Medical Ozone Treatment Attenuates Male Reproductive Toxicity Induced by Bleomycin, Etoposide, and Cisplatin Regimen in an Experimental Animal Model

Abstract

The chemotherapeutic combination of bleomycin, etoposide, and cisplatin (BEP) is well-documented to exert gonadotoxic effects, ultimately leading to impaired fertility. This experimental rat study investigated the potential protective role of repeated medical ozone therapy in mitigating the deleterious effects of BEP treatment in male rats. Thirty-two adult male Sprague Dawley rats were randomly assigned to four groups: (i) a healthy control group, (ii) a group receiving injections of the BEP regimen over nine weeks, (iii) a group receiving the same BEP regimen plus medical ozone (1 mg/kg IP) twice weekly, and (iv) a group receiving only ozone therapy. BEP treatment significantly reduced sperm concentration and increased morphological abnormalities, both of which were partially restored by ozone co-administration. Ozone therapy also elevated testosterone and thyroid-stimulating hormone (TSH) levels when co-administered with BEP compared to BEP treatment alone. Oxidative stress analysis demonstrated that total oxidative status (TOS) and total antioxidant status (TAS) levels were significantly improved in the BEP + ozone group. Histopathological analysis revealed that ozone treatment ameliorated BEP-induced testicular damage, as evidenced by improved Johnsen scores and increased thickness of the seminiferous tubule epithelium. In conclusion, repeated medical ozone therapy appears to mitigate BEP-induced reproductive toxicity by preserving sperm quality, endocrine function, and redox homeostasis.

Keywords: bleomycin; cancer; chemotherapy; cisplatin; etoposide; infertility; medical ozone; oxidative stress; rat; sperm.

Figure 1

Change in body weight (A) and testis weight index (B) of (i) control, (ii) bleomycin, etoposide, and cisplatin (BEP)-treated, (iii) BEP- and ozone-treated, and (iv) ozone-alone-treated rats. Data are presented as mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant).

Figure 2

Change in sperm concentration (A), abnormal morphology (B), abnormal head (C), abnormal neck (D), abnormal tail (E), and multiple anomalies (F) of (i) control, (ii) bleomycin, etoposide, and cisplatin (BEP)-treated, (iii) BEP- and ozone-treated, and (iv) ozone-alone-treated rats. Data are presented as mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant).

Figure 3

Change in sperm viability of (i) control, (ii) bleomycin, etoposide, and cisplatin (BEP)-treated, (iii) BEP- and ozone-treated, and (iv) ozone-alone-treated rats. Data are presented as mean ± SD (* p < 0.05, **** p < 0.0001, ns: not significant).

Figure 4

Photomicrographs in the testis sections of control (A), bleomycin, etoposide, and cisplatin (BEP)-treated (B), BEP- and ozone-treated (C), and ozone-alone-treated rats (D); n: normal tubule, arrowhead: thinning of the epithelium, arrow: vacuole, black *: loss of integrity of the interstitial space, red *: desquamation. Change in Johnsen scoring (E) and the mean thickness of germinal epithelium (F) of (i) control, (ii) bleomycin, etoposide, and cisplatin (BEP)-treated, (iii) BEP- and ozone-treated, and (iv) ozone-alone-treated rats. Representative images under (A–D) 20× magnification. Data are presented as mean ± SD (* p < 0.05, *** p < 0.001, **** p < 0.0001, ns: not significant).

Figure 5

Changes in TAS (A), TOS (B), testosterone (C), and TSH (D) levels of (i) control, (ii) bleomycin, etoposide, and cisplatin (BEP)-treated, (iii) BEP- and ozone-treated, and (iv) ozone-only-treated rats. Data are presented as mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant).

Figure 6

Experimental protocols for the BEP regimen and ozone therapy. Schematic representation of the study design, including group allocation and treatment timeline (created with BioRender.com).