Advertisement

Syncytiotrophoblast stress in preeclampsia: the convergence point for multiple pathways

Published:February 02, 2021DOI:https://doi.org/10.1016/j.ajog.2020.09.047
      Preeclampsia evolves in 2 stages: a placental problem that generates signals to the mother to cause a range of responses that comprise the second stage (preeclampsia syndrome). The first stage of early-onset preeclampsia is poor placentation, which we here call malplacentation. The spiral arteries are incompletely remodeled, leading to later placental malperfusion, relatively early in the second half of pregnancy. The long duration of the first stage (several months) is unsurprisingly associated with fetal growth restriction.
      The first stage of late-onset preeclampsia, approximately 80% of total cases, is shorter (several weeks) and part of a process that is common to all pregnancies. Placental function declines as it outgrows uterine capacity, with increasing chorionic villous packing, compression of the intervillous space, and fetal hypoxia, and causes late-onset clinical presentations such as “unexplained” stillbirths, late-onset fetal growth restriction, or preeclampsia.
      The second stages of early- and late-onset preeclampsia share syncytiotrophoblast stress as the most relevant feature that causes the maternal syndrome. Syncytiotrophoblast stress signals in the maternal circulation are probably the most specific biomarkers for preeclampsia. In addition, soluble fms-like tyrosine kinase-1 (mainly produced by syncytiotrophoblast) is the best-known biomarker and is routinely used in clinical practice in many locations.
      How the stress signals change over time in normal pregnancies indicates that syncytiotrophoblast stress begins on average at 30 to 32 weeks’ gestation and progresses to term. At term, syncytiotrophoblast shows increasing markers of stress, including apoptosis, pyroptosis, autophagy, syncytial knots, and necrosis. We label this phenotype the “twilight placenta” and argue that it accounts for the clinical problems of postmature pregnancies.
      Senescence as a stress response differs in multinuclear syncytiotrophoblast from that of mononuclear cells. Syncytiotrophoblast irreversibly acquires part of the senescence phenotype (cell cycle arrest) when it is formed by cell fusion.
      The 2 pathways converge on the common pathologic endpoint, syncytiotrophoblast stress, and contribute to preeclampsia subtypes. We highlight that the well-known heterogeneity of the preeclampsia syndrome arises from different pathways to this common endpoint, influenced by maternal genetics, epigenetics, lifestyle, and environmental factors with different fetal and maternal responses to the ensuing insults. This complexity mandates a reassessment of our approach to predicting and preventing preeclampsia, and we summarize research priorities to maximize what we can learn about these important issues.

      Key words

      Introduction

      Preeclampsia is recognized and currently defined as a syndrome, meaning a set of clinical signs that tend to occur together. A key feature is that the same syndrome may be recognized by varying combinations of features. Some associations may be so closely linked with a pathogenesis or cause that they may be interchangeably termed a syndrome, disease, or disorder. When the pathogenesis is known, it is better to label the condition more specifically than simply labeling it as a syndrome, which may arise from different pathogenic processes. Here, we present implications of the syndromic nature of preeclampsia. We (C.W.G.R. and A.C.S.) present a specific hypothesis that early-onset and late-onset preeclampsia use different pathophysiological pathways. It is likely that there are additional routes to the syndrome and suggestions are made as to how these subtypes might be deciphered. Together, we consider the research and clinical implications of these concepts.

      The Components of the Syndrome

      A mandatory feature of preeclampsia is its association with pregnancy, antepartum, intrapartum, or, over a short period, postpartum. It is usually symptomless. When symptoms occur, they can be severe and are a feature of a terminal crisis, which may be fatal to both the mother and fetus. Maternal seizures or eclampsia were recognized more than 2000 years in late pregnancy and delivery. Only recently were they distinguished from epilepsy.
      • Chesley L.C.
      A short history of eclampsia.
      When it became possible to measure urinary protein and blood pressure, proteinuria and hypertension were recognized, first as features of eclampsia and later as symptomless precursors of it, termed “toxemia of pregnancy” or preeclampsia.
      Hence, there emerged 4 components of an ill-defined maternal syndrome, pregnancy itself, hypertension, proteinuria, and eclampsia, of which the last was not mandatory. For some time, generalized edema was included but discounted 40 years ago. Until recently, these presentations were deemed primary maternal problems.
      It is unclear when the term “preeclampsia” was first used. Eclampsia itself was so dangerous and unpredictable that it dominated management until at least the middle of the last century. Even as late as the 1930s, hypertension was considered an inconsequential feature and therefore not sought assiduously. It was only in the 1950s that it was appreciated that severe hypertension caused cerebral hemorrhages in nonpregnant cases. The systematic treatment of pregnancy hypertension began in the 1960s and remains an essential part of its management.

      How Placental Involvement in Preeclampsia was First Recognized

      Preeclampsia or eclampsia was originally thought to be a toxemia secondary to waste products produced by the fetus, from which the concept of eclamptogenic toxemia was derived to indicate the maternal syndrome that warned of the ensuing convulsions.
      However, as early as 100 years ago, the placenta was recognized as central to this process. Very early observations indicated specific and initially unexplained placental features. Possibly the first was the association of preeclampsia with hydatidiform mole (recounted in Chesley 1978),
      • Chesley L.C.
      • Preece J.
      Hydatidiform mole, with special reference to recurrence and associated eclampsia.
      citing the first report in 1866 and then of the discovery of trophoblast embolism, or “deportation,” to the lungs of women dying of eclampsia (in Chesley 1978).
      • Chesley L.C.
      Hypertensive disorders of pregnancy.
      The importance of the placenta to the occurrence of eclampsia was recognized more than 100 years ago (Figure 1).
      • Young J.
      The aetiology of eclampsia and albuminuria and their relation to accidental haemorrhage: (an anatomical and experimental investigation.).
      The author stated, “. . . one can be left in no doubt whatever that the disease in the placenta is secondary to the deprivation of the maternal blood supply.” He highlighted that eclampsia did not require a fetus (as in hydatidiform mole) and resolved after delivery when the placenta was delivered. He recognized the conundrum of postpartum eclampsia and acknowledged that he could not explain it. The role of the placenta was further elaborated in a landmark paper in 1939, in the hypothesis that the preeclampsia syndrome was secondary to factors released by an ischemic placenta.
      • Page E.W.
      The relation between hydatid moles, relative ischemia of the gravid uterus, and the placental origin of eclampsia.
      The causes of placental ischemia, now termed “uteroplacental malperfusion,” are still being defined.
      Figure thumbnail gr1
      Figure 1Historic recognition of placental malperfusion in preeclampsia
      Image of a placenta from a case of preeclampsia. It shows a retroplacental clot and a recent infarction. Young in 1914 wrote “. . . one can be left in no doubt whatever that the disease in the placenta is secondary to the deprivation of the maternal blood supply.” Reproduced with permission from Young.
      • Young J.
      The aetiology of eclampsia and albuminuria and their relation to accidental haemorrhage: (an anatomical and experimental investigation.).
      Redman. Syncytiotrophoblast stress in preeclampsia. Am J Obstet Gynecol 2022.
      Here, we use the term preeclampsia to indicate the maternal syndrome, not its placental antecedents or its major risk factors. Syndromes are imprecise concepts. They are composed of a cluster of features that overlap variably. Gestational hypertension, proteinuria, and thrombocytopenia are all recognized as isolated presentations in pregnancy but signify more pathology when they are part of the syndromic cluster. These considerations are important because they indicate that mothers respond variably to the placental causes of preeclampsia. We address this problem of heterogeneity later in this review.
      It is difficult to clearly define syndromes. Inevitably, alternative clusters are grouped with an expanded range of disease definitions, for example, nonproteinuric preeclampsia. More than 40 years ago, we suggested that there may even be normotensive preeclampsia
      • Redman C.W.
      • Denson K.W.
      • Beilin L.J.
      • Bolton F.G.
      • Stirrat G.M.
      Factor-VIII consumption in pre-eclampsia.
      in which a cluster of preeclampsia features is present except hypertension.
      The diagnosis of a syndrome, including preeclampsia, has 1 aspect that is often disregarded. A positive test result will identify cases, whereas a negative test result can never exclude one. Furthermore, there is no guarantee that the components of the syndrome identify a single pathophysiological entity.

      Development of the 2-Stage Model of Preeclampsia

      The pathology of the placental bed in preeclampsia was described in detail from cesarean hysterectomy samples.
      • Brosens I.
      • Robertson W.B.
      • Dixon H.G.
      The physiological response of the vessels of the placental bed to normal pregnancy.
      ,
      • Robertson W.B.
      • Brosens I.
      • Dixon H.G.
      The pathological response of the vessels of the placental bed to hypertensive pregnancy.
      These authors found that the distal ends of the spiral arteries in normal pregnancy are remodeled and enlarged in association with invasion by extravillous cytotrophoblast derived from the base of the anchoring columns. This feature of placentation occurred from 8 to 16 weeks of pregnancy but was restricted in preeclampsia.
      • Brosens I.A.
      • Robertson W.B.
      • Dixon H.G.
      The role of the spiral arteries in the pathogenesis of pre-eclampsia.
      From there, the concept was developed that malplacentation (early pregnancy), by its effect on spiral artery remodeling, could adversely affect placental perfusion and be a cause of preeclampsia. It seemed that events early in an apparently normal pregnancy determined disease much later.
      The 2-stage model of preeclampsia was then formalized.
      • Redman C.W.
      Current topic: pre-eclampsia and the placenta.
      Stage 1 was composed of malplacentation, whereas stage 2 was the clinical syndrome secondary to poor uteroplacental perfusion. Numerous studies sought to determine the mechanisms of cytotrophoblast invasion and spiral artery remodeling. Although the 2-stage model has persisted, it has always been imperfect. First, the focus on trophoblast invasion restricted the interest to the end stages of placentation, whereas it represents a more profound problem of inadequate expansion of the primary extravillous cytotrophoblast, from as early as 2 weeks after conception. This leads to a spectrum of clinical problems from early pregnancy failure to preeclampsia and growth restriction.
      • Burton G.J.
      • Jauniaux E.
      The cytotrophoblastic shell and complications of pregnancy.
      In the first 6 to 8 weeks of pregnancy, the spiral arteries are blocked by intraluminal extravillous cytotrophoblast so that the placenta is sustained at low oxygen tensions by histiotrophic nutrition.
      • Burton G.J.
      • Watson A.L.
      • Hempstock J.
      • Skepper J.N.
      • Jauniaux E.
      Uterine glands provide histiotrophic nutrition for the human fetus during the first trimester of pregnancy.
      Unblocking of the spiral arteries begins at approximately 8 weeks and relatively quickly imposes an increased oxygen tension on the villous tree, bringing the associated risks of damage from oxidative stress. These are countered by increasing the effectiveness of antioxidant defenses with gestational maturity. However, if the arteries unplug prematurely, which is likely if the mass of extravillous cytotrophoblast is reduced, then placental growth is impaired at this critical time because of oxidative stress, not malperfusion.
      • Burton G.J.
      Oxygen, the Janus gas; its effects on human placental development and function.
      Hence, placentation is not just spiral artery invasion but the whole spectrum of placental growth (from the start of pregnancy), at and after implantation (Figure 2). In essence, the development of the placenta starts poorly and never catches up. This is the major reason why early-onset disease is easier to predict than late-onset condition, in which the placenta grows normally to begin with but has end of pregnancy problems.
      Figure thumbnail gr2
      Figure 2Placentation is more than spiral artery remodeling
      Placentation begins at conception or even preimplantation and is largely complete at 18 weeks’ gestation. The figure summarizes the stages in the middle (yellow), how it proceeds normally on the right (blue), and where it might go wrong on the left (orange).
      Redman. Syncytiotrophoblast stress in preeclampsia. Am J Obstet Gynecol 2022.
      The original 2-stage hypothesis described preeclampsia as the sequel to poor placentation. However, malplacentation cannot be the sole cause of preeclampsia because fetal growth restriction (FGR) is not a consistent part of the syndrome.
      • Ness R.B.
      • Roberts J.M.
      Heterogeneous causes constituting the single syndrome of preeclampsia: a hypothesis and its implications.
      In fact, most cases show little or no FGR. This issue was first addressed by the concept that preeclampsia may be maternal or placental in origin.
      • Ness R.B.
      • Roberts J.M.
      Heterogeneous causes constituting the single syndrome of preeclampsia: a hypothesis and its implications.
      Maternal preeclampsia typically would occur at term and result from an interaction between a healthy placenta and a mother with preexisting disorders such as cardiovascular disease (CVD) or obesity. It has always been known that maternal medical problems contribute to the risk of preeclampsia, but the placenta is a mandatory component. The concept was consistent with reports that there was little obvious placental pathology in late-onset disease.
      • Sebire N.J.
      • Goldin R.D.
      • Regan L.
      Term preeclampsia is associated with minimal histopathological placental features regardless of clinical severity.
      Because preeclampsia at term is not associated with FGR,
      • Xiong X.
      • Demianczuk N.N.
      • Saunders L.D.
      • Wang F.L.
      • Fraser W.D.
      Impact of preeclampsia and gestational hypertension on birth weight by gestational age.
      early- and late-onset diseases were deemed distinct subtypes.
      • von Dadelszen P.
      • Magee L.A.
      • Roberts J.M.
      Subclassification of preeclampsia.
      In addition to FGR, early-onset disease is more severe than late-onset disease in other aspects, such as maternal mortality The threshold between the 2 types was set to be before or after 34 weeks’ gestation. In practice, the time of onset of preeclampsia is rarely known precisely, so the time of delivery serves as a surrogate. It should be remembered that the natural outcome of preeclampsia has been heavily modified by clinical practice for the last 50 to 60 years. Early features of preeclampsia at term can be quickly and safely resolved by elective delivery. Without such care, late-onset disease would be less benign than it is now. However, even today, it can still kill
      • Kenneth L.
      • Hall D.R.
      • Gebhardt S.
      • Grové D.
      Late onset preeclampsia is not an innocuous condition.
      and does so in low-resource settings.
      By the turn of the millennium, there were other advances in the 2-stage model. Even if the factors released from the dysfunctional placenta were not identified, a maternal tissue was targeted—the maternal endothelium.
      • Roberts J.M.
      • Taylor R.N.
      • Musci T.J.
      • Rodgers G.M.
      • Hubel C.A.
      • McLaughlin M.K.
      Preeclampsia: an endothelial cell disorder.
      From the mother’s perspective, this was not just a hypertensive disorder, but one of diffuse endothelial dysfunction—later termed “vascular inflammation.” The protean features of preeclampsia could be unified mechanistically, and the association of this dysfunction with an excessive systemic inflammatory response was less surprising. In addition, what was startling was that it was also well developed in the third trimester of pregnancy in normal, pregnant, control women, whose inflammatory profile more closely resembled that of their preeclamptic counterparts than that of matched normal nonpregnant control women.
      • Redman C.W.
      • Sacks G.P.
      • Sargent I.L.
      Preeclampsia: an excessive maternal inflammatory response to pregnancy.
      The functional link between the placenta and maternal endothelial dysfunction was clarified by the seminal discovery of the angiogenic imbalance caused by an excessive circulating soluble receptor, the vascular endothelial growth factor-1 (VEGF-1), also known as the soluble fms-like tyrosine kinase-1 (sFlt-1).
      • Maynard S.E.
      • Min J.Y.
      • Merchan J.
      • et al.
      Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia.
      A decoy factor, it inhibits the actions of the VEGF and that of its cousin, the vascular placental growth factor (PlGF), both essential for vascular endothelial health. Both sFlt-1 and PlGF are produced in large amounts by the placenta. More important to this review, they are, specifically, products of the syncytiotrophoblast (STB).
      Even though this was a critical step and an increased number of villi forward, there was still a major problem. If late-onset preeclampsia is associated with neither FGR nor obvious placental pathology, can uteroplacental malperfusion be the underlying pathology? If it is not, what could be the alternative? Further progress depended on acquiring a better understanding of term and postterm pregnancies.

      “Normal Pregnancy” at Term—Not as Normal as it Seems

      The problems of postterm pregnancies have been documented for 70 years,
      • Clayton S.G.
      Foetal mortality in post-maturity.
      first in the context of apparently unexplained stillbirths and later with what was called “dysmaturity.”
      • Sjöstedt S.
      • Engleson G.
      • Rooth G.
      The latter was a phenotype associated with postterm births, infants born with lower oxygen saturations, lower birthweights, and increased hemoglobin concentrations.
      • Sjöstedt S.
      • Engleson G.
      • Rooth G.
      At this time, the concept of FGR was not formulated, but there is no doubt that this was the problem. Its extreme forms have disappeared because of routine elective delivery of postterm pregnancies. However, milder degrees persist. Just as there is early- and late-onset preeclampsia, there is also early- and late-onset FGR. Furthermore, the incidence of late-onset preeclampsia and eclampsia increases with gestational age, being 3 times more common at 40 than at 37 weeks’ gestation.
      • Caughey A.B.
      • Stotland N.E.
      • Escobar G.J.
      What is the best measure of maternal complications of term pregnancy: ongoing pregnancies or pregnancies delivered?.
      What causes these late pregnancy problems? We can learn more from ultrasound Doppler measurements of the utero- and fetoplacental circulations.
      During the third trimester of pregnancy, uterine artery Doppler pulsatility changes a little, even in postterm pregnancy,
      • Gómez O.
      • Figueras F.
      • Fernández S.
      • et al.
      Reference ranges for uterine artery mean pulsatility index at 11-41 weeks of gestation.
      which suggests that the uteroplacental circulation is stable and not likely to cast light on this problem. However, the fetal circulation clearly changes. The umbilical artery pulsatility index declines steadily throughout the second half of pregnancy compatible with a progressive increase in fetoplacental impedance.
      • Gómez O.
      • Figueras F.
      • Fernández S.
      • et al.
      Reference ranges for uterine artery mean pulsatility index at 11-41 weeks of gestation.
      Even more interesting is that the fetal middle cerebral artery (MCA) pulsatility index peaks at approximately 30 weeks’ gestation and declines after that. The cerebroplacental ratio (CPR) between the indices of the MCA and the umbilical artery follows a similar pattern, interpreted to indicate the increasing redistribution of blood flow to the cerebral circulation
      • Ciobanu A.
      • Wright A.
      • Syngelaki A.
      • Wright D.
      • Akolekar R.
      • Nicolaides K.H.
      Fetal Medicine Foundation reference ranges for umbilical artery and middle cerebral artery pulsatility index and cerebroplacental ratio.
      and an early sign of fetal stress. A low ratio, for example, is associated with an impaired outcome,
      • Khalil A.
      • Morales-Rosello J.
      • Khan N.
      • et al.
      Is cerebroplacental ratio a marker of impaired fetal growth velocity and adverse pregnancy outcome?.
      although its clinical usefulness is not yet clear.
      What underlies these major changes in the fetal circulation? They develop during a time of massive growth in the chorionic villous tree with an expansion of its villous tree,
      • Jackson M.R.
      • Mayhew T.M.
      • Boyd P.A.
      Quantitative description of the elaboration and maturation of villi from 10 weeks of gestation to term.
      which is associated with a steady decline in umbilical and intervillous partial pressure of oxygen (pO2) (Figure 3).
      • Soothill P.W.
      • Nicolaides K.H.
      • Rodeck C.H.
      • Campbell S.
      Effect of gestational age on fetal and intervillous blood gas and acid-base values in human pregnancy.
      A low pO2 was long ago discovered to be a feature of dysmaturity at term.
      • Sjöstedt S.
      • Engleson G.
      • Rooth G.
      We have confirmed this trend by analyzing umbilical arterial pO2 in elective cesarean delivery in postterm pregnancies (unpublished observations). The cause of this progressive hypoxia is unknown. Therefore, we extended our previous proposal
      • Redman C.W.
      • Staff A.C.
      Preeclampsia, biomarkers, syncytiotrophoblast stress, and placental capacity.
      to suggest that it arises from increasing demands of a placenta expanding into a limited space. First, expansion of the chorionic villous tree increases the resistance of the fetoplacental circulation reflected in the declining umbilical arterial pulsatility index.
      • Ciobanu A.
      • Wright A.
      • Syngelaki A.
      • Wright D.
      • Akolekar R.
      • Nicolaides K.H.
      Fetal Medicine Foundation reference ranges for umbilical artery and middle cerebral artery pulsatility index and cerebroplacental ratio.
      Second, the packing density of the chorionic villi increases with gestational age.
      • Mayhew T.M.
      • Wadrop E.
      Placental morphogenesis and the star volumes of villous trees and intervillous pores.
      Finally, mathematical modeling suggests that the perfusion of a placentome becomes restricted as the packing density increases beyond its optimal value,
      • Serov A.S.
      • Salafia C.M.
      • Brownbill P.
      • Grebenkov D.S.
      • Filoche M.
      Optimal villi density for maximal oxygen uptake in the human placenta.
      perhaps intensified by increasing maternal plasma viscosity at the end of pregnancy.
      • Heilmann L.
      Blood rheology and pregnancy.
      Figure thumbnail gr3
      Figure 3Increasing fetal and placental hypoxia with advancing gestational age
      These data were derived from blood samples taken from 200 cases using fetoscopy or cordocentesis for diagnostic purposes. Cases with fetal growth restriction, hydrops, or rhesus disease were excluded. There was no difference attributable to the method of sampling. Means (95% confidence intervals) are shown for intervillous, umbilical venous, and umbilical arterial samples, plotted by gestational age. Reproduced with permission from Soothill et al.
      • Soothill P.W.
      • Nicolaides K.H.
      • Rodeck C.H.
      • Campbell S.
      Effect of gestational age on fetal and intervillous blood gas and acid-base values in human pregnancy.
      Redman. Syncytiotrophoblast stress in preeclampsia. Am J Obstet Gynecol 2022.

      Placental Pathology

      Generally, the placental pathology of early-onset preeclampsia is more severe than that of late-onset disease, when routine histopathology is usually unhelpful.
      • Sebire N.J.
      • Goldin R.D.
      • Regan L.
      Term preeclampsia is associated with minimal histopathological placental features regardless of clinical severity.
      For the former, gross features include placental infarction spiral artery thrombosis or the occlusive lesions of acute atherosis, which are deemed the sequelae of malplacentation and the associated underlying spiral artery disease.
      • Staff A.C.
      • Dechend R.
      • Pijnenborg R.
      Learning from the placenta: acute atherosis and vascular remodeling in preeclampsia-novel aspects for atherosclerosis and future cardiovascular health.
      They also include pathology in the intervillous space and the chorionic villi, related to hypoxia, summarized in previous reports.
      • Ernst L.M.
      Maternal vascular malperfusion of the placental bed.
      ,
      • Stanek J.
      Hypoxic patterns of placental injury: a review.
      Associated pathologies include distal villous hypoplasia,
      • Fitzgerald B.
      • Kingdom J.
      • Keating S.
      Distal villous hypoplasia.
      villitis of unknown etiology,
      • Kim C.J.
      • Romero R.
      • Chaemsaithong P.
      • Kim J.S.
      Chronic inflammation of the placenta: definition, classification, pathogenesis, and clinical significance.
      and massive intervillous fibrin deposition.
      • Romero R.
      • Whitten A.
      • Korzeniewski S.J.
      • et al.
      Maternal floor infarction/massive perivillous fibrin deposition: a manifestation of maternal antifetal rejection?.
      The more obvious features can be ascribed to spiral artery maladaptation during placentation. However, others involving the STB compartment have been previously overlooked.

      Syncytiotrophoblast Pathology

      Classical histopathology reveals the tip of the iceberg of pathologic changes in STB. Electron microscopy or immunohistochemistry with or without confocal microscopy adds resolution and precision. Their application has revolutionized the understanding of STB in preeclampsia. The studies described below concern the STB in late pregnancy and avoid in vitro work on trophoblast cell lines or choriocarcinoma or extravillous invasive cytotrophoblast in early pregnancy.
      The STB is the major interface between the mother and placenta. Its pathology is superimposed on changes in the underlying villi, which may include accelerated maturation, syncytial knots, perivillous fibrin deposition, and villous agglutination. These are considered to indicate maternal malperfusion and are typical of preeclampsia, especially of early onset.
      • Redline R.W.
      • Boyd T.
      • Campbell V.
      • et al.
      Maternal vascular underperfusion: nosology and reproducibility of placental reaction patterns.
      Electron microscopy reveals patchy necrosis, distorted or scanty microvilli, swollen endoplasmic reticulum (ER) typical of ER stress, and mitochondrial swelling.
      • Jones C.J.
      • Fox H.
      An ultrastructural and ultrahistochemical study of the human placenta in maternal pre-eclampsia.
      There may also be changes in the underlying capillary endothelium. Immunohistochemistry or electron microscopy demonstrates changes that are ascribable to and include oxidative, inflammatory, mitochondrial or ER stress or, possible immune attack, and senescence (Table). Breaks in or denudation of STB, exposing the underlying villous stroma to maternal immune cells and clotting activators, are better documented in normal pregnancy than preeclampsia.
      • Nelson D.M.
      Apoptotic changes occur in syncytiotrophoblast of human placental villi where fibrin type fibrinoid is deposited at discontinuities in the villous trophoblast.
      ,
      • Mayhew T.M.
      Villous trophoblast of human placenta: a coherent view of its turnover, repair and contributions to villous development and maturation.
      TableSyncytiotrophoblast stress lesions of preeclampsia
      Syncytial clubbing and necrosis
      • Jones C.J.
      • Fox H.
      An ultrastructural and ultrahistochemical study of the human placenta in maternal pre-eclampsia.
      Syncytial knots
      • Heazell A.E.
      • Moll S.J.
      • Jones C.J.
      • Baker P.N.
      • Crocker I.P.
      Formation of syncytial knots is increased by hyperoxia, hypoxia and reactive oxygen species.
      Mitochondrial stress
      • Yung H.W.
      • Colleoni F.
      • Dommett E.
      • et al.
      Noncanonical mitochondrial unfolded protein response impairs placental oxidative phosphorylation in early-onset preeclampsia.
      Apoptosis
      • Can M.
      • Guven B.
      • Bektas S.
      • Arikan I.
      Oxidative stress and apoptosis in preeclampsia.
      Complement deposition
      • Rampersad R.
      • Barton A.
      • Sadovsky Y.
      • Nelson D.M.
      The C5b-9 membrane attack complex of complement activation localizes to villous trophoblast injury in vivo and modulates human trophoblast function in vitro.
      Senescence
      • Coppé J.P.
      • Patil C.K.
      • Rodier F.
      • et al.
      Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor.
      Oxidative stress
      • Cui X.L.
      • Brockman D.
      • Campos B.
      • Myatt L.
      Expression of NADPH oxidase isoform 1 (Nox1) in human placenta: involvement in preeclampsia.
      Endoplasmic reticulum stress
      • Burton G.J.
      • Yung H.W.
      Endoplasmic reticulum stress in the pathogenesis of early-onset pre-eclampsia.
      Inflammatory stress
      • Aban M.
      • Cinel L.
      • Arslan M.
      • et al.
      Expression of nuclear factor-kappa B and placental apoptosis in pregnancies complicated with intrauterine growth restriction and preeclampsia: an immunohistochemical study.
      Autophagy
      • Pan Y.J.
      • He L.
      • Zhou S.J.
      • Zhang L.J.
      • Zhang A.H.
      • Zhao Y.Y.
      Expression of urotensin II is associated with placental autophagy in patients with severe preeclampsia.
      Pyroptosis
      • Liu H.
      • Cheng F.
      • Xu Q.
      • et al.
      Lipoxin A4 suppresses angiotensin II type 1 receptor autoantibody in preeclampsia via modulating caspase-1.
      Perivillous fibrin deposition
      • Weel I.C.
      • Baergen R.N.
      • Romão-Veiga M.
      • et al.
      Association between placental lesions, cytokines and angiogenic factors in pregnant women with preeclampsia.
      Redman. Syncytiotrophoblast stress in preeclampsia. Am J Obstet Gynecol 2022.
      The pathology can be simulated ex vivo by cultures of placental explants exposed to hypoxia-reoxygenation as would occur in vivo with ischemia reperfusion.
      • Hung T.H.
      • Skepper J.N.
      • Charnock-Jones D.S.
      • Burton G.J.
      Hypoxia-reoxygenation: a potent inducer of apoptotic changes in the human placenta and possible etiological factor in preeclampsia.
      Breaks in STB continuity are relatively common in normal term placenta,
      • Jones L.M.
      • Chen Y.
      • van Oosten-Hawle P.
      Redefining proteostasis transcription factors in organismal stress responses, development, metabolism, and health.
      but their incidence in preeclamptic placentas is unknown.

      Cellular Stress

      Cellular survival is a balancing act that sustains normal functions and coexistence with neighboring cells in terms of 2-way signaling and integration with the total physiology of the organism. It is inevitably complex. Many factors constantly threaten homeostasis; if the threat is substantial, it constitutes cell stress and may lead to cell death.
      There are different stresses, both internal and external, ranging from perturbations of energy supply to trauma or infection. The molecular mechanisms by which these stresses are countered to restore homeostasis constitute the cellular stress response. Because of their intricacy, they are commonly studied in a compartmentalized way to make progress. In fact, a major cellular stress will change or impede many cellular functions and stimulate various responses at the same time. There are 4 outcomes to a stress response (Figure 4): normality is restored; the cell does not recover and becomes chronically stressed, which is characteristic of senescence; or it undergoes programmed cell death or, in the most extreme case, disintegrates (necrosis).
      Figure thumbnail gr4
      Figure 4Cell stressors and the outcomes of their stress responses
      On the left are lists of some of the many stresses to which cells must respond to maintain homeostasis. On the right are the final outcomes that may ensue.
      Redman. Syncytiotrophoblast stress in preeclampsia. Am J Obstet Gynecol 2022.
      Stress sensors may be on the cell surface, in the cell membrane, in the cytoplasm, or in cell organelles. Stress responses are orchestrated by a variety of mechanisms, often in a hierarchical way and always across networks, which are still being defined. Transcription factors, microRNAs (miRNAs), and long noncoding RNAs are among the many regulators.
      • Jones L.M.
      • Chen Y.
      • van Oosten-Hawle P.
      Redefining proteostasis transcription factors in organismal stress responses, development, metabolism, and health.
      • Du J.
      • Li M.
      • Huang Q.
      • et al.
      The critical role of microRNAs in stress response: therapeutic prospect and limitation.
      • Valadkhan S.
      • Valencia-Hipólito A.
      lncRNAs in stress response.
      They can redirect metabolic programs or change their overall endpoints.
      Here, we limit our discussion to examples relevant to STB, beginning with its special aspects.

      A Stressed Syncytium is not the Same as a Stressed Single Cell

      As far as it is known, STB is 1 huge cell covering the entire folded microvillous surface of the placenta.
      • Castellucci M.
      • Kauffmann P.
      Basic structure of the villous trees.
      Its cellular responses have to be localized by mechanisms other than by intercellular partitions. Second, its life expectancy (37–42 weeks) is 2 orders of magnitude less than the 70 to 90 years of other adult human tissues. It has 2 circulations and is an epithelium that functions as an endothelium in the intervillous space. It lives within its genetically disparate mother; hence, it must evade aggression from the maternal immune system. Therefore, it is to be expected that it encounters and must respond to a unique pattern of stresses. Here, we describe this complexity and how it contributes to and is changed by preeclampsia. We focus on the pathology that has been demonstrated to involve the syncytium in relation to preeclampsia in term or postterm pregnancy. It is technically challenging. Study of whole placental tissue (minced) does not resolve STB from other cell types. Immunohistochemistry with confocal microscopy allows higher resolution to demonstrate specific DNA, RNA, or proteins in subcellular organelles. These can be confirmed in experiments with cultured placental explants that are exposed to specific stresses, such as hypoxia-reoxygenation or molecular inducers of ER stress. A major challenge is to be able to distinguish cytotrophoblast from STB. The latter is formed by fusion with the former, which may donate markers to STB, which reflect its own problems rather than those of the STB. They are in close proximity and may be difficult to distinguish for that reason.
      • Longtine M.S.
      • Chen B.
      • Odibo A.O.
      • Zhong Y.
      • Nelson D.M.
      Villous trophoblast apoptosis is elevated and restricted to cytotrophoblasts in pregnancies complicated by preeclampsia, IUGR, or preeclampsia with IUGR.

      Syncytiotrophoblast Stress

      Sprouts and knots—unique syncytial features

      Syncytial sprouts and knots are 2 types of syncytial multinuclear aggregates
      • Burton G.J.
      • Jones C.J.
      Syncytial knots, sprouts, apoptosis, and trophoblast deportation from the human placenta.
      that are readily seen by simple microscopy. The former are composed of outgrowths from the syncytial surface, are transcriptionally active and normal structures, and are present from the early stages of pregnancy. Syncytial knots are clusters of transcriptionally inactive nuclei (a marker of stress) that increase with gestational age
      • Loukeris K.
      • Sela R.
      • Baergen R.N.
      Syncytial knots as a reflection of placental maturity: reference values for 20 to 40 weeks’ gestational age.
      and with disorders of maternal malperfusion, such as preeclampsia.
      • Tomas S.Z.
      • Prusac I.K.
      • Roje D.
      • Tadin I.
      Trophoblast apoptosis in placentas from pregnancies complicated by preeclampsia.
      They can be induced in vitro by hypoxia or reactive oxygen species
      • Heazell A.E.
      • Moll S.J.
      • Jones C.J.
      • Baker P.N.
      • Crocker I.P.
      Formation of syncytial knots is increased by hyperoxia, hypoxia and reactive oxygen species.
      and, as such, are a type of stress response.
      • Fogarty N.M.
      • Ferguson-Smith A.C.
      • Burton G.J.
      Syncytial knots (Tenney-Parker changes) in the human placenta: evidence of loss of transcriptional activity and oxidative damage.
      How they are formed and what their pathophysiological function are unknown. Some consider that they are a type of STB apoptosis (programmed cell death—see the section ‘Autophagy, Apoptosis and Necrosis’ below.).
      • Huppertz B.
      IFPA Award in Placentology lecture: biology of the placental syncytiotrophoblast—myths and facts.
      Others consider that they have other functions because some knots are transcriptionally active and that the STB nuclei do not turn over but accumulate in the syncytium.
      • Jones L.M.
      • Chen Y.
      • van Oosten-Hawle P.
      Redefining proteostasis transcription factors in organismal stress responses, development, metabolism, and health.
      Syncytial knots are important to this review because they clearly demonstrate that placental dysfunction and pathology are not confined to early-onset preeclampsia. Because they increase with gestational age and are commonplace in term placentas without preeclampsia or FGR, they have, for many years, been disregarded as “normal.” In fact, they are important as indicators of the hidden stresses of an apparently uncomplicated term pregnancy.

      Syncytiotrophoblast and Organellar Stress

      Stressed organelles can include the ER, mitochondria, nuclei, or nucleoli. STB organelles can be stressed in preeclampsia or FGR. This required a definition of the molecular processes and their specific localization. Nucleolar,
      • Yang K.
      • Yang J.
      • Yi J.
      Nucleolar stress: hallmarks, sensing mechanism and diseases.
      lysosomal,
      • Burton T.D.
      • Fedele A.O.
      • Xie J.
      • Sandeman L.Y.
      • Proud C.G.
      The gene for the lysosomal protein LAMP3 is a direct target of the transcription factor ATF4.
      or Golgi
      • Sasaki K.
      • Yoshida H.
      Golgi stress response and organelle zones.
      stress has been characterized in other tissues but not yet in STB. However, because the cell is a highly integrated system, there is no reason to suppose that the problem of, say oxidative stress, will not be shared across its organelles in STB as in other cells.

      Syncytiotrophoblast and Oxidative Stress

      The mitochondria and ER are, respectively, the powerhouse and protein factories of the cell, and both generate reactive oxygen species (ROS). ROS are unstable, highly reactive free radicals based on oxygen that are generated under conditions of stress. The 2 organelles communicate closely and can transfer ROS to each other.
      • Fan Y.
      • Simmen T.
      Mechanistic connections between endoplasmic reticulum (ER) redox control and mitochondrial metabolism.
      ROS are a normal byproduct of oxygen metabolism and, at low concentrations, contribute to cell signaling. However, when a cell must respond quickly to stress, ROS levels can increase dramatically and oxidatively damage its contents.
      Cells are powered by oxidative phosphorylation to produce adenosine triphosphate (ATP), a process that is expedited by high energy electron transfers within the mitochondria. Electrons are agents of redox reactions. If a molecule loses an electron, it is oxidized; if it gains an electron, it is reduced. This is the basis of oxidative phosphorylation. Because of their high energy, electrons must be contained within the mitochondrial matrix, shielded from other cell contents by a double membrane. Free radicals, containing unpaired electrons, are powerful oxidants by virtue of electron donation. If they leak out of the safe confines of the mitochondria, they can damage cell molecules in a relatively indiscriminate way. This may reduce or enhance molecular functions or even induce new (and unwanted) functions. This is oxidative stress. It is not simply hypoxia. As is well known, cellular antioxidants neutralize these activities, but when overwhelmed, oxidative stress occurs. Nitric oxide is another hyperreactive free radical that can cause damage by nitrosative stress, analogous to oxidative stress.

      Syncytiotrophoblast, Oxidative Stress, and Preeclampsia

      The concept of oxidative stress in preeclampsia began to evolve 30 years ago and was expanded as the techniques for its study improved. Oxidative stress of the placenta will extend to all cells in the chorionic villus and, in most instances, include the STB. Thus, one of the first studies showed the content of lipid peroxides (products of oxidative stress) in STB membranes as measured by its marker, malondialdehyde, to be higher in preeclampsia samples at term than in normal control samples.
      • Cester N.
      • Staffolani R.
      • Rabini R.A.
      • et al.
      Pregnancy induced hypertension: a role for peroxidation in microvillus plasma membranes.
      Expanded interest lead to various therapeutic trials of antioxidants to counter the stress and help prevent the disorder.
      • Tenório M.B.
      • Ferreira R.C.
      • Moura F.A.
      • Bueno N.B.
      • Goulart M.O.F.
      • Oliveira A.C.M.
      Oral antioxidant therapy for prevention and treatment of preeclampsia: meta-analysis of randomized controlled trials.
      Now, oxidative stress is well documented as part of the pathology of the placenta
      • Myatt L.
      • Cui X.
      Oxidative stress in the placenta.
      What is important for this review is that STB is affected. This is shown in terms of several markers, such as 4-hydroxynonenal,
      • Takagi Y.
      • Nikaido T.
      • Toki T.
      • et al.
      Levels of oxidative stress and redox-related molecules in the placenta in preeclampsia and fetal growth restriction.
      hydroxy-deoxyguanosine,
      • Cindrova-Davies T.
      • Fogarty N.M.E.
      • Jones C.J.P.
      • Kingdom J.
      • Burton G.J.
      Evidence of oxidative stress-induced senescence in mature, post-mature and pathological human placentas.
      which indicates oxidative stress in DNA, mostly in the syncytial nuclei or, finally, advanced glycation end products,
      • Chekir C.
      • Nakatsuka M.
      • Noguchi S.
      • et al.
      Accumulation of advanced glycation end products in women with preeclampsia: possible involvement of placental oxidative and nitrative stress.
      which are formed by oxidative stress in the context of hyperglycemia. The causes of such stress are likely to include intermittent oxygenation intrinsic to the complexities of perfusion through the crevices of the intervillous space in which the placental villi are packed at increasing density as gestation progresses.
      • Serov A.S.
      • Salafia C.M.
      • Brownbill P.
      • Grebenkov D.S.
      • Filoche M.
      Optimal villi density for maximal oxygen uptake in the human placenta.

      Syncytiotrophoblast, Organellar Stress, and Preeclampsia

      A histologic hallmark of ER stress is dilatation of the organelle, which was noted 40 years ago in the STB of preeclamptic placentas.
      • Jones C.J.
      • Fox H.
      An ultrastructural and ultrahistochemical study of the human placenta in maternal pre-eclampsia.
      It was confirmed in molecular studies of placentas from early-onset FGR with or without preeclampsia in terms of what is called the unfolded protein response (UPR).
      • Yung H.W.
      • Calabrese S.
      • Hynx D.
      • et al.
      Evidence of placental translation inhibition and endoplasmic reticulum stress in the etiology of human intrauterine growth restriction.
      Most translational protein synthesis occurs in the ER. The rough ER synthesizes the proteins for secretion or incorporation in the plasma membranes. The smooth ER synthesizes lipids. The ER communicates directly with the mitochondria through mitochondria-associated membranes (MAMs), and its cavity is in continuity with the nucleus. Newly formed proteins are linear polypeptides that need to be folded correctly. Folding depends, in part, on glycosylation and the formation of sulfhydryl bonds, which is an oxidative process. Generally, folding buries hydrophobic sequences in the interior of the nascent protein. ER stress occurs when the protein is incorrectly folded and cannot be exported to the Golgi. Thus, the protein production line is blocked and stops working (Figure 5). The UPR is a switch to a new transcriptional program, which generates rescue factors such as chaperones and antioxidants that promote the clearance of malfolded proteins. In addition, the cell stops dividing (cell cycle arrest).
      • Carreras-Sureda A.
      • Pihán P.
      • Hetz C.
      Calcium signaling at the endoplasmic reticulum: fine-tuning stress responses.
      Because the ER is a major reservoir of intracellular calcium, which is a powerful intracellular signal, its calcium homeostasis is affected, spreading the stress signal to other cellular functions.
      Figure thumbnail gr5
      Figure 5ER stress—unfolded protein response
      A, The endoplasmic reticulum is the main site of cellular protein synthesis, which involves translation of mRNA to create a polypeptide that is then folded into its final tertiary shape. Under conditions of stress, for example, oxidative stress, the ER loses its ability to fold newly formed proteins and export them to the Golgi. B, Misfolded proteins accumulate in the ER where they are detected by stress sensors. These rapidly trigger a process termed the “integrated stress response” to restore homeostasis. The baseline program of protein synthesis is stopped, and the removal of the polypeptide debris is stimulated. New protein synthesis is established, but it is respecified to producing rescue molecules, such as cell chaperones and antioxidants. Nonetheless, in ER stress, the stress response is extended to stimulate apoptosis.
      ER, endoplasmic reticulum.
      Redman. Syncytiotrophoblast stress in preeclampsia. Am J Obstet Gynecol 2022.
      More recently, nuclear
      • Cindrova-Davies T.
      • Fogarty N.M.E.
      • Jones C.J.P.
      • Kingdom J.
      • Burton G.J.
      Evidence of oxidative stress-induced senescence in mature, post-mature and pathological human placentas.
      and mitochondrial
      • Yung H.W.
      • Colleoni F.
      • Dommett E.
      • et al.
      Noncanonical mitochondrial unfolded protein response impairs placental oxidative phosphorylation in early-onset preeclampsia.
      stresses have also been detected in STB. The mitochondria have their own DNA and synthesize mitochondrial proteins. Under stress, they develop a mitochondrial UPR, which signals the problem to other organelles. Nuclear stress is shown by the presence of an oxidized purine, 8-hydroxy-deoxyguanosine demonstrated by immunohistochemistry.
      In preeclampsia, the STB mitochondria are swollen and show diminished activity in various assay systems summarized elsewhere,
      • Holland O.
      • Dekker Nitert M.
      • Gallo L.A.
      • Vejzovic M.
      • Fisher J.J.
      • Perkins A.V.
      Review: placental mitochondrial function and structure in gestational disorders.
      including cases with late-onset disease.
      • Muralimanoharan S.
      • Maloyan A.
      • Mele J.
      • Guo C.
      • Myatt L.G.
      • Myatt L.
      MIR-210 modulates mitochondrial respiration in placenta with preeclampsia.
      A mitochondrial UPR (see above) is activated. The mitochondria and ER coordinate their signaling through their MAMs. Hence, mitochondrial and ER stress often, if not always, occur together and, in combination, contribute to the overall level of cellular stress.
      DNA damage (nuclear stress) is typically caused by oxidative stress and may be lethal. If the cell cannot repair the DNA, it loses its ability to divide (cell cycle arrest) and its normal function to renew the tissue of which it is a part. Alternatively, the cell may survive with a malignant mutation that could kill the organism. Hence, the DNA damage response is critical. It is well characterized particularly in terms of the tumor suppressor factors, of which p53 is the best known. Their main function is to stop the faulty cell from proliferating (cell cycle arrest) or to remove the cell by apoptosis.
      • Reed S.M.
      • Quelle D.E.
      p53 acetylation: regulation and consequences.
      Tumor suppressor proteins are a subset of transcription factors that bind to one or several DNA sequences, in 1 or more genes, to regulate their transcription, frequently coordinated with other transcription factors regulating the same gene. They can therefore promote pleomorphic responses that vary according to context. By inhibiting cell division and promoting apoptosis, they limit the development of mutated malignant cells. These attributes are demonstrated by p53, which apart from inhibiting cell proliferation can promote apoptosis, autophagy, or other parts of the stress response
      • Hu W.
      • Chen S.
      • Thorne R.F.
      • Wu M.
      TP53, TP53 target genes (DRAM, TIGAR), and autophagy.
      and is up-regulated in STB under stress conditions.
      • Heazell A.E.
      • Sharp A.N.
      • Baker P.N.
      • Crocker I.P.
      Intra-uterine growth restriction is associated with increased apoptosis and altered expression of proteins in the p53 pathway in villous trophoblast.
      Apart from the UPR, the increasingly stressed cell may resort to autophagy or, in extreme cases, undergo apoptosis.

      Autophagy, Apoptosis, and Necrosis

      As part of a rescue program, the ER
      • Bhardwaj M.
      • Leli N.M.
      • Koumenis C.
      • Amaravadi R.K.
      Regulation of autophagy by canonical and non-canonical ER stress responses.
      or mitochondrial
      • Galluzzi L.
      • Yamazaki T.
      • Kroemer G.
      Linking cellular stress responses to systemic homeostasis.
      stress can stimulate autophagy, which is a form of cellular retrenchment to cope with troubled times. It is an intracellular process. Damaged organelles and other dysfunctional protein structures are removed through degradation in lysosomes. These can be recycled to conserve energy or excreted from the cell. Autophagy has been demonstrated in the STB of preeclamptic placentas
      • Oh S.Y.
      • Choi S.J.
      • Kim K.H.
      • Cho E.Y.
      • Kim J.H.
      • Roh C.R.
      Autophagy-related proteins, LC3 and Beclin-1, in placentas from pregnancies complicated by preeclampsia.
      and in the related condition of normotensive FGR.
      • Curtis S.
      • Jones C.J.
      • Garrod A.
      • Hulme C.H.
      • Heazell A.E.
      Identification of autophagic vacuoles and regulators of autophagy in villous trophoblast from normal term pregnancies and in fetal growth restriction.
      It is therefore part of the syncytial stress response. Mitophagy, autophagy of the mitochondria, is an important subset of autophagy,
      • Zimmermann M.
      • Reichert A.S.
      How to get rid of mitochondria: crosstalk and regulation of multiple mitophagy pathways.
      which has not yet been fully investigated in relation to normal or preeclamptic pregnancies.
      If normality cannot be restored, then the cell may be directed to apoptosis, as summarized elsewhere,
      • Hetz C.
      The unfolded protein response: controlling cell fate decisions under ER stress and beyond.
      which is a type of programmed cell death that may be triggered by extracellular or intracellular mechanisms. Apoptosis involves a cascade of proteolytic enzymes (caspases) that deconstruct the cell in a controlled way into subcellular membrane-bound vesicles (including pieces of fragmented nuclear DNA) that are released as apoptotic bodies. Because they retain the outer cell membrane, they are relatively noninflammatory, which facilitates simple removal by phagocytic cells. Apoptosis needs energy to be completed. In extremis, if the energy supply fails, then the cell falls apart in an uncontrolled way (necrosis), spilling its remnants into its extracellular environment.
      ER and mitochondrial stress are potent causes of apoptosis. This involves the intrinsic pathway of apoptosis and is mediated by what is called the mitochondrial permeability transition. The mitochondria swell, and the outer membrane may rupture with the loss of cytochrome C into the cytoplasm. This is a strong apoptotic signal, especially as it is combined with depletion of ATP because of loss of mitochondrial function.
      • Wu C.C.
      • Bratton S.B.
      Regulation of the intrinsic apoptosis pathway by reactive oxygen species.

      Syncytiotrophoblast and Apoptosis

      It seems counterintuitive that one vast syncytium can self-destruct in an organized way. However, there seem to be mechanisms that limit apoptosis to a small segment of the syncytium. This has been documented in villous trophoblast and considered to involve STB
      • Austgulen R.
      • Isaksen C.V.
      • Chedwick L.
      • Romundstad P.
      • Vatten L.
      • Craven C.
      Pre-eclampsia: associated with increased syncytial apoptosis when the infant is small-for-gestational-age.
      ,
      • Ishihara N.
      • Matsuo H.
      • Murakoshi H.
      • Laoag-Fernandez J.B.
      • Samoto T.
      • Maruo T.
      Increased apoptosis in the syncytiotrophoblast in human term placentas complicated by either preeclampsia or intrauterine growth retardation.
      and syncytial knots.
      • Fogarty N.M.
      • Ferguson-Smith A.C.
      • Burton G.J.
      Syncytial knots (Tenney-Parker changes) in the human placenta: evidence of loss of transcriptional activity and oxidative damage.
      There are different markers for histologic identification of apoptosis, including fragmented DNA. Syncytiotrophoblast changes can be difficult to distinguish clearly from those involving cytotrophoblast.
      • Longtine M.S.
      • Chen B.
      • Odibo A.O.
      • Zhong Y.
      • Nelson D.M.
      Villous trophoblast apoptosis is elevated and restricted to cytotrophoblasts in pregnancies complicated by preeclampsia, IUGR, or preeclampsia with IUGR.
      Because the nuclei of STB and cytotrophoblast are in close juxtaposition, they might be inadvertently grouped together as a multinuclear cluster in a chorionic villous. There is evidence that mononuclear cytotrophoblast is more susceptible than STB to apoptosis
      • Longtine M.S.
      • Chen B.
      • Odibo A.O.
      • Zhong Y.
      • Nelson D.M.
      Caspase-mediated apoptosis of trophoblasts in term human placental villi is restricted to cytotrophoblasts and absent from the multinucleated syncytiotrophoblast.
      and, being mononuclear cells, they can complete a typical apoptotic sequence. It is also possible that an apoptotic cytotrophoblast could fuse with, and deliver apoptotic markers to, its syncytial neighbor, inappropriately suggesting primary apoptotic activity in STB.
      • Burton G.J.
      • Skepper J.N.
      • Hempstock J.
      • Cindrova T.
      • Jones C.J.
      • Jauniaux E.
      A reappraisal of the contrasting morphological appearances of villous cytotrophoblast cells during early human pregnancy; evidence for both apoptosis and primary necrosis.
      The current evidence is that the STB displays markers of apoptotic activity, such as neoantigen M30,
      • Longtine M.S.
      • Chen B.
      • Odibo A.O.
      • Zhong Y.
      • Nelson D.M.
      Caspase-mediated apoptosis of trophoblasts in term human placental villi is restricted to cytotrophoblasts and absent from the multinucleated syncytiotrophoblast.
      positive TUNEL labeling,
      • Ishihara N.
      • Matsuo H.
      • Murakoshi H.
      • Laoag-Fernandez J.B.
      • Samoto T.
      • Maruo T.
      Increased apoptosis in the syncytiotrophoblast in human term placentas complicated by either preeclampsia or intrauterine growth retardation.
      apoptosis protease activating factor-1 (APAF-1),
      • Can M.
      • Guven B.
      • Bektas S.
      • Arikan I.
      Oxidative stress and apoptosis in preeclampsia.
      caspases 3 and 9,
      • De Falco M.
      • Fedele V.
      • Cobellis L.
      • et al.
      Immunohistochemical distribution of proteins belonging to the receptor-mediated and the mitochondrial apoptotic pathways in human placenta during gestation.
      and electron microscopic evidence of nuclear fragmentation.
      • Ishihara N.
      • Matsuo H.
      • Murakoshi H.
      • Laoag-Fernandez J.B.
      • Samoto T.
      • Maruo T.
      Increased apoptosis in the syncytiotrophoblast in human term placentas complicated by either preeclampsia or intrauterine growth retardation.
      Such activity becomes more evident toward term in normal placentas
      • Ishihara N.
      • Matsuo H.
      • Murakoshi H.
      • Laoag-Fernandez J.B.
      • Samoto T.
      • Maruo T.
      Increased apoptosis in the syncytiotrophoblast in human term placentas complicated by either preeclampsia or intrauterine growth retardation.
      ,
      • Halperin R.
      • Peller S.
      • Rotschild M.
      • Bukovsky I.
      • Schneider D.
      Placental apoptosis in normal and abnormal pregnancies.
      ,
      • Smith S.C.
      • Baker P.N.
      • Symonds E.M.
      Placental apoptosis in normal human pregnancy.
      or with preeclampsia
      • Leung D.N.
      • Smith S.C.
      • To K.F.
      • Sahota D.S.
      • Baker P.N.
      Increased placental apoptosis in pregnancies complicated by preeclampsia.
      or with FGR.
      • Smith S.C.
      • Baker P.N.
      • Symonds E.M.
      Increased placental apoptosis in intrauterine growth restriction.
      There are different forms of programmed cell death other than apoptosis. They include pyroptosis, netosis, and oncosis,
      • D’Arcy M.S.
      Cell death: a review of the major forms of apoptosis, necrosis and autophagy.
      all of which concern the fate of single mononuclear cells. Of these variants only pyroptosis has been studied in preeclampsia in relation to STB. Markers of pyroptosis are up-regulated in early-onset preeclampsia, can be induced by oxidative stress experimentally, and are correlated also with STB and ER stress in terms of an UPR.
      • Cheng S.B.
      • Nakashima A.
      • Huber W.J.
      • et al.
      Pyroptosis is a critical inflammatory pathway in the placenta from early onset preeclampsia and in human trophoblasts exposed to hypoxia and endoplasmic reticulum stressors.
      The question of how the pyroptotic program might be executed in a trophoblastic syncytium is not addressed.

      Syncytiotrophoblast, Apoptosis, and Fibrin Deposition

      In an ex vivo model of STB apoptosis, the importance of limiting the process was demonstrated.
      • Longtine M.S.
      • Barton A.
      • Chen B.
      • Nelson D.M.
      Live-cell imaging shows apoptosis initiates locally and propagates as a wave throughout syncytiotrophoblasts in primary cultures of human placental villous trophoblasts.
      In this context, a single focus of apoptosis spreads centrifugally and stops only when it reaches the edges of the syncytial structure. In vivo, such unrestrained STB apoptosis could cause widespread damage to the chorionic villi. However, small syncytial breaks are not uncommon in term placentas, so something limits their spread.
      • Nelson D.M.
      • Crouch E.C.
      • Curran E.M.
      • Farmer D.R.
      Trophoblast interaction with fibrin matrix. Epithelialization of perivillous fibrin deposits as a mechanism for villous repair in the human placenta.
      This seems to be achieved by perivillous deposits of fibrin or fibrinoid with which the breaks are associated. They cordon off the damage to allow the STB to reestablish its continuity.
      • Nelson D.M.
      • Crouch E.C.
      • Curran E.M.
      • Farmer D.R.
      Trophoblast interaction with fibrin matrix. Epithelialization of perivillous fibrin deposits as a mechanism for villous repair in the human placenta.
      The association of fibrinoid with STB showing apoptotic features without breaks has been shown by other investigators.
      • Mayhew T.M.
      Villous trophoblast of human placenta: a coherent view of its turnover, repair and contributions to villous development and maturation.
      ,
      • Austgulen R.
      • Chedwick L.
      • Vogt Isaksen C.
      • Vatten L.
      • Craven C.
      Trophoblast apoptosis in human placenta at term as detected by expression of a cytokeratin 18 degradation product of caspase.
      In summary, limited apoptosis of the STB is possible because it is restricted behind a barrier of fibrin or fibrinoid. The involvement of apoptosis with fibrin or fibrinoid has been confirmed by EM histology.
      • Nelson D.M.
      Apoptotic changes occur in syncytiotrophoblast of human placental villi where fibrin type fibrinoid is deposited at discontinuities in the villous trophoblast.
      However, apoptosis, as it is classically defined, does not occur.
      A break in the syncytium implies that its interior has been briefly exposed to the extracellular environment. This is an extreme form of cellular stress,
      • Draeger A.
      • Monastyrskaya K.
      • Babiychuk E.B.
      Plasma membrane repair and cellular damage control: the annexin survival kit.
      associated with a rapid influx of Ca2+ ion from the extracellular fluid that activates many stress signals. Membrane repair has been documented in other cell systems. It depends on annexins.
      • Draeger A.
      • Monastyrskaya K.
      • Babiychuk E.B.
      Plasma membrane repair and cellular damage control: the annexin survival kit.
      Annexin A5 (ANXA5) fills this specific role in STB.
      • Carmeille R.
      • Degrelle S.A.
      • Plawinski L.
      • et al.
      Annexin-A5 promotes membrane resealing in human trophoblasts.
      Apart from apoptosis, it is also possible that syncytium is damaged when a multinuclear aggregate (sprout or knot) breaks from the villous surface. Syncytial breaks have not been assessed in preeclampsia cases. However, because apoptosis is more prominent in preeclampsia placentas,
      • Can M.
      • Guven B.
      • Bektas S.
      • Arikan I.
      Oxidative stress and apoptosis in preeclampsia.
      ,
      • Ishihara N.
      • Matsuo H.
      • Murakoshi H.
      • Laoag-Fernandez J.B.
      • Samoto T.
      • Maruo T.
      Increased apoptosis in the syncytiotrophoblast in human term placentas complicated by either preeclampsia or intrauterine growth retardation.
      it would be predicted that the occurrence of breaks would be proportionally more common. An alternative possibility is that they are secondary to maternal immune attack. In favor of this is their association with complement deposition.
      • Rampersad R.
      • Barton A.
      • Sadovsky Y.
      • Nelson D.M.
      The C5b-9 membrane attack complex of complement activation localizes to villous trophoblast injury in vivo and modulates human trophoblast function in vitro.
      Not in favor is their relatively rare focal distribution. Maternal immune attack on the placental would likely be more widespread and destructive.
      During apoptosis, phosphatidylserine is exteriorized in the plasmalemma. It is strongly procoagulant but binds to ANXA5, which is an anticoagulant and uniformly present in the plasma membrane of normal STB.
      • Gourvas V.
      • Soulitzis N.
      • Konstantinidou A.
      • et al.
      Reduced ANXA5 mRNA and protein expression in pregnancies complicated by preeclampsia.
      ANXA5 forms an anticoagulant protective surface. In preeclampsia, STB labeling for ANXA5 is reduced,
      • Gourvas V.
      • Soulitzis N.
      • Konstantinidou A.
      • et al.
      Reduced ANXA5 mRNA and protein expression in pregnancies complicated by preeclampsia.
      implying that the STB is less well protected, possibly as a consequence of its stress.

      Secondary Pathology of Syncytiotrophoblast Stress with Apoptosis

      Perivillous fibrin deposition is a well-documented placental lesion. It has been classified as part of the pathology of maternal placental malperfusion
      • Parks W.T.
      Placental hypoxia: the lesions of maternal malperfusion.
      and is associated with early-onset preeclampsia.
      • Ogge G.
      • Chaiworapongsa T.
      • Romero R.
      • et al.
      Placental lesions associated with maternal underperfusion are more frequent in early-onset than in late-onset preeclampsia.
      Stasis in intervillous blood flow and maternal hypercoagulability may contribute, but it is reasonable to speculate that the fibrin deposition that seals off focal syncytial damage, secondary to syncytial stress, also contributes. In contrast, massive perivillous fibrin deposition (maternal floor infarction) seems to be an unrelated condition, not specifically associated with preeclampsia but with severe FGR. There is evidence that it represents maternal immune rejection of the fetus.
      • Romero R.
      • Whitten A.
      • Korzeniewski S.J.
      • et al.
      Maternal floor infarction/massive perivillous fibrin deposition: a manifestation of maternal antifetal rejection?.
      The STB is an immune frontier that separates a competent maternal immune system from her alloimmune child. Immunohistochemistry has demonstrated that, if it is breached, maternal leukocytes can gain access to the stroma of the chorionic villus and its resident fetal macrophages (Hofbauer cells).
      • Myerson D.
      • Parkin R.K.
      • Benirschke K.
      • Tschetter C.N.
      • Hyde S.R.
      The pathogenesis of villitis of unknown etiology: analysis with a new conjoint immunohistochemistry-in situ hybridization procedure to identify specific maternal and fetal cells.
      The alloimmune reactivity that ensues is the basis of villitis of unknown etiology. The sequence of events is under discussion in relation to the role of complement. Complement attack might be the primary cause of syncytial breaches
      • Kim C.J.
      • Romero R.
      • Chaemsaithong P.
      • Kim J.S.
      Chronic inflammation of the placenta: definition, classification, pathogenesis, and clinical significance.
      or secondary to maternal immune access to the alloimmune fetal stromal cells as proposed here. In favor of the latter proposal is the fact that the breeches as originally described were not associated with leukocyte infiltration but fibrin deposition and STB apoptosis,
      • Nelson D.M.
      • Crouch E.C.
      • Curran E.M.
      • Farmer D.R.
      Trophoblast interaction with fibrin matrix. Epithelialization of perivillous fibrin deposits as a mechanism for villous repair in the human placenta.
      which suggests that complement is involved secondarily.

      Syncytiotrophoblast-Derived Extracellular Vesicles

      Overall cell stress responses are wide ranging not only in their causes but also in their expression. A relatively recent addition to the repertoire is that of extracellular vesicle release.
      Membrane-bound extracellular vesicles (ECVs) are produced by most cell types. They are heterogeneous and include exosomes (approximately 100 nm in diameter), microvesicles (0.1–1 microm), and apoptotic bodies (0.5–5 microm). Exosomes and microvesicles are released constitutively or after activation in relation to pathologies, such as cancer, CVD, or cellular stress. In the latter regard, they are an intrinsic part of the cellular stress response. Any form of cell activation that increases intracellular calcium potently stimulates ECV release.
      • Berridge M.J.
      • Lipp P.
      • Bootman M.D.
      The versatility and universality of calcium signalling.
      ,
      • Morel O.
      • Morel N.
      • Jesel L.
      • Freyssinet J.M.
      • Toti F.
      Microparticles: a critical component in the nexus between inflammation, immunity, and thrombosis.
      Hence, cell stress activates the shedding of microvesicles from the cell surface, and if stress progresses to apoptosis, these will include apoptotic bodies.
      • Morel O.
      • Morel N.
      • Jesel L.
      • Freyssinet J.M.
      • Toti F.
      Microparticles: a critical component in the nexus between inflammation, immunity, and thrombosis.
      The vesicles have signaling functions and the ability to dock with other cells
      • French K.C.
      • Antonyak M.A.
      • Cerione R.A.
      Extracellular vesicle docking at the cellular port: extracellular vesicle binding and uptake.
      to which they can carry and donate cargo
      • van Niel G.
      • D’Angelo G.
      • Raposo G.
      Shedding light on the cell biology of extracellular vesicles.
      to be taken up and used by the recipient cell. The cargoes include regulatory molecules, such as miRNAs, lipids, and DNA. Their purpose is to alert neighboring and distant cells to a problem—to convert local stress into systemic stress for severe problems.
      • Galluzzi L.
      • Yamazaki T.
      • Kroemer G.
      Linking cellular stress responses to systemic homeostasis.
      STB releases ECVs into the maternal circulation, in increasing quantities toward term and substantially more if the mother has preeclampsia, as previously summarized.
      • Tannetta D.
      • Masliukaite I.
      • Vatish M.
      • Redman C.
      • Sargent I.
      Update of syncytiotrophoblast derived extracellular vesicles in normal pregnancy and preeclampsia.
      They are potential circulating biopsies of the STB
      • Tannetta D.
      • Collett G.
      • Vatish M.
      • Redman C.
      • Sargent I.
      Syncytiotrophoblast extracellular vesicles - circulating biopsies reflecting placental health.
      but are a minor part of the total circulating ECVs, of which most are derived from maternal platelets. Although the technology to isolate them from peripheral blood samples is improving, it is still unreliable. A unique feature of pregnancy is the release of multinuclear syncytial vesicles. These are not well studied because they are retained in the maternal pulmonary vasculature. Apart from postmortem studies, they can be detected in uterine vein blood samples taken at cesarean delivery. They may represent deported syncytial sprouts (which are physiological) or detached syncytial knots (indicators of STB stress). Their numbers are increased in preeclampsia and include vesicles with the phenotype of syncytial knots.
      • Johansen M.
      • Redman C.W.
      • Wilkins T.
      • Sargent I.L.
      Trophoblast deportation in human pregnancy--its relevance for pre-eclampsia.
      This is important because it may indicate the shedding of unwanted materials for disposal elsewhere and reflect STB stress.
      The potential for STB ECVs to signal to maternal tissues is large. Current research touches only a small fraction of an issue of undetermined complexity. They can deliver their cargoes to endothelial cells.
      • Cronqvist T.
      • Tannetta D.
      • Mörgelin M.
      • et al.
      Syncytiotrophoblast derived extracellular vesicles transfer functional placental miRNAs to primary human endothelial cells.
      For example, we find that in preeclampsia, the STB ECVs bear reduced nitric oxide synthase
      • Motta-Mejia C.
      • Kandzija N.
      • Zhang W.
      • et al.
      Placental vesicles carry active endothelial nitric oxide synthase and their activity is reduced in preeclampsia.
      ; increased neprilysin
      • Gill M.
      • Motta-Mejia C.
      • Kandzija N.
      • et al.
      Placental syncytiotrophoblast-derived extracellular vesicles carry active NEP (neprilysin) and are increased in preeclampsia.
      ; a peptidase that can cause hypertension, increased trophoblast glycoprotein,
      • Alam S.M.K.
      • Jasti S.
      • Kshirsagar S.K.
      • et al.
      Trophoblast glycoprotein (TPGB/5T4) in human placenta: expression, regulation, and presence in extracellular microvesicles and exosomes.
      which is an example of an oncofetal protein; and, finally, a novel family of noncoding RNA, namely, T halves,
      • Cooke W.R.
      • Cribbs A.
      • Zhang W.
      • et al.
      Maternal circulating syncytiotrophoblast-derived extracellular vesicles contain biologically active 5'-tRNA halves.
      which are induced by stress by angiogenin that has not yet been fully characterized in relation to preeclampsia.
      • Su Z.
      • Kuscu C.
      • Malik A.
      • Shibata E.
      • Dutta A.
      Angiogenin generates specific stress-induced tRNA halves and is not involved in tRF-3-mediated gene silencing.
      ECVs are an important component of the STB stress response so they need to be well-characterized preeclampsia as do their cargoes of miRNAs.
      • Aplin J.D.
      • Myers J.E.
      • Timms K.
      • Westwood M.
      Tracking placental development in health and disease.

      Senescence

      Cellular senescence is a state of permanent cell cycle arrest. The cell cannot reenter the cell cycle in response to external stimuli, such as growth factors, and resists apoptotic stimuli.
      • Sultana Z.
      • Maiti K.
      • Dedman L.
      • Smith R.
      Is there a role for placental senescence in the genesis of obstetric complications and fetal growth restriction?.
      Senescent cells continue to be metabolically active. They maintain a low-grade inflammatory response (typically secretion of interleukin [IL-6 and IL-8), with what is called the senescence-associated secretory phenotype (SASP), which underlies chronic inflammatory conditions of old age.
      • Coppé J.P.
      • Patil C.K.
      • Rodier F.
      • et al.
      Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor.
      Senescent tissues have to “make do” with a population of dysfunctional cells that are damaged but retain some useful functions. The lay perception of senescence is of a weakened state. In fact, senescent cells are stable and persistent and can have important barrier functions in, for example, neoplastic tissues.
      • Narita M.
      • Nũnez S.
      • Heard E.
      • et al.
      Rb-mediated heterochromatin formation and silencing of E2F target genes during cellular senescence.
      Premature senescence occurs with a long-lasting stress response to external stimuli, which wear the system out faster. Replicative senescence occurs because each cell has its “cell by date” determined by telomeres, which are caps that stabilize the ends of individual chromosome. The caps are progressively eroded during cell division.
      • Childs B.G.
      • Durik M.
      • Baker D.J.
      • van Deursen J.M.
      Cellular senescence in aging and age-related disease: from mechanisms to therapy.
      The degree of shortening depends on how many divisions the cell has completed.
      • Childs B.G.
      • Durik M.
      • Baker D.J.
      • van Deursen J.M.
      Cellular senescence in aging and age-related disease: from mechanisms to therapy.
      Increased shortening exposes chromosomal ends to damage or end-to-end fusion with other telomeres either of which can activate a DNA damage response. Telomeres are sensitive to oxidative stress, which accelerates their dysfunction. This is because they are rich in guanine nucleotides, which are unusually vulnerable to oxidative stress.
      • Shen J.
      • Gammon M.D.
      • Terry M.B.
      • et al.
      Telomere length, oxidative damage, antioxidants and breast cancer risk.
      STB is formed by two processes—fusion and senescence. Cell fusion occurs in other contexts as a stress response, for example, to infection from the measles virus.
      • Chuprin A.
      • Gal H.
      • Biron-Shental T.
      • et al.
      Cell fusion induced by ERVWE1 or measles virus causes cellular senescence.
      The human trophoblast depends on a retroviral gene ERVWE1 encoding syncytin-1. Trophoblast fusion itself triggers cell cycle arrest, conventionally deemed a feature of senescence.
      • Chuprin A.
      • Gal H.
      • Biron-Shental T.
      • et al.
      Cell fusion induced by ERVWE1 or measles virus causes cellular senescence.
      Several markers of senescence (p53, p21, p16, and senescence-associated beta galactosidase [SA-βGal]) have been documented in normal STB
      • Chuprin A.
      • Gal H.
      • Biron-Shental T.
      • et al.
      Cell fusion induced by ERVWE1 or measles virus causes cellular senescence.
      of which p16, p21, and SA-βGal progressively increase with maturity, although they remain stable in villous cytotrophoblast, except for SA-βGal, which declines.
      • Higuchi S.
      • Miyamoto T.
      • Kobara H.
      • et al.
      Trophoblast type-specific expression of senescence markers in the human placenta.
      The endpoint is that the placenta is coated in a resilient and relatively stable cellular barrier and that it is senescence that appears to be essential for its formation and functions. Increased production of inflammatory cytokines is certainly a feature of the preeclamptic placenta, but whether they are produced specifically by the third-trimester STB itself and could be classified as SASP is unclear. In other contexts, long-standing cellular senescence is associated with senescence-associated heterochromatic foci (SAHF), which are clusters of cells with characteristic heterochromatic nuclei.
      • Swanson E.C.
      • Rapkin L.M.
      • Bazett-Jones D.P.
      • Lawrence J.B.
      Unfolding the story of chromatin organization in senescent cells.
      Presumably, the equivalent structures in STB are the syncytial knots, but further characterization is needed. The generation of SAHF depends on a senescence pathway mediated by 2 tumor suppressor proteins, retinoblastoma protein (Rb) and p16.
      • Narita M.
      • Nũnez S.
      • Heard E.
      • et al.
      Rb-mediated heterochromatin formation and silencing of E2F target genes during cellular senescence.
      As stated above, p16 is expressed in STB. However, there is only indirect evidence for that of Rb.
      • Chuprin A.
      • Gal H.
      • Biron-Shental T.
      • et al.
      Cell fusion induced by ERVWE1 or measles virus causes cellular senescence.
      If the STB must be senescent to perform its functions, further stress that comes with aging, with the associated damage to the nuclei, which provokes a DNA damage response and impaired organellar functions, such as that of the mitochondria, may contribute to a decline in STB function.
      These aspects are being better characterized now
      • Sultana Z.
      • Maiti K.
      • Dedman L.
      • Smith R.
      Is there a role for placental senescence in the genesis of obstetric complications and fetal growth restriction?.
      and together constitute the phenotype of what we call the “twilight placenta.”

      The “Twilight Placenta”: What is it, What are its Clinical Consequences, and how is it Diagnosed?

      We previously reviewed the evidence that term preeclampsia results from a late decline in placental function.
      • Redman C.W.
      • Staff A.C.
      Preeclampsia, biomarkers, syncytiotrophoblast stress, and placental capacity.
      The evolution of this decline has now been clearly documented in samples taken from normal pregnancies at 37 weeks of gestation to postterm pregnancy. Progression from term to postterm pregnancy is associated with larger changes in the STB in terms of lipid and DNA or RNA oxidation and the distribution and size of autophagosomes. The most extreme changes, the phenotype of the twilight placenta, were seen in samples from cases with unexplained stillbirths. The patterns could be replicated in placental explants stressed by culture in a serum-free medium.
      • Maiti K.
      • Sultana Z.
      • Aitken R.J.
      • et al.
      Evidence that fetal death is associated with placental aging.
      A similar pattern was also clearly shown in another study using different markers, including the tumor suppressor protein p21, lipofuscin (formed from oxidized lipids and protein), 8-hydroxy-2'-deoxyguanosine (a marker for DNA and RNA oxidation), and activation of the DNA damage response.
      • Cindrova-Davies T.
      • Fogarty N.M.E.
      • Jones C.J.P.
      • Kingdom J.
      • Burton G.J.
      Evidence of oxidative stress-induced senescence in mature, post-mature and pathological human placentas.
      That this decline is caused by “senescence” is informative but incomplete. It is clear that senescence progresses in STB, but in terms of cell cycle arrest, STB is always senescent from the moment it forms. Thus, when “markers” of senescence increase toward term, as they do, what does this signify and what causes the added stress? It has been suggested that the stress markers may be “donated” by cytotrophoblast, during fusion. As cytotrophoblast does replicate and must have its own senescence program, this may be reflected in the markers detected in STB. Term is also characterized by increasing hypoxia.
      • Soothill P.W.
      • Nicolaides K.H.
      • Rodeck C.H.
      • Campbell S.
      Effect of gestational age on fetal and intervillous blood gas and acid-base values in human pregnancy.
      This may be caused by, and is certainly associated with, physical constraints on placental growth, which is restricted by the size of the uterus, by increasing the compression of the chorionic villi, and by limits on uteroplacental perfusion. We have previously called this restricted placental capacity.
      • Redman C.W.
      • Staff A.C.
      Preeclampsia, biomarkers, syncytiotrophoblast stress, and placental capacity.
      These limits would create the stresses that we have highlighted and cause cytotrophoblast senescence.
      We propose the hypothesis that the twilight phase immediately precedes STB failure when its renewal by fusion with cytotrophoblast is no longer enough. There is circumstantial evidence that we have cited, however, direct evidence needs to be sought. The clinical correlates are clear: increasing preeclampsia, late-onset FGR, and “unexplained” stillbirth are all well-documented consequences in postterm pregnancies.
      How many pregnancies end with a twilight placenta is not known. Much clinical research is based on comparisons between normal and abnormal. However, at term, what is a normal placenta? At best, it is a declining placenta delivered before maternal preeclampsia occurs. Today’s normal placenta may be tomorrow’s preeclamptic placenta.

      Exceptional Causes of Syncytiotrophoblast Stress and Preeclampsia

      So far, we have considered malplacentation and senescence as the two main causes of STB stress and preeclampsia. However, there are other unusual causes, including infection with parvovirus and immune attack by antiphospholipid autoantibodies. The former may be associated with the mirror syndrome, comprising edema that affects both the mother and fetus.
      • Braun T.
      • Brauer M.
      • Fuchs I.
      • et al.
      Mirror syndrome: a systematic review of fetal associated conditions, maternal presentation and perinatal outcome.
      In addition, the mother may develop preeclampsia, which regresses after recovery from the viral infection. In such cases, the STB demonstrates apoptosis,
      • Jordan J.A.
      • Butchko A.R.
      Apoptotic activity in villous trophoblast cells during B19 infection correlates with clinical outcome: assessment by the caspase-related M30 CytoDeath antibody.
      while maternal sFlt-1 and PlGF, biomarkers of STB stress, increase and decrease, respectively,
      • Goa S.
      • Mimura K.
      • Kakigano A.
      • et al.
      Normalisation of angiogenic imbalance after intra-uterine transfusion for mirror syndrome caused by parvovirus B19.
      as they do in preeclampsia of other causes. Antiphospholipid autoantibodies may be directed against cardiolipin located in the mitochondria or annexin of which the latter is a protective layer for STB. They are mostly associated with early pregnancy loss, but associations with preeclampsia have been documented. Anticardiolipin antibodies
      • Zussman R.
      • Xu L.Y.
      • Damani T.
      • et al.
      Antiphospholipid antibodies can specifically target placental mitochondria and induce ROS production.
      bind to purified placental mitochondria and activate the ROS production. Hence, there is a possible mechanism to explain their adverse effects on pregnancy outcomes, but so far, it has not been possible to invoke participation of STB directly. It is likely that there are other unrecognized causes of STB stress.

      Syncytiotrophoblast Stress, Prediction, Diagnosis, and Perinatal Outcome

      Stress causes STB to activate a new and wide-ranging transcriptional program of protein synthesis through the UPR. Proteins that promote homeostasis are prioritized, and the production of others are restricted (“negative” stress response proteins). Some enter the maternal circulation and contribute to the preeclampsia syndrome. They are the “biomarkers” of the condition. Some increase and some decline. Of special interest are those that are exclusively produced by STB or at least with a minimal contribution from maternal systems. A comprehensive list is not appropriate here, but sFlt-1,
      • Maynard S.E.
      • Min J.Y.
      • Merchan J.
      • et al.
      Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia.
      activin A and inhibin A,
      • Muttukrishna S.
      • Knight P.G.
      • Groome N.P.
      • Redman C.W.
      • Ledger W.L.
      Activin A and inhibin A as possible endocrine markers for pre-eclampsia.
      corticotropin-releasing hormone,
      • Perkins A.V.
      • Linton E.A.
      • Eben F.
      • Simpson J.
      • Wolfe C.D.
      • Redman C.W.
      Corticotrophin-releasing hormone and corticotrophin-releasing hormone binding protein in normal and pre-eclamptic human pregnancies.
      soluble endoglin,
      • Venkatesha S.
      • Toporsian M.
      • Lam C.
      • et al.
      Soluble endoglin contributes to the pathogenesis of preeclampsia.
      and plasminogen activator-1
      • Estellés A.
      • Gilabert J.
      • Aznar J.
      • Loskutoff D.J.
      • Schleef R.R.
      Changes in the plasma levels of type 1 and type 2 plasminogen activator inhibitors in normal pregnancy and in patients with severe preeclampsia.
      are some examples. Of these, the first is used in clinical practice often coupled with measures of PlGF.
      • Torry D.S.
      • Wang H.S.
      • Wang T.H.
      • Caudle M.R.
      • Torry R.J.
      Preeclampsia is associated with reduced serum levels of placenta growth factor.
      In trophoblast, various stressors, including tumor necrosis factor alpha (TNFα), angiotensin II, or hypoxia, induce sFlt-1.
      • Xiong Y.
      • Liebermann D.A.
      • Holtzman E.J.
      • Jeronis S.
      • Hoffman B.
      • Geifman-Holtzman O.
      Preeclampsia-associated stresses activate Gadd45a signaling and sFlt-1 in placental explants.
      This depends on a stress sensor GADD45a, which responds to DNA damage within cells, including STB.
      • Xiong Y.
      • Liebermann D.A.
      • Holtzman E.J.
      • Jeronis S.
      • Hoffman B.
      • Geifman-Holtzman O.
      Preeclampsia-associated stresses activate Gadd45a signaling and sFlt-1 in placental explants.
      PlGF declines with the stress of preeclampsia and is an example of a negative stress factor. Its measurement in maternal blood may be complicated by the partition between free and bound PlGFs.
      • Lecarpentier E.
      • Zsengellér Z.K.
      • Salahuddin S.
      • et al.
      Total Versus free placental growth factor levels in the pathogenesis of preeclampsia.
      However, researchers agree that its production by trophoblast is reduced with hypoxia.
      • Torry D.S.
      • Mukherjea D.
      • Arroyo J.
      • Torry R.J.
      Expression and function of placenta growth factor: implications for abnormal placentation.
      • Debiève F.
      • Depoix C.
      • Gruson D.
      • Hubinont C.
      Reversible effects of oxygen partial pressure on genes associated with placental angiogenesis and differentiation in primary-term cytotrophoblast cell culture.
      • Chang M.
      • Mukherjea D.
      • Gobble R.M.
      • Groesch K.A.
      • Torry R.J.
      • Torry D.S.
      Glial cell missing 1 regulates placental growth factor (PGF) gene transcription in human trophoblast.
      Maternal circulating levels of sFlt-1 and PlGF are therefore biomarkers of STB stress when they are increased and decreased, respectively. The time courses of their circulating measures in normal pregnancies are clear.
      • Levine R.J.
      • Maynard S.E.
      • Qian C.
      • et al.
      Circulating angiogenic factors and the risk of preeclampsia.
      From about 30 to 32 weeks’ gestation, something causes STB stress, which progressively increases toward term (Figure 6). Remember that these are pregnancies with normal outcomes but display relatively universal and consistent changes ascribable to STB stress. This is consistent with the concept of the limited lifespan of the placenta. At term, its function declines, leading to increasing rates of preeclampsia, late-onset FGR, and unexplained term stillbirths.
      Figure thumbnail gr6
      Figure 6Syncytiotrophoblast becomes stressed in normal pregnancies after 32 weeks' gestation
      Measurements in normal pregnancy of serum PlGF (blue) and sFlt-1 (red). A, At 30 to 32 weeks’ gestation, the sFlt-1 begins to rise, whereas PlGF falls, both signs of syncytiotrophoblast stress. B, The stress patterns of established preeclampsia are shown, which are more extreme after the diagnosis has been made. C, By the end of normal pregnancy, the stress pattern begins to resemble that of preeclampsia. Adapted from Levine et al.
      • Levine R.J.
      • Maynard S.E.
      • Qian C.
      • et al.
      Circulating angiogenic factors and the risk of preeclampsia.
      PE, preeclampsia; PlGF, placental growth factor; sFlt-1, soluble fms-like tyrosine kinase-1.
      Redman. Syncytiotrophoblast stress in preeclampsia. Am J Obstet Gynecol 2022.
      In summary, a blood-borne biomarker that is a stress product of STB reflects the health of the STB. sFlt-1 and PlGF are the first to be brought into practice. However, there are many more that await investigation. Of these, STB microvesicles are promising. They carry many signals, which can be delivered in a targeted way. They are not easy to study in blood samples, but the techniques are rapidly improving. If they fulfill the promise of enabling peripheral biopsy of STB, we speculate that it could become possible to determine the “biological age” of a placenta rather than its gestational age. Some placentas at 40 weeks’ gestation may have a potential life expectancy of 3 to 4 more weeks. Others at 38 weeks’ gestation may be in the final stages of STB failure.

      Competing Odds

      As STB stress increases with advancing gestational age so does the likelihood of an adverse outcome (preeclampsia, late-onset FGR, stillbirth). If delivery never occurred, all pregnancies would presumably be eventually affected. However, they do not, because delivery supervenes. In most instances, it does so in good time, and the outcome is normal. In effect, there is a race between 2 biological processes: spontaneous labor or decompensated STB stress. Current theories of what triggers spontaneous labor concern senescence in the amniochorion.
      • Phillippe M.
      Cell-free fetal DNA, telomeres, and the spontaneous onset of parturition.
      ,
      • Menon R.
      Initiation of human parturition: signaling from senescent fetal tissues via extracellular vesicle mediated paracrine mechanism.
      Senescent amniochorionic cells activate an inflammatory SASP, which in time stimulates myometrial contractility and labor. Hence at the end of pregnancy, there are 2 clocks counting down to competing outcomes.
      This can be expressed in statistical terms as competing odds.
      • Wright D.
      • Akolekar R.
      • Syngelaki A.
      • Poon L.C.
      • Nicolaides K.H.
      A competing risks model in early screening for preeclampsia.
      In this study of nearly 66,000 pregnancies, the mean time for getting preeclampsia was estimated to be 55 weeks. Hence, only a small proportion (2.7%) succumbed. Postmaturity pregnancy beyond 41 to 42 weeks is now rare with modern antenatal care, and induction at an earlier gestational age is commonplace if hypertension begins to develop (Figure 7). This practice developed over the last 40 years has prevented an unknown but not insubstantial number of cases of preeclampsia.
      Figure thumbnail gr7
      Figure 7Preeclampsia, competing odds and censorship of outcome by delivery
      At the end of pregnancy, there is a race between spontaneous labor and preeclampsia, which can preempt or censor the occurrence or preeclampsia and can be modeled statistically as competing odds. Women who get early-onset preeclampsia (A) fully express that outcome without censorship. At term (B), as the rate of spontaneous delivery increases, the evolution of incipient preeclampsia is curtailed in an unknown proportion, but many women show early signs of disease (see text). In modern clinical practice, delivery beyond 42 weeks’ gestation is avoided, there is no censorship, and abnormal outcomes are prevented by induced delivery (C). Adapted from Wright et al.
      • Wright D.
      • Akolekar R.
      • Syngelaki A.
      • Poon L.C.
      • Nicolaides K.H.
      A competing risks model in early screening for preeclampsia.
      Redman. Syncytiotrophoblast stress in preeclampsia. Am J Obstet Gynecol 2022.

      The Preeclampsia Syndrome—more than a Single Disorder?

      One of the clear messages that comes from the delineation of the pathophysiology of early- and late-onset preeclampsia as using different pathways to arrive at the common endpoint, STB stress (Figure 8), is that there may be more than “one kind” of preeclampsia. Are there subsets of preeclampsia other than early and late onset? This likelihood is supported by several lines of evidence.
      Figure thumbnail gr8
      Figure 8Two-stage model of preeclampsia: early and late placental dysfunction
      Early-onset disease is characterized by a long first stage and more severe placental and fetal sequelae. Late-onset disease has a shorter first stage and less severe sequelae if delivery supervenes normally. They both cause syncytiotrophoblast stress, associated with the specific stresses as listed. Early-onset preeclampsia is based predominantly on spiral artery dysfunction that causes focal oxidative stress in the relevant territory of the artery. Late-onset disease is more diffuse and affects syncytial health in a less focused way. Adapted from Redman and Redman and Staff.
      • Redman C.W.
      Current topic: pre-eclampsia and the placenta.
      ,
      • Redman C.W.
      • Staff A.C.
      Preeclampsia, biomarkers, syncytiotrophoblast stress, and placental capacity.
      Redman. Syncytiotrophoblast stress in preeclampsia. Am J Obstet Gynecol 2022.
      The clinical presentation of preeclampsia is remarkably diverse. The condition may evolve relatively slowly or may progress explosively to severe illness in 24 hours. Organ involvement is also quite variable. In some women, hepatic and coagulation changes predominate with hemolysis, elevated liver enzymes, and low platelet count; in others, cortical blindness and cardiac or CNS findings are of major impact.
      The long-range implications of preeclampsia are quite different in subsets of women. With preeclampsia delivering at term in only 1 pregnancy, the risk of later life CVD is about doubled.
      • Bellamy L.
      • Casas J.P.
      • Hingorani A.D.
      • Williams D.J.
      Pre-eclampsia and risk of cardiovascular disease and cancer in later life: systematic review and meta-analysis.
      However, having had preeclampsia in more than 1 pregnancy results in a 3.5-fold
      • Funai E.F.
      • Friedlander Y.
      • Paltiel O.
      • et al.
      Long-term mortality after preeclampsia.
      risk, and the women with preeclampsia before 34 weeks’ gestation have almost a 10-fold risk.
      • Mongraw-Chaffin M.L.
      • Cirillo P.M.
      • Cohn B.A.
      Preeclampsia and cardiovascular disease death: prospective evidence from the child health and development studies cohort.
      Laboratory findings are also inconsistent with considerable overlap between women defined as preeclamptic and those who have normal outcomes. This is more of a problem at term when normality is being replaced by the problems of the aging placenta.
      Differences in laboratory findings also include measurement of factors considered as important parts of the pathophysiological pathways to preeclampsia, such as inflammation, oxidative stress,
      • Hubel C.A.
      • McLaughlin M.K.
      • Evans R.W.
      • Hauth B.A.
      • Sims C.J.
      • Roberts J.M.
      Fasting serum triglycerides, free fatty acids, and malondialdehyde are increased in preeclampsia, are positively correlated, and decrease within 48 hours post partum.
      and angiogenic imbalance.
      • Powers R.W.
      • Roberts J.M.
      • Plymire D.A.
      • et al.
      Low placental growth factor across pregnancy identifies a subset of women with preterm preeclampsia: type 1 versus type 2 preeclampsia?.
      In a study from our group of 50 women with preeclampsia followed up longitudinally through pregnancy, PlGF was minimally different from concentrations in normal pregnant women in about half of the women, whereas the other half had PlGF concentrations below the fifth centile of normal pregnancy throughout gestation.
      • Powers R.W.
      • Roberts J.M.
      • Plymire D.A.
      • et al.
      Low placental growth factor across pregnancy identifies a subset of women with preterm preeclampsia: type 1 versus type 2 preeclampsia?.
      This may have been an index of malplacentation, which would explain the higher incidence of preterm preeclampsia in this group.
      Although not as convincing, further critical evidence is the nearly universal observation that attempts at preventive treatment for preeclampsia follow a common pathway. Early, small studies are successful,
      • Chappell L.C.
      • Seed P.T.
      • Briley A.L.
      • et al.
      Effect of antioxidants on the occurrence of pre-eclampsia in women at increased risk: a randomised trial.
      whereas larger studies stimulated by these successes are not effective.
      • Roberts J.M.
      • Myatt L.
      • Spong C.Y.
      • et al.
      Vitamins C and E to prevent complications of pregnancy-associated hypertension.
      An obvious explanation is, of course, publication bias with small successful studies being reported, whereas small unsuccessful studies are not. Although this is a likely explanation, an alternative is the possibility that small studies succeed because of a homogeneous population and larger multicenter studies are more heterogeneous and more likely to include different subsets of preeclampsia.
      Accepting preeclampsia as a multisystem syndrome with different subtypes raises other variations on the theme. Recently in the United States, the rate of hypertensive disorders during pregnancy has increased as has chronic hypertension.
      • Lo J.O.
      • Mission J.F.
      • Caughey A.B.
      Hypertensive disease of pregnancy and maternal mortality.
      However, despite this, deaths from preeclampsia, formerly the third leading cause of maternal deaths, are now the sixth most common precursor of maternal mortality.
      • Creanga A.A.
      • Syverson C.
      • Seed K.
      • Callaghan W.M.
      Pregnancy-related mortality in the United States, 2011-2013.
      The leading cause of maternal mortality in the United States is CVD. Interestingly, there is a compelling relationship between preeclampsia and several CVDs. The odds of maternal mortality from heart failure are 10- to 30-fold higher for women with preeclampsia than for women with normal pregnancy outcome.
      • Mogos M.F.
      • Piano M.R.
      • McFarlin B.L.
      • Salemi J.L.
      • Liese K.L.
      • Briller J.E.
      Heart failure in pregnant women: a concern across the pregnancy continuum.
      The long-term implication of preeclampsia for ischemic CVD is well recognized. Less appreciated are later life deaths from heart failure, in particular heart failure with preserved ejection fraction.
      • Honigberg M.C.
      • Zekavat S.M.
      • Aragam K.
      • et al.
      Long-term cardiovascular risk in women with hypertension during pregnancy.
      There is also a strong association of peripartum cardiomyopathy with preeclampsia.
      • Lindley K.J.
      • Conner S.N.
      • Cahill A.G.
      • Novak E.
      • Mann D.L.
      Impact of preeclampsia on clinical and functional outcomes in women with peripartum cardiomyopathy.
      Interestingly, in an assessment of gene variations associated with CVD, these variants were overrepresented in women with previous preeclampsia.
      • Gammill H.S.
      • Chettier R.
      • Brewer A.
      • et al.
      Cardiomyopathy and preeclampsia.
      Finally, there is evidence of subclinical cardiac dysfunction on echocardiographic assessment of women with preeclampsia and women with resolved preeclampsia.
      • Melchiorre K.
      • Sharma R.
      • Thilaganathan B.
      Cardiovascular implications in preeclampsia: an overview.
      It is important to remember that the clinical findings, which for years characterized the preeclampsia syndrome, hypertension, and proteinuria, were not chosen as the most common or most important. However, these were identified serendipitously in the early 20th century as being associated with eclampsia, which at that time was considered a pregnancy-specific seizure disorder. Although proteinuria has been abandoned as a requirement of preeclampsia, is it possible that these CVDs may be a part of the preeclampsia syndrome without hypertension?
      With this consideration, is it possible that preterm birth, growth restriction (without maternal findings), and stillbirth, which share many placental pathologic and epidemiologic findings with preeclampsia may also share pathophysiological pathways but with a different clinical phenotype?
      In summary, identifying different variants of preeclampsia could have a major impact on the prevention and prediction of the syndrome. These advances might also be shared with other disorders not considered part of the preeclampsia syndrome.

      Identifying Subtypes of the Preeclampsia Syndrome

      To begin to search for subtypes, it would require that we determine how such subtypes arise. Although there is a common endpoint, STB stress, in the pathogenic pathway to preeclampsia, there is heterogeneity in its antecedents: in possible additional routes to STB stress, in the multiple uncharted signals that stressed STB sends to the mother, and in the range of responses by different mothers (Figure 9).
      Figure thumbnail gr9
      Figure 9Evolution of the 2-stage model
      Here, we consider the evolving concept of the original 2-stage model. In the original model, impaired placentation secondary to failed spiral artery remodeling resulted in the production of a placental product, which caused diffuse maternal endothelial dysfunction and the maternal preeclampsia syndrome. A, We present a current concept. In this model, we proposed that the crucial change in the placenta is SBT stress because of failed vascular remodeling or placental growth or compression and senescence and rarely by parvovirus or antiphospholipid antibodies, with subsequent release of SBT products. These factors lead to the maternal preeclampsia syndrome. They are proposed to be influenced by the maternal constitution (genetics and epigenetics) and lifestyle (eg, environment, diet, obesity). This is represented by the blue shading, indicating an interaction with all components of the model (detailed in ). B, A speculative hypothesis extending this model is illustrated (red). The hypothesis proposes that unknown factors may also lead to SBT stress. It posits increased complexity in the number and pathways of SBT signals that could adversely affect the mother. The maternal response is proposed to also be complex with effects of different signals and pathways predominating in different women. These may result in preeclampsia in some women or, in others, different placentally mediated abnormalities, fetal growth restriction without preeclampsia, stillbirth, or other disorders. Adapted from Redman and Roberts et al.
      • Redman C.W.
      Current topic: pre-eclampsia and the placenta.
      ,
      • Roberts J.M.
      • Taylor R.N.
      • Musci T.J.
      • Rodgers G.M.
      • Hubel C.A.
      • McLaughlin M.K.
      Preeclampsia: an endothelial cell disorder.
      SBT, syncytiotrophoblast.
      Redman. Syncytiotrophoblast stress in preeclampsia. Am J Obstet Gynecol 2022.
      Maternal factors contribute to this heterogeneity of preeclampsia. Chronic hypertension, obesity, nutrition, environmental toxins, genetic predisposition, and other factors known and unknown contribute to an increased risk of preeclampsia and influence pathophysiological responses. The question to be answered ultimately resolves into the question of the mechanisms of the interaction between maternal factors and the STB stress (Figure 10). Are these effects secondary to increasing propensity to STB stress, increased maternal response to this insult, or perhaps both are at play for some of these factors?
      Figure thumbnail gr10
      Figure 10Maternal and paternal interactions and preeclampsia and SBT stress
      Four ways in which maternal factors influence the expression of SBT stress. A, Placentation is influenced by maternal and paternal (fetal) immunologic interactions. There are also maternal-specific determinants, including decidual preparation and preconditioning of vessels perfusing the placental bed by previous pregnancies. B, There are other recognized and not yet identified maternal factors that contribute to SBT stress. C, Maternal factors influence the rate of placental senescence or SBT stress. D, The response to the SBT stress signals, both qualitatively and quantitatively, will be different in different women.
      SBT, syncytiotrophoblast.
      Redman. Syncytiotrophoblast stress in preeclampsia. Am J Obstet Gynecol 2022.
      There are 2 ways to search for different disease pathways. The first is the traditional hypothesis testing driven by questions generated by clinical observation. Why are early- and late-onset preeclampsia so different in clinical features of long-range morbidity epidemiologic outcomes? We present the evidence for different pathways to STB stress.
      • Redman C.W.
      • Sargent I.L.
      • Staff A.C.
      IFPA Senior Award lecture: making sense of pre-eclampsia - two placental causes of preeclampsia?.
      Does obesity result in a different subtype of preeclampsia? Is the same disorder associated with undernutrition in low-resource settings? Do women with preeclampsia with markedly different biomarkers (eg, PlGF) pursue a different pathway to preeclampsia?
      • Powers R.W.
      • Roberts J.M.
      • Plymire D.A.
      • et al.
      Low placental growth factor across pregnancy identifies a subset of women with preterm preeclampsia: type 1 versus type 2 preeclampsia?.
      Are women in whom aspirin is effective different from women in whom it is not? These questions generate testable hypotheses.
      The second approach is discovery science. The availability of powerful analytical approaches permits investigators to perform nondirected studies of analytes assayed by state-of-the-art techniques (proteomics, metabolomics, genomics, etc.). From these approaches, systems analyze group findings into clusters, indicating potential pathways to disease, once again generating testable hypotheses. An especially powerful strategy, again taking advantage of currently available analytical power, is the attempt to combine different analytes from different “omics” strategies with linked demographic and clinical findings.

      Clinical Implications

      The problem of late-onset preeclampsia has received minimal attention not only in terms of the pathophysiology but also in the prediction and prevention of preeclampsia. Thus, prediction has been largely considered unimportant because delivery is considered a “safe” option. Interestingly, this approach has even been extended to normal pregnancy, in which the increase in stillbirths after 39 weeks’ gestation has stimulated studies demonstrating the safety of delivering nulliparous women who are at low risk at 39 weeks’ gestation leading to the adoption of this approach in several settings.
      • Grobman W.A.
      • Rice M.M.
      • Reddy U.M.
      • et al.
      Labor induction versus expectant management in low-risk nulliparous women.
      These attitudes are perhaps relevant to high-resource settings, but for low-resource settings, in which late-onset preeclampsia remains a substantial maternal and infant risk, such management is not consistent with available resources. In these settings, identifying women at risk and marshaling resources to deliver women at risk would be a major advance. Even in high-resource settings, delivery near term but before an increase in maternal and infant morbidity is desirable. Furthermore, because the biomarkers marking the genesis of preeclampsia are signs of placental senescence and stress, they are likely signaling the premature expression of normal placental stress accompanying the biological termination of pregnancy. Carefully ovulation dated pregnancies indicate that pregnancy duration is 268±9 days (282±9 days by menstrual dating) with a range of 37 days.
      • Jukic A.M.
      • Baird D.D.
      • Weinberg C.R.
      • McConnaughey D.R.
      • Wilcox A.J.
      Length of human pregnancy and contributors to its natural variation.
      Thus, arbitrary delivery at 39 weeks’ gestation results in 80% of pregnancies being delivered before their biologically determined endpoint. The ability to date the biologically programmed end of pregnancy rather than making decisions based on variable temporal endpoints has obvious advantages.
      We have advanced the hypotheses that there are different pathways to the convergence point of the pathophysiology of preeclampsia, STB stress, and different maternal responses to the resulting placental response. If this is the case, there will never be a 1-way strategy to prevent all cases of preeclampsia as we first suggested in 1999.
      • Redman C.W.
      • Sacks G.P.
      • Sargent I.L.
      Preeclampsia: an excessive maternal inflammatory response to pregnancy.
      This prediction will be at best extraordinarily complex and at worst highly inaccurate. However, the recognition of different subtypes of preeclampsia with clinical-demographic and analytical markers would allow directed assessment of risks and specific treatments. With this knowledge, we will be able to predict, and hopefully prevent, preeclampsia (perhaps even other pregnancy-specific disorders not currently considered part of the preeclampsia syndrome).

      Laboratory Research

      This discussion does not apply to the scope and content of research of placentation and its defects. Laboratory research into the health and dysfunction of STB in the third trimester of pregnancy has special facets that need to be recognized. Analysis of a minced whole placental tissue is too imprecise. However, confocal or electron microscopy with immunocytochemistry can be exceptionally informative in terms of discriminating between the STB and cytotrophoblast in close proximity. Conclusions from experiments with trophoblast cell lines, which can be induced to syncytialize, will always need to be validated on primary tissue. Ex vivo preparations of primary trophoblast cells are expensive and limited because they can only be used in 1 set of experiment. Careful culture of placental explants are easier and more robust. With either method, the tissue may be altered in variable ways by stresses induced by labor,
      • Cindrova-Davies T.
      • Yung H.W.
      • Johns J.
      • et al.
      Oxidative stress, gene expression, and protein changes induced in the human placenta during labor.
      so that mode of delivery should be controlled. Research on delivered placentas is confounded by the need to match for gestational age and normality. As discussed, the concept of normality at term is, to an extent, spurious. Hence, 1 of the top priorities is to determine the biological age of a pregnancy in terms of measures of STB stress, which can be used to grade the “normality” of placentas delivered to apparently normal women at term. This may be feasible by analysis of circulating STB-derived microvesicles. Their cargoes are complex and differ if there is preeclampsia.
      • Pillay P.
      • Vatish M.
      • Duarte R.
      • Moodley J.
      • Mackraj I.
      Exosomal microRNA profiling in early and late onset preeclamptic pregnant women reflects pathophysiology.
      This issue merits detailed investigation in relation to the time course of the end-stage placenta. Large quantities of pure STB microvesicles can be prepared by dual perfusion of single placental lobes to allow their properties in vitro to identify the features of highest interest. These can be used to distinguish preeclampsia from normal or to grade biological age and hence distinguish “twilight” vesicles from those that are biologically younger.
      • Tannetta D.
      • Collett G.
      • Vatish M.
      • Redman C.
      • Sargent I.
      Syncytiotrophoblast extracellular vesicles - circulating biopsies reflecting placental health.
      Finally, STB cannot be considered in isolation of its supporting cytotrophoblast. In terms of all the issues discussed in this paper, detailed analysis of cytotrophoblast condition and stress is urgently needed.

      Clinical Research

      Innovative discovery strategies supported by hypothesis-guided research now have the capacity to unravel the complexity of abnormal pregnancies. Use of these tools to decipher the pathophysiologies of preeclampsia should be a major priority of future studies. All the authors have been frustrated by being involved in clinical trials to prevent preeclampsia, which have had minimal or no success. These trials were guided by conventional wisdom of the time. However, in retrospect, we can see that they also shared the major conceptual shortcoming of considering preeclampsia to be a single disorder, which could be solved by a single “magic bullet.” We recommend that further large clinical trials should await the recognition of subsets that provide specific targets for the test intervention and are tested exclusively in that subgroup.

      Big Data

      For successful understanding of preeclampsia, we must understand the syndrome and the pathophysiology of its subtypes. This mandates a new emphasis on collaborative research where sharing is the norm and a major factor in the study design, data collection, and biospecimen accrual. Although powerful analytical tools are now available, their effective use demands large amounts of carefully collected and harmonized data and biological materials. This is crucial for the big data approach to understanding any disease.
      • Redman C.W.
      • Sacks G.P.
      • Sargent I.L.
      Preeclampsia: an excessive maternal inflammatory response to pregnancy.
      However, sharing of data and biological materials has not been a priority in preeclampsia research. In fact, for many years, academic departments, funders, and publishers have discouraged investigators from being part of large studies in which they are not a primary investigator. New efforts to change this institutional mindset to facilitate data sharing by standardizing sample collection, data fields, and outcome recording have begun.
      • Roberts J.M.
      • Mascalzoni D.
      • Ness R.B.
      • Poston L.
      Global Pregnancy Collaboration
      Collaboration to understand complex diseases: preeclampsia and adverse pregnancy outcomes.
      • Myers J.E.
      • Myatt L.
      • Roberts J.M.
      • Redman C.
      Global Pregnancy Collaboration (CoLab)
      COLLECT, a collaborative database for pregnancy and placental research studies worldwide.
      • Duffy J.
      • Rolph R.
      • Gale C.
      • et al.
      Core outcome sets in women’s and newborn health: a systematic review.
      • Myatt L.
      • Redman C.W.
      • Staff A.C.
      • et al.
      Strategy for standardization of preeclampsia research study design.

      Conclusions

      Preeclampsia is not just a placental disorder but specifically one of STB stress. STB stress may be caused by maternal malperfusion secondary to malplacentation, in early pregnancy, or to late pregnancy factors related to placental growth and compression, which cause malperfusion and hypoxia. Malplacentation is not a stage in the progression of all preeclampsia or is preeclampsia the only outcome of malplacentation. It is a separate condition and, in addition to being a powerful risk factor for early-onset preeclampsia, predisposes to FGR without preeclampsia or some cases of preterm birth. STB stress, secondary to damage caused by antiphospholipid autoantibodies or parvovirus, is an atypical cause of preeclampsia. There are likely other routes to STB stress that are currently not recognized.
      The role of senescence in late pregnancy is complex. STB is always semisenescent because it cannot replicate or can it undergo apoptosis as it is characterized in mononuclear cells. This stable state may be perturbed by superimposed stresses, which stimulate autophagy, shedding of multinuclear fragments, breaks in the STB, or necrosis. STB stress can explain many aspects of the placental pathology of preeclampsia and its downstream consequences, such as syncytial knots and perivillous fibrin deposition. It could also account for some cases of villitis of unknown etiology.
      The decline in health, at and after term, which we call the “twilight placenta,” affects all pregnancies but with different time courses. The degree to which villous cytotrophoblast undergoes senescence is not known. It could be the final determinant of STB failure, when it ceased to be able to renew the overlying STB.
      The best biomarkers of preeclampsia are generated by STB stress. They indicate that on average, STB stress begins at 30 to 32 weeks’ gestation in normal pregnancies. The outcome of term pregnancy depends on competing risks as to which comes first: spontaneous (or induced) delivery or preeclampsia with or without STB failure (unexplained stillbirth). STB-derived microvesicles are complex signals to the mother, which are stimulated by stress. As circulating biomarkers of syncytial health, they have the potential to determine biological rather than gestational age, to monitor the twilight placenta at the end of pregnancy.
      Maternal risk factors modulate the generation, evolution, and consequences of STB stress. They are not the cause of preeclampsia, because the risk factors persist, whereas the syndrome remits.
      The several pathways leading to STB stress, the multiple insults generated by STB stress, and the variable responses to these insults, all modified by maternal factors, mandate a new approach to studying and understanding preeclampsia. It is a complex disorder with multiple targets for prevention that will not be appropriate in all women. The identification of these subsets requires coordinated and collaborative research efforts.

      References

        • Chesley L.C.
        A short history of eclampsia.
        Obstet Gynecol. 1974; 43: 559-602
        • Chesley L.C.
        • Preece J.
        Hydatidiform mole, with special reference to recurrence and associated eclampsia.
        Am J Obstet Gynecol. 1946; 52: 311-320
        • Chesley L.C.
        Hypertensive disorders of pregnancy.
        Appleton-Century-Crofts, New York, NY1978
        • Young J.
        The aetiology of eclampsia and albuminuria and their relation to accidental haemorrhage: (an anatomical and experimental investigation.).
        Proc R Soc Med. 1914; 7: 307-345
        • Page E.W.
        The relation between hydatid moles, relative ischemia of the gravid uterus, and the placental origin of eclampsia.
        Am J Obstet Gynecol. 1939; 37: 291-298
        • Redman C.W.
        • Denson K.W.
        • Beilin L.J.
        • Bolton F.G.
        • Stirrat G.M.
        Factor-VIII consumption in pre-eclampsia.
        Lancet. 1977; 2: 1249-1252
        • Brosens I.
        • Robertson W.B.
        • Dixon H.G.
        The physiological response of the vessels of the placental bed to normal pregnancy.
        J Pathol Bacteriol. 1967; 93: 569-579
        • Robertson W.B.
        • Brosens I.
        • Dixon H.G.
        The pathological response of the vessels of the placental bed to hypertensive pregnancy.
        J Pathol Bacteriol. 1967; 93: 581-592
        • Brosens I.A.
        • Robertson W.B.
        • Dixon H.G.
        The role of the spiral arteries in the pathogenesis of pre-eclampsia.
        J Pathol. 1970; 101: Pvi
        • Redman C.W.
        Current topic: pre-eclampsia and the placenta.
        Placenta. 1991; 12: 301-308
        • Burton G.J.
        • Jauniaux E.
        The cytotrophoblastic shell and complications of pregnancy.
        Placenta. 2017; 60: 134-139
        • Burton G.J.
        • Watson A.L.
        • Hempstock J.
        • Skepper J.N.
        • Jauniaux E.
        Uterine glands provide histiotrophic nutrition for the human fetus during the first trimester of pregnancy.
        J Clin Endocrinol Metab. 2002; 87: 2954-2959
        • Burton G.J.
        Oxygen, the Janus gas; its effects on human placental development and function.
        J Anat. 2009; 215: 27-35
        • Ness R.B.
        • Roberts J.M.
        Heterogeneous causes constituting the single syndrome of preeclampsia: a hypothesis and its implications.
        Am J Obstet Gynecol. 1996; 175: 1365-1370
        • Sebire N.J.
        • Goldin R.D.
        • Regan L.
        Term preeclampsia is associated with minimal histopathological placental features regardless of clinical severity.
        J Obstet Gynaecol. 2005; 25: 117-118
        • Xiong X.
        • Demianczuk N.N.
        • Saunders L.D.
        • Wang F.L.
        • Fraser W.D.
        Impact of preeclampsia and gestational hypertension on birth weight by gestational age.
        Am J Epidemiol. 2002; 155: 203-209
        • von Dadelszen P.
        • Magee L.A.
        • Roberts J.M.
        Subclassification of preeclampsia.
        Hypertens Pregnancy. 2003; 22: 143-148
        • Kenneth L.
        • Hall D.R.
        • Gebhardt S.
        • Grové D.
        Late onset preeclampsia is not an innocuous condition.
        Hypertens Pregnancy. 2010; 29: 262-270
        • Roberts J.M.
        • Taylor R.N.
        • Musci T.J.
        • Rodgers G.M.
        • Hubel C.A.
        • McLaughlin M.K.
        Preeclampsia: an endothelial cell disorder.
        Am J Obstet Gynecol. 1989; 161: 1200-1204
        • Redman C.W.
        • Sacks G.P.
        • Sargent I.L.
        Preeclampsia: an excessive maternal inflammatory response to pregnancy.
        Am J Obstet Gynecol. 1999; 180: 499-506
        • Maynard S.E.
        • Min J.Y.
        • 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
        • Clayton S.G.
        Foetal mortality in post-maturity.
        BJOG. 1941; 48: 450-460
        • Sjöstedt S.
        • Engleson G.
        • Rooth G.
        Dysmaturity. Arch Dis Child. 1958; 33: 123-130
        • Caughey A.B.
        • Stotland N.E.
        • Escobar G.J.
        What is the best measure of maternal complications of term pregnancy: ongoing pregnancies or pregnancies delivered?.
        Am J Obstet Gynecol. 2003; 189: 1047-1052
        • Gómez O.
        • Figueras F.
        • Fernández S.
        • et al.
        Reference ranges for uterine artery mean pulsatility index at 11-41 weeks of gestation.
        Ultrasound Obstet Gynecol. 2008; 32: 128-132
        • Ciobanu A.
        • Wright A.
        • Syngelaki A.
        • Wright D.
        • Akolekar R.
        • Nicolaides K.H.
        Fetal Medicine Foundation reference ranges for umbilical artery and middle cerebral artery pulsatility index and cerebroplacental ratio.
        Ultrasound Obstet Gynecol. 2019; 53: 465-472
        • Khalil A.
        • Morales-Rosello J.
        • Khan N.
        • et al.
        Is cerebroplacental ratio a marker of impaired fetal growth velocity and adverse pregnancy outcome?.
        Am J Obstet Gynecol. 2017; 216: 606.e1-606.e10
        • Jackson M.R.
        • Mayhew T.M.
        • Boyd P.A.
        Quantitative description of the elaboration and maturation of villi from 10 weeks of gestation to term.
        Placenta. 1992; 13: 357-370
        • Soothill P.W.
        • Nicolaides K.H.
        • Rodeck C.H.
        • Campbell S.
        Effect of gestational age on fetal and intervillous blood gas and acid-base values in human pregnancy.
        Fetal Ther. 1986; 1: 168-175
        • Redman C.W.
        • Staff A.C.
        Preeclampsia, biomarkers, syncytiotrophoblast stress, and placental capacity.
        Am J Obstet Gynecol. 2015; 213 (S9–11): S9.e1
        • Mayhew T.M.
        • Wadrop E.
        Placental morphogenesis and the star volumes of villous trees and intervillous pores.
        Placenta. 1994; 15: 209-217
        • Serov A.S.
        • Salafia C.M.
        • Brownbill P.
        • Grebenkov D.S.
        • Filoche M.
        Optimal villi density for maximal oxygen uptake in the human placenta.
        J Theor Biol. 2015; 364: 383-396
        • Heilmann L.
        Blood rheology and pregnancy.
        Baillieres Clin Haematol. 1987; 1: 777-799
        • Staff A.C.
        • Dechend R.
        • Pijnenborg R.
        Learning from the placenta: acute atherosis and vascular remodeling in preeclampsia-novel aspects for atherosclerosis and future cardiovascular health.
        Hypertension. 2010; 56: 1026-1034
        • Ernst L.M.
        Maternal vascular malperfusion of the placental bed.
        APMIS. 2018; 126: 551-560
        • Stanek J.
        Hypoxic patterns of placental injury: a review.
        Arch Pathol Lab Med. 2013; 137: 706-720
        • Fitzgerald B.
        • Kingdom J.
        • Keating S.
        Distal villous hypoplasia.
        Diagn Histopathol. 2012; 18: 195-200
        • Kim C.J.
        • Romero R.
        • Chaemsaithong P.
        • Kim J.S.
        Chronic inflammation of the placenta: definition, classification, pathogenesis, and clinical significance.
        Am J Obstet Gynecol. 2015; 213: S53-S69
        • Romero R.
        • Whitten A.
        • Korzeniewski S.J.
        • et al.
        Maternal floor infarction/massive perivillous fibrin deposition: a manifestation of maternal antifetal rejection?.
        Am J Reprod Immunol. 2013; 70: 285-298
        • Redline R.W.
        • Boyd T.
        • Campbell V.
        • et al.
        Maternal vascular underperfusion: nosology and reproducibility of placental reaction patterns.
        Pediatr Dev Pathol. 2004; 7: 237-249
        • Jones C.J.
        • Fox H.
        An ultrastructural and ultrahistochemical study of the human placenta in maternal pre-eclampsia.
        Placenta. 1980; 1: 61-76
        • Heazell A.E.
        • Moll S.J.
        • Jones C.J.
        • Baker P.N.
        • Crocker I.P.
        Formation of syncytial knots is increased by hyperoxia, hypoxia and reactive oxygen species.
        Placenta. 2007; 28: S33-S40
        • Yung H.W.
        • Colleoni F.
        • Dommett E.
        • et al.
        Noncanonical mitochondrial unfolded protein response impairs placental oxidative phosphorylation in early-onset preeclampsia.
        Proc Natl Acad Sci U S A. 2019; 116: 18109-18118
        • Can M.
        • Guven B.
        • Bektas S.
        • Arikan I.
        Oxidative stress and apoptosis in preeclampsia.
        Tissue Cell. 2014; 46: 477-481
        • Rampersad R.
        • Barton A.
        • Sadovsky Y.
        • Nelson D.M.
        The C5b-9 membrane attack complex of complement activation localizes to villous trophoblast injury in vivo and modulates human trophoblast function in vitro.
        Placenta. 2008; 29: 855-861
        • Coppé J.P.
        • Patil C.K.
        • Rodier F.
        • et al.
        Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor.
        PLoS Biol. 2008; 6: 2853-2868
        • Cui X.L.
        • Brockman D.
        • Campos B.
        • Myatt L.
        Expression of NADPH oxidase isoform 1 (Nox1) in human placenta: involvement in preeclampsia.
        Placenta. 2006; 27: 422-431
        • Burton G.J.
        • Yung H.W.
        Endoplasmic reticulum stress in the pathogenesis of early-onset pre-eclampsia.
        Pregnancy Hypertens. 2011; 1: 72-78
        • Aban M.
        • Cinel L.
        • Arslan M.
        • et al.
        Expression of nuclear factor-kappa B and placental apoptosis in pregnancies complicated with intrauterine growth restriction and preeclampsia: an immunohistochemical study.
        Tohoku J Exp Med. 2004; 204: 195-202
        • Pan Y.J.
        • He L.
        • Zhou S.J.
        • Zhang L.J.
        • Zhang A.H.
        • Zhao Y.Y.
        Expression of urotensin II is associated with placental autophagy in patients with severe preeclampsia.
        J Hum Hypertens. 2018; 32: 759-769
        • Liu H.
        • Cheng F.
        • Xu Q.
        • et al.
        Lipoxin A4 suppresses angiotensin II type 1 receptor autoantibody in preeclampsia via modulating caspase-1.
        Cell Death Dis. 2020; 11: 78
        • Weel I.C.
        • Baergen R.N.
        • Romão-Veiga M.
        • et al.
        Association between placental lesions, cytokines and angiogenic factors in pregnant women with preeclampsia.
        PLoS One. 2016; 11e0157584
        • Nelson D.M.
        Apoptotic changes occur in syncytiotrophoblast of human placental villi where fibrin type fibrinoid is deposited at discontinuities in the villous trophoblast.
        Placenta. 1996; 17: 387-391
        • Mayhew T.M.
        Villous trophoblast of human placenta: a coherent view of its turnover, repair and contributions to villous development and maturation.
        Histol Histopathol. 2001; 16: 1213-1224
        • Hung T.H.
        • Skepper J.N.
        • Charnock-Jones D.S.
        • Burton G.J.
        Hypoxia-reoxygenation: a potent inducer of apoptotic changes in the human placenta and possible etiological factor in preeclampsia.
        Circ Res. 2002; 90: 1274-1281
        • Jones L.M.
        • Chen Y.
        • van Oosten-Hawle P.
        Redefining proteostasis transcription factors in organismal stress responses, development, metabolism, and health.
        Biol Chem. 2020; 401: 1005-1018
        • Du J.
        • Li M.
        • Huang Q.
        • et al.
        The critical role of microRNAs in stress response: therapeutic prospect and limitation.
        Pharmacol Res. 2019; 142: 294-302
        • Valadkhan S.
        • Valencia-Hipólito A.
        lncRNAs in stress response.
        Curr Top Microbiol Immunol. 2016; 394: 203-236
        • Castellucci M.
        • Kauffmann P.
        Basic structure of the villous trees.
        in: Benirschke K. Kaffmann P. Baergen P. Pathology of the human placenta. Springer, New York, NY2006: 50-120
        • Longtine M.S.
        • Chen B.
        • Odibo A.O.
        • Zhong Y.
        • Nelson D.M.
        Villous trophoblast apoptosis is elevated and restricted to cytotrophoblasts in pregnancies complicated by preeclampsia, IUGR, or preeclampsia with IUGR.
        Placenta. 2012; 33: 352-359
        • Burton G.J.
        • Jones C.J.
        Syncytial knots, sprouts, apoptosis, and trophoblast deportation from the human placenta.
        Taiwan J Obstet Gynecol. 2009; 48: 28-37
        • Loukeris K.
        • Sela R.
        • Baergen R.N.
        Syncytial knots as a reflection of placental maturity: reference values for 20 to 40 weeks’ gestational age.
        Pediatr Dev Pathol. 2010; 13: 305-309
        • Tomas S.Z.
        • Prusac I.K.
        • Roje D.
        • Tadin I.
        Trophoblast apoptosis in placentas from pregnancies complicated by preeclampsia.
        Gynecol Obstet Invest. 2011; 71: 250-255
        • Fogarty N.M.
        • Ferguson-Smith A.C.
        • Burton G.J.
        Syncytial knots (Tenney-Parker changes) in the human placenta: evidence of loss of transcriptional activity and oxidative damage.
        Am J Pathol. 2013; 183: 144-152
        • Huppertz B.
        IFPA Award in Placentology lecture: biology of the placental syncytiotrophoblast—myths and facts.
        Placenta. 2010; 31: S75-S81
        • Yang K.
        • Yang J.
        • Yi J.
        Nucleolar stress: hallmarks, sensing mechanism and diseases.
        Cell Stress. 2018; 2: 125-140
        • Burton T.D.
        • Fedele A.O.
        • Xie J.
        • Sandeman L.Y.
        • Proud C.G.
        The gene for the lysosomal protein LAMP3 is a direct target of the transcription factor ATF4.
        J Biol Chem. 2020; 295: 7418-7430
        • Sasaki K.
        • Yoshida H.
        Golgi stress response and organelle zones.
        FEBS Lett. 2019; 593: 2330-2340
        • Fan Y.
        • Simmen T.
        Mechanistic connections between endoplasmic reticulum (ER) redox control and mitochondrial metabolism.
        Cells. 2019; 8: 1071
        • Cester N.
        • Staffolani R.
        • Rabini R.A.
        • et al.
        Pregnancy induced hypertension: a role for peroxidation in microvillus plasma membranes.
        Mol Cell Biochem. 1994; 131: 151-155
        • Tenório M.B.
        • Ferreira R.C.
        • Moura F.A.
        • Bueno N.B.
        • Goulart M.O.F.
        • Oliveira A.C.M.
        Oral antioxidant therapy for prevention and treatment of preeclampsia: meta-analysis of randomized controlled trials.
        Nutr Metab Cardiovasc Dis. 2018; 28: 865-876
        • Myatt L.
        • Cui X.
        Oxidative stress in the placenta.
        Histochem Cell Biol. 2004; 122: 369-382
        • Takagi Y.
        • Nikaido T.
        • Toki T.
        • et al.
        Levels of oxidative stress and redox-related molecules in the placenta in preeclampsia and fetal growth restriction.
        Virchows Arch. 2004; 444: 49-55
        • Cindrova-Davies T.
        • Fogarty N.M.E.
        • Jones C.J.P.
        • Kingdom J.
        • Burton G.J.
        Evidence of oxidative stress-induced senescence in mature, post-mature and pathological human placentas.
        Placenta. 2018; 68: 15-22
        • Chekir C.
        • Nakatsuka M.
        • Noguchi S.
        • et al.
        Accumulation of advanced glycation end products in women with preeclampsia: possible involvement of placental oxidative and nitrative stress.
        Placenta. 2006; 27: 225-233
        • Yung H.W.
        • Calabrese S.
        • Hynx D.
        • et al.
        Evidence of placental translation inhibition and endoplasmic reticulum stress in the etiology of human intrauterine growth restriction.
        Am J Pathol. 2008; 173: 451-462
        • Carreras-Sureda A.
        • Pihán P.
        • Hetz C.
        Calcium signaling at the endoplasmic reticulum: fine-tuning stress responses.
        Cell Calcium. 2018; 70: 24-31
        • Holland O.
        • Dekker Nitert M.
        • Gallo L.A.
        • Vejzovic M.
        • Fisher J.J.
        • Perkins A.V.
        Review: placental mitochondrial function and structure in gestational disorders.
        Placenta. 2017; 54: 2-9
        • Muralimanoharan S.
        • Maloyan A.
        • Mele J.
        • Guo C.
        • Myatt L.G.
        • Myatt L.
        MIR-210 modulates mitochondrial respiration in placenta with preeclampsia.
        Placenta. 2012; 33: 816-823
        • Reed S.M.
        • Quelle D.E.
        p53 acetylation: regulation and consequences.
        Cancers (Basel). 2014; 7: 30-69
        • Hu W.
        • Chen S.
        • Thorne R.F.
        • Wu M.
        TP53, TP53 target genes (DRAM, TIGAR), and autophagy.
        Adv Exp Med Biol. 2019; 1206: 127-149
        • Heazell A.E.
        • Sharp A.N.
        • Baker P.N.
        • Crocker I.P.
        Intra-uterine growth restriction is associated with increased apoptosis and altered expression of proteins in the p53 pathway in villous trophoblast.
        Apoptosis. 2011; 16: 135-144
        • Bhardwaj M.
        • Leli N.M.
        • Koumenis C.
        • Amaravadi R.K.
        Regulation of autophagy by canonical and non-canonical ER stress responses.
        Semin Cancer Biol. 2020; 66: 116-128
        • Galluzzi L.
        • Yamazaki T.
        • Kroemer G.
        Linking cellular stress responses to systemic homeostasis.
        Nat Rev Mol Cell Biol. 2018; 19: 731-745
        • Oh S.Y.
        • Choi S.J.
        • Kim K.H.
        • Cho E.Y.
        • Kim J.H.
        • Roh C.R.
        Autophagy-related proteins, LC3 and Beclin-1, in placentas from pregnancies complicated by preeclampsia.
        Reprod Sci. 2008; 15: 912-920
        • Curtis S.
        • Jones C.J.
        • Garrod A.
        • Hulme C.H.
        • Heazell A.E.
        Identification of autophagic vacuoles and regulators of autophagy in villous trophoblast from normal term pregnancies and in fetal growth restriction.
        J Matern Fetal Neonatal Med. 2013; 26: 339-346
        • Zimmermann M.
        • Reichert A.S.
        How to get rid of mitochondria: crosstalk and regulation of multiple mitophagy pathways.
        Biol Chem. 2017; 399: 29-45
        • Hetz C.
        The unfolded protein response: controlling cell fate decisions under ER stress and beyond.
        Nat Rev Mol Cell Biol. 2012; 13: 89-102
        • Wu C.C.
        • Bratton S.B.
        Regulation of the intrinsic apoptosis pathway by reactive oxygen species.
        Antioxid Redox Signal. 2013; 19: 546-558
        • Austgulen R.
        • Isaksen C.V.
        • Chedwick L.
        • Romundstad P.
        • Vatten L.
        • Craven C.
        Pre-eclampsia: associated with increased syncytial apoptosis when the infant is small-for-gestational-age.
        J Reprod Immunol. 2004; 61: 39-50
        • Ishihara N.
        • Matsuo H.
        • Murakoshi H.
        • Laoag-Fernandez J.B.
        • Samoto T.
        • Maruo T.
        Increased apoptosis in the syncytiotrophoblast in human term placentas complicated by either preeclampsia or intrauterine growth retardation.
        Am J Obstet Gynecol. 2002; 186: 158-166
        • Longtine M.S.
        • Chen B.
        • Odibo A.O.
        • Zhong Y.
        • Nelson D.M.
        Caspase-mediated apoptosis of trophoblasts in term human placental villi is restricted to cytotrophoblasts and absent from the multinucleated syncytiotrophoblast.
        Reproduction. 2012; 143: 107-121
        • Burton G.J.
        • Skepper J.N.
        • Hempstock J.
        • Cindrova T.
        • Jones C.J.
        • Jauniaux E.
        A reappraisal of the contrasting morphological appearances of villous cytotrophoblast cells during early human pregnancy; evidence for both apoptosis and primary necrosis.
        Placenta. 2003; 24: 297-305
        • De Falco M.
        • Fedele V.
        • Cobellis L.
        • et al.
        Immunohistochemical distribution of proteins belonging to the receptor-mediated and the mitochondrial apoptotic pathways in human placenta during gestation.
        Cell Tissue Res. 2004; 318: 599-608
        • Halperin R.
        • Peller S.
        • Rotschild M.
        • Bukovsky I.
        • Schneider D.
        Placental apoptosis in normal and abnormal pregnancies.
        Gynecol Obstet Invest. 2000; 50: 84-87
        • Smith S.C.
        • Baker P.N.
        • Symonds E.M.
        Placental apoptosis in normal human pregnancy.
        Am J Obstet Gynecol. 1997; 177: 57-65
        • Leung D.N.
        • Smith S.C.
        • To K.F.
        • Sahota D.S.
        • Baker P.N.
        Increased placental apoptosis in pregnancies complicated by preeclampsia.
        Am J Obstet Gynecol. 2001; 184: 1249-1250
        • Smith S.C.
        • Baker P.N.
        • Symonds E.M.
        Increased placental apoptosis in intrauterine growth restriction.
        Am J Obstet Gynecol. 1997; 177: 1395-1401
        • D’Arcy M.S.
        Cell death: a review of the major forms of apoptosis, necrosis and autophagy.
        Cell Biol Int. 2019; 43: 582-592
        • Cheng S.B.
        • Nakashima A.
        • Huber W.J.
        • et al.
        Pyroptosis is a critical inflammatory pathway in the placenta from early onset preeclampsia and in human trophoblasts exposed to hypoxia and endoplasmic reticulum stressors.
        Cell Death Dis. 2019; 10: 927
        • Longtine M.S.
        • Barton A.
        • Chen B.
        • Nelson D.M.
        Live-cell imaging shows apoptosis initiates locally and propagates as a wave throughout syncytiotrophoblasts in primary cultures of human placental villous trophoblasts.
        Placenta. 2012; 33: 971-976
        • Nelson D.M.
        • Crouch E.C.
        • Curran E.M.
        • Farmer D.R.
        Trophoblast interaction with fibrin matrix. Epithelialization of perivillous fibrin deposits as a mechanism for villous repair in the human placenta.
        Am J Pathol. 1990; 136: 855-865
        • Austgulen R.
        • Chedwick L.
        • Vogt Isaksen C.
        • Vatten L.
        • Craven C.
        Trophoblast apoptosis in human placenta at term as detected by expression of a cytokeratin 18 degradation product of caspase.
        Arch Pathol Lab Med. 2002; 126: 1480-1486
        • Draeger A.
        • Monastyrskaya K.
        • Babiychuk E.B.
        Plasma membrane repair and cellular damage control: the annexin survival kit.
        Biochem Pharmacol. 2011; 81: 703-712
        • Carmeille R.
        • Degrelle S.A.
        • Plawinski L.
        • et al.
        Annexin-A5 promotes membrane resealing in human trophoblasts.
        Biochim Biophys Acta. 2015; 1853: 2033-2044
        • Gourvas V.
        • Soulitzis N.
        • Konstantinidou A.
        • et al.
        Reduced ANXA5 mRNA and protein expression in pregnancies complicated by preeclampsia.
        Thromb Res. 2014; 133: 495-500
        • Parks W.T.
        Placental hypoxia: the lesions of maternal malperfusion.
        Semin Perinatol. 2015; 39: 9-19
        • Ogge G.
        • Chaiworapongsa T.
        • Romero R.
        • et al.
        Placental lesions associated with maternal underperfusion are more frequent in early-onset than in late-onset preeclampsia.
        J Perinat Med. 2011; 39: 641-652
        • Myerson D.
        • Parkin R.K.
        • Benirschke K.
        • Tschetter C.N.
        • Hyde S.R.
        The pathogenesis of villitis of unknown etiology: analysis with a new conjoint immunohistochemistry-in situ hybridization procedure to identify specific maternal and fetal cells.
        Pediatr Dev Pathol. 2006; 9: 257-265
        • Berridge M.J.
        • Lipp P.
        • Bootman M.D.
        The versatility and universality of calcium signalling.
        Nat Rev Mol Cell Biol. 2000; 1: 11-21
        • Morel O.
        • Morel N.
        • Jesel L.
        • Freyssinet J.M.
        • Toti F.
        Microparticles: a critical component in the nexus between inflammation, immunity, and thrombosis.
        Semin Immunopathol. 2011; 33: 469-486
        • French K.C.
        • Antonyak M.A.
        • Cerione R.A.
        Extracellular vesicle docking at the cellular port: extracellular vesicle binding and uptake.
        Semin Cell Dev Biol. 2017; 67: 48-55
        • van Niel G.
        • D’Angelo G.
        • Raposo G.
        Shedding light on the cell biology of extracellular vesicles.
        Nat Rev Mol Cell Biol. 2018; 19: 213-228
        • Tannetta D.
        • Masliukaite I.
        • Vatish M.
        • Redman C.
        • Sargent I.
        Update of syncytiotrophoblast derived extracellular vesicles in normal pregnancy and preeclampsia.
        J Reprod Immunol. 2017; 119: 98-106
        • Tannetta D.
        • Collett G.
        • Vatish M.
        • Redman C.
        • Sargent I.
        Syncytiotrophoblast extracellular vesicles - circulating biopsies reflecting placental health.
        Placenta. 2017; 52: 134-138
        • Johansen M.
        • Redman C.W.
        • Wilkins T.
        • Sargent I.L.
        Trophoblast deportation in human pregnancy--its relevance for pre-eclampsia.
        Placenta. 1999; 20: 531-539
        • Cronqvist T.
        • Tannetta D.
        • Mörgelin M.
        • et al.
        Syncytiotrophoblast derived extracellular vesicles transfer functional placental miRNAs to primary human endothelial cells.
        Sci Rep. 2017; 7: 4558
        • Motta-Mejia C.
        • Kandzija N.
        • Zhang W.
        • et al.
        Placental vesicles carry active endothelial nitric oxide synthase and their activity is reduced in preeclampsia.
        Hypertension. 2017; 70: 372-381
        • Gill M.
        • Motta-Mejia C.
        • Kandzija N.
        • et al.
        Placental syncytiotrophoblast-derived extracellular vesicles carry active NEP (neprilysin) and are increased in preeclampsia.
        Hypertension. 2019; 73: 1112-1119
        • Alam S.M.K.
        • Jasti S.
        • Kshirsagar S.K.
        • et al.
        Trophoblast glycoprotein (TPGB/5T4) in human placenta: expression, regulation, and presence in extracellular microvesicles and exosomes.
        Reprod Sci. 2018; 25: 185-197
        • Cooke W.R.
        • Cribbs A.
        • Zhang W.
        • et al.
        Maternal circulating syncytiotrophoblast-derived extracellular vesicles contain biologically active 5'-tRNA halves.
        Biochem Biophys Res Commun. 2019; 18: 107-113
        • Su Z.
        • Kuscu C.
        • Malik A.
        • Shibata E.
        • Dutta A.
        Angiogenin generates specific stress-induced tRNA halves and is not involved in tRF-3-mediated gene silencing.
        J Biol Chem. 2019; 294: 16930-16941
        • Aplin J.D.
        • Myers J.E.
        • Timms K.
        • Westwood M.
        Tracking placental development in health and disease.
        Nat Rev Endocrinol. 2020; 16: 479-494
        • Sultana Z.
        • Maiti K.
        • Dedman L.
        • Smith R.
        Is there a role for placental senescence in the genesis of obstetric complications and fetal growth restriction?.
        Am J Obstet Gynecol. 2018; 218: S762-S773
        • Narita M.
        • Nũnez S.
        • Heard E.
        • et al.
        Rb-mediated heterochromatin formation and silencing of E2F target genes during cellular senescence.
        Cell. 2003; 113: 703-716
        • Childs B.G.
        • Durik M.
        • Baker D.J.
        • van Deursen J.M.
        Cellular senescence in aging and age-related disease: from mechanisms to therapy.
        Nat Med. 2015; 21: 1424-1435
        • Shen J.
        • Gammon M.D.
        • Terry M.B.
        • et al.
        Telomere length, oxidative damage, antioxidants and breast cancer risk.
        Int J Cancer. 2009; 124: 1637-1643
        • Chuprin A.
        • Gal H.
        • Biron-Shental T.
        • et al.
        Cell fusion induced by ERVWE1 or measles virus causes cellular senescence.
        Genes Dev. 2013; 27: 2356-2366
        • Higuchi S.
        • Miyamoto T.
        • Kobara H.
        • et al.
        Trophoblast type-specific expression of senescence markers in the human placenta.
        Placenta. 2019; 85: 56-62
        • Swanson E.C.
        • Rapkin L.M.
        • Bazett-Jones D.P.
        • Lawrence J.B.
        Unfolding the story of chromatin organization in senescent cells.
        Nucleus. 2015; 6: 254-260
        • Maiti K.
        • Sultana Z.
        • Aitken R.J.
        • et al.
        Evidence that fetal death is associated with placental aging.
        Am J Obstet Gynecol. 2017; 217: 441.e1-441.e14
        • Braun T.
        • Brauer M.
        • Fuchs I.
        • et al.
        Mirror syndrome: a systematic review of fetal associated conditions, maternal presentation and perinatal outcome.
        Fetal Diagn Ther. 2010; 27: 191-203
        • Jordan J.A.
        • Butchko A.R.
        Apoptotic activity in villous trophoblast cells during B19 infection correlates with clinical outcome: assessment by the caspase-related M30 CytoDeath antibody.
        Placenta. 2002; 23: 547-553
        • Goa S.
        • Mimura K.
        • Kakigano A.
        • et al.
        Normalisation of angiogenic imbalance after intra-uterine transfusion for mirror syndrome caused by parvovirus B19.
        Fetal Diagn Ther. 2013; 34: 176-179
        • Zussman R.
        • Xu L.Y.
        • Damani T.
        • et al.
        Antiphospholipid antibodies can specifically target placental mitochondria and induce ROS production.
        J Autoimmun. 2020; 111: 102437
        • Muttukrishna S.
        • Knight P.G.
        • Groome N.P.
        • Redman C.W.
        • Ledger W.L.
        Activin A and inhibin A as possible endocrine markers for pre-eclampsia.
        Lancet. 1997; 349: 1285-1288
        • Perkins A.V.
        • Linton E.A.
        • Eben F.
        • Simpson J.
        • Wolfe C.D.
        • Redman C.W.
        Corticotrophin-releasing hormone and corticotrophin-releasing hormone binding protein in normal and pre-eclamptic human pregnancies.
        Br J Obstet Gynaecol. 1995; 102: 118-122
        • Venkatesha S.
        • Toporsian M.
        • Lam C.
        • et al.
        Soluble endoglin contributes to the pathogenesis of preeclampsia.
        Nat Med. 2006; 12: 642-649
        • Estellés A.
        • Gilabert J.
        • Aznar J.
        • Loskutoff D.J.
        • Schleef R.R.
        Changes in the plasma levels of type 1 and type 2 plasminogen activator inhibitors in normal pregnancy and in patients with severe preeclampsia.
        Blood. 1989; 74: 1332-1338
        • Torry D.S.
        • Wang H.S.
        • Wang T.H.
        • Caudle M.R.
        • Torry R.J.
        Preeclampsia is associated with reduced serum levels of placenta growth factor.
        Am J Obstet Gynecol. 1998; 179: 1539-1544
        • Xiong Y.
        • Liebermann D.A.
        • Holtzman E.J.
        • Jeronis S.
        • Hoffman B.
        • Geifman-Holtzman O.
        Preeclampsia-associated stresses activate Gadd45a signaling and sFlt-1 in placental explants.
        J Cell Physiol. 2013; 228: 362-370
        • Lecarpentier E.
        • Zsengellér Z.K.
        • Salahuddin S.
        • et al.
        Total Versus free placental growth factor levels in the pathogenesis of preeclampsia.
        Hypertension. 2020; 76: 875-883
        • Torry D.S.
        • Mukherjea D.
        • Arroyo J.
        • Torry R.J.
        Expression and function of placenta growth factor: implications for abnormal placentation.
        J Soc Gynecol Investig. 2003; 10: 178-188
        • Debiève F.
        • Depoix C.
        • Gruson D.
        • Hubinont C.
        Reversible effects of oxygen partial pressure on genes associated with placental angiogenesis and differentiation in primary-term cytotrophoblast cell culture.
        Mol Reprod Dev. 2013; 80: 774-784
        • Chang M.
        • Mukherjea D.
        • Gobble R.M.
        • Groesch K.A.
        • Torry R.J.
        • Torry D.S.
        Glial cell missing 1 regulates placental growth factor (PGF) gene transcription in human trophoblast.
        Biol Reprod. 2008; 78: 841-851
        • Levine R.J.
        • Maynard S.E.
        • Qian C.
        • et al.
        Circulating angiogenic factors and the risk of preeclampsia.
        N Engl J Med. 2004; 350: 672-683
        • Phillippe M.
        Cell-free fetal DNA, telomeres, and the spontaneous onset of parturition.
        Reprod Sci. 2015; 22: 1186-1201
        • Menon R.
        Initiation of human parturition: signaling from senescent fetal tissues via extracellular vesicle mediated paracrine mechanism.
        Obstet Gynecol Sci. 2019; 62: 199-211
        • Wright D.
        • Akolekar R.
        • Syngelaki A.
        • Poon L.C.
        • Nicolaides K.H.
        A competing risks model in early screening for preeclampsia.
        Fetal Diagn Ther. 2012; 32: 171-178
        • Bellamy L.
        • Casas J.P.
        • Hingorani A.D.
        • Williams D.J.
        Pre-eclampsia and risk of cardiovascular disease and cancer in later life: systematic review and meta-analysis.
        BMJ. 2007; 335: 974
        • Funai E.F.
        • Friedlander Y.
        • Paltiel O.
        • et al.
        Long-term mortality after preeclampsia.
        Epidemiology. 2005; 16: 206-215
        • Mongraw-Chaffin M.L.
        • Cirillo P.M.
        • Cohn B.A.
        Preeclampsia and cardiovascular disease death: prospective evidence from the child health and development studies cohort.
        Hypertension. 2010; 56: 166-171
        • Hubel C.A.
        • McLaughlin M.K.
        • Evans R.W.
        • Hauth B.A.
        • Sims C.J.
        • Roberts J.M.
        Fasting serum triglycerides, free fatty acids, and malondialdehyde are increased in preeclampsia, are positively correlated, and decrease within 48 hours post partum.
        Am J Obstet Gynecol. 1996; 174: 975-982
        • Powers R.W.
        • Roberts J.M.
        • Plymire D.A.
        • et al.
        Low placental growth factor across pregnancy identifies a subset of women with preterm preeclampsia: type 1 versus type 2 preeclampsia?.
        Hypertension. 2012; 60: 239-246
        • Chappell L.C.
        • Seed P.T.
        • Briley A.L.
        • et al.
        Effect of antioxidants on the occurrence of pre-eclampsia in women at increased risk: a randomised trial.
        Lancet. 1999; 354: 810-816
        • Roberts J.M.
        • Myatt L.
        • Spong C.Y.
        • et al.
        Vitamins C and E to prevent complications of pregnancy-associated hypertension.
        N Engl J Med. 2010; 362: 1282-1291
        • Lo J.O.
        • Mission J.F.
        • Caughey A.B.
        Hypertensive disease of pregnancy and maternal mortality.
        Curr Opin Obstet Gynecol. 2013; 25: 124-132
        • Creanga A.A.
        • Syverson C.
        • Seed K.
        • Callaghan W.M.
        Pregnancy-related mortality in the United States, 2011-2013.
        Obstet Gynecol. 2017; 130: 366-373
        • Mogos M.F.
        • Piano M.R.
        • McFarlin B.L.
        • Salemi J.L.
        • Liese K.L.
        • Briller J.E.
        Heart failure in pregnant women: a concern across the pregnancy continuum.
        Circ Heart Fail. 2018; 11: e004005
        • Honigberg M.C.
        • Zekavat S.M.
        • Aragam K.
        • et al.
        Long-term cardiovascular risk in women with hypertension during pregnancy.
        J Am Coll Cardiol. 2019; 74: 2743-2754
        • Lindley K.J.
        • Conner S.N.
        • Cahill A.G.
        • Novak E.
        • Mann D.L.
        Impact of preeclampsia on clinical and functional outcomes in women with peripartum cardiomyopathy.
        Circ Heart Fail. 2017; 10e003797
        • Gammill H.S.
        • Chettier R.
        • Brewer A.
        • et al.
        Cardiomyopathy and preeclampsia.
        Circulation. 2018; 138: 2359-2366
        • Melchiorre K.
        • Sharma R.
        • Thilaganathan B.
        Cardiovascular implications in preeclampsia: an overview.
        Circulation. 2014; 130: 703-714
        • Redman C.W.
        • Sargent I.L.
        • Staff A.C.
        IFPA Senior Award lecture: making sense of pre-eclampsia - two placental causes of preeclampsia?.
        Placenta. 2014; 35: S20-S25
        • Grobman W.A.
        • Rice M.M.
        • Reddy U.M.
        • et al.
        Labor induction versus expectant management in low-risk nulliparous women.
        N Engl J Med. 2018; 379: 513-523
        • Jukic A.M.
        • Baird D.D.
        • Weinberg C.R.
        • McConnaughey D.R.
        • Wilcox A.J.
        Length of human pregnancy and contributors to its natural variation.
        Hum Reprod. 2013; 28: 2848-2855
        • Cindrova-Davies T.
        • Yung H.W.
        • Johns J.
        • et al.
        Oxidative stress, gene expression, and protein changes induced in the human placenta during labor.
        Am J Pathol. 2007; 171: 1168-1179
        • Pillay P.
        • Vatish M.
        • Duarte R.
        • Moodley J.
        • Mackraj I.
        Exosomal microRNA profiling in early and late onset preeclamptic pregnant women reflects pathophysiology.
        Int J Nanomedicine. 2019; 14: 5637-5657
        • Roberts J.M.
        • Mascalzoni D.
        • Ness R.B.
        • Poston L.
        • Global Pregnancy Collaboration
        Collaboration to understand complex diseases: preeclampsia and adverse pregnancy outcomes.
        Hypertension. 2016; 67: 681-687
        • Myers J.E.
        • Myatt L.
        • Roberts J.M.
        • Redman C.
        • Global Pregnancy Collaboration (CoLab)
        COLLECT, a collaborative database for pregnancy and placental research studies worldwide.
        BJOG. 2019; 126: 8-10
        • Duffy J.
        • Rolph R.
        • Gale C.
        • et al.
        Core outcome sets in women’s and newborn health: a systematic review.
        BJOG. 2017; 124: 1481-1489
        • Myatt L.
        • Redman C.W.
        • Staff A.C.
        • et al.
        Strategy for standardization of preeclampsia research study design.
        Hypertension. 2014; 63: 1293-1301