Tocolytic therapy with fenoterol induces selective down-regulation of beta-adrenergic receptors in human myometrium.

Tocolytic therapy with beta-adrenergic receptor agonists is a standard regimen to prevent preterm birth. Agonists exposure of beta-adrenergic receptors causes receptor desensitization in other organs, and this may limit the therapeutic value of beta-adrenergic receptor agonists. To study the effects of prolonged beta-adrenergic agonist treatment in human myometrium, we obtained biopsies during Caesarean section of 14 pregnant patients who had received fenoterol for at least 5 days and 14 untreated pregnant controls. The densities of total beta-adrenergic receptors, which are mainly of the beta 2-subtype as assessed by [125I]iodo-cyanopindolol binding in crude membrane fractions, were more than 50% smaller in women receiving fenoterol, whereas alpha 2-adrenergic receptor densities were similar. Gs and Gi G-protein alpha-subunit densities were unaltered as assessed by Western blotting and pertussis toxin-catalyzed [32P]ADP-ribosylation. beta-Adrenergic receptor kinase (beta ARK) activity, as determined using bovine rhodopsin as the substrate, was the same in the two groups. Adenylyl cyclase activities in the presence of guanine nucleotides, NaF, forskolin, or Mn+2 were also not altered by fenoterol treatment. The messenger RNA (mRNA) concentrations of beta 2-adrenergic receptors, beta ARK-I and glyceraldehyde-3-phosphate dehydrogenase (as a reference), as determined by quantitative PCR, were unaffected by fenoterol treatment. We conclude that tocolysis with fenoterol results in a selective down-regulation of myometrial beta-adrenergic receptors, which is not associated with a reduction in the respective mRNA concentrations or alterations of alpha 2-adrenergic receptors, Gs and Gi alpha-subunits, or beta ARK activity or mRNA.

Optimized sensitivity of allele-specific PCR for prenatal typing of human platelet alloantigen single nucleotide polymorphisms.

PCR using sequence-specific primers (PCR-SSP) is widely employed for the genotyping of single nucleotide polymorphisms (SNPs) in both routine diagnosis and medical research. The human platelet alloantigens (HPAs) represent SNPs in platelet-specific glycoproteins, and HPA-1, -2, -3, and -5 are the most relevant in immunohematology. In most protocols, the respective HPA-SNPs are analyzed in allele-specific reactions, each with at least 100 ng DNA. In many cases, prenatal HPA typing in the diagnosis of neonatal alloimmune thrombocytopenia is often limited by the restricted amounts of fetal DNA that are obtainable. We developed a novel PCR-SSP technique to achieve accurate HPA genotypes using only 1 ng DNA per reaction. The concentration of HPA-specific primers was increased to 1 microM each and exhibited a higher sensitivity compared to a commercial PCR-SSP kit. The modified PCR-SSP technique enabled the identification of fetal HPA genotypes using only 0.5 mL amniotic fluid (from week 16 of gestation) and from a maternal plasma sample (from week 38 of gestation). The principle of the modified PCR-SSP technique may also be applied for the genotyping of other SNPs from limited amounts of DNA.

5% Me2SO is sufficient to preserve stem cells derived from cord blood.

On the basis of historical findings, 10% dimethylsulfoxide is still used to cryopreserve stem cells. We studied a final concentration of 5% dimethylsulfoxide in cryopreservation of cord blood-derived stem cells in autologous plasma. In our opinion, a final concentration of 5% dimethylsulfoxide in autologous plasma without further additives is sufficient for cryopreservation of cord blood stem cells in a banking routine.

Proliferative Merkel cells were not detected in human skin.

The fetal development of Merkel cells-neuroendocrine cells of the skin - has been a matter of debate for a long time. Recent results have helped to confirm their intraepidermal development in humans. Simple epithelial cytokeratins (CK) nos. 8, 18, 19 and 20 are well established markers at the light microscopic level. These cells could be detected from fetal week 8 within the epidermis with an enormous increase during the following weeks. This gives rise to the question as to whether Merkel cells are undergoing mitoses or whether they are derived from basal keratinocytes. We studied fetal and adult skin using antibodies to simple epithelial CK and to Ki67, a human nuclear cell proliferation-associated antigen in an attempt to answer these questions. In human adult and fetal skin of various stages we could not detect any Merkel cells undergoing cell division. These results suggest that Merkel cells are postmitotic cells to be renewed from undifferentiated keratinocytes with stem cell characteristics.

Effect and duration of gestagen influence on adrenal cortex and ovary. A morphological study in the Syrian golden hamster.

Perinatal androgen application prevents the cyclic secretion of gonadotropins and leads to sterility. Androgenic side effects were recorded in some synthetic gestagens. In order to determine their extent, different synthetic gestagens (dydrogesterone acetate, medroxyprogesterone acetate, cyproterone acetate) and one androgen hormone (testosterone propionate) were administered to ten separate groups of male and female hamsters. Started perinatally, the treatment was continued with increasing doses till the end of the eight week. Matched groups of animals and controls were killed at the age of 8 weeks (end of treatment), 12 weeks, and 16 weeks. Gestagenic effects on the adrenals and ovaries were studied by gravimetry, light microscopy, and histometry. The adrenals are influenced by the glucocorticoid-like and androgenic side-effects of MP and CA, but DY produces no effects of this kind. All gestagens reduce the number of corpora lutea, but the androgenic side-effects do not prevent the production of corpora lutea. Adrenal and ovarian changes are reversible. The female hamster adrenal is a sensitive substrate for androgenic and glucocorticoid-like side effects of synthetic gestagens.

Prenatal HLA typing of uncultured amniocytes prior to the collection of related allogeneic cord blood.

DNA samples isolated from corresponding uncultured amniotic fluid, cord blood and maternal blood (n=5) were subjected to low resolution typing of the HLA-A, -B and -DRB loci by the polymerase chain reaction using sequence-specific primers (PCR-SSP). Furthermore, the effect of ethylene diamine tetraacetate disodium salt (EDTA) on the quality of genomic DNA isolated from amniotic fluid samples after long-term storage was evaluated. Unambiguous results of HLA typing could be achieved from all amniotic fluid samples stabilized with EDTA. PCR-SSP typing failed in DNA samples from amniotic fluid without the addition of EDTA. In all cases the fetal HLA type could be confirmed by the result from the corresponding cord blood typing. Contamination with maternal DNA led to additional weak PCR-SSP bands in one case, but data interpretation was still unambiguous. Reliable fetal HLA typing can be achieved directly from amniotic fluid and culturing of amniocytes is not required.

[Torsion of the pregnant uterus]. / Torsion des schwangeren Uterus.

Pathological version of the pregnant uterus is a rare complication. A case of 180 degrees sinistrotorsion of the uterus in the 39th gestational week is reported. The case was asymptomatic with the exception of preterm labor in the 30th week of pregnancy and was discovered during a cesarean section carried out because of myoma obstructing the birth canal. Mother and child were discharged in perfect health.

[Uterine rupture without predisposing factors after a single vaginal PgE2 administration in prolonged pregnancy]. / Uterusruptur ohne prädisponierende Faktoren nach einmaliger vaginaler PgE2-Applikation bei Terminüberschreitung.

The case of a 38-year old 3/1 gravida with prolonged pregnancy is discussed. Labour was induced with a prostaglandin (PgE2-) vaginal tablet 4 days after an oxytocin stress test had failed. After rapid labour development, imminent fetal asphyxia suddenly occurred, leading to an emergency cesarean section. A rupture of the left uterus wall rupture with laceration of uterine vessels was demonstrated. This is the first case report of a uterus rupture that happened in prolonged pregnancy without predisposing risk factors after a single PgE2 dose that was correctly placed into the posterior fornix.

[Internal drainage of urine in cases of complicated urinary stasis caused by pregnancy]. / Le drainage interne de l'urine en cas de stase urinaire compliquée provoquée par une grossesse.

In 14 women with sympthomatic hydronephrosis due to pregnancy (calyx diameter 1-2,6 cm) an internal urinary drainage was carried out during the second half of pregnancy. Retrograde stenting was placed per cystoscopy in all cases. Follow-up examinations were taken weekly for the first two weeks and further on biweekly. Primary stenting was possible in 12 out of 14 cases, and in two patients dilatation of the ureteral orifice was necessary. 11 out of 14 patients suffered from complications such as severe dysuria (9), urinary tract infection (7), persisting lumbar pain plus catheter lumen obstruction (6 each) as well as catheter migration (3). Long-term follow-up showed that urinary tract obstruction was relieved by stenting in only 6 out of 14 patients. Sufficient urinary drainage by so called double-J ureteral stents was achieved in less than half of the cases. Moreover, there was a complication rate of more than 75%. Taking these results into consideration, internal drainage of complicated pregnancy hydronephrosis needs careful evaluation.

[Effect of co-medication with magnesium sulfate in beta-mimetic tocolysis on parameters of water-electrolyte balance]. / Zum Einfluss der Komedikation mit Magnesiumsulfat bei betamimetischer Tokolyse auf Parameter des Salz-Wasser-Haushalts.

With 2 groups of 10 patients the influence of an additional therapy with 1 g magnesium sulfate/h during i.v. tocolysis with the betamimetic fenoterol (2 micrograms/min) upon parameters of water and electrolyte balance has been investigated. The whole of the magnesium administered during the 24 hours investigational period has been eliminated via the kidneys. Most probably due to a competition within the distal tubulus hypermagnesemia was associated with hypocalcemia and hypercalciuria, followed by a rise in parathyroid hormone. As PTH is able to compensate hypocalcemia not only by means of bone mobilisation but also by an increase in enteral Ca absorption, estimated losses of calcium are minimal. These may be neglected, as additional therapy with magnesium sulfate--besides the advantages yet known (cardioprotection, saving of betamimetic dosage, reduction of drug tolerance development)--reduces betamimetic induced water retention, thus significantly diminishing lung edema hazard during tocolytic therapy.

[Varicella infection at birth]. / Varizellen-Infektion am Geburtstermin.

At birth maternal infection with the Varizella-Zoster-Virus (VZV) is very rare but poses a truly life-threatening risk to the newborn. The neonatal mortality rate is up to 20-30%, if the maternal VZV-infection occurs between day 4 ante partum and day 2 post partum. The mortality can be decreased, if labour is successfully delayed by tocolytic agents until VZV-antibodies produced by maternal immune response have passed the placental barrier. There are indications that the mortality may also be lowered by passive immunisation of the newborn, but further research is needed. We recommend strongly to check VZV-IgG-antibodies-titres promptly, if VZV-infection is suspected. Labour then should be delayed by tocolytic agents as described in 2 case reports to allow maternal IgG-antibodies to cross the placental barrier to the foetus.

[Aspects of donation and processing of stem cell transplants from umbilical cord blood]. / Präparative und arzneimittelrechtliche Aspekte bei der Sammlung von Stammzellpräparaten aus Plazentarestblut 1.

BACKGROUND: In 1996, the Institute of Transfusion Medicine and Immunology started the banking of unrelated cord blood (CB) transplants in collaboration with the Department of Gynecology and Obstetrics at the Mannheim University Hospital. Up to now, more than 2000 placental blood samples have been cryopreserved. We analyze the results of CB collection and processing, and describe the legal background of CB collection in Germany. MATERIALS AND METHODS: CB is only collected with informed consent of the mothers, and without evidence of neither maternal infection nor deformity of the baby. Units with a volume of > 60 ml or > 5 x 10(8) total nucleated cells (NC) are processed as potential transplants. All specimens are evaluated for cell count, HLA typing, sterility testing, ABO/Rh blood groups and clonogenic capacity of hematopoietic progenitor cells. The maternal blood is screened for various infectious disease markers. To reduce the volume of the transplants, we perform buffy coat processing by centrifugation. Finally, we cryopreserve the transplant using a 5.5 percent final concentration of DMSO, and store it in the vapour phase of liquid nitrogen. Results We collected on average 86 +/- 21 ml CB containing 8.87 +/- 3.41 x 10(8) NC (mean +/- SD). 45 percent of the collected units had to be discarded for various reasons. We achieved a mean mononucleated cell recovery by the buffy coat preparation method of 93.4 percent. Defining a threshold dose of 3.0 x 10(7) NC per kg body weight (BW) for transplantation, the mean BW of patients to be sufficiently treated with the stored transplants showed to be 29.6 kg. Conclusions In a very close collaboration between transfusion medicine and obstetricians, a routine collection program for unrelated and related allogeneic CB transplants was established. The CB donation needs to be in accordance with the German drug law. On the other hand, in terms of evidence based medicine, it seems not to be reasonable to collect autologous placental blood for a future transplantation.

Is prenatal HLA typing of uncultured amniocytes before the collection of related allogenic cord blood helpful?

The collection of related allogenic cord blood is gaining increasing importance in families with one child affected by haematopoietic disease. Within a family, there is only a 25% chance of a full HLA match between siblings. 50% of all collected cord blood samples cannot be used because of poor quality. Because of this, the determination of HLA type is useful for planning the collection of related allogenic cord blood transplants. We studied whether HLA typing is possible during late pregnancy if amniocentesis has not been performed during the first trimester. HLA -A, -B and -DRB loci were detected in amniotic fluid, as well as in corresponding cord blood and maternal blood using PCR-SSP. For the first time, HLA typing was performed from uncultured amniocytes. Unambiguous results were obtained from all samples. Fetal HLA-genotype in amniotic fluid was confirmed by typing results from corresponding cord blood. HLA typing of uncultured amniocytes during late pregnancy is a reliable and fast method. For the first time, prenatal HLA typing by amniocentesis after week 38 of gestation is possible in less than 8 h and without fetal risk.

[Determination of cytokines and cytokine receptors in premature labor]. / Bestimmung der Zytokine und Zytokinrezeptoren bei Frühgeburtlichkeit.

One of the leading causes of preterm labour is a subclinical intrauterine infection. The diagnosis of this inapparent infection is an unsolved problem, because clinical parameters show the infection mostly too late and cannot be used for early detection of women at high risk for preterm labour. A prospective clinical study measuring cytokines and cytokine receptor concentration in amniotic fluid from patients with preterm labour and preterm rupture of membranes (PROM) was undertaken to answer the question whether the amount of cytokines is positively correlated to the risk of preterm delivery. In 78 patients (43 controls, 23 patients with PROM, 7 with preterm labour and delivery < 37 weeks of gestation, 7 with preterm labour and delivery at term) amniotic fluid was collected and the following cytokines were measured by an ELISA: IL1 beta, IL2, IL6, TNF alpha, IFN gamma, TNF receptor 55 und 75, IFN gamma receptor, IL2 receptor. We found significant, in some cases highly significant differences in the cytokine content of amniotic fluid of patients with PROM compared with controls and patients with preterm labour and delivery before 37 weeks of gestation compared with controls. There were no differences between patients with preterm labour and delivery at term and controls. Amniotic fluid cultures were positive only in 20-25% of the cases and therefore not a predictive sign. The determination of cytokines in amniotic fluid of patients with preterm labour seems to be a good marker to predict the risk of preterm delivery.(ABSTRACT TRUNCATED AT 250 WORDS)

[Uterine rupture during induced abortion with prostaglandins in the second trimester]. / Uterusruptur bei Abortinduktion mit Prostaglandinen im zweiten Trimenon.

Based on our own experiences and on the literature of the past 14 years the variety of the presenting symptoms in patients suffering from ruptured uterus during the second trimenon are discussed. focussing especially on the first symptoms of a so-called "silent" uterus rupture. A 41-year old second gravida, first para--the healthy full-term child was delivered by Caesarean section--suffered a "silent" uterus rupture after termination of pregnancy at 20th/21st weeks' gestation. As more than 50 per cent of patients with "silent" uterus rupture are diagnosed with considerable delay, early and repeated ultrasound examinations should be performed in all patients with unexplained symptoms or if despite abortion induction for several days no progression of birth occurs. In an artificially induced abortion, prostaglandins should be topically applied to enhance cervix ripening, preferably as a biphasic treatment (first for cervix ripening, later induction of contractions). It is not yet clear whether a single or total dose reduction of prostaglandins used in labour induction in the second trimenon may help to prevent uterus rupture in patients at risk. Predisposing risk factors must be taken into account before applying prostaglandins. Uterus rupture should always be considered as differential diagnosis if problems occur in patients after induced abortion in the second trimenon.

Mild course of fetal RhD haemolytic disease due to maternal alloimmunisation to paternal HLA class I and II antigens.

The patient is a pregnant women of African origin with a prior history of spontaneous abortion and newborn dystrophy. The investigation showed an anti-Rh D antibody (IgG isotypes 1 and 3) with an indirect antiglobulin tube test (IAT) titre of 1:512. The monocyte monolayer assay (MMA) proved clearly the interaction of Fc receptors with the maternal anti-D, and so a clinical significance was expected. In spite of this, no signs of severe haemolysis in the Rh-D-positive and direct antiglobulin test-positive fetus could be observed. Furthermore, two HLA class I and II alloantibodies (anti-A10, anti-DR13) directed against paternal and fetal antigens were detected in the serum of the gravida. Both antibodies showed an inhibitory effect on the in vitro phagocytosis capacity of mononuclear cells expressing at least one of the corresponding HLA antigens (immunophagocytosis inhibition (IPI) test). Thus, the mild course of haemolytic disease may be explained by an effective inhibition of the fetal mononuclear phagocyte system by maternal HLA class I and/or class II antibodies resulting in a diminished destruction of anti-D-coated fetal red blood cells.

Cord blood as a source of autologous RBCs for transfusion to preterm infants.

BACKGROUND: This prospective study was conducted to gain experience as to whether it is technically possible to produce autologous RBCs in additive solution from cord blood (CB), to optimize the blood supply for preterm infants. STUDY DESIGN AND METHODS: CB was collected from 47 infants with a mean (+/- SD) birth weight of 1717 (+/- 699) g. Whenever possible, RBC components were prepared by standard centrifugation using a six-bag system. All samples were put in sterility testing quarantine for 5 days, and a maximum storage of 14 days from collection to transfusion was specified. The babies were given either the autologous RBCs or standard allogeneic RBC concentrates, if autologous blood was not available. RESULTS: In 81 percent of the samples, autologous RBC components could be processed (vol, 7-87 mL; Hct, 31-82%). But within the group of extremely low birth weight infants (body weight <1000 g), a mean CB net volume of only 37 mL was collected, and the RBC preparation was successful only in exceptional cases. Three CB samples (8.6%) tested positive in sterility testing. Of the 47 infants, 21 were treated with a total of 62 allogeneic and 4 autologous RBC transfusions. Most infants with a body weight over 1400 g did not need any RBC transfusion. CONCLUSION: The preparation of autologous RBCs from the CB of preterm infants is technically possible in principle. However, major concerns must be raised as to whether such preparations are of benefit in ensuring safe care of neonates with blood components, with respect to the high rate of bacterial contamination and the limited availability in babies with low birth weight.