Small Noncoding RNA Signatures for Determining the Developmental Potential of an Embryo at the Morula Stage

As part of the optimization of assisted reproductive technology programs, the aim of the study was to identify key small noncoding RNA (sncRNA) molecules that participate in maternal-to-zygotic transition and determine development potential and competence to form a healthy fetus. Small RNA deep sequencing followed by quantitative real-time RT-PCR was used to profile sncRNAs in 50 samples of spent culture medium from morula with different development potentials (no potential (degradation/developmental arrest), low potential (poor-quality blastocyst), and high potential (good/excellent quality blastocyst capable of implanting and leading to live birth)) obtained from 27 subfertile couples who underwent in vitro fertilization. We have shown that the quality of embryos at the morula stage is determined by secretion/uptake rates of certain sets of piRNAs and miRNAs, namely hsa_piR_011291, hsa_piR_019122, hsa_piR_001311, hsa_piR_015026, hsa_piR_015462, hsa_piR_016735, hsa_piR_019675, hsa_piR_020381, hsa_piR_020485, hsa_piR_004880, hsa_piR_000807, hsa-let-7b-5p, and hsa-let-7i-5p. Predicted gene targets of these sncRNAs included those globally decreased at the 8-cell–morula–blastocyst stage and critical to early embryo development. We show new original data on sncRNA profiling in spent culture medium from morula with different development potential. Our findings provide a view of a more complex network that controls human embryogenesis at the pre-implantation stage. Further research is required using reporter analysis to experimentally confirm interactions between identified sncRNA/gene target pairs

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

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

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%

Preeclampsia: The Interplay between Oxygen-Sensitive miRNAs and Erythropoietin

Changes in the oxygen partial pressure caused by a violation of uteroplacental perfusion are considered a powerful inducer of a cascade of reactions leading to the clinical manifestation of preeclampsia (PE). At the same time, the induction of oxygen-dependent molecule expression, in particular, miRNA and erythropoietin, is modulated. Therefore, the focus of our study was aimed at estimating the miRNA expression profile of placental tissue and blood plasma in pregnant women with preeclampsia using deep sequencing and quantitative RT-PCR, as well as determining the concentration of erythropoietin. The expression of miR-27b-3p, miR-92b-3p, miR-125b-5p, miR-181a-5p, and miR-186-5p, as regulated by hypoxia/reoxygenation, was significantly increased in blood plasma during early-onset preeclampsia. The possibility of detecting early PE according to the logistic regression model (miR-92b-3p, miR-125b-5p, and miR-181a-5p (AUC = 0.91)) was evaluated. Furthermore, the erythropoietin level, which is regulated by miR-125b-5p, was significantly increased. According to PANTHER14.1, the participation of these miRNAs in the regulation of pathways, such as the hypoxia’s response via HIF activation, oxidative stress response, angiogenesis, and the VEGF signaling pathway, were determined

Diagnostic Potential of Exosomal HypoxamiRs in the Context of Hypoxia–Sumoylation–HypoxamiRs in Early Onset Preeclampsia at the Preclinical Stage

As the search for non-invasive preclinical markers of preeclampsia (PE) expands, the number of studies on the diagnostic potential of exosomes is growing. Changes in the partial pressure of oxygen caused by impaired uteroplacental perfusion in PE are a powerful inducer of increased production and release of exosomes from cells, which also determine their cargo. At the same time, the expression pattern of oxygen-dependent microRNAs (miRNAs), called “hypoxamiRs”, is modulated, and their packing into exosomes is strictly regulated by sumoylation. In connection therewith, we emphasize the evaluation of exosomal hypoxamiR expression (miR-27b-3p, miR-92b-3p, miR-181a-5p, and miR-186-5p) using quantitative RT-PCR, as well as SUMO 1–4 and UBC9 (by Western blotting), in pregnant women with early-onset PE. The findings show that miR-27b-3p and miR-92b-3p expression was significantly changed at 11–14 and 24–26 weeks of gestation in the blood plasma of pregnant women with early-onset PE, which subsequently manifested. High sensitivity and specificity (AUC = 1) were demonstrated for these miRNAs in the first trimester, and significant correlations with a decrease in hemoglobin (r = 0.71, p = 0.002; r = −0.71, p = 0.002) were established. In mid-pregnancy, the miR-27b-3p expression was found to correlate with an increase in platelets (r = −0.95, p = 0.003), and miR-92b-3p was associated with a decrease in the prothrombin index (r = 0.95, p = 0.003). Specific exomotifs of studied miRNAs were also identified, to which the sumoylated ribonucleoprotein hnRNPA2/B1 binds, carrying out their packaging into exosomes. The expression of conjugated SUMO 1 (p = 0.05), SUMO 2/3/4 (p = 0.03), and UBC9 (p = 0.1) was increased in exosomes at early-onset PE, and the expression of free SUMO 1 (p = 0.03) and SUMO 2/3/4 (p = 0.01) was significantly increased in the placenta, as an adaptive response to hypoxia. Moreover, SUMO 2/3/4 was negatively correlated with miR-27b-3p expression in the placenta. In conclusion, the diagnostic potential of exosomal hypoxamiRs mediated by sumoylation may form the basis for the development of combined specific targets for the treatment of early-onset PE, as hnRNPA2/B1 is a target of miR-27b-3p, and its sumoylation creates miR-27b-3p–hnRNPA2/B1–SUMO 1–4 cross-talk

Interrelation between miRNAs Expression Associated with Redox State Fluctuations, Immune and Inflammatory Response Activation, and Neonatal Outcomes in Complicated Pregnancy, Accompanied by Placental Insufficiency

Redox disbalance in placental cells leads to the hyperproduction of reactive oxygen species (ROS), it mediates the dysregulation of the maternal immune tolerance to a semi-allogenic fetus, inducing pro-inflammatory reactions, and it plays a central role in perinatal complications and neonatal disease programming. Microvesicles, which provide transplacental communication between a mother and fetus, contain microRNAs (miRNAs) that are sensitive to oxidative stress (OS) mediators and can control the balance of ROS production and utilization in target cells. In the context of this paradigm, we evaluated the markers of redox balance—MDA and 4-HNE for OS and GPx, and SOD, CAT, and GSH for the antioxidant system in the cord blood plasma of newborns diagnosed with fetal growth restriction (FGR)—by using polarography, spectrophotometry, and Western blotting. The expression of miRNAs associated with OS, immune and inflammatory responses in the blood plasma of newborns with intrauterine pneumonia (IP), neonatal sepsis (NS) and respiratory distress syndrome (RDS) was evaluated by a quantitative RT-PCR. Significant differences in the MDA level and reduced GPx and CAT activity were co-found for early-onset FGR (i.e., p ≤ 0.03 and >32 GA; p ≤ 0.009), IP (>32 GA; p ≤ 0.0001), and RDS (>32 GA; p ≤ 0.03). At the same time, the expression of miR-25-3p (p ≤ 0.03) was increased only in newborns with NS (>32 GA; p ≤ 0.03). The risk of developing IVH for premature newborns with IP (AUC = 0.8; cutoff—0.6) and NS (AUC = 0.68; cutoff—0.49) was assessed based on the miR-25-3p and miR-127-3p expression. Several key transcription factors were identified as the targets of studied miRNA since they are involved in the regulation of OS (NRF2), signaling and activation of the immune response (PRDM1, CCL26) and, also, inflammatory responses (NFKB1). The study of these miRNAs showed that they are involved in the modulation of processes leading to perinatal complications. Moreover, miR-127-3p is related to pro-inflammatory reactions and the formation of the macrophage phenotype in newborns with IP, NS, and RDS, while miR-25-3p is associated with an inhibition of macrophage migration and activation of antioxidant enzymes, which may prevent the development of oxidative damage in newborns with NS

The Impact of Maternal SARS-CoV-2 Infection Next to Pre-Immunization with Gam-COVID-Vac (Sputnik V) Vaccine on the 1-Day-Neonate’s Blood Plasma Small Non-Coding RNA Profile: A Pilot Study

The antenatal and postnatal effects of maternal SARS-CoV-2 on the fetus outcomes, especially in the case of maternal pre-vaccination against this infection, are still under investigation. Such effects may be due to placental insufficiency caused by maternal hypoxia and inflammatory response associated with SARS-CoV-2, and/or be a direct cytopathic effect of the virus. In this work, we studied the profile of small non-coding RNAs (sncRNAs) in the blood plasma of a newborn from a mother who had SARS-CoV-2 at the 22nd week of gestation after immunization with Gam-COVID-Vac (Sputnik V). The fetus had ultrasound signs of hypertrophy of the right heart and hydropericardium 4 weeks after infection of the mother with SARS-CoV-2, as well as cysts of the cerebral vascular plexuses by the time of birth. Taking this into account, we compared the sncRNA profile of this newborn on the first postpartum day with that of neonates born to COVID-19-negative women with different perinatal outcomes: severe cardiovascular and/or neurological disorders, or absence of any perinatal complications. According to next-generation sequencing data, we found that the fetus born to a COVID-19-affected mother pre-immunized with Gam-COVID-Vac (Sputnik V) vaccine differs from newborns with severe cardiovascular and/or nervous system abnormalities either in multidirectional changes in circulating sncRNAs or in less pronounced unidirectional changes in the level of sncRNAs relative to control samples. Considering this, it can be concluded that maternal vaccination against SARS-CoV-2 before pregnancy has a protective effect in preventing antenatal development of pathological processes in the cardiovascular and nervous systems of the neonate associated with COVID-19