Human miscarriage is associated with increased number of CD26 decidual lymphocytes.

Dipeptidyl peptidase-IV (DPP-IV, CD26), a serine protease with broad distribution in mammalian tissues and known activity in serum, participates in T-cell activation and promotes a Th1-like cytokine response. Previous data on murine abortion indicate that DPP-IV may play a critical role in pregnancy failure by inducing a Th1 local response. Here, we investigated the possible participation of DPP-IV in the onset of human spontaneous abortion (SA). The systemic (peripheral blood) and local (decidua) percentages of CD4(+), CD8(+), CD26(+) and CD56(+) cells as well as the number of Th1 lymphocytes (CCR5(+) cells) were assessed in samples from women after SAs (n = 20) and from women with normally progressing pregnancies (NPs) (n = 27) using flow cytometry and immunohistochemistry. We further measured the DPP-IV activity and concentrations of Th1 (interferon-gamma and tumour necrosis factor-alpha), Th2 [interleukin-4 (IL-4), IL-10] and Th3 (transforming growth factor-beta2) cytokines in serum samples. We could not find any difference in the number of CD4(+), CD8(+), CD26(+), CD26(+)/CD4(+) or CD8(+)/CD26(+) blood cells between NP and SA patients. No differences in the Th1, Th2 or Th3 cytokine levels could be observed between both groups. However, the percentages of decidual CD26(+) lymphocytes as well as the number of decidual Th1 cells were significantly higher in SA samples compared to samples from patients with NP. Our data support the hypothesis that CD26(+) decidual lymphocytes with DPP-IV activity may play a critical role in SAs, as previously suggested in an abortion mice model. This abortive effect may be mediated by enhancing the levels of Th1 abortogenic cytokines only locally.

Distribution of immunocompetent cells in decidua of controlled and uncontrolled (choriocarcinoma/hydatidiform mole) trophoblast invasion.

PROBLEM: Pregnancy has been considered as a model of successfully controlled tissue invasion where trophoblast cells infiltrate the maternal decidua without being rejected or without destroying the tissue. In choriocarcinoma (CC) and hydatidiform mole (HM), a dysregulation of invasive (malignant/benign) trophoblast cells is present. Immunocompetent cells (IC) are known to be involved in rejection pathways of malignant cells and can also be identified in early pregnancy decidua. The aim of the present study was to identify the phenotype of IC in decidua of women with normal pregnancy (NP), CC and HM. METHODS: Immunocompetent cells were detected by immunohistochemistry in decidual tissue from first trimester NP (n = 10), CC (n = 12) and HM (n = 11) using antibodies against CD8+, CD3+, CD56+, CD68+ cell surface markers and mast cell tryptase (MCT). A scaled eye piece was used for cell counting to obtain semiquantitative results. Statistical analysis was performed using Wilcoxon rank/Mann-Whitney tests. RESULTS: We observed a significantly increased number of lymphocytes positive for CD8, CD3 and MCT positive granulocytes in CC and HM compared with the samples from NP (all P < or = 0.001). Lymphocytes positive for natural killer (NK) cell marker CD56 were significantly decreased in CC and HM versus NP (P < or = 0.001). The number of CD68 positive cells (macrophages) were not significantly different among the tissue pools. CONCLUSION: The increase of CD8/CD3 T cells and mast cells in CC and HM and the decrease of CD56 cells, compared with NP, suggests the necessity of a balance between T and NK cells in controlling trophoblast invasion.

Heme oxygenases in pregnancy II: HO-2 is downregulated in human pathologic pregnancies.

PROBLEM: We previously reported a diminished expression of the heme-degrading enzymes heme oxygenases (HO)-1 and HO-2 in decidua and placenta from mice undergoing Th1-mediated abortion, strongly indicating the protective effect of HO in murine pregnancy maintenance. Here we investigated whether the expression of HO-1 and HO-2 is also reduced at the feto-maternal interface of pathologic human pregnancies. METHOD OF STUDY: Immunohistochemistry was used to detect HOs expression in placental and decidual first-trimester tissue from patients with: spontaneous abortion (n = 14), choriocarcinoma (n = 14), hydatidiform mole (H-mole) (n = 12), compared with normally progressing pregnancies (n = 15). Further, we investigated early third-trimester decidual and placental tissue from patients with pre-eclampsia (n = 13) compared with fetal growth retardation (n = 14) as age-matched controls. RESULTS: In first trimester tissue, we observed a significant reduction of HO-2 expression in invasive trophoblast cells, endothelial cells, and syncytiotrophoblasts in samples from patients with spontaneous abortion compared with normal pregnancy. H-mole samples showed a diminished expression of HO-2 in invasive trophoblast cells and endothelial cells in comparison with NP, whereas choriocarcinoma samples showed no significant differences compared with the control. In third trimester tissue, HO-2 was also reduced in syncytiotrophoblasts and invasive trophoblast cells from pre-eclampsia compared with samples from fetal growth retardation. HO-1 expression was diminished in all pathologies investigated; however, the differences did not reach levels of significance. CONCLUSIONS: Our data indicate that HOs play a crucial role in pregnancy and low expression of HO-2, as observed in pathologic pregnancies, may lead to enhanced levels of free heme at the feto-maternal interface, with subsequent upregulation of adhesion molecules, allowing enhanced inflammatory cells migration to the feto-maternal interface.

Th1 cytokines and the prothrombinase fgl2 in stress-triggered and inflammatory abortion.

PROBLEM: The immune system contributes to the outcome of pregnancy by complex immunological interactions. Cytokines especially influence the immune milieu pro or contra pregnancy. T helper 1 (Th1) cytokines [tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma)] cause inflammation and together are thought to threaten the maintenance of pregnancy. It has been proposed that increased levels of these Th1 cytokines activate coagulation via up-regulating the novel prothrombinase, fgl2. This study further investigates the Th1 cytokine up-regulation of fgl2 expression in a pathophysiological, stress induced abortion model, and an inflammatory, interleukin-12 (IL-12) triggered abortion model. METHOD: The DBA/2J-mated CBA/J female mice were exposed to sonic sound stress or were injected with IL-12 during early gestation. On day 13.5 of pregnancy the uteri were removed and the resorption rate was calculated. We evaluated TNF-alpha, IFN-gamma, fgl2 as well as IL-12 messenger RNA (mRNA) expression in decidual samples of all mice by quantitative, real-time polymerase chain reaction (PCR). RESULTS: A similar resorption rate of 24% was detected in stressed mice, as well as in IL-12 injected mice compared with approximately 11% in non-stressed, non-injected control mice. In stressed mice compared with controls, we observed on day 13.5 up-regulated TNF-alpha, unchanged IFN-gamma down-regulated fgl2, and a slightly increased levels of IL-12. In the IL-12 triggered abortion model, we observed up-regulated levels of TNF-alpha, IFN-gamma and fgl2. CONCLUSION: These novel data suggest two distinct cytokine patterns leading to similar abortion rates. A physiological cascade associated with up-regulation of TNF-alpha, and an IL-12-triggered cascade characterized by persistent up-regulation of TNF-alpha and IFN-gamma as well as a persistent increase in fgl2.

Activation of the novel prothrombinase, fg12, as a basis for the pregnancy complications spontaneous abortion and pre-eclampsia.

PROBLEM: Impaired trophoblast invasion during the first trimester of pregnancy is linked to spontaneous abortion, and defective invasion in the second trimester to hypertension + proteinuria (pre-eclampsia). Hypertension developing during the third trimester of human pregnancy represents, in part, a corrective response in the mother to provide adequate placental perfusion for fetal growth when trophoblast has not to invaded and converted the myometrial porprtion of maternal spiral arteries into to low resistance-high capacitance conduits. Deportation of vesicles from hypoxemic trophoblast is thought to cause hypertension plus proteinuria, vascular damage and a systemic coagulopathy. Trophoblast invasion may be inhibited by local cytokines, such as TGF-betas but Thl-type cytokines associated with pre-eclapmsia and spontaneous abortions (e.g., IL-1, TNF-alpha, IFN-gamma) are not known to inhibit migration at in situ concentrations. Trophoblast invasion is also inhibited by the binding of surface integrins to fibronectin and fibrin, and fibrin production is stimulated by these Th1 cytokines via up-regulation of prothrombinases(s) such as fg12 which directly and via TNF-alpha-facilitated inflamation compromise trophoblast cell integrity. We, therefore, asked if fg12 expression and TNF-alpha are increased in first trimester human miscarriage and in third trimester pre-eclampsia. METHODS: fg12 mRNA was detected using in situ hybridization and fg12 protein by immunohistochemistry. TNF-alpha mRNA and protein were similarly tested. The techniques were validated using uterine sections from day 8.5 of CBA x DBA/2 pregnancies, and then were applied to sections of placentae from normal and pre-eclamptic pregnancies with and without intrauterine fetal growth restriction (IUGR). Fibrin was detectectd by immunohistochemistry. RESULTS: Expression of fg12 protein correlated with fg12 mRNA expression in mouse uteri and in placentae from normal human pregnancies. Increased expression of fg12 and TNF-alpha mRNA and protein, and increased fibrin deposition was detected in placental trophoblast. CONCLUSIONS: Activation of fg12 prothrombinase by Th1-type cytokines in pregnancy may lead to spontaneous abortion, or in ongoing pregnancy, to pre-eclampsia and/or IUGR.

Stress and immune mediators in miscarriage.

BACKGROUND: Stress is thought to be abortogenic and psycho-neuro-immunological pathways have been suggested to be involved in triggering miscarriages. From experiments in pregnant mice exposed to stress some insights into the underlying mechanisms have been gained, delineating immunological imbalances as a cause of pregnancy failure. In order to test the validity of the conclusions drawn from murine experiments and the role of stress in human pregnancy loss, the following study was performed. METHODS: We used an established perceived stress questionnaire and measured the stress score of women with a confirmed diagnosis of first trimester spontaneous abortion (n = 94). Decidual tissue was investigated by immunohistochemistry and in-situ hybridization to detect the presence and distribution of immunocompetent decidual cells [CD56(+) natural killer (NK) cells, CD8(+)and CD3(+) T cells, tryptase(+) mast cells (MCT(+)) and tumour necrosis factor (TNF)-alpha(+) cells]. The patient cohort was divided into women experiencing low or high levels of stress. RESULTS: In the decidua of women with high stress scores we observed significantly higher numbers of MCT(+), CD8(+) T cells and TNF-alpha(+) cells per mm(2) tissue (P < or = 0.05). No significant differences between individuals with lower or higher stress scores could be observed with respect to decidual CD56(+) NK and CD3(+) T cells. CONCLUSIONS: Using a questionnaire to score perceived stress in humans may be a valid approach to assess non-biased stress scores. Stress-triggered abortion in humans, identified by a questionnaire, can be linked to immunological imbalances.

Pregnancy as a model of controlled invasion might be attributed to the ratio of CD3/CD8 to CD56.

PROBLEM: Pregnancy can be considered as a model of successfully controlled tissue invasion. Cellular mediated immunity appears to regulate the controlled invasion of fetal trophoblast cells. In endometrium cancer, a dysregulation of invasive malignant cells can be observed. Since immunocompetent cells are known to be involved in recognition and rejection of 'non-self' antigens, we investigated the presence and distribution pattern of CD3, CD8, CD56, and CD68 positive cells in decidua from normal and failing pregnancies, compared with malignant and benign endometrium. METHOD OF STUDY: Decidual tissue from first trimester normal pregnancies (NP; n = 15) and abortion (AB; n = 12), endometrial samples from premenopausal women (NE; n = 8), and endometrioid adenocarcinoma (EA; n = 8) were examined by immunohistochemistry using monoclonal antibody against large spectrum cytokeratin, and against the receptors CD3, CD8, CD56 and CD68, respectively. RESULTS: In NP, we observed 32.5% CD3, 44.7% CD56, and 22.9% CD68+ cells. In AB, we found 36.9% CD3, 45.3% CD56, and 17.8% CD68+ cells. The differences in ratio between normal pregnancy and abortion were not statistically significant. In NE, we counted 39.5% CD3, 30.2% CD56 and 30.2% CD68 cells. In EA, we observed 47.9% CD3, 12.4% CD56 and 39.7% CD68+ cells. The decrease of CD56 positive cells in endometrioid adenocarcinoma was statistically significant. Interestingly, we found 4.1% of cells positive for CD8 in NP, 4.9% in AB, 22.7% in NE, and 48.2% in EA. CONCLUSIONS: The increase of CD8 cells in NE, and particularly in EA, and decrease of CD56 cells, compared with NP or AB, suggests an interaction between CD8 cells and CD56 cells. Studying different pathological situations in the uterus, such as malignancies or ectopic pregnancies, might help us to understand the mechanisms involved in the maintenance of pregnancy.

Erythropoietin in amniotic fluid as a marker of chronic fetal hypoxia.

OBJECTIVE: To determinate the erythropoietin concentration in amniotic fluid in normal pregnancies and pregnancies with suspected hypoxia. METHOD: The erythropoietin concentration of 164 samples of amniotic fluid was determined by ELISA. The samples were taken by amniotomy during birth, as well as amniocentesis conducted during prenatal care. RESULTS: A distribution of 1.07-7.29 U/l (10th-90th percentile) within the normal group (n = 106) was determined. Significantly elevated erythropoietin levels in amniotic fluid were determined in maternal hypertension (P = 0.039) and low birth-weight children (P = 0.0032). A correlation with the child's sex could be excluded. CONCLUSION: Elevated erythropoietin levels in amniotic fluid indicated chronic fetal hypoxia.

Computerized cardiotocography following vibroacoustic stimulation of premature fetuses.

OBJECTIVE: The purpose of this study was to examine the effect of vibroacoustic stimulation (VAS) on the fetal heart rates (FHRs) in a group of premature fetuses. STUDY DESIGN: The FHRs were analyzed using the Oxford Sonicaid Computer System 8000, 30 min preceding and then 3 times following VAS. RESULTS: The changes of the mean FHR after VAS in the premature group and the control group of term fetuses occurred in the first 10 min after VAS. The greatest increase in the number of accelerations occurred in both groups during the 11-20 min following VAS. The long-term variability increased significantly in all 3 study periods in premature fetuses and only in the periods of 0-10 and 11-20 min in the control group. The increase in short-term variability was similar in both groups and it was greatest in the period of 11-20 min. CONCLUSION: Most of the heart rate changes of premature fetuses occur between 11 and 20 min following VAS.

[Detection of erythropoietin in amniotic fluid]. / Nachweis von Erythropoietin im Fruchtwasser.

OBJECTIVE: To investigate the concentrations of amniotic fluid erythropoietin in normal and risk pregnancies. MATERIAL AND METHODS: The concentrations of erythropoietin were measured in 150 samples of amniotic fluid. The samples were obtained by amniocentesis and amniotomia and were analysed using an enzyme-linked immunosorbent assay (ELISA). Results were available within 6 hours. The intra-assay variation was 6.4%, the inter-assay variation 7.2%. RESULTS: The range of erythropoietin concentration in all samples was between 0.23 and 80 U/L and in a defined group of normal pregnancies between 1.20 and 6.53 U/L (10%-90% percentile). Correlation was found between the concentration of erythropoietin and maternal hypertension (p = 0.0159), amnion infection syndrome in combination with premature birth (p = 0.0593), fetal growth retardation (p = 0.784), and base-excess (p = 0.0487). Elevated erythropoietin concentrations were found in a defined risk group with Apgar scores below 7 after 1 minute (p = 0.072) and after 5 minutes (p = 0.0037). There is a connection between postpartal transfer to the intensive-care unit and elevated erythropoietin concentrations (p = 0.073). No influence is exercised by the child's sex on the concentration of erythropoietin. No significant connection was found between the level of erythropoietin and smoking during pregnancy, volume of amniotic fluid and maturity at birth. A critical erythropoietin concentration could be postulated at 12 U/L. Children with higher levels showed heavy and severe disorders. CONCLUSION: Elevated erythropoietin levels in amniotic fluid indicate prolonged fetal hypoxaemia. Using the ELISA-technique a rapid prepartal determination of such situations is possible and might be helpful in the clinical procedure.