Do miRNAs Play a Role in Fetal Growth Restriction? A Fresh Look to a Busy Corner

Placenta is the crucial organ for embryo and fetus development and plays a critical role in the development of fetal growth restriction (FGR). There are increasing evidences on the role of microRNAs (miRNAs) in a variety of pregnancy-related complications such as preeclampsia and FGR. More than 1880 miRNAs have been reported in humans and most of them are expressed in placenta. In this paper, we aimed to review the current evidence about the topic. According to retrieved data, controversial results about placental expression of miRNAs could be due (at least in part) to the different experimental methods used by different groups. Despite the fact that several authors have demonstrated a relatively easy and feasible detection of some miRNAs in maternal whole peripheral blood, costs of these tests should be reduced in order to increase cohorts and have stronger evidence. In this regard, we take the opportunity to solicit future studies on large cohort and adequate statistical power, in order to identify a panel of biomarkers on maternal peripheral blood for early diagnosis of FGR.


Introduction
Fetal growth restriction (FGR) refers to a condition in which there is the stop or the decrease of the genetic determined potential growth of a fetus during pregnancy. FGR is due to different causes, including maternal smoking, undernutrition, infection, and congenital abnormalities; conversely, if it is not possible to individuate a clear cause, it is defined as idiopathic. FGR is often associated with preeclampsia [1] and represents the most common pregnancy complication, accounting for about a third of all preterm births. Usually FGR is diagnosed when, through ultrasound, fetal weight is estimated to be less than the 10th percentile for gestational age using a validated fetal growth scale [2]. The most studied cause of FGR in animal models is maternal calorie restriction during gestation, but while much is known about the consequences of this deprivation, molecular mechanisms that underlie these conditions still remain unclear.
Placenta is the crucial organ for embryo and fetus development and plays a critical role in the development of FGR. In this regard, FGR could be considered as a placentation disorder, derived from a dysregulation in trophoblast invasion with characteristic tissue morphology that leads to uteroplacental insufficiency. This condition would greatly benefit from the availability of early diagnostic tests to give an opportunity for early intervention or prevention, to improve maternal-fetal outcomes, and to substantially contain the public health costs.

BioMed Research International
There are increasing evidences on the role of microRNAs (miRNAs) in a variety of pregnancy-related complications such as preeclampsia and fetal growth restriction. More than 1880 miRNAs have been reported in humans and most of them are expressed in placenta. This kind of nucleic acid belongs to the family of small noncoding RNAs of on average 22 nucleotides in length, which regulates gene expression at the posttranscriptional level, inhibiting translation or promoting specific mRNAs degradation through interaction with the 3 untranslated region [3,4]. In detail, miRNAs seem to modulate cell development, differentiation, and proliferation, cell type-specific function, and are involved in the pathogenesis of many human diseases [5]. In several cases, miRNA expression is tissue-specific and, in addition, is significantly different between physiology and pathological conditions: for these reasons, investigations about miRNAs gained increasing attention for the possibility of future application in clinical diagnostics [6,7].
Starting from these considerations, we aimed to review the current literature focusing on the role of miRNAs in FGR.

Materials and Methods
We performed a selective literature search of articles in English language, published from 2002 to 2017 and indexed in PubMed. We searched the following Medical Subject Headings (MeSH): "MicroRNAs" AND "Fetal Growth Retardation". The initial database screening was performed by three authors (Laganà AS, Vaiarelli A, La Rosa VL), who were blinded to the aim of the study. Subsequently, other three authors (Chiofalo B, Rossetti D, Vitale SG) selected relevant information from the screened literature. We considered eligible all original manuscripts (randomized, observational, and retrospective studies), case series, and case reports. Furthermore, we extracted relevant information from selected reviews.
Other authors studied miRNA-424 and its target gene (mitogen-activated protein kinase) that play a role in endothelial cell proliferation through fibroblast growth factor receptor 1 and regulate vascular endothelial growth factor [11]. According to their data analysis, the levels of this miRNA are increased in placentas from 25 FGR pregnancies compared with 25 placentas from uncomplicated pregnancies, suggesting that miRNA-424 is involved in placental disorders. Another study by Su et al. [12] searched, in a cohort of placentas, the miRNAs that regulate endocrine gland derived vascular endothelial growth factor (EG-VEGF) expression: they concluded that miR-346 and miR-582-3p regulate EG-VEGF-induced trophoblast invasion through repressing metalloproteinases 2 and 9. In addition, FGR placental tissues show an aberrant high expression level of miR-141, suggesting that this miRNA might play important roles in the pathogenesis of the disease by suppressing E2F transcription factor 3 and pleomorphic adenoma gene 1 [13].
To date, many studies focused their attention on chromosome 19 miRNA cluster (C19MC) [14][15][16]. In detail, C19MC comprises 46 miRNAs and is the largest gene cluster of miRNAs in humans, exclusively expressed in undifferentiated cells and in placenta. In this regard, comparing 14 placentas from FGR pregnancies with 14 from normal pregnancies, it was recently found that hypoxic stress does not affect C19MC miRNA expression, except for downregulation of miR-500c-3p [14]. Similarly, Hromadnikova et al. [17] detected a downregulation of 6 miRNAs (miR-517-5p, miR-518f-5p, miR-519a, miR-519d, miR-520a-5p, and miR-525) in placental tissues of 36 FGR pregnancies: compared to the previous studies, these results seem more robust since that authors investigated more types of miRNAs and used those that were previously demonstrated to be exclusively expressed or highly expressed in placental tissues. The significantly decreased expression of miR-519d, but not of miR-520a-5p and miR-525, was also confirmed by others on a larger cohort (50 healthy pregnancies compared with 45 FGR cases) [15]. Nevertheless, other experiments found that the expression of miR-518b was decreased, whereas miR-519a was significantly increased, in 30 FGR placentas [16]. Some of these miRNAs studied in human placentae were also studied in animal models.

Circulating miRNAs.
During pregnancy, due to a normal extravillous trophoblast invasion, nucleic acids of the placental compartment are released into the maternal circulation: this release occurs through the migration of microvesicles from apoptotic/necrotic cells and active cellular communication system, involving also nanovesicles/exosomes and subcellular fragments [18,19]. Due to placental continuous remodeling, these extracellular nucleic acids may be detected in maternal blood during the course of gestation and can be measured to monitor placental function and allow early diagnosis of pregnancy complications [20][21][22][23].
Some of these miRNAs, such as miR-141, miR-200c, and miR-205, were studied also in animal models [48,49]. In particular, it was found that they play important roles in the maintenance of the integrity of the folded trophoblastendometrial epithelial bilayer in porcine placentas [48].

Discussion
Based on the abovementioned data, miRNAs seem to be involved in placental development and consequently in placenta related disorders. As showed in Table 1, controversial results among these studies in placental expression of miRNAs could be due, at least in part, to the different experimental methods used by different groups. Despite the fact that several authors have demonstrated a relatively easy and feasible detection of some miRNAs in maternal whole peripheral blood [44][45][46][47], costs of these tests should be reduced in order to increase cohorts and have stronger evidence.
In this regard, we acknowledge that it may be extremely important to address future research directions taking into account the already available data from in vitro experiments and animal models: indeed, accumulating evidence suggests that miR141-3p and miR-200a-3p play a pivotal role for placental development in mouse and regulate the expression of insulin-like growth factor 2 [50]. Interestingly, upregulation of miR-125b was found to reduce significantly ethanol-induced caspase-3 activation and to diminish ethanol-induced growth retardation in mouse embryos [51], suggesting a possible protective role that is worthy of further investigation. Conversely, miR-24 and miR-103-2, which are related to adipocyte development, were both increased in low birth weight male guinea pig pups [52]. Probably this last element could be further confirmed in future studies, since several sex-specific effects were already found to be more pronounced in males with respect to females [53]. Last but not least, recent data showed that FGR is associated with increased lung miR-126-3p levels, which is known to modulate the expression of angiogenic factor, in rats [54]. The importance of angiogenic regulatory pathways was also highlighted by the abnormal upregulated expression of miR-127, miR-21, and miR-16 in placentas of deceased cloned sheep with respect to controls [55]. These data are extremely fascinating, since miR-21 expression was associated with increased vascular resistance also in growth-restricted human pregnancies [8,56].
As suggested by accumulating evidence, miRNAs play also a pivotal role in epigenetic processes [57,58]. Epigenetic mechanisms include DNA methylation, imprinting, and RNA transcriptional regulation through RNA molecules, such as miRNAs. These processes are influenced by multiple factors: intrauterine nutrient availability (determined by maternal nutrition and placental function) [59][60][61][62], maternal age [63,64], use of drugs [65,66], endocrine disruptors [67], toxins, and infectious agents. For this reason, integrated assessment of early pregnancy should evaluate a combination of biomarkers and ultrasound [68][69][70][71][72][73]. In addition, we take the opportunity to stress how future investigations about miRNA levels in both sera and placentas should evaluate the possible overlapping among preeclampsia, FGR, and gestational diabetes, since they all have in common placental vascular alterations due to angiogenic disbalance [74].
It is however clear that epigenetic information is transmitted, and potentially inherited, across generations through the remodeling of chromatin states. In this regard, selective miRNA expression may be involved in FGR through epigenetic mechanism.

Conclusion
Understanding which miRNAs are associated with the onset/ progression of FGR seems mandatory to improve early diagnosis and management of the disease. In this regard, we take the opportunity to solicit future studies on large cohort and adequate statistical power, in order to identify a panel of biomarkers on maternal peripheral blood for early diagnosis of FGR.