Effect of ondansetron for preventing of ovarian hyperstimulation syndrome: in an experimental rat model

Abstract Objective: Ovarian hyperstimulation syndrome is an iatrogenic condition that occurs in the treatment of infertility. There is no specific treatment available for OHSS. Cabergoline is a dopamine receptor 2 agonist and VEGF-VEGF2 receptor antagonist . Recently, cabergoline has been widely used to prevent the development of OHSS and reduce its severity Serotonin is known as a neurotransmitter and thought to have a role in the mechanism of angiogenesis and in signaling in endothelial cells. Serotonin is said to have similar effects to VEGF . Ondansetron is Selective Serotonin (5-HT3) Receptor Antagonist . It works by blocking the action of serotonin, a natural substance that may cause nausea and vomiting. In the clinical practice today, there is no choice other than cabergoline, to prevent occurrence and reduce severity of OHSS, and sometimes its effects are limited. Methods: In our study, we compared the effect of cabergoline and ondansetron. 32 immature rats were used and the OHSS model was created. Parameters such as hematocrit value , ovarian size, the number of follicles in the ovary, endometrial capillary congestion and thickness values were evaluated and compared. Results: As a result, in our study, it was seen that ondansetron was effective on OHSS in many parameters. It is thought to be as effective as cabergoline. When we look at the literature, this is the first study in which ondansetron was evaluated for this purpose. It would be good to show this effect of ondansetron with other studies.


Introduction
About 10-15% of couples in society have infertility problems [1]. Ovulatory disorders are identified in 18 to 25 percent of couples presenting with infertility [2]. The use of gonadotropins for ovulation induction in ovulation dysfunction has increased considerably in the last 30 years. However, ovarian hyperstimulation syndrome (OHSS), which is a serious iatrogenic complication, can develop during the use of these drugs [3]. The clinical manifestations of OHSS are massive extravascular fluid accumulation and hemoconcentration. Renal failure, hypovolemic shock, thromboembolic attacks, and adult respiratory distress syndrome (ARDS) may occur in these patients. In this syndrome, an increase in capillary permeability occurs with the secretion of vasoactive substances by the ovaries under stimulation of human chorionic gonadotropin (hCG). Agents such as hCG, Vascular endothelial growth factor (VEGF), estradiol, progesterone, renin-angiotensin system, kinin-kallikrein system, prostaglandins, cytokines, and nitric oxide have been implicated in the development of OHSS in humans [4]. OHSS occurs with ovarian hypersecretion because of increased vascular permeability (VP) due to VEGF activating vascular endothelial growth factor receptor-2 (VEGFR-2) [5]. There is no specific treatment available for OHSS. Although the pathophysiology is not fully understood, there is an increase in permeability in the peri ovarian vessels [6].
Recently, cabergoline has been widely used to prevent the development of OHSS and reduce its severity [9].
Serotonin is known as a neurotransmitter and thought to have a role in the mechanism of angiogenesis and in signaling in endothelial cells. Serotonin is said to have similar effects to VEGF [10]. Studies showing the relationship between serotonin and OHSS are very limited [11,12].
Ondansetron is Selective Serotonin (5-HT3) Receptor Antagonist [13]. It works by blocking the action of serotonin, a natural substance that may cause nausea and vomiting.
In the clinical practice today, there is no choice other than cabergoline, to prevent occurrence and reduce severity of OHSS, and sometimes its effects are limited. The purpose of this study was to investigate whether ondansetron could truly be used as a new treatment option for the prevention and treatment of OHSS in rat model.

Animals
A total of 32 Female Wistar rats (22 d old, weighing 45-75 g) were obtained from Kırıkkale University and were maintained in laboratory on a 12-h light, 12-h dark regimen (lights on 07:00-19:00 h) with free access to water and a standard diet.
The animals were grouped in standard cages with 3-4 rats per cage and housed under standard laboratory conditions. The Animal Care and Use Committee of the Kırıkkale University School of Medicine approved all procedures in this study, which were in accordance with the standards of the National Institutes of Health Guide for Care and Use of Laboratory Animals.

Study and groups
The study was carried out after the approval of the local ethics committee (Date of 21.10.2020 and 06/35 decision no.) in Kırıkkale University Scientific and Technological Research Laboratories. 'This work was supported by Scientific Research Projects Coordination Unit of Kırıkkale University. Project number 2020/092' .
Rat models of OHSS may be valuable tools for developing intervention strategies based on the observations [14][15][16]. For OHSS model, we used a previously published protocol [17]. Rats in groups 2, 3, and 4 were injected with 10 IU of pregnant mare serum gonadotropin in 0.1 ml 0.9% saline for four consecutive days from the 22nd to the 25th day of life, followed by subcutaneous injection of 30 IU hCG on day 26. The study was performed with 32 rats in four main groups (one non-stimulated control and three OHSS positive groups: ondansetron, cabergoline and placebo). The rats were randomly assigned to four experimental groups (eight rats per group): 1. Control group (n = 8): The rats were injected with the same volume of saline instead of the drug as in the same treatment procedure. As a non-stimulated control, rats were given 0.1 ml 0.9% saline for 5 consecutive days (OHSS negative). The doses of the drugs we used in rats were kept the same as the doses used in previous studies [18,19].
Rats were monitored daily, and no evidence of toxicity was reported based on body weight, food consumption, grooming behavior or activity levels compared with controls.
All animals were sacrificed 48 h after hCG administration Anesthesia was induced by ketamine 80 mg/kg (Ketalar, Eczacıbası, Istanbul, Turkey) and xylazine 10 mg/kg (Rompun Vet, Bayer AB, Istanbul, Turkey) by an intramuscular injection. On the 28th d, rats were killed by dislocation. Blood was collected by cardiac puncture. Rats' abdomens were exposed under anesthesia to see the ovaries and uterus were removed and weighed. Blood from rats was centrifuged on hematocrit sticks.

Histopathological evaluation
The histopathological evaluations were performed by a pathologist who was blinded to the groups. Both the right and left ovaries of the rats were blocked in paraffin. Sections of 4-6 µm were taken from the paraffin blocks using a microtome and then deparaffinized. They were stained with hematoxylin-eosin to examine the number of corpora lutea, primer and pre antral follicle as an indicator of ovarian stimulation under a light microscope. Thereafter, uterus specimens were fixed in 4% buffered formaldehyde and embedded in paraffin before 5 μm histological sections were made, stained with hematoxylin-eosin order to reveal the presence of stroma, glandular epithelium, endometrial thickness, endometrial capillary congestion and micro-vessel in endometrial tissue.

Statistical analysis
The statistical analysis was performed using the Statistical Package for the Social Sciences version 21.0 (SPSS, Chicago, IL, USA). Data with normal distributed is presented as the mean ± the standard-deviation (SD), while the non-normally distributed variables are presented as median with interquartile range. The normalities of the distributions of the continuous variables were assessed with Shapiro-Wilk tests. The normally distributed variables were examined with one-way analyses of variance followed by Tukey's post-hoc tests, and the non-normally distributed variables were analyzed with Kruskal-Wallis tests and Mann-Whitney U tests with post hoc Bonferroni corrections. Statistical significance was considered with p values <0.05.

Results
Our date is presented Table 1. Hematocrit, ovarian volume, utero-ovarian weight, lutea corpora were significantly higher in the OHSS groups than the control group. The values lutea corpora, ovarian volume, endometrial capillary congestion, and endometrial thickness counts were found to be lower in the ondansetron group compared to the other groups. Post-study differences between groups and comparison of treatment efficacy are shown in Table 1.
In the comparison between the control and OHSS + placebo groups, significant differences were observed in all parameters (p < 0.05). This result showed that the OHSS rat experimental model was successful.
When we look at the hematocrit and the ovarian volume values in our study, the 1st group was different from the 2nd and 4th groups (p < 0.05). No difference was observed with the 3rd group.
When we look at the lutea corpora and the utero-ovarian weight values in our study, the first group significant difference was observed in all groups (p < 0.05).
Endometrial thickness was statistically significantly higher in the OHSS group than in the control and ondansetron groups ( Figure 1).
There was no difference in any category between cabergoline, which was shown to be effective on OHSS, and ondansetron, which was used in our study.
Data are presented as mean ± SD if normally distributed, or median (interquartile range) if not normally distributed a indi-

Discussion
Ovarian hyperstimulation syndrome is an iatrogenic condition that occurs in the treatment of infertility. Cabergoline has been widely used to prevent the development of OHSS and reduce its severity. In the clinical practice today, there is no choice other than cabergoline, to prevent occurrence and reduce severity of OHSS, and sometimes its effects are limited. In our study, we compared the effect of cabergoline, whose therapeutic efficacy is known, and ondansetron, which we think has an effect. 32 immature rats were used and the OHSS model was created.
OHSS is an iatrogenic condition caused by VEGF. It is a dimeric glycoprotein involved in the control of angiogenesis and vascular permeability [20]. Cabergoline is dopamine agonist and VEGF-VEGF2 receptor antagonist, has been shown to prevent OHSS and is in clinical use [9]. Serotonin is thought to have VEGF-like effects [10]. Ondansetron is Selective Serotonin (5-HT3) Receptor Antagonist [13]. It works by blocking the action of serotonin, a natural substance that may cause nausea and vomiting.
Atılgan R. et al. mentioned in their study that some inflammatory factors are also effective in the development of OHSS [21]. In yet another study, Motavallian-Naeini A et al. mentioned that ondansetron may have anti-inflammatory effects [22]. The anti-inflammatory effect of ondansetron can be considered in the improvement of OHSS.
Animal studies related to OHSS are available in the literature. Lindenbaum et al. have established an OHSS model in adult rats [23]. Another model has been documented in immature rats [24,25]. We used the immature rat model of OHSS because immature animals are not influenced by corpus luteum of preceding cycles and the ovarian physiology can be simplified. As seen in Table 1, the significant difference between the control group, and the OHSS group shows that we have created a successful experimental rat model.
Hematocrit value and ovarian size are important in the diagnosis and treatment follow-up of OHSS [26]. In our study, no difference was observed in hematocrit and ovarian volume values between the control group and the group treated with ondansetron, but a significant difference was observed between the other OHSS groups. This finding is suggestive that ondansetron is effective on OHSS. No difference was observed between the cabergoline group and the ondansetron group, showing that it was at least as effective as it. Although there was no significant difference in the hematocrit value between the ondansetron treatment group and the OHSS group, the value was very close (p = 0.057).
The number of follicles in the ovary were significantly different between the OHSS group and the ondansetron-treated group. This finding is suggestive that ondansetron is effective on OHSS. Engin-Ustun Y et al. used the number of eggs in an OHSS experimental rat study [18]. Elia EM et al. evaluated primary follicle, primordial follicle, preantral follicle and lutea corpora values for evaluation and comparison purposes in their OHSS rat study [27]. endometrial capillary congestion: The number of congested (dilated and filled with erythrocytes) capillaries in the high magnification field (HMF). The average of 10 HMF areas was taken. in the table, the notation as a, b, c, d, a1, b1, c1, d1 indicates the binary statistics correspondences and the results are written below. As expected, there was a significant difference in the number of lutea corpora in all OHSS groups compared to the control group. Although there was no significant difference in the lutea corpora value between the ondansetron treatment group and the OHSS group, the value was very close (p = 0.099).
Endometrial capillary congestion and thickness values were also found to be significantly different between the OHSS group and the ondansetron group. these parameters are used in OHSS studies as in the studies of Sanlı C et al. [28].
As a result, in our study, it was seen that ondansetron was effective on OHSS in many parameters. It is thought to be as effective as cabergoline. When we look at the literature, this is the first study in which ondansetron was evaluated for this purpose. It would be good to show this effect of ondansetron with other studies.

Disclosure statement
No potential conflict of interest was reported by the authors.