Effect Of Gonadotropin Releasing Hormone Antagonists Vs Agonists On Expression Of Vascular Endothelial Growth Factor And Its Receptors In Ovarian Hyperstimulated Rats

BackgroundOvarian hyperstimulation syndrome(OHSS) is one of the most life-threatening and potentially fatal complications of ART. The increasing demand and access to infertility treatments worldwide due to rising infertility rate have led to the increased prevalence of OHSS, which is thought to be associated with ovarian enlargement and an increase in capillary permeability which leads to an extensive accumulation of fluids, predominantly in the peritoneal compartments. Although the pathophysiology of the syndrome has not been completely elucidated yet, OHSS is believed to be triggered by the action of several vasoactive factors secreted by the hyperstimulated ovaries mediated by human chorionic gonadotrophin.Vascular endothelial growth factor(VEGF), also known as permeability factor, is a potent mitogen for vascular endothelial cells acting through stimulating receptor tyrosine kinase, namely flt-1 and flk-1/KDR. Previous study by Mcclure et al indicated the level of VEGF was increased in ascites of OHSS patients, and the vascular permeability reduced approximately 70% when treatment with anti-VEGF antibody, so it was suggested that the main substance inducing vascular hyperpermeability was VEGF. The expression of VEGF was also observed in human luteinized granulose cells in vitro by RT-PCR in OHSS patients, in response to human chorionic gonadotrophin in a dosage and time dependent fashion. Thus VEGF is thought to be the main angiogenic substance responsible for increased capillary permeability leading to extravasation of protein-rich fluid and subsequently OHSS.Recently, kitajima et al reported administration of GnRHa in hyperstimulated ratsreduced vascular permeability, which may be related to a decrease of VEGF and its receptors. Compared to agonist, GnRHanta induce a direct block of GnRH receptor with a rapid decrease in LH and FSH, preventing LH surge. Smaller doses of gonadotrophins, shorter stimulation period and lower OHSS occurrence are reported in literature using GnRH antagonists. The aim of present study is to compare the efficacy of hyperstimulation treatment in an immature rat early OHSS model, in order to investigate the possible mechanism of reducing the occurrence of OHSS by administration of GnRHanta and its specification of action compared to long and short term agonists. MethodsImmature female Wistar rats . All studies were begun when the animals were 22 days old (38.0-42.0g) .forty rats were randomly allocated into five groups, (l)OHSS group was given 10 IU PMSG/day from day 22 to day 25 and 30 IU hCG On day 26 to induce OHSS. (4)GnRHanta treated hyperstimulated rat group was given injections of GnRHanta for two consecutive days (cetrolix, 25ug/kg/d, from day 26 to day 27,twice a day at 8am and 4pm);(2)short-term GnRHa treated group(S-GnRHa) was given injections of GnRHa(decapetyl, lOOug/kg/d, from day 26 to day 27,twice a day at 8am and 4pm );(3)long-term GnRHa treated group(L-GnRHa) was given injections of GnRHa for six consecutive days from the start of PMSG treatment(decapetyl, lOOug/kg/d, from day 22 to day 27,twice a day at 8am and 4pm );(5) control group received 0.1 ml i.p. saline from day 22 to day 25, thus constituting a control group.48h after administration of hCG or saline, the animals were killed. In each set of experiments, one ovary of animals was frozen in nitrogen for mRNA analysis, whereas the animal's other ovary for frozen slices.The capillary permeability was evaluated by Evans-blue content of peritoneal fluid and autofluorescence and RT-polymerase chain reaction (RT-PCR) was used to detect the expression of vascular endothelial growth factor and its receptors flt-l,KDR mRNA. Conclusion1. Our experiment established an immature OHSS rat model after the ovarian stimulation with PMSG/hCG, we investigated the ovarian weight and the vascular permeability by using the level of extravasated evans blue dye in peritoneal fluid injected into the femoral vessel, the ovarian content of evans blue was observed by fluorescence microscope to locate in regions such as the surface of the ovaries, surroundings of blood vessels, andcorpora lutea. These results revealed that hyperstimulation treatment provided a statistically significant increase in ovarian weight, vascular permeability and VEGF, fit-1 and KDR mRNA expression.2. Both GnRHanta and GnRHa treatment resulted in statistically significant reductions in peritoneal vascular permeability, with a decreased productions of ovarian VEGF and its receptors, fit-1 and KDR expression in hyperstimulated rats, which is parallel to clinical data. It is concluded GnRHanta and GnRHa may prevent early OHSS by reducing the vascular permeability through down regulation of vascular endothelial growth factor and its receptors. Long-term GnRHa treatment provided a lower level in flt-1 and KDR mRNA expression in the hyperstimulated rats compared to antagonists treatment, and only the reduction in VEGFR-l(flt-l) expression was statistically different. They exhibited a similar effect in reducing the vascular permeability, this result may be due to the two isoforms VEGF 164 and VEGF 120 mainly acting through interaction with receptor-2, KDR. During two VEGF receptors, KDR undergoes strong ligand-dependent tyrosine phosphorylation in intact cells, while phosphorylation of flt-1 is weak. Therefore, interaction with KDR is a critical requirement to induce the full spectrum of VEGF biological responses, the receptor 2 appears to be mainly involved in regulating VP, angiogenesis, and vasculogenesis.3. GnRHanta induced a rapid decrease in vascular permeability and VEGF receptors expression, but the decrease of ovarian weight not as good as the change of capillary permeability, when severe OHSS is already present ,the administration of GnRHanta is probably useful to shorten the period and ease the symptoms of OHSS which may act through reducing the vasoactive substances such as VEGF.