Omics and coagulation disorders in pregnancy
Sara Ornaghi and Michael J. Paidas
Omic technologies and biomarkers development have been largely based on advances in vascular biology, improved understanding of the molecular basis and biochemical pathways responsible for the clinically relevant diseases, and increasingly robust large cohort and/or registry-based studies.
Omic technologies adopt a holistic view of the molecules that make up a cell, tissue, or organism. They are aimed primarily at the universal detection of genes (genomics), mRNA (transcriptomics), proteins (proteomics), and metabolites (metabolomics) in a specific biological sample in a nontargeted and nonbiased manner. This can also be referred to as high-dimensional biology; the integration of these techniques is called systems biology (Figure 24.1) (1,2).
Figure 24.1 Omic sciences: interaction and related definitions. Numbers are approximate quantity at each functional level. (Source: Ref. 1.)
The basic aspect of these approaches is that a complex system can be understood more thoroughly if considered as a whole. Systems biology and omics experiments differ from traditional studies, which are largely hypothesis driven. By contrast, systems biology experiments are hypothesis generating, using holistic approaches where no hypothesis is known or prescribed but all data are acquired and analyzed to define a hypothesis that can be further tested (3). Thus, the emergence of omic technologies allowing the global analysis of a given biological or molecular system rather than the study of its individual components has revolutionized biomedical research in the past decade. These developments raised the prospect that classical, hypothesis-driven, single gene-based approaches may soon become obsolete. The experience accumulated so far, however, indicates that omic technologies only represent tools similar to those classically used by scientists to make hypotheses and construct models, with the main difference that they generate large amounts of unbiased information. Thus, omics- and classical hypothesis-driven research are rather complementary approaches with the potential to synergistically advance research in many fields, including obstetric medicine (4).
Omic technology can be applied not only for the greater understanding of normal physiological processes but also in disease processes where they play a role in screening, diagnosis, and prognosis as well as in improving our understanding of the etiology of diseases by elucidating underlying molecular mechanisms. Omic research in the field of obstetrics has focused on several pregnancy complications, such as preeclampsia and fetal growth restriction (FGR), and a wide range of biological samples has been used in this research, including plasma/serum, urine, saliva, amniotic fluid, cultured trophoblasts, myometrium, and cervicovaginal fluid (1).
Coagulation disorders in pregnancy and role of omics
Coagulation disorders in pregnancy include different clinical conditions, ranging from maternal thromboembolism to a wide spectrum of placenta-mediated complications such as preeclampsia, FGR, stillbirth, and placental abruption (PA). This cohort of pregnancy complications manifests a shared etiopathogenesis and predisposing risk factors and represents a leading cause of maternal and fetal-neonatal morbidity and mortality in both low- and high-income countries (5–9).
Pregnancy is an example of Virchow’s triad: hypercoagulability, venous stasis, and vascular damage. Altogether these factors lead to an increased incidence of venous thromboembolism (VTE), which includes deep vein thrombosis (DVT) and pulmonary embolism (Figure 24.2) (10,11).
Figure 24.2 Virchow’s triad in pregnancy. (VTE, venous thromboembolism, which includes deep vein thrombosis and pulmonary embolism.)
While postpartum hemorrhage is the leading cause of maternal mortality in the developing world, VTE is the primary cause in the developed world (12). Wen and colleagues have recently performed a retrospective cohort study evaluating risk factors and timing of postpartum VTE readmissions after delivery hospitalization discharge in the United States. In a 2-year period, 2,975 cases of readmission for any VTE were identified (4.7 per 10,000 delivery hospitalizations), with approximately 70% of these readmissions occurring during the first 20 days after discharge. History of VTE and hemorrhage with transfusion were associated with the largest odds of readmission, followed by thrombophilia, cesarean delivery, longer delivery hospitalization, and hypertensive disorders of pregnancy (13). The mechanisms of action of the above-mentioned risk factors is linked to the pathophysiology of VTE by means of hypercoagulable state (thrombophilia, obesity), venous stasis (immobilization), or vascular injury (delivery) (14,15).
The multifactorial etiology of VTE greatly limits the clinical value of genetic analysis due to the presence of several confounding variables, i.e., different processes regulating protein expression and gene-to-gene interactions, which result in a wide range of variability in the link between genotype and phenotype (16). In addition, diagnosis of VTE is still challenging because of the lack of suitable biological assays with sufficient specificity and positive predictive value. In this context, proteomic technology has gained growing attention in the analysis of cellular and plasma components involved in thrombosis and hemostasis, and several biomarkers have been identified with the ability to discriminate VTE cases from healthy controls in pilot studies (17). However, independent clinical validation of these newly identified potential VTE biomarkers is still lacking, thus making the road to their routine clinical application for diagnosis and risk stratification long. Anyhow, the use of an integrated omic approach has the potential to provide substantial data in the quest for VTE biomarker and therapeutic target discovery, thus highlighting the importance of establishing multidisciplinary collaboration between clinicians and omic experts. This importance has been proved in a recently published case-control study in which advanced proteomic techniques were used to assess blood samples collected from 200 individuals before occurrence of VTE (18). Two proteins, transthyretin (a vitamin K–dependent protein Z) and protein/nucleic acid deglycase DJ-1, were identified as the strongest predictive biomarker candidates, supporting their further investigation in larger clinical studies (19).
Placenta-mediated complications are pregnancy-speciﬁc disorders that have in common abnormal placental implantation, excessive placental thrombosis, and altered immune tolerance at the maternal-fetal interface (20). As previously mentioned, they include preeclampsia, FGR, fetal loss, and PA. Several studies suggest that although these conditions may differ in their clinical manifestations, they may be considered as one disease process that clinically manifests itself in an underlying continuum of mild disease to more severe disease states as pregnancy approaches term (21). In addition, placental histological findings support the classification of these clinical entities in a single category referred to as “ischemic placental disease” (22–24).
A maternal predisposition to endothelial dysfunction and an impaired trophoblast invasion and maternal spiral arteries remodeling have been proposed as underlying pathogenic mechanisms for these obstetric diseases, by contributing to shallow placental implantation and eventually placental insufficiency (25–33). In turn, placental insufficiency leads to decreased uteroplacental blood perfusion and impaired materno-fetal exchange of nutrients, gases, and waste products (34). Hypoperfusion and endothelial dysfunction appear to be both a cause and a consequence of abnormal placental development, as suggested by the following examples: (1) successful animal models of preeclampsia have involved mechanically reducing uteroplacental blood flow (35,36), and (2) medical conditions associated with vascular insufficiency and endothelial dysfunction (e.g., chronic hypertension, diabetes mellitus) increase the risk of abnormal placentation and development of placentally related complications (20).
The hypoxic and oxidatively damaged placenta worsens the systemic endothelial dysfunction by releasing antiangiogenic factors, namely. soluble fms-like tyrosine kinase 1 (sFlt-1) and soluble endoglin (sEng), which act by either sequestering or antagonizing proangiogenic molecules, such as vascular endothelial growth factor (VEGF), placental growth factor (PlGF), and transforming growth factor-β (TGF-β) (37). VEGF, PlGF, and TGF-β are all required for normal endothelial function because of their ability to activate the nitric oxide (NO) pathway, which is crucial for angiogenesis (Figure 24.3) (38,39,40).
Figure 24.3 Endothelial dysfunction in preeclampsia. A functional endothelial monolayer requires VEGF, PlGF, and TGF-β for normal function via activation of NO. In preeclampsia, VEGF and TGF-ß protective signals are compromised due to an excess of sFlt-1 and sEng, which is compounded by a decrease in the expression of PlGF. (EngR, Eng receptor; eNOS, endothelial NO synthases; NO, nitric oxide; PlGF, placental growth factor; sEng, soluble endoglin; sFlt-1, soluble fms-like tyrosine kinase 1; TGF-ß, transforming growth factor-ß; TGFR, TGF-ß receptor; VEGF, vascular endothelial growth factor; VEGFR-1, VEGF receptor 1.) (Source: Ref. 41, with permission.)
Maternal serum levels of sFlt-1 and sEng have been reported to be two- to fivefold higher in preeclamptic women as compared with controls (38,42). Low first-trimester levels of PlGF and sFlt-1, as well as high sEng values, have been associated with an increased risk of later onset of preeclampsia, FGR, stillbirth, and preterm birth (43). A similar association has been identified for low concentrations of pregnancy-associated plasma protein A (PAPP-A), a protease that acts on insulin-like growth factor binding proteins (43–48).
Strategies aimed at improving prediction of placenta-mediated complications often incorporate the previously mentioned serum biomarkers with hemodynamic blood flow data obtained with noninvasive imaging technologies of the uteroplacental and maternal circulation early in pregnancy.
In a recent review by Poon and Nicolaides, the effectiveness of screening at 11–13 weeks of pregnancy for early preeclampsia by specific maternal risk factors alone or in combination with several biophysical and biochemical markers, including PAPP-A, PlGF, the uterine artery pulsatility index (UtA PI), and mean arterial pressure (MAP), was evaluated. They reported a detection rate of 93% for maternal risk factors alone and of 96% when specific biomarkers were added to the prediction model (49,50).
Crovetto et al. recently published a nested case-control study within a cohort of nearly 6,000 pregnancies, including 28 cases of early preeclampsia. They looked at maternal characteristics and serum markers and found that significant contributors to early preeclampsia prediction were a maternal history of chronic hypertension and/or preeclampsia, and values of MAP, UtA PI, PlGF, and sFlt-1 (51).
Gaccioli and coworkers published the results of a prospective cohort study aiming to investigate the predictive ability of the association between elevated sFlt-1/PlGF ratio (>85th percentile) and ultrasonically suspected growth restricted fetus (<10th percentile), evaluated at 28 and 36 weeks of gestational age, for preterm delivery with FGR (28 weeks) and for term delivery of a growth restricted fetus with maternal preeclampsia or perinatal morbidity or mortality (52). The authors found that the positive likelihood ratios at both gestational ages were higher than previously described definitions of suspected FGR using purely ultrasonic assessment, thus suggesting that screening and intervention based on an approach combining ultrasound and biomarkers of placental insufficiency could result in net benefit, thus being worth it to be explored in a randomized controlled trial. In a subsequent review, the same authors stressed the role of omic technologies in accelerating discovery of novel and more potent biomarkers for FGR screening by means of next-generation sequencing with unbiased interrogation of genomes or transcriptomes in clinical specimens (53).
Aiming to identify novel serological protein markers to predict preeclampsia with a multi-omic-based discovery approach, Liu and coworkers identified three upregulated and six downregulated biomarkers in preeclamptic sera, associated with lipid homeostasis (APO-E), inflammation (α-2-macroglobin, A2M), and coagulation (disintegrin and ADAM12) (54). Two optimal biomarker panels were then developed for early and late-onset preeclampsia assessment, showing a superior detection rate as compared with the well-known sFlt-1/PIGF ratio.