Diagnostic Role of Cell-Free miRNAs in Identifying Placenta Accreta Spectrum during First-Trimester Screening.

Placenta accreta spectrum (PAS) is a severe complication of pregnancy associated with excessive invasion of cytotrophoblast cells at the sites of the endometrial-myometrial interface and the myometrium itself in cases of adherent (creta) and invasive (increta and percreta) forms, respectively. This leads to a high risk of massive blood loss, maternal hysterectomy, and preterm birth. Despite advancements in ultrasound protocols and found associations of alpha-fetoprotein, PAPP-A, hCG, PLGF, sFlt-1, IL-8, and IL-33 peripheral blood levels with PAS, there is a high need for an additional non-invasive test to improve the diagnostic accuracy and to select the real PAS from the suspected ones in the first-trimester screening. miRNA signatures of placental tissue, myometrium, and blood plasma from women with PAS in the third trimester of pregnancy, as well as miRNA profiles in exosomes from the blood serum of women in the first trimester with physiologically progressing pregnancy, complicated by PAS or pre-eclampsia, were obtained using deep sequencing. Two logistic regression models were constructed, both featuring statistically significant parameters related to the levels of miR-26a-5p, miR-17-5p, and miR-101-3p, quantified by real-time PCR in native blood serum. These models demonstrated 100% sensitivity in detecting PAS during the first pregnancy screening. These miRNAs were identified as specific markers for PAS, showing significant differences in their blood serum levels during the first trimester in the PAS group compared to those in physiological pregnancies, early- or late-onset pre-eclampsia groups. Furthermore, these miRNAs exhibited differential expression in the PAS placenta and/or myometrium in the third trimester and, according to data from the literature, control angiogenesis. Significant correlations were found between extracellular hsa-miR-101-3p and nuchal translucency thickness, hsa-miR-17-5p and uterine artery pulsatility index, and hsa-miR-26a-5p and hsa-miR-17-5p with PLGF. The developed test system for early non-invasive PAS diagnosis based on the blood serum level of extracellular miR-26a-5p, miR-17-5p, and miR-101-3p can serve as an auxiliary method for first-trimester screening of pregnant women, subject to validation with independent test samples.

Extracellular vesicles of human glial cells exert neuroprotective effects via brain miRNA modulation in a rat model of traumatic brain injury.

Stem cell-based therapeutic approaches for neurological disorders are widely studied. Paracrine factors secreted by stem cells in vitro and delivered intranasally might allow bypassing the disadvantages associated with a surgical cell delivery procedure with likely immune rejection of a transplant. In this study, we investigated the therapeutic effect of the extracellular vesicles secreted by glial progenitor cells (GPC-EV) derived from human induced pluripotent stem cell in a traumatic brain injury model. Intranasal administration of GPC-EV to Wistar rats for 6 days improved sensorimotor functions assessed over a 14-day observation period. Beside, deep sequencing of microRNA transcriptome of GPC-EV was estimate, and was revealed 203 microRNA species that might be implicated in prevention of various brain pathologies. Modulation of microRNA pools might contribute to the observed decrease in the number of astrocytes that inhibit neurorecovery processes while enhancing neuroplasticity by decreasing phosphorylated Tau forms, preventing inflammation and apoptosis associated with secondary damage to brain tissue. The course of GPC-EV administration was promoted the increasing protein levels of NF-κB in studied areas of the rat brain, indicating NF-κB dependent mechanisms as a plausible route of neuroprotection within the damaged area. This investigation showed that GPC-EV may be representing a therapeutic approach in traumatic brain injury, though its translation into the clinic would require an additional research and development.

Blood Plasma Small Non-Coding RNAs as Diagnostic Molecules for the Progesterone-Receptor-Negative Phenotype of Serous Ovarian Tumors.

The expression level of the progesterone receptor (PGR) plays a crucial role in determining the biological characteristics of serous ovarian carcinoma. Low PGR expression is associated with chemoresistance and a poorer outcome. In this study, our objective was to explore the relationship between tumor progesterone receptor levels and RNA profiles (miRNAs, piwiRNAs, and mRNAs) to understand their biological characteristics and behavior. To achieve this, we employed next-generation sequencing of small non-coding RNAs, quantitative RT-PCR, and immunohistochemistry to analyze both FFPE and frozen tumor samples, as well as blood plasma from patients with benign cystadenoma (BSC), serous borderline tumor (SBT), low-grade serous ovarian carcinoma (LGSOC), and high-grade serous ovarian carcinoma (HGSOC). Our findings revealed significant upregulation of MMP7 and MUC16, along with downregulation of PGR, in LGSOC and HGSOC compared to BSC. We observed significant correlations of PGR expression levels in tumor tissue with the contents of miR-199a-5p, miR-214-3p, miR-424-3p, miR-424-5p, and miR-125b-5p, which potentially target MUC16, MMP7, and MMP9, as well as with the tissue content of miR-16-5p, miR-17-5p, miR-20a-5p, and miR-93-5p, which are associated with the epithelial-mesenchymal transition (EMT) of cells. The levels of EMT-associated miRNAs were significantly correlated with the content of hsa_piR_022437, hsa_piR_009295, hsa_piR_020813, hsa_piR_004307, and hsa_piR_019914 in tumor tissues. We developed two optimal logistic regression models using the quantitation of hsa_piR_020813, miR-16-5p, and hsa_piR_022437 or hsa_piR_004307, hsa_piR_019914, and miR-93-5p in the tumor tissue, which exhibited a significant ability to diagnose the PGR-negative tumor phenotype with 93% sensitivity. Of particular interest, the blood plasma levels of miR-16-5p and hsa_piR_022437 could be used to diagnose the PGR-negative tumor phenotype with 86% sensitivity even before surgery and chemotherapy. This knowledge can help in choosing the most effective treatment strategy for this aggressive type of ovarian cancer, such as neoadjuvant chemotherapy followed by cytoreduction in combination with hyperthermic intraperitoneal chemotherapy and targeted therapy, thus enhancing the treatment's effectiveness and the patient's longevity.

Prediction of Early- and Late-Onset Pre-Eclampsia in the Preclinical Stage via Placenta-Specific Extracellular miRNA Profiling.

Pre-eclampsia (PE) is one of the severe complications of pregnancy in 3-8% of all cases and is one of the leading causes of maternal and perinatal mortality. The fundamental role in the pathogenesis of PE is assigned to maternal and/or placental factors, whereby the combination and manifestation of which determines the time of onset of the clinical symptoms of PE (before or after 34 weeks of gestation) and their severity. It is known that the expression level of miRNAs, the regulators of signaling cascades in the cell, depends on gestational age. In the present study, we focused on the identification of the placenta-specific miRNAs that differentiate between early- and late-onset pre-eclampsia (ePE and lPE) throughout pregnancy, from the first to the third trimester. A total of 67 patients were analyzed using small RNA deep sequencing and real-time quantitative PCR, which resulted in a core list of miRNAs (let-7b-5p, let-7d-3p, let-7f-5p, let-7i-5p, miR-22-5p, miR-451a, miR-1246, miR-30e-5p, miR-20a-5p, miR-1307-3p, and miR-320e), which in certain combinations can predict ePE or lPE with 100% sensitivity and 84-100% specificity in the 1st trimester of pregnancy. According to the literature data, these miRNA predictors of PE control trophoblast proliferation, invasion, migration, syncytialization, the endoplasmic reticulum unfolded protein response, immune tolerance, angiogenesis, and vascular integrity. The simultaneous detection of let-7d-3p, miR-451a, and miR-1307-3p, resistant to the repeated freezing/thawing of blood serum samples, in combination with biochemical (b-hCG and PAPP-A) and ultrasound (UAPI) parameters, allowed us to develop a universal model for the prediction of ePE and lPE onset (FPR = 15.7% and FNR = 9.5%), which was validated using a test cohort of 48 patients and demonstrated false-positive results in 26.7% of cases and false negatives in 5.6% of cases. For comparison, the use of the generally accepted Astraia program in the analysis of the test cohort of patients led to worse results: FPR = 62.1% and FNR = 33.3%.

Search for New Participants in the Pathogenesis of High-Grade Serous Ovarian Cancer with the Potential to Be Used as Diagnostic Molecules.

Recent studies have attempted to develop molecular signatures of epithelial ovarian cancer (EOC) based on the quantitation of protein-coding and non-coding RNAs to predict disease prognosis. Due to the heterogeneity of EOC, none of the developed prognostic signatures were directly applied in clinical practice. Our work focuses on high-grade serous ovarian carcinoma (HGSOC) due to the highest mortality rate relative to other types of EOC. Using deep sequencing of small non-coding RNAs in combination with quantitative real-time PCR, we confirm the dualistic classification of epithelial ovarian cancers based on the miRNA signature of HGSOC (type 2), which differs from benign cystadenoma and borderline cystadenoma-precursors of low-grade serous ovarian carcinoma (type 1)-and identified two subtypes of HGSOC, which significantly differ in the level of expression of the progesterone receptor in the tumor tissue, the secretion of miR-16-5p, miR-17-5p, miR-93-5p, miR-20a-5p, the level of serum CA125, tumor size, surgical outcome (optimal or suboptimal cytoreduction), and response to chemotherapy. It was found that the combined determination of the level of miR-16-5p, miR-17-5p, miR-20a-5p, and miR-93-5p circulating in blood plasma of patients with primary HGSOC tumors makes it possible to predict optimal cytoreduction with 80.1% sensitivity and 70% specificity (p = 0.022, TPR = 0.8, FPR = 0.3), as well as complete response to adjuvant chemotherapy with 77.8% sensitivity and 90.9% specificity (p = 0.001, TPR = 0.78, FPR = 0.09). After the additional verification of the obtained data in a larger HGSOC patient cohort, the combined quantification of these four miRNAs is proposed to be used as a criterion for selecting patients either for primary cytoreduction or neoadjuvant chemotherapy followed by interval cytoreduction.

Clinical Relevance of Secreted Small Noncoding RNAs in an Embryo Implantation Potential Prediction at Morula and Blastocyst Development Stages.

Despite the improvements in biotechnological approaches and the selection of controlled ovarian hyperstimulation protocols, the resulting pregnancy rate from in vitro fertilization (IVF) protocols still does not exceed 30-40%. In this connection, there is an acute question of the development of a non-invasive, sensitive, and specific method for assessing the implantation potential of an embryo. A total of 110 subfertile couples were included in the study to undergo the IVF/ICSI program. Obtained embryos for transfer into the uterine cavity of patient cohort 1 (n = 60) and cohort 2 (n = 50) were excellent/good-quality blastocysts, and small noncoding RNA (sncRNA) content in the corresponding spent culture medium samples at the morula stage (n = 43) or at the blastocyst stage (n = 31) was analyzed by deep sequencing followed by qRT-PCR in real time. Two logistic regression models were developed to predict the implantation potential of the embryo with 100% sensitivity and 100% specificity: model 1 at the morula stage, using various combinations of hsa_piR_022258, hsa-let-7i-5p, hsa_piR_000765, hsa_piR_015249, hsa_piR_019122, and hsa_piR_008112, and model 2 at the blastocyst stage, using various combinations of hsa_piR_020497, hsa_piR_008113, hsa-miR-381-3p, hsa_piR_022258, and hsa-let-7a-5p. Protein products of sncRNA potential target genes participate in the selective turnover of proteins through the ubiquitination system and in the organization of the various cell cytoskeleton and nucleoskeleton structures, regulating the activity of the Hippo signaling pathway, which determines the fate specification of the blastomers.

Clusterin and Its Potential Regulatory microRNAs as a Part of Secretome for the Diagnosis of Abnormally Invasive Placenta: Accreta, Increta, and Percreta Cases.

Magnetic resonance imaging (MRI) and ultrasound methods used for the diagnosis of an abnormally invasive placenta (AIP) have a wide range of sensitivity (Se, 33-93%) and specificity (Sp, 71-100%) levels, which results in a high risk of unfavorable maternal and perinatal outcomes. The relevance of optimizing the diagnosis of AIP is beyond doubt. Given the epigenetic nature of trophoblast invasion, we aimed to quantitate microRNAs and proteins of their target genes that are potentially associated with AIP in blood plasma samples from 64 pregnant women at gestation weeks 30-34 by reverse transcription coupled with polymerase chain reaction (RT-PCR) and Western blotting, respectively. Statistically significant increases in the expression levels of hsa-miR-17-5p, hsa-miR-21-5p, hsa-miR-25-3p, hsa-miR-92a-3p, and hsa-miR-320a-3p were revealed in the groups of women with AIP (accreta, increta, percreta) relative to the group of women with scars on the uterus or to the group with placenta previa. Opposite changes in the expression level of "gene-target protein/miRNA" pairs were found for the α-subunit of the clusterin secretory form and any of the hsa-miR-21-5p, hsa-miR-25-3p, hsa-miR-92a-3p, hsa-miR-320a-3p, and hsa-miR-17-5p in all cases of AIP. The developed logistic regression models to diagnose AIP cases of various severity gave Se values of 88.8-100% and Sp values of 91.6-100% using a combination of hsa-miR-21-5p, hsa-miR-92a-3p, hsa-miR-320a-3p, or clusterin levels.

miRNAs and Their Gene Targets-A Clue to Differentiate Pregnancies with Small for Gestational Age Newborns, Intrauterine Growth Restriction, and Preeclampsia.

Despite the differences in the clinical manifestations of major obstetric syndromes, such as preeclampsia (PE) and intrauterine growth restriction (IUGR), their pathogenesis is based on the dysregulation of proliferation, differentiation, and invasion of cytotrophoblast cells that occur in the developing placenta, decidual endometrium, and myometrial parts of the spiral arteries. To understand the similarities and differences in the molecular mechanisms of PE and IUGR, samples of the placental bed and placental tissue were analyzed using protein mass spectrometry and the deep sequencing of small RNAs, followed by validation of the data obtained by quantitative RT-PCR in real time. A comparison of the transcriptome and proteomic profiles in the samples made it possible to conclude that the main changes in the molecular profile in IUGR occur in the placental bed, in contrast to PE, in which the majority of molecular changes occurs in the placenta. In placental bed samples, significant changes in the ratio of miRNA and its potential target gene expression levels were revealed, which were unique for IUGR (miR-30c-5p/VIM, miR-28-3p/VIM, miR-1-3p/ANXA2, miR-30c-5p/FBN1; miR-15b-5p/MYL6), unique for PE (miR-185-3p/FLNA), common for IUGR and PE (miR-30c-5p/YWHAZ and miR-654-3p/FGA), but all associated with abnormality in the hemostatic and vascular systems as well as with an inflammatory process at the fetal‒maternal interface.