Tyroxine hydroxylase (TH) downregulation in hyperstimulated ovaries reveals the dopamine agonist bromocriptine (Br2) as an effective and specific method to block increased vascular permeability (VP) in OHSS

Abstract

Objective: Ovarian hyperstimulation syndrome (OHSS) is induced by the ovarian release of vasoactive-angiogenic substances which increase vascular permeability (VP). Vascular Endothelial Growth Factor (VEGF) plays a major role in this regard. Inhibition of VEGF action has been accompanied by decrease in VP. However, the use of such strategy as antiangiogenic tumoral drugs in women has been abandoned due to toxic effects. Aiming to find a specific-non toxic treatment for OHSS, we have looked at the expression of gene products regulated under OHSS conditions, and developed new approaches employing the D-2 dopamine agonist Bromocriptine (Br2). Design: In the first series of experiments, we compared ovarian gene expression in animals (n = 8 per group) subjected to 3 different regimens of gonadotropins: Pregnant Mare′s Serum Gonadotropin (PMSG) 10 IU/day for 4 days and hCG (30 IU) the fifth day (OHSS group); PMSG (10 IU) plus hCG (10 IU) 2 days later (MILD group); and PMSG (1 IU) plus hCG (1 IU) 48 hrs later (CONTROL group). Fourty-eight hours after hCG, VP was measured, and mRNA from ovaries extracted to perform gene expression profiles in macroarray filters containing 14,000 genes. Only genes with a 3-fold up or down regulation were considered to be significantly regulated. The hybridization results were confirmed by quantitative Fluorescence(QF)-PCR, immunostaining, and cluster analysis. In the second series of experiments, Br2 was employed in OHSS animals (40 IU PMSG+30IU hCG). Different doses of Br2: 1.6, 4, 10 and 25 mg/kg were intraperitoneally administered the day of hCG and 24 hrs later. VP was measured 48 hrs after hCG. Materials and Methods: Immature female Wistar rats were employed. In the macroarray technology, RNA was reverse transcribed and transferred to the nylon membrane filters under radioactive conditions. Clusters of 5×5 spot signals were analyzed using the AIDA software, and its expression paired to the other groups after background signal substraction. To confirm gene expression, we used the same RNA and performed QF-PCR in an ABIPRISM 7700 thermocycler to amplify 3 up-regulated and 3 down-regulated genes. To measure VP, 0.2 ml 5mM Evans blue(EB) were injected via the femoral vein. After 30 min, the peritoneal cavity was irrigated with saline. Light absorption was measured in the recovered fluid at 595 nm and the VP expressed as μg EB/100 g body weight. Results: Gene expression in the 3 groups showed 80 upregulated and 7 downregulated genes in OHSS as compared to MILD and CONTROLS. Upregulated genes were grouped in 5 families: Cholesterol synthesis,VEGF signal transduction, Prostaglandins synthesis, oxidative stress process and Prolactin synthesis. The down-regulation of Tyrosine-hydroxylase (TH, enzyme responsible for Dopamine synthesis) was considered a characteristic of OHSS too. As a result, the D2 dopamine agonist Br2 was employed. Increased VP in OHSS animals (30.7±5.8 μg EB/100 g body weight) was significantly reduced when 1.6 mg Br2 (15.3±3.3; p = 0.04), 4 mg Br2 (14.2±2.2; p = 0.04), 10 mg Br2 (10.1±2.6; p = 0.009), and 25 mg Br2 (8.0±1.9; p = 0.001) were employed. Conclusion: OHSS is associated with the up and down regulation of several families of genes in the ovaries. In addition to VEGF, the up and down regulation of genes encoding for prolactin and dopamine expression, encouraged the successful use of Br2 to prevent increased VP. These experiments provide the rationale to test Br2 as a safe and possible effective medication in the prevention/treatment of OHSS.