Despite promising findings, quantitative PCR (qPCR)-based tests for RNA quantification have experienced serious limitations in their clinical application. The noticeable lack of technical standardization remains a huge obstacle in the translation of qPCR-based tests. The incorporation of qPCR-based tests into the clinic will benefit from guidelines for clinical research assay validation. This will ultimately impact the clinical management of the patient, including diagnosis, prognosis, prediction, monitoring of the therapeutic response, and evaluation of toxicity. However, clear assay validation protocols for biomarker investigation in clinical trials using molecular assays are currently lacking. Here, we will focus on the necessary steps, including sample acquisition, processing and storage, RNA purification, target selection, assay design, and experimental design, that need to be taken toward the appropriate validation of qRT-PCR assays in clinical research. These recommendations can fill the gap between research use only (RUO) and in vitro diagnostics (IVD). Our contribution provides a tool for basic and clinical research for the development of validated assays in the intermediate steps of biomarker research. These guidelines are based on the current understanding and consensus within the EU-CardioRNA COST Action consortium (www.cardiorna.eu). Their applicability encompasses all clinical areas.

Gestational diabetes mellitus (GDM) is a severe pregnancy complication for both the woman and the child. Women who suffer from GDM have a greater risk of developing Type 2 diabetes mellitus (T2DM) later in life. Identification of any potential biomarkers for the early prediction of gestational diabetes can help prevent the disease in women with a high risk. Studies show microRNA (miRNA) as a potential biomarker for the early discovery of GDM, but there is a lack of clarity as to which miRNAs are consistently altered in GDM. This study aimed to perform a systematic review and meta-analysis to investigate miRNAs associated with GDM by comparing GDM cases with normoglycemic controls. The systematic review was performed according to PRISMA guidelines with searches in PubMed, Web of Science, and ScienceDirect. The primary search resulted in a total of 849 articles, which were screened according to the prior established inclusion and exclusion criteria. Following the screening of articles, the review was based on the inclusion of 35 full-text articles, which were evaluated for risk of bias and estimates of quality, after which data were extracted and relative values for miRNAs were calculated. A meta-analysis was performed for the miRNA species investigated in three or more studies: MiR-29a, miR-330, miR-134, miR-132, miR-16, miR-223, miR-155, miR-122, miR-17, miR-103, miR-125, miR-210, and miR-222. While some miRNAs showed considerable between-study variability, miR-29a, miR-330, miR-134, miR-16, miR-223, and miR-17 showed significant overall upregulation in GDM, while circulating levels of miR-132 and miR-155 were decreased among GDM patients, suggesting further studies of these as biomarkers for early GDM discovery.

Background: Women with polycystic ovary syndrome (PCOS) often change their metabolic profile over time to decrease levels of androgens while often gaining a propensity for the development of the metabolic syndrome. Recent discoveries indicate that microRNAs (miRNAs) play a role in the development of PCOS and constitute potential biomarkers for PCOS. We aimed to identify miRNAs associated with the development of an impaired metabolic profile in women with PCOS, in a follow-up study, compared with women without PCOS. Methods and materials: Clinical measurements of PCOS status and metabolic disease were obtained twice 6 years apart in a cohort of 46 women with PCOS and nine controls. All participants were evaluated for degree of metabolic disease (hypertension, dyslipidemia, central obesity, and impaired glucose tolerance). MiRNA levels were measured using Taqman^® Array cards of 96 pre-selected miRNAs associated with PCOS and/or metabolic disease. Results: Women with PCOS decreased their levels of androgens during follow-up. Twenty-six of the miRNAs were significantly changed in circulation in women with PCOS during the follow-up, and twenty-four of them had decreased, while levels did not change in the control group. Four miRNAs were significantly different at baseline between healthy controls and women with PCOS; miR-103-3p, miR-139-5p, miR-28-3p, and miR-376a-3p, which were decreased in PCOS. After follow-up, miR-28-3p, miR-139-5p, and miR-376a-3p increased in PCOS women to the levels observed in healthy controls. Of these, miR-139-5p correlated with total testosterone levels (rho = 0.50, p_adj = 0.013), while miR-376-3p correlated significantly with the waist-hip ratio at follow-up (rho = 0.43, p_adj = 0.01). Predicted targets of miR-103-3p, miR-139-5p, miR-28-3p, and miR-376a-3p were enriched in pathways associated with Insulin/IGF signaling, interleukin signaling, the GNRH receptor pathways, and other signaling pathways. MiRNAs altered during follow-up in PCOS patients were enriched in pathways related to immune regulation, gonadotropin-releasing hormone signaling, tyrosine kinase signaling, and WNT signaling. Conclusions: These studies indicate that miRNAs associated with PCOS and androgen metabolism overall decrease during a 6-year follow-up, reflecting the phenotypic change in PCOS individuals towards a less hyperandrogenic profile.

In this study, we conducted a systematic review and meta-analysis to summarize and evaluate the global research potential of different circulating miRNAs as an early diagnostic biomarker for OC. A systematic literature search for relevant studies was conducted in June 2020 and followed up in November 2021. The search was conducted in English databases (PubMed, ScienceDirect). The primary search resulted in a total of 1887 articles, which were screened according to the prior established inclusion and exclusion criteria. We identified 44 relevant studies, of which 22 were eligible for the quantitative meta-analysis. Statistical analysis was performed using the Meta-package in Rstudio. Standardized mean differences (SMD) of relative levels between control subjects and OC patients were used to evaluate the differential expression. All studies were quality evaluated using a Newcastle–Ottawa Scale. Based on the meta-analysis, nine miRNAs were identified as dysregulated in OC patients compared to controls. Nine were upregulated in OC patients compared to controls (miR-21, -125, -141, -145, -205, -328, -200a, -200b, -200c). Furthermore, miR-26, -93, -106 and -200a were analyzed, but did not present an overall significant difference between OC patients and controls. These observations should be considered when performing future studies of circulating miRNAs in relation to OC: sufficient size of clinical cohorts, development of consensus guidelines for circulating miRNA measurements, and coverage of previously reported miRNAs.

Obesity is an important risk factor for cardiovascular disease and type 2 diabetes mellitus. Even a modest weight loss of 5–15% improves metabolic health, but circulating markers to indicate weight loss efficiency are lacking. MicroRNAs, small non-coding post-transcriptional regulators of gene expression, are secreted from tissues into the circulation and may be potential biomarkers for metabolic health. However, it is not known which specific microRNA species are reproducibly changed in levels by weight loss. In this study, we performed a systematic review and meta-analysis to investigate the microRNAs associated with weight loss by comparing baseline to follow-up levels following intervention-driven weight loss. This systematic review was performed according to the PRISMA guidelines with searches in PubMed and SCOPUS. The primary search resulted in a total of 697 articles, which were screened according to the prior established inclusion and exclusion criteria. Following the screening of articles, the review was based on the inclusion of 27 full-text articles, which were evaluated for quality and the risk of bias. We performed systematic data extraction, whereafter the relative values for miRNAs were calculated. A meta-analysis was performed for the miRNA species investigated in three or more studies: miR-26a, miR-126, and miR-223 were overall significantly increased following weight loss, while miR-142 was significantly decreased after weight loss. miR-221, miR-140, miR-122, and miR-146 were not significantly changed by intervention-driven weight loss. These results indicate that few miRNAs are significantly changed during weight loss.

Aim: Ovarian cancer (OC) is the leading cause of gynecological cancer deaths. Current biomarkers of OC are not specific or sensitive enough. Extracellular vesicles (EVs), EV surface proteins and their cargo microRNA (miRNA) show potential as biomarkers. This study aimed to characterize the ability of EVs to identify early OC-biomarkers among blood donors six months before their diagnosis. Methods: Study groups of OC patients, benign tumor patients (B), healthy blood donors (Control), and blood donors with incident OC diagnosis within six months of the last blood draw (Pre-diagnostic; PD) were established. Small EVs were enriched from plasma using ultracentrifugation. EVs were characterized by Dynamic Light Scattering (DLS), EV Array, NanoFlow Cytometry, Nanoparticle Tracking Analysis, and Western blots. RNA from EVs was isolated. A discovery study was performed on OC and B patients using the TaqMan Array Human MicroRNA A card. A validation study of 9 specific miRNAs was performed using RT-qPCR. Results: With DLS, it was identified that the OC patients’ EVs were more heterogeneous in size compared to the other groups. Western blot identified CD63 and TSG101 in the EV enrichments. EV Array assessed 22 known protein biomarkers. TaqMan MicroRNA Array cards indicated a differential miRNA abundance between OC and B; however, technical replication and validation could not validate this pattern. Conclusion: This study has analyzed EVs in OC, B, Control, and PD women. More extensive investigations of EV CD9, CD151, and CD81 in conjunction with other risk factors and well-known biomarkers like CA125 or HE4 should be the main objectives of future research.