Construction of an Adverse Outcome Pathway Framework for Glyphosate-Induced Female Reproductive Toxicity Based on Toxicity Pathways

Premature ovarian insufficiency (POI) is characterized by insufficient ovarian sex hormone production and a reduced ovarian reserve, leading to a rapid decline in ovarian function and early menopause in women. Findings from mammalian studies on GLY-induced reproductive toxicity are consistent with POI. Ren et al. observed significant changes in ovarian morphology and hormone levels in mice exposed to 5 g/L GLY in drinking water, consistent with POI pathology. Specifically, they reported an increase in atretic follicles, a decrease in mature follicles, and ovarian stromal fibrosis. Molecular analyses revealed upregulated LHR expression and downregulated FSHR expression in ovarian tissue, potentially resulting in a pathological feedback loop that exacerbated ovarian dysfunction. Further supporting this association, Novbatova et al. demonstrated that chronic low-dose GLY exposure (2 mg/kg/day for 10 weeks beginning at 7 weeks of age) significantly reduced the population of secondary follicles in mouse. This selective follicular depletion was associated with a decline in pregnancy success rates from 75% to 55%, highlighting the detrimental impact of GLY on reproductive outcomes. Uterine (endometrial) adenocarcinoma (UA), the most prevalent form of uterine cancer, originates from the epithelial cells lining the inner layer of the uterus (endometrium) and is classified as type I (estrogen-dependent) or type II (nonestrogen-dependent) UA. Type I UA, the most common subtype, has been implicated in studies of GLY-induced female reproductive toxicity. Guerrero et al. reported that exposure to GBHs (2 mg of GLY/kg/day) during the developmentally sensitive postnatal period [postnatal day (PND) 1–7] induced multiple uterine lesions in aged rats (PND 600). The observed pathologies comprised adenomyosis, formation of estrogen-induced precancerous daughter glands, atypical endometrial hyperplasia (a UA precursor), and uterine leiomyomas. As discussed in Section , GLY may trigger endometrial epithelial proliferation through hormonal imbalance and uterine structural changes mediated by estrogen signaling pathways, leading to estrogen-dependent precancerous lesions such as adenomyosis and endometrial atypical hyperplasia. , Similarly, GLY-induced ERα upregulation was accompanied by depletion of tumor suppressors ( PTEN and p27 ) in hyperplastic endometrium - hallmarking endometrial cancer initiation. Furthermore, Gastiazoro et al. documented the progression from benign hyperplasia to atypical endometrial hyperplasia and adenomyosis, highlighting the potential of GBHs to induce EMT in endometrial carcinoma cells, characterized by E-cadherin suppression and enhanced migratory capacity. Notably, AOP 503 in the AOP-wiki describes a complete pathway in which ERα activation promotes epigenetic modification of proliferative factors, ultimately leading to endometrial cancer. Given that GLY activates ERα and induces epigenetic processes associated with similar AOs, further research is needed to determine whether GLY contributes to endometrial cancer through the direct epigenetic modification of proliferative factors. Impaired ovarian function and reproductive system diseases, such as endometrial cancer, significantly reduce the likelihood of successful pregnancy and severely compromise fertility. , , , , As previously discussed, exposure to GLY or GBHs can lead to outcomes such as POI and UA, thereby adversely affecting reproductive capacity. Ingaramo et al. found that the number of resorption sites was significantly increased on GD 19, after the newborn female rats were exposed to 2 mg/kg/day GBH on PND1, 3, 5, and 7. Lorenz et al. reported that exposure to GBHs (350 mg GLY/kg bw/day) increased the rate of implantation failure in F1 generation rats during early pregnancy. Furthermore, Milesi et al. observed that perinatal exposure to GBHs resulted in a higher rate of embryo implantation failure in rats. F2 generation offspring in the GBH-exposed group exhibited reduced body weight and body length, while a high-dose GBH (200 mg of GLY/kg of bw/day) exposure group led to structural congenital malformations in F2 fetuses, potentially causing severe long-term health consequences.

Reactive oxygen species (ROS), including superoxide anion radical (O 2–• ), hydrogen peroxide (H 2 O 2 ), hydroxyl radical (OH • ), and singlet oxygen, are crucial regulators of cell energy metabolism and proliferation. While physiologically essential, ROS imbalance, particularly excessive accumulation, can induce cellular dysfunction and irreversible damage. Both exogenous stressors and normal endogenous cellular processes, notably mitochondrial ETC activity, contribute to ROS generation. Evidence indicates that GLY induced ROS overproduction through ETC impairment and mitochondrial dysfunction, resulting in DNA damage and cell cycle arrest in MCF-7 (a hormone-dependent human breast cancer cell line) and MDA-MB-468 (a hormone-independent human breast cancer cell line). Studies demonstrate that GLY chelates intracellular zinc in mice oocytes, compromising antioxidant defenses and exacerbating mitochondrial dysfunction, which dose-dependently elevates ROS levels. , These alterations impair oocyte competence and postfertilization embryo development. In addition, GLY exposure increases uterine ROS levels in piglets, alters uterine and ovarian tissue morphology and ultrastructure, and disrupts hormone balance. Notably, even at concentrations below agricultural application levels (0.9 ppm Roundup, containing 5.33 μM GLY), GBH induced apoptosis via ROS-mediated pathways, compromising early bovine embryonic development. Oxidative stress, characterized by an imbalance between ROS production and antioxidant defense capacity, involves key enzymes including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione (GSH), which cooperatively maintain the reduction–oxidation homeostasis of cells. These enzymatic systems, crucial for cellular protection against oxidative damage, can serve as biomarkers of oxidative stress induced by exogenous substances. GLY exposure increased ROS levels, eliciting compensatory SOD/CAT activation in mouse ovaries, , piglets uterus, and oocytes of mice and cattle. , , Prolonged exogenous oxidative effects can induce mitochondrial ROS accumulation beyond cellular clearance capacity, leading to cumulative oxidative damage that impairs mitochondrial function, reduces antioxidant system efficiency, and exacerbates cellular oxidative stress. Treated with high-dose GLY (105 μg/kg bw GLY) for 28 days impaired antioxidant function of rat’s ovaries, by suppressing ovarian CAT and SOD activity and downregulated glutathione reductase (Gsr) gene expression in GLY-exposed female rats, concomitant with endocrine disruption and impaired folliculogenesis. Similar dose-dependent decreases in CAT, SOD, GPx, and GSH levels, accompanied by apoptosis and steroidogenesis dysregulation, were observed in rat and bovine ovaries following GLY exposure. GBH-induced antioxidant depletion exacerbates oxidative and endoplasmic reticulum stress, mitochondrial dysfunction, and apoptosis in placental cells, leading to developmental abnormalities in fetal mice and piglets. In vitro studies corroborated these findings, demonstrating that GLY exposure during porcine oocytes meiosis elevated intracellular ROS levels while disrupting mitochondrial dynamics and calcium homeostasis, the expression of antioxidant-related genes ( SOD1 , GPx , CAT ) was observably suppressed, suggesting oxidative stress in mitochondria and cytoplasm, ultimately compromising oocyte quality. Taken together, GLY-induced female reproductive toxicity primarily involves ROS overproduction, oxidative stress, and apoptotic pathway activation, manifesting as ovarian dysfunction, embryonic developmental abnormalities, and hormonal dysregulation, which are the most frequently observed key molecular events in GLY-induced toxicity. DNA damage, including nucleotide modifications, single-strand breaks (SSBs), and double-strand breaks (DSBs), occurs during cellular processes and can be directly induced by endogenous or exogenous stressors (e.g., ROS, chemical agents, and ionizing radiation). , GLY-mediated female reproductive toxicity may involve two DNA damage mechanisms, including direct interference with DNA-associated proteins and repair machinery and/or ROS-mediated indirect genotoxicity. Experimental evidence from ER-dependent genotoxicity studies revealed that HEC1A cells (ER-positive endometrial cancer cell line) showed increased sensitivity, characterized by decreased cell viability, elevated DNA fragmentation, mitochondrial depolarization, and early apoptosis. However, MDA-MB-231 cells (ER-insensitive breast cancer cell line) exhibited less toxicity but still detectable DNA damage. , Mechanistic studies indicated that GBHs may dysregulate DNA damage repair pathways, particularly the base excision repair (BER) system, through downregulation of key genes (e.g., OGG1 , XRCC1 ). This suppression may occur through altered oxygen consumption, ROS elevation, and subsequent hypoxia which may promote DNA damage, G1/S phase arrest, and subsequent apoptosis. In mouse oocytes, GLY-induced ROS overproduction was associated with oxidative DNA damage (particularly DSBs), impairing maturation rates and developmental potential while triggering autophagy and premature apoptosis. Lipid peroxidation is a process that involves the oxidative degradation of membrane lipids, compromising the structural integrity of the cellular and organelle membranes. This pathological process is primarily driven by ROS, which target the unsaturated carbon–carbon bonds within fatty acids that constitute lipids. Lipid peroxidation may disrupt cellular homeostasis ultimately leading to membrane destabilization and dysfunction. Accumulating evidence suggests that GLY and GBHs induced female reproductive toxicity via oxidative stress-induced lipid peroxidation. Studies described in section 4.4.1 demonstrated that GLY-induced oxidative stress, manifested by elevated ROS levels, was invariably accompanied by lipid peroxidation in ovarian and uterine tissues. This was evidenced by increased malondialdehyde (MDA) levels. , , Lipid peroxidation may impair membrane protein function and increase membrane permeability, leading to mitochondrial swelling and the collapse of membrane potential. These effects may compromise organelle membrane integrity, potentially causing nuclear and mitochondrial membrane destabilization in porcine uterine cells, structural disorganization of mitochondrial endoplasmic reticulum in porcine oocytes, and endoplasmic reticulum stress in mouse placenta following exposure to GLY and/or GBH. , The consequent structural damage may activate apoptotic pathways via Bax/Bcl-2 imbalance and caspase-3/9 activation, ultimately causing ovarian follicular atresia and placental hypoplasia. , These findings indicate that GLY exposure may cause female reproductive toxicity through oxidative stress-mediated lipid peroxidation, which disrupts cellular homeostasis and triggers apoptotic cascades. This pathological process is characterized by upstream redox imbalance, downstream organelle dysfunction, and terminal ovarian failure and developmental abnormalities. The endometrium serves as a primary target tissue for estrogen, orchestrating critical physiological functions in embryo implantation and maintenance through tightly regulated hormonal signaling. Estrogen exerts its reproductive effects predominantly via two classical nuclear receptors, ERα and estrogen receptor β (ERβ), which coordinate cyclical endometrial proliferation and differentiation. Aberrant ER expression patterns in the endometrium are associated with various pathological conditions including endometriosis, endometrial hyperplasia, and endometrial cancer. Mechanistic studies have demonstrated that dysregulation of ER subtype stoichiometry (ERα/ERβ ratio) and spatiotemporal expression dynamics may contribute to several pathogenic processes, including uncontrolled cellular proliferation, impaired apoptotic regulation, and compromised decidualization, all of which represent characteristic features of endometrial pathophysiology. In vitro studies using a human endometrial adenocarcinoma cell line (Ishikawa) demonstrated that GBH enhanced cell migration and invasion, concomitant with the suppression of epithelial-mesenchymal transition (EMT) markers such as E-cadherin mRNA. These pro-metastatic effects were fully blocked by the ER antagonist fulvestrant, suggesting that GBH may promote malignant transformation and metastasis of endometrial cancer via ER-dependent mechanisms. In vivo developmental exposure to GBH disrupted uterine homeostasis, inducing endometrial hyperplasia in juvenile rats and predisposing adult rats to subsequent uterine pathologies, such as adenomyosis. These effects correlated with aberrant Hoxa10 epigenetic silencing and sustained estrogen dominance via elevated E2/P4 ratios. Parallel studies showed that perinatal GBH exposure dysregulated uterine ER isoform dynamics, downregulating epithelial ERα while upregulating stromal ERβ and progesterone receptor. Receptor imbalance is associated with endometriosis and endometrial carcinogenesis. In addition, studies suggest that GLY and/or GBHs upregulated ERα gene expression epigenetically through hypomethylation and histone modification shifts of the ERα promoter. ERα overexpression may contribute to embryo implantation failures. These findings implicate GLY/GBH as ER-modulating endocrine disruptors, driving female reproductive toxicity through EMT activation, epigenetic dysregulation of developmental genes, and hormonal imbalance, ultimately promoting uterine dysfunction and carcinogenic progression. Mitochondrial dysfunction is a central mechanism in GLY-induced female reproductive toxicity, primarily driven by oxidative stress and impaired ETC activity. As described in Sections and 4.1.1 , GLY directly inhibits ETC complexes II and III, leading to excessive ROS production and oxidative stress. Mitochondria are both the primary source of ROS within cells and the most vulnerable organelle to ROS-induced damage. Excessive ROS production disrupts mitochondrial function through multiple pathways, including oxidizing iron–sulfur clusters and heme groups within ETC complexes I, III, and IV, leading to impaired electron transport and reduction of ATP synthesis. Increased electron leakage may further contribute to a regenerative cycle known as ″ROS-induced ROS release″. ROS also directly affect mitochondrial DNA (mtDNA), causing base deletion, strand breaks, and mutations. These changes may affect the expression of coding genes of the mitochondrial respiratory chain complex, further disrupting oxidative phosphorylation. The inner mitochondria membrane, rich in unsaturated fatty acids, is particularly susceptible to ROS-induced lipid peroxidation, which compromises the membrane integrity. In addition, lipid peroxidation products can activate the mitochondrial permeability transition pore (mPTP), triggering the release of Cytochrome c and other pro-apoptotic factors, which initiate apoptosis. , Substantial evidence supports the role of mitochondrial dysfunction in GLY-induced female reproductive toxicity. For instance, studies on GLY-exposed porcine oocytes observed elevated ROS, reduced mitochondrial DNA copy numbers, and extensive mitochondrial damage that is evidenced by reduced distribution of mitochondria in the oocyte cortex, decreased copy number of mtDNA, and downregulated expression of PGC1α and ATP5B genes. These changes were associated with impaired meiotic progression, decreased oocyte maturation rates, and reduced oocyte quality. Similarly, in bovine and mice oocytes, GLY exposure led to abnormal intracellular ROS accumulation, decreased mitochondrial membrane potential, and upregulation of apoptosis-related genes ( Caspase-3/Caspase-4, BAX ), initiating early apoptosis and autophagy. , , In vivo murine studies have shown that GLY-induced oxidative stress and mitochondrial dysfunction are associated with multiple pathological effects, including ovarian cell apoptosis, reduced ATP production, endometrial glandular atrophy, follicular atresia, as well as dyshomeostasis of thyroid hormone and hypothalamic-pituitary-ovarian (HPO) axis hormones. , Furthermore, GBH-induced mitochondrial dysfunction in porcine placental tissues was linked to impaired vascular formation, barrier integrity, and nutrient transport, potentially affecting neonatal development. In summary, mitochondrial dysfunction may play a critical role in GLY-induced female reproductive toxicity, linking upstream oxidative stress and ETC impairment to downstream pathological outcomes, such as apoptosis, oocyte maturation defects, and developmental abnormalities. Epigenetic modifications, defined as heritable changes in gene expression without alterations to the underlying DNA sequence, include DNA methylation, histone modification, nucleosome assembly/remodeling, and noncoding RNA-mediated regulation. Among these, DNA methylation and histone modifications may be central mechanisms in GLY-induced female reproductive toxicity. DNA methylation involves the covalent addition of methyl groups to cytosine residues within CpG dinucleotides and is catalyzed by DNA methyltransferases (DNMTs). Modifications predominantly occur in gene promoter regions, where hypermethylation typically suppresses transcription. , Emerging evidence indicates that GLY and GBHs may disrupt female reproductive function through epigenetic reprogramming, particularly via DNA methylation and histone modification. Alterations of this nature can impair key gene networks essential for endometrial receptivity and uterine development, ultimately leading to adverse outcomes, such as embryo implantation failure and estrogen-dependent disorders. Lorenz et al. showed that gestational and lactational exposure to GLY at a dose of 2 mg/kg bw/day significantly upregulated DNA methyltransferase DNMT3a in rats. This upregulation was associated with hypermethylation at CpG islands within the promoter and regulatory regions of Hoxa10 , a critical regulator of endometrial receptivity. Hypermethylation correlated with significant downregulation of Hoxa10 mRNA, and impaired embryo implantation. Additionally, 2 mg/kg/day GLY exposure in rats induced the same aberrant epigenetic modifications in the Hoxa10 gene, resulting in hyperplasia of the endometrium and myometrium. Similarly, Almiron et al. observed hypermethylation at a CpG island in the Lif promoter in rats following exposure to either 3.8 mg GLY/kg/day GBH or 3.9 mg GLY/kg/day pure GLY, which resulted in a 60% reduction in Lif mRNA levels. Lif encodes a cytokine essential for embryo-uterine crosstalk, and its suppression compromises endometrial receptivity. Furthermore, a recent study showed that prenatal GBH exposure downregulated the mRNA levels of Dnmt1 and Dnmt3b genes in the jejunum of offspring piglets, which was associated with diminished DNA methylation, which may impair intestinal development and barrier function in newborn piglets. Histones, particularly the H3 variant, play a pivotal role in epigenetic regulation by organizing DNA into nucleosomes and modulating chromatin accessibility. PTMs of histone tails, such as methylation, acetylation, and phosphorylation, regulate chromatin compaction states and transcriptional activity. For example, methylation of histone H3 at lysine 4 (H3K4me1/me2/me3), lysine 36 (H3K36me), or lysine 79 (H3K79me) is associated with open chromatin and transcriptional activation. In contrast, di- or trimethylation of H3K9 (H3K9me2/me3) and H3K27 (H3K27me2/me3) promotes heterochromatin formation and gene silencing. The functional consequences of histone methylation depend on both the specific lysine residue modified and the degree of methylation (mono- vs polymethylation). These modifications are dynamically regulated by histone methyltransferases (HMTs) and demethylases (HDMs), maintaining a balance critical for epigenetic homeostasis. GLY/GBHs elevated repressive histone modifications, such as H3K27me3 (catalyzed by EZH2) and H3K9me3, in the Hoxa10 and Lif regulatory regions. These modifications alter chromatin accessibility, further silencing genes critical for implantation. , Lorenz et al. demonstrated that GBH reduced DNA methylation at the ERα-O promoter and increased activating histone marks (e.g., H4Ac↑, H3K27me3↓), leading to a 2.5-fold increase in ERα expression. ERα hyperactivation can cause uterine hyperplasia and estrogen-dependent pathologies, including adenomyosis and atypical endometrial hyperplasia. Integrating these findings, MIEs may involve binding of GLY/GBH to ERα or antioxidant enzymes, activating DNMTs/EZH2, and driving DNA and histone hypermethylation. Alternatively, exposure may cause oxidative stress, disrupting the balance between histone acetyltransferase (HAT) and histone deacetylase (HDAC). These KEs may perturb gene networks ( Hoxa10 ↓ , Lif ↓ , ER α↑), culminating in AOs at both tissue (e.g., impaired implantation) and organismal (e.g., infertility, neoplasia) levels. The spindle apparatus, a dynamic cytoskeletal structure essential for eukaryotic cell division, plays a vital role in mitosis and meiosis by ensuring precise chromosome separation. In mammalian oocytes, which lack centrioles and centrosomes, meiotic spindle formation assembly depends on microtubule-organizing centers (MTOCs) that substitute for conventional centrosomes. Proper spindle assembly and organization are critical to normal chromosome dynamics, including the accurate alignment of chromosomes during metaphase and their segregation into daughter cells during anaphase. Maintaining normal spindle structure and function is indispensable for ensuring the fidelity of chromosome segregation during meiosis, particularly during the phases of spindle assembly and chromosome alignment. Evidence suggests that exposure to GLY and GBHs induces female reproductive toxicity by causing spindle abnormalities and chromosomal dysregulation during oocyte meiosis. GLY/GBHs trigger intracellular oxidative stress and redox imbalance, potentially initiating a cascade of cellular perturbations, including the suppression of p-MAPK expression, which plays a critical role in regulating microtubule dynamics and spindle assembly. , , Mouse oocytes exposed to 50–300 μM GLY exhibited profound cytoskeletal disorganization characterized by shortened or disrupted spindle fibers, abnormal MTOC formation, and chaotic chromosomal alignment. The concentration-dependent degradation of spindle structure suggests cumulative damage to microtubule polymerization processes, which directly impairs meiotic progression. This is evidenced by reduced polar body extrusion rates and metaphase II arrest, indicating compromised chromosomal segregation fidelity. In addition, DNA double-strand breaks, caused by other MIEs such as oxidative stress, likely exacerbate chromosomal instability during forced segregation attempts. Mitochondrial dysfunction, another consequence of oxidative stress, further diminishes oocyte quality by depleting ATP reserves essential for spindle checkpoint surveillance and apoptosis regulation. The downstream developmental consequences are severe and diverse. Impaired cytoplasmic maturation in GLY/GBH-exposed oocytes resulted in reduced fertilization competence and compromised embryonic potential, as demonstrated by reduced 2-cell embryo formation. , In vivo studies confirmed these findings, with GLY/GBH-exposed mice showing parallel declines in oocyte maturation rates and early embryonic developmental capacity. Collectively, these findings outline a pathogenic pathway in which oxidative stress-induced MAPK suppression disrupts spindle morphogenesis, leading to chromosomal missegregation, meiotic failure, and ultimately reduced reproductive success. In the studies of GLY/GBHs-induced female reproductive toxicity, cell death represents an end point of cytotoxic effects at the cellular level. Evidence from both in vivo and in vitro studies indicates that GLY or GBH exposure reduced the cellular viability in several female reproductive cell types, including oocytes, granulosa cells, placental cells, embryonic cells, and endometrial carcinoma cells. Notably, the specific modes of cell death vary significantly depending on exposure conditions and cell type. Cell death mechanisms are broadly classified into programmed cell death (PCD) and nonprogrammed cell death (NPCD). Unlike uncontrolled necrotic processes, which typically induce inflammatory responses, PCD involves genetically regulated, self-executing pathways for active cell termination. The predominant PCD modalities associated with GLY toxicity include apoptosis and autophagy. Activation of apoptosis through the intrinsic mitochondrial pathway is the most commonly reported cytotoxic mechanism of GLY exposure. GLY and GBHs dose-dependently elevated intracellular ROS levels and subsequently upregulate pro-apoptotic markers (Bax, caspase-3/9) while suppressing antiapoptotic Bcl-2 expression. , , The critical shift in the Bax/Bcl-2 ratio toward the initiation of apoptosis resulted in collapse of the mitochondrial membrane potential, release of Cytc, and activation of the apoptotic cascade. Characteristic apoptotic markers, including annexin-V and elevated caspase-3/9 expression, have been observed in GLY-exposed oocytes and embryonic cells. , In addition to apoptotic activation, GLY exposure induced autophagic responses, potentially as a cytoprotective adaptation to cellular stress. This compensatory mechanism is supported by upregulation of autophagy-related genes ( LC3, Beclin-1, ATG12 ) and corresponding protein markers in reproductive tissues, suggesting enhanced removal of damaged cellular components. , , However, prolonged oxidative stress can turn this adaptive mechanism into a maladaptive response, exacerbating mitochondrial dysfunction and contributing to reproductive impairment through excessive autophagic activity. High-dose or prolonged GLY exposure promotes necrotic cell death progression. Subchronic exposure to GLY at a high dose of 315 mg/kg has been shown to induce characteristic necrotic alterations in rats, including oocyte nuclear pyknosis. In vitro , the cytotoxic effects of GBHs on three human cell types (HUVECs, HEK293, and JEG-3) revealed that GBH exposure caused cell membrane damage within 24 h, as evidenced by the release of cytosolic adenylate kinase (AK), a marker of cell membrane rupture associated with late-stage necrosis. Overall, cell injury and death induced by GLY/GBHs through apoptosis, autophagy, and necrosis may represent key mechanisms underlying GLY-induced female reproductive toxicity. These molecular injuries can lead to functional impairments in reproductive tissues, with ovaries exhibiting follicular atresia, granulosa cell necrosis, and oocyte vacuolization, while placental dysfunction may result in growth-restricted fetuses. , The disruption of hormonal homeostasis may also contribute to GLY-induced female reproductive toxicity, serving as both a consequence and an amplifier of cellular dysfunction across multiple regulatory levels. GLY and GBHs can impair steroidogenesis through oxidative stress-mediated pathways and/or the inhibition of aromatase, directly targeting ovarian granulosa cellsthe primary site of 17β-estradiol (E2) and progesterone (P4) synthesis. Studies across species, including porcine, bovine, and murine models, have consistently demonstrated GLY’s capacity to suppress granulosa cell proliferation and metabolic activity while disrupting steroidogenic enzyme function. For example, in porcine granulosa cells, 0.2–16 μg/mL GLY significantly reduced E2 secretion while paradoxically increasing P4 levels, indicating dysregulation of steroidogenic enzyme cascades. Similarly, bovine granulosa cells exposed to GLY at 0.5–5 μg/mL exhibited dose-dependent inhibition of E2 synthesis without affecting P4 secretion, likely due to direct interference with aromatase activity. In addition, another in vitro study showed that GBH (containing 10 μg/mL GLY) exposure significantly inhibited granulosa cell proliferation and steroid production (E2 and P4). Selective disruption of E2 production was associated with GLY-induced oxidative stress and apoptosis, which may drive steroidogenic dysfunction in murine ovaries, ultimately leading to hormonal imbalance and reduced fertility. However, higher concentrations of GBHs (10–300 mg/mL) caused broader suppression of both E2 and P4 in bovine models, suggesting concentration-dependent toxicity that may overwhelm compensatory mechanisms. Reduced E2 levels disrupted the positive feedback loop necessary for follicular selection and ovulation, while abnormal P4 dynamics, as observed in porcine oocytes, correlated with diminished blastocyst formation rates and compromised embryonic viability. These findings collectively position disrupted steroid hormone synthesis as an upstream key event that triggers downstream consequences, such as follicular atresia (via loss of E2-mediated survival signals) and ovulation failure (due to aberrant P4-regulated luteinization). , Paradoxically, some in vivo studies by Lorenz et al. and Guerrero et al. reported elevated serum E2 levels and decreased P4, suggesting complex systemic feedback mechanisms that may override local ovarian dysfunction. , This apparent contradiction likely arises from multilevel endocrine disruption spanning the HPO axis. GLY exposure induced upstream dysregulation of GnRH and pituitary gonadotropins, as demonstrated by the downregulation of GnRH gene expression and upregulation of FHSβ and LHβ expression. Compensatory increases in LH secretion could transiently stimulate residual ovarian steroidogenesis while simultaneously accelerating follicular depletion through excessive luteinization. Furthermore, the estrogenic activity of GLY via ERα upregulation may establish pseudohyperestrogenic conditions despite impaired ovarian E2 production. The observed serum hormone fluctuations likely reflect disrupted negative feedback mechanisms, wherein ovarian resistance to gonadotropins, combined with direct hypothalamic-pituitary effects and brain aromatase inhibition, generates oscillatory hormonal patterns. The divergent P4 responses observed between granulosa cells and whole-organism studies may be attributed to differences in the temporal and spatial dynamics. Acute in vitro exposure primarily affected the steroidogenic capacity of luteinized granulosa cells, whereas chronic in vivo exposure additionally impacts corpus luteum formation and maintenance, as well as extra-ovarian steroid metabolism. GLY-induced disruption of thyroid axis could indirectly modulate sex hormone binding globulin (SHBG) levels, thereby altering hormone bioavailability. Oxidative stress emerges as a unifying upstream mechanism, which may compromise mitochondrial function in steroidogenic cells while activating stress-responsive signaling pathways that dysregulate HPOA communication. Downstream consequences include impaired folliculogenesis, reduced oocyte competence, and uterine receptivity defects mediated by altered implantation-related genes ( Hoxa10, Lif ). These multifaceted disruptions may collectively contribute to diminished reproductive capacity across multiple biological levels. Oocyte maturation is a critical process in the female reproductive system involving the progression of oocytes from the germinal vesicle stage to metaphase II, culminating in the release of a mature ovum capable of fertilization. This process is essential for successful reproduction as it ensures the genetic integrity and developmental potential of the oocyte. GLY and GBHs have been shown to impair oocyte maturation and ovulation across multiple mammalian models through interconnected molecular mechanisms and endocrine disruptions. Chronic low-dose GLY exposure (2 mg/kg/day for 10 weeks beginning at 7 weeks of age) may selectively deplete secondary follicle populations via oxidative stress-mediated mechanisms, demonstrating stage-specific vulnerability during follicular maturation. Exposure to GLY/GBHs induces oxidative stress as a primary upstream event, which may trigger mitochondrial dysfunction, DNA damage, and the subsequent activation of apoptosis and autophagy pathways in oocytes. This cascade of cellular stress responses may induce meiotic arrest, as evidenced by decreased germinal vesicle breakdown (GVBD) rates and diminished polar body extrusion (PBE) efficiency across multiple species, including mice ( in vivo treatment with 250 mg/kg/day GLY), cattle ( in vitro treatment with 50 mM GLY), and pigs ( in vitro treatment with 400 μmol/L GLY). , , Compromised meiotic progression may further be exacerbated by structural disorganization of MTOCs and chromosomal misalignment, coupled with zinc depletion, a critical regulator of oocyte maturation. Additionally, GLY/GBHs reduce E2 and P4 production in granulosa cells, which may contribute to meiotic arrest and disrupt the final stages of follicular maturation and ovulation. , Ren et al. observed increased atretic follicles and decreased mature follicles in mice exposed to 5 g/L GLY in drinking water from the gestational day (GD) 1 to GD 19, along with ovarian stromal fibrosis. These changes were associated with a significant decline in serum P4 levels and a paradoxical increase in estrogen levels. Hormone imbalance may further lead to follicle atresia and reduced follicle surface area, as reported in other studies. Collectively, these toxicological and hormonal alterations establish a microenvironment wherein GLY exposure potentially triggers oxidative stress as an upstream initiating event, subsequently inducing meiotic arrest via impaired germinal vesicle breakdown and polar body extrusion. Ovarian reserve, defined as the number of oocytes within the ovary, declines over time. The finite pool of oocytes available for maturation and fertilization determines the length of a female’s reproductive lifespan. An intact follicle pool is essential for female fertility, and disruption of its formation can lead to subfertility or infertility. Emerging cross-species evidence identifies ovarian reserve depletion as a critical aspect of GLY-induced female reproductive toxicity mediated through molecular perturbations of folliculogenesis and endocrine dysregulation. For instance, Alarcon et al. revealed that 1 mg/kg/bw/day GBH exposure disrupts follicular development by downregulating key regulatory molecules, including AMH, BMP15, and FSHR, while impairing estrogen and progesterone receptor signaling pathways in peripubertal ewes. This molecular interference was associated with a pathological feedback loop in which reduced AMH expressiona biomarker of ovarian reserve, coincides with primordial follicle pool depletion and compromised follicle recruitment. Upstream endocrine disruption may further amplify follicular depletion. GBHs induce paradoxical steroidogenic shifts characterized by elevated levels of ovarian Hsd3b1 expression, potentially mediated through oxidative stress. This hormonal imbalance may promote premature luteinization of developing follicles, as evidenced by increased corpus luteum formation concurrent with diminished primary follicle counts. Simultaneously, GLY-induced oxidative stress may disrupt HPOA homeostasis, leading to endometrial gland atrophy and ovarian follicular shrinkage in prepubertal swine. These structural alterations may synergize with molecular disruptions that impair follicular nourishment and oocyte-granulosa cell communication. The downstream reproductive consequences may include diminished pregnancy success rates, reflecting both quantitative loss of ovarian reserve and qualitative impairment of remaining follicles. Endometrial hyperplasia (EH) is characterized by abnormal proliferation of endometrial glands relative to the stroma, resulting in an increased gland-to-stroma ratio compared to normal proliferative endometrium, and is strongly associated with prolonged estrogenic stimulation. Accumulating evidence suggests that EH serves as a key event in GLY/GBHs-induced female reproductive toxicity, linking molecular disturbances to pathological progression. As previously discussed, GLY may initiate endocrine disruption through direct activation of ERα and subsequent estrogen signaling pathways, leading to a significant hormonal imbalance characterized by an increased E2/P4 ratio. This establishes a hyperestrogenic microenvironment that can promote endometrial proliferation. , Sustained proliferative stimulus is measured by increased uterine wall thickness, luminal epithelial hyperplasia, and glandular abnormalities, including subgland formationa recognized precursor to estrogen-induced precancerous lesions. , , , Collectively, these findings suggest that EH may serve as both a biomarker of GLY-induced endocrine disruption and a functional link between MIEs and the downstream neoplastic progression in endometrial carcinogenesis.

Estrogen Receptor α (ERα) is a nuclear receptor that binds estrogens and is predominantly expressed in tissues such as the uterus, ovary (theca cells), testes (Leydig cells), breast, brain, and liver. After dimerization, ERα translocates to the nucleus where it binds to estrogen response elements (EREs) on DNA and recruits coactivators or corepressors, which subsequently regulate the expression of estrogen-responsive genes that play critical roles in reproductive processes such as follicular development, ovulation, and endometrial proliferation. Estrogenic effects can also occur through ligand-independent activation of ERα, wherein cellular signaling pathways induce ERα phosphorylation via protein kinase regulation and second messenger system modifications. , Multiple studies have shown that GLY and GBHs, such as Roundup, can activate ERα through direct or indirect mechanisms and disrupt estrogen signaling pathways. Dose-dependent phosphorylation at the Ser118 site, nuclear translocation of ERα, and upregulation of estrogen-responsive genes have been observed, which promote the proliferation of breast cancer cells (e.g., MCF-7 and T47D cells). GLY has also been shown to upregulate ERα expression in T47D cells. ERE-luciferase reporter gene assays confirmed its xenoestrogenic activity via ERα-mediated mechanisms, as both enhanced ERE transcriptional activity and T47D cell proliferation were abolished by treatments with an ERα antagonist. , Although molecular dynamic simulations suggest that GLY may interact with the ligand-binding domain of ERα by forming a complex with zinc ions, another study revealed significantly weak binding energy between GLY and ERα (−4.10 kcal/mol) compared to 17β estradiol (E2) (−25.79 kcal/mol), indicating an unstable interaction. Additionally, the same study reported that IBMX, a cAMP-PKA signaling activator, induced ERE-mediated reporter gene expression, suggesting that GLY may modulate ERα activity via ligand-independent pathways, such as cAMP-dependent PKA pathway. Nevertheless, the absence of direct binding evidence and in vivo validation limit conclusions regarding ligand-independent activation. These studies suggest that ERα is a potential initial target molecule for GLY/GBH, which may lead to adverse female reproductive outcomes such as abnormal endometrial proliferation, uterine lesions, and impaired embryonic development. Aromatase (Cyp19a1, estrogen synthase), a member of the cytochrome P450 superfamily, is a key enzyme in estrogen biosynthesis. In the specialized cells of the ovary, hypothalamus, and placenta, aromatase plays a crucial role in mammalian reproduction by catalyzing the conversion of androstenedione and testosterone to estrone (E1) and E2, respectively. In particular, ovarian aromatase generates both systemic and locally active estrogen. , Brain aromatase regulates the hypothalamic-pituitary–gonadal (HPG) axis through modulation of local estrogen synthesis, subsequently influencing gonadotropin-releasing hormone (GnRH) and kisspeptin release. In humans, aromatase is encoded by a single gene CYP19 , and targeted disruption of this gene or inhibition of its product can effectively eliminate estrogen biosynthesis. Much attention has been paid to understanding the regulation of the aromatase gene and its role in the development and progression of estrogen-dependent diseases such as breast cancer, endometrial cancer, and endometriosis. As a result, aromatase has been identified as a key molecular target for many environmental endocrine disruptors. Numerous in vitro studies have demonstrated that GLY and GBHs can directly suppress aromatase activity. Richard et al. first reported that GLY and GBHs dose-dependently inhibited aromatase activity in human JEG-3 placental cells. The IC 50 was 0.04% Roundup (equivalent to 0.84 mM GLY), which represents a concentration lower than that used in typical agricultural applications (1–2% Roundup, containing 21–42 mM GLY). Mechanistically, GLY and GBHs inhibited aromatase activity by binding directly to the active site of the enzyme, as evidenced by characteristic spectral changes (type II spectrum) resulting from interactions between GLY/GBHs and the heme iron atom of aromatase in purified aromatase systems. The data also indicated that GBHs exerted a stronger inhibitory effect on aromatase than GLY alone. The authors hypothesized that adjuvants in GBHs, such as polyoxyethylene amine (POEA), significantly improved cell membrane permeability and bioaccumulation, thereby increasing intracellular bioavailability and enhancing aromatase inhibition through targeted delivery to endocrine active tissues. Additionally, GLY and GBHs may reduce aromatase activity by downregulating the expression of the Cyp19 gene. Benachour et al. further demonstrated that GLY and GBHs not only interact directly with the activity site of aromatase, but also affect its auxiliary enzymesNADPH, a cytochrome P450 reductase. This dual mechanism enhances the inhibitory effect of GLY on aromatase in the cellular environment. GLY and GBHs exhibited more potent aromatase inhibition in human embryonic kidney 293 cells transfected with the aromatase gene than in microsomes, suggesting that the cellular environment may amplify their inhibitory effects. These findings suggest that GLY and its commercial formation may disrupt aromatase activity at concentrations below typical agricultural application levels, thereby interfering with estrogen synthesis. Such hormonal disturbances could potentially lead to adverse effects on fetal development and reproductive health. Although current in vivo studies in female mammals meeting the screening criteria remain insufficient to validate the aforementioned in vitro findings, a recent investigation on the GLY-induced reproductive toxicity in adult female climbing perch has produced results consistent with in vitro experimental conclusions. The study demonstrated that GLY binds to brain-type aromatase at residues MET424, THR423, and PRO479 (binding energy: −10.685 kcal/mol) and to ovarian-type aromatase at residues ASN479, THR477, among others (binding energy: −10.685 kcal/mol). All GLY-treated groups (2.6, 3.9, and 7.8 mg/L) exhibited ovarian follicular wall rupture and oocyte atresia, with additional vacuolization observed in the low-concentration group (2.6 mg/L). Notably, Cyp19A1A (ovarian-type) and Cyp19A1B (brain-type) expression was significantly downregulated in the highest concentration group (7.8 mg/L GLY). The authors hypothesize that GLY may impair reproductive function through aromatase inhibition and disruption of the HPG axis. Importantly, direct aromatase inhibition by GLY may contribute to ovarian dysfunction, hormone dysregulation, and impaired fetal development. The electron transport chain (ETC), also known as the respiratory chain, consists of large protein complexes (CI, CII, CIII, CIV, CV) and two mobile electron carriers, ubiquinone and cytochrome c (Cytc), located in the inner mitochondrial membrane cristae. Inhibition of the ETC triggers a cascade of mitochondrial events, including excessive production of reactive oxygen species (ROS), impaired oxidative phosphorylation leading to reduced ATP synthesis, a decreased ATP/ADP ratio, release of Cytc from the mitochondrial cristae, and loss of mitochondrial membrane potential (MMP). , Peixoto et al. were the first to demonstrate that 15 mM GBHs directly inhibit mitochondrial respiratory chain complexes II (succinate dehydrogenase, SDH) and III (succinate-Cytc reductase), thereby disrupting ETC function in isolated rat liver mitochondria. This inhibition triggered a series of adverse mitochondrial effects, including reduced mitochondrial membrane potential, uncoupled oxidative phosphorylation, suppressed ATP synthase activity, and consequent ATP synthesis reduction. Subsequent observations revealed mitochondrial swelling (15 mM GBH treatment) and increased membrane permeability (10 mM GBH treatment). The inhibition of ETC complexes has been identified as a key molecular mechanism contributing to GLY-induced female reproductive toxicity, as evidenced in human-derived in vitro models representing various components of the female reproductive system. Exposure to GLY or GBHs significantly suppressed SDH activity in human placental cells (JEG-3), human umbilical vein endothelial cells (HUVEC), and human embryonic kidney cells (HEK 293). Notably, GLY alone inhibited mitochondrial SDH activity across all tested cell types, showing partial inhibition observed at 7.2 g/L and significant inhibition at 360 g/L. However, GBH demonstrated more potent SDH inhibition at substantially lower concentration (0.5% Roundup, containing 1.8 g/L GLY equivalent), as formulation adjuvants (e.g., POEA) enhanced cellular uptake and destabilized mitochondrial membranes, thereby potentiating SDH inhibition. This mitochondrial respiratory chain impairment was consistently associated with increased apoptotic cell death in all tested cell lines. In vivo studies have reported that GBH exposure (3–10% GLY as Touchdown Hitech, containing 30–100 g/L GLY equivalent) inhibits mitochondrial SDH activity in Caenorhabditis elegans and reduces respiratory efficiency. However, research on GLY-induced placental mitochondrial dysfunction in mammals remains limited. Bai et al. reported that high-dose GBH exposure (100 mg/kg) during pregnancy significantly upregulated the mRNA expression of mitochondrial fission gene Fis1 , fusion gene MFN2 and SDHA in both porcine placenta and piglet jejunums, consequently impairing placental angiogenesis and mitochondrial function. , These findings suggest an evolutionarily conserved mechanism of mitochondrial dysfunction across cell types and underscore the potential role of ETC impairment in GLY-induced female reproductive toxicity.

A comprehensive literature search was conducted using the PubMed and Web of Science databases, covering articles published in English-language journals up to March 2025. The search used keywords such as “glyphosate,” “glyphosate-based herbicides,” “female reproduction,” “reproductive,” “oocyte,” and “fertility”. All retrieved articles were initially screened using the title and abstracts. Studies were excluded if they were: (1) nonoriginal research, including reviews or commentaries, (2) studies that did not focus on female reproductive toxicity, and (3) studies that did not use mammalian or cell culture models of the reproductive system. The screening process is shown in Figure . Following this process, 49 original studies were selected, all of which investigated the effects of GLY or GBHs on female reproductive toxicity using mammalian or cell culture models of the female reproductive system. Flow diagram for searching and selecting studies. This review establishes a qualitative AOP framework by synthesizing the available evidence in accordance with the development principles outlined in the OECD handbook. During the evidence synthesis process, we prioritized mammalian experimental evidence as well as studies using mammalian cell lines and human-derived cell lines, while nonmammalian model studies were excluded from the material scope of the AOP framework construction. The graphical linear flow diagram was employed to illustrate the proposed AOP network. The reported end points of GLY or GBH-induced female reproductive toxicity were classified into MIEs, KEs, and AOs according to the AOP framework. The KEs were further categorized into three biological levels ( Tables and S1 ): (1) molecular, (2) cellular, and (3) tissue or organic level. Furthermore, we aligned information on GLY/GBH-induced female reproductive toxicity mechanisms with existing MIEs, KEs, and AOs according to the AOP-wiki. The biological plausibility, the essentiality, and empirical support for each KER were evaluated as high, moderate, or low based on the Bradford-Hill criteria and OECD guidelines, , and the details of the evaluation criteria are provided in the Supporting Text 1 . These criteria were used to assess all KERs ( Table ). The following sections provide a comprehensive description of the KEs associated with GLY-induced female reproductive toxicity at each biological level. To clearly distinguish between hazard identification under high-dose exposure and risk characterization under realistic exposure scenarios, we calculated the maximum daily human exposure dose to GLY as 0.22 μg/kg/day based on the highest reported GLY concentration in human urine samples (7.6 μg/L) (see Supporting Text 2 for detailed calculation procedures). The widely accepted 10-fold safety factor approach was employed to derive human exposure limit standards, incorporating a threshold safety factor when extrapolating experimental data (including animal studies and in vitro test results) to establish a safe reference dose for humans. , As shown in Table S1 , all in vivo studies are classified into two categories: the first category is human-relevant exposure dose studies that can be used for risk characterization, defined as experimental doses ≤10× the estimated maximum daily human exposure dose (0.22 μg/kg/day), i.e., ≤2.2 μg/kg/day; the second category is exceed human-relevant exposure dose studies for hazard identification only, defined as experimental doses >10× the estimated maximum daily human exposure dose (0.22 μg/kg/day), i.e., >2.2 μg/kg/day. This classification method provides clear boundary standards for risk assessment.

This review systematically elucidated the mechanisms by which GLY may cause female reproductive toxicity across multiple biological levels by using the AOP framework. By integrating core concepts such as MIEs, KEs, and AOs, this review highlights toxicological end points triggered by GLY and its formulated herbicides (GBHs) in the female reproductive system. The AOP framework, constructed based on the AOP-wiki database and publicly available data, identifies three primary MIEs through which GLY may cause toxicity: (1) activation of ERα, (2) inhibition of aromatase activity, and (3) disruption of mitochondrial ETC complexes. These MIEs may trigger a cascade of KEs at the molecular and cellular levels, including increased ROS levels, oxidative stress, DNA damage, lipid peroxidation, disruption of estrogen receptor signaling pathways, mitochondrial dysfunction, abnormal epigenetic modifications, spindle apparatus defects, and altered chromosome dynamics, ultimately leading to cell death. At the tissue/organic level, critical events included hormonal homeostasis imbalance (e.g., reduced granulosa cell steroidogenesis and disrupted estradiol-to-progesterone ratio), impaired oocyte maturation, ovulatory dysfunction, depletion of ovarian reserve, and abnormal hyperplasia of the endometrial epithelium. These events may culminate in clinical conditions such as POI and endometrial cancer, resulting in diminished reproduction capacity. A summary of the MIEs, KEs, and AOs discussed is provided in Table . The KERs were assessed using Bradford-Hill criteria and OECD guidelines, , as shown in Table . All of the studies included in the assumed AOP framework construction in this article were based on mammalian models or mammalian/human-derived cell lines. Due to insufficient experimental data from mammalian studies, while two categories of nonmammalian models are cited in Sections and 3.3 for reference, these data were not included in our actual AOP development process. This study systematically organizes and constructs an AOP network of GLY-induced female reproductive toxicity ( Figure ), providing a scientific basis for establishing a health risk assessment system based on early KEs. It also lays a theoretical foundation for the development of prevention and control strategies and the identification of potential intervention targets. However, several limitations remain: (1) limited research has been conducted on the biological mechanisms of GLY and its alternative herbicides, as well as the human exposure-relevant doses that can be used for risk characterization. The hazard identification of GLY is mostly based on high-dose experiments, while risk characterization should rely on actual human-relevant exposure doses. Many existing toxicological in vivo studies use doses several orders of magnitude higher than human-relevant exposure levels, lacking in vivo studies of low-dose GLY exposure, which may lead to misjudgment of the effects of chronic low-dose exposure and make it impossible to characterize and assess the actual exposure risks. (2) The current evidence supporting the AOP mainly comes from animal and in vitro experiments, while epidemiological studies directly linking GLY exposure to human endometrial cancer, POI, and fertility decline remain scarce. The available data are insufficient to adequately translate experimental findings into human health risk assessments. There is an urgent need for more population-based studies (e.g., long-term cohort follow-ups) to elucidate the specific effects of GLY on female reproductive health. (3) Due to the lack of a recognized standardized conversion method across species/exposure conditions, heterogeneous data (e.g., interspecies differences, in vivo vs in vitro variations, exposure condition discrepancies, etc.) have not been formally weighted. (4) While qualitative AOPs can effectively integrate mechanistic evidence and identify hazards, they lack the predictive ability required for health risk assessment. For example, the current model lacks quantitative information on response-response relationships, cannot determine the dose threshold for AOs (such as POI onset), and cannot account for population differences in susceptibility. In contrast, quantitative AOPs (qAOPs) can address these limitations through computational models (such as Bayesian networks, physiologically based pharmacokinetic models), but their development faces challenges including high data requirements and modeling complexity. The qualitative approach was chosen for this study due to the current insufficient dose–response data for GLY across biological levels and the parametrization difficulties caused by interstudy heterogeneity (e.g., species differences, exposure protocols). The qualitative AOP framework provides a rapidly available conceptual basis under resource-limited conditions, although future upgrades to qAOPs will be necessary to support precise risk assessment. Adverse outcome pathway (AOP) network related to GLY/GBHs-associated female reproductive toxicity. All KEs, except “compromised endometrial receptivity”, are documented in the AOP-wiki and are identified by the respective KE IDs. Descriptions of some KE may differ from those in the AOP-wiki. Color coding: Green represents molecular initiating events; blue represents key events involved in molecular or cellular levels; yellow represents key events involved in tissue or organic levels; pink represents adverse outcomes. Solid lines indicate adjacent or strongly supported evidence relationships; dashed lines indicate nonadjacent or weaker evidence supporting relationship. Future research should focus on: (1) In-depth elucidation of the biological mechanisms of GLY and alternative herbicides toxicity, particularly their effects on epigenetic regulation and mitochondrial function, using human-relevant exposure concentrations to bridge hazard identification and risk characterization. (2) Large-scale epidemiological studies to corroborate findings from experimental models and assess real-world exposure scenarios. (3) To advance the development of quantitative AOPs (qAOPs), it is essential to systematically integrate in vivo dose–response data from mammalian studies to establish computable data sets while developing more systematic standardized approaches including the formulation of species extrapolation factors and unified dose measurement metrics. Priority should be given to validating high-impact KERs through in vitro experiments or in silico simulations. Furthermore, quantitative analysis of the associations between mechanistic events and AO probabilities using methods such as benchmark dose (BMD) modeling is crucial for supporting model development. Integrating physiologically based pharmacokinetic (PBPK) models with epidemiological data to calibrate qAOP parameters will ultimately enable the comprehensive enhancement of predictive accuracy. Addressing these research priorities will provide critical theoretical support for refining the health risk evaluation framework of GLY and formulating precise environmental management strategies.

Glyphosate (GLY), an aminophosphonic analogue of the natural amino acid glycine, has been extensively used as an herbicide since the early 1970s due to its ability to inhibit the biosynthesis of essential aromatic amino acids in plants. The development of genetically modified (GM) GLY-resistant crops in 1996 greatly expanded the agricultural use of GLY-based herbicides (GBHs), making them the most widely used herbicides worldwide. In 2024, GBHs were applied to over 350 million hectares globally, consuming approximately 8.6 billion kilograms. Adoption of GM GLY-tolerant crops is responsible for 56% of worldwide GLY consumption. As a result of its widespread use, GLY and its primary metabolite, aminomethylphosphonic acid (AMPA), are now environmentally ubiquitous, and detectable in human urine, serum, as well as breast milk. , In high-use regions, mean urinary concentrations of GLY reached 7.6 μg/L, exceeding levels in the general population (below 4 μg/L). A 2015–2016 Indiana cohort of 71 pregnant women showed that 93% had detections of urinary GLY (mean: 3.4 μg/L), with rural residents exhibiting elevated concentrations (4.19 μg/L) relative to 3.17–3.47 μg/L in suburban/urban subgroups. Similarly, in nonfarming households of Iowa, 88% of children and 65% of mothers had detectable urinary GLY with mean concentrations of 2.5 and 1.2 μg/L, respectively. Given its pervasive environmental presence and controversial toxicity, risk associated with GLY exposure has become a critical public health issue. The US Environmental Protection Agency (EPA) has established a chronic reference dose (cRfD) for GLY of 1.75 mg/kg/day, while the European Union (EU) has set an acceptable daily intake (ADI) of 0.3 mg/kg/day. In addition, the no-observed-adverse-effect level (NOAEL) for reproductive toxicity is set at 50 mg/kg. However, growing evidence from both in vitro and in vivo studies suggests that GLY and its commercial formulations (GBHs) may act as endocrine disruptors at doses near or below regulatory safety thresholds (e.g., cRfD or ADI), with adverse effects on reproduction. , Observed effects include hormonal imbalances, − proliferation/mitotic index alterations, , dysregulation of genes and proteins involved in endocrine signaling pathways, , , induction of oxidative stress, and epigenetic modifications such as changes in DNA methylation levels and histone posttranslational modifications (PTMs) in genes associated with endocrine function. Epidemiological studies have found that GLY exposure levels are associated with adverse birth outcomes such as preterm birth and birth defects, as well as alterations in sex hormone levels in adults and children. , − These findings highlight the endocrine-disrupting potential of GLY, primarily affecting sex hormones and the female reproductive organs, including the uterus and ovary. , , Notably, some studies suggest that GLY may induce multigenerational effects, potentially transmitting adverse outcomes across generations. , The toxicity of GLY has been extensively studied in the past few decades. Research on GLY-induced female reproductive toxicity has used various in vivo and in vitro models in toxicology and environmental health studies. Multiple molecular signaling pathways have been implicated in adverse reproduction effects in females. However, inconsistencies in findings often arise because of differences in models and exposure patterns. Given these complexities, there is an urgent need for a framework that integrates molecular and cellular events with adverse reproductive outcomes in individuals and populations. Such a framework would provide a holistic perspective for assessing the toxicity of GLY to the female reproductive system. The Adverse Outcome Pathway (AOP) framework is a conceptual model that links molecular initiating events (MIEs) to adverse outcomes (AOs) through a series of key events (KEs) connected by key event relationships (KER). MIEs, which serve as the starting point for an AOP, represent specific types of effects triggered by stressors or chemicals that interact with biological systems at the molecular level. KEs are defined as measurable changes in cellular or organic function that are linked by KERs and ultimately lead to AOs at the organism level. Since 2014, 35 AOPs have been officially published in the OECD iLibrary ( www.oecd-ilibrary.org ), with more than 500 additional AOPs currently under development on the AOP-wiki ( https://aopwiki.org/aops ). In recent years, AOP frameworks have been increasingly used to assess the safety and risk of chemical and environmental exposures, enabling the development of risk management strategies based on mechanistic toxicity data. , To date, AOPs have not been associated with GLY-induced female reproductive toxicity in the OECD iLibrary, AOP-wiki, or published literature. This review integrates in vivo and in vitro evidence to summarize current toxicological knowledge and potential mechanisms underlying GLY-induced female reproductive toxicity. Using the AOP-wiki and existing studies, we developed an AOP framework to elucidate the causal mechanism of GLY-induced female reproductive toxicity at multiple biological levels, from the molecular and cellular to the organ, individual, and population levels. This framework not only supports early risk assessment of GLY exposure but also identifies potential intervention targets and provides strategies to mitigate adverse effects.