Ovarian ischemia is an emergency resulting from a sprain (torsion) of the ovaries [1]. Ovarian torsion is more common in patients with ovulation induction, pregnant women, women of reproductive age, patients with a benign or malignant ovarian mass greater than 5 cm, and women who have experienced a previous ovarian torsion [2, 3]. Delays in the diagnosis and treatment of ovarian torsion may result in permanent tissue damage and organ loss [3, 4]. Therefore, it is recommended that patients receive reperfusion of the torsioned ovaries with detorsion [5]. However, detorsion of torsioned ovaries can cause more severe tissue damage. This event is called ischemia-reperfusion (IR) injury [6]. Studies show that oxidative stress is significant in IR damage. As is known, nicotinamide adenine dinucleotide is used in the metabolism of hypoxanthine (HX) with xanthine dehydrogenase (XDH) in an anaerobic environment. Therefore, reactive oxygen species (ROS) are not produced as intermediates [7]. However, XDH produced in an aerobic environment is converted to xanthine oxidase (XO) in an anaerobic environment [8]. Since molecular oxygen (O${}_2$) is used in the metabolism of HX with XO, ROS are produced as an intermediate product. Therefore, the metabolism of HX by the XO enzyme cannot occur unless there is reperfusion in ischemic tissue [9]. Providing reoxygenation in reperfusion causes the metabolism of HX and excessive ROS formation [10]. These ROS, known as reperfusion mediators, cause the formation of toxic products such as aldehyde and malondialdehyde (MDA) from lipids by oxidizing cell membrane lipids [11]. It is known that reduced glutathione (GSH), known as an endogenous antioxidant, decreases in ovarian tissue with high MDA content [12].

Another event that is thought to contribute to the exacerbation of IR injury is inflammation. Inflammation mediators induce an inflammatory response. Recently, proinflammatory interleukin 1 beta (IL-1$\beta$) and tumor necrosis factor-alpha (TNF-$\alpha$) have been shown to increase in tissues exposed to IR [13]. In addition, ROS is known to stimulate the expression of nuclear factor kappa B (NF-$\kappa$B), which controls the production of proinflammatory cytokines. Fatma et al. [14] indicated that the rising in oxidative stress parameters in ovarian IR was associated with a considerable rising the expression of NF-$\kappa$B and TNF-$\alpha$. Unlubilgin et al. [15] reported in their study that the increase of oxidant and proinflammatory cytokines in IR-treated ovarian tissue was associated with infertility. This information obtained from the literature reveals the importance of the early diagnosis and treatment of ovarian torsion in preserving ovarian function and preventing future infertility [1]. As mentioned above, detorsion of the torsioned ovaries causes more severe damage to the ovarian tissue. Therefore, a traditional operation option with detorsion is mainly suggested [16].

Carvacrol is a monoterpene phenol produced from Origanum vulgare and Thymus vulgaris [17]. Research has been conducted on the clinical use of carvacrol. In vitro and in vivo researches have indicated that carvacrol has antioxidant, antibacterial, antifungal, anticancer, anti-inflammatory and spasmolytic effects [18]. It has also been reported that carvacrol has an inhibitory effect on proinflammatory cytokine and MDA production and a stimulant effect on total glutathione (tGSH) production [19]. This information suggests that carvacrol may protect ovarian tissue from the oxidative and inflammatory damage of IR. The goal of our research is to analyze the impact of carvacrol against IR-induced ovarian damage and reproductive dysfunction in female rats.

The experimental results were expressed as mean value $\pm$ standard deviation ($\overline{x}$ $\pm$ SD). The significance of the differences between groups was determined using the one-way ANOVA test followed by post-hoc Tukey tests. Histopathological grading and the number of offspring are presented as the median (min–max) value. While comparing fertility between groups Chi Square was used. While comparing the number of offspring in groups, Kruskal Wallis test and then Dunn post hoc test were used and adjusted p values were presented. All statistical processes were performed using SPSS for Windows version 18.0 (IBM Corp., Chicago, IL, USA), and p 0.05 value was considered significant. While performing histopathological scoring, the median of the values was calculated.

Our study investigated the protective effect of two different doses of carvacrol against IR-induced ovarian damage and reproductive dysfunction in female rats. Our biochemical test results showed that carvacrol at 50 and 100 mg/kg doses equally prevented the increase of MDA in ovarian tissue by IR. As is known, lipids are the biomolecules most affected by oxidative stress [29]. ROSs, by lipid peroxidation (LPO), lead to the formation of toxic aldehydes that exacerbate a variety of oxidative damage [30]. MDA is one of the toxic products of the LPO reaction, which is most commonly used in determining oxidative stress [31]. MDA creates a cytotoxic effect by causing cross-linking and polymerization of cell membrane components and inactivation of receptor and membrane-bound enzymes [32]. Meeran et al. [33] reported that carvacrol and its isomer, thymol, inhibited LPO; they also found that the inhibitory effect of thymol on LPO was stronger than that of carvacrol. It has been suggested that this is due to the greater steric hindrance of thymol. Moreover, the high potential of phenolic compounds to scavenge radicals may be explained by their ability to donate a hydrogen atom from their phenolic hydroxyl groups [34]. From the literature, it is understood that the protective effects of carvacrol and thymol on ovarian tissue are due to their common antioxidant and anti-inflammatory properties [35]. Another common feature of thymol, carvacrol and other plant-derived terpenes in their biological activities is the inhibition of voltage-dependent Na${}^{+}$ ion channels [36]. Ingec et al. [37] reported that the increase in the amount of MDA was associated with the severity of the ovarian tissue injury. Kurt et al. [38], on the other hand, argued that suppressing the increase in the amount of MDA is important in reducing IR-related ovarian damage. Dursun et al. [39] showed that carvacrol suppressed the increase of MDA in testicular tissue after torsion detorsion in rats, they also stated that can prevent apoptotic development and signaling of spermatogenic cells with strong mitotic activity in the basement membranes of seminiferous tubules. Taxifolin, another antioxidant that prevents the increase of oxidants and the decrease of antioxidants in testicular tissue applied torsion/detorsion, has been shown to alleviate histopathological damage. Taxifolin has been proven to prevent the reduction in germinal epithelial thickness caused by the torsion/detorsion, detached germinal cell lines, cell necrosis of the seminiferous tubule, and the development of degenerated germinal giant cells [40]. The present study shows that the IR procedure caused a decrease in tGSH in the ovarian tissue. GSH is known to be a tripeptide antioxidant consisting of L-glutamate, L-cysteine ​​and glycine [41]. GSH has been proven to play a critical role in protecting cells from oxidative damage and the toxicity of xenobiotic electrophiles while maintaining redox homeostasis [42]. Isaoglu et al. [43] demonstrated that an IR event causes a decrease in the amount of tGSH in the ovarian tissue. Another study found that drugs that prevent the decrease of tGSH levels protect ovarian tissue from IR damage [44]. Kadioglu et al. [12] emphasized the importance of keeping MDA low as well as keeping tGSH high in reducing IR-related damage. It has been reported that carvacrol significantly prevents the depletion of cellular GSH content by hydrogen peroxide (H${}_2$O${}_2$) [45]. It has also been reported that carvacrol is a good scavenger of peroxyl radicals and inhibits the peroxidation of phospholipid liposomes in the presence of iron (III) and ascorbate [46].

In our study, TOS and TAS levels were measured to gain an understanding of the way that IR in ovarian tissue changes the oxidant-antioxidant balance in favor of oxidants. MDA, tGSH and other oxidant-antioxidant parameters are known to be used to determine the oxidant-antioxidant balance. TOS and TAS reflect the total effects of all antioxidants and oxidants in tissues [25, 26]. Therefore, TOS levels are used for practical measurement of ROS, and TAS levels are used to evaluate total antioxidant status. In a study supporting our TOS and TAS findings, it was observed that the IR procedure increased TOS levels in ovarian tissue and decreased TAS levels [47].

Our study showed that proinflammatory cytokine levels such as NF-$\kappa$B, TNF-$\alpha$ and IL-1$\beta$ were increased in the IR-applied ovarian tissue. As noted in the literature, the production of NF-$\kappa$B, TNF-$\alpha$, IL-1$\beta$ and oxidant is increased in tissues exposed to IR [48]. Keskin et al. [49] showed that histopathological damage developed in IR-induced testicular tissue with high NF-$\kappa$B, TNF-$\alpha$ and oxidant levels and low antioxidants. NF-$\kappa$B plays an important role in regulating the expression of other proinflammatory cytokine genes [50]. In addition, it has been reported that NF-$\kappa$B activation is induced by ROS, and oxidative stress triggers inflammation [51, 52]. However, in our study, although carvacrol inhibited the increase of IR-related oxidants at 50 and 100 mg/kg doses to the same extent, it was able to inhibit the increase of NF-$\kappa$B, TNF-$\alpha$ and IL-1$\beta$ only at a dose of 100 mg/kg. No information was found in the literature to support these findings. However, carvacrol is known to protect lung tissue from damage due to oxidative stress and proinflammatory cytokine increase [53], and carvacrol has been shown to have a stronger inhibitory effect on oxidant and proinflammatory cytokines at high doses [53, 54]. Li et al. [55] suggested that the anti-inflammatory effect of carvacrol is due to inhibition of the pro-inflammatory NF-$\kappa$B pathway. This information indicates that the IR process causes damage in different tissues with a similar mechanism.

Carvacrol at a dose of 100 mg/kg, which significantly inhibited the overproduction of NF-$\kappa$B, TNF-$\alpha$ and IL-1$\beta$, reduced ovarian IR injury histopathologically better than the 50 mg/kg dose. In addition, inflammatory symptoms such as edema, congested and dilated blood vessels and PNL infiltration were observed in the ovarian tissue of the carvacrol group administered a dose of 50 mg/kg, while these histopathological findings were not observed in the rats that received a dose of 100 mg/kg. Previous studies have found that IR caused histopathologically dilated and congested blood vessels, PNL infiltration, hemorrhage, edema, follicular degeneration and necrosis in the ovarian tissue [56, 57]. Unlubilgin et al. [15] explained that this IR-related histopathological damage leads to infertility. In our study, proinflammatory cytokine production was significantly suppressed, and the number of infertile animals decreased significantly in the carvacrol group (at a dose of 100 mg/kg), in which, histopathologically, no inflammatory symptoms were observed. Bhandari et al. [58] found that the development of infertility was associated with an increase in proinflammatory cytokines and a decrease in anti-inflammatory cytokines. which supports our experimental results. As a result, the IR event caused an increase in oxidant and proinflammatory cytokines and a decrease in antioxidants in ovarian tissue. Ovarian IR caused tissue degeneration, necrosis, vascular damage and inflammatory events histopathologically. At a dose of 100 mg/kg, carvacrol, which inhibits the overproduction of oxidant and proinflammatory cytokines increased by IR in ovarian tissue, prevented the development of inflammation in the tissue. The 50 mg/kg dose of carvacrol, which inhibited the IR-related oxidant release in ovarian tissue to the same extent, did not inhibit the increase of proinflammatory cytokines and the development of inflammation. Carvacrol significantly prevented IR-related infertility at a dose of 100 mg/kg but not at a dose of 50 mg/kg. This indicates that the anti-inflammatory effect of carvacrol at a dose of 100 mg/kg is not related to its antioxidant activity. It also shows that inflammation is an important factor in the pathogenesis of ovarian IR-related infertility. Induction of inflammation in IR-related ovarian tissue suggests that it is not limited to oxidants.

Our experimental results suggest that effective doses of antioxidants and anti-inflammatory drugs may be beneficial in the clinical treatment of ovarian IR injury due to torsion and detorsion and in preventing the development of infertility. In addition, the information obtained from our study and literature indicates that it may be beneficial to apply antioxidant and anti-inflammatory treatment before and after reperfusion.

IR, Ischemia-reperfusion; HX, Hypoxanthine; XDH, Xanthine dehydrogenase; ROS, Reactive oxygen species; XO, Xanthine oxidase; MDA, Malondialdehyde; tGSH, Total glutathione; TOS, Total Oxidant Status; TAS, Total Antioxidant Status; IL-1$\beta$, Interleukin 1 beta; TNF-$\alpha$, Tumor necrosis factor-alpha; NF-$\kappa$B, Nuclear factor kappa B; PNL, Polymorphous nuclear leukocyte; OIR, Ovary ischemia-reperfusion; IRC-50, IR +50 mg/kg carvacrol; IRC-100, IR +100 mg/kg carvacrol; SG, Sham operation group.

NS, IBD and UI designed the research study. BS performed the research. HS provided help and advice on the experiments. GNY and TAC, VA analyzed the data. BS, HS and KU wrote the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript.

The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Atatürk University Animal Experiments Local Ethics Committee, Erzurum, Turkey (Date: 23.03.2021, meeting no: 236643897-000). All animals received care in compliance with the institution’s guidelines, as outlined in the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health.

Thanks to all the peer reviewers for their opinions and suggestions.

This research received no external funding.

The authors declare no conflict of interest.