Abstract
Objective
Evaluating the therapeutic effect of detorsion, resveratrol, and nifedipine on ovarian viability assessed by biochemical, histopathological and immunohistochemical parameters and markers of oxidative stress.
Materials and Methods
Twenty-four Sprague-Dawley rats were included in 4 groups, namely: sham operation, ischemia-reperfusion (I/R), I/R+10 mg/kg nifedipine (NIF), I/R+100 mg/kg resveratrol (RSV). In the study groups, bilateral 720º ovarian torsion was performed and continued for 3 hours, followed by detorsion for another 3 hours. Thirty minutes before the detorsion, NIF and RSV groups received respective treatments. Adnexectomy was performed, and evaluations were made for the expression of anti-müllerian hormone (AMH), vascular endothelial growth factor receptor 2 (VEGFR-2), markers of oxidative stress, and follicle counts. Blood AMH levels were measured.
Results
No change in AMH levels was detected. Although the expression of AMH was significantly reduced following I/R alone, it remained similar to the control group in the NIF group. Meanwhile, the RSV group exhibited slightly lower expression than the control, although it was still higher than that observed with the I/R injury group. VEGFR-2 staining was similar in the I/R and NIF groups, but reduced in the RSV group. Markers of oxidative stress were similar between groups. Primordial follicle count was lower in the untreated I/R injury group compared to the control group (p<0.05). The NIF group had more secondary follicles than the I/R injury and RSV groups (p<0.05).
Conclusion
Nifedipine and resveratrol treatments did not influence AMH levels and antioxidant-oxidant system parameters in rats exposed to I/R injury. However, nifedipine had a positive effect on AMH expression and secondary follicle count, and resveratrol decreased the expression of VEGFR-2 in tissues, which can be an indicator of their clinical potential.
PRECIS: We evaluated the protective effect of nifedipine and resveratrol in an experimental rat adnexal I/R injury model by creating subsequent adnexal torsion and detorsion.
Introduction
Adnexal torsion is a serious gynecological emergency defined by the ovary rotating partially or fully around its pedicle or vascular axis. Torsion creates lymphatic and arterial blockage that may result in gangrene and necrosis in adnexa(1). Ovarian cysts represent the predominant cause of ovarian torsion(2). A sudden occurrence of abdominal pain, often with nausea or vomiting, frequently indicates ovarian torsion(3). In case of a suspected ovarian torsion, the initial imaging technique used is ultrasound. Impaired or absent blood flow in an enlarged and edematous ovary on ultrasound is findings that assist in the diagnosis of ovarian torsion(4). Historically, adnexectomy was considered to be the only option. Nevertheless, results from contemporary studies suggest that detorsion of the adnexa can be a more conservative treatment option with similar success(1). However, concerns have been raised about increased production of reactive oxygen radicals due to reperfusion after detorsion, which can inflict further damage to the adnexa(5). Various agents have been suggested to reduce this ischemia-reperfusion (I/R) injury, including nifedipine and resveratrol(6-8).
Resveratrol, naturally a phytoalexin, is typically found in plants such as grapes and berries, and is thought to have an antioxidant effect due to the hydroxyl group it contains(9). Meanwhile, nifedipine is a calcium channel blocker, and its effect on reducing oxygen radicals formed after I/R injury has been tested in several studies on the heart and testis(7, 10-12). Few studies have examined the antioxidant effect of resveratrol in protecting the adnexa from I/R injury following a detorsion, and none have investigated nifedipine, to the best of our knowledge(8).
Our objective was to examine the effect of nifedipine and resveratrol on ovarian viability in a rat model of adnexal I/R injury, induced through consecutive adnexal torsion and detorsion procedures. Ovarian viability was evaluated with biochemical, histopathological and immunohistochemical parameters and markers of oxidative stress.
Materials and Methods
The protocol was reviewed and approved by the Gazi University Animal Experiments Local Ethics Committee of animal studies (approval number: G.Ü.ET-20.034, date: 09.07.2020). All experiments were conducted in accordance with the ARRIVE guidelines, the U.K. Animals (Scientific Procedures) Act of 1986 and its associated guidelines, EU Directive 2010/63/EU for animal experiments, and the National Research Council’s Guide for the Care and Use of Laboratory Animals.
In the study, we included 24 female Sprague-Dawley rats weighing 255-290 grams and about 3 months old, divided into 4 groups with the same number of rats in each group using the RAND function in Microsoft Excel (v16.0, Microsoft Corporation, Redmond, U.S.). Rats were taken under standard laboratory conditions with a temperature of 22±2° C, 60% relative humidity, and 12 hour light and dark photoperiod before the experiment at a standard temperature of 22° C for at least 48 hours prior to randomization, being fed with tap water ad libitum and standard rat chow.
Allocation concealment was maintained until allocation. Animal care staff were unaware of allocation groups. The surgeon performing the adnexectomy was blind to which treatment each rat received. During the analysis of the outcome, investigators knew which rats were grouped to enable group comparisons; however, they were unaware of which specific treatment each group received.
Groups were determined as follows: Group I, control (sham operation); Group II, torsion-detorsion (I/R); Group III, torsion-detorsion + nifedipine (I/R + NIF); Group IV, torsion-detorsion + resveratrol (I/R + RSV).
Surgical Procedure
After weighing, anesthesia was performed with xylazine (5 mg/kg) and ketamine (45 mg/kg) intramuscular injections. The operation field was sterilized, and all rats were placed in the operation area on their backs. Blood samples were taken from all rats preoperatively for anti-müllerian hormone (AMH) measurement. The torsion-detorsion model was performed according to Parlakgumus et al.’s(13) study. We twisted ovaries two complete circles around their pedicle as described in the method. Laparotomy started with a 2-cm midline abdominal incision, and then intestines were gently retracted. The subsequent procedures were implemented for each group:
• Group I (sham operation): Bilateral adnexal exploration was performed without intervention following the midline laparotomy. Six hours after the laparotomy, a blood sample for AMH measurement was collected, and bilateral ovaries were surgically removed.
• Group II (I/R): After midline laparotomy, both adnexa were explored and then rotated clockwise by 720°. Then, both twisted adnexa were sutured to the lateral abdominal wall with a 5.0 polyglactin suture. The skin was closed with a 4.0 poliglecapron suture. Three hours after torsion, a secondary laparotomy was performed to achieve detorsion of the adnexa to provide reperfusion (Figure 1). The abdomen was closed after detorsion. Three hours following reperfusion, blood samples were taken for AMH measurement, and bilateral adnexa were surgically removed.
• Group III (I/R + NIF): Exact steps from Group II were followed, except, 30 minutes before detorsion, this group received intraperitoneal nifedipine (10 mg/kg). The dosage of nifedipine was based on Chander and Chopra(14) study, which assessed its protective effect against cyclosporine-induced oxidative stress in rats.
• Group IV (I/R + RSV): Exact steps from Group II were followed, except, 30 minutes before detorsion, this group received intraperitoneal resveratrol (100 mg/kg). The dosage of resveratrol was determined according to Aydın et al.’s(15) study, which evaluated its protective effect against sepsis-induced oxidative stress in rats.
Biochemical AMH Evaluation
Serum samples were obtained by centrifuging blood samples at 4000 rpm for 10 minutes. Then all samples were preserved at -80 ºC in Eppendorf tubes in an ultra-low temperature freezer until analysis. AMH levels were analyzed on the same day with an automatic ELISA kit.
Histopathological Examination
Ovaries were placed in fixation fluid containing 10% neutral buffered formalin. Afterwards, samples were embedded in paraffin and were processed using routine histological light microscopy techniques. Once embedded, sections were taken from each paraffin block to create 4-micron-thick slices. Ovary sections were stained with hematoxylin and eosin. Histomorphological analyses and follicle counting were performed using a light microscope and a computer-enhanced visualization system (Leica DM 4000, Germany). Primordial follicles, antral follicles, atretic follicles, and corpus luteum were counted. The number of follicles from five randomly selected cross-sections from both experimental and control groups was quantified, and the results were compared.
Immunohistochemical Procedure
Anti-AMH antibody and anti-vascular endothelial growth factor receptor 2 (VEGFR-2) antibody were used for immunohistochemical evaluation based on the avidin-biotin peroxidase method. Ovarian tissue blocks were sectioned with a microtome into 4 micrometer-thick cross-sections. After deparaffinization, the cross sections underwent retrieval processing by incubation in citrate buffer (pH 6.0) (Lab Vision, Thermo Scientific) and exposure to 3% hydrogen peroxide (Lab Vision, Thermo Scientific) to block endogenous hydrogen peroxidase enzyme activity. Ultra V block (Lab Vision, Thermo Scientific, Fremont) was used to restrict non-selective interaction of enzymes or primary antibodies with tissue. Following the blocking stage, primary antibody (VEGFR-2 polyclonal antibody, bs10412R, Bios, AMH polyclonal antibody, D1201A, AFG Bioscience, respectively) was applied to tissue sections at a dilution ratio of 1:150 for a duration of 90 minutes. Following the primary antibody incubation, sections were washed with phosphate-buffered saline (PBS) three times for three minutes each. Afterwards, the secondary antibody (Lab Vision, Thermo Scientific, Fremont) was used for 10 minutes. The tissues were washed again with PBS three times for three minutes each. The immunohistochemical reaction components were made visible using streptavidin peroxidase complex (Lab Vision, Thermo Scientific, Fremont) with DAB. Counter-staining was performed with Mayer’s hematoxylin. The immunostaining intensity in adnexal sections was evaluated semi-quantitatively as follows: (0) very weak; (1) weak; (2) moderate; and (3) strong(16, 17).
Evaluation of Oxidative Stress
Total antioxidant status (TAS) and total oxidant status (TOS) of ovarian tissues were measured using Erel’s method(18). The oxidative stress index (OSI) was calculated as OSI=0.25 TOS/TAS.
Statistical Analysis
SPSS for Windows 22.0 (Statistical Package for the Social Sciences) software was utilized for statistical analysis. Normal distribution was evaluated by the Shapiro-Wilk test. Standard deviations were used to present normally distributed data, while medians and percentiles were used for the rest. Categoric data were reported as percentages. Independent groups were compared with independent samples t-test for normally distributed data and with Mann-Whitney U test for non-parametric data. ANOVA was used to compare multiple groups, and post-hoc tests were conducted. The threshold for statistical significance was established at p<0.05.
Results
Biochemical AMH Evaluation
Preoperative to postoperative AMH level change was similar among all groups (p=0.058; p=0.089; p=0.756; p=0.206, respectively) (Table 1).
Histopathological Examination
Number of follicles was similar, except that in primordial and secondary follicles (p=0.047, p=0.031, respectively) (Table 2). Post-hoc tests showed that the control group had a higher primordial follicle count than the I/R group (p=0.014). The nifedipine-treated group had a higher secondary follicle count compared to the I/R and I/R+RSV groups (p<0.013, p<0.017, respectively) (Table 3). Visualization of changes in follicle morphology and vascular dilatation is presented in Figure 2.
Immunohistochemical Procedure
Immunohistochemical evaluation showed a remarkable decrease in AMH reactivity in the I/R group (Figure 3). Although AMH staining was more prominent in the resveratrol-treated group compared to the I/R group, it was observed to be weaker than in the control group. It was noted that in the nifedipine-treated group, tissue-wide uptake in the AMH immunostainings was similar to that in the control group.
Immunostaining for VEGFR-2 showed significantly increased immunoreactivity in all regions of the I/R group (Figure 4). VEGFR-2 levels were low in the resveratrol group, as in the control group. Conversely, levels in the nifedipine-treated group were close to those in the I/R group.
Evaluation of TAS, TOS, and OSI
Assessment of TAS, TOS, and OSI in harvested ovarian tissues yielded no significant difference between groups (p=0.094, p=0.089, p=0.162, p=0.611) (Table 4).
Discussion
We investigated the effects of nifedipine and resveratrol on enhancing ovarian viability following I/R injury due to torsion-detorsion. Histopathological findings indicated that the nifedipine-treated group exhibited a higher secondary follicle count compared to the I/R and I/R+RSV groups. Additionally, the AMH immunostaining grade in the nifedipine group was similar to the control group. Shown by VEGFR-2 staining, resveratrol also demonstrated protective attributes.
AMH level is an important parameter for predicting the reproductive period and evaluating long-term ovarian function in women; however, in many studies in the literature, only the postoperative AMH level was evaluated(19, 20). We hypothesized that evaluating the change of AMH level from the preoperative to the postoperative period can better portray the extent of I/R injury. Like our study, Karakaş et al.(21) investigated the effect of metformin on I/R injury in ovaries, focusing on blood AMH levels. The researchers found a higher AMH change in the torsion/detorsion group, while the addition of metformin maintained the levels of the control. In the study of Sakin et al.(19), the effect of phosphodiesterase-5 inhibitors on ovarian I/R injury was investigated, and the postoperative AMH level was measured 6 hours after surgery, without measuring the preoperative AMH level. They reported that administration of vardenafil was associated with higher postoperative AMH levels; however, it could be argued that this result may be unreliable due to preoperative AMH levels being unknown.
Follicle count is an important parameter in most of the studies evaluating ovarian I/R injury. The increase in oxidative stress during ovarian torsion causes tissue damage, and reduction in follicle numbers in almost all stages of oocyte maturation(22). Shokri et al.(23) analyzed the effect of Galea officinalis extract on I/R injury and reported the primary, antral, graafian, and atretic follicle count were higher, indicating a positive effect. Santa-Helena et al.(24), also observed that nifedipine reduced reactive oxygen radicals and increased the viability of cardiomyoblasts after I/R injury. This indicates that nifedipine may have a role against I/R injury. In another study on the same topic, the effect of enoxaparin was investigated, and although primordial, preantral, corpus luteum and atretic follicle counts were not found to be different, the number of small and large antral follicles was significantly lower in the torsion/detorsion group compared to the control(25). We found that the nifedipine-treated group had a higher secondary follicle count than groups 2 and 4, a finding which warrants further investigation in clinical studies.
We performed AMH and VEGFR-2 immunostaining in ovarian tissues. Parlakgumus et al.(13) studied atorvastatin and expression of AMH and VEGF-A in I/R damaged ovaries, and they found that AMH was higher in the torsion/detorsion + atorvastatin group. The expression of AMH originates in granulosa cells of early antral follicles, and the follicles expressing the most AMH are preantral and small antral follicles(26, 27). Ischemia is a factor that reduces AMH expression. Cells expressing higher AMH are thought to have higher proliferative and steroidogenic activity(28). In our study, the staining of preantral and antral follicles for AMH was weak in groups II and IV, although it was close to control in the nifedipine-treated group. This suggests that nifedipine may preserve the proliferative capacity of cells.
Hypoxia is one of the best stimulators of angiogenesis, thus increased expression of VEGF receptors during I/R can be expected. Kanellis et al.(29) showed that after renal I/R, VEGFR-2 immunostaining surged in renal tissue. In our study, although the staining of VEGFR-2 was increased in group 2, it was decreased in the resveratrol-treated group. The lower VEGF receptor level in group 4 suggests that resveratrol can help in tolerating oxidative stress and its effects. Evidence suggests that calcium channel blockers may increase VEGF concentration(30, 31). Our findings indicate that the staining of VEGFR-2 in the nifedipine group was similar to that in the I/R group alone. Therefore, it is suggested that nifedipine has no significant effect on VEGF.
Oxidative stress was similar among groups in the current study. Gungor et al.(32) evaluated the possible beneficial effects of a clinically available omega-3 fatty acid emulsion on I/R injury by measuring TAS, TOS, and OSI, and they did not find any positive effect. Although resveratrol and nifedipine were shown to have antioxidant effects in several studies, we did not observe any evidence of this effect in our study(33).
Study Limitations
There are several limitations that should be noted. First, we performed torsion for only 3 hours. However, in clinical ovarian torsion scenarios, the exact duration from torsion to detorsion is often unknown. As the torsion duration increases, the damage to the ovary might become irreparable. Moreover, we administered the agents at doses used in previously published studies and have not tested other doses, which may have allowed more optimal dosing regimens.
Conclusion
Although nifedipine and resveratrol did not demonstrate a concrete protective effect on every parameter evaluating I/R injury after ovarian torsion/detorsion, increased immunohistochemical AMH expression and secondary follicle count in the nifedipine treated group, and decreased VEGFR-2 expression in the resveratrol treated group, can be regarded as indicators of their potential.
