A Dormant Microbial Component in the Development of Preeclampsia
- 1School of Chemistry, The University of Manchester, Manchester, UK
- 2The Manchester Institute of Biotechnology, The University of Manchester, Manchester, UK
- 3Centre for Synthetic Biology of Fine and Speciality Chemicals, The University of Manchester, Manchester, UK
- 4The Irish Centre for Fetal and Neonatal Translational Research (INFANT), University College Cork, Cork, Ireland
- 5Department of Obstetrics and Gynecology, University College Cork, Cork, Ireland
Preeclampsia (PE) is a complex, multisystem disorder that remains a leading cause of morbidity and mortality in pregnancy. Four main classes of dysregulation accompany PE and are widely considered to contribute to its severity. These are abnormal trophoblast invasion of the placenta, anti-angiogenic responses, oxidative stress, and inflammation. What is lacking, however, is an explanation of how these themselves are caused. We here develop the unifying idea, and the considerable evidence for it, that the originating cause of PE (and of the four classes of dysregulation) is, in fact, microbial infection, that most such microbes are dormant and hence resist detection by conventional (replication-dependent) microbiology, and that by occasional resuscitation and growth it is they that are responsible for all the observable sequelae, including the continuing, chronic inflammation. In particular, bacterial products such as lipopolysaccharide (LPS), also known as endotoxin, are well known as highly inflammagenic and stimulate an innate (and possibly trained) immune response that exacerbates the inflammation further. The known need of microbes for free iron can explain the iron dysregulation that accompanies PE. We describe the main routes of infection (gut, oral, and urinary tract infection) and the regularly observed presence of microbes in placental and other tissues in PE. Every known proteomic biomarker of “preeclampsia” that we assessed has, in fact, also been shown to be raised in response to infection. An infectious component to PE fulfills the Bradford Hill criteria for ascribing a disease to an environmental cause and suggests a number of treatments, some of which have, in fact, been shown to be successful. PE was classically referred to as endotoxemia or toxemia of pregnancy, and it is ironic that it seems that LPS and other microbial endotoxins really are involved. Overall, the recognition of an infectious component in the etiology of PE mirrors that for ulcers and other diseases that were previously considered to lack one.
Preeclampsia is a multisystem disorder of pregnancy, characterized and indeed defined by the presence of hypertension after 20 weeks’ gestation and before the onset of labor, or postpartum, with either proteinuria or any multisystem complication (1–9). It is a common condition, affecting some 3–5% of nulliparous pregnant women (8, 10) and is characterized by high mortality levels (11–14). There is no known cure other than delivery, and consequently, preeclampsia (PE) also causes significant perinatal morbidity and mortality secondary to iatrogenic prematurity. There are a variety of known risk factors (Table 1) that may be of use in predicting a greater likelihood of developing PE, albeit there are so many, with only very modest correlations, that early-stage (especially the first-trimester) prediction of late-stage PE remains very difficult (8, 15–17).
It is striking that most of the “risk factors” of Table 1 are, in fact, risk factors for multiple vascular or metabolic diseases, i.e., they merely pre-dispose the individual to a greater likelihood of manifesting the disease or syndrome (in this case PE). Indeed, some of them are diseases. This would be consistent with the well-known comorbidities, e.g., between PE and later cardiovascular disease [e.g., Ref. (55–65)], between PE and intracerebral hemorrhage during pregnancy [OR 10.39 (66)], and between PE and stroke postpartum (67, 68). The penultimate row of Table 1 lists a series of diseases that amount to comorbidities, although our interest was piqued by the observation that one-third of patients with antiphospholipid syndrome have PE, and infectious agents with known cross-reacting antigens are certainly one original (external) source of the triggers that cause the antiphospholipid antibodies (43, 44, 47, 69) (and see below). Similarly, in the case of urinary tract infection (UTI), the “risk” factor is a genuine external trigger, a point [following the call by Mignini and colleagues (70) for systematic reviews] that we shall expand on considerably here. [A preprint has been lodged at bioRxiv (71).]
In recent decades, intense investigation has led to the development of a two-stage etiological model for PE, first proposed by Redman (72), in which inadequate remodeling of the spiral arteries in early gestation results in poor placental development (stage one) and the resultant ischemia/reperfusion injury and oxidative stress (73) eventually leads to maternal vascular endothelial cell dysfunction and the maternal manifestations of the disease (stage 2) (72, 74–77). However, many clinical inconsistencies challenge the simplicity of this model. For example, while the association between poor placentation and PE is well established, it is not specific. Poor placentation and fetal growth restriction (FGR) frequently present without maternal signs of PE. Moreover, FGR is not a consistent feature of PE. While it is commonly seen in PE presenting at earlier gestations, in PE presenting at term, neonates are not growth restricted and may even be large for dates (78).
Thus, the two-stage model has been further refined by Roberts and others (77, 79, 80) to take into account the heterogeneous nature of PE and the varying contribution from mother and infant to the disorder. We now appreciate that normal pregnancy is characterized by a low-grade systemic inflammatory response and specific metabolic changes, and that virtually all of the features of normal pregnancy are simply exaggerated in PE (81–83). There is also widespread acceptance that maternal constitutional and environmental factors (such as obesity) can interact to modulate the risk of PE. Thus, with profoundly reduced placental perfusion (or significant “placental loading”), the generation of stage 2 may require very little contribution from the mother to provide sufficient stress to elicit the maternal syndrome. In this setting, almost any woman will develop PE. Conversely, the woman with extensive predisposing constitutional sensitivity could develop PE with very little reduced perfusion, or minimal “placental loading.” As with many complex disorders, multiple factors can affect disease development positively or negatively, with a convenient representation of the two main negative sources (fetal and maternal) being that of a see-saw (84), as in Figure 1.
While this explains the inconsistencies of the two-stage model, the precise mechanisms (1) underlying the initial poor placentation and (2) linking placental stress and the maternal syndrome have still not been fully elucidated.
Much recent research in PE has focused on various angiogenic factors, including the pro-angiogenic factors vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) and the two anti-angiogenic proteins, such as soluble endoglin (sEng) and soluble fms-like tyrosine kinase 1 (sFlt-1). Recent data suggest that alterations in circulating angiogenic factors play a pathogenic role in PE. These angiogenic factors tightly regulate angiogenesis and are also essential for maintenance of normal vessel health. Consequently, the synthesis and action of these factors and their receptors in the uterine bed and placenta are essential for normal placental development and pregnancy (85, 86). In PE, increased levels of the anti-angiogenic sFlt-1 and sEng trap circulating VEGF, PlGF, and transforming growth factor-β (TGF-β), respectively. A myriad of data support the idea that circulating levels of these factors alone, or in combination, can be used to predict PE (87–89) (and see below under PE biomarkers), but in line with the heterogeneous nature of PE, these data are somewhat inconsistent and their performance as biomarkers seems limited to disease with significant placental loading (8). Therefore, angiogenic dysregulation would appear unlikely to be the sole link between the stressed placenta and endothelial dysfunction and the clinical manifestations of the disease.
Notwithstanding these many inconsistencies, the central role of the placenta as a source of “toxin,” in a condition regarded, and indeed often named, as “toxemia of pregnancy” (90–92) cannot be refuted. The uncertainty regarding the nature of the toxin continues, and other placental sources of endothelial dysfunction include syncytiotrophoblast basement membrane fragments (STBM) (93) and endothelial progenitor cells (EPC) (94); an increase of reactive oxygen species over scavenging by antioxidants (95, 96) has also been promoted.
The Bradford Hill criteria for causation of a disease Y by an environmental factor X (97) are as follows: (1) strength of association between X and Y, (2) consistency of association between X and Y, (3) specificity of association between X and Y, (4) experiments verify the relationship between X and Y, (5) modification of X alters the occurrence of Y, and (6) biologically plausible cause and effect relationship.
In general terms (98), if we see that two things (A and B) co-vary in different circumstances, we might infer that A causes B, that B causes A, or that something else (C) causes both B and A, whether in series or parallel. To disentangle temporal relations requires a longitudinal study. The job of the systems biologist doing systems medicine is to uncover the chief actors and the means by which they interact (99), in this way fulfilling the Bradford Hill postulates, a topic to which we shall return at the end.
In infection microbiology, and long predating the Bradford Hill criteria, the essentially equivalent metrics are known [widely, but somewhat inaccurately (100)] as the Koch or Henle–Koch postulates (i.e., criteria). They involve assessing the correlation of a culturable organism with the presence of a disease, the cure of the disease (and its symptoms) upon removal of the organism, and the development of the disease with (re)inoculation of the organism. They are of great historical importance but present us with three main difficulties here. The first is that we cannot apply the third of them to humans for obvious ethical reasons. The second (see also below) and related one is that we cannot usefully apply them in animal models because none of the existing models recapitulates human PE well. Finally, as widely recognized (100–107), they cannot be straightforwardly applied when dealing with dormant bacteria or bacteria that are otherwise refractory to culture.
Our solution to this is twofold: (i) we can assess the first two using molecular methods if culturing does not work and (ii) we exploit the philosophy of science principle known as “coherence” (108–112). This states that if a series of ostensibly unrelated findings are brought together into a self-consistent narrative and that narrative is thereby strengthened. Our systems approach purposely represents a “coherence” in the sense given.
Overall, known biochemical associations with PE come into four main categories, such as abnormal trophoblast invasion, oxidative stress, inflammation and altered immune response, and anti-angiogenic responses (Figure 2). Each of these can contribute directly to PE, and although they can interact with each other (black arrows), no external or causal source is apparent. Figure 2 has been redrawn from a very nice review by Pennington and colleagues (113), which indicates four main generally accepted “causes” (or at least accompaniments) of PE as the four outer colored circles. As illustrated with the black two-way arrows, many of these also interact with each other. What is missing, in a sense, is then what causes these causes, and that is the nub of our argument here. Since we now know (and describe below) that microbes can affect each of these four general mechanisms, we have added these routes to Figure 1 (using pink arrows) where dormant, resuscitating, or growing microbes are known to contribute.
Figure 2. There are four main “causes” of preeclampsia, represented by the colored outer circles, and these can also interact with each other. That part of the figure is redrawn from Pennington et al. (113). In addition, we note here, as the theme of this review, that microbes can themselves cause each of the features in the outer colored circles to manifest.
In a similar vein, Magee and colleagues (114) have nicely set down their related analysis of the causes and consequences of PE, with a central focus (redrawn in Figure 3) on endothelial cell activation. While bearing much similarity in terms of overall content to the analysis of Pennington and colleagues (113), and ours above, it again lacks a microbial or infection component as a causative element, but importantly does note that infection and/or inflammation can serve to lower the threshold for PE in cases of inadequate placentation. In our view, microbes can also enter following normal placentation if their dormant microbiome begins to wake up and/or to shed inflammagens.
Figure 3. Another detailed representation of factors known to cause or accompany PE, redrawn from Magee et al. (114).
The question of the extent of heritability of PE (susceptibility) is of interest. Although this seems to vary widely in different studies (Table 1), a number of candidate gene studies (37, 115–118) imply that a susceptibility to PE is at least partly heritable, consistent with the variance in all the other “risk factors” of Table 1 [and see Ref. (6)]. As with all the other gene association studies where phenotypic (“lifestyle”) information is absent (119–121), it is not possible to ascribe the heritability to genetics alone, as opposed to an interaction of a genetic susceptibility (e.g., in the HLA system) with environmental factors (117), such as cytomegalovirus infection (122).
Preeclampsia is accompanied by oxidative stress (123, 124) and inflammation and, thus, shares a set of observable properties with many other (and hence related) inflammatory diseases, be they vascular (e.g., atherosclerosis), neurodegenerative (e.g., Alzheimer’s, Parkinson’s), or “metabolic” (type 1 and 2 diabetes). It is thus at least plausible that they share some common etiologies, as we argue here, and that knowledge of the etiology of these diseases may give us useful clues for PE.
As well as raised levels of inflammatory cytokines that constitute virtually a circular definition of inflammation, we and others have noted that all of these diseases are accompanied by dysregulation of iron metabolism (84, 125, 126), hypercoagulability and hypofibrinolysis (127, 128), blood microparticles (126), and changes in the morphology of fibrin fibers [e.g., Ref. (129–134)] and of erythrocytes [e.g., Ref. (127, 132–137)].
In addition, we and others have recognized the extensive evidence for the role of a dormant blood and/or tissue microbiome in these (138–143) and related (144–147) diseases, coupled in part with the shedding of highly inflammagenic bacterial components such as Gram-negative lipopolysaccharide (LPS) and their Gram-positive cell wall equivalents such as lipoteichoic acids (148). (We shall often use the term “LPS” as a “shorthand,” to be illustrative of all of these kinds of highly inflammagenic molecules.)
The purpose of this review, outlined as a “mind map” in Figure 4, is thus to summarize the detailed and specific lines of evidence suggesting a very important role of a dormant microbial component in the etiology of PE [and see also Ref. (138)]. To do this, we must start by rehearsing what is meant by microbial dormancy.
In microbiology, we usually consider microbes as being in one of the three “physiological macrostates” (Figure 5). The definition of a “viable” bacterium is normally based on its ability to replicate, i.e., “viability” = culturability (149–151). In this sense, classical microbiology has barely changed since the time of Robert Koch, with the presence of a “viable” microorganism in a sample being assessed via its ability to form a visible colony on an agar plate containing suitable nutrients. However, it is well known, especially in environmental microbiology [“the great plate count anomaly” (152)], that only a small percentage of cells observable microscopically is typically culturable on agar plates. In principle, this could be because they are/were “irreversibly” non-culturable (operationally “dead”), or because our culture media either kill them (153) or such media lack nutrients or signaling molecules necessary for their regrowth (154, 155) from an otherwise dormant state (156, 157). These statements are true even for microbes that appear in culture collections and (whose growth requirements) would be regarded as “known.”
However, it is common enough in clinical microbiology that we detect the existence or presence of “novel” microbial pathogens with obscure growth requirements before we learn to culture them; this is precisely what happened in the case of Legionella pneumophila (158–161), Tropheryma whipplei [Whipple’s disease (162, 163)], and Coxiella burnetii [the causative agent of Q fever (164, 165)]. Even Helicobacter pylori was finally brought into culture on agar plates only because an unusually long Easter holiday break meant that the plates were incubated for an extended period of 5 days (rather than the normal 2) before being thrown out (166, 167)! Consequently, there is ample precedent for the presence of “invisible” microbes to go unremarked before they are discovered as the true cause of a supposedly non-infectious disease, even when they are perfectly viable (culturable) according to standard analyses.
Dormancy for a microbe is defined operationally as a state, commonly of low metabolic activity, in which the organism appears not to be viable in that it is unable to form a colony but where it is not dead in that it may revert to a state in which it can do so, via a process known as resuscitation (156, 157). However, an important issue (and see above) is that dormant bacteria do not typically fulfill the Koch–Henle postulates (100–103), and in order for them to do so it is necessary that they be grown or resuscitated. This is precisely what was famously done by Marshall and Warren when they showed that the supposedly non-infectious disease of gastric ulcers was, in fact, caused by a “novel” organism called H. pylori (168, 169). One of the present authors showed in laboratory cultures of actinobacteria that these too could enter a state of true dormancy (170, 171) [as is well known for Mycobacterium tuberculosis, e.g., Ref. (172–176)], and could be resuscitated by a secreted growth factor called Rpf (177–181). This RPF family has a very highly conserved motif that is extremely immunogenic (182, 183), and it is presently under trials as a vaccine against M. bovis.
Prevalence of Dormant, Persistent, or Latent Bacteria in Infection Microbiology
It is worth stressing here that the presence of dormant or latent bacteria in infection microbiology is well established; one-third of humans carry dormant M. tuberculosis [e.g., Ref. (175, 184–187)], most without reactivation, while probably 50–100% are infected with H. pylori, most without getting ulcers or worse (188, 189). As with the risk factors in Table 1, the organisms are merely or equivalently “risk factors” for these infectious diseases and are effectively seen as causative only when the disease is actually manifest.
In a similar vein, so-called persisters are phenotypic variants of infectious microbes that resist antibiotics and can effectively lie in hiding to resuscitate subsequently. This is also very well established [e.g., Ref. (139, 190–203)]. In many cases, they can hide intracellularly (204), where antibiotics often penetrate poorly (205) because the necessary transporters (206–209) are absent. This effectively provides for reservoirs of reinfection, e.g., for Staphylococcus aureus (210), Bartonella spp. (211), and – most pertinently here – for the Escherichia coli involved in urinary tract (re)infection (212–215). The same intracellular persistence is true for parasites such as Toxoplasma gondii (216).
Thus, the main point of the extensive prevalence of microbial dormancy and persistence is that microbes can appear to be absent when they are, in fact, present at high concentrations. This is true not only in cases where infection is recognized as the cause of disease but, as we here argue, such microbes may be an important part of diseases presently thought to lack an infectious component.
Iron and Inflammation
It is well known that [with the possible exception of Borrelia (217, 218)] a lack of free iron normally limits microbial growth in vivo [e.g., Ref. (219–243)], and we have reviewed previously (84, 125, 126) the very clear iron dysregulation accompanying PE [e.g., Ref. (90, 244–256)].
This has led to the recognition (128, 139, 141) that the source of the continuing inflammation might be iron-based resuscitation of dormant microbes that could release well known and highly potent inflammagens such as LPS. Indeed, we have shown that absolutely tiny (highly substoichiometric) amounts of LPS can have a massive effect on the blood clotting process (257), potentially inducing β-amyloid formation directly (258, 259) [something, interestingly, that can be mimicked in liquid crystals (260, 261)]. The overall series of interactions envisaged [see also Kell et al. (139)] is shown in Figure 6.
Figure 6. An 11-stage systems biology model of the factors that we consider cause initially formant microbes to manifest the symptoms (and disease) of preeclampsia.
As pointed out by a referee, worldwide, iron deficiency anemia is associated with increased perinatal morbidity and mortality, predominantly in low resource settings. This is most recently and comprehensively reviewed by Rahman and colleagues (262). This meta-analysis (262) found that in low- and middle-income countries, maternal hemoglobin concentrations <10 or <11 g dL−1 or hematocrit values <33 or <34% accounted for 12% of low birth weight, 19% of preterm births (PTBs), and 18% of perinatal mortality. However, the independent contribution of iron deficiency is difficult to define, especially since in low resource settings anemia is often not found in isolation but as part of a wider spectrum of nutritional insufficiencies (especially vitamins and folic acid) or as a result of parasitic infection. These can increase susceptibility to other insults such a hemorrhage or infection, also common in low resources settings. In addition, the exact speciation and hence availability of iron may be important (not just the total amount). Thus, the hypothesis presented here is that iron in excess or under certain physiological conditions can be, and indeed is, equally detrimental to maternal health. There is a likely a “Goldilocks” concentration of iron which is optimal for maternal and neonatal health, and the authors are proposing the selected use of treatments such as statins or iron chelators in the at-risk population, rather than as a broad panacea. Furthermore, the use of statins to prevent pregnancy complications is far from controversial (see also below). There are at least two registered current trials of statins to prevent PE (ISRCTN17787139 and ISRCTN23410175).
Detecting Dormant Microbes
By definition, dormant bacteria escape detection by classical methods of assessing viability that involve replication on agar plates. Other growth-associated methods include measurements involving changes in turbidity (263), including an important but now rather uncommon technique referred to as the “most probable number” (MPN). The MPN involves diluting samples serially and assessing by turbidity changes in the presence of growth/no growth. Look-up tables based on Poisson statistics enable estimation of the number of cells or propagules that were present. A particular virtue is that they allow dormant and “initially viable” cells to be discriminated via “dilution to extinction” (171), thereby avoiding many artifacts (157). As mentioned earlier, preincubation in a weak nutrient broth (171, 264) was instrumental in allowing the discovery (177) of an autocrine “wake-up” molecule necessary for the growth of many actinobacteria.
Other more classical means of detecting microbes, but not whether they were culturable, involved microscopy (190, 265–268) or flow cytometry (269) with or without various stains that reflected the presence or otherwise of an intact cell wall/membrane (170, 270–277). These stains are sometimes referred to as “viability” stains, but this is erroneous as they do not measure “culturability.” Readers may also come upon the term “viable-but-not-culturable”; however, since viable = culturable, this is an oxymoron that we suggest is best avoided (157). Other methods involved measurement of microbial products, e.g., CO2 (278, 279), or changes in the conductivity or impedance of the growth medium (263, 280–282).
Most importantly, however, dormant (as well as culturable) cells may be detected by molecular means, nowadays most commonly through PCR and/or sequencing of the DNA encoding their small subunit ribosomal RNA (colloquially “16S”) (283–297) or other suitable genes. It is clear that such methods will have a major role to play in detecting, identifying, and quantifying the kinds of microbes that we argue lie at the heart of PE etiology.
A Dormant Blood Microbiome
Of course, actual bacteremia, the presence of replicable bacteria in blood, is highly life-threatening (298), but – as emphasized – viability assays do not detect dormant bacteria. When molecular detection methods are applied to human blood, it turns out that blood does indeed harbor a great many dormant bacteria [e.g., Ref. (299–309)]; they may also be detected ultramicroscopically [e.g., Ref. (139–141, 190, 267, 300, 310)] or by flow cytometry (311), and dormant blood and tissue microbes probably underpin a great many chronic, inflammatory diseases normally considered to lack a microbial component (139–141, 144–147, 190, 267, 268, 302, 312–321). Multiple arguments serve to exclude “contaminants” as the source of the bacterial DNA (141): (1) there are significant differences between the blood microbiomes of individuals harboring disease states and nominally healthy controls, despite the fact that samples are treated identically; (2) the morphological type of organism (e.g., coccus vs. bacillus) seems to be characteristic of particular diseases; (3) in many cases, relevant organisms lurk intracellularly, which is hard to explain by contamination; (4) there are just too many diseases where bacteria have been found to play a role in the pathogenesis, that all of them may be caused by contamination; (5) the actual numbers of cells involved seem far too great to be explicable by contamination; given that blood contains ~5 × 109 erythrocytes mL−1, if there was just one bacterial cell per 50,000 erythrocytes this will equate to 105 bacteria mL−1. These are big numbers, and if the cells were culturable, that number of cells would be the same as that ordinarily defining bacteriuria.
A recent study by Damgaard and colleagues (306) is of particular interest here. Recognizing the strong mismatch between the likelihood of an infection post-transfusion [very high (306)] and the likelihood of detecting culturable microbes in blood bank units (negligible, ca. 0.1%) (306, 322), Damgaard et al. reasoned that our methods of detecting and culturing these microbes might be the problem. Certainly, taking cells from a cooled blood bag and placing them onto an agar plate at room temperature that is directly exposed to atmospheric levels of gaseous O2 is a huge stress leading to the production of “reactive oxygen species” (125, 323), that might plausibly kill any dormant, injured, or even viable microbes. Thus, they incubated samples from blood on a rich medium (trypticase soy agar) for a full week, both aerobically and anaerobically. Subsequent PCR and sequencing allowed them to identify specific microbes in some 35–53% of the samples. Thus, very careful methods need to be deployed to help resuscitate bacteria from physiological states that normally resist culture, even when these bacteria are well-established species. This is very much beginning to happen in environmental microbiology [e.g., Ref. (154, 324–326)], and such organisms are rightly seen as important sources of novel bioactives (327, 328).
As reviewed previously (139–143), the chief sources of these blood microbes are the gut microbiome, the oral microbiome [periodontitis (329)], and via UTIs. Consequently, if we are to argue that there is indeed a microbial component to PE, we should expect to see some literature evidence for it (53, 54, 138, 330–332). In what follows we shall rehearse the fact that it is voluminous.
Direct Evidence for a Role of Infectious Agents in PE
Although we recognize that many of the more molecular methods cannot distinguish culturable from dormant microbes, quite a number of studies have explicitly identified infection as a cause of PE (Table 2). The commonest microbe seems to be H. pylori; while it is most famously associated with gastric ulcers (168, 169, 333), there are many other extragastric manifestations [e.g., Ref. (334–342)]. The odds ratio of no less than 26 in PE vs. controls when the strains can produce CagA antigens is especially striking, not least because it provides a mechanistic link to poor trophoblast invasion via a mechanism involving host antibodies to CagA cross-reacting with trophoblasts (343, 344), and circulating (345) in microparticles (346) or endosomes (347, 348).
Table 2. Many studies have identified a much greater prevalence of infectious agents in the blood or urine of these exhibiting PE than in matched controls.
In contrast to the situation in PE, albeit severe PE is associated with iatrogenic PTBs, there is a widespread recognition [e.g., Ref. (383–410)] that infection is a common precursor to PTB in the absence of PE. The failure of antibiotics to help can be ascribed to their difficulty of penetrating to the trophoblasts and placental regions. Unfortunately, no proteomic biomarkers have yet been observed as predictive of PTB (411, 412). In a similar vein, and if we are talking about a time of parturition that is very much more “preterm,” we are in the realm of miscarriages and spontaneous abortions and stillbirths, where infection again remains a major cause (413–416). Here, we note that early or pre-emptive antibiotic therapy has also proved of considerable value in improving outcomes after multiple spontaneous abortions (417).
Vaginal, Placental, and Amniotic Fluid Microbiomes in PE
It might be natural to assume that the placenta is a sterile organ, like blood is supposed to be. However, various studies [modulo the usual issues of contamination (418)] have shown the presence of microbes in tissues including the placenta (400, 409, 419–432), vagina (393, 433–440), uterus (391, 441, 442), amniotic fluid (430, 443–448), and follicular fluid (449, 450), and how these may vary significantly in PE [we do not discuss other pregnancy disorders such as small for gestational age (SGA) and intrauterine growth restriction (IUGR)]. We list some of these in Table 3.
Origins of a Blood and Tissue Microbiome
As assessed previously (139–141) over a large literature, the chief source of blood microbes is the gut (426), with another major entry point being via the oral microbiome (especially in periodontitis, see below). For rheumatoid arthritis (142, 458–460) and diseases of pregnancy, UTI (see below and Table TT) also provides a major source.
Gut Origins of Blood Microbes and LPS
We have recently rehearsed these issues elsewhere (139–141), so a brief summary will suffice. Clearly, the gut holds trillions of microbes, with many attendant varieties of LPS (461), so even low levels of translocation [e.g., Ref. (462–464)], typically via Peyer’s patches and M cells, provide a major source of the blood microbiome. This may be exacerbated by intra-abdominal hypertension and overeating (465–467) that can indeed stimulate the translocation of LPS (468). For reasons of space and scope, we do not discuss the origins and translocation of microbes in breast milk (469) nor the important question of the establishment of a well-functioning microbiome in the fetus and neonate (470), and the physiological role of the mother therein.
Preeclampsia and Periodontal Disease
One potential origin of microbes that might be involved in, or represent a major cause of, PE is the oral cavity, and in particular when there is oral disease (such as periodontitis and gum bleeding) that can allow microbes to enter the bloodstream. If this is a regular occurrence one would predict that PE would be much more prevalent in patients with pre-existing periodontitis [but cf. Ref. (471) for those in pregnancy] than in matched controls; this is indeed the case (Table 4). As with many of the tables herein, the odds ratios are far beyond anything that might weakly be referred to as an “association.”
Urinary Tract Infections
A particular feature of UTIs is the frequency of reinfection (497–504). This is because the organisms can effectively “hide” in bladder epithelial cells as the so-called “quiescent intracellular reservoirs” (212, 501, 503, 505–509) of (presumably) dormant cells that can resuscitate. This is why reinfection is often from the same strains that caused the original infection (510–514). Other complications can include renal scarring (515). Bacteriuria (often asymptomatic) is a frequent occurrence in pregnancy [e.g., Ref. (373, 375, 473, 516–522)], and the frequency of UTI as a source of microbes causing PE is clear from Table 2.
From Blood to and from the Placenta: A Role for Microparticles
We and others have noted the fact that many chronic, inflammatory disease are accompanied by the shedding of various antigens and other factors; typically they pass through the bloodstream as microparticles (126, 140, 523–530), sometimes known as endosomes (345, 347, 348, 524, 531) [and see later under microRNAs (miRNAs)]. Similarly, LPS is normally bound to proteins such as the LPS-binding protein and apoE (140). Given their prevalence, their role in simply finding their way from maternal blood to placenta, and the fact that we discussed them extensively in two previous reviews (126, 140), we do not discuss them further here.
Evidence from Antibiotic Therapies
Antibiotic drug prescriptions (532–534) may be seen as a proxy for maternal infection, so if dormant (and resuscitating and growing) bacteria are a major part of PE etiology one might imagine an association between antibiotic prescriptions and PE. According to an opposite argument, antibiotics and antibiotic prescriptions given for nominally unrelated infections (UTI, chest, etc., and in particular diseases requiring long-term anti-infective medication that might even last throughout a pregnancy) might have the beneficial side-effect of controlling the proliferation of dormant cells as they seek to resuscitate. There is indeed some good evidence for both of these, implying that it is necessary to look quite closely at the nature, timing, and duration of the infections and of the anti-infective therapy relative to pregnancy. A summary is given in Table 5. A confounding factor can be that some (e.g., the antiretroviral) therapies are themselves quite toxic (535, 536); while the OR for avoiding PE was 15.3 in one study of untreated HIV-infected individuals vs. controls, implying (as is known) a strong involvement of the immune system in PE, the “advantage” virtually disappeared upon triple-antiretroviral therapy (537). Overall, it is hard to draw conclusions from antiretrovirals (538, 539). However, we have included one HIV study in the table. Despite a detailed survey, we found no reliable studies with diseases such as Lyme disease or tuberculosis, where treatment regimens are lengthy, that allowed a fair conclusion as to whether antibiotic treatment was protective against PE. However, we do highlight the absolutely stand-out study of Todros and colleagues (540), who noted that extended spiramycin treatment (of patients with T. gondii) gave a greater than 10-fold protection against PE, when the parasite alone had no effect (541). This makes such an endeavor (assessing the utility of early or pre-emptive antibiotics in PE) potentially highly worthwhile.
Role of LPS in PE
It is exceptionally well known that LPS (sensu lato) is highly inflammagenic, and since one of us recently reviewed that literature in extenso (140) this is not directly rehearsed here. However, since we are arguing that it has a major role in PE naturally or in vivo, we do need to ask whether the literature is consistent with this more focused question. The answer is, of course, a resounding “yes.” Notwithstanding that only primates, and really only humans, are afflicted by “genuine” PE, so the genuine utility of rodent models is questionable (543), even if some can recapitulate elements of the disease (544, 545). Hence, it is somewhat ironic that there are a number of animal models in which LPS (also known as “endotoxin”) is used experimentally to induce a condition resembling PE [e.g., Ref. (546–551) and also see Ref. (552)]. We merely argue that it is not a coincidence that exogenous administration of LPS has these effects, because we consider that it is, in fact, normally one of the main mediators of PE. Also note, in the context of gestational diabetes, that serum levels of LPS are raised significantly in both type 1 (466, 553) and type 2 (554–556) diabetes.
The standard sequelae of LPS activation, e.g., TLR signaling and cytokine production, also occur in PE (557–559), bolstering the argument that this is precisely what is going on. In a similar vein, double-stranded RNA-mediated activation of TLR3 and TLR7/8 can play a key role in the development of PE (560–562). What is new here is our recognition that LPS and other inflammagens [e.g., Ref. (563–565)] may continue to be produced and shed by dormant and resuscitating bacteria that are generally invisible to classical microbiology.
Effects of LPS and Other Microbial Antigens on Disrupting Trophoblast Invasion and/or Stimulating Parturition
As with other cases of cross-reactivity such as that of various antigens in Proteus spp. that can cause disease in rheumatoid arthritis (458–460), the assumption is that various microbial antigens can lead to the production of (auto-)antibodies that attack the host, in the present case of interest by stopping the placentation by trophoblasts. This is commonly referred to as “molecular mimicry” [e.g., Ref. (566–569)] and may extend between molecular classes, e.g., peptide/carbohydrate (570, 571). Table 6 shows some molecular examples where this has been demonstrated.
Table 6. Molecular examples of bacterial antigens that can elicit antibodies that stop successful trophoblast implantation or stimulate parturition.
In many cases, the actual (and possibly microbial) antigens are unknown, and clearly the microbial elicitation of antibodies to anything that might contribute to PE points to multiple potential origins. To this end, we note that PE has also been associated with antibodies to angiotensin receptors (577–590), to smooth muscle (591, 592) [such blocking may be anti-inflammatory (593–595)], to adrenoceptors (596), to the M2 muscarinic receptor (597), and to Th17 (598) [and see Ref. (599)]. It is not unreasonable that epitope scanning of the antibody targets coupled with comparative sequence analysis of potential microbes might light up those responsible. In the case of angiotensin II type 1 receptor antibodies, the epitope is considered (600) to be AFHYESQ, an epitope that also appears on parvovirus B19 capsid proteins; in the event, parvoviruses seem not to be the culprits here (601). However, the role of these antibodies in activating the angiotensin receptor is also considered to underpin the lowering of the renin–angiotensin system that is commonly seen in PE (602–605), but which is typically raised during normal pregnancy.
Th-17 is of especial interest here, since these are the helper T (Th)-cell subset that produces IL-17. IL-17 is probably best known for its role in inflammation and autoimmunity (599, 606–610). However, it also has an important role in induction of the protective immune response against extracellular bacteria or fungal pathogens at mucosal surfaces (608, 611–623). Th17 cells seem to participate in successful pregnancy processes and can be lower in PE (624–626), although more studies show them as higher (599, 627–635) or unchanged (636, 637). One interpretation, consistent with the present thesis, is that the antimicrobial effects of placental IL-17 relative to Treg cells are compromised during PE (599, 633, 638).
A Note on the Terminology of Sepsis
As one may suppose from the name, sepsis (and the use of words like “antiseptic”) was originally taken to indicate the presence of culturable organisms in (or in a sample taken from) a host, e.g., as in bacteremia. Recognizing that it is the products of bacteria, especially cell wall components, that cause the cytokine storms that eventually lead to death from all kinds of infection (639–643), “sepsis” nowadays has more come to indicate the latter, as a stage (in the case of established infection) on a road that leads to septic shock and (eventually) to death [with a shockingly high mortality, and many failures of initially promising treatments, e.g., Ref. (644, 645), and despite the clear utility of iron chelation (84, 125, 646–648)]. In most cases, significant numbers of culturable microbes are either unmeasured or absent, and like most authors, we shall use “sepsis” to imply the results of an infection whether the organisms are detected or otherwise. Overall, it is possible to see the stages of PE as a milder form of the sepsis cascade on the left-hand side of Figure 7. Figure 7 compares the classical route of sepsis-induced death with the milder versions that we see in PE; they are at least consistent with the idea that PE is strongly related to the more classical sepsis in degree rather than in kind.
Figure 7. Preeclampsia bears some similarities to and may be considered as a milder form of, the changes that occur during genuine sepsis leading to a systematic inflammatory response syndrome, septic shock, and multiple organ dysfunction.
Preeclampsia and Neonatal Sepsis
If PE is really based on infectious agents, it is reasonable that one might expect to see a greater incidence of neonatal sepsis (i.e., infection) following PE. While there are clearly other possible explanations (e.g., simply a weakened immune system, sometimes expressed as neutropenia, after PE), there is certainly evidence that this is consistent with this suggestion (649–653).
PE Biomarkers and Infection
Because of the lengthy development of PE during pregnancy, there has long been a search for biomarkers (somewhat equivalent to the “risk factors” discussed earlier) that might have predictive power, and some of these, at both metabolome (15, 654–661) and proteome (662–667) level, are starting to come forward. The typical experimental design is a case–control, in which markers that are raised or lowered significantly relative to the age-matched controls are considered to be candidate markers of PE. However, just as noted with leukocyte markers (81) and polycystic ovary syndrome (PCOS) (668) that does not mean that they might not also be markers for other things too, such as infection (669)!
Thus, one prediction is that if dormant and resuscitating bacteria are responsible for PE then at least some of these biomarkers should also be (known to be) associated with infection. However, one obvious point is that the markers may appear only after infection, and this may itself be after the first-trimester; clearly then these would not then be seen as “first-trimester” biomarkers! There are many well-known inflammatory biomarkers that are part of the innate [and possibly trained (670)] immune response, such as the inflammatory cytokines CRP [cf. Ref. (671, 672)], IL-6 (673), IL-1β (674), TNF-α (675), and macrophage migration inhibitory factor (MIF) (676), which are also all biomarkers of infection (677–681). Certainly, the fact that these increase in PE is consistent with a role for an infectious component. However, we shall mainly look at other biomarkers that are known to increase with PE, and see if they are also known to be biomarkers for (or at least changed in the presence of) infection (and see Th17/IL-17 above), and we next examine this. We shall see that pretty well every biomarker that is changed significantly in PE is also known to be changed following infection, a series of findings that we consider adds very strong weight to our arguments.
Proteomic and Similar Biomarkers – Circulating and Placental
What is really needed is a full systems biology strategy [see, e.g., Ref. (99, 682–684)] that brings together the actors that interact then parametrizes the nature of these interactions in a suitable encoding [e.g., SBML (685)] that permits their modeling, at least as an ODE model using software such as CellDesigner (686), COPASI (687), or Cytoscape (688). Thus, to take a small example, “agonistic autoantibodies against the angiotensin II type 1 receptor autoantibodies (AT1-AA) are described. They induce NADPH oxidase and the MAPK/ERK pathway leading to NF-κB and tissue factor activation. AT1-AA are detectable in animal models of PE and are responsible for elevation of soluble fms-related tyrosine kinase-1 (sFlt1) and soluble endoglin (sEng), oxidative stress, and endothelin-1, all of which are enhanced in pre-eclamptic women. AT1-AA can be detected in pregnancies with abnormal uterine perfusion” (589). Many such players have been invoked, and we next list some.
In a proteomic study of preclamptic vs. normal placentas (696), calretinin was one of the most differentially upregulated proteins (p = 1.6 × 10−13 for preterm PE vs. controls, p = 8.9 × 10−7 for term PE vs. controls), and in a manner that correlated with the severity of disease. While calretinin [normally more expressed in neural tissue and mesotheliomas (697)] is not normally seen as a marker of infection, it is, in fact, raised significantly when Chlamydia pneumoniae infects human mesothelial cells (698).
Chemerin is a relatively recently discovered adipokine, whose level can increase dramatically in the first-trimester of preeclamptic pregnancies (699), and beyond (700). Its levels are related to the severity of the PE (701–703). Specifically, an ROC curve (704) analysis showed that a serum chemerin level >183.5 ng mL−1 predicted PE with 87.8% sensitivity and 75.7% specificity (AUC, 0.845; 95% CI, 0.811–0.875) (699). Papers showing that chemerin is also increased by infection (hence inflammation) include (705, 706); it even has antibacterial properties (707, 708) and was protective in a skin model of infection (709, 710). In a study of patients with sepsis (711), circulating chemerin was increased 1.69-fold compared with controls (p = 0.012) and was also protective as judged by survival. These seem like particularly potent argument for a role of chemerin as a marker of infection rather than of PE per se, and for the consequent fact that PE follows infection and not vice versa.
Copeptin, a glycosylated polypeptide consisting of the 39 C-terminal amino acids of arginine vasopressin, has been suggested as “a new biomarker that is specific for preeclampsia” (712), and certainly changes during its development (713). However, it turns out that it is also essentially a measure of all kinds of stresses and adverse events (714–719), including those caused by infection (720–729).
Not least because kidney function is impaired in PE, low molecular weight (MW) proteins may serve as biomarkers for it. To this end, cystatin C (13 kDa) has been found to be raised significantly in PE (730–736); it also contributed to the marker set in the SCOPE study (8, 17). Notably, although it certainly can be raised during infection (737), it seems to be more of a marker of inflammation or kidney function (738, 739).
“d-dimer” is a term used to describe quite varying forms of fibrin degradation products (740). Given that PE is accompanied by coagulopathies, it is probably not surprising that d-dimer levels are raised in PE (741–745), although this is true for many conditions (746), and some of the assays would bear improvement (747, 748). Needless to say, however, raised d-dimer levels are also a strong marker for infection (749, 750).
Endothelial cell-specific molecule-1 (ESM-1), known as endocan, is a cysteine-rich dermatan sulfate proteoglycan expressed (and sometimes released) by endothelial cells. It has been suggested to be a new biomarker for endothelial dysfunction and PE (751, 752). It would appear, however, to be a rather less specific inflammatory biomarker (753, 754) and is associated with a variety of diseases, including chronic kidney disease (755, 756) and cardiovascular disease (755). Most pertinently from our perspective, it is also raised strongly during sepsis (757–760).
Endoglin is the product of a gene implicated (761, 762) in the rare disease Hereditary Hemorrhagic Telangiectasia. The role of endoglin remains somewhat enigmatic (763). However, endoglin levels were 2.5-fold higher in preeclamptic placentas compared to normal pregnancies (15.4 ± 2.6 vs. 5.7 ± 1.0, p < 0.01). After the onset of clinical disease, the mean serum level of sEng in women with preterm PE was 46.4 ng mL−1, as compared with 9.8 ng mL−1 in controls (p < 0.001) (88). Women with a particular endoglin polymorphism (AA) were 2.29 times more likely to develop PE than those with the GG genotype (p = 0.008) (764), and endoglin is seen as a reasonably good marker for PE (88, 691, 765–768) [cf. Ref. (769)]. Again, endoglin levels are raised following infection by a variety of organisms (770–773), with a particularly clear example that it is a marker of infection coming from the fact that there is raised endoglin only in infected vs. aseptic loosening in joints following arthroplasty (774). In general, it seems likely that these circulating (anti)angiogenic factors are more or less markers of endothelial cell damage, just as we have described for serum ferritin (126).
The natural iron transporter in blood is transferrin [e.g., Ref. (775–780)], present at ca. 1–2 g L−1, with ferritin being an intracellular iron storage molecule, so one is led to wonder why there is even any serum ferritin at all (126, 781). The answer is almost certainly that it is a leakage molecule from damaged cells (126), and when in serum it is found to have lost its iron content (782–785). Serum ferritin is, as expected, raised during PE (244, 246, 249, 253, 255, 786, 787) and in many other inflammatory diseases (126), including infection [e.g., Ref. (788, 789) and above].
microRNAs are a relatively novel and highly important class of ~22 nt non-coding, regulatory molecules (790–793). Some are placenta specific, and those in the circulation [often in endo/exosomes (794–796)] can be identified during pregnancy (797–800), potentially providing a minimally invasive readout of placental condition (801–803). There is aberrant expression of placenta-specific miRNAs in PE including miR-517a/b and miR-517c (804–810) and miR-1233 (811). C19MC is one of the largest miRNA gene clusters in humans, maps to chromosome 19q13.41, and spans a ~100 kb long region. C19MC miRNAs are processed from the cluster (812), are primate-specific, conserved in humans, and comprise 46 miRNA genes, including the miR-517 family (813). miR-517 is known to be antiviral (814, 815), while miR-517a overexpression is apoptotic (816) and can inhibit trophoblast invasion (817). Importantly for our argument, miR-517 molecules are overexpressed following infection (818, 819).
Although, as its name suggests, neuropeptide Y is a neurotransmitter, it is also correlated with stress. Certainly, it is related to noradrenaline (see below) that may itself be responsible for the raised blood pressure (BP) in PE (820). It is also raised in sepsis, where it is considered to counterbalance the vasodilation characteristic of septic shock [e.g., Ref. (821, 822)]. The apparent paradox of a raised BP in PE and a lowered one in septic shock is considered to be related to the very different concentrations of endotoxin involved (Figure 7).
NGAL (Lipocalin 2, Siderocalin)
Neutrophil gelatinase-associated lipocalin (NGAL) is a lipocalin that is capable of binding catecholate-based siderophores (125, 823, 824). As such it is antimicrobial and is also an inflammatory or sepsis biomarker (825, 826). Given our interest in iron, it is not surprising that it is changed during PE. While one study suggested it to be decreased in PE (827), a great many other studies showed it to be increased significantly in PE, and typically in a manner that correlated with PE severity (735, 828–837). Pertinently to PE, it is also well established as an early biomarker of acute kidney injury (AKI) (838–841). However, it is not a specific biomarker for AKI vs. sepsis (839, 842–850) and its origin in sepsis differs (851, 852). Of course, it can be the sepsis that leads to the AKI (853, 854). Fairly obviously, while it does tend to be increased during PE, we again see its direct role as an antimicrobial and marker of sepsis as highly supportive of our present thesis.
Placental Growth Factor
This is a member of the VEGF Family that despite its name has a great many activities (855). It is often considered in parallel with endoglin and sFlt, with a high sFlt:PlGF ratio being considered as especially discriminatory for PE (89, 856–869), i.e., a lower PlGF can be diagnostic of PE (769, 870–873). PlGF tends to be raised in sepsis unrelated to pregnancy (874, 875), while its lowering in PE may be due to the excess sFLT that decreases it (855, 876, 877). In one study of a patient with CMV infection and PE, it was, in fact, raised (878), while, in the case of IUGR, it was massively lowered (879). PlGF alone is thus probably not a useful general marker for either PE or sepsis if one is trying to disentangle them, although it has clear promise when PE is superimposed on CKD (872, 880).
Procalcitonin is the 116 amino acid polypeptide precursor of calcitonin, a calcium regulatory hormone. It is another marker that has been observed to be raised (according to severity) in preeclamptics (742, 881, 882) [but cf. Ref. (713)]. However, it is also a known marker of bacterial infections or sepsis (881, 883–891).
Serum Amyloid A
This is an inflammatory biomarker, which was shown to increase fourfold in PE in one study (892), was significantly raised in another (882), but not in a third (893). However, it is a well-established (and potent) biomarker for infection/sepsis [e.g., Ref. (894–907)]. Defective amyloid processing may be a hallmark of PE more generally (908), and of course amyloid can be induced by various microbes (317, 319, 909, 910) and their products (257).
Soluble fms-Like Tyrosine Kinase 1
The sFlt receptor is a splice variant of the VEGF receptor (766). It is raised considerably in PE (691, 767, 856, 862, 864, 911–914) and may be causal (545, 590, 915–918). Needless to say, by now, we can see that it is also a very clear marker of infection (767, 919, 920), whose levels even correlate with the severity of sepsis (921–923). Of particular note is the fact that sFLT is actually anti-inflammatory (922).
Soluble thrombomodulin was recognized early as an endothelial damage biomarker and is raised in PE (924–934). Interestingly, it has been found to have significant efficacy in the treatment of sepsis [-based disseminated intravascular coagulation (DIC)] (935–943).
TLR4 upregulation in preeclamptic placentas (944) is entirely consistent with infection and the “danger model” as applied to PE (945). As well as LPS activation [reviewed in Kell and Pretorius (140)], the heat shock protein 60 of Chlamydia also activates TLR4 (138).
Visfatin is another adipokine that is raised in PE, approximately 2-fold in the study of Fasshauer and colleagues (946), and 1.5-fold in that of Adali and colleagues (947). However, it was little different in a third study (948), while in a different study, it was rather lower in PE than in controls (949). This kind of phenomenon rather lights up the need for excellent quality studies, including ELISA reagents, when making assessments of this type.
Fairly obviously, the conclusion that this long list of biomarkers that are raised in PE might be specific “PE” biomarkers is challenged very strongly by the finding that they are, in fact, all known markers of infection, a finding that in our view strongly bolsters the case for an infectious component in PE.
In a similar vein, there are a number of other sepsis markers (where sepsis is varied via, or occurs as, an independent variable) that we would predict are likely to be visible as raised in PE patient. These might include (680, 950) PAI-1, sE-selectin (951), and sVCAM-1 (921). In particular, Presepsin looks like a potentially useful marker for sepsis (888, 889, 952–961), but we can find no literature on its use as a PE biomarker, where we predict that it may also be raised.
For fundamental reasons connected with metabolic control and its formal, mathematical analysis (962–966), changes in the metabolome are both expected (967) and found (968–971) to be amplified relative to those in the transcriptome and proteome. For similar reasons, and coupled with evolution’s selection for robustness (972–978) (i.e., homeostasis) in metabolic networks, we do not normally expect to find single metabolic biomarkers for a complex disease or syndrome. Since our initial metabolomic analyzes (654), the technology has improved considerably (979–982), a full human metabolic network reconstruction has been published (978, 983–985) in the style of that done for yeast (986), and a number of candidate metabolomics biomarkers for PE have been identified reproducibly on an entirely separate validation set (15, 655).
This latter, LC-MS-based, study (15) found a cohort of 14 metabolites from the first-trimester that when combined gave an OR of 23 as being predictive of third-trimester PE. For convenience, we list them in Table 7. Note that because they were characterized solely via their mass, there are some uncertainties in the exact identification in some cases, and that untargeted metabolomics of this type has a moderately high limit of detection (maybe 10 μM) such that many potentially discriminatory metabolites are below the limit of detection.
Table 7. Fourteen metabolites contributing to a preeclamptic “signature” (15).
A number of features of interest emerge from this:
1. All the markers save 5-hydroxytryptophan and adipic/methylglutaric acid that were raised in PE; 5-hydroxytryptophan is a precursor of serotonin [which in some studies (987) has been seen to be mildly elevated in PE].
2. Markers came from multiple classes of metabolite or areas of metabolism, including amino acids, carbohydrates, carnitines, dicarboxylic acids, fatty acids (especially), (phospho)lipids, and sterols.
4. In common with many other inflammatory diseases (145), Vitamin D3 levels [usually measured as 25(OH)vitD or calcidiol] are often lower in PE (990–994) [cf. Ref. (995–997)], consistent with the levels of their derivatives being raised. However, the direction of causality inflammation ←→ vitamin D levels is not yet known (998) [see also Ref. (143, 145, 996)].
As well as the non-targeted metabolomics noted above, a number of other small molecule biomarkers have been turned up by more conventional measurements.
An interesting early study (1002) found that venous plasma noradrenaline was raised by 67% in preeclamptics vs. controls. Similar data were found by others (1003). This is of particular interest in the present context since noradrenaline is well established as highly growth stimulatory to Gram-negative microorganisms [e.g., Ref. (1004–1008)], in part by acting as a siderophore (1009–1011). It also raises the levels of neuropeptide Y (820), and as a stress hormone (1012), is of course well known for its role in raising BP, a hallmark of PE.
There is relatively little metabolomics work in sepsis, but in one study, carnitine and sphingolipid metabolism were also modified during sepsis (1013), while in another (1014), a suite of molecules were decreased during acute sepsis. However, the patients involved here were quite close to death, so it is not clear that comparisons between the metabolome in PE and in dying patients are that worthwhile.
We also note a recent and rather interesting suggestion by Eggers (1015) that the maternal release of adrenaline (rather than noradrenaline) may have an important etiological role in PE, although as with the rest of our thesis here it is not there indicated as to what causes the adrenaline to rise (although infection and inflammation can of course do so).
Hyperuricemia is a moderately common finding in preeclamptic pregnancies and may even be involved in its pathogenesis [see, e.g., Ref. (1016–1022)]. However, it does not seem to be very specific (1023–1027) and is seemingly not an early biomarker [and it did not appear in our own study (15)]. Its lack of specificity is illustrated by the fact that there is considerable evidence for the roles of purinergic signaling (1028), and especially the role of uric acid, in Alzheimer’s and Parkinson’s disease (1029–1031), as well as in a variety of other kinds of inflammatory processes, including pro-inflammatory cytokine production (1032, 1033), the Plasmodium falciparum-induced inflammatory response (1034), the mechanistic basis for the action of alum as an adjuvant (1035), and even peanut allergy (1036–1038). As is common in case–control studies when just one disease (e.g., PE) is studied, artificially high levels of sensitivity and (especially) specificity may appear when other patients with other diseases are not considered.
Clotting, Coagulopathies, and Fibrinogen in PE
In much of our previous work [e.g., Ref. (126–134)], we have noted that each of these chronic, inflammatory diseases is accompanied by changes in fibrin fiber morphologies, coagulopathies, and changes in erythrocytes that are both substantial and characteristic. They can variously be mimicked by adding unliganded iron or LPS. As is well known, LPS itself is a strong inducer of coagulation, whether via tissue factor or otherwise [e.g., Ref. (1039–1048)], and will bind to fibrin strongly (259, 1049). The morphological methods have not yet, to our knowledge, been performed on blood from preeclamptics, whether as a diagnostic or a prognostic, although we note that clotting factors came top in one GWAS looking for gene–PE associations (117). Fibrinogen itself is a TLR4 ligand (1050), is raised in PE (1051–1055), and we note the extensive evidence for coagulopathies during pregnancies with PE [e.g., Ref. (64, 128, 527, 741, 1056–1068)]. In the worst cases, these are the very frightening DIC (1047, 1069–1073) that can, of course, also emerge as a consequence of sepsis (1074–1080). Variations in the plasminogen activator inhibitor-1 may contribute to the hypofibrinolysis observed (1081–1083).
We recently showed that LPS can potently induce amyloid formation in fibrin (258, 259, 1084, 1085). Thus, in addition, we note the increasing recognition that amyloid proteins themselves, that may occur as a result of coagulopathies, are themselves both inflammatory [e.g., Ref. (565, 669, 1086–1091)] and cytotoxic [e.g., Ref. (257, 1092–1096)], and that this can of itself contribute strongly to the death of, e.g., trophoblasts.
Related to clotting parameters are three other “old” but easily measured variables that probably reflect inflammation (1097), that have been suggested to differ in PE from normotensives, and may have some predictive power. The first two are the erythrocyte sedimentation rate (ESR) (1098, 1099) and the red cell distribution width (RDW) (1100) [but cf. Ref. (1101)]. Interestingly, the former was the only variable that was predictive of a subsequent stroke following sub-arachnoid hemorrhage (1102). The third relates to the morphology of erythrocytes (that may in part underpin the other two). We and others have shown in a series of studies [e.g., Ref. (134–136, 1103–1106)] that erythrocyte morphology diverges very considerably from that “classical” discoid shape adopted by normal healthy cells, and that this can be a strong indicator of disease (137). In extreme cases [e.g., Ref. (133, 1107–1112)], including following infection (1113), this results in eryptosis, the suicidal death of erythrocytes. It is of interest that ceramide, a precursor of sphingosine-1-phosphate (S1P) (Table 7), is raised in various diseases such as Parkinson’s and may serve to stimulate eryptosis (1114). Although we know of no direct measurements to date, there is evidence that eryptosis may play a significant role in PE (1115).
Apart from low-dose aspirin [that may have little effect (1116–1119) unless initiated relatively early in pregnancy (1120–1124)], and low-dose calcium (1125), there are relatively few treatment options in present use (1126–1129). [Magnesium sulfate (1130–1132) has been used as a treatment for eclampsia and, interestingly, prevents LPS-induced cell death in an in vitro model of the human placenta (1133).]
In the history of science or medicine, some treatments are empirical, while others are considered to have a mechanistic basis. The general assumption is that the more we know about the originating etiology of a disease or syndrome the more likely we are to be able to treat its causes effectively, and not just its symptoms. Clearly, also, clinicians are rightly loth to give complex and potentially teratogenic treatments to pregnant women when this can be avoided (1134–1137). However, the surprising lack of systematic data with antibiotics (1138), modulo one particularly spectacular success (540), suggests that we ought to be performing trials with safe antibiotics on women at special risk (1139). These must take care to avoid any Jarisch–Herxheimer reaction (1140–1143) due to the release from microbes induced by antibiotics of inflammagens such as LPS (1144–1147). A related strategy recognizes that some FDA-approved drugs can actually exert powerful antibiotic effects in vivo (but not on petri plates) by modifying the host (1148).
Because of the known oxidative stress accompanying PE, it had been assumed that antioxidants such as vitamin C (ascorbate) might be preventive; however, this turned out not to be the case (even the opposite) for ascorbate (1118, 1149). Probably, this is because in the presence of unliganded iron, ascorbate is, in fact, pro-oxidant (125). However, polyphenolic antioxidants that actually act by chelating iron (84, 125) seem to be more effective (1150).
Another area that we and others have previously highlighted recognizes the ability of non-siderophoric iron chelators to act as iron-withholding agents and thereby limit the growth of bacteria. Again, a prediction is that women with iron overload diseases should be more susceptible to PE, a prediction that is borne out for α-thalassemia (1151, 1152) though not apparently for hereditary hemochromatosis (1153). However, the extent of use of chelators and degree of control of free iron, thereby obtained is rarely recorded in any detail, so in truth it is difficult to draw conclusions.
How significant coagulopathies are to the etiology of PE development (as opposed to providing merely an accompaniment) is not entirely clear, but on the basis that they are then anticoagulants would potentially assist, just as thrombomodulin does in DIC accompanying sepsis (942, 943, 1079, 1080). Of course, one of many effects of low-dose aspirin is to act as an anticoagulant. There is also evidence for the efficacy of heparin (6, 1127, 1155–1160), which is especially interesting given our highlighting of the role of coagulopathies in PE. These anticoagulants that avoid bleeding (1161) are obviously of particular interest, while anything stopping the fibrin forming β-amyloid (258, 259) should serve as an especially useful anti-inflammatory anticoagulant.
With a change in focus from function-first to target-first-based drug discovery (976), there has been an assumption that because a drug is (i) found to bind potently to a molecular target and (ii) has efficacy at a physiological level in vivo, the first process is thus responsible for the second. This has precisely no basis in logic [it is a logical fault known variously as “affirming the consequent” or “post hoc ergo propter hoc” (1162)]. This is because the drug might be acting physiologically by any other means, since drug binding to proteins is typically quite promiscuous [e.g., Ref. (1163–1167)]. Indeed, the average known number of binding sites for marketed drugs is six (208, 1168). In particular, it is likely, from a network or systems pharmacology perspective [e.g., Ref. (978, 1169–1172)], that successful drugs (like aspirin) are successful precisely because they hit multiple targets. The so-called “statins” provide a particularly good case in point (125).
It had long been known that the enzyme HMGCoA reductase exerted strong control on the biosynthetic flux to cholesterol, and that inhibiting it might lower the flux and steady-state cholesterol levels (as indeed it does). Notwithstanding that cholesterol alone is a poor predictor of cardiovascular disease (1173–1175), especially in the normal range, HMGCoA reductase inhibitors have benefits in terms of decreasing the adverse events of various types of cardiovascular disease (1176). Following an original discovery of natural products such as compactin (mevastatin) and lovastatin containing a group related to hydroxymethylglutaric acid (rather than a CoA version) that inhibited the enzyme (1177), many variants with this (hydroxyl)methylglutaric substructure came to be produced, with the much larger “rest” of the molecule being considerably divergent [see Figure 8, where the MW values vary from 390.5 (mevastatin) to 558.6 (atorvastatin)]. Despite this wide structural diversity (Figure 8), they are still collectively known as “statins,” and despite the wildly illogical assumption that they might all work in the same way(s). The fact that different statins can cause a variety of distinct expression profiles (1178) is anyway utterly inconsistent with a unitary mode of action. In particular, in this latter study, statins clustered into whether they were (fluvastatin, lovastatin, and simvastatin) or were not (atorvastatin, pravastatin, and rosuvastatin) likely to induce the side-effect of rhabdomyolysis or any other myopathy. Clearly, any choice of “statin” should come from the latter group, with pravastatin and rosuvastatin being comparatively hydrophilic.
The epidemiological fact of improved survival despite the comparative irrelevance of cholesterol levels to atherosclerotic plaque formation and heart disease in the normal range provides an apparent paradox (1179). This is easily solved by the recognition [e.g., Ref. (1180–1193), and many other references and reviews] that “statins” are, in fact, anti-inflammatory. They may also be antimicrobial/antiseptic, whether directly or otherwise (1194–1198), and we also note the role of cholesterol in mopping up endotoxin (1199). Finally, here, it needs to be recognized that statins do themselves serve to lower iron levels (1200–1202), and (while oddly this seems not to have been tested directly) simple inspection of their structures (Figure 8) implies that the better ones (with their multiple OH groups) might, in fact, chelate iron directly.
In consequence, a number of authors have indicated the potential utility of statins in treating PE (113, 545, 1203–1213), and pravastatin has been the subject of a number of favorable studies (545, 1204, 1206, 1209, 1211, 1214, 1215), including in humans (1204, 1216–1218). Pravastatin seems more than ripe for a proper, randomized clinical trial (1203).
Another “vascular” class of drugs that has been proposed for treating PE is represented by those of the family of vasodilatory phosphodiesterase 5 inhibitors such as sildenafil (Viagra) and vardenafil (Levitra), as it is reasonable that they might improve endothelial function, especially if started early in pregnancy (1219). Thus, vardefanil restores endothelial function by increasing PlGF (1220), and sildenafil has shown promise in a number of animal studies (1221–1226) and in human tissues (1227, 1228), with a clinical trial ongoing (1229). In particular (1226), it was able to normalize the metabolomics changes observed in a mouse model (the COMT−/− model) of PE.
Anti-hypertensive therapy for PE has been reviewed by Abalos and colleagues (1230) and Magee and colleagues (114). Anti-hypertensives did halve the incidence of hypertension but had no effect on PE. Methyldopa is one of the most commonly used anti-hypertensives in pregnancy, but it may also stimulate eryptosis (1231); alternative drugs were considered to be better (1230) for hypertension. Nifedipine (1232) and labetalol (1233) are considered a reasonable choice. There was also a slight reduction in the overall risk of developing proteinuria/PE when beta blockers and calcium channel blockers considered together (but not alone) were compared with methyldopa (1230). In mice, olmesartan (together with captopril) proved usefully anti-hypertensive (1234); this is of interest because olmesartan is also an agonist of the vitamin D receptor (1235). However, it was not mentioned in either Ref. (1230) or Ref. (114).
Lipopolysaccharide itself has long been recognized as a target of inflammatory diseases. Unfortunately, despite initially promising trials of an anti-LPS antibody known as centoxin (1236), it was eventually withdrawn, apparently because of a combination of ineffectiveness (1237, 1238) and toxicity (1239, 1240). LPS is rather hydrophobic, and thus it is hard to make even monoclonal antibodies very selective for such targets, such that the toxicity was probably because of its lack of specificity between lipid A and other hydrophobic ligands (1241). Other possible treatments based on LPS, such as “sushi peptides” (1242–1249) [or variants (1250, 1251)], and LPS-binding protein were covered elsewhere (140).
If an aberrant or dysbiotic gut microbiome is the source of the microbes that underpin PE, it is at least plausible that the gut microbiome should be predictive of PE (378), but we know of no suitably powered study that has been done to assess this, and this would clearly be worthwhile. However, in a study of primiparous women, the OR for getting severe PE was only 0.6 if probiotic milk drinks containing lactobacilli were consumed daily (1252). This is a very significant effects, such that this too seems an area well worth following up.
From a metabolomics point of view, the molecules seen to be raised in PE may either be biomarkers of the disease etiology or of the body’s attempts to respond to the disease [and this is true generally (1253)]. Thus, it is of great interest that S1P was raised in PE [see Kenny (15) and Table 7]. S1P is mainly vasoconstrictive (1254, 1255), but agonists of the S1P-1 receptor (that is involved in endothelial cell function) seemed to have considerable value in combatting the cytokine storm that followed infection-driven sepsis (1256–1261). The detailed mechanism seems not to be known, but in the context of infection, a need for S1P and other sphingolipids for successful pregnancies (1262, 1263) [see also Parkinson’s (1001)], and the induction of PE by its disruption (1000, 1264–1268), some serious investigation of the potential protective effects of S1PR1 agonists seems highly warranted.
Among other small molecules, melatonin has shown some promise in the treatment of septic shock, by lowering inflammatory cytokine production (1269) [and see Gitto et al. (1270) for neonatal oxidative stress], and a trial is in prospect for PE (1271).
Lipoxin A4 (LXA4) is considered to be an endogenous stop signal in inflammation. While recognizing the difficulties with rodent PE models (above), we note that in one study, the effect of BML-111 (a synthetic analog of LXA4) was tested on experimental PE induced in rats by low-dose endotoxin (LPS), and showed highly beneficial effects (549).
Coda – A Return to the Bradford Hill Criteria
Returning to the Bradford Hill criteria for ascribing causation of a disease to an environmental factor (97), we can now ask whether a detectable (if largely dormant) microbiome X, that is more likely to replicate with free iron, and that can anyway secrete or shed a variety of inflammatory components such as LPS, represents a plausible and major etiological factor for PE (Y):
(1) what is the strength of association between X and Y? We found an overwhelming co-occurrence of microbes or their products and PE.
(2) what is the consistency of association between X and Y? Almost wherever we looked, whether via periodontal disease (PD), UTI, or other means of ingress, we could find a microbial component in PE.
(3) what is the specificity of association between X and Y? Insufficient data are available to ascribe PE solely to one type of organism; however, these data clearly indicate that a variety of microbes, each capable of shedding inflammatory molecules such as LPS, can serve to stimulate or exacerbate PE.
(4) experiments verify the relationship between X and Y. It is unethical to do these in humans in terms of purposely infecting pregnant women, but data from antibiotics show the expected improvements.
(5) modification of X alters the occurrence of Y; this is really as (4).
(6) biologically plausible cause and effect relationship. Yes, this is where we think the ideas set down here are entirely consistent with current thinking on the main causes of PE. What we add in particular is the recognition that bacteria (and other microbes) that may be invisible to culture are both present and responsible, by established means, for the inflammation and other sequelae (and especially the coagulopathies) seen as causative accompaniments to PE.
Classical clinical microbiology, involving mainly replication-based methods, is evolving rapidly to assess the microbial content of samples on the basis of DNA sequences (296, 1272), including 16S rDNA (287, 288, 290, 292, 293, 295, 297, 1273), suitable protein-encoding housekeeping genes [e.g., Ref. (1274–1279)], and, increasingly, full genome sequences (1280). In the future, we can thus expect a considerable increase in molecular assessments of the microbiological content of blood, urine, and tissues, and this will obviously be a vital part of the experimental assessment and development of the ideas presented here. Molecular methods will also be used to assess maternal circulating DNA (1281–1283) and RNA (1284) in terms of both its presence and sequencing, as well as the use of digital PCR (1285).
Since PE has such a strong vascular component, we also predict that measurements designed to detect coagulopathies will increase in importance, for both diagnosis and prognosis, and for assessing treatments.
Finally, we consider that real progress in understanding PE from a systems biology perspective means that it must be modeled accordingly, and this must be a major goal.
We have brought together a large and widely dispersed literature to make the case that an important etiological role in PE is played by dormant microbes, or at least ones that are somewhat refractory to culture, and that these can awaken, shed inflammagens such as LPS, and thereby initiate inflammatory cascades. (The sequelae of these, involving cytokines, coagulopathies, and so on, are well enough accepted.) The case is founded on a large substructure of interlocking evidence, but readers might find the following elements as discussed above, especially persuasive and/or worthy of follow-up:
• the fact that endotoxin (LPS) can act as such a mimic for invoking PE in experimental models.
• the fact that every known proteomic biomarker suggested for PE has also been shown to increase during infection.
• the almost complete absence (one case) of PE in patients treated with spiramycin (540).
This is the paper number 8 of the series “The dormant blood microbiome in chronic, inflammatory diseases”. The other articles of the series can be found in the reference list with the numbers 141; 139; 140; 257; 147; 143; 1084; 258; 142.
Both authors made substantial, direct, and intellectual contributions to the work and approved it for publication.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
DK thanks the Biotechnology and Biological Sciences Research Council (grant BB/L025752/1) for financial support. LK is a Science Foundation Ireland Principal Investigator (grant number 08/IN.1/B2083). LK is also The Director of the Science Foundation Ireland-funded INFANT Research Centre (grant no. 12/RC/2272).
3. North RA, Mccowan LM, Dekker GA, Poston L, Chan EH, Stewart AW, et al. Clinical risk prediction for pre-eclampsia in nulliparous women: development of model in international prospective cohort. BMJ (2011) 342:d1875. doi:10.1136/bmj.d1875
8. Kenny LC, Black MA, Poston L, Taylor R, Myers JE, Baker PN, et al. Early pregnancy prediction of preeclampsia in nulliparous women, combining clinical risk and biomarkers: the screening for pregnancy endpoints (SCOPE) international cohort study. Hypertension (2014) 64:644–52. doi:10.1161/HYPERTENSIONAHA.114.03578
10. Abalos E, Cuesta C, Grosso AL, Chou D, Say L. Global and regional estimates of preeclampsia and eclampsia: a systematic review. Eur J Obstet Gynecol Reprod Biol (2013) 170:1–7. doi:10.1016/j.ejogrb.2013.05.005
14. Abalos E, Cuesta C, Carroli G, Qureshi Z, Widmer M, Vogel JP, et al. Pre-eclampsia, eclampsia and adverse maternal and perinatal outcomes: a secondary analysis of the World Health Organization Multicountry Survey on Maternal and Newborn Health. BJOG (2014) 121(Suppl 1):14–24. doi:10.1111/1471-0528.12629
15. Kenny LC, Broadhurst DI, Dunn W, Brown M, Francis-Mcintyre S, North RA, et al. Robust early pregnancy prediction of later preeclampsia using metabolomic biomarkers. Hypertension (2010) 56:741–9. doi:10.1161/HYPERTENSIONAHA.110.157297
16. Bartsch E, Medcalf KE, Park AL, Ray JG; High Risk of Pre-Eclampsia Identification Group. Clinical risk factors for pre-eclampsia determined in early pregnancy: systematic review and meta-analysis of large cohort studies. BMJ (2016) 353:i1753. doi:10.1136/bmj.i1753
21. Bdolah Y, Elchalal U, Natanson-Yaron S, Yechiam H, Bdolah-Abram T, Greenfield C, et al. Relationship between nulliparity and preeclampsia may be explained by altered circulating soluble fms-like tyrosine kinase 1. Hypertens Pregnancy (2014) 33:250–9. doi:10.3109/10641955.2013.858745
22. Saftlas AF, Olson DR, Franks AL, Atrash HK, Pokras R. Epidemiology of preeclampsia and eclampsia in the United-States, 1979-1986. Am J Obstet Gynecol (1990) 163:460–5. doi:10.1016/0002-9378(90)91176-D
24. Lamminpää R, Vehvilainen-Julkunen K, Gissler M, Heinonen S. Preeclampsia complicated by advanced maternal age: a registry-based study on primiparous women in Finland 1997-2008. BMC Pregnancy Childbirth (2012) 12:47. doi:10.1186/1471-2393-12-47
27. Coonrod DV, Hickok DE, Zhu KM, Easterling TR, Daling JR. Risk-factors for preeclampsia in twin pregnancies – a population-based cohort study. Obstet Gynecol (1995) 85:645–50. doi:10.1016/0029-7844(95)00049-W
29. Bdolah Y, Lam C, Rajakumar A, Shivalingappa V, Mutter W, Sachs BP, et al. Twin pregnancy and the risk of preeclampsia: bigger placenta or relative ischemia? Am J Obstet Gynecol (2008) 198:428.e1–6. doi:10.1016/j.ajog.2007.10.783
30. Bodnar LM, Ness RB, Harger GF, Roberts JM. Inflammation and triglycerides partially mediate the effect of prepregnancy body mass index on the risk of preeclampsia. Am J Epidemiol (2005) 162:1198–206. doi:10.1093/aje/kwi334
35. Ros HS, Lichtenstein P, Lipworth L, Cnattingius S. Genetic effects on the liability of developing pre-eclampsia and gestational hypertension. Am J Med Genet (2000) 91:256–60. doi:10.1002/(SICI)1096-8628(20000410)91:4<256::AID-AJMG3>3.0.CO;2-T
36. Williams PJ, Broughton Pipkin F. The genetics of pre-eclampsia and other hypertensive disorders of pregnancy. Best Pract Res Clin Obstet Gynaecol (2011) 25:405–17. doi:10.1016/j.bpobgyn.2011.02.007
38. Boyd HA, Tahir H, Wohlfahrt J, Melbye M. Associations of personal and family preeclampsia history with the risk of early-, intermediate- and late-onset preeclampsia. Am J Epidemiol (2013) 178:1611–9. doi:10.1093/aje/kwt189
39. Roten LT, Thomsen LCV, Gundersen AS, Fenstad MH, Odland ML, Strand KM, et al. The Norwegian preeclampsia family cohort study: a new resource for investigating genetic aspects and heritability of preeclampsia and related phenotypes. BMC Pregnancy Childbirth (2015) 15:319. doi:10.1186/s12884-015-0754-2
43. Harel M, Aron-Maor A, Sherer Y, Blank M, Shoenfeld Y. The infectious etiology of the antiphospholipid syndrome: links between infection and autoimmunity. Immunobiology (2005) 210:743–7. doi:10.1016/j.imbio.2005.10.004
51. O’Gorman N, Wright D, Syngelaki A, Akolekar R, Wright A, Poon LC, et al. Competing risks model in screening for preeclampsia by maternal factors and biomarkers at 11-13 weeks gestation. Am J Obstet Gynecol (2016) 214:103.e1–12. doi:10.1016/j.ajog.2015.08.034
52. Schieve LA, Handler A, Hershow R, Davis F. Urinary-tract infection during pregnancy – its association with maternal morbidity and perinatal outcome. Am J Public Health (1994) 84:405–10. doi:10.2105/Ajph.84.3.405
54. Rustveld LO, Kelsey SF, Sharma R. Association between maternal infections and preeclampsia: a systematic review of epidemiologic studies. Matern Child Health J (2008) 12:223–42. doi:10.1007/s10995-007-0224-1
56. Bellamy L, Casas JP, Hingorani AD, Williams DJ. Pre-eclampsia and risk of cardiovascular disease and cancer in later life: systematic review and meta-analysis. BMJ (2007) 335:974. doi:10.1136/bmj.39335.385301.BE
57. Craici IM, Wagner SJ, Hayman SR, Garovic VD. Pre-eclamptic pregnancies: an opportunity to identify women at risk for future cardiovascular disease. Womens Health (Lond Engl) (2008) 4:133–5. doi:10.2217/17455057.4.2.133
59. Powe CE, Levine RJ, Karumanchi SA. Preeclampsia, a disease of the maternal endothelium: the role of antiangiogenic factors and implications for later cardiovascular disease. Circulation (2011) 123:2856–69. doi:10.1161/CIRCULATIONAHA.109.853127
60. Skjaerven R, Wilcox AJ, Klungsøyr K, Irgens LM, Vikse BE, Vatten LJ, et al. Cardiovascular mortality after pre-eclampsia in one child mothers: prospective, population based cohort study. BMJ (2012) 345:e7677. doi:10.1136/bmj.e7677
64. Tannetta DS, Hunt K, Jones CI, Davidson N, Coxon CH, Ferguson D, et al. Syncytiotrophoblast extracellular vesicles from pre-eclampsia placentas differentially affect platelet function. PLoS One (2015) 10:e0142538. doi:10.1371/journal.pone.0142538
66. Bateman BT, Schumacher HC, Bushnell CD, Pile-Spellman J, Simpson LL, Sacco RL, et al. Intracerebral hemorrhage in pregnancy: frequency, risk factors, and outcome. Neurology (2006) 67:424–9. doi:10.1212/01.wnl.0000228277.84760.a2
67. Kajantie E, Eriksson JG, Osmond C, Thornburg K, Barker DJP. Pre-eclampsia is associated with increased risk of stroke in the adult offspring: the Helsinki birth cohort study. Stroke (2009) 40:1176–80. doi:10.1161/STROKEAHA.108.538025
70. Mignini LE, Villar J, Khan KS. Mapping the theories of preeclampsia: the need for systematic reviews of mechanisms of the disease. Am J Obstet Gynecol (2006) 194:317–21. doi:10.1016/j.ajog.2005.08.065
73. Genc H, Uzun H, Benian A, Simsek G, Gelisgen R, Madazli R, et al. Evaluation of oxidative stress markers in first trimester for assessment of preeclampsia risk. Arch Gynecol Obstet (2011) 284:1367–73. doi:10.1007/s00404-011-1865-2
77. Redman CW, Sargent IL, Staff AC. IFPA Senior Award Lecture: making sense of pre-eclampsia – two placental causes of preeclampsia? Placenta (2014) 35(Suppl):S20–5. doi:10.1016/j.placenta.2013.12.008
78. Xiong X, Demianczuk NN, Saunders LD, Wang FL, Fraser WD. Impact of preeclampsia and gestational hypertension on birth weight by gestational age. Am J Epidemiol (2002) 155:203–9. doi:10.1093/aje/155.3.203
81. Sacks GP, Studena K, Sargent K, Redman CWG. Normal pregnancy and preeclampsia both produce inflammatory changes in peripheral blood leukocytes akin to those of sepsis. Am J Obstet Gynecol (1998) 179:80–6. doi:10.1016/S0002-9378(98)70254-6
84. Kell DB. Towards a unifying, systems biology understanding of large-scale cellular death and destruction caused by poorly liganded iron: Parkinson’s, Huntington’s, Alzheimer’s, prions, bactericides, chemical toxicology and others as examples. Arch Toxicol (2010) 577:825–89. doi:10.1007/s00204-010-0577-x
88. Levine RJ, Lam C, Qian C, Yu KF, Maynard SE, Sachs BP, et al. Soluble endoglin and other circulating antiangiogenic factors in preeclampsia. N Engl J Med (2006) 355:992–1005. doi:10.1056/NEJMoa055352
89. Palomaki GE, Haddow JE, Haddow HR, Salahuddin S, Geahchan C, Cerdeira AS, et al. Modeling risk for severe adverse outcomes using angiogenic factor measurements in women with suspected preterm preeclampsia. Prenat Diagn (2015) 35:386–93. doi:10.1002/pd.4554
93. Meziani F, Tesse A, David E, Martinez MC, Wangesteen R, Schneider F, et al. Shed membrane particles from preeclamptic women generate vascular wall inflammation and blunt vascular contractility. Am J Pathol (2006) 169:1473–83. doi:10.2353/ajpath.2006.051304
94. Sugawara J, Mitsui-Saito M, Hayashi C, Hoshiai T, Senoo M, Chisaka H, et al. Decrease and senescence of endothelial progenitor cells in patients with preeclampsia. J Clin Endocrinol Metab (2005) 90:5329–32. doi:10.1210/jc.2005-0532
99. Kell DB. Metabolomics, modelling and machine learning in systems biology: towards an understanding of the languages of cells. The 2005 Theodor Bücher lecture. FEBS J (2006) 273:873–94. doi:10.1111/j.1742-4658.2006.05136.x
114. Magee LA, Pels A, Helewa M, Rey E, Von Dadelszen P; Canadian Hypertensive Disorders of Pregnancy Working Group. Diagnosis, evaluation, and management of the hypertensive disorders of pregnancy. Pregnancy Hypertens (2014) 4:105–45. doi:10.1016/j.preghy.2014.01.003
115. Goddard KAG, Tromp G, Romero R, Olson JM, Lu Q, Xu Z, et al. Candidate-gene association study of mothers with pre-eclampsia, and their infants, analyzing 775 SNPs in 190 genes. Hum Hered (2007) 63:1–16. doi:10.1159/000097926
116. Jebbink J, Wolters A, Fernando F, Afink G, van der Post J, Ris-Stalpers C. Molecular genetics of preeclampsia and HELLP syndrome – a review. Biochim Biophys Acta (2012) 1822:1960–9. doi:10.1016/j.bbadis.2012.08.004
121. Zuk O, Hechter E, Sunyaev SR, Lander ES. The mystery of missing heritability: genetic interactions create phantom heritability. Proc Natl Acad Sci U S A (2012) 109:1193–8. doi:10.1073/pnas.1119675109
122. Carreiras M, Montagnani S, Layrisse Z. Preeclampsia: a multifactorial disease resulting from the interaction of the feto-maternal HLA genotype and HCMV infection. Am J Reprod Immunol (2002) 48:176–83. doi:10.1034/j.1600-0897.2002.01076.x
125. Kell DB. Iron behaving badly: inappropriate iron chelation as a major contributor to the aetiology of vascular and other progressive inflammatory and degenerative diseases. BMC Med Genomics (2009) 2:2. doi:10.1186/1755-8794-2-2
128. Kell DB, Pretorius E. The simultaneous occurrence of both hypercoagulability and hypofibrinolysis in blood and serum during systemic inflammation, and the roles of iron and fibrin(ogen). Integr Biol (2015) 7:24–52. doi:10.1039/c4ib00173g
129. Pretorius E, Oberholzer HM, van der Spuy WJ, Swanepoel AC, Soma P. Qualitative scanning electron microscopy analysis of fibrin networks and platelet abnormalities in diabetes. Blood Coagul Fibrinol (2011) 22:463–7. doi:10.1097/MBC.0b013e3283468a0d
130. Pretorius E, Steyn H, Engelbrecht M, Swanepoel AC, Oberholzer HM. Differences in fibrin fiber diameters in healthy individuals and thromboembolic ischemic stroke patients. Blood Coagul Fibrinolysis (2011) 22:696–700. doi:10.1097/MBC.0b013e32834bdb32
131. Pretorius E, Swanepoel AC, Oberholzer HM, van der Spuy WJ, Duim W, Wessels PF. A descriptive investigation of the ultrastructure of fibrin networks in thrombo-embolic ischemic stroke. J Thromb Thrombolysis (2011) 31:507–13. doi:10.1007/s11239-010-0538-5
132. Pretorius E, Du Plooy J, Soma P, Gasparyan AY. An ultrastructural analysis of platelets, erythrocytes, white blood cells, and fibrin network in systemic lupus erythematosus. Rheumatol Int (2014) 34:1005–9. doi:10.1007/s00296-013-2817-x
134. Pretorius E, Bester J, Vermeulen N, Alummoottil S, Soma P, Buys AV, et al. Poorly controlled type 2 diabetes is accompanied by significant morphological and ultrastructural changes in both erythrocytes and in thrombin-generated fibrin: implications for diagnostics. Cardiovasc Diabetol (2015) 13:30. doi:10.1186/s12933-015-0192-5
135. Bester J, Buys AV, Lipinski B, Kell DB, Pretorius E. High ferritin levels have major effects on the morphology of erythrocytes in Alzheimer’s disease. Front Aging Neurosci (2013) 5:00088. doi:10.3389/fnagi.2013.00088
136. Pretorius E, Bester J, Vermeulen N, Lipinski B, Gericke GS, Kell DB. Profound morphological changes in the erythrocytes and fibrin networks of patients with hemochromatosis or with hyperferritinemia, and their normalization by iron chelators and other agents. PLoS One (2014) 9:e85271. doi:10.1371/journal.pone.0085271
137. Pretorius E, Olumuyiwa-Akeredolu OO, Mbotwe S, Bester J. Erythrocytes and their role as health indicator: using structure in a patient-orientated precision medicine approach. Blood Rev (2016) 30:263–74. doi:10.1016/j.blre.2016.01.001
139. Kell DB, Potgieter M, Pretorius E. Individuality, phenotypic differentiation, dormancy and ‘persistence’ in culturable bacterial systems: commonalities shared by environmental, laboratory, and clinical microbiology. F1000Research (2015) 4:179. doi:10.12688/f1000research.6709.1
140. Kell DB, Pretorius E. On the translocation of bacteria and their lipopolysaccharides between blood and peripheral locations in chronic, inflammatory diseases: the central roles of LPS and LPS-induced cell death. Integr Biol (2015) 7:1339–77. doi:10.1039/C5IB00158G
142. Pretorius E, Akeredolu O-O, Soma P, Kell DB. Major involvement of bacterial components in rheumatoid arthritis and its accompanying oxidative stress, systemic inflammation and hypercoagulability. Exp Biol Med (Forthcoming 2016).
143. Pretorius E, Bester J, Kell DB. A bacterial component to Alzheimer-type dementia seen via a systems biology approach that links iron dysregulation and inflammagen shedding to disease. J Alzheimers Dis (2016) 53:1237–56. doi:10.3233/JAD-160318
152. Staley JT, Konopka A. Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats. Annu Rev Microbiol (1985) 39:321–46. doi:10.1146/annurev.mi.39.100185.001541
153. Tanaka T, Kawasaki K, Daimon S, Kitagawa W, Yamamoto K, Tamaki H, et al. A hidden pitfall in the preparation of agar media undermines microorganism cultivability. Appl Environ Microbiol (2014) 80:7659–66. doi:10.1128/AEM.02741-14
154. Nichols D, Cahoon N, Trakhtenberg EM, Pham L, Mehta A, Belanger A, et al. Use of ichip for high-throughput in situ cultivation of “uncultivable” microbial species. Appl Environ Microbiol (2010) 76:2445–50. doi:10.1128/AEM.01754-09
155. Browne HP, Forster SC, Anonye BO, Kumar N, Neville BA, Stares MD, et al. Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation. Nature (2016) 533:543–6. doi:10.1038/nature17645
157. Kell DB, Kaprelyants AS, Weichart DH, Harwood CL, Barer MR. Viability and activity in readily culturable bacteria: a review and discussion of the practical issues. Antonie Van Leeuwenhoek (1998) 73:169–87. doi:10.1023/A:1000664013047
164. Omsland A, Cockrell DC, Howe D, Fischer ER, Virtaneva K, Sturdevant DE, et al. Host cell-free growth of the Q fever bacterium Coxiella burnetii. Proc Natl Acad Sci U S A (2009) 106:4430–4. doi:10.1073/pnas.0812074106
166. Marshall BJ. One hundred years of discovery and rediscovery of Helicobacter pylori and its association with peptic ulcer disease. In: Mobley HLT, Mendz GL, Hazell SL, editors. Helicobacter pylori: Physiology and Genetics. Washington, DC: ASM Press (2001). p. 19–24.
171. Kaprelyants AS, Mukamolova GV, Kell DB. Estimation of dormant Micrococcus luteus cells by penicillin lysis and by resuscitation in cell-free spent medium at high dilution. FEMS Microbiol Lett (1994) 115:347–52. doi:10.1111/j.1574-6968.1994.tb06662.x
173. Kana BD, Gordhan BG, Downing KJ, Sung N, Vostroktunova G, Machowski EE, et al. The resuscitation-promoting factors of Mycobacterium tuberculosis are required for virulence and resuscitation from dormancy but are collectively dispensable for growth in vitro. Mol Microbiol (2008) 67:672–84. doi:10.1111/j.1365-2958.2007.06078.x
176. Shleeva M, Kondratieva T, Rubakova E, Vostroknutova G, Kaprelyants A, Apt A. Reactivation of dormant “non-culturable” Mycobacterium tuberculosis developed in vitro after injection in mice: both the dormancy depth and host genetics influence the outcome. Microb Pathog (2015) 78:63–6. doi:10.1016/j.micpath.2014.11.016
178. Mukamolova GV, Turapov OA, Kazarian K, Telkov M, Kaprelyants AS, Kell DB, et al. The rpf gene of Micrococcus luteus encodes an essential secreted growth factor. Mol Microbiol (2002) 46:611–21. doi:10.1046/j.1365-2958.2002.03183.x
179. Mukamolova GV, Turapov OA, Young DI, Kaprelyants AS, Kell DB, Young M. A family of autocrine growth factors in Mycobacterium tuberculosis. Mol Microbiol (2002) 46:623–35. doi:10.1046/j.1365-2958.2002.03184.x
180. Mukamolova GV, Murzin AG, Salina EG, Demina GR, Kell DB, Kaprelyants AS, et al. Muralytic activity of Micrococcus luteus Rpf and its relationship to physiological activity in promoting bacterial growth and resuscitation. Mol Microbiol (2006) 59:84–98. doi:10.1111/j.1365-2958.2005.04930.x
181. Mukamolova GV, Turapov O, Malkin J, Woltmann G, Barer MR. Resuscitation-promoting factors reveal an occult population of tubercle bacilli in sputum. Am J Respir Crit Care Med (2010) 181:174–80. doi:10.1164/rccm.200905-0661OC
182. Yeremeev VV, Kondratieva TK, Rubakova EI, Petrovskaya SN, Kazarian KA, Telkov MV, et al. Proteins of the Rpf family: immune cell reactivity and vaccination efficacy against tuberculosis in mice. Infect Immun (2003) 71:4789–94. doi:10.1128/IAI.71.8.4789-4794.2003
183. Zvi A, Ariel N, Fulkerson J, Sadoff JC, Shafferman A. Whole genome identification of Mycobacterium tuberculosis vaccine candidates by comprehensive data mining and bioinformatic analyses. BMC Med Genomics (2008) 1:18. doi:10.1186/1755-8794-1-18
184. Dye C, Scheele S, Dolin P, Pathania V, Raviglione RC. Global burden of tuberculosis – estimated incidence, prevalence, and mortality by country. JAMA (1999) 282:677–86. doi:10.1001/jama.282.7.677
194. Conlon BP. Staphylococcus aureus chronic and relapsing infections: evidence of a role for persister cells: an investigation of persister cells, their formation and their role in S. aureus disease. Bioessays (2014) 36:991–6. doi:10.1002/bies.201400080
195. Kester JC, Fortune SM. Persisters and beyond: mechanisms of phenotypic drug resistance and drug tolerance in bacteria. Crit Rev Biochem Mol Biol (2014) 49:91–101. doi:10.3109/10409238.2013.869543
196. Levin BR, Concepción-Acevedo J, Udekwu KI. Persistence: a copacetic and parsimonious hypothesis for the existence of non-inherited resistance to antibiotics. Curr Opin Microbiol (2014) 21:18–21. doi:10.1016/j.mib.2014.06.016
198. Rank RG, Yeruva L. Hidden in plain sight: chlamydial gastrointestinal infection and its relevance to persistence in human genital infection. Infect Immun (2014) 82:1362–71. doi:10.1128/IAI.01244-13
201. Stepanyan K, Wenseleers T, Duéñez-Guzmán EA, Muratori F, Van Den Bergh B, Verstraeten N, et al. Fitness trade-offs explain low levels of persister cells in the opportunistic pathogen Pseudomonas aeruginosa. Mol Ecol (2015) 24:1572–83. doi:10.1111/mec.13127
205. Prideaux B, Via LE, Zimmerman MD, Eum S, Sarathy J, O’Brien P, et al. The association between sterilizing activity and drug distribution into tuberculosis lesions. Nat Med (2015) 21:1223–7. doi:10.1038/nm.3937
207. Kell DB, Dobson PD, Oliver SG. Pharmaceutical drug transport: the issues and the implications that it is essentially carrier-mediated only. Drug Discov Today (2011) 16:704–14. doi:10.1016/j.drudis.2011.05.010
208. Kell DB, Dobson PD, Bilsland E, Oliver SG. The promiscuous binding of pharmaceutical drugs and their transporter-mediated uptake into cells: what we (need to) know and how we can do so. Drug Discov Today (2013) 18:218–39. doi:10.1016/j.drudis.2012.11.008
209. Kell DB, Oliver SG. How drugs get into cells: tested and testable predictions to help discriminate between transporter-mediated uptake and lipoidal bilayer diffusion. Front Pharmacol (2014) 5:231. doi:10.3389/fphar.2014.00231
212. Mysorekar IU, Hultgren SJ. Mechanisms of uropathogenic Escherichia coli persistence and eradication from the urinary tract. Proc Natl Acad Sci U S A (2006) 103:14170–5. doi:10.1073/pnas.0602136103
214. Chen SL, Wu M, Henderson JP, Hooton TM, Hibbing ME, Hultgren SJ, et al. Genomic diversity and fitness of E. coli strains recovered from the intestinal and urinary tracts of women with recurrent urinary tract infection. Sci Transl Med (2013) 5:184ra160. doi:10.1126/scitranslmed.3005497
215. Goneau LW, Hannan TJ, Macphee RA, Schwartz DJ, Macklaim JM, Gloor GB, et al. Subinhibitory antibiotic therapy alters recurrent urinary tract infection pathogenesis through modulation of bacterial virulence and host immunity. MBio (2015) 6:e00356–15. doi:10.1128/mBio.00356-15
218. Aguirre JD, Clark HM, Mcilvin M, Vazquez C, Palmere SL, Grab DJ, et al. A manganese-rich environment supports superoxide dismutase activity in a Lyme disease pathogen, Borrelia burgdorferi. J Biol Chem (2013) 288:8468–78. doi:10.1074/jbc.M112.433540
224. Reid DW, Anderson GJ, Lamont IL. Role of lung iron in determining the bacterial and host struggle in cystic fibrosis. Am J Physiol Lung Cell Mol Physiol (2009) 297:L795–802. doi:10.1152/ajplung.00132.2009
226. Chu BC, Garcia-Herrero A, Johanson TH, Krewulak KD, Lau CK, Peacock RS, et al. Siderophore uptake in bacteria and the battle for iron with the host; a bird’s eye view. Biometals (2010) 23:601–11. doi:10.1007/s10534-010-9361-x
231. Deriu E, Liu JZ, Pezeshki M, Edwards RA, Ochoa RJ, Contreras H, et al. Probiotic bacteria reduce Salmonella typhimurium intestinal colonization by competing for iron. Cell Host Microbe (2013) 14:26–37. doi:10.1016/j.chom.2013.06.007
232. Leal SM Jr, Roy S, Vareechon C, Carrion S, Clark H, Lopez-Berges MS, et al. Targeting iron acquisition blocks infection with the fungal pathogens Aspergillus fumigatus and Fusarium oxysporum. PLoS Pathog (2013) 9:e1003436. doi:10.1371/journal.ppat.1003436
236. Diaz-Ochoa VE, Jellbauer S, Klaus S, Raffatellu M. Transition metal ions at the crossroads of mucosal immunity and microbial pathogenesis. Front Cell Infect Microbiol (2014) 4:2. doi:10.3389/fcimb.2014.00002
237. Potrykus J, Ballou ER, Childers DS, Brown AJP. Conflicting interests in the pathogen-host tug of war: fungal micronutrient scavenging versus mammalian nutritional immunity. PLoS Pathog (2014) 10:e1003910. doi:10.1371/journal.ppat.1003910
239. Nairz M, Ferring-Appel D, Casarrubea D, Sonnweber T, Viatte L, Schroll A, et al. Iron regulatory proteins mediate host resistance to Salmonella infection. Cell Host Microbe (2015) 18:254–61. doi:10.1016/j.chom.2015.06.017
247. Hubel CA, Kozlov AV, Kagan VE, Evans RW, Davidge ST, McLaughlin MK, et al. Decreased transferrin and increased transferrin saturation in sera of women with preeclampsia: implications for oxidative stress. Am J Obstet Gynecol (1996) 175:692–700. doi:10.1053/ob.1996.v175.a74252
248. Lao TT, Tam KF, Chan LY. Third trimester iron status and pregnancy outcome in non-anaemic women; pregnancy unfavourably affected by maternal iron excess. Hum Reprod (2000) 15:1843–8. doi:10.1093/humrep/15.8.1843
252. Bhatla N, Kaul N, Lal N, Kriplani A, Agarwal N, Saxena R, et al. Comparison of effect of daily versus weekly iron supplementation during pregnancy on lipid peroxidation. J Obstet Gynaecol Res (2009) 35:438–45. doi:10.1111/j.1447-0756.2008.00972.x
256. Negi R, Pande D, Karki K, Kumar A, Khanna RS, Khanna HD. Association of oxidative DNA damage, protein oxidation and antioxidant function with oxidative stress induced cellular injury in pre-eclamptic/eclamptic mothers during fetal circulation. Chem Biol Interact (2014) 208:77–83. doi:10.1016/j.cbi.2013.11.010
257. Bester J, Soma P, Kell DB, Pretorius E. Viscoelastic and ultrastructural characteristics of whole blood and plasma in Alzheimer-type dementia, and the possible role of bacterial lipopolysaccharides (LPS). Oncotarget (2015) 6:35284–303. doi:10.18632/oncotarget.6074
258. Pretorius E, Mbotwe S, Bester J, Robinson C, Kell DB. Acute induction of anomalous blood clotting by highly substoichiometric levels of bacterial lipopolysaccharide (LPS). bioRxiv (2016) 053538. doi:10.1101/053538
259. Pretorius E, Mbotwe S, Bester J, Robinson CJ, Kell DB. Acute induction of anomalous and amyloidogenic blood clotting by molecular amplification of highly substoichiometric levels of bacterial lipopolysaccharide. J R Soc Interface (2016) 123:20160539. doi:10.1098/rsif.2016.0539
260. Lin IH, Miller DS, Bertics PJ, Murphy CJ, De Pablo JJ, Abbott NL. Endotoxin-induced structural transformations in liquid crystalline droplets. Science (2011) 332:1297–300. doi:10.1126/science.1195639
261. Miller DS, Abbott NL. Influence of droplet size, pH and ionic strength on endotoxin-triggered ordering transitions in liquid crystalline droplets. Soft Matter (2013) 9:374–82. doi:10.1039/C2SM26811F
262. Rahman MM, Abe SK, Rahman MS, Kanda M, Narita S, Bilano V, et al. Maternal anemia and risk of adverse birth and health outcomes in low- and middle-income countries: systematic review and meta-analysis. Am J Clin Nutr (2016) 103:495–504. doi:10.3945/ajcn.115.107896
273. Nebe-Von-Caron G, Stephens PJ, Hewitt CJ, Powell JR, Badley RA. Analysis of bacterial function by multi-colour fluorescence flow cytometry and single cell sorting. J Microbiol Methods (2000) 42:97–114. doi:10.1016/S0167-7012(00)00181-0
275. Müller S, Nebe-Von-Caron G. Functional single-cell analyses: flow cytometry and cell sorting of microbial populations and communities. FEMS Microbiol Rev (2010) 34:554–87. doi:10.1111/j.1574-6976.2010.00214.x
281. Harris CM, Todd RW, Bungard SJ, Lovitt RW, Morris JG, Kell DB. The dielectric permittivity of microbial suspensions at radio frequencies: a novel method for the estimation of microbial biomass. Enzyme Microbiol Technol (1987) 9:181–6. doi:10.1016/0141-0229(87)90075-5
286. Woo PCY, Lau SKP, Teng JLL, Tse H, Yuen KY. Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories. Clin Microbiol Infect (2008) 14:908–34. doi:10.1111/j.1469-0691.2008.02070.x
287. Cherkaoui A, Emonet S, Ceroni D, Candolfi B, Hibbs J, Francois P, et al. Development and validation of a modified broad-range 16S rDNA PCR for diagnostic purposes in clinical microbiology. J Microbiol Methods (2009) 79:227–31. doi:10.1016/j.mimet.2009.09.014
288. Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Lozupone CA, Turnbaugh PJ, et al. Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc Natl Acad Sci U S A (2011) 108(Suppl 1):4516–22. doi:10.1073/pnas.1000080107
289. Kramski M, Gaeguta AJ, Lichtfuss GF, Rajasuriar R, Crowe SM, French MA, et al. Novel sensitive real-time PCR for quantification of bacterial 16S rRNA genes in plasma of HIV-infected patients as a marker for microbial translocation. J Clin Microbiol (2011) 49:3691–3. doi:10.1128/JCM.01018-11
292. Langille MGI, Zaneveld J, Caporaso JG, McDonald D, Knights D, Reyes JA, et al. Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences. Nat Biotechnol (2013) 31:814–21. doi:10.1038/nbt.2676
293. Mizrahi-Man O, Davenport ER, Gilad Y. Taxonomic classification of bacterial 16S rRNA genes using short sequencing reads: evaluation of effective study designs. PLoS One (2013) 8:e53608. doi:10.1371/journal.pone.0053608
295. Yarza P, Yilmaz P, Pruesse E, Glöckner FO, Ludwig W, Schleifer KH, et al. Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences. Nat Rev Microbiol (2014) 12:635–45. doi:10.1038/nrmicro3330
296. Zumla A, Al-Tawfiq JA, Enne VI, Kidd M, Drosten C, Breuer J, et al. Rapid point of care diagnostic tests for viral and bacterial respiratory tract infections – needs, advances, and future prospects. Lancet Infect Dis (2014) 14:1123–35. doi:10.1016/S1473-3099(14)70827-8
297. D’Amore R, Ijaz UZ, Schirmer M, Kenny JG, Gregory R, Darby AC, et al. A comprehensive benchmarking study of protocols and sequencing platforms for 16S rRNA community profiling. BMC Genomics (2016) 17:55. doi:10.1186/s12864-015-2194-9
300. McLaughlin RW, Vali H, Lau PC, Palfree RGE, De Ciccio A, Sirois M, et al. Are there naturally occurring pleomorphic bacteria in the blood of healthy humans? J Clin Microbiol (2002) 40:4771–5. doi:10.1128/JCM.40.12.4771-4775.2002
301. Moriyama K, Ando C, Tashiro K, Kuhara S, Okamura S, Nakano S, et al. Polymerase chain reaction detection of bacterial 16S rRNA gene in human blood. Microbiol Immunol (2008) 52:375–82. doi:10.1111/j.1348-0421.2008.00048.x
302. Amar J, Serino M, Lange C, Chabo C, Iacovoni J, Mondot S, et al. Involvement of tissue bacteria in the onset of diabetes in humans: evidence for a concept. Diabetologia (2011) 54:3055–61. doi:10.1007/s00125-011-2329-8
303. Gaibani P, Mariconti M, Bua G, Bonora S, Sassera D, Landini MP, et al. Development of a broad-range 23S rDNA real-time PCR assay for the detection and quantification of pathogenic bacteria in human whole blood and plasma specimens. Biomed Res Int (2013) 2013:264651. doi:10.1155/2013/264651
304. Dinakaran V, Rathinavel A, Pushpanathan M, Sivakumar R, Gunasekaran P, Rajendhran J. Elevated levels of circulating DNA in cardiovascular disease patients: metagenomic profiling of microbiome in the circulation. PLoS One (2014) 9:e105221. doi:10.1371/journal.pone.0105221
305. Sato J, Kanazawa A, Ikeda F, Yoshihara T, Goto H, Abe H, et al. Gut dysbiosis and detection of “live gut bacteria” in blood of Japanese patients with type 2 diabetes. Diabetes Care (2014) 37:2343–50. doi:10.2337/dc13-2817
306. Damgaard C, Magnussen K, Enevold C, Nilsson M, Tolker-Nielsen T, Holmstrup P, et al. Viable bacteria associated with red blood cells and plasma in freshly drawn blood donations. PLoS One (2015) 10:e0120826. doi:10.1371/journal.pone.0120826
307. Gyarmati P, Kjellander C, Aust C, Kalin M, Öhrmalm L, Giske CG. Bacterial landscape of bloodstream infections in neutropenic patients via high throughput sequencing. PLoS One (2015) 10:e0135756. doi:10.1371/journal.pone.0135756
308. Gyarmati P, Kjellander C, Aust C, Song Y, Öhrmalm L, Giske CG. Metagenomic analysis of bloodstream infections in patients with acute leukemia and therapy-induced neutropenia. Sci Rep (2016) 6:23532. doi:10.1038/srep23532
309. Païssé S, Valle C, Servant F, Courtney M, Burcelin R, Amar J, et al. Comprehensive description of blood microbiome from healthy donors assessed by 16S targeted metagenomic sequencing. Transfusion (2016) 56:1138–47. doi:10.1111/trf.13477
311. Belstrøm D, Holmstrup P, Damgaard C, Borch TS, Skjødt MO, Bendtzen K, et al. The atherogenic bacterium Porphyromonas gingivalis evades circulating phagocytes by adhering to erythrocytes. Infect Immun (2011) 79:1559–65. doi:10.1128/IAI.01036-10
313. Price WA. Dental Infections Oral and Systemic, Being a Contribution to the Pathology of Dental Infections, Focal Infections and the Degenerative Diseases, Parts I and II. Cleveland: Penton Press (1923).
314. Miklossy J, Kis A, Radenovic A, Miller L, Forro L, Martins R, et al. Beta-amyloid deposition and Alzheimer’s type changes induced by Borrelia spirochetes. Neurobiol Aging (2006) 27:228–36. doi:10.1016/j.neurobiolaging.2005.01.018
317. Nicolson GL, Haier J. Role of chronic bacterial and viral infections in neurodegenerative, neurobehavioural, psychiatric, autoimmune and fatiguing illnesses: part 1. Br J Med Pract (2009) 2:20–8.
319. Nicolson GL, Haier J. Role of chronic bacterial and viral infections in neurodegenerative, neurobehavioural, psychiatric, autoimmune and fatiguing illnesses: part 2. Br J Med Pract (2010) 3:301–10.
325. Bollmann A, Lewis K, Epstein SS. Incubation of environmental samples in a diffusion chamber increases the diversity of recovered isolates. Appl Environ Microbiol (2007) 73:6386–90. doi:10.1128/AEM.01309-07
326. D’Onofrio A, Crawford JM, Stewart EJ, Witt K, Gavrish E, Epstein S, et al. Siderophores from neighboring organisms promote the growth of uncultured bacteria. Chem Biol (2010) 17:254–64. doi:10.1016/j.chembiol.2010.02.010
332. Von Dadelszen P, Magee LA. Could an infectious trigger explain the differential maternal response to the shared placental pathology of preeclampsia and normotensive intrauterine growth restriction? Acta Obstet Gynecol Scand (2002) 81:642–8. doi:10.1034/j.1600-0412.2002.810710.x
337. Figura N, Franceschi F, Santucci A, Bernardini G, Gasbarrini G, Gasbarrini A. Extragastric manifestations of Helicobacter pylori infection. Helicobacter (2010) 15(Suppl 1):60–8. doi:10.1111/j.1523-5378.2010.00778.x
342. Testerman TL, Morris J. Beyond the stomach: an updated view of Helicobacter pylori pathogenesis, diagnosis, and treatment. World J Gastroenterol (2014) 20:12781–808. doi:10.3748/wjg.v20.i36.12781
343. Ponzetto A, Cardaropoli S, Piccoli E, Rolfo A, Gennero L, Kanduc D, et al. Pre-eclampsia is associated with Helicobacter pylori seropositivity in Italy. J Hypertens (2006) 24:2445–9. doi:10.1097/HJH.0b013e3280109e8c
344. Franceschi F, Di Simone N, D’Ippolito S, Castellani R, Di Nicuolo F, Gasbarrini G, et al. Antibodies anti-CagA cross-react with trophoblast cells: a risk factor for pre-eclampsia? Helicobacter (2012) 17:426–34. doi:10.1111/j.1523-5378.2012.00966.x
345. Shimoda A, Ueda K, Nishiumi S, Murata-Kamiya N, Mukai SA, Sawada S, et al. Exosomes as nanocarriers for systemic delivery of the Helicobacter pylori virulence factor CagA. Sci Rep (2016) 6:18346. doi:10.1038/srep18346
351. El-Shourbagy MAA, El-Refaie TA, Sayed KKA, Wahba KAH, El-Din ASS, Fathy MM. Impact of seroconversion and antichlamydial treatment on the rate of pre-eclampsia among Egyptian primigravidae. Int J Gynaecol Obstet (2011) 113:137–40. doi:10.1016/j.ijgo.2010.11.014
352. Haggerty CL, Klebanoff MA, Panum I, Uldum SA, Bass DC, Olsen J, et al. Prenatal Chlamydia trachomatis infection increases the risk of preeclampsia. Pregnancy Hypertens (2013) 3:151–4. doi:10.1016/j.preghy.2013.03.002
353. Haggerty CL, Panum I, Uldum SA, Bass DC, Olsen J, Darville T, et al. Chlamydia trachomatis infection may increase the risk of preeclampsia. Pregnancy Hypertens (2013) 3:28–33. doi:10.1016/j.preghy.2012.09.002
354. Xie F, Hu Y, Magee LA, Money DM, Patrick DM, Krajden M, et al. An association between cytomegalovirus infection and pre-eclampsia: a case-control study and data synthesis. Acta Obstet Gynecol Scand (2010) 89:1162–7. doi:10.3109/00016349.2010.499449
355. Xie F, Von Dadelszen P, Nadeau J. CMV infection, TLR-2 and -4 expression, and cytokine profiles in early-onset preeclampsia with HELLP syndrome. Am J Reprod Immunol (2014) 71:379–86. doi:10.1111/aji.12199
357. Mosbah A, Nabiel Y. Helicobacter pylori, Chlamydiae pneumoniae and trachomatis as probable etiological agents of preeclampsia. J Matern Fetal Neonatal Med (2016) 29:1607–12. doi:10.3109/14767058.2015.1056146
358. Tersigni C, Franceschi F, Todros T, Cardaropoli S, Scambia G, Di Simone N. Insights into the role of Helicobacter pylori infection in preeclampsia: from the bench to the bedside. Front Immunol (2014) 5:484. doi:10.3389/fimmu.2014.00484
359. Üstün Y, Engin-Üstün Y, Ozkaplan E, Otlu B, Sait Tekerekoğlu M. Association of Helicobacter pylori infection with systemic inflammation in preeclampsia. J Matern Fetal Neonatal Med (2010) 23:311–4. doi:10.3109/14767050903121456
360. Aksoy H, Ozkan A, Aktas F, Borekci B. Helicobacter pylori seropositivity and its relationship with serum malondialdehyde and lipid profile in preeclampsia. J Clin Lab Anal (2009) 23:219–22. doi:10.1002/jcla.20330
362. Pugliese A, Beltramo T, Todros T, Cardaropoli S, Ponzetto A. Interleukin-18 and gestosis: correlation with Helicobacter pylori seropositivity. Cell Biochem Funct (2008) 26:817–9. doi:10.1002/cbf.1503
363. Cardaropoli S, Giuffrida D, Piazzese A, Todros T. Helicobacter pylori seropositivity and pregnancy-related diseases: a prospective cohort study. J Reprod Immunol (2015) 109:41–7. doi:10.1016/j.jri.2015.02.004
364. Cardaropoli S, Rolfo A, Piazzese A, Ponzetto A, Todros T. Helicobacter pylori’s virulence and infection persistence define pre-eclampsia complicated by fetal growth retardation. World J Gastroenterol (2011) 17:5156–65. doi:10.3748/wjg.v17.i47.5156
365. McDonnold M, Dunn H, Hester A, Pacheco LD, Hankins GD, Saade GR, et al. High risk human papillomavirus at entry to prenatal care and risk of preeclampsia. Am J Obstet Gynecol (2014) 210(138):138.e1–5. doi:10.1016/j.ajog.2013.09.040
369. Easter SR, Cantonwine DE, Zera CA, Lim KH, Parry SI, Mcelrath TF. Urinary tract infection during pregnancy, angiogenic factor profiles, and risk of preeclampsia. Am J Obstet Gynecol (2016) 214:387.e1–7. doi:10.1016/j.ajog.2015.09.101
370. Mazor-Dray E, Levy A, Schlaeffer F, Sheiner E. Maternal urinary tract infection: is it independently associated with adverse pregnancy outcome? J Matern Fetal Neonatal Med (2009) 22:124–8. doi:10.1080/14767050802488246
371. Minassian C, Thomas SL, Williams DJ, Campbell O, Smeeth L. Acute maternal infection and risk of pre-eclampsia: a population-based case-control study. PLoS One (2013) 8:e73047. doi:10.1371/journal.pone.0073047
373. Karmon A, Sheiner E. The relationship between urinary tract infection during pregnancy and preeclampsia: causal, confounded or spurious? Arch Gynecol Obstet (2008) 277:479–81. doi:10.1007/s00404-008-0643-2
374. Villar J, Carroli G, Wojdyla D, Abalos E, Giordano D, Ba’aqeel H, et al. Preeclampsia, gestational hypertension and intrauterine growth restriction, related or independent conditions? Am J Obstet Gynecol (2006) 194:921–31. doi:10.1016/j.ajog.2005.10.813
375. Bánhidy F, Ács N, Puhó EH, Czeizel AE. Pregnancy complications and birth outcomes of pregnant women with urinary tract infections and related drug treatments. Scand J Infect Dis (2007) 39:390–7. doi:10.1080/00365540601087566
376. López-Jaramillo P, Herrera JA, Arenas-Mantilla M, Jauregui IE, Mendoza MA. Subclinical infection as a cause of inflammation in preeclampsia. Am J Ther (2008) 15:373–6. doi:10.1097/MJT.0b013e318164c149
377. Ide M, Papapanou PN. Epidemiology of association between maternal periodontal disease and adverse pregnancy outcomes – systematic review. J Periodontol (2013) 84:S181–94. doi:10.1902/jop.2013.134009
378. Dunlop AL, Mulle JG, Ferranti EP, Edwards S, Dunn AB, Corwin EJ. Maternal microbiome and pregnancy outcomes that impact infant health: a review. Adv Neonatal Care (2015) 15:377–85. doi:10.1097/ANC.0000000000000218
385. Cassell GH, Waites KB, Watson HL, Crouse DT, Harasawa R. Ureaplasma urealyticum intrauterine infection: role in prematurity and disease in newborns. Clin Microbiol Rev (1993) 6:69–87. doi:10.1128/CMR.6.1.69
386. McGregor JA, French JI, Jones W, Milligan K, Mckinney PJ, Patterson E, et al. Bacterial vaginosis is associated with prematurity and vaginal fluid mucinase and sialidase: results of a controlled trial of topical clindamycin cream. Am J Obstet Gynecol (1994) 170:1048–59; discussion 1059–60. doi:10.1016/S0002-9378(94)70098-2
389. Gerber S, Vial Y, Hohlfeld P, Witkin SS. Detection of Ureaplasma urealyticum in second-trimester amniotic fluid by polymerase chain reaction correlates with subsequent preterm labor and delivery. J Infect Dis (2003) 187:518–21. doi:10.1086/368205
390. Gardella C, Riley DE, Hitti J, Agnew K, Krieger JN, Eschenbach D. Identification and sequencing of bacterial rDNAs in culture-negative amniotic fluid from women in premature labor. Am J Perinatol (2004) 21:319–23. doi:10.1055/s-2004-831884
391. Espinoza J, Erez O, Romero R. Preconceptional antibiotic treatment to prevent preterm birth in women with a previous preterm delivery. Am J Obstet Gynecol (2006) 194:630–7. doi:10.1016/j.ajog.2005.11.050
395. Johnson HL, Ghanem KG, Zenilman JM, Erbelding EJ. Sexually transmitted infections and adverse pregnancy outcomes among women attending inner city public sexually transmitted diseases clinics. Sex Transm Dis (2011) 38:167–71. doi:10.1097/OLQ.0b013e3181f2e85f
396. Rours GIJG, Duijts L, Moll HA, Arends LR, De Groot R, Jaddoe VW, et al. Chlamydia trachomatis infection during pregnancy associated with preterm delivery: a population-based prospective cohort study. Eur J Epidemiol (2011) 26:493–502. doi:10.1007/s10654-011-9586-1
398. Lee SYR, Leung CW. Histological chorioamnionitis – implication for bacterial colonization, laboratory markers of infection, and early onset sepsis in very-low-birth-weight neonates. J Matern Fetal Neonatal Med (2012) 25:364–8. doi:10.3109/14767058.2011.579208
401. Joergensen JS, Kjaer Weile LK, Lamont RF. The early use of appropriate prophylactic antibiotics in susceptible women for the prevention of preterm birth of infectious etiology. Expert Opin Pharmacother (2014) 15:2173–91. doi:10.1517/14656566.2014.950225
402. Allen-Daniels MJ, Serrano MG, Pflugner LP, Fettweis JM, Prestosa MA, Koparde VN, et al. Identification of a gene in Mycoplasma hominis associated with preterm birth and microbial burden in intraamniotic infection. Am J Obstet Gynecol (2015) 212:779.e1–13. doi:10.1016/j.ajog.2015.01.032
403. de Andrade Ramos B, Kanninen TT, Sisti G, Witkin SS. Microorganisms in the female genital tract during pregnancy: tolerance versus pathogenesis. Am J Reprod Immunol (2015) 73:383–9. doi:10.1111/aji.12326
404. Kacerovsky M, Vrbacky F, Kutova R, Pliskova L, Andrys C, Musilova I, et al. Cervical microbiota in women with preterm prelabor rupture of membranes. PLoS One (2015) 10:e0126884. doi:10.1371/journal.pone.0126884
406. Ueno T, Niimi H, Yoneda N, Yoneda S, Mori M, Tabata H, et al. Eukaryote-made thermostable DNA polymerase enables rapid PCR-based detection of mycoplasma, ureaplasma and other bacteria in the amniotic fluid of preterm labor cases. PLoS One (2015) 10:e0129032. doi:10.1371/journal.pone.0129032
409. Vinturache AE, Gyamfi-Bannerman C, Hwang J, Mysorekar IU, Jacobsson B; Preterm Birth International Collaborative (Prebic). Maternal microbiome – a pathway to preterm birth. Semin Fetal Neonatal Med (2016) 21:94–9. doi:10.1016/j.siny.2016.02.004
410. Yoneda S, Shiozaki A, Yoneda N, Ito M, Shima T, Fukuda K, et al. Antibiotic therapy increases the risk of preterm birth in preterm labor without intra-amniotic microbes, but may prolong the gestation period in preterm labor with microbes, evaluated by rapid and high-sensitive PCR system. Am J Reprod Immunol (2016) 75:440–50. doi:10.1111/aji.12484
412. Kacerovsky M, Lenco J, Musilova I, Tambor V, Lamont R, Torloni MR, et al. Proteomic biomarkers for spontaneous preterm birth: a systematic review of the literature. Reprod Sci (2014) 21:283–95. doi:10.1177/1933719113503415
414. Menezes EV, Yakoob MY, Soomro T, Haws RA, Darmstadt GL, Bhutta ZA. Reducing stillbirths: prevention and management of medical disorders and infections during pregnancy. BMC Pregnancy Childbirth (2009) 9(Suppl 1):S4. doi:10.1186/1471-2393-9-S1-S4
415. Nigro G, Mazzocco M, Mattia E, Di Renzo GC, Carta G, Anceschi MM. Role of the infections in recurrent spontaneous abortion. J Matern Fetal Neonatal Med (2011) 24:983–9. doi:10.3109/14767058.2010.547963
417. Toth A, Lesser ML, Brooks-Toth CW, Feiner C. Outcome of subsequent pregnancies following antibiotic therapy after primary or multiple spontaneous abortions. Surg Gynecol Obstet (1986) 163:243–50.
418. Lauder AP, Roche AM, Sherrill-Mix S, Bailey A, Laughlin AL, Bittinger K, et al. Comparison of placenta samples with contamination controls does not provide evidence for a distinct placenta microbiota. Microbiome (2016) 4:29. doi:10.1186/s40168-016-0172-3
419. Onderdonk AB, Delaney ML, Dubois AM, Allred EN, Leviton A; Extremely Low Gestational Age Newborns Study Investigators. Detection of bacteria in placental tissues obtained from extremely low gestational age neonates. Am J Obstet Gynecol (2008) 198(110):e111–7. doi:10.1016/j.ajog.2007.05.044
421. Stout MJ, Conlon B, Landeau M, Lee I, Bower C, Zhao Q, et al. Identification of intracellular bacteria in the basal plate of the human placenta in term and preterm gestations. Am J Obstet Gynecol (2013) 208(226):e221–7. doi:10.1016/j.ajog.2013.01.018
424. Doyle RM, Alber DG, Jones HE, Harris K, Fitzgerald F, Peebles D, et al. Term and preterm labour are associated with distinct microbial community structures in placental membranes which are independent of mode of delivery. Placenta (2014) 35:1099–101. doi:10.1016/j.placenta.2014.10.007
427. Antony KM, Ma J, Mitchell KB, Racusin DA, Versalovic J, Aagaard K. The preterm placental microbiome varies in association with excess maternal gestational weight gain. Am J Obstet Gynecol (2015) 212: 653.e651–616. doi:10.1016/j.ajog.2014.12.041
428. Garmi G, Okopnik M, Keness Y, Zafran N, Berkowitz E, Salim R. Correlation between clinical, placental histology and microbiological findings in spontaneous preterm births. Fetal Diagn Ther (2015) 40:141–9. doi:10.1159/000441518
430. Collado MC, Rautava S, Aakko J, Isolauri E, Salminen S. Human gut colonisation may be initiated in utero by distinct microbial communities in the placenta and amniotic fluid. Sci Rep (2016) 6:23129. doi:10.1038/srep23129
432. Prince AL, Ma J, Kannan PS, Alvarez M, Gisslen T, Harris RA, et al. The placental microbiome is altered among subjects with spontaneous preterm birth with and without chorioamnionitis. Am J Obstet Gynecol (2016) 214:627.e1–16. doi:10.1016/j.ajog.2016.01.193
433. Viniker DA. Hypothesis on the role of sub-clinical bacteria of the endometrium (bacteria endometrialis) in gynaecological and obstetric enigmas. Hum Reprod Update (1999) 5:373–85. doi:10.1093/humupd/5.4.373
434. Svare JA, Schmidt H, Hansen BB, Lose G. Bacterial vaginosis in a cohort of Danish pregnant women: prevalence and relationship with preterm delivery, low birthweight and perinatal infections. BJOG (2006) 113:1419–25. doi:10.1111/j.1471-0528.2006.01087.x
436. Aagaard K, Riehle K, Ma J, Segata N, Mistretta TA, Coarfa C, et al. A metagenomic approach to characterization of the vaginal microbiome signature in pregnancy. PLoS One (2012) 7:e36466. doi:10.1371/journal.pone.0036466
437. Walther-António MR, Jeraldo P, Berg Miller ME, Yeoman CJ, Nelson KE, Wilson BA, et al. Pregnancy’s stronghold on the vaginal microbiome. PLoS One (2014) 9:e98514. doi:10.1371/journal.pone.0098514
438. Huang YE, Wang Y, He Y, Ji Y, Wang LP, Sheng HF, et al. Homogeneity of the vaginal microbiome at the cervix, posterior fornix, and vaginal canal in pregnant Chinese women. Microb Ecol (2015) 69:407–14. doi:10.1007/s00248-014-0487-1
439. Witkin SS. The vaginal microbiome, vaginal anti-microbial defence mechanisms and the clinical challenge of reducing infection-related preterm birth. BJOG (2015):213–8. doi:10.1111/1471-0528.13115
442. Verstraelen H, Vilchez-Vargas R, Desimpel F, Jauregui R, Vankeirsbilck N, Weyers S, et al. Characterisation of the human uterine microbiome in non-pregnant women through deep sequencing of the V1-2 region of the 16S rRNA gene. PeerJ (2016) 4:e1602. doi:10.7717/peerj.1602
444. Bearfield C, Davenport ES, Sivapathasundaram V, Allaker RP. Possible association between amniotic fluid micro-organism infection and microflora in the mouth. BJOG (2002) 109:527–33. doi:10.1111/j.1471-0528.2002.01349.x
445. Combs CA, Gravett M, Garite TJ, Hickok DE, Lapidus J, Porreco R, et al. Amniotic fluid infection, inflammation, and colonization in preterm labor with intact membranes. Am J Obstet Gynecol (2014) 210:125.e1–15. doi:10.1016/j.ajog.2013.11.032
447. Combs CA, Garite TJ, Lapidus JA, Lapointe JP, Gravett M, Rael J, et al. Detection of microbial invasion of the amniotic cavity by analysis of cervicovaginal proteins in women with preterm labor and intact membranes. Am J Obstet Gynecol (2015) 212:482.e481–e412. doi:10.1016/j.ajog.2015.02.007
449. Pelzer ES, Allan JA, Cunningham K, Mengersen K, Allan JM, Launchbury T, et al. Microbial colonization of follicular fluid: alterations in cytokine expression and adverse assisted reproduction technology outcomes. Hum Reprod (2011) 26:1799–812. doi:10.1093/humrep/der108
451. Barak S, Oettinger-Barak O, Machtei EE, Sprecher H, Ohel G. Evidence of periopathogenic microorganisms in placentas of women with preeclampsia. J Periodontol (2007) 78:670–6. doi:10.1902/jop.2007.060362
453. Amarasekara R, Jayasekara RW, Senanayake H, Dissanayake VH. Microbiome of the placenta in pre-eclampsia supports the role of bacteria in the multifactorial cause of pre-eclampsia. J Obstet Gynaecol Res (2015) 41:662–9. doi:10.1111/jog.12619
456. Vanterpool SF, Been JV, Houben ML, Nikkels PG, De Krijger RR, Zimmermann LJ, et al. Porphyromonas gingivalis within placental villous mesenchyme and umbilical cord stroma is associated with adverse pregnancy outcome. PLoS One (2016) 11:e0146157. doi:10.1371/journal.pone.0146157
457. Chaparro A, Blanlot C, Ramirez V, Sanz A, Quintero A, Inostroza C, et al. Porphyromonas gingivalis, Treponema denticola and toll-like receptor 2 are associated with hypertensive disorders in placental tissue: a case-control study. J Periodontal Res (2013) 48:802–9. doi:10.1111/jre.12074
461. Vatanen T, Kostic AD, D’Hennezel E, Siljander H, Franzosa EA, Yassour M, et al. Variation in microbiome LPS immunogenicity contributes to autoimmunity in humans. Cell (2016) 165:842–53. doi:10.1016/j.cell.2016.04.007
462. Ferrier L, Mazelin L, Cenac N, Desreumaux P, Janin A, Emilie D, et al. Stress-induced disruption of colonic epithelial barrier: role of interferon-gamma and myosin light chain kinase in mice. Gastroenterology (2003) 125:795–804. doi:10.1016/S0016-5085(03)01057-6
466. Lassenius MI, Pietiläinen KH, Kaartinen K, Pussinen PJ, Syrjänen J, Forsblom C, et al. Bacterial endotoxin activity in human serum is associated with dyslipidemia, insulin resistance, obesity, and chronic inflammation. Diabetes Care (2011) 34:1809–15. doi:10.2337/dc10-2197
467. Vors C, Pineau G, Drai J, Meugnier E, Pesenti S, Laville M, et al. Postprandial endotoxemia linked with chylomicrons and lipopolysaccharides handling in obese versus lean men: a lipid dose-effect trial. J Clin Endocrinol Metab (2015) 100:3427–35. doi:10.1210/JC.2015-2518
469. Perez PF, Dore J, Leclerc M, Levenez F, Benyacoub J, Serrant P, et al. Bacterial imprinting of the neonatal immune system: lessons from maternal cells? Pediatrics (2007) 119:e724–32. doi:10.1542/peds.2006-1649
471. Newnham JP, Newnham IA, Ball CM, Wright M, Pennell CE, Swain J, et al. Treatment of periodontal disease during pregnancy: a randomized controlled trial. Obstet Gynecol (2009) 114:1239–48. doi:10.1097/AOG.0b013e3181c15b40
472. Huang X, Wang J, Liu J, Hua L, Zhang D, Hu T, et al. Maternal periodontal disease and risk of preeclampsia: a meta-analysis. J Huazhong Univ Sci Technolog Med Sci (2014) 34:729–35. doi:10.1007/s11596-014-1343-8
473. Gilbert GL, Garland SM, Fairley KF, Mcdowall DM. Bacteriuria due to ureaplasmas and other fastidious organisms during pregnancy: prevalence and significance. Pediatr Infect Dis (1986) 5:S239–43. doi:10.1097/00006454-198611010-00007
476. Boggess KA, Edelstein BL. Oral health in women during preconception and pregnancy: implications for birth outcomes and infant oral health. Matern Child Health J (2006) 10:S169–74. doi:10.1007/s10995-006-0095-x
477. Boggess KA, Berggren EK, Koskenoja V, Urlaub D, Lorenz C. Severe preeclampsia and maternal self-report of oral health, hygiene, and dental care. J Periodontol (2013) 84:143–51. doi:10.1902/jop.2012.120079
481. Lachat MF, Solnik AL, Nana AD, Citron TL. Periodontal disease in pregnancy: review of the evidence and prevention strategies. J Perinat Neonatal Nurs (2011) 25:312–9. doi:10.1097/JPN.0b013e31821072e4
482. Politano GT, Passini R, Nomura ML, Velloso L, Morari J, Couto E. Correlation between periodontal disease, inflammatory alterations and pre-eclampsia. J Periodontal Res (2011) 46:505–11. doi:10.1111/j.1600-0765.2011.01368.x
483. Nabet C, Lelong N, Colombier ML, Sixou M, Musset AM, Goffinet F, et al. Maternal periodontitis and the causes of preterm birth: the case-control Epipap study. J Clin Periodontol (2010) 37:37–45. doi:10.1111/j.1600-051X.2009.01503.x
485. Bobetsis YA, Barros SP, Offenbacher S. Exploring the relationship between periodontal disease and pregnancy complications. J Am Dent Assoc (2006) 137(Suppl):7S–13S. doi:10.14219/jada.archive.2006.0403
486. Herrera JA, Parra B, Herrera E, Botero JE, Arce RM, Contreras A, et al. Periodontal disease severity is related to high levels of C-reactive protein in pre-eclampsia. J Hypertens (2007) 25:1459–64. doi:10.1097/HJH.0b013e3281139ea9
487. Ruma M, Boggess K, Moss K, Jared H, Murtha A, Beck J, et al. Maternal periodontal disease, systemic inflammation, and risk for preeclampsia. Am J Obstet Gynecol (2008) 198:389.e1–5. doi:10.1016/j.ajog.2007.12.002
489. Zi MY, Longo PL, Bueno-Silva B, Mayer MP. Mechanisms involved in the association between periodontitis and complications in pregnancy. Front Public Health (2014) 2:290. doi:10.3389/fpubh.2014.00290
490. Swati P, Thomas B, Vahab SA, Kapaettu S, Kushtagi P. Simultaneous detection of periodontal pathogens in subgingival plaque and placenta of women with hypertension in pregnancy. Arch Gynecol Obstet (2012) 285:613–9. doi:10.1007/s00404-011-2012-9
493. Ha JE, Oh KJ, Yang HJ, Jun JK, Jin BH, Paik DI, et al. Oral health behaviors, periodontal disease, and pathogens in preeclampsia: a case-control study in Korea. J Periodontol (2011) 82:1685–92. doi:10.1902/jop.2011.110035
494. Komine-Aizawa S, Hirohata N, Aizawa S, Abiko Y, Hayakawa S. Porphyromonas gingivalis lipopolysaccharide inhibits trophoblast invasion in the presence of nicotine. Placenta (2015) 36:27–33. doi:10.1016/j.placenta.2014.10.015
496. Oettinger-Barak O, Barak S, Ohel G, Oettinger M, Kreutzer H, Peled M, et al. Severe pregnancy complication (preeclampsia) is associated with greater periodontal destruction. J Periodontol (2005) 76:134–7. doi:10.1902/jop.2005.76.1.134
499. Marrs CF, Zhang L, Foxman B. Escherichia coli mediated urinary tract infections: are there distinct uropathogenic E. coli (UPEC) pathotypes? FEMS Microbiol Lett (2005) 252:183–90. doi:10.1016/j.femsle.2005.08.028
500. Hannan TJ, Mysorekar IU, Hung CS, Isaacson-Schmid ML, Hultgren SJ. Early severe inflammatory responses to uropathogenic E. coli predispose to chronic and recurrent urinary tract infection. PLoS Pathog (2010) 6:e1001042. doi:10.1371/journal.ppat.1001042
503. Hannan TJ, Totsika M, Mansfield KJ, Moore KH, Schembri MA, Hultgren SJ. Host-pathogen checkpoints and population bottlenecks in persistent and intracellular uropathogenic Escherichia coli bladder infection. FEMS Microbiol Rev (2012) 36:616–48. doi:10.1111/j.1574-6976.2012.00339.x
505. Justice SS, Hung C, Theriot JA, Fletcher DA, Anderson GG, Footer MJ, et al. Differentiation and developmental pathways of uropathogenic Escherichia coli in urinary tract pathogenesis. Proc Natl Acad Sci U S A (2004) 101:1333–8. doi:10.1073/pnas.0308125100
507. Rosen DA, Hooton TM, Stamm WE, Humphrey PA, Hultgren SJ. Detection of intracellular bacterial communities in human urinary tract infection. PLoS Med (2007) 4:e329. doi:10.1371/journal.pmed.0040329
508. Dhakal BK, Kulesus RR, Mulvey MA. Mechanisms and consequences of bladder cell invasion by uropathogenic Escherichia coli. Eur J Clin Invest (2008) 38(Suppl 2):2–11. doi:10.1111/j.1365-2362.2008.01986.x
509. Schwartz DJ, Chen SL, Hultgren SJ, Seed PC. Population dynamics and niche distribution of uropathogenic Escherichia coli during acute and chronic urinary tract infection. Infect Immun (2011) 79:4250–9. doi:10.1128/IAI.05339-11
511. Russo TA, Stapleton A, Wenderoth S, Hooton TM, Stamm WE. Chromosomal restriction fragment length polymorphism analysis of Escherichia coli strains causing recurrent urinary tract infections in young women. J Infect Dis (1995) 172:440–5. doi:10.1093/infdis/172.2.440
512. Ikäheimo R, Siitonen A, Heiskanen T, Kärkkäinen U, Kuosmanen P, Lipponen P, et al. Recurrence of urinary tract infection in a primary care setting: analysis of a 1-year follow-up of 179 women. Clin Infect Dis (1996) 22:91–9. doi:10.1093/clinids/22.1.91
513. Rosen DA, Pinkner JS, Jones JM, Walker JN, Clegg S, Hultgren SJ. Utilization of an intracellular bacterial community pathway in Klebsiella pneumoniae urinary tract infection and the effects of FimK on type 1 pilus expression. Infect Immun (2008) 76:3337–45. doi:10.1128/IAI.00090-08
514. Luo Y, Ma Y, Zhao Q, Wang L, Guo L, Ye L, et al. Similarity and divergence of phylogenies, antimicrobial susceptibilities, and virulence factor profiles of Escherichia coli isolates causing recurrent urinary tract infections that persist or result from reinfection. J Clin Microbiol (2012) 50:4002–7. doi:10.1128/JCM.02086-12
515. Özlü T, Alçelik A, Çalışkan B, Dönmez ME. Preeclampsia: is it because of the asymptomatic, unrecognized renal scars caused by urinary tract infections in childhood that become symptomatic with pregnancy? Med Hypotheses (2012) 79:653–5. doi:10.1016/j.mehy.2012.08.002
521. Gilbert NM, O’Brien VP, Hultgren S, Macones G, Lewis WG, Lewis AL. Urinary tract infection as a preventable cause of pregnancy complications: opportunities, challenges, and a global call to action. Glob Adv Health Med (2013) 2:59–69. doi:10.7453/gahmj.2013.061
523. Germain SJ, Sacks GP, Sooranna SR, Sargent IL, Redman CWG. Systemic inflammatory priming in normal pregnancy and preeclampsia: the role of circulating syncytiotrophoblast microparticles. J Immunol (2007) 178:5949–56. doi:10.4049/jimmunol.178.9.5949
527. Redman CWG, Tannetta DS, Dragovic RA, Gardiner C, Southcombe JH, Collett GP, et al. Review: does size matter? Placental debris and the pathophysiology of pre-eclampsia. Placenta (2012) 33(Suppl):S48–54. doi:10.1016/j.placenta.2011.12.006
529. Niccolai E, Emmi G, Squatrito D, Silvestri E, Emmi L, Amedei A, et al. Microparticles: bridging the gap between autoimmunity and thrombosis. Semin Thromb Hemost (2015) 41:413–22. doi:10.1055/s-0035-1549850
531. Mitchell MD, Peiris HN, Kobayashi M, Koh YQ, Duncombe G, Illanes SE, et al. Placental exosomes in normal and complicated pregnancy. Am J Obstet Gynecol (2015) 213:S173–81. doi:10.1016/j.ajog.2015.07.001
532. de Jonge L, Bos HJ, Van Langen IM, De Jong-Van Den Berg LTW, Bakker MK. Antibiotics prescribed before, during and after pregnancy in the Netherlands: a drug utilization study. Pharmacoepidemiol Drug Saf (2014) 23:60–8. doi:10.1002/pds.3492
534. Palmsten K, Hernández-Díaz S, Chambers CD, Mogun H, Lai S, Gilmer TP, et al. The most commonly dispensed prescription medications among pregnant women enrolled in the U.S. Medicaid program. Obstet Gynecol (2015) 126:465–73. doi:10.1097/AOG.0000000000000982
536. Suy A, Martínez E, Coll O, Lonca M, Palacio M, De Lazzari E, et al. Increased risk of pre-eclampsia and fetal death in HIV-infected pregnant women receiving highly active antiretroviral therapy. AIDS (2006) 20:59–66. doi:10.1097/01.aids.0000198090.70325.bd
537. Wimalasundera RC, Larbalestier N, Smith JH, De Ruiter A, Mc GTSA, Hughes AD, et al. Pre-eclampsia, antiretroviral therapy, and immune reconstitution. Lancet (2002) 360:1152–4. doi:10.1016/S0140-6736(02)11195-0
539. Adams JW, Watts DH, Phelps BR. A systematic review of the effect of HIV infection and antiretroviral therapy on the risk of pre-eclampsia. Int J Gynaecol Obstet (2016) 133:17–21. doi:10.1016/j.ijgo.2015.08.007
540. Todros T, Verdiglione P, Oggè G, Paladini D, Vergani P, Cardaropoli S. Low incidence of hypertensive disorders of pregnancy in women treated with spiramycin for Toxoplasma infection. Br J Clin Pharmacol (2006) 61:336–40. doi:10.1111/j.1365-2125.2005.02572.x
541. Alvarado-Esquivel C, Vázquez-Alaníz F, Sandoval-Carrillo AA, Salas-Pacheco JM, Hernández-Tinoco J, Sanchez-Anguiano LF, et al. Lack of association between Toxoplasma gondii infection and hypertensive disorders in pregnancy: a case-control study in a Northern Mexican population. Parasit Vectors (2014) 7:167. doi:10.1186/1756-3305-7-167
545. Kumasawa K, Ikawa M, Kidoya H, Hasuwa H, Saito-Fujita T, Morioka Y, et al. Pravastatin induces placental growth factor (PGF) and ameliorates preeclampsia in a mouse model. Proc Natl Acad Sci U S A (2011) 108:1451–5. doi:10.1073/pnas.1011293108
546. Faas MM, Schuiling GA, Baller JF, Visscher CA, Bakker WW. A new animal model for human preeclampsia: ultra-low-dose endotoxin infusion in pregnant rats. Am J Obstet Gynecol (1994) 171:158–64. doi:10.1016/0002-9378(94)90463-4
547. Faas MM, Schuiling GA, Linton EA, Sargent IL, Redman CWG. Activation of peripheral leukocytes in rat pregnancy and experimental preeclampsia. Am J Obstet Gynecol (2000) 182:351–7. doi:10.1016/S0002-9378(00)70223-7
548. Sakawi Y, Tarpey M, Chen YF, Calhoun DA, Connor MG, Chestnut DH, et al. Evaluation of low-dose endotoxin administration during pregnancy as a model of preeclampsia. Anesthesiology (2000) 93:1446–55. doi:10.1097/00000542-200012000-00017
549. Lin F, Zeng P, Xu ZY, Ye DY, Yu XF, Wang N, et al. Treatment of lipoxin A4 and its analogue on low-dose endotoxin induced preeclampsia in rat and possible mechanisms. Reprod Toxicol (2012) 34:677–85. doi:10.1016/j.reprotox.2012.09.009
550. Cotechini T, Komisarenko M, Sperou A, MacDonald-Goodfellow S, Adams MA, Graham CH. Inflammation in rat pregnancy inhibits spiral artery remodeling leading to fetal growth restriction and features of preeclampsia. J Exp Med (2014) 211:165–79. doi:10.1084/jem.20130295
551. Xue PP, Zheng MM, Gong P, Lin CM, Zhou JJ, Li YJ, et al. Single administration of ultra-low-dose lipopolysaccharide in rat early pregnancy induces TLR4 activation in the placenta contributing to preeclampsia. PLoS One (2015) 10:e0124001. doi:10.1371/journal.pone.0124001
552. Kalkunte S, Boij R, Norris W, Friedman J, Lai Z, Kurtis J, et al. Sera from preeclampsia patients elicit symptoms of human disease in mice and provide a basis for an in vitro predictive assay. Am J Pathol (2010) 177:2387–98. doi:10.2353/ajpath.2010.100475
553. Nymark M, Pussinen PJ, Tuomainen AM, Forsblom C, Groop PH, Lehto M, et al. Serum lipopolysaccharide activity is associated with the progression of kidney disease in finnish patients with type 1 diabetes. Diabetes Care (2009) 32:1689–93. doi:10.2337/dc09-0467
554. Al-Attas OS, Al-Daghri NM, Al-Rubeaan K, Da Silva NF, Sabico SL, Kumar S, et al. Changes in endotoxin levels in T2DM subjects on anti-diabetic therapies. Cardiovasc Diabetol (2009) 8:20. doi:10.1186/1475-2840-8-20
555. Hawkesworth S, Moore SE, Fulford AJC, Barclay GR, Darboe AA, Mark H, et al. Evidence for metabolic endotoxemia in obese and diabetic Gambian women. Nutr Diabetes (2013) 3:e83. doi:10.1038/nutd.2013.24
556. Zaman GS, Zaman F. Relationship between postprandial endotoxemia in nonobese postmenopausal women and diabetic nonobese postmenopausal women. J Nat Sci Biol Med (2015) 6:89–93. doi:10.4103/0976-9668.149098
557. Rusterholz C, Hahn S, Holzgreve W. Role of placentally produced inflammatory and regulatory cytokines in pregnancy and the etiology of preeclampsia. Semin Immunopathol (2007) 29:151–62. doi:10.1007/s00281-007-0071-6
559. Anton L, Brown AG, Parry S, Elovitz MA. Lipopolysaccharide induces cytokine production and decreases extravillous trophoblast invasion through a mitogen-activated protein kinase-mediated pathway: possible mechanisms of first trimester placental dysfunction. Hum Reprod (2012) 27:61–72. doi:10.1093/humrep/der362
561. Pineda A, Verdin-Terán SL, Camacho A, Moreno-Fierros L. Expression of toll-like receptor TLR-2, TLR-3, TLR-4 and TLR-9 is increased in placentas from patients with preeclampsia. Arch Med Res (2011) 42:382–91. doi:10.1016/j.arcmed.2011.08.003
562. Chatterjee P, Weaver LE, Doersch KM, Kopriva SE, Chiasson VL, Allen SJ, et al. Placental toll-like receptor 3 and toll-like receptor 7/8 activation contributes to preeclampsia in humans and mice. PLoS One (2012) 7:e41884. doi:10.1371/journal.pone.0041884
563. Gallo PM, Rapsinski GJ, Wilson RP, Oppong GO, Sriram U, Goulian M, et al. Amyloid-DNA composites of bacterial biofilms stimulate autoimmunity. Immunity (2015) 42:1171–84. doi:10.1016/j.immuni.2015.06.002
564. Rapsinski GJ, Wynosky-Dolfi MA, Oppong GO, Tursi SA, Wilson RP, Brodsky IE, et al. Toll-like receptor 2 and NLRP3 cooperate to recognize a functional bacterial amyloid, curli. Infect Immun (2015) 83:693–701. doi:10.1128/IAI.02370-14
570. Jain D, Kaur KJ, Goel M, Salunke DM. Structural basis of functional mimicry between carbohydrate and peptide ligands of con A. Biochem Biophys Res Commun (2000) 272:843–9. doi:10.1006/bbrc.2000.2871
571. Goel M, Krishnan L, Kaur S, Kaur KJ, Salunke DM. Plasticity within the antigen-combining site may manifest as molecular mimicry in the humoral immune response. J Immunol (2004) 173:7358–67. doi:10.4049/jimmunol.173.12.7358
572. Uh A, Nicholson RC, Gonzalez GV, Simmons CF, Gombart A, Smith R, et al. Lipopolysaccharide stimulation of trophoblasts induces corticotropin-releasing hormone expression through MyD88. Am J Obstet Gynecol (2008) 199(317):e311–6. doi:10.1016/j.ajog.2008.06.091
573. Chen Q, Viall C, Kang Y, Liu B, Stone P, Chamley L. Anti-phospholipid antibodies increase non-apoptotic trophoblast shedding: a contribution to the pathogenesis of pre-eclampsia in affected women? Placenta (2009) 30:767–73. doi:10.1016/j.placenta.2009.06.008
574. Chen Q, Guo F, Hensby-Bennett S, Stone P, Chamley L. Antiphospholipid antibodies prolong the activation of endothelial cells induced by necrotic trophoblastic debris: implications for the pathogenesis of preeclampsia. Placenta (2012) 33:810–5. doi:10.1016/j.placenta.2012.07.019
575. Pantham P, Rosario R, Chen Q, Print CG, Chamley LW. Transcriptomic analysis of placenta affected by antiphospholipid antibodies: following the TRAIL of trophoblast death. J Reprod Immunol (2012) 94:151–4. doi:10.1016/j.jri.2012.03.487
576. Tong M, Viall CA, Chamley LW. Antiphospholipid antibodies and the placenta: a systematic review of their in vitro effects and modulation by treatment. Hum Reprod Update (2015) 21:97–118. doi:10.1093/humupd/dmu049
577. Dechend R, Homuth V, Wallukat G, Kreuzer J, Park JK, Theuer J, et al. AT(1) receptor agonistic antibodies from preeclamptic patients cause vascular cells to express tissue factor. Circulation (2000) 101:2382–7. doi:10.1161/01.CIR.101.20.2382
579. Wallukat G, Neichel D, Nissen E, Homuth V, Luft FC. Agonistic autoantibodies directed against the angiotensin II AT1 receptor in patients with preeclampsia. Can J Physiol Pharmacol (2003) 81:79–83. doi:10.1139/y02-160
580. Dechend R, Muller DN, Wallukat G, Homuth V, Krause M, Dudenhausen J, et al. AT1 receptor agonistic antibodies, hypertension, and preeclampsia. Semin Nephrol (2004) 24:571–9. doi:10.1016/j.semnephrol.2004.07.006
581. Dechend R, Homuth V, Wallukat G, Muller DN, Krause M, Dudenhausen J, et al. Agonistic antibodies directed at the angiotensin II, AT1 receptor in preeclampsia. J Soc Gynecol Investig (2006) 13:79–86. doi:10.1016/j.jsgi.2005.11.006
582. Hubel CA, Wallukat G, Wolf M, Herse F, Rajakumar A, Roberts JM, et al. Agonistic angiotensin II type 1 receptor autoantibodies in postpartum women with a history of preeclampsia. Hypertension (2007) 49:612–7. doi:10.1161/01.HYP.0000256565.20983.d4
584. Herse F, Staff AC, Hering L, Müller DN, Luft FC, Dechend R. AT1-receptor autoantibodies and uteroplacental RAS in pregnancy and pre-eclampsia. J Mol Med (Berl) (2008) 86:697–703. doi:10.1007/s00109-008-0332-4
587. Parrish MR, Murphy SR, Rutland S, Wallace K, Wenzel K, Wallukat G, et al. The effect of immune factors, tumor necrosis factor-alpha, and agonistic autoantibodies to the angiotensin II type I receptor on soluble fms-like tyrosine-1 and soluble endoglin production in response to hypertension during pregnancy. Am J Hypertens (2010) 23:911–6. doi:10.1038/ajh.2010.70
590. Siddiqui AH, Irani RA, Zhang W, Wang W, Blackwell SC, Kellems RE, et al. Angiotensin receptor agonistic autoantibody-mediated soluble fms-like tyrosine kinase-1 induction contributes to impaired adrenal vasculature and decreased aldosterone production in preeclampsia. Hypertension (2013) 61:472–9. doi:10.1161/HYPERTENSIONAHA.111.00157
593. Fliser D, Buchholz K, Haller H, Olmesartan EUTO, Pravastatin In I, Atherosclerosis I. Antiinflammatory effects of angiotensin II subtype 1 receptor blockade in hypertensive patients with microinflammation. Circulation (2004) 110:1103–7. doi:10.1161/01.CIR.0000140265.21608.8E
595. Platten M, Youssef S, Hur EM, Ho PP, Han MH, Lanz TV, et al. Blocking angiotensin-converting enzyme induces potent regulatory T cells and modulates TH1- and TH17-mediated autoimmunity. Proc Natl Acad Sci U S A (2009) 106:14948–53. doi:10.1073/pnas.0903958106
596. Ma G, Li Y, Zhang J, Liu H, Hou D, Zhu L, et al. Association between the presence of autoantibodies against adrenoreceptors and severe pre-eclampsia: a pilot study. PLoS One (2013) 8:e57983. doi:10.1371/journal.pone.0057983
597. Li Y, Ma G, Zhang Z, Yue Y, Yuan Y, Wang Y, et al. Association of autoantibodies against the M2-muscarinic receptor with perinatal outcomes in women with severe preeclampsia. J Transl Med (2013) 11:285. doi:10.1186/1479-5876-11-285
599. Darmochwał-Kolarz D, Kludka-Sternik M, Tabarkiewicz J, Kolarz B, Rolinski J, Leszczynska-Gorzelak B, et al. The predominance of Th17 lymphocytes and decreased number and function of Treg cells in preeclampsia. J Reprod Immunol (2012) 93:75–81. doi:10.1016/j.jri.2012.01.006
600. Wenzel K, Rajakumar A, Haase H, Geusens N, Hubner N, Schulz H, et al. Angiotensin II type 1 receptor antibodies and increased angiotensin II sensitivity in pregnant rats. Hypertension (2011) 58:77–84. doi:10.1161/HYPERTENSIONAHA.111.171348
601. Stepan H, Wallukat G, Schultheiss HP, Faber R, Walther T. Is parvovirus B19 the cause for autoimmunity against the angiotensin II type receptor? J Reprod Immunol (2007) 73:130–4. doi:10.1016/j.jri.2006.08.084
602. Irani RA, Zhang Y, Zhou CC, Blackwell SC, Hicks MJ, Ramin SM, et al. Autoantibody-mediated angiotensin receptor activation contributes to preeclampsia through tumor necrosis factor-alpha signaling. Hypertension (2010) 55:1246–53. doi:10.1161/HYPERTENSIONAHA.110.150540
605. Verdonk K, Visser W, Van Den Meiracker AH, Danser AH. The renin-angiotensin-aldosterone system in pre-eclampsia: the delicate balance between good and bad. Clin Sci (Lond) (2014) 126:537–44. doi:10.1042/CS20130455
614. Annunziato F, Cosmi L, Liotta F, Maggi E, Romagnani S. The phenotype of human Th17 cells and their precursors, the cytokines that mediate their differentiation and the role of Th17 cells in inflammation. Int Immunol (2008) 20:1361–8. doi:10.1093/intimm/dxn106
617. Eyerich K, Pennino D, Scarponi C, Foerster S, Nasorri F, Behrendt H, et al. IL-17 in atopic eczema: linking allergen-specific adaptive and microbial-triggered innate immune response. J Allergy Clin Immunol (2009) 123:59–66.e4. doi:10.1016/j.jaci.2008.10.031
621. Weber A, Zimmermann C, Kieseier BC, Hartung HP, Hofstetter HH. Bacteria and their cell wall components uniformly co-activate interleukin-17-producing thymocytes. Clin Exp Immunol (2014) 178:504–15. doi:10.1111/cei.12414
623. Kumar P, Monin L, Castillo P, Elsegeiny W, Horne W, Eddens T, et al. Intestinal interleukin-17 receptor signaling mediates reciprocal control of the gut microbiota and autoimmune inflammation. Immunity (2016) 44:659–71. doi:10.1016/j.immuni.2016.02.007
624. Saito S, Nakashima A, Ito M, Shima T. Clinical implication of recent advances in our understanding of IL-17 and reproductive immunology. Expert Rev Clin Immunol (2011) 7:649–57. doi:10.1586/eci.11.49
626. Ozkan ZS, Simsek M, Ilhan F, Deveci D, Godekmerdan A, Sapmaz E. Plasma IL-17, IL-35, interferon-gamma, SOCS3 and TGF-beta levels in pregnant women with preeclampsia, and their relation with severity of disease. J Matern Fetal Neonatal Med (2014) 27:1513–7. doi:10.3109/14767058.2013.861415
627. Santner-Nanan B, Peek MJ, Khanam R, Richarts L, Zhu E, Fazekas De St Groth B, et al. Systemic increase in the ratio between Foxp3+ and IL-17-producing CD4+ T cells in healthy pregnancy but not in preeclampsia. J Immunol (2009) 183:7023–30. doi:10.4049/jimmunol.0901154
628. Jianjun Z, Yali H, Zhiqun W, Mingming Z, Xia Z. Imbalance of T-cell transcription factors contributes to the Th1 type immunity predominant in pre-eclampsia. Am J Reprod Immunol (2010) 63:38–45. doi:10.1111/j.1600-0897.2009.00763.x
630. Toldi G, Rigó J, Stenczer B, Vásárhelyi B, Molvarec A. Increased prevalence of IL-17-producing peripheral blood lymphocytes in pre-eclampsia. Am J Reprod Immunol (2011) 66:223–9. doi:10.1111/j.1600-0897.2011.00987.x
631. Cornelius DC, Hogg JP, Scott J, Wallace K, Herse F, Moseley J, et al. Administration of interleukin-17 soluble receptor C suppresses TH17 cells, oxidative stress, and hypertension in response to placental ischemia during pregnancy. Hypertension (2013) 62:1068–73. doi:10.1161/HYPERTENSIONAHA.113.01514
637. Vargas-Rojas MI, Solleiro-Villavicencio H, Soto-Vega E. Th1, Th2, Th17 and Treg levels in umbilical cord blood in preeclampsia. J Matern Fetal Neonatal Med (2016) 29:1642–5. doi:10.3109/14767058.2015.1057811
639. Suntharalingam G, Perry MR, Ward S, Brett SJ, Castello-Cortes A, Brunner MD, et al. Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412. N Engl J Med (2006) 355:1018–28. doi:10.1056/NEJMoa063842
640. Wang H, Ma S. The cytokine storm and factors determining the sequence and severity of organ dysfunction in multiple organ dysfunction syndrome. Am J Emerg Med (2008) 26:711–5. doi:10.1016/j.ajem.2007.10.031
643. Singer M, Deutschman CS, Seymour CW, Shankar-Hari M, Annane D, Bauer M, et al. The third international consensus definitions for sepsis and septic shock (sepsis-3). JAMA (2016) 315:801–10. doi:10.1001/jama.2016.0287
645. Lai PS, Matteau A, Iddriss A, Hawes JC, Ranieri V, Thompson BT. An updated meta-analysis to understand the variable efficacy of drotrecogin alfa (activated) in severe sepsis and septic shock. Minerva Anestesiol (2013) 79:33–43.
647. Messaris E, Antonakis PT, Memos N, Chatzigianni E, Leandros E, Konstadoulakis MM. Deferoxamine administration in septic animals: improved survival and altered apoptotic gene expression. Int Immunopharmacol (2004) 4:455–9. doi:10.1016/j.intimp.2004.01.012
648. Islam S, Jarosch S, Zhou J, Del Carmen Parquet C, Toguri JT, Colp P, et al. Anti-inflammatory and anti-bacterial effects of iron chelation in experimental sepsis. J Surg Res (2016) 200:266–73. doi:10.1016/j.jss.2015.07.001
649. Doron MW, Makhlouf RA, Katz VL, Lawson EE, Stiles AD. Increased incidence of sepsis at birth in neutropenic infants of mothers with preeclampsia. J Pediatr (1994) 125:452–8. doi:10.1016/S0022-3476(05)83294-9
650. Kocherlakota P, La Gamma EF. Preliminary report: rhG-CSF may reduce the incidence of neonatal sepsis in prolonged preeclampsia-associated neutropenia. Pediatrics (1998) 102:1107–11. doi:10.1542/peds.102.5.1107
652. Procianoy RS, Silveira RC, Mussi-Pinhata MM, Rugolo LMSS, Leone CR, Lopes JMD, et al. Sepsis and neutropenia in very low birth weight infants delivered of mothers with preeclampsia. J Pediatr (2010) 157:434–U118. doi:10.1016/j.jpeds.2010.02.066
653. Namdev S, Bhat V, Adhisivam B, Zachariah B. Oxidative stress and antioxidant status among neonates born to mothers with pre-eclampsia and their early outcome. J Matern Fetal Neonatal Med (2014) 27:1481–4. doi:10.3109/14767058.2013.860521
654. Kenny LC, Dunn WB, Ellis DI, Myers J, Baker PN; The Gopec Consortium, et al. Novel biomarkers for pre-eclampsia detected using metabolomics and machine learning. Metabolomics (2005) 1:227–34. doi:10.1007/s11306-005-0003-1
655. Kenny LC, Broadhurst D, Brown M, Dunn WB, Redman CWG, Kell DB, et al. Detection and identification of novel metabolomic biomarkers in preeclampsia. Reprod Sci (2008) 15:591–7. doi:10.1177/1933719108316908
656. Odibo AO, Goetzinger KR, Odibo L, Cahill AG, Macones GA, Nelson DM, et al. First-trimester prediction of preeclampsia using metabolomic biomarkers: a discovery phase study. Prenat Diagn (2011) 31:990–4. doi:10.1002/pd.2822
657. Bahado-Singh RO, Akolekar R, Mandal R, Dong E, Xia J, Kruger M, et al. Metabolomics and first-trimester prediction of early-onset preeclampsia. J Matern Fetal Neonatal Med (2012) 25:1840–7. doi:10.3109/14767058.2012.680254
658. Dunn WB, Brown M, Worton SA, Davies K, Jones RL, Kell DB, et al. The metabolome of human placental tissue: investigation of first trimester tissue and changes related to preeclampsia in late pregnancy. Metabolomics (2012) 8:579–97. doi:10.1007/s11306-011-0348-6
659. Woodham PC, O’Connell T, Grimes J, Haeri S, Eichelberger K, Baker A, et al. Metabolomics to predict severe preeclampsia in early pregnancy. Am J Obstet Gynecol (2012) 206:S348–348. doi:10.1016/j.ajog.2011.10.809
660. Kuc S, Koster MPH, Pennings JLA, Hankemeier T, Berger R, Harms AC, et al. Metabolomics profiling for identification of novel potential markers in early prediction of preeclampsia. PLoS One (2014) 9:e98540. doi:10.1371/journal.pone.0098540
661. Koster MPH, Vreeken RJ, Harms AC, Dane AD, Kuc S, Schielen PCJI, et al. First-trimester serum acylcarnitine levels to predict preeclampsia: a metabolomics approach. Dis Markers (2015) 2015:857108. doi:10.1155/2015/857108
662. Myers JE, Hart S, Armstrong S, Mires GJ, Beynon R, Gaskell SJ, et al. Evidence for multiple circulating factors in preeclampsia. Am J Obstet Gynecol (2007) 196(266):e261–6. doi:10.1016/j.ajog.2006.10.875
663. Kolla V, Jenö P, Moes S, Lapaire O, Hoesli I, Hahn S. Quantitative proteomic (iTRAQ) analysis of 1st trimester maternal plasma samples in pregnancies at risk for preeclampsia. J Biomed Biotechnol (2012) 2012:305964. doi:10.1155/2012/305964
664. Myers JE, Kenny LC, Mccowan LM, Chan EH, Dekker GA, Poston L, et al. Angiogenic factors combined with clinical risk factors to predict preterm pre-eclampsia in nulliparous women: a predictive test accuracy study. BJOG (2013) 120:1215–23. doi:10.1111/1471-0528.12195
665. Myers JE, Tuytten R, Thomas G, Laroy W, Kas K, Vanpoucke G, et al. Integrated proteomics pipeline yields novel biomarkers for predicting preeclampsia. Hypertension (2013) 61:1281–8. doi:10.1161/HYPERTENSIONAHA.113.01168
667. Hahn S, Lapaire O, Than NG. Biomarker development for presymptomatic molecular diagnosis of preeclampsia: feasible, useful or even unnecessary? Expert Rev Mol Diagn (2015) 15:617–29. doi:10.1586/14737159.2015.1025757
668. Khan GH, Galazis N, Docheva N, Layfield R, Atiomo W. Overlap of proteomics biomarkers between women with pre-eclampsia and PCOS: a systematic review and biomarker database integration. Hum Reprod (2015) 30:133–48. doi:10.1093/humrep/deu268
669. Paulus P, Jennewein C, Zacharowski K. Biomarkers of endothelial dysfunction: can they help us deciphering systemic inflammation and sepsis? Biomarkers (2011) 16(Suppl 1):S11–21. doi:10.3109/1354750X.2011.587893
670. Netea MG, Joosten LAB, Latz E, Mills KHG, Natoli G, Stunnenberg HG, et al. Trained immunity: a program of innate immune memory in health and disease. Science (2016) 352:aaf1098. doi:10.1126/science.aaf1098
671. Savvidou MD, Lees CC, Parra M, Hingorani AD, Nicolaides KH. Levels of C-reactive protein in pregnant women who subsequently develop pre-eclampsia. BJOG (2002) 109:297–301. doi:10.1111/j.1471-0528.2002.01130.x
672. Kashanian M, Aghbali F, Mahali N. Evaluation of the diagnostic value of the first-trimester maternal serum high-sensitivity C-reactive protein level for prediction of pre-eclampsia. J Obstet Gynaecol Res (2013) 39:1549–54. doi:10.1111/jog.12105
674. Rinehart BK, Terrone DA, Lagoo-Deenadayalan S, Barber WH, Hale EA, Martin JN Jr, et al. Expression of the placental cytokines tumor necrosis factor alpha, interleukin 1beta, and interleukin 10 is increased in preeclampsia. Am J Obstet Gynecol (1999) 181:915–20. doi:10.1016/S0002-9378(99)70325-X
675. Serin ÝS, Özçelik B, Bapbuð M, Kýlýç H, Okur D, Erez R. Predictive value of tumor necrosis factor alpha (TNF-alpha) in preeclampsia. Eur J Obstet Gynecol Reprod Biol (2002):143–5. doi:10.1016/S0301-2115(01)00484-5
676. Todros T, Bontempo S, Piccoli E, Ietta F, Romagnoli R, Biolcati M, et al. Increased levels of macrophage migration inhibitory factor (MIF) in preeclampsia. Eur J Obstet Gynecol Reprod Biol (2005) 123:162–6. doi:10.1016/j.ejogrb.2005.03.014
678. Roger T, Glauser MP, Calandra T. Macrophage migration inhibitory factor (MIF) modulates innate immune responses induced by endotoxin and Gram-negative bacteria. J Endotoxin Res (2001) 7:456–60. doi:10.1179/096805101101533089
681. Su H, Chang SS, Han CM, Wu KY, Li MC, Huang CY, et al. Inflammatory markers in cord blood or maternal serum for early detection of neonatal sepsis-a systemic review and meta-analysis. J Perinatol (2014) 34:268–74. doi:10.1038/jp.2013.186
682. Kell DB, Knowles JD. The role of modeling in systems biology. In: Szallasi Z, Stelling J, Periwal V, editors. System Modeling in Cellular Biology: From Concepts to Nuts and Bolts. Cambridge: MIT Press (2006). p. 3–18.
685. Hucka M, Finney A, Sauro HM, Bolouri H, Doyle JC, Kitano H, et al. The systems biology markup language (SBML): a medium for representation and exchange of biochemical network models. Bioinformatics (2003) 19:524–31. doi:10.1093/bioinformatics/btg015
686. Funahashi A, Matsuoka Y, Jouraku A, Morohashi M, Kikuchi N, Kitano H. CellDesigner 3.5: a versatile modeling tool for biochemical networks. Proc IEEE (2008) 96:1254–65. doi:10.1109/JPROC.2008.925458
689. Madazli R, Kuseyrioglu B, Uzun H, Uludag S, Ocak V. Prediction of preeclampsia with maternal mid-trimester placental growth factor, activin A, fibronectin and uterine artery Doppler velocimetry. Int J Gynaecol Obstet (2005) 89:251–7. doi:10.1016/j.ijgo.2005.02.008
690. Diesch CH, Holzgreve W, Hahn S, Zhong XY. Comparison of activin A and cell-free fetal DNA levels in maternal plasma from patients at high risk for preeclampsia. Prenat Diagn (2006) 26:1267–70. doi:10.1002/pd.1606
691. Reddy A, Suri S, Sargent IL, Redman CW, Muttukrishna S. Maternal circulating levels of activin A, inhibin A, sFlt-1 and endoglin at parturition in normal pregnancy and pre-eclampsia. PLoS One (2009) 4:e4453. doi:10.1371/journal.pone.0004453
692. Phillips DJ, Jones KL, Scheerlinck JY, Hedger MP, De Kretser DM. Evidence for activin A and follistatin involvement in the systemic inflammatory response. Mol Cell Endocrinol (2001) 180:155–62. doi:10.1016/S0303-7207(01)00516-0
693. Hodges R, Salvador L, D’Antona D, Georgiou HM, Wallace EM. Activin A as a marker of intrauterine infection in women with preterm prelabour rupture of membranes. J Perinatol (2010) 30:22–6. doi:10.1038/jp.2009.109
694. Rosenberg VA, Buhimschi IA, Dulay AT, Abdel-Razeq SS, Oliver EA, Duzyj CM, et al. Modulation of amniotic fluid activin-A and inhibin-A in women with preterm premature rupture of the membranes and infection-induced preterm birth. Am J Reprod Immunol (2012) 67:122–31. doi:10.1111/j.1600-0897.2011.01074.x
695. Petrakou E, Fotopoulos S, Anagnostakou M, Anatolitou F, Samitas K, Semitekolou M, et al. Activin-A exerts a crucial anti-inflammatory role in neonatal infections. Pediatr Res (2013) 74:675–81. doi:10.1038/pr.2013.159
697. Lugli A, Forster Y, Haas P, Nocito A, Bucher C, Bissig H, et al. Calretinin expression in human normal and neoplastic tissues: a tissue microarray analysis on 5233 tissue samples. Hum Pathol (2003) 34:994–1000. doi:10.1053/S0046-8177(03)00339-3
698. Rizzo A, Carratelli CR, De Filippis A, Bevilacqua N, Tufano MA, Buommino E. Transforming activities of Chlamydia pneumoniae in human mesothelial cells. Int Microbiol (2014) 17:185–93. doi:10.2436/20.1501.01.221
699. Xu QL, Zhu M, Jin Y, Wang N, Xu HX, Quan LM, et al. The predictive value of the first-trimester maternal serum chemerin level for pre-eclampsia. Peptides (2014) 62:150–4. doi:10.1016/j.peptides.2014.10.002
700. Stepan H, Philipp A, Roth I, Kralisch S, Jank A, Schaarschmidt W, et al. Serum levels of the adipokine chemerin are increased in preeclampsia during and 6 months after pregnancy. Regul Pept (2011) 168:69–72. doi:10.1016/j.regpep.2011.03.005
703. Wang LQ, Yang TL, Ding YL, Zhong Y, Yu L, Peng M. Chemerin plays a protective role by regulating human umbilical vein endothelial cell-induced nitric oxide signaling in preeclampsia. Endocrine (2015) 48:299–308. doi:10.1007/s12020-014-0286-y
705. Kukla M, Zwirska-Korczala K, Gabriel A, Waluga M, Warakomska I, Szczygiel B, et al. Chemerin, vaspin and insulin resistance in chronic hepatitis C. J Viral Hepat (2010) 17:661–7. doi:10.1111/j.1365-2893.2009.01224.x
706. Kukla M, Mazur W, Buldak RJ, Żwirska-Korczala K. Potential role of leptin, adiponectin and three novel adipokines-visfatin, chemerin and vaspin-in chronic hepatitis. Mol Med (2011) 17:1397–410. doi:10.2119/molmed.2010.00105
707. Kulig P, Kantyka T, Zabel BA, Banas M, Chyra A, Stefanska A, et al. Regulation of chemerin chemoattractant and antibacterial activity by human cysteine cathepsins. J Immunol (2011) 187:1403–10. doi:10.4049/jimmunol.1002352
708. Banas M, Zabieglo K, Kasetty G, Kapinska-Mrowiecka M, Borowczyk J, Drukala J, et al. Chemerin is an antimicrobial agent in human epidermis. PLoS One (2013) 8:e58709. doi:10.1371/journal.pone.0058709
710. Banas M, Zegar A, Kwitniewski M, Zabieglo K, Marczynska J, Kapinska-Mrowiecka M, et al. The expression and regulation of chemerin in the epidermis. PLoS One (2015) 10:e0117830. doi:10.1371/journal.pone.0117830
711. Horn P, Metzing UB, Steidl R, Romeike B, Rauchfuß F, Sponholz C, et al. Chemerin in peritoneal sepsis and its associations with glucose metabolism and prognosis: a translational cross-sectional study. Crit Care (2016) 20:39. doi:10.1186/s13054-016-12090-5
713. Birdir C, Janssen K, Stanescu AD, Enekwe A, Kasimir-Bauer S, Gellhaus A, et al. Maternal serum copeptin, MR-proANP and procalcitonin levels at 11-13 weeks gestation in the prediction of preeclampsia. Arch Gynecol Obstet (2015) 292:1033–42. doi:10.1007/s00404-015-3745-7
714. Jochberger S, Morgenthaler NG, Mayr VD, Luckner G, Wenzel V, Ulmer H, et al. Copeptin and arginine vasopressin concentrations in critically ill patients. J Clin Endocrinol Metab (2006) 91:4381–6. doi:10.1210/jc.2005-2830
717. Nickel CH, Bingisser R, Morgenthaler NG. The role of copeptin as a diagnostic and prognostic biomarker for risk stratification in the emergency department. BMC Med (2012) 10:7. doi:10.1186/1741-7015-10-7
719. Odermatt J, Bolliger R, Hersberger L, Ottiger M, Christ-Crain M, Briel M, et al. Copeptin predicts 10-year all-cause mortality in community patients: a 10-year prospective cohort study. Clin Chem Lab Med (2016) 54:1681–90. doi:10.1515/cclm-2016-0151
720. Jochberger S, Luckner G, Mayr VD, Wenzel V, Morgenthaler NG, Friesenecker BE, et al. Course of vasopressin and copeptin plasma concentrations in a patient with severe septic shock. Anaesth Intensive Care (2006) 34:498–500.
721. Müller B, Morgenthaler N, Stolz D, Schuetz P, Muller C, Bingisser R, et al. Circulating levels of copeptin, a novel biomarker, in lower respiratory tract infections. Eur J Clin Invest (2007) 37:145–52. doi:10.1111/j.1365-2362.2007.01762.x
722. Jochberger S, Dörler J, Luckner G, Mayr VD, Wenzel V, Ulmer H, et al. The vasopressin and copeptin response to infection, severe sepsis, and septic shock. Crit Care Med (2009) 37:476–82. doi:10.1097/CCM.0b013e3181957532
723. Krüger S, Ewig S, Kunde J, Hanschmann A, Marre R, Suttorp N, et al. C-terminal provasopressin (copeptin) in patients with community-acquired pneumonia – influence of antibiotic pre-treatment: results from the German competence network CAPNETZ. J Antimicrob Chemother (2009) 64:159–62. doi:10.1093/jac/dkp148
724. Limper M, Goeijenbier M, Wagenaar JF, Gasem MH, Isbandrio B, Kunde J, et al. Copeptin as a predictor of disease severity and survival in leptospirosis. J Infect (2010) 61:92–4. doi:10.1016/j.jinf.2010.03.029
725. Fluri F, Morgenthaler NG, Mueller B, Christ-Crain M, Katan M. Copeptin, procalcitonin and routine inflammatory markers-predictors of infection after stroke. PLoS One (2012) 7:e48309. doi:10.1371/journal.pone.0048309
726. Kolditz M, Halank M, Schulte-Hubbert B, Bergmann S, Albrecht S, Höffken G. Copeptin predicts clinical deterioration and persistent instability in community-acquired pneumonia. Respir Med (2012) 106:1320–8. doi:10.1016/j.rmed.2012.06.008
727. Du JM, Sang G, Jiang CM, He XJ, Han Y. Relationship between plasma copeptin levels and complications of community-acquired pneumonia in preschool children. Peptides (2013) 45:61–5. doi:10.1016/j.peptides.2013.04.015
728. Masajtis-Zagajewska A, Kurnatowska I, Wajdlich M, Nowicki M. Utility of copeptin and standard inflammatory markers in the diagnostics of upper and lower urinary tract infections. BMC Urol (2015) 15:67. doi:10.1186/s12894-015-0061-2
730. Strevens H, Wide-Swensson D, Grubb A, Hansen A, Horn T, Ingemarsson I, et al. Serum cystatin C reflects glomerular endotheliosis in normal, hypertensive and pre-eclamptic pregnancies. BJOG (2003) 110:825–30. doi:10.1111/j.1471-0528.2003.02051.x
732. Kristensen K, Wide-Swensson D, Schmidt C, Blirup-Jensen S, Lindström V, Strevens H, et al. Cystatin C, beta-2-microglobulin and beta-trace protein in pre-eclampsia. Acta Obstet Gynecol Scand (2007) 86:921–6. doi:10.1080/00016340701318133
733. Guo HX, Wang CH, Li ZQ, Gong SP, Zhou ZQ, Leng LZ, et al. The application of serum cystatin C in estimating the renal function in women with preeclampsia. Reprod Sci (2012) 19:712–7. doi:10.1177/1933719111431001
736. Yalcin S, Ulas T, Eren MA, Aydogan H, Camuzcuoglu A, Kucuk A, et al. Relationship between oxidative stress parameters and cystatin C levels in patients with severe preeclampsia. Medicina (Kaunas) (2013) 49:118–23.
737. Odden MC, Scherzer R, Bacchetti P, Szczech LA, Sidney S, Grunfeld C, et al. Cystatin C level as a marker of kidney function in human immunodeficiency virus infection: the FRAM study. Arch Intern Med (2007) 167:2213–9. doi:10.1001/archinte.167.20.2213
738. Randers E, Kornerup K, Erlandsen EJ, Hasling C, Danielsen H. Cystatin C levels in sera of patients with acute infectious diseases with high C-reactive protein levels. Scand J Clin Lab Invest (2001) 61:333–5. doi:10.1080/00365510118007
739. Gupta SK, Kitch D, Tierney C, Melbourne K, Ha B, Mccomsey GA, et al. Markers of renal disease and function are associated with systemic inflammation in HIV infection. HIV Med (2015) 16:591–8. doi:10.1111/hiv.12268
740. Walker JB, Nesheim ME. The molecular weights, mass distribution, chain composition, and structure of soluble fibrin degradation products released from a fibrin clot perfused with plasmin. J Biol Chem (1999) 274:5201–12. doi:10.1074/jbc.274.8.5201
741. Bellart J, Gilabert R, Anglès A, Piera V, Miralles RM, Monasterio J, et al. Tissue factor levels and high ratio of fibrinopeptide A:d-dimer as a measure of endothelial procoagulant disorder in pre-eclampsia. Br J Obstet Gynaecol (1999) 106:594–7. doi:10.1111/j.1471-0528.1999.tb08330.x
742. Gulec UK, Ozgunen FT, Guzel AB, Buyukkurt S, Seydaoglu G, Urunsak IF, et al. An analysis of C-reactive protein, procalcitonin, and d-dimer in pre-eclamptic patients. Am J Reprod Immunol (2012) 68:331–7. doi:10.1111/j.1600-0897.2012.01171.x
743. Pinheiro MDB, Junqueira DRG, Coelho FF, Freitas LG, Carvalho MG, Gomes KB, et al. d-dimer in preeclampsia: systematic review and meta-analysis. Clin Chim Acta (2012) 414:166–70. doi:10.1016/j.cca.2012.08.003
745. Rahman R, Begum K, Khondker L, Majumder NI, Nahar K, Sultana R, et al. Role of d-dimer in determining coagulability status in pre-eclamptic and normotensive pregnant women. Mymensingh Med J (2015) 24:115–20.
746. Di Castelnuovo A, De Curtis A, Costanzo S, Persichillo M, Olivieri M, Zito F, et al. Association of d-dimer levels with all-cause mortality in a healthy adult population: findings from the MOLI-SANI study. Haematologica (2013) 98:1476–80. doi:10.3324/haematol.2012.083410
747. Jennings I, Woods TAL, Kitchen DP, Kitchen S, Walker ID. Laboratory d-dimer measurement: improved agreement between methods through calibration. Thromb Haemost (2007) 98:1127–35. doi:10.1160/TH07-05-0377
748. Khalafallah AA, Morse M, Al-Barzan AM, Adams M, Dennis A, Bates G, et al. d-dimer levels at different stages of pregnancy in Australian women: a single centre study using two different immunoturbidimetric assays. Thromb Res (2012) 130:e171–7. doi:10.1016/j.thromres.2012.05.022
749. Rodelo JR, De La Rosa G, Valencia ML, Ospina S, Arango CM, Gómez CI, et al. d-dimer is a significant prognostic factor in patients with suspected infection and sepsis. Am J Emerg Med (2012) 30:1991–9. doi:10.1016/j.ajem.2012.04.033
750. Khalafallah A, Jarvis C, Morse M, Albarzan AM, Stewart P, Bates G, et al. Evaluation of the innovance d-dimer assay for the diagnosis of disseminated intravascular coagulopathy in different clinical settings. Clin Appl Thromb Hemost (2014) 20:91–7. doi:10.1177/1076029612454936
751. Hentschke MR, Lucas LS, Mistry HD, Pinheiro Da Costa BE, Poli-De-Figueiredo CE. Endocan-1 concentrations in maternal and fetal plasma and placentae in pre-eclampsia in the third trimester of pregnancy. Cytokine (2015) 74:152–6. doi:10.1016/j.cyto.2015.04.013
752. Cakmak M, Yilmaz H, Bağlar E, Darcin T, Inan O, Aktas A, et al. Serum levels of endocan correlate with the presence and severity of pre-eclampsia. Clin Exp Hypertens (2016) 38:137–42. doi:10.3109/10641963.2015.1060993
753. Mosevoll KA, Lindås R, Wendelbo Ø, Bruserud Ø, Reikvam H. Systemic levels of the endothelium-derived soluble adhesion molecules endocan and E-selectin in patients with suspected deep vein thrombosis. Springerplus (2014) 3:571. doi:10.1186/2193-1801-3-571
754. Yuksel MA, Tuten A, Oncul M, Acikgoz AS, Temel Yuksel I, Toprak MS, et al. Serum endocan concentration in women with pre-eclampsia. Arch Gynecol Obstet (2015) 292:69–73. doi:10.1007/s00404-014-3605-x
755. Balta S, Mikhailidis DP, Demirkol S, Ozturk C, Celik T, Iyisoy A. Endocan: a novel inflammatory indicator in cardiovascular disease? Atherosclerosis (2015) 243:339–43. doi:10.1016/j.atherosclerosis.2015.09.030
756. Pawlak K, Mysliwiec M, Pawlak D. Endocan – the new endothelial activation marker independently associated with soluble endothelial adhesion molecules in uraemic patients with cardiovascular disease. Clin Biochem (2015) 48:425–30. doi:10.1016/j.clinbiochem.2015.01.006
758. Scherpereel A, Depontieu F, Grigoriu B, Cavestri B, Tsicopoulos A, Gentina T, et al. Endocan, a new endothelial marker in human sepsis. Crit Care Med (2006) 34:532–7. doi:10.1097/01.CCM.0000198525.82124.74
759. Mihajlovic DM, Lendak DF, Brkic SV, Draskovic BG, Mitic GP, Novakov Mikic AS, et al. Endocan is useful biomarker of survival and severity in sepsis. Microvasc Res (2014) 93:92–7. doi:10.1016/j.mvr.2014.04.004
760. Pauly D, Hamed S, Behnes M, Lepiorz D, Lang S, Akin I, et al. Endothelial cell-specific molecule-1/endocan: diagnostic and prognostic value in patients suffering from severe sepsis and septic shock. J Crit Care (2016) 31:68–75. doi:10.1016/j.jcrc.2015.09.019
761. Shovlin CL, Hughes JMB, Scott J, Seidman CE, Seidman JG. Characterization of endoglin and identification of novel mutations in hereditary hemorrhagic telangiectasia. Am J Hum Genet (1997) 61:68–79. doi:10.1086/513906
764. Bell MJ, Roberts JM, Founds SA, Jeyabalan A, Terhorst L, Conley YP. Variation in endoglin pathway genes is associated with preeclampsia: a case-control candidate gene association study. BMC Pregnancy Childbirth (2013) 13:82. doi:10.1186/1471-2393-13-82