DOI: https://doi.org/10.21203/rs.3.rs-1399403/v1
This study sought to investigate the protective effect of 2-mercaptoethane sodium sulfonate (mesna) on the ovarian reserve of rats being treated with cyclophosphamide.
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.
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.
Mesna could decrease follicular atresia and increase the expression of AMH receptors in the follicles following acute cyclophosphamide toxicity.
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.
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 cm2 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.
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.
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.
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).
| 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).
| 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 |
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.
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. Approval was granted by the Ethics Committee of University of Health Sciences, Bagcılar State Hospital, Animal Laboratory with the approval number: 2020-18.