Monitoring and treatment of anti-D in pregnancy.

Prophylactic anti-D is a very safe and effective therapy for the suppression of anti-D immunization and thus prevention of haemolytic disease of the foetus and newborn. However, migration from countries with low health standards and substantial cuts in public health expenses have increased the incidence of anti-D immunization in many "developed" countries. Therefore, this forum focuses on prenatal monitoring standards and treatment strategies in pregnancies with anti-D alloimmunization. The following questions were addressed, and a response was obtained from 12 centres, mainly from Europe.

[Pregnant women's assessment and level of knowledge of prenatal counseling]. / Wie beurteilen Schwangere die pränatale Beratung und was wissen sie im Anschluss daran?

PURPOSE: Informed decision making and informed consent prior to any intervention are crucial in the ethically and psychologically complex field of prenatal diagnosis (PND). The aim of this study was to investigate whether and to what extent pregnant women understand the information provided by their physicians. MATERIALS AND METHODS: Fifty pregnant women in the first trimester answered a structured questionnaire after their first visit between 7 to 10 weeks of gestation that routinely includes basic prenatal counseling. A special focus was put on information transfer, knowledge about and understanding of prenatal tests, as well as previous experiences with PND. The results were analyzed with regard to differences due to background, educational level and previous experiences with PND. RESULTS: The maternal mean age was 31.1 years (SD 6.7). 38 patients (76 %) had at least one previous pregnancy and two thirds of them had experiences with PND. Their experience was mainly positive. About three quarters of the women stated that they had been informed about the test methods during the consultation and had understood the explanations. Uncertainty was reported in 12.2 % and 23.3 % of the women said they had further questions. The percentage of questions related to appropriate understanding that were answered correctly was only 44 % to 77.5 %. The percentage of correct answers was lower in women without experience with PND, with a lower educational level and born in countries outside the EU and Switzerland. CONCLUSION: Pregnant women are relatively well informed about prenatal tests. Their actual knowledge of the meaning of the tests, however, seems to be incomplete. Especially in the case of immigrants and women without previous experience with PND, it is therefore doubtful whether the preconditions for an informed consent are met. Further research needs to focus on more helpful information and individually adapted counseling concepts for decision making in PND.

Metastatic involvement of the urinary tract in patients with advanced ovarian carcinoma: lessons from the autopsy for an interdisciplinary treatment approach.

PURPOSE OF INVESTIGATION: Frequency and extent of metastases in urologic organs found at autopsy of ovarian carcinoma patients were evaluated. METHODS: Autopsy reports from 170 patients who died of advanced ovarian carcinoma between 1975 and 2005 were studied. The distribution of abdominal metastatic sites with particular attention to the involvement of the urologic organs, and hydronephrosis was analyzed. RESULTS: The distribution of metastatic sites was as follows: kidney (n = 6, 3.5%), urinary bladder (n = 38, 22.4%), and ureter (n = 20, 11.8%). In 36 patients, hydronephrosis was observed (21.2%); of these patients, 20 (55.6%) also had ureteral involvement. All patients with ureteral involvement had hydronephrosis. CONCLUSION: Hydronephrosis in late stages of ovarian carcinoma, usually attributed to extrinsic compression of the ureter by an abdominal tumor, may also be explained by ureteral metastases. This fact must be considered in the clinical management of these patients, particularly in the restoration of luminal patency through an endoscopically placed internal ureteral stent.

Target and reality of adjuvant endocrine therapy in postmenopausal patients with invasive breast cancer.

Previous research evaluating the use of adjuvant endocrine therapy among postmenopausal breast cancer patients showed with 15-50% wide ranges of non-adherence rates. We evaluated this issue by analysing an unselected study group comprising of 325 postmenopausal women, diagnosed from 1997 to 2003 with hormonal receptor-positive invasive breast cancer. The different clinical situations that led to the discontinuation of adjuvant endocrine therapy were clearly defined and differentiated: non-adherence was not simply the act of stopping medication, but rather the manifestation of an intentional behaviour of the patient. Of the 287 patients who initiated endocrine therapy, 191 (66.6%) fully completed this treatment. Thirty-one patients (10.8%) showed non-adherence to therapy. Patients who had follow-up with a general practitioner, rather than in an oncologic unit, were more likely to be non-adherent (P=0.0088). Of 25 patients who changed medication due to therapy-related adverse effects, 20 (80%) patients fully completed the therapy after drug change. In adjuvant endocrine therapy, a lowering of the non-adherence rate to 10.8%, the lowest reported in the literature, is realistic when patients are cared for by a specialised oncologic unit focusing on the individual needs of the patients.

The effect of labour and placental separation on the shedding of syncytiotrophoblast microparticles, cell-free DNA and mRNA in normal pregnancy and pre-eclampsia.

The clinical features of the maternal syndrome of pre-eclampsia can be explained by generalised maternal endothelial cell dysfunction, which is a part of a more global maternal systemic inflammatory response. There is growing evidence that these effects are associated with the shedding of cellular debris, including syncytiotrophoblast microparticles (STBM), cell-free DNA and mRNA, from the surface of the placenta (syncytiotrophoblast) into the maternal circulation. The increased shedding of this debris seen in pre-eclampsia is believed to be caused by placental ischaemia, reperfusion and oxidative stress. This study was carried out to determine whether uterine contractions during labour and subsequent placental separation lead to an acute increase in the release of placental debris into the maternal circulation. To assess the effects of labour, samples were taken from 10 normal pregnant (NP) and 10 pre-eclamptic (PE) women at varied time points. Similarly to assess the effects of placental delivery, plasma samples were taken from 10 NP and 10 PE women undergoing elective caesarean section. There was a significant increase in the shedding of STBM in pre-eclampsia which was not seen in normal pregnancy and there was a small rise in STBM levels at placental separation in both normal pregnant and pre-eclamptic women undergoing caesarean section, but the differences were not significant. However, levels of placental cell-free corticotrophin releasing hormone mRNA were significantly increased in labour in both normal pregnancy and pre-eclampsia and were still high 24 h after delivery in the pre-eclamptic women. There was no significant increase in fetal or total DNA in labour, but the overall levels of total DNA (maternal and fetal) was increased in labour in pre-eclampsia compared to normal labour. The enhanced shedding of STBM and CRH mRNA in pre-eclampsia labour may have a role in cases of postpartum worsening of pre-eclampsia.

Parallel assessment of circulatory cell-free DNA by PCR and nucleosomes by ELISA in breast tumors.

OBJECTIVES: In order to assess the potential biomolecules for breast cancer, we analyzed in parallel the levels of cell-free glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and cell-free nucleosomes in serum samples from patients with benign and malignant breast tumors. The levels of cell-free DNA obtained by quantitative PCR were compared with those obtained by enzyme-linked immunosorbent assay (ELISA). METHODS: Twenty-three patients with benign breast tumors, 27 patients with breast cancer, and 32 age-matched healthy women were recruited. The amounts of serum nucleosomes were analyzed by ELISA and the levels of cell-free GAPDH were measured by real-time quantitative PCR. The correlation between nucleosome and cell-free GAPDH levels was examined using the Spearman rank test. RESULTS: The levels of cell-free GAPDH were significantly higher in the serum samples of patients with benign and malignant breast tumors than in those of the control group (median 37,966 GE/mL, range 3,802-130,104 versus 11,770 GE/mL, range 2,198-73,522, p=0.035 and median 40,698 GE/mL, range 3,644-192,482 versus 11,770 GE/mL range 2,198-73,522, p=0.001). The concentration of cell-free GAPDH correlated significantly with the quantities of nucleosomes in serum samples (r=0.451, p=0.000). There was, however, no significant difference between healthy individuals and women with benign breast tumors or breast cancer in terms of nucleosomes determined by ELISA. CONCLUSION: Our data suggest that the cell-free serum GAPDH DNA assayed by quantitative PCR is a better biomarker than nucleosomes assayed by ELISA in patients with breast tumors.

Georg Schmorl on trophoblasts in the maternal circulation.

Trafficking of cells between the fetus and its mother provides indirect clues to the underlying pathophysiology of pregnancy. Georg Schmorl first documented the presence of fetal cells in the maternal body and emphasized the importance of the placenta in eclampsia. Although his classic paper, written in 1893, is widely cited today, few investigators have actually read the paper, as it was published in German [Schmorl G., Pathologisch-anatomische Untersuchungen über Puerperal-Eklampsie. Verlag FCW Vogel, Leipzig; 1893]. Our goal was to translate the paper into English and critically re-evaluate its conclusions from a 21st century perspective. Schmorl was remarkably astute in his assessment of the pathologic changes that were seen in the 17 women on whom he performed complete autopsies. He found similar severe changes in all of the women, implying a common pathogenesis. This was in direct contrast to the then current doctrine. He was the first to observe the presence of thrombi containing multinucleated syncytial giant cells in the lungs of the women and speculated that they were of placental origin. To support his hypothesis he performed animal experiments. He also recognized that feto-maternal trafficking occurred in normal gestations but was increased in pregnancies affected by eclampsia. Using sophisticated molecular techniques we can now precisely confirm what Schmorl so elegantly described.

Levels of circulating cell-free serum DNA in benign and malignant breast lesions.

PURPOSES OF THE STUDY: We analyzed circulating cell-free DNA in the serum of patients with benign and malignant breast disease and in healthy individuals to determine its diagnostic value. BASIC PROCEDURES: Serum samples were obtained from 50 healthy individuals, 33 patients with malignant breast disease and 32 patients with benign breast disease. Circulatory DNA was extracted from serum samples. Cell-free DNA was quantified by real-time quantitative PCR for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Tissue samples from patients with malignant and benign breast lesions were histopathologically examined. MAIN FINDINGS: The mean levels of circulating cell-free DNA in serum samples were 41,149 genome equivalents (GE)/mL in patients with malignant disease, 30,826 GE/mL in patients with benign disease, and 13,267 GE/mL in healthy individuals. Healthy individuals had significantly lower levels of cell-free DNA than patients with malignant or benign breast disease (p=0.001, p=0.031). No significant difference was observed between malignant and benign disease. There was a correlation between cell-free DNA levels and tumor size but not with other tumor characteristics. PRINCIPAL CONCLUSION: Our results suggest that levels of circulating cell-free DNA in serum could have diagnostic value to discriminate between healthy individuals and patients with breast lesions but not between patients with malignant and benign breast lesions.

Breast cancer sagittal/horizontal plane location influences axillary lymph node involvement.

AIM: To assess the influence of tumour location on axillary lymph node involvement (ALNI) and prognosis in breast cancer by evaluating the significance of the sagittal/horizontal alignment. METHODS: We compared 57 patients with superficially located breast carcinomas up to 3.0 cm with patients having lesions in posterior planes of the breast. Both groups were matched according to age, time of diagnosis, tumour size, grade, hormonal receptor status and tumour site within the frontal plane. Histologic evidence of skin involvement, excluding tumours fulfilling the criteria for pT4b, was defined as inclusion criteria and reference plane for superficial tumour location. RESULTS: Tumours situated in the superficial region of the breast, compared to those located in deeper planes, have an increased risk of ALNI (p=0.023), whereas no difference was observed with reference to disease-specific survival (p=0.203). CONCLUSION: This study shows that ALNI is dependent on sagittal/horizontal as well as frontal tumour location. Clinicians should be aware that tumours lying posteriorly may be at increased risk of occult spread outside axillary lymph nodes.

Oral misoprostol vs. intravenous oxytocin in reducing blood loss after emergency cesarean delivery.

OBJECTIVE: To compare the effectiveness of oral misoprostol and intravenous oxytocin in reducing blood loss in women undergoing indicated or elective cesarean delivery (CD) under spinal anesthesia. METHODS: In this prospective, double-blind pilot study, 56 parturients who received 5 IU of intravenous oxytocin after cord clamping were randomized to further receive either misoprostol orally and a placebo infusion intravenously or placebo orally and an oxytocin infusion intravenously. RESULTS: After adjustment was made for the sonographically estimated amniotic fluid volume, there was no statistical difference in blood loss between the 2 groups (mean+/-S.D., 1083+/-920 mL in the oxytocin group vs. 970+/-560 mL in the misoprostol group; P=.59). CONCLUSION: Oxytocin followed by oral misoprostol is as effective as an oxytocin injection followed by an oxytocin infusion in reducing postoperative blood loss after CD, and the protocol may be a safe, valuable, and cost-effective alternative to oxytocin alone. Visual estimation of intraoperative blood loss undervalues the effective value of misoprostol use by 30%.

Prognostic value of genomic alterations in invasive cervical squamous cell carcinoma of clinical stage IB detected by comparative genomic hybridization.

The clinical behavior of invasive cervical carcinoma of clinical stage IB varies considerably in tumors presenting without regional lymph node metastases. The early identification of patients at higher risk for poor outcome may prove useful because these patients would benefit from aggressive adjuvant treatments. In this study, comparative genomic hybridization was applied to evaluate whether genomic aberrations have prognostic significance in cervical carcinoma. Genomic alterations were evaluated in 62 cervical carcinomas of clinical stage IB. DNA sequence losses were most prevalent at chromosomes 4q (53%), 3p (52%), 13q (45%), 4p (44%), Xq (44%), 5q (40%), 18q (37%), and 6q (35%). Several genomic alterations were associated with poor clinical outcome or metastasis. The total number of DNA aberrations/tumor (P < 0.02) and the number of DNA sequence losses/tumor (P < 0.04) were associated with disease-specific survival. 9p deletions were significantly more frequent in carcinomas with lymph node metastasis than in node-negative tumors (P < 0.03). Losses of chromosome 11p (P < 0.0001) and 18q (P < 0.01) were associated with poor prognosis in cervical carcinomas without lymph node metastasis. These data suggest that inactivation of tumor suppressor genes on chromosomes 9p, 11p, and 18q may play a role in the progression of cervical carcinoma.

[Ethical considerations in the management of infants born at the limit of viability]. / Ethische Aspekte bei Schwangerschaften an der Grenze der kindlichen Lebensfähigkeit.

Despite ongoing progress in perinatal care over the past decade, mortality rates of infants born before 24 completed weeks of gestation have remained high, and the majority of survivors have at least some degree of neurosensory impairment. With increasing knowledge of long-term follow-up data, quality of life aspects have become more important in treatment decisions for infants born at the limit of viability. Many countries have adopted an individualized approach to the care of these infants. Provisional intensive care is initiated in the delivery room and continued in the neonatal intensive care unit as long as there is a reasonable chance of survival and the expected quality of life appears to justify the patient's pain and suffering. On the other hand, redirection of care becomes an ethically justifiable option once the burdens have begun to outweigh the benefits. The published recommendations from different countries show considerable variability. For example, the gestational age below which preference should be given to palliative care ranges between 22 (Germany, Japan) and 25 completed weeks of gestation (Netherlands). Similarly, parental participation in surrogate decision making varies from country to country. Recommendations which emphasize quality of life aspects tend to encourage parental participation more than recommendations which are primarily based on a sanctity of life ideology. The quest to improve care for infants born at the limit of viability will continue. Trying to push the limit of viability towards even lower gestational ages is not a priority. Research efforts should focus on improving long-term outcome for survivors and on developing high quality palliative care for non-survivors.

[New options in prenatal and preimplantation diagnosis of genetic disorders]. / Neue Möglichkeiten der pränatalen genetischen Diagnostik inklusive Präimplantationsdiagnostik.

During recent years the progress with the most important practical impact in prenatal diagnosis has been the implementation of first trimester risk screening for common aneuploidies leading to a much improved identification of pregnancies at risk. Molecular methods for a rapid, cost-effective, but selective aneuploidy diagnosis such as interphase FISH or QF-PCR have been around for years, do have their specific indications, but will unlikely replace conventional cytogenetic tools in routine diagnosis. They apparently do also play a role as marketing instruments in the competition among cytogenetic laboratories. The most thrilling issue for all cytogeneticists in the years to come will be the introduction of array-based methods in the prenatal routine diagnosis of chromosomal abnormalities. Polar body diagnosis has been the only option available for preimplantation genetic diagnosis in german speaking countries. The overwhelming majority of all professionals involved and many families concerned share the hope that the legal situation will improve in these countries to allow the examination of early embryos in high risk situations.

[Recent advances in prenatal diagnostics]. / Neue Möglichkeiten in der pränatalen Diagnostik.

During the last years, technical improvements have increased the possibilities in prenatal ultrasound. During the eighties and nineties, fetal malformations were increasingly detected and specified. Since a few years, the measurement of the fetal nuchal translucency between 11 and 14 weeks of gestation has been implemented to calculate the individual risk, in combination with most recent biochemical markers. Today, the sonographic measurement of the nuchal translucency is regarded as a valuable screening tool for chromosomal anomalies in prenatal medicine. Beside standardized examinations, a profound information and counseling of the pregnant women should be emphasized. With the improvement of the specific maternal risk calculation, using the sonographic measurement of the nuchal translucency, the biochemical markers and the maternal age, unnecessary invasive examinations may be prevented and their overall number can significantly be reduced. The same trend is seen in the whole field of prenatal medicine, illustrated by the detection of the fetal rhesus D status from the maternal blood and the use of Doppler ultrasound in the management of fetal anemia.

Fetal cells and cell free fetal nucleic acids in maternal blood: new tools to study abnormal placentation?

The analysis of fetal cells, and more recently cell free fetal nucleic acids, in maternal blood has to date largely been concerned with the development of risk free methods for prenatal diagnosis. Although elevated feto-maternal cell trafficking has long been associated with preeclampsia, it has only recently been shown that this perturbation is an early event in these pregnancies, occurring well in advance of the onset of symptoms. In a separate development, analogous observations have been made concerning the levels of circulatory fetal nucleic acids in maternal plasma. Subsequent studies have shown that changes in these two parameters may also occur in other pregnancy related disorders including preterm labour, intra-uterine growth retardation, hyperemesis gravidarum or even pregnancies at high altitude. A striking finding of these examinations was that preterm labour was associated with an elevated release of cell free fetal nucleic acids but not with an increment in feto-maternal cell trafficking. This suggested that the analysis of the mechanisms regulating trans-placental cell trafficking or liberation of circulatory fetal nucleic acids may provide key insights into the fundamentally different placental lesions underlying these disorders. As such, circulatory fetal cells and nucleic acids may be viewed as new tools to study alterations in placentation.

A comparative study of the effect of three different syncytiotrophoblast micro-particles preparations on endothelial cells.

Pre-eclampsia is a pregnancy-associated multi-system disorder of unknown etiology, characterized by damage to the maternal endothelium. The latter facet has been suggested to be mediated in part by elevated shedding of inflammatory placental syncytiotrophoblast micro-particles (STBM) into the maternal circulation. In this study, we have examined STBM prepared by three different methods: mechanical dissection, in vitro placental explant culture and perfusion of placental cotyledons. All three preparations yielded morphologically similar STBM, as confirmed by scanning electron microscopy, and all contained syncytiotrophoblast-specific proteins as determined by the presence of placental alkaline phosphatase. The functional properties of the three STBM preparations were examined on human umbilical vein endothelial cells (HUVEC), where the mechanically prepared particles were found to inhibit proliferation to the greatest extent. Furthermore, only mechanically prepared STBM lead to the detachment and apoptosis of HUVEC cells. Our study, therefore, suggests that STBM prepared from placental perfusion or in vitro explant culture are biologically different from mechanically prepared ones, and may provide a better approximation of physiologically produced placental micro-particles.

Umbilical cord blood from preterm human fetuses is rich in committed and primitive hematopoietic progenitors with high proliferative and self-renewal capacity.

Human umbilical cord blood (CB) has been recognized as a source of hematopoietic stem cells for transplantation. While hematopoietic properties of neonatal CB from full-term pregnancies have been well characterized, little is known about CB from early gestational ages. We analyzed the content and the growth properties of primitive and committed hematopoietic progenitors in preterm CB from second trimester (week 16-28; n = 17) and early third trimester (week 29-34; n = 17) in comparison with term CB (n = 18). The frequency of CD34+ and CD34+CD38- cells was significantly higher in preterm than in term CB (mean, 2.51% and 0.56% vs 0.88% and 0.13%;p < 0.002). The number of colony forming units (CFU) in preterm CB was about twofold higher (230 +/- 6 vs 133 +/- 14/ 10(5) mononuclear cells; p < 0.05) and correlated with the content of CD34+ progenitors (r = 0.73). Long-term culture initiating cells (LTC-IC) were enriched about 2.5-fold (6.7 +/- 2.9 vs 2.6 +/- 1.2/10(5) cells; p < 0.05). Progenitors from preterm CB could be expanded in stroma-free liquid cultures supplemented with hematopoietic growth factors as efficiently as progenitors from term neonates. In short-term cultures containing erythropoietin (Epo), interleukin (IL)-1, IL-3, and IL-6, or granulocyte- (G-) and granulocyte-macrophage colony-stimulating factor (GM-CSF) together with stem cell factor (SCF) or Flt3 ligand (FL), expansion of CFUs was six- to eightfold at week 1. In long-term cultures containing thrombopoietin (TPO) and FL, an approximately 1000-fold expansion of multilineage progenitors was observed at week 10. In summary, we show that preterm CB compared with term CB is richer in hematopoietic progenitors, and that precursors from preterm CB can be extensively expanded ex vivo. This may have implications for the development of transplantation and gene transfer strategies targeting circulating fetal stem cells.

Stable transduction with lentiviral vectors and amplification of immature hematopoietic progenitors from cord blood of preterm human fetuses.

Umbilical cord blood (CB) from the early gestational human fetus is recognized as a rich source of hematopoietic stem cells. To examine the value of fetal CB for gene therapy of inborn immunohematopoietic disorders, we tested the feasibility of genetic modification of CD34(+) cells from CB at weeks 24 to 34 of pregnancy, using lentiviral vector-mediated transfer of the green fluorescent protein (GFP) gene. The transduction rate of CD34(+) cells was 42 +/- 9%, resulting in GFP expression in 23 +/- 4% of colonies derived from colony-forming units (CFUs) and 11 +/- 1% from primitive long-term culture-initiating cells (LTC-ICs). Cell cycle analysis demonstrated transduction and GFP expression in cells in the G(0) phase, which contains immature hematopoietic progenitors. Transduced fetal CD34(+) cells could be expanded 1000-fold in long-term cultures supplemented with megakaryocyte growth and development factor along with Flt-3 ligand. At week 10, expression of GFP was observed in 40.5 +/- 11.7% of CFU-derived colonies. While prestimulation of CD34(+) cells with cytokines prior to transduction increased the efficiency of GFP transfer 2- to 3-fold, long-term maintenance of GFP-expressing CFUs occurred only in the absence of prestimulation. The GFP gene was found integrated into the genomic DNA of 35% of LTC-IC-derived colonies initiated at week 10, but GFP expression was not detectable, suggesting downregulation of transgene activity during the extended culture period. These results indicate that human fetal CB progenitors are amenable to genetic modification by lentiviral vectors and may serve as a target for gene therapy of hematopoietic disorders by prenatal autologous transplantation.

Severely reduced presence of tissue macrophages in the basal plate of pre-eclamptic placentae.

Pre-eclampsia is a disorder of unknown aetiology peculiar to human pregnancy. A well-described pathological feature being shallow trophoblast invasion into the spiral arteries during placenta development. Epidemiological studies have revealed an increased risk in pregnancies of primipaternity, and an association with the maternal-fetal HLA-DR relationship, both suggesting the involvement of an immunological component. We were therefore interested in the distribution of HLA-DR expressing myeloid cells in the decidua of healthy and pre-eclamptic placentae. We have studied the monocytes in maternal and fetal peripheral blood as well as in the placenta and identified the cluster of differentiation (CD) 14(+)myeloid cells in the basal plate as mannose receptor (ManR) positive tissue macrophages. In a comparison between peripheral blood monocytes from healthy pregnant and pre-eclamptic women we found no significant difference in the subpopulation size of CD14(+)/CD16(+)monocytes. The number and location of macrophages in the placental villi was similar. However, while the basal plate of the normal decidua contained numerous CD14(+), HLA-DR(bright), ManR(+)tissue macrophages, this compartment was virtually void of these phagocytic cells in the pre-eclamptic placenta. This novel finding suggests that in pre-eclampsia not only the migration of endovascular cytotrophoblasts is disturbed, but that also maternal macrophage migration is affected.