Caffeic Acid Phenethyl Ester | Histone Demethylase Signal

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Investigation of the preventive effects of dehydroepiandrosterone (DHEAa) and Caffeic acid phenethyl ester (CAPE) on cisplatin-induced ovarian damage in rats

Ali Doğukan Anğın, Ismet Gün, Önder Sakin, Muzaffer Seyhan Çıkman, Engin Ersin Şimşek, Resul Karakuş, Kayhan Başak & Asuman Orçun Kaptanağası

To cite this article: Ali Doğukan Anğın, Ismet Gün, Önder Sakin, Muzaffer Seyhan Çıkman, Engin Ersin Şimşek, Resul Karakuş, Kayhan Başak & Asuman Orçun Kaptanağası (2020): Investigation of the preventive effects of dehydroepiandrosterone (DHEA) and Caffeic acid phenethyl ester (CAPE) on cisplatin-induced ovarian damage in rats, Ultrastructural Pathology,
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ImageImageULTRASTRUCTURAL PATHOLOGY

Investigation of the preventive effects of dehydroepiandrosterone (DHEA) and Caffeic acid phenethyl ester (CAPE) on cisplatin-induced ovarian damage in rats
ImageAli Doğukan Anğın a, Ismet Günb, Önder Sakina, Muzaffer Seyhan Çıkmana, Engin Ersin Şimşekc, Resul Karakuşd,
Kayhan Başake, and Asuman Orçun Kaptanağasıf
aDepartment of Obstetrics and Gynecology, University of Health Sciences, Dr Lütfi Kırdar Kartal Training and Research Hospital, İstanbul, Turkey; bDepartment of Obstetrics and Gynecology, University of Health Sciences, Sultan Abdülhamid Han Training and Research Hospital, İstanbul, Turkey; cDepartment of Family Medicine, University of Health Sciences, Dr Lütfi Kırdar Kartal Training and Research Hospital, İstanbul, Turkey; dDepartment of Obstetrics and Gynecology, University of Health Sciences, Zeynep Kamil Women’s and Children’s Disease Training and Research Hospital, İstanbul, Turkey; eDepartment of Pathology, University of Health Sciences, Dr Lütfi Kırdar Kartal Training and Research Hospital, İstanbul, Turkey; fDepartment of Biochemistry, University of Health Sciences, Dr Lütfi Kırdar Kartal Training and Research Hospital, İstanbul, Turkey

ABSTRACT
To investigate whether Dehydroepiandrosterone (DHEA) and Caffeic acid phenethyl ester (CAPE) had any preventive effect against the ovarian damage caused by cisplatin (CP) (cis- diamminedichloroplatinum) in rats. On the first day ovaries were removed, Anti-Müllerian hormone (AMH) was measured (Group1, n:6), in the other groups 7.5 mg/kg cisplatin was administered intraperitoneally. In Group 2 (n = 6), 0.1 ml saline, in Group 3 (n = 5), 20 umol/kg CAPE, in Group 4 (n = 7), DHEA 6 mg/kg were administered every day. On the 10th day, ovaries were removed, AMH was measured. Ovary reserve (primordial/primary/secondary/ tertiary/atretic follicles, AMH), ovarian damage scores (follicular degeneration, congestion, hemorrhage, edema, inflammation) were compared. The number of tertiary follicles were statistically high in the CAPE group (p = .015), the inflammation score in the DHEA group (p = .012), AMH level (p = .009) in the control group. The lowest number of atretic follicles (AF) was in the control group, while the highest number of AF was in the DHEA group (p = .002). Significant decreases in AF were the case in the cisplatin and DHEA groups compared to the control group (p < .008). The AMH values had the highest positive correlation with the number of primordial follicles and the highest negative correlation with the number of AF. The cut off point for AMH was 1.57 ng/ml as an indicator of low ovarian reserve. Cisplatin causes total damage and increased numbers of AF on the ovary. Depending on this, AMH levels fall. These negative effects of cisplatin are not obstructed by CAPE or DHEA, and may even be increased by DHEA.
ARTICLE HISTORY
Received 27 March 2019
Revised 7 December 2019
Accepted 31 December 2019
KEYWORDS
DHEA; CAPE; cisplatin; rat; ovary; AMH

Introduction
Ovarian cancer is one of the most common female cancers.1 CP is one of the most commonly used agents in the medical treatment of ovarian cancer.2 Cisplatin has certain undesirable risks, such as its adverse effects on ovarian reserve (OR) or the fact that it causes decreased fertility, some of these effects are the decrease in the number of primordial follicles and in the levels of AMH.3 Fertility in the young-age group patients is extremely important. For this reason, the adverse effects of cisplatin on OR have been investigated for many years.4–10
AMH is a hormone released from the preantral and antral follicles from granulosa cells.11 It is considered to be the best indicator of OR, regard- less of menstrual variables.12–14 It has also been stated that AMH is a useful biomarker to assess OR in case of the implementation of various chemotherapeutics.15
CAPE is one of the most active compounds of propolis obtained from bee honey. It has extensive biological characteristics. It has antioxidant, anti- inflammatory, anticarcinogenic characteristics and regulates apoptosis.16–18 The successful effects of CAPE on oxidative damage have been demon- strated in many tissues in the body. Its effects

ImageImageCONTACT Ali Doğukan Anğın [email protected] Department of Obstetrics and Gynecology, University of Health Sciences, Dr Lütfi Kırdar Kartal Training and Research Hospital, İstanbul, Turkey
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/iusp.
© 2020 Taylor & Francis Group, LLC

have been investigated in the brain, heart, kidneys, testicles, genital organs, ovaries and many different tissues.19–21 In a recent literature review, the importance of CAPE in improving the pathologies due to oxidative damage has been highlighted, but it has been stated that further studies are needed to determine its correct use.22
DHEA, on the other hand, is one of the most frequently used agents to increase OR in infertility patients. In previous studies, I has been stated that DHEA has shown encouraging results to increase oocyte and embryo yield and live birth rates in those with diminished ovarian reserve (DOR).23–26 A meta-analysis which recently conducted indicated that DHEA supplementation could improve out- comes of pregnancy in patients with decreased ovary response. Nevertheless, it has been stated that further randomized controlled trials are needed.27 In a study investigating the effect of DHEA on follicu- logenesis, it has been indicated that it provided increased number of primordial, primary and grow- ing follicles, but it has been stated that more infor- mation can be obtained by examining AMH.28 The effects of DHEA on follicles have never been evalu- ated in the patient group using cisplatin, either.
Through this research study, we primarily aimed to assess OR by evaluating the cisplatin-induced ovarian damage in the group of patients whose desire for fertility was extremely high. And then, we aimed to investigate whether DHEA and CAPE had preventive properties against the damage.

Materials and methods
The study was carried out at the Animal Experimental Laboratory of University, and approval was received from the Animal Experiments Local Ethics Committee of University. The procedures were in accordance with the ethical standards of the respon- sible committee on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. For the experiment, 30 3–6-month-old, 200–300gr, Wistar Albino type female rats were used. Four groups were formed by random assignment. At first, the study was initiated with a total of 30 mice, 8 of them in all groups except the control group (n: 6). As a result of the mice lost during the study, the experi- ment was completed with 6 mice in Group 2, 5 mice in Group 3 and 7 mice in Group 4.
Experimental groups
Group1 – CONTROL Group (n = 6): A direct opera- tion was performed on the first day, and ovaries were bilaterally excised and sent to pathology. Blood sam- ples were obtained for AMH quantification.
Group2 – CISPLATIN Group (n = 6): Each rat was given 7.5 mg/kg (~1.8 ml) cisplatin intraper- itoneally once on the first day.29 Every day, 0.1 ml of physiological saline solution was administered intraperitoneally. At the end of the 10th day, the ovaries of the remaining 6 rats were excised bilat- erally, and blood samples were obtained for AMH quantification.
Group3 – CAPE Group (n = 5): Each rat was given
7.5 mg/kg cisplatin intraperitoneally once on the first day. Additionally, 20 umol/kg CAPE (Sigma Aldrich® Caffeic Acid Phenyl Ester 99% 25mg, Biostar, Ankara, Turkey) was administered intraper- itoneally every day for 10days.20 At the end of the 10th day, the ovaries of the remaining 5 rats were excised bilaterally by surgery, and blood samples were obtained for AMH quantification.
Group4 – DHEA Group (n = 7): Each rat was given
7.5 mg/kg cisplatin intraperitoneally once on the first day. Additionally, DHEA (CAYMAN®, Dehydroepiandrosterone %97 1gr, Farmasina, İstanbul/Turkey) with a dose of 6 mg/kg dissolved in
0.1 ml sesame oil (by dissolving it in 0.01 ml 95% ethanol and mixing it with 0.09 ml sesame oil) was administered intraperitoneally every day for 10 days.30 At the end of the 10th day, the ovaries of the remaining 7 rats were excised bilaterally by surgery, and blood samples were obtained for AMH quantification.
The drugs were stored on a refrigerator shelf at
+4°C for daily application. The rats in a controlled environment (at 21°C room temperature and 60% humidity) with 12h light/dark cycles, and were fed ad libitum. After excision of their ovaries and blood draw, the animals were decapitated and dumped through red medical waste bins. Rats were caged individually in a room and fed ad libitum at 21°C room temperature and 60% humidity level with 12 hour light/dark cycle.

Form of surgery
The rats were starved to empty and relieve their passage 6 hours before the surgeries, but water

consumption was not restricted. All surgical pro- cedures were performed using sterile, powder-free, latex gloves. After the rats were given 80 mg/kg of 10% ketamine hydrochloride (Ketalar; Eczacıbaşı¸ Warner Lambert, Istanbul, Turkey) and 15 mg/kg of 2% xylazine hydrochloride (Rompun; Bayer Health Care LCC, Kansas, KS) anesthesia, laparot- omy was performed. The rats were laid in the supine position on the operating table. Before the surgery, the outer face of the abdomen was shaved. The abdomen was cleaned using povidone-iodine 10% solution (Batticon; Adeka Laboratories, Istanbul, Turkey). The abdomen was entered through a 5-cm midline incision (between xiphoid and pubis).

Amh examinations
The first plan in the study was to examine AMH through the tail vein of each group before the opera- tion (on the 1st day). However, it was not possible to draw blood from different regions such as the tail vein and the femoral veins, accompanied by a veterinary surgeon. To prevent possible damage, such as the loss of animals, the blood was drawn through injection at the end of the 1st day in the control group and at the end of the 10th day in the other groups intracardially during the operation, after the ovaries were excised. The blood samples were placed in heparinized tubes (venotect®Italy). Blood samples were centrifuged in 30 minutes after it was drawn (15 minutes 1000 × g). The serum was removed, and the remaining plasma was placed in an Eppendorf tube and kept frozen at −20°C until the day to be analyzed. AMH levels were measured from plasma in the form of “ng/ml” by a biochemist blind to the groups in our university laboratory by using the ELISA method. All the samples were tested in the same experiment. A kit with a sensitivity of
0.10 ng/mL, a detection range of 0.16–10 ng/mL and a variability coefficient of less than 10% was used for the rat AMH kit (Elabscience, Rat AMH kit; Houston, Texas, ABD).

Histopathological examinations
The removed ovaries were secured in 10% formal in. All pathological examinations were examined by the same expert pathologist physician (K.B.)
blindly at the Pathology Clinic of Kartal Training and Research Hospital. Paraffin blocking was per- formed 24 hours after the oophorectomy. Afterward, 5 micrometer sections were taken from both ovaries. The sections taken from both ovaries were evaluated in terms of follicular activ- ity and ovarian damage and recorded as the aver- age of the data obtained. Samples were stained with hematoxylin eosin and the light was assessed by using a light microscope. All follicles in the ovaries (primordial, primary, secondary, tertiary, and atretic) were counted and the ovary reserve was assessed (Figure 1). Ovarian damage – includ- ing follicular cell degeneration, vascular conges- tion, hemorrhage, edema and inflammation (neutrophil infiltration) – was scored histologically using a graduated scale (0 = none, 1 = mild, 2 = moderate, and 3 = severe) (Figure 2).

Statistical analysis
For statistical studies, the IBM SPSS statistics version
24 was used. The Shapiro-Wilk and Kolmogorov Smirnov tests were used to test the normality of dis- tributions. The one-way analysis of variance (ANOVA) was used when comparing three or more groups with a normal distribution, whereas the Kruskal Walls test was used when comparing three or more groups with a non-normal distribution. Following that, the Mann Whitney U test with Bonferroni correction was used for pair wise compar- isons. After the Bonferroni correction, p < .008 was considered statistically significant. The chi-square test was used to compare the categorical changes. ROC analysis was performed to determine the predictive value for AMH, considering that Cisplatin would harm the ovary in all conditions between the control and total other groups. DOR group and normal group were created via the calculated prediction point. Subsequently, a binary logistic regression analysis was performed to investigate how total ovarian damage and its subgroups, and total number of folli- cles and its subgroups affect AMH, and an attempt was made to find an appropriate model. A correlation analysis was carried out to investigate whether there was a linear relationship between our dependent vari- able AMH and other continuous variables. Following that, if conditions were appropriate, a Univariate Covariate Analysis of Variance (ANCOVA) test was

Imagea
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c
d

Figure 1. a: Primordial (white arrow heads) and primary (black arrow heads) follicles in cortical area (H&E, ×400). b: Degenerated follicle and the defragmented oocyte in it (H&E, ×400). c: Secondary (black arrow head) and tertiary (white arrow heads) follicles (H&E, ×200). d: Atretic follicle degenerated before completion of maturation and defragmented oocyte in it (H&E, ×400).

Imagea
b
c
d

Figure 2. a: Inflammatory cells in the ovarian cortical area (arrow heads) (H&E, ×400). b: Edema in ovarian stroma (H&E, ×200). c: Widespread congestion in vessels (H&E, ×100). d: Bleeding in ovarian stroma (H&E, ×400).

carried out using appropriate models to determine whether the difference in the dependent variable between the groups was caused by the independent variables. A p value less than .05 was considered as statistically significant.

Results
In this study, When the groups were compared in terms of OR (follicular counts and AMH values) and ovarian damage scores, the tertiary follicle count in the CAPE group (p = .015), and the inflammation score in DHEA group (p = .012) and the AMH level (p = .009) in the control group were statistically sig- nificantly higher as seen in Table 1. The number of
atretic follicles was the lowest in the control group and the highest in the DHEA group (p = .002). AMH values and total follicle counts among groups seen in Figure 3.
During the pairwise comparisons of AMH, ter- tiary follicle count and atretic follicle count between the groups, as seen in Table 2, there was a significant decrease in the AMH level between Group 1 and Group 4 (p = .003), and the atretic follicle count was significantly higher in Group 2 and Group 4 than in the control group (p = .003 and 0.002, respectively). When Spearman’s correlation analyzes were car- ried out to reveal the linear relationship between AMH and ovarian follicular values and between AMH and ovarian damage parameters, the strongest

Table 1. Intergroup comparisons of follicle counts, ovarian damage scores and AMH values.

Parameter/score Group1 (n = 6) Group2 (n = 6) Group3 (n = 5) Group4 (n = 7) Total (n = 24)
p
Primordial follicle 14.17 ± 4.17 10.17 ± 8.66 8.60 ± 2.97 7.71 ± 5.82 10.13 ± 6.08 .263a
Primary follicle 15.17 ± 4.96 16.00 ± 7.93 14.20 ± 3.70 15.43 ± 8.75 15.25 ± 6.46 .978a
Secondary follicle 7.17 ± 3.37 5.50 ± 1.87 6.00 ± 3.00 4.71 ± 1.60 5.79 ± 2.52 .384a
Tertiary follicle 3.83 ± 1.33 3.33 ± 1.97 9.80 ± 2.86 5.29 ± 2.29 5.38 ± 3.16 .015b
Atretic follicle .50 ± .55 3.33 ± 2.07 2.20 ± .84 3.71 ± .95 2.50 ± 1.75 .002b
Total follicle 40.33 ± 8.78 35.00 ± 15.52 38.60 ± 5.23 33.14 ± 14.15 36.54 ± 11.61 .927a
Hemorrhage 0 83.3% (5) 100% (6) 80% (4) 100% (7) 91,7% (22) .454 c
1 16.7% (1) 0.0% (0) 20% (1) 0.0% (0) 8.3% (2)
Vascular congestion 0-1 83.3% (5) 33.3% (2) 40% (2) 28.6% (2) 45.8% (11) .196 c
2-3 16.7% (1) 66.7% (4) 60% (3) 71.4% (5) 54.2% (13)
Cellular degeneration 0 50% (3) 50% (3) 0.0% (0) 42.9% (3) 37.5% (9) .274 c
1 50% (3) 50% (3) 100% (5) 57.1% (4) 62.5% (15)
Inflammatory cell infiltration 0 33.3% (2) 83.3% (5) 100% (5) 100% (7) 79.2% (19) .012 c
1 66.7% (4) 16.7% (1) 0.0% (0) 0.0% (0) 20.8% (5)
Edema 0 66.7% (4) 50% (3) 40% (2) 28.6% (2) 45.8% (11) .572 c

Total damage 1-2 33.3% (2)
2.83 ± 1.84 50% (3)
3.33 ± 1.51 60% (3)
4.20 ± 1.30 71.4% (5)
3.43 ± 1.13 54.2% (13)
3.42 ± 1.44 .344 b
AMH (ng/ml) 5.03 ± 4.32 1.91 ± 3.00 .78 ± .38 .47 ± .15 2.03 ± 3.08 .009 b
AMH (ng/ml)* 4.77 ± 1.40 2.24 ± 1.12 .13 ± 1.20 .87 ± 1.12 2.03 ± 2.38 .094d
The data are presented in average ± SD, or in average ± Std.Error (*) or in % (n). For statistics, a: ANOVA, b: Kruskal Wallis Test, c: x2 test and d: Univariate test were used. *For corrected AMH values, ANCOVA test was used (total damage = 3.42 and atretic follicle = 2.50). Ovarian damage – including follicular cell degeneration, vascular congestion, hemorrhage, edema and inflammation (neutrophil infiltration) – was scored histolo- gically using a graduated scale (0 = none, 1 = mild, 2 = moderate, and 3 = severe)

Image
Figure 3. AMH values and total follicle counts among groups.

Table 2. Pairwise intergroup comparisons of AMH, tertiary fol- licle and atretic follicle.
Groups and p 1–2 1–3 1–4 2–3 2–4 3–4
p1 .037 .011 .003 1.000 .352 .167
p2 .666 .011 .236 .011 .210 .026
p3 .003 .010 .002 .548 .455 .024
p4 1.000 .109 .442 1.000 1.000 1.000
1,2,3Mann-Whitney Test (p < .008 was considered significant), 4Univariate test. p1 AMH, p2 Tertiary follicle, p3 Atretic follicle, p4 AMH corrected by using ANCOVA.

association was between the primordial follicle and AMH (r = 0.541 and p = .006) in the positive direc- tion, and between atretic follicle and AMH in the negative direction (r = −0.473 and p = .020) (Figure 4).
The estimation value for AMH indicating a low ovarian reserve was found as 1.57 ng/ml (sensitiv- ity = 83.3% and sensitivity = 94.4%, area under the curve = .944, p = .001) (Figure 5). After the test for each independent variable, it was seen that the best modeling was the number of atretic follicles (OR = 0.025 and 95% CI 0.002–0.337; the probabil- ity of making the right decision for DOR = 87.5%) and the ovarian total damage score, respectively (OR = 0.100 and 95% CI 0.012–0.818; the probabil- ity of making the right decision for DOR = 79.2%). When the both independent variables were used together, the probability of making the right deci- sion for DOR was 87.5%. When the total damage score and atretic follicle count were fixed to 3.42 and 2.50, respectively, it was seen that the difference between the groups in terms of the dependent vari- able AMH diminished (p = .094). Considering

Image
Figure 5. ROC Curve in determining low ovarian reserve.

Table 1, it is seen that the corrected AMH values increased in Group 2 and Group 4 and decreased in Group 1 and Group 3.

Discussion
AMH and antral follicle count are key markers that are commonly used to reveal OR.31 The ovarian mechanism of action of AMH, which is released from the granulosa cells and plays a role in follicu- logenesis, is still unclear.32 There is a strong correla- tion between AMH and the primordial and preantral follicles.7,33 As seen in Figure 4, we also found that the AMH value decreased significantly as the num- ber of primordial follicles decreased (p = .006). There

Image

Figure 4. The linear relationship between AMH and the primordial follicle and between MH and the atretic follicle.

are studies stating that AMH is an ovarian growth factor that prevents follicular atresia.34,35 As opposed to this finding, we found that AMH decreased as the number of atretic follicles increased, and we think that it is due to follicular apoptosis caused by CP (Figure 4). Although AMH has an effect that pre- vents follicular atresia, this effect may be playing a protective role for the follicles occurring during the normal cycle overtime, but it appears not to be effective against a chemotherapeutic agent.
It is known that CP, which is a chemothera- peutic agent, damages ovaries, decreases primor- dial follicles, reduces the AMH levels, and increases ovarian damage.33,36 These effects of CP were also observed in our study as an expected outcome, and the primordial follicle counts as well as total follicle counts decreased, total ovary damage increased and AMH levels decreased in all groups where CP was adminis- tered (Table 1, Figure 3). When CP is compared with the control group, it is seen that almost all values were impaired; in addition, the atretic follicle count was significantly higher in the CP group compared to that of the control group (p = .003) (Table 2). In their study, Atli et al. have observed a significant decrease in the num- ber of follicles only in the group given CP com- pared to the control group, but there was no significant decrease in AMH values, and basal AMH levels were also measured.3 Erbaş et al. have stated that CP significantly reduces AMH, but it is a study that was carried out without basal AMH levels.7 CP has been reported to reduce AMH levels through the damage it causes on AMH-secreting follicles.37 When we fixed the total ovarian damage and the atretic follicle level to eliminate this effect (the ANCOVA test), the AMH levels in the control and CAPE groups decreased whereas they increased in the other groups; and the difference between the groups was eliminated (p = .094) (Table 2). The elimina- tion of this difference suggests that CP reduced AMH levels by acting on the ovary through the follicular effect and ovarian damage. At the same time, the difference between group averages was measured based on the covariance analysis, while the relationship between the dependent and independent variables was revealed closer to rea- lity through the regression analysis and variance
analysis. Thus, the type 2 error due to the reduc- tion of the error variance was reduced. In our study, the inability to measure the basal AMH levels in the drug groups could have affected these results.
CAPE, which has many biological and pharmaco- logical effects and is prominent with its antioxidant and cytoprotective characteristics, is protective against ischemia/reperfusion damages and has been shown to have this protective effect in the ovary.20,21 It is also protective against different chemotherapeu- tic agents, one of which is CP.19 However, its effect against the ovarian-associated chemotherapeutic agents is not known, especially against the impact of CP on the ovary, as in our study. The beneficial effects of CAPE which have regulatory effects on apoptosis have been reported.38 Nevertheless, it was not able to inhibit the apoptosis caused by CP in our study. In our CAPE group, as with all groups, the total number of follicles decreased. However, com- pared to those of the CP and DHEA groups, this decline was higher although it was not significant (Table 2). The tertiary follicle count was statistically significantly highest in the CAPE group among the 4 groups (9.80 ± 2.86, p = .015). The tertiary follicle, or the antrum follicle, is an important indicator in OR.39 Therefore, CAPE contributed positively to OR in this manner, but this contribution was not reflected in AMH values. There was no significant difference in the pairwise comparisons of tertiary follicle numbers, either (p > .008) (Table 2). Again, the fact that the number of atretic follicles in the CP group was significantly increased compared to the control group (p = .003) and this increase was not observed in the CAPE group could be considered as a protective effect against CP damage on the ovary. Even though such useful properties of it have been demonstrated, CAPE, when considered in its entirety, appears to be ineffective against CP damage in the end. Perhaps CAPE is effective only in cases where oxidative stress occurs due to ischemia/reper- fusion damage such as torsion. Further research is needed in this manner.
It has been shown many times that, with the use of DHEA, the level of AMH increases, DOR is improved and the rates of pregnancy are increased.24,40–43 Hasan et al. have reported that when they damaged the ovary in rats by 4-vinylcyclohexene diepoxide, the total fol- licle count has significantly increased with the

administration of DHEA.28 There are also many find- ings in the opposite direction. Recent studies have shown that in humans, AMH, antral follicle counts and OR are not increased when DHEA is administered.44–46 Qin et al. have also noted that there is no significant difference in pregnancy rates and oocyte counts considering randomized controlled trials.27 It is also known that androgens trigger folli- cular development.23 However, it is still unclear how DHEA increases OR and what the mechanism of action in the ovary is.24,27 In our study, the numbers of primordial follicles, secondary follicles and total follicles were the lowest in the DHEA group although not significantly different. In addition to that, the group with the highest number of atretic follicles (p = .002) and the group with the highest decrease in AMH levels was again the DHEA group (Tables 1, 2). Moreover, compared to the control group, it reduced AMH significantly (p = .003) and increased atretic follicles in pairwise comparisons (p = .002). However, when the number of tertiary follicles was fixed to 5.38 and the AMH value was compared among the groups by using the ‘Bonferroni test,’ the only significant decline was in the DHEA group (p = .006). In other words, rather than protecting against the harmful effects of cisplatin, DHEA almost increased the harmful effects. DHEA may be increas- ing OR by itself, but DHEA seems to be ineffective against the damage caused by CP and DOR, which were our aim in our current study. As far as we know, our current study is the first study to review the effects of CP, DHEA and CAPE, and further research needs to be done to verify the data.
In our study, we investigated the CAPE, DHEA, and CP relationship based on the final results that emerged on the ovary. In order to be able to reveal this relationship more clearly, comparative studies are needed to examine the agents used by the mechanisms they affect, where basal AMH levels are also measured. Moreover, the contribution of studies where long-term results can be followed-up will be important. Different results may emerge. The fact that a control group from one ovary and an experimental group from the other ovary of the same rat were not created was a limiting factor in our study. The AMH of a subject from the control group was approximately 12 ng/ml, and that of a sub- ject from the CP group was around 8 ng/ml. In
PCOS (polycystic ovary syndrome), the AMH value increases 2–3 times. It is likely that the rats had PCOS.47 These values increased the averages in the aforementioned groups, but they were included in the study to keep the data realistic. Furthermore, in our study, the menstrual cycle period in rats was not taken into account. Since follicles may vary accord- ing to menstrual cycle day, the results could be affected. All of these may be counted among the limitations of our study.
The AMH values were substantially reduced in all groups where CP was administered. The decline in the groups that were given CAPE and DHEA in addition to CP was greater than that in the group that was given CP only. Statistically the maximum decline was observed between the control group and Group 4 involving DHEA. While the greatest positive correlation was observed between AMH and the number of primordial follicles, the nega- tive correlation was between AMH and the num- ber of atretic follicles. The number of atretic follicles was also significantly elevated in all CP groups compared to that of the control group and was statistically highest in both the CP and CP
+DHEA groups. The two most important in dependent factors of the decline in AMH levels were the total ovarian damage and increased atre- tic follicle count. In conclusion, CP causes total damage and increased numbers of atretic follicles on the ovary. These adverse effects of CP are not prevented by CAPE or DHEA, and may even be increased by DHEA.

Acknowledgments
Acknowledgement that all authors have contributed significantly, and that all authors are in agreement with the content of the manuscript. Authors give copyright permission which is required to reproduce any material in an article, and confirm that such permission has been obtained from the copyright holder.

Declaration Of Interest Statement
Authors Declare that there is no financial support or relation- ships that may pose potential conflict of interest.

ORCID
Ali Doğukan Anğın Image http://orcid.org/0000-0003-1954-8546

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