Biomedical subjects

Wolfgang Holzgreve

Publications and source records attributed to Wolfgang Holzgreve.

At least 19 recordsLinked to original sources

PubMed2007

Non-invasive prenatal detection of achondroplasia in size-fractionated cell-free DNA by MALDI-TOF MS assay.

Achondroplasia is the most common form of short-limbed dwarfism in humans and is caused by mutations in the FGFR3 gene. Currently, prenatal diagnosis of this disorder relies on invasive procedures. Recent studies have shown that fetal single gene point mutations could be detected in cell-free DNA (cf-DNA) from maternal plasma by either the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) assay with single allele base extension reaction (SABER) approach or the size fractionation of cf-DNA in maternal plasma. Here, we combined the two approaches to non-invasively examine the fetal G1138A mutation in maternal plasma. cf-DNA was extracted from maternal plasma samples obtained from two pregnant women at risk for achondroplasia. The fetal G1138A mutation was determined by the analysis of size-fractionated cf-DNA in maternal plasma using MALDI-TOF MS with SABER approach and homogenous MassEXTEND (hME) assay, respectively. The fetal G1138A mutation was detectable in the two achondroplasia-affected pregnancies by the analysis of cf-DNA in maternal plasma using MALDI-TOF MS. However, the size-fractionation approach led to a more precise detection of the fetal mutation in both analyses. This analysis would be suitable for non-invasive prenatal diagnosis of diseases caused by fetal single gene point mutations.

Achondroplasia↗

PubMed2006

Maternal and neonatal infections and obstetrical outcome in water birth.

OBJECTIVES: The goal of our study was to assess the effect of water birth on obstetrical outcome, the maternal and neonatal infection rate in a selected low risk collective. STUDY DESIGN: In this prospective observational study (1998-2002) 513 women, wished to have a water birth. The study was approved by the local ethical committee, informed consent was obtained. According to the course of delivery, we compared three groups: woman who had a water birth, a normal vaginal delivery after immersion and a normal vaginal delivery without immersion. Outcome measurements were maternal and fetal infection rate, obstetrical outcome parameters and relevant laboratory parameters. RESULTS: The groups were comparable in terms of demographic and obstetric data. The maternal and neonatal infection rate and laboratory parameters showed no significant difference among the groups. There was no maternal infection related to water birth. There were five water born neonates and three neonates after normal vaginal delivery preceded by immersion with conjunctivitis. Significant differences were observed in obstetrical outcome parameters: less use of analgesia, shorter duration of first and second stage of labor, smaller episiotomy rate in water birth. In contrast no differences were seen in all observed fetal outcome parameters: APGAR score, arterial and venous pH, admission rate to neonatal intensive care unit. CONCLUSIONS: Water birth is a valuable alternative to traditional delivery. The maternal and fetal infection rate was comparable to traditional deliveries. A careful selection of a low risk collective is essential to minimize potential risks.

Adult↗

PubMed2006

Is the quantity of circulatory cell-free DNA in human plasma and serum samples associated with gender, age and frequency of blood donations?

Circulatory cell-free DNA (cf-DNA) is increased in a variety of clinical pathologic conditions; therefore, these markers could be widely used as markers for detecting and monitoring several disorders. To better understand the biology of this molecule, we analysed the relationship between the level of circulatory cf-DNA and physiological parameters such as gender, age and frequency of blood donations. Paired plasma and serum samples were obtained from 87 blood donors and 50 healthy adults who had never donated blood. Cf-DNA was extracted from plasma and serum samples using the MagNA Pure LC Instrument. Quantity determination of circulatory cf-DNA was performed by TaqMan real-time PCR for the ubiquitous GAPDH gene. Our data showed that the concentration of cf-DNA in serum was about eightfold higher than that in plasma. Regarding the level of these circulatory species, no significant differences were observed between the age-matched men and women and gender-matched, different-age cohorts, except in women who were older than 60 years of age. Frequent blood donations did not increase the circulatory species. Circulatory cf-DNA in plasma and serum samples is not correlated with human gender and human age except in women who are older than 60 years of age. Frequent blood donation did not affect the quantity of circulatory cf-DNA. The explanation for the latter most likely is the short half-life time of free fetal DNA in maternal circulation.

Adult↗

PubMed2006

Quantitative analysis of intact fetal cells in maternal plasma by real-time PCR.

OBJECTIVE: Fetal genetic materials in maternal circulation might be potentially used for non-invasive prenatal diagnosis (NIPD). In this study, we quantitatively analysed the intact fetal cells existing in maternal plasma, which would be a special method of NIPD. STUDY DESIGN: Eleven samples from preeclamptic patients, two samples from patients with RhD incompatibilities, and 30 samples from normal pregnancies as controls were collected. The intact cells in the plasma fraction were separated by centrifugation at high speed. The intact cell pellets were washed with PBS to remove any cell-free DNA. RESULTS: We were able to detect intact fetal cells in 3 out of 11 preeclamptic plasma samples from women carrying male fetuses, and from both plasma samples of RhD incompatibility affected pregnancies. However, intact fetal cells were not detected in all the 16 normal pregnancies with a male fetus, although cell-free fetal DNA was detected in all these cases. CONCLUSIONS: Intact fetal cells might be present in maternal plasma. However, the rarity of these cells limits their use for reliable, non-invasive prenatal diagnosis. The detection of such cells was successful in some preeclamptic and RhD incompatibility samples, not in the normal controls. Therefore, as opposed to free fetal DNA in maternal blood the use of intact fetal cells does not provide a reliable mode for NIPD.

Cell Separation↗

PubMed2006

In utero haematopoietic stem cell transplantation. Experiences in mice, sheep and humans.

Early prenatal diagnosis and in utero therapy of certain fetal diseases have the potential to reduce fetal morbidity and mortality. The intrauterine transplantation of stem cells provides in some instances a therapeutic option before definitive organ failure occurs. Clinical experiences show that certain diseases, such as immune deficiencies or inborn errors of metabolism, can be successfully treated using stem cells derived from bone marrow. However, a remaining problem is the low level of engraftment that can be achieved. Efforts are made in animal models to optimise the graft and study the recipient's microenvironment to increase long-term engraftment levels. Our experiments in mice show similar early homing of allogeneic and xenogeneic stem cells and reasonable early engraftment of allogeneic murine fetal liver cells (17.1% donor cells in peripheral blood 4 weeks after transplantation), whereas xenogeneic HSC are rapidly diminished due to missing self-renewal and low differentiation capacities in the host's microenvironment. Allogeneic murine fetal liver cells have very good longterm engraftment (49.9% donor cells in peripheral blood 16 weeks after transplantation). Compared to the rodents, the sheep model has the advantage of body size and gestation comparable to the human fetus. Here, ultrasound-guided injection techniques significantly decreased fetal loss rates. In contrast to the murine in utero model, the repopulation capacities of allogeneic ovine fetal liver cells are lower (0.112% donor cells in peripheral blood 3 weeks after transplantation). The effect of MHC on engraftment levels seems to be marginal, since no differences could be observed between autologous and allogeneic transplantation (0.117% donor cells vs 0.112% donor cells in peripheral blood 1 to 2 weeks after transplantation). Further research is needed to study optimal timing and graft composition as well as immunological aspects of in utero transplantation.

Animals↗

PubMed2006

Water birth, more than a trendy alternative: a prospective, observational study.

OBJECTIVE: To prospectively assess the effect of water birth on maternal and fetal outcomes in a selected low-risk collective of a tertiary obstetrical unit. METHOD: In this prospective observational study, 513 patients of a low-risk collective, who requested a water birth, were studied during the years 1998-2002. Primary outcome measurements included the maternal and fetal parameters. Secondary outcome measurements comprised data on the incidence of water births in an interested, low-risk population in an academic hospital. RESULT: All groups were similar in terms of demographic and obstetric data. Significant differences were observed in maternal outcome parameters, which included the use of analgesia/anesthesia during labor, the duration of first and second stages of labor, perineal tears and episiotomy rate. No differences were seen in all observed fetal outcome parameters including APGAR scores, arterial and venous pH, admission rate to neonatal intensive care unit and infection rate. CONCLUSION: Water birth is a valuable and promising alternative to traditional delivery methods. The maternal and fetal outcomes were similar to traditional land births. However, currently there still exist some deficits in the scientific evaluation of its safety. Therefore, the selection of a low-risk collective is essential to minimize the risks with the addition of strictly maintained guidelines and continuous intrapartum observation and fetal monitoring. Based on our results and the literature, water births are justifiable when certain criteria are met and risk factors are excluded.

Apgar Score↗

PubMed2006

T4 category revision enhances the accuracy and significance of stage III breast cancer.

BACKGROUND: Because of the considerable heterogeneity in breast carcinoma with noninflammatory skin involvement (T4b/Stage IIIB), a revision was proposed of the TNM staging system that would classify these tumors exclusively based on their tumor size and lymph node status. In the current study, the authors evaluated how implementation of this proposal will affect Stage III noninflammatory breast cancer. METHODS: Two hundred seven patients who were classified with noninflammatory Stage III breast cancer were treated consecutively between 1990 and 1999 at the University Hospital Basel, Switzerland. To assess the extent of T4b/Stage IIIB tumors independent of the clinicopathologic feature of skin involvement, the reclassification was undertaken. RESULTS: Of 68 patients who had nonmetastatic T4b breast cancer, 37 patients (54.4%) had a tumor extent in accordance with Stage I/II and had improved disease-specific survival (DSS) compared with patients who had Stage III breast cancer (P = .008). Excluding those patients from Stage III led to a 17.9% reduction of the number of patients in this group (n = 170 patients). The 10-year DSS declined from 48.5% to 42.9%. CONCLUSIONS: Considerable numbers of patients who are classified with noninflammatory Stage IIIB breast cancer show only a limited disease extent. Through a revision of the T4 category, these low-risk patients were excluded from the highest nonmetastatic TNM stage, and overstaging could be avoided. This procedure decreased the degree of heterogeneity of the entire Stage III group and may result in a more precise assessment of this disease entity.

Breast Neoplasms↗

PubMed2006

Increased peri- and post-elective cesarean section morbidity in women infected with human immunodeficiency virus-1: a case-controlled multicenter study.

OBJECTIVE: Although elective cesarean section (ECS) is the currently recommended modality for delivering women infected with the human immunodeficiency virus (HIV), historical evidence suggests that they are at higher risk of postoperative complications than noninfected women. Those risks have to be carefully balanced against the presumed minimal benefit of ECS, especially in the case of low viral load and high CD4 counts. We therefore compared the incidence and type of post-ECS complications in HIV-infected women, most with low viral loads and high CD4 cell counts, with those in matched noninfected women treated by the same surgical teams. STUDY DESIGN: A Swiss 8-center, prospective, matched case-control study compared minor and major post-ECS complication prevalence, hospital stay and confounding factors (surgeon experience) between HIV-infected and noninfected women. RESULTS: Minor complications in the 53 matched pairs were eightfold more frequent overall in infected women. More frequent specific minor complications were anemia, blood loss and urinary tract infection. Yet the surgeons performing ECS in infected women were more experienced. Complications prolonged hospital stay in infected women. Major complication rates did not significantly differ between the groups. CONCLUSION: HIV-positive women have a higher risk of post-ECS morbidity, even with high CD4 counts and low viral load. Therefore, the blanket recommendation of ECS in HIV-infected women requires a review.

Adult↗

PubMed2006

Disturbances in placental immunology: ready for therapeutic interventions?

Recent studies have provided new insight into aberrations in the immunological interplay between mother and fetus and their potential role in the development of recurrent fetal loss and preeclampsia. The action of anti-phospholipid antibodies in recurrent fetal loss is now proposed to involve the complement system, neutrophil activation and the production of TNFalpha by immune bystander cells. A clear involvement of the immune system is emerging in preeclampsia, involving mainly the innate arm, especially neutrophils. The activation of peripheral neutrophils by placentally released inflammatory debris triggers the induction of neutrophil extracellular traps (NETs), which may lead to an occlusion of the intervillous space, thereby further promoting a condition of placental hypoxia. It has, hence, been suggested that new therapeutic strategies be developed, including the possible use of TNFalpha antagonists in cases of recurrent miscarriage. These strategies need to be addressed with caution due to the possible induction of fetal congenital abnormalities.

Abortion, Habitual↗

PubMed2006

Spermatogenesis in long-term survivors after allogeneic hematopoietic stem cell transplantation is associated with age, time interval since transplantation, and apparently absence of chronic GvHD.

Factors associated with spermatogenesis after allogeneic hematopoietic stem cell transplantation (HSCT) were assessed in this prospective, single-center, cross-sectional study. All consecutive men aged 18 years or older and in complete remission 2 years or longer after HSCT were invited to participate. Seminal fluid analysis was performed on freshly collected samples according to World Health Organization guidelines. Between April 2003 and June 2004, 39 patients were included. The median age at semen analysis was 34 years (range, 20-59 years), and the median time interval between HSCT and sperm analysis 9 years (range, 2-20 years). Thirty-two patients (82%) underwent total body irradiation (TBI; >or= 10 Gy) as part of their conditioning regimen. Eleven of 39 (28%) patients showed some spermatogenesis. Patients with detectable spermatozoa in the ejaculate were younger at HSCT (median age, 19 versus 28 years; P = .004), had a longer interval since HSCT (median time, 12 versus 7 years; P = .01), and were more often without chronic graft-versus-host disease (GvHD; 2 of 11 patients versus 16 of 28; P = .03). Nine of 16 patients (56%) undergoing transplantation when younger than age 25 years showed some degree of spermatogenesis. In conclusion, men who are long-term survivors, who were younger than 25 years at HSCT, and who apparently do not have chronic GvHD have a reasonable likelihood of spermatogenesis even when conditioned with standard-dose TBI.

Adult↗

PubMed2006

Induction of neutrophil extracellular DNA lattices by placental microparticles and IL-8 and their presence in preeclampsia.

Preeclampsia, a severe pregnancy-related disorder, involves an overt activation of the maternal innate immune system, proposed to result from the elevated release of inflammatory syncytiotrophoblast microparticles (STBM) and cytokines from underlying placental anomaly involving abnormal trophoblast differentiation. Activation of circulating neutrophils has recently been shown to lead to the generation of fibrous extracellular lattices containing DNA, termed NETs (neutrophil extracellular traps). Therefore, we examined whether placentally derived factors activated peripheral neutrophils to generate NETs, and whether NETs formation was increased in preeclamptic placenta. Activation of isolated circulatory neutrophils on treatment with placentally derived factors (interleukin-8 and STBM) was assessed by measuring CD11b expression using Flow cytometry. Furthermore, NETs generation by these activated neutrophils in vitro and in vivo in the placental tissue sections were examined using electron microscopy and fluorescence microscopy. We report that placentally-derived interleukin-8 and STBM efficiently activated neutrophils and triggered NETs formation. Large numbers of NETs were present directly in the intervillous space of preeclamptic placentae. NETs, therefore, appear to be an integral part of neutrophil activation, and their increased presence in preeclampsia suggests that NETs may play a role in the underlying pathology.

Cell-Derived Microparticles↗

PubMed2006

Genotyping fetal paternally inherited SNPs by MALDI-TOF MS using cell-free fetal DNA in maternal plasma: influence of size fractionation.

The determination of fetal point mutations from fetal cell-free DNA (cf-DNA) in maternal plasma is technically challenging due to the preponderance of maternal sequences. It has recently been shown that fetal cf-DNA sequences are smaller than maternal ones and that the selection of small cf-DNA fragments by size fractionation by agarose gel electrophoresis leads to the enrichment of fetal cf-DNA sequences, thereby permitting the detection of otherwise masked fetal point mutations. In a separate development, the use of MALDI-TOF MS has also been shown to facilitate the detection of fetal point mutations from cf-DNA in maternal plasma. In this study, a combination of these approaches was examined. cf-DNA was extracted from 18 maternal plasma samples, 10 taken at term and 8 obtained early in the second trimester. A total of 41 SNP loci were examined in size-fractionated and total cf-DNA using either a conventional homogeneous MassEXTEND (hME) assay or a nucleotide-specific single allele base extension reaction (SABER) assay. The analysis of total cf-DNA indicated that size fractionation considerably enhanced the sensitivity of the standard hME assay, especially for samples taken early in pregnancy. Size fractionation also rendered the signals obtained by the SABER assay more precise.

Alleles↗

PubMed2006

Direct quantification of fetal cells in maternal blood by real-time PCR.

BACKGROUND: Fetal cells in maternal blood still present an enticing alternative for the development of a safe and efficacious non-invasive method for prenatal diagnosis. However, most enrichment methods are very tedious and have failed to realise this long sought after goal. We developed a simple, robust TaqMan real-time PCR assay to directly quantify male fetal cells in maternal blood using the multi-copy DYS14, without the need for any additional enrichment procedure. METHODS: The sensitivity of the DYS14 assay was evaluated by using female genomic DNA spiked with male DNA or male cells. The specificity of the DYS14 assay was evaluated by examining 40 adult blood samples and 44 maternal blood samples from pregnant women with known fetal gender. Direct quantification of fetal cells in maternal blood was performed in 14 of the maternal blood samples by the DYS14 assay. The results were compared to those by SRY assay. RESULTS: The sensitivity of DYS14 PCR assay was found to be higher than that of SRY assay for the detection of fetal cells. The number of fetal cells in maternal blood did not exceed 2/mL blood. The presence of cell-free fetal DNA in maternal blood could lead to erroneous quantification of fetal cell DNA. The number of fetal cells detected in blood cell pellets, which had been unwashed to remove the cell-free fetal DNA, was indeed significantly higher than those in extensively washed samples. CONCLUSIONS: We quantified the fetal cells in maternal blood without fetal cell enrichment procedures by TaqMan real-time PCR for a multi-copy DYS14 locus. Our assay is sensitive and also suitable for automation, and may be a useful tool for the determination of fetal-maternal cell trafficking, such as microchimerism.

Chromosomes, Human, Y↗

PubMed2006

Comparison of activin A and cell-free fetal DNA levels in maternal plasma from patients at high risk for preeclampsia.

OBJECTIVES: We examined the concentration of activin A in a prospective manner before the clinical manifestation of preeclampsia and compared the data with those of cell-free fetal DNA in the maternal plasma. METHODS: The levels of activin A were analysed by enzyme-linked immunosorbent assay (ELISA) for pregnant women: (1) with preeclampsia (n = 34) in the third-trimester and normal controls (n = 44); and (2) at-risk of preeclampsia in the second-trimester (n = 15) as indicated by uterine artery Doppler and normal controls (n = 68). Correlation between activin A level and cell-free fetal DNA level was examined using the Spearman rank test. RESULTS: The level of plasma activin A was significantly higher in the preeclamptic samples (12.056 vs 7.068 ng/mL, p = 0.000). The increase in the activin A concentration was observed prior to the onset of preeclampsia (3.483 vs 1.324 ng/mL, p = 0.000). This increase in activin A correlated significantly with the increased level of cell-free fetal DNA, in the maternal circulation prior to the onset of preeclampsia (r = 0.977, p = 0.000). CONCLUSION: Our data suggest that circulatory activin A could be an independent biomarker for the early identification and monitoring of preeclampsia.

Activins↗

PubMed2006

High levels of circulatory erythroblasts and cell-free DNA prior to intrauterine fetal death.

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Adult↗

PubMed2006

Placental mesenchymal stem cells as potential autologous graft for pre- and perinatal neuroregeneration.

OBJECTIVE: Mesenchymal stem cells (MSCs) have a broad differentiation potential. We aimed to determine if MSCs are present in fetal membranes and placental tissue and to assess their potential to differentiate into neurogenic and mesodermal lineages. STUDY DESIGN: MSCs isolated from first and third trimester chorion and amnion and first trimester chorionic villi and characterized morphologically and by flourescence-activated cell sorting analysis. Their ability to mature under different culture conditions into various cells of mesodermal and neuroectodermal cell lines was assessed by immuno- and cytochemical staining. RESULTS: Independent of gestational age, cells isolated from fetal membranes and placenta showed typical MSC phenotype (positive for CD166, CD105, CD90, CD73, CD49e, CD44, CD29, CD13, MHC I; negative for CD14, CD34, CD45, MHC II) and were able to differentiate into mesodermal cells expressing cell markers/cytologic staining consistent with mature chondroblasts, osteoblasts, adipocytes, or myocytes and into neuronal cells presenting markers of various stages of maturation. The differentiation pattern was mainly dependent on cell type. CONCLUSION: Mesenchymal cells from chorion, amnion, and villous stroma can be differentiated into neurogenic, chondrogenic, osteogenic, adipogenic, and myogenic lineage. Placental tissue obtained during prenatal chorionic villous sampling or at delivery might be an ideal source for autologous stem cell graft for peripartum neuroregeneration and other clinical issues.

Cell Differentiation↗

PubMed2006

Correlation and significance of histopathological and clinical features in breast cancer with skin involvement (T4b).

A retrospective review was performed to investigate the prognostic significance and validity of the pathological and clinical TNM staging of noninflammatory skin involvement in breast cancer. In 128 tumors with histologically proven skin involvement and a size up to 5 cm (64% of the entire group), we distinguished clearly between group A) cases showing the classical clinical signs (cT4b) and those that do not, and between group B) carcinomas infiltrating the epidermis (pT4b) and those infiltrating only the dermis. We found only moderate concordance (kappa = 0.44) between the pathological and clinical TNM staging system. In the analysis of 80 patients with a tumor size from 2.1 to 5.0 cm, neither the appearance of classical clinical signs nor the histological diagnosis of infiltration of the epidermis was shown to be a relevant factor. In comparison to the control groups, similar clinicopathologic entities without significant differences in long-term outcome were observed. After regrouping of the patients having tumor infiltration of the papillary dermis from the control group into the study group (pT4), the study group showed a significant higher number of involved axillary lymph nodes (P = .014) and a more extensive lymph node involvement (pN3; P = .025). The combination epidermis-papillary dermis seems to be more a functional unit than the epidermis alone that is defined as the crucial and delineating factor in the TNM Classification. Our results challenge the validity of the TNM rules and recommendations concerning T4b breast cancer because it leads, in the majority of cases, to tumors of comparable extent and prognosis being placed in different categories.

Adenocarcinoma↗

PubMed2006

A new approach in breast cancer with non-inflammatory skin involvement.

The widely accepted image of breast cancer with non-inflammatory skin involvement (T4b) is determined by the tenet that all these tumors are locally advanced (Stage IIIB). The study addresses the question whether this view is justified. Data from 453 non-metastatic breast cancer patients were collected retrospectively. Eighty-one patients had T4b disease. To assess the malignant potential of tumors independent of the feature skin involvement, a reclassification only considering tumor size was undertaken. We compared the clinical course of three study groups (A: Stage II; B: Stage IIIA; C: Stage IIIC) with control groups of 372 patients without skin involvement. In the study groups, we found a broad distribution among the stages (A:36.2%; B:33.7%; and C:27.7%) with significant differences in disease-specific survival (DSS) (A/B: p = 0.032; B/C: p = 0.048). There were no significant differences in DSS between the study and the corresponding control group. In multivariate analysis, skin involvement was not a significant predictor of DSS. Heterogeneity of the T4b category and a lack of prognostic significance expand the widely accepted image of breast cancer with non-inflammatory skin involvement. The highest T category, or Stage III, is not the appropriate classification for a considerable number of patients having this clinicopathologic entity.

Adult↗