Investigation of the Effect of Vitamin D on Testicular Damage Caused by Cisplatin in Rats

Investigation of the Effect of Vitamin D on Testicular Damage Caused by Cisplatin in Rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Investigation of the Effect of Vitamin D on Testicular Damage Caused by Cisplatin in Rats Rümeyza Hilal Cirik, Elif Taslidere Karaca, Nigar Vardi, Elif Gürel, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5402589/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 Aim: In our study, we aimed to investigate the effects of VD against testicular damage caused by cisplatin by histopathological, immunohistochemical and biochemical methods. Material and Method: 28 rats were divided into four groups, control, VD, cisplatin and cisplatin+ VD groups. At the end of the 10-day experiment, the rats were sacrificed under ketamine/xylazine anesthesia. Right testicles were used for biochemical analyzes and left testicles were used for histological analyses. Sperm vitality and morphology were evaluated from semen samples obtained from the cauda part of the epididymis. Number of sperms was also counted. Biochemically, TOS, TAS, FSH, LH, testosterone, estrogen, VD, Ca and P levels were measured by ELISA test kits using spectrophotometric methods. Results: In histopathological analysis; a decrease in seminiferous tubule diameter and germinal epithelial thickness, decrease in Johnsen score, and degenerative changes in germinal cells were observed in the cisplatin group. While caspase-3 immune positivity increased, VDR immunostaining decreased. In biochemical analysis; while a significant increase was observed in TOS in the cisplatin group, no significant difference was found in terms of TAS. It was observed that VD application reduced histological damage and caused a significant increase in Johnsen score. In this group, caspase-3 immunostaining decreased while VDR immunostaining increased. Biochemically, a significant decrease in TOS level and a significant increase in VD level were detected. Conclusion: In conclusion, this study shows that VD administration alleviates testicular damage in rats with cisplatin-induced testicular damage. However, further studies are needed to add VD to clinical treatment protocols. Vitamin D Rat Cisplatin Testis Immunohistochemistry Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION Cisplatin is a broad-spectrum chemotherapeutic agent widely used in the treatment of many cancer types, including head and neck, bladder, prostate, testis, breast, cervix, ovarian cancers, osteogenic sarcoma, and neuroblastoma ( 1 ). The availability of anticarcinogenic drugs is of great importance in terms of prolonging life expectancy and improving quality of life. However, when the side effects of these drugs and studies on male fertility are analyzed, there will likely be a significant infertility problem in the future ( 2 ). It has been reported that long-term azoospermia and permanent infertility may be observed as a result of the gonadotoxic effect of cisplatin chemotherapy ( 3 ). Excessive cell proliferation, especially during spermatogenesis, makes the testes more sensitive to this agent ( 4 ). Today, the most widely used alternative treatment method to minimize tissue damage that occurs during the use of chemotherapeutics is the use of anti-oxidant substances. Therefore, it is thought that cisplatin-induced testicular toxicity may be reduced by anti-oxidants ( 5 ). Vitamin D (VD) is a steroid prohormone synthesized mainly in the skin with the effect of sunlight. It needs to be hydroxylated in the liver and kidney to become active ( 6 ). VD is generally known for its function in maintaining and regulating calcium (Ca) and phosphorus (P) homeostasis. However, in addition to its known mineral metabolism effects, it also has various cytoprotective effects such as anti-fibrotic, anti-oxidative, and anti-inflammatory effects. It has also been shown to play a role in modulating both male and female reproductive processes. In the last decade, the relationship between VD and the reproductive system has been increasingly investigated, and new information has become available, demonstrating the presence of vitamin D receptors (VDR) in both male and female reproductive organs. Recently, it has also been demonstrated that VD deficiency is associated with erectile dysfunction, decreased sperm concentration, and low sperm motility ( 6 , 7 ). This study aimed to investigate the effects of VD on male reproductive toxicity due to cisplatin, a commonly used chemotherapeutic drug, by histopathologic, immunohistochemical, and biochemical evaluation. MATERIAL AND METHOD Chemicals Koçsel's Cipintu 100 mg/100 ml was used for cisplatin, and Deva's 300,000 IU/ml Devit-3 ampoules were used for VD. Experimental Animals A total of 28 12-week-old male Wistar albino rats weighing 230-300 g were obtained from Malatya İnönü University Experimental Animal Research Center. Rats were housed in a well-ventilated room with clean plastic cages at constant room temperature (23℃-24℃) and relative humidity (50-55%) on a 12/12 h light/dark cycle. Feed and water were changed daily, and ad libitum was provided. All procedures were performed in accordance with the animal research guidelines of the National Institute of Health. Ethical Declaration This study was approved by İnonu University Faculty of Medicine Animal Research Ethics Committee (HAYBIS NO: 11409) and funded by İnonu University Scientific Research Project Coordination Unit with the project code TTU-2021-2533. Experimental Design The animals were randomly divided into four groups: Six animals in the control and VD groups and eight animals in the cisplatin and cisplatin+VD groups (since cisplatin is a toxic substance, two more animals were used in the cisplatin groups than the number determined by biostatistics). The weights of the animals at the beginning of the experiment were recorded, and after a one-week acclimatization period, the following treatments were performed in the groups. Control Group: Solvent agent was applied to this group. VD Group: In this group, 1000 IU/kg intraperitoneal (i.p) VD was administered every other day. The study of Abbaszadeh et al. (8) and the study of BaSalamah et al. (6) were taken as a reference for VD dose and administration times. Cisplatin Group: A total of 14 mg/kg cisplatin i.p. was administered to this group in two doses. In addition, the solvent was administered i.p. every other day to the rats in this group. Öztürk et al. study (9) Hassan et al. study (10) , and Ekmekçi et al. study (11) were taken as references for cisplatin dose and administration times. Cisplatin +VD Group: This group received a total of 14 mg/kg cisplatin i.p in two doses and 1000 IU/kg i.p VD every other day. Collection of Tissue and Blood Samples After ten days of the experimental period, the body weights of the rats were measured, and then 5 mg/kg xylazine and 50 mg/kg ketamine i.p. were administered. Under general anesthesia, their abdomen was opened through a midline incision. Blood samples were taken from the inferior vena cava. Blood samples were centrifuged at 3000 xg for 10 minutes on the same day, and the serum obtained was collected in eppendorf tubes and stored at -80 C° for biochemical evaluations. After weighing both testicular tissues taken from the subjects, the left testis was placed in 10% formaldehyde solution for histological analysis, while the right testis was stored at -80 C° for biochemical studies. Sperm samples were obtained from the caudae of the epididymis for sperm parameter evaluations. After the collection of tissue and blood samples, the animals were euthanized under high-dose anesthesia. Histologic Tissue Monitoring, Staining and Examination For histopathologic evaluation, tissues were subjected to fixation and follow-up procedures. The tissue was fixed in 10% formaldehyde solution. The tissue samples were then trimmed and placed in plastic tissue tracking cassettes, and the formaldehyde solution was renewed. After 24 hours of fixation, the tissue samples were subjected to routine tissue tracking steps in the Tissue-Tek VIP/SAKURA tissue tracking device. Tissue samples were embedded in paraffin blocks after tissue tracking procedures were completed. Sections of 4 μm each were taken from the paraffin blocks using a Leica RM2145 microtome. The sections were stained with hematoxylin-eosin (HE) to observe the general histologic structure and periodic acid schiff (PAS) staining method to examine the basement membrane changes. Sections were also immunohistochemically stained with caspase-3 and VDR primary antibodies. The preparations were examined, scored, and photographed with a Leica DFC280 light microscope and Leica Q (Leica Micros Imaging Solution Ltd, Cambridge, UK) image analysis system. Histologic Evaluation Diameter Measurement of Seminiferous Tubules Measurement was performed under X200 magnification on 20-round or nearly round seminiferous tubules from each preparation. The short and long side lengths were measured for each tubule, and the mean value was recorded. Measurement of Epithelial Thickness of Seminiferous Tubules The measurement was performed under X200 magnification at four different locations, including the thinnest and thickest parts of 20 seminiferous tubules in stages 7-8 for each preparation. The average of these four measurements was recorded for each seminiferous tubule. Johnsen Score Johnsen testicular histologic damage score method was used in the evaluation of the tissues examined by HE staining (12). Immunohistochemical Evaluation To evaluate positive immunoreactivity in testicular tissue, ten different fields from each sample were examined at X400 magnification. The mean number of caspase-3 (SantaCruz) and VDR (SantaCruz) immunopositive cells was measured. Sperm Morphology and Vitality Assessment The cauda portion of the epididymis separated from the right testis was used for sperm analysis. Morphologically, sperms were evaluated by staining dried semen smear slides with sperm morphology stain. The examination was performed under a Leica DFC280 light microscope at X400 magnification. Six samples from each group and 100 spermatozoa per sample were analyzed. The spermatozoa were morphologically categorized into three classes: normal, head-neck anomaly, and tail anomaly by Immunohistochemical Evaluation. For viability, spermatozoa were evaluated under Leica DFC280 light microscope at X400 magnification after drying of smears mixed with eosin-nigrosin. Stained spermatozoa were considered dead, while unstained spermatozoa were considered viable. For each sample, 100 spermatozoa were analyzed, six spermatozoa from each group. Percent viability was recorded for each sample. Sperm Counting The cauda portion of the epididymis, separated from the left testicle, was used for sperm count. In a petri dish containing 1 milliliter of G-IVF solution, the tissue was cut into small pieces and left for incubation. Afterward, the dissected tissue was taken into eppendorf tubes together with the G-IVF solution. After centrifugation at 5000 xg for one minute, the supernatant was sampled with a micropipette on a Thoma slide. Sperm counting was performed under the Leica DFC280 light microscope at X400 magnification. Six samples from each group were evaluated, and sperm count was recorded. Biochemical Analysis FSH, LH, Testosterone, Estrogen, Ca, VD Blood collected from rats under anesthesia was placed in a gel biochemistry tube and centrifuged at 3000 rpm for ten minutes. The serum obtained after centrifugation was collected in eppendorf tubes and stored at -80 °C. SunRed brand rat-specific ELISA kits based on a double-antibody sandwich technique were used to test rat FSH, LH, testosterone, estrogen, VD, and Ca levels in the samples. For FSH, SunRed brand rat-specific FSH ELISA kit with catalog number 201-11-0183 LOT number 202201 was used. For LH, SunRed brand rat-specific LH ELISA kit with catalog number 201-11-0180 LOT number 202201 was used. For testosterone, SunRed brand rat-specific testosterone ELISA kit with catalog number 201-11-0735 and LOT number 202201 was used. For estrogen, SunRed brand rat-specific estrogen ELISA kit with catalog number 201-11-0177 and LOT number 202203 was used. For VD, the SunRed brand rat-specific VD ELISA kit with catalog number 201-11-0016 was used. For Ca, SunRed brand rat-specific Ca ELISA kit with catalog number 201-11-0949, LOT number 202201, was used. Inorganic Phosphate Rel Assay branded kit with LOT number EK2101P was used for inorganic phosphate measurement. A direct method was preferred for P determination. P reacts with ammonium molybdate in an acid medium to form a yellow phosphomolybdate complex. The intensity of the color formed is proportional to the P concentration in the sample at 340 nm (13). The reading on the spectrophotometer was recorded for each sample. Total Oxidant Level LOT number KM21136O Rel Assay branded kit was used for total oxidant level (TOL) measurement. The application was performed by following the sequential steps specified in the kit operating procedure. The working principle of the kit is based on oxidants in the sample medium oxidizing the ferrous ion-chelator complex to ferric ion. Measurement of the intensity of the color formed as a result of ferric ions forming a colored complex with chromogen in an acidic environment at a wavelength of 530 nm in a spectrophotometer gives the total amount of oxidant molecules present in the sample as micromolar hydrogen peroxide equivalent per liter (14). The spectrophotometer reading for each sample was recorded. Total Anti-oxidant Level Rel Assay branded kit with LOT number EK21122A was used for total anti-oxidant level (TAL) measurement. The application was performed by following the sequential steps specified in the kit operating procedure. The working principle of the kit is based on the fact that anti-oxidants in the sample medium convert the dark blue-green ABTS radical into colorless reduced ABTS form. The absorbance change at 660 nm wavelength in the spectrophotometer gives the total anti-oxidant molecule level in the sample with Trolox Equivalent, a vitamin E analog (15). The spectrophotometer reading was recorded for each sample. Statistical Analysis Numerical data were summarized with median, minimum, and maximum values. The Kruskal-Wallis test, followed by the Conover pairwise comparison method, was used for independent group comparisons. Two dependent measures were compared with the Wilcoxon paired two-sample test. The significance level was accepted as 0.05 in all tests. RESULTS Weight Assessment of Experimental Animals When cisplatin and control groups were compared in terms of body weights at the beginning and end of the experiment, it was found that the difference between the body weights of the rats was statistically significant (p = 0.046). No change was observed in body weight loss as a result of VD administration to the cisplatin group (p > 0.05). Animal weights at the beginning and end of the experiment are presented in Table 1 . Table 1 Comparison of Animal Weights at the Beginning and End of the Experiment Initial Weight Final Weight Median Min Max Median Min Max p Control ª 220,00 195 230 246,50 209 254 0,075 Cis b 281,50 275 292 201,00 148 282 0,046 Cis + VD b 247,00 223 267 203,50 156 242 0,028 VD ª 228,50 210 239 243,50 221 271 0,116 * Different letters indicate statistical significance of differences between groups. Assessment of Testicular Weights When cisplatin and the control group were compared in terms of testicular weights at the end of the experiment, it was determined that the difference between the testicular weights of the rats was not statistically significant (p > 0.05). Testicular weights are presented in Table 2 . Table 2 Comparison of Testicular Weights Control Cis Cis + VD VD p Testicular Weights 1,295 (1,08 − 1,44) 1,225 (0,7 − 1,35) 1,255 (1,02 − 1,39) 1,245 (1,06 − 1,29) 0,709 No significant difference was found between groups (p > 0,05). Histologic Evaluation Control Group In HE-stained sections, the seminiferous tubules in the testis had a normal histologic appearance (Fig. 1A). The tubules were composed of seminiferous epithelium sitting on a prominent basal lamina (Fig. 1B). Sertoli cells and spermatogenic series cells were clearly distinguished in the seminiferous epithelium. Spermatozoa were observed in the lumen of the majority of seminiferous tubules. (Fig. 1C). In this group, the mean seminiferous tubule diameter was 169.588 µm, and the mean germinal epithelial height was 85.814 µm. In addition, the mean Johnsen score was 8.75. The comparison of seminiferous tubule diameter, germinal epithelial thickness, and Johnsen score of the groups is shown in Table 3 . Table 3 Comparison of Seminiferous Tubule Diameter, Germinal Epithelial Thickness and Johnsen Score Control Cis Cis + VD VD p Seminiferous Tubule Diameter 169,588 a 147,195 b 151,792 b 173,539 a 0,002 Germinal Epithelial Thickness 85,814 a 69,519 b 71,662 b 85,508 a < 0,001 Johnsen Score 8.75 a (8.4-9) 7.07 b (5.42–7.36) 7.42 c (7.28–7.56) 7.55 c (7.12–7.7) < 0.001 VDR 1.05 ab (0.8–1.5) 0.925 a (0.55–1.05) 1.4 b (1.15–1.6) 1.375 b (1.05–1.8) 0.008 Caspase-3 0 a (0–0) 9 b (0–15) 6 c (0–9) 0 a (0–0) < 0.001 * Different letters indicate statistical significance of differences between groups. VD Group In this group, the testis had a generally normal histologic appearance. Similar to the control group, most of the seminiferous tubules had smooth borders, and spermatogenic germ cells were regularly arranged. (Fig. 1D). The tubules contained germ cells at all stages of spermatogenesis, from spermatogonium to spermatid. The mean seminiferous tubule diameter was 173.539 µm, and the mean germinal epithelial height was 85.508 µm. In addition, the mean Johnsen score was 7.55. When compared with the control group in terms of seminiferous tubule diameter and germinal epithelial thickness, the difference between them was not found to be statistically significant (p > 0.05). However, Johnsen score decreased statistically significantly in this group compared to the control group (p < 0.001). Cisplatin Group In this group, some seminiferous tubule structures were disorganized, and large gaps were observed in the interstitial space between the seminiferous tubules (Fig. 2A). The mean seminiferous tubule diameter was 147.195 µm, and the mean germinal epithelial height was 69.519 µm. When compared with the control group, a statistically significant decrease in tubule diameter and epithelial height was observed in this group (p < 0.01). The mean Johnsen score of this group was 7.07. Compared to the control group, Johnsen score was significantly decreased in this group (p < 0.001). In some seminiferous tubules, it was observed that the cells forming the spermatogenetic series were decreased and irregularly arranged (Fig. 2B). In addition, spermatids with annular chromatin were found in some seminiferous tubules ((Fig. 2C, D). In some tubules, degenerated cells with eosinophilic cytoplasm paused at certain stages of meiosis and were observed in different shapes (Fig. 2E). Another striking finding was the presence of giant cells with multiple nuclei in the tubules (Fig. 2F). In addition, in some tubules, cells belonging to the spermatogenic series that separated from the germinative epithelium and did not complete their development were found to accumulate in the lumen (Fig. 2G, H). Cisplatin + VD Group In this group, the mean seminiferous tubule diameter was 151.792 µm, and the mean germinal epithelial height was 71.662 µm. The increase in these values was not statistically significant when compared with the cisplatin group (p > 0.05). On the other hand, when compared with the control group, the difference in tubule diameters and germinal epithelial height was statistically significant (p < 0.01). The mean Johnsen score of this group was 7.42. Compared to the cisplatin group, Johnsen score increased statistically significantly in this group (p < 0.01). Spermatids with annular chromatin and spermatogenic cells arrested at certain stages of meiosis were also observed in this group (Fig. 3A). Similar to the cisplatin group, cells forming the spermatogenic series were found to be decreased and irregularly arranged in some seminiferous tubules (Fig. 3B). PAS staining In PAS-stained sections, Leydig cells were present in the interstitial connective tissue between the tubules in control and VD groups. The basement membranes of the seminiferous tubules were stained positively for PAS, were thin and smooth, and were found to have a normal histologic structure (Fig. 4A,B). In the cisplatin group, the basement membranes of the seminiferous tubules were corrugated, and PAS positivity was increased (Fig. 4C). In contrast, in the cisplatin + VD group, the corrugation and PAS positivity in the basement membranes of the seminiferous tubules were similar to the cisplatin group (Fig. 4D). Immunohistochemical assessment Caspase-3 Immunohistochemical Staining In the control and VD groups, especially the cytoplasmic residues of spermatids in the seminiferous tubules stained positively with caspase-3 No positively stained spermatogenic cells were observed in these groups (Fig. 5AB). In the cisplatin group, the intensity of positively stained spermatogenic cells was statistically significantly increased compared to the control group (Fig. 5C). In the cisplatin + VD group, the intensity of positively stained spermatogenic cells was statistically significantly decreased (p < 0.001) compared to the cisplatin group (Fig. 5D). A comparison of caspase-3 positivity of the groups is shown in Table 3 . VDR Immunohistochemical Staining In the control and VD groups, positively stained seminiferous tubules were observed in the VDR staining sections (Fig. 6AB). When the VD group was compared with the control group, the increase in the number of positively stained tubules was not statistically significant (p > 0.05). In the cisplatin group, the intensity of positively stained spermatogenic cells decreased in this group compared to the control group, but there was no statistically significant difference (p > 0.05) (Fig. 6C). In the VD group, the intensity of positively stained spermatogenic cells increased statistically significantly in this group compared to the cisplatin group (p < 0.01) (Fig. 6D). A comparison of VDR positivity of the groups is presented in Table 3 Sperm Analysis Findings Table 4 . shows the sperm count, vitality, and abnormal sperm count of the groups. Cisplatin treatment significantly decreased sperm count compared to the control group (p 0.05). Table 4 Comparison of Sperm Analyzes Control Cis Cis + VD VD p Sperm Count 20 a (16–150) 6 b ( 4 – 10 ) 10 b ( 5 – 12 ) 18.5 a (13–200) < 0.001 Sperm Vitality 0.965 a (0.95–0.99) 0.13 b (0.11–0.24) 0.32 c (0.24–0.57) 0.79 d (0.63–0.82) < 0.001 Sperm Total Anomaly 0.085 a (0.05–0.15) 0.23 b (0.18–0.5) 0.16 c (0.11–0.25) 0.21 bc (0.15–0.24) 0.003 Head-Neck Anomaly 0.01 a (0-0.02) 0.095 b (0.03–0.15) 0.015 a (0-0.02) 0.005 a (0-0.02) 0.002 Tail Anomaly 0.07 a (0.05–0.14) 0.175 b (0.08–0.4) 0.145 b (0.1–0.23) 0.2 b (0.14–0.24) 0.011 * Different letters indicate statistical significance of differences between groups. When the cisplatin group was compared with the control group, it was found that sperm vitality decreased statistically significantly in this group (p < 0.001). On the other hand, when the Cisplatin + VD group was compared with the Cisplatin group, it was observed that sperm vitality increased statistically significantly in this group (p < 0.001) (Fig. 7). In the VD-only group, a significant decrease in sperm vitality was detected compared to the control group (p < 0.001). It was determined that cisplatin treatment caused a statistically significant increase in the amount of abnormal sperm (head-neck and tail anomaly), and this increase decreased with VD treatment (p 0.05). There was a significant increase in sperm anomaly in the VD-only group compared to the control group (p < 0.05) (Fig. 8). Biochemical Findings The values of TOL, TAL, FSH, LH, testosterone, estrogen, VD, Ca, and P levels are presented in Table 5 . Table 5 Comparison of biochemical analyzes Control Cis Cis + VD VD p TAS 1.034 1.025 1.119 1.004 0.637 TOS 3.963 a 8.243 b 5.296 c 5.185 c 0.001 FSH 11,008 11,01 10,922 11,591 0,398 LH 8,624 7,197 8,439 7,796 0,198 Testosterone 439,297 410,33 421,423 442,011 0,307 Estrogen 29,938 a 24,669 b 26,77 b 30,794 a 0,001 VD 879.865 ab 768.263 b 845.244 ac 967.212 c 0.015 Ca 2,458 a 2,528 a 2,884 ab 3,683 b 0,016 P 8,304 8,904 8,475 8,412 0,765 * Different letters indicate statistical significance of differences between groups. As a result of the evaluations, it was determined that TOL, which is an indicator of oxidative damage, increased statistically significantly in cisplatin-treated rats compared to control and all other groups (p < 0.01). However, it was determined that VD administration decreased the increase in TOL caused by cisplatin at a statistically significant level (p < 0.01). In the VD-only group, there was a statistically significant increase in TOL level compared to the control group (p 0.05). While no difference was observed between the groups in terms of serum FSH, LH, and testosterone levels (p > 0.05), serum estrogen levels were significantly decreased in cisplatin-treated rats compared to the control group (p 0.05). Serum VD levels decreased in cisplatin-treated rats compared to the control group, but there was no statistically significant difference (p > 0.05). When VD-administered groups were compared with cisplatin and control groups, it was found that VD levels increased statistically significantly (p < 0.05). In terms of serum Ca level, no difference was observed between cisplatin and control groups, while Ca level increased in VD-treated groups when compared with cisplatin and control groups (p 0.05). DISCUSSION Cisplatin is a potent antineoplastic agent and is used in the treatment of many solid organ cancers, mainly head and neck, lung, testicular, ovarian, and breast ( 16 ). However, cisplatin has side effects, including ototoxicity, neurotoxicity, nephrotoxicity, hepatotoxicity, and testicular toxicity, in addition to its anti-tumoral effects ( 17 ). Adverse effects of short- and long-term cisplatin treatment on testicular function have been reported in many studies ( 18 ). Studies have shown that cisplatin leads to excessive production of reactive oxygen species (ROS) as well as direct DNA damage ( 1 ). Therefore, substances with anti-oxidant capacity have been tried to protect tissues against toxic substances ( 18 , 19 , 20 ). VD has anti-oxidative and anti-inflammatory effects, as well as regulates Ca and P homeostasis ( 21 ). However, there are not enough studies on the effects of VD on cisplatin-induced testicular and epididymal damage. Therefore, in our study, we aimed to evaluate the effects of VD on cisplatin-induced male reproductive toxicity by histopathologic, immunohistochemical, and biochemical parameters. In our study, TOL and TAL parameters, which are indicators of oxidative damage and anti-oxidant activity, were evaluated in serum samples to determine the oxidative damage caused by cisplatin in the testis and the protective properties of VD. In previous studies, it was reported that cisplatin administration caused an increase in lipid peroxidation levels and a decrease in anti-oxidant parameters in general ( 22 , 23 ). In our study, it was observed that the TOL level significantly increased in the cisplatin group compared to the control group and other groups, whereas the TAL level was similar to that of the other groups. In a ten-day study in which a single dose of 7 mg/kg i.p. cisplatin was administered, Yücel et al. did not find a significant difference between the groups in the TAL level similar to our study but found that TOL and OSI levels increased in the cisplatin group. Yücel et al. ( 5 ) thought that the reason for the lack of difference in TAL level may be increased anti-oxidant molecules to balance the high levels of prooxidant molecules in damaged testicular tissue. In their 21-day study conducted with three different VD doses (100 IU/kg, 1000 IU/kg, 10.000 IU/kg), Mokhatari-Zaer et al. ( 24 ) reported that VD supplementation decreased the increased MDA level in the damage group and increased CAT and SOD levels among anti-oxidative parameters compared to the damage group. Similarly, in our study, it was found that TOL level decreased significantly in the cisplatin + VD group compared to the cisplatin group. In our study, seminiferous tubule diameters and seminiferous tubule epithelial thickness were measured in HE-stained histologic sections and compared between the groups. Many researchers have shown that damage to the seminiferous tubules develops after cisplatin administration in rats. It has been reported that cisplatin causes a decrease in seminiferous tubule diameter, disorganization in germinative epithelium, and shedding of germ cells at different stages of meiosis into the lumen ( 23 , 25 ). Beytur et al.( 26 ) reported a significant decrease in seminiferous tubule diameter and germinal epithelial thickness in the cisplatin-treated groups in a ten-day study in which a single dose of 7 mg/kg i.p cisplatin was administered. Similarly, we observed a significant decrease in seminiferous tubule diameter and germinal epithelial thickness in the cisplatin-treated groups in our study. We think that this decrease may be due to the decrease in cell diameters and/or the absence of seminiferous epithelial cells that are expelled into the lumen without maturing due to DNA damage caused directly by cisplatin. However, although there was an increase in seminiferous tubule diameter and germinal epithelial thickness in the cisplatin + VD group, this difference was not statistically significant. In contrast to our results, Yalçın et al. ( 27 ) investigated the effect of VD on methotrexate damage and reported that VD significantly increased seminiferous tubule diameter and germinal epithelial thickness compared to the damage group. Mohamed et al. reported that VD caused a significant increase in seminiferous tubule diameter and germinal epithelial height compared to the damage group in their study on torsion-detorsion testicular injury ( 28 ). We think that the reason for this difference from the results of other studies is that our experimental period was limited to ten days or the variability in the drug sensitivity of rats. Mohammadnejad et al. ( 29 ) reported that atrophy in the seminiferous tubules, damage in the germinal epithelium, loss of connection between the basal lamina and spermatogonium and inflammation in the peritubular area were observed in their study investigating the effect of cisplatin on the testis. In our study, we evaluated seminiferous tubule damage with Johnsen score and found that Johnsen score decreased significantly in the cisplatin-treated group. In addition, histologically, we found degenerated cells with eosinophilic cytoplasm, paused at certain stages of meiosis and observed in different shapes, spermatids with multiple annular chromatin in some seminiferous tubules, and giant cells containing more than one nucleus. Ilbey et al. ( 30 ) administered 7 mg/kg i.p cisplatin daily for five days to rats and reported that the spermatogenesis process was negatively affected, and the Johnsen score was significantly decreased in the cisplatin-treated group compared to the control group. Azarbarz et al. ( 23 ) also found that cisplatin caused a significant decrease in Johnsen score in a 15-day study in which they administered a single dose of 5 mg/kg cisplatin. When the cisplatin + VD group was compared with the cisplatin group, we found that the Johnsen score increased significantly. Similar to our study, Yalçın et al. ( 27 ) reported that VD application significantly increased the Johnsen score in their study in which testicular damage was induced by methotrexate. In our study, the basement membrane structure surrounding the seminiferous tubules was evaluated using the PAS staining method. PAS-stained sections in the cisplatin group showed increased corrugation and PAS positivity in the basement membrane of the seminiferous tubules. Similar to our study, Sakr et al. reported that the basal laminae and interstitial areas of the seminiferous tubules of rats treated with cytotoxic agents were stained strongly PAS positive ( 31 ). In the literature review, no studies evaluating the effect of VD on the testis with PAS staining were found. In many studies, it was reported that cisplatin administration caused a decrease in body and testicular weights ( 5 , 32 ). In our study, when the pre- and post-experimental body weights were compared, it was found that there was a statistically significant weight loss in the cisplatin-treated groups compared to the control group, but there was no significant difference between the groups in terms of testicular weights. It is thought that the reason for the loss in body weight in the cisplatin-treated groups may be the toxic effect of the drug on the gastrointestinal system. In our study, there was no significant difference in body weight and testicular weight in the cisplatin + VD group compared to the cisplatin group. Yalçın et al.( 27 ) investigated the effect of VD on methotrexate-induced testicular damage and reported that, similar to our study, there was no significant difference between methotrexate and methotrexate + VD groups in terms of body weights. Cisplatin, a chemotherapeutic agent, is a genotoxic substance that causes anomalies in the head of spermatozoon and damage to spermatozoon DNA ( 18 ). In their study showing that the destructive effects of cisplatin treatment on testicular tissue and spermatogenesis were reduced by retinoic acid administration, Yücel et al. reported that cisplatin given at a dose of 7 mg/kg significantly decreased sperm motility and sperm count in rats ( 27 ). Similarly, Shati et al. reported that cisplatin increased abnormal sperm count and decreased sperm motility and sperm count ( 33 ). In our study, we found that sperm count and sperm vitality decreased significantly in the cisplatin-treated groups compared to the control group. In addition, we found that sperm vitality increased and sperm head-neck abnormality decreased in the cisplatin + VD group compared to the cisplatin group. Abbaszadeh et al. ( 8 ) investigated the effect of VD against lead damage and found that additional VD administration caused improvement in semen parameters (sperm concentration, motility, and vitality), similar to our results. Testosterone, which plays an important role in the initiation and maintenance of spermatogenesis, is secreted by Leydig cells, and testosterone level is directly related to Leydig cell function and number ( 32 ). Unlike germ cells, Sertoli and Leydig cells, which do not proliferate in adults, are resistant to cytotoxic agents, and most of them survive after cytotoxic therapy. However, these cells may undergo functional damage ( 34 ). Previous studies have shown that cisplatin administration decreased serum testosterone levels ( 25 , 35 ). Similarly, Afsar et al. reported in their study that cisplatin caused damage in Leydig cells, resulting in a decrease in cell number and a decrease in testosterone level ( 36 ). It has also been reported that chemotherapy causes Leydig cell damage despite normal testosterone levels; however, a decrease in testosterone level and an increase in LH level are expected to indicate complete Leydig cell failure. Ateşşahin et al. ( 18 ) reported that there was no significant difference between the groups in terms of testosterone hormone in a ten-day study in which a single dose of 7 mg/kg i.p cisplatin was administered. In our study, serum testosterone levels were measured to determine Leydig cell damage, and it was observed that testosterone levels decreased in the cisplatin group compared to the control group, but the decrease was not statistically significant. In addition, there was no difference between the groups when FSH and LH results were evaluated. Our results showed that cisplatin did not cause complete Leydig cell failure, similar to some previous studies. It is known that estradiol in men is mainly derived from the extra gonadal conversion of testosterone. However, studies have shown that Sertoli cells also have aromatase expression. It has also been reported that VD increases aromatase expression in Sertoli cells. Therefore, VD is thought to affect estrogen levels systemically and locally by regulating aromatase enzyme expression ( 37 ). In our study, we found that estrogen levels decreased significantly in the cisplatin-treated group compared to the control group. When cisplatin and cisplatin + VD groups were compared, we observed that estrogen levels increased, but there was no significant difference. We think that the reason why cisplatin decreases estrogen levels in male rats may be the decrease in aromatase expression due to Sertoli cell damage and/or decreased adipose tissue with decreased body weight in cisplatin-treated animals. Because it is known that adipose tissue has aromatase enzyme expression. Since there are no studies on the effect of cisplatin on estrogen levels in male rats in the literature review, further studies are needed to understand the mechanism fully. Recent studies have shown that testicular germ cell death occurs through apoptosis. Spontaneous apoptosis is a physiologic process that occurs during the development of germ cells and leads to the elimination of damaged germ cells, and some external stimuli, such as ionizing radiation, chemotherapy, and hormonal manipulations, also lead to apoptosis ( 38 ). There are several molecular mechanisms by which cisplatin treatment causes cell apoptosis ( 39 , 40 ). The first of these is oxidative stress, which is one of the most important mechanisms in cisplatin toxicity. Increased reactive oxygen species trigger apoptosis both intrinsically and extrinsically. The second is its effect on Ca hemostasis. Activated cisplatin causes uncoupling of oxidative phosphorylation and inhibition of mitochondrial respiration. This results in Ca influx from mitochondria and, consequently, a transient increase in intracellular Ca levels. Disruption of Ca homeostasis by cisplatin initiates primary events such as lipid and enzyme inhibition peroxidation. As a result, apoptosis or necrosis occurs ( 1 , 41 ). Apoptosis has a critical role in the removal of damaged spermatogenic cells to prevent abnormal sperm formation ( 42 ). It has also been shown that spermatocytes that fail to complete mitotic division are removed by apoptosis ( 43 ). Findings such as the separation of spermatogonial cells from each other and from the basal lamina, nuclear condensation, and the appearance of spermatids with annular chromatin are indicators of apoptosis in the seminiferous tubules ( 29 ). In our study, we found a large number of spermatids with annular chromatin in some seminiferous tubules by light microscopy and apoptotic cells, which were identified by immunohistochemistry using caspase-3 activity. Caspases are cysteine-proteinase group enzymes that play an important role during apoptosis, which is known as cell suicide, and some cellular changes that occur during apoptosis depend on the activity of these enzymes ( 44 ). Simsek et al. reported that caspase-3 positivity increased significantly in the cisplatin group in a ten-day study in which they administered a total of 36 mg/kg cisplatin ( 45 ). Aly et al. found a significant increase in caspase-3 activity in cisplatin-treated rats in a four-week study in which they administered 7.5 mg/kg cisplatin weekly ( 46 ). Gholami et al. administered a single dose of 5 mg/kg cisplatin in a seven-day study investigating the effect of methylene blue on cisplatin damage and reported an increase in caspase-3 immunostaining in the cisplatin group ( 32 ). In our study, it was found that the number of caspase-3 positive cells increased significantly in the cisplatin group compared to the control group. In our study, when cisplatin and cisplatin + VD groups were compared, it was observed that the number of caspase-3 positive cells decreased. Similarly, Helal et al. reported a significant decrease in caspase-3 in rats given 1000 IU/kg VD supplementation daily compared with the damage group in their 90-day study ( 47 ). In their study on VD supplementation in diabetic rats, Ding et al. reported a significant decrease in caspase-3 in the VD-supplemented group compared to the damage group ( 48 ). Similarly, Yalçın et al. ( 27 ) investigated the effect of VD on methotrexate damage and found a significant decrease in caspase-3 in the methotrexate + VD group compared to the methotrexate group. Vitamin D is known for its function in Ca and P homeostasis and is mainly synthesized in vivo in the skin under the influence of sunlight. However, it is an atypical vitamin due to its binding to the steroid hormone receptor and is considered a prohormone. The demonstration that VDR and VD metabolizing enzymes (CYP2R1, CYP27B1, and CYP24A1) are expressed in Sertoli cells, germ cells, Leydig cells, spermatozoa, and male genital tract epithelial cells in the testis suggests that VD may be important for spermatogenesis and sperm. Many studies have shown that VD deficiency negatively affects semen quality and can be compensated by VD supplementation ( 21 , 37 , 49 ). Jensen et al. ( 50 ) stated that VDR level is a positive sign for sperm quality and reported that 1α,25-dihydroxyvitamin D3 may cause increased sperm motility by triggering VDR-mediated increase in intracellular Ca concentration in human spermatozoa. Sood et al. ( 51 ) reported a decrease in Sertoli cell function, degenerative changes in germinal epithelium, and a decrease in Leydig cells in rats with VD deficiency. In addition, there are also studies showing that high VD levels negatively affect semen parameters. In an animal experiment in which VD was administered intramuscularly at three different doses, low-dose VD injection was shown to improve testicular function compared to high-dose administration. This study concluded that hypervitaminosis D alters mitochondrial activity by increasing intracellular Ca levels and has negative effects on the spermatogenesis process. This finding shows the importance of optimal VD dose in the regulation of male fertility ( 52 ). In our study, it was observed that serum VD levels decreased in cisplatin-treated rats compared to the control group, but there was no significant difference, but VD levels increased statistically significantly in VD-treated groups compared to cisplatin and control groups. There was no difference between cisplatin and control groups in terms of serum Ca level, but Ca level increased statistically significantly in the VD-treated group when compared with the other groups. In the cisplatin group, VDR positivity statistically decreased significantly compared to the control group. In the cisplatin + VD group, VDR positivity was significantly increased compared to the cisplatin group. Similarly, Jeremy et al. reported that VDR immunostaining decreased in the D-galactose-induced rat group, and VDR immunostaining increased in the VD-supplemented group compared to the damage group ( 7 ). Liu et al. ( 53 ) investigated the effect of VD in diabetic rats and reported that there was a decrease in VDR expression in the diabetic group, and VD supplementation significantly increased VDR expression compared to the diabetic group. The decrease in VDR in the cisplatin group may be associated with increased TOL levels or increased apoptosis. In conclusion, this study shows that VD administration attenuates testicular damage in rats with cisplatin-induced testicular injury. We suggest that VD may be used in combination with cisplatin in cisplatin-treated patients due to its anti-oxidative and anti-inflammatory effects as well as its regulation of Ca and P homeostasis. However, further studies are needed to add VD to clinical treatment protocols. Declarations Author contribution RHC, data collected and histologically analyzed, and interpreted the data, and contributed signifcantly to the writing of the article. ETK and NV, performed histological analysis and interpreted them. EG, analyzed the biochemical data. MG, assisted in histological analysis. HG, performed a statistical analysis of the data. ETK, performed the histological analysis. Disclosure statement No potential conflict of interest was reported by the author(s) Funding The present study was supported by the Inonu University Scientific Research Projects coordination (Grant No. TTU-2021-2533) Data availability statement The data that has been used is confidential. References Dasari S, Bernard Tchounwou P. Cisplatin in cancer therapy: Molecular mechanisms of action. European Journal of Pharmacology 2014; 740:364–78. Aslan Ö, Vural H, Kömürcü Ş, Özet A. 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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-5402589","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":381777290,"identity":"eb1dfc1b-2e3e-48ed-80a5-394037cbac44","order_by":0,"name":"Rümeyza Hilal Cirik","email":"","orcid":"","institution":"Konya City Hospital IVF Center","correspondingAuthor":false,"prefix":"","firstName":"Rümeyza","middleName":"Hilal","lastName":"Cirik","suffix":""},{"id":381777291,"identity":"289c526a-4b66-4082-ad49-f61bae4c3b40","order_by":1,"name":"Elif Taslidere Karaca","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEElEQVRIie3RMWrDMBSA4SceyIupVxtaegWFQNKSEF9FJuAs3boYEqhEwV4SuvYauUFB4CwiXQNZUnoBd2uXUMXx0EEiawf9i4Tg40k2gM/3HwuIaE7rFQIzyzjsztFNkMhX4AD0TPI/hLoIkDOBlii4SCJEKcj3JKUBrj+L4v16GKzqGIpRJm5XBxtJnokUwKdZifSxp/U+vF9u8xj0LBM0YDbCFKkOwJFTDAeJLPch2z0MYlIqQ+w3S1U75Sk1ZPgjj9uOHN2EYUsUKc0Us33riHCT+DSF55v2LYmopyHTun/H61m/pLn9i1XVh2jG8zSK1PpLLCYp2yx7u2YxunnB2krauPXE9Sd9Pp/Pd7lf8lpTqxHmbjkAAAAASUVORK5CYII=","orcid":"","institution":"Inonu University","correspondingAuthor":true,"prefix":"","firstName":"Elif","middleName":"Taslidere","lastName":"Karaca","suffix":""},{"id":381777292,"identity":"313d2fb7-2910-43b7-b372-5ddfa4e23d03","order_by":2,"name":"Nigar Vardi","email":"","orcid":"","institution":"Inonu University","correspondingAuthor":false,"prefix":"","firstName":"Nigar","middleName":"","lastName":"Vardi","suffix":""},{"id":381777293,"identity":"2d53fbeb-6647-4465-9b3c-316252ea0411","order_by":3,"name":"Elif Gürel","email":"","orcid":"","institution":"Inonu University","correspondingAuthor":false,"prefix":"","firstName":"Elif","middleName":"","lastName":"Gürel","suffix":""},{"id":381777294,"identity":"90714151-1b16-4cca-ad83-e1ecb85ffb42","order_by":4,"name":"Harika Gözükara Bağ","email":"","orcid":"","institution":"Inonu University","correspondingAuthor":false,"prefix":"","firstName":"Harika","middleName":"Gözükara","lastName":"Bağ","suffix":""},{"id":381777295,"identity":"af50085c-0aae-47bb-883e-42a2800b0b28","order_by":5,"name":"Mehmet Gül","email":"","orcid":"","institution":"Inonu University","correspondingAuthor":false,"prefix":"","firstName":"Mehmet","middleName":"","lastName":"Gül","suffix":""}],"badges":[],"createdAt":"2024-11-06 12:08:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5402589/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5402589/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":70040189,"identity":"b48b0aec-68ab-455e-8550-0400d6142102","added_by":"auto","created_at":"2024-11-27 17:53:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1544722,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eControl group\u003c/strong\u003e. (A) Normally structured seminiferous tubules (star), interstitial space (arrow) and spermatogenic cells (line) are observed. HE, X100. (B) the interstitial area, Leydig cells and spermatogenic series cells were observed to have a normal histological appearance. HE, X400. (C) Sertoli (S) and spermatogonia cells (Sg) arranged on the basement membrane, primary spermatocytes (Ps), early (Es) and late spermatids (Gs) located close to the lumen are observed. HE, X1000. \u003cstrong\u003eD Vit group\u003c/strong\u003e (D) it is observed that the seminiferous tubule borders are smooth and spermatogenic germ cells (lines) are lined up regularly. HE, X400.\u003c/p\u003e","description":"","filename":"FigureI.png","url":"https://assets-eu.researchsquare.com/files/rs-5402589/v1/e4b42b24a6c6f6a58df1dec0.png"},{"id":70040187,"identity":"645bcc9c-71f3-4e36-aaaf-3c2e36c1fc6d","added_by":"auto","created_at":"2024-11-27 17:53:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2411708,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCis group.\u003c/strong\u003e (A) Seminiferous tubules (arrow) with irregular structure and large gaps (star) in the interstitial area are observed. HE, X100. (B) It is observed that the cells forming the spermatogenic series in the seminiferous tubules are reduced (asterisk) and are located irregularly. HE, X400. (C, D) Spermatids with annular chromatin are observed (arrow). HE, X400, X1000. (E) Cells with eosinophilic cytoplasm, paused in certain stages of meiosis and degenerated in different shapes are observed (arrow). HE, X400. (F) Giant cells containing more than one nucleus are observed (arrow). HE, X1000. (G, H) Cells belonging to the spermatogenic series that have separated from the germinative epithelium and have not completed their development are observed in the lumen (asterisk). HE, X200, X400.\u003c/p\u003e","description":"","filename":"FigureII.png","url":"https://assets-eu.researchsquare.com/files/rs-5402589/v1/086e3af2a07c727dd1768f0c.png"},{"id":70040193,"identity":"57b144ea-b9ca-41f8-89a7-f23532720c80","added_by":"auto","created_at":"2024-11-27 17:53:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":729399,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVD group\u003c/strong\u003e (A) Spermatids with annular chromatin (arrow) and cells with eosinophilic cytoplasm, paused at certain stages of meiosis and degenerating in different shapes (arrowhead) are observed. (B) It is observed that the cells forming the spermatogenic series decrease and are irregularly located (asterisk). HE, X400.\u003c/p\u003e","description":"","filename":"FigureIII.png","url":"https://assets-eu.researchsquare.com/files/rs-5402589/v1/cf31e5e86144e1af44e78e50.png"},{"id":70040797,"identity":"719b8988-f9e6-489b-b7e6-491875d6ff23","added_by":"auto","created_at":"2024-11-27 18:01:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1412236,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eControl group\u003c/strong\u003e (A) The basement membranes of the seminiferous tubules were observed to be PAS positive, thin and smooth (arrow). \u003cstrong\u003eVD group\u003c/strong\u003e(B) Similar to the control group, the basement membranes of the seminiferous tubules are PAS positive, thin and smooth (arrow). \u003cstrong\u003eCis group\u003c/strong\u003e (C) Undulation and increased PAS positivity are observed in the seminiferous tubule basement membrane (arrow). (D) \u003cstrong\u003eCis +VD group\u003c/strong\u003e Undulation and PAS positivity are observed in the seminiferous tubule basement membrane (arrow). PAS, X400.\u003c/p\u003e","description":"","filename":"FigureIV.png","url":"https://assets-eu.researchsquare.com/files/rs-5402589/v1/bdef283520de2f8505164b66.png"},{"id":70040194,"identity":"30c3f457-e8f5-48fc-a796-aa36260d1f05","added_by":"auto","created_at":"2024-11-27 17:53:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1362326,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eControl group\u003c/strong\u003e (A) \u003cstrong\u003eVD group\u003c/strong\u003e (B) Caspase-3 positive spermatogenic cells were not found. \u003cstrong\u003eCis group\u003c/strong\u003e (C), \u003cstrong\u003eCis +VD group\u003c/strong\u003e (D) Caspase-3 positive cells are observed (arrow). IHC, X400.\u003c/p\u003e","description":"","filename":"FigureV.png","url":"https://assets-eu.researchsquare.com/files/rs-5402589/v1/0ffe09f5f44a22669539f6dd.png"},{"id":70041585,"identity":"20b6fc33-ce44-4873-9f1e-966c6b0f40d8","added_by":"auto","created_at":"2024-11-27 18:09:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1245480,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eControl group\u003c/strong\u003e (A) \u003cstrong\u003eVD group\u003c/strong\u003e (B) \u003cstrong\u003eCis group\u003c/strong\u003e (C), \u003cstrong\u003eCis +VD group\u003c/strong\u003e (D) VDR positively stained seminiferous tubules are observed (asterisk). IHC, X200\u003c/p\u003e","description":"","filename":"FigureVI.png","url":"https://assets-eu.researchsquare.com/files/rs-5402589/v1/ac2008cc717da32fe81d5fcd.png"},{"id":70040190,"identity":"c6720212-d864-4d4a-a5d6-0bbd8e40a90c","added_by":"auto","created_at":"2024-11-27 17:53:28","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":599278,"visible":true,"origin":"","legend":"\u003cp\u003eSperm vitality assessment. Live sperm (A, B) (arrow), dead sperm (B, D) (arrowhead).A. Control group, B. VD group, C. Cisplatin group, D. Cisplatin+VD group. Eosin-nigrozin, X1000.\u003c/p\u003e","description":"","filename":"FigureVII.png","url":"https://assets-eu.researchsquare.com/files/rs-5402589/v1/5d0e8453e0c46ee406bea706.png"},{"id":70040798,"identity":"fdcdca13-69b5-45eb-a3fe-15448bc1d17a","added_by":"auto","created_at":"2024-11-27 18:01:28","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":534817,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological evaluation of sperm. a. normal sperm, b. bobtail, c. curled tail, d. headless sperm, e. bent neck, f. tuber head, g. hookless head, h. pin head, i. amorphous head, j. double-headed sperm. Sperm morphology stain, X400.\u003c/p\u003e","description":"","filename":"FigureVIII.png","url":"https://assets-eu.researchsquare.com/files/rs-5402589/v1/43f135da3e08002a50f805cc.png"},{"id":70940585,"identity":"37f1df61-0f9e-4c3c-b79b-b2c5567b6764","added_by":"auto","created_at":"2024-12-09 11:47:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12252745,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5402589/v1/baf2cb0d-dd6c-450b-a21f-4938c5be2771.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Investigation of the Effect of Vitamin D on Testicular Damage Caused by Cisplatin in Rats","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eCisplatin is a broad-spectrum chemotherapeutic agent widely used in the treatment of many cancer types, including head and neck, bladder, prostate, testis, breast, cervix, ovarian cancers, osteogenic sarcoma, and neuroblastoma (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). The availability of anticarcinogenic drugs is of great importance in terms of prolonging life expectancy and improving quality of life. However, when the side effects of these drugs and studies on male fertility are analyzed, there will likely be a significant infertility problem in the future (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). It has been reported that long-term azoospermia and permanent infertility may be observed as a result of the gonadotoxic effect of cisplatin chemotherapy (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Excessive cell proliferation, especially during spermatogenesis, makes the testes more sensitive to this agent (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eToday, the most widely used alternative treatment method to minimize tissue damage that occurs during the use of chemotherapeutics is the use of anti-oxidant substances. Therefore, it is thought that cisplatin-induced testicular toxicity may be reduced by anti-oxidants (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Vitamin D (VD) is a steroid prohormone synthesized mainly in the skin with the effect of sunlight. It needs to be hydroxylated in the liver and kidney to become active (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). VD is generally known for its function in maintaining and regulating calcium (Ca) and phosphorus (P) homeostasis. However, in addition to its known mineral metabolism effects, it also has various cytoprotective effects such as anti-fibrotic, anti-oxidative, and anti-inflammatory effects. It has also been shown to play a role in modulating both male and female reproductive processes. In the last decade, the relationship between VD and the reproductive system has been increasingly investigated, and new information has become available, demonstrating the presence of vitamin D receptors (VDR) in both male and female reproductive organs. Recently, it has also been demonstrated that VD deficiency is associated with erectile dysfunction, decreased sperm concentration, and low sperm motility (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study aimed to investigate the effects of VD on male reproductive toxicity due to cisplatin, a commonly used chemotherapeutic drug, by histopathologic, immunohistochemical, and biochemical evaluation.\u003c/p\u003e"},{"header":"MATERIAL AND METHOD","content":"\u003ch2\u003e\u003cstrong\u003eChemicals\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eKo\u0026ccedil;sel\u0026apos;s Cipintu 100 mg/100 ml was used for cisplatin, and Deva\u0026apos;s 300,000 IU/ml Devit-3 ampoules were used for VD.\u003c/p\u003e\n\u003ch2\u003e \u003cstrong\u003eExperimental Animals\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eA total of 28 12-week-old male \u003cem\u003eWistar albino\u003c/em\u003e rats weighing 230-300 g were obtained from Malatya İn\u0026ouml;n\u0026uuml; University Experimental Animal Research Center. Rats were housed in a well-ventilated room with clean plastic cages at constant room temperature (23℃-24℃) and relative humidity (50-55%) on a 12/12 h light/dark cycle. Feed and water were changed daily, and ad libitum was provided. All procedures were performed in accordance with the animal research guidelines of the National Institute of Health.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eEthical Declaration\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThis study was approved by İnonu University Faculty of Medicine Animal Research Ethics Committee (HAYBIS NO: 11409) and funded by İnonu University Scientific Research Project Coordination Unit with the project code TTU-2021-2533.\u003c/p\u003e\n\u003ch2\u003e \u003cstrong\u003eExperimental Design\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe animals were randomly divided into four groups: Six animals in the control and VD groups and eight animals in the cisplatin and cisplatin+VD groups (since cisplatin is a toxic substance, two more animals were used in the cisplatin groups than the number determined by biostatistics). The weights of the animals at the beginning of the experiment were recorded, and after a one-week acclimatization period, the following treatments were performed in the groups. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eControl Group:\u003c/strong\u003e Solvent agent was applied to this group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVD Group: \u003c/strong\u003eIn this group, 1000 IU/kg intraperitoneal (i.p) VD was administered every other day. The study of Abbaszadeh et al. \u003cstrong\u003e(8) \u003c/strong\u003eand the study of BaSalamah et al. \u003cstrong\u003e(6)\u003c/strong\u003e were taken as a reference for VD dose and administration times. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCisplatin Group: \u003c/strong\u003eA total of 14 mg/kg cisplatin i.p. was administered to this group in two doses. In addition, the solvent was administered i.p. every other day to the rats in this group. \u0026Ouml;zt\u0026uuml;rk et al. study \u003cstrong\u003e(9) \u003c/strong\u003eHassan et al. study \u003cstrong\u003e(10)\u003c/strong\u003e, and Ekmek\u0026ccedil;i et al. study \u003cstrong\u003e(11) \u003c/strong\u003ewere taken as references for cisplatin dose and administration times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCisplatin\u003c/strong\u003e\u003cstrong\u003e+VD Group: \u003c/strong\u003eThis group received a total of 14 mg/kg cisplatin i.p in two doses and 1000 IU/kg i.p VD every other day. \u003c/p\u003e\n\u003ch2\u003e \u003cstrong\u003eCollection of Tissue and Blood Samples\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eAfter ten days of the experimental period, the body weights of the rats were measured, and then 5 mg/kg \u003cem\u003exylazine\u003c/em\u003e and 50 mg/kg \u003cem\u003eketamine\u003c/em\u003e i.p. were administered. Under general anesthesia, their abdomen was opened through a midline incision. Blood samples were taken from the\u003cem\u003e inferior vena cava.\u003c/em\u003e Blood samples were centrifuged at 3000 xg for 10 minutes on the same day, and the serum obtained was collected in eppendorf tubes and stored at -80 C\u0026deg; for biochemical evaluations. After weighing both testicular tissues taken from the subjects, the left testis was placed in 10% formaldehyde solution for histological analysis, while the right testis was stored at -80 C\u0026deg; for biochemical studies. Sperm samples were obtained from the caudae of the epididymis for sperm parameter evaluations. After the collection of tissue and blood samples, the animals were euthanized under high-dose anesthesia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistologic Tissue Monitoring, Staining and Examination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor histopathologic evaluation, tissues were subjected to fixation and follow-up procedures. The tissue was fixed in 10% formaldehyde solution. The tissue samples were then trimmed and placed in plastic tissue tracking cassettes, and the formaldehyde solution was renewed. After 24 hours of fixation, the tissue samples were subjected to routine tissue tracking steps in the Tissue-Tek VIP/SAKURA tissue tracking device. Tissue samples were embedded in paraffin blocks after tissue tracking procedures were completed. Sections of 4 \u0026mu;m each were taken from the paraffin blocks using a Leica RM2145 microtome. The sections were stained with hematoxylin-eosin (HE) to observe the general histologic structure and periodic acid schiff (PAS) staining method to examine the basement membrane changes. Sections were also immunohistochemically stained with caspase-3 and VDR primary antibodies. The preparations were examined, scored, and photographed with a Leica DFC280 light microscope and Leica Q (Leica Micros Imaging Solution Ltd, Cambridge, UK) image analysis system.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eHistologic Evaluation\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003e\u003cstrong\u003eDiameter Measurement of Seminiferous Tubules\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMeasurement was performed under X200 magnification on 20-round or nearly round seminiferous tubules from each preparation. The short and long side lengths were measured for each tubule, and the mean value was recorded.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of Epithelial Thickness of Seminiferous Tubules\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe measurement was performed under X200 magnification at four different locations, including the thinnest and thickest parts of 20 seminiferous tubules in stages 7-8 for each preparation. The average of these four measurements was recorded for each seminiferous tubule.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJohnsen Score\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJohnsen testicular histologic damage score method was used in the evaluation of the tissues examined by HE staining \u003cstrong\u003e(12). \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemical Evaluation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate positive immunoreactivity in testicular tissue, ten different fields from each sample were examined at X400 magnification. The mean number of caspase-3 (SantaCruz) and VDR (SantaCruz) immunopositive cells was measured.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSperm Morphology and Vitality Assessment \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cauda portion of the epididymis separated from the right testis was used for sperm analysis. Morphologically, sperms were evaluated by staining dried semen smear slides with sperm \u003cem\u003emorphology\u003c/em\u003e stain. The examination was performed under a Leica DFC280 light microscope at X400 magnification. Six samples from each group and 100 spermatozoa per sample were analyzed. The spermatozoa were morphologically categorized into three classes: normal, head-neck anomaly, and tail anomaly by Immunohistochemical Evaluation. For viability, spermatozoa were evaluated under Leica DFC280 light microscope at X400 magnification after drying of smears mixed with eosin-nigrosin. Stained spermatozoa were considered dead, while unstained spermatozoa were considered viable. For each sample, 100 spermatozoa were analyzed, six spermatozoa from each group. Percent viability was recorded for each sample.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSperm Counting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cauda portion of the epididymis, separated from the left testicle, was used for sperm count. In a petri dish containing 1 milliliter of G-IVF solution, the tissue was cut into small pieces and left for incubation. Afterward, the dissected tissue was taken into eppendorf tubes together with the G-IVF solution. After centrifugation at 5000 xg for one minute, the supernatant was sampled with a micropipette on a Thoma slide. Sperm counting was performed under the Leica DFC280 light microscope at X400 magnification. Six samples from each group were evaluated, and sperm count was recorded.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiochemical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFSH, LH, Testosterone, Estrogen, Ca, VD\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlood collected from rats under anesthesia was placed in a gel biochemistry tube and centrifuged at 3000 rpm for ten minutes. The serum obtained after centrifugation was collected in eppendorf tubes and stored at -80 \u0026deg;C. SunRed brand rat-specific ELISA kits based on a double-antibody sandwich technique were used to test rat FSH, LH, testosterone, estrogen, VD, and Ca levels in the samples. For FSH, SunRed brand rat-specific FSH ELISA kit with catalog number 201-11-0183 LOT number 202201 was used. For LH, SunRed brand rat-specific LH ELISA kit with catalog number 201-11-0180 LOT number 202201 was used. For testosterone, SunRed brand rat-specific testosterone ELISA kit with catalog number 201-11-0735 and LOT number 202201 was used. For estrogen, SunRed brand rat-specific estrogen ELISA kit with catalog number 201-11-0177 and LOT number 202203 was used. For VD, the SunRed brand rat-specific VD ELISA kit with catalog number 201-11-0016 was used. For Ca, SunRed brand rat-specific Ca ELISA kit with catalog number 201-11-0949, LOT number 202201, was used. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInorganic Phosphate \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRel Assay branded kit with LOT number EK2101P was used for inorganic phosphate measurement. A direct method was preferred for P determination. P reacts with ammonium molybdate in an acid medium to form a yellow phosphomolybdate complex. The intensity of the color formed is proportional to the P concentration in the sample at 340 nm \u003cstrong\u003e(13). \u003c/strong\u003eThe reading on the spectrophotometer was recorded for each sample.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTotal Oxidant Level\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLOT number KM21136O Rel Assay branded kit was used for total oxidant level (TOL) measurement. The application was performed by following the sequential steps specified in the kit operating procedure. The working principle of the kit is based on oxidants in the sample medium oxidizing the ferrous ion-chelator complex to ferric ion. Measurement of the intensity of the color formed as a result of ferric ions forming a colored complex with chromogen in an acidic environment at a wavelength of 530 nm in a spectrophotometer gives the total amount of oxidant molecules present in the sample as micromolar hydrogen peroxide equivalent per liter \u003cstrong\u003e(14). \u003c/strong\u003eThe spectrophotometer reading for each sample was recorded. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTotal Anti-oxidant Level \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRel Assay branded kit with LOT number EK21122A was used for total anti-oxidant level (TAL) measurement. The application was performed by following the sequential steps specified in the kit operating procedure. The working principle of the kit is based on the fact that anti-oxidants in the sample medium convert the dark blue-green ABTS radical into colorless reduced ABTS form. The absorbance change at 660 nm wavelength in the spectrophotometer gives the total anti-oxidant molecule level in the sample with \u003cem\u003eTrolox \u003c/em\u003eEquivalent, a vitamin E analog \u003cstrong\u003e(15). \u003c/strong\u003eThe spectrophotometer reading was recorded for each sample. \u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eNumerical data were summarized with median, minimum, and maximum values. The Kruskal-Wallis test, followed by the Conover pairwise comparison method, was used for independent group comparisons. Two dependent measures were compared with the Wilcoxon paired two-sample test. The significance level was accepted as 0.05 in all tests.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003eWeight Assessment of Experimental Animals\u003c/h2\u003e \u003cp\u003eWhen cisplatin and control groups were compared in terms of body weights at the beginning and end of the experiment, it was found that the difference between the body weights of the rats was statistically significant (p\u0026thinsp;=\u0026thinsp;0.046). No change was observed in body weight loss as a result of VD administration to the cisplatin group (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Animal weights at the beginning and end of the experiment are presented in 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\u003eComparison of Animal Weights at the Beginning and End of the Experiment\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eInitial Weight\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eFinal Weight\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedian\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMax\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMedian\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMax\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003ep\u003c/b\u003e\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\u003eControl \u0026ordf;\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e220,00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e195\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e230\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e246,50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e209\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e254\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0,075\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCis\u003c/b\u003e \u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e281,50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e275\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e292\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e201,00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e148\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e282\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0,046\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCis\u0026thinsp;+\u0026thinsp;VD\u003c/b\u003e \u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e247,00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e223\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e267\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e203,50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e156\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e242\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0,028\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVD \u0026ordf;\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e228,50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e210\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e239\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e243,50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e221\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e271\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0,116\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003e* Different letters indicate statistical significance of differences between groups.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of Testicular Weights\u003c/h2\u003e \u003cp\u003eWhen cisplatin and the control group were compared in terms of testicular weights at the end of the experiment, it was determined that the difference between the testicular weights of the rats was not statistically significant (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Testicular weights are presented in 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\u003eComparison of Testicular Weights\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCis\u0026thinsp;+\u0026thinsp;VD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eVD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ep\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTesticular Weights\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,295 (1,08\u0026thinsp;\u0026minus;\u0026thinsp;1,44)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1,225 (0,7\u0026thinsp;\u0026minus;\u0026thinsp;1,35)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1,255 (1,02\u0026thinsp;\u0026minus;\u0026thinsp;1,39)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1,245 (1,06\u0026thinsp;\u0026minus;\u0026thinsp;1,29)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,709\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eNo significant difference was found between groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0,05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eHistologic Evaluation\u003c/h2\u003e \u003cdiv id=\"Sec26\" class=\"Section4\"\u003e \u003ch2\u003eControl Group\u003c/h2\u003e \u003cp\u003eIn HE-stained sections, the seminiferous tubules in the testis had a normal histologic appearance (Fig.\u0026nbsp;1A). The tubules were composed of seminiferous epithelium sitting on a prominent basal lamina (Fig.\u0026nbsp;1B). Sertoli cells and spermatogenic series cells were clearly distinguished in the seminiferous epithelium. Spermatozoa were observed in the lumen of the majority of seminiferous tubules. (Fig.\u0026nbsp;1C). In this group, the mean seminiferous tubule diameter was 169.588 \u0026micro;m, and the mean germinal epithelial height was 85.814 \u0026micro;m. In addition, the mean Johnsen score was 8.75. The comparison of seminiferous tubule diameter, germinal epithelial thickness, and Johnsen score of the groups is shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of Seminiferous Tubule Diameter, Germinal Epithelial Thickness and Johnsen Score\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCis\u0026thinsp;+\u0026thinsp;VD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eVD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ep\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSeminiferous Tubule Diameter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e169,588\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e147,195\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e151,792\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e173,539\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGerminal Epithelial Thickness\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e85,814\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e69,519\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e71,662\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e85,508\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0,001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eJohnsen Score\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.75\u003csup\u003ea\u003c/sup\u003e (8.4-9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.07\u003csup\u003eb\u003c/sup\u003e (5.42\u0026ndash;7.36)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.42\u003csup\u003ec\u003c/sup\u003e (7.28\u0026ndash;7.56)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.55\u003csup\u003ec\u003c/sup\u003e (7.12\u0026ndash;7.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVDR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.05\u003csup\u003eab\u003c/sup\u003e (0.8\u0026ndash;1.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.925\u003csup\u003ea\u003c/sup\u003e (0.55\u0026ndash;1.05)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.4\u003csup\u003eb\u003c/sup\u003e (1.15\u0026ndash;1.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.375\u003csup\u003eb\u003c/sup\u003e (1.05\u0026ndash;1.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.008\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCaspase-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003csup\u003ea\u003c/sup\u003e\u0026nbsp;(0\u0026ndash;0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9\u003csup\u003eb\u003c/sup\u003e\u0026nbsp;(0\u0026ndash;15)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003csup\u003ec\u003c/sup\u003e\u0026nbsp;(0\u0026ndash;9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003csup\u003ea\u003c/sup\u003e\u0026nbsp;(0\u0026ndash;0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e* Different letters indicate statistical significance of differences between groups.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eVD Group\u003c/h2\u003e \u003cp\u003eIn this group, the testis had a generally normal histologic appearance. Similar to the control group, most of the seminiferous tubules had smooth borders, and spermatogenic germ cells were regularly arranged. (Fig.\u0026nbsp;1D). The tubules contained germ cells at all stages of spermatogenesis, from spermatogonium to spermatid. The mean seminiferous tubule diameter was 173.539 \u0026micro;m, and the mean germinal epithelial height was 85.508 \u0026micro;m. In addition, the mean Johnsen score was 7.55. When compared with the control group in terms of seminiferous tubule diameter and germinal epithelial thickness, the difference between them was not found to be statistically significant (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). However, Johnsen score decreased statistically significantly in this group compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eCisplatin Group\u003c/h2\u003e \u003cp\u003eIn this group, some seminiferous tubule structures were disorganized, and large gaps were observed in the interstitial space between the seminiferous tubules (Fig.\u0026nbsp;2A). The mean seminiferous tubule diameter was 147.195 \u0026micro;m, and the mean germinal epithelial height was 69.519 \u0026micro;m. When compared with the control group, a statistically significant decrease in tubule diameter and epithelial height was observed in this group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The mean Johnsen score of this group was 7.07. Compared to the control group, Johnsen score was significantly decreased in this group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In some seminiferous tubules, it was observed that the cells forming the spermatogenetic series were decreased and irregularly arranged (Fig.\u0026nbsp;2B). In addition, spermatids with annular chromatin were found in some seminiferous tubules ((Fig.\u0026nbsp;2C, D). In some tubules, degenerated cells with eosinophilic cytoplasm paused at certain stages of meiosis and were observed in different shapes (Fig.\u0026nbsp;2E). Another striking finding was the presence of giant cells with multiple nuclei in the tubules (Fig.\u0026nbsp;2F). In addition, in some tubules, cells belonging to the spermatogenic series that separated from the germinative epithelium and did not complete their development were found to accumulate in the lumen (Fig.\u0026nbsp;2G, H).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eCisplatin\u0026thinsp;+\u0026thinsp;VD Group\u003c/h2\u003e \u003cp\u003eIn this group, the mean seminiferous tubule diameter was 151.792 \u0026micro;m, and the mean germinal epithelial height was 71.662 \u0026micro;m. The increase in these values was not statistically significant when compared with the cisplatin group (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). On the other hand, when compared with the control group, the difference in tubule diameters and germinal epithelial height was statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The mean Johnsen score of this group was 7.42. Compared to the cisplatin group, Johnsen score increased statistically significantly in this group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Spermatids with annular chromatin and spermatogenic cells arrested at certain stages of meiosis were also observed in this group (Fig.\u0026nbsp;3A). Similar to the cisplatin group, cells forming the spermatogenic series were found to be decreased and irregularly arranged in some seminiferous tubules (Fig.\u0026nbsp;3B).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePAS staining\u003c/h3\u003e\n\u003cp\u003eIn PAS-stained sections, Leydig cells were present in the interstitial connective tissue between the tubules in control and VD groups. The basement membranes of the seminiferous tubules were stained positively for PAS, were thin and smooth, and were found to have a normal histologic structure (Fig.\u0026nbsp;4A,B). In the cisplatin group, the basement membranes of the seminiferous tubules were corrugated, and PAS positivity was increased (Fig.\u0026nbsp;4C). In contrast, in the cisplatin\u0026thinsp;+\u0026thinsp;VD group, the corrugation and PAS positivity in the basement membranes of the seminiferous tubules were similar to the cisplatin group (Fig.\u0026nbsp;4D).\u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical assessment\u003c/h2\u003e \u003cdiv id=\"Sec32\" class=\"Section3\"\u003e \u003ch2\u003eCaspase-3 Immunohistochemical Staining\u003c/h2\u003e \u003cp\u003eIn the control and VD groups, especially the cytoplasmic residues of spermatids in the seminiferous tubules stained positively with caspase-3 No positively stained spermatogenic cells were observed in these groups (Fig.\u0026nbsp;5AB). In the cisplatin group, the intensity of positively stained spermatogenic cells was statistically significantly increased compared to the control group (Fig.\u0026nbsp;5C). In the cisplatin\u0026thinsp;+\u0026thinsp;VD group, the intensity of positively stained spermatogenic cells was statistically significantly decreased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) compared to the cisplatin group (Fig.\u0026nbsp;5D). A comparison of caspase-3 positivity of the groups is shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cdiv id=\"Sec33\" class=\"Section4\"\u003e \u003ch2\u003eVDR Immunohistochemical Staining\u003c/h2\u003e \u003cp\u003eIn the control and VD groups, positively stained seminiferous tubules were observed in the VDR staining sections (Fig.\u0026nbsp;6AB). When the VD group was compared with the control group, the increase in the number of positively stained tubules was not statistically significant (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In the cisplatin group, the intensity of positively stained spermatogenic cells decreased in this group compared to the control group, but there was no statistically significant difference (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;6C). In the VD group, the intensity of positively stained spermatogenic cells increased statistically significantly in this group compared to the cisplatin group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;6D). A comparison of VDR positivity of the groups is presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e \u003ch2\u003eSperm Analysis Findings\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. shows the sperm count, vitality, and abnormal sperm count of the groups. Cisplatin treatment significantly decreased sperm count compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). There was no statistically significant difference in sperm count between the Cisplatin group and the Cisplatin\u0026thinsp;+\u0026thinsp;VD group (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of Sperm Analyzes\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCis\u0026thinsp;+\u0026thinsp;VD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eVD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ep\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSperm Count\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20\u003csup\u003ea\u003c/sup\u003e (16\u0026ndash;150)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6\u003csup\u003eb\u003c/sup\u003e(\u003cspan additionalcitationids=\"CR5 CR6 CR7 CR8 CR9\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003csup\u003eb\u003c/sup\u003e (\u003cspan additionalcitationids=\"CR6 CR7 CR8 CR9 CR10 CR11\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18.5\u003csup\u003ea\u003c/sup\u003e (13\u0026ndash;200)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSperm Vitality\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.965\u003csup\u003ea\u003c/sup\u003e (0.95\u0026ndash;0.99)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.13\u003csup\u003eb\u003c/sup\u003e (0.11\u0026ndash;0.24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.32\u003csup\u003ec\u003c/sup\u003e (0.24\u0026ndash;0.57)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.79\u003csup\u003ed\u003c/sup\u003e (0.63\u0026ndash;0.82)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSperm Total Anomaly\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.085\u003csup\u003ea\u003c/sup\u003e (0.05\u0026ndash;0.15)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.23\u003csup\u003eb\u003c/sup\u003e (0.18\u0026ndash;0.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.16\u003csup\u003ec\u003c/sup\u003e (0.11\u0026ndash;0.25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.21\u003csup\u003ebc\u003c/sup\u003e (0.15\u0026ndash;0.24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.003\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHead-Neck Anomaly\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.01\u003csup\u003ea\u003c/sup\u003e (0-0.02)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.095\u003csup\u003eb\u003c/sup\u003e (0.03\u0026ndash;0.15)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.015\u003csup\u003ea\u003c/sup\u003e (0-0.02)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.005\u003csup\u003ea\u003c/sup\u003e (0-0.02)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTail Anomaly\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.07\u003csup\u003ea\u003c/sup\u003e (0.05\u0026ndash;0.14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.175\u003csup\u003eb\u003c/sup\u003e (0.08\u0026ndash;0.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.145\u003csup\u003eb\u003c/sup\u003e (0.1\u0026ndash;0.23)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.2\u003csup\u003eb\u003c/sup\u003e (0.14\u0026ndash;0.24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.011\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e* Different letters indicate statistical significance of differences between groups.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWhen the cisplatin group was compared with the control group, it was found that sperm vitality decreased statistically significantly in this group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). On the other hand, when the Cisplatin\u0026thinsp;+\u0026thinsp;VD group was compared with the Cisplatin group, it was observed that sperm vitality increased statistically significantly in this group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;7). In the VD-only group, a significant decrease in sperm vitality was detected compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eIt was determined that cisplatin treatment caused a statistically significant increase in the amount of abnormal sperm (head-neck and tail anomaly), and this increase decreased with VD treatment (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This decrease was statistically significant in terms of total sperm anomaly and head-neck anomaly, but the difference was not statistically significant in terms of tail anomaly (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). There was a significant increase in sperm anomaly in the VD-only group compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;8).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eBiochemical Findings\u003c/h3\u003e\n\u003cp\u003eThe values of TOL, TAL, FSH, LH, testosterone, estrogen, VD, Ca, and P levels are presented in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of biochemical analyzes\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCis\u0026thinsp;+\u0026thinsp;VD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eVD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ep\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\u003eTAS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.034\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.025\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.119\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.637\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTOS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.963\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.243\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.296\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.185\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFSH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11,008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11,01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10,922\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11,591\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,398\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8,624\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7,197\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8,439\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7,796\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,198\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTestosterone\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e439,297\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e410,33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e421,423\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e442,011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,307\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEstrogen\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29,938\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24,669\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26,77\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e30,794\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e879.865\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e768.263\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e845.244\u003csup\u003eac\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e967.212\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.015\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCa\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,458\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2,528\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2,884\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3,683\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,016\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8,304\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8,904\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8,475\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8,412\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,765\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e* Different letters indicate statistical significance of differences between groups.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAs a result of the evaluations, it was determined that TOL, which is an indicator of oxidative damage, increased statistically significantly in cisplatin-treated rats compared to control and all other groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, it was determined that VD administration decreased the increase in TOL caused by cisplatin at a statistically significant level (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In the VD-only group, there was a statistically significant increase in TOL level compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). No difference was observed between the groups in terms of TAL (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eWhile no difference was observed between the groups in terms of serum FSH, LH, and testosterone levels (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), serum estrogen levels were significantly decreased in cisplatin-treated rats compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, it was determined that VD administration did not affect the estrogen decrease caused by cisplatin (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eSerum VD levels decreased in cisplatin-treated rats compared to the control group, but there was no statistically significant difference (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). When VD-administered groups were compared with cisplatin and control groups, it was found that VD levels increased statistically significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In terms of serum Ca level, no difference was observed between cisplatin and control groups, while Ca level increased in VD-treated groups when compared with cisplatin and control groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). No difference was observed between the groups in terms of serum P level (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eCisplatin is a potent antineoplastic agent and is used in the treatment of many solid organ cancers, mainly head and neck, lung, testicular, ovarian, and breast (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). However, cisplatin has side effects, including ototoxicity, neurotoxicity, nephrotoxicity, hepatotoxicity, and testicular toxicity, in addition to its anti-tumoral effects (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Adverse effects of short- and long-term cisplatin treatment on testicular function have been reported in many studies (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Studies have shown that cisplatin leads to excessive production of reactive oxygen species (ROS) as well as direct DNA damage (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Therefore, substances with anti-oxidant capacity have been tried to protect tissues against toxic substances (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). VD has anti-oxidative and anti-inflammatory effects, as well as regulates Ca and P homeostasis (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). However, there are not enough studies on the effects of VD on cisplatin-induced testicular and epididymal damage. Therefore, in our study, we aimed to evaluate the effects of VD on cisplatin-induced male reproductive toxicity by histopathologic, immunohistochemical, and biochemical parameters.\u003c/p\u003e \u003cp\u003eIn our study, TOL and TAL parameters, which are indicators of oxidative damage and anti-oxidant activity, were evaluated in serum samples to determine the oxidative damage caused by cisplatin in the testis and the protective properties of VD. In previous studies, it was reported that cisplatin administration caused an increase in lipid peroxidation levels and a decrease in anti-oxidant parameters in general (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). In our study, it was observed that the TOL level significantly increased in the cisplatin group compared to the control group and other groups, whereas the TAL level was similar to that of the other groups. In a ten-day study in which a single dose of 7 mg/kg i.p. cisplatin was administered, Y\u0026uuml;cel et al. did not find a significant difference between the groups in the TAL level similar to our study but found that TOL and OSI levels increased in the cisplatin group. Y\u0026uuml;cel et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) thought that the reason for the lack of difference in TAL level may be increased anti-oxidant molecules to balance the high levels of prooxidant molecules in damaged testicular tissue. In their 21-day study conducted with three different VD doses (100 IU/kg, 1000 IU/kg, 10.000 IU/kg), Mokhatari-Zaer et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e) reported that VD supplementation decreased the increased MDA level in the damage group and increased CAT and SOD levels among anti-oxidative parameters compared to the damage group. Similarly, in our study, it was found that TOL level decreased significantly in the cisplatin\u0026thinsp;+\u0026thinsp;VD group compared to the cisplatin group.\u003c/p\u003e \u003cp\u003eIn our study, seminiferous tubule diameters and seminiferous tubule epithelial thickness were measured in HE-stained histologic sections and compared between the groups. Many researchers have shown that damage to the seminiferous tubules develops after cisplatin administration in rats. It has been reported that cisplatin causes a decrease in seminiferous tubule diameter, disorganization in germinative epithelium, and shedding of germ cells at different stages of meiosis into the lumen (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Beytur et al.(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) reported a significant decrease in seminiferous tubule diameter and germinal epithelial thickness in the cisplatin-treated groups in a ten-day study in which a single dose of 7 mg/kg i.p cisplatin was administered. Similarly, we observed a significant decrease in seminiferous tubule diameter and germinal epithelial thickness in the cisplatin-treated groups in our study. We think that this decrease may be due to the decrease in cell diameters and/or the absence of seminiferous epithelial cells that are expelled into the lumen without maturing due to DNA damage caused directly by cisplatin. However, although there was an increase in seminiferous tubule diameter and germinal epithelial thickness in the cisplatin\u0026thinsp;+\u0026thinsp;VD group, this difference was not statistically significant. In contrast to our results, Yal\u0026ccedil;ın et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) investigated the effect of VD on methotrexate damage and reported that VD significantly increased seminiferous tubule diameter and germinal epithelial thickness compared to the damage group. Mohamed et al. reported that VD caused a significant increase in seminiferous tubule diameter and germinal epithelial height compared to the damage group in their study on torsion-detorsion testicular injury (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). We think that the reason for this difference from the results of other studies is that our experimental period was limited to ten days or the variability in the drug sensitivity of rats.\u003c/p\u003e \u003cp\u003eMohammadnejad et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e) reported that atrophy in the seminiferous tubules, damage in the germinal epithelium, loss of connection between the basal lamina and spermatogonium and inflammation in the peritubular area were observed in their study investigating the effect of cisplatin on the testis. In our study, we evaluated seminiferous tubule damage with Johnsen score and found that Johnsen score decreased significantly in the cisplatin-treated group. In addition, histologically, we found degenerated cells with eosinophilic cytoplasm, paused at certain stages of meiosis and observed in different shapes, spermatids with multiple annular chromatin in some seminiferous tubules, and giant cells containing more than one nucleus. Ilbey et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e) administered 7 mg/kg i.p cisplatin daily for five days to rats and reported that the spermatogenesis process was negatively affected, and the Johnsen score was significantly decreased in the cisplatin-treated group compared to the control group. Azarbarz et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) also found that cisplatin caused a significant decrease in Johnsen score in a 15-day study in which they administered a single dose of 5 mg/kg cisplatin. When the cisplatin\u0026thinsp;+\u0026thinsp;VD group was compared with the cisplatin group, we found that the Johnsen score increased significantly. Similar to our study, Yal\u0026ccedil;ın et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) reported that VD application significantly increased the Johnsen score in their study in which testicular damage was induced by methotrexate.\u003c/p\u003e \u003cp\u003eIn our study, the basement membrane structure surrounding the seminiferous tubules was evaluated using the PAS staining method. PAS-stained sections in the cisplatin group showed increased corrugation and PAS positivity in the basement membrane of the seminiferous tubules. Similar to our study, Sakr et al. reported that the basal laminae and interstitial areas of the seminiferous tubules of rats treated with cytotoxic agents were stained strongly PAS positive (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). In the literature review, no studies evaluating the effect of VD on the testis with PAS staining were found.\u003c/p\u003e \u003cp\u003eIn many studies, it was reported that cisplatin administration caused a decrease in body and testicular weights (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). In our study, when the pre- and post-experimental body weights were compared, it was found that there was a statistically significant weight loss in the cisplatin-treated groups compared to the control group, but there was no significant difference between the groups in terms of testicular weights. It is thought that the reason for the loss in body weight in the cisplatin-treated groups may be the toxic effect of the drug on the gastrointestinal system. In our study, there was no significant difference in body weight and testicular weight in the cisplatin\u0026thinsp;+\u0026thinsp;VD group compared to the cisplatin group. Yal\u0026ccedil;ın et al.(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) investigated the effect of VD on methotrexate-induced testicular damage and reported that, similar to our study, there was no significant difference between methotrexate and methotrexate\u0026thinsp;+\u0026thinsp;VD groups in terms of body weights.\u003c/p\u003e \u003cp\u003eCisplatin, a chemotherapeutic agent, is a genotoxic substance that causes anomalies in the head of spermatozoon and damage to spermatozoon DNA (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). In their study showing that the destructive effects of cisplatin treatment on testicular tissue and spermatogenesis were reduced by retinoic acid administration, Y\u0026uuml;cel et al. reported that cisplatin given at a dose of 7 mg/kg significantly decreased sperm motility and sperm count in rats (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Similarly, Shati et al. reported that cisplatin increased abnormal sperm count and decreased sperm motility and sperm count (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). In our study, we found that sperm count and sperm vitality decreased significantly in the cisplatin-treated groups compared to the control group. In addition, we found that sperm vitality increased and sperm head-neck abnormality decreased in the cisplatin\u0026thinsp;+\u0026thinsp;VD group compared to the cisplatin group. Abbaszadeh et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) investigated the effect of VD against lead damage and found that additional VD administration caused improvement in semen parameters (sperm concentration, motility, and vitality), similar to our results.\u003c/p\u003e \u003cp\u003eTestosterone, which plays an important role in the initiation and maintenance of spermatogenesis, is secreted by Leydig cells, and testosterone level is directly related to Leydig cell function and number (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Unlike germ cells, Sertoli and Leydig cells, which do not proliferate in adults, are resistant to cytotoxic agents, and most of them survive after cytotoxic therapy. However, these cells may undergo functional damage (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Previous studies have shown that cisplatin administration decreased serum testosterone levels (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Similarly, Afsar et al. reported in their study that cisplatin caused damage in Leydig cells, resulting in a decrease in cell number and a decrease in testosterone level (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). It has also been reported that chemotherapy causes Leydig cell damage despite normal testosterone levels; however, a decrease in testosterone level and an increase in LH level are expected to indicate complete Leydig cell failure. Ateşşahin et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e) reported that there was no significant difference between the groups in terms of testosterone hormone in a ten-day study in which a single dose of 7 mg/kg i.p cisplatin was administered. In our study, serum testosterone levels were measured to determine Leydig cell damage, and it was observed that testosterone levels decreased in the cisplatin group compared to the control group, but the decrease was not statistically significant. In addition, there was no difference between the groups when FSH and LH results were evaluated. Our results showed that cisplatin did not cause complete Leydig cell failure, similar to some previous studies.\u003c/p\u003e \u003cp\u003eIt is known that estradiol in men is mainly derived from the extra gonadal conversion of testosterone. However, studies have shown that Sertoli cells also have aromatase expression. It has also been reported that VD increases aromatase expression in Sertoli cells. Therefore, VD is thought to affect estrogen levels systemically and locally by regulating aromatase enzyme expression (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). In our study, we found that estrogen levels decreased significantly in the cisplatin-treated group compared to the control group. When cisplatin and cisplatin\u0026thinsp;+\u0026thinsp;VD groups were compared, we observed that estrogen levels increased, but there was no significant difference. We think that the reason why cisplatin decreases estrogen levels in male rats may be the decrease in aromatase expression due to Sertoli cell damage and/or decreased adipose tissue with decreased body weight in cisplatin-treated animals. Because it is known that adipose tissue has aromatase enzyme expression. Since there are no studies on the effect of cisplatin on estrogen levels in male rats in the literature review, further studies are needed to understand the mechanism fully.\u003c/p\u003e \u003cp\u003eRecent studies have shown that testicular germ cell death occurs through apoptosis. Spontaneous apoptosis is a physiologic process that occurs during the development of germ cells and leads to the elimination of damaged germ cells, and some external stimuli, such as ionizing radiation, chemotherapy, and hormonal manipulations, also lead to apoptosis (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). There are several molecular mechanisms by which cisplatin treatment causes cell apoptosis (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). The first of these is oxidative stress, which is one of the most important mechanisms in cisplatin toxicity. Increased reactive oxygen species trigger apoptosis both intrinsically and extrinsically. The second is its effect on Ca hemostasis. Activated cisplatin causes uncoupling of oxidative phosphorylation and inhibition of mitochondrial respiration. This results in Ca influx from mitochondria and, consequently, a transient increase in intracellular Ca levels. Disruption of Ca homeostasis by cisplatin initiates primary events such as lipid and enzyme inhibition peroxidation. As a result, apoptosis or necrosis occurs (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Apoptosis has a critical role in the removal of damaged spermatogenic cells to prevent abnormal sperm formation (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). It has also been shown that spermatocytes that fail to complete mitotic division are removed by apoptosis (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Findings such as the separation of spermatogonial cells from each other and from the basal lamina, nuclear condensation, and the appearance of spermatids with annular chromatin are indicators of apoptosis in the seminiferous tubules (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). In our study, we found a large number of spermatids with annular chromatin in some seminiferous tubules by light microscopy and apoptotic cells, which were identified by immunohistochemistry using caspase-3 activity. Caspases are cysteine-proteinase group enzymes that play an important role during apoptosis, which is known as cell suicide, and some cellular changes that occur during apoptosis depend on the activity of these enzymes (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Simsek et al. reported that caspase-3 positivity increased significantly in the cisplatin group in a ten-day study in which they administered a total of 36 mg/kg cisplatin (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Aly et al. found a significant increase in caspase-3 activity in cisplatin-treated rats in a four-week study in which they administered 7.5 mg/kg cisplatin weekly (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Gholami et al. administered a single dose of 5 mg/kg cisplatin in a seven-day study investigating the effect of methylene blue on cisplatin damage and reported an increase in caspase-3 immunostaining in the cisplatin group (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). In our study, it was found that the number of caspase-3 positive cells increased significantly in the cisplatin group compared to the control group. In our study, when cisplatin and cisplatin\u0026thinsp;+\u0026thinsp;VD groups were compared, it was observed that the number of caspase-3 positive cells decreased. Similarly, Helal et al. reported a significant decrease in caspase-3 in rats given 1000 IU/kg VD supplementation daily compared with the damage group in their 90-day study (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). In their study on VD supplementation in diabetic rats, Ding et al. reported a significant decrease in caspase-3 in the VD-supplemented group compared to the damage group (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). Similarly, Yal\u0026ccedil;ın et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) investigated the effect of VD on methotrexate damage and found a significant decrease in caspase-3 in the methotrexate\u0026thinsp;+\u0026thinsp;VD group compared to the methotrexate group.\u003c/p\u003e \u003cp\u003eVitamin D is known for its function in Ca and P homeostasis and is mainly synthesized in vivo in the skin under the influence of sunlight. However, it is an atypical vitamin due to its binding to the steroid hormone receptor and is considered a prohormone. The demonstration that VDR and VD metabolizing enzymes (CYP2R1, CYP27B1, and CYP24A1) are expressed in Sertoli cells, germ cells, Leydig cells, spermatozoa, and male genital tract epithelial cells in the testis suggests that VD may be important for spermatogenesis and sperm. Many studies have shown that VD deficiency negatively affects semen quality and can be compensated by VD supplementation (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). Jensen et al. (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e) stated that VDR level is a positive sign for sperm quality and reported that 1α,25-dihydroxyvitamin D3 may cause increased sperm motility by triggering VDR-mediated increase in intracellular Ca concentration in human spermatozoa. Sood et al. (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e) reported a decrease in Sertoli cell function, degenerative changes in germinal epithelium, and a decrease in Leydig cells in rats with VD deficiency. In addition, there are also studies showing that high VD levels negatively affect semen parameters. In an animal experiment in which VD was administered intramuscularly at three different doses, low-dose VD injection was shown to improve testicular function compared to high-dose administration. This study concluded that hypervitaminosis D alters mitochondrial activity by increasing intracellular Ca levels and has negative effects on the spermatogenesis process. This finding shows the importance of optimal VD dose in the regulation of male fertility (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). In our study, it was observed that serum VD levels decreased in cisplatin-treated rats compared to the control group, but there was no significant difference, but VD levels increased statistically significantly in VD-treated groups compared to cisplatin and control groups. There was no difference between cisplatin and control groups in terms of serum Ca level, but Ca level increased statistically significantly in the VD-treated group when compared with the other groups. In the cisplatin group, VDR positivity statistically decreased significantly compared to the control group. In the cisplatin\u0026thinsp;+\u0026thinsp;VD group, VDR positivity was significantly increased compared to the cisplatin group. Similarly, Jeremy et al. reported that VDR immunostaining decreased in the D-galactose-induced rat group, and VDR immunostaining increased in the VD-supplemented group compared to the damage group (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Liu et al. (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e) investigated the effect of VD in diabetic rats and reported that there was a decrease in VDR expression in the diabetic group, and VD supplementation significantly increased VDR expression compared to the diabetic group. The decrease in VDR in the cisplatin group may be associated with increased TOL levels or increased apoptosis.\u003c/p\u003e \u003cp\u003eIn conclusion, this study shows that VD administration attenuates testicular damage in rats with cisplatin-induced testicular injury. We suggest that VD may be used in combination with cisplatin in cisplatin-treated patients due to its anti-oxidative and anti-inflammatory effects as well as its regulation of Ca and P homeostasis. However, further studies are needed to add VD to clinical treatment protocols.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRHC, data collected and histologically analyzed, and interpreted the data, and contributed signifcantly to the writing of the article. ETK and NV, performed histological analysis and interpreted them. EG, analyzed the biochemical data. MG, assisted in histological analysis. HG, performed a statistical analysis of the data. ETK, performed the histological analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo potential conflict of interest was reported by the author(s)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe present study was supported by the Inonu University Scientific Research Projects coordination (Grant No.\u0026nbsp;TTU-2021-2533)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that has been used is confidential.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDasari S, Bernard Tchounwou P. 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Available from: https://onlinelibrary.wiley.com/doi/full/10.1111/jdi.12886\u003c/li\u003e\n\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":"Vitamin D, Rat, Cisplatin, Testis, Immunohistochemistry","lastPublishedDoi":"10.21203/rs.3.rs-5402589/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5402589/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eAim: \u003c/strong\u003eIn our study, we aimed to investigate the effects of VD against testicular damage caused by cisplatin by histopathological, immunohistochemical and biochemical methods.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterial and Method: \u003c/strong\u003e28 rats were divided into four groups, control, VD, cisplatin and cisplatin+ VD groups. At the end of the 10-day experiment, the rats were sacrificed under ketamine/xylazine anesthesia. Right testicles were used for biochemical analyzes and left testicles were used for histological analyses. Sperm vitality and morphology were evaluated from semen samples obtained from the cauda part of the epididymis. Number of sperms was also counted. Biochemically, TOS, TAS, FSH, LH, testosterone, estrogen, VD, Ca and P levels were measured by ELISA test kits using spectrophotometric methods.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eIn histopathological analysis; a decrease in seminiferous tubule diameter and germinal epithelial thickness, decrease in Johnsen score, and degenerative changes in germinal cells were observed in the cisplatin group. While caspase-3 immune positivity increased, VDR immunostaining decreased. In biochemical analysis; while a significant increase was observed in TOS in the cisplatin group, no significant difference was found in terms of TAS. It was observed that VD application reduced histological damage and caused a significant increase in Johnsen score. In this group, caspase-3 immunostaining decreased while VDR immunostaining increased. Biochemically, a significant decrease in TOS level and a significant increase in VD level were detected.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eIn conclusion, this study shows that VD administration alleviates testicular damage in rats with cisplatin-induced testicular damage. However, further studies are needed to add VD to clinical treatment protocols.\u003c/p\u003e","manuscriptTitle":"Investigation of the Effect of Vitamin D on Testicular Damage Caused by Cisplatin in Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-27 17:53:21","doi":"10.21203/rs.3.rs-5402589/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":"729ae208-8989-4361-9344-2be27c815e49","owner":[],"postedDate":"November 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-09T11:38:58+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-27 17:53:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5402589","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5402589","identity":"rs-5402589","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}