Circular RNAs in Pregnancy and the Placenta

The emerging field of circular RNAs (circRNAs) has identified their novel roles in the development and function of many cancers and inspired the interest of many researchers. circRNAs are also found throughout the healthy body, as well as in other pathological states, but while research into the function and abundance of circRNAs has progressed, our overall understanding of these molecules remains primitive. Importantly, recent studies are elucidating new roles for circRNAs in pregnancy, particularly in the placenta. Given that many of the genes responsible for circRNA production in cancer are also highly expressed in the placenta, it is likely that the same genes act in the production of circRNAs in the placenta. Furthermore, placental development can be referred to as ‘controlled cancer’, as it shares many key signalling pathways and hallmarks with tumour growth and metastasis. Hence, the roles of circRNAs in this field are important to study with respect to pregnancy success but also may provide novel insights for cancer progression. This review illuminates the known roles of circRNAs in pregnancy and the placenta, as well as demonstrating differential placental expressions of circRNAs between complicated and uncomplicated pregnancies.


Introduction
The placenta, a product of conception with a transient existence, uniquely supports pregnancy. It plays a critical role in nutrient, waste and gas exchange between the mother and fetus. Correct placentation underpins fetal development, as well as coordinating maternal adaptations to pregnancy to maintain maternal and fetal health. In pregnancy complications characterised by aberrant placentation such as preeclampsia (PE) [1] and intrauterine growth restriction [2], there is an altered placental transcriptome. Emerging evidence demonstrates the roles of novel RNA species in pregnancy complications, particularly circular RNAs (circRNAs).
The first identified circRNA, the hepatitis D viroid, was reported in 1977 [3]. Following this, circRNAs were found in mammalian cells using electron microscopy in 1979 [4]. Initially, due to their low abundance, they were disregarded as products of misplicing. Then, in 1991, circular transcripts were found in a variety of normal and neoplastic human cells [5]. As technology improved, primarily in sequencing capabilities and bioinformatics, so has the study of the structure and functionality of circRNAs [6].
Many circRNAs have evaded detection until now for two main reasons. Unlike other small RNAs, circRNAs are not able to be easily separated from mRNA through size fractionation or electrophoretic mobility as often they differ from their linear form only in circular structure. They are also easily destroyed by molecular techniques requiring amplification or fractionation due to their circular form and, as they lack polyadenylation, they are often discarded when analysing sequencing data [7]. These covalently closed circular RNA structures remain enigmatic, with a plethora of reported functions and methods of biogenesis. This review will detail what is currently known about circRNAs, their implications for placental development and function, and their broader consequences for pregnancy. It has been shown that the biogenesis of circRNAs, while via backsplicing, still in volves canonical splicing signals and spliceosomal mechanisms [12]. The experimenta use of isoginkgetin, a splicing inhibitor, inhibits the formation of circRNAs, as well a linear RNAs [14,15]. Moreover, mutations in the canonical splicing sites of exons inhib circularisation and circRNA biogenesis [16][17][18]. The biogenesis of circRNAs is in constan competition with linear RNA production through canonical splicing machinery [15]. It ha been shown that the elongation velocity of RNA polymerase II positively correlates wit It has been shown that the biogenesis of circRNAs, while via backsplicing, still involves canonical splicing signals and spliceosomal mechanisms [12]. The experimental use of isoginkgetin, a splicing inhibitor, inhibits the formation of circRNAs, as well as linear RNAs [14,15]. Moreover, mutations in the canonical splicing sites of exons inhibit circularisation and circRNA biogenesis [16][17][18]. The biogenesis of circRNAs is in constant competition with linear RNA production through canonical splicing machinery [15]. It has been shown that the elongation velocity of RNA polymerase II positively correlates with backsplicing efficiency [19]. This has been corroborated by several studies in which mutations in the RNA polymerase II large subunit significantly reduced RNA pol II elongation velocity, and thus, backsplicing efficiency and circRNA production [20][21][22].
There are several ways in which circRNAs can be produced ( Figure 2). Complementary base-pairing ( Figure 2A like structure which can then be cleaved to form a circRNA. This structure promotes spatial reduction in splice signals required for backsplicing and thus contributes to RNA circularization [23,24]. Specifically, Jeck et al. [25] first reported on the importance of inverted ALU repeat elements in backsplice-flanking introns in facilitating circRNA biogenesis. ALU repeats are short nucleotide sequence repeats that comprise approximately 11% of the genome and are primate-specific [26]. These inverted ALU repeats are five times more enriched in sites of human exonic circRNAs formation. There are also examples of exonic circRNAs where the entire gene is circularised and no upstream or downstream exons are leftover for alternatively spliced transcript production, such as SRY, the male sex-determining gene found on the Y chromosome, which abundantly produces circRNAs [27].
backsplicing efficiency [19]. This has been corroborated by several studies in which tations in the RNA polymerase II large subunit significantly reduced RNA pol II elo tion velocity, and thus, backsplicing efficiency and circRNA production [20][21][22].
There are several ways in which circRNAs can be produced ( Figure 2). Comple tary base-pairing ( Figure 2A) occurs when complementary inverted sequences in in flanking backsplice junctions facilitate circularisation by base-pairing to form a stemlike structure which can then be cleaved to form a circRNA. This structure promotes tial reduction in splice signals required for backsplicing and thus contributes to RNA cularization [23,24]. Specifically, Jeck et al. [25] first reported on the importance of inv ALU repeat elements in backsplice-flanking introns in facilitating circRNA biogen ALU repeats are short nucleotide sequence repeats that comprise approximately 11 the genome and are primate-specific [26]. These inverted ALU repeats are five times enriched in sites of human exonic circRNAs formation. There are also examples of e circRNAs where the entire gene is circularised and no upstream or downstream exon leftover for alternatively spliced transcript production, such as SRY, the male sex-d mining gene found on the Y chromosome, which abundantly produces circRNAs [2 Complementary base-pairing (e.g., via Alu repeats) promotes backsp due to spatial reduction in the splice sites. (B) RBP-driven circularisation occurs when RBPs flanking introns and bridge them together for splicing. (C) ciRNA formation: ciRNAs are fo from lariat introns that escape debranching. C-rich (red) and GU-rich (blue) sequence bind sufficient for the intron to avoid debranching and generate a ciRNA. (D) The lariat-driven mo circularisation. Exon-skipping occurs to bring splice sites into close proximity. Complementary base-pairing (e.g., via Alu repeats) promotes backsplicing due to spatial reduction in the splice sites. (B) RBP-driven circularisation occurs when RBPs bind flanking introns and bridge them together for splicing. (C) ciRNA formation: ciRNAs are formed from lariat introns that escape debranching. C-rich (red) and GU-rich (blue) sequence binding is sufficient for the intron to avoid debranching and generate a ciRNA. (D) The lariat-driven model of circularisation. Exon-skipping occurs to bring splice sites into close proximity.
Certain RNA binding proteins (RBPs) are also able to facilitate RNA circularisation ( Figure 2B). For example, the RBP Quaking (QKI), which is highly expressed in the placenta, aids the biogenesis of circRNAs which are involved in epithelial-mesenchymal transition (EMT), a process common to placental development and many cancers. Furthermore, QKI knockdown subsequently inhibits the production of EMT-related circRNAs [18]. However, for correct functioning QKI requires the assistance of binding sites in introns flanking the exons to facilitate circRNA biogenesis [18]. Alternatively, the RBP Muscleblind (MBL), which is also highly expressed in the placenta, facilitates the biogenesis of the circRNA (circMbl) from its own cognate RNA by binding to specific MBL conserved sites in flanking introns [15]. circRNAs can also be formed from RNA lariats (lasso-shaped by-products of RNA splicing), termed circular intronic RNAs (ciRNAs). Distinct from exonic circRNAs, which feature a 3 -5 carbon linkage at the splicing branchpoint, lariat RNAs feature 2 -5 linkages [23]. They can be formed utilising a consensus motif with a GU-rich region, located near the 5 splicing point, and a C-rich region, near the branchpoint site in ciRNA-producing introns, which allow for intron lariat escape from debranching. These regions then facilitate the circularisation of this intron [28,29] ( Figure 2C) and the 3 'tail' downstream from the branch point is trimmed to stabilise the ciRNA and protect from exonucleases. This motif is not enriched in regular introns [23] and has been suggested as an essential RNA element to expedite intron lariat escape from debranching.
The lariat-driven model of circularisation ( Figure 2D) can encompass a variety of the above techniques for circRNA biogenesis. Middle exons of a linear transcript are 'skipped' to allow an upstream 3 splice donor to covalently bond to a downstream 5 splice acceptor. The spliceosome then removes the introns to form the final circRNA product.

circRNA Function
There are several functions for circRNAs that have been identified to date. A small number of circRNAs are able to be translated ( Figure 3A) (e.g., the Hepatitis δ agent, a circular RNA satellite virus of the Hepatitis B virus [30]) while engineered circRNAs can undergo translation if an internal ribosomal entry site (IRES) is included in the design [7]. However, the majority of circRNAs appear to be non-coding.
Certain RNA binding proteins (RBPs) are also able to facilitate RNA circularisa ( Figure 2B). For example, the RBP Quaking (QKI), which is highly expressed in the centa, aids the biogenesis of circRNAs which are involved in epithelial-mesenchymal tr sition (EMT), a process common to placental development and many cancers. Furth more, QKI knockdown subsequently inhibits the production of EMT-related circRN [18]. However, for correct functioning QKI requires the assistance of binding sites in trons flanking the exons to facilitate circRNA biogenesis [18]. Alternatively, the RBP M cleblind (MBL), which is also highly expressed in the placenta, facilitates the biogenesi the circRNA (circMbl) from its own cognate RNA by binding to specific MBL conser sites in flanking introns [15]. circRNAs can also be formed from RNA lariats (lasso-shaped by-products of R splicing), termed circular intronic RNAs (ciRNAs). Distinct from exonic circRNAs, wh feature a 3′-5′ carbon linkage at the splicing branchpoint, lariat RNAs feature 2′-5′ linka [23]. They can be formed utilising a consensus motif with a GU-rich region, located n the 5′ splicing point, and a C-rich region, near the branchpoint site in ciRNA-produc introns, which allow for intron lariat escape from debranching. These regions then fa tate the circularisation of this intron [28,29] ( Figure 2C) and the 3′ 'tail' downstream fr the branch point is trimmed to stabilise the ciRNA and protect from exonucleases. T motif is not enriched in regular introns [23] and has been suggested as an essential R element to expedite intron lariat escape from debranching.
The lariat-driven model of circularisation ( Figure 2D) can encompass a variety of above techniques for circRNA biogenesis. Middle exons of a linear transcript are 'skipp to allow an upstream 3′ splice donor to covalently bond to a downstream 5′ splice acc tor. The spliceosome then removes the introns to form the final circRNA product.

circRNA Function
There are several functions for circRNAs that have been identified to date. A sm number of circRNAs are able to be translated ( Figure 3A) (e.g., the Hepatitis δ agen circular RNA satellite virus of the Hepatitis B virus [30]) while engineered circRNAs undergo translation if an internal ribosomal entry site (IRES) is included in the design However, the majority of circRNAs appear to be non-coding.  Some specific, highly expressed circRNAs function as miRNA sponges ( Figure 3B). The exonic circRNAs from CDR1as [31], cerebellum-related antigen 1, and SRY [12], the testis-determining factor, have been shown to bind miRNAs without degrading them, inhibiting their function. Each of these circRNAs also has multiple miRNA binding sites in its sequence. The circRNA for CDR1as has 74 confirmed sites for miR-7 binding, as well as being densely seeded with Argonaute protein binding sites which allow for Argonaute-miRNA complexes to bind. The circRNA for SRY has 16 binding sites for miR-138 and coprecipitates with Argonaute 2. However, the concept that circRNAs act as miRNA sponges has recently been debated.
Whilst it is true that some circRNAs function efficiently as miRNA sponges, such as ciRS-7 [12], the notion of circRNAs functioning as sponges has been questioned due to the stoichiometric ratio of circRNA to miRNA molecules within the cell [32]. Given that the majority of circRNAs are produced at less than 2.5 copies per cell [18], it is improbable that they are able to significantly regulate the expression of miRNAs, which are often produced at 900-80,000 copies per cell [33]. The potential for circRNAs to mediate miRNA expression is likely to be reserved only for circRNAs with unusually high expression within cells, and multiple miRNA binding sites per molecule. Thus, new studies to examine the potential function of circRNAs as miRNA sponges may need to seek further validation through experiments that involve more than dual luciferase assays. However, this is not to suggest that many circRNAs do not have important cellular functions. As the majority of circRNAs are produced at~2.5 copies per cell, this indicates an approximate 1:1 ratio with the DNA transcripts in each cell. Indeed, interaction with DNA is another function of circRNAs that has important implications in molecular biology (this is explored further below).
circRNAs can also function as transcriptional regulators, termed "mRNA traps" ( Figure 3C). One example of this is the exonic circRNA produced from the Fmn (flavin mononucleotide) gene in mice, which is proposed to sequester the translation start site on the mRNA, reducing protein synthesis [34]. circRNAs can also bind proteins ( Figure 3D), as previously mentioned, circMbl can sequester the Muscleblind RBP [15]. Furthermore, cir-cANRIL, a circRNA in the antisense non-coding RNA in the INK4 locus (ANRIL) long noncoding RNA, regulates the maturation of precursor ribosomal RNA, therefore controlling ribosome biogenesis [15]. circRNAs have also been shown to facilitate the phosphorylation, ubiquitylation and acetylation [15] of proteins ( Figure 3E), and participate as structural components of protein complexes [15]. Importantly, circRNAs have been shown to bind to, and facilitate breakages in DNA (see below) ( Figure 3F). circRNAs have also been reported to recruit proteins to specific subcellular loci [15] and influence host transcript promoter regions ( Figure 3G). With their many attributed functions, it is no surprise that circRNAs have been implicated to play a role in many pathophysiological and physiological states; this review will focus on their role in pregnancy.

The Role of circRNAs in Pregnancy
circRNAs are expressed throughout reproductive tissues in healthy pregnancy and are differentially expressed between healthy and complicated pregnancy. However, the question of whether this is cause or effect requires further research. Studies that have examined circRNAs expressed in reproductive tissues and detected in maternal serum in pregnancy are limited and summarised in Table 1. Table 1. Summary of research on circRNAs in female reproductive tissues and blood during pregnancy (limited to studies using primary tissue).

Author
Year

Tissue Pregnancy Status Key Findings Limitations
Fan X, et al. [35] 2015 Mouse-oocytes and preimplantation embryos Uncomplicated (assumed) Detected 2891 circRNAs from 1316 host genes. A majority of these circRNAs are unique to the preimplantation stage and a large proportion of them exhibit dynamic expression patterns during this developmental process.
Sequencing using the SUPeR-seq method could possibly limit depth of circRNA sequencing due to no enrichment using RNase R.
Szabo L, et al. [36] 2015 Fetal tissue-unspecified, various species Uncomplicated (assumed) Developed an algorithm to compare established data sets in human, rat and mouse tissue and cell lines, with their generated circRNA data from RNA-seq on fetal tissue.
This study assumes a degree of conservation between species.
Zhang YG, et al. [37] 2016 Human maternal red blood cells PE vs. uncomplicated The levels of circ_101222 in red blood cells of patients with PE were significantly higher than in healthy women. Using ENG in combination with circ_101222 improved confidence for the prediction of PE.
Qian Y, et al. [38] 2016 Human placenta PE vs. preterm birth (PTB) 143 circRNAs were up-regulated and 158 were down-regulated in PE samples compared with preterm.
Use of microarray is not as comprehensive as sequencing techniques. PTB placenta is used as a gestational age control but PTB can occur for multiple reasons and is a pathology of pregnancy.
Caution on interpretation is required. Use of microarray is not as thorough as sequencing techniques. Significant differences in BMI, gestational age at delivery and % caesarean sections between severe PE and control groups. Circ_0005243 was identified using RNase R (determining circular form). Circ_0005243 expression was downregulated in placenta and maternal plasma in GDM. Knockdown of circ_0005243 in HTR-8/SVneo cells suppressed cell proliferation and migration and increased secretion of inflammatory factors (TNF-α and IL-6). It also reduced β-catenin expression and increased nuclear NF-κB p65 nuclear translocation.
Zhang Y, et al. [53] 2020 Human placenta and in vivo rat model PE vs. uncomplicated CircSFXN1 was identified using RNase R (determining circular form). CircSFXN1 was elevated in PE placenta. Knockdown of circSFXN1 promoted TEV-1 cell invasion and HUVEC angiogenesis-this effect was opposed with circSFXN1 overexpression. Pregnant rats injected with sFLT1-expressing adenovirus had in increased blood pressure and proteinuria; si-circSFXN1 reversed this. CircSFXN1 recruits sFLT1, validated by RNA-protein pulldown, RNA immunoprecipitation and dual-luciferase reporter assays.
Use of microarray is not as thorough as sequencing techniques, although the study validates these results with qRT-PCR.   CircVEGFC regulates glucose metabolism-higher incidence of GDM in patients with high circVEGFC levels. Elevated circVEGFC levels in GDM plasma. High circVEGFC level group showed higher incidence rates of fetal malformation and hypertension.
Correlation established but further mechanistic studies required to establish whether this is causative.
Zou H, et al. [75] 2022 Human placenta PE vs. uncomplicated Circ_0037078 is upregulated in PE placentae. Knockdown of circ_0037078 increases trophoblast cell proliferation, migration, invasion and angiogenesis. Circ_0037078 also sponges miR-576-5p and increases IL1RAP expression. In PE placentae, circHIPK3 and KCMF1 were downregulated and miR-346 was upregulated. CircHIPK3 overexpression promotes trophoblast cell proliferation, migration and invasion, as well as decreasing cell cycle arrest and apoptosis. CircHIPK3 also targets miR-346 and regulates KCMF1 expression.
Zhang Y, et al. [83] 2021 Human placenta and maternal plasma PE vs. uncomplicated PE predictive power was greatest when plasma sFLT1 and circBRAP levels were combined with uterine pulsatility index. CircBRAP was increased in PE placentae and may regulate miR-106b to decrease TEV-1 cell proliferation, invasion and apoptosis. The paper uses publicly available datasets instead of completing their own sequencing. As such, data quality cannot be ascertained. No luciferase assays to validate the potential axes listed.
In animals, research conducted in murine models has described circRNA profiles in oocytes and pre-implantation embryos [35] and in both implantation and inter-implantation sites in the endometrium [41]. The sizable differences in the profiles between these cells and tissues indicate that circRNAs play a role in the reproductive process. Interestingly, one study was completed in both in vitro cell lines and in vivo rat experiments to demonstrate the effect of circSFXN1 (sideroflexin 1) in PE pathology [53]. sFLT1-expressing adenovirus injections into rats induced a PE-like phenotype, which was abated by treatments with si-circSFXN1. This clearly demonstrates the pathological potential for aberrant circRNA expression. Another study examined atretic follicles in porcine ovaries, determining that a circSLC41A1-miR-9820-5p-SRSF1 axis regulates follicular granulosa cell apoptosis [85].

circRNAs in Gestational Diabetes Mellitus
Studies on placental circRNA expression in GDM focused mainly on profiling differences between GDM and uncomplicated pregnancies. In one study, first and early second-trimester maternal blood samples were collected to compare circRNA differential expression. These measures were then used to determine possible circRNA predictors for GDM development [50]. Other studies showed that circ_0008285 [60], circ_0026497 [71], circ_0039480 [71], circ-PNPT1 [63] and circVEGFC [74] were elevated in maternal plasma and whole blood from women with GDM. In vitro experiments using high glucose media for HTR-8/SVneo cell culture promoted proliferation and migration, which was reversed with circ_0008285 knockdown. Similarly, high glucose-induced arrest of cell viability and migration was reversed upon circ-PNPT1 knockdown. High levels of circVEGFC occurred with higher incidence rates of fetal malformation and hypertension. circ_0074673 [69] was upregulated in exosomes isolated from umbilical cord blood of GDM cases.
In contrast, other studies showed that circ_0001173 [60], circ_0005243 [52] and circ_102682 [73] were downregulated in placentae and maternal plasma from women with GDM. In vitro knockdown of circ_0005243 in HTR-8/SVneo trophoblast cells suppressed cell proliferation and migration, while circ_0001173 levels were positively correlated with glycated haemoglobin.

circRNAs in Other Pregnancy Complications
Other pregnancy complications have also been briefly studied with respect to cir-cRNAs. One study reported almost 600 differentially expressed circRNAs in placentae from women with recurrent spontaneous abortion (RSA) compared with uncomplicated pregnancy [39]. Another study observed that circ_0050703 was downregulated in the placental villous tissue of patients with unexplained RSA (URSA), and circ_0050703 silencing in vivo reduced the number of successfully implanted embryos [64]. A circFOXP1/miR-143-3p/S100A11 axis was suggested in the RSA placentae [72]. Furthermore, circ-SETD2 was implicated in placental growth, with elevated circ-SETD2 in placentae of patients with fetal macrosomia [51]. In vivo overexpression experiments in HTR-8/SVneo cells showed increased cell proliferation and invasion. A circ_0074371/miR-582-3p/LRP6 axis was suggested in the context of fetal growth restriction [65]. Finally, granulosa cells from non-pregnant advanced age (≥38 years) compared with young age (≤30 years) women determined different circRNAs expression profiles depending on maternal age [40]. Whilst the number of studies into circRNAs in pregnancy is low, clearly circRNAs play many roles in pregnancy health, waiting to be discovered.

Limitations of circRNA Research
Research surrounding circRNAs in pregnancy is certainly still in its infancy. Several studies (Table 1) reported only the results of their profiling without any qPCR validation in independent samples. Whilst these data could be useful to other researchers, many of these profiling techniques have now been superseded with novel technologies. Methods such as RPAD [86], along with the use of Li + ions in reaction buffers [87], in RNA-sequencing (RNA-seq) are proving much more reliable than the outdated circRNA arrays. circRNA detection through RNA-seq can be accurate assuming that one of these above methods is employed to prepare the samples. Importantly, many of the studies described in this review lacked RNase R enrichment prior to sequencing. The addition of this exonuclease to a sample results in the digestion of all linear RNAs present, leaving an enriched population of circRNA transcripts. Not using this treatment prior to sequencing means that the depth of sequencing for circRNAs will be limited given that the sample is still composed primarily of linear RNA products. This likely results in the detection of only the most highly expressed circRNAs, leaving many transcripts undetected.
Finally, in studies using delivered placentae, authors should be cautious to declare specific circRNAs as causes of a pregnancy state. Given that these placentae have been collected after birth, without further mechanistic studies, it is unclear whether circRNAs in disease states are causal or an effect. However, these few studies provide enticing evidence to inspire further research into circRNAs in pregnancy and placental growth.

The Potential Importance of circRNAs in the Placenta: What We Can Apply from Our Knowledge of Cancer
The placenta can be considered a 'controlled cancer', as there are many parallels that have been previously drawn between placental development and cancer metastasis [88][89][90]. Some basic principles of cancer progression include tumour growth and tissue invasiveness (through epithelial to mesenchymal transition) [91], immune evasion and stimulation of angiogenesis [92], all of which are essential for successful placentation [93,94]. It is therefore unsurprising that many of the key molecular pathways are common to both placental development and cancer (Table 2). Importantly, the extensive research demand in the field of cancer has yielded a wealth of information about the molecular biology of cancers that can also be applied to the study of the placenta due to their many similarities. Extensive mRNA profiling has been undertaken in placentae from the first trimester, second trimester and term [120][121][122][123]. Utilising this dataset shows that many of the genes responsible for circRNA production in cancer are also highly expressed in the placenta [124]. Hence, it is likely that circRNAs are also produced from these genes in the placenta. For example; the circRNA produced from MYLK has been shown to interfere with VEGFA signalling in bladder cancer [125]. The importance of the VEGF signalling pathway is well established in placentation [107], being most important for early angiogenesis and maintaining vascular health in the mother. Conversely, MYLK expression in the placenta increases across gestation. It is highly likely that circRNAs from the MYLK gene are also produced in the placenta and could impact the VEGF signalling pathway. There appear to be endless examples of genes which are known to produce circRNAs in cancer that are relevant to, and highly expressed in, placental development. We are currently profiling several of these circRNAs in the placenta and examining their roles in its development.
Importantly, circRNAs are not only able to affect development through their interactions with other molecules but they can also facilitate genomic instability through translocations. Rapid proliferation and consequent replication stress are common [126], both in cancer and in the placenta, resulting in possible DNA damage. Evidence has been provided for R-loop formation in plants, where a circRNA forms an RNA:DNA hybrid with its cognate DNA locus, stalling transcription and resulting in DNA breaks [127]. This was also shown to coincide with the recruitment of splicing factors, as well as alternative splicing. This genomic manipulation by circRNAs is likely to also occur in the eukaryotic tissues, although this is yet to be confirmed. If this is the case, dysregulation of circRNAs, particularly in early gestation, could result in genomic alterations that could affect both placental and fetal development and pregnancy health in general.
circRNAs have been shown to accumulate in a number of different tissues over time [128,129] and have been suggested to be a marker of tissue ageing. As the placenta is also known to undergo ageing [130], it is possible that circRNA accumulation in the placenta could occur. The implications of circRNA accumulation are still not well understood, but if these circRNAs continue to exert their functions as they accumulate this could lead to exaggerated circRNA action in the tissue.

Conclusions
Understanding the functions of circRNAs, particularly their involvement in placental development and pregnancy health, is in its infancy. However, these unique molecules are evidently the result of careful regulation, with multiple roles in physiological and pathophysiological conditions. Evidence that circRNAs may be involved in regulating placental development, and that differential circRNA profiles are found between healthy and complicated pregnancies, provides an imperative for further research.