Routine Fetal Rhd Genotyping with Maternal Plasma: A Four-Year Experience in Belgium

peer reviewedBACKGROUND: The objective was to evaluate the diagnostic value of RHD fetal genotyping from the plasma of D- mothers as soon as 10 weeks' gestation in a routine clinical practice in Belgium. STUDY DESIGN AND METHODS: A prospective study was conducted between November 2002 and December 2006. DNA extraction was performed in an automated closed tube system. Fetal RHD/SRY genotypes were detected in the plasma of 563 pregnant mothers by real-time polymerase chain reaction (PCR) targeting multiple exons 4, 5, and 10 of the RHD gene and targeting an SRY gene sequence. These were compared to the D phenotypes determined in the 581 babies they delivered. RESULTS: By combining amplification of three exons, the concordance rate of fetal RHD genotypes in maternal plasma and newborn D phenotypes at delivery was 100 percent (99.8% including one unusual false-positive). The presence of nonfunctional RHD genes and the absence of a universal fetal marker, irrespective of fetal sex, did not influence the accuracy of fetal RhD status prediction. The RHD genotyping from 18 twin pregnancies was also assessed. Five weak D women were excluded from the RHD fetal genotyping prediction. Three discrepant results (0.5%) between predicted fetal genotype and cord blood phenotype were not confirmed by the baby phenotypes from venipuncture blood. CONCLUSION: Prenatal prediction of fetal RHD by targeting multiple exons from the maternal plasma with real-time PCR is highly sensitive and accurate. Over 4 years, this experience has highly modified our management of D- pregnant women

Assay of matrix metalloproteinases types 1, 2, 3 and 9 in breast cancer.

Matrix metalloproteinases (MMPs) are zinc dependent endopeptidases implicated in cancer invasion and metastasis. Gelatin zymography was performed on 84 human breast carcinomas and seven normal breast tissues. The precursor form of MMP-2 (72 kDa) was found in 11 (12%) samples, while its two activated forms, i.e. 62 kDa and 59 kDa, were found in three (6%) and 34 (40%) samples respectively. In contrast to MMP-2, most of the samples (52%) contained MMP-9 in its precursor form. Using ELISA, MMP-1 levels were found in 12% of the samples while MMP-3 levels were found in only 2% of the samples. Levels of MMP-2, -3 and -9 correlated inversely with numbers of nodal metastases. Neither MMP-2 nor -9 levels were significantly related to patient outcome. However, patients with high levels of a 50-kDa gelatinase band after zymography had a significantly better survival than patients with low levels. This species was never observed in normal breast tissue

Enhancement of tumorigenicity of human breast adenocarcinoma cells in nude mice by matrigel and fibroblasts.

The failure of MCF7 cells to induce the formation of tumours after sub-cutaneous inoculation into athymic nude mice can be obviated by the simultaneous injection of an extract of basement membrane proteins (matrigel). Tumour growth is promoted and the latency period is low (2 to 4 weeks). In the absence of matrigel, the simultaneous inoculation of fibroblasts and MCF7 cells also resulted in the development of tumours, but with a longer latency period (about 2 months). The tumorigenic synergy between matrigel and fibroblasts was evidenced by co-inoculating MCF7 cells MDA-MB 231 cells with fibroblasts and matrigel. This co-inoculation decreased the delay of appearance of the tumours and/or accelerated the tumour growth, depending upon the number of fibroblasts injected. Repeated injections of fibroblasts conditioned medium, at the site of inoculum of tumour cells also enhanced tumour growth, suggesting the involvement of soluble factors secreted by fibroblasts. Histologically, tumours induced by co-inoculation of tumour cells and fibroblasts contained more stromal structures including vimentin-positive cells, fibronectin and interstitial collagens. These data suggest that human tumours may be reconstituted and grown in athymic nude mice using basement membrane components and fibroblasts as inductors

Induction of Endothelial Cell Apoptosis by Solid Tumor Cells

The mechanisms by which tumor cells extravasate to form metastasis remain controversial. Previous studies performed in vivo and in vitro demonstrate that the contact between tumor cells and the vascular wall impairs endothelium integrity. Here, we investigated the effect of breast adenocarcinoma MCF-7 cells on the apoptosis of human umbilical vein endothelial cells (HUVEC). TUNEL labeling, nuclear morphology, and DNA electrophoresis indicated that MCF-7 cells induced a two- to fourfold increase in HUVEC apoptosis. Caspase-3 activity was significantly enhanced. Neither normal cells tested (mammary epithelial cells, fibroblasts, leukocytes) nor transformed hematopoietic cells tested (HL60, Jurkat) induced HUVEC apoptosis. On the contrary, cells derived from solid tumors (breast adenocarcinoma, MDA-MB-231 and T47D; fibrosarcoma, HT 1080) had an effect similar to that of MCF-7 cells. The induction of apoptosis requires cell-to-cell contact, since it could not be reproduced by media conditioned by MCF-7 cells cultured alone or cocultured with HUVEC. Our results suggest that cells derived from solid tumors may alter the endothelium integrity by inducing endothelial cell apoptosis. On the contrary, normal or malignant leukocytes appear to extravasate by distinct mechanisms and do not damage the endothelium. Our data may lead to a better understanding of the steps involved in tumor cell extravasation

Human chondrocytes in tridimensional culture.

peer reviewedCartilage was taken from the macroscopically normal part of human femoral heads immediately after orthopedic surgical operations for total prothesis consecutive to hip arthrosis. After clostridial collagenase digestion and repeated washings, chondrocytes (10(6) cells) were cultivated in a gyrotory shaker (100 rpm). Under these conditions, cells were kept in suspension and after 3 to 5 d formed a flaky aggregate which, on Day 10, became dense. These chondrocytes were morphologically differentiated: they had a round shape, were situated inside cavities, and were surrounded by a new matrix. Histochemical methods showed the presence of collagen and polysaccharides in cell cytoplasm and in intercellular matrix, and the immunofluorescence method using specific antisera (anticartilage proteoglycans and anti-type II collagen) showed that these two constituents were in intercellular matrix. The measurement of the amounts of proteoglycans (PG) released into culture medium and those present in chondrocyte aggregate (by a specific PG radioimmunoassay) showed a maximum production on Days 3 to 5 of culture, then the production decreased and stabilized (from Day 10 to the end of culture). The observed difference between the amounts of PG in aggregates after 20 d and those after 2 h of culture demonstrated that PG neosynthesis did occur during cultivation. This conclusion was supported by other results obtained by [14C]glucosamine incorporation in chondrocyte aggregates. Moreover, the aggregate fresh weight related to cell number (appreciated by DNA assay) increased significantly with culture duration. Three-dimensional chondrocyte culture represents an interesting model: chondrocytes were differentiated morphologically as well as biosynthetically and synthesized a new cartilage matrix

Whole Slide Quantification of Stromal Lymphatic Vessel Distribution and Peritumoral Lymphatic Vessel Density in Early Invasive Cervical Cancer: A Method Description

Peritumoral Lymphatic Vessel Density (LVD) is considered to be a predictive marker for the presence of lymph node metastases in cervical cancer. However, when LVD quantification relies on conventional optical microscopy and the hot spot technique, interobserver variability is significant and yields inconsistent conclusions. In this work, we describe an original method that applies computed image analysis to whole slide scanned tissue sections following immunohistochemical lymphatic vessel staining. This procedure allows to determine an objective LVD quantification as well as the lymphatic vessel distribution and its heterogeneity within the stroma surrounding the invasive tumor bundles. The proposed technique can be useful to better characterize lymphatic vessel interactions with tumor cells and could potentially impact on prognosis and therapeutic decisions

Restoring testosterone levels by adding dehydroepiandrosterone to a drospirenone containing combined oral contraceptive: II Clinical effects

Objectives: Combined oral contraceptives (COCs) decrease androgen levels, including testosterone (T), which may be associated with sexual dysfunction and mood complaints in some women. We have shown that co-administration of dehydroepiandrosterone (DHEA) to a drospirenone (DRSP) containing COC restored total T levels to baseline and free T levels by 47%. Here we describe the effects on sexual function, mood and quality of life of such an intervention. Study design: This was a randomized, double-blind, placebo-controlled study in 99 healthy COC starters. A COC containing 30 μg ethinylestradiol (EE) and 3 mg DRSP was used for 3 cycles, followed by 6 cycles of the same COC combined with 50 mg/day DHEA or placebo. Subjects completed the Moos Menstrual Distress Questionnaire (MDQ), the McCoy Female Sexuality Questionnaire (MFSQ) and the short form of the Quality of Life Enjoyment and Satisfaction Questionnaire (Q-LES-Q). Safety and tolerability, including effects on skin were evaluated. Results: The addition of DHEA induced small, but significant improvements compared to placebo in the MDQ score for: Autonomic reactions during the menstrual (- 2.0 vs 0.71; P = 0.05) and the pre-menstrual phase (- 3.1 vs 2.9; P = 0.01); and for Behavior during the inter-menstrual phase (- 1.4 vs 3.6; P = 0.02). A significant difference was found in the MDQ score for arousal during the pre-menstrual phase in favor of placebo (- 5.0 vs 1.0; P = 0.01). There were no statistically significant differences between groups for the MSFQ and Q-LES-Q scores. DHEA co-administration resulted in an acceptable safety profile. DHEA negated the beneficial effect of the COC on acne according to the subjects' self-assessment. Conclusions: Co-administration with DHEA did not result in consistent improvements in sexual function, mood and quality of life indicators in women taking EE/DRSP. Retrospectively, the 50 mg dose of DHEA may be too low for this COC. Implications: A well-balanced judgment of the clinical consequences of normalizing androgens during COC use may require complete normalization of free T

Restoring testosterone levels by adding dehydroepiandrosterone to a drospirenone containing combined oral contraceptive: I Endocrine effects

Objectives: Combined oral contraceptives (COCs) decrease testosterone (T) levels. This study investigated restoration of T and other androgen concentrations during COC use by co-administration of dehydroepiandrosterone (DHEA). Study design: In this randomized, double-blind, placebo-controlled study in 99 new COC starters (18-35 years old with BMI range 18-34 kg/m2), a COC containing 30 μg ethinylestradiol (EE) and 3 mg drospirenone (DRSP) was used for 3 cycles, followed by 6 cycles of the same COC combined with either 50 mg/day DHEA or placebo. Total T, albumin, sex hormone-binding globulin (SHBG), DHEA-sulfate (DHEA-S), Δ4-androstenedione (AD), 3α-androstanediol glucuronide (ADG) and estradiol (E2) were measured, whereas free T and the free T index (FTI) were calculated. Assessments took place at baseline (no COC use), after the run-in period (COC use alone) and during the treatment period (DHEA or placebo). Results: During COC use alone androgen levels decreased, especially total T by 62% and free T by 86%, and SHBG increased by 243%. Total T increased with DHEA compared to placebo (change from end of run-in period to end of treatment period: 1.3 ± 1.2 nmol/L vs 0.0 ± 0.4 nmol/L; P < 0.0001), and was restored to baseline levels. Free T and the FTI increased significantly (P < 0.0001), but the free T level was still 53% below baseline levels. DHEA-S, AD and ADG increased significantly to levels above baseline (P < 0.0001 for each). DHEA had no effect on SHBG, albumin and E2. Conclusions: An EE/DRSP containing COC strongly suppressed endogenous androgen concentrations in all users. The addition of 50 mg DHEA to a COC regimen containing EE/DRSP restored total T to baseline levels, but free T levels were restored by only 47% as most of the T remains bound to SHBG. Implications: When using a COC that increases SHBG considerably, a daily dose of 50 mg DHEA is insufficient to normalize free T levels completely

Development of an animal experimental model to study the effects of levonorgestrel on the human endometrium

BACKGROUND: This study was designed to develop an animal model to test the response of endometrium to local progestin delivery. METHODS: Proliferative human endometrium was subcutaneously grafted in two groups of SCID mice that received, 2 days before, a subcutaneous estradiol (E2) pellet and, for half of them, an additional implant of levonorgestrel (LNG). Mice were sacrificed 1, 2, 3 or 4 weeks after endometrial implantation and grafts were histologically analysed. Proliferation, steroid hormone receptors, blood vessels and stromal decidualization in both groups (E2 and LNG) were immunohistologically evaluated and compared with proliferative endometrium and endometrium from women with an LNG intrauterine device. RESULTS: Grafts presented normal morphological endometrial characteristics. The expression of progesterone receptors was significantly decreased in glands and stroma of the LNG group as compared with the E2 group at all times. A significant decrease was also observed in the stromal expression of estrogen receptor- in the LNG group. At 4 weeks, the mean cross-sectional area of vessels was significantly higher after LNG treatment. CONCLUSIONS: These morphological and immunohistochemical characteristics are similar to those observed in women treated with local LNG. This mouse model might facilitate further investigations needed to understand the mechanisms responsible for the breakthrough bleeding frequently observed in progestin users