Effects of Gallic Acid on Cyclophosphamide-Induced Experimental Ovarian Injury in Rats

Effects of Gallic Acid on Cyclophosphamide-Induced Experimental Ovarian Injury in Rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects of Gallic Acid on Cyclophosphamide-Induced Experimental Ovarian Injury in Rats Elif Koyun Alvuroğlu, Derya Öztürk Okatan, Elif Şahin, Ahmet Alver This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8146263/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 15 You are reading this latest preprint version Abstract Background The potential harmful effects of cancer treatments on reproductive function have now been clearly established. Exposure to chemotherapy is considered a risk factor for premature ovarian failure and causes infertility. This study investigated the prophylactic effects of gallic acid (GA) against cyclophosphamide(CP)-induced ovarian damage in rats. Methods No procedure was applied to the control group. The CP group received 150mg/kg CP via the intraperitoneal (i.p.) route on day 7 of the experiment. The GA group received 20mg/kg GA daily for seven days from day 1 of the experiment via oral gavage. The CP + GA received 20mg/kg GA daily for seven days from day 1 of the experiment via oral gavage and 150mg/kg i.p. CA on day 7 of the experiment. Histopathological examination of ovarian tissues and follicle counting were performed. Glutathione peroxidase, superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), tissue anti-müllerian hormone (AMH), and serum AMH levels were examined at biochemical investigation. Results CP caused follicular cell degeneration, increased the apoptotic index, reduced the numbers of primordial and unilaminar primary follicle cells, and increased the numbers of atretic follicles (p < 0.05). Follicular cell degeneration and the apoptotic index decreased while the numbers of primordial follicles increased in the CP + GA group compared to the CP group (p < 0.05). CAT, SOD and tissue AMH decreased, while MDA levels increased in the CP group compared to the control group. In the CP + GA group, CAT and SOD levels increased while MDA levels decreased compared to the CP group (p < 0.05). Serum AMH levels were higher in the GA group than in the other groups (p < 0.05). Conclusions GA can be effective in preventing follicle cell damage caused by CP in the ovarium. Ovarium toxicology gallic acid cyclophosphamide apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Significant advances in the diagnosis and treatment of cancer have improved survival rates among children and young adults. However, the potential deleterious effects on reproductive functions of these therapies are now clearly established ( 1 ). Exposure to chemotherapy is regarded as a risk factor for premature ovarian failure (POF) and leads to menstrual disorders, metabolic abnormalities, and infertility ( 2 ). Various fertility-preserving methods, such as oocyte, embryo, and ovarian cortex cryopreservation are recommended in order to protect gametes from the gonadotoxic effects of chemotherapy or radiotherapy. However, the application of these methods can be limited by the patient’s age, pubertal status, disease, and emergency conditions ( 1 ). Cyclophosphamide (CP), one such chemotherapeutic, is an alkylating agent indicated for use in malignancies such as Hodgkin and non-Hodgkin lymphoma, lymphocytic lymphoma, Burkitt lymphoma, and multiple myeloma, and in autoimmune diseases such as multiple sclerosis ( 3 , 4 ). CP generally exhibits its antineoplastic effect through its metabolite phosphoramide mustard, which forms as a result of metabolism of the drug by the cytochrome P-450 enzyme in the liver ( 5 ). Another metabolite, acrolein, increases the production of reactive oxygen species (ROS) by inhibiting the antioxidant system, thus giving rise to CP side-effects (MacAllister). CP has been shown to cause cardiotoxicity, urotoxicity, gonodal toxicity, hematological toxicity, and lung damage ( 6 , 7 ). Studies have shown that the induction of oxidative stress in tissues may be one of the causes of this damage resulting from CP ( 8 ). Cyclophosphamide causes a decrease in primordial, primary, secondary, and antral follicles in the ovary, and an increase in atretic follicles ( 9 , 10 ). It is thought to cause irreversible follicle loss through the induction of apotosis in follicles and reducing microvascularization in the corpus luteum and follicles ( 11 ). CP causes decreased antioxidant enzyme activity in the ovary and an increase in ROS and malondialdehyde levels (MDA) ( 12 , 13 ). In addition to a decrease in serum anti-müllerian hormone (AMH) values, CP also causes significant diminutions in body and ovarian weights ( 14 , 15 ). Recent studies have shown that CP causes ovarian damage by increasing granulosa cell apoptosis ( 16 ). Studies have suggested that granulosa cell apoptosis triggered by an impaired antioxidant mechanism and excessive ROS production represents the main cause of follicular atresia ( 17 , 18 ). The primordial follicle pool is exhausted in association with CP-induced damage, and early menopause and infertility are seen as a result ( 19 ). Gallic acid is a polyphenolic benzoic acid known as 3,4,5 trihydroxybenzoic acid ( 20 ). Its chemical formula is C6H2(OH)3COOH ( 21 ). It is widely present in hazelnuts, fruits such as cherries, raspberries, pomegranates, and grapes, and in vegetables. It is also found in onion, green tea, and honey. Experimental and theoretical studies have identified it as a powerful antioxidant ( 22 – 24 ). It is also an important component of plants with anticancer properties, and its derivatives have been shown to induce cancer cell apoptosis ( 20 ). This property of GA is associated with its role as a pro-oxidant, meaning it is both an antioxidant and pro-oxidant ( 25 ). Due to the three alcoholic OH groups found in GA, it produces radicals for the redox reaction and acts as a radical scavenger ( 26 ). A powerful antioxidant, GA also exhibits anticancer, antimicrobial, antiviral, anti-inflammatory, antidiabetic, and antihypertensive properties and acts against neurodegenerative disorders and aging ( 27 – 35 ). It is an important molecule in biomedical practice and drug design ( 36 – 38 ). This study examined the potential ameliorating effects of GA against ovarian damage that may develop in association with the use of CP. We encountered no previous study investigating the protective or therapeutic effect of GA, a potent antioxidant, in CP-induced ovarian damage. We think that this study will contribute to filling this gap in the literature and will be if use to future studies on the subject. MATERIALS AND METHODS Animals, Experimental procedures Approval for the study was granted by the Karadeniz Techincal University (KTU) animal experiments ethical committee (no. 2021/46 dated 21.09.2021). All procedures were performed in accordance with the principles of the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Thirty-two female Sprague Dawley rats were used in the research. These were procured from the KTU Medical Faculty Surgical Application and Research Center and were housed in a 12-hour dark:light cycle at 50% humidity and a temperature of 22 ± 1° C. The rats were housed in standard type III cages throughout the experiment with ad libitum access to standard rat chow (Bayramoğlu Yem ve Un Sanayi Tic. A.Ş. Erzurum, Türkiye) and tap water. All the experimental procedures, animal care, and sacrifice were carried out in the KTU Surgical Application and Research Center in order to avoid environmental stress and adaptation problems. The rats were divided into four groups. No procedure was performed on the control group (n:8). The CP group (n:8) received intraperitoneal (i.p.) CP (Endoxan, Baxter-Eczacıbaşı İlaç Pazarlama, Istanbul, Türkiye) at a dose of 150 mg/kg on day 7 of the experiment. The GA group (n:8) received 20 mg/kg GA (Sigma-Aldrich Chemie Gmbh) (1 g GA dissolved in 100 ml saline solution) by oral gavage for seven days from day 1 of the experiment. The CP + GA group (n:8) received 20 mg/kg GA via oral gavage for seven days from day 1 of the experiment and 150 mg/kg CP i.p. on day 7 of the experiment ( 39 , 40 ). All rats were weighed on days 1 and 8 of the experiment. The animals were finally sacrificed by exsanguination at the end of the experiment (day 8) under anesthesia (ketamine 90 mg/kg). Blood specimens were collected and placed into ethylene diamine acetic acid (EDTA) tubes for biochemical parameter analysis. A midline incision was promptly made to the lower abdomen region. The right ovary was first removed, weighed, and placed into 10% formalin solution for tissue fixation. The left ovaries were placed into Eppendorf tubes and stored at -80° C for biochemical investigation. Histopathological Analysis The right ovaries were fixed in 10% formaldehyde solution. A routine tissue processing program was applied on the tissue processing device (Thermo Scientific Excelsior AS, UK). After processing, the tissues were removed from the cassettes, embedded in paraffin blocks on a blocking device (Leica HistoCare Arcadia H, China), and cooled. Five micron-thick sections were ten taken using a fully automatic microtome (Leica RM 2255, Germany). Serial 5-µm sections were taken from the right ovaries. Sections collected at 50 µm intervals were stained with hematoxylin-eosin H&E using an automatic staining device (Leica Autostainer XL, Germany) ( 41 ). One part of the other sections was stained with Masson’s trichrome ( Masson Trichrome Staining Kit, GBL, Türkiye), Periodic-Acid Schiff (PAS) ( PAS McManus Staining Kit, GBL, Türkiye), and deoxyuridine triphosphate nick end labeling assay (TUNEL) (in situ cell death detection POD kit, Roche, 11684817910, Berlin, Germany) in line with the manufacturer’s instructions. All histopathological examinations were carried out using a light microscope (Olympus, BX51, and Japan) equipped with a DP 71 (Olympus, Japan) digital camera in the KTU Medical Faculty Department of Histology and Embryology. The sections were photographed, the images being transferred to a digital environment. Ovarian Follicle Evaluation Primordial follicles, unilaminar primary follicles, multilaminar primary follicles, secondary follicles, Graafian follicles, and atretic follicles were counted in the entire area of ​​the 5 µm H&E-stained sections taken at 50 µm intervals for each ovary, according to the definitions given below ( 42 ). Primordial follicle: The primary oocyte is covered by a single layer of flat follicle cells. Unilaminar primary follicle: The primary oocyte is surrounded by a single layer of cubic follicle cells. Multilaminar primary follicle: The primary oocyte is surrounded by multilayer cubic follicle cells (zona granulosa). Theca externa and theca interna layers may be present around the zona granulosa. Secondary follicle: The primary oocyte is covered by multilayer cubic follicle cells (zona granulosa). The theca externa and interna layers are present around the zona granulosa. The zona granulosa contains spaces filled with follicular fluid. Graafian follicle: Corona radiata and cumulus cells are present around the eccentrically located primary oocyte. The spaces in the zona granulosa combine to form a single space. Theca layers surround the zona granulosa. Atretic follicle: Apoptosis in granulosa cells in large follicles. Invasion of the granulosa layer by macrophages, neutrophils, and vascularized connective tissue cords. Granulosa cell shedding into the antrum. Follicle and zona pellucida collapse. Vitreous membrane appearance ( 43 ). Vascular congestion, follicular cell degeneration, hemorrhage around the corpus luteum, germinal epithelium degeneration, and theca layer thinning were evaluated and scored at histopathological examinations (0: no damage; 1: mild damage, focal and mild changes; 2: moderate damage, highly focused, significant changes, and 3: severe damage, widespread changes) ( 44 ). The structure of the zona pellucida and basal membrane was evaluated in sections stained with PAS, and connective tissue and collagen density in sections stained with Masson’s trichrome. TUNEL staining Apoptosis in ovarian tissue was evaluated on TUNEL-stained sections. Apoptotic and normal cells were counted in the granulosa and theca cells of follicles in five randomly selected areas in a section from each subject. Cells with brown-stained nuclei were defined as TUNEL (+) apoptotic cells ( 45 ). The apoptotic index (AI) was calculated using the formula number of TUNEL (+) cells /total number of cells x 100) ( 46 ). Biochemical Analysis Tissue Glutathione Peroxidase (GPX) Measurement GPX activity in supernatants obtained from ovarian tissues was determined using a commercial ELISA kit (catalog no. E1242Ra, BT LAB, Zhejiang, China) according to the manufacturer’s instructions. The results were expressed as ng/mg protein. AMH Measurement AMH levels in serum specimens and supernatants obtained from ovarian tissues were determined using a commercial ELISA it (catalog no. BT LAB, Zhejiang, China) in line with the manufacturer’s instructions. The results were expressed as ng/mL for serum specimens and ng/mg for tissue specimens. Tissue Superoxide Dismutase (SOD) Activity Measurement SOD enzyme activity was determined using a modification of the method developed by Sun and Oberley ( 47 ). This method is based on measuring the absorbance at 560 nm of the purple formazan molecule that forms as a result of the reduction of nitroblue tetrazolium by O2.- generated by the xanthine-xanthine oxidase system. SOD activities determined were divided by the total amount of protein, the results being calculated as U/mg protein. Catalase (CAT) Enzyme Activity Measurement A modified version of the Aebi method was employed to determine CAT activity, which catalyzes the hydrolysis of hydrogen peroxide into water and oxygen ( 48 ). This method is based on the decrease in absorbance at 240 nm as a result of the breakdown of H 2 O 2 by CAT. Care was taken to ensure that the decrease in absorbance in 30 seconds was no less than 0.03 and no greater than 0.2. The first order reaction rate constant (k) was employed as the unit for catalase activity. Activity was calculated using the equation below for a 10-second time interval. A1 = Initial absorbance, A2 = absorbance after 10 seconds, k=(2.3/10) x log (A1/A2) seconds − 1 Tissue Malondialdehyde (MDA) Measurement MDA was measured using the method described by Mihara and Uchiyama ( 49 ). This is based on measuring the absorbance at 523 nm of the color formed by MDA with thiobarbituric acid in an acidic environment. The results were expressed as nmol/g protein. Protein Assay Protein assay in the tissue homogenates was performed using the Bradford method ( 50 ). This is based on the principle that Coomassie Brilliant Blue G250, an organic dye, binds to proteins in a phosphoric acid environment, the resulting blue complex exhibiting maximum absorbance at 600 nm. The results were expressed as mg/ml. Statistical Analysis The study data were analyzed on SPSS version 23.0 software and were expressed as mean ± standard deviation. Kruskal Wallis analysis of variance was applied for group comparisons. The Mann Whitney U test was used with post-hoc Bonferroni correction. p values < 0.05 were regarded as statistically significant. RESULTS Body and Ovarian Weight Findings Mean plus standard deviation values for body and ovarian weights on days 1 and 8 of the experiment are shown in Table 1 0. No statistically significant difference was observed between the groups in terms of rat body weights at the end of the experiment. The rats’ right ovaries were compared, and no significant difference was determined between them (Table 1 ). Table 1 Body and ovarian weight K Mean ± SD CP Mean ± SD GA Mean ± SD CP + GA Mean ± SD Body Weight Day 1 245 ± 20 247,5 ± 27,9 225 ± 36,6 255 ± 37 Body Weight 8th Day 252,5 ± 20,7 235,6 ± 31,6 227 ± 15 246 ± 27,3 Ovarian Weight 0,073 ± 0,017 0,068 ± 0,019 0,061 ± 0,008 0,061 ± 0,008 Data are given as mean ± standard deviation (Mean ± SD). K: Control group, CP: Cyclophosphamide group, GA: Gallic acid, CP + GA: Cyclophosphamide + Gallic acid Histopathological analysis Primordial follicle numbers decreased significantly in the CP group compared to the control group (p = 0.015), while increasing significantly in the CP + GA group compared to the CP group (p = 0.029). Unilaminar primary follicle numbers also decreased in the CP group compared to the control and GA groups (p < 0.001 and p = 0.008, respectively), and in the CP + GA group compared to the control group (p = 0.009). No significant intergroup differences were observed in multilaminar primary follicle (p = 0.065), secondary follicle (p = 0.72), or Graafian follicle (p = 0.44) numbers. Atretic follicle numbers increased significantly in the CP group compared to the control and GA groups (p < 0.001 for both). Atretic follicle numbers were also higher in the CP + GA group than in the control and GA (p = 0.009 and p = 0.008, respectively) (Table 2 ). Table 2 Follicle numbers K Ort ± SD CP Ort ± SD GA Ort ± SD CP + GA Ort ± SD Primordial follicle 128,12 ± 21,37 101,25 ± 14,17 a 119 ± 7,48 123,71 ± 18,55 b Unilaminar primary ollicle 65,62 ± 5,62 41,87 ± 13,06 a,c 59,3 ± 10,4 51 ± 9,98 a Multilaminar primary follicle 34,37 ± 2,77 34,25 ± 4,74 34,5 ± 1,04 34 ± 1,41 Secondary follicle 31,37 ± 6,9 29,37 ± 5,8 32,66 ± 5 28,42 ± 3,55 Graafian follicle 31,37 ± 5,47 30,75 ± 6,94 31,33 ± 5,08 33,85 ± 2,73 Atretic follicle 312,75 ± 41,44 438,75 ± 69,33 a,c 261,83 ± 31,16 397,57 ± 63,66 a,c Data are given as mean ± standard deviation (Mean ± SD). K: Control group, CP: Cyclophosphamide group, GA: Gallic acid, CP + GA: Cyclophosphamide + Gallic acid ᵃ p < 0.05 compare control group. ᵇ p < 0.05 compare CP group. ᶜ p < 0.05 compare GA group. The cortex and medulla in the control group exhibited a normal appearance. Normal morphologies were also observed in the germinal epithelium, follicular cells, theca layers, and corpus luteum. Follicular cell degeneration was significantly greater in the CP compared to the control and GA groups (p < 0.001). Follicular cell degeneration was lower in the CP + GA group compared to the CP group (p = 0.003). Follicular cell degeneration was higher in the CP + GA group than in the control and GA groups (p = 0.005 and p = 0.015, respectively). The morphology of the GA group was similar to that of the control group. No significant differences between the groups were observed in terms of vascular congestion, hemorrhage around the corpus luteum, germinal epithelium degeneration, or thinning of the theca layer. No marked findings of inflammation were present in any group (Fig. 1 ). Connective tissue architecture and collagen density were similar in all the groups. No difference was also determined in terms of fibrosis (Fig. 2 ). The zona pellucida exhibited a normal architecture in all the groups. PAS positivity in the basal membrane surrounding the follicles exhibited the same intensity in all groups (Fig. 3 ). Apoptotic Index (AI) The AI was higher in the CP group than in the control group (p = 0.032). However, the AI in the CP + GA group was lower than that in the CP group (p = 0.032) (Table 3 ) (Fig. 4 ). Table 3 Histopathological analysis results and apoptotic index values K Ort ± SD CP Ort ± SD GA Ort ± SD CP + GA Ort ± SD Vascular Congestion 1,25 ± 0,46 1,5 ± 0,53 1,12 ± 0,35 1,25 ± 0,46 Follicular Cell Degeneration 0,25 ± 0,46 2,3 ± 0,51 a,c 0,37 ± 0,51 1,25 ± 0,46 a,b,c Hemorrhage Around The Corpus Luteum 0,75 ± 0,46 1,25 ± 0,35 1,12 ± 0,35 1,25 ± 0,46 Germinal Epithelium Degeneration 0,87 ± 0,35 1,12 ± 0,35 0,87 ± 0,35 1,12 ± 0,35 Thinning Of The Theca Layer 0,62 ± 0,51 1,12 ± 0,35 0,62 ± 0,51 1,25 ± 0,46 AI (%) 32,78 ± 3,57 46,8 ± 8,66 a 33,34 ± 9,03 33,48 ± 6,79 b Data are given as mean ± standard deviation (Mean ± SD).K: Kontrol grubu, CP: Siklofosfamid grubu, GA: Gallik asit, CP + GA: Siklofosfamid + Gallik asit ᵃ p < 0.05 compare control group. ᵇ p < 0.05 compare CP group. ᶜ p < 0.05 compare GA group. Biochemical analysis results Tissue MDA levels were higher in the CP group than in the control and GA groups (p = 0,003 and p = 0.001, respectively). However, lower MDA levels were observed in the CP + GA group compared to the CP group (p = 0.002). Tissue SOD activity decreased in the CP group compared to the control and GA groups (p = 0.003). SOD activity in the CP + GA group increased compared to the CP group but was at a similar level to that in the control group (p = 0.006). No difference was observed between the groups’ tissue GPX values. Tissue CAT activity was lower in the CP group than in the control group (p = 0.038). Tissue CAT activity increased in the CP + GA group compared to the control and CP groups (p < 0.001). Tissue CAT activity also increased in the GA group compared to the control and CP groups (p = 0.001). Tissue AMH values decreased in the CP group compared to the control group (p = 0.015). Serum AMH values were also higher in the GA group than in the control, CP, and CP + GA groups (p = 0.003, p = 0.003, and p = 0.022, respectively) (Fig. 5 ). DISCUSSION One of the oldest anti-cancer drugs, CP is also used in the treatment of various epithelial tumors, such as breast, ovarian, and small cell lung cancers in addition to hematological malignancies such as lymphoma and leukemia ( 6 , 51 ). CP itself is a prodrug, metabolized in the liver by cytochrome P-450 to form 4-hydroxycyclophosphamide, which is subsequently converted to phosphoramide mustard and acrolein. Phosphoramide mustard is a primary active metabolite that inhibits DNA replication by alkylating DNA ( 52 , 53 ). Phosphoramide mustard also affects the mitochondria, resulting in a decreased transmembrane potential and cytosolic cytochrome c accumulation ( 54 ). The effects of CP are independent of the cell cycle. In addition, as with all alkylating agents, rapidly proliferating cells are most sensitive to CP ( 55 ). Despite its pharmacological benefits, CP has been linked to gonadal toxicity ( 56 , 57 ). The reason for the gonadal toxicity caused by CP is still unclear, although the literature seems to support the idea that CP causes severe oxidative stress, nitrative stress inflammation, apoptosis, and genomic changes ( 9 ). CP causes testis damage in males by reducing sperm concentration and motility, testosterone levels, and testicular antioxidant capacity and increasing abnormal sperm production ( 57 ). Experimental studies have shown that CP causes a decrease in both testis and body weights ( 58 , 59 ). The application of CP also causes significant damage to Sertoli cells, alters the gene expression of several important enzymes, causes musculoskeletal damage, and adversely impacts on spermatozoa growth, proliferation, and differentiation ( 60 ). In women, CP causes marked dose-dependent damage to the ovaries ( 61 , 62 ). The ovarian damage induced by CP has been linked to facilitation of granulosa cell apoptosis ( 16 ). The existing evidence shows that CP exerts a direct effect on the follicles, lading to primordial follicle depletion following exposure to it ( 61 ). CP is the first chemotherapeutic drug to be linked to amenorrhea, premature ovarian failure, and ovarian function disorder ( 63 – 65 ). It also directly damages DNA, induces follicular apoptosis, and produces ROS harmful to ovarian cells ( 66 , 67 ). Several studies in the literature have used different substances with antioxidant properties, such as spirulina, quercetin, rosmarinic acid, and zinc to ameliorate or prevent gonadal damage caused by CP ( 2 , 8 , 40 , 68 ). However, we encountered few studies using GA, present in several natural sources including tea, fruits, red grape, nuts, and medicinal plants, as an antioxidant against ovarian damage ( 69 , 70 ). The present study evaluated the antioxidant effects of GA on CP-induced damage in the rat ovary. CP also exhibits suppressive effects on cell division, a phenomenon more marked in rapidly dividing cells. It can therefore cause loss of both body and organ weights ( 71 , 72 ). In an experiment involving female mice, 100 mg/kg CP was administered i.p. six times for two weeks, and a decrease in body weight was observed ( 15 , 73 ). In another study investigating the protective effects of resveratrol against CP-induced ovarian damage, the rats in the CP group received an initial dose of 50 mg/kg followed by daily doses of 8 mg/kg for 14 days. Significant decreases were subsequently detected in both body and ovarian weights ( 15 ). Decreases were also observed in body and ovarian weights in the CP group in the present study, although these were not statistically significant. We think that this may be due to CP being applied on more days in other studies and to dosage differences. Several studies have revealed that oxidative stress can activate various inflammatory and apoptotic pathways. Uncontrolled oxidative stress poses a severe threat to the female reproductive system, and rising oxidative stress has a deleterious impact on follicle development ( 72 ). Nair et al. (2020) reported that CP exhibits its toxicity by disrupting the antioxidant defense mechanism, thus encouraging apoptosis by triggering the proinflammatory process. They also reported that this led to degenerative damage in ovarian follicles ( 74 ). CP has also been reported to be capable of directly causing follicular degeneration and oocyte losses by impairing microvascularization in the corpus luteum and follicles in the ovary, DNA damage, and apoptosis ( 72 , 75 ). In the present study, CP caused a high level of follicular cell degeneration in ovarian tissue. Mazloom et al. suggested that GA is a potent antioxidant, anti-inflammatory, and natural polyphenol that increases antioxidant enzyme activities and reduces ovarian proinflammatory cytokine concentrations, oxidative DNA damage, and lipid peroxidation in an estradiol valerate-induced polycystic ovary phenotype rat model. Those authors also reported that GA may play an important role in reducing inflammatory disorders deriving from oxidative stress ( 76 ). Ayazoglu et al. showed that the application of GA to rat ovaries exposed to cisplatin reduced follicular degeneration, increased the numbers of primordial, primary, and secondary follicles, and reduced the numbers of atretic follicles ( 77 ). The administration of GA also reduced CP-derived follicular degeneration in the present study. Pu et al. (2023) induced a model of premature ovarian failure through the administration of CP and busulfan. Those authors reported a decrease in primordial, primary, and secondary follicles, and an increase in atretic follicle numbers, in their premature ovarian failure group ( 9 ). Similarly to the previous literature, we observed a decrease in primordial and unilaminar primary follicles in the CP group in this study, with an increase in atretic follicle numbers. Primordial follicle numbers increased significantly in the CP + GA group compared to the CP group. This may be attributable to GA preventing apoptosis. Although an increase in unilaminar primary follicle numbers and a decrease in those of atretic follicles were observed in our CP + GA group compared to the CP group, these were not statistically significant. Dos et al. (2023) investigated the protective activity of GA against the toxic effects of doxorubicin in the ovary, and reported decreased primordial, primary, secondary, and antral follicle numbers in the doxorubicin group, these losses being attenuated in the group receiving GA ( 10 ). Those findings are also consistent with the present study. DNA damage is a widely recognized cause of CP toxicity. Liu et al. (2019) showed that CP exhibits an apoptotic effect on ovarian tissue. They also reported that CP induced granulosa cell apoptosis, followed by oocyte death ( 72 ). Similarly in the present study, we detected an increase in the AI in the CP group. We think that this increase may have occurred secondary to the damage caused by CP to DNA, in addition to the oxidative stress induced by CP. Dos et al. investigated the protective effect of GA against the toxic effect of doxorubicin in ovarian tissue, and showed that GA reduced apoptosis in such tissue ( 10 ). Consistent with the literature, AI also decreased in our treatment group compared to the CP group. The evidence in the literature supports our findings concerning CP ( 74 , 78 , 79 ). A study of CP-induced premature ovarian failure reported decreased in the antioxidant parameters GPx, CAT, and SOD in a CP group, with an increase in the lipid peroxidation marker MDA ( 13 ). Hamzeh et al. reported that ovarian damage caused by CP was associated with increased ROS production ( 12 ). Tissue MDA increased in the CP group in the present study, while SOD and CAT activity in tissue decreased. Our findings confirm that CP can lead to ovarian damage by increasing ROS production. Ayazoğlu et al. (2023) reported that the application of GA in CP-induced ovarian damage resulted in a decrease in MDA and oxidative stress index levels and increases in total antioxidant status and CAT levels ( 77 ). Another study reported that GA administered as a protective agent in letrozole-induced polycystic ovary syndrome raised SOD and CAT levels while reducing those of MDA ( 80 ). Tissue MDA increased in the CP group in the present study, while decreasing in the treatment group compared to the damage group. SOD levels were significantly higher in the treatment group compared to the damage group, but similar to those in the control group. Tissue CAT levels rose significantly in the CP + GA and GA groups compared to both the CP and control groups. This shows that GA is capable of reducing the ovarian damage caused by CP by enhancing the activities of antioxidant enzymes such as SOD and CAT. The fact that CAT activity was significantly higher in the groups receiving GA compared to the other groups suggested that GA may have a powerful effect, especially in terms of increasing CAT activity. AMH is essentially produced by granulosa cells in secondary and antral follicles. It is an important negative regulator of the transition of the primordial follicle primordial into a primary follicle. It inhibits the activation of primordial follicles and reduces the sensitivity to follicle-stimulating hormone (FSH) of antral follicles, thus stabilizing a specific number of primordial follicles and the numbers of follicles developing in the ovary. Feng et al. observed a decrease in serum AMH in experimental subjects given CP compared to a control group and attributed that decrease to increasing apoptosis in granulosa cells ( 14 ). Zheng et al. (2021) also showed a decrease in serum AMH values in rats administered CP ( 13 ). In the present study, although CP yielded some degree of diminution of serum AMH levels, this was not statistically significant. However, CP caused a significant decrease in tissue AMH. We think that this may be attributable to the degeneration caused by CP in follicular cells. We encountered no studies investigating the effect of GA on AMH. Xu X et al. (2024) reported an increase in FSH and luteinizing hormone levels in rats with premature ovarian failure induced by means of 4-vimylcyclohexene diepoxide, and suppression of serum estradiol and AMH levels. They also reported that these findings in the damage group were significantly reversed with the application of Bu-Shen-Ning-Xin decoction (BSNXD), which contains six chemical components including GA. Finally, those authors observed an improvement in both irregular hormones and ovarian morphology following the administration of BSNXD ( 81 ). Chen et Tan (2020) reported significant increases in estradiol and AMH levels, ovarian volume, and antral follicle numbers following the application of BSNXD ( 81 , 82 ). In the present study, serum AMH levels in the GA group increased compared to the other groups. One of the limitations of this study is that although the number of subjects in the groups based on previous similar studies and in accordance with the 3R rule, the number of subjects was still low. Another is that the effects observed at histopathological examination were not confirmed by using biochemical markers (such as Caspase-3). Larger numbers of subjects should therefore be employed in future studies in order to enhance statistical significance, and biochemical markers should be included in the evaluation of antiapoptotic and anti-inflammatory effects. CONCLUSION In conclusion, CP has the potential to cause significant biochemical and histological impairments in ovarian tissue. However, GA can reduce CP-induced oxidative damage in the ovary. However, we think that this ameliorative effect can be better evaluated with the planning of new studies involving different doses and application durations. Declarations Conflict of interests No author has any potential or actual conflict of interest to disclose/None of the authors disclose any potential conflict of interest related to the the present article. Data sharing statement The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Funding This study was supported by the KTU Scientific Research Project Coordination Unit (project no. TTU-2021-9805). Author Contribution EKA, DÖO contributed substantially to the conception and design of the study. AKA, DÖO, EŞ, AA contributed substantially to the acquisition of data. AKA, DÖO contributed substantially to the analysis and interpretation of data. AKA, DÖO contributed to drafting the manuscript and critically revising the manuscript. Acknowledgment This study appeared as an oral presentation at the 16th National Congress of Histology and Embryology (niche 2024), 26–28 September, 2024, Sakarya, Türkiye. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8146263","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":554116907,"identity":"5a332cc8-b5ce-4c83-8081-4940eefe8ac0","order_by":0,"name":"Elif Koyun Alvuroğlu","email":"","orcid":"","institution":"Necip Fazıl City Hospital","correspondingAuthor":false,"prefix":"","firstName":"Elif","middleName":"Koyun","lastName":"Alvuroğlu","suffix":""},{"id":554116910,"identity":"8e382704-a106-40bc-8fa7-2352ae052eec","order_by":1,"name":"Derya Öztürk 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Alver","email":"","orcid":"","institution":"Karadeniz Technical University","correspondingAuthor":false,"prefix":"","firstName":"Ahmet","middleName":"","lastName":"Alver","suffix":""}],"badges":[],"createdAt":"2025-11-18 13:53:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8146263/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8146263/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":97667633,"identity":"be2802a3-a61a-4fc3-82b6-54c1917c80d9","added_by":"auto","created_at":"2025-12-08 09:23:56","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":54132,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-8146263/v1/c3d8f21b296a6b1b917d8739.docx"},{"id":97424370,"identity":"42b8723f-5527-4ff2-8356-9fafc65677d7","added_by":"auto","created_at":"2025-12-04 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08:55:38","extension":"xml","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":192343,"visible":true,"origin":"","legend":"","description":"","filename":"7b63d85035c445cf9f06c5de3b2607a81structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8146263/v1/f53f4468c54ffa8020769968.xml"},{"id":97424398,"identity":"00a7f5a5-79e0-43bb-b959-9921d9fa2097","added_by":"auto","created_at":"2025-12-04 08:55:38","extension":"html","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":209151,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8146263/v1/7eee85545b926ebb8fb7e8ba.html"},{"id":97666863,"identity":"da1b9372-4692-4e99-8d12-a3468e5b8ca5","added_by":"auto","created_at":"2025-12-08 09:22:16","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":250537,"visible":true,"origin":"","legend":"\u003cp\u003eOvarian tissue sections stained with H\u0026amp;E. The control group (A) exhibited a normal structure. Sections from the CP group (B) exhibited degeneration in follicular cells (red arrowhead). Sections from the CP+GA group (C) exhibited decreased follicular cell degeneration. The GA group (D) exhibited similar characteristics to those of the control group. All panels × 400\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8146263/v1/b0ec96f5605e977e1b49fb10.jpg"},{"id":97667809,"identity":"10b675ef-02b4-44c5-9c0b-a420d3531a90","added_by":"auto","created_at":"2025-12-08 09:24:17","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":392983,"visible":true,"origin":"","legend":"\u003cp\u003eOvarian tissue sections stained with Masson's trichrome. Collagen fiber (yellow notched arrow head) densities were similar in all the study groups. Control group (A), CP group (B), CP+GA group (C), GA group (D). All panels × 200\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8146263/v1/f9c8bae44dbb2f2bc65e6187.jpg"},{"id":97424371,"identity":"cbc062f1-a2e4-49b6-a082-d65da355ba45","added_by":"auto","created_at":"2025-12-04 08:55:38","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":210334,"visible":true,"origin":"","legend":"\u003cp\u003eOvarian tissue sections stained with PAS. The zona pellucida structure (blue arrow) was intact in all the study groups. All panels × 200\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8146263/v1/a681a99a36c61682ef70ba7b.jpg"},{"id":97667826,"identity":"2a366682-a995-4442-aabe-b452b7b7d2de","added_by":"auto","created_at":"2025-12-08 09:24:20","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":313190,"visible":true,"origin":"","legend":"\u003cp\u003eOvarian tissue sections stained with TUNEL. Normal cell (red notched arrow head), TUNEL (+) cell (blue arrow head). Control group (A), CP group (B), CP+GA group (), GA group. All panels × 400\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8146263/v1/d7849ddd048323d659d6d9e5.jpg"},{"id":97424379,"identity":"8f94dccb-e3ad-411c-a947-8e23773f10a2","added_by":"auto","created_at":"2025-12-04 08:55:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":151588,"visible":true,"origin":"","legend":"\u003cp\u003eMalondialdehyde(MDA), superoxide dismutase (SOD), Glutathione peroxidase (GPX), catalase (CAT), anti-müllerian hormone (AMH) levels of ovarian tissue and AMH levels of serum\u003c/p\u003e\n\u003cp\u003eᵃ p \u0026lt; 0.05 compare control group.\u003c/p\u003e\n\u003cp\u003eᵇ p \u0026lt; 0.05 compare CP group.\u003c/p\u003e\n\u003cp\u003eᶜ p \u0026lt; 0.05 compare GA group.\u003c/p\u003e\n\u003cp\u003eᵈ p \u0026lt; 0.05 compare CP+GA group.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8146263/v1/209deaa8c74d9abecd49e50e.png"},{"id":97677668,"identity":"8d543521-09e7-4e47-a17a-a6b4a86e7a89","added_by":"auto","created_at":"2025-12-08 09:53:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2345916,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8146263/v1/4160723e-aebb-4ed4-8ffa-50ef03856138.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of Gallic Acid on Cyclophosphamide-Induced Experimental Ovarian Injury in Rats","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eSignificant advances in the diagnosis and treatment of cancer have improved survival rates among children and young adults. However, the potential deleterious effects on reproductive functions of these therapies are now clearly established (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Exposure to chemotherapy is regarded as a risk factor for premature ovarian failure (POF) and leads to menstrual disorders, metabolic abnormalities, and infertility (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Various fertility-preserving methods, such as oocyte, embryo, and ovarian cortex cryopreservation are recommended in order to protect gametes from the gonadotoxic effects of chemotherapy or radiotherapy. However, the application of these methods can be limited by the patient\u0026rsquo;s age, pubertal status, disease, and emergency conditions (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCyclophosphamide (CP), one such chemotherapeutic, is an alkylating agent indicated for use in malignancies such as Hodgkin and non-Hodgkin lymphoma, lymphocytic lymphoma, Burkitt lymphoma, and multiple myeloma, and in autoimmune diseases such as multiple sclerosis (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). CP generally exhibits its antineoplastic effect through its metabolite phosphoramide mustard, which forms as a result of metabolism of the drug by the cytochrome P-450 enzyme in the liver (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Another metabolite, acrolein, increases the production of reactive oxygen species (ROS) by inhibiting the antioxidant system, thus giving rise to CP side-effects (MacAllister). CP has been shown to cause cardiotoxicity, urotoxicity, gonodal toxicity, hematological toxicity, and lung damage (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Studies have shown that the induction of oxidative stress in tissues may be one of the causes of this damage resulting from CP (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCyclophosphamide causes a decrease in primordial, primary, secondary, and antral follicles in the ovary, and an increase in atretic follicles (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). It is thought to cause irreversible follicle loss through the induction of apotosis in follicles and reducing microvascularization in the corpus luteum and follicles (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). CP causes decreased antioxidant enzyme activity in the ovary and an increase in ROS and malondialdehyde levels (MDA) (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). In addition to a decrease in serum anti-m\u0026uuml;llerian hormone (AMH) values, CP also causes significant diminutions in body and ovarian weights (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Recent studies have shown that CP causes ovarian damage by increasing granulosa cell apoptosis (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Studies have suggested that granulosa cell apoptosis triggered by an impaired antioxidant mechanism and excessive ROS production represents the main cause of follicular atresia (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). The primordial follicle pool is exhausted in association with CP-induced damage, and early menopause and infertility are seen as a result (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGallic acid is a polyphenolic benzoic acid known as 3,4,5 trihydroxybenzoic acid (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Its chemical formula is C6H2(OH)3COOH (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). It is widely present in hazelnuts, fruits such as cherries, raspberries, pomegranates, and grapes, and in vegetables. It is also found in onion, green tea, and honey. Experimental and theoretical studies have identified it as a powerful antioxidant (\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). It is also an important component of plants with anticancer properties, and its derivatives have been shown to induce cancer cell apoptosis (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). This property of GA is associated with its role as a pro-oxidant, meaning it is both an antioxidant and pro-oxidant (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Due to the three alcoholic OH groups found in GA, it produces radicals for the redox reaction and acts as a radical scavenger (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). A powerful antioxidant, GA also exhibits anticancer, antimicrobial, antiviral, anti-inflammatory, antidiabetic, and antihypertensive properties and acts against neurodegenerative disorders and aging (\u003cspan additionalcitationids=\"CR28 CR29 CR30 CR31 CR32 CR33 CR34\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). It is an important molecule in biomedical practice and drug design (\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThis study examined the potential ameliorating effects of GA against ovarian damage that may develop in association with the use of CP. We encountered no previous study investigating the protective or therapeutic effect of GA, a potent antioxidant, in CP-induced ovarian damage. We think that this study will contribute to filling this gap in the literature and will be if use to future studies on the subject.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAnimals, Experimental procedures\u003c/h2\u003e\u003cp\u003eApproval for the study was granted by the Karadeniz Techincal University (KTU) animal experiments ethical committee (no. 2021/46 dated 21.09.2021). All procedures were performed in accordance with the principles of the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Thirty-two female Sprague Dawley rats were used in the research. These were procured from the KTU Medical Faculty Surgical Application and Research Center and were housed in a 12-hour dark:light cycle at 50% humidity and a temperature of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg; C. The rats were housed in standard type III cages throughout the experiment with ad libitum access to standard rat chow (Bayramoğlu Yem ve Un Sanayi Tic. A.Ş. Erzurum, T\u0026uuml;rkiye) and tap water. All the experimental procedures, animal care, and sacrifice were carried out in the KTU Surgical Application and Research Center in order to avoid environmental stress and adaptation problems.\u003c/p\u003e\u003cp\u003eThe rats were divided into four groups. No procedure was performed on the control group (n:8). The CP group (n:8) received intraperitoneal (i.p.) CP (Endoxan, Baxter-Eczacıbaşı İla\u0026ccedil; Pazarlama, Istanbul, T\u0026uuml;rkiye) at a dose of 150 mg/kg on day 7 of the experiment. The GA group (n:8) received 20 mg/kg GA (Sigma-Aldrich Chemie Gmbh) (1 g GA dissolved in 100 ml saline solution) by oral gavage for seven days from day 1 of the experiment. The CP\u0026thinsp;+\u0026thinsp;GA group (n:8) received 20 mg/kg GA via oral gavage for seven days from day 1 of the experiment and 150 mg/kg CP i.p. on day 7 of the experiment (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). All rats were weighed on days 1 and 8 of the experiment. The animals were finally sacrificed by exsanguination at the end of the experiment (day 8) under anesthesia (ketamine 90 mg/kg). Blood specimens were collected and placed into ethylene diamine acetic acid (EDTA) tubes for biochemical parameter analysis. A midline incision was promptly made to the lower abdomen region. The right ovary was first removed, weighed, and placed into 10% formalin solution for tissue fixation. The left ovaries were placed into Eppendorf tubes and stored at -80\u0026deg; C for biochemical investigation.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eHistopathological Analysis\u003c/h3\u003e\n\u003cp\u003eThe right ovaries were fixed in 10% formaldehyde solution. A routine tissue processing program was applied on the tissue processing device (Thermo Scientific Excelsior AS, UK). After processing, the tissues were removed from the cassettes, embedded in paraffin blocks on a blocking device (Leica HistoCare Arcadia H, China), and cooled. Five micron-thick sections were ten taken using a fully automatic microtome (Leica RM 2255, Germany).\u003c/p\u003e\u003cp\u003eSerial 5-\u0026micro;m sections were taken from the right ovaries. Sections collected at 50 \u0026micro;m intervals were stained with hematoxylin-eosin H\u0026amp;E using an automatic staining device (Leica Autostainer XL, Germany) (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). One part of the other sections was stained with Masson\u0026rsquo;s trichrome \u003cb\u003e(\u003c/b\u003eMasson Trichrome Staining Kit, GBL, T\u0026uuml;rkiye), Periodic-Acid Schiff (PAS) \u003cb\u003e(\u003c/b\u003ePAS McManus Staining Kit, GBL, T\u0026uuml;rkiye), and deoxyuridine triphosphate nick end labeling assay (TUNEL) (in situ cell death detection POD kit, Roche, 11684817910, Berlin, Germany) in line with the manufacturer\u0026rsquo;s instructions. All histopathological examinations were carried out using a light microscope (Olympus, BX51, and Japan) equipped with a DP 71 (Olympus, Japan) digital camera in the KTU Medical Faculty Department of Histology and Embryology. The sections were photographed, the images being transferred to a digital environment.\u003c/p\u003e\n\u003ch3\u003eOvarian Follicle Evaluation\u003c/h3\u003e\n\u003cp\u003ePrimordial follicles, unilaminar primary follicles, multilaminar primary follicles, secondary follicles, Graafian follicles, and atretic follicles were counted in the entire area of ​​the 5 \u0026micro;m H\u0026amp;E-stained sections taken at 50 \u0026micro;m intervals for each ovary, according to the definitions given below (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Primordial follicle: The primary oocyte is covered by a single layer of flat follicle cells. Unilaminar primary follicle: The primary oocyte is surrounded by a single layer of cubic follicle cells. Multilaminar primary follicle: The primary oocyte is surrounded by multilayer cubic follicle cells (zona granulosa). Theca externa and theca interna layers may be present around the zona granulosa. Secondary follicle: The primary oocyte is covered by multilayer cubic follicle cells (zona granulosa). The theca externa and interna layers are present around the zona granulosa. The zona granulosa contains spaces filled with follicular fluid. Graafian follicle: Corona radiata and cumulus cells are present around the eccentrically located primary oocyte. The spaces in the zona granulosa combine to form a single space. Theca layers surround the zona granulosa. Atretic follicle: Apoptosis in granulosa cells in large follicles. Invasion of the granulosa layer by macrophages, neutrophils, and vascularized connective tissue cords. Granulosa cell shedding into the antrum. Follicle and zona pellucida collapse. Vitreous membrane appearance (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eVascular congestion, follicular cell degeneration, hemorrhage around the corpus luteum, germinal epithelium degeneration, and theca layer thinning were evaluated and scored at histopathological examinations (0: no damage; 1: mild damage, focal and mild changes; 2: moderate damage, highly focused, significant changes, and 3: severe damage, widespread changes) (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). The structure of the zona pellucida and basal membrane was evaluated in sections stained with PAS, and connective tissue and collagen density in sections stained with Masson\u0026rsquo;s trichrome.\u003c/p\u003e\n\u003ch3\u003eTUNEL staining\u003c/h3\u003e\n\u003cp\u003eApoptosis in ovarian tissue was evaluated on TUNEL-stained sections. Apoptotic and normal cells were counted in the granulosa and theca cells of follicles in five randomly selected areas in a section from each subject. Cells with brown-stained nuclei were defined as TUNEL (+) apoptotic cells (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). The apoptotic index (AI) was calculated using the formula number of TUNEL (+) cells /total number of cells x 100) (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eBiochemical Analysis\u003c/h3\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eTissue Glutathione Peroxidase (GPX) Measurement\u003c/h2\u003e\u003cp\u003eGPX activity in supernatants obtained from ovarian tissues was determined using a commercial ELISA kit (catalog no. E1242Ra, BT LAB, Zhejiang, China) according to the manufacturer\u0026rsquo;s instructions. The results were expressed as ng/mg protein.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAMH Measurement\u003c/h3\u003e\n\u003cp\u003eAMH levels in serum specimens and supernatants obtained from ovarian tissues were determined using a commercial ELISA it (catalog no. BT LAB, Zhejiang, China) in line with the manufacturer\u0026rsquo;s instructions. The results were expressed as ng/mL for serum specimens and ng/mg for tissue specimens.\u003c/p\u003e\n\u003ch3\u003eTissue Superoxide Dismutase (SOD) Activity Measurement\u003c/h3\u003e\n\u003cp\u003eSOD enzyme activity was determined using a modification of the method developed by Sun and Oberley (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). This method is based on measuring the absorbance at 560 nm of the purple formazan molecule that forms as a result of the reduction of nitroblue tetrazolium by O2.- generated by the xanthine-xanthine oxidase system. SOD activities determined were divided by the total amount of protein, the results being calculated as U/mg protein.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eCatalase (CAT) Enzyme Activity Measurement\u003c/h2\u003e\u003cp\u003eA modified version of the Aebi method was employed to determine CAT activity, which catalyzes the hydrolysis of hydrogen peroxide into water and oxygen (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). This method is based on the decrease in absorbance at 240 nm as a result of the breakdown of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e by CAT. Care was taken to ensure that the decrease in absorbance in 30 seconds was no less than 0.03 and no greater than 0.2. The first order reaction rate constant (k) was employed as the unit for catalase activity. Activity was calculated using the equation below for a 10-second time interval.\u003c/p\u003e\u003cp\u003eA1\u0026thinsp;=\u0026thinsp;Initial absorbance, A2\u0026thinsp;=\u0026thinsp;absorbance after 10 seconds, k=(2.3/10) x log (A1/A2) seconds\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eTissue Malondialdehyde (MDA) Measurement\u003c/h2\u003e\u003cp\u003eMDA was measured using the method described by Mihara and Uchiyama (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). This is based on measuring the absorbance at 523 nm of the color formed by MDA with thiobarbituric acid in an acidic environment. The results were expressed as nmol/g protein.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eProtein Assay\u003c/h2\u003e\u003cp\u003eProtein assay in the tissue homogenates was performed using the Bradford method (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). This is based on the principle that Coomassie Brilliant Blue G250, an organic dye, binds to proteins in a phosphoric acid environment, the resulting blue complex exhibiting maximum absorbance at 600 nm. The results were expressed as mg/ml.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eThe study data were analyzed on SPSS version 23.0 software and were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Kruskal Wallis analysis of variance was applied for group comparisons. The Mann Whitney U test was used with post-hoc Bonferroni correction. p values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were regarded as statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eBody and Ovarian Weight Findings\u003c/h2\u003e\u003cp\u003eMean plus standard deviation values for body and ovarian weights on days 1 and 8 of the experiment are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e0. No statistically significant difference was observed between the groups in terms of rat body weights at the end of the experiment. The rats\u0026rsquo; right ovaries were compared, and no significant difference was determined between them (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBody and ovarian weight\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eK\u003c/p\u003e\u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCP\u003c/p\u003e\u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGA\u003c/p\u003e\u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCP\u0026thinsp;+\u0026thinsp;GA\u003c/p\u003e\u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBody Weight Day 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e245\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e247,5\u0026thinsp;\u0026plusmn;\u0026thinsp;27,9\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e225\u0026thinsp;\u0026plusmn;\u0026thinsp;36,6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e255\u0026thinsp;\u0026plusmn;\u0026thinsp;37\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBody Weight 8th Day\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e252,5\u0026thinsp;\u0026plusmn;\u0026thinsp;20,7\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e235,6\u0026thinsp;\u0026plusmn;\u0026thinsp;31,6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e227\u0026thinsp;\u0026plusmn;\u0026thinsp;15\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e246\u0026thinsp;\u0026plusmn;\u0026thinsp;27,3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOvarian Weight\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e0,073\u0026thinsp;\u0026plusmn;\u0026thinsp;0,017\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0,068\u0026thinsp;\u0026plusmn;\u0026thinsp;0,019\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e0,061\u0026thinsp;\u0026plusmn;\u0026thinsp;0,008\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0,061\u0026thinsp;\u0026plusmn;\u0026thinsp;0,008\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eData are given as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD). K: Control group, CP: Cyclophosphamide group, GA: Gallic acid, CP\u0026thinsp;+\u0026thinsp;GA: Cyclophosphamide\u0026thinsp;+\u0026thinsp;Gallic acid\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eHistopathological analysis\u003c/h2\u003e\u003cp\u003ePrimordial follicle numbers decreased significantly in the CP group compared to the control group (p\u0026thinsp;=\u0026thinsp;0.015), while increasing significantly in the CP\u0026thinsp;+\u0026thinsp;GA group compared to the CP group (p\u0026thinsp;=\u0026thinsp;0.029). Unilaminar primary follicle numbers also decreased in the CP group compared to the control and GA groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and p\u0026thinsp;=\u0026thinsp;0.008, respectively), and in the CP\u0026thinsp;+\u0026thinsp;GA group compared to the control group (p\u0026thinsp;=\u0026thinsp;0.009). No significant intergroup differences were observed in multilaminar primary follicle (p\u0026thinsp;=\u0026thinsp;0.065), secondary follicle (p\u0026thinsp;=\u0026thinsp;0.72), or Graafian follicle (p\u0026thinsp;=\u0026thinsp;0.44) numbers. Atretic follicle numbers increased significantly in the CP group compared to the control and GA groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for both). Atretic follicle numbers were also higher in the CP\u0026thinsp;+\u0026thinsp;GA group than in the control and GA (p\u0026thinsp;=\u0026thinsp;0.009 and p\u0026thinsp;=\u0026thinsp;0.008, respectively) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eFollicle numbers\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eK\u003c/p\u003e\u003cp\u003eOrt\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCP\u003c/p\u003e\u003cp\u003eOrt\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGA\u003c/p\u003e\u003cp\u003eOrt\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCP\u0026thinsp;+\u0026thinsp;GA\u003c/p\u003e\u003cp\u003eOrt\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePrimordial follicle\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e128,12\u0026thinsp;\u0026plusmn;\u0026thinsp;21,37\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e101,25\u0026thinsp;\u0026plusmn;\u0026thinsp;14,17\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e119\u0026thinsp;\u0026plusmn;\u0026thinsp;7,48\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e123,71\u0026thinsp;\u0026plusmn;\u0026thinsp;18,55\u003c/b\u003e\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUnilaminar primary ollicle\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e65,62\u0026thinsp;\u0026plusmn;\u0026thinsp;5,62\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e41,87\u0026thinsp;\u0026plusmn;\u0026thinsp;13,06\u003c/b\u003e\u003csup\u003e\u003cb\u003ea,c\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e59,3\u0026thinsp;\u0026plusmn;\u0026thinsp;10,4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e51\u0026thinsp;\u0026plusmn;\u0026thinsp;9,98\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMultilaminar primary follicle\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e34,37\u0026thinsp;\u0026plusmn;\u0026thinsp;2,77\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e34,25\u0026thinsp;\u0026plusmn;\u0026thinsp;4,74\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e34,5\u0026thinsp;\u0026plusmn;\u0026thinsp;1,04\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e34\u0026thinsp;\u0026plusmn;\u0026thinsp;1,41\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSecondary follicle\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e31,37\u0026thinsp;\u0026plusmn;\u0026thinsp;6,9\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e29,37\u0026thinsp;\u0026plusmn;\u0026thinsp;5,8\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e32,66\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e28,42\u0026thinsp;\u0026plusmn;\u0026thinsp;3,55\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGraafian follicle\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e31,37\u0026thinsp;\u0026plusmn;\u0026thinsp;5,47\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e30,75\u0026thinsp;\u0026plusmn;\u0026thinsp;6,94\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e31,33\u0026thinsp;\u0026plusmn;\u0026thinsp;5,08\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e33,85\u0026thinsp;\u0026plusmn;\u0026thinsp;2,73\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAtretic follicle\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e312,75\u0026thinsp;\u0026plusmn;\u0026thinsp;41,44\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e438,75\u0026thinsp;\u0026plusmn;\u0026thinsp;69,33\u003c/b\u003e\u003csup\u003e\u003cb\u003ea,c\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e261,83\u0026thinsp;\u0026plusmn;\u0026thinsp;31,16\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e397,57\u0026thinsp;\u0026plusmn;\u0026thinsp;63,66\u003c/b\u003e\u003csup\u003e\u003cb\u003ea,c\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eData are given as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD). K: Control group, CP: Cyclophosphamide group, GA: Gallic acid, CP\u0026thinsp;+\u0026thinsp;GA: Cyclophosphamide\u0026thinsp;+\u0026thinsp;Gallic acid\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eᵃ p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 compare control group.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eᵇ p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 compare CP group.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eᶜ p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 compare GA group.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe cortex and medulla in the control group exhibited a normal appearance. Normal morphologies were also observed in the germinal epithelium, follicular cells, theca layers, and corpus luteum. Follicular cell degeneration was significantly greater in the CP compared to the control and GA groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Follicular cell degeneration was lower in the CP\u0026thinsp;+\u0026thinsp;GA group compared to the CP group (p\u0026thinsp;=\u0026thinsp;0.003). Follicular cell degeneration was higher in the CP\u0026thinsp;+\u0026thinsp;GA group than in the control and GA groups (p\u0026thinsp;=\u0026thinsp;0.005 and p\u0026thinsp;=\u0026thinsp;0.015, respectively). The morphology of the GA group was similar to that of the control group. No significant differences between the groups were observed in terms of vascular congestion, hemorrhage around the corpus luteum, germinal epithelium degeneration, or thinning of the theca layer. No marked findings of inflammation were present in any group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eConnective tissue architecture and collagen density were similar in all the groups. No difference was also determined in terms of fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The zona pellucida exhibited a normal architecture in all the groups. PAS positivity in the basal membrane surrounding the follicles exhibited the same intensity in all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eApoptotic Index (AI)\u003c/h2\u003e\u003cp\u003eThe AI was higher in the CP group than in the control group (p\u0026thinsp;=\u0026thinsp;0.032). However, the AI in the CP\u0026thinsp;+\u0026thinsp;GA group was lower than that in the CP group (p\u0026thinsp;=\u0026thinsp;0.032) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eHistopathological analysis results and apoptotic index values\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eK\u003c/p\u003e\u003cp\u003eOrt\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCP\u003c/p\u003e\u003cp\u003eOrt\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGA\u003c/p\u003e\u003cp\u003eOrt\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCP\u0026thinsp;+\u0026thinsp;GA\u003c/p\u003e\u003cp\u003eOrt\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVascular Congestion\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e1,25\u0026thinsp;\u0026plusmn;\u0026thinsp;0,46\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e1,5\u0026thinsp;\u0026plusmn;\u0026thinsp;0,53\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e1,12\u0026thinsp;\u0026plusmn;\u0026thinsp;0,35\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e1,25\u0026thinsp;\u0026plusmn;\u0026thinsp;0,46\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFollicular Cell Degeneration\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e0,25\u0026thinsp;\u0026plusmn;\u0026thinsp;0,46\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e2,3\u0026thinsp;\u0026plusmn;\u0026thinsp;0,51\u003c/b\u003e\u003csup\u003e\u003cb\u003ea,c\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e0,37\u0026thinsp;\u0026plusmn;\u0026thinsp;0,51\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e1,25\u0026thinsp;\u0026plusmn;\u0026thinsp;0,46\u003c/b\u003e\u003csup\u003e\u003cb\u003ea,b,c\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHemorrhage Around The Corpus Luteum\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e0,75\u0026thinsp;\u0026plusmn;\u0026thinsp;0,46\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e1,25\u0026thinsp;\u0026plusmn;\u0026thinsp;0,35\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e1,12\u0026thinsp;\u0026plusmn;\u0026thinsp;0,35\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e1,25\u0026thinsp;\u0026plusmn;\u0026thinsp;0,46\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGerminal Epithelium Degeneration\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e0,87\u0026thinsp;\u0026plusmn;\u0026thinsp;0,35\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e1,12\u0026thinsp;\u0026plusmn;\u0026thinsp;0,35\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e0,87\u0026thinsp;\u0026plusmn;\u0026thinsp;0,35\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e1,12\u0026thinsp;\u0026plusmn;\u0026thinsp;0,35\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eThinning Of The Theca Layer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e0,62\u0026thinsp;\u0026plusmn;\u0026thinsp;0,51\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e1,12\u0026thinsp;\u0026plusmn;\u0026thinsp;0,35\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e0,62\u0026thinsp;\u0026plusmn;\u0026thinsp;0,51\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e1,25\u0026thinsp;\u0026plusmn;\u0026thinsp;0,46\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAI (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e32,78\u0026thinsp;\u0026plusmn;\u0026thinsp;3,57\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e46,8\u0026thinsp;\u0026plusmn;\u0026thinsp;8,66\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e33,34\u0026thinsp;\u0026plusmn;\u0026thinsp;9,03\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e33,48\u0026thinsp;\u0026plusmn;\u0026thinsp;6,79\u003c/b\u003e\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eData are given as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD).K: Kontrol grubu, CP: Siklofosfamid grubu, GA: Gallik asit, CP\u0026thinsp;+\u0026thinsp;GA: Siklofosfamid\u0026thinsp;+\u0026thinsp;Gallik asit\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eᵃ p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 compare control group.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eᵇ p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 compare CP group.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eᶜ p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 compare GA group.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eBiochemical analysis results\u003c/h2\u003e\u003cp\u003eTissue MDA levels were higher in the CP group than in the control and GA groups (p\u0026thinsp;=\u0026thinsp;0,003 and p\u0026thinsp;=\u0026thinsp;0.001, respectively). However, lower MDA levels were observed in the CP\u0026thinsp;+\u0026thinsp;GA group compared to the CP group (p\u0026thinsp;=\u0026thinsp;0.002). Tissue SOD activity decreased in the CP group compared to the control and GA groups (p\u0026thinsp;=\u0026thinsp;0.003). SOD activity in the CP\u0026thinsp;+\u0026thinsp;GA group increased compared to the CP group but was at a similar level to that in the control group (p\u0026thinsp;=\u0026thinsp;0.006). No difference was observed between the groups\u0026rsquo; tissue GPX values. Tissue CAT activity was lower in the CP group than in the control group (p\u0026thinsp;=\u0026thinsp;0.038). Tissue CAT activity increased in the CP\u0026thinsp;+\u0026thinsp;GA group compared to the control and CP groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Tissue CAT activity also increased in the GA group compared to the control and CP groups (p\u0026thinsp;=\u0026thinsp;0.001). Tissue AMH values decreased in the CP group compared to the control group (p\u0026thinsp;=\u0026thinsp;0.015). Serum AMH values were also higher in the GA group than in the control, CP, and CP\u0026thinsp;+\u0026thinsp;GA groups (p\u0026thinsp;=\u0026thinsp;0.003, p\u0026thinsp;=\u0026thinsp;0.003, and p\u0026thinsp;=\u0026thinsp;0.022, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOne of the oldest anti-cancer drugs, CP is also used in the treatment of various epithelial tumors, such as breast, ovarian, and small cell lung cancers in addition to hematological malignancies such as lymphoma and leukemia (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). CP itself is a prodrug, metabolized in the liver by cytochrome P-450 to form 4-hydroxycyclophosphamide, which is subsequently converted to phosphoramide mustard and acrolein. Phosphoramide mustard is a primary active metabolite that inhibits DNA replication by alkylating DNA (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). Phosphoramide mustard also affects the mitochondria, resulting in a decreased transmembrane potential and cytosolic cytochrome c accumulation (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). The effects of CP are independent of the cell cycle. In addition, as with all alkylating agents, rapidly proliferating cells are most sensitive to CP (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDespite its pharmacological benefits, CP has been linked to gonadal toxicity (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). The reason for the gonadal toxicity caused by CP is still unclear, although the literature seems to support the idea that CP causes severe oxidative stress, nitrative stress inflammation, apoptosis, and genomic changes (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). CP causes testis damage in males by reducing sperm concentration and motility, testosterone levels, and testicular antioxidant capacity and increasing abnormal sperm production (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). Experimental studies have shown that CP causes a decrease in both testis and body weights (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). The application of CP also causes significant damage to Sertoli cells, alters the gene expression of several important enzymes, causes musculoskeletal damage, and adversely impacts on spermatozoa growth, proliferation, and differentiation (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e). In women, CP causes marked dose-dependent damage to the ovaries (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e). The ovarian damage induced by CP has been linked to facilitation of granulosa cell apoptosis (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). The existing evidence shows that CP exerts a direct effect on the follicles, lading to primordial follicle depletion following exposure to it (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e). CP is the first chemotherapeutic drug to be linked to amenorrhea, premature ovarian failure, and ovarian function disorder (\u003cspan additionalcitationids=\"CR64\" citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e). It also directly damages DNA, induces follicular apoptosis, and produces ROS harmful to ovarian cells (\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSeveral studies in the literature have used different substances with antioxidant properties, such as spirulina, quercetin, rosmarinic acid, and zinc to ameliorate or prevent gonadal damage caused by CP (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e). However, we encountered few studies using GA, present in several natural sources including tea, fruits, red grape, nuts, and medicinal plants, as an antioxidant against ovarian damage (\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e). The present study evaluated the antioxidant effects of GA on CP-induced damage in the rat ovary.\u003c/p\u003e\u003cp\u003eCP also exhibits suppressive effects on cell division, a phenomenon more marked in rapidly dividing cells. It can therefore cause loss of both body and organ weights (\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e). In an experiment involving female mice, 100 mg/kg CP was administered i.p. six times for two weeks, and a decrease in body weight was observed (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e). In another study investigating the protective effects of resveratrol against CP-induced ovarian damage, the rats in the CP group received an initial dose of 50 mg/kg followed by daily doses of 8 mg/kg for 14 days. Significant decreases were subsequently detected in both body and ovarian weights (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Decreases were also observed in body and ovarian weights in the CP group in the present study, although these were not statistically significant. We think that this may be due to CP being applied on more days in other studies and to dosage differences.\u003c/p\u003e\u003cp\u003eSeveral studies have revealed that oxidative stress can activate various inflammatory and apoptotic pathways. Uncontrolled oxidative stress poses a severe threat to the female reproductive system, and rising oxidative stress has a deleterious impact on follicle development (\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e). Nair et al. (2020) reported that CP exhibits its toxicity by disrupting the antioxidant defense mechanism, thus encouraging apoptosis by triggering the proinflammatory process. They also reported that this led to degenerative damage in ovarian follicles (\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e). CP has also been reported to be capable of directly causing follicular degeneration and oocyte losses by impairing microvascularization in the corpus luteum and follicles in the ovary, DNA damage, and apoptosis (\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e). In the present study, CP caused a high level of follicular cell degeneration in ovarian tissue.\u003c/p\u003e\u003cp\u003eMazloom et al. suggested that GA is a potent antioxidant, anti-inflammatory, and natural polyphenol that increases antioxidant enzyme activities and reduces ovarian proinflammatory cytokine concentrations, oxidative DNA damage, and lipid peroxidation in an estradiol valerate-induced polycystic ovary phenotype rat model. Those authors also reported that GA may play an important role in reducing inflammatory disorders deriving from oxidative stress (\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e). Ayazoglu et al. showed that the application of GA to rat ovaries exposed to cisplatin reduced follicular degeneration, increased the numbers of primordial, primary, and secondary follicles, and reduced the numbers of atretic follicles (\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e). The administration of GA also reduced CP-derived follicular degeneration in the present study.\u003c/p\u003e\u003cp\u003ePu et al. (2023) induced a model of premature ovarian failure through the administration of CP and busulfan. Those authors reported a decrease in primordial, primary, and secondary follicles, and an increase in atretic follicle numbers, in their premature ovarian failure group (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Similarly to the previous literature, we observed a decrease in primordial and unilaminar primary follicles in the CP group in this study, with an increase in atretic follicle numbers. Primordial follicle numbers increased significantly in the CP\u0026thinsp;+\u0026thinsp;GA group compared to the CP group. This may be attributable to GA preventing apoptosis. Although an increase in unilaminar primary follicle numbers and a decrease in those of atretic follicles were observed in our CP\u0026thinsp;+\u0026thinsp;GA group compared to the CP group, these were not statistically significant. Dos et al. (2023) investigated the protective activity of GA against the toxic effects of doxorubicin in the ovary, and reported decreased primordial, primary, secondary, and antral follicle numbers in the doxorubicin group, these losses being attenuated in the group receiving GA (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Those findings are also consistent with the present study.\u003c/p\u003e\u003cp\u003eDNA damage is a widely recognized cause of CP toxicity. Liu et al. (2019) showed that CP exhibits an apoptotic effect on ovarian tissue. They also reported that CP induced granulosa cell apoptosis, followed by oocyte death (\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e). Similarly in the present study, we detected an increase in the AI in the CP group. We think that this increase may have occurred secondary to the damage caused by CP to DNA, in addition to the oxidative stress induced by CP. Dos et al. investigated the protective effect of GA against the toxic effect of doxorubicin in ovarian tissue, and showed that GA reduced apoptosis in such tissue (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Consistent with the literature, AI also decreased in our treatment group compared to the CP group.\u003c/p\u003e\u003cp\u003eThe evidence in the literature supports our findings concerning CP (\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e). A study of CP-induced premature ovarian failure reported decreased in the antioxidant parameters GPx, CAT, and SOD in a CP group, with an increase in the lipid peroxidation marker MDA (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Hamzeh et al. reported that ovarian damage caused by CP was associated with increased ROS production (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Tissue MDA increased in the CP group in the present study, while SOD and CAT activity in tissue decreased. Our findings confirm that CP can lead to ovarian damage by increasing ROS production.\u003c/p\u003e\u003cp\u003eAyazoğlu et al. (2023) reported that the application of GA in CP-induced ovarian damage resulted in a decrease in MDA and oxidative stress index levels and increases in total antioxidant status and CAT levels (\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e). Another study reported that GA administered as a protective agent in letrozole-induced polycystic ovary syndrome raised SOD and CAT levels while reducing those of MDA (\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e). Tissue MDA increased in the CP group in the present study, while decreasing in the treatment group compared to the damage group. SOD levels were significantly higher in the treatment group compared to the damage group, but similar to those in the control group. Tissue CAT levels rose significantly in the CP\u0026thinsp;+\u0026thinsp;GA and GA groups compared to both the CP and control groups. This shows that GA is capable of reducing the ovarian damage caused by CP by enhancing the activities of antioxidant enzymes such as SOD and CAT. The fact that CAT activity was significantly higher in the groups receiving GA compared to the other groups suggested that GA may have a powerful effect, especially in terms of increasing CAT activity.\u003c/p\u003e\u003cp\u003eAMH is essentially produced by granulosa cells in secondary and antral follicles. It is an important negative regulator of the transition of the primordial follicle primordial into a primary follicle. It inhibits the activation of primordial follicles and reduces the sensitivity to follicle-stimulating hormone (FSH) of antral follicles, thus stabilizing a specific number of primordial follicles and the numbers of follicles developing in the ovary. Feng et al. observed a decrease in serum AMH in experimental subjects given CP compared to a control group and attributed that decrease to increasing apoptosis in granulosa cells (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Zheng et al. (2021) also showed a decrease in serum AMH values in rats administered CP (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). In the present study, although CP yielded some degree of diminution of serum AMH levels, this was not statistically significant. However, CP caused a significant decrease in tissue AMH. We think that this may be attributable to the degeneration caused by CP in follicular cells.\u003c/p\u003e\u003cp\u003eWe encountered no studies investigating the effect of GA on AMH. Xu X et al. (2024) reported an increase in FSH and luteinizing hormone levels in rats with premature ovarian failure induced by means of 4-vimylcyclohexene diepoxide, and suppression of serum estradiol and AMH levels. They also reported that these findings in the damage group were significantly reversed with the application of Bu-Shen-Ning-Xin decoction (BSNXD), which contains six chemical components including GA. Finally, those authors observed an improvement in both irregular hormones and ovarian morphology following the administration of BSNXD (\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e). Chen et Tan (2020) reported significant increases in estradiol and AMH levels, ovarian volume, and antral follicle numbers following the application of BSNXD (\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e). In the present study, serum AMH levels in the GA group increased compared to the other groups.\u003c/p\u003e\u003cp\u003eOne of the limitations of this study is that although the number of subjects in the groups based on previous similar studies and in accordance with the 3R rule, the number of subjects was still low. Another is that the effects observed at histopathological examination were not confirmed by using biochemical markers (such as Caspase-3). Larger numbers of subjects should therefore be employed in future studies in order to enhance statistical significance, and biochemical markers should be included in the evaluation of antiapoptotic and anti-inflammatory effects.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eIn conclusion, CP has the potential to cause significant biochemical and histological impairments in ovarian tissue. However, GA can reduce CP-induced oxidative damage in the ovary. However, we think that this ameliorative effect can be better evaluated with the planning of new studies involving different doses and application durations.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflict of interests\u003c/h2\u003e\u003cp\u003eNo author has any potential or actual conflict of interest to disclose/None of the authors disclose any potential conflict of interest related to the the present article.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eData sharing statement\u003c/h2\u003e\u003cp\u003eThe data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis study was supported by the KTU Scientific Research Project Coordination Unit (project no. TTU-2021-9805).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eEKA, D\u0026Ouml;O contributed substantially to the conception and design of the study. AKA, D\u0026Ouml;O, EŞ, AA contributed substantially to the acquisition of data. AKA, D\u0026Ouml;O contributed substantially to the analysis and interpretation of data. AKA, D\u0026Ouml;O contributed to drafting the manuscript and critically revising the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgment\u003c/h2\u003e\u003cp\u003eThis study appeared as an oral presentation at the 16th National Congress of Histology and Embryology (niche 2024), 26\u0026ndash;28 September, 2024, Sakarya, T\u0026uuml;rkiye.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSonigo C, Beau I, Binart N, Grynberg M. The impact of chemotherapy on the ovaries: molecular aspects and the prevention of ovarian damage. 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J N Chin Med. 2020;52:76\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-womens-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bmwh","sideBox":"Learn more about [BMC Women's Health](http://bmcwomenshealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bmwh/default.aspx","title":"BMC Women's Health","twitterHandle":"","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Ovarium, toxicology, gallic acid, cyclophosphamide, apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-8146263/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8146263/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eThe potential harmful effects of cancer treatments on reproductive function have now been clearly established. Exposure to chemotherapy is considered a risk factor for premature ovarian failure and causes infertility. This study investigated the prophylactic effects of gallic acid (GA) against cyclophosphamide(CP)-induced ovarian damage in rats.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eNo procedure was applied to the control group. The CP group received 150mg/kg CP via the intraperitoneal (i.p.) route on day 7 of the experiment. The GA group received 20mg/kg GA daily for seven days from day 1 of the experiment via oral gavage. The CP\u0026thinsp;+\u0026thinsp;GA received 20mg/kg GA daily for seven days from day 1 of the experiment via oral gavage and 150mg/kg i.p. CA on day 7 of the experiment. Histopathological examination of ovarian tissues and follicle counting were performed. Glutathione peroxidase, superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), tissue anti-m\u0026uuml;llerian hormone (AMH), and serum AMH levels were examined at biochemical investigation.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eCP caused follicular cell degeneration, increased the apoptotic index, reduced the numbers of primordial and unilaminar primary follicle cells, and increased the numbers of atretic follicles (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Follicular cell degeneration and the apoptotic index decreased while the numbers of primordial follicles increased in the CP\u0026thinsp;+\u0026thinsp;GA group compared to the CP group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). CAT, SOD and tissue AMH decreased, while MDA levels increased in the CP group compared to the control group. In the CP\u0026thinsp;+\u0026thinsp;GA group, CAT and SOD levels increased while MDA levels decreased compared to the CP group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Serum AMH levels were higher in the GA group than in the other groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eGA can be effective in preventing follicle cell damage caused by CP in the ovarium.\u003c/p\u003e","manuscriptTitle":"Effects of Gallic Acid on Cyclophosphamide-Induced Experimental Ovarian Injury in Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-04 08:55:33","doi":"10.21203/rs.3.rs-8146263/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-20T08:05:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-10T22:33:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-07T15:10:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"107451301938563535547493936911270615552","date":"2026-03-07T15:02:37+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-05T09:10:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"292679978096622214849168271312145416482","date":"2026-03-05T08:59:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"294199101678579722710097116862553899621","date":"2026-03-05T05:00:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-06T09:14:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"260832952132846951914502329694674921284","date":"2025-12-02T14:04:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"277348826301599174506040317348292253367","date":"2025-12-02T10:45:22+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-02T09:03:23+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-19T18:30:40+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-19T09:20:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-19T09:19:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Women's Health","date":"2025-11-18T13:44:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-womens-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bmwh","sideBox":"Learn more about [BMC Women's Health](http://bmcwomenshealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bmwh/default.aspx","title":"BMC Women's Health","twitterHandle":"","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"67899f4b-9708-46ea-a610-ab905ee93325","owner":[],"postedDate":"December 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-29T09:24:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-04 08:55:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8146263","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8146263","identity":"rs-8146263","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}