER.
      Endometrial decidualization: the primary driver of pregnancy health.
      We suggest that low circulating PZP levels in the second half of pregnancy may reflect such placentation issues and that low PZP may identify women with particularly poor placentation and subsequent high risk of early-onset placental dysfunction.
      Amyloid has been identified in atherosclerotic plaques.
      • Hellberg S
      • Silvola JMU
      • Liljenbäck H
      • et al.
      Amyloid-targeting PET tracer [18F]flutemetamol accumulates in atherosclerotic plaques.
      SAA1 and SAA2—nearly identical isoforms—may play a role in atherosclerosis, both locally and systemically. In a murine atherosclerosis model, SAA expression was increased in both early and late atherosclerosis lesions.
      • O'Brien KD
      • McDonald TO
      • Kunjathoor V
      • et al.
      Serum amyloid A and lipoprotein retention in murine models of atherosclerosis.
      SAA possibly acts as a chemoattractant for leukocytes in lesions.
      • Badolato R
      • Wang JM
      • Murphy WJ
      • et al.
      Serum amyloid A is a chemoattractant: induction of migration, adhesion, and tissue infiltration of monocytes and polymorphonuclear leukocytes.
      Increased circulating SAA levels have been associated with recurrent coronary events and stroke, as have elevated levels of hsCRP.
      • Koenig W.
      High-sensitivity C-reactive protein and atherosclerotic disease: from improved risk prediction to risk-guided therapy.
      In a mouse model, even a short period of elevated SAA1 increased atherosclerosis lesion size.
      • Thompson JC
      • Jayne C
      • Thompson J
      • et al.
      A brief elevation of serum amyloid A is sufficient to increase atherosclerosis.
      We speculate that elevated SAA1 in women with preeclampsia may promote accelerated atherosclerosis progression even if the SAA1 elevation were temporary and restricted to some months during pregnancy. Although acute atherosis and early atherosclerosis share morphologic features,
      • Staff AC
      • Redman CW.
      IFPA Award in Placentology Lecture: preeclampsia, the decidual battleground and future maternal cardiovascular disease.
      we found no association between SAA1 and acute atherosis. This may be owing to different mechanisms for the development of acute atherosis, affecting small-caliber spiral arteries, and atherosclerosis, affecting larger arteries and also owing to large time differences for their development.
      Further studies are required to elucidate protein-protein interactions between SAA1 and PZP, their possible role in placental (dys)function, and in turn, their impact on circulating angiogenic proteins. Future longitudinal pregnancy studies may reveal whether circulating SAA1 and PZP could serve as predictors for placental dysfunction. Placenta tissue studies may reveal whether increased circulating SAA1, as in EO-PE, leads to placental amyloid-A deposits. Identifying predictors of future maternal cardiovascular health during pregnancy merits further investigation. Longitudinal studies are needed to identify whether SAA1 shows prolonged elevation following preeclampsia as hsCRP does,
      • Kvehaugen AS
      • Dechend R
      • Ramstad HB
      • Troisi R
      • Fugelseth D
      • Staff AC.
      Endothelial function and circulating biomarkers are disturbed in women and children after preeclampsia.
      ,
      • van Rijn BB
      • Bruinse HW
      • Veerbeek JH
      • et al.
      Postpartum circulating markers of inflammation and the systemic acute-phase response after early-onset preeclampsia.
      placing these women at risk of postpartum amyloid-A deposition and subsequent organ damage.

      Strengths and limitations

      This study is the first to examine circulating SAA1 and PZP—potential markers of protein misfolding—in phenotypically well-characterized subgroups of women with hypertensive disorders of pregnancy and diabetes mellitus in relation to different proxies for placental function and uteroplacental acute atherosis. The population size (549 pregnancies) and well-described pregnancy groups are strengths of our study, which establishes that circulating SAA1 is increased in EO-PE and placental dysfunction. The distinction between EO-PE and LO-PE as shown in our study (delivery <34 or ≥34 weeks’ gestation, respectively) could explain why some previous studies report increased SAA1 in preeclampsia
      • Engin-Ustün Y
      • Ustün Y
      • Karabulut AB
      • Ozkaplan E
      • Meydanli MM
      • Kafkasli A.
      Serum amyloid A levels are increased in pre-eclampsia.
      ,
      • Can M
      • Sancar E
      • Harma M
      • Guven B
      • Mungan G
      • Acikgoz S.
      Inflammatory markers in preeclamptic patients.
      ,
      • Uckan K
      • Sahin HG.
      Serum amyloid A, procalcitonin, highly sensitive C reactive protein and tumor necrosis factor alpha levels and acute inflammatory response in patients with hemolysis, elevated liver enzymes, low platelet count (HELLP) and eclampsia.
      whereas others do not,
      • Kristensen K
      • Wide-Swensson D
      • Lindstrom V
      • Schmidt C
      • Grubb A
      • Strevens H.
      Serum amyloid A protein and C-reactive protein in normal pregnancy and preeclampsia.
      ,
      • Reimer T
      • Rohrmann H
      • Stubert J
      • et al.
      Angiogenic factors and acute-phase proteins in serum samples of preeclampsia and HELLP patients: a matched-pair analysis.
      ,
      • Güngör ZB
      • Tüten A
      • Ekmekçi H
      • et al.
      Possible effects of lipoprotein-associated phospholipase A2 single-nucleotide polymorphisms on cardiovascular risk in patients with preeclampsia.
      as none have made a similar differentiation. One study quantified circulating SAA1, sFlt-1, and PlGF in preeclampsia but did not study correlations between SAA1 and sFlt-1/PlGF.
      • Reimer T
      • Rohrmann H
      • Stubert J
      • et al.
      Angiogenic factors and acute-phase proteins in serum samples of preeclampsia and HELLP patients: a matched-pair analysis.
      PZP has been indirectly studied (by immunodiffusion gel) in 2 smaller studies of preeclampsia
      • Horne CH
      • Briggs JD
      • Howie PW
      • Kennedy AC.
      Serum -macroglobulins in renal disease and preeclampsia.
      ,
      • Griffin JF.
      Pregnancy-associated plasma protein levels at term in normal pregnancy, preeclampsia and essential hypertension.
      and directly studied (by ELISA) in 1 very small study where plasma from 9 women with preeclampsia was pooled before assay.
      • Cater JH
      • Kumita JR
      • Zeineddine Abdallah R
      • et al.
      Human pregnancy zone protein stabilizes misfolded proteins including preeclampsia- and Alzheimer's-associated amyloid beta peptide.
      We are, to the best of our knowledge, among the first to study circulating levels of PZP by ELISA in individual pregnancy samples from uncomplicated, hypertensive, and diabetic pregnancies.
      We chose to overrepresent HDP and DM pregnancies in this study to obtain as much data on these pregnancy complications as possible while maintaining a reasonably large (n=258) control group. Most of the included women (n=468) were delivered by cesarean, either owing to medically complicated pregnancies (HDP, DM), owing to maternal request (including previous psychologically traumatic vaginal delivery or tocophobia), or because of breech presentation (the control group).
      Prepregnancy BMI, early pregnancy blood pressure, gestational age at sampling, and fetal sex did not consistently correlate with SAA1 or PZP levels. Thus, we did not perform regression analyses. Gestational age at sampling correlated with SAA1 and PZP, but only in EO-PE, likely reflecting a more severe placental dysfunction with a clinical phenotype requiring earlier delivery. Our results are likely not confounded by our lack of gestational age matching, as gestational age did not correlate with SAA1 or PZP in any other groups.
      A limitation to our study is the lack of longitudinal samples to establish whether SAA1 and/or PZP show prolonged dysregulation during and/or after pregnancy complications and lack of placental tissue samples for amyloid staining.

      Conclusions

      Understanding and identifying early risk markers for cardiovascular disease in women is essential for achieving targeted primary prevention, optimized health outcomes, and healthier societies. Our finding of increased SAA1 and hsCRP in women with EO-PE, fetal growth restriction, and other signs of placental dysfunction may provide further explanation as to the known epidemiologic link between these pregnancy complications and maternal cardiovascular disease in later life. Dysregulated SAA1 and PZP in EO-PE may point to amyloid-A as a mediator of the observed risk of cardiovascular disease in women with a history of preeclampsia or other forms of placental dysfunction, though longitudinal and mechanistic studies are needed to test this hypothesis.

      Glossary

      Acute atherosis: pregnancy-specific foam cell lesions in spiral arteries; may affect any pregnancy, but is most frequent in preeclampsia.
      Protein misfolding: process by which proteins lose or fail to maintain normal structure, or fail to fold correctly during protein synthesis, thus losing physiological properties.
      Amyloid: fibrillar aggregates of misfolded proteins; very stable compounds because of their cross-β sheet structure, involved in numerous human diseases.

      Acknowledgments

      The authors gratefully acknowledge the pregnant women who participated in the study, Lise Øhra Levy, BSc for biobank management and patient recruitment, and previous doctoral candidates and medical students for patient recruitment to Oslo Pregnancy Biobank.

      Appendix. Supplementary materials

      References

        • Brown MA
        • Magee LA
        • Kenny LC
        • et al.
        Hypertensive disorders of pregnancy: ISSHP classification, diagnosis, and management recommendations for international practice.
        Hypertension. 2018; 72: 24-43
        • Staff AC
        • Redman CW
        • Williams D
        • et al.
        Pregnancy and long-term maternal cardiovascular health: progress through harmonization of research cohorts and biobanks.
        Hypertension. 2016; 67: 251-260
        • Irgens HU
        • Reisaeter L
        • Irgens LM
        • Lie RT.
        Long term mortality of mothers and fathers after pre-eclampsia: population based cohort study.
        BMJ. 2001; 323: 1213-1217
        • Wu P
        • Haththotuwa R
        • Kwok CS
        • et al.
        Preeclampsia and future cardiovascular health: a systematic review and meta-analysis.
        Circ Cardiovasc Qual Outcomes. 2017; 10e003497
        • Vogel B
        • Acevedo M
        • Appelman Y
        • et al.
        The Lancet Women and Cardiovascular Disease Commission: reducing the global burden by 2030.
        Lancet. 2021; 397: 2385-2438
        • Staff AC.
        The two-stage placental model of preeclampsia: an update.
        J Reprod Immunol. 2019; (1–10): 134-135
        • Redman CWG
        • Staff AC
        • Roberts JM.
        Syncytiotrophoblast stress in preeclampsia: the convergence point for multiple pathways.
        Am J Obstet Gynecol. 2022; 226: S907-S927
        • Burton GJ
        • Yung HW
        • Cindrova-Davies T
        • Charnock-Jones DS
        Placental endoplasmic reticulum stress and oxidative stress in the pathophysiology of unexplained intrauterine growth restriction and early onset preeclampsia.
        Placenta. 2009; 30: S43-S48
        • Brosens IA
        • Robertson WB
        • Dixon HG.
        The role of the spiral arteries in the pathogenesis of preeclampsia.
        Obstet Gynecol Annu. 1972; 1: 177-191
        • Kwiatkowski S
        • Dołęgowska B
        • Kwiatkowska E
        • et al.
        Do the physiological aging of the placenta and the changes in angiogenesis marker sFlt-1 and PlGF concentrations predispose patients to late-onset preeclampsia?.
        J Matern Fetal Neonatal Med. 2019; 32: 11-20
        • Mayne BT
        • Leemaqz SY
        • Smith AK
        • Breen J
        • Roberts CT
        • Bianco-Miotto T.
        Accelerated placental aging in early onset preeclampsia pregnancies identified by DNA methylation.
        Epigenomics. 2017; 9: 279-289
        • Staff AC
        • Fjeldstad HE
        • Fosheim IK
        • et al.
        Failure of physiological transformation and spiral artery atherosis: their roles in preeclampsia.
        Am J Obstet Gynecol. 2022; 226: S895-S906
        • Pitz Jacobsen DP
        • Fjeldstad HE
        • Johnsen GM
        • et al.
        Acute atherosis lesions at the fetal-maternal border: current knowledge and implications for maternal cardiovascular health.
        Front Immunol. 2021; 12791606
        • Robertson WB
        • Brosens I
        • Dixon HG.
        The pathological response of the vessels of the placental bed to hypertensive pregnancy.
        J Pathol Bacteriol. 1967; 93: 581-592
        • Maynard SE
        • Min JY
        • Merchan J
        • et al.
        Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia.
        J Clin Invest. 2003; 111: 649-658
        • Shinohara S
        • Uchida Y
        • Kasai M
        • Sunami R.
        Association between the high soluble fms-like tyrosine kinase-1 to placental growth factor ratio and adverse outcomes in asymptomatic women with early-onset fetal growth restriction.
        Hypertens Pregnancy. 2017; 36: 269-275
        • Sack Jr., GH
        Serum amyloid A (SAA) proteins.
        Subcell Biochem. 2020; 94: 421-436
        • Chiti F
        • Dobson CM.
        Protein misfolding, amyloid formation, and human disease: a summary of progress over the last decade.
        Annu Rev Biochem. 2017; 86: 27-68
        • Teoh CL
        • Griffin MD
        • Howlett GJ.
        Apolipoproteins and amyloid fibril formation in atherosclerosis.
        Protein Cell. 2011; 2: 116-127
        • Buhimschi IA
        • Nayeri UA
        • Zhao G
        • et al.
        Protein misfolding, congophilia, oligomerization, and defective amyloid processing in preeclampsia.
        Sci Transl Med. 2014; 6 (245ra92)
        • Kalkunte SS
        • Neubeck S
        • Norris WE
        • et al.
        Transthyretin is dysregulated in preeclampsia, and its native form prevents the onset of disease in a preclinical mouse model.
        Am J Pathol. 2013; 183: 1425-1436
        • Cheng S
        • Banerjee S
        • Daiello LA
        • et al.
        Novel blood test for early biomarkers of preeclampsia and Alzheimer's disease.
        Sci Rep. 2021; 11: 15934
        • Valtanen RS
        • Buhimschi CS
        • Bahtiyar MO
        • et al.
        Conformation-dependent anti-Aβ monoclonal antibody signatures of disease status and severity in urine of women with preeclampsia.
        Pregnancy Hypertens. 2022; 28: 51-59
        • Gan XW
        • Wang WS
        • Lu JW
        • et al.
        De novo synthesis of SAA1 in the Placenta Participates in parturition.
        Front Immunol. 2020; 11: 1038
        • Page MJ
        • Thomson GJA
        • Nunes JM
        • et al.
        Serum amyloid A binds to fibrin(ogen), promoting fibrin amyloid formation.
        Sci Rep. 2019; 9: 3102
        • Ekelund L
        • Laurell CB.
        The pregnancy zone protein response during gestation: a metabolic challenge.
        Scand J Clin Lab Invest. 1994; 54: 623-629
        • Wyatt AR
        • Cater JH
        • Ranson M.
        PZP and PAI-2: structurally diverse, functionally similar pregnancy proteins?.
        Int J Biochem Cell Biol. 2016; 79: 113-117
        • Than GN
        • Csaba IF
        • Szabó DG
        • Karg NJ
        • Novák PF.
        Quantitative immunological study of pregnancy-associated alpha2-globulin antigen.
        Vox Sang. 1976; 30: 134-138
        • Cater JH
        • Kumita JR
        • Zeineddine Abdallah R
        • et al.
        Human pregnancy zone protein stabilizes misfolded proteins including preeclampsia- and Alzheimer's-associated amyloid beta peptide.
        Proc Natl Acad Sci U S A. 2019; 116: 6101-6110
        • Eren MA
        • Vural M
        • Cece H
        • et al.
        Association of serum amyloid A with subclinical atherosclerosis in women with gestational diabetes.
        Gynecol Endocrinol. 2012; 28: 1010-1013
        • Engin-Ustün Y
        • Ustün Y
        • Karabulut AB
        • Ozkaplan E
        • Meydanli MM
        • Kafkasli A.
        Serum amyloid A levels are increased in pre-eclampsia.
        Gynecol Obstet Invest. 2007; 64: 117-120
        • Kristensen K
        • Wide-Swensson D
        • Lindstrom V
        • Schmidt C
        • Grubb A
        • Strevens H.
        Serum amyloid A protein and C-reactive protein in normal pregnancy and preeclampsia.
        Gynecol Obstet Invest. 2009; 67: 275-280
        • Can M
        • Sancar E
        • Harma M
        • Guven B
        • Mungan G
        • Acikgoz S.
        Inflammatory markers in preeclamptic patients.
        Clin Chem Lab Med. 2011; 49: 1469-1472
        • Reimer T
        • Rohrmann H
        • Stubert J
        • et al.
        Angiogenic factors and acute-phase proteins in serum samples of preeclampsia and HELLP patients: a matched-pair analysis.
        J Matern Fetal Neonatal Med. 2013; 26: 263-269
        • Güngör ZB
        • Tüten A
        • Ekmekçi H
        • et al.
        Possible effects of lipoprotein-associated phospholipase A2 single-nucleotide polymorphisms on cardiovascular risk in patients with preeclampsia.
        J Matern Fetal Neonatal Med. 2018; 31: 3119-3127
        • Uckan K
        • Sahin HG.
        Serum amyloid A, procalcitonin, highly sensitive C reactive protein and tumor necrosis factor alpha levels and acute inflammatory response in patients with hemolysis, elevated liver enzymes, low platelet count (HELLP) and eclampsia.
        J Obstet Gynaecol Res. 2018; 44: 440-447
        • Horne CH
        • Briggs JD
        • Howie PW
        • Kennedy AC.
        Serum -macroglobulins in renal disease and preeclampsia.
        J Clin Pathol. 1972; 25: 590-593
        • Griffin JF.
        Pregnancy-associated plasma protein levels at term in normal pregnancy, preeclampsia and essential hypertension.
        Aust N Z J Obstet Gynaecol. 1983; 23: 11-14
      1. World Health Organization. (1999). Definition, diagnosis and classification of diabetes mellitus and its complications: report of a WHO consultation. Part 1, Diagnosis and classification of diabetes mellitus. World Health Organization.

      2. Friis CM, Roum EMS, Holm HO, Toft JH, Roland MCP, Thordarson HB. Svangerskapsdiabetes. Norsk gynekologisk forening Veileder i fødselshjelp. 2020. Available at:https://www.legeforeningen.no/foreningsledd/fagmed/norsk-gynekologisk-forening/veiledere/veileder-i-fodselshjelp/svangerskapsdiabetes/. Accessed March 24, 2022.

        • Johnsen SL
        • Rasmussen S
        • Wilsgaard T
        • Sollien R
        • Kiserud T.
        Longitudinal reference ranges for estimated fetal weight.
        Acta Obstet Gynecol Scand. 2006; 85: 286-297
        • Harsem NK
        • Staff AC
        • He L
        • Roald B.
        The decidual suction method: a new way of collecting decidual tissue for functional and morphological studies.
        Acta Obstet Gynecol Scand. 2004; 83: 724-730
        • Alnaes-Katjavivi P
        • Lyall F
        • Roald B
        • Redman CW
        • Staff AC.
        Acute atherosis in vacuum suction biopsies of decidua basalis: an evidence based research definition.
        Placenta. 2016; 37: 26-33
        • Moe K
        • Heidecke H
        • Dechend R
        • Staff AC.
        Dysregulation of circulating autoantibodies against VEGF-A, VEGFR-1 and PlGF in preeclampsia - a role in placental and vascular health?.
        Pregnancy Hypertens. 2017; 10: 83-89
        • Moe K
        • Alnaes-Katjavivi P
        • Størvold GL
        • et al.
        Classical cardiovascular risk markers in pregnancy and associations to uteroplacental acute atherosis.
        Hypertension. 2018; 72: 695-702
        • Koenig W.
        High-sensitivity C-reactive protein and atherosclerotic disease: from improved risk prediction to risk-guided therapy.
        Int J Cardiol. 2013; 168: 5126-5134
        • Braekke K
        • Holthe MR
        • Harsem NK
        • Fagerhol MK
        • Staff AC.
        Calprotectin, a marker of inflammation, is elevated in the maternal but not in the fetal circulation in preeclampsia.
        Am J Obstet Gynecol. 2005; 193: 227-233
        • Ridker PM
        • Rifai N
        • Pfeffer MA
        • et al.
        Inflammation, pravastatin, and the risk of coronary events after myocardial infarction in patients with average cholesterol levels. Cholesterol and recurrent events (CARE) Investigators.
        Circulation. 1998; 98: 839-844
        • Westermark GT
        • Fändrich M
        • Westermark P.
        AA amyloidosis: pathogenesis and targeted therapy.
        Annu Rev Pathol. 2015; 10: 321-344
        • Lalic-Cosic S
        • Dopsaj V
        • Kovac M
        • et al.
        Evaluation of global haemostatic assays and fibrin structure in patients with pre-eclampsia.
        Int J Lab Hematol. 2020; 42: 322-330
        • Gustavsson CG
        • Agardh CD.
        Inflammatory activity increases with haemoglobin A1c in patients with acute coronary syndrome.
        Scand Cardiovasc J. 2009; 43: 380-385
        • McEneny J
        • Daniels JA
        • McGowan A
        • et al.
        A cross-sectional study demonstrating increased serum amyloid A related inflammation in high-density lipoproteins from subjects with type 1 diabetes mellitus and how this association was augmented by poor glycaemic control.
        J Diabetes Res. 2015; 2015351601
        • Ng SW
        • Norwitz GA
        • Pavlicev M
        • Tilburgs T
        • Simón C
        • Norwitz ER.
        Endometrial decidualization: the primary driver of pregnancy health.
        Int J Mol Sci. 2020; 21: 4092
        • Hellberg S
        • Silvola JMU
        • Liljenbäck H
        • et al.
        Amyloid-targeting PET tracer [18F]flutemetamol accumulates in atherosclerotic plaques.
        Molecules. 2019; 24: 1072
        • O'Brien KD
        • McDonald TO
        • Kunjathoor V
        • et al.
        Serum amyloid A and lipoprotein retention in murine models of atherosclerosis.
        Arterioscler Thromb Vasc Biol. 2005; 25: 785-790
        • Badolato R
        • Wang JM
        • Murphy WJ
        • et al.
        Serum amyloid A is a chemoattractant: induction of migration, adhesion, and tissue infiltration of monocytes and polymorphonuclear leukocytes.
        J Exp Med. 1994; 180: 203-209
        • Thompson JC
        • Jayne C
        • Thompson J
        • et al.
        A brief elevation of serum amyloid A is sufficient to increase atherosclerosis.
        J Lipid Res. 2015; 56: 286-293
        • Staff AC
        • Redman CW.
        IFPA Award in Placentology Lecture: preeclampsia, the decidual battleground and future maternal cardiovascular disease.
        Placenta. 2014; 35: S26-S31
        • Kvehaugen AS
        • Dechend R
        • Ramstad HB
        • Troisi R
        • Fugelseth D
        • Staff AC.
        Endothelial function and circulating biomarkers are disturbed in women and children after preeclampsia.
        Hypertension. 2011; 58: 63-69
        • van Rijn BB
        • Bruinse HW
        • Veerbeek JH
        • et al.
        Postpartum circulating markers of inflammation and the systemic acute-phase response after early-onset preeclampsia.
        Hypertension. 2016; 67: 404-414