Impact of vitrification on granulosa cell survival and gene expression.

INTRODUCTION: Cryopreservation of ovarian tissue is an essential step in Ovarian Tissue Banking. In order to prevent the formation of ice crystals, typically the tissue is slowly frozen using a cryoprotectant. As an alternative the method of ultra-fast freezing by vitrification becomes more attention for freezing ovarian tissue because it has successfully been used for oocytes, embryos and sperm. However the impact of vitrification on granulosa cells, which are an essential part of ovarian tissue is uncertain. AIM: In this study, we have therefore analysed the influence of vitrification on the survival rates of granulosa cells, the impact of DMSO or ethylenglycol containing vitrification protocols and investigated to what extent the gene expression of apoptosis- and temperature-sensitive genes changes. MATERIAL AND METHODS: We used the human granulosa cell line KGN as a model for human granulosa cells and determined the survival rate and cell cycle stages by FACS analyses. The change in gene expression was determined by quantitative PCR analyses. RESULTS: Our results show that vitrification is possible in granulosa cells but it reduces cell viability and leads to fluctuations in the cell cycle. The DMSO containing protocol results in a lower amount of dead cells than the ethylenglycol containing protocol. Gene expression analysis reveals that TNF-alpha expression is strongly increased after vitrification, while other apoptosis or temperature-related genes seem to stay unaffected. CONCLUSION: We conclude that vitrification influences the viability of human granulosa cells. Furthermore, our results suggest that this could be mediated by a change in TNF-alpha gene expression.

Expression of insulin-like growth factor-1 receptor in circulating tumor cells of patients with breast cancer is associated with patient outcomes.

In patients with breast cancer, markers of aggressiveness such as dysregulation of the insulin-like growth factor receptor (IGF1R) system and E-cadherin loss are commonly observed. Reduced IGF1R expression is correlated with decreased E-cadherin levels and increased cell motility. We assessed IGF1R and E-cadherin expression in circulating tumor cells (CTCs) in patients with breast cancer. Peripheral blood mononuclear cells of early (n = 87)- and metastatic (n = 126)-stage breast cancer patients (obtained prior to adjuvant and first-line chemotherapy) were evaluated using double immunofluorescence (IF) staining for cytokeratin (CK) and IGF1R. Triple IF using CK, IGF1R, and E-cadherin antibodies was performed in selected CTC(+) patients. IGF1R(+) CTCs were more frequently observed in early disease than in metastatic disease (86% vs 68% of CTCs, P = 0.04) stage, whereas IGF1R(-) CTCs were more common in metastatic than in early disease (32% vs 14% of CTCs, P = 0.002). 100% of CTC(+) patients with early disease, compared to 79% of those with metastatic disease, harbored IGF1R(+) CTCs (P = 0.007). Patients with early disease and exclusively IGF1R(+) CTCs had longer disease-free (P = 0.02) and overall survival (P = 0.001) compared to patients with both IGF1R(+) and IGF1R(-) CTC populations. 67% of early-stage CTC(+) patients evaluated had exclusively IGF1R(+)/E-cadherin(+) CTCs, 33% also had IGF1R(-)/E-cadherin(-) CTCs, and none had exclusively IGF1R(-)/E-cadherin(-) CTCs compared to 17%, 75%, and 8% of metastatic patients, respectively (P = 0.027). Similarly, in paired samples of patients with early disease that progressed to metastatic disease, the proportion of IGF1R(+)/E-cadherin(+) CTCs was reduced and IGF1R(-)/E-cadherin(-) CTCs were increased in the metastatic stage compared to early disease stage. IGF1R(+) CTCs are commonly detected in breast cancer, and their frequency decreases in the metastatic disease stage. IGF1R(+)/E-cadherin(+) CTCs also decrease in metastatic patients. IGF1R(+) CTCs are associated with favorable outcomes in early disease stage, suggesting that IGF1R expression is correlated with reduced metastatic potential in breast cancer.