Inhibition of cyclooxygenase-2 (COX-2) by meloxicam decreases the incidence of ovarian hyperstimulation syndrome in a rat model Ramiro Quintana, M.D.,a Laura Kopcow, M.D.,a Guillermo Marconi, M.D.,a Edgardo Young, Ph.D.,a Carola Yovanovich, B.Sc.,b and Dante A. Paz, Ph.D.b,c a Instituto de Ginecologıa y fertilidad (IFER); b Biodiversidad y Biologıa Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, and c Instituto de Fisiologıa, Biologıa Molecular y Neurociencias (IFIBYNE–CONICET), Buenos Aires, Argentina
Objective: To investigate the effects of selective cyclooxygenase-2 (COX-2) inhibition on the ovarian hyperstimulation syndrome (OHSS) in an experimental model. Design: Controlled laboratory study. Setting: University-affiliated fertility center. Animal(s): Female Wistar rats. Intervention(s): Female Wistar rats (22 days old) were divided into four groups: group 1 (control group; n ¼ 10) received 0.1 mL of intraperitoneal (IP) saline from days 22–26; group 2 (mild-stimulated group; n ¼ 10) received 10 IU of pregnant mare serum gonadotropin (PMSG) on day 24 and 10 IU of hCG 48 hours later (day 26); group 3 (OHSS group; n ¼ 10) was given 10 IU of PMSG for 4 consecutive days from day 22 and 30 IU hCG on the fifth day to induce OHSS; group 4 was treated the same as group 3, but received 2 mL (15 mg/mL) of meloxicam 2 hours before the PMSG injection for 4 consecutive days, and 2 hours before the hCG injection on the fifth day. All groups were killed on day 26. Main Outcome Measure(s): Number of antral and luteinized follicles, ovarian weight, semiquantitative vascular endothelial growth factor (VEGF) and COX-2 immunohistochemistry. Result(s): There were no differences in the ovarian weight between groups 1 and 2. Group 3 showed significantly increased ovarian weight that was suppressed, in group 4, by meloxicam. There was no difference in the number of antral follicles among the four groups. In the mild-stimulated and OHSS groups, the granulosa cells (GC) of preovulatory follicles and the stromal cells showed intense VEGF immunoreactivity. The ovaries from the meloxicam-treated group showed less immunoreactivity than the OHSS group, indicating diminished VEGF expression associated with meloxicam treatment. Group 3 (OHSS group) showed increased COX-2 immunoreactivity that was diminished in the meloxicam-treated group. Meloxicam treatment did not affect the hormone-induced increase in serum E2 levels seen in OHSS rats. Conclusion(s): Our results in a rat model suggest that meloxicam has a beneficial effect on OHSS by reducing the increases in ovarian weight and VEGF expression associated with OHSS. These effects may be mediated by the COX-2 inhibitory capacity of meloxicam. (Fertil Steril 2008;90:1511–6. 2008 by American Society for Reproductive Medicine.) Key Words: Ovarian hyperstimulation syndrome, VEGF, cyclooxygenase-2 inhibitor, meloxicam
Ovarian hyperstimulation syndrome (OHSS) is the most serious complication of ovulation induction with hMG and hCG. This iatrogenic condition is potentially lethal and occurs in 0.3%–5% of stimulated ovarian cycles (1). Clinical manifestations of OHSS are massive extravascular fluid accumulation and hemoconcentration similar to that in syndromes due to capillary leakage. Renal failure, hypovolemic shock, thromboembolic episodes, and adult respiratory distress syndrome are potential complications of OHSS. Because of the
peripheral arteriolar vasodilatation and the increase in capillary permeability seen in patients with OHSS, it is believed that OHSS is mediated by ovary-produced vasoactive substances in response to gonadotropin stimulation (2, 3). Vascular endothelial growth factor (VEGF) is considered a prime causative agent in OHSS progression (4, 5). Recent experiments in rodents have clearly shown a cause–effect relationship between ovarian VEGF expression and increased vascular permeability (3, 6).
Received June 2, 2007; revised September 6, 2007; accepted September 18, 2007. Supported in part by the CONICET (PIP 5842) to D.A.P. Reprint requests: Dante A. Paz, Ph.D., Facultad de Ciencias Exactas y n 2, Naturales, Depto. Biodiversidad y Biologıa Experimental, Pabello piso 4, Cdad. Universitaria, (1428) Buenos Aires, Argentina (FAX: 5411-4576-3384; E-mail:
[email protected]).
Alternative splicing of the single human VEGF gene generates seven isoforms of VEGF protein (110, 115, 121, 145, 165, 189, and 206 amino acids) (7). Rodent VEGF is shorter by one amino acid than human VEGF (8), and although six VEGF isoforms (VEGF 110, 120, 144, 164, 188, and 205) have been detected in rat tissues (9), only two isoforms have been detected in the ovary (10). The detailed biological differences among these isoforms in the ovary remain unknown.
0015-0282/08/$34.00 doi:10.1016/j.fertnstert.2007.09.028
Fertility and Sterility Vol. 90, Suppl 2, October 2008 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.
1511
Vascular endothelial growth factor binds to two known receptors, VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1, KDR), thereby activating their intracellular tyrosine kinase domains, and in turn influencing several downstream signaling pathways (11). VEGFR-2 appears to transduce most VEGFinduced effects, such as cell proliferation, chemotaxis, changes in protein expression, and antiapoptotic activity, whereas VEGFR-1 has a weak signaling activity and serves as a physiological negative regulator of VEGF action (12). Cyclooxygenase (COX) is a key enzyme in the conversion of arachidonic acid to prostaglandins and other eicosanoids. Two isoforms of COX have been identified: COX-1 is constitutively expressed in many tissues, whereas COX-2 is induced by a variety of factors, including cytokines, growth factors, and tumor promoters. Experimental overexpression of COX-2 in a colon cancer cell line results in the overproduction of several proangiogenic factors, including VEGF (13). In addition, the inhibition of COX-2 suppresses prostate cancer growth and angiogenesis in vivo. Tumors treated with COX-2 inhibitor were smaller, had higher levels of apoptosis, had decreased microvessel density, and had decreased tumor VEGF levels (14). Similarly, COX-2 inhibition prevents hypoxic up-regulation of VEGF in human prostate cancer cells (15, 16). Although the importance of COX-2 in the ovulatory process has been documented, the exact functions of prostaglandins in human ovary, fertilization, and implantation are not completely understood (17). In this study we evaluated the effects of the COX-2 inhibitor meloxicam on the development of OHSS in the rat model. MATERIALS AND METHODS Immature female Wistar rats were obtained from Bioterio Central, Facultad de Ciencias Exactas y Naturales, UBA. All research animals were treated in compliance with the guidelines for the care and use of animals approved by our institutions in accordance to principles of laboratory animal care (NIH Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council, Washington, D.C.). Rats were fed with standard diet, allowed free access to water, and had a 12hour light cycle (lights-on 7 AM to 7 PM). Twenty-two–day-old female rats (weight 44–50 g) were divided into four groups: the control group (n ¼ 10) received 0.1 mL of intraperitoneal (IP) saline from days 22–26; the mild-stimulated group (n ¼ 10) received 10 IU of pregnant mare serum gonadotropin (PMSG; Sigma. St. Louis, MO) on day 24 and 10 IU of hCG (Sigma) 48 hours later (day 26) to mimic a routine ovarian stimulation; the OHSS group (n ¼ 10) was given 10 IU of PMSG for 4 consecutive days and 30 IU hCG on the fifth day to induce OHSS (3); the fourth group (OHSS þ meloxicam; n ¼ 10) was treated similarly as the OHSS group, with the addition of meloxicam (Boeringer, Buenos Aires, Argentina) (0.35 mg/rat) 2 hours before the PMSG injection for 4 consecutive days, and 2 hours before the hCG injection on the fifth day. 1512
Quintana et al.
Four hours after the final hCG injection, the females were decapitated. The ovaries were weighed, the right one was fixed in Bouin’s liquid for immunohistochemistry and follicular count, and the left ovary was frozen for Western blotting. Immunohistochemistry Fixed samples were dehydrated and embedded in paraffin. Six-micron sections were mounted on gelatin-chromalumcoated glass slides. Sections were deparaffined in xylene and hydrated through a series of graded alcohols, and washed in phosphate-buffered saline (PBS). The tissue sections were treated with 3% hydrogen peroxide (H2O2) solution to quench endogenous peroxidase activity. Nonspecific binding sites were blocked by treating the tissues with TNB blocking reagent (FP1020, NEN Life Science Products, Boston, MA) and subsequently incubated with the primary antibodies for 24 hours at 4 C in a dark moist chamber. Antibodies used were rabbit anti-VEGF (sc-507, Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit anti-COX-2 (sc-7950, Santa Cruz). After incubation with primary antibody, the sections were washed with PBS and treated with the appropriate biotinylated antibody (Vector Laboratories, Burlingame, United Kingdom) followed by avidin-horseradish peroxidase-biotin complex (Vectastain ABC kit, Vector Laboratories). The color reaction was visualized by exposure to 30 ,30 -diaminobenzidine tetrahydrochloride staining kit (Dako Cytomation, Carpinteria, CA). For COX-2 immunohistochemistry the reaction was amplified with a Tyramide signal amplification kit (CSA Kit, Dako Cytomation) following the manufacturer’s instructions. The slides were washed twice in PBS, dehydrated, and mounted in permount (Fisher, Pittsburgh, PA). Morphometric Analysis of VEGF and COX-2 Immunoreactivity The immunoreactivity on ovary sections was quantified using Meta Morph software (Universal Imaging Corporation, Downingtown, PA). All sections were processed simultaneously under identical conditions. Three to five sections containing each ovary region of interest were analyzed from each animal. Digital images were captured at 100 or 200 magnification for densitometric analysis. A random procedure was carried out throughout the image analysis. The ovary regions were analyzed using a hand-made frame defining the area of interest, and the number of particles stained above a standard density threshold in the selected area was counted automatically. The mean density for each animal was calculated as the total positive staining area on the multiple sections divided by the total selected area. Statistics The observed mean densities of VEGF and COX-2-immunoreactivity in the ovary were analyzed by Student’s t-test using the SPSS statistical package for Windows (SPSS Inc., Chicago, IL). Data are expressed as the mean SEM. Student’s and Mann-Whitney tests were performed to assess differences between the means using the InStat software package
COX-2 inhibition in ovarian hyperstimulation
Vol. 90, Suppl 2, October 2008
(GraphPad Software Inc., San Diego, CA). Significance was accepted at P
