Identification of Metabolomic Signatures for Ischemic Hypoxic Encephalopathy Using a Neonatal Rat Model.

A study was performed to determine early metabolomic markers of ischemic hypoxic encephalopathy (HIE) using a Rice-Vannucci model for newborn rats. Dried blood spots from 7-day-old male and female rat pups, including 10 HIE-affected animals and 16 control animals, were analyzed by liquid chromatography coupled with mass spectrometry (HPLC-MS) in positive and negative ion recording modes. Multivariate statistical analysis revealed two distinct clusters of metabolites in both HPLC-MS modes. Subsequent univariate statistical analysis identified 120 positive and 54 negative molecular ions that exhibited statistically significant change in concentration, with more than a 1.5-fold difference after HIE. In the HIE group, the concentrations of steroid hormones, saturated mono- and triglycerides, and phosphatidylcholines (PCs) were significantly decreased in positive mode. On the contrary, the concentration of unsaturated PCs was increased in the HIE group. Among negatively charged molecular ions, the greatest variations were found in the categories of phosphatidylcholines, phosphatidylinositols, and triglycerides. The major metabolic pathways associated with changed metabolites were analyzed for both modes. Metabolic pathways such as steroid biosynthesis and metabolism fatty acids were most affected. These results underscored the central role of glycerophospholipid metabolism in triggering systemic responses in HIE. Therefore, lipid biomarkers' evaluation by targeted HPLC-MS research could be a promising approach for the early diagnosis of HIE.

Integrating Proteomics and Lipidomics for Evaluating the Risk of Breast Cancer Progression: A Pilot Study.

Metastasis is a serious and often life-threatening condition, representing the leading cause of death among women with breast cancer (BC). Although the current clinical classification of BC is well-established, the addition of minimally invasive laboratory tests based on peripheral blood biomarkers that reflect pathological changes in the body is of utmost importance. In the current study, the serum proteome and lipidome profiles for 50 BC patients with (25) and without (25) metastasis were studied. Targeted proteomic analysis for concertation measurements of 125 proteins in the serum was performed via liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM MS) using the BAK 125 kit (MRM Proteomics Inc., Victoria, BC, Canada). Untargeted label-free lipidomic analysis was performed using liquid chromatography coupled to tandem mass-spectrometry (LC-MS/MS), in both positive and negative ion modes. Finally, 87 serum proteins and 295 lipids were quantified and showed a moderate correlation with tumor grade, histological and biological subtypes, and the number of lymph node metastases. Two highly accurate classifiers that enabled distinguishing between metastatic and non-metastatic BC were developed based on proteomic (accuracy 90%) and lipidomic (accuracy 80%) features. The best classifier (91% sensitivity, 89% specificity, AUC = 0.92) for BC metastasis diagnostics was based on logistic regression and the serum levels of 11 proteins: alpha-2-macroglobulin, coagulation factor XII, adiponectin, leucine-rich alpha-2-glycoprotein, alpha-2-HS-glycoprotein, Ig mu chain C region, apolipoprotein C-IV, carbonic anhydrase 1, apolipoprotein A-II, apolipoprotein C-II and alpha-1-acid glycoprotein 1.

Transplacental Therapeutic Drug Monitoring in Pregnant Women with Fetal Tachyarrhythmia Using HPLC-MS/MS.

Fetal arrhythmia develops in 0.1-5% of pregnancies and may cause fetal heart failure and fetal hydrops, thus increasing fetal, neonatal, and infant mortality. The timely initiation of transplacental antiarrhythmic therapy (ART) promotes the conversion of fetal tachycardia to sinus rhythm and the regression of the concomitant non-immune fetal hydrops. The optimal treatment regimen search for the fetus with tachyarrhythmia is still of high value. Polymorphisms of these genes determines the individual features of the drug pharmacokinetics. The aim of this study was to study the pharmacokinetics of transplacental anti-arrhythmic drugs in the fetal therapy of arrhythmias using HPLC-MS/MS, as well as to assess the effect of the multidrug-resistance gene ABCB1 3435C > T polymorphism on the efficacy and maternal/fetal complications of digoxin treatment. The predisposition to a decrease in the bioavailability of the digoxin in patients with a homozygous variant of the CC polymorphism showed a probable association with the development of ART side effects. A pronounced decrease in heart rate in women with the 3435TT allele of the ABCB1 gene was found. The homozygous TT variant in the fetus showed a probable association with an earlier response to ART and rhythm disruptions on the digoxin dosage reduction. high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) methods for digoxin and sotalol therapeutic drug monitoring in blood plasma, amniotic fluid, and urine were developed. The digoxin and sotalol concentrations were determined in the plasma blood, urine, and amniotic fluid of 30 pregnant women at four time points (from the beginning of the transplacental antiarrhythmic therapy to delivery) and the plasma cord blood of 30 newborns. A high degree of correlation between the level of digoxin and sotalol in maternal and cord blood was found. The ratio of digoxin and sotalol in cord blood to maternal blood was 0.35 (0.27 and 0.46) and 1.0 (0.97 and 1.07), accordingly. The digoxin concentration in the blood of the fetus at the moment of the first rhythm recovery episode, 0.58 (0.46, 0.8) ng/mL, was below the therapeutic interval. This confirms the almost complete transplacental transfer of sotalol and the significant limitation in the case of digoxin. Previously, ABCB1/P-glycoprotein had been shown to limit fetal exposure to drugs. Further studies (including multicenter ones) to clarify the genetic features of the transplacental pharmacokinetics of antiarrhythmic drugs are needed.

Specific changes in amino acid profiles in monocytes of patients with breast, lung, colorectal and ovarian cancers.

Introduction: Immunometabolism is essential factor of tumor progression, and tumor-associated macrophages are characterized by substantial changes in their metabolic status. In this study for the first time, we applied targeted amino acid LC-MS/MS analysis to compare amino acid metabolism of circulating monocytes isolated from patients with breast, ovarian, lung, and colorectal cancer. Methods: Monocyte metabolomics was analyzed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/ MS) analysis of amino acid extracts. The targeted analysis of 26 amino acids was conducted by LCMS/MS on an Agilent 6460 triple quadrupole mass spectrometer equipped with an electrospray ionization source and an Agilent 1260 II liquid chromatograph. Results: Comparison of monocytes of cancer patients with monocytes of healthy control individuals demonstrated that in breast cancer most pronounced changes were identified for tryptophan (AUC = 0.76); for ovarian cancer, aminobutyric acid was significantly elevated (AUC= 1.00); for lung cancer significant changes we indented for citrulline (AUC = 0.70). In order to identify key amino acids that are characteristic for monocytes in specific cancer types, we compared each individual cancer with other 3 types of cancer. We found, that aspartic acid and citrulline are specific for monocytes of patients with colorectal cancer (p<0.001, FC = 1.40 and p=0.003, FC = 1.42 respectively). Citrulline, sarcosine and glutamic acid are ovarian cancer-specific amino acids (p = 0.003, FC = 0.78, p = 0.003, FC = 0.62, p = 0.02, FC = 0.78 respectively). Glutamine, methionine and phenylalanine (p = 0.048, FC = 1.39. p = 0.03, FC = 1.27 and p = 0.02, FC = 1.41) are lung cancer-specific amino acids. Ornithine in monocytes demonstrated strong positive correlation (r = 0.63) with lymph node metastasis incidence in breast cancer patients. Methyl histidine and cysteine in monocytes had strong negative correlation with lymph node metastasis in ovarian cancer patients (r = -0.95 and r = -0.95 respectively). Arginine, citrulline and ornithine have strong negative correlation with tumor size (r = -0.78, citrulline) and lymph node metastasis (r = -0.63 for arginine and r = -0.66 for ornithine). Discussion: These alterations in monocyte amino acid metabolism can reflect the reaction of systemic innate immunity on the growing tumor. Our data indicate that this metabolic programming is cancer specific and can be inhibiting cancer progression. Cancer-specific differences in citrulline, as molecular link between metabolic pathways and epigenetic programing, provide new option for the development and validation of anti-cancer therapies using inhibitors of enzymes catalyzing citrullination.

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.

Non-Invasive Differential Diagnosis of Cervical Neoplastic Lesions by the Lipid Profile Analysis of Cervical Scrapings.

Cervical cancer is one of the most common cancers in women with pronounced stages of precancerous lesions. Accurate differential diagnosis of such lesions is one of the primary challenges of medical specialists, which is vital to improving patient survival. The aim of this study was to develop and test an algorithm for the differential diagnosis of cervical lesions based on lipid levels in scrapings from the cervical epithelium and cervicovaginal canal. The lipid composition of the samples was analyzed by high-performance chromato-mass spectrometry. Lipid markers were selected using the Mann-Whitney test with a cutoff value of 0.05 and by projections to latent structures discriminant analysis, where a projection threshold of one was chosen. The final selection of variables for binomial logistic regressions was carried out using the Akaike information criterion. As a result, a final neoplasia classification method, based on 20 logistic regression sub-models, has an accuracy of 79% for discrimination NILM/cervicitis/LSIL/HSIL/cancer. The model has a sensitivity of 83% and a specificity of 88% for discrimination of several lesions (HSIL and cancer). This allows us to discuss the prospective viability of further validation of the developed non-invasive method of differential diagnosis.

Shotgun Lipidomics for Differential Diagnosis of HPV-Associated Cervix Transformation.

A dramatic increase in cervical diseases associated with human papillomaviruses (HPV) in women of reproductive age has been observed over the past decades. An accurate differential diagnosis of the severity of cervical intraepithelial neoplasia and the choice of the optimal treatment requires the search for effective biomarkers with high diagnostic and prognostic value. The objective of this study was to introduce a method for rapid shotgun lipidomics to differentiate stages of HPV-associated cervix epithelium transformation. Tissue samples from 110 HPV-positive women with cervicitis (n = 30), low-grade squamous intraepithelial lesions (LSIL) (n = 30), high-grade squamous intraepithelial lesions (HSIL) (n = 30), and cervical cancers (n = 20) were obtained. The cervical epithelial tissue lipidome at different stages of cervix neoplastic transformation was studied by a shotgun label-free approach. It is based on electrospray ionization mass spectrometry (ESI-MS) data of a tissue extract. Lipidomic data were processed by the orthogonal projections to latent structures discriminant analysis (OPLS-DA) to build statistical models, differentiating stages of cervix transformation. Significant differences in the lipid profile between the lesion and surrounding tissues were revealed in chronic cervicitis, LSIL, HSIL, and cervical cancer. The lipids specific for HPV-induced cervical transformation mainly belong to glycerophospholipids: phosphatidylcholines, and phosphatidylethanolamines. The developed diagnostic OPLS-DA models were based on 23 marker lipids. More than 90% of these marker lipids positively correlated with the degree of cervix transformation. The algorithm was developed for the management of patients with HPV-associated diseases of the cervix, based on the panel of 23 lipids as a result. ESI-MS analysis of a lipid extract by direct injection through a loop, takes about 25 min (including preparation of the lipid extract), which is significantly less than the time required for the HPV test (several hours for hybrid capture and about an hour for PCR). This makes lipid mass spectrometric analysis a promising method for express diagnostics of HPV-associated neoplastic diseases of the cervix.

LC-MS Analysis Revealed the Significantly Different Metabolic Profiles in Spent Culture Media of Human Embryos with Distinct Morphology, Karyotype and Implantation Outcomes.

In this study we evaluated possible differences in metabolomic profiles of spent embryo culture media (SECM) of human embryos with distinct morphology, karyotype, and implantation outcomes. A total of 153 samples from embryos of patients undergoing in vitro fertilization (IVF) programs were collected and analyzed by HPLC-MS. Metabolomic profiling and statistical analysis revealed clear clustering of day five SECM from embryos with different morphological classes and karyotype. Profiling of day five SECM from embryos with different implantation outcomes showed 241 significantly changed molecular ions in SECM of successfully implanted embryos. Separate analysis of paired SECM samples on days three and five revealed 46 and 29 molecular signatures respectively, significantly differing in culture media of embryos with a successful outcome. Pathway enrichment analysis suggests certain amino acids, vitamins, and lipid metabolic pathways to be crucial for embryo implantation. Differences between embryos with distinct implantation potential are detectable on the third and fifth day of cultivation that may allow the application of culture medium analysis in different transfer protocols for both fresh and cryopreserved embryos. A combination of traditional morphological criteria with metabolic profiling of SECM may increase implantation rates in assisted reproductive technology programs as well as improve our knowledge of the human embryo metabolism in the early stages of development.

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 balance­MDA 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., <34 gestational age). Significant correlations were found with a low birth weight by Apgar scores with reduced levels of antioxidant enzymes. Indeed, the level of OS markers increased in early-onset FGR in newborns with an extremely low body weight and high echogenicity of the periventricular zones, and reduced in late-onset FGR in newborns with IP, hyperbilirubinemia, intraventricular hemorrhage (IVH) and cerebral cysts. A prognostic model (AUC = 1; cutoff­0.5) was developed to assess the risk of IVH in newborns diagnosed with FGR based on the assessment of the OS markers (i.e., MDA + 4 HNE + CAT + GSH). A significant increase in the miR-127-3p expression was found in the plasma of newborns with NS (<32 GA; 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; cutoff­0.6) and NS (AUC = 0.68; cutoff­0.49) 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.

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.

Alterations in lipid profile upon uterine fibroids and its recurrence.

Uterine fibroids (UF) is the most common (about 70% cases) type of gynecological disease, with the recurrence rate varying from 11 to 40%. Because UF has no distinct symptomatology and is often asymptomatic, the specific and sensitive diagnosis of UF as well as the assessment for the probability of UF recurrence pose considerable challenge. The aim of this study was to characterize alterations in the lipid profile of tissues associated with the first-time diagnosed UF and recurrent uterine fibroids (RUF) and to explore the potential of mass spectrometry (MS) lipidomics analysis of blood plasma samples for the sensitive and specific determination of UF and RUF with low invasiveness of analysis. MS analysis of lipid levels in the myometrium tissues, fibroids tissues and blood plasma samples was carried out on 66 patients, including 35 patients with first-time diagnosed UF and 31 patients with RUF. The control group consisted of 15 patients who underwent surgical treatment for the intrauterine septum. Fibroids and myometrium tissue samples were analyzed using direct MS approach. Blood plasma samples were analyzed using high performance liquid chromatography hyphened with mass spectrometry (HPLC/MS). MS data were processed by discriminant analysis with projection into latent structures (OPLS-DA). Significant differences were found between the first-time UF, RUF and control group in the levels of lipids involved in the metabolism of glycerophospholipids, sphingolipids, lipids with an ether bond, triglycerides and fatty acids. Significant differences between the control group and the groups with UF and RUF were found in the blood plasma levels of cholesterol esters, triacylglycerols, (lyso) phosphatidylcholines and sphingomyelins. Significant differences between the UF and RUF groups were found in the blood plasma levels of cholesterol esters, phosphotidylcholines, sphingomyelins and triacylglycerols. Diagnostic models based on the selected differential lipids using logistic regression showed sensitivity and specificity of 88% and 86% for the diagnosis of first-time UF and 95% and 79% for RUF, accordingly. This study confirms the involvement of lipids in the pathogenesis of uterine fibroids. A diagnostically significant panel of differential lipid species has been identified for the diagnosis of UF and RUF by low-invasive blood plasma analysis. The developed diagnostic models demonstrated high potential for clinical use and further research in this direction.

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.

Normalization methods for reducing interbatch effect without quality control samples in liquid chromatography-mass spectrometry-based studies.

Data normalization is an essential part of a large-scale untargeted mass spectrometry metabolomics analysis. Autoscaling, Pareto scaling, range scaling, and level scaling methods for liquid chromatography-mass spectrometry data processing were compared with the most common normalization methods, including quantile normalization, probabilistic quotient normalization, and variance stabilizing normalization. These methods were tested on eight datasets from various clinical studies. The efficiency of the data normalization was assessed by the distance between clusters corresponding to batches and the distance between clusters corresponding to clinical groups in the space of principal components, as well as by the number of features with a pairwise statistically significant difference between the batches and the number of features with a pairwise statistically significant difference between clinical groups. Autoscaling demonstrated the most effective reduction in interbatch variation and can be preferable to probabilistic quotient or quantile normalization in liquid chromatography-mass spectrometry data.

Comparison of the effectiveness of variable selection method for creating a diagnostic panel of biomarkers for mass spectrometric lipidome analysis.

Hundreds of compounds are detected during untargeted lipidomics analysis. The potential efficacy of lipids as disease markers makes it important to select the species with the most discriminative potential. Datasets based on a selected class of lipids allow the development of a high-quality diagnostic model using orthogonal projection on latent structure. The combination of selection of lipids by variable importance in projection and by Akaike information criteria makes it possible to build a reliable diagnostic model based on logistic regression.

Immunoendocrine Markers of Stress in Seminal Plasma at IVF/ICSI Failure: a Preliminary Study.

We have previously shown that high level of seminal interleukin (IL)-18 is positively associated with a greater risk of pregnancy failure in women exposed to their partners' seminal plasma (SP) during the in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) cycle. Since IL-18 and IL-1ß considered to be the key immune markers of stress, here we ask whether their increase in SP may be due to the stress experienced by men engaged in the IVF programs. Therefore, we correlated seminal IL-18 with IL-1ß and both cytokines with the seminal steroids, whose increase indicates the activation of neuroendocrine stress response systems. Retrospective analysis of stored seminal samples was performed. Based on previously identified cutoff level for content of IL-18 per ejaculate, samples with high IL-18 content from IVF failure group (n = 9), as well as samples with low IL-18 content from IVF success group (n = 7), were included in the study. Seminal cytokines were evaluated using FlowCytomix™ technology. A set of 16 biologically active steroids in SP was quantified by liquid chromatography coupled with mass spectrometry. Concentrations and total amounts per ejaculate of cytokines and steroids were determined. A positive significant correlation was found between the levels of IL-18 and IL-1ß. There was also a positive correlation between IL-18 or IL-1ß and 17-α-hydroxypregnenolone, 17-α-hydroxyprogesterone, dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS), androstenedione, testosterone, dihydrotestosterone, progesterone, corticosterone, 11-deoxycorticosterone, and the ratio of DHEAS/cortisol. We suggested that stress-related overexpression of immune and hormonal factors in SP may be the key link between male stress and embryo implantation failure.

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.

Genetic diversity of SAD and FAD genes responsible for the fatty acid composition in flax cultivars and lines.

BACKGROUND: Flax (Linum usitatissimum L.) is grown for fiber and seed in many countries. Flax cultivars differ in the oil composition and, depending on the ratio of fatty acids, are used in pharmaceutical, food, or paint industries. It is known that genes of SAD (stearoyl-ACP desaturase) and FAD (fatty acid desaturase) families play a key role in the synthesis of fatty acids, and some alleles of these genes are associated with a certain composition of flax oil. However, data on genetic polymorphism of these genes are still insufficient. RESULTS: On the basis of the collection of the Institute for Flax (Torzhok, Russia), we formed a representative set of 84 cultivars and lines reflecting the diversity of fatty acid composition of flax oil. An approach for the determination of full-length sequences of SAD1, SAD2, FAD2A, FAD2B, FAD3A, and FAD3B genes using the Illumina platform was developed and deep sequencing of the 6 genes in 84 flax samples was performed on MiSeq. The obtained high coverage (about 400x on average) enabled accurate assessment of polymorphisms in SAD1, SAD2, FAD2A, FAD2B, FAD3A, and FAD3B genes and evaluation of cultivar/line heterogeneity. The highest level of genetic diversity was observed for FAD3A and FAD3B genes - 91 and 62 polymorphisms respectively. Correlation analysis revealed associations between particular variants in SAD and FAD genes and predominantly those fatty acids whose conversion they catalyze: SAD - stearic and oleic acids, FAD2 - oleic and linoleic acids, FAD3 - linoleic and linolenic acids. All except one low-linolenic flax cultivars/lines contained both the substitution of tryptophan to stop codon in the FAD3A gene and histidine to tyrosine substitution in the FAD3B gene, while samples with only one of these polymorphisms had medium content of linolenic acid and cultivars/lines without them were high-linolenic. CONCLUSIONS: Genetic polymorphism of SAD and FAD genes was evaluated in the collection of flax cultivars and lines with diverse oil composition, and associations between particular polymorphisms and the ratio of fatty acids were revealed. The achieved results are the basis for the development of marker-assisted selection and DNA-based certification of flax cultivars.

MiRNAs Regulating Oxidative Stress: A Correlation with Doppler Sonography of Uteroplacental Complex and Clinical State Assessments of Newborns in Fetal Growth Restriction.

Overproduction of reactive oxygen species (ROS) and, as a result, uncontrolled oxidative stress (OS) can play a central role in disorders of fetal hemodynamics and subsequent development of adverse perinatal outcomes in newborns with fetal growth restriction (FGR). Given the epigenetic nature of such disorders, the aim of our study was to evaluate the expression of miRNAs associated with OS and endothelial dysfunction (miR-27a-3p, miR-30b-5p, miR-125b-5p, miR-221-3p, miR-451a and miR-574-3p) in umbilical cord blood using real-time quantitative RT-PCR. ΜiRNA expression was evaluated in patients with FGR delivery before (n = 9 pregnant) and after 34 weeks of gestation (n = 13 pregnant), and the control groups corresponding to the main groups by gestational age (13 pregnant women in each group, respectively). A significant increase in miR-451a expression was detected in late-onset FGR and correlations with fetoplacental and cerebral circulation were established (increase of resistance in the umbilical artery (pulsatility index, PI UA (umbilical artery): r = -0.59, p = 0.001) and a decrease in cerebral blood flow (CPR: r = 0.48, p = 0.009)). The change in miR-125b-5p expression in the placenta is associated with reduced Doppler of cerebral hemodynamics (CPR: r = 0.73, p = 0.003; PI MCA (middle cerebral artery) : r = 0.79, p = 0.0007), and newborn weight (r = 0.56, p = 0.04) in early-onset FGR. In addition, significant changes in miR-125b-5p and miR-451a expression in umbilical cord blood plasma were found in newborns with neonatal respiratory distress syndrome (NRDS) (in early-onset FGR) and very low birth weight (VLBW) (in late-onset FGR). A number of key signaling pathways have been identified in which the regulation of the studied miRNAs is involved, including angiogenesis, neurotrophin signaling pathway and oxidative stress response. In general, our study showed that changes of the redox homeostasis in the mother-placenta-fetus system in FGR and subsequent perinatal outcomes may be due to differential expression of oxidative stress-associated miRNAs.