Progesterone decreases the extent of ovarian damage caused by cisplatin in an experimental rat model

Material and methods. Forty rats were randomly divided into 4 groups; the control group (the saline group), the cisplatin-treated group (rats were injected with 5 mg/kg/week cisplatin intraperitoneally (i.p.)), the cisplatin + progesterone-treated group (the rats were pretreated with 8 mg/kg progesterone intramuscularly (i.m.) (8 mg/kg) before they were injected with 5 mg/kg/week cisplatin i.p.), and the progesteronetreated group (the rats were treated with 8 mg/kg progesterone i.m.). The ovaries were removed from the rats in all groups 5 days after the final injection of cisplatin.


Introduction
Chemotherapy and radiotherapy are the most common modalities of cancer treatment and they have substantially increased the survival period and quality of life in patients with cancer.One of the most important chemotherapeutic agents is cisplatin (cis-diamminedichloroplatinum(II) -CDDP), which is currently used in the treatment of a wide range of human tumors, and particularly solid tumors occurring in reproductive structures, like the testes and ovaries. 1 Unfortunately, chemotherapy treatment strategies give rise to other problems, especially in the reproductive age group, where prolonged chemotherapy exposes a large proportion of these patients to complications that can include infertility, ovarian dysfunction and increased follicular apoptosis. 2The use of cisplatin may, therefore, lead to side effects, such as ovarian failure, which limits proper administration of this anticancer drug. 3However, the mechanisms that give rise to cisplatin side effects are not clearly understood.For this reason, experimental studies have been initiated to explore methods for preventing reproductive damage in patients who must undergo extensive chemotherapy. 4The current literature indicates that the use of antioxidants might be helpful in preventing infertility caused by cisplatin-induced ovarian damage. 5rogesterone, a steroid hormone, is necessary for the retention of reproductive functions, such as ovulation and implantation, in females and it organizes the reproductive system.Besides this role in reproductive physiology, progesterone administration is reported to have a protective function against damage occurring during cerebral ischemia reperfusion and due to neurologic defects. 6Progesterone is well-documented to protect against injuries of various organs, including the kidney. 7For example, exogenously administered progesterone has a protective effect against acute kidney injury in rats subjected to ischemia reperfusion. 8Therefore, progesterone may have a similar protective effect in the ovary, and particularly on the ovarian reserve, during chemotherapy.
The status of the ovarian reserve is commonly monitored by anti-Müllerian hormone (AMH). 9Also known as Müllerian inhibiting substance, AMH is a 140 kDa polypeptide responsible for multiple reproductive functions.Yeh et al. recently showed that AMH is a better indicator of the ovarian reserve during the aging process than certain other ovarian hormones, as it is a necessary hormone for the growing follicular pool. 10Recent AMH studies suggest that chemotherapeutic drugs might cause ovarian damage and play a role in the failure of the ovarian reserve. 3However, in humans, few approaches are available that can protect patient's fertility and ovaries while applying chemotherapy. 11or these reasons, we hypothesized that progesterone might exert a protective effect on cisplatin-induced ovarian damage.The protective effects of progesterone have been previously studied, but no animal model has yet been developed for the study of the potential protective effect of progesterone on cisplatin-induced ovarian damage.Therefore, this study aimed to establish a rat model for the assessment of the effects of progesterone on cisplatin-induced ovarian damage via histopathological evaluation.The overall goal of the present study was to investigate whether progesterone can protect the ovaries of female rats against follicular damage during cisplatin chemotherapy.

Animals and drug administration
All experimental protocols were conducted in accordance with the institutional guidelines for the experimentation on animals at Erciyes University, Faculty of Medicine (Kayseri, Turkey), and the study was approved by the research ethics board of Erciyes University.For this study, 36 healthy female adult Wistar rats were obtained from Hakan Cetinsaya Experimental and Clinical Research Center of Erciyes University.All the animals were housed in plastic cages placed in a well-ventilated rat house, given ad libitum access to food and water, and subjected to a natural photoperiod of a 12-hour light/12-hour dark cycle.The animals were treated in accordance with the Guidelines for Animal Experimentation of Jichi Medical University (Shimotsuke, Japan), based on the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
The rats were randomly assigned to 4 groups: the saline group (n = 8), the cisplatin-treated group (n = 10), the progesterone + cisplatin-treated group (n = 10), and the progesterone-treated group (n = 8).The rats in the cisplatin group were administered a single intraperitoneal (i.p.) injection of cisplatin (5 mg/kg; Eczacibasi, Istanbul, Turkey).The rats in the control group were similarly administered the same volume of 0.9% sodium chloride (NaCL).Rats subjected to progesterone treatment were administered progesterone (8 mg/kg; Kocak Farma, Istanbul, Turkey) intramuscularly (i.m.) 30 min before the injection of cisplatin in the progesterone + cisplatin-treated group, or saline in the progesterone-treated group.All injections were repeated 1 week later.Five days after the 2 nd injection, the rats were sacrificed by decapitation under i.p. ketamine (50 mg/kg) xylazine (10 mg/ kg) anesthesia.After decapitation, the right and left ovaries were immediately excised.

Histological analysis
A morphological overview of the structure of the ovarian tissue was obtained using routine histological methods.Specimens were fixed in 10% formalin solution for 24-48 h, embedded in paraffin wax and cut into 5-µm thick sections.The slides were cleared in xylene, dehydrated in an ascending alcohol chain, and stained with hematoxylin-eosin (H&E) for the evaluation of the tissue morphology and structure.Photographs were taken with a photomicroscope (Olympus BX51; Olympus, Tokyo, Japan) and analyzed.
The evaluation scale suggested by Li et al. was applied, with some modifications, for the histopathological evaluation of the ovarian tissues. 12The histological sections were examined for the presence of vascular congestion, hemorrhage, follicular degeneration, leukocyte infiltration, and interstitial edema.The histological changes were scored from 0 to 3 according to the histological findings, where 0 represents no pathological findings, and 1, 2 and 3 represent pathological findings in <33%, 33-66% and >66% of the ovary, respectively.The scores for each parameter were calculated and the total scores were obtained.
Follicle counting and classification were performed on every 12 th section stained with H&E.All measurements were carried out only in follicles with an obvious nucleus, using ImageJ software (ImageJ, Bethesda, USA).The assessment of follicular quality was based on the cellular density and the integrity of the basement membrane and the oocyte.The follicles were classified as morphologically normal and only normal follicles were quantified according to these criteria.They were then classified in the histological preparations, based on stage, into primordial, primary, secondary, or antral follicles.Briefly, primordial follicles were defined as an oocyte surrounded by 1 single epithelial cell layer; primary follicles were characterized by a single cell layer with cubic or high-prismatic cells; secondary follicles consisted of multiple layers of granulosa cells; and Graafian follicles were defined as those with antrum folliculi, stratum granulosum and cumulus oophorus.

Immunohistochemistry
The immunoexpression of AMH within the follicles of the ovaries was detected using the avidin-biotin-peroxidase method (ImmunoCruz ™ Staining System, Sc:2053; Santa Cruz Biotechnology Inc., Santa Cruz, USA), following the manufacturer's recommendations.Briefly, serial 5-μm thick paraffin-embedded sections were deparaffinized, rehydrated in graded alcohol and incubated for 5 min in phosphate buffered saline (PBS) at room temperature.Antigen retrieval was carried out by microwave treatment in 0.01M sodium citrate buffer (pH 6.0) at 95°C for 15 min.The specimens were washed several times with PBS and endogenous peroxidase activity was inhibited with 3% hydrogen peroxide in methanol for 10 min.The specimens were incubated with a serum-blocking agent to block nonspecific staining.The histological sections were then incubated overnight at 4°C with a 1/50 dilution of goat polyclonal anti-AMH antibody (MIS (C-20): sc-6886; Santa Cruz Biotechnology Inc.).For immunostaining assays, primary antibodies were omitted as a negative control.After washing with PBS, the sections were incubated for 15 min with the biotinylated secondary antibodies.The sections were visualized by treatment for 3-5 min at room temperature with 3,3-P-diaminobenzidine tetrahydrochloride (DAB) as a chromogen and then lightly counterstained with hematoxylin.Images were taken using an immunofluorescence microscope (Olympus BX51; Olympus).We selected primordial, primary, preantral, secondary, and mature or Graafian follicles in sections from each experimental group at the same magnification (×40).The mean immunoreactivity intensity for each follicle in an ovarian section was measured using ImageJ software (ImageJ).

TUNEL staining
In situ detection of apoptosis was performed in the sections by terminal deoxynucleotide transferase-mediated 20-deoxyuridine 50-triphosphate nick end-labeling (TU-NEL), using an In situ Cell Death Detection Kit (Chemicon, Temecula, USA), according to the manufacturer's instructions.Briefly, paraffin sections (5-μm) from the ovarian tissues were deparaffinized, rehydrated and washed twice in PBS for 5 min.The TUNEL reaction mixture was added to each slide and incubated in a humidified atmosphere for 60 min at 37°C in the dark.After washing with PBS, the sections were then incubated with a converter reagent for 30 min.Color development for the localization of cells containing labeled DNA strand breaks was performed by incubating the slides with Fast Red Substrate System (F4648; Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) for 5 min.Finally, the association with the number of TUNEL-positive cells in the rats from the experimental groups was evaluated by identifying apoptotic cells by morphology.At least 3 randomly chosen fields in each slide were counted at the original ×20 magnification.All slides were examined and photographed using an Olympus BX51 microscope (Olympus).The numbers of apoptotic cells were determined by carefully counting the TUNELpositive cells, using ImageJ software (ImageJ) at the same magnification. 2

Statistical analysis
Statistical significance was evaluated using GraphPad Prism v. 5.01 (Graph Pad Software Inc., San Diego, USA).The one-way analysis of variance (ANOVA) was followed by an appropriate post hoc comparison (depending on the given Gaussian distribution) and all the data was presented as the mean of normalized data ± standard error of the mean (SEM).The results were considered significant if the p-value was <0.05.

Results
The histological sections stained with H&E revealed that the control and progesterone-treated groups had normal ovarian architecture with no considerable pathologic alteration (Fig. 1).Ovarian follicles in various stages of development and with normal appearance were observed in the ovarian cortex, and the cuboid germinal or follicular cells around the oocyte were examined.The cisplatininjected group showed the absence of Graafian follicles and the presence of the corpus luteum (CL), whereas the sections taken from the control tissues exhibited the presence of follicles at different stages of maturation.Cisplatin treatment also resulted in microscopy findings of hemorrhage and vascular congestion, as well as follicular degeneration and edema in the ovarian tissue when compared to the control group.The group that was administered cisplatin + progesterone showed more moderate, but morphologically similar, histological findings of degeneration, hemorrhage and edema.Overall, progesterone markedly decreased cisplatin-induced ovarian damage.
For follicle counting at different stages in each experimental group, unhealthy follicles were distinguished from healthy follicles by nuclear pyknosis and the disappearance of granulosa cells, whereas healthy follicles were classified considering their different developmental stages.The control group showed a number of follicles at various stages, whereas significantly fewer primordial, secondary and Graafian follicles were found in the cisplatin group than in the control group (p < 0.05), indicating substantial damage to the quality of the follicle in the cisplatin group.Primordial and primary follicle counts were higher in the control group than in the cisplatin group, and this difference was statistically significant (p < 0.05).Primordial and primary follicle counts were similar in the cisplatin + progesterone-treated group and the cisplatin group (p < 0.05).The numbers of preantral, secondary and Graafian follicles did not differ among the groups.Preantral, secondary and Graafian follicle counts were lower in the cisplatin + progesterone-treated group than in the cisplatin-treated group.No statistically significant differences were seen between the control and progesterone groups regarding the follicle numbers (Fig. 1E).All histological scores are listed in Table 1.The histopathological differences were marked as follows: 0 -normal; 1 -moderate (moderate vascular congestion, edema, no hemorrhage, and no follicular degeneration); 2 -moderate (moderate vascular congestion, edema, no hemorrhage, and no follicular degeneration); and 3 -severe (severe vascular congestion, edema, hemorrhage, follicular degeneration, and leukocyte infiltration).
The influence of progesterone on AMH immunoreactivity intensity in the ovaries of the rats in all experimental groups is shown in Fig. 2. Anti-Müllerian hormone immunohistochemistry was analyzed by follicle class.No AMH was detected in atretic follicles, theca cells and the oocytes of follicles in the ovarium.Anti-Müllerian hormone was primarily expressed in granulosa cells of primary, preantral and small antral follicles.Anti-Müllerian hormone immunoreactivity intensity progressively decreased in the subsequent phases of follicle development in the control and progesterone-treated groups, whereas the immunoreactivity intensity of AMH was consistently lower in the ovaries of the cisplatin-treated group.The immunoreactivity intensity of AMH was lower, especially in primordial and primary follicles at early development stages, in the cisplatin-administered group than in the control group, but the differences were not statistically significant.The immunoreactivity intensity of AMH did not differ between secondary and tertiary follicles (Fig. 2E).
The TUNEL evaluations for DNA fragmentation in cell nuclei in each group are shown in Fig. 3.The control group showed no increases in the numbers of apoptotic cells in follicles at different stages.By contrast, the numbers of TUNEL-positive cells were significantly higher in the cisplatin-treated groups than in the control group (p < 0.001).The cisplatin group showed badly damaged follicle structures, resulting in a significant rise in the number of TUNEL-positive cells.Markedly lower numbers of TUNEL-positive cells were seen in follicles of the progesterone-treated groups than in the cisplatin-treated group (p < 0.001).Thus, the induction of large numbers of TUNEL-positive cells in the ovarian tissue by cisplatin treatment was significantly suppressed by pretreatment with progesterone (Fig. 3).

Discussion
The present study investigated whether progesterone could protect against cisplatin-induced ovarian damage in a rat model.We demonstrated that an injection of progesterone 30 min prior to cisplatin administration attenuated the cisplatin-induced ovarian injury by reducing cellular apoptosis.Progesterone also positively modulated AMH expression, indicating a possible protection of the ovarian reserve against cisplatin-induced injury.In addition, administration of progesterone led to partial protection.Evaluation of the ovarian reserve following chemotherapy is vital to ensure adequate subsequent fertility.
The targets of the chemotherapeutic drugs might be the granulosa cells in developing follicles, which may affect oocyte maturation and cause follicular destruction, followed by ovarian failure as the result of any damage to these cells.Follicle damage induced by chemotherapy is likely the main cause of infertility.For example, Meirow et al. reported that chemotherapy, in a drug-or dose-addiction, causes   C -effects of progesterone on TUNEL-positive cells in cisplatin injury in rats, a marked decrease was evident in the number of TUNEL-positive cells when compared with the cisplatin-treated group; D -the progesterone-treated group had also fewer TUNEL-positive cells than were observed in the control group; E -graph showing quantification of the different groups.The number of TUNEL-positive cells was higher in the ovarian tissues of the cisplatin-treated group than in the control group and in other groups Arrow -TUNEL-positive cell; star -preantral follicle; head of arrow -primary follicle; thick arrow -secondary follicle; TUNEL -terminal deoxynucleotide transferase-mediated 20-deoxyuridine 50-triphosphate nick end-labeling; Con -control; Cis -cisplatin; Pro -progesterone; TUNEL staining ×200; * p < 0.001.a depletion of the primordial follicle pool. 13Treatment with cisplatin causes an increased prevalence of premature ovarian failure in humans. 14In rats, cisplatin can cause ovarian damage, decreased immunoreactivity intensity of AMH and changes in the estrous cycle. 15isplatin is the commonly preferred anticancer agent for treatment of a wide range of cancer types, ranging from sarcomas and small cell lung cancer to ovarian cancer, cervical cancer and lymphomas. 16The antitumor effect of cisplatin depends on the formation within the cell of a platinum complex that participates in DNA cross-linking and apoptosis. 17In addition, cisplatin is thought to trigger DNA damage, failure of mitochondrial function and a hindrance in protein synthesis. 1,18,19However, these effects on dividing cancer cells may also adversely occur in healthy dividing cells, such as the granulosa cells of follicles, thereby leading to ovarian failure, and ultimately, infertility. 12 positive role of progesterone has been indicated in some disorders, such as traumatic brain and spinal cord injury, neuroinflammation, stroke, ischemia, diabetic neuropathy, and neurodegeneration. 20,21The granulosa cells and the CL in the ovary produce progesterone, which is necessary in mammals for the establishment and maintenance of pregnancy. 22The production of interleukin (IL)-6 can TUNEL-positive cell number also be inhibited by progesterone, suggesting a probable immunosuppressive and anti-inflammatory role as well. 23e et al. showed that progesterone reduces brain edema, ameliorates brain complications with its anti-inflammatory effects and reduces the levels of inflammatory cytokines. 24Progesterone also suppresses the production of proinflammatory cytokines and chemokines in monocytes, and appears to play an important anti-inflammatory role. 25,26Ghasemi et al. demonstrated a beneficial and dose-dependent effect of progesterone on cisplatininduced nephrotoxicity. 7However, the protective effect of progesterone against cisplatin-caused ovarian damage has not been studied extensively.
In the present study, AMH expression was significantly increased in the progesterone + cisplatin-treated group when compared to the cisplatin-treated group, indicating that progesterone can preserve the granulosa cells in growing follicles.No significant increase was observed in AMH immunoreactivity intensity in the progesterone-treated group when compared to the control group.Our results indicated that pretreatment with progesterone before the administration of cisplatin could have protected the follicle cells from the adverse effects of cisplatin, as indicated by the higher AMH immunoreactivity intensity in the progesterone + cisplatin-treated group as compared to the cisplatin-treated group..Anti-Müllerian hormone knockout mouse studies have shown that AMH functions as a negative stimulator of follicular maturation and an inhibitor of follicle-stimulating hormone (FSH) sensitivity. 27nalyzing the apoptotic mechanism is crucial in explaining the adverse cisplatin effects and the mechanisms underlying the action of possible protective agents against these effects.The ideal preventative or protective agents also must not disturb the antitumor effects of chemotherapeutics.Cisplatin can cause the generation of oxygen-free radicals, which then induce oxidative stress that gives rise to cisplatin-induced tissue damage. 28Progesterone used together with chemotherapeutic drugs clearly decreased apoptosis in the present study.Devarajan et al. demonstrated that cisplatin induced both the intrinsic and the extrinsic apoptotic pathways. 29Progesterone may decrease apoptosis by decreasing pro-apoptotic enzymes and increasing anti-apoptotic proteins. 30In the present study, the antiapoptotic effect of progesterone on cisplatin-induced ovarian damage was confirmed by the reduction in the number of TUNEL-positive apoptotic cells in the progesterone + cisplatin-treated group.
This study provides additional details about the damage caused by cisplatin to the ovary.It also presents methods for determining the protective effect of progesterone against this damage and would appear to be the first report on the effect of progesterone on cisplatin-induced ovarian damage and the immunoreactivity intensity of AMH.Taken together, our results indicate that progesterone partially prevents the damage caused by cisplatin.This finding could be crucial for women cancer patients to maintain their fertility.
In summary, we obtained data from a rat model that support the hypothesis that apoptosis can be regulated by the natural sex steroid progesterone.Additional clinical and immunohistochemical research should be carried out to confirm this conclusion about the ameliorating effect of progesterone on ovarian tissue damage, and to evaluate the efficacy, safety and potential use of progesterone.

Fig. 1 .
Fig.1.Representative photomicrographs of H&Estained sections of rat ovaries in each experimental group: A -light microscopy of the ovarian tissue with developing follicles in many different stages in the control group; B -histopathologic injury was more obvious in the cisplatin-treated groups than in other groups, degenerative changes, such as hemorrhage, follicular degeneration and edema, were evident; C -a histological image of the progesterone + cisplatin-treated group shows a significant amelioration of the ovarian histoarchitecture when compared to the disrupted histology observed in the cisplatin-treated group; D -the progesterone-treated group displayed an improved histological appearance with an orderly arrangement of follicles; E -quantification of histopathological scores and number of each type of follicles in the ovarian sections from the experimental groups Original magnification ×200; star -follicular degeneration; arrow -hemorrhage; head of arrow -edema (E); H&E -hematoxylin/eosin; Con -control; Cis -cisplatin; Pro -progesterone; * p < 0.05.

Fig. 2 .
Fig.2.Photomicrographs of rat ovarian sections and immunostaining of AMH in the different groups: A -the control group showed AMH staining in granulosa cells surrounding the oocyte in early primary, preantral and small antral follicles; B -the positive staining for AMH was decreased in the cisplatin-treated group; C -AMH-positive staining of the ovary in the progesterone + cisplatin-treated group; D -similar AMH immunoreactivity was evident in the progesterone-treated group and in the control group; E -quantification of AMH immunoreactivity intensity in ovarian sections from the experimental groups Original magnification ×200; AMH -anti-Müllerian hormone; Con -control; Cis -cisplatin; Pro -progesterone.

Fig. 3 .
Fig.3.Progesterone pretreatment inhibits the development of TUNEL-positive cells in the rat ovary due to cisplatin-induced ovarian damage: A -only a few TUNEL-positive cells were evident in the ovaries in the control group; B -the TUNEL-positive cell number increased in the cisplatin-treated group; C -effects of progesterone on TUNEL-positive cells in cisplatin injury in rats, a marked decrease was evident in the number of TUNEL-positive cells when compared with the cisplatin-treated group; D -the progesterone-treated group had also fewer TUNEL-positive cells than were observed in the control group; E -graph showing quantification of the different groups.The number of TUNEL-positive cells was higher in the ovarian tissues of the cisplatin-treated group than in the control group and in other groups

Table 1 .
Histopathological grading of rat ovary lesions in the treatment groups