An updated look into reactive oxygen species and cyclophosphamide-induced ovarian damage

Abstract

It is estimated that 70–75% of cancer survivors are interested in parenthood with an astounding 80% struggling with reduced fertility. Cancer treatments can deteriorate the ovarian primordial follicular pool and reduce the quality of fertilizable eggs through cytotoxicity and induction of premature ovarian insufficiency (POI). Cyclophosphamide is one of the most commonly used chemotherapeutic agents and is also utilized as an immunosuppressant in autoimmune diseases and in bone marrow transplants. Accordingly, those treated with cyclophosphamide face increased risks of POI and infertility, potentially arising from the toxic effects of its metabolites acrolein and phosphoramide mustard. The downstream accumulation of oxidative stress mediated by reactive oxygen species (ROS) from mitochondrial damage, activation of apoptotic pathways, and the disruption of enzymatic and non-enzymatic machinery may facilitate premature follicle activation, follicular apoptosis, and oocyte quality deterioration resulting in the loss of the ovarian reserve and POI. Furthermore, direct disruption of key antioxidants, such as superoxide dismutase (SOD) and glutathione (GSH), by acrolein and myeloperoxidase (MPO) may play a key role in ovarian disruption and the worsening oxidative state in those seeking treatment with cyclophosphamide.

Introduction

Ovarian aging is a natural process beginning with the gradual decline of the ovarian reserve in the female embryo at 20 weeks gestation and the inevitable loss of fertility at menopause [1]. The declining ovarian reserve is influenced by both apoptotic factors as well as autophagy that promote follicular atresia, the degradation of ovarian follicles [2]. This process includes a decline in not only the quantity of oocytes but in the quality of the remaining oocytes [1], which accelerates over the age of 35, allowing for menopausal transition (with the average age of menopause around 51 years old). Premature ovarian insufficiency (POI) is defined as the irreversible loss of predictable ovarian function before the age of 40 years [3], which can be induced by treatments such as chemotherapy via ovarian cytotoxicity that deteriorates the ovarian primordial follicular pool and reduces the quality of fertilizable eggs [4]. One chemotherapeutic agent of note is cyclophosphamide, which has been associated with increased risks of POI and infertility [4–7]. Cyclophosphamide is an antineoplastic, broad-spectrum anti-tumor and anti-cancer alkylating agent and is also used as an immunosuppressant for treatment of autoimmune diseases and in bone marrow transplants [8].

The mechanism of POI following cancer treatment is still a matter of debate with several theories proposing pathways involving follicular cell apoptosis, oxidative stress, ovarian atrophy, cortical fibrosis, and damage to blood-vessels [4]. This is of utmost importance as cancer incidence in women 15–39 years old (i.e. childbearing age) is 52.3 per 100,000, with 70–75% of cancer survivors interested in parenthood and an astounding 80% facing reduced fertility [9]. Moreover, the process of ovarian aging, follicular atresia, and the loss of fertility are accompanied by endocrine dysfunction, menstrual cycle abnormalities, and often increased risk for chronic conditions such as diabetes, heart disease, cancer, and osteopenia/osteoporosis, among others [10–12].

Previously, our lab investigated the effects of cyclophosphamide and its metabolite, acrolein, on inducing oxidative stress and diminishing oocyte quality. Through these studies we concluded that exposure to acrolein produced more significant oocyte quality deterioration and a higher amount of reactive oxygen species (ROS) production at a lower concentration as compared to treatment with cyclophosphamide [13, 14]. This suggests acrolein is the modulating factor inducing oxidative stress mediated deterioration of oocyte quality when cyclophosphamide is administered. Interestingly, ovarian aging and POI have been associated with the overproduction of ROS that mediate ovarian tissue damage and follicular depletion [15–17] via granulosa cell apoptosis, DNA breaks, mitochondrial dysfunction, and chromosomal and meiotic abnormalities [15, 18].

Myeloperoxidase (MPO), a pro-inflammatory enzyme, is upregulated in inflammation and cancer where it is associated with pro- and anti-tumor properties, tumor initiation, and metastasis through MPO-derived oxidants that facilitate DNA modification, apoptosis, and regulation of cellular growth [19–22]. Oxidants such as superoxide (O₂^•−), hydrogen peroxide (H₂O₂), hydroxyl radical (^•OH), peroxynitrite (ONOO^–), and hypochlorous acid (HOCl) have been shown to deteriorate oocyte quality, evidenced by a disruption in scaffolding proteins that alters microtubule arrangement and chromosomal alignment [23–26] and can induce oocyte granulosa cell apoptosis and protein nitration in the oocyte [27, 28]. These adverse events are produced in a concentration-dependent manner and are especially prevalent in increased inflammatory states such as in endometriosis and aging or following exposure to toxins like heavy metals or pesticides, in which ROS causes follicular granulosa cell apoptosis and protein nitration of the oocyte microenvironment [26–31]. Thus, the already pro-inflammatory state seen during cancer and subsequently produced oxidative stress may be exacerbated by the use of cyclophosphamide, particularly through acrolein accumulation, that accelerates the activation of primordial follicles, apoptosis of primordial follicles, and increased atresia of growing follicles contributing to POI and infertility. Furthermore, the burden of oxidative stress on the ovarian environment may be ameliorated via the use of antioxidants [32], suggesting a potential protective measure during treatment. This review discusses the current research surrounding POI following cyclophosphamide exposure and expands the role of oxidative stress and antioxidant activity on oocyte quality and ovarian damage.

Methods

An extensive literature review was conducted through the online databases PubMed, Science Direct, and Springer Link up to August 2025, using the keywords “cyclophosphamide”, “acrolein”, “ovarian failure”, “premature ovarian insufficiency/POI”, “oxidative stress”, “fertility preservation”, “cancer”, “myeloperoxidase/MPO”, “glutathione/GSH”, “superoxide dismutase/SOD”, “reactive oxygen species/ROS”, and “antioxidants”. Retrieved articles were then screened, and only peer-reviewed articles written in English were considered. Additional hand searches of references from the retrieved literature were performed to ensure comprehensive coverage. One hundred thirty original articles discussing oxidative stress, loss of the ovarian reserve and depletion of oocyte quality, and premature ovarian insufficiency after treatment with cyclophosphamide and/or acrolein as well as fertility preservation methods and antioxidant therapies were included in this review.

Cyclophosphamide metabolites

Cyclophosphamide dosage is determined as the maximum amount tolerated, ranging from 1 to 3 mg/kg/day, and is available either orally in 25 and 50 mg tablets or administered intravenously. As a chemotherapeutic agent, dosage is typically 500–1200 mg/m² cumulatively every 21 days; however, dose reduction is necessary in patients with hepatic and/or renal dysfunction [8]. Previously, it was found that the metabolites of cyclophosphamide can be far more toxic than the drug itself [4, 13, 14, 33, 34]. In vivo, cyclophosphamide is metabolized in the liver by cytochrome P450 into acrolein- an α, β-unsaturated aldehyde- and phosphoramide mustard (Fig. 1). During metabolism, there is formation of the unstable equilibrated transient intermediates, 4-hydroxycyclophosphamide and its tautomer aldophosphamide that can freely diffuse into the cell and release acrolein and phosphoramide mustard [35]. Phosphoramide mustard undergoes nonenzymatic degradation into nor-nitrogen mustard that can alkylate DNA, produce alkyl radicals, and cross-link DNA thus inhibiting replication, altering mitochondrial transmembrane potential, and eventually lead to cell death [36]. This mechanism of action allows for the inhibition of cancer cell growth in actively proliferating cells.

Fig. 1.

Model of cyclophosphamide induced oxidative stress and antioxidant depletion. Cyclophosphamide is metabolized via cytochrome p450 (Cyc P450) into 4-hydroxycyclophosphamide in equilibrium with its ring-opened tautomer aldophosphamide which undergoes 𝛽-elimination to the final toxic metabolites acrolein and phosphoramide mustard (PM). PM generates nor-nitrogen mustard that can generate DNA alkylation, alkyl radicals, and DNA cross-link reactions. Acrolein conjugates with cellular glutathione (GSH) and depletes it through an alkylation reaction and deactivates superoxide dismutase (SOD). Acrolein also directly induces ROS production through mitochondrial damage exhausting the antioxidant defense and further inducing oxidative stress. Subsequent ROS accumulation can deplete nitric oxide (NO), which is essential for oocyte meiotic arrest and fertilization competency.

Conversely, acrolein is produced not only as a byproduct of cyclophosphamide but also through lipid peroxidation of polyunsaturated fatty acids, enzymatic oxidation of polyamine metabolites, and biotransformation of allyl alcohols. β-unsaturated aldehydes (alkenals) like acrolein are highly reactive with lysine, cysteine, and histidine through a Michael-type addition as well as with serine, histidine, arginine, threonine, and lysine residues [37, 38]. In vivo, acrolein can deplete intracellular glutathione (GSH) and copper/zinc superoxide dismutase (Cu/Zn-SOD) and be a large contributor of ROS through activation of the PI3K/Akt/mTOR pathway (a cell cycle regulating, intracellular signaling network) that increases ROS and induces mitochondrial membrane hyperpolarization [39–41]. The accumulation of ROS can lead to enzyme inactivation, DNA breaks and damage, and disruption of lipid peroxidation resulting in a large span of health problems.

Premature ovarian insufficiency

In addition to the obvious fertility challenges, chemotherapy-induced premature ovarian failure can result in a progressive decline of estrogen levels and side-effects including hot flashes, osteoporosis, sexual dysfunction, and cognitive decline [42]. Signs used to diagnose chemotherapy-induced ovarian failure include amenorrhea lasting more than 12 months and follicle stimulating hormone levels (FSH) of ≥ 30 MIU/mL with a negative pregnancy test [43].

Several theories dominate the mechanisms behind POI, namely: accelerated ovarian follicle maturation and direct quiescent follicle DNA damage. Cyclophosphamide has been shown to trigger apoptosis through a signaling axis that involves DNAPK/(ATM), CHK2, p53, and TAp63α [44] and apoptosis of mature follicles as well as up-regulation of the PI3K-PTEN-AKT pathway in the ovaries [45]. Alteration in these pathways may cause dysregulation in autophagy, a method for managing follicular development, primordial follicles, and follicular atresia [2]. Accordingly, the downstream effects of the altered PI3K-PTEN-AKT pathway in ovaries include the rise of gonadotropins and acceleration of premature ovarian follicle maturation that then are exposed to chemotherapy resulting in an accelerated loss of the primordial follicles [45]. Acrolein is highly water soluble and can permeate ovarian tissue through passive diffusion where it can directly induce irreversible damage to follicles and oocytes [46, 47]. Moreover, alkylating agents including chemotherapeutics can cause DNA damage in dormant oocytes through induction of cross-link formation, which may not be able to be repaired [48–50]. The accumulation of DNA strand breaks results in the activation of intracellular pro-apoptotic pathways that further contribute to the rate of apoptosis. It has also been suggested that in addition to direct follicular damage, chemotherapeutic agents have also been noted to disrupt vascularization. One study by Bar-Joseph et al. [51], found a 33% decrease in ovarian blood volume after doxorubicin injection; however, these results have not been reported with the use of other agents such as cyclophosphamide. Regardless of the pathway, cyclophosphamide-induced POI can be detrimental to the future fertility of cancer patients as shown in Fig. 2.

Fig. 2.

Summary of the pathways in which cyclophosphamide and its metabolites, phosphoramide mustard and acrolein, induce mitochondrial disruption and reactive oxygen species (ROS) production, oocyte damage, and alteration of ovarian function (Created in https://BioRender.com)

ROS in chemotherapy-induced ovarian damage

Under normal conditions, macrophages and other immune cells play important physiological roles in reproduction and can be found in the differentiated tissues of the reproductive system [52, 53]. Activated macrophages and neutrophils are key sources of ROS and MPO under inflammatory conditions, which are upregulated in cancer [54–56]. Generally, MPO and other mammalian peroxidases produce hypohalous acids (HOX; where X = Cl^-, F^-, I^-, and SCN^-) and protect against cellular destruction and tissue damage [57, 58]. MPO generates HOCl using H₂O₂ and Cl^- through a 2 e^– pathway which is the most effective way to kill invading pathogens and digest bacteria [58]. HOCl subsequently can directly oxidize reactive groups, including sulfhydryls, iron–sulfur centers, and hemes, or react with amines forming chloramines. The overproduction of ROS such as HOCl may lead to heme destruction and the generation of free iron. Free iron can be used in the Fenton reaction with H₂O₂ to generate ^•OH consequently contributing to further enhancement of oxidative stress, lipid peroxidation events, and ovarian damage [21, 59]. In a recent study, Zhang et al. suggest that local ROS-induced apoptosis of follicular granulosa cells likely impairs follicular function and ultimately diminishes oocyte quality following chemotherapy treatment [60]. This proposed mechanism of action is via mitochondrial dysfunction and increased O₂•⁻ production in granulosa cells, leading to ferroptosis, a form of programmed cell death characterized by lipid peroxidation and iron accumulation [60].

Chen et al. suggested that abnormal granulosa cell death was increased after cyclophosphamide treatment and found upregulation of heme-oxygenase 1, iron overload, disrupted ROS homeostasis, mitochondrial dysfunction, and a downregulation in glutathione peroxidase (GP) [61]. It was concluded that ROS play a key role in cyclophosphamide induced ferroptosis and is a likely mechanism in the subsequent ovarian damage and POI [61]. Acrolein has been suggested to induce mitochondrial dysfunction and has been shown to generate significant amounts of ROS at low concentrations [13, 14, 40, 47, 62, 63]. Previously, our lab and others have shown oocytes and ovarian granulosa cells exposed to acrolein have a significant decrease in their mitochondrial membrane potential with enhancement of caspase activation leading to apoptosis [14, 64, 65]. In the oocyte, the disruption of the mitochondria can induce the release of intracellular calcium and subsequently activate xanthine oxidase and produce O₂^•− that can react to produce several other ROS such as H₂O₂, singlet oxygen, ^•OH, and ONOO^-. In addition to impairment of ovarian function, as mentioned above mitochondrial disruption can lead to the release of calcium that may result in premature oocyte activation and subsequent oocyte aging phenomena, thus contributing to decreased oocyte quality and fertilization potential [14]. Consequently, the toxic effects of cyclophosphamide are likely due to acrolein that, when coupled with the already increased state of inflammation and oxidative stress in cancer, may exacerbate the oxidative state producing ovarian damage both on the level of the follicles and the oocytes. Furthermore, acrolein may serve as a disruptor to the protective antioxidant machinery essential for mitigating these toxic oxidative effects.

Disruption of the antioxidant defense

It has been found that in POI the activity of key antioxidant enzymes are significantly decreased [66]. Of note is cellular GSH, a nonenzymatic antioxidant that can foster detoxification of electrophilic xenobiotics, storage and transport of cysteine, and redox-regulated signal transduction as well as assist in cellular proliferation, minimizing lipid peroxidation of cellular membranes, deoxyribonucleotide synthesis, immune responses, and leukotriene and prostaglandin metabolism regulation [67]. As an antioxidant, GSH can either function as a substrate in the cytosolic GSH redox cycle or as a direct scavenger of ROS including O₂^•−, HOCl, H₂O₂, and ^•OH [68]. Acrolein has been shown to deplete cellular GSH [69], while HOCl has been shown to inhibit GP and effectively scavenge GSH through a rapid reaction with its thiols [70, 71]. In reproduction, GSH has several noted functions including spindle maintenance and other cell cycle events necessary to create a fertilizable oocyte, as concentrations have been shown highest after hormonally induced oocyte maturation and continued through ovulated, metaphase II oocytes [72]. GSH levels have been found to significantly decrease following cyclophosphamide treatment in the ovary [73] as well as in other organ systems [47, 69, 74, 75]; thus it is plausible that one mechanism in which acrolein activates apoptotic pathways and POI may be through disruption in both oxidant and antioxidant levels within the ovarian environment. An in vitro study found a decline in total intracellular GSH after cyclophosphamide treatment in a human granulosa cell line, with subsequent induction of apoptosis [76]. Furthermore, they reported cyclophosphamide treatment with an inhibitor of GSH synthesis increased apoptosis [76]. Accordingly, in cancer when MPO-HOCl production is already increased coupled with cyclophosphamide treatment, depletion of GSH via both HOCl and acrolein is likely and may result in substantial oxidative damage inducing both follicular apoptosis and oocyte deterioration (Fig. 1).

Acrolein has also been noted to covalently modify the lysine and histidine residues of Cu/Zn-SOD, resulting in loss of enzymatic activity. Cu/Zn-SOD facilitates the dismutation of O₂^•− to H₂O₂, and is found to be expressed highest in the theca interna cells in the antral follicles in humans where it is believed to be essential in steroidogenesis [77]. The advanced lipid peroxidation products generated from acrolein induced mitochondrial dysfunction can further deplete antioxidants such as SOD, GSH, and glutathione peroxidase impairing the antioxidant defense. In this state, key molecules essential for folliculogenesis, steroidogenesis, oocyte development and arrest, and ovulation are disturbed, contributing to infertility. One key molecule of note would be nitric oxide (NO), which we have previously shown to be essential for maintaining oocyte quality and has been noted in ovarian steroidogenesis and ovulation [27, 78–80]. It has been found that after administration of cyclophosphamide, cAMP decreases and NO is excessively produced reflecting the heightened inflammatory response [73]. It was also concluded that excess NO can influence granulosa cell apoptosis through inhibition of progesterone synthesis and alters estradiol secretion via inhibition of aromatase [81]. Under inflammatory conditions and in the presence of excess ROS generation, NO may be rapidly consumed in this pathway by both MPO as a one e^- substrate and by O₂^•−, which can accumulate with the loss of SOD activity [27, 78, 79, 82–84]. This consumption of NO not only depletes bioavailable NO from the follicular and oocyte microenvironment but results in the over-production of harmful nitration products such as NO₂^-, NO₃^-, and ONOO^- [27]. Moreover, ONOO^- and HOCl both possess the capability to oxidize nitric oxide synthase (NOS), the enzyme that produces NO, resulting in enzymatic uncoupling and further generation of O₂^•− [30, 85]. Consequently, it has been found that reducing the state of oxidative stress in the ovary induced by cyclophosphamide and its metabolites improves ovarian damage and associated parameters such as improving NO levels, reducing inflammatory markers, and increasing antioxidant activity [73, 81, 86]. Table 1 provides a comprehensive summary of studies aimed at improving the oxidative state and ovarian function altered by cyclophosphamide and their proposed mechanisms of action, organized by in vitro and in vivo animal data and human trials.

Table 1.

Methods for ROS reduction and/or improvement of antioxidant capacity in Cyclophosphamide-Induced ovarian damage

Data Type	Model or Sample	Reference	Treatment	Results	Proposed Mechanism
In vitro	Mouse oocytes	[111]	Alpha lipoic acid 100 µM	Reversal of metaphase II oocyte meiotic maturation failure	Suppression of ROS-mediated DNA damage and apoptosis
	Human Breast cancer cells (MCF-7 and T47D) and Ovarian Cancer Cells (OVCAR or COV434)	[112]	Alpha tocopherol (αToc) 100 µM or gamma tocopherol (γToc) 35.1 µM	γToc was not cytotoxic to the ovarian lines and reduced ROS and cytotoxicity in the COV434 but increased ROS and killed ~ 25% of both breast cancer cell lines	γToc may improve fertility by decreasing ovarian granulosa cell ROS and decreasing condensed nuclei while enhancing chemotherapeutic effects in breast cancer tumor cells due to difference in physiological redox status
In vivo	Rat	[104]	Melatonin 10 or 20 mg/kg	Preservation of hormone levels, follicular morphology and granulosa cell proliferation, upregulation in CYR6/CTGF expression, and reduced apoptosis	Melatonin inhibits LATS1, Mps1-One binder (MOB1), and YAP phosphorylation, thereby activating the Hippo signal pathway
	Mouse	[105]	Melatonin 50 mg/kg	Reduction of oxidative stress, inhibition of follicular apoptosis, Anti-apoptotic effects, AMH stabilization	Reduces over-activation of primordial follicles through inhibiting granulosa cell apoptosis and maintaining AMH
	Mouse	[106]	Melatonin 10, 20, or 30 mg/kg	Improved follicle morphology, decreased primordial follicle loss, reduced mitochondrial damage, and increased GSH levels	Action through the MT1 receptor and regulation of the PTEN/Akt/FOXO3a signaling pathway
	Rat	[113]	Melatonin and human mesenchymal stem cells	Combination melatonin + stem cell therapy showed the highest E2 and AMH values, lowest FSH value, highest level of recovery, and highest pregnancy rates.	Stem cell treatment including a strong antioxidant increases effectiveness of fertility treatment
	Rat	[114]	Combined melatonin (10 mg/kg/day) and vitamin D3 (60.000 IU)	Co-treatment preserved follicular function, restored hormonal balance, reduced stromal fibrosis, and attenuated apoptosis and inflammation markers	Involvement of multiple cellular pathways including apoptosis, oxidative stress, inflammation, fibrosis, and necroptosis
	Mouse	[115]	15 mg/kg pyrroloquinoline quinone (PQQ) and i.p. injections of 200 µg mitochondria derived from mesenchymal stem cells	Combined treatment restored ovarian function and antioxidant capacity and reduced follicular loss	In vitro experiments determined increased mitochondrial biogenesis via SIRT1 and PGC-1α and inhibited ATM/p53 pathways
	Mouse	[45]	Anti-Müllerian hormone (AMH); single dose of 5 mg/kg or 0.5 mg/kg once/week for 4 weeks	Recombinant AMH prevented primordial follicle loss through reduced primordial follicle recruitment. AMH improved ovulation rates without difference in litter numbers compared to cyclophosphamide only group	Inhibition of primordial follicle recruitment potentially through preventing PI3K signaling pathway and decreased phosphorylation of FOXO3A
	Rat	[116]	Glutathione 100 mg/kg or 200 mg/kg	High dose glutathione (200 mg/kg) improved all measures of follicle counts and serum AMH and decreased atretic follicle count	Preservation of antioxidant activity protects against oxidative stress mediated ovarian damage
	Rat	[117]	Capsaicin 0.5 mg/kg/day, quercetin 100 mg/kg/day, or combination	All three treatments restored ovarian function, hormonal levels, and serum total antioxidant capacity and improved follicle counts	Upregulation of BAX gene and decrease in apoptosis inducing genes (BCL-2 and P53)
	Mouse	[103]	Quercetin 12.5, 25, or 50 mg/kg or Coenzyme 10 1.25 mg/kg	Quercetin increased serum AMH/E2/progesterone, decreased FSH/LH, improved ovarian histology, reduced mitochondrial membrane potential, and upregulated expression of PGC1α, mitochondrial transcription factor A, and SOD	Protection against ovarian ROS damage through an anti-pyroptosis pathway
	Mouse	[118]	N-acetylcysteine (NAC) 150 mg/kg Or Epigallocatechin-3-gallate (EGCG) 5, 25, or 50 mg/kg	Pretreatment with NAC or EGCG (25/50 mg/kg) increased glutathione concentration, preserved follicular morphology, prevented follicle loss, and reduced atresia, inflammation and mitochondrial damage	EGCG regulates phosphorylated Akt, FOXO3a and rpS6 and reduces oxidative damage
	Rat	[119]	Vitamin E 200 mg/kg, NAC 200 mg/kg, or combination	The combination group showed the most significant differences in total antioxidant capacity (increase), MDA and serum proinflammatory markers (decrease), FSH/LH (decrease), estrogen (increase), and ovarian function (increase)	Replenishing of GSH, reduction of lipid peroxidation, scavenging free radicals, and restores oxidative balance
	Rat	[120]	Atorvastatin 10 mg/kg	Decrease in oxidative stress biomarkers and ROS, decreased caspace-3, increased cell viability, increased hormone levels, and mitigated inflammation and ovarian damage	Anti-apoptotic and anti-oxidative activity that inhibited lipid and protein peroxidation
	Rat	[73]	Cilostazol 10 mg/kg/day	Restoration of hormonal levels (FSH/LH/E2/AMH) and ovarian damage, and decrease in ovarian oxidative stress, inflammatory biomarkers, and caspase-3	Increased ovarian cAMP levels and upregulation of HO-1
In vivo and In vitro	Mouse	[121]	Humanin analogue S14G-Humanin (HNG)	Increased follicle development, oocyte quality, and litter size; reduction of ovarian tissue apoptosis and ROS; MMP restoration;	Upregulated PGC-1α expression and enhanced AMPK phosphorylation
	Rat	[122]	Stachydrine 20 or 40 mg/kg/d	Improved menses, serum sex hormones, reduced oxidative stress and apoptosis in granulosa cells.	In vitro treatment of granulosa-like cells with 1 µM following cyclophosphamide activated Nrf2/HO-1 pathway and inhibited oxidative stress and apoptosis.
	Mouse	[123]	Umbilical cord-derived mesenchymal stem cells	Suppression of cyclophosphamide induced ferroptosis in granulosa cells	Activation of antioxidant pathway via NRF2
Clinical Trial	40 Untreated stage II breast cancer patients	[124]	Vitamin C 500 mg tablet and vitamin E 400 mg gelatin capsule	Increased levels of GSH and antioxidant enzymes (SOD, catalase, glutathione reductase), and reduced MDA and DNA damage compared to chemotherapy alone or pretreatment levels	Restoration of antioxidant status that is lowered by both cancer and chemotherapy

Protection of ovarian function

Currently, fertility preservation efforts before treatment with cyclophosphamide, including oocyte, embryo, and ovarian cortex cryopreservation, have aided in those achieving pregnancy post-treatment [87–89]. Yet, even in the presence of an effective therapeutic option for preservation of the ovarian reserve, the cost of banking, access to fertility care providers, and treatment urgency are substantial burdens affecting this method. Several studies have investigated the use of stem cell therapies following cyclophosphamide treatment, noting improvement in mitochondrial damage, granulosa cell apoptosis rate, vascular formation, genetic stability, hormone levels, and ovarian function (including increased ovarian reserve, improved folliculogenesis, and reduced follicle atresia) [90–93]. While there is promising potential as a treatment for POI, data is still limited to animal studies and the mechanistic actions will need to be further evaluated to ensure safety and quality in clinical applications.

The use of gonadotropin releasing hormone (GnRH) analogues is one effort that has been given considerable attention and has been shown in clinical trials to reduce POI development in breast cancer patients [94, 95]; however, several other trials using this method have been conducted with no definitive outcome regarding pregnancy, POI, and subsequent consequences surrounding early menopause [96–98]. A summary of clinical trials evaluating the use of GnRH analogues with cyclophosphamide treatment from the last 15 years can be found in Table 2. The development of efficient and targeted pharmacological therapies that could protect and prolong female fertility creates the need for research aimed at understanding the mechanisms underlying the action of chemotherapy compounds on the various components of the ovary. Consequently, the use of antioxidants may provide a therapeutic approach to aid in preserving ovarian quality during treatment.

Table 2.

Clinical trials on GnRH treatments for ovarian damage induced by cyclophosphamide-based chemotherapy

Study Design and Reference	Sample	Intervention	Dosage regimen	Finding
Randomized Clinical Trial [125]	330 premenopausal women with operable stage I to III breast cancer receiving cyclophosphamide-containing chemotherapy treatment	Additional treatment with or without GnRH analog (GnRHa)	3.6 mg goserelin or 3.75 mg leuprorelin, subcutaneous injection once every 28 days 2 weeks prior to first cycle of chemotherapy through 4 weeks after last cycle of chemotherapy.	POI rate 12 months after treatment completion was 10.3% (15 of 146) in the GnRHa group and 44.5% (69 of 155) in the control group.
Long-Term Follow up of Prospective Randomized Trial [126]	129 female patients 18–45 years old treated for Hodgkin or non-Hodgkin lymphoma with alkylating agents. Final data included 67 patients.	Additional treatment with GnRH agonist (GnRHa) or control during all chemotherapy, random assignment	GnRHa group: Intramuscular injection (IM) of 11.25 mg of triptorelin every 12 weeks and 5 mg norethisterone daily Control: 5 mg of norethisterone acetate alone once per day	Premature ovarian failure at 2 year follow up: 19.4% (6/31) GnRHa group and 25% (8/32) control group Pregnancy achievement 2, 3, 4, and 5–7 year follow up: 53.1% GnRHa group and 42.8% control group. AMH/FSH levels similar in both groups.
Phase II Randomized trial [127]	220 premenopausal patients with breast cancer who received Cyclophosphamide-doxorubicin-based chemotherapy prior to randomization	Cyclophosphamide-doxorubicin-based chemotherapy only (chemotherapy group) or chemotherapy plus GnRH analogue (GnRHa Group)	GnRHa group: 3.75 mg subcutaneous injection of leuprolide acetate 2 weeks before chemotherapy with confirmation of ovarian suppression, then dosage every 4 weeks during chemotherapy treatment.	Menses resumption: 27 patients in chemotherapy group and 15 in GnRHa group. Restoration of premenopausal FSH/E2 levels: 7 patients in chemotherapy group and 14 in GnRHa Early menopause: 28.7% in chemotherapy group and 16.9% in GnRHa group
Randomized Control Trial [128]	100 hormone-insensitive breast cancer participants 18–40 years old	Group 1: Chemotherapy given alone (control) or with GnRH antagonist and agonist cotreatment Group 2: Chemotherapy alone (control) or with GnRH agonist	Group 1: GnRH antagonist Cetrotide, cetrorelix 0.25 mg and GnRH agonist Decapeptyl CR, triptorelin 3.75 mg given daily until confirmed ovarian suppression then GnRH agonist only every 4 weeks until end of chemotherapy. Group 2: GnRH agonist Decapeptyl CR, triptorelin 3.75 mg given daily until confirmed ovarian suppression then every 4 weeks until end of chemotherapy	No difference in menstruation resumption or hormonal/ultrasound markers between GnRH treated and controls in either group
Prospective Multicenter Randomized Control Trial [129]	60 patients > 46 years old with hormone-insensitive breast cancer	Cyclophosphamide treatment with or without GnRH agonist	Subcutaneous injection of 3.6 mg goserelin 2 weeks before chemotherapy and every 4 weeks until end of chemotherapy.	No significant differences in temporary amenorrhea or menstruation resumption in controls vs. GnRH agonist groups.
Randomized Control Trial [130]	285 premenopausal breast cancer patients treated with cyclophosphamide-based chemotherapy	Chemotherapy with or without (control) endocrine therapy	3.6 mg subcutaneous GnRH agonist (goserelin) alone every 28 days, 40 mg/day orally tamoxifen alone, or goserelin + tamoxifen	Menstruation resumption 1 year after treatment conclusion: 36% goserelin group, 7% goserelin + tamoxifen, 13% tamoxifen alone, 10% control

2-mercaptoethane sodium sulfonate (mesna) has been suggested to prevent hemorrhagic cystitis in patients undergoing chemotherapy treatment [99, 100] and has also been suggested, when used with cisplatin, to prevent the loss of AMH-positive follicles and increase the activity of antioxidants such as GSH. Importantly, treatment with both cyclophosphamide and mesna or acrolein and mesna can cause quality deterioration in metaphase II mouse oocytes in a concentration dependent manner through microtubule and chromosomal disruption thus making it a potentially unsuitable method for the preservation of fertility in chemotherapy treatment [101, 102]. Chen et al. using a murine model, showed that administration of quercetin, a natural flavonoid that exhibits antioxidant activity, reverses mitochondrial dysfunction, activates mitochondrial biogenesis through the PGC1-α pathway, and downregulates pyroptosis (a pro-inflammatory form of programmed cell death) thereby protecting ovarian function following cyclophosphamide induced POI [103]. Other antioxidants, such as melatonin, have been shown to reduce the adverse effects of chemotherapeutic drugs through reduction in ROS (Table 1). Studies have shown melatonin receptors are present in the oocyte and granulosa cells and administration during cyclophosphamide treatment improved sex hormone levels, follicular morphology, and granulosa cell proliferation and reduced apoptosis in rats [104]. Feng et al. found treatment with 50 mg/kg melatonin maintained the plasma AMH which significantly prevented over-activation of primordial follicles and prevented the mitochondrial apoptotic pathway and subsequent granulosa cell loss in cyclophosphamide treated mice [105]. Another study found pre-treatment with 20 mg/kg of melatonin improved follicular morphology, primordial follicle loss, and mitochondrial damage and increased GSH [106]. As melatonin is both an inhibitor of MPO and a potent scavenger of HOCl [107, 108], administration of melatonin may protect GSH through depletion of MPO-HOCl production.

Conclusion

Acceleration of ovarian aging from follicular damage and deterioration of oocyte quality during cancer treatment poses a significant burden on individuals of childbearing age. The toxic metabolites of cyclophosphamide induce apoptotic pathways and irreversible damage to ovarian tissue generating overwhelming oxidative stress. The direct depletion of antioxidants, like GSH and SOD, exacerbates the state of oxidative stress through minimizing the body’s natural defenses. The direct scavenging of acrolein combined with heightened HOCl production can reduce the bioavailability of GSH in the ovarian environment. Consequently, the use of antioxidant therapy may be a promising option to mitigate the oxidative damage the ovary incurs during treatment thus preventing POI through preservation of ovarian function and oocyte quality. Research on therapies that focus on protection of the ovarian microenvironment such as stem cell therapy, free radical scavenging and antioxidant therapy, immunomodulation, senolytherapies (the use of senolytic drugs that selectively kill senescent cells [109]), and proangiogenic factors provide a more broad and less invasive approach than current methods of cryopreservation and, with more clinical data, may have potential therapeutic applications [110]. Although the current research relies mainly on animal models providing significant limitations on human clinical application, these studies provide essential groundwork to inspire future directions investigating cyclophosphamide-induced POI. Further studies should include a focus on the mechanisms of oxidative stress in the ovarian microenvironment arising from cyclophosphamide therapy and treatments aimed at mitigating the downstream effects leading to oocyte damage and POI.

Abbreviations

POI

Premature ovarian insufficiency

ROS

Reactive oxygen species

MPO

Myeloperoxidase

O₂^•−

Superoxide

H₂O₂

Hydrogen peroxide

^•OH

Hydroxyl radical

ONOO^–

Peroxynitrite

HOCl

Hypochlorous acid

GSH

Glutathione

SOD

Superoxide dismutase

FSH

Follicle stimulating hormone

GP

Glutathione peroxidase

NO

Nitric oxide

NOS

Nitric oxide synthase

Authors’ contributions

O.C. wrote the original draft and H.A.S, M.B., and O.C. prepared Figs. 1 and 2. C.C., J.K., and O.C. prepared Table 1, and 2. All authors reviewed and approved of the manuscript.

Funding

N/A.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

N/A.

Consent for publication

N/A.

Competing interests

The authors declare no competing interests.