Effects of high and moderate intensity exercise training on sex hormones and testicular damage induced by cisplatin in rats

Abstract

Background

Cisplatin, a potent chemotherapeutic agent, induces oxidative stress, inflammation, and apoptosis in the testes, reduces sex hormones (testosterone, LH, FSH), and impairs sperm quality and structure. MICT typically increases testicular function and hormone levels, whereas HIIT may negatively affect steroidogenesis and the HPG axis, although findings vary. This study investigates the effects of HIIT and MICT on reducing cisplatin-induced testicular damage in rat, providing a noninvasive approach to preserve fertility for chemotherapy patients.

Methods

In this randomized controlled experimental study twenty-four eight-week-old Sprague Dawley rats (200–240 g) were randomly assigned into four groups (n = 6 per group): healthy control (HC), cisplatin control (CC), Cisplatin-moderate intensity continuous training (C-MICT) and Cisplatin-high intensity interval training (C-HIIT). Except for the HC group, in other groups, 5 mg/kg of cisplatin was injected intraperitoneally as a single dose. HIIT involved alternating high-speed intervals (54 m/min, 30s) with recovery (40 m/min, 3 min), progressing to 20 intervals per session; MICT consisted of continuous running at 27 m/min for up to 60 min, both performed 6 days/week. Sex hormones, histological parameters of testicular tissue, sperm parameters and oxidant/lipid-peroxide were measured.

Results

Cisplatin injection caused a significant decrease in LH, FSH, and testosterone compared to the HC group (P ≤ 0.0001). C-MICT and C-HIIT significantly increased LH (P ≤ 0.0001), FSH (P = 0.001), and testosterone (P ≤ 0.003) compared to CC groups. C-HIIT significantly increased LH (P ≤ 0.001) and testosterone (P ≤ 0.0001) compared to C-MICT groups. C-HIIT and C-MICT significantly improve sperm parameters such as sperm count, sperm morphology and progressively motile sperm, testicular weight and volume, lipid peroxide, and antioxidant level (P ≤ 0.05) and reverse the cisplatin injection effects.

Conclusion(s)

HIIT and MICT both effectively mitigate cisplatin-induced testicular damage by improving sex hormone levels, sperm quality, and histological parameters. HIIT demonstrated comparable or superior benefits to MICT in certain outcomes, suggesting that exercise training may serve as a valuable non-pharmacological strategy to preserve testicular function during chemotherapy.

Introduction

Chemotherapy is an essential cancer treatment but is often accompanied by severe off-target toxicities. Cisplatin (cis-diamminedichloroplatinum II) is highly effective but induces irreversible gonadal dysfunction, including reduced testosterone, azoospermia, and structural testicular damage [1]. Although cisplatin has been legalized for the treatment of various tumors, its use is limited due to cell resistance that varies according to the dose and duration [2]. Moreover, its unspecific action causes adverse effects on the kidneys, testes, and liver. Cisplatin’s toxicity in the testicular region is severe and irreversible. It has the potential to cause azoospermia and decrease testosterone levels [3, 4]. Despite the lack of complete understanding of the mechanisms, various pathways, including oxidative stress, inflammation, and ischemic injury, have been linked [5]. The development and maintenance of homeostasis is greatly impacted by apoptosis. However, excessive or inconvenient apoptosis in the testis can lead to abnormal spermatogenesis or testicular tumors [6]. There is consensus amongst the scientific and clinical communities that exercise improves health and quality of life. Increasingly, exercise training is being promoted by physicians as primary lifestyle prevention for several diseases, particularly those of the cardiovascular system [7]. It is more challenging to recommend exercise training as a preventive or therapeutic strategy for testicular dysfunction due to the conflicting data regarding its impact on testicular function. The increase in testicular function impairment in industrialized countries has resulted in a decrease in fertility rates and negative clinical outcomes that compromise life quality. Moreover, there are other physiological functions that could be affected by chronic testosterone levels [8]. Thus, decreased levels of testosterone may increase the propensity of developing serious disease states [9]. Moderate-intensity aerobic exercise training was found to have a positive correlation with testicular function parameters, such as sperm concentration and hormonal serum levels, according to studies [9]. Serum levels of total and free testosterone, sperm count, motility, and morphology were all still high in trained subjects [8]. Serum levels of total and free testosterone, sperm count, motility, and morphology were all still high in trained subjects [8]. As increased levels of LH and FSH indicate increased stimulation of Leydig and Sertoli cell functions, the HPG axis could be the cause of the observed changes [10, 11]. However, other studies found that moderate-intensity exercise training had no impact or negative effects on testicular function [11, 12]. In a longitudinal study, lower serum levels of free and total testosterone, LH, and FSH were observed in moderate-intensity trained men, suggesting increases testis function [13]. Unlike moderate-intensity exercises, there is a general awareness that high-intensity exercise training is harmful to testicular function. Animal studies have shown that HIIT can have a detrimental effect on sex hormones and sperm parameters [14, 15]. However, the role of the HPG axis in causing these changes has not been fully elucidated, and the findings are conflicting. Some report that no appreciable change in FSH levels occurs following LH decline, while others report high LH concentrations [14]. In total, the evidence shows that the injection of the chemotherapeutic substance cisplatin causes dysfunction and damage to the testes tissue. On the other hand, the main goal of all drug treatments is to improve the testes condition in people undergoing chemotherapy with Cisplatin is not entirely practical. Currently, the available drugs, in addition to high costs, have side effects, including hypoglycemia, liver toxicity, and dyslipidemia [4], and the only hope to reduce such responses is long-term use and adaptation to them. Therefore, low-cost methods without side effects, such as well physical activity to reduce these side effects, can be a good goal. Despite evidence of exercise’s protective effects, no studies have directly compared HIIT and MICT in cisplatin-induced testicular damage, limiting insights into optimal intensity for hormonal and histological recovery. This study aimed to investigate and compare the effects of 10-week HIIT and MICT protocols on sex hormones (LH, FSH, testosterone), testicular stereology, sperm parameters, and oxidative markers in a rat model of cisplatin-induced testicular toxicity.

Methods

Animals and experimental protocols

Twenty-four male Sprague-Dawley rats (8 weeks old, 200–240 g) were housed in a controlled environment (22 ± 2 °C, 12-hour light/dark cycle) with ad libitum access to food and water. The sample size (n = 24) was calculated a priori using G*Power software (version 3.1.9.7) based on prior studies on exercise and testicular function, assuming an effect size of 0.8 (large, derived from pilot data on testosterone changes), alpha of 0.05, and power of 0.85, yielding n = 6 per group [15]. Rats were included if they were healthy males aged 8 weeks (200–240 g) with no signs of illness; exclusion occurred for any displaying abnormal behavior or weight loss >10% during acclimation. No animals were excluded, and there was no sample loss. The Animal Care and Use Committee at Arak University of Medical Sciences approved the study (IR.ARAKMU.REC.1401.014). Rats were randomly assigned to four groups (n = 6/group): Healthy Control (HC; 1 cc normal saline, i.p.), Cisplatin Control (CC), Cisplatin + Moderate-Intensity Continuous Training (C-MICT), and Cisplatin + High-Intensity Interval Training (C-HIIT). Testicular toxicity was induced over 60 days via intraperitoneal injection of cisplatin (5 mg/kg in normal saline; Sobhan Oncology Pharmaceutical Company, Iran) following a 12-hour fast [16]. One-week post-cisplatin injection, the training protocols began across the designated groups. To prioritize animal welfare and minimize distress or pain, euthanasia was carried out under deep anesthesia. Two days following the exercise protocol, rats were anesthetized with an intraperitoneal injection of ketamine (80 mg/kg) combined with xylazine (12 mg/kg). This combination was chosen for its ability to provide profound sedation and effective pain relief, ensuring the animals remained unconscious and pain-free throughout the procedure. The selected doses (ketamine: 80 mg/kg; xylazine: 12 mg/kg) align with established standards for surgical anesthesia in rats, reliably inducing unconsciousness and analgesia [17]. After anesthesia was achieved, blood samples were obtained via cardiac puncture, which likely served as the final step, as this method is recognized as a humane euthanasia technique for anesthetized rats. This approach was carefully selected to reduce stress and discomfort, adhering to ethical principles. All procedures complied with protocols approved by the Animal Ethics Committee of Arak University of Medical Sciences (approval number: IR.ARAKMU.REC.1401.014) and followed the Helsinki Guidelines and the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals, ensuring adherence to international ethical standards for animal research. In addition, rats were obtained from the Pasteur Institute of Tehran, with their consent for use in this research.

Training protocol

The moderate-intensity continuous training (MICT) and high-intensity interval training (HIIT) protocols were executed on a five-lane motorized rodent treadmill (Model TRD19, Pishro Andishe Sanat Company, IR), ensuring accurate management of speed and duration. The rats in the exercise groups engaged in training six days a week for a total duration of 10 weeks, in accordance with the schedule detailed in Table 1 [18]. Treadmill speed and duration were based on maximal oxygen uptake equivalents from prior rat studies (MICT at ~ 60–70% VO2max; HIIT at ~ 90–100% with recovery at ~ 50%); inclination was 0° to minimize orthopedic stress. Animals underwent a 1-week adaptation phase (10–15 min/day at 10–15 m/min) to familiarize with the treadmill. Compliance was encouraged via mild air puffs and gentle prodding; no electrical shocks were used to avoid stress confounding. Protocols were not individually adjusted but monitored for fatigue, with sessions halted if distress was observed [18].

Table 1.

Moderate-intensity continuous training (MICT) and high-intensity interval training (HIIT) protocols

Week	Day	MICT	HIIT
			Odd day	Even day
Week1	1	20 min, 27 m/min	2intervals, 40 m/min, 3 min
	2	22 min, 27 m/min		3intervals, 54 m/min, 30s
	3	24 min, 27 m/min	2intervals, 40 m/min, 3 min
	4	24 min, 27 m/min		5intervals, 54 m/min, 30s
	5	28 min, 27 m/min	2intervals, 40 m/min, 3 min
	6	30 min, 27 m/min		7intervals, 54 m/min, 30s
Week2	1	32 min, 27 m/min	3intervals, 40 m/min, 3 min
	2	34 min, 27 m/min		9intervals, 54 m/min, 30s
	3	36 min, 27 m/min	3intervals, 40 m/min, 3 min
	4	38 min, 27 m/min		11intervals, 54 m/min, 30s
	5	40 min, 27 m/min	3intervals, 40 m/min, 3 min
	6	42 min, 27 m/min		13intervals, 54 m/min, 30s
Week3	1	44 min, 27 m/min	2intervals, 40 m/min, 3 min
	2	46 min, 27 m/min		15intervals, 54 m/min, 30s
	3	48 min, 27 m/min	4intervals, 40 m/min, 3 min
	4	50 min, 27 m/min		17intervals, 54 m/min, 30s
	5	52 min, 27 m/min	4intervals, 40 m/min, 3 min
	6	54 min, 27 m/min		19intervals, 54 m/min, 30s
Week4	1	56 min, 27 m/min	5intervals, 40 m/min, 3 min
	2	58 min, 27 m/min		19intervals, 54 m/min, 30s
	3	60 min, 27 m/min	5intervals, 40 m/min, 3 min
	4	60 min, 27 m/min		20intervals, 54 m/min, 30s
	5	60 min, 27 m/min	6intervals, 40 m/min, 3 min
	6	60 min, 27 m/min		20intervals, 54 m/min, 30s
Week5-10	1–6	60 min, 27 m/min to the end of the 10th week	6intervals, 40 m/min, 3 min to the end of the 10th week	20intervals, 54 m/min, 30s to the end of the 10th week

Determination of serum level of sex hormones, antioxidant capacity, and oxidative stress biomarkers

Blood samples (5 cc) were collected via cardiac puncture, centrifuged at 3500 rpm for 10 min, and the extracted serum was stored at −80 °C for analysis. Hormonal and biochemical analyses were performed using ELISA kits: testosterone (Rat ELISA Kit, Eastbiopharm, China, Catalog No: Ck-E90243; sensitivity: 0.25 nmol/L, range: 0.5–100 nmol/L), luteinizing hormone (LH) (Rat ELISA Kit, Eastbiopharm, China, Catalog No: Ck-E90904; sensitivity: 0.11 mIU/L, range: 0.2–60 mIU/L), and follicle-stimulating hormone (FSH) (Rat ELISA Kit, Eastbiopharm, China, Catalog No: Ck-E30597; sensitivity: 0.12 mIU/L, range: 0.2–60 mIU/L). Oxidative stress biomarkers were assessed using kits from Teb Pajohan Razi Company (Iran): malondialdehyde (MDA) (range: 0–50 µM, intra-assay CV: 6.7%, inter-assay CV: 7.2%), total antioxidant capacity (TAC) (range: 45–420 µM, intra-assay CV: 5.7%, inter-assay CV: 3.7%), and catalase enzyme (CAT) (range: 5–75 µM, intra-assay CV: 4.1%, inter-assay CV: 9.9%). All assessments followed manufacturer’s instructions.

Histological examination

Tissue shrinkage was measured by taking round samples from testicular sections with a trocar and recording initial radii. After tissue processing and staining, radii were measured again to determine shrinkage. The initial testicular volume was multiplied by the shrinkage factor to obtain the final volume. Immediately after dissection, the testes were fixed in 10% neutral buffered formalin for 48 h to preserve tissue morphology. Following fixation, the samples were dehydrated through a graded series of ethanol (70%, 80%, 95%, and 100%), cleared with xylene, and embedded in paraffin wax. The paraffin blocks were then sectioned at a thickness of 5 μm using a rotary microtome. For the assessment of general morphology and structural changes, the sections were deparaffinized, rehydrated, and stained with the standard hematoxylin and eosin (H&E) method [19]. Stained slides were then examined under a light microscope. Tissue shrinkage was measured by taking round samples from testicular sections with a trocar and recording initial radii. After tissue processing and staining, radii were measured again to determine shrinkage.

Seminiferous tubule and interstitial tissue volumes were quantified by examining an average of 5 random fields of view at 100x magnification using an Olympus DP12 (Olympus Optical Co., Tokyo, Japan) microscope.

A point probe was randomly applied to tissue sections to count points intersecting with the entire tissue, seminiferous tubules, and interstitial tissue, estimating the volumetric density of each component. Component volumes were calculated by multiplying volumetric density by the final testicular volume. The average seminiferous tubule diameter was ascertained by applying a counting probe to fields of view at 100x magnification. Between 130 and 150 tubules were counted and measured using Motic software (Image Motic2000), and the mean diameter was calculated (Fig. 1) [15].

Fig. 1.

Microscopic images of rat testicular tissue in different groups (5-micron slices, with hematoxylin and eosin (H&E), 200x magnification)

Evaluation of epididymal sperm parameters

Following removal and weighing of the left testis, the dissected epididymis was placed in 5 cc DMEM. The tissue was minced to promote sperm diffusion into the medium. After 10 min at 27 °C, 1 ml of the solution was diluted in 9 ml formaldehyde fixative. Sperm heads were then counted manually in a Neubauer hemocytometer according to WHO guidelines, and sperm concentration was expressed as sperm per milliliter (Fig. 2a) [20]. Sperm motility was assessed according to WHO guidelines. A 10 µl sperm suspension was placed on a slide, covered, and at least five fields of view were analyzed, evaluating a minimum of 200 sperm per animal. The percentage of motile sperm was then calculated. Sperm viability was evaluated using eosin-nigrosin staining (Fig. 2b) [18]. For viability assessment, sperm suspensions were mixed with 1% eosin (Merck, Germany) and 10% nigrosin (Merck, Germany). After 30 s, thin smears were prepared and examined at 100X magnification using light microscopy. Viable sperm remained white, while non-viable sperm stained purple. The percentage of live sperm was then calculated. For morphology, Papanicolaou-stained smears were analyzed at 100X magnification, assessing 100 sperm per smear according to WHO guidelines. Morphological abnormalities were recorded as a percentage (Fig. 2c) [18].

Fig. 2.

a. sperm count by homocytometric method, b. Stained viability by eosin–nigrosin,c. Papanicolaou stain morphology method

Statistical analysis

The normality of the data was checked with the Shapiro-Wilk test, and Levene’s test confirmed equal variances. A one-way ANOVA was then used to determine if there were any differences between the groups. The Tukey test was used to identify significant group differences when variances were equal, while Dunnett’s T3 test was used when variances were unequal. Effect size was determined using Eta Squared (η²), with values of 0.01, 0.06, and 0.14 indicating small, medium, and large effects, respectively. SPSS Statistics (Version 27.0 for Windows, SPSS Inc., Chicago, IL, USA) was used for data analysis, and GraphPad Prism 10 was used for generating graphs. Results are presented as means ± SD or SEM, and statistical significance was set at p < 0.05.

Result

Body weight and testicular metrics

While no significant differences in baseline body weight were observed among the groups (P = 0.992), significant differences emerged following the intervention in final body weight (F_3,20=3.65, P = 0.009), left testicular weight (F_3,20=18.21, P ≤ 0.0001, η²=0.732), left testicular volume (F_3,20=8.94, P = 0.001, η²=0.572), and the Gonado-Somatic Index (GSI) (F_3,20=12.20, P ≤ 0.001, η²=0.802). Cisplatin administration (CC group) led to a significant decrease in left testicular weight (P = 0.001), testicular volume (P ≤ 0.001), and GSI (P ≤ 0.0001) compared to the healthy control (HC) group, indicating significant testicular atrophy. Both exercise interventions successfully mitigated these atrophic changes. The C-MICT and C-HIIT groups exhibited significantly greater testicular weights (P ≤ 0.001 for both) and a restored GSI (P ≤ 0.001 for both) compared to the CC group. Testicular volume was also significantly restored in the C-HIIT group (P = 0.034) relative to the CC group. Notably, the final body weight of the C-HIIT group was significantly higher than that of both the HC (P = 0.009) and C-MICT (P = 0.049) groups (Table 2).

Table 2.

Between-group comparisons of body weight, left testicular weight, and left testicular volume

Groups	Body Weight (g)		Left testicular weight (g)	Left testicular volume (mm3)	GSI (%)
	Baseline	Post-test	Final	Final	Final
HC	221.5(± 24)	273.6‏(± 22)	1.655‏(± 0.128)	1136.6‏(± 128)	0.60(± 0.07)
CC	221.6‏(± 13)	309.7‏(± 15)	1.151‏(± 0.045)‏a	573.8‏(± 70)a	0.37(± 0.02)ᵃ
C-MICT	218.1‏(± 8)	286.2‏(± 33)	1.538(± 0.153)b	832.1‏(± 116)	0.54(± 0.08)ᵇ
C-HIIT	219.2‏(± 14)	332.2‏(± 37)ac	1.575‏(±‏0.109)b	917.8(± 64)b	0.47(± 0.02)ᵃᵇ
P-value between group	0.992	0.009	0.0001	0.001	0.001

Data presented as Mean (± SEM). HC Healthy Control group, CC Cisplatin Control group, C-MICT Cisplatin-Moderate Intensity Continuous Training group, C-HIIT Cisplatin-High Intensity Interval Training group. ^aThe significant difference with the HC group. ^bThe significant difference with the CC group

GSI was calculated as: (Left testicular weight/Final body weight) × 100

Serum levels of sex hormones

Significant between-group differences were observed for the serum levels of LH (F_3,20=43.51, P ≤ 0.0001, η²=0.904), FSH (F_3,20=64.69, P ≤ 0.0001, η²=0.907), and testosterone (F_3,20=34.07, P ≤ 0.0001, η²=0.836) (Fig. 3). Cisplatin administration caused a profound decline in all three hormones compared to the HC group (P ≤ 0.0001 for all). Exercise training effectively reversed this hormonal suppression. Both C-MICT and C-HIIT groups displayed significantly elevated levels of LH (P ≤ 0.0001), FSH (P = 0.001), and testosterone (P ≤ 0.003) relative to the CC group. Furthermore, the C-HIIT protocol demonstrated superior efficacy for certain hormones, inducing significantly greater increases in both LH (P ≤ 0.001) and testosterone (P ≤ 0.0001) compared to the C-MICT protocol.

Fig. 3.

Mean (±SEM) of Luteinizing Hormone (LH)(mIU/ml), Follicle Stimulating Hormone (FSH) (mIU/ml), and testosterone in different groups. HC: Data presented as Mean (±SEM). HC: Healthy Control group; CC: Cisplatin Control group; C-MICT: Cisplatin-Moderate Intensity Continuous Training group; C-HIIT: Cisplatin-High Intensity Interval Training group. ns: No significant difference at P≤0.05. *: The significant difference at P≤0.05.**. The significant difference at P≤0.01. ***: The significant difference at P≤0.001. ****: The significant difference at P≤0.0001

Histological examination

Analysis of testicular histology revealed significant group differences in seminiferous tubules diameter (STD) (F_3,20=10.432, P = 0.002, η²=0.610) and seminiferous epithelium thickness (SET) (F_3,20=16.123, P ≤ 0.0001, η²=0.707). However, no significant differences were observed for other parameters like interstitial tissue volume (P = 0.721) (Table 3). Specifically, cisplatin treatment induced a significant reduction in SET compared to the HC group (P = 0.005). Moderate-intensity training (C-MICT) effectively restored this epithelial thickness (P = 0.007 vs. CC). Additionally, both C-MICT (P = 0.008) and C-HIIT (P = 0.012) resulted in a significant increase in STD compared to the CC group.

Table 3.

Between-group comparisons of ITV, ITV, STV, STL, STD and STE in different groups

Groups	Interstitial tissue volume (ITV) (mm2)	Seminiferous tubules volume (STV) (mm2)	Seminiferous tubules length (STL) (µm)	Seminiferous tubules diameter (µm) (STD)	Seminiferous epithelium thickness (SET) (µm)
HC	201.7(± 16.1)	693.8 (± 167.1)	16.1(± 3.8)	223.4 (± 9.8)	235.3 (± 38.9)
CC	177.5(± 60.3)	567.8 (± 155.5)	12.1 (± 3.43)	218.5 (± 4.6)	176.3 (± 12.1)a
C-MICT	208.1(± 36.2)	684.8 (± 213.5)	15.11 (± 2.8)	255.1 (± 23.6)b	232.4 (± 22.3)b
C-HIIT	200.4(± 64.7)	765.7 (± 171.5)	20.4 (± 8.6)	252.8 (± 9.2)b	195.3 (± 24.22)
P-value Between group	0.721	0.313	0.080	0.002	0.0001

Data presented as Mean (± SEM)

HC Healthy Control group, CC Cisplatin Control group, C-MICT Cisplatin-Moderate Intensity Continuous Training group, C-HIIT Cisplatin-High Intensity Interval Training group

^aThe significant difference with the HC group. ^bThe significant difference with the CC group

The histological analysis presented in Fig. 1 provided clear qualitative support for the quantitative data. In the HC group, the testicular tissue appeared entirely normal, with a well-structured layout featuring healthy seminiferous tubules packed with germ cells at multiple stages of maturation. By comparison, the CC group revealed extensive testicular injury, including shrunken tubules, prominent vacuoles throughout, detachment and disorder in the germinal epithelium, along with a sharp decline in mature sperm counts. Notably, both exercise regimens offered substantial protection against these effects. In the C-MICT and C-HIIT groups, we observed a clear recovery in seminiferous tubule integrity, marked by enhanced epithelial thickness, orderly cellular arrangements, and robust signs of spermatogenesis, ultimately countering the harm inflicted by cisplatin.

Sperm parameters

Cisplatin administration severely compromised sperm quality and quantity, with significant between-group differences detected in sperm count (F_3,20=274.459, P ≤ 0.0001, η²=0.976), viability (F_3,20=3.969, P = 0.023, η²=0.373), and morphology (F_3,20=12.545, P ≤ 0.0001, η²=0.653) (Table 4). Compared to the HC group, the CC group showed a drastic reduction in sperm count (P ≤ 0.0001), viability (P = 0.013), and the percentage of morphologically normal sperm (P ≤ 0.0001).

Table 4.

Between-group comparisons of sperm count, sperm motility, sperm viability, and sperm morphology in different groups

Groups	Sperm Count (106)	Sperm Viability (%)	Sperm Morphology (%)	Sperm Motility (%)
				Progressively (%)	No progressively (%)	No motile (%)
HC	34.1 (± 1.3)	61.9 (± 2.3)	93.7 (± 0.7)	57.3 (± 2.2)	15.6 (± 1.7)	27.1 (± 1.4)
CC	19.6 (± 0.8)a	58.5 (± 1.1)a	89.9 (± 0.4)a	46.0 (± 0.7)a	18.8 (± 0.8)a	35.9 (± 0.4)a
C-MICT	21.5 (± 0.8)b	60.6 (± 1.5)	91.9 (± 1.3)b	48.0 (± 1.6)	21.6 (± 0.9)b	30.4 (± 1.5)b
C-HIIT	22.0 (± 0.9)b	60.3 (± 1.5)	91.8 (± 1.4)b	47.8 (± 1.7)	19.6 (± 2.6)	32.6 (± 1.3)b
P-value Between group	0.0001	0.023	0.0001	0.0001	0.0001	0.0001

Data presented as Mean (± SEM).

HC Healthy Control group, CC Cisplatin Control group, C-MICT Cisplatin-Moderate Intensity Continuous Training group, C-HIIT Cisplatin-High Intensity Interval Training group

^aThe significant difference with the HC group. ^bThe significant difference with the CC group

Similarly, sperm motility was significantly affected across groups, including progressively motile (F_3,20=54.590, P ≤ 0.0001, η²=0.891) and non-motile sperm (F_3,20=49.417, P ≤ 0.0001, η²=0.881). The CC group exhibited a significant decline in progressively motile sperm (P ≤ 0.0001) and a sharp increase in non-motile sperm (P ≤ 0.0001) relative to controls. Both exercise interventions significantly counteracted these deficits, leading to a significant recovery in sperm count (P ≤ 0.012) and normal morphology (P ≤ 0.028), and a significant decrease in the percentage of non-motile sperm (P ≤ 0.001) compared to the CC group.

Lipid peroxide and antioxidant status measures

The interventions resulted in significant changes to systemic redox balance, with significant between-group differences for MDA (F_3,20=24.483, P ≤ 0.0001, η²=0.786), TAC (F_3,20=3.563, P = 0.033, η²=0.348), and CAT (F_3,20=19.613, P ≤ 0.0001, η²=0.746) (Fig. 4). Cisplatin induced a state of significant systemic oxidative stress, evidenced by elevated serum MDA (P ≤ 0.0001) and depleted TAC (P = 0.0001) and CAT (P = 0.001) in the CC group compared to the HC group. Exercise training effectively restored this balance. Both C-MICT and C-HIIT protocols led to a significant reduction in serum MDA (P ≤ 0.0001) and a significant enhancement of TAC (P ≤ 0.047) and CAT (P ≤ 0.009) levels compared to the CC group. Notably, the C-HIIT group exhibited significantly higher CAT activity than the C-MICT group (P = 0.001), suggesting a superior effect on this antioxidant enzyme.

Fig. 4.

Mean (±SEM) of serum malondialdehyde (μM), total antioxidant capacity (μM), and catalase (μM) levels in different groups. Data presented as Mean (±SEM). HC: Healthy Control group; CC: Cisplatin Control group; C-MICT: Cisplatin-Moderate Intensity Continuous Training group; C-HIIT: Cisplatin-High Intensity Interval Training group. ns: Not significant.*: significant difference at P≤0.05. **. significant difference at P≤0.01. ***: significant difference at P≤0.001. ****: significant difference at P≤0.0001

Discussion

One key point to consider is whether the stress from our forced treadmill setup might have played a role in the testicular damage we observed. It’s true that making animals run like that can be a source of stress, but our study design really clears this up. The group that only got cisplatin without any exercise the CC group showed by far the worst testicular issues. Meanwhile, the groups that combined cisplatin with either moderate intensity continuous training or high intensity interval training (C MICT and C HIIT) actually saw big improvements across every metric we checked, way better than the CC folks. That really highlights how exercise delivers a strong protective and healing punch, easily overriding any slight stress the workouts might have caused. On top of that, we built in a gentle ramp-up period to ease the animals into it and keep stress low while ensuring they followed through something that’s pretty standard in rodent exercise research. All in all, the data make it clear that cisplatin was the real driver of the damage, and the training stepped in as an effective way to dial it back [21]. This study examined the potential of moderate-intensity continuous training (MICT) and high-intensity interval training (HIIT) to mitigate cisplatin-induced testicular damage and hormonal imbalances in adult male rats. The results indicated that cisplatin significantly reduced sex hormone levels (HL, HSL, testosterone), impaired stereological parameters (left testicular volume and weight, seminiferous tubule diameter and interstitial tissue volume), and diminished sperm quality (concentration, motility, viability, and morphology), consistent with previous research [20, 22–24]. Exercise training, particularly HIIT, ameliorated these effects, improving sex hormone levels, stereological parameters, and sperm parameters. The mechanism by which cisplatin induces testicular damage is multifactorial but is centrally linked to oxidative stress. Cisplatin accumulates in testicular tissue and triggers the excessive production of reactive oxygen species (ROS), which overwhelms the endogenous antioxidant defense systems [16]. This leads to lipid peroxidation of sperm plasma membranes, causing impaired motility and abnormal morphology, and induces DNA damage in spermatogonia stem cells, leading to apoptosis and a reduction in sperm count. Our findings of elevated MDA and depleted TAC and CAT in the CC group confirm this systemic oxidative state. Exercise training, particularly HIIT, likely confers its protective effects by upregulating the body’s own antioxidant machinery. Regular physical activity is known to enhance the expression of key antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) through pathways like the Nrf2-ARE signaling cascade [25, 26]. By bolstering these defenses, exercise creates an environment where testicular cells are better equipped to neutralize cisplatin-induced ROS, thus preserving the integrity of seminiferous tubules and germ cells.

Furthermore, the hormonal improvements observed are intrinsically linked to this reduction in oxidative stress. Leydig cells, which are responsible for testosterone production, are highly susceptible to ROS-mediated damage. Oxidative stress can impair steroidogenic enzymes like 3β-HSD and 17β-HSD, leading to suppressed testosterone synthesis [27]. By mitigating oxidative stress, both MICT and HIIT likely restored Leydig cell function, leading to the observed recovery in testosterone levels. The superior effect of HIIT on testosterone and LH may suggest a more potent stimulus for enhancing the sensitivity of Leydig cells to LH or a stronger central effect on the hypothalamic-pituitary-gonadal (HPG) axis.

Also, this improvement likely stems from exercise-induced interleukin-6 release from skeletal muscles, which triggers anti-inflammatory cytokine production (IL-10, IL-1ra) and inhibits pro-inflammatory cytokine production (IL-1b, TNF) [28]. Furthermore, endurance training can induce heme oxygenase-1 (HO-1) expression in skeletal muscle cells [29], reducing inflammation and apoptosis [30]. Cisplatin, a widely used anticancer drug known for its testicular cancer sensitivity, induces apoptosis via DNA damage [31]. As chemotherapy drugs cannot differentiate between normal and cancerous cells, cisplatin causes toxicity in various organs, including the gonads [20, 22]. In this study, cisplatin treatment resulted in significant tissue damage, including reduced seminiferous tubule diameter, length, and epithelial thickness, along with decreased left testicular weight and volume compared to controls, aligning with prior findings [32, 33]. Cisplatin’s testicular damage manifests as deformed spermatogenic tubules, reduced epithelial thickness, and impaired spermatogenesis, potentially stemming from germ cell loss and impaired maturation of basal germ cells [34]. Weight reduction is attributed to seminiferous tubule epithelial layer damage [16]. Cisplatin also dilates blood vessels in interstitial and sub-capsular testicular tissue [20, 35]. Cisplatin accumulation in mitochondria leads to excessive ROS production, disrupting the respiratory chain and causing oxidative stress and apoptosis [35, 36]. Desquamation of germ cells and reduced epithelial thickness may result from cisplatin’s disruption of Sertoli cell microtubules and interstitial filaments or reduced cadherin expression [37, 38]. Free oxygen species disrupt junctional complex integrity by affecting cadherin/catenin complexes and oxidative phosphorylation [39].

Exercise training, particularly HIIT, improves seminiferous tubule diameter, length, epithelium thickness, and volume. The mean left testicular volume of rats in the HIIT group was significantly different from other groups, indicating that HIIT improves seminiferous tubule epithelial tissue. This proliferation of spermatogonia at the G1/S phases is regulated by Cyclin D1/Cdk4 and Cyclin D1/Cdk6. Reduced Cdk4 and Cyclin D1 expression inhibits CyclinD1/Cdk4 complex formation, subsequently inhibiting germ cell development. Severe DNA damage increases p53 expression, overstimulating p21 protein, which arrests the cell cycle by interacting with Cdk4 to form the p21/Cdk4 complex, leaving Cyclin D1 free and halting cell cycle progression [40]. Similar findings in other tissues show that HIIT significantly increases Cyclin D1 and Cdk4 expression compared to sedentary obese animals [41], and that both MICT and HIIT induce changes in signaling associated with muscle ribosome biogenesis through cyclin D1 and cdk4 interactions [42].

Exercise training, particularly HIIT, improves seminiferous tubule diameter, length, epithelium thickness, and volume. The mean left testicular volume of rats in the HIIT group was significantly different from other groups, indicating that HIIT improves seminiferous tubule epithelial tissue. Our study demonstrated that cisplatin significantly decreased sperm parameters (concentration, motility, viability) while increasing morphological abnormalities. Similar studies have shown that cisplatin injection leads to azoospermia and reduced motility and survival in the testicles of rabbits [20], Rats [23, 24, 40], and humans [43]. Cisplatin consumption increases ROS production, leading to peroxidation of unsaturated fatty acids in the plasma membrane, disruption of cadherin/catenin complexes, and oxidative phosphorylation of the sperm cell membrane. This damage to plasma membrane integrity and the binding complex reduces sperm motility and increases morphological abnormalities [16, 39]. In our study, sperm parameters (concentration, motility, viability) in the HIIT and MICT groups were significantly higher than in the cisplatin control group. Interval training has also been shown to significantly increase Bcl-2/BAX compared to sedentary controls [44]. Furthermore, studies using adipose-derived mesenchymal cells after cisplatin treatment showed significant increases in sperm motility and count [45, 46]. The therapeutic potential of mesenchymal cells stems from their anti-inflammatory, anti-fibrotic, repair, antioxidant properties, and ROS inhibition [20, 47]. Animals previously exercised also exhibited lower levels of cytokines interleukin-10 and TNF, indicating reduced inflammation [30]. Consistent with other studies [20, 23], we found that cisplatin significantly decreased testosterone levels. Leydig cells, responsible for testosterone secretion, are located in the testicular interstitial tissue. Our study confirmed that testosterone levels in the cisplatin control group were significantly lower than in the healthy control group, aligning with research showing that chemotherapy drugs reduce testosterone levels [48]. Cisplatin intake causes impairment and death of Leydig cells, subsequently decreasing testosterone secretion. The reduction in testosterone levels in cisplatin-treated rats is directly associated with decreased LH receptors on Leydig cells [49]. Testosterone is essential for spermatogenesis maintenance, testicular weight, and cell number [40]. Decreased testosterone affects protein synthesis in all spermatogenic cells, disrupting germ cell protein synthesis, causing destructive effects, and affecting spermatogenesis [20, 23, 50].

These effects appear to be consistent with what we have observed in humans, where HIIT is more effective at activating the Nrf2 pathway for antioxidants compared to MICT, which can actually help protect against oxidative damage caused by chemotherapy [25]. In practical terms, we could adapt similar routines for people undergoing cancer treatment. For example, HIIT settings such as four 4-minute high-intensity cycling sessions at 85–95% of maximum heart rate, with 3-minute rests in between, have been shown to be effective in increasing cardiopulmonary fitness while reducing fatigue in men with testicular cancer receiving cisplatin [51]. That said, any human studies need to be careful about things like chemotherapy doses, how vulnerable patients are, and also about overexertion, as overdoing HIIT can worsen problems like low testosterone in people who are already at risk [52]. Looking forward, it would be great to do some proper randomized trials with men taking cisplatin, looking at things like sperm health and hormone balance to see if these exercise approaches actually hold up.

Our studies showed that testosterone levels in the MICT group, and especially the HIIT group, increased significantly compared to the cisplatin control group. Treatment with mesenchymal cells significantly increased testosterone levels in the cisplatin group, likely because mesenchymal cells repair tissue by releasing growth factors and cytokines that promote the proliferation and completion of division of remaining spermatogenic cells. Mesenchymal cells protect the testis by modulating the inflammatory immune status and exerting anti-apoptotic effects [53].

Limitations

Our research comes with a few limitations that we need to own up to. One major one is that we looked at oxidative stress through systemic serum markers, instead of diving straight into measurements from the testicular tissue itself. For instance, checking things like glutathione (GSH) levels in the tissue could have given us a clearer picture of the antioxidant defenses that shield against harm to sperm production, while nitrite levels might have shed light on nitric oxide availability and how it ties into steroid hormone creation. That kind of approach would’ve helped forge a stronger, more direct connection between the benefits from exercise and the actual protection of the testes. Looking ahead, it’d be great if future work incorporated these tissue specific tests to really unpack the molecular mechanisms at work and bolster the evidence for cause and effect relationships.

Conclusions

These preclinical findings suggest potential protective roles of exercise against cisplatin toxicity, but translation to humans requires caution due to species differences. In rats, HIIT may offer comparable or superior benefits to MICT in mitigating cisplatin-induced testicular damage, warranting further mechanistic and clinical studies.

Abbreviations

3β-HSD

3β-Hydroxysteroid Dehydrogenase

17β-HSD

17β-Hydroxysteroid Dehydrogenase

BAX

Bcl-2 Associated X protein

Bcl-2

B-cell lymphoma 2

Cdk4

Cyclin-Dependent Kinase 4

H&E

Hematoxylin and Eosin

HO-1

Heme Oxygenase-1

HPG

Hypothalamic-Pituitary-Gonadal

LH

Luteinizing Hormone

MDA

Malondialdehyde

NIH

National Institutes of Health

Nrf2-ARE

Nuclear factor erythroid 2-related factor 2 - Antioxidant Response Element

ROS

Reactive Oxygen Species

Authors’ contributions

Authors declare their gratitude to all those who helped us in this project.

Funding

This study did not receive any funding.

Data availability

The datasets generated and analyzed during the current study are not publicly available due to ongoing data analysis but are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

All actions performed on the animals followed the Helsinki Guidelines and the guidelines of the Ethics Committee Arak University of Medical Sciences (permission number: IR.ARAKMU.REC.1401.014). The protocol followed the ARRIVE guidelines and the National Institutes of Health guidelines for the care and use of laboratory animals.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.