Department of Obstetrics and Gynecology, University of Milan, H San Raffaele Scientific Institute, Via Olgettina 60, 20132 Milano, Italy 1 To whom correspondence should be addressed
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Abstract |
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Key words: albumin/granulosa cells/ovarian hyperstimulation syndrome/vascular endothelial growth factor
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Introduction |
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Recently, the use of human i.v. albumin at the time of oocyte retrieval has been described in patients at risk of OHSS with different results (Asch et al., 1993; Shoham et al., 1994
; Mukherjee et al., 1995
; Lewit et al., 1996
). Because the pathophysiology of OHSS is not clearly elucidated, the mechanism of albumin action remains elusive.
The primary object of this study was to analyse the in-vitro effects of albumin on VEGF expression in luteinizing granulosa cells.
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Materials and methods |
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Granulosa cell cultures
Human luteinizing granulosa cells were obtained by ultrasound-guided transvaginal follicular aspiration of individual follicles that was accomplished 35 h after HCG injection from three groups of patients with increasing serum oestradiol: group 1 (n = 7) oestradiol <1000 pg/ml, group 2 (n = 7) oestradiol 1000 <2000 pg/ml, group 3 (n = 6) oestradiol
2000 pg/ml.
After removing the oocytes, the remaining cells from each patient were pooled and washed twice with medium 199 (Flow Laboratories, Milan, Italy). Granulosa cells and red blood cells were transferred to a 12 ml tube containing 3.5 ml lymphocyte separation medium (Flow Laboratories, Milan, Italy) and separated by centrifugation at 600 g for 5 min. Granulosa cells were dispersed by gentle shaking at 37°C for 30 min in 5 ml culture medium containing 0.1% collagenase and 20 mg DNase/ml. The dispersed cells were washed in culture medium, counted and plated at a density of 45x105 cells/10 cm plastic culture dish (Falcon) in serum-free medium 199 containing 2 mM glutamine and 50 mg/ml gentamycin. Cells were cultured at 37°C in a 95% air5% CO2 humidified environment. After 2 days, the cells had attached to the wells. At this time the medium was removed and 24 h incubations with and without 10 mg/ml of human albumin in medium 199 were initiated. Granulosa cells were washed twice with medium 199 and then frozen immediately at 80°C until RNA extraction.
RNA extraction and analysis
Undegraded total RNA was prepared from frozen granulosa cells by guanidine thiocyanate/phenol chloroform single-step extraction (RNAfast®; Molecular Systems, San Diego, CA, USA). For dot blot hybridization, 5 µg of total RNA from each sample were denatured in 6% formaldehyde and 50% deionized formamide in x1 standard saline citrate buffer (SSC; 0.75 mol/l NaCl and 0.075 mol/l sodium citrate).
The RNA was then transferred to nitrocellulose and nylon membranes (Hybond-N®; Amersham). The cDNA probe used for hybridizations of VEGF was a 930 bp EcoRI fragment coding for human VEGF (Genentech, South San Francisco, CA, USA). Hybridizations with cDNA probe (VEGF) were performed in Northern hybridization buffer (Celbio, Milan, Italy) in 50% formamide and 100 µg/ml salmon sperm DNA. Hybridizations were performed at 42°C for 16 h with 24x106 cpm/ml labelled with [-32P]dCTP cDNA probe. After hybridization the blots were washed twice with x2 SSC with 0.1% sodium dodecyl sulphate (SDS), then with x0.1 SSC with 0.1% SDS for 15 min at room temperature. After washing, the blots were exposed to X-ray film (Kodak XAR 5®; Eastman Kodak, Rochester, NY, USA) at 80°C with intensifying screens. The relative amount of mRNA examined was determined by reprobing all blots for mRNA encoding actin. Between consecutive hybridizations, the blots were stripped in 0.1% SDS at 100°C for 30 min before rehybridizing. The relative intensity of signals for VEGF on the dot blots was compared to that for actin using computer-assisted densitometry. The ratio of VEGF to actin was used because no up-regulation of actin was observed in the presence of albumin. The ratios between VEGF intensity and actin intensity are indicated as arbitrary units. Variations in arbitrary units between samples are indicated as the SD. Statistical analyses were performed by using Student's paired t-test for comparison within groups.
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Results |
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Discussion |
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VEGF has been shown to be produced by granulosa cells and to increase vascular permeability followed by extravasation of plasma (Senger et al., 1983). Excessive VEGF production by granulosa cells enhances von Willebrand factor serum concentrations and increases the risk of thrombo-embolism (Brock et al., 1991
). Therefore, the most eminent symptoms of OHSS are VEGF related. Asch et al. were the first to administer i.v. human albumin at the time of oocyte retrieval to prevent the development of severe OHSS (Asch et al., 1993
). It has been suggested that human albumin may prevent OHSS by increasing plasma oncotic pressure and by binding the factors responsible for the development of the syndrome. Although VEGF may be involved in the pathogenesis of OHSS, it is not an important clinical marker of the course of OHSS (Ludwig et al., 1998
). Several authors showed that i.v. albumin does not prevent the occurrence of OHSS especially in cases associated with pregnancy (Mukherjee et al., 1995
; Orvieto et al., 1995
; Lewit et al., 1996
; Ndukwe et al., 1997
).
The fact that the increased HCG associated with pregnancy induces an elevated VEGF production may explain the increased incidence of OHSS in these cases (Neulen et al., 1995; Rizk et al., 1997
). VEGF is known to increase vascular permeability and extravasation of plasma. Our results demonstrate that human albumin increases VEGF gene expression in human luteinizing granulosa cells and the maximum expression is present in cultured granulosa cells obtained from patients with serum oestradiol concentrations >2000 pg/ml on the day of HCG injection. This study is an in-vitro observation and because the pathogenesis is still poorly understood and complex its clinical relevance is not known.
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References |
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Submitted on April 6, 1998; accepted on January 27, 1999.