Hartmut Hopp

Diagnosis and treatment of peripartum bleeding

Abstract Severe peripartum hemorrhage (PPH) contributes to maternal morbidity and mortality and is one of the most frequent emergencies in obstetrics, occurring at a prevalence of 0.5–5.0%. Detection of antepartum risk factors is essential in order to implement preventive measures. Proper training of obstetric staff and publication of recommendations and guidelines can effectively reduce the frequency of PPH and its resulting morbidity and mortality. Therefore, an interdisciplinary expert committee was formed, with members from Germany, Austria, and Switzerland, to summarize recent scientific findings. An up-to-date presentation of the importance of embolization and of the diagnosis of coagulopathy in PPH is provided. Furthermore, the committee recommends changes in the management of PPH including new surgical options and the off-label use of recombinant factor VIIa.

Up-regulation of endothelial stretch-activated cation channels by fluid shear stress.

OBJECTIVE Stretch-activated cation channels (SAC) have been suggested to act as endothelial mechanosensors for hemodynamic forces. Ca(2+) influx through SAC could induce an intracellular Ca(2+) signal stimulating Ca(2+)-dependent synthesis of vasodilators like NO, prostacyclin, or EDHF. In the present study we tested whether laminar shear stress (LSS) regulates SAC function. METHODS Electrophysiological properties of SAC were investigated in human umbilical vein endothelial cells (HUVEC) subjected to defined levels of LSS in a flow-cone apparatus. RESULTS In HUVEC, we identified a Ca(2+) permeable SAC that was activated by membrane stretch. Single-channel current densities of SAC in cell-attached patches were significantly increased in HUVEC exposed to an LSS of 5 dyn/cm(2) for 4 h (1.15+/-0.17 SAC/patch) compared to HUVEC kept in stationary culture (0.46+/-0.07 SAC/patch). Exposure of HUVEC to a higher LSS of 15 dyn/cm(2) for 4 h induced similar up-regulation of SAC (1.27+/-0.21 SAC/patch). After 24 h exposure to LSS of 15 dyn/cm(2), single-channel current densities of SAC remained up-regulated (1.07+/-0.18 SAC/patch) compared to controls. In addition, stretch-sensitivity of SAC (channel activity NP(o) at -30 mmHg) significantly increased after 2 h of exposure to LSS of 5 and 15 dyn/cm(2) and remained up-regulated after 24 h. Inhibition of protein kinases and tyrosine kinases by H7 and genistein, respectively, prevented LSS-induced alteration of SAC function. CONCLUSION Single-channel current density and mechanosensitivity of SAC in HUVEC is up-regulated by LSS. Up-regulation of SAC function leads to enhanced mechanosensitive Ca(2+) influx, and represents a novel adaptive mechanism of the endothelium in the presence of altered hemodynamic forces.

Stretch-activated cation channel in human umbilical vein endothelium in normal pregnancy and in preeclampsia

Objective To determine whether stretch-activated cation channels (SAC) are present in intact human umbilical vein endothelium (HUVE) and in an endothelial cell line (EA.hy) and whether they act as endothelial mechanosensors, and to determine whether endothelial SAC in HUVE from women with pregnancies complicated by preeclampsia undergo functional changes compared with those in HUVE from women with normotensive pregnancies. Methods and results By use of the patch-clamp technique we identified a SAC in intact HUVE and in an endothelial cell line. The SAC had mean conductances of 29 ± 5 pS (n = 38) for K+ and 12 ± 2 pS (n = 4) for Ca2+. Administration of 50 μmol/l gadolinium, a blocker of mechanosensitive ion channels, completely blocked activity of this channel. We found from single-channel recordings that influx of Ca2+ through SAC directly activated high-conductance Ca2+-dependent potassium channels, proving that a significant influx of Ca2+ through SAC occurs at physiologic concentrations of Ca2+. In a comparative study, apparent channel density of SAC (percentage of patches with SAC activity) in HUVE from women with pregnancies complicated by preeclampsia (36.2 ± 4.3%) was twofold higher than that in HUVE from women with normal pregnancies (17.9 ± 2.9%, P < 0.01). Channel conductance and sensitivity to stretching of SAC were not altered by preeclampsia. Conclusions Since SAC are capable of acting as endothelial mechanosensors, the greater than normal density of SAC associated with preeclampsia might reflect an alteration of mechanotransduction.

Antibiotic therapy for preterm premature rupture of membranes – results of a multicenter study

Abstract Aims: To evaluate whether primary application of mezlozillin in preterm premature rupture of membranes (pPROM) prolongs pregnancy and lowers neonatal morbidity. Methods: In this prospective, randomized, double blind, placebo-controlled multicenter study a total of 105 pregnant women with pPROM between 24+0 and 32+6 weeks of gestation were examined receiving i.v. injections of corticoids and tocolytics as well as mezlocillin (3×2 g/d) or placebo. Assessed factors were prolongation of pregnancy and neonatal morbidity such as neonatal infection, respiratory distress syndrome (RDS), intraventricular hemorrhage (IVH) and necrotizing enterocolitis (NEC). Results: Prolongation of pregnancy by more than 7 days was achieved in 63.8% of the mezlocillin group versus 44.8% of the placebo group (P<0.05). The babies of mothers treated with anibiotics had fewer neonatal infections, RDS, IVH and NEC. Total morbidity was significantly lowered in the verum group (P=0.02). Conclusions: Antibiotic administration following preterm premature rupture of membranes is associated with a prolongation of pregnancy and a reduction of neonatal infectious morbidity.

Preeclampsia is associated with loss of neuronal nitric oxide synthase expression in vascular smooth muscle cells of the human umbilical cord

Aims : Umbilical blood vessels are not innervated and regulation of blood flow to the placenta must depend on structural changes and the effect of vasoactive factors. Failure to achieve these adaptations may result in reduced fetoplacental perfusion. The purpose of this study was to determine whether neuronal nitric oxide synthase (nNOS) is expressed in human vascular smooth muscle cells (VSMCs) of the fetoplacental circulation. nNOS has been described as a non‐endothelial NOS counterregulating vasoconstriction only in the VSMCs of animal models. Therefore, we investigated nNOS expression in the fetoplacental unit from preeclamptic and healthy pregnancies.

Binding of antibodies against high and low molecular weight cytokeratin proteins in the human placenta with special reference to infarcts, proliferation and differentiation processes

Recent immunocytochemical studies have shown that placental villous trophoblasts contain the high molecular weight cytokeratin (CK) proteins 5/6 and 17. In the case of CK 17, trophoblastic immunostaining was positive in villi covered by fibrinoid. CKs 5/6 and 17 are expressed by hyperproliferative cells. The aim of this investigation was to examine the location of these CKs in placental infarcts, known to be demarcated by fibrinoid and hyperproliferative trophoblasts. The results were compared with those obtained by immunostaining against Ki-67, tenascin and α1-, α6- and β1- integrins, which are involved in cell proliferation, differentiation and regenerative processes. Furthermore, the expression of the single CKs 7, 8, 10, 13, 14, 18 and 19 was investigated by immunocytochemistry and immunoblotting. While low and high molecular weight CKs were present in villous and extravillous trophoblasts, only low molecular weight CKs were detected in vascular and extravascular placental smooth muscle cells. Placental infarcts revealed different immunoreactivities in the infarct margin and centre: high molecular CKs, tenascin, Ki-67 and oncofoetal fibronectin predominated in the infarct margin, low molecular CKs, fibrin and integrins in the centre. The expression of tenascin and a defined change in the expression of CK 17 indicates villous repair and hyperproliferative mechanisms in placental infarcts.

Histochemical evaluation of placental angiotensinase A in pre-eclampsia: Enzyme activity in villous trophoblast indicates an enhanced likelihood of gestational proteinuric hypertension

The aim of this study was to examine whether differences in placental angiotensinase A (glutamyl aminopeptidase, EC 3.4.11.7) activities occurred in hypertensive complications of pregnancy compared with uncomplicated pregnancies. Biochemical and semiquantitative histochemical methods were used and compared for their applicability. Angiotensinase A activity was detected using L-alpha glutamyl-4-methoxy-2-naphthylamide (alpha-Glu-MNA) as substrate and Fast Blue B salt for simultaneous azo-coupling in cryostat sections of placental tissue samples from 32 patients with pre-eclampsia, 11 patients with pregnancy-induced hypertension and 44 participants with uncomplicated pregnancies. The graduated intensity of reaction product in the villous trophoblast and in fetal blood vessels was evaluated semiquantitatively in a double-blind study by light microscopy (semiquantitative score method). Score levels were related to relative frequencies of hypertensive disorders (proportional odds model) and correlated to the severity of gestational hypertension (Spearmans rank correlation). After detection of enzyme activity, the same tissue samples were homogenized and used for kinetic fluorometric measurements under the same substrate and buffer conditions as in enzyme histochemistry. Enhanced villous trophoblastic angiotensinase A activity was significantly associated with an increased frequency of pre-eclampsia in pregnant women (cumulative odds ratio x 0(1) 6.37; P < 0.001) and showed significant correlations with the severity of gestational hypertensive disorders, represented by systolic (r = 0.31; P < 0.05) and diastolic (r = 0.34; P < 0.05 blood pressure and by concomitant proteinuria (r = 044; P < 0.01). Histochemical evaluation of fetal blood vessels and biochemical measurements revealed no statistically significant results. In conclusion this study demonstrates for the first time that increased villous trophoblastic angiotensinase A activity indicates an increased likelihood of the presence of pre-eclampsia and the severity of hypertensive disorders in pregnancy.

Antibodies to cytokeratins bind to epitopes in human uterine smooth muscle cells in normal and pathological pregnancies

Cytokeratin antibodies have been widely used for the identification of trophoblast cells in the placental bed, following their invasion from the developing conceptus. Their identification centres upon the expression of cytokeratin in epithelial cells, from which trophoblast cells are derived. Our recent observations indicate that this strict relationship may be more complex than was thought. Cryostat and paraffin sections of human decidua and myometrium, taken from the placental bed and the uterotomy cut, were examined immunocytochemically for cytokeratins using ten antibody clones selected to identify different cytokeratin proteins and antigenic epitopes. Biopsy specimens were obtained from normal and pathological pregnancies (pre‐eclampsia, fetal retardation, amnioninfection, hysterorrhexis, placenta praevia) at the time of caesarean section (26–41 weeks of pregnancy). Antibodies against nine clones, CAM 5.2, MNF 116, AE1/AE3, CK5, KS‐B17.2, CY‐90, M20, E3, and 34βE12 identified, as expected, syncytial giant cells and mononuclear trophoblasts within the placental bed and glandular epithelial cells throughout the uterus. In addition, they stained numerous fusiform cells that were classified by established criteria to represent smooth muscle cells, both within blood vessels and myometrium. No staining differences were observed between normal and pathological disorders. These results indicate that cytokeratin antibodies CAM 5.2, MNF 116 and AE1/AE3, and other antibodies targeting proteins 8 and 18, cross‐react with epitopes expressed in cells other than giant trophoblastic cells and mononuclear trophoblasts in the uterus and, thus, caution has to be used when such antibodies are used for the diagnostic characterization of tissues related to the placental bed.

Interleukin-8 in urine: a new diagnostic parameter for intra-amniotic infection after premature rupture of the membranes

Interleukin‐8 (IL‐8), a 72 amino acid peptide secreted by cells of the immune system and of the amnion, chorion and decidua, was measured in women in late pregnancy. IL‐8 was detected in the urine of 91 of 104 women with premature rupture of the fetal membranes, with values exceeding 1000 ng/L in cases of severe intra‐amniotic infection. Women with urinary tract infections were excluded. The routine measurement of IL‐8 in urine, together with C‐reactive protein in serum, thus provides a low risk and technically simple approach to the assessment of intra‐amniotic infection.

Indication and results of insulin therapy for gestational diabetes mellitus

The aim of this study was to determine whether amniotic fluid insulin concentration (AFI) is a better parameter than mean maternal blood glucose values (MBG) for deciding about insulin therapy in patients with gestational diabetes. MBGs were calculated on the base of 9 blood glucose levels during a 24 hour period after one week of diet therapy. In a prospective trial between 1987 and 1989 in Karlsburg, 123 gestational diabetic patients were randomized into two groups. Treatment was either based on the concentration of AFI or MBG levels. In a second series in Berlin, 103 patients were offered amniocentesis. 81 patients agreed and 22 refused. Treatment was then analogous to that in Karlsburg. In both groups of the randomized population, strict metabolic control was achieved. There was no difference regarding pregnancy complications. Earlier labor induction and higher cesarean section rates were seen in the non-invasive group (p < 0.05). The incidence of diabetic fetopathy and neonatal hypoglycemia was significantly lower in the invasive group (p < 0.01), even though the metabolic control parameters did not differ between the two groups. The results in Berlin correspond to these findings. In conclusion, AFI enables the recognition of any hyperinsulinism reaction to the maternal metabolic situation. We recommend the additional measurement of the AFI concentration between 28 and 36 weeks as the direct fetal parameter for deciding about insulin treatment.