http://informahealthcare.com/gye ISSN: 0951-3590 (print), 1473-0766 (electronic) Gynecol Endocrinol, Early Online: 1–5 ! 2015 Informa UK Ltd. DOI: 10.3109/09513590.2014.1000849
ORIGINAL ARTICLE
Comparison of montelukast and cabergoline for prevention of ovarian hyperstimulation syndrome: in an experimental rat model Levent Akman1,2, Gulnaz Sahin3, Oytun Erbas4, Huseyin Aktug5, Aysin Akdogan3, Ege Nazan Tavmergen Goker1,3, Dilek Taskiran4, and Erol Tavmergen1,3
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1
Department of Obstetrics and Gynecology, Ege University Medical School, Izmir, Turkey, 2Department of Stem Cell, Institute of Health Sciences, Ege University, Izmir, Turkey, 3Family Planning And Infertility Research And Treatment Center, Ege University, Izmir, Turkey, 4Department of Physiology, and 5Department of Histology and Embryology, Ege University Medical School, Izmir, Turkey
Abstract
Keywords
Ovarian hyperstimulation syndrome (OHSS) is a serious iatrogenic complication that can occur during assisted reproductive techniques. The aim of this study is to investigate the effects of the leukotriene receptor antagonist (montelukast) treatment in prevention of OHSS and compare to cabergoline treatment. Twenty-four immature female Wistar rats were assigned to four groups. Group 1 was the control group. In the remaning three groups, OHSS was induced through ovarian stimulation with gonadotropins. No treatment was given to Group 2. Group 3 was administered a low-dose 100 mg/kg cabergoline treatment and Group 4 was received 20 mg/kg montelukast. Body weight, ovarian weight, vasculary permability (VP), peritoneal fluid vascular endothelial growth factor (VEGF) values and VEGF immune-expression were compared between the groups. Both cabergoline and montelukast prevented progression of OHSS compared to the OHSS group. Body weight, ovarian weight, VP, peritoneal fluid VEGF values and VEGF expression were significantly lower in both cabergoline- and montelukast-treated rats than in those not treated OHSS group. In conclusion, montelukast is an effective option for prevention of OHSS, as well as cabergoline. Montelukast may be a new treatment option to prevent and control the OHSS.
Cabergoline, montelukast, ovarian hyperstimulation syndrome, rat model
Introduction Ovarian hyperstimulation syndrome (OHSS) is a serious iatrogenic complication that can occur during assisted reproductive techniques, and sometimes can lead to mortality. As a result of increased vascular permeability (VP) in OHSS, intravascular fluid shifts into the extravascular space. Consequently, ascites, hydrothorax, hemoconcentration, hepatic and renal failure due to reduced organ perfusion may occur. This potentially lifethreatening situation can be seen in 0.3–5% of all stimulated ovarian cycles [1]. Human chorionic gonadotropin (hCG) does not possess any direct vasoactive effects. In the development of OHSS, some vasoactive substances are released from the ovaries, such as cytokines and growth factors (IL-2, IL-6, IL-8, IL-10, IL-18, vascular endothelial growth factor [VEGF]), histamine, prolactin, prostaglandins and renin-angiotensin, which have been investigated by Alvarez et al. [2]. As a result of previous studies, VEGF, which is a powerful vasoactive mediator, has been recognized to play the most important role, such that OHSS development may decrease via inhibition of VEGF [3]. Among experimental rat models, the development of an OHSS model by gonadotropin stimulation is well-established. Ovarian
History Received 30 July 2014 Revised 10 December 2014 Accepted 17 December 2014 Published online 20 January 2015
VEGF mRNA and vascular permeability increases before hCG administration [4]. However, VEGF mRNA, VEGF receptor-2 mRNA and VP increase, particularly following hCG administration and reach a peak after 48 h [3–5]. Similar to rats, VEGF expression is increased following hCG administration in humans, which is associated with the development of OHSS [6–9]. There is no strategy that will totally prevent the development of OHSS following HCG administration. Recently, cabergoline, which is a dopamine agonist, has been widely used to prevent the development of OHSS and reduce its severity [10]. Cabergoline inhibits VEGFR-2 phosphorylation and low-dose treatment of cabergoline affects OHSS without interfering with angiogenesis [11]. Montelukast, which is a cysteinyl leukotriene receptor antagonist (CysLTRs), has been used in the treatment of asthma. In experimental models, CysLT receptor antagonists have been shown to modulate VP by reducing the expression of VEGF receptor antagonists [12]. In the clinical practice today, there is no choice other than cabergoline, to prevent occurrence and reduce severity of OHSS. The aim of this study was to investigate whether montelukast can truly be used as a new treatment choice in OHSS and to make a comparison between the use of montelukast or cabergoline.
Materials and methods Address for correspondence: Dr Levent Akman, MD, Department of Obstetrics and Gynecology, Ege University Medical School, 35100, Bornova, Izmir, Turkey. Tel: +90 232 3901700. E-mail: [email protected]
Animals In this study, 28 immature female Wistar rats 22-day old, weighing 45–50 g, were used. Animals were fed ad libitum and
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housed in pairs in steel cages having a temperature-controlled environment (22 ± 2 C) with 12-h light/dark cycles. The experimental procedures were approved by the Committee for Animal Research of Ege University. All animal studies are strictly conformed to the animal experiment guidelines of the Committee for Human Care.
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Experimental protocol For the OHSS model, we used a previously published protocol [13]. A total of 24 rats completed the study. Twenty-two-day-old female rats were assigned to four groups (one control and three OHSS groups): Group 1, control group (n ¼ 6) received 0.1 mL of intraperitoneal (i.p.) saline from days 22–26; Group 2, OHSS group (n ¼ 6) was subcutaneously given 10 IU of pregnant mare serum gonadotropin (PMSG-FolligonÕ -Intervet; Schering-Plough Animal Health, Pune, India) for 4 consecutive days and 30 IU _ Schering-Plough Animal Health, hCG (ChorulonÕ -Intervet; Boxmeer, The Netherlands) on the fifth day to induce OHSS (3); Group 3 (OHSS and cabergoline; n ¼ 6) was treated similarly as the OHSS group, with the addition of cabergoline by oral gavage (DostinexÕ -Pharmacia SpA, Ascoli Piceno, Italy) (100 mg/ kg) before the hCG injection on the fifth day. The selected dose and route for cabergoline for prevention of OHSS in the study were determined according to Saylan et al. [14]. Group 4 (OHSS and montelukast; n ¼ 6) was similarly treated as the OHSS group, with an addition of montelukast by oral gavage (SingulairÕ 5 mg, Merck, Haarlem, Holland) (20 mg/kg) 2 h before the PMSG injection for 4 consecutive days, and 2 h before the hCG injection on the fifth day. Permability assay All rats were weighed again and rats were anesthetized by the mixture of ketamine hydrochloride (80 mg/kg, AlfamineÕ , Ege Vet, Alfasan International B.V., Woerdam, Holland) and xylazine hydrochloric (4 mg/kg, AlfazyneÕ , Ege Vet, Alfasan International B.V., Woerdam, Holland), i.p. 48 h after hCG administration, in order to evaluate vascular permeability. To measure VP, a previously described method was employed [5]. Then, 5 mM Evans Blue (EB) dye was diluted in distilled water and a predetermined amount (0.2 mL) was intravenously (i.v.) introduced by means of an insulin syringe through the femoral vein. After a 30-min waiting period, the peritoneal cavity was filled with 5 mL 0.9% NaCl solution (21 C) and was shaken for 30 s. Then, the peritoneal washing liquid was carefully removed by means of a vascular catheter without causing damage to the tissues or vessels. Peritoneal liquid divided into two tubes. For VP measurement, peritoneal liquid was put into tubes containing 0.05 mL 0.1 N NaOH. After centrifugation at 900 g for 12 min, EB concentration was measured at 600 nm on a spectrophotometer (Multiskan GO Microplate Spectrophotometer, Thermo Fisher Scientific Inc., Waltham, MA, USA), the level of extravaseted dye present in the recovered fluid was expressed as micrograms per 100 g body weight. Afterwards, intracardiac blood was drawn from the animals before performing euthanasia. Finally, bilateral ovaries of all the sacrificed animals were removed and weighed. Histopathological examination was then performed. Measurement of peritoneal fluids VEGF values For VEGF measurements, peritoneal fluids were centrifuged at 3000 rpm for 10 min at room temperature and stored at 20 C until assay. Cytokine levels were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits for VEGF (Invitrogen, Camarillo, CA, USA). Samples from each animal were determined in duplicate according to manufacturer’s guide.
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Histopathological evaluation Each ovary was placed in a separate container containing formaldehyde and stained immunohistochemically in order to evaluate VEGF expression. Tissues were fixed in 10% buffered formalin and, after a routine tissue follow-up procedure. Sections of 4 mm were taken from the paraffin blocks using a microtome, and then deparaffinized. They were stained with hematoxylineosin for evaluation. VEGF immunoexpression For immunohistochemistry, sections were incubated with H2O2 (10%) for 30 min to eliminate endogenous peroxidase activity and blocked with 10% normal goat serum (Invitrogen, Camarillo, CA, USA) for 1 h at room temperature. Subsequently, sections were incubated with primary antibodies (VEGF, Bioss Inc.; dilution 1/ 100) for 24 h at 4 C. Antibody detection was performed with the Histostain-Plus Bulk kit (Bioss Inc., Woburn, MA, USA) against rabbit IgG, and 3,30 -diaminobenzidine (DAB) was used to visualize the final product. All sections were washed in PBS and photographed with an Olympus C-5050 digital camera attached to Olympus BX51 microscope, Hamburg, Germany. Brown cytoplasmic staining was scored positive for VEGF. The number of VEGF positive cells was assessed systematically by scoring at least 100 ovarian cells per 10 fields of tissue sections at 100 magnification by two histologists. Statistical analysis Statistical analysis of the data was performed with the software package SPSS for windows 15.0 (Statistical Package for Social Sciences; SPSS Inc., Chicago, IL). Data were expressed as a means ± standard deviation. The Kolmogorov-Smirnov test was used for checking the normal distributions. ANOVA was used for the analyses of the groups of normally distributed continous variables. Bonferroni’s corrections for multiple comparisons were performed. Alfa ¼ 0.05 was shown as a significance level.
Results Measurement of ovarian weight, body weight and vascular permability The effects of hyperstimulation, cabergoline and montelukast on the ovarian weight, body weight and vascular permability in OHSS are summarized in Table 1. The ovarian hyperstimulation caused a significant increase in body weight, ovarian weight and VP compared to the control group (p50.005). Both cabergoline and montelukast prevented progression of OHSS compared to the OHSS group; both also significantly reduced body weight, ovarian weight and VP (p50.05). However, these differences between the cabergoline and montelukast treatment group are not statistically significant. Ovarian VEGF expression and peritoneal fluid VEGF values The effects of hyperstimulation, cabergoline and montelukast on the ovarian VEGF expression and peritoneal VEGF values are shown in Figure 1. The control group showed no staining in either the granulosa cell or theca cells. The OHSS group showed strong staining in the granulosa cells. Both cabergoline and montelukast treatment groups showed slight staining in the granulosa cells. However, a statistically significant decrease in staining was detected in comparison to the OHSS group (Figure 2). VEGF expression percent and peritoneal VEGF levels were significantly lower in both cabergoline- and montelukasttreated rats than in those not treated (p50.05).
Montelukast and cabergoline for prevention of OHSS
DOI: 10.3109/09513590.2014.1000849
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Table 1. The comparision of the ovarian weight, body weight and vascular permability among the groups.
Ovary weight (mg) Body weight (g) Vascular permeability (Evans Blue mM/100 g)
Control
OHSS
OHSS + Cabergoline
OHSS + Montelukast
25.65 ± 2.78 48.16 ± 3.12 12.15 ± 0.36
45.64 ± 5.72* 73.27 ± 5.54y 29.8 ± 2.23*
26.46 ± 2.12y 58.23 ± 6.45y 19.5 ± 0.18y
28.84 ± 3.45y 61.34 ± 4.86y 23.75 ± 0.63y
*p50.005, control group compared OHSS group. yp50.05, OHSS group compared OHSS + cabergoline or OHSS + montelukast group.
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Figure 1. The comparison of the ovarian VEGF expression and the peritoneal VEGF values among the groups. *p50.0001, control group compared OHSS group; #p50.05, OHSS group compared with OHSS + cabergoline or OHSS + montelukast group.
Figure 2. Immunohistochemistry staining of ovarian VEGF. (A) Control group showed no staining either granulosa cell or theca cells; (B) OHSS group showed strongly staining in the granulosa cells; (C) Both cabergoline and montelukast; (D) groups showed slight staining in granulosa cells. However, there was statistical significantly decreased staining compared to the OHSS group.
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Discussion In this study, body weight, ovarian weight, VP, peritoneal fluid VEGF levels and expressions of VEGF were found to be significantly increased in the untreated OHSS group compared to the control group. We detected a significant decrease in body weight, ovarian weight, VP, peritoneal fluid VEGF levels and expressions of VEGF both with cabergoline and montelukast treatment in the OHSS developed groups compared to the untreated group. Both treatments achieved similar efficacy. Many different strategies have been tested for the prevention of OHSS in patients undergoing in vitro fertilization (IVF). Some of these are cycle cancellation, coasting, intravenous albumin administration at the time of ovum pick-up, GnRH agonist for triggering final oocyte maturation, natural-cycle IVF and in vitro oocyte maturation [15–17]. However, today there is no strategy that completely prevents the occurrence of OHSS after hCG administration. The OHSS model has been well-established in animal models. Using immature rats in the OHSS models provide a simplified ovarian physiology [13]. VEGF is thought to be the major factor in the progression of OHSS [18]. Recent experimental rat studies clearly demonstrated the cause and effect relationship between VEGF expression and increased VP [5,19]. VEGF is expressed, produced and released from the ovaries [5]. Also, observational studies in humans clearly indicate that the target and the source of VEGF are the ovaries [5,20,21]. VEGF has two receptors, VEGFR-1 and VEGFR-2. VEGFR-2 mRNA expression has been defined in human granulosa cells, and it has been associated primarily with VP regulation of angiogenesis and vasculogenesis [9,22]. The research by Gomez et al. [4] has established an animal model for OHSS and determined that the ovary is the main source of VEGF; this group has pointed out the importance of the relationship between the mediation of the VEGFR2 pathway and the increase in VP [4]. They have also indicated that specific VEGF receptor-2 inhibitor (SU5416) administration may cause a decline in the increased VP. In another study with a similar OHSS model, Gomez et al. [5] have revealed that administering both high and moderate doses of gonadotropin to hyper-stimulated animals caused excessive expressions of VEGF and VEGFR-2 mRNA, and also showed a correlation with increased VP [5]. In the light of this information, VEGF expression begins before hCG administration and is present in follicles before ovulation [23]. Then, the granulosa-luteinized cells express VEGF mRNA during ovum collection in IVF patients [6,7]. At the same time, granulosa-luteinized cells express VEGFR-2 receptors [9]. Besides the studies mentioned above, Gomez et al. [11] have also reported the results of the effects of the low doses of cabergoline, the dopamine receptor 2 agonist (Dp-r2), in a rat model of OHSS. It was determined that 100 mg/kg Dp-r2 administrations can reverse VEGFR-2-mediated VP by partially inhibiting ovarian VEGFR-2 phosphorylation, but luteal angiogenesis can remain unaffected. Furthermore, cabergoline administration did not affect VEGF/VEGFR-2 ovarian mRNA levels. Based on these results, which demonstrated the safe clinical profile of Dp-r2, administering cabergoline to oocyte donors having a high risk of developing OHSS was encouraged. Dopamine agonists have been widely used in preventing and reducing the severity of OHSS. Moreover, Youssef et al. [24] have evaluated the results of using dopamine agonists in the treatment of IVF in a meta-analysis. In different randomized-controlled trials, prophylactic cabergoline treatment has been tested in different doses (0.25 mg/day and 0.5 mg/day) and at different initiation days in IVF protocols for its efficacy in the prevention of the development of OHSS and various comparisons with
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untreated patients have been performed. As a result of the metaanalysis, prophylactic treatment with cabergoline was indicated to reduce the incidence of OHSS but not OHSS severity, without compromising pregnancy outcomes. In a different randomizedcontrolled study, cabergoline has been compared with intravenous albumin in the prevention of OHSS [25]. Prophylactic oral lowdoses of cabergoline was found more effective and less costly compared to intravenous human albumin in the prevention of OHSS in high-risk patients. However, it was noted that welldesigned, further studies are required to determine the best time and optimum dose for drug administration. Recently, in OHSS rat models, cabergoline has been compared with different treatment options. First, Saylan et al. [14] have compared cabergoline with cyclooxygenase-2 inhibitor, meloxicam. They reported that, both low dose cabergoline and meloxicam were ineffective in decreasing VEGF expression, VP, ovary weight and ovarian diameter in serious OHSS cases. However, Quintana et al. [26] have demonstrated the beneficial effects of meloxicam in decreasing OHSS-related VEGF expression. In another study, Engin-Ustun et al. [27] have compared cabergoline with bevacizumab (recombinant humanized monoclonal immunoglobulin antibody) for treatment of OHSS. Peritoneal VEGF level was measured by the ELISA method. Compared to the untreated group, bevacizumab, like cabergoline, has resulted in less production of VEGF and less ovarian weight. Also, Elia et al. [28] reported that metformin has a beneficial effect in preventing OHSS by reducing the increase in body weight, circulating progesterone and estradiol and vascular permeability. In the prevention of OHSS and decreasing its severity, the most important point for a prophylactic treatment, is the side-effect profile. Meloxicam produces dose-dependent inhibition of ovulation and may cause a delay in ovulation in healthy women [29,30]. For this reason, the impact of meloxicam on oocyte viability and reproductive health is not clear [26]. Systemic use of bevacuzimab may have serious side effects, such as hypertension, proteinuria, neutropenia, thrombosis and stroke [31]. Leukotrienes (LTs) are synthesized by 5-lipoxygenase enzyme, which catalyzes the conversion of arachidonic acid to LTA4. Afterwards, LTA4 hydrolyzes to form LTB4 or conjugates with glutathione into LTC4 and its metabolites, LTD4 and LTE4 [32]. LTC4, LTD4 and LTE4 are known as cysteinyl leukotrienes (cysLTs). LTs receptors are divided into two groups: BLT for LTB4 and cysLT for cysLTs. Both in vivo and in vitro studies have demonstrated that the cysLTs may cause plasma extravasation [33,34]. The cysLTs receptor antagonist montelukast is used for the treatment of asthma. In the mouse asthma model established by Lee et al. [35], using cysLT receptor antagonist (montelukast or pranlukast) has resulted in a decrease in the expression of VEGF. These data show that cysLT receptor signaling may be correlated with the regulation of VEGF expression, and treatment with cysLTs receptor antagonists may decrease the vascular permeability by inhibition of upregulation VEGF expression. LT metabolites are considered to play a major role in the regulation of ovarian functions [36]. In the experimental study by Korzekwa et al. [36], LTB4-induced progesterone and prostaglandin E2 secretion has a luteotropic effect on the corpus luteum, while LTC4-induced luteolitic cascade with prostaglandin F2a secretion in the bovine corpus luteum. Gaytan et al. [19] have investigated the effects of selective inhibition of cyclooxygenase and lipooxygenase pathways in follicle rupture and ovulation in a rat model and showed that rats treated with lipooxygenase inhibitor displayed no change in ovulation. We did not investigate circulating serum estrodiol and progesterone levels and histological examination of the corpus lutum for functional apoptosis in this present study. Further
Montelukast and cabergoline for prevention of OHSS
DOI: 10.3109/09513590.2014.1000849
studies are being designed to investigate the effects of montelukast on luteal function. To our knowledge, there is no study in the literature investigating the effects of montelukast treatment in a rat OHSS model. In this study, we observed in a rat model of OHSS that montelukast treatment, similar to cabergoline treatment, can significantly decrease expression of VEGF, body weight, ovary weight and VP. As a result, montelukast appears to be a new treatment option to prevent OHSS and control the severity of the disease, and appears to work as well like cabergoline. Due to the regulatory effect of LTS on ovarian functions, further studies are needed to evaluate its effects on oocyte viability and reproductive health.
Declaration of interest
15. 16. 17. 18. 19. 20.
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The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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ORIGINAL ARTICLE
Comparison of montelukast and cabergoline for prevention of ovarian hyperstimulation syndrome: in an experimental rat model Levent Akman1,2, Gulnaz Sahin3, Oytun Erbas4, Huseyin Aktug5, Aysin Akdogan3, Ege Nazan Tavmergen Goker1,3, Dilek Taskiran4, and Erol Tavmergen1,3
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1
Department of Obstetrics and Gynecology, Ege University Medical School, Izmir, Turkey, 2Department of Stem Cell, Institute of Health Sciences, Ege University, Izmir, Turkey, 3Family Planning And Infertility Research And Treatment Center, Ege University, Izmir, Turkey, 4Department of Physiology, and 5Department of Histology and Embryology, Ege University Medical School, Izmir, Turkey
Abstract
Keywords
Ovarian hyperstimulation syndrome (OHSS) is a serious iatrogenic complication that can occur during assisted reproductive techniques. The aim of this study is to investigate the effects of the leukotriene receptor antagonist (montelukast) treatment in prevention of OHSS and compare to cabergoline treatment. Twenty-four immature female Wistar rats were assigned to four groups. Group 1 was the control group. In the remaning three groups, OHSS was induced through ovarian stimulation with gonadotropins. No treatment was given to Group 2. Group 3 was administered a low-dose 100 mg/kg cabergoline treatment and Group 4 was received 20 mg/kg montelukast. Body weight, ovarian weight, vasculary permability (VP), peritoneal fluid vascular endothelial growth factor (VEGF) values and VEGF immune-expression were compared between the groups. Both cabergoline and montelukast prevented progression of OHSS compared to the OHSS group. Body weight, ovarian weight, VP, peritoneal fluid VEGF values and VEGF expression were significantly lower in both cabergoline- and montelukast-treated rats than in those not treated OHSS group. In conclusion, montelukast is an effective option for prevention of OHSS, as well as cabergoline. Montelukast may be a new treatment option to prevent and control the OHSS.
Cabergoline, montelukast, ovarian hyperstimulation syndrome, rat model
Introduction Ovarian hyperstimulation syndrome (OHSS) is a serious iatrogenic complication that can occur during assisted reproductive techniques, and sometimes can lead to mortality. As a result of increased vascular permeability (VP) in OHSS, intravascular fluid shifts into the extravascular space. Consequently, ascites, hydrothorax, hemoconcentration, hepatic and renal failure due to reduced organ perfusion may occur. This potentially lifethreatening situation can be seen in 0.3–5% of all stimulated ovarian cycles [1]. Human chorionic gonadotropin (hCG) does not possess any direct vasoactive effects. In the development of OHSS, some vasoactive substances are released from the ovaries, such as cytokines and growth factors (IL-2, IL-6, IL-8, IL-10, IL-18, vascular endothelial growth factor [VEGF]), histamine, prolactin, prostaglandins and renin-angiotensin, which have been investigated by Alvarez et al. [2]. As a result of previous studies, VEGF, which is a powerful vasoactive mediator, has been recognized to play the most important role, such that OHSS development may decrease via inhibition of VEGF [3]. Among experimental rat models, the development of an OHSS model by gonadotropin stimulation is well-established. Ovarian
History Received 30 July 2014 Revised 10 December 2014 Accepted 17 December 2014 Published online 20 January 2015
VEGF mRNA and vascular permeability increases before hCG administration [4]. However, VEGF mRNA, VEGF receptor-2 mRNA and VP increase, particularly following hCG administration and reach a peak after 48 h [3–5]. Similar to rats, VEGF expression is increased following hCG administration in humans, which is associated with the development of OHSS [6–9]. There is no strategy that will totally prevent the development of OHSS following HCG administration. Recently, cabergoline, which is a dopamine agonist, has been widely used to prevent the development of OHSS and reduce its severity [10]. Cabergoline inhibits VEGFR-2 phosphorylation and low-dose treatment of cabergoline affects OHSS without interfering with angiogenesis [11]. Montelukast, which is a cysteinyl leukotriene receptor antagonist (CysLTRs), has been used in the treatment of asthma. In experimental models, CysLT receptor antagonists have been shown to modulate VP by reducing the expression of VEGF receptor antagonists [12]. In the clinical practice today, there is no choice other than cabergoline, to prevent occurrence and reduce severity of OHSS. The aim of this study was to investigate whether montelukast can truly be used as a new treatment choice in OHSS and to make a comparison between the use of montelukast or cabergoline.
Materials and methods Address for correspondence: Dr Levent Akman, MD, Department of Obstetrics and Gynecology, Ege University Medical School, 35100, Bornova, Izmir, Turkey. Tel: +90 232 3901700. E-mail: [email protected]
Animals In this study, 28 immature female Wistar rats 22-day old, weighing 45–50 g, were used. Animals were fed ad libitum and
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housed in pairs in steel cages having a temperature-controlled environment (22 ± 2 C) with 12-h light/dark cycles. The experimental procedures were approved by the Committee for Animal Research of Ege University. All animal studies are strictly conformed to the animal experiment guidelines of the Committee for Human Care.
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Experimental protocol For the OHSS model, we used a previously published protocol [13]. A total of 24 rats completed the study. Twenty-two-day-old female rats were assigned to four groups (one control and three OHSS groups): Group 1, control group (n ¼ 6) received 0.1 mL of intraperitoneal (i.p.) saline from days 22–26; Group 2, OHSS group (n ¼ 6) was subcutaneously given 10 IU of pregnant mare serum gonadotropin (PMSG-FolligonÕ -Intervet; Schering-Plough Animal Health, Pune, India) for 4 consecutive days and 30 IU _ Schering-Plough Animal Health, hCG (ChorulonÕ -Intervet; Boxmeer, The Netherlands) on the fifth day to induce OHSS (3); Group 3 (OHSS and cabergoline; n ¼ 6) was treated similarly as the OHSS group, with the addition of cabergoline by oral gavage (DostinexÕ -Pharmacia SpA, Ascoli Piceno, Italy) (100 mg/ kg) before the hCG injection on the fifth day. The selected dose and route for cabergoline for prevention of OHSS in the study were determined according to Saylan et al. [14]. Group 4 (OHSS and montelukast; n ¼ 6) was similarly treated as the OHSS group, with an addition of montelukast by oral gavage (SingulairÕ 5 mg, Merck, Haarlem, Holland) (20 mg/kg) 2 h before the PMSG injection for 4 consecutive days, and 2 h before the hCG injection on the fifth day. Permability assay All rats were weighed again and rats were anesthetized by the mixture of ketamine hydrochloride (80 mg/kg, AlfamineÕ , Ege Vet, Alfasan International B.V., Woerdam, Holland) and xylazine hydrochloric (4 mg/kg, AlfazyneÕ , Ege Vet, Alfasan International B.V., Woerdam, Holland), i.p. 48 h after hCG administration, in order to evaluate vascular permeability. To measure VP, a previously described method was employed [5]. Then, 5 mM Evans Blue (EB) dye was diluted in distilled water and a predetermined amount (0.2 mL) was intravenously (i.v.) introduced by means of an insulin syringe through the femoral vein. After a 30-min waiting period, the peritoneal cavity was filled with 5 mL 0.9% NaCl solution (21 C) and was shaken for 30 s. Then, the peritoneal washing liquid was carefully removed by means of a vascular catheter without causing damage to the tissues or vessels. Peritoneal liquid divided into two tubes. For VP measurement, peritoneal liquid was put into tubes containing 0.05 mL 0.1 N NaOH. After centrifugation at 900 g for 12 min, EB concentration was measured at 600 nm on a spectrophotometer (Multiskan GO Microplate Spectrophotometer, Thermo Fisher Scientific Inc., Waltham, MA, USA), the level of extravaseted dye present in the recovered fluid was expressed as micrograms per 100 g body weight. Afterwards, intracardiac blood was drawn from the animals before performing euthanasia. Finally, bilateral ovaries of all the sacrificed animals were removed and weighed. Histopathological examination was then performed. Measurement of peritoneal fluids VEGF values For VEGF measurements, peritoneal fluids were centrifuged at 3000 rpm for 10 min at room temperature and stored at 20 C until assay. Cytokine levels were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits for VEGF (Invitrogen, Camarillo, CA, USA). Samples from each animal were determined in duplicate according to manufacturer’s guide.
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Histopathological evaluation Each ovary was placed in a separate container containing formaldehyde and stained immunohistochemically in order to evaluate VEGF expression. Tissues were fixed in 10% buffered formalin and, after a routine tissue follow-up procedure. Sections of 4 mm were taken from the paraffin blocks using a microtome, and then deparaffinized. They were stained with hematoxylineosin for evaluation. VEGF immunoexpression For immunohistochemistry, sections were incubated with H2O2 (10%) for 30 min to eliminate endogenous peroxidase activity and blocked with 10% normal goat serum (Invitrogen, Camarillo, CA, USA) for 1 h at room temperature. Subsequently, sections were incubated with primary antibodies (VEGF, Bioss Inc.; dilution 1/ 100) for 24 h at 4 C. Antibody detection was performed with the Histostain-Plus Bulk kit (Bioss Inc., Woburn, MA, USA) against rabbit IgG, and 3,30 -diaminobenzidine (DAB) was used to visualize the final product. All sections were washed in PBS and photographed with an Olympus C-5050 digital camera attached to Olympus BX51 microscope, Hamburg, Germany. Brown cytoplasmic staining was scored positive for VEGF. The number of VEGF positive cells was assessed systematically by scoring at least 100 ovarian cells per 10 fields of tissue sections at 100 magnification by two histologists. Statistical analysis Statistical analysis of the data was performed with the software package SPSS for windows 15.0 (Statistical Package for Social Sciences; SPSS Inc., Chicago, IL). Data were expressed as a means ± standard deviation. The Kolmogorov-Smirnov test was used for checking the normal distributions. ANOVA was used for the analyses of the groups of normally distributed continous variables. Bonferroni’s corrections for multiple comparisons were performed. Alfa ¼ 0.05 was shown as a significance level.
Results Measurement of ovarian weight, body weight and vascular permability The effects of hyperstimulation, cabergoline and montelukast on the ovarian weight, body weight and vascular permability in OHSS are summarized in Table 1. The ovarian hyperstimulation caused a significant increase in body weight, ovarian weight and VP compared to the control group (p50.005). Both cabergoline and montelukast prevented progression of OHSS compared to the OHSS group; both also significantly reduced body weight, ovarian weight and VP (p50.05). However, these differences between the cabergoline and montelukast treatment group are not statistically significant. Ovarian VEGF expression and peritoneal fluid VEGF values The effects of hyperstimulation, cabergoline and montelukast on the ovarian VEGF expression and peritoneal VEGF values are shown in Figure 1. The control group showed no staining in either the granulosa cell or theca cells. The OHSS group showed strong staining in the granulosa cells. Both cabergoline and montelukast treatment groups showed slight staining in the granulosa cells. However, a statistically significant decrease in staining was detected in comparison to the OHSS group (Figure 2). VEGF expression percent and peritoneal VEGF levels were significantly lower in both cabergoline- and montelukasttreated rats than in those not treated (p50.05).
Montelukast and cabergoline for prevention of OHSS
DOI: 10.3109/09513590.2014.1000849
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Table 1. The comparision of the ovarian weight, body weight and vascular permability among the groups.
Ovary weight (mg) Body weight (g) Vascular permeability (Evans Blue mM/100 g)
Control
OHSS
OHSS + Cabergoline
OHSS + Montelukast
25.65 ± 2.78 48.16 ± 3.12 12.15 ± 0.36
45.64 ± 5.72* 73.27 ± 5.54y 29.8 ± 2.23*
26.46 ± 2.12y 58.23 ± 6.45y 19.5 ± 0.18y
28.84 ± 3.45y 61.34 ± 4.86y 23.75 ± 0.63y
*p50.005, control group compared OHSS group. yp50.05, OHSS group compared OHSS + cabergoline or OHSS + montelukast group.
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Figure 1. The comparison of the ovarian VEGF expression and the peritoneal VEGF values among the groups. *p50.0001, control group compared OHSS group; #p50.05, OHSS group compared with OHSS + cabergoline or OHSS + montelukast group.
Figure 2. Immunohistochemistry staining of ovarian VEGF. (A) Control group showed no staining either granulosa cell or theca cells; (B) OHSS group showed strongly staining in the granulosa cells; (C) Both cabergoline and montelukast; (D) groups showed slight staining in granulosa cells. However, there was statistical significantly decreased staining compared to the OHSS group.
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Discussion In this study, body weight, ovarian weight, VP, peritoneal fluid VEGF levels and expressions of VEGF were found to be significantly increased in the untreated OHSS group compared to the control group. We detected a significant decrease in body weight, ovarian weight, VP, peritoneal fluid VEGF levels and expressions of VEGF both with cabergoline and montelukast treatment in the OHSS developed groups compared to the untreated group. Both treatments achieved similar efficacy. Many different strategies have been tested for the prevention of OHSS in patients undergoing in vitro fertilization (IVF). Some of these are cycle cancellation, coasting, intravenous albumin administration at the time of ovum pick-up, GnRH agonist for triggering final oocyte maturation, natural-cycle IVF and in vitro oocyte maturation [15–17]. However, today there is no strategy that completely prevents the occurrence of OHSS after hCG administration. The OHSS model has been well-established in animal models. Using immature rats in the OHSS models provide a simplified ovarian physiology [13]. VEGF is thought to be the major factor in the progression of OHSS [18]. Recent experimental rat studies clearly demonstrated the cause and effect relationship between VEGF expression and increased VP [5,19]. VEGF is expressed, produced and released from the ovaries [5]. Also, observational studies in humans clearly indicate that the target and the source of VEGF are the ovaries [5,20,21]. VEGF has two receptors, VEGFR-1 and VEGFR-2. VEGFR-2 mRNA expression has been defined in human granulosa cells, and it has been associated primarily with VP regulation of angiogenesis and vasculogenesis [9,22]. The research by Gomez et al. [4] has established an animal model for OHSS and determined that the ovary is the main source of VEGF; this group has pointed out the importance of the relationship between the mediation of the VEGFR2 pathway and the increase in VP [4]. They have also indicated that specific VEGF receptor-2 inhibitor (SU5416) administration may cause a decline in the increased VP. In another study with a similar OHSS model, Gomez et al. [5] have revealed that administering both high and moderate doses of gonadotropin to hyper-stimulated animals caused excessive expressions of VEGF and VEGFR-2 mRNA, and also showed a correlation with increased VP [5]. In the light of this information, VEGF expression begins before hCG administration and is present in follicles before ovulation [23]. Then, the granulosa-luteinized cells express VEGF mRNA during ovum collection in IVF patients [6,7]. At the same time, granulosa-luteinized cells express VEGFR-2 receptors [9]. Besides the studies mentioned above, Gomez et al. [11] have also reported the results of the effects of the low doses of cabergoline, the dopamine receptor 2 agonist (Dp-r2), in a rat model of OHSS. It was determined that 100 mg/kg Dp-r2 administrations can reverse VEGFR-2-mediated VP by partially inhibiting ovarian VEGFR-2 phosphorylation, but luteal angiogenesis can remain unaffected. Furthermore, cabergoline administration did not affect VEGF/VEGFR-2 ovarian mRNA levels. Based on these results, which demonstrated the safe clinical profile of Dp-r2, administering cabergoline to oocyte donors having a high risk of developing OHSS was encouraged. Dopamine agonists have been widely used in preventing and reducing the severity of OHSS. Moreover, Youssef et al. [24] have evaluated the results of using dopamine agonists in the treatment of IVF in a meta-analysis. In different randomized-controlled trials, prophylactic cabergoline treatment has been tested in different doses (0.25 mg/day and 0.5 mg/day) and at different initiation days in IVF protocols for its efficacy in the prevention of the development of OHSS and various comparisons with
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untreated patients have been performed. As a result of the metaanalysis, prophylactic treatment with cabergoline was indicated to reduce the incidence of OHSS but not OHSS severity, without compromising pregnancy outcomes. In a different randomizedcontrolled study, cabergoline has been compared with intravenous albumin in the prevention of OHSS [25]. Prophylactic oral lowdoses of cabergoline was found more effective and less costly compared to intravenous human albumin in the prevention of OHSS in high-risk patients. However, it was noted that welldesigned, further studies are required to determine the best time and optimum dose for drug administration. Recently, in OHSS rat models, cabergoline has been compared with different treatment options. First, Saylan et al. [14] have compared cabergoline with cyclooxygenase-2 inhibitor, meloxicam. They reported that, both low dose cabergoline and meloxicam were ineffective in decreasing VEGF expression, VP, ovary weight and ovarian diameter in serious OHSS cases. However, Quintana et al. [26] have demonstrated the beneficial effects of meloxicam in decreasing OHSS-related VEGF expression. In another study, Engin-Ustun et al. [27] have compared cabergoline with bevacizumab (recombinant humanized monoclonal immunoglobulin antibody) for treatment of OHSS. Peritoneal VEGF level was measured by the ELISA method. Compared to the untreated group, bevacizumab, like cabergoline, has resulted in less production of VEGF and less ovarian weight. Also, Elia et al. [28] reported that metformin has a beneficial effect in preventing OHSS by reducing the increase in body weight, circulating progesterone and estradiol and vascular permeability. In the prevention of OHSS and decreasing its severity, the most important point for a prophylactic treatment, is the side-effect profile. Meloxicam produces dose-dependent inhibition of ovulation and may cause a delay in ovulation in healthy women [29,30]. For this reason, the impact of meloxicam on oocyte viability and reproductive health is not clear [26]. Systemic use of bevacuzimab may have serious side effects, such as hypertension, proteinuria, neutropenia, thrombosis and stroke [31]. Leukotrienes (LTs) are synthesized by 5-lipoxygenase enzyme, which catalyzes the conversion of arachidonic acid to LTA4. Afterwards, LTA4 hydrolyzes to form LTB4 or conjugates with glutathione into LTC4 and its metabolites, LTD4 and LTE4 [32]. LTC4, LTD4 and LTE4 are known as cysteinyl leukotrienes (cysLTs). LTs receptors are divided into two groups: BLT for LTB4 and cysLT for cysLTs. Both in vivo and in vitro studies have demonstrated that the cysLTs may cause plasma extravasation [33,34]. The cysLTs receptor antagonist montelukast is used for the treatment of asthma. In the mouse asthma model established by Lee et al. [35], using cysLT receptor antagonist (montelukast or pranlukast) has resulted in a decrease in the expression of VEGF. These data show that cysLT receptor signaling may be correlated with the regulation of VEGF expression, and treatment with cysLTs receptor antagonists may decrease the vascular permeability by inhibition of upregulation VEGF expression. LT metabolites are considered to play a major role in the regulation of ovarian functions [36]. In the experimental study by Korzekwa et al. [36], LTB4-induced progesterone and prostaglandin E2 secretion has a luteotropic effect on the corpus luteum, while LTC4-induced luteolitic cascade with prostaglandin F2a secretion in the bovine corpus luteum. Gaytan et al. [19] have investigated the effects of selective inhibition of cyclooxygenase and lipooxygenase pathways in follicle rupture and ovulation in a rat model and showed that rats treated with lipooxygenase inhibitor displayed no change in ovulation. We did not investigate circulating serum estrodiol and progesterone levels and histological examination of the corpus lutum for functional apoptosis in this present study. Further
Montelukast and cabergoline for prevention of OHSS
DOI: 10.3109/09513590.2014.1000849
studies are being designed to investigate the effects of montelukast on luteal function. To our knowledge, there is no study in the literature investigating the effects of montelukast treatment in a rat OHSS model. In this study, we observed in a rat model of OHSS that montelukast treatment, similar to cabergoline treatment, can significantly decrease expression of VEGF, body weight, ovary weight and VP. As a result, montelukast appears to be a new treatment option to prevent OHSS and control the severity of the disease, and appears to work as well like cabergoline. Due to the regulatory effect of LTS on ovarian functions, further studies are needed to evaluate its effects on oocyte viability and reproductive health.
Declaration of interest
15. 16. 17. 18. 19. 20.
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The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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