Hypoxic–ischemic encephalopathy (HIE) is one of the most common causes of childhood disability. Hypothermic therapy is currently the only approved neuroprotective approach. However, early diagnosis of HIE can be challenging, especially in the first hours after birth when the decision to use hypothermic therapy is critical. Distinguishing HIE from other neonatal conditions, such as sepsis, becomes a significant problem in diagnosis. This study explored the utility of a metabolomic-based approach employing the NeoBase 2 MSMS kit to diagnose HIE using dry blood stains in a Rice–Vannucci model of HIE in rats. We evaluated the diagnostic fidelity of this approach in a range between 3 and 6 h after the onset of HIE, including in the context of systemic inflammation and concomitant hypothermic therapy. Discriminant analysis revealed several metabolite patterns associated with HIE. A logistic regression model using glycine levels achieved high diagnostic fidelity with areas under the receiver operating characteristic curve of 0.94 at 3 h and 0.96 at 6 h after the onset of HIE. In addition, orthogonal partial least squares discriminant analysis, which included five metabolites, achieved 100% sensitivity and 80% specificity within 3 h of HIE. These results highlight the significant potential of the NeoBase 2 MSMS kit for the early diagnosis of HIE and could improve patient management and outcomes in this serious illness.

In this study, we sought to determine fecal cytokine levels in very preterm newborns at the onset of non-specific clinical symptoms of necrotizing enterocolitis (NEC) and decreased gastrointestinal (GI) motility. The study was approved by the Ethics Committee and the Scientific Council of the National Medical Research Center for Obstetrics, Gynecology and Perinatology named after the Academician V. I. Kulakov of Ministry of Healthcare of the Russian Federation. Each patient’s parents gave their informed consent to their child’s participation in the study. The study was conducted at the National Medical Research Center for Obstetrics, Gynecology and Perinatology named after the Academician V. I. Kulakov over the period from June 2020 to December 2022. Fecal samples from preterm neonates with gestational age ≤ 32 weeks treated at the A.G. Antonov ICU were collected daily over their first 14 days of life. Samples from 46 newborns were selected for analysis: fecal samples collected on the day of an enteral feeding intolerance episode and fecal samples from controls who had not developed non-specific clinical symptoms of NEC or decreased GI motility, collected on the day when enteral intake reached 100 ml/kg/day. Based on the results of NEC and decreased GI motility diagnosis, stool samples were retrospectively divided into 3 groups: an NEC group (n = 8), a decreased GI motility group (n = 14) and a control group (n = 24). In the fecal samples of the very preterm newborns with NEC stage ≥ II, there was a significant increase in IL-6, IL-8, IL-10, TNF-a levels at the onset of initial symptoms of the disease. At the same time, the cytokine profile of the feces of the decreased GI motility patients did not differ significantly from the control group in any of the parameters. In cases of NEC, high IL-6, IL-8, IL-10 and TNF-a levels were detected in the patients’ stool at the onset of enteral feeding intolerance, suggesting that the method under investigation (aimed at determining the pro- and anti-inflammatory profile of fecal cytokines) may be a promising new tool for differentiating NEC from decreased GI motility in very preterm newborns.

The DNA methylation profile of breast cancer differs from that in healthy tissues and can be used as a diagnostic and prognostic biomarker. Aim of this study: To compare the levels of gene methylation in small malignant breast cancer tumors (<2 cm), in healthy tissue, and in fibroadenoma, and to evaluate the effectiveness of the modified Methylation Sensitive-High Resolution Melting (MS-HRM) method for this analysis. Analysis was performed using the modified MS-HRM method. For validation, the methylation levels of five genes were confirmed by pyrosequencing. The main study group included 96 breast cancer samples and the control group included 24 fibroadenoma samples and 24 healthy tissue samples obtained from patients with fibroadenoma. Breast cancer samples were divided into two subgroups (test set and validation set). The methylation of the following 15 genes was studied: MAST1, PRDM14, ZNF177, DNM2, SSH1, AP2M1, CACNA1E, CPEB4, DLGAP2, CCDC181, GCM2, ITPRIPL1, POM121L2, KCNQ1, and TIMP3. Significant differences in the validation set of samples were found for seven genes; the combination of the four genes GCM2, ITPRIPL1, CACNA1E, DLGAP2 (AUC = 0.99) showed the highest diagnostic value based on logistic regression for all breast cancer samples. Our modified MS-HRM method demonstrated that small breast cancer tumors have a specific DNA methylation profile that distinguishes them from healthy tissues and benign proliferative lesions.

The aim of this study was to evaluate the levels of ten energy metabolism factors: C-peptide, ghrelin, GIP, GLP-1, glucagon, insulin, leptin, PAI-1 (total), resistin, and visfatin, and to determine the expression of GLP1R receptors, CD10, CD26 proteases, and pro-inflammatory marker CD86 by macrophages in the peritoneal fluid (PF) in patients with endometriosis. The study included 54 women with endometriosis and a control group of 30 women with uterine myoma without signs of endometriosis. The levels of factors in PF were assessed by a multiplex method. Expression of GLP1R receptors, CD10, CD26 proteases, and CD86 by macrophages was evaluated using flow cytometry. It was found that in women with endometriosis, the concentrations of ghrelin, GLP-1, glucagon, and visfatin in PF were reduced (p = 0.007, p = 0.009, p = 0.002, p = 0.008, respectively). At the same time, there was a noted increase in the CD10 protease expression by peritoneal macrophages (p = 0.044). Correlation analysis showed a positive correlation of ghrelin and GLP-1 levels with CD86 macrophage expression (p = 0.044, p = 0.022, respectively) in the study group; a positive correlation was also found between the levels of GLP-1, glucagon, and visfatin with CD26 macrophage expression (p = 0.041, p = 0.048, p = 0.015, respectively) in PF. No correlations were found in the control group. These results indicate that a decrease in the levels of ghrelin, GLP-1, glucagon, and visfatin in PF may contribute to endometriosis development through their impact on the expression of pro-inflammatory markers of PF macrophages.

The content of eight different cytokines, cell-free DNA (cfDNA) and cell-free fetal DNA (cffDNA) in women’s plasma during preterm birth (PB) was studied. The purpose of this study was to identify the relationships between the investigated factors and determine their prognostic significance. Venous blood samples were collected from 45 women with PB and 35 women with full-term labor at 22–31 and 32–36 weeks of gestation, as well as from 17 women during labor at 39–40 weeks of gestation. The concentration of IL-2, IL-4, IL-6, IL-8, IL-10, GM-CSF, IFN-γ and TNF-α cytokines in peripheral blood plasma was measured by multiplex method. The level of cfDNA and cffDNA was evaluated using PCR analysis. It was found that, the level of IL-6, IL-8 and cfDNA in the blood was significantly increased in women with PB at 22–31 weeks of gestation (p = 0.044, p = 0.001, p < 0.001) and 32–36 weeks of gestation (p = 0.025, p = 0.001, p = 0.002) compared to women with physiological pregnancy at the same terms. The level of cffDNA (p = 0.014) was significantly increased in women with PB at 32–36 weeks of gestation. The IL-8 content had a significant correlation with the cfDNA level in women with PB at all stages of labor and with the cffDNA level in the group who gave birth at 32–36 weeks of gestation. There was no correlation between IL-8, cfDNA and cffDNA, but there was consistency with other cytokines at all studied terms and during delivery in the term-delivery group. The results of the study suggest that cfDNA is a potential marker of PB and show that the aberrant relationship between cfDNA and IL-8 may be important in the genesis of PB.

The DNA methylation profile of breast cancer differs from that in healthy tissue and can be used as a diagnostic and prognostic biomarker. Aim of the study: to compare gene methylation in small malignant breast tumors less than 2 cm and in healthy tissue and fibroadenoma. Methylation of the following 15 genes was studied: MAST1, PRDM14, ZNF177, DNM2, SSH1, AP2M1, CACNA1E, CPEB4, DLGAP2, CCDC181, GCM2, ITPRIPL1, POM121L2, KCNQ1, TIMP3. Methods: analysis was made by our modified MS-HRM method followed confirmation of the results by pyrosequencing. The genes were selected from publications that studied DNA methylation in breast cancer with high genome coverage. The study group included 48 samples of breast cancer, the control group included 24 samples of fibroadenoma and 24 samples of healthy tissue. Results: significant differences were found in methylation of 8 genes: CCDC181, GSM2, ITPRIPL1, ZNF177, CACNA1E, DLGAP2, TIMP3 (all р<0.001), and PRDM14 (р=0.002). The most accurate diagnostic value, based on logistic regression, was shown with the compound of two genes – CCDC181 and ZNF177 (AUC=0.99) in pyrosequencing analysis. Conclusion: small breast cancer tumors have a specific DNA methylation profile that distinguishes them from healthy tissue and benign proliferative lesions.

Preeclampsia (PE) is a leading cause of maternal complications and is diagnosed by clinical manifestation. The aim of the study was to evaluate changes of cell-free DNA (cfDNA) and cell-free foetal DNA (cffDNA) concentration during uncomplicated pregnancy and PE in one group of women, and define the predictive value for PE. A total of 580 women were prospectively evaluated, 20 of them developed PE and were included in laboratory analysis, and 22 healthy pregnant with uncomplicated pregnancy were included as the laboratory control group. We determined cfDNA and cffDNA in maternal blood at 11–14, 24–26, and 30–32 weeks. Level of cfDNA was evaluated by determining the RASSF1A gene using PCR analysis, cffDNA—by determining the hypermethylated part of RASSF1A gene. The concentration of cfDNA did not differ in the first and second trimesters but significantly increased at 30–32 weeks in both groups. During uncomplicated pregnancy, median cffDNA level increased from 14.15 GE/ml to 24.87 GE/ml (p = 0.002) and 32.62 GE/ml (p = 0.005). In the PE group, an elevation in cffDNA level was significant only in the second half of pregnancy (from 54.85 to 96.72 (p > 0.05) and 158.30 GE/ml (p = 0.031) at 11–14, 24–26, and 30–32 weeks, respectively). At all studied periods, cffDNA level in the PE group was significantly higher compared to uncomplicated pregnancy (р < 0.001). ROC analysis showed that a cut-off value of cffDNA concentration 22.54 GE/ml in maternal blood at 11–14 weeks of pregnancy had the greatest predictive value for PE prediction, with 85.0% sensitivity and 81.8% specificity. CffDNA is a promising marker for PE prediction from the first trimester of pregnancy.

Concentrations of eight different cytokines and the level of expression of CD86 and CD163 macrophages were studied in peritoneal fluid in women with endometriosis. It was found that the concentration of both inflammatory (IL-6, IL-8, TNF-α) and anti-inflammatory cytokines (IL-4) as well as the level of macrophage expression of the proinflammatory marker CD86 and anti-inflammatory marker CD163 increased in women with mild external genital endometriosis (1-2 stage), and did not differ from the control group in women with severe endometriosis (3-4 stage). The content of IL-2, IL-10, CM-CSF and IFN-γ in the peritoneal fluid of women with endometriosis did not differ significantly from the control group. The results of the study indicate that the development of external genital endometriosis may be based on insufficient both inflammatory and anti-inflammatory activity of macrophages in the peritoneal fluid.

We evaluated the content of cell-free fetal DNA in maternal blood and expression of ZBP-1 receptors in the placental tissue of women with uncomplicated pregnancy, preeclampsia, and preterm labor. The study included 16 women with preeclampsia (early and late-onset preeclampsia, 8 cases each), 16 women with preterm labor, and 21 women with uncomplicated pregnancy. The concentration of cell-free fetal DNA was measured by PCR by detecting hypermethylated region of the RASSF1A gene. Immunohistochemistry was performed on paraffin-embedded sections of the placenta samples using primary polyclonal antibodies to ZBP-1. Significant increase in the level of cell-free fetal DNA was found in women with preeclampsia (both early and late-onset form) in comparison with uncomplicated pregnancy. The concentration of cell-free fetal DNA in preterm labor group did not differ from the control group; however, it was significantly lower than in early-onset preeclampsia, but not late preeclampsia. Immunohistochemical study showed higher expression of ZBP-1 in the villus syncytiotrophoblast in early-onset preeclampsia in comparison with that in preterm labor group (p=0.006). Fragments of damaged placental cells, predominantly trophoblast, enter maternal circulation and are the source of cell-free fetal DNA and a potential ligand for ZBP-1, which leads to further cell damage and the formation of a vicious circle. The increase in the content of cell-free fetal DNA in maternal blood and ZBP-1 expression in the syncytiotrophoblast in preeclampsia are interrelated processes reflecting impaired morphofunctional state of the placenta.

We analyzed the concentration of extracellular DNA and its fractions in the dynamics of uncomplicated pregnancy Thirty women with singleton pregnancy were examined. The concentration of total, maternal, and fetal cell-free DNA in maternal blood was measured at gestation weeks 11-14, 24-26, and 30-32. The level of total cell-free DNA was evaluated by measuring the concentration of RASSF1A gene using quantitative PCR analysis, the level of cell-free fetal DNA was assessed by determining the hypermethylated part of RASSF1A gene. The concentration of total cell-free DNA and cell-free maternal DNA did not change during the first half of pregnancy, but increased after 24-26 weeks. The level of cell-free fetal DNA increased from the first to the second and third trimester: 14.15 (2.32-36.25), 24.87 (6.29-129.32), and 32.62 (8.97-133.52) GE/ml ( p <0.05), respectively. Our results characterize the dynamics of the content of cell-free DNA and its fractions during pregnancy, which should be taken into account when using cell-free DNA for prediction of placenta-associated complications.

We performed a complex analysis of total and fetal extracellular DNA, 8 cytokines (IL-2, IL-4, IL-6, IL-8, IL-10, granulocyte-macrophage CSF, IFNγ, and TNFα) in blood plasma obtained from women with preeclampsia prior to labor onset. Total (sensitivity 89.47%, specificity 93.75%) and fetal extracellular DNA (sensitivity 73.68%, specificity 87.5%) were the most accurate parameters determining preeclampsia. We revealed a high correlation (p=3×10—6) between total and fetal extracellular DNA levels in the group of preeclampsia. Preeclampsia significantly increased the levels of macrophage factors IL-10 and IL-6. These cytokines significantly correlated with the levels of total and fetal extracellular DNA in the preeclampsia group. In the control group, such correlations were not observed. These findings obtained suggest that preeclampsia develops upon increased macrophage activity, leading to destruction of the placenta trophoblast cells.

α7-Nicotinic acetylcholine receptors (α7nAChR) have been discovered and studied in the human endometrium for the first time. The expression of these receptors at the level of mRNA has been shown in the endometrial tissue by polymerase chain reaction and at the protein level by Western blot. It has been found that the addition of carbachol, an agonist of acetylcholine receptors, or PHA 543613, a selective activator of α7nAChR, to the primary culture of endometrial cells leads to regulated pulse secretion of matrix metalloproteinase- 9.