Ketoprofen and meloxicam impair spermatogenesis and alter testicular histomorphology in ratsAbstract
This study evaluated the effects of the nonsteroidal anti-inflammatory drugs (NSAIDs) ketoprofen and meloxicam on testicular morphology and spermatogenic dynamics in rats. Adult Wistar rats were treated by oral gavage once daily for 15 days with meloxicam (1 mg/kg) or ketoprofen (5 mg/kg). No significant differences were observed among groups regarding testicular biometric and volumetric parameters. However, both treatments significantly reduced Sertoli cell numbers (8.1% in ketoprofen and 9.2% in meloxicam group). Ketoprofen induced more pronounced reductions in type A spermatogonia (35.1%), primary spermatocytes (36.9%), and round spermatids (42.2%) number, whereas meloxicam caused milder decreases (12.2%, 12.2% and 14.3%, respectively). Only ketoprofen significantly impaired spermatogenic dynamics, reducing mitotic, meiotic, and spermatogenic yields, as well as Sertoli cell efficiency. Histometric analysis revealed a marked increase in tubular lumen area (54% in ketoprofen and 113% in meloxicam groups) and a reduction in seminiferous epithelium height (25% and 43%, respectively) in treated groups. In addition, cytoplasmic vacuolization in Sertoli cells was observed in both treated groups. These findings indicate that subacute NSAID exposure compromises seminiferous epithelium architecture and spermatogenesis in rats, with ketoprofen inducing more pronounced deleterious effects on male reproductive function.
Key words
Testis; seminiferous epithelium; spermatogenesis; meloxicam; ketoprofen
INTRODUCTION
The structural and functional integrity of the testes is essential for proper reproductive performance in mammals (Tirpák et al. 2021). To evaluate these aspects, it is necessary to assess the dynamics of spermatogenesis, a highly synchronized and regulated process that occurs within the seminiferous tubules of the gonads and can be affected by various chemical agents, including commonly used medications (Semet et al. 2017, Mo et al. 2023). Among these agents, nonsteroidal anti-inflammatory drugs (NSAIDs) have raised increasing concern, particularly due to their prolonged and often indiscriminate use across different age groups (Kristensen et al. 2016).
NSAIDs exert their effects primarily through inhibition of cyclooxygenase enzymes (COX-1 and COX-2), thereby blocking the synthesis of prostaglandins, which are lipid mediators essential not only in inflammatory processes but also in several physiological functions, including the regulation of steroidogenesis and spermatogenesis (Drobnis & Nangia 2017, Parekattil et al. 2020). Although the ability of NSAIDs to cross the blood-testis barrier and reach the testicular compartment remains incompletely understood, experimental data from reproductive toxicity studies suggest that at least some NSAIDs distribute to the testicular microenvironment at pharmacologically or toxicologically relevant concentrations (Drobnis & Nangia 2017). For instance, both in vitro and in vivo studies indicate that the use of certain NSAIDs, such as ibuprofen and diclofenac, may negatively interfere with testicular physiology by reducing testosterone levels and inducing histological alterations in the seminiferous tubules (Albert et al. 2013, Drobnis & Nangia 2017, Kristensen et al. 2018). Ketoprofen, a preferential COX-1 inhibitor (Cryer & Feldman 1998), and meloxicam, a preferential COX-2 inhibitor (Del Tacca et al. 2002), are widely used NSAIDs in both veterinary and human clinical practice, particularly for painful and inflammatory conditions (EMA 2020, De La Puente et al. 2024). Although these drugs are well recognized for their effectiveness in reducing pain and inflammation, data regarding their adverse effects on the male reproductive system remain scarce and, in some cases, contradictory (Uzun et al. 2015).
In this context, investigating the effects of these drugs on testicular morphological and functional parameters is particularly relevant, especially through histological, histometric, and spermatogenic efficiency analyses. The use of the seminiferous epithelium cycle as a reference, combined with quantitative histological approaches, allows for a detailed assessment of testicular cellular dynamics, including both germ cell populations and Sertoli cell function (França & Russell 1981, Russell et al. 1990).
Therefore, the present study aimed to evaluate the effects of oral treatment with ketoprofen or meloxicam on the morphological, histometric, and functional parameters of Wistar rat testes using standardized cellular and histological analyses. We further hypothesized that both ketoprofen and meloxicam would impair spermatogenesis and induce structural alterations in the seminiferous epithelium, potentially exhibiting distinct effects due to their different COX selectivity profiles. The findings of this study may contribute to a better understanding of the reproductive risks associated with these widely prescribed drugs and provide experimental support for safer therapeutic use, particularly in individuals of reproductive age.
MATERIALS AND METHODS
Ethics committee
This study was previously submitted to the Ethics Committee on Animal Use (CEUA) of the Biosciences Center at the Federal University of Rio Grande do Norte (UFRN), Natal Campus, and approved under protocol No. 239.005/2021. All experimental procedures followed the recommendations of Brazilian Law No. 11.794 of October 8, 2008 (Arouca Law).
Animals
A total of 18 male albino Wistar rats, three months old and weighing 200–400 g, were obtained from the Central Animal Facility of the Biosciences Center, UFRN. The animals were housed in the Animal Facility of the Department of Biophysics and Pharmacology (DBF), UFRN, in standard cages (five animals per cage), with free access to water and food (Nuvilab chow, Quimtia®), under a 12 h light/dark cycle (lights on from 6:00 a.m. to 6:00 p.m.) and controlled temperature (23°C ± 1°C). The cages were lined with wood shavings and cleaned every two days.
Treatment, experimental groups, and animal euthanasia
After an acclimatization period of at least 30 days, the animals were divided into experimental groups: CG (control group; n = 6), KG (ketoprofen group; n = 6), and MG (meloxicam group; n = 6). Treatments were administered daily by gavage for 15 days (subacute treatment) with 0.9% saline (control group), meloxicam 1 mg/kg, or ketoprofen 5 mg/kg. The doses, routes of administration, and treatment duration were based on the BSAVA Small Animal Formulary (Hedley 2020). Animals were monitored daily for body weight, physical condition, and behavior.
At the end of the treatment, all animals were weighed and euthanized by anesthetic overdose with ketamine (300 mg/kg, intraperitoneal) combined with xylazine (30 mg/kg, intraperitoneal).
Testicular biometry
Before euthanasia, biometric parameters were measured using an analog caliper, including testicular length and width. After euthanasia, the testes were carefully excised, dissected, and weighed on a digital balance to determine absolute testicular weight. Relative testicular weight was calculated using the formula: absolute testicular weight / 100 g body weight. All measurements were subjected to quantitative analysis.
Testicular volume
Testicular volume was determined according to the method described by Scherle (1970). The total volume was calculated using the formula: Volume = Density × Mass. For each experimental and control group, the mean total testicular volume was obtained from the arithmetic mean of the volumes of both testes per animal.
Histological processing
Samples were fixed in Bouin’s solution for 24 h. Subsequently, they were subjected to four to five successive washes in 70% ethanol, with tissues remaining immersed for 12 h in each wash. The ethanol was replaced at each interval to ensure complete removal of excess fixative, until no detectable residues remained. The tissues were embedded in paraffin, and sectioned at 3 μm thickness using a microtome. The sections were stained with hematoxylin and eosin (HE) for subsequent qualitative and quantitative histological analyses.
Counting of seminiferous tubule cell types
Analyses were performed using a light microscope (Olympus BX40) with an Image Analysis System (Image Pro-Express v.6.0). Images of seminiferous tubules were captured, and nuclear diameters of ten cells of each type were measured per testis. For Sertoli cells, nucleolar diameters were measured due to the high variability and difficulty in precisely delimiting their nuclei.
For each testis, three slides were analyzed, totaling six slides per animal. Both qualitative and quantitative analyses were performed. Stage VII of the Seminiferous Epithelium Cycle (SEC) was identified based on tubular morphology, as described by Cardoso (1981) and Ortavante et al. (1984), considering nuclear morphology of spermatids, meiotic divisions, and the position of spermatids within the seminiferous epithelium. Cell counts were performed in 20 transverse sections of seminiferous tubules per animal, preferably those with a circular cross-sectional profile. The evaluated cell types included type A spermatogonia, primary spermatocytes at pre-leptotene/leptotene stage, round spermatids, and Sertoli cells (identified by their nucleoli). During analysis, the evaluator was blinded to group assignment.
Cell counts were corrected using the formula proposed by Abercrombie (1946) and modified by Amann & Almquist (1962): Corrected number = obtained count × section thickness / [section thickness + √(DM/2)² – (DM/4)²], where DM corresponds to the mean nuclear or nucleolar diameter and section thickness.
Spermatogenesis yield and Sertoli cell efficiency
These parameters were estimated from quantitative analysis of cell population data obtained from 20 transverse sections of seminiferous tubules, using the following indices:
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Mitotic index or efficiency coefficient: calculated as the ratio between primary spermatocytes at pre-leptotene/leptotene stage and type A spermatogonia.
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Meiotic index: calculated as the ratio between round spermatids and primary spermatocytes at pre-leptotene/leptotene stage.
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Overall spermatogenic yield: calculated as the ratio between round spermatids and type A spermatogonia.
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Sertoli cell efficiency: calculated as the ratio between the total number of round spermatids and the total number of Sertoli cells.
Testicular histometry
Twenty transverse sections of seminiferous tubules at stage VII of the seminiferous epithelium cycle were analyzed, preferably those with a circular morphology, in order to measure tubule diameter and area, lumen area, and germinal epithelium height. The evaluator was blinded to group allocation during the analyses. Images were obtained using a light microscope (Olympus BX40) with a 40× objective, equipped with a digital camera and image analysis system (Image Pro-Express v.6.0). All parameters were evaluated through quantitative morphometric analysis.
Statistical analysis
Quantitative data were expressed as mean ± standard error of the mean (SEM). For statistical comparison among groups, one-way analysis of variance (ANOVA) was performed, followed by Dunnett’s test. In all analyses, a significance level of 5% (p < 0.05) was considered. Statistical analyses were conducted using GraphPad Prism v.6 (San Diego, CA, USA).
RESULTS
No differences in testicular biometric and volumetric values among groups
The animals in the experimental groups were monitored daily, with no deaths recorded during the experimental period. In addition, all animals remained clinically healthy and exhibited behavioral characteristics within the normal range for the species. No statistical differences (p > 0.05) were found in body weight among the control (395.5 ± 5.18 g, n = 6), ketoprofen (394.2 ± 14.04 g), and meloxicam (377.0 ± 10.97 g) groups at the end of treatment. Moreover, absolute and relative testicular weights, as well as testicular volume, length, and width, did not differ significantly (p > 0.05) among experimental groups (Table I).
Absolute and relative weights, volume, length, and width of testes from control (CG), ketoprofen- (KG), and meloxicam-treated albino rats (MG).
NSAID treatment affected the number and dynamics of the seminiferous epithelium cell population
The count of different cell types in the seminiferous epithelium showed that treatment with ketoprofen (KG) and meloxicam (MG) for 15 days promoted significant reductions in the population of germ cells and Sertoli cells in the seminiferous tubules of rats compared with the control group (CG). The total number of Sertoli cells was reduced by 8.1% and 9.2% in KG and MG, respectively (Table II). While the counts of type A spermatogonia and pre-leptotene and leptotene primary spermatocytes decreased similarly, by 35.1% and 36.9% in GK, and by 12.2% for both cell types in MG (Table II). Reinforcing the numerical discrepancy between the treated groups and between these and the control group, the total number of round spermatids decreased by 42.2% in KG and 14.3% in MG (Table II).
Cell counts* of the seminiferous epithelium in control (CG), ketoprofen- (KG), and meloxicam-treated albino rats (MG).
From the total number of seminiferous epithelium cells, it was possible to estimate spermatogenic dynamics among the studied groups. Accordingly, mitotic, meiotic, and spermatogenic yields, as well as Sertoli cell efficiency, were calculated (Figure 1). Only the KG showed significant reductions in all evaluated parameters, with decreases of 3.0% in mitotic yield, 8.2% in meiotic yield, 11.0% in spermatogenic yield, and 47.5% in Sertoli cell efficiency. In contrast, the MG did not show significant differences (p > 0.05) in any of the evaluated indices compared with the CG (Figure 1).
Mitotic, meiotic, and spermatogenic yields, as well as Sertoli cell efficiency, in control (CG), ketoprofen- (KG), and meloxicam-treated albino rats (MG). Bars represent mean ± standard error of the mean (SEM) (n = 6 per group). The ketoprofen-treated group showed significant reductions in all evaluated parameters. *p < 0.05 compared with the control group (CG), one-way ANOVA followed by Dunnett’s test.
Changes were observed in testicular histometric parameters
The variables of testicular histometry are presented as arithmetic means ± standard error of the mean for the CG, KG, and MG. The morphometric data included seminiferous tubule diameter (STD), seminiferous tubule area (STA), tubular lumen area (TLA), and seminiferous epithelium height (SEH) (Table III). Regarding the diameter and area of the seminiferous tubules (STD and STA, respectively), no statistically significant differences were observed in KG and MG compared with CG (Table III).
Seminiferous tubule diameter (STD), seminiferous tubule area (STA), tubular lumen area (TLA), and seminiferous epithelium height (SEH) in control (CG), ketoprofen- (KG), and meloxicam-treated albino rats (MG).
For the variables related to the lumen area and epithelium height of the seminiferous tubules, statistically significant differences were observed. Concerning the tubular lumen area (TLA), we observed a 54% increase in the KG and a 113% increase in the MG compared with the CG (Table IV). Both KG and MG also showed a significant reduction in germinal epithelium height compared with the control group, of approximately 25% and 43%, respectively (Table III).
The morphological alterations in the seminiferous tubules, evidenced by the increase in tubular lumen area (KG) and the reduction in seminiferous epithelium height, in addition to the presence of vacuolization in the cytoplasm of Sertoli cells in KG and MG, are illustrated in Figure 2. In these representations, the main structural components of the seminiferous tubules were highlighted, allowing a comparative visualization of the alterations induced by the treatments.
Photomicrograph of cross-sections of seminiferous tubules from the rat testes of control (a), ketoprofen- (b), and meloxicam-treated albino rats (c). Hematoxylin–Eosin staining. (a) On the left, an intact seminiferous tubule is observed, and on the right, testicular parenchyma with preserved morphology. (b) On the left, marked vacuolization in the seminiferous epithelium is evident, and on the right, an increase in tubular lumen, as well as an increase in interstitial space. (c) On the left, marked desquamation of the seminiferous epithelium is observed, and on the right, vacuolization in the seminiferous epithelium and increased interstitial space. Identified structures: spermatogonia (SPG); primary spermatocyte (PS); round spermatid (RS); Sertoli cell (SC); interstitial tissue (IT); tubular lumen (TL); vacuolization (V); basal lamina (BL); and cellular debris resulting from seminiferous epithelium desquamation (ED).
DISCUSSION
In the present study, we demonstrated that the administration of ketoprofen and meloxicam to Wistar rats for 15 days (subacute administration) was associated with relevant morphological alterations in the testicular parenchyma, although these alterations were not uniformly observed across all evaluated parameters. Altogether, these findings suggest that these drugs may exert harmful effects on testicular tubular architecture and, consequently, on spermatogenesis.
The absence of significant changes in body weight between the treated and control groups is consistent with previous studies that also reported no alterations in this parameter in animals treated with different NSAIDs (Osawe & Farombi 2018, Barbosa et al. 2020, Asadi et al. 2022). The absence of weight variation suggests that the drugs used did not cause systemic effects severe enough to compromise food intake or nutrient absorption during the experimental period. It is well-established that long-term NSAID use can induce adverse gastrointestinal effects, including ulceration, bleeding, vomiting, and diarrhea, which disrupt nutritional homeostasis (Tai & McAlindon 2021, Cion et al. 2025). However, the subacute 15-day protocol may have been insufficient for the manifestation of such alterations, as suggested by Hiware et al. (2018), who reported significant weight loss only after six weeks of ibuprofen exposure.
Testicular weight is widely recognized as a parameter associated with spermatogenic activity, being used as an indirect indicator of sperm production and male fertility (Mo et al. 2023, Montes-Garrido et al. 2023). In addition, it is considered an important parameter to assess the degree of cellular loss in the germinal epithelium (Veloso et al. 2018). Nevertheless, no significant differences were identified in absolute or relative testicular weight, volume, or macroscopic testicular dimensions among the analyzed groups. Similarly, previous studies reported no alterations in testicular weight in rats treated with indomethacin (Saeed et al. 2009) or nimesulide (Ugochukwu et al. 2011), as well as in gonadal weight and size with ibuprofen (Sharma et al. 2020). These findings suggest that NSAID-induced adverse effects on the testes may occur in a localized and functional manner without necessarily being reflected in detectable macroscopic alterations (Tran-Guzman & Culty 2022, Walker 2024). Moreover, although testicular weight is a useful parameter, it is not a precise indicator when evaluated in isolation and should be analyzed alongside other indicators (Mo et al. 2023).
The quantification of seminiferous epithelium cells revealed a reduction in germ and Sertoli cell numbers in the treated groups, with a more pronounced effect in the ketoprofen group. Although the percentage reduction of Sertoli cells was similar between groups, a marked decrease was observed in the populations of spermatogonia, primary spermatocytes, and round spermatids in ketoprofen group compared to meloxicam-treated animals. These findings suggest that, at least in part, the differential testicular effects observed between treatments may be related to differences in cyclooxygenase (COX) inhibition profiles, as ketoprofen acts predominantly on COX-1, whereas meloxicam shows greater selectivity for COX-2 (Cáceres-Cabana et al. 2018).
Both COX isoforms are described in the seminiferous tubules and Leydig cells, with COX-1 showing constitutive expression, while COX-2 expression is dependent on gonadotropins and androgens (Neeraja et al. 2003). Inhibition of testicular prostaglandins, via COX blockade has already been associated with Sertoli cell dysfunction, and impaired spermatogenesis (Frungieri et al. 2015, Tran-Guzman & Culty 2022, Walker 2024). However, further studies are needed to better define the impact of selective COX isoform inhibition on rodent testicular physiology.
Differences in pharmacokinetic properties between ketoprofen and meloxicam may also contribute to the distinct testicular effects observed in the present study. Previous investigations have demonstrated in chukar partridge that these drugs differ in parameters such as volume of distribution, clearance, and elimination half-life, which directly influence tissue exposure. Notably, ketoprofen has been reported to exhibit a higher volume of distribution compared to meloxicam, suggesting a greater capacity to penetrate peripheral tissues (Cetin et al. 2022), potentially including the testicular microenvironment. This increased tissue availability may enhance local inhibition of prostaglandin synthesis and contribute to the more pronounced spermatogenic impairment observed in the ketoprofen-treated group. However, it is important to note that these pharmacokinetic data were obtained in a different species, and interspecies variations may significantly influence drug disposition (Toutain et al. 2010). Therefore, further studies evaluating the pharmacokinetic profiles of these drugs in rodents are warranted to better support this interpretation.
Spermatogenesis involves the differentiation of spermatogonia into spermatocytes, which undergo meiosis to form spermatids that subsequently mature into spermatozoa (Maroto et al. 2025). Thus, the reduction of one cell lineage directly impacts its successors, a phenomenon known as maturation depletion (Creasy 2001). Maturation depletion induced by other NSAIDs has also been reported. For instance, Arslan et al. (2016) observed a reduction in the number of spermatogonia and Sertoli cells after intrauterine exposure to diclofenac in neonatal rats. Similarly, Vyas et al. (2018) reported a dose-dependent decrease of all germ cells in adult rats, reaching complete interruption of spermatogenesis at the highest doses of diclofenac. In the case of ibuprofen, Sharma et al. (2020) reported a slight reduction in germ cells, except for spermatogonia. Conversely, another study in mice that were intrauterinely exposed to acetylsalicylic acid or ibuprofen found no differences in the total number of Sertoli or germ cells per seminiferous tubule (Rossitto et al. 2019). These discrepancies suggest that the effects of NSAIDs may vary widely according to the experimental model, species, drug, dose, and exposure period (Uzun et al. 2015).
The reduction of approximately 8 - 9% in the number of Sertoli cells, although moderate, is a relevant finding considering the essential role of these cells in supporting and regulating the tubular microenvironment. Consequently, this depletion may compromise the testis’ ability to sustain efficient spermatogenesis (Frungieri et al. 2015). In addition, there is evidence that Sertoli cell dysfunction results in germ cell loss and disorganization of testicular tissue (Vyas et al. 2018). Significant decreases, higher than 35%, in type A spermatogonia and primary spermatocytes observed in the ketoprofen-treated group indicate a marked impact on the early stages of spermatogenesis (Maroto et al. 2025). This finding, combined with the drastic reduction in round spermatid numbers in the same group, suggests that ketoprofen may impair both mitotic and meiotic proliferation during spermatogenesis. This reflects a greater deleterious effect on spermatogenesis compared with meloxicam, which also induced adverse effects, albeit to a lesser extent. This hypothesis is reinforced by the data on spermatogenesis-associated yields, which were significantly affected only in the ketoprofen-treated group.
In fact, the significant alterations in the cellular composition of the seminiferous epithelium were reflected functionally only in the ketoprofen-treated group. This group exhibited statistically significant reductions in mitotic, meiotic, and overall spermatogenic yields, as well as in Sertoli cell efficiency, compared to the control. In contrast, no significant changes in these functional parameters were observed in the meloxicam-treated animals, indicating a more detrimental and specific impact of ketoprofen on spermatogenic dynamics.
This pattern is consistent with previous studies evaluating the effects of different NSAIDs on spermatogenesis. Rossitto et al. (2019) reported that intrauterine exposure of mice to combinations of paracetamol with acetylsalicylic acid or ibuprofen, both preferential COX-1 inhibitors, induced early mitotic arrest of germ cells, resulting in reduced cell proliferation in embryonic testes. Conversely, Winnall et al. (2009) demonstrated that a five-week treatment with the selective COX-2 inhibitor celecoxib did not significantly affect spermatogenesis. This evidence supports the hypothesis that NSAIDs which preferentially inhibit the constitutive COX-1 isoform, such as ketoprofen, are more likely to disrupt spermatogenesis. This is particularly plausible given that COX-2 expression in the testis appears to be largely restricted to pathological conditions (Frungieri et al. 2007).
Histometric analysis demonstrated that ketoprofen significantly disrupted seminiferous tubule architecture, as evidenced by a reduction in epithelial height coupled with relative lumen expansion. Meloxicam similarly induced a significant decrease in epithelial height, along with an increase in tubular lumen area. These morphometric alterations, combined with the observed reductions in seminiferous epithelium cell populations, suggest a partial degeneration of cellular components and possible disorganization of the spermatogenic cycle (Aitken 2017, Sharma et al. 2020). Expansion of the lumen area may result either from cell loss (Russell et al. 1990) or from changes in secretion and intratubular reabsorption of fluids released by Sertoli cells (Clulow & Jones 2004, Olawuyi et al. 2019), reflecting an imbalance in the microenvironment necessary to support spermatogenesis (Creasy et al. 2012). Seminiferous epithelium height is a sensitive marker of spermatogenic activity, and significant reductions in this parameter, as observed in the treated groups, reflect impaired proliferation and cell differentiation within the seminiferous tubule (Russell et al. 1990, Creasy 2001, Creasy et al. 2012).
It is also worth to mentioning that this pattern of epithelial alterations induced by ketoprofen and meloxicam aligns with findings from other NSAIDs, although the specific histometric outcomes appear to be highly dependent on the drug, species, and protocol. For example, Asadi et al. (2022) reported reductions in luminal diameter, epithelial height, and tubule diameter in rats treated with acetylsalicylic acid for 5 weeks. In another study, Hartley et al. (2019) did not observe significant changes in tubular diameter or epithelial height but identified a reduction in luminal diameter after intraperitoneal administration of ketoprofen 25 mg/kg in mice. On the other hand, Cáceres-Cabana et al. (2018) found significant reductions in both diameter and epithelial height in rats exposed to meloxicam 1 mg/kg or ibuprofen 120 mg/kg for 30 days. Rats submitted to the treatment with naproxen 20 mg/kg for 30 days presented an increase in the tubular lumen without altering tubule diameter or tubular area (Odum et al. 2021). Ommati et al. (2018) showed that sulfasalazine for 60 days reduced thickness and tubule diameter.
The identified histopathological alterations, although not accompanied by macroscopic testicular atrophy, suggest a subclinical dysfunction that may precede more pronounced declines in sperm quality and fertility (Uzun et al. 2015, Aitken 2017). Key microscopic changes, including reduced epithelial height, increased luminal and interstitial area, marked epithelial desquamation, and cellular vacuolization, were consistently observed in the treated groups. This spectrum of damage is consistent with previous reports for several NSAIDs (Temamoğulları et al. 2018, Vyas et al. 2018, Hartley et al. 2019, Haroun et al. 2020, Asadi et al. 2022, El-Bahr et al. 2022), reinforcing that such structural deficits are a common consequence of this drug class.
The most frequently observed lesions in animal models exposed to NSAIDs are seminiferous tubule degeneration and a reduced number of intraluminal spermatozoa (Nasser et al. 2019). Vascular alterations, such as interstitial vessel congestion and edema, are also recurrent findings, as reported by several authors (Uzun et al. 2015, Cáceres-Cabana et al. 2018, Nasser et al. 2019, Haroun et al. 2020, Owumi et al. 2020, Asadi et al. 2022, El-Bahr et al. 2022, Qureshi et al. 2024). In addition, several authors have reported marked damage to germ cells (Saeed et al. 2009, Uzun et al. 2015, Cáceres-Cabana et al. 2018, Temamoğulları et al. 2018, Vyas et al. 2018, Asadi et al. 2022), tubules with irregular or distorted architecture (Bagoji et al. 2017), and necrotic areas (Bagoji et al. 2017, Qureshi et al. 2024). Increased connective tissue between tubules (Uzun et al. 2015, Cáceres-Cabana et al. 2018, Odum et al. 2021), germ cell desquamation (El-Bahr et al. 2022), hyaline material deposition (Haroun et al. 2020), vacuolization (Temamoğulları et al. 2018, Hartley et al. 2019, Haroun et al. 2020), tubular atrophy (Hartley et al. 2019) and enlargement of the seminiferous tubule lumen (Odum et al. 2021) have also been documented.
The testicular tissue abnormalities observed in the animals treated with ketoprofen or meloxicam are indicative of an adverse impact that, if persistent or cumulative, may compromise reproductive capacity. Notably, this study provides compelling preclinical evidence of differential testicular effects of NSAIDs based on their COX inhibition profile. The finding that ketoprofen, a preferential COX-1 inhibitor (Cryer & Feldman 1998), consistently elicited more severe morphological and functional damage than the COX-2-preferential meloxicam (Del Tacca et al. 2002) carries direct clinical relevance. Differences in pharmacokinetic properties, particularly in volume of distribution, may further contribute to this disparity by influencing tissue exposure and drug availability within the testicular microenvironment (Cetin et al. 2022). This suggests that in clinical practice, especially for patients requiring chronic or repeated analgesic or anti-inflammatory therapy, the choice of NSAID could influence male reproductive health. Moreover, the concomitant use of NSAIDs with other drugs known to impair spermatogenesis, such as valproate, ketoconazole, and spironolactone (Asghar et al. 2024, Pandiyan 2000), may potentiate these adverse effects, representing a potential risk in long-term therapeutic regimens. These results not only advocate for a more cautious and selective prescription of these drugs but also highlight the need for further preclinical and clinical studies to fully elucidate the long-term reproductive of different NSAIDs.
CONCLUSIONS
In conclusion, this study provides compelling evidence that commonly used NSAIDs can adversely affect testicular health, with the profile of these effects likely influenced by COX inhibition selectivity and, potentially, by pharmacokinetic properties. These findings underscore the necessity for a more discerning use of these drugs in clinical practice and underscore the imperative for further research to define safe-use parameters, ultimately guiding safer therapeutic choices for men requiring anti-inflammatory treatment.
Acknowledgements
Thanks are due to Carla Rodrigues de Lima Silva, Mario Massashi Gonçalves Sumida, and Flávio Maurílio dos Santos Lima for technical support.
Data availability
References
- ABERCROMBIE M. 1946. Estimation of nuclear population from microtome sections. Anat Rec 92: 239-247.
- AITKEN RJ. 2017. Reactive oxygen species as mediators of sperm capacitation and pathological damage. Mol Reprod Dev 84: 1039-1052.
- ALBERT O, DESDOITS-LETHIMONIER C, LESNÉ L, LEGRAND A, GUILLÉ F, BENSALAH K, DEJUCO-RAINSFORD N & JÉGOU B. 2013. Paracetamol, aspirin and indomethacin display endocrine disrupting properties in the adult human testis in vitro. Hum Reprod 28: 1890-1898.
- AMANN RP & ALMQUIST JO. 1962. Reproductive capacity of dairy bull. VIII. Direct and indirect measurement of testicular sperm production. J Dairy Sci 45: 774-781.
- ARSLAN H, AKTAŞ A, ELIBOL E, ESENER OB, TÜRKMEN AP, YURT KK, ONGER ME, ALTUNKAYNAK BZ & KAPLAN S. 2016. Effects of prenatal diclofenac sodium exposure on newborn testis: a histomorphometric study. Biotech Histochem 91: 277-282.
- ASADI M, RAHMANI M, SAMADI A & HESARI AK. 2022. Acetylsalicylic acid-induced alterations in male reproductive parameters in Wistar rats and the effect of sprint interval training. Andrologia 54: e14339.
- ASGHAR MA, TANG S, WAN B, HAN H, WONG LP, ZHANG X & ZHAO Q. 2024. Understanding the impact of valproate on male fertility: insights from preclinical and clinical meta-analysis. BMC Pharmacol Toxicol 25(1): 69.
- BAGOJI IB, HADIMANI GA, YENDIGERI SM & DAS KK. 2017. Sub-chronic indomethacin treatment and its effect on the male reproductive system of albino rats: possible protective role of black tea extract. J Basic Clin Physiol Pharmacol 28: 201-207.
- BARBOSA MG, JORGE BC, STEIN J, SANTOS FERREIRA DA, BARRETO ACDS, REIS ACC, MOREIRA SDS, INOCENCIO LCL, MACORINI LFB & ARENA AC. 2020. Pre-pubertal exposure to ibuprofen impairs sperm parameters in male adult rats and compromises the next generation. J Toxicol Environ Health A 83: 559-572.
- CÁCERES-CABANA E, RONDÓN-ALÍ A, DEL-CARPIO-TEJADA G, ARENAZAS-RODRIGUEZ A, COLQUE-RONDÓN W & CÁCERES-CABANA Z. 2018. Efecto tóxico del ibuprofeno y meloxicam en la estrutura histológica de testículos y calidad seminal de ratas albinas (Rattus norvegicus) variedad Sprague Dawley. Rev Toxicol 35: 89-102.
- CARDOSO FM. 1981. Morfologia, cinética e quantificação da espermatogênese em zebus (Bos indicus). Tese de Doutorado. Universidade Federal de Minas Gerais, Belo Horizonte, 208 p.
- CETIN G, CORUM O, CORUM DD, ATIK O, ALTAN F, TURK E, TEKELI IO, FAKI HE & UNEY K. 2022. Pharmacokinetics of intravenous meloxicam, ketoprofen and tolfenamic acid in chukar partridge (Alectoris chukar). Br Poult Sci 63(1): 14-20.
- CION RIA, JUYAD IGA & YASAY EB. 2025. Efficacy of rebamipide in the prevention of nonsteroidal anti-inflammatory drug-induced gastrointestinal mucosal breaks: a systematic review and meta-analysis. Am J Gastroenterol 121(2): 526-533.
- CLULOW J & JONES RC. 2004. Composition of luminal fluid secreted by the seminiferous tubules and after reabsorption by the extratesticular ducts of the Japanese quail, Coturnix coturnix japonica. Biol Reprod 71: 1508-1516.
- CRYER B & FELDMAN M. 1998. Cyclooxygenase-1 and cyclooxygenase-2 selectivity of widely used nonsteroidal anti-inflammatory drugs. Am J Med 104: 413-421.
- CREASY D ET AL. 2012. Proliferative and nonproliferative lesions of the rat and mouse male reproductive system. Toxicol Pathol 40: 40S-121S.
- CREASY DM. 2001. Pathogenesis of male reproductive toxicity. Toxicol Pathol 29: 64-76.
- DE LA PUENTE R, DIEZ R, DIEZ MJ, FERNANDEZ N, SAHAGUN AM, RODRIGUEZ JM, GARCIA JJ & LOPEZ C. 2024. Pharmacokinetics of meloxicam in different animal species: a comprehensive review. Vet Sci 11: 519.
- DEL TACCA M, COLUCCI R, FORNAI M & BLANDIZZI C. 2002. Efficacy and tolerability of meloxicam, a COX-2 preferential nonsteroidal anti-inflammatory drug. Clin Drug Investig 22: 799-818.
- DROBNIS EZ & NANGIA AK. 2017. Impacts of medications on male fertility. In: Advances in Experimental Medicine and Biology, Cham: Springer.
- EL-BAHR SM, ELZOGHBY RR, ALFATTAH MA, KANDEEL M & HAMOUDA AF. 2022. Aqueous ginger (Zingiber officinale) extract ameliorates the harmful effects of high-dose lornoxicam in albino male rats. Biomed Res Int 2022: 1546734.
- EMA - EUROPEAN MEDICINES AGENCY. 2020. European public MRL assessment report: ketoprofen (bovine, porcine and equidae). Amsterdam: EMA, 12 p.
- FRANÇA LR & RUSSELL LD. 1981. The testis of domestic animals. In: Mann T & Lutwak-Mann C (Eds), Male reproductive function and semen: themes and trends in physiology, biochemistry and investigative andrology. Berlin: Springer-Verlag, p. 197-219.
- FRUNGIERI MB, CALANDRA RS, MAYERHOFER A & MATZKIN ME. 2015. Cyclooxygenase and prostaglandins in somatic cell populations of the testis. Reproduction 149: R169-R180.
- FRUNGIERI MB, GONZALEZ-CALVAR SI, MATZKIN ME, MAYERHOFER A & CALANDRA RS. 2007. Sources and functions of prostaglandins in the testis: evidence for their relevance in male (in)fertility. Anim Reprod 4: 63-69.
- HAROUN M, ELDIN A, EL-KHOULY S & MOSTAFA I. 2020. The protective effect of ascorbic acid and curcumin on the chronic toxicity of meloxicam on the testis of adult albino rats. Benha J Appl Sci 5: 189-207.
- HARTLEY R, CONEI D, TORRES L & ROJAS M. 2019. Effects of ketoprofen in spermatogenesis of mice exposed to continuous hypobaric hypoxia. Int J Morphol 37: 1572-1577.
- HEDLEY J. 2020. BSAVA Small Animal Formulary. Part B: Exotic Pets. 10th ed. Quedgeley, Gloucester: British Small Animal Veterinary Association.
- HIWARE SD, GUJAR V, BOKARIYA P, TARNEKAR AM, SHENDE MR & WANKHEDE VR. 2018. Effect of use of NSAID-ibuprofen in prolonged therapeutic doses on mucosa of lower respiratory tract of mice: a histological study. Ann Int Med Dent Res 4: AT01-AT07.
- KRISTENSEN DM ET AL. 2018. Ibuprofen alters human testicular physiology to produce a state of compensated hypogonadism. Proc Natl Acad Sci U S A 115: E715-E724.
- KRISTENSEN DM, MAZAUD-GUITTOT S, GAUDRIULT P, LESNÉ L, SERRANO T, MAIN KM & JÉGOU B. 2016. Analgesic use: prevalence, biomonitoring and endocrine and reproductive effects. Nat Rev Endocrinol 12: 381-393.
- MAROTO M, TORVISCO SN, GARCÍA-MERINO C, FERNÁNDEZ-GONZÁLEZ R & PERICUESTA E. 2025. Mechanisms of hormonal, genetic, and temperature regulation of germ cell proliferation, differentiation, and death during spermatogenesis. Biomolecules 15: 500.
- MO P, ZHAO Z, KE X, FAN Y & LI C. 2023. Effects of clinical medications on male fertility and prospects for stem cell therapy. Front Cell Dev Biol 11: 1258574.
- MONTES-GARRIDO R, ANEL-LOPEZ L, RIESCO MF, NEILA-MONTERO M, PALACIN-MARTINEZ C, SORIANO-ÚBEDA C, BOIXO JC, DE PAZ P, ANEL L & ALVAREZ M. 2023. Does size matter? Testicular volume and its predictive ability of sperm production in rams. Animals (Basel) 13: 3204.
- NASSER A, ELGENDY A, BOGZIL A & MOGODA O. 2019. Adverse effects of carprofen and meloxicam in male rats. Br J Pharm Med Res 4: 1979-1990.
- NEERAJA S, SREENATH AS, REDDY PRK & REDDANNA P. 2003. Expression of cyclooxygenase-2 in rat testis. Reprod Biomed Online 6: 302-309.
- ODUM OP, ADEYANJU OA, OLAWUYI TS, SOLADOYE AO & AKINOLA OB. 2021. Effect of naproxen on testicular morphometry and serum gonadotropin level of cadmium intoxication in rats. Eur J Anat 25: 523-531.
- OLAWUYI TS, UKWENYA VO, JIMOH AGA & AKINOLA KB. 2019. Histomorphometric evaluation of seminiferous tubules and stereological assessment of germ cells in testes following administration of aqueous leaf-extract of Lawsonia inermis on aluminium-induced oxidative stress in adult Wistar rats. JBRA Assist Reprod 23: 24-32.
- OMMATI MM ET AL. 2018. Dual effects of sulfasalazine on rat sperm characteristics, spermatogenesis, and steroidogenesis in two experimental models. Toxicol Lett 284: 46-55.
- ORTAVANTE R, COUROT M & HOCHEREAU-DE-REVIERS MT. 1984. Espermatogénesis en los mamíferos domésticos. In: Cole HH & Cupps PT (Eds), Reproducción de los animales domésticos. 3rd ed. Zaragoza: Acribia, p. 171-190.
- OSAWE SO & FAROMBI EO. 2018. Quercetin and rutin ameliorates sulphasalazine-induced spermiotoxicity, alterations in reproductive hormones and steroidogenic enzyme imbalance in rats. Andrologia 50: e12981.
- OWUMI SE, ALIYU-BANJO NO & ODUNOLA OA. 2020. Selenium attenuates diclofenac-induced testicular and epididymal toxicity in rats. Andrologia 52: e13669.
- PANDIYAN N. 2000. Medical management of male infertility. Reprod Biomed Online 1(1): 13-6.
- PAREKATTIL SJ, ESTEVES SC & AGARWAL A. 2020. Male infertility: contemporary clinical approaches, andrology, ART and antioxidants. 2nd ed., Cham: Springer, 914 p.
- QURESHI IZ, RAZZAQ A & NAZ SS. 2024. Testing of acute and sub-acute toxicity profile of novel naproxen sodium nanoformulation in male and female mice. Regul Toxicol Pharmacol 150: 105650.
- ROSSITTO M, MARCHIVE C, PRUVOST A, SELLEM E, GHETTAS A, BADIOU S, SUTRA T, POULAT F, PHILIBERT P & BOIZET-BONHOURE B. 2019. Intergenerational effects on mouse sperm quality after in utero exposure to acetaminophen and ibuprofen. FASEB J 33: 339-357.
- RUSSELL LD, ETTLIN RA, HIKIM APS & CLEGG ED. 1990. Histological and histopathological evaluation of the testis. Clearwater, FL: Cache River Press.
- SAEED SA, ANWAR N, KHAN KM & SARFRAZ N. 2009. Effect of chronic treatment with a cyclooxygenase inhibitor on reproductive parameters in male rat. J Ayub Med Coll Abbottabad 21: 66-71.
- SCHERLE W. 1970. A simple method for volumetry of organs in quantitative stereology. Mikroskopie 26: 57-63.
- SEMET M, PACI M, SAÏAS-MAGNAN J, METZLER-GUILLEMAIN C, BOISSIER R, LEJEUNE H & PERRIN J. 2017. The impact of drugs on male fertility: a review. Andrology 5: 640-663.
- SHARMA P, KAUR P, GHANGHAS P, KAUR J & KAUSHAL N. 2020. Selenium ameliorates ibuprofen-induced testicular toxicity by redox regulation. Reprod Toxicol 96: 349-358.
- TAI FWD & MCALINDON ME. 2021. Non-steroidal anti-inflammatory drugs and the gastrointestinal tract. Clin Med (Lond) 21: 131-134.
- TEMAMOĞULLARI F, ARAL F & YILMAZ R. 2018. Royal jelly protection on flunixin meglumine-induced spermiotoxicity and testicular degeneration in mice. Pol J Vet Sci 21: 497-506.
- TIRPÁK F, GREIFOVÁ H, LUKÁČ N, STAWARZ R & MASSÁNYI P. 2021. Exogenous factors affecting the functional integrity of male reproduction. Life (Basel) 11: 213.
- TOUTAIN PL, FERRAN A & BOUSQUET-MÉLOU A. 2010. Species Differences in Pharmacokinetics and Pharmacodynamics. In: Cunningham F, Elliott J & Lees P (Eds), Comparative and Veterinary Pharmacology. Handbook of Experimental Pharmacology, vol. 199. Springer, Berlin, Heidelberg.
- TRAN-GUZMAN A & CULTY M. 2022. Eicosanoid biosynthesis in male reproductive development: effects of perinatal exposure to NSAIDs and analgesic drugs. Front Toxicol 4: 842565.
- UGOCHUKWU AP, ORISAKWE EO & ABANOBI O. 2011. Effects of nimesulide on testicular functions in prepubertal albino rats. J Basic Clin Physiol Pharmacol 22: 137-140.
- UZUN B, ATLI O, PERK BO, BURUKOGLU D & ILGIN S. 2015. Evaluation of the reproductive toxicity of naproxen sodium and meloxicam in male rats. Hum Exp Toxicol 34: 415-429.
- VELOSO EVL, FIGUEIRÊDO LCM, MENEZES DJA, SOUSA OB & SANTOS JRS. 2018. Efeitos de sulfato de vincristina sobre os testículos de ratos albinos tratados na fase pré-púbere. Pesq Vet Bras 38: 1217-1224.
- VYAS A, PUROHIT A & RAM H. 2018. Assessment of dose-dependent reproductive toxicity of diclofenac sodium in male rats. Drug Chem Toxicol 42: 478-486.
- WALKER T. 2024. Progress in the study of toxic effects of drugs on the male reproductive system. Asia Pac J Pharmacother Toxicol 4: 76-84.
- WINNALL WR, MUIR JA, LIEW S, HIRST JJ, MEACHEM SJ & HEDGER MP. 2009. Effects of chronic celecoxib on testicular function in normal and lipopolysaccharide-treated rats. Int J Androl 32: 542-555.
