[Significance of evaluation of CD69 expression by peripheral blood lymphocytes for predicting pregnancy outcome in women with recurrent pregnancy loss]. / Znachimost' otsenki ékspressii CD69 limfotsitami perifericheskoi krovi dlia prognoza iskhodov beremennosti u zhenshchin s privychnym vykidyshem.

The aim of this work was to characterize phenotypically peripheral blood T- and NK lymphocytes expressing an early marker of activation, CD69, and assess the significance of CD69 expression for predicting pregnancy outcome in women with idiopathic reccurent pregnancy loss (IRP) before and after immunocytotherapy (ICT). The study group consisted of 36 patients with IRP who became pregnant after pre-gestational allimmunization, in 30 patients the pregnancy was prolonged to the full term and ended with the birth of a viable baby, in 6 - it was terminated before 12 weeks of gestation. In the control group, 15 fertile women outside pregnancy and 11 women at 12 weeks of physiological pregnancy were examined. Assessment of the CD69 expression in women with prolonged pregnancy revealed the absence of significant differences with the control group in the content and proportion of activated lymphocytes (CD69+). In women with aborted pregnancy after pre-gestational ICT, an increase in the number of almost all analyzed lymphocyte subpopulations responding to the activation stimulus, with a clear tendency to increase the proportion of activated T- but not NK-lymphocytes was found. At 5-6 weeks, the proportion of activated lymphocytes among a subpopulation of cytotoxic T-lymphocytes (CD3+CD8+/CD3+CD8+CD69+) in these women was significantly higher than in women with prolonged pregnancy, which confirms the leading role of effector cytotoxic T-lymphocytes in rejection reactions. Thus, the studies showed the promise of evaluating the expression of the early activation marker CD69 as an additional laboratory criterion for the personable appointment of immunocytotherapy to women with a common reccurent pregnancy loss.

Lymphocyte Activation in the Development of Immune Tolerance in Women with Recurrent Pregnancy Loss.

Association between lymphocyte activation and formation of immune tolerance, as well as pregnancy outcome, in the case of immunocytotherapy (ICT) was studied in women with idiopathic recurrent pregnancy loss (IRPL). The content and phenotypic characteristics of activated T lymphocytes and NK cells were investigated in the peripheral blood of IRPL patients with different pregnancy outcomes (pregnancy prolongation to the full term and habitual miscarriage). The fraction of activated cells in the subpopulation of cytotoxic T lymphocytes (CD3+CD8+/CD3+CD8+CD69+) before ICT was significantly lower in women who lost the pregnancy. After ICT, the fraction of these cells during weeks 5-6 of pregnancy in woman with miscarriage was higher than in women with pregnancy prolonged to the full-term. Excessive content of activated cytotoxic lymphocytes can be a mechanism underlying impaired maternal immunotolerance to fetal alloantigens, which is a leading factor of early pregnancy loss. The obtained data confirm the involvement of activated Th17 cells and FOXP3+ Treg cells in the formation of tolerance to paternal antigens of the fetus. Comparison of the decrease in the fraction of CD4+CD25highRORγt+ lymphocytes at the early gestation period (5-6 weeks) and significant upregulation of the IL-17 production by in vitro stimulated whole blood cells in women with miscarriage with the same parameters in women with prolonged pregnancy suggested an imbalance between pro-inflammatory Th17 cells and Treg cells. No such imbalance in the content effector T lymphocytes was observed in women with the full-term pregnancy. Taken together, our data indicate an important role of gestational activation of lymphocytes in the formation of maternal immune response to fetal alloantigens necessary for the prolongation of pregnancy.

Expression of Early Activation Marker CD69 on Peripheral Blood Lymphocytes from Pregnant Women after First Trimester Alloimmunization.

We studied the expression of an early activation marker CD69 in peripheral blood lymphocytes of pregnant women with a history of recurrent pregnancy loss after immunization with paternal lymphocytes. Spontaneous and phytohemagglutinin-stimulated expression of CD69 on the surface of T cells and NK cells isolated from the peripheral blood was analyzed. On gestation week 5-6, the number of T cells expressing CD69 spontaneously and after stimulation was significantly higher in women with miscarriage than in woman with prolonged pregnancy. However, the number of cells with CD56(+) phenotype expressing CD69 did not differ in these groups. No differences were found in the number of cells of all subpopulations expressing CD69 after stimulation on gestation week 12 in woman with full-term current pregnancy and in woman with physiological pregnancy.

Detection of Antileukocytic Antibodies in Blood Serum using Lymphocytes and Latex Microspheres Carrying HLA-Antigens upon Alloimmunization of Women with Recurrent Pregnancy Loss.

Anti-HLA-antibodies were detected using cross-reaction of blood serum with allogenic T and B cells and latex microspheres coated with HLA-I and HLA-II antigens. HLA+ and HLA-sera obtained from women before and after allogeneic immunization were tested. The results obtained by these methods significantly differed. The test with latex microspheres detected antibodies to HLA-I and HLA-II antigens with high sensitivity and specificity and can be used for assessment of clinical significance of alloantibody detection when using alloimmunization in the therapy of gestation disorders.

Detection of Antibodies In Vitro Binding to Endothelial Cells in the Sera from Women with Normal Pregnancy and Preeclampsia.

Activity of serum antibodies in vitro binding to endothelial cells in women with normal pregnancy and preeclampsia was studied. Flow cytometry detected peculiarities of antibody binding to endothelial cells in health and disease. Detection of antiendothelial antibodies in trimesters II and III can be diagnostically important in preeclampsia.

Functional deficit of sperm and fertility impairment in men with antisperm antibodies.

Autoimmune reactions against the sperm cells play an ambiguous role in fertility impairment. The objective of this study was to characterize functional deficit of sperm conditioned by antisperm immune response in normozoospermic men. This was a multi-centric, cross-sectional, сase-control study. The study subjects were 1060 infertile normozoospermic men and 107 fertile men. The main outcome measures were clinical examination, semen analysis including MAR test for antisperm antibodies (ASA), computer-aided sperm analysis, acrosome reaction (AR) detected with flow cytometry, DNA fragmentation measured with sperm chromatin dispersion, reactive oxygen species (ROS) assessed using the luminol-dependent chemiluminescence method. 2% of the fertile men had MAR-IgG ≥ 50%, but all subjects with MAR-IgG>12% were outliers; 16% infertile men had MAR-IgG ≥ 50% (p<0.0001). There was a direct correlation between the infertility duration and MAR-IgG (R=0.3; р<0.0001). The ASA-positive infertile men had AR disorders 2.1 times more frequently (р<0.02), predominantly inductivity disorders. We found signs of hyperactivation proportionate to the ASA level (p<0.001). DNA fragmentation was more highly expressed and was 1.6 and 1.3 times more frequent compared with the fertile and the ASA-negative patients, respectively (p<0.001 and p<0.05). We found signs of oxidative stress (OS): ROS generation by washed ASA-positive spermatozoa was 3.7 times higher than in the fertile men (p<0.00001) and depended on the ASA levels (R=0.5; p<0.0001). The ASA correlation with ROS generation in native sperm was weak (R=0.2; р<0.001). We concluded that autoimmune reactions against spermatozoa are accompanied by a fertility decrease in normozoospermia. This results from AR and capacitation disorders and DNA fragmentation. The pathogenesis of sperm abnormalities in immune infertility is associated with the OS of spermatozoa.

The role of the antisperm antibodies in male infertility assessment after microsurgical varicocelectomy.

Antisperm antibodies (ASA) are a cause of male infertility. ASA are often found in varicocele patients. The study objective was to assess the ASA role in fertility recovery after varicocelectomy. The longitudinal study involved 99 patients with varicocele. Patients were examined according to the WHO recommendations; ASA level was measured using the direct method of Sperm MAR test: 66 patients were ASA-negative, 33 had MAR-IgG ≥ 10%. All patients underwent microsurgical varicocelectomy. Student's t-test, Wilcoxon test, Chi-squared test and signed rank test were used for data analysis. The retrospective analysis of all operated patients data showed that the patients without spermiogram improvement after varicocelectomy had higher ASA levels. 3 months after the surgery, the initially ASA-negative varicocele patients demonstrated 2.5 times increase in number of progressive motile spermatozoa in the ejaculate (p < 0.001), accompanied by 6% decrease in abnormal sperm count (p < 0.05); the spermiogram parameters improved in 77% of cases (p < 0.01). After the surgery, ASA developed in 16% of cases (Max--MAR-IgG = 12%). The patients who were initially ASA-positive demonstrated ASA decrease only in half of the cases (16 of 33; p > 0.05). The main outcome in this group was a favourable response to the surgery (ASA level decrease) vs. no reduction in autoimmune process. The improvement in the ASA-positive group was demonstrated in the patients with higher varicocele grade (median--2 vs. 1; p < 0.05) and lower ASA level (MAR-IgG = 48% vs. 92%; p < 0.01). The pregnancy rate within a year after surgery was 2.8 times more frequent in couples with ASA-negative men: 39% (25 of 65) in the ASA-negative group compared to 14% (4 of 28) in the ASA-positive group (p < 0.05). Thus, antisperm immune response decreases the varicocelectomy efficacy for reproductive function recovery: the higher percentage of ASA and lower grade of varicocele are associated with an unfavourable prognosis.

Time course of the cytokine profiles during the early period of normal pregnancy and in patients with a history of habitual miscarriage.

The time course of the peripheral blood cytokine profiles was studied in patients with a history of habitual miscarriages and in normal gestation. Low levels of anti-inflammatory cytokines during the early periods of gestation were characteristic of patients with a history of habitual miscarriages; however, an anti-inflammatory shift of the cytokine spectrum developed by the end of the first trimester.

Optimal detection of serum antipaternal antileukocytic antibodies after injection of allogenic lymphocytes in women with habitual abortions.

The level of antipaternal antileukocytic antibodies detected by flow cytometry is a parameter reflecting the efficiency of alloimmunization of women with reproductive disorders during preparation to pregnancy. The results of evaluation of antipaternal antileukocytic antibodies by two modifications of the method are presented. The optimal method for detection of antipaternal antileukocytic antibodies after immunocytotherapy is selected.

Relationship between spermatozoon movement velocity and expression of testicular isoform of angiotensin-converting enzyme on their surface.

The expression of testicular isoform of angiotensin-converting enzyme on the surface of human spermatozoa was evaluated by flow cytometry with monoclonal antibodies to epitopes of C-terminal domain of human angiotensin-converting enzyme and the mean path, mean curvilinear and straight-line velocities were determined by computer analysis of spermatozoon movement. A positive correlation between the expression of testicular isoform of angiotensin-converting enzyme on the cell surface and spermatozoon movement velocity was revealed.

Quantitative study of testicular angiotensin-converting enzyme on the surface of human spermatozoa.

Expression of testicular angiotensin-converting enzyme on the surface of human spermatozoa was studied by means of flow cytometry with monoclonal antibodies. Expression of testicular angiotensin-converting enzyme on the cell surface depended on functional and morphological characteristics of spermatozoa.

Free radical generation in ejaculate samples from infertile patients.

Generation of free radicals in ejaculate samples from infertile patients was evaluated using chemiluminescent technique. The presence of antisperm antibodies in samples increased the possibility of damages to spermatozoon plasma membranes due to excessive generation of free radicals.

Antisperm antibodies and sterility: insoluble problem or perspective trend of research?

Conflicting opinions about the effects of antisperm antibodies on fertilization can be due to inadequacy of experimental approaches in evaluating the antisperm immunity. Detection of antisperm antibodies bound to the surface of live spermatozoa can be associated with aggregation of surface antigen-antibody complexes followed by metabolic activation of spermatozoa and acrosomal reaction, which impair cell resistance. New concept of antisperm immunity and its influence on reproduction can be formulated only after comprehensive studies of the mechanisms of spermatozoon response to binding of antisperm antibodies. Further improvement of quantitative assays of antisperm antibodies and evaluation of their effect on spermatozoon function should be aimed at selection of experimental conditions preventing changes in spermatozoa coated with antisperm antibodies during in vitro manipulations.

Antisperm antibodies detection by flow cytometry is affected by aggregation of antigen-antibody complexes on the surface of spermatozoa.

Flow cytometry (FCM) analysis of live antibody-coated spermatozoa subjected to immunofluorescence staining (FCM test) is considered an objective method for the quantitative detection of antisperm antibodies (ASA). But the cross-linking of cell surface antigen (Ag) with bivalent antibodies and/or antigen-antibody (Ag-Ab) complexes with second antibodies may induce the reorganization of surface components (patching and capping) and result in their shedding from the sperm surface. The present study estimates the relationship between aggregation of Ag-Ab complexes on the sperm surface and the results of indirect FCM test. Swim-up spermatozoa of normozoospermic men were incubated with ASA-positive sera from infertile patients and with second antibodies fluorescein isothiocyanate (FITC)-labelled goat anti-human IgG polyclonal antiserum under different conditions and then analysed by FCM and fluorescence microscopy. It was shown that low temperature, cytochalasin B, excess or lack of the primary and/or secondary antibodies and sperm fixation by paraformaldehyde may inhibit aggregation and shedding of Ag-Ab complexes and dramatically increase ASA quantity determined on the sperm surface. However, inhibition of aggregation on the live sperm surface was observed only in a minority of ASA-positive samples and was poorly reproducible using semen of different donors. A high probability of Ag-Ab complex shedding from the sperm surface during experimental manipulation limits the use of indirect FCM test for quantitative ASA determination.

Evaluation of stimulus-induced acrosome reaction by two-colour flow cytometric analysis.

Acrosome status in human spermatozoa from 20 normozoospermic men was evaluated by flow cytometry following the induction of the acrosome reaction with the ionophore A23187. Dual fluorescence staining of methanol fixed spermatozoa incubated with and without (control) the ionophore A23187 was performed with probes which targeted the outer acrosomal membrane (OAM) (rhodamine-labelled Arachis hypogaea agglutinin) or constituents of the acrosomal vesicle (fluorescein-labelled Pisum sativum agglutinin). Flow cytometry analysis revealed two major subpopulations of cells: acrosome-intact and acrosome-reacted spermatozoa after induction of the acrosome reaction. The intensity of green and red fluorescence in acrosome-reacted spermatozoa was significantly lower than that of the acrosome-intact control spermatozoa (P < 0.0001). The intensity of green fluorescence in the acrosome-intact subpopulation of spermatozoa was significantly higher than that of the control population (P < 0.002). Exposure of spermatozoa to the ionophore A23187 resulted in reliable enhancement of the number of spermatozoa with very high intensity of green and/or red fluorescence compared with the control (P < 0.03). An inverse correlation between the number of acrosome-reacted spermatozoa and spermatozoa with a very high intensity of green and/or red fluorescence was demonstrated (r = -0.631, P < 0.01). This method provides an objective and efficient procedure for quantitative estimation of the acrosomal status of human spermatozoa.

Flow cytometry study on the effect of serum and peritoneal fluid of women on sperm-binding activity of immunoglobulin G antisperm antibodies.

OBJECTIVE: To investigate the influence of sera and peritoneal fluids (PFs) from fertile and infertile women on the binding of antisperm antibodies to the surface of spermatozoa. DESIGN: The immunoglobulin (Ig) G antisperm antibodies binding to the surface of liver spermatozoa was evaluated after their incubation in antisperm antibodies-positive serum from an infertile male in the presence and absence of female sera or PFs. SETTING: Russian Scientific Center for Obstetrics, Gynaecology, and Perinatology. PATIENT(S): Serum and PF samples from fertile and infertile women; antisperm antibodies-positive serum from infertile men; high-quality fresh semen from healthy donors. INTERVENTION(S): Serum samples were obtained from fertile and infertile women and from infertile men. Peritoneal fluids were collected during routine laparoscopy. MAIN OUTCOME MEASURE(S): The proportion of spermatozoa positive for IgG antibodies and the quantity of antisperm antibodies on the sperm surface measured by flow cytometry (FCM). RESULT(S): The addition of sera from fertile or infertile women with endometriosis or pelvic adhesion disease to an IgG antisperm antibodies-positive male serum resulted in significant inhibition of the antisperm antibodies binding to the sperm surface. CONCLUSION(S): Sera of fertile as well as infertile woman contain factors that block IgG antisperm antibodies binding to the surface of live spermatozoa.