Vol 24, No 3 (2021)

There is currently no clear understanding of the molecular participants providing cytotoxic and/or cytostatic activity of neutrophils (Nph) in relation to tumor cells. Cytokines produced by neutrophils are necessary for their paracrine and autocrine interactions with surrounding cells. In order to assess the effect of regulatory neutrophilokines on the morphofunctional state of circulating Nph in benign ovarian tumors and ovarian cancer, the ELISA method was used to assess the level of IL-4, IL-6, IL-10, IL-8, IL-18, IFNγ, MCP-1 and MMP-1in neutrophils, expression of CD11b, CD63, CD16, CD95. Determined the rigidity of the membrane and the ability of neutrophils to form NET. Statistical processing of the obtained data was carried out using the software Statistica 13.0, Jamovi 1.6.5.0. It was found that in ovarian cancer the rigidity of neutrophils depends on the level of IL-10, MCP-1, IL-18, IL-8, IL-4, IFNγ, IL-6 in neutrophils. The method of multiple regression revealed the dependence of the ability to form NETs on the level of IL-4 and IL-6 in Nph. Revealed an inverse correlation between the rigidity of the membrane Nph and their ability to form traps in ovarian cancer. In benign ovarian tumors, a noticeable direct correlation was found between the rigidity of the neutrophil membrane and the adhesive marker CD11b. In ovarian cancer, a correlation was found between the rigidity of the Nph membrane and the CD63 degranulation marker. At benign ovarian tumors, no correlations were found between the number of activated neutrophils and the level of intracellular cytokines in Nph. In ovarian cancer, correlations were found between the number of CD11b+Nph and the level of IL-6, IL-8 in them; between the amount of CD63, CD95 and intracellular IL-8. The amount of CD16+Nph correlated with the level of MMP-1 and IL-8 in Nph. The amount of CD95+Nph correlated with the level of IL-18 in Nph. Thus, the change in the level of neutrophilokines in benign ovarian tumors did not correlate with changes in the ability to NETosis, expression of activation markers, but was accompanied by an increase in the rigidity of the membrane of circulating neutrophils. In ovarian cancer, an increase in IL-8 correlated with a decrease in CD16 expression and an increase in CD63; a decrease in CD16 correlated with an increase in MMP-1. An increase in membrane rigidity in ovarian cancer was associated with changes in all considered neutrophilokines (IL-4, IL-6, IL-8, IL-10, IL-18, MCP-1, IFNγ). The combination of IL-4, IL-6, IL-18 indices, NET number and membrane rigidity of circulating Nph (according to the results of multivariate analysis) can be used for differential diagnosis of ovarian cancer.

High incidence of newborns with inborn heart defects (HD) and sufficient contribution of this disorder into perinatal, infant and pediatric mortality, as well as disability levels, even after radical surgical treatment determine significance of search for novel methods of prediction and prevention of appropriate HD risks at the stage of pregnancy planning. HLA-G is among key molecules participating in immune interactions between maternal microenvironment and embryos. Maximal antibody titers for HLA antigens is detected in multiparous women. However, the question remains open, which HLA molecules participate in blockage of immune inflammation in the mother-embryo system: either by maternal antibodies, or by donor immune globulins. Hence, the aim of our study was to obtain enriched γ-globulin preparation from blood of multiparous women and evaluation of its activity towards female HLA-G molecules. The γ-globulin fraction (GGF) was obtained from blood plasma of muliparous women by means of affine chromatography using DEAE Affi-Gel Blue system (BioRad, USA). We have formed 2 groups: a control group (14 healthy males), and experimental group of 14 women who gave birth to the children with inborn heart defects.
The changes of HLA-G expression on lymphocytes exposed to GGF were evaluated according to a flow cytometry protocol with calculation of percentage values using appropriate quotients. Evaluation of functional activity exerted by the GGF concentrate in control group showed inhibition of HLA-G expression on CD3+ and CD--lymphocytes against effects of autologous serum. GGF effects in experimental group upon HLA-G expression were differently directed, e.g., GGF sufficiently inhibited membrane HLA-G expression on the CD3-negative lymphocytes, compared to control group. Meanwhile, GGF showed stimulatory effect upon CD3⁺-lymphocytes, or it did not show any inhibitory action. The preparation obtained may serve as prototype for intravenous infusion. Moreover, in future one may use such immunoglobulin preparations, both for immune prophylaxis of non-syndromal sporadic HDs at the pregragravidary stage, as well as at early terms of pregnancy. The therapeutic effect will achieved due to blockage of embryoblast HLA molecules available to recognition. 

This study aims for assessing relationships between maternal HLA-G gene polymorphisms (rs41551813, rs12722477, rs41557518) and intrauterine infection with the risk of congenital malformations (CM) in infants. We studied 331 women who had offspring with CMs, and 408 women with one or more healthy children. Influence of the intrauterine infection was analyzed on the basis of laboratory tests. Diagnostics of bacterial vaginosis and vulvovaginal candidiasis by microscopic examination were conducted. Viral infections (herpes simplex virus type 2, cytomegalovirus, human papilloma virus type 16/18) as well as Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum, Gardnerella vaginalis; Trichomonas vaginalis and Toxoplasma gondii were detected by enzyme-linked immunoassay or polymerase chain reaction (PCR) techniques. The data were obtained from the medical cards of the surveyed women. The gene polymorphisms were typed for Thr31Ser (rs41551813, HLA-G*01:03) in exon 2, Leu110Ile (rs12722477, HLA-G*01:04) and 1597 delС (rs41557518, HLA-G*01:05N) in exon 3 HLA-G using real-time PCR followed by melting analysis. The study showed that maternal age was not a significant risk factor for CMs in the fetus/newborns. Meanwhile, the maternal intrauterine infections were shown to be a significant risk factor for CMs in their infants (OR = 1.57 (1.08-2.29); p = 0.002). It was found that the 110 Ile allele (HLA-G *01:04) was a risk factor for CMs incidence in the fetus/newborns (OR = 1.57 (1.08-2.29), p = 0.01). No association was found between the maternal rs41551813 and rs41557518 HLA-G genetic polymorphisms and CMs in the infants. Hence, intrauterine infections and maternal 110 Ile allele (HLA-G *01:04) may be suggested as risk factors for birth defects in the children. Our results will be useful in understanding the molecular mechanisms of immune disorders in feto-maternal interface.

Increasing evidence shows that seminal plasma is among the most important immunoregulatory factors in female reproductive function. We suggest that the favorable effect of the partner’s seminal plasma (SP) upon pregnancy occurence in women during the cycle of in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) may be provided by the balanced content of Th1/Th2-dependent seminal cytokines. Otherwise, in case of pathologic changes in SP composition, it may negatively affect the IVF efficiency. Our aim was to determine whether the levels of seminal IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-18, IFNγ, TNFα, TNFβ and TGF-β1 are associated with pregnancy establishment in female patients exposed to seminal plasma during IVF/ICSI cycle.
Twenty-eight female patients were exposed to seminal plasma via sexual intercourse before the day of oocyte retrieval, and also underwent intravaginal application of seminal plasma just after transvaginal puncture and oocyte retrieval. Quantitative measurement of seminal cytokines was performed by FlowCytomix™ technology. IL-1, IL-18 and TNFβ concentrations were significantly higher in non-pregnant group (p = 0.011; p = 0.030 and p = 0.008, respectively). The contents of IL-2, IL-6, IL-18 and TNFβ per ejaculate were also significantly higher in non-pregnant group (p = 0.020; р = 0.042; р = 0.030 and р = 0.004, respectively). We conclude that elevated concentrations of proinflammatory cytokines in seminal plasma, as well as their total excessive content per ejaculate may have an adverse effect on implantation and pregnancy establishment. 

External genital endometriosis (EGE) is currently considered one of the most common disorders in women at their reproductive age. EGE is a chronic disease that affects about 10% of women of reproductive age, leading to functional and structural changes in reproductive system, infertility, pelvic pain, and affects their quality of life. Moreover, the patients with endometriosis often suffer from depression, especially, due to pain syndrome. Endometriosis is associated with dysregulation of T-cell immune response, which presents as altered interactions between T-cells, macrophages, NK (natural killer cells), B-cells, thus allowing ectopic implantation of endometrial cells. The pronounced negative effect of EGE on the quality of life of patients, their reproductive function, impaired adaptation of the patients in community, and growing costs of treatment determine social significance of the disease and importance of appropriate studies. It is obvious that disorders of the immune system play a significant role in IGE development. However, despite extensive research in this area, the processes of apoptosis in IGE have not been sufficiently studied. Therefore, the aim of our study was to assess the origin of apoptosis disorders in women with stage I-II and III-IV of external genital endometriosis. We examined 71 patients with EGE which were divided into 2 groups: group 1 included the patients with EGE stage I-II (n = 31), and group 2, the patients with stage III-IV EGE (n = 40). The control group included 24 patients without EGE. The population profile of peripheral blood lymphocytes was determined by laser flow cytometry using Immunotex reagents, i.e., FITC-labeled CD8+, CD16+, and PE-labeled CD95+ antibodies. The number of lymphocytes entering apoptosis was detected using a diagnostic kit for AnnexinV+ (FITC) and PI+ from Caltag. The results were read on a BECKMAN COULTER EPICS XL-II flow cytometer using standard protocols.
An imbalance between activation and apoptosis of immunocompetent cells may be a significant cause of EGE. Induction of apoptosis by the Fas-FasL pathway in patients with external genital endometriosis leads to decreased cytotoxicity mediated by T-lymphocytes and NK-cells. A statistically significant decrease in the content of AnnexinV+ presenting lymphocytes suggests impairment of their receptor-dependent apoptosis.
Altered apoptosis processes may be responsible for the development of IGE by promotion of proliferation and growth of endometrioid implants.

The development of effective methods for prediction, diagnostics and treatment of female reproductive disorders is an urgent task. Natural antiglycan antibodies (AGAT) are of great interest in both diagnostic and therapeutic aspects, since AGATs are very diverse, and their specificities were selected in the course of natural evolution. In this work, we investigated the possibility of using glycoarray technique, as well as the signature approach to predict effectiveness of therapy in breast cancer (BC), as well as a targeted search for natural antibodies with therapeutic potential.
We studied blood serum samples of apparently healthy female donors (n = 27), and patients with established diagnosis of metastatic breast cancer prior to starting therapy (n = 29). The median age of the patients was 48 years, 41% had “ER/PR+”-status, 59% – “ER/PR-“-status. The median age of healthy subjects was 50 years. The patients received combined therapy with doxorubicin and herceptin with different outcomes: 11 patients did not respond to treatment and 18 patients showed clinical response (the tumor was not revealed). For the study with AGAT, glycoarray was used, on which more than 200 different glycans were printed. The antibodies bound to the ligands were detected using biotinylated goat antibodies against human Ig (G+M+A). To search for a combination of diagnostically significant AGATs (signatures), the previously developed calculation tool “Immunoruler” was used.
An opportunity of using glycoarray to predict efficiency of therapy was studied in breast cancer patients. The study included patients receiving combination therapy with doxirubicin and herceptin, with clinical response monitored at 18-24 weeks. A signature consisting of 10 AGATs with high sensitivity and specificity (90 and 91%, respectively) proved to predict efficiency of the administered therapy.
The possibility of breast cancer diagnosis using AGAT has been further confirmed. The specified signature included five antibodies: the level of two AGATs was significantly higher in patients than in donors, which could be adaptive antibodies developed in response to emerging malignancy. For three other antibodies, the registered signals in patients were lower than in healthy controls, thus, probably, indicating depletion of humoral immunity during the development of breast cancer. Hence, such AGATs may have some therapeutic potential, and, by usage of glycoarray screening technology, they could be searched in purposeful manner.

One of the new trends in the study of the pathogenesis of preeclampsia (PE) is the study of the development of glycopathology in the functional mother-placenta-fetus system. Given the importance of carbohydrate-protein interactions for the morphogenesis of the placenta, interactions in the immune system, and the formation of tolerance to fetal alloantigens, anti-glycan antibodies (AgAbs), which can interfere with these interactions, changing them, may play a special role in the pathogenesis of PE. Since the production of antibodies occurs against the background of existing natural antibodies, as well as adaptive ones acquired during life, it is obvious that there are a significant number of factors that are interrelated with AgAbs, which is important for the pathogenesis and identification of risk factors for the disease. Objective: to determine the relationship between the content of AgAbs in the blood and clinical and laboratory parameters in patients with physiological pregnancy and PE.
The study includes 146 pregnant women: the main group I consisted of 51 patients with moderate PE, the comparison group – 95 conditionally healthy pregnant women. Clinical and anamnestic data, peculiarities of the course of pregnancy, data of laboratory examinations, data of a representative spectrum of AgAbs were studied. AgAbs (IgG and IgM) were studied in serum using a glycoarray containing 473 glycans and 216 polysaccharides. To determine the relationship between the variables, the nonparametric Spearman rank correlation method was used for the analysis of quantitative data, and the Wilcoxon T-test for the analysis of qualitative data.
It was found that in the main group there were more correlations between the level of AgAbs of various specificities and clinical and laboratory parameters than in the comparison group. A burdened gynecological and infectious history, complications during pregnancy are associated with changes in the profile of AgAbs of both classes in patients whose pregnancy is complicated by moderate PE, which indicates the pathogenetic significance of these antibodies. In healthy pregnant women, the level of antibodies to a number of glycans is reciprocally related to the number of lymphocytes, platelets, and ALT, which may indicate the regulatory role of these antibodies, since lymphocytosis, thrombocytopenia, and increased transaminases in the blood are pathological conditions.
The revealed relationships between the AgAbs level and clinical-anamnestic and laboratory parameters indicate different patterns of correlation relationships in health and disease, which apparently indicates the pathogenetic