Blood Of Healthy Pregnant Women Research Articles

Evaluation of preadipocyte factor-1 (Pref-1) level in cord blood of newborns born by mothers with gestational diabetes mellitus (GDM)

BackgroundGestational diabetes mellitus (GDM) is the most common metabolic complication, which leads to short and long-term consequences in both mother and fetus exposed to hyperglycemia. The aetiology of this condition is proposed to be based on the dysfunction of the adipose tissue, which is characterised by the aberrant generation of adipokines. One of them is preadipocyte factor-1 (Pref-1), which could mediate controlling the adaptation of the maternal metabolism to pregnancy.AimsThe study aims to examine the level of Pref-1 in the cord blood of healthy pregnant women’s neonates and fetuses born to mothers with GDM.Materials and methodsCord blood samples were collected from 30 newborns of mothers with GDM and 40 newborns of healthy pregnant women. Pref-1 concentrations were measured with an ELISA kit.ResultsFetal Pref-1 concentrations were significantly lower in newborns of mothers with GDM compared to the normal pregnancy group children (5.32 ± 0.29 vs. 7.38 ± 0.53; p < 0.001). Mothers with GDM had a significantly higher index of BMI before pregnancy, maternal gestational weight gain, and maternal fasting glucose. In-depth analysis through multiple variant linear regression revealed a significant association between fetal serum Pref-1 levels, exposure to GDM, and gestational age.ConclusionThese findings contribute valuable insights into maternal-fetal health and pave the way for more targeted and effective clinical interventions.

ROLE OF GLYCODELIN IN REGULATION OF MYELOIDDERIVED SUPPRESSOR CELL DIFFERENTIATION

Glycodelin (PP14, PAEP, alpha-2-microglobulin, dimeric glycoprotein with molecular weight of 42 to 56 kDa) is considered as a reproductive tissue receptivity marker. Despite that glycodelin immunosuppressive effects are well-known there still remains uncovered its role in myeloid suppressor cell (MDSC) regulation. MDSC represent the heterogeneous population of immature myeloid cells that acquire suppressor phenotype while inhibiting the immune response under the pathological states. MDSC are known to play an essential role in supporting the immune tolerance in pregnancy and at transplantation. Our hypothesis suggests that glycodelin is capable of inducing the MDSC formation as the level of these cells is elevated during the successful pregnancy, whereas the spontaneous abortion and progression of eclampsia are associated with low circulating glycodelin. Therefore, the aim of the work was to analyze the role of recombinant glycodelin in physiological concentrations in regulation of MDSC differentiation. Peripheral blood mononuclear cells of donor volunteers were separated via centrifugation on density gradient of 1,077 g/cm3 (Ficoll-Hypaque, Sigma-Aldrich) to obtain MDSC generation in vitro. Then cells obtained were cultured in 24-well plate at a concentration of 1 × 106 cell/ml in complete medium with cytokines IL-6 (20 ng/ml), GM-CSF (40 ng/ml) therein for 14 days at 37 °C and 5% CO2. Medium replacement was made by 7th day in culture followed by cytokine re-introduction, and on the 11th day recombinant glycodelin in physiological concentrations (0,2; 2 mkg/ml) was applied while the pharmacological concentration was 50 mkg/ml. The M-MDSC (LinHLA-DRCD33+CD11b+CD14+CD66b- ) and PMN-MDSC (LinHLA-DRCD33+CD11b+CD14- CD66b+) level was evaluated in cultures using flow cytometry (СytoFlexS (Beckman Coulter)) and “R&amp;D Systems” antibodies according to standard protocol. Statistical data processing was realized with GraphPad Prizm software using Friedman test. It was found that glycodelin did not significantly affect cell viability being assessed with flow cytometry (PI). It was revealed that high GdA concentration (50 mkg/ml) being pharmacological did not render significant effect on MDSC differentiation. Meanwhile, glycodelin in concentrations correspanding the healthy pregnancy (0,2; 2 mkg/ml) was stated to increase the MDSC percentage in induced cultures of human mononuclear cells. When analyzing the subsets it was disclosed that this effect was conditioned by the increase in PMN-MDSC level while the M-MDSC level remained significantly unchanged. This result could be interpreted as glycodelin fetoprotective effect as the increase of the PMN-MDSC level is associated with the suppression of the immune response to paternal antigens. The PMN-MDSC level is known to be elevated in peripheral blood of healthy pregnant women at all the stages of pregnancy as compared to nonpregnant subjects whereas the M-MDSC amount remains unaltered. Meanwhile, patients with miscarriage demonstrated more that by 30% lowering in the MDSC amount in blood and endometrium and in I trimester, in particular. During the physiological pregnancy PMN-MDSC accumulate in placenta, but at spontaneous abortion their number is found to be declined. Placental PMN-MDSC efficiently suppress the T-cell response while concurrently polarizing the CD4+ lymphocytes in Th2 phenotype. PMN-MDSC are suggested to play an essential role in inducing and supporting the tolerance to fetal antigens that allows considering these as promising target of therapeutical manipulation in pregnancy complications. As a whole, we have originally demonstrated the GdA effect on MDSC differentiation.

Evaluation of elabela, apelin and nitric oxide findings in maternal blood of normal pregnant women, pregnant women with pre-eclampsia, severe pre-eclampsia and umbilical arteries and venules of newborns

Pre-eclampsia is multisystem metabolic diseases, commonly accompanied by hypertension and proteinuria, which are among the important causes of maternal and perinatal mortality and morbidity worldwide. In a pre-eclampsia animal model study in the last year, Elabela (ELA) infusion was reported to correct hypertension and proteinuria and to normalise the birth weights of the offspring. Therefore, our main goal in this human study is to compare ELA, apelin (APLN) and nitric oxide (NO) levels in the maternal blood of pregnant women with pre-eclampsia and severe pre-eclampsia and in their newborns’ venous-arterial cord blood with maternal blood of healthy pregnant women and their newborns’ venous-arterial cord blood. Thirty controls, 28 pre-eclampsia and 24 severe pre-eclampsia cases and their newborns participated in this study. Maternal blood and newborn venous-arterial cord blood samples were collected from these patients. ELA, APLN and NO levels in these samples were measured by ELISA method. When the maternal blood ELA, APLN and NO amounts were compared with control groups, there was a significant decrease in both pre-eclamptic and severe pre-eclamptic women and this was more prominent in the women with severe pre-eclampsia. When ELA, APLN and NO levels in the newborn venous-arterial cord blood of control group was compared with that of severe pre-eclamptic and pre-eclamptic women; it was parallel with maternal findings. ELA, APLN and NO levels appear to play a role in the pathophysiology of pre-eclampsia. It is predicted that if these molecules, which are reduced due to pre-eclampsia and severe pre-eclampsia, are brought to physiological limits in the future; pre-eclampsia related maternal and perinatal mortality and morbidity can be reduced.Impact StatementWhat is already known on this subject? There are two studies (one human and one animal) in the literature evaluating only maternal elabela (ELA) levels in pre-eclamptic pregnancies. The animal study demonstrated decreased blood ELA levels in pre-eclamptic animals and the human study found increased blood ELA levels in pre-eclamptic patients. There are no studies evaluating maternal ELA levels in severe pre-eclampsia patients and also there are no studies evaluating maternal ELA levels in pre-eclampsia and severe pre-eclampsia patients. There are no studies evaluating newborns’ venous-arterial blood APLN and NO levels. Apelin (APLN) and nitric oxide (NO) results were controversial in pre-eclampsia and severe pre-eclampsia patients.What the results of this study add? The present study, for the first time, demonstrates that decreased blood ELA, APLN and NO levels in maternal blood of pregnant women with pre-eclampsia and severe pre-eclampsia and in their newborns’ venous-arterial blood. Furthermore, we have also demonstrated for the first time that decreased ELA, APLN and NO are also related with low birth weights.What the implications are of these findings for clinical practice and/or further research? The low levels of ELA, APLN and NO in maternal blood and newborns’ venous-arterial blood may be the result or the cause of pathologic changes in pre-eclampsia and severe pre-eclampsia. Also, ELA, APLN and NO may be new indicator parameters of systemic endothelial dysfunction together. More studies are needed to evaluate the relationship between of ELA, APLN and NO and pre-eclampsia and severe pre-eclampsia and in newborns’ venous-arterial blood.

Kinetics of Helios(+) and Helios(-) T regulatory cell subsets in the circulation of healthy pregnant women.

Regulatory T cells (Tregs) play a critical role in the maintenance of a pregnancy. While the kinetics of the number of peripheral blood Tregs has been satisfactorily described in mouse models, analysis of these cell populations in human pregnancy is complicated by high variability in the quantity of Tregs and inconsistencies in the markers used for detecting different types of Treg. In the light of this, we set out to investigate the kinetics of various types of Treg, including CD45RA, GARP and PD-1(+) Tregs, in the peripheral blood of pregnant women in the first, second and third trimester, and at the time of delivery. Tregs, defined as a CD4(+)CD25(++)CD127(dim)Foxp3(+) population of leucocytes, were detected using flow cytometry. Natural thymus-derived Tregs and induced Tregs in the peripheral blood were distinguished by the expression or absence of a Helios marker, respectively. Our results showed that during normal pregnancy the sizes of various Treg subpopulations varied across women and also in an individual woman did not remain constant but varied significantly, most notable being the decrease observed at the time of delivery. Helios(-) cells were significantly less frequent in the peripheral blood of healthy pregnant women than Helios(+) cells, and the majority of Tregs were Helios(+)PD-1(+) Tregs.

The CYCLOCALYX study: Ovulatory cycle affects circulating compartments of the endothelial glycocalyx in blood.

The endothelial glycocalyx (EGX) plays an important role in vascular integrity. Recently, increased levels of EGX components were detected in the circulating blood of healthy pregnant women and were related to the increased tendency to edema formation during gestation. However, the EGX has not yet been systematically studied in non-pregnant women during ovulatory cycles. Serum levels of EGX components syndecan-1, heparan sulfate, and hyaluronan in healthy women (n=16) at 3 phases of the ovulatory cycle (early follicular phase, at ovulation, and mid-luteal phase) were compared with a control group of healthy men (n=10). Using immunofluorescence microscopy in cultured human umbilical vein endothelial cells, the effects of progesterone and estrogen on the EGX were measured. Syndecan-1 increased from 11.1±2.4ng/mL at ovulation to 12.6±2.3ng/mL in mid-luteal phase (P=.031) and of heparan sulfate from 663±35ng/mL to 782±55ng/mL (P=.011). In contrast to estrogen, there was a detrimental effect of progesterone on the EGX in HUVECs. The relationship between the natural menstrual cycle and the EGX as an indicator of vascular permeability may provide a new explanation for premenstrual edema in healthy women. This may be an attendant phenomenon of a regular physiological process, the hormonal downregulation of the vascular barrier during pregnancy.

Blood Ferritin Levels in Pregnant Women and Prediction of the Development of Fetal Intrauterine Growth Restriction

Blood Ferritin Levels in Pregnant Women and Prediction of the Development of Fetal Intrauterine Growth RestrictionIntrauterine growth restriction is one of the leading causes of perinatal morbidity and mortality. Prediction of intrauterine growth restriction is one of the priority tasks of perinatal protection. The purpose of this study was to evaluate the levels of serum ferritin in pregnant women, which could point to a group of patients in whom possible development of fetal growth restriction could have been expected. In this investigation, we conducted a prospective study of healthy pregnant women between 30 and 32 gestational weeks, who were estimated for ferritin values. Newborn infants of low birth weight for gestational age were recorded in 8.1%. Anemia was not present in any of the patients who delivered low birth weight babies. Ferritin serum levels in mothers of the babies with low birth weight were on average 6.42 μg/L higher than in the mothers with appropriate for gestational age babies (p&lt;0.005). ROC analysis of newborn infants birth weight and maternal blood ferritin levels showed that blood ferritin level had good predictive value. In case the recorded maternal blood ferritin values are above 13.6 μg/L, we can assume with the sensitivity of 64.7% and specificity of 91.7%, that the pregnant woman will develop a condition of intrauterine growth restriction. The missing decrease of ferritin values, erythrocytes, hemoglobin and hematocrit in the blood of healthy pregnant women between 30 and 32 gestational weeks, can with high probability point to the development of fetal intrauterine growth restriction.

Gamma/deltaT-cell subsets, NKG2A expression and apoptosis of Vdelta2+ T cells in pregnant women with or without risk of premature pregnancy termination.

Potentially cytotoxic Vdelta2+ T lymphocytes recognize human leukocyte antigen-E on the trophoblast via their CD94/NKG2A receptors. This study aims at determing the percentage of gamma/delta T-cell subsets, their NKG2A and Annexin V positivity in peripheral blood of healthy pregnant women and women at risk of premature pregnancy termination. Peripheral Vdelta2+ cells from healthy pregnant women and from women at risk of premature pregnancy termination were tested for the KIR NKG2A and Annexin V positivity by flow cytometry. The percentage of viable Vdelta2+ T cells was higher, that of Vdelta1+ T cells was lower in women at risk of premature pregnancy termination than in healthy pregnant women. The percentage of NKG2A + Vdelta2+ T cells was significantly lower in pregnant women at risk of premature pregnancy termination than in normal pregnancy. These data suggest the involvement of gamma/delta T lymphocytes in the pathogenesis of premature pregnancy termination.

Dynamics of antibody nuclease activity in blood of women during pregnancy and lactation.

In human milk we previously found catalytic antibodies (abzymes) catalyzing hydrolysis of DNA, RNA, NMP, NDP, and NTP and also phosphorylation of proteins and lipids. In the present study we have analyzed nuclease activities of antibodies in blood of women during pregnancy and lactation. Blood of healthy male and female volunteers lacked catalytically active antibodies, whereas antibodies from blood of pregnant women hydrolyzed DNA and RNA and their relative activity varied over a wide range. Relative blood abzyme activities significantly increased after delivery and at the beginning of lactation. The highest abzyme activity was observed in blood of parturient women. Although the dynamics of changes in antibody DNase activity during pregnancy was rather individual for each woman, there was a common trend in the increase in antibody activity in the first and/or third trimester of the pregnancy. The DNase activity of IgG and IgM from blood of healthy pregnant women was 4-5 times less than that from pregnant women with pronounced autoimmune thyroiditis.

Recognition of Nonclassical HLA Class I Antigens by γδ T Cells During Pregnancy

The healthy trophoblast does not express classical HLA-A and HLA-B products; therefore, an MHC-restricted recognition of trophoblast-presented Ags is unlikely. In the decidua and also in peripheral blood of healthy pregnant women, gammadelta T cells significantly increase in number. We investigated the possible role of gammadelta T cells in recognition of trophoblast-presented Ags. PBL and isolated gammadelta T cells from healthy pregnant women as well as from those at risk for premature pregnancy termination were conjugated to choriocarcinoma cells (JAR) transfected with nonclassical HLA Ags (HLA-E, HLA-G). To investigate the involvement of killer-inhibitory/killer-activatory receptors in trophoblast recognition, we tested the effect of CD94 block on cytotoxic activity of Vdelta2(+) enriched gammadelta T cells to HLA-E- and/or HLA-G-transfected targets. Lymphocytes from healthy pregnant women preferentially recognized HLA(-) choriocarcinoma cells, whereas those from pathologically pregnant patients did not discriminate between HLA(+) and HLA(-) cells. Normal pregnancy Vdelta2(+) T cells conjugated at a significantly increased rate to HLA-E transfectants, whereas Vdelta2(+) lymphocytes from pathologically pregnant women did not show a difference between those and HLA(-) cells. Blocking of the CD94 molecule of Vdelta2(+) lymphocytes from healthy pregnant women resulted in an increased cytotoxic activity to HLA-E-transfected target cells. These data indicate that Vdelta2(+) lymphocytes of healthy pregnant women recognize HLA-E on the trophoblast, whereas Vdelta1 cells react with other than HLA Ags. In contrast to Vdelta2(+) lymphocytes from healthy pregnant women, those from women with pathological pregnancies do not recognize HLA-E via their killer-inhibitory receptors and this might account for their high cytotoxic activity.

The role ofγ / δT cells in the feto-maternal relationship

Polymorphic MHC is absent from the trophoblast, therefore, it resists NK as well as CTL-mediated lysis in vitro. Activated γ / δ TCR positive cells are significantly enriched in the decidua as well as in peripheral blood of healthy pregnant women. Human peripheral γ / δ lymphocytes preferentially express the V γ 9/V δ 2 TCR, whereas those of the decidua use the V δ 1 chain. These subpopulations are functionally polarized, the former being Th1, the latter Th2. Potentially cytotoxic V δ 2+ lymphocytes recognize HLA-E on the trophoblast via the CD94/NKG2A receptor, which induces an inhibitory signal, thus potentially inhibiting Th1 type cytokine production.

PAI-2 inhibits the chemiluminescence of phagocytes and suppresses autoimmunity

Activated phagocytes participate in inflammation and in the lysis of tissue or fibrin. An important inflammation mediator is singlet molecular oxygen ( 1O 2), an excited oxidant that emits light when it reverts to its ground state. Other products of activated macrophages are urokinase (u-PA) and plasminogen activator inhibitor (PAI)-2. PAI-2 in physiological concentrations inhibited the hydrogen peroxide-dependent chemiluminescence (CL) of human phagocytes: 1 unit/ml (about 10 ng/ml) of PAI-2 decreased the CL of 2 × 10 5neutrophil granulocytes to about 50%. Preincubation of PAI-2 with excess amounts of urokinase, resulting in complexed PAI-2, also decreased CL. 5 units of PAI-2 injected intraperitoneally in mice suppressed, 2 or 24 h post injection, the CL of stimulated peritoneal macrophages by 48 or 67% respectively. Rats with experimental allergic encephalomyelitis (EAE), an animal model for the human disorder multiple sclerosis (MS), were treated with PAI-2. The inhibitor was injected intraperitoneally (1) at days 0–9 or (2) at days 7–16 after EAE induction. At 17.4 ± 1.5 days after EAE induction, non-treated control rats died. In contrast, 40 out of 40 animals treated with ≥50 units of PAI-2/ animal/ day survived. Treatment with 100 units of PAI-2/ animal/day resulted in less paralytic complications, especially when PAI-2 was injected at days 7–16 after EAE induction. Women with MS have a decreased disease intensity in pregnancy. PAI-2 is elevated in blood of healthy pregnant women (about 30 units/ml PAI-2 at term). Imitation of this physiologic immunsuppression by therapeutic application of PAI-2 could be indicated in a broad range of human diseases of autoimmune character, such as MS. The present findings could have relevance for the understanding of the pathogenesis of autoimmunity.

The role of gamma/delta T-cell receptor-positive cells in pregnancy: part II.

We have previously demonstrated a significantly increased ratio of gamma/delta T-cell receptor (TCR)-positive progesterone receptor(PR)-positive cells in the peripheral blood of healthy pregnant women compared to that of recurrent aborters or non-pregnant individuals. Treatment of pregnancy lymphocytes with a pan anti-gamma/delta TCR antibody inhibits progesterone-induced blocking factor (PIBF) production, increases natural killer (NK) activity, and alters the cytokine profile. The present study was aimed at investigating the role of the different gamma/delta subpopulations in these phenomena. Peripheral blood lymphocytes from healthy pregnant women were incubated with either anti-gamma1.4 and delta1, or anti-gamma9 and delta2 antibodies. The effect of these treatments on PR induction and interleukin (IL)-10 and IL-12 expression were tested by immunocytochemistry. NK activity of anti-gamma/delta treated lymphocytes was also determined. In peripheral blood of healthy pregnant women, the most frequently occurring chain combination was gamma1.4/delta1, whereas in recurrent aborters, the gamma9/delta2 combination was predominant. Treatment of normal pregnancy lymphocytes with a mixture of gamma1.4 and delta1 antibodies resulted in a significantly reduced NK activity and increased PR and IL-10 expression, whereas treatment with a mixture of gamma9 and delta2 antibodies significantly reduced IL-10 production and slightly increased IL-12 production and NK activity. These data suggest the presence of two functionally distinct subpopulations in the peripheral blood of pregnant women.

The role of gamma/delta T cell receptor positive cells in pregnancy.

Due to the lack of classical HLA antigens on the trophoblast, fetal antigens are possibly presented in a non major histocompatibility complex (MHC) restricted way. Decidual gammadelta T cells, which significantly increase in number during pregnancy, might play a role in recognition of fetal antigens and also in determining the quality of the response to these antigens. Our study was aimed at investigating the role of this cell population in progesterone-dependent immunomodulation. Peripheral lymphocytes from healthy pregnant women and from habitual aborters were tested by immunocytochemistry for the presence of gamma/delta T cell receptor (TCR) and progesterone receptor. To investigate the effect of treatment with a pan anti gamma/delta antibody, lymphocytes were incubated for 3 hr with the antibody, and then interleukin (IL)-10, IL-12 and progesterone-induced blocking factor (PIBF) expression (by immuno-cytochemistry) as well as natural killer (NK) cell activity were determined. In peripheral blood of healthy pregnant women the percentage of gamma/delta TCR+ cells was significantly higher (P < 0.001) than in that of recurrent aborters or of non-pregnant individuals. Ninety-seven percent of gamma/delta TCR+ pregnancy lymphocytes expressed progesterone receptor. Binding of a specific antibody to the gamma/delta TCR inhibited PIBF- as well as IL-10 production, whereas it increased NK activity and IL-12 expression. These data suggest the role of gamma/delta TCR-bearing lymphocytes in progesterone-dependent immunomodulation.

The expression of a progesterone-induced immunomodulatory protein in pregnancy lymphocytes.

The immunological effects of progesterone are mediated by a protein, named the progesterone-induced blocking factor (PIBF). The PIBF blocks NK activity in vitro and therefore prevents the abortive effect on high NK activity in mice. Increased NK activity has been suggested to play a role in pregnancy termination; thus NK inhibitory effect of the PIBF should contribute to the maintenance of normal gestation. This study was designed to investigate the relationship between n vivo PIBF-producing capacity and in vitro cytotoxic activity of pregnancy lymphocytes, as well as the clinical status or the outcome of pregnancy. Lymphocytes of 168 pregnant women (96 normal pregnancies, 16 showing clinical symptoms of threatened preterm pregnancy termination, 46 recurrent aborters, and 10 women sampled at the onset of spontaneous abortion or preterm delivery) were isolated on Ficoll-Paque gradient. The lymphocytes were tested for reactivity with a PIBF-specific antibody by immunocytochemistry, and simultaneously for cytotoxic activity to human embryonic fibroblast targets. The percentage of PIBF-positive lymphocytes in peripheral blood of healthy pregnant women was significantly higher than in that of women at risk for premature pregnancy termination. In peripheral blood of patients undergoing spontaneous pregnancy termination at the time of sampling, and in those of women showing symptoms of premature pregnancy termination we found lower than normal percentage of PIBF-positive cells. PIBF expression of the lymphocytes showed an inverse correlation with NK activity, and the rate of PIBF positive lymphocytes was related to the outcome of pregnancy. These data suggest a strong relationship between PIBF producing capacity as well as NK activity of the lymphocytes and the success of gestation.

Androgens in the peripheral venous blood of healthy pregnant women and of those with late gestoses

Androgens in the peripheral venous blood of healthy pregnant women and of those with late gestoses