We have investigated the biological characteristics of an immortalized granulosa cell line (COV434), which may be used to study follicular and oocyte maturation in vitro. Granulosa cell function was defined as consisting of three distinct properties: (i) production of 17beta-oestradiol in response to follicle stimulating hormone (FSH); (ii) presence of specific molecular markers of apoptosis enabling the induction of follicular atresia; and (iii) capacity to form intercellular connections with cells surrounding an oocyte. The addition of FSH to the culture medium supplemented with 10% fetal calf serum and 4-androstene-3,17-dione resulted in proliferation of the COV434 granulosa cells and in an increased synthesis of 17beta-oestradiol, indicating the presence of the FSH receptor and cytochrome P450 aromatase in these cells. The receptor for luteinizing hormone (LH) was undetectable. Similar expression of various apoptosis-associated genes was found in COV434 granulosa cells and in granulosa cells of patients stimulated with gonadotrophins for in-vitro fertilization, thus indicating that the immortalized COV434 granulosa cells were able to sustain apoptosis. Multiple intercellular connections were formed during co-culture of COV434 granulosa cells with cumulus cells containing an immature oocyte but not with cumulus cells devoid of an oocyte. Detailed morphological analysis of the intercellular connections with scanning electron microscopy and confocal light microscopy demonstrated the presence of long slender structures. It is concluded that the immortalized human granulosa cell line COV434 may be useful for experimental studies on follicular development.

Background We analysed cell-free DNA (cfDNA) in the plasma of patients with both malignant and benign breast lesions by real-time quantitative PCR to determine whether the Wnding may have diagnostic and prognostic implications. Methods Plasma samples were obtained from 33 patients with breast cancer, 32 patients with benign breast lesions and 50 healthy women as normal controls. Circulatory cfDNA was extracted from the plasma samples and quantiWed by real-time quantitative PCR for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Results The mean concentrations of cfDNA in the plasma samples from patients with breast cancer, patients with benign breast lesions and normal controls were 2,285, 1,368 and 1,489 genome equivalents (GE) per millilitre, respectively. The level of cfDNA in the breast cancer group was signiWcantly higher than those in the benign lesion group and control group (P = 0.007 and 0.013, respectively). These Wndings were associated with malignant tumour size. The levels of the cfDNA were high in patients with lymph node involvement and distant metastasis. Conclusions Our results suggest that levels of cfDNA in the plasma are elevated in malignant breast cancer and correlated with tumour size. These Wndings could have diagnostic and prognostic value for malignant breast tumours.

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Objectives: Preeclampsia has been shown to be associated with an increased number of fetal and maternal erythroblasts in the maternal circulation, suggesting that preeclampsia involves increased leakage of fetal cells across the placental barrier, as well as increased erythropoiesis. We examined the relationship between circulatory erythroblast levels with maternal plasma concentrations of erythropoietin and activin A. Methods: In a case-control study, we examined 15 pregnancies affected by preeclampsia and 10 matched controls. Erythroblasts were enriched from maternal blood samples by magnetic cell sorting, enumerated and correlated with corresponding plasma activin A and erythropoietin concentrations. Results: The proportion of erythroblast was elevated in preeclampsia (0.8 vs. 0.1%, p = 0.023). Erythropoietin and activin A concentrations were significantly elevated in preeclampsia (100.4 vs. 44.5 pg/ml, p = 0.023, and 7.4 vs. 1.85 ng/ml, p = 0.029, respectively). Circulatory erythroblast numbers were found to correlate with plasma activin A concentrations (r = 0.76, p = 0.01) in cases with preeclampsia. No such relationship existed for erythropoietin. Conclusions: Our data suggest that increased concentrations of activin A promote enhanced levels of erythropoiesis in preeclampsia. As the placenta is one of the major sources of activin A in pregnancy, this increase in activin A-dependent erythropoiesis in preeclampsia may be a reflection of an underlying placental hypoxic condition.

Postpartum urinary retention (PUR) is a clinical condition that is neither well-recognized nor defined by standardized means but normally has a good prognosis. We present the case of a woman with a history of PUR who demonstrated recurrent PUR. Prolonged first and second stage of labor, isolated prolonged second stage of labor, forceps delivery or vacuum extraction, perineal laceration, nulliparity and epidural anesthesia can act as independent risk factors for the development of PUR. In this case, epidural anesthesia, which was administered in both deliveries, was the only risk factor, suggesting that its application in a woman with a history of PUR should be carefully considered and discussed with the patient. Bladder drainage resolves PUR, after which there seems to be no cumulative risk for voiding dysfunction. An initial smaller post-void residual bladder volume may have a predictive value concerning the time to resolution of PUR.

609 Background: The sentinel lymph node (SLN) biopsy has emerged as the standard of care in evaluating the axillary lymph node status in early stage breast cancer patients. It is well known that step sectioning and immunohistochemistry of the SLN allow for a more accurate histopathologic examination. Conversely, it remains to be elucidated whether or not the more accurate SLN staging is associated with improved survival. Therefore, the objective of the present investigation was to evaluate if patients undergoing a SLN biopsy have an improved disease-free and overall survival compared to those undergoing ALND. Methods: From our prospective database 355 node negative patients with early stage breast cancer (pT1 und pT2 <=3cm, pN0/pNSN0) were assessed. Patients underwent either ALND (n=178) in the years 1990–1997 or a SLN biopsy (n=177) in 1998–2004. Long-term disease-free and overall survival were analysed for both groups. Log-rank tests were used for unadjusted analyses, a Cox proportional hazard regression model for risk-adjusted analyses. Results: The median follow- up was 48.2 months in the SLN group and 120.0 months in the ALND group. Patients in the SLN group had a significantly better disease-free (p=0.012) and overall survival (p=0.04) compared to the ALND group. In Cox proportional hazard regression analysis, the performed procedure (SLN compared to ALND) was an independent predictor for improved disease-free survival (hazard ratio 0.28, 95% confidence interval 0.11–0.75, p=0.011) and overall survival (hazard ratio 0.36, 95% confidence interval 0.14–0.89, p=0.027). Conclusion: The present analysis - the first one in the literature - provides compelling evidence that patients with a negative SLN have a significantly improved disease-free and overall survival compared to node negative patients undergoing ALND. This relevant finding is most likely due to an improved histopathologic staging accuracy. The significant survival benefit of node negative patients having SLN biopsy highlights yet another important advantage of the SLN concept in breast cancer. No significant financial relationships to disclose.

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