Recent Updates on microRNA as Potential Biomarkers for Diagnosis of Gestational Diabetes Mellitus

AbstractAbstractAbstractAbstract Background/Aims:Background/Aims:Background/Aims:Background/Aims:Background/Aims: Screening for gestational diabetes mellitus (GDM) are currently done at 24 - 28 weeks of conception, missing out on the most vulnerable period of organogenesis and thus preventing clinicians from starting treatments until the late second or third trimester. MicroRNAs (miR) are small non-coding RNA molecules that could aid in detecting or predicting GDM through establishing a novel non-invasive prenatal testing (NIPT) tool. The objective of this study was to summarize the most recent updates on plasma microRNAs as GDM diagnostic biomarkers. Methods:Methods:Methods:Methods: Methods: Between April and June 2021, a PubMed literature search was undertaken to review recent articles on human plasma miR associated with GDM. Animal studies and papers that are written in languages other than English were excluded. Only plasma miRNAs were used to avoid coagulation biases. Results:Results:Results:Results:Results: A total of 31 miRNAs were found significantly upregulated in the plasma samples of patients with GDM. It was found mainly during the 2nd or 3rd trimester except for miR-223 and miR-23a that were upregulated at 9 – 11 weeks of gestation. Conclusion:Conclusion:Conclusion:Conclusion:Conclusion: Though extensive prospective cohort studies are required, miR-223 and miR-23a should be considered the most promising to develop a successful NIPT tool because they were found to be upregulated earliest, during the first trimester.


Introduction
Gestational diabetes mellitus (GDM), a medical condition with increasing incidence worldwide, affects 7% of all pregnancies 1 . Moreover, implementing the new International Association of Diabetes and Pregnancy Study Groups (IADPSG) criteria compared to prior GDM criteria resulted in a 75% increase in the number of women with GDM who had evidence of heterogeneity. 2 GDM is defined as any degree of glucose intolerance that begins or is first recognized during pregnancy and is associated with many complications, including infant macrosomia, hypoglycemia, and an increased risk of type 2 diabetes (T2D) development in both the mother and the infant. 3,4 GDM can be divided into two types: A1GDM and A2GDM.
The A1GDM can be managed without medications and responds to nutritional therapy, while A2GDM must be handled with medications to achieve adequate glycemic control. 4 At present, screening of GDM is usually done in a single step through 75 g OGTT at 24 -28 weeks of gestation, which means that treatments cannot begin until the late second or third trimester, hence carries a high risk of maternal and fetal morbidity and mortality. 1,5 As a result, early screening in the first or early second trimester of pregnancy is crucial to promptly establish an adequate therapy that normalizes blood glucose levels, reducing GDM adverse pregnancy outcomes 1 .
It should be emphasized that miR are released from intact cells or platelets during RNA extraction, so the use of plasma rather than serum to prevent coagulation biases is preferred 12 . Therefore, the objectives of this study were to find out the recent updates of plasma miR as GDM diagnostic biomarkers, the expression of those miR throughout pregnancy, and the specific correlation between those miR and known GDM risk factors.

Data Sources and Search Strategies
A PubMed literature search was conducted between April and June 2021. Different types of research were studied for this project, including original articles, systematic reviews, meta-analyses, reviews, and randomized controlled trials. Animal studies and articles written in a language other than English were excluded. We have filtered the search to recently published articles, between 2015 and 2021. The search was not limited to a particular population or ethnicity or a specific category of age. The following keywords in various combinations were employed to search the literature: "gestational diabetes", "diabetes mellitus", "type 2 diabetes", "diabetes in pregnancy", "glucose tolerance test", "early pregnancy screening", "pregnancy biomarker", "microRNA". Furthermore, the reference lists of the chosen publications and related reviews were manually searched.

Data Extraction and Analysis
One hundred twenty-one articles initially appeared from the PubMed search. Three reviewers analyzed the search results by reading the titles, abstracts, and objectives. Disagreements were solved through discussions, and the overall records identified by common acceptance were then narrowed down based on inclusion criteria. The articles selected were the ones with the objective of using miR as a diagnostic tool for diabetes in pregnancy. The data extracted included the GDM diagnostic criteria, adjustments made, pregnancy outcomes, study design, country, and the number of study participants.

Discussions
Biogenesis of miRNAs: miRs are non-coding RNA molecules with an approximate length of 19-22 nucleotides. miRs are primarily transcribed by RNA polymerase II, resulting in a primary miRNA (called pri-miRNA) with a length of 500-3000 nucleotides. A microprocessor complex then cleaves the pri-miRNA into a premature miR (called pre-miRNA), which is around 70-80 nucleotides long. Furthermore, pre-miRNA is transported into the cytoplasm by exportin 5, a nuclear export transporter that processes a 22-nucleotide "miRNA duplex" by engaging with the RNase III endonuclease dicer protein and a cofactor which is an RNA binding protein having doublestrands with responsiveness to transactivation. After binding to an argonaute and glycine tryptophan repeatcontaining protein, the miRNA duplex is integrated into an "RNA-induced silencing complex," where it attaches to partial or full-complementary sequences in the 3' untranslated region (3'-UTR) or 5'-UTR of the target mRNA, and thus participates in the regulatory gene expression procedure 7,12 . The process of miR biogenesis is displayed as a schematic in Figure 1. MicroRNAs play an essential role in regulating the physiology of insulin-producing â-cells. They contribute not only to the differentiation of these cells but also to developing their distinct secretory properties. Furthermore, miR assists â-cells in adapting to increasing insulin demand, and specific microRNAs, when produced in excess, can cause cellular dysfunction, and hasten the development of certain kinds of diabetes mellitus 13 . Several researchers have looked at the expression of circulating microRNAs (plasma/serum) in diabetes to see if they may be used as early indicators for this group of chronic metabolic illnesses and their role in the pathogenetic pathways 14 . Previously, the expression of circulating microRNAs in type 1 and type 2 diabetes was discussed in some articles. Still, only a limited number of studies investigated plasma microRNAs as a candidate biomarker of GDM.

Expression of miR in gestational diabetes:
Zhu et al used the next-generation sequencing technique to examine microRNA expression in the plasma of 10 patients with GDM and 10 controls without GDM at the 16th-19th week of pregnancy 15 . As a result, they discovered upregulated microRNA signature in GDM patients comprised of five microRNAs, namely miR-16-5p, miR-17-5p, miR-19a-3p, miR-19b-3p, and miR-20a-5p. The microRNAs discovered by them have been linked to the different signaling pathways implicated in insulin secretion, for instance, the MAPK, insulin, TGF, and mTOR signaling pathways.

Expression of miR in other gestational conditions:
It is to be noted here that miR-16-5p is highly expressed and enriched in erythrocytes, so its expression level may vary during sample hemolysis. This characteristic makes measuring circulating miR-16-5p expression levels to monitor the progression of GDM potentially misleading, hence cannot be used as a reliable biomarker, though having a potentially pivotal role in the pathophysiology of GDM. The involvement of the miR-17-miR-20b microRNA family in smooth muscle cell proliferation has previously been reported, potentially indicating a specific part for these microRNAs in diabetic vascular complications. Moreover, a prior study linked miR-17 and miR-20b to preeclampsia that impairs perinatal outcomes and is substantially correlated with GDM in terms of severity of glucose intolerance and its risk factors. Due to the reported overlaps of various pathologic conditions during pregnancy, it is not unlikely that diverse pregnancy complications, like preeclampsia, may share similar circulating microRNA changes 12 .
The development of novel analytical methodologies is a step forward. For example, next-generation sequencing (NGS) is a novel tool for the identification as well as absolute quantification of RNA. Significantly, new cDNA library preparation chemistries will enable researchers to work with as small as 1ng of plasma 12 . It was observed in several pieces of research that preanalytical conditions such as methods of blood sampling and lack of standardized collection as well as processing techniques of blood samples may have a significant impact on the stability of microRNA, resulting in their variances in expression.

Clinical Implications
Regarding GDM, limited information is available on the expression of circulating plasma microRNAs. The findings of the preceding studies point to some potential candidates for circulating biomarkers for GDM. Developing a NIPT tool, based on miRNA to detect or predict GDM during the embryonic and early fetal period of pregnancy should be considered as an additional measure to prevent associated congenital anomalies.

Future Directions
Additional research is needed to understand the physiological and pathological patterns of expression of these molecules during pregnancy. Furthermore, to establish miR as an early GDM biomarker, standard operating procedures (SOPs) for collecting plasma, sample processing to extract RNA, quality control evaluation, measuring circulating microRNAs, and analyzing their expression profile must be characterized.

Conclusion
GDM reduces pancreatic-cell expansion during pregnancy, and screening for GDM is usually done at 24-28 weeks of gestation. This poses a risk to fetal health, and clinicians miss an opportunity to intervene and manage GDM in a timely manner. According to the available research, some specific miRNAs can be used as a potential biomarker for early diagnosis, as discussed above. Even though there are numerous preanalytical and analytical challenges for clinical use of plasma miRNAs, standardization of these methods may aid in reproducibility among studies. Large prospective cohort studies are needed to determine the diagnostic and or prognostic potential of plasma miR.