Liron Yoffe, Meitar Grad, Avital Luba Polsky, Moshe Hod, and Noam Shomron
Small noncoding RNAs (ncRNAs) are a diverse family of untranslated RNA molecules composed of less than 200 nucleotides (1,2). The most commonly studied small ncRNAs are microRNA (miRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), and transfer RNA (tRNA) (3). A growing number of small noncoding transcripts were identified to play important roles in gene regulation and RNA processing (4). Recent studies have shown that dysregulation of small ncRNAs has functional relevance in numerous human diseases, including cancer (5), neurological disorders, and cardiovascular diseases (6). Therefore, small ncRNAs may be of use in diagnosing and treating these disorders (7,8). Specifically, a plethora of research has been directed at the possible role of miRNA in the identification and diagnosis of various diseases. This chapter focuses on the involvement of miRNA in two prevalent pregnancy complications: gestational diabetes mellitus and preeclampsia.
miRNAs are an abundant class of small (∼22 nucleotides) ncRNAs that are estimated to downregulate the expression of more than 60% of protein-coding genes (6,9). miRNAs play pivotal post-transcriptional regulatory roles in various physiological functions. They bind messenger RNAs (mRNAs) at their 3′ untranslated region (UTR) and initiate either the mRNA degradation or translational repression (9,10). miRNAs are transcribed by RNA polymerase II from either specific miRNA gene loci or through splicing of mirtrons between exons of a host gene (6,9,11,12). Both transcription processes result in the formation of primary miRNAs (pri-miRNAs) that are processed by ribonuclease III (RNase III) enzymes Drosha and Dicer (along with other enzymes and aiding proteins) to form precursor miRNAs (pre-miRNAs) and then mature miRNAs (6,9,11,12). Mature miRNAs are then loaded into the RNA-induced silencing complex (RISC) to downregulate mRNA translation (6,9,11,12). A single miRNA can regulate tens to hundreds of downstream genes from a wide spectrum of biological pathways (13) and may also interact with other miRNAs to regulate gene expression (6). Thus, some miRNAs can be considered master regulators of various biological processes, such as cell proliferation, differentiation, apoptosis, and development (6,9).
The altered expression of specific miRNAs has been reported to be associated with a variety of diseases, including diabetes (14), cancer (15,16), neurological disorders (17,18), and cardiovascular diseases (19). Moreover, miRNA expression profiles were found to be altered by acute or chronic exposure to environmental factors such as cigarette smoking, pesticides, and air pollution (20,21).
Small RNAs as biomarkers
In recent years, many studies have been conducted to identify naturally occurring molecules that may be indicative of the presence or absence of various diseases (i.e., biomarkers) (3,10). Ideally, these biomarkers should be incorporated into a reliable, cost-effective, and noninvasive method for early identification of the disease or for estimation of its risk, enabling physicians to administer prophylactic treatment to assuage or prevent disease progression (20). Small ncRNAs are a promising class of biomarkers. Recent studies have shown that circulating cell-free small ncRNAs can be purified from plasma in sufficient amounts for accurate identification and quantification, implicating them as potential biomarkers for noninvasive identification of various diseases (3,20). Specifically, miRNA levels in plasma have been found to be relatively stable (22) and consistent among individuals of the same species (8,10). Studies have shown that altered physiology can be indicated by aberrant cell-free circulating miRNA expression profiles in patients’ plasma (23–25). For instance, Gilad et al. demonstrated that increased placenta-derived microRNA levels in sera of pregnant women correlated with pregnancy stage (23). Certainly, biomarkers have the potential to revolutionize the diagnosis and treatment of various medical conditions, and studies indicate that miRNAs present in plasma may have the potential to be used as noninvasive biomarkers of various diseases.
Gestational diabetes mellitus
The vast majority (84%) of pregnancies with high blood sugar (hyperglycemia) are due to gestational diabetes mellitus (GDM) (26,27). GDM is defined as glucose intolerance that begins during pregnancy (28). This disease is associated with a heightened risk of maternal morbidity, including subsequent development of diabetes and cardiovascular disease, as well as perinatal and neonatal morbidities (26). GDM results from the inability of the maternal pancreas to produce and secrete enough insulin to accommodate the fetus’s needs (26). Although the involvement of the fetus-placenta-mother interaction in increasing insulin resistance is known, the precise mechanisms underlying GDM remain a mystery (10).
Despite the high prevalence of the disease, there is no standard test for GDM (29). The most commonly used screening and diagnostic test is the oral glucose tolerance test (10,26), which is usually performed at 24–28 weeks of pregnancy, although it is often delayed until 32 weeks (30). As a result of the relatively late administration of the test, women found to have an increased risk of developing GDM have little time to take precautionary measures that could minimize symptoms or even prevent disease onset (29). A recent study conducted by Sovio et al. found that current GDM screening and diagnostic measures do not reduce certain adverse outcomes for the fetus (such as childhood obesity), suggesting that disease onset in the fetus is likely much earlier than the earliest possible detection by existing tests (31). The likelihood of early disease onset intimates that detection should be possible earlier in pregnancy.
Despite relatively high rates of GDM in many Western populations (1.7%–11.6%) (32), education and awareness of the risk factors (e.g., age, BMI, and ethnicity) are inadequate (26). Notwithstanding their poor detection sensitivity, low and middle resource countries prefer selective testing based on known risk factors (26). However, complex testing protocols adjusted for each risk group often lead to lower compliance among healthcare providers and pregnant women (26). The high prevalence of GDM and the variety of risk factor-oriented testing leads to a plethora of unstandardized testing results, thus increasing the probability of a false-negative diagnosis (26). Therefore, to facilitate consistent and uniform testing and results, an easy and reliable method of diagnosis is needed.
Over the years, there have been various attempts to find a reliable biomarker for GDM detection. Several potential biochemical markers have been identified (33–36); however, the subsets of patients used in these studies were too small to demonstrate a clinically sufficient diagnostic yield (10). It is, therefore, important to develop a novel and accurate approach for early GDM detection.
Biomarkers for GDM
Cell-free circulating miRNAs have gained traction as potential biomarkers, mainly for the diagnosis of type 1 and type 2 diabetes (31,37), and have been associated with several mechanisms related to diabetes pathogenesis (38–40). In recent years, the expression of cell-free circulating miRNAs in the blood of GDM patients was investigated, as well as their potential to serve as GDM diagnostic biomarkers (29,41–43). Nevertheless, all studies focused on later stages of pregnancy (early second trimester to full-term pregnancy), rather than on the first trimester.
Among the first to investigate the potential role of miRNAs as GDM diagnostic biomarkers were Zhao et al. (42). They collected serum samples from women at 16–19 gestational weeks, and discovered that the expression levels of three miRNAs (miR-132, miR-29a, and miR-222) were significantly decreased in patients later diagnosed with GDM compared to controls who did not develop the disease (42). However, along with the small sample size (24 patients in each group), the sensitivity (66.7%) and specificity (63.3%) obtained in this study were not sufficient to clearly differentiate between GDM and healthy patients (44).
In 2015, Zhu et al. assessed miRNA expression in maternal plasma at 16–19 weeks gestation, and discovered five miRNAs (miR-16-5p, miR-17-5p, miR-19a-3p, miR-19b-3p, and miR-20a-5p) that were upregulated in GDM-affected pregnancies compared to healthy pregnancies (29). However, this study was conducted on a particularly small sample size (10 patients in each group) and therefore requires further investigation to establish confidence.
Recently, Wander et al. (43) found the levels of circulating miRNAs previously linked to pregnancy complications (such as GDM and preeclampsia) to be associated with GDM during early to mid-pregnancy (7–23 weeks). However, these correlations were significant only among women belonging to established (i.e., pre-pregnancy overweight/obese) or potential (e.g., male fetal sex) risk groups (43). This limitation was probably due to the decision to specifically assess miRNAs known to be involved in pathways linked to both obesity and type 2 diabetes, thus precluding their utilization as biomarkers for GDM alone. Furthermore, Wander and her colleagues found miR-29a to be upregulated in GDM patients, in contrast to the downregulation observed by Zhao et al.
The steadily increasing published studies with contradictory results and/or biased research procedures indicate that further research into identifying specific miRNAs as early GDM diagnostic biomarkers should be conducted. Moreover, even though specifically identified miRNAs have yet to be sufficiently validated as reliable biomarkers, they can still inspire new research directions to better elucidate the mechanisms involved in GDM pathophysiology.
Preeclampsia (PE) is a multisystem disorder affecting 3%–8% of pregnancies, making it a leading cause of maternal and neonatal morbidity and mortality worldwide (45). PE is typically diagnosed after 20 weeks of gestation, with the appearance of hypertension combined with either proteinuria or at least one severe feature (thrombocytopenia, renal insufficiency, impaired liver function, pulmonary edema, and cerebral or visual symptoms) (30,46). A small percentage of preeclamptic women develop eclampsia, a progressive condition that involves life-threatening seizures (46) and a possible progression to HELLP syndrome (hemolysis, elevated liver functions, and low platelets) (47). PE can be classified into early (<34 weeks gestation) and late (>34 weeks gestation) onset, while the former increases the risk of maternal and fetal severe outcomes (48).
PE is often referred to as “the disease of theories,” reflecting the lack of understanding of its pathogenesis (3,46). Several pathophysiological mechanisms in the development of preeclampsia have been proposed, including endothelial dysfunction (49), inflammatory responses (50,51), oxidative stress (52), and dysregulation of angiogenic factors (53). It is commonly believed that preeclampsia originates from abnormal placentation, which may include inadequate blood supply to the placenta, leading to a hypoxic environment (54–56). After 20 weeks of gestation in preeclamptic pregnancies, the placental hypoxia develops into placental ischemia, oxidative stress, release of small placental particles into the maternal circulation, and ultimately presentation of classic maternal PE symptoms (57). Unfortunately, a valid and reliable method for PE diagnosis prior to the appearance of symptoms is not currently available (3