Helmut Pschera

Neurochemical differentiation of human bulbospinal monoaminergic neurons during the first trimester

The neurochemical differentiation of bulbospinal noradrenergic and serotonergic neurons has been followed in first trimester human fetuses. Analysis of microdissected CNS regions revealed detectable levels of noradrenaline (NA) and serotonin (5-HT) in pons, medulla oblongata and throughout the spinal cord from 5-6 weeks of gestation. In all regions there was a pronounced increase in tissue levels of the monoamines, especially from 8-9 weeks on. 5-HT levels were lower than NA levels except for pons, where the opposite was true. With increasing fetal age, the results seemed less consistent because of considerable interindividual variations. Using immunohistochemical localization of tyrosine hydroxylase (TH), a marker for noradrenergic neurons, immature cell bodies were seen in the brain stem at the earliest stage studied, that is at 4 weeks of gestation. Several TH and 5-HT-immunoreactive (IR) cell groups were found in pons and medulla oblongata at 5 weeks. Significant structural differentiation of TH- and 5-HT-IR cell bodies was seen during the first trimester. Immunoreactive fibers began to appear at 5 weeks in the cervical spinal cord. At 6 weeks both types of fibers could be found in the white matter throughout the entire spinal cord while fibers in gray matter appeared at 9 weeks. The number of TH-IR fibers was considerably larger than the number of 5-HT-IR fibers. This is the first time the biochemical development of human bulbospinal monoaminergic neurons during the first trimester has been described. Continued investigations of the ontogenetic growth and differentiation of these human bulbospinal monoaminergic neurons will gain necessary insight into the genetically determined capacity for plasticity, potentially possible to activate later in life in response to spinal cord injury. Further, intraspinal transplantation of CNS tissue relevant to the severed spinal cord would by necessity entail selection of embryonic cell populations. Using such therapeutic strategies, detailed knowledge of the inherent capacities of the donor tissues will be crucial.

Influence of the Maturity of the Vaginal Epithelium upon the Absorption of Vaginally Administered Estradiol-17β and Progesterone in Postmenopausal Women

Serum concentrations of estradiol-17 beta and its metabolites estrone (unconjugated) and total estrone (mainly estrone sulfate) and of progesterone were measured before and after application of one vaginal suppository containing 250 micrograms estradiol-17 beta and 10 mg progesterone to 10 previously unsubstituted healthy postmenopausal women with normal pretreatment serum steroid levels. Vaginal hormone cytology was performed prior to administration of the estrogen-progesterone combination. An atrophic vaginal mucosa was found in 5 and a moderate proliferation in the other 5 women. A significant association was found between proliferation and pretreatment values of estrone, but not with other steroids. There was a clear, significant association between steroid absorption kinetics and the condition of the vaginal mucosa as indicated by cytological data: significantly higher absorption was found in women with an atrophic vaginal mucosa.

Testing and Training of the Pelvic Floor Muscles After Childbirth

In a prospective study of 83 women, two different physiotherapy methods for strengthening the pelvic floor muscles after childbirth were evaluated. The training program was carried out by the patients at home for 12 weeks, starting 8 weeks after spontaneous uneventful delivery. Forty‐two women did pelvic floor exercises in accordance with the method presented by Kegel (1). Forty‐one women used standard vaginal cones with weights increasing in 10 g stages from 20 to 100 g, to be retained in the vagina both when standing erect and moving. Pelvic floor muscle strength, defined as the weight in grams of the heaviest cone that could be retained in the vagina, was recorded before and after the 12‐week training period. Training with vaginal cones produced significantly better pelvic floor muscle strength than did exercise without cones.

Establishment of a tissue bank for fetal stem cell transplantation

Study Objective. To analyse the yield of fetal liver tissue in first trimester abortions and to evaluate the number of nucleated cells obtained from each fetal liver during the sixth to twelfth week of gestation.

Fetal beta cell function in diabetic pregnancy. Amniotic fluid concentrations of proinsulin, insulin, and C-peptide during the last trimester of pregnancy.

Proinsulin, insulin C-peptide, insulin-binding antibody, and glucose concentrations were measured in amniotic fluid samples from 43 insulin-treated diabetic patients and 17 nondiabetic control patients between the thirty-six and thirty-ninth weeks of gestation. Insulin-binding antibodies in amniotic fluid were present in only three diabetic patients, although antibodies in maternal serum were found in 22 of the diabetic subjects. In the diabetic group, maternal serum insulin-binding antibodies were statistically unrelated to levels of C-peptide in amniotic fluid. The mean amniotic fluid concentrations of proinsulin (0.07 nmole/L), insulin (0.08 nmole/L), C-peptide (1.17 nmoles/L), and glucose (2.09 mmoles/L) were markedly elevated (p less than 0.001) in diabetic patients, as compared to nondiabetic control patients, thus suggesting exaggerated fetal beta cell function. C-peptide was correlated to both insulin (r = 0.69) and proinsulin (r = 0.35) in the diabetic group only. Infant birth weight and amniotic fluid C-peptide was significantly correlated in both the control group (r = 0.54) and the diabetic group (r = 0.38). Diabetic pregnancies associated with neonatal morbidity (n = 25) had significantly higher mean amniotic fluid concentrations of both insulin and C-peptide than did pregnancies without neonatal morbidity (n = 18). The amniotic fluid values of C-peptide and insulin in these latter two subgroups were overlapping and, therefore, could not serve to predict neonatal outcome in the individual case.

First Trimester Development of the Human Nigrostriatal Dopamine System

The aim of the present study was to characterize the morphological and neurochemical differentiation of mesencephalic dopaminergic neurons in human embryos, derived from elective first trimester abortions. Embryonic brain tissue was taken for analysis of tyrosine hydroxylase (TH) by immunohistochemistry and Western blot, and for analysis of endogenous dopamine (A) content using HPLC-ED. TH expression was first detected at 3.5 weeks of gestational age (Carnegie stage 11) by immunohistochemical staining of the primordial sympathetic trunk along both sides of the neural tube. In sagittal sections of the intact 4.5-week-old embryo, a small, distinct population of rounded, densely packed TH-immunoreactive perikarya with short primary processes was seen in the midbrain. During the latter half of the first trimester, the number of TH-stained cells as well as the length and number of axonal processes projecting toward and into the developing neostriatum increased rapidly. At the end of the first trimester, varicose fibers could be detected in the striatal anlage. In order to verify that TH was the antigen recognized by the antibodies used for immunohistochemistry on human tissue specimens, mesencephalic tissue of 5-10 weeks gestation was analyzed by Western blot technique. A single, homogeneous band with the apparent molecular weight of approximately 60 kDa was clearly detected at 5 weeks of age. The amount of TH/mg total protein increased at least 10-fold between 5-10 weeks of gestation. For comparison, the mesencephalon and the forebrain/basal ganglia were analyzed for endogenous DA content using HPLC-ED. DA was first detected at 5.5 weeks of gestational age in both mid- and forebrain, and DA levels were found to increase exponentially from 7 to 7.5 weeks of age, reaching 4-5.5 ng DA/mesencephalon and 50-75 ng DA/g caudate nucleus-putamen anlage at the end of the first trimester. Together, morphological and biochemical data presented here constitute evidence for a very early appearance, migration, and differentiation as well as functional development of human mesencephalic dopaminergic neurons and their projections into target areas during the first trimester.

Morphology and Growth of Embryonic, Human Dorsal Root Ganglion Explants in Long-Term Culture: Expression of Cell Type-Specific Markers during Early Differentiation

Embryonic, human spinal ganglion explants were plated at 5-12 weeks postconceptional age and cultured for 5-50 days on a semisynthetic substrate in a serum-containing culture medium without addition of antibiotics or preconditioned medium. The growth pattern in vitro was found to be age dependent. Five- to 6-week ganglia showed a characteristic semicircular growth pattern with bidirectional extension of neurites on top of a monolayer of supportive cells. Explanted 9- to 10-week ganglia showed an extensive, multidirectional neurite outgrowth with less pronounced proliferation of nonneuronal cells. Neurite extension, fasciculation, cell migration and morphology were studied immunohistochemically with antibodies to neurofilament (NF), S-100, and the Thy-1 glycoprotein. Both NF and S-100 were expressed at 5 weeks gestational age in ganglionic neurons and in proliferating Schwann cells in contact with axonal processes, respectively. NF was homogeneously distributed in both cell somata and neurites, whereas S-100 immunoreactivity showed an intense nuclear and a weaker cytoplasmic distribution in spindle-shaped, bipolar Schwann cells. This staining pattern was conserved during differentiation in long-term culture. Thy-1 was expressed on ganglionic neurites forming fascicles by the third week in culture. However, Thy-1 was never expressed until the total age of 10 weeks. In addition, Thy-1 was found on fibroblasts from the first week in culture. The distribution of Thy-1 on the cytoplasmic membrane was similar in both cell types, showing a coarsely granulated membrane staining. The temporal as well as the spatial expression of differentiation antigens in tissue sections of early embryonic spinal cord and spinal ganglia were very similar to what was observed in vitro.

Haematocervix after conization diagnosed by ultrasonography.

Haematocervix is an uncommon complication after conization. This report deals with 1 case that presented with clinical signs of progressive stenosis and was diagnosed by ultrasonography. Based on analysis of the 3 previously reported cases the pathogenesis of this condition is discussed.

Screening of fetal stem cells for infection and cytogenetic abnormalities.

Fetal stem cell transplantation may rely on material from therapeutic abortions. It is essential that the stem cell transplant does not transmit any microorganisms that may affect the fetus and that genetically abnormal cells are avoided. To evaluate such contamination, human fetal stem cells collected February 1992 - December 1993 were analyzed for bacterial and fungal growth, and the placentas were karyotyped. Four samples of 70 were positive for different pathogens. Serological screening of 43 women during this period resulted in five seroconversions and revealed one carrier of anti-HCV. Karyotyping revealed two abnormal findings out of 72 samples. Thus, the concept of using material from therapeutic abortions is safe.

Plasminogen Activators and Inhibitors in Amniotic Fluid

Ten amniotic fluid samples obtained at elective caesarian section were examined for the presence of tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA) and the two specific plasminogen activator inhibitors, PAI-1 and PAI-2. u-PA antigen was demonstrated in all samples (mean concentration equivalent to 0.35 iu/ml), but its bioactivity was very low (mean activity 0.13 iu/ml) when assessed by a bioimmuno-assay (BIA) using chromogenic substrate. None of the samples analysed by ELISA technique had measurable levels of t-PA, neither could any activity of t-PA be demonstrated by a sensitive BIA or fibrin plate technique. Significant amounts of both PAI-1 and PAI-2 antigen were present in all samples, and the mean values were 12.2 and 43.4 ng/ml, respectively. The average inhibitory capacity per ml of amniotic fluid measured by assessing the residual t-PA activity following addition of a known amount of single chain t-PA to the samples was 17.3. Thus, this study demonstrates a fibrinolytic system in amniotic fluid, which is characterised by absence of t-PA activity, low bioactivity of u-PA and excess of the two specific inhibitors, PAI-1 and PAI-2. This inhibitory capacity of amniotic fluid may play a role in preventing premature rupture of membranes.