0015doi:1

Inhibition of cyclooxygenase-2 (COX-2) by meloxicamdecreases the incidence of ovarian hyperstimulationsyndrome in a rat modelRamiro 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 Ginecolog�ıa y fertilidad (IFER); b Biodiversidad y Biolog�ıa Experimental, Facultad de Ciencias Exactas y Naturales,

Universidad de Buenos Aires, and c Instituto de Fisiolog�ıa, Biolog�ıa Molecular y Neurociencias (IFIBYNE–CONICET), Buenos

Aires, Argentina

Objective: To investigate the effects of selective cyclooxygenase-2 (COX-2) inhibition on the ovarian hyperstim-ulation 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) received10 IU of pregnant mare serum gonadotropin (PMSG) on day 24 and 10 IU of hCG 48 hours later (day 26); group 3(OHSS group; n¼ 10) was given 10 IU of PMSG for 4 consecutive days from day 22 and 30 IU hCG on the fifth dayto induce OHSS; group 4 was treated the same as group 3, but received 2 mL (15 mg/mL) of meloxicam 2 hoursbefore the PMSG injection for 4 consecutive days, and 2 hours before the hCG injection on the fifth day. All groupswere killed on day 26.Main Outcome Measure(s): Number of antral and luteinized follicles, ovarian weight, semiquantitative vascularendothelial 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 significantlyincreased ovarian weight that was suppressed, in group 4, by meloxicam. There was no difference in the number ofantral follicles among the four groups. In the mild-stimulated and OHSS groups, the granulosa cells (GC) ofpreovulatory follicles and the stromal cells showed intense VEGF immunoreactivity. The ovaries from the melox-icam-treated group showed less immunoreactivity than the OHSS group, indicating diminished VEGF expressionassociated with meloxicam treatment. Group 3 (OHSS group) showed increased COX-2 immunoreactivity that wasdiminished in the meloxicam-treated group. Meloxicam treatment did not affect the hormone-induced increase inserum 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 theincreases in ovarian weight and VEGF expression associated with OHSS. These effects may be mediated by theCOX-2 inhibitory capacity of meloxicam. (Fertil Steril� 2008;90:1511–6. �2008 by American Society for Repro-ductive Medicine.)

Key Words: Ovarian hyperstimulation syndrome, VEGF, cyclooxygenase-2 inhibitor, meloxicam

Ovarian hyperstimulation syndrome (OHSS) is the most seri-ous complication of ovulation induction with hMG and hCG.This iatrogenic condition is potentially lethal and occurs in0.3%–5% of stimulated ovarian cycles (1). Clinical manifes-tations of OHSS are massive extravascular fluid accumula-tion and hemoconcentration similar to that in syndromesdue to capillary leakage. Renal failure, hypovolemic shock,thromboembolic episodes, and adult respiratory distress syn-drome are potential complications of OHSS. Because of the

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

Naturales, Depto. Biodiversidad y Biolog�ıa Experimental, Pabell�on 2,

piso 4, Cdad. Universitaria, (1428) Buenos Aires, Argentina (FAX:

5411-4576-3384; E-mail: dante@bg.fcen.uba.ar).

-0282/08/$34.000.1016/j.fertnstert.2007.09.028 Copyright ª2008 American

peripheral arteriolar vasodilatation and the increase in capil-lary permeability seen in patients with OHSS, it is believedthat OHSS is mediated by ovary-produced vasoactive sub-stances in response to gonadotropin stimulation (2, 3). Vascu-lar endothelial growth factor (VEGF) is considered a primecausative agent in OHSS progression (4, 5). Recent experi-ments in rodents have clearly shown a cause–effect relation-ship between ovarian VEGF expression and increasedvascular permeability (3, 6).

Alternative splicing of the single human VEGF gene gener-ates seven isoforms of VEGF protein (110, 115, 121, 145, 165,189, and 206 amino acids) (7). Rodent VEGF is shorter by oneamino acid than human VEGF (8), and although six VEGFisoforms (VEGF 110, 120, 144, 164, 188, and 205) havebeen detected in rat tissues (9), only two isoforms havebeen detected in the ovary (10). The detailed biological differ-ences among these isoforms in the ovary remain unknown.

Fertility and Sterility� Vol. 90, Suppl 2, October 2008 1511Society for Reproductive Medicine, Published by Elsevier Inc.

Vascular endothelial growth factor binds to two known re-ceptors, VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1, KDR),thereby activating their intracellular tyrosine kinase domains,and in turn influencing several downstream signaling path-ways (11). VEGFR-2 appears to transduce most VEGF-induced effects, such as cell proliferation, chemotaxis,changes in protein expression, and antiapoptotic activity,whereas VEGFR-1 has a weak signaling activity and servesas a physiological negative regulator of VEGF action (12).

Cyclooxygenase (COX) is a key enzyme in the conversionof arachidonic acid to prostaglandins and other eicosanoids.Two isoforms of COX have been identified: COX-1 is consti-tutively expressed in many tissues, whereas COX-2 is in-duced by a variety of factors, including cytokines, growthfactors, and tumor promoters. Experimental overexpressionof COX-2 in a colon cancer cell line results in the overpro-duction of several proangiogenic factors, including VEGF(13). In addition, the inhibition of COX-2 suppresses prostatecancer growth and angiogenesis in vivo. Tumors treated withCOX-2 inhibitor were smaller, had higher levels of apoptosis,had decreased microvessel density, and had decreased tumorVEGF levels (14). Similarly, COX-2 inhibition prevents hyp-oxic up-regulation of VEGF in human prostate cancer cells(15, 16). Although the importance of COX-2 in the ovulatoryprocess has been documented, the exact functions of prosta-glandins in human ovary, fertilization, and implantation arenot completely understood (17).

In this study we evaluated the effects of the COX-2 inhib-itor meloxicam on the development of OHSS in the ratmodel.

MATERIALS AND METHODS

Immature female Wistar rats were obtained from BioterioCentral, Facultad de Ciencias Exactas y Naturales, UBA.All research animals were treated in compliance with theguidelines for the care and use of animals approved by our in-stitutions in accordance to principles of laboratory animalcare (NIH Guide for the Care and Use of LaboratoryAnimals, Institute of Laboratory Animal Resources, NationalResearch Council, Washington, D.C.). Rats were fed withstandard diet, allowed free access to water, and had a 12-hour light cycle (lights-on 7 AM to 7 PM).

Twenty-two–day-old female rats (weight 44–50 g) weredivided into four groups: the control group (n ¼ 10) received0.1 mL of intraperitoneal (IP) saline from days 22–26; themild-stimulated group (n ¼ 10) received 10 IU of pregnantmare serum gonadotropin (PMSG; Sigma. St. Louis, MO)on day 24 and 10 IU of hCG (Sigma) 48 hours later (day26) to mimic a routine ovarian stimulation; the OHSS group(n ¼ 10) was given 10 IU of PMSG for 4 consecutive daysand 30 IU hCG on the fifth day to induce OHSS (3); the fourthgroup (OHSSþmeloxicam; n¼ 10) was treated similarly asthe OHSS group, with the addition of meloxicam (Boeringer,Buenos Aires, Argentina) (0.35 mg/rat) 2 hours before thePMSG injection for 4 consecutive days, and 2 hours beforethe hCG injection on the fifth day.

1512 Quintana et al. COX-2 inhibition in ovarian hyperstim

Four hours after the final hCG injection, the females weredecapitated. The ovaries were weighed, the right one wasfixed in Bouin’s liquid for immunohistochemistry and follic-ular 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-chromalum-coated glass slides. Sections were deparaffined in xyleneand hydrated through a series of graded alcohols, and washedin phosphate-buffered saline (PBS). The tissue sections weretreated with 3% hydrogen peroxide (H2O2) solution toquench endogenous peroxidase activity. Nonspecific bindingsites were blocked by treating the tissues with TNB blockingreagent (FP1020, NEN Life Science Products, Boston, MA)and subsequently incubated with the primary antibodies for24 hours at 4�C in a dark moist chamber. Antibodies usedwere rabbit anti-VEGF (sc-507, Santa Cruz Biotechnology,Santa Cruz, CA) or rabbit anti-COX-2 (sc-7950, SantaCruz). After incubation with primary antibody, the sectionswere washed with PBS and treated with the appropriate bio-tinylated antibody (Vector Laboratories, Burlingame, UnitedKingdom) followed by avidin-horseradish peroxidase-biotincomplex (Vectastain ABC kit, Vector Laboratories). Thecolor reaction was visualized by exposure to 30,30-diamino-benzidine tetrahydrochloride staining kit (Dako Cytomation,Carpinteria, CA). For COX-2 immunohistochemistry the re-action was amplified with a Tyramide signal amplification kit(CSA Kit, Dako Cytomation) following the manufacturer’sinstructions. The slides were washed twice in PBS, dehy-drated, and mounted in permount (Fisher, Pittsburgh, PA).

Morphometric Analysis of VEGF and COX-2Immunoreactivity

The immunoreactivity on ovary sections was quantified usingMeta Morph software (Universal Imaging Corporation,Downingtown, PA). All sections were processed simulta-neously under identical conditions. Three to five sectionscontaining each ovary region of interest were analyzedfrom each animal. Digital images were captured at �100 or�200 magnification for densitometric analysis. A randomprocedure was carried out throughout the image analysis.The ovary regions were analyzed using a hand-made framedefining the area of interest, and the number of particlesstained above a standard density threshold in the selectedarea was counted automatically. The mean density for eachanimal was calculated as the total positive staining area onthe multiple sections divided by the total selected area.

Statistics

The observed mean densities of VEGF and COX-2-immuno-reactivity in the ovary were analyzed by Student’s t-test usingthe SPSS statistical package for Windows (SPSS Inc., Chi-cago, IL). Data are expressed as the mean � SEM. Student’sand Mann-Whitney tests were performed to assess differ-ences between the means using the InStat software package

ulation Vol. 90, Suppl 2, October 2008

(GraphPad Software Inc., San Diego, CA). Significance wasaccepted at P<.05.

RESULTS

Ovarian Weight

The hyperstimulation treatment caused a significant increasein ovarian weight compared with control animals. The weightincrease observed in the OHSS group was suppressed in theOHSS þmeloxicam group after 6 days of treatment (Fig. 1).

Number of Follicles

Figure 2 summarizes the number of antral follicles (largestfollicles with a defined cumulus granulosa cell (GC) layer)detected in each group of rats. The number of antral folliclesdid not differ in the three treated groups, but there was a ten-dency toward a reduction in the number of antral follicles ob-served in the OHSS þ meloxicam group as compared withthe OHSS group (Fig. 2).

VEGF and COX-2 Protein Localization

The results of immunohistochemistry analysis revealed mod-erate VEGF staining in thecal cells, stromal cells, and GCsfrom developed follicles in all groups analyzed. On the otherhand, the ovaries of the OHSS group showed moderate to in-tense VEGF immunoreactivity in the GCs of preovulatoryfollicles and the corpus luteum (CL). In the OHSS þ melox-icam group, VEGF immunoreactivity was diminished whencompared with the OHSS group (Fig. 3A–D), suggestingthat meloxicam affected VEGF protein expression. Densito-metric analysis revealed significant difference between theVEGF immunoreactivity of the OHSS group and the OHSS

FIGURE 1

Right ovary weights of OHHS rats � meloxicam.Control ¼ saline treated. Mild ¼ treated with 10 IU ofPMSG and 10 IU of hCG 48 hours later. OHSS ¼treated with 10 IU of PMSG for 4 consecutive daysand 30 IU hCG. OHSS þM¼ treated similarly as theOHSS group plus 2 mL (15 mg/mL) of meloxicamfor 5 consecutive days. Data are presented as themean � SD (n ¼ 10 rats). Superscript lettersdesignate statistically significant differences(P%.05). See text for abbreviations.

Quintana. COX-2 inhibition in ovarian hyperstimulation. Fertil Steril 2008.

Fertility and Sterility�

þmeloxicam group (Fig. 3E). There was stronger VEGF im-munoreactivity in the endothelium of blood vessels of theovaries of hormone-treated groups, and this immunoreactiv-ity was not diminished with the meloxicam treatment (datanot shown).

The COX-2 expression in the ovaries localized to the inter-stitial cells and the ovarian blood vessels. In addition, smoothCOX-2 immunostaining was observed in follicular thecablood vessels and ovarian stroma (Fig. 4A). There was noCOX-2 staining in GCs. The ovarian COX-2 expressionwas increased in the hormone-treated ovaries, mainly at theGC level (Fig. 4B); however, meloxicam treatment sup-pressed this increase (Fig. 4C). Densitometric analysis ofCOX-2 immunoreactivity (COX-2-ir) confirms a quantitativediminution of COX-2-ir in the ovaries of the meloxicam-treated group as compared to the OHSS group (Fig. 4E).

Hormone Assay

Serum E2 levels in hyperstimulated rats were approximately10 times greater that those of the control group. Neither 2days nor 6 days of meloxicam treatment had any effect onthe hyperstimulation-induced increase in serum E2 levels(data not shown).

DISCUSSION

Our results confirm that VEGF plays a pivotal role in OHSS.In addition, our results suggest that inhibition of COX-2 by

FIGURE 2

Number of luteinized follicles of OHHS rats �meloxicam. Data are presented as the mean � SD(n ¼ 10 rats). Mild ¼ treated with 10 IU of PMSG and10 IU of hCG 48 hours later. OHSS ¼ treated with10 IU of PMSG for 4 consecutive days and30 IU hCG. OHSS þ M ¼ treated similarly as theOHSS group plus 2 mL (15 mg/mL) of meloxicam for 5consecutive days. Superscript letters designatestatistically significant differences (P%.05). See textfor abbreviations.

Quintana. COX-2 inhibition in ovarian hyperstimulation. Fertil Steril 2008.

1513

FIGURE 3

Immunohistochemistry analysis of VEGF immunolocalization in the ovaries of OHHS rats � meloxicam.(A) A representative ovarian section showing the immunolocalization of VEGF in a control rat (�100).(B) A representative ovarian section from a mild-treated rat (�200). (C) A representative ovarian sectionfrom an OHSS rat (�100). (D) A representative ovarian section from an OHSS plus meloxicam-treated rat (�100).(E) Densitometric analyses of VEGF immunoreactivity in the rat ovaries. Superscript letters designatestatistically significant differences (P%.05). See text for abbreviations.

Quintana. COX-2 inhibition in ovarian hyperstimulation. Fertil Steril 2008.

meloxicam may inhibit development of OHSS, as demon-strated by inhibition of ovarian weight increase and COX-2expression.

Research suggests that VEGF is responsible for the in-creased capillary permeability leading to the extravasationof protein-rich fluid and subsequent OHSS progression (3,17). In addition, high levels of VEGF have been detected inboth serum and follicular fluids (FF) of women with severeOHSS (18). We found increased VEGF immunoreactivityin the ovaries of rats with induced OHSS, and found that

1514 Quintana et al. COX-2 inhibition in ovarian hypersti

co-treating rats with meloxicam during OHSS induction re-duced this intense VEGF immunoreactivity. Our findingsagree with other reports showing that suppressing VEGF ex-pression by COX-2 inhibition causes regression of endome-triosis in an animal model (19).

The effects of meloxicam are likely mediated specificallyby COX-2. Meloxicam has a greater inhibitory effect onCOX-2 than COX-1 (20). In addition, COX-2 is involved inovulatory processes (21). Our results also coincide with pre-vious reports about COX-2 and VEGF interplay in the ovary,

mulation Vol. 90, Suppl 2, October 2008

FIGURE 4

Immunohistochemical detection of COX-2 in ovaries of OHHS rats � meloxicam. (A) A representativeovarian section showing the immunolocalization of COX-2 in a control rat (�100). (B) A representative ovariansection from an OHSS rat (�100). (C) A representative ovarian section from an OHSS plus meloxicam-treated rat(�250). (D) A representative ovarian section from a control rat where the anti-COX-2 antisera was omitted (�100).(E) Densitometric analyses of COX-2 immunoreactivity in the rat ovaries. Superscript letters designatestatistically significant differences (P%.05). See text for abbreviations.

Quintana. COX-2 inhibition in ovarian hyperstimulation. Fertil Steril 2008.

mainly in ovarian tumors. Significant correlation betweenCOX-2 and VEGF expression in ovarian carcinoma hasbeen reported recently (22).

We used immunohistochemical methods to detect the ex-pression of COX-2 and VEGF. This technique preserves spa-tial information, and the protein expression can be studied inits appropriate morphologic context. We found similar spatialdistributions of COX-2 and VEGF in the ovary, suggestinginterrelated activity. Ovarian COX-2 localized in the GCsof growing follicles as well as in the ovarian vessels. Stav-reus-Evers et al. (23) reported similar COX-2 localization

Fertility and Sterility�

in the human ovary of women in luteal and pregnancy cyclephases. In addition they showed an intense COX-2 immuno-reactivity in the outer ovarian surface. We did not detectCOX-2 expression on the ovarian surface, perhaps due to in-terspecies variation. The location of COX-2 in the folliclemay be related to its function in the normal ovarian cycleas COX-2 plays an important role in ovulation. Meloxicamis a dose-dependent ovulation inhibitor in rabbits (24), andpharmacological doses of meloxicam can delay ovulationin healthy women (21). In light of the multiple reproductiveeffects of meloxicam (25), the oocyte viability and the repro-ductive health of meloxicam-treated rats may be in question.

1515

Further research to investigate oocyte viability after meloxi-cam treatment is warranted.

The COX-2 is the major cyclooxygenase implied in ovula-tory processes; however, COX-1 may have a minor role inthis process (22). There is controversy regarding the specific-ity of meloxicam for COX-2. Some studies report preferentialactivity of meloxicam for COX-2 (26), but Flower (27) con-siders meloxicam a nonselective inhibitor of both COX iso-enzymes. More pharmacological studies are necessary. Aknown selective COX-2 inhibitor, such as rofecoxib, shouldbe assayed to identify more precisely the contribution ofeach isoenzyme to OHSS pathology.

In conclusion, our results demonstrate that increasedVEGF and COX-2 immunoreactivity is related to hormone-induced OHSS, and that meloxicam treatment partially sup-presses OHSS, possibly by specific inhibition of COX-2.

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