Ibuprofen is among the most frequently used nonsteroidal anti-inflammatory drugs worldwide and is valued for its analgesic, antipyretic, and anti-inflammatory properties [1,2]. Its wide over-the-counter availability, together with a marked increase in use during the coronavirus disease 2019 (COVID-19) pandemic, has further amplified global consumption [3,4]. As a consequence, ibuprofen is now recognized as a contaminant of emerging concern, characterized by environmental persistence and recurrent detection across multiple environmental matrices [5,6]. Entry into the environment occurs through several pathways, including municipal and industrial wastewater discharges, hospital effluents, agricultural runoff, and irrigation with reclaimed water. Additional contributions arise from the application of sewage sludge or biosolids to agricultural land, as well as from the improper disposal of unused or expired medications [7]. Once released, both ibuprofen and its metabolites have been detected in surface waters, soils, sediments, and even within food chains. Reported concentrations range from ng/L to µg/L in aquatic environments, while levels in solid matrices range from ng/g to µg/g on a dry-weight basis [8,9]. Conventional wastewater treatment technologies often fail to achieve complete removal, allowing ibuprofen residues to persist in the environment. Even at low concentrations, chronic exposure has been associated with ecological disturbances and potential risks to human health [10].

The ovarian reserve, which represents the finite population of primordial follicles established during fetal life, is a central determinant of female reproductive potential [11]. Its natural decline occurs primarily through follicular atresia, a physiological process in which most ovarian follicles undergo degeneration, thereby ensuring that only high-quality follicles reach ovulation and maintaining overall ovarian homeostasis [12]. This predictable depletion of the ovarian reserve has long been a subject of scientific investigation. Research addressing this phenomenon has generally followed two complementary directions. The first focuses on elucidating the mechanisms underlying follicular atresia, with the goal of identifying potential targets to delay or modulate this physiological process [13]. The second explores strategies to improve or restore the ovarian reserve, such as enhancing follicular survival or generating new gametes from somatic cells through in vitro approaches [14]. Together, these lines of investigation provide important avenues for preserving female fertility. However, not all follicular loss occurs within this physiological framework. The ovarian reserve may also be compromised by pathological, environmental, and lifestyle-related factors, which can disrupt follicular development and hormonal balance and ultimately lead to diminished ovarian reserve [15,16]. Among these factors, environmental exposures are particularly concerning, as individuals may be unknowingly subjected to them through air, water, food, or soil, where chronic low-dose exposure over extended periods may silently impair ovarian health and reproductive potential [17].

Although numerous studies have investigated the impact of emerging environmental exposures on ovarian reserve [18], ibuprofen warrants particular attention because it is not only a frequently detected environmental contaminant but also an indispensable pharmaceutical consumed daily by millions of individuals worldwide. Unlike plastics or pesticides, which are widely recognized as toxic [19,20], pharmaceuticals are intentionally designed to preserve health. This dual role makes their presence in the environment especially concerning. In Algeria, environmental monitoring programs have reported widespread contamination of aquatic environments by ibuprofen, with concentrations reaching up to 312.1 ng/L in drinking water [21]. These findings underscore both the limited efficiency of wastewater treatment plants and the likelihood of chronic, low-dose exposure within the population. Despite this evidence, no studies to date have evaluated the biological or reproductive effects of ibuprofen exposure in Algeria, representing a critical gap in current knowledge. Addressing such risks is therefore essential not only to raise public awareness but also to provide scientific evidence capable of informing the development of preventive strategies. In this study, we evaluate the long-term effects of environmentally relevant ibuprofen exposure in female Wistar rats, with particular emphasis on the mechanisms underlying ovarian follicular atresia.

This study aimed to assess the effects of chronic ibuprofen exposure on ovarian function under environmentally relevant conditions. Ibuprofen was selected due to its widespread global use as a nonsteroidal anti-inflammatory drug and its frequent detection in aquatic environments resulting from incomplete removal during wastewater treatment processes. In Algeria, environmental monitoring programs have reported ibuprofen concentrations exceeding 300 ng/L in drinking water, highlighting the potential for continuous low-dose human exposure. To approximate realistic environmental conditions while accounting for possible improvements in water treatment efficiency, the experimental concentration was set at approximately half of the highest reported level. Although extrapolation from environmental water concentrations to internal human exposure has inherent limitations, the use of a chronic low-dose rat model provides a controlled and sensitive system for identifying potential ovarian toxic effects and elucidating underlying mechanisms associated with prolonged ibuprofen exposure. These findings therefore contribute experimental evidence relevant to environmental health risk assessment, while explicitly acknowledging species- and dose-translation constraints.

Our findings clearly demonstrate that chronic exposure to environmentally relevant concentrations of ibuprofen promotes ovarian follicular atresia in female rats. Ibuprofen exposure resulted in a significant increase in the proportion of atretic follicles, indicating a shift in follicular dynamics toward degeneration. These alterations are consistent with previous toxicological studies demonstrating that emerging environmental contaminants disrupt folliculogenesis and compromise ovarian integrity [17]. By accelerating follicular loss, ibuprofen exposure may ultimately reduce ovarian reserve and shorten the reproductive lifespan, thereby highlighting a potential risk to female fertility.

Histological evaluation further revealed that ibuprofen disrupted the ovarian microenvironment by inducing vascular congestion, interstitial edema, hemorrhage, dilated blood vessels, inflammatory cell infiltration, follicular cell degeneration, and cyst formation. Such pathological alterations are known to impair granulosa–theca–oocyte communication, ultimately disrupting follicular development and steroidogenic activity [29]. These tissue-level disruptions were accompanied by systemic effects, as evidenced by reduced body weight gain and a lower ovarian index in ibuprofen-exposed rats, both of which are well-established indicators of general toxicity and ovarian dysfunction. Collectively, these findings demonstrate that ibuprofen compromises both the structural and functional integrity of the ovary.

The fate of granulosa cells is central to the mechanism of follicular atresia. Programmed cell death of granulosa cells represents the critical determinant of follicle degeneration and may occur through apoptosis, autophagy, or other regulated cell death pathways [30].

While these processes are tightly regulated under physiological conditions, environmental contaminants can accelerate their activation. The excessive follicular atresia observed in the present study suggests that ibuprofen disrupts key regulatory networks, thereby driving granulosa cells toward premature programmed cell death, primarily through apoptosis. Consistent with this interpretation, the biochemical profile following ibuprofen exposure revealed increased lipid peroxidation, as indicated by elevated MDA levels, alongside reduced CAT activity, confirming a state of redox imbalance. These findings align with earlier studies demonstrating that ibuprofen induces oxidative stress [31]. Elevated reactive oxygen species levels in granulosa cells are known to damage mitochondrial membranes and suppress the expression of anti-apoptotic genes. In line with this mechanism, immunohistochemical analysis demonstrated reduced Bcl-2 expression in ibuprofen-treated ovaries, providing molecular evidence that ibuprofen downregulates anti-apoptotic defenses and promotes granulosa cell apoptosis. Although multiple signaling pathways regulate Bcl-2 expression, our results suggest that oxidative stress represents the principal driver of its downregulation in this context [32]. In addition, we observed a significant increase in circulating IL-6 levels, indicating activation of inflammatory pathways. IL-6 is a pleiotropic cytokine that not only contributes to systemic inflammation but also promotes apoptotic and pyroptotic signaling in granulosa cells. Thus, inflammation likely acts synergistically with oxidative stress to amplify granulosa cell death and accelerate follicular atresia following ibuprofen exposure [33].

Another key effect of ibuprofen exposure was endocrine disruption, as reflected by the marked reduction in E2 concentrations in treated animals. This decline indicates impaired granulosa cell function and reduced aromatase activity, both of which are essential for follicle survival and oocyte maturation [34]. While previous studies in fish have suggested that ibuprofen may alter endocrine signaling, such models are not fully representative of mammalian reproductive physiology. Our findings, obtained in rats, a model more relevant to human reproductive function, support the hypothesis that ibuprofen can disrupt ovarian steroidogenesis. The precise mechanism underlying this disruption remains to be clarified and may involve direct interference with E2 biosynthesis or indirect effects mediated by oxidative stress and granulosa cell apoptosis. These uncertainties underscore the complexity of ibuprofen’s endocrine-disrupting potential and highlight the need for further studies to elucidate the dose- and sex-dependent mechanisms through which ibuprofen affects steroid hormone regulation.

In summary, our results integrate histological, biochemical, hormonal, and molecular evidence into a coherent mechanistic framework. Ibuprofen, even at environmentally relevant concentrations, promotes oxidative stress and inflammation, which converge to suppress anti-apoptotic defenses and induce granulosa cell apoptosis, thereby promoting follicular atresia. The concurrent reduction in E2 levels reflects impaired granulosa cell function and endocrine imbalance, further compromising ovarian homeostasis. Collectively, these findings identify ibuprofen as a potential environmental risk factor for female reproductive health. Future research should not only delineate the precise molecular pathways involved but also assess the potential reversibility of ovarian damage and explore whether protective interventions, such as antioxidants or anti-inflammatory agents, can mitigate these adverse outcomes. Expanding this line of investigation to epidemiological and clinical studies will be essential to clarify the reproductive risks associated with chronic low-dose ibuprofen exposure in human populations.

Several limitations should be acknowledged. Although the ibuprofen dose was selected based on previously reported environmental concentrations, precise evaluation of current human exposure levels was not possible due to the lack of routine analytical monitoring. As a result, the administered dose represents an environmentally relevant experimental approximation rather than an exact human-equivalent exposure. Furthermore, while follicular atresia was strongly associated with oxidative stress and dysregulation of apoptotic markers, the limited range of pathological pathways examined restricted a more comprehensive mechanistic interpretation. Additional mechanisms potentially involved in follicular atresia, including autophagy, mitochondrial dysfunction, and alterations in endocrine signaling, were not evaluated and should be addressed in future studies.