Does mesna (mercapto-ethanesulphonate sodium) protect ovarian reserve after cyclophosphamide treatment: an in vivo study with rats

Does mesna (mercapto-ethanesulphonate sodium) protect ovarian reserve after cyclophosphamide treatment: an in vivo study with 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 Does mesna (mercapto-ethanesulphonate sodium) protect ovarian reserve after cyclophosphamide treatment: an in vivo study with rats Hayriye Sema Baghaki, Cihan Kaya, Erdem Soztutar, Nilay Aksoy, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1399403/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose This study sought to investigate the protective effect of 2-mercaptoethane sodium sulfonate (mesna) on the ovarian reserve of rats being treated with cyclophosphamide. Methods Twenty-four adult female Wistar albino rats were equally divided into three groups. Group A (n = 8) received saline injections, Group B (n = 8) received cyclophosphamide, and Group C (n = 8) received cyclophosphamide + mesna. Preoperative blood samples (1 ml) were taken from all of the rats prior to any medication being given. The rats in all of the groups underwent bilateral oophorectomy and 1 ml of blood samples were taken 24 hours after the surgery. The difference in the anti-Müllerian hormone (AMH) level between the pre- and postoperative blood samples was determined. The rats’ ovaries were histomorphologically evaluated. Immune staining was performed to detect the AMH receptor expression level in the ovarian tissue. Finally, a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis was performed to assess the level of apoptosis. Results A considerable increase was noted in the AMH receptor expression level in Group C when compared with Group B (122 vs. 117, respectively; p: 0.007). In addition, the number of atretic follicles was found to be significantly lower in Group C than in Group B (10 vs. 5 respectively; p: 0.002). However, the TUNEL analysis revealed no significant difference between Group B and Group C with regard to apoptosis. Conclusion Mesna could decrease follicular atresia and increase the expression of AMH receptors in the follicles following acute cyclophosphamide toxicity. chemotherapy cyclophosphamide mesna ovarian reserve Figures Figure 1 Figure 2 Figure 3 Introduction In recent decades, advancements in cancer therapies have led to increased survival rates worldwide. As a result, an increasing number of cancer patients of reproductive age are surviving the disease. Yet, various sequelae related to the use of cytotoxic chemotherapeutic regimens have been observed in this population, with premature ovarian failure(POF) being a common side effect of such treatments. Cyclophosphamide is a chemotherapeutic drug that is widely used in the treatment of multiple cancers [Calabresi, P., and Parks, R 1980 ], including breast cancer [Koyama, H et al. 1977],, Hodgkin’s disease, acute lymphoblastic leukemia[Sirus, E. S. et al. 1976, Himmelstein-Braw et al. R.1978], and Burkitt’s lymphoma[Fosdick, W. M.et al, 1968]. Moreover, it can be used for the treatment of connective tissue disorders and autoimmune diseases. However, cyclophosphamide has detrimental effects on the gonads in male and female patients [Fairley KF et al. 1968, Sobrinho LG et al. 1972 ]. Thus, patients who need to undergo chemotherapy should be counseled regarding their fertility preservation options. The gonad protective effects of various agents, including GnRH analogs, recombinant anti-Müllerian hormone (AMH) [Roness H et al. 2019 ,Sonigo C et al,2019], progesterone [Ozdamar S et al,2019], antioxidants [Unal F et al.2016, Melekoglu R et al, 2018 ], and antiapoptotic molecules such as sphingomyelin metabolites (e.g., sphingosine-I-phosphate, ceramide-I-phosphate) [Morita Y et al. 2000 , Pascuali N et al.2018] have previously been investigated. However, there is no solid evidence concerning the benefits of co-treatment with GnRH analogs and chemotherapy agents [Blumenfeld Z et al. 1996 , Imai A et al. 2007 ] in relation to fertility preservation[Oktay K et al. 2018 , Fertility preservation in patients undergoing gonadotoxic therapy or gonadectomy: a committee opinion,2019, Fertility preservation and reproduction in patients facing gonadotoxic therapies: an Ethics Committee opinion,2018, Elgindy EA et al.2013, Gerber B. et al. 2011 , .Munster PN MA et al. 2012, Elgindy E et al. 2015 , Bedaiwy MA et al. 2011 ]. To date, oocyte or embryo cryopreservation options have been considered the most reliable treatments for preserving fertility, although the need for such treatments could delay the start of anti-cancer treatments. It has been shown that cyclophosphamide could cause ovarian damage by increasing the levels of free oxygen radicals such as tissue malondialdehyde (MDA) and decreasing the levels of anti-oxidant enzymes such as superoxide dismutase (SOD), thereby causing lipid peroxidation and, consequently, cell death [Yener et al. 2013]. In addition, two primary active metabolites of cyclophosphamide, namely phosphoramide mustard and acrolein, are known to be potent cytotoxic molecules that can be eliminated by another anti-oxidant, glutathione (GSH) [ Lopez et al.2004]. In light of this, an agent with antioxidative properties could serve to protect the ovaries from cyclophosphamide-related gonadotoxicity. Mesna (2-mercaptoethane sodium sulfonate) is an anti-oxidant drug that is used to prevent cyclophosphamide- and ifosfamide-related urotoxicity [Haselberger MB et al. 1995]. It binds the cytotoxic metabolites of cyclophosphamide and exerts protective effects on the bladder and intestinal mucosa [Ypsilantis P et al.2004, Rybak LP et al. 2007 ]. Mesna has also been shown to protect the ovaries following cisplatin treatment [Yeh J et al 2008 , Li X. et al 2013 ]. Yet, although mesna has been reported to be a protective agent, it has not previously been assessed in relation to cyclophosphamide, despite it being one of the most commonly used chemotherapeutic and cytotoxic agents in women of reproductive age [Haselberger MB, Schwinghammer TL.1995, Ypsilantis P et al. 2004 , Rybak LP et al. 2007 , Yeh J et al. 2008 , Li X et al.2013] . The present study sought to investigate the protective effect of mesna on the ovarian reserve of women being treated with cyclophosphamide. Materials And Methods Ethical approval to conduct this study was obtained (approval number: 2020-18), and all of the experiments were conducted in accordance with the requirements of the Declaration of Helsinki concerning animal research. The study was conducted with 24 adult female Wistar albino rats (aged 14–16 weeks, weighing 200–250 gr). All of the rats were kept in a minimum cage area of 350 cm 2 and a minimum cage height of 14 cm. They were fed ad libitum with rat pellets. The mean cage temperature was 21°C with a 12-hour day-night cycle and 50% humidity. The rats were randomly divided into three groups. Prior to the drug administration, all of the rats received anesthesia with 50 mg/kg of intramuscular 10% ketamine hydrochloride (Ketasol; Richter Pharma) and 5 mg/kg of i.m. 2% xylazine (Rompun; Bayer Health Care). A venous blood sample (1 ml) was taken from the jugular vein of each rat and centrifuged at 3000 rpm. The dosage and route of administration of the cyclophosphamide (Nair AB, Jacob S. 2016) and mesna (Kanat, Ozkan, et al. 2006, Morais et al. 1999) were determined according to the recommendations of prior studies. The rats in Group A received intraperitoneal saline injections of the same amount as the cyclophosphamide and mesna doses. The rats in Group B were given 150 mg/kg of cyclophosphamide (Endoxan; Eczacibasi, TR), while the rats in Group C were given 150 mg/kg of cyclophosphamide (Endoxan; Eczacibasi, TR) + mesna (Uromitexan; Eczacibasi, TR) intraperitoneally. The 150 mg/kg of mesna was divided into three equal amounts and each amount of the drug was given at 30 minutes before, four hours after, and eight hours after the cyclophosphamide injection. Twenty-four hours after the drug treatments, oophorectomy was performed and the ovaries were fixed in 10% formaldehyde. A postoperative intracardiac blood sample was taken from each rat, and then the rats were sacrificed using high-dose anesthetic medication. Measurement of the serum AMH level The serum parts of the centrifuged blood samples were transferred into Eppendorf tubes and then stored at -80°C until the day of analysis. The AMH level was measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (MyBioSource, catalog number: MBS726534), which worked based on the quantitative sandwich ELISA principle. Rat AMH antibody precoated ELISA microplates were used. The standards or samples were added to the ELISA microplate wells and the AMH/MIS molecules bound to the specific antibody. Then, a biotinylated detection antibody specific for the rat AMH and avidin-horseradish peroxidase (HRP) conjugate were added to each microplate well and incubated. The free components were washed away. The substrate solution was added to each well. Only those wells that contained the rat AMH, biotinylated detection antibody, and avidin-HRP conjugate turned blue. The enzyme–substrate reaction was terminated via the addition of a stop solution, which caused a color change to yellow. The optical density (OD) was measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value was proportional to the concentration of the rat AMH, which meant that it was possible to calculate the serum AMH levels according to the standard curve of the OD values. Histomorphological analysis All of the tissue samples were evaluated by a histologist (E.S.) who was blinded to the purpose of the study. The ovaries were embedded in paraffin blocks and then cut into 4-µm slices using a microtome. The groups (control, cyclophosphamide only, and cyclophosphamide + mesna) were stained with hematoxylin-eosin staining, immunohistochemical staining for AMH receptors, and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining, respectively. Hematoxylin-eosin staining The first group of slices were deparaffinized with xylene, stained with hematoxylin-eosin, and then evaluated by means of photomicrography (Olympus BX51; Olympus, Tokyo, Japan) to determine the histomorphological features. The follicle count was performed according to the criteria described by Oktay et al. (Oktay K et al, 1995 ). Immunohistochemical staining for AMH receptor expression The second group of slices were deparaffinized using xylene, rehydrated with alcohol, and exposed to 1/10 diluted citrate buffer (PH: 6) to unmask the target antigens and epitopes. Next, the plates were added to an immunohistochemistry staining system (Sequenza Immunostaining Center Each 73300001; Shandon/Thermo Scientific) and washed with phosphate-buffered saline (PBS) buffer for five minutes. The endogenous peroxidase activity was blocked with 3% hydrogen peroxide (TA-125-HP; Thermo Scientific), while the proteins were blocked for five minutes (TA-125-PBQ; Thermo Scientific). The plates were then incubated with anti-AMHR2 antibody (ab197148) for the AMH receptors (AMHR) for one hour and stained with 3,3′-diaminobenzidine (DAB) chromogen (TA-125-HA; Thermo Scientific). The expression of the immunoreactivity of the AMHR in each follicle was measured using ImageJ software (Fiji). TUNEL analysis The final group of slices were reserved for the TUNEL analysis. Following deparaffinization with xylene, they were rehydrated with alcohol. Then, the slices were exposed to proteinase-K for 15 minutes. The endogenous peroxidase activity was blocked using 3% hydrogen peroxide (TA-125-HP; Thermo Scientific) and the slices were washed with PBS. After the administration of equilibration buffer, the slices were added to the TUNEL solution (ApopTag® Peroxidase In Situ Apoptosis Detection Kit; Millipore, catalog number: S7101) and incubated for one hour in darkness. Next, the reaction was stopped using a working stop/wash buffer solution and digoxigenin peroxidase was applied for 30 minutes. The slices were then stained with DAB chromogen and hematoxylin (HHS32, Sigma). The rusty brown-stained nuclei were determined to be TUNEL-positive apoptotic cells. Statistical analysis All of the statistical analyses in this study were performed using Statistical Package for the Social Sciences (SPSS) version 20.0 software (IBM Corp., Armonk, NY, USA). The descriptive data were expressed as the median and interquartile range (IQR). The Kolmogorov-Smirnov test was used to evaluate the normality of the data, while the Mann-Whitney U test was used to compare the nonparametric variables. The Kruskal-Wallis test and Bonferroni correction were used to compare the study groups. The Wilcoxon test was used to determine the changes in the pre- and postoperative AMH levels of each group. A value of p < 0.05 was considered to be statistically significant. Results The histomorphological evaluation of the slices revealed there to be a significant decrease in the stromal edema and hemorrhage, as well as less follicular atresia, in Group C (cyclophosphamide + mesna) when compared with Group B (cyclophosphamide only) (p: 0.002) (Fig. 1 ). However, the follicle count showed no difference between the groups with regard to the amount of primordial, early growing, antral, or total follicles (Table 1 ). Table 1 TUNEL Analysis, the expression of AMH receptors and follicle count Group A Group B Group C p value Primordial follicle count 671(282) 513(189) 588(293) 0.01 The count of growing (primary, secondary, preantral) follicles 366(97) 436(84) 384(68) 0.06 The count of antral follicles 102(63) 84(49) 89(58) 0.66 Total follicle count 1131(371) 1060(237) 992(390) 0.12 AMH expression 123(8.75) 117(4.5) 122(3.5) 0.01 The level of apoptosis in Tunel Analysis 2.92(1.78) 6.73(1.93) 4.77(1.44) 0.004 In terms of the immunohistochemical analysis, the AMHR expression level of the follicles was found to be higher in Group C when compared with both the control group (Group A) and the rats that were treated with only cyclophosphamide (Group B) (p: 0.007) (Fig. 2 ). The apoptosis level was found to be significantly increased in Group B when compared with Group A (P: 0.002), although no statistically significant difference was found between Group B and Group C regarding the apoptosis level (p: 0.052) (Fig. 3 ). There was also no statistically significant difference observed in relation to the serum AMH levels between Group B and Group C (P: 0.75). However, the serum AMH levels were seen to significantly increased in both groups following the respective treatment (P < 0.001) (Table 2 ). Table 2 The comparison of Preop-Postop AMH levels of each group (IQR) Group A Group B Group C p value Preoperative AMH 337.93(130.03) 387.38(167.89) 318.02(134.29) 0.174 Postoperative AMH 935.16(337.52) 1572.81(450.75) 1626.45(788.39) 0.001 Preop-Postop AMH change ( p value) < 0.001 < 0.001 < 0.001 Discussion The present study found that the rats treated with mesna (Group C) had less atretic follicles and stromal edema. They also exhibited better expression of the AMH receptors in their follicles than the rats that were treated with only cyclophosphamide (Group B). This identified effect of mesna on the follicles is in accordance with the findings of previous studies that investigated the ovarian protective effects of mesna in cisplatin-exposed rats [Yeh J et al 2008 , Li X et al, 2013 ]. Yeh et al. found that the AMH levels in the serum and ovarian lysate samples, as well as the number of AMH-positive follicles, were higher in the group treated with mesna + cisplatin [Yeh J et al 2008 ]. Li et al. determined that the rate of AMH-positive follicles [Li X et al, 2013 ] was 70% in the rats that were given mesna and low-dose cisplatin, whereas it was 55% in the rats that were given only cisplatin. Therefore, they proposed that mesna could serve as a protective agent with regard to chemotherapy-induced ovarian failure. The present study found a similar result, as an increase was noted in the number of AMH-positive follicles in the rats that were treated with cyclophosphamide + mesna. This finding supports the findings of Yeh et al.’s and Li et al.’s studies in terms of the ovarian protection offered by mesna, which possibly occurs through antioxidant mechanisms, as both groups of researchers suggested. A number of antioxidant agents have been evaluated with regard to their potential protection of the ovaries. For instance, curcumin and capsaicin were investigated in experimental studies in which the groups that were treated with curcumin and capsaicin showed an increase in the serum AMH levels (5.91 ng/mL in the curcumin group and 6.56 ng/mL in the capsaicin group vs. 3.29 ng/mL in the POF group). Moreover, Melekoglu et al. identified a significant improvement in terms of histomorphological parameters such as stromal hemorrhage, follicular atresia, and vascular congestion in the ovaries of both the curcumin and capsaicin groups [Melekoglu R et al. 2018 ]. Although the present study did not identify a significant change in the serum AMH levels following the addition of mesna, the results concerning follicular atresia and stromal hemorrhage support the findings of Melekoglu et al.’s study. In a study conducted with N-acetyl cysteine (NAC), there was also no change observed between the serum AMH levels of the cyclophosphamide only and NAC + cyclophosphamide groups [Unal et al. 2016 ]. The authors concluded that the dose and time period of the NAC protocol may not have been sufficient to exert the potential function of NAC as an antioxidant agent. In another study, spirulina, an anti-oxidant molecule derived from blue-green algae, was shown to decrease the oxidative molecules in rat ovaries that had been exposed to cyclophosphamide[ Yener et al. 2013 ]. However, no change was seen in the number of follicles, including the primordial follicles and atretic follicles, in the group that was given spirulina. The present study also found no significant change in either the total number of follicles or the number of primordial follicles in the cyclophosphamide + mesna group, although less atretic follicles were detected in that group (i.e., cyclophosphamide + mesna). This finding indicated that cyclophosphamide does not specifically harm primordial follicles during the acute stage, while mesna does not contribute to the primordial follicle pool within the same time frame. Yet, mesna improves the overall follicular atresia following an acute cyclophosphamide insult. Both the curcumin/capsaicin and spirulina studies mentioned above confirmed that cyclophosphamide induces oxidative stress, with an increased level of MDA and oxidative stress-related apoptosis serving as the main pathway for cyclophosphamide gonadotoxicity [Melekoglu 2018, Yener et al. 2013 , Yeh J et al 2008 , Li X et al, 2013 ]. However, these studies did not reveal the level of apoptosis. The present study, therefore, assessed the apoptosis level by means of a TUNEL analysis. The results confirmed that cyclophosphamide causes an increase in apoptosis, although there was no statistically significant difference in terms of the level of apoptosis between the cyclophosphamide and cyclophosphamide + mesna groups. This result might indicate that the mesna dose used in this study may not have been sufficiently high to prevent oxidation-related apoptosis. It is important to acknowledge that the present study had a number of limitations. First, an escalation of serum AMH level was identified in all of the groups following the experiment. This AMH flare could be related to the surgical stress that the animals underwent, as there was no significant difference noted among the groups with regard to the level of the increase. Second, the fact that mesna increased the AMH receptors of the follicles but not the serum AMH level was interpreted as a natural result of the acute cytotoxicity model of cyclophosphamide, although it may also be a result of an insufficient dose of mesna. A study design that allowed for different doses of mesna to be administered to several groups of rats could overcome this limitation. Finally, as this study sought to determine the role of mesna in the setting of the acute toxicity of cyclophosphamide, it did not assess the efficacy of mesna in the case of chronic exposure to cyclophosphamide. The key strength of the present study concerned the methods implemented to detect the effects of mesna at the cell and tissue levels. The immunohistochemistry analysis of the AMH receptors was important in terms of understanding how a medicine effects the up- or down-regulation of receptors on the cell membrane, which provides insight into the rapid changes in the follicles following exposure of a certain drug. Similarly, the TUNEL analysis provided in-depth information concerning the influence of process of apoptosis in relation to the setting of the acute cytotoxicity of cyclophosphamide. In conclusion, the results of this study showed there to be less follicular atresia and increased AMH receptor expression in the follicles of rats that had been treated with concomitant mesna. However, this change was not reflected in the serum AMH levels. Longer periods of mesna administration or a change in the dose could prove beneficial when it comes to understanding the long-term efficacy of mesna. Declarations Acknowledgments The Scientific Research Project Funding Council in University of Health Sciences, Turkey has funded for this study. Statements and Declarations This work was supported by Scientific Research Project Funding Council in University of Health Sciences, Turkey. Competing Interests The authors declare that none of them has financial interest. Author Contributions Sema Baghaki and Cihan Kaya conceived and designed the research. Sema Baghaki, Erdem Soztutar, Nilay Aksoy conducted the experiments. Levent Yasar and Cihan Kaya analyzed the data. Sema Baghaki and Murat Ekin wrote the manuscript. Deniz Ekin edited the manuscript. All authors read and approved the manuscript. Ethics Approval This study was performed in line with the principles of the Declaration of Helsinki. 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Abali5 Unal F, Yuksel MA, Boran B, Yuksel IT, Abali R (2016) The role of N-Acetylcysteine in preventing cyclophosphamide-induced gonadotoxicity: An experimental study in rats. J Obstet Gynaecol 36(3):372–375 Yeh J, Kim BS, Peresie J (2008) Protection against cisplatin-induced ovarian damage by the antioxidant sodium 2-mercaptoethanesulfonate (mesna) in female rats. Am J Obstet Gynecol 198(4):463 e1-6; discussion e6-7 Ypsilantis P, Rybak LP, Yeh J, Li X, Tentes I, Assimakopoulos SF, Kortsaris A, Scopa CD, Simopoulos C et al (2004) Mesna ameliorates intestinal mucosa damage after ifosfamide administration in the rabbit at a dose-related manner. J Surg Res. 2004;121(1):84–91 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-1399403","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":90675929,"identity":"cc0a80a0-4ea1-4d98-9d9b-64366e0f5fc7","order_by":0,"name":"Hayriye Sema Baghaki","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYJACZiBOYOBhbHwM4TI3EK2l2ZiBwQDIZSRaCwObNFgLAwEt8u3diZ8Lamrz+HsOt1UXVPyJ5m8HavlRsQ2nFoMzZzdLzzh2vFjibGPb7RlnDHJnHGZsYOw5cxu3FoncDdI8bMcSG84ztt3mbTPIbQBqYQaycTts/tvNv3n+HUucD9RSDNIyn5AWhhu826R522oSNwAdxgzSsoGQFoMzudusZ/YdSNx45mCzNM8Z49yNQC0H8flFvv3s5tsF3+oS551Jf/iZp0Iud975wwcf/KjA4zAIOIzKPUBIPRDUEaFmFIyCUTAKRiwAAHPrYA3+Lk2YAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-3981-6069","institution":"University of Health Sciences: Saglik Bilimleri Universitesi","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Hayriye","middleName":"Sema","lastName":"Baghaki","suffix":""},{"id":90675930,"identity":"9a3ccba3-7cd4-4cd7-a53c-9b9abcc0ced6","order_by":1,"name":"Cihan Kaya","email":"","orcid":"","institution":"Acıbadem Mehmet Ali Aydınlar Üniversitesi: Acibadem Universitesi","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Cihan","middleName":"","lastName":"Kaya","suffix":""},{"id":90675931,"identity":"fafdd1ed-62d9-4e05-9be8-b85edd7e9c66","order_by":2,"name":"Erdem Soztutar","email":"","orcid":"","institution":"Yeditepe University: Yeditepe Universitesi","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Erdem","middleName":"","lastName":"Soztutar","suffix":""},{"id":90675932,"identity":"b72c1d11-3abe-4c85-9ede-ab1325df1425","order_by":3,"name":"Nilay Aksoy","email":"","orcid":"","institution":"Altınbaş University: Altinbas Universitesi","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nilay","middleName":"","lastName":"Aksoy","suffix":""},{"id":90675933,"identity":"0f55a651-ed58-4d6e-9932-88b9281de4cb","order_by":4,"name":"Levent Yasar","email":"","orcid":"","institution":"University of Health Sciences: Saglik Bilimleri Universitesi","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Levent","middleName":"","lastName":"Yasar","suffix":""},{"id":90675934,"identity":"d0b70287-677d-471b-ad36-04a5aed10215","order_by":5,"name":"Murat Ekin","email":"","orcid":"","institution":"University of Health Sciences: Saglik Bilimleri Universitesi","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Murat","middleName":"","lastName":"Ekin","suffix":""},{"id":90675935,"identity":"1b781ea6-3874-4d3e-b1d8-14eaea29017f","order_by":6,"name":"Deniz Ekin","email":"","orcid":"","institution":"American College","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Deniz","middleName":"","lastName":"Ekin","suffix":""}],"badges":[],"createdAt":"2022-02-26 18:19:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1399403/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1399403/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":19247037,"identity":"fa4163f2-9aac-48a9-b0d8-f575292ee11b","added_by":"auto","created_at":"2022-03-15 15:14:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":678614,"visible":true,"origin":"","legend":"\u003cp\u003eHistomorphologic examination of slices with Hemotoxylin-Eosin stain. Normal ovarian histology is seen in the control group. Arrows show normal follicles in the group A and C. The areas of stromal hemorrhage are shown in Group B with thin arrows. The\u0026nbsp;atretic follicles and stromal hemorrhage are more prominent in Group B than Group C. All pictures are captured in x4 objective of microscope and the scalebar of image was 200 micrometer.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-1399403/v1/bec2a656ec889069c7282815.png"},{"id":19247039,"identity":"f1b38bc6-f920-4e88-aecf-6d68eaf13c99","added_by":"auto","created_at":"2022-03-15 15:14:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":580732,"visible":true,"origin":"","legend":"\u003cp\u003eThe slices stained with AMH receptor (AMHR) immunohistochemistry. The follicles (thick arrows) stain more than corpus luteum (thin arrow). The AMHR stain expression decreased in group B whereas the AMHR expression is significantly stressed in Group C which were the rats given Mesna. and cyclophosphamide. All pictures are captured in x4 objective of microscope and the scalebar of image was 200 micrometer.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-1399403/v1/a415b683f91fae63540bca32.png"},{"id":19247038,"identity":"24b74d3f-7af4-4acf-a10d-8dd9246fc98c","added_by":"auto","created_at":"2022-03-15 15:14:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1003033,"visible":true,"origin":"","legend":"\u003cp\u003eThe slices of control, cyclophosphamide and cyclophosphamide+ Mesna given groups with TUNEL staining. Arrows indicate the pyknotic cells in each picture. The pycnotic cells predominant in Group B compared with Group C, however the difference was not statistically significant. All pictures are captured in x4 objective of microscope and the scalebar of image was 100 micrometer.\u003c/p\u003e","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-1399403/v1/b7fd466fef2293c7f3bc86af.png"},{"id":20835716,"identity":"ee3a4f99-9981-409b-bea0-01e715a83fd1","added_by":"auto","created_at":"2022-04-27 16:14:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2318493,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1399403/v1/ab513964-1bb7-4a9a-ba9a-6cf0b54f6ae7.pdf"}],"financialInterests":"","formattedTitle":"Does mesna (mercapto-ethanesulphonate sodium) protect ovarian reserve after cyclophosphamide treatment: an in vivo study with rats","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn recent decades, advancements in cancer therapies have led to increased survival rates worldwide. As a result, an increasing number of cancer patients of reproductive age are surviving the disease. Yet, various sequelae related to the use of cytotoxic chemotherapeutic regimens have been observed in this population, with premature ovarian failure(POF) being a common side effect of such treatments.\u003c/p\u003e \u003cp\u003eCyclophosphamide is a chemotherapeutic drug that is widely used in the treatment of multiple cancers [Calabresi, P., and Parks, R \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1980\u003c/span\u003e], including breast cancer [Koyama, H et al. 1977],, Hodgkin\u0026rsquo;s disease, acute lymphoblastic leukemia[Sirus, E. S. et al. 1976, Himmelstein-Braw et al. R.1978], and Burkitt\u0026rsquo;s lymphoma[Fosdick, W. M.et al, 1968]. Moreover, it can be used for the treatment of connective tissue disorders and autoimmune diseases. However, cyclophosphamide has detrimental effects on the gonads in male and female patients [Fairley KF et al. 1968, Sobrinho LG et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1972\u003c/span\u003e]. Thus, patients who need to undergo chemotherapy should be counseled regarding their fertility preservation options.\u003c/p\u003e \u003cp\u003eThe gonad protective effects of various agents, including GnRH analogs, recombinant anti-M\u0026uuml;llerian hormone (AMH) [Roness H et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e,Sonigo C et al,2019], progesterone [Ozdamar S et al,2019], antioxidants [Unal F et al.2016, Melekoglu R et al, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e], and antiapoptotic molecules such as sphingomyelin metabolites (e.g., sphingosine-I-phosphate, ceramide-I-phosphate) [Morita Y et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2000\u003c/span\u003e, Pascuali N et al.2018] have previously been investigated. However, there is no solid evidence concerning the benefits of co-treatment with GnRH analogs and chemotherapy agents [Blumenfeld Z et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1996\u003c/span\u003e, Imai A et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2007\u003c/span\u003e] in relation to fertility preservation[Oktay K et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Fertility preservation in patients undergoing gonadotoxic therapy or gonadectomy: a committee opinion,2019, Fertility preservation and reproduction in patients facing gonadotoxic therapies: an Ethics Committee opinion,2018, Elgindy EA et al.2013, Gerber B. et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, .Munster PN MA et al. 2012, Elgindy E et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Bedaiwy MA et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2011\u003c/span\u003e]. To date, oocyte or embryo cryopreservation options have been considered the most reliable treatments for preserving fertility, although the need for such treatments could delay the start of anti-cancer treatments.\u003c/p\u003e \u003cp\u003eIt has been shown that cyclophosphamide could cause ovarian damage by increasing the levels of free oxygen radicals such as tissue malondialdehyde (MDA) and decreasing the levels of anti-oxidant enzymes such as superoxide dismutase (SOD), thereby causing lipid peroxidation and, consequently, cell death [Yener et al. 2013]. In addition, two primary active metabolites of cyclophosphamide, namely phosphoramide mustard and acrolein, are known to be potent cytotoxic molecules that can be eliminated by another anti-oxidant, glutathione (GSH) [ \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eLopez\u003c/span\u003e et al.2004]. In light of this, an agent with antioxidative properties could serve to protect the ovaries from cyclophosphamide-related gonadotoxicity.\u003c/p\u003e \u003cp\u003eMesna (2-mercaptoethane sodium sulfonate) is an anti-oxidant drug that is used to prevent cyclophosphamide- and ifosfamide-related urotoxicity [Haselberger MB et al. 1995]. It binds the cytotoxic metabolites of cyclophosphamide and exerts protective effects on the bladder and intestinal mucosa [Ypsilantis P et al.2004, Rybak LP et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2007\u003c/span\u003e]. Mesna has also been shown to protect the ovaries following cisplatin treatment [Yeh J et al \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Li X. et al \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e]. Yet, although mesna has been reported to be a protective agent, it has not previously been assessed in relation to cyclophosphamide, despite it being one of the most commonly used chemotherapeutic and cytotoxic agents in women of reproductive age [Haselberger MB, Schwinghammer TL.1995, Ypsilantis P et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, Rybak LP et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2007\u003c/span\u003e, Yeh J et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Li X et al.2013] .\u003c/p\u003e \u003cp\u003eThe present study sought to investigate the protective effect of mesna on the ovarian reserve of women being treated with cyclophosphamide.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e \u003cstrong\u003eEthical approval\u003c/strong\u003e \u003cp\u003e to conduct this study was obtained (approval number: 2020-18), and all of the experiments were conducted in accordance with the requirements of the Declaration of Helsinki concerning animal research.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eThe study was conducted with 24 adult female Wistar albino rats (aged 14\u0026ndash;16 weeks, weighing 200\u0026ndash;250 gr). All of the rats were kept in a minimum cage area of 350 cm\u003csup\u003e2\u003c/sup\u003e and a minimum cage height of 14 cm. They were fed ad libitum with rat pellets. The mean cage temperature was 21\u0026deg;C with a 12-hour day-night cycle and 50% humidity.\u003c/p\u003e \u003cp\u003eThe rats were randomly divided into three groups. Prior to the drug administration, all of the rats received anesthesia with 50 mg/kg of intramuscular 10% ketamine hydrochloride (Ketasol; Richter Pharma) and 5 mg/kg of i.m. 2% xylazine (Rompun; Bayer Health Care). A venous blood sample (1 ml) was taken from the jugular vein of each rat and centrifuged at 3000 rpm. The dosage and route of administration of the cyclophosphamide (Nair AB, Jacob S. 2016) and mesna (Kanat, Ozkan, et al. 2006, Morais et al. 1999) were determined according to the recommendations of prior studies.\u003c/p\u003e \u003cp\u003eThe rats in Group A received intraperitoneal saline injections of the same amount as the cyclophosphamide and mesna doses. The rats in Group B were given 150 mg/kg of cyclophosphamide (Endoxan; Eczacibasi, TR), while the rats in Group C were given 150 mg/kg of cyclophosphamide (Endoxan; Eczacibasi, TR)\u0026thinsp;+\u0026thinsp;mesna (Uromitexan; Eczacibasi, TR) intraperitoneally. The 150 mg/kg of mesna was divided into three equal amounts and each amount of the drug was given at 30 minutes before, four hours after, and eight hours after the cyclophosphamide injection. Twenty-four hours after the drug treatments, oophorectomy was performed and the ovaries were fixed in 10% formaldehyde. A postoperative intracardiac blood sample was taken from each rat, and then the rats were sacrificed using high-dose anesthetic medication.\u003c/p\u003e \u003cp\u003eMeasurement of the serum AMH level\u003c/p\u003e \u003cp\u003eThe serum parts of the centrifuged blood samples were transferred into Eppendorf tubes and then stored at -80\u0026deg;C until the day of analysis. The AMH level was measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (MyBioSource, catalog number: MBS726534), which worked based on the quantitative sandwich ELISA principle. Rat AMH antibody precoated ELISA microplates were used. The standards or samples were added to the ELISA microplate wells and the AMH/MIS molecules bound to the specific antibody. Then, a biotinylated detection antibody specific for the rat AMH and avidin-horseradish peroxidase (HRP) conjugate were added to each microplate well and incubated. The free components were washed away. The substrate solution was added to each well. Only those wells that contained the rat AMH, biotinylated detection antibody, and avidin-HRP conjugate turned blue. The enzyme\u0026ndash;substrate reaction was terminated via the addition of a stop solution, which caused a color change to yellow. The optical density (OD) was measured spectrophotometrically at a wavelength of 450 nm\u0026thinsp;\u0026plusmn;\u0026thinsp;2 nm. The OD value was proportional to the concentration of the rat AMH, which meant that it was possible to calculate the serum AMH levels according to the standard curve of the OD values.\u003c/p\u003e \u003cp\u003eHistomorphological analysis\u003c/p\u003e \u003cp\u003eAll of the tissue samples were evaluated by a histologist (E.S.) who was blinded to the purpose of the study. The ovaries were embedded in paraffin blocks and then cut into 4-\u0026micro;m slices using a microtome. The groups (control, cyclophosphamide only, and cyclophosphamide\u0026thinsp;+\u0026thinsp;mesna) were stained with hematoxylin-eosin staining, immunohistochemical staining for AMH receptors, and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining, respectively.\u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eHematoxylin-eosin staining\u003c/h2\u003e \u003cp\u003eThe first group of slices were deparaffinized with xylene, stained with hematoxylin-eosin, and then evaluated by means of photomicrography (Olympus BX51; Olympus, Tokyo, Japan) to determine the histomorphological features. The follicle count was performed according to the criteria described by Oktay et al. (Oktay K et al, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1995\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eImmunohistochemical staining for AMH receptor expression\u003c/p\u003e \u003cp\u003eThe second group of slices were deparaffinized using xylene, rehydrated with alcohol, and exposed to 1/10 diluted citrate buffer (PH: 6) to unmask the target antigens and epitopes. Next, the plates were added to an immunohistochemistry staining system (Sequenza Immunostaining Center Each 73300001; Shandon/Thermo Scientific) and washed with phosphate-buffered saline (PBS) buffer for five minutes. The endogenous peroxidase activity was blocked with 3% hydrogen peroxide (TA-125-HP; Thermo Scientific), while the proteins were blocked for five minutes (TA-125-PBQ; Thermo Scientific). The plates were then incubated with anti-AMHR2 antibody (ab197148) for the AMH receptors (AMHR) for one hour and stained with 3,3\u0026prime;-diaminobenzidine (DAB) chromogen (TA-125-HA; Thermo Scientific). The expression of the immunoreactivity of the AMHR in each follicle was measured using ImageJ software (Fiji).\u003c/p\u003e \u003cp\u003eTUNEL analysis\u003c/p\u003e \u003cp\u003eThe final group of slices were reserved for the TUNEL analysis. Following deparaffinization with xylene, they were rehydrated with alcohol. Then, the slices were exposed to proteinase-K for 15 minutes. The endogenous peroxidase activity was blocked using 3% hydrogen peroxide (TA-125-HP; Thermo Scientific) and the slices were washed with PBS. After the administration of equilibration buffer, the slices were added to the TUNEL solution (ApopTag\u0026reg; Peroxidase In Situ Apoptosis Detection Kit; Millipore, catalog number: S7101) and incubated for one hour in darkness. Next, the reaction was stopped using a working stop/wash buffer solution and digoxigenin peroxidase was applied for 30 minutes. The slices were then stained with DAB chromogen and hematoxylin (HHS32, Sigma). The rusty brown-stained nuclei were determined to be TUNEL-positive apoptotic cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll of the statistical analyses in this study were performed using Statistical Package for the Social Sciences (SPSS) version 20.0 software (IBM Corp., Armonk, NY, USA). The descriptive data were expressed as the median and interquartile range (IQR). The Kolmogorov-Smirnov test was used to evaluate the normality of the data, while the Mann-Whitney U test was used to compare the nonparametric variables. The Kruskal-Wallis test and Bonferroni correction were used to compare the study groups. The Wilcoxon test was used to determine the changes in the pre- and postoperative AMH levels of each group. A value of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to be statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe histomorphological evaluation of the slices revealed there to be a significant decrease in the stromal edema and hemorrhage, as well as less follicular atresia, in Group C (cyclophosphamide\u0026thinsp;+\u0026thinsp;mesna) when compared with Group B (cyclophosphamide only) (p: 0.002) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, the follicle count showed no difference between the groups with regard to the amount of primordial, early growing, antral, or total follicles (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\u003eTUNEL Analysis, the expression of AMH receptors and follicle count\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGroup A\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGroup B\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGroup C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePrimordial follicle count\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e671(282)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e513(189)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e588(293)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.01\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eThe count of growing (primary, secondary, preantral) follicles\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e366(97)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e436(84)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e384(68)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eThe count of antral follicles\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e102(63)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e84(49)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e89(58)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.66\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal follicle count\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1131(371)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1060(237)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e992(390)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAMH expression\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e123(8.75)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e117(4.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e122(3.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.01\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eThe level of apoptosis in Tunel Analysis\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.92(1.78)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.73(1.93)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.77(1.44)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.004\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn terms of the immunohistochemical analysis, the AMHR expression level of the follicles was found to be higher in Group C when compared with both the control group (Group A) and the rats that were treated with only cyclophosphamide (Group B) (p: 0.007) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe apoptosis level was found to be significantly increased in Group B when compared with Group A (P: 0.002), although no statistically significant difference was found between Group B and Group C regarding the apoptosis level (p: 0.052) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThere was also no statistically significant difference observed in relation to the serum AMH levels between Group B and Group C (P: 0.75). However, the serum AMH levels were seen to significantly increased in both groups following the respective treatment (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (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\u003eThe comparison of Preop-Postop AMH levels of each group (IQR)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGroup A\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGroup B\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGroup C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePreoperative AMH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e337.93(130.03)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e387.38(167.89)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e318.02(134.29)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.174\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePostoperative AMH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e935.16(337.52)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1572.81(450.75)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1626.45(788.39)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePreop-Postop AMH change (\u003c/b\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ep\u003c/span\u003e \u003cb\u003evalue)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study found that the rats treated with mesna (Group C) had less atretic follicles and stromal edema. They also exhibited better expression of the AMH receptors in their follicles than the rats that were treated with only cyclophosphamide (Group B). This identified effect of mesna on the follicles is in accordance with the findings of previous studies that investigated the ovarian protective effects of mesna in cisplatin-exposed rats [Yeh J et al \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Li X et al, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eYeh et al. found that the AMH levels in the serum and ovarian lysate samples, as well as the number of AMH-positive follicles, were higher in the group treated with mesna\u0026thinsp;+\u0026thinsp;cisplatin [Yeh J et al \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2008\u003c/span\u003e]. Li et al. determined that the rate of AMH-positive follicles [Li X et al, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e] was 70% in the rats that were given mesna and low-dose cisplatin, whereas it was 55% in the rats that were given only cisplatin. Therefore, they proposed that mesna could serve as a protective agent with regard to chemotherapy-induced ovarian failure. The present study found a similar result, as an increase was noted in the number of AMH-positive follicles in the rats that were treated with cyclophosphamide\u0026thinsp;+\u0026thinsp;mesna. This finding supports the findings of Yeh et al.\u0026rsquo;s and Li et al.\u0026rsquo;s studies in terms of the ovarian protection offered by mesna, which possibly occurs through antioxidant mechanisms, as both groups of researchers suggested.\u003c/p\u003e \u003cp\u003eA number of antioxidant agents have been evaluated with regard to their potential protection of the ovaries. For instance, curcumin and capsaicin were investigated in experimental studies in which the groups that were treated with curcumin and capsaicin showed an increase in the serum AMH levels (5.91 ng/mL in the curcumin group and 6.56 ng/mL in the capsaicin group vs. 3.29 ng/mL in the POF group). Moreover, Melekoglu et al. identified a significant improvement in terms of histomorphological parameters such as stromal hemorrhage, follicular atresia, and vascular congestion in the ovaries of both the curcumin and capsaicin groups [Melekoglu R et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e]. Although the present study did not identify a significant change in the serum AMH levels following the addition of mesna, the results concerning follicular atresia and stromal hemorrhage support the findings of Melekoglu et al.\u0026rsquo;s study. In a study conducted with N-acetyl cysteine (NAC), there was also no change observed between the serum AMH levels of the cyclophosphamide only and NAC\u0026thinsp;+\u0026thinsp;cyclophosphamide groups [Unal et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2016\u003c/span\u003e]. The authors concluded that the dose and time period of the NAC protocol may not have been sufficient to exert the potential function of NAC as an antioxidant agent.\u003c/p\u003e \u003cp\u003eIn another study, spirulina, an anti-oxidant molecule derived from blue-green algae, was shown to decrease the oxidative molecules in rat ovaries that had been exposed to cyclophosphamide[\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eYener et al. 2013\u003c/span\u003e]. However, no change was seen in the number of follicles, including the primordial follicles and atretic follicles, in the group that was given spirulina. The present study also found no significant change in either the total number of follicles or the number of primordial follicles in the cyclophosphamide\u0026thinsp;+\u0026thinsp;mesna group, although less atretic follicles were detected in that group (i.e., cyclophosphamide\u0026thinsp;+\u0026thinsp;mesna). This finding indicated that cyclophosphamide does not specifically harm primordial follicles during the acute stage, while mesna does not contribute to the primordial follicle pool within the same time frame. Yet, mesna improves the overall follicular atresia following an acute cyclophosphamide insult.\u003c/p\u003e \u003cp\u003eBoth the curcumin/capsaicin and spirulina studies mentioned above confirmed that cyclophosphamide induces oxidative stress, with an increased level of MDA and oxidative stress-related apoptosis serving as the main pathway for cyclophosphamide gonadotoxicity [Melekoglu 2018, \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eYener et al. 2013\u003c/span\u003e, Yeh J et al \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Li X et al, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e]. However, these studies did not reveal the level of apoptosis. The present study, therefore, assessed the apoptosis level by means of a TUNEL analysis. The results confirmed that cyclophosphamide causes an increase in apoptosis, although there was no statistically significant difference in terms of the level of apoptosis between the cyclophosphamide and cyclophosphamide\u0026thinsp;+\u0026thinsp;mesna groups. This result might indicate that the mesna dose used in this study may not have been sufficiently high to prevent oxidation-related apoptosis.\u003c/p\u003e \u003cp\u003eIt is important to acknowledge that the present study had a number of limitations. First, an escalation of serum AMH level was identified in all of the groups following the experiment. This AMH flare could be related to the surgical stress that the animals underwent, as there was no significant difference noted among the groups with regard to the level of the increase. Second, the fact that mesna increased the AMH receptors of the follicles but not the serum AMH level was interpreted as a natural result of the acute cytotoxicity model of cyclophosphamide, although it may also be a result of an insufficient dose of mesna. A study design that allowed for different doses of mesna to be administered to several groups of rats could overcome this limitation. Finally, as this study sought to determine the role of mesna in the setting of the acute toxicity of cyclophosphamide, it did not assess the efficacy of mesna in the case of chronic exposure to cyclophosphamide.\u003c/p\u003e \u003cp\u003eThe key strength of the present study concerned the methods implemented to detect the effects of mesna at the cell and tissue levels. The immunohistochemistry analysis of the AMH receptors was important in terms of understanding how a medicine effects the up- or down-regulation of receptors on the cell membrane, which provides insight into the rapid changes in the follicles following exposure of a certain drug. Similarly, the TUNEL analysis provided in-depth information concerning the influence of process of apoptosis in relation to the setting of the acute cytotoxicity of cyclophosphamide.\u003c/p\u003e \u003cp\u003eIn conclusion, the results of this study showed there to be less follicular atresia and increased AMH receptor expression in the follicles of rats that had been treated with concomitant mesna. However, this change was not reflected in the serum AMH levels. Longer periods of mesna administration or a change in the dose could prove beneficial when it comes to understanding the long-term efficacy of mesna.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgments\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe Scientific Research Project Funding Council in University of \u0026nbsp;Health Sciences, Turkey has funded for this study.\u0026nbsp;\u003c/p\u003e\u003cp\u003eStatements and Declarations\u003c/p\u003e\n\u003cp\u003eThis work was supported by Scientific Research Project Funding Council in University of Health Sciences, Turkey.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompeting Interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that none of them has financial interest.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSema Baghaki \u0026nbsp;and Cihan Kaya conceived and designed the research. Sema Baghaki, Erdem Soztutar, Nilay Aksoy conducted the experiments. Levent Yasar and Cihan Kaya analyzed the data. Sema Baghaki and Murat Ekin \u0026nbsp;wrote the manuscript. Deniz Ekin edited the manuscript. All authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of University of Health Sciences, Bagcılar State Hospital, Animal Laboratory with the approval number: 2020-18.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBedaiwy MA, Abou-Setta AM, Desai N, Hurd W, Starks D, El-Nashar SA et al (2011) Gonadotropin-releasing hormone analog cotreatment for preservation of ovarian function during gonadotoxic chemotherapy: a systematic review and meta-analysis. Fertil Steril 95(3):906\u0026ndash;914 e1-4\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlumenfeld Z, Avivi I, Eckman A, Epelbaum R, Rowe JM, Dann EJ (2008) Gonadotropin-releasing hormone agonist decreases chemotherapy-induced gonadotoxicity and premature ovarian failure in young female patients with Hodgkin lymphoma. Fertil Steril 89(1):166\u0026ndash;173\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlumenfeld Z, Avivi I, Linn S, Epelbaum R, Ben-Shahar M, Haim N (1996) Prevention of irreversible chemotherapy-induced ovarian damage in young women with lymphoma by a gonadotrophin-releasing hormone agonist in parallel to chemotherapy. Hum Reprod 11(8):1620\u0026ndash;1626\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlumenfeld Z, Shapiro D, Shteinberg M, Avivi I, Nahir M (2000) Preservation of fertility and ovarian function and minimizing gonadotoxicity in young women with systemic lupus erythematosus treated by chemotherapy. Lupus 9(6):401\u0026ndash;405\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCalabresi P, Parks R (1980) Antiproliferative agents and drugs used for immunosuppression. In: Goodman-Gillman A, Goodman LS, Gillman A (eds) The Pharmacologie Basis of Therapeutics. Macmillan Publishing Co., Inc., New York, pp 1256\u0026ndash;1306\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDel Mastro L, Catzeddu T, Boni L, Bell C, Sertoli MR, Bighin C et al (2006) Prevention of chemotherapy-induced menopause by temporary ovarian suppression with goserelin in young, early breast cancer patients. Ann Oncol 17(1):74\u0026ndash;78\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEffects of Cyclophosphamide and Buthionine Sulfoximine on Ovarian Glutathione and Apoptosis, Sarah G (2004) Lopez 1, Ulrike Luderer PMID: \u003cb\u003e15135172\u003c/b\u003e DOI: 10.1016/j.freeradbiomed.2004.02.067 Free Radic Biol Med. 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Nese Arzu Yener, \u003csup\u003e1,*\u003c/sup\u003e Orhun Sinanoglu, \u003csup\u003e2\u003c/sup\u003e Erdin Ilter, \u003csup\u003e3\u003c/sup\u003e Aygen Celik, \u003csup\u003e3\u003c/sup\u003e Gulbuz Sezgin, \u003csup\u003e4\u003c/sup\u003e Ahmet Midi, \u003csup\u003e1\u003c/sup\u003e Ugur Deveci, \u003csup\u003e5\u003c/sup\u003e and Fehime Aksungar \u003csup\u003e6\u003c/sup\u003e Biochem Res Int (2013) ; 2013: 764262.doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1155/2013/764262\u003c/span\u003e\u003c/span\u003e PMID: 23762559\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEfficacy of mesna for prevention of hemorrhagic cystitis after high-dose cyclophosphamide therapy, Haselberger MB, Schwinghammer TL (1995 Sep) Ann Pharmacother 29(9):918\u0026ndash;921. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1177/106002809502900914\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElgindy E, Sibai H, Abdelghani A, Mostafa M (2015) Protecting Ovaries During Chemotherapy Through Gonad Suppression: A Systematic Review and Meta-analysis. Obstet Gynecol 126(1):187\u0026ndash;195\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElgindy EA, El-Haieg DO, Khorshid OM, Ismail EI, Abdelgawad M, Sallam HN et al (2013) Gonadatrophin suppression to prevent chemotherapy-induced ovarian damage: a randomized controlled trial. Obstet Gynecol 121(1):78\u0026ndash;86\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEthics Committee of the American Society for Reproductive Medicine (2018) Electronic address Aao. Fertility preservation and reproduction in patients facing gonadotoxic therapies: an Ethics Committee opinion. Fertil Steril 110(3):380\u0026ndash;386\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFairley KF, Barrie JV, Johnson W (1972) Sterility and testicular atrophy related tocyclophosphamide therapy. Lancet 1:568\u0026ndash;569\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFosdick WM, Parsons JL, Hill DF (1968) Preliminary report: long-term cyclophosphamide therapy in rheumatoid arthritis. Arthritis Rheum., //: 151\u0026ndash;161,\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFranke HR, Smit WM, Vermes I (2005) Gonadal protection by a gonadotropin-releasing hormone agonist depot in young women with Hodgkin's disease undergoing chemotherapy. Gynecol Endocrinol 20(5):274\u0026ndash;278\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGerber B, von Minckwitz G, Stehle H, Reimer T, Felberbaum R, Maass N et al (2011) Effect of luteinizing hormone-releasing hormone agonist on ovarian function after modern adjuvant breast cancer chemotherapy: the GBG 37 ZORO study. J Clin Oncol 29(17):2334\u0026ndash;2341\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHimmelstein-Braw R, Peters II, Faber I (1978) Morphologic study of the ovaries of leukemic children. Br J Cancer 38:82\u0026ndash;87\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuser M, Crha I, Ventruba P, Hudecek R, Zakova J, Smardova L et al (2008) Prevention of ovarian function damage by a GnRH analogue during chemotherapy in Hodgkin lymphoma patients. Hum Reprod 23(4):863\u0026ndash;868\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eImai A, Furui T (2007) Chemotherapy-induced female infertility and protective action of gonadotropin-releasing hormone analogues. J Obstet Gynaecol 27(1):20\u0026ndash;24\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eImai A, Sugiyama M, Furui T, Tamaya T, Ohno T (2007) Direct protection by a gonadotropin-releasing hormone analog from doxorubicin-induced granulosa cell damage. Gynecol Obstet Invest 63(2):102\u0026ndash;106\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKanat O et al (2006) Comparison of uroprotective efficacy of mesna and amifostine in Cyclophosphamide-induced hemorrhagic cystitis in rats. Indian J Cancer 43(1):12\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoyama H, Wada T, Nishizawa Y Cyclophosphamide-induced ovarian failure and its therapeutic significance in patients with breast cancer.Cancer (Phila.), 39:1403\u0026ndash;1404, 1977.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi X, Yang S, Lv X, Sun H, Weng J, Liang Y et al (2013) The mechanism of mesna in protection from cisplatin-induced ovarian damage in female rats. J Gynecol Oncol 24(2):177\u0026ndash;185\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMelekoglu R, Ciftci O, Eraslan S, Cetin A, Basak N (2018) Beneficial effects of curcumin and capsaicin on cyclophosphamide-induced premature ovarian failure in a rat model. J Ovarian Res 11(1):33\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorita Y, Perez GI, Paris F, Miranda SR, Ehleiter D, Haimovitz-Friedman A et al (2000) Oocyte apoptosis is suppressed by disruption of the acid sphingomyelinase gene or by sphingosine-1-phosphate therapy. Nat Med 6(10):1109\u0026ndash;1114\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMunster PNMA, Ismail-Khan R et al (2012) Randomized trial using gonadotropin-releasing hormone agonist triptorelin for the preservation.J Clin Oncol; 30(533)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNair AB, Jacob S (2016) A simple practice guide for dose conversion between animals and human. J Basic Clin Pharm 7(2):27\u0026ndash;31\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOktay K, Schenken RS, Nelson JF (1995) Proliferating cell nuclear antigen marks the initiation of follicular growth in the rat. Biol Reprod 53:295\u0026ndash;301\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOktay K, Harvey BE, Partridge AH, Quinn GP, Reinecke J, Taylor HS et al (2018) Fertility Preservation in Patients With Cancer: ASCO Clinical Practice Guideline Update. J Clin Oncol 36(19):1994\u0026ndash;2001\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOzdamar S, Taskin MI, Onder GO, Kaymak E, Baran M, Yay A (2019) Progesterone decreases the extent of ovarian damage caused by cisplatin in an experimental rat model. Adv Clin Exp Med 28(1):25\u0026ndash;33\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePascuali N, Scotti L, Di Pietro M, Oubina G, Bas D, May M et al (2018) Ceramide-1-phosphate has protective properties against cyclophosphamide-induced ovarian damage in a mice model of premature ovarian failure. Hum Reprod 33(5):844\u0026ndash;859\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eof cyclophosphamide-induced hemorrhagic cystitis - comparison of the effects of dexamethasone and Mesna M M Morais 1, Pharmacological and histopathological study, Belarmino-Filho JNG A Brito, R A Ribeiro\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePractice Committee of the American Society for Reproductive Medicine (2019) Electronic address aao. Fertility preservation in patients undergoing gonadotoxic therapy or gonadectomy: a committee opinion. Fertil Steril 112(6):1022\u0026ndash;1033\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoness H, Spector I, Leichtmann-Bardoogo Y, Savino AM, Dereh-Haim S, Meirow D (2019) Pharmacological administration of recombinant human AMH rescues ovarian reserve and preserves fertility in a mouse model of chemotherapy, without interfering with anti-tumoural effects. J Assist Reprod Genet 36(9):1793\u0026ndash;1803\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRybak LP, Whitworth CA, Mukherjea D, Ramkumar V (2007) Mechanisms of cisplatin-induced ototoxicity and prevention. Hear Res 226(1\u0026ndash;2):157\u0026ndash;167\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSirus ES, Leventhal BG, Vaitukaitis JL (1976) Effects of childhood leukemia and chemotherapy on puberty and reproductive function in girls. N Engl J Med 294:1143\u0026ndash;1146\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSobrinho LG, Levine RA, Deconti RC (1972) Amenorrhea in patients with Hodgkin's disease treated with anti-neoplastic agents. Am J Obstet Gynecol 109:135\u0026ndash;139\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSonigo C, Beau I, Grynberg M, Binart N (2019) AMH prevents primordial ovarian follicle loss and fertility alteration in cyclophosphamide-treated mice. FASEB J 33(1):1278\u0026ndash;1287\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThe role of N-Acetylcysteine in preventing cyclophosphamide-induced gonadotoxicity: An experimental study in rats F. Unal 1, M. Aytac Yuksel2, B. Boran3, I. Temel Yuksel4, \u0026amp; R. Abali5\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUnal F, Yuksel MA, Boran B, Yuksel IT, Abali R (2016) The role of N-Acetylcysteine in preventing cyclophosphamide-induced gonadotoxicity: An experimental study in rats. J Obstet Gynaecol 36(3):372\u0026ndash;375\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYeh J, Kim BS, Peresie J (2008) Protection against cisplatin-induced ovarian damage by the antioxidant sodium 2-mercaptoethanesulfonate (mesna) in female rats. Am J Obstet Gynecol 198(4):463 e1-6; discussion e6-7\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYpsilantis P, Rybak LP, Yeh J, Li X, Tentes I, Assimakopoulos SF, Kortsaris A, Scopa CD, Simopoulos C et al (2004) Mesna ameliorates intestinal mucosa damage after ifosfamide administration in the rabbit at a dose-related manner. J Surg Res. 2004;121(1):84\u0026ndash;91\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"chemotherapy, cyclophosphamide, mesna, ovarian reserve","lastPublishedDoi":"10.21203/rs.3.rs-1399403/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1399403/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eThis study sought to investigate the protective effect of 2-mercaptoethane sodium sulfonate (mesna) on the ovarian reserve of rats being treated with cyclophosphamide.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eTwenty-four adult female Wistar albino rats were equally divided into three groups. Group A (n\u0026thinsp;=\u0026thinsp;8) received saline injections, Group B (n\u0026thinsp;=\u0026thinsp;8) received cyclophosphamide, and Group C (n\u0026thinsp;=\u0026thinsp;8) received cyclophosphamide\u0026thinsp;+\u0026thinsp;mesna. Preoperative blood samples (1 ml) were taken from all of the rats prior to any medication being given. The rats in all of the groups underwent bilateral oophorectomy and 1 ml of blood samples were taken 24 hours after the surgery. The difference in the anti-M\u0026uuml;llerian hormone (AMH) level between the pre- and postoperative blood samples was determined. The rats\u0026rsquo; ovaries were histomorphologically evaluated. Immune staining was performed to detect the AMH receptor expression level in the ovarian tissue. Finally, a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis was performed to assess the level of apoptosis.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eA considerable increase was noted in the AMH receptor expression level in Group C when compared with Group B (122 vs. 117, respectively; p: 0.007). In addition, the number of atretic follicles was found to be significantly lower in Group C than in Group B (10 vs. 5 respectively; p: 0.002). However, the TUNEL analysis revealed no significant difference between Group B and Group C with regard to apoptosis.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eMesna could decrease follicular atresia and increase the expression of AMH receptors in the follicles following acute cyclophosphamide toxicity.\u003c/p\u003e","manuscriptTitle":"Does mesna (mercapto-ethanesulphonate sodium) protect ovarian reserve after cyclophosphamide treatment: an in vivo study with rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-03-15 15:14:10","doi":"10.21203/rs.3.rs-1399403/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"41d6ab05-035c-4cc0-a276-48e21c4f10fd","owner":[],"postedDate":"March 15th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-04-27T16:14:04+00:00","versionOfRecord":[],"versionCreatedAt":"2022-03-15 15:14:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1399403","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1399403","identity":"rs-1399403","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}