Involvement of Angiopoietin-1 (angpt-1) in Ovarian Hyperstimulation Syndrome (ohss) Pathogenesis

PARBORELL FERNANDA; SCOTTI LEOPOLDINA; ABRAMOVICH DALHIA; PASCUALI NATALIA; DE ZUÑIGA IGNACIO; SOBRAL FABIO; TESONE MARTA

Congreso; 45th SSR Annual Meeting and the 18th Ovarian Workshop; 2012

OHSS is an iatrogenic complication associated with ovarian stimulation for the treatment of infertility.  Risk factors include low body weight, high follicle count and elevated serum estradiol. Vasoactive substances such as vascular endothelial growth factor (VEGF) and angiopoietin-1 (ANGPT1) are involved in the regulation of vascularization. The ANGPTs/Tie-2 system acts in concert with VEGF. ANGPT-1 stabilizes blood vessels while ANGPT2 and the soluble receptor Tie-2 (sTie-2) act as natural antagonists for ANGPT1. The balance between ANGPT1, ANGPT2, sTie-2 and VEGF is central for ovarian angiogenesis. We have previously demonstrated that levels of ANGPT1 are increased while levels of sTie-2 are invariant in fluid follicular (FF) from women at risk of OHSS in contrast with FF from normoresponders. Our aims were to analyze: 1) the levels of ANGPT2 in FF from patients at risk of OHSS and 2) the effect of ANGPT1 neutralizing antibody on endothelial cell migration and claudin-V expression in the presence of FF from patients at risk of developing OHSS. This study was performed in 51 patients aged 25-39 years old undergoing ART. Patients with pelvic pathologies such as endometriosis, uterine fibroids or pelvic inflammatory disease were excluded. The patients were classified into: control group (n=20) and OHSS group (n=19). The criteria to consider a patient at risk of developing OHSS were: serum E2 levels >3,000 pg/ml on the day of hCG administration and a retrieval of >20 oocytes. The FF was centrifuged for 10 min at 2000xg to remove cellular components. The supernatant was stored at –80º C until assayed. The levels of ANGPT2 and claudin-V (tight junction protein) in FF were measured by western blot. To assess the effect of ANGPT1 on ovarian angiogenesis, we evaluated the effect of FF from OHSS patients on endothelial cell migration in the presence of a neutralizing antibody against ANGPT1. For this purpose, a wound healing assay using the EA.hy926 endothelial cell line was performed. The levels of ANGPT2 were similar in patients at risk of OHSS compared to control patients (p<0.01). In addition, the increased levels of ANGPT1 confirmed those obtained by ELISA in our previous study (p<0.05). There was a marked increase in the ANGPT1:ANGPT2  ratio in FF from patients at risk of OHSS compared to control group. FF from patients at risk of OHSS induced endothelial cell migration at a higher extent than in normoresponder patients (p<0.001). Incubation with FF from OHSS group in the presence of an ANGPT1 antibody resulted in a significant decrease in cell migration compared to those FF without antibody (p<0.05). Moreover, incubation with FF from OHSS patients in presence of ANGPT-1 antibody produced an increase in the endothelial levels of claudin-V, in comparison with FF in absence of antibody (p<0.01). In conclusion, the ANGPTs/Tie-2 system is associated with the pathogenesis of OHSS. The balanced levels of these proteins are required for proper ovarian vascular function. Furthermore, the increased endothelial levels of claudin-V in presence of FF from patients at high risk of OHSS and incubated with ANGPT1 antibody suggest a decrease in the vascular permeability, partially regulated by ANGPT1. Therefore, the results described regarding endothelial cell migration may provide new insights into the mechanisms by which ANGPT1 has an effect on ovarian disorders such as OHSS.